IE66524B1 - A process for the preparation of peptides by solid-phase synthesis - Google Patents
A process for the preparation of peptides by solid-phase synthesisInfo
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- IE66524B1 IE66524B1 IE305191A IE305191A IE66524B1 IE 66524 B1 IE66524 B1 IE 66524B1 IE 305191 A IE305191 A IE 305191A IE 305191 A IE305191 A IE 305191A IE 66524 B1 IE66524 B1 IE 66524B1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- 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
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- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A process for the production of peptides of the formula (X)n-A-NH2 in which X is a natural or unnatural amino acid, azaamino acid or imino acid, n is an integer from 1 to 50, preferably 1 to 30, and A is an azaamino acid, and the physiologically tolerated salts thereof is described and is characterised in that a spacer is converted into a form which can be acylated, and the latter is reacted with a suitable formic acid derivative and subsequently with an appropriate amino acid hydrazide, where appropriate the protective group is converted into a protective group which is base-labile or labile to weak acids, the spacer obtained in this way is coupled to a resin, the required peptide is built up stepwise from the C-terminal end, and subsequently the peptide is cleaved off the resin and, where appropriate, converted into its physiologically tolerated salts.
Description
Description
A process for the preparation of peptides by solid-phase synthesis
The invention relates to a process for the preparation of peptides with C-terminal asa-amino amide by solid-phase synthesis.
The object of the invention Is to develop a low-racemisation process for the preparation of peptides with C10 terminal aza-amino amides by solid-phase synthesis.
This object Is achieved according to the Invention by the process for the preparation of peptides of the formula 1 (X)„ - A - NHa (I) in which
X is a natural or unnatural amino acid, aza-amino acid or imino acid, n is an Integer from 1 to 50, preferably 1 to 30, and
A is an aza-amino acid, and the physiologically tolerated salts thereof, which comprises converting a spacer into a form capable of acylation, reacting the latter with a suitable formic acid derivative and subsequently with axx appropriate amino hydrazide, where appropriate converting the protective group into a protective group which Is baselabile or labile to weak acids, coupling the spacer obtained in this way to a resin, synthesizing the required peptide stepwise from the C-terminal end, subsequently cleaving the peptide off the resin and, where appropriate, converting it into physiologically tolerated salts thereof.
Natural or unnatural amino acids can, If chiral, b© in the D or L fora. c-Amino acids are preferred.
Examples which may be mentioned ares Aad, Abu, -yAtau, ABz, 2ABz, eAca, Ach, Acp, Adpd, Ahb, Aib, 0Aib, Ala, jSAla, AAla, Alg, All, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Axe, Azi, Bai, Bph, Can, Cit, Cys, (Cys)2, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Pel, Gin, Glu, Gly, Guv, hAla, hAxg, hCys, hGln, hGlu, His, hlle, hLeu, hLys, hMet, hPhe, hPro, hSer, hThr, hTrp, hTyx, Hyl, Hyp, 3Hvp, lie, Ise, Iva, Kvn, Lant, Lea, Leu, Lsg, Lys, jBLys, ALys, Met, Min, Min, nAxg, Hie, Nva, Oly, Ora, Paa, Pec, Pea, Phe, Phg, Pic, Pro, nPro, Pse, Pya, Pyx, Psa, Qin, Ros, Sax, Sec, Sen, Sex, Thi, £Th±, Thr, Thy, Thx, Tia, Tie, Tly, Trp, Txta, Tyr, Val, Mai, Tbg, Mpg, Cha, Chg, Thia (cf., for example, Houben-Weyl, Methoden der organischen Chemie (Methods of organic chemistry). Volume XV/1 and 2, Stuttgart, 1974).
Aza-amino acids are derived from natural or unnatura* amino acids with the central -CHR- or ~GH2“ unit being replaced by -MR- or -NH- respectively. Examples which may be mentioned are azaglycine, azavaline, azaleucine, azaisoleucine and azaphenylalanine.
