Compounds and their use in the treatment of cancer
阅读说明：本技术 化合物及其在治疗癌症中的用途 (Compounds and their use in the treatment of cancer ) 是由 R·本希达 S·洛奇 C·朗科 E·乔恩 O·格里塞 N·特卡亚 于 2018-12-21 设计创作，主要内容包括：本公开涉及式(I)的双胍衍生物及其重排产物。本公开还涉及这些化合物在用于治疗癌症,特别是黑色素瘤的方法中的用途。<Image he="259" wi="700" file="DDA0002685419930000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present disclosure relates to biguanide derivatives of formula (I) and rearrangement products thereof. The disclosure also relates to the use of these compounds in methods for treating cancer, particularly melanoma.)
1. A compound of formula (I)
Wherein the content of the first and second substances,
R1and R2Independently selected from H, halogen, C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
or R1And R2Together with the carbon-carbon double bond between them form a 6-10 membered aryl or heteroaryl ring, said aryl and heteroaryl optionally substituted with one or more groups independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2、-CN and- (CO) -R;
y is-O-or-S-;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl radical ofAlkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl are optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl radical, C1-C6Alkoxy and-NR "R'";
each R 'and R' is independently selected from H and C1-C6An alkyl group.
2. A compound of formula (I) according to claim 1, wherein
R1And R2Independently selected from H, C1-C6Alkyl and aryl having 6 to 10 ring atoms, said aryl being optionally substituted by one or more-NO2Substitution;
or R1And R2Together with the carbon-carbon double bond between them form a 6-membered aryl ring, optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl and C1-C6The substituent of the alkoxy group is substituted,
y is-S-;
R3is selected from C1-C6Haloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and C7-C16Aralkyl, said heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R;
each R is independently selected from C1-C6An alkyl group.
3. A compound of formula (I) according to claim 1 or 2, wherein the compound is selected from
4. A compound of formula (I) according to any one of claims 1 to 3, wherein the compound is selected from
1.
5. A compound of formula (II) or (III)
Wherein
Y' is-SR4OR-OR5，
R4Is selected from C1-C6An alkyl group and a protecting group selected from any group linked by a disulfide functionality, thioesters, alkyl, alkenyl and alkynyl sulfides, benzyl sulfides, alkylaryl methyl sulfides and triaryl methyl sulfides;
R5selected from the group consisting of H and protecting groups selected from the group consisting of ester, alkenyl and alkynyl ethers, silylated ethers, alkoxymethyl ethers, benzyl ethers, tetrahydropyranyl ethers, pentose and hexose sugars;
each R'Independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2, -CN, and- (CO) -R;
n is 0 to 4;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl radical, C1-C6Alkoxy and-NR "R'";
each R 'and R' is independently selected from H and C1-C6An alkyl group.
6. The compound of formula (II) or (III) according to claim 5, wherein
Y' is-SR4，R4Is selected from C1-C6An alkyl group;
each R' is independently selected from halogen and C1-C6An alkoxy group;
n is 0 to 1;
R3is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkoxy and-CN.
7. The compound of formula (III) according to claim 5 or 6, wherein the compound is
2.
8. The compound of formula (III) according to any one of claims 5 to 7, wherein the compound is selected from
9. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1-4 or a compound of formula (II) or (III) according to any one of claims 5-8, and a pharmaceutically acceptable carrier.
10. Use of a compound of formula (I) according to any one of claims 1 to 4, or a compound of formula (II) or (III) according to any one of claims 5 to 8, as a medicament.
11. Use of a compound according to claim 10 in a method of treating cancer.
12. Use of a compound according to claim 10 or 11 in a method of treating melanoma.
13. Use of a compound according to claim 10 or 11 for a method of treating melanoma resistant to BRAF inhibitors.
Technical Field
The present disclosure relates to biguanide derivatives and their rearrangement products. The disclosure also relates to the use of these compounds in methods of treating cancer, particularly melanoma.
Background
In france, cancer is one of the most important causes of death. Among the cancers, melanoma is a skin cancer that is common in france, with about 8000 new cases diagnosed each year and over 1000 deaths. Thus, this cancer is a major public health problem. Melanoma is a malignant tumor that develops from melanocytes responsible for the synthesis of melanin as a photoprotective pigment. Melanoma is a very aggressive tumor with a high potential for metastasis to lymph nodes, liver, lung, central nervous system and skin. Once metastasis occurs, the prognosis of life (visual prognosis) becomes unfavorable due to the inefficiency of all current therapies.
Recently, BRAF inhibitors (vemurafenib (PLX4032) and dabrafenib) have obtained encouraging results, targeting only B-Raf mutant melanoma (about 50% of metastatic melanoma). Unfortunately, after a brief remission, melanoma develops resistance to these drugs in almost all cases, and metastasis again develops, extending the life expectancy of the patient by about 2 months. Immunotherapy has also recently been developed. They are based on anti-CTLA 4 and anti-PD 1 antibodies that can reactivate the immune response. However, immunotherapy produces an objective response in only 15% to 30% of patients.
Metformin, a commonly used drug for the treatment of diabetes, has also been reported to inhibit melanoma Cell growth (Tomic et al, Cell Death and Disease, 1; 2: e199,2011) and metastasis development (Cerezo et al, Molecular Cancer Therapeutics,12(8): 1605-. However, high doses of metformin may be required to induce cell death (IC50 ═ 10mM, ten millimolar).
Therefore, there is a need for compounds and compositions with anti-proliferative properties that can be particularly useful for treating patients with melanoma, e.g., patients with melanoma resistant to BRAF inhibitors.
Disclosure of Invention
The inventors have surprisingly found that biguanide derivatives and rearrangement products thereof comprising a heteroaryl moiety have high biological activity against cancer lines (such as melanoma cell lines), including melanoma cell lines resistant to BRAF inhibitors.
Thus, in a first aspect, the present disclosure relates to compounds of formula (I)
Wherein the content of the first and second substances,
R1and R2Independently selected from H, halogen, C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
or R1And R2Together with the carbon-carbon double bond between them form a 6-10 membered aryl or heteroaryl ring, said aryl and heteroaryl optionally substituted with one or more groups independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
y is-O-or-S-;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl radical, C1-C6Alkoxy and-NR "R'";
each R 'and R' is independently selected from H and C1-C6An alkyl group.
In a second aspect, the disclosure relates to compounds of formula (II) or (III)
Wherein
Y' is-SR4OR-OR5，
R4Selected from H, C1-C6Alkyl and protecting groups;
R5selected from H and protecting groups;
each R' is independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2、-CN and- (CO) -R;
n is 0 to 4;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl radical, C1-C6Alkoxy and-NR "R"'；
Each R 'and R' is independently selected from H and C1-C6An alkyl group.
In a third aspect, the present disclosure relates to a pharmaceutical composition comprising a compound of formula (I), (II), or (III) and a pharmaceutically acceptable carrier.
In a fourth aspect, the present disclosure relates to the use of a compound of formula (I), (II) or (III) for a method of treating cancer.
In a fifth aspect, the present disclosure relates to a method for treating cancer, the method comprising administering to a subject a therapeutically effective amount of
(i) A compound of the formula (I),
(ii) a compound of formula (II) or (III), or
(iii) A pharmaceutical composition as described herein.
In a sixth aspect, the present disclosure relates to the use of a compound of formula (I), (II) or (III) in the manufacture of a medicament for the treatment of cancer.
In a seventh aspect, the present disclosure relates to the use of a compound of formula (I), (II) or (III) as a medicament.
Detailed Description
Definition of
As used herein, the term "C1-C6Alkyl "by itself or as part of another substituent means a straight or branched chain alkyl functionality having 1 to 6 carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl and its isomers (e.g. n-pentyl, isopentyl) and hexyl and its isomers (e.g. n-hexyl, isohexyl).
As used herein, the term "C3-C6Cycloalkyl "refers to a saturated or unsaturated cyclic group having 3 to 6 carbon atoms. Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term "halogen" as used herein refers to a fluorine (-F), chlorine (-Cl), bromine (-Br) or iodine (-I) group.
As used herein, the term "C1-C6Haloalkyl group"refers to C as defined herein substituted with one or more halogen groups as defined herein1-C6An alkyl group. Suitable C1-C6Haloalkyl includes trifluoromethyl and dichloromethyl.
As used herein, the term "C2-C6Alkenyl "refers to a straight or branched chain hydrocarbon moiety having at least one carbon-carbon double bond. For example, alkenyl groups include ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl and butadienyl.
As used herein, the term "C2-C6Alkynyl "refers to a straight or branched hydrocarbon moiety having at least one carbon-carbon triple bond. Examples of "alkynyl" groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and propargyl (allyl groups), and the like.
As used herein, the term "C1-C6Alkoxy "refers to an-O-alkyl group, wherein the alkyl group is C as defined herein1-C6An alkyl group. Suitable C1-C6Alkoxy groups include methoxy, ethoxy, propoxy.
As used herein, the term "aryl group having 6 to 10 ring atoms" refers to a polyunsaturated aromatic hydrocarbon group containing a single ring of 6 to 10 ring atoms or multiple aromatic rings fused together, wherein at least one ring is aromatic. The aromatic ring may optionally include 1-2 additional rings (cycloalkyl, heterocyclyl, or heteroaryl as defined herein) fused thereto. Suitable aryl groups include phenyl, naphthyl and phenyl rings fused to heterocyclic groups, such as benzopyranyl, benzodioxazolyl, benzodioxanyl and the like.
As used herein, the term "heteroaryl having 5-10 ring atoms" refers to a polyunsaturated aromatic ring system containing a single ring of 5-10 atoms or multiple aromatic rings fused together or linked covalently, wherein at least one ring is aromatic and at least one ring atom is a heteroatom selected from N, O and S. The nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aromatic, cycloalkyl or heterocyclyl ring. Non-limiting examples of such heteroaryl groups include: furyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, dioxanyl, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, and quinoxalinyl.
As used herein, the term "heterocyclyl having 5-10 ring atoms" refers to a saturated or unsaturated cyclic group having 5-10 ring atoms, wherein at least one ring atom is a heteroatom selected from N, O and S. The nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Examples of heterocycles include, but are not limited to, tetrahydropyridinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperazinyl, azepinyl, imidazolinyl, 1, 4-dioxanyl, and the like.
As used herein, the term "C7-C16Aralkyl "refers to an alkyl group as defined herein substituted with one or more aryl groups as defined herein. For example, aralkyl includes benzyl.
Various embodiments of the present disclosure are described herein. It will be appreciated that features defined in each embodiment may be combined with other defined features to provide further embodiments.
The present disclosure encompasses compounds of formula (I), (II), (III), tautomers, enantiomers, diastereomers, racemates or mixtures thereof, and hydrates, solvates or pharmaceutically acceptable salts thereof.
The term "pharmaceutically acceptable salt" refers to salts that retain the biological potency and properties of the disclosed compounds and are typically not biologically or otherwise undesirable.
Any formulae given herein are also intended to represent unlabeled and isotopic forms of the compounds, examplesSuch as deuterium-labelled compounds or14C labeled compound.
A compound of formula (I)
The present disclosure relates to compounds of formula (I)
Wherein the content of the first and second substances,
R1and R2Independently selected from H, halogen, C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
or R1And R2Together with the carbon-carbon double bond between them form a 6-10 membered aryl or heteroaryl ring, said aryl and heteroaryl optionally substituted with one or more groups independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2、-CN and- (CO) -R;
y is-O-or-S-;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl radical of said alkaneOptionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2、-CN and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl radical, C1-C6Alkoxy and-NR "R'";
each R 'and R' is independently selected from H and C1-C6An alkyl group.
In one embodiment, the disclosure relates to compounds of formula (I), wherein
R1And R2Independently selected from H, halogen, C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R;
or R1And R2Together with the carbon-carbon double bond between them form a 6-membered aryl or heteroaryl ring, said aryl and heteroaryl optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R;
y is-O-or-S-;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, aryl having 5 to 10 ring atomsHeteroaryl of a ring atom and C7-C16Aralkyl, said alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl and C1-C6An alkoxy group.
In one embodiment, R1Selected from H, C1-C6Alkyl and aryl having 6 to 10 ring atoms, said alkyl and aryl optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy and-NO2Is substituted with the substituent(s).
In another embodiment, R2Selected from H and C1-C6Alkyl optionally substituted with one or more substituents independently selected from halogen.
In another embodiment, R1And R2Independently selected from H, C1-C6Alkyl and aryl having 6 to 10 ring atoms, said aryl being optionally substituted by one or more-NO2And (4) substitution.
In another embodiment, R1And R2Together with the carbon-carbon double bond between them form a 6-membered aryl ring, optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R; each R is independently selected from H, C1-C6Alkyl and C1-C6An alkoxy group.
In another embodiment, R3Is selected from C1-C6Haloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms and heteroaryl having 5 to 10 ring atoms, said heterocyclyl, aryl and heteroaryl being optionally substituted by oneOr more are independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R; each R is independently selected from C1-C6An alkyl group.
In another embodiment, Y is-S-.
In another embodiment, the present disclosure provides a compound of formula (I), wherein
R1And R2Together with the carbon-carbon double bond between them form a 6-membered aryl ring;
y is-O-;
R3selected from aryl groups having 6 to 10 ring atoms, said aryl groups being optionally substituted with one or more substituents independently selected from halogen.
In another embodiment, the present disclosure provides a compound of formula (I), wherein
R1And R2Independently selected from H, C1-C6Alkyl and aryl having 6 to 10 ring atoms, said aryl being optionally substituted by one or more-NO2Substitution;
or R1And R2Together with the carbon-carbon double bond between them form a 6-membered aryl ring, optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl and C1-C6The substituent of the alkoxy group is substituted,
y is-S-;
R3is selected from C1-C6Haloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and C7-C16Aralkyl, said heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R;
each R is independently selected from C1-C6An alkyl group.
In another embodiment, the present disclosure provides a compound of formula (I), wherein
R1And R2And the carbon-carbon double bond between them forms a 6-membered aryl ring,
y is-S-;
R3selected from aryl having 6 to 10 ring atoms and heteroaryl having 5 to 10 ring atoms, said aryl and heteroaryl being optionally substituted by one or more groups independently selected from halogen, C1-C6Haloalkyl, C1-C6Alkoxy, -CN and- (CO) -R; each R is independently selected from C1-C6An alkyl group.
In another embodiment, the compound of formula (I) is selected from
According to one embodiment, the compound of formula (I) is selected from
In a preferred embodiment, the compound of formula (I) is selected from
In some embodiments, the compounds of formula (I) as described herein have antiproliferative activity against melanoma cell lines. Thus, they may be advantageously used in a method of treating cancer, in particular melanoma.
Without being bound by this theory, the inventors postulate that, in some embodiments, the compounds of formula (I) induce activation of AMPK (AMP-activated protein kinase) involved in regulating apoptosis, thereby inducing cancer cell death.
A compound of formula (II) or (III)
A compound of formula (I) (wherein R1And R2And the carbon-carbon double bond between them together form an optionally substituted 6-membered aryl ring) may be rearranged to form a compound of formula (II) either spontaneously or under specific conditions (depending on the biguanide structure) by opening the benzothiazole or benzoxazole moiety and forming a triazine ring. For example, in some cases, rearrangement to a compound of formula (II) occurs when a compound of formula (I) wherein Y is-S-is treated under oxidizing conditions. When Y' is-SH, the compound of formula (II) may dimerize to form the compound of formula (III) by forming an S-S bond.
The invention therefore also relates to compounds of the formula (II) or (III)
Wherein
Y' is-SR4OR-OR5，
R4Selected from H, C1-C6Alkyl and protecting groups;
R5selected from H and protecting groups;
each R' is independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN and- (CO) -R;
n is 0 to 4;
R3is selected from C1-C6Alkyl radical, C3-C6Cycloalkyl radical, C1-C6Haloalkyl, C2-C6Alkenyl radical, C2-C6Alkynyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, cycloalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxygen, halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -OH, -NR "R'", -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl radical, C1-C6Alkoxy and-NR "R'";
each R 'and R' is independently selected from H and C1-C6An alkyl group.
In one embodiment, the disclosure relates to a compound of formula (II) or (III), wherein
Y' is-SR4OR-OR5，
R4Selected from H, C1-C6Alkyl and protecting groups;
R5selected from H and protecting groups;
each R' is independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN and- (CO) -R;
n is 0 to 4;
R3is selected from C1-C6Alkyl radical, C1-C6Haloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said alkyl, heterocyclyl, aryl, heteroaryl and aralkyl groups are optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy radical, C1-C6Haloalkyl, -NO2CN, -CN, and- (CO) -R;
each R is independently selected from H, C1-C6Alkyl and C1-C6An alkoxy group.
In one embodiment, n is 1 and R' is selected from halogen and C1-C6An alkoxy group. In another embodiment, n is 1 and R' is selected from halogen, C1-C6Alkyl radical, C1-C6Alkoxy and-NO2。
In another embodiment, n is 0.
In another embodiment, Y' is-SR4And R is4Is selected from C1-C6Alkyl groups and protecting groups. Preferably, the protecting group is selected from any group linked by a disulfide functionality, thioesters, alkyl, alkenyl and alkynyl sulfides, benzyl sulfides, alkylaryl methyl sulfides and triaryl methyl sulfides.
In another embodiment, Y' is-OR5And R is5Selected from H and protecting groups. Preferably, the protecting group is selected from the group consisting of ester, alkenyl and alkynyl ethers, silylated ethers, alkoxymethyl ethers, benzyl ethers, tetrahydropyranyl ethers, pentose sugars and hexose sugars.
In one embodiment, Y 'is-OH, n is 1, and R' is selected from the group consisting of halogen and-NO2。
In another embodiment, R3Is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl group of the aryl groupHeteroaryl and aralkyl optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkoxy radical, C1-C6Haloalkyl and-CN.
In another embodiment, the present disclosure provides a compound of formula (II) or (III), wherein
Y' is-SR4，R4Is selected from C1-C6An alkyl group;
each R' is independently selected from halogen and C1-C6An alkoxy group;
n is 0 to 1;
R3is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkoxy and-CN.
In another embodiment, the present disclosure provides a compound of formula (II), wherein
Y' is-SR4，R4Is selected from C1-C6An alkyl group;
each R' is independently selected from halogen and C1-C6An alkoxy group;
n is 0 to 1;
R3is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkoxy and-CN.
In another embodiment, the present disclosure provides a compound of formula (II), wherein
Y' is-OR5And R is5Is H;
each R' is independently selected from halogen, C1-C6Alkyl and-NO2；
n is 0 to 1;
R3is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms and heteroaryl having 5 to 10 ring atoms, said aryl and heteroaryl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkoxy and-CN.
In another embodiment, the present disclosure provides a compound of formula (II), wherein
Each R' is independently selected from halogen and C1-C6An alkoxy group;
n is 0 to 1;
R3is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6 alkoxy and-CN.
In another embodiment, the present disclosure provides a compound of formula (II) selected from
In another embodiment, the present disclosure provides a compound of formula (II) selected from
In another embodiment, the present disclosure provides a compound of formula (III), wherein
Each R' is independently selected from halogen and C1-C6An alkoxy group;
n is 0 to 1;
R3is selected from C1-C6Haloalkyl, aryl having 6 to 10 ring atoms, heterocyclyl having 5 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms and C7-C16Aralkyl, said aryl, heteroaryl, heterocycle and aralkyl being optionally substituted with one or more substituents independently selected from halogen, C1-C6Alkoxy radical, C1-C6Alkyl, -NR "R'", -NO2CN, -CN, and- (CO) -R;
each R is independently selected from C1-C6An alkyl group;
r 'and R' are H.
In another embodiment, the present disclosure provides a compound of formula (III) selected from
In another embodiment, the present disclosure provides a compound of formula (III) selected from
In a preferred embodiment, the present disclosure provides a compound selected from the following (III)
In the compounds of formula (II), the hydroxyl or thiol functional group may be protected with a protecting group. As used herein, a protecting group refers to any group that is cleavable in a biological medium, particularly by hydrolysis or removal via a plasma enzyme or a bio-organic nucleophile such as glutathione. Suitable hydroxyl protecting groups include, but are not limited to, esters, alkyl, alkenyl and alkynyl ethers, silylated ethers, alkoxymethyl ethers, benzyl ethers, tetrahydropyranyl ethers, pentose sugars, hexose sugars. Suitable protecting groups for a thiol group include, but are not limited to, any group linked through a disulfide functionality, thioesters, alkyl, alkenyl, and alkynyl sulfides, benzyl sulfides, alkylaryl methyl sulfides, triaryl methyl sulfides.
