(Nampt) Inhibitors - American Chemical Society

Apr 25, 2013 - ABSTRACT: Nicotinamide phosphoribosyltransferase (Nampt) is a promising ... intermediate in the biosynthesis of nicotinamide adenine...
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Structure-Based Identification of Ureas as Novel 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,‡ Angela Oh,‡ Weiru Wang,‡ Mark Zak,‡ Janet Gunzner-Toste,‡ 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

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‡

S Supporting Information *

ABSTRACT: Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 μM; A2780 IC50 = 0.032 μM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).

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INTRODUCTION Nicotinamide phosphoribosyltransferase (Nampt, NAMPTase, PBEF, Visfatin, EC 2.4.2.12) catalyzes the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-pyrophosphate (PRPP) to form nicotinamide mononucleotide (NMN), an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). In mammalian cells, there are multiple pathways for NAD biosynthesis: a de novo pathway using tryptophan (Trp) as the precursor and three pathways using exogenous vitamin sources as the precursors: NAM, nicotinic acid (NA), and nicotinamide riboside (NR). Nampt is the ratelimiting enzyme in the NAD biosynthetic pathway using NAM. This NAM-dependent pathway is used in cells to recycle the NAM produced by NAD-consuming enzymes such as polyADP-ribose polymerases (PARPs) and sirtuins (Figures 1 and 2).1 Tumors have elevated PARP activity and thereby consume NAD at a higher rate than normal tissues and are thus more dependent upon Nampt to maintain required NAD levels.2 NAD is an essential cofactor in multiple energy producing processes such as mitochondrial oxidative phosphorylation, β oxidation, glycolysis, and the citric acid cycle. Cancer cells have an increased demand for ATP and thus are more sensitive to perturbations of these processes. Rapidly proliferating cancers also have additional biosynthetic requirements that require NAD as a cofactor.3 In addition, NAD is essential in maintaining the reductive environment that protects cells from reactive oxygen species (ROS), which are elevated in cancers.4 The combination of increased NAD consumption and © 2013 American Chemical Society

dependence upon NAD-driven biosynthetic and cellular redox homeostatic processes may make many cancer cells highly dependent on Nampt activity for proliferation and/or survival. Nampt thus represents an attractive target for the development of new cancer therapies. At the outset of our Nampt program, only three classes of Nampt inhibitors were known in the literature. Fujisawa/ TopoTarget’s 15 (APO866) and Leo/Gemin-X/Cephalon/ Teva’s 26 (GMX1778) were the first two published Nampt inhibitors. 1 is currently in phase 2 clinical trials against cutaneous T-cell lymphoma (CTCL), while 2 is in phase 1/2 trials for metastatic melanoma. Triazole-containing compounds were also reported to be potent Nampt inhibitors,7 and a representative example, compound 3, exhibited an SH-SY5Y cytotoxicity IC50 value of 93 nM. More recently, Myrexis,8 Sanofi-Aventis,9 and Abbott10 disclosed additional classes of potent Nampt inhibitors. For example, compound 4 from Myrexis showed a Nampt enzyme IC50 of 0.5 nM and HCT116 cytotoxicity of 3.0 nM (Figure 3). In this paper, we report the identification of highly potent urea-containing small-molecule inhibitors of Nampt, which were designed using the known crystal structure of the enzyme with an inhibitor present. During the course of this work, Myrexis and Sanofi independently reported their own related urea-containing Nampt inhibitors.8a,9 Received: February 6, 2013 Published: April 25, 2013 4921

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Figure 1. Nampt catalyzed biochemical reaction.

Figure 2. Schematic representation of NAD biosynthetic pathways found in mammalian cells.

Figure 3. Reported Nampt inhibitors.

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RESULTS AND DISCUSSION Structure-Based Identification of a New Chemical Scaffold to Target Nampt. Nampt acts both as a phosphoribosyltransferase and as an ATPase, with phosphorylation of His247 increasing the enzyme’s catalytic activity in a stoichiometric manner.11 Structurally, Nampt functions as a homodimer, with two identical catalytic sites located at opposite ends of the dimer interface.11 The binding site of ATP overlaps with the PRPP and NAM binding sites. All three sites combine to form a protein surface of >1000 Å2, which represents a large area that a potential competitive inhibitor could target. The NAM binding site lies at the entrance to a tunnel through the protein that further extends along the dimer interface to a funnel shaped opening. 1 occupies this tunnel, with the piperidyl moiety overlapping with the NAM binding site14 (Figure 4). To pinpoint locations likely to be druggable within these regions, we utilized computational solvent mapping12 techniques employing CS-Map13 software. The consensus sites13 in

Figure 4. Slice through the cocrystal structure of 1 (yellow) in complex with Nampt (important residues and solvent-excluded-surface in gray) (PDB ID 2GVJ).14 The crystallographically determined locations of the Nampt substrates NAM (2E5C)15 and PRPP (2E5D)15 are also shown (green).

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compound 5 was completely reversed by the addition of 0.33 mM of NMN, which proved that the antiproliferation effect was mechanism based. Thus, compound 5 represented an ideal starting point for additional SAR studies with respect to ligand efficiency (LE = 0.4) and lipophilicity efficiency (LipE = 5.7).17 SAR Discussion. To quickly expand SAR around compound 5, we identified analogues by chemical fingerprint similarity searches18 and purchased an additional 121 compounds from various commercial sources. Table 1 summarizes the biological data generated for selected analogues. Moving the pyridyl nitrogen atom from the 3- to the 4-position (6) was highly detrimental to the anti-Nampt activity. At the other end of the inhibitor structure, removal of the piperidine group led to a 100-fold loss in biochemical potency (7: IC50 = 0.63 μM), while removal of the entire sulfonamide group (8) led to complete loss of activity. Simultaneously, in an attempt to better understand how compound 5 bound to Nampt and to use this information to guide our further SAR optimization, a cocrystal structure of 5 in complex with the protein was generated (Figures 6 and 7). In the NAM-binding region (corresponding to pharmacophore points A, B, and C), the binding mode of compound 5 was noted to be similar to that observed for 1 with the pyridyl group stacked between the aromatic side chains of Nampt residues Phe193 and Tyr18′. A crystallographic water was also observed mediating a hydrogen bonding network between the thiourea nitrogens, the backbone carbonyl of Val242, and the Asp219 and Ser241 side chains. The hydroxyl group of Ser275 was located nearly perpendicular to the thiourea moiety of 5, and this conformation did not allow for the formation of a typical hydrogen bond with the nearby sulfur atom. Instead, hydrophobic contacts between the sulfur and the side chains of

the Nampt binding region were used to define six pharmacophore points (A−F, Figure 5) with A and B−C ranking significantly higher than the other three (see Supporting Information for details).

Figure 5. Nampt crystal structure depicting virtual fragments which comprise CS-Map consensus sites (carbons in green) and derived pharmacophore points (gray spheres). A = pyridine, B = hydrogen bond acceptor, C = hydrogen bond donor, D = any ring, E = any polar atom, F = any polar atom. Sphere sizes indicate radius of each point.

