HDAC (histone deacetylase) inhibitor as well as preparation and application thereof

Abstract

The invention provides an HDAC (histone deacylase) inhibitor as well as preparation and application thereof, specifically, the inhibitor has a structure as shown in a formula I. The compound can inhibit the activity of histone deacylase (HDAC) and can be used for preparing the HDAC inhibitor and / or medicines for treating and / or preventing and relieving malignant tumors and immune diseases related to HDAC. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a histone deacetylase (HDAC) inhibitor and its preparation and use. Background Art

[0002] Protein acetylation is one of the most common protein post-translational modifications, which is produced by the transfer of acetyl groups from acetyl-CoA to the ε-amino side chain of lysine by lysine acetyltransferase (KAT). Protein acetylation is a dynamic post-translational modification, and the process is reversible. Its deacetylation process is catalyzed by histone deacetylases (HDACs), also known as lysine deacetylases (KDAC). Histones are one of the main substrates for acetylation. Acetylation can neutralize positive charges, reduce the affinity between histones and DNA helices, lead to chromatin relaxation and chromatin conformation opening, increase the binding of DNA to transcription complexes, and promote gene activation. On the other hand, non-histone proteins can also be modified by acetylation. Acetylation of non-histone proteins is involved in regulating protein stability, activity, subcellular localization and function, thereby regulating physiological and pathological processes such as cell division, signal transduction, autophagy and metabolism.

[0003] So far, 18 HDACs have been identified in mammals, which are divided into four categories according to their domain structure and homology with yeast protein sequences: Class I (HDACs 1, 2, 3, and 8) are homologous to yeast Rpd3, are mainly located in the nucleus, and have high enzymatic activity; Class II is homologous to yeast Hda1, of which Class IIa (HDACs 4, 5, 7, and 9) are characterized by shuttling between the nucleus and the cytoplasm in a phosphorylation-dependent manner, and Class IIb (HDAC6 and 10) have two catalytic domains; Class III (also called sirtuins, including SIRT1-7) are homologous to yeast SIR2 and are NAD+-dependent enzymes, while other classes of HDACs are Zn+-dependent. 2+ Class IV (HDAC 11) has a conserved sequence shared with class I and II HDACs, but has greater tissue specificity and is highly expressed in the brain, heart, testis, and kidney.

[0004] Currently, several HDAC inhibitors have been approved by the FDA for the clinical treatment of cutaneous T-cell lymphoma, peripheral T-cell lymphoma and multiple myeloma. In addition, cedabenamide has been approved by China's NMPA for the treatment of peripheral T-cell lymphoma and breast cancer. Clinical trials exploring the tumor indications of HDAC inhibitors also include acute myeloid leukemia, diffuse large B-cell lymphoma, glioblastoma, non-small cell lung cancer, pancreatic cancer, etc. In addition to anti-tumor activity, HDAC inhibitors also have immunomodulatory effects, enhance T cell activation, and promote T cell secretion of cytokines (Blood 2012; 119(11):2443-51.doi:10.1182 / blood-2011-10-29200). HDAC inhibitors can promote tumor antigen presentation and regulate the tumor microenvironment, thereby enhancing the anti-tumor effect of immune checkpoint inhibitors (J Clin Invest. 2021; 131(16): e138560.doi: 10.1172 / JCI138560; Clin Cancer Res. 2017; 23(17) doi: 10.1158 / 1078-0432.CCR-17-0741). The anti-tumor effects of multiple HDAC inhibitors combined with immune checkpoint inhibitors are under clinical research and evaluation. However, the clinical toxicity of pan-HDAC inhibitors and class I HDAC inhibitors leads to a narrow therapeutic window, which is the main reason restricting their indication expansion and combination therapy. Therefore, the development of more selective HDAC1 / 2 inhibitors that have both anti-tumor and immunomodulatory functions and reduce the side effects caused by targeting other HDAC subtypes is expected to bring more benefits to cancer patients in clinical practice.

[0005] Therefore, it is necessary to provide a class of novel and selective HDAC inhibitors. Summary of the invention

[0006] The purpose of the present invention is to provide a histone deacetylase (HDAC) inhibitor with a novel structure.

[0007] In the first aspect of the present invention, there is provided a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, or isotope thereof,

[0008]

[0009] in, is a single bond or a double bond;

[0010] X1 is selected from N, NH, NR a R b , CR a R b , CHR a R b ;

[0011] X2 is selected from O, NH, CR a R b , CHR a R b ;

[0012] R a , R b Each is independently selected from the following group: H, deuterium, tritium, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C4 alkoxy; or R a , R b The atoms connected thereto together form a substituted or unsubstituted group selected from the group consisting of a C3-C8 cycloalkyl group, a C6-C8 aromatic ring, a 5-8 membered aromatic heterocycle, or a 3-8 membered heterocycle;

[0013] Ring A is a 5-10 membered heterocyclic ring or a 5-10 membered aromatic heterocyclic ring; the heterocyclic ring may be saturated or partially unsaturated;

[0014] R1 is each independently halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy;

[0015] q is each independently 0, 1, 2 or 3;

[0016] Ar is independently selected from: C6-C10 aromatic ring, 5-12 membered heteroaromatic ring;

[0017] R2 and R3 are each independently selected from the group consisting of NH2, substituted or unsubstituted C1-C4 alkyl, halogen;

[0018] m is 0, 1, 2, 3, 4, 5 or 6;

[0019] n is 0, 1, 2, 3, 4, 5 or 6;

[0020] k is 0 or 1;

[0021] Cy is a group selected from the group consisting of a C1-C8 alkyl group, a C6-C10 aromatic ring, a C3-C10 cycloalkyl group, a 5-12 membered heteroaromatic ring, and a 5-12 membered heterocyclic ring;

[0022] Unless otherwise specified, the heterocyclic ring or aromatic heterocyclic ring includes 1, 2 or 3 heteroatoms selected from N, S or O as the ring skeleton; the heterocyclic ring or aromatic heterocyclic ring may be substituted or unsubstituted;

[0023] In the above definitions of each substituent, the substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: deuterium, tritium, halogen, hydroxyl, amino, cyano, C2-6 alkynyl, -SF5, halogenated or unhalogenated C1-C4 alkyl, C3-C10 cycloalkyl, C1-C4 alkoxy, C1-C6 amide, C1-C6 alkylamino, C6-C10 aryl, five-membered or six-membered heteroaryl, five-membered or six-membered heterocyclic group.

[0024] In another preferred embodiment, the compound is a structure shown in Formula I-1, I-2, I-3 and I-4:

[0025]

[0026] Y is selected from the group consisting of: (CH2) p , CH=CH;

[0027] p is each independently 1, 2, 3, 4 or 5;

[0028] The remaining substituents are as described above.

[0029] In another preferred embodiment, the compound is a structure represented by Formula IA, IB, IC, ID or IE:

[0030]

[0031]

[0032] Where R c Each is independently H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or substituted C3-C6 cycloalkyl;

[0033] Y is selected from the group consisting of: (CH2) p , CH=CH;

[0034] p is each independently 1, 2, 3, 4 or 5;

[0035] The remaining substituents are as described above.

[0036] In another preferred embodiment, in the compound, Ar is a group selected from the following group: phenyl, pyridyl, pyrimidinyl, pyridazinyl, tetrazinyl, triazinyl, pyrrolyl, thienyl, furanyl, tetrazolyl, triazolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazole, isoxazolyl, oxadiazolyl, thiadiazolyl, naphthyl, indolyl, indazolyl, quinolyl, isoquinolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzotriazolyl, and morpholinyl.

[0037] In another preferred embodiment, the Ar is a phenyl group.

[0038] In another preferred embodiment, the R2 is an amino group.

[0039] In another preferred embodiment, Cy in the compound is selected from the following group: benzene ring, thiophene ring; and / or

[0040] R3 is each independently halogen; n is 0, 1, 2, 3 or 4.

[0041] In another preferred embodiment, the compound is a structure represented by formula I-AA, I-BB, I-CC, I-DD or I-EE:

[0042]

[0043] Where R c Each is independently H, substituted or unsubstituted C1-C4 alkyl, substituted or substituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy;

[0044] Y is selected from the group consisting of: (CH2) p , CH=CH;

[0045] p is each independently 1, 2, 3, 4 or 5;

[0046] Cy is selected from the group consisting of benzene ring, thiophene;

[0047] The remaining substituents are as defined above.

[0048] In another preferred embodiment, X1 is selected from N, NH or CR a R b ;

[0049] X2 is selected from O, NH, CR a R b ;

[0050] R a , R b Each is independently selected from the following group: H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C4 alkoxy;

[0051] R1 is each independently halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy;

[0052] q is each independently 0, 1, 2 or 3;

[0053] R2 and R3 are each independently selected from the group consisting of NH2, substituted or unsubstituted C1-C4 alkyl, and halogen.

[0054] In another preferred embodiment, the compound has a structure as shown in formula I-AAA, I-AAB and I-AAC:

[0055]

[0056] The remaining substituents are as described above.

[0057] In another preferred embodiment, X1 is selected from N, NH or CR a R b ;

[0058] X2 is independently selected from O, NH, CR a R b ;

[0059] R a , R b Each is independently selected from the following group: H, substituted or unsubstituted C1-C8 alkyl;

[0060] R1 is each independently halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy;

[0061] q is each independently 0, 1, 2 or 3;

[0062] R2 and R3 are each independently selected from the group consisting of NH2, substituted or unsubstituted C1-C4 alkyl, and halogen.

[0063] In another preferred embodiment, the compound has a structure as shown in the formula I-DDA, I-DDB, I-DDC:

[0064]

[0065] In another preferred embodiment, the compound can be selected from the following group:

[0066]

[0067]

[0068] In the second aspect of the present invention, a pharmaceutical composition is provided, comprising: (A) a therapeutically effective amount of a compound of Formula I, its enantiomers, diastereomers, racemates, isotopes and mixtures thereof, and one or more of their pharmaceutically acceptable salts, crystalline hydrates and solvates; and (B) a pharmaceutically acceptable carrier.

[0069] In the third aspect of the present invention, there is provided a use of the compound as described in the first aspect of the present invention or the composition as described in the second aspect of the present invention, for preparing: (a) a histone deacetylase (HDAC) inhibitor; and / or (b) a drug for treating and / or preventing or alleviating HDAC-related malignant tumors; and / or (c) a drug for treating malignant tumors in combination with an immune checkpoint inhibitor.

[0070] In another preferred embodiment, the drug used in combination with an immune checkpoint inhibitor to treat malignant tumors is used for immunomodulation, enhancing T cell activation, promoting T cell secretion of cytokines, promoting tumor antigen presentation, and regulating the tumor microenvironment.

[0071] In another preferred embodiment, the malignant tumor is selected from the following group: cutaneous T-cell lymphoma, peripheral T-cell lymphoma, T-lymphocytic leukemia, multiple myeloma, breast cancer, acute myeloid leukemia, diffuse large B-cell lymphoma, glioblastoma, non-small cell lung cancer, pancreatic cancer, neuroblastoma, malignant peripheral nerve sheath tumor (MPNST), esophageal cancer, bladder cancer, meningeal cancer, mesothelioma, melanoma, astrocytoma, undifferentiated pleomorphic sarcoma, head and neck cancer, gastric adenocarcinoma, myxofibrosarcoma, bile duct cancer, as well as brain cancer, gastric cancer, kidney cancer, endometrial cancer, cervical cancer, urethral cancer, liver cancer, lung cancer, soft tissue cancer, pleural cancer and colorectal cancer or sarcoma.

[0072] In another preferred embodiment, the malignant tumor is selected from the following group: cutaneous T-cell lymphoma, peripheral T-cell lymphoma, T-lymphocytic leukemia, multiple myeloma, breast cancer, acute myeloid leukemia, diffuse large B-cell lymphoma, glioblastoma, non-small cell lung cancer, pancreatic cancer.

[0073] In another preferred embodiment, the malignant tumor is selected from the following group: T-cell lymphoma, T-lymphocytic leukemia, and non-small cell lung cancer.

[0074] In another preferred embodiment, the HDAC is HDAC1 or HDAC2.

[0075] In another preferred embodiment, the combined immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.

[0076] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is a Western Blot image of H3K27Ac, ​​H3K9Ac and H3 in A549 cells.

[0078] Figure 2 The graph of Example 4, compound 4A and 4B concentration and IL-2 concentration is shown in FIG. DETAILED DESCRIPTION

[0079] After extensive and in-depth research, a large number of screenings and tests, the inventors have provided a compound as shown in Formula I, which is an HDAC inhibitor and can be further prepared as a drug for treating and / or preventing and alleviating HDAC-related malignant tumors. On this basis, the present invention was completed.

[0080] the term

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0082] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0083] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0084] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0085] As used herein, the term "alkyl" includes straight or branched alkyl groups. For example, C1-C8 alkyl groups represent straight or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.

[0086] As used herein, the term "alkynyl" includes straight or branched alkynyl groups. For example, C2-C6 alkynyl refers to a straight or branched alkynyl group having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

[0087] As used herein, the term "cycloalkyl" refers to a cyclic saturated or partially unsaturated aliphatic hydrocarbon group having a specific number of carbon atoms. 3-10 Cycloalkyl refers to a cyclic saturated or partially unsaturated aliphatic hydrocarbon group having 3 to 10 carbon atoms. It can be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a similar group. It can also be a bicyclic form, such as a bridged ring or a spirocyclic form.

[0088] As used herein, the term "alkylamino" refers to an amine group substituted by an alkyl group. For example, "C1-C8 alkylamino" refers to an amine group substituted by a C1-C8 alkyl group, which may be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino, etc.

[0089] As used herein, the term "alkoxy" refers to a group having an alkyl-oxy structure. For example, "C1-C8 alkoxy" refers to a straight or branched alkoxy group having 1 to 8 carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, etc.

[0090] As used herein, the term "haloalkyl" represents an alkyl group wherein one or more hydrogen atoms are replaced by halogen, wherein alkyl is as defined above.

[0091] As used herein, the term "haloalkoxy" represents an alkoxy group having one or more hydrogen atoms replaced by a halogen, wherein alkoxy is as defined above.

[0092] As used herein, the term "heterocyclyl" or "heterocycloalkyl" refers to a saturated or partially saturated cyclic group having a specific number of ring atoms (e.g., 3-10 ring atoms), wherein 1-3 atoms are heteroatoms selected from N, S and O. It can be a monocyclic, bicyclic or polycyclic form, such as a bridged ring or a spirocyclic form. Specific examples can be oxetanyl, azetidinyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl and pyrrolidinyl, etc.

[0093] As used herein, the term "aryl" refers to an aromatic ring group having a specific number of carbon atoms, such as C6-C 10 The aryl group represents an aromatic ring group having 6 to 10 carbon atoms, for example, phenyl or naphthyl or the like.

[0094] As used herein, the term "heteroaromatic ring" refers to a cyclic aromatic group having a specific number of atoms, wherein 1 to 3 atoms are heteroatoms selected from N, S and O, for example, a 5-12 membered heteroaromatic ring represents an aromatic ring group having 5 to 12 carbon atoms. It may be a monocyclic ring or a condensed ring. Specific examples may include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, etc.

[0095] Unless otherwise specified as "substituted or unsubstituted", the groups described in the present invention may be substituted by substituents selected from the following groups: deuterium, tritium, halogen, hydroxyl, amino, cyano, C2-6 alkynyl, -SF5, halogenated or unhalogenated C1-C4 alkyl, C3-C10 cycloalkyl, C1-C4 alkoxy, C1-C6 amide, C1-C6 alkylamino, C6-C10 aryl, five-membered or six-membered heteroaryl, five-membered or six-membered non-aromatic heterocyclic group.

[0096] As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halogenated" means substituted with an atom selected from F, Cl, Br, and I.

[0097] Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0098] As used herein, the term "tautomer" means that structural isomers with different energies can interconvert across a low energy barrier. For example, proton tautomers (i.e., prototropic) include interconversions via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversions via reorganization of some bonding electrons.

[0099] As used herein, the term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0100] As used herein, the term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

[0101] Active ingredients

[0102] In the present invention, an active ingredient that can effectively inhibit histone deacetylase (HDAC) is provided. The active ingredient is a compound represented by general formula (I), and the active ingredient can effectively prevent, treat and / or alleviate HDAC-related diseases.

[0103] Experiments have shown that the active ingredients of the present invention can effectively inhibit HDAC, thereby preventing, treating and / or alleviating HDAC-related diseases.

[0104] It should be understood that the active ingredients of the present invention include compounds represented by general formula (I), or pharmaceutically acceptable salts thereof, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include crystalline forms, amorphous compounds, and deuterated compounds of the compounds of general formula (I).

[0105] The term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0106] Pharmaceutical compositions and methods of administration

[0107] Since the compounds of the present invention have excellent HDAC inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to prevent, treat and / or alleviate HDAC-related diseases, such as treating cancer.

[0108] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0109] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0110] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration or subcutaneous administration).

[0111] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, 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 may also comprise buffering agents.

[0112] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0113] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0114] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0115] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0116] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0117] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0118] When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable therapeutic agents. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable therapeutic agents can be used simultaneously, separately or sequentially with the compound of the present invention to prevent, treat and / or alleviate HDAC-mediated diseases.

[0119] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0120] The main advantages of the present invention include:

[0121] The compounds of the present invention have excellent subtype selectivity: the compounds of the present invention are selective for HDAC subtypes and can highly inhibit the activity of HDAC1, but at the same time do not affect the activity of HDAC3 and maintain the high activity of HDAC3, thereby reducing the onset of adverse reactions.

[0122] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0123] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0124] The definitions of each abbreviation are as follows:

[0125]

[0126]

[0127] The raw materials can be obtained from commercial sources or prepared by methods known or disclosed in the art.

[0128] All reagents and solvents were purchased from Aldrich, TCI or other domestic suppliers and used without purification. Unless otherwise stated, the reactions were carried out without excluding air or moisture. Analytical thin layer chromatography was performed on silica gel GF-254 plates (domestic) and developed under ultraviolet light. Flash column chromatography was performed on Rf 150 is used in MPLC system Silica Flash Column-CS (Agela, 40-60 μm mesh, 60A) was used. 1 H and 13 C NMR spectra were recorded on a Bruker Ascend TM-400 MHz spectrometer. Chemical shifts are expressed in parts per million (ppm) and referenced to the residual solvent peak. Analytical LC-MS was performed on an Agilent MSD mass spectrometer connected to an Agilent 1260 system.

[0129] Chiral SFC separation, chromatographic column: DAICEL CHIRALCEL OJ (250mm*30mm, 10μm); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 60%, isocratic elution mode; or chromatographic column: ChiralPak IH, 250*30mm, 10μm; mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 40%, isocratic elution mode

[0130] Preparative LC was performed on a Gilson system using a column: Boston Prime C18 150*30mm*5μm. The purity of all final compounds was assessed by LC-MS analysis to be >95%.

[0131] The main analytical instruments and chiral analysis methods are shown in the table below:

[0132]

[0133]

[0134] Synthesis of Example 1

[0135]

[0136] Step 1: (5-Bromo-2-methylthio-phenyl)methanol (1-2)

[0137] DIBALH (1M, 22.9 mL, 3 eq) was added dropwise to a DCM (10 mL) solution of 5-bromo-2-methylsulfanyl-benzoic acid methyl ester (1-1) (2 g, 7.66 mmol, 1 eq) at -70 to -60°C N2. The mixture was stirred for 2 hours at -70 to -60°C N2. The reaction was quenched with MeOH (10 mL) solution at -65°C (temperature rose to -55°C), the reaction solution was returned to room temperature, and then MTBE and diatomaceous earth were added. The mixture was stirred at 25°C for 1 hour. The mixture was filtered through diatomaceous earth, washed with MTBE (60 mL*3) at 20°C, and concentrated under reduced pressure to obtain a residue. The crude product was purified by column chromatography ( 12g+20g Silica gel flash column, eluent 0-16.7% ethyl acetate / petroleum ether gradient elution @40 mL / min, ethyl acetate / petroleum ether = 1 / 4, Rf = 0.60). The fractions of the desired product were combined and concentrated to give (5-bromo-2-methylsulfanyl-phenyl)methanol (1-2) (1.27 g, 5.45 mmol, yield 71.1%, purity 100%) as a white solid.

[0138] LC-MS: Rt = 0.810 min, (ESI) m / z. [M+H] + 233.0; purity: 100%.

[0139] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.58 (d, J = 2.0Hz, 1H), 7.42 (dd, J = 2.1, 8.4Hz, 1H), 7.14 (d, J = 8.3Hz, 1H), 4.75 (s, 2H), 2.49 (s, 3H)

[0140] Step 2: 2-(Azidomethyl)-4-bromo-1-methylthio-benzene (1-3)

[0141] To a solution of (5-bromo-2-methylsulfanyl-phenyl)methanol (1-2) (1.52 g, 6.52 mmol, 1 eq) and DBU (1.19 g, 7.82 mmol, 1.18 mL, 1.2 eq) in THF (30 mL) was added DPPA (2.15 g, 7.82 mmol, 1.69 mL, 1.2 eq). The mixture was stirred at 20 °C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The crude product was purified by column chromatography ( 20g+20g Silica gel flash column, eluent 0-5%-20% ethyl acetate / petroleum ether gradient elution @ 40 mL / min, ethyl acetate / petroleum ether = 1 / 6, Rf = 0.70). The fractions of the desired product were combined and concentrated to give 2-(azidomethyl)-4-bromo-1-methylsulfanyl-benzene (1-3) (1.68 g, 6.51 mmol, yield 100%, purity 100%) as a yellow oil.

[0142] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.40-7.56 (m, 2H), 7.18 (d, J = 8.3Hz, 1H), 4.48 (s, 2H), 2.50 (s, 3H)

[0143] Step 3: 2-(Azidomethyl)-4-bromo-1-methylsulfinyl-benzene (1-4)

[0144] To a solution of 2-(azidomethyl)-4-bromo-1-methylsulfanyl-benzene (1-3) (1.3 g, 5.04 mmol, 1 eq) in DCM (20 mL) was added m-CPBA (1.33 g, 6.55 mmol, 85% purity, 1.3 eq) at 0°C (ice bath), then the ice bath was removed and the mixture was stirred at 20°C for 1 hour. Saturated Na2S2O3 solution (20 mL) was added to the reaction mixture to quench, H2O (60 mL) was added at 20°C to quench the mixture, then diluted with DCM (30 mL) and extracted with DCM (50 mL*2). The combined organic layers were washed with brine (50 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography ( 20g+20g

[0145] Silica gel flash column, eluent 0-35% ethyl acetate / petroleum ether gradient elution @40 mL / min, ethyl acetate / petroleum ether = 1 / 6, Rf = 0.30). The fractions of the desired product were combined and concentrated to give 2-(azidomethyl)-4-bromo-1-methylsulfinyl-benzene (1-4) (1 g, 3.61 mmol, yield 71.7%, purity 99%) as a white oil.

[0146] LC-MS: Rt = 0.575 min, (ESI) m / z. [M+H] + 274.0; purity: 99%.

[0147] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.98 (d, J = 8.4Hz, 1H), 7.77 (dd, J = 2.0, 8.3Hz, 1H), 7.57 (d, J = 1.8Hz, 1H), 4.52 (d, J = 2.4Hz, 2H), 2.81 (s, 3H)

[0148] Step 4: 5-Bromo-1-methyl-3H-1,2-benzothiazole 1-oxide (1-5)

[0149] A mixture of 2-(azidomethyl)-4-bromo-1-methylsulfinyl-benzene (1-4) (500 mg, 1.82 mmol, 1 eq) and iron (II) phthalocyanine (51.8 mg, 91.2 μmol, 0.05 eq) was dissolved in toluene (9 mL), degassed and purged with N2 for 3 minutes, and then the mixture was stirred at 100 ° C for 16 hours. The mixture was purified by column chromatography ( 12g+12g Silica gel flash chromatography column, eluent 0-1.5% dichloromethane / methanol gradient elution @35mL / min, dichloromethane:methanol=15:1, Rf=0.40). The fractions of the desired product were combined and concentrated to give 5-bromo-1-methyl-3H-1,2-benzothiazole 1-oxide (1-5) (420 mg, 1.47 mmol, yield 80.4%, purity 86%) as a light green solid.

[0150] LC-MS: Rt = 0.360 min, (ESI) m / z. [M+H] + 245.9; purity: 86%.

[0151] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.70 (s, 3H), 4.83-5.02 (m, 1H), 4.63-4.80 (m, 1H), 3.40 (s, 3H)

[0152] Step 5: 1-Methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid ethyl ester (1-6)

[0153] A mixture of 5-bromo-1-methyl-3H-1,2-benzothiazole 1-oxide (1-5) (340 mg, 1.38 mmol, 1 eq), Mo(CO)6 (200 mg, 759 μmol, 102 μL, 0.55 eq), DBU (315 mg, 2.07 mmol, 312 μL, 1.5 eq) and tri-tert-butylphosphonium tetrafluoroborate (40.0 mg, 138 μmol, 0.1 eq) in EtOH (5 mL) was degassed and purged with N2 three times, then Pd(OAc)2 (31.0 mg, 138 μmol, 0.1 eq) was added and the mixture was stirred at 80 °C (IKA) for 2 hours. The reaction was cooled to 20 °C, filtered and concentrated to give a residue. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (15 mL*3). The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography ( 4g+12g Silica gel flash column, eluent 0-0.6% dichloromethane / methanol gradient @35 mL / min, dichloromethane:methanol=15:1, Rf=0.6). The fractions of the desired product were combined and concentrated to give 1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid ethyl ester (1-6) (0.165 g, 572 μmol, yield 41.4%, purity 83%) as a brown oil.

[0154] LC-MS: Rt = 0.465 min, (ESI) m / z. [M+H] + 240.1; purity: 83%.

[0155] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.14-8.36 (m, 2H), 7.91 (d, J = 8.1Hz, 1H), 4.93-5.13 (m ,1H),4.72-4.88(m,1H),4.46(q,J=7.2Hz,2H),3.45(s,3H),1.45(t,J=7.1Hz,3H)

[0156] Step 6: 1-Methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid (1-7)

[0157] LiOH-H2O (2M, 1.03 mL, 3 eq) was added to a solution of 1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid ethyl ester (1-6) (165 mg, 689 μmol, 1 eq) in MeOH (1.5 mL) and THF (1.5 mL). The mixture was stirred at 25°C (IKA, heating) for 4 hours. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL). The pH value of the aqueous layer was adjusted to 3-4 with AcOH and then lyophilized. The residue was purified by preparative HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; gradient: 0%-20% B in 11 minutes). The fractions of the desired product were combined and lyophilized to give 1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid (1-7) (65 mg, 292 μmol, 42.4% yield, 95% purity) as a white solid.