The compounds of formula (II) or (III) described herein have high biological activity against melanoma, including melanoma which is resistant to BRAF inhibitors. Thus, they may be used in methods of treating cancer, particularly melanoma.
Without being bound by this theory, the inventors postulate that the compounds of formula (II) and (III) are prodrugs of the compounds of formula (I). After administration to a subject, the compounds of formula (II) and (III) are modified to compounds of formula (I) by in vivo physiological effects.
Pharmaceutical composition
The disclosure also relates to pharmaceutical compositions comprising a compound of formula (I), (II), or (III) and a pharmaceutically acceptable carrier.
"pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when properly administered to a mammal, particularly a human. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.
The form, route of administration, dosage and regimen of the pharmaceutical composition naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.
The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, intranasal, intraocular, intravenous, intramuscular, or subcutaneous administration, and the like.
The pharmaceutical compositions may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, emulsions, syrups, elixirs, aerosols, or any other suitable composition; and comprises at least one compound according to the present disclosure.
Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. Tablets may be sugar coated or enteric coated, if desired, by standard techniques. The tablets or pills may be coated to provide a dosage form having the advantage of prolonged action. For example, a tablet or pill may comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components may be separated by an enteric layer that serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, including a number of polymeric acids, as well as materials such as shellac, cetyl alcohol and cellulose acetate.
The compounds and other agents of the present disclosure may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
Preferably, the pharmaceutical composition contains a pharmaceutically acceptable carrier for a formulation that can be injected. In particular, these may be isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride, etc., or mixtures of such salts), or in the case of compositions which can be formulated as injectable solutions by addition of sterile or physiological saline, dry (especially lyophilized) compositions.
Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; the preparation comprises oleum Sesami, oleum Arachidis Hypogaeae or propylene glycol water solution; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must have some fluidity to allow for easy injection. It must be stable under the conditions of manufacture and storage and must be preserved against contamination by microorganisms such as bacteria and fungi. Solutions of the free base or pharmacologically acceptable salts of the active compounds may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating (such as lecithin), by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. By formulation, the solution will be administered in a manner and in a therapeutically effective amount compatible with the dosage form. The formulation is readily administered in a variety of dosage forms, such as the types of injectable solutions described above, but drug-releasing capsules and the like may also be used.
For example, for parenteral administration in aqueous solution, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, one skilled in the art will be aware of the sterile aqueous media that may be employed in light of this disclosure. For example, a dose may be dissolved in 1ml of isotonic NaCl solution, or added to 1000ml of subcutaneous injection, or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15 th edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage to be appropriate for the individual subject.
For aerosol administration, the compounds of the present disclosure and other agents are preferably provided in finely divided form together with surfactants and propellants. The surfactant must, of course, be non-toxic and preferably soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, oleic acid and oleic acid containing aliphatic polyols or cyclic anhydrides thereof. Mixed esters, such as mixed or natural glycerides, may be employed. If desired, a carrier, such as lecithin for intranasal delivery, may also be included. One example includes a solution comprising 7.5mg NaCl, 1.7mg citric acid monohydrate, 3mg disodium phosphate dihydrate, and 0.2mg benzalkonium chloride solution (50%) per ml (Gozes et al, J Mol neurosci.19(1-2):167-70 (2002)).
Suitable compositions for topical administration include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations, e.g., aerosol administration.
The dosage for administration can be adjusted according to various parameters, in particular according to the mode of administration used, the pathology concerned or the duration of treatment desired. It will be appreciated that the appropriate dosage of the compound, as well as the composition comprising the compound, may vary from patient to patient. Determining the optimal dosage will generally involve a balance between the level of therapeutic benefit and any risk or deleterious side effects of the treatment described herein. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds and/or materials used in combination, and the age, sex, weight, condition, general health and prior medical history of the patient. The amount of compound and the route of administration will ultimately be at the discretion of the physician, although in general the dosage will be to achieve a local concentration at the site of action which will achieve the desired effect without causing substantial deleterious or deleterious side effects.
According to one embodiment, the pharmaceutical composition comprises a non-ionic emulsifier, preferably Kolliphor EL. The presence of such non-ionic emulsifiers reduces the final concentration of DMSO in the composition. The compound of formula (I), (II) or (III) may be dissolved in a non-ionic emulsifier, for example Kolliphor EL.
Application method
As shown by in vitro and in vivo tests provided in the examples, the compounds of formula (I), (II) or (III) exhibit valuable pharmaceutical properties and are therefore indicated for use in therapy.
The disclosure also relates to the use of a compound of formula (I), (II) or (III) as a medicament.
The disclosure also relates to the use of compounds of formula (I), (II), or (III) for methods of treating cancer.
As used herein, the term "cancer" has its ordinary meaning in the art and includes abnormal states or conditions characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growths or carcinogenic processes; tissue or malignantly transformed cells, tissues or organs are transferred regardless of histopathological type or invasive stage. The term cancer includes malignancies of various organ systems, such as adenocarcinomas affecting the skin, lung, breast, thyroid, lymph, gastrointestinal and genito-urinary tract, as well as adenocarcinomas including malignancies, such as most colon, renal cell, prostate and/or testicular tumors, lung non-small cell, small intestine and esophagus cancers.
Examples of cancers include, but are not limited to, hematologic malignancies, such as B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma (NHL), B-NHL, T-NHL, Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), Mantle Cell Lymphoma (MCL), NK cell lymphoid tumors, and myeloid lineage tumors. Examples of non-hematologic cancers include, but are not limited to, skin cancer, colon cancer, breast cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, and testicular cancer.
In certain embodiments, the disclosure relates to the use of a compound of formula (I), (II), or (III) for a method of treating melanoma. In a particular embodiment, the disclosure also relates to the use of a compound of formula (I), (II), or (III) for a method of treating melanoma resistant to BRAF inhibitors.
In a particular embodiment, the compound of formula (I), (II) or (III) induces the activation of AMPK. Metformin, a commonly used drug for the treatment of type II diabetes, also induces the activation of AMPK, and it has been shown that metformin also inhibits the growth of melanoma cells. Thus, similar to metformin, in one embodiment, the invention also relates to the use of a compound of formula (I), (II) or (III) for a method of treating type II diabetes.
The present disclosure relates to methods for treating cancer comprising administering to a subject a therapeutically effective amount of
(i) A compound of the formula (I),
(ii) a compound of formula (II) or (III), or
(iii) A pharmaceutical composition as described herein.
The term "therapeutically effective amount" of a compound refers to an amount of a compound that will elicit the biological or medical response of a subject, e.g., to ameliorate symptoms, alleviate symptoms, slow or delay disease progression, or prevent disease.
The present disclosure also relates to the use of a compound of formula (I), (II) or (III) in the manufacture of a medicament for the treatment of cancer. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is melanoma resistant to BRAF inhibitors.
Drawings
In all figures, bars represent mean ± SEM: p < 0.05; p < 0.01; p < 0.001.
FIG. 1A shows cell viability of A375S cells treated with 5 μ M CRO15 or PLX4032 at the times shown on the graph.
FIG. 1B shows cell viability of A375S cells treated with different concentrations of CRO15 or with 5. mu.M PLX4032 for 48 hours.
Figure 1C shows the viability of different melanoma cells with various mutations after 48 hours of treatment with 5 μ M CRO 15. Mutations are designated next to the melanoma cell line name. The mutated proteins are indicated with an ". mark".
FIG. 1D shows the viability of melanoma cells from patients treated with 5 μ M CRO15 for 48 hours. The mutated proteins are indicated with an ". mark".
FIG. 1E shows the viability of normal cells treated with 5 μ M CRO15 for 48 hours.
FIG. 2 shows the results of Western blot analysis.
Figure 3A shows the evolution of tumor volume in xenograft mice inoculated subcutaneously with a375 sensitive melanoma cells and treated with CRO15 or PLX 4032.
FIG. 3B shows the tumor weight of xenograft mice subcutaneously inoculated with A375 sensitive melanoma cells and treated with CRO15 or PLX4032 after euthanasia.
Figure 4A shows the viability of sensitive and resistant a375 melanoma cells treated with CRO15 or PLX 4032.
Figure 4B shows the evolution of tumor volume in xenograft mice subcutaneously inoculated with a375 resistant melanoma cells and treated with CRO15 or PLX 4032.
Figure 4C shows tumor weight of xenograft mice subcutaneously inoculated with a375 resistant melanoma cells and treated with CRO15 or PLX4032 after mouse euthanasia.
FIG. 5 shows cell viability of DR6 cells treated with different concentrations of CRO15 or 1. mu.M Vemurafenib and 0.5. mu.M cobitinib for 24 hours.
Figure 6A shows the evolution of tumor volume in xenograft mice subcutaneously inoculated with a375 resistant melanoma cells and treated with MTF319 or PLX 4032.
Figure 6B shows tumor weight of xenograft mice subcutaneously inoculated with a375 resistant melanoma cells and treated with MTF319 or PLX4032 after euthanasia.
Figure 7A shows the evolution of tumor volume in allograft mice subcutaneously inoculated with mouse melanoma cells and treated with CRO 15.
FIG. 7B shows the cell viability of WM9 cells after 8 weeks of treatment with PLX4032, CRO15 or MTF255 and 48 hours of stimulation with 10. mu.M of each drug.
Detailed Description
Experimental procedures
Chemical Synthesis and characterization
Methanol, ethyl acetate, diethyl ether and dichloromethane were purchased from Carlo Erba and used as received. Anhydrous DMF (98.8%, septum)Off-stored) was purchased from Sigma Aldrich and used as received. All chemicals were purchased from Aldrich, Fisher or Alfa Aesar and used without further purification. Thin Layer Chromatography (TLC) was performed on pre-coated Merck 60GF254 silica gel plates, first visualized by UV light (254nm and 360nm) and recorded on a Bruker Advance200MHz spectrometer or a Bruker Advance 400MHz or Bruker Advance 500MHz spectrometer1H and13CNMR spectroscopy. Mass spectra (ESI-MS) were recorded on Bruker (Daltonics Esquire 3000 +). HRMS spectra were recorded at m/
Synthesis procedure and characterization:
general procedure (a) for forming biguanides of formula I. To a solution of the corresponding guanidine (1 equivalent) in N, N-dimethylformamide (25mL/g guanidine) was added sodium hydride (60% dispersion in mineral oil, 1.5 equivalents) and the mixture was stirred at room temperature for 30 minutes. To this solution the corresponding nitrile (1 eq) was added in one portion. The reaction was stirred at rt overnight and monitored by TLC. After completion of the reaction, the mixture was poured into water (200mL/g guanidine), and the precipitate was collected and washed with water, methanol and ether.
General procedure (B) for the formation of N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6-aryl/heteroaryl-1, 3, 5-triazine-2, 4-diamine) of formula II and III (B). A solution of the corresponding biguanide (1 equivalent) in technical grade ethanol (about 25mL/100mg biguanide) was stirred at reflux temperature and monitored by LCMS. After complete conversion (about 6-7 hours), the precipitate formed is filtered and washed with technical grade ethanol.
General procedure (C) for the formation of N2, N2' - (dithiodiylbis (4-alkyl/hal-2, 1-phenylene)) bis (6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) of formulae II and III. To a solution of the corresponding guanidine (1 equivalent) in technical grade ethanol (about 10mL/g guanidine) was added trichloroacetonitrile (2 equivalents), and the reaction mixture was stirred at reflux temperature and monitored by LCMS. After complete conversion (about 6-7 hours), the precipitate formed is filtered and washed with technical grade ethanol and diethyl ether.
General procedure (D) for the formation of 2- ((6-imino-4- (trichloromethyl) -1, 6-dihydro-1, 3, 5-triazin-2-yl) amino) phenol. To a solution of the corresponding guanidine (1 equivalent) in technical grade ethanol (25mL/g guanidine) was added trichloroacetonitrile (10 equivalents) and the reaction mixture was stirred in a sealed tube under an argon atmosphere at 60 ℃. After complete conversion (about 18 hours), the mixture was concentrated to dryness. The residue was purified by flash chromatography on silica gel.
General procedure (E) for the formation of 2- ((4-amino-6-phenyl-1, 3, 5-triazin-2-yl) amino) phenol. To a solution of the corresponding guanidine (1 equivalent) in N, N-dimethylformamide (25mL/g guanidine) was added sodium hydride (60% dispersion in mineral oil, 1.1 equivalents) and the reaction mixture was stirred under an argon atmosphere in a sealed tube. When the evolution of gas into the solution was stopped, the corresponding nitrile (1 eq) was added and the tube was sealed. Then, the resulting solution was stirred at 80 ℃. After complete conversion (about 18 hours), the mixture was concentrated to dryness. The residue was purified by flash chromatography on silica gel.
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) (CRO 15). 1- (benzo [ d ]) is reacted with]A solution of thiazol-2-yl) guanidine (10.0g, 52mmol) and trichloroacetonitrile (10.0mL, 1.92mmol) in technical grade ethanol (100mL) was stirred at 75 ℃. After 1 hour, a large amount of white precipitate appeared in the yellow solution. After completion of the reaction (TLC monitoring, about 3 hours), the suspension was cooled to room temperature and passedAnd (5) filtering. The precipitate was washed with a small amount of cold ethanol and dried in air. Recrystallization from acetone/diethyl ether gave the desired compound as a white solid (10.03g, 57.5%). TLC Rf(Et2O/PE,1/1,v/v)＝0.23。1H NMR (200MHz, acetone-d 6):8.54(s,1H),7.96(d, J ═ 8.0Hz,1H),7.58(dd, J ═ 7.8,1.6Hz,1H),7.35(td, J ═ 7.8,1.6Hz,1H), 7.22-6.93 (m, 3H).13C NMR (101MHz, acetone-d 6):174.00,168.98,166.37,139.05,133.76,130.58,129.92,126.33,125.34, 97.43.HRMS-ESI (M/z) [ M + H ]]+For C20H15Cl6N10S2 +Calculated value, 668.90482; found 668.90497. HPLC (. lamda.)280) The purity is 97.4 percent; t is tR7.958 minutes (method 2).
N2, N2' - (dithiodiylbis (4-methoxy-2, 1-phenylene)) bis (6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) (MTF-232). Following general procedure C, 1- (6-methoxybenzo [ d ] was used]Thiazol-2-yl) guanidine (300mg, 1.35mmol) was synthesized to give the title compound as a green powder (418mg, 85%).1H NMR(400MHz,DMSO-d6):9.64(s,1H),7.59(s,1H),7.45(s,1H),7.20(d,J＝8.7Hz,1H),7.10(s,1H),6.84(dd,J＝8.7,2.8Hz,1H),3.70(s,3H)。13C NMR(50MHz,DMSO-d6):172.28,167.38,166.07,158.26,135.44,129.22,128.28,113.08,112.20,96.77,55.48。HPLC(λ280) The purity is 100.0 percent; t is tR7.433min (method 3).
N2, N2' - (dithiodiylbis (4-chloro-2, 1-phenylene)) bis (6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) (MTF-233). Following general procedure C, 1- (6-chlorobenzo [ d ] was used]Thiazol-2-yl) guanidine (300mg, 1.32mmol) was synthesized to give the title compound (454mg, 93%) as a white powder.1H NMR(400MHz,DMSO-d6):9.66(s,1H),7.71(s,1H),7.58(s,2H),7.40(d,J＝8.5Hz,1H),7.35(dd,J＝8.5,2.3Hz,1H)。13C NMR(50MHz,DMSO-d6):172.39,167.34,165.59,135.71,134.89,131.29,129.12,127.91,127.52,96.63.HPLC(λ280) The purity is 95.1 percent; t is tR13.767min (method 1).
