Antibacterial Inhibitors of Gram-Positive Thymidylate Kinase: Structure

Article pubs.acs.org/jmc

Antibacterial Inhibitors of Gram-Positive Thymidylate Kinase: Structure−Activity Relationships and Chiral Preference of a New Hydrophobic Binding Region Sameer P. Kawatkar,*,†,§ Thomas A. Keating,†,∥ Nelson B. Olivier,‡ John N. Breen,†,‡ Oluyinka M. Green,† Satenig Y. Guler,† Martin F. Hentemann,†,⊥ James T. Loch,† Andrew R. McKenzie,† Joseph V. Newman,† Linda G. Otterson,† and Gabriel Martínez-Botella†,# †

Infection Innovative Medicines and ‡Discovery Sciences, AstraZeneca, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States S Supporting Information *

ABSTRACT: Thymidylate kinase (TMK), an essential enzyme in bacterial DNA biosynthesis, is an attractive therapeutic target for the development of novel antibacterial agents, and we continue to explore TMK inhibitors with improved potency, protein binding, and pharmacokinetic potential. A structureguided design approach was employed to exploit a previously unexplored region in Staphylococcus aureus TMK via novel interactions. These efforts produced compound 39, with 3 nM IC50 against S. aureus TMK and 2 μg/mL MIC against methicillin-resistant S. aureus (MRSA). This compound exhibits a striking inverted chiral preference for binding relative to earlier compounds and also has improved physical properties and pharmacokinetics over previously published compounds. An example of this new series was efficacious in a murine S. aureus infection model, suggesting that compounds like 39 are options for further work toward a new Gram-positive antibiotic by maintaining a balance of microbiological potency, low clearance, and low protein binding that can result in lower efficacious doses.

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INTRODUCTION Resistance to antibiotic agents represents one of the most pressing challenges to the infectious disease community1,2 as it has rendered marketed antibiotics less effective against many common bacterial pathogens. The problem of widespread and increasing antibiotic resistance is only getting worse with the dearth of novel classes of antibiotics, creating a significant unmet medical need in the treatment of serious bacterial infections. One of the strategies to effectively tackle the problem of antibacterial resistance is to develop agents that have a novel mode of action or act through a novel target. In this respect, bacterial thymidylate kinase (TMK), which catalyzes the phosphorylation of dTMP to dTDP using ATP as a cosubstrate, represents a very attractive therapeutic target that is essential for bacterial survival.3−5 This is also an area of active antiviral research.6 Recently, we described the discovery of a potent and specific inhibitor of TMK (1, TK-666; Figure 1) from a thymine-based lead (2).7 Compound 1 has excellent solubility, moderate clearance, broad-spectrum Gram-positive TMK enzyme inhibition, and rapid bactericidal activity. It was also efficacious in a murine model of Staphylococcus aureus infection.4 However, the series was also highly protein bound, and this, coupled with moderate clearance, led to a relatively © XXXX American Chemical Society

high single dose of 150 mg/kg to achieve infection stasis in the mouse. As we continue to explore inhibitors of Gram-positive TMK, we aim for overall improvements in potency, protein binding, and/or clearance through specific design and testing of new compounds. Herein, we describe the design, synthesis, and structure− activity relationships for a separate chemical series derived from the core scaffold of 2 by targeting a previously unexplored region adjacent to ring C (Figure 1B). This new series is distinct from previous compounds in three ways. First, X-ray crystal structures of bound complexes showed newly formed interactions, as well as the steric limits of the functional groups that could be added to the inhibitor scaffold at position C-2. Second, selected examples also possessed significantly reduced protein binding compared to previously reported compounds. Last, and most strikingly, the lead compounds reported here also have a different diastereomeric preference for optimal enzyme potency as compared to the earlier TMK inhibitor series. These features offer new avenues for exploration of quality inhibitors that can be optimized for clinical candidacy. In profiling, this new series yielded TMK inhibitors with Received: December 19, 2013

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dx.doi.org/10.1021/jm500463c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Table 1. Activity of C-2-Substituted Analogues of 7 and 8a

a

compd

R

R1

R2

S. aureus TMK IC50 (nM)

7 8 9 10 11 12 13 14 15 16

H F F F F F H F F H

Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl

H H Me Pr OMe OEt F Cl Br CN

1170 2060 9260 67400 474 625 86 779 581 1190

R2, the C-2 substituent, is highlighted in blue.

Table 2. Activity of Carboxylic Acid Analogues of C-2Methoxy- and C-2-Fluoro-Substituted Compoundsa

Figure 1. (A) Structures of thymidylate kinase (TMK) inhibitor 1 (TK-666), an efficacious inhibitor of Gram-positive TMK, and lead compound 2.4,7 The rings are lettered from A to D for the discussion in the text. Compounds with C-2 substitution are the focus of this work. (B) X-ray structure of 2 in complex with S. aureus TMK. The hydrophobic patch formed by conserved residues Val51, Leu52, and Phe66 was targeted with C-2 substitutions (right panel). In particular, additional hydrophobic interactions (left arrow) and novel dipole− dipole/C−H···O interactions with Phe66 (right arrow) were sought.

compd

R2

S. aureus TMK IC50 (nM)

Sau/SauMRQRb MIC (μg/mL)

log D

17 18 19

H OMe F

73 10 12

32/32 8/16 32/16

−0.55 −0.77 −0.59

excellent activity against Gram-positive TMK, efficacy in a S. aureus murine thigh infection model, and somewhat improved pharmacokinetics versus compounds reported earlier.7

a

R2, the C-2 substituent, is highlighted in blue. bBacterial strains from AstraZeneca collection: S. aureus ARC516/S. aureus ARC517 methicillin-resistant (MR) and quinolone-resistant (QR).

CHEMISTRY The overall approach to prepare the analogues in Tables 1 and 2 required the formation of the carbon−nitrogen bond linking rings B and C.7,8 The key chiral amine 6 and the appropriate aldehyde or mesylate were the main synthetic targets (Schemes 1−3). The chiral amine 6 has previously been described.7,8 For the analogues in Table 1, variation of the C-2 substituent was the main goal, and those compounds were readily accessed by starting with commercially available 3-methoxybenzaldehyde 3 and then by installing ring D via copper-mediated coupling of phenylboronic acids (Scheme 1). The final compound was assembled by standard reductive amination with amine 6. The approach to install the carboxylic acid in ring C for both methoxy and fluoride analogues was analogous (Schemes 2 and 3), involving a palladium-mediated formation of the cyanophenyl ring using zinc cyanide and the corresponding bromophenyl intermediate 24 or 30. Standard alkaline hydrolysis provided the carboxylic acids in good yields. For the C-2 methoxy analogues, the installation of ring A and the alkyl side chain on the carbon linking rings B and C was analogous to the routes described by us previously7,8 (Scheme 2). In this instance, the route was modified to prepare the C-2 fluoro analogues (Scheme 3). First, the aliphatic side chain was

introduced in the initial step of the synthesis by metalation and addition of an aldehyde to 1,4-dibromo-2,3-difluorobenzene (27). Mesylate formation from the resulting alcohol 28, followed by alkylation of amine 6, yielded the bromide intermediate 30, which was converted to the corresponding cyano intermediate 31. Activation of the C-3 fluoride by the adjacent cyano group in 31 allowed the introduction of ring D in 32 by microwave-mediated etherification. Standard basic hydrolysis of the nitrile provided the carboxylic acid 33. Finally, single diastereomers of analogues in Table 2 were obtained by chiral separation. While the individual chemical steps described here are not optimized for yield, all yields were sufficient to obtain the desired compounds in amounts suitable for the profiling described.

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RESULTS AND DISCUSSION As disclosed previously,7 our efforts to design inhibitors of Gram-positive TMK originated in thymine-based lead compound 2, which showed moderate enzymatic activity against S. aureus thymidylate kinase (Figure 1). Further exploration of ring D substituents led to compounds 7 and 8 with improved biochemical potency against S. aureus TMK (Table 1). The Xray structure7 of 2 bound to S. aureus TMK (Figure 1) showed B



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Scheme 1. Synthesis of Compound 13a

Reagents and conditions: (a) HBr, 80 °C, 45 min, 46%; (b) 3-chlorophenylboronic acid, Cu(OAc)2, DEA, 3 Å molecular sieves, rt, 72 h, 48%; (c) polymer-supported cyanoborohydride, DMF/AcOH (18:1), rt, 18 h, 35%.

a

Scheme 2. Synthesis of Compounds 38 and 39a

Reagents and conditions: (a) 3-chlorophenylboronic acid, Cu(OAc)2, pyridine, 1,2-DCE, 72 h, 55 °C, 35%; (b) EtMgBr, Et2O, 2 h, −78 °C, 36%; (c) MsCl, Et3N, DCM, 2 h, 0 °C, >95%; (d) 6, ACN, 12−36 h, 80 °C, 16%; (e) Zn(CN)2, Pd(PPh3)4, DMF, 45 min, microwave, 110 °C; (f) NaOH, EtOH, 12 h, reflux, 31% (two steps); (g) chiral separation, 30% each. a

the thymine moiety (ring A) making hydrogen bond interactions with residues Arg70 and Gln101 and π−π stacking interactions with Phe66. Ring D also makes an edge-to-face interaction with Phe66. The phenyl (ring C) and piperidine (ring B) rings did not appear to make any specific interactions with the enzyme and instead executed a U-turn to properly position rings A and D for their direct interactions. Analysis of the X-ray structure also revealed an additional small hydrophobic region formed by Val51 and Leu52, close to the C-2 carbon of ring C (Figure 1B). We hypothesized that small aliphatic residues could form favorable, buried hydrophobic interactions with these side chains. In addition to targeting this hydrophobic patch with aliphatic substituents, the

proximity of Phe66 to C-2 also prompted us to evaluate alkoxy and halogen substituents for potential interactions with Phe66. We thus embarked on a design and modeling effort to explore this opportunity. In the design and testing phases, we paid close attention to log D as this parameter was found to be critical for S. aureus microbiological activity.7 Molecular modeling of C-2 alkyl substituents suggested that small, aliphatic moieties could positively interact with the side chains of Val51 and Leu52 without altering the position of the remainder of the inhibitor. Many studies have shown that hydrophobic interactions play a crucial role in stabilizing protein structure,9,10 protein−protein interactions,11 and protein−ligand complexes.12−14 While increasing hydrophobicC



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Scheme 3. Synthesis of Compounds 46 and 47a

iPrMgCl−LiCl, 3-methylbutanal, THF, −20 °C, 100%; (b) MsCl, TEA, DCM; (c) DIEA, MeCN, 80 °C, 16% (for two steps); (d) Zn(CN)2, Pd(PPh3)4, 140 °C, microwave, 67%; (e) 3-chlorophenol, K2CO3, 160 °C, microwave, 71%; (f) NaOH, MeOH, H2O, 80 °C, 62%; (g) chiral separation, 38% each. a

moieties in alkyl ethers and protein residues in the targeted region (Supporting Information). Specificially, methoxy was accommodated in the model, but ethoxy appeared too large and forced a flip in modeled ring C. Similar to the alkyl ethers, modeling predicted favorable interactions between halogen atoms and Phe66 protons. While such interactions are popularly known as “halogen bonds”, theoretical analysis has revealed that the interaction is more similar to a dipole−dipole interaction than a hydrogen bond22 and that C−F···H−C interactions are favorable with an interaction energy of up to 1.6 kcal/mol.22 Moreover, a systematic study of several protein−ligand complexes involving C−F bonds interacting with the phenyl side chain of phenylalanine and tyrosine residues showed that the favored interaction of fluorine is with aromatic protons (edge interaction), with very few systems showing fluorine approaching the π-cloud of the aromatic ring (face interaction).22 To test these potential interactions with C-2 substituents (R2 in Table 1), a matched set of analogues of 7 and 8 were tested for biochemical inhibition of S. aureus TMK. As shown in Table 1, replacement of hydrogen with alkyl groups at C-2 was disfavored and resulted in a loss of activity (compounds 9 and 10). As predicted by modeling, the size of the substituent was important for the enzymatic potency. While the loss in activity

ity too much can be deleterious to solubility and protein binding and increase off-target effects, our TMK inhibitors are generally extremely soluble and very specific for the target.4,7 Past efforts required the addition of hydrophobic groups to moderate a very low log D and gain microbiological activity aganst S. aureus.7 These compounds had high serum protein binding, and here it was hoped that by altering the distribution of hydrophobic moieties in the inhibitor, a different proteinbinding profile could be realized. For the specific designs, modeling also indicated that aliphatic substitutions much larger than a methyl group, including branched aliphatics, would not be tolerated. Thus, only methyl and propyl substitutions were prioritized for synthesis and evaluation. In the case of proposed C-2 alkyl ethers, the oxygen atom was predicted to form a new interaction with aromatic protons of Phe66, with the alkyl moiety contributing an interaction with Val51 and Leu52. The interaction between aromatic C−H and oxygen atoms, traditionally termed a C−H···O hydrogen bond, has been studied theoretically15−17 as well as experimentally18−21 and is estimated to contribute between 0.3 and 1.5 kcal/mol in favorable interaction energy depending on the electronic environment of the atoms involved.15−17 Methoxy and ethoxy substitutions were selected as, again, modeling predicted potential steric clashes between longer aliphatic D



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Figure 2. X-ray structures of compounds 18 (A) and 19 (B) in complex with S. aureus TMK at 1.7 and 1.9 Å resolution, respectively. In addition to interactions observed previously (hydrogen bonds with Arg70 and Gln101 and a salt-bridge interaction with Arg48, left panels), the C-2 substituents form predicted interactions with Val51, Leu52, and Phe66 (right panels). In the case of 18, the ether oxygen forms a C−H···O hydrogen bond with the aromatic proton of C-4′Phe66, while the methyl group forms hydrophobic interactions with Val51 and Leu52 (A). In the case of 19, the fluorine atom forms a dipolar interaction with the aromatic proton of C-4′Phe66 (B). The distances shown are between heavy atoms.

for 9 was only about 5-fold, compound 10 with a much larger propyl substituent was more than 30-fold less potent compared to 8. We believe that the loss of potency for the methyl substituent was a result of unfavorable steric and electronic interactions with Phe66 (methyl hydrogens too closely approach aromatic protons on Phe66). Thus, the proximity of the methyl substituent to Phe66 resulted in an unfavorable interaction, and the propyl substituent in addition was too large for the available space. Alkoxy groups and halogens at C-2, however, led to a 3−10-fold improvement in activity, suggesting favorable interactions with Phe66. It was somewhat surprising to see that the ethoxy analogue 12 was about 3-fold better than 8, since the docking model indicated that an ethoxy group, like the propyl substitution, was too large for the space. Among halogens, chlorine and bromine substitution resulted in a 3−5fold improvement over the parent compound (8). Fluorine substitution resulted in the best example with a more than 10fold improvement in inhibition. Cyano substitution (16) was neutral with regard to potency. These results allowed us to focus on the methoxy and fluorine analogues for further development. To test this advance in more elaborate compounds, we incorporated a previously identified C-4 carboxylate substituent7 (Table 2). As described previously,7 the carboxylate moiety was particularly critical for improving enzyme potency by forming a salt-bridge interaction with Arg48. Moreover, introduction of a carboxylate also improved the MICs and

physical properties and avoided human ether-a-go-go-related gene (hERG) ion channel inhibition. Thus, maintenance of this interaction with Arg48 in conjunction with any C-2 substitution was paramount. We also at this time decided on the 3-chloro substituent for ring D over the corresponding 3-bromo of advanced compound 1. While 1 was extremely useful for in vivo target validation,4 bromine is not favored nor well-represented in drug candidates and in the case of 1 offered only small advantages in potency and protein binding over the corresponding 3-chloro analogues. As anticipated, the carboxylates 18 and 19 resulted in moderate improvements (up to 7-fold, Table 2) in IC50 and represent an advance over the prior compound 17. To verify the interactions afforded by the C-2 substituents, X-ray structures of 18 and 19 bound to S. aureus TMK were solved. The structures of both compounds confirmed previously observed binding modes with all the interactions formed by the A and D rings retained (Figure 2, left panels). Importantly, the X-ray structures showed the C-2 substituents in 18 and 19 forming predicted interactions with Val51, Leu52, and Phe66 (Figure 2, right panels). In the case of 18, the ether oxygen was 3.3 Å from the nearest aromatic carbon of Phe66 (C-4′Phe66), forming a C−H···O hydrogen bond, whereas the methyl carbon was equidistant at 3.7 Å from the Val51 and Leu52 side chains. A recent analysis17 of observed C−H···O interactions in systems deposited in the Cambridge Structural Database suggests that the optimal distance of oxygen from the centroid E



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aureus. For example, compound 45 with a log D value of 0.51 had an MIC = 0.5 μg/mL against methicillin-resistant/ quinolone-resistant (MRQR) S. aureus. On the other hand, compound 41 (log D = 0.13) had an MIC 8-fold less potent than that of 45 in spite of a similar IC50. The best compounds, 46 and 47, showed excellent MICs of 0.25−0.5 μg/mL against S. aureus. It was striking that the compounds in Table 3 also revealed an unexpected, altered preference for chirality of the R3 substituent. In the case of previously reported compounds7 (and including 34−37) with no substitution at C-2, R,S diastereomers were consistently 2−10-fold more potent than the corresponding S,S diastereomers. In the specific instance of 1 and its S,S diastereomer, 1 possessed a 3.5-fold better IC50 against S. aureus TMK and a 4-fold better MIC against MRQR S. aureus.7 However, for the C-2-methoxy-substituted compounds, the S,S diastereomer seemed to be significantly more potent than the respective R,S diastereomer (Table 3). For example, compound 39 has a ∼20-fold better IC50 than 38 and has a >8-fold improvement in MIC against MRQR S. aureus. The high-resolution 1.83 Å X-ray structure of 38 in complex with S. aureus TMK displayed a very conserved binding mode relative to the 1.67 Å structure of complexed 18 (RMSDligand heavy atoms = 0.16 Å, Figure 3). The methoxy moiety formed expected interactions with Val51, Leu52, and Phe66; the oxygen of the methoxy group was 3.5 Å from the nearest aromatic carbon of Phe66, whereas the methyl carbon was 3.8 and 3.6 Å from Leu51 and Val52, respectively. The X-ray structure also revealed a very small movement of the methoxy carbon compared to that in 18, resulting in a slightly shorter distance (3.6 Å compared to 3.8 Å) between the methoxy carbon and Val51 side chain. The ethyl moiety at R3, however, did not form the anticipated hydrophobic interactions with Val51 and Leu52; the terminal carbon of the ethyl group was 4.6 Å from the Leu52 side chain. Since 38 bound in an unperturbed binding mode (relative to 18), we hypothesize that the observed lower biochemical potency of 38 relative to its diastereomer 39 was due to the strain arising from a steric clash between the methoxy group and the R3 substituents in the R configuration (we did consider that these di-ortho-substituted compounds could possibly be subject to restricted rotation and atropisomerism, but the relatively small differences in potency observed between diastereomers 38 and 39, along with the very conserved binding mode of 38, argued against this phenomenon; atropisomerism is typically associated with much higher barriers to rotation23). Such a clash can be avoided in the S configuration while retaining the entropic gain achieved via stabilization of the ligand conformation. The R-substituted C-2 fluoro analogues showed improved activity compared to their OMe counterparts, which we propose was due to the much smaller size of fluorine. In fact, the diastereomeric pairs of fluoro analogues have similar IC50 values against S. aureus TMK (Table 2), suggesting that the chirality of the R3 substituents does not affect the binding and potency of the fluoro analogues. This notion is further supported by the 1.62 and 1.78 Å X-ray structures of 46 and 47, respectively, which revealed very similar binding modes (RMSDligand heavy atoms = 0.18 Å) for the core scaffolds (Figure 4). The isobutyl moiety in both X-ray structures, however, did not form any hydrophobic interactions with Val51 and Leu52 residues. Taken together, these data have deepened our understanding of the role of the R3 substituent in the binding and potency of

