Journal of Medicinal Chemistry J
J
@ Copyright I966
the American Chemienl Soeiety
Synthesis of l-~-Penici~~amine-oxytocin, l-D-PeniCillamine-OXytOCin, and 1-Deaminopenicillamine-oxytocin, Potent Inhibitors of the Oxytocic Response of Oxytocin' HORSTSCHCLZ AND
171XCENT DU
VIGNEllUD
Department of Biochemistry, Cornell University Medtcal College, Sew Y o r k , .17ew York 10021 Received M a y 18, 1966 1-GPenicillamine-oxytocin has been prepared by reaction of p-nitrophenyl N-carbobenzoxy-S-benzyl-tpenicillaminate with L-tyrosyl-L-isoleucyl-~-glutaminyl-casparagin~~l-S-benzyl-~-cysteinyl-~prolyl-cleucylglycinamide followed by reduction of the resulting protected nonapeptide with sodium in liquid ammonia and oxidation to the cyclic disulfide. I n the 1-cpenicillamine-oxytocin the /3 carbon of the half-cystine residue a t position 1 of the hormone is substituted by two methyl groups. 1-LPenicillamine-oxytocin and I-D-penicillamine-oxytocin were both obtained by separation through partition chromatography on Sephadex of the diastereoisomeric analogs resulting from a similar series of reactions starting with N-carbobenzoxy-Dcpenicillaminate. I n addition, 1-deaminopenicillamine-oxytocinwas synthesized, with the use of P-benzylmercaptoisovaleric acid in place of N-carbobenzoxy-S-benzylpenicillamine. All three of these analogs were found to be devoid of avian vasodepressor, oxytocic, and pressor activities. On the other hand, they possessed inhibitory activity on the avian depressor and oxytocic activities of oxytocin. The I-bpenicillamine-oxytocin and l-deaminopenicillamineoxytocin were particularly potent inhibitors of the oxytocic activity of the hormone. The D analog was less potent than the L compound.
benzyl-DL-penicillamine was allowed to react wit'h the As a further study of the importance of the specificity free S-benzyloctapeptide amide (11) obtained from t,he of the structure of the amino acid residue a t position 1 protected octapeptide amide (I) by removal of the PI;in oxytocin (Figure 1) to its pharmacological behavior, carbobenzoxy and 0-benzyl groups with hydrogen we thought it would be of interest to ascertain the bromide in trifluoroethanol. The resulting mixture of pharmacological properties of an analog of oxytocin in the two diastereoisomeric prorected nonapeptides was which the half-cystine residue a t position 1 of oxytocin is replaced by that of L-penicillamine. Thus, the two subjected to treatment with sodium in liquid ammonia by the method of Sifferd and du Vigneaud,4 as used in hydrogens attached to the 0 carbon of the half-cystine the synthesis of oxytocin5to cleave the hT-carbobenzoxy residue at position 1would be substituted by two methyl and S-benzyl groups. The resulting disulfhydryl comgroups. We have therefore prepared 1-L-penicillaminepounds were oxidized to the cyclic disulfides in neutral oxytocin along with its diastereoisomer, 1-D-penicillamine-oxytocin. I n addition, we have synthesized 1aqueous solution with potassium ferricyanide.6 After removal of the ferricyanide and ferrocyanide ions, the deaminopenicillamine-oxytocin (I-0-mercaptoisovaleric crude mixture of the diastereoisomeric cyclic octaacid-oxytocin) . peptide amides was freed of gross impurities by subjecSince it has been found in this laboratory that oxytion to countercurrent distribution' in t'he solvent systocin and its diastereoisomer, 1-hemi-&cystinetem 1-butanol-pyridine-benzene-O.l% aqueous acetic oxytocin, can be separated from one another2 both by acid (6:1:2:9). The material in the peak with a K partition chromatography on Sephadex and by countervalue of 1.4 representing the two octapeptide amide current distribution, there seemed to be a good possianalogs was isolated and subjected to partition chromability that one might be able to obtain both 1-L-peniciltography on Sephadex G-2s8in the solvent' system 3.5% lamine-oxytocin and 1-D-penicillamine-oxytocin starting from DL-penicillamine. For the preparation of these aqueous acetic acid (containing 1.5% pyridine)-1butanol (1: 1) in which a separation of the two diastereotwo diastereoisomeric analogs, the protected octapeptide amide N-carbobenzoxy-0-benzyl-L-tyrosyl-L-iso- isomeric analogs was achieved. Two peaks were obleucyl-L-glutaminyl-L-asparaginyl- S - benzyl- L - cystein(4) R . H. Sifferd and V. du Vigneaud, J . B i d . Chem.. 108, i 5 3 (1935). yl-L-prolyl-L-leucylglycinamide (I), prepared by the (5) V. du Vigneaud, C. Ressler, J. &I. Swan, C. W.Roberts, P. G. Katstepwise nitrophenyl ester method, served as starting soyannis, and S. Gordon, J . A m . Chem. Soc., 76, 4879 (1953); V. du Vigneaud, C. Ressler. J. M . Swan, C. W. Roberts, and P. G . Katsoyannis, ibid., 76, material. The nitrophenyl ester of N-carbobenzoxy-S(1) This work was supported in part by Grant HE-01675 from the National
Heart Institute, U S. Public Health Service (2) D. Yamashiro, D. Gillessen, and V du Vlgneaud, J A m . Chem SOC., 88, 1310 (1966). (3) M. Rodansaky and V. du Vlgneaud, zbzd , 81, 5688 (1959).
