Synthesis and Biological Evaluation of Novel ...

Subscriber access provided by The Open University

Article

Synthesis and Biological Evaluation of Novel Bisbenzimidazoles as Escherichia coli Topoisomerase IA Inhibitors and Potential Antibacterial Agents Hemlata Nimesh, Souvik Sur, Devapriya Sinha, Pooja Yadav, Prachi Anand, Priyanka Bajaj, Jugsharan Singh Virdi, and Vibha Tandon J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5003028 • Publication Date (Web): 23 May 2014 Downloaded from http://pubs.acs.org on May 30, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Journal of Medicinal Chemistry

Synthesis and Biological Evaluation of Novel Bisbenzimidazoles

as

Topoisomerase

Inhibitors

IA

Escherichia and

coli Potential

Antibacterial Agents Hemlata Nimesh, †χ Souvik Sur,†χ Devapriya Sinha,†χ Pooja Yadav,† Prachi Anand,†† Priyanka Bajaj,§ Jugsharan S. Virdi,§ Vibha Tandon†*,ҡ †

Department of Chemistry, University of Delhi, Delhi-110 007, India.††Department of Chemistry

& Biochemistry, CUNY-Hunter College, New York, NY 10065 USA. §

Department of Microbiology, University of Delhi, Delhi-110 021, India.

ҡ

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi – 110 067.

χ

First, second and third authors have equal contributions.

1 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2

KEYWORDS Bisbenzimidazoles, Piperazine, E. coli topoisomerase, Docking, Antibacterial agent ABSTRACT Novel bisbenzimidazole inhibitors of bacterial type IA topoisomerase are of interest for the development of new antibacterial agents that are impacted by target-mediated cross resistance with fluoroquinolones. The present study demonstrates the successful synthesis and evaluation of bisbenzimidazole analogs as E. coli topoisomerase IA inhibitors. 5-(4-Propylpiperazin-1-yl)-2[2’-(4-ethoxyphenyl)-5’-benzimidazolyl]benzimidazole (12b) showed significant relaxation inhibition activity against EcTopo 1A (IC50 = 2 ± 0.005 µM) and a tendency to chelate metal ion. Interestingly these compounds did not show significant inhibition of E. coli DNA gyrase and hTop 1 even upto 100 µM. The compound 12b has shown lowest MIC against E. coli strains among 24 compounds evaluated. The binding affinity constant and binding free energy of 12b with EcTopo 1A was observed 6.8 x 106 M-1 and -10.84 Kcal mol- 1from isothermal titration calorimetry (ITC) respectively. In vivo mouse systemic infection and neutropenic thigh model experimental results confirmed the therapeutic efficacy of 12b, suggesting further development of this class of compounds as antibacterial agents.

INTRODUCTION To confront global need of antibacterial resistance; identification of new antibacterial compounds targeting existing targets through new mode of action has emerged as an important strategy in the development of new therapies.1,2 DNA topoisomerases are ubiquitous enzymes which utilize an active site tyrosine residue for nucleophilic attack on DNA phosphodiester


Page 2 of 73

Page 3 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

linkage resulting in formation of covalent protein-DNA intermediate.3-5 Topoisomerase poison inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death.6 Bacterial topoisomerase I is a target for discovery of new antimicrobials as it plays an important role in transcription of stress gene during bacterial stress response. Bacterial topoisomerases are potent drug targets due to their essential bactericidal effect through their inhibition and the presence of multiple sites for drug interaction which are exploited by various antibacterial agents.7,8 Topoisomerase I targeting poisons may be particularly effective when the bacterial pathogen is responding to host defense, or in the presence of other antibiotics that induce the bacterial stress response.9 Every bacterium has at least one DNA topoisomerase IA that could potentially be targeted by a new class of topoisomerase poisons that has the capability to initiate the cell killing process by either stabilizing or increasing the accumulation of the covalent complex formed between the enzyme and cleaved DNA.10-12 Recently anziac acid, an aqueous Lichen Depside obtained from hypotrachyma species was identified as a potent Bacillus subtillis and E. coli topoisomerase I (EcTopo 1A) inhibitor.13 Tse-Dinh et al. screened 49268 small molecules, out of these, Stephenanthrene, Indenophalazinone and Indolopteridine derivatives showed considerable increase in DNA cleavage product formed by both Escherichia coli and Yersinia pestis topoisomerase IA.14 Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in the antibacterial drug discovery in order to address the increasing multidrug resistance. Novel bacterial topoisomerase inhibitors (NBTIs), recently developed by Glaxo Smith Kline (GSK) and Aventis independently are devoid of cross resistance with


4


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fluoroquinolones as they have different mechanism of inhibition. But the major lead compound of Aventis

program

was

(3R,4R)-4-[(S)-3-(3-ﬂuoro-6-methoxy-quinolin-4-yl)-3-hydroxy-

propyl]-1-[2-(thiophen-2-ylsulfanyl)-ethyl]-piperidine-3-carboxylic acid (NXL-101)15 having quinoline nucleus had to be discontinued due to QT interval prolongation.16-17 QT prolonging drugs, can cause cardiac arrhythmias which is a major risk factor in public health issue and thus this drug is not approved by FDA. On the basis of crystallographic studies of novel NBTIs, researchers could rationalize the reason behind the lack of cross-resistance between NBTIs and fluroquinolones and paved the way for the developement of novel non fluoroquinolone inhibitors.18 Fluoroquinolones are proposed to inhibit type II topoisomerase by magnesium ion chelation forming a water metal ion coordination that facilitate quinolone interactions with the conserved serine and acidic residues of catalytic site of enzyme. Ser 83 mutation commonly found in bacterial gyrase, results in partial disruption of the bridge and thus causes resistance against these drugs.19 In continuation of above efforts novel class of substituted 4,5’-bithiazoles and 5-(2hydroxybenzylidene) rhodanine were observed as potent DNA gyrase inhibitor.20,21 Recently, eighteen alkaloids were semisynthesized and screened against topoisomerase IA and cell growth of S. pneumoniae antibiotics targeting topoisomerase 1A enzyme of S. pneumoniae. Two phenanthrenes inhibited Topo 1A enzyme activity and bacterial growth of S. pneumoniae and E.coli efficiently. The above study included molecular docking on topoisomerase 1A enzyme of S. pneumoniae taking homologous structure EcTopo 1A as model in consideration.22 Our

group

has

reported

a

bisbenzimidazole,

2-(3,4-dimethoxyphenyl)-5-[5-(4

methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole (DMA), a synthetic analogue of Hoechst 33342, less cytotoxic which does not affect cell viability in mammalian cells up to a


Page 4 of 73

Page 5 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5

concentration of 100 µM, inhibits growth of E. coli cultures even at a concentration of 2 µM when grown in co-culture with human cells.23,24 We have reported that DMA efficiently inhibited EcTopo 1A by enhancing DNA cleavage product and identified DMA as poison inhibitor of type IA topoisomerase. It was found that DMA has no significant inhibitory effect on hTop 1, Human Topoisomerase IIα (HuTopoIIα) and E. coli gyrase enzyme.25 The parent analogue Hoechst 33258 & 33342 (Figure 1) were found to be mutagenic, clastogenic and cytotoxic to human cells.26,27 but DMA appeared to be a safer and effective molecule.23 These findings proved that DMA act as potential antibacterial agent, selectively targeting EcTopo 1A over hTop 1. Hoechst 33258 & 33342 are sequence specific DNA binding agents that specifically bind AT rich sequences of DNA.28 The finding that bisbenzimidazole can be developed as antibiotics targeting topoisomerase enzyme driven us to synthesize 24 novel bisbenzimidazoles and explored their antibacterial action in vitro and in vivo. These bisbenzimidazoles can be divided into three sub classes (synthetic analogues of Hoechst 33258, 33342 and DMA) with different electron withdrawing and donating substituents on the piperazine ring. We observed that novel compounds inhibit EcTopo 1A selectively over E.coli gyrase. These compounds showed good antibacterial activity against standard, MDR (Multi Drug Resistant) E. coli strains isolated from patients with UTI and different water sources. Hoechst 33258 and 33342 were reported as cytotoxic and mutagenic in nature, limiting its use as drug, inhibition of human topoisomerase enzymes were given as one possible cause of its cytotoxicity. Although, EcTopo 1A and hTop 1 have different structure and mechanism of action, hence it might not be correct to compare activity of single molecule against 1A and 1B class of enzyme, but we performed the enzyme inhibition study on hTop 1 with six newly synthesized molecules to compare them with parent



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6

analogues, Hoechst 33342 and 33258, known hTop1 inhibitors. It was imperative to confirm safety index of 11b, 11g-h, 12a-c in humans to develop them as future antibiotics. We performed ITC experiments to understand the binding affinity of these compounds with EcTopo 1A which is further supported by docking studies. A detailed study including in vivo results confirmed that compound 12b is a promising antibacterial compound, especialy to combat the infections caused by drug resitant pathogens. Scientific approach is summarized in a flow chart (Figure 2).

RESULTS AND DISCUSSION Chemistry Synthetic approaches for the preparation of target bisbenzimidazole compounds and their intermediates are depicted in Schemes 1-3. Total 24 bisbenzimidazoles, 10a-j, 11a-i and 12a-e were synthesized by reacting two intermediates; o-phenylenediamine derivatives 5a-j and the respective aldehydes 9a-c (Scheme 3) in the presence of sodium metabisulfite (Na2S2O5) as oxidizing agent in 40-60% yield using reported procedure.29 The Na2S2O5 in water forms sodium bisulfite (NaHSO3), which forms an adduct with aldehyde, and reacts with diamine to form the product. The diamine intermediates 5a-j were prepared by nucleophilic substitution reaction of 5-chloro-2-nitroacetanilide 1 with the N-substituted piperazines 2a-j to provide 2-nitroacetanilide derivatives 3a-j in > 90% yields. Deacetylation of 3a-j was done using 10% sulphuric acid to get 4a-j in excellent yields (Scheme 1). All the deacetylated intermediates were then reduced by catalytic hydrogenation using 10% Pd/C at ambient temperature to provide diamine derevatives 5a-k in good yield (Scheme 1). 2-Aryl-5-cyano-1H-benzimidazole 8a-c were synthesized in 7590% yield by reacting 4-cyano-1,2-phenylenediamine 6 with the appropriate substituted benzaldehydes 7a-c using reported (Scheme 2).30,31 Catalytic reduction of 8a-c using Ni-Al alloy


Page 6 of 73

Page 7 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7

in the presence of 75% formic acid in water, provided intermediates 9a-c in good yield (Scheme 2).32,33 All final compounds were then converted to respective hydrochloride salts to make them water soluble by passing the dried HCl gas in the methanolic solution of respective bisbenzimidazoles. The synthetic analogues of bisbenzimidazoles 11a-i and 12a-e have shown good antibacterial potential in comparison to 10a-j suggesting that substitution at phenyl ring play an important role in antibacterial activity of bisbenzimidazoles. The analogues having strong electron donating methoxy and ethoxy groups on phenyl ring showed higher antibacterial potency. This might be either due to the diferential uptake of compounds or due to electronic effects of substituents. The compounds 10a-j, with hydroxyl group at para position have less +R effect as compared to 11a-i which have two methoxy group, whereas compounds 12a-e having ethoxy group at para position will show higher +R effect, increasing electron density at imidazolic nitrogen, with increased hydrogen bonding interaction with acidic groups of amino acids lying in the vicinity of compounds, preferentially with Arg, His, Lys, Asp and Glu amino acids. Although compound 11a-i have 3,4-disubstituted methoxy group, but methoxy group at 3-position have more stronger -I effect (electron withdrawing effect) as compared to total +R effect of methoxy group. It has been reported earlier that piperazine ring of Hoechst 33342 is important for its interaction with DNA.34-37 But, we observed through docking of bisbenzimidazoles in EcTopo 1A-ds DNA complex, that bisbenzimidazoles do not bind in the minor groove of DNA in binary complex, rather these compounds dock in the vicinity of active site of EcTopo 1A and positively charged nitrogen of piperazine ring interact with acidic groups of amino acids of topoisomerase IA. Our results suggested that piperazine with longer alkyl chain that is propyl group increased electron density at N-6 of imidazole ring, increasing the chances of protonation of nitrogen.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8

Protonated nitrogens would interact strongly with negatively charged amino acids in topoisomerase IA as compared to phenyl, pyrimidyl or pyridyl substituted piperazine containing bisbenzimidazole. Phenyl substitution might reduce the protonation of N-6, as the lone pair of nitrogen will be shared by phenyl ring as well as piperazine, hence, will not be available for interaction with amino acids. That might be a plausible reason behind the increased binding energy of 12b with topoisomerase IA. Biological Evaluation Effect of Bisbenzimidazoles on Susceptibility of Standard and Clinical E. coli Strains. A series of novel bisbenzimidazole compounds were screened for antibacterial activity against clinical and reference strains of E. coli (DH5α and ATCC 25922). The results showed comparatively lower MIC and MBC values for DMA and Hoechst 33342 analogues than 33258 analogues (Table 1). Out of 24 molecules, several compounds showed MIC90 values in the range of 0.1-8µg/mL with the best results observed in case of 11g, 12a, and 12b ranging from 0.14µg/mL against two standard E. coli strains (Table 1). The position and identity of terminal substituent at phenyl ring appeared to be crucial for the activity of the compounds. Presence of terminal hydroxyl group at phenyl ring leading to the abrogation of activity in case of Hoecsht 33258 analogues.

Based on the results related to Ec Topo1A inhibition and bacterial

susceptibility against standard E. coli strains, six compounds 11b, 11g-h, 12a-c were selected for bacterial susceptibility testing against eleven clinical E. coli strains 72, 118, 151, 360, 451, 59, 81, 85, 132, 401 and 555, obtained from UTI patients from Institute of Pathology, Safdarjung Hospital New Delhi (Table 2) and 10 water borne E. coli strains IS54, WB31, KP21, NG3, IST, IP18, IP9, KK45, KK16 and MKNJ (Table 3). MIC of these clinical and water borne strains were evaluated against standard antibiotics and observed that these strains were resistant to few


Page 8 of 73

Page 9 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9

standard antibiotics (Table S2 and S3). But it was interesting to note that all strains were susceptible to the novel bisbenzimidazoles. Above mentioned six compounds (11b, 11g-h, 12ac) showed good inhibitory activity against all the strains tested with comparatively lower MIC50, MIC90 and MBC values. Among these six compounds, 12b was found to be the best with significantly low MIC values and MBC in the range of 0.1-8 µg/mL when screened against standard, clinical and water-borne E. coli strains. Inhibition of DNA Relaxation Activity of EcTopo 1A by bisbenzimidazoles. Bisbenzimidazole derivatives were proven to inhibit EcTopo 1A significantly.25 Thus in search of potent EcTopo 1A inhibitors, a conventional DNA relaxation assay was done with newly synthesized bisbenzimidazoles. In these experiments, supercoiled plasmid DNA was incubated with Ec Topo IA at increasing concentrations of the compounds (1, 5, 10, 25, 50, 75 and 100 µM) and DNA relaxation products were then resolved by gel electrophoresis on 1% agarose gel. Out of the total 24 bisbenzimidazoles, compounds 10a, 10c, 10j, 11b-e, 11g-h, 12a-c showed lower IC50 values in the range of 1-10 µM concentration (Table 1). Considering the results of bacterial susceptibility test and EcTopo 1A relaxation inhibition, finally six compounds were listed to be the most potent (Figure 3A & B). Compound 12b was observed to be the most poitent and inhibited relaxation activity of EcTopo 1A at 2µM concentration. Metal ion particularly magnesium play an indispensable role in promoting DNA cleavage and rejoining activity of topoisomerase IA by assistance in the appropriate structural changes to assemble and disassemble the active site. These species are consistently bound to a specific region in the active site forming a very organized ionic network to the transphosphorylation centre connecting the anionic centres in the toprim and the anionic centre in the nucleic acids.38 Yuk Ching et. al. have shown Mg2+ dependence on DNA cleavage patterns of Topoisomerase


10


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 73

1A.39 There are literature reports showing metal chelation activity of bisbenzimidazoles.40 In view of above it becomes obligatory to perform metal dependent relaxation assay with EcTopo 1A using 12b (Figure 3C). The probable reason behind this may be that higher concentration of Mg2+ destabilize the active structure of EcTopo 1A leading to its inactive conformation with progressive changes in the plasmid conformation (winding) due to efficient binding of nucleic acid and metal ion.

