Structure-Based Discovery of Novel Amide-Containing Nicotinamide

Jul 16, 2013 - Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) p...
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Structure-Based Discovery of Novel Amide-Containing Nicotinamide Phosphoribosyltransferase (Nampt) Inhibitors Xiaozhang Zheng,*,† Paul Bauer,† Timm Baumeister,† Alexandre J. Buckmelter,† Maureen Caligiuri,† Karl H. Clodfelter,† Bingsong Han,† Yen-Ching Ho,† Nikolai Kley,† Jian Lin,† Dominic J. Reynolds,† Geeta Sharma,† Chase C. Smith,† Zhongguo Wang,† Peter S. Dragovich,‡ Janet Gunzner-Toste,‡ Bianca M. Liederer,‡ Justin Ly,‡ Thomas O’Brien,‡ Angela Oh,‡ Leslie Wang,‡ Weiru Wang,‡ Yang Xiao,‡ Mark Zak,‡ Guiling Zhao,‡ Po-wai Yuen,§ and Kenneth W. Bair† †

Forma Therapeutics, Inc., 500 Arsenal Street, Watertown, Massachusetts 02472, United States Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States § Pharmaron Beijing, Co. Ltd., 6 Tai He Road, BDA Beijing, 100176, P. R. China ‡

S Supporting Information *

ABSTRACT: Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (7, Nampt BC IC50 = 9.0 nM, A2780 cell proliferation IC50 = 10 nM). The Nampt−7 cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of multiple cancer cell lines (IC50 100fold loss of biological activities. The weak biological activities exhibited by 11 may result from its inability to form the corresponding PRPP adduct, although we did not confirm this possibility via mass spectrometry.16 Introducing an additional nitrogen atom into the azaindole system drastically diminished biological activity (12), likely due to ionization of the new heterocycle under physiological conditions, thus causing an electrostatically unfavorable interaction with Asp219 (cf., Figure 4; JChem predicts an NH pKa value of 7.7 for compound 12 and its tautomeric form as compared to 10.8 for compound 8). Similarly, methylation of the pyrrole nitrogen of 8, which was expected to disrupt a hydrogen bonding interaction with the side chain of Asp219 (cf., Figure 4), led to significant loss of biological activities (compare to 13). In contrast, the Nmethylated compound 14 exhibited slightly improved biochemical activity as compared with the unmethylated analog 11. This result was consistent with an altered Nampt binding mode for 11 relative to 8 which did not involve formation of a hydrogen bond between the azaindole NH of 11 and Asp219 (cf., Figure 4).16 Not unexpectedly, changing the amide attachment location to the 3-position of the azaindole, and thereby likely significantly altering the positioning of this moiety relative to that observed in the 7−Nampt cocrystal structure, was also highly detrimental to biological activity (compare 15 with 8). Satisfied with the potent biochemical and cellular activity exhibited by the azaindole-containing compounds 7 and 8, we explored incorporation of other fused heterobiaryl systems into the amide-based inhibitor design. We were particularly F

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liver microsomes consistently indicated that biaryl sulfones exhibited improved stability properties relative to the corresponding piperidine-derived phenyl-sulfonamides (e.g., compare 17 with 18). The biaryl sulfone motif was therefore also incorporated into many subsequent inhibitor designs. As a prelude to conducting in vivo efficacy experiments, we next focused our inhibitor optimization efforts on identifying highly potent cell-active compounds which also exhibited attractive in vitro DMPK properties. This work was initially conducted using the azaindole present in 8 with the expectation that promising modifications so identified could be transferred to compounds containing other fused heterocycles. Previous work with related urea-containing molecules indicated that alterations to the biarylsulfone portion of the inhibitors could greatly impact potency and biopharmaceutical properties.9,10 Similar modifications were expected to be tolerated in the amide-containing Nampt inhibitor series, since the biarylsulfone terminus was anticipated to be highly solvent-exposed (cf., Figure 4). As shown in Table 3, derivitization of the terminal phenyl ring contained in 8 afforded a variety of compounds that all displayed potent biochemical and cell-based anti-Nampt properties (41−51). Among these molecules, those bearing terminal 3-Me-Ph (42), 3-CF3-Ph (44), 3,5-di-F-Ph (49), and 4-morpholino-3-pyridyl (51) substituents exhibited A2780 cell IC50 values of less than 10 nM. The latter three compounds also showed good in vitro stability toward both murine and human liver microsomes. Those three moieties were therefore chosen for inclusion in amide-containing Nampt inhibitors bearing nonazaindole fused heterocycles. As shown in Table 4, all such combinations successfully afforded very potent cellactive Nampt inhibitors, and most of these compounds exhibited good microsomal stability properties (exceptions: 52, 57, 59, and 62). Crystallographic rationalizations for the structure−activity relationships depicted in Tables 3 and 4 are provided below. To enable further optimization of the described amidecontaining Nampt inhibitors, a cocrystal structure of compound 58 in complex with Nampt was solved (Figure 5). The binding mode of the ligand was very similar to that observed for compound 7 with a hydrogen bond noted between the amide NH and Asp219. A water-mediated (W1) hydrogen bond was also observed between the amide carbonyl and Ser275. The bicyclic imidazopyridine ring of 58 was stacked between the side chains of Nampt residues Tyr18′ and Phe193 in the NAM binding region with the imidazole nitrogen pointing toward the PRPP binding site. Interestingly, this nitrogen atom was positioned in a location that was very similar to that observed for the pyridyl N-atom contained in 7 (Figure 5). This positioning was maintained in spite of the differing connectivities between the bicyclic rings present in 7 and 58 and the associated carboxamide moieties of two molecules, and it may reflect an optimal orientation for the formation of the corresponding PRPP adducts in the Nampt active site.15,17 Consistent with this hypothesis, the locations of the exposed heteroaryl N-atoms present in 7 and 58 were also very similar to those observed crystallographically for the pyridine nitrogen of the NMN product that results from Nampt-catalyzed condensation of NAM with PRPP (cf., Figure 1).18 Many of the atoms present in the bicyclic heterocycles contained in 7 and 58 were observed in close spatial proximity (95% for all final compounds, as assessed by LCMS analysis at UV 220 nm. Details regarding the LCMS conditions can be found in the Supporting Information. N-[4-(Piperidine-1-sulfonyl)phenyl]-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (6). To a suspension of 1H-pyrrolo[3,2c]pyridine-2-carboxylic acid (65a, 81 mg, 0.50 mmol) in DMF (8 mL) were added HATU (285 mg, 0.75 mmol) and EtN(i-Pr)2 (0.131 mL, 0.749 mmol), and the resulting mixture was stirred for 25 min at room M

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9.27 (m, 2H), 9.13−9.09 (m, 1H), 7.96−7.88 (m, 4H), 7.85−7.72 (m, 2H), 7.69−7.53 (m, 5H), 4.54 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.64 min, m/z = 393.1 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}isoquinoline-6-carboxamide (36). Substituting the corresponding reagents with isoquinoline-6-carboxylic acid (65l) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 96% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.40 (s, 2H), 8.58 (s, 1H), 8.47 (s, 1H), 8.22−8.18 (m, 1H), 8.05−8.02 (m, 1H), 7.98−7.82 (m, 5H), 7.70−7.50 (m, 5H), 4.60 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.15 min, m/z = 403.1 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}isoquinoline-7-carboxamide (37). Substituting the corresponding reagents with isoquinoline-7-carboxylic acid (65m) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 37% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.40 (s, 2H), 8.68 (s, 1H), 8.57 (s, 1H), 8.20 (d, J = 8.6 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.98−7.82 (m, 5H), 7.70−7.50 (m, 5H), 4.60 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.16 min, m/z = 403.1 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}quinazoline-6-carboxamide (38). Substituting the corresponding reagents with quinazoline-6-carboxylic acid (65n) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 15% yield. 1H NMR (300 MHz, CD3OD) δ: 9.61 (br s, 1H), 9.31 (s, 1H), 8.60 (d, J = 2.1 Hz, 1H), 8.43 (d, J = 2.4 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.07 (d, J = 9.3 Hz, 1H), 7.95 (s, 2H), 7.92 (s, 2H), 7.62−7.51 (m, 5H), 4.69 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.77 min, m/z = 404.1 [M + H]+. N-({4-[3-(Trifluoromethyl)benzenesulfonyl]phenyl}methyl)1H-pyrrolo[3,2-c]pyridine-2-carboxamide (44). Substituting the corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i), the title product was obtained in 25% yield. 1H NMR (300 MHz, DMSO-d6) δ: 12.35 (s, 1H), 9.37 (t, J = 6.0 Hz, 1H), 9.05 (s, 1H), 8.28−8.21 (m, 3H), 8.10−8.01 (m, 3H), 7.85 (t, J = 6.0 Hz, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 6.0 Hz, 1H), 7.39 (s, 1H), 4.58 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.73 min, m/z = 460.2 [M + H]+. N-{[4-(4-Fluorobenzenesulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (47). Substituting the corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and (4-((4-fluorophenyl)sulfonyl)phenyl)methanamine (64k), the title product was obtained in 26% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.40 (s, 1H), 9.10 (s, 1H), 8.29 (d, J = 5.9 Hz, 1H), 8.16 (s, 1H), 8.07−7.92 (m, 5H), 7.58−7.40 (m, 6H), 4.57 (s, 2H). LCMS (method LCMS1, ESI): RT = 0.99 min, m/z = 410.17 [M + H]+. N-{[4-(3,5-Difluorobenzenesulfonyl)phenyl]methyl}-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (49). Substituting the corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and (4-((3,5-difluorophenyl)sulfonyl)phenyl)methanamine (64h), the title product was obtained in 39% yield. 1H NMR (300 MHz, DMSO-d6) δ: 12.0 (s, 2H), 9.24 (s, 1H), 8.90 (s, 1H), 8.20 (d, J = 5.8 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.80−7.50 (m, 5H), 7.38− 7.23 (m, 2H), 4.60 (s, 2H). LCMS (method LCMS1, ESI) RT = 1.03 min, m/z = 428.0 [M + H]+. N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)methyl]-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (51). Substituting the corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2carboxylic acid (65a) and (4-((6-morpholinopyridin-3-yl)sulfonyl)phenyl)methanamine (64j), the title product was obtained in 57% yield. 1H NMR (300 MHz, DMSO-d6) δ: 12.10 (br s, 1H), 9.28 (s, 1H), 8.89 (d, J = 1.0 Hz, 1H), 8.56 (d, J = 2.2 Hz, 1H), 8.17 (d, J = 5.8 Hz, 1H), 7.90−7.84 (m, 3H), 7.52 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 5.8 Hz, 1H), 7.25 (s, 1H), 6.88 (d, J = 9.2 Hz, 1H), 4.54 (d, J = 4.7 Hz, 2H), 3.65−3.55 (m, 8H). LCMS (method LCMS3, ESI): RT = 4.78 min, m/z = 478.1 [M + H]+. N-{[4-(3,5-Difluorobenzenesulfonyl)phenyl]methyl}imidazo[1,2-a]pyridine-6-carboxamide (58). Substituting the corresponding reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h22) and (4-((3,5-difluorophenyl)sulfonyl)phenyl)methanamine (64h), the title product was obtained in 57% yield. 1H NMR (300 MHz, DMSO-

