Synthetic method of 6-carbonyl estradiol and intermediate thereof

Abstract

The invention discloses a synthesis method of 6-carbonyl estradiol and an intermediate thereof, and the synthesis method of the intermediate of 6-carbonyl estradiol comprises the following steps: A1, dissolving an intermediate IN1 in a mixture of an organic solvent and water, adding phthalic anhydride, hydrogen peroxide and a catalyst 1, and reacting to obtain an intermediate IN2; a2, the intermediate IN2 is dissolved in an organic solvent, an intermediate IN3 is obtained through an oxidation reaction, TEMPO, iodobenzene diacetate and a catalyst 2 are added for a reaction in the oxidation reaction, or a Jones reagent is added for a reaction, and the intermediate IN3 is an intermediate of 6-carbonyl estradiol; the invention further discloses a synthesis method of the 6-carbonyl estradiol. The target product obtained through the synthesis method is high in yield and purity, short in reaction time, safe and efficient in reaction and beneficial to industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and specifically relates to a method for synthesizing 6-carbonyl estradiol and an intermediate thereof. Background Art

[0002] Fulvestrant is an intramuscular injection drug developed by AstraZeneca and approved by the U.S. Food and Drug Administration in April 2002. Fulvestrant is a new class of estrogen receptor antagonists, estrogen receptor downregulators, for the treatment of metastatic advanced breast cancer in postmenopausal women who have failed to respond to anti-estrogen therapy, whose disease has progressed, or whose hormone receptors are positive. The compound 6-carbonyl estradiol is a key intermediate in the synthesis of fulvestrant.

[0003] The existing synthesis method of 6-carbonyl estradiol is to use estradiol as a raw material and synthesize it through a 4-step reaction. The specific synthesis route is as follows:

[0004]

[0005] However, the synthesis process has the following problems:

[0006] 1) When compound 1 is used to synthesize compound 2, dangerous and expensive reagents such as butyl lithium need to be used first.

[0007] 2) When compound 2 is used to synthesize compound 3, expensive and dangerous IBX is used as an oxidant, and non-recyclable DMSO is used as a solvent.

[0008] 3) The starting material is highly active estradiol, and all intermediates are steroidal structures with aromatized A ring, which have high physiological activity and are not conducive to the safety of industrial production.

[0009] Therefore, on the basis of the existing technology, a synthesis route of 6-carbonyl estradiol is studied, which has low synthesis cost, high process safety, is suitable for industrial production, and has significant advantages over the existing technology. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a method for synthesizing 6-carbonyl estradiol and its intermediates, which has high yields of intermediates and target products, high purity, short reaction time, safe and efficient reaction, and is conducive to realizing industrial production.

[0011] The present invention includes: a method for synthesizing an intermediate of 6-carbonyl estradiol, comprising the following steps:

[0012] A1: Intermediate IN1 is dissolved in a mixture of an organic solvent and water, and phthalic anhydride, hydrogen peroxide, and catalyst 1 are added to react to obtain intermediate IN2;

[0013] A2: Intermediate IN2 is dissolved in an organic solvent and subjected to an oxidation reaction to obtain intermediate IN3. The oxidation reaction is carried out by adding TEMPO, iodobenzene acetate and catalyst 2, or by adding Jones reagent. The intermediate IN3 is an intermediate of 6-carbonylestradiol.

[0014] The specific synthesis route is as follows:

[0015]

[0016] The R group is =0 or OAc.

[0017] In one embodiment, the organic solvent in step A1 is one or more of ethyl acetate, tetrahydrofuran, methanol, and ethanol, more preferably ethyl acetate; the organic solvent in step A2 is one or more of ethyl acetate, dichloromethane, tetrahydrofuran, and acetone, more preferably ethyl acetate or acetone.

[0018] In one embodiment, the catalyst 2 in step A2 is concentrated sulfuric acid.

[0019] In one embodiment, the catalyst 1 in step A1 is an organic base, preferably triethylamine or pyridine.

[0020] In one embodiment, the amount of phthalic anhydride added in step A1 is 0.5 to 1 times by weight of the amount of intermediate IN1, and the amount of hydrogen peroxide added is 0.5-1 times by weight of the amount of intermediate IN1; the amount of catalyst 1 added is 0.05 times by weight of the amount of intermediate IN1; the amount of catalyst 1 added is 0.005-0.15 times by weight of the amount of intermediate IN1; the volume ratio of the organic solvent to water is 5:1 to 100:1, and the amount of the organic solvent used is 6 to 20 times by volume of the amount of intermediate IN1 added;

[0021] In the step A2, the amount of TEMPO added is 1%-10% by weight of the amount of intermediate IN2 added, the amount of iodobenzene acetate added is 1.1 to 2 times by mole of the amount of intermediate IN2 added, and the amount of Jones reagent added is 1.1 to 2 times by mole of the amount of intermediate IN2 added.

