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September 1969 Angiotensin I1 Analogs. 11. cy clo-(-Val-Ty r-Ile-His-Pro-Phe-) E ~ G E Nc.E P i l T O N g_c__; _J_O R G I A S E ~ and WALTER
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Depai tment o j Pharmaceutzcal Chetntstry, School of Pharmacy, Cnmerszty of Caltjornza, S a n Fianczsco, Calzjoinm 94122
Received Maich 24, 1969
The hexapeptide, Val-Tyr-Ile(or Tal)-His-Pro-Phe, representing a carboxyl terminal portion of the tissue hormone, angiotensin 11, has been reported variously as < O . l - l ~ o as potent in pressor assays as the parent ~ c t a p e p t i d e . * - ~This low activity of the hexapeptide has been significantly enhanced by extension of the peptide at the amino end by a variety of amino acids or their analogs, among the most effective being the hspArg- dipeptide of natural angiotensin II.5 Significant alteration of the carboxyl end resulted in virtual loss of activity. One possible role of peptide backbone extension is the stabilization of a preferred conformation for the essential functional groups of the hexapeptide sequence, such as the strained a helix proposed by Smeby.6a h variety of physical measurements on the hormone and its analogs have been interpreted in support of,6aand in opposition to,' such an ordered structure for angiotensin 11. Thin-film dialysis studies by CraigGbtChave indicated a preferred time-average structure with a rather spherical conformation of near-minimal size at neutral or acidic pH. Since the cyclic hexapeptide represents a more rigid and compact structure, its synthesis and biological comparison with the linear hexapeptide and with some related cyclic peptides was undertaken. The importance of the tyrosine and histidine residues for the biological effects of angiotensin IP made it of interest to examine for angiotensin-like activities the cyclic peptides of Kopple8 containing these amino acids spaced at various intervals, primarily by glycine. ?\Iyotropic activity was tested in the guinea pig ileum. Xnalogb showing a significant response were tested for pressor activity in nephrectomized pentolinium-treated male rats ariehthetized with pentobarbital.I0 Rebults of the bioaisays are presented in Table I.
(1) P a r t I: E. C. Jorgensen. G. C. Windridge, W. P a t t o n , and T. C. Lee, J . .Ked. C h e m . . 12, 733 (1969). This investigation mas supported in part by Public Health Service Research Grant .$AT 08066 from t h e Xational Institute of Arthritis and Metabolic Diseases. T h e abbreviations used t o denote amino acid derivatives and peptides are those recommended by t h e IUPAC-IUB Commission, Biochemistry, 6, 2485 (1966). (2) R.Schvyzer, H e h . C h i m . Acta, 44, 667 (1961). (3) K. Arakawa, R . R. Smeby, and F. 31. Bumpus, J . A m e r . Chum. Sur., 84, 1424 (1962). (4) C. Schattenkerk and E. Havinga, R e c . Trau. Chim. P a y s - B u s , 84, 853 (1965). (5) E. Schrbder and E;. Lubke, "The Peptides," Vol. 2 , .\cademic Press, Inc.. New York, N. Y., 1966, Chapter 1. (6) (a) R. R . Smeby, K. Arakawa. F. PI. Bumpus, and h l . hl. Marsh, B i o c h i m . B i o p h y s . A c t a , 58, 550 (1962); (b) L. C. Craig, E. J. Harfenist, a n d A . C. Paladini, Biochemistry, 3, 764 (1964); (0) L. C. Craig, Proc. Natl. A c a d . Sci. c'. S . ,81, 152 (1968). (7) T. B. Paiva, A. C. h1. Paiva, a n d H. A. Scheraga, Biochemistry, 2, 1327 (1963). (8) (a) K. D. Kopple and D . E. Xitecki, J . A m e r . C h e m . Soc., 83, 4103 (1961); 84, 41.537 ( 1 9 6 2 ) ; (1)) t i . D. Kopplr, R . R. .Taral,zk, P. I,. Rhatia, Biuchemi.&y, 2 , 958 (lY83). (9) D. Regoli and J. R.Vane, B r i t . J . I'harmncol., 23, 351 (1964). (10) R. 13oiicller, R . Veyrat. J. de Champlain, and J. Genest, C a n . .Wed. Bssoc. J . , 90, 194 (1964).