It was interesting to find out that beyond 10 mM concentration of

magnesium the enzymatic activity of topoisomerase 1A was regained. In addition, when concentration of 12b was increased, it could inhibit relaxation activity of the enzyme even at 25mM concentration of Mg2+ (Figure S1). The above results suggest, that 12b is acting as metal ion chelators suggesting metal ion chelation might be a probable reason causing inhibition of enzymatic activity. Effect of Bisbenzimidazoles on the Supercoiling Activity of E. coli DNA Gyrase. To check whether these compounds targets type II topoisomerase enzyme or these compounds have any effect on DNA supercoiling, E. coli gyrase supercoiling inhibition assay was performed. As per our observations, these six compounds 11b, 11g-h, 12a-c did not show significant inhibition of E. coli gyrase activity even up to 100 µM concentration, suggesting that these bisbenzimidazole derivatives do not inhibit DNA gyrase (Figure 3D). Effect of Bisbenzimidazoles on the hTop 1 Activity.

Futher, these molecules were

screened against type IB topoisomerase to check whether they have any effect on hTop 1 activity as parent analogues Hoechst 33342 and 33258. DNA relaxation inhibition assays were performed. As per our result only compounds 11b, 11h and 12a show few states of supercoiling indicating a very minute inhibition at 100 µM (Figure 3E), but the other three compounds 11g, 12b and 12c did not show any significant inhibition even at 100 µM concentration .


Page 11 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11


DNA Binding Study using Fluorescence Spectrscopy. We performed fluorescence titrations of 12b with poly(AT) sequence and observed high binding constant (7 x 107M-1), suggesting that 12b is a DNA binding molecule and have higher affinity with AT rich DNA (Figure S7). Isothermal

Titration

Calorimetry.

Microcalorimetry

provides

an

accurate

thermodynamic evaluation of the interaction in terms of enthalpy change, entropy change and Gibbs Free energy change along with the stoichiometry and binding affinity from a titration. ITC is an effective tool to thermodynamically characterize the binding of small molecules to macromolecules.42,43 Figure 4A depicts the representative ITC thermogram for the complexation of the 12b with the EcTopo 1A. In the upper panel, the raw data for the sequential injection of the EcTopo 1A into the 12b are depicted. Appropriate heat corrections derived from the injection of identical amounts of the EcTopo 1A into the buffer alone were effected to the raw data. In the lower panel the corrected integrated heat is presented. 12b bound to EcTopo 1A exhibited single binding event (Figure 4B). The binding was characterized to be exothermic. The single binding event guided fitting of the data to a model of single set of identical sites. All the thermodynamic parameters deduced from the single site fitting protocol for the complexation of the 12b to the EcTopo 1A are collated in Table 4. The binding affinity value of 12b is 6.8 x 106 M-1 for EcTopo 1A and binding free energy (∆G) was -10.84 kcal mol-1 of 12b, when it was titrated with EcTopo 1A (Table 4). The reaction was found to be exothermic with negative enthalpy of reaction that concludes the binding is entropically unfavorable, but strong favorable enthalpic contributions making binding free energy (∆G) favorable. The hydrogen and van der Waals bonding networks between 12b and EcTopo 1A might be responsible for the favorable enthalpy. A large enthalpy change (∆H) of -22.78 kcal mol-1 and less entropy change (T∆S) of -11.94 kcal



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

12

Page 12 of 73

mol-1 was obtained from experiment of 12b with EcTopo 1A with a moderate binding free energy for effective interaction. We might conclude that 12b showed significant binding affinity with EcTopo 1A. Docking and Molecular modeling studies with EcTopo 1A-dsDNA complex. Six different docking experiments were carried out in order to understand mode of interaction of bisbenzimidazoles with EcTopo 1A-dsDNA complex. In all experiments, docking has been carried out, considering the structure of free protein in an open conformation in absence as well as presence of DNA as a receptor, as these six compounds showed higher inhibition upon its preincubation with the enzyme only (without DNA), that is taken as an index of a direct bisbenzimidaozle-enzyme interaction. The three-dimensional structure of the open conformation has been obtained from a PDB 1ECL. The binding free energies of the 300 docked structures were in the range between -6.79 and -8.57 Kcal mol-1. The docking results revealed a structure of ternary complex, in which compound is docked near the active site of enzyme, in particular at the bottom of the catalytic residues Glu9, Pro11, His33, Asp113, Glu115, Arg169, Arg321, Ala498, Ser499, Ser502 (Figure 5 and S2-S3). All six ligands used in the study bind identically at EcTopo 1A active site (Figure S4). Earlier it was reported that acidic triad Asp111, Asp113 and Glu115 are conserved through out type IA DNA topoisomerases, are located near the active site Tyr 319. It was also confirmed that acidic triad participate in metal coordination, which is important for religation of DNA.43 Recently, a crystal structure of covalent complex of a mutant EcTopo 1A and DNA was elucidated, a clear conformational change was observed in the structure of covalent intermediate with DNA form the previously determined structures of EcTopo 1A.44 The Glu9, Asp113, Glu115 are responsible for DNA breakage and rejoining. Glu9, Asp111, Asp113 and Tyr319


Page 13 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13

make a tetrad in the vicinity of DNA double helix.45 We may hypothesize on the basis of docked poses that in covalent complex Tyr319 is situated quite close to helix (1.76Å), increasing the feasibility to attack through its lone pair of electrons on oxygen to phosphorous of DNA strands, causing strand cleavage. The electron flow was initiated from Glu9, then transferred to the neighbouring Asp111 to Asp113 and finally to Tyr319 (Figure S5). It was also proposed that cleaved 5’-phosphate at the cleavage site is buried in a deep groove, whereas sugar and phosphates with the help of Mg2+ are held through a network of hydrogen bonds and salt bridges in the cavity. Earlier it was proved that religation requires conformational change in EcTopo 1A and Mg2+ ions near the religation site.46 We have observed earlier that DMA causes increase in cleavage product and acts as topoisomerase IA poison. Once again on the basis of docking results we can hypothesize that bisbenzimidazoles inhibit religation-step in two ways, 1) benzimidazoles might not be allowing the conformational change in EcTopo 1A-ds DNA complex 2)benzimidazole being a positively charged molecule at physiological pH increase the positive electrostatic potential significantly and decrease the negative electrostatic potential47 around the side chains of the acidic triad of Asp111, Asp113 and Glu115 responsible for binding of Mg2+ ion necessary for religation (Figure S6). The potential is positive throughout, with small negative regions at the nitrogens and the ethoxy oxygen (magenta lines). The electrostatic potential is a measure of the stabilization of a positive charge at the given point. Conversely, negatively charged species would be stabilized everywhere else on the cationic ligands, implying that the positive charge is not localized at the piperazine nitrogen. The contour of the electrostatic lines corresponds roughly to the shape of the molecule. Hence, the molecule is narrower at the ethoxy end and broader at the piperazine end. Out of these compounds, 12b fitting perfectly in the active site of EcTopo 1A-dsDNA complex. Hence, we can hypothesize



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

14

Page 14 of 73

that 12b is a potent EcTopo 1A inhibitor trapping the covalent complex and not allowing religation, resultant to increased bacterial cell killing. Six compounds 11b, 11g-h, 12a-c used for docking study bind in the similar pocket of EcTopo 1A48, i.e.; at the junction of domain I, III and IV (Figure 5 and S3). We have observed four other interacting sites, Ser495, Ser499, Thr496 and Ser502 in docking study, which were not proposed earlier. The roles of these amino acids are not clear yet. The hydrophilic residues i.e.; Glu9, Asp113, Glu115 Arg321, Ala498 and Ser499 have major interactions with benzimidazoles. Arg321 and Tyr497 showed interactions with N-alkyl piperazine portion. The core structure of type IA topoisomerases, typically of 67 kDa conserved portion, has characteristic toroidal fold formed by four domains. The toroidal fold was first observed in EcTopo 1A. In the case of EcTopo 1A, the C-terminal domain is essential for activity. Although bisbenzimidazole did not interact directly with Tyr319 but it has shown substantial interaction with neighboring amino acids; Glu9, Asp113, Glu115 and Arg321 present at active site of major interactions with compounds. These amino acids form an extensive network of hydrogen bonding, and may water molecules were also attached to the network. Hence, we can say that, bisbenzimidazoles bind to the active site of EcTopo 1A. Based on ranking of the docked complexes, the docking scores were found -7.43, -6.99, -6.79, -7.31,-8.57 and -7.92 for 11b, 11g, 11h, 12a, 12b and 12c respectively (Table S4). Out of above six compounds, 12b the most potent one, (-8.57, docking score) binds strongly with EcTopo 1A. Unexpectedly, these compounds did not show much interaction with DNA and do not indicate interference with DNA binding site on enzyme. The docked structures of 11b, 11g-h, 12a-c were superimposed and found (Figure S4) that all six ligands bind in the same pocket in Ec Topo IA-dsDNA, suggesting uniformity of docking protocol. The pharmacophore was created using Ligand Scout application


Page 15 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15

framework and concluded the detection of directed hydrogen bonding interactions, lipophilic regions, three hydrophobic spheres, charge interactions and steric exclusions . Exclusion volume spheres were also included to account for the protein environment.49,50 Mouse Systemic Infection Model. Systemic infection model of Balb/c mice was developed by injecting E. coli ATCC 25922 (0.5 X 108 CFU in 0.1ml saline) through intraperitoneal injection. Figure 6A shows the survival of infectious mice when treated with different doses (3, 5 & 7 mg/kg body weight) of 12b dissolved in sterile water as vehicle at a volume of 0.1 ml given single bolus intravenous injection in the tail vein. Compound 12b demonstrated dose dependent effects on the survival against E. coli ATCC 25922 bearing mice with 75% survival achieved given the dose 7mg/kg body weight. Mice injected with vehicle alone showed 100% mortality in this model within 4 days post-infection. Treatment of mice with compound 12b led to the survival of the mice infected with E. coli ATCC 25922 with an ED50 of 5mg/kg body weight. Neutropenic Thigh Model. The excellent in vitro activity of compound 12b extends to promising in vivo efficacy. Our result demonstrated compound 12b has dose dependent efficacy in neutropenic thigh model against E. coli ATCC 25922 strain (Figure 6B). A one log bacterial burden reduction was observed at 3 mg/kg bw/day and three log differences were observed at 5 mg/kg body weight/day. Dose of 5mg/kg body weight/day was sufficient to reduce the bacterial load to below the static level (Table 5). CONCLUSIONS In conclusion, we have described synthesis, identification and development of bisbenzimidazoles, a novel class of efficient EcTopo 1A inhibitors, as potent antibacterial agents



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16

Page 16 of 73

showing low MIC and MBC against E. coli strains. Out of 24, six compounds 11b, 11g-h, 12a-c showed significant inhibition of EcTopo 1A with low MIC50, MIC90 and MBC values against various resistant E. coli strains. We confirmed by ITC that the higher affinity of 12b is one of the parameters determining its higher potency to inhibit EcTopo 1A enzyme activity. The docking study of 12b in EcTopo 1A-dsDNA complex revealed that bisbenzimidazoles used in this study are positively charged molecules which significantly increased positive electrostatic potential at the active site in EcTopo 1A enzyme affecting the Mg2+ affinity of the ternary complex and inhibited DNA religation, this fact was further proven by Mg2+chelating ability of 12b. These compounds have dual edge over other EcTopo 1A inhibitors 1) Topo IA as target make it a lead compound of choice against resistant strains to type II Topoisomerase inhibitors, 2) poor activity of these compounds against hTop 1 make it safer molecule for human use. The compound 12b not only proved most potent antibacterial agent in in vitro assays but also displayed promising in vivo efficacy in animal infection models. Our results support further evaluation of safety and efficacy of these compounds for clinical studies. EXPERIMENTAL SECTION Chemicals. All melting points were measured in BUCHI B540 apparatus and are uncorrected. The 1H (300/400/500 MHz) and 13C NMR (300/400 MHz) spectra were recorded in CDCl3, MeOD and DMSO-d6 as the solvent on an ECX-400P, Jeol/Bruker NMR spectrometers using CDCl3, MeOD and DMSO-d6 as an internal standard. Chemical shifts are reported in ppm by assigning the CDCl3, DMSO-d6, and MeOD resonance in the 1H NMR spectrum as 7.26, 2.50 and 3.31 ppm and in the

13

C NMR spectrum as 77.23, 39.50 and 49.00 ppm. All coupling

constant (J) are reported in Hertz (Hz). HRMS spectra were measured on Agilent 6520


Page 17 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17

Accurate Mass Q-TOF LC/MS mass spectrometer and IR spectra were taken on a Perkin-Elmer, FT-IR system, spectrum BX. The progress of all reactions was monitored by thin layer chromatography (TLC), which was performed on 20 × 20 cm aluminum sheets precoated with silica gel 60 (F-254, Merck) to a thickness of 0.25 mm. The developed TLC plates were viewed under ultraviolet light (254−366nm) and treated with iodine vapor. Silica gel column chromatography was performed for the purification of intermediates and final compounds. Analytical HPLC was used for compound purity determinations using Dionex Ultimate 3000 controlled using YMC ODS-AQ analytical column (4.6 × 250 mm) and a Dionex ultimate 3000 photo diode array detector at 340 nm wavelength. The solvent system used for HPLC analysis was Methanol: Water. The gradient HPLC mode was used, and the flow rate was 1.0 mL/min. The purity of compounds was found to be greater than 95%. Elemental analyses of tested compounds were found within ±0.4% of the theoretical values. Commercially available reagents were used without further purification, unless otherwise stated. The anhydrous organic solvents (e.g. Et2O, EtOAc, CHCl3, MeOH, EtOH, CH3CN, DMF, hexane, toluene, etc.) were purchased from commercial sources and used as stated. General Procedure for the Synthesis of 2-Nitroacetanilide Derivatives (3a-j): Synthesis of 2-nitroacetanilide derivatives was done using the reported procedure.24 To a stirred solution of 5-chloro-2-nitroacetanilide (4.65 mmol) in DMSO (10ml), respective N-substituted piperazine (1.5 equiv) and triethylamine (1.0 equiv) were added. The resulting reaction mixture was heated at 120ºC for 2-4 h. The reaction was followed by TLC till completion of reaction; the reaction mixture was poured onto the crushed ice to yield yellow solid. The resulting solid was then filtered, washed with water and dried in vacuum. The crude mixture was purified by column chromatography


18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 73

over silica gel (60-120 mesh size) using ethylacetate: petroleum ether as mobile phase in quantitative yield of the title compound. 5-(4-Ethyl-1-piperazinyl)-2-nitroacetanilide (3a). Yellow solid; (95% yield, 1.29g); mp 109−110 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 1.13 (t, J = 7.2 Hz, 3H), 2.28 (s, 3H), 2.46 (q, J = 7.5 Hz, 2H), 2.51−2.57 (m, 4H), 3.49−3.53 (m, 4H), 6.52 (dd, J = 2.7, 9.6 Hz, 1H), 8.14 (d, J = 9.6 Hz, 1H), 8.32 (d, J = 3.0 Hz, 1H), 11.01 (bs, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 11.9, 26.0, 41.0, 46.6, 52.2, 102.61, 107.7, 126.0, 128.5, 137.9, 155.5, 169.7. FTIR (KBr,cm–1): 3317.49, 2971.10, 2818.73, 1700.08, 1612.64, 1576.40, 1540.74, 1492.36, 1451.13, 1384.29, 1312.15, 1242.00, 1187.89, 1157.70, 1127.48, 1103.13, 1022.22, 978.24, 856.82, 806.79, 750.43, 717.53, 687.76. HRMS (ESI): m/z calcd for C14H20N4O3 [M + H]+ 293.1613 obsd 293.1618. 5-(4-Allyl-1-piperazinyl)-2-nitroacetanilide (3b). Yellow solid; (98% yield, 1.38g); mp 143.5−144.5 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 2.14 (s, 3H), 2.45−2.48 (m, 4H), 2.98 (d, J = 5.88 Hz, 2H), 3.38−3.42 (m, 4H), 5.14−5.22 (m, 2H), 5.78−5.88 (m, 1H), 6.79 (dd, J = 2.2, 9.52 Hz, 1H), 7.67 (s, 1H), 7.97 (d, J = 9.52 Hz, 1H), 10.43 (s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 24.7, 46.3, 52.0, 60.7, 104.7, 108.9, 117.9, 127.8, 128.1, 135.1, 136.0, 154.7, 169.0. FTIR (KBr,cm–1): 3448.34, 3313.86, 2854.91, 2797.80, 1695.96, 1615.78, 1578.18, 1536.15, 1458.11, 1329.67, 1237.63, 1136.63, 941.21, 687.75, 442.09. HRMS (ESI): m/z calcd for C14H18N4O3 [M + H]+ 305.1613 obsd 305.1615. 5-(4-(2-N-Trifluoroacetylaminoethyl)-1-piperazinyl)-2-nitroacetanilide

(3c).

Yellow

solid; (93% yield, 1.74%); mp 181.9−182.9 ºC.1H NMR (400 MHz, CDCl3) δ ppm 2.27 (s, 3H), 2.55−2.63 (m, 6H), 3.47−3.50 (m, 6H), 6.52 (dd, J = 2.68, 9.4Hz, 1H), 6.93 (bs, 1H), 8.14 (d, J = 10.72 Hz, 1H), 8.33 (d, J = 2.68 Hz, 1H), 10.97 (bs, 1H). 13C NMR (100 MHz, CDCl3) δ ppm


Page 19 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19

25.9, 36.0, 46.5, 52.1, 55.4, 102.8, 107.7, 126.1, 128.4, 137.8, 155.3, 156.5, 156.9, 157.2, 157.6, 169.6. FTIR (KBr,cm–1):

3297.65, 3117.79, 2926.86, 2809.71, 2510.86, 2374.97, 2345.70,

1702.03, 1611.18, 1578.40, 1542.34, 1467.94, 1388.72, 1318.87, 1295.81, 1247.99, 1178.11, 1100.84, 976.01, 876.25, 725.11. HRMS (ESI): m/z calcd for C16H20F3N5O4 [M + H]+ 404.1545 obsd 404.1553. 5-[4-(2-(N, N-Dimethylaminoethyl)-1-piperazinyl]-2-nitroacetanilide (3d). Yellow solid; (80% yield, 1.24g); mp 167.2−168.7 ºC.1H NMR (400 MHz, CDCl3) δ ppm 1.73 (s, 6H), 2.27 (s, 4H), 2.61 (s, 3H), 2.98−3.01 (m, 4H), 3.44−3.47 (m, 4H), 6.51 (m, 1H), 8.14 (d, J = 10.08 Hz, 1H), 8.32 (d, J = 2.68 Hz, 1H), 11.01 (bs, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 26.4, 41.3, 46.0, 48.1, 102.9, 108.0, 126.3, 128.9, 138.3, 156.2, 170.1. FTIR (KBr,cm–1): 3439.34, 3294.69, 3159.61, 2836.94, 1618.69, 1567.04, 1420.31, 1384.63, 1250.59, 1114.24, 1002.01, 935.15, 789.70, 805.56, 438.24. HRMS (ESI): m/z calcd for C16H25N5O3 [M + H]+ 336.2035 obsd 336.2032. 5-(4-Phenyl-1-piperazinyl)-2-nitroacetanilide (3e). Orangish yellow solid; (90% yield, 1.42g); mp 151.6−152.3ºC.1H NMR (500 MHz, CDCl3) δ ppm 2.28 (s, 3H), 3.33−3.34 (m, 4H), 3.62−3.66 (m, 4H), 6.56 (dd, J = 3.0, 10.0 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 8.16 (d, J = 10.0 Hz, 1H), 8.37 (d, J = 3.0 Hz, 1H), 11.01 (bs, 1H). 13

C NMR (100 MHz, CDCl3) δ ppm 26.0, 40.9, 46.6, 48.8, 102.8, 107.8, 116.2, 120.4, 126.2, 128.6,

129.3, 137.9, 150.6, 155.4, 169.7. FTIR (KBr,cm–1): 3307.09, 3137.25, 2826.19, 1694.49, 1615.83, 1578.58, 1543.59, 1495.36, 1449.07, 1314.14, 1273.31, 1220.42, 1186.21, 1083.64, 1029.40, 977.17, 925.14, 850.54, 801.50, 769.34, 750.14, 695.13, 652.62, 537.39. HRMS (ESI): m/z calcd for C18H20N4O3 [M + H]+ 341.1613 obsd 341.1606.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20

Page 20 of 73

5-(4-(2-Cyanophenyl)-1-piperazinyl)-2-nitroacetanilide (3f). Yellow solid; (94% yield, 1.59g); mp 175.9−176.6 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 2.36 (s, 1H), 3.31−3.34 (m, 4H), 3.66−3.69 (m, 4H), 6.55 (dd, J = 2.2, 9.52 Hz, 1H), 7.02 (d, J = 8.08 Hz, 1H), 7.06 (t, J =7.32 Hz, , 1H), 7.51 (t, J =7.36 Hz, 1H), 7.58 (d, J = 8.08 Hz, 1H), 8.14 (d, J = 9.52 Hz, 1H), 8.36 (d, J = 2.92 Hz, 1H), 10.95 (br s, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 25.9, 46.8, 50.9, 102.9, 106.2, 107.8, 118.06, 118.6, 122.5, 126.3, 128.4, 133.9, 134.3, 137.7, 154.8, 155.3, 169.6. FTIR (KBr,cm–1): 3331.48, 2855.23, 2217.76, 1697.97, 1615.07, 1595.95, 1576.18, 1543.13, 1489.72, 1445.77, 1384.01, 1368.53, 1313.80, 1245.55, 1217.10, 1184.25, 1145.97, 1034.51, 980.07, 928.33, 751.87. HRMS (ESI): m/z calcd for C19H19N5O3 [M + H]+ 366.1566 obsd 366.1556. 5-(4-(4-Fluorophenyl)-1-piperazinyl)-2-nitroacetanilide (3g). Yellow solid; (92% yield, 1.53g); mp 130.2−132.5 ºC. 1H NMR (500 MHz, CDCl3) δ ppm 2.29 (s, 3H), 3.22−3.24 (m, 4H), 3.63−3.65 (m, 4H), 6.56−6.58 (m, 1H), 6.89−6.92 (m, 2H), 6.98−7.02 (m, 2H), 8.17 (d, J = 9.5 Hz, 1H), 8.38 (d, J = 3.0 Hz, 1H), 11.01 (br s, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 25.9, 46.7, 49.8, 102.8, 107.8, 115.6, 115.8, 118.1, 118.2, 126.2, 128.5, 137.8, 147.2, 155.3, 156.3, 158.7, 169.6. FTIR (KBr,cm–1): 3308.42, 2925.51, 2852.50, 1701.49, 1615.19, 1579.55, 1543.74, 1510.30, 1454.43, 1376.22, 1309.63, 1276.54, 1228.64, 1182.62, 1088.11, 1035.07, 975.98, 923.05, 830.81, 751.85, 714.78, 579.02, 528.74. HRMS (ESI): m/z calcd for C18H19FN4O3 [M + H]+ 359.1519 obsd 359.1511. 5-(4-(4-Pyridyl)-1-piperazinyl)-2-nitroacetanilide (3h). Yellow solid; (80% yield, 1.27g); mp 179.2−180.9 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 2.28 (s, 3H), 3.56−3.59 (m, 4H), 3.68−3.71 (m, 4H), 6.51 (dd, J = 2.76, 9.64 Hz, 1H), 6.66 (dd, J = 1.36, 5.04 Hz, 2H), 8.18 (d, J = 9.64 Hz, 1H), 8.30 (d, J = 6.4 Hz, 1H), 8.33 (d, J = 2.76 Hz, 2H), 10.98 (br s, 1H). 13C NMR


Page 21 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

21


(100 MHz, CDCl3) δ ppm 26.0, 44.9, 45.7, 102.5, 107.3, 107.8, 126.5, 128.6, 137.8, 149.5, 154.2, 154.8, 169.7. FTIR (KBr,cm–1): 3325.90, 2924.84, 2851.54, 1742.65, 1700.42, 1612.90, 1578.34, 1539.44, 1460.45, 1316.47, 1279.83, 1221.32, 1186.01, 1086.14, 1029.76, 808.43, 748.59, 540.79. HRMS (ESI): m/z calcd for C17H19N5O3 [M + H]+ 342.1566 obsd 342.1568. 5-(4-(2-Pyrimidyl)-1-piperazinyl)-2-nitroacetanilide (3i). Yellow solid; (94% yield, 1.49g); mp 226.2−227.2 ºC. 1H NMR (300 MHz, CDCl3) δ ppm 2.29 (s, 3H), 3.60−3.63 (m, 4H), 3.97−4.01 (m, 4H), 6.53−6.58 (m, 2H), 8.17 (d, J = 9.6 Hz, 1H), 8.35 (m, 3H), 11.02 (br s, 1H). 13

C NMR (100 MHz, CDCl3) δ ppm 26.0, 42.9, 46.3, 102.6, 107.6, 110.5, 126.2, 128.6, 137.9,

155.4, 157.8, 161.3, 169.6. FTIR (KBr,cm–1): 3432.23, 3318.76, 3134.83, 2906.68, 2855.88, 1701.16, 1619.01, 1587.59, 1557.49, 1489.34, 1363.71, 1315.43, 1222.59, 1238.12, 1195.60, 982.00, 955.84, 797.96, 653.44. HRMS (ESI): m/z calcd for C16H18N6O3 [M + H]+ 343.1518 obsd 343.1519. 5-(4-(4-Nitrophenyl)-1-piperazinyl)-2-nitroacetanilide (3j). Yellow solid; (97% yield, 1.79g); mp 196.2−197.1 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 2.16 (s, 3H), 3.64−3.67 (m, 4H), 3.69−3.72 (m, 4H), 6.78 (dd, J = 2.2, 9.52 Hz, 1H), 6.98 (d, J = 9.52 Hz, 2H), 7.69 (d, J = 2.92 Hz, 1H), 8.02 (d, J = 9.52 Hz, 1H), 8.09 (d, J = 9.52 Hz, 2H) 10.49 (br s, 1H). 13C NMR (75 MHz, CDCl3) δ ppm 26.0, 45.7, 45.9, 102.4, 107.2, 112.1, 112.5, 126.1, 126.5, 128.7, 137.9, 138.8, 153.8, 154.7, 169.7. FTIR (KBr,cm–1): 3312.85, 2922.88, 2856.09, 1694.87, 1596.26, 1542.52, 1492.05, 1461.41, 1386.67, 1301.24, 1216.61, 1186.73, 1113.72, 1030.10, 994.55, 976.54, 921.92, 851.15, 817.51, 751.84, 691.51, 583.73, 519.96. HRMS (ESI): C18H19N5O5 [M + H]+ 386.1464 obsd 386.1482.


m/z

calcd for

22


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 73

General Procedure for the Synthesis of 2-Nitroaniline Derivatives (4a−j). Synthesis of 2-nitroaniline derivatives was done using the reported procedure. Acetylated compound 3a−j was taken in round bottom flask and 10% H2SO4 solution was added to it (10mL/g). The resulting reaction mixture was the heated at 80 ºC till the reaction completion. After reaction completion, the reaction mixture was brought to room temperature and the poured onto the crushed ice. This resulted in yellow colored solid deacetylated compound. The pH was made neutral for complete precipitation of the compound with 30% ammonia solution.The resulting solid was then filtered, washed with water and dried to yield the title compound in quantitative yield. 5-(4-Ethyl-1-piperazinyl)-2-nitroaniline (4a). Yellow solid; (98% yield, 0.83g); mp 122.9−123.6 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 1.12 (t, J = 7.04 Hz, 3H), 2.46 (q, J = 7.92 Hz, 2H), 2.54−2.56 (m, 4H), 3.36−3.39 (m, 4H), 5.94 (d, J = 2.68 Hz, 1H), 6.13 (br s, 2H), 6.28 (dd, J = 2.68, 9.4 Hz, 1H), 8.01 (d, J = 10.08 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ ppm 10.9, 45.6, 51.0, 51.2, 97.1, 104.3, 122.9, 126.6, 147.2, 154.3. FTIR (KBr,cm–1): 3531.66, 3404.48, 3289.61, 3173.73, 2968.03, 2832.84, 1615.72, 1569.54, 1502.51, 1477.37, 1413.75, 1386.39, 1347.71, 1324.13, 1233.43, 1108.89, 1072.60, 1018.81, 970.18, 872.59, 828.78, 751.15, 651.95, 460.54. HRMS (ESI): m/z calcd for C12H18N4O2 [M + H]+ 251.1508 obsd 251.1507. 5-(4-Allyl-1-piperazinyl)-2-nitroaniline (4b). Yellow solid; (97% yield, 0.83g); mp 83.6−85.4 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 2.43−2.45 (m, 4H), 2.97 (d, J = 6.58 Hz, 2H), 3.29−3.32 (m, 4H), 5.13−5.22 (m, 2H), 5.79−5.86 (m, 1H), 6.19 (d, J = 2.96 Hz, 1H), 6.38 (dd, J = 2.2, 9.52 Hz, 1H), 7.26 (s, 2H), 7.80 (d, J = 9.56 Hz, 1H).

