Design, Synthesis and Evaluation of Novel 2,5,6-Trisubstituted Benzimidazoles Targeting FtsZ as Antitubercular Agents

Abstract

Filamenting temperature-sensitive protein Z (FtsZ), an essential cell division protein, is a promising target for the drug discovery of new-generation antibacterial agents against various bacterial pathogens. As a part of SAR studies on benzimidazoles, we have synthesized a library of 376 novel 2,5,6-trisubstituted benzimidazoles, bearing ether or thioether linkage at the 6-position. In a preliminary HTP screening against Mtb H37Rv, 108 compounds were identified as hits at a cut off concentration of 5 μg/mL. Among those hits, 10 compounds exhibited MIC values in the range of 0.63–12.5 μg/mL. Light scattering assay and TEM analysis with the most potent compound 5a clearly indicate that its molecular target is Mtb-FtsZ. Also, the Kd of 5a with Mtb-FtsZ was determined to be 1.32 μM.

1. Introduction

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a leading single infectious disease killer. In 2012, the World Health Organization (WHO) estimated that there were 8.6 million new cases of TB globally (13% co-infected with HIV) resulting in 1.3 million deaths.¹ In addition, multidrug-resistant (MDR-TB) and extensively drug resistant TB (XDR-TB) are a significant public health threat for TB control efforts.^2,3 Emergence of drug resistant strains of Mtb makes many of the currently available anti-TB drugs much less effective.³ Despite efforts in last 50 years, development of new TB treatments have been limited to drug targets like cell wall biosynthesis, ATP synthesis, RNA synthesis etc, leading to resistance in these areas.^{4, 5} Hence, we need to discover novel drugs that target other bacterial processes in order to counter the developed bacterial resistance.

Filamenting temperature-sensitive protein Z (FtsZ), a tubulin homologue⁶, is an essential and the most abundant bacterial cell division protein. FtsZ polymerizes in the presence of GTP to form a highly dynamic structure, the Z-ring at the division site.^{7, 8} With the recruitment of several other cell division proteins, Z-ring constriction proceeds resulting in septum formation and subsequent cell division.⁹ Due to the crucial role of FtsZ in bacterial cytokinesis, inactivation of FtsZ is an attractive target for novel drug discovery.^{10, 11}

Since FtsZ is a homologue of tubulin with less than 10% sequence identity¹², known tubulin inhibitors could be a good starting point for developing FtsZ specific inhibitor. Previously, various groups have explored known tubulin inhibitors based on the importance of FtsZ assembly in cell division to identify their ability to inhibit FtsZ polymerization or depolymerization.^{10, 13, 14}

Following on this principle, albendazole and thiabendazole, known tubulin inhibitors, were tested for their anti-TB activities.¹⁵ Slayden et al. found that these two compounds inhibit FtsZ polymerization that led to the absence of septum formation based on gene expression profile. As both of these compounds share a common benzimidazole moiety, we chose benzimidazole as the scaffold for development of novel anti-TB agents. In our previous work,^{16, 17} based on rational drug design, libraries of 2,5,6- and 2,5,7-trisubstituted benzimidazoles were synthesized and evaluated for anti-TB activities. A large number of compounds were identified with MICs in the range of 0.38–6.2 strains μg/mL against drug sensitive as well as drug resistant Mtb (Fig. 1). In the light scattering experiment, some of these novel lead compounds exhibited inhibition of FtsZ assembly in a dose dependent manner while enhancing the GTPase activity¹⁸ of Mtb-FtsZ. These results confirmed the hypothesis that the lead benzimidazoles target FtsZ.

Figure 1.

Previously reported anti-TB 2,5,6-trisubstituted benzimidazoles

The preliminary SAR studies of lead compounds indicate that cyclohexyl group at the 2-position and diethyl amino/dimethyl amino group at the 6-position play important role for antibacterial activity.^{17, 18} Building upon three representative compounds bearing alkyl carbamate or benzamide at the 5-position, we planned to expand our novel trisubstituted benzimidazole libraries with a substitution pattern different from the previous series for high throughput (HTP) screening.¹⁹ [Note: In the 6-amino series, we have very recently found that the 6-dimethylamino series exhibit excellent activities up to the MIC value of 0.06 μg/mL.¹⁷]

In order to investigate the effect of substituents other than amines at the 6-postion on antibacterial activity, a new series of 2,5,6-trisubstituted benzimidazole library was designed and synthesized with ether/thioether groups at the 6-position (Fig. 2). Based on previous SAR studies, the cyclohexyl group at the 2-positon was fixed and various substituents at the 5-position were examined.

Figure 2.

Novel 2,5,6-trisubstituted benzimidazoles bearing an ether or thioether substituent at the 6 position.

2. Chemical Synthesis

General procedure for the synthesis of 2,5,6-trisubstituted benzimidazoles bearing an ether/thioether moiety at the 6-position is illustrated in Scheme 1.²⁰ The first step was the nucleophilic aromatic substitution of commercially available 2,4-dinitro-5-fluoroaniline (1) with various alkyl or aryl alcohol/thiols. Compounds 2a, 2b and 2e were treated with 1 M KOH while 2c, 2d and 2f-2h were reacted with 1 M K₂CO₃ to afford compounds 2a-2h in 91–100 % yields. The acylation of compounds 2a-h with the cyclohexanecarbonyl chloride gave 3a-h in 82–89 % yields. Compounds 3a-3d were treated with tin(II) chloride dehydrate while 3e-3h were reacted with tin(II) chloride dihydrate and 4 M HCl to afford benzimidazoles 4a-h in 56–69 % yields. 5-Aminobenzimidazoles 4a-h (0.01 mM) were dissolved in dichloromethane and transferred into 96 well plates. Then, 47 different acyl chlorides, hydroxysuccinimide esters of chloroformates, isocyanates, isothiocyanates and sulfonyl chlorides (1.1 eq.) in dichloromethane were added to the individual wells. These 47 different reagents are shown in the Supporting Information. The plates were gently shaken for a day. Then, aminomethylated polystyrene resin EHL/2% DVB (200–400 mesh) (10 eq.) was added to scavenge excess or unreacted acyl chloride, isocyanates, isothiocyanate and sulfonyl chlorides. After reacting for 24 h, the resin was filtered to afford a library of 376 novel 2,5,6-tribsustituted benzimidazoles 5.

Scheme 1.

Library synthesis of 2,5,6-trisubstituted benzimidazoles. Reagents and conditions: a) R¹OH/R¹SH, 1 M KOH, THF, or K₂CO₃, Acetone, room temperature (RT), 1 h; b) cyclohexanecarbonyl chloride, pyridine, reflux, overnight; c) (i) SnCl₂·2H₂O, EtOH, reflux, 1–6 h or (ii) SnCl₂·2H₂O, 4 M HCl, EtOH, reflux, 1–6 h; d) (i) RC(O)Cl, RC(O)OSu, R₂NC(O)Cl, R₂NC(S)Cl, RSO₂Cl, RN=C=O or RN=C=S (1.0 eq.), CH₂Cl₂, overnight, RT

3. Results and Discussion

3.1. In vitro preliminary screening of the library of 2,5,6-trisubstituted benzimidazoles 5 against Mtb-H37Rv

The library of 2,5,6-trisubstituted benzimidazoles 5 (376 compounds) was screened against drug sensitive Mtb H37Rv strain using the “Microplate Alamar Blue Assay (MABA)”¹⁵ and then, the growth inhibition was measured in percentage. Among these compounds, 108 compounds were identified to inhibit the growth of Mtb H37Rv by 22–79 % at 5 μg/mL concentration and 22 compounds (see Table 1) exhibited 28–65 % growth inhibition at 1.0 μg/mL concentration. From the preliminary screening, the butylthio group, followed by the benzylthio group at the 6 position appeared to be rather preferred, but 4-fluorophenoxy, 4-fluorophenylthio, and phenylthio groups did not seem to be much different. However, no compounds with a phenoxy group at the 6 position were included in the hit list. Also, no benzimidazoles bearing sulfoxide, urea or thiourea groups at the 5 position were found in the hit list. Thus, only amide or carbamate groups appear to be preferred at this position.

Table 1.

Hit compounds 5 from the preliminary screening against Mtb H37Rv strain at 1.0 μg/mL concentration.

