CN109384793A - A kind of sulfur alcohol compound and its application with HDAC6 inhibitory activity - Google Patents

Abstract

The invention belongs to the technical field of medicine, and relates to a thiol compound with antitumor activity, in particular to a thiol compound containing 6(7)-substituted-N-(6-mercaptohexyl)-pyrazolo[3,4-e]indium Thiol compounds of azole-3-carboxamide fragments and pyrazoloquinoline/isoquinoline fragments and pharmaceutically acceptable salts, hydrates thereof, and thiols comprising the compounds and pharmaceutically acceptable salts thereof, Pharmaceutical compositions with hydrates as active ingredients and their use in the preparation of histone deacetylase inhibitors for the preparation of medicines for the treatment and/or prevention of cancer. The structure of the compound of the present invention is shown in formula I: wherein, A, X and R are as described in the claims and description.

Description

A kind of thiol compound with HDAC6 inhibitory activity and its application

Technical field:

The invention belongs to the technical field of medicine, and relates to a thiol compound with antitumor activity, in particular to a thiol compound containing 6(7)-substituted-N-(6-mercaptohexyl)-pyrazolo[3,4-e]indium Thiol compounds of oxazole-3-carboxamide fragments and pyrazoloquinoline/isoquinoline fragments, and pharmaceutically acceptable salts, hydrates thereof, and compounds comprising the compounds and pharmaceutically acceptable salts thereof, Pharmaceutical compositions with hydrates as active ingredients and their use in the preparation of histone deacetylase inhibitors for the preparation of medicines for the treatment and/or prevention of cancer.

Background technique:

Epigenetic research is becoming the hope for mankind to conquer tumors. Epigenetic changes mostly occur in the early stage of tumorigenesis, when tumor cells have not caused substantial harm to the human body. Intervention at this time is likely to kill them in the cradle. In addition, compared to genetic modification, which is almost irreversible, epigenetic modification abnormalities can be reversed, and tumor cells return to a normal state. Therefore, epigenetic research has broader application prospects. Histone modification is an important way of epigenetic modification. Most human tumor cells have abnormal histone modification, which can cause tumor suppressor gene silencing and lead to tumor formation. Histone deacetylase (HDAC) is a family of enzymes that contains multiple members. There are currently 18 known subtypes, which are divided into the following four categories according to their phylogeny and homology with yeast: Rpd3, HoS1, HoSt2 homologous class I, including HDAC1, HDAC2, HDAC3, HDAC8; homologous to yeast Hda1, HoS3 class IIa, including HDAC4, HDAC5, HDAC7, HDAC9, class IIb including HDAC6, HDAC10; and yeast Sir2 Homologous class III, including SIRT1~SIRT7; and class I and II HDACs have partial homology, but their germlines are different, class IV, including HDAC11. Among them, class I, II, and IV are classical Zn ²⁺ -dependent HDACs, while class III belongs to the Sirtuin family and is NAD ⁺ -dependent HDACs. Studies have shown that class I and class II HDACs can inhibit tumor cell differentiation and apoptosis, promote tumor cell proliferation, etc., which are closely related to tumor occurrence and development. One of the hot spots.

Based on the references, the present invention designed and synthesized a series of fragments containing 6(7)-substituted-N-(6-mercaptohexyl)-pyrazolo[3,4-e]indazole-3-carboxamide Thiol compounds of and pyrazoloquinoline/isoquinoline fragments, and their pharmaceutically acceptable salts and hydrates, the in vitro antitumor activity test results show that they have good antitumor activity and exhibit excellent HDAC inhibition.

Invention content:

The present invention aims to provide a thiol compound with HDAC6 inhibitory activity and good antitumor activity, a preparation method thereof, and application of the compound as a histone deacetylase inhibitor in preventing and/or treating tumors . The compound specifically contains the following fragments: 6(7)-substituted-N-(6-mercaptohexyl)-pyrazolo[3,4-e]indazole-3-carboxamide fragment and pyrazoloquinoline/isoquinoline Fragment.

The present invention relates to compounds represented by formula I, and pharmaceutically acceptable salts and hydrates thereof:

in,

The A ring is a 5-6 membered aromatic ring or a heteroaromatic ring, and the heteroaromatic ring contains 1-3 heteroatoms of N, O or S; the A ring can be substituted by a substituent, and the substituent is H, halogen, (C ₁ -C ₆ )alkyl, halo(C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkoxy, (C ₁ -C ₆ )alkoxy (C ₁ -C ) ₆ ) alkyl, (C ₃ -C ₄ ) cycloalkyl, (C ₆ -C ₁₀ ) aryl, (C ₁ -C ₆ ) alkyl substituted phenyl, (C ₁ -C ₆ ) alkoxy substituted phenyl or benzyl, halogenated (C ₆ -C ₁₀ ) aryl, (C ₆ -C ₁₀ ) phenol aryl;

"---" represents single bond or double bond;

X is NH or O;

R is selected from one or more of the following substituents: H, (C ₂ -C ₄ ) acyl, (C ₁ -C ₆ ) alkyl, (C ₆ -C ₁₀ ) aryl substituted ester; (C ₆ -C ) ₁₀ ) Aryl; (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl; the aryl group may be replaced by halogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ ) Alkoxy, nitro, amino, hydroxyl substitution;

The present invention preferably defines the compound represented by formula I, and its pharmaceutically acceptable salts, hydrates:

in,

Ring A is selected from one or more of the following ring systems:

R ₁ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₄ )cycloalkyl, (C ₆ -C ₁₀ )aryl, (C ₁ -C ₄ )alkyl substituted Phenyl, (C ₁ -C ₄ )alkoxy-substituted phenyl or benzyl, halogenated (C ₆ -C ₁₀ ) aryl, (C ₆ -C ₁₀ )phenolaryl;

