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An attempt to develop a water-soluble carbonic anhydrase inhibitor focused on exploring structure-activity relationships in the thienothiopyransulfonamide class. The strategy to influence water solubility while retaining carbonic anhydrase activity involved the introduction of a hydroxyl moiety and adjusting the oxidation state of the sulfur on the thiopyran portion of the molecule. Compounds 4 and 17 best fit the criteria of aqueous solubility and inhibitory potency vs. human carbonic anhydrase II and are candidates for evaluation as topically effective antiglaucoma agents.

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The 3-dimensional structure of human carbonic anhydrase I1 (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, l a , l b , and I C , has been determined by X-ray crystallographic methods. The 3 inhibitors ( l a = C8H,2N204S3) vary only in the length of the substituent on the 4-amino group: l a , proton; Ib, methyl; and IC, ethyl. The binding constants (Ki's) for la, Ib, and I C to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors l a and l b , electron density can be observed for His-64 and a bound water molecule in the native positions. When inhibitor IC is bound, the side chain attached to the 4-amino group is positioned so that His-64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason IC binds tighter to HCAII than does l a or l b appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His-64 side chain in a higher energy state.

Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogendependent platelet aggregation stimulated by ADP with ICso = 3.3 x 10-8 M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg2"-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor-a heterodimeric complex composed of glycoproteins Ub and lila. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 X 10-7 M. Substitution with Ala-24 gave IC50 = 6.1 x 10-7 M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 X 10-6 M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10-4 M), and Ala-Gly-AspPhe (IC50 = 5.0 x 10-4 M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin.