HDAC inhibitor, and preparation and use thereof

Abstract

Provided are an HDAC inhibitor, and a preparation and use thereof. Specifically, the inhibitor is a compound having a structure as shown in formula I. The compound inhibits histone deacetylase (HDAC) activity, and can be used in the preparation of HDAC inhibitors and / or drugs for treating and / or preventing and / or alleviating malignancies and immune diseases associated with HDAC.

Description

An HDAC inhibitor and its preparation and uses Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a histone deacetylase (HDAC) inhibitor and its preparation and uses. Background Technology

[0002] Protein acetylation is one of the most common post-translational modifications of proteins, produced by the transfer of an acetyl group from acetyl-CoA to the ε-amino side chain of lysine via lysine acetyltransferase (KAT). Protein acetylation is a dynamic and reversible post-translational modification, with deacetylation catalyzed by histone deacetylases (HDACs), also known as lysine deacetylases (KDACs). Histones are one of the main substrates for acetylation. Acetylation neutralizes positive charges, reduces the affinity between histones and DNA helices, leading to chromatin relaxation and conformational opening, increasing the binding of DNA to the transcription complex, and promoting gene activation. On the other hand, non-histone proteins are also acetylated. The acetylation of non-histone proteins participates in regulating protein stability, activity, subcellular localization, and function, thereby modulating physiological and pathological processes such as cell division, signal transduction, autophagy, and metabolism.

[0003] To date, 18 HDACs have been identified in mammals, and based on their domains and homology with yeast protein sequences, they are divided into four classes: Class I (HDACs 1, 2, 3, and 8) are homologous to yeast Rpd3, mainly located in the nucleus, and possess high enzymatic activity; Class II are homologous to yeast Hda1, with Class IIa (HDACs 4, 5, 7, and 9) characterized by phosphorylation-dependent migration between the nucleus and cytoplasm, and Class IIb (HDACs 6 and 10) possessing two catalytic domains; Class III (also known as sirtuins, including SIRT1-7) are homologous to yeast SIR2 and are NAD+-dependent enzymes, while the other classes of HDACs are all Zn-dependent. 2+ HDAC 11 is a type-IV HDAC that shares a conserved sequence with HDACs of types I and II, but has higher tissue specificity and is highly expressed in the brain, heart, testes and kidneys.

[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, chidamide has been approved by the China 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, and pancreatic cancer. 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 effects 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 combination therapies of multiple HDAC inhibitors and immune checkpoint inhibitors are currently under clinical investigation and evaluation. However, the clinical toxicity of pan-HDAC inhibitors and class I HDAC inhibitors narrows their therapeutic window, which is the main reason limiting their indication expansion and combination therapy. Therefore, developing more selective HDAC1 / 2 inhibitors that combine anti-tumor and immunomodulatory functions, while reducing the side effects of targeting other HDAC subtypes, is expected to bring more benefits to cancer patients in clinical practice.

[0005] Therefore, there is a need to provide a class of structurally novel and selective HDAC inhibitors. Summary of the Invention

[0006] The purpose of this invention is to provide a novel histone deacetylase (HDAC) inhibitor.

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

[0008] in, It can be a single bond or a double bond;

[0009] X1 is selected from N, NH, NR a R b CR a R b CHR a R b ;

[0010] X2 is selected from O, NH, and CR. a R b CHR a R b ;

[0011] R a R b Each is independently selected from the group consisting of: H, deuterium, tritium, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C4 alkoxy; or R a R b The atoms attached to it together form substituted or unsubstituted groups selected from the group consisting of C3-C8 cycloalkyl, C6-C8 aromatic ring, 5-8 membered aromatic heterocycle, or 3-8 membered heterocycle;

[0012] Ring A is a 5-10 membered heterocycle or a 5-10 membered aromatic heterocycle; the heterocycle may be saturated or partially unsaturated;

[0013] R1 is independently a halogen, hydroxyl group, substituted or unsubstituted C1-C4 alkyl group, or substituted or unsubstituted C1-C4 alkoxy group;

[0014] Each q can be 0, 1, 2, or 3 independently;

[0015] Ar is independently selected from: C6-C10 aromatic rings and 5-12 heterocyclic aromatic rings;

[0016] R2 and R3 are each independently selected from the following group: NH2, substituted or unsubstituted C1-C4 alkyl groups, and halogens;

[0017] m can be 0, 1, 2, 3, 4, 5, or 6;

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

[0019] k is 0 or 1;

[0020] Cy is a group selected from the following group: C1-C8 alkyl, C6-C10 aromatic ring, C3-C10 cycloalkyl, 5-12 membered heteroaromatic ring, 5-12 membered heterocycle;

[0021] Unless otherwise specified, the heterocycles and aromatic heterocycles comprise one, two, or three heteroatoms selected from N, S, or O as the ring skeleton; the heterocycles and aromatic heterocycles may be substituted or unsubstituted;

[0022] In the above definitions of substituents, substitution refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of: 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 alkylamine, C6-C10 aryl, five- or six-membered heteroaryl, and five- or six-membered heterocyclic group.

[0023] In another preferred embodiment, the compound has the structure shown in formulas I-1, I-2, I-3, and I-4:

[0024] Y is selected from the following group: (CH2) p CH = CH;

[0025] p can be 1, 2, 3, 4 or 5 independently;

[0026] The remaining substituents are as described above.

[0027] In another preferred embodiment, the compound has the structure shown in formula IA, IB, IC, ID, or IE:

[0028] Rc is independently H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, or substituted or substituted C3-C6 cycloalkyl;

[0029] Y is selected from the following group: (CH2) p CH = CH;

[0030] p can be 1, 2, 3, 4 or 5 independently;

[0031] The remaining substituents are as described above.

[0032] In another preferred embodiment, Ar in the compound is a group selected from the group consisting of: phenyl, pyridinyl, pyrimidinyl, pyridazinyl, tetrazinyl, triazinyl, pyrroleyl, thiopheneyl, furanyl, tetrazolyl, triazolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, thiazolyl, naphthyl, indolyl, inzolyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiopheneyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoisoxazolyl, benzotriazolyl, and morpholinyl.

[0033] In another preferred embodiment, Ar is phenyl.

[0034] In another preferred embodiment, R2 is an amino group.

[0035] In another preferred embodiment, Cy in the compound is selected from the group consisting of: benzene rings, thiophene rings; and / or

[0036] R3 is an independent halogen; n is 0, 1, 2, 3 or 4.

[0037] In another preferred embodiment, the compound has the structure shown in formula I-AA, I-BB, I-CC, I-DD, or I-EE:

[0038] Each of Rc is independently H, a substituted or unsubstituted C1-C4 alkyl, a substituted or substituted C3-C6 cycloalkyl, or a substituted or unsubstituted C1-C4 alkoxy.

[0039] Y is selected from the following group: (CH2) p CH = CH;

[0040] p can be 1, 2, 3, 4 or 5 independently;

[0041] Cy is selected from the following group: benzene ring, thiophene;

[0042] The definitions of the remaining substituents are as described above.

[0043] In another preferred embodiment, X1 is selected from N, NH or CR. a R b ;

[0044] X2 is selected from O, NH, and CR. a R b ;

[0045] R a R b Each is independently selected from the group consisting of: H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C4 alkoxy;

[0046] R1 is independently a halogen, hydroxyl group, substituted or unsubstituted C1-C4 alkyl group, or substituted or unsubstituted C1-C4 alkoxy group;

[0047] Each q can be 0, 1, 2, or 3 independently;

[0048] R2 and R3 are each independently selected from the following group: NH2, substituted or unsubstituted C1-C4 alkyl groups, and halogens.

[0049] In another preferred embodiment, the compound has a structure as shown in formulas I-AAA, I-AAB, and I-AAC:

[0050] The remaining substituents are as described above.

[0051] In another preferred embodiment, X1 is selected from N, NH or CR.a R b ;

[0052] X2 is independently selected from O, NH, and CR. a R b ;

[0053] R a R b Each is independently selected from the following group: H, substituted or unsubstituted C1-C8 alkyl groups;

[0054] R1 is independently a halogen, hydroxyl group, substituted or unsubstituted C1-C4 alkyl group, or substituted or unsubstituted C1-C4 alkoxy group;

[0055] Each q can be 0, 1, 2, or 3 independently;

[0056] R2 and R3 are each independently selected from the following group: NH2, substituted or unsubstituted C1-C4 alkyl groups, and halogens.

[0057] In another preferred embodiment, the compound has a structure as shown in formulas I-DDA, I-DDB, and I-DDC:

[0058] In another preferred embodiment, the compound may be selected from the following group:

[0059] In a second aspect of the invention, a pharmaceutical composition is provided, the composition comprising: (A) a therapeutically effective amount of a compound of formula I, including one or more of its enantiomers, diastereomers, racemates, isotopes, and mixtures thereof, as well as pharmaceutically acceptable salts, hydrates, and solvates; and (B) a pharmaceutically acceptable carrier.

[0060] In a third aspect of the invention, the use of the compounds as described in the first aspect of the invention or the compositions as described in the second aspect of the invention is provided for the preparation of: (a) histone deacetylase (HDAC) inhibitors; and / or (b) medicaments for the treatment and / or prevention and relief of HDAC-related malignancies; and / or (c) medicaments for the treatment of malignancies in combination with immune checkpoint inhibitors.

[0061] In another preferred embodiment, the drug used in combination with an immune checkpoint inhibitor to treat malignant tumors is used for immune regulation, enhancing T cell activation, promoting T cell secretion of cytokines, promoting tumor antigen presentation, and regulating the tumor microenvironment.

[0062] In another preferred embodiment, the malignant tumor is selected from the group consisting of: 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 schwannoma (MPNST), esophageal cancer, bladder cancer, visceral mesothelioma, mesothelioma, melanoma, astrocytoma, undifferentiated pleomorphic sarcoma, head and neck cancer, gastric adenocarcinoma, myxoid fibrosarcoma, 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.

[0063] In another preferred embodiment, the malignant tumor is selected from the group consisting of: 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, and pancreatic cancer.

[0064] In another preferred embodiment, the malignant tumor is selected from the group consisting of: T-cell lymphoma, T-lymphoblastic leukemia, and non-small cell lung cancer.

[0065] In another preferred embodiment, the HDAC is HDAC1 or HDAC2.

[0066] In another preferred embodiment, the combined immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.

[0067] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0068] Figure 1 shows the Western blot of H3K27Ac, ​​H3K9Ac, and H3 in A549 cells.

[0069] Figure 2 is a graph showing the concentrations of compounds 4A and 4B versus IL-2 concentrations in Example 4. Detailed Implementation

[0070] Through extensive and in-depth research, and through numerous 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 or alleviating HDAC-related malignancies. This invention is based on this.

