WO2008044890A1 - A method for preparing peptides using by solid phase synthesis - Google Patents
A method for preparing peptides using by solid phase synthesis Download PDFInfo
- Publication number
- WO2008044890A1 WO2008044890A1 PCT/KR2007/004989 KR2007004989W WO2008044890A1 WO 2008044890 A1 WO2008044890 A1 WO 2008044890A1 KR 2007004989 W KR2007004989 W KR 2007004989W WO 2008044890 A1 WO2008044890 A1 WO 2008044890A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fmoc
- resin
- group
- peptide
- leu
- 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.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
Definitions
- the present invention relates to novel process for preparing goserelin, buserelin and leuprolide peptide having potent pharmacological activity.
- Buserelin a synthetic gonadotropin-releasing hormone (GRH) agonist, specifically binds to GRH receptor presented at anter iorpituitary and increases or decreases the number of receptors in hypophysis through auto- regulation mechanism (G. Tolis et al., Tumor Growth Inhibition in Patients with Prostatic Carcinoma Treated with Luteinizing Hormone-Feleasing Hormone Agonists, Proc. Natl. Acad. Sci. , 79, pl658, 1982).
- G. Tolis et al. Tumor Growth Inhibition in Patients with Prostatic Carcinoma Treated with Luteinizing Hormone-Feleasing Hormone Agonists, Proc. Natl. Acad. Sci. , 79, pl658, 1982.
- Buserelin PyrHis-TrrSerTyrD-SerfiBiiJ-Leu-ArrPrO-NH-CHi-CHj (acetate salt)
- amino acid derivatives disclosed herein comprises the derivatives protected by the protecting group at N-termina? group and side chain groups thereof selected from Boc UerHDutoxycarbonyl), Fmoc (9- fluorenylmethoxycarbonyl) or Cbz (benzy1oxycarbony1) group, preferably, Fmoc group.
- the reaction temperature disclosed herein ranges from ⁇ about 0 to 70°C, preferably, about 20 to 50 ° C, and the reaction period disclosed herein ranges from 10 mins to 48 hours, preferably, 1 hour to 24 hours considering both of the reactivity of each reactants and the productivity of final product, but it dose not limited thereto. However, the reaction may be performed repeatedly, twice to five times to obtain purposed increased reaction yield.
- the "link" group in the rink amide resin may be preferably rinkamide group having following chemical formulae (d); [Chemistry Figure 4] resin
- the peptide of step 4 is released from the resin by adding weak acidic cleavage solution such as 2% TFA (trifluoroacetic acid)/DCM (dichloromethane) to the peptide resin in the 5 step; after adding hydrazine solution thereto, the side-chain protecting groups, i.e., benzyl group, nitro group, Cbz group etc are removed through catalyst hydrogen transfer reaction using by Pd/C and cyclohexadiene etc to obtain peptide and the peptide is performed to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed goserelin acetate salt in the 6 step.
- weak acidic cleavage solution such as 2% TFA (trifluoroacetic acid)/DCM (dichloromethane)
- the peptide of step 4 is released from the resin by adding weak acidic cleavage solution such as 2% TFA (trifluoroacetic acid)/DCM (dichloromethane) to the peptide resin to obtain peptide derivative, i.e., TFA (trifluoroacetic acid)/DCM (dichloromethane)
- weak acidic cleavage solution such as 2% TFA (trifluoroacetic acid)/DCM (dichloromethane
- the peptide of step 4 is released from the resin by adding weak acidic cleavage solution such as 2%-50% TFA (trifluoroacetic acid)/DCM (dichloromethane) to the peptide resin to obtain peptide derivative, i.e., Pyr-His-Try-Ser(Bzl)-Tyr(Bzl)-DLeu-Leu-Arg(N0 2 ) and then the derivative is reacted with PrO-NH-CH 2 CH 3 and coupling reagent to obtain Pyr-His-Try-
- weak acidic cleavage solution such as 2%-50% TFA (trifluoroacetic acid)/DCM (dichloromethane
- chain protecting groups i.e., benzyl group, nitro group etc are removed through catalytic hydrogen transfer reaction using by Pd/C and cyclohexadiene etc to obtain peptide and the peptide is performed to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed leuprolide acetate salt in the 6 step.
- link 2-chlorotrityl chloride represented by following chemical formulae (e) can be preferably used as the "link" in the 2-chlorotrityl chloride type link-arginine type resin disclosed in the above-described synthetic methods for buserelin and leuprolide: [Chemistry Figure 5]
- the coupling reaction of amino acid disclosed in above-described synthetic methods for buserelin and leuprolide can be performed using by at least one activator selected from the group consisting of; the activated ester of each amino acid, DCC (Dicyclohexyl carbodiimide), DIC (Diisopropyl carbodiimide), BOP (Benzotriazole-lyl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) , PyBOP (Benzotriazol-1-yl-oxytripyrrol idinenophosphonium hexafluorophosphate), HBTU (0-Benzotriazole-N,N,N'N'tetramethyluronium hexafluorophosphate), TBTU (0-(Benzotriazol-lyl)-N,N,N'N'tetramethyluronium tetrafluoroborate), HATU (2-(lH
- inventive method of the present invention characterized in coupling the amino acid derivatives of which amino-terminal group as well as all the side chains are protected, with high molecular support s.de-by side, can provide more favorable advantages over the conventionally known synthetic methods for goserelin, buserelin and leuprolide, for example, high yield and high purity etc. [Advantageous Effects]
- ⁇ 70> Present invention related to the preparation method of goserelin, buserelin and leuprolide using by solid phase synthesis, specifically, reacting amino acid derivatives on rink amide linker introduced modified polystyrene resin side by side to obtain peptide resin and releasing the peptide from the resin to obtain purposed peptides. Accordingly, the inventive preparation methods exhibit more advantageous effects, such as easiness to synthesis, mass production, high yield, high purity of final product etc over the conventionally known synthetic methods showing unsolved problems, such as high cost, limitation to mass production etc.
- the resulted resin - was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 615 mg of Fmoc-Leu-0H (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 670 mg of Fmoc-D-Ser(tBu)-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 768 mg of Fmoc-Arg(N02)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method. After washing the resin, the reaction mixture mixed with 384 mg of Fmoc-Arg(N02)-0H (0.87 mM) and 136 microliter of DIC (0.87 mM) was added thereto again to react together and the resin was washed again.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc- Leu-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 670 mg of Fmoc-D- Ser (tBu)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) vas added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method.
- the resulted r.esin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 742 mg of Fmoc-Trp-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.13g of Fmoc-His(Mmt)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (174 mM) was added thereto again to react together with a similar way to the above-described method.
- the benzyl group and Cbz group among the side chain protecting group in the peptide were removed through catalytic hydrogen transfer reaction using by Pd/C and cyclohexadiene in the presence of methanol.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 670 mg of Fmoc-D- SeKtBu)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc-D- Leu-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc ⁇ TyKOBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method.
