WO2012174730A1

WO2012174730A1 - Preparative method and use of zyj-d08a and its epimers as histone deacetylase inhibitors

Info

Publication number: WO2012174730A1
Application number: PCT/CN2011/076250
Authority: WO
Inventors: 徐文方; 张颖杰; 宋伟国
Original assignee: 寿光富康制药有限公司; 潍坊博创国际生物医药研究院; 方浩; 杨磊; 宋成刚
Priority date: 2011-06-21
Filing date: 2011-06-24
Publication date: 2012-12-27
Also published as: CN102807526B; CN102807526A

Abstract

The present invention discloses ZYJ-D08a and its epimer ZYJ-D08ae as histone deacetylase inhibitors in pharmacy field, and their preparative methods and medical use for inhibiting tumor. ZYJ-D08a and its epimer ZYJ-D08ae are represented by formula (I).

Description

Method and application for preparation of histone deacetylase inhibitor ZYJ-D08a and its epimer

The invention relates to a preparation method of histone deacetylase inhibitor ZYJ-D08a and its epimer ZYJ-D08ae and its use in anti-tumor treatment, and belongs to the technical field of medicine.

Background technique

The earliest discovered histone deacetylases (HDACs) are a class of zinc ion-dependent hydrolases whose main function is to hydrolyze the acetyl group on the ε-amino group of the N-terminal lysine residue of histone nucleosomes. Deacetylated histones increase in positive charge density, which results in tighter binding to negatively charged DNA, hinders binding of various transcription factors to DNA, and ultimately inhibits transcription of various genes (see Christian, AH, et al. Curr. Opin. Chem. Biol, 1997, 1, 300; Kouzarides, T., Curr. Opin. Genet. Dev., 1999, 9, 40; Wolffe, AP Sci. Washington, 1996, 272, 371). In addition, histone deacetylation by HDACs is also closely related to other genomic functions, such as: chromatin assembly, DNA repair and recombination (see Polo, SE, et al. Cancer Lett., 2005, 220, 1; Vidanes, GM, et al. Cell, 2005, 121, 973).

In recent years, the in-depth study of HDACs has greatly expanded the family of HDACs. Currently, the HDACs family includes at least 18 subtype members (see Gregoretti, IV, et al. J. Mol. Biol, 2004, 338, 17.; Annemieke JM, et al. Biochem. J., 2003, 370, 737) . Based on differences in function, location, and homology between these members, they can be divided into four subfamilies: HDACs I, HDACs II, HDACs III, and HDACs IV. Among them, HDACs I subfamily (HDAC1, HDAC2, HDAC3 and HDAC8), HDACs II subfamily (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10) and HDACs IV family (ie HDAC11) belong to zinc ion-dependent metalloproteinases, all The catalytically active sites of zinc-dependent HDACs have high homology, and this homology is more pronounced among members of each subfamily; while the HDACs III subfamily (including Sirl-Sir7) is dependent on NAD+. With the expansion of HDACs family members, more and more non-histone proteins have been identified as substrates for HDACs, such as: transcription factors, microtubules, molecular chaperones (HSP90) and nuclear transporters (see Glozak, MA, et al. Gene, 2005, 363, 15).

The diversity of HDACs' substrates determines the complexity of their functions. Therefore, the dysfunction of HDACs can cause many diseases, including cancer, neurodegenerative diseases, viral infections, inflammation, leukemia, malaria and diabetes. Cancer is undoubtedly human life. The number one killer of health. Studies have shown that excessive deacetylation of histone H4 occurs early in cancer and is a prominent marker of cancer development (see Fraga, M. F., et al. Nature Genet., 2005, 37, 391). In addition, overexpression of the HDACs I subfamily and the HDACs II subfamily, particularly the HDACs I subfamily, has been found in many cancer cases (see Witt, 0., et al. Cancer Letter., 2009, 277, 8).

HDACs play a major role in the development and progression of cancer: promoting cancer cell proliferation and invasion and migration; promoting neovascularization of cancer tissues; enhancing cancer cell resistance to chemotherapeutic drugs; inhibiting cancer cell differentiation and apoptosis, etc. (See Witt, 0., et al. Cancer Letter., 2009, 277, 8). Therefore, designing inhibitors targeting HDACs has become one of the hotspots in antitumor drug research. Based on the pharmacophore model of histone deacetylase inhibitors, we first designed and synthesized a series of tyrosine derivatives of histone deacetylase inhibitors (see Bioorg. Med. Chem., 2010, 18, 1761). -1772), and applied for a related patent (publication number CN 101723896 A) to protect a compound having the structure of the general formula (II). On this basis, we conducted further structural diversity studies on the R ₂ group in the formula (Π) and found a series of more active histone deacetylase inhibitors (see J. Med. Chem.). , 2011, 54, 2823-2838), wherein the compound ZYJ-D08 as shown in formula (III) shows in vivo anticancer activity with certain development prospects. In order to further improve the possible instability of gastric acid in ZYJ-D08, we further modified its structure to obtain histone deacetylase inhibitors ZYJ-D08a and ZYJ-D08ae with the structure of formula (IV). Both of these compounds show strong histone deacetylase inhibitory activity and anti-cancer activity in vitro and in vivo, and are expected to become a new class of antitumor drugs.