Compound	R1X	R2	% Growth inhibition
1	EtO	4-MeC6H4CO	65
2	BuO	CH2=CH(CH2)2CO	28
3	4-FC6H4O	2,4-F2C6H3CO	44
4	4-FC6H4O	4-MeC6H4CO	36
5	4-FC6H4O	CH2=CH(CH2)2CO	54
6	BuS	2,4-F2C6H3CO	51
7	BuS	CH3(CH2)2OCO	38
8	BuS	PhSO2	42
9	BuS	4-t-BuC6H4CO	45
10	BuS	4-MeC6H4CO	53
11	BuS	CH2=CH(CH2)2CO	52
12	BuS	Ph(CH2)2CO	33
13	PhS	4-MeBuC6H4CO	31
14	PhS	4-t-BuC6H4CO	42
15	PhS	CH2=CH(CH2)2CO	56
16	4-FC6H4S	CH3(CH2)2OCO	46
17	4-FC6H4S	PhSO2	30
18	4-FC6H4S	CH2=CH(CH2)2CO	41
19	PhCH2S	CH3(CH2)2OCO	28
20	PhCH2S	PhSO2	30
21	PhCH2S	4-t-BuC6H4CO	52
22	PhCH2S	CH2=CH(CH2)2CO	64

These hit compounds were resynthesized in analytically pure form and examined for their accurate MIC values. Then, it turned out that the MIC values did not necessarily correlate the percent inhibition at the fixed concentration of the test compounds, as anticipated, e,g,, inaccuracy in the actual weight and purity of a test compound in a 96-well, as well as false positives in the HTP screening. As Table 2 shows, some of the hit compounds with a 4-fluorophenoxy or buthylthio group exhibit promising activities, but those with a 6-phenylthio or 6-benzylthio group appear to be less potent among the compounds examined so far.

Table 2.

MIC of selected hit compounds against Mtb H37Rv strain

compound	R1X	R2	MIC (μg/mL) Mtb H37Rv	cytotoxicity (μM) Vero Cells
5a			0.63	60
5b			3.13	40
5c			1.56	26
5d			12.5	> 200
5e			12.5	> 200
5f			6.25	> 200
5g			12.5	> 200
5h			1.25	> 200
5i			1.25	> 200
5j			1.25	75

Although 5a, bearing a n-butoxycarbonylamino group at the 5 position, was not among the 22 hit compounds, we added this compound for the MIC determination, since this carbamate group gave the best potency in the 6-dialkyamino series of 2,5,6-trisubstituted benzimidazoles in our another study,^{17, 18} Indeed, 5a exhibited the best potency (MIC 0.63 μg.mL) against Mtb H37Rv in this series (Table 2).

The cytotoxicity of 5a-5j was evaluated in vitro against Vero cells using the MTT assay.²¹ Compounds 5a, 5b, 5c and 5j showed cytotoxicity with IC₅₀ values in the range of 26–75 μM. However, most of the analytically pure compounds did not show appreciable cytotoxicity against Vero cells.

3.2. FtsZ polymerization assay

Two benzimidazoles 5a and 5d were evaluated for their ability to inhibit the Mtb-FtsZ polymerization.²² A light scattering assay was carried out to examine the effect of these compounds on inhibition of the FtsZ polymerization.^{17, 18} The amount of the FtsZ polymer formed after addition of GTP was monitored by the intensity of light scattered by the sample. As Figure 3 shows, 5a and 5d inhibited FtsZ polymerization in a dose-dependent manner.

Figure 3.

Inhibition of FtsZ polymerization by (A) 5a, (B) 5d

3.3. Transmission Electron Microscopy (TEM) imaging of FtsZ with compound 5a

Transmission electron microscopy (TEM) imaging of Mtb-FtsZ treated with 5a exhibited the ability of the compound to inhibit FtsZ polymerization and aggregation.^{17, 18} Mtb-FtsZ (5 μM) incubated with 5a at 40 μM and 80 μM concentrations in the presence of GTP (25 μM) showed shorter and thinner FtsZ polymers as compared to Mtb-FtsZ in the absence of compound 5a. In the absence of inhibitor, Mtb-FtsZ formed a dense network of long polymers which tend to aggregate (Fig. 4A,B) while in the presence of 5a (40 μM), the length, density and aggregation was visibly reduced (Fig. 4C,D). The effect was more apparent at 80 μM treatment where very short and dispersed FtsZ polymers were observed (Fig. 4E,F). Together with the light scattering assay, TEM images confirm the target of 5a as Mtb-FtsZ and gives insight into the mode of action of the new series of trisubstituted benzimidazoles bearing an ether or thioether linkage at 6-postion.

Figure 4.

Transmission Electron Microscopy (TEM) Images of FtsZ.

FtsZ (5 μM) was polymerized by GTP (25 μM) in the absence (A, B) and presence of 5a at 40 μM (C, D) and 80 μM (E, F). Images (A, C, E) are at 23,000x magnification (scale bar 500 nm) and (B, D, F) are at 49,000x magnification (scale bar 500 nm).

3.4. Dissociation constant of compound 5a with FtsZ

The fluorescence anisotropy of 5a, the most active compound, was used to determine its dissociation constant (Kd) with Mtb-FtsZ.²³ Compound 5a has an emission maximum at 427.9 nm with the excitation maximum at 316 nm. The fluorescence anisotropy of 5a was measured in the presence of increasing FtsZ concentrations to plot the resulting anisotropy profile (Fig. 5), which showed a concentration-dependent change. The change in fluorescence (ΔF) at 427.9 nm was applied to the standard equation ΔF = (ΔFmax × L)/(Kd +L) and the Kd of 5a was determined to be 0.72±0.2 μM.

Figure 5.

Determination of binding parameter of 5a with Mtb-FtsZ. (A) Fixed concentration of 5a (100 μM) was excited at 316 nm with varying concentration of Mtb-FtsZ (as shown in the graph) and fluorescence monitored at 427.9 nm and plotted against wavelength. Increase in fluorescence intensity observed on addition of increasing concentration of protein. (B) Fluorescence profiles of emission intensity at 427 nm for the titration of Mtb-FtsZ. The peak saturation was observed at 2.5 μM and then there is a progressive decrease in fluorescence emission. The Kd of 5a was calculated to be 1.32 ± 0.5 μM/L

3. Conclusion

New library of 2,5,6-trisubstituted benzimidazoles bearing sulfide and ether linkage at the 6 position have been synthesized. A number of hit compounds have been identified against Mtb H37Rv strain with good MIC values in the range of 0.63–12.5 μg/mL. Compound 5a and 5d were chosen for FtsZ polymerization assays and compound 5a was used for TEM images for target validation. These results showed that the two selected compounds inhibit FtsZ assembly in a dose dependent manner. The dissociation constant (Kd) of 5a was determined to be 1.32 μM based on its fluorescent anisotropy. This result provides direct evidence for the binding interaction between this benzimidazole and Mtb-FtsZ protein, which could be applicable to all potent benzimidazoles in this series. Further SAR study is necessary to obtain more detailed information for the substituent effects at 5 and 6 positions of the 2,5.6-trisubstituted benzimidazoles in this series. Nevertheless, 4-fluorophenoxy and butylthio groups were found to be preferred substituents at the 6 position. For the compounds, bearing a 4-fluorophenoxy group at the 6 position, a carbamate group at the 5 position gave the most potent compound (5a), but there is no difference between a carbamate group and benzamide groups for their potency (5j vs 5h and 5i) for the compounds, bearing a butylthio group at the 6 position. This is a unique feature in this series of beznimidazoles since a carbamate group at the 5 position provides, in general, more potent compounds than the corresponding 5-amidobenzimidazoles in the 5-dialkylamino-benzimidazole series.^{17, 18} Further optimization of the lead compounds for their anti-TB activities is actively underway in our laboratory. Also biological evaluations of the hit/lead compounds of this series against various other pathogens will be carried out to investigate their pathogen specific as well as broad spectrum antibacterial activities.