(m, 4H), 1.68−1.58 (m, 4H), 1.45−1.38 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.89 min, m/z = 400.0 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}thieno[2,3-b]pyridine-2-carboxamide (22). Substituting the corresponding reagents with thieno[2,3-b]pyridine-2-carboxylic acid (65d) and (4(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 62% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.52 (t, J = 6.0 Hz, 1H), 8.66−8.63 (m, 1H), 8.37 (d, J = 3.3 Hz, 1H), 8.13 (s, 1H), 7.77−7.64 (m, 2H), 7.61−7.55 (m, 2H), 7.51−7.47 (m, 1H), 4.60 (d, J = 6.0 Hz, 2H), 2.87−2.82 (m, 4H), 1.52−1.47 (m, 4H), 1.35−1.31 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.40 min, m/z = 416.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}thieno[3,2-b]pyridine-2-carboxamide (23). Substituting the corresponding reagents with thieno[3,2-b]pyridine-2-carboxylic acid (65e) and (4(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained with 22.1% yield. 1H NMR (300 MHz, CDCl3) δ: 8.72 (d, J = 4.5 Hz, 1H), 8.27−8.19 (m, 1H), 8.04 (s, 1H), 7.98−7.90 (m, 2H), 7.86−7.83 (m, 2H), 7.73−7.43 (m, 5H), 7.34−7.31 (m, 1H), 7.16 (t, J = 5.4 Hz, 1H), 4.70 (d, J = 6.0 Hz, 2H). LCMS (LCMS10, ESI): RT = 1.74 min, m/z = 409.0 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1,3-benzothiazole-6-carboxamide (25). Substituting the corresponding reagents with benzo[d]thiazole-6-carboxylic acid (65f) and (4-(piperidin-1ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 83% yield. 1H NMR (300 MHz, CDCl3) δ: 9.12 (s, 1H), 8.56 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.99−7.94 (m, 1H), 7.65−7.60 (m, 2H), 7.49−7.43 (m, 2H), 7.18−7.10 (m, 1H), 4.76 (d, J = 6.0 Hz, 2H), 2.97−2.91 (m, 4H), 1.66−1.57 (m, 4H), 1.44−1.35 (m, 2H). LCMS (method LCMS1, ESI): RT = 2.18 min, m/z = 416.0 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1H-1,3-benzodiazole-5-carboxamide (26). Substituting the corresponding reagents with 1H-benzo[d]imidazole-6-carboxylic acid (65g) and (4(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 77% yield. 1H NMR (300 MHz, CDCl3) δ: 8.18 (br s, 1H), 8.03 (s, 1H), 7.73−7.66 (m, 2H), 7.62−7.56 (m, 2H), 7.45−7.39 (m, 2H), 4.69 (d, J = 6.0 Hz, 2H), 2.94−2.87 (m, 4H), 1.63−1.53 (m, 4H), 1.42−1.32 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.81 min, m/z = 399.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,2-a]pyridine-6-carboxamide (28). Substituting the corresponding reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h22) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 79% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.19 (t, J = 6.0 Hz, 1H), 9.12 (s, 1H), 8.04 (s, 1H), 7.95−7.89 (m, 4H), 7.69−7.52 (m, 8H), 4.53 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.69 min, m/z = 391.9 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,2-a]pyridine-7-carboxamide (31). Substituting the corresponding reagents with imidazo[1,2-a]pyridine-7-carboxylic acid (65i) and (4(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 66% yield. 1H NMR (300 MHz, CDCl3) δ: 8.23−8.19 (m, 1H), 8.16 (s, 1H), 7.74−7.65 (m, 4H), 7.53−7.49 (m, 2H), 7.39− 7.35 (m, 2H), 4.78 (d, J = 6.0 Hz, 2H), 3.00−2.95 (m, 4H), 1.68−1.59 (m, 4H), 1.47−1.38 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.81 min, m/z = 399.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,2-a]pyrimidine-6-carboxamide (33). Substituting the corresponding reagents with imidazo[1,2-a]pyrimidine-6-carboxylic acid (65j) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 3% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.45 (s, 1H), 9.33 (t, J = 5.4 Hz, 1H), 8.91 (d, J = 2.4 Hz, 1H), 8.00 (s, 1H), 7.94− 7.90 (m, 4H), 7.79 (s, 1H), 7.68−7.56 (m, 5H), 4.55 (d, J = 5.7 Hz, 1H). LCMS (method LCMS1, ESI): RT = 0.99 min, m/z = 393.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}-[1,2,4]triazolo[4,3a]pyridine-6-carboxamide (35). Substituting the corresponding reagents with [1,2,4]triazolo[4,3-a]pyridine-6-carboxylic acid (65k) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 20% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.35− N

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d6) δ: 9.20 (s, 1H), 9.10 (s, 1H), 8.06−7.96 (m, 3H), 7.75 (s, 2H), 7.75−7.50 (m, 5H), 4.60 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.03 min, m/z = 428.04 [M + H]+. N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)methyl]imidazo[1,2-a]pyridine-6-carboxamide (60). Substituting the corresponding reagents with imidazo[1,2-a]pyridine-6carboxylic acid (65h22) and (4-((6-morpholinopyridin-3-yl)sulfonyl)phenyl)methanamine (64j), the title product was obtained in 82% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.22−9.16 (m, 1H), 9.13− 9.11 (m, 1H), 8.56 (d, J = 2.3 Hz, 1H), 8.12 (s, 1H), 8.04 (s, 1H), 7.90−7.85 (m, 2H), 7.66−7.50 (m, 5H), 6.89 (d, J = 9.2 Hz, 1H), 4.52 (d, J = 5.9 Hz, 2H), 3.65−3.56 (m, 8H). LCMS (method LCMS3, ESI): RT = 4.63 min, m/z = 478.1 [M + H]+. N-{[4-(3,5-Difluorobenzenesulfonyl)phenyl]methyl}imidazo[1,2-a]pyrimidine-6-carboxamide (61). Substituting the corresponding reagents with imidazo[1,2-a]pyrimidine-6-carboxylic acid (65j) and (4-((3,5-difluorophenyl)sulfonyl)phenyl)methanamine (64h), the title product was obtained in 66% yield. 1H NMR (300 MHz, DMSO-d6) δ: 1H NMR (300 MHz, DMSO-d6) δ: 9.48 (d, J = 2.4 Hz, 1H), 9.38 (t, J = 6.0 Hz, 1H), 8.93 (d, J = 2.4 Hz, 1H), 8.03− 7.99 (m, 3H), 7.80 (d, J = 1.5 Hz, 1H), 7.77−7.72 (m, 2H), 7.69−7.60 (m, 3H), 4.58 (d, J = 5.7 Hz, 2H). LCMS (method LCMS3, ESI) RT = 5.64 min, m/z = 429.0 [M + H]+. N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)methyl]imidazo[1,2-a]pyrimidine-6-carboxamide (63). Substituting the corresponding reagents with imidazo[1,2-a]pyrimidine-6carboxylic acid (65j) and (4-((6-morpholinopyridin-3-yl)sulfonyl)phenyl)methanamine (64j), the title product was obtained in 75% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.46 (d, J = 2.5 Hz, 1H), 9.34 (t, J = 5.6 Hz, 1H), 8.92 (d, J = 2.5 Hz, 1H), 8.57 (d, J = 2.6 Hz, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.91−7.86 (m, 3H), 7.79 (d, J = 1.5 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 9.3 Hz, 1H), 4.55 (d, J = 5.7 Hz, 2H), 3.65−3.55 (m, 8H). LCMS (method LCMS3, ESI): RT = 4.94 min, m/z = 478.9 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}-1-methyl-1Hpyrrolo[2,3-c]pyridine-2-carboxamide (14). A solution of 1methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (65o,23 200 mg, 1.14 mmol), EDCI (400 mg, 2.09 mmol), HOBt (300 mg, 2.22 mmol), (4-(phenylsulfonyl)phenyl)methanamine (64o, 500 mg, 2.02 mmol), and EtN(i-Pr)2 (0.8 g 12.5 mmol) in DMF (50 mL) was stirred at room temperature for 2 days. The resulting mixture was concentrated under vacuum. The residue was first purified on a silica gel column eluted with CH2Cl2/MeOH (10:1). The semipurified product (300 mg) was repurified by Flash-Prep-HPLC using the following conditions (IntelFlash-1): column, silica gel; mobile phase, CH3CN/H2O = 1 5% increasing to CH3CN/H2O = 64% within 30 min; detector, UV 254 nm to yield the title product as an off-white solid in 4% yield. 1H NMR (300 MHz, DMSO-d6) δ: 8.97 (s, 1H), 8.19 (d, J = 5.7 Hz, 1H), 7.96−7.93 (m, 4H), 7.68−7.56 (m, 6H), 7.15 (s, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.06 (s, 3H). LCMS (LCMS4, ESI): RT = 1.48 min, m/z = 406.0 [M + H]+. Compounds 15, 27, 30, 39−40, 52, 54, 59, and 62 were synthesized using conditions (EDCI/HOBt coupling) similar to those employed to make compound 14. N-{[4-(Benzenesulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]pyridine-3-carboxamide (15). Substituting the corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-3-carboxylic acid (65p) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 15% yield. 1H NMR (300 MHz, DMSO-d6): δ 11.96 (s, 1H), 9.31 (s, 1H), 8.74 (t, J = 6.0 Hz, 1H), 8.35−8.22 (m, 1H), 8.14 (s, 1H), 7.94 (d, J = 5.7 Hz, 4H), 7.70−7.56 (m, 5H), 7.43 (d, J = 5.7 Hz, 1H), 4.54 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.55 min, m/z = 391.9 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,2-a]pyridine-6-carboxamide (27). Substituting the corresponding reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h22) and (4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 76% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.23 (s, 1H), 9.16 (s, 1H), 8.07 (s, 1H), 7.71−7.54 (m, 7H), 4.59 (d, J = 6.0 Hz, 1H), 2.86−2.81 (m, 4H), 1.51 (br s, 4H), 1.32 (br s, 2H).

LCMS (method LCMS1, ESI): RT = 1.64 min, m/z = 399.2 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,5-a]pyridine-7-carboxamide (30). Substituting the corresponding reagents with imidazo[1,5-a]pyridine-7-carboxylic acid (65q) and (4(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 11% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.15 (t, J = 5.9 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.34 (d, J = 7.5 Hz, 1H), 8.18 (s, 1H), 7.76−7.51 (m, 5H), 7.08−7.05 (m, 1H), 4.53 (d, J = 6.0 Hz, 2H), 2.81 (t, J = 5.2 Hz, 4H), 1.59−1.45 (m, 4H), 1.40−1.19 (m, 2H). LCMS (LCMS4, ESI): RT = 1.43 min, m/z = 399.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}-5-(pyridin-3-yl)-1Hpyrazole-3-carboxamide (39). Substituting the corresponding reagents with 3-(pyridin-3-yl)-1H-pyrazole-5-carboxylic acid (65r) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 29% yield. 1H NMR (300 MHz, DMSO-d6) δ: 12.8 (s, 1H), 9.00 (s, 1H), 8.95 (s, 1H), 8.18 (s, 1H), 8.00−7.80 (m, 4H), 7.70−7.40 (m, 7H), 7.20 (s, 1H), 4.50 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.13 min, m/z = 419.1 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}-3-(pyridin-3-yl)-1,2oxazole-5-carboxamidee (40). Substituting the corresponding reagents with 3-(pyridin-3-yl)isoxazole-5-carboxylic acid (65s) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 40% yield. 1H NMR (300 MHz, CDCl3): δ 9.05 (d, J = 2.1 Hz, 1H), 8.72−8.71 (m, 1H), 8.12−8.06 (m, 1H), 7.93 (dd, J = 7.5, 1.8 Hz, 4H), 7.57−7.47 (m, 6H), 7.37 (d, J = 6.0 Hz, 1H), 7.07 (s, 1H), 4.70 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.93 min, m/z = 420.06 [M + H]+. N-({4-[3,5-Difluorobenzenesulfonyl]phenyl}methyl)furo[2,3c]pyridine-2-carboxamide (52). Substituting the corresponding reagents with furo[2,3-c]pyridine-2-carboxylic acid (65t) and (4-((3,5difluorophenyl)sulfonyl)phenyl)methanamine (64h), the title product was obtained in 51% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.65 (t, J = 6.0 Hz, 1H), 9.09 (s, 1H), 8.50 (d, J = 6.0 Hz, 1H), 8.04 (d, J = 9.0 Hz, 2H), 7.62 (m, 7H), 4.59 (d, J = 6.0 Hz, 2H). LCMS (LCMS11, ESI): RT = 1.92 min, m/z = 429.2 [M + H]+. N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)methyl]furo[2,3-c]pyridine-2-carboxamide (54). Substituting the corresponding reagents with furo[2,3-c]pyridine-2-carboxylic acid (65t) and (4-((6-morpholinopyridin-3-yl)sulfonyl)phenyl)methanamine (64j), the title product was obtained in 26% yield. 1H NMR (300 MHz, DMSO-d6): δ 9.60 (m, 1H), 9.01 (s, 1H), 8.48 (m, 1H), 8.44 (m, 1H), 7.90−7.79 (m, 4H), 7.61 (s, 1H), 7.55 (m, 1H), 6.85 (d, J = 8.4 Hz, 1H), 4.51 (d, J = 6.0 Hz, 2H), 3.64−3.48 (m, 8H). LCMS (method LCMS3, ESI): RT = 1.63 min, m/z = 478.98 [M + H]+. N-({4-[3-(Trifluoromethyl)benzenesulfonyl]phenyl}methyl)imidazo[1,2-a]pyridine-6-carboxamide (59). Substituting the corresponding reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h 22 ) and (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i), the title product was obtained in 46% yield. 1H NMR (300 MHz, DMSO-d6): δ 9.20 (t, J = 6.0 Hz, 1H), 9.13−9.12 (m, 1H), 8.28−8.23 (m, 2H), 8.09−8.00 (m, 4H), 7.86 (t, J = 7.8 Hz, 1H), 7.67−7.57 (m, 5H), 4.56 (d, J = 6.0 Hz, 2H). LCMS (method LCMS3, ESI): RT = 5.72 min, m/z = 460.25 [M + H]+. N-({4-[3-(Trifluoromethyl)benzenesulfonyl]phenyl}methyl)imidazo[1,2-a]pyrimidine-6-carboxamide (62). Substituting the corresponding reagents with imidazo[1,2-a]pyrimidine-6-carboxylic acid (65j) and (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i), the title product was obtained in 23% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.46 (d, J = 2.4 Hz, 1H), 9.35 (t, J = 2.6 Hz, 1H), 8.92 (d, J = 2.4 Hz, 1H), 8.25−8.23 (m, 2H), 8.06−8.00 (m, 4H), 7.86−7.79 (m, 2H), 7.60 (d, J = 8.4 Hz, 2H), 4.57 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 5.88 min, m/z = 461.0 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}furo[2,3-c]pyridine-2-carboxamide (17). A solution of (4-(piperidin-1ylsulfonyl)phenyl)methanamine (64n, 500 mg, 1.93 mmol), furo[2,3-c]pyridine-3-carboxylic acid (65t, 355 mg, 2.13 mmol), EtN(iPr)2 (762 mg, 5.79 mmol), and BOP (1044 mg, 2.31 mmol) in DMF (5 mL) was stirred overnight at room temperature. The reaction was O

