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Articles Structure-Activity Relationships and Pharmacokinetic Analysis for a Series of Potent, Systemically Available Biphenylsulfonamide Matrix Metalloproteinase Inhibitors Patrick M. O’Brien,* Daniel F. Ortwine, Alexander G. Pavlovsky, Joseph A. Picard, Drago R. Sliskovic, Bruce D. Roth, Richard D. Dyer,† Linda L. Johnson,† Chiu Fai Man,† and Hussein Hallak‡ Departments of Chemistry, Biochemistry, and Pharmacokinetics/Drug Metabolism, Parke-Davis Pharmaceutical Research, Division of Warner Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105 Received June 18, 1999
A series of biphenylsulfonamide derivatives of (S)-2-(biphenyl-4-sulfonylamino)-3-methylbutyric acid (5) were prepared and evaluated for their ability to inhibit matrix metalloproteinases (MMPs). For this series of compounds, our objective was to systematically replace substituents appended to the biphenyl and R-position of 5 with structurally diverse functionalities to assess the effects these changes have on biological and pharmacokinetic activity. The ensuing structure-activity relationship (SAR) studies showed that biphenylsulfonamides substituted with bromine in the 4′-position (11c) significantly improved in vitro activity and exhibited superior pharmacokinetics (Cmax, t1/2, AUCs), relative to compound 5. Varying the lipophilicity of the R-position by replacing the isopropyl group of 11c with a variety of substituents, in general, maintained potency versus MMP-2, -3, and -13 but decreased the oral systemic availability. Subsequent evaluation of its enantiomer, 11c′, showed that both compounds were equally effective MMP inhibitors. In contrast, the corresponding hydroxamic acid enantiomeric pair, 16a (S-isomer) and 16a′ (R-isomer), stereoselectivity inhibited MMPs. For the first time in this series, 16a′ provided nanomolar potency against MMP-1, -7, and -9 (IC50’s ) 110, 140, and 18 nM, respectively), whereas 16a was less potent against these MMPs (IC50’s ) 24, 78, and 84 µM, respectively). However, unlike 11c, compound 16a′ afforded very low plasma concentrations following a single 5 mg/kg oral dose in rat. Subsequent X-ray crystal structures of the catalytic domain of stromelysin (MMP-3CD) complexed with inhibitors from closely related series established the differences in the binding mode of carboxylic acid-based inhibitors (11c,c′) relative to the corresponding hydroxamic acids (16a,a′). Introduction Matrix metalloproteinases (MMPs) are a family of zinc-dependent, calcium-containing endopeptidases that have been shown to play a significant physiological role in tissue remodeling in normal growth and development.1 Regulation of their proteolytic activity in tissue remodeling is controlled by a variety of mechanisms, including inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPS).2 An imbalance in the MMP/TIMP ratio favoring the overproduction of MMPs has been implicated in a number of pathological processes, including the destruction of cartilage and bone in rheumatoid arthritis and osteoarthritis, tumor growth and metastasis in both human and animal cancers, degeneration of the aortic wall in abdominal aortic aneurysms, and progressive cardiac dilation in patients with congestive heart failure.3-6 The accumulation of evidence suggesting that upregulation of MMPs is involved in these, and other disease states, has led to a * To whom correspondence should be addressed. Tel: 734-622-7076. Fax: 734-622-3107. E-mail:
[email protected]. † Department of Biochemistry. ‡ Department of Pharmacokinetics/Drug Metabolism.
growing number of pharmaceutical companies attempting to design and develop orally active inhibitors that may restore the balance of MMP regulation in these pathological processes. The majority of the MMPs are divided into four main groups that include collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -1) and membrane-type MMPs (MMP-14, -15, -16, -17), while matrilysin (MMP-7) and metalloelastase (MMP12) are included separately as members of the metalloproteinase family.7 The primary structures for most of the human MMPs have been determined, demonstrating that each possesses similar structural domains, which include an N-terminal propeptide region, a zinccontaining catalytic domain, and, with the exception of matrilysin, a C-terminal domain.8a A number of X-ray and NMR structures of MMP catalytic domain/inhibitor complexes have now been reported and show that inhibitor interactions at the active-site zinc play a critical role in defining the binding mode and relative inhibitor potency.9 Thus, the majority of MMP inhibitors reported in the literature,10 including those reported to be in clinical trials (Figure 1), contain an effective zinc
10.1021/jm9903141 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/04/2000
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Scheme 1a
Figure 1. MMP inhibitors reported to be evaluated in clinical trials.
a (a) NEt , THF/H O; (b) Pd(PPh ) , Na CO , H O/ toluene, 3 2 3 4 2 3 2 reflux; (c) anisole, TFA, 25 °C.
Figure 2. Proposed biphenylsulfonamide SAR studies.
binding group (e.g. hydroxamic acid, carboxylic acid, sulfhydryl group) that is either generally substituted with a peptide-like structure that mimics the substrates that they cleave or appended to smaller side chains that may interact with specific subsites (e.g, P1′, P2′, P3′) within the active site.10,11 To further assess the role of MMPs in vivo, our goal was to identify an inhibitor possessing a suitable pharmacokinetic profile for chronic oral dosing in animal models of arthritis, atherosclerosis, heart failure, and multiple sclerosis. As part of our search for novel MMP inhibitors, we screened our library of compounds and identified (S)-2-benzenesulfonylamino-4-methylpentanoic acid and 4-biphenyl-4-yl-4-oxobutyric acid (Fenbufen) as selective inhibitors of human MMP-2 and -3 (micromolar potency), having no inhibitory activity against MMP-1, -7, and -9. Replacement of the benzene group of the sulfonamide with Fenbufen’s biphenyl led to the identification compound 5 (Figure 2), a biphenylsulfonamide derivative that, unlike the benzenesulfonamides or Fenbufen derivatives, exhibited nanomolar potency vs MMP-2, -3, and -13 and micromolar potency vs MMP-1, -7, and -9. In contrast to our results, Shionogi Research Laboratories reported nanomolar potency vs MMP-9 for a closely related series of N-sulfonylamino acid derivatives, several of which exhibited oral activity in animal models of tumor growth and metastasis.12 As a tool to complement the in vitro screening, we examined the possible binding modes of 5 with the catalytic domain of MMP-3 through molecular modeling methods based on an X-ray structure previously described.13 From this study, we found that the major interactions contributing to the tight binding of 5 with MMP-3CD include catalytic zinc-carboxylate coordina-
tion, hydrogen bonding between the sulfonamide moiety and protein amino acid residues, and hydrophobic interactions plus aromatic stacking of the biphenyl ring system within the P1′ pocket. On the basis of these results, we examined structure-activity relationships (SARs) based on 5, assessing the effects that structural changes made to the biphenyl ring system, the R-position, and the carboxylate moiety have on biological activity. In this paper, we describe the synthesis, structural analysis, SARs, and pharmacokinetics for analogues of compound 5 and the events leading to the selection of 11c as a candidate for evaluation in preclinical efficacy models. Chemistry The synthesis of carboxylic acid derivatives 11a,b, g,h,j-l (Tables 1 and 2) is presented in Scheme 1 (method A). Commercially available 4-bromobenzenesulfonyl chloride (6) was coupled to L-valine tert-butyl ester (7) in the presence of triethylamine to give the bromobenzenesulfonamide intermediate 8. Utilizing reaction conditions developed by Suzuki, compound 8 was coupled with a variety of substituted benzeneboronic acids 9 via palladium catalysis in refluxing toluene to yield substituted biphenylsulfonamide derivatives 10a,b,g,h,j-l.14 Hydrolysis of the tert-butyl ester using trifluoroacetic acid in the presence of anisole gave the corresponding carboxylic acid derivatives 11a,b,g,h,jl. Alternatively, biphenylsufonamide carboxylic acids 11c-f,i,m-v (Tables 1, 2, 4, and 5) were prepared from appropriately substituted biphenyl starting materials 12 as shown in Scheme 2 (method B). Commercially available substituted biphenyls 12 were reacted with chlorosulfonic acid in chloroform to give the biphenylsulfonic acids 13, which were converted to the corresponding sulfonyl chlorides 14 in refluxing thionyl chloride. Analogues of 14 were coupled with a variety of amino acid esters 15 in aqueous tetrahydrofuran to yield compounds 10c-f,m,p,q as tert-butyl esters and
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Scheme 2a
O’Brien et al.
ase’ among the family.15 Mutation of this residue to a nonionizable residue (Gln) resulted in catalytically active protein with a broad pH optimum similar to that of other MMPs.17,18 Testing at MMP-3’s pH optimum allowed us to better differentiate potent inhibitors of this enzyme and to more accurately determine which compounds might be selective for MMP-3. Results and Discussion
a (a) ClSO H, CHCl , 25 °C; (b) SOCl , reflux; (c) NEt , THF/ 3 3 2 3 H2O; (d) anisole, TFA, 25 °C; (e) LiOH, H2O, p-dioxane.
Scheme 3a
a (a) Oxalyl chloride, CH Cl , 25 °C; (b) NH OTHP, NEt , THF/ 2 2 2 3 H2O; (c) HCl.
10n,o,r-v as the corresponding methyl esters. Esters 10c-f,m,p,q required trifluoroacetic acid hydrolysis to the corresponding acids as previously described, whereas methyl esters 10n,o,r-v were saponified to the carboxylic acids 11n,o,r-v using lithium hydroxide in aqueous tetrahydrofuran. The hydroxamic acid analogues presented in Table 5 (16a,a′,b,b′) were synthesized as shown in Scheme 3 (method C). The carboxylic acids 11c,c′,k,k′ were reacted with oxalyl chloride in dichloromethane and coupled with O-tetrahydro-2H-pyran-2-hydroxylamine to give the protected hydroxamic acids. Acidic hydrolysis of the protecting group gave pure hydroxamic acids (16a,a′,b,b′). Biological Evaluation All compounds were tested in vitro against a panel of six matrix metalloproteinases, MMP-1, -2, -3, -7, -9, and -13; see the Experimental Section for details on the protocols used. Assays used the catalytic domains of the proteins, with the exceptioon of MMP-1 and -9, where the full-length forms were employed. Results are shown in Tables 1, 2, 4, and 6 for different classes of compounds. All testing was performed at pH 7, except for MMP3, where a pH of 6 was used. Among MMPs, MMP-3 exhibits a unique acidic pH optimum of 5.5-6.5 for activity,15-17 which is due to the presence of a protonated histidine (residue 224) in the S1′ site (see Figure 3).16,18,19 This histidine is specific to MMP-3 and allowed this protein to be identified as the ‘acid metalloprotein-
Initial SAR studies based on 5 were designed to examine the interaction of the biphenyl ring with the P1′ specificity site within the MMPs of interest. As can be seen from Table 1, 5 exhibits selectivity for MMP-2, -3, and -13 over MMP-1, -7, and -9. Consistent with the in vitro profile for 5, the S1′ channels of MMP-1 and -7 have been reported to be occluded by arginine and tyrosine residues, respectively, and would therefore have difficulty accommodating the distal phenyl ring of 5.13,20 Homology models of MMP-9 suggest an arginine may also play a role in reducing the size of the S1′ channel and therefore reducing the potency of the inhibitor.21 However, it appears that if enough binding energy is gained from the zinc ligating and sulfonamide portions of the molecule, a reorganization of residues in the channel may occur to allow binding of large S1′ groups with less of a reduction in potency than might be expected.22 With this in mind, we prepared analogues of 5 substituting the distal phenyl ring of the biphenyl with various halogens (11a-f), electron-withdrawing groups (11j-m), and electron-donating groups (11g-i) (Tables 1 and 2), utilizing the synthetic routes shown in Schemes 1 and 2, and assessed the effects that these changes have on MMP inhibitory activity. As can be seen in Table 1, halogens appended to the 4′-position of the biphenyl ring (11a,c,e), in general, increased potency against MMP-2, -3, -7, and -13 relative to 5. This magnitude of the increase did not change as the size of the halo substituent was increased from F to Cl to Br. However, substitution at the 3′-position resulted in a decrease in inhibitory activity, particularly for MMPs with narrow or shallow S1′ channels (MMP-1, -7, and -9, respectively), when compared to the corresponding 4′-isomers (compare 11a vs 11b and 11c vs 11d). Incorporation of a fluorine atom in the 2′-position in 11c (11f) was tolerated and provided a modest increase in potency against MMP-1, -7, and -9 and comparable in vitro activity vs MMP-2 and -3. Electron-withdrawing groups (11j-m, Table 2) and moderate electron-donating groups (11g,h) significantly improved inhibitory activity against MMP -2, -3, and -13 relative to the unsubstituted biphenyl derivative 5, while a strongly electron-donating substituent (11i) did not. To help understand the observed SAR and to rationalize the specificity profile, we examined the interaction of compound 11c with the active site of MMP-3CD. Since suitable crystals of an MMP-3CD/ complex of compound 11c could not be obtained for X-ray studies, we modeled 11c into the protein using coordinates from recently published crystal structures of MMP-3CD complexed with a series of related diphenylpiperidine derivatives.13 In this model (Figures 3 and 4), the 4′-bromobiphenyl moiety resides in the S1′ specificity site, which allows for large substituents in the 4′-position, but not in the 2′- or 3′-position where
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Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2 159
Table 1. In Vitro Activity of Halogenated Biphenylsulfonamide Derivatives
MMPs IC50 (µM)b compd 5 11a 11b 11c 11d 11e 11f
R
formulaa
mp (°C)
H 4-F 4-F 4-Br 3-Br 4-Cl 2-F,4-Br
C17H19NO4S C17H18FNO4Se C17H18FNO4S C17H18BrNO4S C17H18BrNO4S C17H18ClNO4S C17H17BrFINO4S
164-166 165-166 145-147 192-193 foam 187-188 175-177
method
1c
2
3d
7
9c
13
B A A B B B B
5.4 4.2 8.6 6.0 100 6.5 3.6
0.040 0.039 0.049 0.004 0.535 0.011 0.005
0.038 0.010 0.017 0.007 0.290 0.009 0.016
71 4.8 22 7.2 100 7.5 2.1
26 64 65 7.9 100 16 4.9
0.062 0.043 0.150 0.008 0.710 0.048 0.007
a Analytical results are within (0.4% of the theoretical values unless otherwise noted. b MMP inhibition in vitro. Except where noted, assays were run at pH 7 against the catalytic domains of the enzymes. See the Experimental Section for complete protocols. c Full-length version of the enzyme was used. d Assay was run at pH 6. Unlike other MMPs, MMP-3 displays a strong pH dependence and is maximally active at pH 5.5-6.15-17 See the Biological Evaluation and Experimental Section for details. e Anal. C: calcd 58.11, found 57.66; H: calcd 5.16, found 5.18; N: calcd 3.99, found 3.69.
Table 2. In Vitro Activity of Biphenylsulfonamide Derivatives Substituted with Electron-Donating or Electron-Withdrawing Functionalities
MMPs IC50 (µM)b compd 5 11g 11h 11i 11j 11k 11l 11m a-d
R
formulaa
mp (°C)
method
1c
2
3d
7
9c
13
H 4-CH3 4-OCH3 4-NH2 4-CF3 4-CN 4-CHO 4-NO2
C17H19NO4S C18H21NO4S C18H21NO5S C17H20N2O4Se C18H18F3NO4S C18H18N2O4S C18H19NO5S C17H18N2O6S
164-166 185-186 180-181 >230 183-184 182-183 189-190 167-169
B A A B A A A B
5.4 2.2 1.5 26 4.2 18 3.2 12
0.040 0.002 0.003 0.036 0.013 0.033 0.012 0.061
0.038 0.003 0.008 0.036 0.009 0.006 0.008 0.015
71 4.5 7.2 31 7.9 7.0 4.5 5.6
26 3.9 2.2 20 20 59 17 38
0.062 0.011 0.006 0.105 0.023 0.037 0.016 0.102
Refer to footnotes in Table 1. e Anal. C: calcd 58.60, found 58.42; H: calcd 5.79, found 5.23; N: calcd 8.04, found 7.65.
the channel narrows. Major interactions contributing to the tight binding of 11c include carboxylic acid-zinc ligation, the carboxylate hydrogen bonding with Glu 202, and hydrogen bonding between the sulfonamide moiety and Ala 165 and Leu 164. In addition, substitution by electron-withdrawing substituents produces an electron-deficient phenyl ring, which in turn can improve aryl-aryl stacking interactions with His 201 (sandwich stack; distance between ring centroids ) 3.5 Å) and Tyr 223 (edge-to-face interaction; average distance between His ring centroid and the two proximal Tyr ring carbons ) 4.6 Å) in the active site. The reduction in binding energy resulting from a reduced π-π stack by phenyl rings para-substituted by moderate electron-releasing groups (CH3, OCH3) appears to be more than offset by the increase in steric bulk/lipophilic contacts these groups provide. MMP-3 and -13 have similar S1′ pockets, which explains why 11c potently inhibits these enzymes. In contrast, the MMPs possessing either shallow (MMP-1 and -7) or narrow (MMP-9) S1′ pockets8b cannot easily accommodate the 4′-bromosubstituted biphenyl ring system, resulting in a loss in binding energy and a decrease in potency. To identify the best possible candidate for chronic dosing in vivo, we determined the pharmacokinetic parameters in rat for selected compounds from Tables 1 and 2. As summarized in Table 3, it appears that biphenyl derivatives substituted in the 4′-position with
nonmetabolizable groups (11a,c,e,f, j) provide compounds that achieve high plasma concentrations with long elimination half-lives (t1/2 ) 34-117 h), compared to 5 (t1/2 ) 3.35 h), whereas easily metabolized groups (11g,h) gave potent inhibitors with very short t1/2 (
