On the same scaffold, a P1' butynyloxy group, designed to fit in the TACE S1'-S3' channel, affords 17 which inhibits activity in THP-1 cells (IC5o -100 nM) and provides 98% inhibition of TNF-a production in the murine LPS model after a 50 mg/kg po dose (46). 5-Hydroxy-pipecolic acid derivative 18 is a low nanomolar inhibitor of TACE enzyme with an IC50 of 1 nM in HWB. Halogen or alkyl substitution at the orthoposition of the terminal phenyl ring greatly enhances activity in HWB. The 4-hydroxy-pipecolates are of equivalent potency against cell-free enzyme, but less potent in HWB. An X-ray crystal structure of 18 bound to TACE shows that several residues in the S1" pocket must move in order to accommodate the ligand's bulky ortho-iodo group. These 3,3-unsubstituted pipecolates with P1' benzyl groups are greater than 100-fold selective for TACE over MMP-1 (47). In contrast, pipecolate hydroxamate CP-661,631,19, with geminal substitution at the 3-position, is a potent broad spectrum MMP/TACE inhibitor. Importantly, in human primary neuron cultures 19 inhibits the regulated secretion of amyloid precursor protein (APP), but does not result in higher levels of Ap (48). Deposition of Ap has been associated with Alzheimer's disease.
The butynyl P1' group of 17 has been shown to enhance activity in TACE enzyme and cellular assays when applied to a variety of other MMP inhibitor scaffolds. Optimization of the TACE activity of the anthranilate MMP inhibitors using a homology model of TACE generated from adamalysin II and the TACE X-ray structure resulted in hydroxamate 20, an MMP-1 selective inhibitor of TACE (IC50 = 25 nM) with cellular activity in the range of 300 nM. Anthranilate 20 gives greater than 50% inhibition of TNF-a for over six hours in the mouse LPS model (49, 50). Lengthening the P1' butynyl group, or replacing it with a benzyl or picolyl moiety results In increased selectivity over MMP-1, but diminished cellular potency (51). Quinoline hydroxamates, and other bicyclic heteroaryl analogs of the anthranilates, are also potent inhibitors of TACE enzyme and have shown efficacy in a mouse collagen-induced arthritis (CIA) model (52). The P1' butynyloxy group of benzodiazepine 21 increases TACE activity by 7-fold over the analogous P1' methoxy analog In cell-free enzyme and affords low micromolar level potency in THP-1 cells (53).
Variations on the sulfonamide hydroxamates include sulfamide 22 and phosphonamide 23. The piperazine hydroxamate 22 is potent in MonoMac-6 cells (IC50 = 0.2 nM), the rat LPS model (ED50 = 11 mg/kg po) and the mouse CIA model. Solubility and pharmacokinetic properties in this series are improved for NH-sulfonamide analogs of 22 derived from acyclic amino acids such as leucine and serine (54). Phosphonamide 23 is a 3 nM inhibitor of cell-free TACE with excellent selectivity over MMP-1. In addition, it is a potent inhibitor (IC50 = 70 nM) of heparin-binding epidermal growth factor-like growth factor (HB-EGF) shedding (55).
Another novel scaffold is represented by IK-682, 24, a lactam hydroxamate, designed from modeling a sulfonamide hydroxamate into MMP-3 (56, 57). An extra methylene spacer, relative to the sulfonamides, is required between the amide carbonyl and the P1' phenyl group to allow the carbonyl to attain the proper orientation for forming a hydrogen bond with the protein. The lactam a-methyl group serves to hold the phenyl group pseudoaxial. Key to obtaining selectivity and cellular potency is the P1' 2-methylquinoline group. The quinoline can be replaced with a 3,5-disubstituted phenyl ring with little loss in enzyme potency or selectivity, since this group is still too wide to be accommodated by the MMPs, but activity in HWB is greatly diminished. The P1' quinolinylmethoxy and benzyloxy moieties have been applied to additional hydroxamic acid inhibitor scaffolds, including thiomorpholines, piperidines, and benzothiadiazepines (57, 58). In this series HWB potency appears to correlate with enzyme activity and the degree of protein binding. IK-682 24 has a K, of 0.6 nM against porcine TACE and is greater than 100-fold selective over a wide spectrum of MMPs. It has low clearance and long half-life (>7h) at 4 mg/kg iv, and good bioavailability (>30%) in rats and dogs at 8 mg/kg po. Optimization of IK-682 24 yielded DPC 333 (BMS-561392), a clinical candidate with potent activity in a variety of animal models including mouse LPS (ED5o = 6 mg/kg po), mouse collagen antibody-induced arthritis, and rat CIA (59).
Finally, a structurally novel series of propenohydroxamate TACE inhibitors with nanomolar potency against cell-free enzyme and excellent selectivity over many MMPs, exemplified by W-3646, 25, has recently been disclosed. This series of inhibitors arose from optimization of 3,3-biphenyl propenohydroxamic acid, an HTS hit. The P2' pyridyl ring and E-olefin configuration are essential for optimizing the moderate THP-1 activity (IC5o = 2.4 nM) attained by this series. Lengthier alkyl groups on the sulfonamide nitrogen are detrimental to enzyme activity. Despite the relatively low potency of 25 in cells it is active in the mouse LPS model at 30 mg/kg po (60).
Summary - The most important unresolved issues impacting the design of future TACE inhibitors are the frequent discrepancies between activity measured in cell-free TACE assays and cellular assays of LPS-stimulated TNF-a, and the desired TACE/MMP selectivity profile to provide adequate efficacy and safety. Many factors, most notably cell permeability and protein binding have been considered in obtaining highly active
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