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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7.65 (m, 1H), 7.51−7.45 (m, 2H), 7.39−7.34 (m, 2H), 4.69 (d, J = 6.0 Hz, 2H), 2.95−2.85 (m, 4H), 1.65−1.55 (m, 4H), 1.42−1.33 (m, 2H). LCMS (method LCMS1, ESI) RT = 2.10 min, m/z = 416.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}thieno[2,3-c]pyridine-2-carboxamide (20). Substituting the corresponding reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e24) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 85% yield. 1H NMR (300 MHz, DMSO-d6) δ 9.60−9.55 (m, 1H), 9.29 (s, 1H), 8.50 (d, J = 6.0 Hz, 1H), 8.15 (s, 1H), 7.95− 7.89 (m, 5H), 7.68−7.54 (m, 5H), 4.55 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI) RT = 1.65 min, m/z = 409.0 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}thieno[3,2-c]pyridine-2-carboxamide (21). Substituting the corresponding reagents with methyl thieno[3,2-c]pyridine-2-carboxylate (66f24) and (4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 39% yield. 1H NMR (300 MHz, CDCl3) δ 8.99 (s, 1H), 8.48 (d, J = 6.0 Hz, 1H), 8.00 (s, 1H), 7.82−7.74 (m, 2H), 7.54− 7.49 (m, 2H), 7.42−7.37 (m, 2H), 4.70 (d, J = 6.0 Hz, 2H), 2.94−2.88 (m, 4H), 1.65−1.55 (m, 4H), 1.43−1.34 (m, 2H). LCMS (method LCMS1, ESI) RT = 2.07 min, m/z = 416.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}-[1,3]thiazolo[5,4-c]pyridine-2-carboxamide (2). Substituting the corresponding reagents with methyl thiazolo[5,4-c]pyridine-2-carboxylate (66g) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 48% yield. 1H NMR (300 MHz, DMSO-d6) δ 10.01 (t, J = 6.0 Hz, 1H), 9.50 (s, 1H), 8.73−8.70 (m, 1 H), 8.11−8.07 (m, 1H), 7.95−7.89 (m, 4H), 7.69−7.54 (m, 5 H), 4.55 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI) RT = 1.91 min, m/z = 410.2 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,5-a]pyridine-6-carboxamide (29). Substituting the corresponding reagents with methyl imidazo[1,5-a]pyridine-6-carboxylate (66h22) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained with 28% yield. 1H NMR (300 MHz, DMSO-d6) δ: 8.71 (s, 1H), 8.26 (s, 1H), 7.92−7.84 (m, 4H), 7.60−7.45 (m, 7H), 7.05− 6.97 (m, 2H), 4.69 (d, J = 6.3 Hz, 2H). LCMS (LCMS4, ESI): RT = 1.36 min, m/z = 392.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,2-a]pyrazine-6-carboxamide (34). Substituting the corresponding reagents with methyl imidazo[1,2-a]pyrazine-6-carboxylate (66b) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 28% yield. 1H NMR (400 MHz, DMSO-d6): δ 9.45 (m, 1H), 9.26 (d, J = 1.2 Hz, 1H), 9.10 (s, 1H), 8.29 (s, 1H), 7.95− 7.91 (m, 5H), 7.66 (d, J = 7.2 Hz, 1H), 7.63−7.55 (m, 4H), 4.56, 4.56 (d, J = 6.4 Hz, 2H). LCMS (LCMS4, ESI): RT = 2.08 min, m/z = 393.0 [M + H]+. N-(4-(3-(Trifluoromethyl)phenylsulfonyl)benzyl)furo[2,3-c]pyridine-2-carboxamide (53). Substituting the corresponding reagents with ethyl furo[2,3-c]pyridine-2-carboxylate (66d) and (4((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i), the title product was obtained in 60% yield. 1H NMR (300 MHz, DMSOd6) δ: 8.90 (s, 1H), 8.52−8.47 (m, 1H), 8.20 (s, 1H), 8.16−8.10 (m, 1H), 8.00−7.92 (m, 2H), 7.70−7.60 (m, 2H), 7.58−7.50 (m, 3H), 7.25 (s, 1H), 7.20 (s, 1H), 4.76−4.73 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.35 min, m/z = 460.99 [M + H]+. N-{[4-(3,5-Difluorobenzenesulfonyl)phenyl]methyl}thieno[2,3-c]pyridine-2-carboxamide (55). Substituting the corresponding reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e24) and (4-((3,5-difluorophenyl)sulfonyl)phenyl)methanamine (64h), the title product was obtained in 76% yield. 1H NMR (300 MHz, DMSOd6) δ: 9.60 (s, 1H), 9.30 (s, 1H), 8.50 (s, 1H), 8.17 (s, 1H), 8.00−7.82 (m, 2H), 7.90 (s, 1H), 7.75−7.64 (m, 2H), 7.64−7.52 (m, 3H), 4.60 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.31 min, m/z = 445.02 [M + H]+. N-({4-[3-(Trifluoromethyl)benzenesulfonyl]phenyl}methyl)thieno[2,3-c]pyridine-2-carboxamide (56). Substituting the corresponding reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e 24 ) and ((4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i), the title product was obtained in 55% yield. 1H NMR (300 MHz, DMSO-d6): δ 9.58 (t, J = 6.0 Hz, 1H), 9.29 (s, 1H), 8.51 (d, J = 5.4 Hz, 1H), 8.27−8.22 (m, 2H), 8.15 (s, 1H), 8.08−8.01 (m, 3H), 7.92−7.83 (m, 2H), 7.59 (d, J = 8.7 Hz, 2H), 4.57 (d, J = 5.7

quenched by the addition of 20 mL of water, and the precipitate was collected by filtration. The solid was purified by recrystallization from ethanol to give the title product as a light brown solid in 40% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.65 (t, J = 6.0 Hz, 1H), 9.06 (s, 1H), 8.49 (d, J = 3.0 Hz, 1H), 7.84 (t, J = 3.0 Hz, 1H), 7.70 (d, J = 4.0 Hz, 3H), 7.58 (d, J = 6.0 Hz, 2H), 4.60 (d, J = 6.0 Hz, 2H), 2.89−2.84 (m, 4H), 1.56−1.51 (m, 4H), 1.35−1.34 (m, 2H). LCMS (LCMS10, ESI): RT = m/z = 400.0 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,2-a]pyrimidine-6-carboxamide (32). This compound was synthesized using conditions (BOP coupling) similar to those employed to make compound 17. Substituting the corresponding reagents with imidazo[1,2-a]pyrimidine-6-carboxylic acid (65j) and (4-(piperidin-1ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 50% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.52 (d, J = 2.4 Hz, 1H), 9.39 (t, J = 5.6 Hz, 1H), 8.98 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 1.2 Hz, 1H), 7.83 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 4.63 (d, J = 6.0 Hz, 2H), 2.88−2.85 (m, 4H), 1.53−1.51 (m, 4H), 1.36−1.34 (m, 2H). LCMS (LCMS5, ESI): RT = 1.88 min, m/z = 400.0 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}-3H-imidazo[4,5-c]pyridine-2-carboxamide (12). To a cooled solution (water bath) of (4-(phenylsulfonyl)phenyl)methanamine (64o, 154 mg, 0.621 mmol) in toluene (20 mL) was added AlMe3 (1.13 mL, 2.26 mmol), and the mixture was stirred for 40 min at room temperature. A solution of methyl 3H-imidazo[4,5-c]pyridine-2-carboxylate (66a, 100 mg, 0.564 mmol) in toluene (10 mL) was then added via syringe. The flask was heated to 80 °C for 4 h, during which the solution became dark yellow with heavy precipitate. The mixture was cooled to room temperature, quenched with saturated Na−K tartrate (25 mL), and diluted with EtOAc (25 mL) and water (20 mL). The mixture was stirred vigorously for 1 h and then was filtered. The biphasic filtrate layers were separated, and the organic layer was dried (MgSO4), filtered, and concentrated to afford a yellow solid. Purification by preparative HPLC [Waters Autopurification MS-directed HPLC prep fraction collection with the following conditions: Column, Xbridge C18 RP 18, 5 μm, 19*50 mm; flow rate 20 mL/min; mobile phase, water with 0.1% ammonium hydroxide (A) and methanol with 0.1% ammonium hydroxide (B) running the following gradient 0 to 2 min (15%B), 2 to 6 min (15−100%B); detector ZQ Mass Detector in electrospray ionization mode] afforded 14 mg (6.3%) of the title compound. 1H NMR (300 MHz, DMSO-d6) δ: 8.30 (s, 1H), 8.15 (s, 1H), 7.95−7.85 (m, 5H), 7.65−7.50 (m, 6H), 7.32−7.27 (m, 1H), 4.90 (s, 2H). LCMS (method LCMS1, ESI): RT = 0.92 min, m/z = 393.02 [M + H]+. Compounds 13, 18−21, 24, 29, 34, 53, and 55−57 were synthesized using conditions (AlMe3-mediated amide coupling) similar to those employed to make compound 12. N-{[4-(Benzenesulfonyl)phenyl]methyl}-1-methyl-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (13). Substituting the corresponding reagents with methyl 1-methyl-1H-pyrrolo[3,2-c]pyridine-2-carboxylate (66c) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 92% yield. 1H NMR (300 MHz, DMSO-d6) δ: 9.26 (s, 1H), 8.91 (s, 1H), 8.28 (s, 1H), 7.94−7.92 (m, 4H), 7.67−7.53 (m, 6H), 7.27 (s, 1H), 4.52 (s, 2H), 3.32 (s, 3H). LCMS (method LCMS1, ESI): RT = 0.93 min, m/z = 406.01 [M + H]+. N-{[4-(Benzenesulfonyl)phenyl]methyl}furo[2,3-c]pyridine2-carboxamide (18). Substituting the corresponding reagents with ethyl furo[2,3-c]pyridine-2-carboxylate (66d) and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was obtained in 68% yield. 1H NMR (300 MHz, DMSO-d6): δ 9.60 (s, 1H), 9.00 (s, 1H), 8.45−8.40 (m, 1H), 7.96−7.88 (m, 4H), 7.80−7.76 (m, 1H), 7.70− 7.50 (m, 6H), 4.52 (d, J = 5.24 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.11 min, m/z = 392.97 [M + H]+. N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}thieno[2,3-c]pyridine-2-carboxamide (19). Substituting the corresponding reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e20) and (4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product was obtained in 37% yield. 1H NMR (300 MHz, CDCl3) δ 9.13 (s, 1H), 8.49 (d, J = 6.0 Hz, 1H), 7.93 (s, 1H), 7.83−7.75 (m, 1H), 7.70− P