[0158] Step 7: N-[4-(4-fluorophenyl)-2-[(1-methyl-1-oxo-3H-1,2-benzothiazole-5-carbonyl)amino]phenyl]carbamate (1-8)

[0159] To a solution of 1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid (1-7) (55 mg, 260 μmol, 1 eq) in pyridine (1.1 mL) were added tert-butyl (3-amino-4'-fluoro-[1,1'-biphenyl]-4-yl) carbamate (78.7 mg, 260 μmol, 1 eq) and EDCI (59.9 mg, 312 μmol, 1.2 eq). The mixture was stirred at 50 °C for 2 hours. 10 mL of H2O was added to the reaction mixture and extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography ( 4g Silica gel flash chromatography column, eluent 0-1.2% dichloromethane / methanol gradient elution @20 mL / min, dichloromethane / methanol = 15 / 1, Rf = 0.60). The fractions of the desired product were combined and concentrated to give tert-butyl N-[4-(4-fluorophenyl)-2-[(1-methyl-1-oxo-3H-1,2-benzothiazole-5-carbonyl)amino]phenyl]carbamate (1-8) (110 mg, 219 μmol, yield 84.4%, purity 99%) as a white solid.

[0160] LC-MS: Rt = 1.202 min, (ESI) m / z. [M+H] + 496.1; purity: 99%.

[0161] Step 8: N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide

[0162] (Example 1)

[0163] Tert-butyl N-[4-(4-fluorophenyl)-2-[(1-methyl-1-oxo-3H-1,2-benzothiazole-5-carbonyl)amino]phenyl]carbamate (1-8) (110 mg, 222 μmol, 1 eq) was dissolved in TFA (614 mg, 5.39 mmol, 0.4 mL, 24.2 eq) and DCM (2 mL). The mixture was stirred at 20 ° C for 1 hour. The mixture was concentrated under reduced pressure to obtain a residue. The residue was dissolved in MeCN and H2O, and NH3.H2O was added to adjust pH = 8. Then a solid was precipitated and filtered to obtain a white solid. MeCN and H2O were added to the white solid, followed by lyophilization to give N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (Example 1) (18 mg, 44.6 μmol, yield 20.1%, purity 98%) as a yellow solid.

[0164] LC-MS: Rt=0.557min, (ESI)m / z.[M+H]+396.0;

[0165] HPLC: Rt = 3.547 min; purity: 98.2%.

[0166] 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.95 (s, 1H), 8.13-8.25 (m, 3H), 7.59 (dd, J = 5.5, 8.6Hz, 2H), 7.52 (d, J = 1. 8Hz,1H),7.33(dd,J=2.0,8.4Hz,1H),7.23(t,J=8.8Hz,2H),6.87(d,J=8.4Hz,1H),5.76(s,1H),5.19(br s,2H),4.77-4.89(m,1H),4.61-4.73(m,1H),3.49(s,3H). 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.45.

[0167] Step 9: (1S)-N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (Example 1A) and (1R)-N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (Example 1B)

[0168] N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (22 mg, 55.6 μmol, 1 eq) was further separated by SFC (chromatographic column: DAICEL CHIRALCEL OJ (250 mm*30 mm, 10 um); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 60%, isocratic elution mode). The target product fractions were combined and concentrated, MeCN and H2O were added, and freeze-dried to obtain the target product. According to the SFC peak order, (1S)-N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (Example 1A) (5.3 mg, 13.4 μmol, yield 24.0%, purity 100%) was first obtained as a white solid. Then (1R)-N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (Example 1-B) (4.3 mg, 10.8 μmol, yield 19.5%, purity 100%) was obtained as a white solid. The chirality of Example 1A and Example 1B was randomly assigned.

[0169] Example 1A Characterization:

[0170] LC-MS: Rt=0.822min, (ESI)m / z.[M+H]+396.2;

[0171] SFC: Rt=1.105min;ee%=100%;

[0172] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.94 (s, 1H), 8.08-8.32 (m, 3H), 7.59 (dd, J = 5.6, 8.5Hz, 2H), 7.52 (s, 1H), 7.33 (dd, J = 1.9, 8.3Hz,1H),7.23(t,J=8.9Hz,2H),6.87(d,J=8.4Hz,1H),5.19(s,2H),4.74-4.90(m,1H),4.60-4.72(m,1H),3.49(s,3H)

[0173] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.462

[0174] Example 1B Characterization:

[0175] LC-MS: Rt=0.822min, (ESI)m / z.[M+H]+396.2;

[0176] SFC: Rt=1.783min;ee%=99.3%;

[0177] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.94 (s, 1H), 8.10-8.28 (m, 3H), 7.59 (dd, J=5.5, 8.6Hz, 2H), 7.52 (d, J=2.0Hz, 1H), 7.33 (dd, J= 2.2,8.4Hz,1H),7.23(t,J=8.8Hz,2H),6.87(d,J=8.4Hz,1H),5.19(s,2H),4.76-4.89(m,1H),4.62-4.73(m,1H),3.49(s,3H)

[0178] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.456

[0179] SFC method: Chromatographic column: Chiral OJ-350×4.6mm ID, 3um Mobile phase: A: CO2 B: Ethanol (0.05% DEA) Gradient: 50% B Flow rate: 2.5mL / min Column temperature: 35℃ ABPR: 1500psi

[0180] Synthesis of Example 2

[0181]

[0182] Step 1: Methyl benzothiophene-5-carboxylate (2-2)

[0183] To a solution of benzothiophene-5-carboxylic acid (2-1) (1 g, 5.61 mmol, 1 eq) in MeOH (15 mL) was added SOCl2 (1.00 g, 8.42 mmol, 611 μL, 1.5 eq), and the reaction was stirred at 60 ° C for 5 hours. LC-MS showed that the starting material was completely consumed and a major peak with the desired mass was detected. The reaction was concentrated under reduced pressure to obtain a residue. The residue was used in the next step without purification. Benzothiophene-5-carboxylic acid methyl ester (1-2) (1.06 g, 5.51 mmol, yield 98.27%) was obtained as a gray solid.

[0184] LC-MS: Rt=0.648min, (ESI)m / z.[M+H] + =193.1.

[0185] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.54 (d, J = 1.3Hz, 1H), 8.15 (d, J = 8.6Hz, 1H), 7.83-7.95 (m, 2H), 7.63 (d, J = 5.5Hz, 1H), 3.90 (s, 3H)

[0186] Step 2: 1-Oxobenzothiophene-5-carboxylic acid methyl ester (2-3)

[0187] To a solution of methylbenzothiophene-5-carboxylate (2-2) (650 mg, 3.38 mmol, 1 eq) in DCM (3.5 mL) and TFA (3.5 mL) was added H2O2 (610 mg, 5.38 mmol, 517 μL, 30% purity, 1.59 eq), and the reaction was stirred at 20°C for 5 hours. LC-MS showed that the starting material was completely consumed and a major peak with the desired mass was detected. The reaction was quenched with Na2CO3 (aq., 2 mL) and Na2S2O3 (715 mg, 4.52 mmol, 1.34 eq), extracted with DCM (10 mL*3), and the combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give a residue. The residue was purified by flash silica gel chromatography ( 12g Silica gel flash chromatography column, eluent: 0-50% ethyl acetate / petroleum ether gradient elution @35 mL / min, dichloromethane / ethyl acetate = 1 / 2, Rf = 0.5). 1-Oxobenzothiophene-5-carboxylic acid methyl ester (2-3) (236 mg, 1.13 mmol, yield 33.52%) was obtained as a white solid.

[0188] LC-MS: Rt=0.757min, (ESI)m / z.[M+H]+209.0.

[0189] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.25 (d, J = 1.0 Hz, 1H), 8.12-8.17 (m, 1H), 8.06-8.11 (m, 1H), 7.53-7.65 (m, 2H), 3.91 (s, 3H).

[0190] Step 3: 1-tert-Butyloxycarbonylimino-1-oxo-benzothiophene-5-carboxylic acid methyl ester (2-4)

[0191] To a mixture of methyl 1-oxobenzothiophene-5-carboxylate (2-3) (210 mg, 1.01 mmol, 1 eq) and NH2Boc (236 mg, 2.02 mmol, 2.0 eq) in DCM (3.5 mL) were added PhI(OAc)2 (487 mg, 1.51 mmol, 1.5 eq), MgO (162 mg, 4.03 mmol, 45.4 μL, 4 eq) and Rh2(OAc)4 (11.1 mg, 25.2 μmol, 0.025 eq), and the reaction was stirred at 40 °C for 16 hours. LC-MS showed that the starting material was completely consumed and a major peak with the desired mass was detected. The reaction was diluted with DCM (2 mL), filtered and concentrated to give a residue. The residue was purified by silica gel flash chromatography ( 12g Silica gel flash chromatography column, eluent 0-25% ethyl acetate / petroleum ether gradient elution @30 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.6). The fractions of the desired product were combined and concentrated to obtain 1-tert-butoxycarbonylimino-1-oxo-benzothiophene-5-carboxylic acid methyl ester (2-4) (270 mg, 835 μmol, yield 82.79%) as a white solid.

[0192] LC-MS: Rt=0.925min, (ESI)m / z.[M+H-Boc] + 224.0.

[0193] 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.14-8.35 (m, 3H), 7.88 (d, J = 6.7Hz, 1H), 7.68 (d, J = 6.6Hz, 1H), 3.93 (s, 3H), 1.40 (s, 9H).

[0194] Step 4: 1-tert-Butyloxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid methyl ester (2-5)

[0195] 1-tert-Butyloxycarbonylimino-1-oxo-benzothiophene-5-carboxylic acid methyl ester (2-4) (165 mg, 510.26 μmol, 1 eq) was dissolved in EtOAc (10 mL), Pd / C (80 mg) was added, and the reactants were degassed under reduced pressure and purged with H2 several times. The reactants were stirred at 25 ° C for 16 hours under a H2 balloon. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The reactants were filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 4g Silica gel flash chromatography column, eluent: 0-20% ethyl acetate / petroleum ether gradient elution @25 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.5). The fractions of the desired product were combined and concentrated to obtain 1-tert-butyloxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid methyl ester (2-5) (140 mg, 430 μmol, yield 84.32%) as a white foam.

[0196] LC-MS: Rt=0.880min, (ESI)m / z.[M+H]+326.1.

[0197] 1 H NMR(400MHz,DMSO-d6)δ(ppm)8.14(s,1H),8.05-8.12(m,2H),4.04-4.11(m,1H),3 .92(s,3H),3.80-3.89(m,1H),3.51-3.63(m,1H),3.39-3.50(m,1H),1.37(s,9H).

[0198] Step 5: 1-tert-Butyloxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-6)

[0199] 1-tert-Butyloxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid methyl ester (2-5) (120 mg, 369 μmol, 1 eq) was dissolved in THF (1 mL), MeOH (1 mL) and H2O (1 mL), and LiOH.H2O (40 mg, 953 μmol, 2.58 eq) was added, and then stirred at 25 ° C for 2 hours. LC-MS showed that the reaction was complete and the desired product mass was detected. The reactants were concentrated to remove THF and MeOH, acidified to pH = 6-7 with AcOH, and a white solid was precipitated. The filter cake was filtered and washed with H2O (2 mL*3), and dried under reduced pressure to obtain 1-tert-Butyloxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-6) (92 mg, 295 μmol, yield 80.12%) as a white solid.

[0200] LC-MS: Rt=0.818min, (ESI)m / z.[M+H]+312.0

[0201] Step 6: tert-Butyl N-[5-[[2-(tert-butoxycarbonylamino)-4-(4-fluorophenyl)phenyl]carbamoyl]-1-oxo-2,3-dihydrobenzothiophene-1-methylene]carbamate (2-7)

[0202] EDCI (55 mg, 287 μmol, 1.28 eq) was added to a solution of 1-tert-butyloxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-6) (70 mg, 225 μmol, 1 eq) and tert-butyl N-[2-amino-4-(4-fluorophenyl)phenyl]carbamate (70 mg, 232 μmol, 1.03 eq) in Py (1.5 mL), and the reaction was stirred at 50 ° C for 2 hours. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The reactants were combined, diluted with H2O (2 mL), extracted with EtOAc (3 mL*3), and the combined organic layers were dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 12g Silica gel flash chromatography column, eluent 0-50% ethyl acetate / petroleum ether gradient elution @35 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.55). The fractions of the desired product were combined and concentrated to give tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-4-(4-fluorophenyl)phenyl]carbamoyl]-1-oxo-2,3-dihydrobenzothiophene-1-methylene]carbamate (2-7) (135 mg, 227 μmol, yield 88.42%) as white foam.

[0203] LC-MS: Rt=1.085min, (ESI)m / z.[M+H] + 596.3.

[0204] Step 7: N-[2-amino-4-(4-fluorophenyl)phenyl]-1-imino-1-oxo-2,3-dihydrobenzothiophene-5-carboxamide (Example 2)

[0205] Tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-4-(4-fluorophenyl)phenyl]carbamoyl]-1-oxo-2,3-dihydrobenzothiophene-1-methylene]carbamate (2-7) (125 mg, 209 μmol, 1 eq) was dissolved in TFA (0.5 mL) and DCM (2.5 mL) and stirred at 25 ° C for 1 hour. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The solvent was removed to obtain a residue. The residue was purified by preparative HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; gradient: 30%-50% B, 11 minutes). The fractions of the desired product were combined and lyophilized to give N-[2-amino-4-(4-fluorophenyl)phenyl]-1-imino-1-oxo-2,3-dihydrobenzothiophene-5-carboxamide (Example 2) (25 mg, 63.2 μmol, 30.13% yield, 100% purity) as a white solid.

[0206] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.87 (s, 1H), 7.96-8.16 (m, 2H), 7.72-7.84 (m, 1H), 7.58 (dd, J = 5.6, 8.4Hz, 2H), 7.50 (d, J = 1.5Hz, 1H), 7. 31(dd,J=2.0,8.3Hz,1H),7.22(t,J=8.8Hz,2H),6.86(d,J=8.3Hz,1H),5.15(s,2H),4.70(s,1H),3.50-3.59(m,2H),3.37-3.43(m,2H)

[0207] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.46(br s,1F)

[0208] LC-MS: Rt=0.783min, (ESI)m / z.[M+H]+396.1

[0209] Synthesis of Example 3

[0210]

[0211] Step 1: 5-Bromo-2,3-dihydro-1,2-benzothiazole 1,1-dioxide (3-2)

[0212] To a solution of 5-bromo-1,1-dioxo-1,2-benzothiazol-3-one (3-1) (900 mg, 3.4 mmol, 1 eq) in THF (23 mL) was added LiAlH4 (2.5 M, 1.8 mL, 1.3 eq) dropwise at 0°C for 3 minutes. The resulting mixture was stirred at 25°C under N2 atmosphere for 2 hours. LC-MS showed that the reactant was completely consumed and a major peak with the desired m / z appeared. EtOAc (25 mL) and H2SO4 (10%, 9 mL) were added to the reactant, extracted with EtOAc / THF (1 / 1, 40 mL*3), dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 20g Silica gel flash chromatography column, eluent 0-35%, ethyl acetate / petroleum ether @20 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.45). 5-bromo-2,3-dihydro-1,2-benzothiazole 1,1-dioxide (3-2) (510 mg, 2 mmol, yield 59.8%) was obtained as a white solid.

[0213] LC-MS: Rt=0.514min, (ESI)m / z.[M+H]+247.9;

[0214] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 7.89-7.95 (m, 1H), 7.83-7.86 (m, 1H), 7.74-7.81 (m, 2H), 4.35-4.45 (m, 2H).

[0215] Step 2: 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylic acid ethyl ester (3-3)

[0216] A mixture of 5-bromo-2,3-dihydro-1,2-benzothiazole 1,1-dioxide (3-2) (510 mg, 2.06 mmol, 1 eq), Mo(CO)6 (298 mg, 1.13 mmol, 152 μL, 0.55 eq), DBU (469 mg, 3.08 mmol, 464 μL, 1.5 eq) and tri-tert-butylphosphonium tetrafluoroborate (59.6 mg, 205 μmol, 0.1 eq) in EtOH (10 mL) was degassed and purged with N2 for 3 times, then Pd(OAc)2 (46 mg, 205 μmol, 0.1 eq) was added and the mixture was stirred at 90 °C for 2 hours. LC-MS showed that the reactants were completely consumed and the desired m / z was detected. The reaction was cooled to room temperature, filtered and concentrated to obtain a residue. The reaction mixture was diluted with water 10 mL and extracted with EtOAc (15 mL*3). The combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography ( 12g Purification by silica gel flash chromatography, eluent 0-30%, ethyl acetate / petroleum ether @30 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.58) gave 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylic acid ethyl ester (3-3) (200 mg, 828 μmol, yield 40.3%) as a white solid.

[0217] LC-MS: Rt=0.534min, (ESI)m / z.[M+H]+242.0;

[0218] 1 H NMR(400MHz,DMSO-d6)δ(ppm)8.13-8.17(m,1H),8.07-8.12(m,1H),7.94-7.99(m,1 H),7.75-7.81(m,1H),4.48(d,J=4.8Hz,2H),4.33-4.39(m,2H),1.34-1.37(m,3H).

[0219] Step 3: 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylic acid (3-4)

[0220] LiOH.H2O (62.6 mg, 1.49 mmol, 2 eq) was added to a solution of ethyl 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylate (3-3) (180 mg, 746 μmol, 1 eq) in MeOH (2 mL). The mixture was stirred at 20 °C for 2 hours. LC-MS showed that the starting material had been consumed. The solvent was removed to give a residue. The pH of the residue was adjusted to 1-2 with 2M HCl. The suspension was filtered and the combined filtrate was dried by freeze drying. The crude product was used in the next step without further purification. 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylic acid (3-4) (169 mg, crude) was obtained as a white solid.

[0221] LC-MS: Rt=0.283,0.359min, (ESI)m / z.[M+H]+214.0;

[0222] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 7.96-8.08 (m, 2H), 7.82-7.92 (m, 1H), 7.69-7.78 (m, 1H), 4.38-4.45 (m, 2H).

[0223] Step 4: tert-Butyl (3-(1,1-dioxido-2,3-dihydrobenzo[d]isothiazole-5-carboxamido)-4'-fluoro-[1,1'-biphenyl]-4-yl)carbamate (3-5)

[0224] To a solution of 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylic acid (3-4) (130 mg, 365 μmol, 1 eq) in pyridine (2 mL) was added EDCI (84.1 mg, 439 μmol, 1.2 eq) and tert-butyl N-[2-amino-4-(4-fluorophenyl)phenyl]carbamate (110 mg, 365 μmol, 1 eq). The mixture was stirred at 50 °C for 2 hours. LC-MS showed that the reactant 3-4 was completely consumed and a major peak with the desired m / z appeared. The reaction mixture was diluted with 2 mL of H2O and extracted with EtOAc (3 mL*2). The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography ( 12g

[0225] Silica gel flash chromatography column, eluent 0-40%, ethyl acetate / petroleum ether @30 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.38). Yellow solid tert-butyl (3-(1,1-dioxido-2,3-dihydrobenzo[d]isothiazole-5-carboxamido)-4'-fluoro-[1,1'-biphenyl]-4-yl)carbamate (3-5) (100 mg, 201 μmol, yield 54.9%) was obtained.

[0226] Step 5: N-[2-amino-5-(4-fluorophenyl)phenyl]-1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxamide (Example 3)

[0227] Tert-butyl N-[2-[(1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carbonyl)amino]-4-(4-fluorophenyl)phenyl]carbamate (3-5) (90 mg, 180 μmol, 1 eq) was dissolved in DCM (1 mL) and TFA (0.1 mL). The mixture was stirred at 20°C for 2 hours. LC-MS showed that the reactant 3-5 was completely consumed and a major peak with the desired m / z appeared. The reaction mixture was concentrated under reduced pressure to remove the solvent. The pH value of the residue was adjusted to 8-9 with NaHCO3 (aq). The residue was purified by preparative HPLC (column: Boston Prime C18150*30mm*5um; mobile phase: [phase A water (FA)-phase B ACN]; gradient: 33%-53% B in 10 minutes). N-[2-amino-5-(4-fluorophenyl)phenyl]-1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxamide (Example 3) was obtained as a white solid (25 mg, 62.9 μmol, yield 34%, purity 100%).

[0228] 1 H NMR(400MHz,DMSO-d6)δ(ppm)9.90-9.99(m,1H),8.12-8.19(m,2H),7.94-8.06(m,2H),7.54-7.63(m,2H),7.47- 7.53(m,1H),7.28-7.36(m,1H),7.17-7.26(m,2H),6.86(d,J=8.4Hz,1H),5.08-5.26(m,2H),4.44-4.55(m,2H).

[0229] LC-MS: Rt=0.79min, (ESI)m / z.[M+H]+398.1; purity of 100%.

[0230] Synthesis of Example 4

[0231]

[0232] Step 1: Methyl 3-amino-4-(methylsulfanylmethyl)benzoate (4-2)

[0233] Under N2 atmosphere, DMSO (4.65 g, 59.5 mmol, 4.65 mL, 1.8 eq) was dissolved in a mixture of anhydrous CH3CN (10 mL) and DCM (10 mL), the reaction mixture was cooled to -70 °C, TFAA (8.34 g, 39.7 mmol, 5.52 mL, 1.2 eq) was added dropwise, methyl 3-aminobenzoate (5 g, 33.1 mmol, 1.0 eq) (4-1) was dissolved in CH3CN (10 mL) and slowly added to the solution. After the reaction mixture was stirred at -70 °C for 5 hours, CH3ONa (5.4 M, 18.4 mL, 3.0 eq) was added dropwise at -70 °C for 12 minutes, the reaction mixture was warmed to 25 °C and stirred for 12 hours. LC-MS showed that the starting material was completely consumed. After adding NaOH (60 mL, 2.8 M), the aqueous layer was extracted with dichloromethane (65 mL*3), dried over anhydrous MgSO4, and the solvent was removed under reduced pressure. The residue was dissolved in acetonitrile (100 mL), triethylamine (10 mL) was added, and after stirring at 90 ° C for 16 hours, the solvent was removed under reduced pressure. The residue was purified by flash silica gel chromatography ( 120g+40g Silica gel flash chromatography column, eluent 0-6% ethyl acetate / petroleum ether gradient elution @60mL / min, petroleum ether / ethyl acetate = 4 / 1, Rf = 0.68, Rf = 0.63). 3-amino-2-(methylsulfanylmethyl)benzoic acid methyl ester (4-b1) (2.5 g, 11.8 mmol, yield 35.8%) was obtained as a light yellow solid; 3-amino-4-(methylsulfanylmethyl)benzoic acid methyl ester (4-2) (1.8 g, 8.52 mmol, yield 25.8%) was obtained as a light yellow solid.

[0234] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.29-7.37 (m, 2H), 7.00 (d, J = 8.1Hz, 1H), 4.00-4.50 (m, 2H), 3.82 (s, 3H), 3.64 (s, 2H), 1.90 (s, 3H)

[0235] Step 2: 2-Methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid methyl ester (4-3)

[0236] Methyl 3-amino-4-(methylsulfanylmethyl)benzoate (1 g, 4.73 mmol, 1.0 eq) (4-2) was dissolved in DCM (15 mL) and the solution was cooled to -40 °C. A solution of NCS (758 mg, 5.68 mmol, 1.2 eq) in DCM (15 mL) was added dropwise over 60 min, and after 15 min, an aqueous solution of NaOH (10%, 3 mL) was added and the temperature was raised to 25 °C. After adding water (30 mL), the organic layer was separated and cooled to -40 °C, and then m-CPBA (1.15 g, 5.68 mmol, 85% purity, 1.2 eq) was added in small batches over 5 min. The reaction mixture was stirred at -40 °C for 0.5 h. LC-MS showed that the starting material was completely consumed and the target compound was detected. The mixture was warmed to 25°C and washed with saturated aqueous Na2SO3 solution (20 mL) and aqueous NaHCO3 solution (20 mL*2) in sequence, dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. The residue was purified by flash silica gel chromatography ( 12g+4g Silica gel flash column, eluent: 0-40% ethyl acetate / petroleum ether gradient elution @ 60 mL / min, petroleum ether / ethyl acetate = 0 / 1, Rf = 0.53). 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid methyl ester (4-3) (0.43 g, 1.81 mmol, yield 38.3%, purity 95%) was obtained as a light yellow solid.

[0237] LC-MS: Rt=0.447min, (ESI)m / z.[M+H]+226.1;

[0238] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.53 (s, 1H), 7.44 (dd, J = 1.4, 7.8Hz, 1H), 7.21 (s, 1H), 4.40-4.55 (m, 2H), 3.83 (s, 3H), 3.36 (s, 3H)

[0239] Step 3: 2-Methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid (4-4)

[0240] To a solution of methyl 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylate (4-3) (260 mg, 1.15 mmol, 1.0 eq) in MeOH (2 mL) and THF (2 mL) was added a solution of LiOH.H2O (72.7 mg, 1.73 mmol, 1.5 eq) in H2O (2 mL). The mixture was stirred at 25 °C for 16 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. The reaction mixture was concentrated under vacuum to remove THF and MeOH, and then the pH was adjusted to 4-5 with 50% HOAc aq. The aqueous phases were combined for further purification. Half of the crude product was used directly in the next step without further purification to obtain 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid (d2) (200 mg, 568 μmol, 35.7% yield, 60% purity) as a light yellow solid (200 mg crude product after freeze-drying). The other half was purified by preparative HPLC (FA conditions) (chromatographic column: Boston Prime C18150*30mm*5um; mobile phase: [water (FA)-ACN]; gradient: 0%-40% B for 11 min). 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid (4-4) (100 mg, 469 μmol, 29.4% yield, 99% purity) was obtained as a white solid.

[0241] LC-MS: Rt=1.232min, (ESI)m / z.[M+H]+211.9;

[0242] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 12.76 (br s, 1H), 7.30-7.36 (m, 2H), 7.25 (s, 1H), 5.03 (d, J = 17.9Hz, 1H), 4.61 (d, J = 18.0Hz, 1H), 3.52 (s, 3H)

[0243] Step 4: tert-Butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-3H-2,1-benzothiazole-6-carbonyl)amino]phenyl]carbamate (4-5)

[0244] To a solution of 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid (4-4) (90.0 mg, 426 μmol, 1.0 eq) in pyridine (3 mL) were added tert-butyl (3-amino-4'-fluoro-[1,1'-biphenyl]-4-yl) carbamate (142 mg, 469 μmol, 1.1 eq) and EDCI (98.0 mg, 511 μmol, 1.2 eq). The mixture was stirred at 50 °C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. 5 mL of H2O was added, the aqueous phase was extracted with ethyl acetate (5 mL*3), the combined organic phases were washed with brine (5 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography ( 4g+4g Silica gel flash chromatography column, eluent 0-25% ethyl acetate / petroleum ether gradient elution @20 mL / min, petroleum ether / ethyl acetate = 2 / 1, Rf = 0.53). Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-3H-2,1-benzothiazole-6-carbonyl)amino]phenyl]carbamate (4-5) (211 mg, 426 μmol, yield 99.9%, purity 100%) was obtained as a white solid.

