Benzothiadiazine compounds for inhibiting HDAC6 and preparation method and application thereof

Abstract

This invention provides a benzothiadiazine compound that inhibits histone deacetylases, its preparation method, and its applications. The general structural formula is shown in Formula (I), or an isomer thereof, or a pharmaceutically acceptable salt, ester, or prodrug thereof. The benzothiadiazine compound of this invention has a novel structure and, as an inhibitor of histone deacetylases HDAC6 and HDAC1, can effectively inhibit the proliferation of K562 (human chronic myeloid leukemia cells) and NCI-H929 (human myeloma cells). It exhibits low cytotoxicity to normal cells and low potential cardiac risk, and holds promise as a highly effective and low-toxicity antitumor therapeutic agent.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a benzothiadiazine compound that inhibits histone deacetylase, its preparation method, and its application. Background Technology

[0002] Histone deacetylases (HDACs) catalyze the deacetylation of histones and non-histone proteins, and together with histone acetyltransferases (HATs), they regulate intracellular acetylation levels, thereby controlling gene expression. Currently, 18 subtypes of mammalian HDACs are known, divided into four classes: Class I (HDAC1, HDAC2, HDAC3, HDAC8); Class II: IIa (HDAC4, HDAC5, HDAC7, HDAC9) and IIb (HDAC6, HDAC10); Class III (Sirt1–Sirt7); and Class IV (HDAC11).

[0003] Currently, there are five marketed histone deacetylase inhibitors (HDACi): vorinostat, belinostat, panobinostat, romidepsin, and chidamide. Vorinostat and romidepsin are used to treat cutaneous T-cell lymphoma (CTCL), belinostat and chidamide are used to treat relapsed and refractory peripheral T-cell lymphoma (PTCL), and panobinostat is used in combination with bortezomib and dexamethasone to treat multiple myeloma (MM). Although HDAC inhibitors have achieved good clinical efficacy, they still have the following drawbacks: (1) strong toxic side effects, such as nausea, vomiting, and bone marrow suppression; (2) genotoxicity; and (3) poor pharmacokinetic characteristics, low bioavailability, and short half-life. Therefore, developing novel HDAC inhibitors with high efficacy and low toxicity remains a challenge.

[0004] HDAC6 is the largest member of the HDAC family. Unlike other HDAC members, HDAC6 is the only histone deacetylase containing two catalytic domains (CD1 and CD2). It is mainly distributed in the cytoplasm rather than the nucleus and plays no significant role in post-translational modifications of histones. HDAC6 possesses strong histone deacetylase activity and can mediate the deacetylation process of non-histone proteins. Its main substrates include α-tubulin, heat shock protein 90 (HSP90), and cortactin. Due to its unique structure and substrate diversity, HDAC6 participates in various intracellular physiological processes, including cell movement, endocytosis, autophagy, apoptosis, and protein transport and degradation. Numerous studies have confirmed that HDAC6 overexpression is closely related to various diseases, such as cancer, autoimmune diseases, and neurodegenerative diseases.

[0005] Celgene's HDAC6 inhibitor, Ricolinostat (ACY-1215), has entered Phase II clinical trials. Phase Ib results show that Ricolinostat is a safe and well-tolerated selective HDAC6 inhibitor that can be used alone or in combination with lenalidomide and dexamethasone for the treatment of relapsed or refractory multiple myeloma.

[0006] Summary of the Invention

[0007] This invention provides a novel benzothiadiazine compound that inhibits HDAC6 activity, its preparation method, and its application. It exhibits strong inhibitory activity against both HDAC6 and HDAC1, good selectivity for tumor cells, and low potential cardiotoxicity, and is expected to serve as a highly effective and low-toxicity antitumor therapeutic agent.

[0008] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides a benzothiadiazine compound with the general structural formula shown in formula (I), or an isomer thereof, or a pharmaceutically acceptable salt, ester or prodrug thereof;

[0009]

[0010] in,

[0011] R1 is selected from hydrogen, halogen, alkyl, alkoxy, benzyl, aryl, heteroaryl, cyano, acyl, amide, alkylamine, sulfonyl, or a 5-7 membered ring containing one or more heteroatoms from 0-2 N, O, and S, which may optionally be substituted.

[0012] R2 is selected from hydrogen, alkyl, acyl, amide, sulfonyl, or a 5-7 membered ring containing one or more heteroatoms of N, O, and S, which may optionally be substituted.

[0013] Preferably, the alkyl group is selected from C1-C6 alkyl groups, which may optionally be substituted with 1-3 halogens;

[0014] Preferably, the halogen is selected from F, Cl, or Br;

[0015] Preferably, the 5-7 membered ring is selected from pyrrole, morpholino, piperidinyl, piperazine ring, tetrahydroquinolino, tetrahydrotriazolylpyrazine, diazacycloheptyl or piperazine, which may optionally be substituted;

[0016] Preferably, the heteroaryl group is selected from naphthyl, anthracene, pyridyl, pyrimidinyl, pyrazinyl, indolyl, imidazolyl, benzoxazolyl, benzofuranyl, benzothiophene, benzothiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrroleyl, pyrazolyl or 5,6,7,8-tetrahydroisoquinoline, which may optionally be substituted;

[0017] Preferably, the acyl group is selected from C1-C4 alkyl-substituted acyl groups or aryl-substituted acyl groups;

[0018] Preferably, the sulfonyl group is selected from C1-C4 alkyl-substituted sulfonyl groups or aryl-substituted sulfonyl groups;

[0019] Preferably, the alkylamine group is selected from dimethylaminoalkyl, methylaminoalkyl, piperazinealkyl, or piperidinylalkyl, and may optionally be substituted.

[0020] Preferably, pharmaceutically acceptable salts of the compound of formula (I) include anionic salts formed by reacting the compound of formula (I) with hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, malic acid, p-toluenesulfonic acid or oxalic acid; or cationic salts formed by reacting the compound of formula (I) with sodium ion solution or potassium ion solution.

[0021] More preferably, R1 is selected from hydrogen, halogen, C1-C6 alkyl or alkoxy containing 1-6 C atoms, which may optionally be substituted;

[0022] More preferably, R2 is selected from hydrogen, benzyl, C1-C6 alkyl or acyl containing 1-6 C atoms.

[0023] In some specific embodiments of the present invention, the benzothiadiazine compound may be the following compound or its isomers, or its pharmaceutically acceptable salts, esters, or prodrugs:

[0024]

[0025]

[0026] A second aspect of the present invention provides a method for preparing the benzothiadiazine compounds described in the above-mentioned technical solution, comprising the following steps:

[0027] S1, Compound (II) reacts with chlorosulfonyl isocyanate via Friedel-Crafts acylation and condensation cyclization to yield Compound (III);

[0028]

[0029] R1 is selected from hydrogen, halogen, alkyl, alkoxy, benzyl, aryl, heteroaryl, cyano, acyl, amide, alkylamine, sulfonyl or a 5-7 membered ring containing one or more heteroatoms of 0-2 N, O and S, which may optionally be substituted.

[0030] S2, Compound (III) reacts with methyl 4-bromomethylbenzoate via a first substitution reaction to give Compound (IV);

[0031]

[0032] In compound (VI), X is a halogen;

[0033] S3, if R2 is hydrogen, then the compound of formula (IV) undergoes a hydroxylamine reaction with hydroxylamine to obtain the compound of formula (I);

[0034]

[0035] If R2 is not hydrogen, then compound (Ⅳ) undergoes a second substitution reaction with compound (Ⅴ) to obtain compound (Ⅵ), and compound (Ⅵ) undergoes a hydroxylamine reaction to obtain compound (Ⅰ);

[0036]

[0037] Wherein, R2 is a non-hydrogen group as shown in claim 1, 2, 4 or 5;

[0038] In compound (V), X is a halogen.

[0039] Preferably, in step S1, the Friedel-Crafts acylation reaction includes the following steps: mixing the compound of formula (II) with chlorosulfonyl isocyanate at -45°C to -30°C for a substitution reaction, and using aluminum chloride as a catalyst for a condensation cyclization reaction at 105°C to 120°C.

[0040] Preferably, in step S2, the first substitution reaction includes the following steps: using potassium carbonate as a catalyst, the compound of formula (III) undergoes a substitution reaction with methyl 4-bromomethylbenzoate, followed by dehydration, to obtain the compound of formula (IV).

[0041] Preferably, in step S3, the hydroxylation reaction uses an alkyl alkoxide as a catalyst; the second substitution reaction uses potassium carbonate as a catalyst, and the reaction temperature is -5℃ to 5℃.

[0042] A third aspect of the present invention provides an intermediate compound for preparing the benzothiadiazine compounds described in the foregoing technical solutions, comprising a compound of formula (III) or an isomer thereof, a pharmaceutically acceptable salt, an ester, or a prodrug:

[0043]

[0044] And / or, compounds of formula (IV) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0045]

[0046] And / or, compounds of formula (VI) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0047]

[0048] R1 and R2 are as shown in the aforementioned technical solution.

[0049] The fourth aspect of the present invention provides the use of the benzothiadiazine compound described in the foregoing technical solution, the benzothiadiazine compound obtained by the preparation method described in the foregoing technical solution, or the intermediate compound described in the foregoing technical solution in the preparation of histone deacetylase inhibitors or antitumor drugs.

[0050] Preferably, the histone deacetylase inhibitor is an HDAC6 and / or HDAC1 inhibitor.

[0051] Preferably, the antitumor drug includes drugs for treating breast cancer, colon cancer, liver cancer, multiple myeloma, sarcoma, lung cancer, prostate cancer, rectal cancer, kidney cancer, pancreatic cancer, leukemia, neuroblastoma, glioma, head cancer, cervical cancer, thyroid cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, testicular cancer, bladder cancer, esophageal cancer, gastric cancer, nasopharyngeal cancer, buccal cancer, oral cancer, gastrointestinal stromal tumor, and skin cancer; more preferably, the antitumor drug includes drugs for treating multiple myeloma.

[0052] A fifth aspect of the present invention provides a pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable excipients; said active ingredient comprising benzothiadiazine compounds as described in the foregoing technical solutions or benzothiadiazine compounds prepared by the foregoing technical solutions.

[0053] Preferably, the pharmaceutically acceptable excipients include one or more of the following: diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, flavorings, and sweeteners.

[0054] The pharmaceutical composition of this invention can be formulated into various forms such as tablets, powders, granules, capsules, oral liquids, and injectable drugs. All of these dosage forms can be prepared using conventional methods in the pharmaceutical field. The active component in the pharmaceutical composition of this invention can also be combined with other effective ingredients that have therapeutic effects or enhance therapeutic effects, reduce toxic side effects, or prolong metabolic time to form a pharmaceutical composition.

[0055] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0056] 1. The benzothiadiazine compounds provided by this invention have novel structures and high inhibitory activity (nM level) against HDAC6 and HDAC1. Some compounds show a certain selective inhibitory effect on HDAC6 (compared to HDAC1).

[0057] 2. The benzothiadiazine compounds provided by this invention exhibit strong anti-proliferative activity against human chronic myeloid leukemia cells K562 and human myeloma cells NCI-H929, while also showing good selective inhibitory effect on human embryonic lung fibroblasts MRC-5.

[0058] 3. The benzothiadiazine compounds provided by this invention have weak inhibitory activity against hERG and low potential cardiotoxicity. Detailed Implementation

[0059] In this invention, the term "isomer" includes, but is not limited to, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, tautomers, mixtures of racemic mixtures and diastereomers, and their pharmaceutically acceptable salts. Unless otherwise stated, when an isomer component is not specifically specified, all possible isomers are included.

[0060] In this invention, "pharmaceutically acceptable salt" refers to a compound modified by forming an acidic or basic salt of the benzothiadiazine compound described in this invention, including but not limited to salts of inorganic acids selected from, for example, hydrochlorides, phosphates, hydrogen phosphates, hydrobromic acids, sulfates, sulfites, and nitrates; and salts of organic salts selected from, for example, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, alkanoates such as acetates, and salts of HOOC-(CH2)n-COOH, where n can be any integer from 0 to 4. If the compound is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acidic salt. Conversely, if the product is a free base, the addition salt (e.g., a pharmaceutically acceptable addition salt) can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with acid, consistent with the conventional process for preparing acid addition salts from basic compounds. Those skilled in the art will understand the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts without excessive experimentation. Similarly, "pharmaceutically acceptable ester" refers to an ester derivative formed by the formation of the small molecule inhibitors of the present invention, and "pharmaceuticalally acceptable prodrugs" include precursor compounds having the ability to form the small molecule inhibitors of the present invention in vitro and in vivo.

[0061] In this invention, the term "aromatic ring" or "aryl" refers to a monocyclic or fused polycyclic group with 5-12 carbon atoms, possessing a fully conjugated π-electron system. Non-limiting examples of aromatic rings include benzene rings, biphenyl rings, naphthyl rings, and anthracene rings. Aromatic rings can be unsubstituted or substituted. Substituents in aromatic rings can be selected from halogens, nitro groups, amino groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, halo-C1-C6 alkyl groups, halo-C1-C6 alkoxy groups, C3-C6 cycloalkyl groups, and halo-C3-C6 cycloalkyl groups.

[0062] In this invention, "heteroaryl" refers to an unsaturated carbon ring with 5-12 ring atoms, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. The heteroaryl ring can be monocyclic or bicyclic, i.e., formed by the fusion of two rings. Specific heterocyclic aryl groups can be: pyrrole, pyrazolyl, imidazolyl, furanyl, thiophene, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrazinyl, pyrrole, morpholinyl, piperidinyl or piperazinyl, thiophene, benzothiophene, pyrazolyl, benzopyrazolyl, indolyl, dioxopentyl, benzo[1,3]dioxopentyl, oxazolyl, benzooxazolyl, furanyl, benzofuranyl, thiazolyl or benzothiazolyl, etc. Heterocyclic aryl groups can be unsubstituted or substituted. The substituents of the heterocyclic aryl group can be selected from halogen, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, C3-C6 cycloalkyl, and halo-C3-C6 cycloalkyl.

[0063] In this invention, "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined above. Examples of "alkoxy" as used in this invention include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy, and the alkoxy group may be unsubstituted or substituted.

[0064] In this invention, "halogen" or "halogenated" means fluorine, chlorine, bromine or iodine.

[0065] In this invention, the pharmaceutically acceptable excipients include, but are not limited to, conventional pharmaceutical diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., and flavoring agents, sweeteners, etc., may be added if necessary. The pharmaceutical compositions of this invention can be formulated into various forms such as tablets, powders, granules, capsules, oral liquids, and injectable drugs, and all of the above dosage forms can be prepared according to conventional pharmaceutical methods.

[0066] In some specific embodiments of the present invention, the benzothiadiazine compound can be prepared according to the following general formula:

[0067]

[0068] Using aniline with R1 as a substituent as a starting material, chlorosulfonyl isocyanate is reacted with chlorosulfonyl isocyanate via Friedel-Crafts acylation and condensation to obtain the key intermediate compound benzothiadiazine dioxide (compound of formula (III)). The N at the 4-position of benzothiadiazine dioxide undergoes a nucleophilic substitution reaction with methyl 4-bromomethylbenzoate to generate compound of formula (IV). Compound of formula (IV) is directly hydroxylated or nucleophilically substituted at the 2-position to form an ester, which is then hydroxylated to obtain compound of formula (I).

[0069] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. All raw materials without specified synthesis methods were purchased from manufacturers such as Exploration Platform, Aladdin, and Sigma-Aldrich, and were of analytical grade.

[0070] Example 1: Preparation of 4-((1,1-dioxide-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T1)

[0071] T1 was prepared according to the general synthetic method, and the synthetic route is as follows:

[0072]

[0073] The specific synthesis steps are as follows:

[0074] 1) At -40°C, aniline (270 mmol) was dissolved in 100 mL of nitromethane, and then added dropwise to a 200 mL solution of chlorosulfonyl isocyanate (26 mL, 300 mmol) in nitromethane using a dropper funnel. After the addition was complete, the reaction mixture was stirred for another 30 minutes and aluminum chloride (39 g, 300 mmol) was added. The mixture was then heated to 110°C with stirring and maintained for 1 hour. The crude material was then poured into ice, the precipitate was collected by vacuum filtration, washed with cold water and anhydrous diethyl ether, and slurried with anhydrous ethanol to give intermediate 2a.

[0075] 2) Intermediate 2a (10 mmol) and potassium carbonate (12 mmol) were added to 30 mL of N,N-dimethylformamide, and an acetonitrile solution of methyl 4-bromomethylbenzoate (10 mmol / 20 mL) was added dropwise. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction solution was poured into 200 mL of water, and a solid precipitated. The solid was separated by filtration. The filter cake was washed with water and dried under vacuum to produce a mixture of regioisoalkylated products. The mixture was then subjected to column chromatography (petroleum ether: ethyl acetate system) to give intermediate methyl 4-((1,1-dioxo-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazin-2-yl)methyl)benzoate (3a). ESI-MS (m / z): 346.90 [M+H]+. 1H NMR(600MHz,DMSO)δ11.54(s,1H),7.92(dd,J =22.7,8.0Hz,3H),7.77–7.72(m,1H),7.48(d,J=8.1Hz,2H),7.38–7.30(m,2H),5.06(s,2H),3.84(s,3H).

[0076] 3) Intermediate 3a (2 mmol) was added to a methanol solution of hydroxylamine / sodium methoxide (20 mmol). Sodium methoxide methanol solution (10 mmol) was added dropwise under ice-water bath conditions, and the reaction was allowed to proceed for 4-6 hours. After the reaction was complete, twice the volume of water was added, and the pH was adjusted to 7-8 with 2M HCl aqueous solution. A solid precipitated out; this was filtered, slurried in methanol, and the target compound T1 was obtained. mp>250℃. ESI-MS (m / z): 348.50 [M+H]+. 1H NMR (400MHz, DMSO) δ 7.80 (d, J=7.9Hz, 1H), 7.75– 7.62 (m, 3H), 7.38 (p, J=6.8, 5.6Hz, 2H), 7.24 (t, J=7.6Hz, 2H), 4.98 (s, 2H).

[0077] Example 2: Preparation of 4-((7-fluoro-1,1-dioxy-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T2)

[0078]

[0079] T2 was prepared according to the general method. mp 170.6–172.1 °C. ESI-MS (m / z): 366.60 [M+H]+. 1H NMR (400 MHz, DMSO) δ 12.57 (s, 1H), 7.71 (dd, J = 11.7, 8.4 Hz, 3H), 7.54 (td, J = 8.8, 3.0 Hz, 1H), 7.37 (dd, J = 13.5, 7.9 Hz, 2H), 7.26 (dd, J = 9.1, 4.4 Hz, 1H), 4.98 (s, 2H).

[0080] Example 3: Preparation of N-hydroxy-4-((7-methyl-1,1-dioxy-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)benzamide (T3)

[0081]

[0082] T3 was prepared according to the general method. mp 204.1–208.8 °C. ESI-MS (m / z): 377.90 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.05 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 2.8 Hz, 2H), 7.25 (d, J = 8.6 Hz, 1H), 5.01 (s, 2H), 3.84 (s, 3H).

[0083] Example 4: Preparation of 4-((7-chloro-1,1-dioxy-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T4)

[0084]

[0085] T4 was prepared according to the general method. mp 164.1–166.2 °C. ESI-MS (m / z): 381.80 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.06 (s, 1H), 7.93 (d, J = 2.4 Hz, 1H), 7.74 (dd, J = 8.8, 2.5 Hz, 1H), 7.71–7.68 (m, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1H), 5.00 (s, 2H).

[0086] Example 5: Preparation of N-hydroxy-4-((7-methoxy-1,1-dioxy-3-oxy-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)benzamide (T5)

[0087]

[0088] T5 was prepared according to the general method. mp 204.1–208.8 °C. ESI-MS (m / z): 377.90 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.05 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 2.8 Hz, 2H), 7.25 (d, J = 8.6 Hz, 1H), 5.01 (s, 2H), 3.84 (s, 3H).

[0089] Example 6: Preparation of 4-((7-bromo-1,1-dioxy-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T6)

[0090]

[0091] T6 was prepared according to the general method. mp 197.6–202.2 °C. ESI-MS (m / z): 425.70 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 12.44 (s, 1H), 11.27 (s, 1H), 9.07 (s, 1H), 7.93 (d, J = 2.2 Hz, 1H), 7.83 (dd, J = 8.8, 2.2 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.8 Hz, 1H), 5.42 (s, 2H).

[0092] Example 7: Preparation of 4-((4-benzyl-7-chloro-1,1-dioxy-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T7)

[0093]

[0094] T7 was prepared according to the general method. mp 135.8-137.8℃. ESI-MS (m / z): 445.90 [M+H]+. 1H NMR (600MHz, DMSO-d6) δ 7.66 (d, J = 7.8Hz, 2H), 7.53 (d, J = 2.6Hz, 1H), 7.32 (d, J = 6.0Hz, 4H), 7.24 (d, J = 7.5Hz, 3H), 6.71 (t, J = 5.6Hz, 1H), 6.62 (d, J = 9.0Hz, 1H), 4.43 (d, J = 5.7Hz, 2H), 4.03 (s, 2H).

[0095] Example 8: Preparation of 4-((7-chloro-4-(cyclopropylmethyl)-1,1-dioxy-3-oxy-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T8)

[0096]

[0097] T8 was prepared according to the general method. mp 164.4-166.4℃. ESI-MS (m / z): 410.00 [M+H]+. 1H NMR (400MHz, DMSO-d6) δ9.41 (s, 1H), 7.63 (d, J = 7.8Hz, 2H), 7.50 (d, J=2.7Hz,1H),7.33(dd,J=8.9,2.7Hz,1H),7.23(d,J=7.9Hz,2H),6.74(d,J =9.0Hz,1H),6.17(t,J=5.2Hz,1H),4.02(s,2H),3.00(t,J=5.9Hz,2H),1.09 (t,J=7.0Hz,1H),0.51–0.46(m,2H),0.24(t,J=5.0Hz,2H).

[0098] Example 9: Preparation of 4-((4-acetyl-1,1-dioxy-3-oxy-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T9)

[0099]

[0100] T9 was prepared according to the general method. mp 150.1-152.4℃. ESI-MS (m / z): 390.02 [M+H]+. 1H NMR (400MHz, DMSO-d6) δ 11.25 (s, 1H), 9.05 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 2.8 Hz, 2H), 7.25 (d, J = 8.6 Hz, 1H), 5.01 (s, 2H), 3.84 (s, 3H).

[0101] Example 10: Preparation of 4-((6-chloro-1,1-dioxy-3-oxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-2-yl)methyl)-N-hydroxybenzamide (T10)

[0102]

[0103] T10 was prepared according to the general method. mp 168.2–170.1℃. ESI-MS (m / z): 381.66 [M+H]+. 1H NMR (600MHz, DMSO-d6) δ 11.13 (s, 1H), 9.06 (s, 1H), 7.93 (d, J = 2.4 Hz, 1H), 7.74 (dd, J = 8.8, 2.5 Hz, 1H), 7.71–7.68 (m, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1H), 4.98 (s, 2H).

[0104] Example 11 Compound's HDAC Inhibition Activity

[0105] The inhibitory effects of the compounds on HDAC6 and HDAC1 targets were detected using the established experimental platform and testing conditions, with Rocilinostat (ACY-1215) used as a positive control compound.

[0106] 1) Reagents and consumables

[0107]

[0108] 2) Instruments

[0109]

[0110]

[0111] 3) HDAC enzyme preparation

[0112] HDAC6 enzyme inhibition assay: Prepare a 40mM DMSO solution from the sample and store it in the dark for later use.

[0113] HDAC1 enzyme inhibition experiment: The compounds were prepared as 20 mM DMSO solutions and stored in the dark for later use.

[0114] 4) HDAC6 enzymatic reaction process

[0115] a. Prepare a 1× reaction solution.

[0116] b. Preparation of compound concentration gradients: The final concentration of the analyte was initially 80 nM, diluted 4-fold, resulting in 5 concentrations, and a single-well assay was performed. The final concentration of the positive compound was initially 3 μM, diluted 3-fold, resulting in 10 concentrations, and a replicate assay was performed. Solutions were serially diluted to the corresponding 100-fold final concentrations in a 384-well Source plate, and then 250 nL was transferred to the 384-well reaction plate using an Echo 550. 250 nL of 100% DMSO was transferred to both the Max and Min wells.

[0117] c. Prepare a 1.67× enzyme solution using a 1× reaction solution.

[0118] Add 15 μL of 1.67× enzyme solution to each well; add 15 μL of 1.6× reaction solution to each Min well. Incubate at room temperature for 15 minutes.

[0119] e. Prepare a 2.5× substrate mixture using 1× reaction solution.

[0120] Add 10 μL of a 2.5× substrate mixture to each well of the reaction plate to initiate the reaction.

[0121] g uses Synergy to continuously read fluorescence signals.

[0122] 5) HDAC1 enzymatic reaction process

[0123] a. Prepare a 1× reaction solution.

[0124] b. Preparation of compound concentration gradients: The final concentration of the analyte was initially 2 μM, diluted 10-fold, and four concentrations were set up for single-well detection; the positive control concentration was initially 3 μM, diluted 3-fold, and ten concentrations were set up for replicate detection. Solutions were serially diluted to the corresponding 100-fold final concentrations in a 384-well Source plate, and then 250 nmol was transferred to the 384-well reaction plate using an Echo 550. 250 nmol of 100% DMSO was transferred to both the Max and Min wells.

[0125] c. Prepare a 1.67× enzyme solution using a 1× reaction solution.

[0126] Add 15 μL of 1.67× enzyme solution to each well; add 15 μL of 1.6× reaction solution to each Min well. Incubate at room temperature for 15 minutes.

[0127] e. Prepare a 2.5× substrate mixture using 1× reaction solution.

[0128] Add 10 μL of a 2.5× substrate mixture to each well of the reaction plate to initiate the reaction.

[0129] g uses Synergy to continuously read fluorescence signals.

[0130] 6) Data Analysis

[0131] The slope is obtained by selecting the linear response segment. The percentage inhibition rate is then calculated using the following formula:

[0132]

[0133] Where: Mean(Max) is the mean slope value of each Max well (containing DMSO and enzyme); Mean(Min) is the mean slope value of each Min well (without enzyme); Sample Signal is the slope value of the compound well.

[0134] Fitting dose-response curves: Using the log value of compound concentration as the X-axis and the corresponding percentage inhibition rate as the Y-axis, the dose-response curves were fitted using the log(inhibitor) vs. response-variable slope function of GraphPad Prism 5 to obtain the IC50 of each compound inhibiting enzyme activity. 50 value.

[0135] The experimental results are shown in the table:

[0136]

[0137]

[0138] Experimental results show that the compound of this invention exhibits strong inhibitory activity against HDAC6 (IC50). 50 <50 nM), and also showed inhibitory activity against HDAC1. Except for compounds T7 and T8, the inhibitory activity of the compounds against HDAC1 was better than that of the positive control Rocilinostat (ACY-1215).

[0139] Example 12: Antiproliferative activity of the compound against tumor cells and normal human cells

[0140] The antiproliferative activity of the compounds of this invention against K562 (human chronic myeloid leukemia cells), NCI-H929 (human myeloma cells), and MRC-5 human normal embryonic lung fibroblasts was tested using the CCK-8 assay at a single concentration (10 μM), with Rocilinostat (ACY-1215) selected as a positive control.

[0141] 1) Cell lines

[0142] K562 (human chronic myeloid leukemia cells) (Source: ATCC);

[0143] NCI-H929 (human myeloma cells) (Source: ATCC);

[0144] MRC-5 human normal embryonic lung fibroblasts (Source: ATCC)

[0145] 2) Reagents and consumables

[0146] Cell Counting Kit-8(Cat#CK04-13, Dojindo)

[0147] 96-well culture plate (Cat#3599, Corning Costar)

[0148] Fetal bovine serum (Cat#10099-141, GIBCO)

[0149] Culture medium (Invitrogen)

[0150] SpectraMax M5 Microplate Reader (Molecular Devices) - Desktop Microplate Reader

[0151] 3) Experimental steps

[0152] (1) Cell Culture

[0153] a) Collect cells in the logarithmic growth phase, count them, and resuspend the cells in complete culture medium.

[0154] b) Adjust the cell concentration to a suitable level (determined based on the cell density optimization test results in the allicin pharmacodynamics experiment), and seed 100 μl of cell suspension into each well of a 96-well plate.

[0155] c) Incubate the cells at 37°C, 100% relative humidity, and 5% CO2 for 12-24 hours.

[0156] (2) Filtering

[0157] a) Collect cells in the logarithmic growth phase, count them, resuspend the cells in complete culture medium, adjust the cell concentration to a suitable level (determined according to the results of cell density optimization experiments), and seed them in 96-well plates, adding 100 μl of cell suspension to each well. Incubate the cells at 37°C, 100% relative humidity, and 5% CO2 for 24 hours.

[0158] b) After the cells have fully adhered, dilute the test compound to 1 mM and 10 mM with DMSO. Add 1 μl / well to the cells to achieve final compound concentrations of 10 μM and 100 μM. The final DMSO concentration is 1%.

[0159] c) The cells were incubated at 37°C, 100% relative humidity, and 5% CO2 for 48 hours.

[0160] d) Add 10 μl of CCK-8 solution and incubate at 37°C for 4 hours.

[0161] e) After gentle shaking, measure the absorbance at 450 nm wavelength on a SpectraMax M5 Microplate Reader, and use the absorbance at 650 nm as a reference to calculate the suppression rate.

[0162] (3) Data processing

[0163] The inhibition rate of drug on tumor cell growth is calculated using the following formula: Tumor cell growth inhibition rate % = [(Ac-As) / (Ac-Ab)] × 100%

[0164] As: OA of the sample (cells + CCK-8 + test compound)

[0165] Ac: Negative control OA (cells + CCK-8 + DMSO)

[0166] Ab: Positive control OA (culture medium + CCK-8 + DMSO)

[0167] The results of the anti-cell proliferation experiment are shown in the table (unit: Inh%in 10μM):

[0168]

[0169]

[0170] A:>90%; B:80~90%; C:70~80%; D:50~70%E:<50%

[0171] As can be seen from the table above, compared with the positive control Rocilinostat, the compounds of the present invention showed better in vitro anti-tumor cell proliferation activity against tumor cells. The anti-proliferative activity against K562 (human chronic myeloid leukemia cells) was better than that against the positive control, and the anti-proliferative activity against NCI-H929 (human myeloma cells) was better than or equivalent to that against the positive control.

[0172] Meanwhile, compared with Rocilinostat, the compound of the present invention has weaker inhibitory activity against MRC-5 human normal embryonic lung fibroblasts and has lower toxicity, revealing that the compound of the present invention has better selectivity in inhibiting the proliferation of tumor cells and normal cells, suggesting that it may have lower toxicity when used as an anti-tumor drug.

[0173] Example 13: Experiment on the effect of compounds on hERG potassium channels

[0174] The potential cardiotoxic side effects of some compounds T1–T6, T9, and T10 of this invention were preliminarily investigated in vitro using an hERG potassium channel inhibition assay. The experimental procedure is as follows:

[0175] 1) Cell preparation

[0176] CHO-hERG cells were cultured in 175 cm² culture flasks. When the cell density reached 60-80%, the culture medium was removed, the cells were washed once with 7 mL PBS, and then 3 mL Detachin was added for digestion.

[0177] After complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and add 5 mL of culture medium to resuspend the cells to ensure a cell density of 2–5 × 10⁶ / mL.

[0178] 2) Electrophysiological recording process

[0179] The entire process of single-cell high-impedance sealing and whole-cell pattern formation was automated by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +20 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds, and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular fluid was applied for another 2 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2 minutes. After all concentrations were administered, 10 μM Cisapride, a positive control compound, was administered. At least 3 cells (n≥3) were tested for each concentration.

[0180] 3) Compound preparation

[0181] The stock solution of the compound was diluted with extracellular fluid. 2 μL of the stock solution was added to 998 μL of extracellular fluid, and then the solution was serially diluted 5-fold in extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested. Experimental data were analyzed using XLFit software.

[0182] The experimental results are shown in the table below:

[0183]

[0184] hERG experimental results showed that compounds T1–T6, T9, and T10 all exhibited inhibitory activity against hERG potassium ion channels greater than 20 μM, suggesting that the compounds of this invention have low potential cardiotoxicity.

[0185] Example 14 Pharmaceutical Composition 1

[0186] The compound T4 prepared in Example 4 was mixed with a filler, a disintegrant, and a lubricant, granulated, and tableted to obtain a pharmaceutical composition 1 with compound T4 as the active ingredient.

[0187] Example 15 Pharmaceutical Composition 2

[0188] The compound T5 prepared in Example 5 was mixed with a solvent and a stabilizer, filtered, and packaged to obtain pharmaceutical composition 2 with compound T5 as the active ingredient.

[0189] Example 16 Pharmaceutical Composition 3

[0190] Compound T2 prepared in Example 2 and compound T10 prepared in Example 10 were mixed with filler, disintegrant, and lubricant, granulated, and tableted to obtain pharmaceutical composition 3 with compounds T2 and T10 as active ingredients.

[0191] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A benzothiadiazine compound, characterized by, The compounds include the following compounds or pharmaceutically acceptable salts thereof:

2. The benzothiadiazine compound according to claim 1, characterized in that, The pharmaceutically acceptable salts include anionic salts of compounds T1-T10 reacting with hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, malic acid, p-toluenesulfonic acid or oxalic acid; or cationic salts of compounds T1-T10 reacting with sodium ion solution or potassium ion solution.

3. A method for preparing the benzothiadiazine compound according to any one of claims 1-2, comprising the following steps: S1, a compound of formula (II) is subjected to a Friedel-Crafts acylation reaction and a cyclization reaction with chlorosulfonyl isocyanate to obtain a compound of formula (III); wherein R1 is selected from hydrogen, halogen, methyl or methoxy; S2, a compound of formula (III) is subjected to a first substitution reaction with methyl 4-bromomethylbenzoate to obtain a compound of formula (IV); wherein R1 is selected from hydrogen, halogen, methyl or methoxy; S3, if R2 is hydrogen, a compound of formula (IV) is subjected to a hydroxylamine reaction with hydroxylamine to obtain a compound of formula (I); if R2 is not hydrogen, a compound of formula (IV) is subjected to a second substitution reaction with a compound of formula (V) to obtain a compound of formula (VI), and a compound of formula (VI) is subjected to a hydroxylamine reaction with hydroxylamine to obtain a compound of formula (I); wherein R2 is selected from benzyl, cyclopropylmethyl or acetyl; wherein X in the compound of formula (V) is halogen.

4. The production method according to claim 3, characterized by, In step S1, a compound of formula (II) is mixed with chlorosulfonyl isocyanate at -45℃ to -30℃ to perform a substitution reaction, and aluminum chloride is used as a catalyst to perform a cyclization reaction at 105℃ to 120℃; and / or, in step S2, the first substitution reaction comprises the following steps: a compound of formula (III) is subjected to a substitution reaction with methyl 4-bromomethylbenzoate using potassium carbonate as a catalyst, and then dehydrated to obtain a compound of formula (IV); and / or, in step S3, the hydroxylamine reaction uses an alkyl alcohol salt as a catalyst; and the second substitution reaction uses potassium carbonate as a catalyst, and the reaction temperature is -5℃ to 5℃.

5. Use of the benzothiadiazine compound according to any one of claims 1-2 or the benzothiadiazine compound obtained by the method according to any one of claims 3-4 in the preparation of a histone deacetylase inhibitor or an anti-tumor drug.

6. Use according to claim 5, characterized in that, The histone deacetylase inhibitor is an HDAC6 and / or HDAC1 inhibitor.

7. Use according to claim 6, characterized in that, The anti-tumor drug includes drugs for treating breast cancer, colon cancer, liver cancer, multiple myeloma, sarcoma, lung cancer, prostate cancer, rectal cancer, renal cancer, pancreatic cancer, blood cancer, neuroblastoma, glioma, head cancer, neck cancer, thyroid cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, testicular cancer, bladder cancer, esophageal cancer, gastric cancer, nasopharyngeal cancer, buccal cancer, oral cancer, gastrointestinal stromal tumor and skin cancer.

8. A pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable adjuvants; the active ingredient comprises the benzothiadiazine compound according to any one of claims 1-2 or the benzothiadiazine compound obtained by the method according to any one of claims 3-4.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutically acceptable excipients include one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorptive carriers, lubricants, flavoring agents, and sweetening agents.

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