Discovery and SAR of hydantoin TACE inhibitors

We disclose inhibitors of TNF-α converting enzyme (TACE) designed around a hydantoin zinc binding moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydantoin to the zinc. SAR, X-ray, and modeling designs are described. To our knowledge, these are the first reported X-ray structures of TACE with a hydantoin zinc ligand.     

Hydantoin based inhibitors of MMP13—Discovery of AZD6605

Piperidine ether and aryl piperazine hydantoins are reported as potent inhibitors of MMP13. A medicinal chemistry campaign focused on replacing the reverse hydroxamate zinc binding group associated with historical inhibitors with a hydantoin zinc binding group then optimising MMP13 potency, solubility and DMPK properties whilst maintaining good selectivity over MMP14. A number of high quality candidates were progressed and following rat and dog safety evaluation, AZD6605 (3m) was identified as a candidate drug.     

Discovery of novel spirocyclopropyl hydroxamate and carboxylate compounds as TACE inhibitors

We have discovered nanomolar inhibitors of TNF-α convertase (TACE) comprised of a novel spirocyclic scaffold and either a carboxylate or hydroxamate zinc binding moiety. X-ray crystal structures and computer models of selected compounds binding to TACE explain the observed SAR. We report the first TACE X-ray crystal structure for an inhibitor with a carboxylate zinc ligand.     

A molecular modeling analysis of novel non-hydroxamate inhibitors of TACE

Recently, an X-ray co-crystal structure of our hydroxamate inhibitor IK682 and TACE [Niu, X.; Umland, S.; Ingram, R.; Beyer, B. M.; Liu, Y.-H.; Sun, J.; Lundell, D.; Orth, P. Arch. Biochem. Biophys. 2006, 451, 43-50] was published that explicitly shows the orientation of the hydroxamate and the TACE-selective 4-[(2-methyl-4-quinolinyl)methoxy]phenyl P1' group in the S1' and S3' sites. The preceding paper described a novel series of potent and TACE-selective hydantoins and we previously described pyrimidinetrione (barbiturate) inhibitors of TACE, both of which contain the same P1' group as IK682. Using this TACE-selective P1' group as an anchor, stereochemical and conformational constraints in the inhibitors, and restrictions to the active site Zn coordination geometry, we developed a highly plausible and predictive pharmacophore model that rationalizes the observed TACE activity of all three inhibitors.     

The C-terminal domains of TACE weaken the inhibitory action of N-TIMP-3

Tumor necrosis factor-alpha converting enzyme (TACE) is an ADAM (a disintegrin and metalloproteinases) that comprises an active catalytic domain and several C-terminal domains. We compare the binding affinity and association rate constants of the N-terminal domain form of wild-type tissue inhibitor of metalloproteinase (TIMP-3; N-TIMP-3) and its mutants against full-length recombinant TACE and the truncated form of its catalytic domain. We show that the C-terminal domains of TACE substantially weaken the inhibitory action of N-TIMP-3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C-terminal domains of ADAM proteinases in influencing TIMP interactions.     

Protease inhibitors: Synthesis of L-alanine hydroxamate sulfonylated derivatives as inhibitors of clostridium histolyticum collagenase

L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides. Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety. The obtained compounds were assayed as inhibitors of Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') sites, in order to achieve tight binding to the enzyme.     

Acetylenic TACE inhibitors. Part 2: SAR of six-membered cyclic sulfonamide hydroxamates

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing a butynyloxy P1' group was explored. In particular, compound 5k has excellent in vitro potency against TACE enzyme and in cells, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.     

Synthesis and activity of quinolinylmethyl P1′ α-sulfone piperidine hydroxamate inhibitors of TACE

A series of α-sulfone piperidine hydroxamate TACE inhibitors 11a–n bearing a quinolinyl methyl P1′ group was prepared, and their activity was compared to analogous α- and β-sulfone piperidine hydroxamates with a butynyloxy P1′ group. The quinolinyl methyl P1′ group affords increased inhibitory enzyme activity relative to the corresponding butynyloxy P1′ analogs in the α-sulfone piperidine hydroxamate series, and greater selectivity than the corresponding butynyloxy P1′ analogs in the β-sulfone piperidine hydroxamate series.     

Structure-based design of TACE selective inhibitors: manipulations in the S1'-S3' pocket

A series of beta-sulfonyl hydroxamate TACE inhibitors, bearing a butynylamino or a butynyloxy P1' group, was designed and synthesized. Of the compounds investigated, 22 has excellent potency against isolated TACE enzyme, shows good selectivity over MMP-2 and MMP-13, and oral activity in an in vivo mouse model of TNF-alpha production.     

Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.     

Synthesis and structure-activity relationship of a novel, non-hydroxamate series of TNF-alpha converting enzyme inhibitors

A novel series of TNF-alpha converting enzyme (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds use a triazolethione moiety as the zinc binding ligand and exhibit IC50 values from 1.5 to 100 nM in a porcine TACE assay. They also have excellent selectivities over other MMPs.     

Acetylenic TACE inhibitors. Part 1. SAR of the acyclic sulfonamide hydroxamates

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P1' group, was explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.     

Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 2: SAR of the acetylenic P1' group

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.     

Biaryl substituted hydantoin compounds as TACE inhibitors

We disclose further optimization of hydantoin TNF-α convertase enzyme (TACE) inhibitors. SAR with respect to the non-prime region of TACE active site was explored. A series of biaryl substituted hydantoin compounds was shown to have sub-nanomolar K_i, good rat PK, and good selectivity versus MMP-1, -2, -3, -7, -9, and -13.     

Protease Inhibitors: Synthesis of L-Alanine Hydroxamate Sulfonylated Derivatives as Inhibitors of Clostridium Histolyticum Collagenase

L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides. Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety. The obtained compounds were assayed as inhibitors of Clostridium histolyticum collage-nase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl-and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P₂, and P₃ sites, in order to achieve tight binding to the enzyme.     

Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 1: Structure-based design of novel acetylenic P1' groups

The structure-based design of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups has led to compounds with excellent in vitro potency against TACE and selectivity over MMP-1.     

Reversed hydroxamate-bearing thermolysin inhibitors mimic a high-energy intermediate along the enzyme-catalyzed proteolytic reaction pathway

A series of inhibitors that bear a reversed hydroxamate moiety have been evaluated as transition state analogue inhibitors for thermolysin. A linear correlation is observed between the K(i) values of these inhibitors and the kinetic parameters (K(M)/k(cat)) of the parallel series of related substrates, satisfying the criterion stipulated for transition state analogue inhibitors by Bartlett and Marlowe. Furthermore, examination of the binding mode of a related reversed hydroxamate bearing thermolysin inhibitor, in comparison with a transition state postulated for the enzyme-catalyzed proteolytic reaction revealed that the inhibitors under study mimic the electronic as well as the geometric characteristics of the transition state. On the basis of these results it may be concluded that the hydroxamate-bearing zinc protease inhibitors are a new type of transition state analogue inhibitors.     

2-(2-Aminothiazol-4-yl)pyrrolidine-based tartrate diamides as potent, selective and orally bioavailable TACE inhibitors

TNF-α converting enzyme (TACE) inhibitors are promising agents to treat inflammatory disorders and cancer. We have investigated novel tartrate diamide TACE inhibitors where the tartrate core binds to zinc in a unique tridentate fashion. Incorporating (R)-2-(2-N-alkylaminothiazol-4-yl)pyrrolidines into the left hand side amide of the tartrate scaffold led to the discovery of potent and selective TACE inhibitors, some of which exhibited good rat oral bioavailability.     

Structural insights into the binding of MMP9 inhibitors

Matrix Metalloproteinase are family of enzymes responsible for degradation of extracellular matrix. MMP9 (gelatinase B) is one of the common matrix metalloproteinase that is associated with tissue destruction in a number of disease states such as rheumatoid arthiritis, fibrotic lung disease, dilated cardiomyopathy, as well as cancer invasion and metastasis. Recent study demonstrates that increased expression of MMP9 results in augmentation of myopathy with increased inflammation and fibernecrosis. Previous studies do not provide any conclusive information related to structural specificity of MMP9 inhibitors towards its active site, but with the availability of experimental structures it is now possible to study the structural specificity of MMP9 inhibitors. In light of availability of this information, we have applied docking and molecular dynamics approach to study the binding of inhibitors to the active site of MMP9. Three categories of inhibitor consisting of sulfonamide hydroxamate, thioester, and carboxylic moieties as zinc binding groups (ZBG) were chosen in the present study. Our docking results demonstrate that thioester based zinc binding group gives favourable docking scores as compared to other two groups. Molecular Dynamics simulations further reveal that tight binding conformation for thioester group has high specificity for MMP9 active site. Our study provides valuable insights on inhibitor specificity of MMP9 which provides valuable hints for future design of potent inhibitors and drugs.     

Discovery of novel hydantoins as selective non-hydroxamate inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

A series of novel hydantoins was designed and synthesized as structural alternatives to hydroxamate inhibitors of TACE. 5-Mono- and di-substituted hydantoins exhibited activity with IC50 values of 11-60 nM against porcine TACE in vitro and excellent selectivity against other MMPs.