Structure activity relationships of 5-, 6-, and 7-methyl-substituted azepan-3-one cathepsin K inhibitors.

The syntheses, in vitro characterizations, and rat and monkey in vivo pharmacokinetic profiles of a series of 5-, 6-, and 7-methyl-substituted azepanone-based cathepsin K inhibitors are described. Depending on the particular regiochemical substitution and stereochemical configuration, methyl-substituted azepanones were identified that had widely varied cathepsin K inhibitory potency as well as pharmacokinetic properties compared to the 4S-parent azepanone analogue, 1 (human cathepsin K, K(i,app) = 0.16 nM, rat oral bioavailability = 42%, rat in vivo clearance = 49.2 mL/min/kg). Of particular note, the 4S-7-cis-methylazepanone analogue, 10, had a K(i,app) = 0.041 nM vs human cathepsin K and 89% oral bioavailability and an in vivo clearance rate of 19.5 mL/min/kg in the rat. Hypotheses that rationalize some of the observed characteristics of these closely related analogues have been made using X-ray crystallography and conformational analysis. These examples demonstrate the potential for modulation of pharmacological properties of cathepsin inhibitors by substituting the azepanone core. The high potency for inhibition of cathepsin K coupled with the favorable rat and monkey pharmacokinetic characteristics of compound 10, also known as SB-462795 or relacatib, has made it the subject of considerable in vivo evaluation for safety and efficacy as an inhibitor of excessive bone resorption in rat, monkey, and human studies, which will be reported elsewhere.

Azepanone-based inhibitors of human cathepsin L.

The extension of a previously reported cathepsin K azepanone-based inhibitor template to the design and synthesis of potent and selective inhibitors of the homologous cysteine protease cathepsin L is detailed. Structure-activity studies examining the effect of inhibitor selectivity as a function of the P3 and P2 binding elements of the potent cathepsin K inhibitor 1 revealed that incorporation of either a P3 quinoline-8-carboxamide or a naphthylene-1-carboxamide led to increased selectivity for cathepsin L over cathepsin K. Substitution of the P2 leucine of 1 with either a phenylalanine or a beta-naphthylalanine also resulted in an increased selectivity for cathepsin L over cathepsin K. Molecular modeling studies with the inhibitors docked within the active sites of both cathepsins L and K have rationalized the observed selectivities. Optimization of cathepsin L binding by the combination of the P3 naphthylene-1-carboxamide with the P2 beta-naphthylalanine provided 15, which is a potent, selective, and competitive inhibitor of human cathepsin L with a K(i) = 0.43 nM.

Chicken collagen X regulatory sequences restrict transgene expression to hypertrophic cartilage in mice.

Collagen X is produced by hypertrophic cartilage undergoing endochondral ossification. Transgenic mice expressing defective collagen X under the control of 4.7- or 1.6-kb chicken collagen X regulatory sequences yielded skeleto-hematopoietic defects (Jacenko O, LuValle P, Olsen BR: Spondylometaphyseal dysplasia in mice carrying a dominant-negative mutation in a matrix protein specific for cartilage-to-bone transition. Nature 1993, 365:56-61; Jacenko O, Chan D, Franklin A, Ito S, Underhill CB, Bateman JF, Campbell MR: A dominant interference collagen X mutation disrupts hypertrophic chondrocyte pericellular matrix and glycosaminoglycan and proteoglycan distribution in transgenic mice. Am J Pathol 2001, 159:2257-2269; Jacenko O, Roberts DW, Campbell MR, McManus PM, Gress CJ, Tao Z: Linking hematopoiesis to endochondral ossification through analysis of mice transgenic for collagen X. Am J Pathol 2002, 160:2019-2034). Current data indicate that the hematopoietic abnormalities do not result from extraskeletal expression of endogenous collagen X or the transgene. Organs from mice carrying either promoter were screened by immunohistochemistry, in situ hybridization, and Northern blot; transgene and mouse collagen X proteins and messages were detected only in hypertrophic cartilage. Likewise, reverse transcriptase-polymerase chain reaction revealed both transgene and mouse collagen X amplicons only in the endochondral skeleton of mice with the 4.7-kb promoter; however, in mice with the 1.6-kb promoter, multiple organs were transgene-positive. Collagen X and transgene amplicons were also detected in marrow, but likely resulted from contaminating trabecular bone; this was supported by reverse transcriptase-polymerase chain reaction analysis of rat tibial zones free of trabeculae. Our data demonstrate that in mice, the 4.7-kb chicken collagen X promoter restricts transcription temporo-spatially to that of endogenous collagen X, and imply that murine skeleto-hematopoietic defects result from transgene co-expression with collagen X. Moreover, the 4.7-kb hypertrophic cartilage-specific promoter could be used for targeting transgenes to this tissue site in mice.

Phenylbutyrates as potent, orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists.

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.

Linking hematopoiesis to endochondral skeletogenesis through analysis of mice transgenic for collagen X.

Each skeletal element where marrow develops is first defined by a hypertrophic cartilage blueprint. Through programmed tissue substitution, the cartilaginous skeletal model is replaced by trabecular bone and marrow, with accompanying longitudinal tissue growth. During this process of endochondral ossification, hypertrophic cartilage expresses a unique matrix molecule, collagen X. Previously we reported that transgenic mice with dominant interference collagen X mutations develop variable skeleto-hematopoietic abnormalities, manifested as growth plate compressions, diminished trabecular bone, and reduced lymphatic organs (Nature 1993, 365:56). Here, histology and flow cytometry reveal marrow hypoplasia and impaired hematopoiesis in all collagen X transgenic mice. A subset of mice with perinatal lethality manifested the most severe skeletal defects and a reduction of marrow hematopoiesis, highlighted by a lymphocyte decrease. Thymic reduction is accompanied by a paucity of cortical immature T cells, consistent with the marrow's inability to replenish maturing cortical lymphocytes. Diminished spleens exhibit indistinct lymphatic nodules and red pulp depletion; the latter correlates with erythrocyte-filled vascular sinusoids in marrows. All mice display reduced B cells in marrows and spleens, and elevated splenic T cells. These hematopoietic defects underscore an unforeseen link between hypertrophic cartilage, endochondral ossification, and establishment of the marrow microenvironment required for blood cell differentiation.