Xoveinber 1969
OXYTOCIN ANALOGS
10%
The Synthesis and Some Pharmacological Activities of [4-~-Norvaline]-oxytocin and [4-~-Norleucine]-oxytocinand Their Deamino Analogs""'
[4-So~valiiie]-osyto~.in,[l-deamino,4-iiorvaliiie!-ox~toci11, [4-1ioi~leuciiie]-c~xyt~~~~iti, a i d [l-dearuitlo,i-tt~~rleucine]-oxytocin have been synthesized fiorn the requisite protected polypeptide precwaors, which were prep:ired by the stepwise p-nitrophenyl ester method. The cornpoutids were isolated by couiitnrctureiit di~.t,iit)iiti(~ti followed by partition chromatography on Sephadex G-25 and then tested for a number of phat macological activities. The aviaii vasodepressor potencies were found to be 99, 128, 51, and 5 3 uriits mg, respectively: the oxytocic potencies 61, 56, 20.5, and 12.8 unit mg, respectively. aiid the milk-ejecting potencies 182, 1:34, 87, and 50 uriits/rng, respectively. The antidiure and pressor a d v i t i e s of all foiir analogs were extremely low or iiegligible. n
The present communicatioIi is an extension of our work 011 the replacement of the glutamine residue a t position 4 of oxytocin (Figure 1) by aliphatic amino
according to the procedure employed by ~IuIisick, et u Z . ~ Oxytocic assays were performed o n isolated uteri from rats in natural estrus according to the method of H ~ l t o n as , ~ modified by lIunsick,s with the use of magnesium-free van Dyke-Hastings solut'ion as the CsHiOH CzHs I bathing fluid. :\Iilk-ejecting activity was measured on 1 CH-CHI NHz 0 CHz 0 ariesthet'ixed rabbits by the met'hod of Cross and I II I /I I H a r r i ~ ,as~ modified by van Dyke, et uZ.,l0 and by C H1-C H-C-NH-CH-C-NH-C H 31 Chan.l' Kat, pressor assays were carried out on anes2 c=o sI 1 thetized male rats as described in the U. S. Pharmaco1 I peia. l 2 Assays for antidiuretic activity were performed 0 0 N H S I 6 ll 6 I1 4 I on anesthetized male rat,s according to the met'hod of C H2-C H-NH-C-C H-NH-C-CH-( C H2)t-C OKHz ,Jeffers, et aZ.,13as modified by Sawyer.14 The potencies I I so obtained for [4-norvaline]-oxytocin, [4-norleucine]I C Hz c=o I oxytocin, [ l-deamino,4-norvaline]-oxyt~ocin, and [ 1I CONHz deamino.4-norleucine]-oxytocin are presented in Table I C Hz-N 0 0 11 9 :dong with corresponding data for related analogs of CH-C-NH-CH-C-NH-CHz-CONHz oxytocin arid deaminooxytocin. I I / .Is can be seen from Table I, as t,he length of the C Hz-C Hz C Hz I st'raight-chain amino acid substituent' increases from ~H(CH$)~ glycine to a-aminobut'yric acid, the avian vasodepressor, oxytocic, and milk-eject'ing potencies of t'he analogs Pigitre l.-Striicture of oxyt.ociii with nnmbers indicating the position of the individual amino acid residues. also increase. Further increases in chain length result in decreased potencies. The [4-norleucine]-oxytocin is even less potent t'han [4-alanine]-oxyt~ocin. I t will also acid residues. We present here the synthesis and pharbe noted that in the corresponding deamino series of macological activitief of [4-norvaline]-oxytocin, [4analogs there is a progressive decrease in pot,ericies as norleucine]-oxytocin, and their deamino analogs. For the chain lengt'h is increased from that of a-aminothe synthesis of these compounds the methods used butyric acid to that of norleucine. were patterned after those employed for the preparation of [4-valine]-oxytocin and [l-deamino,4-valine]-oxy- Of the three oxytocin analogs in Table I wit,h branched-chain amino acids in position 4, the [4tocin.2 The present analogs were isolated by countervaline]-oxytocin is far more pot'ent t,han [4-isoleucine]current distribution3 and were further purified by oxytocin and [4-leucine]-oxyt80cinwit'h respect' to avian partition chromatography4 on Sephadex G-25. vasodepressor, oxytocic, and milk-eject'ing activities, The four-point assay design5 was used for measuremerit of the pharmacological activities against the USP with [4-leucine]-oxytocin being t,he least potent. [3Valine]-oxytocin is also considerably more potent. thaii posterior pituitary reference standard. Avian vasothe straight-chain analog [4-norvaline]-oxytocin. 011 depressor absays were performed on conscious chickens (1) (a) This work \vas supported in part b) Grants HE-016i5 and H E 11680 from the Xational Heart Institute, E. S. Public Health Service. (b) All optically active amino acid residues are of the L variety. (c) Author
t o whom correspondence and reprint requests should be sent a t the Department of Chemistry, Cornell University. Ithaca. N. T. 14850. (2) 2'. d u Yigneaud. G. Flouret. and R . Walter, J . B i d . C h e m . , 241, 2093 (1966). (3) L. C. Craig, W. Hausmann, E. €1. Ahrnns. J r . , and E. J. Harfenist. A n a l . Chem.. 23, 1236 (1951). (4) D. Yamashiro, .Yrrlure, 201, 76 (1964): 11. Yamashiro, D. Gillessen, and 2'. d i i Vinneaud. J . Amer. Cliem. Soc.. 88, 1310 (1966). ( 5 ) H. 0.Scliilri, d . Physiol. (London), 101, 115 (1942).
(6) R. -1.Nunsick, \2'. H . Sawyer, and H . B. van Dyke, E n d o c r i n o l o g y . 66, 860 (1960).
( 7 ) P. Holton, Brit. J . Pharmacol., 3, 328 (1948). (8) R. A. hlunsick, Endocrinology. 66,451 (1960). (9) B. A. Cross and G. W.Harris, J . Endocrinol, 8, 148 (1952). (10) H. B. v a n Dyke, K . Adamsons, Jr.. and S. L. Engel, Recent Propr. H o r m o n e R e s . . 11, 1 (1955). (11) W.Y.Chan, J . Pharmacol. E x p l l . l'herup., 14'7, 48 (1965). (12) "The Pharmacopeia of t h e United States of America," 17th rev, Mack Publishing Co., Easton, Pa., 1965,p 749. (13) W. .4.Jeffers, Jf. Jf. Livezey, and J. H. .\ustin, Proc. Sot. E x p . Biol. .Wed., 60, 184 (1942). (14) T2'. H.Sawyer, Endocrinolooy, 63, 694 (1958).
1037
Sovember 196'3
N-Benzyloxycarbonyl-O-benzyltyrosylisoleucylnorvalylaspara- solved i i i :3 nil of D l I F atid treated \vith I X T nig of p-iiitrophenyl S-beiizyl-p-niei~captopl",pi"""te.*0 l i t e r 2 days the solution wah ginyl-S-benzylcysteinylprolylleucylglycinamide.-.