Design and Synthesis of Matrix Metalloproteinase Inhibitors Guided by

Picking the S1 Pocket Using Conformationally Constrained. Inhibitors. Stephen Hanessian,* D. Bruce MacKay, and Nicolas Moitessier. Department of Chemi...
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Design and Synthesis of Matrix Metalloproteinase Inhibitors Guided by Molecular Modeling. Picking the S1 Pocket Using Conformationally Constrained Inhibitors Stephen Hanessian,* D. Bruce MacKay, and Nicolas Moitessier Department of Chemistry, Universite´ de Montre´ al, C. P. 6128, Succursale Centre-Ville, Montre´ al, P. Q., Canada H3C 3J7 Received March 3, 2001

Conformationally constrained MMP inhibitors based on a D-proline scaffold were designed using AutoDock as a modeling program. Thus a family of D-proline hydroxamic acids, having differentiated functionality at the site of binding to the S1 pocket, was synthesized. Biological evaluation showed low nanomolar activity and modest selectivity toward different MMP subclasses, delineating the importance of binding to the S1 pocket for both activity and selectivity. Introduction The matrix metalloproteinases (MMPs) are recognized as promising drug targets as evidenced by the disclosure of several potent inhibitors in recent years.1 MMP inhibitors can be roughly organized into two categories, namely succinate-type structures, exemplified by Batimastat (1)2 and Ro32-3555 (2),3 and sulfonamides, including CGS 27023A (3)4 and 45 (Figure 1). X-ray crystallography and NMR conformational studies have been used extensively to better understand the binding of inhibitors to these enzymes. For instance, CGS 27023A (3) complexed with MMP-3 was studied by NMR,6 and the tertiary structure of MMP-8 cocrystallized with 1 has been elucidated by X-ray crystallography.7 Although many broad spectrum MMP inhibitors have been disclosed, selectivity toward specific MMP subtypes remains an important issue. We now report the design, synthesis, and in vitro MMP inhibitory activity of conformationally constrained hydroxamic acids related to D-proline. Results and Discussion Molecular Modeling and Design. Previous studies have shown that selectivity can be achieved by optimizing the length of the P1′ subsite according to the difference in depth of the S1′ pocket for different MMP subtypes.8 As observed from X-ray crystal structures,7,9 the S1′ pocket is long and narrow for MMP-2, MMP-3, and MMP-8, and short and narrow for MMP-1 and MMP-9. By contrast, less information is available concerning the exploitation the S1 pocket for selectivity purposes. A survey of reported X-ray crystallographic structures of MMP-inhibitor complexes7,9 revealed that although the tertiary structures were quite similar, a few mutations of amino acids occurred at the S1 pocket (Table 1).10 For example, MMP-1 and MMP-8 featured the same His, Phe, Ser triad of amino acids in this pocket. Similarly, His, Phe, Tyr were found in MMP-2, MMP9, and MMP-13, while MMP-3 presented a distinct * To whom correspondence should be addressed. Fax: (514) 3435728. Tel: (514) 343-6738. E-mail: [email protected].

Figure 1. MMP inhibitors. Table 1. Amino Acids Present in the S1′ Pocket of MMPs MMPs

aa 1

aa 2

aa 3

MMP-1 MMP-8 MMP-2 MMP-9 MMP-13 MMP-3

His-183 His-162 His-166 His-183 His-187 His-166

Phe-185 Phe-164 Phe-168 Phe-185 Phe-189 Tyr-168

Ser-172 Ser-151 Tyr-155 Tyr-172 Tyr-176 Tyr-155

combination of amino acids (His, Tyr, Tyr). It is possible that these differences, which are mainly related to the aromaticity and hydrophobicity of these side chains, could reflect on inhibitor binding and enzymatic activity. Previous studies in our laboratories were concerned with probing the S1, S1′, and S2′ pockets with acyclic inhibitors such as 4 and uncovered subnanomolar inhibition of some MMPs.5 In an effort to further improve our understanding of the bioactive conformation of such acyclic motifs and to validate the potential for selectivity at the S1 pocket, we turned our attention to a constrained scaffold. Using the AutoDock suite of molecular modeling programs,11 we designed the D-

