CN100455564C - Preparation and application of histone deacetylase inhibitor and its pharmaceutical preparation - Google Patents

Abstract

The present invention discloses a histone deacetylase inhibitor for treating tumors, endocrine disturbance, immune system diseases, hereditary diseases and nerve involutional diseases, a preparing method of medicinal preparations thereof and applications thereof. The structure of the histone deacetylase inhibitor is shown as general formula (I), wherein the definitions of Ar1, Ar2, Q1, Q2, Q3, J, and Z are disclosed in the specification.

Description

Preparation and application of histone deacetylase inhibitor and its pharmaceutical preparation

technical field

The present invention relates to the synthesis of a new small molecular compound with therapeutic effect and its clinical application in the treatment of tumors, endocrine disorders, immune system diseases, genetic diseases and neurodegenerative diseases

Background technique

Abnormal gene expression plays an important role in the pathogenesis of many diseases, including tumors, endocrine disorders, immune system diseases, genetic diseases and neurodegenerative diseases. The human genome exists as a chromatin structure packaged with DNA, histones and non-histones, and the chromatin structure plays an important role in determining whether a specific gene is expressed or not. Overall, condensed chromatin represses transcription, whereas transcriptionally active genes tend to be located in open chromatin.

The basic repeating unit of chromatin, the nucleosome, consists of double strands of DNA surrounding a histone core containing four histones. This histone core contains one H3-H4 tetramer and two H2A-H2B dimers. Histone H1 attaches to the junction between nucleosomes and maintains the stability of the chromatin structure by neutralizing the negative charges on the DNA strand through its positively charged carboxyl terminus. This highly ordered structure of the nucleosome defines the relationship between chromatin composition and gene activation (Ricky W. Johnstone, "Histone deacetylase inhibitors: novel drugs for the treatment of cancer", Nature Reviews Drug Discovery 2002, 1:287). Histone N-termini can be post-translationally modified and thus can alter chromatin structure and function. One such modification is the reversible acetylation and deacetylation of lysine residues in histone tails. The level of histone acetylation is jointly controlled by histone acetylases (HATs) and histone deacetylases (Histone deacetylases, HDACs). In addition to being modified by acetylation, the N-terminus of histones can also be phosphorylated, methylated and ADP-ribosylated. These modifications affect the electrical properties and functions of histones, thereby changing the structure of chromatin and gene expression (Current Opinion in Oncology 2001, 13: 477-483).

Recent studies have revealed the close connection between histone acetylation, chromatin remodeling and gene regulation. Many transcriptional activator complexes have intrinsic histone acetylase activity, whereas transcriptional repressor complexes have the activity of recruiting histone deacetylases to target gene promoters (Bioassays 1998, 20:615). Some specific transcriptional activators, such as nuclear receptor superfamily, cAMP effector binding protein (CREB), signal transduction activated transcription factor 1 (STAT-1), etc., can interact with various coactivators and corepressors in different tissues and Selective action in genes constitutes a regulatory network for selective expression of genes. These regulatory networks control the balance of our bodily functions, and interference with these networks can cause disease or affect the course of disease. Therefore, regulating the interaction between these transcription complex proteins provides new methods for the treatment of tumors, endocrine disorders, immune system diseases, genetic diseases and neurodegenerative diseases (E.Korzus, Transcription Factor-specific Requirements for Coactivator and Their Acetyltransferase Functions .Science 1998, 279: 703-707; N.J.McKenna and B.W.O'Malley, Combinatorial Control of Gene Expression by Nuclear Receptors and Coregulators. Cell 2002, 108(4): 465-474; M.J.Pazin and J.T.Kadonaga, What's Up Downwith Histone Deacetylation and Transcription? Cell 1997, 89(3): 325-328; H.Zhong, R.E.Voll and S.Ghosh, Phosphorylation of NF-B p65 byPKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent CovalentInteraction/with the CBP0 .Molecular Cell 1998, 1(5):661-671; J.S.Steffan, Histone deacetylase inhibitors arrestpolyglutamine-dependent neurodegeneration in Drosophila, Nature 2001, 413:691-694; US20020115716A1, WO0056153A1).

For example, cell development and differentiation are regulated by the expression of gene programs, which are regulated at the level of chromatin structure. Genetic variations or mutations that cause constitutive activation of oncogenes such as RAS, or inactivation of tumor suppressor genes such as p53, will affect a series of molecular processes including transcription. In addition, some genetic variants that cause abnormal actions of histone acetylases and sirtuins, such as misplacement of their target genes, or inactivation of histone acetylase function, or overexpression of histone sirtuins, etc. Break the process of normal cell development and differentiation, causing the occurrence and development of tumors (Current Opinion Genet. Development 1999, 9: 40-48 and 175-184). Some human tumors are associated with dysregulation of histone acetylase and sirtuin activity. An example of this is the common translocation of chromosomes 15 and 17 in patients with human acute myeloid leukemia. The translocation results in A fusion protein comprising three protein molecules of RARα, PML and PLZF. This abnormal fusion protein binds to the cis-acting element of RAR and recruits a high-affinity histone deacetylase by strongly binding to the SMRT corepressor, resulting in sustained repression of RAR target gene expression and Loss of response to tretinoin (Oncogene 2001, 20:7204-7215). Retinoic acid receptor (RAR) is a ligand-dependent transcription factor that plays a very important role in the differentiation of bone marrow. The heterodimer composed of RAR and RXR can bind to the retinoic acid response element in the promoter region of the target gene. In the absence of retinoic acid, RAR/RXR can recruit SIN/HDAC through co-repressors NCOR and SMRT to inhibit transcription; and when ligand is added, HDAC is released, and then RAR/RXR can interact with TIF2, CBP, etc. to have HAT Active cofactors bind to activate transcription. Therefore, activation or repression of genes containing retinoic acid response elements is important for the differentiation of myeloid cells. Moreover, the addition of HDAC inhibitors can restore the ability of acute myeloid leukemia cells to retinoic acid-induced differentiation, implying that abnormal histone deacetylation is a key factor in the pathogenesis of leukemia.

It has been reported that the expression of some tumor suppressor genes, such as p53, can be inhibited when histone deacetylase is overexpressed. p53 is a key regulator of cell proliferation, signaling to genes that control the cell cycle and inducing apoptosis in the presence of external stress. The main function of p53 is that it can directly bind to specific DNA sequences and activate transcription. If its DNA binding region is mutated and the function is inactivated, it will often lead to cancer. There is evidence that CBP/p300 can upregulate p53 by acetylating histones and p53 (W.Gu and R.G.Roeder, Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain. Cell 1997, 90(4) : 595-606.). In contrast, HDAC-1, HDAC-2, and HDAC-3 in mammals can down-regulate p53 by deacetylating histones and p53 (L.-J. Juan, et al., Histone Deacetylases Specifically Down-regulate p53- dependent Gene Activation. The Journal of Biological Chemistry 2000, 275(27): 20436-20443).

The above experiments show that the abnormal transcriptional repression mediated by HDACs can change the structure of chromatin, interfere with normal cell differentiation, and lead to the occurrence of tumors and other proliferative diseases. Therefore, inhibition of HDAC activity may be an effective approach for the treatment of tumors and other proliferative diseases.

Several classes of histone deacetylase inhibitors have been discovered, including (1) short-chain fatty acids such as butyric acid and phenylbutyric acid; (2) organic hydroxamic acids such as suberoylanilidehydroxamic acid (SAHA) and trichostatin A ( TSA); (3) cyclic tetrapeptides containing 2-amino-8-oxo-9,10-epoxydecanoyl, such as trapoxin and HC-toxon; (4) without 2-amino-8-oxo-9, cyclic tetrapeptides of 10-epoxydecanoyl, such as Apicidin and FK228; (5) benzamide compounds, such as MS-275 (EP0847992A1, US2002/0103192A1, WO02/26696A1, WO01/70675A2, WO01/18171A2).

As an inhibitor of cell proliferation and inducer of cell differentiation, butyric acid is mainly derived from the inhibition of histone deacetylase (A.Nudelman and A.Rephaeli, Novel Mutual Prodrug of Retinoic and Butyric Acids with Enhanced Anticancer Activity. J. Med. Chem. 2000, 43(15): 2962-2966.). Phenylbutyric acid can be used to treat thalassemia, toxoplasmosis, malaria and other diseases alone, and can also be combined with vitamin A acid to treat acute myeloid leukemia (R.P.Warrell.et al., Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J. Natl. Cancer Inst. 1998, 90(21): 1621-1625.). Valproic acid is an anticonvulsant drug that has also been found to act by directly inhibiting histone deacetylases (C.J.Phiel et al., Histone Deacetylase Is a Direct Target of Valproic Acid, a Potent Anticonvulsant, Mood Stabilizer, and Teratogen .The Journal of Biological Chemistry 2001, 276(39): 36734-36741; EP1170008A1).

Some benzamide compounds have histone sirtuin inhibitor activity at low micromolar concentrations. Among them, the lead compound MS-275 developed by Mitsui Chemicals is the first histone deacetylase inhibitor proven to have oral anticancer activity in animals without serious side effects (A.Saito et al., A syntheticinhibitor of histone deacetylase, MS-27-275, with marked in vivoantitumor activity against human tumors. Proceedings of the National Academy of Sciences of the United States of America 1999, 96(8):4592-4597; EP 0847992 A1). Currently, MS-275 is conducting clinical research on leukemia at the Greenebaum Cancer Center of the University of Maryland, and at the same time conducting clinical research on solid tumors at the National Cancer Institute (EBLevit, Clinical Trials in Leukemiafocus on New Treatment Approaches.2001 Release-University of Maryland Medical News 2001 Maryland http://www.umm.edu/news/releases/karp.html , A Phase I Study of an Oral Histone Deacetylase Inhibitor, MS-275, in Refractory Solid Tumors and Lymphomas. 2001 , National Cancer Institute). However, some new compounds with better properties are still to be developed in order to obtain drugs with stronger HDAC inhibitory activity and lower side effects.

technical content

One of the purposes of the present invention is to disclose a class of compounds designed based on histone deacetylases to treat tumors, endocrine disorders, immune system diseases, genetic diseases and neurodegenerative diseases;

The second object of the present invention is to disclose the preparation method of the compound described in this class;

The third purpose of the present invention is to disclose the clinical application of the above-mentioned compounds in the treatment of tumors, endocrine disorders, immune system diseases, genetic diseases and neurodegenerative diseases.

Said compound of the present invention, its chemical structure is as shown in general formula (I):

Among them, ^Ar is aryl or heterocyclic aryl, which can contain one or more substituents, and its substituents can be halogen, amino, hydroxyl, nitro, cyano, alkyl, alkoxy, aminoalkyl , alkylamino, acyl, amido, thioalkyl, perfluoroalkyl, perfluoroalkoxy, carboxyl, phenyl or heterocyclic substituents;

Ar ² is an arylene or heterocyclic arylene, which may contain one or more substituents, and its substituents may be halogen, amino, hydroxyl, nitro, cyano, alkyl, alkoxy, aminoalkyl, alkane Amino, acyl, amido, thioalkyl, perfluoroalkyl, perfluoroalkoxy, carboxyl, phenyl or heterocyclic substituents;

Q ¹ , Q ² , and Q ³ are covalent bonds or C _1-7 alkylene, which can be linear or cyclic, saturated or unsaturated, and can contain one or more substituents , whose substituents can be halogen, amino, hydroxyl, nitro, cyano, alkyl, alkoxy, aminoalkyl, alkylamino, acyl, amido, thioalkyl, perfluoroalkyl, perfluoro Alkoxy, carboxyl, phenyl or heterocyclic substituents;

J is one of the following structures:

Wherein, ^R is H, alkyl, aralkyl, heterocyclic aralkyl, heterocyclic substituent, aryl or heterocyclic aryl;

Z is -OH or 2-aminocyclohexyl.

One of the preferred methods of the present invention for treating diseases related to differentiation and proliferation, such as cancer and psoriasis, is represented by the general formula ( ¹ ), wherein Ar is a heterocyclic aryl group;

Ar ² is an aromatic support;

Q ¹ , Q ² , and Q ³ are covalent bonds or C _1-2 alkylene, respectively;

J is one of the following structures:

Wherein, R ¹ is H or alkyl;

Z is -OH or 2-aminocyclohexyl.

The second preferred method of the compounds of the present invention for treating diseases related to differentiation and proliferation such as cancer and psoriasis is shown in general formula ( ¹ ), wherein Ar is pyridine;

Ar ² is a benzene ring;

Q ¹ , Q ² , and Q ³ are covalent bonds or C _1-2 alkylene, respectively;

J is one of the following structures:

Wherein, R ¹ is H;

Z is -OH or 2-aminocyclohexyl.

The "halogen" mentioned in the present invention refers to fluorine, chlorine, bromine and iodine;

"Alkyl" in the present invention includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc.;

The "alkoxy" in the present invention includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, etc.;

The "aminoalkyl" in the present invention includes aminoethyl, 1-aminopropyl, 1-aminopropyl, etc.;

The "alkylamino group" mentioned in the present invention includes N-methylamino group, N-ethylamino group, N-isopropylamino group, etc.;

The "acyl" in the present invention includes acetyl, propionyl, isobutyryl, etc.;

The "amide group" mentioned in the present invention includes acetamide group, propionamide group, butyramide group, isobutyramide group, etc.;

The "thioalkyl" mentioned in the present invention includes methylthio, ethylthio, propylthio, etc.;

The "perfluoroalkyl" mentioned in the present invention includes trifluoromethyl, pentafluoroethyl, etc.;

The "perfluoroalkoxy" mentioned in the present invention includes trifluoromethoxy, pentafluoroethoxy, etc.;

The "C _1-7 alkylene" in the present invention includes -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH=CH-, -CH=CH-CH=CH -wait.

"Aralkyl" in the present invention includes benzyl, phenethyl, 3-phenylpropyl, 1-naphthylmethyl, etc.;

The "heterocyclic aralkyl" mentioned in the present invention includes 2-furylmethyl, 3-furylmethyl, 2-pyridylmethyl, etc.;

The "heterocycle" mentioned in the present invention refers to a saturated or unsaturated heterocycle containing one or more heteroatoms (nitrogen, oxygen or sulfur), including tetrahydropyrrole, dihydropyrrole, dihydropyrazole, piperidine , morpholine, imidazole, pyridine, etc.;

The "aryl" mentioned in the present invention includes aromatic rings with or without substituents, and the substituents can be halogen, amino, hydroxyl, alkyl or alkoxy, such as phenyl, naphthyl, etc.;

The "heterocyclic aryl" mentioned in the present invention refers to five-membered, six-membered monocyclic or nine-membered, ten-membered bicyclic aromatic substituents containing one or more heteroatoms (O, S or N atoms), such as furan , thiophenol, pyrrole, imidazole, triazole, pyridine, pyrazine, pyrimidine, quinoline, indole, benzimidazole, etc.;

The synthetic method of said compound of the present invention is as follows:

(a) carry out condensation reaction with the compound of general formula (II) and the compound of general formula (III) to obtain the compound of general formula (IV):

Ar ¹ -Q ¹ -R ²

(II)

Among them, Ar ¹ , Ar ² , Q ¹ , Q ² , Q ³ , and J are as described above; R ² is -OH or -NH ₂ ; when R ² is -OH, R ³ is -NH ₂ ; when R In the case of ² -NH ₂ , R ³ is -OH.

This condensation reaction is with N, N-carbonyl diimidazole (CDI) as catalyst, first N, N-carbonyl diimidazole is reacted with general formula (II) compound to prepare active intermediate, and then with general formula (III ) compound is reacted to obtain the compound of general formula (IV);

(b) Condensing the compound of general formula (IV) with the compound of general formula (V) to obtain the target compound (I):

H ₂ NZ

(V)

Wherein, Z is the same as mentioned above.

The condensation reaction takes ethyl chloroformate as a catalyst, first reacts ethyl chloroformate with the compound of general formula (IV) to obtain an active intermediate, and then reacts with the compound of general formula (V) to obtain the compound of general formula (I) compound.

The reaction temperature of the above-mentioned condensation reactions (a) and (b) is -10 to 80° C., and the reaction time is 1 to 72 hours. The solvent used in the reaction is a common solvent, such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N,N-dimethylformamide and the like. If necessary, a base such as sodium hydroxide, triethylamine, pyridine, etc., or an acid such as hydrochloric acid, acetic acid, trifluoroacetic acid, etc. may be added.

The compound described in the general formula (I) can be purified by common separation methods, such as extraction, recrystallization, column chromatography and the like.

The compound described in the present invention has curative effect on treating tumor, endocrine disorder, immune system disease, genetic disease and neurodegenerative disease.

The compound of the present invention can be made into common pharmaceutical preparations, such as tablets, capsules, powders, syrups, liquids, suspensions, injections, spices, sweeteners, liquid or solid fillers or diluents, etc. can be added Commonly used carrier substances (see "Handbook of Pharmaceutical Excipients", American Pharmaceutical Association, October 1986). The preparation usually contains 1-70% of active ingredients, preferably 5-50%, and the remaining components are carrier fillers, diluents or solvents.

The compounds of the present invention can be administered to mammals (including humans) clinically by oral or injection, especially oral administration. The dosage is 0.0001-200mg/kg body weight per day. The optimal dose depends on the individual, usually starting with a small dose and gradually increasing the dose.

Further illustrate content of the present invention below in conjunction with example, but protection scope of the present invention is not only limited to these examples. Unless otherwise specified, the percentages described in the present invention are all percentages by weight.

Specific implementation method Example 1

Preparation of 4-[N-(4-fluorobenzyloxycarbonyl)aminomethyl]benzoic acid

Add 0.81 g (5.0 mmol) of N,N-carbonyldiimidazole and 10 ml of tetrahydrofuran into the reaction flask, cool to 0° C., add dropwise 10 ml of tetrahydrofuran solution dissolved in 0.63 g (5.0 mmol) of 4-fluorobenzyl alcohol, The reaction was stirred at room temperature for 3 hours, then 5 ml of 1M sodium hydroxide solution in which 0.76 g (5.0 mmol) of 4-aminomethylbenzoic acid was dissolved was added dropwise, and the reaction was continued to stir at room temperature for 5 hours. The reaction mixture was concentrated in vacuo, 5 ml of saturated sodium chloride solution was added to the residue, and the pH was adjusted to 7 with concentrated hydrochloric acid. The precipitated solid was filtered, washed with ice water, and dried to obtain 1.19 g (78.8%) of the target compound.

Example 2

Preparation of N-(2-aminocyclohexyl)-4-[N-(4-fluorobenzyloxycarbonyl)aminomethyl]benzamide (HYM0513)

Add 0.21 g (0.70 mmol) of 4-[N-(4-fluorobenzyloxycarbonyl) aminomethyl]benzoic acid and 4 ml of tetrahydrofuran to the reaction flask, cool to 0°C, add 0.21 g (2.10 mmol) Triethylamine and 0.12 g (1.10 mmol) of ethyl chloroformate were reacted at 0° C. for 30 min. Remove the solid generated by filtration, transfer the filtrate to another reaction flask, add 0.32 g (2.80 mmol) cyclohexanediamine, react at room temperature for 10 hours, concentrate in vacuo to obtain the crude product, and chromatographically separate (developing solvent: chloroform: methanol = 8:1) yielded 0.15 g (53.7%) of the target compound, mp 178-180°C. IR(KBr)cm ^-1 : 3328, 2926, 1691, 1629, 1540, 1266, 1140. ¹ HNMR (300MHz, DMSO-d ₆ ): δppm: 1.12-1.24 (m, 6H), 1.66 (m, 2H), 1.86 (m, 2H), 2.61 (m, 1H), 3.47 (m, 1H), 4.24(d, 2H), 5.03(s, 3H), 7.18(t, 2H), 7.30(d, 2H), 7.41(t, 2H), 7.75(d, 1H), 7.80(d, 2H), 8.04 (m, 1H). HRMS ( _C22H26FN3O3 ) calcd (%): 399.4629; found _{( %} ): 399.4627 _.

Example 3

Preparation of N-hydroxy-4-[N-(4-fluorobenzyloxycarbonyl)aminomethyl]benzamide (HYM0512)

Add 0.21 g (0.70 mmol) of 4-[N-(4-fluorobenzyloxycarbonyl) aminomethyl]benzoic acid and 4 ml of tetrahydrofuran to the reaction flask, cool to 0°C, add 0.21 g (2.10 mmol) Triethylamine and 0.12 g (1.1 mmol) of ethyl chloroformate were reacted at 0° C. for 30 min, the resulting solid was removed by filtration, and the filtrate was collected for future use. Add 0.21 grams (3.0 mmol) of hydroxylamine hydrochloride, 0.17 grams (3.0 mmol) of potassium hydroxide and 5 milliliters of methanol into another reaction flask, stir and react at room temperature for 30 minutes, add the above-mentioned filtrate dropwise, stir and react at room temperature for 20 hours, and concentrate in vacuo to obtain crude The product was separated by chromatography (developing solvent: chloroform:methanol=4:1) to obtain 0.12 g (53.9%) of the target compound. IR(KBr)cm ^-1 : 3331, 2914, 1632, 1629, 1518, 1263. ¹ HNMR (300MHz, DMSO-d ₆ ): δppm: 4.24 (d, 2H), 5.03 (s, 3H), 7.18 (t, 2H), 7.30 (d, 2H), 7.41 (t, 2H), 7.75 ( d, 1H), 7.80 (d, 2H), 8.68 (s, 1H), 10.34 (s, 1H). HRMS (C ₁₆ H ₁₅ FN ₂ O ₄ ) calcd (%): 318.3023; found (%): 318.3020.

Example 4

Preparation of 4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzoic acid

Add 0.81 g (5.0 mmol) of N,N-carbonyldiimidazole and 10 ml of tetrahydrofuran into the reaction flask, cool to 0°C, add dropwise 10 ml of tetrahydrofuran solution in which 0.54 g (5.0 mmol) of 3-hydroxymethylpyridine is dissolved , stirred and reacted at room temperature for 3 hours, then added dropwise 5 milliliters of 1M sodium hydroxide solution in which 0.76 grams (5.0 mmol) of 4-aminomethylbenzoic acid was dissolved, and continued stirring and reacting at room temperature for 5 hours. The reaction mixture was concentrated in vacuo, 5 ml of saturated sodium chloride solution was added to the residue, and the pH was adjusted to 7 with concentrated hydrochloric acid. The precipitated solid was filtered, washed with ice water, and dried to obtain 1.20 g (83.9%) of the target compound.

Example 5

Preparation of N-(2-aminocyclohexyl)-4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzamide (HYM0504)

Add 0.20 g (0.70 mmol) of 4-[N-(pyridine-3-methoxycarbonyl) aminomethyl]benzoic acid and 4 ml of tetrahydrofuran to the reaction flask, cool to 0°C, add 0.21 g (2.10 mmol) Triethylamine and 0.12 g (1.10 mmol) of ethyl chloroformate were reacted at 0° C. for 30 min. Remove the solid generated by filtration, transfer the filtrate to another reaction flask, add 0.32 g (2.80 mmol) cyclohexanediamine, react at room temperature for 10 hours, concentrate in vacuo to obtain the crude product, and chromatographically separate (developing solvent: chloroform: methanol = 8:1) yielded 0.12 g (44.9%) of the target compound, mp 159-160°C. IR(KBr)cm ^-1 : 3303, 2933, 1694, 1595, 1549, 1421, 1227, 1137. ¹ H NMR (300MHz, DMSO-d ₆ ): δppm: 1.25 (m, 4H), 1.69 (m, 2H), 1.90 (m, 2H), 2.92 (m, 1H), 4.22 (m, 2H), 5.09 (s, 3H), 7.00(s, 1H), 7.27(d, 1H), 7.39(s, 1H), 7.62(s, 1H), 7.77(s, 1H), 7.86(m, 3H), 8.55( m, 1H). HRMS ( _C21H26N4O3 ) calcd (%): _382.4602 ; found (%): _{382.4603 .}

Example 6

Preparation of N-hydroxy-4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzamide (HYM0505)

Add 0.20 g (0.70 mmol) of 4-[N-(4-fluorobenzyloxycarbonyl) aminomethyl]benzoic acid and 4 ml of tetrahydrofuran into the reaction flask, cool to 0°C, add 0.21 g (2.10 mmol) Triethylamine and 0.12 g (1.1 mmol) of ethyl chloroformate were reacted at 0° C. for 30 min, the resulting solid was removed by filtration, and the filtrate was collected for future use. Add 0.21 grams (3.0 mmol) of hydroxylamine hydrochloride, 0.17 grams (3.0 mmol) of potassium hydroxide and 5 milliliters of methanol into another reaction flask, stir and react at room temperature for 30 minutes, add the above-mentioned filtrate dropwise, stir and react at room temperature for 20 hours, and concentrate in vacuo to obtain crude The product was separated by chromatography (developing solvent: chloroform:methanol=4:1) to obtain 0.10 g (47.5%) of the title compound. IR(KBr)cm ^-1 : 3308, 2911, 1682, 1583, 1549, 1229. ¹ H NMR (300MHz, DMSO-d ₆ ): δppm: 4.22(m, 2H), 5.09(s, 3H), 7.00(s, 1H), 7.27(d, 1H), 7.39(s, 1H), 7.62 (s, 1H), 7.77 (s, 1H), 7.86 (m, 3H), 8.72 (s, 1H), 10.40 (s, 1H). HRMS (C ₁₅ H ₁₅ N ₃ O ₄ ) calcd (%): 301.2996; found (%): 301.2998.

Example 7

Compound N-(2-aminocyclohexyl)-4-[N-(4-fluorobenzyloxycarbonyl)aminomethyl]benzamide (HYM0513), N-hydroxy-4-[N-(4-fluoro Benzyloxycarbonyl)aminomethyl]benzamide (HYM0512), N-(2-aminocyclohexyl)-4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzamide ( Inhibition of histone deacetylase activity in vitro by N-hydroxy-4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzamide (HYM0505) and HYM0504)

The experiment was completed with HDAC colorimetric assay kit (BIOMOL Research Laboratories, PA, USA). All operations were performed according to the experimental manual. Firstly, the tested compounds were added to the 96-well plate at different concentrations, then mixed with the nuclear extract of HeLa cells and added with the substrate of histone deacetylase, left at 37°C for 10 minutes, and then terminated with the stop color development solution , and then read the results on a microplate reader with a wavelength of 405 nm. The calculation of the inhibition rate was carried out with reference to the instructions. The experimental results are shown in Table 1 (NA: no activity at 10 μM).

Example 8

Compound N-(2-aminocyclohexyl)-4-N-(4-fluorobenzyloxycarbonyl)aminomethyl]benzamide (HYM0513), N-hydroxy-4-[N-(4-fluorobenzene Methoxycarbonyl)aminomethyl]benzamide (HYM0512), N-(2-aminocyclohexyl)-4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzamide (HYM0504 ), and N-hydroxy-4-[N-(pyridine-3-methoxycarbonyl)aminomethyl]benzamide (HYM0505) inhibited the growth of cancer cells in vitro

The growth inhibition rate was determined by MTS method. Inoculate 5,000 test cells per well in a 96-well plate (the inoculation amount of cells with different growth rates is different). After culturing for 24 hours, different concentrations of the compounds to be tested were added. After culturing for 48 hours, 20 μl of MTS detection substrate (Promega) was added to each well. After incubation at 37°C for 2 hours, the results were read on a microplate reader with a wavelength of 490 nm. The relative amount of viable cells was calculated as experimental group/control group × 100%. The concentration of the compound that inhibited the cell growth by 50% was designated as _GI50 . All compounds were dissolved in DMSO and diluted 1:1000 at the time of dosing so that the final concentration of DMSO was ≤0.1%. Each experiment was repeated three times independently, and the experimental results are shown in Table 2.

Table 1. Compound Inhibition of Histone Deacetylases in Vitro

Table 2. Growth inhibitory effect of compounds on tumor cells

^* Cell source:

U2OS: human osteosarcoma cells HeLa: human cervical cancer cells

DU-145: Human prostate cancer cells SGC-7901: Human gastric cancer cells

SMMC-7721: Human liver cancer cells HepG2: Human liver cancer cells

293: Human Embryonic Kidney Cells MCF-7: Human Breast Cancer Cells

MDA-MB-231: Human breast cancer cells H292: Human lung cancer cells

LNCaP: Human Prostate Cancer Cell SK-N-SH: Human Neuroblastoma

PANC-1: Human pancreatic cancer cells SK-OV-3: Human ovarian cancer cells

28SC: Human mononuclear macrophage Raji: Human Burkitt lymphoma

HL-60: Human myeloid leukemia cells Jurkat: Human T lymphocytic leukemia

Claims (8)

1. compound or its salt with following general formula (I):

Wherein, Ar ¹Be pyridine ring or phenyl ring, unsubstituted or substituting group is arranged on the ring, substituting group is halogen, alkyl, alkoxyl group or trifluoromethyl;

Ar ²Be phenyl ring;

Q ¹For-CH ₂-,-CH ₂CH ₂-or-CH ₂CH ₂CH ₂-;

Q ²For-CH ₂-,-CH ₂CH ₂-or-CH ₂CH ₂CH ₂-;

Q ³Be covalent linkage;

J is

R ¹Be H or alkyl;

Z is hydroxyl or 2-aminocyclohexyl.

2. according to the compound of claim 1, wherein:

Ar ¹Be phenyl ring;

Ar ²Be phenyl ring;

Q ¹For-CH ₂-;

Q ²For-CH ₂-;

Q ³Be covalent linkage;

J is

R ¹Be H;

Z is hydroxyl or 2-aminocyclohexyl.

3. according to the compound of claim 1 or 2, wherein:

Ar ¹Be pyridine ring;

Ar ²Be phenyl ring;

Q ¹For-CH ₂-;

Q ²For-CH ₂-;

Q ³Be covalent linkage;

J is

R ¹Be H;

Z is hydroxyl or 2-aminocyclohexyl.

4. the preparation method of the compound of general formula (I), this method comprises the steps:

(a) general formula (II) compound and general formula (III) compound are carried out condensation reaction and obtain general formula (IV) compound:

Ar ¹-Q ¹-R ²

(II)

Wherein, Ar ¹, Ar ², Q ¹, Q ², Q ³, J is identical with the described definition of claim 1; R ²For-OH; R ³For-NH ₂

(b) general formula (IV) compound and logical formula V compound are carried out condensation reaction and obtain general formula (I) compound:

H ₂N-Z

(V)

Wherein, Z is identical with the described definition of claim 1.

5. according to the preparation method of claim 4, wherein, reactions steps (a) is with N, N-phosphinylidyne diimidazole is a catalyzer, make N earlier, N-phosphinylidyne diimidazole and general formula (II) compound reacts and makes active intermediate, and then reacts with general formula (III) compound and to make general formula (IV) compound.

6. according to the preparation method of claim 4, wherein, reactions steps (b) is catalyzer with the Vinyl chloroformate, Vinyl chloroformate and general formula (IV) compound is reacted make active intermediate, and then react with logical formula V compound and to make general formula (I) compound.

7. according to the preparation method of claim 4, wherein, the temperature of reaction of described reactions steps (a) and reactions steps (b) is-10～80 ℃, and the reaction times is 1～72 hour.

8. a NSC 630176 medicinal preparations is characterized in that, said preparation is made up of the compound of the described general formula of claim 1 (I) and pharmaceutical carrier auxiliary material or thinner.