N2, N2' - (dithiodiylbis (4-fluoro-2, 1-phenylene)) bis (6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) (MTF-234). Following the general procedureC, use of 1- (6-fluorobenzo [ d ]]Thiazol-2-yl) guanidine (300mg, 1.42mmol) was synthesized to give the title compound (457mg, 91%) as a white powder.1H NMR(200MHz,DMSO-d6):9.06(br.s.,2H),7.69(br.s.,1H),7.57(br.s.,1H),7.45-7.30(m,2H),7.14(t,1H,J＝8.6Hz,H1).13CNMR(50MHz,DMSO-d6):172.42,167.38,165.94,160.87(d),136.40,131.93,130.04,114.76(d),113.61(d),96.69.HPLC(λ280) The purity is 98.7 percent; t is tR6.858min (method 3).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3-bromobenzamide (MTF-242). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3-bromobenzonitrile (0.95g, 5.20mmol) were synthesized to give the title compound as a white-light yellow powder (708mg, 36%).1H NMR(500MHz,DMSO-d6):10.29(br.s,1H),9.36(br.s,1H),8.94(br.s,1H),8.26(s,1H),8.11(s,1H),8.03(d,J＝7.9Hz,1H),7.80(d,J＝7.7Hz,1H),7.76(dd,J＝7.9,1.1Hz,1H),7.66(d,J＝8.0Hz,1H),7.47(t,J＝7.9Hz,1H),7.39–7.30(m,1H),7.24–7.15(m,1H).13C NMR(50MHz,DMSO-d6):172.23,161.94,160.52,151.47,137.42,134.08,131.23,130.43,126.51,125.66,122.86,121.70,121.15,119.78.HRMS-ESI(m/z):[M+H]+For C15H12BrN5S+Calculated value 374.00696; found 374.00797 HPLC (. lamda.)280) The purity is 99.3 percent; t is tR6.775min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3-chlorobenzamide (MTF-243). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3-chlorobenzonitrile (715mg, 5.20mmol) were synthesized to give the title compound as a yellow powder (206mg, 12%).1H NMR(500MHz,DMSO-d6):10.30(br.s,1H),9.37(br.s,1H),8.90(br.s,1H),8.12(t,br.s,J＝1.8Hz,2H),7.99(d,J＝7.9Hz,1H),7.80(d,J＝7.7Hz,1H),7.66(d,J＝7.9Hz,1H),7.65–7.60(m,1H),7.54(t,J＝7.9Hz,1H),7.38–7.30(m,1H),7.22–7.17(m,1H).13C NMR(50MHz,DMSO-d6):172.22,161.95,160.57,151.47,137.26,133.22,131.23,131.20,130.20,127.53,126.14,125.67,122.87,121.16,119.79.HRMS-ESI(m/z):[M+H]+For C15H12ClN5S+Calculated value 330,05747; found 330,05774 HPLC (. lamda.)280) The purity is 99.2 percent; t is tR6.792min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2-chlorobenzamide (MTF-244). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-chlorobenzonitrile (715mg, 5.20mmol) were synthesized to give the title compound as a white powder (326mg, 19%).1H NMR(500MHz,DMSO-d6):10.14(br.s,1H),9.30(br.s,1H),8.81(br.s,1H),8.09(br.s,1H),7.80(d,J＝7.5Hz,1H),7.65(d,J＝7.7Hz,1H),7.53(br.s,2H),7.50–7.40(m,2H),7.34(t,J＝7.1Hz,1H),7.20(t,J＝7.1Hz,1H).13C NMR(50MHz,DMSO-d6):172.40,162.90,162.00,151.45,136.67,131.23,130.66,130.29,129.56,129.46,126.97,125.63,122.82,121.16,119.74.HRMS-ESI(m/z):[M+H]+For C15H12ClN5S+Calculated value 330.05747; found 330.05783 HPLC (. lamda.)280) The purity is 96.6 percent; t is tR6.500min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4-chlorobenzamide (MTF-245). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 4-chlorobenzonitrile (715mg, 5.20mmol) were synthesized to give the title compound as a white-light yellow powder (721mg, 42%).1H NMR(500MHz,DMSO-d6):10.27(br.s,1H),9.36(br.s,1H),8.85(br.s,1H),8.06(d,br.s,J＝8.6Hz,3H),7.79(d,J＝7.4Hz,1H),7.66(d,J＝7.9Hz,1H),7.59(d,J＝8.6Hz,2H),7.34(t,J＝7.7Hz,1H),7.19(t,J＝7.6Hz,1H).13C NMR(50MHz,DMSO-d6):172.22,162.00,160.95,151.47,136.28,134.00,131.21,129.44,128.36,125.65,122.83,121.15,119.74.HRMS-ESI(m/z):[M+H]+For C15H12ClN5S+Calculated value 330.05747; measured value 330.05743.HPLC(λ280) The purity is 98.8 percent; t is tR6.800min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4-methylbenzamide (MTF-246). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and p-toluenediol (609mg, 5.20mmol) were synthesized to give the title compound as a beige powder (145mg, 9%).1H NMR(500MHz,DMSO-d6):10.28(br.s,1H),9.34(br.s,1H),8.74(br.s,1H),7.96(d,br.s,J＝7.9Hz,3H),7.79(d,J＝7.7Hz,1H),7.65(d,J＝7.9Hz,1H),7.33(dd,J＝18.6,7.7Hz,3H),7.19(t,J＝7.5Hz,1H),2.37(s,3H).13C NMR(50MHz,DMSO-d6):172.21,161.99,161.33,151.46,137.15,134.68,131.21,129.49,125.64,122.82,121.13,119.74,99.13.HRMS-ESI(m/z):[M+H]+For C16H15N5S+Calculated value 310.11209; found 310.11218 HPLC (. lamda.)280) The purity is 97.9 percent; t is tR6.592min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4-iodobenzimidazolamine (MTF-247). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 4-iodobenzonitrile (1.19g, 5.20mmol) were synthesized to give the title compound as a beige powder (832mg, 38%).1H NMR(500MHz,DMSO-d6):10.24(br.s,1H),9.34(br.s,1H),8.82(br.s,1H),8.03(br.s,1H),7.90(d,J＝8.5Hz,2H),7.82(d,J＝8.5Hz,2H),7.79(d,J＝7.8Hz,1H),7.65(d,J＝7.9Hz,1H),7.34(t,J＝7.6Hz,1H),7.19(t,J＝8.0Hz,1H).13CNMR(126MHz,DMSO-d6):172.20,161.97,161.31,151.45,137.13(2C),134.68,131.20,129.47(2C),125.62,122.79,121.11,119.72,99.09.HRMS-ESI(m/z):[M+H]+For C15H12IN5S+Calculated value 421.99309; found 421.99316.HPLC (. lamda.)280) The purity is 98.8 percent; t is tR6.900min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) benzamide (MTF-248). Following general procedure A, using 1- (benzo)[d]Thiazol-2-yl) guanidine (1.00g, 5.20), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 312mg, 7.81mmol) and benzonitrile (0.54mL, 5.20mmol) were synthesized to give the title compound (430mg, 28%) as a beige powder.1H NMR(500MHz,DMSO-d6):10.28(br.s,1H),9.35(br.s,1H),8.80(br.s,1H),8.03(d,br.s,J＝7.3Hz,3H),7.79(d,J＝7.6Hz,1H),7.66(d,J＝7.9Hz,1H),7.60–7.54(m,1H),7.51(t,J＝7.2Hz,2H),7.34(t,J＝7.4Hz,1H),7.19(t,J＝7.4Hz,1H).13C NMR(50MHz,DMSO-d6):172.37,162.29,162.21,151.57,135.28,131.44,131.26,128.28,127.60,125.67,122.82,121.15,119.75.HRMS-ESI(m/z):[M+H]+For C15H13N5S+Calculated value 296.09644; found 296.09659. HPLC (. lamda.)280) The purity is 97.9 percent; t is tR6.358min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2-fluorobenzamide (MTF-249). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-fluorobenzonitrile (0.56mL, 5.20mmol) were synthesized to give the title compound as a red powder (179mg, 11%).1H NMR(400MHz,DMSO-d6):10.16(s,1H),9.31(s,1H),8.69(s,1H),8.05(s,1H),7.80(d,J＝7.7Hz,1H),7.72(td,J＝7.6,1.4Hz,1H),7.65(d,J＝7.9Hz,1H),7.55(ddd,J＝9.4,7.3,1.6Hz,1H),7.37–7.28(m,3H),7.20(t,J＝7.5Hz,1H).13C NMR(101MHz,DMSO-d6):172.30(s),161.95(s),159.34(d,J＝250.6Hz),160.01(s),151.43(s),132.26(d,J＝8.6Hz),131.21(s),130.39(d,J＝2.6Hz),125.62(s),124.59(d,J＝13.0Hz),124.28(d,J＝3.4Hz),122.82(s),121.14(s),119.73(s),116.09(d,J＝21.9Hz).19F NMR(377MHz,DMSO-d6):-114.77.HPLC(λ280) The purity is 97.4 percent; t is tR6.792min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2-bromobenzamide (MTF-250). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-bromobenzonitrile (946mg, 5.20mmol) were synthesized to give the title compound as a beige powderSubstance (136mg, 7%).1HNMR(400MHz,DMSO-d6):9.26(s,1H),7.67(d,J＝7.9Hz,1H),7.54(d,br.s,J＝7.0Hz,2H),7.44(t,J＝7.3Hz,2H),7.35(t,J＝7.1Hz,1H),7.28–7.04(m,4H).13CNMR(101MHz,DMSO-d6):173.15,166.81,165.00,139.47,136.49,133.16,132.85,130.48,130.28,128.84,127.32,127.03,126.48,126.12,120.08.HRMS-ESI(m/z):[M+H]+For C15H12BrN5S+Calculated value 374.00696; found 374.00702.HPLC (. lamda.)280) The purity is 97.8 percent; t is tR4.158min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) pyridinecarboxamide (MTF-251). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-pyridinecarbonitrile (541mg, 5.20mmol) were synthesized to give the title compound as a white-light yellow powder (955mg, 62%).1H NMR(200MHz,DMSO-d6):10.10(br.s,1H),9.40(br.s,1H),8.92(br.s,1H),8.72(ddd,J＝4.7,1.6,0.9Hz,1H),8.37(dt,J＝7.9,1.0Hz,1H),8.03(br.s,td,J＝7.7,1.7Hz,2H),7.81(dd,J＝7.8,0.8Hz,1H),7.73–7.58(m,2H),7.35(td,J＝7.7,1.4Hz,1H),7.20(td,J＝7.6,1.2Hz,1H).13CNMR(101MHz,DMSO-d6):170.26,167.51,165.72,154.24,149.29,136.70,133.47,128.60,127.35,126.57,126.22,125.45,123.31.HRMS-ESI(m/z):[M+H]+Calculation of value for C14H12N6S+291.09169; found 291.09174.HPLC (. lamda.)280) The purity is 98.6 percent; t is tR6.075min (method 1).
N- (N- (N- (benzo [ d ]))]Thiazol-2-yl) carbamoylamino]Nicotinamide (MTF-252). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 312mg, 7.81mmol), 3-pyridinecarbonitrile (541mg, 5.20mmol) to afford the title compound as a white-light yellow powder (1.49g, 97%).1H NMR(200MHz,DMSO-d6):10.20(br.s,1H),9.34(br.s,1H),9.17(dd,J＝2.2,0.6Hz,1H),8.95(br.s,1H),8.74(dd,J＝4.8,1.6Hz,1H),8.37–8.28(m,1H),8.11(br.s,1H),7.66(dd,J＝8.0,0.6Hz,1H),7.55(ddd,J＝8.0,4.8,0.8Hz,1H),7.35(td,J＝7.7,1.4Hz,1H),7.20(td,J＝7.6,1.2Hz,1H).13C NMR(101MHz,DMSO-d6):172.18,161.92,160.34,151.88,151.43,148.75,135.10,131.24,130.86,125.61,123.24,122.82,121.11,19.75.HRMS-ESI(m/z):[M+H]+For C14H12N6S+Calculated value 297.09169; found 297.09174.HPLC (. lamda.)280) The purity is 100.0 percent; t is tR5.592min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) isonicotinamide (MTF-253). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 4-pyridinecarbonitrile (541mg, 5.20mmol) were synthesized to give the title compound as a white-light yellow powder (1.12g, 73%).1H NMR(200MHz,DMSO-d6):10.15(br.s,1H),9.35(br.s,1H),8.96(br.s,1H),8.76(dd,J＝4.6,1.5Hz,2H),8.15(br.s,1H),7.91(dd,J＝4.6,1.5Hz,2H),7.81(dd,J＝7.7,0.7Hz,1H),7.67(d,J＝8.0Hz,1H),7.42–7.30(m,1H),7.27–7.14(m,1H).13C NMR(101MHz,DMSO-d6):172.12,161.90,160.07,151.38,150.07,142.53,131.28,125.64,122.88,121.43,121.13,119.81.HRMS-ESI(m/z):[M+H]+For C14H12N6S+Calculated value 297.09169; found 297.09177 HPLC (. lamda.)280) The purity is 95.9 percent; t is tR5.308min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4-methoxybenzamide (MTF-254). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 4-methoxybenzonitrile (692mg, 5.20mmol) were synthesized to give the title compound as a beige powder (135mg, 8%).1H NMR(500MHz,DMSO-d6):10.27(br.s,1H),9.30(br.s,1H),8.69(br.s,1H),8.03(d,J＝5.9Hz,2H),7.92(br.s,1H),7.78(d,J＝5.4Hz,1H),7.64(d,J＝5.8Hz,1H),7.33(s,1H),7.18(s,1H),7.05(d,J＝5.6Hz,2H),3.83(s,3H).13C NMR(50MHz,DMSO-d6):172.33,162.14,161.96,161.71,151.58,131.16,129.40,127.19,125.62,122.72,121.10,119.64,113.56,55.41.HRMS-ESI(m/z):[M+H]+For C16H15N5OS+Calculated value 326.10701; found 326.10718 HPLC (. lamda.)280) The purity is 95.3 percent; t is tR6.592min (method 1).
3-acetyl-N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) benzamide (MTF-255). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3-acetylbenzonitrile (755mg, 5.20mmol) were synthesized to give the title compound (228mg, 13%) as a red powder.1H NMR(200MHz,DMSO-d6):10.25(br.s,1H),9.33(br.s,1H),8.98(br.s,1H),8.58(t,J＝1.6Hz,1H),8.30–8.22(m,1H),8.20–7.97(m,2H),7.81(dd,J＝7.8,0.8Hz,1H),7.68(t,J＝7.7Hz,2H),7.35(td,J＝7.7,1.4Hz,1H),7.20(td,J＝7.6,1.2Hz,1H),2.66(s,3H).13C NMR(50MHz,DMSO-d6):197.59,172.28,162.07,161.35,151.50,136.86,135.70,132.04,131.24,131.07,128.79,127.31,125.67,122.85,121.16,119.77,26.89.HRMS-ESI(m/z):[M+H]+For C17H15N5OS+Calculated value 338.10701; found 338.10701 HPLC (. lamda.)280) The purity is 95.2 percent; t is tR6.283min (method 1).
3-bromo-N- (N- (4-phenylthiazol-2-yl) carbamoylamino) benzamide (MTF-256). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 3-bromobenzonitrile (833mg, 4.58mmol), synthesized to give the title compound (715mg, 39%) as a yellow powder.1H NMR(500MHz,DMSO-d6):10.36(br.s,1H),9.15(br.s,1H),8.79(br.s,1H),8.25(s,1H),8.02(d,J＝7.8Hz,1H),7.88(d,br.s,J＝7.3Hz,3H),7.76(dd,J＝7.9,1.0Hz,1H),7.47(dd,J＝9.1,6.6Hz,2H),7.43(t,J＝7.7Hz,2H),7.31(t,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):173.11,160.69,160.04,149.88,137.62,134.52,133.94,130.38,128.71,127.61,126.46,125.61,121.73,106.53.HRMS-ESI(m/z):[M+H]+For C17H14BrN5S+Calculated value 400.02261; found 400.02213 HPLC (. lamda.)280) The purity is 98.7 percent; t is tR7.033min (method 1).
3-bromo-N- (N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) benzamide (MTF-257). Synthesized according to general procedure A using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 3-bromobenzonitrile (692mg, 3.80mmol) to give the title compound (711mg, 42%) as a yellow powder.1H NMR(500MHz,DMSO-d6):10.21(s,1H),8.78(s,2H),8.62(s,1H),8.34(d,J＝7.5Hz,1H),8.23(s,1H),8.19(br.s,1H),8.15(d,J＝7.8Hz,1H),8.01(d,J＝7.5Hz,1H),7.80(s,1H),7.76(d,J＝7.7Hz,1H),7.72(t,J＝7.9Hz,1H),7.48(t,J＝7.8Hz,1H).13C NMR(50MHz,DMSO-d6):173.32,160.75,159.98,148.31,147.48,137.51,136.01,133.95,131.81,130.37,130.20,126.47,121.99,121.66,119.77,109.28.HRMS-ESI(m/z):[M+H]+For C17H13BrN6O2S+Calculated value 445.00768; found 445.00806 HPLC (. lamda.)280) The purity is 99.1 percent; t is tR7.100min (method 1).
N- (N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) pyridinecarboxamide (MTF-259). Following general procedure a, using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 2-pyridinecarbonitrile (366mL, 3.80mmol), synthesized to give the title compound as a yellow powder (796mg, 57%).1H NMR(500MHz,DMSO-d6):10.03(br.s,1H),9.08(br.s,1H),8.78(br.s,1H),8.71(d,J＝4.2Hz,1H),8.66–8.61(m,1H),8.38(d,J＝7.9Hz,1H),8.35(d,J＝7.9Hz,1H),8.16(dd,J＝8.1,1.5Hz,1H),8.02(td,J＝7.8,1.6Hz,1H),7.91(br.s,1H),7.82(s,1H),7.73(t,J＝8.0Hz,1H),7.63(ddd,J＝7.4,4.8,1.0Hz,1H).13CNMR(50MHz,DMSO-d6):173.49,161.02,158.37,150.67,148.58,148.29,147.51,137.39,135.99,131.78,130.16,126.39,122.10,121.99,119.78,109.31.HRMS-ESI(m/z):[M+H]+For C16H13N7O2S+Calculated value 368.09242; found 368.09274 HPLC (. lamda.)280) The purity is 99.2 percent; t is tR:6850min (method 1).
N- (N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) nicotinic carboxamide (MTF-260). Following general procedure a, using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 3-pyridinecarbonitrile (395mg, 3.80mmol), synthesized to give the title compound as a yellow powder (656mg, 47%).1H NMR(500MHz,DMSO-d6):10.14(br s,1H),8.84(br.s,2H),8.75(d,J＝5.8Hz,2H),8.63(s,1H),8.34(d,J＝7.8Hz,1H),8.15(dd,J＝8.0,1.4Hz,1H),7.91(d,J＝5.8Hz,2H),7.86(br.s,1H),7.82(s,1H),7.72(t,J＝8.0Hz,1H).13CNMR(50MHz,DMSO-d6):173.23,160.74,159.55,150.07,148.33,147.49,142.64,136.00,131.80,130.20,122.01,121.49,119.79,109.48.HRMS-ESI(m/z):[M+H]+For C16H13N7O2S+Calculated value 368.09242; found 368.09283 HPLC (. lamda.)280) The purity is 95.1 percent; t is tR6.350min (method 1).
N- (N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) isonicotinamide (MTF-261). Following general procedure a, using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 4-pyridinecarbonitrile (395mg, 3.80mmol), synthesized to give the title compound as a yellow powder (544mg, 39%).1H NMR(500MHz,DMSO-d6):10.14(br.s,1H),9.17(d,J＝1.6Hz,1H),8.81(br.s,2H),8.73(dd,J＝4.7,1.4Hz,1H),8.63(s,1H),8.33(t,J＝8.4Hz,2H),8.15(dd,J＝8.1,1.5Hz,1H),7.81(s,1H),7.76(br.s,1H),7.72(t,J＝8.0Hz,1H),7.54(dd,J＝7.8,4.8Hz,1H).13CNMR(50MHz,DMSO-d6):173.30,160.78,159.84,151.81,148.78,148.32,147.48,136.02,135.13,131.79,130.97,130.19,123.25,121.99,119.79,109.31.HRMS-ESI(m/z):[M+H]+For C16H13N7O2S+Calculated value 368.09242; found 368.09283 HPLC (. lamda.)280) The purity is 96.8 percent; t is tR6.367min (method 1).
4-methoxy-N- (N- (4-phenylthiazol-2-yl) carbamoylamino) benzoylAmine (MTF-262). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 4-methoxybenzonitrile (610mg, 4.58mmol), synthesized to give the title compound as a beige powder (32mg, 2%).1H NMR(400MHz,DMSO-d6):10.30(br.s,1H),9.05(br.s,1H),8.55(s,1H),8.01(d,J＝8.9Hz,2H),7.87(d,J＝7.3Hz,2H),7.68(br.s,1H),7.46(s,1H),7.43(t,J＝7.7Hz,1H),7.31(t,J＝7.3Hz,1H),7.04(d,J＝8.9Hz,2H),3.83(s,3H).13C NMR(101MHz,DMSO-d6):173.16,161.77,161.07,160.84,149.72,134.54,129.21,128.64,127.50,127.36,125.51,113.46,106.11,55.37,38.89.HRMS-ESI(m/z):[M+H]+For C18H17N5OS+Calculated value 352.12266; found 352.12268 HPLC (. lamda.)280) The purity is 95.1 percent; t is tR6.842min (method 1).
N- (N- (4-phenylthiazol-2-yl) carbamoylamino) pyridinecarboxamide (MTF-263). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 2-pyridinecarbonitrile (441mL, 4.58mmol), synthesized to give the title compound as a yellow powder (369mg, 25%).1HNMR(500MHz,DMSO-d6):10.16(br.s,1H),9.19(br.s,1H),8.79(br.s,1H),8.70(d,J＝3.6Hz,1H),8.38(d,J＝7.7Hz,1H),8.01(t,J＝7.4Hz,1H),8.19–7.70(m,4H),7.88(d,br.s,J＝7.4Hz,4H),7.66–7.57(m,1H),7.50(s,1H),7.43(t,J＝7.3Hz,2H),7.32(t,J＝7.0Hz,1H).13C NMR(50MHz,DMSO-d6):173.20,160.91,158.35,150.73,149.86,148.61,137.40,134.50,128.70,127.61,126.38,125.59,122.04,106.61.HRMS-ESI(m/z):[M+H]+For C16H14N6S+Calculated value 323.10734; found 323.10770 HPLC (. lamda.)280) The purity is 98.0 percent; t is tR6.692min (method 1).
3-bromo-N- (N- (N- (6-methoxybenzo [ d ]))]Thiazol-2-yl) carbamoylamino) benzamide (MTF-264). Following general procedure A, 1- (6-methoxybenzo [ d ] was used]Thiazol-2-yl guanidine (1.00g, 4.50mmol), sodium hydride (60% dispersion in mineral oil, 1.5 mmQuant, 270mg, 6.75mmol) and 3-bromobenzonitrile (819mg, 4.50mmol) were synthesized to give the title compound as a dark gray powder (146mg, 8%).1H NMR(500MHz,DMSO-d6):10.29(br.s,1H),9.24(br.s,1H),8.84(br.s,1H),8.23(s,1H),8.01(d,br.s,J＝7.5Hz,2H),7.77(d,J＝7.2Hz,1H),7.56(d,J＝8.6Hz,1H),7.48(t,J＝7.8Hz,1H),7.42(s,1H),6.95(d,J＝8.5Hz,1H),3.79(s,3H).13C NMR(50MHz,DMSO-d6):170.29,161.45,160.30,155.69,145.55,137.47,134.03,132.39,130.45,130.36,126.46,121.68,120.37,113.92,104.89,55.55.HRMS-ESI(m/z):[M+H]+For C16H14BrN5OS+Calculated value 404.01752; found 404.01733.HPLC (. lamda.)280) The purity is 95.2 percent; t is tR6.825min (method 1).
3-bromo-N- (N- (6-methylbenzo [ d ]]Thiazol-2-yl) carbamoylamino) benzamide (MTF-265). Following general procedure A, 1- (6-methylbenzo [ d ] was used]Thiazol-2-yl) guanidine (1.00g, 4.85mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 291mg, 7.28mmol) and 3-bromobenzonitrile (883mg, 4.85mmol) were synthesized to give the title compound as a beige powder (207mg, 11%).1H NMR(500MHz,DMSO-d6):10.30(br.s,1H),9.32(br.s,1H),8.87(br.s,1H),8.24(s,1H),8.01(d,br.s,J＝7.6Hz,2H),7.77(d,J＝7.7Hz,1H),7.60(s,1H),7.54(d,J＝8.1Hz,1H),7.48(t,J＝7.8Hz,1H),7.16(d,J＝8.0Hz,1H),2.37(s,3H).13C NMR(50MHz,DMSO-d6):171.42,161.76,160.43,149.40,137.44,134.05,132.22,131.30,130.41,126.86,126.47,121.69,120.95,119.45,20.93.HRMS-ESI(m/z):[M+H]+For C16H14BrN5S+Calculated value 388.02261; found 388.02313.HPLC (. lamda.)280) The purity is 98.3 percent; t is tR6.992min (method 1).