- the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
- the novel method of the present invention related to the preparation method of goserelin, buserelin and leuprolide using by solid phase synthesis, specifically, reacting amino acid derivatives on rink amide linker introduced modified polystyrene resin side by side to obtain peptide resin and releasing the peptide from the resin to obtain purposed peptides.
- the inventive preparation methods exhibit more advantageous effects, such as easiness to synthesis, mass production, high yield, high purity of final product etc over the conventionally known synthetic methods showing unsolved problems, such as high cost, limitation to mass production etc.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the preparation method of goserelin, buserelin and leuprolide having various pharmacological activities, in particular, novel method for preparing the peptides comprising the steps; coupling the amino acids on high molecular solid support step-by-step, and finally releasing the amino acids from the supports to obtain the peptides with high yield and high purity.
Description
[DESCRIPTION] [Invention Title]
A METHOD FOR PREPARING PEPTIDES USING BY SOLID PHASE SYNTHESIS
[Technical Field]
<i> The present invention relates to novel process for preparing goserelin, buserelin and leuprolide peptide having potent pharmacological activity.
<2>
[Background Art]
<3> Goserelin and leuprolide, hormonal anti-caner agents, are Gonadorelin releasing hormone agonists (GnRH analogues). Those analogues which show more stronger stability against peptidase than endogenous GnRH and high affinity against of GnRH receptors reduce the number of GnRH receptors in the cell membrane of hypophysis through down regulation manner, uncoupling the GnRH receptor complex through intracellular process and follow by the desensitization of the receptors, resulting in potent anti-cancer activity (Timothy J. Perren et al., Pharmacokinetic and endocrinological parameters of a slow-release depot preparation of the GnRH analogues ICI 118630 (Zoladex® compared with a subcutaneous bolus and continuous subcutaneous infusion of the same drug in patients with prosthetic cancer, Cancer Chemother. Pharmacol., 18, p39, 1986).
<4> Buserelin, a synthetic gonadotropin-releasing hormone (GRH) agonist, specifically binds to GRH receptor presented at anter iorpituitary and increases or decreases the number of receptors in hypophysis through auto- regulation mechanism (G. Tolis et al., Tumor Growth Inhibition in Patients with Prostatic Carcinoma Treated with Luteinizing Hormone-Feleasing Hormone Agonists, Proc. Natl. Acad. Sci. , 79, pl658, 1982).
<5> The synthetic methods for preparing peptides are divided into two methods, i.e., liquid phase synthesis and solid phase synthesis. The liquid phase peptide synthesis of which all the reagents reacts together under the solution phase by being dissolved in the solution, has been reported to show
rapid reaction rate however it has disadvantages such as the difficulty in separating and purification of the products. In a while, solid phase peptide synthesis which have been developed based on the theory of R. B. Merrifield, has been reported to have various advantages comparing with the former method for example, convenient to isolation and purification, the 'applicability to automation (Bodanszky et al, In Peptide Synthesis, John Wiley & Sons, 1976). Lots of peptide synthetic resins have been developed to synthesize various peptides after the publication of the theory of R. B. Merrifield till now. For example, chloromethyl polystyrene resin had been developed by Merrifield and Wang resin having 4-alkoxybenzyl alcohol had been developed with modifying the former resin to overcome the disadvantages thereof at the early stage. Various resins to improve the disadvantages of conventional resins have been developed after then and the representative resins among those resins are trityl group introduced 2-chlorotrityl resin and rink amide resin which can provide amide group from the carboxyl terminal of peptide under mild cleavage condition, respectively.
<6> At the early stage, the simple structured type-peptides have been synthesized using by the resins however the complex structured type peptides showing various physiological activities have been synthesized mainly. The peptides comprising unnatural amino acids have been synthesized by chemical synthetic method since the peptides could not be prepared by enzymatic synthesis. Among them, the peptides comprising D-amino acid or aza-amino acid have been reported to have potent physiological activities and further to be developed as a medicine (USP Nos. 6,624,290; 6,069,163; 5,965,538; and 4,634,715). However, the novel method for preparing LH-RH such as goserelin or GnRH peptides using by solid phase synthesis has been still need till now since previously known methods, for example, the methods disclosed in USP No. 5,602,231; EP No. 0518655; USP No. 6,879,289; and USP No. 3,914,412, have been reported to have unsolved problems such as a limit to obtain pure product etc.
<7>
<8> Therefore, the present inventors have made extensive researches to discover novel method for preparing peptides with high yield and high purity and finally completed the invention by founding novel preparation for obtaining purposed peptide! i.e., coupling the amino acids of which amine terminal group as well as all the side chains are protected with high molecular support step-by-step, differently from the previously known methods disclosed in the cited references.
<9>
[Disclosure]
[Technical Problem]
<io> Present invention relates to the preparation method of goserelin, buserelin and leuprolide having various pharmacological activities, in particular, novel method for preparing the peptides comprising the steps; coupling the amino acids on high molecular solid support step-by-step, and finally releasing the amino acids from the supports to obtain the peptides with high yield and high purity.
<11>
[Technical Solution]
<i2> Accordingly, in a preferred embodiment of the present invention, the present invention provides novel method for preparing goserelin, buserelin and leuprolide represented by following chemical formula (a)- (c) comprising the steps consisting of: coupling the amino acid derivatives of which amino- terminal group as well as all the side chains are protected, with rink amide linker introduced-high molecular support resin and trityl group introduced
St resin under a reaction solvent side-by-side, at the 1 step! and isolating the peptides from the resins of step 1 and selectively removing the side chain protecting groups from the peptides, at the final step.
<13>
[Chemistry Figure 1]
Goserelin : PrHis-TrrSerTrl>Ser(ffiu)-Leu-ArrPrα-A2agIrNH: (acetate salt)
<14> (a)
[Chemi stry Figure 2]
Buserelin : PyrHis-TrrSerTyrD-SerfiBiiJ-Leu-ArrPrO-NH-CHi-CHj (acetate salt)
(b)
< 15>
[Chemi stry Figure 3]
Leuprolide : I^rHirTrirSerTyrD-Leu-Leu-Arg-PnrNH-CHjCH} (acetate salt)
(c)
<16>
<i7> Wherein said Pyr denotes pyroglutamic acid; His, histidine! Trp, tryptophane; Ser, serine; Tyr, tyrosine; D-Ser(tBu), D-optically active isomer of serine protected with tertiary butyl group on hydroxy1 groupCside chain); D-Leu, D-optically active leucine; Leu, leucine; Arg, arginine; Pro, proline; and Azagly, glycine of which alpha carbon is substituted with nitrogen atom.
<18>
<i9> The term " high molecular support resin" disclosed herein comprises the resin selected from polystyrene, polyamide, glass or silica resin, preferably, polystyrene resin.
<20> The term "amino acid derivatives" disclosed herein comprises the derivatives protected by the protecting group at N-termina? group and side chain groups thereof selected from Boc UerHDutoxycarbonyl), Fmoc (9- fluorenylmethoxycarbonyl) or Cbz (benzy1oxycarbony1) group, preferably, Fmoc group.
<21>
<22> The exemplary reaction solvent used in the present invention comprises the generally used solvent in the chemical synthesis selected from
dichloromethane, chloroform, dichloroethane, dimethylformamide (DMF), dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran (THF), trifluoroacetic acid (TFA), dioxane, or the mixture thereof, preferably, dichloromethane, dimethylformamide (DMF) or trifluoroacetic acid (TFA). <23> The reaction temperature disclosed herein ranges from <about 0 to 70°C, preferably, about 20 to 50°C, and the reaction period disclosed herein ranges from 10 mins to 48 hours, preferably, 1 hour to 24 hours considering both of the reactivity of each reactants and the productivity of final product, but it dose not limited thereto. However, the reaction may be performed repeatedly, twice to five times to obtain purposed increased reaction yield.
<24>
<25> The purposed peptides, i.e., goserelin, buserelin and Ieuprolide of the invention may be chemically synthesized by the methods which will be explained by following reaction schemes hereinafter, which are merely exemplary and in no way limit the invention. The reaction schemes show the representative method for preparing peptides of the present invention, and the other method also may be modified by following the steps with appropriate modifications of reagents and starting materials, which are envisaged by those skilled in the art.
<26>
<27> GENERAL SYNTHETIC PROCEDURE
<28>
<29> Scheme 1
»mm H*G + Fmoc * Ptacxrl» .*
O
Ftaoc-miMJ-X * FtarNHNH-MHAK-
<30>
<31>
<32> As depicted in above Scheme 1, the hydrazine derivative protected by "9-fluorenylmethoxycarbonyl (Fmoc)" protecting group is coupled with carbonyl group and then reacted with rink amide resin therecn to activate N-
St terminal residue to obtain semicarbazide type resin at the 1 step;
St
<33> At the 1 step of the reaction scheme 1, the "X" group may be selectively used among various leaving groups, preferably,1 halogen group, imidazole group or succinimide group and the like.
St
<34> At the 1 step of the reaction scheme 1, the "link" group in the rink amide resin may be preferably rinkamide group having following chemical formulae (d); [Chemistry Figure 4]
resin
Fmoc- protected "Rinkamide'
<35>
(d)
<37> <38> Scheme 2 <39> (A)
Peptide Synthesis
Fmoc-NHNH-CO-NH-Riπk-Sure-
Pyr-Hi^Trp-Ser(Bzl)-Tyr(Bzl)-D-Ser(tBu)-Leu-Arg(N02>Azgly-NH-Rink-Sure- (O
2%TFA/MC
Pyr-Hi&-Trp-Ser(B∑l)-Tyr(Bzl)-D-Ser(tBu)-Lπu-Arg(NO2)-Azgly-NK
■*■ Pyr-His-Trp-Ser-Tyr-C^Ser(tBu)-Leu-Arg-Azgly-NH2 (Goserelin)
Pd1 K
' Sure = surface-layer polystyrene resin manufactured by BeadTech, Inc.
<40>
<41 > (B)
Peptide Synthesis
Fmoc-NHNH-CO-NH-Riπk-Sune-
2%TFA/MC
Pyr-Hi^Tφ-Ser(Bzl)-Tyr(Bzl)-[>Ser(tBu)-L£U-Arg(NO2)-Azgly-NH;
■*■ FVr-His-Trp-Ser-Tyr-[^Ser(tBu)-LeLi-Arg-Azgly-NH2 (Gosemlin)
Pd1 H,
* Su re = su if ace- layer polystyrene resin manufactured by BeadTech, Inc.
<42> <43>
<44> As can be seen in the above scheme 2, two different synthetic methods may be adopted to synthesize goserelin as follows:
<45> st
<46> For example, as the 1 method (A), the Fmoc group attached to the nitrogen atom end of Fmoc-aza-rinkamide resin (azagly: azaglycyl , -NH-NH-CO-) prepared in step 1 is removed in the presence of reaction solvent such as 20% piperidine/DMF etc through deprotecting reaction, in the 2 step! the amino acid derivatives of which amino-terminal group as well as all the side chains are protected, i.e., Fmoc-Arg(N02) (N(V nitro), Fmoc-Leu, Fmoc-D-Ser(tBu)
(tBu; tertiary butyl), Fmoc-Tyr(BzI) (BzI: benzyl), Fmoc-Ser(BzI), Fmoc-Trp, Fmoc-His(Fmoc) , and Pyr(pyroglutamic acid) are serially coupled with the rink amide linker resin in the 3 step! the Fmoc group of said peptide is removed by deprotecting reaction as described in step 2 to obtain peptide resin in th the 4 step.
<47> As an alternative method (B), all the procedure is similar to the method (A) excepting that the Fmoc-His(Mmt) is used instead of Fmoc- His(Fmoc), which could provide more increased yield in the hydrogenation reaction using Pd/C (palladium on carbon, catalyst) since Mmt (4- methoxytrityl) group, a protecting group of His is removed vhere the peptide is released from resin with weak acid. The similar method to the above method (B) may be adopted in the following synthetic method for buserelin.
<48>
<49> The peptide of step 4 is released from the resin by adding weak acidic cleavage solution such as 2% TFA (trifluoroacetic acid)/DCM (dichloromethane) to the peptide resin in the 5 step; after adding hydrazine solution thereto, the side-chain protecting groups, i.e., benzyl group, nitro group, Cbz group etc are removed through catalyst hydrogen transfer reaction using by Pd/C and cyclohexadiene etc to obtain peptide and the peptide is performed to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed goserelin acetate salt in the 6 step.
<50>
<5i> The preparation methods of buserelin and leuprolide are shown in following scheme 3 and scheme 4 similarly to the above-described preparation method of goserelin:
<52> Scheme 3
<53>
PiO-NH-CH2CH3
— COU—ptfitf1^-** PΫr-Ηii-Trp'SirφiD'TyfC&^^StfCliuJ'tjy-ArgCNO^'
Pyr-His-Tw-Sef-B/r-D-Sβrpu^isy-Ars-Pro-MH-CHjCH, (Qusβrβϋn)
<54> <55>
<56> For example, the solid phase synthesis of buserelin is more difficult than that of goserelin since it has ethylamide group at carboxyl terminal end. Accordingly, the rink-arginine type resin linked to arginine derivative could be adopted in case of using solid phase peptide synthesis. 2- Chlorotrityl chloride type rink-arginine type resin is prepared to the similar method to that for preparing rink-amide resin disclosed in the step 1
St of scheme 1 in the 1 step; after coupling the resin with Fmoc-Arg(Nθ2) , the
Fmoc group is removed by deprotecting method using by reaction solvent such as 20% piperidine/DMF etc in the 2 step! the amino acid derivatives, i.e.,
Fmoc-Leu, Fmoc-D-Ser(tBu) , Fmoc-Tyr(Bz1 ) , Fmoc-Ser(Bz1 ) , Fmoc-Trp, Finoc- His(Mmt)(or Fmoc-His (Fmoc)), and Pyr are serially coupled with the resin rd side by side in the 3 step; the Fmoc group of said peptide is removed by deprotecting reaction as described in step 2 to obtain peptide resin in the 4 step! the peptide of step 4 is released from the resin by adding weak
acidic cleavage solution such as 2% TFA (trifluoroacetic acid)/DCM (dichloromethane) to the peptide resin to obtain peptide derivative, i.e.,
Pyr-His-Try-Ser(Bzl)-Tyr(Bzl)-DSer(tBu)-Leu-Arg(NO2) in the 5th step; the
derivative is reacted with PrO-NH-CH2CH3 and coupling reagent to obtain Pyr-
His-Try-Ser(BzI)-Tyr(BzI)-DSer(tBu)-Leu-Arg(N02)-Pro-NH-CH2CH3 in the 5th step!
the side-chain protecting groups, i.e., benzyl group, nitro group, Cbz group etc are removed through catalytic hydrogen transfer reaction using by Pd/C and cyclohexadiene etc to obtain peptide and the peptide is performed to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed buserelin acetate salt in tie 6 step.
<57>
<58> Scheme 4
Pro-NH-CKCH,
— ■" ^ Pvr-Hts-Trp-SffC&MfrfBEll-D-LiU-LSϋ-ΛrolNO^Pro-NH- CHXH^
Pyf-Hi4«Tf#'Sir*Tfr*D-L8U-L6u«Ati'Pr o-NH- CHi)H1 (LeuprctWe)
Pd1 H*
<59>
<60>
<6i> For example, the solid phase synthesis of leuprolide is more difficult than that of goserelin like as buserelin since it has ethylamide group at
carboxyl terminal end. Accordingly, the rink-arginine type resin linked to arginine derivative could be adopted in case of using solid phase peptide synthesis. 2-Chlorotrityl chloride type rink-arginine type resin is prepared to the similar method to that for preparing rink-amide resin disclosed in the
St St
1 step of scheme 1 in the 1 step; after coupling the resin with Fmoc- Arg(N02), the Fmoc group is removed by deprotecting method using by reaction
solvent such as 20% piperidine/DMF etc in the 2 step! the amino acid derivatives, i.e., Fmoc-Leu, Fmoc-D-Leu, Fmoc-Tyr(BzI) , Fmoc-Ser(Bzl), Fmoc- Trp, Fmoc-His(Mmt) (or Fmoc-His(Fmoc)), and Pyr are serially coupled with the rd resin side by side in the 3 step; the Fmoc group of said peptide is removed by deprotecting reaction as described in step 2 to obtain peptide resin in the 4 step! the peptide of step 4 is released from the resin by adding weak acidic cleavage solution such as 2%-50% TFA (trifluoroacetic acid)/DCM (dichloromethane) to the peptide resin to obtain peptide derivative, i.e., Pyr-His-Try-Ser(Bzl)-Tyr(Bzl)-DLeu-Leu-Arg(N02) and then the derivative is reacted with PrO-NH-CH2CH3 and coupling reagent to obtain Pyr-His-Try-
Ser(Bzl)-Tyr(Bzl)-DLeu-Leu-Arg(N02)-Pro-NH-CH2CH3 in the 5th step; the side-
chain protecting groups, i.e., benzyl group, nitro group etc are removed through catalytic hydrogen transfer reaction using by Pd/C and cyclohexadiene etc to obtain peptide and the peptide is performed to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed leuprolide acetate salt in the 6 step.
<62> The above-described methods as shown in Schemes 1-3 can be modified by the well-known methods in the art and the disclosed in the literature {Synthetic Peptides'- A user's Guide, G. R. Grant, Ed., Freeman & Co., 1992, pp77-183) .
<63>
<64> The link 2-chlorotrityl chloride represented by following chemical
formulae (e) can be preferably used as the "link" in the 2-chlorotrityl chloride type link-arginine type resin disclosed in the above-described synthetic methods for buserelin and leuprolide: [Chemistry Figure 5]
dirinylbeflzenθ cress-liked
lystyrene l»adf«»)
<65> <66> (e)
<67> <68> The coupling reaction of amino acid disclosed in above-described synthetic methods for buserelin and leuprolide, can be performed using by at least one activator selected from the group consisting of; the activated ester of each amino acid, DCC (Dicyclohexyl carbodiimide), DIC (Diisopropyl carbodiimide), BOP (Benzotriazole-lyl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) , PyBOP (Benzotriazol-1-yl-oxytripyrrol idinenophosphonium hexafluorophosphate), HBTU (0-Benzotriazole-N,N,N'N'tetramethyluronium hexafluorophosphate), TBTU (0-(Benzotriazol-lyl)-N,N,N'N'tetramethyluronium tetrafluoroborate), HATU (2-(lH-7-Azabenzotriazol-1-yI)-I, 1,3,3- tetramethyluronium hexafluorophosphate) , TATU (2-(lH-7-Azabenzotriazol-l-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate) and CDI (Carbony1 di imidazole), preferably, DIC (Diisopropyl carbodiimide). The amount of the activator ranging from about 1 10 equivalent, preferably, about 1.5 to 3 equivalent amount based on the amount of amino acid can be used and the amount of amino
acid derivatives ranging from about 1 to 10 equivalent, preferably, about 1.5 to 3 equivalent amount based on the substitution rate of the resin may be used in the present invention. As the activated ester of each amino acid, the activating substances at the C-terminal of the amino acid such as the symmetric anhydride form, the mixed anhydride form, the pentafluorophenyl ester form thereof etc can be preferably used in the amount ranging from about 1 to 10 equivalents based on the amount of the coupled amino acid with high molecular resin.
<69> The inventive method of the present invention characterized in coupling the amino acid derivatives of which amino-terminal group as well as all the side chains are protected, with high molecular support s.de-by side, can provide more favorable advantages over the conventionally known synthetic methods for goserelin, buserelin and leuprolide, for example, high yield and high purity etc. [Advantageous Effects]
<70> Present invention related to the preparation method of goserelin, buserelin and leuprolide using by solid phase synthesis, specifically, reacting amino acid derivatives on rink amide linker introduced modified polystyrene resin side by side to obtain peptide resin and releasing the peptide from the resin to obtain purposed peptides. Accordingly, the inventive preparation methods exhibit more advantageous effects, such as easiness to synthesis, mass production, high yield, high purity of final product etc over the conventionally known synthetic methods showing unsolved problems, such as high cost, limitation to mass production etc.
