Preparation method for salmon calcitonin, conjugated preparation of salmon calcitonin and use of conjugated preparation in drugs for osteoporosis

A technology for salmon calcitonin and osteoporosis, which is applied in the field of medicine, can solve the problems of incomplete protection, large impurities in salmon calcitonin, loss of salmon calcitonin, etc., and achieve simplified processing steps, high degree of condensation, and easy operation simple effect

Active Publication Date: 2020-06-19
广东金城金素制药有限公司
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Problems solved by technology

[0004] However, in the synthesis method reported by these two patents, there are relatively large impurities in salmon calcitonin, especially 22-position tyrosine, and tert-butyl (-tBu) is used to protect the hydroxyl group, which is prone to incomplete protection and generates a large amount of impurity E; and Under the above condensation conditions, the 22-position tyrosine cannot be condensed completely with the proline of the 23-position four-membered ring, and it is easy to generate salmon calcitonin impurity C that loses the 22-position tyrosine; in addition, the 17-position histidine (His) Racemization is prone to occur, especially when the protection group such as trityl (Trt) or p-toluenesulfonyl (Tos) is introduced into the amino protecting agent, or when a condensing agent is used to react, the condensation with adjacent amino acids is more likely to cause racemization, forming Impurity F

Method used

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  • Preparation method for salmon calcitonin, conjugated preparation of salmon calcitonin and use of conjugated preparation in drugs for osteoporosis
  • Preparation method for salmon calcitonin, conjugated preparation of salmon calcitonin and use of conjugated preparation in drugs for osteoporosis
  • Preparation method for salmon calcitonin, conjugated preparation of salmon calcitonin and use of conjugated preparation in drugs for osteoporosis

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preparation example Construction

[0039] A preparation method of salmon calcitonin, comprising the steps of:

[0040] The 23-32 position of the salmon calcitonin fragment prepared by the Fmoc-strategy solid-phase method:

[0041] -Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-Resin added Fmoc-Tyr(Alloc)-OH , DFIH, EEDQ and DMSO, reacted, dried with nitrogen, washed with DMSO, dried with nitrogen; added DMSO solution of hexahydropyridine, reacted for 15-25 minutes at 15-25°C, dried with nitrogen, washed with DMSO, dried with nitrogen to obtain -Tyr(Alloc)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-resin, where DFIH and Fmoc The molar ratio of -Tyr(Alloc)-OH is 1-1.5; the molar ratio of EEDQ to Fmoc-Tyr(Alloc)-OH is 0.1-0.5.

[0042] A preparation method of salmon calcitonin, comprising the steps of:

[0043] The 18-32 position of the salmon calcitonin fragment prepared by the Fmoc-strategy solid-phase method:

[0044]-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(Allo...

Embodiment 1

[0156] The steps for the condensation of salmon calcitonin 22 and 23, 17 and 18 are as follows, and the synthesis of other fragments refers to Comparative Example 1.

[0157] (1) The 22nd and 23rd condensation operation steps of salmon calcitonin:

[0158] -Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-Resin added Fmoc-Tyr(Alloc)-OH (MW: 487.51, 74.4mmol) 36.3g, DFIH 23.4g (MW: 262.11, 89.3mmol), EEDQ 1.8g (MW: 247.29, 7.44mmol) and 250g DMSO, reacted, dried with nitrogen, washed three times with DMSO, dried with nitrogen Add 300g25% hexahydropyridine DMSO solution, react for 20 minutes at 20°C, dry with nitrogen, wash with DMSO three times, and dry with nitrogen to obtain -Tyr(Alloc)-Pro-Arg(Pbf)-Thr(tBu)-Asn( Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-resin.

[0159] (2) Condensation steps of 17-position and 18-position of salmon calcitonin:

[0160] -Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(Alloc)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu )-...

Embodiment 2

[0162] The steps for the condensation of salmon calcitonin 22 and 23 are as follows, and the synthesis of other fragments refers to Comparative Example 1.

[0163] Calcitonin 22 and 23 condensation operation steps: -Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)- Add Fmoc-Tyr(Alloc)-OH (MW: 487.51, 74.4mmol) 36.3g, DFIH 29.3g (MW: 262.11, 111.6mmol), EEDQ 9.2g (MW: 247.29, 37.2mmol) and 250gDMSO to Pro-resin, Reaction, dry with nitrogen, wash with DMSO three times, dry with nitrogen; add 300g of 25% hexahydropyridine in DMSO, react at 20°C for 20 minutes, dry with nitrogen, wash with DMSO three times, and dry with nitrogen to obtain -Tyr(Alloc)-Pro -Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-resin.

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Abstract

The invention discloses a preparation method for salmon calcitonin, a preparation of the salmon calcitonin and use of the preparation in drugs for osteoporosis. According to the preparation method, when amino acids of 22 and 23 positions of a fragment of the salmon calcitonin prepared by an Fmoc-policy solid-phase method are condensed, a condensation agent system DFIH / EEDQ is employed, and thus, generation of an impurity E and an impurity C of the salmon calcitonin can be lowered; and when amino acids of 17 and 18 positions of a fragment of the salmon calcitonin prepared by the Fmoc-policy solid-phase method are condensed, DMSO is used as a condensation agent, a weight ratio of the DMSO to Fmoc-His-OH is (10 to 15): 1, and thus, generation of an impurity F of the salmon calcitonin can be lowered. The calcitonin prepared by the preparation method is good in stability and few in impurities; and the calcitonin biological vector conjugated preparation disclosed by the invention has new usein treatment of osteoporosis of the male.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a preparation method of salmon calcitonin and its preparation and its application in medicine for osteoporosis. Background technique [0002] Calcitonin is one of the hormones that regulate calcium metabolism and inhibit parathyroid hormone. It can significantly reduce bone calcium loss in high turnover bone disease, and it can not only inhibit the activity of osteoclasts, but also stimulate the formation of osteoblasts. Calcitonin can also inhibit osteolysis, thereby reducing the pathologically elevated blood calcium concentration and increasing urinary calcium, phosphorus, and serum sodium excretion by reducing renal tubular reabsorption. Salmon calcitonin has a high affinity for its receptor binding site, has a very good clinical effect and has a longer duration of action than synthetic mammalian (including human) calcitonin. [0003] The synthesis method of salmon calcitonin used in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/585C07K1/04A61K38/23A61P19/10
CPCA61K38/00A61P19/10C07K14/585
Inventor 傅苗青周白水李晓飞孟宾杨静李秋荣
Owner 广东金城金素制药有限公司
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