Discovery of Potent, Selective Chymase Inhibitors via Fragment

Article pubs.acs.org/jmc

Discovery of Potent, Selective Chymase Inhibitors via Fragment Linking Strategies Steven J. Taylor,*,† Anil K. Padyana,† Asitha Abeywardane,† Shuang Liang,† Ming-Hong Hao,# Stéphane De Lombaert,∞ John Proudfoot,† Bennett S. Farmer, III,§ Xiang Li,† Brandon Collins,† Leslie Martin,∥ Daniel R. Albaugh,† Melissa Hill-Drzewi,⊥ Steven S. Pullen,‡ and Hidenori Takahashi† †

Department of Medicinal Chemistry, ‡Cardiometabolic Diseases, §IS Scientific Information Management, and ∥IS Research Data Acquisition, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877-0368, United States ⊥ Lead Evaluation Department, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut 06492, United States # H3 Biomedicine, 300 Technology Square, Cambridge, Massachusetts 02139, United States ∞ Karos Pharmaceuticals, 5 Science Park, 401 Winchester Avenue, New Haven, Connecticut 06511, United States

ABSTRACT: Chymase plays an important and diverse role in the homeostasis of a number of cardiovascular processes. Herein, we describe the identification of potent, selective chymase inhibitors, developed using fragment-based, structure-guided linking and optimization techniques. High-concentration biophysical screening methods followed by high-throughput crystallography identified an oxindole fragment bound to the S1 pocket of the protein exhibiting a novel interaction pattern hitherto not observed in chymase inhibitors. X-ray crystallographic structures were used to guide the elaboration/linking of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over cathepsin G and that mitigated a number of liabilities associated with poor physicochemical properties of the series it was derived from.

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INTRODUCTION Chymase is a chymotrypsin-like serine protease contained in the secretory granules of mast cells.1 Although the precise physiological roles of chymase have not been completely revealed, it is known to contribute to activation of TGF-β, matrix metalloproteases, and cytokines.2 Recently, cardiac chymase has been demonstrated to be responsible for the conversion of rat angiotensinogen to angiotensin 2 (Ang II), providing a parallel mechanism to angiotensin converting enzyme (ACE) mediated formation of Ang II.3 Chymase plays an important role in activating TGF-β1 and IL-1β, generating endothelin, altering apolipoprotein metabolism, and degrading the extracellular matrix.4 Inspection of hearts from autopsies of patients with myocardial infarction revealed that Ang II was increased in both the infarcted and noninfarcted areas of hearts, compared to hearts from subjects without cardiac disease, whereas ACE activity was only increased in the infarcted areas of hearts with myocardial infarction.5 Knockdown of the expression of chymase via siRNA has been shown to reduce inflammation in human uterine microvascular endothelial cells via multiple mechanisms of action.6 © 2013 American Chemical Society

Because of its role in the progression of cardiovascular diseases, a number of academic and industrial research organizations have sought to intervene in the modulation of the activity of chymase in vivo.7 The chymase inhibitor NK3201 has recently been shown to suppress oxidative stress, perivascular fibrosis, and the expression of cytokines in a mouse model of hypoxia.8 The same inhibitor has been shown to prevent cardiac fibrosis and dysfunction in rats following myocardial infarct,9 as well as demonstrated preservation of cardiac function and deterred fibrosis after left ventricular repair in rodents.10 A second small molecule chymase inhibitor, TY51469, has shown beneficial effects in animal models of cardiovascular disease, including reducing the progression to heart failure after autoimmune myocarditis initiation in rats.11 The emergence of small-molecule chymase inhibitors in recent years offers the opportunity to specifically define the role of chymase in cardiovascular disease progression via chemical intervention. With this in mind, our goal was to generate a Received: January 28, 2013 Published: May 9, 2013 4465

dx.doi.org/10.1021/jm400138z | J. Med. Chem. 2013, 56, 4465−4481

Journal of Medicinal Chemistry

Article

with the protein at the time were thought to be primarily driven through van der Waals interactions.16 Indeed, the pocket is primarily hydrophobic in nature with the exception of Ser189 and backbone carbonyl Ala190. The X-ray structure of compound 1 exhibits a high B-factor for the butyric acid moiety, most likely due to dynamic disorder with the salt bridge interaction switching with Lys40 and Lys192 in a solvent environment (Figure 2). A salt bridge between the inhibitor

potent inhibitor of chymase while sparing activity against structurally related serine proteases. Early chymase inhibitors, like NK3201, contained an irreversible alkylating moiety that presumably interacted in a covalent manner with the catalytic serine residue (Ser195) in the active site cleft of the protease. More recently, patent applications have appeared in the literature describing seemingly noncovalent inhibitors that replace this covalent interaction with a direct hydrogen bonding interaction to the catalytic serine,12 as well as inhibitors that do not interact directly with the catalytic serine residue of the protein.13 We initiated a lead identification campaign around the literature compound 3-[1-(4-methylbenzo[b]thiophen-3ylmethyl)-1H-benzoimidazol-2-ylsulfanyl]propionic acid (TPC806, Figure 1) that contains no covalent “warhead” and that

Figure 2. Cocrystal structure of compound 1 bound to the active site of chymase. Inhibitor is shown in stick representation with semitransparent rendering of solvent accessible protein surface with key amino acids in stick representation.

Figure 1. Small molecule noncovalent inhibitors of chymase.

does not interact directly with the catalytic serine residue of the protein. We considered that this strategy would result in more selective compounds by relying on potency through interactions outside of the catalytic residue rather than by covalent modification of the protein. A literature-to-lead pharmacophore based campaign was initiated around inhibitor TPC-806 (Figure 1), and benzimidazolone analogue 1 was rapidly identified as a potent proprietary scaffold with comparable chymase potency (120-fold loss of activity, in line with the fragment SAR at this position within the P1 motif. The halogen in the 6-position occupies a lipophilic notch in the S1 pocket that is generated by amino acids Phe191 and Ser218. Replacing the 6-bromoxindole side chain with a 6-bromobenzthiazolone (entry 7, 18) was equally tolerated and was as 4468



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Figure 4. Overlay of chymase X-ray cocomplex structures of fragment 2 (in cyan) with elaborated benzimidazole analogue 1 (in magenta). Inhibitors are shown in stick representation with key amino acids labeled and displayed as sticks on semitransparent rendering of solvent accessible protein surface.

group was reduced with DIBAL-H to provide alcohols 51 and 52. These alcohols were coupled with the benzimidazolone core under Mitsunobu conditions to afford esters 53 and 54. The indolinone was revealed by addition treatment with trifluoroacetic acid, and the ester was saponified with aqueous lithium hydroxide to afford 15 and 17. The preparation of benzthiazolone 18 is shown in Scheme 4. Phenol 57 was nitrated (58) and the acid protected as the ethyl ester 59. The phenol was acylated with dimethylthiocarbamoyl chloride to generate thiocarbamate 60, which rearranged under thermal conditions to give ester 62. The ester was reduced with borane dimethylsulfide, and the resulting alcohol (63) was coupled with the benzimidazolone core to afford the elaborated compound 65. The nitro group was reduced and the amine cyclized in situ generating 66. Ester 66 was hydrolyzed with aqueous lithium hydroxide to afford 18. The synthesis of benzimidazole analogue 19 is shown in Scheme 5. Alcohol 25 was coupled with the benzimidazole core 65 to afford linked analogue 66. The ester and indole were sequentially deprotected with aqueous lithium hydroxide and aqueous HCl to give the indole analogue 67. The indole was oxidized to the corresponding oxindole 19 with pyridinium tribromide/zinc.

water W1. In contrast, there is no directional preference at this position in chymase. Therefore, placing a carbonyl group at this position of Cat-G is energetically less favored than in chymase, thereby rationalizing the better selectivity of the oxindole moiety.

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CHEMISTRY Compounds 1 and 2 were prepared as previously described.23 Compounds 3−11 were purchased from commercial vendors. Preparations of fully elaborated compounds 12−19 are shown in Schemes 1−4. The syntheses of compounds 12 and 13 are shown in Scheme 1. The indoles were protected under mild conditions with di-tert-butyl dicarbonate, and then the esters were reduced to the corresponding alcohols with DIBAL-H. Alcohols 25 and 26 were coupled with the benzimidazolone core under Mitsunobu conditions to provide the linked compounds 27 and 28. Deprotection of the carbamate under acidic conditions revealed the unsubstituted indoles which were oxidized to the corresponding indolinones 33 and 34 with pyridinium tribromide/zinc. Treatment of the esters with aqueous lithium hydroxide provided compounds 13 and 14. The syntheses of analogues 14 and 16 are shown in Scheme 2. Alcohols 25 and 26 were coupled with benzimidazolones 35 and 36 under Mitsunobu conditions to provide the linked analogues 39 and 40. The indoles were deprotected under acidic conditions and converted to the corresponding indolinones 43 and 44 with pyridinium tribromide and zinc. Saponification of esters 43 and 44 provided the linked benzimidazolones 14 and 16. The syntheses of analogues 15 and 17 are shown in Scheme 3. Esters 21 and 22 were oxidized with pyridinium tribromide followed by treatment with zinc to afford indolinones 47 and 48. The indolinones were bis-Boc protected, and then the ester