<71>
[Best Mode]
<72> It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
<73>
[Mode for Invention] <74> Example 1: Preparation of Fmoc-NHNH2
<75> 1-1. Fmoc-NHNHg (1)
<76> 12.6g of Fmoc-ONSu was dissolved in 150 ml of diethyl ether in 100 ml of round bottom flask equipped with magnetic stirrer and the solution (10.Og of NH2NH2-H2O dissolved in 100 ml of diethyl ether) was dropwisely added thereto for 1 hour to react with together for 24 hours. The resultant white solid prepared by removing remaining solvent from the solution by rotary evaporator was dissolved in 250 ml of ethanol with reflux distillation and the reaction solution was cooled to isolate 0.95g of purposed white Fmoc- NHNH2 (1) (yield: 10%).
<77>
<78> 1-2. Fmoc-NHNHg (2)
<79> Wi th the s imi l ar method to the method di sclosed in Example 1-2 except ing us ing 9.7g of Fmoc-Cl and THF as a solvent , 2.47g of Fmoc-NHNH2 (2) was obtained (yield : 26%) .
<80>
<8i> Example 2: Preparation of Semicarbazide resin
<82> 2-1. Semicarbazide resin (1)
<83> To modify the Fmoc~NHNH2 (2) prepared in Example 1-2 with semicarbazide type to be introduced in resin, rink amide resin was activated with 1.1 equivalent amounts of CDI (carbonyl di imidazole) based on the mount of the FmOc-NHNH2 (2) and the Fmoc-NHNH2 was added thereto to obtain semicarbazide resin (1) (substitution rate determined by Fnioc quantity: 0.27 mmol/g; yield
59%) .
<84>
<85> 2-2. Semicarbazide resin (2)
<86> With the similar method to the method disclosed in Example 2-1, rink amide resin was activated with identical amount of disuccinyl carbonate to
the activator used in Example 2-1 and the Fmoc-NHNH2 was added thereto to obtain semicarbazide resin (2) (substitution rate determined by Fmoc quantity: 0.58 mmol/g! yield >95%).
<87>
<88> Example 3: Preparation of Goserelin
<89> 3-1. Use of Fmoc-His(Fmoc) (1)
<90> Ig of the Fmoc-azagly-rink amide resin (2) prepared in Example 2-2 was treated with 20% piperidine/DMF solution twice to remove Fmoc residue and the resin was swollen by being dipped into 10 ml of DMF. The reaction mixture mixed with 585 mg of Fmoc-Pro-OH (1.74 mmol), 271 microliter of DIC (1.74 mmol) and 235 mg of HOBt (1.74 mmol) was added thereto and reacted together for 1.5 hours. The solution was washed with DMF and the former reaction was repeated again. The resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 768 mg of Fmoc-Arg(N(^)-OH
(1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto to react together with a similar way to the above-described method. After washing the resin, the reaction mixture mixed with 384 mg of Fmoc-Arg(N02)-0H (0.87 mmol) and 136 microliter of DIC (0.87 mmol) was added thereto again to react together and the resin was washed again. The resulted resin -was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 615 mg of Fmoc-Leu-0H (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 670 mg of Fmoc-D-Ser(tBu)-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the
above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc group and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mmol) and 271 microliter of DIC (1.74 mmol) was added thereto again to react together with a similar way to the above- described method.
<9i> The resin was washed again and 2ml of 2% TFA (trifluoroacetic acid)/DCM (dichloromethane) per 70mg of peptide resin was added to the resin, eluted to release the peptide from the resin and the elute was co lected with 160 microliter of TEA (triethylamine). The above-described step was repeated three times. The resin was washed with DCM (dichloromethane) and methanol and the elute was collected with the former elute. The elute was concentrated with evaporation and the 4ml of NH2NH2/water (1/200) solution was added to the concentrate to stir for 2 hours. The benzyl group and Cbz group among the side chain protecting group in the peptide were removed through catalytic hydrogen transfer reaction using by Pd/C and cyclohexadiene in the presence of methanol. The resulting peptide was purified with reverse phase column chromatography to isolate pure goserelin (Yield:
<92>
<93> 3-2. Use of Fmoc-His(Mmt)
<94> Ig of the Fmoc-azagly-rink amide resin (2) prepared in Example 2-2 was treated with 20% piperidine/DMF solution twice to remove Fmoc residue and the resin was swollen by being dipped into 10 ml of DMF. The reaction mixture mixed with 585 mg of Fmoc-Pro-OH (1.74 mM), 271 microliter of DIC (1.74 mM) and 235 mg of HOBt (1.74 mM) was added thereto and reacted together for 1.5 hours. The solution was washed with DMF and the former reaction was repeated again. The resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 768 mg of Fmoc-Arg(N02)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method. After washing the resin, the reaction mixture mixed with 384 mg of Fmoc-Arg(N02)-0H (0.87 mM) and 136 microliter of DIC (0.87 mM) was added thereto again to react together and the resin was washed again. The resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc- Leu-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 670 mg of Fmoc-D- Ser (tBu)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) vas added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted r.esin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed
with 742 mg of Fmoc-Trp-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.13g of Fmoc-His(Mmt)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (174 mM) was added thereto again to react together with a similar way to the above-described method.
<95> The resin was washed again and 2ml of 2% TFA (Trifluoroacetic acid)/DCM (dichloromethane) per 70mg of peptide resin was added to the resin, eluted to release the peptide from the resin and the elute was collected with 160 microliter of TEA (Triethylamine) . The above-described step was repeated three times. The resin was washed with DCM (dichloromethane) and methanol and the elute was collected with the former elute. The elute was concentrated with evaporation and the 4ml of NH2NH2/water (1/200) solution was added to the concentrate to stir for 2 hours. The benzyl group and Cbz group among the side chain protecting group in the peptide were removed through catalytic hydrogen transfer reaction using by Pd/C and cyclohexadiene in the presence of methanol. The resulting peptide was purified with reverse phase column chromatography (Shimadzu H-kit, acetonitrile:water= 22:78 → 32:68, 1% increase/min) to isolate pure goserelin (Yield: 65%).
<96>
<97> Example 4: Preparation of buserelin
<98> Ig of 2-chlroro trityl chloride resin showing 0.9 mM/g of substitution rate was swollen with 10ml of DMF and the reaction mixture mixed with 768 mg of Fmoc-Arg (N02)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together. The resulted resin was treated with 20% piperidine
to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc-Leu-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 670 mg of Fmoc-D- SeKtBu)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc-Tyr(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
<99> The resin was washed again and 2ml of 1% TFA (Trifluoroacetic acid)/DCM (dichloromethane) per 70mg of peptide resin was added to the resin, eluted to release the peptide from the resin and the elute was collected with 200
microliter of pyridine. The above-described step was repeated five times. The resin was washed with DCM (dichloromethane) and methanol and the elute was collected with the former elute. The elute was concentrated with evaporation and ether was added thereto to obtain the precipitated peptide. The precipitated peptide was performed to coupling reaction with 305 mg of Pro- NH-CH2CH3 (2.4mM) and 303mg of DIC (2.4 niM) in the presence of DCM
(dichloromethane) solvent. The solution was subjected to concentration with evaporator. The resulting concentrate was dissolved in EtOAc, washed with saturated NaHCOs solution, distilled water, 5% citrate solution and dried with anhydrous MgS(V The remaining MgS04 was discarded with filtration and the filtrate was concentrated with evaporation. The benzyl group and Cbz group among the side chain protecting group in the peptide were removed through catalytic hydrogen transfer reaction using by Pd/C and ammonium formate in the presence of methanol. The resulting peptide was purified with reverse phase column chromatography (Shimadzu H-kit, acetonitrile^water= 22:78 → 32:68, 1% increase/min) to isolate pure buserelin (Yield: 40%).
<ioo>
<ioi> Example 5: Preparation of leuprolide resin
<1O2> Ig of 2-chlroro trityl chloride resin showing 0.9 mM/g of substitution rate was swollen with 10ml of DMF and the reaction mixture mixed with 768 mg of Fmoc-Arg(N02)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together. The resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc-Leu-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 615 mg of Fmoc-D- Leu-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to
remove the Fmoc residue and the reaction mixture mixed with 859 mg of Fmoc~ TyKOBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 726 mg of Fmoc-Ser(OBzI)-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 742 mg of Fmoc-Trp-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 1.078g of Fmoc-His(Fmoc)-0H (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above- described method. After washing the resin, the resulted resin was treated with 20% piperidine to remove the Fmoc residue and the reaction mixture mixed with 244 mg of Pyr-OH (1.74 mM) and 271 microliter of DIC (1.74 mM) was added thereto again to react together with a similar way to the above-described method.
<i03> The resin was washed again and 2ml of 1% TFA (Trifluoroacetic acid)/DCM (dichloromethane) per 70mg of peptide resin was added to the resin, eluted to release the peptide from the resin and the elute was collected with 200 microliter of pyridine. The above-described step was repeated five times. The resin was washed with DCM (dichloromethane) and methanol and the elute was collected with the former elute. The elute was concentrated with evaporation and ether was added thereto to obtain the precipitated peptide. The precipitated peptide was performed to coupling reaction with 305 mg of Pro- NH-CH2CH3 (2.4mM) and 303mg of DIC (2.4 mM) in the presence of DCM
(dichloromethane) solvent. The solution was subjected to concentration with evaporator. The resulting concentrate was dissolved in EtOAc, washed with
saturated NaHCOs solution, distilled water, 5% citrate solution and dried with anhydrous MgSO4. The remaining MgS04 was discarded with filtration and the filtrate was concentrated with evaporation. The benzyl group and Cbz group among the side chain protecting group in the peptide were removed through catalytic hydrogen transfer reaction using by Pd/C and ammonium formate in the presence of methanol. The resulting peptide was purified with reverse phase column chromatography (Shimadzu H-kit, acetonitrile:water= 22:78 → 32:68, 1% increase/min) to isolate pure leuprolide (Yield: 37%).
<104>
[Industrial Applicability]
<iO5> As described above, the novel method of the present invention related to the preparation method of goserelin, buserelin and leuprolide using by solid phase synthesis, specifically, reacting amino acid derivatives on rink amide linker introduced modified polystyrene resin side by side to obtain peptide resin and releasing the peptide from the resin to obtain purposed peptides. Accordingly, the inventive preparation methods exhibit more advantageous effects, such as easiness to synthesis, mass production, high yield, high purity of final product etc over the conventionally known synthetic methods showing unsolved problems, such as high cost, limitation to mass production etc.
<I06>
Claims
[CLAIMS] [Claim 1]
<iO8> A method for preparing goserelin, buserelin and leuprolide represented by following chemical formula (a)- (c) comprising the steps consisting of: coupling the amino acid derivatives of which amino-terminal group as well as all the side chains are protected, with rink amide linker introduced-high molecular support resin and trityl group introduced resin under a reaction solvent side-by-side, at the 1 step; and isolating the peptides from the resins of step 1 and selectively removing the side chain protecting groups from the peptides, at the final step:
<109>
Goserelin : PyrHis-TrprSerTyrD-3er(ffiu)-Leu-ArrPrcrAzaglrNH: (acetate salt)
(a)
Buserelin : IVrHirTrrSerTr^Ser(ffiu)-ku-ArrPr(rHH-CH:-CH} (acetate salt)
(b)
Leuprolide : PyrHis-TrrSerTyrD-Leu-Leu-Arg-PiO-NH-CHrCHj (acetate salt)
(c)
<112> <113>
[Claim 2]
<ii4> The method according to claim 1, said high molecular support resin is selected from polystyrene, polyamide, glass or silica resin.
<115>
[Claim 3]
<ii6> The method according to claim 1, said amino acid derivatives is protected by the protecting group at N-terminal group and side chain groups thereof selected from BOCUe/t-butoxycarbonyl), Fmoc(9- fluorenylmethoxycarbonyl) or CbzCbenzyl oxycarbonyl) group.
<117>
[Claim 4]
<ii8> The method according to claim 1, said reaction solvent is selected from dichloromethane, chloroform, dichloroethane, dimethylformamide(DMF), dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran(THF) , trifluoro- acetic acid (TFA), dioxane, or the mixture thereof.
<119>
[Claim 5]
<12O> The method for preparing goserelin according to claim 1, said method comprise the step consisting of: coupling the hydrazine derivative protected by "9-fluorenylmethoxycarbonyl(Fmoc)" protecting group with carbonyl group and then reacting with link amide resin thereon to activate N-
St terminal residue to obtain semicarbazide type resin at the 1 step! removing the Fmoc group to the nitrogen atom end of Fmoc-aza-rinkamide resin (azagly-" azaglycyl, -NH-NH-CO-) prepared in step 1 in the presence of reaction solvent through deprotecting reaction, in the 2 step; coupling Fmoc-Arg(N02) (N(V
nitro), Fmoc-Leu, Fmoc-D-Ser(tBu) (tBu: tertiary butyl), Fmoc-Tyr(Bzl) (BzI: benzyl), Fmoc-Ser(BzI) , Fmoc-Trp, Fmoc-His(Fmoc), and Pyr(pyroglutamic acid) of which amino-terminal group as well as all the side chains are protected, rd serially with the rink amide linker resin side by side in the 3 step; removing the Fmoc group of said peptide by deprotecting reaction as described th in step 2 to obtain peptide resin in the 4 step; releasing the peptide of step 4 from the resin by adding weak acidic cleavage solution to the peptide th resin in the 5 step! after adding hydrazine solution thereto, removing benzyl group, nitro group and Cbz group among the side-chain protecting groups through catalyst hydrogen transfer reaction to obtain peptide and performing the peptide to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed goserelin acetate th salt in the 6 step.
<121>
[Claim 6]
<122> The method according to claim 5, said Fmoc-His(Fmoc) , is substituted with Fmoc-His(Mmt) .
<123>
[Claim 7]
<124> The method according to claim 5, said link group in the link amide resin is a rink amide group having following chemical formulae (d);
resin 
FnKxr-protected "Rlnkamide"
<125> (d) < 126>
[Claim 8]
<127> The method for preparing buserelin according to claim 1, said method comprise the step consisting of: preparing 2-chlorotrityl chloride type 1 ink—
St arginine type resin in the 1 step; after coupling the resin with Fmoc- Arg(NU2) , removing the Fmoc group by deprotecting method using by reaction nd solvent such as 20% piperidine/DMF etc in the 2 step! coupling Fmoc-Leu, Fmoc-D-Ser(tBu), Fmoc-Tyr(BzI) , Fmoc-Ser(BzI) , Fmoc-Trp, Fmoc-His(Mmt)(or rd
Fmoc-His(Fmoc)), and Pyr serially side by side with the resin in the 3 step; removing the Fmoc group of said peptide by deprotecting reaction as described in step 2 to obtain peptide resin in the 4 step; releasing the peptide of step 4 from the resin to the peptide resin to obtain Pyr-His-Try-Ser(BzI)- Tyr(Bzl)-DSer(tBu)-Leu-Arg(NOa) in the 5 step; reacting the derivative with
PrO-NH-CH2CH3 and coupling reagent to obtain Pyr-His-Try-Ser(BzI )-Tyr(BzI)-
DSer(tBu)-Leu-Arg(NO2)-Pr0-NH-CH2CH3 in the 5 step; removing benzyl group,
nitro group and, Cbz group among the side-chain protecting groups through catalytic hydrogen transfer reaction to obtain peptide and performing the peptide to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed buserelin acetate salt in the 6 step.
<128>
[Claim 9]
<129> The method for preparing leuprolide according to claidi 1, said method comprise the step consisting of: preparing 2-chlorotrityl chloride type link-
St argmine type resin in the 1 step; after coupling the resin with Fmoc- Arg(NO2), removing the Fmoc group by deprotecting method using by reaction nd solvent in the 2 step; coupling Fmoc-Leu, Fmoc-D-Leu, Fmoc-Tyr(Bzl), Fmoc- Ser(Bzl), Fmoc-Trp, Fmoc~His(Mmt)(or Fmoc-His(Fmoc)), and Pyr serially side rd by side with the resin in the 3 step; removing the Fmoc group of said peptide by deprotecting reaction as described in step 2 to obtain peptide th resin in the 4 step; releasing the peptide of step 4 from the resin by adding weak acidic cleavage solution to the peptide resin to obtain Pyr-His- Try-Ser(Bzl)-Tyr(Bzl)-DLeu-Leu-Arg(N02) and then reacting the derivative with PrO-NH-CH2CH3 and coupling reagent to obtain Pyr-His-Try-Ser(Bzl)-Tyr(Bzl)-
DLeu-Leu-Arg(NO2)-Pr0-NH-CH2CH3 in the 5 step; removing benzyl group, nitro
group, and Cbz group among the side-chain protecting group through catalytic hydrogen transfer reaction to obtain peptide and performing the peptide to purification process using by reverse phase column chromatography and ion exchange resin to obtain purposed leuprolide acetate salt in the 6 step.
<130>
[Claim 10]
<i3i> The method according to claim 8 or claim 9, said "link" group in the 2-chlorotrityl chloride type link-arginine type resin is a link 2- chlorotrityl chloride represented by following chemical formulae (e)"
<132>
likβd 
<133> (e)
<134>
[Claim 11]
<i35> The method according to claim 8 or claim 9, said coupling reaction of amino acid is performed using by at least one activator selected from the group consisting of; the activated ester of each amino acid, DCC (dicyclohexyl carbodiimide), DIC (diisopropyl carbodiimide), BOP (benzotriazoIe-1-yl-oxy-tris-(dimethylamino)-phosphoniurn hexafluorophosphate) , PyBOP (benzotriazol-1-yl-oxytripyrrol idinophosphonium hexafluorophosphate) , HBTU (0-benzotriazole-N,N,N'N' tetramethyluroniura hexafluoro-phosphate), TBTU (0-(benzotriazol-l-yl)-N,N,N'N'tetramethyluronium tetrafluoroborate), HATU (2-(lH-7-azabenzotriazol-l-yl)-l,l,3,3-tetra- raethyluronium hexafluorophosphate) , TATU (2-(lH-7-azabenzotriazol-l-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate) and CDI (carbonyl diimidazole).
<136>
<137> <138> <139> <140> <14I> <142>
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20060099224 | 2006-10-12 | ||
| KR10-2006-0099224 | 2006-10-12 | ||
| KR1020070102700A KR101046846B1 (en) | 2006-10-12 | 2007-10-11 | Preparation of Peptides Using Solid Phase Synthesis |
| KR10-2007-0102700 | 2007-10-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008044890A1 true WO2008044890A1 (en) | 2008-04-17 |
Family
ID=39283046
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2007/004989 Ceased WO2008044890A1 (en) | 2006-10-12 | 2007-10-12 | A method for preparing peptides using by solid phase synthesis |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2008044890A1 (en) |
Cited By (14)
| 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 |
| CN102690329A (en) * | 2011-03-25 | 2012-09-26 | 杭州九源基因工程有限公司 | Purification production method of goserelin polypeptide |
| CN102746383A (en) * | 2011-04-21 | 2012-10-24 | 杭州九源基因工程有限公司 | Synthesis method of goserelin |
| CN103554229A (en) * | 2013-11-11 | 2014-02-05 | 宁波市三生药业有限公司 | Solid-phase synthesis method for Buserelin |
| CN103936849A (en) * | 2014-05-05 | 2014-07-23 | 承德医学院中药研究所 | Buserelin preparation method |
| US20150166602A1 (en) * | 2013-12-18 | 2015-06-18 | Scinopharm Taiwan, Ltd. | Process for the preparation of leuprolide and its pharmaceutically acceptable salts |
| CN106432427A (en) * | 2016-10-24 | 2017-02-22 | 合肥国肽生物科技有限公司 | Method for preparing gonadorelin acetate by virtue of specific microwave synthesis |
| US10087221B2 (en) | 2013-03-21 | 2018-10-02 | Sanofi-Aventis Deutschland Gmbh | Synthesis of hydantoin containing peptide products |
| CN108892711A (en) * | 2018-06-29 | 2018-11-27 | 江苏吉泰肽业科技有限公司 | A method of purifying Buserelin |
| CN109293736A (en) * | 2017-07-25 | 2019-02-01 | 齐鲁制药有限公司 | It is a kind of for synthesizing the dipeptides of Rayleigh class drug |
| US10450343B2 (en) | 2013-03-21 | 2019-10-22 | Sanofi-Aventis Deutschland Gmbh | Synthesis of cyclic imide containing peptide products |
| CN114805486A (en) * | 2022-06-02 | 2022-07-29 | 杭州思诺达医药科技有限责任公司 | Synthetic method of leuprorelin acetate impurity |
| CN116655745A (en) * | 2023-07-31 | 2023-08-29 | 杭州湃肽生化科技有限公司 | Application of intermediate in preparation of buserelin |
| CN116675741A (en) * | 2023-07-31 | 2023-09-01 | 杭州湃肽生化科技有限公司 | Application of intermediate in preparation of goserelin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5212288A (en) * | 1990-02-20 | 1993-05-18 | Syntex (U.S.A.) Inc. | Temporary minimal protection synthesis of serine-containing polypeptides |
| US5510460A (en) * | 1991-06-14 | 1996-04-23 | Zeneca Limited | Peptide process |
| US5602231A (en) * | 1991-06-14 | 1997-02-11 | Zeneca Limited | Process for making peptides |
| US6028172A (en) * | 1997-02-11 | 2000-02-22 | Mallinckrodt Inc. | Reactor and method for solid phase peptide synthesis |
| US6897289B1 (en) * | 1999-05-20 | 2005-05-24 | Lipotec, S.A. | Peptide synthesis procedure in solid phase |
-
2007
- 2007-10-12 WO PCT/KR2007/004989 patent/WO2008044890A1/en not_active Ceased
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5212288A (en) * | 1990-02-20 | 1993-05-18 | Syntex (U.S.A.) Inc. | Temporary minimal protection synthesis of serine-containing polypeptides |
| US5510460A (en) * | 1991-06-14 | 1996-04-23 | Zeneca Limited | Peptide process |
| US5602231A (en) * | 1991-06-14 | 1997-02-11 | Zeneca Limited | Process for making peptides |
| US6028172A (en) * | 1997-02-11 | 2000-02-22 | Mallinckrodt Inc. | Reactor and method for solid phase peptide synthesis |
| US6897289B1 (en) * | 1999-05-20 | 2005-05-24 | Lipotec, S.A. | Peptide synthesis procedure in solid phase |
Cited By (20)
| 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 |
| CN102690329A (en) * | 2011-03-25 | 2012-09-26 | 杭州九源基因工程有限公司 | Purification production method of goserelin polypeptide |
| CN102746383A (en) * | 2011-04-21 | 2012-10-24 | 杭州九源基因工程有限公司 | Synthesis method of goserelin |
| US10087221B2 (en) | 2013-03-21 | 2018-10-02 | Sanofi-Aventis Deutschland Gmbh | Synthesis of hydantoin containing peptide products |
| US10450343B2 (en) | 2013-03-21 | 2019-10-22 | Sanofi-Aventis Deutschland Gmbh | Synthesis of cyclic imide containing peptide products |
| CN103554229A (en) * | 2013-11-11 | 2014-02-05 | 宁波市三生药业有限公司 | Solid-phase synthesis method for Buserelin |
| CN103554229B (en) * | 2013-11-11 | 2016-01-06 | 宁波市三生药业有限公司 | The method of solid phase synthesis buserelin |
| US9150615B2 (en) * | 2013-12-18 | 2015-10-06 | Scinopharm Taiwan, Ltd. | Process for the preparation of leuprolide and its pharmaceutically acceptable salts |
| US20150166602A1 (en) * | 2013-12-18 | 2015-06-18 | Scinopharm Taiwan, Ltd. | Process for the preparation of leuprolide and its pharmaceutically acceptable salts |
| CN103936849A (en) * | 2014-05-05 | 2014-07-23 | 承德医学院中药研究所 | Buserelin preparation method |
| CN106432427A (en) * | 2016-10-24 | 2017-02-22 | 合肥国肽生物科技有限公司 | Method for preparing gonadorelin acetate by virtue of specific microwave synthesis |
| CN109293736A (en) * | 2017-07-25 | 2019-02-01 | 齐鲁制药有限公司 | It is a kind of for synthesizing the dipeptides of Rayleigh class drug |
| CN109293736B (en) * | 2017-07-25 | 2023-06-06 | 齐鲁制药有限公司 | Dipeptide for synthesizing relin medicines |
| CN108892711A (en) * | 2018-06-29 | 2018-11-27 | 江苏吉泰肽业科技有限公司 | A method of purifying Buserelin |
| CN114805486A (en) * | 2022-06-02 | 2022-07-29 | 杭州思诺达医药科技有限责任公司 | Synthetic method of leuprorelin acetate impurity |
| CN114805486B (en) * | 2022-06-02 | 2024-03-19 | 杭州思诺达医药科技有限责任公司 | Synthesis method of leuprorelin acetate impurity |
| CN116655745A (en) * | 2023-07-31 | 2023-08-29 | 杭州湃肽生化科技有限公司 | Application of intermediate in preparation of buserelin |
| CN116675741A (en) * | 2023-07-31 | 2023-09-01 | 杭州湃肽生化科技有限公司 | Application of intermediate in preparation of goserelin |
| CN116655745B (en) * | 2023-07-31 | 2023-10-13 | 杭州湃肽生化科技有限公司 | Application of intermediate in preparation of buserelin |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2008044890A1 (en) | A method for preparing peptides using by solid phase synthesis | |
| US11787836B2 (en) | Method for synthesizing peptide containing N-substituted amino acid | |
| KR101046846B1 (en) | Preparation of Peptides Using Solid Phase Synthesis | |
| CN101379075B (en) | Synthesis of glucagon-like peptide | |
| KR20130127443A (en) | Process for the manufacture of degarelix and its intermediates | |
| CN109096388A (en) | A kind of preparation method of Teriparatide | |
| CN108779150B (en) | Preparation method of degarelix and intermediate thereof | |
| EP1179537B1 (en) | Solid phase peptide synthesis method | |
| CA2915439C (en) | Peptide-resin conjugate and use thereof | |
| KR20210102362A (en) | Improved process for making plecanatide | |
| Ruczyński et al. | Problem of aspartimide formation in Fmoc‐based solid‐phase peptide synthesis using Dmab group to protect side chain of aspartic acid | |
| CN105884865A (en) | Synthesis method of goserelin | |
| US9150615B2 (en) | Process for the preparation of leuprolide and its pharmaceutically acceptable salts | |
| KR20190001969A (en) | Process for the Preparation of Triptorelin | |
| KR100591832B1 (en) | Synthesis Process of Peptides Having Tryptophan Residues | |
| CN114945580B (en) | Method for synthesizing south Ji Botai | |
| WO2020202182A1 (en) | Process for the preparation of abaloparatide | |
| CN114805480A (en) | Preparation method of octreotide | |
| CN111233980A (en) | Method for synthesizing goserelin by fragment method | |
| WO2019227612A1 (en) | Method for synthesizing head-to-tail cyclic peptide containing proline | |
| WO2024261319A1 (en) | Methods of chemical synthesis of peptides | |
| CN115433266A (en) | Solid-phase synthesis method of teriparatide | |
| CN114057829A (en) | Solid-phase synthesis method of N-methylated polypeptide | |
| GB2631322A (en) | Methods of chemical synthesis of peptides | |
| CN108148113A (en) | A kind of solid phase synthesis process of nmda receptor adjusting control agent tetrapeptide derivative |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07833300 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 07833300 Country of ref document: EP Kind code of ref document: A1 |