Internal sulfonamide compounds and methods of use thereof

文档序号：1855904 发布日期：2021-11-19 浏览：22次中文

阅读说明：本技术 内磺酰胺化合物及其使用方法 (Internal sulfonamide compounds and methods of use thereof ) 是由祝力段小伟戴丽光杨钊杨艳青张慧胡远东彭勇韩永信赵锐田心王于 2017-03-22 设计创作，主要内容包括：本发明提供内磺酰胺化合物及其使用方法,具体涉及如式I所示的新的具有异柠檬酸脱氢酶1(IDH1)抑制活性的化合物或其药学上可接受的盐、溶剂化物或水合物、其制备方法及包含其的药物组合物。本发明还涉及所述化合物或其药学上可接受的盐、溶剂化物或水合物和包含其的药物组合物治疗IDH1突变诱发的癌症中的用途。(The invention provides an internal sulfonamide compound and a using method thereof, in particular to a novel compound with isocitrate dehydrogenase 1(IDH1) inhibitory activity shown in formula IOr a pharmaceutically acceptable salt, solvate or hydrate thereof, a process for the preparation thereof and a pharmaceutical composition comprising the same. The invention also relates to the use of the compounds or pharmaceutically acceptable salts, solvates or hydrates thereof and pharmaceutical compositions comprising the same for the treatment of IDH1 mutation-induced cancer.)

1. A compound of formula I:

wherein the content of the first and second substances,

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6HeterocycloalkanesOptionally substituted with one or more groups independently selected from R⁶Substituted with a group of (1);

R²selected from phenyl or a 5-6 membered heteroaryl group containing 1-2 heteroatoms selected from N, O or S, which may optionally be substituted with one or more substituents independently selected from R⁷Substituted with a group of (1);

R³selected from phenyl, 5-6 membered heteroaryl containing 1-2 heteroatoms selected from N or O or S, phenyl CH₂Or 5-6 membered heteroaryl CH containing 1-2 heteroatoms selected from N or O or S₂-, which may optionally be substituted by one or more groups independently selected from R⁸Substituted with a group of (1);

R⁴selected from halogen, amino, hydroxy, halogeno C_1-3Alkyl or C_1-6An alkyl group;

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, oxo, or a salt thereof,C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

R⁹selected from H, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_3-6Heterocycloalkyl, phenyl or a 5-6 membered heteroaryl group containing 1-2 atoms selected from N or O or S, which may optionally be substituted with one or more groups independently selected from R¹⁰Substituted with a group of (1);

R¹⁰selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

m is 0 or 1.

2. A compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof,

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R⁴selected from halogen, amino, hydroxy, halogeno C_1-3Alkyl or C_1-6An alkyl group;

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, aminosulfonyl, 4-cyanopyridine-2-aminosulfonyl, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

m is 0 or 1.

3. A compound of formula II:

wherein the content of the first and second substances,

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, oxo, or a salt thereof,C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

R⁹selected from H, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_3-6Heterocycloalkyl, phenyl or 5-6 membered heteroaryl containing 1-2 heteroatoms selected from N or O or S, which may optionally be substituted with one or more substituents independently selected from R¹⁰Substituted with a group of (1);

R¹⁰selected from halogen, amino, hydroxyCyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group.

4. The compound of formula II according to claim 3, or a pharmaceutically acceptable salt thereof,

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, aminosulfonyl, 4-cyanopyridine-2-aminosulfonyl, C_1-6Alkyl or C_3-6A cycloalkyl group; r⁸Selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6Alkynyl.

5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein X is selected from CH₂NH or N (CH)₃)。

6. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereofSalts of (I) a⁵Selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl; preferably, R⁵Selected from hydrogen or methyl.

7. A compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, R¹Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl or piperidinyl, which may optionally be substituted with one or more groups independently selected from R⁶Substituted with a group of (1); preferably, R¹Selected from cyclobutyl or cyclohexyl, which may be optionally substituted by 1 or 2 fluoro; further preferably, R¹Is selected from

8. A compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, R⁶Selected from fluorine, chlorine or bromine.

9. The compound of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, R²Selected from phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl, which may optionally be substituted with one or more groups independently selected from R⁷Substituted with a group of (1); preferably, R²Selected from phenyl or pyridyl, which may optionally be substituted with one or more groups independently selected from R⁷Substituted with a group of (1); further preferably, R²Is selected from

10. The compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein R²Selected from phenyl,Furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl, which may optionally be substituted with one or more groups independently selected from R⁷Substituted with a group of (1); r⁷Selected from fluoro, chloro, bromo, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, monochloromethyl, dichloromethyl, trichloromethyl, aminosulfonyl or 4-cyanopyridine-2-aminosulfonyl; further, R²Selected from phenyl or pyridyl, which may optionally be substituted with one or more groups independently selected from R⁷Substituted with a group of (1); r⁷Selected from fluorine, cyano, trifluoromethyl, -SO₂NH₂Or 4-cyanopyridine-2-aminosulfonyl.

11. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, R⁷Selected from fluoro, chloro, bromo, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, monochloromethyl, dichloromethyl, trichloromethyl, oxo orPreferably, R⁷Selected from fluoro, cyano, trichloromethyl, oxo,

12. The compound of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, R⁹Selected from H, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl, which may optionally be substituted with one or more groups independently selected from R¹⁰Substituted with a group of (1); preferably, R⁹Selected from pyridyl optionally substituted with one or more R independently selected from R¹⁰Is substituted with a group (b).

13. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, R¹⁰Selected from cyano groups.

14. The compound of any one of claims 1-13, or a pharmaceutically acceptable salt thereof,is selected from

15. The compound of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, R³Selected from phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, benzyl, furanylmethylene, thienylmethylene, pyrrolylmethylene, pyrazolyl methylene, imidazolyl methylene, pyridylmethylene, pyrimidinyl methylene, pyridazinylmethylene, pyrazinylmethylene, thiazolyl methylene, isothiazolylmethylene, oxazolylmethylene or isoxazolylmethylene, which may optionally be substituted with one or more groups independently selected from R⁸Substituted with a group of (1); preferably, R³Selected from pyridyl, pyrimidinyl or benzyl, which may optionally be substituted with one or more groups independently selected from R⁸Substituted with a group of (1); further preferably, R³Is selected from

16. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, R⁸Selected from fluoro, chloro, bromo, cyano, ethynyl, 1-propynyl or 1-butynyl; preferably, R⁸Selected from fluoro, cyano or ethynyl.

17. A compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein the compound is preferably:

18. use of a compound of any one of claims 1-17, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a cancer induced by an IDH1 mutation.

19. The use of claim 19, wherein the cancer induced by the IDH1 mutation is selected from the group consisting of: glioblastoma, myelodysplastic syndrome, myeloproliferative neoplasms, acute myelogenous leukemia, sarcoma, melanoma, non-small cell lung cancer, cholangiocarcinoma, or angioimmunoblastic non-hodgkin's lymphoma.

20. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as described in claim 1 or a compound of formula II as described in claim 3, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

Technical Field

The present application relates to internal sulfonamide compounds and methods of use thereof for the treatment of cancer.

Background

IDH (isocitrate dehydrogenase), the most important key enzyme in the intracellular tricarboxylic acid cycle, catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate (i.e., alpha-ketoglutarate). IDH has two distinct subtypes, one of which utilizes NAD (+) as an electron acceptor and the other of which utilizes NADP (+). Five kinds of IDH have been reported, 3 of which are NAD (+) dependent isocitrate dehydrogenase, which are located in the mitochondrial matrix; there are also two types of NADP (+) dependent isocitrate dehydrogenase, one located in the mitochondria and the other in the cytoplasm.

The IDH mutation exists in various tumors (such as glioma, sarcoma, acute granulocytic leukemia and the like), and the mutation site is arginine residue positioned in the catalytic center (IDH1/R132H, IDH2/R140Q, IDH 2/R172K). In 2009, 672 tumors of different origin and 84 different tumor cell lines were examined by Bleeker et al for IDH1 mutation, which was found to occur specifically and intensively in brain gliomas [ Bleeker et al, 2009.IDH1 mutations at rest p.R132(IDH1(R132)) ocur frequency in high-grade gliomas but not in other dissolved tumors. hum Mutat.30:7-11 ]. However, subsequent literature reports show that acute myelocytic leukemia, prostate cancer, paragangliomas, etc. also have mutations in IDH1 [ Green et al, 2010, viral mutations of IDH1 and IDH2 in the leukemia transformation of myeloablative neoplasms. N Engl J Med.362:369-370 ]. Bleeker et al found that R132H accounted for 86.9% of cases with IDH1 mutations. Other types, such as R132C, R132G, R132L, R132V, R132S are in smaller proportions [ Bleeker et al, 2009 ]. The mutated IDH acquires a new capability of catalyzing the conversion of α -ketoglutarate (α -KG) to 2-hydroxyglutarate (2-HG). Studies have shown that α -ketoglutarate is structurally similar to 2-hydroxyglutarate, and that 2-HG competes with α -KG, thereby reducing α -KG-dependent enzyme activity, leading to chromatin hypermethylation, which is thought to interfere with normal cell differentiation, leading to hyperproliferation of immature cells, leading to cancer.

IDH1m inhibitor AG-120 (i.e. ivosidenib) developed by Agios Pharmaceuticals has significant drug effect on acute myeloid leukemia, and meanwhile, researches on other malignant solid tumors such as bile duct cancer, chondrosarcoma and glioma are also in progress.

Disclosure of Invention

In one aspect, the present application provides a compound represented by formula I or a pharmaceutically acceptable salt, solvate, or hydrate thereof:

wherein the content of the first and second substances,

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R³selected from phenyl, 5-6 membered heteroaryl containing 1-2 heteroatoms selected from N or O or S, phenyl CH₂Or contain 1-25-6 membered heteroaryl CH with one heteroatom selected from N, O or S₂-, which may optionally be substituted by one or more groups independently selected from R⁸Substituted with a group of (1);

R⁴selected from halogen, amino, hydroxy, halogeno C_1-3Alkyl or C_1-6An alkyl group;

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, oxo, or a salt thereof,C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

R¹⁰selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

m is 0 or 1.

As a preferred embodiment of the present application, in the compound represented by formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof, the substituents are defined as follows:

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R⁴selected from halogen, amino, hydroxy, halogeno C_1-3Alkyl or C_1-6An alkyl group;

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, aminosulfonyl, N-substituted aminosulfonyl, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

m is 0 or 1.

In one embodiment of the compounds of formula I herein, R⁴Selected from fluorine, chlorine, bromine or trifluoromethyl.

In another aspect, the present application provides a compound represented by the general formula I-1 or a pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein the substituent groups are defined as the compounds in the general formula I.

In another aspect, the present application provides a compound represented by the general formula I-2 or a pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein the substituent groups are defined as the compounds in the general formula I.

As one embodiment of the present application, there is provided a compound represented by general formula II or a pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein the content of the first and second substances,

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl, oxo, or a salt thereof,C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

R¹⁰selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group.

