Investigating the Antibacterial Activity of Biphenylthiazoles against

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Investigating the Antibacterial Activity of Biphenylthiazoles against Methicillin- and Vancomycin-Resistant Staphylococcus aureus (MRSA and VRSA) Mohamed Hagras,† Haroon Mohammad,‡ Mohamed S. Mandour,† Youssef A. Hegazy,‡ Adel Ghiaty,† Mohamed N. Seleem,*,‡,§ and Abdelrahman S. Mayhoub*,†,∥ †

Department of Organic Chemistry, College of Pharmacy, Al-Azhar University, Cairo 11884, Egypt Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907, United States § Purdue Institute for Inflammation, Immunology, and Infectious Diseases, West Lafayette, Indiana 47907, United States ∥ Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology, Giza 12588, Egypt ‡

S Supporting Information *

ABSTRACT: Phenylthiazoles were reported previously as a new scaffold with antibacterial activity against an array of multidrugresistant staphylococci. However, their promising antibacterial activity was hampered in large part by their short half-life due to excessive hepatic clearance. Close inspection of the structure−activity-relationships (SARs) of the phenylthiazoles revealed two important structural features necessary for antibacterial activity (a nitrogenous and a lipophilic component). Incorporating the nitrogenous part within a pyrimidine ring resulted in analogues with a prolonged half-life, while the biphenyl moiety revealed the most potent analogue 1b. In this study, advantageous moieties have been combined to generate a new hybrid scaffold of 5pyrimidinylbiphenylthiazole with the objective of enhancing both anti-MRSA activity and drug-like properties. Among the 37 tested biphenylthiazoles, piperazinyl-containing derivatives 10, 30, and 36 were the most potent analogues with MIC values as low as 0.39 μg/mL. Additionally, 36 exhibited significant improvement in stability to hepatic metabolism.



INTRODUCTION The United Nations recently declared that bacterial resistance to antibiotics poses a “fundamental threat” to global health.1 Over the past decade, it has become apparent that several highly resistant bacterial pathogens have acquired clever mechanisms to negate the effectiveness of numerous therapeutic agents.2 Staphylococcus aureus is one bacterial pathogen that has emerged as a significant concern to healthcare professionals worldwide. Over a period of 8 decades, strains of S. aureus have been isolated that exhibit resistance to several classes of antibacterial drugs including β-lactam antibiotics,3 macrolides,4 fluoroquinolones,4−7 glycopeptides,8 and oxazolidinones.9 The challenge of treating drug-resistant staphylococcal infections was highlighted in a recent report published by the U.S. Centers for Disease Control and Prevention (CDC). Among 23 000 documented fatalities due to antibiotic-resistant infections in the United States alone,10 methicillin-resistant Staphylococcus aureus (MRSA) was responsible for nearly half of these fatalities. Per the CDC report, © 2017 American Chemical Society

nearly 1 in 7 patients that contracted a severe MRSA infection died. This highlights the need for development of new antibacterial agents capable of treating infections caused by multidrug-resistant S. aureus. Our research group has focused on developing new antibacterial agents composed of a novel scaffold (phenylthiazole) that are potent inhibitors of MRSA growth.11−15 The first-generation phenylthiazoles were characterized by two structural groups: a nitrogenous head and a lipophilic side chain linked to the phenyl ring. Subsequent optimization of the lipophilic tail afforded the biphenylthiazole 1b with improved activity against MRSA (Figure 1).12 Recently, we discovered the phenylthiazole compounds exert their antibacterial activity by interfering with bacterial cell wall synthesis. More specifically, we demonstrated that the first-generation lead phenylthiazole compound 1a exerts its antibacterial effect by inhibiting two Received: March 13, 2017 Published: April 24, 2017 4074

DOI: 10.1021/acs.jmedchem.7b00392 J. Med. Chem. 2017, 60, 4074−4085

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1). The presence of two extra doublet signals in the 1H NMR spectrum with J value of 12 Hz confirmed the trans geometry of Scheme 1a

Figure 1. Development progress of phenylthiazole antibiotics. a

enzymes (undecaprenyl pyrophosphate synthase and undecaprenyl pyrophosphate phosphatase) involved in peptidoglycan synthesis.16 The initial series of phenylthiazoles were less potent, in an in vitro assay, than antibiotics frequently used to treat MRSA infections, including vancomycin. However, the phenylthiazoles do possess a unique advantage over vancomycin and linezolid, the two cornerstone antibiotics for treatment of invasive MRSA infections, in that they display rapid killing kinetics.11 Linezolid is a bacteriostatic agent.17 In contrast, vancomycin exhibits a slow bactericidal mode of action18 resulting in difficulty in clearing an infection19 and clinical failure in several reported cases.20 The rapid bactericidal characteristic of the phenylthiazoles observed in vitro against MRSA is posited to be beneficial in helping to clear an infection in vivo, particularly for invasive infections such as endocarditis.21 Prior to examining the anti-MRSA activity of our newly developed compounds in systemic-infected animal models, the PK parameters of 1a were evaluated. Unexpectedly, the lead compound 1a exhibited a very poor PK profile as it was rapidly cleared by human liver microsomes at a high rate (80.3 μL min−1 mg−1) resulting in a short half-life (28.8 min).12 In order to improve the short half-life, the Schiff’s bond (CN) was incorporated within a pyrimidine linker as shown in Figure 1. This strategy furnished compound 1c with enhanced PK properties15 (Figure 1). In this article, the active moieties for 1b and 1c were combined together in one scaffold to generate more potent, metabolically stable phenylthiazole derivatives (Figure 1). In brief, the same strategy of incorporating the C N bond within the more stable pyrimidine was utilized in order to generate a new series of phenylthiazoles bearing a phenyl ring at the lipophilic side chain. These modifications were posited to result in analogues with more potent anti-MRSA activity and improved stability to hepatic metabolism (Figure 1).

Reagents and conditions: (a) DMF−DMA, heat at 80 °C, 8 h; (b) , K2CO3, absolute EtOH, heat at reflux, 3−8 h.

the 4-(dimethylamino)butenone moiety of compound 3. Subsequent reaction of enaminone 3 with guanidine or carboximidine afforded a series of pyrimidine-containing derivatives 4−14 (Scheme 1). Upon pyrimidine formation, the δ value of enaminone with the two distinct doublets was shifted to a higher field, and the value of the coupling constant changed from 12 to 5 Hz indicating a change in configuration from E to Z. To complete the new series of biphenylthiazole derivatives, a second intermediate, 16, was utilized (Scheme 2). First, the methylsulfone 16 was obtained by oxidizing methylmercaptopyrimidine 15 prepared from the reaction between compound 3 and thiourea, followed by methylation (Scheme 2). Hence, the methylsulfonyl moiety was replaced by 24 different nucleophiles to afford the desired final products 17−41 (Scheme 2).



BIOLOGICAL RESULTS AND DISCUSSION Anti-MRSA Activity. As we reported previously, the nitrogenous cationic component in arylthiazoles is essential for anti-MRSA activity.12 In this article, we investigated the structure−activity-relationships (SARs) of the cationic element at pyrimidine position-2 via synthesizing and evaluating the anti-MRSA potency of 37 biphenylthiazolyl analogues. First, removal of the nitrogenous moiety led to compound 4 (R = H), resulting in complete abolishment of anti-MRSA activity (Table 1). In the absence of detailed information about the molecular target, this observation further highlights the importance of the nitrogenous group at positon-2 of the pyrimidine ring. This is in agreement with our previously explored SAR at the cationic part.12,14 With this information in hand, we started exploring the proper nitrogenous moiety at the same position by initially adding an amino group (compound 5, Table 1) as the simplest nitrogenous substituent. As expected, weak antibacterial activity was observed for 5 with a minimum inhibitory concentration (MIC) value of around 6 μg/mL against MRSA (Table 1). Adding one methyl group to



RESULTS AND DISCUSSION Chemistry. The key starting material 4-(dimethylamino)butenone derivative 3 was obtained by treatment of the methyl ketone 2 with DMF−DMA in solvent-free conditions (Scheme 4075

DOI: 10.1021/acs.jmedchem.7b00392 J. Med. Chem. 2017, 60, 4074−4085

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Scheme 2a

Table 1. Minimum inhibitory concentration (MIC in μg/ mL) of newly synthesized biphenylthiazoles vs. MRSA (2658 RCMB)a

a

Reagents and conditions: (a) (i) thiourea, KOH, EtOH, heat at reflux, 8 h; (ii) dimethyl sulfate, KOH, H2O, 23 °C, 2 h; (b) MCPBA, dry DCM, 23 °C, 16 h; (c) appropriate amine, hydrazine, guanidine, or carboximidate, dry DMF, heat at 80 °C for 0.5−8 h.

the pendent amine provided compound 6 with weaker antiMRSA potency (Table 1, entry 5). However, increasing the carbon chain from methyl to ethyl (compound 17) improved the activity by one-fold (Table 1, entry 14); further lengthening of the carbon chain led to a series of derivatives (compounds 18−22) without antibacterial activity, irrespective of the alky side chain (Table 1, entries 15−19). Furthermore, a second alkylation with dimethyl or diethyl groups (compounds 23 and 24) or incorporation of the nitrogen atom within cyclic structures (compounds 8, 9, and 25) furnished inactive derivatives (Table 1, entries 7, 8, and 22). These results collectively suggest that the presence of a hydrophobic region around the pyrimidine position-2 is unfavorable. Taking into consideration the results highlighted above, the next set of structures included adding a second polar atom, HBD or HBA, to the pyrimidine position-2. Hence, the piperazinyl 10, morpholinyl 26, and hydroxyazetidinyl 27 derivatives were synthesized. All three compounds maintained anti-MRSA activity with MIC values 1- or 2-fold better than 5, the lead compound in this series (Table 1, entries 9, 23, and 24). Encouraged by these results, a second amino group was incorporated into the cationic position, and the hydrazinyl analogue 28 was synthesized. For the first time in this series, the MIC of the hydrazide-containing derivative 28 (Table 1, entry 25) was similar to vancomycin (Table 1, entry 31), the drug of choice for treatment of invasive MRSA infections. We extended the structure of 28 further by incorporating the terminal amine within the morpholin or piprazine moieties, the two most active cationic elements obtained so far. This yielded

a

MIC: minimum inhibitory concentration. NA: not applicable. Van.: vancomycin.

compounds 29 and 30 with MIC values of 3.12 and 0.78 μg/ mL, respectively (Table 1, entries 23 and 24). The chemical modifications improved the MIC value of the hydrazidecontaining derivative 28 one-fold and provided the first analogue (compound 30) in this series with a MIC value in the submicrogram level. The 1-aminopiperazino derivative 30 was the first compound in this set to demonstrate antibacterial activity better than vancomycin. Taking into account that drugs that contain a pendent hydrazine and/or hydrazide motif are potent P450 inhibitors and are associated with idiosyncratic hepatotoxicity,22−24 incorporating the hydrazine moiety within a cyclic structure mitigates this issue. Next, the nitrogenous side chain at pyrimidine position-2 was further extended and a series of 12 guanidine- and guanidine4076

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Table 2. Minimum Inhibitory Concentration (MIC in μg/mL) and the Minimum Bactericidal Concentration (MBC μg/mL) of Active Compounds Screened against Additional S. aureus Isolates, Vancomycin-Resistant E. faecium, and P. aeruginosa

compd 30 32 35 36 vancomycin a

S. aureus RN4220

MRSA NRS119

MRSA NRS123 (USA400)

MIC MBC

MIC MBC

MIC MBC

4 8 8 2 1

8 32 32 8 1

4 8 8 2 1

4 16 16 4 1

4 16 16 1 1

4 16 16 2 1

VRS10 (VRSA)

VRS11a (VRSA)

VRS12 (VRSA)

vancomycin-resistant Enterococus faecium ATCC 700221

Pseudomonas aeruginosa ATCC 15442

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MIC

8 32 32 4 512

8 64 64 8 512

8 32 32 4 >512

8 64 64 8 >512

8 64 64 2 16

8 64 64 8 256

>64 NDa 8 16 >64

>64 NDa >64 >64 NA

ND = not determined.

like-containing structures (compounds 14 and 31−41) were prepared. The MIC value of the guanidine-containing 31 (Table 1, entry 28) was similar to vancomycin. Adding one methyl group to the terminal amino group (compound 32) had no effect on the antibacterial activity (Table 1, entry 29). However, increasing the number of carbon units around the terminal amine drastically decreased the anti-MRSA activity, as observed with the pyrrolidinyl and piperidine derivatives 33 and 34. Similar to the SAR with 2-aminopyrimidines (5, 10, and 26) and 2-hydrazinopyrimidines (compounds 28 and 30), adding an extra HBA to the nitrogenous side chain significantly improved the compounds’ MIC value. In brief, the piperazinyl analogue 36 (Table 1, entry 33) inhibited MRSA at a concentration of 0.39 μg/mL. This value is 3 times better than the corresponding guanidinyl derivative 31 and nearly 6fold more potent than the lead compound 1b (Table 1, entry 2). A point worth highlighting is that every compound where the nitrogenous side chain was part of an aromatic structure lacked antibacterial activity, as observed with compounds 11−13 and 37−40 (MIC > 25 μg/mL) We moved to confirm the potent activity of four compounds against six additional clinical isolates of drug-resistant S. aureus (Table 2). The piperazine-1-carboximidamide analogue 36 inhibited the strains tested at concentrations similar to vancomycin. Of note, 36 had a MIC value that was a 100 times more potent than vancomycin against vancomycinresistant S. aureus isolates (VRS10, VRS11a, and VRS12) (Table 2). In order to examine the possible antibacterial spectrum of activity of this new series of compounds, their MIC against one additional Gram-positive bacterial pathogen (vancomycin-resistant Enterococcus faecium (VRE)) and one Gram-negative bacterial pathogen (Pseudomonas aeruginosa) was examined (Table 2). The compounds were less active against vancomycin-resistant E. faecium compared to MRSA and VRSA. Compounds 35 and 36 were able to inhibit growth of VRE ATCC 700221 at concentrations of 8 and 16 μg/mL, respectively. However, the compounds were inactive against Pseudomonas aeruginosa (MIC > 64 μg/mL). In order to investigate if the most potent compound 36 exhibited bactericidal activity against MRSA, the minimum bactericidal concentration (MBC) was determined against six clinical isolates of S. aureus (Table 2). Compound 36 possessed minimum bactericidal concentration (MBC) values close to its MIC values. This observation suggests compound 36 is a bactericidal agent. To confirm this, we assessed how rapidly compound 36 was able to reduce a high inoculum of MRSA using a standard time−kill assay (Figure 2). Unlike vancomycin, a slow bactericidal antibiotic25 that requires 24 h to completely

Figure 2. Time−kill analysis of biphenylthiazoles 35 and 36 against MRSA USA300 and vancomycin over a 24 h incubation period at 37 °C. DMSO served as a negative control. The error bars represent standard deviation values obtained from triplicate samples used for each compound/antibiotic studied.

decrease MRSA CFU to zero, compound 36 exhibited potent bactericidal activity as it completely reduced MRSA CFU to zero within 2 h. This value is 5 times better than the reported value for biphenyl lead compound 1b which requires 10 h to produces a 3 log10 reduction in MRSA CFU mL−1.11 Accordingly, in addition to the superior antibacterial potency against vancomycin-resistant S. aureus (VRSA) strains, compound 36 exhibits an additional advantage over vancomycin in its ability to rapidly eliminate MRSA. The rapid bactericidal activity is important from a clinical perspective to limit/prevent a bacterial infection from spreading.26 Interestingly, compound 35 is structurally similar to 36, but compound 35 behaves differently in the time−kill assay, under the same experimental conditions (Figure 2). Regrowth of bacteria exposed to compound 35 appeared after 12 h, indicating more frequent dosing (at least twice daily) would be necessary in order to effectively treat an infection. One approach to overcome antibiotic resistance is to design inhibitors to target bacterial resistance mechanisms to specific antibiotics (such as inclusion of a β-lactamase inhibitor, like clavulanic acid, with β-lactam antibiotics) or to sensitize resistant strains to the effect of key antibiotics.27 Given vancomycin’s importance clinically for use in treatment of MRSA infections, the rise of VRSA isolates has been deeply concerning. Identifying molecules capable of resensitizing VRSA to the effect of vancomycin is a promising approach to prolong the utility of this key therapeutic. In this regard, we investigated whether three of the most promising piperazinylcontaining compounds would be capable of resensitizing VRSA to the effect of vancomycin.28 In the presence of a subinhibitory concentration (1/2 × MIC), the piperazinyl 10 was superior to the corresponding aminopiperazinyl analogue 30 in resensitiz4077

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ing VRSA to the effect of vancomycin. Briefly, in the presence of compound 10, vancomycin’s MIC against VRS10 dramatically improved (from 512 μg/mL to 1 μg/mL) while compound 30 produced a 128-fold improvement in MIC against the same strain. On the other hand, compound 36, the most potent derivative in this series, was unable to resensitize VRS10 to the effect of vancomycin. In order to confirm that the new analogues exhibit a beneficial relationship when combined with vancomycin against VRSA, a checkerboard assay was utilized. Against VRS10, compound 10 (FIC index range from 0.03 to 0.53) and compound 30 (FIC index range from 0.51 to 0.56) generally exhibited an additive relationship when combined with vancomycin (Table 3). Mindful of these Table 3. Minimum Inhibitory Concentration (MIC) Values and Fractional Inhibitory Concentration Index Range of Vancomycin (VAN) and Biphenylthiazole Compounds 10, 30, and 36 against Vancomycin-Resistant S. aureus (VRS10) compd

MIC of compd alone

MIC of compd in combination

10 30 36

32 8 8

1 4 0.13

10 30 36

32 8 8

1 4 0.13

a

MIC of VAN alone

Replicate 1 128 128 128 Replicate 2 256 512 256

MIC of VAN in combination

FIC index

resulta

64 8 128

0.53 0.56 1.02

ADD ADD ADD

0.5 4 256

0.03 0.51 1.02

SYN ADD ADD

Figure 3. Toxicity analysis of active compounds against human colorectal cells (HRT-18): percent viable mammalian cells (measured as average absorbance ratio (test agent relative to DMSO)) for cytotoxicity analysis of active compounds (tested in triplicate) at different concentrations against HRT-18 cells using the MTS 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay. Dimethyl sulfoxide (DMSO) was used as a negative control to determine a baseline measurement for the cytotoxic impact of each compound. The absorbance values represent an average of a minimum of three samples analyzed for each compound. Error bars represent standard deviation values for the absorbance values. A one-way ANOVA, with post hoc Dunnet’s multiple comparisons test, determined statistical difference between the values obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05).

ADD = additive. SYN = synergistic.

observations, compounds 10 and 30 might serve as potential partners to vancomycin in order to prolong its clinical usage as a therapeutic agent for treatment of severe, drug-resistant staphylococcal infections. After confirming the new biphenylthiazole derivatives exhibit potent, bactericidal activity against MRSA and are capable of resensitizing VRSA to the effect of vancomycin, we moved to examine their toxicity profile against mammalian cells. Compound 10 exhibited the best toxicity profile as it was nontoxic to HRT-18 cells even up to a concentration of 128 μg/mL (Figure 3). Compound 30 was not toxic up to 64 μg/ mL (Figure 3); this represents a 16-fold difference between the MIC values obtained against MRSA for the compound and the concentration where significant toxicity is observed. Compounds 35 and 36 were found to be nontoxic to HRT-18 cells up to a concentration of 32 μg/mL. We suspect that the toxicity observed for these two derivatives at 64 μg/mL was due to the high starting volume of DMSO used in these wells (12.8 μL), given solubility issues noted with 35 and 36. This volume of DMSO was found to be toxic when exposed to HRT-18 cells. Finally, encouraged by the promising results obtained thus far, the antibacterial activity of the most active compound 36, in addition to its analogue 30, was explored using the C. elegans animal model. The results were compared with the lead biphenylthiazole 1b and vancomycin (Figure 4). The C. elegans animal model is an established system for investigating the efficacy of antibacterial agents in vivo in early stages of drug discovery.29 Though C. elegans does not possess a liver, it does express cytochrome P450 enzymes that are homologous to mammalian CYP450s that are responsible for metabolism of xenobiotics.30 Compound 36 retained its potent antibacterial

Figure 4. Antibacterial activity of the lead compound 1b in addition to 30, 36, and vancomycin in vivo against MRSA-infected C. elegans: in vivo examination of antibacterial activity of test agents (at 20 μg/mL) in C. elegans AU37 infected with methicillin-resistant Staphylococcus aureus USA300. Vancomycin served as a positive control. Worms (in L4 stage of growth) were infected with bacteria for 6 h before transferring 20−30 worms to wells of a 96-well plate. Test agents were added and incubated with worms for 18 h. Worms were sacrificed, and the number of viable colony-forming units of MRSA USA300 in infected worms was determined for each treatment regimen. The figure presents the average percent reduction of MRSA USA300 for each treatment condition. Data shown in the graph are obtained from two independent experiments.

activity in vivo as it reduced the burden of MRSA USA300 by more than 65% in infected worms (Figure 4). This value is nearly 6 times better than the lead compound 1b and vancomycin, an antibiotic that exhibits time-dependent bactericidal activity.31 The aminopiperazine analogue 30 reduced the burden of MRSA USA300 by 46% (Figure 4). 4078

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To evaluate the metabolic stability of this new series, the most promising derivative 36 was incubated with human liver microsomes, in addition to two references drugs: one with a very short half-life (verapamil) and another drug with a long duration of action (warfarin). Compound 36 demonstrated more than 5 times improvement in stability to hepatic metabolism; hence, the half-life increased from 29 min in the case of the lead compound 1a to 151 min for the new derivative 36 (Table 4). On the other hand, the intrinsic clearance rate was also reduced by a factor of 5 (Table 4).

Table 6. Evaluation of Apparent Permeability of Tested Compounds, Ranitidine, and Warfarin via the Caco-2 Bidirectional Permeability Assay compd tested

mean Papp(A→B) a (×10−6 cm/s)

mean Papp(B→A) b (×10−6 cm/s)

efflux ratioc

1a 1b 36 ranitidine warfarin

0.0d 0.0d 0.0d 0.2 27.6

1.2 1.2 1.7 1.7 11.1

>2 >2 >2 8.5 0.4

a

Mean Papp(A→B) = mean apparent permeability of test compound from apical to basolateral surface. bMean Papp(B→A) = mean apparent permeability of test compound from basolateral to apical surface. c Efflux ratio = Papp(B→A)/Papp(A→B). dCompound not detected in receiver compartment (peak below limit of detection); permeability may be underestimated.

Table 4. Evaluation of Metabolic Stability of Tested Compounds, Verapamil, and Warfarin, in Human Liver Microsomesa

a

tested compd

NADPHdependent CLint (μL min−1 mg−1)

NADPHdependent t1/2 (min)

NADPH-free Clint (μL min−1 mg−1)

NADPHfree t1/2 (min)

1a 36 verapamil warfarin

80.3 15.3 116 0.3

28.8 151 19.8 >240

240

biphenylthiazoles with a pyrimidine linker at thiazole position-5 was constructed with various nitrogenous side chains at the pyrimidine ring position-2. Analysis of the SAR provided valuable insight about the nature of the active pocket that seems to accommodate large polar moieties. Derivatives containing the piperazine side chain provided better activity than the piperidine analogue; additionally, the aminopiperazine-containing compound 30 was more potent than the corresponding derivative 10 without the amine. Replacement of the amine group with a carboximidate remarkably improved the anti-MRSA activity. The enhancement in antibacterial activity of piperazinylcarboximidate analogue 36 was validated in a C. elegans model of MRSA infection. Compared to the lead compound 1b and vancomycin, compound 36 generated a more pronounced decrease in MRSA burden inside infected worms. Additionally, incorporating the CN bond within a pyrimidine ring notably improved the short half-lives observed with first-generation derivatives. As this series of compounds possessed poor aqueous solubility and limited permeability, they most likely are limited for use in topical formulations for treatment of uncomplicated skin and soft-tissue infections caused by MRSA.

CLint = microsomal intrinsic clearance. t1/2 = half-life.

Finally, adding two extra aromatic rings to the lead compound 1a, i.e., a phenyl group at the lipophilc side and a pyrimidine linker, dramatically deteriorated the aqueous solubility for this series of compounds. The aqueous solubility in phosphate buffered saline (PBS) of the two lead compounds 1a and 1b in addition to compounds 31 and 36 is summarized in Table 5. The three biphenyl-containing derivatives 1b, 31, Table 5. Evaluation of Solubility of Tested Compounds, Reserpine, Tamoxifen, and Verapamil in Phosphate Buffered Saline (PBS)a compd tested

solubility limit (μM)

1a 1b 31 36 reserpine tamoxifen verapamil

62.5 2.7 2.2 2.3 31.3 15.6 >500



EXPERIMENTAL PART

Chemistry. General. The purities of tested compound are ≥95% (elemental analysis). 1H NMR spectra were run at 400 MHz and 13C spectra were determined at 100 MHz in dimethyl sulfoxide (DMSOd6) on a Varian Mercury VX-400 NMR spectrometer. Chemical shifts are given in parts per million (ppm) on the δ scale. Chemical shifts were calibrated relative to those of the solvents. Flash chromatography was performed on 230−400 mesh silica. The progress of reactions was monitored with Merck silica gel IB2-F plates (0.25 mm thickness). The infrared spectra were recorded in potassium bromide disks on Pye Unicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers. Mass spectra were recorded at 70 eV. High resolution mass spectra for all ionization techniques were obtained from a FinniganMAT XL95. Melting points were determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected. All yields reported refer to isolated yields. Compound 2 was prepared as reported elsewhere.12 (E)-1-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-3(dimethylamino)prop-2-en-1-one (3). To compound 2 (3 g, 10.2 mmol), DMF−DMA (2.7 mL, 2.4 g, 20.4 mmol) was added, and the reaction mixture was heated at 80 °C for 8 h. After cooling, the formed solid was collected by filtration, washed with petroleum ether, and crystallized from ethanol to yield the desired product as an orange solid (3.4 g, 95%), mp = 187 °C. 1H NMR (DMSO-d6) δ: 8.04 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.69

a

Solubility limit corresponds to the highest concentration of test compound where no precipitate was detected (OD540).

and 36 were all sparingly soluble in PBS (Saq ranged between 2.2 and 2.7 μM, Table 5). As a result of the poor solubility, compound 36 exhibited a low rate of permeability when examined in a Caco-2 assay (Table 6). The poor aqueous solubility and limited permeability characteristics of 36 suggest it is not a viable drug candidate. However, formulation technology, including particle size reduction, spray drying, and hot melt extrusion, has been shown to be effective at overcoming such limitations in order to propel promising class IV compounds (candidates with low solubility/low permeability) into the market.32



CONCLUSION The recently developed phenylthiazoles represent a promising scaffold for development of new antibacterial agents targeting methicillin- and vancomycin-resistant S. aureus. A new series of 4079

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Journal of Medicinal Chemistry

Article

18.67. MS (m/z) 398. HRMS (EI) m/z 398.1585 M+, calcd for C24H22N4S 398.1565. Anal. Calcd for C24H22N4S: C, 72.33; H, 5.56; N, 14.06%. Found: C, 72.34; H, 5.57; N, 14.08%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(piperidin-1-yl)pyrimidin-4-yl)thiazole (9). Brown solid (0.19 g, 83%), mp = 125 °C. 1H NMR (DMSO-d6) δ: 8.41 (d, J = 5.6 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 6.89 (d, J = 4.8 Hz, 1H), 3.77 (t, J = 5.2 Hz, 4H), 2.72 (s, 3H), 1.63 (p, J = 4.4 Hz, 4H), 1.53 (p, J = 3.6 Hz, 2H). 13C NMR (DMSO-d6) δ: 166.30, 161.32, 159.53, 158.01, 153.67, 142.60, 139.46, 133.23, 132.13, 129.53, 128.55, 127.88, 127.27, 127.15, 106.21, 44.63, 25.70, 24.77, 18.71. MS (m/z) 412. HRMS (EI) m/z 412.1719 M+, calcd for C25H24N4S 412.1722. Anal. Calcd for C25H24N4S (MW = 412): C, 72.78; H, 5.86; N, 13.58%. Found: C, 72.79; H, 4.87; N, 13.58%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(4-methylpiperazin-1yl)pyrimidin-4-yl)thiazole (10). Brown solid (0.15 g, 69%), mp = 130 °C. 1H NMR (DMSO-d6) δ: 8.42 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 7.2 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 5.2 Hz, 1H), 3.75 (t, J = 4.4 Hz, 4H), 2.70 (s, 3H), 2.36 (t, J = 4.4 Hz, 4H), 2.20 (s, 3H). 13 C NMR (DMSO-d6) δ: 166.40, 161.42, 159.53, 158.04, 153.83, 142.63, 139.45, 132.31, 132.11, 129.53, 128.55, 127.87, 127.27, 127.15, 106.81, 54.80, 46.28, 43.72, 18.75. MS (m/z) 427. HRMS (EI) m/z 427.1838 M+, calcd for C25H25N5S 427.1831. Anal. Calcd for C25H25N5S (MW = 427): C, 70.23; H, 5.89; N, 16.38%. Found: C, 70.23; H, 5.90; N, 16.39%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(pyridin-2-yl)pyrimidin-4-yl)thiazole (11). Brown solid (0.15 g, 69%), mp = 182 °C. 1H NMR (DMSO-d6) δ: 8.98 (d, J = 5.4 Hz, 1H), 8.80 (d, J = 4.8 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.99 (t, J = 6.7 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.8 Hz, 2H), 7.50 (t, J = 6.7 Hz, 1H), 7.48 (t, J = 7.2 Hz, 2H), 7.45 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 2.82 (s, 3H). 13C NMR (DMSO-d6) δ: 167.27, 163.54, 159.17, 158.14, 155.08, 154.76, 150.22, 142.76, 139.40, 137.56, 131.96, 131.38, 129.50, 128.63, 127.91, 127.22, 127.13, 125.66, 124.01, 117.00, 18.65. MS (m/z) 406. HRMS (EI) m/z 406.1266 M+, calcd for C25H18N4S 406.1252. Anal. Calcd for C25H18N4S: C, 73.87; H, 4.46; N, 13.78%. Found: C, 73.88; H, 4.47; N, 13.79%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(pyridin-3-yl)pyrimidin-4-yl)thiazole (12). Yellow solid (0.22 g, 96%), mp = 171 °C. 1H NMR (DMSO-d6) δ: 9.55 (s, 1H), 8.97 (d, J = 5.6 Hz, 1H), 8.74 (d, J = 4.8 Hz, 1H), 8.71 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 7.6 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 7.6 Hz, 1H), 7.74 (t, J = 5.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H), 2.88 (s, 3H). 13C NMR (DMSO-d6) δ: 165.51, 163.12, 159.22, 158.13, 156.01, 154.73, 154.52, 151.13, 149.43, 142.56, 139.45, 133.12, 132.16, 129.53, 128.53, 127.86, 127.25, 127.13, 124.37, 116.11, 18.36. MS (m/z) 406. HRMS (EI) m/z 406.1268 M+, calcd for C25H18N4S 406.1252. Anal. Calcd for C25H18N4S: C, 73.87; H, 4.46; N, 13.78%. Found: C, 73.88; H, 4.47; N, 13.79%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(pyridin-4-yl)pyrimidin-4-yl)thiazole (13). Yellow solid (0.2 g, 85%), mp = 200 °C. 1H NMR (DMSO-d6) δ: 8.99 (d, J = 5.6 Hz, 1H), 8.79 (d, J = 4.4 Hz, 2H), 8.26 (d, J = 4.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 5.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.46 (t, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 2.83 (s, 3H). 13C NMR (DMSO-d6) δ: 165.51, 162.97, 159.43, 158.61, 154.71, 154.43, 149.33, 142.62, 139.41, 132.86, 132.16, 129.53, 128.53, 127.62, 127.25, 127.13, 124.39, 117.22, 18.36. MS (m/z) 406. HRMS (EI) m/z 406.1270 M+, calcd for C25H18N4S 406.1252. Anal. Calcd for C25H18N4S: C, 73.87; H, 4.46; N, 13.78%. Found: C, 73.89; H, 4.47; N, 13.78%. 1-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin2-yl}thiourea (14). White solid (0.15 g, 63%), mp = 185 °C. 1H NMR (DMSO-d6) δ: 10.63 (brs, 1H), 10.11 (brs, 1H), 9.21 (s, 1H), 8.70 (d, J = 5.7 Hz, 1H), 8.07 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.2 Hz, 2H), 7.47 (t, J = 7.2 Hz, 1H), 7.42 (d, J = 5.2 Hz, 1H), 2.79 (s, 3H). 13C NMR (DMSO-d6) δ: 181.37, 167.35, 159.33, 158.55, 157.76, 155.75, 143.01, 139.61, 131.82, 130.38, 129.56, 128.02, 127.81, 127.35, 127.19, 112.60, 18.82.

(d, J = 12 Hz, 1H), 7.49 (t, J = 8.7 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 5.4 (d, J = 12.3 Hz, 1H), 3.16 (s, 3H), 2.90 (s, 3H), 2.49 (s, 3H). 13C NMR (DMSO-d6) δ: 179.86, 165.48, 154.73, 154.44, 142.51, 139.43, 134.63, 132.16, 129.48, 128.49, 127.83, 127.22, 127.09, 94.20, 45.04, 37.64, 18.36 MS (m/z) 348. Anal. Calcd for C21H20N2OS (MW = 348): C, 72.38; H, 5.79; N, 8.04%. Found: C, 72.40; H, 5.80; N, 8.05%. Compounds 4−14. General Procedure. To a solution of enaminone 3 (0.2 g, 0.6 mmol) in absolute ethanol (5 mL), proper guanidine or carboximidate (1.25 mmol) and anhydrous potassium carbonate (0.2 g, 1.4 mmol) were added. The reaction mixture was heated at reflux for 8 h, ethanol was evaporated under reduced pressure, and the reaction was quenched with cold water (50 mL). The formed flocculated solid was filtered, washed with water, and purified by crystallization from absolute ethanol or via acid−base extraction using HCl (1 M, 50 mL). Upon neutralization with sodium carbonate to pH 7−8, the desired products were precipitated. The obtained solid was filtered, washed with a copious amount of distilled water, and dried. Physical properties and spectral analysis of isolated products are listed below: 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(pyrimidin-4-yl)thiazole (4). Light brown solid (0.13 g, 70%), mp = 247 °C. 1H NMR (DMSOd6) δ: 9.1 (s, 1H), 8.87 (d, J = 5.6 Hz, 1H), 8.08 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 5.5 Hz, 1H), 7.83 (d, J = 7.8 Hz, 2H), 7.73 (t, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 2.71 (s, 3H). 13 C NMR (DMSO-d6) δ: 167.31, 165.52, 159.10, 158.47, 157.53, 142.85, 142.57, 132.17, 131.96, 129.54, 128.58, 127.86, 127.25, 127.13, 118.13, 18.36. MS (m/z) 329. HRMS (EI) m/z 329.0999 M+, calcd for C20H15N3S 329.0987. Anal. Calcd for C20H15N3S (MW = 329): C, 72.92; H, 4.59; N, 12.76%. Found: C, 72.93; H, 4.59; N, 12.77%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}pyrimidin-2amine (5). Yellowish white solid (0.07 g, 71%), mp = 202 °C. 1H NMR (DMSO-d6) δ: 8.35 (d, J = 5.1 Hz, 1H), 8.06 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 7.8 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 6.73 (brs, 2H), 2.72 (s, 3H). 13C NMR (DMSO-d6) δ: 166.12, 163.83, 159.69, 158.27, 153.42, 142.56, 139.47, 132.38, 132.19, 129.54, 128.54, 127.63, 127.19, 127.13, 107.00, 18.63. MS (m/z) 344. HRMS (EI) m/z 344.1092 M+, calcd for C20H16N4S 344.1096. Anal. Calcd for C20H16N4S: C, 69.74; H, 4.68; N, 16.27%. Found: C, 69.75; H, 4.69; N, 16.29%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-N-methylpyrimidin-2-amine (6). White solid (0.09 g, 89%), mp = 190 °C. 1H NMR (DMSO-d6) δ: 8.37 (d, J = 5.1 Hz, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 8.1 Hz, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 4.5 Hz, 1H), 6.89 (brs, 1H), 2.86 (s, 3H), 2.74 (s, 3H). 13C NMR (DMSO-d6) δ: 166.13, 162.97, 159.47, 158.16, 153.61, 142.55, 139.46, 133.01, 132.17, 129.53, 128.53, 127.87, 127.20, 127.12, 106.62, 28.25, 18.68. MS (m/z) 358. HRMS (EI) m/z 358.1238 M+, calcd for C21H18N4S 358.1252. Anal. Calcd for C21H18N4S: C, 70.36; H, 5.06; N, 15.63%. Found: C, 70.35; H, 5.07; N, 15.64%. N-(4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl)cyanamide (7). Yellow solid (0.16 g, 75%), mp = 195 °C. IR (KBr) cm−1: 3383 (NH), 2160 (CN). 1H NMR (DMSOd6) δ: 8.20 (d, J = 4.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.1 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.42 (t, J = 7.2 Hz, 1H), 6.67 (d, J = 5.1 Hz, 1H), 5.6 (brs, 1H), 2.71 (s, 3H). 13 C NMR (DMSO-d6) δ: 167.21, 165.47, 159.02, 157.89, 152.39, 142.29, 139.55, 133.63, 132.45, 129.52, 128.47, 127.86, 127.12, 127.09, 118.12 105.04, 18.59. MS (m/z) 369. HRMS (EI) m/z 369.1040 M+, calcd for C21H15N5S 369.1048. Anal. Calcd for C21H15N5S: C, 68.27; H, 4.09; N, 18.96%. Found: C, 68.29; H, 4.09; N, 18.97%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)thiazole (8). Brown solid (0.1 g, 93%), mp = 130 °C. 1 H NMR (DMSO-d6) δ: 8.40 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 5.2 Hz, 1H), 3.51 (t, J = 6.4 Hz, 4H), 2.74 (s, 3H), 1.93 (t, J = 6.4 Hz, 4H). 13C NMR (DMSOd6) δ: 166.14, 160.13, 159.33, 157.93, 153.80, 142.56, 139.46, 132.16, 131.55, 129.53, 128.69, 127.98, 127.21, 127.14, 106.08, 46.75, 25.40, 4080

DOI: 10.1021/acs.jmedchem.7b00392 J. Med. Chem. 2017, 60, 4074−4085

Journal of Medicinal Chemistry

Article

0.92 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6) δ: 166.10, 162.58, 159.41, 158.30, 153.47, 142.55, 139.47, 132.92, 132.19, 129.53, 128.53, 127.89, 127.19, 127.13, 106.53, 42.97, 22.59, 18.65, 11.96. MS (m/z) 386. HRMS (EI) m/z 386.1577 M+, calcd for C23H22N4S 386.1565. Anal. Calcd for C23H22N4S: C, 71.47; H, 5.74; N, 14.50%. Found: C, 71.49; H, 5.75; N, 14.51%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-N-butylpyrimidin-2-amine (19). Yellow solid (0.07 g, 76%), mp = 160 °C. 1 H NMR (DMSO-d6) δ: 8.33 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.2 Hz, 2H), 7.49 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 7.27 (brs, 1H), 6.87 (d, J = 5.2 Hz, 1H), 3.22 (t, J = 6.8 Hz, 2H), 2.72 (s, 3H), 1.52 (p, J = 6.8 Hz, 2H), 1.33 (sixt, J = 7.6 Hz, 2H), 0.92 (t, J = 7.6 Hz, 3H). 13C NMR (DMSO-d6) δ: 166.09, 162.55, 159.51, 158.12, 153.53, 142.56, 139.47, 132.98, 132.19, 129.33, 128.53, 127.62, 127.18, 127.13, 106.52, 31.49, 31.13, 20.14, 18.65, 14.26. MS (m/z) 400. HRMS (EI) m/z 400.1715 M+, calcd for C24H24N4S 400.1722. Anal. Calcd for C24H24N4S: C, 71.97; H, 6.04; N, 13.99%. Found: C, 71.97; H, 6.05; N, 13.99%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-N-isopropylpyrimidin-2-amine (20). Yellow solid (0.07 g, 79%), mp = 188.5 °C. 1 H NMR (DMSO-d6) δ: 8.34 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 7.12 (brs, 1H), 6.87 (d, J = 5.2 Hz, 1H), 4.08 (sept, J = 6.8 Hz, 1H), 2.72 (s, 3H), 1.18 (d, J = 6.4 Hz, 6H). 13C NMR (DMSO-d6) δ: 166.11, 161.83, 159.53, 158.14, 153.49, 142.55, 139.47, 132.87, 132.19, 129.53, 128.53, 127.89, 127.19, 127.13, 106.49, 42.54, 22.73, 18.65. MS (m/z) 386. HRMS (EI) m/z 386.1561 M+, calcd for C23H22N4S 386.1565. Anal. Calcd for C23H22N4S: C, 71.47; H, 5.74; N, 14.50%. Found: C, 71.48; H, 5.75; N, 14.51%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-Ncycloproylpyrimidin-2-amine (21). Yellow solid (0.07 g, 74%), mp = 173 °C. 1H NMR (DMSO-d6) δ: 8.33 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 8 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 7.2 Hz, 2H), 7.49 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 7.21 (brs, 1H), 6.85 (d, J = 4.8 Hz, 1H), 4.19 (p, J = 7.2 Hz, 1H), 2.72 (s, 3H), 1.92 (m, 2H), 1.68 (m, 2H), 1.53 (m, 4H). 13C NMR (DMSO-d6) δ: 166.11, 162.20, 159.41, 158.43, 153.49, 142.54, 139.48, 133.01, 132.19, 129.53, 128.53, 127.89, 127.19, 127.13, 106.53, 52.78, 32.63, 23.98, 18.65. MS (m/z) 412. HRMS (EI) m/z 412.1742 M+, calcd for C25H24N4S 412.1722. Anal. Calcd for C25H24N4S: C, 72.78; H, 5.86; N, 13.58%. Found: C, 72.80; H, 5.87; N, 13.60%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-Ncyclohexylpyrimidin-2-amine (22). Brown solid (0.06 g, 57%), mp = 169 °C. 1H NMR (DMSO-d6) δ: 8.33 (d, J = 4.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 7.14 (brs, 1H), 6.85 (d, J = 4.8 Hz, 1H), 3.71 (m, 1H), 2.71 (s, 3H), 1.90 (m, 2H), 1.71 (m, 2H), 1.61 (m, 2H), 1.27 (m, 4H). 13C NMR (DMSO-d6) δ: 166.05, 161.80, 159.11, 158.33, 153.54, 142.56, 139.48, 132.98, 132.19, 129.53, 128.54, 127.91, 127.18, 127.13, 106.54, 32.77, 31.14, 25.85, 25.33, 18.61. MS (m/z) 426. HRMS (EI) m/z 426.1880 M+, calcd for C26H26N4S 426.1878. Anal. Calcd for C26H26N4S: C, 73.21; H, 6.14; N, 13.13%. Found: C, 73.22; H, 6.15; N, 13.14%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-N,Ndimethylpyrimidin-2-amine (23). Yellow solid (0.07 g, 82%), mp = 127 °C. 1H NMR (DMSO-d6) δ: 8.40 (d, J = 5.6 Hz, 1H), 8.03 (d, J = 8 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 7.2 Hz, 2H), 7.49 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 6.88 (d, J = 5.6 Hz, 1H), 3.14 (s, 6H), 2.71 (s, 3H). 13C NMR (DMSO-d6) δ: 166.19, 161.97, 159.28, 157.82, 153.72, 142.54, 139.45, 132.35, 132.14, 129.52, 128.53, 127.95, 127.20, 127.12, 105.95, 36.96, 18.75. MS (m/z) 372. HRMS (EI) m/z 372.1422 M+, calcd for C22H20N4S 372.1409. Anal. Calcd for C22H20N4S: C, 70.94; H, 5.41; N, 15.04%. Found: C, 70.95; H, 5.41; N, 15.05%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-N,N-diethylpyrimidin-2-amine (24). Yellow solid (0.06 g, 62%), mp = 140 °C. 1 H NMR (DMSO-d6) δ: 8.42 (d, J = 5.2 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 5.2 Hz, 1H), 3.15 (q, J = 4.4 Hz, 4H), 2.74 (s, 3H) 1.22 (t, J = 4.4 Hz, 6H). 13C NMR (DMSO-

MS (m/z) 403. HRMS (EI) m/z 403.0930 M+, calcd for C21H17N5S2 403.0925. Anal. Calcd for C21H17N5S2: C, 62.51; H, 4.25; N, 17.36%. Found: C, 62.52; H, 4.27; N, 17.37%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-{2-(methylthio)pyrimidin-4-yl}thiazole (15). To a solution of potassium hydroxide (0.2 g, 3.5 mmol) and thiourea (0.5 g, 6.5 mmol) in ethanol (15 mL), enaminone 3 (1 g, 3 mmol) was added. The reaction mixture was heated to reflux for 8 h and then cooled down in an ice bath to 8 °C. The formed crystals were filtered and washed with diethyl ether to yield the potassium salt intermediate as yellow crystals (1.1 g, 96%), mp >300 °C. 1H NMR (DMSO-d6) δ: 8.74 (d, J = 5.4 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 7.8 Hz, 2H), 7.49 (t, J = 7.8 Hz, 2H), 7.42 (t, J = 7.2 Hz, 1H), 7.13 (d, J = 5.6 Hz, 1H), 2.76 (s, 3H). To a solution of the obtained intermediate (0.8 g, 2.1 mmol) and potassium hydroxide (0.25g, 4.2 mmol) in water (15 mL), dimethyl sulfate (0.5 mL, 4 mmol) was added dropwise with vigorous stirring. After 2 h, the formed solid was filtered and washed with a copious amount of water to yield a yellowish white solid (0.69 g, 89%); mp = 173 °C. 1H NMR (DMSO-d6) δ: 8.68 (d, J = 5.4 Hz, 1H), 8.07 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 8.1 Hz, 2H), 7.49 (t, J = 7.8 Hz, 2H), 7.44 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 6.6 Hz, 1H), 2.78 (s, 3H), 2.57 (s, 3H). 13C NMR (DMSO-d6) δ: 173.77, 169.01, 159.87, 158.89, 156.67, 148.02, 143.18, 139.46, 131.23, 129.55, 128.54, 127.91, 127.37, 127.17, 104.11, 18.95, 14.98. MS (m/z) 375. Anal. Calcd for C21H17N3S2 (MW = 375): C, 67.17; H, 4.56; N, 11.19%. Found: C, 67.17; H, 4.55; N, 11.20%. 2-([1,1′-Biphenyl]-4-yl)-4-methyl-5-(2-(methylsulfonyl)pyrimidin-4-yl)thiazole (16). To a solution of compound 15 (0.5 g, 1.3 mmol) in dry DCM (5 mL), m-CPBA (0.514 g, 2.9 mmol) in DCM (5 mL) was added portionwise with continuous stirring. After the reaction mixture was kept at 23 °C for 16 h, additional DCM (10 mL) was added and the reaction mixture was washed with 25 mL of 5% aqueous solution of sodium metabisulfite and 25 mL of 5% aqueous sodium carbonate. The organic layer was separated, dried, and concentrated under reduced pressure to give the desired product as yellow crystals (0.52 g, 95%), mp = 180 °C. 1H NMR (DMSO-d6) δ: 8.98 (d, J = 5.4 Hz, 1H), 8.08 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 0.5.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.50 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 2.94 (s, 3H), 2.72 (s, 3H). 13C NMR (DMSO-d6) δ: 173.89, 168.13, 166.09, 160.18, 159.31, 156.51, 143.19, 139.33, 131.19, 129.54, 128.53, 127.96, 127.47, 127.17, 118.63, 44.81, 18.91. MS (m/z) 407; calcd for C21H17N3O2S2 407.0762. Anal. Calcd for C21H17N3O2S2 (MW = 407): C, 61.90; H, 4.21; N, 10.31%. Found: C, 61.91; H, 4.22; N, 10.32%. Compounds 17−41. General Procedure. To a solution of 16 (0.1 g, 0.25 mmol) in dry DMF (5 mL), a proper amine hydrazine, guanidine, or carboximidate (0.4 mmol) was added. The reaction mixture was heated at 80 °C for 0.5−8 h and then poured over ice− water (50 mL). The formed solid was filtered and washed with 50% ethanol and recrystallized from absolute ethanol. For 28, the crude solid was washed with boiling water to remove the residual hydrazine. Physical properties and spectral analysis of isolated products are listed below. 4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)-N-ethylpyrimidin-2-amine (17). Yellow solid (0.06 g, 67%), mp = 179.8 °C. 1 H NMR (DMSO-d6) δ: 8.34 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 7.25 (brs, 1H), 6.88 (d, J = 4.8 Hz, 1H), 3.36 (q, J = 6.8 Hz, 2H), 2.73 (s, 3H), 1.14 (t, J = 6.8 Hz, 3H). 13 C NMR (DMSO-d6) δ: 166.11, 162.38, 159.48, 158.49, 153.54, 142.54, 139.47, 132.88, 132.18, 129.52, 128.53, 127.86, 127.18, 127.12, 106.60, 35.89, 18.65, 15.06. MS (m/z) 372. HRMS (EI) m/z 372.1412 M+, calcd for C22H20N4S 372.1409. Anal. Calcd for C22H20N4S: C, 70.94; H, 5.41; N, 15.04%. Found: C, 70.95; H, 5.42; N, 15.05%. 4-{2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl}-N-propylpyrimidin-2-amine (18). Yellow solid (0.07 g, 74%), mp = 179.1 °C. 1 H NMR (DMSO-d6) δ: 8.34 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.2 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 7.30 (brs, 1H), 6.87 (d, J = 4.8 Hz, 1H), 3.23 (t, J = 7.2 Hz, 2H), 2.71 (s, 3H), 1.57 (sixt, J = 7.2 Hz, 2H), 4081

DOI: 10.1021/acs.jmedchem.7b00392 J. Med. Chem. 2017, 60, 4074−4085

Journal of Medicinal Chemistry

Article

d6) δ: 166.11, 162.38, 159.48, 158.49, 153.54, 142.54, 139.47, 132.88, 132.18, 129.52, 128.53, 127.86, 127.18, 127.12, 106.60, 35.89, 18.65, 15.06. MS (m/z) 400. HRMS (EI) m/z 400.1710M+, calcd for C24H24N4S 400.1722. Anal. Calcd for C24H24N4S (MW = 400): C, 71.97; H, 6.0; N, 13.99%. Found: C, 71.99; H, 6.01; N, 14.0%. 2-([1,1′-Biphenyl]-4-yl)-5-(2-(azetidin-1-yl)pyrimidin-4-yl)-4methylthiazole (25). Brown solid (0.06 g, 60%), mp = 125 °C. 1H NMR (DMSO-d6) δ: 8.39 (d, J = 5.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.6 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 6.95 (d, J = 5.6 Hz, 1H), 4.08 (t, J = 7.6 Hz, 4H), 2.72 (s, 3H), 2.31 (p, J = 7.6 Hz, 2H). 13C NMR (DMSO-d6) δ: 166.31, 162.85, 159.32, 158.08, 154.01, 142.60, 139.45, 132.10, 131.83, 129.53, 128.55, 127.87, 127.25, 127.13, 107.14, 50.34, 18.71, 16.30. MS (m/z) 384. HRMS (EI) m/z 384.1395 M+, calcd for C23H20N4S 384.1409. Anal. Calcd for C23H20N4S: C, 71.85; H, 5.24; N, 14.57%. Found: C, 71.86; H, 5.25; N, 14.58%. 4-(4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin2-yl)morpholine (26). Orange solid (0.07 g, 70%), mp = 172.8 °C. 1 H NMR (DMSO-d6) δ: 8.46 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 5.2 Hz, 1H), 3.73 (t, J = 5.2 Hz, 4H), 3.69 (t, J = 5.2 Hz, 4H), 2.73 (s, 3H). 13C NMR (DMSOd6) δ: 166.64, 161.48, 159.53, 158.05, 153.96, 142.63, 139.44, 132.91, 132.09, 129.53, 128.55, 127.86, 127.25, 127.14, 107.16, 66.40, 56.47, 18.78. MS (m/z) 414. HRMS (EI) m/z 414.1518 M+, calcd for C24H22N4OS 414.1514. Anal. Calcd for C24H22N4OS: C, 69.54; H, 5.35; N, 13.52%. Found: C, 69.55; H, 5.35; N, 13.53%. 1-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin2-yl}azetidin-3-ol (27). Orange solid (0.06 g, 61%), mp = 230 °C. 1 H NMR (DMSO-d6) δ: 8.41 (d, J = 4.8 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 4.8 Hz, 1H), 5.62 (brs, 1H), 4.57 (p, J = 4.8 Hz, 1H), 4.28 (dd, J = 4.4 Hz, J = 9.2 Hz, 2H), 3.83 (dd, J = 6.8 Hz, J = 9.2 Hz, 2H), 2.73 (s, 3H). 13C NMR (DMSOd6) δ: 166.38, 162.93, 159.41, 157.82, 154.07, 142.64, 139.45, 132.10, 131.82, 129.54, 128.56, 127.90, 127.28, 127.15, 107.30, 61.27, 60.32, 18.70. MS (m/z) 400. HRMS (EI) m/z 400.1330 M+, calcd for C23H20N4OS 400.1358. Anal. Calcd for C23H20N4OS: C, 68.98; H, 5.03; N, 13.99%. Found: C, 68.99; H, 5.05; N, 13.99%. 2-([1,1′-Biphenyl]-4-yl)-5-(2-hydrazinylpyrimidin-4-yl)-4methylthiazole (28). Yellowish white fluffy powder (0.07 g, 80%), mp = 171 °C. 1H NMR (DMSO-d6) δ: 8.37 (d, J = 4.8 Hz, 1H), 8.30 (brs, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.37 (t, J = 7.2 Hz, 1H), 6.91 (d, J = 5.6 Hz, 1H), 4.21 (brs, 2H), 2.73 (s, 3H). 13C NMR (DMSOd6) δ: 166.24, 164.62, 159.43, 159.10, 153.88, 142.59, 139.45, 132.16, 132.10, 129.53, 128.60, 127.90, 127.20, 127.17, 107.27, 18.75. MS (m/ z) 359. HRMS (EI) m/z 359.1229 M+, calcd for C20H17N5S 359.1205. Anal. Calcd for C20H17N5S: C, 66.83; H, 4.77; N, 19.48%. Found: C, 66.83; H, 4.78; N, 19.50%. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}morpholin-4-amine (29). Yellowish brown solid (0.06 g, 60%), mp = 130 °C. 1H NMR (DMSO-d6) δ: 8.46 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.48 (t, J = 6.8 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 4.8 Hz, 1H), 6.89 (brs, 1H), 4.03 (t, J = 5.6 Hz, 4H), 3.60 (t, J = 5.6 Hz, 4H), 2.71 (s, 3H). 13C NMR (DMSO-d6) δ: 168.66, 166.43, 161.47, 159.29, 158.04, 153.59, 142.63, 139.43, 132.19, 129.51, 128.54, 127.85, 127.47, 127.15, 107.15, 66.40, 44.12, 18.96. MS (m/z) 429. HRMS (EI) m/z 429.1642 M+, calcd for C24H23N5OS 429.1623. Anal. Calcd for C24H23N5OS: C, 67.11; H, 5.40; N, 16.30%. Found: C, 67.12; H, 5.40; N, 16.31%. 4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)-N-(4methylpiperazin-1-yl)pyrimidin-2-amine (30). Yellowish brown solid (0.06 g, 65%), mp = 125 °C. 1H NMR (DMSO-d6) δ: 8.43 (d, J = 4.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 6.88 (brs, 1H), 3.75 (t, J = 4.8 Hz, 4H), 2.71 (s, 3H), 2.36 (t, J = 4.8 Hz, 4H), 2.30 (s, 3H). 13C NMR (DMSO-d6) δ: 166.41, 161.42, 159.55, 158.05, 153.48, 142.63, 139.45, 133.17,

132.11, 129.53, 128.56, 127.89, 127.28, 127.15, 106.82, 54.81, 46.28, 43.72, 18.58. MS (m/z) 442. HRMS (EI) m/z 442.1947 M+, calcd for C25H26N6S 442.1940. Anal. Calcd for C25H26N6S: C, 67.85; H, 5.92; N, 18.99%. Found: C, 67.87; H, 5.93; N, 18.99%. 1-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin2-yl}guanidine (31). Yellowish brown solid (0.09 g, 95%), mp = 234 °C. 1H NMR (DMSO-d6) δ: 8.45 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 5.2 Hz, 1H), 7.01 (brs, 4H), 2.70 (s, 3H). 13C NMR (DMSO-d6) δ: 167.50, 166.11, 159.88, 158.40, 157.72, 153.08, 142.56, 139.48, 133.06, 132.21, 129.54, 128.54, 127.89, 127.22, 127.15, 107.81, 18.58. MS (m/z) 386. HRMS (EI) m/z 386.1325 M+, calcd for C21H18N6S 386.1314. Anal. Calcd for C21H18N6S: C, 65.26; H, 4.69; N, 21.75%. Found: C, 65.27; H, 4.69; N, 21.76%. 1-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin2-yl}-3-methylguanidine (32). Yellowish brown solid (0.06 g, 65%), mp = 176 °C. 1H NMR (DMSO-d6) δ: 8.45 (d, J = 4.8 Hz, 1H), 8.34 (brs, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 7.21 (brs, 1H), 7.01 (brs, 1H), 7.87 (d, J = 5.2 Hz, 1H), 2.86 (s, 3H), 2.71 (s, 3H). 13C NMR (DMSO-d6) δ: 166.14, 166.04, 159.49, 157.56, 153.62, 153.00, 142.55, 139.46, 133.09, 132.21, 129.53, 128.53, 127.89, 127.21, 127.15, 107.80, 18.68, 18.58. MS (m/z) 400. HRMS (EI) m/z 400.1488 M+, calcd for C22H20N6S 400.1470. Anal. Calcd for C22H20N6S: C, 65.98; H, 5.03; N, 20.98%. Found: C, 65.99; H, 5.04; N, 20.99%. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}pyrrolidine-1-carboximidamide (33). Yellowish brown solid (0.09 g, 92%), mp = 220 °C. 1H NMR (DMSO-d6) δ: 8.47 (d, J = 5.2 Hz, 1H), 8.08 (brs, 1H), 8.06 (brs, 1H), 8.02 (d, J = 8 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 5.2 Hz, 1H), 3.41 (t, J = 4.6 Hz, 4H) 2.71 (s, 3H), 1.86 (t, J = 4.8 Hz, 4H). 13C NMR (DMSO-d6) δ: 166.09, 165.96, 158.72, 157.27, 156.60, 152.97, 142.58, 139.46, 133.08, 132.19, 129.53, 128.69, 127.98, 127.20, 127.16, 107.52, 56.46, 30.92, 18.59. MS (m/z) 440. HRMS (EI) m/z 440.1790 M+, calcd for C25H24N6S 440.1783. Anal. Calcd for C25H24N6S: C, 68.16; H, 5.49; N, 19.08%. Found: C, 68.17; H, 4.51; N, 19.09%. N-(4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl)piperidine-1-carboximidamide (34). Yellowish brown solid (0.07 g, 70%), mp = 228 °C. 1H NMR (DMSO-d6) δ: 8.48 (d, J = 5.6 Hz, 1H), 8.27 (brs, 1H), 8.11 (brs, 1H), 8.06 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.2 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 5.2 Hz, 1H), 3.57 (t, J = 5.2 Hz, 4H), 2.70 (s, 3H), 1.60 (p, J = 4.8 Hz, 4H), 1.60 (p, J = 4.8 Hz, 2H). 13C NMR (DMSO-d6) δ: 166.11, 166.06, 158.67, 157.37, 157.35, 153.07, 142.60, 139.45, 132.97, 132.17, 129.53, 128.56, 127.99, 127.92, 127.16, 107.77, 45.13, 25.92, 24.61, 18.59. MS (m/z) 454. HRMS (EI) m/z 454.1943 M+, calcd for C26H26N6S 454.1940. Anal. Calcd for C26H26N6S (MW = 454): C, 68.70; H, 5.77; N, 18.49%. Found: C, 68.72; H, 5.78; N, 18.50%. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}morpholine-4-carboximidamide (35). Yellow solid (0.1 g, 85%), mp = 250 °C. 1H NMR (DMSO-d6) δ: 8.91 (brs, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.46 (brs, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.6 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 5.2 Hz, 1H), 3.62 (m, 4H), 3.57 (m, 4H), 2.72 (s, 3H). 13C NMR (DMSO-d6) δ: 166.16, 166.03, 158.71, 157.75, 157.51, 153.23, 142.63, 139.45, 132.90, 132.16, 129.53, 128.56, 127.92, 127.19, 127.16, 108.28, 66.38, 44.69, 18.62. MS (m/z) 456. HRMS (EI) m/z 456.1718 M+, calcd for C25H24N6OS 456.1732. Anal. Calcd for C25H24N6OS (MW = 456): C, 65.77; H, 5.30; N, 18.41%. Found: C, 65.79; H, 5.31; N, 18.43%. N-(4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl)-4-methylpiperazine-1-carboximidamide (36). Yellow solid (0.07 g, 60%), mp = 215 °C. 1H NMR (DMSO-d6) δ: 8.91 (brs, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.31 (brs, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 5.2 Hz, 1H), 3.58 4082

DOI: 10.1021/acs.jmedchem.7b00392 J. Med. Chem. 2017, 60, 4074−4085

Journal of Medicinal Chemistry

Article

Antimicrobial Testing. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). S. aureus clinical isolates (NRS107, NRS119, NRS123, VRS10, VRS11a, and VRS12), vancomycin-resistant E. faecium ATCC 700221, and P. aeruginosa ATCC 15442 were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program, BEI Resources, and the American Type Culture Collection. In addition, the MRSA (2658 RCMB) strain was obtained from The Regional Center of Mycology & Biotechnology, Cairo, Egypt. The MICs of the newly synthesized compounds, tested against isolates of S. aureus, E. faecium, and P. aeruginosa, were determined using the broth microdilution assay in accordance with the Clinical and Laboratory Standards Institute guidelines.33 Bacteria were cultured in cation-adjusted Mueller−Hinton broth (for S. aureus), brain heart infusion broth (for E. faecium), or tryptic soy broth (for P. aeruginosa) in a 96-well plate. Compounds, using triplicate samples, were added to the plate and serially diluted. Plates were incubated at 37 °C for at least 20 h prior to determining the MIC. Plates were visually inspected, and the MIC was categorized as the concentration at which no visible growth of bacteria was observed. The MBC was determined by transferring a small aliquot (5 μL), from wells where no growth was observed (in the MIC plates), onto tryptic soy agar plates. Plates were incubated at 37 °C for at least 18 h prior to determining the MBC; the MBC was categorized as the lowest concentration where 99.9% of bacterial growth was inhibited. Time−Kill Assay. MRSA USA300 cells in logarithmic growth phase (OD600 > 0.800) were diluted to ∼106 CFU/mL and exposed to concentrations equivalent to 4 × MIC (in triplicate) of tested compounds and vancomycin in tryptic soy broth. Aliquots (100 μL) were collected from each treatment after 0, 2, 4, 6, 8, 10, 12, and 24 h of incubation at 37 °C and subsequently serially diluted in PBS. Bacteria were then transferred to tryptic soy agar plates and incubated at 37 °C for 18−20 h before viable CFU/mL was determined In Vitro Cytotoxicity Analysis of Active Compounds against HRT-18 Cells. Compounds exhibiting anti-MRSA activity were assayed (at concentrations of 16, 32, 64, and 128 μg/mL) against a human colorectal (HRT-18) cell line to determine the potential toxic effect to mammalian cells in vitro. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal horse serum at 37 °C with CO2 (5%). Control cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with compounds (in triplicate) in a 96-well tissue-culture treated plate at 37 °C with CO2 (5%) for 6 h. The assay reagent MTS 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plate was incubated for 4 h. Absorbance readings (at OD490) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data were analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Examining Phenylthiazole Compounds’ Ability To Resensitize Vancomycin-Resistant S. aureus (VRSA) to the Effect of Vancomycin. Tryptic soy broth was inoculated with VRS10 (5 × 105 CFU/mL), as described in a previous study.11 Aliquots (5 mL) of the bacterial suspension were divided into microcentrifuge tubes, and tested compounds (at 1/2 × MIC) were introduced into each tube. After sitting at room temperature for 30 min, samples (1 mL) from each tube were transferred to a new centrifuge tube prior to addition of a subinhibitory concentration of either vancomycin (at a concentration equivalent to 128 μg/mL). Using a 96-well microtiter plate, rows 2−8 were filled with the remaining 4 mL of bacterial suspension (containing the compound). 200 μL aliquots from tubes containing both the compound and vancomycin were transferred to row 1 of the 96-well plate. After aspirating contents in the first row 4− 6 times, 100 μL was transferred from wells in row 1 to row 2. This

(t, J = 4.8 Hz, 4H), 2.71 (s, 3H), 2.33 (t, J = 4.8 Hz, 4H), 2.19 (s, 3H). 13 C NMR (DMSO-d6) δ: 166.12, 166.02, 158.66, 157.59, 157.48, 153.17, 142.61, 139.45, 132.90, 132.17, 129.53, 128.56, 127.92, 127.19, 127.16, 108.08, 54.88, 46.14, 40.63, 18.62. MS (m/z) 469. HRMS (EI) m/z 469.2056 M+, calcd for C26H27N7S 469.2049. Anal. Calcd for C26H27N7S: C, 66.50; H, 5.80; N, 20.88%. Found: C, 66.52; H, 5.81; N, 20.90. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}nicotinimidamide (37). Orange solid (0.08 g, 69%), mp = 170 °C. 1H NMR (DMSO-d6) δ: 9.50 (brs, 1H), 9.20 (s, 1H), 8.90 (brs, 1H), 8.75 (d, J = 5.2 Hz, 1H), 8.71 (d, J = 5.2 Hz, 1H), 8.39 (d, J = 6.8 Hz, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 6.8 Hz, 1H), 7.50 (m, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 1H), 2.76 (s, 3H). 13 C NMR (DMSO-d6) δ: 166.98, 166.07, 159.32, 159.12, 158.28, 154.04, 152.08, 149.09, 142.73, 139.43, 135.66, 132.19, 132.06, 131.92, 129.53, 128.58, 127.92, 127.27, 127.16, 123.81, 111.38, 18.70. MS (m/ z) 448. MS (m/z) 448. HRMS (EI) m/z 448.1481 M+, calcd for C26H20N6S 448.1470. Anal. Calcd for C26H20N6S: C, 69.62; H, 4.49; N, 18.74%. Found: C, 69.63; H, 4.69; N, 18.75%. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}isonicotinimidamide (38). Orange solid (0.07 g, 60%), mp = 180 °C. 1H NMR (DMSO-d6) δ: 9.53 (brs, 1H), 8.93 (brs, 1H), 8.76 (d, J = 6.4 Hz, 2H), 8.51 (d, J = 5.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 5.2 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 5.2 Hz, 1H), 7.47 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 2.76 (s, 3H). 13C NMR (DMSO-d6) δ: 166.77, 166.64, 159.16, 158.99, 158.33, 154.11, 150.55, 143.57, 142.76, 139.43, 132.14, 132.06, 129.53, 128.59, 127.94, 127.28, 127.17, 121.98, 111.60, 18.71. MS (m/z) 448. MS (m/z) 448. HRMS (EI) m/z 448.1482 M+, calcd for C26H20N6S 448.1470. Anal. Calcd for C26H20N6S: C, 69.62; H, 4.49; N, 18.74%. Found: C, 69.62; H, 4.50; N, 18.76%. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}picolinimidamide (39). Orange solid (0.1 g, 92%), mp = 180 °C. 1H NMR (DMSO-d6) δ: 9.51 (brs, 1H), 8.78 (d, J = 5.2 Hz, 1H), 8.71 (brs, 1H) 8.70 (d, J = 5.2 Hz, 1H), 8.47 (d, J = 8 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 8.00 (t, J = 5.2 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.63 (d, J = 8 Hz, 1H), 7.60 (m, 1H), 7.51 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 2.77 (s, 3H). 13 C NMR (DMSO-d6) δ: 166.97, 166.75, 159.10, 158.38, 158.06, 154.05, 151.65, 148.99, 142.74, 139.44, 137.99, 132.21, 132.09, 129.53, 128.57, 127.92, 127.30, 127.17, 126.72, 122.66, 111.44, 18.69. MS (m/ z) 448. HRMS (EI) m/z 448.1460 M+, calcd for C26H20N6S 448.1470. Anal. Calcd for C26H20N6S: C, 69.62; H, 4.49; N, 18.74%. Found: C, 69.64; H, 4.51; N, 18.75%. N-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin-2-yl}-1H-pyrazole-1-carboximidamide (40). Reddish solid (0.06 g, 55%), mp = 211 °C. 1H NMR (DMSO-d6) δ: 8.84 (d, J = 5.2 Hz, 1H), 8.67 (d, J = 6.8 Hz, 1H), 8.09 (d, J = 6.8 Hz, 1H), 8.05 (t, J = 6.8 Hz, 1H), 7.84 (d, J = 8 Hz, 2H), 7.81 (d, J = 8 Hz, 2H), 7.75 (d, J = 6.4 Hz, 2H), 7.64 (d, J = 5.2 Hz, 1H), 7.51 (t, J = 6.4 Hz, 2H), 7.42 (t, J = 6.4 Hz, 1H), 7.39 (brs, 1H), 6.59 (brs, 1H), 2.77 (s, 3H). 13 C NMR (DMSO-d6) δ: 167.73, 166.53, 161.12, 159.98, 159.04, 155.92, 155.74, 150.24, 142.74, 139.37, 132.08, 131.77, 129.55, 128.65, 127.92, 127.32, 127.17, 126.51, 113.98, 18.84. MS (m/z) 437. HRMS (EI) m/z 437.1427 M+, calcd for C24H19N7S 437.1423. Anal. Calcd for C24H19N7S: C, 65.89; H, 4.38; N, 22.41%. Found: C, 65.90; H, 4.39; N, 22.42%. 2-{4-(2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl)pyrimidin2-yl}-1,1,3,3-tetramethylguanidine (41). Yellowish brown solid (0.09 g, 85%), mp = 275 °C. 1H NMR (DMSO-d6) δ: 8.48 (d, J = 5.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 5.6 Hz, 1H), 2.83 (s, 12H), 2.70 (s, 3H). 13C NMR (DMSOd6) δ: 167.01, 166.07, 163.74, 162.04, 159.57, 158.07, 142.13, 139.22, 133.16, 132.51, 129.53, 128.61, 127.89, 127.18, 127.14, 107.80, 26.43, 18.65. MS (m/z) 442. Anal. Calcd for C25H26N6S (MW = 442): C, 67.85; H, 5.92; N, 18.99%. Found: C, 67.85; H, 5.93; N, 18.99%. 4083

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process was repeated to dilute the remaining wells containing no antibiotic. Untreated bacteria served as a control. The plate was incubated at 37 °C for 20−22 h before the MIC was recorded. The MIC was categorized as the concentration at which no visible growth of bacteria was observed in a particular well. A fold reduction was calculated by comparing the MIC of vancomycin alone compared to the MIC of the antibiotic given in combination with each compound. Checkerboard Assay. The relationship of the new analogues and vancomycin against VRS10 was examined using a standard checkerboard assay.28 VRS10, equivalent to a McFarland standard of 0.5, was prepared in PBS. The bacteria were then diluted in TSB to achieve a starting cell density of 1 × 105 CFU/mL. TSB was transferred to all wells of a 96-well microtiter plate. The phenylthiazole compounds and vancomycin were diluted in TSB to achieve a starting concentration equivalent to 2 × MIC or 4 × MIC. Vancomycin was serially diluted along the horizontal axis of the microtiter plate, while the compounds were serially diluted along the vertical axis. Plates were incubated for 20 h at 37 °C. The MIC of the test compound in combination with vancomycin was classified as the lowest concentration of each compound/antibiotic where no visible growth of bacteria was observed. The fractional inhibitory concentration index (FICI) was calculated for each combination, as described previously.11 A synergistic relationship was classified as an FICI less than or equal to 0.50. FIC values above 0.50 but less than 2.00 were characterized as additive; values between 2.00 and 4.00 were characterized as indifference, while FIC values above 4.00 were classified as antagonistic. In Vivo Examination of 1b, 30, 36, and Vancomycin To Kill MRSA USA300 in a Caenorhabditis elegans Animal Model. To examine the efficacy of the thiazole compounds to treat a MRSA infection in vivo, the whole animal model Caenorhabditis elegans (C. elegans) was utilized. The temperature-sensitive sterile mutant strain C. elegans AU37 [sek-1(km4); glp-4(bn2) I] was used, as this strain is sterile at room temperature and capable of laying eggs only at 15 °C. Additionally, this strain is more susceptible to infection due to a mutation in the sek-1 gene of the p38 mitogen-activated protein kinase pathway. Briefly, worms were grown for 5 days at 15 °C (permitting worms to lay eggs) on nematode growth medium (NGM) agar plates seeded with a lawn of Escherichia coli (E. coli) OP50. The eggs were harvested by bleaching and maintained for 24 h at room temperature with gentle agitation for hatching. Hatched larvae were transferred to a new NGM plate seeded with E. coli OP50 and were kept at room temperature for 4−5 days until worms reached the adult stage of growth (L4). Adult worms were collected and washed three times with PBS in a 1:10 ratio to remove E. coli. To test the antibacterial activity of the phenylthiazole compounds against MRSA in vivo, adult worms were transferred to TSA agar plates seeded with a lawn of MRSA USA300 (highly pathogenic to C. elegans) for infection. After 6 h of infection, worms were collected and washed with M9 buffer three times before transferring 25−30 worms to wells in a 96-well microtiter plate. Worms were incubated with 20 μg/mL of tested compounds, vancomycin (positive control), or PBS (negative control) (in triplicate). After treatment for 18 h, worms were washed three times with M9 buffer and then examined microscopically for morphological changes and viability. They were subsequently lysed in microcentrifuge tubes containing 200 mg of 1.0 mm silicon carbide particles (Biospec Products, Bartlesville, OK) that were vortexed for 1 min. Samples were serially diluted and plated onto TSA plates containing 5 μg/mL nalidixic acid to select for MRSA growth. Plates were incubated at 37 °C for 17 h before viable CFU was determined. MRSA USA300 CFU was divided by the number of worms receiving each treatment. The percent reduction in MRSA USA300 growth, relative to PBS-treated worms, was subsequently calculated.





Experimental details of PK analysis and spectral data of all new compounds (PDF) Molecular formula strings and some data (CSV)

AUTHOR INFORMATION

Corresponding Authors

*M.N.S.: phone, 765-494-0763; e-mail, [email protected]. *A.S.M.: phone, 0020-100-771-5002; e-mail, amayhoub@azhar. edu.eg. ORCID

Haroon Mohammad: 0000-0002-8843-8933 Abdelrahman S. Mayhoub: 0000-0002-3987-3680 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by Science & Technology Development Funds (STDF), Grant 5334. ABBREVIATIONS USED Caco-2, heterogeneous human epithelial colorectal adenocarcinoma cells; CFU, colony forming unit; HRT-18, human colorectal cells; Papp, apparent permeability



REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00392. 4084

DOI: 10.1021/acs.jmedchem.7b00392 J. Med. Chem. 2017, 60, 4074−4085

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