Sample size determination in combinatorial chemistry.

Combinatorial chemistry is gaining wide appeal as a technique for generating molecular diversity. Among the many combinatorial protocols, the split/recombine method is quite popular and particularly efficient at generating large libraries of compounds. In this process, polymer beads are equally divided into a series of pools and each pool is treated with a unique fragment; then the beads are recombined, mixed to uniformity, and redivided equally into a new series of pools for the subsequent couplings. The deviation from the ideal equimolar distribution of the final products is assessed by a special overall relative error, which is shown to be related to the Pearson statistic. Although the split/recombine sampling scheme is quite different from those used in analysis of categorical data, the Pearson statistic is shown to still follow a chi2 distribution. This result allows us to derive the required number of beads such that, with 99% confidence, the overall relative error is controlled to be less than a pregiven tolerable limit L1. In this paper, we also discuss another criterion, which determines the required number of beads so that, with 99% confidence, all individual relative errors are controlled to be less than a pregiven tolerable limit L2 (0 < L2 < 1).

Antipneumocystis activity of water-soluble lipopeptide L-693,989 in rats.

Water-soluble lipopeptide L-693,989 was evaluated for its antipneumocystis activity in rats. Rats from colonies with latent Pneumocystis carinii infections were immunosuppressed with dexamethasone for 6 weeks to facilitate the development of acute P. carinii pneumonia (PCP). After 6 weeks, the rats were maintained on dexamethasone and were treated twice daily for 4 days with various concentrations of L-693,989. At a dose of 0.15 mg/kg of body weight, the compound effectively eliminated 90% of the cysts in 4 days. Trophozoite forms of P. carinii were still present in these animals, as determined by using a P. carinii-specific DNA probe. A 3-week therapy study showed that the trophozoite load did not expand during treatment and that the trophozoites already present at the initiation of therapy appeared to persist. This may be a consequence of the stage specificity of the compound for cyst development and the severe immunosuppressive effects of dexamethasone on rats. When evaluated as a daily parenteral prophylactic agent, L-693,989 was effective in preventing the development of both P. carinii cysts and trophozoites, demonstrating its potential for use in prophylaxis and implying that the cyst stage of P. carinii is an obligatory step in trophozoite multiplication. The foamy exudate commonly associated with P. carinii infections was absent in the lungs of rats on prophylaxis. The compound was also evaluated via oral administration and was found to have a 90% effective dose of 32 mg/kg for therapy of acute infections and 5 mg/kg for daily prophylaxis.

Preparation and structure-activity relationships of simplified analogues of the antifungal agent cilofungin: a total synthesis approach.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.

Antioxidant-based inhibitors of leukotriene biosynthesis. The discovery of 6-[1-[2-(hydroxymethyl)phenyl]-1-propen-3-yl]-2,3-dihydro-5- benzofuranol, a potent topical antiinflammatory agent.

The leukotrienes, metabolites of arachidonic acid produced through the action of the enzyme 5-lipoxygenase, are important mediators of immediate hypersensitivity and inflammation. Among the variety of diseases in which the leukotrienes may play a symptomatic or causative role is the dermatological condition psoriasis, a chronic proliferative disease of the skin. This study reports the synthesis and comparative biological activities of various ortho-substituted phenols including 4-methoxyphenols, 6-hydroxy-1,2,3,4-tetrahydrobenzopyrans, 2,3-dihydro-5-benzofuranols, and 5-benzofuranols. The phenols prepared in this study were evaluated for their ability to inhibit the production of leukotriene B4(LTB4) in isolated human polymorphonuclear leukocytes (PMNs) and to inhibit a topical inflammatory response in the topical mouse ear (TME) model. In the former case, when the log IC50 was plotted versus the log of the octanol/water partition coefficient (log P), to eliminate the effect of lipophilicity, the 2,3-dihydro-5-benzofuranol ring system was shown to be more potent than the other ring systems examined throughout the range of partition coefficients studied. The ability to inhibit leukotriene production in vitro in human PMNs can be rationalized on the basis of a model that suggests that the observed inhibition is dependent on the kinetic ability of the inhibitor to reduce a radical species and on the fraction of inhibitor that is partitioned into the cell membrane. While the in vivo antiinflammatory activity as measured by the TME did not correlate with the in vitro data, it was felt that the TME represented a valuable measure of the ability of a compound to penetrate the skin to the site of an ongoing inflammatory response. Of the compounds synthesized in this study, 6-[1-[2-(hydroxymethyl)phenyl]-1-propen-3-yl]-2,3-dihydro-5-benzof uranol (1, L-651896) was chosen for further development.

2,3-Dihydro-5-benzofuranols as antioxidant-based inhibitors of leukotriene biosynthesis.

The enzymes that catalyze the oxidative metabolism of arachidonic acid have provided fertile ground for the development of useful therapeutic agents for nearly a quarter century. Inhibitors of the enzyme cyclooxygenase prevent the formation of the prostaglandins and thromboxanes and are clinically useful antiinflammatories and peripheral analgesics. More recently it has been discovered that the enzyme 5-lipoxygenase is the first step in the formation of a series of biologically important metabolites of arachidonic acid, the leukotrienes. Evidence suggests that an inhibitor of 5-lipoxygenase may be a useful therapeutic agent in the treatment of asthma, immediate hypersensitivity, and inflammation. Various antioxidants have been examined as inhibitors of 5-lipoxygenase in vitro. We were intrigued by recent reports that the 2,3-dihydro-5-benzofuranol ring system maximizes the stereoelectronic effects necessary for efficient hydrogen atom abstraction by peroxyl radicals. In this study we describe the synthesis of over 50 new 2,3-dihydro-5-benzofuranols and their biological evaluation as inhibitors of leukotriene biosynthesis in isolated human polymorphonuclear leukocytes. We show that the 2,3-dihydro-5-benzofuranol ring system, although not a potent inhibitor of leukotriene biosynthesis in itself, can provide a useful template for the design of antioxidant-based inhibitors of leukotriene biosynthesis. Furthermore, within a structural class the potency of a given analogue can be predicted on the basis of its overall calculated lipophilicity (log P). The data are interpreted in terms of a model in which the observed inhibition by this class of inhibitors is dependent on the intrinsic ability of the antioxidant to reduce the enzyme and on the fraction of the inhibitor that is partitioned into the membrane.