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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 January 13.
Published in final edited form as:
PMCID: PMC3071426
NIHMSID: NIHMS256249

Novel Trisubstituted Benzimidazoles, Targeting Mtb FtsZ, As A New Class of Antitubercular Agents

Abstract

Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM) for their antibacterial activity against Mtb H37Rv strain. Moreover, five of the lead compounds also exhibited excellent activity against clinical Mtb strains with different drug-resistance profiles. All lead compounds do not show appreciable cytotoxicity (IC50 >200 μM) against Vero cells, which inhibit Mtb FtsZ assembly in a dose dependent manner. The two lead compounds unexpectedly showed enhancement of the GTPase activity of Mtb FtsZ. The result strongly suggests that the increased GTPase activity destabilizes FtsZ assembly leading to efficient inhibition of FtsZ polymerization and filament formation. The TEM and SEM analyses of Mtb FtsZ and Mtb cells, respectively, treated with a lead compound strongly suggest that lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb FtsZ assembly and Z-ring formation.

Keywords: Mycobacterium tuberculosis, FtsZ, FtsZ inhibitor, drug-resistant Mtb, GTPase, TEM, SEM

Tuberculosis (TB) is one of the leading infectious diseases and remains a major global health problem. According to WHO, 9.2 million new cases and 1.7 million deaths from TB have been reported.1 With the emergence of HIV, TB has become the most common opportunistic infection afflicting patients living with AIDS. There are 0.7 million HIV-positive people infected with TB, contributing to 0.2 million deaths worldwide.1 The lethal combination of TB and HIV is fuelling the TB epidemic in many parts of the world, especially Africa.1 Poor chemotherapeutics and the inadequate administration of drugs have lead to the development of multi-drug resistant TB (MDR-TB),2 treatment of which requires administration of more expensive, second line antibiotics for up to two years. In addition, even more alarming cases of extensively drug resistant strains of TB (XDR-TB) that are resistant to both first and second line drugs have been reported.3 Recent findings by WHO from 2000-2004 suggested that 4% of MDR-TB cases meet the criteria for XDR-TB. Consequently, there is a pressing need for the development of novel TB drugs that are effective against both drug sensitive and resistant Mtb strains.

FtsZ, a tubulin homologue, is a highly conserved and ubiquitous bacterial cell division protein. Similar to the process of microtubule formation by tubulin, FtsZ polymerizes in a GTP-dependent manner, forming a highly dynamic cytokinetic structure, designated as the Z-ring, at the center of the cell. 4-5 The recruitment of the other cell division proteins leads to Z-ring contraction, resulting in septum formation. Due to the requirement of FtsZ in mycobacterial cytokinesis, inhibition of FtsZ is a promising target for antituberculosis drug discovery.6-9 While tubulin and FtsZ share structural and functional homology and tubulin inhibitors are known to affect FtsZ assembly,6-8 the limited sequence homology at the protein level, affords an opportunity to discover FtsZ-specific compounds with limited cytotoxicity to eukaryotic cells.10 Since FtsZ is a novel drug target, compounds targeting FtsZ are expected to be active against drug resistant Mtb strains.11 Furthermore, the validation of FtsZ as a novel antibacterial drug target has been confirmed by the work of various groups including ours.12-24

Researchers at the Southern Research Institute screened known tubulin inhibitors against Mtb and identified several pyridopyrazine and pteridine based FtsZ inhibitors with anti-TB activity.12-14 Later, Slayden et al found that thiabendazole and albendazole, known tubulin inhibitors, interfered and delayed the Mtb cell division processes.8 Taking into account the structural similarity of the pyridopyrazine moiety, pteridine moeity, albendazole and thiabendazole,12-14 we envisioned that the benzimidazole scaffold would be a good starting point for the development of novel FtsZ inhibitors, which will have activity against both drug sensitive and drug resistant Mtb. Therefore, trisubstituted-benzimidazoles were investigated for antibacterial activity, specifically we report the activity of 2,5,6- and 2,5,7-trisubstituted benzimidazoles against Mtb (Figure 1). Importantly, this work substantiates trisubstitutedbenzimidazoles for the development of next generation anti-mycobacterial inhibitors with activity against difficult to treat clinical strains.

Figure 1
Proposed 2,5,6- and 2,5,7-trisubstituted benzimidazoles

The 2,5,6-trisubstituted benzimidazoles were synthesized as outlined in Scheme 1. The aromatic nucleophilic substitution of commercially available 2,4-dinitro-5-fluoroaniline (3) with diethylamine afforded 5-diethylaminodinitroanilines 4a (R1 = R2 = Et) in quantitative yield. The acylation of 4a with different acyl chlorides afforded the corresponding N-acylanilines 5a-1~8 in 75-90% yields. The reduction and subsequent acid-catalyzed cyclization gave 5-aminobenzimidazoles 6a-1~8 in 40-60% yield. The advanced intermediates 6a-1~8 were dissolved in dichloromethane and transferred to 96-well plate. Subsequently, 33 different acyl chlorides and/or alkyl chloroformates were added to 6a-1~8 and reacted at room temperature overnight using a shaker. To the reaction mixture was added an aminomethylated polystyrene resin to scavenge excess or unreacted acyl chlorides and/or alkyl chloroformates. After reacting the mixture overnight with gentle shaking, the resin was filtered to afford a library of 272 2,5,6-trisubstituted benzimidazoles 1a (R1 = R2 = Et) with good to high purity based on LC-MS/UV analysis (see the Supporting Information for the detailed experimental procedures, structures of intermediates and library).

Scheme 1
Synthesis of 2,5,6- Trisubstituted Benzimidazoles a

The 2,5,7-trisubstituted benzimidazoles were synthesized as outlined in Scheme 2. Commercially available 5-amino-2,4-dinitrobenzamide (7) was hydrolyzed to give 4-amino-3,5-dinitrobenzoic acid, which was converted to acyl chloride 8. The reaction of 8 with sodium azide afforded the corresponding acyl azide, which was subjected to the Curtius rearrangement to give the corresponding isocyanate. The isocyanate was subsequently reacted with methanol or ethanol to give the corresponding carbamates 9a (R'1 = Et) or 9b (R'1 = Me) in 78-85% yield (for 5 steps) as a bright red solid. The reduction of 9a,b followed by cyclocondensation with the bisulfite salts of different aldehydes afforded 7-aminobenzimidazoles 10a-A~F and 10b. The final acylation of 10a,b in the same manner as that for the library of 1a gave the 77 compound library of 2,5,7-trisubstituted benzimidazoles 2 with good to high purity (see the Supporting Information for the detailed experimental procedures, structures of intermediates and library).

Scheme 2
Synthesis of 2,5,7-Trisubstituted Benzimidazolesa

The libraries of 2,5,6- and 2,5,7-trisubstituted benzimidazoles 1a and 2 (349 compounds) were screened for their activity against Mtb H37Rv using the “Microplate Alamar Blue assay (MABA)”25 in a 96-well format (single point assay in triplicates). Of these, 26 compounds were identified to inhibit the growth of Mtb H37Rv with MIC values of ≤5 μg/mL. Furthermore, among the 26 hits, resynthesized pure 9 compounds showed MIC values of 0.50-6.1 μg/mL (1.5-15 μM) (Table 1).

Table 1
Antimicrobial Activities of Selected Trisubstituted Benzimidazoles against Drug-Sensitive and Drug-Resistant Strains of Mtba

Preliminary structure–activity relationship studies of hits from the library of 1a indicated that cyclohexyl and diethylamino groups at the 2- and 6-positions, respectively, were critically important for antibacterial activity (Figure 2). Accordingly, another library of 1 (1-G: 238 compounds) was synthesized with different dialkylamino substituents at the 6-position, but retaining the cyclohexyl group at the 2-position using the procedures similar to those for the synthesis of the libraries of 1a and 2 (Scheme 1). (See Supporting Information for the detailed experimental procedures, structures of the intermediates and library.)

Figure 2
Preliminary SAR of 2,5,6-trisubstituted benzimidazole hit compounds 1a

Based on the MABA screening (single point assay in triplicates), 54 compounds were identified as hits, which inhibited the cell growth with MIC values of 5 μg/mL or less. Among the 54 hits, resynthesized pure 1b-G1 and 1b-G2 showed MIC99 values of 0.39 μg/mL (1.0 μM) and 1.56 μg/mL (3.7 μM), respectively (Table 1). It should be noted that all 11 pure lead compounds do not show appreciable cytotoxicity (IC50 >200 μM) against Vero cells (Table 1).

Five lead compounds, 1a-G4, 1a-G7, 1b-G1,1b-G2 and 2b-1, were also assayed for their activities against clinical isolates of Mtb strains W210 (drug-sensitive),26 NHN20 (drug-sensitive),26 NHN335-2 (isoniazide-resistant; KasA G269S mutation),27 NHN382 (isoniazide-resistant; Kat G S315t mutation)26 and TN587 (isoniazide-resistant; KatG 3315T mutation),26 which showed no difference between the drug-sensitive and drug-resistant strains, as anticipated, exhibiting excellent MIC values in the range of 0.39-1.56 μg/mL (1-4.6 μM). These compounds have been advanced to the in vivo rapid model assay in mice.

FtsZ polymerization assay28 was carried out to validate the hypothesis that these lead compounds exhibit antibacterial activity by interacting with FtsZ. Three benzimidazoles, selected from the 11 lead compounds shown in Table 1, have been evaluated for their ability to inhibit the polymerization of the wild-type FtsZ. 1a-G1 (MIC 7.9 μM) inhibited FtsZ polymerization in a dose-dependent manner and ca. 80% inhibition was achieved at 10 μM concentration (FtsZ concentration = 15 μM) (Figure 3a). A similar result was obtained for 1a-G7 (MIC 2 μM) (Figure 3b). 1a-G1 and 1a-G7 inhibited FtsZ polymerization with IC50 values of 6.21 μM and 7.69 μM, respectively. 1a-G4 (MIC 4.2 μM), showed ca. 45% inhibition at 10 μM concentration (Figure 3c).

Figure 3
Inhibition of FtsZ polymerization by (a) 1a-G7; (b) 1a-G1 and (c) 1a-G4

The assembly and disassembly of FtsZ protein has been shown to be GTP dependent. Since FtsZ has GTPase activity, the Malachite Green assay29 was performed to monitor the amount of inorganic phosphate (Pi) released upon treatment of Mtb FtsZ with two lead compounds, 1a-G4 and 1a-G7. On treatment of Mtb FtsZ with these two compounds, an enhancement in GTPase activity was observed (Figure 4). This unique behavior is analogous to the effect that curcumin exhibited on recombinant E.coli FtsZ.28 Enhancement in the GTPase activity together with the fact that these compounds inhibit polymerization of Mtb FtsZ (Figure 3) leads us to conclude that the increased GTPase activity causes the instability of Mtb FtsZ polymer. As a result of the instability, Mtb FtsZ is unable to polymerize normally, leading to the efficient inhibition of the formation of sizable Mtb FtsZ polymers and filaments.

Figure 4
Enhancement of GTPase activity of FtsZ by lead benzimidazoles, 1a-G4 (red) and 1a-G7 (blue)

To further investigate the effect of the hit compounds on polymerization of FtsZ protein, Transmission electron microscopy (TEM) imaging of the treated and untreated Mtb FtsZ protein was carried out. Mtb FtsZ (5 μM) polymerized in the presence of GTP (25 μM) was diluted 5 times with polymerization buffer and immediately transferred onto a carbon coated copper-mesh grid for TEM imaging. Mtb FtsZ protein treated with GTP as well as 40 μM and 80 μM of 1a-G7 were visualized (Figure 5). The protein treated with colchicines, which is known to inhibit FtsZ polymerization,13 was used as a reference in this set of experiments (Figure 5, F and G). As anticipated, there was a considerable reduction in the extent of FtsZ protofilament formation upon treatment with 1a-G7 as compared to the control experiment. At 40 μM concentration of 1a-G7, the density of Mtb FtsZ protofilaments was substantially reduced (Figure 5, B and C). Furthermore, the protofilaments were short and very thin. At 80 μM concentration, the formation of protofilaments was drastically reduced and only numerous tiny aggregates were formed (Figure 5, D and E). In sharp contrast, the Mtb FtsZ protein treated with a high concentration (200 μM) of colchicine showed decreased and thinner protofilaments (Figure 5, G and H), but with more or less similar morphology with that of GTP-initiated polymerization and protofilaments (Figure 5, A and B). These result clearly indicates the highly efficient inhibition of Mtb FtsZ assembly by 1a-G7 in a dose-dependent manner with a novel mechanism of action.

Figure 5
Transmission electron microscope (TEM) images of FtsZ. FtsZ (5μM) was polymerized by GTP in the absence (A, B) and presence of 1a-G7 at 40 μM (C, D) and 80 μM (E, F). TEM images of the inhibition of FtsZ polymerization in the presence ...

Visualization of bacteria treated with 2xMIC and 4xMIC 1a-G7 for 2-days using scanning electron microscopy (SEM) revealed altered cell division lengths and aberrant division indicative of the inhibition of FtsZ assembly and cell division (Figure 6). Specifically, cell division and elongation was inhibited in a dose dependent manner resulting in shorter bacterial cells with increased circumferences and altered polar caps. The observed altered bacterial morphology upon exposure to 1a-G7 is thought to result from inhibition of FtsZ leading to disruption in septum formation and recruitment of septum associated proteins involved in later steps of division and septum resolution.

Figure 6
SEM of Mtb cells treated with 1a-7 (20,000X). (A) Control; (B) 2x MIC; (C) 4 x MIC; (D) distribution of bacterial cell length: Control (black), 2x MIC (grey), 4x MIC (light grey)