13

C NMR (100 MHz,

DMSO−d6) δ ppm 46.2, 52.1, 60.7, 97.5, 105.4 117.9, 122.9, 127.2, 135.2, 148.4, 155.0. FTIR (KBr,cm–1): 3522.49, 3428.39, 3279.30, 3161.40, 2950.52, 2853.74, 2830.74, 1624.21, 1561.45, 1476.81, 1389.36, 1325.99, 1307.93, 1270.38, 1251.79, 1220.85, 1128.23, 999.99, 922.41,


Page 23 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23

811.87, 744.56, 644.54. HRMS (ESI): m/z calcd for C12H16N4O2 [M + H]+ 263.1508 obsd 263.1508. 5-(4-(2-N-Trifluoroacetylaminoethyl)-1-piperazinyl)-2-nitroaniline (4c). Yellow solid; (94% yield, 0.84g); mp 135.6−137.2 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 2.54−2.63 (m, 6H), 3.34−3.37 (m, 4H), 3.48 (q, J = 5.36 Hz, 2H), 5.95 (d, J = 2.68 Hz, 1H), 6.16 (br s, 2H), 6.26 (dd, J = 2.68, 10.08 Hz, 1H), 6.96 (br s, 1H), 8.00 (d, J = 9.44 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 36.1, 46.8, 52.2, 55.5, 98.5, 105.6, 124.9, 128.2, 147.0, 155.3, 156.6, 156.9, 157.3, 157.7. FTIR (KBr,cm–1): 3439.77, 3338.93, 2851.31, 1619.58, 1598.95, 1563.63, 1509.96, 1477.41, 1385.65, 1322.11, 1221.20, 1111.93, 991.67, 804.96, 742.79, 534.26. HRMS (ESI): m/z calcd for C14H18F3N5O3 [M + H]+ 362.1440 obsd 362.1435. 5-[4-(2-(N, N-Dimethylaminoethyl)-1-piperazinyl]-2-nitroaniline (4d). Yellow solid; (95% yield, 0.83g); mp 142.2−143.8 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 2.25 (s, 6H), 2.44−2.47 (m, 2H), 2.50−2.54 (m, 2H), 2.57−2.59 (m, 4H), 3.35−3.38 (m, 4H), 5.92 (d, J = 2.92 Hz, 1H), 6.14 (br s, 2H), 6.25−6.28 (m, 1H), 7.99 (d, J = 9.52 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 45.8, 46.6, 53.0, 56.4, 56.7, 98.1, 105.5, 124.5, 128.1, 147.1, 155.4. HRMS (ESI): m/z calcd for C14H23N5O2 [M + H]+ 294.1930 obsd 294.1910. 5-(4-Phenyl-1-piperazinyl)-2-nitroaniline (4e). Yellow solid; (97% yield, 0.84g); mp 203.1−204.2 ºC. 1H NMR (500 MHz, CDCl3) δ ppm 3.30−3.35 (m, 4H), 3.51−3.54 (m, 4H), 5.99 (s, 1H), 6.17 (bs, 2H), 6.31 (dd, J = 1.48, 9.52 Hz, 1H), 6.89−6.98 (m, 3H), 7.30 (t, J = 7.32 Hz, 2H), 8.04 (d, J = 9.56 Hz, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 46.1, 48.0, 97.5, 105.4, 115.7, 119.5, 123.0, 127.2, 129.1, 148.3, 150.4, 154.8. FTIR (KBr,cm–1): 3574.22, 3479.85, 3365.99, 2832.51, 1616.73, 1594.40, 1445.62, 1496.17, 1477.25, 1389.30, 1322.47, 1227.32,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

24

Page 24 of 73

1183.21, 1110.38, 1030.32, 927.30, 967.90, 894.02, 817.96, 761.80, 692.12, 662.82, 585.17, 446.54. HRMS (ESI): m/z calcd for C16H18N4O2 [M + H]+ 299.1508 obsd 299.1500. 5-(4-(2-Cyanophenyl)-1-piperazinyl)-2-nitroaniline (4f). Yellow solid; (96% yield, 0.84g); mp 175.7−177.7 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.26−3.28 (m, 4H), 3.49−3.51 (m, 4H), 6.29 (s, 1H), 6.44 (dd, J = 2.2, 9.52 Hz, 1H), 7.12 (t, J = 7.32 Hz, 1H), 7.18 (d, J = 8.04 Hz, 1H), 7.30 (s, 2H), 7.61 (t, J = 8.08 Hz, 1H), 7.72 (dd, J = 1.44, 8.04 Hz, 1H), 7.84 (d, J = 9.52 Hz, 1H).13C NMR (100 MHz, DMSO−d6): δ 46.4, 50.6, 97.9, 104.7, 105.4, 118.2, 119.1, 122.3, 123.2, 127.2, 134.3, 134.4, 148.3, 154.8, 154.9. FTIR (KBr, cm–1): 3467.70, 3348.83, 2832.04, 2217.05, 1618.07, 1478.22, 1320.28, 1223.38, 1167.10, 1111.43, 1028.78, 757.56. HRMS (ESI): m/z calcd for C17H17N5O2 [M + H]+ 324.1460 obsd 324.1450. 5-(4-(4-Fluorophenyl)-1-piperazinyl)-2-nitroaniline (4g). Yellow solid; (98% yield, 0.86g); mp 177.2−178.6 ºC. 1H NMR (400 MHz, CDCl3) δ ppm 3.19−3.21 (m, 4H), 3.49−3.51 (m, 4H), 6.0 (d, J = 2.12 Hz, 1H), 6.21 (bs, 2H), 6.28−6.31 (m, 1H), 6.87−6.91 (m, 2H), 6.96−7.00 (m, 2H), 8.01 (d, J = 9.84 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ ppm 46.9, 50.0, 98.5, 105.7, 115.6, 115.8, 118.2, 118.3, 124.9, 128.3, 147.0, 147.7, 155.3. FTIR (KBr,cm–1): 3469.47, 3421.23, 3353.55, 3308.68, 3180.05, 2925.07, 2850.22, 2374.76, 2345.71, 1618.27, 1509.04, 1500.43, 1476.48, 1319.55, 1225.83, 1113.25, 1090.45, 1031.47, 966.79, 926.74, 818.19, 715.86. HRMS (ESI): m/z calcd for C16H17FN4O2 [M + H]+ 317.1414 obsd 317.1404. 5-(4-(4-Pyridyl)-1-piperazinyl)-2-nitroaniline (4h). Yellow solid; (95% yield, 0.83g); mp 177−180.9 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.54−3.57 (m, 4H), 3.67−3.70 (m, 4H), 6.18 (d, J = 2.96 Hz, 1H), 6.39 (dd, J = 2.2, 9.52 Hz, 1H), 7.00 (d, J = 6.6 Hz, 2H), 7.30 (s, 2H), 7.85 (d, J = 10.24 Hz, 2H), 8.23 (s, 2H). 13C NMR (100 MHz, DMSO−d6) δ ppm 44.7, 44.8,


Page 25 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25

96.7, 104.9, 123.0, 127.5, 148.4, 154.3. FTIR (KBr,cm–1): 3445.37, 3335.57, 2918.76, 2851.54, 1619.58, 1598.80, 1563.63, 1511.08, 1476.81, 1402.08, 1322.95, 122.06, 1113.36, 1029.66, 993.11, 937.70, 806.92, 743.71, 657.11. HRMS (ESI): m/z calcd for C15H17N5O2 [M + H]+ 300.1460 obsd 300.1457. 5-(4-(2-Pyrimidyl)-1-piperazinyl)-2-nitroaniline (4i). Yellow solid; (96% yield, 0.84g); mp 168.5−166.8 ºC. 1H NMR (300 MHz, CDCl3) δ ppm 3.47−3.49 (m, 4H), 3.95−3.99 (m, 4H), 5.96 (d, J = 2.4 Hz, 1H), 6.17 (br s, 2H), 6.31 (dd, J = 2.4, 9.6 Hz, 1H), 6.57 (t, J = 4.8 Hz, 1H), 8.05 (d, J = 9.6 Hz, 1H), 8.35 (d, J = 4.8 Hz, 2H).13C NMR (100 MHz, CDCl3) δ ppm 25.6, 42.5, 45.8, 102.1, 107.3, 110.1, 128.1, 137.4, 155.0, 157.4, 169.2. FTIR (KBr,cm–1): 3482.69, 3435.65, 3368.12, 3277.00, 3154.41, 2926.50, 2856.44, 2371.55, 2345.92, 1617.98, 1590.61, 1508.06, 1499.16, 1476.18, 1448.23, 1387.47, 1365.51, 1320.29, 1230.43, 1096.81, 1037.76, 965.80, 868.14, 806.19, 790.68, 751.56, 656.89, 502.80. HRMS (ESI): m/z calcd for C14H16N6O2 [M + H]+ 301.1413 obsd 301.1401. 5-(4-(4-Nitrophenyl)-1-piperazinyl)-2-nitroaniline (4j). Yellow solid; (95% yield, 0.84g); mp 182.3−184.5 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.55−3.57 (m, 4H), 3.60−3.68 (m, 4H), 6.18 (d, J = 2.2 Hz, 1H), 6.38 (dd, J = 2.2, 9.52 Hz, 1H), 7.00 (d, J = 9.52 Hz, 2H), 7.29 (s, 2H), 7.84 (d, J = 9.52 Hz, 1H), 8.08 (d, J = 9.52 Hz, 2H). 13C NMR (100 MHz, DMSO−d6) δ ppm 45.3, 45.5, 96.9, 105.2, 112.3, 123.2, 126.1, 127.7, 137.0, 148.6, 154.4, 154.6. FTIR (KBr,cm–1): 3467.91, 3344.00, 2925.13, 2855.16, 1744.86, 1621.60, 1597.08, 1508.34, 1388.02, 1322.46, 1223.85, 1114.42, 1026.95, 965.92, 930.33, 826.79, 751.52, 693.59, 664.49, 496.69. HRMS (ESI): m/z calcd for C16H17N5O4 [M + H]+ 344.1359 obsd 344.1356.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

General

Procedure

for

the

Synthesis

of

26

Page 26 of 73

4-(4-Substitutedpiperazinyl)-1,2-

phenylenediamine Derivatives (5a−j). A solution of compounds 4a−j in ethylacetate:methanol (4:1) was treated with catalytic amount of 10% Pd/C and mixture was hydrogenated at room temperature under 40 psi H2 pressure until TLC showed the disappearance of starting material and reaction mixture becomes colorless. Reaction mixture was filtered through celite and filtrate was used for next step without further purification and delay.24 General Procedure for Preparation of 2-Aryl-5-cyanobenzimidazoles (8a−c) To the ethanolic solution of the freshly prepared 4−cyano−1,2−phenylenediamine 6 (7.51mmol, 1.0 equiv), a mixture of respective aldehyde (1.5 equiv.) and solution of Na2S2O5 (0.5 equiv) in water (1ml/100mg) were added. The resulting solution was stirred at reflux for 4−6h, then cooled to room temperature and filtered through a bed of celite. The solvents were evaporated under reduced pressure. The crude residue was purified by chromatography on silica gel (60−120 mesh size) in EtOAc/Pet ether as solid in 70−80% yield.24 5-Cyano-2-(4-hydroxyphenyl)benzimidazole (8a). White solid; (80% yield, 1.41g); mp. 190.6−193.2ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 6.94 (d, J = 8.72 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.66 (bs, 1H), 8.04 (d, J = 8.4 Hz, 3H), 10.10 (s, 1H), 13.18 (bs, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 6.93 (d, J = 8.72 Hz, 2H), 7.54 (dd, J = 1.32, 8.4 Hz, 1H), 7.67 (d, J = 8.04 Hz, 1H), 7.98 (d, J = 8.72 Hz, 3H).

13

C NMR (100 MHz, DMSO−d6) δ ppm

103.6, 116.0, 120.1, 120.3, 125.5, 128.9, 155.0, 160.0. FTIR (KBr, cm–1): 3463.07, 3235.04, 2924.36, 2851.54, 2588.23, 2228.91, 1610.10, 1497.78, 1466.59, 1432.01, 1313.71, 1269.93, 1250.70, 1174.47, 1113.28, 966.64, 878.19, 841.98, 816.05, 749.57, 517.20, 439.55. HRMS (ESI): m/z calcd for C14H9N3O [M + H]+ 236.0820 obsd 236.0813.


Page 27 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

27


5-Cyano-2-(4-ethoxyphenyl)benzimidazole

(8b).

White

solid;

(72%

yield,

1.42g);

mp.223.1−224.8ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.36 (t, J = 6.56 Hz, 3H), 4.12 (q, J = 6.6 Hz, 2H), 7.11 (d, J = 8.76 Hz, 2H), 7.57−7.76 (m, 2H), 8.12 (s, 3H), 13.28 (bs, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 1.34 (t, J = 7.32 Hz, 3H), 4.12 (q, J = 7.32 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 8.05 (s, 1H), 8.10 (d, J = 8.76 Hz, 2H).

13

C NMR (100 MHz, DMSO−d6) δ ppm 160.15, 155.29, 129.36, 125.96,

120.65, 120.26, 116.43, 103.92, 64.32, 14.74. FTIR (KBr, cm–1): 3437.66, 2929.80, 2220.87, 1502.97, 1267.03, 1022.70, 813.60. HRMS (ESI): m/z calcd for C16H13N3O [M + H]+ 264.1137 obsd 264.1129. 5-Cyano-2-(3,4-dimethoxyphenyl)benzimidazole (8c). This compound was synthesized by reported procedure.23 Off−white solid; (80% yield, 1.67g); mp 223.1−224.8ºC. General Procedure for the preparation of 2-Aryl-5-formyl benzimidazoles (9a−c). Ni-Al alloy was added to a solution of 8a−8c in 75% aqueous formic acid. The reaction mixture was heated at 95ºC for 30min under inert atmosphere. The hot mixture was filtered through celite bed and the reaction flask and the celite bed were rinsed with water. The aqueous solution was concentrated to dryness. After addition of water to this residue, a precipitate was formed. The pH of this suspension was adjusted to 9.0 by the dropwise addition of 2N NaOH and the product was then extracted into ethylacetate. The organic layers were dried over anhydrous Na2SO4 and filtered. The solvents were evaporated under reduced pressure. The residue was purified by chromatography on silica gel (60−120 mesh size) in MeOH/EtOAc as solid in 65−74% yield.24 5-Formyl-2-(4-hydroxyphenyl)benzimidazole (9a). Off-white solid, (74% yield, 0.74g); mp 315.5−316.2 ºC. 1H NMR (400 MHz, DMSO−d6): δ 6.94 (d, J = 8.72 Hz, 2H), 7.69



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28

Page 28 of 73

(d, J = 8.08 Hz, 1H), 7.74 (d, J = 10.08 Hz, 1H), 8.04 (d, J = 8.76 Hz, 2H), 8.09 (s, 1H), 10.03 (s, 1H), 10.13 (s, 1H). 1H NMR (400 MHz, DMSO−d6+D2O) δ ppm 6.94 (d, J = 8.72 Hz, 2H), 7.69 (s, 1H), 7.74 (d, J = 7.72 Hz, 1H), 8.03 (d, J = 8.76 Hz, 2H), 8.09 (s, 1H), 10.02 (s, 1H).

13

C

NMR (100 MHz, DMSO−d6) δ ppm 115.9, 120.4, 123.0, 128.7, 130.9, 155.0, 159.9, 163.1, 192.5. FTIR (KBr, cm–1): 3447.41, 2924.95, 2851.54, 1609.53, 1454.54, 1356.47, 1272.72, 1019.15, 841.95, 774.82, 528.67, 430.76. HRMS (ESI): m/z calcd for C14H10N2O2 [M + H]+ 239.0820 obsd 239.0813. 5-Formyl-2-(4-ethoxyphenyl)benzimidazole (9b). Off-white solid (70% yield, 0.70g); mp 266.8−268.6ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.36 (t, J = 6.84 Hz, 3H), 4.12 (q, J = 6.88 Hz, 2H), 7.11 (d, J = 8.04 Hz, 2H), 7.75 (s, 1H), 8.02−8.19 (m, 3H), 10.03 (d, J = 2.56 Hz, 1H), 13.20 (bs, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 1.35 (t, J = 6.84 Hz, 3H), 4.10 (q, J = 6.88 Hz, 2H), 7.11 (d, J = 8.72 Hz, 2H), 7.69−7.76 (m, 2H), 8.11 (s, 1H), 8.14 (d, J = 8.68 Hz, 2H), 10.03 (s, 1H).

13


122.9, 128.5, 131.0, 160.5, 192.5. HRMS (ESI): m/z calcd for C16H14N2O2 [M + H]+ 267.1133 obsd 267.1125. 5-Formyl-2-(3,4-dimethoxyphenyl)benzimidazole (9c). This compound was synthesized by reported procedure.23 Crystalline off-white compound; (65% yield, 0.65g); mp 232.6 − 235.4ºC. General Procedure for Preparation of Bisbenzimidazole Derivatives (10a-j, 11a-i and 12a-e). To the ethanolic solution of the freshly prepared diamine (0.5g, 1.0 equiv) 5a−j, a mixture of respective aldehyde 9a−c (1.5 equiv) and solution of Na2S2O5 (0.5 equiv) in water (1mL/100mg) were added. The resulting solution was stirred at reflux for 24h, then cooled to room temperature and filtered through a bed of celite. The solvents were evaporated under reduced


Page 29 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

29


pressure. The crude residue was purified by chromatography on silica gel (100−200 mesh size) in MeOH/DCM as solid title compounds in 40−60% yield. 5-(4-Ethylpiperazin-1-yl)-2-[2’-(4-hydroxyphenyl)-5’-benzimidazolyl]benzimidazole (10a). Orangish brown solid; (55% yield, 0.54g); mp 258.5−259.3 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.05 (t, J = 7.4 Hz, 3H), 2.45 (q, J = 7.4 Hz, 2H), 2.48−2.50 (m, 4H), 3.11−3.15 (m, 4H), 6.90−7.00 (m, 4H), 7.43 (d, J = 8.08 Hz, 1H), 7.60−7.68 (m, 1H), 8.01 (s, 1H), 8.07 (d, J = 8.72 Hz, 2H), 8.27−8.34 (m, 1H).

13


111.4, 115.82, 118.5, 120.4, 120.8, 124.2, 128.5, 147.6, 153.3, 159.5. FTIR (KBr, cm–1): 3113.70, 2823.08, 2361.39, 1610.72, 1443.69, 1240.25, 1173.15, 963.77, 817.12, 443.58. HRMS (ESI): m/z calcd for C26H26N6O [M + H]+439.2246 obsd 439.2233. 5-(4-Propylpiperazin-1-yl)-2-[2’-(4-hydroxyphenyl)-5’-benzimidazolyl]benzimidazole (10b). Yellow colored solid; (52% yield, 0.50g); mp 238.5−239.1 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 0.87 (t, J = 7.32 Hz, 3H), 1.45−1.51 (m 2H), 2.34 (t, J = 7.36 Hz, 2H), 2.58−2.60 (m, 4H), 3.12−3.17 (m, 4H), 6.88−7.00 (m, 5H), 7.43 (d, J = 8.72 Hz, 1H), 7.64 (d, J = 8.72 Hz, 1H), 7.99 (d, J = 8.24 Hz, 1H), 8.06 (d, J = 8.68 Hz, 2H), 8.22−8.28 (m, 1H).

13

C NMR (100 MHz,

DMSO−d6) δ ppm 11.8, 19.3, 49.8, 52.8, 59.6, 113.6, 115.6, 115.8, 120.5, 120.8, 124.2, 126.9, 127.3, 128.4, 130.6, 136.4, 147.6, 151.6, 153.3, 156.8, 159.2, 159.5. FTIR (KBr, cm–1): 3403.35, 2930.07, 2705.51, 2372.79, 1605.83, 1466.67, 1305.37, 1257.42, 1189.98, 1036.66, 962.53, 810.63. HRMS (ESI): m/z calcd for C27H28N6O [M + H]+453.2403 obsd 453.2373. 5-(4-(2-N,N-Dimethylaminoethylpiperazin-1-yl))-2-[2’-(4-hydroxyphenyl)-5’ benzimidazolyl]benzimidazole (10c). Brown crytalline solid; (56% yield, 0.51g); mp 208.5−209.3 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 2.20 (s, 6H), 2.44−2.47 (m, 4H),



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30

Page 30 of 73

2.57−2.59 (m, 4H), 3.09−3.12 (m, 4H), 6.92−6.99 (m, 4H), 6.99 (s, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.08 Hz, 1H), 7.98 (d, J = 9.52 Hz, 1H), 8.04 (d, J = 8.04 Hz, 2H), 8.26 (s, 1H), 12.28 (bs, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 43.8, 43.9, 44.0, 44.1, 44.2, 50.0, 50.5, 51.8, 52.3, 53.7, 54.4, 113.6, 116.4, 118.0, 118.1, 126.4, 126.7, 131.4, 131.8, 133.6, 136.0, 154.1, 165.3. HRMS (ESI): m/z calcd for C28H31N7O [M + H]+482.2668 obsd 482.2640. 5-(4-(2-N-Trifluoroacetylaminoethylpiperazin-1-yl))-2-[2’-(4-hydroxyphenyl)-5’benzimidazolyl]benzimidazole (10d). Creamish yellow solid; (55% yield, 0.45g); mp 248.5−249.8ºC.

1

HNMR (400 MHz, DMSO−d6) δ ppm 3.03−3.16 (m, 4H), 3.19−3.24 (m. 4H),

3.35−3.58 (m, 4H), 6.94 (d, J = 8.76 Hz, 2H), 7.04 (dd, J = 2.0, 8.72 Hz, 1H) 7.11 (s, 1H), 7.52 (d, J = 8.76 Hz, 1H), 7.69 (d, J = 8.72 Hz, 1H), 7.99 (d, J = 8.72 Hz, 1H), 8.05 (d, J = 8.72 Hz, 2H), 8.30 (s, 1H), 9.6 (br s, 1H), 10.07 (bs, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 3.09−3.15 (m, 2H), 3.20−3.26 (m. 2H), 3.32−3.39 (m, 4H), 6.95 (d, J = 8.76 Hz, 2H), 7.03 (dd, J = 2.0, 8.72 Hz, 1H) 7.11 (s, 1H), 7.51 (d, J = 8.72 Hz, 1H), 7.68 (d, J = 8.72 Hz, 1H), 7.98 (d, J = 8.72 Hz, 1H), 8.04 (d, J = 8.72 Hz, 2H), 8.29 (s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 34.8, 47.9, 51.8, 54.3, 100.9, 113.0, 114.5, 114.7, 115.5, 116.0, 117.4, 120.5, 121.0, 122.6, 128.7, 133.2, 138.2, 147.0, 151.4, 153.7, 156.3, 156.7, 156.8, 157.0, 157.1, 157.2, 159.7. HRMS (ESI): m/z calcd for C28H26F3N7O2 [M + H]+ 550.2178 obsd 550.2204. 5-(4-Phenylpiperazin-1-yl)-2-[2’-(4-hydroxyphenyl)-5’-benzimidazolyl]benzimidazole (10e). Creamish yellow solid; (48% yield, 0.43g); mp 218.2−220 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.22−3.26 (m, 4H), 3.28−3.32 (m, 4H), 6.80 (t, J = 7.36 Hz, 1H), 6.93−7.00 (m, 5H), 7.07 (s, 1H), 7.23 (t, J = 8.32 Hz, 2H), 7.47 (d, J = 8.36 Hz, 1H), 7.62−7.69 (m, 1H), 8.02 (bs, 1H), 8.03 (d, J = 8.64 Hz, 2H), 8.25−8,35 (m, 1H), 10.06 (br s, 1H),12.57 (br s, 1H), 12.89 (br s, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 3.14−3.20 (m, 8H), 6.76 (t, J = 7.4 Hz, 1H),


Page 31 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


31

6.89−6.97 (m, 5H), 7.09 (s, 1H), 7.17 (t, J = 8.0 Hz, 2H), 7.47 (d, J = 8.68 Hz, 1H), 7.68 (d, J = 8.32 Hz, 1H), 7.96 (d, J = 8.36 Hz, 1H), 8.00 (d, J = 8.68 Hz, 2H), 8.27 (s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 49.2, 50.8, 115.0, 116.4, 116.5, 120.0, 120.9, 121.4, 124.5, 129.1, 129.7, 148.4, 151.5, 152.1, 154.0, 159.9. HRMS (ESI): m/z calcd for C30H26N6O [M + H]+ 487.2246 obsd 487.2219. 5-(4-(2-Cyanophenylpiperazin-1-yl))-2-[2’-(4-hydroxyphenyl)-5’benzimidazolyl]benzimidazole (10f). Brown crystalline compound; (47% yield, 0.40g); mp 208.2−210 ºC. 1H NMR (400 MHz, MeOD) δ ppm 3.29−3.31 (m, 8H), 6.93 (d, J = 8.72 Hz, 2H), 7.03−7.14 (m, 4H), 7.48 (d, J = 8.72 Hz, 1H), 7.52−7.56 (m, 1H), 7.60 (dd, J = 1.36, 7.76 Hz, 1H), 7.64 (d, J = 8.72 Hz, 2H), 7.88−7.90 (m, 1H), 7.94 (d, J = 8.72 Hz, 2H), 8.19 (s, 1H). 13C NMR (100 MHz, MeOD) δ ppm 52.4, 52.9, 102.1, 107.1, 113.7, 116.12, 116.6, 116.9, 119.3, 120.1, 121.6, 122.4, 123.4, 125.1, 129.7, 135.3, 135.4, 135.6, 139.8, 149.8, 153.5, 155.7, 157.0, 161.4. FTIR (KBr, cm–1): 3400.26, 2926.93, 2369.02, 2219.49, 160.45, 1447.38, 1231.45, 1173.38, 1035.29, 963.40, 840.17, 760.14, 520.09. HRMS (ESI): m/z calcd for C31H25N7O [M + H]+ 512.2199 obsd 512.2181. 5-(4-(4-Fluorophenylpiperazin-1-yl))-2-[2’-(4-hydroxyphenyl)-5’benzimidazolyl]benzimidazole (10g). Yellow solid; (56% yield, 0.49g); mp 228.7−230.2 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.26 (s, 8H), 6.94 (d, J = 8.8 Hz, 2H), 6.98−7.10 (m, 6H), 7.45 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H), 7.99 (d, J = 8.08 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H), 8.27 (s, 1H), 10.03 (s, 1H), 12.86 (s, 1H).

13

C NMR (100 MHz, DMSO−d6) δ ppm 47.9, 51.7, 114.4,

114.6, 115.2, 115.3, 115.9, 117.3, 120.4, 120.9, 122.3, 128.6, 147.0, 150.9, 151.4, 153.7, 156.6, 157.0, 159.7. .HRMS (ESI): m/z calcd for C30H25FN6O [M + H]+ 505.2152 obsd 505.2153.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

32

Page 32 of 73

5-(4-Pyridylpiperazin-1-yl)-2-[2’-(4-hydroxyphenyl)-5’-benzimidazolyl]benzimidazole (10h). Creamish yellow solid; (51% yield, 0.46g); mp 268.6−270 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.22−3.27 (m, 4H), 3.48−3.53 (m, 4H), 6.88−6.99 (m, 5H), 7.17−7.80 (m, 3H), 7.97−8.05 (m, 2H), 8.08−8.34 (m, 3H), 10.03 (br s, 1H), 12.65 (br s, 1H), 12.85 (br s, 1H). 13

C NMR (100 MHz, DMSO−d6): 50.3, 55.7, 109.9, 111.9, 114.2, 116.1, 116.7, 117.8, 119.7, 120.8,

122.3, 123.7, 147.8, 149.0, 150.7, 151.3, 153.1. HRMS (ESI): m/z calcd for C29H25N7O [M + H]+ 488.2199 obsd 488.2175. 5-(4-(2-Pyrimidylpiperazin-1-yl)-2-[2’-(4-hydroxyphenyl) 5’-benzimidazolyl]benzimidazole (10i). Creamish yellow solid; (54% yield, 0.48g); mp 267.1−269.9 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.15−3.17 (m, 4H), 3.89−3.92 (m, 4H), 6.64 (t, J = 5.16 Hz, 1H), 6.94 (d, J = Hz, 2H), 7.00 (d, J = 8.08 Hz, 1H), 7.07 (s, 1H), 7.47 (s, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.97 (d, J = 8.04 Hz, 1H), 8.03 (d, J = 8.8 Hz, 2H), 8.28 (s, 1H), 8.36 (d, J = 4.36 Hz, 2H), 12.75 (br s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 53.4, 56.7,113.5, 116.4, 119.0, 120.3, 124.0, 128.4, 147.7, 151.8, 154.0, 160.7, 162.2. HRMS (ESI): m/z calcd for C28H24N8O [M + H]+ 489.2151 obsd 489.2176. 5-(4-(4-Aminophenylpiperazin-1-yl)-2-[2’-(4-hydroxyphenyl)-5’benzimidazolyl]benzimidazole (10j). Brown solid; (47% yield, 0.41g); mp 255.4−256.9 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.24−3.27 (m, 4H), 3.68−3.75 (m, 4H), 6.94 (d, J = 8.8 Hz, 2H), 6.98 (dd, J = 2.2, 8.8 Hz, 1H), 7.08 (s, 1H), 7.12 (s, 1H), 7.48 (d, J = 8.76 Hz, 1H), 7.65 (d, J = 8.04 Hz, 1H), 8.00 (d, J = 8.04 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 8.21−8.29 (m, 3H), 10.08 (br s, 1H), 12.93 (br s, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 3.23−3.28 (m, 4H), 3.70−3.74 (m, 4H), 6.94 (d, J = 8.8 Hz, 2H), 6.99 (dd, J = 2.2, 8.8 Hz, 1H), 7.09−7.10 (m, 3H), 7.48 (d, J = 8.76 Hz, 1H), 7.65 (d, J = 8.04 Hz, 1H), 7.99 (d, J = 8.04 Hz, 1H), 8.04 (d, J = 8.8


