CN102190709A - Synthesis method of luteinizing hormone releasing hormone derivatives - Google Patents
Synthesis method of luteinizing hormone releasing hormone derivatives Download PDFInfo
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- CN102190709A CN102190709A CN2011100801720A CN201110080172A CN102190709A CN 102190709 A CN102190709 A CN 102190709A CN 2011100801720 A CN2011100801720 A CN 2011100801720A CN 201110080172 A CN201110080172 A CN 201110080172A CN 102190709 A CN102190709 A CN 102190709A
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Abstract
The invention relates to a synthesis method of luteinizing hormone releasing hormone derivatives. The structure of the luteinizing hormone releasing hormone derivatives is pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-R, wherein R=Azgly-NH2 or NHEt. In the invention, goserelin and buserelin are respectively subjected to solid phase synthesis by two sections, the first section is free from the influence of D-Ser(tBu), and the second section just relates to the D-Ser(tBu) in the final step, so that the whole synthesis technique overcomes the defect that the solid phase synthesis of the goserelin and buserelin is incompatible with the traditional solid phase synthesis due to the influence of the D-Ser(tBu); and thus, the whole technique has the advantages of low cost, high yield and high purity.
Description
Technical field
The present invention relates to a kind of synthetic method of luteinizing hormone-releasing hormone derivative.
Background technology
Lutropin (LH) and follicular stimulating hormone (FSH) are discharged by prepituitary gland, be controlled by the LH-RH(luteinizing hormone-releasing hormone that hypothalamus produces) or claim the GnRH(gonadotropin-releasing hormone), hereinafter be referred to as luteinizing hormone-releasing hormone.LH-RH and derivative thereof have the activity that promotes gonadotrophin secretion, administration LH-RH and derivative thereof can suppress gonad function according to the order of sequence, so it can be as medicine to suppress and/or diseases such as treatment such as endometriosis, central precocity, sterile and prostate cancer.LH-RH and derivative thereof as medicine comprises goserelin, buserelin, Leuprolide and nafarelin.Wherein, goserelin (Goserelin) and buserelin (Buserelin) structure can be expressed as follows: pGlu-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-R, work as R=Azgly-NH
2The time, this structural formula is represented goserelin (Goserelin), when R=NHEt, this structure is represented buserelin (Buserelin).
Because it is structural extremely similar, goserelin (Goserelin) can synthesize based on identical synthesis technique with buserelin (Buserelin), and at present the synthetic method of goserelin (Goserelin) and buserelin (Buserelin) comprises that mainly solid phase synthesis and liquid phase synthesize two kinds.
The liquid phase synthetic operation is loaded down with trivial details, and a whenever synthetic step all needs purifying, and synthetic yield is very low, and it is synthetic to be not suitable for surpassing the above polypeptide of 5 amino acid.The raw-material price of liquid phase synthetic has been much higher than the prices of raw and semifnished materials of the Fmoc/tBu solid system of large-scale industrial production, and therefore, liquid phase synthetic method is eliminated gradually by polypeptide compound probability system institute at present.
For solid phase synthesis process, generally adopt the SPPS(a Solid Phase Peptide Synthesis of polypeptide in synthetic) carry out the complete synthesis of entire segment, as reporting among patent EP2119724A1 and the patent US2010/0311946A1.But, goserelin in the luteinizing hormone-releasing hormone (Goserelin) and buserelin (Buserelin) are owing to the Fmoc/tBu solid-phase reaction system that side chain D-Ser contains tBu and routine is incompatible, Fmoc/tBu solid-phase reaction system with routine can't synthesize, so the liquid phase that also has employing to eliminate is synthetic or improved Fmoc/tBu solid-phase reaction system is produced.
For example, reported a kind of solid phase synthesis process of goserelin in the U.S. Pat 5602231, adopted the Trp of side chain no protective, Ser, the Tyr solid-phase reaction system synthesizes goserelin, because the Trp of side chain no protective, Ser, Tyr can cause a lot of side reactions when the Fmoc/tBu solid phase synthesis, cause the synthetic product to be difficult to purifying, yield descends, and adopts the amino acid of not protecting to synthesize the generation that polypeptide is the various side reactions of very difficult control for suitability for industrialized production, so be not suitable for suitability for industrialized production; Reported the Fmoc solid phase synthesis process that adopts the side chain protected system that is of little use among the U.S. Pat 2010/0311946A1, adopted His(trt), Ser (trt), Tyr raw materials such as (2-Cl-trt) synthesizes goserelin, since in the industrial production seldom producer use Ser (trt), these 2 raw materials of Tyr (2-Cl-trt), be difficult to find industrialized supplier, therefore not only cost an arm and a leg, the production cost height brings very big cost pressure for enterprise's scale operation, and when in the end the trt blocking group removes, be difficult to optionally remove trt and do not influence the tBu that D-ser contains, therefore can produce various impurities, cause yield decline; Adopted the method for the Tyr (BrZ) of the Ser of side chain no protective and the side chain protected that is of little use among the patent EP0518656B1 equally; it is the complex body of patent US2010/0311946A1 and patent US5602231; can't separate equally and never protect side reaction and the application that side chain causes and be difficult to find problems such as industrialization supply of raw material merchant, expensive raw material price and production cost height.
Summary of the invention
In order to overcome the defective of prior art, the present invention proposes a kind of synthetic method of luteinizing hormone-releasing hormone derivative, it is by being divided into luteinizing hormone-releasing hormone two fragments solid phase synthesis respectively, be spliced into the method for entire segment again, thereby overcome defective of the prior art, made synthetic method of the present invention have be fit to amplify production, yield height, the advantage that purity is good.
For solving aforementioned technical problem, the technical solution used in the present invention is as follows:
A kind of synthetic method of luteinizing hormone-releasing hormone derivative, described luteinizing hormone-releasing hormone derivant structure are pGlu-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-R, R=Azgly-NH
2Or NHEt, it may further comprise the steps:
(1) the solid phase synthesis first fragment pGlu-His-Trp-Ser-Tyr-OH, wherein, His, Ser, Tyr are side chain protected amino acid;
(2) solid phase synthesis second fragment D-Ser (tBu)-Leu-Arg-Pro-R-solid support, wherein the aminoterminal of D-Ser (tBu) is blocking group Fmoc or Boc;
(3) connect described first fragment and second fragment, and, obtain described luteinizing hormone-releasing hormone derivative pGlu-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-R through cutting peptide, purifying.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, goserelin (Goserelin) and buserelin (Buserelin) are divided into two fragments solid phase synthesis respectively, and first fragment is not subjected to the influence of D-Ser (tBu), the second fragment final step just relates to D-Ser (tBu), thereby can make that the solid phase synthesis of goserelin (Goserelin) and buserelin (Buserelin) and traditional solid phase synthesis can not compatible defectives so that whole synthesis technique has overcome influence because of D-Ser (tBu), thereby it is low to make that integrated artistic has a cost, the yield height, the advantage that purity is good.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, (1) is made up of following steps:
(a1) provide a resin as solid support;
(b1) be Tyr (tBu)-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on (a1) described solid support, then slough blocking group Fmoc or Boc with a deprotecting regent;
(c1) be Ser (tBu)-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Tyr (tBu) on the solid support of (b1), then slough blocking group Fmoc or Boc with a deprotecting regent;
(d1) be the Trp-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Ser (tBu) on the solid support of (c1), then slough blocking group Fmoc or Boc with a deprotecting regent;
(e1) be His (trt)-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Trp on the solid support of (d1), then slough blocking group Fmoc or Boc with a deprotecting regent;
(f1), and be connected on the His (trt) on the solid support of (e1) with activating reagent activation pGlu-OH;
(g1) solid support with (f1) adds in all peptide reagents; make the polypeptide on it break away from from the solid support of (a1); and make that the side chain protected group on Tyr (tBu), Ser (tBu), the His (trt) is sloughed, thereby obtain the described first fragment pGlu-His-Trp-Ser-Tyr-OH.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, (2) are made up of following steps:
(a2) providing coupling coupling described end is that the resin of R group is as solid support;
(b2) be the Pro-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on (a2) described solid support, then slough blocking group Fmoc or Boc with a deprotecting regent;
(c2) be Arg-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Pro on the solid support of (b2), then slough blocking group Fmoc or Boc with a deprotecting regent;
(d2) be the Leu-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Arg on the solid support of (c2), then slough blocking group Fmoc or Boc with a deprotecting regent;
(e2) with an activating reagent activating ammonia cardinal extremity be D-Ser (tBu)-OH of blocking group Fmoc or Boc; and be connected on the Leu on the solid support of (d2), promptly obtain described second fragment D-Ser (tBu)-Leu-Arg-Pro-R-solid support.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, (3) are made up of following steps:
(a3) slough blocking group Fmoc or Boc with a deprotecting regent, activate described first fragment, and be connected on the second fragment solid support of sloughing blocking group with an activating reagent;
(b3) solid support in above-mentioned (a3) is added in all peptide reagents, make that the polypeptide on it breaks away from from the solid support that above-mentioned (a3) obtains, thereby obtain described luteinizing hormone-releasing hormone derivative;
(c3) the luteinizing hormone-releasing hormone derivative that obtains with above-mentioned (b3) utilizes HPLC to carry out purifying.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, described solid support is selected from Fmoc-NH-Rink resin, Wang resin, NovaSyn TGR resin, Rink amide resin, Rink amide mbha resin, Rink amide AM resin, Rink amide PEGA resin, Rink amide NovaGel resin, Sieber amide resin and NovaSyn TG Sieber resin.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, described resin is selected from Fmoc-NH-Rink resin, Wang resin, NovaSyn TGR resin, Rink amide resin, Rink amide mbha resin, Rink amide AM resin, Rink amide PEGA resin, Rink amide NovaGel resin, Sieber amide resin and NovaSyn TG Sieber resin.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, described activating reagent is selected from HoBt, DIC, DCC and DIEA.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention, wherein, described activating reagent is selected from HoBt, DIC, DCC and DIEA.
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention; wherein; described N-terminal blocking group is preferably Fmoc; described deprotecting regent is selected from piperidine, DMF, cyclohexylamine, 1,5-diazabicyclo[5,4; 0] undec-5-ene, ethanolamine, pyrrolidine, 1; 8-diazabicyclo[5,4,0] one or more among undec-7-ene, diethylamine and the morpholine.
The present invention has following beneficial effect:
The synthetic method of luteinizing hormone-releasing hormone derivative of the present invention has following characteristics: simple, and process stabilizing, raw and auxiliary material convenient sources; production cost is low, and is with short production cycle, connects peptide yield height (per step connects peptide efficient 〉=99%); be produced on a large scale, cut peptide yield: 60%-65%.Purification yield: 35%-45%.Total recovery: 20%-30%.
Embodiment
Embodiment 1: goserelin synthetic
(1) Goserelin first is segmental synthetic
(1) Fmoc-Tyr (tBu)-wang resin is synthetic:
Getting wang resin (0.8-1.3mmol/g) soaks with methylene dichloride, make the abundant swelling of resin, the mixture that adds DMF dissolved Fmoc-Tyr (tBu)-OH (MW:459.6), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain, get Fmoc-Tyr (tBu)-wang resin;
(2) Fmoc-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (1), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-Ser (tBu)-OH (MW:383.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Ser (tBu)-Tyr (tBu)-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-Ser (tBu)-OH, HoBt, DIC is 3-5 a times of resin;
(3) Fmoc-Trp-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (2), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-Trp-OH (MW:426.5), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Trp-Ser (tBu)-Tyr (tBu)-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-Trp-OH, HoBt, DIC is 3-5 a times of resin;
(4) Fmoc-His (trt)-Trp-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (3), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-His (trt)-OH (MW:619.7), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-His (trt)-Trp-Ser (tBu)-Tyr (tBu)-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-His (trt)-OH, HoBt, DIC is 3-5 a times of resin;
(5) pGlu-His (trt)-Trp-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (3), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved PGlu-OH (MW:129.1), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain PGlu-His (trt)-Trp-Ser (tBu)-Tyr (tBu)-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of PGlu, HoBt, DIC is 3-5 a times of resin;
(6) acquisition of PGlu-His-Trp-Ser-Tyr-OH:
According to the present invention, cut peptide comprise the steps: with the PGlu-His (trt) in the step (5)-Trp-Ser (tBu)-Tyr (tBu)-wang resin add be chilled in advance 0 ℃ cut peptide reagent (TFA:EDT:TIS:H
2O=9:5:2.5:2.5) in, room temperature reaction 2 hours, ether sedimentation, suction filtration, the ether washing precipitation, vacuum-drying gets thick peptide.
(2) Goserelin second is segmental synthetic
(1) Fmoc-azagly-OSu's is synthetic
(1.1) 20ml hydrazine hydrate/acetonitrile solution (including 760 μ l, 80% hydrazine hydrate solution) is added dropwise in the 100ml Fmoc-OSu acetonitrile solution (includes 6.74g Fmoc-OSu), 16 hours after-filtration of magnetic agitation reaction, with the 1:1(volume ratio) acetonitrile/diethyl ether solution washing three times, diethyl ether solution washing three times, drying;
(1.2) will go up the 8.77g Fmoc-NH-NH that goes on foot gained
2With DSC(9.72g) be dissolved in the 150ml acetonitrile, stirring reaction 20 hours, rotary evaporation, water precipitation is filtered, and solid is with acetic acid ethyl dissolution, saturated NaHCO
3Wash 2 times, ethyl acetate layer is got in saturated common salt water washing twice, with petroleum ether precipitation, washs behind the rotary evaporation, and drying gets solid 11.81g;
(2) preparation of Fmoc-Pro-azagly-NH-Rink resin
Get Fmoc-NH-Rink resin (substitution degree 0.4-0.8mmol/g) and soak with methylene dichloride, fully swelling is drained.Add deprotecting regent (piperidines/DMF=1:5 volume ratio afterwards; the resinite volume concentrations is 10ml/g), room temperature reaction 0.5 hour is drained; use DMF and washed with dichloromethane respectively; drain, add the DMF solution of Fmoc-Azgly-Osu afterwards, (the resinite volume concentrations is 10ml/g; the Fmoc-Azgly-Osu mole number is 2-4 a times of resin); stirring reaction 8-12 hour, drain DMF, washed with dichloromethane.Add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add the mixture of DMF dissolved Fmoc-Pro-OH (MW:337.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Pro-Azgly-NH-Rink resin;
(3) preparation of Fmoc-Arg-Pro-Azgly-NH-Rink resin
In the resin of step (2), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration are 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-Arg-OH (MW:396.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Arg-Pro-Azgly-NH-Rink resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-Arg-OH, HoBt, DIC is 3-5 a times of resin;
(4) preparation of Fmoc-Leu-Arg-Pro-Azgly-NH-Rink resin
In the resin of step (3), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-Leu-OH (MW:353.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Leu-Arg-Pro-Azgly-NH-Rink resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-Leu-OH, HoBt, DIC is 3-5 a times of resin;
(5) preparation of Fmoc-D-Ser (tBu)-Leu-Arg-Pro-Azgly-NH-Rink resin
In the resin of step (4), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-D-Ser (tBu)-OH (MW:383.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-D-Ser (tBu)-Leu-Arg-Azgly-NH-Rink resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-D-Ser (tBu)-OH, HoBt, DIC is 3-5 a times of resin.
(3) butt joint of Goserelin, purifying
(1) Goserelin first fragment and Goserelin second segmental the butt joint
In the Goserelin second segmental resin, add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add the mixture of DMF dissolved Goserelin first fragment (MW:702.8), HoBt (MW:135.1), DIC (MW:167) afterwards, room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain, obtain Goserelin;
(2) acquisition of Goserelin
According to the present invention, cut peptide comprise the steps: with the Goserelin peptide resin add be chilled in advance 0 ℃ cut in the peptide reagent (TFA: methylene dichloride=1:99), room temperature reaction 1 hour, ether sedimentation, suction filtration, the ether washing precipitation, vacuum-drying, thick peptide;
The Goserelin crude product is soluble in water, filter last HPLC purifying (C18 column purification), moving phase: 1 ‰ TFA/H
2O:1 ‰ TFA/ acetonitrile (1:4), flow velocity is 60ml/min, the detection wavelength is 220nm, collects required component, desalination, freeze-drying obtains finished product Goserelin (MW:1269.1), total recovery 10-40%.
Embodiment 2: goserelin synthetic
(1) Goserelin first is segmental synthetic
(1) Fmoc-Tyr (tBu)-wang resin is synthetic:
Getting 50g wang resin (1.3mmol/g) soaks with methylene dichloride, make the abundant swelling of resin, add afterwards DMF dissolved Fmoc-Tyr (tBu)-OH (MW:459.6,59.7g), HoBt (MW:135.1,17.6g), DIC (MW:126.1,21.7ml) mixture, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, get 75g Fmoc-Tyr (tBu)-wang resin approximately;
(2) Fmoc-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (1), add deprotecting regent, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add afterwards 300ml DMF dissolved Fmoc-Ser (tBu)-OH (MW:383.4,53.5g), HoBt (MW:135.1,18.8g), DIC (MW:126.1,23.3ml) mixture, room temperature reaction 2 hours is drained;
(3) Fmoc-Trp-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (2), add deprotecting regent, room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, add 300mlDMF dissolved Fmoc-Trp-OH (MW:426.5 afterwards, 59.5g), HoBt (MW:135.1,17.6g), DIC (MW:126.1, mixture 21.7ml), room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain;
(4) Fmoc-His (trt)-Trp-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (3), add deprotecting regent, room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, add 300mlDMF dissolved Fmoc-His (trt)-OH (MW:619.7 afterwards, 86.4g), HoBt (MW:135.1,17.6g), DIC (MW:126.1, mixture 21.7ml), room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain;
(5) pGlu-His (trt)-Trp-Ser (tBu)-Tyr (tBu)-wang resin is synthetic:
In the resin of step (3), add deprotecting regent, room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, add 300mlDMF dissolved PGlu-OH (MW:129.1 afterwards, 18g), HoBt (MW:135.1,17.6g), DIC (MW:126.1, mixture 21.7ml), room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain;
(6) acquisition of pGlu-His-Trp-Ser-Tyr-OH
PGlu-His (trt) in the step (5)-Trp-Ser (tBu)-Tyr (tBu)-wang resin is added 1 liter of being chilled to 0 ℃ in advance cut peptide reagent (TFA:EDT:TIS:H
2O=9:5:2.5:2.5) in, room temperature reaction 2 hours, ether sedimentation, suction filtration, the ether washing precipitation, vacuum-drying gets the about 30g(yield of thick peptide: 92%).
(2) Goserelin second is segmental synthetic
(1) Fmoc-Azgly-Osu's is synthetic
(1.1) 120ml hydrazine hydrate/acetonitrile solution (including 4.6ml 80% hydrazine hydrate solution) is added dropwise in the 300ml Fmoc-OSu acetonitrile solution (including 40.4g), 16 hours after-filtration of magnetic agitation reaction, with the 1:1(volume ratio) acetonitrile/diethyl ether solution washing three times, diethyl ether solution washing three times, drying;
(1.2) will go up the 52.6g Fmoc-NH-NH that goes on foot gained
2With DSC(58.3g) be dissolved in the 500ml acetonitrile, stirring reaction 20 hours, rotary evaporation, water precipitation is filtered, and solid is with acetic acid ethyl dissolution, saturated NaHCO
3Wash 2 times, ethyl acetate layer is got in saturated common salt water washing twice, with petroleum ether precipitation, washs behind the rotary evaporation, and drying gets solid 70.9g;
(2) preparation of Fmoc-Azgly-NH-Rink resin
Get 50g Fmoc-NH-Rink resin (substitution degree 0.6mmol/g, 30mmol altogether) and soak with methylene dichloride, fully swelling is drained.Add deprotecting regent afterwards, room temperature reaction 0.5 hour is drained, and uses DMF and washed with dichloromethane respectively, drain, add Fmoc-Azgly-Osu (MW:395.3, DMF solution 59.3g) afterwards, stirring reaction 8-12 hour, drain DMF, washed with dichloromethane;
(3) preparation of Fmoc-Arg-Azgly-NH-Rink resin
In the resin of step (2), add deprotecting regent, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add afterwards 300mlDMF dissolved Fmoc-Arg-OH (MW:396.4,35.7g), HoBt (MW:135.1,12.15g), DIC (MW:126.1, mixture 15ml), room temperature reaction 2 hours is drained;
(4) preparation of Fmoc-Leu-Arg-Azgly-NH-Rink resin
In the resin of step (3), add deprotecting regent, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add afterwards 300mlDMF dissolved Fmoc-Leu-OH (MW:353.4,31.8g), HoBt (MW:135.1,12.15g), DIC (MW:126.1, mixture 15ml), room temperature reaction 2 hours is drained;
(5) preparation of Fmoc-D-Ser (tBu)-Leu-Arg-Azgly-NH-Rink resin
In the resin of step (4), add deprotecting regent, room temperature reaction 0.5 hour; drain, use DMF and washed with dichloromethane respectively, drain; add 350mlDMF dissolved Fmoc-D-Ser (tBu)-OH (MW:383.4 afterwards; 34.5g), HoBt (MW:135.1,12.15g), DIC (MW:126.1, mixture 15ml); room temperature reaction 2 hours; drain, use DMF and washed with dichloromethane respectively, drain.
(3) butt joint of Goserelin, purifying
(1) Goserelin first fragment and Goserelin second segmental the butt joint
Add deprotecting regent in the Goserelin second segmental resin, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add the DMF dissolved Goserelin first fragment (MW:702.8 afterwards, 25.3g), HoBt (MW:135.1,4.9g), DIC (MW:126.1, mixture 6ml), room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain, obtain the Goserelin resin;
(2) acquisition of Goserelin
With the Goserelin peptide resin add be chilled in advance 1 liter 0 ℃ cut in the peptide reagent (TFA: methylene dichloride=1:99), room temperature reaction 1 hour, ether sedimentation, suction filtration, the ether washing precipitation, vacuum-drying, thick peptide 16 grams;
The Goserelin crude product is soluble in water, filter last HPLC purifying (C18 column purification), moving phase: 1 ‰ TFA/H
2O:1 ‰ TFA/ acetonitrile (1:4),, the detection wavelength is 220nm, collects required component, desalination, freeze-drying obtains finished product Goserelin (MW:1269.1) 8 grams.
Embodiment 3: buserelin synthetic
(1) Buserelin first is segmental synthetic
Buserelin first fragment is identical with Goserelin first fragment, and concrete implementation content is seen embodiment 1 and embodiment 2;
(2) Buserelin second is segmental synthetic
(1) preparation of Fmoc-Pro-wang resin
Get wang resin (substitution degree 0.4-0.8mmol/g) and soak with methylene dichloride, fully swelling is drained.Use DMF and washed with dichloromethane respectively, drain, add Fmoc-Pro-OH afterwards, DIC, the DMF solution of HOBT, stirring reaction 8-12 hour, to drain, DMF, washed with dichloromethane are drained, and obtain Fmoc-Pro-Wang resin;
(2) preparation of Fmoc-Arg-Pro-wang resin
In the resin of step (1), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration are 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-Arg-OH (MW:396.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Arg-Pro-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-Arg-OH, HoBt, DIC is 3-5 a times of resin;
(3) preparation of Fmoc-Leu-Arg-Pro-wang resin
In the resin of step (2), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration are 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-Leu-OH (MW:353.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-Leu-Arg-Pro-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-Leu-OH, HoBt, DIC is 3-5 a times of resin;
(4) preparation of Fmoc-D-Ser (tBu)-Leu-Arg-Pro-Wang resin
In the resin of step (4), add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour, drain, use DMF and washed with dichloromethane respectively, drain, the mixture that adds DMF dissolved Fmoc-D-Ser (tBu)-OH (MW:383.4), HoBt (MW:135.1), DIC (MW:126.1) afterwards, room temperature reaction 2 hours is drained, and uses DMF and washed with dichloromethane respectively, drain, obtain Fmoc-D-Ser (tBu)-Leu-Arg-wang resin;
Wherein, the weight concentration of resin is 10ml/g in the mixture, and the mole number of Fmoc-D-Ser (tBu)-OH, HoBt, DIC is 3-5 a times of resin.
(3) butt joint of Buserelin, purifying
(1) Buserelin first fragment and Buserelin second segmental the butt joint:
In the Buserelin second segmental resin, add deprotecting regent (piperidines/DMF=1:5 volume ratio, weight resin concentration is 10ml/g), room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add the mixture of DMF dissolved Goserelin first fragment (MW:702.8), HoBt (MW:135.1), DIC (MW:167) afterwards, room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain, obtain Buserelin;
(2) Buserelin cut peptide, purifying:
According to the present invention, cut peptide comprise the steps: with the Buserelin peptide resin add be chilled in advance 0 ℃ cut in the peptide reagent (ethamine: methylene dichloride=1:99), room temperature reaction 24 hours, suction filtration revolves steaming, the ether washing precipitation, vacuum-drying, thick peptide.
The Buserelin crude product is soluble in water, filter last HPLC purifying (C18 column purification), moving phase: 1 ‰ TFA/H
2O:1 ‰ TFA/ acetonitrile (1:4), flow velocity is 60ml/min, the detection wavelength is 220nm, collects required component, desalination, freeze-drying obtains finished product Buserelin (MW:1239.4), total recovery 10-40%.
Embodiment 4:Buserelin's is synthetic
(1) Buserelin first is segmental synthetic
Buserelin first fragment is identical with Goserelin first fragment, and concrete implementation content is seen embodiment 1 and embodiment 2;
(2) Buserelin second is segmental synthetic
(1) preparation of Fmoc-Pro-Wang resin
Get 50g Wang resin (substitution degree 0.6mmol/g, 30mmol altogether) and soak with methylene dichloride, fully swelling is drained.Add deprotecting regent afterwards, room temperature reaction 0.5 hour is drained, and uses DMF and washed with dichloromethane respectively, drain, add Fmoc-Pro-OH 50.6g afterwards, DIC 25 grams, the DMF solution of HOBT20 gram, stirring reaction 8-12 hour, drain DMF, washed with dichloromethane;
(2) preparation of Fmoc-Arg-Pro-wang resin
In the resin of step (2), add deprotecting regent, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add afterwards 300mlDMF dissolved Fmoc-Arg-OH (MW:396.4,35.7g), HoBt (MW:135.1,12.15g), DIC (MW:126.1, mixture 15ml), room temperature reaction 2 hours is drained;
(3) preparation of Fmoc-Leu-Arg-Pro-wang resin
In the resin of step (3), add deprotecting regent, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add afterwards 300mlDMF dissolved Fmoc-Leu-OH (MW:353.4,31.8g), HoBt (MW:135.1,12.15g), DIC (MW:126.1, mixture 15ml), room temperature reaction 2 hours is drained;
(4) preparation of Fmoc-D-Ser (tBu)-Leu-Arg-Pro-wang resin
In the resin of step (4), add deprotecting regent, room temperature reaction 0.5 hour; drain, use DMF and washed with dichloromethane respectively, drain; add 350mlDMF dissolved Fmoc-D-Ser (tBu)-OH (MW:383.4 afterwards; 34.5g), HoBt (MW:135.1,12.15g), DIC (MW:126.1, mixture 15ml); room temperature reaction 2 hours; drain, use DMF and washed with dichloromethane respectively, drain.
(3) butt joint of Buserelin, purifying
(1) Buserelin first fragment and Buserelin second segmental the butt joint:
Add deprotecting regent in the Buserelin second segmental resin, room temperature reaction 0.5 hour is drained, use DMF and washed with dichloromethane respectively, drain, add the DMF dissolved Goserelin first fragment (MW:702.8 afterwards, 25.3g), HoBt (MW:135.1,4.9g), DIC (MW:126.1, mixture 6ml), room temperature reaction 2 hours, drain, use DMF and washed with dichloromethane respectively, drain, obtain the Buserelin resin;
(2) Buserelin cut peptide, purifying:
With the Buserelin peptide resin add be chilled in advance 1 liter 0 ℃ cut in the peptide reagent (TFA: methylene dichloride=1:99), room temperature reaction 24 hours, ether sedimentation, suction filtration, the ether washing precipitation, vacuum-drying, thick peptide 16 grams;
The Buserelin crude product is soluble in water, filter last HPLC purifying (C18 column purification), moving phase: 1 ‰ TFA/H
2O:1 ‰ TFA/ acetonitrile (1:4),, the detection wavelength is 220nm, collects required component, desalination, freeze-drying obtains finished product Buserelin 7.5 grams.
In an embodiment of the present invention, in the building-up process, deprotecting regent is piperidines/DMF=1:5 (volume ratio)
Below be the raw material tabulation in the embodiment of the invention:
| No | The name of an article | Production firm |
| 1 | The Fmoc-NH-Rink resin | Tianjin with become Science and Technology Ltd. |
| 2 | The Wang resin | Tianjin with become Science and Technology Ltd. |
| 3 | Fmoc-Arg-OH | The biochemical company limited of Shanghai gill |
| 4 | Fmoc-Pro-OH | The biochemical company limited of Shanghai gill |
| 5 | Fmoc-Leu-OH | The biochemical company limited of Shanghai gill |
| 6 | Fmoc-D-Ser(tBu)-OH | The biochemical company limited of Shanghai gill |
| 7 | Fmoc-Ser(tBu)-OH | The biochemical company limited of Shanghai gill |
| 8 | Fmoc-Tyr(tBu)-OH | The biochemical company limited of Shanghai gill |
| 9 | Fmoc-Trp-OH | The biochemical company limited of Shanghai gill |
| 10 | Fmoc-His(trt)-OH | The biochemical company limited of Shanghai gill |
| 11 | p-Glu-OH | The biochemical company limited of Shanghai gill |
| ? 12 | HoBt | The biochemical company limited of Shanghai gill |
| ? 13 | TBTU | The biochemical company limited of Shanghai gill |
| ? 14 | DMF | Korea S's Samsung |
| ? 15 | Hexahydropyridine | Chemical plant, Gansu Province, west, Shantou |
| ? 16 | Methylene dichloride | Chemical plant, Gansu Province, west, Shantou |
| ? 17 | Methyl alcohol | Chemical plant, Gansu Province, west, Shantou |
| ? 18 | Acetonitrile | Chemical plant, Gansu Province, west, Shantou |
| ? 19 | Hydrazine hydrate | Chemical plant, Gansu Province, west, Shantou |
| 20 | Fmoc-OSu | The biochemical company limited of Shanghai gill |
Below be the definite title of shortenings among the present invention:
P-Glu-OH: Pyrrolidonecarboxylic acid;
HoBt:1-hydroxyl one benzotriazole;
TBTU:O-(1H-benzotriazole-1-yl)-N, N, N', N'-tetramethyl-isourea tetrafluoride boron;
DMF:N, dinethylformamide;
Fmoc-OSu: fluorenes methoxy carbonyl acyl succinimide.
Claims (9)
1. the synthetic method of luteinizing hormone-releasing hormone derivative, described luteinizing hormone-releasing hormone derivant structure is pGlu-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-R, R=Azgly-NH
2Or NHEt, it is characterized in that comprising:
(1) the solid phase synthesis first fragment pGlu-His-Trp-Ser-Tyr-OH, wherein, His, Ser, Tyr are side chain protected amino acid;
(2) solid phase synthesis second fragment D-Ser (tBu)-Leu-Arg-Pro-R-solid support, wherein the aminoterminal of D-Ser (tBu) is blocking group Fmoc or Boc;
(3) connect described first fragment and second fragment, and, obtain described luteinizing hormone-releasing hormone derivative pGlu-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-R through cutting peptide, purifying.
2. the synthetic method of luteinizing hormone-releasing hormone derivative according to claim 1 is characterized in that: (1) is made up of following steps:
(a1) provide a resin as solid support;
(b1) be Tyr (tBu)-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on (a1) described solid support, then slough blocking group Fmoc or Boc with a deprotecting regent;
(c1) be Ser (tBu)-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Tyr (tBu) on the solid support of (b1), then slough blocking group Fmoc or Boc with a deprotecting regent;
(d1) be the Trp-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Ser (tBu) on the solid support of (c1), then slough blocking group Fmoc or Boc with a deprotecting regent;
(e1) be His (trt)-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Trp on the solid support of (d1), then slough blocking group Fmoc or Boc with a deprotecting regent;
(f1), and be connected on the His (trt) on the solid support of (e1) with activating reagent activation pGlu-OH;
(g1) solid support with (f1) adds in all peptide reagents; make the polypeptide on it break away from from the solid support of (a1); and make that the side chain protected group on Tyr (tBu), Ser (tBu), the His (trt) is sloughed, thereby obtain the described first fragment pGlu-His-Trp-Ser-Tyr-OH.
3. the synthetic method of luteinizing hormone-releasing hormone derivative according to claim 2 is characterized in that: (2) are made up of following steps:
(a2) providing coupling coupling described end is that the resin of R group is as solid support;
(b2) be the Pro-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on (a2) described solid support, then slough blocking group Fmoc or Boc with a deprotecting regent;
(c2) be Arg-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Pro on the solid support of (b2), then slough blocking group Fmoc or Boc with a deprotecting regent;
(d2) be the Leu-OH of blocking group Fmoc or Boc with an activating reagent activating ammonia cardinal extremity, and be connected on the Arg on the solid support of (c2), then slough blocking group Fmoc or Boc with a deprotecting regent;
(e2) with an activating reagent activating ammonia cardinal extremity be D-Ser (tBu)-OH of blocking group Fmoc or Boc; and be connected on the Leu on the solid support of (d2), promptly obtain described second fragment D-Ser (tBu)-Leu-Arg-Pro-R-solid support.
4. according to the synthetic method of claim 1 or 2 or 3 described luteinizing hormone-releasing hormone derivatives, it is characterized in that: (3) are made up of following steps:
(a3) slough blocking group Fmoc or Boc with a deprotecting regent, activate described first fragment, and be connected on the second fragment solid support of sloughing blocking group with an activating reagent;
(b3) solid support in above-mentioned (a3) is added in all peptide reagents, make that the polypeptide on it breaks away from from the solid support that above-mentioned (a3) obtains, thereby obtain described luteinizing hormone-releasing hormone derivative;
(c3) the luteinizing hormone-releasing hormone derivative that obtains with above-mentioned (b3) utilizes HPLC to carry out purifying.
5. the synthetic method of luteinizing hormone-releasing hormone derivative according to claim 1 is characterized in that: described solid support is selected from Fmoc-NH-Rink resin, Wang resin, NovaSyn TGR resin, Rink amide resin, Rink amide mbha resin, Rink amide AM resin, Rink amide PEGA resin, Rink amide NovaGel resin, Sieber amide resin and NovaSyn TG Sieber resin.
6. according to the synthetic method of claim 2 or 3 described luteinizing hormone-releasing hormone derivatives, it is characterized in that: described resin is selected from Fmoc-NH-Rink resin, Wang resin, NovaSyn TGR resin, Rink amide resin, Rink amide mbha resin, Rink amide AM resin, Rink amide PEGA resin, Rink amide NovaGel resin, Sieber amide resin and NovaSyn TG Sieber resin.
7. according to the synthetic method of claim 2 or 3 described luteinizing hormone-releasing hormone derivatives, it is characterized in that: described activating reagent is selected from HoBt, DIC, DCC and DIEA.
8. the synthetic method of luteinizing hormone-releasing hormone derivative according to claim 4 is characterized in that: described activating reagent is selected from HoBt, DIC, DCC and DIEA.
9. the synthetic method of luteinizing hormone-releasing hormone derivative according to claim 3; it is characterized in that: described N-terminal blocking group is Fmoc; described deprotecting regent is selected from piperidines, DMF, cyclohexylamine, 1; 5-diazabicyclo[5; 4,0] undec-5-ene, ethanolamine, pyrrolidine, 1,8-diazabicyclo[5; 4,0] one or more among undec-7-ene, diethylamine and the morpholine.
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| CN106589072A (en) * | 2016-12-22 | 2017-04-26 | 江苏诺泰生物制药股份有限公司 | Synthesis method of goserelin |
| CN110922452A (en) * | 2018-09-20 | 2020-03-27 | 深圳翰宇药业股份有限公司 | Synthesis method of goserelin |
| CN113527438A (en) * | 2021-08-30 | 2021-10-22 | 湖南三太药业有限公司 | Method for synthesizing alarelin in full liquid phase |
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| CN113527438A (en) * | 2021-08-30 | 2021-10-22 | 湖南三太药业有限公司 | Method for synthesizing alarelin in full liquid phase |
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