Heterocyclic compounds as gls1 inhibitors

Abstract

The present application provides a heterocyclic compound as a GLS1 inhibitor, in particular, the present application provides a compound having the structure of formula (II) as shown below, or an optical isomer, a pharmaceutically acceptable salt, a prodrug, a deuterated derivative, a hydrate, a solvate thereof. The above-mentioned compound has GLS1 inhibitory activity and can be used as a therapeutic agent for a disease or disorder related to GLS1.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry; in particular, the present application relates to a novel class of tricyclic heteroaryl-containing derivatives, a method for synthesizing the same, and the use of the same as a GLS1 inhibitor in the preparation of a medicament for treating various diseases related to tumors and the like. BACKGROUND

[0002] The rapid growth of tumor cells requires not only energy, but also nucleic acids, fatty acids and proteins for the generation of new cells. Glutamine, as the most abundant amino acid in the human body, plays a crucial role in the growth and development of tumor cells. Therefore, many tumor cells are described as "addicted to glutamine" cells.

[0003] In the process of glutamine metabolism, one of the important enzymes is glutaminase, which is located in the inner membrane of mitochondria in cells, and can catalyze the reaction of generating glutamic acid from glutamine. Glutamic acid is converted to alpha-ketoglutarate by glutamate dehydrogenase, and enters the tricarboxylic acid cycle as a substrate to provide metabolic intermediates for the synthesis of macromolecules in tumor cells. Glutaminase in the human body can be divided into two types according to gene coding, one is called kidney-type glutaminase (GLS or GLS1); the other is called liver-type glutaminase (GLS2). GLS1 is richly expressed in various parts of the body such as the kidney, brain, intestine, liver, and lymphocytes, and is often highly expressed in tumor cells. Although the two types are highly similar in amino acid sequence, they come from different related genes, have different protein structures and kinetic characteristics, thus perform different functions, and involve different regulatory mechanisms.

[0004] Malignant transformation of cells is accompanied by a significant increase in nucleic acid and protein synthesis. The high rate of protein synthesis in rapidly growing tumor cells requires a constant supply of essential and non-essential amino acids, and glutamine, as the most abundant amino acid in the body, provides this enormous demand. Glutamine metabolism occurs in the mitochondria of the cell, so glutamine must be transported from outside the cell through the cell membrane into the cytoplasm and from the cytoplasm through the mitochondrial membrane into the mitochondria. Studies have shown that tumor cells transport glutamine through the cell membrane much faster than normal cells. Studies on Ehrlich ascites cancer cells have also shown that a special glutamine transport system present in the mitochondrial membrane of the cancer cells can transport glutamine into the mitochondria at a faster rate than normal cells. Because the activity of glutaminease is dependent on the concentration of inorganic phosphorus, and the concentration of inorganic phosphorus in the mitochondria of tumor cells is high, the activity of glutaminease is high. In fact, scientific studies have shown that the high activity of glutaminease is closely related to the rapid growth of tumor cells. Transfection of Ehrlich ascites cancer cells with antisense mRNA of glutaminease not only inhibits their growth but also changes their morphology. Cancer cells transfected with antisense mRNA are completely unable to produce tumors when inoculated into mice, and such mice are completely the same as healthy animals. These scientific findings fully demonstrate that the activity of glutaminease is closely related to the occurrence and development of cancer, and glutaminease has become a target gene that has attracted great attention in anti-cancer therapy.

[0005] The disease and disorder mediated by GLS1 activity or expression level is selected from the group consisting of B-cell lymphoma, monocyte leukemia, liver cancer, rectal cancer, bladder cancer, throat cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, lymphoma, gastric cancer, multiple myeloma and various solid tumors and hematological tumors, and diseases such as allergic asthma, myelofibrosis, rheumatoid arthritis, splenomegaly polycythemia, hypereosinophilic syndrome, primary thrombocytopenia, systemic giant cell disease.

[0006] Therefore, it is important and urgent to develop an effective inhibitor of glutaminease, especially an inhibitor of kidney-type glutaminease. SUMMARY

[0007] The purpose of the present application is to provide a new type of GLS1 inhibitor.

[0008] In a first aspect, the present application provides a compound having the structure shown in formula (II) below, or an optical isomer, a pharmaceutically acceptable salt, a prodrug, a deuterium derivative, a hydrate, or a solvate thereof:

[0009]

[0010] " " indicates a chiral center, which can be R or S;

[0011] R 2 selected from the group consisting of hydrogen, deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-4 haloalkoxy, C 2-6 alkenyl, C 2-6 haloalkenyl, C 2-6 alkynyl, C 2-6 haloalkynyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl;

[0012] R 3 selected from the group consisting of C 2-6 alkenyl, C 2-6 haloalkenyl, C 2-6 alkynyl, C 2-6 haloalkynyl, C 1-4 haloalkoxy C 2-4 alkenyl, C 1-4 haloalkoxy, C 1-4 haloalkenyl, C 1-4 haloalkynyl, C 1-4 haloalkoxy C 1-4 alkoxy, 3-8 membered cycloalkyl, 3-8 membered halocycloalkyl, 3-8 membered heterocyclyl, 3-8 membered cycloalkyl-O-, 3-8 membered halocycloalkyl-O-, 3-8 membered halocycloalkyl-NR i -, C 1-4 haloalkyl 3-8 membered cycloalkyl-O-, C 1-4 haloalkyl 3-8 membered cycloalkyl-NR i -, C 1-4 haloalkoxy 3-8 membered cycloalkyl-O-, C 1-4 haloalkoxy 3-8 membered cycloalkyl-NR i -, C 1-4 haloalkoxy C 1-4 alkyl 3-8 membered cycloalkyl-O-, C 1-4 haloalkoxy C 1-4 alkyl 3-8 membered cycloalkyl-NR i -, C 1-4 haloalkoxy C 1-4 alkoxy 3-8 membered cycloalkyl-O-, CyC=R 5 , -O-CyC=R 5 , -S-CyC=R 5 , or -NR i -CyC=R 5Wherein, CyC is a saturated 3-8 membered cycloalkyl group or a saturated 4-8 membered heterocyclic group, and R... 5 Selected from the following groups: O, CR f R g Among them, R f and R g Each element is independently selected from the following groups: hydrogen, deuterium, halogens, and carbon. 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Alkoxy C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, C 1-4 Halogenated alkoxy groups, C 1-4 Halogenated alkoxy C 1-4 Alkyl; R i Selected from hydrogen or C 1-4 Alkyl group; the CyC may optionally be alkylated by one or more R groups. n Replace; where R n Selected from hydrogen, halogen, or C 1-4 alkyl;

[0013] The prerequisite is that when R 3 Selected from C 2-6 alkenyl, 3-8 membered cycloalkyl, 3-8 membered halocycloalkyl, 3-8 membered heterocyclic, 3-8 membered cycloalkyl-O-, C 1-4 When the alkyl halooxy group is 3-8 membered cycloalkyl-O- or 3-8 membered cycloalkyl-O-, R 2 Selected from C 2-6 Haloalkenyl, C 2-6 alkynyl group, or C 2-6 Halogenated alkynyl group;

[0014] R k Selected from hydrogen, halogen, or C 1-4 alkyl.

[0015] In another preferred embodiment, equation (II) is equivalent to equation (III):

[0016]

[0017] R 2 Selected from the following group: C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkyne group, 3-8 membered cycloalkyl group;

[0018] R 3 Selected from the following group: C 2-6 alkynyl group, C 2-6 Halogenated alkynyl group, C 1-4 Halogenated alkoxy C 2-4alkynyl, 3-8 membered halocycloalkyl-NR i -, C 1-4 haloalkoxyC 1-4 alkoxy, C 1-4 haloalkyl3-8 membered cycloalkyl-O-, C 1-4 haloalkoxyC 1-4 alkyl3-8 membered cycloalkyl-O-, C 1-4 haloalkoxyC 1-4 alkoxy3-8 membered cycloalkyl-O-, CyC=R 5 , -O-CyC=R 5 , -S-CyC=R 5 , or -NR i -CyC=R 5 ; wherein said CyC is a saturated 3-8 membered cycloalkyl, or a saturated 4-8 membered heterocyclyl, and said R 5 is selected from the group consisting of O, CR f R g , wherein R f and R g are each independently selected from the group consisting of hydrogen, deuterium, halogen, C 1-4 alkyl, C 1-4 haloalkyl, C 1-4 alkoxy, C 1-4 alkoxyC 1-4 alkyl, hydroxyC 1-4 alkyl, C 1-4 haloalkoxy, C 1-4 haloalkoxyC 1-4 alkyl; R i is selected from hydrogen or C 1-4 alkyl; said CyC can be optionally substituted with one to more R n ; wherein R n is selected from hydrogen, halogen, or C 1-4 alkyl.

[0019] In another preferred embodiment, R 2 is selected from the group consisting of C 2-6 haloalkenyl, C 2-6 alkynyl, C 2-6 haloalkynyl.

[0020] In another preferred embodiment, R 3 is selected from the group consisting of C 2-6 alkynyl, C 2-6 haloalkynyl, C 1-4 haloalkoxyC 2-4 alkynyl, C 1-4 haloalkoxyC 1-4 alkoxy, C 1-4 haloalkyl3-8 membered cycloalkyl-O-, C1-4 haloalkyl, C 1-4 alkyl 3-8 membered cycloalkyl-O-, C 1-4 haloalkyl, C 1-4 alkoxy 3-8 membered cycloalkyl-O-, CyC=R 5 , or -O-CyC=R 5 ; wherein said CyC is saturated 3-8 membered cycloalkyl, or saturated 4-8 membered heterocyclyl, and said R 5 is selected from the group consisting of O, CR f R g ; wherein R f and R g are each independently selected from the group consisting of hydrogen, deuterium, halogen, C 1-4 alkyl, C 1-4 haloalkyl; said CyC can be optionally substituted with one to more R n ; wherein R n is selected from hydrogen, halogen, or C 1-4 alkyl.

[0021] In another preferred embodiment, formula (II) is formula (IV):

[0022]

[0023] " " indicates a chiral center;

[0024] R 2 is selected from the group consisting of hydrogen, deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 haloalkenyl, C 2-6 alkynyl, C 2-6 haloalkynyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl;

[0025] Y is selected from a bond, O, S, or NR i ; wherein R i is selected from hydrogen or C 1-4 alkyl;

[0026] U is selected from N or CR m ; wherein R m is selected from selected from hydrogen, halogen, or C 1-4 alkyl;

[0027] R f and R g are each independently selected from the group consisting of hydrogen, deuterium, halogen, C 1-4 alkyl, C 1-4 haloalkyl, C 1-4 alkoxy, C 1-4alkoxy C 1-4 alkyl, hydroxy C 1-4 alkyl, C 1-4 haloalkoxy, or C 1-4 haloalkoxy C 1-4 alkyl;

[0028] each R n are each independently selected from the group consisting of hydrogen, halogen, or C 1-4 alkyl;

[0029] R k is selected from the group consisting of hydrogen, halogen, or C 1-4 alkyl;

[0030] c and d are each independently selected from 1, 2, 3, 4, 5, or 6;

[0031] e is selected from 0, 1, 2, 3, or 4.

[0032] In another preferred embodiment, formula (II) is formula (V):

[0033]

[0034] “*” denotes a chiral center;

[0035] R 2 is selected from the group consisting of hydrogen, deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 haloalkenyl, C 2-6 alkynyl, C 2-6 haloalkynyl, 3-8 membered cycloalkyl;

[0036] R f and R g are each independently selected from the group consisting of hydrogen, deuterium, fluorine, C 1-4 alkyl;

[0037] R k is selected from the group consisting of hydrogen, halogen, or C 1-4 alkyl;

[0038] c and d are each independently selected from 1, 2, or 3.

[0039] In another preferred embodiment, formula (II) is formula (VI):

[0040]

[0041] R 2 is selected from the group consisting of C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkynyl, 3-8 membered cycloalkyl.

[0042] In another preferred embodiment, Formula (II) is Formula (VII):

[0043]

[0044] R 2 selected from the group consisting of C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkynyl, 3-8 membered cycloalkyl;

[0045] R x selected from the group consisting of C 1-4 haloalkyl, C 1-4 haloalkoxy C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl;

[0046] f and g are each independently selected from 0, 1, 2, 3, or 4; provided that f and g cannot both be 0. In another preferred embodiment, the fragment is selected from the group consisting of the following structures:

[0047]

[0048] “*” indicates a chiral center.

[0049] In another preferred embodiment, the compound of Formula (II) is selected from the group consisting of:

[0050]

[0051]

[0052] In a second aspect, the present application provides a compound having the following formula:

[0053]

[0054] In a third aspect, the present application provides a pharmaceutical composition comprising, as an active ingredient, a compound according to the first or second aspect of the application, or an optical isomer, a pharmaceutically acceptable salt, a prodrug, a deuterated derivative, a hydrate, a solvate thereof, and a pharmaceutically acceptable carrier.

[0055] In a third aspect, the present application provides use of a compound of the first aspect of the present application, or an optical isomer, a pharmaceutically acceptable salt, a prodrug, a deuterium derivative, a hydrate, a solvate thereof, for the manufacture of a pharmaceutical composition for treating a disease or disorder mediated by GLS1 activity or expression.

[0056] In another preferred embodiment, the disease or disorder mediated by GLS1 activity or expression is selected from the group consisting of B-cell lymphoma, monocyte leukemia, liver cancer, rectal cancer, bladder cancer, throat cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, lymphoma, stomach cancer, multiple myeloma and various solid and hematological tumors, and diseases such as allergic asthma, myelofibrosis, rheumatoid arthritis, splenomegaly polycythemia, hypereosinophilic syndrome, idiopathic thrombocytopenia, systemic giant cell disease, and the like.

[0057] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. DETAILED DESCRIPTION

[0058] The present inventors have made a long-term and in-depth study, and unexpectedly found a class of GLS1 inhibitors containing tricyclic aryl compounds, as well as their preparation methods and applications. The compounds of the present application can be applied to the treatment of various diseases related to the activity of the kinase. Based on the above findings, the inventors completed the present application.

[0059] TERMS

[0060] Unless otherwise indicated, the term "or" as used herein is used to mean "and / or" (i.e., "one or the other or both").

[0061] Unless otherwise indicated, each chiral carbon atom (chiral center) in all of the compounds of the present application can optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

[0062] As used herein, the term "alkyl," alone or in part of other substituents, refers to straight-chain (i.e., unbranched) or branched saturated hydrocarbon groups, or a combination of straight-chain and branched groups. When preceded by a number indicating the number of carbon atoms (e.g., C 1-10 ), it means that the alkyl group contains from 1 to 10 carbon atoms. For example, C 1-8 alkyl refers to an alkyl group containing from 1 to 8 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, or the like.

[0063] As used herein, the term "alkenyl," alone or in combination with other substituents, refers to a straight-chain or branched-chain, carbon-linked group having at least one carbon-carbon double bond. The alkenyl group can be substituted or unsubstituted. When an alkenyl group is preceded by a carbon atom number limitation (e.g., C 2-8 alkenyl), it is intended that the alkenyl group contain 2-8 carbon atoms. For example, the term "C 2-8 alkenyl" refers to a straight-chain or branched-chain alkenyl group having 2-8 carbon atoms, including ethenyl, propenyl, 1,2-butenyl, 2,3-butenyl, butenyl, or the like.

[0064] As used herein, the term "alkynyl," alone or in combination with other substituents, refers to an aliphatic carbon-hydrogen group having at least one carbon-carbon triple bond. The alkynyl group can be straight-chain or branched, or a combination thereof. When an alkynyl group is preceded by a carbon atom number limitation (e.g., C 2-8 alkynyl), it is intended that the alkynyl group contain 2-8 carbon atoms. For example, the term "C 2-8 alkynyl" refers to a straight-chain or branched-chain alkynyl group having 2-8 carbon atoms, including ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, sec-butynyl, t-butynyl, or the like.

[0065] As used herein, the term "cycloalkyl," alone or in combination with other substituents, refers to a monocyclic, bicyclic, or polycyclic (fused, bridged, or spiro) ring system group having saturated or partially saturated units. When a cycloalkyl group is preceded by a carbon atom number limitation (e.g., C 3-10 ), it is intended that the cycloalkyl group contain 3-10 carbon atoms. In some preferred embodiments, the term "C 3-8 cycloalkyl" refers to a saturated or partially unsaturated monocyclic or bicyclic alkyl group having 3-8 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or the like. "Spiro cycloalkyl" refers to a bicyclic or polycyclic group in which the single rings share one carbon atom (termed a spiro atom), which can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. "Fused cycloalkyl" refers to a fully carbon bicyclic or polycyclic group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, in which one or more rings can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. "Bridged cycloalkyl" refers to a fully carbon polycyclic group in which any two rings share two non-adjacent carbon atoms, which can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. The cycloalkyl group contains only carbon atoms. Some examples of cycloalkyl groups are as follows, although the application is not limited to only the cycloalkyl groups described below.

[0066]

[0067] Unless otherwise indicated, the following terms used in the specification and claims have the following meanings. "Aryl" means a completely carbon monocyclic or fused polycyclic (that is, rings which share adjacent pairs of carbon atoms) ring system having a conjugated pi-electron system, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic (including saturated and unsaturated rings) rings, but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent molecule must be on a carbon atom of the ring having the conjugated pi-electron system. The aryl group can be substituted or unsubstituted. Some examples of aryl groups are listed below, and the application is not limited to only the aryl groups listed below.

[0068]

[0069] "Heteroaryl" means a monocyclic or polycyclic ring system having aromaticity, containing one to several heteroatoms (optionally selected from nitrogen, oxygen, and sulfur), or a polycyclic ring system formed by the fusion of a heterocyclyl ring (containing one to several heteroatoms optionally selected from nitrogen, oxygen, and sulfur) with an aryl ring, with the point of attachment being on the aryl ring. The heteroaryl group can be optionally substituted or unsubstituted. Some examples of heteroaryl groups are listed below, and the application is not limited to only the heteroaryl groups listed below.

[0070]

[0071] "Heterocyclyl" means a saturated or partially unsaturated monocyclic or polycyclic ring system in which one or more of the ring atoms is selected from nitrogen, oxygen, or sulfur, with the remaining ring atoms being carbon. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl. Polycyclic heterocyclyl means a heterocyclyl group that includes spiro, fused, and bridged rings. "Spiro heterocyclyl" means a polycyclic heterocyclyl group in which each ring in the system shares one atom (called a spiro atom) with every other ring in the system, in which one or more of the ring atoms is selected from nitrogen, oxygen, or sulfur, with the remaining ring atoms being carbon. "Fused heterocyclyl" means a polycyclic heterocyclyl group in which each ring in the system shares an adjacent pair of atoms with every other ring in the system, one or more of the rings can contain one or more double bonds, but no ring has a fully conjugated pi-electron system, and in which one or more of the ring atoms is selected from nitrogen, oxygen, or sulfur, with the remaining ring atoms being carbon. "Bridged heterocyclyl" means a polycyclic heterocyclyl group in which any two rings share two non-adjacent atoms, which can contain one or more double bonds, but no ring has a fully conjugated pi-electron system, and in which one or more of the ring atoms is selected from nitrogen, oxygen, or sulfur, with the remaining ring atoms being carbon. If both a saturated ring and an aromatic ring are present in a heterocyclyl group (for example, a saturated ring and an aromatic ring are fused together), the point of attachment to the parent molecule must be on the saturated ring. Note: when the point of attachment to the parent molecule is on an aromatic ring, it is referred to as a heteroaryl group, not a heterocyclyl group. Some examples of heterocyclyl groups are listed below, and the application is not limited to only the heterocyclyl groups listed below.

[0072]

[0073] As used herein, the term "halogen" or "halo," alone or in combination with other substituents, refers to F, CI, Br, and I.

[0074] As used herein, the term "substituted" (with or without the modifier "arbitrarily") means that one or more hydrogen atoms on a particular group are replaced with a particular substituent. The particular substituents are those described in the foregoing, or the substituents that appear in each embodiment. Unless otherwise specifically stated, an arbitrarily substituted group can have one substituent selected from a particular group at any substitutable position on the group, which substituents can be the same or different at each position. A cyclic substituent, such as a heterocyclyl group, can be attached to another ring, such as a cycloalkyl group, to form a spiro bicyclic ring system, i.e., two rings that share a common carbon atom. It will be understood by those skilled in the art that combinations of substituents contemplated by the present application are those combinations that are stable or chemically feasible. Such substituents include, but are not limited to: C 1-8 alkyl, C 2-8 alkenyl, C 2-8 alkynyl, C 3-8 cycloalkyl, 3- to 12-membered heterocyclyl, aryl, heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 formyl, C 2-10 acyl, C 2-10 ester, amino.

[0075] For the sake of convenience and in keeping with common understanding, the term "arbitrarily substituted" or "optionally substituted" applies only to positions that can be substituted with a substituent, and does not include those substitutions that are chemically infeasible.

[0076] As used herein, unless otherwise specifically indicated, the term "pharmaceutically acceptable salt" means a salt that is suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, side effects, or interact deleteriously with the tissues. In some embodiments, a pharmaceutically acceptable salt of a compound of the present application includes a salt of a compound of the present application that has an acidic group (e.g., potassium salt, sodium salt, magnesium salt, calcium salt) or a salt of a compound of the present application that has a basic group (e.g., sulfate salt, hydrochloride salt, phosphate salt, nitrate salt, carbonate salt).

[0077] Use:

[0078] The present application provides a use of a compound of Formula (II), or a deuterated derivative thereof, a salt, isomer (enantiomer or diastereomer, if present), hydrate, pharmaceutically acceptable carrier or excipient thereof for inhibiting GLS1.

[0079] The compounds of the present application are useful as a GLS1 inhibitor.

[0080] The expression or activity of the above-mentioned various protein kinases is significantly increased in cancer patients. These overexpressed and / or abnormal protein kinase activity levels are directly related to the occurrence and development of tumors. The compounds of the present application are single and / or dual inhibitors of these protein kinases. By regulating the activity of these protein kinases, diseases are prevented, alleviated or cured. The diseases include B-cell lymphoma, monocyte leukemia, liver cancer, rectal cancer, bladder cancer, throat cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, lymphoma, stomach cancer, multiple myeloma and other solid tumors and hematological tumors, as well as allergic asthma, myelofibrosis, rheumatoid arthritis, splenomegaly polycythemia, hypereosinophilic syndrome, primary thrombocytopenia, systemic giant cell disease and other diseases.

[0081] In some aspects, dual protein kinase inhibitors interfere with two different kinases simultaneously, and the resulting anti-tumor effect is often additive, thus having the potential to more effectively treat various cancers.

[0082] The compounds of the present application can be used as combination drugs with biological agents such as PD-1 inhibitors (e.g. and ) to treat various cancers and related diseases.

[0083] The compounds of the present application, and their deuterated derivatives, pharmaceutically acceptable salts, or isomers thereof, where present, or hydrates and / or compositions thereof, can be formulated with a pharmaceutically acceptable excipient or carrier, and the resulting composition can be administered in vivo to mammals, such as men, women and animals, for the treatment, prevention and alleviation of conditions, symptoms and diseases. The composition can be in the form of tablets, pills, suspensions, solutions, emulsions, capsules, aerosols, sterile injection fluids, sterile powders, and the like. In some embodiments, the pharmaceutically acceptable excipient includes microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate, mannitol, hydroxypropyl-β-cyclodextrin, β-cyclodextrin (increased), glycine, disintegrants (such as starch, croscarmellose sodium, complex silicates, and high molecular weight polyethylene glycols), granulating binders (such as polyvinylpyrrolidone, sucrose, gelatin, and acacia), and lubricating agents (such as magnesium stearate, glycerol, and talc). In preferred embodiments, the pharmaceutical composition is in a dosage form suitable for oral administration, including but not limited to tablets, solutions, suspensions, capsules, granules, and powders. The amount of the compound of the present application or the pharmaceutical composition administered to a patient is not fixed and is generally administered in a pharmaceutically effective amount. Meanwhile, the amount of the compound actually administered can be determined by a physician depending on the actual situation, including the condition to be treated, the selected administration route, the actual compound to be administered, the individual condition of the patient, and the like. The dosage of the compound of the present application depends on the specific use, the administration method, the patient's condition, and the physician's judgment. The proportion or concentration of the compound of the present application in the pharmaceutical composition depends on various factors, including the dosage, the physicochemical properties, the administration route, and the like.

