CN116478111B - Histone methyltransferase inhibitor and preparation method thereof - Google Patents

Histone methyltransferase inhibitor and preparation method thereof Download PDF

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Publication number
CN116478111B
CN116478111B CN202310437364.5A CN202310437364A CN116478111B CN 116478111 B CN116478111 B CN 116478111B CN 202310437364 A CN202310437364 A CN 202310437364A CN 116478111 B CN116478111 B CN 116478111B
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reaction
hours
methanol
histone methyltransferase
compound
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CN116478111A (en
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芦静
范哲
饶淑容
巫鑫屿
谢艳秋
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Yantai University
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Yantai University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention belongs to the technical field of histone methyltransferase. The invention providesA histone methyltransferase inhibitor and its preparing process are disclosed, which have the structural formulaThe present invention provides a histone methyltransferase inhibitor having a novel backbone, which has a half Inhibitory Concentration (IC) against histone methyltransferase 50 ) Up to 127nM.

Description

Histone methyltransferase inhibitor and preparation method thereof
Technical Field
The invention relates to the technical field of histone methyltransferases, in particular to a histone methyltransferase inhibitor and a preparation method thereof.
Background
SMYD2, a member of the SMYD (SET and MYND domain-containing proteins) family, is a class of histone methyltransferases that are widely found in eukaryotic cells. SMYD2 is not only methyltransferase of histone such as H3K4, H3K36, etc., but also non-histone such as EZH2, p53, ER alpha, etc. can be modified; SMYD2 is also closely related to the formation, development, and immune system of various systems such as cardiovascular, nervous, digestive, and motor systems.
Studies prove that SMYD2 can be over-expressed in various tumor tissues, and has sensitivity and specificity in the aspects of expression on cancers such as gastric cancer, gastrointestinal stromal tumor, colon cancer, breast cancer and the like, and part of SMYD2 has a tumor heterologous distribution trend. And the expression level of SMYD2 is correlated with poor prognosis in clinical patients. Therefore, SMYD2 inhibitors are expected to be effective antitumor drugs.
SMYD2 inhibitors AZ505, LLY-507, BAY-598, EPZ033294 and EPZ032597 are substrates and SAM non-competitive inhibitors; PFI-5 is a SAM competitive inhibitor. BAY-598 has a certain anti-tumor cell proliferation activity, but is not effective in animals alone. LLY-507 showed antiproliferative activity against 240 cancer cell lines (whether SMYD2 is highly expressed or not), suggesting that LLY-507 activity is due to off-target effects rather than inhibition of SMYD 2. There is therefore a need in the art for SMYD2 inhibitors with a new backbone to meet clinical needs.
Disclosure of Invention
The invention aims to provide a histone methyltransferase inhibitor and a preparation method thereof, aiming at the defects of the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a histone methyltransferase inhibitor, which has the structural formula ofWherein X is 1 Is a hydrogen atom, methyl or isopropyl group, X 2 Is of the structure of X 2 The para-position of-COOH in (C) is-NH.
The invention also provides a preparation method of the histone methyltransferase inhibitor, when X is 1 In the case of a hydrogen atom, comprising the steps of:
1) Mixing an aniline compound, an amine compound, a substance A and an organic solvent 1, and reacting the mixture with a piperazine compound to obtain an intermediate 1;
2) After reacting the intermediate 1, a solvent and sodium hydroxide, purifying the obtained crude product to obtain an intermediate 2;
3) Reacting the intermediate 2 with HCl/dioxane to obtain a histone methyltransferase inhibitor;
the structural formula of the piperazine compound is
Preferably, the aniline compound in step 1) has the structural formula of
AminesThe compound is triethylamine or triethanolamine, the substance A is tricarbonyl chloride or N, N-carbonyl diimidazole, and the organic solvent 1 is dichloromethane or N, N-dimethylformamide.
Preferably, in the step 1), the molar ratio of the aniline compound, the amine compound, the substance a and the piperazine compound is 0.1 to 3.8:0.5 to 11.5:0.1 to 4.8: 0.2-4, the molar volume ratio of the aniline compound to the organic solvent 1 is 0.1-3.8 mmol: 2.5-10.5 mL; the mixing temperature is 0-30 ℃, the mixing time is 1-2 h, and the mixing is carried out in nitrogen atmosphere; the reaction temperature is 20-30 ℃, and the reaction time is 10-16 h.
Preferably, the solvent in the step 2) is one or more of methanol solution, ethanol and tetrahydrofuran; the volume fraction of the ethanol is 65-75%, and the volume fraction of the methanol solution is 50-75%; the mol volume ratio of the intermediate 1 to the sodium hydroxide to the solvent is 0.4-1.5 mmol:0.5 to 4mmol: 6-12 mL.
Preferably, the temperature of the reaction in the step 2) is 20-80 ℃, and the reaction time is 2-16 h; the purification method is extraction and/or reverse phase chromatography.
Preferably, during the reaction of the intermediate 2 and HCl/dioxane in the step 3), adding an organic solvent 2, wherein the organic solvent 2 is one or more of 1, 4-dioxane, ethyl acrylate and methanol; the mol volume ratio of the intermediate 2, the organic solvent 2 and the HCl/dioxane is 0.3-0.65 mmol: 5-10 mL: 2-10 mL; the reaction temperature is 20-30 ℃, and the reaction time is 1-16 h.
The invention also provides a preparation method of the histone methyltransferase inhibitor, when X is 1 In the case of methyl or isopropyl, comprising the steps of:
(1) Mixing an organic compound, triethylamine, acetic acid, methanol and an organic solvent, reacting the mixture with sodium cyanoborohydride, and carrying out aftertreatment on a reaction product to obtain an intermediate;
(2) Reacting the intermediate, ethanol and sodium hydroxide to obtain a histone methyltransferase inhibitor;
the structural formula of the organic compound is
Preferably, the organic solvent in the step (1) is acetone or formaldehyde solution, and the mass fraction of the formaldehyde solution is 35-40%; the molar ratio of the organic compound to the triethylamine to the acetic acid to the cyano sodium borohydride is 0.8-1.2: 1.8 to 2.2:2.8 to 3.2:1.8 to 2.2; the volume ratio of the methanol to the organic solvent is 8-12: 0.4 to 0.6; the molar volume ratio of the organic compound to the methanol is 0.8-1.2 mmol: 8-12 mL; the mixing temperature is 20-30 ℃, the mixing time is 0.5-1.5 h, and the mixing is carried out in nitrogen atmosphere.
Preferably, the temperature of the reaction in the step (1) is 20-30 ℃, and the reaction time is 10-12 hours; the volume fraction of the ethanol in the step (2) is 65-70%; the mol volume ratio of the intermediate to the sodium hydroxide to the ethanol is 0.5-0.6 mmol:1.5 to 2mmol: 5-7 mL; the reaction temperature is 60-80 ℃, and the reaction time is 12-14 h.
The beneficial effects of the invention are as follows:
the present invention provides a histone methyltransferase inhibitor having a novel backbone, which has a half Inhibitory Concentration (IC) against histone methyltransferase 50 ) Up to 127nM.
