CN112110880B

CN112110880B - Androstane derivative and preparation method and application thereof

Info

Publication number: CN112110880B
Application number: CN202011046305.8A
Authority: CN
Inventors: 贾政; 李宝齐; 田维; 郭嘉林; 刘登科; 孔凯; 董凯; 姚小青
Original assignee: Tianjin Chase Sun Pharmaceutical Co Ltd
Current assignee: Tianjin Chase Sun Pharmaceutical Co Ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-09-09
Anticipated expiration: 2040-09-29
Also published as: CN115160397A; CN115160397B; CN112110880A

Abstract

The invention provides an androstane derivative and a preparation method and application thereof, belonging to the technical field of medicines. The androstane derivative provided by the invention has a very good inhibition effect on BCR-ABL tyrosine kinase and corresponding mRNA level, and the androstane derivative also has a very good inhibition effect on a Hedgehog signal channel, and has a good application prospect in preparation of chronic myelogenous leukemia drugs.

Description

Androstane derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to an androstane derivative and a preparation method and application thereof.

Background

The BCR-ABL fusion gene is formed by mutual translocation of ABL proto-oncogene on chromosome 9 and Bcr gene on chromosome 24 of human cells, and can cause the continuous activation of protein kinase, so that the excessive proliferation of white blood cells can lead to the appearance of Chronic Myelogenous Leukemia (CML).

The BCR-ABL tyrosine kinase inhibitors used globally for the treatment of CML share 17 (including marketed and development-underway drugs), and in addition to the 7 approved drugs on the market, the investigated drugs include Asciminib (Nowa, Clin III), CMLVAXb2a2-25 (Clin II, university of Leibin), PF-114 (Clin II, Fusion Pharma), AN-019 (Clin II, Natco), SUN-K706 (Clin II, Sun Pharmacological advanced Research), Nicotinib (Clin II, Guangzhou institute of Biomedicine and health, Guangzhou, Hipposhu Shunjian plain biomedical Biomedicine), Dasatinib Nanotique formulation (Clin I, Xspray Pharma), ETC-206 (Clin I, Experimential Therapeutics Centre), Adaprostinn (clinical Adhesin, Clin II, clinical Research, McHitachi, and the clinical Research on of cancer, and more inhibitors for the clinical treatment of cancer, and more of BCR-ABL (clinical Adenossac, clinical Research, clinical Adenome Research, clinical Research on the national balance of cancer, clinical Research, McHitachi, has important significance for improving the economic burden of tumor patients and improving the clinical treatment effect of tumors.

Disclosure of Invention

The androstane derivative provided by the invention has a very good inhibition effect on BCR-ABL tyrosine kinase and corresponding mRNA level, has a very good inhibition effect on a Hedgehog signal channel, and has a good application prospect in preparation of chronic myelogenous leukemia medicines.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an androstane derivative which has a structure shown in a formula A or a formula B:

the invention provides a preparation method of the androstane derivative in the technical scheme,

the preparation method of the androstane derivative with the structure shown in the formula A comprises the following steps:

(1) carrying out oxidation reaction on the compound with the structure shown in the formula II to obtain a compound with the structure shown in the formula III;

(2) carrying out condensation reaction on the compound with the structure shown in the formula III and morpholine to obtain a compound with the structure shown in the formula IV;

(3) carrying out esterification reaction on the compound with the structure shown in the formula IV and acetyl chloride to obtain a compound with the structure shown in the formula V;

(4) carrying out quaternization reaction on the compound with the structure shown in the formula V and 3-bromopropionic acid to obtain an androstane derivative with the structure shown in the formula A;

the preparation method of the androstane derivative with the structure shown in the formula B comprises the following steps:

(a) carrying out substitution reaction on a compound with a structure shown in a formula II and ethanolamine to obtain a compound with a structure shown in a formula VI;

(b) carrying out amine ester exchange reaction on the compound with the structure shown in the formula VI and methyl formate to obtain a compound with the structure shown in the formula VII;

(c) carrying out esterification reaction on the compound with the structure shown in the formula VII and acetyl chloride to obtain a compound with the structure shown in the formula VIII;

(d) carrying out quaternization reaction on the compound with the structure shown in the formula VIII and 3-bromopropylene to obtain an androstane derivative with the structure shown in the formula B;

wherein the compounds with the structures shown in formulas II to VIII are shown as follows:

preferably, the oxidizing agent used in the oxidation reaction in the step (1) is hydrogen peroxide, and the compound with the structure shown in the formula II and H in the hydrogen peroxide ₂ O ₂ The molar ratio of (A) to (B) is 10: 200-300;

the mol ratio of the compound with the structure shown in the formula III to morpholine in the step (2) is 1: 1-2;

the molar ratio of the compound with the structure shown in the formula IV to acetyl chloride in the step (3) is 10: 15-30;

the molar ratio of the compound with the structure shown in the formula V to the 3-bromopropionic acid in the step (4) is 1: 1-2.

Preferably, the temperature of the oxidation reaction in the step (1) is 85-95 ℃ and the time is 18-22 h;

the condensation reaction in the step (2) is carried out at the temperature of 15-30 ℃ for 18-20 h;

the temperature of the esterification reaction in the step (3) is 0-10 ℃, and the time is 25-35 min;

the temperature of the quaternization reaction in the step (4) is 0-10 ℃, and the time is 2-5 h.

Preferably, the step (4) further comprises, after the quaternization reaction:

and under the condition of stirring, dropwise adding the reaction liquid obtained after the quaternization reaction into anhydrous ether at the temperature of 0-10 ℃ for crystallization, and then filtering and drying to obtain the androstane derivative with the structure shown in the formula A.

Preferably, the molar ratio of the compound with the structure shown in the formula II to the ethanolamine in the step (a) is 10 to (12-30);

the mol ratio of the compound with the structure shown in the formula VI to the methyl formate in the step (b) is 10 to (20-40);

the molar ratio of the compound with the structure shown in the formula VII to acetyl chloride in the step (c) is 10: 10-30;

the molar ratio of the compound with the structure shown in the formula VIII to the 3-bromopropylene in the step (d) is 10: 10-15.

Preferably, the temperature of the substitution reaction in the step (a) is 100-120 ℃, and the time is 30-40 h;

the temperature of the amine ester exchange reaction in the step (b) is 15-30 ℃, and the time is 10-20 h;

the temperature of the esterification reaction in the step (c) is 0-10 ℃, and the time is 25-35 min;

the temperature of the quaternization reaction in the step (d) is 0-10 ℃, and the time is 2-5 h.

Preferably, the step (d) further comprises, after the quaternization reaction:

and under the condition of stirring, dropwise adding the reaction liquid obtained after the quaternization reaction into anhydrous ether at the temperature of 0-10 ℃ for crystallization, and then filtering and drying to obtain the androstane derivative with the structure shown in the formula B.

The invention provides application of the androstane derivative in the technical scheme in preparation of a medicine for treating chronic myelogenous leukemia.

Preferably, the drug for treating chronic myelogenous leukemia comprises a Hedgehog signaling pathway inhibitor drug, a BCR-ABL tyrosine kinase inhibitor drug or a BCR-ABL tyrosine kinase mRNA inhibitor drug.

The invention provides an androstane derivative having a structure represented by formula A or formula B. The androstane derivative with the structure shown in the formula A or the formula B has a very good inhibition effect on BCR-ABL tyrosine kinase and the corresponding mRNA level, has a very good inhibition effect on a Hedgehog signal channel, and has a good application prospect in preparation of chronic myelogenous leukemia drugs, so that an effective therapeutic drug is provided for chronic myelogenous leukemia patients. The test result of the test example 1 shows that the androstane derivative provided by the invention has obvious inhibition effect on BCR-ABL tyrosine kinase and corresponding mRNA level, and can be used for treating chronic myelogenous leukemia with mutation of BCR-ABL kinase. The test result of test example 2 shows that the average IC50 values of the androstane derivative provided by the invention for inhibiting Hedgehog pathway signaling are respectively 0.019 μ M and 0.018 μ M, which are respectively lower than the average IC50 value of 0.022 μ M of the existing FDA-approved Hedgehog pathway inhibitor Vismodegib, and the average IC50 value of 0.020 μ M of the androstane derivative disclosed in chinese patent 201910706129.7 and having the structure shown in formula I, which are measured by using mouse [ s12] and human [ MZ24] Gli reporter cell lines, and thus the androstane derivative provided by the invention has a very good inhibitory effect on Hedgehog pathway signaling.

The invention also provides a preparation method of the androstane derivative with the structure shown in the formula A or the formula B, and the method provided by the invention has the advantages of short steps, simplicity in operation, high total reaction yield, higher purity of the obtained product, contribution to industrial production and the like.

Drawings

FIG. 1 is a schematic representation of the atomic site identification of a compound having the structure shown in formula A prepared in example 1;

FIG. 2 is a schematic representation of the atomic site identification of the compound having the structure shown in formula B prepared in example 2.

Detailed Description

in the present invention, the androstane derivative having the structure shown in formula a is denoted as compound a, and the chemical name of compound a is: 1- ((2S,3R,3aS,5aS,6S,7S,9aR,9bS) -3-acetoxy-7- (carboxmethyl) -3a, 6-dimethyl-6- (2-morpholino-2-oxoethyl) dodecahydro-1H-cyclopenta [ a ] naphthalen-2-yl) -1- (carboxmethyl) pyrolidin-1-ium bromide; the androstane derivative having the structure shown in formula B is denoted as compound B, which has the chemical name according to IUPAC nomenclature: 1- ((2S,3S,5S,8R,9S,10S,13S,14S,16S,17R) -17-acetoxy-3-hydroxy-2- (N- (2-hydroxyethenyl) formamido) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenyl-16-yl) -1-allylpyrolidin-1-iumbromide.

The present invention provides a process for producing androstane derivatives according to the above technical means, and the following processes for producing compound a and compound B are described below.

In the present invention, the preparation method of compound a comprises the following steps:

wherein, the compounds with the structures shown in the formulas II to V are shown as follows:

the compound (denoted as compound II) with the structure shown in the formula II is subjected to oxidation reaction to obtain the compound (denoted as compound III) with the structure shown in the formula III. The source of the compound II is not particularly limited in the present invention, and commercially available products well known to those skilled in the art may be used; the compound II is named as: (2 α,3 α,5 α,16 β,17 β) -2, 3-epoxy-16- (1-pyrrolidinyl) -androstan-17-ol with CAS number: 119302-19-1.

In the invention, the oxidant adopted in the oxidation reaction is preferably hydrogen peroxide, and the concentration of the hydrogen peroxide is preferably 30 wt%; the oxidation reaction is preferably carried out in the presence of a catalyst, preferably ammonium phosphotungstate trihydrate; wherein, the compound II and hydrogen peroxide are H ₂ O ₂ And the molar ratio of ammonium phosphotungstate trihydrate is preferably 10: 200-300: 1-3, and more preferably 10: 300: 1. In the present invention, the solvent used for the oxidation reaction is preferably water; the invention has no special requirement on the dosage of the solvent, and can dissolve reactants; in the examples of the present invention, the amount ratio of water to the compound II is preferably 1.00L: 100 g.

In the present invention, it is preferable that the compound II and the solvent are mixed with stirring, and then the resultant mixture is mixed with the catalyst and the oxidizing agent, and the system temperature is raised to the temperature of the oxidation reaction to carry out the oxidation reaction. In the invention, the temperature of the oxidation reaction is preferably 85-95 ℃, and more preferably 90 ℃; the time is preferably 18-22 h, and more preferably 20 h. According to the invention, after the components are mixed, the temperature is preferably raised to the temperature of oxidation reaction at a speed of 15-20 ℃/h, so that the phenomenon that the hydrogen peroxide is heated to decompose and release gas to cause material flushing is avoided. The invention preferably adopts hydrogen peroxide as an oxidant to oxidize epoxy groups in the compound II into carboxylic acid, has mild reaction conditions, is environment-friendly, has good tolerance to functional groups of substrates, and has few side reactions.

After the oxidation reaction, the reaction solution is preferably cooled to room temperature (in the embodiment of the present invention, specifically to 25 ℃), extracted with ethyl acetate, the organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated and purified by silica gel column chromatography to obtain a light yellow oil, that is, compound III. After the reaction solution is cooled to room temperature, the reaction solution is preferably tested by using starch potassium iodide test paper, and if the starch potassium iodide test paper does not change color, subsequent treatment is carried out; if the starch potassium iodide test paper is discolored, preferably adding saturated sodium thiosulfate into the reaction solution until the starch potassium iodide test paper is not discolored. In the present invention, the reagent used for purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 5: 1.

After the compound III is obtained, the compound III and morpholine are subjected to condensation reaction to obtain a compound (denoted as a compound IV) with a structure shown in a formula IV. In the invention, the molar ratio of the compound III to morpholine is preferably 1 to (1-2), and more preferably 1 to 1. In the invention, the condensation reaction is preferably carried out in the presence of an organic solvent, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) and triethylamine; the molar ratio of the compound III to the HATU to the triethylamine is 10: 12-20: 20-30, and the preferable molar ratio is 10: 12: 20. In the invention, the organic solvent is preferably Dichloromethane (DCM), and the invention has no special requirement on the dosage of the organic solvent and can dissolve reactants; in the present example, the ratio of the organic solvent to the compound III is preferably 453 mL: 111.1 mmol.

In the invention, preferably, the organic solvent is mixed with the compound III under stirring, the temperature of the system is reduced to 0-10 ℃ (more preferably 0 ℃) under the protection of nitrogen, and triethylamine (Et) is added ₃ N) and HATU, stirring for 10-20 min, adding morpholine, and stirring for 8And (4) about 12min, and then heating the system to the condensation reaction temperature to perform the condensation reaction. In the invention, the condensation reaction temperature is preferably 15-30 ℃, and more preferably 20-25 ℃; in the examples of the present invention, the condensation reaction is specifically carried out at room temperature (25 ℃), i.e. no additional heating or cooling is required; the time of the condensation reaction is preferably 18-20 h, and more preferably 20 h. According to the invention, after the components are mixed and stirred for 8-12 min, the temperature is preferably increased to the condensation reaction temperature at the speed of 10-15 ℃/h, so that the smooth reaction is ensured. According to the invention, the activation of carboxyl in the compound III is preferably realized through HATU, and then morpholine is added for condensation reaction, so that the reaction condition is mild, the selectivity is good, and the reaction activity is high.

After the condensation reaction, preferably extracting the obtained reaction solution with an aqueous solution of sodium hydroxide (the concentration is preferably 1mol/L), continuously extracting the obtained water phase with dichloromethane, adjusting the pH value of the obtained water phase to 5-6 with hydrochloric acid, extracting with dichloromethane, washing the obtained organic phase with saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating the obtained filtrate, and purifying by silica gel column chromatography to obtain a white solid, namely a compound IV; in the present invention, the reagent used for purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 5: 1.

After the compound IV is obtained, the compound IV and acetyl chloride are subjected to esterification reaction to obtain a compound (denoted as a compound V) with a structure shown in a formula V. In the invention, the molar ratio of the compound IV to acetyl chloride is preferably 10 to (15-30), and more preferably 10 to 30. In the invention, the esterification reaction is preferably carried out in the presence of an organic solvent and triethylamine, and the molar ratio of the compound IV to the triethylamine is 10: 15-40, and more preferably 10: 40. In the invention, the organic solvent is preferably dichloromethane, and the invention has no special requirement on the dosage of the organic solvent and can dissolve reactants; in the present example, the amount ratio of the compound IV and the organic solvent is preferably 46.2 mmol: 220 mL.

The organic solvent is preferably mixed under stirringMixing the reagent with a compound IV, reducing the temperature of the obtained system to 0-10 ℃ under the protection of nitrogen, and adding Et ₃ N and acetyl chloride, and carrying out esterification reaction. In the invention, the temperature of the esterification reaction is preferably 0-10 ℃, and more preferably 0 ℃; the time is preferably 25 to 35min, and more preferably 30 min. In the esterification reaction, triethylamine is preferably used as alkali, and the compound IV and acetyl chloride are subjected to esterification reaction to obtain a compound V, wherein the reaction conditions are mild.

After the esterification reaction, preferably, the obtained reaction solution is extracted by water, the obtained water phase is extracted by dichloromethane, the obtained organic phase is washed by saturated saline solution and then dried by anhydrous sodium sulfate, the filtration is carried out, the obtained filtrate is concentrated and then purified by silica gel column chromatography to obtain a white solid, namely a compound V; in the present invention, the reagent used for purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 5: 1.

After the compound V is obtained, the compound V and 3-bromopropionic acid are subjected to quaternization reaction to obtain a compound A. In the invention, the molar ratio of the compound V to the 3-bromopropionic acid is preferably 1: 1-2, and more preferably 1: 1.2. In the invention, the quaternization reaction is preferably carried out in the presence of an organic solvent, the organic solvent is preferably dichloromethane, and the quaternization reaction does not have special requirements on the using amount of the organic solvent and can dissolve reactants; in the examples of the present invention, the amount ratio of the compound V to the organic solvent is preferably 0.0204 mol: 106 mL.

According to the invention, preferably, under the condition of stirring at 0-10 ℃, the compound V and 3-bromopropionic acid are sequentially added into dichloromethane, and then quaternization reaction is carried out. In the invention, the temperature of the quaternization reaction is preferably 0-10 ℃, and more preferably 10 ℃; the time is preferably 2 to 5 hours, and more preferably 3 hours.

After the quaternization reaction, the reaction solution is preferably dropwise added into anhydrous ether at 0-10 ℃ (preferably 5 ℃) under the stirring condition for crystallization, specifically, after dropwise addition, the mixture is stirred for 10-30 min under heat preservation, then filtered, and the obtained filter cake is dried under reduced pressure at normal temperature to obtain a white solid, namely the compound A. The invention preferably utilizes the characteristic that the compound A is almost insoluble in anhydrous ether, the reaction solution is added into the anhydrous ether to separate out the product, and simultaneously the aim of purification is achieved.

In the present invention, the preparation method of compound B comprises the following steps:

wherein, the compound with the structure shown in the formula II is consistent with the technical scheme, and the compounds with the structures shown in the formulas VI to VIII are shown as follows:

according to the invention, a compound with a structure shown in a formula II and ethanolamine are subjected to substitution reaction to obtain a compound with a structure shown in a formula VI (denoted as a compound VI). In the present invention, the starting material required for the preparation of compound B is a compound having a structure represented by formula II (i.e., compound II), which is the same as the starting material described above for the preparation of compound A. In the invention, the molar ratio of the compound II to the ethanolamine is preferably 10 to (12-30), and more preferably 10 to 15. In the invention, the substitution reaction is preferably carried out in the presence of an organic solvent, the organic solvent is preferably n-butyl alcohol, and the invention has no special requirement on the dosage of the organic solvent and can dissolve reactants; in the embodiment of the invention, the dosage ratio of the compound II and the organic solvent is preferably 278 mmol: 500 mL.

According to the invention, preferably, the compound II is mixed with n-butanol, and then ethanolamine is added into the obtained system under the stirring condition at the temperature of 20-25 ℃ for substitution reaction. In the invention, the temperature of the substitution reaction is preferably 100-120 ℃, and more preferably 120 ℃; the time is preferably 30-40 h, and more preferably 30 h. According to the invention, ethanolamine is used as a nucleophilic reagent, the reaction condition is mild, the yield is high, the tolerance to the functional group of the substrate is good, and the side reaction is less.

After the substitution reaction, preferably, the obtained reaction solution is cooled to room temperature, then is mixed with water, the obtained mixture is extracted by methyl tert-butyl ether, an organic layer obtained by washing the obtained mixture by using saturated saline solution is dried by anhydrous sodium sulfate, and is filtered, and the obtained filtrate is concentrated and purified by silica gel column chromatography to obtain a white solid which is marked as a compound VI; in the present invention, the reagent used for purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 5: 1.

After the compound VI is obtained, the compound VI and methyl formate are subjected to amine transesterification reaction to obtain a compound (denoted as a compound VII) with a structure shown in a formula VII. In the present invention, the amine transesterification reaction is preferably carried out in the presence of methanol and sodium methoxide; the mol ratio of the compound VI to the sodium methoxide to the methyl formate is preferably 10: 10-40: 20-40, and more preferably 10: 40; the invention has no special requirement on the dosage of the methanol, and the methanol can be used as an organic solvent to dissolve reactants; in the present example, the ratio between the amount of compound VI and methanol is preferably 252 mmol: 1L of the total amount of the active ingredients.

According to the invention, methanol and a compound VI are preferably mixed under the condition of stirring, the temperature of the obtained system is reduced to 0 ℃, sodium methoxide and methyl formate are added, and then the obtained system is naturally heated to the temperature of amine transesterification reaction for amine transesterification reaction; in the invention, the temperature of the amine ester exchange reaction is preferably 15-30 ℃, and more preferably 20-25 ℃; in the examples of the present invention, the amine transesterification reaction is carried out specifically at room temperature (25 ℃), i.e. without additional heating or cooling; the reaction time of the amine transesterification is preferably 10-20 h, and more preferably 10 h. In the amine transesterification reaction, preferably sodium methoxide is used as alkali, methyl formate is used as an exchange reagent, and the sodium methoxide and the methyl formate perform transesterification reaction with secondary amine in a structure of a compound VI to obtain a compound VII; the method has the advantages of mild reaction conditions, good selectivity and high product yield.

After the amine ester exchange reaction, preferably concentrating the obtained reaction liquid to be dry, dissolving the obtained residual solid with water, extracting with methyl tert-butyl ether, adjusting the pH value of the obtained water phase to 6-7 by adopting hydrochloric acid, extracting with ethyl acetate, washing the obtained organic phase with saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating the obtained filtrate to obtain yellow solid, and purifying by silica gel column chromatography to obtain white solid, namely the compound VII; in the present invention, the reagent used for purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 5: 1.

After a compound VII is obtained, the compound VII and acetyl chloride are subjected to esterification reaction to obtain a compound (named as a compound VIII) with a structure shown in a formula VIII. In the invention, the molar ratio of the compound VII to the acetyl chloride is preferably 10 to (10-30), and more preferably 10 to 15. In the invention, the esterification reaction is preferably carried out in the presence of an organic solvent and triethylamine, and the molar ratio of the compound VII to the triethylamine is 10: 15-40, and more preferably 10: 30. In the invention, the organic solvent is preferably dichloromethane, and the invention has no special requirement on the dosage of the organic solvent and can dissolve reactants; in the embodiment of the invention, the dosage ratio of the compound VII and the organic solvent is preferably 207 mmol: 1L.

The organic solvent and the compound VII are preferably mixed under the stirring condition, the temperature of the obtained system is reduced to 0-10 ℃ under the protection of nitrogen, and Et is added ₃ N and acetyl chloride, intoAnd carrying out esterification reaction. In the invention, the temperature of the esterification reaction is preferably 0-10 ℃, and more preferably 5 ℃; the time is preferably 25 to 35min, and more preferably 30 min. In the esterification reaction, triethylamine is preferably used as an alkali, and the compound VII and acetyl chloride are subjected to esterification reaction to obtain the compound VIII, and the reaction condition is mild.