[0084] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described below (e.g., in the Examples) can be combined with each other to constitute a new or preferred technical solution.

[0085] Pharmaceutical compositions and methods of administration

[0086] Since the compounds of the present application have excellent inhibitory activity against a series of protein kinases, the compounds of the present application and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present application as the main active ingredient can be used for the treatment, prevention, and alleviation of diseases associated with GLS1 activity or expression amount.

[0087] The pharmaceutical composition of the present application contains a safe and effective amount of the compound of the present application or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Among them, "safe and effective amount" refers to the amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present application per dose, more preferably 5-200 mg of the compound of the present application per dose. Preferably, the "dose" is a capsule or a tablet.

[0088] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler substances or gel materials, which are suitable for human use and which must have sufficient purity and low toxicity. "Compatible" in this context means that the components of the composition are capable of being commingled with the compounds of the application, and with each other, without any component adversely affecting the others to an appreciable extent. Examples of some of the components of a pharmaceutically acceptable carrier are celluloses and their derivatives (e.g., sodium carboxymethylcellulose, sodium croscarmellose, ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween 80®, Span 80®, lecithin, etc.), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0089] The mode of administration of the compounds or pharmaceutical compositions of the present application is not narrowly critical, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0090] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such other ingredients as binders, (a) fillers or extenders, e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) absorbents, e.g., kaolin and bentonite clay; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms also can contain buffering agents.

[0091] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings and shells known in the art. They can optionally contain opacifying agents, and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0092] ​Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, or elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, or mixtures thereof.

[0093] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0094] Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar-agar, and the like.

[0095] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyol, and suitable mixtures thereof.

[0096] Dosage forms of the compounds of the present application for topical administration include ointments, powders, sprays, and inhalers. The active compound is admixed under sterile conditions with physiologically acceptable carriers and any preservatives, buffers, or propellants as can be required.

[0097] The compounds of the present application can be administered alone, or in combination with other pharmaceutically acceptable compounds.

[0098] Pharmaceutical compositions are used in a safe and effective amount, which is an amount of the compounds of the present application suitable to treat a mammal (e.g., a human) in need of such treatment, wherein the administration is at a dosage that is pharmaceutically considered an effective amount of administration, typically 1-2000 mg, preferably 5-500 mg, per day for a 60 kg body weight human. Of course, the specific dose will also take into account the route of administration, the patient's health status, and the like, which are all within the skill of a trained physician.

[0099] The main advantages of the present application include:

[0100] 1. A compound of Formula II is provided.

[0101] 2. A novel GLS1 inhibitor is provided, as well as its preparation and use, which inhibits the activity of the above-mentioned protein kinase at very low concentrations.

[0102] 3. A pharmaceutical composition for treating a disease associated with GLS1 activity is provided.

[0103] 4. A GLS1 inhibitor with good oral absorption is provided.

[0104] The application is further described in conjunction with the following specific examples. It should be understood that these examples are intended to illustrate the application and are not intended to limit the scope of the application. The experimental procedures in the following examples, unless otherwise indicated, were carried out under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

[0105] Example 1: Preparation of Compound 1

[0106]

[0107] Compound 1a (465 mg, 4.39 mmol) and sodium carbonate (558 mg, 5.27 mmol) were dissolved in acetonitrile (15 mL) and stirred at 70 °C for 1 h. Then compound 1b (983 mg, 4.39 mmol) diluted in acetonitrile (5 mL) was slowly added to the reaction mixture. The reaction mixture was stirred at 70 °C overnight. The reaction was monitored by TLC. After the reaction mixture was cooled, it was concentrated under reduced pressure. The resulting crude was dissolved in dichloromethane and extracted with dichloromethane (3 x 20 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (dichloromethane:methanol = 15:1) to give compound 1c (448 mg, yield 48%) as a colorless oil. 1 H NMR (500 MHz, CDC13) δ 5.09-4.91 (m, 1H), 3.25 (d, J = 5.5 Hz, 2H), 2.83-2.76 (m, 4H), 2.46-2.36 (m, 2H), 2.00 (t, J = 2.6 Hz, 1H), 1.45 (s, 9H).

[0108] Compound 1d (319 mg, 2.11 mmol) was dissolved in methyl tert-butyl ether (15 mL) at room temperature, and then compound 1c (448 mg, 2.11 mmol) and diisopropylethylamine (1.09 g, 8.44 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. After the reaction was completed as monitored by TLC, the reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 5:1) to give compound 1e (335 mg, yield 49%) as a red solid. 1H NMR (500 MHz, CDC13) δ 4.75 (s, 1H), 4.00-3.90 (m, 4H), 3.46 (d, J = 5.9 Hz, 2H), 2.66-2.60 (m, 2H), 2.00 (s, 1H), 1.39 (s, 9H).

[0109] A mixture of compound 1e (335 mg, 1.03 mmol) and 4-dimethylaminopyridine (25 mg, 0.21 mmol) in tetrahydrofuran (8 mL) was stirred at 60 °C for 2 days. After TLC monitoring the reaction was complete, the reaction mixture was concentrated under reduced pressure. The obtained crude was purified by column chromatography on silica gel (dichloromethane:methanol = 20:1) to give compound 1f (297 mg, yield 97%) as a brown solid. 1 H NMR (500 MHz, CDC13) δ 4.75 (s, 1H), 4.00-3.90 (m, 4H), 3.46 (d, J = 5.9 Hz, 2H), 2.66-2.60 (m, 2H), 2.00 (s, 1H), 1.39 (s, 9H). + .

[0110] To methanol (15 mL) was added compound 1f (235 mg, 0.79 mmol) and palladium on carbon catalyst (10%, 80 mg) at room temperature, the reaction mixture was stirred at room temperature under 1 atmosphere of hydrogen atmosphere for 1 hour. TLC monitoring the reaction was complete. The reaction mixture was filtered through celite, the filtrate was concentrated under reduced pressure to give the crude. The crude was purified by column chromatography on silica gel (dichloromethane:methanol = 20:1) to give the crude compound 1g (222 mg) as a brown solid which was used directly for the next reaction. MS m / z 265.3 [M+H] + .

[0111] Compound 1g (222 mg, 0.84 mmol) was dissolved in 1,4-dioxane (4 mL), then HCl dioxane solution (4 M, 1 mL) was added, the reaction mixture was stirred at room temperature overnight. TLC monitoring the reaction was complete. The reaction mixture was concentrated under reduced pressure. The obtained mixture was dissolved in water, neutralized with saturated sodium bicarbonate solution, extracted with dichloromethane (3 x 10 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (dichloromethane:methanol = 6:1, 2% ammonia water) to give compound 1h (122 mg, yield 94% for two steps) as a yellow solid. MS m / z 165.2 [M+H] + .

[0112] Compound 1h (20 mg, 0.12 mmol) and compound 1i (22 mg, 0.12 mmol) were dissolved in acetonitrile (6 mL), then diisopropylethylamine (24 mg, 0.18 mmol) was added, and the reaction mixture was stirred at 80 °C for 2 h. The reaction was monitored by TLC. After the reaction mixture was cooled to room temperature, it was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1, 2% ammonia water) to give compound 1j (20 mg, yield 62%) as yellow oil. MS m / z 264.4 [M+H] + .

[0113] Compound 1j (20 mg, 0.07 mmol), compound 1k (13 mg, 0.07 mmol), 1H- benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (59 mg, 0.11 mmol), and diisopropylethylamine (15 mg, 0.11 mmol) were mixed in N,N- dimethylformamide (2 mL) and stirred at room temperature for 4 h. After the reaction was completed as monitored by TLC, the reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 15:1, 2% ammonia water) to give compound 1 (9 mg, yield 29%) as white solid. 1 H NMR (500 MHz, CD3OD) δ 8.16 (d, J = 4.5 Hz, 1H), 7.49-7.43 (m, 2H), 7.41-7.33 (m, 3H), 7.06 (d, J = 4.5 Hz, 1H), 4.90 (s, 1H), 3.70-3.62 (m, 6H), 3.42 (s, 3H), 3.06 (t, J = 7.6 Hz, 2H). MS m / z 412.5 [M+H] + .

[0114] Example 2: Preparation of compound 2

[0115]

[0116] Compound 1f (60 mg, 0.20 mmol) was dissolved in methanol (4 mL), then HCl dioxane solution (4 M, 1 mL) was added, and the reaction mixture was stirred at 40 °C for 2 h. The reaction was monitored by TLC. After the reaction mixture was cooled to room temperature, it was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1, 2% ammonia water) to give crude compound 2a (50 mg) as yellow oil, which was directly used in the next reaction. MS m / z 199.2 [M+H] + .

[0117] Compound 2a (50 mg, 0.25 mmol) and compound 1i (45 mg, 0.25 mmol) were dissolved in acetonitrile (3 mL), then diisopropylethylamine (49 mg, 0.38 mmol) was added, and the reaction mixture was stirred at 80 °C for 2 h. The reaction was monitored by TLC. After the reaction mixture was cooled to room temperature, it was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1, 2% ammonia water) to give compound 2b (58 mg, 97% yield for two steps) as an off-white solid. MS m / z 298.4 [M+H] + .

[0118] A mixture of compound 2b (58 mg, 0.19 mmol), compound 1k (32 mg, 0.19 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (152 mg, 0.29 mmol), and diisopropylethylamine (38 mg, 0.29 mmol) in N,N-dimethylformamide (2 mL) was stirred at room temperature for 1 h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 25:1, 2% ammonia water) to give compound 2 (38 mg, 44% yield) as a white solid. 1 H NMR (500 MHz, CD3OD) δ 7.50-7.42 (m, 2H), 7.40-7.32 (m, 3H), 7.05 (s, 1H), 4.89 (s, 1H), 3.72 (t, J = 8.1 Hz, 2H), 3.67-3.56 (m, 4H), 3.41 (s, 3H), 3.05 (t, J = 8.1 Hz, 2H). MS m / z 446.4 [M+H] + .

[0119] Example 3: Preparation of compound 3

[0120]

[0121] Compound 3a (3.0 g, 16.12 mmol) was dissolved in THF (50 mL) and cooled to -78 °C. LDA (2 M in THF, 16 mL) was added under nitrogen atmosphere. The reaction mixture was stirred at -78 °C for 0.5 h, then dimethyl carbonate (1.45 g, 16.12 mmol) was added and stirred for 5 min before being allowed to warm to room temperature and stirred for 2 h. The reaction was checked by TLC. The reaction was quenched with saturated aqueous ammonium chloride solution (100 mL) in an ice bath and extracted with ethyl acetate (3 x 100 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The obtained crude was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) to give compound 3b (1.50 g, 38%) as white solid. MS m / z 244.2, 246.2 [M+H] + .

[0122] Compound 3b (1.50 g, 6.15 mmol) was dissolved in DMF (20 mL) and triethylsilyl acetylene (862 mg, 6.15 mmol), Pd(PPh3)2Cl2(431 mg, 0.61 mmol), cuprous iodide (234 mg, 1.23 mmol) and DIPEA (2.38 g, 18.45 mmol) were added. The reaction mixture was heated and stirred at 90 °C under nitrogen atmosphere for 2 h. The reaction was checked by TLC. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) to give compound 3c (1.20 g, 64%). MS m / z 304.5 [M+H] + .

[0123] Compound 3c (1.20 g, 3.95 mmol) was dissolved in methanol (20 mL) and ammonium hydroxide solution (7 M, 20 mL) was added and stirred at room temperature for 24 h. The reaction was checked by TLC. The reaction was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane: methanol = 20: 1) to give compound 3d (612 mg, 89%). 1 H NMR (500 MHz, CDC13) δ 7.39 (s, 1H), 7.21 (s, 1H), 7.19 (s, 1H), 5.44 (s, 1H), 3.75 (s, 2H), 3.32 (s, 1H), 2.59 (s, 3H). MS m / z 175.2 [M+H] + .

[0124] Cuprous iodide (44 mg, 0.23 mmol), potassium carbonate (317 mg, 2.30 mmol), phenanthroline (83 mg, 0.46 mmol), 3,3-dimethyl-l-(trifluoromethyl)-l,2-benziodoxol-5(2H)-one (568 mg, 1.72 mmol) were dissolved in anhydrous dichloromethane (20 mL) under nitrogen atmosphere. Compound 3d (200 mg, 1.15 mmol) in dichloromethane (20 mL) was added dropwise slowly, after the addition, the reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 3e (85 mg, 31%). MS m / z 243.2 [M+H] + .

[0125] Compound 3f (15 mg, 0.04 mmol, prepared according to the method reported in patent CN201580037629), compound 3e (13 mg, 0.06 mmol), copper powder (3 mg, 0.05 mmol), potassium carbonate (11 mg, 0.08 mmol), N,N'-dimethylethylenediamine (4 mg, 0.05 mmol) were dissolved in toluene (5 mL). The reaction mixture was heated and stirred at 135 °C under nitrogen atmosphere for 1 hour. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature, poured into ice water, extracted with ethyl acetate (3 x 20 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was further purified by preparative thin layer plate (dichloromethane:methanol = 20:1) to give compound 3 (2 mg, 10%) as white solid. 1 H NMR (500 MHz, CDC13) δ 10.75 (s, 1H), 8.39 (d, J = 9.1 Hz, 1H), 8.16 (d, J = 0.9 Hz, 1H), 7.32 (d, J = 9.1 Hz, 1H), 7.28 (s, 1H), 7.25 (s, 1H), 7.13-7.10 (m, 1H), 5.01-4.87 (m, 1H), 4.75-4.65 (m, 1H), 4.64-4.54 (m, 1H), 3.92 (s, 2H), 3.16-3.05 (m, 2H), 3.01 (d, J = 5.0 Hz, 3H), 2.70 (s, 3H), 2.29-2.19 (m, 1H), 2.15-2.06 (m, 1H). MS m / z 519.4 [M+H] + .

[0126] Example 4: Preparation of compound 4

[0127]

[0128] Compound 3d (54 mg, 0.29 mmol), 2-iodo-1,1,1-trifluoroethane (240 mg, 1.14 mmol), Pd2(dba)3(27 mg, 0.03 mmol), triethylenediamine (128 mg, 1.14 mmol), bis(2-diphenylphosphinophenyl)ether (65 mg, 0.12 mmol) were dissolved in toluene (5 mL). The reaction mixture was heated and stirred at 80 °C under nitrogen atmosphere for 4 hours. LCMS showed the reaction was completed. The reaction mixture was cooled to room temperature, filtered through celite, and the filtrate was distilled under reduced pressure. The obtained crude product was purified by silica gel column chromatography (ethyl acetate) to give compound 4a (40 mg, 52%). 1 H NMR (500 MHz, CDC13) δ 7.41 (s, 1H), 7.20 (s, 1H), 7.17 (s, 1H), 5.39 (s, 1H), 3.76 (s, 2H), 3.31 (q, J = 9.4 Hz, 2H), 2.60 (s, 3H). MS m / z 257.3 [M+H] + .

[0129] Compound 4a (30 mg, 0.12 mmol), compound 4b (50 mg, 0.08 mmol, prepared according to the method reported in patent CN201580037629), cuprous iodide (15 mg, 0.08 mmol), potassium phosphate (33 mg, 0.16 mmol), N,N'-dimethylethylenediamine (14 mg, 0.16 mmol) were dissolved in toluene (3 mL). The reaction mixture was heated and stirred at 135 °C under nitrogen atmosphere for 1 hour. TLC showed the reaction was completed. The reaction mixture was cooled to room temperature, poured into ice water, and then extracted with ethyl acetate (3 x 20 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was further purified by preparative thin layer plate (dichloromethane:methanol = 20:1) to give compound 4c (15 mg, 25%) as a white solid. MS m / z 776.8 [M+H] + .

[0130] Compound 4c (18 mg, 0.02 mmol) was dissolved in dichloromethane (5 mL), and then hydrochloric acid dioxane (4 M, 1 mL) was added. The reaction mixture was heated and stirred at 50 °C for 4 hours. LCMS showed the reaction was completed. The reaction mixture was concentrated under reduced pressure to give compound 4d crude (10 mg) which was directly used in the next step. MS m / z 520.4 [M+H] + .

[0131] Compound 4d (10 mg, 0.02 mmol) was dissolved in DMF (2 mL), then methylamine hydrochloride (3 mg, 0.04 mmol), diisopropylethylamine (8 mg, 0.06 mmol), HATU (12 mg, 0.03 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The reaction was monitored by LCMS. The reaction mixture was poured into ice water, extracted with dichloromethane (3 x 20 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The obtained crude was purified by preparative thin layer plate (dichloromethane:methanol = 20:1) to give compound 4 (2 mg, 19%) as a yellow solid. 1 H NMR (500 MHz, CDC13) δ 11.01 (s, 1H), 8.39 (d, J = 9.1 Hz, 1H), 8.16 (d, J = 0.7 Hz, 1H), 7.31 (d, J = 9.2 Hz, 1H), 7.16 (s, 1H), 7.13 (s, 1H), 7.12-7.09 (m, 1H), 5.02-4.87 (m, 1H), 4.75-4.65 (m, 1H), 4.63-4.54 (m, 1H), 3.88 (s, 2H), 3.33-3.26 (m, 2H), 3.15-3.06 (m, 2H), 3.01 (d, J = 5.0 Hz, 3H), 2.65 (s, 3H), 2.31-2.20 (m, 1H), 2.14-2.06 (m, 1H). MS m / z 533.5 [M+H] + .

[0132] Example 5: Preparation of compound 5

[0133]

[0134] Sodium hydride (186 mg, 4.47 mmol) was dissolved in tetrahydrofuran (15 mL), 3,3-difluorocyclobutanol (483 mg, 4.47 mmol) was added portionwise at 0 °C and stirred at 0 °C for 0.25 hours, then compound 5a (1 g, 3.57 mmol) was added to the reaction solution. The reaction mixture was stirred at 25 °C for 4 hours. The reaction was monitored by TLC. Ice water (20 mL) was added to the reaction bottle, extracted with ethyl acetate (3 x 10 mL), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1) to give compound 5b (1.24 g, 100%) as a white solid. 1H NMR (500 MHz, CDC13) δ 6.89 (s, 2H), 4.71 - 4.64 (m, 1H), 3.20 - 3.11 (m, 2H), 2.84 - 2.73 (m, 2H).

[0135] Compound 5b (1.24 g, 3.62 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL) under nitrogen atmosphere, and triisopropylsilyl acetylene (659.4 mg, 3.62 mmol), Pd(PPh3)2Cl2(254.1 mg, 0.362 mmol), cuprous iodide (69 mg, 0.362 mmol) and diisopropylethylamine (5 mL) were added successively. The reaction mixture was stirred at 25 °C overnight. TLC monitoring showed that the reaction was completed, water (30 mL) and ethyl acetate (20 mL*3) were added to extract, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1-20:1) to obtain white solid compound 5c (1.1 g, 68%). 1 H NMR (500 MHz, CDC13) δ 6.86 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 4.73 - 4.65 (m, 1H), 3.19 - 3.09 (m, 2H), 2.83 - 2.71 (m, 2H), 1.19 - 1.06 (m, 21H).

[0136] Compound 5c (1.1 g, 2.47 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere, and zinc bromide ethyl acetate (15 mL, 7.43 mmol), Pd2(dba)3(142 mg, 0.247 mmol), Xphos (117.57 mg, 0.247 mmol) were added successively. The reaction mixture was stirred at 55 °C for 2 hours. TLC monitoring showed that the reaction was completed, and after the reaction mixture was cooled, saturated aqueous ammonium chloride solution (10 mL) and water (10 mL) were added, and extracted with ethyl acetate (3 x 10 mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1-10:1) to obtain yellow solid compound 5d (946 mg, 85%). 1H NMR (500 MHz, CDC13) δ 6.81 (d, J = 2.5 Hz, 1H), 6.72 (d, J = 2.5 Hz, 1H), 4.76 - 4.64 (m, 1H), 4.18 (q, J = 7.0 Hz, 2H), 3.80 (s, 2H), 3.17 - 3.06 (m, 2H), 2.83 - 2.71 (m, 2H), 1.27 (t, J = 7.0 Hz, 3H), 1.19 - 1.10 (m, 21H).

[0137] Compound 5d (946 mg, 2.09 mmol) was dissolved in ammonia methanol solution (7 M, 10 mL) and stirred at 85 °C for 2 hours. TLC monitoring showed the reaction was completed. The reaction solution was cooled to room temperature. The crude product was obtained by concentration under reduced pressure. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 100:1-25:1) to obtain compound 5e (720 mg, 81.5%) as a yellow solid. 1 H NMR (500 MHz, CDC13) δ 6.81 (d, J = 2.5 Hz, 1H), 6.72 (d, J = 2.5 Hz, 1H), 4.76 - 4.64 (m, 1H), 4.18 (q, J = 7.0 Hz, 2H), 3.80 (s, 2H), 3.17 - 3.06 (m, 2H), 2.83 - 2.71 (m, 2H), 1.27 (t, J = 7.0 Hz, 3H), 1.19 - 1.10 (m, 21H).

[0138] Compound 5e (84 mg, 0.2 mmol) was dissolved in anhydrous dioxane (5 mL) under nitrogen atmosphere, and compound 4b (194 mg, 0.3 mmol), allylpalladium (II) dichloride (8 mg, 0.02 mmol), Xantphos (34.8 mg, 0.06 mmol), cesium carbonate (130 mg, 0.4 mmol) were added successively. The reaction mixture was stirred at 80 °C overnight. TLC monitoring showed the reaction was completed. The reaction solution was cooled to room temperature, dichloromethane (5 ml) was added, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1). Compound 5f (60 mg, 32%) was obtained as a yellow solid. 1H NMR (500 MHz, CDC13) δ 10.27 (s, 1H), 8.43 (d, J = 9.5 Hz, 1H), 8.04 (d, J = 1.5 Hz, 1H), 7.87 (d, J = 9.5 Hz, 1H), 6.82 (d, J = 2.5 Hz, 1H), 6.71 (d, J = 2.5 Hz, 1H), 5.18 - 5.02 (m, 1H), 4.80 - 4.60 (m, 3H), 3.90 (s, 2H), 3.22 - 3.09 (m, 3H), 2.83 - 2.72 (m, 2H), 2.56 - 2.46 (m, 1H), 1.59 (s, 9H), 1.45 (s, 9H), 1.41 (s, 9H), 1.17 - 1.13 (m, 21H).

[0139] Compound 5f (50 mg, 0.0675 mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at 50 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residual trifluoroacetic acid was removed by co-evaporation with anhydrous tetrahydrofuran (3 x 10 mL). The yellow solid compound 5g (35 mg, 100%) was obtained and used directly in the next step.

[0140] Compound 5g (35 mg, 0.0675 mmol) was dissolved in tetrahydrofuran (5 mL). Diisopropylethylamine (131 mg, 1.01 mmol), HATU (128 mg, 0.3375 mmol) and methylamine hydrochloride (23 mg, 0.3375 mmol) were added successively. The reaction mixture was stirred at 25 °C for 1 h. The reaction was monitored by TLC. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (20 mL) and washed with water (3 x 20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer preparative plate chromatography (dichloromethane:methanol = 15:1) to give compound 5h (15 mg, 32%) as a white solid. 1H NMR (500 MHz, CDC13) δ 9.98 (s, 1H), 8.34 (d, J = 9.0 Hz, 1H), 8.16 (d, J = 1.0 Hz, 1H), 7.30 (d, J = 9.0 Hz, 1H), 7.12 (q, J = 5.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 5.02 - 4.87 (m, 1H), 4.75 - 4.65 (m, 2H), 4.64 - 4.54 (m, 1H), 3.90 (s, 2H), 3.18 - 3.04 (m, 4H), 3.01 (d, J = 5.0 Hz, 3H), 2.83 - 2.72 (m, 2H), 2.28 - 2.15 (m, 1H), 2.13 - 2.01 (m, 1H), 1.18 - 1.12 (m, 21H).