of the aromatic ring is 4.5 Å and the optimal C−H−O angle is 140°. In the X-ray structure of 18, these parameters were 4.6 Å and 143°, respectively, consistent with the existence of a C− H···O hydrogen bond. Ab initio calculations on similar systems predict such interactions to be favorable, contributing up to 1.5 kcal/mol in interaction energy.17 In the case of 19, the fluorine atom was 3.2 Å away from C-4′Phe66 and the angle C−F···C4′Phe66 was 149°. This geometry is in agreement with previously reported analysis of fluorinated ligands and protein complexes from the Protein Data Bank (PDB)22 in which such C−F··· Caromatic contacts were shown to have a mean distance of 3.2 Å and mean angle of 130° (standard deviation 38°) and is estimated to have 1.6 kcal/mol of positive interaction energy. We next incorporated lipophilic substituents off the methylene linker between the B and C rings (Table 3). Table 3. Activity of Analogues of 18 and 19a

a

Diastereomeric pairs are grouped together in the shaded and white bars. R2, the C-2 substituent, is highlighted in blue. The C-4 substituent is a carboxylic acid. bBacterial strains from AstraZeneca collection: S. aureus ARC516/S. aureus ARC517 methicillin-resistant (MR) and quinolone-resistant (QR). clog D value calculated from measured log D values of the diastereomeric mixture and the opposite diastereomer 38.

Increasing the size and lipophilicity of substituents at the methylene carbon (R3 substituents) resulted in a progressive increase in log D and, as expected, improved MICs against S. aureus (Table 3). In general, methoxy compounds resulted in lower log D values as well as poorer MICs compared to the corresponding fluorinated compounds. We found that changing R3 from ethyl to propyl increased log D by approximately 0.4 for R2 = H and F, but the log D increase was smaller (∼0.15) for the corresponding R2 = OMe compounds. This difference was reflected in the MIC values, which do not improve on going from R3 = ethyl to R3 = propyl when R2 = OMe. The data in Table 3 continue to support the notion that the lipophilicity of the compounds affects cell permeation in S. F



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Figure 3. (A) X-ray structure of 38 in complex with S. aureus TMK. The X-ray structure confirmed previously observed interactions with Arg48, Arg70, and Gln101. (B) Methoxy moiety in 38 with Val51 and Leu52, with the methoxy carbon placed 3.6 and 3.8 Å, respectively, from the Val51 and Leu52 side chains. The methoxy oxygen forms a C−H···O hydrogen bond interaction with the proton of C-4′Phe66. (C) Superimposition of Xray structures of 38 (green) and 18 (yellow) displaying identical binding modes for the core scaffold (RMSDligand heavy atoms = 0.16 Å). No significant changes are observed in either protein structure upon binding of ligands (Cα RMSD = 0.18 Å). Distances between the methoxy group in 18 and Val51, Leu52, and C-4′Phe66 are shown for reference. All distances shown are between heavy atoms.

potency gains from R3 substitution have little to do with direct hydrophobic interaction with Val51 and Leu52, but instead arise from (a) stabilization of the ring B/ring C turn that positions rings A and D for specific interactions and (b) a general hydrophobic effect that both improves the entropy of desolvation and allows the more hydrophobic/higher log D compound to penetrate the cellular membrane of S. aureus more readily. Thus, with no potential steric clash between C-2 and R3 substituents (34−37), there is a minor gain from the R configuration because of slightly positive interactions with the hydrophobic patch. With a small C-2 fluorine in place, preference for R or S disappears, and with a larger methoxy, the preference inverts because the steric repulsion more than cancels any small direct hydrophobic interaction. However, for all molecules, the proposed dominant turn stabilization and desolvation effects are preserved. These results expand the series’ structure−activity relationship (SAR) potential, as new combinations at C-2 and R3 can be explored to improve the overall profile. The free fraction is one key parameter: the chirality of substituents at R3 had an impact on the human plasma protein binding of some C-2methoxy-substituted compounds (Table 4). For example, compound 38 had a free fraction of 1.5% in human plasma, whereas the other, more potent diastereomer 39 showed significantly lower protein binding (11% free). Fluorosubstituted compounds, in general, have higher protein binding in human plasma than the methoxy-substituted compounds, and the corresponding fluoro-substituted diastereomeric pair (42 and 43) had a more uniformly low free fraction. Several studies have reported the effect of chirality on plasma protein binding,24 resulting in significant differences in the amount of free drug present in the plasma.25,26 Moreover, such stereoselective plasma protein binding can be species-dependent.26 Indeed, compounds 38 and 39, which have a significant difference in the human plasma, did not exhibit these differences in mouse plasma, which is important for performance in the infection model (Table 4). We also measured improvements in metabolic clearance; reference compound 1 had a clearance in rat of 42 mL/min/kg (61% of hepatic blood flow). In the series presented here, clearance was dependent on the R3 chirality: for example, rat clearance for 44 and 45 was 64 and 18 mL/min/kg, respectively, and for 46 and 47, it was 84

Figure 4. (A) Superimposition of X-ray structures of diastereomers 46 (green) and 47 (yellow) in complex with S. aureus TMK displaying identical binding modes for the core scaffold of ligands (RMSDligand heavy atoms = 0.18 Å). No significant changes were observed in either protein structure upon binding of ligands (Cα RMSD = 0.24 Å). In neither ligand does the butyl group form hydrophobic interactions with Val51 and Leu52, as the distances between the isobutyl groups and side chains are greater than 4 Å. (B) The experimental electron densities of the ligands (2Fo − Fc map at 1.2σ) clearly show the absolute configuration at the chiral center. The ligands are rotated 90° relative to those in panel A.

this inhibitor series. While the prior series7 exhibited a small, but consistent, preference (2−10 fold) for the R configuration of alkyl substituents at this position, the C-2 methoxy compounds here yielded optimized compounds with the opposite stereochemistry, and the C-2 fluoro eliminated the preference altogether. Taken as a whole, we propose that the G



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aureus using the same procedure as described previously.4 Under selection at 4 times the MIC, resistant colonies were found at a rate of 6 × 10−9. Those mutants found to be stable upon passage showed an 8- to >64-fold increase in MIC. Sequencing of the mutants revealed changes in the TMK protein coding region that produced the single amino acid changes Gly44Ser and Glu37Asp in separate mutants. These mutations map to the inhibitor-binding region (Supporting Information). As a proof-of-principle that efficacy was achievable in this series of compounds, 47 was evaluated in vivo against methicillin-sensitive S. aureus ARC516 in a mouse thigh infection model. The selection of 47 was based on better MICs against S. aureus and other Gram-negative pathogens and improved pharmacokinetics (PK) (Table 4) over 39. 47 was administered once daily via the intraperitoneal route for doses from 5 to 200 mg/kg (Supporting Information); to achieve a total daily dose of 400 mg/kg, 200 mg/kg was administered bid, q12h (twice daily, every 12 h). 47 was found to be efficacious, inhibiting progression of infection at a dose between 100 and 200 mg/kg/day and achieving a 10-fold reduction in the number of colony-forming units (CFU) at a dose of 400 mg/ kg/day (Figure 5A). While it is not indicative of a defined pharmacokinetic/pharmacodynamic relationship, for the 200 and 400 mg/kg/day doses, which resulted in a decrease in bacterial burden below the starting input, the amount of time above the free plasma MIC over 24 h ranged from 27% to 60%. These results were similar to those of 14 when dosed in the same model (the exposures between 5 and 200 mg/kg for 47 are shown in Figure 5B), yielding an efficacious dose for infection stasis of about 150 mg/kg/day. This was not surprising given that 1 and 47 are broadly comparable in MIC, mouse free fraction, and mouse clearance. As the in vivo behavior of 47 was consistent with that of 1, these results validate TMK as an antibacterial target with a second inhibitor and reinforce our focus on improving the key parameters of potency, protein binding, and clearance. These results also underline the potential of C-2-substituted compounds to be developed into an efficacious antibacterial agent against serious bacterial infections caused by Gram-positive pathogens, in particular S. aureus, with low cytotoxicity in humans. The challenge is improving the cellular potency and pharmacoki-

Table 4. Effect of the Chirality of the R3 Substituent (Table 3) on Plasma Protein Binding in Diastereomersa

a

Diastereomeric pairs are grouped together in the shaded and white bars. ND = not determined.

and 38 mL/min/kg, respectively (Table 4). In each case, the S diastereomer at R3 yielded an improved clearance rate. The observed improvement in human plasma protein binding along with reasonable MIC and MIC90 values (Table 5) against MRQR S. aureus established compounds in this series as strong alternate leads. These compounds also had good MICs against Staphylococcus epidermidis and Enterococcus faecium and excellent selectivity against human TMK (>25000fold in IC50) and Gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa (MIC > 64 μg/mL, Table 5). Other microbiological advantages to these inhibitors such as rapid bactericidal activity and a low resistance rate were established earlier4 and were only verified here using simple analogues. Specifically, the D ring bromo analogue of 34 was tested in a time-kill experiment as described previously.4 Against S. aureus, rapid and concentration-dependent killing (>3 log units in 4 h) was observed at 0.5 times the MIC and above. Some regrowth was observed at 24 h at 0.5 and 1 times the MIC. Separately, compound 36 was used to generate resistance mutants in S.