[0071] the term

[0072] Unless otherwise defined, 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 pertains.

[0073] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. 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.).

[0074] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0075] As used herein, the term “room temperature” or “normal temperature” refers to a temperature of 4–40°C, preferably 25 ± 5°C.

[0076] As used herein, the term "alkyl" includes straight-chain or branched alkyl groups. For example, C1-C8 alkyl groups refer to straight-chain or branched alkyl groups having 1-8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.

[0077] As used herein, the term "alkynyl" includes straight-chain or branched alkynyl groups. For example, C2-C6 alkynyl refers to straight-chain or branched alkynyl groups having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or similar groups.

[0078] As used herein, the term "cycloalkyl" refers to a cyclic saturated or partially unsaturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, C 3-10 Cycloalkyl refers to a cyclic saturated or partially unsaturated aliphatic hydrocarbon group having 3-10 carbon atoms. It can be a monocyclic group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be a bicyclic group, such as a bridged ring or a spiro ring.

[0079] As used herein, the term "alkylamino" refers to an amino group substituted with an alkyl group. For example, "C1-C8 alkylamino" refers to an amino group substituted with a C1-C8 alkyl group, which can be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, ditert-butylamino, etc.

[0080] As used herein, the term "alkoxy" refers to a group having an alkyl-oxy group structure. For example, "C1-C8 alkoxy" refers to a straight-chain or branched alkoxy group having 1-8 carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, etc.

[0081] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen, wherein the definition of alkyl is as described above.

[0082] As used herein, the term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by a halogen, wherein the definition of alkoxy is as described above.

[0083] As used herein, the term "heterocyclic group" or "heterocyclic alkyl group" 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 of these atoms are heteroatoms selected from N, S, and O. It can be monocyclic, bicyclic, or polycyclic, such as bridged or spirocyclic forms. Specific examples include oxobutyranyl, azabutyranyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, and pyrrolidinyl, etc.

[0084] As used herein, the term "aryl" refers to an aromatic ring group having a specific number of carbon atoms, such as C6-C. 10 Aryl groups represent aromatic ring groups having 6-10 carbon atoms, such as phenyl or naphthyl groups.

[0085] As used herein, the term "heteroaromatic ring" refers to a cyclic aromatic group having a specific number of atoms, of which 1-3 atoms are heteroatoms selected from N, S, and O. For example, a 5-12 membered heteroaromatic ring indicates an aromatic cyclic group having 5-12 carbon atoms. It can be monocyclic or fused-ring. Specific examples include pyridinyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrroleyl, pyrazolyl, imidazoleyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thiopheneyl, isoxazolyl, thiazolyl, oxazolyl, etc.

[0086] Unless otherwise specified as “substituted or unsubstituted”, the groups described in this invention may be substituted by substituents selected from the group consisting of: 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 alkylamine, C6-C10 aryl, five- or six-membered heteroaryl, and five- or six-membered non-aromatic heterocyclic groups.

[0087] 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 by an atom selected from F, Cl, Br, and I.

[0088] Unless otherwise specified, the structural formulas described in this invention are intended to include all isomers (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers with double bonds, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or geometric isomers (or conformational isomers), is within the scope of this invention.

[0089] As used herein, the term "tautomer" refers to structural isomers with different energies that can cross a low energy barrier and thus interconvert. For example, proton tautomers (i.e., proton shifts) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via some bonding electron recombination.

[0090] As used herein, the term "solvent complex" refers to a complex of the compound of the present invention coordinated with solvent molecules in a specific ratio.

[0091] As used herein, the term "hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0092] Active ingredients

[0093] This invention provides an active ingredient that can effectively inhibit histone deacetylase (HDAC). This active ingredient is a compound represented by general formula (I), and it can effectively prevent, treat, and / or alleviate HDAC-related diseases.

[0094] Experiments have shown that the active ingredients of this invention can effectively inhibit HDAC, thereby preventing, treating and / or alleviating HDAC-related diseases.

[0095] It should be understood that the active ingredients of the present invention include compounds represented by general formula (I), pharmaceutically acceptable salts thereof, or prodrugs thereof. It should also be understood that the active ingredients of the present invention include crystalline forms, amorphous compounds, and deuterated compounds of general formula (I).

[0096] The term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Acids suitable 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 class of salts are salts formed by the compounds of the present invention with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0097] Pharmaceutical Compositions and Administration

[0098] Because 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 cancer.

[0099] The pharmaceutical compositions of the present invention comprise the compound of the present invention within a safe and effective range and a pharmaceutically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0100] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that 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 and with the compounds of the present invention without significantly reducing the efficacy of the compounds. 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0101] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous, intramuscular, or subcutaneous).

[0102] 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 components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0103] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0104] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0105] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0106] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0107] Compositions for parenteral injection may comprise 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.

[0108] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0109] When administered in combination, the pharmaceutical composition further comprises one or more (two, three, four, or more) other pharmaceutically acceptable therapeutic agents. One or more (two, three, four, or more) of these other pharmaceutically acceptable therapeutic agents may be used simultaneously, separately, or sequentially with the compounds of the present invention for the prevention, treatment, and / or relief of HDAC-mediated disease.

[0110] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0111] The main advantages of this invention include:

[0112] The compounds of this invention have superior subtype selectivity: the compounds of this invention are selective for HDAC subtypes, can highly inhibit the activity of HDAC1, but do not affect the activity of HDAC3, maintaining the high activity of HDAC3, which can reduce the occurrence of adverse reactions.

[0113] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0114] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0115] The definitions of each abbreviation are as follows:

[0116] The raw materials can be obtained commercially or prepared using methods known or disclosed in the art.

[0117] All reagents and solvents were purchased from Aldrich, TCI, or other domestic suppliers and required no purification before use. Unless otherwise specified, no precautions were taken to remove air or moisture during the reactions. Analytical thin-layer chromatography was performed on silica gel GF-254 plates (domestic) and developed under UV light. Rapid column chromatography was performed on… Used in Rf 150MPLC system The experiment was conducted using Silica Flash Column-CS (Agela, 40-60μm mesh, 60A). 1 H and 13 C10 NMR spectra were recorded on a Bruker Ascend™ 400 MHz spectrometer. Chemical shifts are expressed in parts per million (ppm) and referenced to residual solvent peaks. Analytical LC-MS was performed on an Agilent MSD mass spectrometer connected to an Agilent 1260 system.

[0118] Chiral SFC separation: Column: DAICEL CHIRALCEL OJ (250mm*30mm, 10μm); Mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 60%, isocratic elution mode; or Column: ChiralPak IH, 250*30mm, 10μm; Mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 40%, isocratic elution mode.

[0119] Preparative LC was performed on a Gilson system using a Boston Prime C18 column: 150 x 30 mm x 5 μm. The purity of all final compounds was assessed as >95% by LC-MS analysis.

[0120] The main analytical instruments and chiral analysis methods are shown in the table below:

[0121] Synthesis of Example 1

[0122] Step 1: (5-Bromo-2-methylthio-phenyl)methanol (1-2)

[0123] DIBALH (1M, 22.9mL, 3eq) was added dropwise to a solution of methyl 5-bromo-2-methylthioalkylbenzoate (1-1) (2g, 7.66mmol, 1eq) in 10mL of DCM under N2 conditions at -70 to -60°C. The mixture was stirred for 2 hours under N2 conditions at -70 to -60°C. The reaction was quenched with MeOH (10mL) solution at -65°C (temperature was raised to -55°C), and the reaction solution was restored to room temperature. Then, MTBE and diatomaceous earth were added. The mixture was stirred for 1 hour at 25°C. The mixture was filtered through diatomaceous earth, washed with MTBE (60mL*3) at 20°C, concentrated under reduced pressure, and the residue was obtained. The crude product was purified by column chromatography. 12g + 20g A rapid silica gel column was used for elution with a gradient of 0–16.7% ethyl acetate / petroleum ether at 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-methylthioalkyl-phenyl)methanol (1-2) (1.27 g, 5.45 mmol, yield 71.1%, purity 100%) as a white solid.

[0124] LC-MS: Rt = 0.810 min, (ESI) m / z. [M+H] + 233.0; Purity: 100%.

[0125] 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)

[0126] Step 2: 2-(azidomethyl)-4-bromo-1-methylthio-benzene (1-3)

[0127] DPPA (2.15 g, 7.82 mmol, 1 eq) was added to a solution of (5-bromo-2-methylthioalkyl-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 30 mL of THF. The mixture was stirred at 20 °C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain the residue. The crude product was purified by column chromatography. 20g+20g A rapid silica gel column was used for elution with a gradient of 0–5%–20% ethyl acetate / petroleum ether at 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-methylthioalkylbenzene (1-3) (1.68 g, 6.51 mmol, 100% yield, 100% purity), as a yellow oil.

[0128] 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)

[0129] Step 3: 2-(azidomethyl)-4-bromo-1-methylsulfinyl-benzene (1-4)

[0130] To a solution of 2-(azidomethyl)-4-bromo-1-methylthiobenzene (1-3) (1.3 g, 5.04 mmol, 1 eq) in DCM (20 mL), m-CPBA (1.33 g, 6.55 mmol, 85% purity, 1.3 eq) was added at 0 °C (ice bath). The ice bath was then removed, and the mixture was stirred at 20 °C for 1 hour. A saturated Na₂S₂O₃ solution (20 mL) was added to the reaction mixture for quenching, followed by the addition of H₂O (60 mL) at 20 °C for further quenching. The mixture was then diluted with DCM (30 mL) and extracted with DCM (50 mL x 2). The combined organic layers were washed with brine (50 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was purified by column chromatography. 20g+20g A rapid silica gel column was used for elution with a gradient of 0–35% ethyl acetate / petroleum ether at 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-methylsulfinylbenzene (1-4) (1 g, 3.61 mmol, yield 71.7%, purity 99%), as a white oil.

[0131] LC-MS: Rt = 0.575 min, (ESI) m / z. [M+H] + 274.0; Purity: 99%.

[0132] 1H 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)

[0133] Step 4: 5-Bromo-1-methyl-3H-1,2-benzothiazole 1-oxide (1-5)

[0134] A mixture of 2-(azidomethyl)-4-bromo-1-methylsulfinylbenzene(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 min. The mixture was then stirred at 100 °C for 16 h. The mixture was purified by column chromatography. 12g+12g A silica gel rapid chromatography column was used, with elution of 0–1.5% dichloromethane / methanol gradient at 35 mL / 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.

[0135] LC-MS: Rt = 0.360 min, (ESI) m / z. [M+H] + 245.9; Purity: 86%.

[0136] 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)

[0137] Step 5: Ethyl 1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylate (1-6)

[0138] 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 three times with N2. 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 mixture was cooled to 20 °C, filtered, and concentrated to give the residue. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (15 mL x 3). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was purified by column chromatography. 4g+12g A rapid silica gel column was used as the eluent, with a 0–0.6% dichloromethane / methanol gradient at 35 mL / min (dichloromethane:methanol = 15:1, Rf = 0.6). The fractions of the desired product were combined and concentrated to give ethyl 1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxylate (1–6) (0.165 g, 572 μmol, yield 41.4%, purity 83%), as a brown oil.

[0139] LC-MS: Rt=0.465min, (ESI)m / z.[M+H]+240.1; Purity: 83%.

[0140] 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)

[0141] Step 6: 1-Methyl-1-oxo-3H-1,2-benzothiazole-5-carboxylic acid (1-7)

[0142] LiOH-H₂O (2M, 1.03mL, 3eq) was added to a solution of ethyl 1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxylate (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, heated) for 4 hours. The reaction mixture was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL). The pH 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*30 mm*5 μm; mobile phase: [water (ammonia v / v)-ACN]; gradient: 0%–20% B, within 11 min). The fractions of the desired product were combined and freeze-dried to give 1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxylic acid (1-7) (65 mg, 292 μmol, yield 42.4%, purity 95%), as a white solid.

[0143] Step 7: N-[4-(4-fluorophenyl)-2-[(1-methyl-1-oxo-3H-1,2-benzothiazol-5-carbonyl)amino]phenyl]carbamate (1-8)

[0144] To a solution of 1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxylic acid (1-7) (55 mg, 260 μmol, 1 eq) in pyridine (1.1 mL), 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) were added. 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 the residue. The crude product was purified by column chromatography. 4g A silica gel rapid chromatography column was used, with a gradient elution of 0–1.2% dichloromethane / methanol at 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-benzothiazol-5-carbonyl)amino]phenyl]carbamate (1-8) (110 mg, 219 μmol, yield 84.4%, purity 99%), as a white solid.

[0145] LC-MS: Rt = 1.202 min, (ESI) m / z. [M+H] + 496.1; Purity: 99%.

[0146] Step 8: N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide

[0147] (Example 1)

[0148] Tert-butyl N-[4-(4-fluorophenyl)-2-[(1-methyl-1-oxo-3H-1,2-benzothiazol-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 the pH was adjusted to 8 by adding NH3·H2O. The solid was then precipitated and filtered to obtain a white solid. MeCN and H2O were added to the white solid, and then the mixture was freeze-dried to obtain N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxamide (Example 1) (18 mg, 44.6 μmol, yield 20.1%, purity 98%), which was a yellow solid.

[0149] LC-MS: Rt=0.557min, (ESI)m / z.[M+H]+396.0;

[0150] HPLC: Rt = 3.547 min; Purity: 98.2%.

[0151] 1 H 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.

[0152] 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)

[0153] N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxamide (22 mg, 55.6 μmol, 1 eq) was further separated by SFC (column: DAICEL CHIRALCEL OJ (250 mm * 30 mm, 10 μm); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 60%, isocratic elution mode). The target product fractions were combined and concentrated, and MeCN and H2O were added. The product was then lyophilized to obtain the target product. Following the SFC eluent sequence, a white solid (1S)-N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazol-5-carboxamide (Example 1A) (5.3 mg, 13.4 μmol, yield 24.0%, purity 100%) was obtained. A white solid (1R)-N-[2-amino-5-(4-fluorophenyl)phenyl]-1-methyl-1-oxo-3H-1,2-benzothiazole-5-carboxamide (Example 1-B) was subsequently obtained (4.3 mg, 10.8 μmol, yield 19.5%, purity 100%). The chirality of Examples 1A and 1B was randomly assigned.

[0154] Characterization of Example 1A:

[0155] LC-MS: Rt=0.822min, (ESI)m / z.[M+H]+396.2;

[0156] SFC: Rt=1.105min;ee%=100%;

[0157] 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)

[0158] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.462

[0159] Characterization of Example 1B:

[0160] LC-MS: Rt=0.822min, (ESI)m / z.[M+H]+396.2;

[0161] SFC: Rt=1.783min;ee%=99.3%;

[0162] 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)

[0163] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.456

[0164] SFC Method: Column: Chiral OJ-350 × 4.6 mm ID, 3 μm; Mobile Phase: A: CO2; B: Ethanol (0.05% DEA); Gradient: 50% B; Flow Rate: 2.5 mL / min; Column Temperature: 35℃; ABPR: 1500 psi

[0165] Synthesis of Example 2

[0166] Step 1: Benzothiophene-5-carboxylic acid methyl ester (2-2)

[0167] To a MeOH (15 mL) solution of benzothiophene-5-carboxylic acid (2-1) (1 g, 5.61 mmol, 1 eq), SOCl2 (1.00 g, 8.42 mmol, 611 μL, 1.5 eq) was added, and the reaction mixture was stirred at 60 °C for 5 h. LC-MS showed that the starting material was completely consumed, and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue could be used for the next step without purification. Benzothiophene-5-carboxylic acid methyl ester (1-2) (1.06 g, 5.51 mmol, 98.27% yield) was given as a gray solid.

[0168] LC-MS: Rt=0.648min, (ESI)m / z.[M+H] + =193.1.

[0169] 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)

[0170] Step 2: Methyl 1-oxobenzothiophene-5-carboxylic acid (2-3)

[0171] H₂O₂ (610 mg, 5.38 mmol, 517 μL, 30% purity, 1.59 eq) was added to a solution of methylbenzothiophene-5-carboxylic acid ester (2-2) (650 mg, 3.38 mmol, 1 eq) in DCM (3.5 mL) and TFA (3.5 mL), and the reaction mixture was stirred at 20 °C for 5 hours. LC-MS showed that the starting material was completely consumed and a main 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), the combined organic layers were washed with brine (10 mL), dried with Na₂SO₄, filtered, and concentrated to give the residue. The residue was purified by rapid silica gel chromatography. 12g A silica gel rapid chromatography column was used, with elution of 0–50% ethyl acetate / petroleum ether gradient at 35 mL / min, dichloromethane / ethyl acetate = 1 / 2, Rf = 0.5. Methyl 1-oxobenzothiophene-5-carboxylic acid (2-3) (236 mg, 1.13 mmol, yield 33.52%) was obtained as a white solid.

[0172] LC-MS: Rt=0.757min, (ESI)m / z.[M+H]+209.0.

[0173] 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).

[0174] Step 3: Methyl 1-tert-butoxycarbonylimino-1-oxo-benzothiophene-5-carboxylic acid (2-4)

[0175] 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) were added to a mixture of methyl 1-oxobenzothiophene-5-carboxylic acid (2-3) (210 mg, 1.01 mmol, 1 eq) and NH₂Boc (236 mg, 2.02 mmol, 2.0 eq) in DCM (3.5 mL). The reaction mixture was then stirred at 40 °C for 16 h. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reaction mixture was diluted with DCM (2 mL), filtered, and concentrated to obtain the residue. The residue was purified by silica gel rapid chromatography (HPLC). 12g A silica gel rapid chromatography column was used, with a gradient elution of 0–25% ethyl acetate / petroleum ether at 30 mL / min (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.6). The fractions of the desired product were combined and concentrated to give methyl 1-tert-butoxycarbonylimino-1-oxo-benzothiophene-5-carboxylic acid (2-4) (270 mg, 835 μmol, yield 82.79%) as a white solid.

[0176] LC-MS: Rt=0.925min, (ESI)m / z.[M+H-Boc] + 224.0.

[0177] 1 H 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).

[0178] Step 4: Methyl 1-tert-butoxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-5)

[0179] Methyl 1-tert-butoxycarbonylimino-1-oxo-benzothiophene-5-carboxylic acid (2-4) (165 mg, 510.26 μmol, 1 eq) was dissolved in EtOAc (10 mL), and Pd / C (80 mg) was added. The reaction mixture was then degassed under reduced pressure and purged several times with H2. The reaction mixture was stirred at 25 °C for 16 hours under an H2 gas chamber. LC-MS showed that the reaction was complete and the desired product mass was detected. The reaction mixture was filtered and concentrated to obtain the residue. The residue was purified by rapid silica gel chromatography. 4g A silica gel rapid chromatography column was used, with a gradient elution of 0–20% ethyl acetate / petroleum ether at 25 mL / min (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.5). The fractions of the desired product were combined and concentrated to give a white, foamy methyl 1-tert-butoxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid ester (2-5) (140 mg, 430 μmol, yield 84.32%).

[0180] LC-MS: Rt=0.880min, (ESI)m / z.[M+H]+326.1.

[0181] 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).

[0182] Step 5: 1-tert-Butoxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-6)

[0183] Methyl 1-tert-butoxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-5) (120 mg, 369 μmol, 1 eq) was dissolved in THF (1 mL), MeOH (1 mL), and H₂O (1 mL). LiOH·H₂O (40 mg, 953 μmol, 2.58 eq) was added, and the mixture was stirred at 25 °C for 2 hours. LC-MS showed the reaction was complete, and the desired product mass was detected. The reactants were concentrated to remove THF and MeOH, and acidified with AcOH to pH 6-7, precipitating a white solid. The precipitate was filtered, washed with H₂O (2 mL * 3), and dried under reduced pressure to obtain 1-tert-butoxycarbonylimino-1-oxo-2,3-dihydrobenzothiophene-5-carboxylic acid (2-6) (92 mg, 295 μmol, yield 80.12%), a white solid.

[0184] LC-MS: Rt=0.818min, (ESI)m / z.[M+H]+312.0

[0185] Step 6: Tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-4-(4-fluorophenyl)phenyl]carbamoyl]-1-oxo-2,3-dihydrobenzothiophene-1-methylene]carbamate (2-7)

[0186] EDCI (55 mg, 287 μmol, 1.28 eq) was added to a Py (1.5 mL) 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), and the mixture was stirred at 50 °C for 2 h. LC-MS showed that the reaction was complete and the desired product mass was detected. The reactants were combined, diluted with H2O (2 mL), extracted with EtOAc (3 mL * 3), dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by rapid silica gel chromatography. 12g A silica gel rapid chromatography column was used, with a gradient elution of 0–50% ethyl acetate / petroleum ether at 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 a white foam.

[0187] LC-MS: Rt=1.085min, (ESI)m / z.[M+H] + 596.3.

