A method for synthesizing HE3286

Abstract

This invention discloses a method for synthesizing HE3286, belonging to the field of pharmaceutical preparation technology. This invention provides a novel method for synthesizing HE3286, using dehydroepiandrosterone acetate as a raw material, and efficiently obtaining high-purity HE3286 in only five steps: acetylation, diacetylation, oxidation, reduction, and deprotection. The HE3286 synthesis method provided by this invention features inexpensive and readily available reagents, simple and safe operation, low cost, no pollution, and easy scale-up for industrial production, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a method for synthesizing HE3286. Background Technology

[0002] HE3286, also known as Bezisterim, is a novel steroidal compound with the chemical name 17α-ethynyl-5-androstene-3β,7β,17β-triol. It is used to treat autoimmune diseases and has shown good efficacy in treating rheumatoid arthritis and obesity-related diabetes. No adverse effects were observed in genotoxicity, systemic toxicity, or neurotoxicity studies. It also showed no significant effects in long-term toxicity or reproductive toxicity, exhibiting good cardiopulmonary safety. In studies on estrogenic effects, a mild estrogenic effect was observed in rats, and its main metabolites also showed no estrogenic effect, demonstrating good safety.

[0003] To date, patents WO2009149392 and CN114478672A have reported four synthetic routes for HE3286.

[0004] In the first route, starting with dehydroepiandrosterone (DHEA), the 3-hydroxyl group is first protected by a trimethylsilyl group. Then, a nucleophilic addition of trimethylsilylacetylene and n-butyllithium introduces an acetylene group at the 17-position. During this process, the trimethylsilyloxy group dissociates, leading to the introduction of an acetyl group to protect the hydroxyl group again. Finally, allylic oxidation occurs under the action of tert-butylhydrogen peroxide and cuprous iodide. After reduction and deprotection, the target product HE3286 is obtained, with an overall yield of 15%. This method is cumbersome and has a low yield; the use of saccharin as a catalyst results in poor selectivity and low catalytic activity, making it generally unsuitable for industrial production; the lithium reagent used is expensive and used in large quantities, leading to high production costs; the acetylation process requires harsh reaction conditions and poses safety risks, making it unsuitable for industrial production. The reaction route of this method is as follows:

[0005]

[0006] In the second route, starting with dehydroepiandrosterone acetate, a ketal structure is introduced to the 17-carbonyl group via the action of p-toluenesulfonic acid and ethylene glycol. This is followed by oxidation under the action of tert-butylhydrogen peroxide and sodium perchlorate, then reduction and deethylene glycol protection, followed by deacetylation protection. The two exposed hydroxyl groups are then further protected with a trimethylsilyl group, and acetylation is achieved using the addition method of trimethylsilylacetylene and n-butyllithium. Finally, the target product is obtained by deprotection. This method introduces too many unnecessary protecting groups, and the multiple introduction and removal of groups makes the route more cumbersome, increases the difficulty of handling unit reactions, and results in a relatively low overall yield of 6%, significantly increasing production costs and making it unsuitable for industrial production. The reaction route of this method is as follows:

[0007]

[0008] In the third route, starting with dehydroepiandrosterone acetate, the C7 position is first oxidized, then the carbonyl group is hydroxylated to an oxime structure for protection 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 manner as the previous route. This method achieves an overall yield of 30%, but it still comes at the cost of production costs and risks, making it unsuitable for industrial production. The hydroxylation reagents have poor selectivity, are toxic and corrosive, and have adverse effects on water, soil, and atmosphere. The reaction conditions are harsh, requiring high standards for equipment and operational control. Sodium thiosulfate has many restrictions on its use, and sulfur-containing compounds pose environmental risks. Using saccharin as a catalyst results in poor selectivity and low catalytic activity, and it is generally not chosen for industrial production. The reaction route of this method is as follows:

[0009]

[0010] In the fourth route, using the trihydroxy compound produced from drospirenone as a starting material, the 7-hydroxyl group undergoes a transposition reaction under strong acid to invert its configuration. Then, acyl groups are introduced at the 3- and 15-hydroxyl groups using pivaloyl chloride. Elimination and hydrogenation complete the synthesis of the androster D ring. An acetylenic group is then introduced using an acetylenic Grignard reagent, and finally, deprotection yields the target compound HE3286. This method uses the intermediate trihydroxy compound, synthesized from drospirenone via bio-fermentation, as the starting material is expensive and difficult to obtain. The palladium-on-carbon catalyst used in the hydrogenation reaction is expensive and costly, and the use of hydrogen poses a high safety risk. The presence of two unsaturated bonds in the compound's structure makes hydrogenation selection difficult and prone to over-hydrogenation or suboptimal selectivity. Similarly, the presence of the trihydroxy group makes the introduction of acyl protection highly selective and challenging. The selection of the acetylenic reagent also presents significant problems; the acetylenic Grignard reagent is expensive, requires stringent operating conditions, and has poor selectivity, leading to difficulties in separating and purifying byproducts. All these issues affect the practicality and industrial applicability of this method. The reaction route is as follows:

[0011]

[0012] In summary, there are many technical problems in the existing HE3286 synthesis routes that urgently need to be solved. In view of this, the present invention aims to provide a synthesis method that is simple, mild, low-cost, easy to separate and purify, highly efficient, and suitable for industrial-scale production. Summary of the Invention

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

[0014] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0015] The first aspect of the present invention provides a method for synthesizing HE3286, wherein the method uses dehydroepiandrosterone acetate as a raw material and prepares HE3286 by acetylation, diacetylation, oxidation, reduction and deprotection, as shown in the following reaction formula.

