CN111205303B

CN111205303B - Thieno[2,3-d]pyrimidinyl hydroxamic acid derivatives and uses thereof

Info

Publication number: CN111205303B
Application number: CN202010140982.XA
Authority: CN
Inventors: 何谷; 李响; 蒋庆琳
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-03-03
Filing date: 2020-03-03
Publication date: 2021-04-27
Anticipated expiration: 2040-03-03
Also published as: CN111205303A

Abstract

The invention relates to thieno [2,3-d ]]Pyrimidinyl radicalsHydroxamic acid derivatives and application thereof, belonging to the technical field of antitumor drugs. The technical problem solved by the invention is to provide a novel BRD4 and HDACs double-target inhibitor. Thieno [2,3-d ] of the invention]The structure of the pyrimidyl hydroxamic acid derivative is shown as a formula I or a formula II. The compound of the invention has novel structure, can be used as a micromolecular BRD4 and HDACs dual inhibitor, has better anti-colorectal cancer proliferation capacity compared with RVX-208 and vorinostat, and is a promising anti-cancer drug by selectively inhibiting BRD4 and HDACs and subsequent autophagic cell death.

Description

Thieno [2,3-d ] pyrimidinyl hydroxamic acid derivatives and uses thereof

Technical Field

The invention relates to a thieno [2,3-d ] pyrimidyl hydroxamic acid derivative and application thereof, belonging to the technical field of antitumor drugs.

Background

Colorectal cancer is one of the most common malignancies worldwide, the second and third leading cause of cancer-related death in adults, both male and female, respectively. In addition, clinically used molecular targeting drugs for colorectal cancer, such as Epidermal Growth Factor Receptor (EGFR) inhibitors, cetuximab, bevacizumab, etc., appear to have limited effect in metastatic colorectal cancer (mCRC) patients, but greatly increase the cost of treatment.

Immunohistochemistry detects histone acetylation levels closely related to staging, metastasis and prognosis of colorectal cancer. During histone acetylation, there are two families of proteins that are considered potential drug targets: one is protein 4 containing a bromo amino group (BRD4), which acts as a histone acetylated lysine "reader"; another class is Histone Deacetylases (HDACs), which act as acetylated lysine "erasers". BRD4 is a member of the Bromodomain and extra-terminal (BET) protein families, playing a central role in the recognition of histone and non-histone substrates, and it is likely to regulate many molecular and cellular processes in epigenetic modification and gene transcription.

Existing BRD4 inhibitors or HDACs inhibitors, both of which are single-acting, require more structurally novel anti-tumor compounds due to the resistance of tumor cells.

Disclosure of Invention

In view of the above drawbacks, the technical problem underlying the present invention is to provide a novel dual-target inhibitor of BRD4 and HDACs.

The structure of the thieno [2,3-d ] pyrimidyl hydroxamic acid derivative is shown as a formula I or a formula II:

wherein R is₁Is hydrogen, C1-C4 alkyl or benzyl; r₂Is C1-C5 alkyl,

R₃Is hydroxy, amino or

Preferably, R₁Is C1-C4 alkyl; preferably R₁Is methyl.

Preferably, R₃Is hydroxy or

Preferably R₃Is a hydroxyl group.

Preferably, the structure is shown as formula I.

Preferably, the thieno [2,3-d ] pyrimidinyl hydroxamic acid derivative of the present invention has a structure represented by any one of the following structural formulae:

the invention also provides isomers, pharmaceutically acceptable salts and hydrates of the thieno [2,3-d ] pyrimidinyl hydroxamic acid derivatives.

The invention also provides application of the thieno [2,3-d ] pyrimidyl hydroxamic acid derivative in preparation of BRD4 inhibitors.

The invention also provides the application of the thieno [2,3-d ] pyrimidyl hydroxamic acid derivative in the preparation of HDACs inhibitors.

The invention also provides application of the thieno [2,3-d ] pyrimidyl hydroxamic acid derivative in preparation of antitumor drugs.

Preferably, the tumor is colorectal cancer.

The invention also provides a pharmaceutical composition.

The pharmaceutical composition comprises an effective component and pharmaceutically acceptable auxiliary materials, wherein the effective component comprises a therapeutically effective amount of the thieno [2,3-d ] pyrimidyl hydroxamic acid derivative or isomer thereof or pharmaceutically acceptable salt or hydrate thereof.

Compared with the prior art, the invention has the following beneficial effects:

the invention designs and synthesizes a series of novel BRD4-HDACs double inhibitors, and carries out subsequent in vitro and in vivo biological evaluation. The compound induces apoptosis and autophagic death by down regulating c-myc, deacetylation of histone H3, inhibiting apoptosis inhibiting protein Bcl-2 and interfering formation of autophagosomal of HCT-116 colorectal cancer cells. In addition, the compounds mainly inhibit HDAC1, 2,3 and 6, and western blotting and IHC analysis show that the compounds have obvious inhibiting effect on deacetylation of histone H3, have good therapeutic effect on HCT-116 colorectal cancer xenograft mice, and increase cytotoxic T cell infiltration by activating IL6-JAK-STAT signal pathway.

