CN102653555B - Method for preparing goserelin by solid phase - Google Patents
Method for preparing goserelin by solid phase Download PDFInfo
- Publication number
- CN102653555B CN102653555B CN201210155366.7A CN201210155366A CN102653555B CN 102653555 B CN102653555 B CN 102653555B CN 201210155366 A CN201210155366 A CN 201210155366A CN 102653555 B CN102653555 B CN 102653555B
- Authority
- CN
- China
- Prior art keywords
- fmoc
- resin
- azagly
- goserelin
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for preparing goserelin by solid phase. According to the technical scheme, the method comprises the following steps of: coupling N,N-2-succinimide-carbonate and Sieber Resin; obtaining Azagly-sieber Resin by a hydrazine hydrate reaction; coupling Fmoc-Pro-OH and Azagly-Sieber Resin; gradually coupling other types of amino acids, and thus obtaining goserelin resin; splitting the goserelin-Sieber resin, eliminating side chain protection, and thus obtaining coarse peptide; and purifying the coarse peptide, and thus obtaining the goserelin. The method has the characteristics of simple reaction operation, simple post-treatment, high yield, low cost and the like and is considerable in economic practical value and wide in application prospect.
Description
Technical field
The present invention relates to a kind of preparation method of polypeptide drugs, particularly relate to the preparation method that a kind of solid phase prepares goserelin.
Background technology
Prostate cancer is the one of the main reasons of cancer death in men America and Europe.Sickness rate is with age growth, prostate cancer morbidity has significantly area and racial difference, Chinese are minimum according to statistics, European is the highest, Africa and Israel are between two parties, China and Japan and other countries are prostate cancer low area, and therefore someone thinks that Oriental's growth of cancer cells is slower than westerner, and clinical case is less.Prostate cancer and environment also have relation in addition.Mammary cancer is the modal a kind of malignant tumours of the mankind, and various countries are because of the difference of geographical environment, living habit, and the sickness rate of mammary cancer has very big-difference.North America and Northern Europe most countries are the districts occurred frequently of women with breast cancer, and South America and some countries of southern Europe are medium, and the most area in Asia, Latin America and Africa is Di Fa district.In North America, Liang Oudeng developed country, the sickness rate of women with breast cancer occupies the first place of female malignant sickness rate.The sickness rate of mammary cancer in China each department is not identical yet, though China belongs to the Di Fa state of women with breast cancer in the world.But the sickness rate of mammary cancer obviously increases in recent years.Especially Shanghai, capital, Tianjin and coastland are the hotspots of China's mammary cancer.
Conventional tumour medicine mainly chemicals, but its chemotherapy side effect side effect is large, and so finding a kind of efficient, low bio-pharmaceutical is trend of the times, current clinical effectiveness is preferably Buserein (LHRH-a).Goserelin advantage is few side effects, absent cardiovascular complication.
Goserelin molecular formula is: Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
2.
Preparation method's mainly liquid phase synthesis process of goserelin and analogue thereof, complicated operation, reactions steps is many, purification process is loaded down with trivial details, is unfavorable for industrial production, and using value is not high, such as: united states drug the Chemicals J. Med. Chem 121(10), 1018-1024,1978 what adopt is 4+6 segment condensation method, first adopts liquid phase synthesizing method synthesis protection six peptide Z-Tyr-(Bzl)-D-Ser-Arg (NO
2)-Pro-AzaGly-NH
2, synthesize tetrapeptide pGlu-His-Trp-Ser-OMe.Trinitride method is again adopted to synthesize goserelin four peptide fragment be synthesized and six peptide fragment.Although above small peptide synthesis in solve arginine protection problem; but when removing six peptide protections, the protecting group on tyrosine side chain phenolic hydroxyl group and arginine side chain guanidine radicals is also removed; a large amount of by product can be produced when in the end a step connects peptide; and by product character is more or less the same, be difficult to be separated.
US4024248 adopts 3+7 segment condensation method.In this route, first, liquid phase synthesis protection tripeptides pGlu-His-Trp-OMe.Resynthesis seven peptide Z-Ser-Tyr-D-Ser (But)-Leu-Arg-Pro-AzaGly-NH
2, respectively by tripeptide fragment pGlu-His-Trp-OMe hydrazinolysis, seven peptide fragment, except after Z protecting group, adopt azide method by tripeptide fragment and seven peptide fragment condensations, namely obtain goserelin.This method segmentation is few, and seven peptides progressively condensation productive rate are very low, and arginine side chain guanidine radicals is not protected, and causes often walking condensation and occurs by product, often walks separation and purification very difficult.
Poland Patent PL194997 reports a kind of 5+5 segment condensation method, first adopts liquid phase synthesizing method, synthesizes dipeptide fragment Z-Ser-Tyr-OMe and Z-Pro-AzaGly-NH respectively
2, then respectively adopt carbochain progressively growth method synthesize pentapeptide fragment pGlu-His-Trp-Ser-Tyr-OMe and D-Ser (But)-Leu-Arg-Pro-AzaGly-NH
2, finally adopt trinitride method to synthesize decapeptide target compound goserelin two pentapeptide fragments.Because amino acid does not all carry out side chain protected, can cause so all producing by product in ensuing condensation course, cause purification difficult.
What US2010311946A1 adopted is 2-CTC resin or Merrifield resin, needs solid-liquid combination method to synthesize goserelin, and finally will use the reagent KOCN of severe toxicity, cause risk to environment and medicine; In addition, Fmoc-Osu and hydrazine hydrate synthesis Fmoc-NH-NH
2, experiment finds Fmoc-NH-NH
2be difficult to dissolve at DMF, very large difficulty is caused to solid phase synthesis.
Summary of the invention
In order to overcome the defect of above-mentioned liquid phase synthesis techniques, as long in reaction time, by product is many, be difficult to be separated, be unfavorable for amplifying and produce etc., the invention provides a kind of high yield, low cost, reaction conditions be gentle, environmental pollution is little, the solid phase preparation technology of the goserelin that is conducive to realizing industrialization.
For achieving the above object, the present invention takes following technical scheme: a kind of method preparing goserelin, is characterized in that:
1) by N, N-two succinimdyl carbonate and substitution degree scope be sensitivity to acid aminoresin coupling under organic weak base catalyzer of 0.2-1.2mmol/g;
2) Azagly-Resin is obtained with hydrazine hydrate and step 1) gained resin reaction;
3) under the effect of coupling agent, Fmoc-Pro-Azagly-Resin is obtained with Fmoc-Pro-OH and Azagly-Resin;
4) by Fmoc-Pro-Azagly-Resin and Fmoc protected amino acid and coupling agent according to goserelin peptide sequence one by one the mode of coupling synthesize and obtain goserelin-Resin;
5) cracking is carried out to goserelin-Resin, remove side chain protected, obtain thick peptide and obtain goserelin through liquid phase purifying, freeze-drying.
Step 1), wherein, described sensitivity to acid aminoresin comprises Sieber Resin, Sieber-AM Resin, the preferred 0.3-0.9mmol/g of substitution degree scope of resin, more preferably 0.4-0.8 mmol/g; Because goserelin has the structure of D-Ser side chain tBu, technical scheme of the present invention requires higher to resin, needs to use sensitivity to acid aminoresin; If use the resin of other types, when cracking, the side chain tBu of D-Ser is easily damaged, and can not realize object of the present invention.The preferred Sieber resin of sensitivity to acid aminoresin, Sieber-AM resin.
Present invention applicant finds, when the substitution degree of resin is lower than 0.2mmol/g, the amount of resin of needs is excessive, and a large amount of resin application makes the cost of product; When the substitution degree of resin is greater than 1.2mmol/g, linked reaction occurs not exclusively, to generate default peptide, and not only make purifying process become very complicated, the purity of final product is not high simultaneously.Storm strongholds in defiance of difficulties through applicant, be surprised to find that when the substitution degree of resin is 0.2-1.2mmol/g, the relation between the purity of the equilibrium product that can be satisfied with in industrialization and cost.Applicant finds further, and when the substitution degree scope of resin is 0.3-0.9mmol/g, product purity and cost meet the requirement of industrialization more; Substitution degree is when 0.4-0.8 mmol/g, more desirable.
N, N-two the consumption of succinimdyl carbonate be that 3-10 is doubly to sensitivity to acid aminoresin;
Applicant is surprised to find that, works as N, and N-two succinimdyl carbonate consumption is less than 3 times, and can cause reaction not exclusively, cause purifying difficulty to strengthen, the simultaneous reactions time is oversize, causes product cost to raise; N, N-two succinimdyl carbonate consumption higher than 10 times time, beyond the consumption required for reaction, remaining N, N-two succinimdyl carbonate add the difficulty of purifying, make technique become complicated.
Described catalyzer is organic weak base, preferred triethylamine, 2，4，6-trimethylpyridine, DMAP, DIPEA; Be more preferably DMAP;
Applicant finds, the present invention is comparatively harsh to the alkalinity requirements of catalyzer, needs to carry out under the condition of organic weak base, be surprised to find that, triethylamine, 2,4,6-trimethylpyridine, the alkalescence of DMAP, DIPEA has good catalytic effect to the present invention, reaction times is obviously shortened, and improve the utilization ratio of reactant, wherein DMAP alkalescence is most suitable, and catalytic effect is best.
Step 2) obtain Azagly-Resin with the resin reaction of hydrazine hydrate and step 1) gained; , the reaction times is 1-5 hour, preferred 2-5 hour.
Applicant finds, the reaction times is less than 1 hour, and reactant reaction does not fully cause waste of raw materials, and reactant is difficult to purifying, can not obtain desirable industrialization result; Reaction times is greater than 5 hours, although such as 6 hours, 10 hours etc. can realize goal of the invention, efficiency of its synthesis does not improve, add the reaction times on the contrary, make the time lengthening of technique, cause the waste of the energy and human cost, the cost of product is increased.So this reactions steps needs to grope the best reaction times, namely need the relation of balanced reaction fully and between product cost.Through research of technique, applicant finds, the reaction times is 1-5 hour is desirable industrialization result, is more preferably at 2-5 hour, the better effects if of this time period inner equilibrium sufficient reacting and product cost.
3) be obtained by reacting Fmoc-Pro-Azagly-Resin with Fmoc-Pro-OH and Azagly-Resin, the coupling agent used is DIC+A or B+A+C, and wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
Fmoc protected amino acid described in step 4) is Fmoc-Arg (R
1)-OH, Fmoc-Leu-OH, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (R
2)-OH, Fmoc-Trp(R
3)-OH, Fmoc-Ser (R
4)-OH, Fmoc-His (Trt)-OH, Pyr-OH.Wherein, R
1=HCl, NO
2, H; R
2=H, 2-ClTrt, 4-NO
2z, Bzl; R3=H, 2-ClTrt; R4=H, Trt.
Wherein, in amino acid whose coupling agent be DIC+A or B+A+C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
Applicant finds that coupling agent has crucial impact to the present invention, and some coupling agent can produce the phenomenon of racemization, and some coupling agent yield is not high.The coupling agent reducing racemization coupling simultaneously yield high is selected to be technical issues that need to address.Through the research of contriver, be surprised to find that coupling agent is DIC+A or B+A+C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP technical requirements according to the invention.
Step 5) lytic reagent is: trifluoroacetic acid, methylene dichloride, water, and wherein the volume ratio (V:V) of trifluoroacetic acid is 1-15%, preferred 3-10%; The volume ratio (V:V) of methylene dichloride is 75-85%, preferred 80-85%; The volume ratio (V:V) of water is 1-5%, preferred 3-5%; More preferably TFA: methylene dichloride: H
2volume ratio=the 10:85:5 of O.
Removing its method of side chain is that the method for hydro-reduction and hydrogen transference removes Side chain protective group, and hydro-reduction adopts palladium carbon, Raney Ni, Pt, Pt/C, PtO
2, Pd (OH)
2, Rd/C, RhCl (PPh
3)
3deng; Hydrogen transference catalysis, the hydrogen donor participating in reaction can be tetrahydrobenzene, cyclohexadiene, formic acid, ammonium formiate, tetraline etc.Reaction can use single hydrogen donor, also can be used in combination.In a preferred embodiment of the present invention, be preferably ammonium formiate, what another embodiment adopted is formic acid.
Compared with the prior art, present invention process has that operation is simple, aftertreatment is easy, raw material is simple and easy to get, cost is low, yield high, has considerable economical and practical value, is with a wide range of applications in polypeptide drugs design and synthesis field simultaneously.
| English abbreviation | Chinese implication |
| Azagly-Resin | Urea,amino-structural resin |
| Sieber Resin | 3-hydroxybenzyl-9H-xanthenes-9-fluorenylmethyloxycarbonyl aminoresin |
| Sieber-AM Resin | 3-hydroxybenzyl acetyl-9H-xanthenes-9-fluorenylmethyloxycarbonyl aminoresin |
| DMAP | 4-lutidine |
| DIC | DIC |
| HOBt | I-hydroxybenzotriazole |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| HBTU | Benzotriazole-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester |
| HATU | O-(7-azo benzotriazole-1-oxygen)-N, N, N ', N '-tetramethyl-urea hexafluorophosphate |
| TBTU | O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid ester |
| PyBOP | (benzotriazole-1-oxygen) tripyrrole alkane subbase phosphorus hexafluorophosphate |
| DIPEA | DIPEA |
| TMP | 2，4，6-trimethylpyridine |
| 2-ClTrt | 2-chlorine trityl |
| 4-NO 2Z | 4-oil of mirbane methoxycarbonyl |
| Bzl | Benzyl |
| Trt | Trityl |
| tBu | The tertiary butyl |
| TFA | Trifluoroacetic acid |
| Pt/C | Palladium carbon |
| Rd/C | Platinum carbon |
| RhCl(PPh 3) 3 | Three (triphenylphosphine) rhodium chloride |
| DCM | Methylene dichloride |
| DMF | DMF |
Accompanying drawing explanation
Fig. 1: adopt embodiment 13 method to obtain the data of the thick peptide spectrogram of goserelin and correspondence;
Fig. 2: schematic flow sheet of the present invention.
Embodiment
Provide embodiment below to be specifically described the present invention; what be necessary to herein means out is that following examples are only for the present invention is further illustrated; limiting the scope of the invention can not be interpreted as; some nonessential improvement and adjustment that the person skilled in the art in this field makes the present invention according to content of the present invention, still belong to protection scope of the present invention.
specific embodiment explanation
embodiment 1: the preparation of Fmoc-Pro-Azagly-Sieber Resin
By 12.5g, substitution degree is the Sieber Resin of 0.2mmol/g, joins in solid state reaction post, adds DCM swellable resins after 30 minutes, and remove Fmoc protection for twice with 20% piperidines/DMF solution, the time is respectively 10 minutes and 15 minutes.DMF washs six times, by 3.12g N, N-two succinimdyl carbonate 30ml DMF dissolve, add 0.32g DMAP, under ice-water bath, pre-activate 5 minutes, adds in solid phase reactor, room temperature reaction 2.5 hours.Reaction end detects with ninhydrin method and is as the criterion.Reaction terminates rear DMF and washs six times.
Add DMF as solvent, slowly add 1.8ml hydrazine hydrate, room temperature reaction 3 hours, after reaction terminates, DMF washs six times.
By 7.76g Fmoc-Pro-OH, 8.30g HATU, 2.98g HOAt is dissolved in DMF in ice bath situation, adds molecular balance in above-mentioned resin and after 10 minutes, adds 2.4ml TMP, room temperature reaction 2 hours.After DMF washs 3 times, DCM washes 3 times, shrinks three times with methyl alcohol, and the time is respectively 3,5,8 minutes, obtains 12.8 grams of Fmoc-Pro-Azagly-Sieber Resin, and detection substitution degree is 0.185mmol/g.
embodiment 2: the preparation of Fmoc-Pro-Azagly-Sieber Resin
Be the Sieber Resin of 1.2mmol/g by 10g substitution degree, join in solid state reaction post, add DCM swellable resins after 30 minutes, protect with 20% piperidines/DMF solution removal Fmoc, the time is respectively 10 minutes and 15 minutes.DMF washs six times, by 31.2g N, N-two succinimdyl carbonate 100ml DMF dissolve, to add under 2.56g DMAP ice-water bath pre-activate 5 minutes, add in solid phase reactor, room temperature reaction 5 hours.Reaction end detects with ninhydrin method and is as the criterion.Reaction terminates rear DMF and washs six times;
Add DMF as solvent, slowly add 10.8ml hydrazine hydrate, room temperature reaction 5 hours, after reaction terminates, DMF washs six times;
By 23g Fmoc-Pro-OH, 28.2g HATU, 1.3g HOAt is dissolved in DMF in ice bath situation, adds molecular balance in above-mentioned resin and after 10 minutes, adds 21.3ml DIPEA, room temperature reaction 2 hours.After DMF washs 3 times, DCM washes 3 times, shrinks three times with methyl alcohol, and the time is respectively 3,5,8 minutes, obtains 16.5 grams of Fmoc-Pro-Azagly-Sieber Resin, and detection substitution degree is 0.78mmol/g.
embodiment 3: the preparation of Fmoc-Pro-Azagly-Sieber Resin
By 10g, substitution degree is the Sieber Resin of 0.7mmol/g, joins in solid state reaction post, adds DCM swellable resins after 30 minutes, and remove Fmoc protection for twice with 20% piperidines/DMF solution, the time is respectively 10 minutes and 15 minutes.DMF washs six times, by 12g N, N-two succinimdyl carbonate 20ml DMF dissolve, add 0.51g DMAP ice-water bath pre-activate 5 minutes, add in solid phase reactor, room temperature reaction 2.5 hours.Reaction end detects with ninhydrin method and is as the criterion.Reaction terminates rear DMF and washs six times;
Add appropriate DMF as solvent, slowly add 14.1ml hydrazine hydrate, room temperature reaction 3 hours, after reaction terminates, DMF washs six times;
Just 1.34g Fmoc-Pro-OH, 5.67g HOAt is dissolved in DMF in ice bath situation, adds molecular balance in above-mentioned resin and after 10 minutes, adds 6.3ml DIC, room temperature reaction 2 hours.After DMF washs 3 times, DCM washes 3 times, shrinks three times with methyl alcohol, and the time is respectively 3,5,8 minutes, obtains 12.1 grams of Fmoc-Pro-Azagly-Sieber Resin, and detection substitution degree is 0.58mmol/g.
embodiment 4: the preparation of Fmoc-Pro-Azagly-Sieber-AM Resin
By 10.0g, substitution degree is the Amide Sieber-AM Resin of 1.0mmol/g, joins in solid state reaction post, adds DCM swellable resins after 30 minutes, and remove Fmoc protection for twice with 20% piperidines/DMF solution, the time is respectively 10 minutes and 15 minutes.DMF washs six times, by 25.6g N, N-two succinimdyl carbonate 50ml DMF dissolve, add 1.22g DMAP ice-water bath pre-activate 5 minutes, add in solid phase reactor, room temperature reaction 2.5 hours.Reaction end detects with ninhydrin method and is as the criterion.Reaction terminates rear DMF and washs six times;
Add appropriate DMF as solvent, slowly add 10ml hydrazine hydrate, room temperature reaction 5 hours, after reaction terminates, DMF washs six times;
7.76g Fmoc-Pro-OH, 2.98g HOBt is dissolved in DMF in ice bath situation, adds molecular balance in above-mentioned resin and after 10 minutes, add 2.4ml DIC, room temperature reaction 2 hours.After DMF washs 3 times, DCM washes 3 times, shrinks three times with methyl alcohol, and the time is respectively 3,5,8 minutes, obtains 13.2 grams of Fmoc-Pro-Azagly-Sieber-AM Resin, and detection substitution degree is 0.68mmol/g.