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SUMMARY Fragment based screening has gained wide acceptance as a tool in drug discovery over the past 15 years. Fragment linking and fragment elaboration have resulted in the development of a number of advanced analogues that have progressed into clinical trails. To our knowledge this is the first example of utilizing the fragment based approach specifically to identify a chemical alternative to a functional group contained on an already established scaffold to mitigate specific liabilities inherent to the molecule. By use of the crystal structure of 4469



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Table 2. In Vitro Inhibition of Chymase with Elaborated Analoguesa

IC50 values represented as μM and averaged from a minimum of two determinations. Ligand efficiency (LE) calculated using −RT ln(IC50)/(no. heavy atoms).

a

liabilities frequently associated with large lipophilic molecules24 diminished.

the parent compound as guidance, a fragment discovered though screening activities was linked successfully to provide a more polar, soluble alternative to a known ligand. This modification resulted in a hybrid compound with excellent potency and greater selectivity against Cat-G when compared to the parent molecule. In addition, compound 14 was profiled for its ability to deplete GSH under oxidative conditions and showed no measurable difference in GSH adduct formation versus control. By incorporating more polar soluble motifs into an established scaffold, we demonstrated that dogma about what is tolerated in terms of potency can be challenged and the

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EXPERIMENTAL SECTION

Chemistry. General Remarks. Starting materials were obtained from commercial suppliers and used without further purification unless otherwise stated. 1H NMR spectra were recorded on a Bruker UltraShield 400 MHz spectrometer operating at 400 MHz in solvents, as noted. Proton coupling constants (J) are rounded to the nearest Hz. All coupling constants are reported in hertz (Hz), and multiplicities are labeled s (singlet), bs, (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), and m 4470



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Solvents: (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid, flow 1.5 mL/min. Photodiode array detector at 190 or 400 nm. (a) Agilent Zorbax Eclipse XDB-C8 5 μm, 4.6 mm × 150 mm column, 4.6 mm × 30 mm, 3.5 μm, from 99% to 5% solvent A over 10 min or (b) Column Agilent Zorbax C18 SB 3.5 μm, 4.6 mm × 30 mm cartridge, from 95% to 5% solvent A over 2.5 min. 3-[3-(6-Chloro-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2oxo-2,3-dihydrobenzoimidazol-1-yl]propionic Acid (12). Ester 20 (9.0 g, 43 mmol) and BOC2O (11g, 52 mmol) were dissolved in acetonitrile (140 mL) and stirred at room temperature for 10 min, at which time DMAP (180 mg, 1.6 mmol) was added in one portion to the solution. The mixture was stirred for 16 h at room temperature, and then the volatiles were evaporated in vacuo. The resulting oil was taken up in ethyl acetate and washed with water, 10% aqueous citric acid, and brine. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to give 6-chloroindole-1,4-dicarboxylic acid 1-tertbutyl ester 4-methyl ester 23 which was used without further purification (13.2 g, 99%). 1H NMR (400 MHz, CDCl3): 8.37 (bs, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 3.6 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 3.91 (s, 3H), 1.61 (s, 9H). Ester 23 (13.2g, 42 mmol) was dissolved in dry THF (200 mL) and cooled to −78 °C (CO2, iPOH), and the mixture was purged with nitrogen. DIBAL-H (1.5 M, in heptanes, 170 mL, 250 mmol) was added dropwise to the stirred mixture, and the mixture was stirred for 2 h at −78 °C. The reaction was quenched at −78 °C by the slow addition of 500 mL of saturated Rochelle’s salt solution, and the suspension was allowed to warm to room temperature and stirred overnight. The resulting biphasic mixture was separated, and the water layer was extracted with DCM. The combined organic layers were dried (MgSO4), filtered, and evaporated in vacuo to give an oil that was purified on silica (hexanes/EtOAc) to afford 6-chloro-4hydroxymethylindole-1-carboxylic acid tert-butyl ester 25 as a clear solid (9.5g, 80%). 1H NMR (400 MHz, CDCl3): δ 8.16 (bs, 1H), 7.61 (d, J = 3.6 Hz, 1H), 7.28 (s, 1H), 6.68 (d, J = 3.6 Hz, 1H), 4.93 (s, 2H), 1.69 (s, 9H). Alcohol 25 (350 mg, 1.2 mmol), triphenylphosphine (360 mg, 1.4 mmol), 3-(2-oxo-2,3-dihydrobenzoimidazol-1-yl)propionic acid methyl ester (300 mg, 1.4 mmol), and THF (6 mL) were combined and cooled to 0 °C in an ice−water bath. Diisopropyl diazodicarboxylate (DIAD, 0.67 mL, 1.4 mmol) was added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 1 h. The solvent was evaporated in vacuo and the product purified on silica (hexanes/EtOAc) to give 6-chloro-4-[3-(2-methoxycarbonylethyl)-2oxo-2,3-dihydrobenzoimidazol-1-ylmethyl]indole-1-carboxylic acid tert-butyl ester 27 as a white solid (280 mg, 47%). 1H NMR (400 MHz, CDCl3): δ 8.07 (bs, 1H), 7.50 (d, J = 3.6 Hz, 1H), 7.08 (s, 1H), 7.02−6.96 (m, 2H), 6.92−6.85 (m, 1H), 6.78−6.69 (m, 2H), 5.17 (s, 2H), 4.17 (t, J = 6.8 Hz, 2H), 3.58 (s, 3H), 2.76 (t, J = 7.2 Hz, 2H), 1.57 (s, 9H). Boc-protected indole 27 was dissolved in DCM (4.5 mL), and trifluororacetic acid (0.5 mL) was added dropwise to the reaction mixture. The reaction was stirred for 1 h at room temperature and then the solvents were removed in vacuo to give 3-[3-(1H-indol-4ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]propionic acid methyl ester 29 as an oil that was used without further purification (450 mg, 100%). Indole 29 (137 mg, 0.36 mmol) was suspended in tert-butanol (5 mL) and 40 °C, and pyridinium tribromide (570 mg, 1.8 mmol) was added portionwise to the stirred mixture. The reaction mixture was stirred at 40 °C for 3 h. The solvent was then removed in vacuo at low pressure and low temperature to afford 3-[3-(3,3-dibromo-2-oxo-2,3dihydro-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]propionic acid methyl ester 31 that was used directly without further purification. Dibromide 31 (362 mg, 0.0.65 mmol) was dissolved in acetic acid (10 mL), and zinc powder was added (211 mg, 3.2 mmol). The suspension was stirred vigorously for 1 h at room temperature and then filtered and evaporated in vacuo. The crude oil was purified using reverse phase HPLC to give 3-[3-(6-chloro-2-oxo-2,3-dihydro-1H-

Figure 5. Chymase X-ray cocomplex structural overlays. (a) Overlay of fragment 2 (in cyan) and compound 15 (in green) shown in stick representation. (b) Overlay of compound 1 (in magenta) and compound 15 (in green). Inhibitors are shown in stick representation with key amino acids labeled and displayed as sticks on semitransparent rendering of solvent accessible protein surface. (multiplet). All NMR spectra were referenced to tetramethylsilane (TMS δH 0, δC 0). All solvents were HPLC grade or higher. The reactions were followed by TLC on precoated Uniplate silica gel plates purchased from Analtech. The developed plates were visualized using 254 nm UV illumination or by PMA stain. Flash column chromatography on silica gel was performed on Redi Sep prepacked disposable silica gel columns using an Isco Combiflash, Biotage SP1 or on traditional gravity columns. Reactions were carried out under an atmosphere of argon at room temperature, unless otherwise noted. Mass spectrometry data were obtained using the Micromass Platform LCZ (flow injection). Purity for all final compounds was >95%, and purity was evaluated by the following: System 1: analytical HPLC using a Varian Dynamax SD-200 pump coupled to a Varian Dynamax UV-1 detector. Solvents: (A) water + 0.05% TFA and (B) acetonitrile + 0.05% TFA, flow 1.2 mL/min. Column: Vydac RP-18, 5 m, 250 mm × 4.6 mm. Photodiode array detector at 220 nm. Gradient: from 95% to 20% solvent A over 25 min. System 2: HP 1110 Agilent LCMS using a quaternary G1311A pump coupled to a Micromass Platform LCZ detector. 4471



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Figure 6. Overlay of the binding site of chymase (Chy, in green) with a bound inhibitor (15) obtained by X-ray crystallography and cathepsin G (Cat-G, in yellow, PDB code 1T32) as a reference with the hydrogen bonds between pairs of residues shown as dashed lines..