Table 5. Gram-Positive Spectrum Data, Population MICs, and Selectivity of Selected Compounds MIC (μg/mL) bacterial straina

1

39 b

46 b

47 c

S. aureus/S. aureus MRQR Streptococcus pneumoniae Streptococcus pyogenes Staphylococcus epidermidis Enterococcus faecium Enterococcus faecalis Escherichia coli Pseudomonas aeruginosa

MIC90 = 1 MIC90 = 0.03b MIC90 = 0.5b 2 0.25 1 >64 >64

MIC90 = 4 MIC90 = 2b MIC90 = 4b 4 8 >8 >64 >64

MIC90 = 2 MIC90 = 0.13c MIC90 = 2c 2 2 8 >64 >64

0.5/0.5 0.13 2 4 2 4 >64 >64

human TMK IC50 (nM) human A549 MIC (μg/mL)

62000 >64

>200000 >64

51000 ND

29000 ND

a

Bacterial strains from AstraZeneca collection: S. aureus ARC516; SauMRQR, S. aureus ARC517 methicillin-resistant (MR) and quinolone-resistant (QR); S. pneumoniae ARC2481; S. pyogenes ARC2834; S. epidermidis ARC323; E. faecium ARC1239 linezolid-resistant (LRE); E. faecalis ARC1618 vancomycin-resistant (VRE); E. coli ARC523; P. aeruginosa ARC545. bS. aureus MIC90 of 22 strains, S. pneumoniae MIC90 of 19 strains, S. pyogenes MIC90 of 21 strains. cS. aureus MIC90 of 23 strains, S. pneumoniae MIC90 of 20 strains, S. pyogenes MIC90 of 21 strains. H



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ences at this position and yielded equipotent diastereomeric compound pairs. These results have shed light on the specific role of the R3 alkyl substituent, showing it to be sensitive to an intramolecular steric clash with C-2 substituents when bound and indicating that a direct hydrophobic interaction with conserved Val51 and Leu52 is likely to be of minor value. These results also open up new options for SAR development, while preserving broad Gram-positive coverage with good MICs against S. aureus, S. pyogenes, S. epidermidis, and Enterococcus species and excellent selectivity against human TMK. An example of this new series was successful in the S. aureus mouse infection model, proving that new C-2- and R3substituted compounds can be efficacious in vivo. We continue to develop SARs for these inhibitors to find the proper balance of MIC, low clearance, and low protein binding that will result in lower efficacious doses. The work presented here not only illustrates an example of a structure-guided design approach, but also highlights how taking advantage of a small region observed in the X-ray structures led to new SAR directions and new insights into the sources of binding potency.

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EXPERIMENTAL SECTION

General Experimental Details. All commercial reagents and anhydrous solvents were obtained from commercial sources and were used without further purification, unless otherwise specified. LC−MS Conditions. Method 1. Samples were analyzed by reversedphase LC−MS using Varian Polaris C18A, 2 mm × 50 mm, 3 μm particle size columns. An Agilent HP1100 (Wilmington, DE) LC system was used with a gradient elution profile of 5−95% B over 4.5 min at 1 mL/min and then re-equilibration at the initial conditions to 6 min. The injection volume was 2 μL and column temperature 30 °C. Mobile phase A was 0.1% formic acid in water and mobile phase B 0.1% formic acid in acetonitrile. Detection was based on electrospray ionization (ESI) in positive and negative polarity using a Waters ZQ mass spectrometer (Milford, MA), diode-array UV detector from 210 to 400 nm, and evaporative light-scattering detector (Sedex 75, Sedere, Alfortville Cedex, France). Method 2. This method was the same as method 1, except mobile phase A was 10 mM ammonium acetate in 5:95 acetonitrile/water and mobile phase B was acetonitrile. Method 3. This method was the same as method 1, except the gradient elution profile was 0−95% B. Method 4. This method was the same as method 2, except the gradient elution profile was 0−95% B. Accurate mass was measured using a hybrid quadrupole time-offlight mass spectrometer (microTOFQ, Bruker Daltonics) in ESI+ mode. Method: 5−95% mobile phase B from 0.0 to 5.0 min, hold at 95% mobile phase B to 5.1 min (mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile), 1.0 mL/min flow, column Varian Polaris C18-A 2.0 mm × 50 mm, 3.0 μm particle size. A 1 μL volume of sample was injected into an HPLC instrument using an externally calibrated instrument for accurate mass measurement. A 50 μL/min flow of eluent from the LC instrument and a 50 μL/min flow of make-up solution (CH3CN/H2O, 50:50) went into the MS instrument. 1 H NMR spectra (δ, ppm) were recorded using a Bruker Advance Ultrashield 300 MHz or Bruker DPX 400 MHz instrument. Column chromatography was performed using Silcycle FLHR10030B Silisep cartridges (12−330 g). Preparative reversed-phase HPLC chromatography was carried out using a Waters Atlantis T3C18 column, 19 × 100 mm, 5 μm, a linear gradient from 10% to 90% CH3CN in H2O over 12 min (0.1% trifluoroacetic acid), and a flow rate of 20 mL/min. Preparative chiral chromatography was carried out as follows: HPLC, Chiralpak IC column, 30 × 250 mm, 5 μm, hexane (60%), methanol/ethanol (1:1) (40%), 0.5% diethylamine, flow rate 40 mL/min; SFC, Chiralpak AD column, 30 × 250 mm, 5 μm, carbon dioxide (60%), 2-propanol (40%), flow rate 120 mL/min; chiral

Figure 5. (A) Dose−response effect on the treatment of mice with 47 in a S. aureus thigh infection model. Error bars represent standard errors in CFU measurements. (B) PK profile of 47 representing a nonlinear exposure in mice with predosed aminobenzotriazole (ABT).

netic profile of compounds like 39 while maintaining the gains achieved in free fraction. We believe that taking advantage of the vector offered by the ethyl group in 39 or substitutions in other parts of the scaffold will allow desired improvements in cellular potency while maintaining or bettering the protein binding and metabolic profile of 39, and new results along these lines will be disclosed in due course.

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CONCLUSIONS In summary, we employed a structure-guided design approach to exploit a previously unexplored binding region in the S. aureus TMK enzyme. Our goals were to provide new options for SAR development that could improve these Gram-positive TMK inhibitors in the areas of potency, serum free fraction, and/or metabolic clearance, which we have identified as the key parameters driving in vivo efficacy.4,7 Compounds with halogens as well as small alkyl and alkyl ether substituents at C-2 were synthesized and tested to probe this area of the enzyme. We found that fluoro and methoxy substituents at C-2 improved potency through new interactions with aromatic hydrogens and hydrophobic side chains in the vicinity. Alkyl groups or larger alkoxy groups did not provide the same benefit. An unexpected discovery was that the methoxy substituent inverted the chiral preference at the R3 (linker) position for binding, a result we attribute to an unfavorable intramolecular steric interaction in the bound form of the inhibitor that overrides the small preference for a direct hydrophobic interaction in the opposite diastereomer in C-2unsubstituted analogues. On the other hand, the best halogen substituent, fluorine, largely eliminated diastereomeric differI



Article

analytical HPLC, Chiralpak IC column, 4.6 × 250 mm, 5 μm, hexane (60%), methanol/ethanol (1:1) (40%), 0.5% diethylamine, flow rate 1 mL/min; chiral analytical SFC, Chiralpak AD column, 4.6 × 250 mm, 5 μm, carbon dioxide (60%), 2-propanol (40%), flow rate 2.8 mL/min. The purity of the final compounds was assessed on the basis of analytical LC−MS, and the results were greater than 95% unless specified otherwise. The diastereomeric ratio of specific compounds was measured by chiral analytical HPLC and was >95:5 unless otherwise indicated. Molecular Modeling. Molecular docking was performed using Glide version 5.027,28 in standard flexible docking mode. X-ray structures of S. aureus TMK protein in complex with related ligands (2, 18, and 19; PDB IDs 4HEJ, 4QG7, and 4QGA, respectively) were used as templates for docking studies. The protein structures were prepared using Protein Preparation Wizard, whereas the ligands were prepared using the Ligprep utility in Maestro 8.5 (Schrodinger, LLC, New York, 2008). Docked poses were minimized in the OPLS 200129 force field as implemented in Maestro 8.5. General experimental procedures for biochemical assays, protein crystallography, and the in vivo infection model are described in the Supporting Information. Screening Pharmacokinetics. In vivo screening pharmacokinetic experiments were performed in male Wistar rats dosed at 5−10 mg/kg iv bolus in a vehicle of DMA/TEG/saline (40:40:20). Bioanalysis of plasma levels was performed as previously described.4 Experimental Synthetic Procedures and Characterization Data of the Compounds. Experimental procedures and characterization data for compounds 1, 2, 17, and 34−37 and intermediate 6 can be found elsewhere.4,7 Compounds presented in Table 1 (9−16) were synthesized using a general procedure exemplified for compound 13 (Scheme 1). 2-Fluoro-3-hydroxybenzaldehyde (4). The title compound was prepared by microwave irradiation of a solution of 2-fluoro-3methoxybenzaldehyde (3; 600 mg, 3.89 mmol) in a solution of hydrogen bromide and acetic acid (33% (w/w), 7.2 mL, 39.64 mmol) at 80 °C for 45 min. The reaction mixture was poured into water (20 mL) and quenched by the addition of excess saturated sodium bicarbonate solution (50 mL). The resulting mixture was extracted with EtOAc (3 × 50 mL), and the combined organic layers were dried with magnesium sulfate, filtered, and concentrated. The crude residue was chromatographed on silica gel employing a 0−20% EtOAc gradient in chloroform. Desired fractions were pooled and concentrated to furnish 250 mg (46%) of 4 as a colorless solid. LC−MS (method 1): tR = 1.23 min, m/z = 139 (M − 1). 1H NMR (300 MHz, DMSO-d6): δ 10.35 (s, 1H), 10.23 (s, 1H), 7.20 (m, 3H) ppm. 3-(3-Chlorophenoxy)-2-fluorobenzaldehyde (5). The title compound was prepared by stirring a mixture of 4 (150 mg, 1.07 mmol), 3chlorophenylboronic acid (250 mg, 1.6 mmol), copper(II) acetate (427 mg, 2.14 mmol), and 3 Å molecular sieves in a 40 mL vial in anhydrous dichloromethane (30 mL). Hunig’s base (0.932 mL, 5.35 mmol) was added in one portion, and the reaction was stirred at room temperature for 72 h. The reaction was diluted with additional dichloromethane (20 mL) and water (20 mL) and then acidified by addition of 2 M HCl. The biphasic mixture was stirred vigorously and filtered, and the organic layer was collected. The acidic layer was extracted twice more with dichloromethane (2 × 30 mL), and then the combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The crude residue was chromatographed employing a 0−100% chloroform gradient in hexanes. The desired fractions were concentrated to provide 129 mg (48%) of 5 as a colorless oil. LC−MS (method 1): tR = 2.88 min, m/z = 249 (M − 1). 1H NMR (300 MHz, MeOH-d4): δ 10.27 (s, 1H), 6.65−7.55 (m, 7H) ppm. (S)-1-(1-(3-(3-Chlorophenoxy)-2-fluorobenzyl)piperidin-3-yl)-5methylpyrimidine-2,4(1H,3H)-dione (13). The title compound was prepared by stirring polymer-supported cyanoborohydride (515 mg, 2.0 mmol/g, 1.03 mmol), 5 (129 mg, 0.51 mmol), and (S)-5-methyl-1(piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (6; 194 mg, 0.93 mmol) in a 50 mL flask in an 18:1 mixture of DMF (9 mL) and acetic acid (0.50 mL). The suspension was stirred for 18 h at room temperature