As a preferred embodiment of the present application, in the compound represented by formula II or a pharmaceutically acceptable salt, solvate or hydrate thereof, the substituents are defined as follows:

x is selected from CH₂Or NR⁵；

R¹Is selected from C_3-6Cycloalkyl or C_3-6Heterocycloalkyl, which may be optionally substituted with one or more substituents independently selected from R⁶Substituted with a group of (1);

R⁵selected from hydrogen or C_1-6An alkyl group;

R⁶selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁷selected from halogen, amino, hydroxy, cyano, halogenGeneration C_1-3Alkyl, aminosulfonyl, N-substituted aminosulfonyl, C_1-6Alkyl or C_3-6A cycloalkyl group;

R⁸selected from halogen, amino, hydroxy, cyano, halogeno C_1-3Alkyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6Alkynyl.

In one embodiment of the compounds of formula II herein, X is selected from CH₂NH or N (CH)₃)。

In one embodiment of the compounds of formula II herein, R⁵Selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl.

In one embodiment of the compounds of formula II herein, R⁵Selected from hydrogen or methyl.

In one embodiment of the compounds of formula II herein, R¹Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl or piperidinyl, which may optionally be substituted with one or more groups independently selected from R⁶Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R¹Selected from cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl or piperidinyl, which may optionally be substituted with one or more groups independently selected from R⁶Substituted with a group of (1); r⁶Selected from fluorine, chlorine or bromine.

In one embodiment of the compounds of formula II herein, R⁶Selected from fluorine, chlorine or bromine.

In one embodiment of the compounds of formula II herein, R¹Selected from cyclobutyl or cyclohexyl, which may be optionally substituted by 1 or 2 fluoro.

In one embodiment of the compounds of formula II herein, R¹Is selected from

As a preferred specific embodiment herein, in the compound represented by formula II or a pharmaceutically acceptable salt, solvate or hydrate thereof,R²selected from phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl or triazinyl, which may optionally be substituted with one or more groups independently selected from R⁷Substituted with a group of (1); r⁷Selected from fluorine, chlorine, bromine, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, monochloromethyl, dichloromethyl, trichloromethyl, aminosulfonyl or N-substituted aminosulfonyl. Further, R²Selected from phenyl or pyridyl, which may optionally be substituted with one or more groups independently selected from R⁷Substituted with a group of (1); r⁷Selected from fluorine, cyano, trifluoromethyl, -SO₂NH₂Or 4-cyanopyridine-2-aminosulfonyl.

In one embodiment of the compounds of formula II herein, R²Selected from phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl or triazinyl, which may optionally be substituted with one or more groups independently selected from R⁷Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R²Selected from phenyl or pyridyl, which may optionally be substituted with one or more groups independently selected from R⁷Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R⁷Selected from fluoro, chloro, bromo, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, monochloromethyl, dichloromethyl, trichloromethyl, oxo or

In one embodiment of the compounds of formula II herein, R⁷Selected from fluoro, cyano, trichloromethyl, oxo,

In one embodiment of the compounds of formula II herein, R⁹Selected from H, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl or triazinyl, which may optionally be substituted with one or more groups independently selected from R¹⁰Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R⁹Selected from pyridyl optionally substituted with one or more R independently selected from R¹⁰Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R¹⁰Selected from cyano groups.

In one embodiment of the compounds of formula II herein,is selected from

In one embodiment of the compounds of formula II herein, R²Is selected from

As a preferred specific embodiment of the present invention, in the compound represented by the formula II or a pharmaceutically acceptable salt, solvate or hydrate thereof, R³Selected from phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl, triazinyl, benzyl, furylmethylene, thienylmethylene, pyrrolylmethylene, pyrazolyl methylene, imidazolyl methylene, pyridylmethylene, pyrimidylmethylene, pyridazinylmethylenesMethyl, pyrazinylmethylene, thiazolylmethylene, isothiazolylmethylene, oxazolylmethylene, isoxazolylmethylene, tetrazolylmethylene, or triazinylmethylene, which may optionally be substituted with one or more groups independently selected from R⁸Substituted with a group of (1); r⁸Selected from hydrogen, fluorine, chlorine, bromine, cyano, ethynyl, 1-propynyl or 1-butynyl, and further, R³Selected from pyridyl, pyrimidinyl or benzyl, which may optionally be substituted with one or more groups independently selected from R⁸Substituted with a group of (1); r⁸Selected from fluoro, cyano or ethynyl.

In one embodiment of the compounds of formula II herein, R³Selected from phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl, triazinyl, benzyl, furanylmethylene, thienylmethylene, pyrrolylmethylene, pyrazolyl methylene, imidazolyl methylene, pyridylmethylene, pyrimidylmethylene, pyridazinylmethylene, pyrazinylmethylene, thiazolyl methylene, isothiazolylmethylene, oxazolylmethylene, isoxazolylmethylene, tetrazolylmethylene, or triazinylmethylene, which may optionally be substituted with one or more groups independently selected from R⁸Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R³Selected from pyridyl, pyrimidinyl or benzyl, which may optionally be substituted with one or more groups independently selected from R⁸Is substituted with a group (b).

In one embodiment of the compounds of formula II herein, R⁸Selected from fluorine, chlorine, bromine, cyano, ethynyl, 1-propynyl or 1-butynyl.

In one embodiment of the compounds of formula II herein, R⁸Selected from fluoro, cyano or ethynyl.

In one embodiment of the compounds of formula II herein, R³Is selected from

As one embodiment of the present application, there is provided a compound represented by general formula II-1 or a pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein the substituents are defined as in the compounds of the general formula II.

As one embodiment of the present application, there is provided a compound represented by general formula II-2 or a pharmaceutically acceptable salt, solvate or hydrate thereof:

wherein the substituents are defined as in the compounds of the general formula II.

The following compounds and pharmaceutically acceptable salts, solvates, or hydrates thereof are preferred for use herein:

another aspect of the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula i, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions of the present application may further contain one or more additional therapeutic agents.

Another aspect of the present application provides a method of treating cancer induced by an IDH1 mutation, the IDH1 mutation having an R132X mutation; in some embodiments, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S, and R132G; in some preferred embodiments, the R132X mutation is selected from R132H and R132C; the method comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula I or formula II, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutical composition thereof.

Another aspect of the present application provides the use of a compound of formula I, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutical composition comprising the same, in the manufacture of a medicament for the treatment of a cancer induced by the IDH1 mutation.

Another aspect of the present application provides a compound of formula I, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutical composition comprising the same, for treating a cancer induced by IDH1 mutation.

In some embodiments of the present application, the cancer induced by the IDH1 mutation is selected from the group consisting of: glioblastoma (glioma), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), Acute Myelogenous Leukemia (AML), sarcoma (preferably chondrosarcoma, fibrosarcoma), melanoma, non-small cell lung cancer, cholangiocarcinoma or angioimmunoblastic non-hodgkin's lymphoma (NHL). In more specific embodiments, the cancer to be treated is glioma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), Acute Myelogenous Leukemia (AML), cholangiocarcinoma, chondrosarcoma, or angioimmunoblastic non-hodgkin's lymphoma (NHL), and the like, preferably including Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), glioma, cholangiocarcinoma, or chondrosarcoma.

The compound of the formula I or the pharmaceutically acceptable salt, solvate or hydrate thereof provided by the application has very good IDH1 enzyme inhibition activity, the activity of the compound is equivalent to or better than that of AG-120, and the compound has very good in vivo metabolism level and very long in vivo half life, and is expected to be a medicine more suitable for treating cancers induced by IDH1 mutation.

The pharmaceutical compositions of the present application can be prepared by combining a compound of the present application or a pharmaceutically acceptable salt, solvate or hydrate thereof with a suitable pharmaceutically acceptable carrier, for example, and can be formulated into solid, semi-solid, liquid or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.

Typical routes of administration of a compound of the present application, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutical composition comprising the same, include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical compositions of the present application can be manufactured by methods well known in the art, such as by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

For oral administration, pharmaceutical compositions may be formulated by mixing the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present application to be formulated as tablets, pills, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid excipients, the resulting mixture is optionally milled, if desired with further suitable auxiliaries, and the mixture is then processed to granules, to give tablets or dragee cores. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like. Such as microcrystalline cellulose, glucose solution, acacia syrup, gelatin solution, sucrose and starch paste; talc, starch, magnesium stearate, calcium stearate or stearic acid; lactose, sucrose, starch, mannitol, sorbitol, or dicalcium phosphate; silicon dioxide; croscarmellose sodium, pregelatinized starch, sodium starch glycolate, alginic acid, corn starch, potato starch, methylcellulose, agar, carboxymethylcellulose, crospovidone, and the like. The dragee cores may optionally be coated, in particular with enteric coatings, according to methods well known in normal pharmaceutical practice.

The pharmaceutical compositions may also be adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms. Suitable excipients, such as fillers, buffers or surfactants can be used.

The compounds of formula i, or pharmaceutically acceptable salts, solvates or hydrates thereof, described herein may be administered by any suitable route and method, for example, orally or parenterally (e.g., intravenously). A therapeutically effective amount of a compound of formula I or II is from about 0.0001 to 20mg/Kg body weight/day, for example from 0.001 to 10mg/Kg body weight/day.

The frequency of administration of the compounds of formula I is determined by the individual requirements of the patient, for example 1 or 2 administrations per day, or more administrations per day. Administration may be intermittent, for example, wherein a patient receives a daily dose of a compound of formula i or formula ii over a period of several days, followed by a period of several days or more in which the patient does not receive a daily dose of a compound of formula i or formula ii.

Related definitions:

the following terms and phrases used herein have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or active ingredient thereof.

The term "optionally" or "optionally" means followingThe description that follows may or may not occur, and includes both the occurrence of the event or circumstance and the non-occurrence of the event or circumstance. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F) Polysubstituted (e.g. CHFCH)₂F、CH₂CHF₂Etc.) or completely substituted (CF)₂CF₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.

C as used herein_m-nMeaning that the moiety has m-n carbon atoms. For example, "C_3-10Cycloalkyl "means that the cycloalkyl group has 3 to 10 carbon atoms. "C_0-6Alkylene "means that the alkylene group has 0 to 6 carbon atoms, and when alkylene has 0 carbon atom, the group is a bond.

Numerical ranges herein refer to each integer in the given range. E.g. "C_1-10By "is meant that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, or that a particular atom donates or accepts an electron pair to form a coordinate bond with another atom, at which time the particular atom is deemed to be substituted with another atom, so long as the valency of the particular atom is normal and the substituted compound is stable. When the substituent is a keto group (i.e., ═ O), meaning that two hydrogen atoms are substituted, the keto substitution does not occur on the aromatic group.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2R, the group may optionally be substituted with up to two R, and there are separate options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, the term "hetero" means a heteroatom or group of heteroatoms (i.e., a group of atoms containing heteroatoms), i.e., atoms other than carbon and hydrogen, or a group of atoms containing such atoms, the heteroatoms being independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, silicon, germanium, aluminum, boron. In embodiments where two or more heteroatoms are present, the two or more heteroatoms may be the same as each other, or some or all of the two or more heteroatoms may be different from each other.

The term "halogen" or "halo" refers to any group of fluorine, chlorine, bromine or iodine.

The term "hydroxy" refers to-OH.

The term "cyano" refers to — CN.

The term "amino" refers to the group-NH₂-NH (alkyl) and-N (alkyl)₂Specific examples of amino groups include, but are not limited to, -NH₂、-NHCH₃、-NHCH(CH₃)₂、-N(CH₃)₂、-NHC₂H₅、-N(CH₃)C₂H₅And the like.

The term "oxo" refers to a substituent on a C atom being a keto group (i.e., ═ O) or a substituent on an N atom being O (i.e., N → O).

The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. The particular alkyl group includes all isomeric forms thereof, e.g., propyl includes-CH₂CH₂CH₃、-CH(CH₃)₂For example, butyl includes-CH₂CH₂CH₂CH₃、-CH(CH₃)(CH₂CH₃)、-C(CH₃)₃、-CH₂CH(CH₃)₂. The term "C_1-6Alkyl "refers to an alkyl group having 1 to 6 carbon atoms. The term "C_1-4Alkyl "refers to an alkyl group having 1 to 4 carbon atoms. The term "C_1-3Alkyl "means having 1-3 carbon atomsAlkyl group of (1). The "alkyl group", "C_1-8Alkyl group "," C_1-6Alkyl "or" C_1-3Alkyl "may be unsubstituted or substituted with one or more substituents selected from hydroxy, halogen or amino.

The term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl; for example, the term "halo C_1-3Alkyl "is intended to include, but not be limited to, trifluoromethyl, 2,2, 2-trifluoroethyl, and 3-bromopropyl, and the like. Examples of haloalkyl groups include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl and pentachloroethyl.

The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2 to 12 carbon atoms and having one or more double bonds. Examples of alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl, and 3-hexenyl. One of the double bond carbons may optionally be the point of attachment of an alkenyl substituent.

The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 12 carbon atoms and characterized by having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of an alkynyl substituent.

The term "cycloalkyl" refers to an all-carbon monocyclic saturated hydrocarbon group consisting of carbon and hydrogen atoms, such as C_3-20Cycloalkyl, preferably C_3-6Cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. The cycloalkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxy, aryl, heteroaryl, amino, halo, sulfonyl, sulfinyl, phosphoryl, and hydroxy.

The term "aryl" refers to an all-carbon monocyclic or polycyclic fused aromatic ring group having a conjugated pi-electron system, preferably having 6 to 14 carbon atoms, more preferably having 6 to 12 carbon atoms, and most preferably having 6 carbon atoms. For example monocyclic aromatic ring groups, such as from phenyl; bicyclic fused aromatic ring groups consist of phenyl fused to a 4-6 membered aromatic or nonaromatic carbocyclic ring, including naphthyl.

The term "heteroaromatic ring" refers to a monocyclic or fused ring of 5 to 12 ring atoms, having 5,6, 7, 8, 9, 10, 11 or 12 ring atoms, containing 1,2, 3 or 4 ring atoms selected from N, O, S, the remaining ring atoms being C, and having a fully conjugated pi-electron system.

The term "heteroaryl" refers to the group remaining after 1 hydrogen atom has been removed from the "heteroaromatic ring" molecule, which may be unsubstituted or substituted, including but not limited to alkyl, alkyloxy, aryl, aralkyl, amino, halo, hydroxy, cyano, nitro, carbonyl, and heteroalicyclic. Non-limiting examples of unsubstituted heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazinyl.

The term "heteroalicyclic" refers to a monocyclic or fused ring of 3 to 12 ring atoms having 3,4, 5,6, 7, 8, 9, 10, 11 or 12 ring atoms wherein 1 or 2 ring atoms are selected from N, O, S, S (O)_n(wherein n is 0, 1 or 2) and the remaining ring atoms are C. Such rings may be saturated or unsaturated (e.g. with one or more double bonds), but do not have a fully conjugated pi-electron system. Examples of 3-membered saturated heteroalicyclic include, but are not limited toExamples of 4-membered saturated heteroalicyclic include, but are not limited toExamples of 5-membered saturated heteroalicyclic include, but are not limited to Examples of 6-membered saturated heteroalicyclic include, but are not limited to Examples of 7-membered saturated heteroalicyclic include, but are not limited to Examples of 5-membered unsaturated heteroalicyclic include, but are not limited toExamples of 6-membered unsaturated heteroalicyclic include, but are not limited to

The term "heterocycloalkyl" refers to a radical of the "heteroalicyclic" molecule that is left after removal of 1 hydrogen atom, which heterocycloalkyl may be unsubstituted or wherein the hydrogen atom is optionally substituted with a substituent including, but not limited to, alkyl, alkoxy, heteroaryl, and the like,Aryl, aralkyl, -COOH, -CN, amino, halogen or hydroxy.

"DMF" refers to N, N-dimethylformamide.

"DIAD" refers to diisopropyl azodicarboxylate.

"Boc-" means t-butyloxycarbonyl.

"TFA" refers to trifluoroacetic acid.

"DCM" means dichloromethane.

"PE" refers to petroleum ether.

"EA" refers to ethyl acetate.

"DCM" means dichloromethane.

"0.5% MC" means that the formulation is formulated to contain 0.5% methylcellulose.

"0.2% Tween 80" means that the formulation contains 0.2% sorbitan monooleate polyoxyethylene ether 80.

“Pd₂(dba)₃"refers to tris (dibenzylideneacetone) dipalladium.

"Xantphos" refers to 4, 5-bis diphenylphosphine-9, 9-dimethylxanthene.

The term "Ugi reaction" refers to a multi-component reaction of one molecule of aldehyde or ketone, one molecule of amine, one molecule of isonitrile, and one molecule of carboxylic acid to form an alpha-acylamidoamide.

The term "Goldberg coupling reaction" refers to a copper-catalyzed bond-forming reaction of an aryl C atom with an N, O, C atom.

The term "Mitsunobu reaction" means that the alcoholic hydroxyl group is displaced by a nucleophile, i.e. a Lewis base, under the action of diethyl azodicarboxylate (DEAD) and triphenylphosphine, with the inversion of the configuration of the carbon atom to which the hydroxyl group is attached.

The term "pharmaceutically acceptable" is intended to refer to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned.

The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

Certain compounds of the present application may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present application. Certain compounds of the present application may exist in polycrystalline or amorphous form.

Certain compounds of the present application may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are included within the scope of the present application.

The enantiomers of ambiscial and scalemic or enantiomerically pure compounds herein are illustrated by Maehr, J.chem.Ed.1985,62: 114-120. Unless otherwise indicated, the absolute configuration of a stereocenter is indicated by wedge bonds and dashed bonds. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, they include the E, Z geometric isomer unless otherwise specified. Likewise, all tautomeric forms are included within the scope of the present application.

The compounds of the present application may exist in specific geometric or stereoisomeric forms. The present application contemplates all such compounds, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present application. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present application.

Optically active (R) -and (S) -isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one of the enantiomers of a compound of the present application is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the diastereomers by fractional crystallization or chromatography, as is well known in the art, and the pure enantiomers are recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines).

The compounds of the present application may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labelled with radioactive isotopes, such as tritium (A), (B), (C) and C)³H) Iodine-125 (¹²⁵I) Or C-14(¹⁴C) In that respect All isotopic variations of the compounds of the present application, whether radioactive or not, are intended to be encompassed within the scope of the present application.

The term "pharmaceutically acceptable carrier" refers to those carriers which do not have a significant irritating effect on the organism into which the carrier is ingested and which do not impair the biological activity and performance of the active compound. By "pharmaceutically acceptable carrier" is meant an inert substance which facilitates administration of the active ingredient in conjunction with administration of the active ingredient, including, but not limited to, any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier acceptable for use in humans or animals (e.g., livestock) as permitted by the national food and drug administration. Non-limiting examples of such carriers include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and the like. For additional information on The vector, reference may be made to Remington, The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams & Wilkins (2005), The contents of which are incorporated herein by reference.

The term "excipient" generally refers to a carrier, diluent, and/or vehicle necessary to formulate an effective pharmaceutical composition.

The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For oral dosage forms herein, an "effective amount" of one active agent in a composition is the amount needed to achieve the desired effect when combined with another active agent in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof well known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The chemical reactions of the embodiments herein are carried out in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

The compounds of formula II herein can be prepared by one skilled in the art of organic synthesis using standard methods in the art by the following routes:

wherein the content of the first and second substances,

X、R¹、R²、R³are as defined above for the compounds of formula II.

The isonitrile compound 1, o-chlorobenzaldehyde 2, amino compound 3 and sulfonamide carboxylic acid 4 are directly subjected to Ugi reaction in a suitable solvent (such as methanol and the like) to obtain the compound of formula II.

The compounds of formula II herein can also be prepared by those skilled in the art of organic synthesis using standard methods in the art by the following routes:

when R is³When H, compound 5 can be obtained by the Ugi reaction described above; carrying out Goldberg coupling reaction on the compound 5 and the halogenated compound 6 under the following reaction conditions: suitable copper salts (e.g., CuI, etc.) are used as catalysts, and a suitable ligand (e.g., N' -dimethylethylenediamine, etc.) and a base (e.g., cesium carbonate, etc.) are added to the mixture and reacted by heating in a suitable solvent (e.g., 1, 4-dioxane, etc.) to obtain the compound of formula II. The compound 5 and the halogenated compound 6 can also undergo palladium-catalyzed arylamidation reaction, and the reaction conditions are as follows: suitable palladium (e.g. Pd)₂(dba)₃) Adding a suitable ligand (such as Xantphos and the like) and a base (such as cesium carbonate and the like) as a catalyst, and heating in a suitable solvent (such as 1, 4-dioxane and the like) to react to obtain the compound of the formula II.

Intermediates 1-6 and 1-7 herein can be prepared by one skilled in the art of organic synthesis using standard methods in the art by the following routes:

reacting the L-homocystine 1-1 with thionyl chloride in a methanol solution to obtain L-homocystine dimethyl ester dihydrochloride 1-2; introducing chlorine gas into the solution 1-2 to obtain (S) -2-amino-4-chlorosulfonyl methyl butyrate hydrochloride 1-3; 1-3 is cyclized under the action of triethylamine to obtain (S) -isothiazolidine-3-methyl formate 1, 1-dioxide 1-4; 1-4 is hydrolyzed to obtain an intermediate (S) -isothiazolidine-3-formic acid 1, 1-dioxide 1-7. 1-4 and 2-bromo-4-cyanopyridine in the presence of CuI or Pd₂(dba)₃Catalytic coupling to obtain a compound 1-5; 1-5 hydrolyzing to obtainIntermediates 1 to 6.

Intermediates 2-6 and 2-10 of the present application can be prepared by one skilled in the art of organic synthesis using standard methods in the art by the following routes:

carrying out reductive amination on the L-serine amino acid methyl ester hydrochloride 2-1 to obtain a benzyl protected compound 2-2; 2-2 is sulfonylated to obtain a compound 2-3; 2-3, carrying out Mistunobu reaction and intramolecular cyclization to obtain a compound 2-4; 2-4 is hydrogenated and debenzylated to obtain a compound 2-5; 2-5 to obtain an intermediate 2-6. 2-4 removing Boc protecting group to obtain compound 2-7; 2-7 are subjected to N-methylation reaction to obtain a compound 2-8; 2-8 is hydrogenated and debenzylated to obtain a compound 2-9; 2-9 to obtain an intermediate 2-10.

