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Acknowledgments This work was supported by NIH/National Human Genome Research Institute (NHGRI) R00 HG006922 and NIH/NHGRI R01 HG008974 (to J.G.), the Huntsman Cancer Institute, and the Women’s Cancers Disease-Oriented Team at the Huntsman Cancer Institute. Research reported in this publication utilized the High-Throughput Genomics Shared Resource at the University of Utah and was supported by NIH/National Cancer Institute award P30 CA042014. A.C.R. was supported by diversity supplement R00 HG006922S1. We thank Ed Grow for providing reagents and we thank K.-T. Varley as well as Gertz and Varley laboratory members for their helpful comments on the study and the manuscript.
Introduction Glucocorticoids (GCs) belong to the most efficient agents currently used for the treatment of allergic and inflammatory diseases. Prednisolone, dexamethasone and fluorocortivazol are notable examples of this class of drugs. Unfortunately their immunosuppressive effects are often accompanied by side effects such as muscle atrophy , steroid-induced osteoporosis , WEHI-539 hydrochloride  and hypertension . We have recently initiated a research program aiming at developing new potential ligands for the glucocorticoid receptors (GRs). We started from the simplified analogues of fluorocortivazol (MK, Scheme 1) uncovered by Ali and coll . which were developed as promising selective glucocorticoid receptor modulators (SEGRMs) . As originally demonstrated, the steroidal rings C and D of fluorocortivazol could be successfully replaced by lipophilic substituents at C11. The introduction of various side-chains R carrying aryl substituents led to the discovery of fluorocortivazol analogs with an IC50 in the low nM range for hGRs. Starting from the MK ligand, we decided to explore a spirocyclic pattern resulting from disconnecting C7 from C7a and reconnecting C7 with the methyl group at C4a (Scheme 1, target structures). The replacement of steroidal fused-rings by a spiranic structure should increase the conformational freedom of the B-D rings with less entropy loss than with a simple alkyl tether. Several low-energy conformations could be available in solution thus offering more possibilities for a better interaction with the receptor.
Synthesis This initial study was intended to validate the synthetic access to this type of compounds and not to fully explore the structure-activity relationships generated by variation of substituents (R), or the size (n) of the spirocyclic ring (Scheme 1). From this perspective, guanine did not require an access to enantiopure compounds. The stereochemical challenge was thus limited to the generation of pure diastereomers. Since the conformational flexibility should increase with the size of the spirocyclic ring, we decided to prepare representative alcohols of the homologous 5- and 6-membered B-rings. These should be easily obtained by addition of an organometallic reagent to the corresponding 5- and 6-membered ring ketone (Scheme 2).
Conclusions Spirocyclic indazoles were designed as potential ligands for the glucocorticoid receptors. Although the synthetic route to these compounds originally appeared as straightforward, in practice the construction of the homologous in 5- and 6-membered spiranic ring systems required specific sequences for each member of the homologous series as a consequence of very different reactivity patterns. Eventually, we were able to establish practical synthetic routes for each member of the homologous series. The last step leading to the alcohols yielded two diastereomers. As examples, we separated both diastereoisomers 11 and 12 and also 13b and 14b. The conformations of representative final products and key intermediates were also studied by DFT calculations and confirmed by X-rays diffraction analyses. These synthetic studies offer chemists an easy access to a wide variety of structural analogs of this new class of spirocycles. They can be easily modulated to conduct to new glucocorticoids. In order to test the validity of our design we tested both diastereoisomers 13b and 14b. If the isomer 13b is devoid of affinity for the GRs, having an IC50 of 5.7 μM, the corresponding diastereoisomer 14b has a promising IC50 of 27 nM. The completely different binding pattern of these isomers could result from the different position of the hydroxyl moiety, expected to mimic the initial C11 alcohol of fluorocortivazol. More examples in this series are necessary in order to define the SAR for this class of derivatives. This will be discussed in a forthcoming publication in a medicinal chemistry journal.