3-chloro-N- (N- (4-phenylthiazol-2-yl) carbamoylamino) benzimidazole amide (MTF-267). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 3-chlorobenzonitrile (630mg, 4.58mmol) gave the title compound as a white-light yellow powder (749mg, 46%).1H NMR(500MHz,DMSO-d6):10.36(br.s,1H),9.15(br.s,1H),8.78(br.s,1H),8.10(t,J＝1.7Hz,1H),7.98(d,J＝7.9Hz,1H),7.88(d,br.s,J＝7.2Hz,3H),7.63(dd,J＝8.0,1.2Hz,1H),7.54(t,J＝7.9Hz,1H),7.48(s,1H),7.43(t,J＝7.7Hz,2H),7.31(t,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):173.05,160.66,160.04,149.85,137.43,134.51,133.20,131.03,130.14,128.69,127.59,127.46,126.08,125.58,106.53.HRMS-ESI(m/z):[M+H]+For C17H14ClN5S+Calculated value 356.07312; found 356.07321 HPLC (. lamda.)280) The purity is 98.0 percent; t is tR7.008min (method 1).
4-chloro-N- (N- (5-phenylthiazol-2-yl) carbamoylamino) benzamide (MTF-268). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 4-chlorobenzonitrile (630mg, 4.58mmol), synthesized to give the title compound as a yellow powder (717mg, 44%).1H NMR(500MHz,DMSO-d6):10.35(br.s,1H),9.14(br.s,1H),8.73(br.s,1H),8.05(d,J＝8.6Hz,2H),7.87(d,J＝7.2Hz,2H),7.73(br.s,1H),7.58(d,J＝8.6Hz,2H),7.47(s,1H),7.43(t,J＝7.7Hz,2H),7.31(t,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):173.07,160.72,160.43,149.82,136.13,134.52,134.17,129.37,128.69,128.31,127.58,125.57,106.47.HRMS-ESI(m/z):[M+H]+For C17H14ClN5S+Calculated value 356.07312; found 356.07318 HPLC (. lamda.)280) The purity is 99.1 percent; t is tR6.925min (method 1).
N- (N- (4-phenylthiazol-2-yl) carbamoylamino) benzamide (MTF-272). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and benzonitrile (0.471mL, 4.58mmol), synthesized to give the title compound as a beige powder (486mg, 33%).1H NMR(500MHz,DMSO-d6):10.33(br.s,1H),9.13(br.s,1H),8.67(br.s,1H),8.02(d,J＝7.2Hz,2H),7.88(d,J＝7.3Hz,2H),7.72(br.s,1H)7.56(t,J＝7.2Hz,1H),7.49(dd,J＝14.9,7.7Hz,3H),7.43(t,J＝7.7Hz,2H),7.31(t,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):173.16,161.66,160.90,149.80,135.41,134.54,131.26,128.71(2C),128.22(2C),127.57,127.50(2C),125.57(2C),106.37.HPLC(λ280) Purity is 82.0%; t is tR6.850min (method 1).
N- (N- (4-phenylthiazol-2-yl) carbamoylamino) nicotinic carboxamide (MTF-273). Following general procedure a, using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 3-cyanopyridine (477mg, 4.58mmol), synthesized as a light yellow powder (694mg, 47%).1H NMR(500MHz,DMSO-d6):10.30(br.s,1H),9.17(d,br.s,J＝1.6Hz,2H),8.82(br.s,1H),8.73(dd,J＝4.7,1.4Hz,1H),8.33(d,J＝8.0Hz,1H),7.88(d,br.s,J＝7.3Hz,3H),7.54(dd,J＝7.8,4.8Hz,1H),7.49(s,1H),7.43(t,J＝7.6Hz,2H),7.32(t,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):173.07,160.68,159.88,151.80,149.86,148.77,135.10,134.52,131.05,128.71,127.60,125.59,123.25,106.58.HRMS-ESI(m/z):[M+H]+For C16H14N6S+Calculated value 323.10734; found 323.10764 HPLC (. lamda.)280) The purity is 96.9 percent; t is tR6.075min (method 1).
N- (N- (4-phenylthiazol-2-yl) carbamoylamino) isonicotinamide (MTF-274). Synthesized according to general procedure A using 1- (4-phenylthiazol-2-yl) guanidine (1.00g, 4.58mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 275mg, 6.87mmol), and 4-pyridinecarbonitrile (477mg, 4.58mmol) to give the title compound as a light yellow powder (871mg, 59%).1H NMR(500MHz,DMSO-d6):10.30(br.s,1H),9.20(br.s,1H),8.86(br.s,1H),8.75(dd,J＝4.5,1.6Hz,2H),7.92(dd,J＝4.5,1.6Hz,2H),7.88(d,br.s,J＝7.2Hz,3H),7.50(s,1H),7.43(t,J＝7.7Hz,2H),7.32(t,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):173.00,160.64,159.63,150.09,149.90,142.74,134.50,128.72,127.63,125.61,121.49,106.75.HRMS-ESI(m/z):[M+H]+For C16H14N6S+Calculated value 323.10734; found 323.10757 HPLC (. lamda.)280) The purity is 95.2 percent; t is tR6.058min (method 1).
2-chloro-N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) benzamide (MTF-276.) synthesized according to general procedure a using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 2-chlorobenzonitrile (523mg, 3.80mmol) to give the title compound as a brown powder (30mg, 2%).1H NMR(400MHz,DMSO-d6):10.08(br.s,1H),8.83(br.s,1H),8.70(br.s,1H),8.62(s,1H),8.34(d,J＝7.7Hz,1H),8.15(d,J＝7.4Hz,1H),8.03-7.64(br.s,1H),7.81(s,1H),7.72(t,J＝7.7Hz,1H),7.52(t,J＝7.8Hz,2H),7.49–7.37(m,2H).13C NMR(101MHz,DMSO-d6):173.55,162.25,160.72,148.36,147.36,136.69,136.00,131.76,130.52,130.25(2C),129.51,129.39,126.87,121.95,119.70,109.25.HRMS-ESI(m/z):[M+H]+For C17H13ClN6O2S+Calculated value 401.05820; found 401.05820 HPLC (. lamda.)280) The purity is 95.2 percent; t is tR6.858min (method 1).
3-chloro-N- (N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) benzamide (MTF-277). Following general procedure a, using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 3-chlorobenzonitrile (523mg, 3.80mmol), synthesized to give the title compound as a brown powder (457mg, 30%).1H NMR(500MHz,DMSO-d6):10.22(br.s,1H),8.77(br.s,1H),8.62(s,1H),8.34(d,J＝7.7Hz,1H),8.23-7.91(br.s,1H),8.15(d,J＝8.0Hz,1H),8.09(s,1H),7.97(d,J＝7.7Hz,1H),7.80(s,1H),7.72(t,J＝8.0Hz,1H),7.63(d,J＝7.9Hz,1H),7.54(t,J＝7.9Hz,1H).13CNMR(50MHz,DMSO-d6):173.31,160.75,160.02,148.34,147.48,137.34,136.02,133.17,131.83,131.05,130.24,130.17,127.45,126.11,122.02,119.78,109.32.HRMS-ESI(m/z):[M+H]+For C17H13ClN6O2S+Calculated value 401.05820; found 401.05835 HPLC (. lamda.)280) The purity is 95.5 percent; t is tR7.025min (method 1).
4-chloro-N- (N- (4- (3-nitrophenyl) thiazol-2-yl) carbamoylamino) benzathinesAmide (MTF-281). Following general procedure a, using 1- (4- (3-nitrophenyl) thiazol-2-yl) guanidine (1.00g, 3.80mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 228mg, 5.70mmol), and 4-chlorobenzonitrile (523mg, 3.80mmol), synthesized to give the title compound as a yellow powder (640mg, 42%).1H NMR(200MHz,DMSO-d6):10.15(br.s,1H),8.74(br.s,2H),8.68–8.60(m,1H),8.55-7.90(br.s,1H),8.39–8.30(m,1H),8.16(ddd,J＝8.2,2.3,0.8Hz,1H),8.10–7.97(m,2H),7.82(s,1H),7.72(t,J＝8.0Hz,1H),7.66–7.48(m,2H).13C NMR(50MHz,DMSO-d6):173.37,160.84,160.46,148.30,147.47,136.17,136.04,134.10,131.77,130.16,129.40(2C),128.29(2C),121.96,119.77,109.19.HRMS-ESI(m/z):[M+H]+For C17H13ClN6O2S+Calculated value 401.05820; found 401.05820 HPLC (. lamda.)280) The purity is 96.7 percent; t is tR7.033min (method 1).
N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-283). Following general procedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol) and benzonitrile (0.66mL, 6.41mmol), synthesized to give the title compound (382mg, 23%) as a brown powder.1H NMR(500MHz,DMSO-d6):10.42(br.s,1H),9.11(br.s,1H),8.61(br.s,1H),8.02–7.96(m,2H),7.57–7.52(m,1H),7.85–7.39(br.s,4H),7.51–7.46(m,2H),6.57(d,J＝1.0Hz,1H),2.24(d,J＝0.9Hz,3H).13C NMR(50MHz,DMSO-d6):172.99,161.63,160.69,147.64,135.50,131.20,128.21,127.45,105.64,17.56.HRMS-ESI(m/z):[M+H]+f for C12H13N5S+Calculated value 260.09644; found 260.09653 HPLC (. lamda.)280) The purity is 95.7 percent; t is tR5.167min (method 1).
2-chloro-N- (N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-284) synthesized according to general procedure A using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol) and 2-chlorobenzonitrile (881mg, 6.41mmol) to give the title compound as a white powderThe title compound (132mg, 7%).1H NMR(400MHz,DMSO-d6):10.27(br.s,1H),9.06(br.s,1H),8.63(br.s,1H),7.63(br.s,1H),7.46(m,4H),6.57(s,1H),2.24(s,3H).13C NMR(101MHz,DMSO-d6):172.87,162.18,160.47,147.49,136.79,130.49,130.27,129.55,129.40,126.88,105.69,17.46.HRMS-ESI(m/z):[M+H]+For C12H12ClN5S+Calculated value 294.05747; found 294.05747 HPLC (. lamda.)280) The purity is 95.0 percent; t is tR5.500min (method 1).
3-chloro-N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-285). Following general procedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol) and 3-chlorobenzonitrile (881mg, 6.41mmol), synthesized to give the title compound as a white-light yellow (1.24g, 66%).1HNMR(500MHz,DMSO-d6):10.43(br.s,1H),9.13(br.s,1H),8.71(br.s,1H),8.11–8.05(m,1H),7.95(d,J＝7.9Hz,1H),7.64–7.59(m,1H),7.58(br.s,1H),7.53(t,J＝7.9Hz,1H),6.59(d,J＝0.9Hz,1H),2.25(d,J＝0.5Hz,3H).13C NMR(50MHz,DMSO-d6):172.82,160.44,159.91,147.64,137.47,133.18,130.97,130.15,127.39,126.00,105.82,17.53.HRMS-ESI(m/z):[M+H]+For C12H12ClN5S+Calculated value 294.05747; found 294.05768 HPLC (. lamda.)280) The purity is 95.0 percent; t is tR5.933min (method 1).
4-chloro-N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-286). Following general procedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol), and 4-chlorobenzonitrile (881mg, 6.41mmol), synthesized to give the title compound as a white-light yellow (923mg, 49%).1H NMR(500MHz,DMSO-d6):10.42(br.s,1H),9.13(br.s,1H),8.67(br.s,1H),8.01(d,J＝8.6Hz,2H),7.57(d,br.s J＝8.6Hz,3H),6.57(d,J＝1.0Hz,1H),2.24(d,J＝0.7Hz,3H).13C NMR(50MHz,DMSO-d6):172.87,160.50,160.37,147.63,136.08,134.23,129.30,128.29,105.74,17.53.HRMS-ESI(m/z):[M+H]+For C12H12ClN5S+Calculated value 294.05747; found 294.05762 HPLC (. lamda.)280) The purity is 95.3 percent; t is tR5.958min (method 1).
2-bromo-N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-287). Following general procedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol), and 2-bromobenzonitrile (1.17g, 6.41mmol), synthesized to give the title compound as a white-light yellow (499mg, 23%).1H NMR(400MHz,DMSO-d6):10.27(br.s,1H),9.07(br.s,1H),8.63(br.s,1H),7.67(d,br.s,J＝7.9Hz,2H),7.46–7.43(m,2H),7.36(ddd,J＝8.1,6.0,3.2Hz,1H),6.57(s,1H),2.24(s,3H).13C NMR(101MHz,DMSO-d6):172.89,163.29,160.49,147.50,138.85,132.49,130.57,129.44,127.37,119.62,105.68,17.46.HRMS-ESI(m/z):[M+H]+For C12H12BrN5S+Calculated value 338.00696; found 338.00705.HPLC (. lamda.)280) The purity is 95.8 percent; t is tR5.192min (method 1).
3-bromo-N- (N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-288) synthesized according to general procedure A using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol) and 3-bromobenzonitrile (1.17g, 6.41mmol) to give the title compound as a white-light yellow solid (1.11g, 51%).1H NMR(500MHz,DMSO-d6):10.42(s,1H),9.12(s,1H),8.72(s,1H),8.21(t,J＝1.7Hz,1H),7.98(d,J＝8.0Hz,1H),7.75(dd,br.s,J＝8.0,1.1Hz,2H),7.46(t,J＝7.9Hz,1H),6.59(d,J＝0.9Hz,1H),2.24(d,J＝0.8Hz,3H).13C NMR(50MHz,DMSO-d6):172.80,160.42,159.83,147.63,137.63,133.86,130.38,130.27,126.35,121.66,105.81,17.53.HRMS-ESI(m/z):[M+H]+For C12H12BrN5S+Calculated value 338.00696; found 338.00760.HPLC (. lamda.)280) The purity is 95.1 percent; t is tR6.042min (method 1).
N- (N- (4-methylthiazol-2-yl) carbamoylamino) pyridinecarboxamide (MTF-289). According to the generalProcedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol), and 2-pyridinecarbonitrile (0.617mL, 6.41mmol) was synthesized to give the title compound as a yellow powder (1.05g, 63%).1H NMR(200MHz,DMSO-d6):10.20(s,1H),9.15(s,1H),8.77(s,1H),8.73–8.65(m,1H),8.34(d,J＝8.5Hz,1H),8.00(td,J＝7.8,1.7Hz,1H),7.61(ddd,J＝7.4,4.8,1.1Hz,2H),6.60(d,J＝1.0Hz,1H),2.25(s,3H).13C NMR(101MHz,DMSO-d6):172.90,160.61,158.17,150.75,148.52,147.60,137.32,126.26,121.87,105.82,17.46.HRMS-ESI(m/z):[M+H]+For C11H12N6S+Calculated value 261.09169; found 261.09174.HPLC (. lamda.)280) The purity is 95.7 percent; t is tR5.708min (method 1).
N- (N- (4-methylthiazol-2-yl) carbamoylamino) nicotinic carboxamide (MTF-290). Following general procedure A, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol) and 3-pyridinecarbonitrile (0.667mg, 6.41mmol), synthesized to give the title compound as a yellow powder (751mg, 45%).1H NMR(500MHz,DMSO-d6):10.39(br.s,1H),9.15(d,br.s,J＝1.7Hz,2H),8.77(br.s,1H),8.71(dd,J＝4.7,1.5Hz,1H),8.36–8.23(m,1H),7.65(br.s,1H),7.52(dd,J＝7.5,4.8Hz,1H),6.59(d,J＝0.7Hz,1H),2.25(s,3H).13C NMR(50MHz,DMSO-d6):172.84,160.46,159.79,151.75,148.72,147.67,135.03,131.09,123.24,105.89,17.54.HRMS-ESI(m/z):[M+H]+For C11H12N6S+Calculated value 261.09169; found 261.09171.HPLC (. lamda.)280) The purity is 97.1 percent; t is tR5.508min (method 1).
N- (N- (4-methylthiazol-2-yl) carbamoylamino) isonicotinamide (MTF-291). Following general procedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 384mg, 9.61mmol) and 4-pyridinecarbonitrile (0.667mg, 6.41mmol), synthesized to give the title compound as a yellow powder (985mg, 59%).1H NMR(500MHz,DMSO-d6):10.37(br.s,1H),9.15(br.s,1H),8.80(br.s,1H),8.74(dd,J＝4.5,1.6Hz,2H),7.89(dd,J＝4.5,1.6Hz,2H),7.68(br.s,1H),6.60(d,J＝0.9Hz,1H),2.25(d,J＝0.8Hz,3H).13C NMR(50MHz,DMSO-d6):172.77,160.43,159.58,150.08,147.74,142.81,121.45,106.08,17.54.HRMS-ESI(m/z):[M+H]+For C11H12N6S+Calculated value 261.09169; found 261.09174.HPLC (. lamda.)280) The purity is 99.5 percent; t is tR4.525min (method 1).
4-methoxy-N- (N- (4-methylthiazol-2-yl) carbamoylamino) benzamide (MTF-292). Following general procedure a, using 1- (4-methylthiazol-2-yl) guanidine (1.00g, 6.41mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 384mg, 9.61mmol), and 4-methoxybenzonitrile (0.853mg, 6.41mmol), synthesized to give the title compound as a beige powder (167mg, 9%).1H NMR(500MHz,DMSO-d6):10.40(br.s,1H),9.06(br.s,1H),8.50(br.s,1H),7.98(d,J＝8.9Hz,2H),7.49(br.s,1H),7.02(d,J＝8.9Hz,2H),6.55(d,J＝0.8Hz,1H),3.83(s,3H),2.24(s,3H).13CNMR(101MHz,DMSO-d6):172.97,161.71,160.99,160.62,147.51,129.13,127.43,113.43,105.33,55.34,17.48.HRMS-ESI(m/z):[M+H]+For C13H15N5OS+Calculated value 290.10701; found 290.10709.HPLC (. lamda.)280) The purity is 100.0 percent; t is tR5.308min (method 1).
2-bromo-N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) benzamide (MTF-295). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol) and 2-bromobenzonitrile (1.07g, 5.88mmol), synthesized to give the title compound as a brown powder (746mg, 36%).1H NMR(500MHz,DMSO-d6):10.28(br.s,1H),9.01(br.s,1H),8.58(br.s,1H),7.70-7.30(br.s,1H),7.67(d,J＝7.9Hz,1H),7.44(dd,J＝8.1,5.2Hz,2H),7.40–7.33(m,1H),2.21(s,3H),2.14(s,3H).13C NMR(101MHz,DMSO-d6):169.26,163.14,160.22,142.62,138.85,132.48,130.54,129.44,127.36,119.64,116.98,14.58,10.71.HRMS-ESI(m/z):[M+H]+For C13H14BrN5S+Calculated value 352.02261; found 352.02289 HPLC (. lamda.)280) The purity is 97.8 percent; t is tR5.717min (method 1).
2-bromo-N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) benzamide (MTF-296). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol) and 3-bromobenzonitrile (1.07g, 5.88mmol), synthesized to give the title compound as a brown powder (746mg, 36%).1H NMR(200MHz,DMSO-d6):10.45(br.s,1H),9.01(br.s,1H),8.70(br.s,1H),8.20(t,J＝1.7Hz,1H),8.06-7.28(br.s,1H),7.97(d,J＝8.0Hz,1H),7.74(ddd,J＝7.9,1.9,0.8Hz,1H),7.46(t,J＝7.9Hz,1H),2.21(s,3H),2.15(s,3H).13C NMR(50MHz,DMSO-d6):169.14,160.12,159.63,142.74,137.65,133.81,130.41,130.18,126.29,121.61,117.13,14.63,10.76.HRMS-ESI(m/z):[M+H]+For C13H15BrN5S+Calculated value 352.02261; found 352.02377 HPLC (. lamda.)280) The purity is 97.3 percent; t is tR5.767min (method 1).
N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) pyridinecarboxamide (MTF-297). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 353mg, 8.82mmol), and 2-pyridinecarbonitrile (0.566mL, 5.88mmol), synthesized to give the title compound as a yellow powder (726mg, 45%).1H NMR(500MHz,DMSO-d6):10.19(br.s,1H),9.09(br.s,1H),8.68(d,br.s.J＝4.1Hz,2H),8.33(d,J＝7.9Hz,1H),7.99(td,J＝7.7,1.3Hz,1H),7.60(dd,br.s,J＝6.5,5.0Hz,2H),2.21(s,3H),2.15(s,3H).13C NMR(101MHz,DMSO-d6):169.29,160.32,158.01,150.80,148.50,142.71,137.28,126.19,121.80,117.16,14.56,10.69.HRMS-ESI(m/z):[M+H]+For C12H14N6S+Calculated value 275.10734; found 275.10742 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR5.475min (method 1).
N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) nicotinic carboxamide (MTF-298). According to aGeneral procedure a was synthesized using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 353mg, 8.82mmol) and 3-pyridinecarbonitrile (612mg, 5.88mmol) to give the title compound as a yellow powder (742mg, 46%).1H NMR(500MHz,DMSO-d6):10.43(br.s,1H),9.04(br.s,1H),8.67(br.s,1H),8.20(t,J＝1.6Hz,1H),7.97(d,J＝8.0Hz,1H),7.80–7.70(m,1H),7.88–7.29(br.s,1H),7.46(t,J＝7.9Hz,1H),2.21(s,3H),2.15(s,3H).13C NMR(101MHz,DMSO-d6):169.13,160.11,159.55,151.63,148.60,142.72,134.89,131.06,123.17,117.16,14.57,10.69.HRMS-ESI(m/z):[M+H]+For C12H14N6S+Calculated value 275.10734; found 275.10736 HPLC (. lamda.)280) The purity is 97.0 percent; t is tR5.458min (method 1).
N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) isonicotinamide (MTF-299). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol) and 4-pyridinecarbonitrile (612mg, 5.88mmol), synthesized to give the title compound as a yellow powder (548mg, 34%).1H NMR(500MHz,DMSO-d6):10.40(br.s,1H),9.09(br.s,1H),8.73(d,br.s.,J＝5.7Hz,3H),7.88(d,J＝5.5Hz,2H),7.61(br.s,1H),2.21(s,3H),2.15(s,3H).13C NMR(101MHz,DMSO-d6):169.05,160.06,159.33,149.98,142.78,121.31,117.38,14.55,10.68.HRMS-ESI(m/z):[M+H]+For C12H14N6S+Calculated value 275.10734; found 275.10739 HPLC (. lamda.)280) The purity is 99.5 percent; t is tR5.108min (method 1).
2-chloro-N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) benzamide (MTF-300). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol) and 2-chlorobenzonitrile (809mg, 5.88mmol), synthesized to give the title compound as a yellow powder (109mg, 6%).1H NMR(500MHz,DMSO-d6):10.30(br.s,1H),9.01(br.s,1H),8.59(br.s,1H),7.46(m,5H),2.21(s,3H),2.14(s,3H).13C NMR(101MHz,DMSO-d6):169.26,162.01,160.22,142.63,136.82,130.47,130.28,129.56,129.39,126.88,117.00,14.58,10.71.HRMS-ESI(m/z):[M+H]+For C13H14ClN5S+Calculated value 308.07312; found 308.07318 HPLC (. lamda.)280) The purity is 95.4 percent; t is tR5.425min (method 1).
3-chloro-N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) benzamide (MTF-301). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol) and 3-chlorobenzonitrile (809mg, 5.88mmol), synthesized to give the title compound as a yellow powder (416mg, 23%).1H NMR(500MHz,DMSO-d6):10.44(br.s,1H),9.04(br.s,1H),8.68(br.s,1H),8.08–8.03(m,1H),7.93(d,J＝7.9Hz,1H),7.61(ddd,J＝8.0,2.1,0.9Hz,1H),7.78–7.39(br.s,1H),7.52(t,J＝7.9Hz,1H),2.21(s,3H),2.15(s,3H).13C NMR(50MHz,DMSO-d6):169.15,160.14,159.71,142.73,137.49,133.13,130.91,130.15,127.31,125.93,117.14,14.62,10.75.HRMS-ESI(m/z):[M+H]+For C13H14ClN5S+Calculated value 308.07312; found 308.07321 HPLC (. lamda.)280) The purity is 95.2 percent; t is tR6.075min (method 1).
4-chloro-N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) benzamide (MTF-302). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol), 4-chlorobenzonitrile (809mg, 5.88mmol), synthesized to give the title compound (90mg, 5%) as a yellow powder.1H NMR(500MHz,DMSO-d6):10.44(br.s,1H),9.04(br.s,1H),8.64(br.s,1H),8.01(d,J＝8.6Hz,2H),7.57(d,br.s,J＝8.6Hz,3H),2.22(s,3H),2.15(s,3H).13C NMR(101MHz,DMSO-d6):169.15,160.16,160.11,142.66,135.93,134.23,129.19(2C),128.22(2C),117.02,14.57,10.70.HRMS-ESI(m/z):[M+H]+For C13H14ClN5S+Calculated value 308.07312; found 308.07315 HPLC (. lamda.)280) Is made ofDegree 98.0%; t is tR6.267min (method 1).
N- (N- (4, 5-dimethylthiazol-2-yl) carbamoylamino) benzamide (MTF-303). Following general procedure a, using 1- (4, 5-dimethylthiazol-2-yl) guanidine (1.00g, 5.88mmol), sodium hydride (60% dispersion in mineral oil, 1.5 eq, 353mg, 8.82mmol) and benzonitrile (0.605mL, 5.88mmol), synthesized to give the title compound (113mg, 7%) as a yellow powder.1H NMR(200MHz,DMSO-d6):10.45(s,1H),8.94(s,1H),8.61(s,1H),7.98(dd,J＝7.9,1.7Hz,2H),7.84–7.31(m,4H),2.21(d,J＝0.6Hz,3H),2.15(s,3H).13C NMR(101MHz,DMSO-d6):169.26,161.31,160.33,142.65,135.47,131.06,128.12,127.30,116.88,14.58,10.70.HRMS-ESI(m/z):[M+H]+For C13H15N5S+Calculated value 274.11209; found 274.11212 HPLC (. lamda.)280) The purity is 97.5 percent; t is tR5.408min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2,2, 2-trichloroacetamide (MTF-305). 1- (benzo [ d ]) is reacted with]A solution of thiazol-2-yl) guanidine (500mg, 2.6mmol) and trichloroacetonitrile (260. mu.L, 2.6mmol) in technical grade ethanol (5mL) was stirred at room temperature under an argon atmosphere for 22 h. The precipitate formed was filtered, washed with a small amount of cold ethanol and then with a small amount of diethyl ether, dried rapidly in air (the compound was oxidized in air over time) and stored under argon. Yellow solid (100mg, 11%). TLC Rf(CHCl3/MeOH,95/5,v/v)＝0.90.1HNMR (400MHz, acetone-d 6):10.71(s,1H),9.84(s,1H),8.39(s,1H),7.80(dd, J ═ 7.9,1.3Hz,1H),7.72(d, J ═ 8.0Hz,1H),7.66(s,1H),7.38(td, J ═ 8.2,7.8,1.3Hz,1H),7.25(td, J ═ 7.6,1.2Hz,1H).13C NMR (101MHz, acetone-d 6):206.12,126.63,124.28,121.93,121.33 HRMS-ESI (M/z): M + H]+For C10H9Cl3N5S+Calculated value 335.96388; found 335.96420 HPLC (. lamda.)254) The purity is 99.7 percent; t is tR7.983min (method 1).
N2- (2- (methylthio) phenyl) -6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine (MTF-316). To N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6-To a suspension of (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) CRO15(150mg, 0.225mmol) in methanol was added 10mL of mercaptoethanol (350 μ L, 4.5mmol), and the mixture was stirred at room temperature. After stirring for 10 minutes, the total solubility was reached. TLC and LCMS showed complete conversion to reduced thiophenol. After stirring at room temperature for 20h, K was added2CO3(62.1mg, 0.45mmol) and the mixture was sonicated until complete dissolution. Then, MeI (28. mu.L, 0.45mmol) was added and the solution was stirred at room temperature for 5h (reaction time not optimized). TLC and LCMS showed complete conversion to two new compounds (
6- (dichloromethyl) -N2- (2- (methylthio) phenyl) -1,3, 5-triazine-2, 4-diamine (MTF-317). To a suspension of N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (trichloromethyl) -1,3, 5-triazine-2, 4-diamine) CRO15(150mg, 0.225mmol) in methanol (10mL) was added mercaptoethanol (350 μ L, 4.5mmol), and the mixture was stirred at room temperature. After stirring for 10 minutes, the total solubility was reached. TLC and LCMS showed complete conversion to reduced thiophenol. After stirring at room temperature for 20h, K was added2CO3(62.1mg, 0.45mmol) and the mixture was sonicated until complete dissolution. Then, MeI (28. mu.L, 0.45mmol) was added and the solution was stirred at room temperature for 5h (reaction time not optimized). TLC and LCMS showed complete conversion to two new compounds (
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-methoxyphenyl) -1,3, 5-triazine-2, 4-diamine (MTF-318) according to general procedure B, using N- (benzo [ d ] B)]Thiazol-2-yl) carbamoylamino) -3-methoxybenzamide (325mg, 1mmol) was synthesized to give the title compound (272mg, 84%) as a white powder.1H NMR(200MHz,DMSO-d6):9.25(s,2H),7.82(dd,J＝8.1,5.1Hz,4H),7.61(dd,J＝7.7,1.4Hz,2H),7.44–7.33(m,4H),7.27(td,J＝7.5,1.4Hz,2H),7.22–6.97(m,8H),3.78(s,6H).13C NMR(50MHz,DMSO-d6):169.95,167.28,165.48,159.19,138.15,136.69,133.74,129.31,128.36,127.63,126.62,126.32,120.25,117.26,112.78,55.10.HRMS-ESI(m/z):[M+H]+ for C15H12BrN5S+Calculated value 374.00696; found 374.00797 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR11.458min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-ethoxyphenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-319). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3-ethoxybenzamide (339mg, 1mmol) was synthesized to give the title compound (291mg, 86%) as a white powder.1H NMR(200MHz,DMSO-d6):9.24(s,2H),7.83(d,J＝8.8Hz,4H),7.62(dd,J＝7.6,1.1Hz,2H),7.37(t,J＝7.8Hz,4H),7.32–7.23(m,2H),7.22–7.15(m,2H),7.14–6.98(m,6H),4.04(q,J＝6.9Hz,4H),1.33(t,J＝6.9Hz,6H).13C NMR(50MHz,DMSO-d6):169.98,167.28,165.48,158.46,138.12,136.73,133.68,129.30,128.39,127.64,126.61,126.29,120.13,117.81,113.27,63.06,14.68.HRMS-ESI(m/z):[M+H]+For C34H33N10O2S2 +Calculated value 677.22239; found 677.22253.HPLC (. lamda.)280) The purity is 100.0 percent; t is tR13.058min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-fluorophenyl) -1,3, 5-triazine-2, 4-diamine (MTF-320.) following general procedure B, N- (N- (benzo [ d ] benzo)]Thiazol-2-yl) carbamoylamino) -3-fluorobenzamide (313mg, 1mmol) was synthesized to give the title compound (237mg, 76%) as a white powder.1H NMR(200MHz,DMSO-d6):9.32(s,2H),8.08(d,J＝7.6Hz,2H),7.93(d,J＝9.7Hz,2H),7.62(d,J＝7.7Hz,2H),7.57–7.45(m,2H),7.45–7.06(m,12H).19F NMR(188MHz,DMSO-d6):-113.29.13C NMR(50MHz,DMSO-d6):168.98,167.26,165.51,164.58,159.75,139.31(d,J＝7.7Hz),136.52,133.76,130.39(d,J＝7.8Hz),128.22,127.66,126.62(d,J＝16.1Hz),123.79,118.23(d,J＝20.5Hz),114.11(d,J＝22.6Hz).HRMS-ESI(m/z):[M+H]+For C30H23F2N10S2 +Calculated value 625.15112; found 625.15125 HPLC (. lamda.)280) The purity is 97.5 percent; t is tR12.817min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (pyridin-2-yl) -1,3, 5-triazine-2, 4-diamine) (MTF-321). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) pyridinecarboxamide (296mg, 1mmol) was synthesized to give the title compound (266mg, 90%) as a white powder.1H NMR(200MHz,DMSO-d6):9.39(s,1H),8.74–8.64(m,1H),8.20(d,J＝7.8Hz,1H),7.93(td,J＝7.7,1.8Hz,1H),7.61(dd,J＝7.6,1.5Hz,1H),7.51(ddd,J＝7.5,4.7,1.2Hz,1H),7.39(dd,J＝7.7,1.4Hz,1H),7.35–7.08(m,4H).13C NMR(50MHz,DMSO-d6):170.30,167.53,165.79,154.32,149.29,136.74,136.57,133.61,128.25,127.64,126.88,126.47,125.48,123.33.HRMS-ESI(m/z):[M+H]+For C28H23N12S2 +CalculatingA value of 591.16048; found 591.16048 HPLC (. lamda.)280) The purity is 95.3 percent; t is tR6.483min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-chlorophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-322). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3-chlorobenzamide (329mg, 1mmol) was synthesized to give the title compound (260mg, 79%) as a white powder.1H NMR(200MHz,DMSO-d6):9.33(s,2H),8.24(s,2H),8.16(d,J＝7.6Hz,2H),7.58(ddd,J＝20.7,9.4,4.5Hz,6H),7.44–7.08(m,10H).13C NMR(50MHz,DMSO-d6):168.82,167.23,165.50,138.80,136.49,133.81,133.18,131.14,130.31,128.22,127.66,127.45,126.79,126.51,126.27.HRMS-ESI(m/z):[M+H]+For C30H23Cl2N10S2 +Calculated value 657.09201; found 657.09222 HPLC (. lamda.)280) The purity is 96.9 percent; t is tR12.867min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-bromophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-323). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3-bromobenzamide (374mg, 1mmol) was synthesized to give the title compound as a white powder (339mg, 91%).1H NMR(200MHz,DMSO-d6):9.34(s,2H),8.40(s,2H),8.20(d,J＝7.8Hz,2H),7.74(ddd,J＝8.0,2.0,1.0Hz,2H),7.62(dd,J＝7.5,1.6Hz,2H),7.46(t,J＝7.9Hz,2H),7.40–7.13(m,10H).13C NMR(50MHz,DMSO-d6):168.73,167.22,165.49,138.97,136.48,134.02,133.83,130.60,130.43,128.22,127.66,126.81,126.63,126.55,121.69.HRMS-ESI(m/z):[M+H]+For C30H23Br2N10S2 +Calculated value 744.99098; found 744.99098 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR17.600min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (4-methoxyphenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-324). Following general procedure B, with N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -4-methoxybenzamide (325mg, 1mmol) was synthesized to give a white powderThe title compound (263mg, 81%).1H NMR(200MHz,DMSO-d6):9.13(s,2H),8.20(d,J＝8.8Hz,4H),7.62(dd,J＝7.8,1.2Hz,2H),7.40(d,J＝7.2Hz,2H),7.32–7.22(m,2H),7.22–7.12(m,2H),7.01(d,J＝8.8Hz,8H),3.82(s,6H).13CNMR(50MHz,DMSO-d6):169.77,167.19,165.36,161.99,136.91,133.33,129.62(2C),128.90,128.61,127.72,126.36,126.10,113.57(2C),55.30.HRMS-ESI(m/z):[M+H]+For C32H29N10O2S2 +Calculated value 649.19109; found 649.19116 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR10.800min (method 2).
3,3' - (((dithiolethylenebis (2, 1-phenylene)) bis (azediyl)) bis (6-amino-1, 3, 5-triazine-4, 2-diyl)) dibenzylnitrile (MTF-325). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3-cyanobenzamide (320mg, 1mmol) was synthesized to give the title compound (291mg, 91%) as a white powder.1HNMR(200MHz,DMSO-d6):9.37(s,2H),8.49(d,J＝10.9Hz,4H),8.02(d,J＝7.7Hz,2H),7.72(t,J＝7.8Hz,2H),7.63(dd,J＝7.6,1.4Hz,2H),7.42–7.15(m,10H).13C NMR(50MHz,DMSO-d6):168.32,167.24,165.49,137.79,136.48,134.74,133.71,132.16,131.22,129.88,128.38,127.79,126.80,126.59,118.56,111.54.HRMS-ESI(m/z):[M+H]+For C32H23N12S2 +Calculated value 639.16046; found 639.16064 HPLC (. lamda.)280) The purity is 96.1 percent; t is tR11.858min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6-phenyl-1, 3, 5-triazine-2, 4-diamine) (MTF-326). According to general procedure B, N- (N- (benzo [ d ]) was used]Thiazol-2-yl) aminoamido) benzamide (295mg, 1mmol) was synthesized to give the title compound (218mg, 74%) as a white powder.1H NMR(200MHz,DMSO-d6):9.23(s,2H),8.24(d,J＝6.5Hz,4H),7.62(d,J＝7.3Hz,2H),7.57–7.34(m,8H),7.33–7.01(m,8H).13C NMR(50MHz,DMSO-d6):170.16,167.29,165.51,136.71,136.61,133.59,131.41,128.37,128.24(2C),127.82(2C),127.66,126.61,126.31.HRMS-ESI(m/z):[M+H]+For C30H25N10S2 +Calculated value 589.16996; found 589.16992 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR11.792min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (4-chlorophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-327). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -4-chlorobenzamide (329mg, 1mmol) was synthesized to give the title compound (283mg, 86%) as a white powder.1H NMR(200MHz,DMSO-d6):9.27(s,2H),8.22(d,J＝8.5Hz,4H),7.66–7.50(m,6H),7.38(dd,J＝7.6,1.0Hz,2H),7.33–7.05(m,8H).13C NMR(50MHz,DMSO-d6):169.18,167.22,165.46,136.62,136.20,135.47,133.52,129.53(2C),128.42(3C),127.73,126.65,126.37.HRMS-ESI(m/z):[M+H]+For C30H23Cl2N10S2 +Calculated value 657.09201; found 657.09210 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR15.633min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (4-iodophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-328). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -4-iodobenzamide (421mg, 1mmol) was synthesized to give the title compound (387mg, 92%) as a white powder.1H NMR(200MHz,DMSO-d6):9.26(s,2H),8.00(d,J＝8.4Hz,4H),7.87(d,J＝8.4Hz,4H),7.61(d,J＝7.6Hz,2H),7.39(d,J＝7.4Hz,2H),7.33–6.99(m,8H).13C NMR(50MHz,DMSO-d6):169.58,167.21,165.45,137.24(2C),136.63,136.19,133.49,129.70(2C),128.47,127.73,126.61,126.34,99.15.HRMS-ESI(m/z):[M+H]+For C30H23I2N10S2 +Calculated value 840.96324; found 840.96289 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR17.833min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (pyridin-4-yl) -1,3, 5-triazine-2, 4-diamine) (MTF-329). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) isonicotinamide (296mg, 1mmol) to give the title product as a white powderCompound (266mg, 90%).1H NMR(200MHz,DMSO-d6):9.41(s,2H),8.73(dd,J＝4.5,1.5Hz,4H),8.05(d,J＝5.8Hz,4H),7.62(dd,J＝7.4,1.2Hz,2H),7.43–7.08(m,10H).13C NMR(50MHz,DMSO-d6):168.77,167.33,165.59,150.23(2C),144.04,136.41,133.74,128.33,127.75,126.86,126.61,121.55(2C).HRMS-ESI(m/z):[M+H]+For C28H23N12S2 +Calculated value 591.16046; found 591.16089 HPLC (. lamda.)280) The purity is 99.2 percent; t is tR7.742min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (pyridin-3-yl) -1,3, 5-triazine-2, 4-diamine) (MTF-330). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) nicotinic carboxamide (296mg, 1mmol) was synthesized to give the title compound (266mg, 90%) as a white powder. (277mg, 94%).1H NMR(200MHz,DMSO-d6):9.35(s,4H),8.71(dd,J＝4.8,1.7Hz,2H),8.47(d,J＝8.1Hz,2H),7.62(dd,J＝7.6,1.2Hz,2H),7.52(dd,J＝7.8,4.6Hz,2H),7.38(dd,J＝7.7,1.4Hz,2H),7.33–7.06(m,8H13C NMR(50MHz,DMSO-d6):168.81,167.12,165.37,151.99,149.09,136.50,135.09,133.66,132.00,128.31,127.69,126.77,126.49,123.51HRMS-ESI(m/z):[M+H]+For C28H23N12S2 +Calculated value 591.16046; found 591.16052 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR7.700min (method 2).