Drawings

FIG. 1 is a graph showing the results of the inhibition of 2-HG in IDH1 mutant HT-1080 cells by the compound of example 2 and control AG-120.

FIG. 2 is the results of the inhibition of the compound of example 2 against IDH1 mutant HT-1080 extracellular 2-HG with control AG-120.

FIG. 3 is the results of the inhibition of 2-HG of the compound of example 2 against IDH1 mutant U87 with control AG-120.

Detailed Description

The following specific examples are presented to enable those skilled in the art to more clearly understand and practice the present application. They should not be considered limiting the scope of the application, but merely as being exemplary illustrations and representative of the application. Those skilled in the art will understand that: there are other synthetic routes to the compounds of the present application, and the following non-limiting examples are provided.

Unless otherwise indicated, temperatures are in degrees celsius. Reagents were purchased from commercial suppliers such as national drug group chemical reagents beijing ltd, Alfa Aesar (Alfa Aesar), or beijing carbofuran technologies ltd, and these reagents were used directly without further purification unless otherwise specified.

Unless otherwise stated, the following reactions are carried out in anhydrous solvents, under positive pressure of nitrogen or argon or using a drying tube; the reaction bottle is provided with a rubber diaphragm so as to add the substrate and the reagent through an injector; glassware was dried and/or heat dried.

Unless otherwise stated, column chromatography purification was performed using 200-300 mesh silica gel from Qingdao oceanic plants; the thin-layer chromatography silica gel precast slab (HSGF254) produced by the institute of chemical industry of cigarette end is used for the preparation of the thin-layer chromatography separation; MS is measured by a Thermo LCQ fly model (ESI) liquid chromatography-mass spectrometer; the optical rotation was measured using an SGW-3 automatic polarimeter available from Shanghai Shenguan instruments Ltd.

Unless otherwise indicated, nuclear magnetic data (¹H-NMR) was run at 400MHz using a Varian apparatus. The solvent used for nuclear magnetic data is CDCl₃、CD₃OD、D₂O、DMSO-d₆Etc., based on tetramethylsilane (0.00ppm) or based on residual solvent (CDCl)₃：7.26ppm；CD₃OD：3.31ppm；D₂O：4.79ppm；DMSO-d₆: 2.50 ppm). When indicating the diversity of the peak shapes, the following abbreviations represent the different peak shapes: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (doublet of doublets), dt (doublet of triplets). If the coupling constant is given, it is given in Hertz (Hz).

Unless otherwise indicated, some of the examples in this application title compounds do not indicate the absolute configuration of the chiral center, some of the compounds obtained in the preparation process are mixtures of all enantiomers, and ordinary column chromatography fails to separate the enantiomers, but does not indicate that no enantiomer exists in some of the compounds.

Example 1: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: l-homocystine dimethyl ester dihydrochloride

Under stirring in ice bath, thionyl chloride (10.64g,89.4mmol) was added dropwise to a methanol suspension of L-homocystine (8.0g,29.8mmol) and the solution was gradually clarified, after addition, the ice bath was removed by stirring for 10min, and the solution was stirred at room temperature overnight to remove the solvent, thereby obtaining L-homocystine dimethyl dihydrochloride (10.6g, yield 100%).

¹H-NMR(400MHz,DMSO-d₆):δ＝8.79(s,6H),3.75(s,6H),2.95-2.80(m,4H),2.52-2.47(m,2H),2.20-2.10(m,4H)。

And B: (S) -2-amino-4-chlorosulfonyl butyric acid methyl ester hydrochloride

While stirring in an ice bath, a mixed solution of L-homocystine dimethyl ester dihydrochloride (10.6g,29.8mmol) in ethanol (40mL) and chloroform (80mL) was purged with chlorine for 20 minutes to form a white solid, which was then filtered and washed with chloroform to give (S) -methyl 2-amino-4-chlorosulfonylbutyrate hydrochloride (7.5g, yield 100%).

¹H-NMR(400MHz,DMSO-d₆):δ＝13.46(s,1H),8.57(s,2H),3.66(s,3H),3.18-2.95(m,2H),2.52-2.45(m,1H),2.22-1.97(m,2H)。

And C: (S) -isothiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide

To a chloroform suspension of (S) -methyl 2-amino-4-chlorosulfonylbutyrate hydrochloride (4.5g, 17.85mmol) was added dropwise a chloroform solution of triethylamine with stirring in an ice salt bath, and after completion of the addition of the ice salt bath, the mixture was stirred at room temperature overnight to remove the solvent, and the mixture was filtered through celite, washed with ethyl acetate, and the solvent was removed to obtain a pale yellow oily substance, i.e., (S) -methyl isothiazolidine-3-carboxylate 1, 1-dioxide (3.2g, yield 100%).

¹H-NMR(400MHz,CDCl₃):δ＝4.98(s,1H),4.21(dd,J＝8.3,4.6Hz,1H),3.84(s,3H),3.30–3.11(m,1H),3.09–2.90(m,1H),2.90–2.73(m,1H),2.60(ddd,J＝18.4,8.9,4.7Hz,1H)。

Step D: (S) -isothiazolidine-3-carboxylic acid 1, 1-dioxide

To a solution of methyl S-isothiazolidine-3-carboxylate 1, 1-dioxide (2.4g, 13.4mmol) in methanol in tetrahydrofuran was added dropwise a suspension of lithium hydroxide under stirring in ice bath, reacted overnight, 1N hydrochloric acid was added dropwise to adjust the pH to less than 5, and the solvent was removed, filtered, washed with methanol and removed to give (S) -isothiazolidine-3-carboxylate 1, 1-dioxide (2.2g, yield 100%).

¹H-NMR(400MHz,CDCl₃):δ＝3.85-3.75(m,1H),3.10-2.85(m,2H),2.55-2.30(m,2H)。

Step E: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide

3-amino-5-fluoropyridine (57mg, 0.508mmol) and o-chlorobenzaldehyde (72mg, 0.512mmol) were dissolved in methanol at room temperature and stirred for 30 minutes, and (S) -isothiazolidine-3-carboxylic acid 1, 1-dioxide (84mg, 0.508mmol) was added to the mixture and stirred for 10 minutes, and 1, 1-difluoro-3-isocyanocyclobutane (60mg, 0.508mmol, prepared by the method described in patent CN 103097340) was added thereto and stirred overnight, the solvent was removed and the product was isolated by silica gel column chromatography (PE: EA ═ 1: 1) to obtain the desired product (60mg, yield 22%).

m/z＝517[M+H]⁺。

Step F: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide

To a closed tube reactor were added (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide (60mg, 0.116mmol), 2-bromo-4-cyanopyridine (22mg, 0.139mmol), cuprous iodide (28mg, 0.147mmol), N' -dimethylethylenediamine (13mg, 0.147mmol) and cesium carbonate (95mg, 0.29mmol), dioxane (8mL) was added, nitrogen was introduced for 5 minutes, the tube was sealed, the reaction was overnight at 80 ℃, the starting material was reacted off, and column chromatography was performed after removal of the solvent (PE: EA ═ 1: 1) to obtain a racemic product, the racemic product was subjected to thin layer chromatography (DCM: EA ═ 8: 1) to give (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide (7mg, yield 10%).

¹H-NMR(400MHz,CDCl₃):δ＝8.56(d,J＝5.1Hz,1H),8.37(s,1H),7.64(s,1H),7.43(d,J＝8.1Hz,1H),7.30-7.05(m,6H),6.35(s,1H),6.20(s,1H),4.65-4.55(m,1H),4.22-4.10(m,1H),3.70-3.60(m,1H),3.42-3.33(m,1H),2.95-2.75(m,2H),2.60-2.50(m,2H),2.35-2.15(m,2H)。

m/z＝619[M+H]⁺。

HPLC conditions: chiral column:IC-3 column (25 cm); mobile phase: n-hexane/ethanol 85/15; flow rate: 0.8 mL/min; column temperature: 40 ℃; wavelength/time: 210nm, 20 min; retention time: example 1 the title compound was 17.60 min.

Example 2: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step F of example 1, (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide was isolated by thin layer chromatography.

¹H-NMR(400MHz,CDCl₃):δ＝8.47(d,J＝5.1Hz,1H),8.31(s,1H),8.08(d,J＝8.1Hz,1H),7.62(s,1H),7.30-6.75(m,6H),6.56(s,1H),6.22(s,1H),4.80-4.70(m,1H),4.40-4.30(m,1H),3.75-3.65(m,1H),3.40-3.33(m,1H),3.10-2.98(m,2H),2.60-2.40(m,4H)。

m/z＝619[M+H]⁺。

Example 3: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 5-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3, 5-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the starting material, 3-amino-5-fluoropyridine, was replaced with 3, 5-difluoroaniline to give the desired product, (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3, 5-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide (yield 13%).

m/z＝619[M+H]⁺。

And B: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 5-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 5-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide was obtained in 35% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.54(d,J＝5.0Hz,1H),7.67(s,1H),7.43-6.76(m,8H),6.11(s,1H),6.01(s,1H),4.80-4.72(m,1H),4.16-4.2(m,1H),3.68-3.65(m,1H),3.38-3.34(m,1H),2.80-2.98(m,2H),2.58-2.54(m,2H),2.28-2.30(m,2H)。

m/z＝636[M+H]⁺。

Example 4: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 5-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 3, the title compound was isolated by thin layer chromatography in 40% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.45(d,J＝5.0Hz,1H),7.67(s,1H),7.35-6.66(m,8H),6.46(s,1H),5.93(d,J＝6.8,1H),4.85-4.83(m,1H),4.40-4.23(m,1H),3.72-3.69(m,1H),3.37-3.34(m,1H),3.03-2.99(m,2H),2.60-2.20(m,4H)。

m/z＝636[M+H]⁺。

Example 5: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the starting material, 3-amino-5-fluoropyridine, was replaced with 3-trifluoromethylaniline to give the desired product, (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide in 50% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.10and 8.00(s,0.5and 0.5H),7.58-6.60(m,7H),6.55and 6.39(s,0.5and 0.5H),5.90(s,1H),5.23and 5.18(d,J＝7.2,0.5and 0.5H),4.40-4.30(m,1H),4.00-3.90(m,1H),3.11-2.96(m,4H),2.59-2.26(m,4H)。

m/z＝566[M+H]⁺。

And B: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide was obtained in 16% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.54(d,J＝5.0Hz,1H),8.40(brs,1H),7.64-7.05(m,9H),6.32(m,2H),4.68-4.65(m,1H),4.22-4.04(m,1H),3.64-3.59(m,1H),3.34-3.28(m,1H),2.98-2.65(m,2H),2.60-2.43(m,2H),2.38-2.15(m,2H)。

m/z＝668[M+H]⁺。

Example 6: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 5, (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-trifluoromethylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide was isolated by thin layer chromatography in 23% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.46(d,J＝5.0Hz,1H),8.14(s,1H),7.69-6.81(m,9H),6.45(s,1H),5.88-5.80(m,1H),4.80-4.65(m,1H),4.40-4.25(m,1H),3.82-3.65(m,1H),3.40-3.25(m,1H),3.10-2.90(m,2H),2.70-2.40(m,4H)。

m/z＝668[M+H]⁺。

Example 7: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-cyano-5-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-cyano-5-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E of example 1, the starting material, 3-amino-5-fluoropyridine was replaced with 3-fluoro-5-cyanoaniline to give the desired product in 17% yield.