Page 33 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

33


Hz, 2H), 8.21−8.29 (m, 3H). 13C NMR (100 MHz, DMSO−d6) δ ppm 45.7, 49.7, 107.9, 113.9, 115.8, 120.6, 120.8, 124.1, 128.4, 143.4, 147.0, 151.9, 153.3, 155.8, 159.4. HRMS (ESI): m/z calcd for C30H27N7O [M + H]+ 502.2355 obsd 501.2189. 5-(4-Ethylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’-benzimidazolyl]benzimidazole (11a). Brown solid; (55% yield, 0.60g); mp 157.3−158.8 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.05 (t, 3H, J = 7.36 Hz), 2.43 (q, 2H, J = 6.6 Hz), 2.57−2.61 (m, 4H), 3.12−3.16 (m, 4H), 3.85 (s, 3H), 3.90 (s, 3H), 6.92−6.94 (m, 2H), 7.15 (d, 1H, J = 8.04 Hz), 7.35−7.51 (m, 1H), 7.60−7.80 (m, 3H), 7.96−8.04 (m, 1H), 8.22−8.34 (m, 1H), 12.58 (br s, 1H), 12.95 (br s, 1H). 13

C NMR (400 MHz, DMSO−d6) δ ppm 11.9, 49.9, 51.6, 52.5, 55.7, 108.8, 109.8, 111.4, 11.8,

116.1, 118.7, 119.6, 120.4, 121.1, 122.4, 124.3, 124.6, 135.4, 136.1, 144.1, 145.0, 147.7, 149.0, 150.6, 152.8, 153.1. FTIR (KBr, cm–1): 3413.50, 2927.71, 2817.92, 1618.41, 1435.90, 1262.74, 1176.69, 1138.44, 1023.14, 965.25, 808.97, 765.26, 720.23. HRMS (ESI):

m/z calcd for

C28H30N6O2 [M + H]+ 483.2508 obsd 483.2489. 5-(4-Propylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’-benzimidazolyl]benzimidazole (11b). Brown solid; (48% yield, 0.50g); mp 254.3−255.8ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 0.93 (t, 3H, J = 7.32 Hz), 1.63−1.73 (m, 1H), 3.02 (t, 3H, J = 8.04 Hz), 3.26−3.66 (m, 8H), 3.85 (s, 3H), 3.90 (s, 3H), 6.99 (d, 1H, J = 8.76 Hz), 7.11 (s, 1H), 7.16 (d, 1H, J = 8.8 Hz), 7.49 (d, 1H, J = 8.76 Hz), 7.69 (s, 1H), 7.78−7.80 (m, 2H), 8.01 (d, 1H, J = 8.76 Hz), 8.30 (s, 1H), 12.96 (bs, 1H).

13

C NMR(400 MHz, DMSO−d6) δ ppm 11.3, 17.7, 48.3, 51.7, 55.6, 55.7, 58.0,

109.9, 111.8, 113.9, 119.7, 120.8, 122.4, 124.3, 146.7, 149.0, 150.6, 151.9, 153.0, 163.4. FTIR (KBr, cm–1): 3393.44, 2927.85, 1603.67, 1499.28, 1438.12, 1263.12, 1176.75, 1135.97, 1020.08, 981.05, 962.71, 869.39, 811.83, 765.09, 715.95, 619.12. HRMS (ESI): C29H32N6O2 [M + H]+ 497.2665 obsd 497.2654.


m/z

calcd for


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

34

Page 34 of 73

5-(4-(2-N,N-Dimethylaminoethylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’benzimidazolyl]benzimidazole (11c). Brown coloured crystalline solid, (61% yield, 0.60g); mp 157.3−158.8ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 2.37 (s, 6H). 2.44−2.52 (m, 6H), 2.70 (t, J = 6.56 Hz, 2H), 3.00−3.04 (m, 4H), 3.76 (s, 3H), 3.82 (s, 3H), 6.83 (d, J = 7.32 Hz, 1H), 6.95 (s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.08 Hz, 1H), 7.74−7.77 (m, 2H), 7.97 (d, J = 8.76 Hz, 1H), 8.28 (s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 14.6, 44.0, 49.9, 52.1, 53.0, 53.8, 54.7, 55.6, 55.7, 109.9, 111.8, 112.7, 113.7, 119.7, 120.8, 122.5, 124.4, 147.6, 148.9, 150.5, 151.6, 153.0, 160.7, 165.5. FTIR (KBr, cm–1): 3403.10, 2928.42, 2670.24, 2368.40, 2345.77, 1628.29, 1466.73, 1277.64, 1236.88, 1157.82, 1013.66, 963.49, 814.24, 717.53.HRMS (ESI): m/z calcd for C30H35N7O2 [M + H]+ 526.2930 obsd 526.2905. 5-(4-Phenylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’-benzimidazolyl]benzimidazole (11d). Yellow solid; (45% yield, 0.44g); mp 225.2−226.4 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.25 −3.31 (m, 8H), 3.85 (s, 3H), 3.90 (s, 3H), 6.81 (t, J = 7.32 Hz, 1H), 7.01 (d, J = 8.08 Hz, 4H), 7.16 (d, J = 8.08 Hz, 1H), 7.24 (t, J = 8.04 Hz, 2H), 7.40−7.63 (m, 1H), 7.73−7.80 (m, 3H), 8.01 (dd, J = 7.32, 23.44 Hz, 1H), 8.23−8.37 (m, 1H), 12.64 (s, 1H), 12.97 (br s, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 3.16−3.22 (m, 8H), 3.82 (s, 3H), 3.88 (s, 3H), 6.76 (t, J = 7.32 Hz, 1H), 6.88−6.95 (m, 2H), 7.08−7.19 (m, 4H), 7.47 (d, J = 8.08 Hz, 1H), 7.71−7.81 (m, 3H), 8.05 (d, J = 8.08 Hz, 1H), 8.36 (s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 48.5, 50.3, 55.6, 109.9, 111.8, 114.0, 115.6, 119.1, 119.6, 120.8, 122.4, 124.4, 129.0, 147.6, 149.0, 150.6, 151.0, 151.7, 153.0. FTIR (KBr, cm–1): 3410.04, 3198.21, 2999.40, 2936.28, 2962.51, 2835.27, 2095.47, 1600.79, 1522.19, 1500.83, 1475.49, 1458.19, 1447.23, 1437.82, 1329.65, 1273.91, 1263.56, 1236.74, 1173.94, 1147.60, 1039.63, 1027.87, 958.75, 870.33, 822.32, 796.60,


Page 35 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


35

760.73, 695.51, 635.39, 614.39, 528.09, 496.97. HRMS (ESI): m/z calcd for C32H30N6O2 [M + H]+ 531.2508 obsd 531.2490. 5-(4-(2-Cyanophenylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’benzimidazolyl]benzimidazole (11e). Brown crystalline compound; (42% yield, 0.39g); mp 202.3−203.6 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.85 (s, 3H), 3.90 (s, 3H), 7.00−7.02 (m, 1H),7.10−7.17 (m, 4H), 7.25 (d, J = 8.04 Hz, 1H), 7.49 (d, J = 8.76 Hz, 1H), 7.62−7.80 (m, 5H), 8.01−8.03 (d, J = 8.04 Hz, 1H), 8.29 (s, 1H). 1H NMR (400 MHz, DMSO−d6 + D2O) δ ppm 3.32 (s, 8H), 3.83 (s, 3H), 3.88 (s, 3H), 6.99−7.02 (m, 1H), 7.09−7.15 (m, 4H), 7.25 (d, J = 8.04 Hz, 1H), 7.48 (d, J = 8.76 Hz, 1H), 7.60−7.78 (m, 5H), 7.99 (d, J = 8.04 Hz, 1H), 8.29 (s, 1H). 13

C NMR (100 MHz, DMSO−d6) δ ppm 49.5, 51.2, 55.7, 55.8, 99.0, 104.9, 110.1, 111.8, 114.6,

115.8, 118.30, 119.2, 120.1, 121.4, 122.3, 133.3, 134.5, 149.0, 149.5, 150.9, 154.0, 155.0. HRMS (ESI): m/z calcd for C33H29N7O2 [M + H]+ 556.2461 obsd 556.2440. 5-(4-(4-Fluorophenylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’benzimidazolyl]benzimidazole (11f). Creamish yellow solid; (42% yield, 0.40g); mp 238.6−239.8 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.24 (s, 8H), 3.85 (s, 3H), 3.90 (s, 3H), 6.97−7.08 (m, 6H), 7.15 (d, J = 8.04 Hz, 1H), 7.48 (s, 1H), 7.63−7.81 (m, 3H), 8.03 (dd, J = 8.08, 21.24 Hz, 1H), 8.26−8.38 (m, 1H), 12.65 (br s, 1H), 13.0 (br s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 49.3, 50.2, 55.6, 108.9, 109.9, 111.5, 111.8, 114.0, 115.2, 115.4, 117.3, 117.3, 118.7, 119.6, 120.6, 121.1, 122.4, 124.4, 147.5, 147.9, 149.0, 150.6, 151.7, 153.0, 154.9, 157.3. FTIR (KBr, cm–1): 3411.47, 2971.27, 2839.56, 1605.65, 1510.80, 1491.80, 1438.69, 1325.30, 1259.30, 1236.17, 1174.34, 1145.73, 1035.86, 1022.28, 982.70, 959.26, 868.92, 831.03, 810.17, 766.16, 720.33, 608.59, 494.73. HRMS (ESI): m/z calcd for C32H29FN6O2 [M + H]+ 549.2414 obsd 549.2414.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

36

Page 36 of 73

5-(4-Pyridylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’-benzimidazolyl]benzimidazole (11g). Yellow solid; (43% yield, 0.42g); mp 256.9−258.3 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.26−3.28 (m, 4H), 3.71−3.74 (m, 4H), 3.84 (s, 3H), 3.90 (s, 3H), 7.00 (dd, J = 2.2, 8.8 Hz, 1H), 7.09−7.16 (m, 4H), 7.49 (d, J = 8.76 Hz, 1H), 7.68−7.70 (m, 1H), 7.79−7.81 (m, 2H), 8.02 (d, J = 8.04 Hz, 1H), 8.23 (d, J = 7.32 Hz, 2H), 8.31 (s, 1H), 13.07 (br s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 50.3, 55.7, 109.9, 111.9, 114.2, 116.1, 117.8, 119.7, 120.8, 122.3, 123.7, 147.8, 149.0, 150.6, 151.3, 153.1. FTIR (KBr, cm–1): 3403.67, 2927.16, 1617.60, 1443.57, 1246.18, 1219.58, 1139.31, 1032.16, 995.80, 808.53, 617.95. HRMS (ESI): m/z calcd for C31H29N7O2 [M + H]+ 532.2461obsd 532.2428. 5-(4-(2-Pyrimidylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)

5’-

benzimidazolyl]benzimidazole (11h). Yellow solid; (51% yield, 0.50g); mp 239−240.8ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.15−3.20 (m, 4H), 3.84 (s, 3H), 3.89 (s, 3H), 3.91−4.93 (m, 4H), 6.65 (t, J = 4.4 Hz, 1H), 7.01 (dd, J = 2.2, 8.8 Hz, 1H), 7.09 (s, 1H), 7.15 (d, J = 8.04 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.68 (s, 1H), 7.75−7.79 (m, 2H), 8.00 (d, J = 8.8 Hz, 1H), 8.29 (s, 1H), 8.39 (d, J = 4.4 Hz, 2H), 13.03 (bs, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 49.2, 52.0, 56.1, 56.2, 110.4, 112.3, 114.3, 120.2, 122.9, 124.8, 147.4, 149.4, 151.0, 152.3, 153.5. FTIR (KBr, cm–1): 3422.55, 2925.17, 2278.84, 1618.01, 1433.08, 1262.63, 1175.89, 1021.83, 982.35, 806.70, 716.34. HRMS (ESI): m/z calcd for C30H28N8O2 [M + H]+ 533.2413 obsd 533.2200. 5-(4-(4-Aminophenylpiperazin-1-yl)-2-[2’-(3,4-dimethoxyphenyl)-5’benzimidazolyl]benzimidazole (11i). Brown solid; (42% yield, 0.40g); mp 249.7−250.6 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 3.13−3.15 (m, 4H), 3.25−3.38 (m, 4H), 3.85 (s, 3H), 3.90 (s, 3H), 6.62 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 7.01 (dd, J = 2.2, 8.8 Hz, 1H), 7.08 (s, 1H), 7.16 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.68−7.80 (m, 3H), 8.00 (d, J = 9.52 Hz,


Page 37 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37

1H), 8.30 (s, 1H). 13C NMR (400 MHz, DMSO−d6) δ ppm 42.3, 47.4, 52.7, 55.4, 114.2, 114.5, 120.9, 122.2, 123.5, 128.3, 146.2, 151.8, 153.0, 160.9. FTIR (KBr, cm–1): 3154.59, 2937.43, 2833.10, 1627.08, 1512.67, 1475.39, 1449.90, 1379.29, 1252.40, 1228.58, 1108.21, 1028.14, 969.84, 931.13, 862.21, 810.74, 766.04, 714.22, 638.19, 614.63, 551.26. HRMS (ESI): m/z calcd for C32H31N7O2 [M + H]+546.2617obsd 546.2579. 5-(4-Ethylpiperazin-1-yl)-2-[2’-(4-ethoxyphenyl)-5’benzimidazolyl]benzimidazole

(12a).

Brown solid; (57% yield, 0.60g); mp 212.3−214.5ºC. 1H NMR (400 MHz, MeOD) δ ppm 1.24 (t, J = 7.32 Hz, 3H), 1.37 (t, J = 6.88 Hz, 3H), 2.83 (q, J = 7.36 Hz, 4H), 2.97−3.02 (m, 4H), 3.25−3.30 (m, 4H), 6.99 (d, J = 6.88 Hz, 2H), 7.10 (s, 1H), 7.47 (d, J = 8.72 Hz, 1H), 7.63 (d, J = 8.24 Hz, 1H), 7.89 (dd, J = 1.8, 8.68 Hz, 1H), 7.98 (d, J = 9.16 Hz, 2H), 8.18 (s, 1H), 8.53 (s, 1H).

13

C NMR (100 MHz, MeOD) δ ppm 10.8, 14.3, 15.1, 50.7, 50.9, 53.2, 53.4, 64.7, 102.6,

115.9, 116.4, 122.4, 122.7, 125.4, 129.5, 140.2, 148.9, 153.8, 155.3, 162.5, 170.3. FTIR (KBr, cm–1): 3435.78, 2979.05, 2829.06, 2451.77, 2368.73, 1609.26, 1491.29, 1474.77, 1432.36, 1387.66, 1292.80, 1252.40, 1179.42, 1122.52, 1041.26, 810.34, 719.26. HRMS (ESI): m/z calcd for C28H30N6O [M + H]+ 467.2559 obsd 467.2556. 5-(4-Propylpiperazin-1-yl)-2-[2’-(4-ethoxyphenyl)-5’-benzimidazolyl]benzimidazole

(12b).

