Structure-activity studies on the C-terminal amide of substance P

Nov 1, 1982 - Olivier Le Marec , Cindy Neveu , Benjamin Lefranc , Christophe Dubessy , Jean A. Boutin , Jean-Claude Do-Régo , Jean Costentin ...
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J. Med. Chem. 1982,25, 1317-1321

Structure-Activity Studies on the C-Terminal Amide of Substance P1 Emanuel Escher,*i? Rgjean Couture,? Constantinos Poulos,* Nikos Pinas,$ Jacques Mizrahi,? Dimitrios Theodoropoulos,* and Domenico Regolit Department of Organic Chemistry, University of Patras, Patras, Greece, and Department of Physiology and Pharmacology, University of Sherbrooke, Sherbrooke, Quebec J I H 5N4, Canada. Received January 22, 1982 Twelve C-terminal heptapeptide analogues of substance P have been synthesized by solid phase and by the classical solution method. The modifications concerned all the C-terminal primary amide of SP and should therefore help to understand the biological significance of this carboxamide, as evaluated by in vivo and in vitro bioassays. From the results it can be seen that not the slightest change of the two amide protons is tolerated without an important loss of activity: replacement of one or two amide protons with alkyl groups, extension of the amide to the hydrazide and its alkyl analogues, and exchange of the amide with an ester or a carboxylic acid all reduce the relative activity/affinity a t least by 2-fold. It is not clear for what reason all these modifications produce such a drastic activity reduction.

Scheme I In a previous study2we have investigated the biological importance of the primary amides on-the glutamineresidues in positions 5 and 6 of substance P (SP, Arg-ProBoc-Gln-Gln-Phe-Phe-Gly-Leu-NIe-O-CH2-C~H4 Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NHJ. No special function could be found for the side-chain amide in position 5, while in position 6 some increase of the in vivo biological activity was observed with more lipophilic amide DEA-MeOH substitutions. Moreover, all attempts to remove the Boc -GI n - G In- P he - Ph e - GIy - Leu - NIe - OC H3 carboxy terminal Met-NHz resulted in peptides devoid of I MeOH 2 NH?NH2 SP-like a ~ t i v i t y . ~ The only modifications that were tolBoc-Gln-Gin-Phe-Phe-Gly-Leu-Nle-NHNHZ erated were replacements of Met by Nle, Ala, and Leu, which, however, produced variable loss of activity. Boc-Gln-Gln-Phe-Phe-Gly- Leu-Nle-NHz Alal1-SP4retains about 10-20% of affinity, compared to SP, and Led1-SP retains 26%6and Nlell-SP retains 50% Boc-Gln-Gin-Phe-Phe -Gly-Leu-Nle-NHEt of affinity, according to the most recent findings.6 The NHEtZ replacement of the carboxamide group by a carboxylate Phe - GI y - Leu -Nle - NEt2 results in an analogue almost inactive in all b i o a s ~ a y , ~ J ~ Boc-Gln-Gin-Phe~~~ but the methyl ester analogue of SP is reported with H-Gln-Gln-Phe-Phe-Gly-Leu-Nle-OH considerable but somewhat divergent biological activities on the same bioassays.8J0 TLC. The identity of the peptides was mainly shown by We therefore decided to carry out a more detailed the reversed-phase TLC, which showed large differences analysis of the C-terminal amide in order to learn more between, for example, the methyl ester 9 and the amide about the significance of this amide and its contribution to the hormone-receptor interaction. The C-terminal amide was modified in two independent series of peptides, (1) Symbols and abbreviations are in accordance with the recommendation of the IUPAC-IUB Commission on Biochemical one derived from the heptapeptide SP(5-11) and the other Nomenclature [J. Biol. Chem., 247, 977 (197111. Other abfrom Nlell-SP(5-11). In both series the primary amide breviations used are: SP; DMF, dimethylformamide; THF, was mono- and dialkylated, replaced by the carboxylate tetrahydrofuran; TFA, trifluoroacetic acid; DCC, dicycloand by an ester, and extended as a hydrazide. The biohexylcarbodiimide; DCU, dicyclohexylurea; HOBt, l-hydroxylogical activities were established with the two standard benzotriazole; DCHA, dicyclohexylamine;ONP, n-nitrophenol; DEA, diisopropylethylamine; TEA, triethylamine; NMM, N tests, in vivo, the hypotensive effect on the rat blood methylmorpholine. pressure, and in vitro on guinea pig ileum. Some of these (2) N. Pinas, C . Poulos, R. Couture, J. Mizrahi, D. Regoli, D. compounds have been already preliminarily communiTheodoropoulos and E. Escher, Eur. J. Med. Chem., in press. cated.ll (3) N. Yanaihara, C. Yanaihara, M. Hirobashi, H. Sato, Y. Lizuka, Syntheses. The C-terminal analogues of MetWP(5T. Hashimoto, and M. Sakagami, "Substance P", U. S. von 11)were synthesized by the classical solution method using Euler and B. Pernow, Eds; Raven Press, New York, 1977, p 22-44. a stepwise chain elongation from the C terminal combined (4) R. Couture, A. Fournier, J. Magnan, S. St-Pierre, and D. Rewith fragment couplings. The analogues of Nle'l-SP(5-11) goli, Can. J. Physial. Pharmacal., 57, 1427 (1979). have been prepared by the solid-phase method; the pro( 5 ) J. Leban, G. Rackur, I. Yamaguchi, K. Folkers, U. Bjorkroth, tected heptapeptide sequence was built up on chloroS. Rosell, N. Yanaihara, and C. Yanaihara, Acta Chem. Scund., methylated polystyrene resin, and, according to the desired Ser. B, 33, 664 (1979). peptide, different methods were used for the peptide-resin (6) E. Escher, R. Couture, G. Champagne, J. Mizrahi, and D. Regoli, J. Med. Chem., 25, 470 (1982). cleavage (see Scheme I). All peptides were purified by (7) E. A. Mroz and S. E. Leeman, Vitam. Horm., 35, 209 (1977). gel filtration and partition chromatography and were (8) M. A. Cascieri, M. M. Goldenberg, and T. Liang, Mol. Pharidentified with the standard analytical procedures. The macol., 20, 457 (1981). purity and identity of the products from the classical so(9) M. Otsuka and M. Yanagisawa, Advances in Pharmacology lution method were shown by amino acid analysis, eleand Therapeutics, Proceedings of the International Congress mentary analysis of products and fragments, and by TLC. of Pharmacology, 7th, Paris, 1978, Pergamon Press, Oxford, The products from the solid-phase synthesis were tested 1979, Vol 2, p 181-190. (10) A Fournier, R. Couture, D. Regoli, M. Gendreau, and S. Stby amino acid analysis, TLC, HPLC, and reversed-phase

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+University of Patras. University of Sherbrooke.

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Pierre, J . Med. Chem., 25, 64 (1982). (11) D. Theodoropoulos, C. Poulos, N. Pinas, R. Couture, D. Regoli, and E. Escher, In "Peptides"; D. Rich and E. Gross, Eds., Pierce Chemical Co., Rockford, IL, 1981, p 603.

0022-2623/82/1825-1317$01.25/0 0 1982 American Chemical Society

1318 Journal of Medicinal Chemistry, 1982, Vol. 25, No. 11

Escher et al.

Table I. Biological Activities of C-Terminally Modified Substance P( 5-11) Analoguesa

no.

modification of SP(5-11) Met'l -NH Met"-OCH, Met"-NHCH, Met"-N(CH,), Me t"-NHNH, Met'l-NHN(CH,), Nle"-NH,

rat blood pressure : in vivo RBP 100 1.0 5 0.2 0.4