m/z＝541[M+H]⁺。

And B: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-cyano-5-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-cyano-5-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide was obtained in 15% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.83(d,J＝6.0Hz,1H),8.57(d,J＝5.0Hz,1H),7.66-7.12(m,8H),6.29(s,1H),6.16(s,1H),4.63-4.61(m,1H),4.21-4.10(m,1H),,3.70-3.60(m,1H),3.40-3.25(m,1H),3.30-2.82(m,2H),2.61-2.42(m,2H),2.40-2.20(m,2H)。

m/z＝643[M+H]⁺。

Example 8: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-cyano-5-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 7, the title compound was isolated by thin layer chromatography in 20% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.47-6.85(m,10H),6.50(d,J＝9.77Hz,1H),5.97(s,1H),4.75-4.73(m,1H),4.40-4.30(m,1H),3.80-3.65(m,1H),3.42-3.27(m,1H),3.15-2.30(m,6H)。

m/z＝643[M+H]⁺。

Example 9: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 4-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3, 4-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the starting material, 3-amino-5-fluoropyridine was replaced with 3, 4-difluoroaniline to give the desired product in 18% yield.

m/z＝534[M+H]⁺。

And B: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 4-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 4-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide was obtained in 30% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.55(s,1H),7.62(s,1H),7.40-7.15(m,8H),6.43(s,1H),6.27(s,1H),4.70-4.68(m,1H),4.15-4.11(m,1H),3.61-3.53(m,1H),3.32-3.26(m,1H),2.89-2.69(m,2H),2.57-2.53(m,2H),2.28-2.11(m,2H)。

m/z＝636[M+H]⁺。

Example 10: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3, 4-difluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 9, the title compound was isolated by thin layer chromatography in 35% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.47-8.43(m,1H),7.66(s,1H),7.35-6.82(m,8H),6.44(d,J＝5.78,1H),5.97(d,J＝6.57,1H),4.81-4.79(m,1H),4.33-4.32(m,1H),3.73-3.69(m,1H),3.36-3.33(m,1H),3.02-2.99(m,2H),2.61-2.40(m,4H)。

m/z＝636[M+H]⁺。

Example 11: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide

To a closed tube reactor were added (S) -isothiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (prepared from example 1, step C, 200mg, 1.11mmol), 2-bromo-4-cyanopyridine (204mg, 1.11mmol), cuprous iodide (105mg, 0.55mmol), N' -dimethylethylenediamine (98mg, 1.11mmol), and cesium carbonate (723mg, 2.22mmol), dioxane (8mL) was added, nitrogen was introduced for 5 minutes, the tube was sealed, the reaction was allowed to proceed overnight at 80 ℃, the starting materials reacted off, and after removal of the solvent, column chromatography (PE: EA ═ 1: 1) was performed to separate the target product (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (230mg, 74%).

¹H-NMR(400MHz,CDCl₃):δ＝8.40(dd,J＝5.2,0.8Hz,1H),7.69(t,J＝1.0Hz,1H),7.19(dd,J＝5.2,1.0Hz,1H),5.01(dd,J＝8.0,3.6Hz,1H),3.78(s,3H),3.64-3.55(m,1H),3.48-3.42(m，1H),2.95-2.84(m,1H),2.65-2.52(m,1H)。

Step B (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid 1, 1-dioxide

While stirring in ice bath, a suspension of lithium hydroxide was added dropwise to a methanolic tetrahydrofuran solution of (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (116mg, 0.41mmol) to react overnight, after completion of the reaction, 10mL of water was added to dilute the reaction solution, followed by extraction with ethyl acetate to remove impurities, addition of 1N hydrochloric acid to the aqueous phase to adjust the pH to less than 5, extraction with ethyl acetate, and removal of the solvent to give (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid 1, 1-dioxide (103mg, 94% yield).

¹H-NMR(400MHz,DMSO-d₆):δ＝13.5(s,1H),8.54(d,J＝5.0,1H),7.51(dd,J＝3.74,4.76Hz,1H),7.45(s,1H),4.95-4.90(m,1H),3.75-3.60(m,2H),2.85-2.72(m,1H),2.46-2.38(m,1H)。

And C: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

With reference to step E in example 1, the starting material (S) -isothiazolidine-3-carboxylic acid 1, 1-dioxide was replaced with (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid 1, 1-dioxide to give the desired product (S) -N- ((R) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide in 30% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.56(d,J＝5.0Hz,1H),7.62(s,1H),7.39(d,J＝8.1Hz,1H),7.21-6.99(m,8H),6.43(s,1H),6.23(s,1H),4.73(dd,J＝6.5,3.1Hz,1H),4.20-4.05(m,1H),3.57(dd,J＝20.0,11.9Hz,1H),3.27(dd,J＝11.9,3.5Hz,1H),2.84-2.72(m,2H),2.55(dd,J＝14.9,9.3Hz,2H),2.28-2.13(m,2H)。

m/z＝618[M+H]⁺。

Example 12: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step C of example 11, the title compound was isolated by thin layer chromatography in 33% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.46(m,1H),7.67(d,J＝8.8Hz,1H),7.63(s,1H),7.22-6.84(m,8H),6.47(d,J＝3.6,1H),6.08(s,1H),4.82(d,J＝6.1Hz,1H),4.33(m,1H),3.68-3.60(m,1H),3.40-3.28(m,1H),3.10-2.98(m,2H),2.68-2.38(m,4H)。

m/z＝618[M+H]⁺。

Example 13: (S) -N- ((R) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (pyrimidin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the reaction starting materials 2, 2-difluorocyclobutyl isocyanide and 3-amino-5-fluoropyridine were replaced with cyclohexyl isocyanide and 3-fluoroaniline to give the desired product in 81% yield.

¹H-NMR(400MHz,CDCl₃):δ＝7.70-6.86(m,8H),6.50-6.25(m,1H),5.41-5.35(m,1H),5.25-5.10(m,1H),4.05-4.95(m,1H),3.90-3.80(m,1H),3.12-2.90(m,2H),2.65-1.00(m,12H)。

m/z＝508[M+H]⁺。

Step B (S) -N- ((R) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (pyrimidin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, (S) -N- ((R) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (pyrimidin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide was obtained in a yield of 26%.

¹H-NMR(400MHz,CDCl₃):δ＝8.61(d,J＝4.9Hz,2H),7.75(brs,1H),7.42-7.37(m,2H),7.26-7.21(m,2H),7.01-7.07(m,1H),7.02-6.96(m,2H),6.85(brs,1H)6.12(s,1H),5.71(d,J＝8.4Hz,1H),4.75(d,J＝5.6Hz,1H),3.76-3.68(m,2H),3.34-3.30(m,1H),2.53-2.46(m,2H),1.86-1.78(m,2H),1.52-1.62(m,4H),1.24-1.30(m,2H),1.12-0.73(m,2H)。

m/z＝586[M+H]⁺。

Example 14: (S) -N- ((S) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (pyrimidin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 13, the title compound was isolated by thin layer chromatography in 30% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.57(d,J＝4.9Hz,2H),7.74(s,1H),7.43-6.88(m,7H),6.49(s,1H),5.40(d,J＝7.9Hz,1H),4.79(s,1H),3.84-3.75(m,2H),3.33-3.30(m,1H),2.62-2.37(m,2H),1.86-1.78(m,2H),1.52-1.62(m,4H),1.24-1.30(m,2H),1.12-0.73(m,2H)。

m/z＝586[M+H]⁺。

Example 15: (S) -N- ((R) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, the title product was obtained in 39% yield from (S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide (prepared by step A in example 13).

¹H-NMR(400MHz,CDCl₃):δ＝8.56(d,J＝5.0Hz,1H),7.70(s,1H),7.67(s,1H),7.35-6.99(m,8H),6.12(s,1H),5.70(d,J＝7.4Hz,1H),4.75-4.74(m,1H),3.76-3.56(m,2H),3.34-3.36(m,1H),2.66-2.43(m,2H),1.85-1.71(m,2H),1.62-1.56(m,4H),1.28-1.24(m,2H),1.12-0.85(m,2H)。

m/z＝610[M+H]⁺。

Example 16: (S) -N- ((S) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 15, the title compound was isolated by thin layer chromatography in 39% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.46(d,J＝5.0Hz,1H),7.75-6.88(m,10H),6.45(s,1H),5.36-5.30(m,1H),4.82-4.81(m,1H),3.82-3.74(m,2H),3.34-3.32(m,1H),2.67-2.65(m,1H),2.45-2.40(m,1H),1.97-1.94(m,2H),1.74-1.59(m,4H),1.38-1.24(m,2H),1.17-1.01(m,2H)。

m/z＝610[M+H]⁺。

Example 17: (S) -N- ((R) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-fluoropyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step F in example 1, the coupling of (S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide (prepared by step A in example 13) with 2-bromo-4-fluoropyridine provided the desired product in 25% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.32(dd,J＝8.4,5.9Hz,1H),7.45-6.73(m,10H),6.03(s,1H),5.73(s,1H),4.88-4.68(m,1H),3.76-3.62(m,2H),3.29(ddd,J＝12.0,6.7,2.6Hz,1H),2.67-2.39(m,2H),1.85-1.75(m,2H),1.62-1.56(m,2H),1.32-1.25(m,3H),1.05-0.91(m,3H)。

m/z＝603[M+H]⁺。

Example 18: (S) -N- ((S) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-fluoropyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 17, the title compound was isolated by thin layer chromatography in 25% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.26(s,1H),7.37-6.72(m,10H),6.45(s,1H),5.37(d,J＝8.1,1H),4.83(d,J＝7.7,1H),3.83-3.70(m,2H),3.29(ddd,J＝12.0,6.7,2.6Hz,1H),2.65-2.62(m,1H),2.42-2.25(m,1H),1.94-1.91(m,2H),1.68-1.56(m,2H),1.32-1.25(m,3H),1.05-0.91(m,3H)。

m/z＝603[M+H]⁺。

Example 19: (3S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -N- (3-fluorophenyl) -2- (4-alkynylpyridin-2-yl) isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: 2-bromo-4- (2, 2-dibromoethenyl) pyridine

Triphenylphosphine (4.23g, 16.13mmol) was added to a solution of carbon tetrabromide (2.68g, 8.08mmol) in dichloromethane at 0 deg.C, stirred for 5min, then 2-bromo-4-formylpyridine (0.50g, 2.69mmol) in methanol was added, warmed to room temperature and stirred for 30 min. After completion of the reaction, water was added to terminate the reaction, and the reaction was extracted with ethyl acetate (30mL × 3), and the organic phase was dried over anhydrous sodium sulfate and separated by silica gel column chromatography (PE: EA ═ 10: 1) to obtain the product (140mg, yield 15%).