After the esterification reaction, preferably, the obtained reaction liquid is extracted by water, the obtained water phase is extracted by dichloromethane, the obtained organic phase is washed by saturated saline solution and then dried by anhydrous sodium sulfate, the filtration is carried out, the obtained filtrate is concentrated and then purified by silica gel column chromatography to obtain a white solid, namely a compound VIII; in the present invention, the reagent used for purification is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 5: 1.

After the compound VIII is obtained, the compound VIII and 3-bromopropylene are subjected to quaternization reaction to obtain the androstane derivative (namely the compound B) with the structure shown in the formula B. In the invention, the molar ratio of the compound VIII to the 3-bromopropylene is preferably 10 to (10-15), and more preferably 10 to 12. In the invention, the quaternization reaction is preferably carried out in the presence of an organic solvent, the organic solvent is preferably dichloromethane, and the invention has no special requirement on the dosage of the organic solvent and can dissolve reactants; in the present example, the amount ratio of the compound VIII and the organic solvent is preferably 89 mmol: 400 mL.

The method preferably comprises the steps of mixing a compound VIII, 3-bromopropylene and an organic solvent at the temperature of 0-10 ℃ under the stirring condition for carrying out quaternization reaction; in the invention, the temperature of the quaternization reaction is preferably 0-10 ℃, and more preferably 10 ℃; the time is preferably 2 to 5 hours, and more preferably 3 hours.

After the quaternization reaction, the reaction solution is preferably dripped into anhydrous ether at the temperature of 0-10 ℃ under the stirring condition for crystallization, specifically, after dripping is finished, the mixture is stirred for 10-30 min under heat preservation, and then filtered, and the obtained filter cake is dried under reduced pressure at normal temperature to obtain a white solid, namely the compound B. The invention preferably utilizes the characteristic that the compound B is hardly dissolved in anhydrous ether, the reaction solution is added into the anhydrous ether to separate out the product, and simultaneously the aim of purification is achieved.

The invention provides application of the androstane derivative in the technical scheme in preparation of a medicine for treating chronic myelogenous leukemia. In the present invention, the drug for treating chronic myelogenous leukemia preferably comprises a Hedgehog signaling pathway inhibitor drug, a BCR-ABL tyrosine kinase inhibitor drug or a BCR-ABL tyrosine kinase mRNA inhibitor drug.

In the present invention, the medicament for treating chronic myelogenous leukemia preferably comprises an active component and a carrier; the active component is a compound A or a compound B; the carrier is a pharmaceutically acceptable carrier, including but not limited to one or more of the following: mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, monovalent alkali metal carbonates, acetates, phosphates or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose or derivatives thereof, alginates, gelatin, polyvinylpyrrolidone, glycerol, tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid-like materials, kaolin, talc, calcium stearate, magnesium stearate. In the invention, the content of the active component in the medicine is preferably 0.1-99.9 wt.%.

The invention has no special requirements on the dosage form of the medicine, and can be specifically tablets, capsules, oral liquid, buccal agents, granules, medicinal granules, pills, powder, ointment, pellets, suspensions, powders, solutions, injections, suppositories, tinctures, ointments, creams, sprays, drops or patches; preferably tablet, powder, granule, tincture, pill, capsule, oral liquid, aerosol inhalant or injection. The invention can be added with other auxiliary materials according to the preparation dosage form to be prepared. The invention has no special requirement on the types of the auxiliary materials, and the auxiliary materials well known in the field can be adopted.

In the present invention, when the dosage form of the drug is a preparation for oral administration, the drug preferably further comprises an excipient, and the present invention does not particularly require the kind of the excipient, and it is sufficient to use an excipient well known in the art; in particular, binders, fillers, diluents, tabletting agents, lubricants, disintegrants, colorants, flavouring agents or wetting agents can be mentioned. In the present invention, the filler preferably comprises one or more of cellulose, mannitol, lactose and other similar fillers, the specific type of which is not specifically required by the present invention, and which may be those known in the art to have similar functions; the disintegrant preferably comprises one or more of starch, polyvinylpyrrolidone and starch derivatives, the present invention does not specifically require the particular type of starch derivative, and starch derivatives well known in the art may be used, such as sodium starch glycolate; the lubricant preferably comprises magnesium stearate; the wetting agent preferably comprises sodium lauryl sulfate.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

An androstane derivative having the structure shown in formula a was prepared according to the following reaction scheme:

(1) under the condition of stirring, sequentially adding water (1.00L) and a compound (100g, 278mmol) with a structure shown in a formula II into a three-mouth reaction bottle, then adding ammonium phosphotungstate trihydrate (83.0g, 27.8mmol) and hydrogen peroxide (945g, 8.34mol) with the concentration of 30 wt%, heating to 90 ℃ at the speed of 18 ℃/h, and reacting for 20 h; after the reaction is finished, cooling the obtained reaction liquid to room temperature (25 ℃, testing the reaction liquid by using a starch potassium iodide test paper, wherein the starch potassium iodide test paper does not change color), extracting for 3 times by using ethyl acetate, wherein the volume of the ethyl acetate used in each extraction is 500mL, combining organic layers, washing by using common salt water, drying an obtained organic phase after washing by using anhydrous sodium sulfate, filtering, concentrating the obtained filtrate, purifying by silica gel column chromatography (a purifying reagent is dichloromethane and methanol, the volume ratio of the dichloromethane to the methanol is 5: 1), obtaining 45.3g of a light yellow oily substance, wherein the yield is 40.0%, and the purity is 95.6% by detecting by an LC-MS method (the detection conditions are shown in Table 1).

The pale yellow oil was characterized by the following specific data:

MS：m/z：408[M+H] ⁺ 。

the above results indicate that the pale yellow oil is the compound having the structure shown in formula III.

TABLE 1 LC-MS method detection conditions

(2) Dichloromethane (DCM, 453mL) and the compound of formula III (45.3g, 111.1mmol) were added to a three-necked flask with stirring, the temperature of the resulting system was reduced to 0 ℃ under nitrogen protection, and triethylamine (Et) was added ₃ N, 22.5g, 222.2mmol) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 50.7g, 133.4mmol), stirring for 15min, adding morpholine (10.7g, 111.1mmol), stirring for 10min, heating to room temperature at the speed of 12 ℃/h, and continuing to stir for reaction for 20 h; after the reaction, the reaction solution was poured into 500mL of 1mol/L aqueous sodium hydroxide solution for extraction, and the aqueous phase was extracted with dichloromethane 2 times, the volume of dichloromethane used for each extraction being200mL of the solid is collected, the pH value of the aqueous phase is adjusted to 5-6 by 2mol/L hydrochloric acid, then dichloromethane is used for extraction for 3 times, the volume of dichloromethane used in each extraction is 500mL, organic phases are combined, the organic phases are washed by saturated saline solution and then dried by anhydrous sodium sulfate, filtration is carried out, the obtained filtrate is concentrated to obtain yellow solid, and the yellow solid is purified by silica gel column chromatography (the purifying reagents are dichloromethane and methanol, the volume ratio of the dichloromethane to the methanol is 5: 1) to obtain 22.0g of white solid, the yield is 42.0%, and the purity is 90.1%.

The white solid was characterized by the following specific characterization data:

MS：m/z：477[M+H] ⁺ 。

the above results indicate that the white solid is a compound having the structure shown in formula IV.

(3) Adding DCM (220mL) and the compound having the structure shown in formula IV (22.0g, 46.2mmol) into a three-neck flask under stirring, cooling the temperature of the obtained system to 0 ℃ under the protection of nitrogen, and adding Et ₃ N (18.7g, 184.8mmol) and acetyl chloride (10.9g, 138.6mmol) at 0 deg.C with stirring for 30 min; after the reaction is finished, the obtained reaction liquid is poured into 500mL of water for extraction, the obtained water phase is continuously extracted for 2 times by using dichloromethane, the volume of dichloromethane used for each extraction is 200mL, organic phases are combined, the organic phases are washed by saturated saline solution and then dried by anhydrous sodium sulfate, the organic phases are filtered, the obtained filtrate is concentrated to obtain yellow solid, and the yellow solid is purified by silica gel column chromatography (the purifying reagents are dichloromethane and methanol, the volume ratio of the dichloromethane to the methanol is 5: 1) to obtain 10.6g of white solid, the yield is 65.0 percent, and the purity is 96.1 percent.

MS：m/z：519[M+H] ⁺ 。

the above results indicate that the white solid is a compound having the structure shown in formula V.

(4) Adding a compound with a structure shown in a formula V (10.6g,0.0204mol) and 3-bromopropionic acid (3.4g,0.0245mol) into 106mL of dichloromethane in turn under the stirring condition at 10 ℃, stirring for reaction for 3h at 10 ℃, keeping the stirring condition, dropwise adding the obtained reaction liquid into 5 ℃ anhydrous ether (1.0kg), stirring for 10min at 5 ℃ after dropwise adding, filtering, drying the obtained filter cake at room temperature under reduced pressure to obtain 8.0g of white solid, wherein the yield is 60.0%, and the purity is more than 96% by an LC-MS method (the detection conditions are shown in Table 1).

high resolution mass spectrometry:

high resolution mass spectrometry: m/z 577.3470M ⁺ ；

A simulated molecular formula: c ₃₁ H ₄₉ N ₂ O ₈ ⁺ ；

Theoretical calculation value: 577.3489, respectively;

unsaturation degree: 9;

nuclear magnetic resonance:

1H-NMR(400MHz,CD ₃ CN∶D ₂ o ═ 1: 1) showed overlap of the fat region signals, and the presence of 3 methyl signals δ H1.149, δ H1.175, δ 0H 2.546 could be determined by HSQC; there are 16 methylene signals, δ 1H 1.787(2H), δ 2H 2.516(4H), δ 3H 1.949 and δ 4H 1.646, δ 5H 2.094 and δ 6H 2.492, δ 7H 1.317 and δ 8H 2.036, δ 9H 2.319 and δ H2.816, δ 0H 2.703(2H), δ 1H 1.724 and δ 2H 2.147, δ 3H 3.851(2H), δ 4H 3.888(2H), δ 5H 4.522(2H), δ 6H 4.039 and δ 7H 4.248, δ 8H 4.088 and δ 9H 4.224, δ H3.972 (2H) and δ 0H 3.989(2H), wherein, delta 1H 3.851(2H), delta 2H 3.888(2H), delta 3H 4.522(2H), delta 4H 4.039 and delta 5H 4.248, delta 6H 4.088 and delta 7H 4.224, delta 8H 3.972(2H) and delta 9H 3.989(2H) are all connected with atoms with stronger electronegativity; there are 6 methine signals δ H1.881, δ 0H 2.749, δ 1H 1.489, δ H2.119, δ H5.000, δ H5.472, where δ H5.000, δ H5.472 are attached to the more electronegative atom.

13C-NMR(100MHz,CD ₃ CN∶D ₂ O1: 1) shows the presence of 4 carbonyl carbon signals δ C166.804, δ C170.354, δ 0C 170.763, δ 1C 176.646; there are 2 quaternary carbon signals δ 2C 39.365 and δ C44.381; there are 6 methine carbon signals δ C33.008, δ C38.585, δ C46.335, δ C46.488, δ C69.388, δ C77.743, where δ C77743 is attached to the oxygen atom, the Δ C69.388 signal is very weak, the peak is particularly low, and the peak shape is broad, indicating that it is affected by the nitrogen cation; the presence of 16 methylene carbon signals δ C21.037, δ 0C 23.107(2), δ 1C 27.001, δ 2C 27.161, δ 3C 30.456, δ 4C 35.756, δ 5C 35.851, δ 6C 37.047, δ 7C 41.202, δ 8C 45.759, δ 9C 60.515 and δ C63.249, δ 0C 65.035, δ 1C 65.852, δ C66.019, δ C60.515 and δ C63.249 is very weak, particularly low in peak appearance, and broad in peak shape, indicating that it is affected by the nitrogen positive ion; there are 3 methyl carbon signals δ C12.099, δ C14.803, δ C20.016, where δ C20.016 is ortho to the carbonyl.

By the formula C ₃₁ H ₄₉ N ₂ O ₈ ⁺ And relative molecular weight 577 identifies the compound as having 4 carbonyl groups with 4 unsaturations, the remaining 5 unsaturations being presumed to have 5 cyclic structures.

FIG. 1 is a schematic diagram of the site-directed labeling of an atom in a compound having the structure shown in formula A prepared in example 1.

Delta H5.472 (H-20), delta H5.000 (H-19), delta H2.492 (18), delta H2.094 (18), delta H1.489 (17), delta H2.119 (16a), delta H1.724 (22), delta H2.147 (22), delta H1.787 (23), delta H1.949 (15), delta H2.749 (24), delta H2.816 (12), delta H2.319 (12), delta H1.317 (14) in 1H1H-COSY, delta H5.472 (H-20) and delta C12.099 (C-32), delta C27.161 (C-18), delta C37.047 (C-22), delta C44.381 (C-21), delta C170.354 (C-36) in HMBC have remote correlation, and the combination of residual unsaturation, the parent nucleus of formula A is presumed to be a steroid, delta H1.149 (H-32) and delta C77.743 (H-20) in HMBC, delta C44.381 (C-21), delta C37.047 (C-22) have remote correlation, delta H1.175 (H-33) and delta C39.365 (25) have remote correlation, and 2 methyl groups respectively belong to the 21-position and the 25-position of a mother nucleus and accord with the structural characteristics of the steroid compounds.

Delta H3.989 (H-31) in 1H1H-COSY is related to delta H3.851, delta H3.989 (H-31) in HMBC is remotely related to delta C65.852 (C-27) and delta C45.759 (C-28), the hydrocarbon data of the methylene group at the 27 position and the methylene group at the 31 position are both shown to be connected with an oxygen atom, and the hydrocarbon data of the methylene group at the 28 position and the methylene group at the 30 position are both shown to be connected with a nitrogen atom, and the existence of a morpholine ring is inferred. In HMBC, delta H3.851 (H-30) and delta H3.888 (H-28) are both remotely related to delta C170.763 (C-10), delta H2.703 (H-9) is remotely related to delta C170.763 (C-10), delta C39.365 (C-25) and delta C14.803 (C-33), methylene hydrocarbon data at the 9-position are shown to be ortho to the amide carbonyl group, and the existence of a 2-morpholino-2-oxyethyl structural fragment is deduced to be located at the 25-position.

In HMBC, delta H2.546 (H-38) is remotely related to delta C170.354 (C-36), the existence of acetyl is proved, the data of the methine hydrocarbon at the 20 th position show that the methylene is connected with an oxygen atom, and in HMBC, delta H5.472 (H-20) is remotely related to delta C170.354 (C-36), and the acetoxyl group is deduced to be positioned at the 20 th position.

The delta H2.319 (H-12) and the delta C176.646 (C-11) in HMBC have long-range correlation, and the existence of a carboxymethyl structural fragment is proved. The delta H2.319 (H-12) and the delta C54.428 (C-24) in HMBC have remote correlation, which indicates that the carboxymethyl is positioned at the 13-position.

