Synthetic method of HE3286

Abstract

The invention discloses a synthesis method of HE3286, belongs to the technical field of medicine preparation, and provides a brand-new synthesis method of HE3286, according to the method, dehydroepiandrosterone acetate is used as a raw material, and high-purity HE3286 is efficiently prepared through only five steps of ethynylation, diacetylation, oxidation, reduction and deprotection. The HE3286 synthesis method provided by the invention has the characteristics of cheap and easily available reagents, simplicity and safety in operation, low cost, no pollution and easiness in large-scale industrial production, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and in particular relates to a method for synthesizing HE3286. Background Art

[0002] HE3286, also known as Bezisterim in English and 17α-ethynyl-5-androstene-3β,7β,17β-triol in chemical form, is a novel steroidal compound for the treatment of autoimmune diseases, with promising therapeutic effects in rheumatoid arthritis and obesity-related diabetes. It has shown no adverse effects in genotoxicity, systemic toxicity, or neurotoxicity studies, and no significant effects in long-term toxicity or reproductive toxicity. It has good cardiopulmonary safety. Studies of estrogenic effects have shown mild estrogenic effects in rats, and its major metabolite also exhibits no estrogenic effects, demonstrating a favorable safety profile.

[0003] So far, patent WO2009149392 and patent CN114478672A have reported four synthetic routes of HE3286.

[0004] In the first route, dehydroepiandrosterone is used as the starting material. The hydroxyl group at position 3 is first protected with a trimethylsilyl group. Then, an acetyl group at position 17 is introduced by nucleophilic addition under the action of trimethylsilyl acetylene and n-butyl lithium. During the process, the trimethylsilyl group dissociates, and an acetyl group is introduced again to protect the hydroxyl group. The allylic position is then oxidized under the action of tert-butyl hydroperoxide and cuprous iodide. After reduction and deprotection, the target product HE3286 is obtained with an overall yield of 15%. This method has a cumbersome route and low yield. The use of saccharin as a catalyst has poor selectivity and low catalytic activity, making it unsuitable for general industrial production. The lithium reagent used is expensive and requires large amounts, resulting in high production costs. The acetylation process has harsh reaction conditions and safety risks, making it unsuitable for industrial production. The reaction route of this method is as follows:

[0005]

[0006] In the second route, dehydroepiandrosterone acetate is used as the starting material. A ketal structure is introduced to the carbonyl group at position 17 via the action of p-toluenesulfonic acid and ethylene glycol. The product is then oxidized with tert-butyl hydroperoxide and sodium perchlorate, followed by reduction and removal of the glycol protection group, followed by deacetylation. The two exposed hydroxyl groups are then further protected by the introduction of trimethylsilyl groups. Acetylation is then performed using the same addition method of trimethylsilyl acetylene and n-butyl lithium, and finally the target product is obtained by deprotection. This method introduces too many unnecessary protecting groups. The multiple introduction and removal of groups makes the route more complicated and the unit reaction processing more difficult. The 6% total yield is also at a relatively low level, which greatly increases production costs and is not suitable for industrial production. The reaction route of this method is as follows:

[0007]

[0008] In the third route, dehydroepiandrosterone acetate is used as the starting material, and the C7 position is first oxidized. Then, the carbonyl hydroxylamine is converted to an oxime structure under the action of hydroxylamine hydrochloride. After reducing the carbonyl group at the 7 position, the hydroxylamine protection is removed by reduction with sodium thiosulfate. Finally, the target compound is obtained by acetylation and deprotection in the same way as the above route. The total yield of this method reaches 30%, but it still sacrifices production costs and production risks. It is not suitable for industrial production: the hydroxylation reagent has poor selectivity, is toxic and corrosive, has adverse effects on water, soil and atmosphere, and has harsh reaction conditions, requiring high equipment and operation control. There are many restrictions on the use of sodium thiosulfate, and sulfur-containing compounds pose environmental risks. The use of saccharin as a catalyst has poor selectivity and low catalytic activity, and is generally not an option for industrial production. The reaction route of this method is as follows:

[0009]

[0010] In the fourth route, the trihydric compound produced by drospirenone is used as the raw material. First, the configuration of the 7-hydroxyl group is reversed by a transposition reaction under strong acid. Then, acyl protection is introduced at the 3- and 15-hydroxyl groups using pivaloyl chloride. The synthesis of the androstane D ring is completed by elimination and hydrogenation. Then, the acetylene group is introduced by an acetylene Grignard reagent. Finally, the protecting group is removed to obtain the target compound HE3286. The raw material selected for this method is the intermediate trihydric compound obtained by synthesizing drospirenone by a biological fermentation method. The raw material is expensive and difficult to obtain. The palladium-carbon catalyst used in the hydrogenation reaction is expensive and costly, the use of hydrogen has high safety risks, and the two unsaturated bonds in the objective structure of the compound make the hydrogenation selectivity high and difficult, which may cause problems such as over-hydrogenation or unsatisfactory selectivity. Similarly, the presence of the trihydroxyl group makes the introduction of acyl protection selectivity high and difficult. There are also major problems in the selection of acetylation reagents. The acetylene Grignard reagent is expensive, has harsh usage conditions, and poor selectivity, which makes the introduction of by-products difficult to separate and purify. All of the above problems may affect the practicality of the actual operation of this method and its applicability in industrial production. The reaction route of this method is as follows:

[0011]

[0012] In summary, the existing HE3286 synthesis route has many technical problems that need to be solved. In view of this, the present invention aims to provide a synthesis method with a simple route, mild conditions, low cost, low separation and purification difficulty, high efficiency, and industrial scalability. Summary of the Invention

[0013] In order to overcome the above-mentioned technical problems existing in the current field, the purpose of the present invention is to provide a method for synthesizing HE3286 with a simple route, which shortens the original 8-step reaction and 6-step reaction to 5 steps through alkynylation, diacetylation, oxidation, reduction, and deprotection, has mild conditions, low costs of raw materials and reagents, does not rely on large-scale silica gel column purification, has low separation and purification difficulty, high efficiency, and can be industrially scaled up for production.