ZYJ-D08a ZYJ-D08ae ( _IV ) SUMMARY OF THE INVENTION

The present invention is directed to the deficiencies of the prior art, and provides a novel histone deacetylase inhibitor ZYJ-D08a and its preparation method for the epimer ZYJ-D08ae and its application in anti-tumor therapy.

The present invention discloses two novel compounds ZYJ-D08a and its epimer ZYJ-D08ae, the structure is as shown in the formula 0V), and the chemical names are (S)-2-"2S,3S)-2- 3,3- Dimethylbutyryl)-3-methyl-n-pentanoyl)-7-(2-(hydroxylamine)-2-carbonylethoxy)-N-(4-methoxyphenyl)-1,2, 3,4-tetrahydroisoquinoline-3-amide and (S)-2-((2R,3S)-2-(3,3-dimethylbutyramido)-3-methyl-n-pentanoyl) -7-(2-(Hydroxyamine)-2-carbonylethoxy 3⁄4)-N-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline-3-amide. The two compounds are histone deacetylase inhibitors, and they have been shown to have significant growth inhibitory effects on human breast cancer cells MDA-MB-231 and colon cancer cells HCT116 grafted in nude mice, and are expected to become a new class of Anti-tumor drugs.

ZYJ-D08a ZYJ-D08ae (IV) The preparation method of the compound, the reaction step and the reaction formula are as follows:

Synthetic route 1: using optically pure 3, 5-diiodo-L-tyrosine as a raw material, followed by Pictet-Spengler cyclization, protecting secondary amine groups, hydrogenation reduction and deiodination, and condensation of polypeptides with p-methoxyanilinyl groups Group, nucleophilic reaction with methyl bromoacetate, removal of tert-butoxycarbonyl protecting group to obtain key intermediate 7; optically pure L-isoleucine (L-ile) as raw material, protected by primary amine group to obtain intermediate 8; Intermediates 7 and 8 were condensed, deprotected, N-acylated, and finally made of hydroxamic acid to give ZYJ-D08a. The reaction formula is as follows:

The synthetic route 2 method is similar to that of the route 1, except that the optically pure D-isoleucine (D-aile) is substituted for L-isoleucine (L-ile) as a raw material, and the same reaction is carried out to obtain ZYJ. - the epimer of the D08a ZYJ-D08ae. The reaction formula is as follows: Synthetic route 2:

The reagents in the above Reaction Schemes 1 and 2 are: (a) paraformaldehyde, 37% hydrochloric acid, ethylene glycol dimethyl ether, 72-75 ° C, reaction for 18 hours; (b) di-tert-butyl carbonate, lmol /L sodium hydroxide solution, tetrahydrofuran; (c) 10% palladium on carbon, hydrogen, methanol; (d) p-methoxyaniline, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, anhydrous tetrahydrofuran (e) methyl bromoacetate, potassium carbonate, anhydrous N,N-dimethylformamide; (f) trifluoroacetic acid, dichloromethane, anhydrous potassium carbonate; ( _§ ) 0-benzotriazole ^^,^^-tetramethylurea tetrafluoroborate, triethylamine, tetrahydrofuran; (h) l) trifluoroacetic acid, dichloromethane, anhydrous potassium carbonate; 2) 3,3-dimethylbutyl Acid, 0-benzotriazole-oxime, oxime, Ν', Ν'-tetramethylurea tetrafluoroborate, triethylamine, tetrahydrofuran; (i) potassium hydroxyamine, anhydrous methanol. Specific synthesis method:

Take ZYJ-D08a as an example:

1) (S)-7-Hydroxy-6, 8-diiodo-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride 2

To 250 mL of concentrated hydrochloric acid, 3,5-diiodo-L-tyrosine (1, 30.0 g, 69.3 mmol), ethylene glycol dimethyl ether (20 mL) and paraformaldehyde (7.8 g, 260.0 mmol) were added. And gradually warmed to 72 ° C. After 0.5 hours, concentrated hydrochloric acid (50 mL), ethylene glycol dimethyl ether (10 mL) and paraformaldehyde (5.2 g, 173.3 mmol) were added, and the reaction was continued at 72-75 ° C for 18 hours in an oil bath. The reaction suspension was cooled in an ice-bath and filtered, and the filter cake was washed with ethyl ether. Yield: 58%, ESI-MS m/z: 446.2[M+H ⁺ ], 3⁄4-NMR (DMSO-^ e) ^ 3.07 (dd, 16.8 Hz, 10.8 Hz, 1H), 3.22 (dd, 16.8 Hz, 4.8 Hz, 1H), 4.02 (d, 16.2 Hz) , 1H), 4.15 (d, 16.2 Hz, 1H), 4.32 (dd, 4.8 Hz, 10.8 Hz, 1H), 7.73 (s, 1H), 9.68 (s, 1H), 10.00 (br s, 2H), 14.17 (br s, 1H).

2) (S)-2-tert-Butoxycarbonyl-7-hydroxy-6,8-diiodo-1, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid 3

Compound 2C 4.81 g, 10.0 mmol) was dissolved in 22 mL of 1 mol/L sodium hydroxide solution, and 5 mL of di-tert-butyl carbonate (2.40 g, 11.0 mmol) in tetrahydrofuran was added. The pH of the reaction solution was controlled at 9-11 with a 1 mol/L sodium hydroxide solution during the reaction. After reacting for 6 hours at room temperature, the tetrahydrofuran in the reaction mixture was distilled off, and the reaction liquid was extracted three times with petroleum ether, and acidified to pH 4-5 with a 1 mol/L citric acid solution, and then extracted three times with ethyl acetate. After the mixture was washed with brine, dried over anhydrous magnesium sulfate and evaporated Yield: 92%, 3⁄4-NMR (DMSO-) J l.34+1.40 (s, 9H, cis/trans), 2.87-3.00 (m, 2H), 4.13-4.41 (m, 2H), 4.61-4.75 (m, 1H), 7.57 (s, 1H), 9.41 (br s, 1H), 12.71 (br s, 1H).

3) (S)-2-tert-Butoxycarbonyl-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 4

Compound 3 ( 2.73 g, 5.0 mmol) was dissolved in 30 mL of anhydrous methanol, and triethylamine (1.11 g, 11.0 mmol) and 10% palladium carbon (0.23 g) were added thereto. After reacting with hydrogen for 5 hours, the catalyst was filtered off with Celite, the methanol was evaporated, and then 1 mol/L citric acid solution was added to pH 4-5, and then extracted three times with ethyl acetate. The mixture was washed with brine, dried over anhydrous magnesium sulfate and evaporated. Yield: 75%, iH-NMR (DMSO-^e)^ 1.39+1.45 (s, 9H, cis/trans), 2.92-3.04 (m, 2H), 4.26-4.51 (m, 2H), 4.57-4.82 (m, 1H), 6.52 (s, 1H), 6.57 (d, ^8.4 Hz, 1H), 6.97 (d, 8.4 Hz, 1H), 9.28 (s, 1H), 12.60 (s, 1H).

4) (S)-2-tert-Butoxycarbonyl-7-hydroxy-3-(N-4-methoxyphenyl-amido)-1,2,3,4-tetrahydroisoquinoline 5 Compound 4 (2.93 g, 10.0 mmol) and 1-hydroxybenzotriazole (1.49 g, 11.0 mmol) were dissolved in 40 mL of anhydrous tetrahydrofuran, and dicyclohexylcarbodiimide (2.27 g, 11.0) was added dropwise under ice bath. Methyl) tetrahydrofuran solution. After 30 minutes, p-methoxyaniline (1.35 g, 11.0 mmol) was added and the reaction was continued at room temperature overnight. After the reaction, the tetrahydrofuran was distilled off, ethyl acetate was added, and the mixture was frozen in a refrigerator overnight to distill the dicyclohexylurea (DCU), dicyclohexylurea (DCU) was filtered off, and the filtrate was successively saturated with sodium carbonate solution, 1 mol. The /L hydrochloric acid solution and a saturated sodium chloride solution were washed, dried over anhydrous magnesium sulfate, and evaporated to give 2.76 g of yellow powder 5a. Yield: 69%, ESI-MS m/z: 399.2 [M+H ⁺ ] , ^! H-NMR (DMSO-^e) δ 1.30+1.45 (s, 9H, cis/trans), 2.85-3.11 (m , 2H), 3.70 (s, 3H), 4.24-4.54 (m, 2H), 4.60-4.78 (m, 1H), 6.58-6.64 (m, 2H), 6.85-6.88 (m, 2H), 7.00-7.03 (m, 1H), 7.39-7.47 (m, 2H), 9.30 (s, 1H), 9.82 (s, 1H).