Results and Discussion The hexapeptide, Val-Tyr-Ile-His-Pro-Phe (2))in the guinea pig ileum myotropic assay, showed an activity of 0.1% relative to 100% for [Asn',Valj]-angiotensin I1 (1). This is much lower than the rat uterus myotropic activity of 2.6y0reported3 for 2, but is close to the rat pressor value (0.3yG)4 for 2, or for its analog, ValTyr-Val-His-Pro-Phe (imple relationship between a spacing of histidine and tyrosine residuei, although apparently of importance in the production of the myotropic response, is not alone a determining factor in the pressor response of angiotensin and its analogs. Experimental Section1'
Val-Tyr-Ile-His-Pro-Phe.-To 250 mg (0.24 mmole) of Z-ValTyr-Ile-His-Pro-Phe-OBzl( X0z)lzin 25 ml of MeOH was added 70 mg of lo$; Pd-C suspended in 6 ml of HOAc. The suspen.-ion was flushed with Nz for 5 min, HZ was bubbled through slonly for 5 min. The catalyst was removed for S hr, followed by and the filtrate was evaporated to dryness in vacuo. The oily residue was dissolved in 50 ml of H20 and lyophilized three times, yielding a white solid, 185 mg (100%). Tlc (BAW) and paper electrophoresis a t pH 3.5 and pH 6.5 showed a single major com(11) Melting points were measured in a Thomas-Hoover apparatus and are uncorrected. .4mino acid analyses were done on a Rpinco 120B analyzer on samples hydrolyzed with twice-distilled hydrochloric acid for 24 111. a t l l O o in evacuated and sealed tubes. Optical rotations were measured i n a Rudolph photoelectric polarimeter. Paper electrophoresis was carried out a t 2000 V a t p H 3.5 (HO.lc-pyridine-HiO, 10: 1 : 190) or p H 6.5 (0.4: 10: 190). Electrophoretic mobilities are reported relative t o the migration of histidine, E= = 1.0. TIC was on silica gel G (31erck) with the solvent system n-BuOH-HOAc-H?O (100: 10:30) (BAT\-) (top layer). (12) K. Arakawa and F. hT. Bumpus, J . A m e r . C h e m . Soc.. 83, i 2 8 (1961).
poiieiit, no startiiig material, a i d foiir minor component>. X azo10nes3 are reported to have similar activities. O u r 170-mg portion was dissolved in AleOH-HL) ( l : l ) $applied i n previous experiericc with S-arylpiperazine derivative, bands on five sheets of Whatnian No. 311hI filter paper, and subhaving sedative, hypotensive, and :mtiadrenergic jected to electrophoresis a t pH 3 The major band a t E H activities led us to study certain 3-w-(-l.-aryl-l-piper0.50 was eluted with water and lyop zed to yield 63 mg of white aziny1)alkyl-.'-methyl- (or ?-phenyl-) 4(SH)-quinazopowder: single spot on paper electrophoresis at pH 3.5 ( E H 0..3)and pH 6.5 ( E M 0.51); 011 paper chromatography, Ki lories (I) (Tableb I and 11). 0.74; ninhydrin and Pauly ; aiuiiio acid aiialysis; 1.d 1.00, These compounds were readilj- prepared by heatiiig T y r 0.57, Ile 0.91, His 1.02, Pro U.I)X, Phe 0.!)4. "methyl- (or 2-phenyl-) 4-oxo-4H-3,l-benxoxazine (11) c.!lcLo-(-Val-Tyr-Ile-His-Pro-Phe-). ---To a >tiired ,s-.oliitioiiiuf 16 111: (0.084 mmole) of l-eth~-l-8-i:~-diinethylariiinopropyl)c~ari~o- with appropriate primary amines (method A) or by tliimide hydrochloride i r i 2 in1 of I ) l I F was added dropwise over treating isatoic atihydride (111) with the amines to give 14 hr 53 nig (0.04 mmole) of ~al-'l'~r-Ile-Ilis-Pi~o-Phe dissolved o-amino-S-substituted benzamideh (IV) which wcrc i n 14 nil of UMF. After 24 hr at rooni ternperatwe in the dark, then benzoylated iiiid cyclized with A c 2 0 (method B) paper electrophoresis showed no starting material at, EH 0..30 (Scheme I). I (R' = CHZ) iz alm prepared by heating (pH 3.5), and in addition to yeveral minor spots, a aiiigle major IV in Ac?O. The detail5 of the preparative cliemihtrj ninhydrin -. The coniponent at Ea 0.40 (pH 3.5); Palily solvent a a s removed in vacuo and the residue was dissolved iii h:tve been tlewrihcxl iri :I r( MeOH, applied in bands on sheets of Whatniaii No. 3 X A 1 filter paper. Follom-ing electrophoresis, the band a t with water and lyophilized to yield 15 m g powder: single spot on paper elect,rophores 0,4:3; Pauly ninhydrin - ; amino acid T y r 0.91, Ile 0.89, His 1.01, Pro 1.04) €'he 1.08.
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Acknowledgments.-Ke are grateful to Ilr. Kennetli 11. Kopple for samples of cyclic peptides, Drs. 1'. A. Lehmaii, T. C. Lee, arid G . \Yindridge for assistance bioassays, Ilrs. €3. Katzung and S. ,J. Feinglass aiice with computer programming, and Dr. D. Sitecki for her helpful discussions and :L sample of thn soluble carbodiimide derivative used in the cyclization rc:ictioti.
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Therapeutics lleseui ch Diviszon, M d e s Laboratories, Inc., Elkhait, Indiana /t6'51.$ \\I)
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