4. Experimental

The chemicals were purchased from Sigma Aldrich Co., Synquest Inc., Alfa Aesar and purified before use by standard methods. Tetrahydrofuran was freshly distilled from sodium metal and benzophenone. Dichloromethane was also distilled immediately prior to use under nitrogen from calcium hydride. ¹H and ¹³C NMR spectra were measured on a Bruker 400 or 500 MHz NMR spectrometer. Melting points were measured on a Thomas Hoover Capillary melting point apparatus and are uncorrected. TLC was performed on Sorbtech with UV254 and column chromatography was carried out on silica gel 60 (Merck; 230–400 mesh ASTM). High- resolution mass spectra were obtained on Agilent-TOF instrument. The optical density was determined from the resulting solutions using the Acsent Multiskan optical density reader. Purity of synthesized compounds was determined by HPLC analysis at 244 and 303 nm wavelengths with a Shimadzu LC-2010A HT series HPLC assembly or Agilent 1100 series HPLC assembly. For the HPLC analysis, an Adsorbosphere silica 5 μm, 250 mm × 4.6 mm column was used with isopropanol-hexanes (5–50% isopropanol in gradient) as eluent at the flow rate of 1 mL/min, t = 0–40 min. Light scattering assays were performed using a PTI-QM4 spectrofluorimeter. FEI Tecnai12 BioTwinG transmission electron microscope with an AMT XR-60 CCD digital camera system was used to acquire transmission electron microscopy images.

5.1. 2,4-Dinitro-5-ethoxyaniline (2a)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline (1) (4.0 g, 19.9 mmol) in 50 mL of THF and excess ethanol was added 1 M KOH aqueous solution dropwise until a yellow precipitate appeared. The reaction mixture was stirred for additional 1 h. The solution was extracted with ethyl acetate. The organic layer was collected, dried over anhydrous magnesium sulfate and concentrated in vacuo to give 2a as a yellow solid (4.8 g, 100 % yield): mp 162–165 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.53 (t, 3 H, J = 7.0 Hz), 4.18 (q, 2 H, J = 7.0 Hz), 6.23 (s, 1 H), 8.95 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 14.3, 66.1, 99.7, 127.2, 148.8, 158.1; HRMS (ESI) m/z calcd for C₈H₁₀N₃O₅⁺ 228.0615 Found: 228.0615 (Δ = 0.0 ppm)

In a similar manner, intermediate 2b, 2e were synthesized and characterized.

5.2. 5-Butoxy-2,4-dinitroaniline (2b)

Yellow solid; 76 % yield; mp 176–178 °C; ¹H NMR (500 MHz, CDCl₃) δ 0.99 (t, 3 H, J = 7.4 Hz), 1.21–1.33 (m, 3 H), 1.41–1.46 (m, 2 H), 1.53 (dd, 2 H, J = 15.1, 7.5 Hz), 1.63–1.66 (m, 2 H), 1.75–1.78 (m, 2 H), 1.86 (t, 2 H, J = 7.6 Hz), 1.92–1.95 (m, 2 H), 2.32–2.36 (m, 1 H), 4.10 (t, 2 H, J = 6.4 Hz), 6.24 (s, 1 H), 8.95 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 13.7, 19.0, 25.3, 25.7, 28.8, 30.6, 42.8, 70.0, 99.7, 124.5, 127.2, 130.2, 148.8, 158.2; HRMS (ESI) m/z calcd for C₁₀H₁₄N₃O₅⁺ 256.0928 Found: 256.0931 (Δ = 1.17 ppm).

5.3. 2,4-Dinitro-5-phenoxyaniline (2c)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline 1 (3.0 g, 14.9 mmol) in 45 mL of acetone were added phenol (1.68 g, 17.9 mmol) and anhydrous K₂CO₃ (4.12 g, 29.8 mmol). The reaction mixture was stirred mechanically at room temperature for at least 16 h until the total disappearance of 1 was confirmed by MS (FIA) analysis. The solution was extracted with ethyl acetate. The organic layer was collected, dried over anhydrous magnesium sulfate, and concentrated in vacuo to give 2c as a yellow solid (3.7 g, 91 % yield): mp 148–150 °C; ¹H NMR (500 MHz, CDCl₃) δ 5.97 (s, 1 H), 7.56 (m, 3 H), 7.60 (dd, 2 H, J = 7.57, 1.83 Hz), 9.05 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 114.6, 126.4, 128.4, 129.7, 130.5, 130.9, 134.7, 136.2, 146.3, 148.7; HRMS (ESI) m/z calcd for C₁₂H₁₀N₃O₅⁺ 276.0615 Found: 275.0358 (Δ = −29.0 ppm).

In a similar manner, intermediate 2d, 2f, 2g, and 2h were synthesized and characterized.

5.4. 2,4-Dinitro-5-(4-fluorophenoxy)aniline (2d)

Yellow solid; 93% yield; mp 164–165.5 °C; ¹H NMR (500 MHz, CDCl₃) δ 6.01 (s, 1 H), δ 7.11–7.19 (m, 4 H), δ 9.05 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 103.7, 117.2, 117.4, 122.5, 122.6, 127.6, 148.5, 149.2, 157.8, 159.5, 161.5; HRMS (ESI) m/z calcd for C₁₂H₉FN₃O₅⁺ 294.0521 Found: 294.0521 (Δ = 0.0 ppm)

5.5. 5-(Butylthio)-2,4-dinitroaniline (2e)

Yellow solid; 99% yield; mp 148–149 °C; ¹H NMR (400 MHz, CDCl₃) δ 0.99 (t, 3 H, J = 7.5 Hz), 1.53–1.57 (m, 2 H), 1.74–2.05 (m, 2 H), 2.01 (t, 2 H, J = 7.5 Hz), 6.55 (s, 1 H), 9.18 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 13.7, 22.3, 29.2, 32.4, 112.6, 126.6, 127.8, 135.4, 146.3, 147.8; HRMS (ESI) m/z calcd for C₁₀H₁₄N₃O₄S⁺ 272.0700 Found: 272.0701 (Δ = 0.35 ppm)

5.6. 2,4-Dinitro-5-(phenylthio)aniline (2f)

Yellow solid; 100% yield; mp 214–217 °C; ¹H NMR (400 MHz, CDCl₃) δ 5.97 (s, 1 H), 7.21–7.24 (m, 3 H), 7.28–7.32 (m, 2 H), 9.02 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 126.4, 127.2, 127.5, 129.1, 130.5, 130.8, 136.2, 137.0; HRMS (ESI) m/z calcd for C₁₂H₁₀N₃O₄S⁺ 292.0387 Found: 292.0387 (Δ = 0.0 ppm).

5.7. 2,4-Dinitro-5-(4-fluorophenylthio)aniline (2g)

Yellow solid; 100 % yield; m,p 217–219 °C; ¹H NMR (500 MHz, CDCl₃) δ 5.96 (s, 1 H), 7.04 (t, 1 H, J = 8.6 Hz), 7.27 (d, 1 H, J = 8.53), 7.49-7.46 (m, 1 H), 7.62 (dd, 1 H, J = 8.5, 5.3 Hz), 9.23 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 114.4, 116.2, 116.4, 117.8, 118.0, 126.5, 131.2, 131.3, 138.3, 138.4, 146.3; HRMS (ESI) m/z calcd for C₁₂H₉FN₃O₄S⁺ 310.0292 Found: 310.0292 (Δ = 0.0 ppm).

5.8. 5-(Benzylthio)-2,4-dinitroaniline (2h)

Yellow solid; 100 % yield; mp 188.5–190 °C; ¹H NMR (400 MHz, CDCl₃) δ 4.17 (s, 2 H), δ 6.62 (s, 1 H), δ 7.36-7.44 (m, 5 H), 9.19 (s, 1 H); ¹³C NMR (100 MHz, CDCl3) δ 37.8, 113.0, 126.5, 128.2, 129.0, 129.1, 133.7; HRMS (ESI) m/z calcd for C₁₂H₁₂N₃O₄S⁺ 306.0543 Found: 306.0543 (Δ = 0.0 ppm).

5.9. 1-(Cyclohexanecarboxamido)-5-ethoxy-2,4-dinitro-benzene (3a)

To a solution of 2a (0.71 g, 3.13 mmol) in 12 mL of pyridine was added cyclohexanecarbonyl chloride (0.54 mL, 1.3 eq.), and the mixture was magnetically stirred and refluxed overnight. After completion of the reaction by TLC analysis, the reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate, and then washed with CuSO₄ solution twice to get rid of the leftover pyridine. The reaction mixture was washed with brine, diluted with ethyl acetate, and washed with water three times. The organic layers were dried over sodium sulfate, filtered, and concentrated to afford 3a as a yellow solid (0.9 g, 89 % yield): mp 109–109.5 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.28–1.31 (m, 1 H), 1.38–1.42 (m, 2 H), 1.51 (t, 3 H, J = 7.41 Hz), 1.59–1.60 (m, 2 H), 1.76–1.79 (m, 1 H), 1.88–1.93 (m, 2 H), 2.05–2.09 (m, 2 H), 2.44 (tt, 1 H, J = 11.6, 3.5 Hz), 3.12 (q, 2 H, J = 7.44 Hz), 9.19 (s, 1 H), 9.24 (s, 1 H), 10.9 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 12.2, 25.4, 25.5, 27.1, 29.4, 47.4, 116.9, 124.9, 130.7, 138.1, 149.7, 176.0; HRMS (ESI) m/z calcd for C₁₅H₂₀N₃O₆⁺ 338.1347 Found: 338.1351 (Δ = 1.18 ppm).

