f'arrish et al.
1132 Journal of Medicinai Chemistrj', 1978, Voi. 21, No. 11
R e f e r e n c e s and N o t e s (1) Istituto di Chimica Farmaceutica e di Chimica Organica, Universita Degli Studi di Camerino, 62032 Camerino (MC),
Italy. (2) Y. Ueda, C. Melchiorre, B. Lippert, B. Belleau, S.Chona, and D. J. Triggle, Farmaco. Ed. Sei.. 33 (7,479 (1978). (3) G. E.Demaree, R. E. Brockenton, M. H. Heiffer, and U'. E. Rothie, J . Pharm. Sci., 60, 1743 (1971). (4) B. Lippert and B. Belleau in "Frontiers in Catecholamine Research", E. Usdin and S. H. Snyder, Eds., Pergamon Press, London and New York, 1973,p 369. (5) \t'. Eschweiler. Ber., 38, 880 (1905); H. T.Clarke, H. B. Gillespie, and S. Z. Reisshaus, J . Am. Chem. Soc.. 55,4571 (1933). (6) D. E. Koshland, Jr., G. Xemethy, and D. Filmer, Biochemistry, 5, 365 (1966). (7) B. G.Benfey and S. A. Grillo. Rr. J . Pharrnacol., 20,528 (1963). (8) H. Boyd, G. Burnstock, G. Campbell, A. Jowett, J. O'Shea, and M. Wood, Rr. J . Pharmacol., 20,418 (1963).
(9) B. G. Benfey and K. Greeff, Br. J . Pharmacol. C'hemother., 17, 232-235 (1961). (10) L. L. Iversen, Br. Med. Bull., 29, 130--135(1973). (11) M. S. Yong and N. Nickerson. J . Pharmucoi. E x p . ?'her., 186, 100-108 (1973). (12) S . C. Harvey and M. Nickerson, J . Pharmacol. Exp. Ther., 112, 274 (1954). (13) A. D'Iorio and J. C. Lague, Can. J . Biochem.. 41,121 (1963). (14) J . M.Goldman and M. E. Hadley, J . Pharrnacol. Exp. Ther., 182, 93 (1972). (15) D. R. Mottram, Hiochem. Pharmacol., 25: 2104 (19%). (16) R. J. Wineman, M. H. Gallis, J. C. James, and A. M. Pomponi, J . Org. Chem., 27, 4222 (1962). (17) J. von Braun and C. Muller, Ber., 38, 2203 (1905). (18) C. H. Elderfield, J. W. Gender, H. T. Bembry, F. Brody, L. Widerhold, and B. Neuman, J . Am. Chem. Soc., 68,1568 (1946). (19) W e have recently obtained concrete evidence that the discriminatory effect of BHC reflects the existence of two a-receptor subspecies displaying different affinities for BHC and consequently for NE and E, respectively.
Structure-Activity Relationships for the Inhibition of Acrosin by Benzamidine Derivatives R. F. Parrish, J. W.Straus, J. D. Paulson, K. L. Polakoski,* Department of Obstetrics and Gj,necology, Washington L'nicersit) School of Medicine, St. Louis, Missouri 631 10
R. R. Tidwell, J. D. Geratz, a n d F. M. Stevens Department of Pathology, C'nicersity of North Carolina School of Medicine, Chapel Hill,North Carolina 27514. Receiued April 24, 1978 A series, consisting of 52 benzamidine derivatives, was evaluated for inhibitory activity against homogeneous boar sperm acrosin. All of the compounds in the series proved to be more potent than benzamidine (Ki = 4.0 X M),with one of the derivatives, cu-(4-amidino-2,6-diiodophenoxy)-3-nitrotoluene (compound 16), showing outstanding potency with a Ki value of 4.5 X M. Although all of the derivatives were effective acrosin inhibitors, structural specificity was observed within homologous groups of compounds. The information gained from this preliminary study should prove extremely beneficial in the design and synthesis of future acrosin inhibitors.
Prior communications have described the development of aromatic amidines as potent a n d specific inhibitors of a number of proteolytic enzymes, i.e., thrombin, trypsin, and kallikrein.' During the course of these studies a sizable series of structurallv diverse amidine derivatives has been prepared. Using many of these known amidines, as well as an equal number of novel derivatives, this paper reports the initial structure-activity studies of another biologically important protease, acrosin. Acrosin (E.C. 3.4.21.10), a trypsin-like protease in spermatozoa, functions to digest a p a t h for the spermatozoon through t h e zona pellucida of t h e ovum.* T h e critical dependency of reproduction on acrosin was clearly demonstrated when both in vivo3i4a n d in vitro5s6 fertilization were blocked by acrosin inhibitors. Thus, acrosin inhibition possesses a powerful potential to serve in an antienzymatic approach to fertility control. Although human acrosin is the ultimate target enzyme for utilization of this potential, only limited studies have been undertaken, due to the small quantities of material available and t o t h e lability of t h e human e n ~ y r n e . ~However, ,~ boar acrosin has recently been purified to homogeneityg from t h e purified zymogen precursor, proacrosin, is now available in sufficient quantities, and is sufficiently stable, so t h a t large-scale testing of synthetic inhibitors is now
feasible. Amidine derivatives were chosen for this study because they were not only proven, potent antiproteolytic agents b u t also because benzamidine has previously been determined to inhibit crude preparations of both rabbitlo a n d bull" acrosin. Results a n d Discussion T h e dissociation constants of the amidine derivatives with acrosin were determined from rate assays employing N-a-benzoyl-L-arginine ethyl ester (BzArgOEt) as the substrate. In each instance the reaction followed Michaelis-Menton kinetics, a n d inhibition was strictly competitive a n d reversible. T h e compounds are listed in Tables I-IV with their structural formulas and respective K , values. T h e Ki values for thrombin, trypsin, and kallikrein have been included for comparison in Table IV. B e n z y l P h e n y l Ethers ( T a b l e I). T h e series of compounds comprising Table I includes 17 novel derivatives a n d five known compounds. This extensive homologous series, with its wide range of antiacrosin activities, serves best to illustrate the specificity of acrosin for amidine-type inhibitors. With the exception of the p-cyano derivative (compound 8) and the p-nitro derivative (compound 9), monosubstitution of the leadoff compound, 1, at either the meta (R5)or para (R4) position of the benzyl
0022-2623/78~1821-1132$01.00~0T 1978 American Chemical Societ)
Journal of Medicinal Chemistry, 1978, Vol. 21, No. 11 1133
Acrosin Inhibition by Benzamidine
Table I. Acrosin Inhibition by Benzyl Phenyl Ether Derivatives R3\
ref
compd no. benzamidine Ama Am 2 Am 3 4 Am Am 5 Am 6 7 Am Am 8 9 Am 10 Am H 11 H 12 13 NO 2 14 NO2 H 15 H 16 17 Am 18 Am 19 Am Am 20 H 21 H 22 H 23 Am = -C( = NH)NH2. 1
H H H H H H H H H H Am Am H H NO1 NO2 H H H H Am Am Am
H H H H H H H H H H H H H I H I H H Br I Br
H H H
H
CH(CH3)2 OCH,
H H H H H NO2 H NO1 H H H H Am H H H H H H
c1
3"
CN NO2 H NO2 H Am Am Am Am H Am Am Am Am Am Am
c1 I
H H H H H H H H H H H H H H H I H H H H H
c1 I
4.0 i 0.1 2.6 i 0.9 1.7 i 0.4 1.9 i 0.2 0.95 5 0.04 1.1 i 0.05 1.6 t 0.3 2.3 i 0.4 3.9 i 0.1 2.6 i 0.5 0.61 i 0.04 0.76 i 0.06 0.86 i 0.05 1.6 i 0.1 0.30 i 0.09 2.4 t 0.2 0.045 i 0.006 0.29 i 0.02 0.48 i 0.02 0.68 i 0.06 0.45 i 0.02 1.1 ? 0.1 0.44 t 0.06 0.19 i 0.01
this work this work this work this work this work this work this work this work this work this work this work this work this work this work this work this work lb lb lb this work lb lb lb
Table 11. Acrosin Inhibition by a,w-Dioxyalkane Derivatives
R
I
R2
compdno.
a
n
R,
Ama 24 3 Am 25 3 Am 26 3 Am 27 3 Am 28 5 Am 29 5 Am 30 5 Am 31 5 32 5 Am Am 33 5 Am 34 5 35 5 H 36 5 H 37 5 Am Am 38 8 Am 39 8 Am 40 8 Am 41 10 Am 42 10 Am 43 12 Am 44 14 Am = -C(=NH)NH,.
R2
R3
H H H H
I
H H
H
H H H H H Am Am H H
I H
H H H H H H
H Br Br H H H I H H I H H I H Br H H
~
O
~
C
H
:
~
O
~
R
~
R7
R3
R4
R,
H H H Br H H H H H H H H H H H H H H H H H
H H Br H H H H H I H H H H H H H I H Br H H
ring (compounds 2-12, 17, and 18) resulted in an increase in antiacrosin potency. The most effective substituent was an amidino group placed in either the meta or para position (compounds 17 and 18), where a nine- and fivefold increase in potency, respectively, was observed when compared to compound 1. Halogenation of compound 18 a t the R3position (compounds 19 and 20) produced little
~
R6
Am Am Am Am H Am NH2 NO2 Am H H NO, NH, H H Am Am Am Am Am Am
R7
H H H H H H H H H NO2 NO2 H H Am
H H H H H H H
Ki, P M
ref
3.5 t 0.3 0.68 i 0.3 1.7 i 0.3 0.57 i 0.3 0.28 * 0.08 2.3 i 1.3 2.0 1. 0.1 0.86 i 0.2 1.0 i 0.1 1.1* 0.2 0.27 * 0.07 0.68 i 0.1 0.56 i 0.07 1.0 i 0.4 1.0 i 0.02 1.2 i 0.2 0.26 i 0.02 0.56 * 0.04 0.26 i 0.09 0.44 i 0.1 0.13 i 0.01
la la la la la this work la this work this work la this work this work this work this work this work la la la la
la la
change in antiacrosin activity. But, when the amidino group a t the R1position of compound 19 is moved t o the R2 position (compound 21), a twofold decrease in antiacrosin potency was observed, relative t o compounds 18-20. Of considerable interest was the diverse effect that nitro substitution had on acrosin binding affinity. For example, replacement of one of the amidino moieties on
1134 Journul of Medicinal Chemistry, 1978, Vol. 21, No. 11
Purrish et ul
Table 111. Acrosin Inhibition by u,a',oi"-Tris(phenoxy)mesitylene Derivatives
R3q qR2 'O\
R6
--__
R5
cH2vcHz
R9
RB
CH2
l
Cr'; 0
R4
Rl
compdno. R , 45 Amu 46 Am 47 NO, 48 NO, 49 H _____ ' Am = -C(=NH)NH,.
R, Am Am Am Am H
R3
R4
Am Am Am Am H
H H H H Am
R, H H H H Am
R6
H H H H Am
R, H I
R; H I
H
H
H H
C1 H
ref
R,
Ki, F M
H I H C1 H
1 . 3 i 0.3 0.64 i 0.3 1.6 * 0.4
Id
Id this work this work Id
1.6 i- 0.2
_ _ _ _0.56 _ _ i- ~0.2_ _ _ _ -
~
Table IV. ComDarison of Acrosin Inhibition with Potent Protease Inhibitors compd no.
-
50
~
51
b b-