Three pharmacophore models were then created by separately combining the ABC points with one of lower rank (D−F). The resulting models (ABCD, EABC, and FABC) were used to virtually screen the eMolecules database,16 resulting in a set of 228 compounds which were subsequently purchased and tested in our biochemical and cell-based assays. Of the three pharmacophore models, only ABCD yielded compounds with submicromolar anti-Nampt activity. In particular, compound 5 (Table 3) showed a biochemical IC50 of 0.007 μM and a cellular IC50 value of 0.19 μM. The cellular activity of

Table 1. Structure−Activity Relationships and Optimization of Hit Compound 5

a

Nampt biochemical inhibition. bA2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN. cUnless otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs (n = 2). dSee Figure 3 for the structures of 1 and 2. See Supporting Information for experimental details. 4923

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Figure 6. Co-crystal structure of compound 5 (green) in complex with Nampt. The ligand binds to a site formed by the interface of two Nampt protein monomers (depicted as white and cyan, respectively). Water molecules are depicted as red spheres, and possible hydrogen bonds are indicated with dashed orange lines. Hydrogen bonds which do not interact with the ligand, either directly or via a water molecule, are not shown. The resolution of the structure is 1.9 Å.

Figure 7. Schematic representation of the binding mode of compound 5 to Nampt. Waters are depicted as “W” and hydrogen bonds as dashed orange lines. Waters and hydrogen bonds which do not interact with the ligand are not shown.

Ile351 and Phe193 were observed. The phenyl ring of 5 occupied the Nampt tunnel region and participated in an edgeto-face interaction with the side chain of His191. The ligand’s sulfone oxygen atoms filled a hydrophobic pocket formed by the side chains of Ala379, Ile351, and Ile309 but also made hydrogen bonds with crystallographic waters which, in turn,

formed other hydrogen bonds to Nampt backbone carbonyl groups. The piperidine moiety of 5 occupied space close to the piperidine present in 1 but was situated in a perpendicular orientation. Hydrophobic contacts were noted between the piperidine C2 carbon atom and the side chain of Ile309, as well 4924

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On the basis of the cocrystal structure information obtained with compound 5, the thiourea sulfur atom did not form a hydrogen bond with the Nampt Ser275 residue (the serine hydroxyl oxygen was within a suitable distance of 3.2 Å but was perpendicular to the thiourea plane). The corresponding urea compound (9) was therefore made to possibly enable a more favorable binding geometry and at the same time reduce compound 5’s lipophilicity (calculated ClogP was reduced from 2.4 to 1.5). In fact, compound 9 exhibited biological potency and microsomal stability similar to that displayed by compound 5 (Table 2) and a significantly improved solubility profile (82 vs 3.1 μM). Accordingly, further SAR exploration was focused on molecules containing the urea linker. In the cocrystal structure of compound 5, the phenyl ring sits in a narrow portion of the tunnel where meta and particularly ortho substitution are sterically restricted by the tunnel walls vs para substitution which points toward the opening. This was confirmed by the meta-attachment of the sulfonamide RHS in compound 10 (Table 2). In the absence of further significant structural guidance for RHS optimization, we initiated general exploratory SAR studies of the RHS with a broad survey of

as between the piperidine C3 and C4 carbons and the Val242 and Pro273 residues. To facilitate further optimization, we investigated three distinct areas of these Nampt inhibitors: the left-hand side (LHS), the right-hand side (RHS), and the linker (Figure 8).

Figure 8. Nampt inhibitor general structure.

Because we were unsure how improvements in one portion would translate to the others, our initial inhibitor design strategy involved concurrent optimization of the various sections to define a preferred pharmacophore. Accordingly, we first chose to optimize the linker region in tandem with the RHS. Table 2. Initial SAR Exploration

a

Nampt biochemical inhibition. bA2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN. cUnless otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs (n = 2). dMouse liver microsomal stability: % remaining at 30 min. eHuman liver microsomal stability: % remaining at 30 min. fAqueous solubility. gThe R-group was linked to the meta-position of the phenyl ring. hAbove limit of quantification (>100 μM). iBelow limit of quantification (3.0 0.171

0.027 0.009 0.021 0.044 0.003

a

Nampt biochemical inhibition. As a result of independent experiments, the IC50 values determined using 1 μM NAM for the indicated compounds differ slightly from those listed in Tables 3−5. bCalculated Ki value for each inhibitor (see ref 21). Results are reported as the arithmetic mean of at least two separate runs (n = 2).

NAM-competitive manner with linear increases in IC50 values observed with increasing NAM concentrations.20 Importantly, varying NAM concentrations had a relatively minor impact on the IC50 value of the cell active compound (50) as compared with effects on IC50 outcomes for the other (cell-inactive) molecules studied. This behavior resulted in compound 50 having the most potent calculated Ki value among these inhibitors (Table 7).21 Thus, the ability of the compounds and/ or their PRPP−ribose adducts to effectively inhibit Nampt in Scheme 1. Urea Derivatives Synthesis: Isocyanate Approacha

a Reagents and conditions: (i) THF, −20 to 25 °C, 2 h, 32−95% yield; (ii) THF, 25 °C, 1 h, 14−60% yield; (iii) toluene−CH2Cl2−DMA, 25−50 °C, 18 h, 17−60% yield; (iv) Na2SO3, NaHCO3, H2O, 80 °C, 3 h, 23% yield; (v) Cu(OAc)2, Et3N, DMSO, 60 °C, 16 h, 31% yield; (vi) LiOH, MeOH, 25 °C, 16 h, 58% yield; (vii) piperidine, BOP, Et3N, DMA, 25 °C, 6 h, 45% yield.

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Scheme 2. Urea and Carbamate Derivatives Synthesis: Triphosgene Approacha

Reagents and conditions: (i) triphosgene, Et3N, CH2Cl2 −10 to 25 °C, 16 h, 16−92% yield; (ii) mCPBA, CH2Cl2−MeOH, 25 °C, 16 h, 15% yield; (iii) TFA, CH2Cl2, 25 °C, 16 h, 58% yield.

a

Scheme 3. Urea and Carbamate Derivatives Synthesis: Chloroformate Approacha

a Reagents and conditions: (i) 4-nitrophenyl chloroformate, toluene, 85 °C, 3 h, 100% yield; (ii) phenyl chloroformate, THF, 25 °C, 20 min, 86% yield; (iii) EtOH, reflux, 3 h, 27% yield; (iv) Et3N, 1,4-dioxane, 60 °C, 1 h, 61−96% yield.

Scheme 4. Synthesis of Compound 30 and 32a

a

Reagents and conditions: (i) Et3N, CH2Cl2, 25 °C, 4 h, 33−52% yield.

Scheme 5. Synthesis of Compound 38a

a

Reagents and conditions: (i) Et3N, CH2Cl2, 0−25 °C, 12 h, 69% yield; (ii) Et3N, CH3CN, 80 °C, 16 h, 14% yield.

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CONCLUSIONS In summary, using structure-based design and leveraging key information from literature-disclosed cocrystal structures, we discovered a structurally novel thiourea-containing Nampt inhibitor (5). Guided by the cocrystal structure of compound 5 in complex with Nampt, several rounds of SAR optimization ultimately yielded the urea-containing compound 50, which possessed excellent cellular potency and in vitro and in vivo ADME properties. Compound 50 demonstrated reasonable PK properties in mice with an oral bioavailability of 26% and also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. In addition, SAR and mechanistic studies indicated that an appropriately

positioned and sufficiently nucleophilic nitrogen atom was required in the LHS of a given inhibitor to enable Namptmediated formation of the corresponding phosphoribosylated (PRPP) adduct and thereby impart potent cellular activity. Further details of related compounds which build on these discoveries will be disclosed in due course.