~siispeiisioii of treated with EIOA~,aiid the i,esiiltiiig precipitate was collected 2.24 g of the precediiig protected heptapeptide iii 13 nil of alihyand witshed with EtOH and Ii:t?O: yield 440 mg, mp 24:3-245", drous .4cOH was treated with 13 ml of 32(,, IIBr iii AcOII. [ a ] " D -51.2' (C 1, DIIF). The hept,apept,ide hydrobromide was isolated and converted to -4solution of 200 ing of this material was iediiced with sodiiini the free heptapeptide by the iisiial procedure. The white powder iti liqiiid ammoiiia by the iisiial method aiid coiiverted to [ l olitaiiied was dissolved in S nil of I l l I F and treated with 1.51 deaitiiiio,4-iic1rv~~liiie]-~~x~t~~ciii by oxidatioii with 0.01 1 .V g of p-nitrophenyl S-beiizyloxyc~arboiiy1-O-beiix~lt~rosiiiat~e.18 K3Fe(CS)+ After removal of ferri- and fei.rocayaiiide ioiis, the The solid mass formed overnight, was broken lip with 100 ml of i,esultiiig soliit ion gave a negative I'llniaii test. 2 8 The soliitioii 13tOA4r, filiered off, and wahhed with Et011 aiid fiiinlly with x a > conceiit rated i o about 15 nil, placed i i i the first five tiibes of &(I; yield 2.4:; g, nip 25:3-25.i", I.11911 -40" (c 2 , 1)31D). LL 400-1 iilie c,c,iiiitei'c,iii'i'eiit disti,ibiitioii app:tratii?, aiid siibjected .Inn/. (C621~P2N10011S) e, IT, s. N-Benzyloxycarbonyl-S-benzylcysteinyltyrosylisoleucylnor- i0 400 il.:rIisfew i i i ihe solveiit system n-BiiOr-I-CsHa-t)..i' ; va I y I a sparaginyl-S-benzylcysteinylprolylleucylglycinamide.--~~ AcOH coiitaiiiiiig 0.1' pyridiiie ( 1 : 3 : 4 ) . The dihti~itiiiti~ii profile, tletei,miiied by the Foliri-Lowy coloi, i,eactioii, i,evealed siihpension of 0 . 5 g of t h e pi.ecwliiig protected oc*tapeplide i t i 25 oiily oiie peak ( K = 0.01). The coiiteiits of the tiibea f r o m the nil of ice-cooled ti.ifliioloetIiariu1 \vas sat iii,ated at 0" with HB1,.4 eelit i,al p o i , t i o i i of the peak wei'e conibiiied, coiiceuti,ated to a The i,esultiiig wlutioii \vas allo\ved t o staiid at, i'ooni temperat iii'e >mall volume, aiid lyophilized t o give 66 mg of pi.odiict. For f o i , L hr and then was evapoixted i o dryiess iiiider va(8iiiini. The further piirificatioti, 47 m y of this material was dissolved i i i .i residiie n-as washed with Et20 aiid dried in r'actio over P 2 0 5 . drops of n-BirOH, .i nil of lower phase, and .i ml of iipper phwe The octapeptide hydrobromide Jva.: dissolved iit 100 in1 of 11eOI.I of the solveiit system n-BiiOH-C6H~-3.5' AcOH coiitaiiiing and neutralized with ion-exchange resiii IItA-410 (OH). The siispeiisioii was filtered, evaporated to dryness, arid dried over P&j in ~ U C Z L O .The free peptide was dissolved in 3 nil of D l I F aiid 233 mg of p-iiitrophenyl S-ben~yloxycarbonyl-~-berizylcyst eiitatela was added. T h e reacBtioii mixt,iii,e, which solidified overnight, was t8riiiuxtedwith 20 in1 of I 3 0 A c aiid filtered. The Folid material w m washed wilh 1CtOlT mid finally u-ith 14:10.4c:, yield 412 rng, nip 24%-244", [ C X ] ~ " I J -5:I.X" ( c 1, 1)311~). dAnal. (C6iHF;x,iO,~s2)e, IT, &-. [4-Norvaline]-oxytocin.--.I sollition of 115 m g of t Ire precwling pi,ot ected noiiapeptide in 400 nil of boiliitg anhydroiin SHswas treated with Na19 i i t i i i l a tilrie coloration per The S H , was removed by evapomtioii in vu The resulting residiie was dissolved i n 200 ml of 0,25C,; AcOH, the pH was adjrist,ed t o 6.8 with 1 ?;H,OH, and the residtiirg solution was aerat,ed with COI-free air overnight. The oxidation was completed by treat,nient with 0.011 .