CN108349882B

CN108349882B - Substituted acrylamide compound and pharmaceutical composition thereof

Info

Publication number: CN108349882B
Application number: CN201780003913.0A
Authority: CN
Inventors: 王义汉; 金剑
Original assignee: Shenzhen Targetrx Inc
Current assignee: Shenzhen Targetrx Inc
Priority date: 2016-03-04
Filing date: 2017-02-22
Publication date: 2020-09-18
Anticipated expiration: 2037-02-22
Also published as: WO2017148318A1; CN108349882A

Abstract

The invention provides a substituted acrylamide compound and a pharmaceutical composition thereof, wherein the substituted acrylamide compound is a compound shown as a formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvate thereof. The compound can inhibit the activity of Histone Deacetylase (HDAC), has better pharmacodynamics/pharmacokinetic performance, good applicability and high safety, can be used for preparing medicines for treating diseases mediated by the HDAC, and has good market development prospect.

Description

Substituted acrylamide compound and pharmaceutical composition thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a substituted acrylamide compound and a pharmaceutical composition thereof, which can be used for treating related diseases mediated by HDAC.

Background

Histone Deacetylases (HDACs) are a class of proteases and play an important role in the structural modification and gene expression regulation of chromosomes. Generally, acetylation of histones facilitates dissociation of DNA from histone octamers and relaxation of nucleosome structure, thereby allowing specific binding of various transcription factors and co-transcription factors to DNA binding sites, activating gene transcription. In the nucleus, histone acetylation and histone deacetylation processes are in dynamic equilibrium and are regulated by Histone Acetyltransferase (HAT) and histone deacetylase together. HAT transfers acetyl group of acetyl-CoA to specific lysine residue at the amino terminal of histone, HDACs deacetylates histone, tightly binds to DNA with negative charge, chromatin is densely coiled, and gene transcription is inhibited.

In cancer cells, overexpression of HDACs results in increased deacetylation, increasing the attraction between DNA and histone by restoring the positive charge of histone, and the relaxed nucleosome becomes very compact and unfavorable for the expression of specific genes, including some tumor suppressor genes. Histone deacetylase inhibitors (HDACi) can increase histone acetylation in specific areas of chromatin, thereby regulating expression and stability of proteins related to apoptosis and differentiation, and inducing apoptosis and differentiation, and become a new class of antitumor drugs. HDACi has not only good therapeutic effects on a variety of hematological and solid tumors, but also the advantage 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 resistant to high concentrations of HDACi, and thus HDACi is considered to be non-toxic, and experiments have shown that low doses of HDACi have neuroprotective and nephroprotective effects under the effects of hypoxia, inflammatory responses or stress. The reported adverse reactions of nonspecific HDACI in clinical trials of stage I and stage II of tumors include nausea, vomiting, abnormal response blood system and QT interval prolongation, nonspecific HDACI such as SAHA, LBH589 and ITF-2357 may cause transient thrombocytopenia or myelosuppression, and whether specific HDACI causes corresponding adverse reactions in the research of tumors is not determined, and in addition, no adverse reaction is reported as low-dose HDACI. Therefore, low-dose HDACi, which has organ protection and better tolerability, is increasingly being used in attempts to treat chronic diseases.

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

Disclosure of Invention

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

In contrast, the technical scheme adopted by the invention is as follows:

a histone deacetylase inhibitor, which is a substituted acrylamide compound shown as a formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvent compound thereof,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹Each independently is hydrogen, deuterium or halogen;

with the proviso that R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹At least one of which is deuterated or deuterium.

As a further improvement of the invention, R¹、R²、R³、R⁴And R⁵Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁶、R⁷、R⁸And R⁹Each independently is deuterium or hydrogen.

As a further improvement of the invention, R¹⁰And R¹¹Each independently is deuterium or hydrogen.

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

by adopting the technical scheme, the shape and the volume of deuterium in a drug molecule are basically the same as those of hydrogen, and if the hydrogen in the drug molecule is selectively replaced by deuterium, the original biological activity and selectivity of the deuterium-substituted drug can be generally kept. Meanwhile, the inventor proves that the combination of carbon and deuterium bonds is more stable than the combination of carbon and hydrogen bonds, and the absorption, distribution, metabolism, excretion and other properties of some medicines can be directly influenced, so that the curative effect, safety and tolerance of the medicines are improved.

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

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 25%At 50%, more preferably at more than 55%, more preferably at more than 60%, more preferably at more than 65%, more preferably at more than 70%, more preferably at more than 75%, more preferably at more than 80%, more preferably at more than 85%, more preferably at more than 90%, more preferably at more than 95%, more preferably at more than 99%.

In another preferred embodiment, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹At least one of R, preferably two of R, more preferably three of R, more preferably four of R, more preferably five of R, more preferably six of R, more preferably seven of R, more preferably eight of R, more preferably nine of R, more preferably ten of R, and more preferably eleven of R.

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

The invention also discloses a pharmaceutical composition which contains a pharmaceutically acceptable carrier and the histone deacetylase inhibitor, or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate, a stereoisomer, a prodrug or an isotopic variant thereof.

As a further improvement of the invention, the pharmaceutically acceptable carrier comprises at least one of a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersing agent, 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, pill, capsule, powder, granule, paste, emulsion, suspension, solution, suppository, injection, inhalant, gel, microsphere or aerosol.

Typical routes of administration of the pharmaceutical compositions of the present 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 may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

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

The active ingredients of the present 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 pharmaceutical properties of the combination. It is also possible to combine any of the compounds of the invention with one or more other active ingredients for simultaneous or sequential administration to a patient in a single dosage form. The combination therapy may be administered on a simultaneous or sequential dosing regimen. When administered sequentially, the combination may be administered in two or more administrations. Combination therapy may provide a "synergistic effect" or "synergy", in other words, the effect obtained when the active ingredients are used together is greater than the sum of the effects obtained when the compounds are used separately. When the active ingredients are: (1) are co-formulated and administered or delivered simultaneously in a combined formulation; (2) as separate formulations administered alternately or in parallel; or (3) by some other dosing regimen, a synergistic effect may be obtained. When delivered in alternating treatments, synergy can be obtained when the compounds are administered or released sequentially, e.g., in separate tablets, pills or capsules, or by different injections with separate syringes. Generally, during alternation therapy, the effective dose of each active ingredient is administered sequentially, i.e. consecutively, whereas in combination therapy, the effective doses of two or more active ingredients are co-administered.

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

The term "treatment" as used herein in the treatment of a disorder generally relates to the treatment of a human or animal (e.g., as used by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibition of the development of the disorder (including reduction in the rate of development, cessation of development), amelioration of the disorder, and cure of the disorder. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by 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 some desired therapeutic effect, while providing a reasonable benefit/risk ratio.

The term "treatment" includes combination therapies in which two or more therapies are used in combination, e.g., sequentially or simultaneously. For example, the compounds described herein may also be used in combination therapy with other drugs (e.g., cytotoxic drugs). Examples of therapies include, but are not limited to, chemotherapy (administration of active drugs including, for example, HDAC inhibitors, antibodies (e.g., in immunotherapy), prodrugs (e.g., photodynamic therapy, GDEPT, ADEPT, etc.), surgery, radiation therapy, and gene therapy.

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

The terms "proliferative disorder" and "proliferative disease" are used interchangeably herein and refer to an undesired or uncontrolled proliferation of cells, such as neoplastic or hyperplastic growth, in excess or abnormal cells (not desired).

In one embodiment, the treatment is the treatment of a proliferative disorder characterized by benign, premalignant, or malignant cell proliferation, including, but not limited to, tumors, hyperplasia, and neoplasia (e.g., histiocytoma, glioma, astrocytoma (astrocyoma), osteoma), cancer (see below), psoriasis, bone disease, fibroproliferative disease (e.g., of connective tissue), pulmonary fibrosis, atherosclerosis, vascular smooth muscle cell proliferation (e.g., stenosis or restenosis following angioplasty).

In one embodiment, the treatment is a treatment of cancer.