embodiment 5: Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (Bzl)-D-Ser (tBu)-Leu-Arg (NO
2) preparation of-Pro-AzaGly-Sieber Resin
Taking 20.2 grams of substitution degrees is that the 10mmol Fmoc-Pro-Azagly-Sieber Resin (10mmol) of 0.495mmol/g adds in reactor; with DCM swelling 0.5 hour; use 20% piperidines/DMF twice to remove Fmoc protection again, the time is respectively 10 minutes and 15 minutes.Fmoc-Arg (NO is connected after washing
2)-OH.By 12.9g Fmoc-Arg (NO
2)-OH, 4.9g HOBt, 6.1ml DIC is dissolved in DCM and (can adds a small amount of DMF hydrotropy), after ice-water bath activates 5 minutes, adds in solid phase reactor, room temperature reaction 2 hours.Reaction end detects with ninhydrin method and is as the criterion.Repeat above step, complete successively, Fmoc-Leu-OH, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (Bzl)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Trp-OH, the coupling of Pyr-OH, obtains Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (Bzl)-Ser (tBu)-Leu-Arg (NO
2)-Pro-AzaGly-Sieber Resin 37.2 grams.
embodiment 6: Pyr-His (Trt)-Trp-Ser (Trt)-Tyr-D-Ser (tBu)-Leu-Arg (NO
2) preparation of-Pro-AzaGly-Sieber Resin
Taking 20.2 grams of substitution degrees is that the 10mmol Fmoc-Pro-Azagly-Sieber Resin (10mmol) of 0.495mmol/g adds in reactor; with DCM swelling 0.5 hour; use 20% piperidines/DMF twice to remove Fmoc protection again, the time is respectively 10 minutes and 15 minutes.Fmoc-Arg (NO is connected after washing
2)-OH.By 12.9g Fmoc-Arg (NO
2)-OH, 4.9g HOBt, 6.1ml DIC is dissolved in DCM and (can adds a small amount of DMF hydrotropy), after ice-water bath activates 5 minutes, adds in solid phase reactor, room temperature reaction 2 hours.Reaction end detects with ninhydrin method and is as the criterion.Repeat above step, complete successively.
Fmoc-Leu-OH, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr-OH, Fmoc-Ser (Trt)-OH, Fmoc-Trp-OH, the coupling of Pyr-OH, obtains Pyr-His (Trt)-Trp-Ser (Trt)-Tyr-Ser (tBu)-Leu-Arg (NO
2)-Pro-AzaGly-Sieber Resin 36.8 grams.
embodiment 7: Pyr-His (Trt)-Trp-Ser (Trt)-Tyr(2-ClTrt)-D-Ser (tBu)-Leu-Arg (NO
2) preparation of-Pro-AzaGly-Sieber Resin
Taking 12.8 grams of substitution degrees is that the 10mmol Fmoc-Pro-Azagly-Sieber Resin (10mmol) of 0.68mmol/g adds in reactor; with DCM swelling 0.5 hour; use 20% piperidines/DMF twice to remove Fmoc protection again, the time is respectively 10 minutes and 15 minutes.Fmoc-Arg (NO is connected after washing
2)-OH.By 12.9g Fmoc-Arg (NO
2)-OH, 4.9g HOBt, 6.1ml DIC is dissolved in DCM and (can adds a small amount of DMF hydrotropy), after ice-water bath activates 5 minutes, adds in solid phase reactor, room temperature reaction 2 hours.Reaction end detects with ninhydrin method and is as the criterion.Repeat above step, complete Fmoc-Leu-OH successively, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (2-ClTrt)-OH, Fmoc-Ser (Trt)-OH, the coupling of Fmoc-Trp-OH, Pyr-OH, obtains Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (2-ClTrt)-Ser (tBu)-Leu-Arg (NO
2)-Pro-AzaGly-Sieber Resin 32.2 grams.
