J.Med. Chem. 1988,31, 1377-1382
1377
Synthesis and Renin Inhibitory Activity of Angiotensinogen Analogues Having Dehydrostatine, Leu$[CHzS]Val, o r Leu$[CH,SO]Val at the P1-P1’Cleavage Site’ Clark W. Smith,*+Hossain H. Saneii,* Tomi K. Sawyer,+Donald T. Pals,+Terrence A. Scahill,§ Bharat V. Kamdar,t and Judy A. Lawsoni Biopolymer Chemistry, Cardiovascular Diseases Research, and Physical and Analytical Chemistry Units, The Upjohn Company, Kalamazoo, Michigan 49001. Received October 13, 1987
The synthesis and in vitro renin inhibitory potencies of angiotensinogen (ANG) analogues having amide (CONH) bond replacements a t Pl-P,’, the Leu-Val cleavage site, corresponding to Leu$[CH,S]Val, Leu$[ CH2SO]Val,and acid (dehydrostatine, Dhs), are reported. the trans olefinic analogue of statine (Sta), 4(S)-amino-6-methyl-2(E)-heptenoic These are compared to P1-P1’ Leu$[CH,NH]Val-, Sta-, or Phe-Phe-substituted analogues of the same template. The Dhs pseudodipeptide was found to be an adequate mimic of a trans CONH bond and gave a peptide, HPro-His-Pro-Phe-His-Dhs-Ile-His-D-Lys-OH, approximately equal in potency to a Phe-Phe-containing inhibitor, but 200-fold less potent than its Sta-substituted congener. That the enhanced potency of the Sta-containing peptide most likely depends on hydrogen bonding as well as tetrahedral geometry is indicated by the 50-100-fold lower potency of the tetrahedral Leu$[CH,S]Val and Leu$[CH,SO]Val analogues as compared to the Leu$[CH,NH]Val-containing congener.
The aspartic proteinase renin catalyzes the first step of Table I. Solid-Phase Synthesis Procedure the biosynthetic pathway leading to the production of step solvent or reagent repetitions time, min angiotensin 11, one of the most potent naturally occurring 1 CH2Cl2 n4 1.5 vasoconstrictive substances known. Angiotensin I1 has 1,5, 25” 2 CF&OPH-CH&12(1:1) 2“ been implicated in the physiological regulation of blood 3 CHZC12 4 1.5 pressure and may also play a role in pathophysiological 5 4 DIPEA-CH&l,(l:B) 3 1.5 5 CHzClz 4 forms of h y p e r t e n ~ i o n . ~ ~ It~follows that inhibition of renin 1.5 6 DMF 4 may be therapeutically beneficial in controlling hypervariable 7 coupling step (see text) 1 tension and, moreover, that renin inhibitors may constitute 1.5 8 DMF 4 a novel class of antihypertensive drugs.4 1.5 9 EtOH 4 Early work on substrate-based inhibitors using systema When Boc-His(Tos) was deprotected, three CF3CO2H-CH2Cl2 atic structure-activity studies5 culminated with the detreatments are performed for 1.5, 25, and 25 min. capeptide, H-Pro-His-Pro-Phe-His-Phe-Phe-Val-TyrLys-OH, which was the first inhibitor active in vivo6 methyl (tripheny1phosphoranylidene)acetate. Saponifi(Figure 1). In this analogue the key to conversion from cation of the methyl ester yielded the expected trans substrate to inhibitor was the substitution of Phe-Phe for the labile Leu-Val sequence at P1-P1’. More recently, Abbreviations of amino acids follow the recommendations of modifications a t P1-P1’ have included changes that may the IUPAC-IUB Commission on Biochemical Nomenclature mimic the transition state amino1 (C[OH],NH) generated Eur. J . Biochem. 1984,158,9. The $[I nomenclature for pepduring hydrolytic cleavage of the peptide bond by renin tide bond modifications follows the suggestion of Spatola, A. (e.g., Leu$[CH,NH]Val and Sta)7-11as well as changes of F. Chemistry and Biochemistry of Amino Acids, Peptides, and the peptide bond to noncleavable olefinic groups (e.g., Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1983; L ~ U $ [ E - C H = C H ] G ~ ~ )In . ’ ~many cases these have led Vol. 7, Chapter 5. All optically active amino acids are of the to extremely potent and specific inhibitors of renin.13-19 L variety unless otherwise specified. The following additional abbreviations are used: ANG, angiotensinogen; Boc, tert-buWe report here the synthesis and in vitro renin inhityloxycarbonyl; DCC, dicyclohexylcarbodiimide; Dhs, dehybitory potencies of compounds with peptide bond redrostatine; DIPEA, (diisopropylethy1)amine; HBT, 1placements a t the Pl-P1’ sites corresponding to Leu$hydroxybenzotriazole; Sta, statine. [ CH,S]Val, Leu$[ CH,SO]Val, and the olefinic analogue Peach, M. J. Physiol. Rev. 1977, 57, 313. of Sta, 4(S)-amino-6-methyl-2(E)-heptenoic acid, (dehyOndetti, M. A.; Cushman, D. W. Annu. Rev. Biochem. 1982, drostatine, Dhs). These are compared to the Phe-Phe-, 51, 283. Haber, E. Clin. Exp. Hypertension-Theory Practice 1983, A5, Leu+[CH,NH]Val-, and statine-containing analogues all within the same Pro-His-Pro-Phe-His-Xaa-Yaa-1le-His-D- 1193. Burton, J.; Poulsen, K.; Haber, E. Polymeric Drugs;Academic: Lys template where Xaa-Yaa is the particular P1-P1’ New York, 1978; pp 219-237. substitution. This template is not optimized for maximum Burton, J.; Cody, R. J., Jr.; Herd, J. A.; Haber, E. Proc. Natl. potency of renin inhibitors and was chosen so that difAcad. Sci. U.S.A. 1980, 77, 5476. ferences in activity affected by the Xaa-Yaa substitutions Szelke, M.; Leckie, B.; Hallet, A.; Jones, D. M.; Sueiras, J.; Atrash, B.; Lever, A. F.; Nature (London) 1982, 299, 555. could be measured without approaching the limit (conSzelke, M.; Jones, D. M.; Atrash, B.; Hallett, A,; Leckie, B. centration of renin in the test plasma) for ICbodetermiPeptides: Structure and Function. Proceedings of the Eighth nations. American Peptide Symposium; Tucson, AZ; Hruby, V. J., Rich, D. H., Eds.; Pierce Chemical Co.: Rockford, IL, 1983; p Results and Discussion 579. Chemistry. Synthesis of Boc-Dhs-OMe containing both Boger, J.; Lohr, N. S.; Ulm, E. H.; Poe, M.; Blaine, E. H.; trans and cis olefinic bonds in an approximate ratio of 7:3 Fanelli, G. M.; Lin, T.-Y.; Payne, L. S.; Schorn, T. W.; LaMont, was accomplished as shown in Scheme I. Boc-L-leucinol B. I.; Vassii, T. C.; Stabilito, I. I.; Veber, D. R.; Rich, D. H.; was oxidized to Boc-L-leucinal by treatment with pyriBoparai, A. S. Nature (London) 1983, 303, 81. Boger, J. Peptides: Structure and Function. Proceedings of dine-sulfur trioxide in DMS020 followed by reaction with Biopolymer Chemistry. t Cardiovascular Diseases 5 Physical and Analytical
Research. Chemistry. 0022-2623/88/1831-1377$01.50/0
the Eighth American Peptide Symposium; Tucson, AZ; Hruby, V. J.; Rich, D. H., Eds.; Pierce Chemical Co.: Rockford,IL, 1983; p 569. Rich, D. H. Proteinase Inhibitors; Barrett, A. J., Salvesen, G., Eds.; Elsevier: New York, 1986; Chapter 5. 0 1988 American Chemical Society
1378 Journal of Medicinal Chemistry, 1988, Vol. 31, No. 7
Smith et al.
Scheme I CH ,I
CH
CH3,
yCH3
I
0
CH3
I I
II
CHJ-C-0-C
CHz
-
I
I CH3-C-0-C I
I1
II
I
-NH-CH-CH
ANG6.13
9
ANG Positions ANG Binding Sites
Figure 1.
olefinic acid and 4- [ (tert-butyloxycarbony1)amino] -4hydroxy-6-methylheptanoicacid lactone (see below). No II
CHa-C-0-C
I
CH3
I a 3
CH3-C-
I
I
0 0-C
II
-
CHz
I
NH- CH-CH=
0 CH-C-
II
0-CH3
78%
I
0
CHzOH
CH3
RENIN
I
I
NH- CH-
0- CH3
II 0
CH3
CHz
-
CH3
97%
8
II
0- C
I
0 3 P = CH- C-
CHZ
CHI
CH3
0
CH /CH3
0
CH3
7
C-
0
I
6
I
CH3-
II
CH3,
0
CH3
NH-CH-C-OH
CH3
CH/
I
BH3 ’ THF + MF
-NH-C
’
CH2
,
/“p‘y
I
L0/C.
‘ 0
CH
CH3
cis olefinic acid was produced. Boc-Leu+[CH2S]Val was synthesized according to the method described by Spatola et a1.,21and the sulfoxide was obtained by peracetic acid oxidation of the pseudodipeptide-containingfinal product. (12) Johnson, R. L. J. Med. Chem. 1984,27, 1351. (13) Sawyer, T. K.; Pals, D. T.; Smith, C. W.; Saneii, H. H.; Epps, D. E.; Duchamp, D. J.; Hester, J. B.; TenBrink, R. E.; Staples, D. J.; deVaux, A. E.; Affholter, J. A.; Skala, G. F.; Kati, W. M.; Lawson, J. A,; Schuette, M. R.; Kamdar, B. V.; Emmert, D. E. Peptides: Structure and Function. Proceedings of the Ninth American Peptide Symposium; Deber, C. M.; Hruby, V. J.; Kopple, K. D., Eds.; Pierce Chemical Co.: Rockford, IL, 1985; p 729. (14) Boger, J.; Payne, L. S.; Perlow, D. S.; Lohr, N. S.; Poe, M.; Blaine, E. H.; Ulm, E. H.; Schorn, T. W.; LaMont, B. I.; Lin, T.-Y.; Kawai, M.; Rich, D. H. J . Med. Chem. 1985, 28, 1779. (15) For a recent review, see: Boger, J. Annu. Rep. Med. Chem. 1985, 20, 257. (16) Thaisrivongs, S.; Schostarez, H. J.; Pals, D. T.; Turner, S. R. J . Med. Chem. 1987,30, 1837. (17) Sham, J. L.; Stein, H.; Rempel, C. A,; Cohen, J.; Plattner, J. J. FEBS Lett. 1987, 220, 299. (18) Bolis, G.; Fung, A. K. L.; Greer, J.; Kleinert, H. D.; Marcotte, P. A.; Perun, T. J.; Plattner, J. J.; Stein, H. H. J . Med. Chem. 1987, 30, 1729. (19) Sawyer, T. K.; Pals, D. T.; Mao, B.; Staples, D. J.; deVaux, A. E.; Maggiora, L. L.; Affholter, J. A.; Kati, W.; Duchamp, D.; Hester, J. B.; Smith, C. W.; Saneii, H. H.; Kinner, J.; Handschumacher, M.; Carlson, W. J. Med. Chem. 1988, 31, 18. (20) Hamada, Y.; Schioiri, T. Chem. Pharm. Bull. 1982, 30, 1921. (21) Spatola, A. F.; Bettag, A. L.; Fok, K.-F.; Saneii, H. H.; Yankeelov, J. A., Jr. Peptides: Structure and Biological Function. Proceedings of the Sixth American Peptide Symposium; Gross, E., Meienhofer, J., Eds.; Pierce Chemical Co.: Rockford, IL, 1979; p 273.
Table 11. Renin Inhibitory Activities of Several ANG-Based PeDtides relative number compound IC,,, M potency 1 H-Pro-His-Pro-Phe-His-Phe1.0 X 1.0 Phe-Val-Tyr-D-Lys-OH 2 H-Pro-His-Pro-Phe-His-Phe1.7 X 0.6 Phe-Ile-His-D-Lys-OH 3 H-Pro-His-Pro-Phe-His-Dhs6.2 X lo4 1.6 Ile-His-D-Lys-OH 4 H-Pro-His-Pro-Phe-His-Sta2.6 X 380 Ile-His-D-Lys-OH 5 H-Pro-His-Pro-Phe-His-Leu$6.0 X lo* 1.6 [ CH2S]Val-I1e-His-~-Lys-OH 6 H-Pro-His-Pro-Phe-His-Leu$2.5 X lo4 4.0 [CH2SO]Val-1le-His-~-Lys-OH 7 H-Pro-His-Pro-Phe-His-Leu$4.9 X lo-@ 200 [ CH2NH]Val-Ile-His-~-LysOH
All of the peptides were synthesized by the solid-phase method of s y n t h e s i ~ ,a~single ~ , ~ ~cycle of which is given in Table I. Similar to the report by Sasaki and we observed that Boc-Leu+[ CH2NH]Val could be incorporated into the peptide and extended upon without protection of the secondary amine function. The peptide was cleaved from the resin, and the side-chain protecting groups were removed simultaneously by treatment with HF-anisole (1O:l). Final products were purified by gel filtration chromatography and preparative reverse-phase chromatography or partition c h r o r n a t ~ g r a p h y . ~ ~ , ~ ~ Structure-Activity Studies. All of the ANG-based analogues (Figure 2) were evaluated for their inhibition of human plasma renin by using an in vitro assay described previously.27 The results shown in Table I1 are compared to H-Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-D-Lys-OH, 1, although the compounds discussed were formally analogues of 2. The comparison to 1 is necessary because the presence of the human Pz’-P3/ sequence, Ile-His, made compound 2 a substrate for renin (K, = 5 X lo* M, k,,, = 10 (22) Merrifield, R. B. J . Am. Chem. Sac. 1963, 85, 2149. (23) Barany, G.; Merrifield, R. B. The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic: New York, 1979; Chapter 1. (24) Sasaki, Y.; Coy, D. H. Peptides 1987, 8, 119. (25) Yamashiro, D. Nature, (London) 1964, 201, 76. (26) Yamashiro, D.; Gillessen, D.; du Vigneaud, V. J. Am. Chem. SOC.1966, 88, 1310. (27) Pals, D. T.; Thaisrivongs, S.; Lawson, J. A.; Kati, W. M.; Turner, S. R.; DeGraaf, S. R.; Harris, D. W.; Johnson, G. A. Hypertension 1986, 8, 1105.
Journal of Medicinal Chemistry, 1988, Vol. 31, No. 7 1379
Synthesis and Renin Inhibitory Activity
V
$H HN CH. CO N
YHz CONH-CH-
-
His
YH CH! ,CH3 CH? ,CH3 CH $H2 YHz I ~ H Z CONH- CH- CONH- CH- CO- NH- CH- CONH-CH CONH-CH- C o b Ile His His
YH
-
Pro
Phe
6
7
8
9
12
p5
p,
p3
p2
p,'
13 p,'
ANG6.13 Residues Positions Binding Sites
CHt ,CHa
H : z': ?H * NH CH-CH CH2.CO*
-
-
sta
% NH-
CH-CH
= CH- COh.
Dhs
F i g u r e 2.
min-l). It appears that the trans olefinic bond in Dhs was a possible mimic of the trans form of the P1-P1' peptide bond since peptide 3 possessed a potency about equal to that of 1. Similar to a report on a series of renin inhibitors having dehydrohydroxyethylene pseudodipeptides a t P1-P