[0014] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0015] A first aspect of the present invention provides a method for synthesizing HE3286. The method uses dehydroepiandrosterone acetate as a raw material, and obtains HE3286 through acetylation, diacetylation, oxidation, reduction, and deprotection. The reaction formula is shown below.

[0016]

[0017] In the present invention, HE3286 is known as Bezisterim in English, 17α-ethynyl-5-androstene-3β,7β,17β-triol in chemical form, and has a CAS number of 1001100-69-1. HE3286 is a synthetic derivative of the natural anti-inflammatory steroid β-AET and an orally active partial inhibitor of NF-κB. HE3286 can reduce pro-inflammatory signals, including IL-6 and matrix metallopeptidase 3. Furthermore, HE3286 can freely cross the blood-brain barrier in mice. HE3286 is useful in the research of ulcerative colitis, arthritis, experimental autoimmune encephalomyelitis, and other neurological diseases.

[0018] In the present invention, the dehydroepiandrosterone acetate is the starting material for synthesizing HE3286 of the present invention. Its corresponding English name is Dehydroisoandrosterone 3-acetate, its chemical name is 3-β-hydroxy-deoxyandrost-5-ene-17-one-3-acetate, its corresponding CAS number is 853-23-6, and its corresponding molecular formula is C 21 H 30 O3, molecular weight is 330.461. It should be noted that the present invention has no particular limitation on the source of the starting material, and those skilled in the art can purchase it through conventional channels.

[0019] Furthermore, the synthesis method comprises the following steps:

[0020] (1) Acetylation reaction

[0021] Compound I (dehydroepiandrosterone acetate) is dissolved in an organic solvent, the temperature is controlled, acetylene gas is introduced, an organic base is added, and the reaction is stirred. After the reaction is completed, alcohol is added at room temperature, and the reaction is continued with stirring. After the reaction is completed, acid is added for neutralization, and the mixture is concentrated to a small amount, eluted with water, filtered, and dried to obtain Compound II.

[0022] (2) Diacetylation reaction

[0023] Compound II is dissolved in an organic solvent, acetic anhydride as an acetylation agent is added, an acid-binding agent is added, a catalyst is added, the temperature is controlled, the reaction is stirred, and after the reaction is completed, the temperature is returned to room temperature, the mixture is concentrated to a small amount, water is added for precipitation, and the mixture is filtered and dried to obtain compound III;

[0024] (3) Oxidation reaction

[0025] Compound III is dissolved in organic solvent A, the feeding temperature is controlled, pyridinium dichromate and tert-butyl hydroperoxide are added for oxidation, diatomaceous earth is added as a catalyst, and the reaction is stirred at room temperature. After the reaction is completed, the diatomaceous earth is filtered, the mixture is concentrated to a small amount, water is added for separation, and the crude product is dried to obtain a crude product. The crude product is slurried with organic solvent B, dried, and recrystallized with organic solvent C to obtain compound IV.

[0026] (4) Reduction reaction

[0027] Compound IV is dissolved in organic solvent D, the feeding temperature is controlled, a methanol solution of cerium chloride heptahydrate is added, sodium borohydride as a reducing agent is added, and the reaction is stirred at low temperature. After the reaction is completed, an acid solution is added to quench the reaction. After the quenching is complete, the reaction is concentrated to a small amount, eluted with water, filtered, and dried to obtain a crude product. The crude product is recrystallized by adding organic solvent E and dried to obtain compound V.

[0028] (5) Alcoholysis reaction

[0029] Compound V was dissolved in organic solvent F, and alcohol was added. A base was added at a controlled low temperature, and the reaction was stirred at room temperature. After the reaction was completed, acid was added for neutralization, and the mixture was concentrated to a small amount. Water was added for precipitating, the mixture was filtered, and the mixture was dried to obtain a crude product. The crude product was recrystallized from organic solvent G to obtain the target compound HE3286.

[0030] In some embodiments, the at least amount refers to, under the premise of satisfying subsequent elutriation operation and whole synthesis technique target, the solvent in the reaction system is concentrated to the least possible amount. Specifically, the at least amount does not have a fixed numerical value or ratio, needs operating personnel to judge and adjust according to actual conditions, usually can be determined by observing the state of solution in the concentration process (such as whether becoming thick, whether having product to start precipitation etc.) and in conjunction with experience suitable degree of concentration. Those skilled in the art can carry out routine adjustment and determination to the at least amount in conjunction with the common knowledge in this area.