[0245] LC-MS: Rt=0.666min, (ESI)m / z.[M+H-56]+440.1;

[0246] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.99 (br s,1H),7.83-7.88(m,1H),7.39-7.47(m,3H),7.24-7.34(m,3H),7.19-7.22(m,1H),7.01(t,J=8.2Hz,2H),6.87(s,1H),4.54(br d,J=17.3Hz,1H),4.43(d,J=17.3Hz,1H),3.35(s,3H),1.45(s,9H)

[0247] 19 F NMR(376MHz,CDCl3)δ(ppm)-115.57(s,1F)

[0248] Step 5: N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxamide (Example 4)

[0249] To a solution of tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-3H-2,1-benzothiazole-6-carbonyl)amino]phenyl]carbamate (4-5) (200 mg, 404 μmol, 1.0 eq) in DCM (4 mL) were added ethoxyethane; trifluoroborane; hydrofluoric acid (261 mg, 807 μmol, 222 μL, 50% purity, 2.0 eq). The mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. The pH value of the reaction mixture was adjusted to 7 with saturated aqueous NaHCO3 solution and then concentrated in vacuo to remove DCM. The residue was purified by preparative HPLC (FA conditions) (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (FA)-ACN]; gradient: 28%-48% B, 11 minutes). N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxamide (Example 4) (75 mg, 190 μmol, yield 47.0%, purity 100%) was obtained as an off-white solid.

[0250] LC-MS: Rt=0.981min, (ESI)m / z.[M+H]+396.0

[0251] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.64 (s, 1H), 7.58 (dd, J = 5.5, 8.6Hz, 2H), 7.48-7.54 (m, 1H), 7.33 -7.41(m,3H),7.30(dd,J=2.1,8.3Hz,1H),7.22(t,J=8.9Hz,2H),6.87(d,J=8.3Hz,1H),5.06(br d,J=17.8Hz,3H),4.62(d,J=17.9Hz,1H),3.54(s,3H)

[0252] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.47(s,1F)

[0253] Synthesis of Example 5

[0254]

[0255] Step 1: 1,3-Dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-2)

[0256] To a solution of sodium sulfanyl sodium nonahydrate (5-1) (1.79 g, 7.45 mmol, 1.25 mL, 1.2 eq) in EtOH (110 mL) was added dropwise a solution of methyl 3,4-bis(bromomethyl)benzoate (2 g, 6.21 mmol, 1.0 eq) in EtOH (30 mL). The mixture was stirred at 25 °C for 16 hours under N2. LC-MS showed that the starting material was completely consumed and the desired product was detected. The reaction mixture was filtered, the filtrate was concentrated to 30 mL under reduced pressure, 50 mL of ethyl acetate was added, and washed with water (3*30 mL). The organic layer was washed with brine (30 mL), dried over MgSO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography ( 20g+12g Silica gel flash chromatography column, eluent 0-4% ethyl acetate / petroleum ether gradient elution, flow rate 30 mL / min, petroleum ether / ethyl acetate = 4 / 1, Rf = 0.64). 1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-2) (1 g, 4.80 mmol, yield 77.3%, purity 100%) was obtained as a light yellow solid.

[0257] LC-MS: Rt=0.667min, (ESI)m / z.[M+H]+209.1;

[0258] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.86 (s, 1H), 7.83 (dd, J = 8.0, 1.4Hz, 1H), 7.24 (d, J = 7.9Hz, 1H), 4.31 (q, J = 7.1Hz, 2H), 4.22 (s, 4H), 1.33 (t, J = 7.1Hz, 3H)

[0259] Step 2: 2-Imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-3)

[0260] A suspension of PhI(OAc)2 (2.71 g, 8.40 mmol, 2.5 eq) was added dropwise to a mixture of 1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-2) (0.7 g, 3.36 mmol, 1.0 eq) and NH3.H2O (2.36 g, 16.8 mmol, 2.59 mL, 25% purity, 5.0 eq) in MeOH (10.5 mL). The mixture was stirred at 25 °C for 16 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. The mixture was concentrated under reduced pressure, saturated Na2SO3 (10 mL) was added, extracted with DCM (10 mL*2), washed with saturated Na2CO3 (10 mL) and concentrated. The residue was purified by flash silica gel chromatography ( 4g+4g Silica gel flash chromatography column, eluent: 0-100% ethyl acetate / petroleum ether gradient elution @20 mL / min, petroleum ether / ethyl acetate = 0 / 1, Rf = 0.3). Obtained black solid 2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-3) (0.32 g, 1.34 mmol, yield 27.8%). LC-MS: Rt = 0.729 min, (ESI) m / z. [M+H] + 240.1

[0261] Step 3: 2-tert-Butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-4)

[0262] Anhydrous THF (3 mL) was added to NaH (100 mg, 2.51 mmol, 60% purity, 2.0 eq) under N2, and the reaction mixture was cooled to 0°C. A solution of ethyl 2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylate (5-3) (300 mg, 1.25 mmol, 1.0 eq) in THF (3 mL) was added dropwise, and the mixture was stirred at 0-25°C for 1 hour, then (Boc)2O (547 mg, 2.51 mmol, 576 μL, 2.0 eq) was added, and the mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired compound was detected. The mixture was quenched with saturated aqueous NH4Cl solution (10 mL) and extracted with DCM (10 mL*3). The organic layers were combined and washed with brine (10 mL), dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by flash silica gel chromatography ( 12g Silica gel flash chromatography column, eluent 0-20% ethyl acetate / petroleum ether gradient elution @30 mL / min, petroleum ether / ethyl acetate = 2 / 1, Rf = 0.4). 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-4) (290 mg, 820 μmol, yield 50.3%, purity 96%) was obtained as a light yellow solid.

[0263] LC-MS: Rt=0.630min, (ESI)m / z.[M+H-56]+284.0;

[0264] Step 4: 2-tert-Butyloxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-5)

[0265] LiOH.H2O (48.2 mg, 1.15 mmol, 1.5 eq) was dissolved in H2O (3 mL) and THF (3 mL), and 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid ethyl ester (5-4) (260 mg, 766 μmol, 1.0 eq) was added and dissolved in MeOH (3 mL) and THF (3 mL). The mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. The reaction mixture was concentrated under vacuum to remove THF and MeOH, and the pH value of the residue was adjusted to 4-5 with 50% HOAc aq. The mixture was purified by preparative HPLC (FA conditions) (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; gradient: 25%-45% B, 11 minutes). 2-tert-Butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-5) (187 mg, 601 μmol, yield 78.4%, purity 100%) was obtained as a white solid.

[0266] LC-MS: Rt=0.593min, (ESI)m / z.[M+H-56]+256.0;

[0267] Step 5: tert-Butyl N-[5-[[2-(tert-butoxycarbonylamino)-5-(4-fluorophenyl)phenyl]carbamoyl]-2-oxo-1,3-dihydro-2-benzothiophene-2-methylene]carbamate (5-6)

[0268] To a solution of 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-5) (70 mg, 225 μmol, 1.0 eq) in pyridine (2 mL) were added tert-butyl (3-amino-4'-fluoro-[1,1'-biphenyl]-4-yl) carbamate (74.8 mg, 247 μmol, 1.1 eq) and EDCI (51.7 mg, 270 μmol, 1.2 eq). The mixture was stirred at 50 °C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. 5 mL of H2O was added, and the aqueous phase was extracted with ethyl acetate (5 mL*3), washed with brine (5 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography ( 4g+4g Silica gel flash chromatography column, eluent 0-25%-40% ethyl acetate / petroleum ether gradient elution @20 mL / min, petroleum ether / ethyl acetate = 2 / 1, Rf = 0.33). Tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-5-(4-fluorophenyl)phenyl]carbamoyl]-2-oxo-1,3-dihydro-2-benzothiophene-2-methylene]carbamate (5-6) (130 mg, 218 μmol, yield 97.1%, purity 100%) was obtained as an off-white solid.

[0269] LC-MS: Rt=0.764min, (ESI)m / z.[M+H-156]+440.1

[0270] Step 6: N-[2-amino-5-(4-fluorophenyl)phenyl]-2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxamide (Example 5)

[0271] Tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-5-(4-fluorophenyl)phenyl]carbamoyl]-2-oxo-1,3-dihydro-2-benzothiophene-2-methylene]carbamate (5-6) (120 mg, 201 μmol, 1.0 eq) was dissolved in TFA / DCM (1 / 10, 3.6 mL) and stirred at 25°C for 3 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. The mixture was concentrated in vacuo. Saturated aqueous NaHCO3 was added to pH 8, and a white solid was separated, filtered and concentrated in vacuo. The crude product was triturated with H2O (5 mL*4) and DCM (5 mL*2). After freeze-drying, N-[2-amino-5-(4-fluorophenyl)phenyl]-2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxamide (Example 5) (70 mg, 177 μmol, yield 87.9%, purity 100%) was obtained as a white solid.

[0272] LC-MS: Rt=0.604min, (ESI)m / z.[M+H]+396.1;

[0273] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.79 (s, 1H), 7.90-8.05 (m, 2H), 7.58 (br dd, J = 5.6, 8.4Hz, 2H), 7.51 (br d,J=6.5Hz,2H),7.31(dd,J=1.8,8.3Hz,1H),7.22(t,J=8.8Hz,2H),6.87(d,J=8.3Hz,1H),5.13(s,2H),4.42-4.53(m,4H),4.34(s,1H)

[0274] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.47(s,1F)

[0275] Synthesis of Example 6

[0276]

[0277] Step 1: Methyl 4-methylsulfanylbenzoate (6-2)

[0278] To a solution of 4-methylsulfanylbenzoic acid (6-1) (5 g, 29.7 mmol, 1 eq) in MeOH (25 mL) was added H2SO4 (145 mg, 1.49 mmol, 79 μL, 0.05 eq) (5 drops). The mixture was stirred at 75°C (oil bath) for 16 hours. Thin layer chromatography showed that the reactant 6-1 remained at 50%, and a major new spot with lower polarity was detected (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.71). The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with NaHCO3.aq (100 mL) and extracted with EtOAc (100 mL*3). The combined organic layers were washed with brine (100 mL), dried over MgSO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography ( 40g Silica gel flash chromatography column, eluent 0-12% ethyl acetate / petroleum ether gradient elution @40 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.71). 4-methylsulfanylbenzoic acid methyl ester (6-2) (3.76 g, 20.5 mmol, yield 69%, purity 99.4%) was obtained as a white solid.

[0279] LC-MS: Rt=0.971min, (ESI)m / z.[M+H]+183.1; purity:99.4%

[0280] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 7.86 (d, J = 8.4Hz, 2H), 7.36 (d, J = 8.4Hz, 2H), 3.79-3.87 (m, 3H), 2.53 (s, 3H)

[0281] Step 2: Methyl 4-(methylsulfonylimino)benzoate (6-3)

[0282] To a solution of methyl 4-methylsulfanylbenzoate (6-2) (1.5 g, 8.23 ​​mmol, 1 eq) in MeOH (30 mL) was added aqueous ammonia, carbamic acid (1.93 g, 24.7 mmol, 3 eq) and PhI(OAc)2 (7.95 g, 24.7 mmol, 3 eq). The mixture was stirred at 25 °C for 16 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with Na2S2O3 (50 mL) and extracted with DCM (50 mL*3). The combined organic layers were washed with brine (100 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography ( 40g Silica gel flash chromatography column, eluent 0-30% (ethyl acetate / EtOH=3 / 1) / petroleum ether gradient elution @40 mL / min, petroleum ether / (ethyl acetate / EtOH=3 / 1)=1 / 1, Rf=0.38). Methyl 4-(methylsulfonylimino)benzoate (6-3) (1.51 g, 7.08 mmol, yield 86.03%) was obtained as a white solid.

[0283] LC-MS: Rt=0.535min, (ESI)m / z.[M+H]+214.0

[0284] 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.12-8.17 (m, 2H), 8.04-8.10 (m, 2H), 4.45 (s, 1H), 3.86-3.94 (m, 3H), 3.11 (s, 3H).

[0285] Step 3: 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid methyl ester (6-4)

[0286] A mixture of methyl 4-(methylsulfonylimino)benzoate (6-3) (2.45 g, 11.5 mmol, 1 eq), dichlororhodium, 1,2,3,4,5-pentamethylcyclopentane (1.01 g, 1.72 mmol, 0.15 eq), silver (V) hexafluoroantimonate (1 g, 2.91 mmol, 0.25 eq) and 2,2-dimethylpropionic acid (293 mg, 2.87 mmol, 330 μL, 0.25 eq) in DCE (40 mL) was degassed and purged with N2 three times, and then 1,3-dioxol-2-one (1.98 g, 22.9 mmol, 2 eq) was added. The mixture was stirred at 85 ° C (oil bath) under N2 atmosphere for 48 hours. LC-MS showed that about 35% of the reactant 6-4 remained. LC-MS showed several new peaks and about 28% of the target compound was detected. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM (50 mL*3). The combined organic layers were washed with 50 mL of brine, dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography ( 80g Silica gel flash chromatography column, eluent: 0-20% (ethyl acetate / EtOH=3 / 1) / petroleum ether gradient elution @60 mL / min, petroleum ether / (ethyl acetate / EtOH=3 / 1)=1 / 1, Rf=0.69). Yellow solid 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid methyl ester (6-4) (440 mg, 1.76 mmol, yield 7.67%, purity 95%) was obtained.