¹H-NMR(400MHz,CDCl₃):δ＝8.39-8.38(m,1H),7.63-7.62(m,1H),7.40-7.38(m,2H)。

And B: 2-bromo-4- ((trimethylsilyl) ethynyl) pyridine

2.4M n-butyllithium (358. mu.L, 0.86mmol) was added dropwise to a solution of 2-bromo-4- (2, 2-dibromovinyl) pyridine (140mg, 0.41mmol) in tetrahydrofuran at-78 ℃ and after stirring for 30 minutes chlorotrimethylsilane (53. mu.L, 0.61mmol) was added further, after stirring for 1 hour at 78 ℃ the temperature was raised to room temperature and stirring was continued for 30 minutes. After completion of the reaction, water was added to terminate the reaction, and the reaction was extracted with ethyl acetate (30mL × 3), and the organic phase was dried over anhydrous sodium sulfate and separated by silica gel column chromatography (PE: EA ═ 20: 1) to obtain the product (30mg, yield 29%).

¹H-NMR(400MHz,CDCl₃):δ＝8.32-8.30(m,1H),7.52-7.51(m,1H),7.27-7.24(m,1H),0.28-0.25(m,9H)。

And C: (3S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -N- (3-fluorophenyl) -2- (4-alkynylpyridin-2-yl) isothiazolidine-3-carboxamide 1, 1-dioxide

(S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide (prepared from example 13, step A, 60mg, 0.12mmol), 2-bromo-4- [ (trimethylsilyl) ethynyl ] pyridine (30mg, 0.12mmol), cuprous iodide ((12mg, 0.06mmol), N' -dimethylethylenediamine (13. mu.L, 0.12mmol), and cesium carbonate (77mg, 0.24mmol) were stirred in 1, 4-dioxane (8mL) overnight at 80 deg.C after the reaction was complete, the mixture was filtered, the mother liquor was concentrated, and the title compound (5mg, yield 7%).

¹H-NMR(400MHz,CDCl₃):δ＝8.36-8.30(m,1H),7.73(m,1H),7.55(m,1H),7.46-7.30(m,2H),7.24-6.88(m,6H),6.46and 6.03(s,1H),5.74and 5.39(d,J＝6.4Hz,1H),4.80and 4.75(m,1H),3.81-3.66(m,3H),3.31-3.28(m,1H),2.62-2.49(m,2H),2.05-1.55(m,6H),1.15-0.84(m,4H)。

m/z＝609[M+H]⁺。

Example 20: (3S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3- (N- (4-cyanopyridin-2-yl) aminosulfonyl) phenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (3S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2-N- (3-aminosulfonyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the starting material, 3-fluoro-5-aminopyridine, was replaced with 3-aminobenzenesulfonamide to give the desired product in 53% yield.

m/z＝569[M+H]⁺。

And B: (3S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3- (N- (4-cyanopyridin-2-yl) aminosulfonyl) phenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Reference was made to step F of example 1 to give the title compound in 25% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.60-6.60(m,14H),6.41-6.39(s,1H),6.18-5.56(s,1H),5.44-5.36(m,1H),4.62-4.59(m,1H),3.90-3.61(m,2H),3.40-3.33(m,1H),2.60-2.24(m,2H),2.00-0.80(m,10H)。

m/z＝773[M+H]⁺。

Example 21: (S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (3-cyanophenylethyl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (S) -2- (3-cyanophenethyl-2-yl) isothiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide

Methyl (S) -isothiazolidine-3-carboxylate 1, 1-dioxide (179mg, 1.0mmol), 3-cyanobenzyl bromide (392mg, 2.0mmol), tetrabutylammonium iodide (37mg, 0.1mmol) were dissolved in DMF (3mL), the reaction solution was stirred overnight at room temperature, diluted with water, extracted with ethyl acetate, dried, filtered, concentrated, and the crude product was isolated by silica gel column chromatography to give the desired product (260mg, 88% yield).

¹H-NMR(400MHz,CDCl₃):δ＝7.66(s,1H),7.65-7.59(m,2H),7.47(t,J＝7.6Hz,1H),4.41(q,J＝15.6Hz,2H),3.85-3.82(m,1H),3.63(s,3H)3.40-3.25(m,1H),3.23-3.16(m,1H),2.66-2.42(m,2H)。

And B: (S) -methyl 2- (3-cyanophenethyl-2-yl) isothiazolidine-3-carboxylic acid 1, 1-dioxide

Referring to step B in example 11, the desired product was obtained in 75% yield.

¹H-NMR(400MHz,CDCl₃):δ＝7.81(s,1H),7.77-7.54(m,3H),4.35(s,2H),3.98-3.96(m,1H),3.38-3.26(m,2H),2.66-2.50(m,1H),2.49-2.26(m,1H)。

And C: (S) -N- { (RS) -1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl } -2- (3-cyanophenylethyl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the title compound was obtained in 79% yield.

¹H-NMR(400MHz,CDCl₃):δ＝7.80-6.75(m,12H),6.41and 6.37(s,0.5and 0.5H),5.41and 5.35(m,0.5and 0.5H),4.62and 4.53(d,J＝16,0.5and 0.5H),4.15and 4.04(d,J＝15.2,16,0.5and 0.5H),3.96-3.78(m,1H),3.65-3.60(m,1H),3.45-3.40(m,1H),3.18-3.02(m,1H),2.40-2.20(m,2H),2.02-1.80(m,2H),1.79-1.45(m,4H),1.40-1.02(m 4H)。

m/z＝623[M+H]⁺。

Example 22: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino)) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Step A: (3S) -N- (1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) isothiazolidine-3-carboxamide 1, 1-dioxide

2-chloro-benzaldehyde (85mg, 0.605mmol) and 3-fluoroaniline (67mg, 0.605mmol) were mixed in MeOH (6.0mL) with stirring at room temperature for 30 min. (S) -isothiazolinone-3-carboxylic acid 1, 1-dioxide (150mg, 0.908mmol) was added and the reaction mixture was stirred for 10 minutes, followed by addition of 1, 1-difluoro-4-isocyancyclohexane (88mg, 0.605mmol), and stirring at room temperature overnight. The solvent was removed in vacuo, and the resulting product was subjected to silica gel column chromatography to give (3S) -N- (1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) isothiazolidine-3-carboxamide 1, 1-dioxide (114mg, yield 34.7%) which was directly charged into the next reaction.

m/z＝544[M+H]⁺。

And B: (S) -N- ((R) -1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino)) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Adding (3S) -N- (1- (2-chlorphenyl) -2- ((4, 4-difluorocyclohexyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) isothiazolidine-3-formamide 1, 1-dioxide (114mg, 0.210mmol), 2-bromo-4-cyanopyridine (47mg, 0.252mmol), cuprous iodide (21mg, 0.11mmol), N' -dimethylethylenediamine (19mg,0.21mmol) and cesium carbonate (206mg, 0.63mmol) into a closed tube reactor, adding dioxane (8mL), introducing nitrogen for 5 minutes, sealing the tube at 80 ℃ for reaction overnight, removing the solvent, performing column chromatography (PE: EA ═ 1: 1) to obtain a racemic product, and performing thin layer chromatography (DCM: EA ═ 8: 1) on the racemic product to obtain a chiral pure compound (S) -N- ((R) -1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino)) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide (39mg, yield 28.7%).

¹H-NMR(400MHz,CDCl₃):δ＝8.55(d,J＝4.7Hz,1H),7.67(s,1H),7.39(d,J＝7.7Hz,1H),7.33-7.18(m,5H),7.07(dd,J＝24.5,16.7Hz,2H),6.95-6.69(m,1H),6.10(s,1H),5.70(d,J＝5.6Hz,1H),4.76(d,J＝6.9Hz,1H),3.72-3.70(m,2H),3.36-3.31(m,1H),2.58-2.54(m,2H),2.07-1.64(m,5H),1.41-1.17(m,3H)。

m/z＝646[M+H]⁺。

Example 23: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino)) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

In step B of example 22, (S) -N- ((R) -1- (2-chlorophenyl) -2- ((4, 4-difluorocyclohexyl) amino)) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide (40mg, yield 29.4%) was obtained by thin layer chromatography.

¹H-NMR(400MHz,CDCl₃):δ＝8.46(s,1H),7.71-7.68(m,2H),7.33(d,J＝13.3Hz,1H),7.08-7.01(m,6H),6.45(s,1H),5.43(d,J＝7.6Hz,1H),4.81(d,J＝7.2Hz,1H),3.98-3.93(m,1H),3.75-3.70(m,1H),3.34(s,1H),2.63(s,1H),2.46(s,1H),2.11-2.05(m,4H),1.89-1.81(m,2H),1.63-1.35(m,2H),0.88(s,1H)。

m/z＝646[M+H]⁺。

Example 24: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -1,2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

Step A: (S) -2- (benzylamino) -3-hydroxypropionic acid methyl ester

Benzaldehyde (13.6g, 128.6mmol) and anhydrous sodium sulfate (6.0g) were added to a 500 ml round bottom flask containing a mixture of L-serine methyl ester hydrochloride (20g, 128.6mmol) and triethylamine (13g, 128.6mmol) in dichloromethane with stirring at room temperature, and the reaction was stirred at room temperature overnight. After the reaction, filtering and concentrating. Methanol was added to the concentrated residue to dissolve it, sodium borohydride (4.86g, 128.6mmol) was added carefully in portions under ice bath, and the reaction was carried out at room temperature for 1 hour. Methanol was removed, diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate, extracted with dichloromethane three times, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give methyl (S) -2- (benzylamino) -3-hydroxypropionate (20.6g, 77% yield).

¹H-NMR(400MHz,CDCl₃):δ＝7.38–7.26(m,5H),4.48(d,J＝12.8Hz,1H),3.8-3.72(m,6H),3.61(dd,J＝10.8,10.8Hz,1H),3.54(dd,J＝6.8,6.4Hz,1H)。

And B: (S) -2- (benzyl (N- (tert-butoxycarbonyl) sulfonyl) amino) -3-hydroxypropionic acid methyl ester

To a solution of (S) -methyl 2- (benzylamino) -3-hydroxypropionate (10.0g, 47.79mmol) in methylene chloride was added triethylamine (7.25g, 71.69mmol) and tert-butylchlorosulfonylcarbamate (10.3g, 47.79mmol) with stirring in an ice bath, and the reaction was stirred at room temperature overnight. After completion of the reaction, it was diluted with dichloromethane, quenched with water, extracted three times with dichloromethane, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to afford methyl (S) -2- (benzyl (N- (tert-butoxycarbonyl) sulfonyl) amino) -3-hydroxypropionate (6.43g, yield 35%) by column chromatography.

¹H-NMR(400MHz,CDCl₃):δ＝7.44-7.28(m,6H),4.67(dd,J＝14.3,6.1Hz,2H),4.56(d,J＝15.6Hz,1H),4.00(d,J＝7.2Hz,2H),3.69(s,3H),2.84(s,1H),1.48(s,9H)。

And C: (S) -2-tert-butyl-4-methyl-5-benzyl-1, 2, 5-thiadiazolidine-2, 4-dicarboxylate 1, 1-dioxide

To a solution of (S) -methyl 2- (benzyl (N- (tert-butoxycarbonyl) sulfonyl) amino) -3-hydroxypropionate (6.42g, 16.54mmol) and triphenylphosphine (5.2g, 19.84mmol) in methylene chloride was added DIAD (4.0g, 19.84mmol) with stirring in an ice bath, and the reaction was carried out at room temperature for 2 hours. After the reaction was complete, it was diluted with dichloromethane, quenched with water, extracted three times with dichloromethane, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to afford the title compound (5.77g, 94.3% yield) which was isolated by column chromatography.

¹H-NMR(400MHz,CDCl₃):δ＝7.43-7.28(m,5H),4.54(d,J＝14.4Hz,1H),4.44(d,J＝14.4Hz,1H),4.07-4.00(m,1H),3.89(t,J＝2.9Hz,2H),3.72(s,3H),1.55(s,9H)。

Step D: (S) -2-tert-butyl 4-methyl-1, 2, 5-thiadiazolidine-2, 4-dicarboxylate 1, 1-dioxide

To a dichloromethane solution containing (S) -2-tert-butyl-4-methyl-5-benzyl-1, 2, 5-thiadiazolidine-2, 4-dicarboxylate (2.97g, 8.02mmol) was added palladium on carbon (1.0g) in an amount of 10% by mass with stirring at room temperature, and the mixture was pumped with a circulating water for 10 times and reacted at room temperature under hydrogen protection overnight. After the reaction, the reaction mixture was filtered, concentrated, and separated by column chromatography to give (S) -2-tert-butyl 4-methyl-1, 2, 5-thiadiazolidine-2, 4-dicarboxylate 1, 1-dioxide (1.49g, yield 66%).

¹H-NMR(400MHz,CDCl₃):δ＝5.17(d,J＝8.4Hz,1H),4.39(dd,J＝16,7.6Hz,1H),4.18(dd,J＝10.0,2.4Hz,1H),3.88-3.83(m,4H),1.52(s,9H)。

Step E: (S) -5- (tert-butyloxycarbonyl) -1,2, 5-thiadiazolidine-3-carboxylic acid 1, 1-dioxide

Lithium hydroxide monohydrate (342mg, 14.27mmol) was added to a solution of (S) -2-tert-butyl 4-methyl-1, 2, 5-thiadiazolidine-2, 4-dicarboxylate 1, 1-dioxide (800mg, 2.854mmol) in 10ml of methanol/water (volume ratio 5/1) with stirring in an ice bath, and reacted at room temperature overnight. After the reaction was complete, the methanol was removed by rotary evaporation, the system pH was adjusted to less than 3 with 4N HCl, extracted three times with ethyl acetate, the combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give (S) -5- (tert-butoxycarbonyl) -1,2, 5-thiadiazolidine-3-carboxylic acid 1, 1-dioxide (759mg, 100% yield) by column chromatography.

¹H-NMR(400MHz,CDCl₃):δ＝5.39-5.33(m,1H),4.46(s,1H),4.23(dd,J＝10,5.6Hz,1H),3.98(dd,J＝9.6,7.6Hz,1H),1.50(s,9H)。

Step F: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) -5-tert-butoxycarbonyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

2-chloro-benzaldehyde (400mg, 2.85mmol) and 3-fluoroaniline (317mg,2.85mmol) were mixed with MeOH (6.0mL) with stirring at room temperature for 30 minutes. (S) -5- (tert-Butoxycarbonyl) -1,2, 5-thiadiazolidine-3-carboxylic acid 1, 1-dioxide (759mg, 2.85mmol) was added and the reaction mixture was stirred for 10 minutes. 1, 1-difluoro-4-isocyanocyclobutane (333mg, 2.85mmol) was then added and the reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was chromatographed on silica gel to give (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) -5-tert-butoxycarbonyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (578mg, yield 32.9%).

m/z＝617[M+H]⁺。

Step G: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -5-tert-butoxycarbonyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

To a closed tube reactor were added (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) -5-tert-butoxycarbonyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (570mg, 0.924mmol), 2-bromo-4-cyanopyridine (186mg, 1.10mmol), cuprous iodide (88mg, 0.462mmol), N' -dimethylethylenediamine (82mg, 0.924mmol), and cesium carbonate (903mg, 2.772mmol), dioxane ((8mL) was added, nitrogen was bubbled for 5 minutes, the tube was sealed at 80 ℃ for overnight, the solvent was removed, and (3S) -N- (1- (2-chlorophenyl) -2-oxoethyl) -N- (3-fluorophenyl) -2-thiadiazolidine-3-carboxamide (PE: EA: 1) was obtained by column chromatography (PE: EA ═ 1: 1) - ((3, 3-Difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -5-tert-butoxycarbonyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (190mg, yield 28.6%).

m/z＝719[M+H]⁺。

Step H: (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -1,2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

Trifluoroacetic acid (2.0mL) was added to a dichloromethane solution containing (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -5-tert-butoxycarbonyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (190mg, 0.264mmol) with stirring in an ice bath, and reacted at room temperature under nitrogen atmosphere overnight. After completion of the reaction, it was concentrated and separated by column chromatography to give (S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2 yl) -N- (3-fluorophenyl) -1,2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (7mg, yield 4.3%).

¹H-NMR(400MHz,CDCl₃):δ＝8.43(t,J＝5.6Hz,1H),7.63(d,J＝8.1Hz,1H),7.56(s,1H),7.40-7.27(m,2H),7.18(d,J＝6.1Hz,2H),7.10-6.83(m,4H),6.47(d,J＝6.7Hz,1H),6.04(d,J＝6.3Hz,1H),5.74(d,J＝28.7Hz,1H),4.83(s,1H),4.25(d,J＝39.4Hz,1H),3.77(d,J＝9.7Hz,1H),3.68-3.46(m,1H),3.17-2.90(m,2H),2.45(m,2H)。

m/z＝619[M+H]⁺。

Example 25: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

Step A: (S) -2-benzyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide

Trifluoroacetic acid (15.0mL) was added to a dichloromethane solution containing (S) -2-tert-butyl 4-methyl-5-benzyl-1, 2, 5-thiadiazolidine-2, 4-dicarboxylate (2.8g, 7.559mmol) with stirring in an ice bath, and the reaction was allowed to proceed overnight at room temperature under nitrogen. After the reaction was completed, the reaction mixture was concentrated and separated by column chromatography to give (S) -2-benzyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (1.54g, yield 75.5%).

And B: (S) -2-benzyl-5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide

Methyl iodide (629mg, 4.44mmol) was added to a solution of (S) -2-benzyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (600mg, 2.22mmol) and potassium carbonate (920mg, 6.66mmol) in DMF (6.0mL) with stirring at room temperature, and reacted overnight at room temperature under nitrogen. After the reaction was complete, it was diluted with ethyl acetate, quenched with water, extracted three times with ethyl acetate, and the combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give (S) -2-benzyl-5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (616mg, yield 97.6%) by column chromatography.

¹H-NMR(400MHz,CDCl₃):δ＝7.40-7.31(m,5H),4.48(d,J＝1.2Hz,2H),3.86(dd,J＝8,7.6Hz,1H),3.66(s,3H),3.54(dd,J＝10,9.6Hz,1H),3.35(dd,J＝10,10Hz,1H),2.77(s,3H)。

And C: (S) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide

While stirring at room temperature, 10% by mass of palladium on carbon (300mg) was added to a dichloromethane solution containing (S) -2-benzyl-5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (616mg, 2.166mmol), and the mixture was purged with water for 10 times and reacted at room temperature under hydrogen protection overnight. After the reaction, the reaction mixture was filtered, concentrated, and separated by column chromatography to give (S) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (342mg, yield 81.4%).

¹H-NMR(400MHz,CDCl₃):δ＝5.19-5.13(m,1H),4.26-4.20(m,1H),3.88(s,3H),3.63(dd,J＝10.4,10.0Hz,1H),3.55(dd,J＝10.0,10.0Hz,1H),2.72(s,3H)。

Step D: (S) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid 1, 1-dioxide

Lithium hydroxide monohydrate (210mg, 8.755mmol) was added to a solution of (S) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid methyl ester 1, 1-dioxide (340mg, 1.751mmol) in 10ml of methanol/water (5/1 volume ratio) with stirring in an ice bath, and reacted at room temperature overnight. After the reaction was complete, the methanol was removed by rotary evaporation, the system pH was adjusted to less than 3 with 4N HCl, extracted three times with ethyl acetate, the combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo, and separated by column chromatography to give (S) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid 1, 1-dioxide (315mg, 100% yield).

Step E: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

2-chloro-benzaldehyde (156mg, 1.11mmol) and 3-fluoroaniline (124mg, 1.11mmol) were mixed in MeOH (6.0mL) with stirring at room temperature for 30 min. (S) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxylic acid 1, 1-dioxide (200mg, 1.11mmol) was added and the reaction mixture was stirred for 10 minutes. 1, 1-difluoro-4-isocyanocyclobutane (130mg, 1.11mmol) was then added and the reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was subjected to silica gel column chromatography to give (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (240mg, yield 41.0%).

¹H-NMR(400MHz,CDCl₃):δ＝7.63-7.57(m,1H),7.42(d,J＝8.0Hz,0.5H),7.32(d,J＝7.6Hz,0.5H),7.29-7.20(m,1H),7.14-6.92(m,4H),6.78-6.6(m,1H),6.55(s,0.5H),6.34(s,0.5H),6.14(d,J＝6.4Hz,0.5H),6.01(d,J＝6.4Hz,0.5H),5.72-5.52(m,1H),4.35-4.29(m,1H),4.20-4.06(m,1H),3.56-3.41(m,1H),3.09-2.80(m,3H),2.66(s,3H),2.61-2.31(m,2H)。

m/z＝531[M+H]⁺。

Step F: (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide

To a closed tube reactor were added (3S) -N- (1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -N- (3-fluorophenyl) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (140mg, 0.264mmol), 2-bromo-4-cyanopyridine (91mg, 0.291mmol), cuprous iodide (50mg, 0.132mmol), N' -dimethylethylenediamine (42mg,0.264mmol) and cesium carbonate (441mg, 0.792mmol), dioxane (8mL) was added, nitrogen was bubbled through for 5 minutes, the tube was sealed at 80 ℃ for overnight, the solvent was removed, and (3S) -N- (1- (2-chlorophenyl) -2- ((ca. 1: 1) was obtained by column chromatography (PE: EA ═ 1: 1) 3, 3-Difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-fluorophenyl) -5-methyl-1, 2, 5-thiadiazolidine-3-carboxamide 1, 1-dioxide (81mg, yield 48.5%).

¹H-NMR(400MHz,CDCl₃):δ＝8.55-8.47(m,1H),7.90-7.65(m,1H),7.51(d,J＝5.2Hz,0.5H),7.34(d,J＝6.4Hz,0.5H),7.29-7.16(m,4H),7.14(m,4H),6.75-6.70(m,1H),6.55(d,J＝4.4Hz,1H),6.16(d,J＝12Hz,1H),4.95-4.73(m,1H),4.19-4.11(m,1H),3.53-3.44(m,1H),3.34(t,J＝7.8Hz,1H),3.0-2.81(m,4H),2.41-2.31(m,2H)。

m/z＝633[M+H]⁺。

Example 26: (3S) -N- (1- (2-chlorophenyl) -2- (cyclohexylamino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (3-aminosulfonylphenyl) -isothiazolidine-3-carboxamide 1, 1-dioxide

Referring to step E in example 1, the reaction starting materials 2, 2-difluorocyclobutylisocyanide, 3-amino-5-fluoropyridine and (S) -isothiazolidine-3-carboxylic acid 1, 1-dioxide were replaced with cyclohexylisocyanide, 3-aminobenzenesulfonamide and (S) -2- (4-cyanopyridin-2-yl) isothiazolidine-3-carboxylic acid 1, 1-dioxide, respectively (prepared in step B of example 11), to give the title compound in 36% yield.