Figure 7. Water map analysis of inhibitor 15 in chymase and cathepsin G active sites: (a) overlay of calculated water-binding sites with a bound inhibitor (15) in chymase; (b) overlay of calculated water-binding sites with a docked inhibitor (15) in cathepsin G. Inhibitor is shown in stick representation. Waters are shown as cyan spheres with their calculated diople moments shown as gray and red sticks. indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]propionic acid methyl ester 33 as a white solid (34 mg, 13%). 1H NMR (400 MHz, CDCl3): δ 8.04 (bs, 1H), 7.33−7.29 (m, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 1.6 Hz, 1H), 6.93−6.85 (m, 2H), 5.00 (s, 2H), 4.26 (t, J = 6.8 Hz, 2H), 3.71 (s, 3H), 3.37 (s, 2H), 2.88 (t, J = 6.8 Hz, 2H). Ester 33 (34 mg, 0.09 mmol) was dissolved in 1,4-dioxane (2 mL) and water (2 mL), and LiOH monohydrate (18 mg, 0.4 mmol) was added. The mixture was stirred for 1 h at room temperature, and then the solvent was evaporated in vacuo. The crude solid was purified using reverse phase HPLC to afford 12 as a white solid (12 mg, 37%). LCMS (ES+) m/z found 386, retention time 0.68 min. C19H16ClN3O4 requires 386. HPLC, retention time 8.82 min. 1H NMR (400 MHz, CD3OD): δ 7.30 (d, J = 7.6 Hz,1H), 7.16 (td, J = 7.6, 1.2 Hz, 1H), 7.09 (td, J = 7.6, 1.2 Hz, 1H), 7.02 (d, J = 7.6 Hz,1H), 6.85 (d, J = 2.4 Hz, 2H), 5.08 (s, 2H), 4.26 (t, J = 6.8 Hz, 2H), 3.43 (s, 2H), 2.80 (t, J = 6.8 Hz, 2H). 3-[3-(6-Bromo-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2oxo-2,3-dihydrobenzoimidazol-1-yl]propionic Acid (13). Indole ester 21 (3.0 g, 14 mmol) and BOC2O (3.0g, 11 mmol) were dissolved in acetonitrile (40 mL) and stirred at room temperature for 10 min, at which time DMAP (40 mg, 0.2 mmol) was added to the mixture in one portion. The mixture was stirred for 16 h at room temperature,

and the volatiles were evaporated in vacuo. The resulting oil was taken up in ethyl acetate and washed with water, 10% aqueous citric acid, and brine. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to give 6-bromoindole-1,4-dicarboxylic acid 1-tertbutyl ester 4-methyl ester 24, which was used without further purification (13.2 g, 99%). 1H NMR (400 MHz, CDCl3): δ 8.53 (bs, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.59 (d, J = 3.6 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 3.90 (s, 3H), 1.61 (s, 9H). Ester 24 (4.1 g, 11.2 mmol) was dissolved in dry THF (35 mL), cooled to −78 °C (CO2, iPOH), and the mixture was purged with nitrogen. DIBAL-H (1.5 M, in heptanes 21 mL, 32 mmol) was added dropwise to the stirred mixture, and the mixture was stirred for 2 h at −78 °C. The reaction was quenched at −78 °C by the slow addition of 500 mL of saturated Rochelle’s salt solution, and the mixture was allowed to warm to room temperature and stirred overnight. The resulting biphasic mixture was separated, and the water layer was extracted with DCM. The combined organic layers were dried (MgSO4), filtered, and evaporated in vacuo to give an oil that was purified on silica (hexanes/EtOAc) to afford 6-bromo-4-hydroxymethylindole-1-carboxylic acid tert-butyl ester 26 as a white solid (3.7, 95%). 1H NMR (400 MHz, CDCl3): δ 8.33 (bs, 1H), 7.59 (d, J = 3.6 4472



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Scheme 1. Synthesis of Elaborated Indolinone Analogues 12 and 13a

Reagents and conditions (a) BOC2O, DMAP, acetonitrile, 99%; (b) DIBAL-H, −78 °C, THF, 80−95%; (c) triphenylphosphine, DIAD, THF, 0 °C, 22−47%; (d) TFA, DCM, 49−100%; (e) pyridinium tribromide, tert-butanol, 40 °C, 100%; (f) Zn (powder), HOAc, 13−42%; (g) aq LiOH, THF, 1,4-dioxane, 32−37%. a

Hz, 1H), 7.41 (s, 1H), 6.67 (d, J = 3.6 Hz, 1H), 4.93 (s, 2H), 1.69 (s, 9H). Alcohol 26 (500 mg, 1.5 mmol), triphenylphosphine (482 mg, 1.8 mmol), 3-(2-oxo-2,3-dihydrobenzoimidazol-1-yl)propionic acid methyl ester (388 mg, 1.7 mmol), and THF (8 mL) were combined and cooled to 0 °C in an ice−water bath. Diisopropyl diazodicarboxylate (0.35 mL, 1.8 mmol) was added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 1 h. The solvent was evaporated in vacuo and the product purified directly on silica (hexanes/EtOAc) to afford 6-bromo-4-[3-(2-methoxycarbonylethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-ylmethyl]indole-1-carboxylic acid tert-butyl ester 28 as a white solid (270 mg, 22%). Protected indole 28 (330 mg, 0.62 mmol) was dissolved in dichloromethane (10 mL), and trifluorocactic acid (5 mL) was added dropwise to the mixture. The mixture was stirred for 3 h at room temperature, and the solvent evaporated in vacuo, and the crude product purified on silica (dichloromethane/MeOH) to give 3-[3-(6bromo-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]propionic acid methyl ester 30 as a white solid (130 mg, 49%). Indole 30 (125 mg, 0.3 mmol) was suspended in tert-butanol (10 mL) at 40 °C, and pyridinium tribromide (187 mg, 0.6 mmol) was added portionwise to the stirred mixture. The reaction mixture was stirred at 40 °C for 3 h, and solvent was then removed in vacuo at low pressure and low temperature to provide the dibromide 32. The crude product was immediately dissolved in acetic acid (10 mL), and zinc powder was added (100 mg, 1.4 mmol) to the solution. The suspension was stirred vigorously for 1 h at room temperature, and the mixture was filtered and evaporated in vacuo. The crude oil was

purified using reverse phase HPLC to give 3-[3-(6-bromo-2-oxo-2,3dihydro-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]propionic acid methyl ester 34 as a white solid (55 mg, 42%). 1H NMR (400 MHz, CDCl3): δ 7.27 (d, J = 7.2 Hz, 1H), 7.19−7.07 (m, 2H), 7.04−6.98 (m, 3H), δ 5.12 (s, 2H), 4.27 (t, J = 6.8 Hz, 2H), 3.60 (s, 3H), 3.39 (s, 2H), 2.84 (t, J = 6.8 Hz, 2H). Ester 34 (55 mg, 0.12 mmol) was dissolved in 1,4-dioxane (1 mL) and water (1 mL), and solid lithium hydroxide (26 mg, 0.6 mmol) was added to the solution. The mixture was stirred for 1 h at room temperature and then acidified to pH 2 with 1 N HCl. The resulting precipitate was collected and purified using reverse phase HPLC to afford 13 as a white solid (15 mg, 32%). LCMS (ES+) m/z found 430, 432; retention time 0.69 min. C19H16BrN3O4 requires 430, 432. HPLC, retention time 8.97 min. 1H NMR (400 MHz, DMSO-d6): δ 12.50 (bs, 1H), 10.57 (s, 1H), 7.28 (d, J 7.68 Hz, 1H), 7.11−6.98 (m, 3H), 6.87 (d, J = 3.2 Hz, 2H), 4.99 (s, 2H), 4.10 (t, J = 6.8 Hz, 2H), 3.46 (s, 2H), 2.67 (t, J = 6.8 Hz, 2H). 3-[3-(6-Chloro-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2oxo-2,3-dihydrobenzoimidazol-1-yl]hexanoic Acid (14). Alcohol 25 (700 mg, 2.4 mmol), triphenylphosphine (716 mg, 2.7 mmol), 3-(2-oxo-2,3-dihydrobenzoimidazol-1-yl)hexanoic acid ethyl ester 35 (755 mg, 2.7 mmol), and THF (16 mL) were combined and cooled to 0 °C in an ice−water bath. Diisopropyl diazodicarboxylate (0.59 mL, 2.7 mmol) was added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 1 h. The volatiles were evaporated in vacuo and the crude product purified on silica (hexanes/ EtOAc) to give 6-chloro-4-[3-(1-ethoxycarbonylmethylbutyl)-2-oxo2,3-dihydrobenzoimidazol-1-ylmethyl]indole-1-carboxylic acid tert4473



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Scheme 2. Preparation of Fragment-Linked Chymase Inhibitors with Benzimidazole Core 14 and 16a

a Reagents and conditions: (a) triphenylphosphine, DIAD, THF, 0 °C, 57−75%; (b) TFA, DCM, 74−94%; (c) pyridinium tribromide, tert-butanol, 40 °C, 100%; (d) Zn (powder), HOAc, 20−87%; (e) aq LiOH, THF, 1,4-dioxane, 26−32%

butyl ester 37 as a white solid (1.0 g, 75%). 1H NMR (400 MHz, CDCl3): δ 8.15 (bs, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.03 (td, J = 7.6, 0.8 Hz, 1H), 6.93 (td, J = 7.6, 0.8 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H), 5.26 (s, 2H), 4.78 (m, 1H), 4.03 (q, J = 7.2 Hz, 2H), 3.27 (m, 1H), 2.87 (dd, J = 11.6, 5.6 Hz, 1H), 2.29 (m, 1H), 1.79 (m, 1H), 1.65 (s, 9H), 1.22−1.33 (m., 2H), 1.08 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.2 Hz, 3H). Boc-protected indole 37 (540 mg, 1.0 mmol) was dissolved in DCM (8 mL), and trifluororacetic acid (1 mL) was added dropwise to the reaction mixture. The mixture was stirred for 1 h at room temperature, and then the solvents were removed in vacuo to give 3[3-(6-chloro-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1yl]hexanoic acid ethyl ester 39 as an oil that was used without further purification (416 mg, 94%). Indole 39 (235 mg, 0.0.5 mmol) was suspended in tert-butanol (20 mL) at 40 °C, and pyridinium tribromide (410 mg, 1.2 mmol) was added portionwise to the stirred reaction. The reaction mixture was stirred at 40 °C for 3 h. The solvent was then removed in vacuo at low pressure and low temperature to give 3-[3-(3,3-dibromo-6-chloro-2oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]hexanoic acid ethyl ester 41 that was used directly without further purification. Dibromide 41 (338 mg, 0.55 mmol) was dissolved in acetic acid (10 mL), and zinc powder was added (180 mg, 2.7 mmol). The suspension was stirred vigorously for 1 h at room temperature and then filtered and evaporated in vacuo. The crude oil was purified using reverse phase HPLC to give 3-[3-(6-chloro-2-oxo-2,3-dihydro-1H-indol-4ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]hexanoic acid ethyl ester 43 as clear oil (50 mg, 20%). 1H NMR (400 MHz, CDCl3): δ 8.45 (bs, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.11 (td, J = 7.6, 0.8 Hz, 1H), 7.03 (7.6 Hz, 1.2 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 1.6 Hz, 1H), 4.98 (s, 2H), 4.77 (m, 1H), 4.06 (q, J = 7.2 Hz, 2H), 3.34 (s, 2H), 3.28 (m, 1H), 2.86 (dd, J = 12, 5.6 Hz, 1H), 2.25 (m, 1H), 1.18 (m, 1H), 1.31 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H). Ester 43 (50 mg, 0.11 mmol) was dissolved in 1,4-dioxane (1 mL) and water (1 mL), and solid lithium hydroxide (26 mg, 0.6 mmol) was

added to the solution. The mixture was stirred for 1 h at room temperature and then acidified to pH 2 with 1 N HCl. The resulting precipitate was collected and purified using reverse phase HPCL to give 14 as a white solid (11 mg, 32%). LCMS (ES+) m/z found 428, retention time 0.84 min. C22H22ClN3O4 requires 428. HPLC, retention time 10.41 min. 1H NMR (400 MHz, CD3OD): δ 7.22 (d, J = 7.60,1H), δ 7.14 (td, J = 1.14, 7.60 Hz, 1H), δ 7.07 (td, J = 1.00, 7.70 Hz, 1H), 6.99 (d, J = 7.81 Hz,1H), 6.84 (d, J = 3.50 Hz, 2H), 5.07 (s, 2H), 4.81 (m, 1H), 3.40 (s, 2H), 3.15 (m, 1H), 2.89 (dd, J = 10.6, 5.6 Hz, 1 H), 2.29−2.19 (m, 1H), 1.87−1.76 (m, 1H), 1.38−1.17 (m, 2H), 0.94 (t, J = 7.24 Hz, 3H). 3-[3-(6-Bromo-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2oxo-2,3-dihydrobenzoimidazol-1-yl]hexanoic Acid (15). 6Bromo-1H-indole-4-carboxylic acid methyl ester 21 (3.5 g, 13 mmol) was suspended in tert-butanol (100 mL) and heated to 40 °C. Pyridinium tribromide (14 g, 44 mmol) was added portionwise to the mixture over 10 min and the mixture stirred for 2 h. The solvents were evaporated in vacuo, and the crude product was taken up in EtOAc and washed with water. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to give 3,3,6-tribromo-2-oxo-2,3dihydro-1H-indole-4-carboxylic acid methyl ester 45 as a red solid that was used without further purification (6.1 g, 88%). 1H NMR (400 MHz, DMSO-d6): δ 11.63 (s, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 3.94 (s, 3H). Dibromide 45 (16g, 37 mmol) was suspended in acetic acid (0.25 L), and zinc powder (24 g, 37 mmol) was added portionwise to the mixture over 5 min. The mixture was stirred for 3 h and then filtered though Celite. The Celite was washed with ethyl acetate, and the combined washings were evaporated in vacuo to give an oil that was purified on silica (DCM/methanol) to give 6-bromo-2-oxo-2,3dihydro-1H-indole-4-carboxylic acid methyl ester 47 a white solid (12.1 g, 50%). Oxindole 47 (1 g, 3.7 mmol), BOC2O (1.7 g, 8 mmol), and DMAP (5 mg, 0.3 mmol) were dissolved in tetrahydrofuran (75 mL) and heated to 50 °C for 3 h. The solvents were evaporated in vacuo and the material was purified on silica (hexanes/EtOAc) to give 6-bromo2-tert-butoxycarbonyloxyindole-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester 49 as a clear oil (850 mg, 49%). 1H NMR (400 MHz, 4474



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Scheme 3. Preparation of Fragment-Linked Chymase Inhibitors with Benzimidazole Core 15 and 17a

Reagents and conditions: (a) pyridinium tribromide, tert-butanol, 40 °C, 88−92%; (b) Zn (powder), HOAc, 39−50%; (c) BOC2O, DMAP, THF, 50 °C, 10−49%; (d) DIBAL-H, −78 °C, THF, 48−89%; (e) triphenylphosphine, DIAD, THF, 0 °C, 15−57%; (f) TFA, DCM 85%; (g) aq LiOH, THF, 1,4-dioxane, 63−97%. a

CDCl3): δ 8.47 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 6.99 (s, 1H), 3.97 (s, 3H), 1.69 (s, 9H), 1.59 (s, 9H). Ester 49 (280 mg, 0.6 mmol) was dissolved in THF (7 mL) and the mixture flask cooled to −78 °C. DIBAL-H (1 M in heptane, 3.8 mL, 3.8 mmol) was added dropwise to the mixture over 5 min. The mixture was stirred at −78 °C for 2 h, and then the reaction was quenched by the addition of 50 mL of saturated Rochelle’s salt. The mixture was warmed to room temperature overnight and the organic layer separated. The aqueous layer was extracted with dichloromethane, and the combined organic layers were dried (MgSO4), filtered, and evaporated in vacuo to give an oil that was purified on silica (hexanes/EtOAc) to give 6-bromo-2-tert-butoxycarbonyloxy-4hydroxymethylindole-1-carboxylic acid tert-butyl ester 51 as a clear oil (125 mg, 48%). 1H NMR (400 MHz, CDCl3): δ 8.02 (d, J = 2.0 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 6.29 (s, 1H), 4.77 (s, 2H), 1.59 (s, 9H), 1.50 (s, 9H). Alcohol 51 (220 mg, 0.49 mmol), triphenylphosphine (156 mg, 0.59 mmol), benzimidazolone 35 (151 mg, 0.55 mmol), and THF (5 mL) were combined and the reaction flask cooled to 0 °C in an ice− water bath. Diisopropyl diazodicarboxylate (0.12 mL, 0.6 mmol) was added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 1 h. The solvent was evaporated in vacuo and the product purified on silica (DCM/methanol) to afford 6bromo-2-tert-butoxycarbonyloxy-4-[3-(1-ethoxycarbonylmethylbutyl)2-oxo-2,3-dihydrobenzoimidazol-1ylmethyl]indole-1-carboxylic acid tert-butyl ester 53 as a white solid (200 mg, 57%). Bis-Boc protected indolenone 53 (200 mg, 0.29 mmol) was dissolved in dichloromethane (5 mL) and cooled to 0 °C. Trifluoroacetic acid (0.25 mL) was added dropwise to the mixture, and the mixture was stirred at 0 °C

for 15 min. The solvents were evaporated in vacuo, and the crude product was purified using reverse phase HPLC to afford 3-[3-(6bromo-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]hexanoic acid methyl ester 55 as a white solid (17 mg, 85%). 1H NMR (400 MHz, CD3OD): δ 7.32 (m 1H), 7.22 (m, 2H), 6.95−7.02 (m, 3H), 5.07 (s, 2H), 4.84 (m, 1H), 3.98 (m, 2H), 3.42 (s, 2H), 3.28 (m, 1H), 2.87 (m, 1H), 2.25 (m, 1H), 1.81 (m, 1H), 1.28 (m, 2H), 1.01 (m, 3H), 0.95 (m, 3H). Ester 55 (80 mg, 0.16 mmol) was dissolved in 1,4-dioxane (3 mL) and water (2 mL), and solid lithium hydroxide (10 mg, 0.6 mmol) was added to the solution. The mixture was stirred for 1 h at room temperature and then acidified to pH 2 with 1 N HCl. The resulting precipitate was collected and purified using reverse phase HPLC to give 15 as a white solid (73 mg, 97%). LCMS (ES+) m/z found 472, 474; retention time 0.85 min. C22H22BrN3O4 requires 472, 474. HPLC, retention time 10.54 min. 1H NMR (400 MHz, DMSO-d6): δ 12.23 (bs, 1H), 10.54 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 6.95−7.08 (m, 3H), 6.86 (s, 1H), 6.76 (s, 1H), 4.98 (s, 2H), 4.72 (m, 1H), 3.44 (s, 2H), 3.06 (m, 1H), 2.85 (m, 1H), 2.10 (m, 1H), 1.70 (m, 1H), 1.12 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). 2-[3-(6-Bromo-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2oxo-2,3-dihydrobenzoimidazol-1-ylmethyl]hexanoic Acid (16). Alcohol 26 (620 mg, 1.9 mmol), triphenylphosphine (548 mg, 2.0 mmol), 2-(2-oxo-2,3-dihydrobenzoimidazol-1-ylmethyl)pentanoic acid ethyl ester 36 (557 mg, 2.1 mmol), and THF (6 mL) were combined and cooled to 0 °C in an ice−water bath. Diisopropyl diazodicarboxylate (0.41 mL, 2.0 mmol) was added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 1 h. The solvent was evaporated in vacuo and the product purified on silica 4475