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EXPERIMENTAL SECTION

General. Unless otherwise indicated, all reagents and solvents were purchased from commercial sources and were used without further purification. Moisture or oxygen sensitive reactions were conducted under an atmosphere of argon or nitrogen gas. Unless otherwise stated, 1H NMR spectra were recorded at 300 or 400 MHz using Varian or Bruker instruments operating at the indicated frequencies. 4932

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SPE column. The vial was rinsed once with CH2Cl2 (500 μL), and the solution was transferred to the SPE column. The column was eluted once with 10:2.5:1 EtOAc/MeOH/Et3N (3 mL), and the eluent was evaporated under reduced pressure. The dark solid obtained was dissolved in DMSO (0.5 mL), and the crude product 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 mm × 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 gradients: 0−2 min (15% B), 2−6 min (15−100% B). Detector ZQ mass detector in electrospray ionization mode] to give the title compound as a white solid (2.5 mg, 17%). LCMS (method LCMS2, ESI): RT = 0.93 min, m/z = 363.2 [M + H]+. Compounds 43−46 were synthesized using the same parallel synthesis conditions employed to make compound 42. 3-(4-[Phenylsulfamoyl]phenyl)-1-(pyridin-3-ylmethyl)urea (43). Yield 60%. 1H NMR (400 MHz, DMSO-d6) δ: 9.97 (s, 1H), 9.12 (s, 1H), 7.82 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.21 (dd, J = 8.4, 7.3 Hz, 2H), 7.12−7.03 (m, 2H), 7.03−6.96 (m, 1H), 6.93 (s, 1H), 4.41 (s, 2H). LCMS (method LCMS3, ESI): RT = 4.43 min, m/z = 383.1 [M + H]+. 3-[4-(Diethylsulfamoyl)phenyl]-1-(pyridin-3-ylmethyl)urea (44). Yield 20%. LCMS (method LCMS2, ESI): RT = 0.93 min, m/z = 363.2 [M + H]+. 3-{4-[(2-Methylpiperidin-1-yl)sulfonyl]phenyl}-1-(pyridin-3ylmethyl)urea (45). Yield 55%. 1H NMR (400 MHz, CDCl3) δ: 8.48 (d, J = 2.0 Hz, 1H), 8.45 (dd, J = 4.8, 1.6 Hz, 1H), 8.28 (s, 1H), 7.66− 7.63 (m, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.23 (dd, J = 7.6, 4.8 Hz, 1H), 6.15 (t, J = 6 Hz, 1H), 4.39 (d, J = 5.6 Hz, 2H), 4.16−4.07 (m, 1H), 3.60 (dd, J = 13.2, 3.2 Hz, 1H), 2.96−2.89 (m, 1H), 1.55−1.23 (m, 6H), 1.02 (d, J = 6.8 Hz, 3H). LCMS (method LCMS2, ESI): RT: = 1.00 min, m/z = 389.2 [M + 1] +. 3-{4-[(3-Methylpiperidin-1-yl)sulfonyl]phenyl}-1-(pyridin-3ylmethyl)urea (46). Yield 20%. LCMS (method LCMS2, ESI): RT = 1.13 min, m/z = 389.2 [M + H]+. 1-(4-{[(Pyridin-3-ylmethyl)carbamoyl]amino}benzenesulfonyl)piperidine-4-carboxamide (48). Yield 14%. 1H NMR (300 MHz, CDCl3) δ: 9.18 (s, 1H), 8.52 (d, J = 3.1 Hz, 1H), 8.48−8.41 (m, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.64−7.52 (m, 4H), 7.38−7.33 (m, 1H), 7.18 (s, 1H), 6.92 (t, J = 6.0 Hz, 1H), 6.78 (s, 1H), 4.34 (d, J = 6.1 Hz, 2H), 3.50 (d, J = 6.8 Hz, 2H), 2.27−2.18 (m, 2H), 2.08−1.95 (m, 1H), 1.82−1.68 (m, 2H), 1.58−1.42 (m, 2H). LCMS (method LCMS1, ESI): RT = 0.63 min, m/z = 418.0 [M + H]+. 1-[4-(Morpholine-4-sulfonyl)phenyl]-3-(pyridin-3-ylmethyl)urea (49). Yield 23%. 1H NMR (300 MHz, CDCl3) δ: 8.52 (s, 1H), 7.99 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.62−7.50 (m 4H), 7.34−7.23 (m, 1H), 5.70 (t, J = 6.3 Hz, 1H), 4.47 (d, J = 6.0 Hz, 2H), 3.78−3.68 (m, 4H), 3.02−2.90 (m, 4H). LCMS (method LCMS1, ESI): RT = 0.63 min, m/z = 377.3 [M + H]+. Ammonium-4-(3-(pyridin-3-ylmethyl)ureido)benzenesulfinate (55). 53 (3.99 g, 12.25 mmol) was added to a solution of sodium sulfite (6.18 g, 49.0 mmol) and NaHCO3 (6.17 g, 73.5 mmol) in water (175 mL), and the resulting mixture was stirred at 80 °C for 3 h. The mixture was then concentrated to dryness under reduced pressure, and the resulting solids were triturated with hot DMF (100 mL) and the solids filtered off. The solvent was then removed under reduced pressure, and the resulting residue was triturated with hot DCM (100 mL) and the off-white crystalline solids were collected by vacuum filtration. The solids were purified by SCX ion-exchange column (eluting with 5:1 CH3CN/NH4OH) to afford the title compound 0.9 g (23%). 1H NMR (400 MHz, DMSO-d6) δ: 9.00 (s, 1H), 8.50 (d, J = 1.2 Hz, 1H), 8.43−8.40 (m, 2H), 8.03 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.15 (s, br, 4H), 7.02 (t, J = 6.0 Hz, 1H), 4.29 (d, J = 6.0 Hz, 2H) LCMS (method LCMS2, ESI): RT = 0.55 min, m/z = 308.1 [M + NH4]+. 1-(4-((1-Methyl-1H-pyrazol-4-yl)sulfonyl)phenyl)-3-(pyridin3-ylmethyl)urea (12). To a solution of 53 (37 mg, 0.12 mmol), 1methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (37 mg, 0.18 mmol), and Et3N (0.056 mL, 0.4 mmol) in DMSO (1

Chemical shifts are expressed in ppm relative to an internal standard; tetramethylsilane (ppm =0.00). The following abbreviations are used: br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, p = pentet, m = multiplet. Purification by silica gel chromatography was carried out using Biotage systems with prepacked cartridges. Chemical purities were >95% for all final compounds as assessed by LCMS analysis at UV 220 nm. 4-(3-(Pyridin-3-ylmethyl)ureido)benzene-1-sulfonyl Chloride (53). 4-Isocyanatobenzene-1-sulfonyl chloride (52a; 125 mg, 0.574 mmol) was dissolved in THF (10 mL) and cooled to −20 °C. To this cooled solution was added dropwise a solution of (pyridin-3yl)methanamine (51a; 62 mg, 0.574 mmol) in THF (6 mL). The mixture was stirred at −20 °C for 5 min and allowed to warm up to room temperature. After stirring for 2 h, Et2O (30 mL) was added. The resulting precipitate was collected by filtration to give the title compound (60 mg, 32%). This material was used in the next step without further purification. 1H NMR (300 MHz, DMSO-d6): δ 9.18 (s, 1H), 8.87−8.76 (m, 2H), 8.60−8.48 (m, 1H), 8.12−8.02 (m, 1H), 7.67−7.58 (m, 1H), 7.48−7.39 (m, 2H), 7.35−7.28 (m, 2H), 4.55− 4.43 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.11 min, m/z = 326.3 [M + H]+. Compound 15 and 56 were synthesized using conditions similar to those employed to make compound 53. 1-(4-Phenoxyphenyl)-3-(pyridin-3-ylmethyl)urea (15). Yield 54%. 1H NMR (300 MHz, DMSO-d6) δ: 8.62 (s, 1H), 8.51 (s, 1H), 8.43 (d, J = 3.3 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.43−7.27 (m, 5H), 7.05 (t, J = 7.2 Hz, 1H), 6.94−6.87 (m, 4H), 6.70 (t, J = 6.0 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.49 min, m/z = 320.0 [M + H]+. Methyl-4-(3-(pyridin-3-ylmethyl)ureido)benzoate (56). Yield 95%. 1H NMR (300 MHz, CDCl3) δ: 8.51−845 (m, 2H), 7.92−7.88 (m, 3H), 7.66−7.79 (m, 1H), 7.42−7.37 (m 2H), 7.29−7.26 (m, 1H), 5.80 (t, J = 6.3 Hz, 1H), 4.42 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H). LCMS (method LCMS1, ESI): RT = 0.71 min, m/z = 286.3 [M + H]+. 1-[4-(Piperidine-1-sulfonyl)phenyl]-3-(pyridin-3-ylmethyl)urea (9). 53 (60 mg, 0.184 mmol) was suspended in THF (10 mL). To this suspension was added piperidine (54a, 147 mg, 1.72 mmol). The mixture was stirred at room temperature for 1 h. The mixture was then diluted with EtOAc (50 mL), washed with brine (2 × 15 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material was crystallized from diethyl ether to afford 40 mg (58%) of the title compound. 1H NMR (300 MHz, CDCl3) δ: 8.52− 8.45 (m, 2H), 7.92 (s, 1H), 7.68 (d, J = 5.0 Hz, 1H), 7.61−7.45 (m, 4H), 7.32−7.27 (m, 1H), 5.80 (t, J = 3.4 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 2.99−2.90 (m, 4H), 1.48−1.75 (m, 4H), 1.42−1.38 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.00 min, m/z = 375.3 [M + H]+. Compounds 39−40 and 48−49 were synthesized using conditions similar to those employed to make compound 9. 3-[4-(Azepane-1-sulfonyl)phenyl]-1-(pyridin-3-ylmethyl)urea (39). Yield 38%. 1H NMR (DMSO-d6): δ 9.13 (s, 1H), 8.52 (d, J = 1.2 Hz, 1H), 8.45 (dd, J = 1.2, 5.7 Hz, 1H), 7.69 (dt, J = 1.2, 9.0 Hz, 1H), 7.61 (m, 4H), 7.34 (dd, J = 6.0, 12.0 Hz, 1H), 6.88 (t, J = 9.0 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 3.14 (t, J = 6.0 Hz, 4H), 1.58−1.52 (m, 4H), 1.47−1.42 (m, 4H). LCMS (method LCMS1, ESI): RT = 1.11 min, m/z = 389.02 [M + H]+ 3-(Pyridin-3-ylmethyl)-1-[4-(pyrrolidine-1-sulfonyl)phenyl]urea (40). Yield 24%. 1H NMR (DMSO-d6): δ 9.16 (s, 1H), 8.52 (d, J = 1.2 Hz, 1H), 8.45 (dd, J = 1.2, 5.8 Hz, 1H), 7.70−7.59 (m, 5H), 7.38 (dd, J = 6.0, 12.0 Hz, 1H), 6.90 (t, J = 9.0 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 3.10−3.05 (m, 4H), 1.62−1.60 (m, 4H). LCMS (method LCMS2, ESI): RT = 0.83 min, m/z = 360.98 [M + H]+ 3-[4-(Butylsulfamoyl)phenyl]-1-(pyridin-3-ylmethyl)urea (42). To a 2 mL reaction vial containing a solution of n-butyl amine in toluene (0.2 M, 400 uL, 80 umol) was add a solution pyridine in CH2Cl2 (1 M, 160 μL, 160 μmol). A solution of (4-(3-(pyridin-3ylmethyl)ureido)benzene-1-sulfonyl chloride in DMA (0.2 M, 200 μL, 40 umol) was added and the vial was shaken at ambient temperature for 2 h then at 50 °C for 16 h. Methanol (200 uL) was added to the vial, and the mixture was shaken well then transferred to a 0.5 g silica 4933

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Journal of Medicinal Chemistry

