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Mature female mice, grouped in the absence of a male stimulus, exhibit a suppressed estrous cycle (the so-called Lee-Boot effect). We have designed a series of experiments to elucidate the involvement of the adrenal gland in this phenomenon. Our initial results indicate that adrenalectomized mice exhibit a regular estrous cycle in either isolated or grouped conditions. A single, intact mouse caged with five adrenalectomized females showed repeated normal cycles. When the urine samples from group-caged intact mice or group-caged adrenalectomized mice were applied to the external nares of singly caged females, estrous cycles were inhibited in the animals receiving urine from the intact mice but not from the adrenalectomized mice. In addition, corticosterone therapy restored the function of estrus suppression in grouped, adrenalectomized mice. We had previously shown that the urinary excretion of several volatile compounds (2-heptanone, trans-5-hepten-2-one, trans-4-hepten-2-one, pentyl acetate, cis-2-penten-1-yl acetate, and 2,5-dimethylpyrazine) was adrenal mediated (Science 1986; 231:722-725). A further testing of these compounds in relation to estrus suppression has now revealed that a mixture of these compounds is effective, but removing 2, 5-dimethylpyrazine from the mixture abolished the biological response. The overall results of this study show conclusively an important role of the adrenal gland and adrenal-mediated urinary metabolites in estrus suppression.

The heterocyclic cycloprop[f]indene 19 was synthesized via cycloaddition of diene 10 to the cyclopropene 11a and subsequent base-induced aromatization. While 19 is isolable, although very short-lived, the oxa analogue 18 decomposed under the conditions required for its preparation. The difluoro derivatives 14 and 15, in which the heterocyclic moiety is saturated, are accessible by the cycloaddition approach, but the corresponding dichlorides are again not isolable. Cycloprop[f]indenes carrying substituents at C(4) have been obtained via cycloaddition of 22b to 1-bromo-2-chlorocyclopropene. The key step of the sequence is a double Curtius degradation of the cycloadduct 23b to the ketone 27a. While aromatization of the alcohol 27b provided the cycloprop[f]indenol 28b, the reaction failed with 27a. Attempts to convert 28b to 1,3-dihydrocycloprop[f]indene (25) via the methanesulfonate 28d failed.

The synthesis of 4,5-dihydro-1H,3H-cycloprop[f]inden-4-ol (1) and diethyl 4,5-dihydro-1H,3H-cycloprop[f]-indene-4,4-dicarboxylate (26) starting from diene 4 is described. The cyclopentene ring is constructed by condensation of diethyl malonate to the dibromide 21. The key-step in the synthesis of 1 consists in a twofold Curtius degradation of 22, with subsequent reduction of the carbonyl group and aromatization. The skeleton of the isomer 31 is synthesized via cycloaddition of butadiene to cyclopent-4-ene-1,3-dione (7) and addition of dichlorocarbene to the adduct 27 after ketalisation. The attempted synthesis of dihydrocycloprop[f]indene (2) by base-induced elimination of several appropriately substituted precursors failed.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.

Two major volatile constituents of the male mouse preputial gland, E,E-alpha-farnesene and E-beta-farnesene, were examined for their role in inducing estrous cycles in grouped female mice. The results indicated that the mixture of the farnesenes was as effective as the homogenate of the intact preputial gland, while the extract of the castrate preputial tissue did not show a pronounced response.