Rational design of potent and selective NH-linked aryl/heteroaryl cathepsin K inhibitors

Prior reports from our laboratories have identified the nonpeptidic inhibitor 2 as a potent and selective Cathepsin K (Cat K) inhibitor. Modelling studies suggested that the introduction of a NH linker between the P3 aryl and P2 leucinamide moieties would allow the formation of a H-bond with the Gly66 residue of Cat K, hopefully increasing potency. Aniline 4 was thus synthesized and showed improved potency over its predecessor 2. Further modelling concluded that a 2-substituted five membered ring could more adequately place the P3 moiety of 4 into the S3 pocket of Cat K. The synthesis of the 2-substituted thiophene 5 confirmed this hypothesis by displaying a slight increase in potency against Cat K (>10-fold increase in potency vs 2) and a good selectivity profile against Cathepsins B, L, and S. This rationally designed inhibitor 5 also displayed increased potency in a functional bone resorption assay (10nM) versus 2 (95 nM).     

Peptidic 1-cyanopyrrolidines: synthesis and SAR of a series of potent,selective cathepsin inhibitors

1-Cyanopyrrolidines have previously been reported to inhibit cysteinyl cathepsins (Falgueyret, J.-P. et al., J. Med. Chem. 2001, 44, 94). In order to optimize binding interactions for a given cathepsin and simultaneously reduce interactions with the other closely related enzymes, small peptidic substituents were introduced to the 1-cyanopyrrolidine scaffold, either at the 2-position starting with proline or at the 3-position of aminopyrrolidines. The resulting novel compounds proved to be micromolar inhibitors of cathepsin B (Cat B) but nanomolar to picomolar inhibitors of cathepsins K, L, and S (Cat K, Cat L, Cat S). Several of the compounds were >20-fold selective versus the other three cathepsins. SAR trends were observed, most notably the remarkable potency of Cat L inhibitors based on the 1-cyano-D-proline scaffold. The selectivity of one such compound, the 94 picomolar Cat L inhibitor 12, was demonstrated at higher concentrations in DLD-1 cells. Although none of the compounds in the proline series that was tested proved to be submicromolar in the in vitro bone resorption assay, two Cat K inhibitors in the 3-substituted pyrrolidine series, 24 and 25 were relatively potent in that assay.     

Beta-substituted cyclohexanecarboxamide cathepsin K inhibitors: modification of the 1,2-disubstituted aromatic core

Further SAR study around the central 1,2-disubstituted phenyl of the previously disclosed Cat K inhibitor (-)-1 has demonstrated that the solvent exposed P2-P3 linker can be replaced by various 5- or 6-membered heteroaromatic rings. While some potency loss was observed in the 6-membered heteroaromatic series (IC(50)=1 nM for pyridine-linked 4 vs 0.5 nM for phenyl-linked (+/-)-1), several inhibitors showed a significantly decreased shift in the bone resorption functional assay (10-fold for pyridine 4 vs 53-fold for (-)-1). Though this shift was not reduced in the 5-membered heteroaromatic series, potency against Cat K was significantly improved for thiazole 9 (IC(50)=0.2 nM) as was the pharmacokinetic profile of N-methyl pyrazole 10 over our lead compound (-)-1.     

Advances in the discovery of cathepsin K inhibitors on bone resorption

Cathepsin K (Cat K), highly expressed in osteoclasts, is a cysteine protease member of the cathepsin lysosomal protease family and has been of increasing interest as a target of medicinal chemistry efforts for its role in bone matrix degradation. Inhibition of the Cat K enzyme reduces bone resorption and thus, has rendered the enzyme as an attractive target for anti-resorptive osteoporosis therapy. Over the past decades, considerable efforts have been made to design and develop highly potent, excellently selective and orally applicable Cat K inhibitors. These inhibitors are derived from synthetic compounds or natural products, some of which have passed preclinical studies and are presently in clinical trials at different stages of advancement. In this review, we briefly summarised the historic development of Cat K inhibitors and discussed the relationship between structures of inhibitors and active sites in Cat K for the purpose of guiding future development of inhibitors.     

Design and synthesis of tetracyclic nonpeptidic biaryl nitrile inhibitors of cathepsin K

The synthesis and biological profile of a novel series of potent and selective inhibitors of cysteine protease cathepsin K (Cat K) are described. Pharmacokinetic evaluation of 12 indicated that some members of this series could be suitable candidates to develop new orally active therapeutic agents for the treatment of osteoporosis.     

Utilization of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold in the design of potent inhibitors of serine proteases: SAR studies using carboxylates

A series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds has been synthesized and the inhibitory profile of these compounds toward human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3) was then determined. Most of the compounds were found to be potent, time-dependent inhibitors of elastase, with some of the compounds exhibiting k(inact)/K1 values as high as 4,928,300 M(-1) s(-1). The inhibitory potency of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide platform was found to be influenced by both the pKa and the inherent structure of the leaving group. Proper selection of the primary specificity group (R(I)) was found to lead to selective inhibition of HLE over Cat G, however, those compounds that inhibited HLE also inhibited PR 3, albeit less efficiently. The predictable mode of binding of these compounds suggests that, among closely-related serine proteases, highly selective inhibitors of a particular serine protease can be fashioned by exploiting subtle differences in their S' subsites. This study has also demonstrated that the degradative action of elastase on elastin can be abrogated in the presence of inhibitor 17.     

Discovery of selective and nonpeptidic cathepsin S inhibitors

Nonpeptidic, selective, and potent cathepsin S inhibitors were derived from an in-house pyrrolopyrimidine cathepsin K inhibitor by modification of the P2 and P3 moieties. The pyrrolopyrimidine-based inhibitors show nanomolar inhibition of cathepsin S with over 100-fold selectivity against other cysteine proteases, including cathepsin K and L. Some of the inhibitors showed cellular activities in mouse splenocytes as well as oral bioavailabilities in rats.     

Identification of a potent and selective non-basic cathepsin K inhibitor

Based on our previous study with trifluoroethylamine as a P2-P3 amide isostere of cathepsin K inhibitor, further optimization led to identification of compound 22 (L-873724) as a potent and selective non-basic cathepsin K inhibitor. This compound showed excellent pharmacokinetics and efficacy in an ovariectomized (OVX) rhesus monkey model. The volumes of distribution close to unity were consistent with this compound not being lysosomotropic, which is a characteristic of basic cathepsin K inhibitors.     

The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.     

3,4-disubstituted azetidinones as selective inhibitors of the cysteine protease cathepsin K. Exploring P2 elements for selectivity

A novel series of 3,4-disubstituted azetidinones based inhibitors of the cysteine protease cathepsin K (Cat K) has been identified. Although not optimized, some of these compounds show at least 100-fold selectivity against other cathepsins. The use of cyclic moieties as P2 elements has proven to be crucial to achieve a high degree of selectivity.     

1,2,4-thiadiazole: a novel Cathepsin B inhibitor

A novel class of Cathepsin B inhibitors has been developed with a 1,2,4-thiadiazole heterocycle as the thiol trapping pharmacophore. Several compounds with different dipeptide recognition sequence (i.e., P1′–P2′=Leu-Pro-OH or P2–P1=Cbz-Phe-Ala) at the C5 position and with different substituents (i.e., OMe, Ph, or COOH) at the C3 position of the 1,2,4-thiadiazole ring have been synthesized and tested for their inhibitory activities. The substituted thiadiazoles 3a–h inhibit Cat B in a time dependent, irreversible manner. A mechanism based on active-site directed inactivation of the enzyme by disulfide bond formation between the active site cysteine thiol and the sulfur atom of the heterocycle is proposed. Compound 3a (K_i=2.6 μM, k_i/K_i=5630 M⁻¹ s⁻¹) with a C3 methoxy moiety and a Leu-Pro-OH dipeptide recognition sequence, is found to be the most potent inhibitor in this series. The enhanced inhibitory potency of 3a is a consequence of its increased enzyme binding affinity (lower K_i) rather than its increased intrinsic reactivity (higher k_i). In addition, 3a is inactive against Cathepsin S, is a poor inhibitor of Cathepsin H and is >100-fold more selective for Cat B over papain.     

Ultrasensitive internally quenched substrates of human cathepsin L

Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO₂)-NH₂ with high specificity constant (k_cat/K_M = 2.6 × 10⁷ M⁻¹ s⁻¹) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.     

Isosteric replacements for benzothiazoles and optimisation to potent Cathepsin K inhibitors free from hERG channel inhibition

The discovery of nitrile compound 4, a potent inhibitor of Cathepsin K (Cat K) with good bioavailability in dog is described. The compound was used to demonstrate target engagement and inhibition of Cat K in an in vivo dog PD model. The margin to hERG ion channel inhibition was deemed too low for a clinical candidate and an optimisation program to find isosteres or substitutions on benzothiazole group led to the discovery of 20, 24 and 27; all three free from hERG inhibition.     

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.     

The amino-acid sequences of the double-headed proteinase inhibitors from cat, lion and dog submandibular glands

Cat and lion submandibular glands each contain a double-headed secretory proteinase inhibitor. Their amino-acid sequences were determined, and the amino-acid sequence of the inhibitor of dog submandibular glands was revised. Extensive homologies were found between these inhibitors in both domains. The trypsin-inhibiting domains of cat and lion inhibitors, however, contain a Lys residue in the reactive site in contrast to an Arg residue in the dog inhibitor. Domains I and II of cat, lion, and dog inhibitors are structurally related both to each other and to the sequenced monovalent secretory pancreatic trypsin inhibitors, Notable differences in inhibitory properties of canine and feline inhibitors are discussed with respect to sequence differences.     

Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, anti-viral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being alpha-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for a hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K(i)=17microM) and selective over reticulocyte 15-hLO-1 (K(i) 15-hLO-1/12-hLO>30).     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

Investigation of ketone warheads as alternatives to the nitrile for preparation of potent and selective cathepsin K inhibitors

Amino ketone warheads were explored as alternatives to the nitrile group of a potent and selective cathepsin K inhibitor. The resulting compounds were potent and selective inhibitors of cathepsin K and these nitrile replacements had a significant effect on metabolism and pharmacokinetics.     

Involvement of nitric oxide and potassium channels in the bradykinin-induced vasodilatation in the rat kidney perfused ex situ

The role of nitric oxide (NO), K(+) channels, and arachidonic acid metabolism, via cytochrome P450 and cyclooxygenase pathways, in the renal vasodilatory effect of bradykinin was examined in the isolated rat kidney perfused ex situ with a blood-free solution. Bradykinin (BK, 0.25-1.0 microM) induced a dose-dependent reduction of 10-35% in the relative renal vascular resistance (rRVR) of isolated kidneys preconstricted with phenylephrine (PHE, 0.17-0.35 microM). The vasodilating effect of 0.5 microM bradykinin was significantly inhibited by the nitric oxide synthase inhibitors, N(G)-nitro-L-arginine (95% inhibition) and N(G)-nitro-L-arginine methyl ester (45-75% inhibition). Clotrimazole, an inhibitor of cytochrome P450 pathway but not indomethacin, a cyclooxygenase inhibitor, reduced the renal vasodilator response to bradykinin by 84%. The nonspecific K(+) channel inhibitor, tetraethylammonium ion (TEA) and the selective inhibitor of Ca(2+)-activated K(+) channels, charybdotoxin (ChTX) greatly attenuated the vasodilator response to bradykinin by approximately 84% and 79%, respectively. These two K(+) channel inhibitors showed similar effects on vasodilatation induced by S-nitroso-acetyl-D,L-penicillamine (1 microM), a nitric oxide donor. The results suggest that bradykinin releases nitric oxide which, by opening potassium channels specifically the Ca(+)-dependent type, mediates the renal vasodilator response to bradykinin in the isolated kidney perfused ex situ.     

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.