Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

Azepanone-based inhibitors of human and rat cathepsin K.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

Design and characterization of orally active Arg-Gly-Asp peptidomimetic vitronectin receptor antagonist SB 265123 for prevention of bone loss in osteoporosis.

The Arg-Gly-Asp (RGD)-binding integrin alpha(V)beta(3) is highly expressed on osteoclasts and has been proposed to mediate cell-matrix adhesion required for osteoclast-mediated bone resorption. Antagonism of this receptor should prevent stable osteoclast adhesion and thereby inhibit bone resorption. We have generated an orally bioavailable, nonpeptide RGD mimetic alpha(v)beta(3) antagonist, SB 265123, which prevents bone loss in vivo when dosed by oral administration. SB 265123 binds alpha(v)beta(3) and the closely related integrin alpha(v)beta(5) with high affinity (K(i) = 3.5 and 1.3 nM, respectively), but binds only weakly to the related RGD-binding integrins alpha(IIb)beta(3) (K(i) >1 microM) and alpha(5)beta(1) (K(i) >1 microM). The compound inhibits alpha(v)beta(3)-mediated cell adhesion with an IC(50) = 60 nM and more importantly, inhibits human osteoclast-mediated bone resorption in vitro with an IC(50) = 48 nM. In vivo, SB 265123 completely blocks bone resorption in a thyroparathyroidectomized rat model of acute bone resorption when dosed at 2.5 mg/kg/h by continuous i.v. infusion. When dosed orally with 3 to 30 mg/kg b.i.d. , in the ovariectomy-induced rat model of osteoporosis, SB 265123 prevents bone resorption in a dose-dependent fashion. This is the first report of an orally active alpha(v)beta(3) antagonist that is effective at inhibiting bone resorption when dosed in a pharmaceutically acceptable fashion. Such a molecule may provide a novel therapeutic agent for the treatment of postmenopausal osteoporosis.

Orally bioavailable nonpeptide vitronectin receptor antagonists with efficacy in an osteoporosis model.

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.

Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase.

Members of three classes of pyridinylimidazoles bind with varying affinities to CSBP (p38) kinase which is a member of a stress-induced signal transduction pathway. Based upon SAR and protein homology modeling, the pharmacophore and three potential modes of binding to the enzyme are presented. For a subset of pyridinylimidazoles, binding is shown to correlate with inhibition of CSBP kinase activity, whereas no significant inhibition of PKA, PKC alpha and ERK kinase activity is observed.

Design of a potent and orally active nonpeptide platelet fibrinogen receptor (GPIIb/IIIa) antagonist.

The direct design of the potent nonpeptide platelet fibrinogen receptor (GPIIb/IIIa) antagonist, 8-[[[4- (aminoiminomethyl)phenyl]amino]carbonyl]-2,3,4,5-tetrahydro-3-oxo- 4- (2-phenylethyl)-1H-1,4-benzodiazepine-2-acetic acid, (3) (SB 207448), based on the structure and conformation of the potent and highly constrained cyclic peptide antagonist SK&F 107260 (2), has been reported [Ku et al., J. Am. Chem. Soc. 1993, 115, 8861]. While 3 displayed in vivo activity in the conscious dog following intravenous administration, it was not active following intraduodenal administration; activity was measured with an ex vivo platelet aggregation assay. The secondary amide in 3 was N-methylated in the expectation of increased absorption and bioavailability. The resulting tertiary amide, 4 (SB 208651), also showed high binding affinity for human GPIIb/IIIa and potent antiaggregatory activity in human platelet-rich plasma. Most importantly, 4 was active in vivo following intravenous and intraduodenal administration. Comparison of the iv and id inhibition curves suggests an apparent bioavailability of approximately 10%. Thus, 4 represents the first orally active compound in this series of potent, nonpeptide fibrinogen receptor antagonists.

The crystal structure, absolute configuration, and phosphodiesterase inhibitory activity of (+)-1-(4-bromobenzyl)-4-(3-(cyclopentyloxy)- 4-methoxyphenyl)-pyrrolidin-2-one.

Chiral HPLC resolution of the phosphodiesterase IV (PDE IV) inhibitor rolipram (1) provided (-)-1, and this enantiomer was converted into its 1-(4-bromobenzyl) derivative, (+)-2. X-ray structural analysis of (+)-2 established the absolute configuration as R, which provides the first direct evidence for a previously assumed assignment of configuration. The crystal structure of (+)-2 and the PDE inhibitory activity of both enantiomers of 2 are discussed in the context of a previously proposed topological model.

Direct high-performance liquid chromatographic separation of enantiomeric peptidoleukotriene antagonists.

Enantiomeric peptidoleukotriene antagonists, SK&F R-106203 and SK&F S-106203 can be effectively separated on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase. The utility of this chiral high-performance liquid chromatographic method in assigning absolute stereochemistry to SK&F S-106203-Z2, a non-crystalline amorphous compound which is not amenable to single crystal X-ray analysis, is demonstrated by correlation with the absolute configuration determined crystallographically for a second salt form.

Identification of fenoldopam prodrugs with prolonged renal vasodilator activity.

Fenoldopam (SK&F 82526) is a short-acting selective dopamine-1 agonist in clinical trials for the treatment of hypertension, congestive heart failure and renal failure. In the present study, we tested various N-ethyl carbamate esters of fenoldopam in the conscious dog instrumented with a femoral arterial Vascular-Access-Port and a renal artery flow probe. Oral administration of SK&F R-82526 at 1 and 3 mumol/kg resulted in transient (30-60 min) dose-dependent increases in plasma fenoldopam levels and renal blood flow. Administration of the 7,8-bis-N-ethyl carbamate ester of R-fenoldopam (SK&F R-106114) and the 4',7,8-tris-N-ethyl carbamate ester of R-fenoldopam (SK&F R-105058) at 1, 3 and 10 mumol/kg p.o. also resulted in dose-dependent increases in plasma fenoldopam levels and renal blood flow; however, both parameters remained elevated for at least 4 hr. Intravenous administration of SK&F R-105058 also resulted in sustained plasma fenoldopam levels and increases in renal blood flow, indicating that slow absorption was not the cause of the sustained effect. The present study indicates that N-ethyl carbamate esters of fenoldopam are fenoldopam prodrugs which result in sustained increases in renal blood flow and plasma fenoldopam levels.

Synthesis and structure-activity relationship studies of a series of 5-aryl-4,6-dithianonanedioic acids and related compounds: a novel class of leukotriene antagonists.

A series of 5-alkynyl- and 5-aryl-4,6-dithianonanedioic acids and related compounds has been prepared for evaluation of leukotriene antagonist activity. The alkynyl compounds were prepared by thioacetal exchange from the corresponding acetylenic acetals. The aryl derivatives were synthesized from the appropriate benzaldehydes, most of which were prepared by one of three general routes: Meyers' oxazolin method, a palladium coupling procedure, and a hydroxybenzaldehyde alkylation. The analogues were examined in vitro for their ability to antagonize an LTD4-induced contraction of isolated guinea pig tracheal smooth muscle and to compete with [3H]LTD4 for receptor sites on guinea pig lung membrane. A number of structure-activity relationships have emerged from this study. There is an optimal chain length of 10-12 atoms (or its equivalent) in the lipid tail and two methylenes in the polar region. In the aromatic series, the ortho- and meta-substituted compounds have comparable activity, whereas the para derivatives are inactive. Substitution in the aromatic ring and lipid tail is generally well tolerated, with the terminal phenyl (6) and acetylene (33) analogues having especially good activity. Conformational restriction of either the polar region or lipid tail produced compounds devoid of activity. A number of selected analogues were also evaluated in vivo as antagonists of LTD4-induced bronchoconstriction in the guinea pig. The data established these compounds as a novel class of leukotriene antagonists with potential utility for the treatment of asthma and other immediate hypersensitivity diseases.

Adrenergic agents. 8.1 Synthesis and beta-adrenergic agonist activity of some 3-tert-butylamino-2-(substituted phenyl)-1-propanols.

Replacement of the benzylic hydroxyl group of N-tert-butylnorepinephrine with a hydroxymethyl substituent affords a propanolamine homologue which retains a high degree of beta-adrenergic agonist activity. As modification of the meta substituent of catecholic ethanolamines, such as N-tert-butylnorepinephrine, often provides compounds that exert a more pronounced effect in relaxing tracheobronchial smooth muscle (beta2-adrenergic agonist) than in stimulating cardiac muscle (beta1-adrenergic response), a series of 3-tert-butylamino-2-(3-substituted 4-hydroxyphenyl)-1-propanols was prepared. The 3-meta substituents included HOCH2 (1b), H2NCONH (1c), MeSO2NH (1d), H (le), and NH2 (1f). These phenylpropanolamine derivatives were compared with their phenylethanolamine counterparts in in vitro tests that measure the ability of these compounds to relax spontaneously contracted guinea pig tracheal smooth muscle (a measure of potential bronchodilating activity) and to increase the rate of contraction of a spontaneously beating guinea pig right atrial preparation (an indicator of potential cardiac stimulating activity). In these tests all of the propanolamine derivatives included in the study were less potent than their ethanolamine relatives. In both series replacement of the catecholic m-hydroxyl group with the indicated substituents usually resulted in compounds with increased selectivity for tracheobronchial vs. cardiac muscle.