J.Med. Chem.
1986,29, 585-589
585
Substituted Isatoic Anhydrides: Selective Inactivators of Trypsin-like Serine ProteasestJ Michael H. Gelb and Robert H. Abeles* Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254. Received May 6, 1985 Derivatives of isatoic anhydride were prepared and tested as inhibitors of serine proteases. A number of isatoic anhydrides with positively charged substituentsirreversibly inactivated several trypsin-like enzymes and preferentially inactivated trypsin over chymotrypsin. Further selectivity was obtained by introduction of an aromatic group on the N-1 position of isatoic anhydride. 7-(Aminomethyl)-l-benzylisatoicanhydride was prepared and was a selective inactivator of thrombin; thus it is possible to prepare derivatives of isatoic anhydride that are highly enzyme selective without attaching peptide recognition structures. We have recently described the inactivation of serine proteases by isatoic anhydride (l),loxazinediones (2),lp2 and benzoxazinones (3).3 The mode of inactivation of 0
H 1
0
H
2
0
3
chymotrypsin by 1 involves the initial attack of the active-site serine onto the anhydride carbonyl group of 1 to produce an enzyme-bound carbamate. This species rapidly decarboxylates to the anthranoyl-enzyme. Because of the presence of the electron-releasing amino group, anthranoylchymotrypsin resists deacylation. We have now explored the possibility of increasing the selectivity of inactivation toward specific proteases by modification of these inhibitors, but without the attachment of polypeptides. We chose to prepare derivatives of isatoic anhydride (1) that are targeted toward thrombin. Thrombin inactivators are of interest as potential antithrombotic drugs. Synthetic Procedures. The synthetic approach to the substituted isatoic anhydrides is illustrated in Scheme I. Commercially available 4 was converted via the Rosenmund-von Braun synthesis to nitrile 5 by heating with CuCN. As reported earlier, bromination of 5 to the benzyl bromide proved to be difficult;* however, when the crude bromide was heated with sodium acetate in acetic acid, a satisfactory yield of acetate 6 was obtained following chromatography on silica. Heating 6 in aqueous HBr produced acid 7. Treatment of 7 with excess ammonia gave amino acid 8, which was treated with di-tert-butyl pyrocarbonate to afford the tert-butyl carbamate amino acid 9. Reduction of 9 with sodium dithionite under alkaline conditions gave the substituted anthranillic acid 10. Reaction of 10 with phosgene gave the isatoic anhydride lla. Compound l l a could be alkylated at N-1 by stirring with an alkyl halide in the presence of Na2C0t to produce 1 lb and 1 IC. Final deprotection with trifluoroacetic acid gave the N-1-substituted 7-aminomethylisatoic anhydrides 12a-c. Inhibition of Serine Proteases by Substituted Isatoic Anhydrides. The design of substituted isatoic anhydrides that selectively inhibited trypsin-like serine proteases was carried out in two stages. First, a substituted isatoic anhydride containing the basic aminomethyl group 'Publication 1578 from the Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254. *This work was supported in part by NIH Grant No. 5R01 GM 12633-22 and by an American Cancer Society Postdoctoral Fellowship to MHG. 0022-2623/86/1829-0585$01.50/0
attached to the aromatic ring was prepared (compound 12a). Previous studies have shown that trypsin-like enzymes are inhibited by aromatic compounds containing positively charged groups, i.e., benzamidine and phenylguanidine.6 Results in Table I show that compounds 12a and 12b containing a positive charge are better inactivators for trypsin and thrombin than for chymotrypsin. For example, compound 12a rapidly inactivates both trypsin and thrombin at 2.5 pM, whereas no detectable inactivation of chymotrypsin was measured under these conditions. In contrast, unsubstituted isatoic anhydride (1) inactivates chymotrypsin rapidly at 2.5 pM, but no inactivation of either trypsin or thrombin occurs under these conditions. Second, additional modifications of 12a were made in order to prepare selective inhibitors for thrombin. The inhibitory properties toward thrombin of a series of ester and amide derivatives of N"-substituted arginine has been reported.' Many of the reported compounds contain an aromatic substituent on the Ne group of arginine. Since this position is somewhat isosteric with N-1 of isatoic anhydride, we attached aromatic substituents to N-1 with the expectation that such derivatives would be salective inactivators of thrombin. The data in Table I show that incorporation of an aromatic residue increases selectivity toward thrombin. The most effective thrombin inhibitor in the series was the N-1-benzylderivative 12b. Compound 12b, at 2.5 pM, inactivated thrombin with a half-time of 2.6 min. No detectable inactivation of trypsin, chymotrypsin, or plasmin was observed under these conditions. The reactivation of thrombin inactivated with 12b was studied. Thrombin was incubated with 10 pM 12b until no activity remained (ea. 5 min). Excess inactivator was removed by dialyzing the solution for several hours at 4 OC. The dialyzed solution was kept at 25 "C, and the return of enzymatic activity was followed by periodically assaying small aliquots of the mixture. No activity was detected after 1h at 25 "C. After 13 h, 30% of the activity had returned. These results demonstrate that thrombin inactivation by 12b is long-lasting but not completely irreversible. Compound 12b was also tested as an anticoagulant. The presence of 500 pM 12b in serum increased the partial thromboplastin time by 2.6-fold. If 12b is preincubated ~~~
(1) Moorman, A. R.; Abeles, R. H. J . Am. Chem. SOC. 1982, 104, 6785. (2) Weidmann, B.; Abeles, R. H. Biochemistry 1984, 23, 2373. (3) Hedstrom, L.; Moorman, A. R.; Dobbs, J.; Abeles, R. H. Biochemistry 1984, 23, 1753. (4) Julia, M.; Chastrette, F. Bull. SOC.Chim. Fr. 1962, 2255. (5) Hardtmann, G. E.; Koletar, G.; Pfister, 0. R. J . HeterocycL Chem. 1975, 12, 565. (6) Mares-Guia, M.; Shaw, E. J . Biol. Chem. 1965, 240, 1579. (7) Okamoto, S.; Kinjo, K.; Hijikata, A. J. Med. Chem. 1980,23, 827. Kikumoto, R.; Tamao, Y.; Ohkubo, K.; Tezuka, T.; Tonamura, S. Zbid. 830. Kikumoto, R.; Tamao, y.;Ohkubo, K.; Tezuka, T.; Tonamura,S.; Okamoto, S.; Hijikata, A. Zbid. 1293.
0 1986 American Chemical
Society
586 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 4
Notes
Scheme I 0
II CH2-O-C-CH3 I
1. Brz/hw 2. C83C02Na/CH3C0zH
’ (6) CN
aq. RBr/li
@,
~
(1)
NR3/cthanol
NoZ
coon 0
II cHz-NH-c-o
+
0
It CHZ-NH-C-0
COOH
+
CboH 0
I1
CHz-NH-C-0
+
0
COCl2/Na2CO3 RBr/Na2C03
’
Table I. T l j 2for Inactivation of Serine Proteases by Substituted Isatoic Anhydrides 0
TI,, for inactivation: min
no.
R1
1 12a
12b
H CH2NH3+ CH3NH3+
12c
CH2NH3+
R2
H H
VI, PM: chymotrypsin 2.5 25 125 2.7 0.5 0.8 NI CK 1.5 NI NI CK
[I], pM: trypsin 2.5 25 125 NIb 3.9 2.0 1.3 0.7 KO.1 NI CK 10
[I], pM: thrombin 2.5 25 125 NI CKc 11.5 3.4 2.6