[0022] In one embodiment, when R is =O, the synthesis method of intermediate IN1 is:

[0023] The raw material SM is subjected to an ene esterification reaction to obtain the intermediate IN1. The reaction conditions are: cooling to 10-15°C, adding an acid catalyst of toluenesulfonic acid or sulfuric acid, and reacting at 10-30°C;

[0024] The reaction route is:

[0025]

[0026] The organic solvent for the olefination reaction is one of ethyl acetate, tetrahydrofuran, and dichloromethane, or no solvent, preferably no solvent, and the amount of the solvent used is 1 to 3 times the volume of the raw material SM, and acetic anhydride is added in an amount of 2 to 5 times the weight of SM;

[0027] When R is OAc, the synthesis method of intermediate IN1 is:

[0028] B1: The raw material SM is subjected to enzymatic reduction to obtain the intermediate INA;

[0029] B2: Intermediate INA is subjected to esterification reaction to obtain intermediate IN1;

[0030] The reaction route is:

[0031]

[0032] In one embodiment, the esterification reaction conditions in step B2 are: adding isopropenyl acetate, the reaction temperature is 40-60° C., the reaction time is 4-12 h, and the reaction solvent is one of ethyl acetate, dichloromethane or tetrahydrofuran.

[0033] Another object of the present invention is to provide a method for synthesizing 6-carbonyl estradiol. When R is =O, the method for synthesizing 6-carbonyl estradiol is:

[0034] C1: The intermediate IN3 synthesized by the above synthesis method is subjected to enzymatic dehydrogenation reaction to obtain the intermediate IN4;

[0035] C2: intermediate IN4 is reduced by enzymatic reaction to obtain 6-carbonyl estradiol;

[0036] The reaction route is:

[0037]

[0038] The C1 reaction conditions are as follows: triethanolamine, potassium dihydrogen phosphate, pyrophosphate, water and isopropanol are added to IN3, and then XE-1 dehydrogenase, catalase and PMS (phenazine methyl sulfate) are added to react under air atmosphere; the amount of triethanolamine added is 20%-40% times the weight of IN3, the amount of potassium dihydrogen phosphate added is 1%-5% times the weight of IN3, the total amount of water and isopropanol added is 5-30 times the volume of IN3, wherein the volume ratio of water to isopropanol is 10:1 to 50:1, the XE-1 dehydrogenase is a steroid 1,2 dehydrogenase, and the amount added is 2 to 8 times the volume of IN3, the amount of catalase added is 1%-8% times the volume of IN3, and the amount of PMS added is 5%-15% times the weight of catalase; the reaction temperature is 28-35°C, and the reaction time is 16-36h.

[0039] The reaction conditions of C2 are as follows: weigh IN4, add glucose, water and isopropanol, stir, adjust the pH value to 7.0-7.5 with dilute hydrochloric acid or sodium carbonate, add glucose dehydrogenase and NADP(+) (nicotinamide adenine dinucleotide phosphate) after the pH is adjusted, and finally add 17-ketoreductase; the amount of glucose added is 0.8-1.5 times the mass of the amount of IN4 added, the total amount of water and isopropanol added is 8 to 30 times the volume of the amount of IN4 added, wherein the amount of water and isopropanol added is 0.8-1.5 times the mass of the amount of IN4 added, and the total amount of water and isopropanol added is 0.8-1.5 times the volume of the amount of IN4 added. The volume ratio is 5:1 to 40:1; the amount of glucose dehydrogenase added is 0.2 to 0.5 times the volume of IN4, the amount of NADP(+) added is 1% to 3% of glucose dehydrogenase, and the amount of 17-ketoreductase added is 2 to 5 times the volume of IN4; the pH value of the reaction is controlled at 6.8-8.0 by a pH meter, and when the pH meter reading decreases, the pH meter is used to automatically add sodium carbonate aqueous solution, the reaction temperature is 28-35°C, and the reaction time is 24-36h.

[0040] When R is OAc, the synthesis method of 6-carbonyl estradiol is:

[0041] D1: The intermediate IN3 synthesized by the above synthesis method is subjected to enzymatic dehydrogenation reaction to obtain the intermediate INE;

[0042] D2: The intermediate INE undergoes ester hydrolysis to obtain 6-carbonyl estradiol;

[0043] The reaction route is:

[0044]

[0045] The D2 ester group hydrolysis reaction is as follows: adding a base to an organic solvent and water, wherein the organic solvent is methanol, ethanol, or tetrahydrofuran, and the base is sodium hydroxide, potassium hydroxide, or potassium carbonate.

[0046] Beneficial effects of the present invention:

[0047] (1) In the reaction process of obtaining intermediate IN2 by hydroxylation reaction of intermediate IN1, monoperoxyphthalic acid is replaced by phthalic anhydride + hydrogen peroxide, which reduces the cost and improves the yield. In addition, the peroxygen reagent is prepared in situ by phthalic anhydride and hydrogen peroxide, and there is no process of separating monoperoxyphthalic acid separately, which simplifies the process operation;

[0048] (2) During the reaction of obtaining intermediate IN2 by hydroxylation of intermediate IN1, a catalyst triethylamine or pyridine is added to catalyze the preparation reaction of the peroxy reagent of phthalic anhydride and hydrogen peroxide, thereby increasing the reaction rate and enabling the reaction to be smoothly and completely converted; and when the reaction solvent is replaced with ethyl acetate, the post-treatment is simplified, and the continuous addition reaction of IN2 and IN3 is achieved;