Conclusions

We have created a library of novel trisubstituted benzimidazoles through rational drug design. As Table 1 shows, a good number of these compounds exhibited promising MIC values in the range of 0.5-6.1 μg/mL (1.0-15 μM) for their antibacterial activity against Mtb H37Rv strain. Moreover, 1b-G4, 1b-G7, 1b-G1, 1b-G2 and 2a-1 were also found to exhibit excellent activity against clinical Mtb strains, W210, NHN20, NHN335, NHN382 and TN587, with different drug-resistance profiles. All lead compounds do not show appreciable cytotoxicity (IC50 >200 μM) against Vero cells. The standard light scattering assay to assess the effect of these compounds on the polymerization of FtsZ, clearly showed the inhibition of FtsZ assembly in a dose dependent manner. As the duration of steady state of FtsZ polymer is dependent on the rate of GTP hydrolysis, the effect of lead compounds, 1a-G4 and 1a-G7, on GTPase activity was examined. Unexpectedly, instead of inhibiting, these compounds enhanced the GTPase activity of Mtb FtsZ by 3-4 folds. The result strongly suggests that the increased GTPase activity destabilizes FtsZ polymer leading to efficient inhibition of FtsZ polymerization and filament formation. The effect of 1a-G7 on FtsZ polymerization was examined by TEM, which revealed an impressive dose-dependent inhibition of Mtb FtsZ assembly and a dramatic suppression of FtsZ protofilament formation at higher concentration of 1a-G7. The SEM images of Mtb cells treated with 1a-G7 showed the absence of septum formation and shorter bacterial cells with increased circumferences and altered polar caps. The unique bacterial morphology indicates that the disruption of septum formation by 1a-G7 may recruit other septum associated proteins involved in later steps of division and septum resolution. The TEM and SEM analyses strongly suggest that 1a-G7 and very likely other lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb FtsZ assembly and Z-ring formation. Further optimization and in vivo evaluation of these highly promising lead compounds are actively underway in these laboratories.

Experimental Section

Methods

1H and 13C NMR spectra were measured on a Varian 300, 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 Merck DCalufolien with Kieselgel 60F-254 and column chromatography was carried out on silica gel 60 (Merck; 230-400 mesh ASTM). High-resolution mass spectra were obtained from Mass Spectrometry Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL. Purity was determined by Agilent 1100 series HPLC assembly. Two analytical conditions were used and noted as a part of the characterization data for resynthesized compounds. HPLC1: Luna PFP, 3 μm, 100 Å (pore size), 3.0 × 150 mm column, Solvent A – H2O:ACN, 95:5 (20 mM Ammonium acetate, pH 6.5), Solvent B = H2O:ACN, 5:95 (20 mM Ammonium acetate, pH 6.5),Temperature 30 °C, flow rate 0.6 mL/min, t = 0-13.5 min, gradient 15-90 % solvent B. HPLC2: Kinetex PFP, 2.6 μm, 100 Å (pore size), 4.6 × 100 mm column, Solvent A – H2O:ACN, 95:5 (25 mM Ammonium acetate, pH 6.5), Solvent B = H2O:ACN, 5:95 (25 mM Ammonium acetate, pH=6.5),Temperature 25 °C, flow rate 1.0 mL/min, t = 0-15 min, gradient 20-95 % solvent B. SensoLtye® MG phosphate assay kit from Anaspec was used for Malachite Green assay. Light scattering assays were performed using a Fluorolog fluorimeter from ISA instruments. Scanning electron micrographs were obtained with a JOEL JSM-6500F scanning electron microscope. FEI Tecnai12 BioTwinG transmission electron microscope with an AMT XR-60 CCD digital camera system was used to acquire transmission electron microscopy images.

Materials

BL21(DE3)pLysS cells, His-Bind protein purification resin and the buffers were purchased from Novagen. BCA kit for protein concentration determination was purchased from Sigma. Buffer salts (reagent grade or better), solvents (HPLC grade or better), and all the other chemicals were purchased from Fisher Scientific Co. (Pittsburgh, PA). The chemicals were purchased from Aldrich Co., Synquest Inc. and Sigma 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. Aminomethylated polystyrene resin EHL (200-400 mesh) 2 % DVB was purchased from Novagenbiochem.

Chemical Synthesis

Novel 2,5,6-trisubstituted benzimidazoles 1 and 2,5,7-trisubstituted benzimidazoles were synthesized in accordance with the general procedures illustrated in Scheme 1 and Scheme 2. The detailed procedures for the syntheses of intermediates, 4, 5, 6, 8, 9 and 10 as well as their characterization data are described in the Supporting Information.

General procedure for the synthesis of the libraries of trisubstituted benzimidazoles 1 and 2

A 5-Aminobenzimidazole 6 or 7-aminobenzimidazole 10 (0.005 mmol) was dissolved in dichloromethane and transferred into a 96-well plate. To these wells were added 33 different acyl chlorides (1.1 eq.) or alkyl chloroformates (1 eq) and reacted overnight on a shaker. Aminomethylated polystyrene resin EHL (200-400 mesh) 2 % DVB (10 eq) was added to scavenge excess or unreacted acyl chlorides or alkyl chloroformates. After reacting overnight using the shaker, the resin was filtered to provide a library of trisubstituted benzimidazoles 1 or 2. The product in each well was analyzed by LC-MS/UV for the purity and confirmation of the structure. The purity was in the range of 80-90 %.

General Procedure for the Synthesis of analytically pure trisubstituted benzimidazoles 1 and 2

A typical procedure is described for the synthesis of 5-butoxycarbonylamino-2-cyclohexyl-6-N,N-diethylamino-1H-benzimidazole (1a-G7).