Yellow solid; (48% yield, 0.49g); mp 205.2−206.2ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 0.87 (t, J = 7.32 Hz, 3H), 1.36 (t, J = 7.32 Hz, 3H), 1.42−1.51 (m, 2H), 2.28 (t, J = 7.32 Hz, 3H), 2.51−2.53 (m, 4H), 3.06−3.15 (m, 4H), 4.12 (q, J = 7.32 Hz, 2H), 6.91 (s, 2H), 7.11 (d, J = 8.8 Hz, 2H), 7.35−7.48 (m, 1H), 7.59−7.73 (m, 1H), 7.96−8.04 (m, 1H), 8.14 (d, J = 7.32 Hz, 2H), 8.22−8.35 (m, 1H).

13

C NMR (100 MHz, DMSO−d6) δ ppm 11.6, 18.5, 49.1, 52.2, 58.8, 113.7,

115.8, 120.6, 120.8, 124.1, 128.4, 147.1, 151.8, 153.3, 159.5, 164.2. FTIR (KBr, cm–1): 3151.43,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

38

Page 38 of 73

2960.43, 2928.41, 2821.53, 2362.41, 1610.81, 1445.77, 1252.41, 1178.95, 1041.69, 963.13, 810.19, 631.80. HRMS (ESI): m/z calcd for C29H32N6O [M + H]+ 481.2716 obsd 481.2707. 5-(4-(2-(N,N-Dimethylaminoethylpiperazin-1-yl)-2-[2’-(4-ethoxyphenyl)-5’benzimidazolyl]benzimidazole (12c). Brown solid; (53% yield, 0.51g) ; mp 205.8−206.2 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.3 (t, J = 7.32 Hz, 3H), 2.45 (s, 6H), 2.55−2.63 (m, 6H), 2.78 (t, J = 6.6 Hz, 2H), 3.11−3.14 (m, 4H), 4.12 (q, J = 7.32 Hz, 2H), 6.93 (dd, J =2.2, 8.8 Hz, 1H), 7.01 (s, 1H), 7.11 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 9.16 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.99 (t, J = 8.04 Hz, 1H), 8.15 (d, J = 8.8 Hz, 2H), 8.94 (s, 1H). 13C NMR (100 MHz, DMSO−d6 ) δ ppm 14.3, 42.3, 45.8, 48.2, 49.2, 49.6, 49.8, 50.0, 51.0, 98.5, 113.3, 114.2, 114.7, 115.2, 116.1, 117.2, 119.2, 124.4, 125.8, 130.3, 131.5, 132.7, 132.9, 135.3, 146.8, 148.3, 149.9, 151.3, 154.2, 162.4. HRMS (ESI): m/z calcd for C30H35N7O [M + H]+ 510.2981 obsd 510.2974. 5-(4-(2-N-trifluoroacetylaminoethylpiperazin-1-yl)-2-[2’-(4-ethoxyphenyl)-5’benzimidazolyl]benzimidazole (12d). Brown solid; (49% yield, 0.42g); mp 225.1−226.2ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.37 (t, J = 7.32 Hz, 3H), 2.61 (t, J = 5.88 Hz, 3H), 2.62−2.66 (m, 4H), 2.99 (t, J = 5.88 Hz, 2H), 3.15−3.19 (m, 4H), 4.11 (q, J = 7.36 Hz, 2H), 6.95 (dd, J = 2.2, 8.8Hz, 1H), 7.03 (s, 1H), 7.11 (d, J = 8.76 Hz, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H), 7.99 (d, J = 7.36 Hz, 1H), 8.13 (d, J = 8.76 Hz, 2H), 8.28 (s, 1H), 12.93 (br s, 1H).

13

C

NMR (100 MHz, DMSO−d6) δ ppm 14.7, 35.8, 49.7, 52.7, 54.3, 63.4, 114.0, 114.9, 120.8, 122.1, 123.9, 128.3, 147.7, 151.5, 153.0, 160.2. FTIR (KBr, cm–1): 3409.90, 3070.02, 2972.48, 2095.82, 1630.26, 1611.75, 1578.91, 1521.89, 1487.83, 1466.41, 1393.91, 1295.81, 1251.82, 1137.28, 988.26, 919.06, 801.40, 737.64, 720.69, 663.02, 637.90, 618.25, 578.16, 525.63, 445.95. HRMS (ESI): m/z calcd for C30H30F3N7O2 [M + H]+ 578.2491 obsd 578.2470.


Page 39 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39

5-(4-(4-pyridylpiperazin-1-yl)-2-[2’-(4-ethoxyphenyl)-5’-benzimidazolyl]benzimidazole (12e). Cream colored solid; (41% yield, 0.39g); mp 215.2−216.7 ºC. 1H NMR (400 MHz, DMSO−d6) δ ppm 1.37 (t, J = 6.96 Hz, 3H), 3.27−3.30 (m, 4H), 3.74−3.77 (m, 4H), 4.13 (q, J = 7.32 Hz, 2H), 7.00 (d, J = 8.8 Hz, 1H), 7.10−7.15 (m, 5H), 7.49 (d, J = 8.04 Hz, 1H), 7.62−7.69 (m, 1H), 8.01 (s, 1H), 8.14 (d, J = 8.8 Hz, 2H), 8.24−8.34 (m, 3H), 12.99 (br s, 1H). 13C NMR (100 MHz, DMSO−d6) δ ppm 14.6, 45.8, 49.7, 54.9, 63.3, 107.7, 114.8, 122.2, 124.2, 128.3, 142.5, 146.9, 151.8, 152.9, 156.0, 160.1. HRMS (ESI): m/z calcd for C31H29N7O [M + H]+ 516.2512 obsd 515.2500. Biology. ATCC 25922 and DH5α was procured from Himedia ltd,India. The water borne strains were obtained from Prof. J. S. Virdi, Deptt. Of Microbiology, who have characterized these strains using biochemical criteria as well as sequencing of partial 16S ribosomal genes (submitted to GenBank) of and clinical E. coli strains were obtained from Institute of Pathology, Safdarganj Hospital, New Delhi, a national facility of Govt. of India. These strians were classified as E.coli strains by biochemical criteria. pHOT1 plasmid DNA was purchased from TopoGen Inc. (Port Orange, FL, USA). E. coli DNA gyrase and its relaxed substrate were purchased from New England Biolabs (GmBH, Germany). All antibiotics were obtained from Sigma (St Louis, MO, USA). All animal experiments were approved by Animal ethical committe of University of Delhi,Delhi, India using ethical guidelines. Animals were maintained under controlled conditions with free access of food and water. Determination of Minimal Inhibitory Concentrations (MICs). MICs were determined by the broth microdilution method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI)51 Inoculants were incubated at 37°C on cation adjusted Mueller Hinton (MH) Broth for 18 to 24 h. Compound were dissolved in culture media to 10 doubling dilutions from



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40

Page 40 of 73

128 to 0.125 µg/mL. All the standard E. coli strains ATCC 25922, DH5α, different pathogenic and commensal water borne E. coli strains and different clinical resistant strains of E. coli from UTI pateints were grown in 96 well plate with low evaporation lid (Falcon, Becton Dickinson) and derivatives of benzimidazoles were present at 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128,256 µg/mL concentrations. The similar experiments were done with standard antibiotics also. Plates were read by spectrometry at 600 nm. MIC50 and MIC90 value was scored as the minimal concentration that inhibited growth to 50% and 90% at 24h respectively. Determination of Minimal Bactericidal Concentrations (MBC). MBC was calculated using literature method.52,53 Reference MBC for each strain was determined by pelleting the culture of MIC50 value and three values beyond MIC50 values. The pellet was resuspended in compound free MH broth and washed thrice to remove the compound. Then the compound free pellet was again resuspended in fresh 100µL MH broth and plated on MH agar plates without compound. MBC endpoints were read as the lowest dilution of compound with no growth (>99.9% killing) after overnight incubation at 37 ºC using culture without compound as control. Relaxation Assay of EcTopo 1A. EcTopo 1A was diluted with a buffer of 10 mM Tris−HCl, pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin, 0.5 mM MgCl2. compounds were added to 10 ng of the diluted enzyme present in 10 µl volume, before addition of 10 µl of the same buffer containing 250 ng of supercoiled pHOT1 plasmid DNA purified by Qiagen Maxiprep Kit. The mixture was incubated at 37oC for 30 min before termination of the reaction and analysed by agarose gel electrophoresis as described previously.The ethidium bromide stained gel was photographed over UV light for densitometry analysis.


Page 41 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41

The percent relaxation was first determined by dividing the distance between the negatively supercoiled band (SC); and the weighted center of the partially relaxed band (PR); by the distance between the supercoiled band (SC) and the fully relaxed band (FR) to obtain percent relaxation = (SC−PR)/(SC−FR)54 Percent inhibition by different compounds was then calculated by subtracting percent relaxation in the presence of compounds from 100% relaxation obtained with enzyme only. In order to check the effect of metal ion and to understand the mechanism of inhibition of compound 12b, relaxation assay was performed in three different conditions. First, relaxation assay was done with increasing concentrations of Mg2+ from 0 to 50 mM, to find out optimum concentration of Mg2+ by EcTopo 1A (Figure S1A). Second, relaxation assay was performed with increasing concentration of Mg2+ in the presence of 5 µM compound 12b (Figure 3C) and in third reaction we have increased the concentration of 12b from 5-100 µM in a buffer with 25 mM of MgCl2 (Figure S1B). DNA Gyrase Supercoiling Assay with E. coli DNA Gyrase Enzyme.

In DNA

supercoiling assay, 0.5 µg of relaxed plasmid DNA pHOTI was incubated with 1 unit of E. coli DNA gyrase at 37 ºC for 30 min in 25 µL reaction containing 1, 5, 10, 25, 50, 75 and 100 µM of compounds. Reactions were terminated by adding 10 mM EDTA, 0.5% SDS, 0.25 µg/mL bromophenol blue, and 15% glycerol. The gels were stained with 5 µg/mL ethidium bromide, destained in water and photographed under UV illumination at alpha imager 2200. Relaxation Assay of Human Topoisomerase IB. Relaxation of negatively supercoiled plasmid DNA by human topoisomerase I was assayed in 20 µl of reaction buffer (10 mM Tris−HCl, pH 7.9, 150 mM NaCl, 0.1 % BSA, 0.1 mM spermidine and 5% glycerol) containing



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

42

Page 42 of 73

250 ng of supercoiled pHOT1 plasmid DNA and 1 unit of enzyme. After incubation at 37oC for 30 min, the reactions were terminated and analyzed by agarose gel electrophoresis as described for Ec Topo IA relaxation assay. Isothermal Titration Calorimetry. The energetics of the binding of EcTopo 1A to 12b analogue was carried out at 25°C and pH 7.9 using 20 mM Tris-Cl, 50 mM KCl, 10% Glycerol, 0.1mM DTT (Dithiothreitol) and in presence of 10 mM MgCl2 using a VP-ITC unit (MicroCal, Inc., Northampton, MA, USA). The enthalpy of binding (∆H), affinity constant (Ka), and molar binding stoichiometry were obtained as follows. Aliquots (5-10 µL) of ligands (4 x 10-4 M) were injected from a 250 µL micro syringe into the 1.4235 mL calorimeter cell containing the protein solution (1.0 x 10-5M) to achieve a complete binding isotherm. Aliquots of ligand solution were injected from the rotating syringe (540 rpm) into the isothermal chamber containing the protein solutions. Corresponding control experiments to determine the heat of dilution of the ligand were performed by injecting identical volumes of same concentration of the ligand into the buffer alone. The area under each heat burst curve was determined by integration using the Origin 7.0 software to give the measure of the heat associated with the injection. The heat of dilution was measured by injecting the ligand into the buffer solution or by additional injections of ligand after saturation; the value that was obtained was subtracted from the heat of reaction to obtain the effective heat of binding. The resulting corrected injection heats were plotted as a function of molar ratio. Titration curves were fitted using MicroCal Origin software, assuming one set of sites. Changes in free energy (∆G) and entropy (∆S) were calculated from the relationship ∆G = -RT ln Ka = ∆H - T∆S. The reported values of Ka and ∆H are the means from at least five measurements with the standard deviation.


Page 43 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

43


Docking

Procedure.

We have done docking experiments

with

six

potent

bisbenzimidazoles only, with minor modifications in the standard protocol of Dessideri et. al.55,56 The 3D structures of 11b, 11g-h, 12a-c have been designed with the program Chem Draw 12.0. Atomic co-ordinates of the receptor for the docking with the E.coli topoisomerase-IA has been extracted from the crystal structure PDB 1ECL. We have chosen dsDNA sequence 5’AATGCGCT-3’ (PDB ID. 3PX7) for docking with EcTopo 1A enzyme. The structures of the compounds and the receptors have been prepared for the docking using Auto Dock Tools suite version 1.3.2.57 All compounds structures were viewed in Viewer Lite software and then brought to their energetically minimized structures by GAUSSIAN utilizing a conjugate gradient method with 6-31G MP2 force field. Autodock tools (ADT) were used to merge nonpolar hydrogens of EcTopo 1A and assign atomic charges. Nonpolar hydrogen of each compound was merged and rotatable bonds were assigned. Grid-maps were generated for each atom type using AUTOGRID. An active site box of 78 x 78 x 78Å with grid spacing of 0.375Å was created and placed at the center of EcTopo 1A structure. Docking calculations were carried out using Lamarckian genetic algorithm. A population of random individuals (population size: 150) was used with 2500000 energy evaluations. A maximum number of 27000 generations with mutation rate of 0.02 were used. Fifty independent runs for each compound were performed with each EcTopo 1A structure. The resulting positions were clustered according to a root mean square criterion of 0.5Å. Two steps of docking was done in each cases, first docking was done using dsDNA as compound and topoisomerase as receptor and in the same context, second docking was done using the docked topoisomerase-dsDNA complex as receptor with various compounds. Stereoview of electrostatic potential maps were generated with Deep View Swiss PDB Viewer (SPDV) 4.158 after energy minimization using solvent dielectric constant 80. The