10.1021/jm010096n CCC: $20.00 © 2001 American Chemical Society Published on Web 08/14/2001

Synthesis of MMP Inhibitors Guided by Molecular Modeling

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Scheme 1a

a (a) LiHMDS, PMPSO Cl, THF, 79%; (b) LDA, THF then 2 PhSeBr; (c) O3, CH2Cl2, 89% (two steps); (d) (vinyl)2CuCN(MgBr)2, TMSCl, Et2O, 71%; (e) LiHMDS, MeI, DMPU, THF (two iterations), 69%; (f) LiAlH4, THF; (g) Et3SiH, BF3‚Et2O, CH2Cl2, 56% (two steps); (h) NaClO2, NaOCl, TEMPO, CH3CN, aqueous phosphate buffer; (i) CH2N2, Et2O, quant. (two steps); (j) O3, CH2Cl2; then Me2S; then NaBH4, EtOH, 69%; (k) NH2OK, NH2OH, MeOH, 65% (5b), 22% (5c).

Figure 2. Designed inhibitors.

proline-derived analogues represented by structures 5a-i (Figure 2).12 AutoDock docking studies confirmed the expected binding mode of 5a to be similar to that of 4 in MMP-3 (Figure 3, panels a and b). Analogues 5a, 5d, and 5f were involved in hydrophobic interactions with the S1 pocket through the phenyl moieties. Although the side chain of 5e was also aromatic, the presence of the methoxy group sterically disfavored a good interaction in this pocket. Interestingly, AutoDock also revealed the presence of a hydrogen bond formed between the hydroxymethyl analogue 5c with residue Ala-165 of the protein backbone in MMP-3 (Figure 3, panel c). This interesting feature led us to design compound 5g in which the hydroxyl and the aromatic groups could interact with the protein (Figures 3, panel d). Synthesis. D-Pyroglutamic acid was converted to the known lactam 6 according to a literature procedure.13 The sulfonamide functionality was introduced as in 7, since it was common to all targeted inhibitors and was expected to be compatible with subsequent chemistry. Elimination via the phenylselenyl derivative afforded the R,β-unsaturated lactam 8, which was subjected to conjugate addition with a vinyl magnesio cuprate to give 9 in good yield and with exclusive stereocontrol. Initially, dimethylation of 9 to give 10 proved quite sluggish, affording the monomethyl product in preponderance. To achieve complete methylation, the reaction

had to be repeated on the crude material. Reduction of 10 with LiAlH4 then with Et3SiH in the presence of BF3‚ Et2O led to product, which was immediately desilylated to afford 11. Oxidation14 and esterification gave 12, which was subjected to ozonolysis followed by reductive workup using sequential treatment with Me2S then NaBH4 to give 13 (Scheme 1). Conversion of the ester functionality of 12 and 13 directly to the hydroxamic acid was effected using NH2OK in MeOH.15 Hydroxamic acid 5c proved to be highly water soluble, and even after extensive extraction with EtOAc from H2O, only modest amounts of product could be recovered. Alcohol 13 was transformed to the thioethers 15a, 15d, and 15e (Scheme 2) by reaction of mesylate 14 with preformed thiolates generated from reaction of thiols with NaH in DMF. Although longer reaction times were required compared with literature procedures,16 the desired thioethers were nevertheless obtained in moderate to excellent yield. Use of DMF was critical for the thiolate substitution, since no reaction was observed in other solvents such as THF, toluene, or acetonitrile. These observations can be rationalized considering the steric hindrance to attack of the thiolates on the mesylate. The alkoxide generated upon treatment of 13 with NaH proved to be unstable, decomposing to a mixture of unidentified products. Performing an in situ quench of the reaction mixture with benzyl bromide at low temperature to afford 16 minimized this decomposi-

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Figure 3. Proposed docked structures for 4 (a), 5a (b), 5c (c), and 5h (d) in the MMP-3 binding site. Arrow in panel d indicates H-bond with residue Ala-165.