[0049] (3) During the reaction of intermediate IN2 to obtain intermediate IN3 by hydroxylation, various oxidants added did not react or reacted incompletely, such as IBX oxidation, manganese dioxide oxidation, etc.; the iodobenzene acetate + TEMPO oxidation used in this application, with concentrated sulfuric acid added for catalysis, or Jones oxidation, the reaction was complete and the reaction rate was greatly improved;

[0050] (4) Synthesis of intermediate IN2 from intermediate INE Compared with the copper salt dehydrogenation aromatization method, the enzymatic dehydrogenation of the present application has the advantages of high yield, almost no by-products, and green environmental protection; in the process of synthesizing intermediate IN1 from raw material SM, the reaction conditions are strictly controlled, the by-products are few, and the intermediate product purity is high; the reaction of obtaining intermediate IN1 from INA by esterification reaction uses isopropenyl acetate instead, the reaction conditions are mild, the use of irritating acetyl chloride and high temperature reaction are avoided, and the post-processing is simple;

[0051] (5) The entire reaction process uses cheap raw materials, a simplified process, and does not require column chromatography separation, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is the H-NMR spectrum of IN-3 (R group is =O);

[0053] Figure 2 is the C NMR spectrum of IN-3 (R group is =O);

[0054] Figure 3 is the H NMR spectrum of IN-4;

[0055] Figure 4 is the C NMR spectrum of IN-4;

[0056] Figure 5 It is the H NMR spectrum of 6-carbonylestradiol;

[0057] Figure 6 It is the NMR carbon spectrum of 6-carbonylestradiol;

[0058] Figure 7 is the H NMR spectrum of IN-1 (R group is =OAc);

[0059] Figure 8 is the C NMR spectrum of IN-1 (R group is =OAc);

[0060] Fig. 9 is the H NMR spectrum of IN-3 (R group is =OAc);

[0061] Fig.10 is the C NMR spectrum of IN-3 (R group is =OAc);

[0062] Fig.11 is the H NMR spectrum of IN-E;

[0063] Fig.12 This is the C NMR spectrum of IN-E. DETAILED DESCRIPTION

[0064] Example 1

[0065] Synthesis of compound IN-1 (R group is =O)

[0066] Take a 500ml three-necked flask, add 100g SM, 200g acetic anhydride, replace with nitrogen three times, cool to about 20℃, add p-toluenesulfonic acid, control the temperature at 20-25℃ and react for 6-8 hours, take the reaction liquid TLC monitoring until the raw material is basically reacted, transfer the reacted system to 5w ice water, control the temperature at 0-5℃ and stir for 1-1.5h, filter, drain as much as possible, wash with water to pH 6-7, filter the product, and put it directly into the next step without drying and refining.

[0067] Example 2

[0068] Synthesis of compound IN-2 (R group is =O)

[0069] 120 mL of ethyl acetate was measured, 15 g of phthalic anhydride and 1 g of triethylamine were added, 14 g of hydrogen peroxide (30% content) was added dropwise at room temperature, and the addition was completed within 10 min. The obtained reaction solution was stirred for 20 min. IN-1 wet product (20 g of SM was used as a raw material and prepared by the method of Example 1) was taken, 80 mL of ethyl acetate was added, and the mixture was stirred at about 15 degrees. The reaction solution of hydrogen peroxide and phthalic anhydride was added dropwise for 20 min, and the reaction was continued at 15 ° C until the reaction was complete. 8 g of sodium sulfite and 15.4 g of sodium carbonate in 120 mL of aqueous solution were added dropwise to quench the reaction, the liquids were separated, the aqueous phase was extracted with 30 mL of ethyl acetate, the organic phases were combined, the organic phases were washed twice with 40 mL of water, dried, and concentrated to obtain IN-2 crude product, 40 mL of ethyl acetate was added to pulp overnight, IN-2 fine product was filtered off, and 18 g was dried. The total yield of the two steps was 90%, and the purity was 95.126% (6α accounted for 22.773%, 6β accounted for 72.353%, and the detection wavelength was 240 nm).

[0070] Example 3

[0071] Synthesis of compound IN-2 (R group is =O)

[0072] Measure 200 mL of ethanol, add wet IN-1 (20 g of SM as a raw material prepared by the method of Example 1), stir, add 1 g of pyridine, add 15 g of phthalic anhydride, control the temperature at about 15 ° C, start to drop 14 g of hydrogen peroxide (30% content), add it for 20 min, continue to react at 15 ° C until the reaction is complete, drop 8 g of sodium sulfite and 15.4 g of sodium carbonate in 120 mL of aqueous solution to quench the reaction, concentrate to remove most of the ethanol, add 200 mL of water for hydrolysis, filter to obtain IN2, dry weight 17.1 g, two-step total yield 85.5%, purity 95.33% (6α accounts for 25.01%, 6β accounts for 70.32%, detection wavelength 240 nm).