In one embodiment, the treatment is of a cancer such as lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, breast cancer, ovarian cancer, prostate cancer, testicular 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 of a HDACs-mediated disorder.

As used herein, the term "HDACs-mediated condition" refers to a condition in which HDAC and/or the effects of HDAC are important or necessary, e.g., for the onset, progression, manifestation, etc. of the condition; or to a condition known to be treatable with an HDAC inhibitor (e.g., trichostatin a). For any particular cell type, one of ordinary skill in the art can readily determine whether an hdac inhibitor candidate would treat an HDACs-mediated disorder. Assays 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 WO02/30879, 2002.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

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

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

Compared with the prior art, the invention has the beneficial effects that: the compound of the present invention has excellent inhibitory activity on histone deacetylase; the deuteration technology changes the metabolism of the compound in organisms, so that the compound has better pharmacokinetic parameter characteristics. In this case, the dosage can be varied and a depot formulation formed, improving the applicability; deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body is improved, and the medicine curative effect is improved; deuterium is used for replacing hydrogen atoms in the compound, so that certain metabolites can be inhibited, and the safety of the compound is improved.

Detailed Description

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 100 ℃, preferably from 0 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

Example 1 preparation of N-hydroxy-3- (3-d 5-phenylsulfamoyl-phenyl) -acrylamide (compound 8) with The following route was used for the synthesis:

step 1 synthesis of compound 3.

3-Chlorosulfonylbenzoic acid (Compound 1, 300mg), d 7-aniline (Compound 2, 0.30mL) were dissolved in 10mL anhydrous dichloromethane and stirred overnight at room temperature under nitrogen. Filtration and filtration of the filter cake with 10mL of ice-cold dichloro-benzeneThe filter cake was collected by three methane washes and dried under vacuum overnight to give the product compound 3 as a white solid (377 mg). LC-MS: 281.1[ M-1 ]]^-。

Step 2 synthesis of compound 4.

Under the protection of nitrogen, compound 3(379mg) is dissolved in 10mL of anhydrous tetrahydrofuran, and BH is slowly added dropwise₃THF (1M, 5.4mL) was added dropwise and reacted at room temperature overnight. And 2mL of methanol is dropwise added under the cooling of an ice water bath, the mixture is stirred for 30 minutes at room temperature after the dropwise addition, the tetrahydrofuran and the methanol are concentrated and evaporated to dryness, the obtained oily substance is dissolved by 10mL of ethyl acetate and 5mL of water and is subjected to shaking liquid separation, and the water phase is extracted by 10mL of ethyl acetate for three times. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give the product compound 4 as a white solid (343 mg). LC-MS: 269.1[ M +1 ]]⁺。

Step 3 synthesis of compound 5.

Compound 4(343mg) was dissolved in anhydrous methylene chloride, and 2, 2, 6, 6-tetramethylpiperidine oxide (TEMPO, 20mg) was added thereto, iodophenylenediacetic acid (436mg) was added in portions, followed by reaction at room temperature for 2 hours. The dichloromethane was concentrated to dryness and column chromatography (petroleum ether/ethyl acetate, 4/1) was carried out to give the product compound 5(313mg) as a pale yellow oily liquid. LC-MS: 266.1[ M +1 ]]⁺。

Step 4 synthesis of compound 7.

Dissolving the compound 5(313mg) and trimethylphosphonoacetic ester (compound 6, 600mg) in 7mL of tetrahydrofuran, dropwise adding 2.5mL of 2.5M NaOH solution in an ice-water bath, reacting at room temperature for 2 hours after dropwise adding, adding 7mL of ethyl acetate, shaking, separating liquid, taking a water phase, adjusting the pH to about 2-3 by using 6N hydrochloric acid solution, precipitating a solid, filtering, and drying in vacuum at 55 ℃ overnight to obtain an off-white solid product, namely the compound 7(253 mg). LC-MS: 308.0[ M-1 ]]^-。

Step 5 compound 8 synthesis.

Taking the compound 7(253mg) and 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 344mg), adding 5mL of N, N-dimethylformamide for dissolving, slowly adding 307 microliter of N, N-diisopropylethylamine in an ice water bath, adding 63mg of hydroxylamine hydrochloride, and reacting at room temperature for 4 hours. Pouring the reaction solution into a container 20mL of 1N hydrochloric acid, ethyl acetate extraction, organic phases combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by column chromatography (petroleum ether/ethyl acetate, 1/2) to give compound 8(62mg) as a pale yellow solid product.¹H NMR(300MHz，DMSO-d₆)10.84(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＝7.7Hz，1H)，7.56(t，J＝7.8Hz，1H)，7.46(d，J＝15.7Hz，1H)，6.49(d，J＝15.8Hz，1H)。

EXAMPLE 2 preparation of N-hydroxy-3- (3- (2, 3, 5-d 3-phenyl) sulfamoyl-phenyl) -acrylamide (Compound 15) The synthesis is carried out by adopting the following route:

step 1 synthesis of compound 10.

Aniline (1.023g) is added into 9mL of deuterium water to be dispersed evenly, deuterated concentrated hydrochloric acid (1.1mL) is added to be sealed, and the microwave reaction is carried out for 2 hours under the condition of 180 ℃. And (3) closing the microwave reaction device, naturally cooling to room temperature, adding sodium bicarbonate solid to adjust the pH value to be about 8-9, extracting with ethyl acetate, washing an organic phase with saturated saline solution, drying with anhydrous sodium sulfate, filtering and concentrating to obtain a light yellow oily liquid product compound 10(876 mg). LC-MS: 96.15[ M +1 ]]⁺；¹H NMR(500MHz，DMSO-d₆)7.46(s，1H)。

Step 2 synthesis of compound 11.

3-Chlorosulfonylbenzoic acid (Compound 1, 300mg), Compound 10(0.30mL) was dissolved in 10mL of anhydrous dichloromethane and stirred overnight at room temperature under nitrogen. Filtration, the filter cake washed three times with 10mL ice-cold dichloromethane, the filter cake collected and dried under vacuum overnight to give the product compound 11 as a white solid (379 mg); LC-MS: 280.0[ M +1 ]]⁺。

Step 3 synthesis of compound 12.

Compound 11(379mg) was dissolved in 10mL of anhydrous tetrahydrofuran and BH 3. THF (1M, 5.4mL) was added dropwise slowly under nitrogen. After the addition was complete, the reaction was carried out overnight at room temperature. 2mL of methanol is dropwise added under the cooling of ice water bathAfter dropping, the mixture is stirred for 30 minutes at room temperature, tetrahydrofuran and methanol are concentrated and evaporated to dryness, the obtained oily substance is dissolved in 10mL of ethyl acetate and 5mL of water and is subjected to shaking liquid separation, and the water phase is extracted three times by 10mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give compound 12 as a white powder (277 mg). LC-MS: 266.1[ M +1 ]]⁺。

Step 4 synthesis of compound 13.

Compound 12(277mg) was dissolved in anhydrous methylene chloride, and 20mg of 2, 2, 6, 6-tetramethylpiperidine oxide (TEMPO) was added to the solution, followed by addition of 355mg of iodophenylenediacetic acid in portions. After the addition was completed, the reaction was carried out at room temperature for 2 hours. The dichloromethane was concentrated to dryness and column chromatography (petroleum ether/ethyl acetate, 4/1) was carried out to give the product compound 12(228mg) as a pale yellow oily liquid. LC-MS: 264.0[ M +1 ]]⁺。

Step 5 synthesis of compound 14.

Dissolving the compound 13(228mg) and trimethylphosphonoacetate (compound 6, 414mg) in 4mL of tetrahydrofuran, dropwise adding 2.5mL of 2.5M NaOH solution in an ice-water bath, reacting at room temperature for 2 hours after dropwise adding, adding 5mL of ethyl acetate, shaking, separating liquid, taking a water phase, adjusting the pH to about 2-3 by using 6N hydrochloric acid solution, precipitating a solid, filtering, and drying in vacuum at 55 ℃ overnight to obtain an off-white solid product, namely the compound 13(218 mg). LC-MS: 304.9[ M-1 ]]^-。

Step 6 synthesis of compound 15.

Taking the compound 14(218mg), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 298mg), adding 5mL of N, N-dimethylformamide for dissolving, slowly adding 266 microliter of N, N-diisopropylethylamine in an ice water bath, adding 55mg of hydroxylamine hydrochloride, and reacting at room temperature for 4 hours. The reaction solution was poured into 20mL of 1N hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by column chromatography (petroleum ether/ethyl acetate, 1/2) to give compound 15(57mg) as a pale yellow solid product.¹H NMR(300MHz，DMSO-d₆)10.83(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＝8.2Hz，1H)，7.56(t，J＝7.8Hz，1H)，7.45(d，J＝15.9Hz，1H)，6.49(d，J＝15.8Hz，1H)。

EXAMPLE 3 preparation of N-hydroxy-3- (3-phenylsulfamoyl-phenyl) -2-d-acrylamide (Compound 21) Using The synthesis was carried out by the following route:

step 1 synthesis of compound 16.

dispersing trimethyl phosphono acetate (compound 6, 1.54g) in 6mL deuterium water, adding 15mg potassium carbonate, sealing at 80 ℃ and carrying out microwave reaction for 30 minutes, closing the microwave reactor, naturally cooling to room temperature, extracting with ethyl acetate (10mL × 2), washing an organic phase with saturated saline, drying with anhydrous sodium sulfate, filtering and concentrating to obtain a colorless oily liquid product compound 16(1.471 g).¹H NMR(300MHz，CDCl₃)3.82(s，3H)，3.78(s，3H)，3.74(s，3H)。

Step 2 synthesis of compound 17.

3-Chlorosulfonylbenzoic acid (Compound 1, 300mg) and aniline (0.30mL) were dissolved in 10mL of anhydrous dichloromethane and stirred overnight at room temperature under nitrogen. Filtration and the filter cake washed three times with 10mL ice-cold dichloromethane, the filter cake collected and dried under vacuum overnight to give the product compound 17(380mg) as a white solid. LC-MS: 277.0[ M-1]^-。

Step 3 synthesis of compound 18.

Dissolving compound 17(380mg) in 10mL of anhydrous tetrahydrofuran, and slowly dropwise adding BH under the protection of nitrogen₃THF (1M, 5.4 mL). After the addition was complete, the reaction was carried out overnight at room temperature. And 2mL of methanol is dropwise added under the cooling of an ice water bath, the mixture is stirred for 30 minutes at room temperature after the dropwise addition, the tetrahydrofuran and the methanol are concentrated and evaporated to dryness, the obtained oily substance is dissolved by 10mL of ethyl acetate and 5mL of water and is subjected to shaking liquid separation, and the water phase is extracted by 10mL of ethyl acetate for three times. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness to give the product compound 18(276mg) as a white solid powder.¹H NMR(300MHz，CDCl₃)7.80(s，1H)，7.68(d，J＝7.8Hz，1H)，7.56(d，J＝7.7Hz，1H)，7.44(t，J＝7.7Hz，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(243mg) was dissolved in anhydrous dichloromethane, and 2, 2, 6, 6-tetramethylpiperidineoxide (TEMPO, 20mg) was added to the solution, followed by addition of 315mg of iodophenylenediacetic acid in portions. After the addition was completed, the reaction was carried out at room temperature for 2 hours. The dichloromethane was concentrated to dryness and column chromatography (petroleum ether/ethyl acetate, 4/1) was carried out to give compound 19(200mg) as a pale yellow oily liquid product. LC-MS: 262.1[ M +1 ]]⁺。

Step 5 synthesis of compound 20.

