A method for preparing an l96 side chain compound

Abstract

The application belongs to the field of medicine synthesis, and provides a preparation method of compound II, which comprises the following steps: reacting monomethyl dodecanedioate, N-hydroxysuccinimide and a condensing agent in an aprotic solvent to generate an active ester intermediate; and reacting compound I and an acid binding agent with the active ester intermediate to obtain compound II. Compound III can be prepared from compound II, the starting material of the steps is cheap and easy to obtain, the conditions are mild, the steps are simple, the yield is high, the product has high purity and stable chemical properties, the defects of high preparation cost and easy generation of impurities by column chromatography of compound III in the prior art are overcome, and the method is suitable for industrialized production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine synthesis, and relates to a preparation method of an L96 side chain compound, in particular to a preparation method of a compound III. BACKGROUND

[0002] The GalNAc conjugation technology can be applied in siRNA drugs and ASO drugs, and is the most effective small nucleic acid drug delivery system so far. GalNAc is a targeting ligand of sialic acid glycoprotein receptor (ASGPR), which has high affinity and rapid internalization capacity with ASGPR on the surface of hepatocytes. After GalNAc binds with ASGPR, it enters the cell to form an endosome through endocytosis, thereby bringing a sufficient amount of nucleic acid drugs into the cell to achieve liver-targeted delivery of nucleic acid drugs. Such siRNA conjugates have good application potential in the treatment of liver-related diseases related to gene overexpression.

[0003] Studies have shown that when the dose of GalNAc-siRNA single conjugate is 1 μg / g, subcutaneous injection can achieve effective silencing of specific target genes in the liver, and the silencing efficiency is much higher than that of siRNA loaded in lipid nanoparticles. GalNAc-siRNA optimized by chemical modification has better stability and interference efficiency, and such siRNA conjugates have good application potential in the treatment of liver-related diseases related to abnormal high expression of genes. The structure of GalNAc-siRNA conjugate is composed of three parts: a three-touch GalNAc target head, a connecting arm and an siRNA molecule. There are four nucleic acid drugs on the market in the world that use GalNAc conjugation delivery system, which cover genetic metabolic diseases and cardiovascular system diseases, and all of them use L96 as a delivery molecule. L96 includes a three-touch GalNAc target head (left half) and a connecting arm (right half, side chain), and its structure is as follows:

[0004]

[0005] The L96 molecule is connected to the siRNA through the hydroxyl group of the side chain. US patent applications US32852808 and US201113326203 disclose a preparation method of L96, both of which use compound ((3R, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol) and compound dodecanedioic acid monomethyl ester as raw materials, and obtain L96 side chain (compound III) through condensation. The synthesis route is as follows:

[0006]

[0007] Most of the current GalNAc-siRNA targeted delivery field adopts the above synthetic route, wherein the synthesis step of ((3R, 5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol) is long and expensive, resulting in high cost of compound III, and during the column chromatography purification process of compound III, silica gel can easily cause the removal of DMT protecting group to produce impurity A, which is difficult to remove in the subsequent purification step, finally affecting the purity of L96. The structure of impurity A is shown below:

[0008]

[0009] Chinese patent application 201780034659.0 discloses another preparation route of L96, as follows:

[0010]

[0011] (3R, 5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) (7.9 g, 47.4 mmol), 12-methoxy-12-oxododecanoic acid (11.5 g, 47.4 mmol), HBTU (36 g, 76 mmol) and TEA (20 mL, 142.2 mmol) were stirred in DCM at room temperature for 16 hours. The precipitate was removed by filtration and the organic was washed with 1M HCl (x2), saturated NaHCO3 (x2), H2O and brine. After drying, the organic was concentrated in vacuo and purified by column chromatography (5% MeOH / DCM) to give 12-((2S, 4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoic acid methyl ester (137) (3.1 g, 18.3%).