By an imino acid are meant in general natural or unnatural amino acids whose amino group is monosubstituted. Particular mention may be mad® in this connection of compounds which are substituted by Gj-Cg-alkyl, which in turn is optionally mono- or diunsatur&ted and can be substituted by up to 3 identical or different radicals from the series comprising mercapto; hydroxyl; Cj,-C7” alkoxy; carbamoyl; Ci-Cg-alkanoyloxy; carboxyl; Cj-C7alkoxycaxhonyl; P; Cl; Bx; 1; amino; amidino* which can optionally be substituted by one, two or three Ci-Cg-alkyl radicals; guanidino which can optionally be substituted by one or two benzyloxycarbonyl radicals or by one, two, three or four Cx-Cg-alkyl radicals; C,-C7-alkylamino; diCx-Cy-alkylamino; Cx-Cg-alkoxycarbonylamino; Cy-C^-ax&lkoxycarbonyl; G7-C13-aralkoxycarbonylamino; phen.yl-Cx-C,%alkoxy; 9-fluorenylmethoxycarbonylamino; Ci-Ce-alkylsulfonyl; Cx-Cs-alkylsulfinyl; Ci-Cg-alkylthio; hydroxyamino;
hydroxyimino; sulfamoyl.
Also suitable are heterocycles from the following groups pyrrolidine-2-carboxylic acid;: piperidine-2-carboxylic acid; 1,2,3,4-tetrahydraisocruinoline-3~c&rho3cylic acid; decahydroisoquinollne-3-carhoxylic acid; octahydroindole2- carboxylic acid; decahydroquixioline-2-carboacylic acid; octahydrocyclopenta[b]pyrrole-2-carboxylie acid; 2-azabicyclo[2.2.2]octane-3-carboxylic acid; 2-azabicyclo[2.2.1]heptane-3-carboxylic acid; 2-azablcyclo[3»i.O]hexane-3-carboxylic acid; 2-azaspixo[4.4]nonane-3-carboxylic acid; 2-azaspiro[4.5]decane-3-carboxylic acid; spiro[(bicyclo[2.2.1]heptane)-2,3-pyrroIidine-S-carboxy lie acid; spiro[(bicyclo[2.2.2]oetane)~2,3-pjrrrolidine-5-carboxylic acid; 2-azatricyclo[4.3.0.16,a]decane3- carboxylic acid; decahydrocyclohepta[b]pyrrole-2carboxylic acid; octahydrocyclopenta[c]pyrrol~ 2 -carboxylic acid; octahydroisoindole-l-carboxylic acid? 2,3,3a,4,6a-hexahydrocyclopenta[b]pyrrole-2-carboxylic acid; 2,3,3a,4,5,7a-hexahydroindole-2-carboxylic acid; tetrahydrothiazole~4-carboxylic acid; isoxazolidine-3carboxylic acid; pyrazolidine-3-carboxylic acid; hydroxyproline-2-carboxylic acid; all of which can optionally be substituted;
By..salts of .compounds of the formula! are-meant, in particular, pharmaceutically utilisable or non-toxic salts. Particularly suitable are alkali metal or alkaline earth metal salts, salts with physiologically tolerated amines and salts with inorganic or organic acids, such as, for example, HCl, HBr, H,SO>, HaPO*, maleic acid, fumaric acid,, citric acid, tartaric acid and acetic acid.
The process is advantageously carried out in such a way that a compound of the formula II pi r·
ΓΪ o.
£ Y4 (II) in which
Y1, Y2, Y3, Ϋ and Y5 are hydrogen, Ci-C^-alkyl, Ci-C^-alkoxy or -Q-(CH2) a~COOH, - (CH2) jj-COOH or -NH-CO- (CH2) a-COOH, where the radicals can he identical or different, but at least one radical is «O~(CH2)a-COQH, ~(CH2)a-C00H or -NH-CO-iCHaJa-COOH, n is an integer from 1 to 6, preferably 1 to 3, and
R1 is hydrogen or C,“C5-alko3^y-C6”C12-aryl s preferably 4~ methoxyphenyl, is reacted with a silylating reagent, for example tert, butyldimethylsilyl chloride, tert. butylphenylsilyl chloride, trimethylchlorosilane, especially trimethylchlorosilane, in a solvent suitable for this purpose, Csuch as, for example, THF, acetonitrile, methylene chloride, dimethylformamide or mixtures thereof, and subsequently the silylated compound Is converted with a chlorofoxmic acid derivative, especially the substituted ester derivatives, into compounds of the formula III
in which
R1, Y1, Y2, Y3, Y* and Y5 are as defined above, and
R2 is a C6=-C12'-aryl radical which is substituted by electron-attracting substituents, preferably nitro and halogen, for example F or Cl, the compounds of the formula III obtained in this way axe reacted with an amino hydraside of the formula IV
R3 · X - CO - NH - NH - R“‘ (IV) in which
X is a natural or unnatural amino acid or imino acid and Is as defined above,
R3 is a protective group which is base-labile or labile to weak acids or hydrogenation, such as, for example, a urethane protective group (see, for example, Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14 to 23), and
R4 is Ci-Ca-alkyl, C3-Cg-cycloalkyl, Cs-C12-aryl, Cs-C12“ aryl-Cj-Ca-alkyl, heteroaryl or heteroaryl-Gj-Cg-alkyl or hydrogen, in a solvent in which the compounds of the formula III and IV are soluble, such as, for example, DMF, to give the compounds of the formula V
in which R1, R3, R4 and Y1, T2, Y3, Y4 and Ύ3 have the abovementioned meanings, if R3 is a protective group which is labile to hydrogenation, preferably bensyloxycarbonyl, this protective group is removed by hydrogenation on a Rd catalyst and, before the subsequent reaction, converted into a base-labile urethane protective group, preferably Fmoc, or a urethane protective group which is labile to weak acids, preferably Bpoc, subsequently the compound of the formula V in which R1, R*, y1, Y2, Y3, Y4 and y5 have the abovementioned meanings, and R3 is a urethane protective group which is labile to bases or weak acids, is coupled with the coupling reagents customary in peptide chemistry via the -O-(CH2)n-COOH, - (CH2) n—COOH or -NH-CO- (CH2) a-GOOH group to a resin, the protective group R3 Is eliminated, natural or unnatural amino, imino or aza-amino acids which have been temporarily protected by amino-protective groups which are base-labile or labile to weak acids and which are optionally in the form of their activated derivatives are coupled on stepwise, and, after synthesis is complete, the peptides of the formula I are liberated from the resin by treatment with a moderately strong acid, with elimination again, simultaneously or by suitable measures subsequent thereto, of temporarily introduced side-chain protective groups.
The compounds of the formula IV are prepared by reacting the natural or unnatural amino acids or imino acid with the appropriate hydrazines by coupling methods customary In peptide chemistry.
Alkyl can be straight-chain or branched. A corresponding statement applies to radicals derived therefrom, such as, for example, alkoxy, alkylthio, alkylamino, dialkylamino and alkanoyl. Alkyl is. In particular, C,-C4“alkyl.
Cycloalkyl also means alkyl-substituted radicals such as, for example, 4-methylcyclohexyl or 2,3-dimethylcyclopentyl.
Halogen Is fluorine, chlorine, bromine or Iodine, ©specially fluorine or chlorine.
C6~G,2“ aryl is, for example, phenyl or naphthyl, preferably phenyl. Hetero&ryl is the radical of a 5- to 7membered monocyclic or 8- to 10-membered bicyclic aromatic ring system which, can be benso-fused and which can contain as hetero elements one, tvo^ three or four different radicals from the group comprising N, Q, S, NO, SO and S02 and which is substituted by 1 to S hydroxyl or by one, two or three identical or different radicals from the series comprising F, Cl, Br, I; hydroxyl, mono-, dior trihydroxy-C^—C^-alkyl; trifluorcmethyl; formyl; carboxamido; mono- or di-C^^-alkylaminocaxbonyl; nitro; .C-j-C^alkary; C^-CLalkyl; C1-C7-alkoxycarbonyl; amino; Cj-Cy-alkylamino ; diIQ C1-C7-alkylamino? carboxyl; carboxymethoxy; amino-Ci.-C,alkyl; Gi-Cy-alkylamino-Ci-Cy-alkvl; di-C1-C7-alkylaminoCi-C7~alkyX; Cj-Cy-alkoxycarbonylmethoxy; carbamoyl; sulfamoyl; C1-C7-alkoxysulfonyl; C1-Cn-alkylsulfonyl; sulfo-C^-Cg-alkyl; guanidino-C^-Cg-alkyl and C^-Cg-alkoxycarbonyl and/or mono-, di- or trisubstituted by oxo. Particular mention may be made of; furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, isoindolyl, indazolyl, phthalazinyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, cinnolinyl, ^-carbolinyl or a faenzo-fused derivative of these radicals.
Xf necessary to prevent side reactions or for the synthesis of specific peptides, the functional groups in the side chain of amino,, aza-amino and imino acids axe additionally protected by suitable protective groups (see, for example, T.W. Greene, Protective Groups in Organic Synthesis, New York, John Wiley & Sons, 1981; Hubbuch, Kontakte (Merck) 1979, No. 3, pages 14-23; Bixllesbach, Kontakte (Merck) 1980, No. 1, pages 23-35), employing primarily Arg(Tos), Arg(Mts), Arg(Mtr), Arg(Pmc), Asp(OBzl), Asp(OtBu), Cys(4-Me3zl), Cys(Acm), Cys(StBu), Glu(OBzl), Glu(OtBu), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(Cl-2), Lys(Boc), M©t(O), Ser(Bzl), Ser(tBu), Thr(Bzl), Thr(tBu). It is also possible for the functional groups in the side chain to be glycosylated as described, for example, in EP-A 2S3 521 (HOE 8S/F 253).
The resins used as support material are commercially available or prepared by the user, such as, for example, alkoxybensvl alcohol-resins, aminomethyl-resins or benshydrylaml.no»·res ins. Aminomethyl-, benzhydrylamino(BHA) and methylbenshydrylamino-resins (MBHA) are preferred. The loading is determined by amino-acid analysis and/or elemental analysis.
By suitable spacers are meant, for example, the spacers described in Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101-117 (Karger, Basel 1981)? EP-A 264 802 (HOE 86/F 259), EP-A 287 882 (HOE 87/F 101) and EP-A 322 348 (HOE 87/E 386K), and derivatives derived therefrom, such as, for example, those whose protective group has been eliminated. 4-Carboxylatopropoxy-4fmethoxybenzhydrylamine and 5-carboxylatoethyl-2,4-dimethoxy-41-methoxybenzhydrylamine are preferred.
It is possible to use as coupling reagent for the spacer of the formula V and the other amino acid derivatives all the possible activating reagents used in peptide synthesis, see, for example, Houben-Tieyl, Methoden der Organischen Chemie, Volume XV/2, Stuttgart 1974, but especially carbodiimides such as, for example, N,NPdicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide or N-ethyl-Be- (3-dlmethylaminopropyl)carbodlimide. This coupling can be carried out directly by addition of amino acid derivative with the activating reagent and, where appropriate, an additive suppressing racemization, such as, for example, 4-dimethylaminopyridine, 1-hydroxybenzotriazole (HOBt) (^. Konig, R. Geiger, Chem. Ber. 103 (1970) 788 - 798) or 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine (HQObt) (W. Konig, R. geiger, Chem. Ber. 103 (1970) 2034 - 2040) to the resin, or else the preactivation of the amino acid derivative as symmetrical anhydride or HOBt or HOObt ester can take place separately and the solution of the activated species in a suitable solvent can be added to the peptide-resin capable of coupling.
The coupling and activation of the spacer of the formula V and of the amino acid derivative with one of the abovementioned activating reagents can be carried out in dimethylformamide or methylene chloride or a mixture of the two. The activated amino acid derivative is normally employed in a 1.5- to 4-fold excess. In cases where incomplete coupling occurs, the coupling reaction is repeated without previously carrying out the deblocking of the cs-amino group of the peptide-resin which is necessary for the coupling of th© next amino acid in sequence.
The success of the coupling reaction can be checked using the ninhydrin reaction as described, for example, by E. Kaiser et al. (Anal. Biochem. 34 (1970) 595). The synthesis can also be carried out automatically, for example with a model 430A peptide synthesiser from Applied Biosystems, it being possible to use either the synthesis programs provided by the equipment manufacturer or else those drawn up by the user himself. The latter are particularly employed when using amino acid derivatives protected by the Fmoc group.
The peptide amides are cleaved off the resin by treatment with moderately strong acids customarily used In peptide synthesis (for example trifluoroacetic acid), adding as cation traps substances such as phenol, cresol, thiocresol, anisole, thioanisole, ethanedithiol, dimethyl sulfide, ©thyl methyl sulfide or similar cation traps customary in solid-phase synthesis, singly or a mixture of two or more of these auxiliaries. In this connection the trifluoroacetic acid can also be used diluted by a suitable solvent such as,· for example, methylene chloride. When the spacer is cleaved off the resin there Is simultaneous elimination of the side-chain protective groups.
The crude peptides obtained In this way are purified by chromatography on *Sephadex, ion exchanger resins or by
HPLC.
A process of solid-phase synthesis for the preparation of Ac-D-Nal (2) -p-CX-D-Phe-D-Trp-Ser-Tyr-D-Ser (2 and pGlu-His-Trp-Ser~Tyr-D-Ser(tBu)Leu-Arg-Pro-Azagly-NH2 (Zoladex) is preferred.
List of
BSA
Cha
Chg
DCC
DIG
DMAP
Fmoc
HOBt
HOObt
Nal
IMpg
Pmc
Tfag
THP
Thia abbreviations usech bis trimethyls ily1acet amide eyelohexy1alanine eyelohexylglycine dicyclohexvlc&rhodi imide di i s opropy1carbodi imide dimethylaminopvridine
9-fluorenylmethoxycarbonyl
1- hydroxybenzotriazole
3-hydroxy-4-oxo-3,4-dihydro-l, 2,3-benzotriazine naphthylalanine neopentylglycine
2,2,5,7,8-pentamethylchroman-6-sulfonyl tert.-butylglycine tetrahydrofuran
2- thienylalanine
The examples which follow serve to illustrate the present invention without intending to restrict it thereto.
Example 1
a) S-Carhoxylatoethyl-2,4-dimethoxy-4 p -methoxybenzhydryl amine
17.5 g of 5-carboxylatoethyl-2f4-dimethoxy-4-methoxybenzophenone oxime were dissolved in 450 ml of a 1:1 mixture of ethanol and DMF, and 2 ml of concentrated NH3 were added. After addition of the Pt/C catalyst, hydrogenation was carried out under atmospheric pressure for 5 days. After completion of the reaction, the catalyst was filtered off with suction, the filtrate was concentrated, and the product was precipitated with ether. It was employed as such without further purification.
lb) N~ (p-Nitrophenyloxycarbonyl) -5-carboxylatoethyl-2 g 45 dimethoxy-4f -methoxybenzhydrylamine g of the title compound from Example la) were introduced Into 100 ml of a 4:1 THE/DMF mixture and, at room temperature, 2.1 ecuiv. of bistrimethylsilylacetamide (BSA) were added. The suspension became completely clear in a short time, and the clear solution was then stirred for 2 hours. 3 g of nitrophenyl chloroformat® were then added and the mixture was stirred for a further hour. After completion of the reaction, the solvent was removed under high vacuum. The residue was mixed with
300 ml of water, and the resulting oil was extracted with ethyl acetate. The ethyl acetate phase was washed with a 1 N KHSO* solution and water. The organic phase was dried over MgSO* and evaporated to dryness. The residue (12 g) is characterized by NMR, IR and MS.
N- (p-Nitrophenyloxycarbonyl)-5-carboxylato®thyl-2, 4dimethoxy-4 ' -methoxybenzhydrylamine was then reacted with amino hydrazides to give the suitable substituted anchors.
lc) 3enzyloxycarbonyl-4-prolylazaglycine (5-carboxylato25 ethyl-2,4-dimethoxy-4e-methoxybenzhydryl) amide.
3.27 g of benzyloxycarbonyl-prolyl hydrazide hydrochloride and 6.94 g of the title compound from Example lb) ..were dissolved in 40 ml of dimethylformamide (DMF), and 3 eguiv. of N-ethylmorpholine and a catalytic amount of dimethyl aminopyridine (DM&P) were added. Reaction was allowed to take place for 16 hours. After completion of the reaction, the mixture was evaporated to dryness. The residue was taken up in ethyl acetate/butanol, and the organic phase was washed with saturated NaHCO3 solution,
-13 1 N KHSOa solution and water- The organic phase was dried over MgSO* and, after filtration, evaporated to dryness. It was possible to recrystallize the residue from pure ethyl acetate. 5.6 g of the title compound were obtained.
FAB-MS: 541 (M+Li+)
IR: CO 1695 cm'1XH-NMR (DMSQ)s S = 3-7 s (6H, OCH3) ppm ld) 9-Fluorenylmethoxyearbonyl-I.~pr0lylaz&glycine (5carboxylatoethyl-2,4~dimethoxy-4 'methoxybenshydryl) amide
26.5 g of the title compound from Example Ic) were dissolved in 300 ml of methanol, and 2 g of Pd/C catalyst were added. The hydrogenation was complete after one hour. The catalyst was filtered off, and the filtrate was evaporated to dryness. The residue (17.5 g) was, without further purification, taken up in a mixture of 80 ml of water and 80 ml of dioxane and mixed with 8 g of sodium bicarbonate and 17 g of N-(9-fluorenylmethoxycarbonyl~ ojcy) succinimide (Fmoc-ONSu). Reaction was allowed to take place for one day. After the reaction was complete, the mixture was filtered through a clarifying filter. The filtrate was adjusted to pH 6 with 2 N H2SO- and evaporated In vacuo to a volume of 80 ml. The mixture was diluted with 100 ml of wafer and extracted with a mixture of ethyl acetate and n-butanol (8.5:1.5). The organic phase was washed with 50 % saturated NaCl solution and then evaporated to dryness. The residue was filtered through 500 g of silica gel with ethyl acetate. 20 g of the title compound were obtained.
