CA1340421C - Sythesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase method - Google Patents
Sythesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase methodInfo
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- CA1340421C CA1340421C CA000549680A CA549680A CA1340421C CA 1340421 C CA1340421 C CA 1340421C CA 000549680 A CA000549680 A CA 000549680A CA 549680 A CA549680 A CA 549680A CA 1340421 C CA1340421 C CA 1340421C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
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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
- C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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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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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
- Pyrane Compounds (AREA)
Abstract
The invention relates to compounds of the formula I
Description
~40 ~2~L
Specification:
The synthesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase method s The introduction of an aminoalkylamide into the C-terminal end of a biologically active peptide has in some cases had beneficial effects on the metabolic stability and activity (EP-A 179 332). The preparation of the peptides modified in this way has made use of the classical coupling of frasments in solution.
In the solid-phase synthesis of peptides (see Patchornik, Cohen in Perspectives in Peptide Chemistry, pages 118 - 1Z8 (Karger, Basle 1981)) the reactive chains are often not grafted directly onto the synthetic resin material, but are bonded to the carrier material by what are called spacers or links. The literature (for example Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101 - 117 (Karger, Basle 1981)) discloses, for example, reagents for introducing such spacers (called "linkage agents") which have the formulae YI, VII and VIII.
HOCH2 -~CH2- CH2- C02H HOCH~ OCH2 - CO2H HOCH2~ C02H
(VI) (VII) (VIII) New linkage agents which allow direct construction, by solid phase synthesis, of peptides modified by C-terminal amino-alkylamide or hydrazide have been found.
Thus the present invention relates to compounds of the tor-mula I
yl y2 ( I ) A- [B]p-NH- [X]m-NH-CO-O-CH2 _~_ W-C02-V
y3--y4 *
in which 1 3 ~ O ~ 2 1 A denotes hydrogen or an amino protective group which is labile to bases or labile to weak acids, B represents identical or different amino acid resi-dues, X denotes (C1-C12)-alkylene or (C6-C10)-arYl-(c1-c12) alkylene, y1, y2~ y3 and Y4 are identical or different and denote hydrogen, methyl, methoxy or nitro, at Least one of these radicals denoting hydrogen, V denotes hydrogen or a carboxyl protective group, ~ denotes -tCH2]n- or ~0-[CH2]n-, m is 0 or 1, n is an integer from 0 to 6, and 15 p is an integer from 0 to 5.
Preferred compounds of the formula I are those in which p is 0, 1 or 2, in particular 0, and/or in which m is 1.
X is preferably ~CCH2]q~~ it being possible for q to be 1 -12, preferably 1 - 8.
Preferably at least 2, in particular at least 3, of the radi-ls y1 y2 y3 and Y4 denote hydrogen-25Protective groups which are labile to bases or labile to weak acids are, in particular, urethane protective groups, such as Fmoc, Ddz, Bpoc, Msc, Peoc, Pse and Tse, preferaDly Fmoc (see, for example, Hubbuch, Kontakte (Merck) 1979, No.
30 3, pages 14 - 23).
B represents the residue of an amino acid, preferably of an ~-amino acid, which, if chiral, can be present in the D or L form. Preference is given to residues of naturally occur-35 ring amino acids, their enantiomers, homologs, derivativesand simple metabolites (see, for example, ~unsch et al., Houben-~eyl 15/1 and 2, Stuttgart, Thieme 1974). Thus, for example, the following are suitable:
_ 3 - 134~'121 Aad, Abu, ~Abu, ABz, 2ABz, ~Aca, Ach, Acp, Adpd, Ahb, Aib, B Aib, ALa, ~ Ala, a Ala, ALg, ALL, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel, Gln, Glu, Gly, Guv, hCys, His, hSer, Hyl, Hyp, 3Hyp, Ile, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg, Lys, ~ Lys, a Lys, Met, Mim, Min, nArg, Nle, Nva, Oly, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, a Pro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Sem, Ser, Thi, ~ Thi, Thr, Thy, Thx, Tia, Tle, Tly, Trp, Trta, Tyr, Val and the residues of the corresponding enantiomeric D-amino acids.
Functional groups in the side chains of the said amino acid residues can be in protected form. Suitable protective groups are described in Hubbuch, Kontakte (Merck) 1979, No.
3, pages 14 - 23, and in Bullesbach, Kontakte (Merck) 198Q, No. 1, pages 23 - 35. The preferred groups are those which are stable to bases and weak acids and can be eliminated using strong acids.
Alkylene can be straight-chain or branched. Examples of definitions of (C6-C10)-aryl are phenyl, tolyl or naph-thyl; phenyl is preferred.
A carboxyl protective group V is, for example, (C1-C6)-aLkyL
or (C7-C11)-araLkyL; preference is given to methyL, ethyL, tert.butyL, benzyL and modified benzyl, such as p-chloro-, p-bromo-, p-nitro- and p-methoxybenzyl and the nitrogen ana-log picolyl. In the wider sense, such protective groups in-clude activated ester groups such as ONSu, OBt, OObt or p-nitrophenoxy.
The invention also relates to a process for the preparation of the compounds of the formula I, which comprises a) reaction of a compound of the formula II
Y ~ y2 R-co-o-cH2~/ ~rW-co2-v ~ ==~ (II) y3 y4 ~ 4 ~ 13~04~1 in which R represents a leaving group which can be detached nucleophilically, V represents a carboxyl protective group, and ~, y1~ y2, y3 and Y4 are as defined i~ claim 1, wit~ a com-pound of the formula III
A- [B]p-NH- [X]m~NH2 ( I I I ) in which A represents an amino protective group which is labile to bases or labile to weak acids, and B, X, p and m are as defined in claim 1, and elimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I, with the formation of the free NH2 and/or C02H group(s), the preferred processes being those in which V is selectively eliminated, for exam-ple by reductive cleavage with Zn/glacial acetic acid, or b) reaction of a compound of the formula I in which A
denotes hydrogen, and B, X, y1, y2~ y3, y4, V, W, m, n and p are as defined in claim 1, with a compound Ot the formula IV
A-[B]s p-OH ~ (IV) in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate elimination of a carboxyl protective group V with the formation of the carboxyl group.
A leaving group R which can be detached nucleophilically is, for example, halogen, such as chlorine, bromine and iodine, or activated aryloxy, such as p-nitrophenoxy.
The reaction of a compound of the formula II with a com-pound of the formula III is preferably carried out in an 13~0~1 aprotic solvent such as, for example, THF, DMF, CHCl3 or CH2Cl2, advantageously in the presence of a base such as, for example, a tertiary amine, for example ethyl triiso-propylamine, triethylamine or pyridine, the addition of an acylation catalyst such as, for example, DMAP, HOObt or HOBt having an advantageous effect, at a temperature between 0~C and the boiling point of the reaction mixt~re, preferably between 0~C and 40~C.
Compounds of the formula I (A = hydrogen) are reacted with compounds of the formula IV, their active ester, ha-lide or azide preferably in an organic solvent, such as DMF, advantageously in the presence of a base such as, for example, a tert.amine, at a temperature between -10~C
and the boiling point of the reaction mixture, preferably at room temperature. Examples of suitable active esters are the ONSu, OBt, OObt and p-nitrophenoxy compounds.
Preferred halogen derivatives are the chlorides. Pyridin-ium perchlorate can be added to improve the solubility.
Compounds of the formula II are prepared by, for example, reacting esters of the formula IX
yl y2 HO-CH2 ~ ~ W-C02-V (IX) y3 y4 in which y1~ y2~ y3, y4, W and V are as defined above, but V does not denote hydrogen, with phosgene or phosgene derivatives such as, for example, nitrophenyl chloroform-ate in an aprotic polar solvent, for example THF or DMF, mixed with a tert. base, for example a tert. amine such as pyridine, preferably in the ratio 1 : 1, at a tempera-ture between -40~C and room temperature, preferably be-tween -20~C and 0~C.
The invention also relates to the use of a compound of the formula I, in which V denotes hydrogen and A does not - 6 - 13404~1 denote hydrogen, in the solid-phase synthesis of compounds of the formula V
P-NH- [X]m-NH2 (V) s in which P represents a peptide residue comprising q < p+1 ~-amino acids, and X, m and p are as defined above, and to a process for the preparation of a peptide of the formula V, in which P, X, m and p are as defined above, by solid-phase synthesis, which comprises coupling a compound of the formula I, in which A does not denote hydrogen, and V represents hydrogen, to a resin, eliminating the protective group A, stepwise coupling on q-p ~-amino acids which are, where appropriate, in the form of their activated derivatives and which have been temporarily protected by amino protective groups which are labile to bases or labile to weak acids and, after construction is complete, liberating the peptide from the resin by treatment with a moderately strong to strong acid, the temporarily introduced side-chain protective groups being eliminated again at the same time or, by suitable measures, subsequent thereto.
If necessary to prevent side reactions or for the synthe-sis of specific peptides, the functional groups in the side chain of amino acids are additionally protected by suitable protective groups (see, for example, T.W. Greene, "Protective Groups in Organic Syntheses", New York, John Wiley & Sons, 1981), those primarily used being Arg(Tos), Arg(Mts), Arg(Mtr), Asp(OBzl), Asp(OBut), Cys(4-Me8zl), Cys(Acm), Cys(SBut), Glu(OBzl), Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(Cl-2), Lys(Boc), Met(0), Ser(Bzl), Ser(But), Thr(Bzl), Thr(But).
The resins used as carrier material are commercially available. BHA and MBHA resins are preferred.
The peptide of the formula V is then cleaved off by treatment with the moderateLy strong to strong acids1 340 42 customarily used in peptide synthesis (for example trifluoracetic acid and HF), there being cleavage of the urethane protective group contained in the spacer.
It is possible to use as coupling reagent for the com-pound of the formula I (V = H) and the other amino acid derivatives all possible activating reagents used in pep-tide synthesis, see, for example, Houben-Weyl, Methoden der organischen Chemie (Methods of organic chemistry), volume 15/2, but in particular carbodiimides such as, for example, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropyl-carbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbo-diimide. This coupling can be carried out directly by addition of the amino acid derivative with the activating reagent and, where appropriate, an additive which supp-resses racemization, such as, for example, 1-hydroxybenzo-triazole (HOBt) (W. Konig, R Geiger, Chem. Ber. 102, 708 (1970)) or 3-hydroxy-4-oxo-3,4-dihydroxybenzotriazine (HOOBt) (W. Konig, R. Geiger, Chem. Ber. 103, 2054 (1970)) to the resin, or the preactivation of the amino acid deri-vative can be carried out separately as the symmetric an-hydride or HOBt or HOObt ester, and the solution of the activated species in a suitable solvent can be added to the peptide-resin which is ready for coupling.
The coupling and activation of the compound of the formula I (V = H) and of the amino acid derivatives 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 nor-mally used in a 1.5- to 4-fold excess. In cases where in-complete coupling occurs, the coupling reaction is re-peated, without previously carrying out the deblocking of the ~-amino group of the peptide-resin which is neces-sary for coupling the next amino acid in the sequence.
Successful completion of the coupling reaction can be checked using the ninhydrin reaction as described, for -- 8 - 1340~421 example, by E. Kaiser et al. Anal. Biochem. 34, 595 (1970).
The synthesis can also be carried out automatically, for example using an Applied Biosystems model 430A peptide S synthesizer, it being possible to use either the synthe-sis programs provided by the apparatus manufacturer or those constructed by the user himself. The latter are particularly employed when amino acid derivatives protec-ted with the Fmoc group are used.
When the peptide amides are cleaved off the resin with hydrogen fluoride and trifluoroacetic acid, it is custo-mary to add substances as cation traps, such as phenol, cresol, thiocresol, thioanisole, anisole, ethanedithiol, dimethyl sulfide, ethyl methyl sulfide or a mixture of two or more of these auxiliaries. In this connection, the trifluoroacetic acid can also be used diluted by sui~-able solvents such as, for example, methylene chloride.
2û Abbreviations used:
Fmoc 9-fluorenylmethyloxycarbonyl Ddz a,~-dimethyl-3,5-dimethoxybenzyloxycarbonyl Bpoc 2-[4-biphenylyl]-2-propyloxycarbonyl Msc Methylsulfonylethyloxycarbonyl Peoc pyridylethyloxycarbonyl Pse phenylsulfonylethyloxycarbonyl Tse tolylsulfonylethyloxycarbonyl HONSu N-hydroxysuccinimide HOBt 1-hydroxybenzotriazole HOObt 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine THF tetrahydrofuran DMF dimethylformamide DMAP dimethylaminopyridine The examples which follow serve to illustrate the present invention without intending to confine it to them.
Example 1: Methyl 4- hydroxyethylphenoxyacetate 18.2 9 of 4-hydroxymethylphenoxyacetic acid are dissolved together with 17.1 ml of N,N-diisopropylethylamine in S 50 ml of DMF, and then 6.1 ml of methyl iodide are added to the stirred solution. The mixture warms slightly dur-ing this. The reaction is complete after 3 h. The sol-vent is removed in vacuo. The residue is taken up in ether, and the solution is extracted once with 0.5 N
hydrochloric acid. The aqueous phase is then extracted three times with ether, and the combined ether phases are washed with aqueous sodium bicarbonate solution and con-centrated. The residue is dissolved in ethyl acetate and filtered through a short silica gel column. The pale yellowish oil which is obtained after concentration crys-tallizes on being left to stand.
NMR and mass spectrum are consistent with the indicated structure.
Example 2: Fmoc-NH-(cH2)4-NH-co-o-cH2 ~ 0-CH2-COOCH3 9.8 9 of methyl 4-hydroxymethylphenoxyacetate are dis-solved in 200 ml of dry CH2Cl2, and then 10.1 9 of p-nitro-phenyl chloroformate and 7 ml of triethylamine are added.The mixture is boiled under reflux for about 6 h, until the precursor has completely reacted. Then a suspension of 15.5 9 of Fmoc-NH-(CH2)4-NH2 (prepared by reaction of Boc-NH-(CH2)4-NH2 with Fmoc-ONSu followed by elimin-ation of Boc) in 100 ml of dry CH2Cl2 and a further 7 mlof triethylamine are added, and the mixture is boiled under reflux. After the reaction is complete, the solvent is removed in vacuo, and the residue is digested with ether and filtered off ~ith suction. The residue on the filter is washed with aqueous 1 N Na2CO3 solution and then with hot water, and is dried under high vacuum in a desic-cator.
Melting point 122-124~C, NMR and mass spectrum are -consistent with the indicated structure. 134G~21 Exa-Ple 3: H2N-(CH2)4-NH-CO-0 CH2 ~ 0-CH2-COOH
5.2 9 of the ester obtained as in Example Z are suspended in 100 ml of methanol, and 6 equivalents of an aqueous 1 N NaOH solution are added. After the reaction is com-plete, the pH is adjusted to 3 with aqueous 1 N HCl, and the methanol is removed in vacuo. The precipitate is fil-tered off with suction, washed with a little H20, and thendigested in ether and again filtered with suction.
Melting point starts at 196~C (decomposition), NMR and mass spectrum are consistent with the indicated structure.
Exa-ple 4: Fmoc-Phe-NH-(CH2)4-NH-CO-O-CH2 ~ 0-CH2-COOH
1.5 9 of the product obtained as in Example 3 are suspen-ded in 50 ml of dry DMF. Then, successively, 0.9 9 of ZO pyridinium perchlorate (to improve the solubility) and 2.6 9 of Fmoc-Phe-OObt and 0.5 ml of triethylamine are added. The mixture is stirred at room temperature. After the reaction is complete, the solvent is removed in vacuo, and the residue is partitioned between ethyl acetate and HzO. The aqueous phase is extracted once more with ethyl acetate, and the combined organic phases are dried and concentrated. The residue is digested with a little CHCl3 and is filtered off with suction. The residue on the fil-ter is washed with a little ether and is dried.
Melting point starts at 140~C (decomposition), NMR and mass spectrum are consistent with the indicated structure.
Exa-ple 5: Fmoc-Phe-NH-(CH2)4-NH-CO-O-CH2 ~ 0-CH2-CO-(4-eethylbenzhydryla-ine resin) 1.4 9 of the Fmoc-phenylalanine spacer acid obtained as in Example 4 are dissolved together with 350 mg of HOBt in 40 ml dry DMF, and the solution is added to 3.66 9 of 4-methylbenzhydrylamine resin (Nova Biochem, loading 0.4 mmol/g). Then 0.6 ml of diisopropylcarbodiimide 3s 21 added, and the reaction is allowed to go to completion, mixing continuously. After the reaction is complete, the product is filtered off with suction, washed with DMF, isopropanoL, CHzClz and tert.-butyl methyl ether and is dried under high vacuum. Loading according to elemental analysis (nitrogen determination): 0.3 mmol/g.
Example 6: Synthesis of [des-Tyr24, des-Arg23]-r-atriopeptinIII-(4-amino)butyla-ide The peptide synthesis is carried out on 1 9 of the above-mentioned resin using OOBt esters of Fmoc-amino acids with an Applied Biosystems model 430A automatic peptide synthe-sizer and synthesis programs modified by ourselves.
For this, 1 mmol of each of the appropriate amino acidderivatives is weighed into the cartridges supplied by the manufacturer, Fmoc-Arg(Mtr)-OH, Fmoc-Asn-OH and Fmoc-Gln-OH are weighed together with 1.5 mmol of HOBt into the cartridges. These amino acids are preactivated direct-ly in the cartridges by dissolving in 4 ml of DMF and adding 2 ml of a 0.55 M solution of diisopropylcarbodi-imide in DMF. The HOObt esters are dissolved in 6 ml of DMF and then pumped, in the same way as the amino acids arginine, asparagine and glutamine which are preactivated in situ, onto the resin which has previously been deblocked with 20% piperidine in DMF. The amino acids which are activated in situ are coupled twice.
After the synthesis is complete, the peptide butylamide is cleaved off the resin, simultaneously removing the side-chain protective groups with trifluoroacetic acid which contains th-ioanisole and m-cresol as cation traps.
The residue obtained after removal of the trifluoroacetic acid in vacuo is subjected to digestion with ethyl ace-tate and centrifugation several times. The remaining crude peptide is treated with tributylphosphine in tri-fluoroethanol to remove the cysteine protective group.
After the solvent has been removed, the residue is again digested with ethyl acetate and centrifuged. The reduced crude peptide is immediately oxidized with iodine in 80%
strength aqueous acetic acid, the excess I2 is removed with ascorbic acid, and the reaction mixture is concentrated to a small volume and then salt is removed on ~ Sephadex G25 with aqueous 1 N acetic acid. The fractions contain-ing the pure peptide are combined and freeze-dried.
According to amino acid analysis, the amino acid composi-tion of the peptide corresponds to the indicated formula.
Example 7: Phenacyl 4-hydroxymethylphenoxyacetate 182 9 of 4-hydroxymethylphenoxyacetic acid and 199 9 of ~-bromoacetophenone are dissolved in 600 ml of dry DMF, and then, at 0~C, 138 ml of triethylamine are rapidly added dropwise. The mixture is allowed to reach room temperature, and is stirred overnight. The DMF solution is poured into 3.5 l of water, and the aqueous phase is extracted with ethyl acetate. The organic phase is washed with water, dried over sodium sulfate and concentrated.
The product precipitates out on evaporation. It is fil-tered off with suction, washed with ethyl acetate/n-hexane 1:1 and dried under high vacuum.
Melting point: 94-95~C, NMR is consistent with the indi-cated structure.
Example 8: O2N ~ O-CO-O-CH2 ~ O-CH2-CO2-CH2-CO
30 9 of phenacyl 4-hydroxymethylphenoxyacetate are dis-solved, under protective gas, in 500 ml of a 1:1 mixture of THF and pyridine, and the solution is cooled to -20~C.
Then 21 9 of p-nitrophenyl chloroformate dissolved in 100 ml of THF are added dropwise. After the mixture has been stirred at this temperature for 30 minutes, it is allowed to warm to 0~C and stirred into 1 l of a half-saturated aqueous NaCl solution at 0~C, and the mixture is then stirred for 30 minutes. The precipitate is filtered 1~0421 off with suction, washed with ice-water and, after drying, triturated with n-hexane.
Melting point: 142-145~C, NMR is consistent with the indi-cated structure.
ExaepLe 9:
oc Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2- C02- CH2- CO
9.3 9 of the compound prepared in Example 8, 12.25 9 of Fmoc-Phe-NH-(CH2)g-NH2 trifluoroacetate and 3.26 9 of HOObt are placed as the solid substances in a flask, and then a mixture of 2.58 9 of ethyl diisopropylamine in 100 ml of dry DMF is poured over. The mixture is then stirred at 40~C for 3.5 hours and then stirred into 500 ml of hal~-saturated aqueous NaCl solution. The precipitate whichseparates out is filtered off with suction, washed with ice-water and, after drying, triturated with ether/ethyl acetate.
Melting point: 147-150~C, NMR and MS are consistent with the indicated formula.
The following compounds (Examples 10 to 14) are prepared in analogy to Example 9:
Exa~ple 10:
Fmoc- Phe- NH- ( CH2 ) 4- NH- CO- O- CH2 ~ O- CH2- C~2 - CH2- CO ~
Melting point 144-147~C, NMR and MS correspond to the indi-cated formula.
Exa-ple 11:
Fmoc- Al a- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2 - C02- CH2 - Co~3 Melting point 179-181~C, NMR and MS correspond to the indi-cated formula.
Exa~ple 12: 134~1 E~oc- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2- C02- CH2- CO ~=~
S Melting point 144-145~C, NMR and MS correspond to the indi-cated formula.
Formu~a 13:
1 ~ E moc-NH- ( CH2 ) 6-NH- CO- O- CH2 ~ O- CH2- C~2- CH2- CO~
Melting point 172-175~C, NMR corresponds to the indicated formula.
ExampLe 14:
Fmoc-NEI-(CH2)4-NH-CO-O-CH2 ~ O-CH2-C02-CH2-CO ~ ) Melting point 165-166~C, NMR corresponds to the indicated formuLa.
Exa~pLe 15:
E'moc- Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ ~- CH2- C02H
8 . 4 9 Fmoc- Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2 - C~2 - CH2 - CO
are suspended in a mixture of 150 ml of glacial acetic acid and 50 ml of dichloromethane, and 12 9 of zinc pow-der which has previously been activated by washing with 1 N HCl and dry ethanol are added in portions. After a few minutes, the suspension becomes more viscous and dif-ficult to stir, while there is slight evolution of heat.
Hence a further 80 ml of glacial acetic acid and 50 ml of dichloromethane are added, and stirring is continued over-night. The mixture is then filtered with suction through a filter with a clarifying layer, washing with glacial acetic acid and dichloromethane. The filtrate is -- 15 - 13~0~21 concentrated, and the oil which remains as residue is - taken up in a little dichloromethane and stirred with ethyl acetate and ether. The precipitated product is filtered off with suction and dried under high vacuum.
Melting point: decomposition above 160~C, NMR and MS are consistent with the indicated formula.
In addition, the compounds of Examples 16 to 18 are pre-pared by the method described in Example 15:
Exacple 16:
E~oc- Phe- NH- ( CH2 ) 4- NH- CO- O- CH2~ O- CH2- C~2H
Melting point: decomposition above 150~C, NMR and MS are consistent with the indicated formula.
Exa~pLe 17:
2 0 Fmoc- Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~o- CH2 - C~2H
Melting point: decomposition above 160~C, NMR and MS are consistent with the indicated formula.
25 Exanple 18:
Fmoc- NH- ( CH2 ) 8- NH- CO- O- CH2-~30- CH2- C02H
Melting point: decomposition above 154~C, NMR and MS are consistent with the indicated formula.
Exa~pLe 19:
Fmoc- NH- ( CH2 ) 6- NH- CO- O- CH2 ~ O- CH2 - C02CH3 The synthesis is carried out in analogy to Example 2.
Melting point: 115-118~C, NMR and MS are consistent wi~h the indicated formula.
,, . .. . . . ~
ExampLe 20: 1340421 NH2- (CH2)6-NH-CO-O-cH2~0-cH2-co2H
was prepared by the method described in Example 3.
Melting point: 184-187~C decomposition, NMR and MS are con-sistent with the indicated formula.
Exa~pLe 21:
Fmoc- Phe- NH- ( CH2 ) 6- NH- CO- O- CH2~0- CH2 - C02H
The synthesis is carried out in analogy to Example 4.
Melting point: deco~position above 120~C, NMR and MS are consistent with the indicated formula.
Specification:
The synthesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase method s The introduction of an aminoalkylamide into the C-terminal end of a biologically active peptide has in some cases had beneficial effects on the metabolic stability and activity (EP-A 179 332). The preparation of the peptides modified in this way has made use of the classical coupling of frasments in solution.
In the solid-phase synthesis of peptides (see Patchornik, Cohen in Perspectives in Peptide Chemistry, pages 118 - 1Z8 (Karger, Basle 1981)) the reactive chains are often not grafted directly onto the synthetic resin material, but are bonded to the carrier material by what are called spacers or links. The literature (for example Atherton, Sheppard in Perspectives in Peptide Chemistry, pages 101 - 117 (Karger, Basle 1981)) discloses, for example, reagents for introducing such spacers (called "linkage agents") which have the formulae YI, VII and VIII.
HOCH2 -~CH2- CH2- C02H HOCH~ OCH2 - CO2H HOCH2~ C02H
(VI) (VII) (VIII) New linkage agents which allow direct construction, by solid phase synthesis, of peptides modified by C-terminal amino-alkylamide or hydrazide have been found.
Thus the present invention relates to compounds of the tor-mula I
yl y2 ( I ) A- [B]p-NH- [X]m-NH-CO-O-CH2 _~_ W-C02-V
y3--y4 *
in which 1 3 ~ O ~ 2 1 A denotes hydrogen or an amino protective group which is labile to bases or labile to weak acids, B represents identical or different amino acid resi-dues, X denotes (C1-C12)-alkylene or (C6-C10)-arYl-(c1-c12) alkylene, y1, y2~ y3 and Y4 are identical or different and denote hydrogen, methyl, methoxy or nitro, at Least one of these radicals denoting hydrogen, V denotes hydrogen or a carboxyl protective group, ~ denotes -tCH2]n- or ~0-[CH2]n-, m is 0 or 1, n is an integer from 0 to 6, and 15 p is an integer from 0 to 5.
Preferred compounds of the formula I are those in which p is 0, 1 or 2, in particular 0, and/or in which m is 1.
X is preferably ~CCH2]q~~ it being possible for q to be 1 -12, preferably 1 - 8.
Preferably at least 2, in particular at least 3, of the radi-ls y1 y2 y3 and Y4 denote hydrogen-25Protective groups which are labile to bases or labile to weak acids are, in particular, urethane protective groups, such as Fmoc, Ddz, Bpoc, Msc, Peoc, Pse and Tse, preferaDly Fmoc (see, for example, Hubbuch, Kontakte (Merck) 1979, No.
30 3, pages 14 - 23).
B represents the residue of an amino acid, preferably of an ~-amino acid, which, if chiral, can be present in the D or L form. Preference is given to residues of naturally occur-35 ring amino acids, their enantiomers, homologs, derivativesand simple metabolites (see, for example, ~unsch et al., Houben-~eyl 15/1 and 2, Stuttgart, Thieme 1974). Thus, for example, the following are suitable:
_ 3 - 134~'121 Aad, Abu, ~Abu, ABz, 2ABz, ~Aca, Ach, Acp, Adpd, Ahb, Aib, B Aib, ALa, ~ Ala, a Ala, ALg, ALL, Ama, Amt, Ape, Apm, Apr, Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys, Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc, Fel, Gln, Glu, Gly, Guv, hCys, His, hSer, Hyl, Hyp, 3Hyp, Ile, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg, Lys, ~ Lys, a Lys, Met, Mim, Min, nArg, Nle, Nva, Oly, Orn, Pan, Pec, Pen, Phe, Phg, Pic, Pro, a Pro, Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sec, Sem, Ser, Thi, ~ Thi, Thr, Thy, Thx, Tia, Tle, Tly, Trp, Trta, Tyr, Val and the residues of the corresponding enantiomeric D-amino acids.
Functional groups in the side chains of the said amino acid residues can be in protected form. Suitable protective groups are described in Hubbuch, Kontakte (Merck) 1979, No.
3, pages 14 - 23, and in Bullesbach, Kontakte (Merck) 198Q, No. 1, pages 23 - 35. The preferred groups are those which are stable to bases and weak acids and can be eliminated using strong acids.
Alkylene can be straight-chain or branched. Examples of definitions of (C6-C10)-aryl are phenyl, tolyl or naph-thyl; phenyl is preferred.
A carboxyl protective group V is, for example, (C1-C6)-aLkyL
or (C7-C11)-araLkyL; preference is given to methyL, ethyL, tert.butyL, benzyL and modified benzyl, such as p-chloro-, p-bromo-, p-nitro- and p-methoxybenzyl and the nitrogen ana-log picolyl. In the wider sense, such protective groups in-clude activated ester groups such as ONSu, OBt, OObt or p-nitrophenoxy.
The invention also relates to a process for the preparation of the compounds of the formula I, which comprises a) reaction of a compound of the formula II
Y ~ y2 R-co-o-cH2~/ ~rW-co2-v ~ ==~ (II) y3 y4 ~ 4 ~ 13~04~1 in which R represents a leaving group which can be detached nucleophilically, V represents a carboxyl protective group, and ~, y1~ y2, y3 and Y4 are as defined i~ claim 1, wit~ a com-pound of the formula III
A- [B]p-NH- [X]m~NH2 ( I I I ) in which A represents an amino protective group which is labile to bases or labile to weak acids, and B, X, p and m are as defined in claim 1, and elimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I, with the formation of the free NH2 and/or C02H group(s), the preferred processes being those in which V is selectively eliminated, for exam-ple by reductive cleavage with Zn/glacial acetic acid, or b) reaction of a compound of the formula I in which A
denotes hydrogen, and B, X, y1, y2~ y3, y4, V, W, m, n and p are as defined in claim 1, with a compound Ot the formula IV
A-[B]s p-OH ~ (IV) in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate elimination of a carboxyl protective group V with the formation of the carboxyl group.
A leaving group R which can be detached nucleophilically is, for example, halogen, such as chlorine, bromine and iodine, or activated aryloxy, such as p-nitrophenoxy.
The reaction of a compound of the formula II with a com-pound of the formula III is preferably carried out in an 13~0~1 aprotic solvent such as, for example, THF, DMF, CHCl3 or CH2Cl2, advantageously in the presence of a base such as, for example, a tertiary amine, for example ethyl triiso-propylamine, triethylamine or pyridine, the addition of an acylation catalyst such as, for example, DMAP, HOObt or HOBt having an advantageous effect, at a temperature between 0~C and the boiling point of the reaction mixt~re, preferably between 0~C and 40~C.
Compounds of the formula I (A = hydrogen) are reacted with compounds of the formula IV, their active ester, ha-lide or azide preferably in an organic solvent, such as DMF, advantageously in the presence of a base such as, for example, a tert.amine, at a temperature between -10~C
and the boiling point of the reaction mixture, preferably at room temperature. Examples of suitable active esters are the ONSu, OBt, OObt and p-nitrophenoxy compounds.
Preferred halogen derivatives are the chlorides. Pyridin-ium perchlorate can be added to improve the solubility.
Compounds of the formula II are prepared by, for example, reacting esters of the formula IX
yl y2 HO-CH2 ~ ~ W-C02-V (IX) y3 y4 in which y1~ y2~ y3, y4, W and V are as defined above, but V does not denote hydrogen, with phosgene or phosgene derivatives such as, for example, nitrophenyl chloroform-ate in an aprotic polar solvent, for example THF or DMF, mixed with a tert. base, for example a tert. amine such as pyridine, preferably in the ratio 1 : 1, at a tempera-ture between -40~C and room temperature, preferably be-tween -20~C and 0~C.
The invention also relates to the use of a compound of the formula I, in which V denotes hydrogen and A does not - 6 - 13404~1 denote hydrogen, in the solid-phase synthesis of compounds of the formula V
P-NH- [X]m-NH2 (V) s in which P represents a peptide residue comprising q < p+1 ~-amino acids, and X, m and p are as defined above, and to a process for the preparation of a peptide of the formula V, in which P, X, m and p are as defined above, by solid-phase synthesis, which comprises coupling a compound of the formula I, in which A does not denote hydrogen, and V represents hydrogen, to a resin, eliminating the protective group A, stepwise coupling on q-p ~-amino acids which are, where appropriate, in the form of their activated derivatives and which have been temporarily protected by amino protective groups which are labile to bases or labile to weak acids and, after construction is complete, liberating the peptide from the resin by treatment with a moderately strong to strong acid, the temporarily introduced side-chain protective groups being eliminated again at the same time or, by suitable measures, subsequent thereto.
If necessary to prevent side reactions or for the synthe-sis of specific peptides, the functional groups in the side chain of amino acids are additionally protected by suitable protective groups (see, for example, T.W. Greene, "Protective Groups in Organic Syntheses", New York, John Wiley & Sons, 1981), those primarily used being Arg(Tos), Arg(Mts), Arg(Mtr), Asp(OBzl), Asp(OBut), Cys(4-Me8zl), Cys(Acm), Cys(SBut), Glu(OBzl), Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt), Lys(Cl-2), Lys(Boc), Met(0), Ser(Bzl), Ser(But), Thr(Bzl), Thr(But).
The resins used as carrier material are commercially available. BHA and MBHA resins are preferred.
The peptide of the formula V is then cleaved off by treatment with the moderateLy strong to strong acids1 340 42 customarily used in peptide synthesis (for example trifluoracetic acid and HF), there being cleavage of the urethane protective group contained in the spacer.
It is possible to use as coupling reagent for the com-pound of the formula I (V = H) and the other amino acid derivatives all possible activating reagents used in pep-tide synthesis, see, for example, Houben-Weyl, Methoden der organischen Chemie (Methods of organic chemistry), volume 15/2, but in particular carbodiimides such as, for example, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropyl-carbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbo-diimide. This coupling can be carried out directly by addition of the amino acid derivative with the activating reagent and, where appropriate, an additive which supp-resses racemization, such as, for example, 1-hydroxybenzo-triazole (HOBt) (W. Konig, R Geiger, Chem. Ber. 102, 708 (1970)) or 3-hydroxy-4-oxo-3,4-dihydroxybenzotriazine (HOOBt) (W. Konig, R. Geiger, Chem. Ber. 103, 2054 (1970)) to the resin, or the preactivation of the amino acid deri-vative can be carried out separately as the symmetric an-hydride or HOBt or HOObt ester, and the solution of the activated species in a suitable solvent can be added to the peptide-resin which is ready for coupling.
The coupling and activation of the compound of the formula I (V = H) and of the amino acid derivatives 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 nor-mally used in a 1.5- to 4-fold excess. In cases where in-complete coupling occurs, the coupling reaction is re-peated, without previously carrying out the deblocking of the ~-amino group of the peptide-resin which is neces-sary for coupling the next amino acid in the sequence.
Successful completion of the coupling reaction can be checked using the ninhydrin reaction as described, for -- 8 - 1340~421 example, by E. Kaiser et al. Anal. Biochem. 34, 595 (1970).
The synthesis can also be carried out automatically, for example using an Applied Biosystems model 430A peptide S synthesizer, it being possible to use either the synthe-sis programs provided by the apparatus manufacturer or those constructed by the user himself. The latter are particularly employed when amino acid derivatives protec-ted with the Fmoc group are used.
When the peptide amides are cleaved off the resin with hydrogen fluoride and trifluoroacetic acid, it is custo-mary to add substances as cation traps, such as phenol, cresol, thiocresol, thioanisole, anisole, ethanedithiol, dimethyl sulfide, ethyl methyl sulfide or a mixture of two or more of these auxiliaries. In this connection, the trifluoroacetic acid can also be used diluted by sui~-able solvents such as, for example, methylene chloride.
2û Abbreviations used:
Fmoc 9-fluorenylmethyloxycarbonyl Ddz a,~-dimethyl-3,5-dimethoxybenzyloxycarbonyl Bpoc 2-[4-biphenylyl]-2-propyloxycarbonyl Msc Methylsulfonylethyloxycarbonyl Peoc pyridylethyloxycarbonyl Pse phenylsulfonylethyloxycarbonyl Tse tolylsulfonylethyloxycarbonyl HONSu N-hydroxysuccinimide HOBt 1-hydroxybenzotriazole HOObt 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine THF tetrahydrofuran DMF dimethylformamide DMAP dimethylaminopyridine The examples which follow serve to illustrate the present invention without intending to confine it to them.