Scheme 2a

Scheme 3a

a (a) MsCl, Et N, CH Cl , quant.; (b) RSNa, DMF, 92% (15a), 3 2 2 49% (15d), 55% (15e); (c) NH2OK, NH2OH, MeOH, 80% (5a), 79% (5d), 52% (5e), 64% (5f); (d) NaH, RBr, DMF, 25%.

tion. The final hydroxamic acids 5a,d-f were prepared as described above. Initially, hydroxamic acid 5g was prepared as a mixture of diastereomers, starting from olefin 12 (Scheme 3). Dihydroxylation of 12 using OsO4 and NMO for 2 days afforded a 1.3:1 mixture of inseparable diastereomers in excellent yield. As expected, selective tosylation of the primary alcohol and subsequent reaction with sodium benzenethiolate afforded 18 in excellent overall

a (a) OsO , NMO, acetone/H O, 87%, dr 1.3:1; (b) TsCl, Et N, 4 2 3 pyridine, CH2Cl2; (c) PhSNa, DMF, 84% (2 steps); (d) NH2OK, NH2OH, MeOH, 66% (5g), 50% (5h), 66% (5i); (e) ∆, vacuum.

yield. Conversion to hydroxamic acids 5g proceeded smoothly to give a 1.3:1 mixture of inseparable dia-

Synthesis of MMP Inhibitors Guided by Molecular Modeling Table 2. Inhibitory Activities of Compounds 4 and 5a-ia IC50, nM compd

MMP-1

MMP-2

MMP-3

MMP-9

MMP-13

4 5a 5b 5c 5d 5e 5f 5h 5i

104 198 25710 8760 654 458 1620 117 301

0.7 1.64 441 143 15.4 4.6 32 2.5 16.2

0.7 6.7 293 136 6.5 16.7 10.1 3.0 18.7

2.5 0.9 138 50 2.8 4.4 9.2 0.9 3.6

12 5.5 519 218 17.0 22.3 44.2 3.7 18.1

a

See Experimental Section for details.

stereomers. The mixture of diastereoisomeric alcohols 18 could be separated by careful flash chromatography to afford the R-stereoisomer 18a and the S-stereoisomer 18b. Conversion of each isomer to the corresponding hydroxamic acid afforded 5h and 5i in diastereomerically pure form, as opposed to the original 5g which consisted of a mixture of the two. Heating 17 in vacuo led to selective cyclization of the S-diastereomer to the corresponding lactone 19, along with recovered 17 which was enriched in the R-diastereomer. 1H NMR analysis of 19 showed that the hydroxyl group occupied an equatorial position, judging by the large coupling constant (J ) 8.5 Hz) between the methine proton R to the alcohol and the proton at the 3-position of the pyrrolidine ring. On the basis of this NMR data on the lactone 19, we were able to assign the configuration of the epimeric alcohols 18a and 18b as R- and S- respectively. Biological Assays. The IC50 values of analogues 5a-i compared to the acyclic counterpart 4 on five different MMPs are listed in Table 2. The initial goal of this work was to constrain the acyclic carbon framework of 4 into the pyrrolidine analogue 5a. Indeed, both were found to be roughly equally active against four out of five MMPs. The AutoDock model (Figure 3, panel c) showed an additional hydrogen bond between the hydroxyl group in 5c and the protein backbone of MMP-3. It is of interest that 5c was between two and three times more active than 5b (Scheme 2) which lacks this hydroxyl group for all MMPs. A loss of activity of approximately 2 orders of magnitude in going from 5a to 5b may be due to the absence of an aromatic P1 moiety. Lengthening the P1 subsite as in 5d resulted in a slightly different orientation of the central core, and for 5e, the exclusion of the p-methoxyphenyl moiety out of the S1 pocket of MMP3. Nevertheless, these analogues did not lose significant inhibitory activity compared to 5a. Substituting the sulfur atom by oxygen (5a to 5f) resulted in a modest loss of potency. The second generation compound, 5h, featured both the aromatic ring at the P1 subsite and the R-hydroxyl group that, according to the proposed binding mode (Figure 3, panel d), was involved in a H-bond with Ala-165. Accordingly, 5h exhibited activities 3-6 times higher than the diastereomer 5i in inhibiting MMP-3, as well as the other MMPs. While we succeeded in discovering highly active compounds, the observed selectivity was somewhat disappointing except that activity against MMP-1 was much weaker compared to other MMPs. The low to subnanomolar enzymatic inhibition of the acyclic analogue 4 against MMP-2, MMP-3, MMP-9, and MMP-