3115 (1954). ( 6 ) D. B. Hope, V. V. S. Murti, and V. du Vigneaud, J . Biol. Chem., 261, 1563 (1962). (7) L. C. Craig, W. Hausmann, E. H . Ahrens, Jr., and E . J . Harfenist, Anal. Chem., 23, 1236 (1951). (8) D. Yamashiro, Nature, 201, 76 11964).
NH2 0
II
I
C~HIOH
CzHs
I CHI 0 I I1
CH-CHI
I I
CHI-CH-C-NH-CH-C-NH-CH 1 1 2 S
I
31
c=o
0
S
I/
I 6 11 6 C H1-C H-NH-C-CH-NH-C-CH-(
I c=o C Hz-N
I /
C Hz-C
4
I CH2)2-C ONHz
CHI
I
CONHz
0 \7
NH
I
I
I
I
0
II
8
0
II
9
CH-C-NH-CH-C-NH-CHz-CONHZ
Hz
I
CI Hz CH(CH~)?
Figtire l.--Structure of osytociii, rvith numbers indicathy the posit ion of the individiinl nniirio a d residues.
t:iined ab determined by the Polin-Lowry color value\," one with an R f of 0.29 and the other with :in R f of 0.4j. In order to establish thc identity of the t1i:Lstereoisonicrh represented by the two peaks, L-pciiic~illaniiri(.oxytocin was prepared. With the use of thii :Lutheritiv saniple the peak with an R f of 0.29 IWS eitabliihed :irepresenting the L andog. Thc. l-r)-peiiic~illaiiii~i(~oxytocin ( R f0.46) was subjected to further purification. The material was rechrom:itogr:iphcd on Sep1i:iclc.i (2-2: in the solvent system #3..;"/c :iqucoui avetir :icltl (containing 1.3% p~ridirie)-l-but:~r~ol-t~ctizc~ie (9 . 1 . 10) (R, 0.31). It is interesting to note that of the two periic~i1l:iiiiitit~ uidogs the diastereoisomer c~ont:iiliing the r) :imino ;wid residue is more soluble in the organic* phaw i n countercurrent distribution :tiid partition c.hroni:itography as in the case of l-henii-D-c.ystiiit.-oxytoc.iii :uid oxytocin.2 This same relatioii5hip with respevt t o the greater Folubility in the organic phase of the 11 diastereoisomer in comparison with that of oxytocw was also found to be true of 2-r~-tyros11ie-oxytocin.~~' 3-n-asparagine-oxytocin," ~-n-glutamine-orytocin,'l (ihenii-D-cystine-oxytorin,12'i-n-prolint.-oxgtoi.in, ' d an(I 8-u-lruc*ine-oxytocin.l 4 I'or the preparation of the l-L-penicillsniirie-~xytoc.iii. p-nitropheiiyl ~-carbobenzoxy-S-be~~zyl-~-penicill:~ni~nate w:ts allowed to react with the aforeiiieritioiied frcc S-benzyloctapeptide amide (11). The resulting nonapeptide S-carbobenzoxy-S-benzyl-~-penicillaniinyl-~tyro,~yl-I,-isoleucyl-L-glutaniinpl-~asparaginyl-S- henzyl-L-cyst einyl-r,-prolyl-L-leucylgly~~~i:~nii~l~~ ~ .:is t cluacd :mcl oxidized as described for the inixture of the t\\ o tliastereoisomeric nonapeptidrs. The purification w i i c:irricd out by countercurrent distribution in the solvelit system l-butanol-pyridine-berlzene-O,l~oaqueous arctic wid (6:1:2:9) ( K , 1.25), followed by partitioil c1iroinntogrn~)liyon SephzLdex G-25 111 the solven t n a -
(9) 0 . 1-1. Lowry, N . J. Roselirough. .\, I,. l ' a r r , a n d K..J. Randall, .I. Uzo?. Chem., 193, 265 (1951). (IO) S. Draharek a n d V. du Vipneaud, .I.Am. C h r m . Suc., 87, 3974 (1905). ( 1 1 ) J. ,J. Ferraro and V. du Vigneaud, unpui~listieddata. [ l a ) bl. AIanning a n d V. (111 Virneaud, J . .lnz. Chem. .*ioc., 87, :iY;h ( I YG5). (1:ij J. J . VerraroandV. du Vigneaurl, i b i d . , 88, 3847 ( l Y B t i ) . ( 1 4 ) (', Sciineider a n d V, du Vignearid, it,
