510
J.Med. Chem. 1988,31, 510-516
Effects of D-Amino Acid Substitution on Antagonist Activities of Angiotensin I1 Analogues? J. Samanen,*$D. Narindray,t W. Adams, Jr.,t T. Cash,t T. Yellin,$ and D. Regolis Smith Kline & French Laboratories, Peptide Chemistry Department, Swedeland, Pennsylvania 19479, and Department of Pharmacology, University of Sherbrooke, Quebec, Canada. Received November 26, 1986 The synthesis and biological activities of angiotensin I1 (AII) analogues are described and compared to the literature. D-Amino acid substitution was employed to search for novel AI1 antagonists that would also display reduced partial agonist activity. Substitution of D-aminO acids into the interior positions 2-7 of [Sar',Iles]-AI1 gave rise to inactive compounds or weak antagonists. Substitution of D-amino acids into position 8 gave rise to potent antagonists in ~ ] (29), vivo including [Sar',D-Phe8] -AI1 8, [Sar',D- (aMe)Phes] -AI1 (35), [ Sar',~-Trp'] -AI1 (32), [S a r ' , ~ - P h g -AI1 analogues (antagonists) [Sar',~-Pe$]-A11 (30), and [Sar1,~-Phes]-AII-NH2 (31). The structural requirements for D-AA~ showed similarities with those of L-AA~analogues (agonists). The latter three analogues, 29-31, were considerably more potent in vivo than their in vitro affinities would indicate, suggesting that these analogues may resist carboxypeptidase-like degradation. While partial agonist activity was not removed by D-AA~substitution, [Sar',DPhe8]-AII-NH2(31) displays lower partial agonist activity than [Sar',Iles]-AII. A receptor model is presented that highlights the difference between [L-AA~I-AII analogue agonist activity and [D-AA'I-AII analogue antagonist activity.
Of the more than 300 analogue of angiotensin I1 (AII) described in the literature,'y2 only a small percentage of these analogues were designed to probe the structural requirements for AI1 antagonists. Potent antagonists to AI1 have been obtained by substituting aliphatic amino acids in place of phenylalanine in position 8. Two of these analogue, [Sar1,Ala8]-AII(saralasin) (3) and [Sar1,11e8]-AII (4), have been shown to lower blood pressure in human hypertensives with high renin levels. These peptides display sufficient residual agonist action to severely limit their use as antihypertensive agenk3v4 In order to eliminate intrinsic agonist activity in AI1 antagonists, alternate strategies are needed in the search for improved antagonists. In the present study we have pursued the approach of D-amino acid substitution into various positions of angiotensin 11. D-Amino acid substitution can be a useful tool for increasing potency in a peptide or for changing its activity from agonist to a n t a g ~ n i s t . ~The potency and duration of action of naturally occurring peptides has often been dramatically increased by D-amino acid substitution. In these cases enhanced properties were ascribed to either increased resistance to proteolysis or stabilization of bioactive conformation. A number of naturally occurring peptides have been transformed into antagonists by substitution of D-amino acids for L-amino acids in certain positions of the native s e q ~ e n c e s . ~In these cases D substitution presumably stabilizes conformations that bind to the receptor but do not provoke receptor stimulation. On the basis of such examples, the substitution of D-amino acids into angiotensin analogues appears to be a reasonable approach in the search for ways to increase antagonist potency and duration and for unique modifications that generate antagonist activity. Analogues of angiotensin I1 bearing D-amino acids in positions 1-8 have been reported previously (analogues + T h e abbreviations for natural amino acids (AAs) and nomenclature for peptide structures follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (J.Biol. Chem. 1971,247,977. Abbreviations for nonnative amino acids include Apb = 3-amino-4-phenylbutyric acid, Apib = 3amino-3-phenylisobutyric acid, Bph = p-(dihydroxybory1)phenylalanine, (2)Nal = 3-(2-naphthyl)alanine, Peg = phenethylglycine, Phg = phenylglycine, and Sar = sarcosine. Other abbreviations in this paper include AA = amino acid, Boc-ON = 2- [ (tert-butoxycarbonyloxy)imino]-2-phenylacetonitrile. Smith Kline & French Laboratories. 3 University of Sherbrooke.