\- &Fe(CN)6 solut8ion.z0 The ferro- and ferricyariide ions were removed by means of A(i3-X4 rexiii (rhloi.ide f o i , i n ) . The soliition was concentrated to appiuximately 20 nil, placed iii the first five tiibeh of a 400-titbe c.ouiiterciirre~it distilibiiti aratii?, arid siibject traiisfers in t,he solvent n-Bii( )II-wPI~II-0. cont,aining 0.1 :>(' pyridii : 8 ) . The Folin-T,t valiiesZ1defiiied a main peak ( K = 2.1). The sohit sponding to 1his peak were comtiiiied, coiiceiilrated l o a small volume, and lyophilized. This niatei,ial (24 mg) was subjected t,o further purification by dissolving it i n 3 ml of the iipper phase of the solvent syblem ~ - B i i o H - c ~ ~ ~A4cOTI 6 - ~ .containing ~~~~ l..i'> pyridine ( 3 : 1 : 4 ) arid siibjecting it to partition chromatography4 on a 2.13 X 110 cm coltinin of Sephadex (3-25. The fract,ions corresponding to the major peak, as determined from Foliii-Lowry color values, x-ere pooled and lyophilized; yield 16 mg, [ c x ] * ~ I ) -22' (c 0.3, 1 .\T-4rOH). :lnal. (C43H6~K1~O&) C, H, K. A sample was hydrolyzed for 22 hr in 6 ,V HCI a t 110" and subjected to aniino acid anal , 2 2 on a Beckrnan,%pirico amino acid aiia.lyzer. The following molar ratios were obtained, with the valiie of glycine taken as 1.0: aspartic acid 1.0, proline 1.0, glycine 1.0, cystine 1.0, iroleiicine 0.9, leiicine I .O, tyrosine 0.87, iiorvaline 1.0, and NH3 2.0. Paper electrophoresis with pyridiiie-AeOH buffer p H 3.6 (19 h r ) showed [4-iioi~aline]-oxytociii to have approximately the same electrophoretic mobility ah that of oxytocin and to travel as a single spot. Desceiiding paper chromatography iri two different solvent8systems, n-BitOH-AcOH-HZO (4: 1: 5) and pyridine-hcOH-H& (10: 7 : 3 ) , showed [4-norvaline]-oxytociii to travel as a single spot (Rf 0.70 and 0.81, respectively). [l-Deamino,4-norvaline]-oxytocin.-.\ riispensioii of 0.64 g of the protected octapept ide ?u'-berixylosycarbonyl-O-beiizyltyrosyli~r~leucylriorval~lasparaginS.l-S-benxylcy,~ teinylprolylleucylglyciiiamide i n 4 ml of arihydroiis .4cOR \vas treated with 4 ml of 32TC HBr i i i AcOIT. The free octapeptide, isolated as u s l i d , was dis(19) R. H. Sifferd a n d V. d u Vigneaud, J . B i d . Chem., 108, 763 (19%). (20) V. d u Vigneaud, G. Winestock, V. V. S. Murti, D. B. Hope, and R. D. Kimbrough, Jr.. J . B i d . Chem., 236, PC64 (1960); D.B. Hope, V. 1 '. S.
Murti, and V. d u Vigneaud, i b i d . , a37, 1563 (1962). ( 2 1 1 0. 11. I , u \ v ~ s . N. J. R ~ ~ ~ ~ I ~.\.w I,. u ~ II:LI.I.. I I , rind I t . .I. I ~ R I I ~ I ~ ~ I . thiri.. 193, 205 (1951). (221 I ) . 11. Sipackrnan, J\-. H. Stein, and S.AIoore, :Ixfd. C h e m . . 30, 1190 (19.i8).
4), and the resiiltiiig soliitioii was subjected tography on a 2.15 X 110 em coliimii of Sephadex G-25 which had been eqiiilibrated with lower phase. Lyi)philiza(ioiiof the coiiteiits of ttibes represented by the c8entra.J p J r t io11 of the rnaiii peak of Foliii-Lowry coloi, valiies yielded :3;3 m g of pro(lilci, [a]*" - 1 l x O ( r o.:, 1 s ArOH). .Inctl. (C,,Hss-
s~oo,ls2) c, TI, N.