In a similar manner, compounds 3b-3h were synthesized and characterized.

5.10. 1-(Cyclohexanecarboxamido)-5-butoxy-2,4-dinitro-benzene (3b)

Yellow solid; 100 % yield; mp 187.5–189°C; ¹H NMR (500 MHz, CDCl₃) δ 0.99 (t, 3 H, J = 7.4 Hz), 1.21–1.33 (m, 3 H), 1.45 (d, 2 H, J = 11.5 Hz), 1.50–1.56 (m, 2 H), 1.6401.66 (m, 1 H), 1.75–1.78 (m, 2 H), 1.86 (dd, 2 H, J = 8.4, 6.8 Hz), 1.93 (d, 2 H, J = 12.8 Hz), 2.32–2.36 (m, 1 H), 4.09 (t, 2 H, J = 6.4 Hz), 6.24 (s, 1 H), 8.95 (s, 1 H);¹³C NMR (125 MHz, CDCl₃) δ 13.7, 19.0, 25.3, 25.3, 28.8, 30.6, 42.8, 70.0, 99.7, 124.5, 127.2, 130.2, 148.8, 158.2; MS (ESI) m/z 366.1 (M+1).

5.11. 1-(Cyclohexanecarboxamido)-5-phenoxyphenyl-2,4-dinitro-benzene (3c)

Yellow solid; 97 % yield; mp 150.5–152 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.21 (tt, 1 H, J = 12.3, 3.2 Hz), 1.26–1.35 (m, 2 H), 1.43 (qd, 2 H, J = 12.3, 3.1), 1.68–1.72 (m, 1 H), 1.81 (dt, 2 H, J = 13.1, 3.3 Hz), 1.95 (dd, 2 H, J = 13.5, 1.9 Hz), 2.23–2.33 (m, 1 H), 7.15–7.17 (m, 2 H), 7.35 (t, 1 H, J = 7.5 Hz), 7.49–7.52 (m, 2 H), 8.53 (s, 1 H), 9.06 (s, 1 H), 10.8 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.5, 29.3, 47.3, 108.8, 120.6, 121.6, 125.6, 126.7, 128.8, 129.4, 130.6, 133.2, 140.4, 153.2, 157.7, 175.4; HRMS (ESI) m/z calcd for C₁₉H₂₀N₃O₆⁺ 386.1347 Found: 386.1345 (Δ = −0.52 ppm).

5.12. 1-(Cyclohexanecarboxamido)-5-(4-fluorophenoxy)-2,4-dinitrobenzene (3d)

Yellow solid; mp 147–149 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.18–1.20 (m, 1 H), 1.27–1.32 (m, 2 H), 1.40 (d, 2 H, J = 11.6 Hz), 1.71 (dd, 1 H, J = 1.69, 1.42 Hz), 1.78–1.83 (m, 2 H), 1.90–1.94 (m, 2 H), 2.22–2.30 (m, 1 H), 7.23–2.30 (m, 2 H), 7.58–7.62 (m, 2 H), 8.47 (s, 1 H), δ 9.21 (s, 1 H), δ 10.6 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.2, 25.4, 29.1, 29.3, 47.2, 117.9, 118.1, 119.0, 123.9, 124.1, 124.7, 138.1, 138.2,150.4, 165.8, 174.8; MS (ESI) m/z 404.0 (M+1)⁺.

5.13. 1-(Cyclohexanecarboxamido)-5-(butylthio)-2,4-dinitro-benzene (3e)

Yellow solid; 100 % yield; mp 118–119 °C; ¹H NMR (500 MHz, CDCl₃) δ 0.99 (t, 3 H, J = 7.3 Hz), 1.25–1.32 (m, 1 H), 1.33–1.42 (m, 2 H), 1.55–1.59 (m, 4 H), 1.74–1.78 (m, 1 H), 1.80–1.83 (m, 2 H), 1.88 (dt, 2 H, J = 13.3, 3.4 Hz), 2.05 (dd, 2 H, J = 13.4, 2.2 Hz), 2.42 (tt, 1 H, J = 11.7, 3.5 Hz), 3.07 (t, 2 H, J = 7.3 Hz), 9.16 (s, 1 H), 9.21 (s, 1 H), 10.9 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 13.7, 22.1, 25.5, 29.5, 32.7, 47.5, 117.0, 124.9, 138.1, 150.0, 175.9; MS (ESI) m/z 382.1 (M+1)⁺.

5.14. 1-(Cyclohexanecarboxamido)-5-(phenylthio)-2,4-dinitrobenzene (3f)

Yellow solid; 100 % yield; mp 217–218 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.18–1.22 (m, 2 H), 1.24–1.32 (m, 2 H), 1.37(qd, 2 H, J = 12.2, 2.8 Hz), 1.67 (dd, 1 H, J = 12.8, 0.8 Hz), 1.78–1.82 (m, 2 H), 1.89–1.92 (m, 2 H), 2.24 (tt, 1 H, J = 11.7, 3.5 Hz), 7.60 (q, 5 H, J = 6.1 Hz), 8.46 (s, 1 H), 9.22 (s, 1 H), 10.6 (s, 1 H); ¹³C NMR (100 MHz, CDCl3) δ 25.4, 29.3, 30.9, 47.3, 119.1, 124.7, 128.6, 130.6, 131.1, 135.9, 137.5, 138.0, 150.7, 174.8; MS (ESI) m/z 402.1 (M+1)⁺.

5.15. 1-(Cyclohexanecarboxamido)-5-(4-fluorophenylthio)-2,4-dinitrobenzene (3g)

Yellow solid; 96 % yield; mp 186–189 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.16–1.23 (m, 1 H), 1.25–1.31 (m, 2 H), 1.34–1.42 (m, 2 H), 1.68–1.74 (m, 1 H), 1.79–1.83 (m, 2 H), 1.94 (dd, 2 H, J = 12.9, 2.1 Hz), 2.25–2.31 (m, 1 H), 7.25–7.29 (m, 2 H), 7.62 (dd, 2 H, J = 8.8, 5.2 Hz), 8.49 (s, 1 H), 9.24 (s, 1 H), 10.6 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 25.5, 29.3, 31.0, 47.3, 119.1, 124.7, 128.7, 130.6, 131.1, 131.5, 135.9, 137.6, 138.1, 150.8, 174.8; MS (ESI) m/z 420.1 (M+1)⁺.

5.16. 1-(Cyclohexanecarboxamido)-5-(benzylthio)-2,4-dinitrobenzene (3h)

Yellow solid; 96 % yield: mp 150–153 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.27–1.30 (m, 1 H), 1.37–1.44 (m, 2 H), 1.52–1.62 (m, 2 H), 1.74–1.77 (m, 1 H), 1.87–1.90 (m, 2 H), 2.04–2.07 (m, 2 H), 2.39–2.47 (m, 1 H), 4.30 (s, 2 H), 7.26–7.37 (m, 3 H), 7.45 (d, 2 H, J = 6.8 Hz), 9.21 (s, 1 H), 9.29 (s, 1 H), 10.9 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.4, 25.5, 29.4, 38.1, 43.3, 47.4, 117.1, 124.8, 127.4, 128.2, 128.5, 128.8, 129.4, 129.6, 130.9, 133.5, 137.4, 138.1, 138.2, 149.1, 175.9; MS (ESI) m/z 416.0 (M+1)⁺.

5.17. 5-Amino-6-ethoxy-2-cyclohexyl-1H-benzo[d]imidazole (4a)

A solution of 3a (100 mg, 0.30 mmol), tin(II) chloride dihydrate (0.47 g, 2.1 mmol) in 10 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen for 1 h. The reaction mixture was cooled, quenched with 30 % KOH, and pH adjusted to ~13. The solution was diluted with dichloromethane and washed with water three times. The organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography on silica gel (gradient 20–40 % ethyl acetate/hexanes) to afford compound 4a as a pale red color solid (69 g, 89 % yield): mp 89–90 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.23 (ddd, 1 H, J = 14.2, 10.8, 3.3 Hz), 1.32 (ddd, 2 H, J = 14.2, 11.1, 3.1 Hz), 1.37–1.42 (m, 3 H), 1.60 (qd, 2 H, J = 12.4, 3.1 Hz), 1.69–1.71 (m, 1 H), 1.79–1.81 (m, 2 H), 2.08 (d, 2 H, J = 12.4 Hz), 2.80 (tt, 1 H, J = 11.8, 3.51 Hz), 3.79 (br, 2 H), 3.99 (dd, 2 H, J = 8.3, 3.3 Hz), 6.78 (s, 1 H), 6.96 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 15.0, 25.9, 26.1, 32.0, 38.5, 64.4, 132.9, 133.0, 144.5, 157.0; HRMS (ESI) m/z calcd for C₁₅H₂₂N₃O⁺ 260.1757 Found: 260.1758 (Δ = 0.38 ppm).

In a similar manner, compounds 4b-4d were synthesized and characterized.

5.18. 5-Amino-6-butoxy-2-cyclohexyl-1H-benzo[d]imidazole (4b)

Pale red solid; 69 % yield; mp 113–115 °C; ¹H NMR (400 MHz, CDCl₃) δ 0.97 (t, 3 H, J = 7.4 Hz), 1.23–1.28 (m, 1 H), 1.35–1.40 (m, 2 H), 1.50 (dd, 2 H, J = 15.0, 7.5 Hz), 1.60 (qd, 2 H, J = 12.4, 3.2 Hz), 1.70–1.75 (m, 1 H), 1.76–1.86 (m, 4 H), 2.08–2.12 (m, 2 H), 2.78–2.84 (m, 1 H), 3.76 (br, 2 H), 3.98 (t, 2 H, J = 6.5 Hz), 6.80 (s, 1 H), 7.00 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 13.9, 19.4, 25.9, 26.1, 31.4, 32.0, 38.4, 68.6, 133.1, 144.6, 156.7; HRMS (ESI) m/z calcd for C₁₇H₂₆N₃O⁺ 288.2070 Found: 288.2071 (Δ = 0.35 ppm).

5.19. 5-Amino-6-phenoxy-2-cyclohexyl-1H-benzo[d]imidazole (4c)

Pale yellow solid; 46 % yield; mp 136–138 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.25–1.32 (m, 1 H), 1.35–1.44 (m, 2 H), 1.56–1.66 (m, 2 H), 1.72–1.77 (m, 1 H), 1.83–1.88 (m, 2 H), 2.09–2.14 (m, 2 H), 2.80–2.86 (m, 1 H), 3.73 (br, 2 H), 6.93–6.97 (m, 2 H), 7.01–7.05 (m, 1 H), 7.12 (s, 1 H), 7.26–7.29 (m, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 25.9, 26.0, 31.9, 38.5, 116.7, 122.4, 129.7, 135.3, 140.3, 158.1; HRMS (ESI) m/z calcd for C₁₉H₂₂N₃O⁺ 308.1757 Found: 308.1758 (Δ = 0.32 ppm).

5.20. 5-Amino-6-(4-fluorophenoxy)-2-cyclohexyl-1H-benzo[d]imidazole (4d)

Beige solid; 70 % yield; mp 195–196 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.26–2.30 (m, 1 H), 1.38–1.43 (m, 2 H), 1.61 (dd, 2 H, J = 12.4, 3.1 Hz), 1.72–1.77( m, 1 H), 1.86 (dt, 2 H, J = 13.1, 3.3 Hz), 2.12 (dd, 2 H, J = 13.6, 2.0 Hz), 2.83 (tt, 1 H, J = 11.8, 3.6 Hz), 3.74 (br, 2 H), 6.95 (m, 5 H), 8.89 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 25.8, 26.0, 31.8, 38.5, 116.0, 116.2, 118.1, 118.2, 154.0, 157.1, 159.5; HRMS (ESI) m/z calcd for C₁₉H₂₃FN₃O⁺ 326.1663 Found: 326.1667 (Δ = 1.2 ppm).

5.21. 5-Amino-6-(butylthio)-2-cyclohexyl-1H-benzo[d]imidazole (4e)

A solution of 3e (1.97 g, 5.16 mmol), tin(II) chloride dihydrate (15.5 g, 36.1 mmol), and 4 M HCl (80 mL) in 200 mL of ethanol was magnetically stirred and refluxed for 4 h. The reaction mixture was cooled, quenched with 1M NaOH, and pH was adjusted to ~10. Tin salts precipitated in solution upon addition of 1 M NaOH. The reaction mixture was filtered to remove the tin salts. The solution was diluted with ethyl acetate and washed with water three times. The organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (gradient 20–40 % ethyl acetate/hexanes) to afford compound 4e as a pale greenish gray solid (0.413g): 71 % yield; mp 110–112 °C; ¹H NMR 500 MHz, CDCl₃) δ 0.88 (t, 3 H, J = 7.3 Hz), 1.25–1.30 (m, 1 H), 1.36–1.42 (m, 4 H), 1.55 (dt, 2 H, J = 14.0, 7.4 Hz), 1.64 (qd, 2 H, J = 12.4, 3.2 Hz), 1.72–1.76 (m, 1 H), 1.85 (dt, 2 H, J = 13.2, 3.3 Hz), 2.13 (dd, 2 H, J = 13.7, 1.9 Hz), 2.72 (t, J = 7.4 Hz), 2.86 (tt, 1 H, J = 11.8, 3.5 Hz), 4.35 (br, 2 H), 6.83 (s, 1 H), 7.66 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 13.7, 21.9, 25.8, 26.0, 31.6, 31.9, 35.5, 38.5, 114.5, 144.0, 158.8; HRMS (ESI) m/z calcd for C₁₇H₂₆N₃S+ 304.1842 Found: 304.1842 (Δ = 0.0 ppm).

In a similar manner, compounds 4f-4h were synthesized and characterized.

5.22. 5-Amino-6-(phenylthio)-2-cyclohexyl-1H-benzo[d]imidazole (4f)

Pale brown solid; 57 % yield; mp 116–118 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.27–1.35 (m, 1 H), 1.42 (qt, 2 H, J = 12.79, 3.29 Hz), 1.66 (qd, 2 H, J = 12.4, 3.29 Hz), 1.76–1.80 (m, 1 H), 1.89 (dt, 2 H, J = 13.3, 3.38 Hz), 2.16 (dd, 2 H, J = 13.7, 2.01 Hz), 2.88 (tt, 1 H, J = 11.8, 3.55 Hz), 4.22 (br, 2 H), 6.92 (s, 1 H), δ 7.08–7.13 (m, 3 H), δ 7.20–7.23 (m, 2 H), δ 7.67 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.8, 26.0, 31.8, 38.5, 110.6, 125.3, 126.1, 128.9, 137.6, 144.6; HRMS (ESI) m/z calcd for C₁₉H₂₂N₃S⁺ 324.1529 Found: 324.1529 (Δ = 0.0 ppm).

5.23. 5-Amino-6-(4-fluorophenylthio)-2-cyclohexyl-1H-benzo[d]imidazole (4g)

Pale green solid; 61 % yield; mp 107–108 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.25 (ddd, 1 H, J = 13.8, 7.1, 3.6 Hz), 1.33–1.40 (m, 2 H), 1.62 (qd, 2 H, J = 12.4, 3.1 Hz), 1.71–1.74 (m, 1 H), 1.82–1.84 (m, 2 H), 2.85 (tt, 1 H, J = 11.8, 3.5 Hz), 6.86–6.89 (m, 3 H), 7.02–7.05 (m, 2 H), 7.70 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.8, 26.0, 31.8, 38.5, 98.8, 111.2, 115.9, 116.1, 124.2, 128.2, 132.4, 144.4, 159.3, 160.2, 162.1; HRMS (ESI) m/z calcd for C₁₉H₂₂N₃S⁺ 342.1435 Found: 342.1435 (Δ = 0.0 ppm).

