WO2017148318A1 - Substituted acrylamide compound and pharmaceutical composition thereof - Google Patents

Abstract

Disclosed are a substituted acrylamide compound and a pharmaceutical composition thereof, the substituted acrylamide compound being a compound of a formula (I), or a crystalline form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate, or solvate thereof. The compound of the present invention can inhibit activity of a histone deacetylase (HDAC) and also has improved pharmacodynamic/pharmacokinetic properties; the compound has high applicability, is safe to use, and can be used for the preparation of a pharmaceutical composition for the treatment of HDAC-mediated diseases, thereby having great market potential.

Description

Substituted acrylamide compound and pharmaceutical composition thereof Technical field

The invention belongs to the technical field of medicine, and in particular relates to a substituted acrylamide compound and a pharmaceutical composition thereof, which are useful for treating HDAC-mediated related diseases.

Background technique

Histone deacetylase (HDACs, HDAC) is a class of proteases that play an important role in the structural modification of chromosomes and regulation of gene expression. In general, acetylation of histones facilitates the dissociation of DNA and histone octamers, and the nucleosome structure is relaxed, allowing various transcription factors and co-transcription factors to specifically bind to DNA binding sites, activating genes. Transcription. In the nucleus, histone acetylation is in a dynamic equilibrium with histone deacetylation and is regulated by histone acetyltransferase (HAT) and histone deacetylase. HAT transfers the acetyl group of acetyl-CoA to the specific lysine residue at the amino terminus of histones. HDACs deacetylate histones, bind tightly to negatively charged DNA, compact chromatin, and inhibit gene transcription. .

In cancer cells, overexpression of HDACs leads to enhanced deacetylation, which restores the gravitational pull between DNA and histones by restoring the positive charge of histones, making the relaxed nucleosomes very tight and detrimental to specific genes. Expression, including some tumor suppressor genes. Histone deacetylase inhibitors (HDACi) can regulate apoptosis and differentiation-related protein expression and stability by inducing histone acetylation in specific regions of chromatin, and induce apoptosis and differentiation. Become a new class of anti-tumor drugs. HDACi not only has a good therapeutic effect on a variety of hematological tumors and solid tumors, but also has the advantages of relatively high selectivity and low toxicity of tumor cells.

In the treatment of malignant tumors, HDACi is effective and well tolerated. Normal cells are highly tolerant to high concentrations of HDACi, so HDACi is considered to be non-toxic, and experiments have shown that low doses of HDACi have neurological and renal protective effects under hypoxia, inflammatory response or load stress. Non-specific HDACi has been reported in stage I and phase II clinical trials for adverse reactions such as nausea, vomiting, abnormal blood system and prolonged QT interval. Non-specific HDACi such as SAHA, LBH589, and ITF-2357 may cause Sexual thrombocytopenia or myelosuppression, and whether specific HDACi causes corresponding adverse reactions in tumor research is inconclusive, and there is no report of adverse reactions as low-dose HDACi. Therefore, low doses of HDACi with organ protection and better tolerance are increasingly being used to try to treat chronic diseases.

Therefore, there is still a need in the art to develop compounds having inhibitory activity or better pharmacodynamic properties against histone deacetylases.

Summary of the invention

In view of the above technical problems, the present invention discloses a substituted acrylamide compound and a composition comprising the same and use thereof, which have better histone deacetylase inhibitory activity and/or have better pharmacodynamics/ Pharmacokinetic properties.

In this regard, the technical solution adopted by the present invention is:

A histone deacetylase inhibitor, such as a substituted acrylamide compound represented by formula (I), or a crystalline form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvent compound,

Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently hydrogen, deuterium or halogen;

An additional condition is that at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ is deuterated or deuterated.

As a further improvement of the present invention, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁶ , R ⁷ , R ⁸ and R ⁹ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹⁰ and R ¹¹ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, the compound may be selected from the following compounds or a pharmaceutically acceptable salt thereof, but is not limited to the following compounds:

With this technical solution, the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.

Preferably, the strontium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, and even more preferably greater than 95. %, more preferably greater than 99%.

Specifically, in the present invention, the strontium isotope content of each of the deuterated positions of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ is at least 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20%, more preferably more than 25%, more preferably more than 30%, more preferably more than 35%, more preferably more than 40 More preferably, more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably more than 70%, more preferably more than 75% More preferably, it is more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 99%.

In another preferred embodiment, at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ of the compound of formula (I) R contains 氘, more preferably two R contains 氘, more preferably three R contains 氘, more preferably four R contains 氘, more preferably five R contains 氘, more preferably six R contains 氘, more Preferably, the seven R-containing strontiums, more preferably eight R-containing hydrazines, more preferably nine R-containing hydrazines, more preferably ten R-containing hydrazines, more preferably eleven R-containing hydrazines.

In another preferred embodiment, the compound does not include a non-deuterated compound.

The present invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the histone deacetylase inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate thereof Or a pharmaceutical composition of a solvate, stereoisomer, prodrug or isotopic variation.

As a further improvement of the present invention, the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant At least one of a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.

As a further improvement of the present invention, the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.

Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, Intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical compositions of the present invention can be produced by methods well known in the art, such as conventional mixing methods, dissolution methods,

Granulation method, sugar-coated pellet method, grinding method, emulsification method, freeze-drying method, and the like.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to a histone deacetylase inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, The hydrate or solvate is mixed to form a pharmaceutical composition.

The active ingredients of the invention may also be used in combination with other active ingredients. The choice of such combination is based on the condition of the treatment, the cross-reactivity of the ingredients, and the combined pharmaceutical properties. It is also possible to administer any of the compounds of the invention in combination with one or more other active ingredients in a single dosage form for simultaneous or sequential administration to a patient. Combination therapies can be administered simultaneously or sequentially. When administered continuously, the combination can be administered in two or more administrations. Combination therapy can provide "synergistic effects" or "synergistic effects", in other words, when the active ingredients are used together, the effect obtained is greater than the sum of the effects obtained by using the compounds separately. When the active ingredient: (1) is co-formulated and administered or delivered simultaneously in a combined formulation; (2) administered as a separate formulation or administered in parallel; or (3) obtained by some other dosage regimen Synergy. When delivered in alternation therapy, synergistic effects can be obtained when the compounds are administered or released sequentially, for example, as separate tablets, pills or capsules, or by separate injections of separate syringes. Generally, during alternation therapy, the effective dose of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the effective dose of two or more active ingredients is administered together.

The invention also discloses the use of a substituted acrylamide histone deacetylase inhibitor as described above, i.e., the compounds of the invention are advantageously useful as therapeutic agents for the treatment of, for example, cell proliferative disorders.

The term "treating" as used in the treatment of a condition of the present invention generally relates to the treatment of a human or animal (e.g., by a veterinarian) wherein certain desired therapeutic effects are achieved, for example, by inhibiting the progression of the condition (including reducing the rate of progression, Development stops, improves the condition and cures the condition. It also includes treatment as a preventive measure (such as prevention). The use of a patient who has not yet developed a condition but is at risk of developing the condition is also included in the term "treatment."

The term "effective dose" as used herein refers to an amount of an HDAC inhibitor that, when administered with a desired therapeutic regimen, produces certain desired therapeutic effects, while having a reasonable benefit/risk ratio.

The term "treatment" includes combination therapy wherein, for example, two or more treatments are used in combination, either sequentially or simultaneously. For example, the compounds described herein can also be used in combination therapy with other drugs, such as cytotoxic drugs. Examples of treatments include, but are not limited to, chemotherapy (administering an active drug including, for example, an HDAC inhibitor, an antibody (eg, in immunotherapy), a prodrug (eg, photodynamic therapy, GDEPT, ADEPT) Etc.), surgery, radiation therapy and gene therapy.

In one embodiment, the treatment is treatment of a proliferative disorder.

The terms "proliferative disorder" and "proliferative disorder" are used interchangeably herein and refer to excess or abnormally fine. Undesired or uncontrolled cell proliferation of cells (not required), such as neoplastic or proliferative growth.

In one embodiment, the treatment is treatment of a proliferative disorder characterized by benign, pre-malignant or worsening cell proliferation, including but not limited to tumors, hyperplasia and neoplasms (eg, histiocytoma, glial) Tumor, astrocyoma, osteoma, cancer (see below), psoriasis, bone disease, fibroproliferative diseases (eg, connective tissue), pulmonary fibrosis, atherosclerosis, vascular smooth muscle Cell proliferation (eg, stenosis or restenosis after angioplasty).

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the treatment is treatment of a cancer such as lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, colon cancer, rectal cancer, colorectal cancer, breast cancer, ovarian cancer, prostate cancer, testis Cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma, malignant melanoma, basal cell tumor or leukemia.

In one embodiment, the treatment is treatment of a condition mediated by HDACs.

As used herein, the term "HDACs mediated disorder" refers to a disorder in which the action of HDAC and/or HDAC is important or necessary, for example, for the onset, progression, performance, etc. of the disorder; or refers to the known use of HDAC inhibitors (eg, , a disease treated with trichostatin A). For any particular cell type, one of ordinary skill in the art can readily determine whether an HDACs inhibitor candidate is capable of treating a HDACs mediated disorder. Experiments that can be conveniently used to evaluate the activity of a particular compound are described, for example, in Watkins et al., International (PCT) Patent Application No. WO 02/30879.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Herein, "halogen" means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.

As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".

As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).

The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.

Compared with the prior art, the beneficial effects of the present invention are: the compound of the present invention has excellent inhibition to histone deacetylase; the technique of deuteration changes the metabolism of the compound in the organism, so that the compound has more Good pharmacokinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability; the substitution of a hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, and improves the therapeutic effect of the drug; Substituting a hydrogen atom in a compound inhibits certain metabolites and increases the safety of the compound.

detailed description

The preparation of the structural compound of the formula (I) of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

Example 1 Preparation of N-hydroxy-3-(3-d5-phenylsulfamoyl-phenyl)-acrylamide (Compound 8), The following routes are synthesized:

Step 1 Synthesis of Compound 3.

3-Chlorosulfonylbenzoic acid (Compound 1, 300 mg), d7-aniline (Compound 2, 0.30 mL) was dissolved in 10 mL of anhydrous dichloromethane. After filtration, the filter cake was washed three times with 10 mL of ice cold dichloromethane. LC-MS: 281.1 [M-1] ^- .

Step 2 Synthesis of Compound 4.

Under a nitrogen atmosphere, the compound 3 (379 mg) was dissolved in 10 mL of anhydrous tetrahydrofuran, and BH ₃ ·THF (1M, 5.4 mL) was slowly added dropwise, and the mixture was allowed to react at room temperature overnight. 2 mL of methanol was added dropwise under ice-cooling, and the mixture was stirred at room temperature for 30 minutes. The mixture was evaporated to dryness and evaporated to ethyl ether. The organic phase was combined, washed with EtOAc EtOAc m. LC-MS: 269.1 [M+1] ⁺ .

Step 3 Synthesis of Compound 5.

Compound 4 (343 mg) was dissolved in anhydrous dichloromethane, and 2,2,6,6-tetramethylpiperidine oxide (TEMPO, 20 mg) was added, and iodobenzenediacetic acid (436 mg) was added in portions. After the reaction at room temperature for 2 hours. Concentration and evaporation of methylene chloride (EtOAc m.) LC-MS: 266.1 [M+1] ⁺ .

Step 4 Synthesis of Compound 7.

Compound 5 (313 mg), trimethylphosphonoacetate (compound 6, 600 mg) was dissolved in 7 mL of tetrahydrofuran, and 2.5 mL of 2.5 M NaOH solution was added dropwise in an ice water bath. After the completion of the dropwise addition, the reaction was carried out for 2 hours at room temperature, and 7 mL of acetic acid was added. The ester was shaken, and the aqueous phase was taken. The pH was adjusted to about 2 to 3 with a 6N hydrochloric acid solution to precipitate a solid, which was filtered, and dried under vacuum at 55 ° C overnight to give the product as a white solid (yield 253 mg). LC-MS: 308.0 [M-1] ^- .

Step 5 Compound 8 was synthesized.

Compound 7 (253 mg), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 344 mg) was added, and 5 mL of N, N was added. - Dimethylformamide was dissolved, and 307 μL of N,N-diisopropylethylamine was slowly added thereto in an ice water bath, and 63 mg of hydroxylamine hydrochloride was added thereto, and the mixture was reacted at room temperature for 4 hours. The reaction mixture was poured into 20 mL of 1N EtOAc EtOAc (EtOAc m. Yellow solid product Compound 8 (62 mg). ^{1 H NMR (300MHz, DMSO-} d 6) δ10.84 (s, 1H), 10.33 (s, 1H), 9.14 (s, 1H), 7.90 (s, 1H), 7.77 (d, J = 7.7Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 15.7 Hz, 1H), 6.49 (d, J = 15.8 Hz, 1H) .