The compound of the invention has novel structure, can be used as a micromolecular BRD4 and HDACs dual inhibitor, has better anti-colorectal cancer proliferation capacity compared with RVX-208 and vorinostat, and is a promising anti-cancer drug by selectively inhibiting BRD4 and HDACs and subsequent autophagic cell death.

Drawings

FIG. 1 is the IC of compound 9c in inhibiting normal human colonic epithelial cells NCM460 and 6 tumor cells₅₀The value is obtained.

FIG. 2 is a cell cycle analysis of HCT-116 cells treated with different concentrations of compound 9c (0.2uM, 0.5 uM).

FIG. 3 is a bivariate flow cytometer scattergram after Annexin V-FITC/PI double staining.

FIG. 4 is a fluorescent micrograph stained by Hoechst 33258.

Fig. 5 is a graph showing the results of the antitumor activity test of compound 9c in a transplanted tumor model.

Detailed Description

wherein R is₁Is hydrogen, C1-C4 alkyl or benzyl; r₂Is C1-C5 alkyl,

R₃Is hydroxy, amino or

In the present invention, the "C1-C5 alkyl group" refers to a straight-chain or branched alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl and the like. "C1-C4 alkyl" refers to a straight or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and the like.

Preferably, R₁Is C1-C4 alkyl; r₂Is C1-C5 alkyl;

R₃is hydroxy, amino or

More preferably, R₁Is methyl, R₂Is C1-C5 alkyl;

R₃is hydroxy, amino or

As a preferred embodiment, R₁Is hydrogen, C1-C4 alkyl or benzyl; r₂Is C1-C5 alkyl;

R₃is hydroxy or

Preferably, R₁Is hydrogen, C1-C4 alkyl or benzyl; r₂Is C1-C5 alkyl;

R₃is a hydroxyl group.

As a preferred embodiment, R₁Is C1-C4 alkyl; r₂Is C1-C5 alkyl;

R₃is hydroxy or

Preferably, R₁Is C1-C4 alkyl; r₂Is C1-C5 alkyl;

R₃is a hydroxyl group.

As a preferred embodiment, R₁Is methyl, R₂Is C1-C5 alkyl;

R₃is hydroxy or

Preferably, R₁Is methyl; r₂Is C1-C5 alkyl;

R₃is a hydroxyl group.

Preferably, the structure of the compound of the invention is shown as formula I.

The following are some preferred structural formulae for the compounds of the present invention:

the invention also relates to an isomer, a pharmaceutically acceptable salt and a hydrate of the compound shown as the general formula I or the general formula II. The pharmaceutically acceptable salts include, but are not limited to, salts of the compounds of the present invention with inorganic acids and salts of the compounds of the present invention with organic acids. Wherein, the inorganic acid includes but is not limited to hydrochloric acid, phosphoric acid, phosphorous acid, sulfuric acid, nitric acid or hydrobromic acid; organic acids include, but are not limited to, malic acid, citric acid, maleic acid, fumaric acid, succinic acid, fumaric acid, tartaric acid, acetic acid, lactic acid, p-toluenesulfonic acid, methanesulfonic acid, palmitic acid, and the like. Some of the compounds of the present invention may be crystallized or recrystallized using water or various organic solvents, in which case various solvates may be formed. The present invention includes those stoichiometric solvates, including hydrates, as well as compounds containing variable amounts of water that are formed when prepared by a low pressure sublimation drying process.

The compound of the invention can be used for preparing BRD4 inhibitors.

The compounds of the invention may also be used to prepare HDACs inhibitors.

The compound can be used for preparing antitumor drugs.

Preferably, the tumor is colorectal cancer, i.e. the compounds of the invention are preferably used against colorectal cancer.

The compound of the present invention or a pharmaceutically acceptable salt thereof may be used alone or in the form of a pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient, and when used in the form of a pharmaceutical composition, a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt or hydrate thereof and one or more pharmaceutically acceptable carriers or diluents are usually combined to make an appropriate administration form or dosage form. Accordingly, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention, all possible isomers thereof or a pharmaceutically acceptable salt or hydrate thereof and at least one pharmaceutically acceptable carrier.

The compound of the invention can be prepared by adopting a conventional chemical synthesis method, and concretely, the compound is synthesized by adopting the following process flow:

reagents and conditions: (a) NCCH₂CO₂Et，S₈，Et₃N, EtOH, refluxing for 12 h; (b) DBU, THF, reflux, 24 h; (c) CH (CH)₃OH, NaOH for 30 min; (d) DCC/HOBt, DCM for 24 h; (e) ethyl chloroformate, N-methylmorpholine, 1, 4-dioxane, 15min, then NH₂OH，CH₃OH, 20 min; (f) compounds 2a-e, HCl-1, 4-dioxane, 25 ℃, 48 h.

