Aziridine derivatives of E-64 have been synthesized, and their characterization against the cysteine proteases cathepsin B, cathepsin L, and papain is reported. The inhibition was found to be strongly pH-dependent, with maximum activity observed at pH 4, indicating that the protonated aziridinium ion form of the inhibitor is the more reactive form. At low pH, the peptide aziridine HO-(L)Az-Leu-NH-iAm inactivated papain with a second-order rate constant, kinac/Ki, of 7.0 x 10(4) M-1 s-1, a value very close to that observed with E-64 or with the corresponding epoxysuccinyl analog HO-(L)Eps-Leu-NH-iAm. This demonstrates that with the correct peptide sequence, aziridine analogs of E-64 can be good irreversible inhibitors of cysteine proteases. Substitution of the epoxysuccinyl moiety by an aziridine does not affect the specificity of inhibition against the three proteases used in this study. The D-diastereomer is the preferred (by 10-fold) diastereomer for the inhibition of cysteine proteases. The reactivity of both diastereomers of iBuNH-Az-LeuPro-OH against cathepsin B was also found to be much lower than that of iBuNH-(L)Eps-LeuPro-OH, which is a potent selective inhibitor of cathepsin B. These differences are attributed mainly to the presence of the protonated aziridine ring, which can modify the binding mode of aziridine analogs at the active site of cysteine proteases.

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Various approaches to 17a-and 17,3-N (CH2CH2C1)2 androstane compounds have been evaluated and of the routes explored, those involving N(CH2CH2OH)2 derivatives proved the most reliable. Using androstanes with no functionality other than the mustard moiety, and also the more sensitive compounds containing oxygen and/or ethylenic groups in ring A, a detailed evaluation of the merits and disadvantages of a range of reagents and procedures for effecting the final, critical, chlorination reaction has been made. The data obtained are considered to be relevant to nonaromatic mustards in general and thus provide a guide for the selection of the most appropriate chlorinating conditions for variously functionalized steroidal and aliphatic mustards. An analysis of the factors to be considered in designing antitumor active steroid mustards has been carried out. No cytotoxic activity was detected when representative mustards were tested against DMBA-induced mammary tumors in Sprague-Dawley rats.

The low-temperature measurements were accomplished by passing precooled nitrogen through a dewar-jacketed probe with monitoring of the temperature by a copper-constantan thermocouple. For the spectra taken with the A-56/60A spectrometer, the Varian V-6057 variable temperature accessory was used. The temperatures were calibrated by replacing the sample tube with a similar one containing another thermocouple, and also by the temperature dependence of the chemical shift of the methanol hydroxyl group. The temperatures are believed to be accurate to +2".The theoretical spectra and the Arrhenius plots were computed by an IBM 7094 I1 computer coupled to a Moseley X-Y plotter.All of the gem-fluorocyclohexanes used in this study (except 1,ldifluorocyclohexane) and 1,1,2,2-tetrafluorocyclohexane were synthesized by the reaction of sulfur tetrafluoride with the corresponding ketones.2n The ketones were synthesized as below or obtained commercially.

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