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Scheme 4. Preparation of Fragment-Linked Chymase Inhibitor with Benzimidazole Corea

Reagents and conditions: (a) H2SO4, HNO3, 0 °C, 85%; (b) EtOH, H2SO4, reflux, 69%; (c) NaH, dimethylthiocarbamoyl chloride, 99%; (d) NMP, 100 °C, 56%; (e) DIBAL-H, −78 °C, THF, 87%; (f) triphenylphosphine, DIAD, THF, 0 °C, 41%; (g) SnCl2, MeOH, EtOAc, 21%; (h) aq LiOH, THF, 1,4-dioxane, 47%.

a

Scheme 5. Preparation of Fragment-Linked Chymase Inhibitors with Benzimidazole Corea

Reagents and conditions: (a) triphenylphosphine, DIAD, THF, 0 °C, 70%; (b) aq LiOH, THF, 1,4-dioxane, water, then aq HCl, 62%; (e) pyridinium tribromide, tert-butanol, 40 °C, then Zn (powder), HOAc, 7%.

a

bromo-1H-indol-4-ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1ylmethyl]pentanoic acid ethyl ester 40 as a white solid (400 mg, 74%). 1 H NMR (400 MHz, DMSO-d6): 11.34 (s, 1H), 7.51 (s, 1H), 7.38 (t, J = 2.8 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 1.6 Hz, 1H), 6.94−7.02 (m, 3H), 6.65 (m, 1H), 5.28 (d, J = 2.0, 2H), 3.94−4.12 (m, 2H), 3.84−3.95 (m, 2H), 2.92 (m, 1H), 1.20−1.64 (m, 4H), 0.95 (t, J = 7.2 Hz, 3H), 0.86 (t, J = 7.2 Hz, 3H).

(DCM/methanol) to afford 6-bromo-4-[3-(2-ethoxycarbonylpentyl)2-oxo-2,3-dihydrobenzoimidazol-1-ylmethyl]indole-1-carboxylic acid tert-butyl ester 38 as a white solid (637 mg, 57%). Protected indole 38 (650 mg, 1.1 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (4 mL) was added dropwise to the solution. The mixture was stirred at room temperature for 2 h, and then the solvent was evaporated in vacuo and the crude product purified using reverse phase HPLC to give 2-[3-(64476



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Indole 42 (200 mg, 0.4 mmol) was dissolved in tert-butanol (20 mL) and warmed to 40 °C. After 15 min, pyridinium tribromide (265 mg, 0.8 mmol) was added portionwise to the mixture, and the mixture was stirred for 30 min. The solvents were evaporated at 40 °C under high vacuum, and the resulting dibromide 42 was suspended in acetic acid (10 mL). Zinc dust (134 mg, 2 mmol) was added to the mixture, and the mixture was stirred for 2 h at room temperature. The reaction mixture was filtered, evaporated in vacuo, and purified using reverse phase HPLC to give 2-[3-(6-bromo-2-oxo-2,3-dihydro-1H-indol-4ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-ylmethyl]pentanoic acid ethyl ester 44 as a white solid (180 mg, 87%). Ester 44 (120 mg, 0.24 mmol) was dissolved in 1,4-dioxane (2 mL) and water (2 mL), and solid lithium hydroxide (80 mg, 3.5 mmol) was added to the solution. The mixture was stirred for 1 h at room temperature and then acidified to pH 2 with 1 N HCl. The resulting precipitate was collected and purified using reverse phase HPLC to afford 16 as a white solid (30 mg, 26%). LCMS (ES+) m/z found 472, 474; retention time 0.83 min. C22H22BrN3O4 requires 472, 474. HPLC, retention time 10.45 min. 1H NMR (400 MHz, DMSO-d6): δ 12.41 (bs, 1H), 10.54 (s, 1H), 7.20 (d, J = 8.0 Hz, 1H), 6.96−7.09 (m, 3H), 6.85 (s, 1H), 6.80 (s, 1H), 4.99 (s, 2H), 4.05 (dd, J = 14.4, 8.0 Hz, 1H), 3.94 (dd, J = 14.4, 8.0 Hz, 1H), 3.45 (s, 2H), 2.84 (m, 1H), 1.51 (m, 1H), 1.20−1.47 (m, 3H), 0.83 (t, J = 7.6 Hz, 3H). 3-[2-Oxo-3-(2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-2,3-dihydrobenzoimidazol-1-yl]hexanoic Acid (17). 1H-Indole-4-carboxylic acid methyl ester 22 (9 g, 51 mmol) was suspended in tertbutanol (90 mL) and heated to 40 °C. Pyridinium tribromide (52 g, 164 mmol) was added portionwise to the mixture over 10 min, and then the mixture was stirred for 2 h. The solvents were evaporated in vacuo, and the crude product was taken up in EtOAc and washed with water. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to give 3,3-dibromo-2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid methyl ester 46 as a red solid (16.5 g, 92%). 1H NMR (400 MHz, DMSO-d6): δ 11.46 (s, 1H), 7.60 (dd, J = 7.2, 0.8 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.18 (dd, J = 8.0, 1.2 Hz, 1H), 3.92 (s, 3H). Dibromide 46 (43g, 0.12 mmol) was suspended in acetic acid (1 L), and zinc powder (80g, 1.2 mmol) was added to the mixture over 5 min. The mixture was stirred for 3 h and then filtered though Celtie. The Celite was washed with EtOAc, and the combined washings were evaporated in vacuo to give an oil that was purified on silica (DCM/ methanol) to give 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid methyl ester 48 as a white solid (9 g, 39%). 1H NMR (400 MHz, DMSO-d6): δ 7.40 (dd, J = 8.0, 0.8 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 3.84 (s, 3H), 3.69 (s, 2H). Oxindole 48 (2 g, 10 mmol), BOC2O (6.8 g, 31 mmol), and DMAP (127 mg, 1 mmol) were dissolved in acetonitrile (52 mL), heated to 50 °C, and stirred for 3 h. The solvents were evaporated in vacuo and the crude material was purified on silica (hexanes/EtOAc) to give 2tert-butoxycarbonyloxyindole-1,4-dicarboxylic acid 1-tert-butyl ester 4methyl ester 50 (400 mg, 10%). 1H NMR (400 MHz, CDCl3): δ 8.24 (d, J = 8.0 Hz, 1H), 7.96 (dd, J = 7.6, 0.8 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.00 (d, J = 0.8 Hz, 1H), 3.95 (s, 3H), 1.66 (s, 9H), 1.57 (s, 9H). Ester 50 (480 mg, 1.2 mmol) was dissolved in THF (7 mL), and the reaction flask was cooled to −78 °C. DIBAL-H (1 M in heptanes, 6.1 mL, 6.1 mmol) was added dropwise to the mixture over 5 min. The mixture was stirred at 78 °C for 2 h, and then the reaction was quenched by the addition of 50 mL of saturated Rochelle’s salt. The mixture was warmed to room temperature, and the organic layer was separated. The aqueous layer was extracted with dichloromethane, and the combined organic layers were dried (MgSO4), filtered, and evaporated in vacuo to give an oil that was purified on silica (hexanes/ EtOAc) to give 2-tert-butoxycarbonyloxy-4-hydroxymethylindole-1carboxylic acid tert-butyl ester 52 as a clear oil (400 mg, 89%). 1H NMR (400 MHz, CDCl3): δ 7.95 (d, J = 8.0 Hz, 1H), 7.20−7.27 (m, 2H), 6.42 (d, J = 0.8 Hz, 1H), 4.87 (s, 2H), 1.66 (s, 9H), 1.57 (s, 9H). Alcohol 52 (166 mg, 0.45 mmol), triphenylphosphine (143 mg, 0.55 mmol), benzimidazolone 35 (126 mg, 0.48 mmol), and THF (2 mL) were combined and cooled to 0 °C in an ice−water bath. Diisopropyl diazodicarboxylate (0.11 mL, 0.55 mmol) was added dropwise to the mixture, and the mixture was warmed to room