[0012] Methyl 12-((2S, 4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) (2.7 g, 45.5 mmol) was prepared from methyl 12-((2S, 4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 9.0 mmol), DMT-Cl (2.8 g, 8.2 mmol) and TEA (1.1 mL, 8.2 mmol) in DCM at room temperature for 16 hours. The reaction was concentrated in vacuo and the residue was purified by column chromatography (5% MeOH / DCM, 0.1% TEA) to give methyl 12-((2S, 4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) (2.7 g, 45.5 mmol);

[0013] The scheme has the following defects:

[0014] The product (138) is 2.7 g, which should be 4.09 mmol, and the yield is 47.2%, and the overall yield is low;

[0015] The condensing reagent is HBTU, which is high in cost and has sensitization, and is not suitable for large-scale application;

[0016] The two synthesis steps take 16 hours respectively, and the reaction time is long, which is not suitable for industrial production;

[0017] The purification method of column chromatography is used, the product will be adsorbed and lost when passing through the column, which affects the yield; and the product may lose the DMTr protective group when passing through the column, which produces impurity A and affects the purity of the product. SUMMARY

[0018] The present application provides a preparation method of L96 side chain compound (compound III) to overcome the shortcomings of the prior art. The starting material of the method is cheap and easy to obtain, the reaction conditions are mild, the reaction rate is fast, the product does not need column chromatography purification, the operation is simple, the yield is high, the product is high in purity and stable in chemical properties, and the method is suitable for industrial production. The preparation route of the method is as follows:

[0019]

[0020] In one aspect, the present application provides a preparation method of compound II, which comprises the following steps:

[0021] A: reacting monomethyl dodecanedioate with N-hydroxysuccinimide and a condensing reagent to generate an active ester intermediate;

[0022] B: reacting compound I and an acid binding agent with the active ester intermediate to obtain compound II.

[0023] In some embodiments, the molar ratio of the condensing reagent to monomethyl dodecanedioate is 1.0-1.3:1.

[0024] In some embodiments, the molar ratio of the compound I to the active ester intermediate is 1.0-1.2:1.

[0025] In some embodiments, the molar ratio of the compound I to the acid binding agent is 1:1.5-3.0.

[0026] In some embodiments, the temperature of the step A reaction is 0-35°C, preferably 15-20°C.

[0027] In some embodiments, the reaction time of the step A is 2-3h.

[0028] In some embodiments, the temperature of the step B reaction is 0-35°C, preferably 15-20°C.

[0029] In some embodiments, the reaction time of step B is 2-4 h.

[0030] wherein the structure of the active ester intermediate is shown below:

[0031]

[0032] In some embodiments, the condensing agent can be one or more of carbodiimide reagents, preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIC). More preferably, the condensing agent is EDCI.

[0033] In some embodiments, the acid scavenger is selected from one or more of pyridine, N,N-diisopropylethylamine (DIEA) and triethylamine.

[0034] Preferably, the acid scavenger is pyridine.

[0035] In some embodiments, the aprotic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide and acetonitrile.

[0036] Preferably, the aprotic solvent is dichloromethane.

[0037] In another aspect, the present application provides a method for preparing compound III, the method comprising the steps of:

[0038] C: coupling the compound II with a protecting monomer in the presence of an acid scavenger in an aprotic solvent to obtain a crude compound III;

[0039]

[0040] In some embodiments, the mass to volume ratio of the compound II to the polar aprotic solvent is 1:5-15, the mass of the compound II is in g and the volume of the polar aprotic solvent is in mL.

[0041] In some embodiments, the molar ratio of the compound II to the protecting monomer is 1:1.0-1.1.

[0042] In some embodiments, the aprotic solvent is a polar aprotic solvent.

[0043] In some embodiments, the aprotic solvent is selected from one or more of dichloromethane, pyridine and N,N-dimethylformamide.

[0044] In some embodiments, the volume ratio of the pyridine to N,N-dimethylformamide or dichloromethane is 1:8-12.

[0045] In some specific embodiments, the aprotic solvent is pyridine.

[0046] In some embodiments, the protecting monomer is DMT-Cl.