1-8) (Table I).ls6-12 D substitution in position 1 of AII, analogue 1, was reported to enhance agonist activity by stabilizing against aminopeptidase action.1° Leduc et aL9 reported that D substitution in position 8 of [Sarll-AI1gave an analogue, 8, that lacked significant agonist activity and displayed potent antagonism. Also previously reported were analogues 2 , 3 , 5 , 6 , and 7 bearing D-amino acids in the interior positions of the AI1 sequence.lpW1' These were weak agonists with no reported antagonist effects. D-Amino acid substitution into [Sar',IIes]-AI1 could conceivably stabilize unique antagonist conformations to give antagonists with increased potency or reduced agonist activity. This paper describes the synthesis and biological activities of such analogues and analyzes the structureactivity requirements for AI1 antagonists, bearing a D residue in position 8. An efficient comparison of new analogue activities with literature analogue activities necessitates construction of data tables bearing both sets of data. Data from the literature is footnoted in each table. The conformational analysis of angiotensin I1 has been thoroughly reviewed.12 (1) Khosla, M. C.; Smeby, R. R.; Bumpus, F. M. Handb. Exp.
Pharrnacol. 1974,37,126-161. (2) Bumpus, F. M.; Khosla, M. C. In Hypertension: Physiopa-
thology and Treatment; Genest, J., Koiw, E., Kuchel, O., Eds.; McGraw-Hill, New York, 1977; Chapters 6 and 7 , pp 183-201. (3) Pals, D. T.; Denning, G. S., Jr.; Kennan, R. E. Kidney Znt. Suppl. 1979, 15 (Suppl 9), 7-11. (4) Hota, T.; Ogihara, T.; Mikami, M.; Nakamura, M.; Maruyama, A.; Mandai, T.; Kumahara, Y. Life Sci. 1978,22, 1955-1962. (5) Samanen, J. Biomedical Polypeptides-A Wellspring of Pharmaceuticals in Bioactive Polymeric Systems; Gebelein, C. G., Carraher, C. E., Jr., Eds.; Plenum: New York, 1985;pp 279-382. (6) Khosla, M. C.; Hall, M. M.; Smeby, R. R.; Bumpus, F. M. J. Med. Chem. 1973, 16, 829-832. (7) Schattenkirk, C.; Havinga, E. Recl. Traul. Chem. Pays-Bas 1965,84,653-658. (8) Jorgensen, E. C.; Rapaka, S. R.; Windridge, G. C.; Lee, T. C. J . Med. Chem. 1971,14, 904. (9) Leduc, R.; Bernier, M.; Escher, E. Helv. Chim. Acta 1983,66, 960-970. (10) Regoli, D.; Rioux, F.; Park, W. K.; Choi, C. Can. J. Physiol. Pharrnacol. 1974, 52, 39-49. (11) Moore, G. J. In Peptides, Synthesis-Structure-Function, Proceedings of the Seventh American Peptide Symposium; Rich, D. H., Gross, E., Eds.; Pierce Chemical Co., Rockford, IL, 1981; pp 245-298. (12) Smeby, R. R.; Khosla, M. C. In Chemistry and Biochemistry of Amino Acids, Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker, New York, 1978; pp 117-162.
0022-2623/88/1831-0510$01.50/00 1988 American Chemical Society
Journal of Medicinal Chemistry, 1988, Vol. 31, No. 3 511
Angiotensin 11 Analogues
Table I. Effects of D-Amino Acid Substitution on Activities of Angiotensin Analogues primary structure 3 4 5 Val Tyr Ile
biological activities in vivo rat blood 7 in vitro rabbit aorta" pressureb Pro Phe AII-likec pA2 AII-liked Phe8 200 Phe8 5 Phe8 0.03 Phe8 10 0.05 Phe8 4.0 D-Pr07 Phe8 0.1 (RU)f 97 0.31 0.47 0.56 D-Phg His Pro Val Ile 29 TYr -4% (0.97) (1.03) f+) (0.92) (0.98) (1.06) (1.04) 25 4.35 >95 0.19 0.56 0.49 His D-Peg Pro Ile Val 30 TYr Arg (1.02) (1.01) (+) (1.01) (0.98) (1.00) (1.01) 2.81 25 >98 0.13 0.64 0.72 His D-Phe-NH, Pro Ile Val 31 TYr -4% (0.99) (1.01) (1.04) (1.00) (0.97) (1.01) (0.99) 20 4.5 90 0.30 0.50 0.61 His D-Trp Pro Ile Val 32 TYr Arg (1.02) (0.97) (0.80) (0.94) (1.02) (1.03) (1.0) 29 4.39 >97 His ~-(2)Nal 0.28 0.67 0.52 Pro Ile Val 33 TYr Arg (1.01) (1.02) (1.00) (1.00) (1.00) (1.00) (0.97) 35 10.9 85 His D-Tyr 0.03 0.08 0.53 Phe Pro Val Ile 34 Arg (1.03) (1.05) (1.03) (0.95) (0.98) (1.04) (0.92) 20 4.60 His Pro >98 Ile D- (aMe)Phe 