temperature and stirred for 1 h. The solvents were evaporated in vacuo and the product was purified on silica (DCM/methanol) to afford 2tert-butoxycarbonyloxy-4-[3-(1-methoxycarbonylmethylbutyl)-2-oxo2,3-dihydrobenzoimidazol-1-ylmethyl]indole-1-carboxylic acid tertbutyl ester 54 as a white solid (40 mg, 15%). 1H NMR (400 MHz, CDCl3): δ 7.92 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 6.8 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 7.00 (td, J = 8.8, 1.2 Hz, 1H), 6.90 (dd, J = 8.8, 1.2 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.56 (s, 1H), 5.29 (s, 2H), 4.77 (m, 1H), 3.57 (s, 3H), 3.24 (dd, J = 12.0, 4.8 Hz, 1H), 2.87 (dd, J = 12.0, 2.0 Hz, 1H), 2.27 (m, 1H), 1.76 (m, 1H), 1.63 (s, 9H), 1.56 (s, 9H), 1.25 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H). Bis-Boc protected indolinone 54 was dissolved in DCM (5 mL) and cooled to 0 °C. Trifluoroacetic acid (0.05 mL) was added dropwise to the mixture, and the mixture was stirred at 0 °C for 15 min. The solvents were evaporated in vacuo and the crude product was purified using reverse phase HPLC to afford 3-[2-oxo-3-(2-oxo-2,3-dihydro1H-indol-4-ylmethyl)-2,3-dihydrobenzoimidazol-1-yl]hexanoic acid methyl ester 56 as a white solid (17 mg, 85%). 1H NMR (400 MHz, CDCl3): δ 8.95 (bs, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.09 (td, J = 8.4, 0.8 Hz, 1H), 7.00 (td, J = 8.8, 1.2 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.83 (t, J = 7.2 Hz, 2H), 5.05 (d, J = 5.2 Hz, 2H), 4.79 (m, 1H), 3.56 (s, 3H), 3.40 (d, J = 4.4 Hz, 2H), 3.26 (dd, J = 16.0, 9.6 Hz, 1H), 2.84 (dd, J = 16.0, 4.8 Hz, 1H), 2.20 (m, 1H), 1.78 (m, 1H), 1.25 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H). Ester 56 (18 mg, 0.11 mmol) was dissolved in 1,4-dioxane (1 mL) and water (1 mL), and solid lithium hydroxide (5 mg, 0.6 mmol) was added to the solution. The mixture was stirred for 1 h at room temperature and then acidified to pH 2 with 1 N HCl. The resulting precipitate was collected and purified using reverse phase HPCL to give 17 as a white solid (11 mg, 63%). LCMS (ES+) m/z found 394, retention time 0.76 min. C22H23N3O4 requires 394. HPLC, retention time 9.59 min. 1H NMR (400 MHz, CD3OD): δ 7.19 (d, J = 8.0 Hz,1H), 7.06 (t, J = 8.0 Hz, 1H), 6.99 (td, J = 7.6, 1.2 Hz, 1H), 6.90 (td, J = 7.6, 1.2 Hz, 1H), 6.82 (J = 8.0 Hz, 1H), 6.74 (d, J = 7.6 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 5.38 (s, 2H), 4.74 (m, 1H), 3.27 (s, 2H), 3.11 (dd, J = 15.6, 9.6 Hz, 1H), 2.77 (dd, J = 16.0, 5.2 Hz, 1 H), 2.12 (m, 1H), 1.70 (m, 1H), 1.20 (m, 2H), 0.82 (t, J=7.6 Hz, 3H)). 3-[3-(5-Bromo-2-oxo-2,3-dihydrobenzothiazol-7-ylmethyl)2-oxo-2,3-dihydrobenzoimidazol-1-yl]hexanoic Acid (18). Phenol 57 (23 g, 105 mmol) was suspended in sulfuric acid (70 mL) and cooled to 0 °C in an ice−water bath. A solution of nitric acid (6.6 mL) in sulfuric acid (15.4 mL) was added dropwise to the mixture over 1 h, and the temperature was monitored so that it did not rise above 10 °C. The ice bath was removed and the mixture stirred at room temperature for an additional 3 h, at which time it was slowly poured onto crushed ice. The resulting bright yellow solid was collected and dried in vacuo to afford 5-bromo-2-hydroxy-3-nitrobenzoic acid 58 as a yellow solid that was used without further purification (24.3 g, 85%). 1 H NMR (400 MHz, DMSO-d6): δ 8.29 (d, J = 2.4 Hz, 1H), 8.12 (d, J = 2.4 Hz, 1H). Acid 58 (26 g, 99 mmol) was suspended in ethanol (200 mL), and concentrated sulfuric acid (20 mL) was added to the suspension. The mixture was diluted with toluene (200 mL), equipped with a Dean− Stark apparatus, and heated to reflux for 72 h with azeotropic removal of water. The volatiles were removed in vacuo, and the resulting oil was taken up in ethyl acetate and washed with water followed by a saturated solution of sodium bicarbonate. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to afford 5-bromo-2hydroxy-3-nitrobenzoic acid ethyl ester 59 as a bright orange solid that was used without further purification (20 g, 69%). 1H NMR (400 MHz, DMSO-d6): δ 8.35 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 2.4 Hz, 1H), 4.36 (q, J= 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). Phenol 59 (5 g, 17 mmol) was dissolved in DMF (50 mL), and sodium hydride (40% in mineral oil, 760 mg, 18 mmol) was added to the solution (CAUTION: HYDORGEN GAS EVOLUTION). The mixture was stirred for an additional 10 min, and an additional 50 mL of DMF was added. Dimethylthiocarbamoyl chloride (2.3 g, 18 mmol) was added portionwise over 5 min to the mixture, and the resulting mixture was stirred overnight at room temperature. The reaction was quenched with water and extracted into EtOAc. The organic layer was 4477



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dried (MgSO4), filtered, and evaporated in vacuo to give 5-bromo-2dimethylthiocarbamoyloxy-3-nitrobenzoic acid ethyl ester 60 (6.4 g, 99%) that was used without further purification. 1H NMR (400 MHz, CDCl3): δ 8.34 (d, J = 2.4 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 4.36 (qd, J = 7.2 Hz, 2.0 Hz, 2H), 3.46 (s, 3H), 3.41 (s, 3H), 1.37 (t, J = 7.6 Hz, 3H). Thiocarbamate 60 (6.2 g, 16 mmol) was dissolved in NMP (60 mL) and the mixture heated to 100 °C for 6 h. The mixture was cooled to room temperature and poured into ether/EtOAc and the organic layer washed with water and brine. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to give 5-bromo-2-dimethylcarbamoylsulfanyl-3-nitrobenzoic acid ethyl ester 62 (3.5g, 56%) as a orange solid that was used without further purification. 1H NMR (400 MHz, CDCl3): δ 8.12 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 3.11 (s, 3H), 2.98 (s, 3H), 1.38 (t, J = 7.2 Hz, 3H). Ester 62 (2 g, 5.3 mmol) was dissolved in THF (12 mL) and ether (12 mL), and the mixture was cooled to 0 °C in an ice bath. Borane− dimethyl sulfide complex (1 M in THF, 11 mL, 11 mmol) was added dropwise to the solution, and the mixture was allowed to warm to room temperature over 1 h. The mixture was heated to 45 °C for 3 h and then cooled to 0 °C and quenched by the addition of saturated aqueous ammonium chloride. The product was extracted into EtOAc and the organic layer dried (MgSO4) filtered, and evaporated in vacuo to give an oil which was purified on silica (DCM/MeOH) to give dimethylthiocarbamic acid 4-bromo-2-hydroxymethyl-6-nitrophenyl ester 63 (1.5 g 87%) as a tan solid. 1H NMR (400 MHz, CD3OD): δ 8.19 (d, J = 2.4 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 4.58 (s, 2H), 3.04 (s, 3H), 2.89 (s, 3H). Alcohol 63 (300 mg, 0.9 mmol), triphenylphosphine (258 mg, 0.9 mmol), benzimidazolone 64 (272 mg, 0.9 mmol), and THF (6 mL) were combined and cooled to 0 °C in an ice−water bath. Diisopropyl diazodicarboxylate (0.19 mL, 0.9 mmol) was added dropwise to the mixture, and the mixture was warmed to room temperature and stirred for 1 h. The solvent was evaporated in vacuo and the product purified on silica (DCM/methanol) to give 2-[3-(5-bromo-2-dimethylcarbamoylsulfanyl-3-nitrobenzyl)-2-oxo-2,3-dihydrobenzoimidazol-1ylmethyl]pentanoic acid ethyl ester 65 (220 mg, 41%) as a white solid. Nitroaryl 65 (240 mg, 0.4 mmol) was suspended in methanol (10 mL) and ethyl acetate (10 mL), and tin(II) chloride (456 mg, 2.0 mmol) was added in one portion. The resulting suspension was heated to 75 °C for 1 h and cooled to room temperature, and the solvents were evaporated in vacuo. The resulting solid was taken up in EtOAc and the suspension filtered though Celite. The filtrates were evaporated in vacuo and the product was purified using reverse phase HPLC to give 2-[3-(5-bromo-2-oxo-2,3-dihydrobenzothiazol-7ylmethyl)-2-oxo-2,3-dihydrobenzoimidazol-1-ylmethyl]pentanoic acid ethyl ester 66 as a white solid (45 mg, 21%). 1H NMR (400 MHz, DMSO-d6): δ 12.14 (s, 1H), 7.31 (d, J = 1.6 Hz, 1H), 7.20 (d, J = 5.6 Hz, 1H), 7.19 (s, 1H), 7.07 (t, J = 7.6 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 5.10 (d, J = 2.4 Hz, 2H), 4.09 (dd, J = 14.0, 8.4 Hz, 1H), 3.88−4.00 (m, 3H), 2.92 (m, 1H), 1.56 (m, 2H), 1.29 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H), 0.86 (J = 7.2 Hz, 3H). Ester 66 (45 mg, 0.1 mmol) was dissolved in 1,4-dioxane (1 mL) and water (1 mL) as lithium hydroxide was added to the solution (20 mg, 0.9 mmol). The mixture was stirred for 1 h at room temperature and acidified to neutral pH with 1 N HCl. The solvents were evaporated in vacuo and the product was purified using reverse phase HPLC to give 18 as a white solid (20 mg, 47%). LCMS (ES+) m/z found 490, 492; retention time 0.87 min. C21H20BrN3O4S requires 490, 492. HPLC, retention time 10.71 min. 1H NMR (400 MHz, DMSO-d6): δ 12.47 (s, 1H), 11.80 (s, 1H), 7.43 (s, 1H), 7.36−6.91 (m, 5H), 5.17 (s, 2H), 5.10 (m, 1H), 4.07 (dd, J = 14.4, 7.6 Hz, 1H), 3.96 (dd, J = 14.4, 7.6 Hz, 1H), 2.85 (m, 1H), 1.61−1.18 (m, 4H), 0.84 (t, J = 7.2 Hz, 3H). 4-[1-(6-Chloro-2-oxo-2,3-dihydro-1H-indol-4-ylmethyl)-1Hbenzoimidazol-2-ylsulfanyl]butyric Acid (19). Alcohol 25 (300 mg, 1.2 mmol), triphenylphosphine (391 mg, 1.5 mmol), and 4-(1Hbenzoimidazol-2-ylsulfanyl)butyric acid ethyl ester 65 (465 mg, 1.7 mmol) were dissolved in THF (8 mL) and cooled to 0 °C. The reaction mixture was stirred for 15 min followed by the addition of