[0188] Step 7: N-[2-amino-4-(4-fluorophenyl)phenyl]-1-imino-1-oxo-2,3-dihydrobenzothiophene-5-carboxamide (Example 2)

[0189] 125 mg, 209 μmol, 1 eq of tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-4-(4-fluorophenyl)phenyl]carbamoyl]-1-oxo-2,3-dihydrobenzothiophene-1-methylene]carbamate (2-7) 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 desired product mass was detected. The solvent was removed to obtain the residue. The residue was purified by preparative HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water (ammonia v / v)-ACN]; gradient: 30%-50% B, 11 min). The fractions of the desired product were combined and freeze-dried to obtain N-[2-amino-4-(4-fluorophenyl)phenyl]-1-imino-1-oxo-2,3-dihydrobenzothiophene-5-carboxamide (Example 2) (25 mg, 63.2 μmol, yield 30.13%, purity 100%), a white solid.

[0190] 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)

[0191] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.46(br s,1F)

[0192] LC-MS: Rt=0.783min, (ESI)m / z.[M+H]+396.1

[0193] Synthesis of Example 3

[0194] Step 1: 5-Bromo-2,3-dihydro-1,2-benzothiazole 1,1-dioxide (3-2)

[0195] At 0 °C, LiAlH4 (2.5 M, 1.8 mL, 1.3 eq) was added dropwise to a THF solution of 5-bromo-1,1-dioxo-1,2-benzothiazol-3-one (3-1) (900 mg, 3.4 mmol, 1 eq) in 23 mL of THF over 3 minutes. The resulting mixture was stirred at 25 °C under a N2 atmosphere for 2 hours. LC-MS showed that the reactants were completely consumed and a main peak with the desired m / z appeared. EtOAc (25 mL) and H2SO4 (10%, 9 mL) were added to the reactants, and the mixture was extracted with EtOAc / THF (1 / 1, 40 mL * 3), dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by rapid silica gel chromatography. 20g A silica gel rapid chromatography column was used with 0–35% eluent, ethyl acetate / petroleum ether @ 20 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.45. The result was 5-bromo-2,3-dihydro-1,2-benzothiazole 1,1-dioxide (3-2) (510 mg, 2 mmol, yield 59.8%), as a white solid.

[0196] LC-MS: Rt=0.514min, (ESI)m / z.[M+H]+247.9;

[0197] 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).

[0198] Step 2: Ethyl 1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylate (3-3)

[0199] 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 phosphorus tri-tert-butyltetrafluoroborate (59.6 mg, 205 μmol, 0.1 eq) in EtOH (10 mL) was degassed and purged three times with N2. Then, Pd(OAc)2 (46 mg, 205 μmol, 0.1 eq) was added, and the mixture was stirred at 90 °C for 2 h. LC-MS showed complete consumption of the reactants and the desired m / z was detected. The reaction mixture was cooled to room temperature, filtered, and concentrated to obtain the residue. The reaction mixture was diluted with 10 mL of water and extracted with EtOAc (15 mL x 3). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was then subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica gel rapid column with 0–30% eluent, ethyl acetate / petroleum ether @ 30 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.58. The result was ethyl 1,1-dioxo-2,3-dihydro-1,2-benzothiazol-5-carboxylate (3-3) (200 mg, 828 μmol, yield 40.3%), as a white solid.

[0200] LC-MS: Rt=0.534min, (ESI)m / z.[M+H]+242.0;

[0201] 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).

[0202] Step 3: 1,1-Dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxylic acid (3-4)

[0203] To a solution of ethyl 1,1-dioxo-2,3-dihydro-1,2-benzothiazol-5-carboxylic acid (3-3) (180 mg, 746 μmol, 1 eq) in MeOH (2 mL), LiOH·H₂O (62.6 mg, 1.49 mmol, 2 eq) was added. 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 obtain the residue. The pH of the residue was adjusted to 1–2 with 2 M HCl. The suspension was filtered, and the combined filtrates were dried by lyophilization. The crude product was ready for the next step without further purification. 1,1-dioxo-2,3-dihydro-1,2-benzothiazol-5-carboxylic acid (3-4) (169 mg, crude product) was given as a white solid.

[0204] LC-MS: Rt=0.283,0.359min, (ESI)m / z.[M+H]+214.0;

[0205] 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).

[0206] Step 4: Tert-butyl(3-(1,1-dioxo-2,3-dihydrobenzo[d]isothiazolyl-5-carbamate)-4'-fluoro-[1,1'-biphenyl]-4-yl)carbamate (3-5)

[0207] To a solution of 1,1-dioxo-2,3-dihydro-1,2-benzothiazol-5-carboxylic acid (3-4) (130 mg, 365 μmol, 1 eq) in pyridine (2 mL), 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) were added. The mixture was stirred at 50 °C for 2 hours. LC-MS showed that reactants 3-4 were completely consumed and a main peak with the desired m / z appeared. The reaction mixture was diluted with 2 mL H2O and extracted with EtOAc (3 mL * 2). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by rapid silica gel chromatography (…). 12g A silica gel rapid chromatography column was used with 0–40% eluent, ethyl acetate / petroleum ether @ 30 mL / min, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.38. The product yielded a yellow solid tert-butyl(3-(1,1-dioxo-2,3-dihydrobenzo[d]isothiazolyl-5-carboxamido)-4'-fluoro-[1,1'-biphenyl]-4-yl)carbamate (3-5) (100 mg, 201 μmol, yield 54.9%).

[0208] Step 5: N-[2-amino-5-(4-fluorophenyl)phenyl]-1,1-dioxo-2,3-dihydro-1,2-benzothiazole-5-carboxamide (Example 3)

[0209] tert-butyl N-[2-[(1,1-dioxo-2,3-dihydro-1,2-benzothiazol-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 reactant 3-5 was completely consumed and a main peak with the desired m / z appeared. The reaction mixture was concentrated under reduced pressure to remove the solvent. The pH of the residue was adjusted to 8-9 with NaHCO3 (aq). The residue was purified by preparative HPLC (column: Boston Prime C18150*30 mm*5 μm; mobile phase: [phase A: water (FA)-phase B: ACN]; gradient: 33%-53% B over 10 min). 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%).

[0210] 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).

[0211] LC-MS: Rt=0.79min, (ESI)m / z.[M+H]+398.1; purity of 100%.

[0212] Synthesis of Example 4

[0213] Step 1: Methyl 3-amino-4-(methylthioalkylmethyl)benzoate (4-2)

[0214] Under a nitrogen 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, and 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 stirring the reaction mixture at -70 °C for 5 hours, CH3ONa (5.4 M, 18.4 mL, 3.0 eq) was added dropwise over 12 minutes at -70 °C. The reaction mixture was then heated to 25 °C and stirred for another 12 hours. LC-MS showed that the starting materials were 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 the mixture was stirred at 90 °C for 16 hours, after which the solvent was removed under reduced pressure. The residue was purified by rapid silica gel chromatography. 120g + 40g A silica gel rapid chromatography column was used, with a gradient elution of 0–6% ethyl acetate / petroleum ether at 60 mL / min (petroleum ether / ethyl acetate = 4 / 1, Rf = 0.68, Rf = 0.63). Methyl 3-amino-2-(methylthioalkylmethyl)benzoate (4-b1) (2.5 g, 11.8 mmol, yield 35.8%) was obtained as a pale yellow solid; methyl 3-amino-4-(methylthioalkylmethyl)benzoate (4-2) (1.8 g, 8.52 mmol, yield 25.8%) was also obtained as a pale yellow solid.

[0215] 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)

[0216] Step 2: Methyl 2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylate (4-3)

[0217] Methyl 3-amino-4-(methylthiomethyl)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 minutes. After 15 minutes, a 10% NaOH aqueous solution (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. Then, m-CPBA (1.15 g, 5.68 mmol, 85% purity, 1.2 eq) was added in small batches over 5 minutes. The reaction mixture was stirred at -40 °C for 0.5 hours. LC-MS showed complete consumption of the starting material and detection of the target compound. The mixture was heated to 25°C and washed successively with saturated Na₂SO₃ aqueous solution (20 mL) and NaHCO₃ aqueous solution (20 mL * 2), dried over anhydrous MgSO₄, and the solvent was removed under reduced pressure. The residue was purified by rapid silica gel chromatography. 12g+4g A rapid silica gel column was used as the eluent, with a gradient of 0–40% ethyl acetate / petroleum ether at 60 mL / min (petroleum ether / ethyl acetate = 0 / 1, Rf = 0.53). The product yielded methyl 2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxylic acid (4-3) (0.43 g, 1.81 mmol, yield 38.3%, purity 95%), as a pale yellow solid.

[0218] LC-MS: Rt=0.447min, (ESI)m / z.[M+H]+226.1;

[0219] 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)

[0220] Step 3: 2-Methyl-2-oxo-3H-2,1-benzothiazole-6-carboxylic acid (4-4)

[0221] A solution of LiOH·H₂O (72.7 mg, 1.73 mmol, 1.5 eq) in H₂O (2 mL) was added to a solution of methyl 2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxylic acid (4-3) (260 mg, 1.15 mmol, 1.0 eq) in MeOH (2 mL) and THF (2 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that the starting material had been 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 reaction without further purification to obtain 2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxylic acid (d2) (200 mg, 568 μmol, 35.7% yield, 60% purity), a pale yellow solid (200 mg crude product after lyophilization). The other half was purified by preparative HPLC (FA conditions) (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (FA)-ACN]; gradient: 0%-40% B for 11 min). This yielded 2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxylic acid (4-4) (100 mg, 469 μmol, 29.4% yield, 99% purity), a white solid.

[0222] LC-MS: Rt=1.232min, (ESI)m / z.[M+H]+211.9;

[0223] 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)

[0224] Step 4: tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-3H-2,1-benzothiazol-6-carbonyl)amino]phenyl]carbamate (4-5)

[0225] To a pyridine (3 mL) solution of 2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxylic acid (4-4) (90.0 mg, 426 μmol, 1.0 eq), 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) were added. 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 under vacuum. The residue was purified by rapid silica gel chromatography. 4g+4g A silica gel rapid chromatography column was used, with a gradient elution of 0–25% ethyl acetate / petroleum ether at 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-benzothiazol-6-carbonyl)amino]phenyl]carbamate (4-5) (211 mg, 426 μmol, yield 99.9%, purity 100%) was obtained as a white solid.

[0226] LC-MS: Rt=0.666min, (ESI)m / z.[M+H-56]+440.1;

[0227] 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)

[0228] 19 F NMR(376MHz,CDCl3)δ(ppm)-115.57(s,1F)

[0229] Step 5: N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxamide (Example 4)

[0230] Ethoxyethane, trifluoroborane, and hydrofluoric acid (261 mg, 807 μmol, 222 μL, 50% purity, 2.0 eq) were added to a solution of tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-3H-2,1-benzothiazol-6-carbonyl)amino]phenyl]carbamate (4-5) (200 mg, 404 μmol, 1.0 eq) in DCM (4 mL). The mixture was stirred at 25 °C for 2 h. LC-MS showed that the starting material was completely consumed and the desired product was detected. The pH of the reaction mixture was adjusted to 7 with a saturated aqueous solution of NaHCO3, and then concentrated under vacuum to remove DCM. The residue was purified by preparative HPLC (FA conditions) (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water (FA)-ACN]; gradient: 28%-48% B, 11 min). 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 a grayish-white solid.

[0231] LC-MS: Rt=0.981min, (ESI)m / z.[M+H]+396.0

[0232] 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)

[0233] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.47(s,1F)

[0234] Synthesis of Example 5

[0235] Step 1: Ethyl 1,3-dihydro-2-benzothiophene-5-carboxylate (5-2)

[0236] A solution of methyl 3,4-bis(bromomethyl)benzoate (2 g, 6.21 mmol, 1.0 eq) in EtOH (30 mL) was added dropwise to a solution of sodium thioalkylene oxide (5-1) nonahydrate (1.79 g, 7.45 mmol, 1.25 mL, 1.2 eq) in EtOH (110 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, and the filtrate was concentrated to 30 mL under reduced pressure. 50 mL of ethyl acetate was added, and the mixture was washed with water (3 x 30 mL). The organic layer was washed with brine (30 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by rapid silica gel chromatography (…). 20g + 12g A silica gel rapid chromatography column was used with a gradient elution of 0–4% ethyl acetate / petroleum ether at a flow rate of 30 mL / min (petroleum ether / ethyl acetate = 4 / 1, Rf = 0.64). Ethyl 1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-2) (1 g, 4.80 mmol, yield 77.3%, purity 100%) was obtained as a pale yellow solid.

[0237] LC-MS: Rt=0.667min, (ESI)m / z.[M+H]+209.1;

[0238] 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)

[0239] Step 2: Ethyl 2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylate (5-3)

[0240] A suspension of PhI(OAc)2 (2.71 g, 8.40 mmol, 2.5 eq) was added dropwise to a mixture of ethyl 1,3-dihydro-2-benzothiophene-5-carboxylate (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, extracted with saturated Na2SO3 (10 mL), and washed with saturated Na2CO3 (10 mL) and concentrated. The residue was purified by rapid silica gel chromatography. 4g+4g A rapid silica gel column was used for elution with a gradient of 0–100% ethyl acetate / petroleum ether at 20 mL / min (petroleum ether / ethyl acetate = 0 / 1, Rf = 0.3). Ethyl 2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-3) (0.32 g, 1.34 mmol, yield 27.8%) was obtained as a black solid. LC-MS: Rt = 0.729 min, (ESI) m / z. [M+H]+240.1

[0241] Step 3: Ethyl 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylate (5-4)

[0242] 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-carboxylic acid (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 complete consumption of the starting material and detection of the desired compound. The mixture was quenched with saturated NH₄Cl aqueous 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 under vacuum. The residue was purified by rapid silica gel chromatography. 12g A silica gel rapid chromatography column was used, with a gradient elution of 0–20% ethyl acetate / petroleum ether at 30 mL / min (petroleum ether / ethyl acetate = 2 / 1, Rf = 0.4). Ethyl 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-4) (290 mg, 820 μmol, yield 50.3%, purity 96%) was obtained as a pale yellow solid.

[0243] LC-MS: Rt=0.630min, (ESI)m / z.[M+H-56]+284.0;

[0244] Step 4: 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-5)

[0245] LiOH·H₂O (48.2 mg, 1.15 mmol, 1.5 eq) was dissolved in H₂O (3 mL) and THF (3 mL), and ethyl 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylate (5-4) (260 mg, 766 μmol, 1.0 eq) was 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 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 μm; mobile phase: [water (FA)-ACN]; gradient: 25%–45% B, 11 min). 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-5) was obtained (187 mg, 601 μmol, yield 78.4%, purity 100%) as a white solid.

[0246] LC-MS: Rt=0.593min, (ESI)m / z.[M+H-56]+256.0;

[0247] 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)

[0248] To a pyridine (2 mL) solution of 2-tert-butoxycarbonylimino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxylic acid (5-5) (70 mg, 225 μmol, 1.0 eq), 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) were added. 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 under vacuum. The residue was purified by rapid silica gel chromatography. 4g+4g A silica gel rapid chromatography column was used, with elution of a gradient of 0–25%–40% ethyl acetate / petroleum ether at 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 a grayish-white solid.

[0249] LC-MS: Rt=0.764min, (ESI)m / z.[M+H-156]+440.1

[0250] Step 6: N-[2-amino-5-(4-fluorophenyl)phenyl]-2-imino-2-oxo-1,3-dihydro-2-benzothiophene-5-carboxamide (Example 5)

[0251] 120 mg (201 μmol, 1.0 eq) of tert-butyl N-[5-[[2-(tert-butoxycarbonylamino)-5-(4-fluorophenyl)phenyl]carbamoyl]-2-oxo-1,3-dihydro-2-benzothiophene-2-methylene]carbamate (5-6) 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 under vacuum. A saturated aqueous solution of NaHCO3 was added to pH 8, and a white solid was separated, filtered, and concentrated under vacuum. The crude product was ground with H2O (5 mL x 4) and DCM (5 mL x 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.

[0252] LC-MS: Rt=0.604min, (ESI)m / z.[M+H]+396.1;

[0253] 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)

[0254] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.47(s,1F)

[0255] Synthesis of Example 6

[0256] Step 1: Methyl 4-methylthioalkylbenzoate (6-2)

[0257] Add 5 drops of H₂SO₄ (145 mg, 1.49 mmol, 79 μL, 0.05 eq) to a solution of 4-methylthioalkylbenzoic acid (6-1) (5 g, 29.7 mmol, 1 eq) in MeOH (25 mL). Stir the mixture at 75 °C (oil bath) for 16 hours. Thin-layer chromatography showed 50% of reactant 6-1 remaining, with a new major spot of low polarity detected (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.71). Concentrate the reaction mixture under reduced pressure to remove MeOH. Dilute the residue with NaHCO₃·aq (100 mL) and extract with EtOAc (100 mL * 3). Wash the combined organic layers with brine (100 mL), dry with MgSO₄, filter, and concentrate under reduced pressure to obtain the residue. Purify the residue by rapid silica gel chromatography (…). 40g A silica gel rapid chromatography column was used, with elution of 0–12% ethyl acetate / petroleum ether gradient at 40 mL / min (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.71). Methyl 4-methylthioalkylbenzoate (6-2) (3.76 g, 20.5 mmol, yield 69%, purity 99.4%) was obtained as a white solid.

[0258] LC-MS: Rt=0.971min, (ESI)m / z.[M+H]+183.1; purity:99.4%

[0259] 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)

[0260] Step 2: Methyl 4-(methylsulfonylimide)benzoate (6-3)

[0261] Ammonia, carbamic acid (1.93 g, 24.7 mmol, 3 eq), and PhI(OAc)2 (7.95 g, 24.7 mmol, 3 eq) were added to a solution of methyl 4-methylthioalkylbenzoate (6-2) (1.5 g, 8.23 ​​mmol, 1 eq) in MeOH (30 mL). 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 obtain the residue. The residue was purified by rapid silica gel chromatography (…). 40g A rapid silica gel column was used for elution with a gradient of 0–30% (ethyl acetate / EtOH = 3 / 1) / petroleum ether at 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.

[0262] LC-MS: Rt=0.535min, (ESI)m / z.[M+H]+214.0

[0263] 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).

[0264] Step 3: 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6), methyl 2,4,7,9-penten-8-carboxylate (6-4)

[0265] A mixture of methyl 4-(methylsulfonylimino)benzoate (6-3) (2.45 g, 11.5 mmol, 1 eq), rhodium dichloro, 1,2,3,4,5-pentamethylcyclopentane (1.01 g, 1.72 mmol, 0.15 eq), silver hexafluoroantimonate (V) (1 g, 2.91 mmol, 0.25 eq), and 2,2-dimethylpropionic acid (293 mg, 2.87 mmol, 330 μL, 0.25 eq) in a DCE (40 mL) was degassed and purged three times with N2, followed by the addition of 1,3-dioxacyclopenten-2-one (1.98 g, 22.9 mmol, 2 eq). The mixture was stirred at 85 °C (oil bath) for 48 hours under a N2 atmosphere. LC-MS showed approximately 35% of reactant 6-4 remaining. LC-MS showed several new peaks, with approximately 28% of the target compound detected. The reaction mixture was diluted with 50 mL of H₂O and extracted with DCM (50 mL x 3). The combined organic layers were washed with 50 mL of brine, dried over MgSO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel chromatography. 80g A silica gel rapid chromatography column was used, with elution of 0–20% (ethyl acetate / EtOH = 3 / 1) / petroleum ether gradient @ 60 mL / min, petroleum ether / (ethyl acetate / EtOH = 3 / 1) = 1 / 1, Rf = 0.69). A yellow solid of 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6), methyl 2,4,7,9-penten-8-carboxylic acid (6-4) was obtained (440 mg, 1.76 mmol, yield 7.67%, purity 95%).

[0266] LC-MS: Rt = 0.818 min, (ESI) m / z. [M+H] + 238.0; Purity: 95%

[0267] Step 4: 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6), 2,4,7,9-penten-8-carboxylic acid(6-5)

[0268] LiOH·H2O (70 mg, 1.67 mmol, 3.96 eq) was added to a solution of methyl 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]decane-1(6),2,4,7,9-penten-8-carboxylic acid methyl ester (6-4) (100 mg, 421 μmol, 1 eq) in THF (1 mL) and H2O (1 mL). The resulting mixture was stirred at 20 °C for 16 h. LC-MS showed that the reaction mixture was complete. 1 mL of MeOH was added to the reaction mixture, and then the mixture was sent to HPLC for preparation. The crude product was purified by reversed-phase HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water(FA)-ACN]; gradient: 10%-50% B, 11 min). The residual aqueous solution was freeze-dried to obtain a yellow solid, which was 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-pentene-8-carboxylic acid (6-5) (35 mg, 139 μmol, yield 33.1%, purity 89%), a yellow solid.

[0269] LC-MS: Rt = 0.700 min, (ESI) m / z. [M+H] + 224.0; Purity: 89%

[0270] Step 5: N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-penten-8-carbonyl)amino]phenyl]tert-butyl carbamate (6-6)

[0271] To a solution of 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-penten-8-carboxylic acid (6-5) (30 mg, 134 μmol, 1 eq) in pyridine (1.5 mL), 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) were added. 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 the residue. The crude product was ready for the next step without further purification. N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-penten-8-carbonyl)amino]phenyl]carbamate tert-butyl ester (6-6) (65 mg, crude product) was obtained as a yellow solid.

[0272] LC-MS: Rt=1.047min, (ESI)m / z.[M+H]+508.1

[0273] 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)

[0274] N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]decacarbon-1(6),2,4,7,9-penten-8-carbonyl)amino]phenyl]tert-butyl 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 obtain the residue. The residue was added to 0.5 mL of DMSO and 0.5 mL of MeOH and then sent to HPLC for preparation. The crude product was purified by reversed-phase HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [phase A: water (NH3H2O-NH4HCO3)-phase B: MeCN]; gradient: 38%-58% B, 11 min) to obtain 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%), which was a yellow solid.

[0275] LC-MS: Rt = 2.372 min, (ESI) m / z. [M+H] + 408.2; Purity: 100%

[0276] 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)

[0277] 19 F NMR(374MHz,DMSO-d6)δ(ppm)-117.44(br s,1F)

[0278] Synthesis of Example 7

[0279] Step 1: 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6), methyl 2,7,9-tetraen-8-carboxylate (7-1)

[0280] Methyl 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,4,7,9-penten-8-carboxylic acid (6-4) (980 mg, 4.13 mmol, 1 eq) was dissolved in MeOH (50 mL) and HCl / MeOH (2 M, 10 mL, 4.84 eq). The solution was purged with Ar and degassed for 5 min, and Pd / C (490 mg, 460 μmol, 10% purity, 0.1 eq) was added. The reaction mixture was purged with Ar and degassed three times, and then purged with H2 (15 Psi) and degassed three times. The reaction mixture was stirred at 50 °C for 16 h under an H2 (50 Psi) atmosphere. The desired product was detected by LC-MS. The reaction mixture was filtered through diatomaceous earth. The filter cake was washed with MeOH (100 mL * 3), and the filtrate was concentrated under vacuum to obtain the residue. The residue was purified by rapid silica gel chromatography. 20g Silica Flash Column, eluent 20%–30% (EtOAc / EtOH = 3 / 1) / petroleum ether gradient elution @ 40 mL / min, petroleum ether / (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-tetraen-8-carboxylic acid methyl ester (7-1) (66 mg, 203 μmol, yield 4.91%, purity 73.6%) was obtained as a yellow solid.

[0281] LC-MS: Rt=0.613min, (ESI)m / z.[M+H]+240.0

[0282] Step 2: 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6), 2,7,9-tetraen-8-carboxylic acid(7-2)

[0283] Methyl 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-8-carboxylic acid ester (7-1) (66 mg, 203 μmol, 1 eq) was dissolved in THF (1 mL) and H2O (1 mL), and LiOH·H2O (46 mg, 1.10 mmol, 5.40 eq) was added. The reaction mixture was then stirred at 25 °C for 16 hours. LC-MS showed the desired product as the main peak. THF was removed by N2 flow, the pH of the mixture was adjusted to 7 with FA, diluted with MeOH (0.5 mL), and filtered. The filtrate was purified by pre-column chromatography (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [phase A: water (FA) - phase B: ACN]; gradient: 0%-40% B, 10 min). 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6), 2,7,9-tetraen-8-carboxylic acid (7-2) (35 mg, 155 μmol, yield 76.5%) was obtained as a white solid.

[0284] LC-MS: Rt=0.298min, (ESI)m / z.[M+H]+226.0

[0285] 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)

[0286] 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-tetraen-8-carbonyl)amino]phenyl]carbamate (7-3)

[0287] 2-Methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(10), 2,6,8-tetraen-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 desired product was the main peak. 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 under vacuum to obtain the residue. The residue was purified by rapid silica gel chromatography. 4g A silica gel rapid chromatography column was used, with 30% EtOAc / petroleum ether gradient elution at 35 mL / min (petroleum ether / (EtOAc / EtOH=3 / 1)=1 / 1, Rf=0.30). Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-8-carbonyl)amino]phenyl]carbamate (7-3) (62 mg, 111 μmol, yield 83.2%, purity 91.1%), was obtained as a white solid.

[0288] LC-MS: Rt=0.852min, (ESI)m / z.[M+H]+510.1

[0289] Step 4: Methyl 2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-8-carboxylic acid (Example 7)

[0290] Tert-butyl N-[4-(4-fluorophenyl)-2-[(2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-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 desired product was the main peak. TFA / DCM was removed by N2 stream, and then DCM (5 mL) and water (3 mL) were added. The pH 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 with MgSO4, filtered, and concentrated under vacuum to obtain the residue. The crude product was ground with MTBE (2 mL) at 20 °C for 10 minutes to obtain N-[2-amino-5-(4-fluorophenyl)phenyl]-2-methyl-2-oxo-2-thia-3-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraene-8-carboxamide (Example 7) (20 mg, 48.8 μmol, yield 40.2%, purity 100%), which was a yellow solid.

[0291] LC-MS: Rt=1.192min, (ESI)m / z.[M+H]+410.1

[0292] 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)

[0293] 19 FNMR(376MHz,DMSO-d6)δ(ppm)-117.44

[0294] Synthesis of Example 8

[0295] Step 1: Methyl 3-bromo-4-(bromomethyl)benzoate (8-2)

[0296] NBS (15.0 g, 84.3 mmol, 1.61 eq) and BPO (1.27 g, 5.24 mmol, 0.1 eq) were added to a solution of methyl 3-bromo-4-methylbenzoate (8-1) (12 g, 52.4 mmol, 1 eq) in 100 mL of DCE, and the reaction mixture was stirred at 80 °C for 16 hours. Thin-layer chromatography showed the formation of the starting material and a new spot (petroleum ether / ethyl acetate = 10 / 1). The solvent was removed to obtain the residue. The residue was analyzed by rapid silica gel chromatography (…). 120g Purification was performed using a silica gel rapid chromatography column with an eluent of 0–2% ethyl acetate / petroleum ether gradient at 80 mL / min. Methyl 3-bromo-4-(bromomethyl)benzoate (8-2) (14 g, 22.7 mmol, yield 43.39%, purity 50%) was obtained, both as colorless oils.

[0297] 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)

[0298] Step 2: Methyl 3-bromo-4-formylbenzoate (8-3)

[0299] NMO (1.00 g, 8.54 mmol, 900 μL, 1.05 eq) was added to a CH3CN (50 mL) solution of methyl 3-bromo-4-(bromomethyl)benzoate (8-2) (5.0 g, 8.12 mmol, 1 eq), and the mixture was stirred at 25 °C for 16 h. TLC showed the reaction was complete. The reactants were diluted with H2O (50 mL), extracted with EtOAc (50 mL x 3), and the combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by rapid silica gel chromatography. 80g A silica gel rapid chromatography column was used, with elution using a gradient of 0–2% ethyl acetate / petroleum ether at 60 mL / min. The fractions of the desired product were combined and concentrated. Methyl 3-bromo-4-carboxymethyl benzoate (8-3) was given as a white solid (1.0 g, 4.11 mmol, yield 50.68%).

[0300] 1H 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)

[0301] Step 3: 3-Methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6), methyl 2,4,7,9-penten-9-carboxylate (8-4)

[0302] Cs₂CO₃ (2.68 g, 8.23 ​​mmol, 2 eq) was added to a Tol. (20 mL) solution of methyl 3-bromo-4-carboxybenzoate (8-3) (1.0 g, 4.11 mmol, 1 eq) and iminodimethyloxothione (766 mg, 8.23 ​​mmol, 2.0 eq), and the reaction was purged with N₂ for 1 min. BINAP (512 mg, 822 μmol, 0.2 eq) and Pd(OAc)₂ (92.4 mg, 411 μmol, 0.1 eq) were added, and the reaction was stirred at 110 °C for 16 h. LC-MS showed that the reaction was complete. The solvent was removed to obtain the residue. The residue was diluted with H₂O (30 mL), extracted with DCM (50 mL * 3), the combined organic layers were dried over Na₂SO₄, filtered, and concentrated to obtain the residue. The residue was ground with EtOAc (10 mL), filtered, and the grayish-white crude solid 8-4 (350 mg) was collected. The mother liquor was concentrated to obtain the residue. The residue was analyzed by rapid silica gel chromatography (…). 24g Purification was performed using a silica gel rapid chromatography column with a gradient elution of 0–50% ethyl acetate / petroleum ether at 35 mL / min. The fractions of the desired product were combined and concentrated to give methyl 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6), methyl 2,4,7,9-penten-9-carboxylic acid (8-4) (475 mg, 2.00 mmol, yield 48.66%) as a yellow solid.

[0303] LC-MS: Rt=0.776min, (ESI)m / z.[M+H]+237.9

[0304] 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)

[0305] Step 4: 3-Methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6), methyl 2,7,9-tetraen-9-carboxylate (8-5)

[0306] Under argon atmosphere, Raney-Ni (400 mg) was added to a MeOH (20 mL) solution of methyl 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6), methyl 2,4,7,9-penten-9-carboxylic acid (8-4) (400 mg, 1.69 mmol, 1 eq) (400 mg, 1.69 mmol, 1 eq). The suspension was degassed under vacuum and purged several times with H2. The mixture was stirred at 50 °C for 16 hours under H2 (50 Psi). TLC showed a small amount of starting material remaining and detected the desired product. The reaction solution was allowed to stand for 20 minutes, the layers were separated, the upper layer was collected, and the residue was concentrated. The residue was purified by rapid silica gel chromatography (…). 12g A silica gel rapid chromatography column was used, with eluents of 0–60 g and a gradient elution of 80% ethyl acetate / petroleum ether at a flow rate of 35 mL / 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), methyl 2,7,9-tetraen-9-carboxylic acid (8-5) (217 mg, 906 μmol, yield 53.79%) was obtained as a grayish-white solid.

[0307] LC-MS: Rt=0.744min, (ESI)m / z.[M+H]+240.0

[0308] 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)

[0309] Step 5: 3-Methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6), 2,7,9-tetraen-9-carboxylic acid(8-6)

[0310] Methyl 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-9-carboxylic acid ester (8-5) (200 mg, 836 μmol, 1 eq) was dissolved in a solution of THF (2 mL) and MeOH (2 mL). LiOH·H₂O (1 M, 2.00 mL, 2.39 eq) was added, and the mixture was stirred at 25 °C for 2 hours. LC-MS showed that the reaction was complete and the desired product mass was detected. The solvent was removed to obtain the residue. The pH of the residue was adjusted to 3-4 with 1 M HCl, precipitating a yellow solid. The solid was filtered, 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-tetraen-9-carboxylic acid (8-6) was obtained as a yellow solid (170 mg, 755 μmol, yield 90.29%).

[0311] LC-MS: Rt=0.490min, (ESI)m / z.[M+H]+226.0

[0312] 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-tetraen-9-carbonyl)amino]phenyl]carbamate (8-7)

[0313] EDCI (61.3 mg, 319 μmol, 1.2 eq) was added to a Py (2 mL) solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-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), and the mixture was stirred at 50 °C for 2 h. LC-MS showed that the reaction was complete and the desired product mass was detected. The reactants were diluted with H2O (3 mL), extracted with EtOAc (5 mL * 3), the combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by rapid silica gel chromatography. 12g A silica gel rapid chromatography column was used, with a gradient elution of 0–90% ethyl acetate / petroleum ether at a flow rate of 30 mL / 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-tetraen-9-carbonyl)amino]phenyl]carbamate (8-7) (140 mg, 274 μmol, yield 88.16%) was obtained as a white foam.

[0314] LC-MS: Rt=0.989min, (ESI)m / z.[M+H]+510.1

[0315] 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-tetraen-9-carboxamide (Example 8)

[0316] tert-butyl N-[4-(4-fluorophenyl)-2-[(3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,7,9-tetraen-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 desired product mass was detected. The solvent was removed with N2 to obtain the residue. The pH of the residue was adjusted to 8 with Na2CO3 (aq.), extracted with DCM (3 mL * 3), the organic layers were combined, dried with Na2SO4, filtered, concentrated, and the residue was obtained. The residue was ground with MTBE (2 mL), filtered, 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.

[0317] LC-MS: Rt=0.833min, (ESI)m / z.[M+H]+410.0

[0318] 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)

[0319] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.49(br s,1F)

[0320] Synthesis of Example 9

[0321] Step 1: 3-Methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6), 2,4,7,9-penten-9-carboxylic acid(9-1)

[0322] Methyl 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-penten-9-carboxylic acid ester (8-4) (300 mg, 935 μmol, 1 eq) dissolved in MeOH (2 mL) and THF (2 mL) was added to LiOH·H₂O (1 M, 1.48 mL, 1.58 eq), and the mixture was stirred at 20 °C for 2 h. LC-MS showed that the reaction was complete and the desired product mass was detected. The solvent was removed to obtain the residue. The residue was diluted with H₂O (3 mL), filtered, and the filtrate was adjusted to pH 3-4 with 1 M HCl, precipitating a yellow solid. The precipitate 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-penten-9-carboxylic acid (9-1) was obtained (50 mg, 224 μmol, yield 23.94%).

[0323] LC-MS: Rt=0.666min, (ESI)m / z.[M+H]+224.0

[0324] 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-penten-9-carbonyl)amino]phenyl]carbamate (9-2)

[0325] EDCI (51.5 mg, 268 μmol, 1.2 eq) was added to a Py (1 mL) solution of 3-methyl-3-oxo-3-thia-2-azabicyclo[4.4.0]dec-1(6),2,4,7,9-penten-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), and the mixture was stirred at 50 °C for 2 h. LC-MS showed that the reaction was complete and the m / z of the desired product was detected. The reactants were diluted with H2O (3 mL), extracted with EtOAc (5 mL * 3), the combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by rapid silica gel chromatography. 12g A silica gel rapid chromatography column was used, with a gradient elution of 0–80% ethyl acetate / petroleum ether at a flow rate of 30 mL / 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-penten-9-carbonyl)amino]phenyl]carbamate (9-2) (85 mg, 167 μmol, yield 74.77%) was obtained as a white solid.

[0326] LC-MS: Rt=1.025min, (ESI)m / z.[M+H]+508.2

[0327] 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-penten-9-carboxamide (Example 9)

[0328] 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-penten-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 desired product mass was detected. The solvent was removed with N2 to obtain the 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, concentrated, and the residue was obtained. The residue was ground with MTBE (2 mL), filtered, 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.

[0329] LC-MS: Rt=0.853min, (ESI)m / z.[M+H]+408.0

[0330] 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)

[0331] 19 F NMR(376MHz,DMSO-d6)δ(ppm)-117.47(br s,1F)

[0332] Synthesis of Example 10

[0333] Step 1: tert-butyl N-[2-[(2-methyl-2-oxo-3H-2,1-benzothiazol-6-carbonyl)amino]-4-(2-thienyl)phenyl]carbamate (10-1)

[0334] To a solution of 2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxylic acid (4-4) (32 mg, 151 μmol, 1.0 eq) in pyridine (1 mL), 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) were added. 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 under vacuum. The residue was purified by rapid silica gel chromatography. 4g A silica gel rapid chromatography column was used, with elution of 0–100% ethyl acetate / petroleum ether gradient at 20 mL / min (petroleum ether / ethyl acetate = 0 / 1, Rf = 0.53). Tert-butyl N-[2-[(2-methyl-2-oxo-3H-2,1-benzothiazol-6-carbonyl)amino]-4-(2-thienyl)phenyl]carbamate (10⁻¹) (75 mg, 149 μmol, yield 98.3%, purity 96%) was obtained as a pale yellow solid.

[0335] LC-MS: Rt=0.869min, (ESI)m / z.[M+H-56]+427.9;

[0336] Step 2: N-[2-amino-5-(2-thienyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazole-6-carboxamide (Example 10)

[0337] Tert-butyl N-[2-[(2-methyl-2-oxo-3H-2,1-benzothiazol-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 h. LC-MS showed that the starting material was completely consumed and the desired product was detected. The pH of the reaction mixture was adjusted to 7 with saturated NaHCO₃ aqueous solution, and then concentrated under N₂ to remove DCM. The residue was purified by preparative HPLC (FA conditions) (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [phase A: water (FA) - phase B: ACN]; gradient: 30%-50% B over 10 min). N-[2-amino-5-(2-thienyl)phenyl]-2-methyl-2-oxo-3H-2,1-benzothiazol-6-carboxamide (Example 10) was obtained as a yellow solid (20 mg, 51.6 μmol, yield 38.4%, purity 99%).

[0338] LC-MS: Rt=1.657min, (ESI)m / z.[M+H]+383.9

[0339] 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).

[0340] The following compounds were synthesized using the methods described in the above examples combined with general methods. The corresponding structures and characterizations of the products are shown in Table 1:

[0341] Table 1

[0342] Biological Test Example 1: HDAC1 Enzyme Activity Inhibition Experiment

[0343] Experimental materials

[0344] HDAC1 (BPS, catalog number 50001), HDAC3 (BPS, catalog number 50003), Trypsin (Sigma, catalog number T1426), Ac-peptide (LGK(Ac)) (GL Biochem, custom), 384-well plate (Perkin Elmer, catalog number 6007279), Echo 650 ultrasonic nanoliter liquid handling system (Labcyte, model Echo 650), microplate reader (Perkin Elmer, model Envision).

[0345] Experimental methods

[0346] (1) Enzyme reaction process

[0347] 1.1 Prepare 1x buffer (modified Tris buffer).

[0348] 1.2 Dilution of the compound: The compound was dissolved in 100% DMSO and the compound solution was added to a 384-well plate using an Echo 650.

[0349] 1.3 Prepare enzyme solution: Prepare enzyme solution using 1x buffer.

[0350] 1.4 Prepare substrate solution: Add Trypsin and Ac-peptide to 1x buffer.

[0351] 1.5 Add 15 μL of enzyme solution to a 384-well plate and add 15 μL of 1x buffer to the negative control well.

[0352] 1.6 Add 10 μL of substrate solution to each well to begin the reaction. Set the microplate reader to 355 nm excitation wavelength and 460 nm emission wavelength to dynamically read the values.

[0353] (2) Data Analysis

[0354] 2.1 Convert the original data into % inhibition rate according to Formula 1:

[0355] Formula 1: % Inhibition Rate = (Max - Signal) / (Max - Min) * 100

[0356] Substituting the % inhibition rate data into XL-Fit Equation 2, we obtain the IC. 50 value:

[0357] Formula 2: Y=Bottom+(Top-Bottom) / (1+(IC 50 / X)*HillSlope)

[0358] Where Y is the % inhibition rate and X is the compound concentration.

[0359] Experimental results

[0360] The bioactivity of some compounds was determined experimentally; "A" indicates IC50. 50 (nM) < 100, "B" indicates 100 <IC 50 (nM) < 1000, “C” means 1000 ≤ IC 50 (nM); HDAC3 activity selectivity for HDAC1 is achieved using HDAC3 IC. 50 / HDAC1 IC 50 The levels are indicated by a plus sign: "+" indicates 3 ≤ fold < 10, "++" indicates 10 ≤ fold < 20, and "+++" indicates 20 ≤ fold (see Table 2).

[0361] Table 2: Inhibitory activity of some compounds

[0362] Experimental results show that the compound described in this invention has an inhibitory effect on HDAC1 but does not affect the activity of HDAC3, exhibiting superior subtype selectivity.

[0363] Biological Test Example 2: Detection Experiment of Intracellular Histone Acetylation Levels

[0364] 2.1 Experimental Materials

[0365] A549 cells (NCACC, catalog number SCSP-503), DMEM medium (Gibco, catalog number 11995065), fetal bovine serum (FBS, Gibco, catalog number A5669701), penicillin-streptomycin 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 TM BCA protein assay kit (Thermo, catalog number 23227), Tanon TM 4-20% Bis-Tris protein pregel (Tianneng, catalog number 180-9115H), SuperBlock TM T20(TBS) blocking buffer (Thermo, catalog number 37536), SuperSignal TM Chemiluminescent substrate (Thermo, catalog number 34577), iBlotTM 2. Gel transfer instrument (Invitrogen, model IB21001), chemiluminescence gel imaging system (Tianneng, model 4600SF).

[0366] 2.2 Experimental Methods

[0367] 2.2.1 Cell treatment with compounds: A549 cells were cultured in DMEM + 10% FBS + 1% penicillin-streptomycin antibiotic complete medium; 2 mL of A549 cell suspension (4 × 10⁶ cells / well) was added to each well of a 6-well plate. 4 Cells / mL), after the cells adhered, the compound was added, and 0.1% (v / v) DMSO was added to the control wells. The cells were incubated at 37°C (containing 5% CO2) for 48 hours.

[0368] 2.2.2 Protein extraction: After 48 hours of compound treatment, the supernatant was removed, SDS lysis buffer was added to lyse the cells, and the supernatant was collected by centrifugation at 4°C. The protein concentration was determined using a BCA protein detection kit.

[0369] 2.2.3 Western Blot

[0370] Gel electrophoresis: Mix 10 μg of total protein with sample buffer, denature at 98°C for 10 minutes, load the protein precast gel, and electrophores at a constant voltage of 100V for 90 minutes.

[0371] Protein transfer: using iBlot TM Use a gel transfer apparatus at 20V for 7 minutes to transfer the protein from the gel to a membrane.

[0372] Blocking and antibody incubation: using SuperBlock TM Block the protein membrane with blocking buffer at room temperature for 1 hour; then dilute the primary antibody (1:1000) with blocking buffer and incubate overnight at 4°C on a shaker. The next day, remove the protein membrane and wash it three times with 1X TBST for 10 minutes each time at room temperature; then incubate with secondary antibody (1:2000) at room temperature for 2 hours, followed by washing three times with 1X TBST for 10 minutes each time.

[0373] Chemiluminescence imaging: Drain the protein membrane, uniformly cover the chemiluminescent substrate on the membrane, and place it in a gel imaging system for imaging.

[0374] Data processing: The grayscale value of the target band was measured using ImageJ 1.53 software.

[0375] 2.3 Experimental Results

[0376] The experimental results are shown in Figure 1. As a substrate of HDAC1 / 2, the acetylation levels of histones H3K27 and H3K9 increased with increasing compound dosage (compared to the internal reference protein H3). These results demonstrate that the compound described in this invention can promote the acetylation levels of histones H3K27 and H3K9 in a dose-dependent manner.

[0377] Biological Test Example 3: Tumor Cell Proliferation Inhibition Experiment

[0378] 3.1 Experimental Materials

[0379] 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 antibiotics (Gibco, catalog number 15140122), trypsin (Gibco, catalog number 25200056), CellTiter-Glo assay kit (Promega, catalog number G7572), 384-well clear flat-bottom black-walled cell culture plate (Corning, catalog number 3764), micropipette (Tecan, model D300e), multi-functional microplate reader (Biotek, model SynergyHTX).

[0380] 3.2 Experimental Methods

[0381] 3.2.1 Cell Culture: A549 cells were cultured in DMEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics; Jurkat cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics; HuT 78 cells were cultured in IMDM medium with 20% fetal bovine serum and 1% penicillin-streptomycin antibiotics; ensuring that the cells were always in the logarithmic growth phase and that the cell viability was greater than 95%.

[0382] 3.2.2 Preparation of compound concentration gradients: The analyte was added to a 384-well plate using an ultra-micro pipette, starting at 10 μM and diluted 3-fold with DMSO, for a total of 9 concentrations, with triplet wells.

[0383] Cell treatment with the compound: Add 40 μL of trypsin-digested A549, Jurkat, or HuT 78 cell suspension to 384-well plates pre-stamped with the test compound, i.e., 100 cells per well, with a final DMSO concentration of 0.4%. Incubate the cell culture plates at 37°C in a 5% CO2 incubator for 6 days.

[0384] 3.2.3 Detection: Add 20 μL of CellTiter-Glo reagent to each well of the cell culture plate and incubate with shaking at room temperature for 30 minutes. Detect the luminescence signal at 578 nm using a multi-mode microplate reader.

[0385] 3.2.4 Data Analysis: GraphPad Prism 8.0 software was used to fit the data using a four-parameter inhibitor-reaction model to obtain the IC50 of the test compound. 50 Value (half-maximal inhibitory concentration).

[0386] 3.3 Experimental Results

[0387] The inhibitory activities of some compounds on the proliferation of A549, Jurkat, and HuT 78 cells were determined experimentally. "A" indicates IC50. 50 (μM)<1, “B” indicates 1 <IC 50 (μM)<10, “C” means 10≤IC 50 (μM), see Table 3.

[0388] Biological Test Example 4: T Cell Activation Experiment

[0389] 4.1 Experimental Materials

[0390] RPMI 1640 medium (Gibco, catalog number 11875119), fetal bovine serum (FBS, BDBIO, catalog number F806-500), penicillin-streptomycin antibody (Gibco, catalog number 15140122), mouse anti-human CD3 antibody (BioLegend, catalog number 300332), human PBMCs (SaiLyBio, catalog number XFB-HP025B), human IL2 ELISA kit (R&D, catalog number DY202), 96-well clear round-bottom microplate (Corning, catalog number 3799), 96-well clear flat-bottom high-binding microplate (Corning, catalog number 3361), 96-well clear flat-bottom white microplate (Corning, catalog number 3764), multi-functional microplate reader (Molecular Devices, model SpectraMax i3x).

[0391] 4.2 Experimental Methods

[0392] 4.2.1 Resuscitate PBMC cells: Resuspend in RPMI 1640 complete medium (RPMI 1640 + 10% FBS + 1% penicillin-streptomycin) to a final volume of 10⁻⁶ cells / mL. 6 Cells were incubated overnight at 37°C in a 5% CO2 incubator.

[0393] 4.2.2 Compound treatment of PBMC cells: Collect PBMC cells and add 2×10⁻⁶ compounds to each well of a 96-well clear round-bottom microplate. 5 Add 100 μL of culture medium containing twice the concentration of the test compound (final concentration 0-10 μM) to each cell (100 μL), mix well, and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0394] 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.

[0395] 4.2.3 The PBMC suspension pretreated for 24 hours was transferred to a 96-well plate coated with CD3 antibody and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0396] 4.2.2 After 24 hours, 100 μL of culture supernatant was taken and the IL-2 concentration was detected by ELISA.

[0397] 4.3 Experimental Results

[0398] The experimental results are shown in Figure 2. It can be observed that after T cells in PBMCs were activated by anti-CD3 antibody for 24 hours, the concentration of IL-2 in the culture supernatant increased with the increase of the concentration of the test compound. This proves that the compound of the present invention can enhance T cell activation in a dose-dependent manner, promote the secretion of cytokines such as IL-10 by T cells, help regulate the tumor microenvironment, and exert a better anti-cancer effect.

[0399] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, isotope thereof, Formula I wherein, It can be a single bond or a double bond; X1is selected from N, NH, NR a R b , CR a R b , CHR a R b ; X2is selected from O, NH, CR a R b , CHR a R b ; R a R b Each is independently selected from the group consisting of: H, deuterium, tritium, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C4 alkoxy; or R a R b The atoms attached to it together form substituted or unsubstituted groups selected from the group consisting of C3-C8 cycloalkyl, C6-C8 aromatic ring, 5-8 membered aromatic heterocycle, or 3-8 membered heterocycle; Ring A is a 5-10 membered heterocycle or a 5-10 membered aromatic heterocycle; the heterocycle may be saturated or partially unsaturated; R1 is independently a halogen, hydroxyl group, substituted or unsubstituted C1-C4 alkyl group, or substituted or unsubstituted C1-C4 alkoxy group; Each q can be 0, 1, 2, or 3 independently; Ar is independently selected from: C6-C10 aromatic rings and 5-12 heterocyclic aromatic rings; R2 and R3 are each independently selected from the following group: NH2, substituted or unsubstituted C1-C4 alkyl groups, and halogens; m can be 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 following group: C1-C8 alkyl, C6-C10 aromatic ring, C3-C10 cycloalkyl, 5-12 membered heteroaromatic ring, 5-12 membered heterocycle; Unless otherwise specified, the heterocycles and aromatic heterocycles comprise one, two, or three heteroatoms selected from N, S, or O as the ring skeleton; the heterocycles and aromatic heterocycles may be substituted or unsubstituted; In the above definitions of substituents, substitution refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of: 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 alkylamine, C6-C10 aryl, five- or six-membered heteroaryl, and five- or six-membered heterocyclic group.

2. The compound of claim 1, wherein The compounds are of the structure 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 can be 1, 2, 3, 4 or 5 independently; The remaining substituents are as described in claim 1.

3. The compound of claim 1, wherein The compounds are of the structure shown in Formula I-A, I-B, I-C, I-D, or I-E: wherein R c each independently H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4alkoxy, substituted or substituted C3-C6cycloalkyl; Y is selected from the group consisting of (CH2) p CH=CH; p can be 1, 2, 3, 4 or 5 independently; The remaining substituents are as described in claim 1.

4. The compound of claim 1, wherein In the compound, Ar is a group selected from the group consisting of: phenyl, pyridinyl, pyrimidinyl, pyridazinyl, tetrazinyl, triazinyl, pyrroleyl, thiopheneyl, furanyl, tetrazolyl, triazolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, thiazolyl, naphthyl, indolyl, inzolyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiopheneyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoisoxazolyl, benzotriazolyl, and morpholinyl.

5. The compound of claim 1, wherein In the compound, Cy is selected from the group consisting of: benzene ring, thiophene ring; and / or R3 is an independent halogen; n is 0, 1, 2, 3 or 4.

6. The compound of any one of claims 1-3, wherein The compounds are of the structure shown in Formula I-AA, I-BB, I-CC, I-DD, or I-EE: wherein R c each independently H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1-C4alkoxy; Y is selected from the group consisting of (CH2) p CH=CH; p can be 1, 2, 3, 4 or 5 independently; Cy is selected from the following group: benzene ring, thiophene; The definitions of the remaining substituents are as described in claim 3.

7. The compound of any one of claims 1-5, wherein The compounds have structures as shown in Formulae I-AAA, I-AAB, and I-AAC: wherein R c is as defined in claim 3.

8. The compound of any one of claims 1-5, wherein X1is selected from N, NH or CR a R b ; X2is selected from O, NH, CR a R b ; R a , R b each independently is selected from the group consisting of H, substituted or unsubstituted C1-C8alkyl; R1 is independently a halogen, hydroxyl group, substituted or unsubstituted C1-C4 alkyl group, or substituted or unsubstituted C1-C4 alkoxy group; Each q can be 0, 1, 2, or 3 independently; R2 and R3 are each independently selected from the following group: NH2, substituted or unsubstituted C1-C4 alkyl groups, and halogens.

9. The compound of any one of claims 1-8, wherein, The compounds can be selected from the group consisting of:

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

11. Use of a compound according to any one of claims 1 to 9 or a composition according to claim 10, characterized in that, Used in the preparation of: (a) histone deacetylase (HDAC) inhibitors; and / or (b) medicines for the treatment and / or prevention and relief of HDAC-related malignancies; and / or (c) medicines for the treatment of malignancies in combination with immune checkpoint inhibitors.

12. The use according to claim 11, characterized in that, The malignant tumors are 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 schwannoma (MPNST), esophageal cancer, bladder cancer, mesothelioma, melanoma, astrocytoma, undifferentiated pleomorphic sarcoma, head and neck cancer, gastric adenocarcinoma, myxoid fibrosarcoma, 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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