2- ((4-amino-6- (3-bromophenyl) -1,3, 5-triazin-2-yl) amino) phenol (MTF-331). Following general procedure A, using 1- (benzo [ d ]]Oxazol-2-yl) guanidine (1.00g, 5.68mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 341mg, 8.52mmol) and 3-bromobenzonitrile (1.034g, 5.68mmol) were synthesized to give the title compound (1.46g, 72%) as a brown powder.1H NMR(400MHz,DMSO-d6):9.97(s,1H),8.44(s,1H),8.36(s,1H),8.26(d,J＝7.5Hz,1H),7.90(d,J＝7.7Hz,1H),7.76(d,J＝7.7Hz,1H),7.48(t,J＝7.8Hz,1H),7.29(s,2H),6.99–6.92(m,1H),6.89(d,J＝7.4Hz,1H),6.82(t,J＝7.1Hz,1H).13C NMR(50MHz,DMSO-d6):168.84,167.06,164.48,148.30,138.75,133.26,131.22,130.38,127.46,126.84,126.29,124.12,122.65,118.99,115.77.HRMS-ESI(m/z):[M+H]+For C15H13BrN5O+Calculated value 358.02980 found 358.03094 HPLC (. lamda.)280) The purity is 97.1 percent; t is tR9.808min (method 1).
2- ((4-amino-6- (3-chlorophenyl) -1,3, 5-triazin-2-yl) amino) phenol (MTF-332). Following general procedure A, using 1- (benzo [ d ]]Oxazol-2-yl) guanidine (1.00g, 5.68mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 341mg, 8.52mmol) and 3-chlorobenzonitrile (781mg, 5.68mmol) were synthesized to give the title compound (1.45g, 82%) as a brown powder.1H NMR(400MHz,DMSO-d6):10.03(s,1H),8.36(s,1H),8.29(s,1H),8.23(d,J＝7.4Hz,1H),7.92(d,J＝7.5Hz,1H),7.62(d,J＝7.5Hz,1H),7.55(t,J＝7.8Hz,1H),7.29(s,2H),6.99–6.92(m,1H),6.89(d,J＝7.3Hz,1H),6.82(t,J＝7.1Hz,1H).13C NMR(50MHz,DMSO-d6):168.74,167.02,164.48,148.21,138.90,134.11,130.67,130.40,126.66(2C),124.17,122.79,121.73,119.10,115.78.HRMS-ESI(m/z):[M+H]+For C15H13ClN5O+Calculated value 314.08031; found 314.08066 HPLC (. lamda.)280) The purity is 97.3 percent; t is tR9.625min (method 1).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6-benzyl-1, 3, 5-triazine-2, 4-diamine (MTF-333.) in a microwave tube under an argon atmosphere]Thiazol-2-yl) guanidine (100mg, 0.52mmol) was dissolved in NMP (2mL), then the tube was cooled to 0 deg.C, and NaH (60% suspension in oil, 23mg, 0.57mmol) was added. After the evolution of gas had ceased, the tube was sealed and the mixture was heated to 110 ℃ under microwave irradiation for 15 minutes. The resulting slurry was dissolved in Et2And O, and filtering. The precipitate was then purified by flash chromatography on silica gel (dichloromethane/MeOH, 10/0-9/1) to give the desired product as a white powder (50mg, 31%). TLC: rf (dichloromethane/MeOH, 9/1, v/v) ═ 0.83.1H NMR(400MHz,DMSO-d6):9.05(s,1H),7.54(dd,J＝7.8,0.9Hz,1H),7.35–7.17(m,7H),7.14(t,J＝7.7Hz,1H),6.95(d,J＝16.0Hz,2H),3.71(s,2H).13C NMR(101MHz,DMSO-d6):176.6,166.9,165.3,151.5,139.2,137.9,136.6,129.1,128.2,128.0,127.6,126.3,124.9,30.4.HRMS-ESI(m/z):[M+H]+For C32H29N10S2 +Calculated 617.20126, found 617.20154 HPLC (. lamda.)254) The purity is 96.2 percent; t is tR10.742min (method 2).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3-methoxybenzamide (MTF-342). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3-methoxybenzonitrile (0.635mL, 5.20mmol) were synthesized to give the title compound (914mg, 54%) as a white powder.1H NMR(200MHz,DMSO-d6):10.26(s,1H),9.33(s,1H),8.82(s,1H),8.05(s,1H),7.80(d,J＝7.6Hz,1H),7.73–7.52(m,3H),7.48–7.29(m,2H),7.17(dd,J＝14.6,7.6Hz,2H),3.83(s,3H).13C NMR spectrum could not be properlyrecorded as this compound rearranges and dimerizes into compound MTF-318during the time of the analysis.HRMS-ESI(m/z):[M+H]+For C16H16N5OS+Calculated value 326.10701; found 326.10706 HPLC (. lamda.)280) The purity is 100.0 percent; t is tR6.558min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3- (trifluoromethyl) benzamide (MTF-343). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3- (trifluoromethyl) benzonitrile (890mg, 5.20mmol) were synthesized to give the title compound (435mg, 23%) as a white powder.1H NMR(200MHz,DMSO-d6):10.33(br.s,1H),9.36(br.s,1H),8.93(br.s,1H),8.39(s,1H),8.31(d,J＝7.9Hz,1H),8.16(br.s,1H),7.95(d,J＝7.9Hz,1H),7.86–7.72(m,2H),7.67(d,J＝8.0Hz,1H),7.35(t,J＝7.6Hz,1H),7.20(t,J＝7.5Hz,1H).19F NMR (376MHz, DMSO-d6): 61.04-this compound was not correctly recorded because it rearranged and dimerized during the analysis13C NMR spectrum. HRMS-ESI (M/z) [ M + H ]]+For C16H13F3N5S+CalculatingA value of 364.08383; found 364.08408 HPLC (. lamda.)280) The purity is 97.9 percent; t is tR6.533min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3-ethoxybenzamide (MTF-344). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3-ethoxybenzonitrile (765mg, 5.20mmol) were synthesized to give the title compound (830mg, 47%) as a white powder.1H NMR(200MHz,DMSO-d6):10.23(br.s,1H),9.30(br.s,1H),8.78(br.s,1H),8.03(br.s,1H),7.80(dd,J＝7.8,0.8Hz,1H),7.65(dd,J＝8.0,0.6Hz,1H),7.62–7.53(m,2H),7.46–7.29(m,2H),7.24–7.07(m,2H),4.09(q,J＝6.9Hz,2H),1.36(t,J＝6.9Hz,3H).13C NMR(50MHz,DMSO-d6):172.24,162.07,161.82,158.40,151.49,136.56,131.16,129.36,125.63,122.78,121.13,119.68,119.65,117.34,113.64,63.24,14.63.HRMS-ESI(m/z):[M+H]+For C17H18N5OS+Calculated value 340.12266; found 340.12296.HPLC (. lamda.)280) The purity is 100.0 percent; t is tR6.733min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) pyrazine-2-carboxymethylamide (MTF-345). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and pyrazinecarbonitrile (0.465mL, 5.20mmol) were synthesized to give the title compound as a yellow powder (959mg, 62%).1H NMR(200MHz,DMSO-d6):10.13(br.s,1H),9.50(s,1H),9.44(br.s,1H),8.99(br.s,1H),8.88(d,J＝2.5Hz,1H),8.79(dd,J＝2.5,1.4Hz,1H),8.26(br.s,1H),7.83(d,J＝7.7Hz,1H),7.69(d,J＝7.9Hz,1H),7.44–7.30(m,1H),7.29–7.15(m,1H).13C NMR(50MHz,DMSO-d6):172.18,161.92,157.68,151.39,147.21,145.85,143.84,143.44,131.22,125.73,123.01,121.22,119.90.HRMS-ESI(m/z):[M+H]+For C13H12N7S+Calculated value 298.08694; found 298.08707.HPLC (. lamda.)280) The purity is 95.7 percent; t is tR5.508min (method 1).
N- (N- (benzene)And [ d ]]Thiazol-2-yl) carbamoylamino) -3-fluorobenzamide (MTF-346). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 3-fluorobenzonitrile (0.56mL, 5.20mmol) were synthesized to give the title compound (895mg, 55%) as a white powder.1H NMR(200MHz,DMSO-d6):10.26(s,1H),9.34(s,1H),8.89(s,1H),8.09(s,1H),7.95–7.76(m,3H),7.70–7.51(m,2H),7.50–7.28(m,2H),7.19(td,J＝7.6,1.2Hz,1H).19F NMR (376MHz, DMSO-d6): 112.99. due to rearrangement and dimerization of the compound to the compound MTF-320 during the analysis, correct recording was not possible13C NMR spectrum. HRMS-ESI (M/z) [ M + H ]]+For C15H13FN5S+Calculated value 314.08702; found 314.08722 HPLC (. lamda.)280) The purity is 98.7 percent; t is tR6.442min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3-cyanobenzamide (MTF-347). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 1, 3-dicyanobenzene (667mg, 5.20mmol) were synthesized to give the title compound as a white-light yellow powder (1.23g, 74%).1H NMR(200MHz,DMSO-d6):10.27(br.s,1H),9.37(br.s,1H),8.97(br.s,1H),8.45(s,1H),8.33(dd,J＝8.0,1.0Hz,1H),8.15(br.s,1H),8.06(dd,J＝7.7,1.0Hz,1H),7.89–7.62(m,3H),7.35(t,J＝7.6Hz,1H),7.20(t,J＝7.5Hz,1H).13C NMR(50MHz,DMSO-d6):172.17,161.83,159.93,151.42,136.30,134.69,132.15,131.40,131.24,129.67,125.67,122.89,121.17,119.81,118.39,111.50.HRMS-ESI(m/z):[M+H]+For C16H13N6S+Calculated value 321.09169; found 321.09167.HPLC (. lamda.)280) The purity is 96.4 percent; t is tR6.342min (method 1).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (pyrazin-2-yl) -1,3, 5-triazine-2, 4-diamine) (MTF-348). Following general procedure B, using N- (N- (benzo [ d ]]Thiazole-2-yl) carbamoylamino) pyrazine-2-carboxyformamide (297mg, 1mmol),the title compound was obtained as a pale yellow powder (279mg, 94%).1H NMR(400MHz,DMSO-d6):9.48(br.s,1H),9.33(br.s,1H),8.77(m,2H),7.61(d,J＝7.7Hz,1H),7.38(d,J＝6.7Hz,2H),7.23(m,3H).13C NMR(50MHz,DMSO-d6):168.83,167.37,165.65,149.51,146.27,144.58,144.38,136.35,133.76,128.18,127.66,127.06,126.68.HRMS-ESI(m/z):[M+H]+For C28H23N12S2 +Calculated value 591.16046; found 591.16052 HPLC (. lamda.)280) The purity is 99.2 percent; t is tR8.875min (method 2).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -6-chloronicotinecarboxamide (MTF-379). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and 6-chloro-3-pyridinecarbonitrile (720mg, 5.20mmol) were synthesized to give the title compound as a beige powder (1.48g, 86%).1H NMR(400MHz,DMSO-d6):10.21(s,1H),9.37(s,1H),9.01(d and br.s,J＝2.1Hz,2H),8.38(dd,J＝8.4,2.3Hz,1H),8.12(s,1H),7.80(d,J＝7.8Hz,1H),7.69(d,J＝8.4Hz,1H),7.66(d,J＝8.1Hz,1H),7.34(t,J＝7.6Hz,1H),7.19(t,J＝7.5Hz,1H).13C NMR(101MHz,DMSO-d6):172.16,161.79,159.12,152.70,151.42,149.28,138.70,131.29,130.36,125.68,123.98,122.91,121.17,119.83.HPLC(λ280) The purity is 97.2 percent; t is tR7.033min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2-chloroisonicotinamide (MTF-380). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-chloro-4-pyridinecarbonitrile (720mg, 5.20mmol) were synthesized to give the title compound as a beige powder (327mg, 19%).1H NMR(400MHz,DMSO-d6):10.22(br.s,1H),9.39(br.s,1H),9.04(br.s,1H),8.60(d,J＝5.1Hz,1H),8.21(br.s,1H),8.05(s,1H),7.94(dd,J＝5.2,1.3Hz,1H),7.81(d,J＝7.3Hz,1H),7.67(d,J＝7.9Hz,1H),7.38–7.31(m,1H),7.24–7.17(m,1H).13C NMR(101MHz,DMSO-d6):172.08,161.68,158.50,151.36,150.84,150.52,146.10,131.31,125.70,122.99,122.27,121.20,120.99,119.90.HPLC(λ280) The purity is 97.8 percent; t is tR7.108min (method 1).
N- (N- (N- (benzo [ d ]))]Thiazol-2-yl) carbamoylamino]Thiophene-2-carboxyformamide (MTF-381). Following the general procedure, 1- (benzo [ d ] is used]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-thiophenecarbonitrile (0.48mL, 5.20mmol) to give the title compound as a light yellow powder (815mg, 52%).1H NMR(400MHz,DMSO-d6):10.22(br.s,1H),9.34(br.s,1H),8.88(br.s,1H),7.94(dd,J＝3.7,0.9Hz,2H),7.79(d,J＝6.2Hz,2H),7.65(d,J＝7.9Hz,1H),7.39–7.28(m,1H),7.24–7.14(m,2H).13C NMR(101MHz,DMSO-d6):172.22,161.71,157.31,151.46,140.40,131.89,131.13,128.97,128.11,125.65,122.79,121.12,119.68.HPLC(λ280) The purity is 99.2 percent; t is tR6.975min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3,4, 5-trimethoxybenzamide (MTF-382). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and 3,4, 5-trimethoxybenzonitrile (1g, 5.20mmol) were synthesized to give the title compound as a beige powder (441mg, 22%).1H NMR(200MHz,DMSO-d6):10.22(br.s,1H),9.29(br.s,1H),8.77(br.s,1H),8.01(br.s,1H),7.80(d,J＝7.0Hz,1H),7.65(d,J＝7.5Hz,1H),7.39(s,2H),7.38–7.27(m,1H),7.19(td,J＝7.7,1.1Hz,1H),3.87(s,6H),3.74(s,3H).13C NMR(50MHz,DMSO-d6):172.28,161.97,161.53,152.51(2C),151.54,140.27,131.19,130.33,125.63,122.75,121.12,119.66,105.29(2C),60.14,56.06(2C).HPLC(λ280) The purity is 96.1 percent; t is tR7.175min (method 1).
N- (N- (N- (benzo [ d ]))]Thiazol-2-yl) carbamoylamino]Benzo [ d ] carbonyl][1,3]Dioxazole-5-carboxyformamide (MTF-383). According to general procedure A, 1- (benzo [ d ] is used]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and piperonyl nitrile (765mg, 5.20mmol) gave the title compound as a beige powder (600mg, 34%).1H NMR(400MHz,DMSO-d6):10.27(s,1H),9.29(s,1H),8.68(s,1H),7.95(s,1H),7.79(d,J＝7.7Hz,1H),7.68–7.62(m,2H),7.60(d,J＝1.6Hz,1H),7.37–7.29(m,1H),7.22–7.15(m,1H),7.04(d,J＝8.2Hz,1H),6.13(s,2H).13CNMR(50MHz,DMSO-d6):172.25,161.95,161.27,151.52,150.06,147.38,131.13,128.99,125.62,122.74,122.56,121.11,119.65,107.85,107.61,101.80.HPLC(λ280) The purity is 96.0 percent; t is tR7.133min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2-naphthamide (MTF-384). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and naphthalene-2-carbonitrile (796mg, 5.20mmol) were synthesized to give the title compound as an off-white powder (1.04g, 58%).1H NMR(400MHz,DMSO-d6):10.38(br.s,1H),9.42(br.s,1H),8.99(br.s,1H),8.64(s,1H),8.16(br.s and dd,J＝8.6,1.4Hz,2H),8.08–7.96(m,3H),7.81(d,J＝7.7Hz,1H),7.67(d,J＝7.9Hz,1H),7.66–7.57(m,2H),7.40–7.31(m,1H),7.25–7.15(m,1H).13C NMR(50MHz,DMSO-d6):172.34,162.16(2C),151.53,134.30,132.66,132.15,131.22,128.83,127.84,127.75(2C),127.66,126.80,125.65,124.55,122.81,121.14,119.73.HPLC(λ280) The purity is 95.7 percent; t is tR7.392min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4- (trifluoromethyl) benzamide (MTF-385). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 4- (trifluoromethyl) benzonitrile (889mg, 5.20mmol) were synthesized to give the title compound as a beige powder (1.38g, 73%).1H NMR(400MHz,DMSO-d6):10.28(br.s,1H),9.38(br.s,1H),8.97(br.s,1H),8.22(d,J＝8.2Hz,2H),8.11(br.s,1H),7.90(d,J＝8.3Hz,2H),7.80(d,J＝7.3Hz,1H),7.67(d,J＝7.7Hz,1H),7.39–7.30(m,1H),7.24–7.16(m,1H).19F NMR(377MHz,DMSO-d6):-61.28.13C NMR(50MHz,DMSO-d6):172.11,161.89,160.69,151.37,139.09,131.19,131.40(q,J＝31.8Hz),128.36(2C),125.57,125.16(q,J＝3.8Hz,2C),123.91(q,J＝67.7Hz),122.79,121.07,119.72.HPLC(λ280) The purity is 96.4 percent; t is tR7.442min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -2-bromoisonicotinamide (MTF-386). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equivalents, 312mg, 7.81mmol) and 2-bromo-4-cyanopyridine (952mg, 5.20mmol) were synthesized to give the title compound (859mg, 44%) as a yellow powder.1H NMR(400MHz,DMSO-d6):10.19(br.s,1H),9.38(br.s,1H),9.04(br.s,1H),8.58(d,J＝5.1Hz,1H),8.18(s and br.s,2H),7.96(dd,J＝5.1,1.3Hz,1H),7.81(d,J＝7.4Hz,1H),7.67(d,J＝7.9Hz,1H),7.39–7.32(m,1H),7.24–7.17(m,1H).13C NMR(50MHz,DMSO-d6):172.08,161.67,158.42,151.36,151.00,145.67,141.81,131.32,125.94,125.71,123.00,121.25,121.21,119.91.HPLC(λ280) The purity is 97.0 percent; t is tR7.192min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4-bromothiophene-3-carboxyformamide (MTF-387). Using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and 4-bromothiophene-3-carbonitrile (978mg, 5.20mmol) were synthesized to give the title compound as a brown powder (969mg, 49%).1H NMR(400MHz,DMSO-d6):10.03(br.s,1H),9.30(br.s,1H),8.60(br.s,1H),7.98(br.s and d,J＝3.4Hz,2H),7.80(d,J＝7.7Hz,1H),7.77(d,J＝3.4Hz,1H),7.64(d,J＝8.0Hz,1H),7.38–7.30(m,1H),7.20(d,J＝7.3Hz,1H).13C NMR(50MHz,DMSO-d6):172.41,161.84,159.08,151.48,137.36,131.24,129.06,125.66,125.44,122.85,121.16,119.75,108.70.HPLC(λ280) The purity is 96.6 percent; t is tR6.967min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino]-2, 2-dimethyl-2H-chromene-6-carboxymethylimine (MTF-388). Following general procedure A, using 1- (benzo [ d ]]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and 2, 2-dimethyl-2H-1-benzopyran-6-carbonitrile (963mg, 5.20mmol) were synthesized as a beige powderTitle compound (118mg, 6%).1H NMR(400MHz,DMSO-d6):10.25(br.s,1H),9.29(br.s,1H),8.66(br.s,1H),7.91(br.s,1H),7.84–7.75(m,3H),7.64(d,J＝7.8Hz,1H),7.37–7.30(m,1H),7.22–7.15(m,1H),6.85(d,J＝8.5Hz,1H),6.45(d,J＝9.9Hz,1H),5.84(d,J＝9.8Hz,1H),1.41(s,6H).13C NMR(101MHz,DMSO-d6):169.68,167.16,165.32,155.42,136.88,133.37,131.25,129.27,129.18,128.53,127.69,126.40,126.16,126.10,121.61,120.27,115.66,76.97,27.93(2C).HPLC(λ280) The purity is 98.4 percent; t is tR7.492min (method 1).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -3, 5-dichloropicolinamide (MTF-389). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and 3, 5-dichloropyridine-2-carbonitrile (900mg, 5.20mmol) to give the title compound as a brown powder (779mg, 41%).1H NMR(400MHz,DMSO-d6):9.73(br.s,1H),9.29(br.s,1H),8.73(br.s,1H),8.67(d,J＝1.5Hz,1H),8.37(s,1H),8.12(br.s,1H),7.77(d,J＝7.7Hz,1H),7.62(d,J＝7.9Hz,1H),7.32(t,J＝7.5Hz,1H),7.18(t,J＝7.5Hz,1H).13CNMR(50MHz,DMSO-d6):172.48,161.60,160.60,151.43,150.95,145.79,137.15,131.46,131.19,129.02,125.59,122.77,121.12,119.67.HPLC(λ280) The purity is 95.3 percent; t is tR7.192min (method 1).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (4- (trifluoromethyl) phenyl) -1,3, 5-triazine-2, 4-diamine) (MTF-394). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) aminoamido) -4- (trifluoromethyl) benzamide (363mg, 1mmol) to give the title compound as a white powder (239mg, 66%).1H NMR(400MHz,DMSO-d6):9.34(s,1H),8.42(d,J＝7.8Hz,2H),7.86(d,J＝8.4Hz,2H),7.64(d,J＝7.7Hz,1H),7.41(d,J＝7.4Hz,1H),7.25(ddd,J＝34.2,11.4,4.1Hz,4H).19F NMR(377MHz,DMSO-d6):-61.24.13C NMR(101MHz,DMSO-d6):168.98,167.28,165.53,140.54,136.56,133.57,131.18,128.46(2C),127.76,126.75,126.48,125.50,125.30(2C),122.80.HPLC(λ254) The purity is 97.0 percent; t is tR16.125min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (thien-2-yl) -1,3, 5-triazine-2, 4-diamine) (MTF-396). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) thiophene-2-carbox-boxamide (301mg, 1mmol) was synthesized to give the title compound as a white powder (250mg, 83%).1H NMR(400MHz,DMSO-d6):9.21(s,1H),7.84(d,J＝2.9Hz,1H),7.75(dd,J＝5.0,1.1Hz,1H),7.61(dd,J＝7.9,1.1Hz,1H),7.37(d,J＝7.4Hz,1H),7.26(td,J＝7.7,1.2Hz,1H),7.21–7.16(m,2H),7.09(d,J＝15.2Hz,2H).13C NMR(101MHz,DMSO-d6):166.91,166.58,165.13,142.40,136.55,133.67,131.03,129.34,128.37,128.08,127.60,126.68,126.35.HPLC(λ280) The purity is 97.1 percent; t is tR11.817min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (4-bromothien-3-yl) -1,3, 5-triazine-2, 4-diamine) (MTF-397). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -4-bromothiophene-3-carboxyformamide (380mg, 1mmol) was synthesized to give the title compound (167mg, 44%) as a white powder.1H NMR(400MHz,DMSO-d6):9.14(s,1H),8.11(d,J＝3.6Hz,1H),7.73(d,J＝3.6Hz,1H),7.57(d,J＝7.3Hz,1H),7.42(d,J＝7.7Hz,1H),7.28–7.22(m,1H),7.12(t,J＝7.4Hz,1H),7.07(s,2H).13C NMR(101MHz,DMSO-d6):167.68,166.88,165.20,137.92,136.60,133.26,130.88,128.44,127.69,126.98,126.29,126.03,109.03.HPLC(λ254) The purity is 97.1 percent; t is tR12.483min (method 2).
N- (N- (benzo [ d ])]Thiazol-2-yl) carbamoylamino) -4-nitrobenzamide (MTF-398). Following general procedure A, using 1- (benzo [ d ]]Thiazol-2-yl) guanidine (1.00g, 5.20mmol), sodium hydride (60% dispersion in mineral oil, 1.5 equiv., 312mg, 7.81mmol) and 4-nitrobenzonitrile (770mg, 5.20mmol) gave the title compound as a yellow powder (212mg, 12%).1H NMR(500MHz,DMSO-d6):10.30(br.s,1H),9.37(br.s,1H),8.90(br.s,1H),8.12(t,br.s,J＝1.8Hz,2H),7.99(d,J＝7.9Hz,1H),7.80(d,J＝7.7Hz,1H),7.66(d,J＝7.9Hz,1H),7.65–7.60(m,1H),7.54(t,J＝7.9Hz,1H),7.38–7.30(m,1H),7.22–7.17(m,1H).13C NMR(50MHz,DMSO-d6):172.22,161.95,160.57,151.47,137.26,133.22,131.23,131.20,130.20,127.53,126.14,125.67,122.87,121.16,119.79.HPLC(λ280) The purity is 99.2 percent; t is tR6.792min (method 1).
2- ((4-amino-6- (trichloromethyl) -1,3, 5-triazin-2-yl) amino) phenol (MTF 373). Following general procedure D, using 1- (benzo [ D ]]Oxazole-2-acyl) guanidine (100mg, 0.6mmol) and trichloroacetonitrile (600 μ L, 6mmol) to give the title compound as a beige powder (61mg, 32%).1H NMR (400MHz, acetone-d 6):9.10(s,1H),8.38(br.s,1H),7.99(br.s,1H),7.28(br.s,1H),7.15(br.s,1H), 7.05-6.92 (m,2H),6.87(td, J ═ 7.8,1.7Hz,1H).13C NMR (101MHz, acetone-d 6):174.0,168.9,165.8,148.6,127.6,125.6,123.2,120.8,117.2,97.4.HRMS-ESI (M/z): M + H]+For C10H9ON5Cl3 +Calculated value 319.98672; found 319.98709.HPLC (. lamda.)254):97.9％；tR9.108min (method 4).
2- ((4-amino-6- (trichloromethyl) -1,3, 5-triazin-2-yl) amino) -4-chlorophenol (MTF 374). Following general procedure D, using 2- (5-chlorobenzo [ D ]]Oxazol-2-yl) guanidine (100mg, 0.48mmol) and trichloroacetonitrile (450 μ L, 4.8mmol) to afford the title compound as a pale pink powder (55mg, 32%).1H NMR (400MHz, acetone-d 6) 9.48(s,1H),8.30(s,1H),8.18(s,1H),7.43(s,1H),7.20(s,1H), 7.01-6.92 (m,2H).13C NMR (101MHz, acetone-d 6):174.0,168.9,165.6,146.6,128.8,124.9,124.3,121.9,117.3,97.3.HRMS-ESI (M/z): M + H]+For C10H8ON5Cl4 +Calculated value 353.94775; found 353.94830 HPLC (. lamda.)254):97.1％；tR10.504min (method 4).
2- ((4-amino-6- (trichloromethyl) -1,3, 5-triazin-2-yl) amino) -5-chlorophenol (MTF 375). Following general procedure D, using 2- (6-chlorobenzo [ D ]]Oxazol-2-yl) guanidine (100mg, 0.48mmol) and trichloroacetonitrile (480 μ L, 4.8mmol) to give the title compound as a pale green powder (59mg, 35%).1H NMR (400MHz, acetone-d 6):9.66(br.s,1H),8.32(br.s,1H),8.04(br.s,1H),7.29(br.s,1H),7.18(br.s,1H),6.99(d, J ═ 2.2Hz,1H),6.90(d, J ═ 2.2Hz,1H)d,J＝8.7,2.1Hz,1H).13C NMR (101MHz, acetone-d 6):174.0,168.9,165.7,149.5,129.5,126.7,124.1,120.4,116.8,97.3.HRMS-ESI (M/z): M + H]+For C10H8ON5Cl4 +Calculated value 353.94775; found 353.94837.HPLC (. lamda.)254):95.3％；tR10.615min (method 4).
2- ((4-amino-6- (trichloromethyl) -1,3, 5-triazin-2-yl) amino) -5-nitrophenol (MTF 376). Following general procedure D, using 2- (6-nitrobenzo [ D ]]Oxazol-2-yl) guanidine (100mg, 0.45mmol) and trichloroacetonitrile (450 μ L, 4.5mmol) to give the title compound as a pale yellow powder (50mg, 30%).1H NMR(400MHz,MeOD):8.72(d,J＝9.1Hz,1H),7.78(dd,J＝9.1,2.5Hz,1H),7.70(d,J＝2.5Hz,1H).13C NMR (101MHz, acetone-d 6):174.3,169.1,165.7,147.1,143.7,134.5,120.3,116.6,110.4,97.1.HRMS-ESI (M/z): M + H]+For C10H8O3N6Cl3 +Calculated value 364.97180; found 364.97208.HPLC (. lamda.)254):95.1％；tR10.623min (method 4).
2- ((4-amino-6- (trichloromethyl) -1,3, 5-triazin-2-yl) amino) -4-nitrophenol (MTF 377). Following general procedure D, using 2- (5-nitrobenzo [ D ]]Oxazol-2-yl) guanidine (100mg, 0.45mmol) and trichloroacetonitrile (450 μ L, 4.5mmol) to give the title compound as a light orange powder (117mg, 71%).1H NMR (400MHz, acetone-d 6):9.29(br.s,1H),8.32(br.s,1H),7.91(dd, J ═ 8.9,2.6Hz,1H),7.54(br.s,1H),7.23(br.s,1H),7.11(d, J ═ 8.9Hz,1H),6.61(br.s,1H).13C NMR (101MHz, acetone-d 6):174.3,168.9,165.6,146.9,143.6,134.3,120.3,116.7,110.3,97.1.HRMS-ESI (M/z): M + H]+For C10H8O3N6Cl3 +Calculated value 364.97180; found 364.97229 HPLC (. lamda.)254):97.7％；tR9.434min (method 4).
2- ((4-amino-6-phenyl-1, 3, 5-triazin-2-yl) amino) -4-chlorophenol (MTF 409). Following general procedure E, 1- (5-chlorobenzo [ d ] was used]Oxazol-2-yl) guanidine (500mg, 2.4mmol), sodium hydride (60% oil suspension, 10%4mg, 2.6mmol) and benzonitrile (0.25mL, 2.4mmol) to give the title compound (210mg, 28%) as a beige powder.1HNMR (400MHz, acetone-d 6):9.88(br.s,1H), 8.41-8.37 (m,2H),8.16(s,2H),7.56(t, J ═ 7.2Hz,1H),7.50(t, J ═ 7.3Hz,2H), 7.01-6.92 (m,2H),6.81(br.s,2H).13C NMR (101MHz, acetone-d 6):172.1,168.6,165.7,147.0,137.3,132.6,129.9,129.1(2C),129.0(2C),124.8,124.1,121.9,118.4.HRMS-ESI (M/z): M + H]+For C15H13OClN5 +Calculated value 314.08031; found 314.08044 HPLC (. lamda.)254):98.1％；tR9.916min (method 4).
2- ((6-imino-4- (trichloromethyl) -1, 6-dihydro-1, 3, 5-triazin-2-yl) amino) -5-methylphenol (MTF 410). Following general procedure D, using 1- (6-methylbenzo [ D ]]Oxazol-2-yl) guanidine (500mg, 2.6mmol) and trichloroacetonitrile (2.6mL, 26mmol) were synthesized to give the title compound as a pale gray powder (480mg, 55%).1H NMR (400MHz, acetone-d 6):9.01(br.s,1H),8.31(br.d, J: 75.1Hz,1H),7.79(br.d, J: 51.9Hz,1H),7.25(br.s,1H),7.14(br.s,1H),6.79(d, J: 0.9Hz,1H),6.69(dd, J: 8.1,1.0Hz,1H),2.25(s,3H).13C NMR (101MHz, acetone-d 6):173.8,168.7,165.6,149.0,135.8,124.7,123.6,121.3,118.0,97.3,20.9.HRMS-ESI (M/z): M + H]+For C11H11OCl3N5 +Calculated value 334.00237; found 334.00250 HPLC (. lamda.)254):100％；tR9.948min (method 4).
2- ((4-amino-6-phenyl-1, 3, 5-triazin-2-yl) amino) -5-methylphenol (MTF 411). Following general procedure E, using 1- (6-methylbenzo [ d ]]Oxazol-2-yl) guanidine (500mg, 2.6mmol), sodium hydride (60% oil suspension, 114mg, 2.9mmol) and benzonitrile (0.27mL, 2.6mmol) were synthesized to give the title compound as a beige powder (410mg, 54%).1H NMR (400MHz, acetone-d 6):9.75(br.s,1H),8.37(d, J ═ 7.4Hz,2H),8.30(br.s,1H),7.59-7.53(m,2H),7.48(t, J ═ 7.3Hz,2H),6.80(s,1H),6.74(br.s,2H),6.69(d, J ═ 8.0Hz,1H),2.26(s,3H).13C NMR (101MHz, acetone-d 6):171.9,168.4,165.6,149.1,137.4,135.4,132.5,129.1,129.0(2C),125.9,123.3,121.3(2C),119.2,20.9.HRMS-ESI (m/z): 2M+H]+For C16H16ON5 +Calculated value 294.13494; found 294.13495.HPLC (. lamda.)254):99.7％；tR8.456min (method 4).
2- ((4-amino-6-phenyl-1, 3, 5-triazin-2-yl) amino) phenol (MTF 412). Following general procedure E, using 1- (benzo [ d ]]Oxazol-2-yl) guanidine (500mg, 2.8mmol), sodium hydride (60% suspension in oil, 123mg, 3.1mmol) and benzonitrile (0.29mL, 2.8mmol) to give the title compound as a light brown powder (53mg, 7%).1HNMR (400MHz, acetone-d 6):9.82(br.s,1H),8.39(d, J ═ 7.1Hz,2H),8.31(br.s,1H),7.83(d, J ═ 7.7Hz,1H), 7.58-7.52 (m,1H), 7.52-7.46 (m,2H), 7.03-6.94 (m,2H), 6.90-6.84 (m,1H),6.77(br.s,2H).13C NMR (101MHz, acetone-d 6):172.0,168.5,165.7,148.9,137.4,132.6,129.1(2C),129.0(2C),128.5,125.3,123.1,120.7,118.3.HRMS-ESI (M/z): M + H]+For C15H14ON5 +Calculated value 280.11929; found 280.11929.HPLC (. lamda.)254):98.9％；tR8.100min (method 4).
2- ((6-imino-4-phenyl-1, 6-dihydro-1, 3, 5-triazin-2-yl) amino) -4-nitrophenol (MTF 413). Following general procedure E, using 1- (6-nitrobenzo [ d ]]Oxazol-2-yl) guanidine (500mg, 2.3mmol), sodium hydride (60% oil suspension, 100mg, 2.5mmol) and benzonitrile (0.24mL, 2.3mmol) to give the title compound as a light yellow powder (62mg, 8%).1H NMR(400MHz,DMSO-d6):11.39(br.s,1H),8.63(d,J＝9.0Hz,1H),8.36–8.30(m,2H),8.26(s,1H),7.80(dd,J＝9.0,2.6Hz,1H),7.70(d,J＝2.6Hz,1H),7.61–7.56(m,1H),7.52(dd,J＝11.4,4.4Hz,2H),7.45(br.s,2H).13C NMR(101MHz,DMSO-d6):170.7,167.2,164.2,146.4,141.5,136.1,134.5,131.9,128.5(2C),128.0(2C),118.9,115.4,108.9.HRMS-ESI(m/z):[M+H]+For C15H13O3N6 +Calculated value 325.10436; found 325.10446 HPLC (. lamda.)254):99.1％；tR10.242min (method 4).
2- ((6-imino-4- (pyrazin-2-yl) -1, 6-dihydro-1, 3, 5-triazin-2-yl) amino) phenol (MTF 439). According to the generalProcedure E, using 1- (benzo [ d ]]Oxazol-2-yl) guanidine (500mg, 2.8mmol), sodium hydride (60% oil suspension, 123mg, 3.08mmol) and pyrazinecarbonitrile (250 μ L, 2.8) to give the title compound as a bright yellow powder (510mg, 65%). The biguanide was then dissolved in DMSO and stirred at room temperature for 8 hours to give the desired product as a bright yellow powder.1H NMR(400MHz,DMSO-d6):10.07(br.s,1H),9.41(s,1H),8.79(s,2H),8.57(s,1H),7.88(dd,J＝8.0,0.6Hz,1H),7.53(br.s,1H),7.41(br.s,1H),6.99–6.93(m,1H),6.90(dd,J＝8.0,1.4Hz,1H),6.85–6.79(m,1H).13C NMR(101MHz,DMSO-d6):168.7,167.2,164.6,149.4,148.4,146.4,144.6,144.5,126.8,124.4,123.0,119.2,116.1.HRMS-ESI(m/z):[M+H]+For C13H12ON7 +Calculated value 282.10978; found 282.10977 HPLC (. lamda.)254):98.8％；tR4.209min (method 4).
N- (N- (benzo [ d ])]Oxazol-2-yl) carbamoylamino) pyrazine-2-carboxymethylamide (MTF 440). Following general procedure E, using 1- (benzo [ d ]]Oxazol-2-yl) guanidine (500mg, 2.8mmol) sodium hydride (60% oil suspension, 123mg, 3.08mmol) and 3-pyridinecarbonitrile (292mg, 2.8mmol) were synthesized to give the title compound (510mg, 65%) as a bright yellow powder.1H NMR(400MHz,DMSO-d6):9.98(s,1H),9.40(d,J＝1.4Hz,1H),8.73(dd,J＝4.8,1.7Hz,1H),8.54(d,J＝8.0Hz,1H),8.34(s,1H),7.93(dd,J＝7.9,1.4Hz,1H),7.54(ddd,J＝8.0,4.8,0.5Hz,1H),7.32(s,2H),6.98–6.93(m,1H),6.91–6.88(m,1H),6.86–6.80(m,1H).13C NMR(101MHz,DMSO-d6):168.8,167.0,164.4,152.1,149.1,148.1,135.2,132.0,126.8,124.1,123.6,122.7,119.1,115.7.HRMS-ESI(m/z):[M+H]+For C14H13ON6 +Calculated value 281.11454; found 281.11450 HPLC (. lamda.)254):99.7％；tR4.165min (method 4).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (naphthalen-2-yl) -1,3, 5-triazine-2, 4-diamine) (MTF 443). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -2-naphthyridinylidene imide (345mg, 1mmol) to give the title compound as a white powder (299mg, 87%).1H NMR(400MHz,DMSO-d6):9.30(s,1H),8.87(s,1H),8.36(d,J＝8.5Hz,1H),7.99(dd,J＝19.5,10.4Hz,3H),7.68(d,J＝7.8Hz,1H),7.63–7.53(m,2H),7.48(d,J＝7.7Hz,1H),7.29(t,J＝7.2Hz,1H),7.25–7.11(m,3H).13C NMR(101MHz,DMSO-d6):170.19,167.33,165.53,136.81,134.58,134.17,133.57,132.40,128.98,128.66,128.16,127.74,127.65(2C),127.51,126.55(2C),126.26,124.75.HPLC(λ280) The purity is 97.3 percent; t is tR15.642min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (2, 2-dimethyl-2H-chromium 6-yl) -1,3, 5-triazine-2, 4-diamine) (MTF 444). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -2, 2-dimethyl-2H-chromene-6-carbox boxamide (377mg, 1mmol) was synthesized to give the title compound (241mg, 64%) as a white powder.1H NMR(400MHz,DMSO-d6):9.10(s,1H),8.00(d,J＝8.5Hz,1H),7.95(s,1H),7.61(d,J＝9.0Hz,1H),7.41(d,J＝7.1Hz,1H),7.27(t,J＝7.0Hz,1H),7.16(t,J＝7.5Hz,1H),6.99(s,2H),6.81(d,J＝8.5Hz,1H),6.45(d,J＝9.9Hz,1H),5.80(d,J＝9.8Hz,1H),1.40(s,6H).13C NMR(101MHz,DMSO-d6):169.67,167.15,165.33,155.42,136.86,133.39,131.25,129.26,129.18,128.51,127.69,126.41,126.16(2C),121.61,120.26,115.65,76.97,27.93(2C).HPLC(λ280) The purity is 95.2 percent; t is tR15.767min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-nitrophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 445). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3-nitrobenzamide (340mg, 1mmol) was synthesized to give the title compound (265mg, 78%) as a white powder.1H NMR(400MHz,DMSO-d6):9.41(s,1H),9.04(s,1H),8.60(d,J＝6.8Hz,1H),8.39(dd,J＝8.2,1.4Hz,1H),7.79(t,J＝8.0Hz,1H),7.64(dd,J＝7.9,0.9Hz,1H),7.39(dd,J＝7.8,0.9Hz,1H),7.36–7.17(m,4H).13C NMR(101MHz,DMSO-d6):168.16,167.23,165.49,147.97,138.33,136.42,133.75(2C),130.10,128.35,127.72,126.82,126.57,125.93,122.27.HPLC(λ280) The purity is 95.3 percent; t is tR13.550min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3,4, 5-trimethoxyphenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 446). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3,4, 5-trimethoxybenzamide (385mg, 1mmol) was synthesized to give the title compound (358mg, 93%) as a white powder.1H NMR(400MHz,DMSO-d6):9.24(s,1H),7.59(s,3H),7.40(d,J＝7.7Hz,1H),7.27(t,J＝7.5Hz,1H),7.12(dd,J＝23.2,15.8Hz,3H),3.79(s,6H),3.73(s,3H).13C NMR(101MHz,DMSO-d6):169.55,167.26,165.29,152.58(2C),140.36,136.92,133.81,131.90,128.71,127.66,126.37,126.11,105.08(2C),60.11,55.74(2C).HPLC(λ280) The purity is 96.6 percent; t is tR10.367min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (benzo [ d ]][1,3]Dioxa-5-yl) -1,3, 5-triazine-2, 4-diamine) (MTF 449). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) benzo [ d][1,3]Dioxazole-5-carboxymethylimine (340mg, 1mmol) was synthesized to give the title compound as a white powder (247mg, 73%).1H NMR(400MHz,DMSO-d6):9.14(s,1H),7.87(d,J＝8.1Hz,1H),7.68(s,1H),7.61(d,J＝7.8Hz,1H),7.39(d,J＝7.7Hz,1H),7.27(t,J＝7.4Hz,1H),7.17(t,J＝7.5Hz,1H),7.08–6.97(m,3H),6.10(s,2H).13C NMR(101MHz,DMSO-d6):169.41,167.15,165.34,150.10,147.37,136.77,133.53,130.76,128.41,127.66,126.47,126.21,122.88,107.97,107.44,101.60.HPLC(λ280) The purity is 95.1 percent; t is tR10.767min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (2-chlorophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 450). The general procedure B was followed using N- (N- (benzo [ d ]]Thiazol-2-yl) aminoamido) -2-chlorobenzamide (329mg, 1mmol) was synthesized to give the title compound (273mg, 83%) as a white powder.1H NMR(400MHz,DMSO-d6):9.30(s,1H),7.58(dt,J＝9.4,4.8Hz,2H),7.51(dd,J＝7.8,1.0Hz,1H),7.45(td,J＝7.7,2.0Hz,1H),7.41(dd,J＝7.4,1.3Hz,1H),7.38(d,J＝6.8Hz,1H),7.28–7.22(m,1H),7.22–7.09(m,3H).13C NMR(101MHz,DMSO-d6):172.24,166.83,165.19,137.48,136.34,133.55,130.93,130.50,130.47,129.82,128.16,127.59,127.08,126.87,126.52.HPLC(λ280) The purity is 95.1%；tR10.517min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (p-tolyl) -1,3, 5-triazine-2, 4-diamine) (MTF 451). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -4-methylbenzamide (309mg, 1mmol) was synthesized to give the title compound (179mg, 58%) as a white powder.1H NMR(400MHz,DMSO-d6):9.15(s,1H),8.14(d,J＝8.0Hz,2H),7.61(d,J＝7.9Hz,1H),7.41(d,J＝7.1Hz,1H),7.26(dd,J＝8.8,4.7Hz,3H),7.17(t,J＝7.5Hz,1H),7.04(s,2H),2.36(s,3H).13C NMR(101MHz,DMSO-d6):170.14,167.25,165.46,141.31,136.80,133.90,133.44,128.86(2C),128.52,127.86(2C),127.71,126.51,126.21,21.09.HPLC(λ280) The purity is 96.7 percent; t is tR12.958min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (2-fluorophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 452). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) aminoamido) -2-fluorobenzamide (313mg, 1mmol) was synthesized to give the title compound (191mg, 61%) as a white powder.1H NMR(400MHz,DMSO-d6):9.26(s,1H),7.87(td,J＝7.7,1.6Hz,1H),7.59(dd,J＝7.9,1.0Hz,1H),7.56–7.49(m,1H),7.39(d,J＝7.6Hz,1H),7.30–7.23(m,3H),7.20–7.10(m,3H).13C NMR(101MHz,DMSO-d6):169.77(d),166.97,165.31,160.33(d),136.49,133.54,132.19(d),131.13(d),128.26,127.65,126.92,126.46,125.89(d),124.09(d),116.50(d).HPLC(λ280) The purity is 95.4 percent; t is tR10.333min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (2, 6-dichlorophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 455). Following general procedure B, with N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -2, 6-dichlorobenzamide (364mg, 1mmol) was synthesized to give the title compound (320mg, 88%) as a white powder.1H NMR(400MHz,DMSO-d6):9.44(s,1H),7.55(dd,J＝13.4,7.9Hz,3H),7.45(dd,J＝9.0,7.1Hz,1H),7.33(d,J＝7.7Hz,1H),7.30–7.22(m,3H),7.18(t,J＝7.1Hz,1H).13C NMR(101MHz,DMSO-d6):170.75,166.93,165.40,136.69,135.98,133.90,132.08(2C),130.63,128.12(2C),127.88,127.50,127.25,126.83.HPLC(λ280) The purity is 95.8 percent; t is tR10.150min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3, 5-dibromopyridin-4-yl) -1,3, 5-triazine-2, 4-diamine) (MTF 456). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -3, 5-dibromoisonicotinamide (454mg, 1mmol) was synthesized to give the title compound as a white powder (412mg, 91%).1H NMR(400MHz,DMSO-d6):9.56(s,1H),8.84(s,2H),7.57(d,J＝7.7Hz,1H),7.39(d,J＝14.8Hz,2H),7.31(d,J＝7.6Hz,1H),7.25(t,J＝7.2Hz,1H),7.20(d,J＝5.4Hz,1H).13C NMR(101MHz,DMSO-d6):170.93,166.78,165.27,150.32(2C),146.43,135.71,133.98,127.70,127.49,127.35,127.02,119.05(2C).HPLC(λ280) The purity is 95.1 percent; t is tR9.700min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (6-bromobenzo [ d ]][1,3]Dioxo-5-yl) -1,3, 5-triazine-2, 4- (MTF 458). Following general procedure B, N- (N- (N- (benzo [ d ]) was used]Thiazol-2-yl) carbamoylamino) -6-bromobenzo [ d][1,3]Bisoxazole-5-carbo-boxamide) (418mg, 1mmol) was synthesized to give the title compound (300mg, 72%) as a white powder.1H NMR(400MHz,DMSO-d6):9.31(s,1H),7.57(d,J＝7.6Hz,1H),7.38(d,J＝7.3Hz,1H),7.28–7.23(m,2H),7.22–7.11(m,4H),6.12(s,2H).13C NMR(101MHz,DMSO-d6):172.43,166.50,165.08,148.68,146.86,136.29,133.50,132.49,128.17,127.63,127.08,126.58,112.81,111.49,109.98,102.31.HPLC(λ280) The purity is 96.1 percent; t is tR10.475min (method 12).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (3-aminophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 460). Following general procedure B, 3-amino-N- (N- (benzo [ d ]) was used]Thiazol-2-yl) carbamoylamino) benzamide (310mg, 1mmol) was synthesized to give the title compound as a yellow powder (241mg, 78%).1H NMR(400MHz,DMSO-d6):9.06(s,1H),7.61(dd,J＝7.9,1.2Hz,1H),7.49(s,1H),7.43(d,J＝7.8Hz,2H),7.27(td,J＝7.7,1.3Hz,1H),7.17(t,J＝7.6Hz,1H),7.09(t,J＝7.8Hz,1H),6.98(s,2H),6.71(dd,J＝7.9,1.4Hz,1H),5.18(s,2H).13C NMR(101MHz,DMSO-d6):170.95,167.23,165.44,148.52,137.33,136.87,133.20,128.59,128.55,127.72,126.43,126.12,116.94,115.86,113.34.HPLC(λ280) The purity is 97.8 percent; t is tR7.167min (method 2).
1,1' - ((((dithiodiylbis (2, 1-phenylene)) bis (azenediyl)) bis (6-amino-1, 3, 5-triazine-4, 2-diyl)) bis (3, 1-phenylene)) bis (ethan-1-one) (MTF 462). Following general procedure B, 3-acetyl-N- (N- (benzo [ d ]) was used]Thiazol-2-yl) carbamoylamino) benzamide (337mg, 1mmol) was synthesized to give the title compound as a white powder (225mg, 67%).1H NMR(400MHz,DMSO-d6):9.32(s,1H),8.83(s,1H),8.46(d,J＝7.4Hz,1H),8.13(d,J＝7.8Hz,1H),7.64(t,J＝7.1Hz,2H),7.40(d,J＝7.1Hz,1H),7.23(dt,J＝15.3,8.0Hz,4H),2.61(s,3H).13C NMR(101MHz,DMSO-d6):197.57,169.41,167.28,165.49,137.06,136.86,136.62,133.74,132.14,131.24,128.80,128.46,127.69,127.32,126.67,126.40,26.77.HPLC(λ280) The purity is 96.9 percent; t is tR10.908min (method 2).
N2, N2' - (dithiodiylbis (2, 1-phenylene)) bis (6- (2-nitrophenyl) -1,3, 5-triazine-2, 4-diamine) (MTF 463). Following general procedure B, using N- (N- (benzo [ d ]]Thiazol-2-yl) carbamoylamino) -2-nitrobenzamide (340mg, 1mmol) was synthesized to give the title compound (278mg, 82%) as a white powder.1H NMR(400MHz,DMSO-d6):9.29(s,1H),7.92(d,J＝7.6Hz,1H),7.86(d,J＝7.3Hz,1H),7.77(t,J＝7.3Hz,1H),7.70(t,J＝7.6Hz,1H),7.57(d,J＝7.8Hz,1H),7.32(d,J＝7.6Hz,1H),7.28–7.07(m,4H).13C NMR(101MHz,DMSO-d6):170.32,166.77,165.14,149.12,136.11,133.49,132.58,132.34,130.89,130.51,128.06,127.58,127.11,126.63,123.88.HPLC(λ280) The purity is 98.9 percent; t is tR10.350min (method 2).
Cell culture
Fresh sterile tissue was obtained at Nice CHU hospital from surgical waste from patients diagnosed with metastatic melanoma. The epidermal cell suspension is obtained from foreskin of caucasian children by the following method: digestion was carried out overnight at 4 ℃ in phosphate buffered saline containing 0.5% dispase grade II, followed by digestion with 0.05% trypsin-0.02% EDTA in phosphate buffered saline for 20 minutes (V/V) at 37 ℃. Human primary melanocytes were grown in MCDB 153 medium supplemented with 2% FCS, 0.4. mu.g/ml hydrocortisone, 5. mu.g/ml insulin, 16 nPhorbol-12 myristate 13-acetate, 1ng/ml basic fibroblast growth factor, 10. mu.g/ml bovine pituitary extract and penicillin/streptomycin (100U/ml/50. mu.g/ml). Human primary keratinocytes were cultured in KSFM medium supplemented with 0.1ng/ml epidermal growth factor, 15. mu.g/ml bovine pituitary extract and penicillin/streptomycin (100U/ml/50. mu.g/ml). Human primary fibroblasts and melanoma cells derived from the corresponding dermis were grown in
Different melanoma cell lines were purchased from the american tissue culture collection: a375 and WM9 cells mutated on B-Raf, CDKN2A and PTEN proteins; SKmel28 cells mutated on B-Raf and P53 proteins; 1205Lu cells mutate on B-Raf and PTEN proteins; g-361 cells mutate on B-Raf and CDKN2A proteins; c8161 cells in B-Raf and K-Ras protein mutations; WM3912 cells mutated on B-Raf and CDKN2A proteins; MeWo cells mutate on P53 and CDKN2A proteins; WM3918 had no mutations in the signature protein.
Drug resistant melanoma cell lines a375 and SKMel28 were given from professor p. marchetti and were described in the article by Corazao-Rozas et al (2013). Cells were grown in RPMI 1640 or DMEM supplemented with 10% Fetal Calf Serum (FCS) and penicillin/streptomycin (100U/ml/50mg/ml) at 37 ℃ and 5
Trypan blue exclusion assay
For trypan blue staining, 200mL cells (melanoma and normal human cells) were aseptically transferred to 1.5mL clear Eppendorf tubes and incubated with an equal volume of 0.4% trypan blue solution for 3 minutes at room temperature. Viable cells were counted and the results expressed as a percentage of the control cell value. All experiments were performed 3 times in triplicate.
Viability test
A375 sensitive cells were treated with different concentrations of synthetic molecule (5.0. mu.M or 10. mu.M) for different times (24 hours or 48 hours) or DMSO (control) for 12 or 48 hours. At the end of the stimulation, viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage compared to control. The results are shown in table 1. They show that the compounds of formulae (I), (II) and (III) induce a reduction in the viability of melanoma cells.
Kinetics of CRO15
A375 sensitive cells were treated with 5 μ M CRO15 for various periods of time (2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, or 48 hours) or with 5 μ M PLX4032(B-Raf inhibitor) for 48 hours or DMSO (control) for 48 hours. At the end of the stimulation, viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage relative to control and mean ± SEM of data from three independent experiments performed in triplicate; p < 0.01; p < 0.001. The results are shown in FIG. 1A. They show that CRO15 induced a decrease in melanoma cell viability, and that the decrease in viability after 48 hours was more pronounced with CRO15 than with PLX 4032.
IC50 of CRO15
A375 sensitive cells were treated with different concentrations of CRO15 (0.5. mu.M, 2.5. mu.M, 5.0. mu.M, 7.5. mu.M, 50. mu.M) for 48 hours or 5. mu.MPLX 4032(B-Raf inhibitor) for 48 hours or DMSO (control) for 48 hours. At the end of the stimulation, viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage relative to control and mean ± SEM of data from three independent experiments performed in triplicate; p < 0.01; p < 0.001. IC50 of CRO15 was determined to be 3.75 μ M at 48 hours. The results are shown in FIG. 1B. They show that CRO15 has a dose-response relationship to melanoma cell viability.
Viability testing Using CRO15
Different melanoma cell lines with various mutations, patient cells with various mutations and normal human cells were treated with 5 μ MCRO15 for 48 hours or DMSO (control). At the end of the stimulation, viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage relative to control and mean ± SEM of data from three independent experiments performed in triplicate; p < 0.01; p < 0.001. The results are shown in fig. 1C (melanoma cell lines with various mutations) and 1D (patient cells with various mutations) and 1E (normal human cells). They show that CRO15 induces a reduced viability of different melanoma cell lines with various mutations and different patient cells with various mutations, whereas CRO15 is not toxic to normal cells.
Activity assay of CRO15 against drug resistant melanoma cells
A375 sensitive and resistant melanoma cells were treated with 5 μ M CRO15 or 45 μ M PLX4032 or DMSO (control) for 48 hours. At 48 hours, viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage relative to control and mean ± SEM of data from three independent experiments performed in triplicate; p < 0.01; p < 0.001. The results are shown in fig. 4A. They show that CRO15 induced a decrease in cell viability in both sensitive and resistant melanoma cells, and that the decrease in viability with CRO15 was more pronounced compared to PLX 4032.
Viability test of CRO15 cells with dual resistance to BRAF and MEK inhibitors
Melanoma cell lines DR6 cells resistant to B-Raf inhibitor (vemurafenib ═ PLX4032) and MEK inhibitor (Cobimetinib) were treated with DMSO (control), with 5 μ M or 10 μ M CRO15, or with a combination of 1 μ M vemurafenib and 0,5 μ M Cobimetinib. After 24 hours of stimulation, viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage compared to control. Error bars represent ± SEM of triplicates.
The results are shown in FIG. 5. They indicate that CRO15 induces a decrease in cell viability in melanoma cell lines resistant to B-Raf inhibitor (PLX4032) and MEK inhibitor (Cobimetinib).
Western blot analysis
Western blot analysis was performed as described (lehraki et al, 2014). The proteins were extracted in a buffer containing 50mmol/l Tris-HCl (pH 7.5), 15mmol/l NaCl, 1% Triton X-100 and 1 Xprotease and phosphatase inhibitors. Briefly, cell lysates (30mg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore) and exposed to appropriate antibodies. Proteins were visualized using the ECL system from Amersham. The western blot analysis shown represents at least three independent experiments.
Western blot analysis: a375 sensitive cells were treated with 5 μ M CRO15 for various periods of time (6 hours, 12 hours or 24 hours) or with DMSO (control) for 24 hours.
The results are shown in fig. 2. They showed that CRO15 induced activation of AMPK.
In vivo mouse cancer model
Animal experiments were carried out according to the declaration of Helsinki and were approved by the local ethical Commission CIEPAL (Commite institute d 'Ethique Pour l' Animal de laboratory-Azur). Female immunodeficient BALB/c nu/nu (nude) mice were obtained from Envigo laboratories (Gannat, France) at 5 weeks of age nude mice were inoculated subcutaneously with A375 sensitive or resistant melanoma cells (1,0x 10)6Individual cells/mouse). After disappearance of the tumor (+ -5 days), animals were injected intraperitoneally with Labrafil (control), PLX4032(0.7 mg/mouse/day) or CRO15(0.7 mg/mouse/day) dissolved in Labrafil. The growing tumor curve was determined by measuring tumor volume using the equation V ═ W2)/2(V ═ tumor volume, W ═ tumor width, L ═ tumor length). At the end of the experiment, mice were euthanized by cervical dislocation and tumors were subjected to Western blot and immunofluorescence (LC3, cleared Caspase 3). TUNEL assay was performed using the in situ cell death detection kit (Roche, Meylan, france). The results are shown in fig. 3A and 3B (a375 sensitive melanoma cells) and 4B and 4C (a375 resistant melanoma cells). They show that CRO15 reduces tumor volume and weight in mice inoculated with sensitive and resistant melanoma cells.
xenograft-A375 resistant cells (Riv)
In vivo murine carcinoma model Animal experiments were performed according to the declaration of helsinki and were approved by the local ethics committee CIEPAL (suite institute of Ethique and force source' Animal de laboratory-Azur). Female immunodeficient BALB/c nu/nu (nude) mice were aged 5 weeks from Envigobtained in the laboratory (Gannat, France). Nude mice were inoculated subcutaneously with a375 resistant melanoma cells (1,0x 10)6Individual cells/mouse). After tumor disappearance (+ 5 days), animals received intraperitoneal injections of Labrafil (control), PLX4032(0.7 mg/mouse/day) or MTF319(0.7 mg/mouse/day) dissolved in Labrafil. The growth tumor curve was determined by measuring tumor volume using the equation V ═ W2)/2(V ═ tumor volume, W ═ tumor width, L ═ tumor length). At the end of the experiment, mice were euthanized by cervical dislocation.
The results are shown in fig. 6A and 6B. When mice were injected with MTF319, they showed a decrease in tumor volume. Tumor weight also indicates that MTF319 has an effect on tumor growth.
CRO15 inhibits tumor growth of murine melanoma BP cells allografted to C57BL6 mice
In vivo murine carcinoma model Animal experiments were performed according to the declaration of helsinki and were approved by the local ethics committee CIEPAL (suite institute of Ethique and force source' Animal de laboratory-Azur). Female C57BL6/J mice were obtained from Envigo laboratories (Gannat, France) at 5 weeks of age. With BP melanoma cells (1X 10)6Individual cells/mouse) were inoculated subcutaneously. After tumor disappearance (+ -5 days), animals received intraperitoneal injections of either Labrafil (vehicle) or CRO15(0.7 mg/mouse/day). The growth tumor curve was determined by measuring tumor volume using the equation V ═ W2)/2(V ═ tumor volume, W ═ tumor width, L ═ tumor length). At the end of the experiment, mice were euthanized by cervical dislocation.
The results are shown in fig. 7A. Bars represent mean ± SEM. P < 0.05; p < 0.01. They showed that immunocompetent mice (C57BL/6) injected subcutaneously with mouse melanoma cells (BP cells) had no tumor growth when treated daily with CRO15 compared to vehicle-treated mice.
To investigate whether melanoma cells WM9 would be resistant to CRO15, these cells were cultured for 8 weeks in the presence of increasing concentrations of DMSO (control), PLX4032, CRO15 or MTF 255. The starting concentration was 0.2. mu.M. As observed in the figure, the drug concentration slightly increased (+0.2 μ M) every 2 passages until resistance to PLX4032 was reached when challenged at an intensity of 10 μ M as shown in figure 7B.
Untreated WM9S (untreated cells) and drug-treated WM9S were then stimulated with 10 μ M of each drug for 48 hours, respectively. Viable cells were counted using trypan blue staining exclusion. Results were normalized to percentage compared to control (DMSO). Error bars represent ± SEM of triplicates.
The results are shown in fig. 7B. They showed that PLX-treated WM9 was resistant to PLX4032, while CRO15 was still able to induce cell death in CRO 15-treated
TABLE 1
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