0.36 0.54 0.69 Val 35 TYr Arg 0.96) (1.04) (1.05) (0.95) (0.98) (1.02) (+) 10 Pro 5.86 >98 His Ala 0.06 0.29 Val Ile 36 TYr Arg (1.04) (1.04) (1.00) (0.99) (1.01) (0.94) (0.97) 20 2.79 91 His Phe Pro 0.19 0.39 0.58 Val Ile 46 TYr -4% (1.03) (0.95) (0.97) (1.05) (0.92) (1.05) (1.03) 25 Pro 2.6 89 42 His 0.18 0.48 0.54 Val Ile Peg TYr (1.03) (0.90) (0.93) (1.07) , , Amino acid analysis expressed in molar ratios of the D,L amino acids in the peptides. (+) a See text for details of analytical procedures. = amino acid present in roughly 1 molar equiv (in cases where quantitation is difficult). * = amino acid present in two positicns. Value expressed is one half the experimental value. \
\-
\-.--,
(+r
In most cases coupling was complete after 2 h. If the ninhydrin test remained positive, a recoupling cycle was performed. After the last coupling and deprotection, the peptide was cleaved from resin by treatment with anhydrous HF containing 50% (v/v) anisole at 0 OC for 60 min. After vacuum evaporation of HF, the resin was rinsed with EhO to remove anisole and then rinsed with glacial HOAc and filtered. The filtrate was diluted with water and lyophilized to a powder of crude peptide material. The crude peptides were purified to homogeneity either by (a) partitioning through 200 transfers of counter-current distribution in n-BuOH-HOAc-H20 (4:1:5), (b) partition ~ h r o r n a t o g r a p h y ~ ~ on Sephadex G-15 in n-BuOH-HOAc-H,O (4:1:5), or (c) reversed-phase semipreparative HPLC35on a Whatman Cl8 column (34) Yamashiro, D. Norm. Proteins Pept. 1980, 11, 26-106.
using the appropriate solvent mixture of CH3CN-0.1 N NH,OAc, p H 4. The volumes of chromatographic fractions containing pure peptide were reduced by partial rotary evaporation and dried to powders by lyophilization to constant weight. Homogeneity of each peptide was determined by the following methods: (a) amino acid analysis of 72 h acid hydrolysate (6 N HCl, 110 OC) performed on a Beckman Model 120C analyzer; (b) (35) Smith, J. A.; McWilliams, R. A. Am. Lab. (Fairfield, Conn.) 1980, 12, 23-29. (36) Rioux, F.; Park, W. K.; Regoli, D. Can. J.Physiol. Pharmacol. 1973,51, 665-672. (37) Regoli, D.; Park, W. K.; Rioux, F. Pharm. Rev. 1974, 26, 69-118. (38) Regoli, D.; Park, W. K. Can. J.Physiol. Pharmacol. 1972,50, 99-112.
J.Med. Chem. 1988, 31, 516-520
516
analytical TLC on silica gel plates with solvent systems (A) nBuOH-AcOH-H,O (4:1:5), (B) n-BuOH-AcOH-H20-EtOAc (l:l:l:l), (C) n-BuOH-AcOH-H20-pyridine (15:3:1210), visualizing spots with Pauly reagent;33(c) analytical reversed-phase HPLC on CI8-silicagel column using the appropriate CH3CN-0.1 N NH,OAc (pH 4) mixture, following elution by UV (250-nm detection). Analytical data for all peptide are listed in Table V.
Registry No. 1, 51833-71-7; 2, 67037-14-3; 3, 3438-23-1; 4, 34305-50-5; 5, 57667-99-9; 6, 49707-73-5; 7, 111821-38-6; 8, 111821-39-7; 9, 37827-06-8; 10, 51887-63-9; 11, 111771-38-1; 12, 111771-39-2; 13, 101713-05-7; 14, 111821-40-0; 15, 111821-41-1;
16,111821-42-2;17, 111821-43-3; 18,49707-74-6;19, 111771-40-5; 20,111771-41-6; 21, 111771-42-7;22,111771-43-8; 23, 111821-44-4; 24,111821-45-5;25,49707-72-4;26,111771-44-9;27,111771-45-0; 28,111771-46-1; 29, 111821-46-6;30,111771-47-2; 31,111771-48-3; 32,95841-12-6;33, 111771-49-4;34, 111771-50-7;35, 111771-51-8; 36, 38027-95-1; 37, 25061-67-0; 38, 90937-06-7; 39, 111771-52-9; 40, 25061-71-6; 41, 53935-04-9; 42, 111821-47-7; 43, 35492-37-6; 44, 6663-62-3; 45, 47917-11-3; 46, 111821-48-8;47, 111771-53-0; 48, 111771-54-1; AII, 11128-99-7; H-DL-Bph-OEt, 111771-55-2; H-D-Bph-OEt, 111771-56-3; H-D-Bph-OH, 111821-49-9; BOC-DBph-OH, 111771-57-4; H-D-(aMe)Phe-OH, 17350-84-4; BOCD-(aMe)Phe-OH, 111771-58-5; BOC-D-(aMe)Phe-OH.DCHA, 111771-59-6.
2,4-Diamino-6,7-dimethoxyquinazolines.4. 2-[4-( Substituted oxyethoxy)piperidino] Derivatives as cyl-Adrenoceptor Antagonists and Antihypertensive Agents Simon
F. Campbell,*
John
C. Danilewicz, Colin W. Greengrass, a n d R h o n a M. Plews
Department of Discovery Chemistry, Pfizer Central Research, Sandwich, Kent, United Kingdom. Received August 4, 1987
A series of 4-amino-6,7-dimethoxy-2-[4-(substituted oxyethoxy)piperidino]quinazolinederivatives (2) was synthesized and evaluated for a-adrenoceptor affinity and antihypertensive activity. Most compounds showed binding affinities within the nanomolar range for a,-receptors, although 25 and 26 showed enhanced potency (Ki, ca. 1.5 X M), equivalent to that of prazosin. Series 2 also displaced [3H]clonidinefrom a2-adrenoceptors,but at relatively high doses of lo4 M, and selectivity for a , sites still predominated. In a rabbit pulmonary artery preparation, 12, 16, and 25 were potent antagonists of the a,-mediated, postjunctional vasoconstrictor activity of norepinephrine with no effect at the prejunctional a2 sites which modulate transmitter release. Physicochemical measurements gave a pK, of 7.63 f 0.10 for 12, and N-1 protonation will be favored (60%) at physiological pH to provide the a,-adrenoceptor plmmacophore, 28. Antihypertensive activity of series 2 was evaluated following oral administration to spontaneously hypertensive rats, and blood pressure was measured after 1 and 6 h. Compounds 12, 13, 16,23, and 37 displayed moderate efficacy and duration of action in lowering blood pressure, but the plasma half-life (ca. 2 h) of 16 in dogs was not compatible with potential once-daily administration in humans.
In previous papers, t h e synthesis and biological activities of two series of 2- [4-(1,4-benzodioxan-2-ylcarbonyl)piperazin-1-yl]- a n d 2444 (substituted amino)carbonyl]piperidinolquinazoline derivatives la a n d l b were reported.lJ I n these studies, t h e roles of t h e relatively rigid
Scheme I
Pfz
n
"2
"2
3
"2
11-27
marked structural variations t h a n those already reported might also be tolerated. I n t h i s paper, t h e carbonyl or heteroaromatic a systems common t o t h e previously disclosed series are replaced by an ethylenedioxy function (2),
b , X = C H , Y = CONRaRb c . X = N, Y = heteroaryl
(la) and flexible (lb) carboxamide moieties were compared with respect t o effects o n a,-adrenoceptor affinity a n d antihypertensive activity. Subsequently, it was also demonstrated t h a t t h e carbonyl function present in la and l b could be replaced by an appropriately substituted heteroaromatic a system (IC)with no adverse effects on in vitro or i n vivo a ~ t i v i t y . ~ T h e s e structure-activity relationship (SAR) studies suggested that, although t h e quinazoline 2-substituents play a n i m p o r t a n t role in modulating a,adrenoceptor affinity a n d antihypertensive activity, more (1) Campbell, S. F.; Davey, M. J.; Hardstone, J. D.; Lewis, B. N.; Palmer, M. J. J. Med. Chem. 1987, 30, 49. (2) Alabaster, V. A.; Campbell, S. F.; Danilewicz, J. C.; Greengrass, C. W.; Plews, R. M. J. Med. Chem. 1987, 30,999. (3) Campbell, S. F.; Plews, R. M. J . Med. Chem. 1987, 30, 1794.
"2
2
a n d further substitution of t h e alkyl chain is explored for effects o n in vitro receptor affinity a n d in vivo antihypertensive activity. Chemistry. All of t h e compounds for pharmacological testing were prepared by condensation of 4-amino-2chloro-6,7-dimethoxyquinazoline(3) with a n appropriate 4-alkoxypiperidine derivative in butanol under reflux (Scheme I).4 I n route A, chromatographic purification of 13-15, 22, a n d 24-27 was required whereas in route B, (4) Campbell, S. F.; Danilewicz, J. C.; Greengrass, C. W. Br. Patent Appl. 2,010,824A, 1979 and 2,041,373A, 1980.
0022-2623/88/1831-0516$01.50/0 0 1988 American Chemical Society