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Hz, 2H). LCMS (method LCMS3, ESI): RT = 6.32 min, m/z = 477.22 [M + H]+. N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)methyl]thieno[2,3-c]pyridine-2-carboxamide (57). Substituting the corresponding reagents with methyl thieno[2,3-c]pyridine-2carboxylate (66e24) and (4-((6-morpholinopyridin-3-yl)sulfonyl)phenyl)methanamine (64j), the title product was obtained in 32% yield. 1H NMR (300 MHz, DMSO-d6) δ 9.57 (t, J = 6.0 Hz, 1H), 9.30 (s, 1H), 8.57−8.55 (m, 1H), 8.51 (d, J = 6.0 Hz, 1H), 8.15 (s, 1H), 7.93−7.85 (m, 4 H), 7.53 (d, J = 6.0 Hz, 2H), 6.88 (d, J = 6.0 Hz, 1H), 4.54 (d, J = 6.0 Hz, 2 H), 3.65−3.55 (m, 8H). LCMS (method LCMS3, ESI) RT = 5.15 min, m/z = 495.0 [M + H]+. N-{[4-(2-Methylbenzenesulfonyl)phenyl]methyl}-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (41). tert-Butyl 4-(2Methylbenzenesulfonyl)benzylcarbamate (69a). To a glass vial was added sodium 4-(((tert-butoxycarbonyl)amino)methyl)benzenesulfinate (67, 0.2 M in DMSO, 400 μL, 80 μmol), 2methylbenzeneboronic acid (68a, 0.2 M in 1,4-dioxane, 600 μL, 120 μmol), Et3N (50 μL, 360 μmol), and Cu(OAc)2 (0.2 M in DMSO, 500 μL, 100 μmol). The vial was capped and heated at 65 °C for 16 h on a heater/shaker. The reaction was cooled to RT, and brine (2 mL) was added followed by concentrated NH4OH (100 μL) and 3:1 EtOAc/CH2Cl2 (3 mL). The layers were separated, and the organic layer was transferred to a new vial. The aqueous layer was extracted twice with 3:1 EtOAc/CH2Cl2 (2 mL), and the combined organic layers were evaporated to dryness in a Genevac evaporator. The crude title compound was obtained as a dark brown solid, which was used in the next step without further purification. (4-(2-Methylbenzenesulfonyl)phenyl)methanamine Hydrochloride (64a). MeOH (500 μL) was added to the vial containing crude 69a (80 μmol), and the mixture was heated to 50 °C for 15 min to dissolve the solid. The solution was cooled to RT, HCl (4.0 M in 1,4dioxane, 400 μL, 1.6 mmol) was added, and the vial was shaken at RT for 30 min and then at 50 °C for 3 h. The solvent was evaporated to dryness in a Genevac evaporator. The light brown solid thus obtained was used in the next step without further purification. N-{[4-(2-Methylbenzenesulfonyl)phenyl]methyl}-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (41). To the vial containing crude 64a was added DMA (250 μL), Et3N (67 μL, 480 μmol), 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a, 0.2 M in DMA, 440 μL, 88 μmol), and BOP (0.2 M in DCE, 440 μL, 88 μmol). The mixture was heated at 50 °C for 4 h and then was stirred at room temperature for 16 h. Brine (1.5 mL), NaOH (1 M in water, 100 μL, 100 μmol), and 3:1 EtOAc/CH2Cl2 (2 mL) were added, and the layers were separated. The aqueous layer was extracted twice with 3:1 EtOAc/CH2Cl2 (2 mL), and the combined organic layers were evaporated to dryness in a Genevac evaporator. The residue was purified by preparative HPLC [Waters Autopurification MS-directed HPLC prep fraction collection with the following conditions: column, Xbridge C18 RP 18, 5 μm, 19*50 mm; flow rate 20 mL/min; mobile phase, water with 0.1% ammonium hydroxide (A) and methanol with 0.1% ammonium hydroxide (B) running the following gradient 0 to 2 min (15%B), 2 to 6 min (15−100%B); detector ZQ Mass Detector in electrospray ionization mode] to give the title compound as a white powder with 6.4% yield in 3 steps. LCMS (method LCMS2, ESI): RT = 0.96 min, m/z = 406.1 [M + H]+. Compounds 42−43, 45−46, 48, and 50 were synthesized using conditions similar to those employed to make compound 41. N-{[4-(3-methylbenzenesulfonyl)phenyl]methyl}-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (42). Substituting the corresponding reagent with 3-methylbenzeneboronic acid (68b), the title product was obtained in 4% yield in 3 steps. 1H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 9.24 (t, J = 6.0 Hz, 1H), 8.92 (d, J = 1.0 Hz, 1H), 8.22 (d, J = 5.8 Hz, 1H), 7.96−7.91 (m, 2H), 7.77−7.74 (m, 1H), 7.74−7.70 (m, 1H), 7.59−7.53 (m, 2H), 7.50−7.46 (m, 2H), 7.36 (dt, J = 5.8, 1.1 Hz, 1H), 7.29 (s, 1H), 4.57 (d, J = 5.9 Hz, 2H), 2.37 (s, 3H). LCMS (method LCMS2, ESI): RT = 0.98 min, m/z = 406.1 [M + H]+. N-({4-[2-(Trifluoromethyl)benzenesulfonyl]phenyl}methyl)imidazo[1,2-a]pyrimidine-6-carboxamide (43). Substituting the

corresponding reagent with (2-(trifluoromethyl)phenyl)boronic acid (68c), the title product was obtained in 7% yield in 3 steps. LCMS (method LCMS2, ESI): RT = 0.96 min, m/z = 460.1 [M + H]+. N-({4-[4-(Trifluoromethyl)benzenesulfonyl]phenyl}methyl)1H-pyrrolo[3,2-c]pyridine-2-carboxamide (45). Substituting the corresponding reagent with (4-(trifluoromethyl)phenyl)boronic acid (68d), the title product was obtained in 11% yield in 3 steps. 1H NMR (400 MHz, DMSO-d6): δ 12.61 (br s, 1H), 9.45 (t, J = 6.0 Hz, 1H), 9.16 (s, 1H), 8.31 (d, J = 6.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 2H), 8.00− 7.97 (m, 4H), 7.60−7.57 (m, 3H), 7.47 (s, 1H), 4.59 (d, J = 5.2 Hz, 2H). LCMS (method LCMS2, ESI): RT = 0.96 min, m/z = 460.3 [M + H]+. N-{[4-(3-Fluorobenzenesulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (46). Substituting the corresponding reagent with 3-fluorobenzeneboronic acid (68e), the title product was obtained in 11% yield in 3 steps. 1H NMR (400 MHz, DMSO-d6) δ 12.01 (s, 1H), 9.26 (t, J = 6.0 Hz, 1H), 8.93 (d, J = 1.2 Hz, 1H), 8.22 (d, J = 5.8 Hz, 1H), 8.02−7.95 (m, 2H), 7.83−7.76 (m, 2H), 7.71− 7.63 (m, 1H), 7.62−7.52 (m, 3H), 7.36 (dt, J = 5.8, 1.1 Hz, 1H), 7.30 (s, 1H), 4.59 (d, J = 5.9 Hz, 2H). LCMS (method LCMS2, ESI): RT = 0.95 min, m/z = 410.0 [M + H]+. N-{[4-(3,4-Difluorobenzenesulfonyl)phenyl]methyl}-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (48). Substituting the corresponding reagent with 3,4-difluorobenzeneboronic acid (68f), the title product was obtained in 8% yield in 3 steps. LCMS (method LCMS2, ESI): RT = 1.00 min, m/z = 428.1 [M + H]+. N-{[4-(pyridine-3-sulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (50). Substituting the corresponding reagent with pyridin-3-ylboronic acid (68g), the title product was obtained in 5% yield in 3 steps. LCMS (method LCMS2, ESI) RT = 0.69 min, m/z = 393.1 [M + H]+. N-(3-Aminopyridin-4-yl)-2,2,2-trichloroacetamide (72). Pyridine-3.4-diamine (70, 2.5 g, 22.91 mmol) was dissolved in CF3COOH (5.3 mL, 68.7 mmol). Methyl 2,2,2-trichloroacetimidate (71, 4.45 g, 25.2 mmol) was added over 30 min at 0 °C. The mixture was then stirred at room temperature for 2 days. The mixture was filtered, and the solid was washed with diethyl ether and dried to afford 5.7 g of desired product as a white solid in 98% yield. 1H NMR (300 MHz, DMSO-d6) δ: 7.80 (d, J = 5.4 Hz, 1H), 7.70 (s, 1H), 7.06 (s, 1H), 6.60 (d, J = 5.4 Hz, 1H), 5.33 (s, 2H). Methyl 3H-Imidazo[4,5-c]pyridine-2-carboxylate (66a). A mixture of H2O (1.21 mL, 67.2 mmol), Na2CO3 (7.12 g, 67.2 mmol), and 72 (5.7 g, 22.40 mmol) in MeOH (80 mL) was heated at reflux overnight. The mixture was cooled to room temperature, diluted with EtOAc, washed with saturated NaHCO3, dried, and concentrated. Biotage purification of the residue (10% MeOH/CH2Cl2) afforded 100 mg of the title product in 3% yield. 1H NMR (300 MHz, CD3OD) δ: 8.97 (s, 1H), 8.34 (s, 1H), 7.23 (s, 1H), 4.10 (s, 3H). 6-Bromoimidazo[1,2-a]pyrazine (75). To a solution of 5bromopyrazin-2-amine (73, 20 g, 114.9 mmol) and 2-bromo-1,1dimethoxyethane (74, 29 g, 171.6 mmol) in EtOH (500 mL) maintained under argon was added 40% HBr (50 mL). The reaction mixture was stirred at 80 °C for 12 h and then cooled to room temperature. The mixture was concentrated under vacuum to remove the excess EtOH. The pH value of the solution was adjusted to 9 with 1 N NaOH. The solution was extracted with 3 × 100 mL of EtOAc. The organic layers were combined and then washed with 2 × 300 mL of water and 1 × 500 mL of brine. The organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified on a silica gel column eluted with 45% EtOAc in petroleum ether to give the title product as a light-yellow solid in 15% yield. LCMS (LCMS4, ESI): RT = 0.94 min, m/z = 197.9 [M + H]+. Imidazo[1,2-a]pyrazine-6-carbonitrile (76). A mixture of 75 (2 g, 10.10 mmol), Zn(CN)2 (1.4 g, 12.2 mmol), and Pd(PPh3)4 (700 mg, 0.61 mmol) in DMF (30 mL) was stirred under nitrogen at 120 °C for 12 h. The solid was removed by filtration, and the filtrate was diluted with 200 mL of water. The resulting solution was extracted with 3 × 100 mL of CH2Cl2. The combined organic layers were washed with 2 × 300 mL of brine, were dried over anhydrous sodium sulfate, and were concentrated under vacuum. The residue was purified Q

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

4-(4-Fluorophenylsulfonyl)benzonitrile (81). To a solution of 4-fluorobenzonitrile (79, 5.98 g, 49.4 mmol) in 20 mL of DMSO at 120 °C was added dropwise of sodium 4-fluorobenzenesulfinate (80, 3 g, 16.47 mmol) in 20 mL of DMSO. The resulting mixture was stirred at 120 °C overnight and then was cooled to room temperature. 400 mL of ice water was added into the reaction mixture, and the resulting precipitate was filtered, washed with water (2 × 30 mL), and dried. Biotage purification with EtOAc/hexanes afforded 1.30 g of the title product in 30% yield. 1H NMR (300 MHz, DMSO-d6) δ: 8.04−8.26 (m, 6H), 7.44−7.52 (m, 2H). (4-(4-Fluorophenylsulfonyl)phenyl)methanamine (64k). To a solution of 81 (2.3 g, 8.8 mmol) in 150 mL of 2 N NH3/MeOH was added Raney Ni (500 mg), and the mixture was hydrogenated on a Parr apparatus (45 psi) overnight. Nitrogen was bubbled through the crude mixture to purge any residual H2, and the mixture was filtered through Celite, rinsing with MeOH (2 × 30 mL). The filtrate was concentrated to dryness and triturated with Et2O, and the solid was collected by vacuum filtration to give 2.2 g of the title product in 94% yield which was used for next step without further purification. LCMS (method LCMS2, ESI): RT = 0.87 min, m/z = 266.0 [M + H]+. 4-(Piperidin-1-ylsulfonyl)benzaldehyde (84). In a 100 mL round-bottomed flask was added piperidine (83, 233 mg, 2.74 mmol) and EtN(i-Pr)2 (0.85 mL, 4.88 mmol) in CH2Cl2 (10 mL), followed by a solution of 4-formylbenzene-1-sulfonyl chloride (82, 501 mg, 2.448 mmol) in 5 mL of CH2Cl2. The reaction mixture was stirred at room temperature for 16 h and then was diluted with CH2Cl2 (30 mL) and was washed successively with water (10 mL) and brine (10 mL). The organic layer was dried over MgSO4, was filtered through a short plug of silica gel, and was concentrated to afford the title product as a colorless solid in 100% yield. 1H NMR (300 MHz, CDCl3) δ: 10.11 (s, 1H), 8.06−7.90 (m, 4H), 3.06−3.01 (m, 4H), 1.69−1.61 (m, 4H), 1.48−1.40 (m, 2H). (E)-2-Methyl-N-(4-(piperidin-1-ylsulfonyl)benzylidene)propane-2-sulfinamide (85). To a 0.5 M solution of 84 (712 mg, 2.81 mmol) in THF (6 mL) under nitrogen was added titanium(IV) ethoxide (1.2 mL, 5.62 mmol). tert-Butanesulfinamide (341 mg, 2.81 mmol) was then added, and the resulting mixture was stirred for 2 h at room temperature. The reaction was poured into an equal volume of sat. NaHCO3 with rapid stirring and immediately filtered through Celite. The filter cake was washed with EtOAc (2 × 30 mL), and the aqueous layer was separated and washed once with EtOAc (20 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated. The title product was obtained as a colorless solid by quick purification using silica gel chromatography (EtOAc/hexanes, 40/60) in 59% yield (the product slowly hydrolyzes on silica gel). 1H NMR (300 MHz, CDCl3) δ: 8.64 (s, 1H), 8.01−7.84 (m, 4H), 3.05− 3.01 (m, 4H), 1.69−1.62 (m, 4H), 1.47−1.43 (m, 2H), 1.29 (s, 9H). 2-Methyl-N-(1-(4-(piperidin-1-ylsulfonyl)phenyl)ethyl)propane-2-sulfinamide (86). A solution of 85 (73 mg, 0.21 mmol) was dissolved in 5:1 Et2O/THF (6 mL) and cooled to −78 °C. Methyllithium (1.6 M in Et2O, 1.5 mL, 2.4 mmol) was added dropwise via syringe and the mixture was stirred 1 h at −78 °C and then was warmed to room temperature for 16 h. The mixture was neutralized with a minimal amount of sat. Na2SO4, diluted with EtOAc (15 mL). The organic layer was dried (MgSO4) and concentrated. The title product was obtained in 47% yield after silica gel chromatography (EtOAc/hexanes, 40/60). 1H NMR (300 MHz, CDCl3) δ: 7.73 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.65−4.57 (m, 1H), 3.46 (d, J = 3.4 Hz, 1H), 2.99 (t, J = 5.4 Hz, 4H), 1.68−1.60 (m, 4H), 1.53 (d, J = 5.6 Hz, 3H), 1.47−1.39 (m, 2H), 1.24 (s, 9H). 1-(4-(Piperidin-1-ylsulfonyl)phenyl)ethanamine Hydrochloride (64l). A solution of 86 (36 mg, 0.1 mmol) in MeOH (0.5 mL) was treated with 4 M HCl in 1,4-dioxane (0.5 mL), and the mixture was stirred for 30 min before being concentrated to 30% of its original volume. Et2O (2 mL) was added, forming a precipitate. The mixture was again concentrated to 30% of the original volume, and Et2O (4 mL) was added. The title product was obtained as a colorless solid in 68% yield after vacuum filtration and washing with cold Et2O (2 mL). This compound was used directly in the next step (synthesis of compound 9) without further purification.

on a silica gel column eluted with 70% EtOAc in petroleum ether to give the title product as a white solid in 24% yield. LCMS (LCMS5, ESI): RT = 0.81 min, m/z = 145.0 [M + H]+. Methyl Imidazo[1,2-a]pyrazine-6-carboxylate (66b). Hydrogen chloride gas was bubbled into a solution of 76 (200 mg, 1.39 mmol) in MeOH (20 mL) for 15 min. The resulting solution was stirred at 80 °C for 2 h. The reaction mixture was concentrated under vacuum. The residue was dissolved in 50 mL of EtOAc and then was washed with 1 × 50 mL of water and 1 × 50 mL of brine. The organic layer was dried over anhydrous sodium sulfate, was filtered, and was concentrated under vacuum. The residue was purified on a silica gel column eluted with EtOAc/petroleum ether (1:4) to give the title product as a white solid in 12% yield. LCMS (LCMS6, ESI): RT = 0.88 min, m/z = 178.0 [M + H]+. N-(4-(3,5-Difluorophenylsulfonyl)benzyl)acetamide (78h). In a 1 L round-bottomed flask was added sodium 4-(acetamidomethyl)benzenesulfinate (77, 7.45 g, 31.7 mmol), 3,5-difluorophenylboronic acid (68h, 5 g, 31.7 mmol), copper(II) acetate (6.33 g, 34.8 mmol), potassium carbonate (9.63 g, 67.9 mmol), and 100 mL of DMSO followed by 10 g of regular 4 Å molecular sieves. The reaction mixture was stirred at room temperature for overnight, and then the solution was filtered through Celite. The filtrate was concentrated under reduced pressure, diluted with EtOAc (300 mL), and washed with water (3 × 50 mL). The organic layer was dried over Na2SO4, was filtered, and was concentrated. Biotage purification of the residue (50% EtOAc/hexanes) afforded 4.07 g of the title product in 40% yield. 1H NMR (300 MHz, DMSO-d6) δ: 8.42 (t, J = 5.8 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.73−7.61 (m, 5H), 7.48 (d, J = 8.3 Hz, 1H), 4.29 (d, J = 6.0 Hz, 2H), 1.82 (s, 3H). LCMS (method LCMS3, ESI) RT = 5.79 min, m/z = 326.12 [M + H]+. Compounds 78i − 78j were synthesized using conditions similar to those employed to make compound 78h. N-(4-((3-(Trifluoromethyl)phenyl)sulfonyl)benzyl)acetamide (78i). Substituting the corresponding reagent with 3-(trifluoromethyl)phenylboronic acid (68i), the title product was obtained in 40% yield. 1 H NMR (300 MHz, DMSO-d6) δ: 8.45−37 (m, 1H), 8.27−8.21 (m, 2H), 8.10−8.05 (m, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.86 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 8.5 Hz, 2H), 4.29 (d, J = 5.9 Hz, 2H), 1.85 (s, 3H). N-(4-((6-Morpholinopyridin-3-yl)sulfonyl)benzyl)acetamide (78j). Substituting the corresponding reagent with 6-morpholinopyridin-3-ylboronic acid (68j), the title product was obtained in 16% yield. 1H NMR (300 MHz, DMSO-d6) δ: 8.57−8.54 (m, 1H), 8.43− 8.36 (m, 1H), 7.89−7.82 (m, 3H), 7.41 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 9.2 Hz, 1H), 4.26 (d, J = 6.0 Hz, 2H), 3.65−3.55 (m, 8H), 1.84 (s, 3H). (4-(3,5-Difluorophenylsulfonyl)phenyl)methanamine (64h). The mixture of 78h (4 g, 12.30 mmol) and 12 M HCl (102 mL, 3 M, 307 mmol) in 2-propanol was heated at 100 °C overnight. The reaction mixture was cooled to room temperature, and the 2-propanol was removed under reduced pressure to afford a white solid. The solid was then suspended in 3 M NaOH, filtered, washed with water (60 mL), and dried to give 3.14 g of the title product in 90% yield. 1H NMR (300 MHz, DMSO-d6) δ: 7.96 (d, J = 8.5 Hz, 2H), 7.74−7.62 (m, 4H), 7.57 (d, J = 8.6 Hz, 2H), 3.76 (s, 2H). LCMS (method LCMS1, ESI): RT = 0.91 min, m/z = 284.0 [M + H]+. Compounds 64i−64j were synthesized using conditions similar to those employed to make compound 64h. (4-((3-(Trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i). Substituting the corresponding reagent with 78i, the title product was obtained in 86% yield. 1H NMR (300 MHz, CDCl3) δ: 8.19 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 3.94 (s, 2H), 1.48 (s, 2H). (4-((6-Morpholinopyridin-3-yl)sulfonyl)phenyl)methanamine (64j). Substituting the corresponding reagent with 78j, the title product was obtained in 100% yield. 1H NMR (300 MHz, DMSO-d6) δ: 8.57−8.55 (m, 1H), 7.90−7.81 (m, 3H), 7.51 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 9.2 Hz, 1H), 3.73 (s, 2H), 3.65−3.56 (m, 8H), 1.85 (s, 2H). R

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Methyl 3-(4-((tert-Butoxycarbonylamino)methyl)phenylthio)propanoate (89). A 200 mL flask equipped with a magnetic stir bar and containing tert-butyl-4-bromobenzylcarbamate (87, 6.72 g, 23.5 mmol) was charged with toluene (50 mL), EtN(i-Pr)2 (8.21 mL, 47.0 mmol), Xantphos (0.680 g, 1.175 mmol), Pd2(dba)3 (0.538 g, 0.588 mmol), and methyl 3-mercaptopropanoate (88, 2.60 mL, 23.50 mmol). The mixture was heated to 100 °C under nitrogen for 2.5 h. After cooling, the mixture was loaded directly on a short silica gel column. Elution with 5:1 to 3:1 hexanes−EtOAc afforded the title product as a colorless oil in 94% yield. 1H NMR (400 MHz, CDCl3): δ 7.32 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 4.84 (br s, 1H), 4.28 (d, J = 5.8 Hz, 2H), 3.67 (s, 3H), 3.14 (t, J = 7.4 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.45 (s, 9H). LCMS (method LCMS2, ESI) m/z = 348.15 [M + Na]+. Methyl 3-(4-((tert-Butoxycarbonylamino)methyl)phenylsulfonyl)propanoate (90). A 500 mL flask equipped with a magnetic stir bar was charged with Oxone (23.61 g, 38.4 mmol) and water (82 mL). The mixture was stirred at RT for 5 min to dissolve the Oxone, and a solution of 89 (5.0 g, 15.36 mmol) in CH3CN (45 mL) was then added at RT with rapid stirring. The mixture was stirred at RT for 3 h. Water (60 mL) and EtOAc (100 mL) were added, and the layers were separated. The aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed with halfsaturated brine (2 × 50 mL), dried (Na2SO4), and concentrated under reduced pressure to afford the title product as a white solid in 94% yield. 1H NMR (400 MHz, CDCl3): δ 7.86 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 5.03 (br s, 1H), 4.41 (d, J = 6.2 Hz, 2H), 3.64 (s, 3H), 3.41 (t, J = 7.7 Hz, 2H), 2.74 (t, J = 7.8 Hz, 2H), 1.46 (s, 9H). LCMS (method LCMS2, ESI) m/z = 380.07 [M + Na]+. S o d i u m 4 - ( ( ( t e r t- B u t o xy c a r b o n y l ) a m i n o)m e t h y l)benzenesulfinate (67). To a solution of 90 (358 mg, 1 mmol) in MeOH (5 mL) was added EtONa (21% in ethanol, 0.38 mL, 1.02 mmol), and the mixture was heated at 50 °C for 1 h. After cooling, the solvent was removed under reduced pressure and the residue was dried under high vacuum to give the title product as a light yellow solid in 97% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.35 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 4.07 (d, J = 5.6 Hz, 2H), 1.37 (s, 9H).

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efficacy data. We thank the DMPK group at Genentech for their contribution to the generation of the DMPK data, especially Erlie Marie Delarosa and Jonathan Cheong. We also acknowledge Agilent Technologies (Woburn, MA) for generating the Rapidfire LCMS data, Tandem Laboratories (Woburn, MA) for performing the phosphoribosylated adduct study, Yigong Shi’s group (Tsinghua University, Beijing, China) for providing Nampt DNA construct and Nampt protein, and James Kyranos and Jaime Escobedo for helpful discussions.

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ABBREVIATIONS USED MLM, mouse liver microsome; HLM, human liver microsome; MDCK, Madin−Darby canine kidney; SRB, sulforhodamine B; CyQuant, cell counting via DNA content to assess proliferative effects; Cmax, maximum concentration; t1/2, half-life; Tmax, time to reach the maximum concentration; CL, clearance; Vss, volume of distribution; HATU, (O-(7-azabenzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate); HOBt, 1-hydroxybenzotriazole; DIAD, diisopropyl azodicarboxylate; Xantphose, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; SCX, strong cationic exchange; THPP, tris(hydroxypropyl)phosphine; D5W, 5% dextrose in water

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(1) (a) Belenky, P.; Bogan, K. L.; Brenner, C. NAD+ metabolism in health and disease. Trends Biochem. Sci. 2007, 32, 12−19. (b) Koppenol, W. H.; Bounds, P. L.; Dang, C. V. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat. Rev. Cancer 2011, 11, 325−337. (c) Ward, P. S.; Thompson, C. B. Metabolic reprogramming: A cancer hallmark even Warburg did not anticipate. Cancer Cell 2012, 21, 297−308. (2) (a) Galli, M.; Van Gool, F.; Rongvaux, A.; Andris, F.; Leo, O. The nicotinamide phosphoribosyltransferase: A molecular link between metabolism, inflammation, and cancer. Cancer Res. 2010, 70, 8−11. (b) Revollo, J. R.; Grimm, A. A.; Imai, S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J. Biol. Chem. 2004, 279, 50754− 50763. (c) Garten, A.; Petzold, S.; Korner, A.; Imai, S.-I.; Kiess, W. Nampt: Linking NAD Biology, Metabolism and Cancer. Trends Endocrinol. Metab. 2009, 20, 130−138. (d) Burgos, E. S.; Schramm, V. L. Weak coupling of ATP hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyltransferase. Biochemistry 2008, 47, 11086−11096. (3) (a) Zaremba, T.; Ketzer, P.; Cole, M.; Coulthard, S.; Plummer, E. R.; Curtin, N. J. Poly(ADP-ribose) polymerase-1 polymorphisms, expression and activity in selected human tumour cell lines. Br. J. Cancer 2009, 101, 256−262. (b) Khan, J. A.; Forouhar, F.; Tao, X.; Tong, L. Nicotinamide adenine dinucleotide metabolism as an attractive target for drug discovery. Expert Opin. Ther. Targets 2007, 11, 695−705. (4) (a) Cerna, D.; Li, H.; Flaherty, S.; Takebe, N.; Coleman, C. N.; Yoo, S. S. Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by the small molecule GMX1778 regulates ROSmediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner. J. Biol. Chem. 2012, 287, 22408−22417. (b) Vander Heiden, M. G.; Cantley, L. C.; Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009, 324, 1029−1033. (5) Schou, C.; Ottosen, E. R.; Petersen, H. J.; Bjorkling, F.; Latini, S.; Hjarnaa, P. V.; Bramm, E.; Binderup, L. Novel cyanoguanidines with potent oral antitumor activity. Bioorg. Med. Chem. Lett. 1997, 7, 3095− 3100. (6) Biedermann, E.; Hasmann, M.; Loser, R.; Rattel, B.; Reiter, F.; Schein, B.; Seibel, K.; Vogt, K. Preparation and formulation of pyridine

ASSOCIATED CONTENT

S Supporting Information *

Details of computational methods; details of analytical LCMS methods; in vitro ADME and experimental procedures; experimental details for PK and efficacy study of compound 58 in the mouse; protein expression/purification and crystallographic methods and procedures for 7 and 58 (in complex with Nampt); details of Nampt biochemical and cell-based assays. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

PDB nos.: 4KFN for 7 complexed with Nampt; 4KFO for 58 complexed with Nampt.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: 857-209-2382. E-mail: xzheng@formatherapeutics. com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Forma’s high speed synthesis group and George Luke for their synthetic contributions, ADME group for generating the microsomal and solubility data, Rashida GarciaDancey and Shohini Ganguly for helping generate the biochemical and cellular data, and Lakshmanan Manikandan, Saradhi Vijay, and Danilal C. Sharma for generating the in vivo S