Dissolving the compound 19(264mg) and the compound 16(424mg) in 4.5mL of tetrahydrofuran, dropwise adding 3.0mL of 2.5M NaOH solution in an ice-water bath, reacting at room temperature for 2 hours after dropwise adding, adding 5mL of ethyl acetate, shaking, separating liquid, taking an aqueous phase, adjusting the pH to about 2-3 by using a 6N hydrochloric acid solution, precipitating a solid, filtering, and performing vacuum drying at 55 ℃ overnight to obtain an off-white solid compound 20(171 mg).¹H NMR(300MHz，DMSO-d₆)7.72(s，1H)，7.56(d，J＝7.4Hz，1H)，7.40(d，J＝7.8Hz，1H)，7.28(t，J＝7.6Hz，1H)，7.05(s，1H)，6.86(t，J＝7.6Hz，2H)，6.74(d，J＝7.8Hz，2H)，6.41(t，J＝7.3Hz，1H)。

Step 6 synthesis of compound 21.

taking the compound 20(171mg), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 235mg) and adding 5mL of N, N-dimethylformamide to dissolve, slowly adding 232 microliter of N, N-diisopropylethylamine in an ice-water bath, adding 43mg of hydroxylamine hydrochloride, reacting for 4 hours at room temperature, pouring the reaction solution into 20mL of 1N hydrochloric acid, adding ethyl acetate (20mL × 3) to extract, combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and concentrating column chromatography (petroleum ether/ethyl acetate, 1/2) to obtain a light yellow solid product, namely the compound 21(54 mg).¹H NMR(300MHz，DMSO-d₆)10.79(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＝7.8Hz，1H)，7.45(d，J＝15.8Hz，1H)，7.27-7.17(m，2H)，7.13-7.05(m，2H)，7.02(td，J＝7.1，1.2Hz，1H)，6.49(d，J＝15.8Hz，1H)。

Biological activity assay

(1) Assay for histone deacetylase inhibitory Activity

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

Reagent: 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. k 340-100); MTS assay 96-well plates (CORNING, cat. No. 3599); 96-well plates (GREINER, CAT. NO. 655076).

Experimental methods

Compound formulation for HDACs experiments: test compounds were dissolved in DMSO to make 20mM stock. Dilutions were made in DMSO at 50-fold final concentration. When adding medicine, diluting with ultrapure water to obtain 2 times of diluted solution.

Compound IC₅₀And (3) detection: compounds prepared in pre-dilution at different concentrations were added to 96-well plates, with duplicate wells set up for each concentration. Subsequently, a mixture of HeLa Nuclear Extract and HDAC Substrae was added. After mixing, incubation was carried out at 37 ℃ for 30 minutes, and Lysine Developer was added. After mixing, incubation is carried out for 30 minutes at 37 ℃, fluorescence detection is carried out by an enzyme-linked immunosorbent assay (BioTek, Synergy2) under the excitation light of 330nm and the emission light of 440nm, and data are collected. In addition, negative and positive controls were set up with Trichostatin A as a positive reference.

Compound formulation for MTS experiments: test compounds were dissolved in DMSO to make 20mM stock solutions. The stock solutions were diluted in DMSO in steps to 200-fold final concentration. For dosing, 4-fold final concentration of the stock solution (4. mu.L of 200-fold gradient compound to 196. mu.L complete medium) was diluted with cell culture medium and 50. mu.L of 4-fold final concentration compound was added to 150. mu.L of the cell-containing plates.

MTS cell viability assay: collecting cell suspension in logarithmic growth phase, centrifuging to collect cells, inoculating 150 μ L of the suspension in 96-well plate according to optimized density, and adding after 24 hrAdd 4 times the concentration of compound diluted in medium 50. mu.L/well. Wells to which the same volume of 2% DMSO was added served as controls, with a final DMSO concentration of 0.5%. After the cells were cultured for 72 hours, the MTS measured the cell viability. The specific method comprises the following steps: adding 20 mu LMTS into each well, placing into an incubator, continuously culturing for 1-4 hours, and detecting OD₄₉₀In OD₆₅₀The values are used as reference. Dose-response curves were generated and IC calculated using GraphPad Prism software₅₀. A represents IC₅₀< 100nM, B means 100 nM. ltoreq.IC₅₀Less than or equal to 200nM, C means 200nM < IC₅₀Less than or equal to 300nM, D represents IC₅₀> 300 nM. (as shown in table 1 below).