[0047] In some embodiments, the mass to volume ratio of the compound II to the aprotic solvent is 1:5-15, the mass of the compound II is in gram, and the volume of the polar aprotic solvent is in milliliter.

[0048] In some embodiments, the molar ratio of the compound II to DMT-Cl is 1:1.0-1.1.

[0049] In some embodiments, the reaction temperature of the coupling protecting group is 0°C-30°C, preferably 10°C-20°C.

[0050] In some embodiments, the method further comprises the following steps:

[0051] The solvent is added and stirred to wash, the liquid is separated, the organic phase is dried with a drying agent, filtered, and concentrated.

[0052] Preferably, the solvent is selected from one or more of water, ethyl acetate, dichloromethane, butyl acetate.

[0053] In some embodiments, the method further comprises:

[0054] The compound III crude product is purified by recrystallization, and the recrystallization comprises the following steps:

[0055] D: After the compound III crude product is mixed with a recrystallization solvent, the mixture is heated to a first temperature until it is completely dissolved, to obtain a solution;

[0056] E: The solution is cooled to a precipitation temperature, and a solid is precipitated;

[0057] F: The solid is filtered and vacuum dried, to obtain the purified compound III.

[0058] In some embodiments, the first temperature is 40°C-55°C.

[0059] The precipitation temperature is -30°C--20°C.

[0060] Preferably, the E comprises the following steps:

[0061] The solution is first cooled to 30°C-40°C, preferably 35°C, and maintained for 2-4 hours, and then cooled to the precipitation temperature.

[0062] More preferably, the E comprises the following steps:

[0063] The feed liquid is first cooled to 30-40°C, preferably 35°C, and maintained for 2-4 hours, and then cooled to the precipitation temperature at a cooling gradient of 0.1-0.3°C / min, preferably 0.1°C / min.

[0064] Preferably, the recrystallization solvent is an ether reagent.

[0065] More preferably, the recrystallization solvent is selected from one or more of methyl tert-butyl ether, isopropyl ether and diethyl ether.

[0066] In some embodiments, the mass-volume ratio of the compound III crude product to the recrystallization solvent is 1:1-6, the mass of the compound III crude product being measured in grams, and the volume of the recrystallization solvent being measured in milliliters.

[0067] In some embodiments, the temperature of the vacuum drying is 25-40°C, preferably 30°C.

[0068] The present application has the following advantages and effects:

[0069] (1) The condensation reagent is selected from EDCI, DCC or DIC, which is lower in cost and non-sensitizing;

[0070] (2) The reaction rate is fast, the reaction time is shortened, and it is suitable for industrial application;

[0071] (3) The crude compound III is purified by recrystallization, which has a high yield and effectively avoids the generation of impurity A, and improves the purity of the compound III;

[0072] (4) The chemical stability of the compound III purified by recrystallization is excellent, and the chemical properties remain stable under the conditions of light, high temperature and high humidity for up to 10 days or more, and the purity does not decrease significantly. DETAILED DESCRIPTION

[0073] The present application will be further described below in conjunction with specific examples, and the advantages and features of the present application will become more apparent as the description proceeds. However, these examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0074] The present application provides a preparation method of compound III, which does not need column chromatography, can effectively remove impurity A generated in the synthesis process, and the chemical properties of the obtained compound III are stable.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein.

[0076] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0077] The term "purity" herein refers to the percentage of a substance having a particular chemical structure in a sample relative to the total mass of the sample. Preferably, the term "purity" as used herein can refer to the purity of a compound as measured by HPLC.

[0078] The term "acid-binding agent" in the present application refers to an agent that can chemically react with acidic substances to form salts or acid anhydrides. The acid-binding agent reduces the acidity by binding the acidic components in the system.

[0079] The term "aprotic solvent" in the present application refers to a solvent that has very weak or no tendency to donate protons.

[0080] Instrument used: Agilent 1260, Bruker NMR 400MHz.

