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The synthesis and biological evaluation of substituted exo-1-(4-pyridyl)-1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene s as inhibitors of estrogen biosynthesis is described [H (1); 4-OCH3 (2); 5-OCH3 (3); 6-OCH3 (4); 1-CH3, 6-OCH3 (5); 4-OCH3, 7-Br (6); 6-OCH3, 5-Br (7); 4-OH (8); 5-OH (9); 6-OH (10)]. The synthetic key step--the formation of the cyclopropyl ring--was accomplished using the conditions of a modified Wolff-Kishner reduction (N2H5OH/KOH; delta T) and yielded exclusively the exo-configurated diastereomers. The racemic compounds 1-10 showed an inhibition of human placental aromatase (P450 arom) exhibiting relative potencies (rp) from 3.7 to 303 (compounds 8 and 4, respectively; rp of aminoglutethimide (AG) identical to 1, fadrozole = 359). The enantiomers of 4 and 7 were separated by LPLC on tribenzoyl cellulose and by crystallization of the diastereomeric tartrates (4). (1aS,2S,7bS)-(+)-4 (absolute configuration determined by X-ray crystallographic analysis) is the active P450 arom inhibiting enantiomer of 4 and shows a rp value of 617. Compound 4 is a reversible inhibitor showing a competitive type of inhibition and a type II difference spectrum. In vitro 4 influenced other steroidogenic P450 enzymes either not at all (bovine adrenal P450 scc) or only marginally (rat testicular P450 17, bovine adrenal P450 18). In ACTH-stimulated rat adrenal tissue, 4 was less active, inhibiting corticosterone and aldosterone formation compared to AG and fadrozole, respectively. In vivo 4 was not superior to AG as far as the inhibition of the uterotrophic activity of androstenedione (juvenile SD rats) and the reduction of the plasma estradiol concentration (pregnant mares' serum gonadotropin-primed SD rats) are concerned. Compound 4 shows marked antitumor activity in the dimethylbenzanthracene-induced mammary carcinoma of the SD rat: in the postmenopausal model it is at least as active as AG; in the premenopausal experiment it is clearly superior to AG. No induction of hepatic P450 enzymes was observed in the latter experiment. The rp value of 4 toward rat ovarian P450 arom, i.e., 23 (rp of AG identical to 1), is markedly decreased compared to the human enzyme (rp value of 303). From this fact it must be concluded that 4 should be more active in the human than in the rat.

An active site mapping of human cathepsin B with dipeptide nitrile inhibitors was performed for a combinatorial approach by introducing several points of diversity and stepwise optimizing the inhibitor structure. To address the occluding loop of cathepsin B by a carboxylate moiety, click chemistry to generate linker-connected molecules was applied. Inhibitor 17 exhibited K i values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of the assay. Kinetic data were discussed with respect to the conformational selection and induced fit models. KEYWORDS:Copper-catalyzed azide−alkyne cycloaddition, cysteine proteases, human cathepsin B, nitrile inhibitors C athepsin B is a papain-like cysteine protease that is ubiquitously expressed and involved in various physiological processes. 1 In nontumor cells, it is localized within endosomes/lysosomes, while in transformed cells it adopts a peripheral distribution in the cytoplasm and is, moreover, associated with the plasma membrane. 2,3 At the surface of invasive tumor cells, cathepsin B was found to bind to annexin II or caveolin-1, the structural protein of caveolae. Caveolae serve as a platform for a proteolytic cascade for the degradation of the extracellular matrix (ECM), executed by several proteases, including cathepsin B. 2−4 Cathepsin B affects the ECM either directly by extracellular proteolytic degradation of its components or indirectly via activation of other proteases. Partially degraded ECM components can be internalized to intracellular endosomal/lysosomal vesicles where the proteolysis is continued. This ECM breakdown remodels the tumor environment, promotes tumor invasion, and enables angiogenesis and metastasis. Cathepsin B also contributes to angiogenesis by degrading metalloprotease inhibitors and releasing growth factors that are bound to ECM components. 2,5,6 The crucial roles of cathepsin B at multiple points of the tumor development have been established in several in vitro and in vivo models and cathepsin B was proposed to be a prognostic marker in patients with various types of cancer. 1,2,7 Cathepsin B shows endopeptidase and exopeptidase activity, a unique feature for cysteine cathepsins, arousing from a flexible structural element, referred to as the occluding loop. The occluding loop is a 19−20 amino acid section that blocks the active site cleft at the end of the primed site. This event leaves only space for two amino acid residues of the substrate C-terminal of the scissile bond, that is, in the P1′ and P2′ positions. In this socalled closed conformation, two salt bridges, His110-Asp22 and Arg116-Asp224, hold the occluding loop over the primed subsites of the substrate binding cleft, preventing extended binding of large endopeptidase substrates and conferring an exopeptidase activity to cathepsin B. Mutations of His110 and Asp22 or the deletion of 12 amino acids of the occluding loop to enforce an open conformation reduced the exopeptidase activity of cathepsin B in favor of its endopeptidase activity. 8,9 His110...

Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 μM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe's concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 μM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes’ lipophilicity, and the obtained

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