¹H-NMR(400MHz,CDCl₃):δ＝8.58(d,J＝5.0Hz,1H),8.14-6.84(m,12H),6.52(s,1H),5.53(m,1H),4.76(m,1H),3.88-3.78(m,1H),3.80-3.65(m,1H),3.39-3.36(m,1H),2.69-2.63(m,1H),2.50-2.42(m,1H),1.95-1.90(m,2H),1.74-1.59(m,4H),1.38-1.24(m,2H),1.17-0.94(m,2H)。

m/z＝671[M+H]⁺。

Example 27- ((S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -1, 1-dioxidoisothiazolidine-3-carboxamido) -5-fluoropyridin-1-oxide

(S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -N- (5-fluoropyridin-3-yl) -isothiazolidine-3-carboxamide 1, 1-dioxide (50mg, 0.081mmol, prepared in example 2) and m-chloroperoxybenzoic acid (28mg, 0.16mmol) were added to dichloromethane (10mL) and stirred at room temperature overnight. The solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel thin layer chromatography (EA) to give 3- ((S) -N- ((S) -1- (2-chlorophenyl) -2- ((3, 3-difluorocyclobutyl) amino) -2-oxoethyl) -2- (4-cyanopyridin-2-yl) -1, 1-dioxidoisothiazolidine-3-carboxamido) -5-fluoropyridine-1-oxide (22mg, yield 43%).

¹H-NMR(400MHz,CDCl₃):δ8.72(s,0.5H),8.13(s,0.5H),8.48-8.41(m,1H),7.94(s,1H),7.80-7.68(m,1H),7.41(d,J＝8.0Hz,1H),7.26-7.15(m,4H),7.05-6.95(m,1H),6.49(s,1H),6.01(s,0.5H),5.90(s,0.5H),4.86-4.82(m,1H),4.34(s,1H),3.78-3.68(m,1H),3.45-3.35(m,1H),3.10-3.00(m,2H),2.61-2.38(m,4H)。

m/z＝635[M+H]⁺。

Biological Activity assay

Enzyme assay:

resazurin is a conventional redox dye, which can be reduced from non-fluorescent blue Resazurin to pink resorufin, which can be measured and quantified by Relative Fluorescence Units (RFU) of a fluorescence photometer (Ex 530-570nm, Em 590-620 nm). At present, resazurin is widely used for activity determination of bacteria, cells and the like and enzyme activity detection of oxidoreductase. We determined the inhibitory activity of compounds against IDH1m by measuring the depletion of the cofactor NADPH, and against IDH WT by measuring the production of the cofactor NADPH. The compound is preincubated with IDH1m and NADPH, then the reaction is started by adding alpha-KG, and after a certain time of reaction under linear conditions, Diaphorase (lipoamide dehydrogenase) and a corresponding substrate Resazurin (Resazurin) are added for detection. Lipoamide dehydrogenase terminates the IDH1m reaction by depleting the available cofactor NADPH, which oxidizes NADPH to NADP, and reduces resazurin to highly fluorescent resorufin, the amount of cofactor NADPH remaining after a particular reaction time being quantified by a readily detectable fluorophore.

The compound is preincubated with IDH-WT and NADP, then the reaction is started by adding isocitrate, Diaphorase (lipoamide dehydrogenase) and the corresponding substrate Resazurin (Resazurin), and after a certain time of reaction under linear conditions, the amount of fluorescent substance is detected. This experiment reduced NADP to NADPH, which reduced Resazurin to highly fluorescent resorufin under the action of lipoamide dehydrogenase, and the amount of the cofactor NADPH generated after a certain reaction time was quantified by the detected fluorophore, thereby calculating the inhibitory effect of the compound on IDH-WT.

The specific operation mode is as follows: mu.L of 3 Xgradient diluted compounds were added to a 384-well plate, followed by addition of 5. mu.L of a reaction buffer (20mM Tris-HCl, pH 7.5; 150mM NaCl; 10mM MgCl) containing 40nM IDH1(R132H/R132C) and 20. mu.M NADPH₂(ii) a 0.4mg/mL BSA (bovine serum albumin) and 2mM DTT (dithiothreitol). The above test mixture was then incubated at 23 ℃ for 16 hours, after which the reaction was initiated by adding 2.5. mu.L of reaction buffer containing 4mM α -KG. After 60 minutes incubation at room temperature, 5. mu.L of a stop mixture in reaction buffer (0.4U/ml Diaphorase and 20. mu.M Resazurin) was added and the Resazurin was converted to resorufin to measure the remaining NADPH. After incubation at 23 ℃ for 10 minutes, fluorescence measurements were carried out by Flexstation 3 at Ex535/Em 595. The activity of the enzyme was measured at 12 concentrations for each compound, and the data was calculated using GraFit6.0 Software (Erithocus Software) to obtain the IC of the compound₅₀The value is obtained.

2-HG assay:

phosphoglycerate dehydrogenase PHGDH is capable of reducing NAD to NADPH in the presence of 2-HG, which can be quantitatively determined by lipoamide dehydrogenase and its substrate Resazurin (Resazurin).

HT-1080 cells are IDH1(R132C) mutated human fibrosarcoma cell line, U87 cells are IDH1(R132H) mutated human glioblastoma cell line, cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100units/mL penicillin, 0.1mg/mL streptomycin.

Cells were digested by pancreatin and treated at 5X 10⁵Was inoculated in 6-well plates and incubated overnight in an incubator at 37 ℃. The next day, test compound (final DMSO concentration of 0.1%) was added and incubation was continued for 24 hours. The culture solution of each sample was aspirated, centrifuged at 1000rpm for 10 minutes, and the supernatant was aspirated and the 2-HG content was measured. In addition, after the cells were washed with PBS (phosphate buffered saline), the cells were collected by trypsinization, and the 2-HG content in the cells was measured after one washing with PBS.

The intracellular 2-HG assay was as follows: the cells were resuspended in 300. mu.L of reaction buffer (40mM Tris-HCl, pH 8.5; 150mM NaCl) and disrupted by sonication. The resultant was centrifuged at 12,000rpm at 4 ℃ for 10 minutes to remove insoluble matter. 25 μ L of the supernatant was aspirated for protein concentration determination by BCA kit, and 200 μ L of the supernatant was transferred to a new set of centrifuge tubes, 4 μ L of 3M HCl was added to each tube, and the tubes were left at room temperature for 5 minutes and centrifuged at 12,000rpm at room temperature for 5 minutes. Pipette 100. mu.L of the supernatant into a 96-well "V" plate, add 3.6. mu.L of 2M Tris base (tromethamine) per well, and allow to stand at room temperature for 5 minutes. Centrifuge at 12,000rpm for 2 minutes. The pH paper detects a pH equal to about 8.0.

2-HG Standard Curve preparation: the 2-HG stock was diluted to 500. mu.M with reaction buffer, after which 200. mu.L was diluted in a two-fold gradient for a total of 10 points. The following operations are the same as above, and include the steps of acid treatment and alkali neutralization.

The above sample cell sample to be tested or the standard was diluted 5X, and then 5. mu.L of the diluted sample was added to a 384-well plate, 10. mu.L of the assay mixture (8. mu.M PHGDH (phosphoglycerate dehydrogenase); 0.5mM NAD; 0.1U/ml Diaphorase and 10. mu.M Resazurin) was added to each well, reacted at 23 ℃ for 60 minutes, and the fluorescence value was measured by Flexstation 3 under Ex535/Em 595.

The measured fluorescence values are calibrated with the protein concentration of the corresponding sample and compared.

The extracellular 2-HG assay was as follows: mu.L of each culture supernatant was added to 10. mu.L of 3M HCl per tube and left at room temperature for 5 minutes, and then 18. mu.L of 2M Tris base was added to each tube and left at room temperature for 5 minutes. Centrifuge at 12,000rpm for 2 minutes. The pH paper detects a pH equal to about 8.0. 2-HG Standard Curve preparation: the 2-HG stock was diluted to 500. mu.M with complete medium, after which 500. mu.L was taken for a two-fold gradient dilution for a total of 10 points. The following operations are the same as above, and include the steps of acid treatment and alkali neutralization. The above sample culture supernatant sample or standard to be tested was diluted 5X, and then 5. mu.L of the diluted sample was added to a 384-well plate, 10. mu.L of the assay mixture (8. mu.M PHGDH; 0.5mM NAD; 0.1U/mL Diaphorase and 10. mu.M Resazurin) was added to each well, reacted at 23 ℃ for 60 minutes, and the fluorescence value was measured by Flexstation 3 at Ex535/Em 595.

Selected compounds prepared as described above were analyzed according to the biological methods described herein, with the following results:

1. inhibitory Activity (IC) of Compounds on IDH1 mutant (R132H and R132C)₅₀) As shown in table 1.

TABLE 1

Note: - - -means not determined.

2. The results of the compound of example 2 inhibition of 2-HG in HT-1080 cells exhibiting the IDH1 mutation are shown in FIG. 1. The results of the inhibition of 2-HG extracellular to HT-1080 cells exhibiting the IDH1 mutation by the compound of example 2 are shown in FIG. 2.

3. The results of the inhibition of 2-HG by the compound of example 2 on U87 cells exhibiting the IDH1 mutation are shown in figure 3.

4. Inhibitory Activity of the example 2 Compounds on U87 cells exhibiting the IDH1 mutation (IC)₅₀) As shown in table 2 below.

TABLE 2

Compound (I)	U87/IDH1(R132H)IC₅₀(nM)
		AG-120	46
Example 2 Compounds	6.7

Pharmacokinetics experiment

Male SD rats are from Beijing Wittingle laboratory animal technology, Inc., the rats are grouped into 3 groups, and the suspension (5mg/kg) of the sample to be tested is respectively orally taken for single gavage. Animals were fasted overnight prior to the experiment, with the fasting time ranging from 10 hours prior to dosing to 4 hours post-dosing. Blood was collected at 0.25, 0.5, 1,2, 4, 6, 8 and 24 hours post-dose. After isoflurane anesthesia by using a small animal anesthesia machine, 0.3mL of whole blood is collected through an eyeground venous plexus, the whole blood is placed in a heparin anticoagulation tube, a sample is centrifuged at 4000rpm for 5min at 4 ℃, and the plasma is transferred to a centrifugal tube and stored at-80 ℃ until analysis. Samples in plasma were extracted using protein precipitation and the extracts were analyzed by LC-MS/MS, where HPLC conditions are flow rate: 0.4 mL/min; mobile phase A: water/formic acid (99.9/0.1, v/v); mobile phase B: acetonitrile/formic acid (99.9/0.1, v/v); sample introduction amount: 5 mu L of the solution; column temperature: room temperature; autosampler temperature: room temperature run time: 2.5 minutes.

The PK data for the compound of example 12 and AG-120 are shown in Table 3:

TABLE 3

According to PK data, the plasma exposure amount of the compound in the example 12 is far higher than that of AG-120 under the same oral dose, the half-life period reaches 10.7 hours, and the pharmacokinetic property of the compound is obviously better than that of AG-120.

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