5) (S)-2-tert-Butoxycarbonyl-7-(2-(methoxy)-2-carbonylethoxy)-3-(N-4-methoxyphenyl-amido)-1 , 2, 3, 4-tetrahydroisoquinoline 6

Add potassium carbonate powder (1.91 g, 13.86 mmol) and methyl bromoacetate (2.12 g, 13.86 mmol) to a solution of compound 5 (2.76 g, 6.93 mmol) in 40 mL of N, N-dimethylformamide. After the reaction for 3 hours, the reaction mixture was poured into 300 mL of water, and a large amount of the precipitate was precipitated, and the mixture was extracted with ethyl acetate. The mixture was combined with ethyl acetate and washed with saturated brine. The crude product was separated with a silica gel column ( petroleum ether: ethyl acetate = 3:1) to yield 1. Yield: 40%, ESI-MS m/z: 471.2 [M+H ⁺ ], -NMR (DMSO-^e) δ 1.33+1.45 (s, 9H, cis/trans), 2.91-3.14 (m, 2H), 3.70 (s, 3H), 3.71 (s, 3H), 4.32-4.37 (m, 2H), 4.54-4.65 (m, 1H), 4.77 (s, 2H), 6.72-6.88 (m, 4H), 7.08-7.16 (m, 1H), 7.39-7.46 (m, 2H), 9.85 (s, 1H).

6) (S)-7-(2-(Methoxy)-2-carbonylethoxy)-3-(N-4-methoxyphenyl-amido)-1, 2, 3, 4- Tetrahydroisoquinoline 7

To a solution of compound 6 (1.32 g, 2.8 mmol) in 10 mL of dichloromethane was added 4 mL of trifluoroacetic acid. After the reaction was finished, a saturated sodium carbonate solution was added to pH 7-8. The mixture was quenched and the organic layer was washed three times with distilled water, dried over anhydrous magnesium sulfate and evaporated. Yield: 85%, ESI-MS m/z -. 371.3 [M+H]+, ^! H-NMR (DMSO-^e) δ 2.72-2.76 (m, 1H), 2.88-2.91 (m, 1H) , 3.52-3.55 (m, 1H), 3.69 (s, 3H), 3.72 (s, 3H), 3.87-3.95 (m, 2H), 4.74 (s, 2H), 6.64-6.65 (m, 1H), 6.71 -6.72 (m, 1H), 6.88 (d, J = A Hz, 2H ), 7.04-7.06 (m, 1H), 7.57 (d, 8.4 Hz, 2H ), 9.75 (s, 1H).

7) (2S, 3S)-2-((tert-Butoxycarbonyl)amino)-3-methylpentanoic acid 8

L-isoleucine ( 1.31 g, 10.0 mmol) was dissolved in 11 mL of 1 mol/L sodium hydroxide solution, and 3 mL of di-tert-butyl carbonate (2.40 g, 11.0 mmol) in tetrahydrofuran was added. The pH of the reaction solution was controlled at 9-11 with a 1 mol/L sodium hydroxide solution during the reaction. After reacting at room temperature for 6 hours, the tetrahydrofuran in the reaction liquid was distilled off, and the reaction liquid was extracted three times with petroleum ether, and acidified to pH 4-5 with a 1 mol/L citric acid solution, and then extracted three times with ethyl acetate. After the mixture was washed with brine, dried over anhydrous magnesium sulfate Yield: 92%, ESI-MS m/z 232.2 [M+H ⁺ ], 3⁄4-NMR (DMSO-^e) 0.83-0.86 (m, 6H), 0.95 (s, 9H), 1.16-1.22 (m , 1H), 1.38-1.44 (m, 1H), 1.72-1.78 (m, 1H), 4.15 (dd, J= 8.4 Hz, J= 6.6 Hz, 1H), 7.83 (d, J= 8.4 Hz, 1H) , 12.46 (s, 1H).

8) 2-(((S)-2-((2S, 3S)-2-((tert-Butoxycarbonyl)amino)-3-methylpentanoyl)-3-((4-methoxyphenyl) Carbamoyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)acetic acid methyl ester 9

To a solution of the compound 8 (2.12 g, 9.2 mmol) in 40 mL of dry THF, EtOAc (EtOAc, EtOAc) Methylurea tetrafluoroborate (3.24 g, 10 mmol) was stirred at room temperature for 10 min then compound 7 (3.4 g, 9.2 mmol). After the completion of the reaction, the tetrahydrofuran was distilled off, ethyl acetate was added, and the mixture was washed with a saturated sodium carbonate solution, a 1 mol/L hydrochloric acid solution and a saturated sodium chloride solution, dried over anhydrous magnesium sulfate and evaporated to give 3.65 g of white powder. 9. Yield: 68%, ESI-MS m/z: 584.3 [M+H ⁺ ], 3⁄4-NMR (DMSO-) J 0.84-0.91 (m, 6H), 0.97 (s, 9H), 1.09-1.15 (m , 1H), 1.33-1.37 (m, 1H), 1.73-1.76 (m, 1H), 2.95-3.26 (m, 2H), 3.69 (s, 3H), 3.72 (s, 3H), 4.78 (s, 2H ), 4.83 (d, J = 15.6 Hz, 1H), 4.90 (d, J = 15.6 Hz, 1H), 4.64-4.67 (m, 1H), 5.14-5.16 (m, 1H), 6.73-6.89 (m, 4H), 7.08-7.09 (m, 1H), 7.50-7.52 (m, 2H), 8.14 (d, J = 8.4 Hz, 1H), 9.35 (s, 1H).

9) 2-(((S)-2-((2S, 3S)-2-((3,3-Dimethyl-butanoyl)amino)-3-methylpentanoyl)-3-((4) -Methoxyphenyl)carbamoyl)methyl 1,1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)acetate 10

To a solution of Compound 9 (3.65 g, 6.23 mmol) in 30 mL of dichloromethane was added 12 mL of trifluoroacetic acid. After the reaction was finished, an excess of triethylamine was added to the mixture until the mixture was used. To a solution of 3,3-dimethylbutyric acid (0.72 g, 6.23 mmol) in 10 mL of dry EtOAc (EtOAc) ^-Tetramethylurea tetrafluoroborate (1.58 g, 4.93 mmol), after stirring at room temperature for 10 minutes, the reaction solution was poured into dichloromethane. After reacting overnight, the solvent was evaporated, ethyl acetate was added, and washed with a saturated sodium carbonate solution, a 1 mol/L hydrochloric acid solution and a saturated sodium chloride solution, dried over anhydrous magnesium sulfate and evaporated to give a crude product. Column separation (petroleum ether: ethyl acetate = 1 : 1) gave 2.1 g of white powder. Yield: 58%, ESI-MS m/z-. 582.3 [M+H ⁺ ], <RTIgt;>H-NMR (DMSO-^e) O.83-0.91 (m, 6H), 0.96 (s, 9H), 1.09-1.15 (m, 1H), 1.33-1.37 (m, 1H), 1.73-1.76 (m, 1H), 2.01-2.05 (m, 1H), 2.16-2.20 (m, 1H), 2.95-3.22 (m , 2H), 3.69 (s, 3H), 3.71 (s, 3H), 4.77 (s, 2H), 4.83 (d, J = 15.6 Hz, 1H), 4.90 (d, J = 15.6 Hz, 1H), 4.64 -4.68 (m, 1H), 5.14-5.17 (m, 1H), 6.74-6.89 (m, 4H), 7.08-7.10 (m, 1H), 7.50-7.52 (m, 2H), 8.14 (d, J= 8.4 Hz, 1H), 9.34 (s, 1H).

10) (S)-2-((2S,3S)-2-(3,3-dimethylbutyramide)-3-methylpentanoyl)-7-(2-(hydroxylamine)-2-oxoethyl Oxy) _ _N _ ₍₄ _ methoxyphenyl) 4,2,3,4-tetrahydroisoquinoline-3-carboxamide ZYJ-D08a

Preparation of potassium hydroxyamine (NH ₂ OK) solution: 14 mL of saturated potassium hydroxide solution of potassium hydroxide was added dropwise to 24 mL of anhydrous methanol solution containing 4.67 g (67 mmol) of hydroxylamine hydrochloride to control the internal temperature below 40 °. C, after the dropwise addition is completed, the reaction solution is cooled, and the white potassium chloride precipitate is filtered off, and the obtained filtrate is sealed and stored for use.

After compound 10 (2.1 g, 3.61 mmol) was dissolved in 30 mL of anhydrous methanol, 6 mL of the above-mentioned potassium hydroxyamine (NH ₂ OK) solution was added thereto. After 0.5 hours, the methanol was distilled off, and the mixture was evaporated to dryness with EtOAc (EtOAc). The crude product was obtained, and the crude product was crystallized from ethanol to give a white powder (yield: 0.95 g) of white powder ZYJ-D08a. Yield: 45%, mp: 123-125 ° C; ^! H-NMR (DMSO-) (t, J =, 2 Hz, 3H), 0.92 (d, = 6.0 Hz,

3H), 0.97 (s, 9H), 1.09-1.15 (m, 1H), 1.33-1.37 (m, 1H), 1.73-1.76 (m, 1H), 2.03 (d, J = 12.6 Hz, 1H), 2.18 (d, J = 12.6 Hz, 1H), 2.95-2.99 (m, 1H), 3.21-3.26 (m, 1H), 3.69 (s, 3H), 4.42 (s, 2H), 4.83 (d, J = 15.6 Hz, 1H), 4.90 (d, J = 15.6 Hz, 1H), 4.64-4.67 (m, 1H), 5.14-5.16 (m, 1H), 6.73-6.89 (m, 4H), 7.08-7.09 (m, 1H), 7.50-7.52 (m, 2H), 8.14 (d, J= 8.4 Hz, 1H), 8.98 (s, 1H), 9.35 (s, 1H), 10.83 (s, 1H) ; HRMS (AP-ESI Scaled for C31H43N4O7 [M+H] ⁺ 583.3132, found 583.3165.

The synthesis method of the epimer ZYJ-D08ae of ZYJ-D08a is similar to that of ZYJ-D08a except that the optically pure D-isolysine (D-aile) is substituted for L-isoleucine (L). -ile) reacts as a raw material. ZYJ-D08ae mp: 116-118. C; 3⁄4-NMR (DMSO-ώέ) ^0.85 (t, J = 12 Hz, 3H), 0.90 (s, 9H), 0.93 (d, J = 6.6 Hz, 3H), 1.15-1.20 (m, 1H) , 1.54-1.57 (m, 1H), 1.74-1.90 (m, 1H), 1.96 (d, J = 12.6 Hz, 1H), 2.04 (d, J = 12.6 Hz, 1H), 2.92-3.06 (m, ( m, 2H), 3.70 (s, 3H), 4.43 (s, 2H), 4.70 (d, J = 15.6 Hz, 1H), 4.74-4.77 (m, 1H), 4.92-4.94 (m, 1H), 5.15 (d, J = 15.6 Hz, 1H), 6.74-6.91 (m, 4H), 7.11-7.14 (m, 1H), 7.40-7.41 (m, 2H), 8.01 (d, J = 8.4 Hz, 1H), 8.96 (s, 1H), 9.86 (s, 1H), 10.82 (s, 1H); HRMS (AP-ESI) / S caled for C31H43N4O7 [M+H] ⁺ 583.3132, found 583.3165.

Those skilled in the art can make changes to the above steps to improve the yield. They can determine the route of synthesis according to the basic knowledge in the field, such as selecting reactants, solvent and temperature, and can use various conventional protections. The base is protected from side reactions to increase the yield. These conventional methods of protection can be found, for example, in T. Greene, Protecting Groups in Organic Synthesis.

Obviously, the above route is a stereoselective synthesis, and an optically active peptoid compound can also be obtained by the above route. For example, the starting material 3, 5-diiodo-L-tyrosine is replaced by its optical isomer (D configuration). Various other isomers are readily available to those skilled in the art and can be purified by conventional separation means such as chiral salts or chiral columns.

The pharmacodynamic effects of the histone deacetylase inhibitors of the present invention are evaluated by an in vitro HDACs inhibitory assay, an in vitro anti-tumor cell proliferation assay, and an anti-human breast cancer and anti-colon cancer proliferation assay in nude mice. Specifically, there are the following steps:

Inhibition of histone deacetylase activity by target compounds in vitro

Histone deacetylase (HDACs) activity fluorescence analysis method is mainly divided into two steps: First step, fluorinated substrate of lysine HDACs containing one acetylated side chain (Boc-Lys (acetyl)-AMC), with group A sample of protein deacetylase (human cervical cancer Hela cell cells and extracts, HDAC6 and B HDAC8) is incubated to deacetylate the substrate and activate the substrate. In the second step, Boc-Lys-AMC is hydrolyzed by trypsin to produce 4-amino-7-methylcoumarin (AMC), a fluorescent group (ie, chromophore) at the emission wavelength/excitation wavelength (390 _n The fluorescence intensity was measured at m/460 nm), and the inhibition rate was calculated from the fluorescence intensity of the inhibitor group and the control group, and the IC _5Q value was calculated. The experimental results are shown in Table 1. Table 1. Results of in vitro inhibition studies of compounds

The value in table a is the average of three trials.

The SAHA trade name is Zolinza, commonly known as Vorinostat, a histone deacetylase inhibitor approved by the US Food and Drug Administration (FDA) in 2006.

The above test results show that the compound ZYJ-D08a exhibits better inhibitory activity against histone deacetylase than the positive control drug Vorinostat (SAHA), which has good development prospects and can be used as a novel high-efficiency histone deacetylase inhibitor. Lead compound of the agent. Activity test of target compound inhibiting cell proliferation in vitro

Compounds ZYJ-D08 and ZYJ-D08ae were tested for their activity in inhibiting cancer cell proliferation in vitro. The results are shown in Table 2.

Explanation of terms:

MDA-MB-231, MDA-MB-468, MDA-MB-435, HBL-100 are human breast cancer cell lines of different strains; HCT116 is a human colon cancer cell line.

SAHA: The trade name Zolinza, commonly known as Vorinostat, is a histone deacetylase inhibitor approved by the US Food and Drug Administration (FDA) in 2006.

DMSO: dimethyl sulfoxide.

ICso : half the inhibitory concentration.

1. [Materials] MDA-MB-231 , MDA-MB-468 , MDA-MB-435, HBL-100, HCT116 Cell line, MTT, MTT, 10% fetal bovine serum, 96-well plate

2. [Method]

Cell culture MDA-MB-231, MDA-MB-468, MDA-MB-435, HBL-100, HCT 116 Five tumor cell lines were cultured in a conventional manner. Logarithmic growth phase cells were used during the experiment.

Cell growth assay (MTT assay) MDA-MB-231, MDA-MB-468, MDA-MB-435, HBL-100, HCT116 cell suspension were adjusted to lxlOVml and seeded in 96-well plates (50 μΐ/well) , 5000 cells/well. After 4 hours of plating, 50 ul of medium containing different concentrations of compound was added to each well to make the final concentration of the compound in the well: 诵, 200, 40, 8, 1.6, 0.32 ug/ml, and three duplicate wells per concentration. When the cell-free well reading was taken, a blank was added, and the well without the compound was added as a compound blank, and SAHA was used as a compound positive control. Incubate for 48 h at 37 ° C in 5% carbon dioxide, add 10 μΐ 0.5% MTT staining solution to each well, continue to incubate for 4 h, centrifuge at 2500 rpm for 30 min, then discard the medium in the well and add dimethyl Sulfone, 200 ul / well. The absorbance OD value of each well was measured at 570 nm on a microplate reader, and the cell growth inhibition rate was calculated by the following formula:

Inhibition rate (%) ₌ entanglement average ODi straight - average shouting super

Control hole average OD value Table 2 Cell proliferation experiment results

The values in table a are the average of three trials.

The above table test data showed that the compounds ZYJ-D08a and ZYJ-D08ae showed comparable or even better activity to the positive control SAHA in the anti-tumor cell proliferation test in vitro, and had good development prospects. Proliferative activity of anti-human breast cancer cells MDA-MB-231 and anti-human colon cancer cells HCT116 in nude mice

In the present invention, the target compound is subjected to an in vivo anti-human breast cancer cell MDA-MB-231 proliferation activity test (see Fig. 2, Table 3) and an anti-human colon cancer cell HCT116 proliferation activity test (see Fig. 3, Table 4).

Human breast cancer MDA-MB-231 and human colon cancer cell HCT116 nude mouse xenograft tumor model were used to observe the target compound at different doses of oral (po) or intraperitoneal (ip) administration, and with the positive drug SAHA. The antitumor effect was compared.

An experimental grouping and dose design:

Test drugs: ZYJ-D08a and ZYJ-D08ae

Positive control: SAHA

Negative control group: equal amount of drug solvent

Tumor strain: human breast cancer cell line MDA-MB-231 , human colon cancer cell HCT116

Animals: BALB/c-nu mice, ? , 4 to 5 weeks old Experimental procedure: After the animal was purchased, the laboratory was balanced for 1 week, human breast cancer cell line MDA-MB-231 and one. £) J3⁄4: 3US-OIsasum US human. Colon cancer cell HCT116 was routinely cultured, 1640 medium It is cultured with 10% FBS, 5% CO 2, and 37 °C. Nude mice were inoculated with cells at 5 to 6 weeks old, ⁵ ×10 ⁶ cells/only, subcutaneously inoculated. Ten days after the inoculation, the nude mice were randomly divided into groups of 6 (each group of 6) when the tumor grew to near 100 mm ³ and administration was started. The drug was dissolved in PBS: DMSO (60:40), and the test group was administered in the dose and administration mode as shown in the following table. The DMSO control group was given an equal volume of solvent and administered orally for a period of time. At the end of the experiment, the mice were sacrificed by cervical vertebrae, the tumor was removed, the tumor weight was weighed, and the tumor inhibition rate was compared. The t test method was used to compare the statistical differences of tumor weight, tumor volume, RTV and other indicators in each group. Negative control group mean tumor weight - treatment group mean tumor weight

Tumor inhibition rate TGI ( % ) =

Negative control group mean tumor weight relative to tumor volume (RTV ) = Vt / Vo

Vt: tumor volume at the end of the experiment; Vo: tumor volume at the start of the experiment The evaluation index of antitumor activity is the relative tumor growth rate T/C (%),

Treatment group (T) RTV

T/C ( % ) =

Negative control group (C) RTV

1

7 10 13 16 19 22

Davs Figure 1 Growth curve of human breast cancer MDA-MB-231 in nude mice Table 3 Results of anti-human breast cancer MDA-MB-231 in vivo Compd tumor inhibition rate TGI relative tumor growth rate T/C

SAHA (90 mg/kg, ip) 43% 58%

SAHA (90 mg/kg, po) 43% 57%

ZYJ-D08a (90 mg/kg, ip) 51% 40%

ZYJ-D08ae (60 mg/kg, ip) 45% 62%

ZYJ-D08a (90 mg/kg, po) 66% 30% a Compared with the negative control group, the effect of all drug treatment groups was statistically significant by t test (PO.05)

1 4 7 10 13 16 19

Davs Figure 2 Growth curve of human colon cancer HCT116 in nude mice

'Compared with the negative control group, the effect of all drug treatment groups was statistically significant by t test (PO.05). The above test results indicated that ZYJ-D08a and its epimer ZYJ-D08ae act as histone deacetylase. Inhibitors have strong anti-tumor activity in vitro and in vivo, and have certain development and application prospects.

Claims

Rights request

a compound of the formula (I),

Among them, * is a stereo configuration of S or R optically pure or its racemate.

2. A compound according to claim 1 which is one of the following compounds:

Compound ZYJ-D08a, chemical name (S)-2-((2S,3S)-2-(3,3-dimethylbutyryl)-3-methyl-n-pentanoyl)-7-(2- (hydroxylamine)-2-carbonylethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline-3-amide, and its epimer ZYJ -D08ae, chemical name (S)-2-((2R,3S)-2-(3,3-dimethylbutyryl)-3-methyl-n-pentanoyl)-7-(2-(hydroxylamine) )-2-carbonylethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline-3-amide.

3. A method of preparing a compound according to claim 1, characterized by comprising the steps of:

Synthetic route 1:

Synthetic route 2: Optically pure 3, 5-diiodo-L-tyrosine is used as a raw material, followed by Pictet-Spengler cyclization to protect secondary amine groups, hydrogenation reduction and deiodination, and polypeptide condensation to p-methoxyaniline Group, nucleophilic reaction with methyl bromoacetate, removal of tert-butoxycarbonyl protecting group to obtain key intermediate 7; optically pure L-isoleucine (L-ile) as raw material, protected by primary amine group to obtain intermediate 11; Intermediates 7 and 11 were condensed, deprotected, N-acylated, and finally made of hydroxamic acid to give ZYJ-D08ae. The reaction formula is as follows:

Synthetic route 2:

The reagents in the above two synthetic routes: (a) paraformaldehyde, 37% hydrochloric acid, ethylene glycol dimethyl ether, 72-75 ° C, reaction for 18 hours; (b) di-tert-butyl carbonate, lmol / L sodium hydroxide solution, tetrahydrofuran; (c) 10% palladium carbon, hydrogen, methanol; (d) p-methoxyaniline, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, anhydrous tetrahydrofuran; (e) methyl bromoacetate, potassium carbonate, anhydrous N,N-dimethylformamide; (f) trifluoroacetic acid, dichloromethane, anhydrous potassium carbonate; ( _§ ) 0-benzotriazole ^,^^-tetramethylurea tetrafluoroborate, triethylamine, tetrahydrofuran; (h) l) trifluoroacetic acid, dichloromethane, anhydrous potassium carbonate; 2) 3,3-dimethylbutyric acid , 0-benzotriazole-oxime, oxime, Ν', Ν'-tetramethylurea tetrafluoroborate, triethylamine, tetrahydrofuran; (i) potassium hydroxyamine, anhydrous methanol.

Use of the compound of claim 1 for the manufacture of a medicament for preventing or treating a mammalian disease associated with aberrant expression of histone deacetylase activity; said mammal associated with abnormal expression of histone deacetylase activity Diseases include: cancer, neurodegenerative diseases, viral infections, inflammation, malaria and diabetes.