TABLE 1 example Compounds for Histone deacetylase inhibitory Activity

Numbering	HDACs IC₅₀(nM)	Jurkat IC₅₀(nM)
			Belinostat	C	C
8	C	B
			15	C	C
21	B	A

As shown in the above table, compared with a new drug, belinostat, developed by Spectrum biomedical corporation for treating Peripheral T Cell Lymphoma (PTCL), compound 15 of the present invention has activity equivalent to that of belinostat, and compound 21 of the present invention has activity superior to that of belinostat, indicating that the compound of the present invention can significantly inhibit Histone Deacetylase (HDAC), and thus is more suitable for preparing drugs for treating diseases related to histone deacetylase, such as lymphoma and the like.

(2) Liver microparticle metabolism test

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

Preparing a stock solution: an amount of the compound powder of example was weighed out precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

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

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L 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. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

Table 2 evaluation of liver microparticle metabolism of the compounds of the examples

The experimental results are shown in table 2, compared with belinostat, the compound of the invention has the advantages of obviously increased half-life, smaller clearance rate, excellent metabolic stability in human liver microsome experiments and rat liver microsome experiments, and is more suitable for being used as a medicament for inhibiting histone deacetylase.

(3) Pharmacokinetic experiment of rat

Purpose of the experiment: study the pharmacokinetic behaviour of the compounds of the invention was examined after administration of N-hydroxy-3- (3-phenylsulfamoyl-phenyl) acrylamide, the compounds of examples 1-6, to rats.

Experimental animals:

species and strain: SD rat grade: SPF stage

Sex and amount: male, 6

Body weight range: 180 to 220g (actual weight range 187 to 197g)

The source is as follows: shanghai Xipulbikai laboratory animals Co., Ltd

The experimental process comprises the following steps:

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

Rats, males, weighing 187-197 g, were randomized into 2 groups, fasted overnight but with free access to water starting the afternoon of the day before the experiment, and given food 4h after administration. Group A was administered 3mg/kg of N-hydroxy-3- (3-phenylsulfamoyl-phenyl) acrylamide, group B was administered 3mg/kg of the compound of example 1-6, and about 100-200L of blood was collected from orbital veins of rats 15min, 30min, 1, 2, 3, 5, 8, 10h after administration, and placed in 0.5mL Eppendorf tubes anticoagulated with EDTA-K2, immediately mixed, after anticoagulation, the tubes were gently inverted and mixed 5-6 times as soon as possible, the blood was collected and placed in an ice box, blood samples were centrifuged at 4000rpm for 30min at 10min and 4 ℃ to separate plasma, and all plasma was collected and immediately stored at-20 ℃. Plasma concentrations were determined in plasma at each time point after sample collection at all time points.

Based on the mean plasma concentration-time data obtained above after administration, pharmacokinetic-related parameters after i.g administration of 9N-hydroxy-3- (3-phenylsulfamoyl-phenyl) acrylamide (3mg/kg) and the compound of examples 1 to 6 (3mg/kg) were calculated for male SD rats, respectively, by non-atrioventricular statistical moment theory using Winnonin software.

Experiments show that compared with N-hydroxy-3- (3-phenylsulfamoyl-phenyl) acrylamide, the compound has better activity and excellent pharmacokinetic property, so the compound is more suitable to be used as a compound for inhibiting histone deacetylase and is further suitable to be used for preparing medicines for treating cell proliferative diseases and cancers.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof,

with the proviso that R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰And R¹¹At least one of which is deuterium.

2. The compound of claim 1, wherein: r¹、R²、R³、R⁴And R⁵Each independently is deuterium or hydrogen.

3. The compound of claim 1, wherein: r⁶、R⁷、R⁸And R⁹Each independently is deuterium or hydrogen.

4. The compound of claim 1, wherein: r¹⁰And R¹¹Each independently is deuterium or hydrogen.

5. The compound of claim 1, wherein: the compound may be selected from the following compounds or pharmaceutically acceptable salts thereof:

6. a pharmaceutical composition characterized by: comprising a pharmaceutically acceptable carrier and a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 5 or a pharmaceutical composition as claimed in claim 6 in the manufacture of a medicament for the treatment and/or prevention of a disease associated with expression of HDACs.