Detailed Description
The invention provides a histone methyltransferase inhibitor, which has the structural formula ofWherein X is 1 Is a hydrogen atom, methyl or isopropyl group, X 2 Is of the structure of
X 2 The para-position of-COOH in (C) is-NH.
The invention also provides a preparation method of the histone methyltransferase inhibitor, when X is 1 In the case of a hydrogen atom, comprising the steps of:
1) Mixing an aniline compound, an amine compound, a substance A and an organic solvent 1, and reacting the mixture with a piperazine compound to obtain an intermediate 1;
2) After reacting the intermediate 1, a solvent and sodium hydroxide, purifying the obtained crude product to obtain an intermediate 2;
3) Reacting the intermediate 2 with HCl/dioxane to obtain a histone methyltransferase inhibitor; the structural formula of the piperazine compound is
In the invention, the preparation method of the piperazine compound preferably comprises the following steps:
a) Reacting the intermediate 1-1, acetonitrile, isoamyl nitrite and cuprous bromide to obtain an intermediate 1-2;
b) Reacting the intermediate 1-2, isopropanol, the intermediate 1-3 and triethylamine to obtain an intermediate 1-4;
c) And (3) reacting the intermediate 1-4, methanol, ammonium chloride solution and iron powder to obtain the piperazine compound.
In the present invention, the intermediate 1-1 of step A) preferably has the formula
In the invention, the molar volume ratio of the intermediate 1-1, the isoamyl nitrite, the cuprous bromide and the acetonitrile in the step A) is preferably 11-12 mmol: 110-120 mmol: 110-120 mmol:140 to 160mL, more preferably 11.2 to 11.8mmol: 112-118 mmol: 112-118 mmol:145 to 155mL, more preferably 11.4 to 11.6mmol: 114-116 mmol: 114-116 mmol: 148-152 mL.
In the present invention, the temperature of the reaction in step A) is preferably 70 to 90 ℃, more preferably 75 to 85 ℃, still more preferably 80 ℃; the reaction time is preferably 12 to 18 hours, more preferably 14 to 16 hours, and still more preferably 15 hours; the reaction is preferably carried out under a nitrogen atmosphere; after the reaction, the solution is preferably concentrated, and the crude product is washed, and the washed reagent is preferably ethyl acrylate.
In the present invention, the structural formula of the intermediate 1-2 in the step B) is preferablyThe structural formula of the intermediates 1 to 3 is preferably +.>
In the present invention, the molar volume ratio of the intermediate 1-2, the intermediate 1-3, triethylamine and isopropanol in the step B) is preferably 8 to 10mmol: 8-10 mmol: 16-20 mmol:25 to 35mL, more preferably 8.5 to 9.5mmol:8.5 to 9.5mmol: 17-19 mmol: 28-32 mL, more preferably 9mmol:9mmol:18mmol:30mL.
In the present invention, the temperature of the reaction in step B) is preferably 70 to 90 ℃, more preferably 75 to 85 ℃, still more preferably 80 ℃; the reaction time is preferably 12 to 18 hours, more preferably 14 to 16 hours, and still more preferably 15 hours; the reaction is preferably carried out under a nitrogen atmosphere; after the reaction, the solution is preferably concentrated, and the crude product is purified, preferably by silica gel column chromatography.
In the present invention, the structural formula of the intermediates 1 to 4 in step C) is preferablyThe concentration of the ammonium chloride solution is preferably 25 to 30mmol/L.
In the invention, the molar volume ratio of the intermediate 1-4, the ammonium chloride solution, the iron powder and the methanol in the step C) is preferably 2.5-3 mmol: 13-14 mmol: 13-14 mmol:11 to 13mL, more preferably 2.6 to 2.8mmol:13.2 to 13.8mmol:13.2 to 13.8mmol:11.5 to 12.5mL, more preferably 2.7mmol:13.4 to 13.6mmol:13.4 to 13.6mmol:12mL.
In the present invention, the temperature of the reaction in step C) is preferably 70 to 90 ℃, more preferably 75 to 85 ℃, and even more preferably 80 ℃; the reaction time is preferably 1 to 3 hours, more preferably 1.5 to 2.5 hours, and still more preferably 2 hours; the reaction is preferably carried out under a nitrogen atmosphere; after the reaction, the solution is preferably filtered and concentrated, and the crude product is purified, preferably by silica gel column chromatography.
In the present invention, the structural formula of the aniline compound in step 1) is preferably
The amine compound is preferably triethylamine or triethanolamine, the substance A is preferably tricarbonyl chloride or N, N-carbonyl diimidazole, and the organic solvent 1 is preferably dichloromethane or N, N-dimethylformamide.
In step 1) of the present invention, the aniline compoundPreferably comprising the steps of:
(A) Reacting the intermediate 2-1, dimethyl sulfoxide, paraformaldehyde and sodium phenolate to obtain an intermediate 2-2;
(B) Mixing and reacting the intermediate 2-2, isoindoline-1, 3-dione, triphenylphosphine, tetrahydrofuran and diethyl azodicarboxylate to obtain an intermediate 2-3;
(C) Mixing the intermediate 2-3, ethanol and hydrazine hydroxide, and then reacting to obtain an intermediate 2-4;
(D) Mixing the intermediate 2-4, methanol and di-tert-butyl dicarbonate, and then reacting to obtain an intermediate 2-5;
(E) Mixing intermediate 2-5, methanol and palladium carbon, and reacting to obtain aniline compound
In the present invention, the intermediate 2-1 in the step (A) is preferably represented by the formulaIntermediate 2-2 has the structural formula +.>
In the invention, the molar volume ratio of the intermediate 2-1, paraformaldehyde, sodium phenolate and dimethyl sulfoxide in the step (A) is preferably 23-24 mmol:23.5 to 24.5mmol:26 to 26.5mmol:75 to 85mL, more preferably 23.5mmol:24mmol:26.4mmol:80mL; the reaction temperature is preferably 70 to 90 ℃, more preferably 72 to 85 ℃, still more preferably 74 to 80 ℃, and the reaction time is preferably 2 to 4 hours, more preferably 2.5 to 3.5 hours, still more preferably 3 hours, and the reaction is preferably carried out under a dark condition.
In the present invention, after the reaction in step (a) is completed, the reaction mixture and water are preferably mixed, then extracted, and then the organic phase is sequentially dried, filtered, concentrated and purified; the volume ratio of the water to the dimethyl sulfoxide is preferably 95-105: 75 to 85, more preferably 100:80; the reagent for extraction is preferably ethyl acetate, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the method comprises the steps of carrying out a first treatment on the surface of the The dried reagent is preferably magnesium sulfate and the purification method is preferably silica gel column chromatography.