[0141] Compound 5h (15 mg, 0.021 mmol) was dissolved in tetrahydrofuran (2.4 mL), and tetrabutylammonium fluoride (1 M, 0.6 mL) was added. The reaction mixture was stirred at 25 °C for 0.25 h, and the reaction was monitored by TLC. It was diluted with dichloromethane (20 mL), washed with water (3 x 20 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer preparative plate chromatography (dichloromethane:methanol = 15:1) to give compound 5 (8 mg, 70%) as a white solid. 1 H NMR (500 MHz, CDC13) δ 9.98 (s, 1H), 8.34 (d, J = 9.0 Hz, 1H), 8.16 (d, J = 1.0 Hz, 1H), 7.30 (d, J = 9.0 Hz, 1H), 7.12 (q, J = 5.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 5.02 - 4.87 (m, 1H), 4.75 - 4.65 (m, 2H), 4.64 - 4.54 (m, 1H), 3.90 (s, 2H), 3.18 - 3.04 (m, 4H), 3.01 (d, J = 5.0 Hz, 3H), 2.83 - 2.72 (m, 2H), 2.28 - 2.15 (m, 1H), 2.13 - 2.01 (m, 1H), 1.18 - 1.12 (m, 21H). + .

[0142] Example 6: Preparation of compound 6

[0143]

[0144] Compound 6a (2.00 g, 10.0 mmol) was dissolved in DMF (17 ml) and a solution of potassium tert-butoxide (1.69 g, 15.05 mmol) in 20 ml of DMF was added portionwise at -40 °C and stirred for 0.25 h at -40 °C, then 5 ml of saturated ammonium chloride and 13 ml of 3 M hydrochloric acid were added to the reaction mixture. The reaction mixture was slowly brought to room temperature. The reaction was monitored by TLC. Ice water (100 mL) was added to the reaction flask and the mixture was extracted with tert-butyl methyl ether (3 x 30 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 10:1) to give compound 6b (1.76 g, 90%) as a white solid.

[0145] Compound 6b (1.757 g, 7.53 mmol) was dissolved in 3 M HCl / EA and stirred at 25 °C for 2 h. The reaction was monitored by TLC. The mixture was filtered, washed with DCM and dried to give compound 6c (0.986 g, 77%) as a white solid.

[0146] Compound 6c (2.04 g, 12 mmol) was dissolved in anhydrous DMF (20 mL) under a nitrogen atmosphere and sodium hydride (4.0 g, 100 mmol) was added portionwise at 0 °C. The reaction mixture was stirred at 25 °C for 10 min. 2,6-Dimethyl-4-nitropyridine (2.82 g, 10 mmol) was added and the reaction was stirred at room temperature overnight. The reaction was monitored by TLC. Water (50 mL) was added to the reaction mixture and the mixture was extracted with tert-butyl methyl ether (3 x 20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 10:1) to give compound 6d (0.98 g, 26%) as a white solid.

[0147] Compound 6d (257 mg, 0.70 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) under a nitrogen atmosphere and n-butyllithium (0.5, 0.768 mmol) was added at -78 °C. The reaction mixture was stirred at -78 °C for 0.5 h. Iodomethane (298 mg, 2.10 mmol) was added and the reaction mixture was stirred at -78 °C for 2 h. The reaction was monitored by TLC. Saturated aqueous ammonium chloride solution (5 mL) and water (5 mL) were added to the reaction mixture and the mixture was extracted with ethyl acetate (3 x 5 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 5:1) to give compound 6e (135 mg, 67%) as a yellow liquid. 1H NMR (500 MHz, CDC13) δ 6.66 (d, 1 H), 6.46 (d, 1 H), 3.40 (m, 4 H), 2.41 (s, 3 H), 2.27 (m, 4 H),

[0148] Compound 6e (325 mg, 1.07 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere, zinc bromide ethyl acetate (6.5 mL, 3.22 mmol), Pd2(dba)3(61.64 mg, 0.11 mmol), Xphos (51.03 mg, 0.11 mmol) were added successively. The reaction mixture was stirred at 55 °C for 2 hours. TLC monitoring showed the reaction was completed. After the reaction mixture was cooled, saturated aqueous ammonium chloride solution (10 mL) and water (10 mL) were added, and the mixture was extracted with ethyl acetate (3 x 10 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 6f (530 mg). It was used directly for the next step.

[0149] Compound 6f (333 mg, 1.07 mmol) was dissolved in ammonium methylate solution (7 M, 10 mL) and stirred at 85 °C for 2 hours. TLC monitoring showed the reaction was completed. The reaction mixture was cooled to room temperature. Concentration under reduced pressure gave the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 100:1-25:1) to give compound 6g (97 mg, 32% yield over two steps) as a yellow solid.

[0150] Compound 6g (42 mg, 0.15 mmol) was dissolved in anhydrous dioxane (3 mL) under nitrogen atmosphere, compound 4b (146 mg, 0.225 mmol), allylpalladium (II) dichloride (6 mg, 0.015 mmol), Xantphos (26 mg, 0.045 mmol), cesium carbonate (98 mg, 0.3 mmol) were added successively. The reaction mixture was stirred at 80 °C overnight. TLC monitoring showed the reaction was completed. The reaction mixture was cooled to room temperature, dichloromethane (5 mL) was added, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 25:1). Compound 6h (35 mg, 30%) was obtained as a yellow solid. 1H NMR (500 MHz, CDC13) δ 8.49 (d, 1H), 8.08 (s, 1H), 7.88 (d, 1H), 6.48 (dd, 2H), 5.06-5.19 (m, 1H), 4.70 (m, 2H), 3.79 (s, 2H), 3.44 (m, 4H), 3.25 (dd, 1H), 2.58 (dd, 1H), 2.56 (s, 3H), 2.28 (m, 4H), 1.46 (s, 9H), 1.42 (s, 9H), 1.46 (s, 9H), 1.26 (s, 9H),

[0151] Compound 6h (35 mg, 0.044 mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at 50 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a crude product. The crude product was dried under reduced pressure with anhydrous tetrahydrofuran (3 x 10 mL) and trifluoroacetic acid. The yellow solid compound 6i (25 mg, 100%) was obtained.

[0152] Compound 6i (25 mg, 0.044 mmol) was dissolved in tetrahydrofuran (2 mL), and diisopropylethylamine (131 mg, 1.01 mmol), HATU (128 mg, 0.3375 mmol), and methylamine hydrochloride (23 mg, 0.3375 mmol) were added sequentially. The reaction mixture was stirred at 25 °C for 1 h. The reaction was monitored by TLC. Tetrahydrofuran was removed under reduced pressure, and the residue was diluted with dichloromethane (20 mL) and washed with water (3 x 20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography (dichloromethane:methanol = 15:1) to give compound 6 (5 mg, 20%) as a white solid. 1 H NMR (500 MHz, CDC13) δ 8.49 (d, 1H), 8.08 (s, 1H), 7.88 (d, 1H), 6.48 (dd, 2H), 5.06-5.19 (m, 1H), 4.70 (m, 2H), 3.79 (s, 2H), 3.44 (m, 4H), 3.25 (dd, 1H), 2.58 (dd, 1H), 2.56 (s, 3H), 2.28 (m, 4H), 1.46 (s, 9H), 1.42 (s, 9H), 1.46 (s, 9H), 1.26 (s, 9H), + .

[0153] Example 7: Preparation of compound 7

[0154]

[0155] To a solution of t-BuOK (1.68 g, 15.0 mmol) in anhydrous DMF (15 mL) was added dropwise a solution of compound 7a (1.76 g, 10.0 mmol) and difluoromethyl(2-pyridyl)sulfone (1.61 g, 8.33 mmol) in DMF (5 mL) at -50 °C under nitrogen atmosphere. The mixture was stirred at -45 °C for 30 min. Saturated ammonium chloride (15 mL) and 3 M hydrochloric acid (15 mL) were added dropwise, then slowly raised to room temperature and reacted for 3 h, extracted with water (60 mL) and MTBE (3 x 50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 5: 1) to give compound 7b as a colorless liquid, 423 mg, yield 20%. 1 H NMR (500 MHz, CDC13) δ 7.38-7.29 (m, 5H), 4.44 (s, 2H), 4.16 (m, 1H), 2.91 (m, 2H), 2.68 (m, 2H).

[0156] To a suspension of sodium hydride (96 mg, 2.40 mmol) in anhydrous tetrahydrofuran (10 mL) was added dropwise a solution of compound 7c (198 mg, 1.65 mmol) in anhydrous tetrahydrofuran (5 mL) at 0 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 30 min. A solution of 2-bromo-6-methyl-4-nitropyridine (434 mg, 2.00 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise, the mixture was raised to 25 °C and stirred overnight. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 10: 1) to give compound 7d as a yellowish liquid, 418 mg, yield 87%.

[0157] To a suspension of sodium hydride (96 mg, 2.40 mmol) in anhydrous tetrahydrofuran (10 mL) was added dropwise a solution of compound 7c (198 mg, 1.65 mmol) in anhydrous tetrahydrofuran (5 mL) at 0 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 30 min. A solution of 2-bromo-6-methyl-4-nitropyridine (434 mg, 2.00 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise, the mixture was raised to 25 °C and stirred overnight. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 10: 1) to give compound 7d as a yellowish liquid, 418 mg, yield 87%. 1H NMR (500 MHz, CDC13), δ 6.71 (d, 1 H), 6.53 (d, 1 H), 4.78 (m, 1 H), 3.20 (m, 2 H), 2.85 (m, 2 H), 2.47 (s, 3 H).

[0158] Compound 7d (400 mg, 1.38 mmol), X-Phos (66 mg, 0.138 mmol) and Pd2(dba)3(127 mg, 0.138 mmol) were dissolved in tetrahydrofuran (5 mL) under nitrogen atmosphere and deoxygenated by bubbling nitrogen gas for 5 minutes. Then freshly prepared ethyl acetate zinc bromide reagent (8.3 mL, 4.15 mmol) was added and deoxygenated by bubbling nitrogen gas for another 10 minutes. The mixture was heated to 55 °C and stirred for 1 hour. After the reaction was cooled to room temperature, it was diluted with ethyl acetate (50 mL) and cooled to 0 °C. The reaction was quenched with saturated ammonium chloride solution (50 mL). The aqueous phase was extracted with ethyl acetate and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue gave compound 7e (285 mg) as a yellow liquid which was used directly for the next step.

[0159] Compound 7e (270 mg, 0.908 mmol) was dissolved in 7 M ammonium hydroxide solution (8 mL) in a sealed tube and heated to 80 °C and stirred for 2 h. The reaction was concentrated and the residue was purified by column chromatography (dichloromethane:methanol = 15:1) to give compound 7f (177 mg, 48% yield over two steps) as a white solid. 1 H NMR (500 MHz, CDC13), δ 6.71 (d, 1 H), 6.53 (d, 1 H), 4.78 (m, 1 H), 3.20 (m, 2 H), 2.85 (m, 2 H), 2.47 (s, 3 H).

[0160] Compound 7f (77 mg, 0.29 mmol) was dissolved in anhydrous dioxane (3 mL) under nitrogen atmosphere. 4b (223 mg, 0.34 mmol), allylpalladium (II) dichloride (5.3 mg, 0.0057 mmol), XantPhos (33 mg, 0.057 mmol) and Cs2CO3(187 mg, 0.57 mmol) were added successively. The reaction mixture was stirred at 80 °C overnight. The reaction was monitored by TLC and was complete. The reaction was cooled to room temperature, diluted with dichloromethane (5 mL), filtered and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (dichloromethane:methanol = 35:1) to give compound 7g (78 mg, 34% yield) as a yellow solid. MS m / z 788.7 [M+H] + .

[0161] Compound 7g (78 mg, 0.099 mmol) was dissolved in TFA (3 mL) and stirred at 50 °C for 1.5 h. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a crude product. The TFA was removed by co-evaporation with anhydrous tetrahydrofuran (3 x 10 mL). The residue was dried under reduced pressure to give 7h (59 mg, 100% yield). MS m / z 532.4 [M+H] + .

[0162] Compound 7h (59 mg, 0.1 mmol) was dissolved in THF (3 mL) and DIPEA (129 mg, 1.0 mmol), HATU (190 mg, 0.5 mmol), and methylamine hydrochloride (35 mg, 0.5 mmol) were added sequentially. The reaction mixture was stirred at 25 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure and extracted with dichloromethane (3 x 20 mL) and water (20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse phase column chromatography and lyophilized to give compound 7 (0.82 mg, 2% yield). 1 H NMR (500 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.52 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.23 (d, J = 9.2 Hz, 1H), 7.60 (d, J = 9.2 Hz, 1H), 6.72 (d, J = 2.1 Hz, 1H), 6.66 (d, J = 2.1 Hz, 1H), 5.12 - 4.97 (m, 1H), 4.97 - 4.89 (m, 1H), 4.78 - 4.70 (m, 2H), 3.95 (m, 2H), 3.86 (s, 2H), 3.20 (m, 2H), 3.03 (m, 2H), 2.76 (d, J = 4.7 Hz, 3H), 2.38 (s, 3H), 2.00 - (m, 2H). MS m / z 545.6 [M+H]+.

[0163] Example 8: Preparation of compound 8

[0164]

[0165] To a suspension of NaH (186 mg, 4.64 mmol) in anhydrous tetrahydrofuran (10 mL) was added dropwise a solution of compound 2,2,2-trifluoroethanol (446 mg, 4.46 mmol) in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere and ice bath 0 °C. The mixture was stirred at 0 °C for 30 min. A solution of 2,6-dibromo-4-nitropyridine (1.00 g, 3.57 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise and the mixture was allowed to warm to 25 °C and stirred overnight. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The organic phase was combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to give compound 8b (986 mg, 83%) as a yellow solid.

[0166] Compound 8b (800 mg, 2.388 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere, followed by the addition of Pd(PPh3)2Cl2(167.8 mg, 0.238 mmol), CuI (45.5 mg, 0.238 mmol), DIPEA (925.87 mg, 164 mmol) and finally triisopropylsilyl acetylene (480 mg, 2.63 mmol). The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with THF (3 x 5 mL), the filtrate was collected and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (PE:EA = 20:1) to give compound 8c (735 mg, 70%) as a white solid. 1 H NMR (500 MHz, CDC13) δ 7.00 (m, 2H), 4.40 (m, 2H), 1.13 (s, 18H), 1.12 (s, 3H)

[0167] Compound 8c (735 mg, 1.68 mmol), XPhos (80 mg, 0.168 mmol) and Pd2(dba)3(96.6 mg, 0.168 mmol) were dissolved in tetrahydrofuran (5 mL) under nitrogen atmosphere and the mixture was bubbled with nitrogen for 5 min to remove oxygen. Then, freshly prepared ethyl acetate zinc bromide reagent (10.1 mL, 5.04 mmol) was added and the mixture was bubbled with nitrogen for another 10 min. The mixture was heated to 55 °C and stirred for 2 hours. After the mixture was cooled to room temperature, it was diluted with ethyl acetate (50 mL) and cooled to 0 °C. The mixture was quenched with saturated ammonium chloride solution (50 mL). The aqueous phase was extracted with ethyl acetate and the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (PE:EA = 20:1-10:1) to give compound 8d (590 mg, 82%) as a yellow liquid. 1HNMR (500 MHz, CDC13), δ 6.94 (d, 1H), 6.87 (d, 1H), 4.39 (q, 2H), 4.17 (q, 2H), 3.83 (s, 2H), 1.27 (t, 3H), 1.14 (s, 18H), 1.13 (s, 3H).

[0168] Compound 8d (590 mg, 1.3 mmol) was dissolved in 7M ammonia methanol solution (8 mL), the reaction tube was sealed and heated to 80 °C and stirred overnight. The reaction solution was concentrated, and the residue was separated and purified by column chromatography (dichloromethane:methanol = 20:1) to obtain a yellow solid compound, which was stirred with petroleum ether (5 mL) to obtain 8e (316 mg, 55%). 1 H NMR (500 MHz, CDC13), δ 7.20 (s, 1H), 6.95 (d, 1H), 6.83 (d, 1H), 5.65 (s, 1H), 4.41 (q, 2H), 3.70 (s, 2H), 1.14 (s, 18H), 1.13 (s, 3H).

[0169] Compound 8e (100 mg, 0.24 mmol) was dissolved in anhydrous dioxane (5 mL) under a nitrogen atmosphere, and 4b (466 mg, 0.48 mmol), allylpalladium (II) dichloride (10 mg, 0.024 mmol), XantPhos (41.65 mg, 0.072 mmol), and Cs2CO3 (160 mg, 0.48 mmol) were added in sequence. The reaction mixture was stirred at 80 °C overnight. The reaction was monitored by TLC, and the reaction solution was cooled to room temperature, dichloromethane (5 mL) was added, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 15:1). Compound 8f (200 mg, yield 89%) was obtained as a yellow solid.

[0170] Compound 8f (200 mg, 0.215 mmol) was dissolved in TFA (3 mL) and stirred at 50 °C for 1 hour. The reaction was monitored by TLC, and the reaction solution was cooled to room temperature, concentrated under reduced pressure to obtain a crude product, and TFA was removed with anhydrous tetrahydrofuran (3 x 10 mL). Compound 8g (131 mg, yield 90%) was obtained by rotary evaporation under reduced pressure.

[0171] Compound 8g (131 mg, 0.1935 mmol) was dissolved in THF (3 mL), DIPEA (375.12 mg, 2.9 mmol), HATU (367.88 mg, 0.9675 mmol), and methylamine hydrochloride (67 mg, 0.97 mmol) were added sequentially. The reaction mixture was stirred at 25 °C for 1 h. TLC monitoring showed the reaction was complete. The THF was evaporated under reduced pressure, and the residue was extracted with dichloromethane (3 x 20 mL) and water (20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by PTLC (dichloromethane:methanol = 15:1) to give 8h (48 mg, 36% yield).

[0172] Compound 8h (48 mg, 0.069 mmol) was dissolved in THF (5 mL), and TBAF / THF (1 M, 0.4 mL) was added. The reaction mixture was stirred at 25 °C for 0.25 h. TLC monitoring showed the reaction was complete. The residue was extracted with dichloromethane (10 mL) and water (3 x 10 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by PTLC (dichloromethane:methanol = 17:1), and lyophilized to give compound 8 (2.75 mg, 8% yield). 1 H NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1H), 8.53 (s, 1H), 8.50 (d, J = 4.5 Hz, 1H), 8.21 (d, J = 9.2 Hz, 1H), 7.61 (d, J = 9.2 Hz, 1H), 7.25 (d, J = 1.8 Hz, 1H), 7.17 (d, J = 1.8 Hz, 1H), 5.12-4.96 (m, 1H), 4.95 (m, 2H), 4.85-4.69 (m, 2H), 4.37 (s, 1H), 3.95 (s, 2H), 3.02 (m, 2H), 2.76 (d, J = 4.6 Hz, 3H), 2.00 (m, 2H). MS m / z 535.07 [M+H] + .

[0173] Example 9: Preparation of compound 9

[0174]

[0175] To a suspension of sodium hydride (104 mg, 2.60 mmol) in anhydrous tetrahydrofuran (10 mL) was added dropwise a solution of compound 3-(difluoromethyl)cyclobutan-1-ol (293 mg, 2.40 mmol) in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere and ice bath at 0 °C. The mixture was stirred at 0 °C for 30 min, and a solution of 9a (434 mg, 2.00 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise. The mixture was allowed to warm to 25 °C and stirred overnight. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 10: 1) to give two stereoisomeric compounds, 9b-cis: 9b-trans (4: 1), in which the major product was the cis isomer 9b-cis, 416 mg, yield 71%. 1 H NMR (500 MHz, CDC13) δ 6.71 (d, 1H), 6.53 (d, 1H), 5.80 (t, 1H), 4.64 (m, 1H), 2.62 (m, 2H), 2.46 (s, 3H), 2.47 (m, 1H), 2.23 (m, 2H). 9b-trans, 110 mg, yield 19%. 1 H NMR (500 MHz, CDC13) δ 6.71 (d, 1H), 6.53 (d, 1H), 5.80 (t, 1H), 4.64 (m, 1H), 2.62 (m, 2H), 2.46 (s, 3H), 2.47 (m, 1H), 2.23 (m, 2H). 9b-trans, 110 mg, yield 19%.

[0176] Compound 9b-cis (410 mg, 1.52 mmol), X-Phos (109 mg, 0.228 mmol) and Pd2(dba)3(139 mg, 0.152 mmol) were dissolved in tetrahydrofuran (5 mL) under nitrogen atmosphere and deoxygenated by bubbling nitrogen gas for 5 min. Then freshly prepared ethyl acetate zinc bromide reagent (9.1 mL, 4.55 mmol) was added and deoxygenated by bubbling nitrogen gas for another 10 min. The mixture was heated to 55 °C and stirred for 1 h. After the reaction was cooled to room temperature, it was diluted with ethyl acetate (50 mL) and cooled to 0 °C. The reaction was quenched with saturated ammonium chloride solution (50 mL). The aqueous phase was extracted with ethyl acetate and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography to give compound 9c-cis as a yellow liquid (189 mg, 42%).

[0177] Compound 9c-cis (180 mg, 0.60 mmol) was dissolved in 7M ammonia methanol solution (8 mL), the reaction tube was sealed and heated to 80 °C for 2 h. The reaction was concentrated and the residue was purified by column chromatography (dichloromethane:methanol = 15:1) to give compound 9d-cis as a white solid (98 mg, 60%). 1 H NMR (500 MHz, CDC13) δ 7.51 (s, 1H), 6.50 (m, 1H), 6.48 (d, 1H), 5.80 (m, 1H), 5.42 (s, 1H), 4.65 (m, 1H), 3.63 (s, 2H), 2.61 (m, 2H), 2.48 (s, 3H), 2.46 (m, 1H), 2.22 (m, 2H).

[0178] Compound 9d-cis (38 mg, 0.14 mmol) was dissolved in anhydrous dioxane (3 mL) under nitrogen atmosphere, 4b (183 mg, 0.282 mmol), allylpalladium (II) dichloride (3 mg, 0.0071 mmol), XantPhos (16.3 mg, 0.028 mmol), Cs2CO3(92 mg, 0.282 mmol) were added successively. The reaction mixture was stirred at 80 °C overnight. TLC monitoring showed that the reaction was completed. The reaction was cooled to room temperature, dichloromethane (5 mL) was added, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 35:1). Compound 9e-cis was obtained as a yellow solid (41 mg, 37% yield).

[0179] Compound 9e-cis (41 mg, 0.052 mmol) was dissolved in TFA (3 mL) and stirred at 50 °C for 1.5 h. TLC monitoring showed that the reaction was completed. The reaction was cooled to room temperature and concentrated under reduced pressure to give a crude product. TFA was removed by anhydrous tetrahydrofuran (3 x 10 mL). 9f-cis was obtained by rotary evaporation under reduced pressure (35 mg, 100% yield).

[0180] Compound 9f-cis (27 mg, 0.0519 mmol) was dissolved in THF (3 mL), DIPEA (67.1 mg, 0.519 mmol), HATU (98.67 mg, 0.259 mmol), and methylamine hydrochloride (18 mg, 0.259 mmol) were added successively. The reaction mixture was stirred at 25 °C for 1 h. TLC monitoring showed that the reaction was completed. THF was removed by rotary evaporation under reduced pressure, and the residue was extracted with dichloromethane (3 x 20 mL) and water (20 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase column chromatography, and 9 (11.5 mg, 40% yield) was obtained by freeze-drying. 1H NMR (500 MHz, DMSO-d6) δ 11.31 (s, 1H), 8.51 (s, 1H), 8.46 (d, J = 4.7 Hz, 1H), 8.21 (d, J = 9.2 Hz, 1H), 7.60 (d, J = 9.2 Hz, 1H), 6.73 (d, J = 2.1 Hz 1H), 6.66 (d, J = 2.1 Hz 1H), 6.10 (td, J = 57.2 Hz, J = 4.1 Hz, 1H), 5.11 - 4.95 (m, 1H), 4.81 (m, 1H), 4.78 - 4.70 (m, 2H), 3.86 (s, 2H), 3.35 (m, 2H), 3.03 (m, 2H), 2.76 (d, J = 4.7 Hz, 3H), 2.60 (m, 2H), 2.48 (m, 1H), 2.38 (s, 3H), 2.00 (m, 2H). MS m / z 547.29 [M+H] + .

[0181] Example 10: Preparation of compound 10

[0182]

[0183] Compound 3, 3-difluorocyclobutylamine (107 mg, 1.00 mmol) was dissolved in anhydrous dioxane (5 mL) under nitrogen atmosphere, Pd2(dba)3(92 mg, 0.1 mmol), 10a (366 mg, 1.5 mmol), XantPhos (116 mg, 0.2 mmol), Cs2CO3(651 mg, 2 mmol) were added successively. The reaction mixture was stirred at 80 °C overnight. TLC monitoring showed that the reaction was completed. The reaction mixture was cooled to room temperature, quenched by saturated aqueous ammonium chloride solution, extracted with water (20 mL) and THF (3 x 10 mL), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 10b (109 mg, yield 40%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 6.28 (d, 1H), 6.18 (d, 1H), 4.55 (s, 1H), 3.89 (m, 1H), 3.71 (m, 5H), 3.07 (m, 2H), 2.46 (m, 2H), 2.42 (s, 3H).

[0184] Compound 10b (364 mg, 1.34 mmol) was dissolved in 7M ammonia methanol solution (5 mL) and the reaction tube was sealed and heated to 53 °C with stirring overnight. The reaction was concentrated and the residue was purified by column chromatography (dichloromethane:methanol = 20:1) to give a yellow solid which was slurried in petroleum ether (10 mL) to give a white solid compound 10c (230 mg, 67%). 1 H NMR (500 MHz, CDC13) δ 7.65 (s, 1H), 6.21 (d, 1H), 6.16 (d, 1H), 5.36 (s, 1H), 4.41 (m, 1H), 3.89 (m, 1H), 3.56 (s, 2H), 3.08 (m, 2H), 2.46 (m, 2H), 2.42 (s, 3H).

[0185] Compound 10c (60 mg, 0.235 mmol) was dissolved in dry dioxane (5 mL) under nitrogen atmosphere, followed by the addition of 4b (306 mg, 0.48 mmol), allylpalladium (II) dichloride (4.3 mg, 0.0116 mmol), XantPhos (26.9 mg, 0.047 mmol), and Cs2C03(153 mg, 0.47 mmol). The reaction mixture was stirred at 80 °C overnight. The reaction was monitored by TLC and upon completion, the reaction was cooled to room temperature and quenched with saturated aqueous ammonium chloride solution, extracted with water (20 mL) and dichloromethane (3 x 10 mL), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (dichloromethane:methanol = 15:1-10:1). Compound 10d (100 mg, 55% yield) was obtained as a yellow solid.

[0186] Compound 10d (100 mg, 0.129 mmol) was dissolved in TFA (2 mL) and stirred at 50 °C for 1 hour. The reaction was monitored by TLC and upon completion, the reaction was cooled to room temperature and concentrated under reduced pressure to give a crude product. The TFA was taken out with dry tetrahydrofuran (3 x 10 mL). Compound 10e (68 mg, 100% yield) was obtained after concentration under reduced pressure.

[0187] Compound 10e (68 mg, 0.129 mmol) was dissolved in THF (5 mL), DIPEA (166.7 mg, 1.29 mmol), HATU (245.25 mg, 0.645 mmol), methylamine hydrochloride (45 mg, 0.645 mmol) were added successively. The reaction mixture was stirred at 25 °C for 2 h. The reaction was monitored by TLC and upon completion, the reaction mixture was extracted with dichloromethane (3 x 20 mL) and water (20 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse phase column separation and lyophilized to obtain compound 10 (4.5 mg, 7% yield). 1 H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.52 (s, 1H), 8.49 (q, J = 4.7 Hz, 1H), 8.24 (d, J = 9.15 Hz, 1H), 7.61 (d, J = 9.15 Hz, 1H), 6.38 (s, 1H), 6.27 (s, 1H) 5.10-4.96 (m, 1H), 4.85-4.70 (m, 2H), 3.87-3.79 (m, 1H), 3.73 (s, 2H), 3.13-2.96 (m, 5H), 2.76 (d, J = 4.7 Hz, 3H), 2.47-2.42 (m, 1H), 2.29 (s, 3H), 2.18-2.07 (m, 1H), 2.05-1.94 (m, 2H). MS m / z 532.04 [M+H] + .

[0188] Example 11: Preparation of compound 11

[0189]

[0190] A mixture of 2-benzyloxyethanol (3.60 g, 23.65 mmol), (bromodifluoromethyl)trimethylsilane (9.60 g, 47.30 mmol), potassium acetate (9.28 g, 94.60 mmol) was suspended in a mixture of DCM (20 mL) and water (20 mL) and stirred at 25 °C overnight. The reaction mixture was extracted with water (100 mL) and DCM (3 x 50 mL), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 10: 1) to give compound, 1-benzyloxy 2-difluoromethoxyethane as a yellow liquid, 3.02 g, 63% yield. 1 H NMR (500 MHz, CDCl3) δ 7.35 (m, 4H), 7.30 (m, 1H), 6.28 (t, 1H), 4.58 (s, 2H), 4.02 (t, 2H), 3.68 (t, 2H).

[0191] Compound 1-benzyloxy 2-difluoromethoxyethane (3.02 g, 14.94 mmol), Pd (1.0 g) were dissolved in tetrahydrofuran (70 mL) and methanol (30 mL) under hydrogen atmosphere and the reaction was stirred for 16 h, filtered and concentrated. This resulted in compound 2-difluoromethoxyethanol (1.60 g, 90%) as a yellow liquid. 1 H NMR (500 MHz, CDC13) δ 6.28 (t, 1H), 3.98 (t, 2H), 3.83 (t, 2H).

[0192] To a suspension of sodium hydride (260 mg, 6.50 mmol) in anhydrous tetrahydrofuran (10 mL) was added a solution of compound 2-difluoromethoxyethanol (672 mg, 6.00 mmol) in anhydrous tetrahydrofuran (5 mL) dropwise under nitrogen atmosphere and ice bath 0 °C. The mixture was stirred at 0 °C for 30 min. A solution of 11a (1.09 g, 5.00 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise and the mixture was stirred at 25 °C overnight. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The organic phase was combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 8: 1) to give compound 11b as a yellow liquid, 1.326 g, yield 92%. 1 H NMR (500 MHz, CDC13) δ 6.85 (d, 1H), 6.65 (d, 1H), 6.29 (t, 1H), 4.20 (s, 4H), 2.48 (s, 3H).

[0193] Compound 11b (1.15 g, 3.97 mmol), X-Phos (283 mg, 0.596 mmol) and Pd2(dba)3 (364 mg, 0.397 mmol) were dissolved in tetrahydrofuran (5 mL) under nitrogen atmosphere and deoxygenated by bubbling nitrogen gas for 5 min. Zinc reagent (30 ml, 11.93 mmol) was then added and deoxygenated by bubbling nitrogen gas for another 10 min. The mixture was heated to 55 °C and stirred for 45 min. The reaction was cooled to room temperature, diluted with ethyl acetate (50 mL) and cooled to 0 °C. The reaction was quenched with saturated ammonium chloride solution (50 mL). The aqueous phase was extracted with ethyl acetate and the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 11c (1.5 g) as a yellow liquid. It was used directly in the next step.

[0194] Compound 11c (70 mg, 0.242 mmol) was dissolved in 7M ammonia methanol solution (8 mL), the reaction tube was sealed and heated to 80 °C for 2 h, the reaction was concentrated, and the residue was purified by column chromatography (dichloromethane:methanol = 15:1) to give compound 11d (42 mg) as a white solid. 1 H NMR (500 MHz, CDC13) δ 7.47 (s, 1H), 6.64 (d, 1H), 6.61 (d, 1H), 6.29 (t, 1H), 5.59 (q, 1H), 4.20 (s, 4H), 3.64 (s, 2H), 3.49 (s, 3H).

[0195] Compound 11d (48 mg, 0.185 mmol) was dissolved in anhydrous dioxane (3 mL) under nitrogen atmosphere, 4b (240 mg, 0.37 mmol), allylpalladium (II) dichloride (3.4 mg, 0.009 mmol), XantPhos (21.4 mg, 0.037 mmol), and Cs2C03(121 mg, 0.37 mmol) were added successively. The reaction mixture was stirred at 80 °C overnight. The reaction was monitored by TLC, and the reaction mixture was cooled to room temperature, dichloromethane (15 mL) was added, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (dichloromethane:methanol = 25:1). Compound 11e (46 mg, 32% yield) was obtained as a yellow solid.

[0196] Compound 11e (45 mg, 0.052 mmol) was dissolved in TFA (3 mL) and stirred at 50 °C for 1.5 h. The reaction was monitored by TLC, and the reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a crude product. TFA was removed by rotary evaporation under reduced pressure with anhydrous tetrahydrofuran (3 x 10 mL). Compound 11f (31 mg, 100% yield) was obtained after rotary evaporation under reduced pressure.

[0197] Compound 11f (31 mg, 0.059 mmol) was dissolved in THF (3 mL), and DIPEA (70 mg, 0.59 mmol), HATU (112 mg, 0.295 mmol), and methylamine hydrochloride (21 mg, 0.295 mmol) were added successively. The reaction mixture was stirred at 25 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure, extracted with dichloromethane (3 x 20 mL) and water (20 mL), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase column chromatography, and 11 (4.86 mg, 17% yield over two steps) was obtained after lyophilization. 1H NMR (500 MHz, DMSO-d6) δ 11.31 (s, 1H), 8.52 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 8.28 (s, 0.5H), 8.22 (d, J = 9.1 Hz, 1H), 7.60 (d, J = 9.1 Hz, 1H), 6.84 (s, 1H), 6.79 (s, 1H), 6.74 (t, J = 75.6 Hz, 0.5H), 5.10-4.95 (m, 1H), 4.85-4.70 (m, 2H), 4.43 (m, 1H), 4.28 (m, 1H), 4.25 (m, 1H), 4.17 (m, 1H), 3.88 (s, 2H), 3.02 (m, 2H) 2.76 (d, J = 5.25 Hz, 3H), 2.39 (s, 3H), 2.06 (m, 2H). MS m / z = 537.6 [M+H] +

[0198] Example 12: Preparation of compound 12

[0199]

[0200] To a suspension of NaH (111 mg, 2.77 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise a solution of compound 1-hydroxycyclopropanecarboxylic acid methyl ester (322 mg, 2.77 mmol) in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere and ice bath 0 °C, the mixture was allowed to warm to room temperature and stirred for 30 minutes. Then it was cooled to 0 °C and a solution of 2-bromo-6-methyl-4-nitropyridine (500 mg, 2.31 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise, the mixture was allowed to warm to 25 °C and stirred overnight. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (3 x 50 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated and purified by column chromatography (petroleum ether: ethyl acetate = 6: 1) to give compound 12b (255 mg, 39%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 6.85 (d, J = 1.9 Hz, 1H), 6.64 (d, J = 1.9 Hz, 1H), 3.75 (s, 3H), 2.48 (s, 3H), 1.68 - 1.64 (m, 2H), 1.36 - 1.32 (m, 2H).

[0201] Compound 12b (255 mg, 0.89 mmol) was dissolved in dry tetrahydrofuran (5 mL) and DIBAL-H (1 M, 2.2 mL, 2.2 mmol) in n-hexane was added dropwise at 0 °C under nitrogen atmosphere. The mixture was stirred at room temperature for 1 h. It was quenched by the addition of 0.5 M aqueous potassium sodium tartrate (3 mL) at 0 °C, diluted with ethyl acetate (50 mL) and stirred at room temperature for 15 min. It was filtered, the solid was washed with ethyl acetate and the filtrate was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by separation on a preparative thin layer silica gel plate (petroleum ether: ethyl acetate = 2:1) to give compound 12c (210 mg, 92%) as a white solid. 1 H NMR (500 MHz, CDC13) δ 6.98 (d, J = 1.8 Hz, 1H), 6.73 (d, J = 1.8 Hz, 1H), 3.86 (s, 2H), 2.46 (s, 3H), 1.09 - 1.05 (m, 2H), 1.01 - 0.97 (m, 2H).

[0202] To a solution of oxalyl chloride (0.13 mL, 1.56 mmol) in dry dichloromethane (5 mL) was added dropwise a solution of dimethyl sulfoxide (0.65 mL) in dry dichloromethane (0.5 mL) at -60 °C under nitrogen atmosphere. After stirring for 5 min, a solution of compound 12c (200 mg, 0.78 mmol) in dry dichloromethane (2 mL) was added and stirring was continued for 15 min. Then triethylamine (0.54 mL, 3.89 mmol) was added and the mixture was slowly allowed to warm to room temperature and stirred for 1 h. The organic phase was diluted with dichloromethane (50 mL) and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by separation on a preparative thin layer silica gel plate (petroleum ether: ethyl acetate = 2:1) to give compound 12d (151 mg, 76%) as a yellow oily liquid. 1 H NMR (500 MHz, CDC13) δ 6.98 (d, J = 1.8 Hz, 1H), 6.73 (d, J = 1.8 Hz, 1H), 3.86 (s, 2H), 2.46 (s, 3H), 1.09 - 1.05 (m, 2H), 1.01 - 0.97 (m, 2H).

[0203] Compound 12d (220 mg, 0.86 mmol) was dissolved in dichloromethane (2 mL) and cooled to -10 °C in a ice-salt bath. BAST (667 mg, 3.02 mmol) was added. The mixture was stirred at 0 °C for 1 h and then allowed to warm to room temperature for 2 h. The reaction was diluted with dichloromethane (50 mL) and quenched with saturated aqueous sodium bicarbonate solution (50 mL) at 0 °C. The aqueous phase was extracted with dichloromethane (50 mL), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer silica gel chromatography (petroleum ether: ethyl acetate = 3: 1) to give compound 12e (198 mg, 83% yield, 50% purity) as a white solid. 1 H NMR (500 MHz, CDC13) δ 6.99 (d, J = 1.8 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H), 6.04 (t, J = 56.1 Hz, 1H), 2.48 (s, 3H), 1.32 - 1.28 (m, 2H), 1.17 - 1.12 (m, 2H).

[0204] Preparation of zinc reagent: To a suspension of zinc dust (516 mg, 8.08 mmol) in tetrahydrofuran (2 mL) was added TMSC1 (44 mg, 0.4 mmol) under nitrogen atmosphere. The mixture was stirred at room temperature for 15 min. Then ethyl bromoacetate (664 mg, 4.0 mmol) in tetrahydrofuran (6 mL) was added and the mixture was stirred at 30 °C for 30 min. The resulting light green zinc reagent was about 0.5 M in tetrahydrofuran and was used directly in the next step by filtration through a frit.

[0205] Compound 12e (313 mg, 1.13 mmol), XPhos (28 mg, 0.06 mmol) and Pd2(dba)3 (26 mg, 0.03 mmol) were dissolved in tetrahydrofuran (5 mL) and degassed with a stream of nitrogen for 5 min. Then the above prepared zinc reagent (6.8 mL, 3.39 mmol) was added and the mixture was degassed with a stream of nitrogen for another 10 min. The mixture was heated to 50 °C and stirred for 2 h. The reaction was diluted with ethyl acetate (50 mL) and quenched with saturated ammonium chloride solution (50 mL) at 0 °C. The aqueous phase was extracted with ethyl acetate and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer silica gel chromatography (dichloromethane:methanol = 20: 1) to give compound 12f (240 mg, 75% yield, 50% purity) as a yellow liquid. 1H NMR (500 MHz, CDC13) δ 6.79 (s, 1H), 6.71 (s, 1H), 6.08 (t, J = 56.3 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.75 (s, 2H), 2.49 (s, 3H), 1.29 - 1.24 (m, 5H), 1.16 - 1.10 (m, 2H).

[0206] Compound 12f (85 mg, 0.3 mmol) was dissolved in 7M ammonia methanol solution (5 mL), the reaction tube was sealed and heated to 60 °C and stirred overnight. The reaction was concentrated and the residue was purified by preparative thin layer silica gel plate (dichloromethane:methanol = 15:1) to give compound 12g (43 mg, yield 56%, 66% purity) as a yellow solid. 1 H NMR (500 MHz, CDC13) δ 6.79 (s, 1H), 6.71 (s, 1H), 6.08 (t, J = 56.3 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.75 (s, 2H), 2.49 (s, 3H), 1.29 - 1.24 (m, 5H), 1.16 - 1.10 (m, 2H).

[0207] Compound 12g (18 mg, 0.07 mmol) was dissolved in dry dioxane (3 mL) under nitrogen atmosphere, 4b (54.58 mg, 0.084 mmol), allylpalladium (II) dichloride (1.3 mg, 0.003 mmol), XantPhos (8.1 mg, 0.014 mmol), Cs2C03(45.6 mg, 0.14 mmol) were added sequentially. The reaction mixture was stirred at 80 °C overnight. The reaction was monitored by TLC and upon completion, the reaction was cooled to room temperature, quenched with saturated aqueous ammonium chloride solution and extracted with water (20 mL) and ethyl acetate (3 x 10 mL), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 12h (25 mg, yield 36%) as a yellow solid.

[0208] Compound 12h (50 mg, 0.0645 mmol) was dissolved in TFA (3 mL) and stirred at 50 °C for 2 hours. The reaction was monitored by TLC and upon completion, the reaction was cooled to room temperature and concentrated under reduced pressure to remove TFA. Compound 12i (30 mg, yield 100%) was obtained.

[0209] Compound 12i (30 mg, 0.0645 mmol) was dissolved in THF (5 mL), DIPEA (84 mg, 0.645 mmol), HATU (122.6 mg, 0.3225 mmol), methylamine hydrochloride (23 mg, 0.3225 mmol) were added successively. The reaction mixture was stirred at 25 °C for 2 h. TLC monitoring reaction was completed, extracted with dichloromethane (3 x 20 mL) and water (20 mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse phase column separation, and compound 12 (2.2 mg, yield 7%) was obtained by freeze-drying. 1 H NMR (500 MHz, DMSO-d6) δ 11.32 (s, 1H), 8.53 (s, 1H), 8.50 (q, J = 4.7 Hz, 1H), 8.23 (d, J = 9.1 Hz, 1H), 7.61 (d, J = 9.1 Hz, 1H), 6.92 (d, J = 1.9 Hz, 1H), 6.86 (d, J = 1.8 Hz, 1H), 6.23 (t, J = 54.5 Hz, 1H), 5.11 - 4.96 (m, 1H), 4.86 - 4.70 (m, 2H), 3.89 (s, 2H), 3.04 (m, 2H), 2.77 (d, J = 4.7 Hz, 3H), 2.41 (s, 3H), 2.00 (m, 2H), 1.27 (m, 2H), 1.16 (m, 2H). MS m / z 533.05 [M+H] + .

[0210] Example 13: Bioactivity test

[0211] 1. GLS1 enzyme activity test

[0212] The glutaminase GLS1 activity was determined by glutaminase / Amplex Red coupling method, and the ability of the compound to inhibit GLS1 activity was detected. L-glutamic acid can be oxidized to a-ketoglutaric acid, NH3 and H2O2 by glutamate oxidase. When horseradish peroxidase (HRP) is present in the system, Amplex Red reagent can react with the generated H2O2 in a 1:1 stoichiometric ratio to form a highly fluorescent resorufin, and the fluorescence intensity of resorufin is detected to reflect the enzyme catalytic reaction rate and enzyme activity. The ability of the compound to inhibit the GLS1 activity of the cells was evaluated by determining the glutamate consumption of the cells using the glutaminase in A549 cells and coupling analysis. The specific method is as follows: 3-5 x 10 3A549 cells / well were seeded into 96-well plates at a density of [number] cells / well, and different concentrations of the test compound solutions were added. The plates were incubated at 37°C for 24 hours. After incubation, the supernatant was discarded, and the cells were washed 1-2 times with PBS. 20 μL of cell lysis buffer (containing 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM MEGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate) was added, and the cells were lysed on ice for 30 minutes. The cell lysis buffer was collected into 1.5 ml centrifuge tubes and centrifuged at 12000 rpm for 10 minutes. After centrifugation, 4 μL of the supernatant cell lysis buffer was transferred to a 384-well plate, and 35 μL of assay solution (containing 27.5 μm Amplex Red, 0.1375 U / mL horseradish peroxidase, 0.044 U / mL glutamate oxidase, 100 mM Tris, pH 7.5) was added. After incubation at room temperature in the dark for 30 minutes, the fluorescence values ​​of the wells were read at 535 / 590 nm using a fluorescence microplate reader. The raw data were analyzed and the IC50 was calculated. 50 Values. The GLS1 enzyme activity test results for representative compounds are listed in Table 1.

[0213] 2. GLS1 cell viability assay

[0214] The CCK-8 assay was used to detect cell proliferation and the inhibitory effects of compounds on cell growth. The basic principle is that the CCK-8 reagent contains WST-8, a water-soluble tetrazolium salt, chemically named 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt. This reagent can be reacted with NAD+ produced in cells in the presence of electron-coupling reagents. + It is reduced to a water-soluble yellow formazan product. The more live cells there are, the more formazan is produced, and the darker the color will be. Finally, the absorbance value can be measured colorimetrically to calculate the proportion of live cells. The specific method is as follows: 3–5 × 10⁻⁶ 3 Cell density per well: A549 cells were seeded into 96-well plates and cultured for 24 hours. Different concentrations of the test compound solutions were added, and the plates were incubated at 37°C for 48 hours. After incubation, 10 μL of LCK-8 solution was added to each well, and incubation continued for 3-4 hours. The culture plates were then removed and placed in a microplate reader to measure absorbance at 450 nm. A blank control group was also included. The raw data were analyzed, and the IC50 was calculated. 50 value.

[0215] The results of GLS1 cell viability assays for representative compounds are listed in Table 1.

[0216] Table 1. GLS1 enzyme and GLS1 cell inhibitory activity

[0217]

[0218]

[0219] Positive control compound IPN-60090

[0220] (prepared according to the method reported in patent CN201580037629)

[0221] From the data in Table 1, it can be seen that the representative compounds of the present application (such as compounds 4, 7, 8, 9, 11 and 12) are significantly better than the positive control compound IPN-60090 in inhibiting GLS1 enzyme and cell activity.

[0222] 3. Inhibition of non-small cell lung cancer H2122 cell proliferation

[0223] Select H2122 cells in good growth state, trypsinize. Add fresh culture medium, mix well, and centrifuge at 800 rpm for 3 minutes. Seed in 96-well plates at a density of 2000 cells per well, and incubate in a 37°C incubator overnight. The next day, remove the culture plate, dilute the compounds in a four-fold gradient, administer the dose, and place in a 37°C incubator for 72 hours.

[0224] Place the cell culture plate at room temperature for 30 minutes; add 100 μL of CellTiter Glo detection reagent to each well, mix well on a plate shaker for 2 minutes to induce cell lysis; place the 96-well plate at room temperature for 10 minutes to stabilize the luminescent signal; attach a white bottom film to the bottom of the culture plate, and use Enspire to detect the chemiluminescence value. The IC50 value of each compound is analyzed by nonlinear regression method using XLFit software.

[0225] The results of the representative compounds in inhibiting non-small cell lung cancer H2122 cell proliferation are shown in Table 2.

[0226] Table 2. Inhibition of non-small cell lung cancer H2122 cell proliferation

[0227] Compound IC 50 (nM) <!-- 31 -->]]> IPN-60090 160.5 7 75 9 58.5 12 72

[0228] From the data in Table 2, it can be seen that the representative compounds of the present application (such as compounds 7, 9, 12) are better than the positive control compound IPN-60090 in inhibiting non-small cell lung cancer H2122 cell proliferation.

[0229] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. A compound with the structure shown in the following formula, or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises, as an active ingredient, the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. The use of the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Used to prepare pharmaceutical compositions for treating diseases or conditions mediated by GLS1 activity or expression.

4. The use as described in claim 3, characterized in that, The diseases and conditions mediated by GLS1 activity or expression are selected from the following group: B-cell lymphoma, monocytic leukemia, liver cancer, rectal cancer, bladder cancer, pharyngeal cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, lymphoma, gastric cancer, multiple myeloma, allergic asthma, myelofibrosis, rheumatoid arthritis, splenomegaly with polycythemia, eosinophilic leukocytosis, primary thrombocytopenia, and systemic giant cell disease.

5. The use as described in claim 3, characterized in that, The diseases and conditions mediated by GLS1 activity or expression level are selected from the following group: lung adenocarcinoma, lung squamous cell carcinoma.

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