[0031] Furthermore, in step (1), the organic solvent is N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methyl ethyl ketone, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, dioxane, diethyl ether, methanol and / or ethanol;

[0032] Optionally, in step (1), the organic solvent is tetrahydrofuran;

[0033] Optionally, in step (1), the volume of the solvent used is 8 to 20 times the mass of the substrate;

[0034] Optionally, in step (1), the volume of the solvent used is 11 times the mass of the substrate;

[0035] Optionally, in step (1), the controlled temperature is -5°C to 10°C;

[0036] Optionally, in step (1), the controlled temperature is 5°C;

[0037] Optionally, in step (1), the organic base is potassium tert-butoxide, potassium isobutoxide, potassium isopropoxide and / or potassium ethoxide;

[0038] Optionally, in step (1), the organic base is potassium tert-butoxide;

[0039] Optionally, in step (1), the molar ratio of compound I to potassium tert-butoxide is 1:1 to 1:3;

[0040] Optionally, in step (1), the molar ratio of compound I to potassium tert-butoxide is 1:2;

[0041] Optionally, in step (1), the stirring reaction time is 1.5 to 3 hours;

[0042] Optionally, in step (1), the stirring reaction time is 2 h;

[0043] Optionally, in step (1), the alcohol is methanol, ethanol, tert-butanol and / or isopropanol;

[0044] Optionally, in step (1), the alcohol is methanol;

[0045] Optionally, in step (1), the molar ratio of methanol to compound I is 5:1 to 20:1;

[0046] Optionally, in step (1), the molar ratio of methanol to compound I is 6:1;

[0047] Optionally, in step (1), the stirring reaction time is 20 min to 1 h;

[0048] Optionally, in step (1), the stirring reaction time is 0.5h;

[0049] Optionally, in step (1), the acid used for acid neutralization is dilute hydrochloric acid, dilute sulfuric acid and / or glacial acetic acid; Optionally, in step (1), the acid used for acid neutralization is dilute hydrochloric acid;

[0050] Optionally, in step (1), the mass fraction of the dilute hydrochloric acid is 5% to 25%;

[0051] Optionally, in step (1), the mass fraction of the dilute hydrochloric acid is 20%.

[0052] Further, in step (2), the organic solvent is dichloromethane and / or tetrahydrofuran;

[0053] Optionally, in step (2), the organic solvent is dichloromethane;

[0054] Optionally, in step (2), the volume of the solvent used is 10 to 20 times the mass of the substrate;

[0055] Optionally, in step (2), the volume of the solvent used is 10 times the mass of the substrate;

[0056] Optionally, in step (2), the reaction temperature is 25°C to the reflux temperature of the solvent;

[0057] Optionally, in step (2), the reaction temperature is 38°C;

[0058] Optionally, in step (2), the acid binding agent is triethylamine and / or pyridine;

[0059] Optionally, in step (2), the acid binding agent is triethylamine;

[0060] Optionally, in step (2), the molar ratio of compound II to the acetylating agent acetic anhydride is 1:2 to 1:4; Optionally, in step (2), the molar ratio of compound II to the acetylating agent acetic anhydride is 1:3;

[0061] Optionally, in step (2), the molar ratio of compound II to the acid binding agent is 1:2 to 1:4;

[0062] Optionally, in step (2), the molar ratio of compound II to the acid binding agent is 1:3;

[0063] Optionally, in step (2), the catalyst is p-toluenesulfonic acid or 4-dimethylaminopyridine;

[0064] Optionally, in step (2), the amount of the catalyst p-toluenesulfonic acid or 4-dimethylaminopyridine is 20% to 30% of the substrate weight;

[0065] Optionally, in step (2), the amount of the catalyst p-toluenesulfonic acid or 4-dimethylaminopyridine is 30%;

[0066] Optionally, in step (2), the stirring reaction time is 15 to 24 hours;

[0067] Optionally, in step (2), the stirring reaction time is 20 hours.

[0068] Furthermore, in step (3), the organic solvent A is dichloromethane, ethyl acetate or acetone: n-heptane = 1:1;

[0069] Optionally, in step (3), the organic solvent A is ethyl acetate;

[0070] Optionally, in step (3), the volume of the solvent used is 10 to 20 times the mass of compound III;

[0071] Optionally, in step (3), the volume of the solvent used is 15 times the mass of compound III;

[0072] Optionally, in step (3), the feeding temperature is 0°C to 5°C;

[0073] Optionally, in step (3), the feeding temperature is 5°C;

[0074] Optionally, in step (3), the molar ratio of compound III to pyridinium dichromate is 1:1 to 1:4;

[0075] Optionally, in step (3), the molar ratio of compound III to pyridinium dichromate is 1:3.7;

[0076] Optionally, in step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:2 to 1:8;

[0077] Optionally, in step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:7.4;

[0078] Optionally, in step (3), the stirring reaction time is 14 to 24 hours;

[0079] Optionally, in step (3), the stirring reaction time is 20 h;

[0080] Optionally, in step (3), the organic solvent B is a combination of methanol and ethyl acetate or a combination of ethanol and ethyl acetate;

[0081] Optionally, in step (3), the organic solvent B is a combination of methanol and ethyl acetate;

[0082] Optionally, in step (3), the ratio of methanol to ethyl acetate in the organic solvent B is 8:1 to 10:1;

[0083] Optionally, in step (3), the recrystallization solvent organic solvent C is methanol, methanol:ethyl acetate = 5:1, methanol:ethyl acetate = 10:1, ethanol, ethanol:ethyl acetate = 5:1 or ethanol:ethyl acetate = 10:1;

[0084] Optionally, in step (3), the organic solvent C used as the recrystallization solvent is methanol:ethyl acetate = 5:1.