5.24. 5-Amino-6-(benzylthio)-2-cyclohexyl-1H-benzo[d]imidazole (4h)

Pale beige solid; 56 % yield; mp 129.5–131 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.18–1.23 (m, 1 H), 1.27–1.35 (m, 2 H), 1.60 (qd, 2 H, J = 12.4, 3.2 Hz), 1.69 (d, 1 H, J = 12.7 Hz), 1.78 (dt, 2 H, J = 13.1, 3.0 Hz), 2.07 (dd, 2 H, J = 14.2, 2.5 Hz), 2.80-1.85 (m, 1 H), 3.86 (s, 2 H), 6.77 (s, 1 H), 7.10–7.12 (m, 2 H), 7.17 (td, 3 H, J = 6.5, 2.8 Hz), 7.49 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.7, 26.0, 31.7, 38.4, 40.7, 64.4, 98.4, 114.0, 123.1, 126.9, 128.3, 128.7, 132.8, 138.2, 139.1, 144.2, 159.1, 176.3; HRMS (ESI) m/z calcd for C₂₀H₂₄N₃S⁺ 338.1685 Found: 338.1686 (Δ = 0.3 ppm).

5.25. 5-Butoxycarbonylamino-2-cyclohexyl-6-(4-fluorophenoxy)-1H-benzo[d]imidazole (5a)

To a solution of 4a (100 mg, 0.31 mmol) in 6 mL of dichloromethane was added N-butoxycarbonyloxysuccinimide (68 mg, 0.31 mmol) in 6 mL of dichloromethane and the mixture was magnetically stirred under nitrogen atmosphere in an ice bath. The reaction mixture was slowly warmed up to room temperature and stirred for 16 h. The solution was diluted with dichloromethane and basified with NaHCO₃ and then washed with water three times. The organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography on silica gel (gradient 20–40 % ethyl acetate/hexanes) to afford compound 5a as an off-white solid (54 mg, 47 % yield): mp 91–92 °C; ¹H NMR (400 MHz, CDCl₃) 0.97 (t, 3 H, J = 7.4 Hz), 1.28–1.30 (m, 1 H), 1.39–1.45 (m, 4 H), 1.61–1.65 (m, 2 H), 1.67 (t, 2 H, J = 7.5 Hz), 1.76 (d, 1 H, J = 12.5 Hz), 1.85–1.88 (m, 2 H), 2.12 (d, 2 H, J = 12.5 Hz), 2.83–2.88 (m, 1 H), 4.19 (t, 2 H, J = 6.7 Hz), 6.96–7.05 (m, 4 H), 7.13 (s, 1 H), 8.23 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 13.8, 19.1, 25.8, 26.0, 31.0, 31.8, 38.5, 65.3, 116.5, 119.5, 125.6, 142.3, 153.1, 154.0, 157.8, 159.5, 159.6, 159.7; HRMS (ESI) m/z calcd for C₂₄H₂₉FN₃O₃⁺ 426.2187 Found: 426.2187 (Δ = 0.0 ppm). HPLC: t= 7.2 min, purity > 98%.

In a similar manner, compound 5j was synthesized and characterized.

5.26. 6-(Butylthio)-2-cyclohexyl-5-propoxycarbonylamino-1H-benzo[d]imidazole (5j)

Off-white solid; 62 % yield; mp 133–134.5 °C; ¹H NMR (500 MHz, CDCl₃) δ 0.87 (t, 3 H, J = 7.32 Hz), 1.00 (t, 3 H, J = 7.43 Hz), 1.30 (dt, 1 H, J = 3.51, 12.5 Hz), 1.38–1.43 (m, 3 H), 1.50–1.53 (m, 2 H), 1.59–1.67 (m, 4 H), 1.74 (q, 2 H, J = 7.11 Hz), 1.88 (dt, 2 H, J = 3.41, 13.3 Hz), 2.13 (dd, 2 H, J = 2.29, 13.4 Hz), 2.69 (t, 2 H, J = 7.35 Hz), 2.84–2.89 (m, 1 H), 4.26 (t, 2 H, J = 6.74 Hz), 7.88 (s, 1 H), 8.20 (s, 1 H), 8.27 (s, 1 H), 8.95 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 10.4, 13.6, 21.8, 22.3, 25.8, 26.0, 29.7, 31.4, 31.7, 36.9, 38.4, 66.8, 98.5, 100.2, 126.5, 126.6, 134.9, 153.9; HRMS (ESI) m/z calcd for C₂₁H₃₂N₃O₂S⁺ 390.2210 Found: 390.2214 (Δ = 1.0 ppm). HPLC: t= 11.3 min, purity> 94 %.

5.27. 2-Cyclohexyl-6-(4-fluorophenoxy)-5-(4-methoxy-benzamido)-1H-benzo[d]imidazole (5b)

To a solution of 4d (100 mg, 0.31 mmol) in 6 mL of dichloromethane was added 4-methoxybenzoyl chloride (42 μL, 0.31 mmol) in 6 mL of dichloromethane, and magnetically stirred in the ice bath. The reaction mixture was slowly warmed up to room temperature and stirred for 16 h. The solution was diluted with dichloromethane, basified with NaHCO₃ and then washed with water three times. The organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography on silica gel (gradient 20–40 % ethyl acetate/hexanes) to afford compound 5b as an off-white solid (152 mg, 92 % yield): mp > 230 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.22–1.33 (m, 3 H), 1.59 (dd, 2 H, J = 12.1, 2.9 Hz), 1.68–1.71 (m, 1 H), 1.78–1.81 (m, 2 H), 2.01–2.08 (m, 2 H), 2.76–2.82 (m, 1 H), 3.87 (s, 3 H), 6.96 (d, 2 H, J = 8.8 Hz), 7.03 (d, 3 H, J = 6.3 Hz), 7.78 (d, 2 H, J = 8.8 Hz), 8.55 (s, 1 H), 8.83 (s, 1 H), 9.81 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 13.8, 19.1, 25.8, 26.0, 31.0, 31.8, 38.5, 65.3, 116.5, 119.5, 125.6, 142.3, 153.1, 154.0, 157.8, 159.5, 159.6, 159.8; HRMS (ESI) m/z calcd for C₂₇H₂₇FN₃O₃⁺ 460.2031 Found: 460.2028 (Δ = −0.7 ppm). HPLC: t= 9.4 min, purity> 99 %.

In a similar manner, compounds 5c-5g were synthesized and characterized.

5.28. 2-Cyclohexyl-6-(4-fluorophenoxy)-5-(4-methxyl-benzamido)-1H-benzo[d]imidazole (5c)

Off-white solid; 47 % yield; mp 166–168 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.81-1.23 (m, 1 H), 1.26–1.34 (m, 2 H), 1.56–1.64 (qd, 2 H, J = 12.4, 2.6 Hz), 1.71 (m, 1 H), 1.78–1.81 (d, 2 H, J = 12.8 Hz), 2.01 (dd, 2 H, J = 12.5, 0.6 Hz), 2.45 (t, 3 H), 2.79 (t, 1 H, J = 11.5 Hz), 7.05 (d, 3 H, J = 6.4 Hz), 7.24 (s, 1 H), 7.31 (d, 2 H, J = 7.8 Hz), 7.74 (d, 2 H, J = 8.1 Hz), 8.63 (s, 1 H), 8.90 (s, 1H), 10.2 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 21.5, 25.7, 25.9, 29.7, 31.7, 38.5, 116.4, 116.6, 119.4, 119.5, 125.3, 126.9, 132.2, 142.6, 153.0, 153.1, 137.9, 159.9, 160.2, 165.8; HRMS (ESI) m/z calcd for C₂₇H₂₇FN₃O₂⁺ 442.2082 Found: 442.2082 (Δ = 0.0 ppm). HPLC: t= 7.7 min, purity> 99 %.

5.29. 2-Cyclohexyl-5-(2,4-difluorobenzamido)-6-(4-fluorophenoxy)-1H-benzo[d]imidazole (5d)

Off-white solid; 92 % yield; mp 184–185 °C; ¹H NMR (500 MHz, CDCl₃) 0.83–0.88 (m, 1 H), 1.25–1.32 (m, 2 H), 1.59 (dd, 2 H, J = 12.3, 3.0 Hz), 1.68–1.70 (m, 1 H), 1.78–1.80 (m, 2 H), 2.07 (d, 2 H, J = 12.8 Hz), 2.80–2.84 (m, 1 H), 6.90 (ddd, 1 H, J = 11.7, 8.8, 2.6 Hz), 6.99 (d, 3 H, J = 6.3 Hz), 7.02–7.06 (m, 1 H), 7.21 (s, 1 H), 8.21 (td, 1 H, J = 8.9, 6.6 Hz), 8.81 (s, 1 H), 9.26 (d, 1 H, J = 15.7 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 25.9, 26.2, 31.9, 38.2, 101.1, 105.2, 112.5, 112.6, 112.7, 116.5, 116.7, 120.8, 120.9, 126.4, 128.8, 138.4, 144.8, 151.7, 158.4, 161.0, 163.1; HRMS (ESI) m/z calcd for C₂₆H₂₃F₃N₃O₂⁺ 466.1737 Found: 466.1743 (Δ = 1.29 ppm). HPLC: t= 4.6 min, purity> 96 %.

5.30. 2-Cyclohexyl-6-(4-fluorophenoxy)-5-(pent-4-enimido)- 1H-benzo[d]imidazole (5e)

White solid; 63 % yield; mp 180.5–181.5 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.24–1.30 (m, 2 H), 1.36–1.44 (m, 2 H), 1.59–1.64 (m, 2 H), 1.73–1.76 (m, 1 H), 1.86 (dq, 2 H, J = 3.34, 9.98 Hz), 2.11 (dd, 2 H, J = 2.28, 13.3 Hz), 2.48 (dt, 3 H, J = 5.88, 11.7 Hz), 2.82–2.87 (m, 1 H), 4.99 (d, 1 H, J = 10.2 Hz), 5.07 (d, 1 H, J = 16.1 Hz), 5.82 (ddt, 1 H, J = 6.27, 10.5, 16.9 Hz), 6.95–7.04 (m, 3 H), 7.17 (s, 1 H), 7.87 (s, 1 H), 8.58 (s, 1 H), 9.58 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 14.2, 25.8, 26.0, 29.5, 31.7, 37.3, 38.5, 60.4, 102.7, 108.0, 116.1, 116.3, 116.5, 119.5, 125.2, 136.4, 142.4,157.9, 159.8, 170.7; HRMS (ESI) m/z calcd for C₂₄H₂₆FN₃O₂⁺ 408.2082 Found: 408.2090 (Δ = 1.96 ppm). HPLC: t= 10.3 min, purity> 97 %.

5.31. 2-Cyclohexyl-5-(4-methxylbenzamido)-6-(phenylthio)-1H-benzo[d]imidazole (5f)

Off-white solid; 79 % yield; mp 184.5–186 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.06–1.20 (m, 3 H), 1.53–1.63 (m, 3 H), 1.70 (d, 2 H, J = 9.5 Hz), 2.00 (d, 2 H, J = 11.3 Hz), 2.44 (s, 3 H), 2.73 (t, 1 H, J = 9.6 Hz), 7.14 (t, 2 H, J = 7.62 Hz), 7.22–7.27 (m, 4 H), 7.59 (d, 2 H, J = 8.0 Hz), 8.06 (s, 1 H), 9.01 (s, 1 H), 9.39 (s, 1 H), 11.0 (s, 1 H);¹³C NMR (125 MHz, CDCl₃) δ 21.5, 25.6, 25.9, 29.7, 31.6, 38.5, 103.3, 113.6, 126.0, 126.3, 126.4, 126.9, 129.0, 129.3, 129.6, 132.2, 134.2, 136.6, 142.6, 160.9, 166.1; HRMS (ESI) m/z calcd for C₂₆H₃₄N₃OS⁺ 436.2417 Found: 436.2417 (Δ = 0.0 ppm). HPLC: t= 4.8 min, purity> 99 %.

5.32. 6-(Benzylthio)-2-cyclohexyl-5-(4-tert-butylbenzamido)-1H-benzo[d]imidazole (5g)

Off-white solid; 92 % yield; mp 173–173.5 °C; ¹H NMR (500 MHz, CDCl₃) δ 1.06–1.23 (m, 3 H), 1.40 (s, 9 H), 1.51–1.59 (m, 3 H), 1.68 (d, 2 H, J = 11.3 Hz), 1.97 (d, 2 H, J = 11.8 Hz), 2.67–2.72 (m, 1 H), 3.90 (s, 2 H), 6.98 (dd, 2 H, J = 7.1, 2.3 Hz), 7.04 (dd, 3 H, J = 5.0, 1.9 Hz), 7.52 (d, 2 H, J = 8.4 Hz), 7.70 (d, 2 H, J = 8.4 Hz), 7.96 (s, 1 H), 8.93 (s, 1 H), 9.38 (s, 1 H), 11.1 (s, 1 H); ¹³C NMR (125 MHz, CDCl₃) δ 25.6, 25.9, 31.2, 31.7, 35.1, 38.5, 43.4, 102.2, 116.4, 125.8, 126.9, 127.1, 127.3, 128.5, 132.3, 135.3, 138.0, 140.2, 155.5, 160.8, 165.8; HRMS (ESI) m/z calcd for C₃₁H₃₆N₃OS⁺ 498.2574 Found: 498.2574 (Δ = 0.0 ppm). HPLC: t= 13.8 min, purity> 95 %.

5.33. 6-(Butylthio)-5-(2,4-difluorobenzamido)-2-cyclohexyl-1H-benzo[d]imidazole (5h)

Off-white solid; 93 % yield; mp 170–170.5 °C; ¹H NMR (400 MHz, CDCl₃) δ 0.83 (t, 3 H, J = 7.33 Hz), 1.17–1.29 (m, 3 H), 1.36 (dd, 2 H, J = 15.0, 7.37 Hz), 1.49–1.60 (m, 4 H), 1.67 (d, 1 H, J = 12.1 Hz), 1.77 (d, 2 H, J = 12.3 Hz), 2.06 (d, 2 H, J = 11.9 Hz), 2.73 (t, 2 H, J = 7.42 Hz), 2.79 (m, 1 H), 6.96–7.09 (m, 2 H), 7.93 (s, 1 H), 8.23 (td, 1 H, J = 8.85, 6.54 Hz), 8.87 (s, 1 H), 10.1 (s, 1 H), 10.3 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 13.6, 21.7, 25.7, 25.9, 31.3, 31.7, 37.0, 38.5, 104.7, 112.5, 112.7, 117.8, 118.3, 118.4, 133.8, 134.3, 160.5; HRMS (ESI) m/z calcd for C₂₄H₂₈F₂N₃OS⁺ 444.1916 Found: 444.1922 (Δ = 1.35 ppm). HPLC: t= 7.0 min, purity> 97 %.

5.34. 6-(Butylthio)-5-(4-metylbenzamido)-2-cyclohexyl-1H-benzo[d]imidazole (5i)

Off-white solid; 34 % yield; mp 155–156 °C; ¹H NMR (400 MHz, CDCl₃) δ 0.81 (t, 3 H, J = 7.38 Hz), 1.05–1.15 (m, 3 H), 1.24 (s, 1 H), 1.34 (q, 2 H, J = 7.34 Hz), 1.49–1.60 (m, 4 H), 1.66 (d, 2 H, J = 12.1 Hz), 1.97 (d, 2 H, 11.9 Hz), 2.46 (s, 3 H), 2.71–2.75 (m, 3 H), 7.36 (d, 2 H, J = 7.79 Hz), 7.92 (d, 3 H, J = 7.73 Hz), 8.99 (s, 1 H), 9.84 (s, 1 H), 11.4 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ; 13.5, 21.5, 21.8, 25.5, 25.8, 29.6, 31.4, 31.6, 37.2, 38.4, 102.7, 117.1, 126.2, 127.0, 129.7, 132.3, 133.8, 135.2, 140.1, 142.6, 160.7, 165.7; HRMS (ESI) m/z calcd for C₂₅H₃₂N₃OS⁺ 422.2261 Found: 422.2265 (Δ = 0.9 ppm). HPLC: t= 5.2 min, purity> 97 %.

5.35. Bacterial Strains and Growth

For evaluation of drug sensitivity, Mtb H37Rv was grown in 7H9 media containing 10% oleic acid/albumin/catalase (OADC) enrichment and 0.05% Tween-80 and assessed at mid log phase growth.

5.36. Antibacterial Activity

The minimum inhibitory concentration (MIC) was determined by the microplate Alamar Blue assay (MABA)¹⁹ as described previously. Briefly, stock solutions of the compounds were prepared in DMSO and were serially diluted 2-fold in 96-well microtiter plates, and Mtb H37Rv strain was added to each well to an OD600 of 0.005. Plates were incubated for 6 days at 37 °C. Alamar Blue (Invitrogen) was added to the plates, and the plates were incubated for an additional 24 h at 37 °C. Plates were monitored for color change, and MIC₉₉ was determined in triplicate.

5.37. Cytotoxicity Assay

The cytotoxicity of the compounds was tested against Vero cells using MTT assay.^{21, 24} The cells were grown in DMEM media supplemented with 5 % Bovine Serum and 1 % Penn Strip and incubated at 37 °C with 5 % CO₂. Then, 5,000 cells were added to each well of a 96-well plate in 200 μL aliquots. The cells were incubated at 37 °C for 1–2 days. A serial dilution of benzimidazoles 5 dissolved in sterile DMSO was added to the 96-well plates in 200 μL aliquots. The plates were incubated at 37 °C for 3 days. The medium was aspirated and then 40 μL of 0.5 mg/mL MTT in DPBS was added to each well. The plates were then incubated at 37 °C for 3 h. Then, 40 μL of 0.8 M HCl solution were added to dissolve the remaining crystals. Each experiment was run in triplicate. The optical density data was used to calculate IC₅₀ values using the Hill slope equation. The IC₅₀ values and their standard errors were calculated from the viability-concentration curve using the Four Parameter Logistic Model of Sigmaplot.

5.38. Mtb-FtsZ Protein expression and Preparation^{18, 25}

E. coli expression plasmid encoding the ftsz gene (pET 15b vector) was transformed into 100 μL of BL21 (DE3) cells. The transformed cells were plated onto LB plates, containing 100 μg/mL ampicilin. The antibiotic concentration was kept the same for the following steps. The plates were incubated overnight at 37 °C. The colonies were picked and grown in 10 mL of LB media at 37 °C at 250 rpm shake rate. The inoculum was transferred to 1 L of LB media in a 4 L flask and grown to an OD of 0.6 at A600. Then, 1 mM IPTG was added to induce protein expression overnight at 20 °C at 250 rpm shake rate. Cells were then pelleted by centrifugation at 3000 × g, flash frozen in liquid nitrogen, and stored at −80 °C until further purification steps. Thawed cells were suspended in 40 mL 50 mM Tris pH 7.5, 500 mM NaCl, 100 mM KCl, 0.1% NP-40 per liter cell culture growth and passed through 3 rounds of cell disrupter (French press) at 27 psi to disrupt cells. Lysed cells were centrifuged at 27,000 × g for 20 min to clear insoluble cellular components and cell wall fractions.

For polymerization assay, protein was purified as follows. Thawed cells were re-suspended in 40 mL of buffer containing 50 mM sodium phosphate pH 7.5, 300 mM sodium chloride, 10 mM imidazole, per liter cell culture growth and sonicated at 15 W 6 times for 30 seconds each with 1 minute pauses in between to disrupt cells. Lysed cells were centrifuged at 44,000 × g for one hour to clear insoluble cellular components. Cleared lysate was applied to Ni⁺² charged His-bind resin and washed with double the volume of resuspension buffer of 50 mM sodium phosphate pH 7.5, 300 mM sodium chloride, then double the volume of re-suspension buffer of 50 mM sodium phosphate pH 7.5, 300 mM sodium chloride, 60 mM imidazole. FtsZ protein was eluted with 4 × 5 mL portions of 50 mM sodium phosphate pH 7.5, 300 mM NaCl, 500 mM imidazole. Protein was loaded onto a Sephadex G25 size exclusion column to remove excess imidazole and to exchange protein into 25 mM HEPES pH 7.2, 1mM DTT, 0.1 mM EDTA, 10% glycerol (or other buffer as indicated). Resulting protein fractions were pooled and the N-terminal 6xHis affinity tag was removed by biotin tagged thrombin treatment overnight at 4 °C (0.25 Units biotinylated thrombin per mg tagged FtsZ protein). Successive passes through streptavidin agarose and fresh Ni⁺² charged His-bind resin removed biotinylated thrombin, uncut FtsZ protein, and free cut off affinity tag. A final cleanup was performed through an Akta driven Sephadex 200 60/16 size exclusion column in 25mM HEPES pH 7.2, 1 mM DTT, 0.1 mM EDTA, 10% glycerol or other buffer as indicated. Protein was then concentrated to 10 mg/mL (~250 μM) with centrifugal 30 kDa molecular weight cutoff filters, aliquoted 150 μL, flash frozen in liquid nitrogen, and stored at −80°C.

For Kd studies the protein was purified as follows. Cleared lysate was applied to Ni⁺² charged His-bind resin and made to equilibrate for one hour followed by washing in two volumes of wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 100 mM KCl, 0.1% NP-40, and 10 mM Imidazole). Bound protein was eluted with 10 mL of Elution buffer (50 mM Tris pH 7.5, 500 mM NaCl, 100 mM KCl, and 500 mM imidazole). The eluted protein was first dialyased against 2 L Storage buffer (50 mM Tris pH 7.8, 200 mM NaCl, 100 mM KCl,) overnight followed by 3 hrs dialysis against Storage buffer with 10% glycerol. Post dialysis, the concentration of the protein was checked by Bradford assay and purity of the purified protein determined by SDS page. If necessary, protein was further concentrated to 10 mg/mL (~250 μM) with centrifugal 10 kDa molecular weight cutoff filters, aliquoted 500 μL, and flash frozen in liquid nitrogen, and stored at −80 °C till further use.

For TEM analysis the following procedure was followed for protein purification. The cells were suspended in approximately 20–30 mL binding buffer (500 mM NaCl, 20 mM sodium phosphate, pH 7.8) and lysed using cell disruptor. The lysate was centrifuged in an ultracentrifuge at 126603 × g, 4 °C for 90 min. The supernatant was filtered and loaded onto Ni²⁺-NTA column, washed with 50 mL of binding buffer and eluted using a gradient of binding buffer with 30–500 mM imidazole. The eluted protein was dialyzed against buffer containing 50 mM Tris, 5mM MgCl₂, 50 mM KCl, pH 7.2 followed by buffer containing 10 % v/v glycerol. The protein after dialysis was concentrated and stored at −80 °C for further use. Since the number of aromatic residues in Mtb-FtsZ protein are low (Tyr: 1, Trp: 0), it is not reliable to follow concentration of protein by scanning at A280. The concentration of protein was therefore ascertained using the Bradford kit from Sigma.

5.39. Mtb-FtsZ Polymerization Inhibitory Assay

The inhibitory activity of lead benzamidazoles for Mtb-FtsZ polymerization was determined by means of light scattering on a PTI-QM4 Fluorescence Master system. The 90° light scattering was measured at 30 °C, using excitation and emission wavelength of 400 nm with slit width of 2 nm. The gain was set at 875 V. Mtb-FtsZ (15 μM) was incubated in the polymerization buffer (50 mM MES pH 6.5, 100 mM KCl, 5m M MgCl₂) for up to 300 sec. Polymerization was initiated with 100 μM GTP and monitored for up to 30 min. Benzimidazole stocks were prepared in DMSO and incubated with FtsZ enzyme prior to initiation of polymerization with GTP.

5.40. Transmission Electron Microscopy (TEM) Analysis

Stock solution of compound 5a was prepared in ethanol. Mtb-FtsZ (5 μM) was incubated with 40 or 80 μM of 5a in the polymerization buffer (50 mM MES, 5 mM MgCl₂, 100 mM KCl, pH 6.5) for 15 min on ice. To each solution was added GTP to the final concentration of 25 μM. The resulting solution was incubated at 37 °C for 30 min. The incubated solution was diluted 2 times with the polymerization buffer and immediately transferred to carbon coated 300 mesh formvar copper grid and negatively stained with 1% uranyl acetate. The samples were viewed with a FEI Tecnai12 BioTwinG transmission electron microscope at 80 kV. Digital images were acquired with an AMT XR-60 CCD digital camera system.¹⁸

5.41. Binding Studies of 5a with Mtb-FtsZ

The fluorescence anisotropy of the compound was measured in the presence of increasing FtsZ concentrations, in 50 mM MES, 100 mM KCl, 5 mM MgCl₂ in absence of GTP by exciting the compound at 316 nm and monitoring the change of fluorescence at 427.9 nm using a PTI-QM4 spectrofluorometer. The change in fluorescence (ΔF) at 427.9 nm was fitted into the equation ΔF = (ΔFmax × L)/(Kd +L).²³