[0016]

[0017] In this invention, HE3286 is referred to as Bezisterim in English, 17α-ethynyl-5-androstene-3β,7β,17β-triol in chemical form, and has the CAS number 1001100-69-1. HE3286 is a synthetic derivative of the natural anti-inflammatory steroid β-AET and is a partially active NF-κB inhibitor. HE3286 can reduce pro-inflammatory signaling, including IL-6 and matrix metallopeptidase 3. Furthermore, HE3286 can freely cross the mouse blood-brain barrier. HE3286 can be used in research on ulcerative colitis, arthritis, experimental autoimmune encephalomyelitis, and neurological diseases.

[0018] In this invention, dehydroepiandrosterone acetate is the starting material for the synthesis of HE3286. Its English name is Dehydroisoandrosterone 3-acetate, its chemical name is 3-β-hydroxy-dehydroandroster-5-en-17-one-3-acetate, its CAS number is 853-23-6, and its molecular formula is C3. 21 H 30 O3, with a molecular weight of 330.461. It should be noted that this invention does not impose any particular restrictions on the source of this starting material, which can be obtained by those skilled in the art through conventional means.

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

[0020] (1) Acrylation reaction

[0021] Compound I (dehydroepiandrosterone acetate) was dissolved in an organic solvent. Under controlled temperature, acetylene gas was introduced, an organic base was added, and the reaction was stirred. After the reaction was completed, an alcohol was added at room temperature, and the reaction was stirred again. After the reaction was completed, an acid was added to neutralize the mixture, the volume was concentrated to a minimum, water was added for precipitation, the mixture was filtered, and the product was dried to obtain compound II.

[0022] (2) Diacetylation reaction

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

[0024] (3) Oxidation reaction

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

[0026] (4) Reduction reaction

[0027] Compound IV was dissolved in organic solvent D. The feeding temperature was controlled, and a methanol solution of cerium chloride heptahydrate was added. Sodium borohydride was added as a reducing agent. The reaction was stirred at low temperature. After the reaction was completed, an acid solution was added to quench the reaction. After complete quenching, the mixture was concentrated to a minimum volume. Water was added for precipitation, and the mixture was filtered and dried to obtain a crude product. The crude product was added to organic solvent E for recrystallization and dried to obtain compound V.

[0028] (5) Alcohololysis reaction

[0029] Compound V was dissolved in organic solvent F, alcohol was added, alkali was added under controlled low temperature, and the reaction was stirred at room temperature. After the reaction was completed, acid was added to neutralize, the mixture was concentrated to a minimum, water was added for precipitation, filtered, and dried to obtain crude product. The crude product was recrystallized with organic solvent G to obtain the target compound HE3286.

[0030] In some implementations, the "minimum amount" refers to concentrating the solvent in the reaction system to the lowest possible quantity while still meeting the objectives of subsequent water separation operations and the overall synthesis process. Specifically, the "minimum amount" does not have a fixed value or ratio and needs to be judged and adjusted by the operator based on the actual situation. The appropriate degree of concentration is typically determined by observing the state of the solution during concentration (e.g., whether it becomes thick, whether product begins to precipitate, etc.) and combining this with experience. Those skilled in the art can routinely adjust and determine the "minimum amount" based on common knowledge in the field.

[0031] Further, 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 solvent used is 8 to 20 times the mass of the substrate;

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

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

[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 continued stirring reaction time is 20 min to 1 h;

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

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

[0050] Optionally, in step (1), the acid used for acid neutralization is dilute hydrochloric acid;

[0051] Optionally, in step (1), the mass fraction of the dilute hydrochloric acid is 5% to 25%;

[0052] Optionally, in step (1), the mass fraction of the dilute hydrochloric acid is 20%.