embodiment 8: Pyr-His (Trt)-Trp-Ser (Trt)-Tyr(2-ClTrt)-D-Ser (tBu)-Leu-Arg (NO
2) preparation of-Pro-AzaGly-Sieber-AM Resin
Taking 14.7 grams of substitution degrees is that 10mmol Fmoc-Pro-Azagly-Sieber AM Resin (10mmol) of 0.68mmol/g adds in reactor; with DCM swelling 0.5 hour; use 20% piperidines/DMF twice to remove Fmoc protection again, the time is respectively 10 minutes and 15 minutes.Fmoc-Arg (NO is connected after washing
2)-OH.By 12.9g Fmoc-Arg (NO
2)-OH, 4.9g HOBt, 6.1ml DIC is dissolved in DCM and (can adds a small amount of DMF hydrotropy), after ice-water bath activates 5 minutes, adds in solid phase reactor, room temperature reaction 2.2 hours.Reaction end detects with ninhydrin method and is as the criterion.Repeat above step, complete Fmoc-Leu-OH successively, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (2-ClTrt)-OH, Fmoc-Ser (Trt)-OH, the coupling of Fmoc-Trp-OH, Pyr-OH, obtains Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (2-ClTrt)-Ser (tBu)-Leu-Arg (NO
2)-Pro-AzaGly-Sieber AM Resin 34.2 grams.
embodiment 9: Pyr-His (Trt)-Trp-Ser (Trt)-Tyr(2-ClTrt) preparation of-D-Ser (tBu)-Leu-Arg (HCl)-Pro-AzaGly-Sieber Resin
Taking 16.7 grams of substitution degrees is that the 10mmol Fmoc-Pro-Azagly-Sieber Resin (10mmol) of 0.6mmol/g adds in reactor; with DCM swelling 0.5 hour; use 20% piperidines/DMF twice to remove Fmoc protection again, the time is respectively 10 minutes and 15 minutes.Fmoc-Arg (HCl)-OH is connected after washing.12.9g Fmoc-Arg (HCl)-OH, 4.9g HOBt, 6.1ml DIC are dissolved in DCM and (a small amount of DMF hydrotropy can be added), after ice-water bath activates 5 minutes, add in solid phase reactor, room temperature reaction 2 hours.Reaction end detects with ninhydrin method and is as the criterion.Repeat above step, complete successively
Fmoc-Leu-OH, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (2-ClTrt)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Trp-OH, the coupling of Pyr-OH, obtains Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (2-ClTrt)-Ser (tBu)-Leu-Arg (HCl)-Pro-AzaGly-Sieber Resin 36.2 grams.
embodiment 10: Pyr-His-Trp-Ser-Tyr (Bzl)-D-Ser (tBu)-Leu-Arg (NO
2)-Pro-Azagly-NH
2preparation
Preparation lytic reagent 380ml, wherein trifluoroacetic acid 38ml, water 19ml, DCM 323ml, put into refrigerator precooling 30 minutes; The peptide resin 37.2g that method described in embodiment 5 obtains is joined in 500ml round-bottomed flask; Joined by lytic reagent in resin, ice-water bath while stirring, passes into nitrogen.React and withdraw ice bath after 30 minutes, continue reaction under room temperature 2 hours.Filter resin, collect filtrate.With a small amount of DCM washing resin, merging filtrate.Concentrated solution, to 60ml, slowly adds in 600ml ice ether and precipitates by concentrating under reduced pressure.Centrifugal, ice washed with diethylether 5 times, drying under reduced pressure obtains thick peptide Pyr-His-Trp-Ser-Tyr (Bzl)-D-Ser (tBu)-Leu-Arg (NO
2)-Pro-Azagly-NH
216.2g.
embodiment 11:the preparation of the thick peptide of goserelin
Thick peptide embodiment 10 obtained 16.2 grams is molten adds 2ml formic acid in 160ml methyl alcohol, 10 gram of 10% palladium carbon, be pressurized to 10 normal atmosphere, room temperature reaction 5 hours, detect raw material and disappear, filter palladium carbon, filtrate inspection is concentrated into 30ml, joins 500ml ice ether sedimentation, centrifugal, dry, obtain the thick peptide of goserelin 11.2 grams, thick peptide yield 88.3%, HPLC purity 83.8%.
HPLC purity is 83.2%.By mass spectrum determination structure be: Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
2.
embodiment 12: the preparation of the thick peptide of goserelin
Thick peptide embodiment 10 obtained 16.2 grams is molten adds 0.63 ammonium formiate in 160ml methyl alcohol, 16.2 gram of 10% palladium carbon, room temperature reaction 3 hours, detects raw material and disappears, filter palladium carbon, filtrate inspection is concentrated into 30ml, join 500ml ice ether sedimentation, centrifugal, dry, obtain the thick peptide of goserelin 11.8 grams, thick peptide yield 92.9%, HPLC purity 85.2%, by mass spectrum determination structure is: Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
2.
embodiment 13: the preparation of the thick peptide of goserelin
Thick peptide embodiment 10 obtained 16.2 grams is molten in 160ml 4.4% formic acid methanol solution, 16.2 gram of 10% palladium carbon, room temperature reaction 0.5 hour, detection raw material disappears, filter palladium carbon, filtrate inspection is concentrated into 30ml, joins 500ml ice ether sedimentation, centrifugal, drying, obtains the thick peptide of goserelin 11.0 grams, thick peptide yield 86.7%, HPLC purity 93.7%, by mass spectrum determination structure is: Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
2.
embodiment 14: the preparation of the thick peptide of goserelin
Preparation lytic reagent 360ml, wherein trifluoroacetic acid 36ml, water 18ml, DCM 306ml, put into refrigerator precooling 30 minutes; Peptide resin 36.2g embodiment 9 obtained joins in 500ml round-bottomed flask; Joined by lytic reagent in resin, ice-water bath while stirring, passes into nitrogen.React and withdraw ice bath after 30 minutes, continue reaction under room temperature 2 hours.Filter resin, collect filtrate.With a small amount of DCM washing resin, merging filtrate.Concentrated solution, to 60ml, slowly adds in 600ml ice ether and precipitates by concentrating under reduced pressure.Centrifugal, after ice washed with diethylether 5 times, drying under reduced pressure obtains thick peptide Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
210.2g.Thick peptide yield 80.4%, HPLC purity 73.8%, by mass spectrum determination structure is: Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
2.
embodiment 15:the preparation of goserelin essence peptide
By Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH
2thick peptide 11.0g, thick peptide 10% acetonitrile solution dissolves, and filters; Purifier apparatus: the logical permanent PT600 of innovation, chromatographic column: C18,10 μ, 5cm*25cm, determined wavelength: 230nm, mobile phase A phase: the 0.1%TFA aqueous solution, Mobile phase B phase: acetonitrile; Gradient: 10-40%, the time is 30 minutes, through HPLC purifying, turns salt, obtains 5.7g essence peptide.Purity 99.68%, total recovery 45.2%.Determine that structure is by MS:
Pyr-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-Azagly-NH
2。
Claims (3)
1. prepare a method for goserelin, comprise following steps:
1) by N, N-bis-succinimdyl carbonate and substitution degree scope be sensitivity to acid aminoresin coupling under organic weak base catalyzer of 0.2-1.2mmol/g, N, N-bis-the consumption of succinimdyl carbonate be that 3-10 is doubly to sensitivity to acid aminoresin; Wherein sensitivity to acid aminoresin is Sieber Resin, Sieber-AM Resin, and described organic weak base catalyzer is DMAP;
2) by hydrazine hydrate and step 1) resin reaction of gained obtains Azagly-Resin, and the reaction times is 1-5 hour;
3) under the effect of coupling agent, Fmoc-Pro-Azagly-Resin is obtained with Fmoc-Pro-OH and Azagly-Resin;
4) by Fmoc-Pro-Azagly-Resin and Fmoc protected amino acid and coupling agent according to goserelin peptide sequence one by one the mode of coupling synthesize and obtain goserelin-resin, i.e. Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (Bzl)-D-Ser (tBu)-Leu-Arg (NO
2)-Pro-Azagly-Resin; step 3) and step 4) described in coupling agent be DIC+A or B+A+C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP; C is DIPEA or TMP, and described Fmoc protected amino acid is Fmoc-Arg (R
1)-OH, Fmoc-Leu-OH, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (R
2)-OH, Fmoc-Trp (R
3)-OH, Fmoc-Ser (R
4)-OH, Fmoc-His (Trt)-OH; And amino acid Pyr-OH; Wherein, R
1=HCl, NO
2or H; R
2=H, 2-ClTrt, 4-NO
2z or Bzl; R
3=H or 2-ClTrt; R
4=H or Trt;
5) cracking is carried out to goserelin-resin, remove side chain protected, obtain thick peptide and obtain goserelin through purifying.
2. preparation method as claimed in claim 1, is characterized in that: step 5) in cracking lytic reagent used be: the mixed solution of trifluoroacetic acid, methylene dichloride, water, wherein trifluoroacetic acid: methylene dichloride: the volume ratio=10:85:5 of water.
3. preparation method as claimed in claim 1, is characterized in that: step 5) described in remove side chain protected, the method wherein removing side chain is that the method for hydro-reduction and hydrogen transference removes Side chain protective group; hydro-reduction adopts palladium carbon, Raney Ni, Pt; Pt/C, PtO
2, Pd (OH)
2, Rd/C or RhCl (PPh
3)
3; Hydrogen transference catalysis, participate in reaction hydrogen donor be tetrahydrobenzene, cyclohexadiene, formic acid, ammonium formiate or tetraline, institute use hydrogen donor be single hydrogen donor or mix hydrogen donor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210155366.7A CN102653555B (en) | 2012-05-18 | 2012-05-18 | Method for preparing goserelin by solid phase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210155366.7A CN102653555B (en) | 2012-05-18 | 2012-05-18 | Method for preparing goserelin by solid phase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102653555A CN102653555A (en) | 2012-09-05 |
| CN102653555B true CN102653555B (en) | 2015-04-22 |
Family
ID=46729300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210155366.7A Active CN102653555B (en) | 2012-05-18 | 2012-05-18 | Method for preparing goserelin by solid phase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102653555B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875639A (en) * | 2012-09-26 | 2013-01-16 | 深圳翰宇药业股份有限公司 | Solid-phase synthetic method of peptide and peptide synthesized by same |
| WO2014047822A1 (en) * | 2012-09-27 | 2014-04-03 | 深圳翰宇药业股份有限公司 | Method for preparation of buserelin |
| GB201310921D0 (en) * | 2013-06-19 | 2013-07-31 | Chemical & Biopharmaceutical Lab Of Patras S A | Peptide-resin conjugate and use thereof |
| CN107540727B (en) * | 2016-06-28 | 2021-03-16 | 深圳翰宇药业股份有限公司 | Preparation method of buserelin or goserelin |
| CN108383896B (en) * | 2018-05-31 | 2021-06-04 | 中肽生化有限公司 | Method for synthesizing goserelin by fragment method |
| CN110922453B (en) * | 2018-09-20 | 2023-12-26 | 深圳翰宇药业股份有限公司 | Method for synthesizing goserelin |
| CN110922452B (en) * | 2018-09-20 | 2023-12-26 | 深圳翰宇药业股份有限公司 | Method for synthesizing goserelin |
| CN113735940B (en) * | 2020-05-31 | 2023-08-29 | 深圳市健元医药科技有限公司 | Solid phase synthesis method of peptide |
| CN116675741B (en) * | 2023-07-31 | 2023-10-31 | 杭州湃肽生化科技有限公司 | Application of intermediate in preparation of goserelin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721370A (en) * | 2008-10-10 | 2010-06-09 | 深圳清华大学研究院 | Goserelin release microsphere preparation, preparation method and detecting method thereof |
| CN101759777A (en) * | 2010-01-05 | 2010-06-30 | 江苏诺泰制药技术有限公司 | Method for synthesizing goserelin |
| CN102746383A (en) * | 2011-04-21 | 2012-10-24 | 杭州九源基因工程有限公司 | Synthesis method of goserelin |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010141276A1 (en) * | 2009-06-03 | 2010-12-09 | Mallinckrodt Inc. | Solid phase peptide synthesis process for the production of goserelin |
| CN101935339B (en) * | 2010-08-17 | 2013-07-03 | 深圳翰宇药业股份有限公司 | Solid-phase preparation method for buserelin |
-
2012
- 2012-05-18 CN CN201210155366.7A patent/CN102653555B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721370A (en) * | 2008-10-10 | 2010-06-09 | 深圳清华大学研究院 | Goserelin release microsphere preparation, preparation method and detecting method thereof |
| CN101759777A (en) * | 2010-01-05 | 2010-06-30 | 江苏诺泰制药技术有限公司 | Method for synthesizing goserelin |
| CN102746383A (en) * | 2011-04-21 | 2012-10-24 | 杭州九源基因工程有限公司 | Synthesis method of goserelin |
Non-Patent Citations (1)
| Title |
|---|
| 宋扬.戈舍瑞林的液相合成研究.《中国优秀硕士学位论文全文数据库》.2012,全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102653555A (en) | 2012-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102653555B (en) | Method for preparing goserelin by solid phase | |
| CN102286076B (en) | Preparation method for bivalirudin | |
| CN103864918A (en) | Solid-phase synthesis method for liraglutide | |
| CN103497245A (en) | Method for synthesizing thymalfasin | |
| CN107857797A (en) | The liquid-phase fragment synthetic method of one species snake venom tripeptides | |
| CN102260326A (en) | Method for preparing carbetocin | |
| CN101104638B (en) | Solid phase synthetic technique for thymosin alpha1 | |
| CN101372505B (en) | Method for preparing desmopressin acetate | |
| CN102702320A (en) | Method for preparing eptifibatide | |
| CN101519444B (en) | Method for preparing Nesiritide | |
| CN107540727B (en) | Preparation method of buserelin or goserelin | |
| CN101747426A (en) | Method for synthesizing pramlintide | |
| CN110128505A (en) | A kind of synthetic method of Goserelin impurity | |
| CN105037496B (en) | A kind of preparation method of eptifibatide | |
| CN101857629A (en) | Solid-phase synthesis method of Bremelanotide | |
| CN106478805A (en) | A kind of preparation method of GLP-1 derivant | |
| CN105622727A (en) | Method for synthesizing leuprorelin by solid phase and liquid phase | |
| CN109306366B (en) | Method for synthesizing PT141 | |
| CN102190709A (en) | Synthesis method of luteinizing hormone releasing hormone derivatives | |
| CN103467573A (en) | Carbetocin preparation method | |
| CN107629111B (en) | Liquid phase synthesis method of acetyl tetrapeptide-2 | |
| CN103242443A (en) | Preparation method for thymosin [alpha]1 and analogues thereof | |
| CN102304167B (en) | Novel method for synthesizing polypeptide containing aspartic acid-arginine and derivate units | |
| CN108383896A (en) | A kind of method of segment method synthesis Goserelin | |
| CN102875657B (en) | Method for preparing telomerase polypeptide vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |