HMOX1 inducers
阅读说明：本技术 Hmox1诱导剂 (HMOX1 inducers ) 是由 M·毕斗 A·克鲁格 S·萨斯墨 B·拉古 吴新元 T·荻山 E·贝尔 于 2020-04-08 设计创作，主要内容包括：本发明涉及作为血红素加氧酶1(HMOX 1)诱导剂的结构(I)的化合物。(式I)本发明还涉及一种控制哺乳动物个体中的血红素加氧酶1的活性或其量或活性和其量两者的方法。本文中提供了变量的定义。(The present invention relates to compounds of structure (I) that are inhibitors of heme oxygenase 1(HMOX 1). (formula I))
1. A compound represented by structural formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
ring A is a 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclyl, 5-6 membered heteroaryl or phenyl;
x is-S-, -O-or-NRb-；
When X is-S-and ring a is phenyl, then R is 3-7 membered monocyclic heterocyclyl optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy and-NRaRa；
When X is-S-and Ring A is 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclyl or 5-6 membered heteroaryl; or when X is-O-or-NRb-time of day; then R is-H, - (C)1-C4) Alkyl or- (CH)2)i3-7 membered monocyclic heterocyclic group, wherein said- (C) represented by R1-C4) Alkyl or 3-7 membered monocyclic heterocyclyl is optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C) 1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy, -SO2Raand-NRaRa；
Each R1Independently is-H, halogen, - (CH)2)kCOOH、-(CH2)kCO(C1-C4) Alkyl, - (CH)2)kCOO(C1-C4) Alkyl, - (CH)2)pC(＝O)NRaR3、-CH(CF3)NRaR3、-C(＝NOH)CF3or-CH (CF)3)OR3Wherein is represented by R1Said (C) in the group represented1-C4) Alkyl is optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy and-NRaRa；
Each R2Independently is-H, halo, CN, - (C)1-C4) Alkyl, -OH, - (C)1-C4) Alkoxy, -COOH, -C (═ O) (C)1-C4) Alkyl, -C (═ O) O (C)1-C4) Alkyl, -C (═ O) NRa(C1-C4) Alkyl, -NRaRa3-6 membered monocyclic cycloalkyl, -O (CH)2)i3-7 membered monocyclic heterocyclic group, 3-7 membered monocyclic heterocyclic group or 5-6 membered heteroaryl group, wherein R is substituted with R2Is represented by R2Said- (C) in the group represented1-C4) Alkyl, - (C)1-C4) Alkoxy, heterocyclyl or heteroaryl groups are optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Haloalkoxy and-NRaRa；
Each R3Independently H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy, - (CH)2)p13-7 membered monocyclic heterocyclic group, - (CH)2)p1NH3-7 membered monocyclic heterocyclyl, 5-6 membered heteroaryl, -O (CH) 2)p2NRaC(＝O)(C1-C4) Alkyl, - (CH)2)p2O(CH2)p2O(C＝O)(C1-C4) Alkyl or (CH)2)p2NRaC(＝O)(C1-C4) Alkyl radical, wherein R3Is represented by R3Said alkyl, alkoxy, heteroaryl or heterocyclyl group in the groups represented are optionally substituted by one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Haloalkoxy, - (C)1-C4) Carboxyalkoxy, -S (O)2(C1-C4) Alkyl, - (C)1-C4) Hydroxyalkoxy and-NRaRa；
Each RaIndependently is-H or- (C)1-C4) An alkyl group;
each RbIndependently is-H or- (C)1-C4) An alkyl group; wherein is represented by RbThe above- (C) of1-C4) Alkyl is optionally substituted with one or more groups selected from: halo, -CN, -OH, - (C)3-C6) Cycloalkyl, phenyl, 3-7 membered monocyclic heterocyclyl and 5-6 membered heteroaryl;
i is 0 or 1;
k is 0, 1, 2, 3 or 4;
m is 0, 1, 2, 3 or 4;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
p1is 0, 1, 2, 3 or 4; and
p2is 2, 3 or 4.
2. The compound of claim 1, wherein the compound is represented by structural formula (II):
or a pharmaceutically acceptable salt thereof.
3. The compound of claim 1, wherein the compound is represented by structural formula (II'):
or a pharmaceutically acceptable salt thereof.
4. The compound of claim 1 or 2, wherein the compound is represented by structural formula (II-a):
or a pharmaceutically acceptable salt thereof.
5. The compound of claim 1 or 2, wherein the compound is represented by structural formula (II-B):
or a pharmaceutically acceptable salt thereof.
6. The compound of claim 1 or 2, wherein the compound is represented by structural formula (II-C):
or a pharmaceutically acceptable salt thereof.
7. The compound of claim 1 or 3, wherein the compound is represented by structural formula (II' -a):
or a pharmaceutically acceptable salt thereof.
8. A compound according to claim 6, or a pharmaceutically acceptable salt thereof, wherein Rbis-H or- (C)1-C4) Alkyl radical, wherein RbThe above- (C) of1-C4) Alkyl is optionally substituted with one or more groups selected from halo, -CN, and-OH.
9. According to claims 1 to 3 or 5 to 8The compound of any one of, or a pharmaceutically acceptable salt thereof, wherein R is optionally- (C)1-C4) Alkoxy substituted- (C)1-C4) An alkyl group; or- (CH) optionally substituted by one or more groups selected from the group consisting of2)i3-6 membered monocyclic heterocyclic group: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C) 1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy and-NRaRa。
10. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R is azetidinyl, azepanyl, morpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxetanyl, tetrahydrofuranyl or tetrahydropyranyl, each of which is optionally substituted with one or more groups selected from the group consisting of: halo, -OH, - (C)1-C4) Alkyl and-NH2。
11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein R1Is- (CH)2)kCOOH, - (CH) optionally substituted by halo2)kCO(C1-C4) Alkyl, - (CH)2)pC(＝O)NRaR3、-C(＝NOH)CF3or-CH (CF)3)NRaR3。
12. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R2Is H; halogen; CN; optionally halogen-substituted- (C)1-C4) An alkyl group; - (C) optionally substituted by halo, hydroxy, methoxy or ethoxy1-C4) An alkoxy group; -C (═ O) (C)1-C4) An alkyl group; 3-4 membered monocyclic cycloalkyl; -O (CH)2)i3-7 membered monocyclic heterocyclic group, 3-7 membered monocyclic ringHeterocyclyl or 5-6 membered heteroaryl, wherein R is2Is represented by R2Said heterocyclyl or heteroaryl of a group represented by (A) is optionally substituted by halo, hydroxy or- (C) 1-C4) Alkoxy substitution.
13. The compound according to any one of claims 1 to 3, 5 to 9, 11 or 12, or a pharmaceutically acceptable salt thereof, wherein R is optionally substituted with methoxy or-SO2CH3Substituted- (C)1-C4) An alkyl group; - (CH)2) A tetrahydrofuranyl group; - (CH) optionally substituted by hydroxy2) An oxetanyl group; a pyrrolidinyl group; a piperidinyl group; or tetrahydropyranyl; wherein said pyrrolidinyl or piperidinyl is optionally substituted with- (C)1-C4) Alkyl substitution.
14. The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein R1is-COOH, -C (═ O) CF3、-CH(CF3)(NH2)、-C(＝NOH)CF3or-C (═ O) NHR3。
15. The compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein R3Is (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (CH)2)p13-6 membered monocyclic heterocyclic group or (CH)2)p2NHC(＝O)(C1-C4) Alkyl radical, wherein R3Is represented by R3Said alkyl, alkoxy or heterocyclyl group in the groups represented are optionally substituted by one or more groups selected from the group consisting of: halo, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy, -S (O)2(C1-C4) Alkyl, - (C)1-C4) Hydroxyalkoxy and-NRaRa。
16. The compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein R 2Is H; halogen; CN; optionally halogen-substituted- (C)1-C4) An alkyl group; - (C) optionally substituted by halo, hydroxy, methoxy or ethoxy1-C4) An alkoxy group; -C (═ O) (C)1-C4) An alkyl group; a cyclopropyl group; o-tetrahydropyranyl; an N-pyrrolidinyl group; or a thiazolyl group.
17. The compound of claim 5, wherein the compound is represented by structural formula (III-B):
or a pharmaceutically acceptable salt thereof.
18. A compound according to any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, wherein
R1is-COOH or-C (═ O) NHR3，
R3Is- (C)1-C4) Alkyl, -hydroxy (C)1-C4) Alkyl, -methoxy (C)1-C4) Alkyl, amino (C)1-C4) Alkyl, - (C)1-C4) Hydroxyalkoxy (C)1-C4) Alkyl or- (CH)2)p2NHC (═ O) (dimethylamino (C)1-C4) Alkyl groups).
19. The compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, wherein R2Is H, halo, - (C)1-C4) Haloalkyl, - (C)1-C4) Haloalkoxy, optionally methoxy-substituted- (C)1-C4) Alkoxy or-N-pyrrolidinyl.
20. The compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, wherein R2Is H, F, CF3、OCHF2、OCF3、OCH2CH2OCH3or-N-pyrrolidinyl.
21. The compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein
R2Is H, R1Is optionally substituted by-OH, NH2Oxetanyl or- (C)1-C4) Hydroxyalkoxy substituted-C (═ O) NH (C)1-C4) An alkyl group; or-C (═ O) NH (CH)2)p2NHC (═ O) (dimethylamino (C)1-C4) Alkyl groups); or
R2Is F, R1is-C (═ O) NH (methoxy (C)1-C4) Alkyl groups); or
R2is-N-pyrrolidinyl, R1is-C (═ O) NH ((C)1-C4) Hydroxyalkoxy (C)1-C4) An alkyl group),
R2is CF3、OCHF2Or OCF3，R1is-COOH; or
R2Is OCH2CH2OCH3，R1is-C (═ O) NH ((C)1-C4) Hydroxyalkoxy (C)1-C4) Alkyl), -C (═ O) NH (hydroxy (C)1-C4) Alkyl), -C (═ O) NH (methoxy (C)1-C4) Alkyl) or-C (═ O) NH (hydroxy (C)1-C4) Alkoxy groups).
22. A compound according to 17 or a pharmaceutically acceptable salt thereof, wherein
R1Is optionally substituted by-OH, NH2、-(C1-C4) -alkoxy or- (C)1-C4) Hydroxyalkoxy substituted-C (═ O) NH (C)1-C4) Alkyl, and
R2is H or OCH2CH2OCH3。
23. A compound according to 22 or a pharmaceutically acceptable salt thereof, wherein
R2Is H.
24. A compound according to 23 or a pharmaceutically acceptable salt thereof, wherein
R1Is a quilt- (C)1-C4) Hydroxyalkoxy substituted-C (═ O) NH (C)1-C4) An alkyl group.
25. The compound of claim 24, wherein the compound is:
2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide or a pharmaceutically acceptable salt thereof.
26. A compound according to 22 or a pharmaceutically acceptable salt thereof, wherein
R1Is a quilt- (C)1-C4) Alkoxy substituted-C (═ O) NH (C)1-C4) Alkyl, and
R2is OCH2CH2OCH3。
27. The compound of claim 26, wherein the compound is:
2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide or a pharmaceutically acceptable salt thereof.
28. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is
29. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof.
30. A method of increasing the activity of HMOX1 or an amount thereof in a human subject comprising: administering to a human subject an effective amount of a compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition according to claim 29.
31. A method of activating the transcription factor Nrf2 in a human individual, comprising: administering to a human subject an effective amount of a compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition according to claim 29.
32. A method of reducing the amount of ROS in a human subject, comprising: administering to a human subject an effective amount of a compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition according to claim 29.
33. A method of treating a disease, disorder, or condition, comprising administering to a human subject an effective amount of a compound of any one of claims 1-28, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition of claim 29, wherein the disease, disorder, or condition is: (i) fibrotic diseases including pulmonary fibrosis diseases, Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis, sarcoidosis, hepatic fibrosis diseases including those caused by alcoholic cirrhosis, steatosis, cholestasis, drug side effects and viral infections, dermal fibrotic diseases, scleroderma or psoriasis; (ii) neurodegenerative diseases, including Friedreich's ataxia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, cerebral neurodegenerative diseases or Charcot-Marie-Tooth syndrome; (iii) cardiovascular disease, including hypertension, hypercholesterolemia, atherosclerosis, arteriosclerosis, thrombosis, acute coronary thrombosis, deep vein thrombosis, peripheral vascular disease, congestive heart failure, acute coronary syndrome, arterial fistula dialysis failure, ischemia reperfusion injury, primary pulmonary hypertension, or secondary pulmonary hypertension; (iv) renal diseases including acute kidney injury, polycystic kidney disease, Alport syndrome (Alport syndrome), diabetic nephropathy, glomerulonephritis, lupus nephritis, sickle cell nephropathy, and acute tubular necrosis; (v) inflammatory diseases, including asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, inflammatory bowel syndrome, Crohn's disease, celiac disease, ulcerative colitis, chronic inflammatory bowel disease, scleroderma, dermatitis, systemic lupus erythematosus, esophagitis, vasculitis, pancreatitis, tendonitis, osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, or chronic inflammation of the brain; (vi) liver diseases including drug-induced hepatotoxicity, non-alcoholic steatohepatitis, hepatitis b infection or hepatitis c infection; (vii) eye diseases including conjunctivitis, glaucoma, uveitis, eye trauma, corneal transplants, Focus 'endothelial dystrophy (Fuchs' endokelial corneal dystrophy), macular degeneration, cataracts, mild retinopathy, retinitis pigmentosa, diabetic retinopathy and retinopathy of prematurity; (viii) thyroid disorders including Graves' disease, follicular adenoma, or papillary and follicular carcinoma; (ix) viral infections, including human immunodeficiency virus, hepatitis b, hepatitis c, or herpes virus infections; (x) Osteoporosis; (xi) A pregnancy disorder; (xii) Endometriosis; (xiii) Diabetes, including type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, hyperglycemia, metabolic syndrome, or a secondary condition resulting from a diabetic condition; (xiv) Cancer; (xv) Skin diseases including dermatitis, scleroderma or psoriasis; (xvi) Mitochondrial diseases such as mitochondrial myopathy, Leber's Hereditary Optic Neuropathy (LHON), myoclonic epilepsy with fragmented red fibers (MERFF), mitochondrial encephalomyopathy, lactic acidosis and stroke-like attacks (MELAS) or Leigh's syndrome; (xvii) Hematological diseases such as Dabby anemia (Diamond Blackfan anemia), myelodysplastic syndrome, sickle cell disease, and beta-thalassemia; or (xviii) muscle diseases such as Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy and rhabdomyolysis.
34. A compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 29, for use in treating a disease, disorder or condition of claim 33 in a human subject, comprising administering to the subject in need thereof.
35. Use of a compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 29, for the manufacture of a medicament for treating a disease, disorder or condition of claim 33 in a human subject, comprising administering to the subject in need thereof.
36. Use of a compound according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 29, for treating a disease, disorder or condition of claim 33 in a human subject, comprising administering to the subject in need thereof.
Technical Field
The present application relates to heme oxygenase 1(HMOX1) inducers and methods of use thereof, such as for controlling the activity or the amount or both the activity and the amount of heme oxygenase in a mammalian subject.
Background
Oxidative stress represents an imbalance between cellular Reactive Oxygen Species (ROS) production and cellular responses to ROS, such as the degradation of ROS species and the production of endogenous antioxidant molecules.
ROS provide critical cell signaling requirements, but can have deleterious effects if overproduced or allowed to develop. Increased levels of ROS in cells can result in damage to components such as lipids, proteins, polysaccharides, and DNA. Chronic oxidative stress is also associated with chronic diseases affecting almost every major organ system. For example, chronic oxidative stress involves the onset or progression of disease conditions such as neurodegenerative diseases, lung diseases, cardiovascular diseases, renal diseases, diabetes, inflammatory pain, and cancer. Therefore, strategies to alleviate oxidative stress are desirable for many therapeutic situations.
Under normal physiological conditions, ROS production is balanced by a well-defined and conserved set of cellular pathways that respond to, limit, and repair damage caused by ROS. This adaptive genome is called phase II system. It encodes enzymes that directly degrade ROS as well as increase the levels of cellular endogenous antioxidant molecules comprising glutathione and bilirubin.
In the phase II enzyme system, HMOX1 (a human gene encoding the enzyme heme oxygenase 1) has been found to be a key component. HMOX1 functions to metabolize heme into bilirubin, carbon monoxide and free iron by a two-step process. The first and rate limiting step is the production of biliverdin and carbon monoxide from heme by HMOX 1. The second step is the production of bilirubin from biliverdin by biliverdin reductase. Bilirubin and carbon monoxide have been shown to scavenge ROS and have potent antioxidant and anti-inflammatory activities.
Agents that induce HMOX1 production have been shown to have beneficial activity in models of diabetes, cardiovascular disease, hypertension, and lung function. Heme, heavy metal ions (e.g., arsenite, cadmium, iron, lead, chromium, and mercury), and electrophilic reagents (e.g., natural products such as sulforaphane and curcumin) can all induce HMOX1 production. Induction of HMOX1 and other phase II genes (phase II genes) is controlled by a variety of transcription factors that respond to heavy metals, heme, and electrophilic reagents. Transcription factors Nrf2, Bach1 and small Maf proteins are particularly important in this process. For example, a common sequence called Antioxidant Responsive Element (ARE) is present in the promoter of each gene of the phase II enzyme, and its expression is induced by the transcription factor Nrf2 (NF-E2-related factor 2).
HMOX1 was also induced as part of a general stress response to stimuli such as: thermal shock; oxidative stress; and cytokines such as interleukin-1 (IL-1), tumor necrosis factor, and interleukin-6 (IL-6). This stress response is considered beneficial because it protects vulnerable cells from multiple insults.
It was reported that HMOX1 can be induced by small molecules that bind to the transcription factor Bach 1. Heme binding to Bach1 has been shown to reduce the DNA binding activity of Bach1 and induce gene transcription. See Ogawa K et al, EMBO J (EMBO J) (2001)20: 2835-. Furthermore, it was reported that small molecules induce HMOX1 by binding to Bach 1. See Attucks OC et al, public science library Integrated services (PLOS ONE) (2014)9(7) e101044, WO2011/103018 and WO 2012/094580.
Therefore, there is a need for new HMOX1 inducers and/or Bach1 binders/inhibitors for the above-referenced therapeutic indications.
Disclosure of Invention
Applicants have discovered novel compounds that are potent inducers of HMOX1 (see examples 1-142). In particular, certain compounds of the invention have been shown to effectively induce the production of HMOX1 (see example 143). Furthermore, certain compounds of the invention have a combination of desirable properties, including potent HMOX 1-inducing activity (example 143), significantly reduced hERG inhibition compared to certain comparative compounds (see example 145), good solubility (example 144), and potent effect on HMOX1 protein expression in vivo (example 151). In addition, applicants have also found that the compounds disclosed herein bind to Bach1 (see example 146).
In one embodiment, provided herein is a compound represented by the following structural formula (I):
or a pharmaceutically acceptable salt thereof, the definitions of each variable being provided below.
Also disclosed herein are pharmaceutical compositions of the compounds of the invention. Particular embodiments include a pharmaceutically acceptable carrier or diluent and one or more of the compounds of the invention or a pharmaceutically acceptable salt thereof.
Compounds of formula (I) and pharmaceutically acceptable salts thereof are useful as agents that induce the production of HMOX1 and/or increase its activity, and thus are useful in the treatment of various chronic diseases associated at least in part with oxidative stress, including but not limited to: fibrotic diseases, neurodegenerative diseases, cardiovascular diseases, kidney diseases, inflammatory diseases, liver diseases, eye diseases, thyroid diseases, viral infections, osteoporosis, pregnancy disorders, endometriosis, diabetes, cancer, skin diseases, mitochondrial diseases, blood disorders and muscle diseases.
Another embodiment of the invention includes treating the above-mentioned disease or condition in a subject by administering to the subject an effective amount of one or more compounds of the invention, or pharmaceutically acceptable salts thereof, or a pharmaceutical composition including the disclosed compounds.
Also provided herein is the use of one or more compounds of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds of the invention, for the manufacture of a medicament for the treatment of the above-mentioned diseases or conditions.
In another embodiment, provided herein is a compound of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more of the disclosed compounds, for use in treating the above-mentioned diseases or conditions.
Drawings
Fig. 1A and 1B depict graphs showing significant fold changes in gene-specific mRNA in HepG2 cells treated with the compound of example 17 or the Nrf2 activator DMF. Figure 1A depicts that the compound of example 17 induces HMOX1 expression more strongly than DMF. Figure 1B depicts that the compound of example 17 and DMF induced FTH1 expression in comparable fold increases.
Figure 2 depicts a graph showing that the compound of example 17 does responsively inhibit microvascular stasis in Townes-SS sickle cell disease mice. Townes-SS mice were gavaged once daily for 8 days with vehicle or compound of example 17. After the last gavage, the dorsal skinfold chamber was transplanted on mice, and 20 to 22 flowing venules were selected and located in the subcutaneous skin using in vivo microscopy. Mice were then infused with pan-heme (panhemin) (3.2 μmol heme/kg body weight) via the tail vein. One hour after infusion, each venule was examined for microvascular stasis (no flow) and the data were expressed as% stasis. The fouling value is the mean + SD. One-way ANOVA with Dunnett's multiple comparison to vehicle, P <0.0001
Figure 3 depicts a graph showing the percentage of F cell increase in Townes-SS mice treated with the compound of example 17. Blood smears were made using heparinized whole blood for F cell staining. Counting stained F cells and total red blood cells in four fields of view on a blood smear; average 77 erythrocytes/field. F cells were expressed as a percentage of total red blood cells. The value is mean + SD. One-way ANOVA with Dunnett multiple comparison with vehicle, p ≦ 0.01, p ≦ 0.001, p ≦ 0.0001
Fig. 4 depicts a graph showing Glutathione (GSH) levels in primary human endothelial cells. Compared with the two-tailed unpaired t test of DMSO, the # p is less than or equal to 0.0001; one-way ANOVA (GSH pattern) with Dunnett multiple comparison to hemin ≦ 0.0001
Fig. 5 depicts a graph showing fold-change in gene expression in primary human endothelial cells. # p ≦ 0.0001, one-way ANOVA (VCAM plot) in case of Dunnett multiple comparison with TNFa ≦ 0.0001
Detailed Description
In response to higher levels of Reactive Oxygen Species (ROS), cells induce the expression of oxidative stress responsive genes, such as genes encoding proteins that degrade ROS or increase the levels of endogenous antioxidant molecules in the unit. One such gene is HMOX 1. Induction of expression of HMOX1 and other oxidative stress response genes is regulated in part by the transcription factor Nrf 2. On the basis, the linker protein Keap1 forms a heterodimer with Nrf2, targets Nrf2 for proteolysis and inhibits Nrf 2-regulated transcription. When cells are exposed to a chemical electrophile or agent that elevates ROS, Keap1 interaction with Nrf2 is attenuated and Nrf2 levels in the cells are increased, which in turn increases Nrf2 levels in the nucleus and causes induction of oxidative stress response genes. Nrf2 activity is also regulated by a transcriptional repressor Bach1, which Bach1 blocks the binding of Nrf2 to the promoter region of oxidative stress responsive genes.
In order to mitigate the effects of oxidative stress in cells, for example in disease situations, it is therefore desirable to identify compounds that promote the induction of the expression of oxidative stress response genes, for example compounds that modulate the interaction of Nrf2 with Keap1 or Bach1 with Maf recognition elements (MAREs) to increase transcription of cytoprotective genes. However, it is also desirable that such compounds do not act as electrophiles or otherwise stimulate stress in the cell.
Compounds of the invention
Disclosed herein are examples of compounds having the general structural formula (I).
In a first embodiment, the present invention provides a compound represented by the following structural formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
ring A is a 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclyl, 5-6 membered heteroaryl or phenyl;
x is-S-, -O-or-NRb-；
When X is-S-and ring a is phenyl, then R is 3-7 membered monocyclic heterocyclyl optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy and-NRaRa；
When X is-S-and Ring A is 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclyl or 5-6 membered heteroaryl; or when X is-O-or-NR b-time of day; then R is-H, - (C)1-C4) Alkyl or- (CH)2)i3-7 membered monocyclic heterocyclic group, wherein said- (C) represented by R1-C4) Alkyl or 3-7 membered monocyclic heterocyclyl is optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy, -SO2Raand-NRaRa；
Each R1Independently is-H, halogen, - (CH)2)kCOOH、-(CH2)kCO(C1-C4) Alkyl, - (CH)2)kCOO(C1-C4) Alkyl, - (CH)2)pC(＝O)NRaR3、-CH(CF3)NRaR3、-C(＝NOH)CF3or-CH (CF)3)OR3Wherein is represented by R1Said (C) in the group represented1-C4) Alkyl is optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy and-NRaRa；
Each R2Independently is-H, halo, CN, - (C)1-C4) Alkyl, -OH, - (C)1-C4) Alkoxy, -COOH, -C (═ O) (C)1-C4) Alkyl, -C (═ O) O (C)1-C4) Alkyl, -C (═ O) NRa(C1-C4) Alkyl, -NRaRa3-6 membered monocyclic cycloalkyl, -O (CH)2)i3-7 membered monocyclic heterocyclic group, 3-7 membered monocyclic heterocyclic group or 5-6 membered heteroaryl group, wherein R is substituted with R2Is represented by R2Said- (C) in the group represented1-C4) Alkyl, - (C)1-C4) Alkoxy, heterocyclyl or heteroaryl groups are optionally substituted with one or more groups selected from the group consisting of: halo, -CN, -OH, - (C) 1-C4) Alkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Haloalkoxy and-NRaRa；
Each R3Independently H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy, - (CH)2)p13-7 membered monocyclic heterocyclic group, - (CH)2)p1NH3-7 membered monocyclic heterocyclyl, 5-6 membered heteroaryl, -O (CH)2)p2NRaC(＝O)(C1-C4) Alkyl, - (CH)2)p2O(CH2)p2O(C＝O)(C1-C4) Alkyl or (CH)2)p2NRaC(＝O)(C1-C4) Alkyl radical, wherein R3Is represented by R3Said alkyl, alkoxy, heteroaryl or heterocyclyl group in the groups represented are optionally substituted by one or more groups selected from the group consisting of: halo, -CN, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy, - (C)1-C4) Carboxyalkoxy, -S (O)2(C1-C4) Alkyl, - (C)1-C4) Hydroxyalkoxy and-NRaRa；
Each RaIndependently is-H or- (C)1-C4) An alkyl group;
each RbIndependently is-H or- (C)1-C4) An alkyl group; wherein is represented by RbThe above- (C) of1-C4) Alkyl is optionally substituted with one or more groups selected from: halo, -CN, -OH, - (C)3-C6) Cycloalkyl, phenyl, 3-7 membered monocyclic heterocyclyl and 5-6 membered heteroaryl;
i is 0 or 1;
k is 0, 1, 2, 3 or 4;
m is 0, 1, 2, 3 or 4;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
p1is 0, 1, 2, 3 or 4; and
p2is 2, 3 or 4.
In a second embodiment, the present disclosure provides a compound according to the previous embodiment, wherein the compound is represented by the following structural formula (II):
or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In a third embodiment, the present disclosure provides a compound according to the first embodiment, wherein the compound is represented by structural formula (II'):
or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In a fourth embodiment, the present disclosure provides a compound according to the first or second embodiment, wherein the compound is represented by structural formula (II-a):
or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In a fifth embodiment, the present disclosure provides a compound according to the first or second embodiment, wherein the compound is represented by structural formula (II-B):
or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In a sixth embodiment, the present disclosure provides a compound according to the first or second embodiment, wherein the compound is represented by structural formula (II-C):
Or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In a seventh embodiment, the present disclosure provides a compound according to the first or third embodiment, wherein the compound is represented by structural formula (II' -a):
or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In an eighth embodiment, the present invention provides a compound according to the sixth embodiment or a pharmaceutically acceptable salt thereof, wherein Rbis-H or- (C)1-C4) Alkyl radical, wherein RbThe above- (C) of1-C4) Alkyl is optionally substituted with one or more groups selected from: halo, -CN and-OH. Wherein the remainder of the variables are as defined in the first embodiment. In a preferred embodiment, Rbis-H.
In a ninth embodiment, the present invention provides a compound according to the first, second, third, fifth, sixth, seventh and eighth embodiments, or a pharmaceutically acceptable salt thereof, wherein R is optionally- (C)1-C4) Alkoxy substituted- (C)1-C4) An alkyl group; or- (CH) optionally substituted by one or more groups selected from the group consisting of2)i3-6 membered monocyclic heterocyclic group: halo, -CN, -OH, - (C) 1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy and-NRaRaWherein the remainder of the variables are as defined in the first or eighth embodiment.
In a tenth embodiment, the present disclosure provides a compound according to the first, second, third, fourth, fifth, sixth, seventh or eighth embodiment, or a pharmaceutically acceptable salt thereof, wherein R is azetidinyl, azepanyl, morpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxetanyl, tetrahydrofuranyl or tetrahydropyranyl, each of which is optionally substituted with one or more groups selected from the group consisting of: halo, -OH, - (C)1-C4) Alkyl and-NH2Wherein the remainder of the variables are as defined in the first or eighth embodiment.
In an eleventh embodiment, the present disclosure provides a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth embodiment orA pharmaceutically acceptable salt thereof, wherein R1Is- (CH)2)kCOOH, - (CH) optionally substituted by halo2)kCO(C1-C4) Alkyl, - (CH)2)pC(＝O)NRaR3、-C(＝NOH)CF3or-CH (CF)3)NRaR3Wherein the remainder of the variables are as defined in the first, eighth, ninth or tenth embodiments.
In a twelfth embodiment, the present disclosure provides a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or eleventh embodiment or a pharmaceutically acceptable salt thereof, wherein R is2Is H; halogen; CN; optionally halogen-substituted- (C)1-C4) An alkyl group; - (C) optionally substituted by halo, hydroxy, methoxy or ethoxy1-C4) An alkoxy group; -C (═ O) (C)1-C4) An alkyl group; 3-4 membered monocyclic cycloalkyl; -O (CH)2)i3-7 membered monocyclic heterocyclic group, 3-7 membered monocyclic heterocyclic group or 5-6 membered heteroaryl group, wherein R is substituted with R2Is represented by R2Said heterocyclyl or heteroaryl of a group represented by (A) is optionally substituted by halo, hydroxy or- (C)1-C4) Alkoxy, wherein the remainder of the variables are as defined in the first, eighth, ninth, tenth or eleventh embodiments. In a preferred embodiment, R2Is H or-OCF3。
In a thirteenth embodiment, the present invention provides a compound according to the first, second, third, fifth, sixth, seventh, eighth, ninth, eleventh or twelfth embodiment or a pharmaceutically acceptable salt thereof, wherein R is optionally substituted with methoxy or-SO2CH3Substituted- (C)1-C4) An alkyl group; - (CH)2) A tetrahydrofuranyl group; - (CH) optionally substituted by hydroxy 2) An oxetanyl group; a pyrrolidinyl group; a piperidinyl group; or tetrahydropyranyl; wherein said pyrrolidinyl or piperidinyl is optionally substituted with- (C)1-C4) Alkyl substitution, wherein the remainder of the variables are as first, eighth, ninth, tenth, eleventh orAs defined in the twelfth embodiment. In a preferred embodiment, pyrrolidinyl or piperidinyl is optionally substituted with-CH3And (4) substitution.
In a fourteenth embodiment, the present invention provides a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth embodiment or a pharmaceutically acceptable salt thereof, wherein R is1is-COOH, -C (═ O) CF3、-CH(CF3)(NH2)、-C(＝NOH)CF3or-C (═ O) NHR3Wherein the remainder of the variables are as defined in the first, eighth, ninth, tenth, eleventh, twelfth or thirteenth embodiment.
In a fifteenth embodiment, the present disclosure provides a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth embodiment or a pharmaceutically acceptable salt thereof, wherein R is3Is (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (CH) 2)p13-6 membered monocyclic heterocyclic group or (CH)2)p2NHC(＝O)(C1-C4) Alkyl radical, wherein R3Is represented by R3Said alkyl, alkoxy or heterocyclyl group in the groups represented are optionally substituted by one or more groups selected from the group consisting of: halo, -OH, - (C)1-C4) Alkyl, - (C)1-C4) Haloalkyl, - (C)1-C4) Hydroxyalkyl, - (C)1-C4) Alkoxy, - (C)1-C4) Haloalkoxy, -S (O)2(C1-C4) Alkyl, - (C)1-C4) Hydroxyalkoxy and-NRaRaWherein the remainder of the variables are as defined in the first, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth embodiment.
In a sixteenth embodiment, the present disclosure provides a computer readable medium according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or ninth embodimentsA compound of the twelfth, thirteenth, fourteenth or fifteenth embodiment or a pharmaceutically acceptable salt thereof, wherein R2Is H; halogen; CN; optionally halogen-substituted- (C)1-C4) An alkyl group; - (C) optionally substituted by halo, hydroxy, methoxy or ethoxy1-C4) An alkoxy group; -C (═ O) (C)1-C4) An alkyl group; a cyclopropyl group; o-tetrahydropyranyl; an N-pyrrolidinyl group; or thiazolyl, wherein the remainder of the variables are as defined in the first, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth embodiments.
In a seventeenth embodiment, the present disclosure provides a compound according to the fifth embodiment, wherein the compound is represented by structural formula (III-B):
or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as defined in the first embodiment.
In an eighteenth embodiment, the present disclosure provides a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth or seventeenth embodiment or a pharmaceutically acceptable salt thereof, wherein R is1is-COOH or-C (═ O) NHR3；R3Is- (C)1-C4) Alkyl, -hydroxy (C)1-C4) Alkyl, -methoxy (C)1-C4) Alkyl, -amino (C)1-C4) Alkyl, - (C)1-C4) Hydroxyalkoxy (C)1-C4) Alkyl or- (CH)2)p2NHC (═ O) (dimethylamino (C)1-C4) Alkyl), wherein the remainder of the variables are as defined in the first, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth or seventeenth embodiment.
In the nineteenth embodiment, the inventionThere is provided a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenth embodiment or a pharmaceutically acceptable salt thereof, wherein R is hydrogen 2Is H, halo, - (C)1-C4) Haloalkyl, - (C)1-C4) Haloalkoxy, optionally methoxy-substituted- (C)1-C4) Alkoxy or-N-pyrrolidinyl, wherein the remainder of the variables are as defined in the first, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenth embodiments. In a preferred embodiment, R2Is H, F, CF3、OCHF2、OCF3、OCH2CH2OCH3or-N-pyrrolidinyl.
In a twentieth embodiment, the present disclosure provides a compound according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth or nineteenth embodiment or a pharmaceutically acceptable salt thereof, wherein
R2Is H, R1Is optionally substituted by-OH, NH2Oxetanyl or- (C)1-C4) Hydroxyalkoxy substituted-C (═ O) NH (C)1-C4) An alkyl group; or-C (═ O) NH (CH)2)p2NHC (═ O) (dimethylamino (C)1-C4) Alkyl groups); or
R2Is F, R1is-C (═ O) NH (methoxy (C)1-C4) Alkyl groups); or
R2is-N-pyrrolidinyl, R1is-C (═ O) NH ((C)1-C4) Hydroxyalkoxy (C)1-C4) An alkyl group),
R2is CF3、OCHF2Or OCF3，R1is-COOH; or
R2Is OCH2CH2OCH3，R1is-C (═ O) NH ((C) 1-C4) Hydroxyalkoxy (C)1-C4) Alkyl), -C (═ O) NH (hydroxy (C)1-C4) Alkyl), -C (═ O) NH (methoxy (C)1-C4) Alkyl) or-C (═ O) NH (hydroxy (C)1-C4) Alkoxy groups);
wherein the remainder of the variables are as defined in the first, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth or nineteenth embodiment.
In a twenty-first embodiment, the present disclosure provides a compound according to the seventeenth embodiment or a pharmaceutically acceptable salt thereof, wherein
R1Is optionally substituted by-OH, NH2、-(C1-C4) Alkoxy or- (C)1-C4) Hydroxyalkoxy substituted-C (═ O) NH (C)1-C4) Alkyl radicals, and
R2is H or OCH2CH2OCH3。
In a twenty-second embodiment, the present disclosure provides a compound according to the twenty-first embodiment or a pharmaceutically acceptable salt thereof, wherein
R2Is a compound of formula (I) wherein the compound is H,
wherein the remainder of the variables are as defined in the twenty-first embodiment.
In a twenty-third embodiment, the present disclosure provides a compound according to the twenty-second embodiment or a pharmaceutically acceptable salt thereof, wherein
R1Is a quilt- (C)1-C4) Hydroxyalkoxy substituted-C (═ O) NH (C)1-C4) An alkyl group, a carboxyl group,
wherein the remainder of the variables are as defined in the twenty-second embodiment.
In a twenty-fourth embodiment, the present disclosure provides a compound according to the twenty-first embodiment or a pharmaceutically acceptable salt thereof, wherein
R1Is a quilt- (C)1-C4) Alkane (I) and its preparation methodoxy-substituted-C (═ O) NH (C)1-C4) Alkyl, and
R2is OCH2CH2OCH3。
In one embodiment, the compound or pharmaceutically acceptable salt thereof is selected from the group consisting of the compounds disclosed in the examples and table 1.
Definition of
The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, and allergic response, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, s.m. berge et al in journal of pharmacy (j.pharm.sci.,1977,66,1-19) describe pharmaceutically acceptable salts.
The present teachings encompass pharmaceutically acceptable salts of the compounds disclosed herein. Compounds having a basic group can form pharmaceutically acceptable salts with pharmaceutically acceptable acids. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include salts of inorganic acids (e.g., hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric) and organic acids (e.g., acetic, benzenesulfonic, benzoic, ethanesulfonic, methanesulfonic and succinic). The compounds of the present teachings having an acidic group (e.g., carboxylic acid) can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. Suitable pharmaceutically acceptable basic salts include ammonium salts, alkali metal salts (e.g., sodium and potassium salts), and alkaline earth metal salts (e.g., magnesium and calcium salts).
As used herein, the term "halo" means halogen and includes chlorine, fluorine, bromine, and iodine.
The term "alkyl", alone or as part of a larger moiety (moiety) such as "alkoxy" or "haloalkyl", means a saturated aliphatic straight or branched chain monovalent hydrocarbon group. Unless otherwise specified, alkyl groups typically have 1 to 5 carbon atoms, i.e. (C)1-C5) An alkyl group. As used herein, "(C)1-C4) Alkyl "means a group having 1 to 4 carbon atoms in a straight or branched chain arrangement. Example bagContains methyl, ethyl, n-propyl, isopropyl, etc.
The term "alkoxy" means an alkyl group attached through an oxygen linking atom, denoted as-O-alkyl. For example, "(C)1-C4) Alkoxy "includes methoxy, ethoxy, propoxy and butoxy.
The terms "haloalkyl" and "haloalkoxy" mean alkyl or alkoxy, as the case may be, substituted with one or more halogen atoms.
The term "cycloalkyl" refers to a monocyclic saturated hydrocarbon ring system. Unless otherwise specified, cycloalkyl groups have 3 to 7 carbon atoms. For example, C3-C6Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Unless otherwise described, "cycloalkyl" has three to six carbon atoms.
The terms "heteroaryl", "heteroaromatic group", "heteroaromatic ring", "heteroaryl", "heteroaromatic ring" or "heteroaromatic group" are used alone or as part of a larger moiety as in "heteroaralkyl" or "heteroaralkoxy" to refer to monocyclic aromatic ring groups having five or six ring atoms (i.e., "5-6 membered") selected from carbon and at least one (typically 1 to 4, more typically 1 or 2) heteroatom (e.g., oxygen, nitrogen or sulfur).
Examples of monocyclic heteroaryls include furyl (e.g., 2-furyl, 3-furyl), imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl (e.g., 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), oxadiazolyl (e.g., 2-oxadiazolyl, 5-oxadiazolyl), oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrazolyl (e.g., 3-pyrazolyl, 4-pyrazolyl), pyrrolyl (e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (e.g., 2-pyrimidinyl, pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl (e.g., 3-pyridazinyl), thiazolyl (e.g., 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), triazolyl (e.g., 2-triazolyl, 5-triazolyl), tetrazolyl (e.g., tetrazolyl), thienyl (e.g., 2-thienyl, 3-thienyl), pyrimidinyl, pyridyl, and pyridazinyl.
The term "heterocyclyl" refers to a monocyclic non-aromatic cyclic group containing 3 to 7 ring atoms (i.e., "3-7 membered") selected from carbon atoms and 1 or 2 heteroatoms. Each heteroatom is independently selected from nitrogen, quaternary nitrogen, nitrogen oxide (e.g., NO); oxygen; and sulfur, including sulfoxides and sulfones. Representative heterocyclyl groups include morpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactam, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. A "substituted heterocyclyl" is substituted at any one or more substitutable ring atoms that are ring carbon or ring nitrogen atoms bonded to a hydrogen.
As used herein, many moieties (e.g., alkyl, alkylene, cycloalkyl, cycloalkylene, aryl, arylene, heteroaryl, heteroarylene, heterocyclyl, or heterocyclylene) are referred to as "substituted" or "optionally substituted". When a moiety is modified by one of these terms, it indicates that any moiety in the moiety known to those skilled in the art to be useful for substitution may be substituted, including one or more substituents, unless otherwise indicated. In the case when more than one substituent is present, then each substituent may be independently selected. Such means for substitution are well known in the art and/or taught by the present disclosure. The optional substituent may be any substituent suitable for attachment to the moiety.
Suitable substituents are those that do not have a significant adverse effect on the ability of the compound to induce HMOX 1. When suitable substituents are not specifically listed, exemplary substituents include, but are not limited to: (C)1-C5) Alkyl, (C)1-C5) Hydroxyalkyl group, (C)1-C5) Haloalkyl, (C)1-C5) Alkoxy group, (C)1-C5) Haloalkoxy, halogen, hydroxy, -CN, -NH2、-NO2、-ORc1、-NRa1Rb1、-S(O)iiRa1、-NRa1S(O)iiRb1、-S(O)iiNRa1Rb1、-C(＝O)ORa1、-OC(＝O)ORa1、-C(＝S)ORa1、-O(C＝S)Ra1、-C(＝O)NRa1Rb1、-NRa1C(＝O)Rb1、-C(＝S)NRa1Rb1、-NRa1C(＝S)Rb1、-NRa1(C＝O)ORb1、-O(C＝O)NRa1Rb1、-NRa1(C＝S)ORb1、-O(C＝S)NRa1Rb1、-NRa1(C＝O)NRa1Rb1、-NRa1(C＝S)NRa1Rb1、-C(＝S)Ra1、-C(＝O)Ra1Phenyl or 5-6 membered heteroaryl. Each Ra1And each Rb1Independently selected from-H and optionally substituted by hydroxy or (C)1-C3) Alkoxy-substituted (C)1-C5) An alkyl group; rc1is-H, (C)1-C5) Haloalkyl or (C)1-C5) Alkyl group, wherein (C) is1-C5) Alkyl optionally substituted by hydroxy or (C)1-C3) Alkoxy substitution; and ii is 1 or 2.
Pharmaceutical composition
The compounds disclosed herein are HMOX1 inducers. The pharmaceutical compositions of the present invention comprise one or more inducers of HMOX1, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or diluent.
"pharmaceutically acceptable carrier" and "pharmaceutically acceptable diluent" refer to a substance that facilitates formulation and/or administration of an active agent to and/or absorption by a subject, and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect to the subject. Non-limiting examples of pharmaceutically acceptable carriers and/or diluents include water, NaCl, standard saline solution, lactated Ringer's solution, common sucrose, common glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavor meters, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g., lactose, amylose, or starch), hydroxymethylcellulose, fatty acid esters, polyvinylpyrrolidine, pigments, and the like. Such formulations can be sterilized and, if necessary, mixed with adjuvants that do not deleteriously react with or interfere with the activity of the compounds provided herein, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, and/or aromatic substances, and the like. One skilled in the art will recognize that other pharmaceutical excipients are suitable for use with the disclosed compounds.
The pharmaceutical compositions of the present invention optionally comprise one or more of their pharmaceutically acceptable carriers and/or diluents, such as lactose, starch, cellulose and dextrose. Other excipients, such as flavoring agents, sweeteners, and preservatives, such as methyl paraben, ethyl paraben, propyl paraben, and butyl paraben, may also be included. A more complete list of suitable Excipients can be found in Handbook of Pharmaceutical Excipients (5 th edition, Pharmaceutical Press (2005)). One skilled in the art will know how to prepare formulations suitable for different types of routes of administration. Conventional procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003-20 th edition) and in the United states Pharmacopeia published in 1999: the National Formulary (USP 24NF19) is The United States Pharmacopeia (National Formulary). Carriers, diluents, and/or excipients are "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.
Method of treatment
In certain embodiments, the present invention provides methods of increasing the activity of HMOX1 or an amount thereof in a human subject, comprising: administering to a human subject an effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition thereof.
In certain embodiments, the present invention provides a method of activating the transcription factor Nrf2 in a human individual, comprising: administering to a human subject an effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition thereof.
In certain embodiments, the present invention provides a method of reducing the amount of ROS in a human subject, comprising: administering to a human subject an effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition thereof.
In certain embodiments, the present invention provides methods of using an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, or an effective amount of a pharmaceutical composition thereof. The compounds of the present invention and pharmaceutical compositions thereof may be useful in a variety of therapeutic applications, including, for example, the treatment and/or reduction of a wide variety of diseases and disorders including, for example, fibrotic diseases, neurodegenerative diseases, cardiovascular diseases, renal diseases, inflammatory diseases, liver diseases, ocular diseases, thyroid diseases, viral infections, osteoporosis, pregnancy disorders, endometriosis, diabetes, cancer, skin diseases, mitochondrial diseases, hematological disorders, and muscle diseases. The methods comprise administering to a subject in need thereof a pharmaceutically effective amount of one or more compounds of the present invention, pharmaceutically acceptable salts thereof, and/or pharmaceutical compositions thereof.
Compounds that increase the level or activity of HMOX1 may be useful for treating diseases or conditions that may be associated, at least in part, with oxidative stress, such as, but not limited to, fibrotic diseases, neurodegenerative diseases, cardiovascular diseases, renal diseases, inflammatory diseases, liver diseases, eye diseases, thyroid diseases, viral infections, osteoporosis, pregnancy disorders, endometriosis, diabetes, cancer, skin diseases, mitochondrial diseases, hematologic disorders, and muscle diseases. As used herein, a disease or condition associated with oxidative stress also includes chronic effects (e.g., tissue damage, chronic inflammation) associated with an increase in persistent or chronic oxidative stress due to the disease or condition described herein.
Fibrotic diseases associated with oxidative stress include, but are not limited to, pulmonary fibrotic diseases such as Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis, and sarcoidosis; liver fibrosis diseases, including those caused by alcoholic cirrhosis, steatosis, cholestasis, drug side effects, and viral infections; and skin fibrotic diseases, including autoimmune diseases such as scleroderma and psoriasis.
Neurodegenerative diseases associated with oxidative stress include, but are not limited to, Friedreich's ataxia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, cerebral neurodegenerative diseases, and Charcot-Marie-Tooth syndrome.
Cardiovascular diseases associated with oxidative stress include, but are not limited to, hypertension, heart failure, hypercholesterolemia, atherosclerosis, arteriosclerosis, thrombosis, acute coronary thrombosis, deep vein thrombosis, peripheral vascular disease, congestive heart failure, acute coronary syndrome, failed arterial fistula dialysis, ischemia reperfusion injury, primary pulmonary hypertension, and secondary pulmonary hypertension.
Kidney diseases associated with oxidative stress include, but are not limited to, acute kidney injury, polycystic kidney disease, Alport syndrome, diabetic nephropathy, glomerulonephritis, lupus nephritis, sickle cell nephropathy, and acute tubular necrosis.
Inflammatory diseases associated with oxidative stress include, but are not limited to, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, inflammatory bowel syndrome, Crohn's disease, celiac disease, ulcerative colitis, chronic inflammatory bowel disease, scleroderma, dermatitis, systemic lupus erythematosis, esophagitis, vasculitis, pancreatitis, tendonitis, osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and chronic encephalitis.
Liver diseases associated with oxidative stress include, but are not limited to, drug-induced hepatotoxicity, non-alcoholic steatohepatitis, and hepatitis, such as hepatitis b infection and hepatitis c infection.
Ocular diseases and conditions associated with oxidative stress include, but are not limited to, conjunctivitis, glaucoma, uveitis, wound healing (e.g., after surgery such as LASIK), ocular trauma, corneal transplantation, fox's corneal endothelial dystrophy, macular degeneration, cataracts, mild retinopathy, retinitis pigmentosa, diabetic retinopathy, and retinopathy of prematurity, as well as inflammation and tissue damage associated with these diseases.
Thyroid disorders associated with oxidative stress include, but are not limited to, Graves' disease, follicular adenoma, and papillary and follicular carcinoma.
Pulmonary diseases associated with oxidative stress include, but are not limited to, bronchitis, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary bronchitis, bronchiectasis, pulmonary edema, and emphysema.
Skin diseases associated with oxidative stress include, but are not limited to, dermatitis, scleroderma, and psoriasis.
Viral infections associated with oxidative stress include viral replication of the virus and tissue damage (e.g., fibrosis) due to oxidative stress caused by chronic viral infection, for viruses including, but not limited to, human immunodeficiency virus, hepatitis b, hepatitis c, and herpes virus.
Diabetic conditions include, but are not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, hyperglycemia, and metabolic syndrome, as well as secondary conditions resulting from diabetic conditions (e.g., congestive heart failure and renal disease).
Mitochondrial diseases associated with oxidative stress include, but are not limited to, mitochondrial myopathy, Leber's Hereditary Optic Neuropathy (LHON), myoclonic epilepsy with fragmented red fibers (MERFF), mitochondrial encephalomyopathy, lactic acidosis and stroke-like attacks (MELAS) or Leigh's Syndrome.
Hematologic disorders associated with oxidative stress include, but are not limited to, Dabby anemia (Diamond Blackfan anemia), myelodysplastic syndrome, sickle cell disease, and beta-thalassemia.
Muscle diseases associated with oxidative stress include, but are not limited to, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, and rhabdomyolysis.
Cancers associated with oxidative stress include, but are not limited to, breast cancer, colorectal cancer, lung cancer, ovarian cancer, uterine cancer, prostate cancer, leukemia, lymphoma, brain cancer (including glioblastoma multiforme and neuroblastoma), head and neck cancer, pancreatic cancer, melanoma, hepatocellular carcinoma, renal cancer, and soft tissue sarcoma. In one embodiment, the cancer is breast cancer, colon cancer, and ovarian cancer. In one embodiment, the cancer is selected from leukemia, acute myeloid leukemia, chronic myeloid leukemia, breast cancer, brain cancer, colon cancer, colorectal cancer, head and neck cancer, hepatocellular cancer, lung adenocarcinoma, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer, and renal cancer. In one embodiment, the cancer is lung cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme, or ovarian cancer. In another embodiment, the cancer is lung cancer, breast cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme, or ovarian cancer. In yet another embodiment, the cancer is breast cancer, colon cancer, and lung cancer. In another embodiment, the cancer is breast cancer. In yet another embodiment, the cancer is a basal subtype breast cancer or a tubular B subtype breast cancer. In yet another embodiment, the cancer is a basal subtype breast cancer. In yet another embodiment, the underlying subtype breast cancer is an Estrogen Receptor (ER), HER2, and Progesterone Receptor (PR) negative breast cancer. In yet another embodiment, the cancer is a soft tissue cancer. "Soft tissue cancer" is a art-recognized term that encompasses tumors derived from any soft tissue of the body. Such soft tissues connect, support or surround various structures and organs of the body, including but not limited to smooth muscle, skeletal muscle, tendons, fibrous tissue, adipose tissue, blood and lymphatic vessels, perivascular tissue, nerves, mesenchymal cells, and synovial tissue. Thus, soft tissue cancers may be adipose tissue, muscle tissue, neural tissue, joint tissue, blood vessels, lymphatic vessels, and fibrous tissue. Soft tissue cancers may be benign or malignant. In general, malignant soft tissue cancers are referred to as sarcomas or soft tissue sarcomas. There are many types of soft tissue tumors, including lipoblastoma, hibernate tumor, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyosarcoma, neurofibroma, schwannoma/neurilemoma, neuroma, malignant schwannoma, neurofibrosarcoma, neurosarcoma, nodular tenosynovitis, synovial sarcoma, hemangioma, hemangioblastoma, extravascular dermatoma, angioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma, fibroma, elastofibroma, superficial fibromatosis, fibrosarcoma, fibromatosis, dermatofibrosarcoma, DFSP, Malignant Fibrosarcoma (MFH), myxoma, granulomatoma, malignant mesenchyma, alveolar soft tissue sarcoma, epithelioid sarcoma, hyaline cell sarcoma, and desmoplastic small cell tumor. In a particular embodiment, the soft tissue cancer is a sarcoma selected from the group consisting of: fibrosarcoma, gastrointestinal sarcoma, leiomyosarcoma, dedifferentiated liposarcoma, liposarcoma polymorpha, malignant fibrous histiocytoma, round cell sarcoma, and synovial sarcoma.
Accordingly, the present invention provides a method of treatment comprising administering to a subject a compound of formula (I), or a pharmaceutically acceptable salt thereof, in order to treat at least one of the diseases or conditions listed above.
An "individual" is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).
Methods of administration and dosage forms
The precise amount of the compound that is administered to provide an "effective amount" to an individual will depend on the mode of administration, the type and severity of the disease or condition, and on individual characteristics such as general health, age, sex, weight, and drug tolerance. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. When administered in combination with other therapeutic agents, for example when administered in combination with an anti-cancer agent, the "effective amount" of any additional therapeutic agent will depend on the type of drug used. Suitable dosages are known for approved therapeutics and can be adjusted by one of skill in the art depending on the condition of the individual, the type of condition being treated, and the amount of the compounds of the invention for use, for example, in: the dosages reported in the literature and suggested in physicians' Desk Reference (57 th edition, 2003).