[0053] Furthermore, in step (2), the organic solvent is dichloromethane and / or tetrahydrofuran;

[0054] Optionally, in step (2), the organic solvent is dichloromethane;

[0055] Optionally, in step (2), the volume of solvent used is 10 to 20 times the mass of the substrate;

[0056] Optionally, in step (2), the volume of solvent used is 10 times the mass of the substrate;

[0057] Optionally, in step (2), the reaction temperature is 25°C to the solvent reflux temperature;

[0058] Optionally, in step (2), the reaction temperature is 38°C;

[0059] Optionally, in step (2), the acid-binding agent is triethylamine and / or pyridine;

[0060] Optionally, in step (2), the acid-binding agent is triethylamine;

[0061] Optionally, in step (2), the molar ratio of compound II to the acetylation reagent acetic anhydride is 1:2 to 1:4;

[0062] Optionally, in step (2), the molar ratio of compound II to the acetylation reagent acetic anhydride is 1:3;

[0063] Optionally, in step (2), the molar ratio of compound II to the acid-binding agent is 1:2 to 1:4;

[0064] Optionally, in step (2), the molar ratio of compound II to the acid-binding agent is 1:3;

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

[0066] Optionally, in step (2), the amount of catalyst p-toluenesulfonic acid or 4-dimethylaminopyridine is 20% to 30% of the substrate mass;

[0067] Optionally, in step (2), the amount of catalyst p-toluenesulfonic acid or 4-dimethylaminopyridine is 30%;

[0068] Optionally, in step (2), the stirring reaction time is 15-24 h;

[0069] Optionally, in step (2), the stirring reaction time is 20 h.

[0070] Furthermore, in step (3), the organic solvent A is dichloromethane, ethyl acetate, or acetone: n-heptane = 1:1;

[0071] Optionally, in step (3), the organic solvent A is ethyl acetate;

[0072] Optionally, in step (3), the volume of solvent used is 10 to 20 times the mass of compound III;

[0073] Optionally, in step (3), the volume of solvent used is 15 times the mass of compound III;

[0074] Optionally, in step (3), the feeding temperature is 0℃~5℃;

[0075] Optionally, in step (3), the feeding temperature is 5°C;

[0076] Optionally, in step (3), the molar ratio of compound III to pyridinium dichromate is 1:1 to 1:4;

[0077] Optionally, in step (3), the molar ratio of compound III to pyridinium dichromate is 1:3.7;

[0078] Optionally, in step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:2 to 1:8;

[0079] Optionally, in step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:7.4;

[0080] Optionally, in step (3), the stirring reaction time is 14~24 h;

[0081] Optionally, in step (3), the stirring reaction time is 20 h;

[0082] Optionally, in step (3), the organic solvent B is a combination of methanol and ethyl acetate or a combination of ethanol and ethyl acetate;

[0083] Optionally, in step (3), the organic solvent B is a combination of methanol and ethyl acetate;

[0084] Optionally, in step (3), the ratio of methanol to ethyl acetate in the organic solvent B is 8:1 to 10:1;

[0085] 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;

[0086] Optionally, in step (3), the recrystallization solvent C is methanol: ethyl acetate = 5:1.

[0087] Furthermore, in step (4), the organic solvent D is methanol, ethanol, and / or tetrahydrofuran;

[0088] Optionally, in step (4), the organic solvent D is tetrahydrofuran;

[0089] Optionally, in step (4), the volume of solvent used is 5 to 15 times the mass of compound IV;

[0090] Optionally, in step (4), the volume of solvent used is 10 times the mass of compound IV;

[0091] Optionally, in step (4), the feeding temperature and reaction temperature are -20℃ to 0℃.

[0092] Optionally, in step (4), the feeding temperature and the reaction temperature are -5°C;

[0093] Optionally, in step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1 to 1:2;

[0094] Optionally, in step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1.5;

[0095] Optionally, in step (4), the molar ratio of compound IV to the catalyst cerium chloride heptahydrate is 1:1 to 1:2;

[0096] Optionally, in step (4), the molar ratio of compound IV to the catalyst cerium chloride heptahydrate is 1:1.

[0097] Furthermore, in step (4), the stirring reaction time is 1.5~2.5 h;

[0098] Optionally, in step (4), the stirring reaction time is 2 h;

[0099] Optionally, in step (4), the acid solution used for quenching is dilute hydrochloric acid or acetic acid;

[0100] Optionally, in step (4), the acid solution used for quenching is dilute hydrochloric acid;

[0101] Optionally, in step (4), the mass fraction of dilute hydrochloric acid is 5% to 20%;

[0102] Optionally, in step (4), the mass fraction of dilute hydrochloric acid is 10%;

[0103] Optionally, in step (4), the organic solvent E used for recrystallization is methanol, ethanol or ethyl acetate;

[0104] Optionally, in step (4), the organic solvent E used for recrystallization is methanol.

[0105] Furthermore, in step (5), the organic solvent F is tetrahydrofuran and / or methanol;

[0106] Optionally, in step (5), the organic solvent F is tetrahydrofuran;

[0107] Optionally, in step (5), the volume of solvent used is 10 to 25 times the mass of compound V;

[0108] Optionally, in step (5), the volume of solvent used is 20 times the mass of compound V;

[0109] Optionally, in step (5), the alcohol is methanol, ethanol, or isopropanol;

[0110] Optionally, in step (5), the alcohol is methanol;

[0111] Optionally, in step (5), the volume of alcohol used is 30 to 50 times the mass of compound V;

[0112] Optionally, in step (5), the volume of alcohol used is 40 times the mass of compound V;

[0113] Optionally, in step (5), the base is sodium hydroxide, potassium hydroxide, sodium methoxide and / or potassium tert-butoxide;

[0114] Optionally, in step (5), the base is potassium tert-butoxide.