DIAD (0.29 mL, 1.5 mmol). The mixture was stirred for an additional 30 min, then evaporated in vacuo and purified on slica (hexanes/ EtOAc) to afford 6-chloro-4-[2-(3-ethoxycarbonylpropylsulfanyl)benzoimidazol-1-ylmethyl]indole-1-carboxylic acid tert-butyl ester 66 as a white solid (460 mg, 70%). Ester 66 (370 mg, 0.7 mmol) was dissolved in THF (2 mL) and water (2 mL), and solid lithium hydroxide (84 mg, 3.5 mmol) was added to the solution in one portion. The resulting solution was stirred at room temperature for 16 h and acidified to pH 2 with 6 M HCl. The resulting solution was stirred for 1 h, and then the product was extracted into EtOAc. The combined organic layers were dried (MgSO4), filtered, and evaporated in vacuo to give 4-[1-(6-chloro-1Hindol-4-ylmethyl)-1H-benzoimidazol-2-ylsulfanyl]butyric acid 67 as a clear solid that was used without further purification (175 mg, 62%). 1 H NMR (400 MHz, CD3OD): δ 7.64 (d, J = 8.0 Hz, 1H), 7.35 (bs, 1H), 7.16−7.03 (m, 4H), 6.51 (m, 1H), 6.47 (dd, J = 3.2, 1.2 Hz, 1H), 5.70 (s, 2H), 3.38 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.30 (m, 2H). Indole 67 (220 mg, 0.56 mmol) was suspended in t-BuOH (1 mL) and THF (2 mL) and warmed to 40 °C. Pyridinium tribromide (571 mg, 1.7 mmol) was added portionwise to the mixture over 5 min and the resulting solution stirred for 5 h. The reaction mixture was poured into water/EtOAc, and the organic layer was washed with water and brine. The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to give an oil that was taken up in 12 mL of acetic acid. Zinc powder (360 mg, 5.5 mmol) was added to the mixture and the suspension stirred for 15 min and then filtered though Celite . The Celite was washed with EtOAc, and the combined washings were evaporated in vacuo to give an oil that was purified using reverse phase HPLC to afford 19 as a white solid (16 mg, 7%). LCMS (ES+) m/z found 416, retention time 0.70 min. C20H18ClN3O3S requires 416. HPLC, retention time 7.79 min. 1H NMR (400 MHz, CD3OD): 7.54 (d, J = 7.6 Hz,1H), 7.22 (d, J = 7.2 Hz, 1H), 7.11−7.19 (m, 2H), 6.74 (s, 1H), 6.39 (s, 1H), 5.31 (s, 2H), 3.29 (t, J = 7.2 Hz, 2H), 3.16 (s, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.93 (m, 2H). Biological Assays. Chymase and Cathepisn G Enzyme Inhibition. Chymase was assayed in a 15 μL volume containing 500 pM recombinant human chymase and 100 nM rhodamine 110, bis(succinoyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanylamide) (American Peptide) in a buffer containing 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 100 μM TCEP, 0.01% CHAPS, and 1% DMSO for 1 h at 28 °C/80% humidity, and fluorescence was read at 485 nm excitation and 530 nm emission. Cathepsin G was assayed as described for chymase in 15 μL containing 4 nM cathepsin G (Athens Research and Technology) and 300 nM chymase substrate in a buffer containing 1× phosphate-buffered saline, 10 mM HEPES, pH 7.4, 100 μM TCEP, 0.01% CHAPS, and 1% DMSO. Reactive Metabolite Formation. To assess reactive metabolite formation, compound 1 (50 μM) was added to human liver microsomes (1 mg/mL) in phosphate buffer (100 mM, pH 7.4). Pooled human liver microsomes were purchased from BD Biosciences (San Jose, CA). After preincubation at 37 °C for 3 min, the reaction was initiated by the addition of NADPH (2.5 mM). The final incubation volume was 1 mL. Samples without compound or NADPH were used as negative controls. After incubation for 2 h, samples were quenched with 2 mL of acetonitrile. Samples were centrifuged at 2095g for 5 min, and the supernatant was transferred to a glass tube and the solvent was evaporated using a Caliper TurboVap LV (Hopkinton,MA). Samples were redissolved in 500 μL of water/acetonitrile (1:1, v/v) and analyzed by mass spectrometry for the detection of glutathione adducts. A glutathione adduct of compound 1 was detected after analysis of the microsomal incubate. X-ray Crystallographic Methods: Crystallization, Soaking, and Cryofreezing. Purified chymase (5 mg/mL) in storage buffer of 10 mM MOPS, pH 6.8, 50 mM NaCl, 5 mM DTT was used for setting up crystallization trays. Protein was mixed in 1:1 ratio and equilibrated in a hanging drop vapor diffusion setup with a well solution of 26− 33% PEG8000, 0.1 M Tris, pH 8.0, 2 mM ZnSO4 at room temperature. Mature crystals, grown in 5 days, were harvested and incubated in a soaking buffer of 30% PEG8000, 0.1 M Tris, pH 8.0, 2 4478



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Table 3. Summary of X-ray Data Collection and Refinement Statistics compound wavelength (Å) resolution range (Å)a space group unit cell dimentions a, b, c (Å) α, β, γ (deg) total reflections unique reflectionsa multiplicitya completeness (%)a mean I/σ(I) a Wilson B-factor Rmerge a,b Rwork a,c Rfree a no. of atoms macromolecules ligands waters protein residues rms bonds (Å) rms angles (deg) Ramachandran favored (%) Ramachandran outliers (%) clashscore average B-factor macromolecules solvent PDB code

2

3

11

15

1.5418 20.64−2.30 (2.38−2.30) P43

0.9099 33.37−1.50 (1.55−1.50) P43

1.5418 74.61−1.80 (1.86−1.80) P43

1.5418 33.26−1.50 (1.55−1.50) P43

74.41, 74.41, 49.70 90, 90, 90 39 447 11 117 (1125) 3.54 (3.46) 90.76 (91.84) 5.50 (2.50) 25.86 0.153 (0.441) 0.178 (0.214) 0.251 (0.291) 1922 1689 26 207 217 0.007 1.07 98 0 7.70 15.00 14.10 20.50 4K2Y

74.62, 74.62, 49.74 90, 90, 90 265 131 43 872 (4385) 6.04 (5.98) 99.87 (100.00) 10.82 (2.81) 15.45 0.077 (0.426) 0.181 (0.294) 0.201 (0.298) 2117 1753 26 338 219 0.006 1.11 98 0 5.37 19.40 17.30 29.60 4K60

74.61, 74.61, 49.90 90, 90, 90 87 854 24 996 (2554) 3.43 (2.64) 97.57 (99.96) 11.46 (2.56) 20.78 0.079 (0.28) 0.223 (0.261) 0.263 (0.326) 1968 1719 26 223 220 0.008 1.15 99 0 7.46 25.60 24.70 31.10 4K5Z

74.37, 74.37, 49.47 90, 90, 90 123 950 43 089 (4241) 2.87 (2.21) 99.25 (98.06) 11.26 (2.60) 15.59 0.060 (0.239) 0.197 (0.214) 0.223 (0.244) 2021 1696 46 279 215 0.006 1.11 99 0 4.35 20.20 18.70 28.60 4K69

Statistics for the highest-resolution shell are shown in parentheses. bRmerge = ∑hkl∑i|I(hkl)i − ⟨I(hkl)⟩|/∑hkl∑i⟨I(hkl)i⟩. cRwork = ∑hkl∑i|Fo(hkl)i − Fc(hkl)|/∑hklFo(hkl), where Fo and Fc are observed and calculated structure factors, respectively. a

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mM ZnSO4, and 10 mM or 2 mM compound. Soaked crystals were harvested in 3−5 days for data collection by plunge freezing in liquid nitrogen. Data Collection, Refinement, and Deposition. Diffraction data were collected either using an in-house X-ray source from Rigaku FR-E generator with mar345 imageplate/Saturn92 CCD detector system or using synchrotron Swiss Light Source PXI beamline with marCCD detector under standard cryogenic conditions. For compound 3, a single wavelength anomalous dispersion experiment was conducted at an X-ray wavelength 0.91 Å near the bromine K edge absorption peak. Data were reduced using d*TREK. Structures of ligand cocomplexes were determined by using in-house determined apo-chymase as a starting model and calculating difference Fourier synthesis map to determine the electron density for the ligand. Iterative rounds of manual model building using COOT25 combined with refinement using CNX and PHENIX26 generated final models. All crystal structure figures were generated using PyMOL. The cocomplex structure of chymase with compound 2 was determined at a resolution limit of 2.30 Å and refined to Rfree/Rwork statistics of 0.25/0.18. The cocomplex structure of chymase with compound 3 was determined at a resolution limit of 1.50 Å and refined to Rfree/Rwork statistics of 0.20/ 0.18. The cocomplex structure of chymase with compound 11 was determined at a resolution limit of 1.80 Å and refined to Rfree/Rwork statistics of 0.26/0.22. The cocomplex structure of chymase with compound 15 was determined at a resolution limit of 1.50 Å and refined to Rfree/Rwork statistics of 0.22/0.19. Atomic coordinates and experimental structure factors for all four costructure complexes discussed in this manuscript have been deposited at the Protein Data Bank (PDB, www.rcsb.org). The accession codes along with the X-ray data collection and refinement statistics are tabulated in Table 3.

AUTHOR INFORMATION

Corresponding Author

*Phone: 203-791-6371. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Drs. Neil Farrow and Ho-Yin Lo for their careful reading of the manuscript. ABBREVIATIONS USED Cat-G, cathepsin G; Chy, chymase; HTS, high throughput screening; ALI, alternative lead identification; FBS, fragment based screening; MW, molecular weight; AMU, atomic mass units; clogP, calculated partition coefficient in octanol/water; NMR, nuclear magnetic resonance; SPR, surface plasmon resonance; MSS, muscular skeletal syndrome; STD-NMR, saturation transfer difference nuclear magnetic resonance; SECMS, size exclusion chromatography mass spectrometry; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; LE, ligand efficiency −RT ln(IC50)/(no. heavy atoms); PAMPA, parallel artificial membrane permeability assay; PSA, polar surface area; DMF, dimethylformamide; GSH, glutathione; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium; TBTU, O-(benzo4479



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Expert. Opin. Ther. Targets 2011, 15, 519−527. (c) Guo, X.; Man, C. C.; Takahashi, H. Preparation of Azaquinazolinediones Useful as Therapeutic Chymase Inhibitors. WO2010088195, 2010. (d) Emmanuel, M. J.; Guo, X.; Kim, J. M.; Lo, H. Y.; Nemoto, P. A.; Qian, K. C. Preparation of Aza-benzimidazolone Compounds as Chymase Inhibitors for Therapeutic Applications. WO2010030500, 2010. (e) Hawkins, M. J.; Greco, M. N.; Powell, E.; Garavilla, L.; Maryanoff, B. E. Preparation of Phosphonic Acid and Phosphinic Acid Compounds as Novel Chymase Inhibitors. US20100048513, 2010. (f) Abeywardane, A.; Hao, M.-H.; Taylor, S. J. Preparation of 1Oxo-1H-phthalazine Compounds As Chymase Inhibitors for Therapeutic Applications. WO2010019417, 2010. (g) Abeywardane, A.; Cook, B. N.; De Lombaert, S.; Emmanuel, M. J.; Guo, X.; Kim, J. M.; Hao, M.-H.; Lo, H. Y.; Man, C. C.; Morwick, T.; et al.et al. Benzimidazolone Derivatives as Chymase Inhibitors and Their Preparation, Pharmaceutical Compositions and Use in the Treatment of Diseases. WO2008147697, 2008. (h) Banner, D.; Mauser, H.; Minder, R. E.; Wessel, H. P. Preparation of Sulfonamide Derivatives as Chymase Inhibitors. US20080167348, 2008. (i) Banner, D.; Hilpert, H.; Kuhn, B.; Mauser, H. Preparation of Hydroxyoxocyclobutenylbenzylindoles and Related Compounds as Chymase Inhibitors. WO2008043698, 2008. (j) Tsuchiya, N.; Mizuno, T.; Saitou, H.; Matsumoto, Y.; Takeuchi, S.; Hase, N. Preparation of Thiobenzimidazole Derivatives as Chymase Inhibitors. US20050267148, 2005. (k) Hoover, D. J. Preparation of Peptide Inhibitors of Angiotensin I chymase(s) Including Human Heart Chymase. WO9325574 and US 8193214, 1993. (8) Hoashi, T.; Matsumiya, G.; Miyagawa, S.; Ichikawa, H.; Ueno, T.; Ono, M.; Saito, A.; Shimizu, T.; Okano, T.; Kawaguchi, N.; Matsuura, N.; Sawa, Y. Chymase plays an important role in left ventricular remodeling induced by intermittent hypoxia in mice. Hypertension 2009, 54, 164−171. (9) Kanemitsu, H.; Takai, S.; Tsuneyoshi, H.; Nishina, T.; Yoshikawa, K.; Miyazaki, M.; Ikeda, T.; Komeda, M. Chymase inhibition prevents cardiac fibrosis and dysfunction after myocardial infarction in rats. Hypertens Res. 2006, 29, 57−64. (10) Kanemitsu, H.; Takai, S.; Tsuneyoshi, H.; Yoshikawa, E.; Nishina, T.; Miyazaki, M.; Ikeda, T.; Komeda, M. European results with a continuous-flow ventricular assist device for advanced heartfailure patients. Eur. J. Cardiol. Surg. 2008, 33, 25−31. (11) Palaniyandi, S. S.; Nagai, Y.; Watanabe, K.; Ma, M.; Veeraveedu, P. T.; Prakash, P.; Kamal, F. A.; Abe, Y.; Yamaguchi, K.; Tachikawa, H.; Kodama, M.; Aizawa, Y. Chymase inhibition reduces the progression to heart failure after autoimmune myocarditis in rats. Exp. Biol. Med. (Maywood, NJ, U. S.) 2007, 232, 1213−1221. (12) Michael, N.; Greco, M. J.; Hawkins, E. T.; Powell, H. R.; Almond, L. G.; Jeffrey, H.; Minor, L. K.; Yuanping, W.; Corcoran, T. W.; Di Cera, E.; Cantwell, A. M.; Savvides, S. N.; Damiano, B. P.; Maryanoff, B. E. Discovery of potent, selective, orally active, nonpeptide inhibitors of human mast cell chymase. J. Med. Chem. 2007, 50, 1727−1730. (13) (a) Refer to home page of Teijin Pharma Limited: www.teijinpharma.co.jp./English/. (b) Tsuchiya, N.; Mizuno, T.; Saitou, H.; Matsumoto, Y.; Takeuchi, S.; Hase, N. Preparation of Thiobenzimidazole Derivatives as Chymase Inhibitors. US20050267148, 2005. (c) Yajima, N.; Hiroki, Y.; Yoshino, H.; Koizumi, T. Preparation of 3Hydroxymethylbenzo[b]thiophene Derivatives. WO2007049812, 2007. (14) Lo, H. Y.; Nemoto, P. A.; Kim, J.-M.; Hao, M.-H.; Qian, K. C.; Farrow, N. A.; Albaugh, D. A.; Fowler, D. M.; Schneiderman, R. D.; August, E. M.; Martin, L.; Hill-Drzewi, M.; Pullen, S. S.; Takahashi, H.; De Lombaert, S. Benzimidazolone as potent chymase inhibitor: modulation of reactive metabolite formation in the hydrophobic (P1) region. Bioorg. Med. Chem. Lett. 2011, 21, 4533−4539. (15) Walsh, J. S.; Miwa, G. T. Bioactivation of drugs: risk and drug design. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 145−167. (16) Arooj, M.; Thangapandian, S.; John, S.; Hwang, S.; Park, J. K.; Lee, K. W. 3D QSAR pharmacophore modeling, in silico screening,

triazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; DIEA, N,N-diisopropylethylamine; THF, tetrahydrofuran; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; SEM-Cl, 2(trimethylsilyl)ethoxymethyl chloride; TBAF, tetra-n-butylammonium fluoride; HPLC, high performance liquid chromatography; BNC, bovine nasal cartilage; h/r LM, human/rat liver microsome stability; DIBAL-H, diisobutylaluminum hydride; BOC2O, di-tert-butyl dicarbonate; DMAP, 4-dimethylaminopyridine; DIAD, diisopropyl azodicarboxylate; LiOH, lithium hydroxide; EtOAc, ethyl acetate; PMA, phosphomolybdic acid; MeOH, methanol; HOAc, acetic acid; TFA, trifluoroacetic acid; SAR, structure−activity relationship; compd no., compound number; PDB, protein data bank

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REFERENCES

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Discovery of Potent, Selective Chymase Inhibitors via Fragment

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