A preparation method of salmon calcitonin, its combined preparation and the application of the combined preparation in medicine for osteoporosis

The technology of salmon calcitonin and condensing agent, 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 achieves simplified processing steps, high degree of condensation, and simple operation. Effect

CN111303273BActive Publication Date: 2020-12-22广东金城金素制药有限公司
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2020-12-22

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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.
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Description

Technical field

[0001] The invention relates to the field of medicine, in particular to a preparation method and preparation of salmon calcitonin and its use in osteoporosis medicine.Background technique

[0002] Calcitonin is one of the hormones that regulate calcium metabolism and inhibit parathyroid hormone. It can significantly reduce the loss of bone calcium in high turnover bone diseases. It can inhibit osteoclast activity and stimulate osteoblast formation. Calcitonin can also inhibit osteolysis, thereby reducing the pathologically elevated blood calcium concentration and increasing urinary calcium, phosphorus and sodium excretion by reducing renal tubular reabsorption. Salmon calcitonin has a high affinity to 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 patent CN1865283B is Fmoc strategy solid-phase method, the main synthes...

Examples

preparation example Construction

[0039]A preparation method of salmon calcitonin includes the following steps:

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

[0041]-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-Fmoc-Tyr(Alloc)-OH , DFIH, EEDQ and DMSO, react, dry with nitrogen, wash with DMSO, and dry with nitrogen; add hexahydropyridine in DMSO solution, react at 15-25℃ for 15-25 minutes, dry with nitrogen, wash with DMSO, 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, 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 includes the following steps:

[0043]There will be Fmoc-strategy solid-phase preparation of salmon calcitonin fragment 18-32:

[0044]-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(Alloc)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu )-Gly-Thr(tB...

Embodiment 1

[0156]The operation 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) Condensation steps of 22 and 23 positions of salmon calcitonin:

[0158]-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-Fmoc-Tyr(Alloc)-OH (MW: 487.51, 74.4mmol) 36.3g, DFIH23.4g (MW: 262.11, 89.3mmol), EEDQ1.8g (MW: 247.29, 7.44mmol) and 250g DMSO, react, dry with nitrogen, wash with DMSO three times, and dry with nitrogen ; Add 300g of 25% hexahydropyridine DMSO solution, react at 20°C for 20 minutes, blow dry with nitrogen, wash three times with DMSO, and blow 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 salmon calcitonin 17 and 18 positions:

[0160]-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(Alloc)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu )-Gly-Thr(tBu)-Pro-resin, add 28.1g o...

Embodiment 2

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

[0163]Condensation steps of salmon calcitonin 22 and 23: -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), EEDQ9.2g (MW: 247.29, 37.2mmol) and 250g DMSO to Pro-resin, For reaction, dry with nitrogen, wash with DMSO three times, and dry with nitrogen; add 300g of 25% hexahydropyridine DMSO solution, react at 20°C for 20 minutes, blow dry with nitrogen, wash with DMSO three times, blow dry with nitrogen to obtain -Tyr(Alloc)-Pro -Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tBu)-Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-resin.