Synthesis method of Itecatide key intermediate

Abstract

The invention discloses a synthesis method of an Itecatide key intermediate, and relates to the technical field of chemical synthesis of drugs, Fmoc-Cys (Bzl)-OH is used as a starting material, Fmoc-Cys (Bzl)-OH and tert-butyl alcohol are subjected to an esterification reaction to obtain a compound A1, the compound A1 is subjected to a reduction reaction to obtain a compound A2, and the compound A2 is subjected to a reaction with tert-butyl alcohol to obtain the Itecatide key intermediate. Carrying out oxidation reaction on the compound A2 and N-acetyl-D-cysteine to obtain an Itecatide key intermediate; the synthesis method not only greatly simplifies the synthesis operation, but also can obtain the high-purity and high-yield Itecatide key intermediate.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemical synthesis, and particularly relates to a method for synthesizing a key intermediate of etelcalcetide. Background Art

[0002] Primary hyperparathyroidism (PHPT) is a relatively common endocrine disease, which is caused by excessive synthesis and secretion of parathyroid hormone (PTH) due to lesions of the parathyroid gland itself. Etelcalcetide is a calcimimetic agent developed by Amgen Europe B.V., which has the effect of regulating the secretion of parathyroid hormone by the calcium-sensing receptor (CaSR) on the surface of parathyroid chief cells, thereby reducing the PTH level and can be used to treat hyperparathyroidism. Etelcalcetide is also applicable to secondary hyperparathyroidism (SHPT) in adult patients with chronic kidney disease (CKD) undergoing hemodialysis, and can be administered intravenously while undergoing hemodialysis, which is a blessing for chronic kidney disease patients who need to use a large amount of drugs every day.

[0003] The formation of disulfide bonds is a key step in the synthesis process of etelcalcetide. Currently, the synthesis methods of etelcalcetide mainly include liquid-phase synthesis method and solid-phase synthesis method.

[0004] Patent CN105504012A uses Rink amino resin as a solid-phase carrier, and gradually couples to obtain a linear peptide resin by traditional solid-phase synthesis method, then forms disulfide bonds by air oxidation on the solid phase, and then obtains the target product through cleavage, purification, and salt conversion. However, this method forms disulfide bonds in a solid-phase manner, and the reaction time is too long, generally more than 24 hours, which is not conducive to large-scale production.

[0005] Patent CN 111925418A is easy to control the purity by using Cbz-protected amino acids for liquid-phase coupling, effectively avoiding the missing peptides; and by using the active intermediate Boc-L-Cys(S-S-Py)-OtBu formed by disulfide dipyridine and cysteine to connect with the peptide chain, it is convenient to accurately control the synthesis of disulfide bonds. However, the 7-peptide intermediate with exposed thiol groups in this method needs to be prepared by HPLC purification, and the whole process requires two HPLC purifications, increasing the cost. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for synthesizing a key intermediate of etelcalcetide, which uses Fmoc-Cys(Bzl)-OH as the starting material, and prepares a disulfide bond compound through esterification, reduction, and oxidation reactions as an intermediate for the synthesis of etelcalcetide.

[0007] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0008] A synthetic method for a key intermediate of etelcalcetide, using Fmoc-Cys(Bzl)-OH as the starting material. Fmoc-Cys(Bzl)-OH undergoes an esterification reaction with tert-butanol to obtain compound A1. Compound A1 undergoes a reduction reaction to obtain compound A2. Compound A2 undergoes an oxidation reaction with N-acetyl-D-cysteine to obtain compound A3.

[0009] The synthetic route is as follows:

[0010]

[0011] Furthermore, the esterification reaction is carried out in the presence of a dehydrating agent and a catalyst. Further, the dehydrating agent includes but is not limited to one or more of dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC); the catalyst includes but is not limited to one or more of 4-dimethylaminopyridine (DMAP), imidazole, 1,2,4-triazole (TMAJ). The role of the dehydrating agent is to remove the water generated during the esterification reaction and promote the reaction of the acid with the alcohol to form an ester. The role of the catalyst is to promote the esterification reaction, increase the reaction rate, and lower the activation energy of the reaction.

[0012] Furthermore, the molar ratio of Fmoc-Cys(Bzl)-OH, tert-butanol, dehydrating agent, and catalyst is 1:(1 - 4):(0.5 - 2):(0.1 - 1). The esterification reaction can be carried out at room temperature or the reaction rate can be increased by heating, but the heating temperature needs to be controlled to prevent the formation of by-products.

[0013] Regarding the reaction solvent for the esterification reaction, an organic solvent that has good solubility for Fmoc-Cys(Bzl)-OH, can be miscible with tert-butanol, and will not react with Fmoc-Cys(Bzl)-OH, tert-butanol, dehydrating agent, and catalyst can be selected, such as dichloromethane, which has a low boiling point and is conducive to recycling and reuse.

[0014] Furthermore, the reduction reaction is carried out under a hydrogen atmosphere. Compound A1 undergoes a reduction reaction under a hydrogen atmosphere to reduce the thioether to form a mercapto group.

[0015] Furthermore, at least one of palladium carbon and palladium hydroxide is used as the catalyst for the reduction reaction. In some specific embodiments, the amount of the catalyst used is 0.01 - 1 times the mass of compound A1.

[0016] Regarding the reaction solvent for the reduction reaction, an organic solvent that has good solubility for compound A1 and will not react with compound A1 can be selected, such as ethanol, which has a low boiling point and is conducive to recycling and reuse; and has low toxicity and high safety in use.

[0017] Further, the oxidation reaction is carried out in the presence of an oxidizing agent. In some specific embodiments, the oxidizing agent is iodine, oxygen or air. The sulfhydryl group of compound A2 and the sulfhydryl group of N-acetyl-D-cysteine form a disulfide bond through oxidation.

[0018] Further, the molar ratio of compound A2 to N-acetyl-D-cysteine is 1:(1-2). When the oxidizing agent is iodine, the molar ratio of compound A2 to iodine is 1:(1-2).

[0019] The beneficial effects of the present invention are as follows: The key intermediate of iticatide is synthesized by using Fmoc-Cys(Bzl)-OH as the starting material, successively through the esterification reaction with tert-butanol, the reduction reaction of thioether and the oxidation reaction with N-acetyl-D-cysteine. This synthesis method not only greatly simplifies the synthesis operation, but also can obtain the key intermediate of iticatide with high purity and high yield. Description of the Drawings

[0020] Figure 1 It is the mass spectrum of compound A3. Detailed Embodiments

[0021] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments and illustrations.

[0022] Example 1

[0023] Synthesis of compound A1:

[0024] To a solution of Fmoc-Cys(Bzl)-OH (11.97 g, 27.6 mmol), tert-butanol (6.14 g, 82.8 mmol) and DMAP (2.70 g, 22.1 mmol) in dichloromethane (100 mL) at 0 °C, DCC (8.54 g, 41.4 mmol) was added in three portions. The mixture was stirred at 0 °C for 15 min, then heated to 25 °C and stirred for 24 h. The reaction was stopped, and the reaction solution was filtered through diatomaceous earth and washed with dichloromethane. The filtrate was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The organic phase was successively washed with saturated aqueous citric acid solution, saturated aqueous sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was purified by column chromatography to obtain compound A1. The purity was 98% and the yield was 92%.

[0025] Synthesis of compound A2:

[0026] Add 10 g of compound A1, 0.5 g of 5% palladium on carbon, and 50 mL of ethanol to a hydrogenation autoclave. Close the autoclave, displace with nitrogen three times, charge 1.0 MPa of hydrogen, and react at room temperature for 12 h. Stop the reaction, filter the reaction solution through diatomaceous earth, distill the filtrate under reduced pressure, and slurry the residue with n-heptane to obtain compound A2. The purity is 99% and the yield is 85%.

[0027] Synthesis of compound A3:

[0028] Dissolve compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (2.04 g, 12.5 mmol) in 100 mL of 30% aqueous acetic acid solution respectively, and dropwise add a methanol solution of iodine (10 g / L, 12.5 mmol). After the addition is complete, react for 40 min. Stop the reaction, add the reaction solution to 100 mL of ethyl acetate, wash successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution, take the organic phase, dry it over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and subject the residue to column chromatography to obtain compound A3. The purity is 95% and the yield is 50%.

[0029] Example 2

[0030] Synthesis of compound A1:

[0031] Add DCC (11.39 g, 55.2 mmol) to a solution of Fmoc-Cys(Bzl)-OH (11.97 g, 27.6 mmol), tert-butanol (2.05 g, 27.6 mmol), and DMAP (3.37 g, 27.6 mmol) in dichloromethane (100 mL) in three portions at 0 °C. Stir at 0 °C for 15 min, then heat to 30 °C and stir for 24 h. Stop the reaction, filter the reaction solution through diatomaceous earth, wash with dichloromethane, concentrate the filtrate under reduced pressure, extract the residue with ethyl acetate, wash the organic phase successively with saturated citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and subject the residue to column chromatography to obtain compound A1. The purity is 97% and the yield is 90%.

[0032] Synthesis of compound A2:

[0033] Add 10 g of compound A1, 0.1 g of palladium hydroxide, and 50 mL of ethanol to a hydrogenation autoclave. Close the autoclave, displace with nitrogen three times, charge 1.2 MPa of hydrogen, and react at room temperature for 18 h. Stop the reaction, filter the reaction solution through diatomaceous earth, distill the filtrate under reduced pressure, and slurry the residue with n-heptane to obtain compound A2. The purity is 99% and the yield is 81%.

[0034] Synthesis of compound A3:

[0035] Compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (4.08 g, 25 mmol) were separately dissolved in 100 mL of 30% aqueous acetic acid solution. An ethanol solution of iodine (15 g / L, 25 mmol) was added dropwise. After the addition was complete, the reaction was carried out for 30 min. The reaction was stopped, and the reaction solution was added to 100 mL of ethyl acetate, washed successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain compound A3. The purity was 93% and the yield was 55%.

[0036] Example 3

[0037] Synthesis of compound A1:

[0038] To a solution of Fmoc-Cys(Bzl)-OH (11.97 g, 27.6 mmol), tert-butanol (8.18 g, 110.4 mmol) and DMAP (0.34 g, 2.76 mmol) in dichloromethane (100 mL) at 0 °C, DCC (2.85 g, 13.8 mmol) was added in three portions. The mixture was stirred at 0 °C for 15 min, then heated to reflux and stirred for 12 h. The reaction was stopped, and the reaction solution was filtered through diatomaceous earth and washed with dichloromethane. The filtrate was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The organic phase was washed successively with saturated aqueous citric acid solution, saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain compound A1. The purity was 98% and the yield was 87%.

[0039] Synthesis of compound A2:

[0040] 10 g of compound A1, 1 g of 5% palladium on carbon and 50 mL of ethanol were added to a hydrogenation autoclave. The hydrogenation autoclave was closed, purged with nitrogen three times, filled with 1.0 MPa of hydrogen, and reacted at room temperature for 12 h. The reaction was stopped, and the reaction solution was filtered through diatomaceous earth. The filtrate was distilled under reduced pressure, and the residue was slurried with n-heptane to obtain compound A2. The purity was 99% and the yield was 89%.

[0041] Synthesis of compound A3:

[0042] Compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (3.06 g, 18.75 mmol) were separately dissolved in 100 mL of 30% aqueous acetic acid solution. A methanol solution of iodine (12 g / L, 12.5 mmol) was added dropwise. After the addition was complete, the reaction was carried out for 15 min. The reaction was stopped, and the reaction solution was added to 100 mL of ethyl acetate, washed successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain compound A3. The purity was 90% and the yield was 46%.

[0043] As can be seen from Examples 1 to 3, the yield of synthesizing Compound A3 from Compound A2 is very low, resulting in a small amount of Compound A3 produced. In order to improve the yield of Compound A3, the inventors further studied the reaction conditions of this oxidation reaction. On the premise of using iodine as the oxidant, catalysts that can substantially improve the reaction activity were screened to increase the conversion rates of Compound A2 and N-acetyl-D-cysteine and promote the formation of disulfide bonds. Finally, the inventors screened out oxotitanium phthalocyanine as the catalyst for this oxidation reaction. When its dosage is 5% of the mass of Compound A2, the yield of Compound A3 can reach more than 70%. Further increasing the dosage of this catalyst, the yield of Compound A3 can still increase slightly. However, based on cost control considerations, the dosage of this catalyst in the present invention is controlled at 5-8% of the mass of Compound A2. That is to say, the present invention also provides a method for synthesizing a key intermediate of itcatibant. Using Fmoc-Cys(Bzl)-OH as the starting material, Fmoc-Cys(Bzl)-OH undergoes an esterification reaction with tert-butanol to obtain Compound A1. Compound A1 is obtained by a reduction reaction to obtain Compound A2. Compound A2 undergoes an oxidation reaction with N-acetyl-D-cysteine to obtain Compound A3. Among them, the oxidation reaction is carried out in the presence of an oxidant and a catalyst; the catalyst is oxotitanium phthalocyanine, and the dosage is 5-8% of the mass of Compound A2.

[0044] Example 4

[0045] Synthesis of Compound A3:

[0046] Compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (3.06 g, 18.75 mmol) were respectively dissolved in 100 mL of 30% aqueous acetic acid solution, and 0.25 g of oxotitanium phthalocyanine was added and dispersed evenly. Subsequently, a methanol solution of iodine (12 g / L, 12.5 mmol) was added dropwise. After the addition was completed, the reaction was carried out for 15 min. The reaction was stopped, the reaction solution was filtered, 100 mL of ethyl acetate was added to the filtrate, and it was washed successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution. The organic phase was taken and dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain Compound A3. The purity was 94% and the yield was 72%.

[0047] Example 5

[0048] Synthesis of Compound A3:

[0049] Dissolve compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (3.06 g, 18.75 mmol) separately in 100 mL of 30% aqueous acetic acid solution, add 0.3 g of titanium phthalocyanine oxide, disperse evenly by ultrasonic wave, and then dropwise add a methanol solution of iodine (12 g / L, 12.5 mmol). After the addition is complete, react for 15 min. Stop the reaction, filter the reaction solution, add it to 100 mL of ethyl acetate, wash successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution, take the organic phase, dry it with anhydrous sodium sulfate, filter, concentrate under reduced pressure, subject the residue to column chromatography to obtain compound A3. The purity is 95% and the yield is 75%.

[0050] Example 6

[0051] Synthesis of compound A3:

[0052] Dissolve compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (3.06 g, 18.75 mmol) separately in 100 mL of 30% aqueous acetic acid solution, add 0.4 g of titanium phthalocyanine oxide, disperse evenly by ultrasonic wave, and then dropwise add a methanol solution of iodine (12 g / L, 12.5 mmol). After the addition is complete, react for 15 min. Stop the reaction, filter the reaction solution, add it to 100 mL of ethyl acetate, wash successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution, take the organic phase, dry it with anhydrous sodium sulfate, filter, concentrate under reduced pressure, subject the residue to column chromatography to obtain compound A3. The purity is 95% and the yield is 78%.

[0053] Comparative Example 1

[0054] Synthesis of compound A3:

[0055] Dissolve compound A2 (5 g, 12.5 mmol) and N-acetyl-D-cysteine (3.06 g, 18.75 mmol) separately in 100 mL of 30% aqueous acetic acid solution, add 0.5 g of titanium phthalocyanine oxide, disperse evenly by ultrasonic wave, and then dropwise add a methanol solution of iodine (12 g / L, 12.5 mmol). After the addition is complete, react for 15 min. Stop the reaction, filter the reaction solution, add it to 100 mL of ethyl acetate, wash successively with saturated sodium thiosulfate solution and saturated sodium chloride aqueous solution, take the organic phase, dry it with anhydrous sodium sulfate, filter, concentrate under reduced pressure, subject the residue to column chromatography to obtain compound A3. The purity is 95% and the yield is 78%.

[0056] It can be seen from Example 6 and Comparative Example 1 that increasing the dosage of titanium oxyphthalocyanine from 8% to 10% will not further increase the yield of Compound A3. However, as the dosage of titanium oxyphthalocyanine increases, the cost will increase. Therefore, in the present invention, the dosage of titanium oxyphthalocyanine is controlled within 8% of the mass of Compound A2.

[0057] The use of titanium oxyphthalocyanine as a catalyst for the oxidation reaction to form disulfide bonds in the present invention is an accidental discovery by the inventor during the research process. Therefore, the catalytic mechanism of titanium oxyphthalocyanine cannot be determined for the time being. In the future, the inventor will conduct in-depth research on its catalytic mechanism, hoping to obtain a catalyst with higher catalytic efficiency to further increase the yield of Compound A3.

[0058] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A method for synthesizing a key intermediate of itkatide, characterized in that: Using Fmoc-Cys(Bzl)-OH as a starting material, Fmoc-Cys(Bzl)-OH undergoes an esterification reaction with tert-butyl alcohol to obtain compound A1, compound A1 undergoes a hydrogenation reaction to obtain compound A2, and compound A2 undergoes an oxidation reaction with N-acetyl-D-cysteine ​​to obtain a disulfide bond intermediate; The synthetic route is as follows:

2. The method for synthesizing the key intermediate of itkatide according to claim 1, characterized in that: The esterification reaction is carried out in the presence of a dehydrating agent and a catalyst.

3. The method for synthesizing the key intermediate of itkatide according to claim 2, characterized in that: The dehydrating agent is one or more of dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

4. The method for synthesizing the key intermediate of itkatide according to claim 2, characterized in that: The catalyst is one or more of 4-dimethylaminopyridine, imidazole and 1,2,4-triazole.

5. The method for synthesizing the key intermediate of itkatide according to claim 1, characterized in that: The molar ratio of the Fmoc-Cys(Bzl)-OH, tert-butyl alcohol, dehydrating agent and catalyst is 1:(1-4):(0.5-2):(0.1-1).

6. The method for synthesizing the key intermediate of itkatide according to claim 1, characterized in that: The reduction reaction is carried out under a hydrogen atmosphere.

7. The method for synthesizing the key intermediate of itkatide according to claim 6, characterized in that: The hydrogenation reaction uses at least one of palladium carbon and palladium hydroxide as a catalyst; preferably, the amount of the catalyst is 0.01 to 1 times the mass of compound A1.

8. The method for synthesizing the key intermediate of itkatide according to claim 1, characterized in that: The oxidation reaction is carried out in the presence of an oxidizing agent.

9. The method for synthesizing the key intermediate of itkatide according to claim 8, characterized in that: The oxidant is iodine, oxygen or air.

10. The method for synthesizing the key intermediate of itkatide according to claim 1, characterized in that: The molar ratio of the compound A2 to N-acetyl-D-cysteine ​​is 1:(1-2).

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