Delta H4.522 (H-6) and delta C166.804 (C-7) in HMBC are related, and the existence of a carboxymethyl structural fragment is proved; delta H2.516 (H-3&4) and delta H4.088 (H-2), delta H4.248 (H-5) in 1H1H-COSY and TOCSY, the 2 methylene hydrocarbon data at position 2/5 showing attachment to the nitrogen atom, combined with the remaining unsaturation, presumably exists as a pyrrolidinyl group; the hydrocarbon data for the methine group at position 19 and the methylene group at position 6 both show attachment to the nitrogen atom, and taken together the presence of the 1-propenylpyrrolidinium group at position 19 is presumed.

The structure shown in the formula A is subjected to hydrocarbon data attribution through 1H-NMR, 13C-NMR, 1H1H-COSY, HSQC, HMBC and TOCSY characterization. From the above characterization results, it is understood that the white solid prepared in example 1 is an androstane derivative having a structure represented by formula a.

Example 2

An androstane derivative having the structure shown in formula B was prepared according to the following reaction scheme:

(1) adding n-butanol (500mL) and a compound (100g, 278mmol) with a structure shown in formula II into a three-mouth reaction bottle in sequence under the condition of stirring, then adding ethanolamine (25.6g, 420mmol) at the temperature of 25 ℃ under the condition of stirring, and heating to 120 ℃ for reaction for 30 hours; after the reaction, the obtained reaction solution was cooled to room temperature, and then poured into 3L of water, and extracted 3 times with methyl tert-butyl ether, wherein the volume of the methyl tert-butyl ether used for each extraction was 500mL, the organic layers were combined and washed with brine, and after washing, the obtained organic phase was dried over anhydrous sodium sulfate, filtered, and purified by silica gel column chromatography (the purification reagents were dichloromethane and methanol, the volume ratio of dichloromethane and methanol was 5: 1) to obtain 106g of a white solid, the yield was 90%, and the purity was 96.0% (the purity detection method was the same as in example 1).

MS：m/z：421[M+H] ⁺ 。

the above results indicate that the white solid is a compound having the structure shown in VI.

(2) Adding MeOH (1.00L) and a compound (106g, 252mmol) with a structure shown in formula VI into a three-necked flask under stirring, reducing the temperature of the obtained system to 0 ℃, adding sodium methoxide (54.4g, 1.00mol) and methyl formate (60.53g, 1.00mol), naturally heating the obtained reaction liquid to room temperature, and continuing stirring for reaction for 10 hours; after the reaction is finished, concentrating the obtained reaction liquid to be dry, dissolving the residual solid in 1.00L of water, extracting for 3 times by using methyl tert-butyl ether, wherein the volume of the methyl tert-butyl ether used for each extraction is 300mL, collecting a water phase, adjusting the pH of the water phase to 6-7 by using 2mol/L hydrochloric acid, extracting for 3 times by using ethyl acetate, the volume of the ethyl acetate used for each extraction is 400mL, washing an organic phase by using saturated saline solution, drying by using anhydrous sodium sulfate, filtering, concentrating the obtained filtrate to obtain a yellow solid, and purifying by silica gel column chromatography (the volume ratio of the dichloromethane to the methanol is 5: 1) to obtain 92.7g of a white solid, wherein the yield is 82.0%, and the purity is 96.1%.

MS：m/z：449[M+H] ⁺ 。

the above results indicate that the white solid is a compound of the structure shown in VII.

(3) Adding DCM (1.00L) and the compound having the structure shown in formula VII (92.7g, 207mmol) into a three-necked flask under stirring, cooling the obtained system to 5 ℃ under the protection of nitrogen, and adding Et ₃ N (62.8g, 621mmol) and acetyl chloride (24.3g, 310mmol) were reacted with stirring for 30 min; after the reaction is finished, the obtained reaction solution is poured into 500mL of water for extraction, the water phase is extracted for 2 times by using dichloromethane, the volume of dichloromethane used for each extraction is 200mL, organic phases are combined, the organic phases are washed by saturated saline solution and then dried by anhydrous sodium sulfate, the filtration is carried out, the filtrate is concentrated to obtain yellow solid, and then the yellow solid is purified by silica gel column chromatography (the purifying reagents are dichloromethane and methanol, the volume ratio of the dichloromethane to the methanol is 5: 1) to obtain 43.7g of white solid, the yield is 43%, and the purity is 97.0%.

MS：m/z：491[M+H] ⁺ 。

the above results indicate that the white solid is a compound having the structure shown in VIII.

(4) DCM (400mL), the compound having the structure shown in formula VIII (43.7g,89mmol) and 3-bromopropene (12.9g,107mol) were added to a three-necked flask with stirring at 10 ℃ and reacted for 3 hours with stirring at 10 ℃; after the reaction is finished, stirring conditions are kept, the obtained reaction solution is dripped into anhydrous ether (3.00kg) at the temperature of 5 ℃, stirring is carried out for 10min at the temperature of 5 ℃ after dripping is finished, filtering is carried out, and the obtained filter cake is dried under reduced pressure at normal temperature to obtain white solid 50.0g, the yield is 90%, and the purity is more than 96% (the purity detection method is the same as that of example 1);

high resolution mass spectrometry:

high resolution mass spectrometry: m/z 531.3789M ⁺ ；

Simulated molecular formula: c ₃₁ H ₅₁ N ₂ O ⁵⁺ ；

Theoretically calculated values are as follows: 531.3798, respectively;

unsaturation degree: 8;

nuclear magnetic resonance:

1H-NMR(600MHz,CD ₃ CN∶D ₂ o1: 1) shows fat region signal overlap, and the presence of 3 methyl signals δ H0.731, δ H0.819, δ 0H 2.121 can be determined by HSQC; there are 15 methylene signals, δ 1H 1.426 and δ 2H 1.278, δ 3H 2.060(4H), δ 4H 2.036 and δ 5H 1.686, δ 6H 1.105 and δ 7H 1.402, δ 8H 0.865 and δ 9H 1.622, δ H1.808 and δ 0H 1.442, δ 1H 1.250 and δ 2H 1.700, δ 3H 1.610 and δ 4H 1.366, δ 5H 3.224 and δ 6H 3.269, δ 7H 3.525 and δ 8H 3.709, δ 9H 3.568(4H), δ H3.850 (2H), δ 0H 5.604 and δ 1H 5.540, where δ 2H 5.604 and δ 3H 5.540 are terminal alkene hydrogen signatures, δ 4H 3.224 and δ 5H 3.269, δ 6H 3.525 and δ 7H 3.709, δ 7H 3.568H 56 (δ 9H) are strongly negative atoms connected to δ H2H 850; there are 10 methine signals δ H1.402, δ 0H 1.585, δ 1H 0.991, δ 2H 0.761, δ 3H 3.593, δ 4H 3.925, δ 5H 3.975, δ 6H 5.024, δ 7H 5.970, δ 8H 7.964, where δ 9H 7.964 is the formyl proton signal, δ H5.970 is the alkene hydrogen signal, δ H3.593, δ H3.925, δ H3.975, δ H5.024 attached to the atom with the stronger electronegativity.

13C-NMR(150MHz,CD ₃ CN∶D ₂ O ═ 1: 1) shows the presence of 2 carbonyl carbon signals δ C165.426, δ C170.304; there are 2 quaternary carbon signals δ 0C 35.004 and δ 1C 44.731; there are 10 methine carbon signals δ 2C 33.064, δ 3C 37.906, δ 4C 46.065, δ 5C 54.428, δ 6C 60.232, δ 7C 64.199, δ 8C 67.560, δ 9C 77.586, δ C125.031, δ 0C 165.426, where δ 1C 165.426 is the formyl carbon signal, δ 2C 77.586 is attached to the oxygen atom, δ 3C 125.031 is the alkene carbon signal, δ 4C 67.560 is the weak signal, the peak appearance is particularly low, and the peak shape is broad, indicating that it is affected by the nitrogen positive ion; there are 15 methylene carbon signals δ 5C 20.158, δ 6C 22.980(2), δ 7C 26.918, δ 8C 27.136, δ 9C 30.418, δ C34.603, δ 0C 37.089, δ 1C 40.451, δ 2C 42.704, δ 3C 58.242, δ 4C 62.631(2), δ 5C 64.695, δ C127.664, where δ C127.664 is the terminal alkene carbon signal; there are 3 methyl carbon signals δ C12.211, δ C16.053, δ C20.020, where δ C20.020 is ortho to the carbonyl.