[0085] Further, in step (4), the organic solvent D is methanol, ethanol and / or tetrahydrofuran;

[0086] Optionally, in step (4), the organic solvent D is tetrahydrofuran;

[0087] Optionally, in step (4), the volume of the solvent used is 5 to 15 times the mass of compound IV;

[0088] Optionally, in step (4), the volume of the solvent used is 10 times the mass of compound IV;

[0089] Optionally, in step (4), the feeding temperature and the reaction temperature are -20°C to 0°C,

[0090] Optionally, in step (4), the feeding temperature and the reaction temperature are -5°C;

[0091] Optionally, in step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1 to 1:2;

[0092] Optionally, in step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1.5;

[0093] Optionally, in step (4), the molar ratio of compound IV to the catalyst cerium chloride heptahydrate is 1:1 to 1:2;

[0094] Optionally, in step (4), the molar ratio of compound IV to the catalyst cerium chloride heptahydrate is 1:1.

[0095] Furthermore, in step (4), the stirring reaction time is 1.5 to 2.5 hours;

[0096] Optionally, in step (4), the stirring reaction time is 2 h;

[0097] Optionally, in step (4), the acid solution used for quenching is dilute hydrochloric acid or acetic acid;

[0098] Optionally, in step (4), the acid solution used for quenching is dilute hydrochloric acid;

[0099] Optionally, in step (4), the mass fraction of dilute hydrochloric acid is 5% to 20%;

[0100] Optionally, in step (4), the mass fraction of dilute hydrochloric acid is 10%;

[0101] Optionally, in step (4), the organic solvent E used in the recrystallization is methanol, ethanol or ethyl acetate;

[0102] Optionally, in step (4), the organic solvent E used in the recrystallization is methanol.

[0103] Further, in step (5), the organic solvent F is tetrahydrofuran and / or methanol;

[0104] Optionally, in step (5), the organic solvent F is tetrahydrofuran;

[0105] Optionally, in step (5), the volume of the solvent used is 10 to 25 times the mass of compound V;

[0106] Optionally, in step (5), the volume of the solvent used is 20 times the mass of compound V;

[0107] Optionally, in step (5), the alcohol is methanol, ethanol or isopropanol;

[0108] Optionally, in step (5), the alcohol is methanol;

[0109] Optionally, in step (5), the volume of alcohol used is 30 to 50 times the mass of compound V;

[0110] Optionally, in step (5), the volume of alcohol used is 40 times the mass of compound V;

[0111] Optionally, in step (5), the base is sodium hydroxide, potassium hydroxide, sodium methoxide and / or potassium tert-butoxide;

[0112] Optionally, in step (5), the base is potassium tert-butoxide.

[0113] Furthermore, in step (5), the stirring reaction time is 15 to 24 hours;

[0114] Optionally, in step (5), the stirring reaction time is 15 h;

[0115] Optionally, in step (5), the acid used for acid neutralization is dilute hydrochloric acid, dilute sulfuric acid and / or glacial acetic acid;

[0116] Optionally, in step (5), the acid used for acid neutralization is dilute hydrochloric acid;

[0117] Optionally, in step (5), the mass fraction of dilute hydrochloric acid is 5% to 20%;

[0118] Optionally, in step (5), the mass fraction of dilute hydrochloric acid is 5%;

[0119] Optionally, in step (5), the organic solvent G used in the recrystallization is methanol, ethanol, water, tetrahydrofuran or any combination thereof;

[0120] Optionally, in step (5), the organic solvent G used in the recrystallization is a combination of methanol and water;

[0121] Optionally, in step (5), the ratio of methanol to water in the organic solvent G used for the recrystallization is methanol:water=10:1.

[0122] The second aspect of the present invention provides a compound prepared by the synthesis method described in the first aspect of the present invention.

[0123] In some embodiments, the products prepared by the synthesis method described in the first aspect of the present invention are also included in the protection scope of the present invention.

[0124] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0125] (1) The novel HE3286 synthesis method provided by the present invention has streamlined steps, shortening the existing synthesis route from 8 steps to 5. Technical problems such as the multiple introduction and removal of protecting groups and the separation and purification of intermediate products involved in the original route will no longer exist, saving man-hours and equipment investment. Compared with the existing technology, the HE3286 synthesis method provided by the present invention is a significant improvement;

[0126] (2) The starting materials and reagents used in the novel HE3286 synthesis method provided by the present invention are economical and readily available, and strictly comply with the residual solvent standards of ICHQ3C, without the use of carcinogenic, toxic, or environmentally hazardous solvents;

[0127] (3) The present invention further adjusts the reaction conditions and catalysts involved in the HE3286 synthesis method, so that the reaction can be carried out in a mild environment of normal temperature and pressure with high yield, and has extremely low requirements for production equipment, reducing energy consumption in the production process, and is suitable for industrial production;

[0128] (4) The novel HE3286 synthesis method provided by the present invention reduces operational difficulty and avoids the use of hydrogen, palladium-carbon catalyst, lithium reagent, and acetylene Grignard reagent, which are more demanding on reaction and equipment conditions in the existing route. The process is stable and controllable, and the safety is improved.