To a solution of 2-cyclohexyl-5-N,N-diethylamino-6-aminobenzimidazole (6a-G) (486 mg, 1.71 mmol) in dichloromethane (20 mL) was added a solution of N-butoxycarbonyloxysuccinimide (340 mg, 1.71 mmol) in dichloromethane (20 mL) dropwise at room temperature. After the addition, the reaction mixture was stirred at room temperature overnight. After the completion of the reaction, the reaction mixture was concentrated. The crude was purified via flash chromatography on silica gel (gradient 20-40% EtOAc/hexanes) to give 1a-G7 as white solid (336 mg, 51 % yield): mp 171-172 °C; 1HNMR (500 MHz, CDCl3) δ 0.91 (t, 6 H, J = 7.2 Hz), 0.96 (t, 3 H, J = 7.5 Hz), 1.23-1.46 (m, 5 H), 1.57-1.72 (m, 5 H), 1.81 (m, 2 H, J = 10 Hz), 2.07 (m, 2 H, J = 10 Hz), 2.85 (m, 1 H), 2.92 (q, 4 H, J = 7.2 Hz), 4.19 (t, 2 H, J = 6.5 Hz), 7.46 (s, 1 H), 8.24 (s, 1 H), 8.51 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 12.72, 13.75, 19.11, 25.81, 26.02, 31.07, 31.78, 38.46, 50.43, 64.87, 98.88, 107.3, 113.3, 131.2, 132.2, 134.3, 138.5, 154.1, 158.8; HRMS (FAB) m/z calcd for C22H34N4O2H+: 387.2763, Found: 387.2760 (Δ = 0.8 ppm). HPLC(2): 10.6 min, purity >99 %.

In a similar manner, other lead benzimidazoles were synthesized and characterized.

5-Benzamido-2-cyclohexyl-6-N,N-diethylamino-1H-benzo[d]imidazole (1a-G1)

White solid; 74 % yield; mp 180-181 °C; 1HNMR (400 MHz, CDCl3) δ 0.97 (t, 6 H, J = 7.2 Hz), 1.16 (m, 3 H), 1.65 (m, 5 H), 1.98 (m, 2 H), 2.71 (m, 1 H), 3.03 (m, 4 H, J = 6.8 Hz), 7.57 (m, 4 H), 7.98 (d, 2 H, J = 6.2 Hz), 8.97 (s, 1 H),10.4 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 13.09, 21.48, 25.64, 25.91, 31.73, 38.40, 50.77, 100.9, 113.1, 126.9, 129.5, 131.6, 132.7, 135.15, 139.5, 159.8, 165.1; HRMS (FAB) m/z calcd for C24H30N4OH+: 391.2486, Found: 391.2498 (Δ = −3.1 ppm). HPLC(2): 9.50 min, purity >99 %.

2-Cyclohexyl-6-N,N-diethylamino-5-propoxycarbonylamino-1H-benzo[d]imidazole (1a-G2)

White solid; 63 % yield; mp 80-181 °C; 1HNMR (300 MHz, CDCl3) δ 0.92 (t, 6 H, J = 7.2 Hz), 0.99 (t, 3 H, J = 7.5 Hz), 1.25-1.41 (m, 4 H), 1.60-1.76 (m, 5 H), 1.86 (m, 2 H), 2.10 (m, 2 H), 2.87 (m, 1 H), 2.92 (m, 4 H, J = 6.9 Hz), 4.14 (t, 2 H, J = 6.6 Hz), 7.47 (s, 1 H), 8.23 (s, 1 H), 8.51 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 10.28, 12.67, 22.28, 25.71, 25.96, 31.75, 38.44, 50.38, 66.56, 100.0, 112.0, 132.1, 132.4, 134.3, 136.92, 154.1, 159.0; HRMS (FAB) m/z calcd for C21H32N4O2H+: 373.2601, Found: 373.2604 (Δ = −0.8 ppm). HPLC(2): 9.98 min, purity >99 %.

2-Cyclohexyl-6-N,N-diethylamino-5-(4-methoxybenzamido)-1H-benzo[d]imidazole (1a-G3)

White solid; 79%; mp 198-199 °C; 1HNMR (400 MHz, CDCl3) δ 0.97 (t, 6 H, J = 7.2 Hz), 1.16 (m, 3 H), 1.53-1.68 (m, 5 H), 1.98 (m, 2 H), 2.71 (m, 1 H), 3.02 (q, 4 H, J = 7.2 Hz), 3.9 (s, 3 H), 7.05 (d, 2 H, J = 8.8 Hz), 7.55 (s, 1 H), 7.95 (d, 2 H, J = 8.8 Hz), 8.95 (s, 1 H), 10.32 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 13.36, 25.85, 26.18, 31.96, 38.67, 51.02, 55.75, 101.2, 113.2, 114.2, 127.9, 128.8, 129.0, 132.1, 139.3, 135.2, 160.0, 162.6, 164.9; HRMS (FAB) m/z calcd for C25H32N4O2H+: 421.2601, Found: 421.2604 (Δ = -0.7 ppm). HPLC(2): 9.5 min, purity >99 %.

5-But-3-enoxycarbonylamino-2-cyclohexyl-6-N,N-diethylamino-1H-benzo[d]imidazole (1a-G4)

White solid; 51 % yield; mp 143-145 °C; 1HNMR (500 MHz, CDCl3) δ 0.91 (t, J = 7 Hz, 6 H), 1.16-1.31 (m, 3 H), 1.56-1.66 (m, 3 H),1.77 (d, 2 H, J = 13.5 Hz), 2.04 (d, 2 H, J = 12.5 Hz), 2.47 (t, 2 H, J = 6.5 Hz), 2.83 (m, 1 H), 2.88 (q, 4 H, J = 7.5 Hz), 4.23 (t, 2 H, J = 7 Hz), 5.11 (m, 2 H), 5.84 (m, 1 H), 7.41 (s, 1 H), 8.24 (s, 1 H), 8.52 (s, 1 H) ; 13C NMR (125 MHz, CDCl3) δ 12.66, 25.72, 25.96, 31.76, 33.47, 50.39, 63.98, 99.97, 112.1, 117.1, 131.9, 132.0, 131.9, 133.9, 134.3, 137.3, 153.9, 159.2; HRMS (FAB) m/z calcd for C22H32N4O2H+: 385.2604, Found: 385.2604 (Δ = 0.0 ppm). HPLC(2): 10.1 min, purity >99 %.

5-Chlorobenzamido-2-cyclohexyl-6-N,N-diethylamino-1H-benzo[d[imidazole (1a-G5)

White solid; 64 % yield; mp 216-217 °C (turned brown); 1HNMR (400 MHz, CDCl3) δ 0.96 (t, 6 H, J = 7.2 Hz), 1.21-1.32 (m, 2 H), 1.59-1.81 (m, 5 H), 2.08 (m, 2 H), 2.79 (m, 1 H), 3.02 (m, 4 H, J = 7 Hz), 7.50 (d, 2 H, J = 6.8 Hz), 7.58 (s, 1 H), 7.88 (d, 2 H, J = 8.8 Hz), 8.82 (s, 1 H),10.31 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 13.12, 25.75, 25.97, 29.69, 31.76, 38.47, 50.78, 100.5, 113.6, 128.3, 129.1, 131.3, 131.8, 133.9, 135.2, 137.9, 139.6, 159.5, 163.7; HRMS (FAB) m/z calcd for C24H29N4OClH+: 425.2108, Found: 425.2108 (Δ = −0.0 ppm). HPLC(2): 10.5 min, purity >99 %.

2-Cyclohexyl-6-N,N-diethylamino-5-(4-methylbenzamido)-1H-benzo[d]imidazole (1a-G6)

White solid; 65 % yield; mp 183-184 °C; 1HNMR (300 MHz, CDCl3) δ 0.97 (t, 6 H, J = 6 Hz), 1.2 (m, 3 H), 1.58-1.78 (m, 5 H), 2.06 (m, 2 H), 2.45 (s, 3 H), 2.76 (m, 1 H), 3.03 (q, 4 H, J = 7.2 Hz), 7.34 (d, 2 H, J = 8.4 Hz) 7.59 (s, 1 H), 7.87 (d, 2 H, J = 8.1 Hz), 8.91 (s, 1 H), 10.31 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 13.09, 21.48, 25.63, 25.92, 31.72, 38.40, 50.75, 100.9, 113.1, 126.8, 129.6, 131.8, 132.7, 135.0, 139.4, 142.1, 159.8, 165.1; HRMS (FAB) m/z calcd for C25H32N4OH+: 405.2654, Found: 405.2654 (Δ = −0.0 ppm). HPLC(2): 10.1 min, purity >99 %.

Benzyloxycarbonylamino-2-cyclohexyl-6-N,N-diethylamino-1H-benzo[d]imidazole (1a-G8)

White solid; 61 % yield; mp 208-209 °C; 1HNMR (300 MHz, CDCl3) δ 0.90 (t, 6 H, J = 7.2 Hz), 1.38 (m, 4 H), 1.84-1.62 (m, 5 H), 2.11 (m, 2 H), 2.84 (m, 1 H), 2.91 (m, 4 H, J =7.2 Hz), 5.22 (s, 2 H), 7.45-7.35 (m, 5 H), 8.25 (s, 1 H), 8.61 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 12.62, 25.77, 25.97, 31.77, 38.46, 50.41, 66.68, 99.02, 113.2, 128.1, 128.5, 131.3, 132.0, 134.2, 136.3, 138.2, 153.7, 159.0; HRMS (FAB) m/z calcd for C25H32N4O2H+: 421.2601, Found: 421.2604 (Δ = −0.5 ppm). HPLC(2): 10.7 min, purity >99 %.