By the formula C ₃₁ H ₅₁ N ₂ O ⁵⁺ And a relative molecular weight of 531 determines that the compound has 2 carbonyls1 olefinic double bond, is 3 unsaturations, the remaining 5 unsaturations presuming the presence of 5 cyclic structures.

Delta H5.024 (H-20), delta H3.975 (H-19), delta H1.686 (H-18), delta H2.036 (H-18), delta H0.991 (H-17), delta H1.402 (H-16), delta H0.865 (H-15), delta H1.105 (H-14), delta H0.761 (H-24), delta H1.278 (H-23), delta H1.700 (H-22), delta H1.585 (H-13), delta H1.610 (H-9), delta H1.366 (H-9), delta H3.593 (H-10), delta H3.925 (H-11), delta H1.808 (H-12) are relevant in 1H1H-COSY and TOCSY; in HMBC delta H0.991 (H-17) and delta C26.918 (C-18), delta C44.731 (C-21) have remote correlation, delta H1.426 (H-23) and delta C35.004 (C-25), delta C54.428 (C-24), delta C37.906 (C-13), delta C44.731 (C-21) have remote correlation, and the parent nucleus of formula I is conjointly assumed to be a steroid in combination with the remaining unsaturation, delta H0.731 (H-30) and delta C44.731 (C-21), delta C37.089 (C-22), delta C46.065 (C-17), delta C77.586 (C-20) have remote correlation, delta H0.819 (H-31) and delta C35.004 (C-25), delta C40.451 (C-9), delta C37.906 (C-13), delta C54.428 (C-24) have remote correlation, it is proved that 2 methyl groups belong to the 21-position and the 25-position of the mother nucleus respectively, and accord with the structural characteristics of the steroid.

H-27 delta H3.525/3.709 in 1H1H-COSY is related to H-28 delta H3.224/3.269, and the data of 2 connected methylene hydrocarbon shows that the methylene hydrocarbon is connected with an oxygen atom and a nitrogen atom respectively; in HMBC, delta H7.964 (H-44) has long-range correlation with delta C60.232 (C-10), delta H3.593 (H-10) has long-range correlation with delta C165.426 (C-44), and delta H3.224/3.269 (H-28) has long-range correlation with delta C58.242 (C-27) and delta C165.426 (C-44), and the existence of an N- (2-hydroxyethyl) formamide structure segment at position 10 is inferred.

The remote correlation between delta H2.121 (H-36) and delta C170.304 (C-34) in HMBC proves that acetyl exists, the methylene hydrocarbon data of the 20 th position shows that the methylene hydrocarbon is connected with an oxygen atom, the remote correlation between delta H5.024 (H-20) and delta C170.304 (C-34) in HMBC deduces that acetoxyl is positioned at the 20 th position.

Delta H5.970 (H-7) and delta H3.850 (H-6) in 1H1H-COSY and TOCSY are related, and delta H5.604 (H-8) proves that allyl is present; 1H1H-COSY and TOCSY δ H3.568 (H-2&5) and δ H2.060 (H-3&4) are related, the 2 methylene hydrocarbon data at position 2/5 showing attachment to the nitrogen atom, combined with the remaining unsaturation, presumably the presence of pyrrolidinyl; the hydrocarbon data of the 19-methine and 6-methylene show that both are linked to the nitrogen atom, and the presence of the 1-propenyl pyrrolidinium group is presumed to be at the 19-position overall;

the structure shown in the formula B is subjected to hydrocarbon data attribution through 1H-NMR, 13C-NMR, 1H1H-COSY, HSQC, HMBC and TOCSY characterization. From the above characterization results, it is understood that the white solid prepared in example 2 is an androstane derivative having a structure represented by formula B.

Test example 1

The inhibition effect of the androstane derivative with the structure shown in the formula A and the formula B on BCR-ABL tyrosine kinase and the corresponding mRNA level is tested and compared with the compound with the structure shown in the formula I in the patent 201910706129.7, and the inhibition effect is as follows:

the K562 cell line (human chronic myelogenous leukemia cell line) was used in the experiment. K562 cells were routinely cultured in RPMI 1640 medium (containing 10% fetal bovine serum, 100. mu.g/mL penicillin and 100. mu.g/mL streptomycin) and placed at 37 ℃ in 5% CO ₂ The culture is carried out in a constant temperature incubator. The cells are subjected to liquid change and passage for 1 time every other day, and K562 cells in the logarithmic growth phase are taken for experiment.

MTT assay for inhibitory effect of compounds on cell proliferation: the K562 cells were seeded in 96-well plates at 1X 10 cells per well, respectively ⁵ The cells, 100. mu.L in volume, were incubated at 37 ℃ in 5% CO ₂ Culturing in a constant temperature incubator for 3 days. After the culture was completed, 100. mu.L of each compound solution having the structures represented by formula I, formula A and formula B, which was diluted with RPMI 1640 medium and had the concentrations of 1. mu. mol/L and 10. mu. mol/L, was added to each fraction, and the blank wells were replaced with medium containing no cells. After 24h, 48h and 72h of incubation, 20. mu.L of MTT solution (5mg/mL) was added to each well, the incubation was continued for 4h, centrifugation was performed, the supernatant was carefully discarded in the absence of light, and 150. mu.L of DMSO was added to each well to dissolve the crystals in the wells sufficiently. And reading the absorbance (A) value of each hole at 490nm and 620nm of the reference wavelength on the microplate reader. The cell proliferation inhibition rate was calculated and the experiment was repeated three times. Cell proliferation inhibition rate (control group a value-test group a value)/pairControl group A value X100%.

Detecting the expression level of BCR/ABL mRNA by using a real-time fluorescent quantitative PCR technology: collecting the total RNA of K562 cells and Trizol reagent extracted cells after 48h of treatment of the compounds with the structures shown in the formula A and the formula B for PCR detection. The DNA sequence of BCR/ABL gene is searched from NCBI database by using human GAPDH as internal reference, and primer premier 5.0 software is used for designing primer.

GAPDH upstream primer: 5'-CTCTGCTCCTCCTGTTCGAC-3', respectively;

a downstream primer: 5'-CACTGTGTTGGCGTACAGG-3', respectively;

BCR/ABL upstream primer: 5'-AATGCCGCTGAGTATCTGCT-3';

a downstream primer: 5'-GCCATCAGAAGCAGTATTGA-3' is added.

The PCR amplification conditions were: pre-denaturation at 94 ℃ for 10min, annealing at 60 ℃ for 60s, and extension at 72 ℃ for 30s for 45 cycles.

The results of the experiment were analyzed using computer software for threshold cycle number (CT) with 3 replicate wells per sample and calculated as an average. The mean value of GAPDH was designated as CT0 and the mean value of the experimental groups as CT (X). Calculating the expression amount relative to GAPDH, and calculating the formula: 2 ^–ΔΔCT ＝2 ^{-[ΔCT(X)-(CT(X)-CT0)]} . Specific test results are shown in tables 2-3.

TABLE 2 inhibition of K562 cell proliferation (%). of test substances at different concentrations

According to the data in the table 2, after selecting different concentrations of formula I, formula A and formula B (0, 1, 10. mu. mol/l) to treat K562 cells for 24h, 48h and 72h, the MTT method is adopted to detect the proliferation inhibition effect on K562 cell lines. The result shows that the compound with the structure shown in the formula I can not inhibit the proliferation of K562 cells, while the compound with the structure shown in the formula A, B has obvious inhibition effect on the proliferation of the K562 cells, and the inhibition effect is increased along with the prolonging of time, so that the compound has certain dose dependence.