44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 73

3D models were constructed using Swiss Pdb Viewer using following protocol; 1) load template pdb file; 2) align primary target sequence with template; 3) submit modeling request to Swiss Model Server. We have created the pharmacophore using Ligand Scout application59 before starting the alignment. Mouse Systemic Infection Model. The invivo efficacy was determined in a septicaemia mice model as described previously.60 Female Balb/c mice weighing 20-25g of 6-8 weeks old were injected with E. coli ATCC 25922 (0.5 x 108 CFU in 0.1ml saline) intraperitoneally. At 30 min post inoculation, compound 12b at different doses (3, 5 and 7 mg/kg body weight) dissolved in sterile water as vehicle at a volume of 0.1 ml given single bolus intravenous injection in the tail vein. Each treatment or control group had 10 mice. Mortality was recorded daily for 7 days post-infection.61 Neutropenic Mouse E. coli Thigh Infection Model. The neutropenic mice thigh infection model, fully described by Craig, was used in this study. This model has been used extensively

for

determination

of

pharmacokinetic/pharmacodynamics

(PK/PD)

index

determination and prediction of antibiotic efﬁcacy in patients.62 Female Balb/c mice (6 mice per dosing group) weighing 20-25g were rendered neutropenic with 2 intraperitoneal injections of cyclophosphamide (150 mg/kg of body weight 4 days prior to bacterial inoculation and 100 mg/kg 1 day before inoculation) 2 h prior to bacterial inoculation. This regimen reliably resulted in transient neutropenia in mice that lasted for at least 3 days after the last dose of cyclophosphamide was given. Bacteria were injected into the right thigh of each mouse at time zero. Inocula were selected on the basis of pilot studies with vehicle-treated animals that determined the maximum number of CFU that could be inoculated without substantial mortality. Neutropenia was defined as an absolute neutrophil count of 500polymorphonuclear


Page 45 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


45

leukocytes/cm3 of blood. The bacterial suspension was diluted to a concentration of 106 CFU/mL with normal saline. Then, 0.1 ml of the bacterial suspension was injected into each posterior thigh muscle 2 h after the second dose of cyclophosphamide was administered.63,64 Infected mice were given compound 12b at different doses (3 and 5 mg/kg body weight) dissolved in sterile water as vehicle at a volume of 0.1 ml by single bolus intravenous injection in the tail vein 2h after bacterial inoculation. Twenty-four hours after drug treatment was begun, the mice were humanely sacrificed. Right thigh muscles from each mouse were aseptically collected, homogenized and serially diluted 1:10 in phosphate buffer saline, and processed for quantitative cultures. ASSOCIATED CONTENT Supporting Information. 1

H NMR, 13C NMR and HRMS copies of compounds 3a-12e; Elemental analysis of compounds

10a-12e; MIC of clinical E. coli strains against standard antibiotics; MIC of water borne E. coli strains against standard antibiotics; Docking Scores along with binding sites; Relaxation Assay; Structure of 12b; The atom based alignment; Docking poses of bisbenzimidazole compounds. (A)11b, (B)11g, (C)11h, (D)12a, (E)12b and (F)12c with EcTopo 1A enzyme; Schematic diagram of (A) DNA cleavage and (B) DNA religation step in EcTopo 1A enzyme; Electrostatic potential maps; Fluorescence Titration of 12b with dsDNA. This material is available free of charge via the Internet at http://pubs.acs.org.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

46

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected] Present Address †ҡ

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi – 110 067

Author Contributions The manuscript was written through contributions of all authors. First, Second and third authors have equal contributions. All authors have given approval to the final version of the manuscript. Funding Sources The authors are greatful to the Council of Scientific and Industrial Research and Indian Council of Medical Research, Delhi, India.

ACKNOWLEDGMENT We gratefully acknowledge the University of Delhi, CSIR and ICMR for financial support and USIC for providing instrumentation facilities. Conflict of interest Disclosure The authors declare no competing financial interest.

ABBREVIATIONS USED MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; EcTopo 1A, E. coli topoisomerase IA; hTop 1, human topoisomerase IB; UTI, urinary tract infection; IC50, Inhibitory concentration; Hoechst 33258, 2-(4-hydoxyphenyl)-5-[5-(4 methylpiperazin-1-


Page 46 of 73

Page 47 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

47


yl)-1H-benzimidazol-2-yl]-1H-benzimidazole;

Hoechst

33342,

2-(4-ethoxyphenyl)-5-[5-(4

methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole.

REFERENCES

1. Reck, F.; Alm, R. A.; Brassil, P.; Newman, J. V.; Ciaccio, P.; McNulty, J.; Barthlow, H.; Goteti, K.; Breen, J.; Comita-Prevoir, J.; Cronin, M.; Ehmann, D. E.; Geng, B.; Godfrey, A. A.; Fisher, S. L. Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II with reduced pK(a): antibacterial agents with an improved safety profile. J. Med. Chem. 2012, 55, 6916-6933. 2. Black, M. T.; Hodgson, J. Novel target sites in bacteria for overcoming antibiotic resistance. Adv. Drug Deliv. Rev. 2005, 57, 1528-1538. 3. Lima, C. D.; Wang, J. C.; Mondragon, A. Three-dimensional structure of the 67K Nterminal fragment of E. coli DNA topoisomerase I. Nature 1994, 367, 138-146. 4. Stewart, L.; Redinbo, M. R.; Qiu, X.; Hol, W. G.; Champoux, J. J. A model for the mechanism of human topoisomerase I. Science 1998, 279, 1534-1541. 5. Champoux, J. J. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001, 70, 369-413. 6. Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 2010, 17, 421-433. 7. Mitscher, L. A. Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial agents. Chem. Rev. 2005, 105, 559-592. 8. Talbot, G. H.; Bradley, J.; Edwards, J. E., Jr.; Gilbert, D.; Scheld, M.; Bartlett, J. G. Bad bugs need drugs: an update on the development pipeline from the Antimicrobial



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

48

Page 48 of 73

Availability Task Force of the Infectious Diseases Society of America. Clin. Infect. Dis. 2006, 42, 657-668. 9. Tse-Dinh, Y. C. Bacterial topoisomerase I as a target for discovery of antibacterial compounds. Nucleic Acids Res. 2009, 37, 731-737. 10. Nitiss, J. L. DNA topoisomerases in cancer chemotherapy: using enzymes to generate selective DNA damage. Curr. Opin. Investig. Drugs 2002, 3, 1512-1516. 11. Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 1989, 58, 351-375. 12. Drlica, K.; Malik, M.; Kerns, R. J.; Zhao, X. Quinolone-mediated bacterial death. Antimicrob. Agents Chemother. 2008, 52, 385-392. 13. Cheng, B.; Cao, S.; Vasquez, V.; Annamalai, T.; Tamayo-Castillo, G.; Clardy, J.; TseDinh, Y. C. Identification of anziaic acid, a lichen depside from Hypotrachyna sp., as a new topoisomerase poison inhibitor. PLoS. One. 2013, 8, e60770. 14. Cheng, B.; Liu, I. F.; Tse-Dinh, Y. C. Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I. J. Antimicrob. Chemother. 2007, 59, 640-645. 15. Black, M. T.; Stachyra, T.; Platel, D.; Girard, A. M.; Claudon, M.; Bruneau, J. M.; Miossec, C. Mechanism of action of the antibiotic NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II topoisomerases. Antimicrob. Agents Chemother. 2008, 52, 3339-3349. 16. Black, M. T.; Coleman, K. New inhibitors of bacterial topoisomerase GyrA/ParC subunits. Curr. Opin. Investig. Drugs 2009, 10, 804-810.


Page 49 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


49

17. Miles, T. J.; Barfoot, C.; Brooks, G.; Brown, P.; Chen, D.; Dabbs, S.; Davies, D. T.; Downie, D. L.; Eyrisch, S.; Giordano, I.; Gwynn, M. N.; Hennessy, A.; Hoover, J.; Huang, J.; Jones, G.; Markwell, R.; Rittenhouse, S.; Xiang, H.; Pearson, N. Novel cyclohexyl-amides as potent antibacterials targeting bacterial type IIA topoisomerases. Bioorg. Med. Chem. Lett. 2011, 21, 7483-7488. 18. Aldred, K. J.; McPherson, S. A.; Turnbough, C. L., Jr.; Kerns, R. J.; Osheroff, N. Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance. Nucleic Acids Res. 2013, 41, 4628-4639. 19. Bax, B. D.; Chan, P. F.; Eggleston, D. S.; Fosberry, A.; Gentry, D. R.; Gorrec, F.; Giordano, I.; Hann, M. M.; Hennessy, A.; Hibbs, M.; Huang, J.; Jones, E.; Jones, J.; Brown, K. K.; Lewis, C. J.; May, E. W.; Saunders, M. R.; Singh, O.; Spitzfaden, C. E.; Shen, C.; Shillings, A.; Theobald, A. J.; Wohlkonig, A.; Pearson, N. D.; Gwynn, M. N. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 2010, 466, 935-940. 20. Brvar, M.; Perdih, A.; Renko, M.; Anderluh, G.; Turk, D.; Solmajer, T. Structure-based discovery of substituted 4,5’-bithiazoles as novel DNA gyrase inhibitors. J. Med. Chem. 2012, 55, 6413-6426. 21. Guo, C.; Pairish, M.; Linton, A.; Kephart, S.; Ornelas, M.; Nagata, A.; Burke, B.; Dong, L.; Engebretsen, J.; Fanjul, A. N. Design of oxobenzimidazoles and oxindoles as novel androgen receptor antagonists. Bioorg. & Med. Chem. Lett. 2012, 22, 2572-2578. 22. Garcia, M. T.; Blazquez, M. A.; Ferrandiz, M. J.; Sanz, M. J.; Silva-Martin, N.; Hermoso, J. A.; de la Campa, A. G. New alkaloid antibiotics that target the DNA topoisomerase I of Streptococcus pneumoniae. J. Biol. Chem. 2011, 286, 6402-6413.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

50

Page 50 of 73

23. Tawar, U.; Bansal, S.; Shrimal, S.; Singh, M.; Tandon, V. Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA. Mol. Cell Biochem. 2007, 305, 221-233. 24. Singh, M.; Tandon, V. Synthesis and biological activity of novel inhibitors of topoisomerase I: 2-aryl-substituted 2-bis-1H-benzimidazoles. Eur. J. Med. Chem. 2011, 46, 659-669. 25. Bansal, S.; Sinha, D.; Singh, M.; Cheng, B.; Tse-Dinh, Y. C.; Tandon, V. 3,4dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I. J. Antimicrob. Chemother. 2012, 67, 2882-2891. 26. Chen, A. Y.; Yu, C.; Bodley, A.; Peng, L. F.; Liu, L. F. A new mammalian DNA topoisomerase I poison Hoechst 33342: cytotoxicity and drug resistance in human cell cultures. Cancer Res. 1993, 53, 1332-1337. 27. Durand, R. E.; Olive, P. L. Cytotoxicity, mutagenicity and DNA damage by Hoechst 33342. J. Histochem. Cytochem. 1982, 30, 111-116. 28. Singh, M.; Sur, S.; Rastogi, G. K.; Jayaram, B.; Tandon, V. Bi and tri-substituted phenyl rings containing bisbenzimidazoles bind differentially with DNA duplexes: a biophysical and molecular simulation study. Mol. BioSyst. 2013, 9, 2541-2553. 29. Ozbey, S.; Kaynak, F. B.; Kus, C.; Goker, H. 2-(2,4-Dichlorophenyl)-5-fluoro-6morpholin-4-yl-1H-benzimidazole monohydrate. Acta Crystallogr., Sect. E 2002, 58, 1062-1064 30. Kelly, D. P.; Bateman, S. A.; Martin, R. F.; Reum, M. E.; Rose, M.; Whittaker,A. R., DNA binding compounds. synthesis and characterization of boron-containing


Page 51 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


51

bibenzimidazoles related to the DNA minor groove binder, Hoechst 33258 Aust. J. Chem. Abstr. 1994, 47, 247-262 31. Kelly, D. P.; Bateman, S. A.; Hook, R. J.; Martin, R. F.; Reum, M. E.;Rose, M.; Whittaker, A. R. D. DNA binding compounds: synthesis and characterization of 2,5'disubstituted bibenzimidazoles related to the DNA minor groove binder Hoechst 33258. Aust. J. Chem. 1994, 47, 1751-1769. 32. Kim, J. S.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J. Substituted 2,5'-bi-1Hbenzimidazoles: topoisomerase I inhibition and cytotoxicity. J. Med. Chem. 1996, 39, 992-998. 33. Es, T. van, Staskun, B. Reductions with Raney Alloy in acid solution. J. Chem. Soc. 1965, 5775-5777. 34. Vega, M. C.; Garcia, S., I; Aymami, J.; Eritja, R.; Van der Marel, G. A.; Van Boom, J. H.; Rich, A.; Coll, M. Three-dimensional crystal structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor-groove-binding drug Hoechst 33258. Eur. J. Biochem. 1994, 222, 721-726. 35. Clark, G. R.; Squire, C. J.; Gray, E. J.; Leupin, W.; Neidle, S. Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2. Nucleic Acids Res. 1996, 24, 4882-4889. 36. Boege, F.; Straub, T.; Kehr, A.; Boesenberg, C.; Christiansen, K.; Andersen, A.; Jakob, F.; Kohrle, J. Selected novel flavones inhibit the DNA binding or the DNA religation step of eukaryotic topoisomerase I. J. Biol. Chem. 1996, 271, 2262-2270. 37. Clark, G. R.; Boykin, D. W.; Czarny, A.; Neidle, S. Structure of a bis-amidinium derivative of hoechst 33258 complexed to dodecanucleotide d(CGCGAATTCGCG)2: the


52


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 73

role of hydrogen bonding in minor groove drug-DNA recognition. Nucleic Acids Res. 1997, 25, 1510-1515. 38. Sissi. C; Palumbo. M. Effect of Magnesium and related divaltent metal ions in topoisomerase structure and function. Nucleic Acids Research, 2009, 37, No. 3. 702–711. 39. Domainico, P.L.; Tse Dinh Y. C. Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherechia coli DNA topoisomerase I: product and rate analysis. Biochemistry, 1988, 27, 6365-6371. 40. Yeung, K.; Meanwell, N.A.;

Qiu, Z.; Hernandez, D.; Zhang, S.;

McPhee, F.;

Weinheimer, S.; Clark. J.M. Structure activity relationship studies of a bisbenzimidazole based, Zn2+ dependent inhibitor of HCV NS3 serine protease. Bioorganic and medicinal Chemistry letters, 2002, 11, 2355-2359. 41. Lafitte, D.; Lamour, V.; Tsvetkov, P.; Makarov, A.; Klich, M.; Deprez, P.; Moras, D.; Briand, C.;‡ and Robert, G. DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5’-methyl group of the noviose. Biochemistry, 2002, 41, 7217-7223. 42. Miller, J. R.; Herberg, J. T.; Tomilo, M.; McCroskey. M. C.; Feilmeier, B. Streptococcus pneumononiae gyrase ATPase: development and validation of an assay for inhibitor discovery and characterization. J. Analytical Biochemistry, 2007, 365, 132–143. 43. Zhu, C. X.; Tse-Dinh, Y. C. The acidic triad conserved in type IA DNA topoisomerases is required for binding of Mg(II) and subsequent conformational change. J. Biol. Chem. 2000, 275, 5318-5322.


Page 53 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


53

44. Zhang, Z.; Cheng, B.; Tse-Dinh, Y. C. Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I. Proc. Natl. Acad. Sci. U. S. A 2011, 108, 6939-6944. 45. Baker, N. M.; Rajan, R.; Mondragon, A. Structural studies of type I topoisomerases. Nucleic Acids Res. 2009, 37, 693-701. 46. Sorokin, E. P.; Cheng, B.; