Diaryl urea compound and its preparation method and medicinal use
阅读说明：本技术 二芳基脲类化合物及其制法和药物用途 (Diaryl urea compound and its preparation method and medicinal use ) 是由 肖志艳 杨亚军 杨颖� 景连栋 张浩超 于 2019-11-05 设计创作，主要内容包括：本发明属于药物化学领域,公开了式(I)化合物所示新的二芳基脲类化合物及其生理上可接受的盐,溶剂化物以及结晶形式,所述化合物的制备方法,含有所述化合物的药物制剂,以及所述化合物在治疗CDK8相关疾病如肿瘤等临床上的应用。(The invention belongs to the field of pharmaceutical chemistry, and discloses a novel diaryl urea compound shown as a compound in a formula (I), and a physiologically acceptable salt, solvate and crystal form thereof, a preparation method of the compound, a pharmaceutical preparation containing the compound, and clinical application of the compound in treatment of CDK8 related diseases such as tumors and the like.)
1. Diaryl urea compounds represented by the following general formula (I) and physiologically acceptable salts thereof,
wherein the content of the first and second substances,
x is selected from CH2Or an oxygen atom;
y is selected from 2H or oxygen atom;
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen;
R3selected from the following structures:
n is 1, 2 or 3.
2. The compound of claim 1, wherein the compound is a compound of formula (IA) and a physiologically acceptable salt thereof:
y is selected from 2H or oxygen atom;
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen;
R3selected from the following structures:
3. a compound according to claim 2, and the physiologically acceptable salts thereof, characterized in that said compound is of the general formula (IAa):
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen.
4. The compound and physiologically acceptable salts thereof according to claim 2, wherein the compound is a compound represented by the general formula (IAb):
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen.
5. The compound and physiologically acceptable salts thereof according to claim 2, wherein the compound is a compound represented by the general formula (IAc):
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substitutedA substituted halogen.
6. A compound having the structure and physiologically acceptable salts thereof, wherein the compound is selected from the group consisting of:
7. a process for the preparation of a compound according to any one of claims 1 to 6, comprising the steps of:
X、Y、n、R1、R2、R3as defined in any one of claims 1 to 6.
8. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 6 and physiologically acceptable salts thereof and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, pills, injections, sustained release formulations, controlled release formulations, and various microparticle delivery systems.
10. Use of a compound according to any one of claims 1 to 6, and physiologically acceptable salts thereof, for the preparation of Cyclin dependent kinase 8 (CDK 8) inhibitors.
11. Use of a compound according to any one of claims 1 to 6 and physiologically acceptable salts thereof for the manufacture of a medicament for the treatment of tumours.
Technical Field
The invention relates to novel diaryl ureas of general formula (I) and physiologically acceptable salts thereof. The application of the compounds in preparing Cyclin dependent kinase 8 (CDK 8) inhibitors and medicaments for treating tumors and pharmaceutical compositions containing the compounds.
Background
Malignant tumor has become a common disease which endangers the life health of people, and since the 21 st century, the malignant tumor is still one of the major diseases which seriously threaten the health of human beings, and along with the change of factors such as living habits, diet and living environment of people, the incidence of the malignant tumor is also obviously changed, but the general incidence trend is still not optimistic. The study shows that 1010 ten thousand of new malignant tumor patients and 620 ten thousand of death patients all over the world in 2000 have been diagnosed as the patients with the diseases 2240 ten thousand. The latest global tumor patient data published by the world health organization in 2012 shows that 1410 ten thousands of newly-increased tumor groups in the world die 820 ten thousands of new tumor groups in the world, wherein the newly-increased tumor patients in China are the first to live in the world.
CDK8 is an important component of the mediator complex and plays a critical role in the functioning of the mediator. CDK8 was thought to be involved in multiple regulatory pathways, high expression of which was found to be present in many colorectal cancer samples, while aberrant expression of CDK8 was also found in some adenocarcinomas. CDK8 is considered as a potential target for the treatment of colorectal, breast and some adenocarcinomas. Therefore, anticancer drugs targeting CDK8 kinase have become the subject of great interest and research for pharmaceutical and biotechnology companies.
The early studies of CDK8 were mostly associated with colorectal cancer, and there have been recent advances in CDK 8's association with cancer. The most prominent of them belongs to breast cancer. CDK8 was found to be expressed in elevated levels in breast cancer, closely linked to the progression of breast cancer, and the highly expressed CDK8 and its interacting proteins were considered to be associated with a shorter progression-free survival of patients. Researchers believe that CDK8 inhibitors may be used as first line agents in combination with selective estrogen receptor down-regulators or aromatase inhibitors in the future to improve therapeutic efficacy and reduce hormone therapy resistance. In other aspects, CDK8 may be involved in malignant tumors such as gastric cancer, ovarian cancer, melanoma, etc., but related studies are not deep enough.
At present, although CDK8 kinase inhibitor small molecules are reported, no drug is successfully marketed due to poor pharmacokinetic property and kinase selectivity. In view of this, the inventors of the present application intend to provide a novel, safe and effective molecular targeted small molecule antitumor drug.
Disclosure of Invention
The invention aims to provide a novel diaryl urea compound shown in a general formula (I).
The invention also aims to provide a method for preparing diaryl urea compounds shown in the general formula (I) and analogues thereof.
The invention also aims to provide application of the compound shown in the general formula (I) in preparing CDK8 inhibitors and in preparing medicaments for treating tumors.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
wherein the content of the first and second substances,
x is selected from CH2Or an oxygen atom;
y is selected from 2H or oxygen atom;
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen;
R3selected from the following structures:
n is 1, 2 or 3.
In a further aspect of the present invention there is provided a compound of formula (IA) and physiologically acceptable salts thereof:
y is selected from 2H or oxygen atom;
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen;
R3selected from the following structures:
a further embodiment of the present invention provides compounds of the general formula (IAa) and physiologically acceptable salts thereof:
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen.
Still another embodiment of the present invention is to provide a compound represented by the general formula (IAb):
R1selected from hydrogen, mono-or poly-substitutedSubstituted halogen, mono-or polysubstituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen.
Still another embodiment of the present invention is to provide a compound represented by the general formula (IAc):
R1selected from hydrogen, mono-or poly-substituted halogen, mono-or poly-substituted trifluoromethyl;
R2selected from hydrogen, mono-or polysubstituted C1-C3Alkyl, mono-or poly-substituted halogen.
In another aspect, the present invention provides the compound and physiologically acceptable salts thereof, wherein the compound is selected from the group consisting of:
the invention also provides a synthesis method of the compound shown in the general formula (I), which comprises the following steps:
X、Y、n、R1、R2、R3as defined in any one of claims 1 to 7.
For the preparation of medicaments, the compounds of the general formula (I) and their physiologically acceptable salts are mixed in a known manner with suitable pharmaceutical carrier substances, fragrances, flavors and colors in a known manner and are formed into tablets or coated tablets, or are suspended or dissolved in water or oil with other additional substances.
The invention also relates to a pharmaceutical composition containing a pharmaceutically effective dose of the compound shown as the general formula I and physiologically acceptable salts thereof and a pharmaceutically acceptable carrier.
The compounds of the invention may be administered orally or parenterally. The oral preparation can be tablet, capsule, and coating agent, and the parenteral preparation can be injection and suppository. These formulations are prepared according to methods well known to those skilled in the art. Adjuvants used for the manufacture of tablets, capsules, coatings are the customary auxiliaries, such as starch, gelatin, gum arabic, silica, polyethylene glycol, solvents for liquid dosage forms, such as water, ethanol, propylene glycol, vegetable oils, such as corn oil, peanut oil, olive oil, etc. The formulations containing the compounds of the present invention may also contain other adjuvants such as surfactants, lubricants, disintegrants, preservatives, flavoring agents, coloring agents, and the like.
The invention also provides application of the compound and the physiologically acceptable salt thereof in preparing CDK8 inhibitors and medicines for treating tumors.
The beneficial technical effects are as follows:
poor specificity of action is one of the major challenges facing the current study of CDK8 inhibitors. The compound has the characteristics and advantages of specifically inhibiting CDK8, and is expected to provide a novel, safe and effective CDK8 inhibitor as a small-molecule antitumor drug
Drawings
FIG. 1. Selective inhibition of CDK8 by Compounds (Single concentration inhibition of kinase by the Compound of interest (10. mu.M)).
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.
The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS) or High Resolution Mass Spectrometry (HRMS). NMR shifts (δ) in parts per millionThe units in (ppm) are given. m.p. is the melting point given in ° c, the temperature is uncorrected. The column chromatography generally uses 200-300 mesh silica gel as a carrier. NMR was measured using INOVA-300 and CDCl as the solvent3、DMSO-D6The internal standard is TMS and the chemical shifts are given in ppm. MS was measured using an Agilent LC/MSD TOF LC/MS spectrometer.
Example 1: synthesis of Compound 1
1) Synthesis of p-nitrophenoxyacetylmorpholine
P-nitrophenoxyacetic acid (788mg, 4mmol), morpholine (348.5mg, 4mmol), HATU (1672mg, 4.4mmol), DIEA (1548mg, 12mmol) were weighed out in 10mL dichloromethane and stirred overnight at room temperature to give a large amount of solid the next day, which was directly filtered to give a yellow solid which was washed several times with dichloromethane and largely faded yellow to give a off-white solid, 37a, about 900mg, 78% yield.
2) Synthesis of p-aminophenoxyacetyl morpholine
P-nitrophenoxyacetylmorpholine (440mg, 1.53mmol) was dissolved in 5mL of methanol, Pd/C (44mg, 10%) was added, and the flask was purged three times with a balloon of hydrogen under normal pressure, followed by stirring at room temperature for about 3 hours. After the reaction, the palladium-carbon was filtered off with celite, the filter cake was washed with methanol several times, and the filtrate was concentrated under reduced pressure to give p-aminophenoxyacetylmorpholine (pale yellow after agricultural standing in air), which was about 390mg, with a yield of 99%.
3) Synthesis of 1- (3, 4-dichlorophenyl) -3- (4- (2-morpholine-2-oxoethoxy) phenyl) urea
3, 4-dichlorobenzene (94mg, 0.5mmol) was dissolved in 3mL dichloromethane, p-aminophenoxyacetyl morpholine (118mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 64%. mp:236-238 deg.C;1H NMR(400MHz,DMSO-d6)δ8.91(s,1H),8.61(s,1H),7.86(d,J＝2.4Hz,1H),7.49(d,J＝8.8Hz,1H),7.32(d,J＝8.9Hz,2H),7.29(d,J＝2.4Hz,1H),6.86(d,J＝8.8Hz,2H),4.76(s,2H),3.55-3.59(m,4H),3.45(s,4H)；HR-ESI-MS:m/z＝424.0826[M+H]+,calcd for C19H20Cl2N3O4:424.0825.
example 2: synthesis of Compound 2
3, 5-bistrifluoromethylphenylisocyanate (127mg, 0.5mmol) was dissolved in 3mL of dichloromethane, p-aminophenoxyacetylmorpholine (118mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 hours. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 70%. mp:240 ℃ and 242 ℃;1H NMR(400MHz,Acetone-d6)δ8.77(s,1H),8.26(s,1H),8.20(s,2H),7.58(s,1H),7.44(d,J＝8.8Hz,2H),6.93(d,J＝9.2Hz,2H),4.79(s,2H),3.53-3.66(m,8H)；HR-ESI-MS:m/z＝492.1352[M+H]+,calcd forC21H20F6N3O4:492.1353.
example 3: synthesis of Compound 3
1) Synthesis of 1- (3-chlorophenyl) -3- (4-hydroxy-2-methylphenyl) urea
Weighing m-chlorobenzene isocyanate (1.535g, 10mmol), dissolving in 20mL of anhydrous dichloromethane, adding 4-amino-3-methylphenol (1.23g, 10mmol) into the system, stirring at room temperature for reaction for 1h, and after the reaction is finished, generating a large amount of white flocculent solid substances in the system. The white solid material was filtered and the filter cake was suction dried to give a white solid, about 2.8g, 86% yield.
2) Synthesis of 1- (3-chlorophenyl) -3- (2-methyl-4- (2-morpholine-2-oxoethoxy) phenyl) urea
1- (3-chlorophenyl) -3- (4-hydroxy-2-methylphenyl) urea (8)0mg, 0.3mmol), 2-chloroacetyl morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol) in 2mL DMF and heated to 80 ℃ for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on a silica gel column to give a white solid, about 50mg, in 41% yield. mp:108-110 ℃;1H NMR(400MHz,Acetone-d6)δ8.40(s,1H),7.81(t,J＝2.0Hz,1H),7.56(d,J＝8.4Hz,1H),7.39(s,1H),7.30-7.34(m,1H),7.25(t,J＝8.0Hz,1H),6.96-6.99(m,1H),6.76-6.83(m,2H),4.78(s,2H),3.50-3.69(m,8H),2.23(s,3H)；HR-ESI-MS:m/z＝404.1366[M+H]+,calcd forC20H23ClN3O4:404.1372.
example 4: synthesis of Compound 4
1- (3-chlorophenyl) -3- (2-fluoro-4-hydroxyphenyl) urea (84.2mg, 0.3mmol), 2-chloroacetyl morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol) dissolved in 2mL of DMF was heated to 80 ℃ for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on a silica gel column to give a white solid, about 50mg, with a yield of 40%. mp 216 ℃ and 218 ℃;1H NMR(400MHz,DMSO-d6)δ9.10(s,1H),8.33(d,J＝1.2Hz,1H),7.80(t,J＝9.2Hz,1H),7.70(t,J＝2.0Hz,1H),7.28(t,J＝8.0Hz,1H),7.20-7.23(m,1H),6.98-7.02(m,1H),6.91(dd,J＝12.8,2.8Hz,1H),6.72-6.76(m,1H),4.82(s,2H),3.54-3.60(m,4H),3.42-3.45(m,4H)；HR-ESI-MS:m/z＝408.1115[M+H]+,calcd for C19H20ClFN3O4:408.1121.
example 5: synthesis of Compound 5
1-(3-chlorophenyl) -3- (3, 5-dichloro-4-hydroxyphenyl) urea (99.5mg, 0.3mmol), 2-chloroacetyl morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol) dissolved in 2mL of DMF was heated to 80 ℃ for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on a silica gel column to give a white solid, about 50mg, in 58% yield. mp:174-176 ℃;1H NMR(400MHz,DMSO-d6)δ9.06(s,1H),9.01(s,1H),7.65-7.68(m,1H),7.58(s,2H),7.25-7.32(m,2H),7.03(dt,J＝6.8,2.0Hz,1H),4.63(s,2H),3.56-3.60(m,4H),3.46-3.52(m,4H)；HR-ESI-MS:m/z＝458.0433[M+H]+,calcd for C19H19Cl3N3O4:458.0436.
example 6: synthesis of Compound 6
1- (3-chlorophenyl) -3- (4-hydroxynaphthalen-1-yl) urea (93.8mg, 0.3mmol), 2-chloroacetyl morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol) dissolved in 2mL of DMF was heated to 80 ℃ for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on silica gel to give a white solid with a yield of 51%. mp 170-172 ℃;1H NMR(400MHz,DMSO-d6)δ9.53(s,1H),8.80(s,1H),8.24-8.26(m,1H),8.10-8.13(m,1H),7.73-7.75(m,1H),7.67(d,J＝8.4Hz,1H),7.53-7.62(m,2H),7.28-7.30(m,2H),6.97-7.01(m,1H),6.92(d,J＝8.4Hz,1H),5.01(s,2H),3.48-3.61(m,8H)；HR-ESI-MS:m/z＝440.1371[M+H]+,calcd forC23H23ClN3O4:440.1372.
example 7: synthesis of Compound 7
1) Synthesis of 1- (3-chlorophenyl) -3- (4-phenoxyacetic acid carbethoxy) urea
1- (3-chlorophenyl) -3- (4-hydroxyphenyl) urea (1310mg, 5mmol), ethyl bromoacetate (1670mg, 10mmol), potassium carbonate (1380mg, 10mmol), potassium iodide (415mg, 2.5mmol) were dissolved in 10mL of DMF and heated to 80 ℃ for about 5 h. After the reaction was completed, saturated brine was added to the reaction solution, extraction was performed several times with ethyl acetate, the ethyl acetate organic layers were combined to obtain a solid containing a part of impurities, and the solid matter was washed with ethyl acetate to obtain a white solid, about 800mg, with a yield of 46%.
2) Synthesis of 1- (3-chlorophenyl) -3- (4-phenoxyacetoxy) urea
1- (3-chlorophenyl) -3- (4-phenoxyacetic acid carbethoxy) urea (348mg, 1mmol) was dissolved in 3mL of ethanol and 3mL of tetrahydrofuran, and 3mL of a 1N aqueous solution of sodium hydroxide was added to the system, followed by stirring at room temperature for about 1 hour. After the reaction, the excess solvent was distilled off, 2mL of distilled water was added, extraction was performed with ethyl acetate, and the aqueous layer was collected and the pH of the aqueous solution was adjusted to weak acidity with 1N hydrochloric acid aqueous solution. A large amount of solid matter was precipitated from the system, which was filtered and dried to obtain a white solid, about 260mg, in a yield of 81%. mp 218-220 ℃;1H NMR(400MHz,DMSO-d6)δ12.97(s,1H),8.95(s,1H),8.70(s,1H),7.68-7.70(m,1H),7.31-7.36(m,2H),7.22-7.29(m,2H),6.97(dt,J＝6.8,2.0Hz,1H),6.81-6.86(m,2H),4.58(s,2H)；HR-ESI-MS:m/z＝321.0638[M+H]+,calcd for C15H14ClN2O4:321.0637.
example 8: synthesis of Compound 8
1- (3-chlorophenyl) -3- (4-phenoxyacetic acid carbethoxy) urea (220mg, 0.63mmol) in 7M NH3The reaction solution was stirred at room temperature overnight, after completion of the reaction, the solvent was distilled off, the reaction solution was extracted with ethyl acetate and saturated brine, and the organic layer was concentrated to give a white solid, about 190mg, in 94% yield. mp 242-244 ℃;1H NMR(400MHz,DMSO-d6)δ8.80(s,1H),8.56(s,1H),7.69(t,J＝2.0Hz,1H),7.47(s,1H),7.36(s,1H),7.32-7.36(m,2H),7.24-7.29(m,1H),7.21-7.25(m,1H),6.96-7.00(m,1H),6.86-6.91(m,2H),4.36(s,2H)；HR-ESI-MS:m/z＝320.0796[M+H]+,calcd for C15H15ClN3O3:320.0796.
example 9: synthesis of Compound 9
3-Chlorobenzene isocyanate (77mg, 0.5mmol) was dissolved in 3mL of dichloromethane, p-aminophenoxyacetylbenzomorpholine (142mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 61%. mp: 126-;1H NMR(400MHz,DMSO-d6)δ8.81(s,1H),8.57(s,1H),7.90(s,1H),7.70(s,1H),7.32-7.37(m,2H),7.23-7.30(m,2H),7.03-7.08(m,1H),6.98-7.01(m,1H),6.85-6.91(m,4H),5.00(s,2H),4.29(t,J＝4.0Hz,2H),3.88(t,J＝4.4Hz,2H)；HR-ESI-MS:m/z＝438.1210[M+H]+,calcd for C23H21ClN3O4:438.1215.
example 10: synthesis of Compound 10
3-Chlorobenzene isocyanate (77mg, 0.5mmol) was dissolved in 3mL of dichloromethane, and 2- (4-aminophenoxy) -N- (pyridin-4-ylmethyl) acetamide (128.6mg, 0.5mmol) was added and the reaction stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 63%. mp 206-208 ℃;1H NMR(400MHz,DMSO-d6)δ8.82(s,1H),8.71(t,J＝6.0Hz,1H),8.59(s,1H),8.45-8.47(m,2H),7.69(t,J＝2.0Hz,1H),7.34-7.39(m,2H),7.22-7.30(m,2H),7.20-7.22(m,2H),6.97-7.00(m,1H),6.91-6.95(m,2H),4.54(s,2H),4.35(d,J＝6.0Hz,2H)；HR-ESI-MS:m/z＝411.1223[M+H]+,calcd for C21H20ClN4O3:411.1218.
example 11: synthesis of Compound 11
M-chlorobenzene isothiocyanate (84.8mg, 0.5mmol) was dissolved in 3mL of dichloromethane, p-aminophenoxyacetylmorpholine (118mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 hours. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 20%. mp:154-156 ℃;1H NMR(400MHz,DMSO-d6)δ9.78(s,2H),7.70(t,J＝2.0Hz,1H),7.37-7.41(m,1H),7.34(d,J＝8.0Hz,1H),7.28-7.32(m,2H),7.14-7.17(m,1H),6.88-6.93(m,2H),4.82(s,2H),3.55-3.61(m,4H),3.46(s,4H)；HR-ESI-MS:m/z＝406.0987[M+H]+,calcd for C19H21ClN3O3S:406.0987.
example 12: synthesis of Compound 12
3, 5-Ditrifluoromethylphenylisocyanate (127.6mg, 0.5mmol) was dissolved in 3mL of dichloromethane, 2- (4-aminophenoxy) -N- (pyridin-4-ylmethyl) acetamide (128.6mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 55%. mp 205-207 deg.C;1H NMR(400MHz,DMSO-d6)δ9.37(s,1H),8.85(s,1H),8.72(t,J＝6.0Hz,1H),8.45-8.47(m,2H),8.12(s,2H),7.61(s,1H),7.37-7.42(m,2H),7.20-7.22(m,2H),6.92-6.97(m,2H),4.55(s,2H),4.35(d,J＝6.0Hz,2H)；HR-ESI-MS:m/z＝513.1367[M+H]+,calcd forC23H19F6N4O3:513.1356.
example 13: synthesis of Compound 13
1- (3, 5-bis (trifluoromethyl) phenyl) -3- (3, 5-dichloro-4-hydroxyphenyl) urea (130mg, 0.3mmol), 2-chloroacetyl morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol) dissolved in 2mL DMF was heated to 80 ℃ for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on silica gel to give a white solid with a yield of 50%. mp:148-150 ℃;1H NMR(400MHz,DMSO-d6)δ9.58(s,1H),9.25(s,1H),8.13(s,2H),7.66(s,1H),7.62(s,2H),4.64(s,2H),3.56-3.62(m,4H),3.45-3.52(m,4H)；HR-ESI-MS:m/z＝560.0574[M+H]+,calcd for C21H18Cl2F6N3O4:560.0573.
example 14: synthesis of Compound 14
3, 5-Ditrifluoromethylphenylisocyanate (127.6mg, 0.5mmol) was dissolved in 3mL of dichloromethane, 2- (4-amino-2, 6-dichlorophenoxy) -N- (pyridin-4-ylmethyl) acetamide (163mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 40%. mp 192 ℃ and 194 ℃;1HNMR(400MHz,DMSO-d6)δ9.59(s,2H),8.84(s,1H),8.51-8.53(m,2H),8.15(s,2H),7.67(s,1H),7.65(s,2H),7.30(d,J＝5.6Hz,2H),4.47(s,2H),4.41(s,2H)；HR-ESI-MS:m/z＝581.0578[M+H]+,calcd for C23H17Cl2F6N4O3:581.0576.
example 15: synthesis of Compound 15
3, 5-bistrifluoromethylphenylisocyanate (127.6mg, 0.5mmol) was dissolved in 3mL of dichloromethane, 3- (4-aminophenyl) -N- (pyridin-4-ylmethyl) propionamide (128mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 60%. mp: 234-;1H NMR(400MHz,DMSO-d6)δ9.40(s,1H),8.95(s,1H),8.38-8.43(m,3H),8.12(s,2H),7.62(s,1H),7.36-7.40(m,2H),7.10-7.16(m,2H),7.04-7.06(m,2H),4.25(d,J＝6.0Hz,2H),2.81(t,J＝7.6Hz,2H),2.46(t,J＝7.6Hz,2H)；HR-ESI-MS:m/z＝511.1563[M+H]+,calcd for C24H21F6N4O2:511.1563.
example 16: synthesis of Compound 16
3, 5-bistrifluoromethylphenylisocyanate (127.6mg, 0.5mmol) was dissolved in 3mL of dichloromethane, 2- (4-aminophenoxy) -N-benzylacetamide (128mg, 0.5mmol) was added, and the reaction was stirred at room temperature for 2 h. Upon completion of the reaction, a large amount of white solid was produced, and the solid was filtered and washed several times with dichloromethane to give a white solid with a yield of about 67%. mp is 182 ℃ and 184 ℃;1H NMR(400MHz,DMSO-d6)δ9.48(s,1H),8.91(s,1H),8.63(t,J＝6.0Hz,1H),8.12(s,2H),7.62(s,1H),7.37-7.41(m,2H),7.29-7.33(m,2H),7.20-7.26(m,3H),6.92-6.97(m,2H),4.52(s,2H),4.35(d,J＝6.0Hz,2H)；HR-ESI-MS:m/z＝512.1401[M+H]+,calcd forC24H20F6N3O3:512.1403.
example 17: synthesis of Compound 17
1- (3-chlorophenyl) -3- (3-hydroxyphenyl) urea (79 mg)0.3mmol), 2-chloroacetyl morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol), dissolved in 2mL DMF, heated to 80 ℃ for reaction for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on silica gel to give a white solid with a yield of 69%. mp 164-166 deg.C;1H NMR(400MHz,DMSO-d6)δ8.87(s,1H),8.74(s,1H),7.70(t,J＝2.0Hz,1H),7.25-7.31(m,1H),7.22-7.25(m,1H),7.16(t,J＝8.0Hz1H),7.12(t,J＝2.0Hz,1H),6.99-7.02(m,1H),6.95-6.98(m,1H),6.53-6.56(m,1H),4.78(s,2H),3.55-3.61(m,4H),3.44-3.47(m,4H)；HR-ESI-MS:m/z＝390.1213[M+H]+,calcd for C19H21ClN3O4:390.1215.
example 18: synthesis of Compound 18
1- (3-chlorophenyl) -3- (4-hydroxyphenyl) urea (79mg, 0.3mmol), 4- (3-chloropropyl) -morpholine (54mg, 0.33mmol), potassium carbonate (83mg, 0.6mmol), potassium iodide (25mg, 0.15mmol), dissolved in 2mL of DMF, was heated to 80 ℃ for about 3 h. After the reaction, the reaction solution was extracted with saturated brine and ethyl acetate, the organic layers were combined and concentrated to give a crude solid, which was chromatographed on silica gel to give a white solid with a yield of 40%. mp at 186 ℃ and 188 ℃;1H NMR(400MHz,DMSO-d6)δ8.78(s,1H),8.52(s,1H),7.69(t,J＝2.0Hz,1H),7.30-7.34(m,2H),7.25-7.29(m,1H),7.21-7.24(m,1H),6.95-6.99(m,1H),6.83-6.87(m,2H),3.95(t,J＝6.4Hz,2H),3.56(t,J＝4.8Hz 4H),2.40(t,J＝7.6Hz,2H),2.35(s,4H),1.80-1.87(m,2H)；HR-ESI-MS:m/z＝390.1568[M+H]+,calcd for C20H25ClN3O3:390.1579.
pharmacological experiments:
experimental example 1: in vitro kinase inhibitory Activity assay
The evaluation of the activity of the compounds on the molecular level of CDK8/cyclin C was entrusted to the company Thermo Fisher scientific (Sammerfei, USA). The compounds were diluted 100-fold in DMSO solution, mixed with buffer (50mM HEPES pH 7.5, 0.01% BRIJ-35, 10mM MgCl2, 1mM EGTA), a mixture of kinase and antibody, and Tracer, shaken for 30s, and then incubated at room temperature for 60 min. Subsequently, the emulsion Ratio (ER, AF647 emulsion (665nm) to Europium emulsion (615 nm)) was read on a microplate reader and analyzed. In the IC50 test, compounds were diluted in 3-fold gradients for a total of 10 concentrations tested. Blank control was set in the test and positive control was sorafenib as reference. The results are given as a percentage of "the difference between the ER of the blank and the ER of the test sample" and "the difference between the ER of the blank and the ER of the positive control", a larger percentage indicating better binding of the compound to the kinase. Table 1 shows the results of in vitro enzyme inhibition experiments for the compounds of the present invention.
TABLE 1 inhibition of CDK8/Cyclin C by compounds of the invention
Experimental example 2: in vitro kinase Selective Activity assay
The present invention selects 8 representative effective inhibitors to selectively test 12 kinases. 4 of them are CDK subtypes, including CDK2, CDK6, CDK7 and CDK 9; the other 8 are aurora a, BRAF, EGFR, FGFR1, FLT3, JAK1, PDGFR α and GSK3 α. The kinase selectivity of the 8 selected compounds of the invention was determined at a concentration of 10 μ M. As shown in figure 1 and table 2, 8 compounds according to the invention all showed selective inhibition of CDK 8.
TABLE 2 inhibition of 12 kinases by 8 compounds of the invention
Cyclin dependent kinase (Cyclin dependent kinase); BRAF sarcoma viral oncogene homolog B1 of filtration virus (v-raf murine sarcococcal oncogene homolog B1); aurora A Aurora kinase A (Aurora kinase A); EGFR, epidermal growth factor receptor (epidermal growth factor receptor); PDGFR alpha Platelet-derived growth factor receptor alpha (Platelet-derived growth factor receptor alpha); JAK1 Janus kinase 1(Janus kinase 1); FGFR1 Fibroblast Growth Factor Receptor 1(Fibroblast Growth Factor Receptor-1); GSK3 α glycogen synthase kinase 3 α (glycogen synthase kinase 3 α); FLT3 FMS-like tyrosine kinase 3(Fms-like tyrosine kinase).
Experimental example 3: in vitro anti-tumor cell proliferation assay
Screening of antitumor activity of target substance Sorafenib (Sorafenib) was used as a control. Cell growth half-inhibitory concentrations IC of structurally representative targets against KB (human nasopharyngeal carcinoma cells), KBvin (KB resistant strain), A549 (human lung carcinoma cells), MCF-7 (human breast carcinoma cells), MDA-MB-231 (triple negative breast carcinoma cells) were determined by the SRB (Sulforhodamine B) method50。
As a result:
separately determining and calculating the IC of several compounds of the invention50Value (. mu.M). The results are shown in Table 3.
TABLE 3 inhibition of various tumor cells by test compounds
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