[0115] Furthermore, in step (5), the stirring reaction time is 15~24 h;

[0116] Optionally, in step (5), the stirring reaction time is 15 h;

[0117] Optionally, in step (5), the acid used for acid neutralization is dilute hydrochloric acid, dilute sulfuric acid, and / or glacial acetic acid;

[0118] Optionally, in step (5), the acid used for acid neutralization is dilute hydrochloric acid;

[0119] Optionally, in step (5), the mass fraction of dilute hydrochloric acid is 5% to 20%;

[0120] Optionally, in step (5), the mass fraction of dilute hydrochloric acid is 5%;

[0121] Optionally, in step (5), the organic solvent G used for recrystallization is methanol, ethanol, water, tetrahydrofuran or any combination thereof;

[0122] Optionally, in step (5), the organic solvent G used for recrystallization is a combination of methanol and water;

[0123] Optionally, in step (5), the ratio of methanol to water in the organic solvent G used for recrystallization is methanol:water = 10:1.

[0124] A second aspect of the present invention provides compounds prepared using the synthetic method described in the first aspect of the present invention.

[0125] In some embodiments, the products prepared by the synthesis method described in the first aspect of the present invention are also included within the scope of protection of the present invention.

[0126] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0127] (1) The new HE3286 synthesis method provided by the present invention has simplified the steps, reducing the existing synthesis route from 8 steps to 5 steps. The technical problems of multiple introduction and removal of protecting groups and multiple separation and purification of intermediate products involved in the original route will no longer exist, saving time and equipment investment. Compared with the existing technology, the HE3286 synthesis method provided by the present invention has significant progress.

[0128] (2) The starting materials and reagents used in the novel HE3286 synthesis method provided by this invention are economical and readily available, and strictly comply with the ICH Q3C residual solvent standard, without the use of carcinogens, toxic and environmentally harmful solvents;

[0129] (3) The present invention further adjusts the reaction conditions and catalysts involved in the synthesis method of HE3286, so that the reaction can be carried out in a high yield under mild ambient temperature and pressure environment, and the requirements for production equipment are extremely low, reducing energy consumption in the production process and making it suitable for industrial production.

[0130] (4) The novel HE3286 synthesis method provided by this invention reduces the difficulty of operation and avoids the use of hydrogen, palladium on carbon catalyst, lithium reagent and acetylene Grignard reagent, which have more stringent requirements for reaction and equipment conditions in the existing route. The process is stable and controllable and the safety is improved.

[0131] (5) The purification of each unit reaction product in the novel HE3286 synthesis method provided by the present invention is carried out by pulping or recrystallization, which does not rely on a large amount of silica gel column purification, does not require complex equipment, reduces the cost of raw materials and equipment, and improves the purity of the target compound. Attached Figure Description

[0132] Figure 1 The mass spectrum of HE3286 prepared using the novel HE3286 synthesis method provided by this invention is shown.

[0133] Figure 2 The hydrogen nuclear magnetic resonance spectrum of HE3286 prepared using the novel HE3286 synthesis method provided by this invention;

[0134] Figure 3 The image shows the carbon NMR spectrum of HE3286 prepared using the novel HE3286 synthesis method provided by this invention.

[0135] Figure 4 The image shows a high-performance liquid chromatogram of HE3286 prepared using the novel HE3286 synthesis method provided by this invention. Detailed Implementation

[0136] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0137] The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0138] This invention synthesizes HE3286 in five steps: acetylation, diacetylation, oxidation, reduction, and deprotection. The synthetic route is shown below, and the specific experimental steps and verification results are described in the following examples.

[0139]

[0140] Example 1: Preparation of Compound II

[0141] 50 g of compound I (dehydroepiandrosterone acetate) was fully dissolved in 550 mL of anhydrous tetrahydrofuran. The temperature was controlled at 5°C, and acetylene gas was introduced. 33.75 g of potassium tert-butoxide was slowly added. After the addition was complete, the reaction was continued at this temperature with stirring for 2 h. After the reaction of substrate compound I was confirmed to be complete by TLC, the mixture was moved to room temperature, and 30 mL of anhydrous methanol was added dropwise. Stirring was continued for 0.5 h. After the reaction of the intermediate was confirmed to be complete by TLC, 20% hydrochloric acid aqueous solution was added dropwise for neutralization. Stirring was continued for 0.5 h. The solvent was concentrated under reduced pressure to a minimum amount (the minimum amount achievable under the premise of meeting experimental or production needs). Water was added to precipitate the solid, which was then filtered under reduced pressure, washed with water, and the filter cake was collected and dried to obtain 47 g of pale yellow solid compound II, with a yield of 99.7% and a melting point of 242.5–244.6°C. 1 HNMR (600 MHz, DMSO-d6) δ 5.26 (s, 2H), 4.59 (d, J = 4.5 Hz, 1H), 3.30 – 3.28(m, 1H), 3.24 (tt, J = 9.9, 5.0 Hz, 1H), 2.17 – 2.12 (m, 1H), 2.11 – 2.03 (m,2H), 1.93 (dd, J = 17.0, 3.3 Hz, 1H), 1.83 (td, J = 12.7, 4.0 Hz, 1H), 1.77(d, J = 13.3 Hz, 1H), 1.70 – 1.64 (m, 1H), 1.58 (d, J = 4.9 Hz, 4H), 1.50 –1.44 (m, 1H), 1.43 – 1.31 (m, 4H), 1.23 (tt, J = 11.9, 6.0 Hz, 1H), 1.00 (dd,J = 13.7, 3.7 Hz, 1H), 0.95 (s, 3H), 0.88 – 0.82 (m, 1H), 0.73 (s, 3H).

[0142] Example 2 Preparation of Compound III

[0143] 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 at 38 °C and refluxed. The mixture was stirred for 20 h. After the reaction was confirmed to be complete by TLC, the temperature was lowered to room temperature, and the solvent was concentrated under reduced pressure to a minimum. Water was added to precipitate the solid, which was then filtered under reduced pressure, washed with water, and the filter cake was collected and dried to obtain 61.5 g of brown solid compound III, with a yield of 96.5% and a melting point of 158.0~161.0 °C. 1 H NMR (600 MHz, DMSO-d6) δ 5.35 (d, J = 2.9 Hz, 1H), 4.47 – 4.41 (m,1H), 3.53 (s, 1H), 2.54 (m, 1H), 2.27 (d, J = 7.7 Hz, 2H), 2.04 – 2.00 (m,1H), 1.98 (d, J = 2.2 Hz, 7H), 1.95 (t, J = 5.3 Hz, 1H), 1.85 (dt, J = 13.4,3.6 Hz, 1H), 1.79 – 1.72 (m, 2H), 1.70 – 1.49 (m, 6H), 1.47 (dt, J = 10.4,5.2 Hz, 1H), 1.40 (td, J = 11.1, 5.9 Hz, 2H), 1.30 (m, 1H), 1.10 (td, J =13.8, 3.7 Hz, 1H), 0.99 (s, 3H), 0.96 – 0.91 (m, 1H), 0.83 (s, 3H).

[0144] Example 3 Preparation of Compound IV

[0145] 50 g of compound III was fully dissolved in 750 mL of ethyl acetate at 5 °C. 174.8 g of pyridinium dichromate and 20 g of diatomaceous earth were added, followed by slow dropwise addition of 41.86 g of tert-butyl hydroperoxide. After the addition was complete, the mixture was stirred at room temperature for 20 h. After TLC detection of complete reaction, the mixture was filtered through diatomaceous earth, and the filtrate was collected, concentrated, and stirred with methanol and ethyl acetate for 24 h. The mixture was then filtered under reduced pressure, the filter cake was collected and dried, and recrystallized in a methanol:ethyl acetate ratio of 5:1. The resulting product was filtered and dried to obtain 32.24 g of white solid compound IV, with a yield of 62.0% and a melting point of 245.4–246.7 °C. 1H NMR (600 MHz, DMSO-d6)δ 5.67 (s, 1H), 4.58 (tt, J = 10.6, 5.9 Hz, 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 = 11.6 Hz, 1H), 1.52 – 1.43 (m,3H), 1.28 (td, J = 13.7, 3.7 Hz, 1H), 1.20 (s, 3H), 0.84 (s, 3H).

[0146] Example 4 Preparation of Compound V

[0147] 50 g of compound IV was dissolved in 500 mL of anhydrous tetrahydrofuran at -5 °C. 45 g of cerium chloride heptahydrate in 900 mL of anhydrous methanol was added, along with 6.9 g of sodium borohydride as a reducing agent. The mixture was stirred for 2 h at this temperature. After the reaction was completed by TLC, 10% hydrochloric acid aqueous solution was slowly added to quench the reaction. After quenching, the solvent was concentrated to a minimum, and water was added for precipitation. The mixture was then filtered under reduced pressure, and the filter cake was collected, dried, and recrystallized from methanol to give 40.2 g of white solid compound V, with a yield of 80.1% and a melting point of 205.2–207.0 °C. 1 H NMR (600 MHz, DMSO) δ 5.22 (s, 1H), 4.48 – 4.42 (m, 1H), 4.37 (d, J = 7.3 Hz, 1H), 3.62 (t, J = 7.7 Hz, 1H), 3.52 (s, 1H), 3.31 (s,1H), 2.29 – 2.24 (m, 2H), 1.98 (d, J = 7.8 Hz, 7H), 1.93 – 1.86 (m, 1H), 1.82 (d, J = 13.3 Hz, 2H), 1.72 (td, J = 12.7, 4.3 Hz, 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, 3.7 Hz, 1H), 1.02 (s, 4H), 0.82 (s, 3H).

[0148] Example 5: Preparation of HE3286

[0149] 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 0 °C. After the addition was complete, the mixture was moved to room temperature and stirred for 15 h. After the reaction was completed by TLC, 5% hydrochloric acid aqueous solution was added dropwise to neutralize the solution. The solvent was concentrated to a minimum, and water was added for precipitation. The filter cake was collected by vacuum filtration and dried. Recrystallization from methanol:water = 10:1 gave 22.5 g of white solid HE3286, with a yield of 56.4% and a melting point of 256.8–258.8 °C. 1 H NMR (600 MHz, DMSO-d6) δ 5.20 (s, 1H), 5.13 (s, 1H), 4.62 (s, 1H), 4.21 (s, 1H), 3.56 (d, J= 8.2 Hz, 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 = 11.6 Hz, 1H), 1.59 – 1.43 (m, 5H), 1.39 –1.29 (m, 3H), 0.99 – 0.88 (m, 5H), 0.71 (s, 3H).

[0150] The mass spectrum, proton NMR spectrum, and carbon NMR spectrum of HE3286 prepared using the novel HE3286 synthesis method provided by this invention are shown below. Figure 1-3 As shown, the results indicate that the molecular weight, fragment ion peaks, chemical shift ranges corresponding to different hydrogen and carbon atoms, and number of peaks of the synthesized product are all consistent with HE3286, indicating that the product synthesized by the method provided in this invention is indeed HE3286.

[0151] Example 6: Detection of the purity of HE3286 synthesized by the above method using high performance liquid chromatography.

[0152] 1. Experimental Methods

[0153] The following are the methods for determining the content using high-performance liquid chromatography (HPLC):

[0154] Chromatograph: ThermoFisher Vanquish Flex;

[0155] Detector: CAD;

[0156] Column: Ultimate XB-C18 (4.6×250 mm, 5 μm);

[0157] Flow rate: 1.0 mL / min;

[0158] Injection volume: 1 μL;

[0159] Sample chamber temperature: 15℃;

[0160] Column temperature: 40℃;

[0161] Mobile phase: A: H2O with 0.1% Formic acid; B: Acetonitrile;

[0162] Atomization temperature: 35℃;

[0163] Data acquisition frequency: 10;

[0164] Filtering: 1.0;

[0165] Gradient elution was performed, and the gradient elution conditions are shown in Table 1 below.

[0166] Table 1 Gradient elution conditions

[0167]

[0168] 2. Experimental Results

[0169] The purity of HE3286 synthesized using the above method was determined by high-performance liquid chromatography (HPLC), and the results are as follows: Figure 4 As shown, the results indicate that the purity of the synthesized HE3286 sample is as high as 98.03%, indicating that the target compound HE3286 accounts for a very large proportion of the sample and the synthesized product has high purity. Combined with the experimental results in the above examples, 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 of synthesis of HE3286, characterized by, The synthesis method takes dehydroepiandrosterone acetate as raw material, and prepares HE3286 through acetylene, diacetylation, oxidation, reduction and deprotection, and the reaction formula is shown in the following, 。 2. The method of synthesis of claim 1, wherein, The synthesis method comprises the following steps: (1) acetylene reaction Compound I dehydroepiandrosterone acetate is dissolved in an organic solvent, temperature is controlled, acetylene gas is introduced, potassium tert-butoxide is added, stirring reaction is carried out, after the reaction is completed, methanol is added at room temperature, continuous stirring reaction is carried out, after the reaction is completed, acid is added for neutralization, the amount is concentrated, water is added for precipitation, filtration is carried out, and drying is carried out to obtain compound II; (2) diacetylation reaction Compound II is dissolved in an organic solvent, acetylation reagent acetic anhydride is added, triethylamine is added, 4-dimethylaminopyridine is added, temperature is controlled, stirring reaction is carried out, after the reaction is completed, the temperature is restored to room temperature, the amount is concentrated, water is added for precipitation, filtration is carried out, and drying is carried out to obtain compound III; (3) oxidation reaction Compound III is dissolved in ethyl acetate, the feeding temperature is controlled, pyridinium dichromate is added, a catalyst diatomite is added, and tert-butyl hydroperoxide is slowly added dropwise, stirring reaction is carried out at room temperature, after the reaction is completed, the diatomite is filtered, the amount is concentrated, the slurry is prepared with an organic solvent B, drying is carried out, and recrystallization is carried out with an organic solvent C, and drying is carried out to obtain compound IV; (4) reduction reaction Compound IV is dissolved in an organic solvent D, the feeding temperature is controlled, cerium chloride heptahydrate methanol solution is added, a reducing agent sodium borohydride is added, stirring reaction is carried out at low temperature, after the reaction is completed, an acid solution is added for quenching, after the quenching is completed, the amount is concentrated, water is added for precipitation, filtration is carried out, and drying is carried out to obtain a crude product, the crude product is recrystallized with an organic solvent E, and drying is carried out to obtain compound V; (5) alcoholysis reaction Compound V is dissolved in tetrahydrofuran, methanol is added, potassium tert-butoxide is added at low temperature, stirring reaction is carried out at room temperature, after the reaction is completed, acid is added for neutralization, the amount is concentrated, water is added for precipitation, filtration is carried out, and drying is carried out to obtain a crude product, and the crude product is recrystallized with an organic solvent G to obtain the target compound HE3286.