FAB-MS ϊ 729 (M+Li+)
IR: CO 1695 cm1 le) Coupling of the title compound from Example Id) to a polystyrene resin
1.0 g of eminomethylpoiystyrene resin (loading 1.07 mmol) and 1.2 g of the title compound from Example Id) were suspended in 10 ml of dimethylformamide, and 2IS mg of Xhydroxybenzotriazole (HOBt) and 0.75 ml of diisopropy1carbodiimide (DIG) were added. Reaction was allowed to take place overnight until the ninhydrin test indicated complete reaction. The resin was filtered off and washed with dimethylformamide and methylene chloride and thoroughly dried in vacuo. The loading of the resin with proline was 0.51 mmol/g.
If) Ac-D-Nal (2) -p~Cl~D-Phe-D-Trp-Ser-Tyr-D-Ser (β-L-Rha) Leu~Arg~?ro~Azagly-NH2
The 9-fluorenylmefho3{ycarbonyl-Se-amino-“protective group of the compound from Example le) was eliminated with a 20 % strength piperidine/dimethylformamide solution (2x3 min, 2x8 ml). The resin was then washed with Nmethylpyrrolidinone (5x10 ml) and the peptide was synthesized on the resin (785 mg of resin from Example Ic)), carrying out the following steps in eyeless elimination of the Fmoc protective group with 20 % piperidine in DMF washing of the resin with DMF/N-methylpyrrolidinone coupling on of the Fmoc-amino acid with in situ activation as HOBt ester using diisopropylcarbodiimide as activating reagent (1.5 mmol of amino acid, 2.25 mmol of HOBt, 1.6 mmol of diisopropylcarbodiimide)
If th© coupling was incomplete (Kaiser test), the coupling step was· repeated. The last amino acid employed was Fmoc-D-Nal(2)-OH. The ^-terminal acetyl group was introduced by reaction with acetic anhydride.
After completion of the solid-phase synthesis, the resin was washed (DMF, CH2C12) and thoroughly dried. 1,,35 g of substituted resin were obtained.
The dried resin was suspended at room temperature in 0.75 ml of ethanedithiol. After 15 minutes, 7.5 ml of trifluoroacetic acid were added and the suspension was stirred for 1.5 hours. After this time, the resin was filtered off and thoroughly washed with 80 % strength trifluoroacetic acid. The filtrate was evaporated in vacuo and taken up in 30 ml of water. NaHCO3 was added to adjust to pH 6-7, and the peptide was extracted by shaking with n-pentanol (4x30 ml). The n-pentanol phase was evaporated and taken up in 10 ml of methanol/Hz0 (9s 1), and 0.5 g of K2C03 was added. The mixture was stirred for 30 minutes and filtered, and the filtrate was concentrated. The residue was taken up In 100 ml of npentanol, and the organic phase was washed with water.
The organic phase was dried with MgSOZl and filtered and then evaporated. 740 mg of crude product were obtained. Chromatography on “'’Sephadex G 25 (1 M acetic acid) and on silica gel resulted in 185 mg of the pure title compound. FAB-MSs 1531 (M+H+)
IS
Claims (6)
1. A process for the preparation of peptides of the formula X (X)„-A-NH a (I) in which Z is a natural or unnatural amino acid, aza-eaaino acid or imino acid, n Is an integer from 1 to 50, preferably 1 to 30, and A Is an aza-amino acid, and the physiologically tolerated salts thereof, which comprises converting a spacer info a form capable of acylation, reacting the latter with a suitable formic acid derivative and subsequently with an appropriate amino hydrazide, where appropriate converting the protective group into a protective group which is baselabile or labile to weak acids, coupling the spacer obtained in this way to a resin, synthesizing the required peptide stepwise from the C-terminal end, subsequently cleaving the peptide off the resin and, where appropriate, converting it into physiologically tolerated salts thereof.
2. The process as claimed in claim 1, wherein a compound of the formula II R-—c: Y 4 Y 5 (IX) In which Y 1 , Y 2 , Y 3 , Y* and Y s are hydrogen, C,-C*-alkyl, Cx-C^-alkoxy or -O-(CH,) a -COOH, -(CH,) n -CGGH or -NH-C0-(CH 2 ) a -C00H, where the radicals can be identical or different, but at least one radical is -O-(CH 2 ) n -COOH, ~(CH 2 ) n ~COOH or -NH~CO-(CH 2 ) n ~COOH, η is an integer from 1 to δ { , preferably 1 to 3, and 5 R 1 is hydrogen or C^Cg-alkoxy-Cg-C^-axyl, is reacted with a silylating reagent in a solvent suitable for this purpose, and subsequently the silylated compound is converted with a ehloroformic acid derivative into compounds of the formula XXI in which R 1 , Y 1 , Y 2 , Y 3 , Y 4 and Y 5 are as defined above, and R 2 is a C 6 -C 12 -aryl radical which is substituted by electron-attracting substituents t preferably nitro 15 · and halogen, the compounds of the formula III obtained in this way are reacted with an amino hydrazide of the formula IV R 3 - X - CO - NH · NH - R* (IV) in which 20 X is a natural or unnatural amino acid or imino acid and is as defined above, R 3 Is a protective group which is base-labile or labile to weak acids or hydrogenation, and R 6 is Ci-Ce-alkyl, C 3 -C 9 -cycloalkyl, Ce-C^-aryl, Cg-Cj,aryl-Cji-Ce-alkyl, heteroaryl or heteroaryl-Cj-Ce-alkyl or hydrogen, in a suitable solvent to give th® compounds of the formula V in which R 1 , R 3 , R 4 and Y 1 , Y 2 , I 3 , Y 4 and Y 5 have the abovementioned meanings, if R 3 is a protective group labile to hydrogenation this protective group is removed by hydrogenation on a Pd catalyst and, before the subsequent reaction, converted into a urethane protective group which is base-labile or labile to weak acids, subsequently the compound of the formula V in which R 1 , R 4 Y 1 , Y 2 , Y 3 , Y 4 and Y 5 have the abovementioned meanings, and R 3 is a urethane protective group which Is labile to bases and weak acids, is coupled with the coupling reagents customary in peptide chemistry via the -O- (CH 2 ) a -COOH, - (CH 2 ) n -COOH or -NH-CO-(CH 2 ) n -COOH group to a resin, the protective group R 3 is eliminated, natural or unnatural amino, imino or aza-amino acids which have been temporarily protected by amino-protective groups which are base-labile or labile to weak acids and which are optionally in the form of their activated derivatives are coupled on stepwise, and, after the synthesis is complete, the peptides of the formula I ar® liberated from the resin by treatment with a moderately strong acid, with elimination again, simultaneously or by suitable measures subsequent thereto, of temporarily Introduced side-chain protective groups.......
3. The process as claimed in either of claims 1 and 2, wherein Ac-D-Nal(2)-p-Cl-D-Phe-D~Trp-Ser-Tyr-D-Ser(c-LRha)-Leu-Arg-Pro-Azagly-NH 2 is prepared.
4. The process as claimed in either of claims 1 and 2, - 19 wherein pGlu-His-Trp-Ser-Tyr-D-Ser (tBu,) -Lau-Arg-Pro Azagly-MH 2 is prepared.
5. A process hereinbefore as claimed in claim 1, substantially as described and exemplified.
6. A peptide of the formula I given and defined in · claim 1 or a physiologically tolerated salt thereof, whenever prepared by a process claimed in a preceding claim.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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DE4027394 | 1990-08-30 |
Publications (2)
Publication Number | Publication Date |
---|---|
IE913051A1 IE913051A1 (en) | 1992-03-11 |
IE66524B1 true IE66524B1 (en) | 1996-01-10 |
Family
ID=6413202
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
IE305191A IE66524B1 (en) | 1990-08-30 | 1991-08-29 | A process for the preparation of peptides by solid-phase synthesis |
Country Status (22)
Country | Link |
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EP (1) | EP0475184B1 (en) |
JP (1) | JP3177269B2 (en) |
KR (1) | KR100203548B1 (en) |
AU (1) | AU646488B2 (en) |
CA (1) | CA2050216C (en) |
CZ (1) | CZ282881B6 (en) |
DE (1) | DE59104538D1 (en) |
DK (1) | DK0475184T3 (en) |
ES (1) | ES2069148T3 (en) |
FI (1) | FI102380B (en) |
GR (1) | GR3015359T3 (en) |
HU (1) | HU208838B (en) |
IE (1) | IE66524B1 (en) |
IL (1) | IL99338A (en) |
NO (1) | NO300216B1 (en) |
NZ (1) | NZ239575A (en) |
PL (1) | PL167504B1 (en) |
PT (1) | PT98813B (en) |
RU (1) | RU2036200C1 (en) |
SK (1) | SK280319B6 (en) |
TW (1) | TW295589B (en) |
ZA (1) | ZA916848B (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
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GB9112825D0 (en) * | 1991-06-14 | 1991-07-31 | Ici Plc | Process for making peptides |
GB9727123D0 (en) * | 1997-12-22 | 1998-02-25 | Int Centre Genetic Eng & Bio | Synthesis of diamines |
ES2154590B1 (en) * | 1999-05-20 | 2001-11-01 | Lipotec Sa | SOLID PHASE SYNTHESIS PROCEDURE |
CA2594477C (en) | 2005-01-21 | 2016-07-12 | Astex Therapeutics Limited | Pharmaceutical compounds |
EP2073803B1 (en) | 2006-10-12 | 2018-09-19 | Astex Therapeutics Limited | Pharmaceutical combinations |
JP5528807B2 (en) | 2006-10-12 | 2014-06-25 | アステックス、セラピューティックス、リミテッド | Compound drug |
AU2014234400B2 (en) | 2013-03-21 | 2017-11-16 | Sanofi-Aventis Deutschland Gmbh | Synthesis of hydantoin containing peptide products |
CN105102427B (en) | 2013-03-21 | 2018-09-07 | 赛诺菲-安万特德国有限公司 | The synthesis of peptide prod containing cyclic imide |
Family Cites Families (1)
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EP0322348B1 (en) * | 1987-12-22 | 1994-02-02 | Hoechst Aktiengesellschaft | Acidically non-stable anchoring groups for solid-phase peptide amide synthesis |
-
1991
- 1991-08-17 TW TW080106522A patent/TW295589B/zh active
- 1991-08-27 DE DE59104538T patent/DE59104538D1/en not_active Expired - Lifetime
- 1991-08-27 DK DK91114332.9T patent/DK0475184T3/en active
- 1991-08-27 EP EP91114332A patent/EP0475184B1/en not_active Expired - Lifetime
- 1991-08-27 ES ES91114332T patent/ES2069148T3/en not_active Expired - Lifetime
- 1991-08-28 IL IL9933891A patent/IL99338A/en not_active IP Right Cessation
- 1991-08-28 NZ NZ239575A patent/NZ239575A/en not_active IP Right Cessation
- 1991-08-28 FI FI914064A patent/FI102380B/en not_active IP Right Cessation
- 1991-08-29 RU SU915001313A patent/RU2036200C1/en active
- 1991-08-29 PT PT98813A patent/PT98813B/en not_active IP Right Cessation
- 1991-08-29 CZ CS912666A patent/CZ282881B6/en not_active IP Right Cessation
- 1991-08-29 KR KR1019910014992A patent/KR100203548B1/en not_active IP Right Cessation
- 1991-08-29 SK SK2666-91A patent/SK280319B6/en not_active IP Right Cessation
- 1991-08-29 NO NO913396A patent/NO300216B1/en not_active IP Right Cessation
- 1991-08-29 AU AU83402/91A patent/AU646488B2/en not_active Expired
- 1991-08-29 JP JP21852591A patent/JP3177269B2/en not_active Expired - Lifetime
- 1991-08-29 PL PL91291561A patent/PL167504B1/en unknown
- 1991-08-29 IE IE305191A patent/IE66524B1/en not_active IP Right Cessation
- 1991-08-29 CA CA002050216A patent/CA2050216C/en not_active Expired - Lifetime
- 1991-08-29 ZA ZA916848A patent/ZA916848B/en unknown
- 1991-08-30 HU HU912825A patent/HU208838B/en unknown
-
1995
- 1995-03-10 GR GR950400517T patent/GR3015359T3/en unknown
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