Example 2 Preparation of N-hydroxy-3-(3-(2,3,5-d3-phenyl)sulfamoyl-phenyl)-acrylamide (Compound 15), Use the following route for synthesis:

Step 1 Synthesis of Compound 10.

The aniline (1.023 g) was added to 9 mL of hydrazine and dispersed uniformly. The mixture was sealed with deuterated concentrated hydrochloric acid (1.1 mL), and the microwave reaction was sealed at 180 ° C for 2 hours. The microwave reaction apparatus was closed, and the mixture was cooled to room temperature. The pH was adjusted to about 8 to 9 with sodium bicarbonate solid, and ethyl acetate was extracted. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate 10 (876 mg). LC-MS: 96.15 [M + 1] +; 1 H NMR (500MHz, DMSO-d 6) δ7.46 (s, 1H).

Step 2 Synthesis of Compound 11.

3-Chlorosulfonylbenzoic acid (Compound 1, 300 mg) was obtained, and Compound 10 (0.30 mL) was dissolved in 10 mL of anhydrous dichloromethane. The filter cake was washed three times with 10mL of ice-cold dichloromethane, filter cake was collected, dried overnight to give a white solid product Compound 11 (379mg) in vacuo; LC-MS: 280.0 [M + 1] +.

Step 3 Synthesis of Compound 12.

Compound 11 (379 mg) was dissolved in 10 mL of anhydrous tetrahydrofuran, and BH ₃ ·THF (1M, 5.4mL) was slowly added dropwise under nitrogen. After the completion of the dropwise addition, the reaction was carried out at room temperature overnight. 2 mL of methanol was added dropwise under ice-cooling, and the mixture was stirred at room temperature for 30 minutes, and the mixture was evaporated to dryness and evaporated to ethyl ether. The organic layer was combined, washed with brine, dried over sodium sulfate LC-MS: 266.1 [M+1] ⁺ .

Step 4 Synthesis of Compound 13.

Compound 12 (277 mg) was dissolved in anhydrous dichloromethane, and 20 mg of 2,2,6,6-tetramethylpiperidine oxide (TEMPO) was added thereto, and 355 mg of iodobenzenediacetic acid was added in portions. After the addition, the reaction was carried out for 2 hours at room temperature. Concentration and evaporation of methylene chloride (m.p.) LC-MS: 264.0 [M+1] ⁺ .

Step 5 Synthesis of Compound 14.

Compound 13 (228 mg), trimethylphosphonoacetate (compound 6,414 mg) was dissolved in 4 mL of tetrahydrofuran, and 2.5 mL of 2.5 M NaOH solution was added dropwise in an ice water bath. After the completion of the dropwise addition, the reaction was carried out for 2 hours at room temperature, and 5 mL of acetic acid was added. The ester was shaken and separated, and the aqueous phase was taken. The pH was adjusted to about 2 to 3 with a 6N hydrochloric acid solution, and the solid was precipitated, filtered, and dried under vacuum at 55 ° C overnight to give the product as a white solid (13 mg). LC-MS: 304.9 [M-1] ^- .

Step 6 Synthesis of Compound 15.

Compound 14 (218 mg), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 298 mg) was added, and 5 mL of N, N was added. - Dimethylformamide was dissolved, and 266 μl of N,N-diisopropylethylamine was slowly added thereto in an ice water bath, and 55 mg of hydroxylamine hydrochloride was added thereto, and the mixture was reacted at room temperature for 4 hours. The reaction mixture was poured into 20 mL of 1N EtOAc EtOAc (EtOAc m. Yellow solid product Compound 15 (57 mg). ^{1 H NMR (300MHz, DMSO-} d 6) δ10.83 (s, 1H), 10.33 (s, 1H), 9.13 (s, 1H), 7.90 (s, 1H), 7.77 (d, J = 7.7Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 15.9 Hz, 1H), 6.49 (d, J = 15.8 Hz, 1H) .

Example 3 Preparation of N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-2-d-acrylamide (Compound 21), using The following routes are synthesized:

Step 1 Synthesis of Compound 16.

Trimethylphosphonoacetate (compound 6, 1.54g) was dispersed in 6mL of hydrazine water, sealed with 15mg of potassium carbonate, sealed at 80 ° C for 30 minutes, closed the microwave reactor, naturally cooled to room temperature, ethyl acetate The mixture was extracted (10 mL × 2). ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.82 (s, 3H), 3.78 (s, 3H), 3.74 (s, 3H).

Step 2 Synthesis of Compound 17.

3-Chlorosulfonylbenzoic acid (Compound 1, 300 mg), aniline (0.30 mL) was dissolved in 10 mL of anhydrous dichloromethane. After filtration, the filter cake was washed three times with 10 mL of ice cold dichloromethane. LC-MS: 277.0 [M-1] ^- .

Step 3 Synthesis of Compound 18.

Compound 17 (380 mg) was dissolved in 10 mL of anhydrous tetrahydrofuran, and BH ₃ ·THF (1M, 5.4mL) was slowly added dropwise under nitrogen. After the completion of the dropwise addition, the reaction was carried out at room temperature overnight. 2 mL of methanol was added dropwise under ice-cooling, and the mixture was stirred at room temperature for 30 minutes, and the mixture was evaporated to dryness and evaporated to ethyl ether. The organic phase was combined, washed with brine, dried over anhydrous sodium sulfate. ¹ H NMR (300MHz, CDCl ₃ ) δ 7.80 (s, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.44 (t, J = 7.7 Hz) , 1H), 7.27–7.23 (m, 1H), 7.18–7.04 (m, 3H), 4.73 (s, 2H).

Step 4 Synthesis of Compound 19.

Compound 18 (243 mg) was dissolved in anhydrous dichloromethane, and 2,2,6,6-tetramethylpiperidine oxide (TEMPO, 20 mg) was added, and 315 mg of iodobenzenediacetate was added portionwise. After the addition, the reaction was carried out for 2 hours at room temperature. The title compound (19 mg) was obtained as a pale yellow oil (yield). LC-MS: 262.1 [M+1] ⁺ .

Step 5 Synthesis of Compound 20.

Compound 19 (264 mg), compound 16 (424 mg) was dissolved in 4.5 mL of tetrahydrofuran, and 3.0 mL of 2.5 M NaOH solution was added dropwise to an ice water bath. After the completion of the dropwise addition, the mixture was reacted at room temperature for 2 hours, and 5 mL of ethyl acetate was added to shake the liquid to obtain an aqueous phase. The pH was adjusted to about 2 to 3 with a 6N hydrochloric acid solution, and a solid was precipitated, which was filtered, and dried under vacuum at 55 ° C overnight to give a white solid compound 20 (171 mg). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.72 (s, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.05 (s, 1H), 6.86 (t, J = 7.6 Hz, 2H), 6.74 (d, J = 7.8 Hz, 2H), 6.41 (t, J = 7.3 Hz, 1H).

The compound of step 6 is the synthesis of 21.

Compound 20 (171 mg), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 235 mg) plus 5 mL of N,N- The dimethylformamide was dissolved, and 232 μl of N,N-diisopropylethylamine was slowly added in an ice water bath, and 43 mg of hydroxylamine hydrochloride was added thereto, and the mixture was reacted at room temperature for 4 hours. The reaction mixture was poured into 20 mL of 1N EtOAc, EtOAc (EtOAc (EtOAc) /2) The product compound 21 (54 mg) was obtained as pale yellow solid. ^{1 H NMR (300MHz, DMSO-} d 6) δ10.79 (s, 1H), 10.38 (s, 1H), 9.15 (s, 1H), 7.90 (t, J = 1.7Hz, 1H), 7.81-7.66 ( m, 2H), 7.56 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 15.8 Hz, 1H), 7.27 - 7.17 (m, 2H), 7.13 - 7.05 (m, 2H), 7.02 (td , J = 7.1, 1.2 Hz, 1H), 6.49 (d, J = 15.8 Hz, 1H).

Biological activity test

(1) Test for histone deacetylase inhibitory activity

Cell line: lymphoma cell JURKAT; CLONE E6-1 was purchased from CAS; cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ML penicillin, 100 ΜG/ML streptomycin.

Reagents: MTS kit (Promega, Cat# G1111), 0.25% trypsin (Gibco, Cat #25200), Rpmi-1640 (Gibco, Cat#A10491-01), bovine serum (Gibco, Cat#10099141), DMSO ( Sigma, Cat#D2650), HDAC inhibitor drug screening kit (Biovision, Cat. No. K340-100); MTS assay 96-well plate (CORNING, CAT. NO. 3599); 96-well plate (GREINER, CAT.NO) .655076).

experimental method

Compound formulation for HDACs experiments: Test compounds were dissolved in DMSO to make a 20 mM stock solution. The dilution was diluted in DMSO to 50 times the final concentration of the dilution. Dilute into 2 times the final concentration of the dilution with ultrapure water.

Compound IC ₅₀ Assay: Compounds of different concentrations prepared in advance dilution were added to 96-well plates with double replicate wells at each concentration. A mixture of HeLa Nuclear Extract and HDAC Substrate was then added. After mixing, incubate for 30 minutes at 37 ° C and add Lysine Developer. After further mixing, the cells were incubated at 37 ° C for 30 minutes, and fluorescence was detected by a microplate reader (BioTek, Synergy 2) at 330 nm excitation light and 440 nm emission light, and data were collected. In addition, negative and positive controls were established with Trichostatin A as a positive reference.

Compound formulation for MTS experiments: Test compounds were dissolved in DMSO to make a 20 mM stock solution. The solution was diluted in DMSO to a final concentration of 200 times. When dosing, use a cell culture medium to dilute to a final concentration of 4 times of the preservation solution (take 4 μL of 200-fold gradient compound into 196 μL of complete medium), and take 50 μL of 4 times the final concentration of the compound into 150 μL of the culture containing cells. In the board.

MTS cell viability assay: Cell suspensions in logarithmic growth phase were collected, cells were harvested by centrifugation, 150 μL of cells were seeded at a defined density in 96-well plates, and 24 μl of compound diluted in culture medium at 50 μL/well was added after 24 hours. A well of the same volume of 2% DMSO was added as a control, and the final concentration of DMSO was 0.5%. After the cells were cultured for 72 hours, MTS was assayed for cell viability. The specific method is as follows: 20 μL LMTS was added to each well, and the OD ₄₉₀ was detected after being cultured for 1-4 hours in an incubator, with the OD ₆₅₀ value as a reference. GraphPad Prism software produced dose-response curve and calculate IC _50. A represents an IC ₅₀ <100nM, B represents 100nM≤IC ₅₀ ≤200nM, C represents 200nM <IC ₅₀ ≤300nM, D represents the IC _50> 300nM. (As shown in Table 1 below).

Table 1 Example compound against histone deacetylase inhibitory activity

Numbering	HDACs IC 50 (nM)	Jurkat IC 50 (nM)
Belista	C	C
8	C	B
15	C	C
twenty one	B	A

As shown in the above table, Compound 15 of the present invention is comparable to belistatin activity compared to a new drug, belixitastat, developed by Spectrum Biopharmaceutical Company for the treatment of peripheral T-cell lymphoma (PTCL). Compound 21 is superior to belistat, indicating that the compound of the present invention can significantly inhibit histone deacetylase (HDAC), and is thus more suitable for the preparation of diseases associated with histone deacetylase, such as lymphoma.

(2) Liver particle metabolism experiment

Microsomal experiments: human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).

Preparation of stock solution: A certain amount of the compound powder of the example was accurately weighed and dissolved to 5 mM with DMSO.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

A solution of NADPH regeneration system (containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride) was prepared and placed on wet ice before use.

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. Place the 96-well incubator and the NADPH regeneration system in a 37 ° C water bath at 100 rpm / Minutes of shock, pre-incubation for 5 minutes. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

Table 2 Evaluation of liver particle metabolism of the compound of the example

The experimental results are shown in Table 2 above. Compared with belistat, the compound of the present invention has a significant increase in half-life and a small clearance rate, and exhibits superior metabolic stability in both human liver microsomes and rat liver microsomes. Sex, more suitable as a drug that inhibits histone deacetylase.