5-Butoxycarbonylamino-2-cyclohexyl-6-(pyrrolidin-1-yl)-1H-benzo[d]imidazole (1b-G1)

White solid; 80 % yield; mp 128-129 °C; 1HNMR (500 MHz, CDCl3) δ 0.94 (t, 3 H, J = 6.5 Hz), 1.26-1.19 (m, 5 H), 1.73-1.55 (m, 7 H), 1.92 (bs, 4 H), 2.00 (m, 2 H), 2.76 (m, 1 H), 2.93 (bs, 4 H), 4.18 (t, J = 6.5 Hz, 2 H), 7.40 (s, 1 H), 7.95 (s, 1 H), 8.17 (s,1 H); 13C NMR (125 MHz, CDCl3) δ 13.68, 19.03, 24.24, 25.71, 25.94,30.99, 31.75, 38.45, 53.41, 64.90, 101.3, 109.80, 129.3, 131.8, 135.6, 137.5, 154.3, 159.1. HRMS (FAB) m/z calcd for C22H32N4O2H+: 385.2594, Found: 385.2604 (Δ = −1.1 ppm). HPLC(2): 10 min, purity >99 %.

5-Benzyloxycarbonylamino-2-cyclohexyl-6-(pyrrolidin-1-yl)-1H-benzo[d]imidazole (1b-G2)

White solid; 68 % yield; mp 86-87 °C 1HNMR (500 MHz, CDCl3) δ 1.21-1.28 (m, 4 H), 1.59-1.65 (m, 4 H), 1.76 (m, 2 H), 1.91 (s, 4 H ), 2.04 (d, 2H, J = 12.5 Hz), 2.82 (m, 1 H), 2.93 (s, 4 H), 5.22 (s, 2 H), 7.34-7.48 ( m, 5 H aromatic), 8.08 (s, 1 H), 8.22 (s, 1 H);13C NMR (125 MHz, CDCl3) δ 24.29, 25.69, 25.91, 31.72, 38.31, 53.54, 66.75, 101.5, 108.7, 128.1, 128.5, 129.6, 131.9, 136.0, 136.2, 153.8, 158.9. HRMS (FAB) m/z calcd for C25H30N4O2H+: 419.2448, Found: 419.2447 (Δ = 0.2 ppm). HPLC(2): 10.1 min, purity >99 %.

7-Acetylamino-5-ethoxycarbonylamino-2-phenyl-1H-benzo[d]imidazole (2a-1)

White solid; 84 % yield: mp 158-160 °C; 1H NMR (400 MHz, CD3OD) δ 1.32 (t, J = 7.2 Hz, 3H), 2.26 (s, 3H), 4.20 (q, J = 7.2 Hz, 2H), 7.54 (m, 3H), 7.69 (bs, 1H), 7.79 (bs, 1H), 8.05 (m, 2H); 13C NMR (100 MHz, DMSO) δ 14.59, 23.99, 59.92, 96.14, 105.46, 126.18, 128.87, 129.16, 129.48, 130.08, 131.28, 131.82, 135.21, 149.61, 153.74, 168.61. HRMS (FAB) m/z calcd for C18H18N4O3H+: 339.1450, Found: 339.1457 (Δ = −2.1 ppm). HPLC(2): 4.95 min, purity >99 %.

Bacterial Strains and Growth

H37Rv, the drug sensitive laboratory strain of Mtb as well as clinical Mtb strains W210, NHN20, HN355, NHN382 and TN587 exhibit different resistant profiles to isoniazid and rifampicin.8, 27 For evaluation of drug sensitivity all strains were grown in 7H9 media containing 10% oleic acid/albumin/dextrose/catalase (OADC) enrichment and 0.05% Tween-80 and assessed at mid log phase growth.

Antibacterial Activity

The minimum inhibitory concentration (MIC) was determined by the Microplate Alamar Blue assay (MABA) as described previously.25 Briefly, stock solutions of the compounds were prepared in DMSO and were serially diluted 2 fold in 96 well microtiter plates and each Mtb strains was added to each well to an OD600 of 0.005. Plates were incubated for 6 days at 37°C. AlamarBlue® (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.

Cytotoxicity Assay

The cytotoxicity of the compounds was tested against VERO cells. Epithelial cells from the kidneys of the African Green Monkey were used to start the VERO cell line. Vero cells were grown in L15 media without CO2. Serial two fold dilutions of the drugs were prepared in the 96 well microtiter plates. The cells were added to the plates in media containing the appropriate amount of AlamarBlue®. The cells were added to a final concentration of 2.5 × 104 / well. The plates were incubated for 3 days at 37°C. The LD50 was calculated according to manufacturer directions.

FtsZ Polymerization Inhibitory Assay12

The inhibitory activity of lead benzamidazoles for Mtb FtsZ polymerization was determined by means of light scattering on a PTI Fluorescence Master Systems. 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 950 V. Mtb FtsZ (15 μM) was incubated in the polymerization buffer (50 mM MES, 5mM MgCl2, 50 mM KCl, pH 6.5) to establish a baseline. Then, 5 mM GTP (20 μL) was added to make the final volume 1,000 μL in a 1.5 mL cuvette. The light scattering was measured for a period of 30-60 min. When DMSO was used as a solvent for the inhibitor, the control also contained the same amount of DMSO. For inhibition studies, a lead benzimidazole at various concentrations was incubated with the polymerization buffer to establish a base line. This was followed by addition of FtsZ (15 μM). Once the baseline was stabilized, 5 mM GTP (20 μL) was added to make the final volume 1,000 μL in a 1.5 mL cuvette and the light scattering was measured.

GTPase Assay

The amount of inorganic phosphate (Pi) released during the assembly of FtsZ was measured using a standard Malachite Green/ammonium molybdate assay.29 Briefly, FtsZ protein (10 μM) was incubated without or with a benzimidazole at different concentrations (0, 20, 40 and 80 μM) in polymerization buffer (50 mM MES, 5 mM MgCl2, 50 mM KCl, pH 6.5) at room temperature for 15 min. Then, 50 μM GTP was added to the reaction mixture and incubated at 37 °C to start the hydrolysis reaction. After 30 min of incubation, Malachite green reagent (20% v/v) was added to the reaction mixtures to quench the reaction. The reaction mixtures were centrifuged at 13,000 rpm for 90 sec to remove the protein debris. The samples (100 μL) were transferred to a 96 well plate and the absorbance of each well was measured at 620 nm. The background absorbance was subtracted from all the readings. A phosphate standard curve was prepared using phosphate standard provided with a Malachite Green assay kit (SensoLyte®). All the solutions were prepared in polymerization buffer.

Transmission Electron Microscopy (TEM) Analysis

Mtb FtsZ (5 μM) was incubated with 40 μM or 80 μM of 1a-G7 in the polymerization buffer (50 mM MES, 5mM MgCl2, 50 mM KCl, pH 6.5) for 15-20 min. 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 5 times with the polymerization of buffer and immediately transferred to carbon coated 300 mesh formvor copper grid and negatively stained with 1 % uranyl acetate. The samples were viewed with a FEI Tecnai12 BioTwinG transmission electron microscope at 80 kV.19 Digital images were acquired with an AMT XR-60 CCD Digital Camera System.

Ultrastructural analysis via scanning electron micrscopy (SEM)

Mtb H37Rv bacteria were exposed to 2 × MIC and 4 × MIC 1a-G7 for 2 days. The bacteria were prepared for scanning electron microscopy as described by Slayden et. al.8 Briefly, bacteria were prepared for SEM treatment with 2.5% glutaraldehyde in 0.1 M sodium cacodyte (pH 7.2), 5mM CaCl2 and 5 mM MgCl2 and incubated at room temperature for 1-2 h. The fixed bacteria were harvested by centrifugation and washed in PBS, and subjected to 2.5% glutaraldehyde overnight at 4 °C. Final preparation for SEM was achieved by treatment with 1% OsO4 in sodium cacodylate buffer and dehydration in a graded alcohol series. The bacteria were then examined using a JOEL JSM-6500F scanning electron microscope. Bacteria from all treatment groups were measured for their lengths. Size frequency graphs were prepared for each treatment group.

Supplementary Material

1_si_001

Acknowledgments

This research is supported by grants from NYSTAR (to I.O.) and the National Institutes of Health (AI078251 to I.O.). The authors acknowledge Dr. Edward Melief for the helpful discussions. The authors gratefully acknowledge the technical support of Ms. Susan Van Horn for TEM operation at the Microscopy Imaging Center at Stony Brook University.

Abbreviations

ACN
Acetonitrile
AIDS
Acquired Immuno Deficiency Syndrome
BCA
bicinchoninic acid
DIPEA
Diisopropyl ethylamine
DMF
N,N-dimethylformamide
DMSO
Dimethyl Sulfoxide
E. coli
Escherichia coli
FtsZ
Filamenting temperature sensitive mutant Z
GTP
Guanosine Triposphate
GTPase
Guanosine Triposphatase
HIV
Human Immuno deficiency Virus
MABA
Microplate Alamar Blue Assay
MDR-TB
Multi Drug Resistant-Tuberculosis
MIC
Minimum Inhibitory Concentration
Mtb
Mycobacterium tuberculosis
OADC
Oleic acid/Albumin/Dextrose/Catalase
PBS
Phosphate Buffered Saline
Pi
Inorganic Phosphate
SAR
Structure activity relationship
SEM
Scanning Electron Microscopy
TB
Tuberculosis
TEM
Transmission Electron Microscopy
THF
Tetrahydrofuran
TLC
Thin Layer Chromatography
WHO
World Health Organization
XDR-TB
Extremely Drug Resistant-Tuberculosis

Footnotes

Supporting Information Available: Synthetic procedures and the characterization data for new benzimidazole intermediates as well as Mtb FtsZ protein preparation. This material is available free of charge via the Internet at http://pubs.acs.org.

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