[0073] Example 4

[0074] Synthesis of compound IN-3 (R group is =O)

[0075] Weigh 20g of IN-2, add 200mL of ethyl acetate, stir at about 20°C, add 0.5g of concentrated sulfuric acid and 0.8g of TEMPO, protect with nitrogen, avoid light, add 25g of iodobenzene acetate in batches, stir, warm to about 25°C for reaction, track until there is no raw material by TLC, add 100mL of aqueous solution containing 4g of sodium sulfite and 9g of sodium carbonate to the reaction solution to quench the reaction, stir, stand, separate, extract the aqueous phase once with 20mL of ethyl acetate, combine the organic phases, wash the organic phase twice with 20mL of water, concentrate to remove most of the ethyl acetate, add 120mL of petroleum ether, stir at room temperature for 2h, cool and filter, wash the filter cake with a small amount of petroleum ether to obtain the crude product of IN-3. Then add 40mL of isopropanol to the crude product of IN-3, stir at 40 degrees for 2h, cool, filter and obtain the fine product of IN-3, 15.2g in drying, yield 76%, purity 96.8%. 1 H NMR(600MHz,Chloroform-d)δ6.50(d,J=2.9Hz,1H),2.82(dd,J=16.7,3.9Hz,1H),2.60(m,1H),2 .55-2.41(m,3H),2.37(m,1H),2.20-2.11(m,2H),2.07(m,1H),2.00(m,1H),1.91(m,2H),1.71(m Hz,1H),1.60(m,1H),1.47(m,1H),1.43-1.32(m,2H),1.27(m,1H),0.95(s,3H). 13 C NMR (101MHz, CDCl3) δ219.38,199.64,199.33,153.58,128.67,50.71,47.64, 45.63,44.66,43.25,37.71,37.14,35.58,30.91,28.36,25.65,21.48,13.67.

[0076] Example 5

[0077] Synthesis of compound IN-3 (R group is =O)

[0078] Weigh 20g of IN-2, add 200mL of dichloromethane, stir at about 20°C, add 0.6g of concentrated sulfuric acid and 1.2g of TEMPO, protect with nitrogen, avoid light, add 25g of iodobenzene acetate in batches, stir, warm to about 25°C and react for about 5h. TLC tracking until there is no raw material, add 100mL of aqueous solution containing 4g of sodium thiosulfate and 9g of sodium carbonate to the reaction solution to quench the reaction, stir, stand, separate, extract the aqueous phase once with 20mL of dichloromethane, combine the organic phases, wash the organic phases twice with 20mL of water, concentrate to remove most of the dichloromethane, add 40mL of n-heptane, concentrate the remaining dichloromethane, add 120mL of n-heptane again, stir at room temperature for 2h, filter, and wash the filter cake with a small amount of n-heptane to obtain the crude product of IN-3. Then add 40 mL of isopropanol to the crude IN-3, stir at 40 degrees for 2 hours, cool, and filter to obtain the fine IN-3, 14.2 g in drying, with a yield of 71% and a purity of 96.3%.

[0079] Example 6

[0080] Synthesis of compound IN-3 (R group is =O)

[0081] Weigh 20g of IN-2, add 140mL of acetone, stir at about 0°C, dropwise add 40mL of Jones reagent (2.5M), track by TLC until there is no raw material, add 10mL of isopropanol to the reaction solution to quench the reaction, stir for 30min, add 200mL of water, concentrate to remove most of the acetone, add 200mL of water again for precipitation, filter, and wash with water to obtain crude IN-3. Add 40mL of isopropanol to the crude IN-3, stir at 40 degrees for 2h, cool, and filter to obtain fine IN-3, 12.4g in drying, with a yield of 62% and a purity of 97.3%.

[0082] Example 7

[0083] Synthesis of compound IN4

[0084] Weigh 5.00g of IN-3, add 1.50g of triethanolamine, 0.10g of potassium dihydrogen phosphate, 1.50g of bubbling, 60ml of water, 2ml of isopropanol, shake evenly, then add 25mL of C1,2 dehydrogenase with a measuring cylinder, shake evenly, add 0.2ml of catalase with a pipette, shake evenly, then drop 0.02g of PMS in 5mL of aqueous solution, shake while dropping. Set the shaker temperature to 30℃, stir at 200 rpm, keep warm for 20h, monitor by TLC, if the reaction is not complete, continue to extend the time until there is basically no raw material left in the reaction, adjust the pH to 8-9, add about 0.1g of alkaline protease at room temperature, heat to 40℃ and continue stirring for 2h, adjust the pH to 5-6, stir for 30min, filter, wash the residue with water, and get crude IN4. The crude IN4 was dissolved in 40 mL of acetone, filtered, concentrated to a small volume, and 40 mL of water was added for precipitation to obtain the fine IN4, which was dried to obtain 4.4 g, with a yield of 88%.

[0085] Example 8

[0086] Synthesis of compound IN4

[0087] Weigh 50.00g of IN-3, add 15g of triethanolamine, 1.0g of potassium dihydrogen phosphate, 15.0g of bubbling enemy, 600ml of water, 20ml of isopropanol, stir, then add 250mL of C1,2 dehydrogenase with a measuring cylinder, add 2ml of catalase with a pipette, and finally drop 0.2g of PMS in 50mL of aqueous solution. Set the temperature to 30℃, stir at 200 rpm, blow air with a bubbler, keep warm for 16h, monitor by TLC, if the reaction is not complete, continue to extend the time until there is basically no raw material left in the reaction, adjust the pH to 4-5, heat to 55℃ and continue stirring for 2h, filter, wash the filter residue with water, and obtain a mixture of IN4 crude product and enzyme, add 400mL of acetone to dissolve, stir for 30min, filter, wash the filter cake with a small amount of acetone, combine the organic phases, concentrate to about 50mL, add 400mL of water to precipitate IN4 fine product, dry to obtain 46g, and the yield is 92%. 1 H NMR (400MHz, DMSO-d6) δ9.63(s,1H),7.32(dd,J=8.7,1.0Hz,1H),7.29(d,J=2.8Hz,1H),7.01(dd,J=8.5,2.9Hz,1H),2.64(dd,J=16 .7,3.5Hz,1H),2.49-2.31(m,3H),2.13-2.03(m,1H),1.96(m,2H),1.83-1.76(m,1H),1.66(m,1H),1.60-1.37(m,3H),0.82(s,3H). 13C NMR (101MHz, DMSO) δ219.68,197.38,156.23,137.86,133.43,127.31,121. 77,112.27,49.64,47.52,43.04,42.42,35.73,31.44,25.14,21.29,13.78.

[0088] Example 9

[0089] Synthesis of 6-Carboxyestradiol

[0090] Weigh IN4 (5 g of IN3 was used to prepare crude IN4 by the method of Example 6), add 5 g of glucose monohydrate, 100 ml of water, and 5 ml of isopropanol, stir, set the temperature to 31°C, adjust the pH to 7.0-7.5, after adjusting the pH, add 1.5 mL of glucose dehydrogenase with a measuring cylinder, stir, weigh 0.03 g of NADP(+), and finally add 20 mL of thawed 17-ketoreductase with a measuring cylinder, measure the pH of the system with a pH meter, observe whether the pH meter reading decreases, and a decrease proves that the enzyme begins to work. The minimum alarm value of the pH meter is set to 7.30, and the peracid is adjusted with 15% sodium carbonate. Keep the reaction at 31°C for 24 hours. The reaction was stopped when TLC monitoring showed that about 5% of the raw materials remained, the pH value was adjusted to 8-9, about 0.1 g of protein hydrolase was added, the temperature was raised to 45°C, stirring was continued for 2 h, cooled, filtered, and washed with water to obtain crude 6-carbonyl estradiol, which was refined by beating with 10 mL of acetone to obtain fine 6-carbonyl estradiol, and dried to obtain 4.0 g, with a two-step yield of 80% and a purity of 99.5%. 1 H NMR (400MHz, Methanol-d4) δ7.36(d,J=2.8Hz,1H),7.31(dd,J=8.6,1.0Hz,1H),7.00(dd,J=8.5,2.9Hz,1H),3.67(t,J=8.6Hz,1H),2.60(dd,J=17.0,3.5H z,1H),2.49-2.31(m,2H),2.19(dd,J=17.0,13.3Hz,1H),2.11-1.95(m,2H), 1.83(m,1H),1.75-1.61(m,1H),1.61-1.45(m,2H),1.34(m,3H),0.76(s,3H). 13 C NMR(101MHz,Methanol-d4)δ199.05,155.72,138.73,133.00,126.44,121.29,111 .95,80.75,49.54,43.48,42.75,42.62,40.27,36.08,29.17,25.30,22.29,10.09.

[0091] Example 10

[0092] Synthesis of 6-Carboxyestradiol

[0093] Weigh 35 g of IN4 prepared by the method of Example 7, add 35 g of glucose monohydrate, 650 ml of water, and 30 ml of isopropanol, stir, set the temperature to 31°C, adjust the pH to 7.0-7.5, after adjusting the pH, add 10 mL of glucose dehydrogenase with a measuring cylinder, stir, weigh 0.2 g of NADP(+), and finally add 150 mL of thawed 17-ketoreductase with a measuring cylinder, measure the pH of the system with a pH meter, observe whether the pH meter reading decreases, and a decrease proves that the enzyme begins to work. The minimum alarm value of the pH meter is set to 6.90, and the peracid is adjusted with 15% sodium carbonate. Keep the reaction at 31°C for 24 hours. The reaction was stopped when TLC monitoring showed that about 5% of the raw materials remained, the pH value was adjusted to 8-9, about 0.1 g of protein hydrolase was added, the temperature was raised to 45°C, stirring was continued for 2 h, cooled, filtered, and washed with water to obtain crude IN5. 350 mL of dichloromethane was used for pulping, and IN5 fine product was filtered and dried to obtain 28.9 g, with a yield of 82.6% and a purity of 95.4%.

[0094] Embodiment 11

[0095] Synthesis of compound IN-A

[0096] To a 1000ml three-necked reaction flask, add 50g SM, 400ml water, 50g glucose and 40ml isopropanol in sequence and stir to obtain a pH of 7-8; add 0.5g NADP+ and measure a pH of 5-6; adjust the pH to 7.5-8.0 with 2% sodium hydroxide solution, add 25ml glucose dehydrogenase and 50ml 17-ketoreductase; measure the system pH with a pH meter and observe whether the pH meter reading decreases. A decrease indicates that the enzyme begins to work. The minimum alarm value of the pH meter is set to 6.90. Adjust the peracid with 15% sodium carbonate and keep the reaction at 31°C for 24h. When TLC monitoring shows that about 1-2% of the raw material remains, the reaction is stopped, the pH value is adjusted to 8-9, about 1 g of protein hydrolase is added, the temperature is raised to 45°C, stirring is continued for 2 h, and the crude INA is obtained after cooling, filtration and washing with water. The product is dissolved in 200 mL of dichloromethane, filtered, concentrated to dryness, and slurried with petroleum ether to obtain the product, which is dried to obtain 47.1 g with a yield of 94.2%.

[0097] Example 12

[0098] Synthesis of compound IN1 (R group is OAc)

[0099] Weigh 20 g of INA, add 100 g of acetic anhydride, stir at 10°C, add 2 g of p-toluenesulfonic acid in batches, react at room temperature for 16 h, and after TLC shows that the reaction is complete, slowly pour it into 300 mL of ice water, stir well, filter it, wash it with water until it is neutral, and dry it to obtain 24 g of IN1, with a weight yield of 120%. 1 H NMR (400MHz, Chloroform-d) δ5.77(d,J=2.4Hz,1H),5.54-5.43(m,1H),4.62(dd,J=9.2,7.7Hz,1H),2.13(s,3H),2.05(s,3H),0.82(s,3H). 13 C NMR (101MHz, CDCl3) δ171.28,169.30,148.71,134.56,123.75,117.59,82.82,50.28,43.53, 42.64,40.58,36.65,36.58,30.90,27.99,27.49,27.24,26.26,23.33,21.22,21.13,11.95.

[0100] Embodiment 13

[0101] Synthesis of compound IN1 (R group is OAc)

[0102] Weigh 20 g of INA, add 60 mL of ethyl acetate, 24 g of isopropyl acetate, stir, add 1 g of p-toluenesulfonic acid, react at 50 ° C for 8 h, add 1 mL of triethylamine after TLC shows that the reaction is complete, concentrate to remove ethyl acetate, add 20 mL of methanol, concentrate again, and finally add 40 mL of methanol, stir well, add dropwise 120 mL of aqueous solution containing 10 g of sodium bicarbonate, stir for 30 min, filter, wash the filter cake with water until neutral, and dry to obtain 25 g of IN1, with a yield of 125%.

[0103] Embodiment 14

[0104] Synthesis of compound IN2 (R group is OAc)

[0105] 180 mL of ethyl acetate was measured, and 25 g of the dry product of IN2 synthesized by the method of Example 12 was added, 14 g of hydrogen peroxide (30% content), 1 g of pyridine, and 6 mL of water were added, and the temperature was cooled to 20°C. 15 g of phthalic anhydride was added in batches. After the addition, the reaction was continued at 20°C until the reaction was complete. 120 mL of an aqueous solution of 5 g of sodium thiosulfate and 15.4 g of sodium carbonate was added dropwise to quench the reaction. The liquids were separated, and the aqueous phase was extracted with 30 mL of ethyl acetate. The organic phases were combined, and the organic phases were washed twice with 40 mL of water, dried, and concentrated to obtain a light yellow oil, which can also be directly put into the next step without concentration and purification.

[0106] Embodiment 15

[0107] Synthesis of compound IN3 (R group is OAc)

[0108] 0.5 g sulfuric acid and 0.8 g TEMPO were added to the ethyl acetate solution of INC prepared by the method of Example 13 (synthesized with 25 g INB as raw material) under stirring at about 20 ° C, nitrogen protection, light protection, 25 g iodobenzene acetate was added in batches, stirred, and the temperature was raised to about 25 ° C for reaction. TLC tracking until there was no raw material, 100 mL of aqueous solution containing 4 g sodium sulfite and 9 g sodium carbonate was added to the reaction solution to quench the reaction, stirred, allowed to stand, separated, the aqueous phase was extracted once with 20 mL ethyl acetate, the organic phases were combined, the organic phases were washed twice with 20 mL water, concentrated to remove most of the ethyl acetate, 120 mL petroleum ether was added, stirred at room temperature for 2 h, cooled and filtered, and the filter cake was washed with a small amount of petroleum ether to obtain a crude IND product. Then 40 mL of isopropanol was added to the crude IND product, stirred at 40 degrees for 2 h, cooled, and filtered to obtain a fine IND product, 15.2 g in drying, and the total yield of the two-step reaction was 60.8%, and the purity was 95.7%. 1 H NMR (400MHz, Chloroform-d) δ6.49 (dd, J=2.9, 1.1Hz, 1H), 4.66 (dd, J=9.2, 7.8H z,1H),2.71(dd,J=16.7,3.9Hz,1H),2.64-2.55(m,1H),2.49-2.30(m,3H),2.22 (m,1H),2.06(s,4H),2.00-1.93(m,1H),1.86(dd,J=9.2,2.7Hz,1H),1.82(s,1H ),1.68(m,2H),1.63-1.50(m,1H),1.43-1.29(m,3H),1.23(m,2H),0.87(s,3H). 13 C NMR (101MHz, CDCl3) δ199.81,199.78,171.09,153.99,128.39,82.08,50.15,45.46, 45.42,43.38,42.67,37.74,37.44,36.15,28.33,27.26,25.93,23.08,21.14,11.93.