[0081] Preparation of compound II of Example 1

[0082] Step A: 1.22 g of dodecanedioic acid monomethyl ester and 0.61 g of N- hydroxysuccinimide (NHS) and 15 mL of dichloromethane were added to a flask, stirred to dissolve, temperature controlled at 15-20 °C. 1.01 g of l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDCI) was added, stirred to react for 2.5 h, TLC monitored the reaction to completion. 10 mL of water was added, stirred to wash, separated, the organic phase was dried over anhydrous sodium sulfate. Filtered to remove the drying agent. Concentrated to give 1.70 g of the active ester intermediate, yield 99.7%.

[0083] Step B: To a flask was added 15 mL of dichloromethane, 0.80 g of (3R, 5S)-5- (hydroxymethyl)pyrrolidin-3-ol hydrochloride (Compound I) and 1.50 g of N,N- diisopropylethylamine (DIEA) and stirred to dissolve. The temperature was controlled between 15°C and 20°C and 1.70 g of the active ester intermediate was added and stirred for 3 h. The reaction was monitored by TLC until completion. Water was added and stirred, allowed to separate and the organic phase was dried over anhydrous sodium sulfate. The drying agent was removed by filtration. Concentration gave Compound II 1.62 g in 94.7% yield.1H NMR (400 MHz, CDCI3): δ = 4.41 (m, 1H), 4.21 (m, 1H), 3.78 (d, 1H), 3.68 (m, 2H), 3.65 (s, 3H), 3.50 (dd, 1H), 2.31 (m, 4H), 2.07 (m, 1H), 1.73 (m, 1H), 1.61 (m, 4H), 1.27 (m, 12H).

[0084] Example 2 Preparation of Compound II

[0085] Step A: To a flask was added 1.22 g of dodecanedioic acid monomethyl ester and 0.61 g of N-hydroxysuccinimide (NHS) and 15 mL of dichloromethane and stirred to dissolve, controlling the temperature between 15°C and 20°C. 0.63 g of diisopropylcarbodiimide (DIC) was added and stirred for 2.5 h. The reaction was monitored by TLC until completion. 10 mL of water was added and stirred, allowed to separate and the organic phase was dried over anhydrous sodium sulfate. The drying agent was removed by filtration. Concentration gave the active ester intermediate 1.63 g in 95.3% yield.

[0086] Step B: To a flask was added 15 mL of N,N-dimethylformamide, 0.77 g of (3R, 5S)-5- (hydroxymethyl)pyrrolidin-3-ol hydrochloride (Compound I) and 1.50 g of N,N- diisopropylethylamine (DIEA) and stirred to dissolve. The temperature was controlled between 15°C and 20°C and 1.63 g of the active ester intermediate was added and stirred for 3 h. The reaction was monitored by TLC until completion. Water and dichloromethane were added and stirred, allowed to separate and the organic phase was dried over anhydrous sodium sulfate. The drying agent was removed by filtration. Concentration gave Compound II 1.6 g in 97.6% yield.

[0087] Example 3 Preparation of Compound II

[0088] Step A: Into a flask was placed 1.22 g of dodecanedioic acid monomethyl ester and 0.61 g of N-hydroxysuccinimide (NHS) and 15 mL of acetonitrile, stirred to dissolve, temperature controlled at 15-20 °C. 1.03 g of dicyclohexyl carbodiimide (DCC) was added, stirred for 2.5 h, TLC monitored for completion of reaction. 10 mL of water was added, stirred to wash, partitioned, organic phase was dried over anhydrous sodium sulfate. Filtered to remove drying agent. Concentrated to give 1.71 g of active ester intermediate, 100% yield.

[0089] Step B: Into a flask was placed 15 mL of acetonitrile, 0.81 g of (3R,5S)-5- (hydroxymethyl)pyrrolidin-3-ol hydrochloride (Compound I) and 1.50 g of N,N- diisopropylethylamine (DIEA), stirred to dissolve, temperature controlled at 15-20 °C. 1.71 g of active ester intermediate was added, stirred for 3 h, TLC monitored for completion of reaction. Washed with water, allowed to stand, partitioned. Organic phase was dried over anhydrous sodium sulfate, filtered to remove drying agent. Concentrated to give 1.65 g of Compound II, 95.9% yield.

[0090] Example 4 Preparation of Compound III

[0091] Step C: Into a flask was placed 1.00 g of Compound II and 1.5 mL of pyridine in 10 mL of dichloromethane, stirred to dissolve, temperature controlled at 10-20 °C. 1.04 g of 4,4-dimethoxytrityl chloride (DMT-Cl) was added, stirred for 1.5 h, TLC monitored for completion of reaction.

[0092] Washed with 20 mL of water, stirred to wash, partitioned, organic phase was dried over anhydrous sodium sulfate. Filtered to remove drying agent. Concentrated to give 2.07 g of Compound III as a crude product.

[0093] Step D: To the flask containing Compound III crude product was added 10 mL of diethyl ether, heated to reflux, stirred to dissolve completely.

[0094] Step E: The solution was cooled to -30 °C to precipitate the solid.

[0095] Step F: Filtered under suction, the filter cake was dried under vacuum at 30 °C to give 1.72 g of white powder as a solid, 91.5% yield, 98.38% purity by HPLC, no impurity A detected.

[0096] Example 5 Preparation of Compound III

[0097] Step C: Add 1.00 g of compound II and 1.5 mL of pyridine into 10 mL of N,N- dimethylformamide, stir to dissolve. Control the temperature at 10-20 °C, add 1.04 g of 4,4- dimethoxytrityl chloride (DMT-Cl), stir to react for 1.5 h, monitor the reaction completion by TLC.

[0098] Add 30 mL of ethyl acetate and 30 mL of water, stir to wash, separate the organic phase, and dry over anhydrous sodium sulfate. Filter to remove the drying agent. Concentrate to obtain 2.11 g of crude compound III.

[0099] Step D: Add isopropyl ether 10 mL into the flask containing the crude compound III, heat to 50 °C, and stir to dissolve until completely dissolved.

[0100] Step E: Cool the solution to -30 °C to precipitate the solid.

[0101] Step F: Filter by suction, dry the filter cake at 30 °C under vacuum to obtain 1.64 g of white powder solid, with a yield of 87.2%, HPLC purity of 97.84%, and no impurity A detected.

[0102] Preparation of compound III in Example 6

[0103] Step C: Add 1.00 g of compound II into 10 mL of pyridine, stir to dissolve. Control the temperature at 10-20 °C, add 1.40 g of 4,4-dimethoxytrityl chloride (DMT-Cl), and stir to react for 1.5 h. Monitor the reaction completion by TLC.

[0104] Add 30 mL of ethyl acetate and 30 mL of water, stir to wash, separate the organic phase, and dry over anhydrous sodium sulfate. Filter to remove the drying agent. Concentrate to obtain 1.88 g of crude compound III.

[0105] Step D: Add methyl tert-butyl ether 10 mL into the flask containing the crude compound III, heat to 45 °C, and stir to dissolve until completely dissolved.

[0106] Step E: Cool to 35 °C and maintain at 35 °C for 2-4 h until solid precipitates. Then, cool the solution to -30 °C.

[0107] Step F: Filter by suction, dry the filter cake at 30 °C under vacuum to obtain 1.69 g of white powder solid, with a yield of 89.9%, HPLC purity of 99.12%, and no impurity A detected.

[0108] Preparation of compound III in Example 7

[0109] Step C: Add 1.00 g of compound II to 10 mL of pyridine and stir to dissolve. Control the temperature at 10-20 °C and add 1.40 g of 4,4-dimethoxytrityl chloride (DMT-Cl) and stir the reaction for 1.5 h. Monitor the reaction completion by TLC.

[0110] Add 30 mL of ethyl acetate and 30 mL of water, stir to wash and separate the layers. Dry the organic phase over anhydrous sodium sulfate. Filter to remove the drying agent. Concentrate to obtain 1.92 g of crude compound III.