The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1 preparation of N- (2-aminophenyl) -4- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydro-pyridine [4',3':4,5] thiophene [2,3-d ] pyrimidin-2-yl) phenoxy) butanamide (8b)

Reaction a: preparation of ethyl 2-amino-6-methyl-4, 5,6, 7-tetrahydrothieno [2,3-c ] pyridine-3-carboxylate (2a)

N-methylpiperidinone (4.90g,50mmol), ethyl cyanoacetate (5.66g,100.0mmol), triethylamine (10mL) and sublimed sulfur (1.60g,50.0mmol) were mixed and reacted in 100mL of anhydrous ethanol under reflux for 12 hours, the reaction solution was concentrated, the residue was extracted with water and ethyl acetate, the organic layer was separated and concentrated, and 2a, recrystallized from 100mL of ethanol, yield 79%, pale yellow powder was obtained.¹H NMR(400MHz,DMSO-d₆)δ7.23(s,2H,NH₂),4.15(q,J＝7.2Hz,2H,OCH₂CH₃),3.22(t,J＝2.0Hz,2H,N-CH₂-Thiophene),2.66(t,J＝5.2Hz,2H,N-CH₂-CH₂),2.53(t,J＝6.0Hz,2H,N-CH₂-CH₂),2.29(s,3H,N-CH₃),1.24(t,J＝7.2Hz,3H,CH₂CH₃).

And b, reaction: preparation of methyl 4- (4-cyano-2, 6-dimethylphenoxy) butyrate (4b)

4-hydroxy-3, 5-dimethylbenzylacetonitrile (1.47g,10mmol), methyl 4-bromobutyrate (2.17g,12mmol) were dissolved in 100ml of THF, DBU (3.04g,20mmol) was added, the reaction was refluxed for 24 hours, the reaction solution was concentrated and extracted with water and ethyl acetate, the organic layer was separated and concentrated, and the residue was subjected to silica gel column chromatography, petroleum ether, ethyl acetate (12:1) to give 4b, yield 70%, as a pale yellow oil.¹H NMR(400MHz,DMSO-d₆)δ7.53(s,2H,Ar-H),3.82(t,J＝6.4Hz,2H,OCH₂),3.62(s,3H,OCH₃),2.55(t,J＝7.4Hz,2H,CH₂COOCH₃),2.23(s,6H,2Ar-CH₃),2.01(p,J＝6.8Hz,2H,OCH₂CH₂).

And c, reaction: preparation of methyl 4- (4-cyano-2, 6-dimethylphenoxy) butyrate (5b)

4b (1.24g,5mmol) was dissolved in 30ml CH₃To OH, 1M NaOH (30ml) was added, reacted at room temperature for 30 minutes, neutralized with hydrochloric acid to pH 3, to precipitate a white solid, which was washed with methanol to give 5b in 76% yield as a white powder.¹H NMR(400MHz,DMSO-d₆)δ12.19(brs,1H,COOH),7.54(s,2H,Ar-H),3.81(t,J＝6.4Hz,2H,OCH₂),2.45(t,J＝7.2Hz,2H,CH₂COOH),2.24(s,6H,2Ar-CH₃),1.97(p,J＝6.8Hz,2H,OCH₂CH₂).

Reaction d: preparation of N- (2-aminophenyl) -4- ((4-cyano-2, 6-dimethylphenoxy) methyl) benzamide (6 b).

5b (281mg,1mmol) and o-phenylenediamine (432mg,4mmol) were dissolved in anhydrous CH₂Cl₂To (15ml) were added EDCI (192mg,1mmol) and HOBt (135mg,1mmol), and the mixture was reacted at room temperature for 24 hours. The crude product was chromatographed on silica gel column with petroleum ether and ethyl acetate (5:1) to give 6b in 67% yield as a white solid. mp is 194.3 to 195.5 ℃,¹H NMR(400MHz,DMSO-d₆)δ9.69(brs,1H,NH),8.03(d,J＝7.8Hz,2H,Ar-H),7.62(d,J＝8.2Hz,2H,Ar-H),7.59(s,2H,Ar-H),7.18(dd,J＝7.8,1.6Hz,1H,Ar-H),6.79(dd,J＝7.8,1.6Hz,1H,Ar-H),6.61(td,J＝7.2,1.4Hz,1H,Ar-H),4.98(s,2H,CH₂),4.91(brs,2H,NH2),2.29(s,6H,2Ar-CH₃).¹³C NMR(100MHz,DMSO-d₆)δ165.47,159.82,143.64,140.69,134.80,133.23(4C),128.44,128.18,127.19,126.99,123.71,119.34,116.71,116.59,106.99,73.53,16.43.HRMS(ESI):calcd for C₂₃H₂₂N₃O₂ ⁺[M+H]⁺,372.1707；found 372.1711.

reaction f: preparation of N- (2-aminophenyl) -4- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydro-pyridine [4',3':4,5] thiophene [2,3-d ] pyrimidin-2-yl) phenoxy) butanamide (8b)