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derivatives as antitumor agents and immunosuppressants. WO 199748397, 1997. (7) (a) Ravaud, A.; Cerny, T.; Terret, C.; Wanders, J.; Bui, B. N.; Hess, D.; Droz, J.-P.; Fumoleau, P.; Twelves, C. Phase I study and pharmacokinetics of CHS-828, a guanidino-containing compound, administered orally as a single dose every 3 weeks in solid tumours: An ECSG/EORTC study. Eur. J. Cancer 2005, 41, 702−707. (b) Hovstadium, P.; Larsson, R.; Jonsson, E.; Skov, T.; Kissmeyer, A.-M.; Krasilnikoff, K.; Bergh, J.; Karlsson, M. O.; Lonnebo, A.; Ahlgren, J. A phase I study of CHS 828 in patients with solid tumor malignancy. Clin. Cancer Res. 2002, 8, 2843−2850. (c) Vig Hjarnaa, P.-J.; Jonsson, E.; Latini, S.; Dhar, S.; Larsson, R.; Bramm, E.; Skov, T.; Binderup, L. CHS828, a novel pyridine cyanoguanidine with potent antitumor activity in vitro and in vivo. Cancer Res. 1999, 59, 5751−5757. (8) (a) Holen, K.; Saltz, L. B.; Hollywood, E.; Burk, K.; Hanauske, A.R. The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor. Invest. New Drugs 2008, 26, 45−51. (b) Olesen, U. H.; Thougaard, A. V.; Jensen, P. B.; Sehested, M. A preclinical study on the rescue of normal tissue by nicotinic acid in high-dose treatment with APO866, a specific nicotinamide phosphoribosyltransferase inhibitor. Mol. Cancer Ther. 2010, 9, 1609−1617. (9) Zheng, X.; Bauer, P.; Baumeister, T.; Buckmelter, A. J.; Caligiuri, M.; Clodfelter, K. H.; Han, B.; Ho, Y.-C.; Kley, N.; Lin, J.; Reynolds, D. J.; Sharma, G.; Smith, C. C.; Wang, Z.; Dragovich, P. S.; Oh, A.; Wang, W.; Zak, M.; Gunzner-Toste, J.; Zhao, G.; Yuen, P.-w.; Bair, K. W. Structure-based identification of ureas as novel nicotinamide phosphoribosyltransferase (Nampt) inhibitors. J. Med. Chem. 2013, 56, 4921−4937. (10) Gunzner-Toste, J.; Zhao, G.; Bauer, P.; Baumeister, T.; Buckmelter, A. J.; Caligiurib, M.; Clodfelter, K. H.; Fu, B.; Han, B.; Ho, Y.-C.; Kley, N.; Liang, X.; Liederer, B.; Lin, J.; Mukadam, S.; O’Brien, T.; Reynolds, D. J.; Sharma, G.; Skelton, N.; Smith, C. C.; Wang, W.; Wang, Z.; Xiao, Y.; Yuen, P.-w.; Zak, M.; Zhang, L.; Zheng, X.; Bair, K. W.; Dragovich, P. S. Discovery of potent and efficacious urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with reduced CYP2C9 inhibition properties. Bioorg. Med. Chem. Lett. 2013, 23, 3531−3538. (11) For a recent and broad review of many different classes of Nampt inhibitors, see: Galli, U.; Travelli, C.; Massarotti, A.; Fakhfouri, G.; Rahimian, R.; Tron, G. C.; Genazzani, A. A. Medicinal chemistry of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. DOI: 10.1021/jm4001049. (12) Related PRPP adducts have also been described for other Nampt inhibitors. See: Watson, M.; Roulston, A.; Belec, L.; Billot, X.; Marcellus, R.; Bedard, D.; Bernier, C.; Branchaud, S.; Chan, H.; Dairi, K.; Gilbert, K.; Goulet, D.; Gratton, M. O.; Isakau, H.; Jang, A.; Khadir, A.; Koch, E.; Lavoie, M.; Lawless, M.; Nguyen, M.; Paquette, D.; Turcotte, E.; Berger, A. The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors. Mol. Cell. Biol. 2009, 29, 5872−5888. (13) The antiproliferative effects exhibited in cell culture by 7 were completely eliminated (“reversed”) when the compound was tested in the presence of 0.33 mM NMN (the product of the Nampt-catalyzed condensation of nicotinamide and PRPP; cf., Figure 1). This result strongly suggests that the observed cell-based effects result from Nampt inhibition. Related NMN experiments were conducted in cellbased assessments with all inhibitors reported in this work and all displayed similar reversals of their antiproliferative effects. (14) The resolution of the 7−Nampt cocrystal structure cannot distinguish the depicted azaindole binding orientation from the rotational isomer which positions the azaindole NH away from Asp219 and toward R311. However, the described orientation optimally locates the azaindole nitrogen atom for condensation with PRPP in the Nampt active site and is thus consistent with the PRPP adduct formation observed for closely related azaindole-containing inhibitors (e.g., 8)

(15) We currently believe that many cell-potent Nampt inhibitors form PRPP-derived phosphoribosylated adducts in the protein’s active site which block the function of the enzyme. This belief is consistent with the repeated observation of these adducts by mass spectrometry in biochemical experiments (this work and ref 9). It is also consistent with the inability to detect similar adduct formation in related experiments with compound 10 (see Supporting Information) and another cell-inactive compound that was nevertheless a potent biochemical Nampt inhibitor (ref 9). Once formed, the PRPP adducts may accumulate intracellularly and thereby enhance cell culture antiproliferation effects (see ref 11 for additional information and discussion). However, there are many other factors that also likely influence Nampt inhibitor cell potency, including the following: biochemical potency, the ability of a given inhibitor and/or its corresponding PRPP-derived ribose adduct to effectively compete with the NAM substrate, cell membreane permeability, and/or protein binding. In addition, performing the PRPP adduct assessments was a relatively resource-intensive task and such determinations were therefore made for only a small number of compounds. (16) Formation of the 11-phosphoribosylated adduct would likely require positioning the inhibitor azaindazole N-atom in the same location occupied by the corresponding atom present in 7 and 8. Such positioning would reorient the azaindazole NH of 11 relative to the other two molecules and possibly thereby weaken interactions with Nampt. (17) As described in this work, compound 8 (a close structural isomer of compound 7) was observed by mass spectrometry to form PRPP-derived adducts in biochemical experiments. Compound 58 also forms an adduct with PRPP, as evidenced by mass spectrometry analysis of biochemical inhibition reaction mixtures, and we recently solved a crystal structure of phosphoribosylated-58 in complex with Nampt. The details associated with the biochemical and crystallography experiments involving the PRPP-derived phosphoribosylated adduct of compound 58 will be reported in a separate disclosure: Oh, A.; Ho, Y.-C.; Zak, M.; Liu, Y.; Liu, Y.; Yuen, P.-w.; Zheng, X.; Dragovich, P. S.; Wang, W. manuscript in preparation. (18) (a) Khan, J. A.; Tao, X.; Tong, L. Molecular basis for the inhibition of human NMPRTase, a novel target for anticancer agents. Nat. Struct. Mol. Biol. 2006, 13, 582−588. (b) Kim, M. K.; Lee, J. H.; Kim, H.; Park, S. J.; Kim, S. H.; Kang, G. B.; Lee, Y. S.; Kim, J. B.; Kim, K. K.; Suh, S. W; Eom, S. H. Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866. J. Mol. Biol. 2006, 362, 66−77. (c) Wang, T.; Zhang, X.; Bheda, P.; Revollo, J. R.; Imai, S.; Wolberger, C. Structure of Nampt/PBEF/visfatin, a mammalian NAD+ biosynthetic enzyme. Nat. Struct. Mol. Biol. 2006, 13, 661−662. (d) Burgos, E. S.; Ho, M. C.; Almo, S. C.; Schramm, V. L. A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 13748−13753. (19) Compound 58 also exhibited robust efficacy in xenograft models derived from the other cell lines listed in Table 6. Detailed descriptions of these efficacy assessments and associated PD effects will be presented elsewhere: O’Brien, T.; Xiao, Y.; Oeh, J.; Liang, X.; Vanderbilt, A.; Qin, A.; Yang, L.; Lee, L. B.; Ly, J.; Cosino, E.; LaCap, J. A.; Ogasawara, A.; Nannini, M.; Liederer, B. M.; Jackson, P.; Williams, S.; Dragovich, P. S.; Sampath, D. Submitted to Cancer Res. (20) Sikazwe, D.; Bondarev, M. L.; Dukat, M.; Rangisetty, J. B.; Roth, B. L.; Glennon, R. A. Binding of sulfonyl-containing arylalkylamines at human 5-HT6 serotonin receptors. J. Med. Chem. 2006, 49, 5217− 5225. (21) Thurkauf, A.; Chen, D.; Phadke, A.; Li, S.; Deshpande, M. Preparation of azabenzofuran substituted thioureas as inhibitors of viral replication. WO 2005067900, 2005. (22) Li, Q.; Woods, K. W.; Claiborne, A.; Gwaltney, S. L., II; Barr, K. J.; Liu, G.; Gehrke, L.; Credo, R. B.; Hui, Y. H.; Lee, J.; Warner, R. B.; Kovar, P.; Nukkala, M. A.; Zielinski, N. A.; Tahir, S. K.; Fitzgerald, M.; Kim, K. H.; Marsh, K.; Frost, D.; Ng, S.; Rosenberg, S.; Sham, H. L. Synthesis and biological evaluation of 2-indolyloxazolines as a new T

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class of tubulin polymerization inhibitors. Discovery of A-289099 as an orally active antitumor agent. Bioorg. Med. Chem. Lett. 2002, 12, 465− 469. (23) Fyfe, M. C. T.; Thomas, G. H.; Gardner, L. S.; Bradley, S. E.; Gattrell, W.; Rasamison, C. M.; Shah., V. K. Preparation of furopyridine and pyrrolopyridine derivatives and analogs thereof as G-protein coupled receptor agonists. WO 2006067532, 2006. (24) Walker, D. P.; Piotrowski, D. W.; Jacobsen, E. J.; Acker, B. A.; Wishka, D. G.; Reitz, S. C.; Groppi, V. E., Jr. Preparation of N(azabicyclyl)arylamides for therapeutic use as nicotinic acetylcholine receptor agonists. WO 2003029252, 2003.

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