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13, which share the common His, Phe, Tyr or His, Tyr, Tyr triads, compared to MMP-1 (His, Phe, Ser) is of interest in the context of a preferred binding of an arylalkylthio P1 appendage. The same trend was observed with the constrained analogue 5a, which encompassed the acyclic skeleton of 4 and approximated its bioactive conformation according to AutoDock modeling, with the same arylalkylthio P1 substituent. Such a constrained analogue has also proven to be a valuable probe to fine-tune the nature of the S1-P1 interaction in the quest for more potent and selective inhibitors. Furthermore, the enhanced activity of the R-hydroxy 1-phenylthioethyl analogue 5h compared to 5i nicely validates the value of capitalizing on observations based on modeling and showed stereochemical dependence in the P1 site. Conclusion Previously reported acyclic MMP inhibitor 4 was constrained into a D-proline hydroxamic acid. Further design of a series of analogues with the help of a systematic docking study with MMP-3 led to analogues with P1 appendages of different sizes, hydrophobicities, and shapes. They were prepared from an advanced common chiron derived from D-pyroglutamic acid and subsequently used to probe the S1 pocket in MMPs. These “designed” compounds exhibited nanomolar activities with a predictable pattern of potencies. Modeling of the complexes with MMP-3 found an extra hydrogen bond in the case of 5c that could explain its enhanced activity compared to 5b and was used to modulate the activity of 5a. On the basis of this observation, we prepared compound 5h which incorporates a new hydrogen bond donor on the hydrophobic phenylthioethyl P1 side chain. Compound 5h proved to be more active than its epimeric analogue 5i. Experimental Section Chemistry. Solvents were distilled under positive pressure of dry argon before use and dried by standard methods; THF and ether, from Na/benzophenone; CH2Cl2 and toluene, from CaH2. All commercially available reagents were used without further purification. All reactions were performed under argon atmosphere. NMR (1H, 13C) spectra were recorded on AMX300 and ARX-400 spectrometers in CDCl3 or CD3OD with solvent resonance as the internal standard. Low- and highresolution mass spectra were recorded on VG Micromass, AEIMS 902, or Kratos MS-50 spectrometers using fast atom bombardement (FAB). Optical rotations were recorded on a Perkin-Elmer 241 polarimeter in a 1 dm cell at ambient temperature. Analytical thin-layer chromatography was performed on Merck 60F254 precoated silica gel plates. Visualization was performed by UV or by development using KMnO4 or FeCl3 solutions. Flash column chromatography was performed using (40-60 µm) silica gel at increased pressure. All melting points are uncorrected. (R)-5-(tert-Butyl-diphenylsilanyloxymethyl)-1-(4-methoxy-benzenesulfonyl)-pyrrolidin-2-one (7). To a solution of lactam 6 (0.80 g, 1.93 mmol) in THF (25 mL) at -25 °C was added LiHMDS (1.0 M in THF, 2.32 mL, 2.32 mmol). The reaction mixture was stirred at -25 °C for 15 min. A solution of PMPSO2Cl (0.490 g, 2.35 mmol) in THF (4 mL) was added, and the reaction mixture was stirred at -25 °C for 1 h, quenched with H2O (70 mL), and taken up in EtOAc (40 mL). The phases were separated, and the organic phase was washed with saturated NH4OH, saturated NaHCO3, and brine (50 mL each), dried over MgSO4, filtered, and concentrated in vacuo.