Placing compound 2a (105.50mg,0.5mmol) and compound 6b (161.50mg,0.5mmol) in a 25mL sealed tube, adding 5mL of saturated HCl solution of 1, 4-dioxane, reacting at room temperature for 48 hr, concentrating the reaction solution, adding 10mL of H₂O, combined use of NaHCO₃Neutralized to pH 7, centrifuged (4500rounds/minute) and washed with ethyl acetate (5ml) and ethanol (5ml) to give 8b, a pale yellow solid in 38% yield. mp 239.8-241.8 ℃.¹H NMR(400MHz,DMSO-d₆)δ12.38(brs,1H,Pyrimidine-H),9.71(s,1H,CONH),8.04(s,2H,Ar-H),7.91(s,2H,Ar-H),7.64(s,2H,Ar-H),7.19(s,1H,Ar-H),6.98(s,1H,Ar-H),6.80(s,1H,Ar-H),6.61(s,1H,Ar-H),4.97(s,2H,O-CH₂),4.92(s,2H,NH₂),3.56(s,2H,N-CH₂-Thiophene),2.97(s,2H,N-CH₂-CH₂),2.67(s,2H,N-CH₂-CH₂),2.38(s,3H,N-CH₃),2.33(s,6H,2Ar-CH₃).¹³C NMR(100MHz,DMSO-d₆)δ165.50,164.01,159.23,158.57,152.52,143.64,141.05,134.69,131.60,130.16,129.37,128.81,128.42,128.09,127.72,127.19,126.97,123.75,120.62,116.71,116.59,73.44,53.50,51.73,45.53,26.24,16.76.HRMS(ESI):calcd for C₃₂H₃₂N₅O₃S⁺[M+H]⁺,566.2220；found 566.2224.

EXAMPLE 2 preparation of N- (2-aminophenyl) -2- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydro-pyridine [4',3':4,5] thiophene [2,3-d ] pyrimidin-2-yl) phenoxy) acetamide (8a)

Referring to the preparation method of example 1, the starting material methyl 4-bromobutyrate in reaction b was changed to methyl 4-bromoacetate to obtain compound 8 a.

Pale yellow solid, yield 35%. mp at 243.7-245.8 deg.C.¹H NMR(400MHz,DMSO-d₆)δ12.39(brs,1H,Pyrimidine-H),9.80(s,1H,CONH),8.15(s,1H,Ar-H),8.02(d,J＝7.6Hz,1H,Ar-H),7.92(s,2H,Ar-H),7.72(d,J＝7.6Hz,1H,Ar-H),7.57(t,J＝7.6Hz,1H,Ar-H),7.19(d,J＝7.8Hz,1H,Ar-H),6.98(t,J＝7.6Hz,1H,Ar-H),6.80(d,J＝8.0Hz,1H,Ar-H),6.61(t,J＝7.6Hz,1H,Ar-H),4.95(s,4H,O-CH₂,NH₂),3.56(s,2H,N-CH₂-Thiophene),2.97(t,J＝5.6Hz,2H,N-CH₂-CH₂),2.67(t,J＝5.6Hz,2H,N-CH₂-CH₂),2.38(s,3H,N-CH₃),2.35(s,6H,2Ar-CH₃).¹³C NMR(100MHz,DMSO-d₆)δ165.61,164.26,160.45,158.33,153.33,143.66,137.97,135.27,131.45,131.28,129.40,129.30,128.88,128.81,128.58,127.93,127.89,127.24,126.93,123.82,120.46,116.68,116.65,73.60,53.56,51.79,45.56,26.33,16.77.HRMS(ESI):calcd for C₃₂H₃₂N₅O₃S⁺[M+H]⁺,566.2220；found 566.2224.

EXAMPLE 3 preparation of N- (2-aminophenyl) -6- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydro-pyridine [4',3':4,5] thiophene [2,3-d ] pyrimidin-2-yl) phenoxy) hexanamide (8c)

Referring to the preparation method of example 1, the starting material methyl 4-bromobutyrate in reaction b was changed to methyl 4-bromohexanoate to obtain compound 8 c.

Yield 37% yellow solid. mp is 222.2-223.8 ℃.¹H NMR(400MHz,DMSO-d₆)δ12.34(brs,1H,Pyrimidine-H),9.13(s,1H,CONH),7.87(s,2H,Ar-H),7.16(dd,J＝8.0,1.6Hz,1H,Ar-H),6.89(td,J＝7.6,1.6Hz,1H,Ar-H),6.71(dd,J＝8.0,1.6Hz,1H,Ar-H),6.54(td,J＝7.6,1.6Hz,1H,Ar-H),4.82(s,2H,NH₂),3.80(t,J＝6.4Hz,2H,O-CH₂),3.55(s,2H,N-CH₂-Thiophene),2.95(d,J＝5.6Hz,2H,N-CH₂-CH₂),2.66(t,J＝5.6Hz,2H,N-CH₂-CH₂),2.39-2.34(m,5H,N-CH_3,CH₂CONH),2.28(s,6H,2Ar-CH₃),1.80(p,J＝6.8Hz,2H,OCH₂CH₂CH₂CH₂CH₂),1.69(p,J＝7.2Hz,2H,OCH₂CH₂CH₂CH₂CH₂),1.54(m,1.60–1.50,2H,OCH₂CH₂CH₂CH₂CH₂).¹³C NMR(100MHz,CF₃COOD)δ178.18,163.31,156.55,150.17,149.69,135.10,129.59,129.27,128.99,128.86,128.31,126.98,126.52,126.35,118.03,114.38,111.56,73.12,51.71,51.56,42.75,35.50,29.00,25.40,24.95,21.93,14.82.HRMS(ESI):calcd for C₃₀H₃₆N₅O₃S⁺[M+Na]⁺,546.2533；found 546.2540.

Example 43- ((2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydropyrido [4',3':4,5] thieno [2,3-d ] pyrimidin-2 yl) phenoxy) methyl) -N-hydroxybenzamide (9a).

By following the procedure of example 2, compound 5a was obtained;

reaction e: hydroxylamine hydrochloride (345mg,5mmol) was added to a 10ml methanol solution of sodium methoxide (270mg,5mmol), stirred at room temperature for 30min, filtered, and the filtrate was reserved.

Compound 5a (281mg,1mmol) was dissolved in 10ml of tetrahydrofuran, N-methylmorpholine (130mg,1.3mmol) was added, and ethyl chloroformate (130mg,1.2mmol), reacting at room temperature for 15min, filtering, adding the filtrate into the newly prepared hydroxylamine methanol solution, and reacting for 20 min. The reaction solution was concentrated, and subjected to silica gel column chromatography, dichloromethane: acetone ═ 5:1 gave compound 7a, yield: 60% of an off-white solid. mp is 137.9-138.6 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.44(brs,1H,OH),8.75(brs,1H,NH),7.53(s,2H,Ar-H),3.80(t,J＝6.4Hz,2H,O-CH₂),2.24(s,6H,2Ar-CH₃),2.20(t,J＝7.6Hz,2H,C＝O-CH₂),1.97(p,J＝6.8Hz,2H,CH₂-CH₂-CH₂).¹³C NMR(100MHz,DMSO-d₆)δ169.02,160.04,133.13,132.98,119.35,106.63,71.81,29.12,26.38,16.21.HRMS(ESI):calcd for C₁₃H₁₆N₂NaO₃ ⁺[M+Na]⁺,271.1053；found 271.1060.

Reaction f: placing compound 2a (105.50mg,0.5mmol) and compound 7a (148mg,0.5mmol) in a 25mL sealed tube, adding 5mL of saturated HCl solution of 1, 4-dioxane, reacting at room temperature for 48 hr, concentrating the reaction solution, adding 10mL of H₂O, combined use of NaHCO₃Neutralized to pH 7, centrifuged (4500rounds/minute) and washed with ethyl acetate (5ml) and ethanol (5ml) to give 9a in 41% yield as a yellow solid. mp is 184.1-186.1 ℃.¹H NMR(400MHz,DMSO-d₆)δ12.39(brs,1H,Pyrimidine-H),11.29(brs,1H,OH),9.07(brs,1H,C＝ONH),7.93(s,1H,Ar-H),7.90(s,2H,Ar-H),7.75(d,J＝7.5Hz,1H,Ar-H),7.65(d,J＝7.4Hz,1H,Ar-H),7.51(t,J＝7.6Hz,1H,Ar-H),4.91(s,2H,OCH₂),3.58(s,2H,N-CH₂-Thiophene),2.96(t,J＝5.7Hz,2H,N-CH₂-CH₂),2.68(t,J＝5.7Hz,2H,N-CH₂-CH₂),2.39(s,3H,N-CH₃),2.32(s,6H,2Ar-CH₃).¹³C NMR(100MHz,DMSO-d₆)δ164.02,159.19,158.50,152.52,138.03,133.46,132.99,131.63,131.12,130.08,129.35,128.99,128.81,127.69,127.07,126.86,120.60,73.57,53.46,51.70,45.48,26.20,16.72.HRMS(ESI):calcd for C₂₆H₂₇N₄O₄S⁺[M+H]⁺,491.1748；found 491.1752.

Example 54- ((2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydropyrido [4',3':4,5] thieno [2,3-d ] pyrimidin-2 yl) phenoxy) methyl) -N-hydroxybenzamide (9b).

Referring to the procedure of example 4, methyl 3-bromomethylbenzoate was changed to methyl 4-bromomethylbenzoate to obtain compound 9 b.

¹H NMR(400MHz,DMSO-d₆)δ7.93(s,2H,Ar-H),7.81(s,2H,Ar-H),7.58(s,2H,Ar-H),4.90(s,2H,OCH₂),3.53(s,2H,N-CH₂-Thiophene),2.97(s,2H,N-CH₂-CH₂),2.66(s,2H,N-CH₂-CH₂),2.37(s,3H,N-CH₃),2.30(s,6H,2Ar-CH₃).¹³C NMR(100MHz,DMSO-d₆)δ164.58,131.22,131.09,129.27,128.80,128.76,128.16,120.31,73.37,53.68,51.95,45.63,16.75.HRMS(ESI):calcd for C₂₆H₂₇N₄O₄S⁺[M+H]⁺,491.1748；found 491.1750.

Example 66- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydropyrido [4',3':4,5] thieno [2,3-d ] pyrimidin-2 yl) phenoxy) -N-hydroxyhexanamide (9c).

Referring to the procedure of example 4, methyl 3-bromomethylbenzoate was changed to methyl 6-bromohexanoate to give compound 9 c.

¹H NMR(400MHz,DMSO-d₆)δ10.42(brs,1H,OH),8.75(s,1H,NH),7.90(s,2H,Ar-H),3.75(t,J＝6.4Hz,2H,ArOCH₂),3.51(s,2H,N-CH₂-Thiophene),2.96(t,J＝5.8Hz,2H,N-CH₂-CH₂),2.64(t,J＝5.8Hz,2H,N-CH₂-CH₂),2.36(s,3H,N-CH₃),2.27(s,6H,2Ar-CH₃),2.03–1.96(m,2H,CH₂C＝O),1.74(p,J＝7.2Hz,2H,ArOCH₂CH₂),1.58(p,J＝7.2Hz,2H,CH₂CH₂C＝O),1.51–1.40(m,2H,ArOCH₂CH₂CH₂).¹³C NMR(100MHz,DMSO-d₆)δ169.45,165.12,157.84,130.51,129.11,128.81,128.70,128.66,126.49,119.87,72.10,53.79,52.10,45.68,32.69,30.12,26.67,25.69,25.48,16.59.HRMS(ESI):calcd for C₂₄H₃₁N₄O₄S⁺[M+H]⁺,471.2061；found 471.2057.

Example 74- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydropyrido [4',3':4,5] thieno [2,3-d ] pyrimidin-2 yl) phenoxy) -N-hydroxyhexanamide (9d).

Referring to the procedure of example 4, methyl 3-bromomethylbenzoate was changed to methyl 4-bromobutyrate to give compound 9 d.

¹H NMR(400MHz,DMSO-d₆)δ12.17(brs,Pyrimidine-H),10.45(s,1H,OH),7.85(s,2H,Ar-H),3.79(t,J＝6.3Hz,2H,OCH₂),3.55(s,2H,N-CH₂-Thiophene),2.95(t,J＝5.7Hz,2H,N-CH₂-CH₂),2.67(t,J＝5.7Hz,2H,N-CH₂-CH₂),2.37(s,3H,N-CH₃),2.28(s,6H,2Ar-CH₃),2.21(t,J＝7.4Hz,2H,CH₂C＝O),1.99(q,J＝6.8Hz,2H,OCH₂CH₂).¹³C NMR(100MHz,DMSO-d₆)δ169.06,164.03,159.22,158.77,152.56,131.35,130.05,129.33,128.72,127.37,120.54,71.65,53.48,51.71,45.51,29.22,26.47,26.23,16.57.HRMS(ESI):calcd for C₂₂H₂₇N₄O₄S⁺[M+H]⁺,443.1748；found 443.1750.

Example 8 (E) -3- (4- ((2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydropyrido [4',3':4,5] thieno [2,3-d ] pyrimidin-2 yl) phenoxy) methyl) phenyl) -N-hydroxyacrylamide (9E).

Referring to the procedure of example 4, methyl 3-bromomethylbenzoate was changed to methyl 4-bromomethyl cinnamate to give compound 9 e.

¹H NMR(400MHz,)δ8.03(d,J＝8.7Hz,2H,Ar-H),7.91(s,2H,Ar-H),7.73(d,J＝20.6Hz,2H,Ar-H),5.18(s,2H,OCH₂Ar),4.20–3.54(m,4H,Piperidline-H),3.36(d,J＝9.6Hz,2H,Piperidline-H),2.56–2.36(m,9H,2Ar-CH₃,N-CH₃).HRMS(ESI):calcd for C₂₈H₂₉N₄O₄S⁺[M+H]⁺,517.1904；found 517.1912.

Example 9 (E) -3- (4- ((2- (2, 6-dimethyl-4- (7-methyl-4-oxo-3, 4,5,6,7, 8-hexahydropyrido [4',3':4,5] thieno [2,3-d ] pyrimidin-2 yl) phenoxy) ethoxy) methyl) phenyl) -N-hydroxyacrylamide (9f).

Referring to the procedure of example 4, methyl 3-bromomethylbenzoate was changed to methyl 4-bromoethoxymethyl cinnamate to give compound 9 f.

¹H NMR(400MHz,DMSO-d₆)δ7.90–7.74(m,3H,Ar-H,Ar-CH＝CH),7.56(s,2H,Ar-H),7.41(s,2H,Ar-H),6.48(s,1H,Ar-CH＝CH),4.61(s,2H,OCH₂Ar),4.01(s,2H,ArOCH₂CH₂),3.78(s,2H,ArOCH₂CH₂),3.56(s,2H,Piperidline-H),2.96(s,2H,Piperidline-H),2.67(s,2H,Piperidline-H),2.42–2.22(m,9H,2Ar-CH₃,N-CH₃).HRMS(ESI):calcd for C₃₀H₃₃N₄O₅S⁺[M+Na]⁺,583.1986；found 583.1995.

Test example 1 in vitro enzyme Activity test and antitumor cell proliferation Activity test

The in vitro enzyme activity and the anti-tumor cell proliferation activity of the compounds prepared in examples 1 to 9 were determined, wherein the BRD4 in vitro enzyme activity assay procedure was as follows:

1) preparation of a buffer solution: the buffer solution containing 50mM of hydroxyethyl piperazine ethanethiosulfonic acid (HEPES), 100mM of NaCl, 0.1% of bovine serum albumin, 0.05% of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt (CHAPS) and pH 7.4 was equilibrated to room temperature.

2) Samples at a concentration of 0-100. mu.M were serially diluted 1:3 for the BRD4 inhibitor to be tested, 4uL was transferred to a small volume 384-well plate, 4uL of glutathione-labeled BRD4 protein was added, and incubation was carried out at room temperature for 30 minutes with RVX-208 as a positive control.

3) Add 4u L biotinylated histone H4 KAc polypeptide concentration, continue the incubation for 30 minutes.

4) Finally 4. mu.L of HTRF donor (25ug/mL) and 4. mu.L of acceptor (25ug/mL) were added, with care taken to avoid light.

5) After incubating the plate for 60 minutes at room temperature in the dark, the fluorescence change was read with a blank control group of DMSO, and IC was calculated₅₀Values and compared to the RVX-208 positive control.

The enzyme activity test procedure of the BRD4/HDAC double-target inhibitor is the same as that of the BRD4 inhibitor, SAHA is selected as a positive control of HDACs, and anti-H3K9 monoclonal antibody and fluorescence-labeled streptavidin are selected as chromogenic markers.

The test results are shown in table 1.

Results of in vitro enzyme Activity test and anti-tumor cell proliferation Activity test for Compounds of Table 1

^aND:Not determined ^bRVX-208 and SAHA molar ratio is 1: 1.

Both RVX-208 and SAHA are present inhibitors and are positive controls.

From the experimental results, it can be seen that: 1)8a-c and 9a-f have good inhibitory activity against BRD4, HDACs and various colorectal cancer cells. 2) The inhibitory activity of compounds 9a-f having hydroxamic acid as the zinc ion binding domain against HDACs is generally higher than that of compounds having o-phenylenediamine as the zinc ion binding domainThe activity is high. 3) All compounds had superior inhibitory activity to HDAC2 and HDAC6 over HDAC7, suggesting that this dual-target inhibitor selectively inhibits class I and class IIb HDACs. 4) All dual-target compounds still had better BRD4 inhibitory activity, suggesting that only a weak effect on BRD4 inhibitory activity was observed after derivatization of the BRD4 backbone. 5) Finally, we screened the most active compound 9c, which inhibits IC of HDAC2 and HDAC6₅₀58nM and 73nM, respectively, relative to HDAC7 (IC)₅₀>10uM) with good selectivity. While compound 9c has good BRD4 inhibitory activity (IC)₅₀710nM), even better than the activity of the positive control RVX-208, its IC for HCT-116, SW620 and DLD1 antiproliferative activity₅₀Respectively 0.45, 1.78 and 2.11uM, which are superior to positive control, and indicate that the compound has strong anti-tumor activity potential.

Test example 2 inhibitory Activity of Compound 9c against HDAC1-3 and HDAC6

Compound 9c was further tested for its selectivity for BRDs and HDACs subtypes, as described in example 1, and the results are shown in Table 2.

TABLE 2 inhibitory Activity of Compound 9c on BRDs and HDACs

^aND:Not determined

As can be seen from Table 2, Compound 9c has very good inhibitory activity against HDAC1-3 and HDAC6, and IC₅₀IC for inhibition of HDAC8 and HDAC10 between 46 and 75nM₅₀Also below micromolar levels, whereas for class IIa and IV HDACs there is no inhibitory activity when the concentration is increased to 10uM, and the selectivity for HDACs is consistent with vorinostat. On the other hand, compound 9c was selective for BRD4 in preference to other BRDs, with selectivity superior to that of the positive control JQ-1. For this we selected compound 9c as a candidate for BRD4/HDAC dual-target inhibitor and further tested it for anti-colorectal cancer activity.

Test example 3 Induction of apoptosis in HCT-116 cells by Compound 9c

By MTT assay, we found the IC of compound 9c in inhibiting NCM460 on normal human colonic epithelial cells₅₀The value is far greater than the IC for inhibiting the rest 6 tumor cells₅₀The values (fig. 1) indicate that 9c has a low ability to inhibit the proliferation of cells in normal tissues and a low toxicity to normal cells, indicating a high safety. Subsequently, in the cell cycle staging assay, it was found that HCT-116 cells were treated with different concentrations of compound 9c (0.2uM, 0.5uM) and that the cells in the sub g1 phase appeared to a different extent than the normal saline control group, and that there was a positive correlation with the concentration, suggesting that compound 9c could induce apoptosis of HCT-116 cells (fig. 2). In the Annexin V-FITC/PI double staining method, Annexin V can be combined with early apoptotic cells and transferred from the interior of a cell membrane to the exterior of the cell membrane to form fatty acyl serine (PS) which is green fluorescence, and Propidium Iodide (PI) can permeate the cell membrane of cells in middle and late apoptosis stages to stain the cell nucleus red. On a bivariate flow cytometer scattergram after Annexin V-FITC/PI double staining (FIG. 3), the lower left quadrant is (FITC-/PI-), and represents live cells; the upper right quadrant is (FITC +/PI +), indicating late apoptotic cells; the lower right panel is (FITC +/PI-), indicating early apoptotic cells. From the figure we know that the NS group of early apoptotic cells is 2.8%, and that of compound 9c is 8.2% and 9.5%, respectively; late apoptotic NS (normal saline) group was 3.51%, compound 9c group was 23.0% and 35.9%, respectively; the total apoptosis compound 9c was 33.2 + -5.30% (0.2uM) and 45.3 + -6.8% (0.5uM), respectively, which were significantly higher than the NS group (6.3 + -0.4%, p)<0.05), further demonstrating that compound 9c has a dose-dependent effect on inducing apoptosis of HCT-116 cells.

By fluorescence micrographs of Hoechst33258 staining (fig. 4), we can see that the cells of the NS group fluoresce diffusely and uniformly; while compound 9c (0.2uM, 0.5uM) clearly showed a large amount of intense, dense, granular, lumpy fluorescence. As Hoechst33258 is a specific DNA blue fluorescent dye which can penetrate through cell membranes, after the cells are subjected to apoptosis, chromatin is subjected to pyknosis dyeing and is in dense and dense dyeing, or the cells are in a broken block shape, the dense and dense dyeing color is slightly whitish, and the cell nucleuses of normal cells are in normal blue, which shows that the cells of the experimental group are actually subjected to apoptosis compared with the cells of the NS group.

Test example 4 test of antitumor Activity of Compound 9c in transplantation tumor model

To evaluate the anti-colorectal cancer activity of compound 9c in vivo, we established a mouse HCT-116 colorectal cancer transplantation tumor model. After continuous administration for 19 days, the compound 9c and SAHA have obvious antitumor activity under the administration dosage, and the average tumor inhibition rate of the compound 9c can reach 68.8%. The reduction in tumor tissue volume of compound 9c was positively correlated with the amount administered compared to the saline control group. Compared with the positive control group, the compound 9c has better anti-tumor effect than RVX-208 and SAHA, and has smaller influence on the whole body weight of mice, which indicates that the compound 9c has certain selectivity on tumor cells. The results of immunohistochemical experiments show that the expression of c-Myc is obviously reduced in the RVX-208 group and the compound 9c group, and the BRD4 can promote the expression of the c-Myc, which indicates that the BRD4 is possibly inhibited; the expression of Ac-H3 was significantly up-regulated in the SAHA group and compound 9c group, indicating that the activity of HDACs was inhibited and that the amount of acetylated histones was increased in vivo; ki-67 in experiments to determine the proliferation rate of tumor cells, most of the G can be used₀The positive rate of the Ki-67 is higher in the cell markers outside the period, which indicates that the proportion of cells in the proliferation period is higher, the tumor grows faster, the positive rate of the Ki-67 in the compound 9c group is obviously reduced, and the proportion of the cells in the proliferation period is reduced, so that the tumor growth is inhibited; in the experiment for detecting the tumor cell apoptosis by the TUNEL method, the 3-OH tail end of the DNA broken by apoptotic cells is labeled and stained, so that the higher the positive rate of TUNEL is, the larger the proportion of apoptotic cells is, the obviously higher the positive rate of TUNEL staining in the compound 9c group is, and the larger the proportion of apoptotic tumor cells after the treatment of the compound 9c is suggested (figure 5). Therefore, we conclude that compound 9c has a good anti-colorectal cancer activity and a certain selectivity on cancer cells in vivo based on the BRD4/HDAC dual-target action mechanism.

Claims

1. Thieno[2,3-d]pyrimidyl hydroxamic acid derivative, characterized in that: its structure is shown in formula I or formula II:

Wherein, R ₁ is hydrogen, C1-C4 alkyl or benzyl;

R ₂ is C1-C5 alkylene,

R ₃ is hydroxyl, amino or

2 . The thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to claim 1 , wherein R ₁ is a C1-C4 alkyl group. 3 .

3. The thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to claim 2, wherein R ₁ is methyl.

4. The thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to claim 1, wherein R ₃ is hydroxyl or

5. The thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to claim 4, wherein R ₃ is a hydroxyl group.

6. The thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to any one of claims 1 to 5, wherein the structure is shown in formula I.

7. The thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to claim 1, wherein its structure is any one of the following structural formulas:

8. A pharmaceutically acceptable salt of the thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to any one of claims 1 to 7.

9. Use of the thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to any one of claims 1 to 7 in the preparation of a BRD4 inhibitor.

10. Use of the thieno[2,3-d]pyrimidinyl hydroxamic acid derivatives according to any one of claims 1 to 7 in the preparation of HDACs inhibitors.

11. Use of the thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to any one of claims 1 to 7 in the preparation of antitumor drugs.

12 . The use of the thieno[2,3-d]pyrimidinyl hydroxamic acid derivative according to claim 11 , wherein the tumor is colorectal cancer. 13 .

13. A pharmaceutical composition, characterized in that it is composed of an active ingredient and a pharmaceutically acceptable excipient, the active ingredient comprising a therapeutically effective amount of the thieno according to any one of claims 1 to 7 [2,3 -d] Pyrimidyl hydroxamic acid derivative or a pharmaceutically acceptable salt thereof.