(3) Rat pharmacokinetic experiments

EXPERIMENTAL OBJECTIVE: To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of N-hydroxy-3-(3-phenylsulfamoyl-phenyl)acrylamide and the compounds of Examples 1-6 in rats.

Experimental animals:

Type and strain: SD rat grade: SPF grade

Gender and quantity: male, 6

Weight range: 180 ~ 220g (actual weight range is 187 ~ 197g)

Source: Shanghai Xipuer Bikai Experimental Animal Co., Ltd.

experiment procedure:

Before the blood sample was collected, 20 L of 2M sodium fluoride solution (esterase inhibitor) was previously added to the EDTA-K2 anticoagulation tube, dried in an 80 degree oven, and stored in a 4 degree refrigerator.

Rats, males, weighing 187-197 g, were randomly divided into 2 groups. They were fasted overnight in the afternoon before the experiment but were free to drink water. Food was given 4 h after administration. Group A was given 3 mg/kg of N-hydroxy-3-(3-phenylsulfamoyl-phenyl)acrylamide, and group B was given 3 mg/kg of the compound of Example 1-6, respectively, 15 min, 30 min, 1 after administration. 2, 3, 5, 8, 10h, take 100-200L of blood from the orbital vein of the rat, place it in a 0.5mL Eppendorf tube that is anticoagulated with EDTA-K2, mix immediately, and after anticoagulation, lighten the test tube as soon as possible. After mixing 5-6 times with light inversion, the blood was taken and placed in an ice box. The blood samples were centrifuged at 4000 rpm, 10 min, 4 ° C for 30 min, and all plasma was collected and stored at -20 ° C immediately. Plasma concentrations in plasma at each time point were determined after sample collection at all time points.

According to the average blood drug concentration-time data obtained after the above, the male SD rats were subjected to ig-administered 9N-hydroxy-3-(3-phenylsulfamoyl) by Winnonin software according to the non-compartmental statistical moment theory. Pharmacokinetic related parameters after phenyl)acrylamide (3 mg/kg) and the compound of Example 1-6 (3 mg/kg).

Experiments have shown that the compounds of the present invention have superior activity and excellent pharmacokinetic properties compared to N-hydroxy-3-(3-phenylsulfamoyl-phenyl)acrylamide, and thus are more suitable as A compound that inhibits histone deacetylase is further suitable for the preparation of a medicament for treating cell proliferative diseases and cancer.

It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples are generally in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (9)

1. A histone deacetylase inhibitor characterized by an acrylamide compound represented by formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or Solvent compound,

   

   Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently hydrogen, deuterium or halogen;

   An additional condition is that at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ is deuterated or deuterated.
2. The histone deacetylase inhibitor according to claim 1, wherein each of R ¹ , R ² , R ³ , R ⁴ and R ^{5 is} independently hydrazine or hydrogen.
3. The histone deacetylase inhibitor according to claim 1, wherein each of R ⁶ , R ⁷ , R ⁸ and R ^{9 is} independently hydrazine or hydrogen.
4. The histone deacetylase inhibitor according to claim 1, wherein each of R ¹⁰ and R ^{11 is} independently hydrazine or hydrogen.
5. The histone deacetylase inhibitor according to claim 1, wherein the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:

   

   

   
6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a histone deacetylase inhibitor according to any one of claims 1 to 5, or a crystalline form thereof, pharmaceutically acceptable A pharmaceutical composition of a salt, hydrate or solvate, stereoisomer, prodrug or isotopic variation.
7. The pharmaceutical composition according to claim 6 wherein said pharmaceutical composition is useful for treating, preventing or ameliorating a condition associated with HDACs. Pharmaceutical compositions comprising these compounds are useful for treating, preventing, or slowing the progression of the disease or disorder in different therapeutic areas, such as cancer.
8. A method of treating a disease comprising administering a compound of claim 1 or a composition of claim 6 to a patient in need of such treatment, the disease being associated with abnormal expression of HDACs, such as a proliferative disease, cancer.
9. A method of treating and/or preventing a disease associated with HDACs expression in a subject, the method comprising administering to the subject a compound of formula (I) according to any one of claims 1 to 5. Or a polymorphic form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or a solvent compound, or a pharmaceutical composition according to any one of claims 6 or 7.