Example 1: Methyl 4- hydroxyethylphenoxyacetate 18.2 9 of 4-hydroxymethylphenoxyacetic acid are dissolved together with 17.1 ml of N,N-diisopropylethylamine in S 50 ml of DMF, and then 6.1 ml of methyl iodide are added to the stirred solution. The mixture warms slightly dur-ing this. The reaction is complete after 3 h. The sol-vent is removed in vacuo. The residue is taken up in ether, and the solution is extracted once with 0.5 N
hydrochloric acid. The aqueous phase is then extracted three times with ether, and the combined ether phases are washed with aqueous sodium bicarbonate solution and con-centrated. The residue is dissolved in ethyl acetate and filtered through a short silica gel column. The pale yellowish oil which is obtained after concentration crys-tallizes on being left to stand.
NMR and mass spectrum are consistent with the indicated structure.
Example 2: Fmoc-NH-(cH2)4-NH-co-o-cH2 ~ 0-CH2-COOCH3 9.8 9 of methyl 4-hydroxymethylphenoxyacetate are dis-solved in 200 ml of dry CH2Cl2, and then 10.1 9 of p-nitro-phenyl chloroformate and 7 ml of triethylamine are added.The mixture is boiled under reflux for about 6 h, until the precursor has completely reacted. Then a suspension of 15.5 9 of Fmoc-NH-(CH2)4-NH2 (prepared by reaction of Boc-NH-(CH2)4-NH2 with Fmoc-ONSu followed by elimin-ation of Boc) in 100 ml of dry CH2Cl2 and a further 7 mlof triethylamine are added, and the mixture is boiled under reflux. After the reaction is complete, the solvent is removed in vacuo, and the residue is digested with ether and filtered off ~ith suction. The residue on the filter is washed with aqueous 1 N Na2CO3 solution and then with hot water, and is dried under high vacuum in a desic-cator.
Melting point 122-124~C, NMR and mass spectrum are -consistent with the indicated structure. 134G~21 Exa-Ple 3: H2N-(CH2)4-NH-CO-0 CH2 ~ 0-CH2-COOH
5.2 9 of the ester obtained as in Example Z are suspended in 100 ml of methanol, and 6 equivalents of an aqueous 1 N NaOH solution are added. After the reaction is com-plete, the pH is adjusted to 3 with aqueous 1 N HCl, and the methanol is removed in vacuo. The precipitate is fil-tered off with suction, washed with a little H20, and thendigested in ether and again filtered with suction.
Melting point starts at 196~C (decomposition), NMR and mass spectrum are consistent with the indicated structure.
Exa-ple 4: Fmoc-Phe-NH-(CH2)4-NH-CO-O-CH2 ~ 0-CH2-COOH
1.5 9 of the product obtained as in Example 3 are suspen-ded in 50 ml of dry DMF. Then, successively, 0.9 9 of ZO pyridinium perchlorate (to improve the solubility) and 2.6 9 of Fmoc-Phe-OObt and 0.5 ml of triethylamine are added. The mixture is stirred at room temperature. After the reaction is complete, the solvent is removed in vacuo, and the residue is partitioned between ethyl acetate and HzO. The aqueous phase is extracted once more with ethyl acetate, and the combined organic phases are dried and concentrated. The residue is digested with a little CHCl3 and is filtered off with suction. The residue on the fil-ter is washed with a little ether and is dried.
Melting point starts at 140~C (decomposition), NMR and mass spectrum are consistent with the indicated structure.
Exa-ple 5: Fmoc-Phe-NH-(CH2)4-NH-CO-O-CH2 ~ 0-CH2-CO-(4-eethylbenzhydryla-ine resin) 1.4 9 of the Fmoc-phenylalanine spacer acid obtained as in Example 4 are dissolved together with 350 mg of HOBt in 40 ml dry DMF, and the solution is added to 3.66 9 of 4-methylbenzhydrylamine resin (Nova Biochem, loading 0.4 mmol/g). Then 0.6 ml of diisopropylcarbodiimide 3s 21 added, and the reaction is allowed to go to completion, mixing continuously. After the reaction is complete, the product is filtered off with suction, washed with DMF, isopropanoL, CHzClz and tert.-butyl methyl ether and is dried under high vacuum. Loading according to elemental analysis (nitrogen determination): 0.3 mmol/g.
Example 6: Synthesis of [des-Tyr24, des-Arg23]-r-atriopeptinIII-(4-amino)butyla-ide The peptide synthesis is carried out on 1 9 of the above-mentioned resin using OOBt esters of Fmoc-amino acids with an Applied Biosystems model 430A automatic peptide synthe-sizer and synthesis programs modified by ourselves.
For this, 1 mmol of each of the appropriate amino acidderivatives is weighed into the cartridges supplied by the manufacturer, Fmoc-Arg(Mtr)-OH, Fmoc-Asn-OH and Fmoc-Gln-OH are weighed together with 1.5 mmol of HOBt into the cartridges. These amino acids are preactivated direct-ly in the cartridges by dissolving in 4 ml of DMF and adding 2 ml of a 0.55 M solution of diisopropylcarbodi-imide in DMF. The HOObt esters are dissolved in 6 ml of DMF and then pumped, in the same way as the amino acids arginine, asparagine and glutamine which are preactivated in situ, onto the resin which has previously been deblocked with 20% piperidine in DMF. The amino acids which are activated in situ are coupled twice.
After the synthesis is complete, the peptide butylamide is cleaved off the resin, simultaneously removing the side-chain protective groups with trifluoroacetic acid which contains th-ioanisole and m-cresol as cation traps.
The residue obtained after removal of the trifluoroacetic acid in vacuo is subjected to digestion with ethyl ace-tate and centrifugation several times. The remaining crude peptide is treated with tributylphosphine in tri-fluoroethanol to remove the cysteine protective group.
After the solvent has been removed, the residue is again digested with ethyl acetate and centrifuged. The reduced crude peptide is immediately oxidized with iodine in 80%
strength aqueous acetic acid, the excess I2 is removed with ascorbic acid, and the reaction mixture is concentrated to a small volume and then salt is removed on ~ Sephadex G25 with aqueous 1 N acetic acid. The fractions contain-ing the pure peptide are combined and freeze-dried.
According to amino acid analysis, the amino acid composi-tion of the peptide corresponds to the indicated formula.
Example 7: Phenacyl 4-hydroxymethylphenoxyacetate 182 9 of 4-hydroxymethylphenoxyacetic acid and 199 9 of ~-bromoacetophenone are dissolved in 600 ml of dry DMF, and then, at 0~C, 138 ml of triethylamine are rapidly added dropwise. The mixture is allowed to reach room temperature, and is stirred overnight. The DMF solution is poured into 3.5 l of water, and the aqueous phase is extracted with ethyl acetate. The organic phase is washed with water, dried over sodium sulfate and concentrated.
The product precipitates out on evaporation. It is fil-tered off with suction, washed with ethyl acetate/n-hexane 1:1 and dried under high vacuum.
Melting point: 94-95~C, NMR is consistent with the indi-cated structure.
Example 8: O2N ~ O-CO-O-CH2 ~ O-CH2-CO2-CH2-CO
30 9 of phenacyl 4-hydroxymethylphenoxyacetate are dis-solved, under protective gas, in 500 ml of a 1:1 mixture of THF and pyridine, and the solution is cooled to -20~C.
Then 21 9 of p-nitrophenyl chloroformate dissolved in 100 ml of THF are added dropwise. After the mixture has been stirred at this temperature for 30 minutes, it is allowed to warm to 0~C and stirred into 1 l of a half-saturated aqueous NaCl solution at 0~C, and the mixture is then stirred for 30 minutes. The precipitate is filtered 1~0421 off with suction, washed with ice-water and, after drying, triturated with n-hexane.
Melting point: 142-145~C, NMR is consistent with the indi-cated structure.
ExaepLe 9:
oc Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2- C02- CH2- CO
9.3 9 of the compound prepared in Example 8, 12.25 9 of Fmoc-Phe-NH-(CH2)g-NH2 trifluoroacetate and 3.26 9 of HOObt are placed as the solid substances in a flask, and then a mixture of 2.58 9 of ethyl diisopropylamine in 100 ml of dry DMF is poured over. The mixture is then stirred at 40~C for 3.5 hours and then stirred into 500 ml of hal~-saturated aqueous NaCl solution. The precipitate whichseparates out is filtered off with suction, washed with ice-water and, after drying, triturated with ether/ethyl acetate.
Melting point: 147-150~C, NMR and MS are consistent with the indicated formula.
The following compounds (Examples 10 to 14) are prepared in analogy to Example 9:
Exa~ple 10:
Fmoc- Phe- NH- ( CH2 ) 4- NH- CO- O- CH2 ~ O- CH2- C~2 - CH2- CO ~
Melting point 144-147~C, NMR and MS correspond to the indi-cated formula.
Exa-ple 11:
Fmoc- Al a- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2 - C02- CH2 - Co~3 Melting point 179-181~C, NMR and MS correspond to the indi-cated formula.
Exa~ple 12: 134~1 E~oc- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2- C02- CH2- CO ~=~
S Melting point 144-145~C, NMR and MS correspond to the indi-cated formula.
Formu~a 13:
1 ~ E moc-NH- ( CH2 ) 6-NH- CO- O- CH2 ~ O- CH2- C~2- CH2- CO~
Melting point 172-175~C, NMR corresponds to the indicated formula.
ExampLe 14:
Fmoc-NEI-(CH2)4-NH-CO-O-CH2 ~ O-CH2-C02-CH2-CO ~ ) Melting point 165-166~C, NMR corresponds to the indicated formuLa.
Exa~pLe 15:
E'moc- Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ ~- CH2- C02H
8 . 4 9 Fmoc- Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~ O- CH2 - C~2 - CH2 - CO
are suspended in a mixture of 150 ml of glacial acetic acid and 50 ml of dichloromethane, and 12 9 of zinc pow-der which has previously been activated by washing with 1 N HCl and dry ethanol are added in portions. After a few minutes, the suspension becomes more viscous and dif-ficult to stir, while there is slight evolution of heat.
Hence a further 80 ml of glacial acetic acid and 50 ml of dichloromethane are added, and stirring is continued over-night. The mixture is then filtered with suction through a filter with a clarifying layer, washing with glacial acetic acid and dichloromethane. The filtrate is -- 15 - 13~0~21 concentrated, and the oil which remains as residue is - taken up in a little dichloromethane and stirred with ethyl acetate and ether. The precipitated product is filtered off with suction and dried under high vacuum.
Melting point: decomposition above 160~C, NMR and MS are consistent with the indicated formula.
In addition, the compounds of Examples 16 to 18 are pre-pared by the method described in Example 15:
Exacple 16:
E~oc- Phe- NH- ( CH2 ) 4- NH- CO- O- CH2~ O- CH2- C~2H
Melting point: decomposition above 150~C, NMR and MS are consistent with the indicated formula.
Exa~pLe 17:
2 0 Fmoc- Phe- NH- ( CH2 ) 8- NH- CO- O- CH2 ~o- CH2 - C~2H
Melting point: decomposition above 160~C, NMR and MS are consistent with the indicated formula.
25 Exanple 18:
Fmoc- NH- ( CH2 ) 8- NH- CO- O- CH2-~30- CH2- C02H
Melting point: decomposition above 154~C, NMR and MS are consistent with the indicated formula.
Exa~pLe 19:
Fmoc- NH- ( CH2 ) 6- NH- CO- O- CH2 ~ O- CH2 - C02CH3 The synthesis is carried out in analogy to Example 2.
Melting point: 115-118~C, NMR and MS are consistent wi~h the indicated formula.
,, . .. . . . ~
ExampLe 20: 1340421 NH2- (CH2)6-NH-CO-O-cH2~0-cH2-co2H
was prepared by the method described in Example 3.
Melting point: 184-187~C decomposition, NMR and MS are con-sistent with the indicated formula.
Exa~pLe 21:
Fmoc- Phe- NH- ( CH2 ) 6- NH- CO- O- CH2~0- CH2 - C02H
The synthesis is carried out in analogy to Example 4.
Melting point: deco~position above 120~C, NMR and MS are consistent with the indicated formula.
Claims (7)
1. A compound of the formula I
in which A denotes hydrogen or an amino protective group which is labile to bases or labile to weak acids, a represents identical or different amino acid residues, X denotes (C1-C12)-alkylene or (C6-C10)-aryl-(C1-C12)-alkylene, y1, y2, y3 and Y4 are identical or different and denote hydrogen, methyl, methoxy or nitro, at least one of these radicals denoting hydrogen, V denotes hydrogen or a carboxyl protective group, ~ denotes -(CH2]n- or -0-[CH2]n-, m is 0 or 1, n is an integer from 0 to 6, and p is an integer from 0 to 5.
in which A denotes hydrogen or an amino protective group which is labile to bases or labile to weak acids, a represents identical or different amino acid residues, X denotes (C1-C12)-alkylene or (C6-C10)-aryl-(C1-C12)-alkylene, y1, y2, y3 and Y4 are identical or different and denote hydrogen, methyl, methoxy or nitro, at least one of these radicals denoting hydrogen, V denotes hydrogen or a carboxyl protective group, ~ denotes -(CH2]n- or -0-[CH2]n-, m is 0 or 1, n is an integer from 0 to 6, and p is an integer from 0 to 5.
2. A compound of the formula I as claimed in claim 1, in which p is 0, 1 or 2.
3. A compound of the formula I as claimed in claim 1, in which m is 1.
4. A compound of the formula I as claimed in claim 1, 2 or 3, in which X denotes -(CH2]q-, and q denotes an integer from 1 to 12.
5. A compound of the formula I as claimed in claim 1, 2 or 3, in which at least 2 of the radicals y1, y2, y3 and Y4 denote hydrogen.
6. A process for the preparation of a compound as claimed in claim 1, 2 or 3, which comprises a) reaction of a compound of the formula II
in which R represents a leaving group which can be detached nucleophilically, V represents a carboxyl protective group, and W, y1, y2, y3 and Y4 are as defined in claim 1, with a compound of the formula III
A-[B]p-NH-[x]m-NH2 (III) in which A represents an amino protective group which is labile to bases or labile to weak acids, and B, X, p and m are as defined in claim 1, and elimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I as defined in claim 1, with the formation of one or both of the free NH2 and CO2 group(s) or b) reaction of a compound of the formula I in which A
denotes hydrogen, and B, X, y1, y2, y3, y4, V, W, m, n and p are as defined in claim 1, with a compound of the formula IV
A-[B]S-p-OH (IV) in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate elimination of a carboxyl protective group V with the formation of the carboxyl group.
in which R represents a leaving group which can be detached nucleophilically, V represents a carboxyl protective group, and W, y1, y2, y3 and Y4 are as defined in claim 1, with a compound of the formula III
A-[B]p-NH-[x]m-NH2 (III) in which A represents an amino protective group which is labile to bases or labile to weak acids, and B, X, p and m are as defined in claim 1, and elimination of, where appropriate, one or both of the protective groups A and/or V in the resulting protected compound of the formula I as defined in claim 1, with the formation of one or both of the free NH2 and CO2 group(s) or b) reaction of a compound of the formula I in which A
denotes hydrogen, and B, X, y1, y2, y3, y4, V, W, m, n and p are as defined in claim 1, with a compound of the formula IV
A-[B]S-p-OH (IV) in which A, B and p are as defined above, but A does not denote hydrogen, or its active ester, halide or azide, and, if V is not hydrogen, where appropriate elimination of a carboxyl protective group V with the formation of the carboxyl group.
7. The use of a compound of the formula I as claimed in claim 1, 2 or 3 in which V denotes hydrogen, and A does not denote hydrogen, in the solid-phase synthesis of compounds of the formula V
P-NH-[X]a-NH2 (V) in which P represents a peptide residue comprising q ~ p+1 .alpha.-amino acids, and X, m and p are defined as in claim 1.
P-NH-[X]a-NH2 (V) in which P represents a peptide residue comprising q ~ p+1 .alpha.-amino acids, and X, m and p are defined as in claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863635670 DE3635670A1 (en) | 1986-10-21 | 1986-10-21 | SYNTHESIS OF PEPTIDE-AMINOALKYLAMIDES AND PEPTIDYHYDRAZIDES BY MEANS OF THE SOLID PHASE METHOD |
| DEP3635670.0 | 1986-10-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1340421C true CA1340421C (en) | 1999-03-09 |
Family
ID=6312082
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000549680A Expired - Fee Related CA1340421C (en) | 1986-10-21 | 1987-10-20 | Sythesis of peptide aminoalkylamides and peptide hydrazides by the solid-phase method |
Country Status (18)
| Country | Link |
|---|---|
| EP (1) | EP0264802B1 (en) |
| JP (1) | JP2540564B2 (en) |
| KR (1) | KR960013073B1 (en) |
| AT (1) | ATE83244T1 (en) |
| AU (1) | AU595390B2 (en) |
| CA (1) | CA1340421C (en) |
| DE (2) | DE3635670A1 (en) |
| DK (1) | DK175126B1 (en) |
| ES (1) | ES2052535T3 (en) |
| FI (1) | FI88031C (en) |
| GR (1) | GR3007231T3 (en) |
| HU (1) | HU197719B (en) |
| IE (1) | IE60864B1 (en) |
| IL (1) | IL84195A (en) |
| NO (1) | NO172895C (en) |
| NZ (1) | NZ222204A (en) |
| PT (1) | PT85952B (en) |
| ZA (1) | ZA877862B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3926822A1 (en) | 1989-08-14 | 1991-02-21 | Hoechst Ag | PEPTIDES WITH BRADYKININ ANTAGONISTIC EFFECT |
| EP0376218B1 (en) * | 1988-12-27 | 1999-02-24 | Perseptive Biosystems, Inc. | Racemization free attachment of amino acids to solid phase |
| DE4408533A1 (en) | 1994-03-14 | 1995-09-28 | Hoechst Ag | PNA synthesis using a base-labile amino protecting group |
| SI0946478T1 (en) * | 1996-12-19 | 2007-06-30 | Aventis Pharma Inc | Process for the solid phase synthesis of aldehydes, ketones and hydroxamic acid compounds |
| GB9727123D0 (en) * | 1997-12-22 | 1998-02-25 | Int Centre Genetic Eng & Bio | Synthesis of diamines |
| US6852789B2 (en) * | 2002-02-15 | 2005-02-08 | Degussa - Ag | Glycols starting materials containing dispersed superfine ceramic powder coagulates capable of forming polyester molded bodies having high mechanical strength and transparency |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4108846A (en) * | 1977-02-01 | 1978-08-22 | Hoffmann-La Roche Inc. | Solid phase synthesis with base N alpha-protecting group cleavage |
-
1986
- 1986-10-21 DE DE19863635670 patent/DE3635670A1/en not_active Withdrawn
-
1987
- 1987-10-14 ES ES87114992T patent/ES2052535T3/en not_active Expired - Lifetime
- 1987-10-14 EP EP87114992A patent/EP0264802B1/en not_active Expired - Lifetime
- 1987-10-14 AT AT87114992T patent/ATE83244T1/en not_active IP Right Cessation
- 1987-10-14 DE DE87114992T patent/DE3783009D1/de not_active Expired - Lifetime
- 1987-10-18 IL IL84195A patent/IL84195A/en not_active IP Right Cessation
- 1987-10-19 FI FI874586A patent/FI88031C/en not_active IP Right Cessation
- 1987-10-19 NZ NZ222204A patent/NZ222204A/en unknown
- 1987-10-20 KR KR1019870011632A patent/KR960013073B1/en not_active IP Right Cessation
- 1987-10-20 JP JP62262950A patent/JP2540564B2/en not_active Expired - Lifetime
- 1987-10-20 HU HU874713A patent/HU197719B/en unknown
- 1987-10-20 DK DK198705482A patent/DK175126B1/en not_active IP Right Cessation
- 1987-10-20 ZA ZA877862A patent/ZA877862B/en unknown
- 1987-10-20 CA CA000549680A patent/CA1340421C/en not_active Expired - Fee Related
- 1987-10-20 AU AU79935/87A patent/AU595390B2/en not_active Expired
- 1987-10-20 PT PT85952A patent/PT85952B/en unknown
- 1987-10-20 IE IE282187A patent/IE60864B1/en not_active IP Right Cessation
- 1987-10-20 NO NO874374A patent/NO172895C/en not_active IP Right Cessation
-
1993
- 1993-03-05 GR GR920402952T patent/GR3007231T3/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DK548287D0 (en) | 1987-10-20 |
| NO874374L (en) | 1988-04-22 |
| FI88031B (en) | 1992-12-15 |
| EP0264802B1 (en) | 1992-12-09 |
| EP0264802A3 (en) | 1989-07-26 |
| DK175126B1 (en) | 2004-06-07 |
| JP2540564B2 (en) | 1996-10-02 |
| ES2052535T3 (en) | 1994-07-16 |
| ZA877862B (en) | 1988-04-22 |
| NO874374D0 (en) | 1987-10-20 |
| HU197719B (en) | 1989-05-29 |
| FI874586A (en) | 1988-04-22 |
| PT85952A (en) | 1987-11-01 |
| KR880005147A (en) | 1988-06-28 |
| GR3007231T3 (en) | 1993-07-30 |
| AU7993587A (en) | 1988-04-28 |
| FI874586A0 (en) | 1987-10-19 |
| FI88031C (en) | 1993-03-25 |
| HUT46706A (en) | 1988-11-28 |
| IL84195A0 (en) | 1988-03-31 |
| AU595390B2 (en) | 1990-03-29 |
| KR960013073B1 (en) | 1996-09-30 |
| JPS63104951A (en) | 1988-05-10 |
| NO172895C (en) | 1993-09-22 |
| IE60864B1 (en) | 1994-08-24 |
| PT85952B (en) | 1990-07-31 |
| DE3783009D1 (en) | 1993-01-21 |
| EP0264802A2 (en) | 1988-04-27 |
| DE3635670A1 (en) | 1988-04-28 |
| ATE83244T1 (en) | 1992-12-15 |
| NO172895B (en) | 1993-06-14 |
| IE872821L (en) | 1988-04-21 |
| DK548287A (en) | 1988-04-22 |
| IL84195A (en) | 1992-08-18 |
| NZ222204A (en) | 1989-10-27 |
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| Date | Code | Title | Description |
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| MKLA | Lapsed |
Effective date: 20140310 |