R ₂ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₄ )cycloalkyl, (C ₆ -C ₁₀ )aryl, (C ₁ -C ₄ )alkyl substituted Phenyl, (C ₁ -C ₄ )alkoxy-substituted phenyl or benzyl, halogenated (C ₆ -C ₁₀ ) aryl, (C ₆ -C ₁₀ )phenolaryl;

R ₃ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₄ )cycloalkyl, (C ₆ -C ₁₀ )aryl, (C ₁ -C ₄ )alkyl substituted Phenyl, (C ₁ -C ₄ )alkoxy-substituted phenyl or benzyl, halogenated (C ₆ -C ₁₀ ) aryl, (C ₆ -C ₁₀ )phenolaryl;

R is selected from one or more of the following substituents: H, (C ₂ -C ₄ ) acyl, (C ₁ -C ₆ ) alkyl, (C ₆ -C ₁₀ ) aryl substituted ester; (C ₆ -C ) ₁₀ ) Aryl; (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl; the aryl group may be replaced by halogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ ) Alkoxy, nitro, amino, hydroxyl substitution;

"---" represents single bond or double bond;

X is NH or O.

The present invention preferably defines compounds of formula I, and their pharmaceutically acceptable salts, hydrates:

in,

Ring A is preferably:

R ₁ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₄ )cycloalkyl, (C ₆ -C ₁₀ )aryl, (C ₁ -C ₄ )alkyl substituted Phenyl, (C ₁ -C ₄ )alkoxy-substituted phenyl or benzyl, halogenated (C ₆ -C ₁₀ ) aryl, (C ₆ -C ₁₀ )phenolaryl;

R ₂ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl, (C ₃ -C ₄ )cycloalkyl, (C ₆ -C ₁₀ )aryl, (C ₁ -C ₄ )alkyl substituted Phenyl, (C ₁ -C ₄ )alkoxy-substituted phenyl or benzyl, halogenated (C ₆ -C ₁₀ ) aryl, (C ₆ -C ₁₀ )phenolaryl;

R is selected from one or more of the following substituents: H, (C ₂ -C ₄ ) acyl, (C ₁ -C ₄ ) alkyl, (C ₆ -C ₁₀ ) aryl substituted ester; (C ₆ -C ) ₁₀ ) Aryl; (C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl; the aryl group may be replaced by halogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ ) Alkoxy, nitro, amino, hydroxyl substitution;

"---" represents single bond or double bond;

X is NH or O.

The present invention also preferably defines compounds of formula I, and their pharmaceutically acceptable salts, hydrates:

in,

Ring A is preferably

R ₁ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl;

R ₂ is selected from one or more of the following substituents: H, halogen, (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, ( C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl;

R is selected from one or more of the following substituents: H, (C ₂ -C ₄ ) acyl, (C ₁ -C ₄ ) alkyl, (C ₆ -C ₁₀ ) aryl substituted ester; phenyl; benzyl ; (C ₁ -C ₄ )alkoxy(C ₁ -C ₄ )alkyl; the phenyl or benzyl group may be replaced by halogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkane Oxy, nitro, amino or hydroxy substitution;

"---" represents single bond or double bond;

X is NH or O.

The present invention also preferably defines compounds of formula I, and their pharmaceutically acceptable salts, hydrates:

in,

Ring A is preferably

R ₁ and R ₂ are the following substituents: H, methyl, ethyl, methoxyethyl, ethoxyethyl, cyclopropylmethyl, benzyl, p-fluorobenzyl, p-methoxybenzyl, p-hydroxyl benzyl;

R is selected from one or more of the following substituents: H, (C ₂ -C ₄ ) acyl, (C ₁ -C ₆ ) alkyl, (C ₆ -C ₁₀ ) aryl substituted ester; phenyl; benzyl ; (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl; the phenyl or benzyl group may be replaced by halogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkane Oxy, nitro, amino or hydroxy substitution;

"---" represents single bond or double bond;

X is NH or O.

The present invention also particularly preferably defines compounds of formula I, and their pharmaceutically acceptable salts, hydrates:

in,

Ring A is preferably

R ₁ and R ₂ are preferably the following substituents: H, methyl, ethyl, methoxyethyl, ethoxyethyl, cyclopropylmethyl, benzyl, p-fluorobenzyl, p-methoxybenzyl, p- hydroxybenzyl;

R is preferably H or acetyl;

"---" represents single bond or double bond;

X is NH or O.

The present invention also particularly preferably defines compounds of formula I, and their pharmaceutically acceptable salts, hydrates:

in,

Ring A is preferably

R ₁ and R ₂ are preferably the following substituents: H, methyl, ethyl, methoxyethyl, ethoxyethyl, cyclopropylmethyl, benzyl, p-fluorobenzyl, p-methoxybenzyl, p- hydroxybenzyl;

R is preferably H or acetyl;

Y is preferably -CH ₂ -CH ₂ - or -CH=CH-;

X is NH or O.

Specifically, the present invention preferably the following compounds:

N-(6-Mercaptohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-5,6-dihydro-4H-isoxazolo[5,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-5,6-dihydro-4H-isoxazolo[5,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-7-methyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-methyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-6-methyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-methyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-7-benzyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-benzyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-6-benzyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-benzyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-7-(4-fluorobenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-(4-fluorobenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-6-(4-fluorobenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-(4-fluorobenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-7-(4-methoxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-(4-methoxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-6-(4-methoxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-(4-methoxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-7-(4-hydroxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-(4-hydroxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-6-(4-hydroxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-(4-hydroxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-6(7)H-isoxazolo[5,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6(7)H-isoxazolo[5,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Methyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Methyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Methyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Methyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-ethyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Ethyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Ethyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Ethyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Methoxyethyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Methoxyethyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Methoxyethyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Methoxyethyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Cyclopropylmethyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

7-Cyclopropylmethyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Cyclopropylmethyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

6-Cyclopropylmethyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Mercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Mercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

N-(6-Acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

N-(6-Mercaptohexyl)-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Acetylmercaptohexyl)-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Mercaptohexyl)-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

N-(6-Acetylmercaptohexyl)-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

N-(6-Mercaptohexyl)-7-ethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-ethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-6-ethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-ethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-7-methoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-methoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-6-methoxyethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-methoxyethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-7-ethoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-ethoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-6-ethoxyethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-ethoxyethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-7-cyclopropylmethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-7-cyclopropylmethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Thiahexyl)-6-cyclopropylmethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-6-cyclopropylmethyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

or its hydrates, solvates, metabolites and pharmaceutically acceptable salts or its prodrugs.

In addition, the present invention also includes prodrugs of the compounds of the present invention. According to the present invention, prodrugs are compounds of formula I, which may themselves have weak or even no activity, but which, after administration, are converted under physiological conditions (for example by metabolism, solvolysis or otherwise) into the corresponding the biologically active form.

The present invention includes a pharmaceutical composition comprising a fragmented thiol compound encompassed by the above formula I and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipients refer to any diluents, adjuvants and/or carriers that can be used in the pharmaceutical field. The compounds of the present invention can be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions.

The pharmaceutical composition of the present invention can be formulated into several dosage forms, which contain some excipients commonly used in the pharmaceutical field, for example, oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as Injectable solutions or suspensions, or injectable dry powders, ready for immediate use by adding water for injection before injection); topical preparations (eg, ointments or solutions).

The carriers used in the pharmaceutical composition of the present invention are common types available in the pharmaceutical field, including: binders, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents, pigments, Flavoring agents, etc.; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for topical preparations. Pharmaceutical formulations may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically), and if certain drugs are unstable under gastric conditions, they may be formulated as enteric-coated tablets.

Through in vitro enzyme inhibition test screening, we found that the compound of the present invention can inhibit the activity of histone deacetylase. Therefore, the compound of the present invention can be used for preparing the application in the treatment of diseases related to the abnormal expression of histone deacetylase activity, such as various kind of cancer.

Through in vitro activity screening and in vivo pharmacodynamic studies, we found that the compound of the present invention has antitumor activity, so the compound of the present invention can be used to prepare medicines for the treatment and/or prevention of various cancers, such as breast, lung, colon, rectum, stomach , prostate, bladder, uterine, pancreatic and ovarian cancers.

The active compounds of the present invention can be used as the sole anti-cancer drug, or in combination with one or more other anti-tumor drugs. Combination therapy is accomplished by administering the individual therapeutic components simultaneously, sequentially or separately.

The examples and preparations provided hereinafter further illustrate and illustrate the compounds of the present invention and methods for their preparation. It should be understood that the scope of the following examples and preparations do not limit the scope of the invention in any way.

The following synthetic schemes describe the preparation of compounds of formula I of the present invention, all starting materials were prepared by methods described in these schemes, by methods well known to those of ordinary skill in the art of organic chemistry, or are commercially available. All final compounds of this invention are prepared by methods described in these schemes or by methods analogous thereto, which are well known to those of ordinary skill in the art of organic chemistry. All variable factors applied in these routes are as defined below or as defined in the claims.

According to the compound of formula I of the present invention, when A ring is a (substituted) pyrazole ring, and "---" is a double bond, the target compound is prepared according to the method of route 1, and each substituent is as defined in the content of the invention.

Reagents and conditions: (a) DMF-DMA, neat, flux, 1h; (b) NH ₂ NH ₂ .HCl, MeOH, flux. 2h; (c) RX, K ₂ CO ₃ or KOH, MeCN, flux, 2h ; (d) dimethyl oxalate, LiHMDs, THF, rt, 4h; (e) NH ₂ NH ₂ .H ₂ O, AcOH, 80°C, 2h; (f) NaOH, H ₂ O, 50°C, 2h; (g) )6-bromohexan-1-amine,EDCI,HOBt,DMF,rt,24h;(h)KSAc,EtOH,reflux,4h;(i)DDQ,dioxane,reflux,4h;(j)LiOH,MeOH,rt, 1h.

When the A ring is a (substituted) pyrazole ring and "---" is a single bond, the target compound is prepared according to the method of route two. Each other substituent is as defined in the Summary of the Invention.

Reagents and conditions: (a) DMF-DMA, neat, flux, 1h; (b) NH ₂ NH ₂ .HCl, MeOH, flux. 2h; (c) RX, K ₂ CO ₃ or KOH, MeCN, flux, 2h ; (d) dimethyl oxalate, LiHMDs, THF, rt, 4h; (e) NH ₂ NH ₂ .H ₂ O, AcOH, 80°C, 2h; (f) NaOH, H ₂ O, 50°C, 2h; (g) ) 6-bromohexan-1-amine, EDCI, HOBt, DMF, rt, 24h; (h) KSAc, EtOH, reflux, 4h; (i) LiOH, MeOH, rt, 1h.

When the A ring is a pyridine ring, and "---" is a single bond or a double bond, the target compound is prepared according to the method of route three. Each other substituent is as defined in the Summary of the Invention.

Reagents and conditions: (a) KMnO ₄ , CH ₃ COOH, rt; (b) dimethyl oxalate, LiHMDs, THF, rt, 4h; (c) NH ₂ NH ₂ .H ₂ O, CH ₃ COOH, 80°C, 0.5 h;(d)NaOH(aq),50℃,1h;(e)6-bromohexan-1-amine,EDCI,HOBt,DMF,rt,overnight;(f)KSAc,C ₂ H ₅ OH,reflux,1h ;(g) _LiOH (aq),CH3OH,rt,4h;(h)DDQ,1,4-dioxane,reflux,2h.

The preparation method of the invention has simple operation and mild conditions, and the obtained compounds all have histone deacetylase inhibitory activity and have remarkable antitumor effect.

Detailed ways:

The present invention will be described in detail below through specific examples, but the purposes and purposes of these exemplary embodiments are only used to illustrate the present invention, and do not constitute any form of limitation on the actual protection scope of the present invention, nor do they limit the present invention. The scope of protection is limited to this.

Example 1: Preparation of N-(6-mercaptohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

Step A: Preparation of 2-(dimethylamino)methylene-1,3-cyclohexanedione

1,3-cyclohexanedione (50 mmol) and N,N-dimethylformamide dimethyl acetal (DMF-DMA) (100 mmol) were added to a 250 mL eggplant-shaped flask, and heated under reflux at 95° C. for 1 h. Subsequently, the unreacted DMF-DMA was recovered by distillation under reduced pressure to obtain a brown solid, which was subjected to the next reaction without purification. Yield 100%. LC-MS m/z: 168.2 [M+H] ⁺ .

Step B: Preparation of 2,5,6,7-tetrahydro-4H-indazol-4-one

2-(dimethylamino)methylene-1,3-cyclohexanedione (50 mmol) was dissolved in methanol (100 mL), then hydrazine hydrochloride (55 mmol) was added, and the reaction was heated at 60° C. for 2 h. Subsequently, the reaction insolubles were removed, and the filtrate was evaporated to dryness to obtain a brown solid, which was subjected to the next reaction without purification. Yield 100%. LC-MS m/z: 137.2[M+H] ⁺ .

Step C: Preparation of 2-triphenylmethyl-6,7-dihydro-2H-indazol-4(5H)-one

2,5,6,7-Tetrahydro-4H-indazol-4-one (10 mmol) was dissolved in dichloromethane (50 mL), followed by addition of triethylamine (20 mmol), triphenylmethyl chloride (11 mmol) ), heated at 80°C for 4h. Subsequently, the reaction solvent was removed, and the petroleum ether was recrystallized to obtain a white solid. Yield 88%. LC-MS m/z: 179.3[M+H] ⁺ .

Step D: Preparation of methyl 1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylate

2-Triphenylmethyl-6,7-dihydro-2H-indazol-4(5H)-one (10 mmol) was dissolved in tetrahydrofuran (50 mL), followed by adding dimethyl oxalate (12 mmol), slowly dropping LiHMDS (1M in THF, 12mmol) was added, and the reaction was carried out at room temperature for 4h. The reaction solution was removed, redissolved in acetic acid (50 mL), hydrazine hydrate (80%, 12 mmol) was added, and the reaction was heated at 80° C. for 4 h. After the reaction solution was cooled to room temperature, the reaction solution was poured into water (100 mL), a large amount of solid was precipitated, and the filter cake was collected by suction filtration to obtain a white solid. Yield 62%. LC-MS m/z: 219.1[M+H] ⁺ .

Step E: Preparation of N-(6-Acetylthiohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

Methyl 1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylate (8 mmol) was dissolved in methanol (10 mmol) and an aqueous solution of sodium hydroxide (10 mmol) was added , heated at 50 ℃ for 4h. After the reaction solution was cooled to room temperature, adjusted to pH=2 with 0.5M hydrochloric acid, a large amount of solid was precipitated, suction filtered, the filter cake was collected, and dried in vacuum. Subsequently, it was redissolved in DMF (50 mL), and HOBt (10 mmol), 6-bromo-1-hexylamine (10 mmol) and EDCI (10 mmol) were successively added under ice bath, and the reaction was carried out at room temperature for 24 h. Then the reaction solution was poured into water (100 mL), extracted with ethyl acetate (60 mL×3), the organic layers were combined, backwashed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Subsequently, it was redissolved in ethanol (50 mL), potassium thioacetate (10 mmol) was added, and the reaction was heated at 60° C. for 4 h. The solvent was removed and separated by silica gel column chromatography to obtain a white solid. Yield 42%. LC-MS m/z: 362.3[M+H] ⁺ .

Step F: Preparation of N-(6-Acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide (5 mmol) was dissolved in dioxane ( 50 mL), added dichlorodicyanobenzoquinone (DDQ, 10 mmol), heated at 100 °C and reacted for 2 h. After the reaction solution was cooled to room temperature, saturated sodium bicarbonate was added to quench the reaction, the reaction solution was poured into water (100 mL), extracted with ethyl acetate (60 mL×3), the organic layers were combined, backwashed with saturated sodium chloride, and anhydrous sulfuric acid. Dry over sodium and separate by silica gel column chromatography to obtain a white solid. Yield 58%. LC-MS m/z: 360.2[M+H] ⁺ .

Step G: Preparation of N-(6-mercaptohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

N-(6-Acetylthiohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide (3 mmol) was dissolved in methanol (10 mL), Lithium hydroxide solution (1.2M, 3.3mmol) was added, and the reaction was carried out at room temperature for 4h. Then adjust pH=2 with 0.5M hydrochloric acid, pour the reaction solution into a large amount of water (100 mL), extract with ethyl acetate (60 mL×3), combine the organic layers, dry over anhydrous sodium sulfate, and separate by silica gel column chromatography. A white solid was obtained. Yield 61%. LC-MS m/z: 320.0[M+H] ⁺ .

According to the preparation method of Example 1, select appropriate raw materials to prepare the compounds of Example 2-Example 60.

Example 2: Preparation of N-(6-acetylthiohexyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 362.3[M+H] ⁺ .

Example 3: Preparation of N-(6-mercaptohexyl)-5,6-dihydro-4H-isoxazolo[5,4-e]indazole-3-carboxamide

LC-MS m/z: 320.1[M+H] ⁺ .

Example 4: Preparation of N-(6-acetylthiohexyl)-5,6-dihydro-4H-isoxazolo[5,4-e]indazole-3-carboxamide

LC-MS m/z: 363.2[M+H] ⁺ .

Example 5: Preparation of N-(6-mercaptohexyl)-7-methyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 333.2[M+H] ⁺ .

Example 6: Preparation of N-(6-acetylthiohexyl)-7-methyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 376.3[M+H] ⁺ .

Example 7: Preparation of N-(6-mercaptohexyl)-6-methyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 333.2[M+H] ⁺ .

Example 8: Preparation of N-(6-acetylthiohexyl)-6-methyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 376.3[M+H] ⁺ .

Example 9: Preparation of N-(6-mercaptohexyl)-7-benzyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 409.2[M+H] ⁺ .

Example 10: Preparation of N-(6-acetylthiohexyl)-7-benzyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 452.3[M+H] ⁺ .

Example 11: Preparation of N-(6-mercaptohexyl)-6-benzyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 409.2[M+H] ⁺ .

Example 12: Preparation of N-(6-acetylthiohexyl)-6-benzyl-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 452.3[M+H] ⁺ .

Example 13: N-(6-Mercaptohexyl)-7-(4-fluorobenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 427.2[M+H] ⁺ .

Example 14: N-(6-Acetylthiohexyl)-7-(4-fluorobenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3- Preparation of formamide

LC-MS m/z: 470.3[M+H] ⁺ .

Example 15: N-(6-Mercaptohexyl)-6-(4-fluorobenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 427.2[M+H] ⁺ .

Example 16: N-(6-Acetylthiohexyl)-6-(4-fluorobenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3- Preparation of formamide

LC-MS m/z: 470.3[M+H] ⁺ .

Example 17: N-(6-Mercaptohexyl)-7-(4-methoxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3- Preparation of formamide

LC-MS m/z: 439.1[M+H] ⁺ .

Example 18: N-(6-Acetylthiohexyl)-7-(4-methoxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole- Preparation of 3-formamide

LC-MS m/z: 482.2[M+H] ⁺ .

Example 19: N-(6-Mercaptohexyl)-6-(4-methoxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3- Preparation of formamide

LC-MS m/z: 439.1[M+H] ⁺ .

Example 20: N-(6-Acetylthiohexyl)-6-(4-methoxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole- Preparation of 3-formamide

LC-MS m/z: 482.2[M+H] ⁺ .

Example 21: N-(6-Mercaptohexyl)-7-(4-hydroxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 425.1[M+H] ⁺ .

Example 22: N-(6-Acetylthiohexyl)-7-(4-hydroxybenzyl)-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3- Preparation of formamide

LC-MS m/z: 468.2[M+H] ⁺ .

Example 23: N-(6-Mercaptohexyl)-6-(4-hydroxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 425.1[M+H] ⁺ .

Example 24: N-(6-Acetylthiohexyl)-6-(4-hydroxybenzyl)-1,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3- Preparation of formamide

LC-MS m/z: 468.2[M+H] ⁺ .

Example 25: Preparation of N-(6-mercaptohexyl)-7-ethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 346.0 [MH] ⁺ .

Example 26: Preparation of N-(6-acetylthiohexyl)-7-ethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 389.1[M+H] ⁺ .

Example 27: Preparation of N-(6-thiohexyl)-6-ethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 346.0[M+H] ⁺ .

Example 28: Preparation of N-(6-acetylthiohexyl)-6-ethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 389.1[M+H] ⁺ .

Example 29: Preparation of N-(6-thiohexyl)-7-methoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 376.0 [MH] ⁺ .

Example 30: N-(6-Acetylthiohexyl)-7-methoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 420.1[M+H] ⁺ .

Example 31: Preparation of N-(6-thiohexyl)-6-methoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 376.0 [MH] ⁺ .

Example 32: N-(6-Acetylthiohexyl)-6-methoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 420.1[M+H] ⁺ .

Example 33: Preparation of N-(6-thiohexyl)-7-ethoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 390.1 [MH] ⁺ .

Example 34: N-(6-Acetylthiohexyl)-7-ethoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 434.2[M+H] ⁺ .

Example 35: Preparation of N-(6-thiohexyl)-6-ethoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 390.1 [MH] ⁺ .

Example 36: N-(6-Acetylthiohexyl)-6-ethoxyethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 434.2[M+H] ⁺ .

Example 37: Preparation of N-(6-thiohexyl)-7-cyclopropylmethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 372.0 [MH] ⁺ .

Example 38: N-(6-Acetylthiohexyl)-7-cyclopropylmethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 416.3[M+H] ⁺ .

Example 39: Preparation of N-(6-thiohexyl)-6-cyclopropylmethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 372.0 [MH] ⁺ .

Example 40: N-(6-Acetylthiohexyl)-6-cyclopropylmethyl-1,4,5,7-tetrahydropyrazolo[3,4-e]indazole-3-carboxamide preparation

LC-MS m/z: 416.3[M+H] ⁺ .

Example 41: Preparation of N-(6-mercaptohexyl)-6(7)H-isoxazolo[5,4-e]indazole-3-carboxamide

LC-MS m/z: 319.0[M+H] ⁺ .

Example 42: Preparation of N-(6-acetylthiohexyl)-6(7)H-isoxazolo[5,4-e]indazole-3-carboxamide

LC-MS m/z: 361.2[M+H] ⁺ .

Example 43: Preparation of N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 318.1[M+H] ⁺ .

Example 44: Preparation of N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 360.2[M+H] ⁺ .

Example 45: Preparation of 7-methyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 332.0[M+H] ⁺ .

Example 46: Preparation of 7-methyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 374.3[M+H] ⁺ .

Example 47: Preparation of 6-methyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 332.1[M+H] ⁺ .

Example 48: Preparation of 6-methyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 374.3[M+H] ⁺ .

Example 49: Preparation of 7-ethyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 346.3[M+H] ⁺ .

Example 50: Preparation of 7-ethyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 388.2[M+H] ⁺ .

Example 51: Preparation of 6-ethyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 346.2[M+H] ⁺ .

Example 52: Preparation of 6-ethyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 388.3[M+H] ⁺ .

Example 53: Preparation of 7-methoxyethyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 376.0[M+H] ⁺ .

Example 54: Preparation of 7-methoxyethyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 418.2[M+H] ⁺ .

Example 55: Preparation of 6-methoxyethyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 376.1[M+H] ⁺ .

Example 56: Preparation of 6-methoxyethyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 418.3[M+H] ⁺ .

Example 57: Preparation of 7-cyclopropylmethyl-N-(6-mercaptohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 372.3[M+H] ⁺ .

Example 58: Preparation of 7-cyclopropylmethyl-N-(6-acetylthiohexyl)-1,7-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 414.2[M+H] ⁺ .

Example 59: Preparation of 6-cyclopropylmethyl-N-(6-mercaptohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 372.2[M+H] ⁺ .

Example 60: Preparation of 6-cyclopropylmethyl-N-(6-acetylthiohexyl)-1,6-dihydropyrazolo[3,4-e]indazole-3-carboxamide

LC-MS m/z: 414.3[M+H] ⁺ .

Example 61: Preparation of N-(6-mercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

Step A: Preparation of 6,7-dihydroisoquinolin-8(5H)-one

Dissolve 5,6,7,8-tetrahydroisoquinoline in acetic acid, add 1.2 equivalents of potassium permanganate, stir at room temperature for 8 hours, add water to the reaction solution, extract with a large amount of dichloromethane, and quickly Medium pressure preparation liquid phase separation and purification to obtain a colorless viscous oily substance with a yield of 15%. LC-MS m/z: 148.1 [M+H] ⁺ .

Step B: Preparation of methyl 4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxylate

6,7-Dihydroisoquinolin-8(5H)-one (10 mmol) was dissolved in tetrahydrofuran (50 mL), then dimethyl oxalate (12 mmol) was added, and LiHMDS (1 M in THF, 12 mmol) was slowly added dropwise, The reaction was carried out at room temperature for 4h. The reaction solution was removed, redissolved in acetic acid (50 mL), hydrazine hydrate (80%, 12 mmol) was added, and the reaction was heated at 80° C. for 1 h. After the reaction solution was cooled to room temperature, the reaction solution was poured into water (100 mL), a large amount of solid was precipitated, suction filtration, and the filter cake was collected to obtain a crude product, which was subjected to liquid phase separation and purification by rapid medium-pressure preparation to obtain a pale yellow solid with a yield of 60 %. LC-MS m/z: 230.2 [M+H] ⁺ .

Step C: Preparation of N-(6-bromohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

Methyl 4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxylate (8 mmol) was dissolved in methanol (10 mmol) and sodium hydroxide (0.1 M, 10 mmol) was added ) solution, heated at 50 °C for 1 h. After the reaction solution was cooled to room temperature, the pH was adjusted to 3 with 0.5M hydrochloric acid, a large amount of solid was precipitated, and the filter cake was collected by suction filtration and dried in vacuum. Subsequently, it was redissolved in DMF (50 mL), the pH was adjusted to weakly basic (7-8) with triethylamine, and HOBt (10 mmol), 6-bromo-1-hexylamine (10 mmol), EDCI (10 mmol) were added successively under ice bath. ) and reacted at room temperature for 24h. Subsequently, the reaction solution was poured into water (100 mL), extracted with ethyl acetate (60 mL×3), the organic layers were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and subjected to rapid medium-pressure preparative liquid phase separation and purification to obtain Pale yellow oily viscous material, yield 55%. LC-MS m/z: 377.1 [M+H] ⁺ .

Step D: Preparation of N-(6-Acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Bromohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide was dissolved in ethanol and potassium thioacetate (10 mmol) was added , heated at 60 °C for 1 h. The solvent was removed, and a light yellow oily viscous substance was obtained by rapid medium pressure preparative liquid phase separation and purification, and the yield was 91%. LC-MS m/z: 373.4 [M+H] ⁺ .

Step E: Preparation of N-(6-Acetylmercaptohexyl)-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide (5 mmol) was dissolved in dioxane ( 50 mL), added dichlorodicyanobenzoquinone (DDQ, 10 mmol), heated at 100 °C and reacted for 2 h. After the reaction solution was cooled to room temperature, saturated sodium bicarbonate was added to quench the reaction, the reaction solution was poured into water (100 mL), extracted with ethyl acetate (60 mL×3), the organic layers were combined, backwashed with saturated sodium chloride, and anhydrous sulfuric acid. Dry with sodium and separate by silica gel column chromatography to obtain a white solid; the yield is 75%. LC-MS m/z: 371.1 [M+H] ⁺ .

Step F: Preparation of N-(6-Mercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

N-(6-Acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide (3 mmol) was dissolved in ethanol (10 mL), Lithium hydroxide solution (1.2M, 3.3mmol) was added, and the reaction was carried out at room temperature for 4h. Then adjust pH=2 with 0.5M hydrochloric acid, pour the reaction solution into a large amount of water (100mL), extract with ethyl acetate (60mL×3), combine the organic layers, dry over anhydrous sodium sulfate, and prepare liquid phase separation and purification by rapid medium pressure, A pale yellow solid was obtained with a yield of 45%. LC-MS m/z: 328.8 [MH] ^- .

According to the preparation method of Example 61, select appropriate raw materials to prepare the compounds of Example 62-Example 68.

Example 62: Preparation of N-(6-acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

LC-MS m/z: 373.2[M+H] ⁺ .

Example 63: Preparation of N-(6-mercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

LC-MS m/z: 329.4 [MH] ^- .

Example 64: Preparation of N-(6-acetylmercaptohexyl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

LC-MS m/z: 373.4[M+H] ⁺ .

Example 65: Preparation of N-(6-mercaptohexyl)-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

LC-MS m/z: 327.0 [MH] ^- .

Example 66: Preparation of N-(6-acetylmercaptohexyl)-1H-pyrazolo[4,3-h]isoquinoline-3-carboxamide

LC-MS m/z: 371.1[M+H] ⁺ .

Example 67: Preparation of N-(6-mercaptohexyl)-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

LC-MS m/z: 372.2 [MH] ^- .

Example 68: Preparation of N-(6-acetylmercaptohexyl)-1H-pyrazolo[4,3-h]quinoline-3-carboxamide

LC-MS m/z: 371.0[M+H] ⁺ .

Example 69. Pharmacological studies on the products of the present invention

A positive control group (vorinostat and ACY-1215) was set up in the experiment. HeLa cell nuclear extract (Enzo LifeSciences, USA) was used as HDACs enzyme source, and recombinant rHDAC1, 2, 3 and rHDAC6 proteins (BPS Bioscience, USA) were used as subtype enzyme source. All reactions were performed in 96-well plates. The reaction buffer contained 25 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl ₂ and 0.1 mg/mL BSA. Compound solution (5 μL) and enzyme (5 μL) with gradient concentration were pre-incubated at 25°C for 15 min, and then the fluorescent substrate Boc–Lys(Ac)–AMC (5 μL) was added to initiate the reaction, and incubated at 37°C for 60 min. Finally, stop buffer (25 μL) containing Trypsin and SAHA was added to the system and incubated for another 10 min. Fluorescence intensity was measured at excitation wavelength 355 nm and emission wavelength 460 nm (Thermo Scientific Varioskan Flash Station). _IC50 was calculated with GraphPad. The inhibitory activities of target compounds on HDAC are shown in Table 1.

Table 1 Inhibitory activity of target compounds on HDAC enzymes

^a ND stands for no activity, no activity detected, or activity for the subtype not tested.

^bMeasured results are the mean of at least two independent determinations, with standard deviation expressed as _IC50 ±SD.

The above experimental results show that the compound of the general formula to be protected by the present invention has good antitumor activity and HDAC inhibitory effect. The compounds of the present invention have good industrial application prospects.

Claims (10)

1. Thiol compounds having HDAC6 inhibitory activity represented by formula I:

wherein,

ring a is a 5-6 membered aromatic or heteroaromatic ring containing 1-3 heteroatoms of N, O or S; the A ring may be substituted with a substituent selected from the group consisting of H, halogen and (C)₁-C₆) Alkyl, halo (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Alkoxy (C)₁-C₆) Alkyl, (C)₃-C₄) Cycloalkyl group, (C)₆-C₁₀) Aryl group, (C)₁-C₆) Alkyl substituted phenyl, (C)₁-C₆) Alkoxy-substituted phenyl or benzyl, halo (C)₆-C₁₀) Aryl group, (C)₆-C₁₀) A phenolic aryl group;

"- - -" represents a single bond or a double bond;

x is NH or O;

r is selected from one or more of the following substituents: H. (C)₂-C₄) Acyl, (C)₁-C₆) Alkyl, (C)₆-C₁₀) An aryl-substituted ester group; (C)₆-C₁₀) An aryl group; (C)₁-C₆) Alkoxy (C)₁-C₆) An alkyl group; the aryl group may be substituted by halogen, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy, nitro, amino and hydroxyl.

2. The thiol compound having HDAC6 inhibitory activity of claim 1 and a pharmaceutically acceptable salt or hydrate thereof:

wherein,

ring a is selected from one or more of the following ring systems:

wherein,

R₁selected from one or more of the following substituents: H. halogen, (C)₁-C₄) Alkyl, halo (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Alkoxy (C)₁-C₄) Alkyl, (C)₃-C₄) Cycloalkyl group, (C)₆-C₁₀) Aryl group, (C)₁-C₄) Alkyl substituted phenyl, (C)₁-C₄) Alkoxy-substituted phenyl or benzyl, halo (C)₆-C₁₀) Aryl group, (C)₆-C₁₀) A phenolic aryl group;

R₂selected from one or more of the following substituents: H. halogen, (C)₁-C₄) Alkyl, halo (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Alkoxy (C)₁-C₄) Alkyl, (C)₃-C₄) Cycloalkyl group, (C)₆-C₁₀) Aryl group, (C)₁-C₄) Alkyl substituted phenyl, (C)₁-C₄) Alkoxy-substituted phenyl or benzyl, halo (C)₆-C₁₀) Aryl group, (C)₆-C₁₀) A phenolic aryl group;

R₃selected from one or more of the following substituents: H. halogen, (C)₁-C₄) Alkyl, halo (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Alkoxy (C)₁-C₄) Alkyl, (C)₃-C₄) Cycloalkyl group, (C)₆-C₁₀) Aryl group, (C)₁-C₄) Alkyl substituted phenyl, (C)₁-C₄) Alkoxy-substituted phenyl or benzyl, halo (C)₆-C₁₀) Aryl group, (C)₆-C₁₀) A phenol aryl group.

3. The thiol compound having HDAC6 inhibitory activity according to claim 1 or 2, and a pharmaceutically acceptable salt or hydrate thereof:

wherein,

r is selected from one or more of the following substituents: H. (C)₂-C₄) Acyl, (C)₁-C₄) Alkyl, (C)₆-C₁₀) An aryl-substituted ester group; a phenyl group; a benzyl group; (C)₁-C₄) Alkoxy (C)₁-C₄) An alkyl group; the phenyl or benzyl group may be substituted by halogen, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy radical,Nitro, amino or hydroxy.

4. A thiol compound having HDAC6 inhibitory activity according to any one of claims 1 to 3 and a pharmaceutically acceptable salt or hydrate thereof:

wherein,

the A ring is:

R₁selected from one or more of the following substituents: H. halogen, (C)₁-C₄) Alkyl, halo (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Alkoxy (C)₁-C₄) An alkyl group;

R₂selected from one or more of the following substituents: H. halogen, (C)₁-C₄) Alkyl, halo (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Alkoxy (C)₁-C₄) An alkyl group.

5. A compound as described below, and pharmaceutically acceptable salts or hydrates thereof: is selected from the group consisting of,

n- (6-mercaptohexyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -5, 6-dihydro-4H-isoxazolo [5,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -5, 6-dihydro-4H-isoxazolo [5,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -7-methyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7-methyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -6-methyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6-methyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -7-benzyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7-benzyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -6-benzyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6-benzyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -7- (4-fluorobenzyl) -1,4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7- (4-fluorobenzyl) -1,4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -6- (4-fluorobenzyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6- (4-fluorobenzyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -7- (4-methoxybenzyl) -1,4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7- (4-methoxybenzyl) -1,4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -6- (4-methoxybenzyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6- (4-methoxybenzyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -7- (4-hydroxybenzyl) -1,4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7- (4-hydroxybenzyl) -1,4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -6- (4-hydroxybenzyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6- (4-hydroxybenzyl) -1,4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -6(7) H-isoxazolo [5,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6(7) H-isoxazolo [5,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-methyl-N- (6-mercaptohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-methyl-N- (6-acetylthiohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-methyl-N- (6-mercaptohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-methyl-N- (6-acetylthiohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-Ethyl-N- (6-mercaptohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-Ethyl-N- (6-acetylthiohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-Ethyl-N- (6-mercaptohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-Ethyl-N- (6-acetylthiohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-methoxyethyl-N- (6-mercaptohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-methoxyethyl-N- (6-acetylthiohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-methoxyethyl-N- (6-mercaptohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-methoxyethyl-N- (6-acetylthiohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-cyclopropylmethyl-N- (6-mercaptohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

7-cyclopropylmethyl-N- (6-acetylthiohexyl) -1, 7-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-cyclopropylmethyl-N- (6-mercaptohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

6-cyclopropylmethyl-N- (6-acetylthiohexyl) -1, 6-dihydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-mercaptohexyl) -4, 5-dihydro-1H-pyrazolo [4,3-H ] isoquinoline-3-carboxamide

N- (6-acetylmercaptohexyl) -4, 5-dihydro-1H-pyrazolo [4,3-H ] isoquinoline-3-carboxamide

N- (6-mercaptohexyl) -4, 5-dihydro-1H-pyrazolo [4,3-H ] quinoline-3-carboxamide

N- (6-acetylmercaptohexyl) -4, 5-dihydro-1H-pyrazolo [4,3-H ] quinoline-3-carboxamide

N- (6-mercaptohexyl) -1H-pyrazolo [4,3-H ] isoquinoline-3-carboxamide

N- (6-acetylmercaptohexyl) -1H-pyrazolo [4,3-H ] isoquinoline-3-carboxamide

N- (6-mercaptohexyl) -1H-pyrazolo [4,3-H ] quinoline-3-carboxamide

N- (6-acetylmercaptohexyl) -1H-pyrazolo [4,3-H ] quinoline-3-carboxamide

N- (6-mercaptohexyl) -7-ethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7-ethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-thiohexyl) -6-ethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6-ethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-thiohexyl) -7-methoxyethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7-methoxyethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-thiohexyl) -6-methoxyethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6-methoxyethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-thiohexyl) -7-ethoxyethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7-ethoxyethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-thiohexyl) -6-ethoxyethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6-ethoxyethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-thiohexyl) -7-cyclopropylmethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -7-cyclopropylmethyl-1, 4,5, 7-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-Thiohexyl) -6-cyclopropylmethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide

N- (6-acetylthiohexyl) -6-cyclopropylmethyl-1, 4,5, 6-tetrahydropyrazolo [3,4-e ] indazole-3-carboxamide.

6. A pharmaceutical composition characterized by: comprising a compound of any one of claims 1-5, and pharmaceutically acceptable salts or hydrates thereof, and a pharmaceutically acceptable excipient.

7. The process for preparing thiol compounds having HDAC6 inhibitory activity according to claim 1, wherein the thiol compounds are represented by formula I,

when ring A is (substituted) pyrazole ring and "- - - -" is double bond, the synthetic route is as follows:

when the ring A is a (substituted) pyrazole ring and the < - - - > is a single bond, the synthetic route is as follows:

when the ring A is pyridine ring and the < - - - > is single bond or double bond, the synthetic route is as follows:

8. use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition according to claim 6, for the manufacture of a medicament for the treatment of a disease associated with aberrant expression of histone deacetylase activity.

9. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition according to claim 6, for the preparation of an anti-neoplastic medicament.

10. Use of a compound according to any one of claims 1 to 5, and pharmaceutically acceptable salts or hydrates thereof, or a pharmaceutical composition according to claim 6, for the manufacture of a medicament for the treatment and/or prophylaxis of prostate cancer, breast cancer, cervical cancer or leukaemia.