[0129] (5) The purification of each unit reaction product in the novel HE3286 synthesis method provided by the present invention is carried out by slurrying or recrystallization, which does not rely on a large number of silica gel column purifications and does not require complex equipment, thereby reducing the cost of raw materials and equipment and improving the purity of the target compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 The mass spectrum of HE3286 prepared by the new HE3286 synthesis method provided by the present invention;

[0131] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of HE3286 prepared by the new HE3286 synthesis method provided by the present invention;

[0132] Figure 3 This is the carbon NMR spectrum of HE3286 prepared by the new HE3286 synthesis method provided by the present invention;

[0133] Figure 4 The figure is a high performance liquid chromatogram of HE3286 prepared by the new HE3286 synthesis method provided by the present invention. DETAILED DESCRIPTION

[0134] The present invention will be further described below with reference to specific embodiments. The following specific embodiments are intended only to illustrate the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0135] The experimental consumables, reagents, and raw materials used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0136] The present invention adopts 5 steps of acetylation, diacetylation, oxidation, reduction and deprotection to synthesize HE3286. The synthesis route of the method is shown below, and the specific experimental steps and verification results are described in the following examples.

[0137]

[0138] Example 1 Preparation of Compound II

[0139] 50 g of compound I (dehydroepiandrosterone acetate) was fully dissolved in 550 mL of anhydrous tetrahydrofuran, the temperature was controlled at 5 ° C, acetylene gas was introduced, and 33.75 g of potassium tert-butoxide was slowly added. After the addition was completed, the temperature was maintained and the reaction was stirred for 2 h. After TLC detection of the complete reaction of the substrate compound I, the mixture was moved to room temperature and 30 mL of anhydrous methanol was added dropwise. The stirring was continued for 0.5 h. After TLC detection of the complete reaction of the intermediate, 20% aqueous hydrochloric acid solution was added dropwise to neutralize the mixture. The stirring was continued for 0.5 h. The solvent was concentrated under reduced pressure to a minimum amount (the amount of the solvent was as small as possible to meet the experimental or production requirements). Water was added to precipitate the solid, which was filtered under reduced pressure, washed with water, and the filter cake was collected and dried to obtain 47 g of light yellow solid compound II with a mass yield of 99.7% and a melting point of 242.5 to 244.6 ° C. 1 H NMR(600MHz,DMSO-d6)δ5.26(s,2H),4.59(d,J=4.5Hz,1H),3.30–3.28(m,1H),3.24(tt,J=9.9,5.0Hz, 1H),2.17–2.12(m,1H),2.11–2.03(m,2H),1.93(dd,J=17.0,3.3Hz,1H),1.83(td,J=12.7,4.0Hz,1H),1 .77(d,J=13.3Hz,1H),1.70–1.64(m,1H),1.58(d,J=4.9Hz,4H),1.50–1.44(m,1H),1.43–1.31(m,4H),1 .23(tt,J=11.9,6.0Hz,1H),1.00(dd,J=13.7,3.7Hz,1H),0.95(s,3H),0.88–0.82(m,1H),0.73(s,3H).

[0140] Example 2 Preparation of Compound III

[0141] 50 g of compound II prepared in Example 1 was dissolved in 500 mL of dichloromethane, 6 g of 4-dimethylaminopyridine (DMAP) was added, 48.3 g of triethylamine was added dropwise, and 48.73 g of acetic anhydride was added dropwise. The temperature was controlled to 38° C. and refluxed, and the reaction was stirred for 20 h. After TLC detection showed that the reaction was complete, the temperature was cooled to room temperature, the solvent was concentrated under reduced pressure to a small amount, water was added to precipitate the solid, and the solid was filtered under reduced pressure and washed with water. The filter cake was collected and dried to obtain 61.5 g of brown solid compound III with a mass yield of 96.5%. The product melting point was 158.0-161.0° C. 1 HNMR(600MHz,DMSO-d6)δ5.35(d,J=2.9Hz,1H),4.47–4.41(m,1H),3.53(s,1H),2.54(m,1H),2.27( d,J=7.7Hz,2H),2.04–2.00(m,1H),1.98(d,J=2.2Hz,7H),1.95(t,J=5.3Hz,1H),1.85(dt,J=13.4, 3.6Hz,1H),1.79–1.72(m,2H),1.70–1.49(m,6H),1.47(dt,J=10.4,5.2Hz,1H),1.40(td,J=11.1,5 .9Hz,2H),1.30(m,1H),1.10(td,J=13.8,3.7Hz,1H),0.99(s,3H),0.96–0.91(m,1H),0.83(s,3H).

[0142] Example 3 Preparation of Compound IV

[0143] 50g of compound III was fully dissolved in 750mL of ethyl acetate. The temperature was controlled at 5°C, and 174.8g of pyridinium dichromate and 20g of diatomaceous earth were added. 41.86g of tert-butyl hydroperoxide was slowly added dropwise. After completion of the addition, the mixture was stirred at room temperature for 20h. After TLC, the reaction was completed, filtered through diatomaceous earth, the filtrate was collected, concentrated, and slurried with methanol and ethyl acetate for 24h. The mixture was filtered under reduced pressure, the filter cake was collected and dried, and recrystallized by adding methanol:ethyl acetate = 5:1 solvent. The mixture was filtered and dried to obtain 32.24g of compound IV as a white solid with a mass yield of 62.0% and a melting point of 245.4-246.7°C. 1H NMR (600MHz, DMSO-d6) δ5.67 (s, 1H), 4.58 (tt, J = 10.6, 5.9Hz, 1H), 3.54 ( s,1H),3.31(s,1H),2.58–2.51(m,3H),2.38–2.29(m,2H),2.01(s,3H),1. 98(s,3H),1.94–1.85(m,2H),1.71–1.64(m,4H),1.60(d,J=11.6Hz,1H),1 .52–1.43(m,3H),1.28(td,J=13.7,3.7Hz,1H),1.20(s,3H),0.84(s,3H).

[0144] Example 4 Preparation of Compound V

[0145] Dissolve 50 g of compound IV in 500 mL of anhydrous tetrahydrofuran, control the temperature at -5°C, add a solution of 45 g of cerium chloride heptahydrate in 900 mL of anhydrous methanol, add 6.9 g of reducing agent sodium borohydride, and stir the reaction at the same temperature for 2 h. After the reaction is completed by TLC, slowly add 10% aqueous hydrochloric acid solution to quench the reaction. After quenching, concentrate the solvent to a small amount, add water for elution, filter under reduced pressure, collect the filter cake, dry it, and recrystallize it with methanol to obtain 40.2 g of white solid compound V with a mass yield of 80.1%. The product has a melting point of 205.2-207.0°C. 1 H NMR (600MHz, DMSO) δ5.22(s,1H),4.48–4.42(m,1H),4.37(d,J=7.3Hz,1H),3.62(t,J=7 .7Hz,1H),3.52(s,1H),3.31(s,1H),2.29–2.24(m,2H),1.98(d,J=7.8Hz,7H),1.93–1.8 6(m,1H),1.82(d,J=13.3Hz,2H),1.72(td,J=12.7,4.3Hz,1H),1.62–1.53(m,4H),1.51– 1.46(m,1H),1.41–1.34(m,2H),1.07(td,J=13.7,3.7Hz,1H),1.02(s,4H),0.82(s,3H).

[0146] Example 5 Preparation of HE3286

[0147] 50 g of compound V was dissolved in 1 L of anhydrous tetrahydrofuran, and 2 L of anhydrous methanol was added. 40.6 g of potassium tert-butoxide was slowly added at low temperature of 0°C. After the addition, the mixture was transferred to room temperature and stirred for 15 h. After the reaction was completed by TLC, 5% aqueous hydrochloric acid was added dropwise to neutralize the mixture. The solvent was concentrated to a small amount, and water was added for elution. The filter cake was collected by filtration under reduced pressure and dried. Recrystallization was carried out in the ratio of methanol to water = 10:1 to obtain 22.5 g of white solid HE3286 with a mass yield of 56.4% and a melting point of 256.8-258.8°C. 1 H NMR (600MHz, DMSO-d6) δ5.20(s,1H),5.13(s,1H),4.62(s,1H),4.21(s,1H),3.56(d,J=8.2Hz,1H),3.29–3.23(m,2H),2.16–2.06(m,2H) ),2.05–2.00(m,1H),1.84–1.71(m,3H),1.67(d,J=11.6Hz,1H),1.59–1.43(m,5H),1.39–1.29(m,3H),0.99–0.88(m,5H),0.71(s,3H).

[0148] The mass spectrum, hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum of HE3286 prepared by the novel HE3286 synthesis method provided by the present invention are as follows: Figure 1-3 As shown, the results show that the molecular weight, fragment ion peaks, chemical shift ranges corresponding to different hydrogen atoms and carbon atoms, and the number of peaks of the synthesized product are consistent with those of HE3286, indicating that the product synthesized by the method provided by the present invention is HE3286.

[0149] Example 6: HPLC detection of the purity of HE3286 synthesized by the above method

[0150] 1. Experimental methods

[0151] The HPLC method for content testing is as follows:

[0152] Chromatograph: ThermoFisher Vanquish Flex;

[0153] Detector: CAD;

[0154] Chromatographic column: Ultimate XB-C18 (4.6×250 mm, 5 μm);

[0155] Flow rate: 1.0 mL / min;

[0156] Injection volume: 1 μL;

[0157] Sample chamber temperature: 15°C;

[0158] Column temperature: 40°C;

[0159] Mobile phase: A: H2O with 0.1% formalin; B: Acetonitrile;

[0160] Atomization temperature: 35℃;

[0161] Data collection frequency: 10;

[0162] Filter: 1.0;

[0163] Gradient elution: The gradient elution conditions are shown in Table 1 below.

[0164] Table 1 Gradient elution conditions

[0165]

[0166] 2. Experimental results

[0167] The purity of HE3286 synthesized by the above method was detected by high performance liquid chromatography. Figure 4 As shown, the results show that the purity of the synthesized HE3286 sample is as high as 98.03%, indicating that the target compound HE3286 accounts for a large proportion in the sample and the synthesized product has a high purity. Combined with the experimental results in the above embodiments, it can be seen that the method provided by the present invention can successfully and efficiently synthesize HE3286 with high purity.

Claims

1. A method for synthesizing HE3286, characterized in that: The synthesis method uses dehydroepiandrosterone acetate as a raw material, and obtains HE3286 through acetylation, diacetylation, oxidation, reduction, and deprotection. The reaction formula is shown below.

2. The synthesis method according to claim 1, wherein The synthesis method comprises the following steps: (1) Acetylation reaction Compound I (dehydroepiandrosterone acetate) is dissolved in an organic solvent, the temperature is controlled, acetylene gas is introduced, an organic base is added, and the reaction is stirred. After the reaction is completed, alcohol is added at room temperature, and the reaction is continued with stirring. After the reaction is completed, acid is added for neutralization, and the mixture is concentrated to a small amount, eluted with water, filtered, and dried to obtain Compound II. (2) Diacetylation reaction Compound II is dissolved in an organic solvent, acetic anhydride as an acetylation agent is added, an acid-binding agent is added, a catalyst is added, the temperature is controlled, the reaction is stirred, and after the reaction is completed, the temperature is returned to room temperature, the mixture is concentrated to a small amount, water is added for precipitation, and the mixture is filtered and dried to obtain compound III; (3) Oxidation reaction Compound III is dissolved in organic solvent A, the feeding temperature is controlled, pyridinium dichromate and tert-butyl hydroperoxide are added for oxidation, diatomaceous earth is added as a catalyst, and the reaction is stirred at room temperature. After the reaction is completed, the diatomaceous earth is filtered, the mixture is concentrated to a small amount, water is added for separation, and the crude product is dried to obtain a crude product. The crude product is slurried with organic solvent B, dried, and recrystallized with organic solvent C to obtain compound IV. (4) Reduction reaction Compound IV is dissolved in organic solvent D, the feeding temperature is controlled, a methanol solution of cerium chloride heptahydrate is added, sodium borohydride as a reducing agent is added, and the reaction is stirred at low temperature. After the reaction is completed, an acid solution is added to quench the reaction. After the quenching is complete, the reaction is concentrated to a small amount, eluted with water, filtered, and dried to obtain a crude product. The crude product is recrystallized by adding organic solvent E and dried to obtain compound V. (5) Alcoholysis reaction Compound V was dissolved in organic solvent F, and alcohol was added. A base was added at a controlled low temperature, and the reaction was stirred at room temperature. After the reaction was completed, acid was added for neutralization, and the mixture was concentrated to a small amount. Water was added for precipitating, the mixture was filtered, and the mixture was dried to obtain a crude product. The crude product was recrystallized from organic solvent G to obtain the target compound HE3286.

3. The synthesis method according to claim 2, characterized in that In step (1), the organic solvent is N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methyl ethyl ketone, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, dioxane, diethyl ether, methanol and / or ethanol; Optionally, in step (1), the organic solvent is tetrahydrofuran; Optionally, in step (1), the volume of the solvent used is 8 to 20 times the mass of the substrate; Optionally, in step (1), the volume of the solvent used is 11 times the mass of the substrate; Optionally, in step (1), the controlled temperature is -5°C to 10°C; Optionally, in step (1), the controlled temperature is 5°C; Optionally, in step (1), the organic base is potassium tert-butoxide, potassium isobutoxide, potassium isopropoxide and / or potassium ethoxide; Optionally, in step (1), the organic base is potassium tert-butoxide; Optionally, in step (1), the molar ratio of compound I to potassium tert-butoxide is 1:1 to 1:3; Optionally, in step (1), the molar ratio of compound I to potassium tert-butoxide is 1:2; Optionally, in step (1), the stirring reaction time is 1.5 to 3 hours; Optionally, in step (1), the stirring reaction time is 2 h; Optionally, in step (1), the alcohol is methanol, ethanol, tert-butanol and / or isopropanol; Optionally, in step (1), the alcohol is methanol; Optionally, in step (1), the molar ratio of methanol to compound I is 5:1 to 20:1; Optionally, in step (1), the molar ratio of methanol to compound I is 6:1; Optionally, in step (1), the stirring reaction time is 20 min to 1 h; Optionally, in step (1), the stirring reaction time is 0.5h; Optionally, in step (1), the acid used for acid neutralization is dilute hydrochloric acid, dilute sulfuric acid and / or glacial acetic acid; Optionally, in step (1), the acid used for acid neutralization is dilute hydrochloric acid; Optionally, in step (1), the mass fraction of the dilute hydrochloric acid is 5% to 25%; Optionally, in step (1), the mass fraction of the dilute hydrochloric acid is 20%.

4. The synthesis method according to claim 2, characterized in that In step (2), the organic solvent is dichloromethane and / or tetrahydrofuran; Optionally, in step (2), the organic solvent is dichloromethane; Optionally, in step (2), the volume of the solvent used is 10 to 20 times the mass of the substrate; Optionally, in step (2), the volume of the solvent used is 10 times the mass of the substrate; Optionally, in step (2), the reaction temperature is 25°C to the reflux temperature of the solvent; Optionally, in step (2), the reaction temperature is 38°C; Optionally, in step (2), the acid binding agent is triethylamine and / or pyridine; Optionally, in step (2), the acid binding agent is triethylamine; Optionally, in step (2), the molar ratio of compound II to the acetylating agent acetic anhydride is 1:2 to 1:4; Optionally, in step (2), the molar ratio of compound II to the acetylating agent acetic anhydride is 1:3; Optionally, in step (2), the molar ratio of compound II to the acid binding agent is 1:2 to 1:4; Optionally, in step (2), the molar ratio of compound II to the acid binding agent is 1:3; Optionally, in step (2), the catalyst is p-toluenesulfonic acid or 4-dimethylaminopyridine; Optionally, in step (2), the amount of the catalyst p-toluenesulfonic acid or 4-dimethylaminopyridine is 20% to 30% of the substrate weight; Optionally, in step (2), the amount of the catalyst p-toluenesulfonic acid or 4-dimethylaminopyridine is 30%; Optionally, in step (2), the stirring reaction time is 15 to 24 hours; Optionally, in step (2), the stirring reaction time is 20 hours.