The aiiali)g was hj-drolyzed iii 6 .\. HCl at IIOo f o r 22 hr and the following molar rat ios of amino ac.idh aiid ammonia were foiiiid, with leiiciiie takeii as 1.0: aspai,tic acid l.Oj pi,oliiie 1.0, glyeiiie 1.0, leiiciiie 1.0, tyi~isiiie1.0, NH, ixed disiilfide*Oof cysteiiie and pPaper chromatography with the solveiit C6H6-pylidiiie-0.1'; A d ) H (1:3: 1:;) and n.\(-OH (1: 3 :4) showed [ l-dearniiio,4-1iorvaliiie]-oxytocin to have oiily one niitjoi, component with Rr 0.76 aiid 0.78, re-pect ively.
p-Nitrophenyl N-benzyloxycarbonylnorleucinate.A soliitioii of 6.46 g of ~-t)eiii.yloxycarboiiyliiorleiiciiie24aiid 4.16 g of p-iiilropheiiol in 60 nil of EtOAc was cooled in ice. h soliitioii of -5.04g of I)CI iii 30 i d of I3tOhc was added t o the stirred reacticin mixtiire. After 3 hr, AcOH (20 dropr) was added, aiid after 30 min the dicyclohexyliirea was filtered off and washed with EtOAc The filtrate and washings were combined aiid evaporated in vacuo, and the residual oil was crystallized from 1.5 ml of EiOH by the addition of hexane t o incipient clouditiess: yield 8.0 g, nip 70.*i-72", [ a ] % -30.8" (c 2, DlIF). d n a l . (C2,Ht>K*Otj)C, H, S .
N-Benzyloxycarbonylnorleucylasparaginyl-S- benzylcysteinylprolylleucylglycinamide.-.\ suspension of i3.04 carboiiyla%paragiiiyl-~-beiizylcyst einylprolyllei 15 ml of dry AcOH was treated with 15 nil of :3 The peritapept ide h>drobivmide u'as i.wlat ed aiid coiiverted as iisital i o the fi,ee peptapeptide, which \\a,< dissolved i i i $1 nil of UlIF atid ii,eateti \ \ i t h 1 .T8 g of p-iiit ri~pheiiyl S-beiixyloxyc~ai~boiiyllic~i~~eiiciii~~te. T h e ivaxy matei,ial n-hich formed overnight was brokeii iip with 100 ml of b:tOAc and filtered. Tritiiratioii with EtOH aiid finally ivith E t & yielded 2.8 g, mp 246-247", [ a ]191i -61.8' (c 1, 1)lIF). d n a l . (C4,H,,S,OgS) C, H, ?;. N-Benzyloxycarbonylisoleucylnorleucylasparaginy1-S-benzylcysteinylprolylleucyIglycinamide.-A siispension of 1.85 g of the preceding protected hesapeptide i i i 12 ml of AcOH \vas converted to the free hexapeptide in the iiaiial niaiiiier. The product \vas disbolved i i i (i nil of 1)1IF and treated with 0.94 g of p-tiitrophenyl S-heirzylosycarhc~iiylis~~leiiciiiate.Xfter 2 day,< the waxy material formed \ \ a < bi,okeii iip with 100 ml of EtOhc and filtered. The material collected wah ivashed with Et,C)H aiid finally with E;trO: yield l.X.i g, nip 255-259". This material wah dissolved i i i 80' 1EtOH arid reprecipitated by additioii to EtOAc, filt,ered off, and washed with EtpO; yield 1.35 g, mp 260-262", [CU]"D -56.3' (C 1, DhIF). A n d . (C,iH69?;gOloS) C, H, S . N-Benzyloxycarbonyl-O-benzyltyrosylisoleucylnorleucylasparaginyl-S-benzylcysteinylprolylleucylglycinamide.-.\ suspension of
