J. Med. Chem. 1982,25,470-475
470
Synthesis and Biological Activities of Photoaffinity Labeling Analogues of Substance P E. Escher,* R. Couture, G. Champagne, J. Mizrahi, and D. Regoli Dgpartement de Pharmacologie, Facult6 de Mgdecine, Universitg de Sherbrooke, Sherbrooke, Qugbec, J l H 5N4, Canada. Received J u n e 16, 1981 Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNHz, SP) is an undecapeptide with important properties as a neurotransmitter and with other functions. No specific antagonists and no long-acting analogues of this peptide hormone are known to date. In order to reach these goals, analogues of SP have been prepared which contain potential affinity, as well as photoaffinity labeling functions, suitable for irreversible attachment to S P receptors. We report here the synthesis of SP analogues which have the Phe residues in positions 7 or 8 replaced with (4’-NOJPhe, (4’-NH2)Phe, (4’-Nz+)Phe,and (4’-N3)Phe. Some of these peptides are used for photoaffinity labeling studies using various bioassays. The synthesis of the (N02)Phe-containingpeptide was carried out on solid phase using Nle instead of Met and the Boc strategy up to residue 4; the remaining amino acids were added using an Fmoc strategy. The protected undecapetide was cleaved by ammonolysis, purified by chromatography on silica gel with chloroform/ methanol, and deprotected afterwards. The amino, diazonium, and azido peptides were obtained in this sequence by chemical modification of the nitro peptides. On guinea pig ileum the modified peptides in position 8 had close to maximal activity, whereas modifications in position 7 produced some reduced activity, especially the nitro modification. No diazonium peptide produced any irreversible effects on guinea pig ileum. Photoinactivation studies were carried out on strips of guinea pig trachea, but no irreversible effects have been observed, neither permanent stimulation nor permanent inactivation. The biological activities and effects are discussed in view of the molecular properties of the synthesized analogues.
Among the classical peptide hormones, such as angiotensin (AT), bradykinin (BK), adrenocorticotropin (ACTH), and vasopressin/oxytocin, substance P (SP) has a special status; it was the first to be discovered (1930, but it is the least understood. No clear physiological role has been established until very recently, and no potent specific and no competitive inhibitors are known. Earlier structureactivity studies showed the low importance of the N-terminal portion and the prominent importance of the Cterminal pentapeptide,l including the two phenylalanines in positions 7 and 8. The apparent similarities between kinins, like BK and SP, in size, chemical nature, and pharmacological actions and the recent results obtained with AT and BK photoaffinity l a b e l ~ , ~encouraged J us to try the same technique on SP in order to develop (a) specific and irreversible antagonists of SP and (b) labeling tools which might help to isolate the unknown SP receptor from target tissues. Syntheses. The syntheses of SP and of its analogues are notorious for solubility and purification problem^.^ This peptide family very readily aggregates in different solvents and renders even the most trivial purification step often into a difficult operation. The pure peptide itself is quickly oxidized, most probably on its Met residue; therefore, it is almost impossible to rely over a longer period of time on a concentrated standard solution of this peptide. We therefore decided to try a new approach which will eliminate these two principal problems. The synthesis was laid out to produce at the end a fully protected peptide, which would be soluble in organic solvents and could be purified with classical silica gel chromatography before a final and mild deprotection. The oxidation problem was eliminated by the already reported substitution of Met by Nle?p6 a change which in the literature ~~~~~
and in another case 80%6 of the in one case gave biological potency of SP. [NlellISP synthesized on several occasions in our laboratories7gave, in the same pharmacological tests, 50% relative affinity if measured in comparison to very freshly prepared SP. [NlellISP did not lose its activity upon storage as SP does: which would also explain the observed activity discrepancies with the above-mentioned literature reports. The Nlel’ substitution was also necessary because the conditions needed to modify the precursor nitro peptides into the amino, diazonium, and azido analogues exclude Met or create serious problems. For example, hydrogenation on palladium/charcoal is difficult because the thioether of Met absorbs to noble metals and poisons the catalyst. The condition of diazotization which is the only reasonable way for obtaining the diazonium and azido analogues8oxidizes Met completely. A subsequent reduction of Met sulfoxide back into Met is always difficult but possible; however, it would certainly reduce also the azido function back to the amine. The synthesis was designed to produce fully protected, soluble peptides; therefore, the synthesis on solid phase was not started on benzhydrylamine resin but on chloromethylated polystyrene. The resulting peptide-resin ester can undergo ammonolysisgand produce the fully protected peptide amide. Deprotection of the final peptide amide required protecting groups which can be cleaved under mild conditions, avoiding harsh conditions like anhydrous hydrogen fluoride (HF). The sequence Arg-Pro-Lys-ProGln-Gln-Phe-Phe-Gly-Leu-Nle-NHz([Nlel’] SP) was prepared up to residue 4 (Pro) by the classical solid-phase method uving the Boc protection group and Boc-(4’NOz)Phe instead of Phe7 or Phe8. After residue 4 the synthesis procedure was switched over to the Fmoc procedurel0 for the next two residues, leaving the N’-Boc protection of Lys intact. Incorporation of the last amino
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(1) Couture, R.; Fournier, A.; Magnan, J.;St-Pierre, S.; Regoli, D. Can. J . Physiol. Pharmacol. 1979, 57, 1427. (2) Escher, E.; Guillemette, G. J. Med. Chem. 1979, 22, 1047.
(3) Escher, E.; Laczko, E.; Guillemette, G.; Regoli, D. J. Med. Chem. 1981,24, 1409. (4) Feldman, J. A,; Cohn, M. L.; Blair, D. J. Liq. Chromatogr., 1, 833 (1978). (5) van Rieschoten, J.; Tregear, G.;Leeman, S.; Powell, D.; Niall, H.; Potts, J. T. in “Peptides”, Proceedings of 13th European Peptide Symposium, Kiryat Anaview, Israel, 1974,Y. Wolman, Ed., Wiley, New York, 1975.
(6) Chipkin, R. E.; stewart, J. M.; Sweeney, V. E.; Harris, K.; Williams, R. Arch. Znt. Pharmacodyn. 1979, 240, 193. (7) Couture, R. Ph.D. Thesis, University of Sherbrooke, Sherbrooke, Quebec, Canada, Jan. 1981. (8) Escher, E.; Schwyzer, R. Helu. Chirn. Acta 1975, 58, 1465. (9) Ives, D. A. J. Can. J . Chem. 1968, 46, 3218. (10) Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J., Makopki, R. C.; Chang, C. D., Int. J . Pept. Protein Res. 1979, 13, 35.
0022-262318211825-0470$01.25/0 0 1982 American Chemical Society
Journal of Medicinal Chemistry, 1982, Vol. 25, No. 4 471
Photoaffinity Labeling Analogues of Substance P
Scheme 1' R
R
R
R
0
0
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-
LOO
CONCENTRATION
fM)
Figure 1. Dose-response curves of [Nle"]SP and analogues. On the left panel are the biological activities of the modifications in position 7, and on the right are the modifications in position 8.
3
a
3
acid, Arg, was attempted as the bis(Boc) or even the tris(Boc) derivative; however, this failed completely. Unluckily, no other mild acid cleavable Arg derivative was available; the syntheses had to be finished with N"-BocNg-Tos-Arg, and the final deprotection after purification had to be carried out in HF anyway. For future syntheses, adamantyloxycarbonyl-protected Argil will be used, but this was not available at that time. Ammonolysis at room temperature in DMF/2-propanol was achieved in about 10 days, and the resulting crude, protected peptide was purified by chromatographyon silica gel, eluted with chloroform/methanol. The pure protected peptide was treated for several minutes with small quantities of HF, and the resulting products were subjected to gel filtration on Sephadex LH20, eluted with DMF. The two analogues [ (4'-NO2)Phe7,Nle'l]SP and [(4'NOz)Phe8,Nle11]SP,respectively, were further modified according to Scheme I, a pathway already well established with angiotensin I1 (AT),12bradykinin (BK),I3and recently neurotensin." The nitro peptide was hydrogenated to the corresponding (4'-NH2)Phe peptide, which in turn was diazotized to the (4'-Nz+)Pheanalogue and converted to the final photolabel [(4'-N3)Phe7,Nle11]SPand [(4'-N3)Phee,Nlel1]SP, respectively (see Scheme I). Biological Activities. All substances were tested on guinea pig ileum for their biological activities in vitro, and the results are presented in Tables I and I1 and Figure 1. All compounds retained much of their relative affinity compared to [NlellISP, the only exception being [(4'N02)Phe7,Nle11]SPwhich had only about 10% of relative affinity; all compounds retained full intrinsic activity. It has been shown earlier that both positions 7 and 8 are important for binding SP to its receptor,' but changes in position 8 reduce activity less than the same modifications (11) Jager, G.; Geiger, R. Chem. Ber. 1970,103,1727. (12) Escher, E.; Nguyen, T. M. D.; Robert, H.; St-Pierre, S.; Regoli, D. J. Med. Chem. 1978,21,860. ( 1 3 ) Laczko, E.; Escher, E. Helu. Chim. Acta 1981,64, 621. (14) Unpublished results, in preparation.
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472 Journal of Medicinal Chemistry, 1982, Vol. 25, No. 4
Escher et al.
Table 11. Comparison of Structure, Biological Activity, and Physicochemical Parameters of [ Nle” ] SP Analogues position 7 position 8 physicochemical p a r a m e t e d modification no. RA,“ of [Nle’l] SP no. RA“ Ue .f MR“
___
Phe 1 100 1 100 0.00 1.96 25.36 (4’-NO, )Phe 2 10 3 93 0.78 1.68 31.69 (4’-NH,)Phe 4 55 5 69 -0.66 0.73 29.75 (4‘-N, +)Phe 6 81 7 72 1.91