In the present invention, the temperature at which the intermediate 2-2, isoindoline-1, 3-dione, triphenylphosphine, tetrahydrofuran and diethyl azodicarboxylate are mixed in step (B) is preferably-1 to 1 ℃, more preferably 0 ℃, and the mixing is preferably performed under a nitrogen atmosphere; the mol volume ratio of the intermediate 2-2, the isoindoline-1, 3-dione, the triphenylphosphine, the diethyl azodicarboxylate and the tetrahydrofuran is preferably 10-11 mmol:
13-14 mmol: 15-16 mmol: 15-16 mmol:35 to 45mL, more preferably 10.2 to 10.8mmol:13.2 to 13.8mmol:15.2 to 15.8mmol:15.2 to 15.8mmol: 38-42 mL, more preferably 10.5mmol:13.5mmol:15.5mmol:15.5mmol:40mL; the reaction temperature is preferably 20 to 30 ℃, more preferably 24 to 26 ℃, still more preferably 25 ℃, and the reaction time is preferably 14 to 18 hours, more preferably 15 to 17 hours, still more preferably 16 hours.
In the invention, after the reaction in the step (B) is finished, the reaction mixture and water are preferably mixed, then extraction is carried out, then the organic phase is washed, dried and filtered in sequence, and the filtrate is concentrated and then purified; the volume ratio of the water to the tetrahydrofuran is preferably 95-105: 35 to 45, more preferably 98 to 102:38 to 42, more preferably 100:40, a step of performing a; the reagent for extraction is preferably methylene chloride, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate and the purification method is preferably silica gel column chromatography.
In the present invention, the intermediate 2-3 in step (C) is preferably of the formulaThe structural formula of the intermediate 2-4 is +.>The volume fraction of ethanol is preferably 90 to 95%, more preferably 95%.
In the present invention, the temperature at which the intermediate 2-3, ethanol and hydrazine hydroxide are mixed in step (C) is preferably-1 to 1 ℃, more preferably 0 ℃, and the mixing is preferably performed in a nitrogen atmosphere; the molar volume ratio of the intermediate 2-3, the hydrazine hydroxide and the ethanol is preferably 8-9 mmol:26 to 26.5mmol:25 to 35mL, more preferably 8.2 to 8.8mmol:26.2 to 26.4mmol: 28-32 mL, more preferably 8.6mmol:26.3mmol:30mL; the reaction temperature is preferably 65 to 75 ℃, more preferably 68 to 72 ℃, still more preferably 70 ℃, and the reaction time is preferably 1.5 to 2.5 hours, more preferably 1.8 to 2.2 hours, still more preferably 2 hours.
In the present invention, after the reaction in step (C) is completed, the reaction mixture is preferably cooled, mixed with water, extracted, and then the organic phase is sequentially washed, dried and filtered, and the filtrate is concentrated and purified; the volume ratio of the water to the ethanol is preferably 95-105: 25 to 35, more preferably 98 to 102:28 to 32, more preferably 100:30; the cooling temperature is preferably 20-25 ℃, more preferably 22-23 ℃, the extracting reagent is preferably dichloromethane, and the extracting times are preferably 2-4 times, more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate and the purification method is preferably silica gel column chromatography.
In the present invention, the mixing sequence of the intermediate 2-4, methanol and di-tert-butyl dicarbonate in the step (D) is preferably that the intermediate 2-4 and methanol are mixed first, and then di-tert-butyl dicarbonate is added for mixing, and the conditions of the intermediate 2-4 and methanol are preferably ice bath and nitrogen atmosphere.
In the present invention, the molar volume ratio of the intermediate 2-4, di-tert-butyl dicarbonate and methanol in the step (D) is preferably 5 to 6mmol:6 to 6.5mmol:16 to 20mL, more preferably 5.2 to 5.8mmol:6.2 to 6.4mmol:17 to 19mL, more preferably 5.4 to 5.6mmol:6.3mmol:18mL; the reaction temperature is preferably 20 to 30 ℃, more preferably 24 to 26 ℃, still more preferably 25 ℃, and the reaction time is preferably 4 to 6 hours, more preferably 4.5 to 5.5 hours, still more preferably 5 hours.
In the present invention, after the reaction in the step (D) is completed, the reaction mixture is preferably mixed with water, and then extracted, and then the organic phase is washed, dried and filtered in sequence, and the filtrate is concentrated and purified; the volume ratio of the water to the methanol is preferably 95-105: 16 to 20, more preferably 98 to 102:17 to 19, more preferably 100:18; the reagent for extraction is preferably methylene chloride, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate and the purification method is preferably silica gel column chromatography.
In the invention, the structural formula of the intermediate 2-5 in the step (E) isThe molar volume ratio of the intermediate 2-5 to the methanol is preferably 4.5-5 mmol:25 to 35mL, more preferably 4.6 to 4.8mmol: 28-32 mL, more preferably 4.7mmol:30mL, the molar mass ratio of the intermediate 2-5 to palladium carbon is preferably 4.5-5 mmol:0.13 to 0.16g, more preferably 4.8mmol:0.15g.
In the present invention, the step (E) of mixing the intermediate 2-5, methanol and palladium-carbon is preferably performed in a hydrogen atmosphere, the reaction temperature is preferably 20 to 30 ℃, more preferably 24 to 26 ℃, more preferably 25 ℃, and the reaction time is preferably 4 to 6 hours, more preferably 5 hours; after the reaction, the reaction mixture is preferably filtered and concentrated sequentially.
In step 1) of the present invention, the aniline compoundPreferably comprising the steps of:
A. mixing the intermediate 6-1, N-bromosuccinimide, dibenzoyl peroxide and carbon tetrachloride and then reacting to obtain an intermediate 6-2;
B. mixing the intermediate 6-2, sodium methoxide and methanol, and then reacting to obtain an intermediate 6-3;
C. mixing intermediate 6-3, methanol and palladium-carbon, and reacting to obtain
In the invention, the structural formula of the intermediate 6-1 in the step A is preferably The structural formula of intermediate 6-2 is preferably +.>The mixing sequence of the intermediate 6-1, the N-bromosuccinimide, the dibenzoyl peroxide and the carbon tetrachloride is preferably that the intermediate 6-1, the N-bromosuccinimide and the carbon tetrachloride are mixed firstly, and then the dibenzoyl peroxide is added for mixing; the temperature at which the intermediate 6-1, N-bromosuccinimide and carbon tetrachloride are mixed is preferably 20 to 30 ℃, more preferably 25 ℃, and the mixing is preferably performed under a nitrogen atmosphere.
In the invention, the molar volume ratio of the intermediate 6-1, the N-bromosuccinimide, the dibenzoyl peroxide and the carbon tetrachloride in the step A is preferably 26-27 mmol:26.5 to 27mmol:2.5 to 3mmol:70 to 90mL, more preferably 26.5mmol:26.8mmol:2.8mmol:80mL; the reaction temperature is preferably 20 to 30 ℃, more preferably 25 ℃, and the reaction time is preferably 14 to 18 hours, more preferably 16 hours.
In the invention, the mixing sequence of the intermediate 6-2, sodium methoxide and methanol in the step B is preferably that the intermediate 6-2 and methanol are mixed first, and then sodium methoxide is added for mixing; the temperature at which the intermediate 6-2 and methanol are mixed is preferably 20 to 30 ℃, more preferably 25 ℃, and the mixing is preferably performed under a nitrogen atmosphere.
In the invention, the molar volume ratio of the intermediate 6-2, sodium methoxide and methanol in the step B is preferably 10.5-11 mmol: 21-22 mmol:28 to 32mL, more preferably 10.8mmol:21.5mmol:30mL; the reaction temperature is preferably 20 to 30 ℃, more preferably 25 ℃, and the reaction time is preferably 14 to 18 hours, more preferably 15 to 17 hours, more preferably 16 hours.
In the invention, after the reaction in the step A and the step B is finished, the independent preference is that the reaction mixture is cooled and then mixed with water, then extracted, and then organic phase is washed, dried and filtered in sequence, and the filtrate is concentrated and purified; the volume ratio of the water to the carbon tetrachloride is independently preferably 95-105: 70 to 90, more preferably 98 to 102:75 to 85, more preferably 100:80; the cooling temperature is preferably 20 to 25 ℃, and more preferably 25 ℃; the reagent for extraction is preferably methylene chloride, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate and the purification method is preferably silica gel column chromatography.
In the present invention, the intermediate 6-3 in the step C preferably has the structural formulaThe molar volume ratio of the intermediate 6-3 to the methanol is preferably 6.5-7 mmol:14 to 16mL, more preferably 6.8mmol:15mL, the molar mass ratio of the intermediate 6-3 to palladium carbon is preferably 6.5-7 mmol:0.14 to 0.16g, more preferably 6.8mmol:0.15g, the mass volume ratio of palladium carbon to methanol is preferably 0.14-0.16 g:14 to 16mL, more preferably 0.15g:15mL.
In the invention, the mixing sequence of the intermediate 6-3, the methanol and the palladium-carbon in the step C is preferably that the intermediate 6-3 and the methanol are mixed firstly, and then the palladium-carbon is added for mixing; the temperature at which the intermediate 6-3 and methanol are mixed is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, still more preferably 24 to 26 ℃, and the mixing is preferably performed in a hydrogen atmosphere.
In the present invention, the temperature of the reaction in the step C is preferably 20 to 25 ℃, more preferably 22 to 24 ℃, still more preferably 23 ℃, and the reaction time is preferably 14 to 18 hours, more preferably 16 hours; after the reaction, the reaction mixture is preferably sequentially filtered, concentrated and purified, and the purification method is preferably silica gel column chromatography.
In the present invention, the molar ratio of the aniline compound, the amine compound, the substance a and the piperazine compound in the step 1) is preferably 0.1 to 3.8:0.5 to 11.5:0.1 to 4.8:0.2 to 4, more preferably 1.1 to 2.8:3.5 to 8.5:1.1 to 3.8:1.2 to 3, more preferably 1.5 to 2.5:5.5 to 6.5:2 to 3.5:2 to 2.5; the molar volume ratio of the aniline compound to the organic solvent 1 is preferably 0.1-3.8 mmol:2.5 to 10.5mL, more preferably 1.5 to 2.6mmol:5.5 to 7.5mL, more preferably 1.8 to 2.2mmol: 6-7 mL; the temperature of the mixing is preferably 0 to 30 ℃, more preferably 10 to 25 ℃, still more preferably 15 to 20 ℃; the mixing time is preferably 1 to 2 hours, more preferably 1.2 to 1.8 hours, still more preferably 1.4 to 1.6 hours; the mixing is preferably carried out under a nitrogen atmosphere; the temperature of the reaction is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, and even more preferably 24 to 26 ℃; the reaction time is preferably 10 to 16 hours, more preferably 12 to 15 hours, and still more preferably 13 to 14 hours.
In the invention, after the reaction of the step 1), the pH value of the solution is preferably adjusted, then the solution is extracted and concentrated, and then the crude product is purified; the reagent used for adjusting the pH value of the solution is preferably sodium bicarbonate solution, and the concentration of the sodium bicarbonate solution is preferably 1.5-2.5 mmol, and more preferably 2mmol; the pH value of the solution is preferably adjusted to 7.5-8, the extracted reagent is preferably ethyl acrylate, and the purification method is preferably silica gel column chromatography.
In the invention, after the reaction in the step 1), the reaction mixture and water are preferably mixed, then extracted, and then the organic phase is washed, dried and filtered in sequence, and the filtrate is concentrated and purified; the volume ratio of the water to the organic solvent 1 in step 1) is preferably 90 to 100:2.5 to 10.5, more preferably 92 to 98:4.5 to 8.5, more preferably 94 to 96:6 to 7; the reagent for extraction is preferably methylene chloride, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate and the purification method is preferably silica gel column chromatography.
In the invention, the solvent in the step 2) is preferably one or more of methanol solution, ethanol and tetrahydrofuran; the volume fraction of the ethanol is preferably 65 to 75%, more preferably 68 to 72%, more preferably 70%, and the volume fraction of the methanol solution is preferably 50 to 75%, more preferably 55 to 70%, more preferably 60 to 65%; the molar volume ratio of the intermediate 1, sodium hydroxide and solvent is preferably 0.4-1.5 mmol:0.5 to 4mmol:6 to 12mL, more preferably 0.8 to 1.2mmol:1.5 to 3mmol:8 to 10mL, more preferably 1mmol:2 to 2.5mmol: 8.5-9 mL.
In the present invention, the temperature of the reaction in step 2) is preferably 20 to 80 ℃, more preferably 30 to 70 ℃, and even more preferably 40 to 60 ℃; the reaction time is preferably 2 to 16 hours, more preferably 5 to 13 hours, and still more preferably 8 to 10 hours; the purification method is preferably extraction and/or reverse phase chromatography.
In the invention, after the reaction of the step 2), the reaction mixture is preferably cooled, neutralized and extracted in sequence, then the organic phase is washed, dried and filtered in sequence, and the filtrate is concentrated and purified; the cooling temperature is preferably-1 ℃, more preferably 0 ℃, the neutralizing agent is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5-1.5 mmol/L, more preferably 1mmol/L; the pH of the neutralized mixture is preferably from 6 to 7, more preferably 7; the reagent for extraction is preferably methylene chloride, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate and the purification method is preferably reverse phase chromatography.
In the present invention, after the reaction of step 2), the reaction mixture is preferably cooled, neutralized and purified sequentially; the cooling temperature is preferably-1 to 1 ℃, more preferably 0 ℃, the neutralizing agent is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5 to 1.5mmol/L, more preferably 1mmol/L; the pH of the neutralized mixture is preferably from 6 to 7, more preferably 7; the method of purification is preferably reverse phase chromatography.
In the present invention, after the reaction of step 2), the reaction mixture is preferably cooled, neutralized, filtered in sequence, and then the solid is washed; the cooling temperature is preferably-1 to 1 ℃, more preferably 0 ℃, the neutralizing agent is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5 to 1.5mmol/L, more preferably 1mmol/L; the pH of the neutralized mixture is preferably from 6 to 7, more preferably 7; the washing is preferably carried out sequentially with methanol and ethyl acrylate.
In the invention, after the reaction of the step 2), the solvent is distilled off under reduced pressure, the pH value of the solution is regulated, and then extraction, drying and concentration are sequentially carried out; the reagent for regulating the pH value of the solution is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5-1.5 mmol/L, and more preferably 1mmol/L; the pH value of the solution is preferably adjusted to 6-7, more preferably 7, the extracted reagent is preferably ethyl acetate, and the extraction times are preferably 2-4 times, more preferably 3 times; the amount of ethyl acetate used in each extraction is preferably 45 to 55mL, more preferably 50mL; the dried reagent is preferably anhydrous sodium sulfate.
In the present invention, during the reaction of intermediate 2 with HCl/dioxane in step 3), it is preferable to add organic solvent 2, and organic solvent 2 is preferably one or more of 1, 4-dioxane, ethyl acrylate and methanol; the molar volume ratio of the intermediate 2, the organic solvent 2 and the HCl/dioxane is preferably 0.3-0.65 mmol: 5-10 mL:2 to 10mL, more preferably 0.4 to 0.55mmol: 6-9 mL:4 to 8mL, more preferably 0.5mmol: 7-8 mL: 5-6 mL; the temperature of the reaction is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, and even more preferably 24 to 26 ℃; the reaction time is preferably 1 to 16 hours, more preferably 5 to 12 hours, and still more preferably 7 to 10 hours.
In the present invention, after the reaction of step 3) is completed, the reaction product is preferably sequentially subjected to solvent removal, recrystallization and filtration, and the solvent used in the recrystallization process is methanol.
In the present invention, after the reaction in step 3) is completed, the reaction product is preferably concentrated, pH-adjusted, filtered and washed sequentially, and the reagent used for adjusting pH is preferably sodium bicarbonate solution, and the concentration of sodium bicarbonate solution is preferably 1.5-2.5 mmol/L, and more preferably 2mmol; the pH is preferably adjusted to 6 to 7, more preferably 7, and the washing is preferably performed sequentially with acetonitrile and water, and the amount of acetonitrile and water is preferably 2 to 4 times, more preferably 3 times the amount of HCl/dioxane, independently.
In the present invention, it is preferable to concentrate the reaction product after the completion of the reaction in step 3).
The invention also provides a preparation method of the histone methyltransferase inhibitor, when X is 1 In the case of methyl or isopropyl, comprising the steps of:
(1) Mixing an organic compound, triethylamine, acetic acid, methanol and an organic solvent, reacting the mixture with sodium cyanoborohydride, and carrying out aftertreatment on a reaction product to obtain an intermediate;
(2) Reacting the intermediate, ethanol and sodium hydroxide to obtain a histone methyltransferase inhibitor;
the structural formula of the organic compound is
In the present invention, the organic solvent in the step (1) is preferably acetone or formaldehyde solution, and the mass fraction of the formaldehyde solution is preferably 35-40%, more preferably 37-39%, and even more preferably 38%; the molar ratio of the organic compound, triethylamine, acetic acid and cyano sodium borohydride is preferably 0.8-1.2: 1.8 to 2.2:2.8 to 3.2:1.8 to 2.2, more preferably 0.9 to 1.1:1.9 to 2.1:2.9 to 3.1:1.9 to 2.1, more preferably 1:2:3:2; the volume ratio of the methanol to the organic solvent is preferably 8-12: 0.4 to 0.6, more preferably 9 to 11:0.45 to 0.55, more preferably 10:0.5; the molar volume ratio of the organic compound to the methanol is preferably 0.8-1.2 mmol:8 to 12mL, more preferably 0.9 to 1.1mmol:9 to 11mL, more preferably 1mmol:10mL; the temperature of the mixing is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, and even more preferably 24 to 26 ℃; the mixing time is preferably 0.5 to 1.5 hours, more preferably 0.8 to 1.2 hours, and still more preferably 1 hour; the mixing is preferably carried out under a nitrogen atmosphere.
In the present invention, the temperature of the reaction in the step (1) is preferably 20 to 30 ℃, more preferably 22 to 28 ℃, and even more preferably 24 to 26 ℃; the reaction time is preferably 10 to 12 hours, more preferably 10.5 to 11.5 hours, and still more preferably 11 hours; the volume fraction of the ethanol in the step (2) is preferably 65-70%, more preferably 66-68%, and even more preferably 67%; the molar volume ratio of the intermediate to the sodium hydroxide to the ethanol is preferably 0.5-0.6 mmol:1.5 to 2mmol:5 to 7mL, more preferably 0.52 to 0.58mmol:1.6 to 1.8mmol:5.5 to 6.5mL, more preferably 0.54 to 0.56mmol:1.7mmol:6mL; the temperature of the reaction is preferably 60 to 80 ℃, more preferably 65 to 75 ℃, and even more preferably 70 ℃; the reaction time is preferably 12 to 14 hours, more preferably 12.5 to 13.5 hours, and still more preferably 13 hours.
In the present invention, after the reaction in step (1) is completed, the reaction mixture is preferably cooled, mixed with water, extracted, and then the organic phase is washed, dried and filtered in sequence, and the filtrate is concentrated and purified; the volume ratio of the water to the methanol is preferably 95-105: 8 to 12, more preferably 98 to 102:9 to 11, more preferably 100:10; the cooling temperature is preferably 20 to 25 ℃, and more preferably 22 to 24 ℃; the reagent for extraction is preferably methylene chloride, and the number of times of extraction is preferably 2 to 4 times, and more preferably 3 times; the amount of methylene chloride used is preferably 45 to 55mL, more preferably 50mL, per extraction; the washing reagent is preferably saturated saline, and the number of washing is preferably 4 to 6, more preferably 5; the amount of the saturated saline used is preferably 90 to 110mL, more preferably 100mL, per washing; the dried reagent is preferably anhydrous sodium sulfate, the purification method is preferably silica gel column chromatography,
In the present invention, after the reaction in step (2) is completed, the reaction mixture is preferably cooled, neutralized and filtered sequentially, and then the solid is washed; the cooling temperature is preferably-1 to 1 ℃, more preferably 0 ℃, the neutralizing agent is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5 to 1.5mmol/L, more preferably 1mmol/L; the pH of the neutralized mixture is preferably from 6 to 7, more preferably 7; the washing is preferably carried out sequentially with methanol and ethyl acrylate.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The preparation method of the piperazine compound in examples 1 to 9 is as follows:
11.3mmol of intermediate 1-1, 150mL of acetonitrile, 113mmol of isoamyl nitrite and 113mmol of cuprous bromide are reacted for 14 hours under the nitrogen atmosphere at 80 ℃, and then the solution is concentrated, and the crude product is washed by adopting ethyl acrylate to obtain intermediate 1-2; reacting 9mmol of intermediate 1-2, 9mmol of intermediate 1-3, 18mmol of triethylamine and 30mL of isopropanol for 16h under nitrogen atmosphere at 80 ℃, concentrating the solution, and purifying the crude product by silica gel chromatography to obtain intermediate 1-4; 2.7mmol, 13.7mmol of ammonium chloride solution with a concentration of 25mmol/L, 13.7mmol of iron powder and 12mL of methanol are reacted for 2h under nitrogen atmosphere at 80 ℃, then the solution is filtered and concentrated, and the crude product is purified by silica gel chromatography to obtain piperazine compound.
Example 1
Will be 0.2mmolMixing 0.6mmol of triethylamine, 0.1mmol of tricarbonyl chloride and 3mL of dichloromethane at 0 ℃ for 2 hours under nitrogen atmosphere, adding 0.27mmol of piperazine compound to react for 12 hours at 25 ℃, adjusting the pH value of the solution to 7.5 by using sodium bicarbonate solution, extracting with ethyl acrylate, concentrating, purifying the crude product by silica gel column chromatography to obtain an intermediate 1->
0.56mmol of intermediate1. 1.25mmol of sodium hydroxide and 6mL of 65% strength by volume ethanol were reacted at 60℃for 14 hours, then the reaction mixture was cooled to 0℃and the pH of the mixture was adjusted to 7 with 1mmol/L of hydrochloric acid, extraction was performed 3 times with methylene chloride (50 mL of methylene chloride was used for each extraction), the organic phase was washed 5 times with saturated brine (100 mL of saturated brine was used for each washing), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by reverse phase chromatography to give intermediate 2
After reacting 0.384mmol of intermediate 2 with 5 mLHCl/dioxane at 25℃for 15h, the reaction product was desolvated and then recrystallized from methanol, filtered to give a histone methyltransferase inhibitor
Example 2
After 23.9mmol of intermediate 2-1, 24mmol of paraformaldehyde, 26.39mmol of sodium phenolate and 80mL of dimethyl sulfoxide are reacted for 3 hours at 80 ℃ under the dark condition, the reaction mixture is mixed with 100mL of deionized water, the mixture is extracted for 3 times by ethyl acetate, the organic phase is dried by magnesium sulfate, and then the organic phase is filtered, concentrated and purified by silica gel column chromatography to obtain intermediate 2-2; 10.45mmol of the intermediate 2-2, 13.59mmol of isoindoline-1, 3-dione, 15.68mmol of triphenylphosphine, 15.68mmol of diethyl azodicarboxylate and 40mL of tetrahydrofuran were reacted at 25℃for 16 hours, the reaction mixture was mixed with 100mL of deionized water, the mixture was extracted 3 times with methylene chloride (the amount of methylene chloride used for each extraction was 50 mL), the organic phase was washed 5 times with saturated brine (the amount of saturated brine used for each washing was 100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give the intermediate 2-3;
8.69mmol of intermediate 2-3, 26.07mmol of hydrazine hydroxide and 30mL of ethanol with volume fraction of 95% are mixed in a nitrogen atmosphere at 0 ℃, the mixture is reacted for 2 hours at 70 ℃, after cooling to 25 ℃, the reaction mixture is mixed with 100mL of deionized water, dichloromethane is adopted for extraction for 3 times (the dosage of the dichloromethane is 50mL in each extraction), an organic phase is washed with saturated saline for 5 times (the dosage of the saturated saline is 100mL in each washing), the mixture is dried by anhydrous sodium sulfate and filtered, and filtrate is concentrated and purified by silica gel column chromatography to obtain intermediate 2-4; under the condition of nitrogen atmosphere and ice bath, 5.52mmol of intermediate 2-4 and 18mL of methanol are mixed, 6.08mmol of di-tert-butyl dicarbonate is added for reaction for 5 hours at 25 ℃, then the reaction mixture is mixed with 100mL of deionized water, dichloromethane is adopted for extraction for 3 times (the dosage of the dichloromethane is 50mL during each extraction), an organic phase is washed for 5 times by saturated saline (the dosage of the saturated saline is 100mL during each washing), the organic phase is dried by anhydrous sodium sulfate and filtered, and the filtrate is concentrated and purified by silica gel column chromatography to obtain intermediate 2-5;
4.59mmol of the intermediate 2-5, 30mL of methanol and 0.15g of palladium on carbon were reacted at 25℃under a hydrogen atmosphere for 5 hours, and then the reaction mixture was filtered and concentrated to obtain
Will be 1.47mmol4.41mmol of triethanolamine, 1.62mmol of N, N-carbodiimidazole and 10mL of dichloromethane are mixed for 1h at 25 ℃ under nitrogen atmosphere, then 1.62mmol of piperazine compound is added to react for 16h at 30 ℃, the reaction mixture is mixed with 100mL of deionized water, the mixture is extracted 3 times with dichloromethane (the usage of dichloromethane is 50mL in each extraction), the organic phase is washed 5 times with saturated saline (the usage of saturated saline is 100mL in each washing), the mixture is dried with anhydrous sodium sulfate and filtered, and the filtrate is concentrated and purified by silica gel column chromatography to obtain the intermediate 1
0.61mmol of intermediate 1, 1.22mmol of sodium hydroxide and 6mL of 67% by volume ethanol were reacted at 70℃for 12 hours, then the reaction mixture was cooled to 0℃and the pH of the mixture was adjusted to 7 with 1mmol/L of hydrochloric acid, extraction was performed 3 times with methylene chloride (50 mL of methylene chloride was used for each extraction), the organic phase was washed 5 times with saturated brine (100 mL of saturated brine was used for each washing), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by reverse phase chromatography to give intermediate 2
After 0.42mmol of intermediate 2, 5mL of LHCl/dioxane and 5mL of 1, 4-dioxane were reacted at 30℃for 16 hours, the reaction product was concentrated, then the pH of the solution was adjusted to 7 with a sodium bicarbonate solution having a concentration of 2mmol/L, the mixture was filtered, and the solid was washed with 20mL of acetonitrile and 20mL of deionized water to give a histone methyltransferase inhibitor
Example 3
Will be 1.12mmol3.35mmol of triethanolamine, 1.17mmol of N, N-carbodiimidazole and 10mL of dichloromethane were mixed at 25℃under nitrogen atmosphere for 1 hour, then 1.23mmol of piperazine compound was added to react at 28℃for 16 hours, the reaction mixture was mixed with 100mL of deionized water, extraction was performed 3 times with dichloromethane (the amount of dichloromethane used in each extraction was 50 mL), the organic phase was washed 5 times with saturated saline (the amount of saturated saline used in each washing was 100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give intermediate 1
0.66mmol of intermediate 1, 0.66mmol of sodium hydroxide and 6mL of 67% by volume ethanol were reacted at 80℃for 10 hours, then the reaction mixture was cooled to 0℃and the pH of the mixture was adjusted to 7 with hydrochloric acid having a concentration of 1mmol/L, and the crude product was purified by reverse phase chromatography to give intermediate 2
After 0.38mmol of intermediate 2, 5mL of LHCl/dioxane and 5mL of ethyl acrylate were reacted at 30℃for 16 hours, the reaction product was concentrated, then the pH of the solution was adjusted to 6 with a sodium bicarbonate solution having a concentration of 2mmol/L, the mixture was filtered, and the solid was washed with 20mL of acetonitrile and 20mL of deionized water to obtain a histone methyltransferase inhibitor
Example 4
Will be 1.34mmol4mmol of triethylamine, 1.5mmol of N, N-carbodiimidazole and 10mL of methylene chloride are mixed for 1h at 24 ℃ under nitrogen atmosphere, then 1.48mmol of piperazine compound is added for reaction at 25 ℃ for 16h, the reaction mixture is mixed with 100mL of deionized water, the mixture is extracted 3 times with methylene chloride (the usage of methylene chloride is 50mL in each extraction), the organic phase is washed 5 times with saturated saline (the usage of saturated saline is 100mL in each washing), the mixture is dried with anhydrous sodium sulfate and filtered, and the filtrate is concentrated and purified by silica gel column chromatography to obtain an intermediate 1
0.49mmol of intermediate 1, 0.98mmol of sodium hydroxide and 6mL of 70% by volume ethanol were reacted at 70℃for 8 hours, then the reaction mixture was cooled to 0℃and the pH of the mixture was adjusted to 7 with 1mmol/L of hydrochloric acid, and after extraction with methylene chloride 3 times (50 mL of methylene chloride was used for each extraction), the crude product was purified by reverse phase chromatography to give intermediate 2
After 0.3mmol of intermediate 2, 5mL of LHCl/dioxane and 5mL of ethyl acrylate were reacted at 28℃for 16 hours, the reaction product was concentrated, then the pH of the solution was adjusted to 7 with a sodium bicarbonate solution having a concentration of 2mmol/L, the mixture was filtered, and the washed solid was washed with 20mL of acetonitrile and 20mL of deionized water to give a histone methyltransferase inhibitor
Example 5
Will 3mmol8.9mmol of triethylamine, 3.25mmol of N, N-carbodiimidazole and 10mL of methylene chloride were mixed at 25℃under nitrogen atmosphere for 1.2 hours, then 3.25mmol of piperazine compound was added to react at 30℃for 10 hours, the reaction mixture was mixed with 100mL of deionized water, extracted 3 times with methylene chloride (the amount of methylene chloride used for each extraction was 50 mL), the organic phase was washed 5 times with saturated brine (the amount of saturated brine used for each washing was 100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give intermediate 1
0.67mmol of intermediate 1, 2.7mmol of sodium hydroxide, 8mL of a 50% volume fraction of methanol solution and 4mL of tetrahydrofuran were reacted at 65℃for 16 hours, then the reaction mixture was cooled to 0℃and the pH of the mixture was adjusted to 6 with 1mmol/L of hydrochloric acid, and the mixture was filtered and the solid was washed with methanol and ethyl acrylate to give intermediate 2
After 0.61mmol of intermediate 2, 10mL of LHCl/dioxane and 10mL of 1, 4-dioxane were reacted at 26℃for 1 hour, the reaction product was concentrated, then the pH of the solution was adjusted to 6 with a sodium bicarbonate solution having a concentration of 2mmol/L, the mixture was filtered, and the solid was washed with 20mL of acetonitrile and 20mL of deionized water to give a histone methyltransferase inhibitor
Example 6
After 26.62mmol of intermediate 6-1, 26.9mmol of N-bromosuccinimide and 80mL of carbon tetrachloride were mixed at 25℃under a nitrogen atmosphere, 2.67mmol of dibenzoyl peroxide was added, the reaction mixture was cooled to 20℃after 16 hours of reaction at 25℃and 100mL of deionized water was added to mix, then the mixture was extracted 3 times with methylene chloride (50 mL of methylene chloride was used for each extraction), the organic phase was washed 5 times with saturated brine (100 mL of saturated brine was used for each washing), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give intermediate 6-2; 10.95mmol of intermediate 6-2 and 30mL of methanol are mixed in a nitrogen atmosphere at 25 ℃, 21.89mmol of sodium methoxide is added for reaction at 25 ℃ for 16 hours, then the reaction mixture is cooled to 20 ℃, 100mL of deionized water is added for mixing, then the mixture is extracted 3 times with dichloromethane (the usage of dichloromethane is 50mL in each extraction), the organic phase is washed 5 times with saturated saline (the usage of saturated saline is 100mL in each washing), dried with anhydrous sodium sulfate and filtered, and the filtrate is concentrated and purified by silica gel column chromatography to obtain intermediate 6-3;
Mixing 6.66mmol of intermediate 6-3 with 15mL of methanol at 25deg.C in hydrogen atmosphere, adding 0.15g of palladium on carbon, reacting at 25deg.C for 16h, filtering the mixture, concentrating the filtrate, and purifying by column chromatography
Will be 3.69mmol11.07mmol of triethylamine, 4.8mmol of N, N-carbodiimidazole and 10mL of methylene chloride were mixed at 25℃under a nitrogen atmosphere for 1 hour, then 3.69mmol of piperazine compound was added to react at 30℃for 10 hours, the reaction mixture was mixed with 100mL of deionized water, extraction was performed 3 times with methylene chloride (the amount of methylene chloride was 50mL for each extraction), the organic phase was washed 5 times with saturated brine (the amount of saturated brine was 100mL for each washing), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give intermediate 1
1.27mmol of intermediate 1, 3.81mmol of sodium hydroxide and 6mL of a 75% by volume methanol solution were reacted at 20℃for 2 hours, then methanol was distilled off under reduced pressure, the pH of the solution was adjusted to 7 with hydrochloric acid having a concentration of 1mmol/L, and then extracted 3 times with ethyl acetate (the amount of ethyl acetate used in each extraction was 50 mL), dried over anhydrous sodium sulfate and concentrated to give intermediate 2
After reacting 0.33mmol of intermediate 2, 2 mLHCl/dioxane and 5mL of methanol at 25℃for 1 hour, the reaction product was concentrated to give a histone methyltransferase inhibitor
Example 7
1mmol in nitrogen atmosphere at 25 DEG C2mmol of triethylamine, 3mmol of acetic acid, 10mL of methanol and 0.5mL of acetone were mixed togetherAfter 1.5h, 2mmol of sodium cyanoborohydride was added to react for 12h at 30℃and then the reaction mixture was cooled to 20℃and mixed with 100mL of deionized water, extracted 3 times with methylene chloride (50 mL of methylene chloride was used for each extraction), the organic phase was washed 5 times with saturated brine (100 mL of saturated brine was used for each washing), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give an intermediate
Reacting 0.58mmol of intermediate, 6mL of 65% ethanol and 1.76mmol of sodium hydroxide at 70 ℃ for 13h, cooling the reaction mixture to 0 ℃, adjusting the pH of the mixture to 7 with 1mmol/L hydrochloric acid, filtering the mixture, washing the solid with methanol and ethyl acrylate to obtain the histone methyltransferase inhibitor
Example 8
Will be 3.69mmol11.07mmol of triethylamine, 4.8mmol of N, N-carbodiimidazole and 10mLN, N-dimethylformamide were mixed at 25℃under a nitrogen atmosphere for 1 hour, then 3.69mmol of piperazine compound was added to react at 25℃for 10 hours, the reaction mixture was mixed with 100mL of deionized water, extracted 3 times with methylene chloride (the amount of methylene chloride used in each extraction was 50 mL), the organic phase was washed 5 times with saturated brine (the amount of saturated brine used in each washing was 100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give intermediate 1
1.27mmol of intermediate 1, 3.81mmol of sodium hydroxide and 8mL of a 75% by volume methanol solution were reacted at 25℃for 2 hours, then methanol was distilled off under reduced pressure, the pH of the solution was adjusted to 6 with hydrochloric acid having a concentration of 1mmol/L, and then extracted 3 times with ethyl acetate (the amount of ethyl acetate used in each extraction was 50 mL), dried over anhydrous sodium sulfate and concentrated to give intermediate 2
After reacting 0.33mmol of intermediate 2, 2 mLHCl/dioxane and 5mL of methanol at 25℃for 1 hour, the reaction product was concentrated to give a histone methyltransferase inhibitor
Example 9
1mmol in nitrogen atmosphere at 25 DEG CAfter 2mmol of triethylamine, 3mmol of acetic acid, 10mL of methanol and 0.5mL of a 37% by mass formaldehyde solution were mixed for 1 hour, 2mmol of sodium cyanoborohydride was added to react at 30℃for 10 hours, then the reaction mixture was cooled to 25℃and mixed with 100mL of deionized water, extraction was performed 3 times with methylene chloride (the amount of methylene chloride used for each extraction was 50 mL), the organic phase was washed 5 times with saturated brine (the amount of saturated brine used for each washing was 100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give an intermediate>
Reacting 0.58mmol of intermediate, 6mL of 65% ethanol and 1.76mmol of sodium hydroxide at 70 ℃ for 14h, cooling the reaction mixture to 0 ℃, adjusting the pH of the mixture to 7 with 1mmol/L hydrochloric acid, filtering the mixture, washing the solid with methanol and ethyl acrylate to obtain the histone methyltransferase inhibitor
The histone methyltransferase inhibitors of examples 1 to 9 were diluted 3-fold with a buffer (buffer consisting of 50mM Tris-HCl (pH 9.0), 0.01% Tween-20 and 1mM DTT) using an Echo machine, 5. Mu.L/well of histone methyltransferase at 100nM was added, and Sinfugin was set as a positive control and dimethyl sulfoxide as a negative control for each test plate. After centrifugation of the test plate at 1000rpm for 15s, incubation at 23℃for 15min, the reaction was initiated by adding 5. Mu.L/well of substrate solution (0.2. Mu.M Biotin-H3 (1-21) peptide and 0.2. Mu.M SAM); after centrifugation of the test plate at 1000rpm for 15s and sealing of the membrane on the test plate, incubation at 23℃for 60min was performed, and detection solution (8.3. Mu.g/mL Anti-methyl-Histone H3 Lysine 4 (H3K 4me 1-2) AlphaLISA Acceptor Beads (PERKIN ELMER, cat#AL 116M) and 8.3. Mu.g/mL Strep-Tactin Alpha Donor beads (PERKIN ELMER, cat#AS 106R)) was added to all wells of the test plate; centrifuging at 1000rpm for 15s, sealing the film on the measuring plate, and incubating at 23deg.C for 60min; assay plates were read on EnVision and the histone methyltransferase inhibitors of examples 1-9 were analyzed for their inhibitory activity on histone methyltransferase using xlit 5 software, the results of which are shown in table 1.
TABLE 1 inhibition of histone methyltransferase by different histone methyltransferase inhibitors
Histone methyltransferase inhibitor numbering IC 50 (nM)
Sinefugin 495
Example 1 905
Example 2 127
Example 3 365
Example 4 1288
Example 5 1508
Example 6 945
Example 7 >10000
Example 8 490
Example 9 >10000
As can be seen from Table 1, the histone methyltransferase inhibitor of the present invention has a half inhibitory concentration (IC 50 ) Up to 127nM.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (6)

1. A histone methyltransferase inhibitor, wherein the histone methyltransferase inhibitorThe structural formula of the agent isWherein X is 1 Is a hydrogen atom, X 2 Is of the structure of X 2 The para-position of-COOH in (C) is-NH.
2. The method for preparing a histone methyltransferase inhibitor according to claim 1, comprising the steps of:
1) Mixing an aniline compound, an amine compound, a substance A and an organic solvent 1, and reacting the mixture with a piperazine compound to obtain an intermediate 1;
2) After reacting the intermediate 1, a solvent and sodium hydroxide, purifying the obtained crude product to obtain an intermediate 2;
3) Reacting the intermediate 2 with HCl/dioxane to obtain a histone methyltransferase inhibitor;
the structural formula of the piperazine compound is
The structural formula of the aniline compound in the step 1) is The amine compound is triethylamine or triethanolamine, the substance A is tricarbonyl chloride or N, N-carbonyl diimidazole, and the organic solvent 1 is dichloromethane or N, N-dimethylformamide;
the solvent in the step 2) is one or more of methanol solution, ethanol and tetrahydrofuran; the volume fraction of the ethanol is 65-75%, and the volume fraction of the methanol solution is 50-75%.
3. The preparation method according to claim 2, wherein the molar ratio of the aniline compound, the amine compound, the substance a and the piperazine compound in the step 1) is 0.1 to 3.8:0.5 to 11.5:0.1 to 4.8: 0.2-4, the molar volume ratio of the aniline compound to the organic solvent 1 is 0.1-3.8 mmol: 2.5-10.5 mL; the mixing temperature is 0-30 ℃, the mixing time is 1-2 h, and the mixing is carried out in nitrogen atmosphere; the reaction temperature is 20-30 ℃, and the reaction time is 10-16 h.
4. A process according to claim 3, wherein in step 2) the molar volume ratio of intermediate 1, sodium hydroxide and solvent is between 0.4 and 1.5mmol:0.5 to 4mmol: 6-12 mL.
5. The preparation method according to claim 4, wherein the reaction temperature in the step 2) is 20-80 ℃ and the reaction time is 2-16 h; the purification method is extraction and/or reverse phase chromatography.
6. The preparation method according to claim 4 or 5, wherein in the step 3), the organic solvent 2 is added during the reaction of the intermediate 2 and HCl/dioxane, wherein the organic solvent 2 is one or more of 1, 4-dioxane, ethyl acrylate and methanol; the mol volume ratio of the intermediate 2, the organic solvent 2 and the HCl/dioxane is 0.3-0.65 mmol: 5-10 mL: 2-10 mL; the reaction temperature is 20-30 ℃, and the reaction time is 1-16 h.
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Citations (3)

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CN113350347A (en) * 2020-03-05 2021-09-07 安徽中科拓苒药物科学研究有限公司 Novel use of indazole compound

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EP1609789A1 (en) * 2004-06-23 2005-12-28 Eli Lilly And Company Ureido-pyrazole derivatives and their use as kinase inhibitors
CN113350347A (en) * 2020-03-05 2021-09-07 安徽中科拓苒药物科学研究有限公司 Novel use of indazole compound

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