Identification of potent water soluble purine-scaffold inhibitors of the heat shock protein 90.

Hsp90 is a chaperone protein that allows cancer cells to tolerate the many components of dysregulated pathways. Its inactivation may result in targeting multiple molecular alterations and, thus, in reverting the transformed phenotype. The PU-class, a purine-scaffold Hsp90 inhibitor series, has been reported to be potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of this class was synthesized and evaluated as inhibitors of the chaperone. The structure-activity relationship and selectivity for tumor Hsp90 of compounds within the series is presented. The study identifies water soluble derivatives (>5 mM in PBS pH 7.4) of nanomolar potency (IC(50) approximately 50 nM) in cellular and animal models of cancer. Binding affinities of these compounds for Hsp90 correlate well with their biological activities. When administered in vivo to mice bearing MDA-MB-468 human breast cancer xenografted tumors, these agents result in pharmacologically relevant concentrations and, accordingly, in modulation of Hsp90-client proteins in tumors.

Evaluation of 8-arylsulfanyl, 8-arylsulfoxyl, and 8-arylsulfonyl adenine derivatives as inhibitors of the heat shock protein 90.

Hsp90 is a chaperone protein with important roles in maintaining transformation and in elevating the survival and growth potential of cancer cells. Currently there is an increasing interest in developing inhibitors of this protein as anticancer therapeutics. One of such inhibitors, the purine-scaffold class, has been reported to be potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of 8-arylsulfanyl, -sulfoxyl, and -sulfonyl adenine members of the purine class was synthesized and evaluated as inhibitors of the chaperone. The structure-activity relationship and selectivity for tumor Hsp90 of compounds within the series is presented. Our results suggest that 8-arylsulfanyl adenine derivatives are good inhibitors of chaperone activity, whereas oxidation of the sulfides to sulfoxides or sulfones leads to compounds of decreased activity. The study identifies derivative 11v as the most potent Hsp90 inhibitor of the purine-scaffold series published to date (EC(50) = 30 nM), and also as the compound of this class with highest selectivity for tumor vs normal cell Hsp90 (700 to 3000-fold). Most rewardingly, this work has allowed for the identification of Hsp90 inhibitors with selective affinities for Hsp90-client protein complexes, derivatives that may represent useful pharmacological tools in dissecting Hsp90-regulated processes.

Comparative study of the reactivities of substituted 3-(benzoyl)benzyl carbanions in water and in DMSO.

Benzyl-substituted carbanions produced by photodecarboxylation of ketoprofen derivatives have been examined in basic aqueous and DMSO solutions. Product studies, combined with kinetic measurements from laser flash photolysis, have allowed the determination of absolute rate constants for protonation and intra-S(N)2 reactions leading to five- and six-membered ring cyclizations; the former are significantly faster. Many of the well-known trends in carbanion reactivity are placed on an absolute rate basis; thus, intra-S(N)2 are favored in polar nonprotic solvents, and the effect is larger for the more hindered carbanion centers. Protonation by water is slightly dependent on the nature of the carbanion center and is approximately 400 times faster in nonhydroxylic solvents, compared with bulk water. As expected, the reactivity for halide leaving groups follows the usual order of decreasing bond strengths, i.e., I(-) > Br(-) > Cl(-).

Development of a fluorescence polarization assay for the molecular chaperone Hsp90.

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90alpha (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90alpha with high affinity. Even at low Hsp90alpha concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z' values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90alpha with EC(50)s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.

General method for the synthesis of 8-arylsulfanyl adenine derivatives.

We report a general method for the synthesis of 8-arylsulfanyl adenine derivatives using a mild protocol of coupling 8-mercaptoadenine with a variety of aryl iodides.

Mapping photogenerated radicals in thin polymer films: fluorescence imaging using a prefluorescent radical probe.

Prefluorescent radical probes, in which fluorescence is activated by radical trapping, and photoinitiators were used to detect radical generation in polymer films using fluorescence spectroscopy and microscopy. Prefluorescent radical probes are the foundation of a fluorescence imaging system for polymer films, that may serve both as a mechanistic tool in the study of photoinitiated radical processes in polymer films and in the preparation of functional fluorescent images.

First determination of absolute rate constants for the reaction of aroyl-substituted benzyl carbanions in water and DMSO.

The prompt generation of carbanions II and III within the duration of the nanosecond laser pulse provides a way of evaluating absolute rate constants for their two decay pathways, protonation and cyclization, the latter resulting from an intramolecular nucleophilic carbanion displacement of iodide tethered at the end of the lateral alkyl chain. Absolute rate constants are given for both carbanions (II and III) and show that the intra-S(N)2 reaction is favored in aprotic media, such as dimethyl sulfoxide (DMSO), while protonation is the dominant reaction in basic aqueous media.

Absolute rate constants for water protonation of 1-(3-benzoylphenyl)alkyl carbanions.

[reaction: see text] Efficient photodecarboxylation of (3-benzoylphenyl)alkanoic acids with formation of carbanions has enabled the determination of their protonation rate constants in water; the values obtained show that the reactivity toward protonation is determined by the size of the alkyl groups attached to the carbanion center.