[0111] Step D: Add methyl tert-butyl ether 10 mL to the flask containing crude compound III and heat to 45 °C and stir to dissolve completely.

[0112] Step E: Cool to 35 °C and maintain at 35 °C for 2-4 h until solid precipitates. Then cool the solution to -20 °C at a cooling gradient of 0.3 °C / min.

[0113] Step F: Filter the solid under suction and dry the filter cake under vacuum at 30 °C to obtain 1.74 g of white powder as solid with 92.5% yield and 99.30% purity by HPLC with no impurity A detected.

[0114] Preparation of compound III of Example 8

[0115] Step C: Add 1.00 g of compound II to 10 mL of pyridine and stir to dissolve. Control the temperature at 10-20 °C and add 1.40 g of 4,4-dimethoxytrityl chloride (DMT-Cl) and stir the reaction for 1.5 h. Monitor the reaction completion by TLC.

[0116] Add 30 mL of ethyl acetate and 30 mL of water, stir to wash and separate the layers. Dry the organic phase over anhydrous sodium sulfate. Filter to remove the drying agent. Concentrate to obtain 1.92 g of crude compound III.

[0117] Step D: Add methyl tert-butyl ether 10 mL to the flask containing crude compound III and heat to 45 °C and stir to dissolve completely.

[0118] Step E: Cool to 35 °C and maintain at 35 °C for 2-4 h until solid precipitates. Then cool the solution to -20 °C at a cooling gradient of 0.3 °C / min.

[0119] Step F: Filter the solid under suction and dry the filter cake under vacuum at 30 °C to obtain 1.74 g of white powder as solid with 92.5% yield and 99.30% purity by HPLC with no impurity A detected.

[0120] Preparation of compound III of Example 9

[0121] Step C: Add 1.00 g of compound II into 10 mL of pyridine, stir to dissolve. Control the temperature at 10-20 °C, add 1.40 g of 4,4-dimethoxytrityl chloride (DMT-Cl), stir the reaction for 1.5 h. Monitor the reaction completion by TLC.

[0122] Add 30 mL of ethyl acetate and 30 mL of water, stir to wash, separate the organic phase, dry over anhydrous sodium sulfate. Filter to remove the drying agent. Concentrate to obtain 1.97 g of compound III as a crude product.

[0123] Step D: Add methyl tert-butyl ether 10 mL into the flask containing the crude compound III, heat to 45 °C, stir to dissolve until fully dissolved.

[0124] Step E: Cool down to 35 °C and maintain at 35 °C for 2-4 h until solid precipitates. Then, cool the solution to -20 °C at a cooling gradient of 0.1 °C / min.

[0125] Step F: Filter with suction, dry the filter cake at 30 °C under vacuum to obtain 1.69 g of white powder as a solid, with a yield of 89.9%, HPLC purity of 99.62%, and no impurity A detected.

[0126] Preparation of compound III (US20120136042A1)

[0127] This example uses the preparation method of compound III disclosed in US20120136042A1 as a comparison.

[0128] Add 3.00 g of (3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol and 1.75 g of dodecanedioic acid monomethyl ester into 50 mL of N,N-dimethylformamide. Add 3.25 g of O-benzotriazol- tetramethyluronium hexafluorophosphate and 3.7 mL of N,N-diisopropylethylamine, stir overnight. Pour the reaction mixture into an ice water mixture, extract with dichloromethane. Wash the organic phase with aqueous sodium bicarbonate solution, water and brine separately, dry over anhydrous sodium sulfate. Concentrate, purify on a silica gel column (eluted with 50% ethyl acetate / n-hexane and 5% methanol / dichloromethane) to obtain 3.97 g of white solid, with a yield of 88.2%, HPLC purity of 96.78%, and impurity A content of 1.33%.

[0129] Preparation of compound III (CN109312342A)

[0130] This example uses the preparation method of compound III disclosed in CN109312342A as a comparison.

[0131] (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) (7.9 g, 47.4 mmol), 12-methoxy-12-oxododecanoic acid (11.5 g, 47.4 mmol), HBTU (36 g, 76 mmol) and TEA (triethylamine) (20 mL, 142.2 mmol) were stirred in DCM at room temperature for 16 hours. The precipitate was removed by filtration and the organics were washed with 1M HCl (x2), saturated NaHCO3 (x2), H2O and brine. After drying, the organics were concentrated in vacuo and purified by column chromatography (5% MeOH / DCM) to give methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 18.3%).

[0132] Methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 9.0 mmol), DMT-C1 (4,4'-dimethoxytrityl chloride)

[0133] (2.8 g, 8.2 mmol) and TEA (1.1 mL, 8.2 mmol) were stirred in DCM at room temperature for 16 hours. The reaction was concentrated in vacuo and the residue was purified by column chromatography (5% MeOH / DCM, 0.1% TEA) to give methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) (2.7 g, 45.5 mmol).

[0134] CN109312342A discloses that the data calculation in the above scheme is wrong, and the product (138) is obtained in an amount of 2.7 g, which should be 4.09 mmol, and the yield is 47.2%.

[0135] Table 1 summarizes the yield, purity and other characterization data of compound III under different purification conditions in Examples 4-9 and Comparative Examples 1-2 for comparison:

[0136] Table 1 Comparison of compound III purification conditions and results

[0137]

[0138]

[0139] Stability test

[0140] The chemical stability of compound III prepared by different methods was explored.

[0141] Compound III prepared by example 4, example 5, example 9 and comparative example 1 was respectively weighed, and a stability test was carried out according to the guidance method of stability test in the fourth part of Chinese Pharmacopoeia. The purity was detected by HPLC method. The specific test results are shown in table 2.

[0142] Table 2: Stability test results of different products under different conditions

[0143]

[0144]

[0145] As shown in table 2, the purity of compound III prepared by the present application does not change obviously under the conditions of light, high temperature and high humidity, and impurity A is not detected. The purity of the product of comparative example 1 prepared by the method disclosed in US20120136042A1 is greatly reduced under the same experimental conditions, and the contents of impurity A and total impurities are obviously increased, that is, the product is deteriorated. Therefore, the compound III prepared by the present application has good chemical stability.

[0146] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing compound III, characterized in that, The method includes the following steps: A: In aprotic solvent 1, monomethyl dodecanoate reacts with N-hydroxysuccinimide and a condensing agent to generate an active ester intermediate; B: Compound I and the acid-binding agent are reacted with the active ester intermediate to obtain compound II; ； C: In aprotic solvent 2, under the condition of the presence of an acid-binding agent, compound II is reacted with a protecting monomer to obtain crude compound III; ； D: After mixing the crude product of compound III with the recrystallization solvent, heat it to the first temperature until it is completely dissolved to obtain the liquid. E: Cool the liquid to the precipitation temperature to precipitate solids; F: Filter and dry to obtain purified compound III; The condensing agent is selected from one or more of EDCI, DCC, and DIC; the acid-binding agent is selected from one or more of pyridine, N,N-diisopropylethylamine, and triethylamine. The recrystallization solvent is selected from one or more of methyl tert-butyl ether, isopropyl ether, and diethyl ether; the first temperature is 40℃~55℃; the precipitation temperature is -30℃~-20℃; The E includes the following steps: first, cool the liquid to 30℃~40℃ and maintain it for 2~4 hours, then cool it to the precipitation temperature at a cooling gradient of 0.1~0.3℃ / min.

2. The method according to claim 1, characterized in that, The aprotic solvent 1 is selected from one or more of dichloromethane, N,N-dimethylformamide, and acetonitrile.

3. The method according to claim 1, characterized in that, The aprotic solvent 2 is a polar aprotic solvent.

4. The method according to claim 3, characterized in that, The aprotic solvent 2 is selected from one or more of dichloromethane, pyridine, and N,N-dimethylformamide.

5. The method according to claim 1, characterized in that, The protective monomer is DMT-Cl.

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