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1-(4-((4-Chlorophenyl)thio)phenyl)-3-(pyridin-3-ylmethyl)urea (14). Yield 43%. 1H NMR (DMSO-d6) δ: 8.90 (s, 1H), 8.52− 8.50 (m, 1H), 8.45−8.43 (m, 1H), 7.71−7.67 (m, 1H), 7.51−7.46 (m, 2H), 7.37−7.31 (m, 5H), 7.11−7.06 (m, 2H), 6.81 (t, J = 6.0 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 2.79 min, m/z = 370.0 [M + H]+. 1-(4-Benzoylphenyl)-3-(pyridin-3-ylmethyl)urea (16). Yield 45%. 1H NMR (DMSO-d6): δ 9.16 (s, 1H), 8.53 (s, 1H), 8.45 (s, 1H), 9.72−7.50 (m, 10H), 7.38−7.31 (m, 1H), 6.88 (t, J = 5.1 Hz, 1H), 4.36 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.61 min, m/z = 332.09 [M + H]+. 3-Benzyl-1-[4-(piperidine-1-sulfonyl)phenyl]urea (18). Yield 46%. 1H NMR (300 MHz, DMSO-d6) δ: 9.11 (s, 1H), 7.64−7.51 (m, 4H), 7.35−7.21 (m, 5H), 6.81 (t, J = 6.0 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H), 2.83−2.76 (m, 4H), 1.56−1.45 (m, 4H), 1.34−1.28 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.50 min, m/z = 374.0 [M + H]+. 3-[(2-Fluorophenyl)methyl]-1-[4-(piperidine-1-sulfonyl)phenyl]urea (20). Yield 78%. 1H NMR (300 MHz, DMSO-d6) δ: 9.14 (s, 1H), 7.62−7.51 (m, 4H), 7.38−7.25 (m, 2H), 7.20−7.12 (m, 2H), 6.82 (t, J = 5.8 Hz, 1H), 4.34 (d, J = 6.0 Hz, 2H), 2.83−2.75 (m, 4H), 1.55−1.45 (m, 4H), 1.34−1.27 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.51 min, m/z = 392.0 [M + H]+. 3-[(3-Fluorophenyl)methyl]-1-[4-(piperidine-1-sulfonyl)phenyl]urea (21). Yield 71%. 1H NMR (300 MHz, DMSO-d6) δ: 9.17 (s, 1H), 7.65−7.51 (m, 4H), 7.40−7.31 (m, 1H), 7.14−7.01 (m, 3H), 6.88 (t, J = 5.8 Hz, 1H), 4.31 (d, J = 5.7 Hz, 2H), 2.83−2.76 (m, 4H), 1.53−1.46 (m, 4H), 1.35−1.29 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.52 min, m/z = 392.0 [M + H]+. 1-[(4-Fluorophenyl)methyl]-3-[4-(piperidine-1-sulfonyl)phenyl]urea (22). Yield 92%. 1H NMR (300 MHz, DMSO-d6) δ: 9.13 (s, 1H), 7.64−7.55 (m, 4H), 7.36−7.31 (m, 2H), 7.18−7.13 (m 2H), 6.85−6.81 (m, 1H), 4.30−4.28 (d, J = 5.7 Hz, 2H), 2.84−2.79 (m, 4H), 1.53−1.50 (m, 4H), 1.34−1.32 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.51 min, m/z = 392.0 [M + H]+. 3-[(5-Fluoropyridin-3-yl)methyl]-1-[4-(piperidine-1sulfonyl)phenyl]urea (23). Yield 92%. 1H NMR (300 MHz, DMSO-d6) δ: 9.23 (s, 1H), 8.42 (dd, J = 2.7 Hz, J = 14.4 Hz, 2H), 7.59−7.53 (m, 5H), 6.95 (t, J = 6 Hz, 1H), 4.36 (d, J = 6 Hz 2H), 2.82−2.78 (m, 4H), 1.56−1.46 (m, 4H), 1.38−1.24 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.31 min, m/z = 393.0 [M + H]+. 1-[4-(Benzenesulfonyl)phenyl]-3-{imidazo[1,2-a]pyridin-6ylmethyl}urea (24). Yield 64%. 1H NMR (300 MHz, DMSO-d6) δ: 9.20 (s, 1H), 8.43 (s, 1H), 7.92−7.86 (m, 3H), 7.81−7.77 (m, 2H), 7.64−7.49 (m, 7H), 7.20−7.15 (m, 1H), 6.90−6.84 (m, 1H), 4.30− 4.26 (m, 2H). LCMS (method LCMS1, ESI) RT = 0.94 min, m/z = 407.2 [M + H]+. 3-[4-(Benzenesulfonyl)phenyl]-1-{[6-(1H-imidazol-1-yl)pyridin-3-yl]methyl}urea (26). Yield 17%. 1H NMR (300 MHz, CDCl3): δ 8.39 (s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 7.89−7.86 (m, 2H), 7.77−7.71 (m, 3H), 7.59 (s, 1H), 7.59−7.43 (m, 5H), 7.21−7.17 (m, 2H), 6.08 (s, 1H), 4.43 (d, J = 6 Hz, 2H). LCMS (method LCMS1, ESI): RT = 0.98 min, m/z = 434.2 [M + H]+ 1-[4-(Benzenesulfonyl)phenyl]-3-(1H-pyrazol-3-ylmethyl)urea (27). Yield 53%. 1H NMR (300 MHz, DMSO-d6) δ: 12.60 (s, 1H), 9.11 (s, 1H), 7.90−7.86 (m, 2H), 7.81−7.77 (m, 2H), 7.65−7.55 (m, 6H), 6.67−6.63 (m, 1H), 6.27−6.24 (m, 1H), 4.25−4.21 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.74 min, m/z = 357.0 [M + H]+. 4-(Piperidine-1-sulfonyl)phenyl N-(Pyridin-3-ylmethyl)carbamate (28). Yield 45%. 1H NMR (300 MHz, CDCl3) δ: 8.60−8.49 (m, 2H), 7.73−7.70 (m, 3H), 7.31−7.26 (m, 3H), 6.09 (s, 1H), 4.45 (d, J = 6.0 Hz, 2H), 2.97−2.94 (m, 4H), 1.75−1.55 (m, 4H), 1.52−1.39 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.43 min, m/z = 376.06 [M + H]+. 3-[4-(Benzenesulfonyl)phenyl]-1-(pyridin-3-yl)urea (34). Yield 34%. 1H NMR (300 MHz, DMSO-d6) δ: 9.36 (s, 1H), 9.00 (s, 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.20 (dd, J = 4.5, 1.2 Hz, 1H), 7.95− 7.82 (m, 5H), 7.69−7.52 (m, 5H), 7.33−7.27 (m, 1H). LCMS (method LCMS1, ESI): RT = 1.07 min, m/z = 354.1 [M + H]+.

mL) was added Cu(OAc)2 (33 mg, 0.18 mmol). The mixture was stirred at 60 °C for 16 h. Water (8 mL) and EtOAc (8 mL) were added, and the layers were separated. The aqueous layer was extracted with EtOAc (8 mL), and the extract was washed with brine (10 mL), dried, and evaporated. The residue was purified by preparative TLC with 100:7.5:0.75 CH 2Cl2 /MeOH/NH4 OH to give the title compound as an off-white solid (14 mg, 31%). 1H NMR (400 MHz, CDCl3) δ: 8.47 (s, 1H), 8.44 (s, 1H), 8.26 (s, 1H), 7.79 (s, 1H), 7.71− 7.65 (m, 4H), 7.43 (d, J = 8.8 Hz, 2H), 7.26−7.23 (m, 1H), 6.12 (t, J = 6.4 Hz, 1H), 4.39 (d, J = 6.0 Hz, 2H), 3.88 (s, 3H). LCMS (method LCMS2, ESI): RT = 0.67 min, m/z = 372.1 [M + H]+. 4-(3-(Pyridin-3-ylmethyl)ureido)benzoic Acid (57). 56 (750 mg, 2.63 mmol) was treated with LiOH (5.26 mL of a 1.0 M aqueous solution, 5.26 mmol) in MeOH (25 mL) at room temperature for 16 h. The solvent was removed, and the residue was suspended in 20 mL of water and then was washed with EtOAc (3 × 20 mL). The aqueous layer was acidified to pH 4−5 with 1 M HCl. The resulting precipitate was collected, washed with water, and dried to give the desired product (410 mg, 58%). 1H NMR (300 MHz, DMSO-d6) δ: 12.45 (br, 1H), 9.01 (s, 1H), 8.53 (s, 1H), 8.40 (s, 1H), 7.83−7.75 (m, 2H), 7.71− 7.68 (m, 1H), 7.50−7.42 (m, 2H), 7.36−7.28 (m, 1H), 6.85−6.72 (m, 1H), 4.30 (d, J = 6.1 Hz, 2H). LCMS (method LCMS1, ESI): RT = 0.37 min, m/z = 272.3 [M + H]+. 1-[4-(Piperidine-1-carbonyl)phenyl]-3-(pyridin-3-ylmethyl)urea (17). 57 (100 mg, 0.369 mmol), piperidine (37.7 mg, 0.442 mmol), and Et3N (0.062 mL, 0.442 mmol) were dissolved in DMA (5 mL). To this solution, BOP (196 mg, 0.442 mmol) was added. The mixture was stirred 6 h at room temperature. The reaction mixture was diluted with water (50 mL), and the aqueous layer was back-extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with saturated, aqueous NH4Cl (3 × 15 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was crystallized from EtOAc to afford the title compound (46 mg, 45%). 1H NMR (300 MHz, CDCl3) δ: 8.56−8.42 (m, 2H), 8.02 (s, 1H), 7.71−7.62 (m, 1H), 7.30−7.22 (m, 3H), 7.18−7.08 (m, 2H), 6.30 (t, J = 5.8 Hz, 1H), 4.41(t, J = 6.3 Hz, 2H), 3.40−3.35 (m, 4H), 2.02−1.70 (m, 4H), 1.72−1.60 (m, 2H). LCMS (method LCMS1, ESI): RT = 0.83 min, m/z = 339.4 [M + H]+. 1-[3-(Piperidine-1-sulfonyl)phenyl]-3-(pyridin-3-ylmethyl)urea (10). A solution of 3-(piperidin-1-ylsulfonyl)aniline (58a; 240 mg, 0.999 mmol) and Et3N (1.28 mmol) in CH2Cl2 (5 mL) was added dropwise to a cooled solution (−10 °C) of triphosgene (98 mg, 0.330 mmol) in CH2Cl2 (6 mL). The mixture was stirred for 1 h at room temperature followed by addition of a solution of pyridin-3ylmethanamine (51a; 108 mg, 0.999 mmol) and Et3N (1.28 mmol) in CH2Cl2 (5 mL). The mixture was then stirred for 16 h at room temperature. The reaction was diluted with CH2Cl2 (25 mL), and the organic layer was washed with 1N NaOH, water, and saturated, aqueous NaCl (15 mL). The organic layer was dried with anhydrous MgSO4, filtered, and concentrated under reduced pressure to give the crude product which was purified by Biotage and eluted with 5% MeOH−CH2Cl2. The collected product was solidified from Et2O to offer the desired compound as a white solid (330 mg, 88% yield). 1H NMR (300 MHz, DMSO-d6) δ: 9.08 (s, 1H), 8.51 (d, J = 1.8 Hz, 1H), 8.45−8.41 (m, 1H), 7.90 (t, J = 1.8 Hz, 1H), 7.72−7.66 (m, 1H), 7.62−7.56 (m, 1H), 7.42 (t, J = 7.5 Hz, 1H), 737−7.31 (m, 1H), 7.23−7.10 (m, 1H), 6.83 (t, J = 5.7 Hz, 1H), 4.31 (d, J = 5.7 Hz, 2H), 2.87−2.80 (m, 4H), 1.62−1.45 (m, 4H), 1.39−1.30 (m, 2H). LCMS (method LCMS1, ESI): RT = 0.98 min, m/z = 375.1 [M + H]+. Compounds 11, 14, 16, 18, 20−24, 26−28, 34−37, 41, 47, 50, and 59 were synthesized using conditions similar to those employed to make compound 10. 1-[4-(Benzenesulfonyl)phenyl]-3-(pyridin-3-ylmethyl)urea (11). Yield 55% yield. 1H NMR (300 MHz, CDCl3) δ: 9.20 (s, 1H), 8.55−8.49 (m, 1H), 8.45−8.40 (m, 1H), 7.90−7.81 (m, 2H), 7.80− 7.65 (m, 2H), 7.70−7.54 (m, 6H), 7.35−7.26 (m, 1H), 6.89 (t, J = 5.7 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H), LCMS (method LCMS1, ESI): RT = 0.95 min, m/z = 368.2 [M + H]+. 4934

dx.doi.org/10.1021/jm400186h | J. Med. Chem. 2013, 56, 4921−4937

Journal of Medicinal Chemistry

Article

3-[4-(Piperidine-1-sulfonyl)phenyl]-1-[2-(pyridin-3-yl)ethyl]urea (35). Yield 57%. 1H NMR (DMSO-d6): δ 9.00 (s, 1H), 8.44− 8.40 (m, 2H), 7.67−7.66 (m, 1H), 7.60−7.52 (m, 4H), 7.34−7.30 (m, 1H), 6.34 (t, J = 5.7 Hz, 1H), 3.39−3.35 (m, 2H), 2.82−2.52 (m, 6H), 1.56−1.48 (m, 4H), 1.32−1.33 (m, 2H). LCMS (method LCMS1, ESI): RT = 0.99 min, m/z = 389.03 [M + H]+ 3-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1-(pyridin-3ylmethyl)urea (36). Yield 20%. 1H NMR (300 MHz, DMSO-d6) δ: 8.45 (s, 1H), 8.49−8.42 (m, 1H), 7.68−7.59 (m, 3H), 7.45 (d, J = 3.0 Hz, 2H), 7.33−7.29 (m, 1H), 6.71−6.60 (m, 2H), 4.31 (d, J = 5.7 Hz, 2H), 4.23 (d, J = 5.7 Hz, 2H), 2.85−2.82 (m, 4H), 1.53−1.48 (m, 4H), 1.35−1.29 (m, 2H). LCMS (method LCMS1, ESI): RT = 0.95 min, m/z = 389.1 [M + H]+. 3-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1-(pyridin-3-yl)urea (37). Yield 68%. 1H NMR (300 MHz, DMSO-d6) δ: 8.90 (s, 1H), 8.54 (m, 1H), 8.11 (dd, J = 1.2, 4.5 Hz, 1H), 7.92−7.85 (m, 1H), 7.70−7.64 (m, 2H), 7.54−7.49 (m, 2H), 7.27−7.20 (m, 1H), 6.91 (t, J = 5.7 Hz, 1H), 4.40 (d, J = 6.0 Hz, 2H), 2.88−2.79 (m, 4H), 1.55− 1.47 (m, 4H), 1.35−1.30 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.01 min, m/z = 375.2 [M + H]+. 3-[4-(Azetidine-1-sulfonyl)phenyl]-1-(pyridin-3-ylmethyl)urea (41). Yield 43%. 1H NMR (300 MHz, DMSO-d6): δ 9.26 (s, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.44 (dd, J = 1.8, 4.8 Hz 1H), 7.66−7.58 (m, 5H), 7.37−7.32 (m, 1H), 6.95 (t, J = 6.3 Hz, 1H), 4.33 (d, J = 6.3 Hz, 2H), 3.58 (t, J = 7.8 Hz 4H), 1.96−1.91 (m, 2H). LCMS (method LCMS1, ESI): RT = 0.72 min, m/z = 347.14 [M + H]+ 3-{4-[(4-Methylpiperidin-1-yl)sulfonyl]phenyl}-1-(pyridin-3ylmethyl)urea (47). Yield 16%. 1H NMR (300 MHz, DMSO-d6): δ 9.17 (s, 1H), 8.51 (s, 1H), 8.45−8.43 (m, 1H), 7.71−7.67 (m, 1H), 7.62−7.53 (m, 4H), 7.36−7.32 (m, 1H), 6.92−6.89 (m, 1H), 4.31 (d, J = 6.3 Hz, 2H), 3.53 (d, J = 11.4 Hz, 2H), 2.11−2.04 (m, 2H), 1.65− 1.57 (m, 2H), 1.22−1.18 (m, 1H), 1.12−1.05 (m, 2H), 0.82 (d, J = 6.3 Hz, 3H). LCMS (method LCMS1, ESI): RT = 0.99 min, m/z 389.03 [M + H]+ 1-(4-{8-Oxa-3-azabicyclo[3.2.1]octane-3-sulfonyl}phenyl)-3(pyridin-3-ylmethyl)urea (50). Yield 78%. 1H NMR (300 MHz, DMSO-d6) δ: 9.20 (s, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.46−8.42 (m, 1H), 7.72−7.67 (m, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H), 7.38−7.30 (m, 1H), 6.92 (t, J = 6.0 Hz, 1H), 4.32 (d, J = 6.0 Hz, 4H), 3.20 (d, J = 11.1 Hz, 2H), 2.38 (d, J = 9.3 Hz, 2H), 1.75 (s, br,, 4H). LCMS (method LCMS1, ESI): RT = 1.11 min, m/z = 403.14 [M + H]+. tert-Butyl-3-((3-(4-(phenylsulfonyl)phenyl)ureido)methyl)phenylcarbamate (59). Yield 85%. 1H NMR (300 MHz, CDCl3) δ: 7.87−7.82 (m, 2H), 7.73−7.65 (m, 2H), 7.52−7.42 (m, 4H), 7.40− 7.37 (m, 2H), 7.29 (s, 1H), 7.15−7.10 (m, 2H), 6.88−6.84 (m, 1H), 6.65 (s, 1H), 5.51 (t, J = 5.7 Hz, 1H), 4.24 (d, J = 5.7 Hz, 2H), 1.47 (s, 9H). LCMS (method LCMS1, ESI): RT = 2.24 min, m/z = 482.1 [M + H]+. 3-[(3-Aminophenyl)methyl]-1-[4-(benzenesulfonyl)phenyl]urea (19). A solution of 59 (390 mg, 0.810 mmol) and trifluoroacetic acid (5 mL, 64.9 mmol) in CH2Cl2 (10 mL) was stirred at ambient temperature for 16 h. The mixture was concentrated and partitioned between CH2Cl2 and saturated NaHCO3. The organic layer was washed with brine (2 × 15 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by Biotage using 10% MeOH/ CH2Cl2 to offer the title compound (180 mg, 58%). 1H NMR (300 MHz, DMSO-d6): δ 9.12 (s, 1H), 8.55−8.49 (m, 2H), 8.45−8.40 (m, 2H), 7.90−7.81 (m, 5H), 6.90 (t, J = 7.6 Hz, 1H), 6.69 (t, J = 5.8 Hz, 1H), 6.50−6.40 (m, 3H), 5.41 (br, s, 2H), 4.13 (d, J = 5.7 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.16 min, m/z = 382.28 [M + H]+. 3-[4-(4-Chlorobenzenesulfonyl)phenyl]-1-(pyridin-3ylmethyl)urea (13). A solution of 14 (50 mg, 0.135 mmol) in 10:1 CH2Cl2:MeOH (3.3 mL) was treated with mCPBA (62 mg, 0.270 mmol), and the solution was stirred for 16 h at ambient temperature. The mixture was then diluted with saturated NaHCO3 (10 mL) and CH2Cl2 (10 mL) and the layers separated. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by preparatory TLC (6% MeOH/CH2Cl2) to obtain the title compound (8 mg, 15%). 1H NMR (300 MHz, DMSO-d6) δ: 9.24 (s, 1H), 8.49 (s,

1H), 8.43−8.42 (m, 1H), 7.90−7.86 (m, 2H), 7.81−7.77 (m, 2H), 7.68−7.58 (m, 5H), 7.35−7.31 (m, 1H), 6.92 (t, J = 6.0 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.69 min, m/z = 402.0 [M + H]+. 4-Nitrophenyl-N-[4-(benzenesulfonyl)phenyl]carbamate (61a). A solution of 4-(benzenesulfonyl)aniline (58b; 300 mg, 1.29 mmol) and 4-nitrophenyl chloroformate (60a; 310.6 mg, 1.54 mmol) in toluene (10 mL) was stirred at 85 °C for 3 h. The resulting mixture was concentrated under vacuum to give 620 mg (>100%) of crude product as an off-white solid, which was used for next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ: 10.97 (s, 1H), 8.35 (d, J = 9.2 Hz, 2H), 8.10−7.90 (m, 4H), 7.80−7.50 (m, 7H). Pyridin-3-ylmethyl N-[4-(Benzenesulfonyl)phenyl]carbamate (29). A solution of pyridin-3-ylmethanol (62a; 80 mg, 0.73 mmol) and 61a (292 mg, 0.73 mmol) in ethanol (4 mL) was refluxed for 3 h. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was purified by PrepHPLC with the following conditions: Column, Xbridge Shield RP 18, 5 um, 19 mm × 150 mm; mobile phase, water with 50 mmol NH4HCO3 and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min). Detector, UV 254 nm to give 73.7 mg (27%) of the desired compound as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 8.58−8.52 (m, 2H), 7.73−7.70 (m, 3H), 7.29−7.26 (m, 3H), 6.09 (s, 1H), 4.45 (d, J = 6 Hz, 2H), 2.97−2.93 (m, 4H), 1.70−1.56 (m. 4H), 1.41−1.38 (m, 2H). LCMS (method LCMS3, ESI): RT = 1.43 min, m/z = 369.1 [M + H]+. Phenyl-(4-(phenylsulfonyl)phenyl)carbamate (61b). To the mixture of 4-(benzenesulfonyl)aniline (58b; 0.6 g, 2.57 mmol) and K2CO3 (1.4 g, 10.3 mmol) in THF (15 mL) was added phenyl carbonochloridate (60b; 2.0 g, 12.9 mmol). The reaction mixture was stirred at room temperature for 20 min and then was concentrated to half volume under vacuum. Water (10 mL) and brine (5 mL) were added to the reaction mixture and the product precipitated as a solid. The crude white solid was collected by filtration, washed twice with Et2O, and dried under high vacuum to yield the title compound (0.78 g, 86%). This material was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ: 10.72 (s, 1H), 7.96− 7.88 (m, 4H), 7.76−7.57 (m, 5H), 7.43 (t, J = 7.7 Hz, 2H), 7.31−7.19 (m, 3H). LCMS (method LCMS4, ESI): RT = 0.83 min, m/z = 234.1 [M + H]+. 3-[4-(Benzenesulfonyl)phenyl]-1-methyl-1-(pyridin-3ylmethyl)urea (31). To N-methyl-1-(pyridin-3-yl)methanamine (62b; 17 mg, 0.140 mmol) and Et3N (0.06 mL, 0.42 mmol) in 1,4dioxane (1 mL) was added 61b (50 mg, 0.140 mmol), and the reaction was heated to 60 °C for 1 h. The compound was purified by preparative HPLC (column, Gemini-NX, 3 cm × 10 cm, 10 μm; detection: UV 254 nm; mobile phase A, H2O containing 0.1% NH4OH; mobile phase B, acetonitrile; flow rate, 60 mL/min; gradient, 0−1 min 5% B, 1−10 min. 5−50% B, 10−11 min, 50% B, 11−11.2 min, 50−95% B, 11.2−13 min, 95% B, 13−13.2 min, 95−5% B, 13.2− 15 min, 5% B). Isolation and concentration of the appropriate fractions afforded the title compound as a white solid (49 mg, 91%). 1 H NMR (400 MHz, DMSO-d6) δ: 8.93 (s, 1H), 8.54−8.44 (m, 2H), 7.90 (dd, J = 7.3, 1.8 Hz, 2H), 7.82 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.9 Hz, 2H), 7.70−7.63 (m, 2H), 7.60 (dd, J = 8.3, 6.7 Hz, 2H), 7.37 (dd, J = 7.8, 4.8 Hz, 1H), 4.57 (s, 2H), 2.96 (s, 3H). LCMS (method LCMS5, ESI): RT = 3.35 min, m/z = 382.0 [M + H]+. 1-[4-(Benzenesulfonyl)phenyl]-3-{5H,6H,7H,8H-imidazo[1,2a]pyridin-6-ylmethyl}urea (25). This compound was synthesized using conditions similar to those employed to make compound 31. Yield 61%. 1H NMR (300 MHz, DMSO-d6) δ: 9.08 (s, 1H), 7.90− 7.85 (m, 2H), 7.80−7.75 (m, 2H), 7.64−7.55 (m, 5H), 6.97 (d, J = 6.0 Hz, 1H), 6.79 (d, J = 6.0 Hz, 1H), 6.57−6.52 (m, 1H), 4.05−3.98 (m, 1H), 3.60−3.52 (m, 1H), 3.18−3.14 (m, 2H), 2.79−2.75 (m, 1H), 2.68−2.63 (m, 1H), 2.16−2.06 (m, 1H), 1.97−1.88 (m, 1H), 1.60− 1.48 (m, 1H). LCMS (method LCMS1, ESI): RT = 1.30 min, m/z = 411.2 [M + H]+. 3-[4-(Benzenesulfonyl)phenyl]-1-[1-(pyridin-3-yl)ethyl]urea (33). This compound was synthesized using conditions similar to 4935