[0109] Example 16

[0110] Synthesis of compound IN3 (R group is OAc)

[0111] The INC oil prepared by the method of Example 13 (synthesized with 25 g INB as raw material) was dissolved in 140 mL acetone, and 43 mL Jones reagent (2.5 M) was added dropwise under an ice bath. The reaction lasted for about 30 min, and 10 mL isopropanol was added. After the reaction was completed, 200 mL of water was added, and most of the acetone was removed by concentration. 200 mL of water was added again, and the mixture was cooled, filtered, and washed with water to obtain a light yellow to white solid. 40 mL of isopropanol was added to pulp for 2 h, filtered, and dried to obtain 14.7 g of the product. The total yield of the two-step reaction was 58.8%, and the purity was 96.1%.

[0112] Embodiment 17

[0113] Synthesis of compound INE

[0114] Weigh 10.00 g of IND synthesized by the method of Example 14, add 3 g of triethanolamine, 0.2 g of potassium dihydrogen phosphate, 3.0 g of folate, 120 ml of water, and 4 ml of isopropanol, stir, then add 50 ml of C1,2 dehydrogenase with a measuring cylinder, add 0.4 ml of catalase with a pipette, and finally add dropwise 10 ml of an aqueous solution of 0.04 g of PMS. The temperature was set at 30°C, the stirring speed was 200 rpm, air was introduced with a bubbler, the reaction was kept warm for 16 hours, and TLC was monitored. If the reaction was not complete, the reaction time was continued to be extended until there was basically no raw material remaining in the reaction, the pH was adjusted to 4-5, the temperature was raised to 55°C, and stirring was continued for 2 hours. The residue was filtered and washed with water to obtain a mixture of crude INE and enzyme, and 300 mL of a mixed solvent of methanol and dichloromethane (1:3) was added to dissolve, stirred for 30 minutes, filtered, and the filter cake was washed with a small amount of methanol-dichloromethane. The organic phases were combined and concentrated to dryness to obtain crude INE, and 20 mL of dichloromethane was added to slurry for 2 hours, filtered, and 8.9 g of fine INE was obtained, with a yield of 89%. 1 H NMR(400MHz,Chloroform-d)δ7.54(d,J=2.9Hz,1H),7.31(dd,J=8.6,1.1Hz,1H),7.08( dd,J=8.5,2.9Hz,1H),4.88(s,1H),4.71(dd,J=9.2,7.8Hz,1H),2.74(dd,J=16.9,3.4Hz ,1H),2.48(td,J=11.1,4.5Hz,1H),2.41-2.32(m,1H),2.29-2.17(m,2H),2.07(s,3H),1 .98-1.93(m,2H),1.75-1.68(m,2H),1.65-1.52(m,2H),1.51-1.34(m,3H),0.84(s,3H). 13C NMR (101MHz, CDCl3) δ198.38,171.33,154.64,139.28,133.29,126.93,121.57,112.80 ,82.28,77.24,49.64,43.98,42.76,39.85,36.44,27.39,25.42,22.97,21.19,11.93.

[0115] Embodiment 18

[0116] Synthesis of 6-Carboxyestradiol

[0117] Weigh 10 g of INE synthesized by the method of Example 16, add 60 mL of methanol, stir at room temperature, add 20 mL of aqueous solution containing 3 g of sodium hydroxide, react for 3 h until the reaction is basically complete, add acetic acid to adjust the pH to neutral, concentrate to remove most of the methanol, add 60 mL of water and stir for 2 h, filter to obtain crude 6-carbonyl estradiol, add 30 mL of ethyl acetate and beat for 4 h to obtain refined 6-carbonyl estradiol, dry to obtain 9.2 g, with a yield of 92% and a purity of 99.2%.

[0118] Comparative Example 1

[0119] Synthesis of compound IN-2 (R group is =O)

[0120] Measure 200 mL of ethyl acetate, add wet IN-1 (20 g of SM as raw material prepared by the method of Example 1), stir at about 15 degrees, add 18.6 g (75% content) of m-chloroperbenzoic acid in batches, add it for 20 minutes, continue to react at 15 ° C until the reaction is complete, add 3 g of sodium thiosulfate and 6 g of sodium carbonate in 100 mL of aqueous solution to quench the reaction, separate the liquids, extract the aqueous phase with 30 mL of ethyl acetate, combine the organic phases, wash the organic phases twice with 40 mL of water, dry, and concentrate to obtain the crude IN-2 product, add 40 mL of ethyl acetate to pulp overnight, filter to obtain the fine IN-2 product, and dry to obtain 15.8 g, with a total yield of 79% in two steps and a purity of 94.9%.

[0121] Comparative Example 2

[0122] Synthesis of compound IN-2 (R group is =O)

[0123] Measure 120mL of ethyl acetate, add 15g of phthalic anhydride, and add 14g of hydrogen peroxide (30% content) dropwise at room temperature for 10 minutes. The reaction solution is stirred for 1 hour. Take IN-1 wet product (20g SM is prepared by the method of Example 1 as a raw material), add 80mL of ethyl acetate, stir at about 15 degrees, and then add the reaction solution of hydrogen peroxide and phthalic anhydride dropwise. After 20 minutes, continue to react at 15°C-20°C. Most of the raw materials are still present after 2 days of reaction.

[0124] Comparative Example 3

[0125] Synthesis of compound IN-3 (R group is =O)

[0126] Weigh 20 g of IN-2, add 200 mL of ethyl acetate, stir at about 20 °C, add 0.5 g of oxalic acid and 0.8 g of TEMPO, protect with nitrogen, protect from light, add 25 g of iodobenzene acetate in batches, stir, raise the temperature to about 25 °C and react for 5 hours. Most of the raw materials are still not converted. If the reaction is allowed to proceed overnight, some raw materials are still not completely converted.

[0127] Comparative Example 4

[0128] Synthesis of compound IN-3 (R group is =O)

[0129] Weigh 20g of IN-2, add 100mL of acetone and 100mL of DMSO, stir at about 20°C, add 26g of IBX (2-iodoacylbenzoic acid) reagent in batches, heat to about 25°C and react overnight. Some raw materials are still not converted. Add 10mL of isopropanol to quench the reaction, concentrate to remove most of the acetone, add 400mL of water to precipitate a sticky solid, which is difficult to filter.

[0130] Comparative Example 5

[0131] Synthesis of compound IN-3 (R group is =O)

[0132] Weigh 20 g of IN-2, add 200 mL of ethyl acetate, stir at about 20 °C, add 0.8 g of TEMPO, protect with nitrogen, avoid light, add 25 g of iodobenzene acetate in batches, stir, raise the temperature to about 25 °C and react for 5 hours. Most of the raw materials are still not converted. If the reaction is allowed to proceed overnight, a large amount of raw materials are still not converted.

[0133] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0134] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. A method for synthesizing an intermediate of 6-carbonyl estradiol, characterized in that: The following steps are involved: A1: Intermediate IN1 is dissolved in a mixture of an organic solvent and water, and phthalic anhydride, hydrogen peroxide, and catalyst 1 are added to react to obtain intermediate IN2; A2: Intermediate IN2 is dissolved in an organic solvent and subjected to an oxidation reaction to obtain intermediate IN3. The oxidation reaction is carried out by adding TEMPO, iodobenzene acetate and catalyst 2, or by adding Jones reagent. The intermediate IN3 is an intermediate of 6-carbonylestradiol. The specific synthesis route is as follows: The R group is =0 or OAc.

2. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 1, characterized in that: The organic solvent in step A1 is one or more of ethyl acetate, tetrahydrofuran, methanol, and ethanol; the organic solvent in step A2 is one or more of ethyl acetate, dichloromethane, tetrahydrofuran, and acetone.

3. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 2, characterized in that: The organic solvent in step A1 is ethyl acetate; the organic solvent in step A2 is ethyl acetate or acetone.

4. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 1, characterized in that: The catalyst 2 in step A2 is concentrated sulfuric acid.

5. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 1, characterized in that: In the step A1, the catalyst 1 is an organic base.

6. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 5, characterized in that: In the step A1, the catalyst 1 is triethylamine or pyridine.

7. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 1, characterized in that: The amount of phthalic anhydride added in step A1 is 0.5 to 1 times the weight of the amount of intermediate IN1, the amount of hydrogen peroxide added is 0.5-1 times the weight of the amount of intermediate IN1; the amount of catalyst 1 added is 0.005-0.15 times the weight of the amount of intermediate IN1; the volume ratio of the organic solvent to water is 5:1 to 100:1, and the amount of the organic solvent is 6 to 20 times the volume of the amount of intermediate IN1 added; In the step A2, the amount of TEMPO added is 1%-10% by weight of the amount of intermediate IN2 added, the amount of iodobenzene acetate added is 1.1 to 2 times by mole of the amount of intermediate IN2 added, and the amount of Jones reagent added is 1.1 to 2 times by mole of the amount of intermediate IN2 added.

8. A method for synthesizing an intermediate of 6-carbonylestradiol according to any one of claims 1 to 7, characterized in that: When R is =O, the synthesis method of intermediate IN1 is: The raw material SM is subjected to an ene esterification reaction to obtain the intermediate IN1. The reaction conditions are: cooling to 10-15°C, adding an acid catalyst, and reacting at 10-30°C; The reaction route is: When R is OAc, the synthesis method of intermediate IN1 is: B1: The raw material SM is subjected to enzymatic reduction to obtain the intermediate INA; B2: Intermediate INA is subjected to esterification reaction to obtain intermediate IN1; The reaction route is:

9. The method for synthesizing an intermediate of 6-carbonylestradiol as claimed in claim 8, characterized in that: The esterification reaction conditions in step B2 are: adding isopropenyl acetate, the reaction temperature is 40-60° C., the reaction time is 4-12 hours, and the reaction solvent is one of ethyl acetate, dichloromethane or tetrahydrofuran.

10. A method for synthesizing 6-carbonyl estradiol, characterized in that: When R is =O, the synthesis method of 6-carbonyl estradiol is: C1: intermediate IN4 is obtained by enzymatic dehydrogenation reaction of the intermediate IN3 synthesized by the synthesis method according to any one of claims 1 to 9; C2: intermediate IN4 is reduced by enzymatic reaction to obtain 6-carbonyl estradiol; The reaction route is: When R is OAc, the synthesis method of 6-carbonyl estradiol is: D1: intermediate IN3 synthesized by the synthesis method according to any one of claims 1 to 9 is subjected to enzymatic dehydrogenation reaction to obtain intermediate INE; D2: The intermediate INE undergoes ester hydrolysis to obtain 6-carbonyl estradiol; The reaction route is:

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