5. The synthesis method according to claim 2, characterized in that In step (3), the organic solvent A is dichloromethane, ethyl acetate or acetone: n-heptane = 1:1; Optionally, in step (3), the organic solvent A is ethyl acetate; Optionally, in step (3), the volume of the solvent used is 10 to 20 times the mass of compound III; Optionally, in step (3), the volume of the solvent used is 15 times the mass of compound III; Optionally, in step (3), the feeding temperature is 0°C to 5°C; Optionally, in step (3), the feeding temperature is 5°C; Optionally, in step (3), the molar ratio of compound III to pyridinium dichromate is 1:1 to 1:4; Optionally, in step (3), the molar ratio of compound III to pyridinium dichromate is 1:3.7; Optionally, in step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:2 to 1:8; Optionally, in step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:7.4; Optionally, in step (3), the stirring reaction time is 14 to 24 hours; Optionally, in step (3), the stirring reaction time is 20 h; Optionally, in step (3), the organic solvent B is a combination of methanol and ethyl acetate or a combination of ethanol and ethyl acetate; Optionally, in step (3), the organic solvent B is a combination of methanol and ethyl acetate; Optionally, in step (3), the ratio of methanol to ethyl acetate in the organic solvent B is 8:1 to 10:1; Optionally, in step (3), the recrystallization solvent organic solvent C is methanol, methanol:ethyl acetate = 5:1, methanol:ethyl acetate = 10:1, ethanol, ethanol:ethyl acetate = 5:1 or ethanol:ethyl acetate = 10:1; Optionally, in step (3), the organic solvent C used as the recrystallization solvent is methanol:ethyl acetate = 5:

1.

6. The synthesis method according to claim 2, characterized in that In step (4), the organic solvent D is methanol, ethanol and / or tetrahydrofuran; Optionally, in step (4), the organic solvent D is tetrahydrofuran; Optionally, in step (4), the volume of the solvent used is 5 to 15 times the mass of compound IV; Optionally, in step (4), the volume of the solvent used is 10 times the mass of compound IV; Optionally, in step (4), the feeding temperature and the reaction temperature are -20°C to 0°C, Optionally, in step (4), the feeding temperature and the reaction temperature are -5°C; Optionally, in step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1 to 1:2; Optionally, in step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1.5; Optionally, in step (4), the molar ratio of compound IV to the catalyst cerium chloride heptahydrate is 1:1 to 1:2; Optionally, in step (4), the molar ratio of compound IV to the catalyst cerium chloride heptahydrate is 1:

1.

7. The synthesis method according to claim 6, characterized in that In step (4), the stirring reaction time is 1.5 to 2.5 hours; Optionally, in step (4), the stirring reaction time is 2 h; Optionally, in step (4), the acid solution used for quenching is dilute hydrochloric acid or acetic acid; Optionally, in step (4), the acid solution used for quenching is dilute hydrochloric acid; Optionally, in step (4), the mass fraction of dilute hydrochloric acid is 5% to 20%; Optionally, in step (4), the mass fraction of dilute hydrochloric acid is 10%; Optionally, in step (4), the organic solvent E used in the recrystallization is methanol, ethanol or ethyl acetate; Optionally, in step (4), the organic solvent E used in the recrystallization is methanol.

8. The synthesis method according to claim 2, characterized in that In step (5), the organic solvent F is tetrahydrofuran and / or methanol; Optionally, in step (5), the organic solvent F is tetrahydrofuran; Optionally, in step (5), the volume of the solvent used is 10 to 25 times the mass of compound V; Optionally, in step (5), the volume of the solvent used is 20 times the mass of compound V; Optionally, in step (5), the alcohol is methanol, ethanol or isopropanol; Optionally, in step (5), the alcohol is methanol; Optionally, in step (5), the volume of alcohol used is 30 to 50 times the mass of compound V; Optionally, in step (5), the volume of alcohol used is 40 times the mass of compound V; Optionally, in step (5), the base is sodium hydroxide, potassium hydroxide, sodium methoxide and / or potassium tert-butoxide; Optionally, in step (5), the base is potassium tert-butoxide.

9. The synthesis method according to claim 8, characterized in that In step (5), the stirring reaction time is 15 to 24 hours; Optionally, in step (5), the stirring reaction time is 15 h; Optionally, in step (5), the acid used for acid neutralization is dilute hydrochloric acid, dilute sulfuric acid and / or glacial acetic acid; Optionally, in step (5), the acid used for acid neutralization is dilute hydrochloric acid; Optionally, in step (5), the mass fraction of dilute hydrochloric acid is 5% to 20%; Optionally, in step (5), the mass fraction of dilute hydrochloric acid is 5%; Optionally, in step (5), the organic solvent G used in the recrystallization is methanol, ethanol, water, tetrahydrofuran or any combination thereof; Optionally, in step (5), the organic solvent G used in the recrystallization is a combination of methanol and water; Optionally, in step (5), the ratio of methanol to water in the organic solvent G used for the recrystallization is methanol:water=10:

1.

10. A compound prepared by the synthesis method according to any one of claims 1 to 9.

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