dx.doi.org/10.1021/jm400186h | J. Med. Chem. 2013, 56, 4921−4937

Journal of Medicinal Chemistry

Article

those employed to make compound 31, 52 mg, 96% yield. 1H NMR (400 MHz, DMSO-d6) δ: 9.05 (s, 1H), 8.56 (d, J = 2.2 Hz, 1H), 8.44 (dd, J = 4.7, 1.4 Hz, 1H), 7.93−7.83 (m, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.73 (dt, J = 8.0, 2.0 Hz, 1H), 7.65 (t, J = 7.2 Hz, 1H), 7.62−7.51 (m, 4H), 7.35 (dd, J = 7.9, 4.8 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 4.84 (p, J = 7.1 Hz, 1H), 1.42 (d, J = 7.0 Hz, 3H). LCMS (method LCMS5, ESI): RT = 3.41 min, m/z = 382.0 [M + H]+. N-[4-(Benzenesulfonyl)phenyl]-2-(pyridin-3-yloxy)acetamide (30). A mixture of 2-(pyridin-3-yloxy)acetyl chloride (110 mg, 0.64 mmol), 4-(benzenesulfonyl)aniline (58b, 150 mg, 0.64 mmol), and Et3N (190 mg, 1.88 mmol) in CH2Cl2 (5 mL) was stirred at room temperature for 4 h. The reaction mixture was quenched with 5 mL of H2O and then extracted with 3 × 10 mL of CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: column, SunFire Prep C18, 19 mm × 150 mm 5 μm; mobile phase, water with 0.05% NH4HCO3 and CH3CN (5% CH3CN up to 43% in 7 min).Detector, UV 254 nm to give 79.1 mg (33%) of the title compound as a white solid. 1H NMR (300 MHz, CD3OD) δ: 8.40 (s, 1H), 8.20 (d, J = 4.5 Hz, 1H), 7.97−7.86 (m, 6H), 7.78−7.50 (m, 4H), 7.39 (m, 1H), 4.81 (s, 2H). LCMS (LCMS6, ESI): RT = 1.83 min, m/z = 369.0 [M + H]+. N-[4-(Benzenesulfonyl)phenyl]-3-(pyridin-3-yl)propanamide (32). A mixture of 4-(benzenesulfonyl)aniline (58b, 100 mg, 0.43 mmol), 3-(pyridin-3-yl)propanoyl chloride hydrochloride (88 mg, 0.43 mmol), and Et3N (130 mg, 1.28 mmol) in CH2Cl2 (10 mL) was refluxed for 3 h. The reaction mixture was concentrated under vacuum, and the crude product was purified by Prep-HPLC with the following conditions: Column, Xbridge Shield RP 18, 5 um, 19 mm × 150 mm; mobile phase, water with 50 mmol NH4HCO3 and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min). Detector, UV 254 nm to give 82.1 mg (52%) of the title compound as an off-white solid. 1H NMR (300 MHz, CDCl3) δ: 8.57 (s, 1H), 8.46 (s, 1H), 8.33 (s, 1H), 7.97−7.87 (m, 4H), 7.68 7.59 (m, 3H), 7.55−7.48 (m, 3H), 7.30−7.37 (m, 1H), 3.06 (t, J = 7.2 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H). LCMS (method LCMS6, ESI): RT = 1.35 min, m/z = 367.0 [M + H]+. 2-Chloroethyl-N-(4-(phenylsulfonyl)phenyl)sulfamoylcarbamate (64). Sulfurisocyanatidic chloride (0.283 g, 2.00 mmol) was dissolved in CH2Cl2 (20 mL) and cooled to 0 °C. To this cooled solution was added 2-chloroethanol (0.161 g, 2.00 mmol), and the mixture was stirred at 0−5 °C for 1.5 h. A suspension of 4(benzenesulfonyl)aniline (58b, 0.467 g, 2.00 mmol) and Et3N (0.279 mL, 2.00 mmol) in CH2Cl2 (10 mL) was added, and the resulting mixture was warmed to room temperature and stirred for 12 h. The reaction was quenched with 1N HCl to pH = 7. The organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure. The crude material thus obtained was used in the next step without further purification (0.76 g, 69% yield). LCMS (method LCMS1, ESI): RT = 1.75 min, m/z = 436.0 [M + NH4]+. N-(Pyridin-3-ylmethyl){[4-(benzenesulfonyl)phenyl]amino}sulfonamide (38). To the solution of compound 64 (0.76 g, 1.814 mmol) in ACN (15 mL), pyridin-3-ylmethanamine (51a; 0.196 g, 1.814 mmol) and TEA (0.506 mL, 3.63 mmol) were added. The mixture was heated to 80 °C for 16 h. After removal of the solvent, the crude product was purified by Biotage using MeOH−CH2Cl2 from 0% to 12% to give the desired product (0.10 g, 14%). 1H NMR (300 MHz, DMSO-d6) δ: 10.47 (s, 1H), 8.50−8.39 (m, 1H), 8.36−8.29 (m, 2H), 7.93−7.87 (m, 2H), 7.84−7.74 (m, 2H), 7.69−7.55 (m, 3H), 7.48−7.43 (m, 1H), 7.20−7.14 (m, 2H), 6.99−6.92 (m, 1H), 4.03 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT = 1.30 min, m/z = 404.0 [M + H]+.

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expression/purification and crystallographic methods and procedures for 5 (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 code 4JR5 for 5 complexed with Nampt.

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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 for their synthetic contributions and the ADME group for generating the microsomal and solubility data, Rashida Garcia-Dancey for helping generating the cellular data, Lakshmanan Manikandan, Saradhi Vijay, and Danilal C. Sharma for generating the in vivo PK and efficacy data, Agilent Technologies (Woburn, MA) for generating the Rapidfire LCMS data, Tandem Laboratories (Woburn, MA) for performing the phosphoribosylated adduct study, Professor Yigong Shi’s group (Tsinghua University, Beijing, China) for providing Nampt DNA construct and Nampt protein, and James Kyranos for helpful discussions.

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ABBREVIATIONS USED SCID, severe combined immunodeficiency; MLM, mouse liver microsome; HLM, human liver microsome; Cmax, maximum concentration; Tmax, time to reach the maximum concentration; Cl, clearance; Vss, volume distribution; Na2SO3, sodium sulfite; NaHCO3, sodium bicarbonate; Cu(OAc)2, copper acetate; Et3N, triethylamine; LiOH, lithium hydroxide; MeOH, methanol; BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; CH2Cl2, dichloromethane; EtOH, ethanol; Et2O, diethyl ether; CH3CN, acetonitrile; EtOAc, ethyl acetate; brine, a saturated aqueous solution of sodium chloride; MgSO4, magnesium sulfate; SCX, strong cationic exchange; NH4OH, ammonium hydroxide; Na2SO4, sodium sulfate; NaOH, sodium hydroxide; NH4HCO3, ammonium bicarbonate; NaCl, sodium chloride; MgCl2, magnesium chloride; THP, tris(hydroxypropyl)phosphine; D5W, 5% dextrose in water

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Details of computational methods, details of analytical LCMS and LC/MS/MS methods, in vitro and in vivo ADME and experimental procedures, experimental details for PK and efficacy study of compound 50 in the mouse, protein 4936

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