[0287] LC-MS: Rt = 0.818 min, (ESI) m / z. [M+H] + 238.0; purity: 95%

[0288] Step 4: 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid (6-5)

[0289] To a solution of 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]decane-1(6),2,4,7,9-pentene-8-carboxylic acid methyl ester (6-4) (100 mg, 421 μmol, 1 eq) in THF (1 mL) and H2O (1 mL) was added LiOH.H2O (70 mg, 1.67 mmol, 3.96 eq). The resulting mixture was stirred at 20°C for 16 hours. LC-MS showed that the reaction mixture was complete. 1 mL of MeOH was added to the reaction mixture and then sent to HPLC preparation. The crude product was purified by reverse phase HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (FA)-ACN]; gradient: 10%-50% B, 11 minutes). The residual aqueous solution was lyophilized to give a yellow solid, affording 2-methyl-2-oxo-2thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid (6-5) (35 mg, 139 μmol, 33.1% yield, 89% purity) as a yellow solid.

[0290] LC-MS: Rt = 0.700 min, (ESI) m / z. [M+H] + 224.0; purity: 89%

[0291] Step 5: tert-Butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carbonyl)amino]phenyl]carbamate (6-6)

[0292] To a solution of 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid (6-5) (30 mg, 134 μmol, 1 eq) in pyridine (1.5 mL) was added tert-butyl N-[2-amino-4-(4-fluorophenyl)phenyl]carbamate (g) (42 mg, 138 μmol, 1.03 eq) and EDCI (31.9 mg, 166 μmol, 1.24 eq). The mixture was stirred at 50 °C for 2 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue. The crude product was used in the next step without further purification. Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carbonyl)amino]phenyl]carbamate (6-6) (65 mg, crude) was obtained as a yellow solid.

[0293] LC-MS: Rt=1.047min, (ESI)m / z.[M+H]+508.1

[0294] Step 6: N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxamide (Example 6)

[0295] Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]deca-1(6),2,4,7,9-pentene-8-carbonyl)amino]phenyl]carbamate (6-6) (65 mg, 128 μmol, 1 eq) was dissolved in DCM (1 mL) and TFA (0.2 mL). The mixture was stirred at 20°C for 2 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was concentrated under reduced pressure to remove the solvent. Na2CO3.aq was added to the residue to adjust the pH to 10-11. The residue was diluted with H2O (10 mL) and extracted with DCM (10 mL*3). The combined organic layers were washed with brine (15 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was added with 0.5 mL DMSO and 0.5 MeOH and then sent to HPLC for preparation. The crude product was purified by reverse phase HPLC (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [A phase water (NH3H2O-NH4HCO3)

[0296] -B phase MeCN]; gradient: 38%-58% B, 11 min) to give N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxamide (Example 6) (12 mg, 29.4 μmol, yield 23%, purity 100%) as a yellow solid.

[0297] LC-MS: Rt = 2.372 min, (ESI) m / z. [M+H] + 408.2; purity: 100%

[0298] 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.93 (s, 1H), 8.27 (d, J = 8.4Hz, 1H), 8.06 (s, 1H), 7.98-8.03 (m, 1H), 7.55-7.64 (m, 2H), 7.52 (d, J = 2.0Hz, 1H ),7.29-7.35(m,1H),7.22(t,J=8.8Hz,2H),7.13(d,J=6.8Hz,1H),6.87(d,J=8.4Hz,1H),6.29(d,J=6.8Hz,1H),5.20(s,2H),3.85(s,3H)

[0299] 19 F NMR(374MHz,DMSO-d6)δ(ppm)-117.44(br s,1F)

[0300] Synthesis of Example 7

[0301]

[0302] Step 1: 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxylic acid methyl ester (7-1)

[0303] A solution of 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid methyl ester (6-4) (980 mg, 4.13 mmol, 1 eq) in MeOH (50 mL) and HCl / MeOH (2M, 10 mL, 4.84 eq) was purged with Ar and degassed for 5 minutes, and Pd / C (490 mg, 460 μmol, 10% purity, 0.1 eq) was added. The reaction mixture was purged and degassed with Ar three times, and then purged and degassed with H2 (15 Psi) three times. The reaction mixture was stirred at 50 ° C under H2 (50 Psi) atmosphere for 16 hours. LC-MS showed that the desired product was detected. The reaction mixture was filtered through diatomaceous earth. The filter cake was washed with MeOH (100 mL*3) and the filtrate was concentrated in vacuo to obtain a residue. The residue was purified by flash silica gel chromatography ( 20g Silica Flash Column, eluent: 20% to 30% (EtOAc / EtOH=3 / 1) / petroleum ether gradient elution @ 40 mL / min, petroleum ether

[0304] / (EtOAc / EtOH=3 / 1)=1 / 1,Rf=0.30). 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxylic acid methyl ester (7-1) (66 mg, 203 μmol, yield 4.91%, purity 73.6%) was obtained as a yellow solid.

[0305] LC-MS: Rt=0.613min, (ESI)m / z.[M+H]+240.0

[0306] Step 2: 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxylic acid (7-2)

[0307] 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxylic acid methyl ester (7-1) (66 mg, 203 μmol, 1 eq) was dissolved in THF (1 mL) and H2O (1 mL), LiOH.H2O (46 mg, 1.10 mmol, 5.40 eq) was added, and the reaction mixture was stirred at 25 °C for 16 hours. LC-MS showed that the main peak was the desired product. THF was removed by N2 flow, the pH value of the mixture was adjusted to 7 with FA, diluted with MeOH (0.5 mL), and filtered. The filtrate was purified by pre-chromatographic column (chromatographic column: Boston Prime C18

[0308] 150*30mm*5um; mobile phase: [A phase water (FA)-B phase ACN]; gradient: 0%-40% B, 10 minutes). 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxylic acid (7-2) (35 mg, 155 μmol, yield 76.5%) was obtained as a white solid.

[0309] LC-MS: Rt=0.298min, (ESI)m / z.[M+H]+226.0

[0310] 1 H NMR(400MHz,DMSO-d6)δ(ppm)8.00-8.06(m,1H),7.92-7.99(m,1H),7.89(s,1H),3.41-3.46(m,2H),3.25(s,3H),2.78-2.86(m,2H)

[0311] Step 3: tert-Butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carbonyl)amino]phenyl]carbamate (7-3)

[0312] 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(10), 2,6,8-tetraene-8-carboxylic acid (7-2) (30 mg, 133 μmol, 1 eq), tert-butyl N-[2-amino-4-(4-fluorophenyl)phenyl]carbamate (Intermediate B-1) (40.3 mg, 133 μmol, 1 eq) and EDCI (30.6 mg, 160 μmol, 1.2 eq) were dissolved in pyridine (0.6 mL) and stirred at 50 °C for 2 hours. LC-MS showed that the starting material had been consumed and the main peak was the desired product. The reaction mixture was diluted with water (5 mL) and extracted with EtOAc (5 mL*3). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by flash silica gel chromatography ( 4g Silica gel flash chromatography column, eluent 30% EtOAc / petroleum ether gradient elution @35mL / min, petroleum ether

[0313] / (EtOAc / EtOH=3 / 1)=1 / 1, Rf=0.30). Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carbonyl)amino]phenyl]carbamate (7-3) (62 mg, 111 μmol, yield 83.2%, purity 91.1%) was obtained as a white solid.

[0314] LC-MS: Rt=0.852min, (ESI)m / z.[M+H]+510.1

[0315] Step 4: 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxylic acid methyl ester (Example 7)

[0316] Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carbonyl)amino]phenyl]carbamate (7-3) (62 mg, 122 μmol, 1 eq) was dissolved in TFA / DCM (1.5 mL) and stirred at 20°C for 2 hours. LC-MS showed that the starting material had been consumed and the main peak was the desired product. TFA / DCM was removed by N2 flow, and then DCM (5 mL) and water (3 mL) were added. The pH value of the mixture was adjusted to 8 with Na2CO3 (saturated aqueous solution) and then separated. The aqueous layer was extracted with DCM (5 mL*2). The combined organic layers were washed with water (5 mL), dried over MgSO4, filtered, and concentrated in vacuo to obtain a residue. The crude product was triturated with MTBE (2 mL) at 20 °C for 10 min to give N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-2thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxamide (Example 7) (20 mg, 48.8 μmol, 40.2% yield, 100% purity) as a yellow solid.

[0317] LC-MS: Rt=1.192min, (ESI)m / z.[M+H]+410.1

[0318] 1 H NMR(400MHz,DMSO-d6)δ(ppm)9.88(s,1H),8.00-8.14(m,2H),7.96(s,1H),7.54-7.63(m,2H),7.4 9(d,J=2.0Hz,1H),7.32(dd,J=2.2,8.3Hz,1H),7.13-7.27(m,2H),6.86(d,J=8.3Hz,1H),5.17(br s,1H),3.43-3.51(m,2H),3.28(s,3H),2.75-2.96(m,2H)

[0319] 19 FNMR(376MHz,DMSO-d6)δ(ppm)-117.44

[0320] Synthesis of Example 8

[0321]

[0322] Step 1: Methyl 3-bromo-4-(bromomethyl)benzoate (8-2)

[0323] To a solution of methyl 3-bromo-4-methylbenzoate (8-1) (12 g, 52.4 mmol, 1 eq) in DCE (100 mL) was added NBS (15.0 g, 84.3 mmol, 1.61 eq) and BPO (1.27 g, 5.24 mmol, 0.1 eq), and the reaction was stirred at 80 ° C for 16 hours. Thin layer chromatography showed the formation of starting material and a new spot (petroleum ether / ethyl acetate = 10 / 1). The solvent was removed to obtain a residue. The residue was purified by flash silica gel chromatography ( 120g Purification was performed by silica gel flash chromatography with 0-2% ethyl acetate / petroleum ether gradient @ 80 mL / min as eluent to obtain methyl 3-bromo-4-(bromomethyl)benzoate (8-2) (14 g, 22.7 mmol, yield 43.39%, purity 50%) as a colorless oil.

[0324] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.27 (d, J = 1.5Hz, 1H), 7.93-8.02 (m, 1H), 7.55 (d, J = 7.9Hz, 1H), 4.63 (s, 2H), 3.95 (s, 3H)

[0325] Step 2: Methyl 3-bromo-4-formylbenzoate (8-3)

[0326] To a solution of methyl 3-bromo-4-(bromomethyl)benzoate (8-2) (5.0 g, 8.12 mmol, 1 eq) in CH3CN (50 mL) was added NMO (1.00 g, 8.54 mmol, 900 μL, 1.05 eq), and then the reaction was stirred at 25 °C for 16 hours. TLC showed that the reaction was complete. The reactant was diluted with H2O (50 mL), extracted with EtOAc (50 mL*3), and the combined organic layers were dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 80g Silica gel flash chromatography column, eluent 0-2% ethyl acetate / petroleum ether gradient elution @ 60 mL / min). The fractions of the desired product were combined and concentrated to obtain methyl 3-bromo-4-formylbenzoate (8-3) as a white solid (1.0 g, 4.11 mmol, yield 50.68%).

[0327] 1 H NMR (400MHz, CDCl3) δ (ppm) 10.43 (s, 1H), 8.34 (d, J = 1.2Hz, 1H), 8.09 (d, J = 8.1Hz, 1H), 7.98 (d, J = 8.1Hz, 1H), 3.99 (s, 3H)

[0328] Step 3: 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid methyl ester (8-4)

[0329] To a Tol. (20 mL) solution of methyl 3-bromo-4-formylbenzoate (8-3) (1.0 g, 4.11 mmol, 1 eq) and iminodimethylsulfoxide (766 mg, 8.23 ​​mmol, 2.0 eq) was added Cs2CO3 (2.68 g, 8.23 ​​mmol, 2 eq), and then purged with N2 for 1 minute. BINAP (512 mg, 822 μmol, 0.2 eq) and Pd(OAc)2 (92.4 mg, 411 μmol, 0.1 eq) were added, and then stirred at 110°C for 16 hours. LC-MS showed that the reaction was complete. The solvent was removed to obtain a residue. The residue was diluted with H2O (30 mL), extracted with DCM (50 mL*3), and the combined organic layer was dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was triturated with EtOAc (10 mL), filtered and the off-white solid crude product 8-4 (350 mg) was collected. The mother liquor was concentrated to give a residue. The residue was purified by flash silica gel chromatography ( 24g Purification was performed by flash chromatography on a silica gel column with a gradient elution of 0-50% ethyl acetate / petroleum ether @ 35 mL / min. The fractions of the desired product were combined and concentrated to give 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid methyl ester (8-4) (475 mg, 2.00 mmol, 48.66% yield) as a yellow solid.

[0330] LC-MS: Rt=0.776min, (ESI)m / z.[M+H]+237.9

[0331] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.93 (s, 1H), 7.57-7.79 (m, 2H), 7.39 (br d, J = 8.1Hz, 1H), 6.69 (br d, J = 9.8Hz, 1H), 3.95 (s, 3H), 3.46 (s, 3H)

[0332] Step 4: 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxylic acid methyl ester (8-5)

[0333] Under argon, Raney-Ni (400 mg) was added to a solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid methyl ester (8-4) (400 mg, 1.69 mmol, 1 eq) in MeOH (20 mL). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred at 50 ° C under H2 (50 Psi) for 16 hours. TLC showed that a small amount of starting material remained and the desired product was detected. The reaction solution was allowed to stand for 20 minutes, the layers were separated, the upper liquid was taken, and it was concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 12g Silica gel flash chromatography column, eluent 0-60, 80% ethyl acetate / petroleum ether gradient elution, flow rate 35mL / min). The fractions of the desired product were combined and concentrated. 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxylic acid methyl ester (8-5) (217mg, 906μmol, yield 53.79%) was obtained as an off-white solid.

[0334] LC-MS: Rt=0.744min, (ESI)m / z.[M+H]+240.0

[0335] 1 H NMR (400MHz, CDCl3) δ (ppm) 7.57 (d, J = 1.6 Hz, 1H), 7.51 (dd, J = 1.6, 7.8 Hz, 1H), 7.09 (d, J = 7. 8Hz,1H),3.89(s,3H),3.40-3.53(m,2H),3.26-3.34(m,1H),3.25(s,3H),3.07-3.19(m,1H)

[0336] Step 5: 3-Methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxylic acid (8-6)

[0337] To a solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxylic acid methyl ester (8-5) (200 mg, 836 μmol, 1 eq) in THF (2 mL) and MeOH (2 mL) was added LiOH.H2O (1 M, 2.00 mL, 2.39 eq), and then stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The solvent was removed to obtain a residue. The pH of the residue was adjusted to 3-4 with 1 M HCl to precipitate a yellow solid, which was filtered, and the filter cake was washed with H2O (2 mL*3) and dried under high vacuum. 3-Methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxylic acid (8-6) was obtained as a yellow solid (170 mg, 755 μmol, yield 90.29%).

[0338] LC-MS: Rt=0.490min, (ESI)m / z.[M+H]+226.0

[0339] Step 6: tert-Butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carbonyl)amino]phenyl]carbamate (8-7)

[0340] EDCI (61.3 mg, 319 μmol, 1.2 eq) was added to a solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxylic acid (8-6) (60 mg, 266 μmol, 1 eq) and tert-butyl N-[2-amino-4-(4-fluorophenyl)phenyl]carbamate (80.5 mg, 266 μmol, 1 eq) in Py (2 mL), and the reaction was stirred at 50 ° C for 2 hours. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The reactant was diluted with H2O (3 mL), extracted with EtOAc (5 mL*3), and the combined organic layers were dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 12g Silica gel flash chromatography column, eluent 0-90% ethyl acetate / petroleum ether gradient elution, flow rate 30mL / min). The fractions of the desired product were combined and concentrated. Tert-butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carbonyl)amino]phenyl]carbamate (8-7) (140mg, 274μmol, yield 88.16%) was obtained as white foam.

[0341] LC-MS: Rt=0.989min, (ESI)m / z.[M+H]+510.1

[0342] Step 7: N-[2-amino-5-(4-fluorophenyl)phenyl]-3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxamide (Example 8)

[0343] Tert-butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carbonyl)amino]phenyl]carbamate (8-7) (130 mg, 255 μmol, 1 eq) was dissolved in TFA (0.5 mL) and DCM (2.5 mL) and stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The solvent was removed with N2 to obtain a residue. The pH of the residue was adjusted to 8 with Na2CO3 (aq.,) and extracted with DCM (3 mL*3). The organic layers were combined, dried with Na2SO4, filtered, and concentrated to obtain a residue. The residue was triturated with MTBE (2 mL), filtered, and the filter cake was washed with MTBE (1 mL*2) and dried under high vacuum. N-[2-amino-5-(4-fluorophenyl)phenyl]-3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-9-carboxamide (Example 8) (75 mg, 181 μmol, yield 71.08%, purity 99%) was obtained as a white solid.

[0344] LC-MS: Rt=0.833min, (ESI)m / z.[M+H]+410.0

[0345] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.60 (s, 1H), 7.57 (dd, J = 5.6, 8.5Hz, 2H), 7.50 (d, J = 1.3Hz, 1H), 7.32-7.42 (m, 2H), 7.28 (dd,J=1.9,8.3Hz,1H),7.13-7.25(m,3H),6.86(d,J=8.3Hz,1H),5.05(s,2H),3.37-3.53(m,2H),3.31(s,3H),3.30(br s,1H),3.08-3.23(m,1H)

[0346] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.49(br s,1F)

[0347] Synthesis of Example 9

[0348]

[0349] Step 1: 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid (9-1)

[0350] To a solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid methyl ester (8-4) (300 mg, 935 μmol, 1 eq) in MeOH (2 mL) and THF (2 mL) was added LiOH.H2O (1 M, 1.48 mL, 1.58 eq), and then stirred at 20°C for 2 hours. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The solvent was removed to obtain a residue. The residue was diluted with H2O (3 mL), filtered, and the filtrate was adjusted to pH = 3-4 with 1 M HCl to precipitate a yellow solid, which was filtered and collected. The yellow solid 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid (9-1) (50 mg, 224 μmol, yield 23.94%) was obtained.

[0351] LC-MS: Rt=0.666min, (ESI)m / z.[M+H]+224.0

[0352] Step 2: tert-Butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carbonyl)amino]phenyl]carbamate (9-2)

[0353] EDCI (51.5 mg, 268 μmol, 1.2 eq) was added to a solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxylic acid (9-1) (50 mg, 224 μmol, 1 eq) and tert-butyl N-[2-amino-4-(4-fluorophenyl)phenyl]carbamate (67.7 mg, 224 μmol, 1 eq) in Py (1 mL), and the reaction was stirred at 50 °C for 2 hours. LC-MS showed that the reaction was complete and the m / z of the desired product was detected. The reactant was diluted with H2O (3 mL), extracted with EtOAc (5 mL*3), and the combined organic layers were dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography ( 12g Silica gel flash chromatography column, eluent 0-80% ethyl acetate / petroleum ether gradient elution, flow rate 30mL / min). The fractions of the desired product were combined and concentrated. Tert-butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carbonyl)amino]phenyl]carbamate (9-2) (85mg, 167μmol, yield 74.77%) was obtained as a white solid.

[0354] LC-MS: Rt=1.025min, (ESI)m / z.[M+H]+508.2

[0355] Step 3: N-[2-amino-5-(4-fluorophenyl)phenyl]-3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxamide (Example 9)

[0356] Tert-butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carbonyl)amino]phenyl]carbamate (9-2) (80 mg, 157 μmol, 1 eq) was dissolved in TFA (0.5 mL) and DCM (2.5 mL) and stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete and the mass of the desired product was detected. The solvent was removed with N2 to obtain a residue. The pH of the residue was adjusted to 8 with Na2CO3 (aqueous solution), extracted with DCM (3 mL*3), the organic layers were combined, dried with Na2SO4, filtered, and concentrated to obtain a residue. The residue was triturated with MTBE (2 mL), filtered, and the filter cake was washed with MTBE (1 mL*2) and dried under high vacuum. N-[2-amino-5-(4-fluorophenyl)phenyl]-3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-9-carboxamide (Example 9) (35 mg, 84.2 μmol, yield 53.41%, purity 98%) was obtained as a white solid.

[0357] LC-MS: Rt=0.853min, (ESI)m / z.[M+H]+408.0

[0358] 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.79 (s, 1H), 7.92 (d, J = 9.9Hz, 1H), 7.73 (s, 1H), 7.43-7.63 (m, 5H) ,7.31(dd,J=2.2,8.4Hz,1H),7.09-7.27(m,3H),6.87(d,J=8.4Hz,1H),5.12(s,2H),3.56(s,3H)

[0359] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.47(br s,1F)

[0360] Synthesis of Example 10

[0361]

[0362] Step 1: tert-Butyl N-[2-[(2-methyl-2-oxo-3H-2,1-benzothiazole-6-carbonyl)amino]-4-(2-thienyl)phenyl]carbamate (10-1)

[0363] To a solution of 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid (4-4) (32 mg, 151 μmol, 1.0 eq) in pyridine (1 mL) were added tert-butyl (2-amino-4-(thiophen-2-yl) phenyl) carbamate (44.0 mg, 151 μmol, 1.0 eq) and EDCI (34.9 mg, 182 μmol, 1.2 eq). The mixture was stirred at 50 °C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. 5 mL of H2O was added and the aqueous phase was extracted with ethyl acetate (5 mL*3). The combined organic phases were washed with brine (5 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography ( 4g Silica gel flash chromatography column, eluent: 0-100% ethyl acetate / petroleum ether gradient elution @20 mL / min, petroleum ether / ethyl acetate = 0 / 1, Rf = 0.53). Tert-butyl N-[2-[(2-methyl-2-oxo-3H-2,1-benzothiazole-6-carbonyl)amino]-4-(2-thienyl)phenyl]carbamate (10-1) (75 mg, 149 μmol, yield 98.3%, purity 96%) was obtained as a light yellow solid.

[0364] LC-MS: Rt=0.869min, (ESI)m / z.[M+H-56]+427.9;

[0365] Step 2: N-[2-amino-5-(2-thienyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxamide (Example 10)

[0366] Tert-butyl N-[2-[(2-methyl-2-oxo-3H-2,1-benzothiazole-6-carbonyl)amino]-4-(2-thienyl)phenyl]carbamate (10-1) (65 mg, 134 μmol, 1.0 eq) was dissolved in TFA / DCM (1 / 5, 2 mL) and stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and the desired product was detected. The pH value of the reaction mixture was adjusted to 7 with saturated aqueous NaHCO3 solution and then concentrated under N2 to remove DCM. The residue was purified by preparative HPLC (FA conditions) (column: Boston Prime C18 150*30mm*5um; mobile phase: [A phase water (FA)-B phase ACN]; gradient: 30%-50% B in 10 minutes). N-[2-amino-5-(2-thienyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxamide (Example 10) was obtained as a yellow solid (20 mg, 51.6 μmol, yield 38.4%, purity 99%).

[0367] LC-MS: Rt=1.657min, (ESI)m / z.[M+H]+383.9

[0368] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.64 (s, 1H), 7.49 (d, J = 1.9Hz, 1H), 7.33-7.40 (m, 4H), 7.30 (dd, J = 2.1, 8.3Hz,1H),7.25(dd,J=0.9,3.5Hz,1H),7.06(dd,J=3.6,5.0,Hz,1H),6.82(d,J=8.4Hz,1H),5.13(br s, 2H), 5.06 (d, J = 17.8Hz, 1H), 4.62 (d, J = 17.9Hz, 1H), 3.52-3.55 (m, 3H).

[0369] The following compounds were synthesized based on the methods of the above examples combined with the general method. The corresponding structures and characterizations of the products are shown in Table 1:

[0370] Table 1

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] Biological test example 1 HDAC1 enzyme activity inhibition experiment

[0377] Experimental Materials

[0378] HDAC1 (BPS, catalog number 50001), HDAC3 (BPS, catalog number 50003), Trypsin (Sigma, catalog number T1426), Ac-peptide (LGK(Ac)) (GL Biochem, custom made), 384-well plate (Perkin Elmer, catalog number 6007279), Echo 650 ultrasonic nanoliter liquid handling system (Labcyte, model Echo 650), microplate reader (PerkinElmer, model Envision).

[0379] Experimental methods

[0380] (1) Enzyme reaction process

[0381] 1.1 Prepare 1x buffer (modified Tris buffer).

[0382] 1.2 Compound dilution: Compounds were dissolved in 100% DMSO and the compound solution was added to a 384-well plate using an Echo 650.

[0383] 1.3 Prepare enzyme solution: Prepare enzyme solution with 1x buffer.

[0384] 1.4 Prepare substrate solution: Add Trypsin and Ac-peptide to 1x buffer.

[0385] 1.5 Add 15 μL of enzyme solution to the 384-well plate and 15 μL of 1x buffer to the negative control wells.

[0386] 1.6 Add 10 μL of substrate solution to each well to start the reaction, and set the microplate reader to 355 nm excitation wavelength and 460 nm emission wavelength to dynamically read the values.

[0387] (2) Data Analysis

[0388] 2.1 Convert the raw data into % inhibition rate according to formula 1:

[0389] Formula 1: % inhibition rate = (Max-Signal) / (Max-Min)*100

[0390] Substitute the % inhibition data into XL-Fit Formula 2 to obtain the IC 50 value:

[0391] Formula 2: Y=Bottom+(Top-Bottom) / (1+(IC 50 / X)*HillSlope)

[0392] Wherein, Y is the % inhibition rate and X is the compound concentration.

[0393] Experimental Results

[0394] The biological activity of some compounds was measured by experimental methods, and "A" represents IC 50 (nM)<100, "B" means 100 <IC 50 (nM)<1000, "C" means 1000≤IC 50 (nM); HDAC3 IC is used to measure the selectivity of HDAC3 activity over HDAC1. 50 / HDAC1 IC 50 Indicated by + sign, the level is indicated by “+”, indicating 3≤fold<10, “++” indicating 10≤fold<20, and “+++” indicating 20≤fold, see Table 2.

[0395] Table 2: Inhibitory activity of some compounds

[0396]

[0397]

[0398] The experimental results show that the compound of the present invention has an inhibitory effect on HDAC1, but does not affect the activity of HDAC3, and has excellent subtype selectivity.

[0399] Biological test example 2: Intracellular histone acetylation level detection experiment

[0400] 2.1 Experimental Materials

[0401] A549 cells (NCACC, catalog number SCSP-503), DMEM medium (Gibco, catalog number 11995065), fetal bovine serum (FBS, Gibco, catalog number A5669701), penicillin-streptomycin double antibody (Gibco, catalog number 15140122), H3K27Ac antibody (CST, catalog number 8173), H3K9Ac antibody (CST, catalog number 9649), H3 antibody (CST, catalog number 4499), secondary antibody (CST, catalog number 7074S), 6-well plate (Costar, catalog number 3516), Pierce TMBCA protein assay kit (Thermo, catalog number 23227), Tanon TM 4-20%, Bis-Tris protein precast gel (Tianneng, catalog number 180-9115H), SuperBlock TM T20 (TBS) blocking buffer (Thermo, catalog number 37536), SuperSignal TM Chemiluminescent substrate (Thermo, catalog number 34577), iBlot TM 2 Gel transfer instrument (Invitrogen, model IB21001), chemiluminescent gel imager (Tianneng, model 4600SF).

[0402] 2.2 Experimental methods

[0403] 2.2.1 Compound-treated cells: A549 cells were cultured in complete medium containing DMEM + 10% FBS + 1% penicillin-streptomycin. 2 mL of A549 cell suspension (4 × 10 4 After the cells adhered to the wall, the compounds were added, and 0.1% (v / v) DMSO was added to the control wells. The cells were incubated at 37°C in an incubator (containing 5% CO2) for 48 hours.

[0404] 2.2.2 Protein extraction: After 48 hours of compound treatment, remove the supernatant, add SDS lysis buffer to lyse the cells, centrifuge at 4°C to obtain the supernatant, and use the BCA protein detection kit to determine the protein concentration.

[0405] 2.2.3Western Blot

[0406] Gel electrophoresis: Take 10 μg of total protein and mix with sample buffer, denature at 98°C for 10 minutes, load the protein precast gel, and perform electrophoresis at a constant voltage of 100 V for 90 minutes.

[0407] Protein transfer: using iBlot TM Transfer the proteins on the gel to the membrane using a gel transfer apparatus at 20V for 7min.

[0408] Blocking and antibody incubation: Use SuperBlock TM The protein membrane was blocked with blocking solution at room temperature for 1 hour; then the primary antibody was diluted with blocking solution (1:1000) and incubated overnight at 4°C on a shaker. The protein membrane was removed the next day and washed three times with 1XTBST at room temperature for 10 minutes each; then the secondary antibody (1:2000) was incubated at room temperature for 2 hours, and then washed three times with 1X TBST for 10 minutes each.

[0409] Chemiluminescent imaging: Drain the protein membrane, evenly cover the chemiluminescent substrate on the membrane, and place it in a gel imager for imaging.

[0410] Data processing: ImageJ 1.53 software was used to measure the gray value of the target band.

[0411] 2.3 Experimental Results

[0412] The experimental results are as follows Figure 1 As shown, as substrates of HDAC1 / 2, the acetylation levels of histones H3K27 and H3K9 increased with increasing compound doses (compared with internal reference protein H3). The above results prove that the compounds of the present invention can promote the acetylation levels of histones H3K27 and H3K9 in a dose-dependent manner.

[0413] Biological test example 3 Tumor cell proliferation inhibition experiment

[0414] 3.1 Experimental Materials

[0415] A549 cells (NCACC, catalog number SCSP-503), Jurkat Clone E6-1 cells (NCACC, catalog number SCSP-513), HuT 78 cells (ATCC, catalog number TIB-161), DMEM medium (Gibco, catalog number 11995065), RPMI 1640 medium (Gibco, catalog number 11875119), IMDM medium (Gibco, catalog number 31980097), fetal bovine serum (FBS, Gibco, catalog number A5669701), penicillin-streptomycin double antibody (Gibco, catalog number 15140122), trypsin (Gibco, catalog number 25200056), CellTiter-Glo detection kit (Promega, catalog number G7572), 384-well transparent flat-bottom black-walled cell culture plate (Corning, catalog number 3764), ultra-micropipette instrument (Tecan, model D300e), multi-function microplate reader (Biotek, model SynergyHTX).

[0416] 3.2 Experimental methods

[0417] 3.2.1 Cell culture: A549 cells were cultured in DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin; Jurkat cells were cultured in RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin-streptomycin; HuT 78 cells were cultured in IMDM medium + 20% fetal bovine serum + 1% penicillin-streptomycin; cells were kept in the logarithmic growth phase and the cell viability was greater than 95%.

[0418] 3.2.2 Preparation of compound concentration gradient: The test compound was added to a 384-well plate using an ultra-micropipette, starting from 10 μM, and diluted 3-fold with DMSO, for a total of 9 concentrations, with triplicate wells.

[0419] Compound-treated cells: Add the trypsin-digested A549, Jurkat or HuT 78 cell suspension to a 384-well plate dotted with the test compound, 40 μL per well, i.e., 100 cells per well, with a final DMSO concentration of 0.4%. The cell culture plate was placed in a 37°C, 5% CO2 incubator for 6 days.

[0420] 3.2.3 Detection: Add 20 μL of CellTiter-Glo reagent to each well of the cell culture plate and incubate at room temperature for 30 minutes with shaking. Use a multifunctional microplate reader to detect the luminescent signal at 578 nm.

[0421] 3.2.4 Data analysis: GraphPad Prism 8.0 software was used to fit the data using a four-parameter inhibitor-response model to obtain the IC values ​​of the test compounds. 50 value (half inhibitory concentration).

[0422] 3.3 Experimental Results

[0423] The proliferation inhibitory activity of some compounds on A549, Jurkat and HuT 78 cells was measured by experimental methods. "A" represents IC 50 (μM)<1, "B" means 1 <IC 50 (μM)<10, "C" means 10≤IC 50 (μM), see Table 3.

[0424]

[0425]

[0426] Biological test example 4T cell activation experiment

[0427] 4.1 Experimental Materials

[0428] RPMI 1640 medium (Gibco, catalog number 11875119), fetal bovine serum (FBS, BDBIO, catalog number F806-500), penicillin-streptomycin double antibody (Gibco, catalog number 15140122), mouse anti-human CD3 antibody (BioLegend, catalog number 300332), human PBMC (SaiLyBio, catalog number XFB-HP025B), human IL2 ELISA kit (R&D, catalog number DY202), 96-well transparent round-bottom microplate (Corning, catalog number 3799), 96-well transparent flat-bottom high-binding microplate (Corning, catalog number 3361), 96-well transparent flat-bottom white microplate (Corning, catalog number 3764), multi-function microplate reader (Molecular Devices, model SpectraMax i3x).

[0429] 4.2 Experimental methods

[0430] 4.2.1 Resuscitation of PBMC cells: Resuspend in RPMI 1640 complete medium (RPMI 1640 + 10% FBS + 1% penicillin-streptomycin double antibody) to 10 / ml. 6 The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0431] 4.2.2 Compound treatment of PBMC cells: PBMC cells were collected and 2 × 10 5 cells (100 μL), add 100 μL of culture medium containing 2-fold concentration of the test compound (final concentration 0-10 μM), mix well, and culture in a 37° C., 5% CO 2 incubator for 24 hours.

[0432] 4.2.3 Add 100 μL of a solution containing 1 μg / mL mouse anti-human CD3 antibody to each well of a 96-well transparent flat-bottom high-binding microplate, seal the plate, incubate overnight at 4°C, then remove the solution and wash twice with DPBS.

[0433] 4.2.3 Transfer the PBMC suspension pretreated for 24 hours to a 96-well plate coated with CD3 antibody and culture it in a 37°C, 5% CO2 incubator for 24 hours.

[0434] 4.2.2 After 24 hours, 100 μL of culture supernatant was collected and the IL-2 concentration was detected by ELISA.

[0435] 4.3 Experimental Results

[0436] The experimental results are as follows Figure 2As shown, it can be observed that after the T cells in PBMC were activated by anti-CD3 antibodies for 24 hours, the concentration of IL-2 in the culture supernatant increased with the increase of the concentration of the test compound, proving that the compounds of the present invention can enhance T cell activation in a dose-dependent manner, promote T cells to secrete cytokines such as IL-10, help regulate the tumor microenvironment, and exert a better anti-cancer effect.

[0437] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, stereoisomer, or isotope thereof, in, is a single bond or a double bond; X1 is selected from N, NH, NR a R b , CR a R b , CHR a R b ; X2 is selected from O, NH, CR a R b , CHR a R b ; R a , R b Each is independently selected from the following group: H, deuterium, tritium, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C4 alkoxy; or R a , R b The atoms connected thereto together form a substituted or unsubstituted group selected from the group consisting of a C3-C8 cycloalkyl group, a C6-C8 aromatic ring, a 5-8 membered aromatic heterocycle, or a 3-8 membered heterocycle; Ring A is a 5-10 membered heterocyclic ring or a 5-10 membered aromatic heterocyclic ring; the heterocyclic ring may be saturated or partially unsaturated; R1 is each independently halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; q is each independently 0, 1, 2 or 3; Ar is independently selected from: C6-C10 aromatic ring, 5-12 membered heteroaromatic ring; R2 and R3 are each independently selected from the group consisting of NH2, substituted or unsubstituted C1-C4 alkyl, halogen; m is 0, 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3, 4, 5 or 6; k is 0 or 1; Cy is a group selected from the group consisting of a C1-C8 alkyl group, a C6-C10 aromatic ring, a C3-C10 cycloalkyl group, a 5-12 membered heteroaromatic ring, and a 5-12 membered heterocyclic ring; Unless otherwise specified, the heterocyclic ring or aromatic heterocyclic ring includes 1, 2 or 3 heteroatoms selected from N, S or O as the ring skeleton; the heterocyclic ring or aromatic heterocyclic ring may be substituted or unsubstituted; In the above definitions of each substituent, the substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: deuterium, tritium, halogen, hydroxyl, amino, cyano, C2-6 alkynyl, -SF5, halogenated or unhalogenated C1-C4 alkyl, C3-C10 cycloalkyl, C1-C4 alkoxy, C1-C6 amide, C1-C6 alkylamino, C6-C10 aryl, five-membered or six-membered heteroaryl, five-membered or six-membered heterocyclic group.

2. The compound according to claim 1, characterized in that The compounds are represented by the structures shown in formulas I-1, I-2, I-3 and I-4: Y is selected from the group consisting of: (CH2) p , CH=CH; p is each independently 1, 2, 3, 4 or 5; The remaining substituents are as described in claim 1.

3. The compound according to claim 1, characterized in that The compound is a structure shown in Formula IA, IB, IC, ID or IE: Where R c Each is independently H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or substituted C3-C6 cycloalkyl; Y is selected from the group consisting of: (CH2) p , CH=CH; p is each independently 1, 2, 3, 4 or 5; The remaining substituents are as described in claim 1.

4. The compound according to claim 1, characterized in that In the compound, Ar is a group selected from the following group: phenyl, pyridyl, pyrimidinyl, pyridazinyl, tetrazinyl, triazinyl, pyrrolyl, thienyl, furyl, tetrazolyl, triazolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazole, isoxazolyl, oxadiazolyl, thiadiazolyl, naphthyl, indolyl, indazolyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzotriazolyl, and morpholinyl.

5. The compound according to claim 1, characterized in that In the compound, Cy is selected from the following group: benzene ring, thiophene ring; and / or R3 is each independently halogen; n is 0, 1, 2, 3 or 4.

6. The compound according to any one of claims 1 to 3, characterized in that The compound has a structure shown in formula I-AA, I-BB, I-CC, I-DD or I-EE: Where R c Each is independently H, substituted or unsubstituted C1-C4 alkyl, substituted or substituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C4 alkoxy; Y is selected from the group consisting of: (CH2) p , CH=CH; p is each independently 1, 2, 3, 4 or 5; Cy is selected from the group consisting of benzene ring, thiophene; The remaining substituents are as defined in claim 3.

7. The compound according to any one of claims 1 to 5, characterized in that The compounds have structures shown in formulas I-AAA, I-AAB and I-AAC: Where R c The definition as set forth in claim 3.

8. The compound according to any one of claims 1 to 5, characterized in that X1 is selected from N, NH or CR a R b ; X2 is selected from O, NH, CR a R b ; R a , R b Each is independently selected from the following group: H, substituted or unsubstituted C1-C8 alkyl; R1 is each independently halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; q is each independently 0, 1, 2 or 3; R2 and R3 are each independently selected from the group consisting of NH2, substituted or unsubstituted C1-C4 alkyl, and halogen.

9. The compound according to any one of claims 1 to 8, characterized in that The compound may be selected from the following group:

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (A) a therapeutically effective amount of a compound of formula I, its enantiomers, diastereomers, racemates, isotopes and mixtures thereof, and one or more of its pharmaceutically acceptable salts, crystalline hydrates and solvates; and (B) a pharmaceutically acceptable carrier.

11. Use of the compound according to any one of claims 1 to 9 or the composition according to claim 10, characterized in that: Used to prepare: (a) histone deacetylase (HDAC) inhibitors; and / or (b) drugs for treating and / or preventing and alleviating HDAC-related malignant tumors; and / or (c) drugs for treating malignant tumors in combination with immune checkpoint inhibitors.

12. The use according to claim 11, characterized in that The malignant tumor is selected from the following group: cutaneous T-cell lymphoma, peripheral T-cell lymphoma, T-lymphocytic leukemia, multiple myeloma, breast cancer, acute myeloid leukemia, diffuse large B-cell lymphoma, glioblastoma, non-small cell lung cancer, pancreatic cancer, neuroblastoma, malignant peripheral nerve sheath tumor (MPNST), esophageal cancer, bladder cancer, meningeal cancer, mesothelioma, melanoma, astrocytoma, undifferentiated pleomorphic sarcoma, head and neck cancer, gastric adenocarcinoma, myxofibrosarcoma, bile duct cancer, as well as brain cancer, gastric cancer, kidney cancer, endometrial cancer, cervical cancer, urethral cancer, liver cancer, lung cancer, soft tissue cancer, pleural cancer and colorectal cancer or sarcoma.

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