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The residue was purified by column chromatography (SiO2, 20-50% EtOAc in hexanes) to afford sulfonamide 7 (0.89 g, 79%) as a colorless oil, which solidified upon standing and was recrystallized from EtOAc to afford colorless needles: mp 110112 °C; [R]D +14.5 (c 1.00, CHCl3); TLC Rf ) 0.17 (20% EtOAc in hexanes); IR (neat liquid): 3074, 1733, 1596, 1498 cm-1; 1 H NMR (300 MHz, CDCl3) δ 7.95 (d, J ) 9.0 Hz, 2H), 7.627.55 (m, 4H), 7.46-7.33 (m, 6H), 6.88 (d, J ) 9.0 Hz, 2H), 4.47-4.38 (m, 1H), 4.02 (dd, J ) 11.0, 4.0 Hz, 1H), 3.84-3.78 (m, 1H), 3.82 (s, 3H), 2.67 (dt, J ) 20.0, 10.0 Hz, 1H), 2.361.94 (m, 3H), 1.03 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 173.94, 163.59, 135.49, 135.37, 132.69, 132.30, 130.33, 130.20, 129.84, 129.80, 127.70, 127.67, 113.87, 65.50, 60.49, 55.48, 31.41, 26.68, 22.32, 19.02; HRMS calcd for C28H34NO5SiS (MH+) 524.1927, found 524.1912. (R)-5-(tert-Butyl-diphenylsilanyloxymethyl)-1-(4-methoxy-benzenesulfonyl)-1,5-dihydro-pyrrol-2-one (8). LDA was prepared as follows: To a solution of diisopropylamine (0.12 mL, 0.86 mmol) in THF (10 mL) at -78 °C was added BuLi (2.5 M in hexanes, 0.340 mL, 0.85 mmol). The solution was allowed to warm by removal of the cooling bath for 5 min and then cooled to -78 °C. To the LDA solution was added a solution of lactam 7 (0.497 g, 0.851 mmol), and the reaction mixture was stirred at -78 °C for 15 min. A solution of PhSeBr (0.250 g, 1.06 mmol) in THF (4 mL) was added dropwise, and the reaction mixture was stirred at -78 °C for 1 h, quenched with H2O (15 mL), and allowed to warm to room temperature. EtOAc (75 mL) was added, the phases were separated, and the organic phase was washed with 2 N HCl (75 mL), saturated NaHCO3 (75 mL), and brine (75 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was taken up in CH2Cl2 (50 mL) and cooled to -78 °C, and the solution was sparged with O3 until a pale blue color persisted. The reaction mixture was sparged with Ar until the blue color was completely lost from solution, pyridine (1.5 mL) was added, and the reaction mixture was allowed to warm to room temperature by removal of the cooling bath. The reaction mixture was diluted with CH2Cl2 (75 mL) and saturated NaHCO3 (75 mL), the phases were separated, and the aqueous phase was extracted with CH2Cl2 (2 × 75 mL). The combined organic phases were dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (10-40% EtOAc in hexanes) to afford unsaturated lactam 8 (0.44 g, 89%) as a pale yellow foam: [R]D +70.6 (c 1.26, CHCl3); TLC Rf ) 0.51 (40% EtOAc in hexanes); IR (neat liquid): 1730, 1595, 1490 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.93 (d, J ) 9.0 Hz, 2H), 7.58-7.56 (m, 4H), 7.50-7.33 (m, 6H), 7.11 (d, J ) 6.0 Hz, 1H), 6.90 (d, J ) 9.0 Hz, 2H), 6.02 (d, J ) 6.0 Hz, 1H), 4.79-4.75 (m, 1H), 4.24 (dd, J ) 10.0, 3.0 Hz, 1H), 3.99 (dd, J ) 10.0, 6.0 Hz, 1H), 3.83 (s, 3H), 0.99 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 169.32, 164.00, 150.90, 135.86, 135.74, 132.88, 132.83, 130.47, 130.30, 130.28, 130.21, 128.07, 128.04, 126.68, 114.31, 65.33, 63.42, 55.83, 26.97, 19.39 (1 C missing); HRMS calcd for C28H32NO5SiS (MH+) 522.1770, found 522.1762. (4R,5R)-5-(tert-Butyl-diphenylsilanyloxymethyl)-1-(4methoxy-benzenesulfonyl)-4-vinyl-pyrrolidin-2-one (9). Caution: CuCN is highly toxic and must be handled with due caution. Contact of reaction byproducts with acid must be avoided. To a suspension of CuCN (3.20 g, 35.7 mmol) in Et2O (100 mL) at -20 °C was added vinylmagnesium bromide (1 M in THF, 73.0 mL, 73.0 mmol) over several minutes. The reaction was stirred for 20 min and then cooled to -78 °C. A solution of lactam 8 (4.83 g, 9.27 mmol) and TMSCl (2.6 mL, 28.0 mmol) in Et2O (35 mL) was added over 5 min, and the reaction mixture was stirred at -78 °C for 1 h. The reaction was quenched by addition of aqueous NH4OH (15%, 10 mL), warmed to room temperature, and filtered through Celite. The residue was triturated with Et2O (2 × 150 mL). The combined organic extracts were washed with aqueous NH4OH (15%, 200 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (0-30% EtOAc in hexanes) to afford unsaturated lactam 9 (3.59 g, 71%) as a clear, colorless oil: [R]D +15.0 (c 0.94, CHCl3); TLC Rf )

0.70 (40% EtOAc in hexanes); IR (neat) 1739, 1596, 1498 cm-1; 1 H NMR (300 MHz, CDCl3) δ 7.90 (d, J ) 9.0 Hz, 2H), 7.657.56 (m, 4H), 7.46-7.29 (m, 6H), 6.90 (d, J ) 9.0 Hz, 2H), 5.74-5.13 (m, 1H), 4.92 (dd, J ) 16.0, 7.0 Hz, 2H), 4.12-4.07 (m, 1H), 4.04-3.82 (m, 2H), 3.78 (s, 3H), 2.93-2.82 (m, 2H), 2.22-2.13 (m, 1H), 1.04 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 173.02, 163.71, 138.14, 135.56, 135.45, 135.30, 135.34, 132.67, 132.28, 130.38, 129.90, 127.77, 127.70, 115.32, 113.86, 66.14, 64.95, 55.52, 37.93, 36.77, 26.74, 19.09; HRMS calcd for C30H36NO5SiS (MH+) 550.2084, found 550.2104. (4R,5R)-5-(tert-Butyl-diphenylsilanyloxymethyl)-1-(4methoxy-benzenesulfonyl)-3,3-dimethyl-4-vinyl-pyrrolidin-2-one (10). To a solution of lactam 9 (103.8 mg, 0.189 mmol) in THF (2 mL) at -20 °C was added LiHMDS (1.0 M in THF, 0.40 mL, 0.40 mmol). The solution was stirred for 10 min at -10 °C, DMPU (0.10 mL, 0.826 mmol) was added, and the solution was stirred for 10 min at -10 °C. MeI (0.100 mL, 1.61 mmol) was added, and the reaction mixture was allowed to warm to room temperature over 2.5 h. The reaction mixture was taken up in Et2O (10 mL), washed with H2O (3 × 10 mL), dried over MgSO4, filtered, and concentrated in vacuo. (Crude NMR at this point shows a mixture of starting material and products from mono- and dialkylation.) The alkylation procedure was repeated exactly for the crude product. The crude product after the second iteration was purified by column chromatography (5-10% EtOAc in hexanes) to afford lactam 10 (74.7 mg, 69%) as a clear, colorless oil: [R]D +17.1 (c 0.97, CHCl3); TLC Rf ) 0.48 (30% EtOAc in hexanes); IR (neat) 1739, 1596, 1498 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.79 (d, J ) 9.0 Hz, 2H), 7.48-7.40 (m, 4H), 7.30-7.15 (m, 6H), 6.70 (d, J ) 9.0 Hz, 2H), 5.37 (dt, J ) 17.0, 10.0 Hz, 1H), 4.92 (d, J ) 10 Hz, 1H), 4.63 (d, J ) 10 Hz, 1H), 4.35 (dd, J ) 11.0, 3.0 Hz, 1H), 3.78 (m, 1H), 3.65 (s, 3H), 3.58 (d, J ) 9.0 Hz, 1H), 2.63 (t, J ) 9.0 Hz, 2H), 0.92 (s, 3H), 0.88 (s, 9H), 0.67 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 179.25, 163.62, 135.72, 135.68, 132.98, 132.87, 132.80, 130.46, 130.16, 129.79, 127.83, 127.69, 120.35, 