Journal of Medicinal Chemistry 0 Copyright
1069 bu the American Chemical Society
VOLUME12, No. 5
SEPTEMBER 1969
Synthesis and Some Pharmacological Activities of [Z-~-Valine]-oxytocinand [2-~-Leucine]-oxytocin~~*" lTICTOR J.
HRUBYAND VINCEXT
DU VIGSEAIJD'~
DPpnrtment of Chemistry, Cornell L'niversity, Zfhaca, &YewYork
[email protected] Received M a y 6, 1969 [2-Valine]-oxytocin and [2-leucine]-oxytocin have been synthesized from the requisite protected nonapeptide intermediates, which were prepared by the stepwise p-iiitrophenyl ester method. The analogs were isolated by partition chromatography arid gel filtratiori o n Sephadex and then tested for a number of pharmacological activities. [%Valine]-oxytociri possesses approximately 7 units/mg of avian vasodepressor, 2 units! mg of oxytocic, 15 units/mg of milk-ejecting, 0.05 uiiitimg of pressor, and less than 0.01 unit/mg of antidiuretic activit,ies. [2-Leucine]-oxytocin has approximately 3 units/mg of avian vasodepressor, less than 0.5 unit/mg of oxytocic, about 7 units/mg of milk-ejecting, 0.05 unit :mg of pressor, and negligible antidiuretic activit,ies. Both of these analogs are much less active than [2-isoleucine]-oxytocin.
It was recently reported2that [2-isoleucine]-oxytocin, an analog in which the aromatic tyrosine residue a t position 2 of oxytocin (Figure 1) is replaced by the aliphatic amino acid isoleucine, possesses a considerable degree of avian vasodepressor, oxytocic, and milkejecting activities. This finding was surprising in view of the extremely low activities reported for [2-leucine]oxytocin by JoBt, et aL3 The striking difference in the biological activities of two such similar analogs of oxytocin has led us to synthesize [2-valine]-oxytocin and examine its pharmacological properties. To make possible the comparison of the pharmacological activities of [2-isoleucine]-oxytocin and [2-valine]oxytocin with those of [Zleucine]-oxytocin under the same assay conditions, the [2-leucine]-oxytocin was also prepared in this laboratory. The stepwise p nitrophenyl ester method as used in the %ynthesis of oxytocin4 and [2-isoleucine]-oxytocin2was utilized for the preparation of the requisite protected nonapeptide intermediates for [%-valine]-oxytocin arid [2-leucine]oxytocin. The preparation of the protected nonapeptide intermediate for the synthesis of [2-leucine]oxytocin by JoSt, et u Z . , ~ was accomplished by means of the azide method with the use of N-tosyl-S-benzylcysteinylleucine hydrazide and isoleucylglutaminylasparaginyl-S- benzylcys t einy lprolylleucylglycinamide. In our synthesis of [2--valine]-oxytocin and [2leucine]-oxytocin, the protected nonapeptides K-benzyl(1) (a) This n o r k v a s supported in p a r t b) Grant HE-11680 from t h e Kational Heart Institute, U S Public Health Service. (b) All optically a c t i l e amino acid residues are of t h e L variety (c) Author t o n h o m correspondence and reprint requests should be sent (2) L 4 Branda, V. J Hrub,, and 1' du Vigneaud, 2fol Phrcrmacol , S, 248 (1967). (3) E; JoSt. J. Rudinger, a n d F. Sorm, Collectton Czech Chem. Commun , as, 1706 (1963). (4) hl. Bodanszky a n d V. d u Vigneaud, S a t u r e , 183, 1324 (19.59), J . Amer Chem Soc , 81,5688 (1959). ( 5 ) E;. JoSt, J Rudinger, and F h r m , Collectton Czech. Chem. Commun , 26, 2496 (1961).
73 1
oxycarboriyl-S-benzylcystein~~lvalylisoleuc~~lglut aminylasparaginyl - S - benzylc ysteinylprolylleucylglycinamide and N- benzyloxycarbonyl - S- benzylcysteinylleucylisoleucylglutaminylasparaginyl - S - benzylcysteinylprolylleucylglycinamide were treated with S a in liquid SH36 to remove the protecting groups, and the resulting disulfhydryl peptides were oxidized to the oxytocin analogs by treatment with potassium ferricyanide.' The [Zvalinel-oxytocin and [2-leucine]-oxytocin were purified b y partition chromatography8 followed by gel filtration on Sephadex G-2Zg with the use of the solvent systems given in the Experimental Section. The four-point assay designlo was used for measurement of the pharmacological activities against the ESP posterior pituitary reference standard. Avian vasodepressor assays were performed on conscious chickens according to the procedure employed by Nunsick, et al." Oxytocic assays were performed on isolated uteri from rats in natural estrus according to the method of Holton,12as modified by JIunsick,13 with the use of magnesium-free van Dyke-Hastings solution as the bathing fluid. Xlk-ejecting activity was measured on anesthetized rabbits by the method of Cross and Harris,14 as modified by van Dyke, et al.,16 and by Chan.16 Rat pressor assays mere carried out on anes(6) R. H. Sifferd and V. d u Vigneaud, J . B i d . Chem., 108, 753 (1935). (7) D. B. Hope, 1'. V. S. Murti, a n d V. d u Vigneaud. ibid., 231, 1563 (1962). (8) D. Yamashiro, Nature. 201, 76 (1964); D.Yamashiro, D. Gillessen, and V. d u Vigneaud, J . Amer. Chem. Soc., 88, 1310 (1966). (9) J. Porath a n d P. Flodin, A'ature, 188, 1657 (1959). (10) H . 0.Schild, J. Physiol. (London), 101, 11.5 (1942). (11) R. 4. Munsick, IT. H. Sawyer, and H. B. van Dyke, Endocrinology, 66, 860 (1960). (12) P. Holton, Brit. J . Pharmacol., 3, 328 (1948). (13) R. A. Munsick, Endocrinology, 6 6 , 451 (1960). (14) B. A. Cross a n d G. W. Harris, J . E n d o c r i d . , 8, 148 (1952). (15) H.B. v a n Dyke, K. .4damsons, Jr., a n d S. L. Engel, Recent Proor. Hormone Res., 11, 1 (1955). (16) W. Y. Chan, J. Pharmacol. E z p f l . T h e r a p . , 141, 48 (1965).
September 1969
lhGIOTESSIN
acid, 1.0: proline, 1.1 ; glycine, 0.9; cystine, 1.0; isoleucine, 1.0; leucine, 2 . 0 ; and ammonia, 3.0.
N-Benzyloxycarbonylvalylisoleucylglutaminylasparaginyl-SbenzylcysteinylprolylleucyIglycinamide.-.4 soliit ion of 1.67 g of crystalline isoleucylglutaminylasparaginyl-S-benzylcysteinylprolyllen~ylglycinamide*~ in 20 ml of DAIF was stirred a t room temperature with 0.73 g of p-nitrophenyl N-beiizyloxycarbonylvaliiiate?j for 20 hr, 3 0 0 ml of EtOAc was added, and the slurry was cooled to -20'. The precipitate was filtered off arid washed with 73 ml of EtOAkc