3. The method of synthesis of claim 2, wherein, 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.

4. The method of synthesis of claim 2, wherein, In step (1), the organic solvent is tetrahydrofuran.

5. The method of synthesis of claim 2, wherein, In step (1), the volume of the organic solvent is 8-20 times the mass of compound I.

6. The method of synthesis of claim 2, wherein, The volume of the organic solvent is 11 times the mass of compound I.

7. The method of synthesis of claim 2, wherein, In step (1), the controlled temperature is -5℃-10℃.

8. The method of synthesis of claim 2, wherein, In step (1), the controlled temperature is 5℃.

9. The method of synthesis of claim 2, wherein, In step (1), the molar ratio of compound I to potassium tert-butoxide is 1:1-1:

3.

10. The method of synthesis of claim 2, wherein, In step (1), the molar ratio of compound I to potassium tert-butoxide is 1:

2.

11. The method of synthesis of claim 2, wherein, In step (1), the stirring reaction time is 1.5-3 h.

12. The method of synthesis of claim 2, wherein, In step (1), the stirring reaction time is 2 h.

13. The method of synthesis of claim 2, wherein, In step (1), the molar ratio of methanol to compound I is 5:1-20:

1.

14. The method of synthesis of claim 2, wherein, In step (1), the molar ratio of methanol to compound I is 6:

1.

15. The method of synthesis of claim 2, wherein, In step (1), the continuous stirring reaction time is 20 min-1 h.

16. The method of synthesis of claim 2, wherein, In step (1), the continuous stirring reaction time is 0.5 h.

17. The method of synthesis of claim 2, wherein, In step (1), the acid used for the acid neutralization is dilute hydrochloric acid, dilute sulfuric acid and / or glacial acetic acid.

18. The method of synthesis of claim 2, wherein, In step (1), the acid used for the acid neutralization is dilute hydrochloric acid.

19. The method of synthesis of claim 18, wherein, In step (1), the mass fraction of the dilute hydrochloric acid is 5% to 25%.

20. The method of synthesis of claim 18, wherein, In step (1), the mass fraction of the dilute hydrochloric acid is 20%.

21. The method of synthesis of claim 2, wherein, In step (2), the organic solvent is dichloromethane and / or tetrahydrofuran.

22. The method of synthesis of claim 2, wherein, In step (2), the organic solvent is dichloromethane.

23. The method of synthesis of claim 2, wherein, In step (2), the volume of the organic solvent is 10 to 20 times the mass of compound II.

24. The method of synthesis of claim 2, wherein, In step (2), the volume of the organic solvent is 10 times the mass of compound II.

25. The method of synthesis of claim 2, wherein, In step (2), the reaction is controlled at a temperature of 25°C to the refluxing temperature of the solvent.

26. The method of synthesis of claim 2, wherein, In step (2), the reaction is controlled at a temperature of 38°C.

27. The method of synthesis of claim 2, wherein, In step (2), the molar ratio of compound II to acetylating agent acetic anhydride is 1:2 to 1:

4.

28. The method of synthesis of claim 2, wherein, In step (2), the molar ratio of compound II to acetylating agent acetic anhydride is 1:

3.

29. The method of synthesis of claim 2, wherein, In step (2), the molar ratio of compound II to triethylamine is 1:2 to 1:

4.

30. The method of synthesis of claim 2, wherein, In step (2), the molar ratio of compound II to triethylamine is 1:

3.

31. The method of synthesis of claim 2, wherein, In step (2), the stirring reaction time is 15 to 24 h.

32. The method of synthesis of claim 2, wherein, In step (2), the stirring reaction time is 20 h.

33. The method of synthesis of claim 2, wherein, In step (3), the volume of ethyl acetate is 10 to 20 times the mass of compound III.

34. The method of synthesis of claim 2, wherein, In step (3), the volume of ethyl acetate is 15 times the mass of compound III.

35. The method of synthesis of claim 2, wherein, In step (3), the feeding temperature is 0°C to 5°C.

36. The method of synthesis of claim 2, wherein, In step (3), the feeding temperature is 5°C.

37. The method of synthesis of claim 2, wherein, In step (3), the molar ratio of compound III to pyridinium dichromate is 1:1 to 1:

4.

38. The method of synthesis of claim 2, wherein, In step (3), the molar ratio of compound III to pyridinium dichromate is 1:3.

7.

39. The method of synthesis of claim 2, wherein, In step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:2 to 1:

8.

40. The method of synthesis of claim 2, wherein, In step (3), the molar ratio of compound III to tert-butyl hydroperoxide is 1:7.

4.

41. The method of synthesis of claim 2, wherein, In step (3), the stirring reaction time is 14 to 24 h.

42. The method of synthesis of claim 2, wherein, In step (3), the stirring reaction time is 20 h.

43. The method of synthesis of claim 2, wherein, In step (3), the organic solvent B is a combination of methanol and ethyl acetate or a combination of ethanol and ethyl acetate.

44. The method of synthesis of claim 2, wherein, In step (3), the organic solvent B is a combination of methanol and ethyl acetate.

45. The method of synthesis of claim 44, wherein, In step (3), the ratio of methanol to ethyl acetate in the organic solvent B is 8:1 to 10:

1.

46. The method of synthesis of claim 2, wherein, 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.

47. The method of synthesis of claim 2, wherein, In step (3), the recrystallization solvent organic solvent C is methanol:ethyl acetate=5:

1.

48. The method of synthesis of claim 2, wherein, In step (4), the organic solvent D is methanol, ethanol and / or tetrahydrofuran.

49. The method of synthesis of claim 2, wherein, In step (4), the organic solvent D is tetrahydrofuran.

50. The method of synthesis of claim 2, wherein, In step (4), the volume of the organic solvent D is 5 to 15 times the mass of compound IV.

51. The method of synthesis of claim 2, wherein, In step (4), the volume of organic solvent D is 10 times the mass of compound IV.

52. The method of synthesis of claim 2, wherein, In step (4), the feeding temperature and reaction temperature are -20℃ to 0℃.

53. The method of synthesis of claim 2, wherein, In step (4), the feeding temperature and reaction temperature are -5℃.

54. The method of synthesis of claim 2, wherein, In step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1 to 1:

2.

55. The method of synthesis of claim 2, wherein, In step (4), the molar ratio of compound IV to reducing agent sodium borohydride is 1:1.

5.

56. The method of synthesis of claim 2, wherein, In step (4), the molar ratio of compound IV to catalyst cerium chloride heptahydrate is 1:1 to 1:

2.

57. The method of synthesis of claim 2, wherein, In step (4), the molar ratio of compound IV to catalyst cerium chloride heptahydrate is 1:

1.

58. The method of synthesis of claim 2, wherein, In step (4), the stirring reaction time is 1.5 to 2.5 h.

59. The method of synthesis of claim 2, wherein, In step (4), the stirring reaction time is 2 h.

60. The method of synthesis of claim 2, wherein, In step (4), the acid solution used for quenching is dilute hydrochloric acid or acetic acid.

61. The method of synthesis of claim 2, wherein, In step (4), the acid solution used for quenching is dilute hydrochloric acid.

62. The method of synthesis of claim 61, wherein, In step (4), the mass fraction of dilute hydrochloric acid is 5% to 20%.

63. The method of synthesis of claim 61, wherein, In step (4), the mass fraction of dilute hydrochloric acid is 10%.

64. The method of synthesis of claim 2, wherein, In step (4), the organic solvent E used for recrystallization is methanol, ethanol or ethyl acetate.

65. The method of synthesis of claim 2, wherein, In step (4), the organic solvent E used for recrystallization is methanol.

66. The method of synthesis of claim 2, wherein, In step (5), the volume of tetrahydrofuran is 10 to 25 times the mass of compound V.

67. The method of synthesis of claim 2, wherein, In step (5), the volume of tetrahydrofuran is 20 times the mass of compound V.

68. The method of synthesis of claim 2, wherein, In step (5), the volume of methanol is 30 to 50 times the mass of compound V.

69. The method of synthesis of claim 2, wherein, In step (5), the volume of methanol is 40 times the mass of compound V.

70. The method of synthesis of claim 2, wherein, In step (5), the stirring reaction time is 15 to 24 h.

71. The method of synthesis of claim 2, wherein, In step (5), the stirring reaction time is 15 h.

72. The method of synthesis of claim 2, wherein, In step (5), the acid used for neutralization is dilute hydrochloric acid, dilute sulfuric acid and / or glacial acetic acid.

73. The method of synthesis of claim 2, wherein, In step (5), the acid used for neutralization is dilute hydrochloric acid.

74. The method of synthesis of claim 73, wherein, In step (5), the mass fraction of dilute hydrochloric acid is 5% to 20%.

75. The method of synthesis of claim 73, wherein, In step (5), the mass fraction of dilute hydrochloric acid is 5%.

76. The method of synthesis of claim 2, wherein, In step (5), the organic solvent G used for recrystallization is methanol, ethanol, water, tetrahydrofuran or any combination thereof.

77. The method of synthesis of claim 2, wherein, In step (5), the organic solvent G used for recrystallization is a combination of methanol and water.

78. The method of synthesis of claim 77, wherein, In step (5), the ratio of methanol to water in the organic solvent G used for recrystallization is methanol:water = 10:1.

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