113.88, 62.46, 61.07, 55.55, 48.30, 44.56, 26.89, 22.69, 20.14, 19.37; HRMS calcd for C32H40NO5SiS (MH+) 578.2396, found 578.2400. (2R,3R)-[1-(4-Methoxy-benzenesulfonyl)-4,4-dimethyl3-vinyl-pyrrolidin-2-yl]-methanol (11). To lactam 10 (55.0 mg, 0.0958 mmol) at room temperature was added LiAlH4 (1 M in THF, 0.40 mL, 0.40 mmol). The reaction mixture was stirred at room temperature for 30 min, quenched with H2O (5 mL), extracted with Et2O (3 × 10 mL), dried over MgSO4, filtered, and concentrated in vacuo. The crude lactamol was used immediately without further purification. To a solution of the lactamol in CH2Cl2 (2 mL) at 0 °C were added Et3SiH (100 µL, 0.626 mmol) and BF3‚Et2O (40 µL, 0.33 mmol). The reaction mixture was allowed to warm to room temperature overnight and concentrated in vacuo. The residue was purified by column chromatography (20-60% EtOAc in hexanes) to afford alcohol 11 (17.4 mg, 56% from 10) as a clear, colorless oil: [R]D +59.3 (c 0.76, CHCl3); TLC Rf ) 0.64 (60% EtOAc in hexanes); IR (neat) 3501 (br), 1596, 1497 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.78 (d, J ) 9.0 Hz, 2H), 6.96 (d, J ) 9.0 Hz, 2H), 5.37 (dt, J ) 16.5, 10.0 Hz, 1H), 5.16-5.04 (m, 2H), 3.903.80 (m, 1H), 3.86 (s, 3H), 3.56 (dd, J ) 12.0, 4.5 Hz, 1H), 3.303.15 (m, 3H), 2.86 (br s, 1H), 2.24 (t, J ) 10.0 Hz, 1H), 0.87 (s, 3H), 0.25 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 163.13, 147.33, 133.20, 129.50, 120.33, 114.25, 65.59, 63.88, 62.73, 56.25, 55.61, 39.90, 24.12, 20.73; HRMS calcd for C16H24NO4S (MH+) 326.1426, found 326.1434. (2R,3R)-1-(4-Methoxy-benzenesulfonyl)-4,4-dimethyl3-vinyl-pyrrolidine-2-carboxylic acid methyl ester (12). A solution of alcohol 11 (0.670 g, 2.06 mmol) and TEMPO (22.6 mg, 0.145 mmol) in CH3CN (10 mL) and sodium phosphate buffer (0.67 M, pH ) 6.5, 7.5 mL) was heated to 35 °C (the heating bath was thermostated to 42 °C). Solutions of NaClO2 (tech. grade, 80%, 0.457 mg, 40.4 mmol) in H2O (2 mL) and aqueous NaOCl (10.3% available chlorine, 0.05 mL, diluted to 2 mL) were added dropwise over 15 min. (Note: the solutions must not be mixed as they are unstable together.) A dark reddish-brown color developed in the reaction mixture during

Synthesis of MMP Inhibitors Guided by Molecular Modeling

Journal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3079

addition. The reaction mixture was stirred at 35 °C overnight, extracted with EtOAc (3 × 40 mL) from aqueous HCl (0.2 M, 40 mL), dried over MgSO4, filtered, and concentrated in vacuo. The crude carboxylic acid was taken up in Et2O (15 mL) and treated at room temperature with CH2N2 until a pale yellow color was observed. The reaction mixture was stirred at room temperature for 5 min, titrated with AcOH in Et2O until the yellow color of CH2N2 was lost, and concentrated in vacuo. The residue was purified by column chromatography (10-30% EtOAc in hexanes) to afford ester 12 (0.736 g, quant.) as a clear, colorless oil: [R]D +85.9 (c 0.80, CHCl3); TLC Rf ) 0.33 (30% EtOAc in hexanes); IR (neat) 1752, 1596, 1497 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.78 (d, J ) 9.0 Hz, 2H), 6.94 (d, J ) 9.0 Hz, 2H), 5.49 (dt, J ) 16.0, 9.0 Hz, 1H), 5.10 (d, J ) 9 Hz, 1H), 5.04 (d, J ) 16.0 Hz, 1H) 3.94 (d, J ) 10.0 Hz, 1H), 3.80 (s, 3H), 3.65 (s, 3H), 3.23-3.16 (m, 2H), 2.46 (t, J ) 9.0 Hz, 1H), 0.87 (s, 3H), 0.43 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.33, 163.02, 131.64, 129.80, 129.59, 120.12, 114.09, 64.46, 60.99, 58.72, 55.56, 52.32, 41.77, 23.75, 20.34; HRMS calcd for C17H24NO5S (MH+) 354.1375, found 354.1375. (2R,3R)-3-Hydroxymethyl-1-(4-methoxy-benzenesulfonyl)-4,4-dimethyl-pyrrolidine-2-carboxylic Acid Methyl Ester (13). A solution of olefin 12 (0.736, 2.06 mmol) in CH2Cl2 (70 mL) at -78 °C was sparged with O3 until a faint blue color persisted in solution. The solution was sparged with Ar at -78 °C until all blue coloration was lost from solution. Me2S (1.0 mL, 14 mmol) was added, and the solution warmed to room temperature over 30 min. NaBH4 (82.2 mg, 2.17 mmol) and EtOH (75 mL) were added, and the reaction mixture was stirred at room temperature for 2 h. The solvents were removed in vacuo. The residue was stirred with 2 N HCl (30 mL) for 15 min, extracted with EtOAc (3 × 30 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (40-80% EtOAc in hexanes) to afford alcohol 13 (0.5128 g, 69%) as a clear colorless oil, which crystallized upon standing, and was recrystallized (EtOAc/hexanes) to afford 13 as colorless needles: mp 115117; [R]D +73.8 (c 0.88, CHCl3); TLC Rf ) 0.21 (50% EtOAc in hexanes); IR (neat liquid): 3528 (br), 1741, 1595, 1497 cm-1; 1H NMR (300 MHz, CDCl ) δ 7.79 (d, J ) 9.5 Hz, 2H), 6.96 (d, 3 J ) 9.5 Hz, 2H), 4.05 (d, J ) 9.0 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 3H), 3.72-3.55 (m, 2H), 3.20 (s, 2H), 2.19 (ddd, J ) 9.0, 7.5, 5.0 Hz, 1H), 1.76 (br s, 1H), 1.05 (s, 3H), 0.57 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 173.46, 163.03, 129.96, 129.61, 114.09, 64.21, 61.48, 60.98, 55.86, 55.34, 52.67, 40.37, 25.15, 20.56; HRMS calcd for C16H24NO6S (MH+) 358.1324, found 358.1325. Representative Procedure for Hydroxamic Acid Formation from Esters Using NH2OK/NH2OH. (2R,3R)-1-(4Methoxy-benzenesulfonyl)-4,4-dimethyl-3-vinyl-pyrrolidine-2-carboxylic Acid Hydroxyamide (5b). Preparation of NH2OK/NH2OH solution (Note: a blast shield was used for this operation): NH2OH‚HCl (0.476 mg, 6.85 mmol) was solubilized in MeOH (2.4 mL) by heating to reflux. Most, but not all of the salt dissolved. The solution was cooled to