185
[2,4-Diisoleucine]-oxytocin. A n Analog of Oxytocin with Natriuretic and Diuretic Activities'"," TICTOR J. HRUBY AND VINCESTD U V ~ G N E A U D ' ~ Department of Chemzstry, Cornell C'nizsersity, Ifhaca, *Yew York ASD
Depaifment
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14860
w.Y. C H ~ L X ' ~
Phal macology, Cornell rnzversLty X e d l c a l College, S e w Y o r k , AYew York
10021
Recezoed October 29, 196.9 [2,4-Diisoleucine]-oxytocinhas been synthesized from the requisite protected nonapeptide intermediate which was prepared by the stepwise p-nitrophenyl ester method. I n this analog of oxytocin, isoleucine residues replace the tyrosine and glutamine residues at, the 2 and 4 positions of the hormone, and thus the isoleucine residue occurs swcessivelv " at *oositions 2. 3, and 4. This conwound possesses a very low level of avian vasodepressor, oxytocic, and pressor activities, and no antidiuretic activity However, [2,4-diisoleucine]-oxytocin has a pronounced natriuretic activity and a mild diuretic effect. I
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A recent communicationZ reported that the replacement of the tyrosine residue a t position 2 of oxytocin (Figure 1) by an isoleucine residue afforded an analog ( [2-isoleucine]-oxytocin) that exhibited much higher levels of oxytocic, avian vasodepressor, and milkejecting activities than did [2-le~cine]-oxytocin.~~~ It was also found that [2-valine]-oxytocin4 is somewhat more potent in these activities than [2-leucine]-oxytocin, though conbiderably less active than [2-isoleucine]-oxtyocin. [2-Glycine]-oxytocin possesses negligible oxytocic arid avian vasodepressor activitie3.j In studies iiivolving replacement of the glutamine residue at po4tion 4 of oxytocin by a variety of aliphatic amino acids including isoleucine,6 alanine,7 valinej3 a-aniiiiobutyric acid,$ and leucine,6 analogs were obtained that possessed varying degrees of avian vasodepre++or, oxytocic, and milk-ejecting activities depending on the nature of the aliphatic side chain. [4-Valine]-oxytocin was found to be the most active, and [4-i-oleucine]-oxytocin was found to be more potent than [4-leucine]-oxytocin in these activities. [4-G1ycine]-oxytocin5 had also been synthesized and was found to posaess very lo^ avian vasodepressor, oxytocic, and milk-ejecting activities and no pressor and antidiuretic activities. All of the analogs containing aliphatic side chains at position 4 possessed small or iiegligible antidiuretic and pressor activities. [4-Leucine]-oxytoci1i possesses considerable diuretic and natriuretic effects, as well as anti-BDH activity.6z10 The aforementioned results led us to prepare [2,4diisoleuciiiel-oxytocin, in which the tyrosine residue (1) (a) This work xvas supported in part by Grants HE-11680 (VduVj and HE-09795 (U-1-C) from t h e Sational Heart Institute, U. S. Public Health Service; (b) .ill optically active amino acid residues are of the L variety; ( c ) T o whom reprint requests should be addressed; (dj Recipient of Career Development .$\yard, l-K4-HE-38,849 USPHS. (2) L. -1.Branda. V. J. Hruhy, and V. du Vigneaud, M o l . Pharmacol., 3, 248 (1967). (3) K . J6St. J . Rudinger, and F. Sorm. Collect. Czech. Chem. C o m m u n . , 28, 1706 (1963). (4) V. J. Hruhy and V. d u Vigneaud, J . M e d . Chem., 12, 731 (1969). (5) S.Drabarek, J . A m e r . Chem. Soc., 86, 4477 (1965). (6) V. J. Hruhy, G. Flouret, and V. du Vigneaud, J . Bid. Chem., 244, 3890 (1969). Boissonnas, H e l r . Chim. A c t a , 46, 1626 (1963). (7) S.Guttmann and R (8) T'. du Vigneaud. G. Flouret, and R. TTaIter, J . Bid. Chem., 241, 2093 (1966). (9) V. du Vigneaud, G. S. Denning, Jr., S.Draharek, and W. T.Chan. i b i d . , Z 3 8 , PC1560 (1963); 239,472 (1964). (10) IT. T.Clian. V. J. Hruhy, G. Flouret, and V. d u Vigneaud, Science, 161,280 (1968).
at position 2 and the glutamine at position 4 are both formally replaced by isoleucine. Thi- highly lipophilic analog contains iioleucine residue5 a t the 2 , 3, and 4 positions of oxytocin, since in the natural hormone the residue a t position 3 iz that of isoleucine. For the synthesis of [2,4-diisoleucine]-oxytocin the protected heptapeptide, N-benzyloxycarbonylisoleucylisoleucylasparaginyl - S - benz ylcyst einylprolylleucylglycinamide, used in the byiithesis of [4-isoleu~ine]-oxytocin,~ served a3 starting material. Following removal of the N-benzyloxycarbonyl group with HBr in *kOH and neutralization of the heptapeptide halt, the next re5idue, isoleucine, was added as p-nitrophenyl N-benzyloxycarboiiylisoleucinate.ll The entire procedure wab repeated and the free octapeptide was treated xvith p-nitrophenyl N-benzyloxycarbonyl-S-benzylcysteiiiatell to give the desired protected nonapeptide, N-beriz\-loxycarbonyl-S-benz~lcy~teiriylisoleucyliioleucyli~oleucyl~sparaginyl-~benzylcysteinylprolyl1euc~-lglycinamide. The nonapeptide was treated with S a in liquid SH312to cleave the benzyloxycarbonyl arid S-benzyl protecting group-, and the resulting dizulfhydryl compound was oxidized in aqueoui solution with &Fe(CS)6.13 The analog was purified as described in the Experimental Sectioii by partition chromatography on Sephadex G-2514 in two different solvent sy-teni. arid by gel filtration'j on Sephadex G-25. The [2,4-diisoleucine]-oxytocinwas assayed for avian vasodepressor,16 oxytocic," pressor,14 and anti-
(11) AI. Bodanszky and V. du Vigneaud, .\-atwe, 183, 1324 (1959): J . Amer. Chem. Soc., 81,5688 (1959). (12) R . H. Sifferd and T'. du Vigneaud, J . B i d . Chem., 108, 753 (1935). (13) D. B. Hope, V. T. S. hInrti, and V. du Vigneaud, i 5 i d . , 237, 1563 (1962). (14) D. Tamashiro. Srhture, 201, 76 (1964); D. Tamashiro, D . Gillessen, and V. du Vigneaud, J . Amer. C h e m . Soc.. 88, 1310 (1966). (15) J. Porath and P. Flodin, S a t u r e , 183, 165i (1939). (16) Avian vasodepressor assays were performed on conscious chickens according t o the method of R. A . hIunsick, W.H . Savyer, and H. R.,-an Dyke, Endocrinology, 66, 860 (1960). (17) Oxytocic assays x e r e performed according t o the method of P. Holton. Brit. J . Pharmncol., 3, 328 (1948j, a s modified by R. -1.llunsick, Endocrinoloau, 66, 451 (1960). on isolated uteri from rats in natural estrus with the use of magnesium-free Yan Dyke-Hastings solution. (18) Pressor assays ivere carried out on anesthetized male rats as described in "The Pharmacopeia of the United States of .Imerica," 17th rev. Mack Publishing Co., Easton, Pa.. 1965, p 749.
PPRIDOXOL ASALOGS
March 1970
pooled and lyophilized to give 45.75 mg of [2,4-diisoleucine]oxyt,ocin as a white powder, [ ( Y ] * ~ D-35.7" ( c 0.47, 1 .I' AcOH). Anul. ( C ~ ~ H ~ ~ X I I O C,I H, O SS ~ .) The sample was hydrolyzed for 90 hr in 6 X HC1 at 110' and analyzed on a Beckman/Spinco amino acid analyzer according The molar to the method of Spackman, Stein, and ratios obtained with glycine taken as 1.0 were: aspartic acid, 1.0; proline, 1.1; glycine, 1.0; cystine, 0.95; isoleucine, 3.0; leucine, 1.0; and SHo, 2.0. Prolonged hydrolysis wa? necessi(24) D. H. Spackman, (1958).
187
tated by the difficulty in the hydrolysis of an isoleucyl-isoleucine peptide bond.* 6 25
Acknowledgments.-The authors n ish to thank A h . H L. Aanning for the preparation of N-benzyloxycarbonylisoleucylasparaginyl - S - benz ylcyst einylprolylleucylglycinamide, A h . Joseph Albert for the elemental analyses, and Mr. Roger Sebbane for the amino acid analysis.
W.H. Stein, and S.Xoore, Anal. Chem., 30, 1190 ( 2 6 ) S Moore and IT, H. Stein, Methods E n r g m o l . , 6, 819 (1963)
Selective Modifications of the cr4-Position of Pyridoxol. and Branching of the 4-Side Chain1
I.
Extension
\v. I and G. Irick, J X e d . P h a r m . Chem., 6, 49 (1962). (10) W.Korytnyk, i b i d . , 8, 112 (1965). (11) H. Ahrens and W.Korytnyk, J . Heterocycl. C h e m . , 4, 625 (1967). (12) R. P. Singh a n d 15'. Korytnyk [ J . .Wed. C h e m . , 8 , 116 (1965)l have been using benzoyl groups for selective blockage of the &hydroxyl group. This approach had serious hmitations because of the instability of the group and other factors. (13) H. 11 JVuest, J A . Bigot, Th. J. d3B.e , a n d J. P. Wibaut, Koninll. l\-ed. A k a d . Wetenschap. Proc , Ser B 61, 160 (1958).