and then filtered to remove the solids. The polymer was rinsed with additional DMF (5 mL), and the combined organic layers were concentrated. The crude residue was redissolved in DMSO and purified by HPLC employing a 10−75% acetonitrile gradient in water (0.1% TFA). The desired fractions were pooled and lyophilized to furnish 80 mg (35%) of 13 as an off-white solid. LC−MS (method 3): tR = 2.00 min, m/z = 444 (M + H). 1H NMR (300 MHz, MeOH-d4): δ 7.44 (m, 2H), 7.33 (m 3H), 7.14 (m, 1H), 7.01 (t, 1H), 6.92 (dd, 1H) 4.73 (m, overlapping w/CD3OD, ∼1H), 4.53 (m, 2H), 3.60 (d, 1H), 3.40 (t, 1H), 3.10 (t, 1H), 1.90−2.25 (m, 4H), 1.87 (s, 3H) ppm. HRMS: m/z 444.1467, calcd 444.1491 (M + H). (S)-1-(1-(3-(3-Chloro-5-fluorophenoxy)-2-methylbenzyl)piperidin3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9). Yield: 75 mg (13%) as an off-white solid. LC−MS (method 3): tR = 2.30 min, m/z = 460 (M + H). 1H NMR (300 MHz, MeOH-d4): δ 7.42 (m, 3H) 7.17 (m, 1H) 6.93 (dt, 1H), 6.72 (m, 1H) 6.62 (dt, 1H), 4.55 (m, 1H), 4.50 (m, 2H) 3.53 (m, 2H), 3.45 (m, 1H), 3.15 (m, 1H) 2.33 (s, 3H), 1.80−2.25 (overlapping s and m, 7H) ppm. HRMS: m/z 458.1641, calcd 458.1647 (M + H). (S)-1-(1-(3-(3-Chloro-5-fluorophenoxy)-2-propylbenzyl)piperidin3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (10). Yield: 19.1 mg (24%) as an off-white solid. LC−MS (method 3): tR = 2.50 min, m/z = 488 (M + H). 1H NMR (300 MHz, MeOH-d4): δ 7.73 (br s, 1H), 7.22 (overlapping m’s, 2H), 6.92 (m, 1H), 6.88 (dt, 1H), 6.67 (m, 1H), 6.57 (dt, 1H), 4.57 (m, 1H), 3.60 (m, 2H), 2.85 (m, 2H), 2.65 (m, 2H), 2.33 (dt, 2H), 1.45−1.90 (s overlapping m’s, 9H), 0.97 (t, 3H) ppm. (S)-1-(1-(3-(3-Chloro-5-fluorophenoxy)-2-methoxybenzyl)piperidin-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (11). Yield: 44 mg (93%) as an off-white solid. LC−MS (method 4): tR = 3.00 min, m/z = 474 (M + H). 1H NMR (300 MHz, CD3OD-d4): δ 7.46 (br s, 1H), 7.43 (m, 1H), 7.29 (m, 2H), 6.95 (dt, 1H), 6.80 (br s, 1H), 6.70 (dt, 1H), 4.75 (m, 1H) 4.45 (m, 2H), 3.93 (s, 3H), 3.57 (m, 2H), 3.07 (m, 1H), 1.93−2.23 (m, 4H), 1.88 (s, 3H) ppm. HRMS: m/z 474.1521, calcd 474.1597 (M + H). (S)-1-(1-(3-(3-Chloro-5-fluorophenoxy)-2-ethoxybenzyl)piperidin3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (12). Yield: 72 mg (44%) as an off-white solid. LC−MS (method 3): tR = 2.32 min, m/z = 490 (M + 3). 1H NMR (300 MHz, MeOH-d4): δ 7.43 (m, 2H), 7.29 (m, 2H), 6.95 (dt, 1H), 6.79 (m, 1H), 6.69 (dt, 1H), 4.73 (m, 1H), 4.45 (m, 1H), 4.17 (q, 2H), 3.57 (m, 2H), 3.37 (m, 1H), 3.10 (m, 1H), 1.83−2.23 (m and s 7H) ppm. HRMS: m/z 488.1761, calcd 488.1753 (M + H). (S)-1-(1-(2-Chloro-3-(3-chloro-5-fluorophenoxy)benzyl)piperidin3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (14). Yield: 32 mg (17%) as an off white solid. LC−MS (method 3): tR = 2.31 min, m/z = 478 (M + H). 1H NMR (300 MHz, MeOH-d4): δ 7.44−7.62 (m, 3 H) 7.36 (dd, 1H) 6.99 (dt, 1H) 6.74−6.82 (m, 1H) 6.70 (dt, 1H) 4.66− 4.77 (m, 1H) 4.51−4.66 (m, 2H) 3.51−3.67 (m, 2H) 3.35−3.48 (m, 1H) 3.04−3.25 (m, 1H) 1.92−2.23 (m, 4H) 1.89 (d, 3H) ppm. HRMS: m/z 478.1114 calcd 478.1101 (M + H). (S)-1-(1-(2-Bromo-3-(3-chloro-5-fluorophenoxy)benzyl)piperidin3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (15). Yield: 53 mg (24%) as an off-white solid. LC−MS (method 3): tR = 2.34 min, m/z = 522 (M + H). 1H NMR (300 MHz, MeOH-d4): δ 7.57−7.65 (m, 2H) 7.52 (d, 1H) 7.31−7.42 (m, 1H) 7.03 (dt, 1H) 6.78−6.85 (m, 1H) 6.73 (dt, 1H) 4.71−4.82 (m, 1H) 4.62−4.70 (m, 2H) 3.59−3.73 (m, 2H) 3.44−3.56 (m, 1H) 3.16−3.30 (m, 1H) 1.95−2.29 (m, 4H) 1.94 (d, 3H) ppm. HRMS: m/z 522.0605, calcd 522.0517 (M + H). (S)-2-(3-Chlorophenoxy)-6-((3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzonitrile (16). Yield: 40 mg (24%) as an off-white solid. LC−MS (method 3): tR = 2.16 min, m/z = 451 (M + H). 1H NMR (300 MHz, MeOH-d4): δ 8.46 (br s, 1H), 7.83 (br s, 1H), 7.59 (t, 1H), 7.42 (t, 1H), 7.36 (d, 1H), 7.25 (m, 1H), 7.12 (t, 1H), 7.02 (dd, 1H) 6.94 (m, 1H) 4.58 (m, 1H) 3.77 (s, 1H), 2.88 (dd, 1H), 2.77 (d, 1H), 2.48 (m, 2H), 1.63−1.93 (s overlapping m, 7H) ppm. HRMS: m/z 451.1531, calcd 451.1538 (M + H). J



Article

Compounds presented in Table 2 were synthesized using a general procedure exemplified for compounds 38, 39, 46, and 47 (Schemes 2 and 3). 4-Bromo-3-(3-chlorophenoxy)-2-methoxybenzaldehyde (21). To a stirred suspension of bromo-3-hydroxy-2-methoxybenzaldehyde (20; 5 g, 21.645 mmol), 3-chlorophenylboronic acid (6.77 g, 43.290 mmol), and pyridine (5.14 g, 64.935 mmol) in 1,2-dichloroethane (50 mL) was added copper(II) acetate (9.8 g, 54.112 mmol). The mixture was then heated at 55 °C for 3 days with continuous oxygen bubbling. The reaction mixture was filtered through a Celite bed, and the filtrate was washed successively with water (2 × 25 mL) and brine (25 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude residue was purified by flash chromatography using 20% ethyl acetate in hexane to yield 2.6 g (35%) of the title product 21. LC−MS (method 1): tR = 3.28 min, m/ z = 341 (M + H). 1H NMR (400 MHz, CD3OD): δ 3.80 (s, 3H), 6.74−6.80 (m, 2H), 7.02−7.06 (m, 1H), 7.26−7.34 (m, 2H), 7.89− 7.94 (m, 1H), 10.32 (s, 1H) ppm. 1-[4-Bromo-3-(3-chlorophenoxy)-2-methoxyphenyl]propan-1-ol (22). To a stirred solution of aldehyde 21 (700 mg, 2.049 mmol) in diethyl ether (10 mL) was added ethylmagnesium bromide (6.15 mL, 6.147 mmol, 1.0 M solution in diethyl ether) at −78 °C, and the resulting mixture was allowed to stir for 2 h. The reaction was quenched with saturated aqueous NH4Cl (10 mL) and then diluted with ethyl acetate (60 mL). The organic layer was washed with water (2 × 20 mL) and brine (20 mL) and dried over Na2SO4. The solvent was evaporated under reduced pressure to obtain the crude product, which was purified by flash chromatography using 20% ethyl acetate in hexane to yield 275 mg (36%) of the title compound 22. LC−MS (method 1): tR = 3.27 min, m/z = 371 (M + H). 1H NMR (400 MHz, CD3OD): δ 0.93 (t, 3H), 1.71−1.76 (m, 2H), 3.80 (s, 3H), 4.88−4.82 (m, 1H), 6.73−6.77 (m, 2H), 7.04−7.06 (m, 1H), 7.25−7.33 (m, 2H), 7.44−7.47 (m, 1H) ppm. 1-[(3S)-1-{1-[4-Bromo-3-(3-chlorophenoxy)-2-methoxyphenyl]propyl}piperidin-3-yl]-5-methylpyrimidine-2,4(1H,3H)-dione (24). To a stirred solution of 22 (450 mg, 1.32 mmol) in dry dichloromethane (5 mL) was added triethylamine (400 mg, 3.96 mmol) followed by mesyl chloride (227 mg, 1.050 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. The mixture was diluted with additional dichloromethane and then washed successively with 10% NaHCO3 (10 mL), water (10 mL), and brine (20 mL), dried over sodium sulfate, and filtered. The resulting solution was concentrated under reduced pressure to obtain the crude mesyl derivative 23 (0.700 mmol), which was dissolved in CH3CN (5 mL). Next 6 (673 mg, 3.30 mmol) was added in one portion, and the reaction mixture was stirred at 80 °C for 12−36 h. The reaction solvent was evaporated, and the mixture was diluted with ethyl acetate (25 mL) and then washed with water (25 mL) and brine (25 mL), dried over Na2SO4, filtered, and concentrated. The obtained crude residue was purified by flash chromatography (50% ethyl acetate in hexane) to yield the title product 24 (100 mg, 16%). LC−MS (method 3): tR = 2.41 min, m/z = 562 (M + H). 1H NMR (400 MHz, DMSO-d6): δ 0.77 (t, 3H), 1.60−1.76 (m, 7H), 1.80−2.02 (m, 4H), 2.74−2.82 (m, 2H), 3.65−3.69 (m, 3H), 3.82−3.88 (m, 1H), 4.30− 4.40 (m, 1H), 6.71−6.92 (m, 2H), 7.10−7.14 (m, 1H), 7.20−7.23 (m, 1H), 7.27−7.38 (m, 1H), 7.52−7.55 (m, 1H), 7.63−7.67 (m, 1H), 11.20−11.22 (m, 1H) ppm. 2-(3-Chlorophenoxy)-3-methoxy-4-{1-[(3S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl]propyl}benzoic Acid (26). In a 2−5 mL microwave vial a solution of 24 (90 mg, 0.159 mmol) in DMF (3 mL) was purged with nitrogen for 10 min. Zn(CN)2 (56 mg, 0.479 mmol) and Pd(PPh3)4 (8.3 mg, 0.007 mmol) were added at room temperature, then the reaction vial was capped, and the reaction mixture was heated at 110 °C for 45 min under microwave irradiation. After being cooled to room temperature, the reaction was quenched with ice−water (20 mL) and extracted with diethyl ether (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude 25 was dissolved in ethanol (8 mL), then solid NaOH (79 mg, 1.972 mmol) was added,

and the resulting mixture was stirred at reflux for 12 h. The mixture was concentrated under reduced pressure, and the residue was dissolved in water (25 mL) and extracted once with diethyl ether to remove neutral impurities. The aqueous portion was acidified with 1.5 N HCl and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (10 mL), dried over sodium sulfate, and evaporated. The crude residue was purified by flash chromatography using 5% methanol in chloroform to furnish 26 (10 mg, 19%). LC−MS (method 3): tR = 2.29 min, m/z = 528 (M + H). 1 H NMR (400 MHz, DMSO-d6): δ 0.65−0.72 (m, 3H), 1.62−1.98 (m, 8H), 2.08−2.20 (m, 1H), 2.24−2.36 (m, 1H), 2.62−2.74 (m, 1H), 2.90−3.01 (m, 1H), 3.60−3.74 (m, 4H), 4.52−4.60 (m, 1H), 4.70− 4.94 (m, 1H), 6.79−6.98 (m, 2H), 7.09 (d, J = 7.96 Hz, 1H), 7.24− 7.34 (m, 1H), 7.51−7.57 (m, 1H), 7.64−7.78 (m, 2H), 11.36−11.40 (m, 1H), 13.20 (br s, 1H) ppm. 2-(3-Chlorophenoxy)-3-methoxy-4-{(1R)-1-[(3S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl]propyl}benzoic Acid (38). A 36 mg sample of 26 was subjected to HPLC preparative chiral separation eluting with a mixture of hexanes (50%), methanol/ ethanol (1:1) (50%), and diethylamine (0.1%). The title compound eluted first. After removal of the solvent, the sample was lyophilized to provide 10 mg of 38 (30%) as a white solid. LC−MS (method 4): tR = 1.55 min, m/z = 528 (M + H). 1H NMR (400 MHz, MeOH-d4): δ 7.83 (d, 1H), 7.50 (d, 1H), 7.40 (br s, 1H), 7.25 (t, 1H), 7.04 (dd, 1H), 6.85 (br s, 1H), 6.74 (dd, 1H), 4.73 (probably overlapping m’s, also overlapping CD3OD, ∼2H), 3.89 (s, 3H), 3.82 (m, 1H), 3.42 (m, 1H), 3.13 (m, 1H), 2.87 (m, 1H), 2.40−1.70 (s overlapping m’s, 9H), 0.85 (t, 1H). HRMS: m/z 528.1896, calcd 528.1902 (M + H). 2-(3-Chlorophenoxy)-3-methoxy-4-{(1S)-1-[(3S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl]propyl}benzoic Acid (39). A 36 mg sample of 26 was subjected to HPLC preparative chiral separation eluting with a mixture of hexanes (50%), methanol/ ethanol (1:1) (50%), and diethylamine (0.1%). The title compound eluted second. After removal of the solvent, the sample was lyophilized to provide 11 mg of 39 as a white solid. LC−MS (method 4): tR = 1.65 min, m/z = 528 (M + H). 1H NMR (400 MHz, MeOH-d4): δ 7.83 (d, 1H), 7.52 (d, 1H), 7.43 (br s, 1H), 7.25 (t, 1H) 7.04 (dd, 1H) 6.85 (br s, 1H), 6.74 (dd, 1H) 4.70 (probably overlapping m’s, also overlapping CD3OD, 2H), 3.92 (s, 3H), 3.77 (m, 1H), 3.44 (m, 1H), 3.10 (m, 1H), 2.87 (m, 1H), 2.40−1.70 (s overlapping m’s, 9H, 0.87 (t, 1H). HRMS: m/z 528.1905, calcd 528.1902 (M + H). 2-(3-Chlorophenoxy)-3-methoxy-4-((R)-1-((S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (40). A 30 mg sample of racemic material was subjected to HPLC preparative chiral separation eluting with a mixture of hexanes (50%), methanol/ethanol (1:1) (50%), and diethylamine (0.1%). The title compound eluted second. After removal of the solvent, the sample was lyophilized to provide 40 as a solid (98:2 diastereomeric ratio) (5.3 mg, 18%). LC−MS (method 4): tR = 2.18 min, m/z = 542 (M + H). 2-(3-Chlorophenoxy)-3-methoxy-4-((S)-1-((S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (41). A 30 mg sample of racemic material was subjected to HPLC preparative chiral separation eluting with a mixture of hexanes (50%), methanol/ethanol (1:1) (50%), and diethylamine (0.1%). The title compound eluted first. After removal of the solvent, the sample was lyophilized to provide 41 as a gum (>99:1 diastereomeric ratio) (9.7 mg, 32%). LC−MS (method 4): tR = 1.98 min, m/z = 542 (M + H). (S)-2-(3-Chlorophenoxy)-3-methoxy-4-((3-(5-methyl-2,4-dioxo3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzoic Acid (18). Yield: 18 mg (28.9%) as an off-white solid. LC−MS (method 3): tR = 2.17 min, m/z = 500 (M + H). 1H NMR (300 MHz, CD3OD): δ 7.76 d 1H; 7.53 d 1H; 7.42 d 1H; 7.25 t 1H; 7.02 dm 1H; 6.84 (t,1H); 6.74 (dm, 1H); 4.70 (m, 1H); 4.47 (q, 1H); 3.90 (s, 3H); 3.53 (m, 2H); 3.33 (m, overlapping CD3OD, 1H); 3.13 (t, 1H); 2.20− 1.90 (m, 4H); 1.87 (s, 3H) ppm. 1-(4-Bromo-2,3-difluorophenyl)-3-methylbutan-1-ol (28). 1,4-Dibromo-2,3-difluorobenzene (27; 4.35 g, 16.00 mmol) was dissolved in THF (70.8 mL) and the solution cooled to −40 °C. Isopropylmagnesium chloride−lithium chloride (17.23 mL, 22.40 mmol) was added slowly, keeping the temperature below −30 °C. The mixture was stirred at about −35 °C for 1 h and warmed to −7 °C for an additional K



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1 h. The reaction mixture was cooled to −40 °C, 3-methylbutanal (3.43 mL, 32.00 mmol) was added in one portion (the temperature rose to −30 °C), and stirring was continued for 4 h at −25 °C. The reaction mixture was poured into 1 M H2SO4 and ice, and the resulting mixture was saturated with solid NaCl and then extracted twice with tert-butyl methyl ether (2 × 150 mL). The combined extracts were dried over sodium sulfate and concentrated in vacuo. After silica gel chromatography (0−100 ethyl acetate in hexane) 28 was isolated as a colorless oil (5.50 g, 123%). The material was taken on to the next step with no additional purification. LC−MS (method 3): tR = 3.24 min, m/z = 279.0 (M − H). 1H NMR (300 MHz, CDCl3): δ 0.83 (dd, 6H), 1.39 (m, 1H), 1.58 (m, 2H), 2.59 (br s, 1H), 4.92 (m, 1H), 7.12 (m, 1H), 7.43 (m, 1H) ppm. 1-(4-Bromo-2,3-difluorophenyl)-3-methylbutyl Methanesulfonate (29). 28 (5.5 g, 19.70 mmol) was dissolved in dichloromethane and the resulting solution then cooled to 0 °C. TEA (8.24 mL, 59.11 mmol) was added followed by methanesulfonyl chloride (2.303 mL, 29.56 mmol), and the reaction mixture was stirred for 2 h at 0 °C. After this time, the reaction mixture was warmed to ambient temperature (starting material did not ionize in LC−MS) and stirred for an additional 12 h at this temperature. At that time, it was washed successively with 1 N HCl, satd bicarbonate solution, and then brine. The organic portion was dried over magnesium sulfate, filtered, and concentrated to give 29 as a yellow oil which was used without further purification. 1-((3S)-1-(1-(4-Bromo-2,3-difluorophenyl)-3-methylbutyl)piperidin-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (30). 29 (7 g, 19.60 mmol) and 6 (6.15 g, 29.39 mmol) were combined in acetonitrile (88 mL), and then Hunig’s base (10.27 mL, 58.79 mmol) was added. The reaction mixture was heated at 80 °C for 24 h, at which time LC−MS indicated that the reaction was clean. At this time, the reaction was cooled to ambient temperature, then diluted with EtOAC, and washed twice with water and once with brine. The organic portion was dried over sodium sulfate, filtered, and concentrated. The resulting oil was purified via silica chromatogaphy (gradient 0−100% ethyl acetate, 80 g column) to give 30 (1.500 g, 16.27%) as a white solid. LC−MS (method 3): tR = 3.10 min, m/z = 470/472 (M + 1). 1H NMR (300 MHz, CD3OD): δ 0.93 (dd, 6H), 1.38−1.91(br m, 10H), 1.95−2.33 (br m, 3H), 2.83 (m, 1H), 2.99 (m, 1H), 4.53 (m, 1H), 7.12 (m, 1H), 7.43 (m, 1H), 7.62 (br s, 1H) ppm. 2,3-Difluoro-4-(3-methyl-1-((S)-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzonitrile (31). 30 (1.5 g, 3.19 mmol), dicyanozinc (1.5 g, 12.76 mmol), and tetrakis(triphenylphosphine)palladium(0) (37 mg, 0.03 mmol) were suspended in acetonitrile (10 mL), and the reaction mixture was heated under microwave irradiation at 140 °C for 105 min. The mixture was filtered, and the resulting filtrate was concentrated via rotary evaporation and then purified via normal-phase chromatography (0−100% ethyl acetate, 40 g column) to isolate the desired product as well as unreacted starting material. The presence of 31 (0.895 g, 67.4%) was confirmed by LC−MS with some unreacted starting material. The material was taken forward without further purification. LC−MS (method 3): tR = 1.93 min, m/z = 416.0 (M − 1). 1H NMR (300 MHz, DMSO-d6): δ 0.86 (dd, 6H), 1.32−2.04 (br m, 12H), 2.69−2.96 (br m, 2H), 4.07 (m, 1H), 4.35 (m, 1H), 7.45 (app t, 1H), 7.59 (m, 1H), 7.77 (app t, 1H) 11.20 (s, 1H) ppm. 2-(3-Chlorophenoxy)-3-fluoro-4-(3-methyl-1-((S)-3-(5-methyl2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzonitrile (32). 31 (0.895 g, 2.15 mmol) was dissolved in NMP (2 mL), and 3-chlorophenol (0.359 g, 2.79 mmol) was added in a microwave vial. Potassium carbonate (0.594 g, 4.30 mmol) was added, and the reaction mixture heated at 160 °C for 15 min under microwave irradiation. At this time, the reaction was diluted with EtOAc (25 mL) and then washed twice with water and once with brine. The organic portion was dried over magnesium sulfate, filtered, concentrated, and then purified via normal-phase chromatography (40 g, 0−100% ethyl acetate). After concentration of the appropriate fractions via rotary evaporation, LC−MS confirmed the presence of 32 (0.800 g, 70.9%). LC−MS (method 3): tR = 2.62 min, m/z = 525.0 (M + H).

2-(3-Chlorophenoxy)-3-fluoro-4-(3-methyl-1-((S)-3-(5-methyl2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (33). 32 (1170 mg, 2.23 mmol) was taken and diluted with water (15 mL) and MeOH (5 mL). NaOH (891 mg, 22.29 mmol) was added, and the mixture was heated at 80 °C for 12 h. At this time, the reaction was cooled to ambient temperautre, then taken up in EtOAc (100 mL), and carefully acidified to pH 5 with concentrated HCl. The aqeuous portion was washed six times with EtOAc, and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was taken up in 1.5 mL of MeOH and purified via reversed-phase chromatography (0−100% ACN). Clean fractions were collected and after lyophilization afforded 33 (750 mg, 61.9%) as a white solid. LC−MS (method 3): tR = 1.99 min, m/z = 544.0 (M + H). 1H NMR (300 MHz, CD3OD): δ 0.82 (dd, 6H), 1.19 (m, 1H), 1.66−1.95 (br m, 7H), 2.04 (m, 1H), 2.19 (dt, 1H), 2.78 (m, 1H), 3.06 (m, 1H), 3.38 (m, 1H), 3.68 (m, 1H), 4.65 (m, 2H), 6.70 (m, 1H), 6.76 (m, 1H), 6.96 (m, 1H), 7.18 (app t, 1H), 7.32 (br s, 1H), 7.51 (dd, 1H), 7.83 (m, 1H) ppm. 2-(3-Chlorophenoxy)-3-fluoro-4-((R)-3-methyl-1-((S)-3-(5-methyl2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (46). A diastereomeric mixture of 33 (328 mg, 0.60 mmol) was subjected to HPLC preparative chiral separation eluting with a mixture of hexanes (50%), methanol/ethanol (1:1) (50%), and diethylamine (0.1%). The title compound 46 eluted first. After removal of the solvent, the title compound 46 was obtained as an offwhite solid (125 mg, 38.1%). 1H NMR (300 MHz, CD3OD): δ 0.82 (dd, 6H), 1.22 (br s, 1H), 1.87 (m, 4H), 2.03 (m, 1H), 2.18 (app t, 1H), 2.75 (br t, 1H), 2.95 (br t, 1H), 3.44 (br d, 1H), 3.56 (br d, 1H), 4.50−4.68 (m, 3H), 6.70 (m, 1H), 6.75 (m, 1H), 6.97 (m, 1H), 7.17 (app t, 1H), 7.31 (br s, 1H), 7.50 (dd, 1H), 7.82 (dd, 1H) ppm. HRMS: m/z 544.2015, calcd 544.2015 (M + H). 2-(3-Chlorophenoxy)-3-fluoro-4-((S)-3-methyl-1-((S)-3-(5-methyl2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (47). A diastereomeric mixture of 33 (328 mg, 0.60 mmol) was subjected to HPLC preparative chiral separation eluting with a mixture of hexanes (50%), methanol/ethanol (1:1) (50%), and diethylamine (0.1%). The title compound 47 eluted second. After removal of the solvent, the title compound 47 was obtained as an off white solid (125 mg, 38.1%). LC−MS (method 3): tR = 2.0 min, m/z = 545.0 (M + H). 1H NMR (300 MHz, CD3OD): δ 0.87 (dd, 6H), 1.21 (br s, 1H), 1.87 (m, 4H), 2.02 (m, 1H), 2.19 (app t, 1H), 2.75 (br t, 1H), 2.95 (br t, 1H), 3.49 (m, 2H), 4.50−4.68 (m, 3H), 6.73 (m, 2H), 6.96 (m, 1H), 7.18 (app t, 1H), 7.32 (br s, 1H), 7.50 (dd, 1H), 7.82 (dd, 1H) ppm. HRMS: m/z 544.2027, calcd 544.2015 (M + H). 2-(3-Chlorophenoxy)-3-fluoro-4-((R)-1-((S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)propyl)benzoic Acid (42). Yield: 28 mg (23.3%) as an off-white solid. LC−MS (method 3): tR = 1.75 min, m/z = 516.2 (M + 1). 1H NMR (300 MHz, CD3OD): δ 0.78 (t, 3H), 1.06 (d, 2H), 1.54−1.86 (br m, 8H), 1.97 (m, 1H), 2.15 (br t, 2H), 2.80 (m, 1H), 2.91 (m, 1H), 3.89 (m, 1H), 4.42 (m, 1H), 6.64−6.75 (m, 2H), 6.92 (m, 1H), 7.15 (t, 1H), 7.27 (dd, 1H) 7.54 (br s, 1H), 7.64 (app d, 1H) ppm. HRMS: m/z 516.1677, calcd 516.1702 (M + H). 2-(3-Chlorophenoxy)-3-fluoro-4-((S)-1-((S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)propyl)benzoic Acid (43). Yield: 12 mg (10%) as an off-white solid. LC−MS (method 3): tR = 1.72 min, m/z = 516.2 (M + 1). 1H NMR (300 MHz, CD3OD): δ 0.75 (t, 3H), 1.47−2.11 (br m, 11H), 2.24 (br t, 1H), 2.76 (m, 1H), 2.96 (m, 1H), 3.82 (m, 1H), 4.47 (m, 1H), 6.70 (m, 2H), 6.92 (m, 1H), 7.13 (m, 1H), 7.28 (m, 1H), 7.51 (br s, 1H), 7.65 (app d, 1H) ppm. HRMS: m/z 516.1675, calcd 516.1702 (M + H). 2-(3-Chlorophenoxy)-3-fluoro-4-((R)-1-((S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (44). Yield: 25 mg (23.8%) as an off-white solid. LC−MS (method 3): tR = 1.84 min, m/z = 532.2 (M + H). 1H NMR (300 MHz, CD3OD): δ 0.77 (br t, 3H), 1.06 (d, 2H), 1.56−1.87 (br m, 8H), 1.97 (m, 1H), 2.17 (br t, 2H), 2.82 (m, 1H), 2.92 (m, 1H), 3.91 (m, 1H), 4.43 (m, 1H), 4.81 (br s, 1H), 6.74 (m, 1H), 6.87 (m, 1H), 7.08 (m, 2H), 7.28 (m, 1H), 7.53 (br s, 1H), 7.66 (br d, 1H) ppm. HRMS: m/z 530.1853, calcd 530.1859 (M + H). L



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2-(3-Chlorophenoxy)-3-fluoro-4-((S)-1-((S)-3-(5-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (45). Yield: 26 mg (24.7%) as an off-white solid. LC−MS (method 3): tR = 1.86 min, m/z = 532.2 (M + H). 1H NMR (300 MHz, CD3OD): δ 0.75 (br t, 3H), 1.05 (d, 2H), 1.50−2.09 (br m, 10H), 2.25 (t, 1H), 2.77 (m, 1H), 2.98 (m, 1H), 3.83 (m, 1H), 4.48 (m, 1H), 4.78 (m, 1H), 6.72 (m, 1H), 6.88 (m, 1H), 7.08 (m, 2H), 7.29 (dt, 1H), 7.52 (br s, 1H), 7.66 (dd, 1H) ppm. (S)-2-(3-Chlorophenoxy)-3-fluoro-4-((3-(5-methyl-2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzoic Acid (19). (S)-2-(3-Chlorophenoxy)-3-fluoro-4-((3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzonitrile was added to ethanol (3 mL) and water (3 mL) followed by NaOH (32.4 mg, 0.81 mmol) pellets. The suspension was heated under nitrogen at reflux for 2 days. The solvents were stripped, and any residual EtOH was chased with water. The residue was then taken up in water, and the pH was adjusted to ∼5 with 2 N HCl. The aqueous portion was then extracted three times with EtOAc. The combined organic layers were dried over MgSO4 and stripped. The crude white solid was taken up in DMSO and a little acetonitrile and purified via preparative HPLC eluting with 15−50% acetonitrile with 0.1% TFA. The fractions containing the major peak were combined and concentrated on the Rotovap to remove acetonitrile. The remaining solution was then frozen on dry ice and placed on the lyophilizer to give the title compound (0.02 g, 50.6%) as a solid. LC−MS (method 3): tR = 1.89 min, m/z = 488 (M + H). 1H NMR (300 MHz, CD3OD): δ 7.87 (dd, 1H), 7.55 (m, 1H), 7.48 (br s, 1H), 7.27 (t, 1H), 7.06 (dm, 1H); 6.88 (t, 1H), 6.80 (dd, 1H), 4.67 (m, 1H), 4.33 (m, 1H), 3.43 (m, 1H), 3.17 (t, 1H), 2.95 (t, 1H), 2.20 1.80 (overlapping m and s, 6H) ppm. HRMS: m/z 488.1383, calcd 488.1389 (M + H).

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Prasad for human TMK IC50 values, and Selvi Pradeepan for coordinating the log D measurements.

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ABBREVIATIONS TMK, thymidylate kinase; MIC, minimum inhibitory concentration

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(1) Fischbach, M. A.; Walsh, C. T. Antibiotics for Emerging Pathogens. Science 2009, 325, 1089−1093. (2) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Drugs for Bad Bugs: Confronting the Challenges of Antibacterial Discovery. Nat. Rev. Drug Discovery 2007, 6, 29−40. (3) Choi, J. Y.; Plummer, M. S.; Starr, J.; Desbonnet, C. R.; Soutter, H.; Chang, J.; Miller, J. R.; Dillman, K.; Miller, A. A.; Roush, W. R. Structure Guided Development of Novel Thymidine Mimetics Targeting Pseudomonas aeruginosa Thymidylate Kinase: From Hit to Lead Generation. J. Med. Chem. 2012, 55, 852−870. (4) Keating, T. A.; Newman, J. V.; Olivier, N. B.; Otterson, L. G.; Andrews, B.; Boriack-Sjodin, P. A.; Breen, J. N.; Doig, P.; Dumas, J.; Gangl, E.; Green, O. M.; Guler, S. Y.; Hentemann, M. F.; JosephMcCarthy, D.; Kawatkar, S.; Kutschke, A.; Loch, J. T.; McKenzie, A. R.; Pradeepan, S.; Prasad, S.; Martinez-Botella, G. In Vivo Validation of Thymidylate Kinase (TMK) with a Rationally Designed, Selective Antibacterial Compound. ACS Chem. Biol. 2012, 7, 1866−1872. (5) Vanheusden, V.; Van Rompaey, P.; Munier-Lehmann, H.; Pochet, S.; Herdewijn, P.; Van Calenbergh, S. Thymidine and Thymidine-5′O-Monophosphate Analogues as Inhibitors of Mycobacterium tuberculosis Thymidylate Kinase. Bioorg. Med. Chem. Lett. 2003, 13, 3045− 3048. (6) Deville-Bonnea, D.; El Amria, C.; Meyer, P.; Chen, Y.; Agrofoglio, L. A.; Janin, J. Human and Viral Nucleoside/Nucleotide Kinases Involved in Antiviral Drug Activation: Structural and Catalytic Properties. Antiviral Res. 2010, 86, 101−120. (7) Martinez-Botella, G.; Breen, J. N.; Duffy, J. E.; Dumas, J.; Geng, B.; Gowers, I. K.; Green, O. M.; Guler, S.; Hentemann, M. F.; Hernandez-Juan, F. A.; Joseph-McCarthy, D.; Kawatkar, S.; Larsen, N. A.; Lazari, O.; Loch, J. T.; Macritchie, J. A.; McKenzie, A. R.; Newman, J. V.; Olivier, N. B.; Otterson, L. G.; Owens, A. P.; Read, J.; Sheppard, D. W.; Keating, T. A. Discovery of Selective and Potent Inhibitors of Gram-Positive Bacterial Thymidylate Kinase (TMK). J. Med. Chem. 2012, 55, 10010−10021. (8) Martinez-Botella, G.; Loch, J. T.; Green, O. M.; Kawatkar, S. P.; Olivier, N. B.; Boriack-Sjodin, P. A.; Keating, T. A. Sulfonylpiperidines as Novel, Antibacterial Inhibitors of Gram-Positive Thymidylate Kinase (TMK). Bioorg. Med. Chem. Lett. 2013, 23, 169−173. (9) Pace, C. N. Contribution of the Hydrophobic Effect to Globular Protein Stability. J. Mol. Biol. 1992, 226, 29−35. (10) Pace, C. N.; Fu, H.; Fryar, K. L.; Landua, J.; Trevino, S. R.; Shirley, B. A.; Hendricks, M. M.; Iimura, S.; Gajiwala, K.; Scholtz, J. M.; Grimsley, G. R. Contribution of Hydrophobic Interactions to Protein Stability. J. Mol. Biol. 2011, 408, 514−528. (11) Vallone, B.; Miele, A. E.; Vecchini, P.; Chiancone, E.; Brunori, M. Free Energy of Burying Hydrophobic Residues in the Interface between Protein Subunits. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6103− 6107. (12) Bissantz, C.; Kuhn, B.; Stahl, M. A Medicinal Chemist’s Guide to Molecular Interactions. J. Med. Chem. 2010, 53, 5061−5084. (13) Böhm, H.-J.; Klebe, G. What Can We Learn from Molecular Recognition in Protein-Ligand Complexes for the Design of New Drugs? Angew. Chem., Int. Ed. Engl. 1996, 35, 2588−2614. (14) Leung, C. S.; Leung, S. S.; Tirado-Rives, J.; Jorgensen, W. L. Methyl Effects on Protein-Ligand Binding. J. Med. Chem. 2012, 55, 4489−4500. (15) Gu, Y.; Kar, T.; Scheiner, S. Fundamental Properties of the CH···O Interaction: Is It a True Hydrogen Bond? J. Am. Chem. Soc. 1999, 121, 9411−9422.

ASSOCIATED CONTENT

* Supporting Information S

Protocols for enzymatic, MIC, protein binding, and log D assays, protein crystallography methods and statistics tables, details of animal care and pharmacokinetic experiments, and S. aureus mouse thigh infection model. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

4QG7 (compound 18), 4QGA (19), 4QGF (38), 4QGG (46), and 4QGH (47).

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (781) 839 4694. Present Addresses §

S.P.K.: AstraZeneca Oncology Innovative Medicines, 35 Gatehouse Dr., Waltham, MA 02451. ∥ T.A.K.: ImmunoGen, Inc., 830 Winter St., Waltham, MA 02451. ⊥ M.F.H.: Novartis Institutes for BioMedical Research, 250 Massachusetts Ave., Cambridge, MA 02139. # G.M.-B.: Sage Therapeutics, 215 First St., Cambridge, MA 02141. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mahalingam Kannan and the team at Syngene (Bangalore, India) for chemistry support. We thank AstraZeneca Infection colleagues Christina Blinn and Krista Farrington for in vivo pharmacology support, Nancy DeGrace and Natascha Bezdenejnih-Snyder for analytical support, Swati M



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