The term "effective amount" means an amount that, when administered to a subject, produces a beneficial or desired result, including a clinical result, e.g., inhibiting, suppressing, or reducing the symptoms of the condition being treated in the subject as compared to a control. For example, a therapeutically effective amount may be given in unit dosage form (e.g., 0.1mg to about 50 g/day, alternatively 1mg to about 5 g/day; and additionally alternatively 10mg to 1 g/day).
As used herein, the term "administration" refers to a method useful for enabling delivery of a composition to a desired site of biological action. These methods include, but are not limited to, intra-articular (in the joint), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, subcutaneous, oral, topical, intrathecal, inhalation, transdermal, rectal, and the like. Administration techniques that may be employed with the agents and methods described herein may be found, for example, in: goodman and Gilman, The Pharmacological Basis of Therapeutics (The Pharmacological Basis of Therapeutics), Current protocols; pergamon and Remington, Pharmaceutical Sciences (Current edition), Mack Publishing Co, of Isston, Pa.
In addition, the disclosed HMOX1 inducers may be co-administered with other therapeutic agents. As used herein, the terms "co-administration," "combined administration with … …," and grammatical equivalents thereof, will be intended to encompass the administration of two or more therapeutic agents to a single individual, and are intended to encompass treatment regimens in which the agents are administered by the same or different routes of administration, or at the same or different times. In some embodiments, one or more compounds described herein will be co-administered with other agents. These terms encompass the administration of two or more agents to an individual such that the agents and/or metabolites thereof are present in the individual at the same time. Comprising simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present. Thus, in some embodiments, the compounds described herein and other agents are administered in a single composition. In some embodiments, the compounds described herein and other agents are incorporated into the composition.
The particular mode of administration and dosing regimen will be selected by the attending clinician in view of the details of the case (e.g., individual, disease condition involved, particular treatment). Treatment may involve administration once or more than once daily or less than once daily (e.g., once weekly or once monthly, etc.) over a period of days to months or even years. However, one of ordinary skill in the art will immediately recognize appropriate and/or equivalent dosing for approved compositions for treating diseases using the disclosed HMOX1 inducers for guidance.
As will be appreciated by those of skill in the art, the compounds taught herein or corresponding pharmaceutical compositions may be administered to a patient in a variety of forms depending on the route of administration selected. The compounds taught by the present invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and pharmaceutical compositions formulated accordingly. Parenteral administration includes modes of intravenous, intraperitoneal, subcutaneous, intramuscular, epithelial, nasal, intrapulmonary, intrathecal, rectal and topical administration. Parenteral administration may be by continuous infusion over a selected period of time.
The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. In one embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to a human. In a preferred embodiment, the pharmaceutical composition is formulated for intravenous administration.
Generally, for oral therapeutic administration, the compounds of the present teachings may be incorporated into excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers (wafers), and the like.
Generally for parenteral administration, solutions of the compounds taught in the present invention can be prepared in water suitable for mixing with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof (with or without alcohol) and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Generally, for injectable use, sterile aqueous solutions or dispersions and sterile powders of the compounds described herein are appropriate for the extemporaneous preparation of sterile injectable solutions or dispersions.
Example
Abbreviations used throughout the specification may be summarized herein below in their specific meaning:
ACN-acetonitrile;
AcOH-acetic acid;
Ac2o-acetic anhydride;
AlCl3-aluminium chloride;
-anhydrous;
aq. -aqueous/water solution;
BOC-tert-butoxycarbonyl;
bs-broad singlet;
conc. -rich;
-degree centigrade;
CDI-carbonyldiimidazole;
CH3MgBr-methyl magnesium bromide;
CS2carbon disulfide
d-doublet;
delta-delta;
DCM-dichloromethane;
DIPEA-N, N-diisopropylethylamine;
DMFA-N, N-dimethylformamide;
DMSO-dimethyl sulfoxide;
DMSO-d6-deuterated dimethyl sulfoxide;
D2o-deuterated water;
DPPA-azidodiphenyl phosphate;
edc.hcl-1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;
EtOH-ethanol;
EtOAc-ethyl acetate;
g-g;
h-hours;
1h-proton;
H2-hydrogen;
HATU-N- [ (dimethylamino) -1H-1,2, 3-triazolo- [4,5-b ] pyridin-1-ylmethylene ] -N-methylmethanaminium hexafluorophosphate N-oxide;
HBTU-N, N' -tetramethyl-O- (1H-benzotriazol-1-yl) uronium hexafluorophosphate;
1HNMR-proton nuclear magnetic resonance;
HNO3-nitric acid;
H2o-water;
HCl-hydrochloric acid;
Hz-Hz;
H2SO4-sulfuric acid;
j-coupling constant;
K2CO3-potassium carbonate;
KOH-potassium hydroxide;
K3PO4-potassium phosphate;
LC-liquid chromatography;
LiOH.H2o-lithium hydroxide monohydrate;
M+-a molecular ion;
m-multiplet;
m-molarity;
m-CPBA-m-chloroperbenzoic acid;
MeI-methyl iodide;
MeOH-methanol;
mg-mg;
min-min;
MHz-megahertz (frequency);
mL-mL;
mM-millimolar concentration;
mmol-millimole;
MS-mass spectrum;
m/z-mass to charge ratio;
n-equivalent concentration;
NaBH4-sodium borohydride;
NaH-sodium hydride;
NaHCO3-sodium bicarbonate;
NaNO2-sodium nitrite;
NaNO3-sodium nitrate;
NaOEt-sodium ethoxide; NaOH-sodium hydroxide;
NaOMe-sodium methoxide;
NBS-N-bromosuccinimide
Pd/C-palladium on carbon;
Pd(OAc)2-palladium (II) acetate;
POCl3-phosphorus oxychloride;
P2S5-phosphorus pentasulfide;
PtO2-platinum dioxide:
percent-percent;
pH-pH value;
psi-pounds per square inch;
q-quartet;
RT-room temperature;
s-singlet;
SOCl2-thionyl chloride;
t-triplet peak;
TBAF-tetra-n-butylammonium fluoride;
TBDMSCl-tert-butyldimethylsilyl chloride;
TEA-triethylamine;
TFA-trifluoroacetic acid;
THF-tetrahydrofuran;
TLC-thin layer chromatography.
Example 1H and 1 i.e. Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and 2- (benzo-oxazol-2-ylamino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide:
conditions are as follows: a) carbon disulfide, KOH and ethanol are refluxed for 16 hours; b) k2CO3Methyl iodide, acetonitrile, 0 ℃ to RT, 5 h; c) m-CPBA, DCM, 0 ℃ -RT, 4 h; d) methylamine, DMF, 60 ℃, 16 h; e) 10% Pd/C, MeOH, H2RT, 5 h; f) cyanogen bromide, THF, H2O, 50 ℃, 16 h; g) NaH, 2- (methylsulfonyl) benzo [ d ]]Oxazole, 1, 4-dioxane, RT, 16 h; h) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; i) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of benzo [ d ] oxazole-2-thiol (1a)
To a solution of 2-aminophenol (4.0g, 36.7mmol) in ethanol (80mL) at Room Temperature (RT) was added powdered potassium hydroxide (3.59g, 64.2mmol) and carbon disulfide (20mL, 330.3mmol), and the reaction mixture was refluxed for 16 hours (h). The reaction mixture was concentrated, diluted with water (100mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound (4.0g, 72%); 1H NMR(400MHz，DMSO-d6)：δ13.85(bs，1H)，7.51-7.49(m，1H)，7.32-7.23(m，3H)；LC-MS：m/z 152.0(M+1)+。
Step-b: synthesis of 2- (methylthio) benzo [ d ] oxazole (1b)
To benzo [ d ] at 0 DEG C]To a solution of oxazole-2-thiol (3.0g, 19.9mmol) in acetonitrile (50mL) was added potassium carbonate (3.01g, 21.9mmol) and methyl iodide (1.36mL, 21.9mmol), and the reaction mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with water (60mL) and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to afford the product, which was used in the next step without any further purification (2.5g, 76%);1H NMR(400MHz,DMSO-d6):δ7.65-7.62(m,2H),7.34-7.30(m,2H),2.76(s,3H)；LC-MS:m/z 166.0(M+1)+。
step-c: synthesis of 2- (methylsulfonyl) benzo [ d ] oxazole (1c)
To 2- (methylthio) benzo [ d ] at 0 DEG C]To a solution of oxazole (500mg, 3.0mmol) in DCM (20mL) was added m-chloroperbenzoic acid (938mg, 9.1mmol) and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with water (40mL) and the aqueous layer was extracted with DCM (2X 100 mL). The combined organic layers were washed with saturated sodium bicarbonate solution (6 × 150mL), water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the product (300mg) which was used in the next step without any further purification; LC-MS M/z 198.0(M +1) +。
Step-d: synthesis of ethyl 4- (methylamino) -3-nitrobenzoate (1d)
To a solution of ethyl 4-chloro-3-nitrobenzoate (30.0g, 131mmol) in DMFA (100mL) at room temperature was added methylamine (26.8mL (40% aq), 262mmol) and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (1000mL) and stirred at room temperature for 1 h. The resulting solid was filtered and dried in vacuo to afford the product as a yellow solid (26.0g, 89%);1H NMR(400MHz,DMSO-d6):δ8.62(d,J＝2.0Hz,1H),8.59(bs,1H),7.99(dd,J＝2.0Hz,J＝8.8Hz,1H),7.07(d,J＝8.8Hz,1H),4.29(q,J＝6.8Hz,2H),3.01(d,J＝5.2Hz,3H),1.31(t,J＝6.8Hz,3H)；LC-MS:m/z 225.0(M+1)+。
step-e: synthesis of ethyl 3-amino-4- (methylamino) benzoate (1e)
To a solution of ethyl 4- (methylamino) -3-nitrobenzoate (13.0g, 58mmol) in methanol (180mL) under a nitrogen atmosphere was added a 10% Pd/C (1.3g in 10mL ethanol) slurry. The flask was held on a Parr shaker at room temperature under hydrogen pressure (60psi) for 5 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (10.0g, 88%);1H NMR(400MHz,DMSO-d6):δ7.23(dd,J＝1.6Hz,J＝8.4Hz 1H),7.16(d,J＝1.6Hz,1H),6.39(d,J＝8.4Hz,1H),5.38-5.37(m,1H),4.66(s,2H),4.18(q,J＝7.2Hz,2H),2.77(d,J＝5.2Hz,3H),1.26(t,J＝7.2Hz,3H)；LC-MS:m/z 195.1(M+1)+。
step-f: synthesis of ethyl 2-amino-1-methyl-1H-benzo [ d ] imidazole-5-carboxylate (1f)
To a solution of ethyl 3-amino-4- (methylamino) benzoate (5.6g, 28.8mmol) in THF (23mL) and water (56mL) at room temperature was added cyanogen bromide (3.7g, 34.6mmol), and the reaction mixture was stirred at 50 ℃ for 16 h. The reaction mixture was cooled to room temperature and diluted with water (50mL) and ethyl acetate (50 mL). The aqueous layer was basified with saturated sodium bicarbonate solution and extracted with EtOAc (2X 250 mL). The combined organic layers were washed with water (100mL), brine (100mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the product (5.2g, 82%) which was used in the next step without further purification; 1H NMR(400MHz,DMSO-d6):δ7.70(d,J＝1.2Hz,1H),7.59(dd,J＝1.2Hz,J＝8.4Hz,1H),7.20(d,J＝8.4Hz,1H),6.64(s,2H),4.27(q,J＝6.8Hz,2H),3.53(s,3H),1.32(t,J＝6.8Hz,3H)。
Step-g: synthesis of ethyl 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate (1g)
To 2-amino-1-methyl-1H-benzo [ d ] at room temperature]To a solution of imidazole-5-carboxylic acid ethyl ester (150mg, 0.68mmol) in 1, 4-dioxane (4mL) was added sodium hydride (96mg, 2.4mmol), and the reaction mixture was stirred for 15 minutes (min), followed by addition of 2- (methylsulfonyl) benzo [ d [ -d ] in]Oxazole (202mg, 1.0 mmol). The reaction mixture was stirred at room temperature for 16h and concentrated, diluted with water (20mL) and acidified with 1N HCl (pH 6). The resulting solid was filtered, dried in vacuo and purified by flash (combiflash) column chromatography using 100% DCM as eluent to give the title compound (50mg, 22%);1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.24(s,1H),7.88(d,J＝8.4Hz,1H),7.53-7.43(m,3H),7.22(t,J＝7.2Hz,1H),7.13(t,J＝7.6Hz,1H),4.33(q,J＝7.2Hz,2H),3.64(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 337.0(M+1)+。
step-h: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid (1H)
To 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (50mg, 0.15mmol) in a solvent mixture of THF (1mL), ethanol (1mL) and water (0.5mL) was added lithium hydroxide monohydrate (19mg, 0.45 mmol). The reaction mixture was heated at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water and acidified with 1N HCl to give a solid which was filtered and dried in vacuo to give the title compound (35mg, 76%); 1H NMR(400MHz,DMSO-d6):δ12.60(bs,2H),8.20(d,J＝1.6Hz,1H),7.87(dd,J＝1.6Hz,J＝8.0Hz,1H),7.51-7.48(m,2H),7.44(d,J＝7.2Hz,1H),7.23-7.20(m,1H),7.15-7.11(m,1H),3.64(s,3H)；LC-MS:m/z 308.6(M+1)+。
Step-i: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide (1i)
To 2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (30mg, 0.1mmol) in DMFA (1.0mL) were added N-ethyldiisopropylamine (0.02mL, 0.1mmol) and diphenylphosphorylazide (0.02mL, 0.1 mmol). The reaction mixture was stirred for 30min, followed by addition of 2-methoxyethylamine (8mg, 0.1 mmol). The reaction mixture was then stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (15mL) and stirred for 15 min. The resulting solid was filtered, washed with diethyl ether and dried in vacuo to give the title compound as a white solid (22mg, 53%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，1H)，8.45(t，J＝4.9Hz，1H)，8.08(s，1H)，7.76(dd，J＝1.5Hz，J＝8.3Hz，1H)，7.47(d，J＝8.3Hz，1H)，7.44-7.39(m，2H)，7.25-7.19(m，1H)，7.13-7.10(m，1H)，3.63(s，3H)，3.48-3.44(m，4H)，3.29(s，3H)；LC-MS：m/z 364.0(M-1)。
examples 2d and 2 e.synthesis of 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and synthesis of N- (2-methoxyethyl) -1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide:
conditions are as follows: a) tetrahydro-2H-pyran-4-amine, K2CO3，DMSO，70℃，16h；b)10％Pd/C，MeOH，H2RT, 5 h; c) 2-amino- (trifluoromethoxy) benzothiazole, 1, 1' -thiocarbonyldiimidazole, edc.hcl, DMF, 100 ℃, 16 h; d) h, LiOH 2O, THF, MeOH, water, 60 ℃, 16 h; e) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 3-nitro-4- ((tetrahydro-2H-pyran-4-yl) amino) benzoate (2a)
To a stirred solution of ethyl 4-chloro-3-nitrobenzoate (1.0g, 4.4mmol) in DMSO (10mL) was added potassium carbonate (1.2g, 8.7mmol) and tetrahydro-2H-pyran-4-amine (0.53g, 5.2mmol) at room temperature. Subsequently, the reaction mixture was heated to 70 ℃ and stirred for 16h, and then cooled to room temperature. The reaction mixture was diluted with cold water and the resulting solid was filtered and dried in vacuo to give the title compound (1.1g, 86%);1H NMR(400MHz，DMSO-d6)：δ8.62(d，J＝2.0Hz，1H)，8.23(d，J＝7.8Hz，1H)，7.97(dd，J＝1.9Hz，J＝8.8Hz，1H)，7.29(d，J＝9.3Hz，1H)，4.29(q，J＝7.3Hz，2H)，3.96-3.94(m，1H)，3.90-3.83(m，2H)，3.51-3.45(m，2H)，1.96-1.93(m，2H)，1.66-1.61(m，2H)，1.31(t，J＝7.3Hz，3H)；LC-MS：m/z 294.8(M+1)+。
step-b: synthesis of ethyl 3-amino-4- ((tetrahydro-2H-pyran-4-yl) amino) benzoate (2b)
The title compound was synthesized using the same procedure as intermediate 1e, using ethyl 3-nitro-4- ((tetrahydro-2H-pyran-4-yl) amino) benzoate as starting material (yield: 100%);1H NMR(400MHz,DMSO-d6):δ7.19(d,J＝8.0Hz,1H),7.17(s,1H),6.52(d,J＝8.3Hz,1H),4.99(d,J＝7.3Hz,1H),4.97(s,2H),4.18(q,J＝7.3Hz,2H),3.89-3.87(m,2H),3.57-3.53(m,1H),3.45-3.40(m,2H),1.92-1.89(m,2H),1.48-1.38(m,2H),1.25(t,J＝7.4Hz,3H)；LC-MS:m/z 264.8(M+1)+。
step-c: synthesis of ethyl 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate (2c)
To a stirred solution of 2-amino-6- (trifluoromethoxy) benzothiazole (177mg, 0.76mmol) in DMFA (55mL) was added 1,1' -thiocarbonyldiimidazole (270mg, 1.5mmol) and the solution was heated at 100 ℃ for 4 h. The reaction mixture was cooled to room temperature. Edc.hcl (291mg, 1.5mmol) was added, and the reaction mixture was heated at 60 ℃ for 10min, followed by the addition of ethyl 3-amino-4- ((tetrahydro-2H-pyran-4-yl) amino) benzoate (200mg, 0.76 mmol). Subsequently, the reaction mixture was heated at 100 ℃ for 16 h. It was cooled to room temperature, diluted with water (50mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% DCM as eluent to give the title compound (120mg, 31%); LC-MS: M/z 505.0 (M-1).
Step-d: synthesis of 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid (2d)
Using the same procedure as intermediate 1H, 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 53%);1H NMR(400MHz,DMSO-d6):δ12.91(bs,1H),12.57(bs,1H),8.24(s,1H),8.02-7.64(m,4H),7.37(s,1H),4.96-4.94(m,1H),4.20-4.05(m,2H),3.59-3.52(m,2H),2.60-2.44(m,2H),1.79-1.76(m,2H)；LC-MS:m/z 477.0(M-1)。
step-e: synthesis of N- (2-methoxyethyl) -1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide (2e)
Using the same procedure as for intermediate 1i, 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 18%);1H NMR(400MHz,DMSO-d6):δ8.25(t,J＝5.4Hz,1H),7.90(d,J＝1.5Hz,1H),7.71-7.64(m,2H),7.33(d,J＝0.9Hz,1H),7.16-7.13(m,1H),6.87(d,J＝8.8Hz,1H),5.23-5.17(m,1H),4.11-4.10(m,2H),3.65(t,J＝11.2Hz,2H),3.35-3.32(m,4H),3.28(s,3H),2.66-2.60(m,2H),1.92-1.90(m,2H)；LC-MS:m/z 536.0(M+1)+。
examples 3d and 3 e.Synthesis of 1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and N- (2-methoxyethyl) -1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzhomo-thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 1-methylpiperidine-4-amine, DIPEA, DMF, 70 ℃, 16 h; b) 10% Pd/C, MeOH, H 2RT, 2 h; c) 2-amino-6- (trifluoromethoxy) benzothiazole, 1, 1' -thiocarbonyldiimidazole, edc.hcl, DMF, 100 ℃, 16 h; d) h, LiOH2O, THF, EtOH, water, 60 ℃, 16 h; e) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 4- ((1-methylpiperidin-4-yl) amino) -3-nitrobenzoate (3a)
To a stirred solution of 4-chloro-3-nitrobenzoic acid ethyl ester (500mg, 2.2mmol) in DMFA (5mL) was added 1-methylpiperidin-4-amine (0.30mL, 2.6mmol) and N-ethyldiisopropylamine (0.8mL, 5.0 mmol). The reaction mixture was heated to 70 ℃ and stirring was continued at the same temperature for 16 h. The reaction mixture was cooled to room temperature, diluted with water (50mL) and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with water (50mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (620mg, 92%);1H NMR(400MHz，DMSO-d6)：δ8.62(s，1H)，8.25(d，J＝8.0Hz，1H)，7.97(d，J＝9.2Hz，1H)，7.23(d，J＝9.2Hz，1H)，4.30(q，J＝7.6Hz，2H)，3.71(bs，1H)，2.69-2.66(m，2H)，2.18(s，3H)，2.16-2.13(m，2H)，1.96-1.93(m，2H)，1.67-1.58(m，2H)，1.33(t，J＝7.6Hz，3H)；LC-MS：m/z 307.7(M+1)+。
step-b: synthesis of ethyl 3-amino-4- ((1-methylpiperidin-4-yl) amino) benzoate (3b)
The title compound was synthesized using the same procedure as intermediate 1e, using ethyl 4- ((1-methylpiperidin-4-yl) amino) -3-nitrobenzoate as starting material and stirring for 2 hours (yield: 100%);1H NMR(400MHz，DMSO-d6)：δ7.19-7.16(m，2H)，6.47(d，J＝8.4Hz，1H)，4.96(d，J＝7.2Hz，1H)，4.78(bs，2H)，4.18(q，J＝7.2Hz，2H)，3.37-3.31(m，1H)，2.86-2.83(m，2H)，2.25(s，3H)，2.19-2.13(m，2H)，1.94-1.91(m，2H)，1.54-1.45(m,2H),1.26(t,J＝6.8Hz,3H)；
LC-MS:m/z 278.2(M+1)+。
step-c: synthesis of ethyl 1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate (3c)
The title compound was synthesized using the same procedure as intermediate 2c, using ethyl 3-amino-4- ((1-methylpiperidin-4-yl) amino) benzoate as starting material (yield: 15%);1H NMR(400MHz,DMSO-d6):δ12.6(bs,1H),8.14(s,1H),7.91(s,1H),7.79(d,J＝8.3Hz,1H),7.62(d,J＝8.3Hz,1H),7.55(s,1H),7.35(d,J＝8.8Hz,1H),4.74-4.68(m 1H),4.34(q,J＝6.8Hz,2H),3.01-2.98(m,2H),2.67-2.56(m,2H),2.30(s,3H),2.20-2.15(m,2H),1.79-1.76(m,2H),1.35(t,J＝6.9Hz,3H)；LC-MS:m/z 520.1(M+1)+。
step-d: synthesis of 1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid (3d)
Using the same procedure as intermediate 1h, 1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 56%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.12(s,1H),7.91(s,1H),7.78(d,J＝8.3Hz,1H),7.60(d,J＝8.3Hz,1H),7.58(bs,1H),7.34(d,J＝8.9Hz,1H),4.79-4.68(m 1H),3.02-2.99(m,2H),2.57-2.54(m,2H),2.30(s,3H),2.21-2.15(m,2H),1.79-1.77(m,2H)；LC-MS:m/z 491.6(M+1)+。
step-e: synthesis of N- (2-methoxyethyl) -1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide (3e)
Using the same procedure as for intermediate 1i, 1- (1-methylpiperidin-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 40%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.44(s,1H),8.09(s,1H),7.91(s,1H),7.70-7.58(m,3H),7.35(d,J＝6.8Hz,1H),4.75-4.65(m,1H),3.50-3.43(m,4H),3.28(s,3H),3.02-2.99(m,2H),2.60-2.54(m,2H),2.31(s,3H),2.20-2.15(m,2H),1.79-1.76(m,2H)；LC-MS:m/z 549.2(M+1)+。
examples 4d and 4 e.synthesis of 1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and synthesis of N- (2-methoxyethyl) -1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide:
Conditions are as follows: a) 1-methylpyrrolidine-3-amine, DIPEA, DMF, 70 ℃, 16 h; b) 10% Pd/C, MeOH, H22 h; c) 2-amino-6- (trifluoromethoxy) benzothiazole, 1, 1' -thiocarbonyldiimidazole, edc.hcl, DMF, 100 ℃, 16 h; d) h, LiOH2O, THF, MeOH, water, 60 ℃, 16 h; e) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 deg.C-RT, 16
Step-a: synthesis of ethyl 4- ((1-methylpyrrolidin-3-yl) amino) -3-nitrobenzoate (4a)
The title compound was synthesized using the same procedure as intermediate 3a, using ethyl 4-chloro-3-nitrobenzoate and 1-methylpyrrolidin-3-amine as starting materials (yield: 79%);1H NMR(400MHz，DMSO-d6)：δ8.62(d，J＝1.9Hz，1H)，8.39(d，J＝6.8Hz，1H)，7.98(dd，J＝2.0Hz，J＝8.8Hz，1H)，7.15(d，J＝9.3Hz，1H)，4.32-4.36(m，3H)，2.80-2.75(m，1H)，2.70-2.59(m，2H)，2.42-2.30(m，2H)，2.29(s，3H)，1.73-1.65(m，1H)，1.31(t，J＝7.4Hz，3H)；LC-MS：m/z 294.1(M+1)+。
step-b: synthesis of ethyl 3-amino-4- ((1-methylpyrrolidin-3-yl) amino) benzoate (4b)
The title compound was synthesized using the same procedure as intermediate 1e, using ethyl 4- ((1-methylpyrrolidin-3-yl) amino) -3-nitrobenzoate as starting material and stirring for 2h (yield: 78%);1H NMR(400MHz，DMSO-d6)：δ7.19-7.15(m，2H)，6.38(d，J＝8.3Hz，1H)，5.16(d，J＝6.9Hz，1H)，4.81(s，2H)，4.18(q，J＝7.3Hz，2H)，3.95(bs，1H)，2.74-2.70(m，1H)，2.44-2.34(m，2H)，2.28-2.22(m，1H)，2.25(s，3H)，1.67-1.63(m，1H)，1.25(t，J＝7.3Hz，3H)；LC-MS：m/z 264.1(M+1)+。
step-c: synthesis of ethyl 1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate (4c)
The title compound was synthesized using the same procedure as intermediate 2c, using ethyl 3-amino-4- ((1-methylpyrrolidin-3-yl) amino) benzoate as starting material. The product obtained was used in the next step without further purification; LC-MS: m/z506.1(M +1) +。
Step-d: synthesis of 1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid (4d)
Using the same procedure as intermediate 1h, 1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material using THF, MeOH, and water (2: 1) as a solvent (yield: 13%);1H NMR(400MHz，DMSO-d6+D2O)：δ8.03(s，1H)，7.83(s，1H)，7.78(d，J＝8.3Hz，1H)，7.59-7.54(m，2H)，7.32(d，J＝8.8Hz，1H)，5.50(bs，1H)，3.86-3.75(m，2H)，3.35(bs，1H)，3.07-3.05(m，1H)，2.91(s，3H)，2.28-2.0(m，2H)；LC-MS：m/z478.1(M+1)+。
step-e: synthesis of N- (2-methoxyethyl) -1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide (4e)
Using the same procedure as for intermediate 1i, 1- (1-methylpyrrolidin-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 30%);1H NMR(400MHz，DMSO-d6): δ 8.10(bs, 1H), 8.04(s, 1H), 7.85(s, 1H), 7.76(bs, 1H), 7.68(d, J ═ 8.3Hz, 1H), 7.62(bs, 1H), 7.30(d, J ═ 8.8Hz, 1H), 5.50(bs, 1H), 3.52-3.44(m, 4H), 3.30(s, 3H), 2.76-2.65(m, 2H), 2.50(s, 3H, combined with DMSO peak), 2.31-2.30(m, 2H), 2.15-2.13(m, 2H); LC-MS: m/z 535.0(M +1) +。
Example 5 Synthesis of N- (2-methoxyethyl) -1-methyl-2- ((4, 5, 6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide:
conditions are as follows: a)4, 5, 6, 7-tetrahydrobenzo [ d ]]Thiazole-2-amine, 1, 1' -thiocarbonyldiimidazole, EDC.HCl, DMF, 100 ℃, 16 h; b) h, LiOH2O, THF, MeOH, water, 60 ℃, 16 h; c) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of methyl 1-methyl-2- ((4, 5, 6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate (5a)
To 4, 5, 6, 7-tetrahydrobenzo [ d ]]To a stirred solution of thiazol-2-amine (216mg, 1.4mmol) in DMFA (5mL) was added 1, 1' -thiocarbonyldiimidazole (494mg, 2.8mmol) and the solution was heated at 100 ℃ for 2 h. The reaction mixture was cooled to room temperature. EDC.HCl (533mg, 2.8mmol) was added,and the reaction mixture was heated at 60 ℃ for 10min, followed by addition of methyl 3-amino-4- (methylamino) benzoate (250mg, 1.4 mmol). The reaction mixture was heated at 100 ℃ for 16 h. It was cooled to room temperature, diluted with water (50mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% DCM as eluent to give the title compound (110mg, 23%); 1H NMR(400MHz,DMSO-d6) δ 12.01(bs,1H),7.97(s,1H),7.73(dd, J ═ 1.6Hz, J ═ 8.4Hz,1H),7.37(d, J ═ 8.4Hz,1H),3.84(s,3H),3.61(s,3H),2.50(bs,4H, combined with DMSO peak), 1.78(bs, 4H); LC-MS: M/z 341.0 (M-1).
Step-b: synthesis of 1-methyl-2- ((4,5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid (5b)
Using the same procedure as intermediate 1h, 1-methyl-2- ((4,5,6, 7-tetrahydrobenzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized using methyl imidazole-5-carboxylate as a starting material and THF, MeOH and water (2:2:1) as solvents (yield: 83%);1H NMR(400MHz,DMSO-d6) δ 12.20(bs,2H),7.95(s,1H),7.73(dd, J ═ 0.8Hz, J ═ 8.4Hz,1H),7.34(d, J ═ 8.4Hz,1H),3.61(s,3H),2.50(bs,4H, combined with DMSO peak), 1.78(bs, 4H); LC-MS: M/z 327.0 (M-1).
Step-c: synthesis of N- (2-methoxyethyl) -1-methyl-2- ((4,5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide (5c)
Using the same procedure as for Compound 1i, 1-methyl-2- ((4,5,6, 7-tetrahydrobenzo [ d ]]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 30%);1H NMR(400MHz,DMSO-d6) δ 12.0(bs,1H),8.34(bs,1H),7.95(s,1H),7.64(d, J ═ 8.4Hz,1H),7.32(d, J ═ 8.4Hz,1H),3.60(s,3H),3.48-3.42(m,4H),3.28(s,3H),2.50(bs,4H, combined with DMSO peak), 1.78(bs, 4H); LC-MS: M/z 384.0 (M-1).
EXAMPLE 6 Synthesis of 2- ((6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) trimethylsilyl trifluoromethanesulfonate, TEA and DCM at 0 ℃ for 1.5 h; b) NBS, sodium acetate, THF, water, RT, 2 h; thiourea, at 80 ℃ for 6 h; RT, 16 h; c) ethyl 3-amino-4- (methylamino) benzoate, 1, 1' -thiocarbonyldiimidazole, edc.hcl, DMF, 100 ℃, 18 h; d) h, LiOH2O, THF, EtOH, water, 80 ℃, 16 h; e)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of ((4, 4-dimethylcyclohex-1-en-1-yl) oxy) trimethylsilane
To a solution of 4, 4-dimethylcyclohex-1-one (3.0g, 23.8mmol) in DCM (180mL) at 0 deg.C was added triethylamine (9.95mL, 71.42 mmol). The reaction mixture was stirred for 5min, then trimethylsilyl trifluoromethanesulfonate (6.5g, 29.28mmol in 54mL DCM) was added. The reaction mixture was stirred at 0 ℃ for 1.5h, and then quenched with saturated sodium bicarbonate solution (30mL) and water (100 mL). The organic layer was separated and washed with water (2 × 50mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (3.8g, 81%) which was used in the next step without further purification; 1H NMR(400MHz，CDCl3)：δ4.74(t，J＝4.0Hz，1H)，1.99-1.97(m，2H)，1.80(d，J＝2.0Hz，2H)，1.39(t，J＝6.8Hz，2H)，1.16(s，6H)，0.15(s，9H)。
Step-b: synthesis of 6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-amine
To a solution of ((4, 4-dimethylcyclohex-1-en-1-yl) oxy) trimethylsilane (3.8g, 19.16mmol) in THF (40mL) and water (40mL) at room temperature was added N-bromosuccinimide (4.09g, 23.0mmol) and sodium acetate (0.22g, 2.7 mmol). The reaction mixture was then stirred for 2 h. Thiourea (1.43g, 18.8mmol) was added and the reaction mixture was heated at 80 ℃ with stirring for 6 h. Subsequently, it was cooled to room temperature and stirring was continued for 16 h. Reaction ofThe mixture was quenched with water (30mL) and extracted with DCM (2X 50 mL). The aqueous layer was basified with 2M sodium hydroxide (pH 9) and stirred at room temperature for 2 h. The precipitated solid was filtered and dried in vacuo to give the title compound (1.3g, 37%) which was used in the next step without further purification;1H NMR(400MHz，DMSO-d6)：δ6.55(bs，2H)，2.35(t，J＝6.4Hz，2H)，2.26(s，2H)，1.46(t，J＝6.0Hz，2H)，0.95(s，6H)；LC-MS：m/z 183.1(M+1)+。
step-c: synthesis of ethyl 2- ((6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To 6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ]]To a stirred solution of thiazol-2-amine (809mg, 4.44mmol) in DMFA (10mL) was added 1,1' -thiocarbonyldiimidazole (1.58g, 8.88mmol) and the solution was heated at 100 ℃ for 2 h. The reaction mixture was cooled to room temperature and edc.hcl (1.70g, 8.88mmol) was added. The reaction mixture was heated at 50 ℃ for 10min, followed by the addition of ethyl 3-amino-4- (methylamino) benzoate (800mg, 4.44 mmol). It was then heated at 100 ℃ for 16h and cooled to room temperature. The reaction mixture was diluted with water (50mL) and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% DCM as eluent to give the title compound (500mg, 32%); 1H NMR(400MHz,DMSO-d6) δ 12.10(bs,1H),7.98(s,1H),7.74(d, J ═ 8.4Hz,1H),7.37(d, J ═ 8.4Hz,1H),4.30(q, J ═ 7.2Hz,2H),3.61(s,3H),2.50(2H, combined with DMSO peak), 2.33(s,2H),1.56(t, J ═ 6.0Hz,2H),1.33(t, J ═ 7.2Hz,3H),1.00(s, 6H); LC-MS M/z 385.1(M +1)+。
Step-d: synthesis of 2- ((6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
To 2- ((6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d)]Thiazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (500mg, 1.30mmol) in a mixture of THF (4mL), ethanol (4mL) and water (2mL) was added lithium hydroxide monohydrate (136mg, 3.25 mmol). The reaction mixture was heated at 80 ℃ for 16h, and then cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water and acidified with 1N HCl to give a solid which was filtered and dried in vacuo to give the title compound (300mg, 65%);1H NMR(400MHz,DMSO-d6) δ 8.00(s,1H),7.86(d, J ═ 8.4Hz,1H),7.54(d, J ═ 8.4Hz,1H),3.64(s,3H),2.50(2H, combined with DMSO peak), 2.38(s,2H),1.58(t, J ═ 6.0Hz,2H),1.01(s, 6H); LC-MS M/z 357.1(M +1)+。
Step-e: synthesis of 2- ((6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To 2- ((6, 6-dimethyl-4, 5,6, 7-tetrahydrobenzo [ d ] at 0 deg.C]Thiazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (100mg, 0.28mmol) in DMFA (2mL) was added N-ethyldiisopropylamine (0.04mL, 0.28mmol) and HBTU (106mg, 0.28 mmol). The reaction mixture was stirred for 30min, then 2- (2-aminoethoxy) ethan-1-ol (0.02mL, 0.28mmol) was added and stirring continued at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (20mL) and stirred for 15 min. The resulting solid was filtered and dried in vacuo. The residue was purified by flash column chromatography using 2% MeOH/DCM as eluent to give the title compound (50mg, 40%);1H NMR(400MHz，DMSO-d6): δ 12.0(bs, 1H), 8.34(s, 1H), 7.95(s, 1H), 7.64(d, J ═ 8.4Hz, 1H), 7.32(d, J ═ 8.4Hz, 1H), 4.60(bs, 1H), 3.60(s, 3H), 3.56-3.42(m, 8H), 2.50(2H, combined with DMSO peak), 2.32(m, 2H), 1.55(t, J ═ 6.0Hz, 2H), 1.00(s, 6H); LC-MS: m/z 444.1(M +1)+。
EXAMPLE 7 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6-methyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) trifluoromethanesulfonic acidTrimethylsilyl ester, TEA, DCM, 0 ℃, 1.5 h; b) NBS, sodium acetate, EtOH, water, RT, 2 h; thiourea, at 80 ℃ for 6 h; RT, 16 h; c) ethyl 3-amino-4- (methylamino) benzoate, 1, 1' -thiocarbonyldiimidazole, edc.hcl, DMF, 100 ℃, 16 h; d) h, LiOH2O, THF, EtOH, water, 60 ℃, 16 h; e)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of trimethyl ((4-methylcyclohex-1-en-1-yl) oxy) silane
To a solution of 4-methylcyclohex-1-one (2.0g, 17.8mmol) in DCM (50mL) at 0 deg.C was added triethylamine (7.5mL, 53.4mmol) and stirred for 5min, followed by trimethylsilyl trifluoromethanesulfonate (4.95g, 22.3mmol in 30mL DCM). The reaction mixture was stirred at 0 ℃ for 1.5h, and then quenched with saturated sodium bicarbonate solution (20mL) and water (100 mL). The organic layer was separated and washed with water (2 × 100mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (3.4g, 100%);1H NMR(400MHz，DMSO-d6)：δ4.74-4.71(m，1H)，2.66-2.49(m，1H)，2.16-1.99(m，2H)，1.89-1.88(m，1H)，1.62-1.57(m，2H)，1.25-1.22(m，1H)，0.89(d，J＝2.4Hz，3H)，0.27(s，9H)。
step-b: synthesis of 6-methyl-4, 5, 6, 7-tetrahydrobenzo [ d ] thiazol-2-amine
To a solution of trimethyl ((4-methylcyclohex-1-en-1-yl) oxy) silane (3.3g, 17.9mmol) in THF (25mL) and water (25mL) at room temperature was added N-bromosuccinimide (3.83g, 21.5mmol) and sodium acetate (0.20g, 2.5mmol), and then stirred for 2 h. Thiourea (1.43g, 18.8mmol) was added and the reaction mixture was heated at 80 ℃ with stirring for 6 h. Subsequently, the reaction mixture was cooled to room temperature and stirring was continued for 16 h. The reaction mixture was quenched with water (30mL) and extracted with DCM (2X 100 mL). The aqueous layer was basified with 2M sodium hydroxide (pH) ～9) And extracted with DCM (3X 100 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (1.9g, 63%);1H NMR(400MHz,DMSO-d6):δ6.55(bs,2H),2.57-2.51(m,1H),2.40-2.36(m,2H),2.12-2.05(m,1H),1.79-1.74(m,2H),1.35-1.34(m,1H),1.00(d,J＝6.4Hz,3H)；LC-MS:m/z 169.2(M+1)+。
step-c: synthesis of ethyl 1-methyl-2- ((6-methyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
To 6-methyl-4, 5,6, 7-tetrahydrobenzo [ d ]]To a stirred solution of thiazol-2-amine (400mg, 2.38mmol) in DMFA (10mL) was added 1,1' -thiocarbonyldiimidazole (848mg, 4.76mmol) and the solution was heated at 100 ℃ for 2 h. The reaction mixture was cooled to room temperature and EDC.HCl (914mg, 4.76mmol) was added. The reaction mixture was heated at 50 ℃ for 10min, followed by addition of ethyl 3-amino-4- (methylamino) benzoate (462mg, 2.38 mmol). The reaction mixture was heated at 100 ℃ for 16 h. It was cooled to room temperature, diluted with water (50mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% DCM as eluent to give the title compound (400mg, 45%);1H NMR(400MHz,DMSO-d6) δ 12.03(bs,1H),7.98(s,1H),7.73(dd, J ═ 1.0Hz, J ═ 8.3Hz,1H),7.37(d, J ═ 8.3Hz,1H),4.30(q, J ═ 7.2Hz,2H),3.62(s,3H),2.67-2.60(m,1H),2.50(2H, combined with DMSO peak), 2.18-2.11(m,1H),1.87-1.84(m,2H),1.46-1.42(m,1H),1.34(t, J ═ 7.1Hz,3H),1.05(d, J ═ 6.3Hz, 3H); LC-MS M/z 371.05(M +1) +。
Step-d: synthesis of 1-methyl-2- ((6-methyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, 1-methyl-2- ((6-methyl-4, 5,6, 7-tetrahydrobenzo [ d)]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 100%);1H NMR(400MHz,DMSO-d6) δ 13.01(bs,1H),12.83(bs,1H),8.01(s,1H),7.87(d, J ═ 8.4Hz,1H),7.56(d, J ═ 8.4Hz,1H),3.67(s,3H),2.67-2.66(m,1H),2.52(2H, combined with DMSO peak), 2.33-2.21(m,1H),1.91-1.86(m,2H),1.48-1.43(m,1H),1.06(d, J ═ 6.4Hz, 3H); LC-MS M/z 343.05(M +1)+。
Step-e: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6-methyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6, step-e, 1-methyl-2- ((6-methyl-4, 5,6, 7-tetrahydrobenzo [ d ]]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 48%);1H NMR(400MHz，DMSO-d6): δ 11.99(bs, 1H), 8.35(t, J ═ 5.6Hz, 1H), 7.95(s, 1H), 7.64(d, J ═ 8.0Hz, 1H), 7.32(d, J ═ 8.4Hz,1H), 4.62(t, J ═ 5.0Hz, 1H), 3.61(s, 3H), 3.55-3.50(m, 4H), 3.47-3.42(m, 4H), 2.67-2.66(m,1H),2.52(2H, combined with DMSO peak), 2.17-2.16(m, 1H), 1.90-1.88(m, 2H), 1.45-1.40(m, 1H), 1.05(d, J ═ 6.4Hz, 3H); LC-MS: m/z 430.10(M +1) +。
EXAMPLE 8 Synthesis of 2- ((5, 6-dihydro-4H-cyclopenta [ d ] thiazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) iodine and thiourea at 110 ℃ for 12 hours; b) ethyl 3-amino-4- (methylamino) benzoate, 1, 1' -thiocarbonyldiimidazole, edc.hcl, DMF, 100 ℃, 16 h; c) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; h) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 5, 6-dihydro-4H-cyclopenta [ d ] thiazol-2-amine
A mixture of cyclopentanone (1.0g, 11.9mmol), thiourea (1.8g, 23.8mmol) and iodine (3.0g, 11.9mmol) was stirred in a sealed tube at 110 ℃ for 12 h. The reaction mixture was cooled to room temperature, and hot water (30mL) was added and stirred for 30 min. The reaction mixture was extracted with diethyl ether (2X 20mL), the aqueous layer basified with solid sodium bicarbonate (pH 8) and extracted with DCM (3X 30 mL). The combined organic layers were washed with brine (20mL), dried over anhydrous sodium sulfate and concentrated in vacuo. By flash chromatography using 10% methanol in DCM andthe resulting residue was purified with 0.1% ammonia as eluent to give the title compound (160mg, 10%);1H NMR(400MHz，CDCl3)：δ4.77(bs，2H)，2.79-2.74(m，2H)，2.68-2.64(m，2H)，2.41-2.34(m，2H)；LC-MS：m/z 141.1(M+1)+。
step-b: synthesis of ethyl 2- ((5, 6-dihydro-4H-cyclopenta [ d ] thiazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 5a, using ethyl 3-amino-4- (methylamino) benzoate and 5, 6-dihydro-4H-cyclopenta [ d ]]The title compound was synthesized with thiazol-2-amine as a starting material (yield: 23%);1H NMR(400MHz,DMSO-d6):δ12.0(bs,1H),8.05(s,1H),7.77(d,J＝8.0Hz,1H),7.38(d,J＝8.4Hz,1H),4.30(q,J＝6.8Hz,2H),3.56(s,3H),2.80-2.72(m,4H),2.36-2.33(m,2H),1.33(t,J＝6.8Hz,3H)；LC-MS:m/z343.0(M+1)+。
step-c: synthesis of 2- ((5, 6-dihydro-4H-cyclopenta [ d ] thiazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, 2- ((5, 6-dihydro-4H-cyclopenta [ d)]Thiazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 55%); LC-MS: m/z 315.1(M +1)+。
Step-d: synthesis of 2- ((5, 6-dihydro-4H-cyclopenta [ d ] thiazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((5, 6-dihydro-4H-cyclopenta [ d)]Thiazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 13%);1H NMR(400MHz,DMSO-d6):δ12.0(bs,1H),8.36(bs,1H),7.95(s,1H),7.67(d,J＝7.6Hz,1H),7.33(d,J＝8.4Hz,1H),3.56(s,3H),3.47-3.42(m,4H),3.28(s,3H),2.77-2.67(m,4H),2.36-2.33(m,2H)；LC-MS:m/z 372.0(M+1)+。
EXAMPLE 9 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((4,5,6, 7-tetrahydrothiazolo [5,4-c ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide hydrochloride
Conditions are as follows: a) i) pyrrolidone hydrobromide, THF, reflux, 10min ii) thiourea, EtOH, reflux, 3 h; b) ethyl 3-amino-4- (methylamino) benzoate, 1, 1' -thiocarbonyldiimidazole, EDC, DMF, 100 ℃ for 18 h; c) h, LiOH 2O，THF，MeOH，H2O, 50 ℃, 16 h; d)2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethan-1-amine, HBTU, DIPEA, DMF, 0 ℃ -RT, 16 h; e) 1, 4-dioxane containing 4M HCl, 1, 4-dioxane, 0 ℃ to RT, 16h
Step-a: synthesis of 2-amino-6, 7-dihydrothiazolo [5, 4-c ] pyridine-5 (4H) -carboxylic acid tert-butyl ester
To a stirred solution of tert-butyl 4-oxopiperidine-1-carboxylate (3.0g, 15.07mmol) in THF (70mL) at room temperature was added pyrrolidone hydrobromide (7.47g, 15.07mmol) and the reaction mixture was refluxed for 10 min. The reaction mixture was cooled to room temperature, the precipitated solid was removed by filtration, and the filtrate was concentrated. The resulting residue was dissolved in ethanol (80mL), followed by the addition of thiourea (1.53g, 20.14mmol) and the reaction mixture refluxed for 3 h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by flash chromatography using 5% methanol/DCM as eluent to give the title compound (450mg, 12%);1H NMR(400MHz，DMSO-d6)：δ6.80(s，2H)，4.29(s，2H)，3.56(t，J＝5.6Hz，2H)，2.43(bs，2H)，1.41(s，9H)；LC-MS：m/z 256.2(M+1)+。
step-b: synthesis of tert-butyl 2- ((5- (ethoxycarbonyl) -1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) -6, 7-dihydrothiazolo [5, 4-c ] pyridine-5 (4H) -carboxylate
To the 2-amino group-6, 7-dihydrothiazolo [5, 4-c ]To a stirred solution of pyridine-5 (4H) -carboxylic acid tert-butyl ester (424mg, 1.66mmol) in DMFA (6mL) was added 1, 1' -thiocarbonyldiimidazole (593mg, 3.33mmol) and the solution was heated at 100 ℃ for 2H. The reaction mixture was cooled to room temperature and edc.hcl (639mg, 3.33mmol) was added. The reaction mixture was heated at 50 ℃ for 10min, followed by the addition of ethyl 3-amino-4- (methylamino) benzoate (300mg, 1.66 mmol). The reaction mixture was heated at 100 ℃ for 16 h. It was cooled to room temperature, diluted with water (50mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 0.4% methanol/DCM as eluent to give the title compound (130mg, 18%);1H NMR(400MHz，DMSO-d6): δ 12.10(bs, 1H), 8.03(bs, 1H), 7.77(d, J ═ 8.4Hz, 1H), 7.39(d, J ═ 8.4Hz, 1H), 4.39(bs, 2H), 4.30(q, J ═ 6.8Hz, 2H), 3.67-3.59(m, 5H), 2.50(2H, combined with DMSO peak), 1.43(s, 9H), 1.34(t, J ═ 6.8Hz, 3H); LC-MS: m/z 458.2(M +1)+。
Step-c: synthesis of 2- ((5- (tert-butoxycarbonyl) -4, 5, 6, 7-tetrahydrothiazolo [5, 4-c ] pyridin-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, using 2- ((5- (ethoxycarbonyl) -1-methyl-1H-benzo [ d)]Imidazol-2-yl) amino) -6, 7-dihydrothiazolo [5,4-c]The title compound was synthesized from pyridine-5 (4H) -carboxylic acid tert-butyl ester as starting material and THF, methanol, water (2:2:1) as solvent. (yield: 70%);1H NMR(400MHz,DMSO-d6):δ12.50(bs,1H),8.00(s,1H),7.78(d,J＝8.4Hz,1H),7.40(d,J＝8.4Hz,1H),4.40(s,2H),3.66(t,J＝4.8Hz,2H),3.60(s,3H),2.66-2.63(m,2H),1.43(s,9H)；LC-MS:m/z 430.1(M+1)+。
step-d: synthesis of tert-butyl 2- ((5- ((2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) carbamoyl) -1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) -6, 7-dihydrothiazolo [5,4-c ] pyridine-5 (4H) -carboxylate
Using the same procedure as example 6 step-e, using 2- ((5- (tert-Butoxycarbonyl)Yl) -4,5,6, 7-tetrahydrothiazolo [5,4-c]Pyridin-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethan-1-amine as starting materials (yield: 40%); LC-MS: m/z 631.2(M +1)+。
Step-e: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((4,5,6, 7-tetrahydrothiazolo- [5,4-c ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide hydrochloride
To a solution of 2- ((5- ((2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) carbamoyl) -1-methyl-1H-benzo [ d ] at 0 deg.C ]Imidazol-2-yl) amino) -6, 7-dihydrothiazolo [5,4-c]To a stirred solution of pyridine-5 (4H) -carboxylic acid tert-butyl ester (50mg, 0.08mmol) in 1, 4-dioxane (0.8mL) was added 4M HCl in 1, 4-dioxane (0.3mL) and the mixture was stirred at room temperature for 16H. The reaction mixture was concentrated under reduced pressure. The residue was wet milled with diethyl ether and the solvent was decanted. The resulting solid was dried in vacuo to give the title compound (12mg, 33%);1H NMR(400MHz,CD3OD):δ8.01(s,1H),7.87(d,J＝8.0Hz,1H),7.55(d,J＝8.4Hz,1H),4.35(s,2H),3.74(s,3H),3.71-3.53(m,10H),3.05(t,J＝6.0Hz,2H)；LC-MS:m/z 417.1(M+1)+。
EXAMPLE 10 Synthesis of 2- ((5-acetyl-4, 5,6, 7-tetrahydrothiazolo [5,4-c ] pyridin-2-yl) amino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) TFA, DCM, 0 deg.C-RT, 16 h; b) ac of2O, pyridine, 0-RT, 16 h; c)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ to RT, 1 deltah
Step-a: synthesis of 1-methyl-2- ((4,5,6, 7-tetrahydrothiazolo [5,4-c ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid trifluoroacetate
To a mixture of 2- ((5- (tert-butoxycarbonyl) -4,5,6, 7-tetrahydrothiazolo [5, 4-c) at 0 deg.C]Pyridin-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid (17)0mg, 0.39mmol) in DCM (5mL) was added TFA (0.1mL, 1.19mmol) and stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, and the obtained residue was wet-milled with diethyl ether, and the solvent was decanted. The residue was dried in vacuo to give the title compound (180mg, 100%); 1H NMR(400MHz，DMSO-d6)：δ9.22(s，2H)，8.04(s，1H)，7.81(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.42(d，J＝8.4Hz，1H)，4.23(s，2H)，3.60(s，3H)，3.48-3.46(m，2H)，2.89(bs，2H)；LC-MS：m/z 330.1(M+1)+。
Step-b: synthesis of 2- ((5-acetyl-4, 5,6, 7-tetrahydrothiazolo [5, 4-c ] pyridin-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
To 1-methyl-2- ((4,5,6, 7-tetrahydrothiazolo [5, 4-c) at 0 deg.C]Pyridin-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid trifluoroacetate (180mg, 0.55mmol) in pyridine (2mL) was added acetic anhydride (0.06mL, 0.6mmol) and stirred at room temperature for 16 h. The reaction mixture was diluted with water (15mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with 1N HCl (30mL), water (20mL), brine (20mL), dried over anhydrous sodium sulfate and concentrated in vacuo, and the obtained residue was wet-milled with diethyl ether and the solvent was decanted. The resulting solid was dried in vacuo to give the title compound (70mg, 46%); LC-MS: m/z 372.1(M +1)+。
Step-c: synthesis of 2- ((5-acetyl-4, 5,6, 7-tetrahydrothiazolo [5, 4-c ] pyridin-2-yl) amino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6, step-e, using 2- ((5-acetyl-4, 5,6, 7-tetrahydrothiazolo [5, 4-c)]Pyridin-2-yl) amino) -1-methyl-iH-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 29%); 1H NMR(400MHz，DMSO-d6): δ 12.0(bs, 1H), 8.38(s, 1H), 7.96(d, J ═ 3.6Hz, 1H), 7.67(d, J ═ 7.6Hz, 1H), 7.35(d, J ═ 8.4Hz, 1H), 4.62-4.49(m, 3H), 3.78-3.73(m, 2H), 3.60-3.41(m, 10H), 3.30(2H, combined with DMSO water peak), 2.09(s, 3H); l isC-MS：m/z 459.1(M+1)+。
EXAMPLE 11 Synthesis of 2- ((6, 7-dihydro-4H-pyrano [4, 3-d ] thiazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 3-amino-4- (methylamino) benzoic acid methyl, 1, 1' -thiocarbonyldiimidazole, EDC and DMF at 100 ℃ for 16 h; b) h, LiOH2O，THF，MeOH，H2O, 60 ℃, 16 h; c) 2-methoxyethyl-1-amine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of methyl 2- ((6, 7-dihydro-4H-pyrano [4, 3-d ] thiazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 5a, using methyl 3-amino-4- (methylamino) benzoate and 6, 7-dihydro-4H-pyrano [4, 3-d ]]Thiazol-2-amine as starting material and stirred for 16h to synthesize the title compound (yield: 20%);1H NMR(400MHz，DMSO-d6): δ 12.20(bs, 1H), 8.02(s, 1H), 7.77(d, J ═ 8.0Hz, 1H), 7.40(d, J ═ 8.4Hz, 1H), 4.58(s, 2H), 3.93(t, J ═ 5.6Hz, 2H), 3.84(s, 3H), 3.60(s, 3H), 2.50(2H, combined with DMSO peaks); LC-MS: m/z 343.0 (M-1).
Step-b: synthesis of 2- ((6, 7-dihydro-4H-pyrano [4, 3-d ] thiazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1H, using 2- ((6, 7-dihydro-4H-pyrano [4, 3-d)]Thiazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized using imidazole-5-carboxylic acid methyl ester as a starting material and THF, methanol and water (2: 2: 1) as solvents (yield: 52%); δ 12.40(bs, 2H), 7.98(s, 1H), 7.75(d, J ═ 8.0Hz, 1H), 7.36(d, J ═ 8.0Hz, 1H), 4.58(s, 2H), 3.92(t, J ═ 4.8Hz, 2H), 3.59(s, 3H), 2.50(2H, combined with DMSO peak); LC-MS: m/z330.7(M +1)+。
Step-c: synthesis of 2- ((6, 7-dihydro-4H-pyrano [4, 3-d ] thiazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzoimidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((6, 7-dihydro-4H-pyrano [4, 3-d) was used]Thiazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials. The crude product was purified by flash chromatography using 1.2% methanol/DCM as eluent (yield: 34%);1H NMR(400MHz，DMSO-d6): δ 12.20(bs, 1H), 8.40(bs, 1H), 7.96(s, 1H), 7.66(bs, 1H), 7.35(d, J ═ 8.4Hz, 1H), 4.58(bs, 2H), 3.93(s, 2H), 3.58(s, 3H), 3.48-3.42(m, 4H), 3.27(s, 3H), 2.50(2H, combined with DMSO peaks); LC-MS: m/z 387.6(M +1) +。
Example 12.2 synthesis of- ((1H-benzo [ d ] imidazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide:
conditions are as follows: a)1, 1' -thiocarbonyldiimidazole, acetonitrile, RT, 16 h; b) 3-amino-4- (methylamino) benzoic acid methyl ester, EDC.HCl, DMF, 100 ℃, 16 h; d) h, LiOH2O, THF, MeOH, water, 60 ℃, 16 h; e) 2-methoxyethyl-1-amine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of N- (1H-benzo [ d ] imidazol-2-yl) -1H-imidazole-1-thiocarbamamide
To 1H-benzo [ d ] at room temperature]To a solution of imidazol-2-amine (1.0g, 7.5mmol) in acetonitrile (10mL) was added 1, 1' -thiocarbonyldiimidazole (1.34g, 7.5mmol) and stirred at room temperature for 16 h. The reaction mixture was filtered and the resulting solid was dried in vacuo to give the title compound (1.1g, 60%);1H NMR(400MHz，DMSO-d6)：δ13.18(bs，2H)，8.55(s，1H)，7.93(d，J＝1.2Hz，1H)，7.63-7.59(m，2H)，7.36-7.32(m，2H)，6.99(d，J＝1.2Hz，1H)。
step-b: synthesis of methyl 2- ((1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To N- (1H-benzo [ d ]]To a stirred solution of imidazol-2-yl) -1H-imidazole-1-thiocarboxamide (188mg, 0.77mmol) in DMFA (5mL) was added edc.hcl (297mg, 1.5mmol) and heated to 60 ℃ for 10 min. The reaction mixture was cooled to room temperature, then methyl 3-amino-4- (methylamino) benzoate (150mg, 0.77mmol) was added and it was heated at 100 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with water (30mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 2% methanol/DCM as eluent to give the title compound (40mg, 16%); 1H NMR(400MHz，DMSO-d6)：δ12.12(bs，2H)，8.05(d，J＝1.6Hz，1H)，7.73(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.37-7.34(m，3H)，7.12-7.08(m，2H)，3.85(s，3H)，3.65(s，3H)；LC-MS：m/z 322.0(M+1)+。
Step-c: synthesis of 2- ((1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as intermediate 1H, using 2- ((1H-benzo [ d)]Imidazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid methyl ester as a starting material and methanol, THF and water as solvents (yield: 92%);1H NMR(400MHz，DMSO-d6)：δ13.0(bs，3H)，7.92(s，1H)，7.89(d，J＝1.6Hz，1H)，7.60(d，J＝8.4Hz，1H)，7.45-7.41(m，2H)，7.29-7.26(m，2H)，3.68(s，3H)；LC-MS：m/z 308.1(M+1)+。
step-d: synthesis of 2- ((1H-benzo [ d ] imidazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((1H-benzo [ d)]Imidazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 14%);1H NMR(400MHz，DMSO-d6)：δ12.10(bs，2H)，8.33(t，J＝4.8Hz，1H)，7.98(d，J＝0.8Hz，1H)，7.62(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.35-7.30(m，3H)，7.11-7.07(m，2H)，3.64(s，3H)，3.50-3.42(m，4H)，3.29(s，3H)；LC-MS：m/z365.0(M+1)+。
examples 13 and 14.1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid synthesis and N- (2-methoxyethyl) -1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide synthesis:
conditions are as follows: a) 10% Pd/C, MeOH, H2RT, 16 h; b)1, 1' -thiocarbonyl diimidazole, acetonitrile, 60 ℃ for 16 h; c)4- (trifluoromethoxy) benzene-1, 2-diamine, EDC.HCl, DMF, 100 ℃, 16 h; d) h, LiOH 2O, THF, EtOH, water, 60 ℃, 16 h; e) 2-methoxyethyl-1-amine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of 4- (trifluoromethoxy) benzene-1, 2-diamine
To a solution of methyl 2-nitro-5- (trifluoromethoxy) aniline (2.0g, 9.0mmol) in methanol (40mL) under a nitrogen atmosphere was added 10% Pd/C (1.0 g). Subsequently, the reaction mixture was stirred under hydrogen for 16 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (1.6g, 93%);1H NMR(400MHz,DMSO-d6):δ6.48(d,J＝8.0Hz,1H),6.45(d,J＝1.6Hz,1H),6.29(dd,J＝2.0Hz,J＝8.0Hz,1H),4.80(s,2H),4.58(s,2H)；LC-MS:m/z 193.0(M+1)+。
step-b: synthesis of ethyl 2- (1H-imidazole-1-carbosulfanyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at room temperature]To a solution of imidazole-5-carboxylic acid ethyl ester (500mg, 2.28mmol) in acetonitrile (10mL) was added 1,1' -thiocarbonyldiimidazole (529mg, 2.97mmol) and the reaction mixture was stirred at 60 ℃ for 16 h. It was cooled to room temperature and stirred for 30 min. The resulting solid was filtered and dried in vacuo to give the title compound (500mg, 66%);1H NMR(400MHz,DMSO-d6):δ13.40(bs,1H),8.68(s,1H),8.40(d,J＝1.2Hz,1H),8.03(s,1H),7.99(dd,J＝1.2Hz,J＝8.4Hz,1H),7.75(d,J＝8.4Hz,1H),7.01(s,1H),4.35(q,J＝7.2Hz,2H),3.79(s,3H),1.36(t,J＝7.2Hz,3H)；LC-MS:m/z 328.2(M-1)。
step-c: synthesis of ethyl 1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as example 12 step-b, using 2- (1H-imidazole-1-carbothioamido) -1-methyl-1H-benzo [ d ]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and 4- (trifluoromethoxy) benzene-1, 2-diamine as starting materials (yield: 27%);1HNMR-VT, at 90 deg.C (400MHz, DMSO-d)6):δ12.0(bs,2H),8.09(s,1H),7.77(d,J＝8.4Hz,1H),7.35(d,J＝8.4Hz,2H),7.28(s,1H),7.0(d,J＝8.4Hz,1H),4.35(q,J＝6.8Hz,2H),3.63(s,3H),1.35(t,J＝6.8Hz,3H)；LC-MS:m/z 420.0(M+1)+。
Step-d: synthesis of 1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as intermediate 1H, 1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d)]Imidazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester the title compound was synthesized (yield: 77%);1H NMR(400MHz,DMSO-d6):δ12.60(bs,3H),8.05(d,J＝1.6Hz,1H),7.82(dd,J＝1.6Hz,J＝8.4Hz,1H),7.45(d,J＝8.4Hz,1H),7.40(d,J＝8.8Hz,1H),7.34(s,1H),7.09(dd,J＝1.2Hz,J＝8.8Hz,1H),3.65(s,3H)；LC-MS:m/z 392.0(M+1)+。
step-e: synthesis of N- (2-methoxyethyl) -1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-methyl-2- ((5- (trifluoromethoxy) -1H-benzo [ d)]Imidazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 43%);1H NMR(400MHz，DMSO-d6)：δ12.20(bs，2H)，8.37(bs，1H)，8.0(d，J＝1.2Hz，1H)，7.68(dd，J＝1.2Hz，J＝8.4Hz，1H)，7.36(d，J＝8.4Hz，2H)，7.30(s，1H)，7.01(d，J＝8.4Hz，1H)，3.63(s，3H)，3.59-3.43(m，4H)，3.29(s，3H)；LC-MS：m/z 449.0(M+1)+。
EXAMPLE 15 Synthesis of 1-methyl-2- ((1-methyl-5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Conditions are as follows: a) NaH, methyl iodide, DMF, 0-RT, 1 h; b) 10% Pd/C, MeOH, H2RT, 6 h; c) n1-methyl-4- (difluoromethoxy) benzene-1, 2-diamine, EDC.HCl, DMF, 100 ℃, 16 h; d) h, LiOH 2O，THF，EtOH，H2O，60℃，16h
Step-a: synthesis of N-methyl-2-nitro-4- (trifluoromethoxy) aniline
To a stirred solution of 2-nitro-4- (trifluoromethoxy) aniline (2.0mg, 9.0mmol) in DMFA (20mL) at 0 deg.C was added sodium hydride (60% dispersion in mineral oil) (0.4g, 10.0mmol) and the reaction mixture was stirred for 10 min. Methyl iodide (0.56mL, 9.0mmol) was added to the reaction mixture and stirred at room temperature for 1 h. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 5% ethyl acetate/hexanes as eluent to give the title compound (1.8g, 85%);1H NMR(400MHz，DMSO-d6)：δ8.28(d，J＝4.0Hz，1H)，8.00(d，J＝2.4Hz，1H)，7.61(dd，J＝2.4Hz，J＝9.2Hz，1H)，7.10(d，J＝9.2Hz，1H)，2.98(d，J＝5.2Hz，3H)；LC-MS：m/z 237.0(M+1)+。
step-b: n is a radical of1Synthesis of (E) -methyl-4- (trifluoromethoxy) benzene-1, 2-diamine
The title compound was synthesized using the same procedure as compound 1e, using N-methyl-2-nitro-4- (trifluoromethoxy) aniline as starting material and stirring for 6h (yield: 92%);1H NMR(400MHz，DMSO-d6)：δ6.44(s，1H)，6.42(d，J＝10.4Hz，1H)，6.33(d，J＝8.4Hz，1H)，4.84(bs，2H)，2.70(s，3H)；LC-MS：m/z 207.0(M+1)+。
step-c: synthesis of ethyl 1-methyl-2- ((1-methyl-5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 12 step-b, using 2- (1H-imidazole-1-carbothioamido) -1-methyl-1H-benzo [ d ]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and N1-methyl-4- (trifluoromethoxy) benzene-1, 2-diamine as starting materials (yield: 37%); LC-MS: m/z 434.2(M +1)+。
Step-d: synthesis of 1-methyl-2- ((1-methyl-5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, 1-methyl-2- ((1-methyl-5- (trifluoromethoxy) -1H-benzo [ d)]Imidazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 53%);1H NMR(400MHz，DMSO-d6)：δ12.5(bs，2H)，8.10(d，J＝1.2Hz，1H)，7.79(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.44(s，1H)，7.41-7.37(m，2H)，7.08(d，J＝8.8Hz，1H)，3.70(s，3H)，3.68(s，3H)；LC-MS：m/z 406.1(M+1)+。
EXAMPLE 16 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
To 2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (50mg, 0.16mmol) in DMFA (1.0mL) were added N-ethyldiisopropylamine (0.03mL, 0.16mmol) and HBTU (62mg, 0.16 mmol). The reaction mixture was stirred for 30min, followed by the addition of 2-aminoethyl-1-Alcohol (10mg, 0.16mmol), and stirring at room temperature was continued for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (20mL) and stirred for 15 min. The resulting solid was filtered, washed with diethyl ether and dried in vacuo to give the title compound as a white solid (25mg, 44%); 1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.36(t，J＝5.4Hz，1H)，8.08(d，J＝0.9Hz，1H)，7.78-7.76(m，1H)，7.48(d，J＝8.3Hz，2H)，7.43(d，J＝7.8Hz，1H)，7.21(t，J＝7.3Hz，1H)，7.12(t，J＝7.3Hz，1H)，4.72(bs，1H)，3.63(s，3H)，3.54(t，J＝6.4Hz，2H)，3.37-3.34(m，2H)；LC-MS：m/z 352.2(M+1)+。
EXAMPLE 17 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) methylamine, DMF, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 3 h; c) cyanogen bromide, THF, H2O, 60 ℃, 16 h; d) NaH, 2-chlorobenzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; f)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of ethyl 4- (methylamino) -3-nitrobenzoate
To a solution of ethyl 4-chloro-3-nitrobenzoate (30.0g, 131mmol) in DMFA (100mL) at room temperature was added methylamine (26.8mL (40% aq), 262mmol) and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (1000mL) and stirred for 1 h. The resulting solid was filtered and dried in vacuo to afford the product as a yellow solid (28.0g, 96%);1H NMR(400MHz，DMSO-d6)：δ8.62(d，J＝2.0Hz，1H)，8.59(bs，1H)，7.99(dd，J＝2.0Hz，J＝8.8Hz，1H)，7.07(d，J＝8.8Hz，1H)，4.29(q，J＝6.8Hz，2H)，3.01(d，J＝5.2Hz，3H)，1.31(t，J＝6.8Hz，3H)；LC-MS：m/z 225.0(M+1)+。
step-b: synthesis of ethyl 3-amino-4- (methylamino) benzoate
To a solution of ethyl 4- (methylamino) -3-nitrobenzoate (27.0g, 120mmol) in methanol (300mL) under a nitrogen atmosphere was added a 10% Pd/C (2.8g in 15mL ethanol) slurry. The flask was held on a Parr shaker at room temperature under hydrogen (60psi) for 3 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (19.0g, 82%); 1H NMR(400MHz，DMSO-d6)：δ7.23(dd，J＝1.6Hz，J＝8.4Hz 1H)，7.16(d，J＝1.6Hz，1H)，6.39(d，J＝8.4Hz，1H)，5.38-5.37(m，1H)，4.66(s，2H)，4.18(q，J＝7.2Hz，2H)，2.77(d，J＝5.2Hz，3H)，1.26(t，J＝7.2Hz，3H)；LC-MS：m/z 195.1(M+1)+。
Step-c: synthesis of ethyl 2-amino-1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To a solution of ethyl 3-amino-4- (methylamino) benzoate (17.0g, 87.6mmol) in THF (68mL) and water (170mL) at room temperature was added cyanogen bromide (11.13g, 105.2mmol), and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and basified with saturated sodium bicarbonate solution. The resulting solid was filtered and dried in vacuo to give the title compound (18.0g, 94%) which was used in the next step without further purification;1H NMR(400MHz，DMSO-d6)：δ7.70(d，J＝1.2Hz，1H)，7.59(dd，J＝1.2Hz，J＝8.4Hz，1H)，7.20(d，J＝8.4Hz，1H)，6.64(s，2H)，4.27(q，J＝6.8Hz，2H)，3.53(s，3H)，1.32(t，J＝6.8Hz，3H)；LC-MS：m/z 220.1(M+1)+。
step-d: synthesis of ethyl 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at 0 DEG C]To a solution of imidazole-5-carboxylic acid ethyl ester (4.0g, 18.3mmol) in 1, 4-dioxane (40mL) was added sodium hydride (60% dispersion in mineral oil) (1.09g, 27.4mmol) portionwise and stirred for 10 min. Reacting 2-chlorobenzo [ d]Oxazole (2.79g, 18.3mmol) was added to the reactionThe mixture was stirred at room temperature for 16 h. The reaction mixture was poured onto cold water (100mL) and stirred at room temperature for 5 to 10 min. The resulting solid was filtered, dried in vacuo and purified by flash column chromatography using 100% DCM as eluent to give the title compound (4.0g, 60%); 1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.24(s,1H),7.88(d,J＝8.4Hz,1H),7.53-7.43(m,3H),7.22(t,J＝7.2Hz,1H),7.13(t,J＝7.6Hz,1H),4.33(q,J＝7.2Hz,2H),3.64(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 337.0(M+1)+。
Step-e: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
To 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (4.0g, 11.90mmol) in a solvent mixture of THF (20mL), ethanol (20mL) and water (10mL) was added lithium hydroxide monohydrate (1.25g, 29.76 mmol). The reaction mixture was heated with stirring at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (60mL), acidified with 1N HCl to give a solid, filtered and dried in vacuo to give the title compound (3.4g, 93%);1H NMR(400MHz,DMSO-d6):δ12.60(bs,2H),8.20(d,J＝1.6Hz,1H),7.87(dd,J＝1.6Hz,J＝8.0Hz,1H),7.51-7.48(m,2H),7.44(d,J＝7.2Hz,1H),7.23-7.20(m,1H),7.15-7.11(m,1H),3.64(s,3H)；LC-MS:m/z 309.1(M+1)+。
step-f: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To 2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (3.0g, 9.74mmol) in DMFA (30mL) was added N-ethyldiisopropylamine (4.36mL, 24.25mmol) and HBTU (4.06g, 10.71 mmol). The reaction mixture was stirred for 30min, then 2- (2-aminoethoxy) ethan-1-ol (1.02g, 9.74mmol) was added and stirring continued at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (300mL) and stirred for 30 min. The resulting solid was filtered, dried in vacuo and chromatographed by flash column chromatography using 5% methanol in DCM Purification as eluent gave the title compound as a white solid (2.9g, 75%);1H NMR(400MHz,DMSO-d6):δ12.20(bs,1H),8.43(t,J＝5.3Hz,1H),8.07(s,1H),7.76(d,J＝8.4Hz,1H),7.48(d,J＝7.9Hz,2H),7.43(d,J＝7.8Hz,1H),7.21(t,J＝7.3Hz,1H),7.11(t,J＝7.3Hz,1H),4.60(bs,1H),3.63(s,3H),3.57-3.42(m,8H)；LC-MS:m/z 396.2(M+1)+。
EXAMPLE 18 Synthesis of N- (2-aminoethyl) -2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide hydrochloride
Conditions are as follows: a) tert-butyl (2-aminoethyl) carbamate, HBTU, DIPEA, DMF, 0 ℃ -RT for 16 h; b) 4N HCl in 1, 4-dioxane, THF, 0 deg.C-RT, 3h
Step-a: synthesis of tert-butyl (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethyl) carbamate
Using the same procedure as example 6 step-e, using 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and tert-butyl (2-aminoethyl) carbamate as starting materials (yield: 51%);1H NMR(400MHz，DMSO-d6): δ 12.20(bs, 1H), 8.41(t, J ═ 5.1Hz, 1H), 8.08(s, 1H), 7.75(d, J ═ 8.3Hz, 1H), 7.48(d, J ═ 8.4Hz, 2H), 7.43(d, J ═ 7.8Hz, 1H), 7.23-7.19(m, 1H), 7.14-7.09(m, 1H), 6.92(d, J ═ 5.4Hz, 1H), 3.64(s, 3H), 3.34-3.33(m, 2H, combined with DMSO water peak), 3.15-3.10(m, 2H), 1.38(s, 9H); LC-MS: m/z 451.55(M +1)+。
Step-b: synthesis of N- (2-aminoethyl) -2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide hydrochloride
To (2- (2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxamido) ethyl) carbamic acid tert-butyl ester (50mg, 0.11mmol) in THF (2mL) was added 4N HCl in 14-dioxane (0.5mL), and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated on a rotary evaporator and stirred in diethyl ether (10 mL). The resulting solid was filtered and dried in vacuo to give the title compound (35mg, 81%);1H NMR(400MHz，DMSO-d6)：δ12.40(bs，1H)，8.70(s，1H)，8.13(s，1H)，7.96(bs，3H)，7.86(d，J＝8.3Hz，1H)，7.56(d，J＝8.3Hz，1H)，7.48(d，J＝7.9Hz，2H)，7.25(t，J＝7.6Hz，1H)，7.17(t，J＝7.8Hz，1H)，3.67(s，3H)，3.55(q，J＝5.7Hz，2H)，3.03-2.99(m，2H)；LC-MS：m/z 351.10(M+1)+。
EXAMPLE 19 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2- (dimethylamino) acetamido) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) tert-butyl (2-aminoethyl) carbamate, HBTU, DIPEA, DMF, 0 ℃ -RT for 16 h; b) TFA, DCM, 0 deg.C-RT, 16 h; c) n- (2-aminoethyl) -2- (dimethylamino) acetamide difluoroacetate, HBTU, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of tert-butyl (2- (2- (dimethylamino) acetamido) ethyl) carbamate
To a stirred solution of dimethylglycine (1.0g, 9.71mmol) in DMFA (10mL) at 0 deg.C was added N-ethyldiisopropylamine (1.69mL, 9.71mmol) and HBTU (3.68g, 9.71 mmol). The reaction mixture was stirred for 30min, followed by addition of tert-butyl (2-aminoethyl) carbamate (1.55g, 9.71 mmol). The reaction mixture was stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (50mL) and extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with water (3 × 30mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting solid was stirred in diethyl ether (20mL) for 5min, filtered and dried in vacuo to give the title compound (600mg, 25%); 1H NMR(400MHz，DMSO-d6)：δ7.77(t，J＝5.6Hz，1H)，6.83(t，J＝5.2Hz，1H)，3.11(q，J＝6.2Hz，2H)，2.99(q，J＝6.1Hz，2H)，2.82(s，2H)，2.18(s，6H)，1.37(s，9H)；LC-MS：m/z 246.15(M+1)+。
Step-b: synthesis of N- (2-aminoethyl) -2- (dimethylamino) acetamide trifluoroacetate
To a solution of tert-butyl (2- (2- (dimethylamino) acetamido) ethyl) carbamate (600mg, 2.45mmol) in DCM (15mL) at 0 ℃ was added TFA (0.56mL, 7.34mmol) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was concentrated on a rotary evaporator and dried in vacuo to give the title compound (600mg, 100%);1H NMR(400MHz，DMSO-d6)：δ9.75(bs，1H)，8.75(t，J＝5.6Hz，1H)，7.90(bs，2H)，3.88(s，2H)，3.38(q，J＝6.2Hz，2H)，2.91(q，J＝5.9Hz，2H)，2.82(s，6H)；LC-MS：m/z 146.20(M+1)+。
step-c: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2- (dimethylamino) acetamido) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To 2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (100mg, 0.32mmol) in DMFA (4mL) was added N-ethyldiisopropylamine (0.17mL, 0.97mmol) and HBTU (123mg, 0.32 mmol). The reaction mixture was stirred for 30min, then N- (2-aminoethyl) -2- (dimethylamino) acetamide trifluoroacetate (145mg, 0.39mmol) was added. The reaction mixture was then stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (15 mL). The resulting solid was filtered, washed with diethyl ether and dried in vacuo to give the title compound as a white solid (40mg, 28%); 1H NMR(400MHz,DMSO-d6) δ 12.20(bs,1H),8.44(t, J ═ 4.9Hz,1H),8.07(s,1H),7.92(bs,1H),7.74(d, J ═ 7.8Hz,1H),7.49-7.47(m,2H),7.43(d, J ═ 7.8Hz,1H),7.21(t, J ═ 7.6Hz,1H),7.12(t, J ═ 7.6Hz,1H),3.63(s,3H),3.37(t, J ═ 5.2Hz,2H),3.35(2H, combined with DMSO water peak), 2.86(s,2H),2.19(s, 6H); LC-MS M/z436.0(M +1)+。
EXAMPLE 20 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-N- (2-morpholinoethyl) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2-morpholinoethan-1-amine and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6) δ 12.21(bs,1H),8.33(s,1H),8.06(s,1H),7.73(d, J ═ 8.4Hz,1H),7.48(d, J ═ 8.4Hz,1H),7.43(d, J ═ 8.0Hz,2H),7.21(t, J ═ 7.2Hz,1H),7.11(t, J ═ 8.0Hz,1H),3.63(s,3H),3.58(t, J ═ 3.6Hz,4H),3.41(q, J ═ 6.4Hz,2H),3.30(2H, combined with DMSO water peak), 2.43(bs, 4H); LC-MS M/z 421.2(M +1)+。
EXAMPLE 21 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (dimethylamino) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using N, N-dimethylethane-1, 2-diamine and 2- (benzo [ d ] ]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.21(bs,1H),8.29(t,J＝5.2Hz,1H),8.07(s,1H),7.74(d,J＝8.4Hz,1H),7.48-7.42(m,3H),7.21(t,J＝7.6Hz,1H),7.11(t,J＝8.0Hz,1H),3.63(s,3H),3.39-3.35(m,2H),2.50-2.42(m,2H),2.19(s,6H)；LC-MS:m/z 379.2(M+1)+。
EXAMPLE 22 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- ((4, 5-dihydro-1H-imidazol-2-yl) amino) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using N1- (4, 5-dihydro-1H-imidazol-2-yl) ethane-1, 2-diamine and 2- (benzo [ d)]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,2H),8.52(bs,1H),8.20(bs,1H),8.09(s,1H),7.75(d,J＝8.4Hz,1H),7.52-7.43(m,3H),7.21(t,J＝7.2Hz,1H),7.12(t,J＝8.0Hz,1H),3.64(s,3H),3.59(s,2H),3.45-3.43(m,2H),3.36-3.34(m,4H)；LC-MS:m/z 417.05(M-1)-。
EXAMPLE 23 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-hydroxypropyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 1-aminopropan-2-ol and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.36(t,J＝5.6Hz,1H),8.19(d,J＝1.2Hz,1H),7.78(dd,J＝1.2Hz,J＝8.4Hz,1H),7.52-7.36(m,3H),7.21-7.19(m,1H),7.14-7.09(m,1H),4.87-4.78(m,1H),3.81(q,J＝6.0Hz,1H),3.64(s,3H),3.24-3.20(m,2H),1.14(d,J＝6.8Hz,3H)；LC-MS:m/z 366.2(M+1)+。
EXAMPLE 24 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2, 3-dihydroxypropyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 3-aminopropane-1, 2-diol and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized. 1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.31(t,J＝5.2Hz,1H),8.08(s,1H),7.77(dd,J＝1.6Hz,J＝8.4Hz,1H),7.49-7.42(m,3H),7.21(t,J＝8.0Hz,1H),7.11(t,J＝7.2Hz,1H),4.82(d,J＝4.4Hz,1H),4.58(bs,1H),3.64(s,3H),3.45-3.37(m,3H),3.26-3.19(m,2H)；LC-MS:m/z 382.1(M+1)+。
EXAMPLE 25 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2-hydroxypropoxy) ethyl) -1, 6-dimethyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 1- (2-aminoethoxy) propan-2-ol and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.42(t,J＝5.2Hz,1H),8.07(s,1H),7.75(dd,J＝1.2Hz,J＝8.4Hz,1H),7.49-7.42(m,3H),7.21(t,J＝7.6Hz,1H),7.11(t,J＝8.0Hz,1H),4.57(bs,1H),3.75(q,J＝5.6Hz,1H),3.63(s,3H),3.57-3.54(m,2H),3.46-3.43(m,2H),3.29-3.23(m,2H),1.03(d,J＝6.4Hz,3H)；LC-MS:m/z 410.2(M+1)+。
EXAMPLE 26 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- ((3-hydroxyoxetan-3-yl) methyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 3- (aminomethyl) oxetan-3-ol and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.55(t,J＝5.2Hz,1H),8.10(d,J＝1.2Hz,1H),7.79(dd,J＝1.6Hz,J＝8.4Hz,1H),7.51-7.42(m,3H),7.21(t,J＝7.6Hz,1H),7.14(t,J＝7.6Hz,1H),5.90(s,1H),4.52(d,J＝6.8Hz,2H),4.41(d,J＝6.4Hz,2H),3.64(s,3H),3.59(d,J＝6.0Hz,2H)；LC-MS:m/z 394.2(M+1)+。
EXAMPLE 27 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2-hydroxy-2-methylpropoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 1- (2-aminoethoxy) -2-methylpropan-2-ol and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.43(t，J＝5.2Hz，1H)，8.07(d，J＝1.2Hz，1H)，7.75(dd，J＝2.4Hz，J＝8.4Hz，1H)，7.49-7.42(m，3H)，7.21(dd，J＝1.2Hz，J＝7.6Hz，1H)，7.12(dd，J＝1.2Hz，J＝7.6Hz，1H)，4.34(bs，1H)，3.63(s，3H)，3.57(t，J＝6.4Hz，2H)，3.47-3.43(m，2H)，3.20(s，2H)，1.09(s，6H)；LC-MS：m/z 424.2(M+1)+。
EXAMPLE 28 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-N- (2- (pyrrolidin-1-yl) ethyl) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- (pyrrolidin-1-yl) ethan-1-amine and 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz，DMSO-d6): δ 12.24(bs, 1H), 8.38(t, J ═ 5.6Hz, 1H), 8.07(s, 1H), 7.74(d, J ═ 8.4Hz, 1H), 7.49-7.42(m, 3H), 7.21(t, J ═ 6.8Hz, 1H), 7.11(t, J ═ 7.2Hz, 1H)3.69(s, 3H), 3.48-3.37(m, 2H), 2.64-2.57(m, 2H), 2.50(4H, incorporated in DMSO peak), 1.66(bs, 4H)；LC-MS：m/z 405.2(M+1)+。
EXAMPLE 29 Synthesis of 2- (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) acetic acid
Conditions are as follows: a) ethyl 2-bromoacetate, NaH, Kl, THF, 0 deg.C-RT, 2 h; b) TFA, DCM, RT, 5 h; c)2- (2-aminoethoxy) ethyl acetate triflate, HBTU, DIPEA, DMF, 0-RT, 16 h; d) h, LiOH2O, ethanol, THF, water, RT, 3h
Step-a: synthesis of ethyl 2- (2- ((tert-butoxycarbonyl) amino) ethoxy) acetate
To a solution of tert-butyl (2-hydroxyethyl) carbamate (1.0g, 6.21mmol) in THF (10mL) at 0 deg.C was added sodium hydride (60% dispersion in mineral oil) (397mg, 9.93mmol) in portions followed by potassium iodide (164mg, 0.99mmol) and ethyl bromoacetate (2.07g, 12.42 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with cold water (60mL) and extracted with ethyl acetate (3X 60 mL). The combined organic layers were washed with water (30mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 1% methanol/dichloromethane as eluent to give the title compound (1.0g, 65%); 1H NMR(400MHz,DMSO-d6):δ6.74(bs,1H),4.14-4.08(m,4H),3.45(t,J＝5.6Hz,2H),3.08(q,J＝6.2Hz,2H),1.37(s,9H),1.19(t,J＝6.8Hz,3H)；LC-MS:m/z 148.1(M-Boc)+。
Step-b: synthesis of ethyl 2- (2-aminoethoxy) acetate trifluoroacetate salt
To a stirred solution of ethyl 2- (2- ((tert-butoxycarbonyl) amino) ethoxy) acetate (1.0g, 4.05mmol) in DCM (5mL) at room temperature was added trifluoroacetic acid (1mL) and stirred at room temperature for 5 h. The reaction mixture was concentrated under reduced pressure to give the title compound (1.1g, 100%) which was used in the next step without any further purification;1H NMR(400MHz,DMSO-d6):δ7.88(bs,3H),4.17(s,2H),4.14(q,J＝6.8Hz,2H),3.68(t,J＝5.0Hz,2H),3.01(q,J＝5.2Hz,2H),1.21(t,J＝7.0Hz,3H)；LC-MS:m/z 148.2(M+1)+。
step-c: synthesis of ethyl 2- (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) acetate
To 2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (150mg, 0.48mmol) in DMFA (2mL) was added N-ethyldiisopropylamine (0.26mL, 1.46mmol) and HBTU (184mg, 0.48 mmol). The reaction mixture was stirred for 30min, then ethyl 2- (2-aminoethoxy) acetate trifluoroacetate salt (129mg, 0.53mmol) was added. The reaction mixture was then stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (6mL) and stirred for 5 min. The precipitated solid was filtered and dried in vacuo to give the title compound (125mg, 59%); 1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.46(bs,1H),8.08(s,1H),7.76(d,J＝8.4Hz,1H),7.49-7.47(m,2H),7.43(d,J＝7.6Hz,1H),7.21(t,J＝8.0Hz,1H),7.14(t,J＝7.6Hz,1H),4.15(s,2H),4.12(q,J＝7.0Hz,2H),3.65-3.62(m,5H),3.47(q,J＝5.8Hz,2H),1.19(t,J＝7.0Hz,3H)；LC-MS:m/z 438.1(M+1)+。
Step-d: synthesis of 2- (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) acetic acid
To 2- (2- (2- (benzo [ d ]))]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxamido) ethoxy) ethyl acetate (110mg, 0.25mmol) was added to a solution of lithium hydroxide monohydrate (26mg, 0.63mmol) in a solvent mixture of THF (1mL), ethanol (1mL) and water (0.5 mL). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in water (5mL) and acidified with 1N HCl to give a solid, which was filtered and dried in vacuo to give the title compound (80mg, 78%);1H NMR(400MHz，DMSO-d6)：δ12.60(bs，1H)，8.47(s，1H)，8.08(s，1H)，7.77(d，J＝8.4Hz，1H)，7.50-7.43(m，3H)，7.22(t，J＝7.6Hz，1H)，7.13(t，J＝7.2Hz，1H)，4.06(s，2H)，3.64(bs，5H)，3.45(q，J＝5.2Hz，2H)；LC-MS：m/z 410.4(M+1)+。
EXAMPLE 30 DL-valine 2- (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) ethyl ester hydrochloride
Conditions are as follows: a) di-tert-butyl dicarbonate, sodium hydroxide, THF, water, RT for 16 h; b) (tert-butoxycarbonyl) valine, HATU, DIPEA, DMF, 0 ℃ -RT, 16 h; c) 4N HCl in 1, 4-dioxane, 10 ℃ to RT, 16h
Step-a: synthesis of (tert-butoxycarbonyl) -DL-valine
To a stirred solution of DL-valine (2.0g, 17.07mmol) in THF (25mL) and water (20mL) at room temperature was added sodium hydroxide (0.82g, 20.5mmol) and di-tert-butyl dicarbonate (4.09g, 18.77mmol) and the reaction mixture was stirred for 16 h. The mixture was cooled to 0 ℃, acidified with 1N HCl and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product (1.8g) was used in the next step without any further purification,
Step-b: synthesis of (tert-butoxycarbonyl) -DL-valine 2- (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) ethyl ester
To 2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxamide (70mg, 0.18mmol) and (tert-butoxycarbonyl) -DL-valine (42mg, 0.19mmol) in DMFA (5mL) were added N-ethyldiisopropylamine (0.04mL, 0.21mmol) and HATU (80mg, 0.21 mmol). The reaction mixture was stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (20mL) and extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with water (20mL), brine solution (20mL), dried over anhydrous sodium sulfate and concentrated in vacuo. By means of flash column chromatography, the liquid phase is separated,the residue was purified using 4.1% methanol/dichloromethane as eluent to give the title compound (40mg, 38%); LC-MS: m/z 595.7(M +1)+。
Step-c: synthesis of DL-valine 2- (2- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) ethyl ester hydrochloride
To (tert-butoxycarbonyl) -DL-valine 2- (2- (2- (benzo [ d ]) at 10 deg.C ]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxamido) ethoxy) ethyl ester (40mg, 0.07mmol) in 1, 4-dioxane (5mL) was added 4N HCl in 1, 4-dioxane (0.03mL, 0.13mmol) and the reaction mixture was stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was concentrated under reduced pressure and lyophilized for 18h to give the title compound as the hydrochloride salt (19mg, 53%);1H NMR(400MHz，DMSO-d6)：δ12.50(s，1H)，8.52(bs，1H)，8.40(bs，3H)，8.09(s，1H)，7.80(d，J＝8.4Hz，1H)，7.54(d，J＝7.6Hz，1H)，7.48(d，J＝7.6Hz，2H)，7.25(t，J＝7.2Hz，1H)，7.17(t，J＝8.0Hz，1H)，4.43-4.40(m，2H)，4.28-4.25(m，2H)，3.95(bs，1H)，3.70-3.67(s，3H)，3.61-3.57(m，2H)，3.45-3.44(m，2H)，2.15-2.11(m，1H)，0.97(d，J＝6.8Hz，3H)，0.92(d，J＝6.8Hz，3H)；LC-MS：m/z 495.1(M+1)+。
examples 31 and 32.Synthesis of 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and N- (2-methoxyethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) HNO3Acetic acid, 0-RT, 16 h; b) 10% Pd/C, MeOH, H2, RT, 16H; c) refluxing potassium ethylxanthate and ethanol for 16 h; d) SOCl2Catalytic DMF, refluxing for 3 h; e) NaH, 2-chloro-6- (trifluoromethyl) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; g) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 2-nitro-5- (trifluoromethyl) phenol
To a solution of 3- (trifluoromethyl) phenol (5.0g, 30.86mmol) in acetic acid (50mL) at 0 deg.C was added dropwise a 60% aqueous nitric acid solution (3.5 mL). Subsequently, the mixture was stirred at this temperature for 1.5h and stirring at room temperature was continued for 16 h. The mixture was poured into ice water (80mL) and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 5% ethyl acetate/hexanes as eluent to give the title compound (1.0g, 16%); 1H NMR(400MHz,DMSO-d6):δ11.75(bs,1H),8.06(d,J＝8.3Hz,1H),7.41(d,J＝1.4Hz,1H),7.32(dd,J＝1.4Hz,J＝8.3Hz,1H)；LC-MS:m/z 206.0(M-1)-。
Step-b: synthesis of 2-amino-5- (trifluoromethyl) phenol
To a solution of 2-nitro-5- (trifluoromethyl) phenol (1.0g, 4.83mmol) in methanol (15mL) under a nitrogen atmosphere was added 10% Pd/C (100 mg). The reaction mixture was stirred under a balloon of hydrogen for 16 h. The reaction mixture was filtered through celite bed and the filtrate was concentrated in vacuo to give the title compound (850mg, 99%);1H NMR(400MHz,DMSO-d6):δ9.58(bs,1H),6.88(s,1H),6.86(s,1H),6.66(d,J＝7.8Hz,1H),5.18(s,2H)；LC-MS:m/z 178.0(M+1)+。
step-c: synthesis of 6- (trifluoromethyl) benzo [ d ] oxazole-2-thiol
To a solution of 2-amino-5- (trifluoromethyl) phenol (850mg, 4.8mmol) in ethanol (10mL) at room temperature was added potassium ethyl-xanthate (1.69g, 10.56mmol) and the reaction mixture was refluxed for 16 h. The reaction mixture was concentrated in vacuo and diluted with cold water (50mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound (1.0g, 95%);1H NMR(400MHz,DMSO-d6):δ14.2(bs,1H),7.98(s,1H),7.66(d,J＝8.4Hz,1H),7.41(d,J＝7.8Hz,1H)；LC-MS:m/z 217.9(M-1)-。
step-d: synthesis of 2-chloro-6- (trifluoromethyl) benzo [ d ] oxazole
To 6- (trifluoromethyl) benzo [ d ] at room temperature]To a solution of oxazole-2-thiol (500mg, 2.28mmol) in thionyl chloride (4mL) was added dimethylformamide (catalytic amount) and the reaction mixture was refluxed for 3 h. The reaction mixture was concentrated, diluted with cold water (30mL) and extracted with EtOAc (2X 30 mL). The combined organic layers were washed with brine solution (20mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (290mg, 57%); 1H NMR(400MHz,DMSO-d6):δ8.31(s,1H),7.97(d,J＝8.3Hz,1H),7.80(d,J＝8.3Hz,1H)。
Step-e: synthesis of ethyl 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at 0 DEG C]To a solution of imidazole-5-carboxylic acid ethyl ester (240mg, 1.09mmol) in 1, 4-dioxane (8mL) was added sodium hydride (60% dispersion in mineral oil) (109mg, 2.74mmol), followed by 2-chloro-6- (trifluoromethyl) benzo [ d []Oxazole (290mg, 1.31 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated, diluted with cold water (15mL) and stirred at room temperature for 30 min. The resulting solid was filtered, dried in vacuo and purified by flash column chromatography using 100% DCM as eluent to give the title compound (150mg, 34%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.25(d,J＝1.5Hz,1H),7.89(dd,J＝1.5Hz,J＝8.3Hz,1H),7.83(s,1H),7.61-7.51(m,3H),4.33(q,J＝6.9Hz,2H),3.66(s,3H),1.36(t,J＝6.9Hz,3H)；LC-MS:m/z 405.30(M+1)+。
step-f: synthesis of 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid (example 31)
To 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (80mg, 0.20mmol) in a solvent mixture of THF (0.5mL), ethanol (0.5mL) and water (0.25mL) was added lithium hydroxide monohydrate (20mg, 0.50 mmol). The reaction mixture was heated at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. Dissolving the residue in water Dissolved in water and acidified with 1N HCl to give a solid which was filtered and dried in vacuo to give the title compound (55mg, 81%);1H NMR(400MHz,DMSO-d6):δ12.80(bs,1H),12.40(bs,1H),8.22(s,1H),7.89(d,J＝8.3Hz,1H),7.84(s,1H),7.63-7.53(m,3H),3.67(s,3H)；LC-MS:m/z 377.05(M+1)+。
step-g: synthesis of N- (2-methoxyethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide (example 32)
To a mixture of 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] at 0 deg.C]Oxazol-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (50mg, 0.13mmol) in DMFA (1.5mL) were added N-ethyldiisopropylamine (0.02mL, 0.13mmol) and diphenylphosphorylazide (0.02mL, 0.13 mmol). The reaction mixture was stirred for 30min, followed by addition of 2-methoxyethylamine (0.01mL, 0.13mmol), and then the reaction mixture was stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (15mL) and stirred for 15 min. The resulting solid was filtered, washed with diethyl ether and dried in vacuo to give the title compound (28mg, 49%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.48(s,1H),8.10(s,1H),7.83(s,1H),7.79(d,J＝8.4Hz,1H),7.61-7.51(m,3H),3.67(s,3H),3.48-3.44(m,4H),3.30(s,3H)；LC-MS:m/z 434.1(M+1)+。
EXAMPLE 33 Synthesis of N- (2-hydroxyethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (trifluoromethyl) benzo [ d) ]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 34%);1H NMR(400MHz，DMSO-d6)：δ12.40(bs，1H)，8.39(t，J＝5.3Hz，1H)，8.10(s，1H)，7.82(s，1H)，7.80(d，J＝8.3Hz，1H)，7.61-7.51(m，3H)，4.72(t，J＝5.2Hz，1H)，3.67(s，3H)，3.54-3.51(m，2H)，3.37-3.34(m，2H)；LC-MS：m/z 420.0(M+1)+。
EXAMPLE 34 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 50%);1H NMR(400MHz，DMSO-d6)：δ12.40(bs，1H)，8.46(bs，1H)，8.10(s，1H)，7.83(s，1H)，7.79(d，J＝8.3Hz，1H)，7.62-7.52(m，3H)，4.60(bs，1H)，3.67(s，3H)，3.58-3.44(m，8H)；LC-MS：m/z 464.20(M+1)+。
EXAMPLE 35 Synthesis of N- (2-hydroxypropyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 1-aminopropan-2-ol and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.35(t，J＝1.2Hz，1H)，8.10(d，J＝1.2Hz，1H)，7.82-7.79(m，2H)，7.61-7.51(m，3H)，4.76(d，J＝4.0Hz，1H)，3.84-3.80(m，1H)，3.67(s，3H)，3.26-3.21(m，2H)，1.09(d，J＝6.4Hz，3H)；LC-MS：m/z 434.2(M+1)+。
EXAMPLE 36 Synthesis of 1-methyl-N- (2- (pyrrolidin-1-yl) ethyl) -2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 2- (pyrrolidin-1-yl) ethan-1-amine and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] ]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6) δ 12.30(bs,1H),8.39(t, J ═ 5.2Hz,1H),8.09(s,1H),7.82(s,1H),7.77(d, J ═ 8.4Hz,1H),7.61-7.51(m,3H),3.66(s,3H),3.43-3.38(m,2H),2.61(t, J ═ 7.2Hz,2H),2.5(m,4H, combined in DMSO peak), 1.69(bs, 4H); LC-MS M/z 473.1(M +1)+。
EXAMPLE 37 Synthesis of 1-methyl-N- (2- (piperidin-1-yl) ethyl) -2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 2- (piperidin-1-yl) ethan-1-amine and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.32(t,J＝5.6Hz,1H),8.07(s,1H),7.82(s,1H),7.75(d,J＝8.0Hz,1H),7.61-7.51(m,3H),3.66(s,3H),3.41-3.36(m,2H),2.43-2.32(m,6H),1.53-1.38(m,6H)；LC-MS:m/z 487.15(M+1)+。
EXAMPLE 38 Synthesis of N- (2- (2-hydroxypropoxy) ethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- (2-aminoethoxy) propan-2-ol and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.46(t,J＝5.6Hz,1H),8.09(s,1H),7.83(s,1H),7.78(d,J＝8.8Hz,1H),7.62-7.52(m,3H),4.58(d,J＝4.4Hz,1H),3.77-3.73(m,1H),3.66(s,3H),3.56(t,J＝6.0Hz,2H),3.45(t,J＝5.2Hz,2H),2.29-2.23(m,2H),1.04(d,J＝6.4Hz,3H)；LC-MS:m/z 478.2(M+1)+。
EXAMPLE 39 Synthesis of N- (2, 3-dihydroxypropyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 3-aminopropane-1, 2-diol and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6) δ 12.40(bs,1H),8.39(t, J ═ 5.2Hz,1H),8.10(s,1H),7.84(s,1H),7.80(d, J ═ 8.4Hz,1H),7.62-7.53(m,3H),4.87(d, J ═ 4.4Hz,1H),4.62(bs,1H),3.67(s,4H),3.45-3.40(m,1H),3.30(2H, combined with DMSO peak), 3.25-3.19(m, 1H); LC-MS M/z 450.15(M +1)+。
EXAMPLE 40 Synthesis of N- (2- (2- (dimethylamino) acetamido) ethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 19 step-c, using N- (2-aminoethyl) -2- (dimethylamino) acetamide trifluoroacetate and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.47(bs，1H)，8.09(s，1H)，7.92(bs，1H)，7.83(s，1H)，7.76(d，J＝8.4Hz，1H)，7.61-7.52(m，3H)，3.66(s，3H)，3.38-3.35(m，4H)，2.86(s，2H)，2.19(s，6H)；LC-S：m/z 504.2(M+1)+。
EXAMPLE 41 Synthesis of 2- (2- (1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) acetic acid
Conditions are as follows: a) ethyl 2- (2-aminoethoxy) acetate trifluoroacetate, HBTU, DIPEA, DMF, 0 ℃ -RT for 16 h; b) h, LiOH 2O, ethanol, THF, water, RT, 3h
Step-a: synthesis of ethyl 2- (2- (1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) acetate
Use ofThe same procedure as in example 29 step-c, using 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid and ethyl 2- (2-aminoethoxy) acetate trifluoroacetate as starting materials the title compound was synthesized (yield: 65%);1H NMR(400MHz，DMSO-d6)：δ12.38(bs，1H)，8.49(bs，1H)，8.10(s，1H)，7.82(s，1H)，7.79(d，J＝8.8Hz，1H)，7.61-7.52(m，3H)，4.15-4.09(m，4H)，3.66-3.62(m，5H)，3.48-3.46(m，2H)，1.19(t，J＝7.0Hz，3H)；LC-MS：m/z 506.1(M+1)+。
step-b: synthesis of 2- (2- (1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamido) ethoxy) acetic acid
Using the same procedure as example 29 step-d, using 2- (2- (1-methyl-2- ((6- (trifluoromethyl) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxamido) ethoxy) ethyl acetate was used as a starting material to synthesize the title compound (yield: 75%);1H NMR(400MHz，DMSO-d6)：δ12.70(bs，1H)，12.30(bs，1H)，8.49(bs，1H)，8.10(s，1H)，7.82-7.78(m，2H)，7.61-7.51(m，3H)，4.06(s，2H)，3.66-3.62(m，5H)，3.48-3.41(m，2H)；LC-MS：m/z 478.2(M+1)+。
EXAMPLE 42 Synthesis of N- (2- (2-hydroxy-2-methylpropoxy) ethyl) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 1- (2-aminoethoxy) -2-methylpropan-2-ol and 1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] ]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz，DMSO-d6)：δ12.40(bs，1H)，8.83(s，1H)，8.08(s，1H)，7.81(s，1H)，7.77(d，J＝8.4Hz，1H)，7.58-7.52(m，3H)，4.32(bs，1H)，3.66(s，3H)，3.58-3.56(m，2H)，3.46-3.44(m，2H)，3.21(s，2H)，1.08(s，6H)；LC-MS：m/z 492.1(M+1)+。
EXAMPLE 43 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((5- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaOH and DMSO at 60 ℃ for 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) refluxing potassium ethylxanthate and ethanol for 16 h; d) SOCl2Catalytic DMF, refluxing for 1 h; e) NaH, 2-chloro-5- (trifluoromethyl) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, methanol, water, 50 ℃ and 16 h; g)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 2-nitro-4- (trifluoromethyl) phenol
To a solution of 1-chloro-2-nitro-4- (trifluoromethyl) benzene (10.0g, 44.3mmol) in DMSO (100mL) at room temperature was added sodium hydroxide (4.44g, 110.7mmol) and the mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with water (250mL) and extracted with EtOAc (200 mL). The aqueous layer was acidified to pH-1 with 3N HCl and extracted with EtOAc (2X 250mL), the organic layer was washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (7.0g, 81%); 1H NMR(400MHz，DMSO-d6)：δ11.99(bs，1H)，8.22(d，J＝1.6Hz，1H)，7.88(dd，J＝2.0Hz，J＝8.8Hz，1H)，7.30(d，J＝8.8Hz，1H)。
Step-b: synthesis of 2-amino-4- (trifluoromethyl) phenol
To a solution of 2-nitro-4- (trifluoromethyl) phenol (5.0g, 24.15mmol) in methanol (50mL) under a nitrogen atmosphere was added 10% Pd/C (2.5 g). The reaction mixture was stirred under a balloon of hydrogen for 16 h. The reaction mixture was filtered through a celite bed, and the filtrate was concentrated in vacuo to give the title compound (3.0g, 70%);1H NMR(400MHz，DMSO-d6)：δ9.85(bs，1H)，6.87(d，J＝2.0Hz，1H)，6.77-6.70(m，2H)，5.0(bs，2H)；LC-MS：m/z 178.1(M+1)+。
step-c: synthesis of 5- (trifluoromethyl) benzo [ d ] oxazole-2-thiol
To a solution of 2-amino-4- (trifluoromethyl) phenol (3.0g, 16.9mmol) in ethanol (45mL) at room temperature was added potassium ethyl-xanthate (6.78g, 42.4mmol) and the reaction mixture was refluxed for 16 h. The reaction mixture was concentrated in vacuo and diluted with cold water (100mL) and acidified with 3N HCl. The precipitated solid was filtered and dried in vacuo to give the title compound (3.0g, 81%);1H NMR(400MHz,DMSO-d6):δ14.2(bs,1H),7.72(d,J＝8.4Hz,1H),7.64(d,J＝8.4Hz,1H),7.51(s,1H)；LC-MS:m/z 220.0(M+1)+。
step-d: synthesis of 2-chloro-5- (trifluoromethyl) benzo [ d ] oxazole
To 5- (trifluoromethyl) benzo [ d ] at room temperature]To a solution of oxazole-2-thiol (2.0g, 9.1mmol) in thionyl chloride (10mL) was added N, N-dimethylformamide (catalytic amount) and the reaction mixture was refluxed for 1 h. The reaction mixture was concentrated, diluted with cold water (60mL) and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (1.3g, 65%); 1H NMR(400MHz,DMSO-d6):δ8.23(s,1H),8.02(d,J＝8.8Hz,1H),7.85(d,J＝8.8Hz,1H)。
Step-e: synthesis of ethyl 1-methyl-2- ((5- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at 0 DEG C]To a solution of imidazole-5-carboxylic acid ethyl ester (200mg, 0.91mmol) in 1, 4-dioxane (10mL) was added sodium hydride (60% dispersion in mineral oil) (91mg, 2.27mmol) and stirred for 15min, followed by addition of 2-chloro-5- (trifluoromethyl) benzo [ d [ -d ] ]]Oxazole (222mg, 1.0mmol) and stirring at room temperature was continued for 16 h. The reaction mixture was concentrated, diluted with cold water (50mL) and acidified with 3N HCl and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water (30mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% dichloromethane as eluent to give the title compound (150mg, 40%);1H NMR(400MHz,DMSO-d6):δ12.41(bs,1H),8.25(s,1H),7.90(dd,J＝1.2Hz,J＝8.4Hz,1H),7.67-7.64(m,2H),7.57(d,J＝8.4Hz,1H),7.49(d,J＝8.4Hz,1H),4.33(q,J＝7.2Hz,2H),3.66(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 405.00(M+1)+。
step-f: synthesis of 1-methyl-2- ((5- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
To 1-methyl-2- ((5- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (150mg, 0.37mmol) in a solvent mixture of THF (2mL), methanol (2mL) and water (2mL) was added lithium hydroxide monohydrate (78mg, 1.85 mmol). The reaction mixture was heated at 50 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (30mL), acidified to pH 2 with 3N HCl and stirred for 1 h. The precipitated solid was filtered and dried in vacuo to give the title compound (140mg, 100%); 1H NMR(400MHz,DMSO-d6):δ12.85(bs，1H)，12.40(bs，1H)，8.18(s，1H)，7.87(d，J＝8.4Hz，1H)，7.67(s，1H)，7.63(d，J＝8.4Hz，1H)，7.52(d，J＝8.4Hz，1H)，7.46(d，J＝8.0Hz，1H)，3.64(s，3H)；LC-MS：m/z 377.1(M+i)+。
Step-g: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((5- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
To a mixture of 1-methyl-2- ((5- (trifluoromethyl) benzo [ d ] at 0 deg.C]Oxazol-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (80mg, 0.21mmol) in DMFA (5mL) were added N-ethyldiisopropylamine (0.04mL, 0.21mmol) and HBTU (81mg, 0.21 mmol). The reaction mixture was stirred for 15min, followed by addition of 2- (2-aminoethoxy) ethan-1-ol (22mg, 0.21 mmol). The reaction mixture was then stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (30mL) and stirred for 1 h. The precipitated solid was filtered and dried in vacuo. The solid was stirred in diethyl ether (15mL), filtered and dried in vacuo to give the title compound (80mg, 85%);1H NMR(400MHz，DMSO-d6)：δ12.33(bs，1H)，8.46(t，J＝5.4Hz，1H)，8.09(d，J＝1.2Hz，1H)，7.78(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.68(s，1H)，7.64(d，J＝8.4Hz，1H)，7.52(d，J＝8.0Hz，1H)，7.48(d，J＝8.4Hz，1H)，4.60(t，J＝5.2Hz，1H)，3.66(s，3H)，3.58-3.43(m，8H)；LC-MS：m/z464.2(M+1)+。
example 44 and 45.1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid Synthesis and N- (2-methoxyethyl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide Synthesis:
conditions are as follows: a) HNO3Acetic acid, 15 ℃ -RT for 16 h; b) 10% Pd/C, MeOH, H 2RT, 5 h; c) carbon disulfide, KOH and ethanol are refluxed for 6 hours; d) k2CO3Methyl iodide, acetonitrile, RT, 16 h; e) m-CPBA, DCM, 0 ℃ -RT, 6 h; f) NaH, 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; g) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; h) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 2-nitro-5- (trifluoromethoxy) phenol
To a solution of 3- (trifluoromethoxy) phenol (1g, 5.6mmol) in acetic acid (10mL) at 10-15 ℃ was added dropwise 60% aqueous nitric acid (1 mL). The mixture was stirred at this temperature for 1.5h and stirring at room temperature was continued for 16 h. The mixture was poured into ice water (20mL) and extracted with EtOAc (2X 25 mL). The combined organic layers were washed with brine solution (25mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 5% EtOAc/hexanes as the eluent to give the title compound (300mg, 24%);1H NMR(400MHz,CDCl3):δ10.73(s,1Η),8.18(d,J＝9.6Hz,1H),6.99(s,1H),6.83(d,J＝9.2Hz,1H)；LC-MS:m/z 222.0(M-1)。
step-b: synthesis of 2-amino-5- (trifluoromethoxy) phenol
Under nitrogen atmosphere, to 2-nitro-5To a solution of (trifluoromethoxy) phenol (300mg, 1.35mmol) in methanol (6mL) was added 10% Pd/C (60 mg). The reaction mixture was stirred under hydrogen for 5 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (250mg, 96%); 1H NMR(400MHz,DMSO-d6):δ9.60(s,1Η),6.59-6.57(m,2H),6.53-6.50(m,1H),4.68(bs,2H)；LC-MS:m/z 194.0(M+1)+。
Step-c: synthesis of 6- (trifluoromethoxy) benzo [ d ] oxazole-2-thiol
To a solution of 2-amino-5- (trifluoromethoxy) phenol (250mg, 1.3mmol) in ethanol (5mL) at room temperature was added powdered potassium hydroxide (127mg, 2.3mmol) and carbon disulfide (1mL, 17.0 mmol). The reaction mixture was refluxed for 6 h. After cooling to room temperature, the mixture was concentrated in vacuo. The residue was diluted with cold water (100mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound (200mg, 66%);1H NMR(400MHz,DMSO-d6):δ14.10(bs,1H),7.73(s,1H),7.32(s,2H)；LC-MS:m/z 235.9(M+1)+。
step-d: synthesis of 2- (methylthio) -6- (trifluoromethoxy) benzo [ d ] oxazole
Using the same procedure as for Compound 1b, using 6- (trifluoromethoxy) benzo [ d ]]Oxazole-2-thiol as a starting material and stirred at room temperature for 16h to synthesize the title compound (yield: 94%);1H NMR(400MHz,DMSO-d6):δ7.85(d,J＝1.6Hz,1H),7.73(d,J＝8.0Hz,1H),7.37-7.34(m,1H),2.77(s,3H)；LC-MS:m/z 250.1(M+1)+。
step-e: synthesis of 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d ] oxazole
Using the same procedure as for Compound 1c, 2- (methylthio) -6- (trifluoromethoxy) benzo [ d ] was used]Oxazole as a starting material and stirred at room temperature for 6h to synthesize the title compound (yield: 94%);1H NMR(400MHz,DMSO-d6):δ8.22(s,1H),8.15(d,J＝8.8Hz,1H),7.62(d,J＝8.8Hz,1H),3.67(s,3H)。
step-f: synthesis of ethyl 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at room temperature]To a solution of imidazole-5-carboxylic acid ethyl ester (100mg, 0.46mmol) in 1, 4-dioxane (2mL) was added sodium hydride (60% dispersion in mineral oil) (64mg, 1.6mmol), followed by 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d ]]Oxazole (167mg, 0.6 mmol). The reaction mixture was stirred at room temperature for 16 h. It was then concentrated, diluted with cold water (15mL) and acidified with 1N HCl. The resulting solid was filtered, dried in vacuo and purified by flash column chromatography using 100% DCM as eluent to give the title compound (30mg, 16%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.24(s,1H),7.89(dd,J＝1.6Hz,J＝8.4Hz,1H),7.58-7.50(m,3H),7.22(d,J＝8.0Hz,1H),4.33(q,J＝7.6Hz,2H),3.65(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 421.0(M+1)+。
step-g: synthesis of 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 72%);1H NMR(400MHz,DMSO-d6):δ12.80(bs,1H),12.30(bs,1H),8.20(s,1H),7.88(d,J＝8.4Hz,1H),7.58(s,1H),7.52(d,J＝8.8Hz,2H),7.22(d,J＝8.0Hz,1H),3.65(s,3H)；LC-MS:m/z 393.0(M+1)+。
step-h: synthesis of N- (2-methoxyethyl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 23%);1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.47(t,J＝5.2Hz,1H),8.08(s,1H),7.78(d,J＝8.4Hz,1H),7.57(s,1H),7.52-7.49(m,2H),7.21(d,J＝8.4Hz,1H),3.64(s,3H),3.48-3.44(m,4H),3.28(s,3H)；LC-MS:m/z 450.0(M+1)+。
EXAMPLE 46 Synthesis of 1-methyl-N- (1H-pyrazol-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1H-pyrazol-4-amine and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.65(bs,1H),12.30(bs,1H),10.40(s,1H),8.17(d,J＝1.2Hz,1H),8.02(bs,1H),7.89(dd,J＝1.2Hz,J＝8.4Hz,1H),7.69(bs,1H),7.58-7.49(m,3H),7.21(d,J＝8.4Hz,1H),3.67(s,3H)；LC-MS:m/z 458.0(M+1)+。
EXAMPLE 47 Synthesis of N- (1, 3-dihydroxy-2- (hydroxymethyl) propan-2-yl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 2-amino-2- (hydroxymethyl) propane-1, 3-diol and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1HNMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.05(s,1H),7.43(d,J＝8.4Hz,1H),7.57(s,1H),7.50(d,J＝5.8Hz,2H),7.26(s,1H),7.21(d,J＝8.4Hz,1H),4.83(bs,3H),3.71(bs,6H),3.65(s,3H)；LC-MS:m/z 496.3(M+1)+。
EXAMPLE 48 Synthesis of N- (1, 3-dihydroxypropan-2-yl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 2-amino-propane-1, 3-diol and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.09(s,1H),7.88(d,J＝7.6Hz,1H),7.81(d,J＝8.4Hz,1H),7.57(d,J＝1.2Hz,1H),7.51(d,J＝8.0Hz,2H),7.21(dd,J＝1.6Hz,J＝8.4Hz,1H),4.66(t,J＝6.0Hz,2H),4.00-3.95(m,1H),3.65(s,3H),3.54(t,J＝6.0Hz,4H)；LC-MS:m/z466.0(M+1)+。
EXAMPLE 49 Synthesis of N- (2- (2- (dimethylamino) acetamido) ethyl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 19 step-c, using N- (2-aminoethyl) -2- (dimethylamino) acetamide trifluoroacetate and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6) δ 12.0(bs,1H),8.45(t, J ═ 4.4Hz,1H),8.07(s,1H),7.91(t, J ═ 5.6Hz,1H),7.75(d, J ═ 8.4Hz,1H),7.56(s,1H),7.50(d, J ═ 8.4Hz,2H),7.21(d, J ═ 8.0Hz,1H),3.64(s,3H),3.36(t, J ═ 5.6Hz,2H),3.30(2H, combined with DMSO water peak), 2.85(s,2H),2.18(s, 6H); LC-MS M/z 520.5(M +1)+。
EXAMPLE 50 Synthesis of 1-methyl-N- (1-methyl-1H-pyrazol-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-methyl-1H-pyrazol-4-amine and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ]]Oxazol-2-yl) amino) -1H-benzo [ d ]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),10.40(s,1H),8.17(s,1H),8.03(s,1H),7.88(d,J＝8.0Hz,1H),7.58-7.51(m,4H),7.22(d,J＝7.6Hz,1H),3.83(s,3H),3.66(s,3H)；LC-MS:m/z472.0(M+1)+。
EXAMPLE 51 Synthesis of 1-methyl-N- (oxetan-3-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, oxetan-3-amine and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),9.07(d,J＝6.0Hz,1H),8.10(s,1H),7.85(d,J＝8.4Hz,1H),7.57(s,1H),7.52(t,J＝8.8Hz,2H),7.21(d,J＝8.4Hz,1H),5.05-5.00(m,1H),4.78(t,J＝6.4Hz,2H),4.62(t,J＝6.4Hz,2H),3.65(s,3H)；LC-MS:m/z 448.0(M+1)+。
EXAMPLE 52 Synthesis of N- ((3-hydroxyoxetan-3-yl) methyl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 3- (aminomethyl) oxetan-3-ol and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.54(t,J＝6.0Hz,1H),8.10(d,J＝1.2Hz,1H),7.80(dd,J＝1.2Hz,J＝8.8Hz,1H),7.57(d,J＝1.6Hz,1H),7.53-7.49(m,2H),7.21(dd,J＝1.6Hz,J＝8.8Hz,1H),5.88(s,1H),4.52(d,J＝6.4Hz,2H),4.41(d,J＝6.4Hz,2H),3.65(s,3H),3.59(d,J＝6.0Hz,2H)；LC-MS:m/z 478.2(M+1)+。
EXAMPLE 53. Synthesis of N- (2-hydroxyethyl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 2-aminoethan-1-ol and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized. 1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.38(t,J＝5.2Hz,1H),8.09(d,J＝1.2Hz,1H),7.78(dd,J＝1.6Hz,J＝8.8Hz,1H),7.57(s,1H),7.52-7.49(m,2H),7.22(d,J＝8.0Hz,1H),4.72(t,J＝5.2Hz,1H),3.64(s,3H),3.54(q,J＝6.0Hz,2H),3.37-3.30(m,2H)；LC-MS:m/z 436.0(M+1)+。
EXAMPLE 54 Synthesis of 1-methyl-N- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 2- (methylsulfonyl) ethan-1-amine hydrochloride and 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.68(t,J＝5.2Hz,1H),8.09(d,J＝1.6Hz,1H),7.76(dd,J＝1.6Hz,J＝8.4Hz,1H),7.57(s,1H),7.53-7.50(m,2H),7.27-7.20(m,1H),3.70(q,J＝6.0Hz,2H),3.65(s,3H),3.40(t,J＝6.8Hz,2H),3.05(s,3H)；LC-MS:m/z 497.9(M+1)+。
Examples 55 and 56.2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid synthesis and 2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide synthesis
Conditions are as follows: a) 2-methoxyethyl-1-ol, sodium, 100 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) refluxing potassium ethylxanthate and ethanol for 16 h; d) SOCl2Catalytic DMF, refluxing for 2 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, ethanol, water, 60 ℃ and 8 h; g) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 5- (2-methoxyethoxy) -2-nitrophenol
To stirred 2-methoxyethon-1-ol (50mL) at 0 deg.C was added sodium metal (2.29g, 95.46mmol) and the reaction mixture was stirred at room temperature for 30min, followed by the addition of 5-fluoro-2-nitrophenol (5.0g, 31.82 mmol). The reaction mixture was heated to 100 ℃ for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was cooled to 0 ℃, diluted with cold water (200mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound as a yellow solid (4.0g, 59%);1H NMR(400MHz，DMSO-d6)：δ10.93(s，1H)，7.96(d，J＝9.3Hz，1H)，6.64-6.58(m，2H)，4.19-4.17(m，2H)，3.67-3.65(m，2H)，3.30(s，3H)。
step-b: synthesis of 2-amino-5- (2-methoxyethoxy) phenol
The title compound was synthesized using the same procedure as compound 1e, using 5- (2-methoxyethoxy) -2-nitrophenol as the starting material (yield: 96%); LC-MS: m/z 184.1(M +1)+。
Step-c: synthesis of 6- (2-methoxyethoxy) benzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32 step-c, using 2-amino-5- (2-methoxyethoxy) phenol as the starting material (yield: 76%);1H NMR(400MHz，DMSO-d6)：613.78(s，1H)，7.29(d，J＝2.4Hz，1H)，7.19(d，J＝8.8Hz，1H)，6.96(dd，J＝2.4Hz，J＝8.8Hz，1H)，4.18-4.16(m，2H)，3.73-3.71(m，2H)，3.36(s，3H)；LC-MS：m/z 226.0(M+1)+。
step-d: synthesis of 2-chloro-6- (2-methoxyethoxy) benzo [ d ] oxazole
Using the same procedure as example 32 step-d, using 6- (2-methoxyethoxy) benzo [ d ]The title compound was synthesized with oxazole-2-thiol as the starting material and stirring for 2h (yield: 69%); LC-MS: m/z 228.05(M +1)+。
Step-e: synthesis of ethyl 2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound example 32, step-e, using 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (2-methoxyethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 24%);1H NMR(400MHz,DMSO-d6):δ12.20(bs,1H),8.21(d,J＝1.5Hz,1H),7.86(dd,J＝1.5Hz,J＝8.3Hz,1H),7.49(d,J＝8.3Hz,1H),7.35(d,J＝8.8Hz,1H),7.14(d,J＝2.4Hz,1H),6.83(dd,J＝2.4Hz,J＝8.8Hz,1H),4.33(q,J＝7.2Hz,2H),4.12(t,J＝4.6Hz,2H),3.67(t,J＝4.6Hz,2H),3.62(s,3H),3.32(s,3H),1.35(t,J＝7.1Hz,3H)；LC-MS:m/z 411.0(M+1)+。
step-f: synthesis of 2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, using 2- ((6- (2-methoxyethoxy) benzo [ d]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 77%);1H NMR(400MHz,DMSO-d6):δ12.15(bs,2H),8.17(d,J＝1.5Hz,1H),7.85(dd,J＝2.0Hz,J＝8.8Hz,1H),7.47(d,J＝8.3Hz,1H),7.36(d,J＝8.3Hz,1H),7.13(d,J＝2.4Hz,1H),6.82(dd,J＝2.4Hz,J＝8.3Hz,1H),4.12(t,J＝4.6Hz,2H),3.67(t,J＝4.6Hz,2H),3.62(s,3H),3.32(s,3H)；LC-MS:m/z 383.0(M+1)+。
step-g: synthesis of 2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((6- (2-methoxyethoxy) benzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 29%);1H NMR(400MHz,DMSO-d6):δ12.15(bs,1H),8.44(t,J＝5.4Hz,1H),8.05(d,J＝1.5Hz,1H),7.75(dd,J＝1.4Hz,J＝8.3Hz,1H),7.45(d,J＝8.3Hz,1H),7.35(d,J＝8.3Hz,1H),7.13(d,J＝2.4Hz,1H),6.82(dd,J＝2.4Hz,J＝8.3Hz,1H),4.12-4.10(m,2H),3.68-3.66(m,2H),3.62(s,3H),3.49-3.43(m,4H),3.32(s,3H),3.28(s,3H)；LC-MS:m/z 440.60(M+1)+。
EXAMPLE 57 Synthesis of N- (2-hydroxyethyl) -2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Using the same procedure as example 6 step-e, using 2- ((6- (2-methoxyethoxy) benzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid as a starting material (yield: 27%);1H NMR(400MHz，DMSO-d6)：δ12.16(bs，1H)，8.35(t，J＝5.4Hz，1H)，8.05(d，J＝1.0Hz，1H)，7.75(dd，J＝1.5Hz，J＝8.3Hz，1H)，7.45(d，J＝8.3Hz，1H)，7.35(d，J＝8.8Hz，1H)，7.12(d，J＝2.4Hz，1H)，6.81(dd，J＝2.5Hz，J＝8.4Hz，1H)，4.72(bs，1H)，4.11(t，J＝4.6Hz，2H)，3.67(t，J＝4.6Hz，2H)，3.61(s，3H)，3.53(t，J＝5.3Hz，2H)，3.35(t，J＝6.0Hz，2H)，3.32(s，3H)；LC-MS：m/z 426.45(M+1)+。
EXAMPLE 58. Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6 step-e, using 2- ((6- (2-methoxyethoxy) benzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 61%);1H NMR(400MHz，DMSO-d6)：δ12.15(bs，1H)，8.41(t，J＝5.4Hz，1H)，8.05(d，J＝0.9Hz，1H)，7.74(dd，J＝1.5Hz，J＝8.3Hz，1H)，7.45(d，J＝8.3Hz，1H)，7.35(d，J＝8.8Hz，1H)，7.12(d，J＝1.9Hz，1H)，6.82(dd，J＝2.4Hz，J＝8.8Hz，1H)，4.60(bs，1H)，4.13(t，J＝4.6Hz，2H)，3.67(t，J＝4.6Hz，2H)，3.62(s，3H)，3.57-3.42(m，8H)，3.32(s，3H)；LC-MS：m/z470.25(M+1)+。
examples 59 and 60.2- ((6-isopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and 2- ((6-isopropylbenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaNO3，NaNO2，3M H2SO4，DCM，RT，24h；b)10％Pd/C，MeOH，H2RT, 16 h; c) refluxing potassium ethylxanthate and ethanol for 16 h; d) SOCl2Catalytic DMF, refluxing for 2 h; e) n is a radical ofaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, ethanol, water, 60 ℃ and 5 hours; g) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 5-isopropyl-2-nitrophenol
To a solution of 3-isopropylphenol (2.0g, 14.7mmol) in DCM (30mL) and 3M sulfuric acid (25mL) was added sodium nitrate (1.37g, 16.18mmol) and sodium nitrite (10mg, 0.14mmol) at room temperature. Subsequently, the mixture was stirred for 24 h. The mixture was poured into ice water (100mL) and extracted with DCM (2X 50 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 5% ethyl acetate/hexanes as the eluent to give the title compound (500mg, 19%);1H NMR (400MHz，DMSO-d6)：δ10.76(s，1H)，7.86(d，J＝8.8Hz，1H)，6.98(d，J＝1.5Hz，1H)，6.89(dd，J＝1.5Hz，J＝8.3Hz，1H)，2.92-2.89(m，1H)，1.19(d，J＝6.9Hz，6H)；LC-MS：m/z 180.0(M-1)。
step-b: synthesis of 2-amino-5-isopropylphenol
The title compound was synthesized using the same procedure as compound 1e, using 5-isopropyl-2-nitrophenol as the starting material (yield: 86%);1H NMR(400MHz，DMSO-d6)：δ8.88(bs，1H)，6.50(dd，J＝1.9Hz，J＝10.7Hz，1H)，6.41(d，J＝2.0Hz，1H)，6.39(d，J＝2.0Hz，1H)，4.25(s，2H)，2.68-2.61(m，1H)，1.10(d，J＝6.8Hz，6H)；LC-MS：m/z 152.15(M+1)+。
step-c: synthesis of 6-isopropylbenzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5-isopropylphenol as a starting material (yield: 68%);1H NMR(400MHz，DMSO-d6)：δ13.74(s，1H)，7.41(s，1H)，7.20-7.13(m，2H)，3.0-2.93(m，1H)，1.21(d，J＝6.9Hz，6H)；LC-MS：m/z 194.15(M+1)+。
step-d: synthesis of 2-chloro-6-isopropylbenzo [ d ] oxazole
Using the same procedure as in example 32, step-d, using 6-isopropylbenzo [ d ]]The title compound was synthesized with oxazole-2-thiol as the starting material and stirring for 2h (yield: 85%);1H NMR(400MHz,DMSO-d6):δ7.17(s,1H),7.02-6.97(m,2H),2.96-2.86(m,1H),1.19(d,J＝6.8Hz,6H)；LC-MS:m/z 196.0(M+1)+。
step-e: synthesis of ethyl 2- ((6-isopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6-isopropylbenzo [ d ]]Oxazole as a starting material the title compound was synthesized (yield: 77%);1H NMR(400MHz,DMSO-d6):δ12.15(bs,1H),8.22(d,J＝1.0Hz,1H),7.86(dd,J＝1.0Hz,J＝8.3Hz,1H),7.50(d,J＝8.3Hz,1H),7.38(d,J＝8.3Hz,1H),7.33(s,1H),7.11(d,J＝7.8Hz,1H),4.33(q,J＝7.0Hz,2H),3.63(s,3H),3.01-2.95(m,1H),1.35(t,J＝7.0Hz,3H),1.25(d,J＝6.8Hz,6H)；LC-MS:m/z 379.1(M+1)+。
step-f: synthesis of 2- ((6-isopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- ((6-isopropylbenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester as starting material and stirred at 60 ℃ for 5h to synthesize the title compound (yield: 92%);1H NMR(400MHz,DMSO-d6):δ12.20(bs,2H),8.18(s,1H),7.86(d,J＝8.3Hz,1H),7.49(d,J＝8.3Hz,1H),7.39(d,J＝7.8Hz,1H),7.33(s,1H),7.10(d,J＝7.8Hz,1H),3.63(s,3H),2.98-2.97(m,1H),1.25(d,J＝6.8Hz,6H)；LC-MS:m/z 351.20(M+1)+。
step-g: synthesis of 2- ((6-isopropylbenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((6-isopropylbenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized (yield: 69%)；1H NMR(400MHz,DMSO-d6):δ12.15(bs,1H),8.44(s,1H),8.07(s,1H),7.75(d,J＝8.3Hz,1H),7.46(d,J＝8.3Hz,1H),7.37(d,J＝8.3Hz,1H),7.32(s,1H),7.09(d,J＝7.8Hz,1H),3.63(s,3H),3.48-3.44(m,4H),3.28(s,3H),2.98-2.97(m,1H),1.25(d,J＝6.8Hz,6H)；LC-MS:m/z 408.0(M+1)+。
EXAMPLE 61. Synthesis of N- (2-hydroxyethyl) -2- ((6-isopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide.
Using the same procedure as example 6, step-e, using 2- ((6-isopropylbenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials.1H NMR(400MHz,DMSO-d6):δ12.50(bs,1H),8.43(bs,1H),8.08(s,1H),7.82(d,J＝8.0Hz,1H),7.55(d,J＝8.2Hz,1H),7.38-7.36(m,2H),7.16(d,J＝8.0Hz,1H),3.67(s,3H),3.54(d,J＝5.6Hz,2H),3.36(q,J＝6.0Hz,2H),3.02-2.99(m,1H),1.25(d,J＝7.2Hz,6H)；LC-MS:m/z 394.0(M+1)+。
EXAMPLE 62 Synthesis of 2- ((6- (difluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Conditions are as follows: a) k2CO3Benzyl bromide, DMF, RT, 16 h; b) KOH, water, 100 ℃, 30 h; c) diethyl phosphonate (bromodifluoromethyl ester), KOH, ACN and water (1: 1), RT, 1 h; d) 10% Pd/C, MeOH, H2RT, 16 h; e) refluxing potassium ethylxanthate and ethanol for 16 h; f) SOCl2Catalytic DMF, refluxing for 2 h; g) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH２O, THF, ethanol, water, 60 ℃ for 16h
Step-a: synthesis of 2- (benzyloxy) -4-fluoro-1-nitrobenzene
To a stirred solution of 5-fluoro-2-nitrophenol (10g, 63.7mmol) in DMFA (100mL) at room temperature was added potassium carbonate (10.54g, 76.43 g)mmol) and benzyl bromide (7.56mL, 63.7 mmol). The reaction mixture was then stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with water (300mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with cold water (2X 200mL), brine solution (100mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 5% EtOAc/hexanes as eluent to give the title compound (12.0g, 76%);1H NMR(400MHz，DMSO-d6)：δ8.04(dd，J＝5.8Hz，J＝9.3Hz，1H)，7.48-7.34(m，6H)，7.02-6.97(m，1H)，5.33(s，2H)。
step-b: synthesis of 3- (benzyloxy) -4-nitrophenol
To a stirred solution of KOH (7.93g, 141.7mmol) in water (70mL) at room temperature was added 2- (benzyloxy) -4-fluoro-1-nitrobenzene (7.0g, 28.3mmol) and the reaction mixture was stirred at 100 ℃ for 30 h. The reaction mixture was cooled to room temperature, diluted with water (100mL), acidified with 3N HCl and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 20% EtOAc/hexanes as eluent to give the title compound (2.0g, 29%); 1H NMR(400MHz，DMSO-d6)：δ10.88(s，1H)，7.90(d，J＝8.8Hz，1H)，7.47(t，J＝7.4Hz，2H)，7.41-7.39(m，2H)，7.36-7.32(m，1H)，6.69(d，J＝2.0Hz，1H)，6.49(dd，J＝2.4Hz，J＝9.3Hz，1H)，5.25(s，2H)；LC-MS：m/z 244.0(M-1)。
Step-c: synthesis of 2- (benzyloxy) -4- (difluoromethoxy) -1-nitrobenzene
To a stirred solution of 3- (benzyloxy) -4-nitrophenol (1.1g, 4.49mmol) in acetonitrile (6mL) at 0 deg.C was added potassium hydroxide (5.02g, 89.79mmol) in water (6mL) and diethyl phosphonate (bromodifluoromethyl ester) (1.58mL, 8.98 mmol). The reaction mixture was stirred at room temperature for 1h and diluted with water (100 mL). The reaction mixture was extracted with ethyl acetate (3X 60 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. By flash column chromatography, using 10% EtOAc in hexanes asThe residue was purified by eluent to give the title compound (1.2g, 91%);1H NMR(400MHz,DMSO-d6):δ8.04(d,J＝9.3Hz,1H),7.64-7.27(m,6H),7.26(d,J＝2.5Hz,1H),6.93(dd,J＝2.5Hz,J＝8.8Hz,1H),5.34(s,2H)。
step-d: synthesis of 2-amino-5- (difluoromethoxy) phenol
To a solution of 2- (benzyloxy) -4- (difluoromethoxy) -1-nitrobenzene (1.2g, 4.07mmol) in methanol (20mL) under a nitrogen atmosphere was added 10% Pd/C (300 mg). The reaction mixture was stirred under a balloon of hydrogen for 16 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (600mg, 84%); LC-MS: m/z 176.0(M +1)+。
Step-e: synthesis of 6- (difluoromethoxy) benzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5- (difluoromethoxy) phenol as the starting material (yield: 81%); 1H NMR(400MHz,DMSO-d6):δ13.96(bs,1H),7.49(d,J＝2.0Hz,1H),7.26(d,J＝8.3Hz,1H),7.21(t,J＝73.2Hz,1H),7.13(dd,J＝2.4Hz,J＝8.8Hz,1H)；LC-MS:m/z 218.0(M+1)+。
Step-f: synthesis of 2-chloro-6- (difluoromethoxy) benzo [ d ] oxazole
Using the same procedure as example 32 step-d, using 6- (difluoromethoxy) benzo [ d ]]The title compound was synthesized with oxazole-2-thiol as the starting material and stirring for 2h (yield: 82%);1H NMR(400MHz,DMSO-d6):δ7.80(d,J＝8.8Hz,1H),7.74(d,J＝2.5Hz,1H),7.29-7.27(m,2H)。
step-g: synthesis of ethyl 2- ((6- (difluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (difluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 27%);1H NMR(400MHz,DMSO-d6):δ12.20(bs,1H),8.23(d,J＝1.0Hz,1H),7.88(dd,J＝1.5Hz,J＝8.4Hz,1H),7.53(d,J＝8.3Hz,1H),7.46(d,J＝8.3Hz,1H),7.38-7.37(m,1H),7.19(s,1H),7.07-7.00(m,1H),4.33(q,J＝7.0Hz,2H),3.64(s,3H),1.35(t,J＝7.1Hz,3H)；LC-MS:m/z 403.0(M+1)+。
step-h: synthesis of 2- ((6- (difluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, using 2- ((6- (difluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 93%);1H NMR(400MHz,DMSO-d6):δ12.80(bs,1H),12.40(bs,1H),8.19(d,J＝1.0Hz,1H),7.87(dd,J＝1.4Hz,J＝8.3Hz,1H),7.52-7.46(m,2H),7.38-7.00(m,3H),3.64(s,3H)；LC-MS:m/z 375.0(M+1)+。
EXAMPLE 63 Synthesis of 2- ((6- (difluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((6- (difluoromethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid and 2-methoxyethyl-1-amine as starting materials to synthesize the title compound;1H NMR(400MHz,DMSO-d6):δ12.25(bs,1H),8.45(t,J＝5.2Hz,1H),8.07(s,1H),7.77(d,J＝8.4Hz,1H),7.49-7.45(m,2H),7.37(s,1H),7.18(s,1H),7.06-7.00(m,1H),3.63(s,3H),3.48-3.44(m,4H),3.28(s,3H)；LC-MS:m/z 432.0(M+1)+。
EXAMPLE 64 Synthesis of 2- ((6- (difluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((6- (difluoromethoxy) benzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials to synthesize the title compound;1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.38(t,J＝5.6Hz,1H),8.08(s,1H),7.78(d,J＝8.4Hz,1H),7.50(d,J＝8.4Hz,1H),7.46(d,J＝8.4Hz,1H),7.38-7.00(m,3H),3.64(s,3H),3.53(t,J＝6.4Hz,2H),3.35(q,J＝6.0Hz,2H)；LC-MS:m/z 418.0(M+1)+。
EXAMPLE 65. Synthesis of N- (2-methoxyethyl) -1-methyl-2- ((5-methylbenzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 10% Pd/C, MeOH, H2RT, 16 h; b) potassium ethyl xanthate, EtOH, refluxing for 16 h; c) k2CO3，MeI，ACN，0℃-RT，3h；d)m-CPBA，DCM，0℃-RT，3h；e)SOCl2Catalytic DMF, DCM, reflux, 4 h; f) 2-methoxyethyl-1-amine, TEA, DCM, 0 deg.C-RT, 16 h; g) THF, DIPEA and DMF containing 2M methylamine at 70 ℃ for 24 h; h) 10% Pd/C, MeOH, H2RT, 16 h; i) cyanogen bromide, THF, H2O, the temperature is 50 ℃ to 60 ℃, and the time is 16 h; j) NaH, 5-methyl-2- (methylsulfonyl) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16h
Step-a: synthesis of 2-amino-4-methylphenol
To a solution of 4-methyl-2-nitrophenol (1.75g, 11.4mmol) in methanol (15mL) under a nitrogen atmosphere was added a 10% Pd/C (500mg in 5mL methanol) slurry. Subsequently, the reaction mixture was stirred under a balloon of hydrogen for 16 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (1.3g, 93%); 1H NMR(400MHz，DMSO-d6)：δ8.62(bs，1H)，6.49(d，J＝8.0Hz，1H)，6.38(s，1H)，6.17(d，J＝8.0Hz，1H)，4.36(bs，2H)，2.07(s，3H)；LC-MS：m/z 124.2(M+1)+。
Step-b: synthesis of 5-methylbenzo [ d ] oxazole-2-thiol
To a solution of 2-amino-4-methylphenol (1.3g, 10.5mmol) in ethanol (15mL) at room temperature was added potassium ethyl-xanthate (3.7g, 22.2mmol) and the reaction mixture was refluxed for 16 h. The reaction mixture was concentrated in vacuo and diluted with cold water (30mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound (1.3g, 75%);1H NMR(400MHz，DMSO-d6)：δ13.78(s，1H)，7.37(d，J＝8.0Hz，1H)，7.07-7.05(m，2H)，2.36(s，3H)；LC-MS：m/z 166.1(M+1)+。
step-c: synthesis of 5-methyl-2- (methylthio) benzo [ d ] oxazole
To 5-methylbenzo [ d ] at 0 DEG C]To a solution of oxazole-2-thiol (1.3g, 7.9mmol) in acetonitrile (15mL) was added potassium carbonate (1.3g, 9.4mmol) and methyl iodide (0.53mL, 8.7mmol), and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo and diluted with water (30mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound (1.4g, 99%);1H NMR(400MHz，DMSO-d6)：δ7.50(d，J＝8.0Hz，1H)，7.43(s，1H),7.11(d,J＝8.4Hz,1H),2.74(s,3H),2.40(s,3H)；LC-MS:m/z 180.1(M+1)+。
step-d: synthesis of 5-methyl-2- (methylsulfonyl) benzo [ d ] oxazole
To 5-methyl-2- (methylthio) benzo [ d ] at 0 DEG C]To a solution of oxazole (350mg, 1.95mmol) in DCM (10mL) was added m-chloroperbenzoic acid (2.14g, 6.8mmol) and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (100mL), washed with saturated sodium bicarbonate solution (2 × 30mL), 1N aqueous sodium hydroxide solution (30mL), water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the product (340mg) which was used in the next step without any further purification; 1H NMR(400MHz,DMSO-d6):δ7.84-7.79(m,2H),7.49(d,J＝8.4Hz,1H),3.65(s,3H),2.48(s,3H)。
Step-e: synthesis of 4-chloro-3-nitrobenzoyl chloride
To a solution of 4-chloro-3-nitrobenzoic acid (15.0g, 74.6mmol) in DCM (150mL) at 10 deg.C was added thionyl chloride (15.9mL, 223.8mmol) and DMFA (1mL) and the reaction mixture was refluxed for 4 h. The reaction mixture was concentrated in vacuo to give the product (15.0g), which was used immediately in the next step without any further purification.
Step-f: synthesis of 4-chloro-N- (2-methoxyethyl) -3-nitrobenzamide
To 2-methoxyethyl-1-amine (5.1g, 68.2mmol) in DCM (60 m) at 0 deg.CL) to a solution was added triethylamine (20mL, 138.6mmol), and stirred for 10 min. Subsequently, a solution of 4-chloro-3-nitrobenzoyl chloride (15.0g, 68.2mmol) in DCM (50mL) was added to the reaction mixture and the contents stirred at rt for 16 h. The reaction mixture was diluted with DCM (150mL) and water (100 mL). The organic layer was separated and washed with water (150mL), brine (100mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (18.5g, 100%);1H NMR(400MHz,DMSO-d6):δ8.88(bs,1H),8.51(d,J＝2.0Hz,1H),8.14(dd,J＝2.0Hz,J＝8.4Hz,1H),7.90(d,J＝8.4Hz,1H),3.49-3.42(m,4H),3.30(s,3H)。
step-g: synthesis of N- (2-methoxyethyl) -4- (methylamino) -3-nitrobenzamide
To a solution of 4-chloro-N- (2-methoxyethyl) -3-nitrobenzamide (18.0g, 69.8mmol) in DMFA (180mL) at 0 deg.C was added DIPEA (12.5mL, 69.8mmol) and 2M methylamine in THF (70mL, 139.3 mmol). The reaction mixture was stirred at 70 ℃ for 24 h. The reaction mixture was cooled to room temperature, diluted with water (200mL) and extracted with EtOAc (2X 250 mL). The combined organic layers were washed with water (100mL), brine (100mL), dried over anhydrous sodium sulfate and concentrated in vacuo to afford the product, which was used in the next step without further purification (17.0g, 96%); 1H NMR(400MHz,DMSO-d6):δ8.65(d,J＝2.0Hz,1H),8.54(t,J＝5.2Hz,1H),8.42(d,J＝4.8Hz,1H),8.02(dd,J＝1.6Hz,J＝9.2Hz,1H),7.03(d,J＝9.2Hz,1H),3.46-3.40(m,4H),2.98(d,J＝3.2Hz,3H),3.26(s,3H)；LC-MS:m/z 254.1(M+1)+。
Step-h: synthesis of 3-amino-N- (2-methoxyethyl) -4- (methylamino) benzamide
To a solution of N- (2-methoxyethyl) -4- (methylamino) -3-nitrobenzamide (6.0g, 23.7mmol) in methanol (100mL) under a nitrogen atmosphere was added a 10% Pd/C (1.2g in 20mL methanol) slurry. The reaction mixture was stirred under a balloon of hydrogen for 16 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (4.0g, 75%);1H NMR(400MHz,DMSO-d6):δ7.88(t,J＝5.2Hz,1H),7.11-7.06(m,2H),6.34(d,J＝8.0Hz,1H),5.07(bs,1H),4.54(bs,2H),3.42-3.34(m,4H)3.27(s,3H),2.75(d,J＝2.8Hz,3H)；LC-MS:m/z 224.2(M+1)+。
step-i: synthesis of 2-amino-N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To a solution of 3-amino-N- (2-methoxyethyl) -4- (methylamino) benzamide (2.0g, 8.92mmol) in THF (10mL) and water (20mL) at room temperature was added cyanogen bromide (1.04g, 9.82mmol) and the reaction mixture was stirred at 50 ℃ to 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and basified with saturated sodium bicarbonate solution and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product, which was purified by flash chromatography using 5% methanol/DCM as eluent to give the title compound (1.3g, 59%); 1H NMR(400MHz,DMSO-d6):δ8.26(bs,1H),7.66(s,1H),7.48(d,J＝7.6Hz,1H),7.14(d,J＝8.0Hz,1H),6.52(bs,2H),3.51(s,3H),3.45-3.41(m,4H),3.26(s,3H)；LC-MS:m/z 249.2(M+1)+。
Step-j: synthesis of N- (2-methoxyethyl) -1-methyl-2- ((5-methylbenzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
To the solution of 2-amino-N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] at 10 deg.C]To a solution of imidazole-5-carboxamide (300mg, 1.2mmol) in 1, 4-dioxane (5mL) was added sodium hydride (60% dispersion in mineral oil) (96mg, 2.4mmol) and stirred for 10min, followed by the addition of 5-methyl-2- (methylsulfonyl) benzo [ d]Oxazole (306mg, 1.45mmol) and was stirred at room temperature for 16 h. The reaction mixture was quenched with cold water (20mL) and extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product, which was purified by flash chromatography using 2% methanol/DCM as eluent to give the title compound (6mg, 1%);1H NMR(400MHz,DMSO-d6):δ12.2(bs,1H),8.45(t,J＝5.2Hz,1H),8.08(d,J＝1.6Hz,1H),7.76(dd,J＝1.2Hz,J＝8.0Hz,1H),7.47(d,J＝8.4Hz,1H),7.30(d,J＝8.4Hz,1H),7.26(s,1H),6.93(d,J＝7.6Hz,1H),3.62(s,3H),3.50-3.42(m,4H),3.29(s,3H),2.49(s,3H)；LC-MS:m/z 380.2(M+1)+。
examples 66 and 67.2 Synthesis of 2- ((5-fluorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and 2- ((5-fluorobenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 10% Pd/C, MeOH, H2RT, 16 h; b) potassium ethyl xanthate, EtOH, refluxing for 16 h; c) k 2CO3MeI, ACN, RT, 16 h; d) m-CPBA, DCM, 0 ℃ -RT, 4 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; g) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 2-amino-4-fluorophenol
The title compound was synthesized using the same procedure as in example 32, step-b, using 4-fluoro-2-nitrophenol as the starting material (yield: 91%);1H NMR(400MHz，DMSO-d6)：δ8.88(bs，1H)，6.56-6.53(m，1H)，6.35(dd，J＝2.0Hz，J＝10.4Hz，1H)，6.13-6.08(m，1H)，4.78(bs，2H)；LC-MS：m/z 128.1(M+1)+。
step-b: synthesis of 5-fluorobenzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32 step-c, using 2-amino-4-fluorophenol as a starting material (yield: 86%);1H NMR(400MHz，DMSO-d6)：δ14.01(bs，1H)，7.55-7.51(m，1H)，7.15-7.08(m，2H)；LC-MS：m/z 170.0(M+i)+。
step-c: synthesis of 5-fluoro-2- (methylthio) benzo [ d ] oxazole
Using the same procedure as for Compound 1b, 5-fluoro-benzo [ d ] is used]Oxazole-2-thiol as a starting material (stirred for 16h) was synthesized as the title compound (yield: 92%);1H NMR(400MHz，DMSO-d6)：δ7.69-7.65(m，1H)，7.53(dd，J＝2.4Hz，J＝8.8Hz，1H)，7.19-7.11(m，1H)，2.76(s，3H)；LC-MS：m/z 184.1(M+1)+。
step-d: synthesis of 5-fluoro-2- (methylsulfonyl) benzo [ d ] oxazole
Using the same procedure as for Compound 1c, 5-fluoro-2- (methylthio) benzo [ d ] is used]Oxazole as a starting material (stirred for 4h) was synthesized the title compound (yield: 99%);1H NMR(400MHz，DMSO-d6)：δ8.05-8.01(m，1H)，7.94(dd，J＝2.4Hz，J＝8.0Hz，1H)，7.57-7.54(m，1H)，3.67(s，3H)；LC-MS：m/z 216.0(M+1)+。
step-e: synthesis of ethyl 2- ((5-fluorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-1-methyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 5-fluoro-2- (methylsulfonyl) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 26%);1H NMR(400MHz，DMSO-d6)：δ12.34(bs，1H)，8.23(s，1H)，7.88(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.49(d，J＝8.0Hz，1H)，7.46-7.42(m，1H)，7.23(dd，J＝2.4Hz，J＝9.2Hz，1H)，6.97-6.91(m，1H)，4.34(q，J＝6.4Hz，2H)，3.64(s，3H)，1.36(t，J＝4.4Hz，3H)；LC-MS：m/z 355.2(M+1)+。
step-f: synthesis of 2- ((5-fluorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- ((5-fluorobenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 58%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.19(s，1H)，7.87(d，J＝8.4Hz，1H)，7.51(d，J＝8.8Hz，1H)，7.45-7.42(m，1H)，7.24(dd，J＝2.4Hz，J＝8.8Hz，1H)，6.96-6.91(m，1H)，3.64(s，3H)；LC-MS：m/z 327.2(M+1)+。
step-g: synthesis of 2- ((5-fluorobenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((5-Fluorobenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 62%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，1H)，8.20(s，1H)，8.06(s，1H)，7.76(d，J＝8.4Hz，1H)，7.44(d，J＝8.4Hz，1H)，7.40-7.37(m，1H)，7.20(d，J＝8.8Hz，1H)，6.89(t，J＝10.0Hz，1H)，3.63(s，3H)，3.50-3.45(m，4H)，3.09(s，3H)；LC-MS：m/z 384.2(M+1)+。
examples 68 and 69.2- ((6-Fluorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid Synthesis and 2- ((6-Fluorobenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide Synthesis
Conditions are as follows: a) 10% Pd/C, MeOH, H 2RT, 16 h; b) potassium ethyl xanthate, EtOH, refluxing for 16 h; c) k2CO3MeI, ACN, 0 ℃ -RT, 5 h; d) m-CPBA, DCM, 0 ℃ -RT, 4 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, ethanol, water, 60 ℃ and 16 h; g) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 2-amino-5-fluorophenol
The title compound was synthesized using the same procedure as in example 32 step-b, using 5-fluoro-2-nitrophenol as the starting material (yield: 92%);1H NMR(400MHz,DMSO-d6):δ9.5(bs,1H),6.54-6.51(m,1H),6.46(dd,J＝2.8Hz,J＝10.0Hz,1H),6.37-6.32(m,1H)；LC-MS:m/z 128.2(M+1)+。
step-b: synthesis of 6-fluorobenzo [ d ] oxazole-2-thiol
The same as in example 32, step-c was usedThe title compound was synthesized using 2-amino-5-fluorophenol as a starting material (yield: 89%);1H NMR(400MHz,DMSO-d6):δ13.94(bs,1H),7.57(dd,J＝2.0Hz,J＝8.4Hz,1H),7.25-7.22(m,1H),7.19-7.14(m,1H)；LC-MS:m/z 170.0(M+1)+。
step-c: synthesis of 6-fluoro-2- (methylthio) benzo [ d ] oxazole
Using the same procedure as for Compound 1b, using 6-fluoro-benzo [ d ]]Oxazole-2-thiol as a starting material (stirred for 5h) was synthesized as the title compound (yield: 99%);1H NMR(400MHz,DMSO-d6):δ7.67-7.63(m,2H),7.24-7.18(m,1H),2.75(s,3H)；LC-MS:m/z 184.0(M+1)+。
step-d: synthesis of 6-fluoro-2- (methylsulfonyl) benzo [ d ] oxazole
Using the same procedure as for Compound 1c, 6-fluoro-2- (methylthio) benzo [ d ] is used]Oxazole as a starting material (stirred for 4h) was synthesized the title compound (yield: 95%); 1H NMR(400MHz,DMSO-d6):δ8.08-8.02(m,1H),8.01-7.99(m,1H),7.56-7.46(m,1H),3.67(s,3H)。
Step-e: synthesis of ethyl 2- ((6-fluorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-1-methyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 6-fluoro-2- (methylsulfonyl) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 28%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.23(d,J＝1.6Hz,1H),7.88(dd,J＝2.0Hz,J＝8.4Hz,1H),7.52(d,J＝8.4Hz,1H),7.45-7.41(m,2H),7.10-7.04(m,1H),4.33(q,J＝6.8Hz,2H),3.64(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 355.1(M+1)+。
step-f: synthesis of 2- ((6-fluorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
To 2- ((6-fluorobenzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (45mg, 0.13mmol) in a solvent mixture of THF (1mL), ethanol (1mL) and water (0.5mL) was added lithium hydroxide monohydrate (10mg, 0.25 mmol). In thatThe reaction mixture was heated at 60 ℃ for 16h with stirring. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (15mL), acidified with 1N HCl to give a solid, filtered and dried in vacuo to give the title compound (30mg, 72%);1H NMR(400MHz,DMSO-d6):δ12.8(bs,1H),12.3(bs,1H)8.19(d,J＝1.6Hz,1H),7.87(dd,J＝1.6Hz,J＝8.4Hz,1H),7.50(d,J＝8.8Hz,1H),7.46-7.41(m,2H),7.09-7.03(m,1H),3.63(s,3H)；LC-MS:m/z 327.05(M+1)+。
step-g: synthesis of 2- ((6-fluorobenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To 2- ((6-fluorobenzo [ d ] at 0 DEG C]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ]To a stirred solution of imidazole-5-carboxylic acid (30mg, 0.09mmol) in DMFA (2mL) were added N-ethyldiisopropylamine (0.03mL, 0.18mmol) and diphenylphosphorylazide (0.02mL, 0.09 mmol). The reaction mixture was stirred for 30min, followed by addition of 2-methoxyethylamine (6mg, 0.09mmol), and then the reaction mixture was stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (6mL) and stirred for 15 min. The resulting solid was filtered, washed with diethyl ether and dried in vacuo to give the title compound as a white solid (20mg, 58%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，1H)，8.45(t，J＝5.2Hz，1H)，8.07(d，J＝1.2Hz，1H)，7.75(dd，J＝1.6Hz，J＝8.8Hz，1H)，7.49-7.40(m，3H)，7.08-7.03(m，1H)，3.63(s，3H)，3.50-3.43(m，4H)，3.39(s，3H)；LC-MS：m/z384.1(M+1)+。
examples 70 and 71.6-fluoro-1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid synthesis and 6-fluoro-N- (2-methoxyethyl) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide synthesis
Conditions are as follows: a) the reaction mixture of cyanogen bromide and hydrogen bromide,THF，H2o, 60 ℃, 16 h; b) NaH, 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; c) h, LiOH2O，THF，MeOH，H2O, 60 ℃, 16 h; d) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 2-amino-6-fluoro-1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid methyl ester
The title compound was synthesized using the same procedure as compound 1f, using 5-amino-2-fluoro-4- (methylamino) benzoic acid methyl ester as starting material and heating to 60 ℃ for 16 h. (yield: 89%);1H NMR(400MHz，DMSO-d6)：δ7.54(d，J＝6.4Hz，1H)，7.16(d，J＝11.6Hz，1H)，6.69(bs，2H)，3.80(s，3H)，3.51(s，3H)；LC-MS：m/z224.1(M+1)+。
step-b: synthesis of methyl 6-fluoro-1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-6-fluoro-1-methyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid methyl ester and 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 18%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.10(d,J＝6.0Hz,1H),7.95-7.51(m,3H),7.22(d,J＝8.4Hz,1H),3.87(s,3H),3.61(s,3H)；LC-MS:m/z 425.1(M+1)+。
step-c: synthesis of 6-fluoro-1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, using 6-fluoro-1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid methyl ester as a starting material and THF, methanol and water (2:2:1) as solvents (yield: 66%);1H NMR(400MHz,DMSO-d6):δ13.0(bs,1H),12.3(bs,1H),8.08(d,J＝6.0Hz,1H),7.59(s,1H),7.53-7.49(m,2H),7.22(d,J＝7.2Hz,1H),3.61(s,3H)；LC-MS:m/z411.1(M+1)+。
step-d: synthesis of 6-fluoro-N- (2-methoxyethyl) -1-methyl-2- ((6- (trifluoromethoxy) -benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, using 6-fluoro-1-methyl-2- ((6- (trifluoromethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 48%);1HNMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.18(d,J＝4.0Hz,1H),7.86(d,J＝6.4Hz,1H),7.58(s,1H),7.52-7.49(m,2H),7.21(d,J＝9.2Hz,1H),3.61(s,3H),3.47-3.44(m,4H),3.29(s,3H)；LC-MS:m/z 468.1(M+1)+。
EXAMPLE 72 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -6-fluoro-N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaH, 2-chlorobenzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O，THF，MeOH，H2O, 60 ℃, 16 h; g)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of methyl 2- (benzo [ d ] oxazol-2-ylamino) -6-fluoro-1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-6-fluoro-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid methyl ester and 2-chlorobenzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 66%);1H NMR(400MHz，DMSO-d6)：δ12.25(bs，1H)，8.09(d，J＝6.0Hz，1H)，7.55-7.44(m，3H)，7.24-7.22(m，1H)，7.17-7.12(m，1H)，3.86(s，3H)，3.60(s，3H)；LC-MS：m/z341.0(M+1)+。
step-b: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -6-fluoro-1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- (benzo [ d ]]Oxazol-2-ylamino) -6-fluoro-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid methyl ester as a starting materialThe title compound was synthesized from the material and methanol, THF and water (2: 1) as solvents (yield: 75%); 1H NMR(400MHz，DMSO-d6)：δ12.80(bs，2H)，8.07(d，J＝6.8Hz，1H)，7.50-7.44(m，3H)，7.22(t，J＝6.8Hz，1H)，7.15-7.11(m，1H)，3.60(s，3H)；LC-MS：m/z 327.0(M+1)+。
Step-c: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -6-fluoro-N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- (benzo [ d ]]Oxazol-2-ylamino) -6-fluoro-1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 49%);1H NMR(400MHz，DMSO-d6)：δ12.20(bs，1H)，8.15(bs，1H)，7.86(d，J＝6.4Hz，1H)，7.50-7.47(m，2H)，7.43(d，J＝8.0Hz，1H)，7.23-7.19(m，1H)，7.14-7.10(m，1H)，4.60(bs，1H)，3.60(s，3H)，3.57-3.51(m，4H)，3.48-3.31(m，4H)；LC-MS：m/z 414.05(M+1)+。
EXAMPLE 73.2 Synthesis of- ((5-fluoro-1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Conditions are as follows: a)1, 1' -thiocarbonyl diimidazole, ACN, 60 ℃, 16 h; b) 4-fluorobenzene-1, 2-diamine, EDC.HCl and DMF at 100 ℃ for 16 h; c) h, LiOH2O，THF，MeOH，H2O，60℃，16h
Step-a: synthesis of ethyl 2- (1H-imidazole-1-carbosulfanyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at room temperature]To a solution of imidazole-5-carboxylic acid ethyl ester (1.0g, 4.5mmol) in acetonitrile (20mL) was added 1, 1' -thiocarbonyldiimidazole (1.06g, 5.9mmol) and stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and stirred for 30 min. The resulting solid was filtered and dried in vacuo to give the title compound (1.1g, 73%) The title compound was used in the next step without further purification; 1H NMR(400MHz，DMSO-d6)：δ13.4(bs，1H)，8.68(s，1H)，8.41(d，J＝1.6Hz，1H)，8.03(s，1H)，7.99(dd，J＝1.2Hz，J＝8.8Hz，1H)，7.75(d，J＝8.4Hz，1H)，7.01(d，J＝1.2Hz，1H)，4.35(q，J＝7.2Hz，2H)，3.79(s，3H)，1.36(t，J＝6.8Hz，3H)。
Step-b: synthesis of ethyl 2- ((5-fluoro-1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To 2- (1H-imidazole-1-carbothioamide) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (900mg, 2.74mmol) in DMFA (15mL) was added EDC & HCl (1.05g, 5.47mmol) and heated to 60 ℃ for 10 min. The reaction mixture was cooled to room temperature, then 4-fluorobenzene-1, 2-diamine (345mg, 2.74mmol) was added and heated at 100 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with water (30mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography using 0.5% methanol/DCM as eluent to give the title compound (200mg, 21%); LC-MS: m/z 354.2(M +1)+。
Step-c: synthesis of 2- ((5-fluoro-1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-iH-benzo [ d ] imidazole-5-carboxylic acid
To 2- ((5-fluoro-1H-benzo [ d)]Imidazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (200mg, 0.57mmol) in a solvent mixture of THF (5mL), methanol (5mL) and water (5mL) was added lithium hydroxide monohydrate (119mg, 2.83 mmol). The reaction mixture was heated with stirring at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (15mL), acidified with 1N HCl to give a solid, filtered and dried in vacuo to give the title compound (150mg, 81%); 1H NMR(400MHz，DMSO-d6)：δ12.2(bs，2H)，8.04(s，1H)，7.75(d，J＝8.0Hz，1H)，7.32-7.26(m，2H)，7.13(d，J＝8.4Hz，1H)，6.88(t，J＝8.0Hz，1H)，3.62(s，3H)；LC-MS：m/z 326.05(M+1)+。
EXAMPLE 74 Synthesis of 1- (2- (dimethylamino) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Conditions are as follows: a) n is a radical of1，N1-dimethylethane-1, 2-diamine, DIPEA, DMF, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) cyanogen bromide, THF, H2O, 60 ℃, 16 h; d) NaH, 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O，60℃，16h
Step-a: synthesis of ethyl 4- ((2- (dimethylamino) ethyl) amino) -3-nitrobenzoate
To a solution of ethyl 4-chloro-3-nitrobenzoate (5.0g, 21.8mmol) in DMFA (40mL) at room temperature was added (9.5mL, 54.4mmol) and N1，N1-dimethylethane-1, 2-diamine (2.87g, 32.7mmol), and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (250mL) and stirred for 10 min. The precipitated solid was filtered and dried in vacuo to give the title compound (4.8g, 92%);1H NMR(400MHz，DMSO-d6)：δ8.65(t，J＝4.4Hz，1H)，8.62(d，J＝2.0Hz，1H)，7.79(dd，J＝2.0Hz，J＝8.8Hz，1H)，7.13(d，J＝9.2Hz，1H)，4.32-4.26(m，2H)，3.47-3.43(m，2H)，2.57-2.52(m，2H)，2.22(s，6H)，1.33-1.28(m，3H)；LC-MS：m/z 282.2(M+1)+。
step-b: synthesis of ethyl 3-amino-4- ((2- (dimethylamino) ethyl) amino) benzoate
The title compound was synthesized using the same procedure as compound 1e, using 4- ((2- (dimethylamino) ethyl) amino) -3-nitrobenzoic acid ethyl ester as starting material and stirring for 16 h. (yield: 67%); 1H NMR(400MHz，DMSO-d6)：δ7.23(dd，J＝2.0Hz，J＝8.0Hz，1H)，7.18(d，J＝2.0Hz，1H)，6.45(d，J＝8.4Hz，1H)，5.09(bs，1H)，4.68(s，2H)，4.18(q，J＝6.8Hz，2H)，3.19-3.15(m，2H)，2.49-2.46(m，2H)，2.18(s，6H)，1.26(t，J＝6.8Hz，3H)；LC-MS：m/z 252.2(M+1)+。
Step-c: synthesis of ethyl 2-amino-1- (2- (dimethylamino) ethyl) -1H-benzo [ d ] imidazole-5-carboxylate
The title compound was synthesized using the same procedure as compound 1f, using ethyl 3-amino-4- ((2- (dimethylamino) ethyl) amino) benzoate as starting material and heating to 60 ℃ for 16 h. (yield: 26%); LC-MS: m/z 277.2(M +1)+。
Step-d: synthesis of ethyl 1- (2- (dimethylamino) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 1g, 2-amino-1- (2- (dimethylamino) ethyl) -1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 2- (methylsulfonyl) -6- (trifluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 26%);1H NMR(400MHz，DMSO-d6)：δ12.4(bs，1H)，8.25(d，J＝1.2Hz，1H)，7.87(dd，J＝1.2Hz，J＝8.4Hz，1H)，7.59-7.57(m，2H)，7.51(d，J＝8.4Hz，1H)，7.22(d，J＝8.8Hz，1H)，4.36-4.26(m，4H)，2.68(q，J＝6.8Hz，2H)，2.22(s，6H)，1.35(t，J＝6.8Hz，3H)；LC-MS：m/z 478.1(M+1)+。
step-e: synthesis of 1- (2- (dimethylamino) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, 1- (2- (dimethylamino) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 18%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.20(d，J＝1.6Hz，1H)，7.86(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.59-7.51(m，3H)，7.21(d，J＝8.4Hz，1H)，4.27(t，J＝6.4Hz，2H)，2.79-2.66(m，2H)，2.21(s，6H)；LC-MS：m/z 450.1(M+1)+。
examples 75 and 76.Synthesis of 1- (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and N, 1-bis (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-methoxyethyl-1-amine, DMSO, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) cyanogen bromide, THF, H2O, 60 ℃, 16 h; d)1, 1' -thiocarbonyl diimidazole, ACN, 60 ℃, 16 h; e)4- (trifluoromethoxy) benzene-1, 2-diamine, EDC.HCl, DMF, 100 ℃, 16 h; f) h, LiOH2O，THF，MeOH，H2O, 60 ℃, 16 h; g) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 4- ((2-methoxyethyl) amino) -3-nitrobenzoate
To a solution of ethyl 4-chloro-3-nitrobenzoate (10g, 43.7mmol) in DMSO (100mL) at room temperature was added 2-methoxyethyl-1-amine (7.5mL g, 87.3mmol) and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (250mL) and stirred for 1 h. The precipitated solid was filtered and dried in vacuo to give the title compound (12g, 100%);1H NMR(400MHz,DMSO-d6):δ8.61(d,J＝2.4Hz,1H),8.54(s,1H),7.97(dd,J＝1.6Hz,J＝8.8Hz,1H),7.18(d,J＝9.2Hz,1H),4.29(q,J＝6.8Hz,2H),3.60(bs,4H),3.31(s,3H),1.31(t,J＝6.8Hz,3H)；LC-MS:m/z 269.1(M+1)+。
step-b: synthesis of ethyl 3-amino-4- ((2-methoxyethyl) amino) benzoate
The title compound was synthesized using the same procedure as compound 1e, using ethyl 4- ((2-methoxyethyl) amino) -3-nitrobenzoate as starting material (yield: 75%);1H NMR(400MHz,DMSO-d6):δ7.22-7.17(m,2H),6.46(d,J＝8.4Hz,1H),5.20(t,J＝7.6Hz,1H),4.74(bs,2H),4.18(q,J＝7.2Hz,2H),3.53(t,J＝5.6Hz,2H),3.30-3.26(m,5H),1.26(t,J＝7.6Hz,3H)；LC-MS:m/z 239.1(M+1)+。
step-c: synthesis of ethyl 2-amino-1- (2-methoxyethyl) -1H-benzo [ d ] imidazole-5-carboxylate
The title compound was synthesized using the same procedure as compound 1f, using ethyl 3-amino-4- ((2-methoxyethyl) amino) benzoate as starting material and heating to 60 ℃ for 16 h. (yield: 73%); LC-MS: m/z 264.1(M +1)+。
Step-d: synthesis of ethyl 2- (1H-imidazole-1-carbothioamide) -1- (2-methoxyethyl) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 12a, 2-amino-1- (2-methoxyethyl) -1H-benzo [ d ]]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and 1,1' -thiocarbonyldiimidazole as starting materials (yield: 60%);1H NMR(400MHz,DMSO-d6):δ13.5(bs,1H),8.66(s,1H),8.42(s,1H),8.01-7.97(m,2H),7.78(d,J＝8.8Hz,1H),7.01(s,1H),4.54(t,J＝4.8Hz,2H),4.35(q,J＝7.2Hz,2H),3.74(t,J＝4.8Hz,2H),3.20(s,3H),1.35(t,J＝7.2Hz,3H)。
step-e: synthesis of ethyl 1- (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 12b, 2- (1H-imidazole-1-carbothioamido) -1- (2-methoxyethyl) -1H-benzo [ d ]]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and 4- (trifluoromethoxy) benzene-1, 2-diamine as starting materials (yield: 38%); LC-MS: m/z 464.0(M +1)+。
Step-f: synthesis of 1- (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, 1- (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d) ]Imidazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester as starting material and THF, methanol and water as solventThe title compound was synthesized. (yield: 65%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,3H),8.08(d,J＝1.2Hz,1H),7.74(dd,J＝1.6Hz,J＝8.4Hz,1H),7.41(d,J＝8.4Hz,1H),7.36-7.31(m,2H),7.02(d,J＝8.0Hz,1H),4.32(t,J＝5.2Hz,2H),3.71(t,J＝6.0Hz,2H),3.25(s,3H)；LC-MS:m/z 436.0(M+1)+。
step-g: synthesis of N, 1-bis (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- (2-methoxyethyl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d)]Imidazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 2%);1H NMR(400MHz，DMSO-d6)：δ12.23(bs，1H)，8.26-8.23(m，1H)，7.58-7.53(m，1H)，7.43-7.38(m，1H)，7.30-6.37(m，5H)，4.50-4.48(m，2H)，3.86-3.80(m，2H)，3.32(s，3H)，3.27-3.25(m，4H)，3.16(s，3H)；LC-MS：m/z 493.1(M+1)+。
examples 77 and 78.Synthesis of 1- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and N, 1-bis (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaH, 2-chloro-6- (trifluoromethoxy) benzo [ d ] oxazole, 1, 4-dioxane, 0 ℃ -90 ℃ for 16 h; b) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 1- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
To the solution of 2-amino-1- (2-methoxyethyl) -1H-benzo [ d ] at 0 deg.C]To a solution of imidazole-5-carboxylic acid ethyl ester (200mg, 0.76mmol) in 1, 4-dioxane (10mL) was added sodium hydride (60% in mineral oil)Dispersion) (107mg, 2.66mmol) and stirred for 10 min. Reacting 2-chloro-6- (trifluoromethoxy) benzo [ d ]]Oxazole (270mg, 1.14mmol) was added to the reaction mixture and stirred at 90 ℃ for 16 h. The reaction mixture was cooled to room temperature. The solvent was evaporated and the residue diluted with cold water (20mL) and acidified with 1N HCl. The reaction mixture was extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 2% methanol/DCM as eluent to give the title compound (70mg, 21%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.20(d，J＝1.6Hz，1H)，7.86(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.58-7.52(m，3H)，7.22(d，J＝7.2Hz，1H)，4.36(t，J＝4.8Hz，2H)，3.74(t，J＝5.6Hz，2H)，3.24(s，3H)；LC-MS：m/z 437.05(M+1)+。
step-b: synthesis of N, 1-bis (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 2%); 1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.45(bs，1H)，8.08(s，1H)，7.75(d，J＝8.4Hz，1H)，7.56(s，1H)，7.53-7.50(m，2H)，7.21(d，J＝8.4Hz，1H)，4.35(t，J＝4.8Hz，2H)，3.73(t，J＝5.2Hz，2H)，3.48-3.43(m，4H)，3.28(s，3H)，3.24(s，3H)；LC-MS：m/z 494.15(M+1)+。
Example 79 and 80.1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzoimidazole-5-carboxylic acid synthesis and N- (2-methoxyethyl) -1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide synthesis
Conditions are as follows: a) cyanogen bromide, THF, H2O, 60 ℃, 16 h; b) NaH, 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; c) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; d) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 2-amino-1- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d ] imidazole-5-carboxylate
The title compound was synthesized using the same procedure as compound 1f, using ethyl 3-amino-4- ((tetrahydro-2H-pyran-4-yl) amino) benzoate as starting material and heating to 60 ℃. (yield: 92%);1H NMR(400MHz，DMSO-d6)：δ7.71(s，1H)，7.55(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.41(d，J＝8.0Hz，1H)，6.65(bs，2H)，4.50-4.48(m，1H)，4.28(q，J＝7.2Hz，2H)，4.05-4.01(m，2H)，3.47(t，J＝11.2Hz，2H)，2.36-2.32(m，2H)，1.72(d，J＝10.4Hz，2H)，1.31(t，J＝6.8Hz，3H)；LC-MS：m/z 290.1(M+1)+。
step-b: synthesis of ethyl 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) -benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
To room temperature 2-amino-1- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d]To a solution of imidazole-5-carboxylic acid ethyl ester (100mg, 0.35mmol) in 1, 4-dioxane (2mL) was added sodium hydride (60% dispersion in mineral oil) (34mg, 0.86mmol), and the mixture was stirred for 10 min. Reacting 2-chloro-6- (trifluoromethoxy) benzo [ d ] ]Oxazole (99g, 0.41mmol) was added to the reaction mixture and stirred at room temperature for 16 h. The reaction mixture was concentrated and diluted with cold water (30mL) and stirred at room temperature for 5 to 10 min. The resulting solid was filtered, dried in vacuo and purified by flash chromatography using 100% DCM as eluent to give the title compound (60mg, 35%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.29(d,J＝1.6Hz,1H),7.86(dd,J＝1.2Hz,J＝8.0Hz,1H),7.74(d,J＝8.4Hz,1H),7.58(d,J＝1.6Hz,1H),7.52(d,J＝8.4Hz,1H),7.21(dd,J＝1.6Hz,J＝8.8Hz,1H),4.97-4.93(m,1H),4.33(q,J＝7.6Hz,2H),4.07-4.03(m,2H),3.56(t,J＝9.2Hz,2H),2.63-2.55(m,2H),1.79(d,J＝9.2Hz,2H),1.35(t,J＝6.8Hz,3H)；LC-MS:m/z 491.1(M+1)+。
step-e: synthesis of 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 71%);1H NMR(400MHz,DMSO-d6):δ12.8(bs,1H),12.50(bs,1H),8.24(d,J＝1.6Hz,1H),7.86(dd,J＝1.2Hz,J＝8.8Hz,1H),7.71(d,J＝8.0Hz,1H),7.58(d,J＝1.2Hz,1H),7.53(d,J＝8.8Hz,1H),7.22(d,J＝10.4Hz,1H),5.01-4.92(m,1H),4.05(dd,J＝4.0Hz,J＝11.2Hz,2H),3.58-3.53(m,2H),2.67-2.57(m,2H),1.78(d,J＝10.0Hz,2H)；LC-MS:m/z 463.1(M+1)+。
step-f: n- (2-methoxyethyl) -1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Synthesis of imidazole-5-carboxamide Using the same procedure as Compound 1i, 1- (tetrahydro-2H-pyran-4-yl) -2- ((6- (trifluoromethoxy) benzo [ d [ -d ]]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 76%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.48(t,J＝5.2Hz,1H),8.13(d,J＝1.2Hz,1H),7.76(dd,J＝1.6Hz,J＝8.8Hz,1H),7.68(d,J＝8.0Hz,1H),7.57(s,1H),7.52(d,J＝8.4Hz,1H),7.21(d,J＝8.8Hz,1H),4.98-4.92(m,1H),4.07-4.04(m,2H),3.56(t,J＝1.2Hz,2H),3.49-3.44(m,4H),3.28(s,3H),2.67-2.58(m,2H),1.78(d,J＝9.6Hz,2H)；LC-MS:m/z 520.2(M+1)+。
EXAMPLE 81 Synthesis of N- (2-methoxyethyl) -1- (tetrahydro-2H-pyran-4-yl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)1, 1' -thiocarbonyl diimidazole, ACN, 60 ℃, 16 h; b)4- (trifluoromethoxy) benzene-1, 2-diamine, EDC.HCl, DMF, 100 ℃, 16 h; c) h, LiOH2O, THF, EtOH, H2O, 60 ℃ for 16H; d) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 2- (1H-imidazole-1-carbosulfanyl) -1- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 12a, 2-amino-1- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d ] was used]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and 1, 1' -thiocarbonyldiimidazole as starting materials (yield: 72%);1H NMR(400MHz，DMSO-d6)：δ13.50(bs，1H)，8.64(s，1H)，8.43(s，1H)，7.98(d，J＝4.4Hz，1H)，7.96-7.94(m，1H)，7.09(s，1H)，6.72(bs，1H)，5.05-5.00(m，1H)，4.36-4.26(m，2H)，4.06-4.02(m，2H)，3.60(t，J＝11.6Hz，2H)，2.54-2.50(m，2H)，1.86(d，J＝9.6Hz，2H)，1.37-1.33(m，3H)。
step-b: synthesis of ethyl 1- (tetrahydro-2H-pyran-4-yl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 12b, 2- (1H-imidazole-1-carbothioamido) -1- (tetrahydro-2H-pyran-4-yl) -1H-benzo [ d ]]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and 4- (trifluoromethoxy) benzene-1, 2-diamine as starting materials (yield: 46%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.12(d，J＝6.4Hz，1H)，7.95(s，1H)，7.74(dd，J＝2.0Hz，J＝8.4Hz，1H)，7.57(d，J＝8.4Hz，1H)，7.37-7.32(m，2H)，7.06(dd，J＝1.6Hz，J＝8.4Hz，1H)，4.97-4.90(m，1H)，4.32(q，J＝6.8Hz，2H)，4.08-4.05(m，2H)，3.51(t，J＝11.6Hz，2H)，2.67-2.55(m，2H)，1.75(d，J＝9.6Hz，2H)，1.07(t，J＝8.4Hz，3H)；LC-MS：m/z490.0(M+1)+。
step-c: synthesis of 1- (tetrahydro-2H-pyran-4-yl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, 1- (tetrahydro-2H-pyran-4-yl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d)]Imidazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 55%);1H NMR(400MHz，DMSO-d6)：δ12.7(bs，3H)，8.04(s，1H)，7.80(d，J＝8.4Hz，1H)，7.65(d，J＝8.8Hz，1H)，7.42-7.35(m，2H)，7.12(d，J＝8.0Hz，1H)，4.93(bs，1H)，4.08-4.05(m，2H)，3.54-3.48(m，2H)，2.61-2.58(m，2H)，1.77(d，J＝10.0Hz，2H)；LC-MS：m/z 462.15(M+1)+。
step-d: synthesis of N- (2-methoxyethyl) -1- (tetrahydro-2H-pyran-4-yl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d ] imidazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- (tetrahydro-2H-pyran-4-yl) -2- ((5- (trifluoromethoxy) -1H-benzo [ d)]Imidazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 42%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，2H)，8.37(t，J＝5.6Hz，1H)，8.02(d，J＝1.6Hz，1H)，7.64(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.52(d，J＝8.8Hz，1H)，7.53-7.30(m，2H)，7.03(d，J＝8.0Hz，1H)，4.98-4.89(m，1H)，4.08-4.05(m，2H)，3.51-3.43(m，6H)，3.28(s，3H)，2.67-2.58(m，2H)，1.75-1.73(m，2H)；LC-MS：m/z 519.1(M+1)+。
examples 82 and 83.2 Synthesis of- ((5-chloro-1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and Synthesis of 2- ((5-chloro-1H-benzo [ d ] imidazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 4-chlorobenzene-1, 2-diamine, EDC.HCl, DMF, 100 ℃, 16 h; b) h, LiOH2O，THF，EtOH，H2O，6At 0 ℃ for 16 h; c) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 2- ((5-chloro-1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo-imidazole-5-carboxylate
Using the same procedure as for Compound 12b, 2- (1H-imidazole-1-carbothioamido) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester and 4-chlorobenzene-1, 2-diamine as starting materials (yield: 24%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，2H)，8.10(s，1H)，7.76(d，J＝8.4Hz，1H)，7.40-7.29(m，3H)，7.09(dd，J＝1.2Hz，J＝8.4Hz，1H)，4.31(q，J＝6.8Hz，2H)，3.64(s，3H)，1.35(t，J＝6.8Hz，3H)；LC-MS：m/z 370.05(M+1)+。
step-b: synthesis of 2- ((5-chloro-1H-benzo [ d ] imidazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1H, using 2- ((5-chloro-1H-benzo [ d)]Imidazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 84%);1H NMR(400MHz，DMSO-d6)：612.3(bs，3H)，8.07(s，1H)，7.76(d，J＝8.0Hz，1H)，7.37-7.29(m，3H)，7.07(d，J＝7.6Hz，1H)，3.63(s，3H)；LC-MS：m/z 342.0(M+1)+。
step-c: synthesis of 2- ((5-chloro-1H-benzo [ d ] imidazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((5-chloro-1H-benzo [ d)]Imidazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 49%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，2H)，8.36(s，1H)，7.99(s，1H)，7.66(d，J＝7.2Hz，1H)，7.35(d，J＝8.4Hz，1H)，7.30-7.28(m，2H)，7.06(d，J＝8.4Hz，1H)，3.62(s，3H)，3.48-3.43(m，4H)，3.17(s，3H)；LC-MS：m/z 399.1(M+1)+。
example 84 and 85.1-Ethyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid Synthesis and 1-Ethyl-N- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide Synthesis
Conditions are as follows: a) ethylamine aqueous solution, DMF, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) cyanogen bromide, THF, H2O, 60 ℃, 16 h; d) NaH, 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; f) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 4- (ethylamino) -3-nitrobenzoate
To a solution of ethyl 4-chloro-3-nitrobenzoate (4.0g, 17.5mmol) in DMFA (100mL) at room temperature was added ethylamine (2.24mL (70% aq), 34.9mmol) and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (200mL) and stirred for 15 min. The precipitated solid was filtered and dried in vacuo to give the title compound (3.0g, 63%);1H NMR(400MHz，DMSO-d6)：δ8.62(d，J＝2.0Hz，1H)，8.51(t，J＝5.2Hz，1H)，7.97(dd，J＝2.0Hz，J＝9.2Hz，1H)，7.14(d，J＝9.2Hz，1H)，4.29(q，J＝6.8Hz，2H)，3.49-3.42(m，2H)，1.33(t，J＝5.2Hz，3H)，1.23(t，J＝7.2Hz，3H)；LC-MS：m/z239.0(M+1)+。
step-b: synthesis of ethyl 3-amino-4- (ethylamino) benzoate
The title compound was synthesized using the same procedure as compound 1e, using 4- (ethylamino) -3-nitrobenzoic acid ethyl ester as starting material (yield: 79%), and the crude compound was used in the next step without any purification.
Step-c: synthesis of ethyl 2-amino-1-ethyl-1H-benzo [ d ] imidazole-5-carboxylate
Use andthe title compound was synthesized using the same procedure as for compound 1f, 3-amino-4- (ethylamino) benzoic acid ethyl ester as a starting material (yield: 74%);1H NMR(400MHz,DMSO-d6):δ7.70(s,1H),7.58(dd,J＝1.6Hz,J＝8.4Hz,1H),7.23(d,J＝8.4Hz,1H),6.66(bs,2H),4.27(q,J＝7.2Hz,2H),4.05(q,J＝6.8Hz,2H),1.31(t,J＝6.8Hz,3H),1.20(t,J＝7.6Hz,3H)；LC-MS:m/z 234.1(M+1)+。
step-d: synthesis of ethyl 1-ethyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-1-ethyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 54%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.25(d,J＝1.6Hz,1H),7.88(dd,J＝2.0Hz,J＝8.8Hz,1H),7.60-7.57(m,2H),7.51(d,J＝8.8Hz,1H),7.22(dd,J＝1.6Hz,J＝8.8Hz,1H),4.33(q,J＝7.2Hz,2H),4.22(q,J＝7.6Hz,2H),1.37-1.31(m,6H)；LC-MS:m/z 435.05(M+1)+。
step-e: synthesis of 1-ethyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, 1-ethyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 75%);1H NMR(400MHz,DMSO-d6):δ12.8(bs,1H),12.3(bs,1H),8.21(s,1H),7.88(d,J＝8.0Hz,1H),7.58-7.57(m,2H),7.52(d,J＝8.4Hz,1H),7.20(d,J＝9.6Hz,1H),4.22(q,J＝7.6Hz,2H),1.33(t,J＝6.8Hz,3H)；LC-MS:m/z 407.0(M+1)+。
step-f: synthesis of 1-ethyl-N- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-ethyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Synthesis of the title Compound starting from imidazole-5-carboxylic acid and 2-methoxyethylamine Substance (yield: 58%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.46(s,1H),8.09(s,1H),7.77(d,J＝8.4Hz,1H),7.56-7.50(m,3H),7.21(d,J＝8.0Hz,1H),4.22(q,J＝7.6Hz,2H),3.48-3.44(m,4H),3.28(s,3H),1.32(t,J＝7.6Hz,3H)；LC-MS:m/z 464.1(M+1)+。
EXAMPLE 86 Synthesis of 1-ethyl-N- (2-hydroxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
To a solution of 1-ethyl-2- ((6- (trifluoromethoxy) benzo [ d ] at 0 deg.C]Oxazol-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (60mg, 0.14mmol) in DMFA (1mL) were added N-ethyldiisopropylamine (0.02mL, 0.14mmol) and HBTU (52mg, 0.14 mmol). The reaction mixture was stirred for 30min, then 2-aminoethan-1-ol (8mg, 0.14mmol) was added and stirring continued at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (15mL) and stirred for 15 min. The resulting solid was filtered, washed with diethyl ether and dried in vacuo to give the title compound as a white solid (20mg, 30%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.37(t，J＝4.8Hz，1H)，8.09(s，1H)，7.78(d，J＝8.4Hz，1H)，7.56-7.49(m，3H)，7.21(d，J＝8.0Hz，1H)，4.72(t，J＝5.6Hz，1H)，4.22(q，J＝7.2Hz，2H)，3.53(q，J＝5.2Hz，2H)，3.39-3.37(m，2H)，1.32(t，J＝8.0Hz，3H)；LC-MS：m/z 450.1(M+1)+。
example 87 and 88.1 Synthesis of- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) -benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid N- (2-methoxyethyl) -1- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) (tetrahydrofuran-3-yl) methylamine, DIPEA, THF, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) cyanogen bromide, THF, H2O, 50 ℃, 16 h; d) NaH, 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; f) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 3-nitro-4- (((tetrahydrofuran-3-yl) methyl) amino) benzoate
To a solution of ethyl 4-chloro-3-nitrobenzoate (2.0g, 8.7mmol) in THF (40mL) at room temperature were added DIPEA (4.6mL, 26.1mmol) and (tetrahydrofuran-3-yl) methylamine (1.06g, 10.5mmol), followed by stirring the reaction mixture at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (200mL) and stirred for 1 h. The precipitated solid was filtered and dried in vacuo to give the title compound (2.4g, 94%);1H NMR(400MHz,DMSO-d6):δ8.62-8.59(m,2H),7.97(dd,J＝2.0Hz,J＝9.2Hz,1H),7.20(d,J＝8.8Hz,1H),4.29(q,J＝7.6Hz,2H),3.82-3.77(m,1H),3.73-3.69(m,1H),3.66-3.60(m,1H),3.53-3.50(m,1H),3.45-3.40(m,2H),2.67-2.58(m,1H),2.03-1.95(m,1H),1.69-1.63(m,1H),1.31(t,J＝7.2Hz,3H)；LC-MS:m/z 295.0(M+1)+。
step-b: synthesis of ethyl 3-amino-4- (((tetrahydrofuran-3-yl) methyl) amino) benzoate
The title compound was synthesized using the same procedure as compound 1e, using ethyl 3-nitro-4- (((tetrahydrofuran-3-yl) methyl) amino) benzoate as starting material (yield: 86%);1H NMR(400MHz,DMSO-d6):δ7.20(dd,J＝2.0Hz,J＝8.0Hz,1H),7.16(d,J＝2.0Hz,1H),6.46(d,J＝8.0Hz,1H),5.26(bs,1H),4.75(s,2H),4.18(q,J＝7.2Hz,2H),3.77-3.73(m,2H),3.64-3.62(m,1H),3.49-3.47(m,1H),3.07(t,J＝6.4Hz,2H),2.50-2.49(m,1H),2.11-1.98(m,1H),1.63-1.48(m,1H),1.26(t,J＝7.2Hz,3H)；LC-MS:m/z 265.1(M+1)+。
step-c: synthesis of ethyl 2-amino-1- ((tetrahydrofuran-3-yl) methyl) -1H-benzo [ d ] imidazole-5-carboxylate
The title compound was synthesized using the same procedure as compound 1f, using ethyl 3-amino-4- (((tetrahydrofuran-3-yl) methyl) amino) benzoate as the starting material (yield: 79%);1H NMR(400MHz,DMSO-d6):δ7.70(s,1H),7.58(dd,J＝1.6Hz,J＝8.4Hz,1H),7.27(d,J＝8.4Hz,1H),6.71(s,2H),4.27(q,J＝6.8Hz,2H),4.03(d,J＝8.0Hz,2H),3.78-3.71(m,1H),3.63-3.59(m,2H),3.48-3.41(m,1H),2.72-2.68(m,1H),1.85-1.79(m,1H),1.65-1.58(m,1H),1.31(t,J＝7.2Hz,3H)；LC-MS:m/z 290.1(M+1)+。
step-d: synthesis of ethyl 1- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
T Using the same procedure as in example 32, step-e, using 2-amino-1-ethyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 44%);1H NMR(400MHz,DMSO-d6):δ12.4(bs,1H),8.26(d,J＝1.6Hz,1H),7.88(dd,J＝1.6Hz,J＝8.4Hz,1H),7.63(d,J＝8.4Hz,1H),7.59(s,1H),7.52(d,J＝8.4Hz,1H),7.22(d,J＝7.6Hz,1H),4.33(q,J＝7.2Hz,2H),4.20-4.18(m,2H),3.88-3.86(m,1H),3.70-3.56(m,3H),3.80-3.78(m,1H),1.94-1.91(m,1H),1.73-1.71(m,1H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 491.15(M+1)+。
step-e: synthesis of 1- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) -benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, 1- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 76%);1HNMR(400MHz,DMSO-d6):δ12.8(bs,1H),12.3(bs,1H),8.21(s,1H),7.87(d,J＝8.0Hz,1H),7.62-7.58(m,2H),7.53(d,J＝8.8Hz,1H),7.21(d,J＝8.4Hz,1H),4.19(d,J＝7.6Hz,2H),3.89-3.84(m,1H),3.70-3.58(m,3H),2.89-2.85(m,1H),1.98-1.90(m,1H),1.76-1.68(m,1H)；LC-MS:m/z 463.1(M+1)+。
step-f: synthesis of N- (2-methoxyethyl) -1- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- ((tetrahydrofuran-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 18%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.47(s,1H),8.09(s,1H),7.77(d,J＝7.6Hz,1H),7.60-7.50(m,3H),7.21(d,J＝8.0Hz,1H),4.18(bs,2H),3.87(bs,1H),3.67-3.57(m,3H),3.47(bs,4H),3.28(s,3H),2.88(bs,1H),1.92(bs,1H),1.73(bs,1H)；LC-MS:m/z520.15(M+1)+。
EXAMPLE 89 and 90.1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid synthesis and N- (2-methoxyethyl) -1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide synthesis
Conditions are as follows: a)2- (methylsulfonyl) ethyl-1-amine hydrochloride, DIPEA, DMF, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) cyanogen bromide, THF, H2O, 50 ℃, 16 h; d) NaH, 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; f) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of ethyl 4- ((2- (methylsulfonyl) ethyl) amino) -3-nitrobenzoate
The title compound was synthesized using the same procedure as compound 3a, using ethyl 4-chloro-3-nitrobenzoate and 2- (methylsulfonyl) ethan-1-amine hydrochloride as starting materials and heating to 60 ℃. (yield:89％)；1H NMR(400MHz，DMSO-d6)：δ8.69(t，J＝6.0Hz，1H)，8.63(d，J＝2.0Hz，1H)，8.02(dd，J＝2.0Hz，J＝8.8Hz，1H)，7.20(d，J＝9.2Hz，1H)，4.30(q，J＝6.8Hz，2H)，3.89(q，J＝5.6Hz，2H)，3.52(t，J＝6.8Hz，2H)，3.08(s，3H)，1.30(t，J＝6.8Hz，3H)；LC-MS：m/z317.0(M+1)+。
step-b: synthesis of ethyl 3-amino-4- ((2- (methylsulfonyl) ethyl) amino) benzoate
The title compound was synthesized using the same procedure as compound 1e, using ethyl 4- ((2- (methylsulfonyl) ethyl) amino) -3-nitrobenzoate as starting material (yield: 86%);1H NMR(400MHz，DMSO-d6)：δ7.24-7.20(m，2H)，6.52(d，J＝8.4Hz，1H)，5.45(t，J＝5.2Hz，1H)，4.72(s，2H)，4.19(q，J＝7.2Hz，2H)，3.58(q，J＝6.4Hz，2H)，3.38(t，J＝6.8Hz，2H)，3.03(s，3H)，1.26(t，J＝6.8Hz，3H)；LC-MS：m/z 287.0(M+1)+。
step-c: synthesis of ethyl 2-amino-1- (2- (methylsulfonyl) ethyl) -1H-benzo [ d ] imidazole-5-carboxylate
The title compound was synthesized using the same procedure as compound 1f, using ethyl 3-amino-4- ((2- (methylsulfonyl) ethyl) amino) benzoate as starting material (yield: 86%);1H NMR(400MHz，DMSO-d6)：δ7.71(d，J＝1.2Hz，1H)，7.61(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.28(d，J＝8.4Hz，1H)，6.75(s，2H)，4.47-4.40(m，2H)，4.30(q，J＝7.6Hz，2H)，3.55(t，J＝6.8Hz，2H)，3.03(s，3H)，1.31(t，J＝6.8Hz，3H)。
step-d: synthesis of ethyl 1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 1g, 2-amino-1- (2- (methylsulfonyl) ethyl) -1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 24%);1H NMR(400MHz,DMSO-d6):δ13.4(bs,1H),8.25(d,J＝1.6Hz,1H),7.89(dd,J＝1.2Hz,J＝8.0Hz,1H),7.63-7.61(m,2H),7.54(d,J＝8.8Hz,1H),7.56-7.51(m,1H),4.61(t,J＝6.8Hz,2H),4.34(q,J＝7.2Hz,2H),3.73(d),J＝6.8Hz,2H),3.14(s,3H),1.35(t,J＝7.6Hz,3H)；LC-MS:m/z513.05(M+1)+。
step-e: synthesis of 1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, using 1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d ]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 17%);1H NMR(400MHz,DMSO-d6):δ12.8(bs,1H),12.4(bs,1H),8.20(d,J＝1.2Hz,1H),7.88(dd,J＝1.2Hz,J＝8.0Hz,1H),7.61-7.59(m,2H),7.54(d,J＝8.4Hz,1H),7.22(d,J＝8.8Hz,1H),4.61(t,J＝6.8Hz,2H),3.72(t,J＝6.8Hz,2H),3.14(s,3H)；LC-MS:m/z 484.9(M+1)+。
step-f: synthesis of N- (2-methoxyethyl) -1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- (2- (methylsulfonyl) ethyl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 32%);1H NMR(400MHz,DMSO-d6) δ 12.30(bs,1H),8.48(t, J ═ 5.2Hz,1H),8.08(s,1H),7.77(d, J ═ 8.4Hz,1H),7.67-7.54(m,3H),7.21(d, J ═ 9.6Hz,1H),4.60(t, J ═ 6.8Hz,2H),3.74(t, J ═ 10.8 confirming coupling constants Hz,2H),3.48-3.44(m,4H),3.28(s,3H),3.14(s, 3H); LC-MS M/z 542.0(M +1)+。
EXAMPLE 91.2 Synthesis of- ((6-bromobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Conditions are as follows: a) SnCl22H2O，EtOH，Refluxing for 4 h; b) potassium ethyl xanthate, EtOH, refluxing for 16 h; c) SOCl2Catalytic DMF, refluxing for 3 h; d) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O，60℃，16h
Step-a: synthesis of 2-amino-5-bromophenol
To a solution of 5-bromo-2-nitrophenol (4.0g, 18.35mmol) in ethanol (60mL) at room temperature was added SnCl2.2H2O (20.6g, 91.74mmol), and the reaction mixture was refluxed for 4 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with water (20mL) and basified with saturated aqueous sodium bicarbonate and extracted with ethyl acetate (2X 100 mL). The combined organic layers were washed with water (50mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (2.4g, 70%);1H NMR(400MHz，DMSO-d6)：δ9.44(bs，1H)，6.75(d，J＝2.4Hz，1H)，6.67(dd，J＝2.0Hz，J＝8.4Hz，1H)，6.51(d，J＝8.4Hz，1H)，4.65(bs，2H)；LC-MS：m/z190.0(M+1)+。
step-b: synthesis of 6-bromobenzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32 step-c, using 2-amino-5-bromophenol as starting material (yield: 98%);1H NMR(400MHz，DMSO-d6)：δ14.0(bs，1H)，7.84(d，J＝1.6Hz，1H)，7.47(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.18(d，J＝8.4Hz，1H)；LC-MS：m/z 227.9(M-1)-。
step-c: synthesis of 6-bromo-2-chlorobenzo [ d ] oxazole
To 6-bromobenzo [ d ] at room temperature]To a solution of oxazole-2-thiol (2.89g, 12.56mmol) in thionyl chloride (20mL) was added N, N-dimethylformamide (0.2mL) and the reaction mixture was refluxed for 3 h. The reaction mixture was concentrated, diluted with cold water (50mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (50mL), brine solution (40mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (2.8g, 96%); 1H NMR(400MHz，DMSO-d6)：δ8.14(s，1H)，7.72(d，J＝8.8Hz，1H)，7.62(dd，J＝2.0Hz，J＝8.8Hz，1H)。
Step-d: synthesis of ethyl 2- ((6-bromobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 6-bromo-2-chlorobenzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 20%);1H NMR(400MHz，DMSO-d6)：δ11.56(bs，1H)，8.23(d，J＝1.6Hz，1H)，7.88(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.72(d，J＝1.6Hz，1H)，7.54(d，J＝8.8Hz，1H)，7.38-7.36(m，2H)，4.33(q，J＝7.2Hz，2H)，3.64(s，3H)，1.35(t，J＝7.2Hz，3H)；LC-MS：m/z 416.9(M+1)+。
step-e: synthesis of 2- ((6-bromobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- ((6-bromobenzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 75%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.19(s，1H)，7.87(d，J＝8.4Hz，1H)，7.71(d，J＝1.6Hz，1H)，7.51(d，J＝8.8Hz，1H)，7.42-7.35(m，2H)，3.64(s，3H)；LC-MS：m/z 388.9(M+1)+。
EXAMPLE 92. Synthesis of N- (2-hydroxyethyl) -2- ((6-methoxybenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaOMe, MeOH, 60 ℃, 40 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) potassium ethyl xanthate, EtOH, refluxing for 16 h; d) SOCl2Catalytic DMF, refluxing for 2 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; g) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Step-a: synthesis of 5-methoxy-2-nitrophenol
To a stirred suspension of sodium methoxide (0.86g, 18.35mmol) in methanol (50mL) at room temperature was added a solution of 5-fluoro-2-nitrophenol (3.0g, 19.1mmol) in methanol (40mL) by syringe and the reaction mixture was heated at 60 ℃ for 40 h. The reaction mixture was cooled to room temperature, quenched with cold water (50mL), acidified with 1N HCl and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 3% ethyl acetate/hexanes as eluent to give the title compound (2.0g, 62%);1H NMR(400MHz，DMSO-d6)：δ10.94(s，1H)，7.97(d，J＝9.6Hz，1H)，6.63(d，J＝2.4Hz，1H)，6.59(dd，J＝3.0Hz，J＝9.2Hz，1H)，3.83(s，3H)；LC-MS：m/z 167.95(M-1)-。
step-b: synthesis of 2-amino-5-methoxyphenol
The title compound was synthesized using the same procedure as compound 1e, using 5-methoxy-2-nitrophenol as the starting material (yield: 96%);1H NMR(400MHz,DMSO-d6):δ8.8(bs,1H),6.49(d,J＝8.4Hz,1H),6.30(d,J＝2.1Hz,1H),6.18-6.14(m,1H),3.58(s,3H)；LC-MS:m/z 140.15(M+1)+。
step-c: synthesis of 6-methoxybenzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32 step-c, using 2-amino-5-methoxyphenol as the starting material (yield: 72%);1H NMR(400MHz,DMSO-d6):δ13.7(s,1H),7.21(d,J＝2.4Hz,1H),7.13(d,J＝8.4Hz,1H),6.88(dd,J＝2.0Hz,J＝8.4Hz,1H),3.77(s,3H)；LC-MS:m/z 182.0(M+1)+。
step-d: synthesis of 2-chloro-6-methoxybenzo [ d ] oxazole
Using the same procedure as example 32, step-d, using 6-methoxybenzo [ d ]Oxazole-2-thiols as herbicidesThe title compound was synthesized as starting material and stirred for 2h (yield: 91%); LC-MS: m/z 184.0(M +1)+。
Step-e: synthesis of ethyl 2- ((6-methoxybenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6-methoxybenzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 18%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.21(d,J＝1.4Hz,1H),7.87(dd,J＝1.2Hz,J＝8.4Hz,1H),7.50(d,J＝8.0Hz,1H),7.36(d,J＝8.4Hz,1H),7.13(d,J＝2.0Hz,1H),6.83(dd,J＝2.4Hz,J＝8.4Hz,1H),4.33(q,J＝6.8Hz,2H),3.82(s,3H),3.62(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 367.0(M+1)+。
step-f: synthesis of 2- ((6-methoxybenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- ((6-methoxybenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 79%);1H NMR(400MHz,DMSO-d6):δ12.5(bs,2H),8.17(s,1H),7.85(dd,J＝1.6Hz,J＝8.4Hz,1H),7.47(d,J＝8.4Hz,1H),7.37(d,J＝8.8Hz,1H),7.12(d,J＝2.0Hz,1H),6.82(dd,J＝2.4Hz,J＝8.8Hz,1H),3.79(s,3H),3.62(s,3H)；LC-MS:m/z 339.0(M+1)+。
step-g: synthesis of N- (2-hydroxyethyl) -2- ((6-methoxybenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((6-methoxybenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials (yield: 59%); 1H NMR(400MHz,DMSO-d6):δ12.18(bs,1H),8.35(t,J＝5.2Hz,1H),8.05(d,J＝1.2Hz,1H),7.75(dd,J＝1.6Hz,J＝8.4Hz,1H),7.45(d,J＝8.4Hz,1H),7.35(d,J＝8.4Hz,1H),7.11(d,J＝2.4Hz，1H)，6.81(dd，J＝2.4Hz，J＝8.8Hz，1H)，4.72(bs，1H)，3.78(s，3H)，3.61(s，3H)，3.53(t，J＝6.0Hz，2H)，3.35-3.30(m，2H)；LC-MS：m/z 382.0(M+1)+。
Examples 93 and 94.1 Synthesis of- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and 1- ((3-hydroxyoxetan-3-yl) methyl) -N- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) nitromethane, TEA, RT, 16 h; b) 10% Pd/C, MeOH, H2RT, 3 h; c)3- (aminomethyl) oxetan-3-ol, DIPEA, DMF, 60 ℃, 16 h; d) 10% Pd/C, MeOH, H2RT, 16 h; e) cyanogen bromide, THF, H2O, 50 ℃, 16 h; f) NaH, 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole, 1, 4-dioxane, RT, 16 h; g) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; h) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 3- (nitromethyl) oxetan-3-ols
To a solution of oxetan-3-one (10.0g, 138.9mmol) in nitromethane (25mL) at 0 deg.C was added triethylamine (5mL, 347.2mmol) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography using 40% ethyl acetate/hexanes as eluent to give the title compound as a light yellow liquid (14.0g, 77%); 1H NMR(400MHz，CDCl3)：δ4.82(s，2H)，4.71(d，J＝8.0Hz，2H)，4.61(d，J＝8.0Hz，2H)，3.53(s，1H)。
Step-b: synthesis of 3- (aminomethyl) oxetan-3-ol
To a solution of 3- (nitromethyl) oxetan-3-ol (5.0g, 37.6) in methanol (80mL) under a nitrogen atmosphere was added a 10% Pd/C (2.0g in 20mL methanol) slurry. Then, under a hydrogen balloonThe reaction mixture was stirred for 3 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (3.8g, 98%); LC-MS: m/z 104.2(M +1)+。
Step-c: synthesis of ethyl 4- (((3-hydroxyoxetan-3-yl) methyl) amino) -3-nitrobenzoate
The title compound was synthesized using the same procedure as compound 3a, using 4-chloro-3-nitrobenzoic acid ethyl ester and 3- (aminomethyl) oxetan-3-ol as starting materials and heating to 60 ℃ (yield: 54%);1H NMR(400MHz,DMSO-d6):δ8.62(d,J＝1.6Hz,1H),8.53(bs,1H),7.99(dd,J＝2.0Hz,J＝9.2Hz,1H),7.29(d,J＝9.2Hz,1H),6.35(s,1H),4.51(d,J＝6.8Hz,2H),4.44(d,J＝6.8Hz,2H),4.29(q,J＝7.2Hz,2H),3.75(d,J＝5.2Hz,2H),1.31(t,J＝6.8Hz,3H)；LC-MS:m/z 297.1(M+1)+。
step-d: synthesis of ethyl 3-amino-4- (((3-hydroxyoxetan-3-yl) methyl) amino) benzoate
The title compound was synthesized using the same procedure as compound 1e, using ethyl 4- (((3-hydroxyoxetan-3-yl) methyl) amino) -3-nitrobenzoate as starting material (yield: 95%);1H NMR(400MHz,DMSO-d6):δ7.23-7.20(m,2H),6.57(d,J＝8.4Hz,1H),5.96(bs,1H),5.04(bs,1H),4.75(s,2H),4.46-4.43(m,4H),4.19(q,J＝7.2Hz,2H),3.40(d,J＝5.6Hz,2H),1.26(t,J＝6.8Hz,3H)；LC-MS:m/z 267.1(M+1)+。
step-e: synthesis of ethyl 2-amino-1- ((3-hydroxyoxetan-3-yl) methyl) -1H-benzo [ d ] imidazole-5-carboxylate
The title compound was synthesized using the same procedure as compound 1f, using ethyl 3-amino-4- (((3-hydroxyoxetan-3-yl) methyl) amino) benzoate as starting material (yield: 70%); 1H NMR(400MHz,DMSO-d6):δ7.72(s,1H),7.59(dd,J＝1.6Hz,J＝8.0Hz,1H),7.35(d,J＝8.0Hz,1H),6.56(s,2H),6.45(s,1H),4.55(d,J＝6.4Hz,2H),4.43(d,J＝6.0Hz,2H),4.34(s,2H),4.28(q,J＝6.8Hz,2H),1.32(t,J＝5.2Hz,3H)；LC-MS:m/z 292.1(M+1)+。
Step-f: synthesis of ethyl 1- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as example 32, step-e, using 2-amino-1- ((3-hydroxyoxetan-3-yl) methyl) -1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (trifluoromethoxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 18%);1H NMR(400MHz,DMSO-d6):δ12.4(bs,1H),8.24(d,J＝1.6Hz,1H),7.85(dd,J＝1.6Hz,J＝8.4Hz,1H),7.60-7.58(m,2H),7.53(d,J＝8.4Hz,1H),7.23(dd,J＝1.2Hz,J＝8.8Hz,1H),6.18(s,1H),4.79(d,J＝6.8Hz,2H),4.50(bs,2H),4.46(d,J＝6.8Hz,2H),4.33(q,J＝6.8Hz,2H),1.35(t,J＝7.6Hz,3H)；LC-MS:m/z 493.0(M+1)+。
step-g: synthesis of 1- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, 1- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 49%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.19(d，J＝1.2Hz，1H)，7.84(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.60-7.52(m，3H)，7.22(dd，J＝1.6Hz，J＝8.4Hz，1H)，6.18(s，1H)，4.79(d，J＝6.4Hz，2H)，4.51(s，2H)，4.46(d，J＝6.4Hz，2H)；LC-MS：m/z 465.1(M+1)+。
step-h: synthesis of 1- ((3-hydroxyoxetan-3-yl) methyl) -N- (2-methoxyethyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials. The crude product was purified by flash chromatography using 2% methanol/DCM as eluent to give the title compound (yield: 28%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.43(t，J＝5.2Hz，1H)，8.07(s，1H)，7.73(d，J＝8.8Hz，1H)，7.58(s，1H)，7.55-7.51(m，2H)，7.21(d，J＝8.4Hz，1H)，6.17(s，1H)，4.79(d，J＝6.8Hz，2H)，4.50(s，2H)，4.46(d，J＝6.4Hz，2H)，3.49-3.43(m，4H)，3.28(s，3H)；LC-MS：m/z522.0(M+1)+。
EXAMPLE 95. Synthesis of N- (2-hydroxyethyl) -1- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Using the same procedure as example 6 step-e, 1- ((3-hydroxyoxetan-3-yl) methyl) -2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials. The crude product was purified by flash chromatography using 3% methanol/DCM as eluent to give the title compound (yield: 15%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.35(t，J＝5.6Hz，1H)，8.07(d，J＝1.2Hz，1H)，7.73(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.58(s，1H)，7.58-7.51(m，2H)，7.22(dd，J＝1.2Hz，J＝10.0Hz，1H)，6.17(s，1H)，4.80(d，J＝6.8Hz，2H)，4.71(t，J＝5.2Hz，1H)，4.51(s，2H)，4.46(d，J＝6.4Hz，2H)，3.55-3.51(m，2H)，3.37-27(m，2H)；LC-MS：m/z 508.45(M+1)+。
examples 96 and 97.2 Synthesis of- ((6-chlorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and 2- ((6-chlorobenzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) potassium ethyl xanthate, EtOH, refluxing for 16 h; b) SOCl 2Catalytic DMF, refluxing for 3 h; c) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; d) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; e) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Step-a: synthesis of 6-chlorobenzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5-chlorophenol as starting material (yield: 69%);1H NMR(400MHz，DMSO-d6)：δ14.0(bs，1H)，7.73(d，J＝2.0Hz，1H)，7.35(dd，J＝1.6Hz，J＝8.0Hz，1H)，7.23(d，J＝8.8Hz，1H)；LC-MS：m/z 184.0(M-1)-。
step-b: synthesis of 2, 6-dichlorobenzo [ d ] oxazole
Using the same procedure as in example 32, step-d, using 6-chlorobenzo [ d]The title compound was synthesized from oxazole-2-thiol as a starting material (yield: 69%);1H NMR(400MHz，DMSO-d6)：δ8.01(d，J＝1.6Hz，1H)，7.77(d，J＝8.4Hz，1H)，7.51(dd，J＝2.0Hz，J＝8.4Hz，1H)。
step-c: synthesis of ethyl 2- ((6-chlorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2, 6-dichlorobenzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 24%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.23(d，J＝1.6Hz，1H)，7.88(d，J＝8.0Hz，1H)，7.60(d，J＝2.0Hz，1H)，7.53(d，J＝8.4Hz，1H)，7.44(d，J＝8.0Hz，1H)，7.25(dd，J＝2.0Hz，J＝8.0Hz，1H)，4.33(q，J＝7.2Hz，2H)，3.64(s，3H)，1.35(t，J＝7.2Hz，3H)；LC-MS：m/z 369.0(M-1)-。
step-d: synthesis of 2- ((6-chlorobenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, using 2- ((6-chlorobenzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 81%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.19(d，J＝1.2Hz，1H)，7.87(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.59(d，J＝2.0Hz，1H)，7.51(d，J＝8.4Hz，1H)，7.45(d，J＝8.4Hz，1H)，7.24(dd，J＝2.0Hz，J＝8.4Hz，1H)，3.64(s，3H)；LC-MS：m/z 342.9(M+1)+。
step-e: synthesis of 2- ((6-chlorobenzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((6-chlorobenzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials (yield: 53%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.37(t，J＝5.6Hz，1H)，8.08(s，1H)，7.78(d，J＝8.4Hz，1H)，7.58(d，J＝2.0Hz，1H)，7.49(d，J＝8.8Hz，1H)，7.44(d，J＝8.4Hz，1H)，7.24(dd，J＝2.0Hz，J＝8.4Hz，1H)，4.72(t，J＝5.2Hz，1H)，3.64(s，3H)，3.55-3.51(m，2H)，3.37-3.33(m，2H)；LC-MS：m/z386.1(M+1)+。
examples 98 and 99.1 Synthesis of 1-methyl-2- ((6- (2, 2, 2-trifluoroethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid and N- (2-methoxyethyl) -1-methyl-2- ((6- (2, 2, 2-trifluoroethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) benzyl bromide, K2CO3DMF, 0 ℃ -RT, 16 h; b) trifluoroethanol, NaH, THF, 0-RT, 3 h; c) 10% Pd/C, MeOH, H2RT, 16 h; d) potassium ethyl xanthate, EtOHRefluxing for 16 h; e) SOCl2Catalytic DMF, refluxing for 2 h; f) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; g) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; h) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 2- (benzyloxy) -4-fluoro-1-nitrobenzene
To a stirred solution of 5-fluoro-2-nitrophenol (8.0g, 50.9mmol) in DMFA (80mL) at 0 deg.C was added potassium carbonate (14.0g, 101.8mmol) and benzyl bromide (5.4mL, 45.8mmol) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (200mL) and extracted with ethyl acetate (2X 200 mL). The combined organic layers were washed with water (2 × 200mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 10% ethyl acetate/hexanes as eluent to give the title compound (8.0g, 64%);1H NMR(400MHz，DMSO-d6)：δ8.06-8.02(m，1H)，7.47-7.36(m，6H)，7.05-6.95(m，1H)，5.33(s，2H)。
step-b: synthesis of 2- (benzyloxy) -1-nitro-4- (2, 2, 2-trifluoroethoxy) benzene
To a solution of trifluoroethanol (0.23mL, 3.24mmol) in THF (10mL) at 0 ℃ was added sodium hydride (60% dispersion in mineral oil) (194mg, 4.86mmol) portionwise and stirred for 1 h. Subsequently, a solution of 2- (benzyloxy) -4-fluoro-1-nitrobenzene (400mg, 1.62mmol) was added to the reaction mixture and stirred at room temperature for 2 h. The reaction mixture was concentrated and diluted with cold water (20 mL). The precipitated solid was filtered and dried in vacuo to give the title compound (350mg, 66%); 1H NMR(400MHz,DMSO-d6):δ8.01(d,J＝9.2Hz,1H),7.48-7.33(m,5H),7.11(d,J＝2.8Hz,1H),6.82(dd,J＝2.4Hz,J＝8.8Hz,1H),5.32(s,2H),4.94(q,J＝8.8Hz,2H)。
Step-c: synthesis of 2-amino-5- (2,2, 2-trifluoroethoxy) phenol
To a solution of 2- (benzyloxy) -1-nitro-4- (2,2, 2-trifluoroethoxy) benzene (350mg, 1.07mmol) in methanol (10mL) under a nitrogen atmosphere was added 10% Pd/C (50 mg). In the presence of hydrogenThe reaction mixture was stirred under a balloon for 16 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (210mg, 95%);1H NMR(400MHz,DMSO-d6):δ6.51(d,J＝8.8Hz,1H),6.38(d,J＝2.4Hz,1H),6.28(dd,J＝2.8Hz,J＝8.8Hz,1H),4.48(q,J＝8.8Hz,2H)；LC-MS:m/z 208.0(M+1)+。
step-d: synthesis of 6- (2,2, 2-trifluoroethoxy) benzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5- (2,2, 2-trifluoroethoxy) phenol as the starting material (yield: 79%);1H NMR(400MHz,DMSO-d6):δ13.8(bs,1H),7.38(d,J＝2.4Hz,1H),7.18(d,J＝8.8Hz,1H),7.01(dd,J＝2.4Hz,J＝8.8Hz,1H),4.79(q,J＝8.8Hz,2H)；LC-MS:m/z 250.0(M+1)+。
step-e: synthesis of 2-chloro-6- (2,2, 2-trifluoroethoxy) benzo [ d ] oxazole
Using the same procedure as example 32 step-d, using 6- (2,2, 2-trifluoroethoxy) benzo [ d]Oxazole-2-thiol was used as starting material and stirred for 2h to synthesize the title compound. (yield: 87%);1H NMR(400MHz,DMSO-d6):δ7.69(d,J＝8.8Hz,1H),7.60(d,J＝2.4Hz,1H),6.15(dd,J＝2.4Hz,J＝8.8Hz,1H),4.85(q,J＝8.8Hz,2H)。
step-f: synthesis of ethyl 1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- (2,2, 2-trifluoroethoxy) benzo [ d ]Oxazole as a starting material the title compound was synthesized (yield: 21%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.22(d,J＝1.6Hz,1H),7.88(dd,J＝1.2Hz,J＝8.0Hz,1H),7.51(d,J＝8.4Hz,1H),7.39(d,J＝8.4Hz,1H),7.29(d,J＝2.4Hz,1H),6.94(dd,J＝2.4Hz,J＝8.4Hz,1H),4.77(q,J＝8.8Hz,2H),4.33(q,J＝7.2Hz,2H),3.63(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 435.35(M+1)+。
step-g: synthesis of 1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, 1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [ d ] is used]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 62%);1H NMR(400MHz，DMSO-d6)：δ12.5(bs，2H)，8.18(d，J＝1.2Hz，1H)，7.86(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.48(d，J＝8.8Hz，1H)，7.40(d，J＝8.8Hz，1H)，7.29(d，J＝2.4Hz，1H)，6.93(dd，J＝2.4Hz，J＝8.8Hz，1H)，4.77(q，J＝9.2Hz，2H)，3.62(s，3H)；LC-MS：m/z 407.0(M+1)+。
step-h: synthesis of N- (2-methoxyethyl) -1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [ d ] is used]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 70%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，1H)，8.4(t，J＝5.6Hz，1H)，8.06(s，1H)，7.75(d，J＝8.0Hz，1H)，7.46(d，J＝8.0Hz，1H)，7.39(d，J＝8.0Hz，1H)，7.28(d，J＝1.6Hz，1H)，6.93(dd，J＝2.4Hz，J＝8.4Hz，1H)，4.77(q，J＝8.8Hz，2H)，3.62(s，3H)，3.48-3.43(m，4H)，3.28(s，3H)；LC-S：m/z 464.0(M+1)+。
EXAMPLE 100 Synthesis of N- (2-hydroxyethyl) -1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (2,2, 2-trifluoroethoxy) benzo [d]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials (yield: 63%);1H NMR(400MHz，DMSO-d6)：δ12.5(bs，1H)，8.42(t，J＝5.2Hz，1H)，8.07(s，1H)，7.81(d，J＝8.4Hz，1H)，7.53(d，J＝8.4Hz，1H)，7.40(d，J＝8.8Hz，1H)，7.32(d，J＝2.4Hz，1H)，6.98(dd，J＝2.4Hz，J＝8.8Hz，1H)，4.80(q，J＝8.8Hz，2H)，3.66(s，3H)，3.54(t，J＝6.4Hz，2H)，3.36(q，J＝6.0Hz，2H)；LC-MS：m/z 450.0(M+1)+。
examples 101 and 102.2 Synthesis of- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and 2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzimidazo-5-carboxamide
Conditions are as follows: a) KOH, water, 100 ℃, 30 h; b) (2-Bromoethoxy) (tert-butyl) dimethylsilane, K2CO3，DMF，130℃，4h；c)10％Pd/C，MeOH，H2RT, 16 h; d) potassium ethyl xanthate, EtOH, refluxing for 16 h; e) k2CO3Mel, ACN, RT, 16 h; f) m-CPBA, DCM, 0 ℃ -RT, 4 h; g) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; h) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; h) 2-methoxyethylamine, DPPA, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of 3- (benzyloxy) -4-nitrophenol
To a stirred suspension of 2- (benzyloxy) -4-fluoro-1-nitrobenzene (4.0g, 16.2mmol) in water (70mL) at room temperature was added potassium hydroxide (4.53g, 80.9mmol) and the reaction mixture was stirred at 100 ℃ for 30 h. The reaction mixture was cooled to room temperature, diluted with water (50mL) and acidified with 1N HCl and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and Concentration in vacuo gave a residue which was purified by flash chromatography using 20% ethyl acetate/hexanes as the eluent to give the title compound (1.5g, 38%);1H NMR(400MHz，DMSO-d6)：δ10.88(s，1H)，7.90(d，J＝8.8Hz，1H)，7.49-7.47(m，2H)，7.43-7.40(m，2H)，7.35(d，J＝6.8Hz，1H)，6.69(d，J＝2.4Hz，1H)，6.49(dd，J＝2.4Hz，J＝9.2Hz，1H)，5.25(s，2H)。
step-b: synthesis of (2- (3- (benzyloxy) -4-nitrophenoxy) ethoxy) (tert-butyl) dimethylsilane
To a stirred solution of 3- (benzyloxy) -4-nitrophenol (1.5g, 6.1mmol) in DMFA (15mL) at room temperature in a sealed tube was added potassium carbonate (2.1g, 15.3mmol) and (2-bromoethoxy) (tert-butyl) dimethylsilane (1.75g, 7.3mmol) and heated at 130 ℃ for 4 h. The reaction mixture was cooled to room temperature, diluted with water (50mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water (2 × 500mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography using 5% ethyl acetate/hexanes as the eluent to give the title compound (8.0g, 64%);1H NMR(400MHz，DMSO-d6)：δ7.96(d，J＝8.8Hz，1H)，7.49-7.47(m，2H)，7.43-7.39(m，2H)，7.36-7.34(m，1H)，6.89(d，J＝2.4Hz，1H)，6.67(dd，J＝2.4Hz，J＝9.2Hz，1H)，5.32(s，2H)，4.17(t，J＝4.4Hz，2H)，3.92(t，J＝4.8Hz，2H)，0.86(s，9H),0.02(s,6H)。
step-c: synthesis of 2-amino-5- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) phenol
The title compound was synthesized using the same procedure as compound 1e, using (2- (3- (benzyloxy) -4-nitrophenoxy) ethoxy) (tert-butyl) dimethylsilane as starting material (yield: 87%); 1H NMR(400MHz,DMSO-d6):δ6.47(d,J＝8.0Hz,1H),6.30(d,J＝2.8Hz,1H),6.15(dd,J＝2.4Hz,J＝8.4Hz,1H),3.84-3.80(m,4H),0.87(s,9H),0.02(s,6H)；LC-MS:m/z 284.1(M+1)+。
Step-d: synthesis of 6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) benzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) phenol as starting material (yield: 77%). The crude compound was used in the next step without any analytical data.
Step-e: synthesis of 6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) -2- (methylthio) benzo [ d ] oxazole
Using the same procedure as for Compound 1b, using 6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) benzo [ d]Oxazole-2-thiol was used as starting material and stirred for 16h to synthesize the title compound. (yield: 80%);1H NMR(400MHz,DMSO-d6):δ7.50(d,J＝8.8Hz,1H),7.28(d,J＝2.4Hz,1H),6.91(dd,J＝2.8Hz,J＝8.8Hz,1H),4.06(t,J＝4.4Hz,2H),3.92(t,J＝5.2Hz,2H),2.27(s,3H),0.86(s,9H),0.2(s,6H)；LC-MS:m/z 340.0(M+1)+。
step-f: synthesis of 6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) -2- (methanesulfonyl) -benzo [ d ] oxazole
Using the same procedure as for Compound 1c, using 6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) -2- (methylthio) benzo [ d]Oxazole as a starting material and stirred for 4h to synthesize the title compound (yield: 100%);1H NMR(400MHz,DMSO-d6):δ7.90-7.89(m,1H),7.55(d,J＝8.0Hz,1H),6.99-6.97(m,1H),4.10(t,J＝4.8Hz,2H),3.95(t,J＝5.2Hz,2H),3.62(s,3H),0.85(s,9H),0.2(s,6H)。
step-g: synthesis of ethyl 2- ((6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-1-methyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) -2- (methanesulfonyl) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 17%);1H NMR(400MHz,DMSO-d6):δ12.2(bs,1H),8.21(d,J＝1.6Hz,1H),7.86(dd,J＝1.6Hz,J＝8.4Hz,1H),7.49(d,J＝8.0Hz,1H),7.35(d,J＝8.4Hz,1H),7.12(d,J＝2.4Hz,1H),6.81(dd,J＝1.6Hz,J＝8.0Hz,1H),4.32(q,J＝7.6Hz,2H),4.06-4.04(m,2H),3.94-3.92(m,2H),3.62(s,3H),1.35(t,J＝6.8Hz,3H),0.88(s,9H),0.2(s,6H)；LC-MS:m/z 511.1(M+1)+。
step-h: synthesis of 2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
To 2- ((6- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) benzo [ d]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (80mg, 0.15mmol) in a solvent mixture of THF (2mL), ethanol (2mL) and water (1mL) was added lithium hydroxide monohydrate (32mg, 0.78 mmol). The reaction mixture was heated with stirring at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (20mL) and acidified with 1n hcl. The resulting solid was filtered and dried in vacuo to give the title compound (50mg, 87%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.3(bs，1H)，8.16(d，J＝1.6Hz，1H)，7.85(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.46(d，J＝8.4Hz，1H)，7.36(d，J＝8.4Hz，1H)，7.12(d，J＝2.4Hz，1H)，6.82(dd，J＝2.4Hz，J＝8.4Hz，1H)，4.85(bs，1H)，4.01(t，J＝4.8Hz，2H)，3.75-3.73(m，2H)，3.66(s，3H)；LC-MS：m/z 369.0(M+1)+。
step-i: synthesis of 2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- ((6- (2-hydroxyethoxy) benzo [ d ] was used ]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials. The crude product was purified by flash chromatography using 4% methanol/DCM as eluent (yield: 17%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，1H)，8.43(t，J＝5.2Hz，1H)，8.05(d，J＝1.2Hz，1H)，7.75(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.45(d，J＝8.8Hz，1H)，7.35(d，J＝8.4Hz，1H)，7.12(d，J＝2.4Hz，1H)，6.82(dd，J＝2.8Hz，J＝8.8Hz，1H)，4.11(t，J＝4.4Hz，2H)，3.66(t，J＝4.4Hz，2H)，3.61(s，3H)，3.49-3.41(m，4H)，3.28(bs，1H)，3.24(s，3H)；LC-MS：m/z 426.4(M+1)+。
EXAMPLE 103 Synthesis of 2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Using the same procedure as example 6 step-e, using 2- ((6- (2-hydroxyethoxy) benzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials. The crude product was purified by flash chromatography using 6% methanol/DCM as eluent to give the title compound (yield: 18%);1H NMR(400MHz，DMSO-d6)：δ12.05(bs，1H)，8.35(t，J＝5.2Hz，1H)，8.05(s，1H)，7.75(d，J＝8.0Hz，1H)，7.45(d，J＝8.4Hz，1H)，7.35(d，J＝8.4Hz，1H)，7.11(d，J＝2.4Hz，1H)，6.82(dd，J＝2.8Hz，J＝8.8Hz，1H)，4.85(bs，1H)，4.72(t，J＝4.8Hz，1H)，4.01(t，J＝4.8Hz，2H)，3.72(q，J＝4.4Hz，2H)，3.61(s，3H)，3.53(q，J＝5.6Hz，2H)，3.37-3.33(m，2H)；LC-MS：m/z 412.0(M+1)+。
EXAMPLE 104 Synthesis of 2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as in example 6 step-e, 2-((6- (2-hydroxyethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials. The crude product was purified by flash chromatography using 10% methanol/DCM as eluent to give the title compound (yield: 18%);1H NMR(400MHz，DMSO-d6)：δ12.05(bs，1H)，8.41(bs，1H)，8.04(s，1H)，7.74(d，J＝8.4Hz，1H)，7.45(d，J＝8.4Hz，1H)，7.35(d，J＝8.8Hz，1H)，7.11(s，1H)，6.82(d，J＝6.8Hz，1H)，4.61(bs，2H)，4.01(t，J＝4.4Hz，2H)，3.72(t，J＝4.8Hz，2H)，3.66(s，3H)，3.57-3.43(m，8H)；LC-MS：m/z456.3(M+1)+。
EXAMPLE 105 Synthesis of N- (2-aminoethyl) -2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide hydrochloride
Conditions are as follows: a) tert-butyl (2-aminoethyl) carbamate, HBTU, DIPEA, DMF, 0 ℃ -RT for 16 h; b) HCl, 1, 4-dioxane, 0 ℃ to RT, 3h
Step-a: synthesis of tert-butyl (2- (2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamido) ethyl) carbamate
To a mixture of 2- ((6- (2-hydroxyethoxy) benzo [ d ] at 0 deg.C]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid (100mg, 0.27mmol) in DMFA (2mL) were added N-ethyldiisopropylamine (0.09mL, 0.54mmol) and HBTU (102mg, 0.27 mmol). The reaction mixture was stirred for 30min, then tert-butyl (2-aminoethyl) carbamate (48mg, 0.30mmol) was added and stirring at room temperature was continued for 16 h. Once the reaction was complete, the reaction mixture was diluted with water (15mL) and stirred for 15 min. The resulting solid was filtered and dried in vacuo to give the crude product, which was purified by flash chromatography using 3% methanol/DCM as eluent to give the title compound (70mg, 50%); 1H NMR(400MHz，DMSO-d6): δ 12.20(bs, 1H), 8.39(bs, 1H), 8.05(s, 1H), 7.73(d, J ═ 8.4Hz, 1H), 7.45(d, J ═ 8.4Hz, 1H), 7.35(d, J ═ 8.0Hz, 1H), 7.11(d, J ═ 2.0Hz, 1H), 6.91(bs, 1H), 6.82(dd, J ═ 2.0Hz, J ═ 8.4Hz, 1H), 4.85(bs, 1H), 4.01(t, J ═ 4.8Hz, 2H), 3.72(bs, 2H), 3.61(s, 3H), 3.30(2H, combined with DMSO water peak), 3.12(d, J ═ 6.0, 2H), 1.38(s, 9H); LC-MS: m/z 511.3(M +1)+。
Step-b: synthesis of N- (2-aminoethyl) -2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide hydrochloride
To (2- (2- ((6- (2-hydroxyethoxy) benzo [ d) at 0 deg.C]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxamido) ethyl) carbamic acid tert-butyl ester (70mg, 0.14mmol) to a stirred solution in 1, 4-dioxane (3mL) was added 4M HCl in 1, 4-dioxane (1mL) and stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure, and the residue was wet-milled with diethyl ether and the solvent was decanted. The resulting solid was dried in vacuo to give the title compound (35mg, 57%);1H NMR(400MHz，DMSO-d6)：δ12.5(bs，1H)，8.78(s，1H)，8.12-8.07(m，4H)，7.90(d，J＝8.0Hz，1H)，7.56(d，J＝8.4Hz，1H)，7.36(d，J＝8.4Hz，1H)，7.18(s，1H)，6.88(d，J＝8.0Hz，1H)，4.02(bs，2H)，3.73(bs，2H)，3.66(s，3H)，3.56(q，J＝5.2Hz，2H)，3.02(q，J＝5.6Hz，2H)；LC-MS：m/z411.2(M+1)+。
EXAMPLE 106 Synthesis of N- (2- ((4, 5-dihydro-1H-imidazol-2-yl) amino) ethyl) -2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) n is a radical of1- (4, 5-dihydro-1H-imidazol-2-yl) ethane-1, 2-diamine, HBTU, DIPEA, DMF, 0 deg.C-RT, 16H
Using the same procedure as example 6 step-e, using 2- ((6- (2-hydroxyethoxy) benzeneAnd [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and N1- (4, 5-dihydro-1H-imidazol-2-yl) ethane-1, 2-diamine as starting materials (yield: 58%);1H NMR(400MHz，DMSO-d6): δ 8.73(bs, 1H), 8.49(s, 1H), 8.05(s, 1H), 7.75(d, J ═ 8.4Hz, 1H), 7.41(d, J ═ 8.0Hz, 1H), 7.31(d, J ═ 8.4Hz, 1H), 7.08(d, J ═ 2.0Hz, 1H), 6.79(dd, J ═ 1.6Hz, J ═ 8.0Hz, 1H), 4.86(bs, 1H), 4.00(t, J ═ 4.8Hz, 2H), 3.72(bs, 2H), 3.60-3.55(m, 7H), 3.43(bs, 2H), 3.30 (2H), combined with DMSO water peak; LC-MS: m/z 479.2(M +1)+。
Examples 107 and 108.2 Synthesis of- ((5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid and 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) HNO3(65-70％)，AcOH，0℃-RT，2h；b)10％Pd/C，MeOH，H2RT, 16 h; c) potassium ethyl xanthate, EtOH, refluxing for 16 h; d) SOCl 2Catalytic DMF, refluxing for 2 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O, THF, EtOH, H2O, 60 ℃ for 16H; g) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Step-a: synthesis of 4-fluoro-2-nitro-5- (trifluoromethyl) phenol
To a solution of 4-fluoro-3- (trifluoromethyl) phenol (1g, 5.6mmol) in acetic acid (20mL) was added dropwise acetic acid (5mL) containing 60% aqueous nitric acid (3mL) at 10-15 ℃. The reaction mixture was stirred at room temperature for 2 h. Subsequently, the mixture was poured into ice water (50mL) and extracted with EtOAc (3X 50 mL). The combined organic layers were washed with water (2X 50mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product, which was purified by flash chromatography using 5% EtOAc in hexanesThe crude product was purified as eluent to give the title compound (700mg, 22%);1H NMR(400MHz，DMSO-d6)：δ11.71(bs，1H)，8.14(d，J＝10.4Hz，1H)，7.44(d，J＝5.6Hz，1H)；LC-MS：m/z 224.0(M-1)-。
step-b: synthesis of 2-amino-4-fluoro-5- (trifluoromethyl) phenol
The title compound was synthesized using the same procedure as compound 1e, using 4-fluoro-2-nitro-5- (trifluoromethyl) phenol as the starting material (yield: 82%);1H NMR(400MHz，DMSO-d6)：δ9.54(s，1H)，6.78(d，J＝7.2Hz，1H)，6.48(d，J＝13.2Hz，1H)，5.5(bs，2H)。
step-c: synthesis of 5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32 step-c, using 2-amino-4-fluoro-5- (trifluoromethyl) phenol as the starting material (yield: 82%);1H NMR(400MHz,DMSO-d6):δ14.4(bs,1H),8.10(d,J＝5.6Hz,1H),7.43(d,J＝9.6Hz,1H)；LC-MS:m/z 236.0(M-1)-。
step-d: synthesis of 2-chloro-5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazole
Using the same procedure as example 32 step-d, using 5-fluoro-6- (trifluoromethyl) benzo [ d]The title compound was synthesized with oxazole-2-thiol as the starting material and stirring for 2h (yield: 99%);1H NMR(400MHz,DMSO-d6):δ7.74(d,J＝6.0Hz,1H),7.30(d,J＝10.0Hz,1H)。
step-e: synthesis of ethyl 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-5-fluoro-6- (trifluoromethyl) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 52%);1H NMR(400MHz,DMSO-d6):δ12.43(bs,1H),8.22(d,J＝1.6Hz,1H),7.88(dd,J＝1.6Hz,J＝8.4Hz,1H),7.83(d,J＝6.0Hz,1H),7.75(d,J＝8.4Hz,1H),7.41(d,J＝11.2Hz,1H),4.33(q,J＝7.2Hz,2H),3.66(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 423.0(M+1)+。
step-f: synthesis of 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 86%);1H NMR(400MHz,DMSO-d6):δ12.4(bs,2H),8.20(s,1H),7.89(d,J＝8.4Hz,1H),7.84(d,J＝6.4Hz,1H),7.55(d,J＝8.8Hz,1H),7.44(d,J＝11.2Hz,1H),3.67(s,3H)；LC-MS:m/z 395.1(M+1)+。
step-g: synthesis of 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials (yield: 72%);1HNMR(400MHz,DMSO-d6):δ12.35(bs,1H),8.40(bs,1H),8.09(s,1H),7.84-7.79(m,2H),7.54(d,J＝8.4Hz,1H),7.44(d,J＝11.6Hz,1H),4.50(bs,1H),3.67(s,3H),3.54(t,J＝6.4Hz,2H),3.38-3.33(m,2H)；LC-MS:m/z 438.0(M+1)+。
EXAMPLE 109 Synthesis of N- (2-methoxyethyl) -1-methyl-2- (oxazolo [5,4-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 10% Pd/C, MeOH, H2RT, 16 h; b) thiophosgene, THF, RT, 16 h; c) SOCl2Catalytic DMF, refluxing for 2 h; d) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; f) 2-methoxyethyl-1-amine, DPPA, DIPEA, DMF, at 0 ℃ -RT，16h
Step-a: synthesis of 3-aminopyridin-2-ol
The title compound was synthesized using the same procedure as compound 1e, using 3-nitropyridin-2-ol as the starting material (yield: 85%);1H NMR(400MHz，DMSO-d6)：δ11.3(bs，1H)，6.59(dd，J＝2.0Hz，J＝6.8Hz，1H)，6.43(dd，J＝2.4Hz，J＝6.8Hz，1H)，5.98(t，J＝6.0Hz，1H)，4.97(bs，2H)；LC-MS：m/z 111.25(M+1)+。
step-b: synthesis of oxazolo [5,4-b ] pyridine-2-thiol
To a solution of 3-aminopyridin-2-ol (500mg, 4.5mmol) in THF (15mL) at room temperature was added thiophosgene (0.41mL, 5.4mmol) slowly over a 15min period and stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous ammonium chloride (9mL) and concentrated. The aqueous layer was basified with 10N sodium hydroxide solution (5mL) and extracted with ethyl acetate (2X 30mL), the aqueous layer was acidified with 1N HCl and extracted with ethyl acetate (2X 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography using 2% methanol/DCM as eluent to give the title compound (200mg, 29%); LC-MS: m/z 153.15(M +1) +。
Step-c: synthesis of 2-chlorooxazolo [5, 4-b ] pyridine
To oxazolo [5, 4-b ] at room temperature]To a solution of pyridine-2-thiol (200mg, 1.31mmol) in thionyl chloride (5mL) was added N, N-dimethylformamide (1 drop) and the reaction mixture was refluxed for 2 h. The reaction mixture was concentrated under reduced pressure and the crude compound (220mg) was used in the next step without any further purification;1H NMR(400MHz，DMSO-d6)：δ8.40(dd，J＝1.6Hz，J＝4.8Hz，1H)，8.26(dd，J＝1.2Hz，J＝7.6Hz，1H)，7.56-7.53(m，1H)；LC-MS：m/z 155.1(M+1)+。
step-d: synthesis of ethyl 1-methyl-2- (oxazolo [5, 4-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-Ethyl formate and 2-chlorooxazolo [5, 4-b ]]Pyridine was used as a starting material to synthesize the title compound (yield: 20%);1H NMR(400MHz，DMSO-d6)：δ8.24(s，1H)，8.02(d，J＝5.2Hz，1H)，7.89(dd，J＝1.6Hz，J＝8.8Hz，1H)，7.79(d，J＝7.2Hz，1H)，7.56(d，J＝8.4Hz，1H)，7.29-7.26(m，1H)，4.33(q，J＝7.2Hz，2H)，3.67(s，3H)，1.35(t，J＝6.8Hz，3H)；LC-MS：m/z 338.15(M+1)+。
step-e: synthesis of 1-methyl-2- (oxazolo [5, 4-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, 1-methyl-2- (oxazolo [5, 4-b)]Pyridin-2-ylamino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 54%);1H NMR(400MHz，DMSO-d6)：δ12.8(bs，1H)，12.4(bs，1H)，8.20(s，1H)，8.01(d，J＝5.2Hz，1H)，7.89(d，J＝8.0Hz，1H)，7.79(d，J＝7.2Hz，1H)，7.54(d，J＝8.0Hz，1H)，7.28-7.25(m，1H)，3.67(s，3H)；LC-MS：m/z310.1(M+1)+。
step-f: synthesis of N- (2-methoxyethyl) -1-methyl-2- (oxazolo [5, 4-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-methyl-2- (oxazolo [5, 4-b)]Pyridin-2-ylamino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 38%);1H NMR (400MHz，DMSO-d6)：δ12.38(bs，1H)，8.47(s，1H)，8.09(s，1H)，8.0(d，J＝3.6Hz，1H)，7.79-7.77(m，2H)，7.52(d，J＝8.4Hz，1H)，7.27-7.24(m，1H)，3.66(s，3H)，3.48-3.44(m，4H)，3.28(s，3H)；LC-MS：m/z 367.2(M+1)+。
EXAMPLE 110 Synthesis of N- (2-hydroxyethyl) -1-methyl-2- (oxazolo [5, 4-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Using the same procedure as in example 6 step-e, using 1-methyl-2- (oxazolo [5, 4-b)]Pyridin-2-ylamino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials (yield: 51%);1H NMR(400MHz，DMSO-d6)：δ12.3(bs，1H)，8.38(bs，1H)，8.09(s，1H)，8.01(d，J＝4.4Hz，1H)，7.80-7.76(m，2H)，7.52(d，J＝8.4Hz，1H)，7.25(t，J＝6.8Hz，1H)，4.72(bs，1H)，3.66(s，3H)，3.53(bs，2H)，3.35(q，J＝6.0Hz，2H)；LC-MS：m/z 353.0(M+1)+。
examples 111 and 112.1 Synthesis of 1-methyl-2- (oxazolo [4, 5-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylic acid and N- (2-methoxyethyl) -1-methyl-2- (oxazolo [4, 5-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) CS2EtOH, reflux, 12 h; b) SOCl2Catalytic DMF, refluxing for 2 h; c) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; d) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; e) 2-methoxyethyl-1-amine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of oxazolo [4, 5-b ] pyridine-2-thiol
To a solution of 2-aminopyridin-3-ol (2.2g, 20mmol) in ethanol (40mL) at room temperature was added potassium hydroxide (1.68g, 30mmol) and carbon disulfide (15 mL). The reaction mixture was refluxed for 12h, then cooled to room temperature, diluted with water (75mL) and neutralized with glacial acetic acid. The precipitated solid was filtered and dried in vacuo to give the title compound (1.8g, 59%);1H NMR(400MHz，DMSO-d6)：δ14.5(s，1H)，8.23(dd，J＝1.2Hz，J＝5.2Hz，1H)，7.88(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.29-7.26(m，1H)；LC-MS：m/z 153.1(M+1)+。
step-b: synthesis of 2-chlorooxazolo [4, 5-b ] pyridine
Using the same procedure as in example 32 step-d, using oxazolo [4, 5-b ]]Pyridine-2-thiol was used as starting material and heated for 2h to synthesize the title compound. (yield: 100%);1H NMR(400MHz，DMSO-d6)：δ8.01(dd，J＝1.6Hz，J＝5.6Hz，1H)，7.61(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.10-7.06(m，1H)；LC-MS：m/z 155.01(M+1)+。
step-c: synthesis of ethyl 1-methyl-2- (oxazolo [4, 5-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chlorooxazolo [4, 5-b ]]Pyridine was used as a starting material to synthesize the title compound (yield: 32%);1H NMR(400MHz，DMSO-d6)：δ7.98(s，1H)，7.94(d，J＝4.4Hz，1H)，7.62(d，J＝9.2Hz，1H)，7.36(d，J＝7.2Hz，1H)，7.21-7.18(m，1H)，6.77-6.74(m，1H)，4.32-4.26(m，2H)，3.57(s，3H)，1.38(t，J＝6.8Hz，3H)；LC-MS：m/z 338.15(M+1)+。
step-d: synthesis of 1-methyl-2- (oxazolo [4, 5-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, 1-methyl-2- (oxazolo [4, 5-b) ]Pyridin-2-ylamino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 73%);1H NMR(400MHz，DMSO-d6)：δ12.83(bs，1H)，12.44(bs，1H)，8.26-8.23(m，2H)，7.89(d，J＝8.4Hz，1H)，7.78(d，J＝7.6Hz，1H)，7.75(d，J＝8.4Hz，1H)，7.12-7.09(m，1H)，3.72(s，3H)；LC-MS：m/z 310.15(M+1)+。
step-e: synthesis of N- (2-methoxyethyl) -1-methyl-2- (oxazolo [4, 5-b ] pyridin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound 1i, 1-methyl-2- (oxazolo)[4，5-b]Pyridin-2-ylamino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 53%);1H NMR (400MHz，DMSO-d6)：δ12.4(bs，1H)，8.48(bs，1H)，8.23(d，J＝4.4Hz，1H)，8.13(s，1H)，7.79-7.75(s，2H)，7.53(d，J＝8.4Hz，1H)，7.11-7.08(m，1H)，3.67(s，3H)，3.48-3.44(m，4H)，3.29(s，3H)；LC-MS：m/z 367.2(M+1)+。
EXAMPLE 113 Synthesis of N- (2-hydroxyethyl) -1-methyl-2- ((5- (trifluoromethyl) oxazolo [5, 4-b ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) (E) -5, 5, 5-trifluoro-4-oxopent-2-enoic acid ethyl ester, NaOEt, EtOH, 90 ℃, 2 h; b) 10% Pd/C, MeOH, H2, RT, 16H; c) thiophosgene, THF, RT, 16 h; d) SOCl2Catalytic DMF, refluxing for 3 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) H2O, THF, EtOH, H2O, 60 ℃ for 16H; g) 2-aminoethan-1-ol, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Step-a: synthesis of 3-nitro-6- (trifluoromethyl) pyridin-2-ol
To a solution of 2-nitroacetamide (5.0g, 47.8mmol) in ethanol (25mL) at room temperature was added (E) -ethyl 5, 5, 5-trifluoro-4-oxopent-2-enoate (11.24g, 57.3mmol) and ethanol (25.9mL, 95.3mmol) containing 25% sodium ethoxide. The reaction mixture was stirred at 90 ℃ for 2 h. The reaction mixture was cooled to room temperature, diluted with water (50mL), acidified with 1N HCl and extracted with EtOAc (2X 150 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue which was purified by flash chromatography using 50% EtOAc/hexanes as the eluent to give the title compound (1.5 g); 1H NMR(400MHz，DMSO-d6)：δ8.58(d，J＝8.4Hz，1H)，7.84(bs，1H)，7.51(d，J＝7.6Hz，1H)。
Step-b: synthesis of 3-amino-6- (trifluoromethyl) pyridin-2-ol
The title compound was synthesized using the same procedure as compound 1e, using 3-nitro-6- (trifluoromethyl) pyridin-2-ol as the starting material (yield: 82%);1H NMR(400MHz,DMSO-d6):δ11.7(bs,1H),6.88(bs,1H),6.69(bs,1H),5.60(s,2H)；LC-MS:m/z 179.1(M+1)+。
step-c: synthesis of 5- (trifluoromethyl) oxazolo [5,4-b ] pyridine-2-thiol
The title compound was synthesized using the same procedure as in example 109, step-b, using 3-amino-6- (trifluoromethyl) pyridin-2-ol as the starting material (yield: 65%);1H NMR(400MHz,DMSO-d6):δ7.81-7.75(m,2H)；LC-MS:m/z219.0(M-1)。
step-d: synthesis of 2-chloro-5- (trifluoromethyl) oxazolo [5,4-b ] pyridine
The title compound was synthesized using the same procedure as in example 32 step-d, using 5- (trifluoromethyl) oxazolo [5,4-b ] pyridine-2-thiol as the starting material (yield: 74%). The crude compound was used in the next step without further purification.
Step-e: synthesis of ethyl 1-methyl-2- ((5- (trifluoromethyl) oxazolo [5,4-b ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-5- (trifluoromethyl) oxazolo [5,4-b]Pyridine was used as a starting material to synthesize the title compound (yield: 43%);1H NMR(400MHz,DMSO-d6):δ12.5(bs,1H),8.27(s,1H),7.93-7.89(m,2H),7.75(d,J＝12.4Hz,1H),7.62(d,J＝8.4Hz,1H),4.34(q,J＝6.8Hz,2H),3.71(s,3H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 406.1(M+1)+。
step-f: synthesis of 1-methyl-2- ((5- (trifluoromethyl) oxazolo [5,4-b ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 1-methyl-2- ((5- (trifluoromethyl) oxazolo [5, 4-b)]Pyridin-2-yl) amino) -1H-benzo [ d]Preparation of imidazole-5-carboxylic acid ethyl esterThe title compound was synthesized for the starting material (yield: 75%);1H NMR(400MHz,DMSO-d6):δ8.20(bs,1H),7.74-7.70(m,2H),7.55(d,J＝6.8Hz,1H),7.09(d,J＝8.4Hz,1H),3.62(s,3H)；LC-MS:m/z 378.1(M+1)+。
step-g: synthesis of N- (2-hydroxyethyl) -1-methyl-2- ((5- (trifluoromethyl) oxazolo [5,4-b ] pyridin-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as in example 6 step-e, using 1-methyl-2- ((5- (trifluoromethyl) oxazolo [5, 4-b)]Pyridin-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-aminoethan-1-ol as starting materials (yield: 28%);1HNMR(400MHz,DMSO-d6):δ8.22(bs,1H),7.78(bs,1H),7.60(d,J＝8.0Hz,1H),7.51(bs,1H),7.31(d,J＝8.4Hz,2H),4.70(t,J＝5.2Hz,1H),3.67(s,3H),3.50(q,J＝5.2Hz,2H),3.36(q,J＝6.0Hz,2H)；LC-MS:m/z 421.1(M+1)+。
EXAMPLE 114 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- (thiazolo [4, 5-b ] pyrazin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) o-ethyl isothiocyanate carbonate, 1, 4-dioxane, RT for 16 h; b)6M HCl, 1, 4-dioxane, 50 ℃ for 4 h; c) NaOH, H2O, 120 ℃, 16 h; d) ethyl 3-amino-4- (methylamino) benzoate, 1, 1' -thiocarbonyldiimidazole, EDC, DMF, 100 ℃ for 18 h; e) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; f)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: 114a Synthesis
To a stirred solution of pyrazin-2-amine (2.0mg, 21.0mmol) in 1, 4-dioxane (20mL) at room temperature was added O-ethyl isothiocyanate carbonate (3.03g, 23.1mmol) and stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo. Ethyl acetate was added to the residue, and the precipitate was filteredThe solid was removed and dried in vacuo to afford the title compound (2.0g, 42%);1H NMR(400MHz，DMSO-d6)：δ12.07(s，1H)，11.76(s，1H)，9.67(s，1H)，8.50(s，2H)，4.24(q，J＝7.2Hz，2H)，1.27(t，J＝7.2Hz，3H)；LC-MS：m/z 227.0(M+1)+。
step-b: synthesis of Thiazolo [4, 5-b ] pyrazin-2-yl Ethylcarbamate
To a stirred solution of 114a (1.8g, 7.96mmol) in 1, 4-dioxane (9mL) was added 6M HCl (18mL) and stirred at 50 ℃ for 4 h. The reaction mixture was cooled to room temperature and the resulting solid was filtered and dried in vacuo to give the title compound (1.5g, 84%);1H NMR(400MHz，DMSO-d6)：δ12.60(bs，1H)，8.58(d，J＝3.2Hz，1H)，8.44(d，J＝2.4Hz，1H)，4.29(q，J＝6.8Hz，2H)，1.30(t，J＝7.6Hz，3H)；LC-MS：m/z 225.0(M+1)+。
step-c: synthesis of thiazolo [4, 5-b ] pyrazin-2-amine
To thiazolo [4, 5-b ]]To a solution of pyrazin-2-ylcarbamate (500mg, 2.23mmol) in water (5mL) was added sodium hydroxide (267mg, 6.69mmol) and heated at 120 ℃ for 16 h. The reaction mixture was cooled to room temperature and acidified with 1n hcl. The resulting solid was filtered and dried in vacuo to give the title compound (300mg, 88%);1H NMR(400MHz，DMSO-d6)：δ8.42(s，2H)，8.23(d，J＝2.8Hz，1H)，8.03(d，J＝2.8Hz，1H)；LC-MS：m/z 153.1(M+1)+。
step-d: synthesis of ethyl 1-methyl-2- (thiazolo [4, 5-b ] pyrazin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for Compound 2c, using ethyl 3-amino-4- (methylamino) benzoate and thiazolo [4, 5-b ]]The title compound was synthesized from pyrazin-2-amine as a starting material (yield: 9%);1H NMR(400MHz，DMSO-d6)：δ12.63(bs，1H)，8.44(d，J＝2.4Hz，1H)，8.33(s，1H)，8.23(d，J＝2.8Hz，1H)，7.91(dd，J＝1.2Hz，J＝8.4Hz，1H)，7.60(d，J＝8.4Hz，1H)，4.33(q，J＝7.2Hz，2H)，3.67(s，3H)，1.35(d，J＝7.2Hz，3H)；LC-MS：m/z 355.0(M+1)+。
step-e: synthesis of 1-methyl-2- (thiazolo [4, 5-b ] pyrazin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxylic acid
To 1-methyl-2- (thiazolo [4, 5-b ]]Pyrazin-2-ylamino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (60mg, 0.17mmol) in a solvent mixture of THF (1mL), ethanol (1mL) and water (0.5mL) was added lithium hydroxide monohydrate (35mg, 0.85 mmol). The reaction mixture was heated with stirring at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude material (40mg) was used directly in the next step; LC-MS: m/z 326.95(M +1)+。
Step-f: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- (thiazolo [4, 5-b ] pyrazin-2-ylamino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6, step-e, 1-methyl-2- (thiazolo [4, 5-b)]Pyrazin-2-ylamino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 59%);1H NMR(400MHz，DMSO-d6)：δ12.60(bs，1H)，8.46-8.41(m，2H)，8.20-8.14(m，2H)，7.78(s，1H)，7.56(s，1H)，4.60(t，J＝4.8Hz，1H)，3.70(s，3H)，3.59-3.43(m，8H)；LC-MS：m/z414.05(M+1)+。
examples 115 and 116.2- ((6-Cyclopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid Synthesis and 2- ((6-Cyclopropylbenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide Synthesis
Conditions are as follows: a) cyclopropylboronic acid, Pd (OAc)2N, N is tricyclohexylphosphine, K3PO4Toluene, H2O，100℃，16h；b)PtO2，EtOH，THF，H2RT, 4 h; c) potassium ethyl xanthate, EtOH, refluxing for 16 h; d) SOCl2Catalytic DMF, refluxing for 3 h; e) the reaction solution of NaH and NaH is added,2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; g) 2-methoxyethyl-1-amine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of 5-cyclopropyl-2-nitrophenol
To a stirred solution of 5-bromo-2-nitrophenol (2.0g, 9.17mmol) and cyclopropylboronic acid (1.02g, 11.92mmol) in toluene (40mL) was added potassium phosphate (6.81g, 32.1mmol), tricyclohexylphosphine (0.25g, 0.91mmol) and water (2 mL). The reaction mixture was purged with nitrogen for 10min, and then palladium diacetate (0.1g, 0.45mmol) was added to the reaction mixture and heated at 100 ℃ for 16 h. The reaction mixture was cooled to room temperature, filtered through a celite bed and washed with ethyl acetate (150 mL). The organic layer was washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography using 100% hexane as eluent to give the title compound (0.9g, 55%); 1H NMR(400MHz,DMSO-d6):δ10.72(bs,1H),7.82(d,J＝8.8Hz,1H),6.82(d,J＝2.0Hz,1H),6.68(dd,J＝2.0Hz,J＝8.8Hz,1H),1.99-1.94(m,1H),1.08-1.03(m,2H),0.78-0.74(m,2H)。
Step-b: synthesis of 2-amino-5-cyclopropylphenol
To a solution of 5-cyclopropyl-2-nitrophenol (1.75g, 11.4mmol) in ethanol (4mL) and THF (4mL) was added PtO2(12 mg). The reaction mixture was stirred under a balloon of hydrogen for 4 h. It was filtered through celite bed and concentrated in vacuo to give the title compound (250mg, 100%);1H NMR(400MHz,DMSO-d6):δ8.99(bs,1H),6.61-6.59(m,1H),6.45(d,J＝2.0Hz,1H),6.43-6.34(m,1H),5.35(bs,2H),1.74-1.65(m,1H),0.80-0.75(m,2H),0.48-0.44(m,2H)。
step-c: synthesis of 6-cyclopropylbenzo [ d ] oxazole-2-thiol
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5-cyclopropylphenol as the starting material (yield: 90%);1H NMR(400MHz,DMSO-d6):δ13.72(bs,1H),7.22(s,1H),7.11-7.04(m,2H),2.02-1.98(m,1H),0.98-0.94(m,2H),0.71-0.67(m,2H)；LC-MS:m/z 190.05(M-1)。
step-d: synthesis of 2-chloro-6-cyclopropylbenzo [ d ] oxazole
Using the same procedure as in example 32, step-d, 6-cyclopropylbenzo [ d ] was used]The title compound was synthesized from oxazole-2-thiol as a starting material (yield: 98%);1H NMR(400MHz,DMSO-d6):δ7.59(d,J＝8.0Hz,1H),7.46(s,1H),7.18(d,J＝8.4Hz,1H),2.09-2.01(m,1H),1.02-0.99(m,2H),0.75-0.74(m,2H)；LC-MS:m/z194.0(M+1)+。
step-e: synthesis of ethyl 2- ((6-cyclopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6-cyclopropylbenzo [ d ]]Oxazole as a starting material the title compound was synthesized (yield: 27%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.22(s,1H),7.87(d,J＝8.4Hz,1H),7.50(d,J＝8.4Hz,1H),7.33(d,J＝8.4Hz，1H)，7.15(s，1H)，6.97(d，J＝6.8Hz，1H)，4.35-4.27(m，2H)，3.62(s，3H)，2.02-1.98(m，1H)，1.36-1.30(m，3H)，0.97-0.92(m，2H)，0.71-0.67(m，2H)；LC-MS：m/z 377.2(M+1)+。
step-f: synthesis of 2- ((6-cyclopropylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, using 2- ((6-cyclopropylbenzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 81%);1H NMR(400MHz，DMSO-d6)：δ13.5-11.2(bs，2H)，8.18(s，1H)，7.91(d，J＝8.4Hz，1H)，7.58(d，J＝8.4Hz，1H)，7.34(d，J＝8.4Hz，1H)，7.22(s，1H)，7.04(d，J＝8.4Hz，1H)，3.67(s，3H)，2.06-2.02(m，1H)，1.00-0.95(m，2H)，0.72-0.68(m，2H)；LC-MS：m/z 349.0(M+1)+。
step-g: synthesis of 2- ((6-cyclopropylbenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as for Compound li, 2- ((6-cyclopropylbenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2-methoxyethylamine as starting materials (yield: 46%);1H NMR(400MHz，DMSO-d6)：δ12.2(bs，1H)，8.44(t，J＝5.2Hz，1H)，8.06(d，J＝1.6Hz，1H)，7.75(d，J＝8.4Hz，1H)，7.46(d，J＝8.4Hz，1H)，7.33(d，J＝8.0Hz，1H)，7.14(s，1H)，6.96(dd，J＝1.6Hz，J＝8.4Hz，1H)，3.62(s，3H)，3.48-3.43(m，4H)，3.28(s，3H)，2.02-1.98(m，1H)，0.96-0.92(m，2H)，0.70-0.66(m，2H)；LC-MS：m/z 406.2(M+1)+。
EXAMPLE 117 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((7- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaNO3，NaNO2，H2SO4，DCM，RT，16h；b)10％Pd/C，MeOH，H2RT, 4 h; c) potassium ethyl xanthate, EtOH, refluxing for 16 h; d) SOCl2Catalytic DMF, refluxing for 2 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; g)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 2-nitro-6- (trifluoromethyl) phenol
To 3M sulfuric acid (25mL) stirring at room temperature was added sodium nitrate (1.73g, 20.37mmol) and sodium nitrite (100mg), followed by a solution of 2- (trifluoromethyl) phenol (3.0g, 18.52mmol) in DCM (40mL) and stirring for 16 h. The mixture was poured into ice water (50mL) and extracted with DCM (2X 100 mL). The combined organic layers were washed with water (50mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. By flash chromatography using 5% acetic acid The residue was purified with ethyl/hexane as eluent to give the title compound (1.3g, 40%);1H NMR(400MHz,DMSO-d6):δ11.34(bs,1H),8.27(dd,J＝1.6Hz,J＝8.4Hz,1H),8.00(dd,J＝1.2Hz,J＝7.6Hz,1H),7.21-7.17(m,1H)。
step-b: synthesis of 2-amino-6- (trifluoromethyl) phenol
The title compound was synthesized using the same procedure as compound 1e, using 2-nitro-6- (trifluoromethyl) phenol as starting material and stirring for 4h (yield: 90%);1H NMR(400MHz,DMSO-d6):δ6.88-6.84(m,1H),6.75-6.70(m,2H),6.50(bs,2H)；LC-MS:m/z 178.05(M+1)+。
step-c: synthesis of 7- (trifluoromethyl) benzo [ d ] oxazole-2 (3H) -thione
The title compound was synthesized using the same procedure as in example 32 step-c, using 2-amino-6- (trifluoromethyl) phenol as the starting material (yield: 65%);1H NMR(400MHz,DMSO-d6):δ14.28(bs,1H),7.59-7.53(m,2H),7.49-7.45(m,1H)；LC-MS:m/z 218.1(M-1)-。
step-d: synthesis of 2-chloro-7- (trifluoromethyl) benzo [ d ] oxazole
Using the same procedure as in example 32 step-d, using 7- (trifluoromethyl) benzo [ d ]]Oxazole-2 (3H) -thione as a starting material and stirred for 2H to synthesize the title compound (yield: 84%);1H NMR(400MHz,DMSO-d6):δ8.11(d,J＝8.0Hz,1H),7.84(d,J＝7.6Hz,1H),7.64(t,J＝7.2Hz,1H)。
step-e: synthesis of ethyl 1-methyl-2- ((7- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-7- (trifluoromethyl) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 36%);1H NMR(400MHz,DMSO-d6):δ12.41(s,1H),8.26(d,J＝1.6Hz,1H),7.90(dd,J＝1.6Hz,J＝8.4Hz,1H),7.75(d,J＝6.4Hz,1H),7.57(d,J＝8.4Hz,1H),7.44-7.38(m,2H),4.33(q,J＝6.8Hz,2H),3.67(s,3H),1.35(d,J＝6.8Hz,3H)；LC-MS:m/z 405.0(M+1)+。
step-f: synthesis of 1-methyl-2- ((7- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, 1-methyl-2- ((7- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 75%);1H NMR(400MHz,DMSO-d6)：δ12.83(bs，1H)，12.40(bs，1H)，8.22(d，J＝1.6Hz，1H)，7.89(dd，J＝1.2Hz，J＝8.4Hz，1H)，7.75(d，J＝8.4Hz，1H)，7.55(d，J＝8.0Hz，1H)，7.43-7.37(m，2H)，3.67(s，3H)；LC-MS：m/z 377.0(M+1)+。
step-g: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((7- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 1-methyl-2- ((7- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 61%);1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.46(t，J＝5.2Hz，1H)，8.10(d，J＝1.2Hz，1H)，7.80-7.73(m，2H)，7.53(d，J＝8.4Hz，1H)，7.42-7.38(m，2H)，4.60(bs，1H)，3.67(s，3H)，3.58-3.43(m，8H)；LC-MS：m/z 464.1(M+1)+。
EXAMPLE 118 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) benzo [ d ] oxazole-5-carboxamide
Conditions are as follows: a) methylamine water solution, DMF, 60 ℃, 16 h; b) 10% Pd/C, MeOH, H2RT, 16 h; c) cyanogen bromide, THF, H2O, 50 ℃, 16 h; d) NaH, 2-chlorobenzo [ d]Oxazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; e) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; f)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of N-methyl-2-nitroaniline
To a solution of 1-fluoro-2-nitrobenzene (1.0g, 7.08mmol) in DMFA (3mL) at room temperature was added methylamine (1mL, 40% aqueous solution, 35.4mmol) and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, diluted with cold water (50mL) and stirred for 2 h. The resulting solid was filtered and dried in vacuo to afford the product as a yellow solid (700mg, 75%);1H NMR(400MHz，DMSO-d6)：δ8.14(bs，1H)，8.06(dd，J＝1.2Hz，J＝8.8Hz，1H)，7.57-7.53(m，1H)，6.98(d，J＝8.8Hz，1H)，6.69-6.66(m，1H)，2.96(d，J＝4.8Hz，3H)；LC-MS：m/z 153.0(M+1)+。
step-b: n is a radical of1Synthesis of (E) -methylbenzene-1, 2-diamine
The title compound was synthesized using the same procedure as in compound 1e, using N-methyl-2-nitroaniline as the starting material (yield: 98%);1H NMR(400MHz,DMSO-d6):δ6.52-6.48(m,2H),6.41-6.34(m,2H),4.45(bs,2H),2.68(s,3H)；LC-MS:m/z 123.2(M+1)+。
step-c: synthesis of 1-methyl-1H-benzo [ d ] imidazol-2-amine
Using the same procedure as for Compound 1f, using N1-methylbenzene-1, 2-diamine as starting material the title compound was synthesized (yield: 83%);1H NMR(400MHz,DMSO-d6):δ7.11-7.09(m,2H),6.94-6.87(m,2H),6.37(s,2H),3.78(s,3H)。
step-d: synthesis of ethyl 2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) benzo [ d ] oxazole-5-carboxylate
Using the same procedure as in example 32, step-e, 1-methyl-1H-benzo [ d ] was used]Imidazol-2-amine and 2-chlorobenzo [ d]Oxazole-5-carboxylic acid ethyl ester was used as a starting material to synthesize the title compound (yield: 31%);1H NMR(400MHz,DMSO-d6):δ12.20(bs,1H),7.99(d,J＝1.6Hz,1H),7.76-7.74(m,1H),7.64-7.62(m,1H),7.52(d,J＝8.0Hz,1H),7.45-7.43(m,1H),7.26-7.23(m,2H),4.32(q,J＝7.6Hz,2H),3.63(s,3H),1.35(t,J＝6.8Hz,3H)；LC-MS:m/z 336.95(M+1)+。
step-e: synthesis of 2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) benzo [ d ] oxazole-5-carboxylic acid
Using the same procedure as for Compound 1H, using 2- ((1-methyl-1H-benzo [ d)]Imidazol-2-yl) amino) benzo [ d]Oxazole-5-carboxylic acid ethyl ester was used as a starting material to synthesize the title compound (yield: 78%);1H NMR(400MHz,DMSO-d6):δ12.80(bs,1H),12.22(bs,1H),7.98(d,J＝1.6Hz,1H),7.76(dd,J＝2.0Hz,J＝8.4Hz,1H),7.67-7.63(m,1H),7.52(d,J＝8.4Hz,1H),7.47-7.45(m,1H),7.28-7.21(m,2H),3.68(s,3H)；LC-MS:m/z309.1(M+1)+。
step-f: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) benzo [ d ] oxazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((1-methyl-1H-benzo [ d)]Imidazol-2-yl) amino) benzo [ d]Oxazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol were used as starting materials to synthesize the title compound. The crude product was purified by flash chromatography using 9% methanol/DCM as eluent (yield: 64%);1H NMR(400MHz,DMSO-d6):δ12.30(bs,1H),8.48(t,J＝5.2Hz,1H),7.92(s,1H),7.65-7.62(m,2H),7.48-7.45(m,2H),7.28-7.22(m,2H),3.63(s,3H),3.57-3.35(m,8H)；LC-MS:m/z 396.2(M+1)+。
EXAMPLE 119 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-methoxyethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 1-bromo-2-methoxyethane, TEA, DMF, 60 ℃, 16 h; b) hydrazine hydrate, MeOH, reflux, 4 h; c) o- (2-methoxyethyl) hydroxylamine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of 2- (2-methoxyethoxy) isoindoline-1, 3-dione
To N-hydroxyphthalic acid at room temperatureTo a stirred solution of dicarboximide (4.6g, 28.2mmol) in DMFA (15mL) was added triethylamine (8.0mL, 56.4mmol) and 1-bromo-2-methoxyethane (4.0mL, 42.3 mmol). The reaction mixture was subsequently stirred at 60 ℃ for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was cooled to room temperature, diluted with cold water (100mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with cold water (2 × 200mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (2.8g, 45%); 1H NMR(400MHz，DMSO-d6)：δ7.86(s，4H)，4.26(t，J＝4.4Hz，2H)，3.65-3.63(m，2H)，3.25(s，3H)；LC-S：m/z222.20(M+1)+。
Step-b: synthesis of O- (2-methoxyethyl) hydroxylamine
To a stirred solution of 2- (2-methoxyethoxy) isoindoline-1, 3-dione (2.8g, 12.6mmol) in methanol (75mL) at room temperature was added hydrazine hydrate (0.95g, 19.0mmol) and refluxed for 4 h. Subsequently, the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated. The resulting residue was stirred in diethyl ether (30mL) and filtered. The filtrate was concentrated in vacuo to give the title compound (550mg, 48%);1H NMR(400MHz，DMSO-d6)：δ5.50(s，2H)，3.84-3.82(m，2H)，3.58-3.56(m，2H)，3.39(s，3H)。
step-c: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-methoxyethoxy) -1-methyl-1H-benzoimidazole-5-carboxamide
Using the same procedure as for Compound 1i, 2- (benzo [ d ]]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid and O- (2-methoxyethyl) hydroxylamine as starting materials the title compound was synthesized (yield: 20%);1H NMR(400MHz，DMSO-d6)：δ12.28(s，1H)，11.73(s，1H)，8.01(s，1H)，7.64(d，J＝8.3Hz，1H)，7.48(d，J＝7.8Hz，2H)，7.44(d，J＝7.8Hz，1H)，7.22(t，J＝7.8Hz，1H)，7.12(t，J＝7.9Hz，1H)，4.04(t，J＝4.2Hz，2H)，3.63(s，3H)，3.59(t，J＝4.2Hz，2H)，3.31(s，3H)；LC-MS：m/z 382.0(M+1)+。
EXAMPLE 120 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-hydroxypropoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) refluxing 2-methyloxirane, DIPEA, tetrabutylammonium bromide and toluene for 5 h; b)6N Aq. HCl, RT, 16 h; c)1- (aminooxy) propan-2-ol hydrochloride, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 2- (2-hydroxypropoxy) isoindoline-1, 3-dione
To a stirred solution of 2-hydroxyisoindoline-1, 3-dione (5.0g, 30.65mmol) and 2-methyloxirane (4.1mL, 61.3mmol) in toluene (50mL) at room temperature were added DIPEA (0.6mL, 3.1mmol) and tetrabutylammonium bromide (0.99g, 3.1 mmol). Subsequently, the reaction mixture was refluxed for 5 h. The mixture was cooled to room temperature, concentrated under reduced pressure and extracted with EtOAc (2X 100mL) and water (50 mL). The combined organic layers were washed with water (30mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% dichloromethane as eluent to give the title compound (4.0g, 59%);1H NMR(400MHz，DMSO-d6)：δ7.85(s，4H)，4.80(d，J＝4.8Hz，1H)，4.03-4.00(m，1H)，3.96-3.93(m，2H)，1.14(d，J＝6.0Hz，3H)；LC-MS：m/z 222.1(M+1)+。
step-b: synthesis of 1- (aminoxy) propan-2-ol hydrochloride
A solution of 2- (2-hydroxypropoxy) isoindoline-1, 3-dione (2.0g, 9.0mmol) in 6N aqueous hydrochloric acid (15mL) was stirred at room temperature for 16 h. The precipitated solid in the reaction mixture was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound as a hydrochloride salt (1.0g), which was used in the next step without further purification.
Step-c: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2-hydroxypropoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To a stirred solution of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid (100mg, 0.32mmol) in DMFA (5mL) at 0 deg.C was added N-ethyldiisopropylamine (0.06mL, 0.32mmol) and HBTU (122mg, 0.32 mmol). The reaction mixture was stirred for 30min, then 1- (aminooxy) propan-2-ol hydrochloride (41mg, 0.32mmol) was added and stirring continued at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (20mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water (30mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by preparative HPLC to give the title compound (35mg, 28%).
Preparative HPLC purification method details:
diluting liquid: THF, ACN and water
Mobile phase A: 100% water
Mobile phase B: 100% acetonitrile
Gradient: 0/10,6/20, 10/80
Flow rate: 15ml/min
Column: luna (250x21.1x5 μm)
1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，11.7(bs，1H)，8.01(d，J＝1.2Hz，1H)，7.63(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.48(d，J＝8.8Hz，2H)，7.43(d，J＝7.6Hz，1H)，7.21(t，J＝7.6Hz，1H)，7.12(t，J＝7.6Hz，1H)，4.90(bs，1H)，3.92-3.89(m，1H)，3.78-3.74(m，2H)，3.63(s，3H)，1.10(d，J＝6.4Hz，3H)；LC-MS：m/z 382.05(M+1)+。
EXAMPLE 121 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2- (dimethylamino) acetamido) ethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) (2-hydroxyethyl) carbamic acid tert-butyl ester, DIAD, triphenylphosphine, THF, 0 deg.C-RT, 16 h; b) TFA, DCM, 0 deg.C-RT, 3 h; c) dimethylglycine, HATU, DIPEA, DMF, 0 ℃ -RT, 16 h; d) hydrazine hydrate and methanol are refluxed for 4 hours; e) n- (2- (aminooxy) ethyl) -2- (dimethylamino) acetamide, HBTU, DIPEA, DMF, 0 ℃ -RT for 16h
Step-a: synthesis of tert-butyl (2- ((1, 3-dioxoisoindolin-2-yl) oxy) ethyl) carbamate
To a stirred solution of 2-hydroxyisoindoline-1, 3-dione (5.0g, 30.67mmol) in THF (150mL) at 0 deg.C was added tert-butyl (2-hydroxyethyl) carbamate (4.93g, 30.67mmol) and triphenylphosphine (8.03g, 30.67 mmol). The reaction mixture was stirred for 10min, then diisopropyl azodicarboxylate (6.19g, 30.67mmol) was added and stirring at room temperature was continued for 16 h. The reaction mixture was diluted with water (50mL) and extracted with ethyl acetate (2X 100 mL). The combined organic layers were washed with water (50mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 10% ethyl acetate/hexane as eluent to give the title compound (12.0g, semi-pure) which was used in the next step without any further purification;1H NMR(400MHz,DMSO-d6):δ7.86(s,4H),6.82(bs,1H),4.13(t,J＝5.6Hz,2H),3.28-3.27(m,2H),1.37(s,9H)；LC-MS:m/z 207.0(M-Boc)+。
step-b: synthesis of 2- (2-aminoethoxy) isoindoline-1, 3-dione trifluoroacetate
To a stirred solution of tert-butyl (2- ((1, 3-dioxoisoindolin-2-yl) oxy) ethyl) carbamate (12.0g, 39.08mmol) in DCM (150mL) at 0 deg.C was added trifluoroacetic acid (20mL) and stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo, the residue was stirred in diethyl ether (50mL), and the precipitated solid was filtered and dried in vacuo to give the title compound (3.8g, 32%); 1H NMR(400MHz,DMSO-d6):δ8.05(bs,3H),7.90(s,4H),4.34(t,J＝5.2Hz,2H),3.38(q,J＝7.2Hz,2H)。
Step-c: synthesis of 2- (dimethylamino) -N- (2- ((1, 3-dioxoisoindolin-2-yl) oxy) ethyl) acetamide
To a stirred solution of 2- (2-aminoethoxy) isoindoline-1, 3-dione trifluoroacetate (1.0g, 4.85mmol) in DMFA (15mL) at 0 deg.C was added dimethyl glycerolAmino acid (499mg, 4.85mmol), N-ethyldiisopropylamine (1.69mL, 9.7mmol) and HATU (2.02g, 5.34mmol), and the reaction mixture was stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with water (50mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water (30mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 2% methanol/dichloromethane as eluent to give the title compound (280mg, 29%); LC-MS: m/z 292.1(M +1)+。
Step-d: synthesis of N- (2- (aminooxy) ethyl) -2- (dimethylamino) acetamide
To a stirred solution of 2- (dimethylamino) -N- (2- ((1, 3-dioxoisoindolin-2-yl) oxy) ethyl) acetamide (280mg, 0.96mmol) in methanol (5mL) at room temperature was added hydrazine hydrate (0.3mL) and refluxed for 4 h. The reaction mixture was cooled to room temperature and concentrated on a rotary evaporator. The resulting residue was stirred in diethyl ether (30mL) and filtered. The filtrate was concentrated in vacuo to give the title compound (120mg, 77%); 1H NMR(400MHz,CDCl3):δ7.39(bs,1H),5.50(bs,2H),3.73(t,J＝5.2Hz,2H),3.57-3.47(m,2H),2.96(s,2H),2.29(s,6H)。
Step-e: synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (2- (dimethylamino) acetamido) ethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To a stirred solution of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid (80mg, 0.26mmol) in DMFA (2mL) at 0 deg.C was added N-ethyldiisopropylamine (0.04mL, 0.26mmol) and HBTU (98mg, 0.26 mmol). The reaction mixture was stirred for 30min, then N- (2- (aminooxy) ethyl) -2- (dimethylamino) acetamide (41mg, 0.26mmol) was added. The reaction mixture was then stirred at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (15mL) and extracted with ethyl acetate (2 × 20 mL). The aqueous layer was concentrated in vacuo. The resulting residue was purified by preparative HPLC to give the title compound (10mg, 9%).
Preparative HPLC purification method details:
diluting liquid: THF, ACN and water
Mobile phase A: 10mM ammonium acetate in water
Mobile phase B: 100% acetonitrile
Gradient: t/% B: 0/10, 10/50, 17/65, 19/90
Flow rate: 15ml/min
Column: phenomenex Luna (250X21.2X5 μm)
1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，11.60(bs，1H)，8.01(bs，2H)，7.64(d，J＝8.8Hz，1H)，7.49-7.42(m，3H)，7.20(t，J＝8.0Hz，1H)，7.12(t，J＝8.0Hz，1H)，3.90(s，2H)，3.63(s，3H)，3.41(q，J＝6.0Hz，2H)，2.89(s，2H)，2.32(s，6H)；LC-MS：m/z 452.2(M+1)+。
EXAMPLE 122 Synthesis of N- (2- (dimethylamino) ethoxy) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) 1-chloro-2-dimethylaminoethane hydrochloride, TEA and DMF at 60 ℃ for 16 h; b) hydrazine hydrate, MeOH, reflux, 4 h; c)2- (aminooxy) -N, N-dimethylethyl-1-amine, HBTU, DIPEA, DMF, 0 deg.C-RT, 16h
Step-a: synthesis of 2- (2- (dimethylamino) ethoxy) isoindoline-1, 3-dione
The title compound was synthesized using the same procedure as in example 119 step-a, using N-hydroxyphthalimide and 1-chloro-2-dimethylaminoethane hydrochloride as starting materials and 4 equivalents of triethylamine (yield: 17%);1H NMR(400MHz，DMSO-d6)：δ7.86(s，4H)，4.21(t，J＝5.4Hz，2H)，2.61(t，J＝5.4Hz，2H)，2.18(s，6H)；LC-MS：m/z 235.1(M+1)+。
step-b: synthesis of 2- (aminoxy) -N, N-dimethylethyl-1-amine
Using the same procedure as in example 119, step-b, use2- (2- (dimethylamino) ethoxy) isoindoline-1, 3-dione as a starting material the title compound was synthesized (yield: 34%);1H NMR(400MHz，DMSO-d6)：δ5.88(bs，2H)，3.58(t，J＝5.9Hz，2H)，2.38(t，J＝5.9Hz，2H)，2.13(s，6H)；LC-MS：m/z 105.20(M+1)+。
step-c: synthesis of N- (2- (dimethylamino) ethoxy) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid and 2- (aminoxy) -N, N-dimethylethyl-1-amine as starting materials the title compound was synthesized (yield: 15%); 1H NMR(400MHz，DMSO-d6)：δ12.40(bs，2H)，8.01(s，1H)，7.82(s，1H)，7.66(d，J＝1.5Hz，1H)，7.64(d，J＝1.5Hz，1H)，7.61-7.51(m，2H)，3.99(t，J＝5.4Hz，2H)，3.66(s，3H)，2.55(t，J＝5.3Hz，2H)，2.22(s，6H)；LC-MS：m/z 463.50(M+1)+。
EXAMPLE 123 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -N- (2- (dimethylamino) ethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
The title compound was synthesized using the same procedure as example 6 step-e, using 2- (aminooxy) -N, N-dimethylethyl-1-amine and 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid as starting materials.
1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，8.00(s，1H)，7.63(d，J＝8.4Hz，1H)，7.48(d，J＝8.4Hz，2H)，7.43(d，J＝8.0Hz，1H)，7.21(t，J＝7.2Hz，1H)，7.12(t，J＝7.6Hz，1H)，3.99(t，J＝6.0Hz，2H)，3.82(s，3H)，2.56(t，J＝5.2Hz，2H)，2.32(s，6H)；LC-MS：m/z 395.1(M+1)+。
EXAMPLE 124 Synthesis of N- (2-methoxyethoxy) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) o- (2-methoxyethyl) hydroxylamine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Using the same procedure as for Compound 1i, 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid and O- (2-methoxyethyl) hydroxylamine as starting materials the title compound was synthesized (yield: 28%);1H NMR(400MHz，DMSO-d6): δ 12.39(bs, 1H), 11.72(s, 1H), 8.03(s, 1H), 7.83(s, 1H), 7.67(d, J ═ 8.3Hz, 1H), 7.61(d, J ═ 8.3Hz, 1H), 7.57-7.52(m, 2H), 4.04(t, J ═ 4.4Hz, 2H), 3.66(s, 3H), 3.59(t, J ═ 4.4Hz, 2H), 3.30(s, 3H, combined in DMSO); LC-MS: m/z 450.15(M +1)+。
EXAMPLE 125 Synthesis of N- (2-hydroxyethoxy) -1-methyl-2- ((6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (aminooxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (aminooxy) ethan-1-ol as starting materials (yield: 51%);1H NMR(400MHz，DMSO-d6)：δ12.39(bs，1H)，11.73(s，1H)，8.04(s，1H)，7.84(s，1H)，7.68(d，J＝8.3Hz，1H)，7.61(d，J＝8.3Hz，1H)，7.57-7.53(m，2H)，4.78(s，1H)，3.94(t，J＝4.9Hz，2H)，3.67(s，3H)，3.66-3.63(m，2H)；LC-MS：m/z 436.15(M+1)+。
EXAMPLE 126 Synthesis of N- (2-hydroxyethoxy) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (aminooxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (aminooxy) ethan-1-ol as starting materials (yield: 58%);1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，11.72(s，1H)，8.02(d，J＝1.0Hz，1H)，7.66(d，J＝6.9Hz，1H)，7.58(s，1H)，7.53-7.51(m，2H)，7.22(d，J＝8.3Hz，1H)，4.78(t，J＝5.6Hz，1H)，3.94(t，J＝4.7Hz，2H)，3.64-3.62(m，5H)；LC-MS：m/z 451.9(M+1)+。
EXAMPLE 127 Synthesis of N- (2-methoxyethoxy) -1-methyl-2- ((6- (trifluoromethoxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) o- (2-methoxyethyl) hydroxylamine, DPPA, DIPEA, DMF, 0 ℃ to RT, 16h
Using the same procedure as for Compound 1i, 1-methyl-2- ((6- (trifluoromethoxy) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d ]Imidazole-5-carboxylic acid and O- (2-methoxyethyl) hydroxylamine as starting materials the title compound was synthesized (yield: 28%);1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，11.70(s，1H)，8.01(d，J＝1.4Hz，1H)，7.65(dd，J＝1.5Hz，J＝8.3Hz，1H)，7.58(d，J＝1.5Hz，1H)，7.53-7.49(m，2H)，7.22(dd，J＝1.5Hz，J＝8.3Hz，1H)，4.05-4.02(m，2H)，3.64(s，3H)，3.61-3.58(m，2H)，3.31(s，3H)；LC-MS：m/z 466.0(M+1)+。
EXAMPLE 128 Synthesis of N- (2-hydroxyethoxy) -2- ((6- (2-methoxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- (aminooxy) ethan-1-ol and 2- ((6- (2-methoxyethoxy) benzo [ d ]]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid as starting material the title compound was synthesized.1H NMR(400MHz，DMSO-d6): δ 12.20(bs, 1H), 11.68(s, 1H), 7.98(s, 1H), 7.63(d, J ═ 8.0Hz, 1H), 7.46(d, J ═ 8.4Hz, 1H), 7.35(d, J ═ 8.8Hz, 1H), 7.12(s, 1H), 6.82(d, J ═ 8.8Hz, 1H), 4.78(s, 1H), 4.11(bs, 2H), 3.93(bs, 2H), 3.67-3.61(m, 7H), 3.32(s, 3H, combined with DMSO water peak); LC-MS: m/z442.2(M +1)+。
EXAMPLE 129 Synthesis of N- (2-hydroxyethoxy) -2- ((6- (2-hydroxyethoxy) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (aminooxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6 step-e, using 2- ((6- (2-hydroxyethoxy) benzo [ d) ]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (aminooxy) ethan-1-ol as starting materials. The crude product was purified by flash chromatography using 10% methanol/DCM as eluent to give the title compound (yield: 14%);1H NMR(400MHz，DMSO-d6)：δ12.15(bs，1H)，11.67(s，1H)，7.98(s，1H)，7.63(d，J＝7.6Hz，1H)，7.45(d，J＝8.8Hz，1H)，7.35(d，J＝8.4Hz，1H)，7.11(s，1H)，6.82(d，J＝8.8Hz，1H)，4.83(bs，1H)，4.77(bs，1H)，4.01(t，J＝4.4Hz，2H)，3.93(t，J＝4.4Hz，2H)，3.72(bs，2H)，3.61(bs，5H)；LC-MS：m/z 428.2(M+1)+。
EXAMPLE 130 Synthesis of 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d ] oxazol-2-yl) amino) -N- (2-hydroxyethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (aminooxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6 step-e, using 2- ((5-fluoro-6- (trifluoromethyl) benzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (aminooxy) ethan-1-ol as starting materials (yield: 61%);1H NMR(400MHz，DMSO-d6)：δ12.45(bs，1H)，11.74(s，1H)，8.03(s，1H)，7.85(d，J＝5.6Hz，1H)，7.68(d，J＝8.4Hz，1H)，7.55(d，J＝8.0Hz，1H)，7.45(d，J＝11.2Hz，1H)，4.50(bs，1H)，3.94(t，J＝4.4Hz，2H)，3.66(s，3H)，3.64-3.63(m，2H)；LC-MS：m/z 454.1(M+1)+。
EXAMPLE 131 Synthesis of N- (2-hydroxyethoxy) -1-methyl-2- ((6-methyl-4, 5, 6, 7-tetrahydrobenzo [ d ] thiazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a)2- (aminooxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Using the same procedure as example 6, step-e, 1-methyl-2- ((6-methyl-4, 5, 6, 7-tetrahydrobenzo [ d ]]Thiazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (aminooxy) ethan-1-ol as starting materials (yield: 43%); 1H NMR(400MHz，DMSO-d6): δ 12.05(bs, 1H), 11.56(s, 1H), 7.83(s, 1H), 7.55(dd, J ═ 1.0Hz, J ═ 8.3Hz, 1H), 7.35(d, J ═ 8.3Hz, 1H), 4.78(bs, 1H), 3.92(t, J ═ 4.9Hz, 2H), 3.63-3.61(m, 5H), 2.67-2.59(m, 1H), 2.50(2H, combined with DMSO peak), 2.18-2.11(m, 1H), 1.87-1.84(m, 2H), 1.46-1.43(m, 1H), 1.05(d, J ═ 6.3Hz, 3H); LC-MS: m/z 402.2(M +1)+。
EXAMPLE 132 Synthesis of 2- ((6-Acetylbenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) refluxing with 10% NaOH aqueous solution for 4 h; b) potassium ethyl xanthate, EtOH, refluxing for 16 h; c) k2CO3，Mel，ACN，RT，3h；d)NaBH4MeOH, 0 deg.C-RT, 1 h; e) TBDMSCl, imidazole, DCM, 0 ℃ -RT for 2 h; f) m-CPBA, DCM, 0 ℃ -RT, 4 h; g) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; h) THF containing TBAF, THF, 0-RT, 16 h; i) dess-martin periodinane, DCM, 0 deg.C-RT, 1 h; j) LiOH H2O，THF，EtOH，H2O, 60 ℃, 16 h; k) o- (2-methoxyethyl) hydroxylamine, HBTU, DIPEA, DMF, 0 ℃ to RT, 16h
Step-a: synthesis of 1- (4-amino-3-hydroxyphenyl) ethan-1-one
Reacting 6-acetylbenzo [ d]A mixture of oxazol-2 (3H) -one (5.0g, 28.2mmol) in 10% aqueous sodium hydroxide (50mL) was refluxed for 4H. The reaction mixture was cooled to room temperature, acidified with 3N HCl and then basified to pH-8 with saturated sodium carbonate solution. The precipitated solid was filtered and dried in vacuo to give the title compound (4.1g, 98%); 1H NMR(400MHz，DMSO-d6)：δ9.34(s，1H)，7.28(dd，J＝2.0Hz，J＝8.4Hz，1H)，7.22(d，J＝2.0Hz，1H)，6.58(d，J＝8.4Hz，1H)，5.44(s，2H)，2.36(s，3H)。
Step-b: synthesis of 1- (2-mercaptobenzo [ d ] oxazol-6-yl) ethan-1-one
The title compound was synthesized using the same procedure as in example 32, step-c, using 1- (4-amino-3-hydroxyphenyl) ethan-1-one (yield: 100%);1H NMR(400MHz，DMSO-d6)：δ14.2(bs，1H)，8.04(s，1H)，7.94(d，J＝8.0Hz，1H)，7.33(d，J＝8.0Hz，1H)，2.60(s，3H)；LC-MS：m/z 192.02(M-1)-。
step-c: synthesis of 1- (2- (methylthio) benzo [ d ] oxazol-6-yl) ethan-1-one
Using the same procedure as for Compound 1b, 1- (2-mercaptobenzo [ d ]]Oxazol-6-yl) ethan-1-one as starting material the title compound was synthesized (yield: 78%);1H NMR(400MHz，DMSO-d6)：δ8.22(s，1H)，7.98(d，J＝8.4Hz，1H)，7.73(d，J＝8.0Hz，1H)，2.80(s，3H)，2.67(s，3H)。
step-d: synthesis of 1- (2- (methylthio) benzo [ d ] oxazol-6-yl) ethan-1-ol
To 1- (2- (methylthio) benzo [ d ] at 0 DEG C]To a stirred solution of oxazol-6-yl) ethan-1-one (4.1g, 19.8mmol) in methanol (80mL) was added sodium borohydride (1.12g, 29.7mmol) and the reaction mixture was stirred for 1 h. The reaction mixture was quenched with cold water (100mL) and extracted with EtOAc (2X 150 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (4.1g, 100%);1H NMR(400MHz,DMSO-d6):δ7.57-7.53(m,2H),7.31(dd,J＝1.2Hz,J＝8.0Hz,1H),5.28(d,J＝3.6Hz,1H),4.85-4.80(m,1H),2.74(s,3H),1.35(d,J＝6.4Hz,3H)。
step-e: synthesis of 6- (1- ((tert-butyldimethylsilyl) oxy) ethyl) -2- (methylthio) benzo [ d ] oxazole
To 1- (2- (methylthio) benzo [ d ] at 0 DEG C]To a stirred solution of oxazol-6-yl) ethan-1-ol (4.1g, 19.6mmol) in DCM (80mL) was added imidazole (2.0g, 29.4mmol) and stirred for 5min, followed by TBDMSCl (3.5g, 23.5mmol) and stirred at room temperature for 2 h. The reaction mixture was diluted with water (100mL) and extracted with DCM (2X 150 mL). The combined organic layers were washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (6.6g, 100%); 1H NMR(400MHz,DMSO-d6):δ7.63-7.61(m,2H),7.36(d,J＝8.0Hz,1H),5.08(q,J＝6.4Hz,1H),2.80(s,3H),1.42(d,J＝6.0Hz,3H),0.91(s,9H),0.2(s,6H)；LC-MS:m/z 324.1(M+1)+。
Step-f: synthesis of 6- (1- ((tert-butyldimethylsilyl) oxy) ethyl) -2- (methanesulfonyl) -benzo [ d ] oxazole
Using the same procedure as for Compound 1c, using 6-(1- ((tert-butyldimethylsilyl) oxy) ethyl) -2- (methylthio) benzo [ d]Oxazole as a starting material and stirred for 4h to synthesize the title compound (yield: 100%);1H NMR(400MHz,DMSO-d6):δ7.96-7.49(m,3H),5.14-5.11(m,1H),3.66(s,3H),1.40(d,J＝6.0Hz,3H),0.88(s,9H),0.2(s,3H),-0.2(s,3H)。
step-g: synthesis of ethyl 2- ((6- (1- ((tert-butyldimethylsilyl) oxy) ethyl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-1-methyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 6- (1- ((tert-butyldimethylsilyl) oxy) ethyl) -2- (methylsulfonyl) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 31%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.23(d,J＝1.6Hz,1H),7.87(dd,J＝1.6Hz,J＝8.4Hz,1H),7.51(d,J＝8.8Hz,1H),7.42-7.39(m,2H),7.20(d,J＝8.0Hz,1H),5.0(d,J＝6.4Hz,1H),4.32(q,J＝7.6Hz,2H),3.63(s,3H),1.40-1.33(m,6H),0.87(s,9H),0.001(s,3H),-0.036(s,3H)。
step-h: synthesis of ethyl 2- ((6- (1-hydroxyethyl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To a solution of 2- ((6- (1- ((tert-butyldimethylsilyl) oxy) ethyl) benzo [ d ] at 0 deg.C]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (1.4g, 2.8mmol) in THF (30mL) was added 1M TBAF in THF (5.7mL, 5.7mmol) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was quenched with saturated ammonium chloride solution (50mL) and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was stirred in pentane for 15min, and the resulting solid was filtered and dried in vacuo to give the title compound (1.1g, 100%); 1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),8.23(s,1H),7.87(d,J＝8.4Hz,1H),7.51(d,J＝8.4Hz,1H),7.41-7.39(m,2H),7.19(d,J＝7.6Hz,1H),5.16(bs,1H),4.80(bs,1H),4.33(q,J＝7.2Hz,2H),3.63(s,3H),1.40-1.35(m,6H)；LC-MS:m/z381.15(M+1)+。
Step-i: synthesis of ethyl 2- ((6-acetylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To a mixture of 2- ((6- (1-hydroxyethyl) benzo [ d ] at 0 DEG C]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (1.1g, 2.9mmol) in DCM (30mL) was added Dess-martin periodinane (1.47g, 3.5mmol) and the reaction mixture was stirred for 1 h. The reaction mixture was diluted with DCM (100mL), water (50 mL). The organic layer was washed with saturated sodium bicarbonate solution (2 × 50mL), water (50), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (540mg, 49%);1H NMR(400MHz,DMSO-d6):δ12.4(bs,1H),8.26(s,1H),8.00(s,1H),7.91-7.89(m,2H),7.58-7.51(m,2H),4.34(q,J＝6.8Hz,2H),3.87(s,3H),2.66(s,3H),1.35(t,J＝6.8Hz,3H)；LC-MS:m/z 379.15(M+1)+。
step-j: synthesis of 2- ((6-acetylbenzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] -imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, using 2- ((6-acetylbenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 68%);1H NMR(400MHz,DMSO-d6):δ12.8(bs,1H),12.4(s,1H),8.21(s,1H),7.99(s,1H),7.82-7.79(m,2H),7.59-7.52(m,2H),3.67(s,3H),2.66(s,3H)；LC-MS:m/z 351.1(M+1)+。
step-k: synthesis of 2- ((6-acetylbenzo [ d ] oxazol-2-yl) amino) -N- (2-methoxyethoxy) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((6-acetylbenzo [ d) ]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid and O- (2-methoxyethyl) hydroxylamine as starting materials the title compound was synthesized (yield: 42%);1H NMR(400MHz,DMSO-d6):δ12.4(bs,1H),11.7(s,1H),8.02-7.99(s,2H),7.99(dd,J＝1.6Hz,J＝8.4Hz,1H),7.68-7.63(m,1H),7.54-7.51(m,2H),4.04(t,J＝4.4Hz,2H),3.66(s,3H),3.60(t,J＝4.4Hz,2H),3.22(s,3H),2.60(s,3H)；LC-MS:m/z 424.15(M+1)+。
EXAMPLE 133 Synthesis of N- (2-hydroxyethyl) -2- ((6- (2-hydroxyprop-2-yl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzimidazo-5-carboxamide
Conditions are as follows: a)2- ((tert-butyldimethylsilyl) oxy) ethan-1-amine, HBTU, DIPEA, DMF, 0 ℃ -RT, 16 h; b) containing CH3Diethyl ether of MgBr, THF, 0 deg.C, 30 min; c) TBAF-containing THF, THF, 0 deg.C-RT, 4h
Step-a: synthesis of 2- ((6-acetylbenzo [ d ] oxazol-2-yl) amino) -N- (2- ((tert-butyldimethylsilyl) oxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 2- ((6-acetylbenzo [ d)]Oxazol-2-yl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- ((tert-butyldimethylsilyl) oxy) ethan-1-amine as starting materials (yield: 48%); LC-MS: m/z 508.25(M +1)+。
Step-b: synthesis of N- (2- ((tert-butyldimethylsilyl) oxy) ethyl) -2- ((6- (2-hydroxypropan-2-yl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
To 2- ((6-acetylbenzo [ d ] at 0 DEG C]Oxazol-2-yl) amino) -N- (2- ((tert-butyldimethylsilyl) oxy) ethyl) -1-methyl-1H-benzo [ d]To a stirred solution of imidazole-5-carboxamide (200mg, 0.39mmol) in THF (5mL) was added diethyl ether (0.13mL, 0.39mmol) containing 3M methyl magnesium bromide and the reaction mixture was stirred for 30 min. The reaction mixture was quenched with saturated aqueous ammonium chloride (20mL) and extracted with ethyl acetate (2X 50 mL). The combined organic layers were washed with brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (100mg, crude material); LC-MS: m/z 524.3(M +1)+。
Step-c: synthesis of N- (2-hydroxyethyl) -2- ((6- (2-hydroxypropan-2-yl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
The title compound was synthesized using the same procedure as example 132 step-H, using N- (2- ((tert-butyldimethylsilyl) oxy) ethyl) -2- ((6- (2-hydroxyprop-2-yl) benzo [ d ] oxazol-2-yl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide as starting material and stirring for 4H. The crude product was purified by preparative HPLC method (yield: 6%).
Preparative HPLC purification method details:
diluting liquid: ACN water
Mobile phase A: 0.1% formic acid/water
Mobile phase B: 100% acetonitrile
Gradient: 0/10,2/10,6/100, 10/100, 11/10, 12/10
Flow rate: 1.0ml/min
Column: kinetex C-18(250X21.1X5 μm)
1H NMR(400MHz，DMSO-d6)：δ12.23(bs，1H)，8.35(bs，1H)，8.06(s，1H)，7.76(d，J＝6.8Hz，1H)，7.51(s，1H)，7.46(d，J＝8.4Hz，1H)，7.37(d，J＝8.4Hz，1H)，7.30(d，J＝8.4Hz，1H)，5.03(bs，1H)，4.73(bs，1H)，3.62(s，3H)，3.53(bs，2H)，3.35-3.34(m，2H)，1.47(s，6H)；LC-MS：m/z 410.2(M+1)+。
EXAMPLE 134 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (thiazol-4-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) AlCl3DMF, 2-bromoacetyl chloride, 0 ℃ -75 ℃ for 2.5 h; b) formamide, P2S51, 4-dioxane, refluxing for 18 h; c) NaOH, H2O, refluxing for 16 h; d) potassium ethyl xanthate, EtOH, refluxing for 16 h; c) k2CO3MeI, ACN, RT, 4 h; f) m-CPBA, DCM, 0 ℃ -RT, 4 h; g) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; h) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; k)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 6- (2-bromoacetyl) benzo [ d ] oxazol-2 (3H) -one
DMFA (5.75mL, 74mmol) was added dropwise to AlCl3(34.5g, 259.3mmol) and the mixture stirred at 45 ℃ for 30 min. Subsequently, benzo [ d ] will]Oxazol-2 (3H) -one (5.0g, 37mmol) and 2-bromoacetyl chloride (4.6mL, 55.5mmol) were added to the reaction mixture and stirred at 75 ℃ for 2H. The reaction mixture was cooled to room temperature and poured onto ice and stirred for 10 min. The precipitated solid was filtered and dried in vacuo to give the title compound (4.0g, 42%); 1H NMR(400MHz,DMSO-d6):δ12.13(bs,1H),7.90-7.87(m,2H),7.23(d,J＝7.6Hz,1H),4.89(s,2H)；LC-MS:m/z 253.9(M-1)-。
Step-b: synthesis of 6- (thiazol-4-yl) benzo [ d ] oxazol-2 (3H) -one
To a stirred solution of formamide (5.49mL, 137.7mmol) in 1, 4-dioxane (100mL) at room temperature was added phosphorus pentasulfide (6.1g, 27.5mmol) and heated at 100 ℃ for 2 h. The precipitated solid was removed by filtration, and the filtrate was added to 6- (2-bromoacetyl) benzo [ d]Oxazol-2 (3H) -one (13.5, 13.7 mmol). The reaction mixture was stirred at 100 ℃ for 16h and concentrated on a rotary evaporator to remove 1, 4-dioxane. The residue was diluted with ethyl acetate (150mL), water (100mL), and extracted. The organic layer was washed with water (50mL), brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 30% ethyl acetate/hexanes as eluent to give the title compound (1.2g, 40%);1H NMR(400MHz,DMSO-d6):δ11.72(s,1H),9.18(d,J＝1.6Hz,1H),8.12(d,J＝1.2Hz,1H),7.88(s,1H),7.83(d,J＝8.0Hz,1H),7.15(d,J＝8.0Hz,1H)；LC-MS:m/z219.0(M+1)+。
step-c: synthesis of 2-amino-5- (thiazol-4-yl) phenol
To 6- (thiazol-4-yl) benzo [ d ] at room temperature]To a stirred solution of oxazol-2 (3H) -one (600mg, 2.75mmol) in water (6mL) was added sodium hydroxide (1.09g, 27.5mmol) and heated at 100 ℃ for 16H. Will be provided withThe reaction mixture was cooled to room temperature, quenched with saturated ammonium chloride solution and extracted with ethyl acetate (100 mL). The organic layer was washed with aqueous brine (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (400mg, 76%); 1H NMR(400MHz,DMSO-d6):δ9.13(bs,1H),9.06(d,J＝1.2Hz,1H),7.65(d,J＝1.6Hz,1H),7.31(d,J＝1.2Hz,1H),7.19(d,J＝7.6Hz,1H),6.61(d,J＝8.4Hz,1H),4.71(bs,2H)；LC-MS:m/z 193.0(M+1)+。
Step-d: synthesis of 6- (thiazol-4-yl) benzo [ d ] oxazole-2 (3H) -thione
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5- (thiazol-4-yl) phenol (yield: 92%);1H NMR(400MHz,DMSO-d6):δ13.99(s,1H),9.22(d,J＝1.6Hz,1H),8.23(d,J＝1.6Hz,1H),8.10(d,J＝1.2Hz,1H),7.99(dd,J＝1.6Hz,J＝8.4Hz,1H),7.31(d,J＝8.4Hz,1H)；LC-MS:m/z 234.95(M+1)+。
step-e: synthesis of 2- (methylthio) -6- (thiazol-4-yl) benzo [ d ] oxazole
Using the same procedure as for Compound 1b, using 6- (thiazol-4-yl) benzo [ d ]]The title compound was synthesized from oxazole-2 (3H) -thione as a starting material by stirring for 4 hours (yield: 84%);1H NMR(400MHz,DMSO-d6):δ9.21(bs,1H),8.24-8.23(m,2H),8.01(d,J＝8.4Hz,1H),8.69(d,J＝8.4Hz,1H),2.78(s,3H)；LC-MS:m/z249.0(M+1)+。
step-f: synthesis of 2- (methylsulfonyl) -6- (thiazol-4-yl) benzo [ d ] oxazole
The title compound was synthesized using the same procedure as compound 1c, using 2- (methylthio) -6- (thiazol-4-yl) benzo [ d ] oxazole as starting material and stirring for 4 h. The crude compound was used in the next step without any analytical data.
Step-g: synthesis of ethyl 1-methyl-2- ((6- (thiazol-4-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as for 1g of Compound, 2-amino-1-methyl-1H-benzo [ d ] is used]Imidazole-5-carboxylic acid ethyl ester and 2- (methylsulfonyl) -6- (thiazol-4-yl) benzo [ d]Oxazoles asStarting Material the title compound was synthesized (yield: 26%);1H NMR(400MHz,DMSO-d6):δ12.4(bs,1H),9.20(d,J＝4.4Hz,1H),8.25(s,1H),8.22(s,1H),8.08(s,1H),7.92-7.87(m,2H),7.54-7.50(m,2H),4.33(q,J＝7.6Hz,2H),3.66(s,3H),1.35(t,J＝6.8Hz,3H)；LC-MS:m/z 420.05(M+1)+。
step-h: synthesis of 1-methyl-2- ((6- (thiazol-4-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1h, 1-methyl-2- ((6- (thiazol-4-yl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester as starting material and stirred for 5h to synthesize the title compound (yield: 71%);1H NMR(400MHz,DMSO-d6):δ9.16(s,1H),8.00(s,1H),7.95(s,1H),7.81(s,1H),7.77(d,J＝8.0Hz,1H),7.66(d,J＝8.4Hz,1H),7.20(d,J＝7.6Hz,1H),7.06(d,J＝8.4Hz,1H),3.59(s,3H)；LC-MS:m/z 392.1(M+1)+。
step-i: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (thiazol-4-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6- (thiazol-4-yl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials. The crude product was purified by flash chromatography using 20% methanol/DCM as eluent (yield: 28%);1H NMR(400MHz,DMSO-d6):δ12.3(bs,1H),9.19(s,1H),8.45(bs,1H),8.13-8.04(m,3H),7.91(d,J＝7.6Hz,1H),7.77(d,J＝8.4Hz,1H),7.51(d,J＝8.0Hz,2H),3.66(s,3H),3.56-3.44(m,8H)；LC-MS:m/z479.05(M+1)+。
EXAMPLE 135 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (3-methyl-2-oxooxazolidin-5-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) NaBH4MeOH, 0 deg.C-RT, 1 h; b) 40% methylamine water solution, RT, 30 min; c) CDI, DMF, 100 ℃, 16 h; d) POCl3TEA, reflux, 5 h; e) NaHCO 23Toluene, acetone, H containing benzyl chloroformate2O，0℃-RT，16h；f)LiOH.H２O，THF，EtOH，H2O, 80 ℃, 16 h; g)2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethan-1-amine, HBTU, DIPEA, DMF, 0 ℃ -RT, 16 h; h) Pd/C, H 2MeOH, RT, 6 h; i)5- (2-chlorobenzo [ d ]]Oxazol-6-yl) -3-methyloxazolidin-2-one, NaH, 1, 4-dioxane, RT, 3 h; j) TBAF, THF, RT 16h
Step-a: synthesis of 6- (2-bromo-1-hydroxyethyl) benzo [ d ] oxazol-2 (3H) -one
To 6- (2-bromoacetyl) benzo [ d ] at 0 deg.C]To a stirred solution of oxazol-2 (3H) -one (4.0g, 15.62mmol) in methanol (50mL) was added sodium borohydride (587mg, 15.62mmol) portionwise and the reaction mixture was stirred for 1H. The reaction mixture was quenched with water (100mL) and concentrated under reduced pressure to remove methanol. The aqueous layer was extracted with EtOAc (3X 100 mL). The combined organic layers were washed with brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (4.0g, 99%); LC-MS: m/z 257.9(M +1)+。
Step-b: synthesis of 6- (1-hydroxy-2- (methylamino) ethyl) benzo [ d ] oxazol-2 (3H) -one
Stirring of 6- (2-bromo-1-hydroxyethyl) benzo [ d ] at room temperature]A mixture of oxazol-2 (3H) -one (4.0g, 15.5mmol) in 40% aqueous methylamine (20mL) for 30 min. The reaction mixture was concentrated in vacuo and the residue was purified by flash chromatography using 5% methanol/DCM as eluent to give the title compound (1.8g, 56%);1H NMR(400MHz，DMSO-d6)：δ11.8(bs，1H)，8.9(bs，1H)，7.47(d，J＝1.2Hz，1H)，7.28(dd，J＝1.2Hz，J＝8.0Hz，1H)，7.17(d，J＝8.0Hz，1H)，5.68(t，J＝4.8Hz，1H)，4.29(t，J＝6.4Hz，1H)，3.81-3.74(m，2H)，1.24(s，3H)；LC-MS：m/z 209.05(M+1)+。
step-c: synthesis of 6- (3-methyl-2-oxooxazolidin-5-yl) benzo [ d ] oxazol-2 (3H) -one
To 6- (1-hydroxy-2- (methylamino) ethyl) benzo [ d ] at room temperature]To a stirred solution of oxazol-2 (3H) -one (1.8g, 8.65mmol) in DMFA (20mL) was added 1, 1' -carbonyldiimidazole (1.54g, 9.52 mmol). The reaction mixture was heated to 100 ℃ for 16h with stirring. The reaction mixture was cooled to room temperature, diluted with water (100mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 1% methanol/DCM as eluent to give the title compound (500mg, 25%);1H NMR(400MHz,DMSO-d6):δ7.22(s,1Η),7.09(d,J＝8.0Hz,1H),7.01(d,J＝8.0Hz,1H),3.57-3.54(m,1H),3.45-3.41(m,1H),3.34-3.30(m,1H),2.16(s,3H)。
step-d: synthesis of 5- (2-chlorobenzo [ d ] oxazol-6-yl) -3-methyloxazolidin-2-one
A50 mL round-bottom flask was charged with 6- (3-methyl-2-oxooxazolidin-5-yl) benzo [ d ] at 0 deg.C]Oxazol-2 (3H) -one (500mg, 2.14mmol) and POCl3(0.98mL, 10.68mmol) followed by slow addition of triethylamine (1.51mL, 10.68 mmol). The reaction mixture was heated to reflux for 5 h. The reaction mixture was cooled to room temperature, poured onto ice and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with saturated sodium bicarbonate solution (2 × 50mL), water (50mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 50% EtOAc/hexanes as the eluent to give the title compound (120mg, 22%); 1H NMR(400MHz,DMSO-d6):δ7.58(s,1Η),7.35(dd,J＝1.6Hz,J＝8.0Hz,1H),7.19(d,J＝7.6Hz,1H),4.64(bs,1H),4.23-4.19(m,1H),4.16-4.11(m,1H),2.46(s,3H)。
Step-e: synthesis of ethyl 2- (bis ((benzyloxy) carbonyl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at 0 DEG C]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (1.0g, 3.24mmol) in acetone (10mL) and water (10mL) was added sodium bicarbonate (722mg, 6.88mmol) and benzyl chloroformate (1.2mL, 4.22mmol, 50% in toluene). The reaction mixture is stirred at room temperatureCompound 16 h. The reaction mixture was diluted with water (100mL) and extracted with ethyl acetate (2X 100 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The crude compound (2.2g, 99%) was used in the next step without any further purification; LC-MS: m/z 488.2(M +1)+。
Step-f: synthesis of 2- (((benzyloxy) carbonyl) amino) -1-methyl-1H-benzo [ d ] imidazole-5-carboxylic acid
To 2- (bis ((benzyloxy) carbonyl) amino) -1-methyl-1H-benzo [ d ]]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (2.2g, 4.52mmol) in a solvent mixture of THF (15mL), ethanol (15mL) and water (10mL) was added lithium hydroxide monohydrate (948mg, 22.6 mmol). The reaction mixture was heated at 80 ℃ for 16h with stirring. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water (30mL), acidified with 1N HCl and extracted with ethyl acetate (2X 150 mL). The combined organic layers were washed with brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give crude compound (1.4g, 95%) which was used in the next step without any further purification; LC-MS: m/z 326.1(M +1) +。
Step-g: synthesis of benzyl (5- ((2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) carbamoyl) -1-methyl-1H-benzo [ d ] imidazol-2-yl) carbamate
Using the same procedure as example 6 step-e, using 2- (((benzyloxy) carbonyl) amino) -1-methyl-1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethan-1-amine as starting materials (yield: 66%);1H NMR(400MHz,DMSO-d6):δ12.20(bs,1H),8.42(t,J＝5.2Hz,1H),7.85(s,1H),7.68(d,J＝8.4Hz,1H),7.41-7.33(m,6H),5.08(s,2H),3.66(t,J＝5.2Hz,2H),3.54-3.38(m,9H),0.81(s,9H),0.03(s,6H)；LC-MS:m/z 527.25(M+1)+。
step-h: synthesis of 2-amino-N- (2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) -1-methyl-1H-benzo [ d ] imidazole-5-carboxamide
Under a nitrogen atmosphere, to (5- ((2- (2- ((tert-butyl-di-tert-butyl)Methylsilyl) oxy) ethoxy) ethyl) carbamoyl) -1-methyl-1H-benzo [ d]Imidazol-2-yl) carbamic acid benzyl ester (320mg, 0.61mmol) to a solution in methanol (8mL) was added 10% Pd/C (40 mg). Subsequently, the reaction mixture was stirred under a balloon of hydrogen for 6 h. The reaction mixture was filtered through celite bed and concentrated in vacuo to give the title compound (170mg, 71%);1H NMR(400MHz,DMSO-d6):δ8.22(t,J＝4.8Hz,1H),7.65(s,1H)),7.45(d,J＝8.0Hz,1H),7.14(d,J＝8.0Hz,1H),6.52(s,2H),3.69(t,J＝4.8Hz,2H),3.55-3.46(m,7H),3.41-3.37(m,2H),0.84(s,9H),0.01(s,6H)；LC-MS:m/z393.65(M+1)+。
step-i: synthesis of N- (2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) -1-methyl-2- ((6- (3-methyl-2-oxooxazolidin-5-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 32 step-e, using 2-amino-N- (2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) -1-methyl-1H-benzo [ d]Imidazole-5-carboxamides and 5- (2-chlorobenzo [ d ]]Oxazol-6-yl) -3-methyloxazolidin-2-one as starting material was stirred for 3h to synthesize the title compound (yield: 74%);1H NMR(400MHz,DMSO-d6):δ11.74(bs,1H),8.43(t,J＝5.2Hz,1H),8.05(d,J＝1.2Hz,1H),7.77(dd,J＝1.6Hz,J＝8.4Hz,1H),7.56(d,J＝8.4Hz,1H),7.42(d,J＝1.2Hz,1H),7.25(dd,J＝1.6Hz,J＝8.4Hz,1H),7.15(d,J＝8.0Hz,1H),4.87(t,J＝7.2Hz,1H),4.35(t,J＝9.2Hz,1H),3.82-3.76(m,5H),3.68(t,J＝5.2Hz,2H),3.54-3.52(m,2H),3.48-3.31(m,2H),2.62(s,3H),0.84(s,9H),-0.01(s,3H),-0.02(s,3H)；LC-MS:m/z 609.3(M+1)+。
step-j: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (3-methyl-2-oxooxazolidin-5-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
To a solution of N- (2- (2- ((tert-butyldimethylsilyl) oxy) ethoxy) ethyl) -1-methyl-2- ((6- (3-methyl-2-oxooxazolidin-5-yl) benzo [ d ] at 0 deg.C]Oxazol-2-yl) amino) -1H-benzo [ d]To a stirred solution of imidazole-5-carboxamide (30mg, 0.05mmol) in THF (2mL) was added 1M TBAF in THF (0.1mL), andthe reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with saturated ammonium chloride solution (10mL) and extracted with EtOAc (2X 30 mL). The combined organic layers were washed with water (20mL), brine (20mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography using 5% methanol/DCM as eluent to give the title compound (14mg, 58%); 1H NMR(400MHz，DMSO-d6)：δ11.8(bs，1H)，8.43(t，J＝5.2Hz，1H)，8.06(s，1H)，7.78(d，J＝8.8Hz，1H)，7.57(d，J＝8.0Hz，1H)，7.38(s，1H)，7.21(d，J＝8.0Hz，1H)，7.13(d，J＝7.6Hz，1H)，4.87(t，J＝8.0Hz，1H)，4.59(s，1H)，4.35(t，J＝9.2Hz，1H)，3.82(t，J＝9.2Hz，1H)，3.78(s，3H)，3.56-3.41(m，8H)，2.63(s，3H)；LC-MS：m/z 495.2(M+1)+。
EXAMPLE 136 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6-morpholinobenzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) morpholine, ACN, 60 ℃, 2 h; b) 10% Pd/C, MeOH, H2RT, 3 h; c) potassium ethyl xanthate, EtOH, refluxing for 16 h; d) SOCl2Catalytic DMF, DCM, RT, 1 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; g)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 5-morpholinyl-2-nitrophenol
To a stirred solution of 5-fluoro-2-nitrophenol (5.0g, 31.84mmol) in acetonitrile (50mL) at room temperature was added morpholine (8.31g, 95.54mmol) and the reaction mixture was heated at 60 ℃ for 2 h. The reaction mixture was cooled to room temperature, diluted with cold water (300mL), and the precipitated solid was filtered and dried in vacuo to give the title compound (5.0g, 70%);1H NMR(400MHz，DMSO-d6)：δ10.9(bs，1H)，7.88(d，J＝9.2Hz，1H)，6.65(dd，J＝2.4Hz，J＝9.6Hz，1H)，6.44(d，J＝2.8Hz，1H)，3.70(t，J＝5.2Hz，4H)，3.41(t，J＝4.8Hz，4H)；LC-MS：m/z225.1(M+1)+。
step-b: synthesis of 2-amino-5-morpholinylphenol
The title compound was synthesized using the same procedure as compound 1e, using 5-morpholinyl-2-nitrophenol as starting material and stirring for 3h (yield: 81%); 1H NMR(400MHz,DMSO-d6):δ8.8(bs,1H),6.48(d,J＝8.0Hz,1H),6.33(d,J＝2.4Hz,1H),6.19(dd,J＝2.8Hz,J＝8.8Hz,1H),3.68(t,J＝5.2Hz,4H),2.84(t,J＝4.8Hz,4H)；LC-MS:m/z 195.1(M+1)+。
Step-c: synthesis of 6-morpholinylbenzo [ d ] oxazole-2 (3H) -thione
The title compound was synthesized using the same procedure as in example 32, step-c, using 2-amino-5-morpholinophenol as the starting material (yield: 75%);1H NMR(400MHz,DMSO-d6):δ13.6(s,1H),7.14(d,J＝2.4Hz,1H),7.09(d,J＝8.8Hz,1H),6.91(dd,J＝2.4Hz,J＝8.8Hz,1H),3.73(t,J＝4.4Hz,4H),3.10(t,J＝4.8Hz,4H)；LC-MS:m/z 237.1(M+1)+。
step-d: synthesis of 2-chloro-6-morpholinylbenzo [ d ] oxazole
To 6-morpholinobenzo [ d ] at room temperature]To a solution of oxazole-2 (3H) -thione (500mg, 1.12mmol) in DCM (10mL) was added thionyl chloride (0.79mL, 10.6mmol) and dimethylformamide (0.2mL), and the reaction mixture was stirred at room temperature for 1H. The reaction mixture was poured onto cold water (50mL), basified with saturated sodium bicarbonate solution and extracted with DCM (2 × 50 mL). The combined organic layers were washed with brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using 30% EtOAc/hexanes as the eluent to give the title compound (300mg, 60%);1H NMR(400MHz,DMSO-d6):δ7.56(d,J＝8.8Hz,1H),7.29(d,J＝2.4Hz,1H),7.09(dd,J＝2.4Hz,J＝8.8Hz,1H),3.75(t,J＝4.8Hz,4H),3.16(t,J＝4.8Hz,4H)；LC-MS:m/z 239.0(M+1)+。
step-e: synthesis of ethyl 1-methyl-2- ((6-morpholinobenzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6-morpholinobenzo [ d ]]Oxazole as a starting material the title compound was synthesized (yield: 37%); 1H NMR(400MHz,DMSO-d6):δ12.2(bs,1H),8.20(d,J＝1.2Hz,1H),7.86(dd,J＝1.6Hz,J＝8.4Hz,1H),7.49(d,J＝8.4Hz,1H),7.33(d,J＝8.4Hz,1H),7.09(d,J＝2.4Hz,1H),6.87(dd,J＝2.4Hz,J＝8.8Hz,1H),4.32(q,J＝8.4Hz,2H),3.76(t,J＝4.4Hz,4H),3.61(s,3H),3.09(t,J＝4.8Hz,4H),1.35(t,J＝7.2Hz,3H)；LC-MS:m/z 422.1(M+1)+。
Step-f: synthesis of 1-methyl-2- ((6-morpholinobenzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as for Compound 1h, 1-methyl-2- ((6-morpholinobenzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 71%);1H NMR(400MHz,DMSO-d6):δ12.5(bs,1H),8.16(d,J＝1.2Hz,1H),7.85(d,J＝8.4Hz,1H),7.46(d,J＝8.4Hz,1H),7.34(d,J＝8.8Hz，1H)，7.08(d，J＝2.0Hz，1H)，6.86(dd，J＝2.0Hz，J＝8.8Hz，1H)，3.76(t，J＝4.4Hz，4H)，3.61(s，3H)，3.09(t，J＝4.4Hz，4H)；LC-MS：m/z 394.0(M+1)+。
step-g: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6-morpholinobenzo [ d ] -oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6 step-e, using 1-methyl-2- ((6-morpholinobenzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials (yield: 98%);1H NMR(400MHz，DMSO-d6)：δ12.4(bs，1H)，8.49(t，J＝4.8Hz，1H)，8.05(d，J＝1.2Hz，1H)，7.78(d，J＝7.2Hz，1H)，7.52(d，J＝7.2Hz，1H)，7.34(d，J＝8.4Hz，1H)，7.15(s，1H)，6.94(d，J＝7.2Hz，1H)，3.86(bs，4H)，3.64(s，3H)，3.56-3.54(m，4H)，3.47-3.45(m，4H)，3.14(bs，4H)；LC-MS：m/z 481.2(M+1)+。
EXAMPLE 137 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (pyrrolidin-1-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) pyrrolidine, acetonitrile, 100 ℃, 12 h; b) 10% Pd/C, MeOH, H2RT, 4 h; c) refluxing potassium ethylxanthate and ethanol for 16 h; d) SOCl2DCM, 0 ℃ for 1 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-ethyl formate, 1, 4-dioxane, 0 ℃ -RT, 4 h; f) h, LiOH 2O, THF, ethanol, water, 60 ℃ and 16 h; g)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 2-nitro-5- (pyrrolidin-1-yl) phenol
To a solution of 5-fluoro-2-nitrophenol (2.0g, 12.73mmol) in acetonitrile 20mL) at room temperature was added pyrrolidine (3.15mL, 38.21mmol) and stirred at 100 ℃ for 12 h. The reaction mixture was concentrated in vacuo and the residue was purified by flash column chromatography using 30% ethyl acetate/hexanes as the eluent to give the title compound (1.5g, 57%);1H NMR(400MHz，DMSO-d6)：δ7.85(d，J＝9.2Hz，1H)，6.28(dd，J＝2.4Hz，J＝9.2Hz，1H)，6.02(d，J＝2.4Hz，1H)，3.39-3.36(m，4H)，1.98-1.94(m，4H)。
step-b: synthesis of 2-amino-5- (pyrrolidin-1-yl) phenol
To a solution of 2-nitro-5- (pyrrolidin-1-yl) phenol (1.5g, 7.20mmol) in methanol (300mL) under a nitrogen atmosphere was added 10% Pd/C (300 mg). The reaction mixture was stirred under a balloon of hydrogen for 4 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated in vacuo to give crude compound (1.3g) which was used in the next step without any further purification and structure confirmation by analytical techniques.
Step-c: synthesis of 6- (pyrrolidin-1-yl) benzo [ d ] oxazole-2 (3H) -thione
To a solution of 2-amino-5- (pyrrolidin-1-yl) phenol (1.3g, 7.30mmol) in ethanol (13mL) at room temperature was added potassium ethyl xanthate (2.3g, 14.6mmol) and the reaction mixture was refluxed for 16 h. The reaction mixture was concentrated in vacuo and diluted with cold water (50mL) and acidified with 1N HCl. The resulting solid was filtered and dried in vacuo to give the title compound (0.8g, 50%) which was used in the next step without further purification; 1H NMR(400MHz,DMSO-d6):δ13.50(bs,1H),7.03(d,J＝8.4Hz,1H),6.65(d,J＝2.0Hz,1H),6.46(dd,J＝2.0Hz,J＝8.4Hz,1H),3.22-3.19(m,4H),1.97-1.94(m,4H)；LC-MS:m/z 220.9(M+1)+。
Step-d: synthesis of 2-chloro-6- (pyrrolidin-1-yl) benzo [ d ] oxazole
To 6- (pyrrolidin-1-yl) benzo [ d ] at 0 deg.C]To a solution of oxazole-2 (3H) -thione (400mg, 1.82mmol) in DCM (10mL) was added thionyl chloride (0.66mL, 9.09mmol) and the reaction mixture was stirred at 0 ℃ for 1H. The reaction mixture was quenched with water (20mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with saturated sodium bicarbonate solution (30mL), brine solution (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (300mg, 74%) which was used in the next step without further purification;1H NMR(400MHz,CD3OD):δ6.91(d,J＝8.4Hz,1H),6.48(d,J＝2.0Hz,1H),6.42(dd,J＝2.4Hz,J＝8.8Hz,1H),3.21-3.16(m,4H),1.96-1.91(m,4H)。
step-e: synthesis of ethyl 1-methyl-2- ((6- (pyrrolidin-1-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
To 2-amino-1-methyl-1H-benzo [ d ] at 0 DEG C]To a stirred solution of imidazole-5-carboxylic acid ethyl ester (200mg, 0.91mmol) in 1, 4-dioxane (5mL) was added sodium hydride (60% dispersion in mineral oil) (73mg, 1.82mmol) and stirred for 30min, followed by addition of 2-chloro-6- (pyrrolidin-1-yl) benzo [ d ] c]Oxazole (243mg, 1.09 mmol). The reaction mixture was stirred at room temperature for 4h and then quenched with cold water (20mL) and extracted with EtOAc (2X 50 mL). Water for combined organic layer (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 3% methanol/DCM as eluent to give the title compound (150mg, 40%);1H NMR(400MHz,DMSO-d6):δ12.10(bs,1H),8.18(d,J＝1.6Hz,1H),7.85(dd,J＝1.2Hz,J＝8.4Hz,1H),7.46(d,J＝8.4Hz,1H),7.29(d,J＝8.8Hz,1H),6.64(d,J＝2.0Hz,1H),6.46(dd,J＝2.0Hz,J＝8.4Hz,1H),4.32(q,J＝6.8Hz,2H),3.60(s,3H),3.26-3.23(m,4H),1.99-1.95(m,4H),1.35(t,J＝6.8Hz,3H)；LC-MS:m/z 406.2(M+1)+。
step-f: synthesis of 1-methyl-2- ((6- (pyrrolidin-1-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
To 1-methyl-2- ((6- (pyrrolidin-1-yl) benzo [ d)]Oxazol-2-yl) amino) -1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester (150mg, 0.37mmol) in [ THF (2mL), ethanol (2mL) and water (1mL)]To a stirred solution of the solvent mixture of (1) was added lithium hydroxide monohydrate (77mg, 1.85 mmol). The reaction mixture was heated at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water and acidified with 1N HCl to give a solid which was filtered and dried in vacuo to give the title compound (120mg, 86%);1H NMR(400MHz,DMSO-d6):δ7.87(s,1H),7.61(d,J＝7.6Hz,1H),7.00(d,J＝8.4Hz,1H),6.95(d,J＝7.6Hz,1H),6.50(s,1H),6.31(d,J＝7.2Hz,1H),3.52(s,3H),3.25-3.21(m,4H),1.97-1.93(m,4H)；LC-MS:m/z 376.0(M-1)。
step-g: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- (pyrrolidin-1-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
To a stirred solution of 1-methyl-2- ((6- (pyrrolidin-1-yl) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid (120mg, 0.32mmol) in DMFA (5mL) at 0 deg.C was added N-ethyldiisopropylamine (0.08mL, 0.48mmol) and HBTU (180mg, 0.48 mmol). The reaction mixture was stirred for 30min, then 2- (2-aminoethoxy) ethan-1-ol (80mg, 0.48mmol) was added and stirring continued at room temperature for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was diluted with cold water (20mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (25mL), brine (25mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by preparative HPLC purification to give the title compound (15mg, 10%);
Preparative HPLC purification method details:
diluting liquid: THF + acetonitrile water (50:50)
Mobile phase A: 0.1% formic acid/water
Mobile phase B: acetonitrile (100%)
Gradient: t/% B: 0/15, 10/35
Flow rate: 15mL/min
Column: agilent ZORBAX XDB C18(150X21.2X5 μm)
1H NMR(400MHz,DMSO-d6):δ12.0(bs,1H),8.40(t,J＝5.2Hz,1H),8.03(s,1H),7.73(d,J＝8.4Hz,1H),7.43(d,J＝8.4Hz,1H),7.28(d,J＝8.4Hz,1H),6.63(bs,1H),6.46(bs,1H),4.63(s,1H),3.59(s,3H),3.57-3.39(m,8H),3.24(bs,4H),1.97(bs,4H)；LC-MS:m/z 465.20(M+1)+。
EXAMPLE 138 Synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Conditions are as follows: a) tetrahydro-2H-pyran-4-ol, NaH, DMF, 60 ℃, 16H; b) 10% Pd/C, MeOH, H2RT, 6 h; c) potassium ethyl xanthate, EtOH, refluxing for 16 h; d) SOCl2Catalytic DMF, refluxing for 3 h; e) NaH, 2-amino-1-methyl-1H-benzo [ d]Imidazole-5-carboxylic acid ethyl ester, 1, 4-dioxane, RT, 16 h; f) h, LiOH2O，THF，EtOH，H2O, 60 ℃, 16 h; g)2- (2-aminoethoxy) ethan-1-ol, HBTU, DIPEA, DMF, 0 ℃ -RT, 16h
Step-a: synthesis of 2-nitro-5- ((tetrahydro-2H-pyran-4-yl) oxy) phenol
To tetrahydro-2H-pyran-4-ol (2.27mL, 22.29mmol) at 10 deg.C in DMFA (DMFA: (TM)) (II)15mL) was added sodium hydride (60% dispersion in mineral oil) (1.27g, 31.84mmol) and stirred for 5min, then 5-fluoro-2-nitrophenol (1.0g, 6.37mmol) was added to the reaction mixture and stirred at 60 ℃ for 16 h. The reaction mixture was cooled to room temperature, quenched with cold water (100mL) and neutralized with 1N HCl. The precipitated solid was filtered and dried in vacuo to give the title compound (1.0g, 33%); 1H NMR(400MHz，DMSO-d6)：δ10.90(bs，1H)，7.95(d，J＝9.2Hz，1H)，6.68(d，J＝2.0Hz，1H)，6.63(dd，J＝2.4Hz，J＝9.2Hz，1H)，4.71-4.68(m，1H)，3.86-3.82(m，2H)，3.52-3.49(m，2H)，1.99-1.97(m，2H)，1.64-1.56(m，2H)。
Step-b: synthesis of 2-amino-5- ((tetrahydro-2H-pyran-4-yl) oxy) phenol
The title compound was synthesized using the same procedure as compound 1e, using 2-nitro-5- ((tetrahydro-2H-pyran-4-yl) oxy) phenol as starting material and stirring for 6H (yield: 87%);1H NMR(400MHz，DMSO-d6)：δ9.0(bs，1H)，6.47(d，J＝8.4Hz，1H)，6.32(d，J＝2.8Hz，1H)，6.21(dd，J＝2.4Hz，J＝8.4Hz，1H)，4.22-4.17(m，1H)，3.83-3.78(m，2H)，3.44-3.40(m，2H)，1.89-1.85(m，2H)，1.54-1.45(m，2H)；LC-MS：m/z209.95(M+1)+。
step-c: synthesis of 6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] oxazole-2 (3H) -thione
The title compound was synthesized using the same procedure as example 32, step-c, using 2-amino-5- ((tetrahydro-2H-pyran-4-yl) oxy) phenol as starting material (yield: 75%);1H NMR(400MHz，DMSO-d6)：δ13.74(bs，1H)，7.30(d，J＝2.4Hz，1H)，7.12(d，J＝8.8Hz，1H)，6.92(dd，J＝2.4Hz，J＝8.4Hz，1H)，4.59-4.53(m，1H)，3.87-3.82(m,2H),3.47-3.40(m,2H),1.98-1.93(m,2H),1.61-1.52(m,2H)；LC-MS:m/z 252.1(M+1)+。
step-d: synthesis of 2-chloro-6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] oxazole
Using the same procedure as example 32, step-d, using 6- ((tetrahydro-2H-pyran-4-yl)) Oxy) benzo [ d]The title compound was synthesized from oxazole-2 (3H) -thione as a starting material (yield: 25%);1H NMR(400MHz,DMSO-d6):δ7.59(d,J＝8.8Hz,1H),7.47(d,J＝2.0Hz,1H),7.02(dd,J＝2.0Hz,J＝8.8Hz,1H),4.62-4.59(m,1H),3.85-3.77(m,2H),3.49-3.43(m,2H),1.97-1.94(m,2H),1.61-1.52(m,2H)；LC-MS:m/z 254.1(M+1)+。
step-e: synthesis of ethyl 1-methyl-2- ((6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylate
Using the same procedure as in example 32 step-e, using 2-amino-1-methyl-1H-benzo [ d ]]Imidazole-5-carboxylic acid ethyl ester and 2-chloro-6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d]Oxazole as a starting material the title compound was synthesized (yield: 23%); 1HNMR(400MHz,DMSO-d6):δ12.20(bs,1H),8.21(d,J＝1.2Hz,1H),7.85(dd,J＝1.6Hz,J＝8.4Hz,1H),7.49(d,J＝8.4Hz,1H),7.35(d,J＝8.8Hz,1H),7.19(d,J＝2.4Hz,1H),6.85(dd,J＝2.0Hz,J＝8.8Hz,1H),4.56-4.54(m,1H),4.32(q,J＝7.2Hz,2H),3.88-3.85(m,2H),3.62(s,3H),3.51-3.44(m,2H),2.00-1.98(m,2H),1.61-1.58(m,2H),1.35(q,J＝6.8Hz,3H)；LC-MS:m/z 437.2(M+1)+。
Step-f: synthesis of 1-methyl-2- ((6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxylic acid
Using the same procedure as Compound 1H, 1-methyl-2- ((6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] is used]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid ethyl ester as a starting material (yield: 67%);1H NMR(400MHz,DMSO-d6):δ12.40(bs,1H),8.17(d,J＝1.2Hz,1H),7.86(dd,J＝1.2Hz,J＝8.4Hz,1H),7.49(d,J＝8.4Hz,1H),7.36(d,J＝5.6Hz,1H),7.20(d,J＝2.4Hz,1H),6.86(dd,J＝2.4Hz,J＝8.8Hz,1H),4.57-4.53(m,1H),3.88-3.84(m,2H),3.62(s,3H),3.51-3.44(m,2H),2.00-1.95(m,2H),1.61-1.58(m,2H)；LC-MS:m/z 409.1(M+1)+。
step-g: synthesis of N- (2- (2-hydroxyethoxy) ethyl) -1-methyl-2- ((6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] oxazol-2-yl) amino) -1H-benzo [ d ] imidazole-5-carboxamide
Using the same procedure as example 6, step-e, 1-methyl-2- ((6- ((tetrahydro-2H-pyran-4-yl) oxy) benzo [ d ] was used]Oxazol-2-yl) amino) -1H-benzo [ d]The title compound was synthesized from imidazole-5-carboxylic acid and 2- (2-aminoethoxy) ethan-1-ol as starting materials. The crude product was purified by flash chromatography using 4% methanol/DCM as eluent (yield: 40%);1H NMR(400MHz，DMSO-d6)：δ12.17(bs，1H)，8.44(d，J＝5.6Hz，1H)，8.05(d，J＝1.2Hz，1H)，7.74(dd，J＝1.6Hz，J＝8.4Hz，1H)，7.46(d，J＝8.4Hz，1H)，7.34(d，J＝8.4Hz，1H)，7.18(d，J＝2.0Hz，1H)，6.84(dd，J＝2.4Hz，J＝8.8Hz，1H)，4.62(bs，1H)，4.57-4.51(m，1H)，3.89-3.84(m，2H)，3.61(s，3H)，3.55-3.33(m，10H)，1.98-1.95(m，2H)，1.61-1.57(m，2H)；LC-MS：m/z496.2(M+1)+
examples 139, 140 and 141 Synthesis of 1- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazol-5-yl) -2, 2, 2-trifluoroethyl-1-one, synthesis of (Z) -1- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazol-5-yl) -2, 2, 2-trifluoroethyl-1-one oxime and N- (5- (1-amino-2, 2, 2-trifluoroethyl) -1-methyl-1H-benzo [ d ] imidazol-2-yl) benzo [ d ] oxazol-2-amine.
Conditions are as follows: a) concentrated H2SO4Fuming nitric acid, 0-RT, 2 h; b) methylamine water solution, DMF, 0-RT, 2 h; c) iron, concentrated HCl, MeOH, 0 deg.C-RT, 1h, 60 deg.C, 1 h; d) cyanogen bromide, THF, water, 60 ℃, 16 h; e) 2-chlorobenzoxazole, sodium hydride, 1, 4-dioxane, 0 ℃ to RT, 16 h; f) hydroxylamine hydrochloride, potassium acetate and ethanol are refluxed for 16 hours; g) LiAlH4(1M solution in THF), THF, 10 ℃ for 4h
Step-a: synthesis of 2, 2, 2-trifluoro-1- (4-fluoro-3-nitrophenyl) ethan-1-one
To 2, 2, 2-trifluoro-1- (4-fluorophenyl) ethan-1-one (3.0g, 15.62mmol) at 0 deg.C in concentrated sulfuric acidTo the solution in (12mL) was added fuming nitric acid (0.9mL) and the mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with cold water (100mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with cold water (2 × 50mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (3.5g, 94%) which was used in the next step without further purification;1H NMR(400MHz，DMSO-d6)：δ8.80(d，J＝6.8Hz，1H)，8.37(d，J＝8.8Hz，1H)，7.54(t，J＝9.4Hz，1H)。
step-b: synthesis of 2, 2, 2-trifluoro-1- (4- (methylamino) -3-nitrophenyl) ethan-1-one
To a stirred solution of 2, 2, 2-trifluoro-1- (4-fluoro-3-nitrophenyl) ethan-1-one (3.5g, 14.7mmol) in DMFA (17.5mL) at 0 ℃ was added 40% aqueous methylamine (3.5mL) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with cold water (100mL) and extracted with ethyl acetate (2X 100 mL). The combined organic layers were washed with water (2 × 50mL), brine solution (50mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (3.3g, 89%) which was used in the next step without further purification; 1H NMR(400MHz,DMSO-d6):δ8.99(bs,1H),8.69(s,1H),8.05(d,J＝9.2Hz,1H),7.19(d,J＝9.2Hz,1H),3.06(d,J＝4.8Hz,3H)；LC-MS:m/z 247.1(M-1)。
Step-c: synthesis of 1- (3-amino-4- (methylamino) phenyl) -2,2, 2-trifluoroethyl-1-one
To a stirred solution of 2,2, 2-trifluoro-1- (4- (methylamino) -3-nitrophenyl) ethan-1-one (2.0g, 8.1mmol) in methanol (20mL) at 0 deg.C was added iron (2.25g, 40.3mmol) and concentrated hydrochloric acid (5.0mL, 40.3 mmol). The reaction mixture was stirred at room temperature for 1h and then heated with stirring at 60 ℃ for a further 1 h. The reaction mixture was cooled to room temperature, diluted with methanol (30mL) and filtered through a pad of celite. The filtrate was concentrated and diluted with water (50mL), basified with saturated sodium bicarbonate solution and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (1.5g, 84%) which was taken on without further treatmentPurification i.e. used in the next step;1H NMR(400MHz,DMSO-d6):δ7.35(d,J＝8.4Hz,1H),7.21(s,1H),6.50(d,J＝8.8Hz,1H),6.30(bs,1H),4.96(s,2H),2.85(d,J＝4.8Hz,3H)；LC-MS:m/z218.90(M+1)+。
step-d: synthesis of 1- (2-amino-1-methyl-1H-benzo [ d ] imidazol-5-yl) -2,2, 2-trifluoroethyl-1-one
To a stirred solution of 1- (3-amino-4- (methylamino) phenyl) -2,2, 2-trifluoroethyl-1-one (1.0g, 4.6mmol) in THF (10mL) and water (10mL) at room temperature was added cyanogen bromide (0.58g, 5.5mmol), and the reaction mixture was stirred at 60 ℃ for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (50mL), basified with saturated sodium bicarbonate solution and extracted with EtOAc (2X 100 mL). The combined organic layers were washed with water (50mL), brine (500mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give the title compound (900mg, 81%) which was used in the next step without further purification; 1H NMR(400MHz,DMSO-d6):δ7.75(s,1H),7.70(d,J＝8.4Hz,1H),7.38(d,J＝8.4Hz,1H),6.96(s,2H),3.58(s,3H)；LC-MS:m/z 244.1(M+1)+。
Step-e: synthesis of 1- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazol-5-yl) -2,2, 2-trifluoroethyl-1-one
To 1- (2-amino-1-methyl-1H-benzo [ d ] at 0 DEG C]To a stirred solution of imidazol-5-yl) -2,2, 2-trifluoroethyl-1-one (900mg, 3.70mmol) in 1, 4-dioxane (20mL) was added sodium hydride (60% dispersion in mineral oil) (518mg, 12.96mmol) and stirred for 15min, followed by addition of 2-chlorobenzoxazole (567mg, 3.70 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with cold water (30mL) and extracted with EtOAc (2X 50 mL). The combined organic layers were washed with water (30mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 100% DCM as eluent to give the title compound (700mg, 52%);1H NMR(400MHz,DMSO-d6):δ12.50(bs,1H),8.35(s,1H),7.97(d,J＝8.8Hz,1H),7.67(d,J＝8.8Hz,1H),7.52-7.46(m,2H),7.25(t,J＝7.8Hz,1H),7.16(t,J＝7.8Hz,1H),3.67(s,3H)；LC-MS:m/z 361.1(M+1)+。
step-f: synthesis of (Z) -1- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazol-5-yl) -2,2, 2-trifluoroethyl-1-one oxime
To 1- (2- (benzo [ d ]) at room temperature]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazol-5-yl) -2,2, 2-trifluoroethyl-1-one (700mg, 1.94mmol) in ethanol (20mL) was added hydroxylamine hydrochloride (405mg, 5.83mmol) and potassium acetate (572mg, 5.83 mmol). The reaction mixture was stirred at 80 ℃ for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was concentrated under reduced pressure and diluted with water (50mL) and extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with water (50mL), brine (30mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 2% methanol/DCM as eluent to give the title compound (500mg, 68%); 1H NMR(400MHz,DMSO-d6):δ13.05(bs,1H),12.40(bs,1H),7.81-7.76(m,1H),7.59-7.53(m,1H),7.48-7.46(m,2H),7.41-7.37(m,1H),7.24(t,J＝7.4Hz,1H),7.15(t,J＝7.6Hz,1H),3.65(s,3H)；LC-MS:m/z 376.1(M+1)+。
Step-g: synthesis of N- (5- (1-amino-2, 2, 2-trifluoroethyl) -1-methyl-1H-benzo [ d ] imidazol-2-yl) benzo [ d ] oxazol-2-amine
To a stirred solution of (Z) -1- (2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazol-5-yl) -2,2, 2-trifluoroethyl-1-one oxime (300mg, 0.80mmol) in THF (5mL) at 10 deg.C was added a 1M solution of lithium aluminum hydride (1.6mL, 1.60 mmol). The reaction mixture was stirred at 10 ℃ for 4 h. Once the reaction was complete (monitored by TLC), the reaction mixture was quenched with 1N sodium hydroxide solution (5mL), diluted with water (10mL) and extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with water (20mL), brine (20mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by preparative HPLC to give the title compound (100mg, 35%).
Preparative HPLC purification method details:
diluting liquid: THF + ACN water (50:50)
Mobile phase A: 0.1% formic acid/water
Mobile phase B: 100% acetonitrile
Gradient: t/% B: 0/15, 10/40
Flow rate: 15mL/min
Column: kinetex C-18(250X21.1X5 μm)
1H NMR(400MHz，DMSO-d6)：δ12.20(bs，1H)，7.73(s，1H)，7.46-7.36(m，4H)，7.20(t，J＝7.2Hz，1H)，7.10(t，J＝7.2Hz，1H)，4.59-4.57(m，1H)，3.61(s，3H)；LC-MS：m/z 362.3(M+1)+。
The two isomers of this compound were separated by chiral HPLC purification.
Details of chiral HPLC purification method:
diluting liquid: IPA: DCM (90: 10)
Mobile phase A: 0.1% DEA/Hexane
Mobile phase B: IPA: DCM (90: 10)%
Isocratic: a: B (40: 60)
Flow rate: 15mL/min
Peak-1 analytical data:
1H NMR(400MHz，DMSO-d6)：δ12.20(bs，1H)，7.73(s，1H)，7.46-7.36(m，4H)，7.20(t，J＝7.2Hz，1H)，7.10(t，J＝7.2Hz，1H)，4.59-4.57(m，1H)，3.61(s，3H)；LC-MS：m/z 362.05(M+1)+。
peak-2 analytical data:
1H NMR(400MHz，DMSO-d6)：δ12.20(bs，1H)，7.73(s，1H)，7.46-7.36(m，4H)，7.20(t，J＝7.2Hz，1H)，7.10(t，J＝7.2Hz，1H)，4.60-4.58(m，1H)，3.61(s，3H)；LC-MS：m/z 362.05(M+1)+。
EXAMPLE 142.1 Synthesis of 2- (benzo [ d ] oxazol-2-ylamino) -1-methyl-1H-benzo [ d ] imidazol-5-yl) -2, 2, 2-trifluoroethyl-1-ol
Conditions are as follows: a) sodium borohydride, MeOH, 0 deg.C-RT, 1h
To 1- (2- (benzo [ d ]) at 0 DEG C]Oxazol-2-ylamino) -1-methyl-1H-benzo [ d]To a stirred solution of imidazol-5-yl) -2, 2, 2-trifluoroethyl-1-one (50mg, 0.14mmol) in methanol (3mL) was added sodium borohydride (6mg, 0.15mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with cold water (10mL) and extracted with EtOAc (2X 20 mL). The combined organic layers were washed with water (15mL), brine (15mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography using 2% methanol/DCM as eluent to give the title compound (40mg, 80%);1H NMR(400MHz，DMSO-d6)：δ12.30(bs，1H)，7.77(s，1H)，7.46-7.34(m，4H)，7.20(t，J＝7.6Hz，1H)，7.10(t，J＝7.6Hz，1H)，6.90(bs，1H)，5.24-5.22(m，1H)，3.62(s，3H)；LC-MS：m/z363.30(M+1)+。
EXAMPLE 143 ELISA assay for measuring human heme Oxygenase-1 (human heme Oxygenase-1; HMOX-1) in HepG2 lysate
Reagents and technical description:
ELISA capture antibodies from R & D systems, standard HO-1, detection antibody and streptavidin-HRP (DYC3776-2) were supplied in DuoSet IC human Total HO-1 ELISA. The substrate mixture was KPL LumigLuO reserve chemiluminescent substrate (54-71-00). PBS is Corning (Corning) Cellgro cell culture (21-040-CV). 10xPBS + 0.05% Tween20 (tween20) (PBST) was from KPL.
All incubations of the plates were closed in drawers at room temperature (20 ℃).
The ELISA plate was processed manually. All loads used multichannel pipettes. The plate was emptied by shaking into the tank and tapping onto a paper towel to remove any remaining reagent. Plate washing was achieved by loading all wells with PBS + 0.05% tween 20(PBST) using a squirt bottle, shaking out the PBST and gently patting the plate onto paper towels.
Preparation of ELISA plate:
1. after reconstitution in PBS, the anti-HO-1 capture antibody was 1440 μ g/ml. This antibody was diluted to 8. mu.g/ml in PBS and added to 384 well greiner "limifrac 200" plates at 50. mu.l per well. The incubation was carried out overnight.
2. The plates were emptied and 100 μ Ι PBS + 1% BSA was added to all wells. Incubate at room temperature for 90 minutes.
Preparation of HepG2 lysate:
1. cells were cultured in 96-well tissue culture plates washed with PBS, and the plates were subsequently frozen at-70 ℃ overnight. The plate was warmed to ice temperature in an ice bucket. Add 20. mu.l PBS, 0.5% Triton X100, 1mM EDTA and 1 XHALT to each wellTMProtease inhibitors, and plates were incubated on ice for 1 hour.
2. The lysate was frozen at-20 ℃ overnight.
Measurement of HMOX1 ("HO-1")
1. Human HO-1 (after reconstitution in PBS + 0.5% Triton +1mM EDTA) was serially diluted 2-fold from 20ng/ml in PBS + 0.5% Triton-X100, 1mM EDTA in polypropylene 384-well plates to prepare 12-point standard curves containing zero points.
2. HepG2 lysate was diluted 1 to 20 fold in diluent No. 4 from the DUOSET kit in polypropylene 96 well plates, where the analytical standards were also diluted.
3. PBS + 1% BSA was emptied from ELISA plates and 30 μ Ι of all samples and standards were added (suggested to be repeated twice). The plates were incubated for 90 minutes.
4. The plates were emptied and washed 4 times with PBST.
5. HO-1 detection antibody was diluted to 200ng/ml in PBS + 1% BSA, and 30. mu.l was added to all wells. Incubate for 90 minutes.
6. The plates were emptied and washed 4 times with PBST.
7. To all wells 30 μ l of streptavidin HRP in 1/200 in PBS + 1% BSA (stock streptavidin concentration not specified) was added. Incubate for 30 minutes.
8. The plates were emptied and washed 4 times with PBST.
9. Lumiglo reserve reagent was diluted, two buffers, one Lumiglo substrate. Add 30 μ l to all wells. Incubate for 5 minutes.
10. Chemiluminescence was measured on spectramax M5 integrated using 150 ms.
Table 1 below lists EC for human HMOX1 protein levels relative to DMSO control after treatment with representative compounds50And fold changes.
Example 144 kinetic solubility
mu.L of a 10mM DMSO stock solution was aliquoted into 1.2mL of 96 well plates in 490. mu.L DMSO solution and 490. mu.L of DPBS solution pH 7.4 Dulbecco's phosphate buffered saline (Dulbecco's phosphate buffer saline), in duplicate. The final concentration of test compound was 200. mu.M. The samples were incubated at 25 ℃ for 16h with shaking at 200 rpm. The samples were centrifuged at 3500rpm for 20min at 25 ℃ and the supernatants submitted for HPLC analysis.
Kinetic solubility was measured using the following equation:
the kinetic solubilities of representative compounds of the invention are provided in table 1 below.
TABLE 1
2 or more EC50Mean value of measurement
ND is not determined
NA is inactive
Example 145 hERG analysis
Cell lines and cell cultures
HEK 293 cell line (catalog No. K1236) stably expressing hERG channel was purchased from Invitrogen. Cells were cultured in 85% DMEM, 10% dialyzed FBS, 0.1mM NEAA, 25mM HEPES, 100U/mL penicillin-streptomycin and 5. mu.g/mL blasticidin, and 400. mu.g/mL geneticin.
Cell use TrypLETMExpress is split approximately three times a week and is maintained between-40% to-80% confluence. Before analysis, at 5X 10 per 6cm cell culture dish5Cells were transferred to coverslips and induced with 1. mu.g/mL doxycycline (doxycycline) for 48 hours.
Preparation of solutions
1) External solution (in mM): 132NaCl, 4KCl, 3CaCl2，0.5MgCl211.1 glucose and 10HEPES (pH adjusted to 7.35 with NaOH)
2) Internal solution (in mM): 140KCl, 2MgCl210EGTA, 10HEPES and 5MgATP (pH adjusted to 7.35 with KOH)
3) The hERG current was tested in the presence of 10. mu.M compound concentration, with the final concentration of DMSO being 0.1%.
Experimental procedures
1) The coverslip was removed from the cell culture dish and placed on the microscope stage in the bath chamber.
2) The desired cells were positioned using a 10 × objective lens. By focusing above the cell plane, the tip of the electrode is positioned under the microscope using a 10 x objective lens once the tip is in focus, the electrode is pushed down towards the cell using a coarsely controlled manipulator while moving the objective lens to keep the tip in focus.
3) When directly above the cell, switch to 40 × objective and use a finely controlled manipulator to access the cell surface in small steps.
4) Gentle suction is applied through the side ports of the electrode clamps to form a gigaohm seal.
5) Cfast is used to remove the capacitance current coinciding with the voltage step. Whole cell configuration was obtained by applying repeated, brief, intense aspirations until the membrane patch ruptured.
6) The membrane potential was set at-60 mV at this time to ensure that the hERG channel was not open. The spike in the capacitor current is then eliminated using Cslow on the amplifier.
7) Setting the holding potential to-90 mV for 500 milliseconds (ms); the current at 50kHz was recorded and the current at 10kHz was filtered. The leakage current was tested at-80 mV for 500 ms.
8) hERG current was induced by depolarization at +30mV for 4.8 seconds, and then voltage was returned to-50 mV for 5.2 seconds to remove the inactivation and observe the deactivation tail current. The maximum amount of tail current magnitude is used to determine the hERG current amplitude.
9) The current was recorded for 120 seconds to analyze the current stability. Only stable cells with parameters above the threshold were recorded for compound administration.
10) Vehicle controls were first applied to the cells to establish a baseline. Once the hERG current was found to be stable for 5 minutes, the test compound was applied. The hERG current in the presence of test compounds was recorded for approximately 5 minutes to reach steady state and 5 scans were subsequently captured. To ensure good performance of the cultured cells and the manipulations, the same batch of cells was also tested using the positive control Dofetilide (Dofetilide) with 5 dose concentrations.
Data analysis
The following criteria were used to determine the acceptability of the data.
1) Initial seal resistance >1G Ω;
2) leakage current < 50% of control peak-to-tail current at any time;
3) peak-to-tail amplitude >250 pA;
4) the membrane resistance Rm is more than 500 MOmega;
5) an access resistance (Ra) <10M Ω;
6) the apparent drop in peak current was < 2.5%/min.
Data that meet the above hERG current quality criteria are further analyzed as follows.
1) Percent hERG current inhibition was calculated using the following equation.
Note that: PatchMaster or Clampfit software was used to extract the peak current from the raw data.
2) Using Graphpad Prism 6.0, dose response curves for test compounds were plotted as percentage of hERG current inhibition versus test compound concentration and fitted to sigmoidal dose response curves with variable slopes.
hERG data for selected compounds in the manual patch clamp assay are provided in table 2 below. Notably, only the hERG values were measured for compounds showing > 5 μ M solubility. For compounds with a solubility below 5 μ M, accurate measurement of hERG activity is not possible.
R: methyl radical
TABLE 2
1. Example 7 is a tetrahydrobenzo [ d ] thiazole compound instead of a benzothiazole compound.
R is ethyl.
R is CH2CH2OMe。
EXAMPLE 146 Microthermophoresis (MST) assay/Bach 1 binding assay
Overview of protein expression/production
Human BACH1 (construct-ID: 10XHIS-GP-BACH1(aa179-736) -Thrombin-FLAG) was expressed in Sf9 insect cells. Protein purification was performed using affinity chromatography followed by size exclusion chromatography. SDS-PAGE analysis was used to determine the quality of the resulting protein.
Principle of analysis
The MST technique is based on the measurement of protein movement along a temperature gradient. Thus, a fluorescently labeled protein is loaded into the capillary where an infrared laser heats a small volume. Before and during heating, the fluorescence intensity of the irradiated site was measured, and the fluorescence loss in the IR laser focus was quantified.
Two main factors contribute to the change in fluorescence signal. First, the so-called TRIC (temperature-dependent intensity variation) effect is caused by the quantum yield of the temperature-dependent fluorophore. Herein, the degree of temperature dependence depends on the chemical environment, which can be altered by binding of ligands (e.g. compounds, peptides) to the target. Furthermore, the Kd value between the ligand and the target may depend on the temperature. Second, thermophoresis, defined as the movement of fluorescent molecules along a temperature gradient, mainly causes fluorescence changes. Thus, protein movement along the gradient is dependent on hydrodynamic radius, charge and hydration of the outer layer. These properties may be changed after the labeled protein has been bound to another species, such as a compound. Thus, MST measures the difference in protein movement as the ligand concentration increases: from this, the fraction of ligand-binding protein and the Kd value can be determined.
MST assay protocol
For the determination of Kd for compound binding to BACH1, the following protocol was applied: 10XHIS-GP-BACH1(aa179-736) -Thrombin-FLAG (expressed and purified on Proteros) was fluorescently labeled by its lysine-residue (2 nd generation NHS-NT.647 dye).
The experiment was performed using the experimental setup of NanoTemper Technologies, Monolith NT.115Pico, with the MST power set to medium and the excitation power set to 2% at a reaction temperature of 25 ℃.
10nM fluorescently labeled BACH1 was applied to a reaction volume of 8. mu.L of reaction buffer containing 50mM HEPES pH 8.0, 100mM NaCl, 5% glycerol, 0.05% Tween 20, and 1mM DTT using a Monolith NT.115 quality capillary. Compounds were applied at a maximum concentration of 103 μ M, followed by 15 2-fold factor dilutions.
Kd data for selected compounds in the MST assay are provided in table 3 below.
Table 3.
Example numbering
Kd(μM)
Kd error (μ M)
17
0.25
0.051
33
0.26
0.051
56
0.25
0.15
112
0.56
0.13
124
0.20
0.089
From the data provided in table 3 above, it can be concluded that the compounds of the present invention also bind to Bach 1.
Example 147. the HMOX1 inducer of the invention strongly increased the expression of HMOX1 in vitro
RNA isolation and quantification of Gene expression
Inducible HMOX1 expression has been shown to reduce the Vascular Obstruction Crisis (VOC) in Sickle Cell Disease (SCD) mice, particularly heme-induced vascular obstruction (Belcher JD et al, J2017, 26:748 762, Krishn Amoorthy S et al, J2017, 2: e96409, J clinical research and insights). HepG2 cells were seeded at 250 k/well in 12-well tissue culture plates coated with collagen (collagen I, rat tail (thin plate coating); ENZO Life Sciences) for approximately 24 hours. Subsequently, the seeded cells were treated with dose titration of the compound of example 17 or dimethyl fumarate (DMF), an Nrf2 activator (Fisher Scientific), with three biological replicates of each treatment, by media exchange. After approximately 24 hours of treatment, the cells were visually inspected to confirm that the compound treatment was not toxic. Prior to RNA isolation, the treated cells were washed with PBS, aspirated, sealed with a sealing membrane and frozen at-80 ℃. RNA isolation kit Using Machery-Nagel ((II)) RNA: catalog 740955.250), and then quantified using a seemer fisher (ThermoFisher) Nanodrop.
Dilution of RNA for Nanostring baseDue to expression analysis. The diluted RNA at the input of 200ng RNA was run on a Nanostring instrument that supportedSPRINT Profiler System and nSolver4.0 software,SPRINT Cartridge、SPRINT Reagent Pack andSPRINT Hybridization Buffer. Analysis was performed manually using raw data obtained from nsolver4.0 software. CLTC, POLR2A, RPL27 and TBP were used as reference genes for normalization. Data are shown as fold changes compared to DMSO treatment.
Relative mRNA levels of the HMOX1 gene in HepG2 cells treated with the compound of example 17 or DMF were measured according to the methods described above. It should be noted that for comparative analysis of gene expression between the compound of example 17 and DMF, HepG2 cells were treated with the test compound for 24 hours (results are shown in figure 1).
The compound of example 17 was found to induce HMOX1 expression much more strongly than the other Nrf2 activator DMF (fig. 1A). For example, 30 μ M of the compound of example 17 induced 4-fold higher expression of HMOX1 than 250 μ M DMF.
The relative mRNA levels of several other Bach 1-responsive genes in HepG2 cells treated with the compound of example 17 and the Nrf2 activator DMF were also compared. In addition, the compound of example 17 and DMF induced FTH1 expression at comparable fold increases (fig. 1B).
EXAMPLE 148 HMOX inducers of the invention reduce vascular occlusion in SCD mice, increase embryonic hemoglobin (HbF) in SCD mice
Dosing regimen of HMOX inducer to HbSS Townes sickle cell disease mice
HbSS towns sickle cell disease mice were orally administered a gavage vehicle or compound of example 17 listed below for 8 days once a day. To minimize experimental inconsistencies, fresh formulations were prepared daily and vortexed before drawing test substances into the syringe for each administration, and administered by the same individual throughout the day. The last day dose was administered by oral gavage for 4 hours, followed by hemin infusion as described below.
1. Vehicle (0.45% w/v methylcellulose with 0.5% w/v Tween 80)
2. The compound of example 17 (10mg/kg PO administration)
3. The compound of example 17 (25mg/kg PO administration)
4. The compound of example 17 (50mg/kg PO administration)
Measurement of vascular occlusion (stasis)
Male and female sickle cell disease mice approximately 12 weeks old were weighed prior to surgery. Animals were anesthetized with a mixture of ketamine (106mg/Kg) and xylazine (7.2mg/Kg) and surgically implanted with a dorsal skin-fold chamber (DSFC). On the day of anesthesia, mice were placed on a special live microscopy table, and flowing subcutaneous venules in 20 to 22 DSFC windows were selected and located. After venule selection and location, hemin chloride (2.677 mM; Frontier Scientific) dissolved in sterile saline containing sodium carbonate (11.36 mM; Sigma-Aldrich) and D-sorbitol (9.59 mM; Sigma-Aldrich) was filtered (0.22 μ M), diluted 1:10 in sterile saline (final 267.7 μ M hemin) and infused into the tail vein of mice (.012ml/g, 3.2 μmol heme/kg body weight). 1h after hemin infusion, all selected venules were rechecked and the number of static (no-flow) venules was counted and expressed as percent stasis. Four hours after hemin infusion, mice were in CO 2Euthanasia in atmosphere and remove liver, spleen and kidney, snap frozen and stored at-85 ℃.
Measurement of F-cells
Heparinized whole blood was collected from the inferior vena cava of HbSS Townes mice dosed with vehicle or compound of example 17 4 hours after hemin infusion. F-cells were stained on whole blood smears by the Kleihauer-Betke method using the embryonic cell staining kit (Simmler) according to the manufacturer's instructions. F-cells and total red blood cells were counted in 4 independent microscopic fields for each mouse at 100X magnification. F-cells are expressed as a percentage of total red blood cells (erythrocytes/red blood cells). Human embryonic umbilical cord blood was used as a positive control.
Vascular occlusion is a hallmark of SCD. To evaluate whether the disclosed compounds are effective in reducing vascular occlusion, the method described above measures heme-induced vascular occlusion (stasis) in the subcutaneous venules of HbSS-Townes sickle cell disease mice implanted with dorsal skinfold chamber (DSFC). As shown in figure 2, microvascular stasis was significantly reduced in those mice administered the compound of example 17 compared to HbSS-Townes mice administered vehicle one hour after heme infusion. In addition, the compound of example 17 dose-responsively inhibited microvascular stasis (fig. 2). For the compound of example 17, stasis was reduced to-17% (10mg/kg dose), to-12% (25mg/kg dose) and to-5% (50mg/kg dose).
At sufficiently high concentrations, HbF can inhibit hemoglobin S (HbS) polymerization and subsequent hemolysis and vessel occlusion (Krishnhamoorchy S et al J. Clin. Res. and insights 2017,2: e 96409). To evaluate whether the HMOX inducer of the present invention can effectively increase HbF, F-cells (i.e., HbF-containing red blood cells) were measured as a percentage of total red blood cells according to the methods described above. As shown in figure 3, the percentage of F-cells was significantly increased in those mice administered the compound of example 17 compared to HbSS-Townes mice administered vehicle. Specifically, the percentage of F-cells increased to 55-70% at all tested doses of the compound of example 17, more than two-fold compared to vehicle-dosed mice. The increase in the percentage of F-cells did not appear to be dose-responsive.
The results shown above demonstrate that the compounds of the present invention are HMOX inducers and Bach 1 binders/inhibitors and can be used to treat SCD at least by binding to Bach 1, increasing HMOX1 activity, increasing HbF, and reducing vascular occlusion.
Example 149 Glutathione (GSH) levels in Primary human endothelial cells
Primary Human Pulmonary Artery Endothelial Cells (HPAEC) (Lonza # CC-2530) cultured in endothelial cell growth medium-2 (EGM2 medium, Lonza # CC-3162) were seeded in white 96-well plates (12.5 k cells per well) #3610) and left at 37 ℃ with 5% CO2In (1). Cells were treated with the compound of example 17 by media change 18 to 24 hours after seeding. After 24 hours of treatment with the compound, cells were treated with freshly prepared stock of hemin (Sigma-Aldrich #51280 in 0.1N NaOH), alone or with the compound of example 17. After 30min of hemin stress, the cells were visually examined for toxicity, where toxicity was not noted. Media was withdrawn and GSH-Glo was performed according to the manufacturer's protocolTMGlutathione assay (Promega # V6911). A white bottom plate seal (PerkinElmer #6005199) was used during reading. Analysis was performed using Softmax Pro.
The results are shown in fig. 4. When hemin induces oxidative stress in primary human pulmonary arterial endothelial cells, thereby reducing GSH levels, pre-incubation with an HMOX inducer/Bach 1 inhibitor (e.g., the compound of example 17) protects these cells from hemin-mediated oxidative stress.
Example 150 Gene expression in Primary human endothelial cells
Inflammatory conditions in endothelial cells increase the expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, by NF-. kappa.B signaling. HMOX 1-/-endothelial cells showed increased expression of VCAM-1 in response to TNF stimulation, compared to HMOX +/+ endothelial cells. See Seldon et al, J Immunol, 2007, 12.1, 179(11), 7840-7851.
Primary Human Pulmonary Artery Endothelial Cells (HPAEC) (Lonza # CC-2530) were seeded at 200k cells per well in 12-well plates (TrueLine # TR5001) and placed at 37 ℃ with 5% CO2In (1). Cells were treated with the compound of example 17 by media change 18 to 24 hours after seeding.After 24 hours of treatment with the compound, 10ng/mL TNF α (Invitrogen # PHC3015 in H) was used alone or with the compound of example 172O) treating the cells. After 4 hours of treatment, the cells were visually examined for toxicity, with no toxicity noted. The medium was aspirated, the cells were washed once with 1 × PBS (Corning #21-040-CV), aspirated to dryness, sealed with a sealing membrane and left at-80 ℃ until RNA isolation was performed. By usingKit (MACHEREY-NAGEL #740955.250, USA) RNA was isolated according to the kit protocol. In a 20 μ L reaction, RNA was used to generate cDNA using a large capacity cDNA reverse transcription kit (seimer feishol #4368814 in the united states). After completion of reverse transcription, the cDNA was diluted 1:10 with water. 20ng of cDNA was mixed with water and iQTM Green Supermix (BioRad # 170-. Raw data was exported from the software (Biorad CFX Manager in usa) and imported into a spreadsheet (Microsoft Excel in usa). To calculate fold change, the Δ Ct from each sample was calculated as Ct (Gene of interest) -Ct (average of reference genes). Subsequently, Δ Δ Ct was calculated as Δ Ct (experimental sample) -average Δ Ct (control). Fold change was calculated as 2 -ΔΔCT。
The results are shown in fig. 5. Specifically, TNF- α stimulation was used to activate human primary endothelial cells. For example, TNF- α induces the expression of Vascular Cell Adhesion Molecules (VCAM) in primary human pulmonary artery endothelial cells. However, pretreatment with an HMOX inducer/Bach 1 inhibitor (e.g., the compound of example 17) reduced this TNF- α mediated endothelial cell activity as evidenced by the reduced expression of the adhesion molecule VCAM-1.
Example 151 PK study
Male C57BL/6 mice 9 to 10 weeks old were orally administered 50mg/kg of the test compound formulated in 5% w/v Tween 80 and 0.5% methyl cellulose. After 0.5 and 4h of dosing, whole blood was collected, animals were sacrificed and brain tissue was collected. Blood samples were immediately placed on ice and centrifuged at 14000rpm for 3min at 4 ℃ within 60min to obtain plasma. The plasma was transferred by pipette to a pre-labeled Eppendorf tube and stored at-80 ℃ until analysis.
Brain tissue samples were collected and rinsed with fresh ice cold 0.9% NaCl solution, flash dried and thereafter frozen on dry ice/liquid nitrogen, and stored at-70 ± 10 ℃ until analysis. Phosphate buffered saline was used for homogenization and used for analysis. Bioanalysis was performed using LC-MS/MS (API 4000).
The test results are provided in table 4 below. The compound of example 56 showed good brain penetration.
TABLE 4
Not applicable
Compounds of example 17 and compounds of example 56 were measured in the same study. Comparative example 1 was measured in a previous study.
Example 152 PD study
Male C57BL/6 mice 9 to 10 weeks old were orally administered 50mg/kg of vehicle or compound formulated in 5% w/v Tween 80 and 0.5% methyl cellulose. At designated time points (3 and 6 hours or 4 and 8 hours post-dose, as shown in the results below), whole blood was collected, animals were sacrificed and liver was collected. Blood samples were transferred to bottles containing 4 μ L of 10% w/v EDTA and centrifuged at 6000rpm for 8in below 10 ℃ to obtain plasma. Samples were frozen and later processed for HMOX1 protein levels as indicated below:
HMOX1 protein levels: mouse liver
Preparation of homogenization/lysis buffer
Homogenization buffers were prepared according to table a.
TABLE A preparation of homogenization buffer
DTT (seegmiel technologies # R0861) was added to a final concentration of 5mM immediately prior to use, and a 100XHalt protease and phosphatase inhibitor cocktail (seegmiel technologies #78440) was added to a final concentration of 1X.
Preparation of mouse liver samples
Liver samples were crushed by mortar and pestle under refrigerated conditions. The crushed tissue was transferred to a pre-cooled bead breaker OMNI tube (OMNI International, Cat. No. 19-628). To the comminuted tissue, 1mL of homogenization buffer (+ DTT and HALT) was added, and the tube was placed on ice. The sample was loaded and passed through the bead breaker at a speed of 5.64m/s for two 20 second cycles with a 10 second dwell between cycles and the sample was immediately returned to ice. All materials were transferred to a freshly cooled 2mL Eppendorf tube. The sample was centrifuged at 2,000rpm at 4 ℃ for 5 minutes. The supernatant was transferred to a freshly cooled 1.7mL Eppendorf tube. The sample was centrifuged at 2,000rpm at 4 ℃ for 5 minutes. The supernatant consisting of cytosol and microsomes was transferred to a freshly cooled 1.7mL Eppendorf tube. The samples were stored at-80 ℃. Microsome samples were quantified using the Pierce 660 assay (seemer fly catalog No. 22660) with pre-diluted BSA standards ranging from 125 to 2000 μ g/mL (seemer fly catalog No. 23208). For an ELISA input of 0.5. mu.g in 50. mu.l, each sample was diluted to 10 ng/. mu.l.
Preparation of analytical reagents
All assay reagents were provided in the heme oxygenase 1(HO1) mouse SimpleStep ELISA kit (Abcam # ab 204524). All reagents were equilibrated to room temperature before use. 1X PT was prepared by diluting 10X wash buffer PT with deionized water at 1: 10. The antibody mixture was prepared by diluting the 10X capture antibody and the 10X detection antibody to 1X in antibody diluent 5 BI. Mouse heme oxygenase 1 protein standards were reconstituted using 500 μ l homogenization buffer (table a), mixed and held at room temperature for 10 minutes before dilution. An eight-point standard curve was generated by diluting the stock standard of the standard curve at 1:2, ranging from 10,000 to 156.3 pg/mL.
Heme oxygenase 1(HO1) mouse SimpleStepELISA
Add 50 μ Ι of all samples and standards to the appropriate wells, repeated twice. To each well was added 50. mu.l of a 1X antibody mix. The plates were sealed with the seals provided and incubated at room temperature on a plate shaker at 400 rpm. After one hour of incubation, the antibody mixture was aspirated, and the wells were washed three times with 350 μ Ι of 1 × wash buffer PT, with adequate aspiration between each step. After the last wash was aspirated to dryness, 100 μ l of TMB substrate was added to each well and the plates were incubated at 400rpm on a plate shaker in the dark at room temperature for 10 minutes. After 10 min incubation, 100 μ l of stop solution was added to each well. The plate was shaken at 400rpm for 1 minute for mixing. Softmax Pro 7.0.3 was used to read OD at 450nm and for analysis.
Heme oxygenase 1 protein levels in mouse plasma
Preparation of analytical reagents
All assay reagents were provided in the heme oxygenase 1(HO1) mouse SimpleStep ELISA kit (Abcam # ab 204524). 1X PT was prepared by diluting 10X wash buffer PT with deionized water at 1: 10. The antibody mixture was prepared by diluting the 10X capture antibody and the 10X detection antibody to 1X in antibody diluent 5 BI. Mouse heme oxygenase 1 protein standards were reconstituted using 500 μ l of sample diluent NS, mixed and held at room temperature for 10 minutes before dilution. An eight point standard curve was generated by diluting the stock standard of the standard curve at 1:2, ranging from 5,000 to 78.1 pg/mL.
Preparation of plasma samples
Prior to ELISA, frozen plasma was thawed on ice. Plasma samples were diluted 1:10 in sample diluent NS, where the final concentration was 10% plasma.
Heme oxygenase 1(HO1) mouse SimpleStep ELISA
Add 50. mu.l of sample and standard to the appropriate wells and repeat twice. To each well was added 50. mu.l of a 1X antibody mix. The plates were sealed with the seals provided and incubated at room temperature on a plate shaker at 400 rpm. After one hour of incubation, the antibody mixture was aspirated, and the wells were washed three times with 350 μ Ι of 1 × wash buffer PT, with adequate aspiration between each step. After the last wash was aspirated to dryness, 100 μ l of TMB substrate was added to each well and the plates were incubated at 400rpm on a plate shaker in the dark at room temperature for 10 minutes. After 10 min incubation, 100 μ l of stop solution was added to each well. The plate was shaken at 400rpm for 1 minute for mixing. Softmax Pro 7.0.3 was used to read OD at 450nm and for analysis.
Representative compounds of the present disclosure were tested and the results are provided in table 5 below. Plasma and liver protein levels were assessed by the mouse HMOX1 ELISA assay (kit) and were fold-induced compared to time-matched vehicle control mice. Carboxylic acids showed a modest increase in HMOX1 protein, but at concentrations much higher than EC50(30 to 100 x).
TABLE 5
Time points were 4 and 8 hours instead of 3 and 6 hours.