Oct. 20, 1964 [ C O X T R I B U ~ K I XFROM THE
4477
ANALOGS OF OXYTOCIN DEPARTMEXT O F BIOCHEMISTRY, CORNELL UXIVERSITY MEDICAL COLLEGE,
NEW Y O R K
21,
XEW YORK]
Analogs of Oxytocin Containing Glycine in place of Tyrosine, Isoleucine, or Glutamine’ BY STEFANIA DRABAREK~ RECEIVED J U N E 5 , 1964 Three analogs of oxytocin in which the tyrosme, isoleucine, and glutamine residues in positions 2, 3, and 4 of the peptide chain are replaced, respectively, by a glycine residue have beeii synthesized and tested for some of the pharmacological properties characteristic of oxytocin. The required protected nonapeptide intermediates were prepared by use of the azide method and in the case of Zglycine-oxytocin and 4-glycine-oxytocin the nonapeptides were also prepared by the stepwise p-nitrophenyl ester method. After removal of the protecting groups from the nonapeptides by means of sodium in liquid ammonia, the dithiols thus obtained were oxidized t o 2-glycine-ou>-tocin,3-glycine-nxytocin, and 4-glycine-oxytocin 4-Glycine-oxytocin possessed approximately 5 units per mg. of avian depressor activity, 3 units per mg. of oxytocic activity, 17 units per mg. of milk-ejecting activity, and negligible pressor and antidiuretic activities. Tne other two analogs were practically inactive.
benzyl-N-carbobenzoxy-L-c y s t ei n yl-L-t y r o s yl-L-i soI n connection with a series of s t dies in this laboraleucylglycyl-L-asp a r a g i n y l-S-b e n z y l-L-c y s t ei n y l-Ltory on the significance of the chemical functional prolyl-L-leucylglycinarnide. The free heptapeptide with groups of oxytocin (Fig. 1) to its biological activities,3 isoleucine a t the amino end of the peptide chain, an analog of the hormone was prepared in which the carboxamide group of the glutamine residue in the 4- obtained during the course of this synthesis, was also coupled with the azide prepared from Sposition of the molecule was replaced by hydrogen, benzyl-N-carbobenzoxy-L-cysteinyl-L-tyrosinehydranamely 4-decarboxamido-oxytocin. In this comzide7 according to the procedure described by pound an L-a-aminobutyric acid residue replaces the Honzl and Rudinger8 to give the same protected L-glutamine residue present in the hormone. T h u s nonapeptide. in effect an ethyl group is attached to the carbon of the glycine residue in position 4 of the backbone strucFor preparation of the protected nonapeptide for the ture of oxytocin. The 4-decarboxamido-oxytocinwas synthesis of 3-glycine-oxytocin the S-benzyl-N-carbofound to possess approximately one-fifth of the avian benzoxy-L-cysteinyl-L-tyrosine azide was allowed t o depressor and one-sixth of the oxytocic activity of react with glycyl-L-glutaminyl-L-asparaginyl-S-benoxytocin, while its pressor and antidiuretic activities zyl-L-cysteinyl-L-prolyl-L-leucylglycinamide.The rewere extremely low. In the light of the relatively high quired heptapeptide was itself prepared from Lavian depressor and oxytocic potencies of this analog, glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyli t was decided to investigate the effect of replacing the L-leucylglycinamideg,loby reaction with p-nitrophenyl ethyl grouping by hydrogen. The present paper recarbobenzoxyglycinate followed by removal of the ports the synthesis and pharmacological properties of carbobenzoxy group. this 4-glycine-oxytocin, as well as two other analogs of L-Isoleucyl-L-glutaminyl-z-asparaginyl-S-benzy1-Loxytocin, 3-glycine-oxytocin and 2-glycine-oxytocin, cysteinyl-L-prolyl-L-leucylglycinamidell served as in which the glycine residue replaces an isoleucine and starting material for preparation of the proa tyrosine residue, respectively. tected nonapeptide intermediate for the synthesis For the preparation of the protected nonapeptide for of 2-glycine-oxytocin. T h e glycine and cysteine resithe synthesis of 4-glycine-oxytocin the protected pentadues were linked to the heptapeptide by the stepwise peptide amide, carbobenzoxy-L-asparaginyl-S-benzylp-nitrophenyl ester method in one route, and in the L-cystein yl-L-pro1yl-L-leucylglycinamide> served as hysecond S-benzyl-N-carbobenzoxy-L-cysteinylglycine starting material. rlfter removal of the carhobenzdrazideI2 was converted to the azide and allowed to oxy group by treatment with HBr in glacial acetic react with the heptapeptide and form the required proacid, the peptide chain was lengthened stepwise by tected nonapeptide intermediate, S-benzyl-N-carbosuccessive reactions with the appropriate protected 9-nitrophenyl amino acid esters in a manner similar benzoxy-L-cysteinylglycyl-L-isoleucyl-L-glu t a m i n y 1-Lto t h a t used by Bodanszky and du Vigneaud for the asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-L-1 e u c y I g 1ysynthesis of oxytocin.j The nitrophenyl esters of cinamide. It should be noted t h a t the free octacarbobenzoxyglycine,6 carbobenzoxy-~-isoleucine~~ 0- peptide and protected nonapeptide intermediates in benzyl-N-carbobenzoxy-L-tyrosine,j and S-benzyl-Nthe synthesis of 2-glycine-oxytocin did not dissolve but carbobenzoxy-~-cysteine~ were employed successively formed a thick gel in dimethylformamide. to obtain the required protected nonapeptide, SThe protecting groups of the nonapeptide interme(1) This work was supported in p a r t hy G r a n t HE-01675 from t h e S a diates were removed by treatment with sodium in liquid tional H e a r t Institute, U. S. Public Health Service. (2) Acknowledgment is made of a grant under the United States Governammonia by the method of Sifferd and d u Vigneaudi3 ment International Exchange Program and of a Fulbright Travel G r a n t . (3) T h e biological activities of a highly purified sample of synthetic oxytocin have been recently reported by Chan and du Vigneaud [Endocrinology, 71, 977 ( 1 9 6 2 ) l . T h e values obtained are given in Table I in t h e present paper, ( 4 ) V . du X‘igneaud, G. S. Denning, J r . , S. Drabarek, and W. Y. C h a n , J . Bioi. C h e m . , 233, PC1560 ( 1 9 6 3 ) . 239, 472 (1964). ( 5 ) M . Bodanszky and V. du Vigneaud, .Va!u?e, 183, 1324 (1959); J . A m . 81, 5688 (1959). ChPm. SOL., ( 6 ) M. Bodanszky and V. du Vigneaud, “Biochemical Preparations,” Vol. 9, M . J. Coon, E d . , John Wiley and Sons, Inc., New York, N . Y . , 1962, p. 110.
(7) C . W. Roberts, J . A m . Chem. Soc., 76, 6203 (1954) ( 8 ) J. Honzl and J. Rudinger, Coiieclion Czrch. Chem. C o m m u n . , 26, 2333 (1961). (9) R . A . Boissonnas, S. G u t t m a n n , P . - A . J a q u e n o u d , and J:P. Waller, H e h . C h i m . Aciu, 38, 1491 (1933). ( I O ) J. Rudinger, J . H o n z l , and M . Zaoral, Goliecison C w c h . C h e m . C o m m u n . , 21, 202 (3956). (11) D . B. Hope and V . d u Vigneaud, J . Bioi. C h e m . , 237, 3146 (1962). (12) K C. Hooper, H. N . R y d o n , J. A. Schofield, and G S. H e a t o n , J . Chem. S o c . , 3148 (1956) (13) R . H. Sifferd and V. du Vigneaud, J . Bioi. C h e m . , 108, i 5 3 (1935).
STEFANIA DRABAREK
4478
I
CsHiOH
CsHs
CHs 0
cn-cHa
I i
o ll
/I
I
CH1-CH-C-NH-CH-C-KH-CH
sI
L o
2
i
I
0
S 1
6
I1
i
"
0
6
11
4
I
CHs-CH-NH-C-CH-NH-C-CH-(CH2)s-CONH, ~
c=o CHs--N
I
I
I
c n2 I
CONHi
\'CH-C-NH-CH-C-NH-CH1-CONH, oI1 ll 9 I /
CHS-CHz
8
0
C H?
I
CH(CH3)s
Fig. 1,-Structure of oxytocin with numbers indicating the position of the individual amino acid residues.
as used in the synthesis of oxytocin.'l T h e dithiols so obtained were oxidized in neutral solution by aeration with carbon dioxide-free air followed by titration with a solution of potassium ferricyanide.'j After removal of ferrous and ferric ions by means of resin the oxidized products were subjected to countercurrent distribution in the solvent system l-butanol-l-propanol-O.5% acetic acid containing 0.1% pyridine ( 6 : 1 :8)l8and then isolated by concentration and lyophilization. 4-Glycine-oxytocin had a partition coefficient ( K ) of approximately 0.8, whereas the partition coefficients of 3-glycine-oxytocin and 2-glycine-oxytocin were very low, approximately 0.11 and 0.18, respectively. The latter two analogs of oxytocin were much less soluble in the organic phase than the 4-glycine-oxytocin and hence moved very slowly on countercurrent distribution. The three analogs were subjected to partition chromatography on Sephadex G-25 by a procedure patterned after t h a t devised by Yamashiro for the purification of o x y t o ~ i n . ' ~The highly purified samples of the three analogs gave the expected amino acid and elemental analyses. The glycine analogs were assayed for pharmacological activity under the direction of Dr. W , U.Chan of this laboratory. The 2-glycine-oxytocin and 3-glycineoxytocin had no appreciable avain depressor, l8 rat uterine-contracting, l 9 or rat pressorzo activity. The 4-glycine-oxytocin, however, was found to possess approximately 5 units per mg. of avian depressor activity, 3 units per mg. of rat uterine-contracting activity, 17 units per mg. of rabbit milk-ejecting activity,21 and negligible rat pressor and antidiuretic22activities. ( 1 4 ) V , du Vigneaud, C . Ressler, J. M Swan, C . W. Roberts, P. G . K a t soyannis, and S. Gordon, J . .Am. Chem. Soc., 76, 4879 (1953); V . d u Vigneaud, C. Ressler, J. M Swan, C . W . Roberts, and P. G. Katsoyannis, i b i d . , 76, 3115 (lLj4). (1.5) V , d u Vigneaud, G. Winestock, V. V. S. Murti, D. B . Hope, and R . I ) . Kimbroiigh, Jr., J Biol. Chem , 236, PC64 (1960); D. B. Hope, V. V , S. hlurti, and V , d u Vigneaud, ibid.,237, 1,563 (1962). (16) T h e use of solvent systems containing pyridine in the countercurrent distribution of oxytocin and related compounds was found by Dr. Derek Jarvis of this laboratory t o be advantageous in avoiding emulsification and consequent time loss. (17) D. Yamashiro, . V a t w e , 201, 76 (1964). (18) R . A . Llunsick, W , H . Sawyer, and H. B. van Dyke, Endocvinology, 6 6 , 860 (1960). (19) P . Holton, Bvtt. J . Phovmacol., 3 , 328 (1948); R . A . Munsick, Endocviiioiopy, 66, 451 (1960) (20) "The Pharmacopeia of the United States of America," 16th Revision, Mack Printing Co., Easton, P a , 1960, p. 793. (21) B.A . Cross and G. I\' Harris, J . Endocrinol , 8, 148 (1952); H . B. van Dyke, K . Adamsons, Jr., and S. L. Engel, Recent Progr. Hovmone R e s . , 11, 1 (19%). ( 2 2 ) W . A . Jeffers, 11. M. Livezey, and J . H . Austin, Pvoc. SOL. E x p t l . Biol. \ f e d . , SO, 181 (1942); U'. H. Sawyer, Endocrinology, 63, 694 (1958).
Vol. SB
Experimental Carbobenzoxyglycyl- L-asparaginyl-S-benzyl-L-cysteinyl-1,prolyl-L-1eucylglycinamide.-A suspension of 5 g. of finely powdered carbobenzoxy-L-as~,araginyl-S-benzylprolyl-L-leucylglycinamidej in 17.5 ml. of glacial acetic ;ic.itl was treated with 17.5 ml. of HBr in glacial acetic acid ( 2 Z f I L V . / \ I - . ) , After 2 hr. a t room temperature the solution was pourctl into 150 ml. of cold dry ether. The precipitated hydrobroniitle ( i f the free base was washed by decantation with three 100-id. p~irtioris of ether. After being dried in w z m o over potassium hydroxide and calcium chloride for several hours the hydrobroiiiitlc IV:LS dissolved in i 5 mi. of dry methanol and passed through a coluinn of ion-exchange resin IRh-410 ( O H form). The column WLS washed with 50 nil. of methanol. The solid (Jbtailied d t c r evaporation of the solvent from the eluate and washing w a s dissolved in 20 ml. of diinethylformamide and 1.99 g. of p-nitrophenyl carbobenzosyglycinate was added. .\fter 2 (lays a t room temperature the product was precipitated with 200 n i l . of ethyl acetate, collected, washed with 100 ml. of ethyl acetate, 50 ml. of ethanol, and 30 ml. of ether, and dried to coiiit:trit weight over PnOi in vacuo a t 56", giving 3.5 g . , m.p. 20:3--20
