Xovember 1967
1037
6-L-PENICILLAMIKE-OXYTOCIN
Synthesis and Some Pharmacological Properties of 6-~-Penicillamine-oxytocin~
6-~-Penicillamine-oxytocin has been synthesized from the protected rioriapept ide intermediate, N-carbobenzoxy-S-benzyl-L-cyateinyl-L-t~grosyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-S-benzyl-I,-penicillamiiiyl-L-prol~l-~.lekicylglycinaniide, by reductio11 with sodium in liquid ammonia and subsequent oxidation of the disiilfhydryl componnd with potassium ferricyanide. The protected nonapeptide intermediate was synthesized frorn L-prolyl-L-leucylglycinamide by use of the stepwise p-iiit'rophenyl ester method. I n contrast to 1-L-petiicillamirie-oxytocin, 6-~-penicillamine-oxytocin does not show an inhibitory effect on the oxytocic act,ivity of oxytocin. 6-~-Penicillamine-oxytocin possesses approximately 2.5 units/mg of oxytocic activity. On the other hand, 6-~-penicillamine-oxytocin exhibits a somewhat greater inhibitory effect on the avian vasodepressor act ivity of oxytocin than that shou-n by 1-L-penicillamine-oxytocin.
with potassium ferricyanideg to the cyclic octapeptide It has been shown that the replacement of the halfamide (I), and the crude material was purified by cystine residue a t position 1 of oxytocin (Figure 1) by countercurrent distribution'O in the solvent system an L-penicillamine residue causes total loss of oxytocic 1-butanol-pyridine-benzene-O.l% aqueous acetic acid and avian vasodepressor activity and that this analog (6 : 1:2 : 9) ( K = 1.O) and also by partition chromatog(1-L-penicillamine-oxytocin) is a highly potent inraphy on Sephadex G-2511 in the solvent system 3.5% hibitor of the oxytocic activity of oxytocin on the rat uterus both in vitro and in v ~ v o .1-L-Penicillamine~ ~ ~ aqueous acetic acid (containing 1.5% pyridine)oxytocin also exhibits a slight inhibitory effect on the 1-butanol (1:1) (& 0.32). The material was then avian vasodepressor activity of oxytocin. It then subjected to gel filtration on Sephadex G-25 in 0.2 N acetic acid, and 6-L-penicillamine-oxytocin (I) emerged became of interest to determine the effect of the rein a peak at the same position as oxytocin. placement of the half-cystine residue at position 8 of oxytocin by an ~-penicillaniineresidue on the oxytocic The 6-~-penicillamine-osytocin was assayed for arid avian vasodepressor activities of the hormone. avian vasodepressor'* and rat oxytocic activites.13 In contrast to 1-L-penicillamine-oxytociii, the G-~-penicilThe present paper reports the synthesis of G-L-penicillamine-oxytocin did riot show any inhibition of the lamine-oxytocin (I), i n which the two hydrogens atoxytocic activity of oxytocin and in fact the 6-~-pentached to the fl-carbon of the half-cystine residue at icillamine-oxytocin possessed 2.7 position 6 of oxytocin are replaced by two methyl 0.5 units/mg of groups. oxytocic activity. On the other hand, the 8 L-penicilFor the synthesis of G-~-penicillamine-oxytocinthe lamine-oxytocin exhibited a somewhat greater inhibiprotected nonapeptide S-carbober~zoxy-S-berlz~-l-~- tory effect on the avian vasodepressor activity of oxgcysteinyl-~-tyros~-l-~-isoleucylL- glutaminyl- L - asparatocin than that shown by the 1-L-penicillamine-oxyt o ~ i n . Thus ~ giriyl-S-be~izyl-~-periicil!aminyl-~-prolyl-~-leuc~-Iglyci~~we have observed somewhat different amide (11) was prepared by the use of the stepeffects on the oxytocic and avian vasodepressor activwise p-nitrophenyl ester method as employed in the ities of oxytocin, depending upon whether the hydrogens synthesis of oxytocin.5 L-I-'rolyl-L-leuc~lgl~cirlamide6 on the /3-carbon of the half-cystine residue at position served as the starting material for the series of reactions, 1 or position 6 are replaced by methyl groups. in which the first step involved the coupling of this tripeptide n-ith p-nitrophenyl K-carbobenzoxy-S-henExperimental SectionI4 zyl-~-penicillaminate.~All of the intermediate peptides containing the S-benzyl-~-periicillamirle residue N-Carbobenzoxy-S-benzyl-~-penicillaminyl-~-pro~yl-~-leucylwere found to be more soluble in organic solvents than glycinamide.-L-Prolyl-L-leucylglycinamide6 (4.2 g) was disthe corresponding peptides containing the S-benzylsolved in 15 ml of dimethylformamide (DMF) and p-nitrophenyl N-carbobenzoxy-S-benzyl-~-penicillaminate~ (8 g) was added. L-cysteine residue. The protected noriapeptide (IT) After the solution was stirred for 3 days at room temperature, 100 was reduced with sodium in liquid ammonia to remove ml of ethyl acetate was added, and the solution was washed ten the protecting groups by the method of Sifferd and du Vigneaud7 as used in the original synthesis of oxytocin.8 The disulfhydryl compound was oxidized a t pH 6.5-7.0
+
( 8 ) V. du Vigneaud, C. Ressler, J. 11. Swan, C . JV. Roberts, P. G. Ratsoyannis. and S.Gordon, J . Am. Chem. Soc., 76, 4879 (1953); V. dii Vigneaud. C. Ressler, J. RZ. Swan, C. IT. Roberts, and P. G. Batsoyannis. (bid., 76,
(1) T h i s work %-as srrpported in part by Grant HE-01675 from the Xa-
tional Heart Institute. U. S. Public Health Service. (2) To whom correspondence should be sent a t Department of Chemistry, Cornell University, Ithaca, N. T. 14850. (3) H. Schulz and V. du Vigneaud. J . .Wed. Chem.. 9, 617 (1966). (4) IT. I-.Chan, R. Fear, a n d V. du Vigneaud, Endocrinology. in press. ( 5 ) 11. Bodanszky and V. du Yigneaud, J . .Im. Chem. Soc., 81, 5688 (1959). (6) XI. Zaoral and J. Rudinger, Collection Czech. Chem. Commun., 2 0 , 1183 (1955); R . .1.Boissonnas, S.G u t t m a n n , P.-A. Jaquenoud, and J.-P. \\-aller. Helu. Chim.Acta, S8, 1491 (1955); M-,B. Lutz, C . Ressler, D. E. Kettleton, Jr., a n d V . d u Vigneaud, J Am. Chem. SOC., 81, 167 (1959). (7) R. H . Sifferd a n d V. d u Vigneaud, J . Biol. Chem., 108, i 5 3 (1935).
3115 (1954). (9) D. B. Hope, 1'. S.hlurti, and V. d u Vigneaud, J . B i d . Chem., 237, 1563 (1962). (10) L. C . Craig, W. Hausmann, E. H. .ihrens, Jr.. and E. J. Harfenist, A n a l . Chem., 23, 1236 (1951). (11) D. Yamashiro, Nature, 201, 76 (1964). (12) .4vian vasodepressor assays were performed on conscious chickens
V.
according t o the procedure employed by R. d.Munsick, W.H . Sawyer, and H. B. van Dyke, Endocrinology, 66, 860 (1960). (13) Oxytocic assays were performed according to the method of P. Holton, Brit. J . Pharmacol., 3, 328 (1948), on uteri from rats in natural estrus with the use of Mg-free van Dyke-Hastings solution aa employed by R. A. Nunsick, Endocrinology, 66, 451 (1960). (14) Capillary melting points were determined and are corrected.
16-HETERO h
Soveniber 196'7
m
1i-0xa-D-Hoaio STEROIDS
1039
The Synthesis, Stereochemistry, and Biology of 16-Hetero and 17-Oxa-D-homo Steroids' JOHX S.l
3 - 4 ~ ~ 4 ~
Ilir i ~ i o no r Chemical Research, G . D Seaile B Co., Skol; w, Illinois
Rerelied A p r ? l , 13, 1967
The synthesis, stereochemistry, and biological activities of lBoxa, 16-aza-, and li-ma-D-homo hteroids and related seco steroids derived from estrone 3-methyl ether, (i)-13-ethyl-3-methosygona-l~3,5( lO)-trien-l i-one, and 14-isoeqidenin are presented.
Stamler, A\Iarmiston, Oliver, and others? have presented evidence that estrogens play a significant role in human female resistance toward atherosclerosis by virtue of their ability t o alter serum lipid concentrations. However, the effect of estrogens on secondary iex characteristics is an obvious deterrent to any therapeutical value they niay have in man. At our laboratories, there has been a conslstent effort t o obtain a qubstance which might inimic estrone (la) or 17pestradiol (lb) in its ability to alter blood fat patterns in animals without affecting the reproductive organs. h program which began with the inveitigation of new ring-D seco bteroidb related to bonie eytrogeriolic an cx:tmplc of which i< doisyiiolic acid (8), led to the byrithesis of 1B-oxaestra-l,B,j (10)-triene-3, li-diol 3methyl ether (14b), a substance nhich. 111 the rat, ha5 significant effect3 on blood lipids arid which i q devoid of
HO la,R=O
b,R'