TABLE 3 relative expression level (x. + -. s) of compounds acting on 48h BCR-ABL gene of K562 cells

According to the data in the table 3, after K562 cells are treated by selecting different concentrations of the compounds shown in the formulas I, A and B (0, 1 and 10 mu mol/L), the expression level of BCR/ABL mRNA is detected by a real-time fluorescence quantitative method, and the result shows that the compound with the structure shown in the formula I can not inhibit the expression of the BCR-ABL mRNA at the concentration of 0 mu mol/L, 1 mu mol/L or 10 mu mol/L, but the compound with the structure shown in the formula A, B has an inhibition effect on the BCR-ABL mRNA; in addition, compared with the blank group, the BCR/ABL mRNA expression quantity is reduced after the treatment of the structural compounds shown in the formula A and the formula B, and the treatment has certain dose dependence.

Test example 2

The inhibition of the Hedgehog signaling pathway by androstane derivatives of the structure shown in formula a and androstane derivatives of the structure shown in formula B were tested according to the method of example 3 of patent invention 201910706129.7 and compared to compounds of the structure shown in formula I of patent invention 201910706129.7, as follows:

mouse reporter cell line-10T 1/2-GliLuc [ s12] cells (derived from cell line C3H10T1/2ATCC # CCL-226); mouse embryonic fibroblasts; growth medium: dulbecco's Modified Eagles Medium (DMEM) with 10% Fetal Bovine Serum (FBS), 10 units/mL penicillin, 100. mu.g/mL streptomycin, 2mM glutamine and 10mM HEPES.

Human reporter cell line-HEPM-GliLuc [ MZ24] -cell (derived from HEPM, human embryonic palatine mesenchyme ATCC # CRL-1486); growth medium: the minimal essential medium (MEM; containing Earle's salts) was used with 10-20% Fetal Bovine Serum (FBS), 10 units/mL penicillin, 100. mu.g/mL streptomycin, 2mM glutamine, and 10mM HEPES pH7.2.

Microtiter plate (MTP): cells were plated in 96-well MTP (white, flat bottom, clear field) for luciferase assay.

Luciferase assay medium: DMEM with 0.5% FBS, 10 units/mL penicillin, 100. mu.g/mL streptomycin, 2mM glutamine and 10mM HEPES pH7.2.

PBS/Ca/Mg mixture: containing 0.5mM CaCl ₂ And 1mM MgCl ₂ Phosphate Buffered Saline (PBS).

Measurement procedure:

s12 and MZ24 cells were seeded separately in separate media and placed at 37 ℃ in 5% CO ₂ The incubator of (2) for cultivation. The S12 and MZ24 cells contained a luciferase reporter driven by the Hedgehog-responsive Gli promoter, respectively. The cell cultures were passaged every 3 days in a sub-confluent state (sub-confluency) in which the mass ratio of S12 to cell cultures was 1: (20-40); the mass ratio of MZ24 to cell culture was 1: (3-10). The cells were harvested, diluted with growth medium, seeded in microtiter plates at 10,000-20,000 cells (S12)/100 μ L/well or 20,000-30,000 cells (MZ24)/100 μ L/well, and incubated at 37 ℃ and 5% CO ₂ The cells were further incubated for about 48h in the incubator of (1).

After 48h incubation, the growth medium in the microtiter plates was replaced with fluorimetry medium containing S12 and a compound having the structure shown in formula I, fluorimetry medium containing S12 and Vismodegib, an FDA-approved Hedgehog pathway inhibitor, and fluorimetry medium containing only S12 (100. mu.L/well), respectively, for the S12 cell line, with the S12 cell line varying concentrations in each medium ranging from 0.1 to 0.3. mu.g/mL. Aiming at MZ24 cell lines, a fluorescence determination culture medium containing MZ24 and a compound with a structure shown in formula A (or formula B), a fluorescence determination culture medium containing MZ24 and Vismodegib (a Hedgehog pathway inhibitor approved by FDA) and a fluorescence determination culture medium (100 mu L/hole) containing only MZ24 are respectively used for replacing the growth culture medium in a microtiter plate, and the change concentration of the MZ24 cell line in each culture medium is 0.5-1.0 mu g/mL. The cells were further incubated for 24 h.

Then using luciferase reporter gene detection kit (LucLite) ^TM ) Detecting the microtiter plate, and testing a series of compounds with different concentrationsActivity at a particular target is then fitted to a curve and IC50 values are calculated according to IC50 methods well known in the art. The assay procedure was modified from the manufacturer's protocol in which the medium was removed and the reaction mixture was run in a 1: 1 PBS/Ca/Mg: the lysis buffer re-dissolves the substrate, rather than directly re-dissolving the substrate with the lysis buffer.

Briefly, the ratio by volume of 1: 1 PBS/Ca/Mg was mixed with lysis buffer and 10mL was added to each substrate vial (1000 assay kits). The assay medium from the microtiter plate is then discarded and 100. mu.L of this substrate mixture is added to each well. Plates were incubated at room temperature for 30min, and then Relative Light Units (RLUS) representing the relative expression levels of luciferase reporter gene were determined using a Topcount reader (Packard) or an Analyst reader (Molecular Devices). And the IC50 values for the corresponding compounds were calculated.

Table 4 is the IC50 values for inhibition of Hedgehog pathway signaling for specific compounds measured using mouse [ S12] and human [ MZ24] Gli reporter cell lines according to the procedure described above.

TABLE 4 test results

Compound (I)	IC50(S12)	IC50(MZ24)	IC50 mean value (μ M)
				A compound having a structure represented by formula A	0.016	0.021	0.019
A compound having a structure represented by formula B	0.016	0.020	0.018
				Vismodegib	0.020	0.024	0.022

According to the data in the table 4, the compounds provided by the invention and having the structures shown in the formulas A and B also have the Hedgehog pathway signal inhibition effect, and the IC50 of the compounds reaches the level equivalent to the Vismodegib.

As can be seen from the test examples, the androstane derivative provided by the invention and having the structure shown in the formula A or the formula B has a very good inhibition effect on BCR-ABL tyrosine kinase and the corresponding mRNA level, and the androstane derivative also has a very good inhibition effect on a Hedgehog signaling pathway, which indicates that the androstane derivative has a remarkable treatment effect on chronic myelogenous leukemia.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. An androstane derivative having the structure of formula a:

2. the process for preparing an androstane derivative according to claim 1 comprising the steps of:

3. the preparation method according to claim 2, wherein an oxidant used in the oxidation reaction in the step (1) is hydrogen peroxide, and the compound having the structure shown in formula II and H in the hydrogen peroxide ₂ O ₂ The molar ratio of (A) to (B) is 10 (200-300);

the mol ratio of the compound with the structure shown in the formula III to morpholine in the step (2) is 1 (1-2);

the molar ratio of the compound with the structure shown in the formula IV to acetyl chloride in the step (3) is 10 (15-30);

the mol ratio of the compound with the structure shown in the formula V to the 3-bromopropionic acid in the step (4) is 1: (1-2).

4. The preparation method according to claim 2 or 3, wherein the temperature of the oxidation reaction in the step (1) is 85-95 ℃ and the time is 18-22 h;

5. The method according to claim 2, wherein the step (4) further comprises, after the quaternization reaction:

6. Use of an androstane derivative according to claim 1 for the preparation of a medicament for the treatment of chronic myelogenous leukemia.

7. The use of claim 6, wherein the medicament for treating chronic myelogenous leukemia is a Hedgehog signaling pathway inhibitor medicament, a BCR-ABL tyrosine kinase inhibitor medicament, or a BCR-ABL tyrosine kinase mRNA inhibitor medicament.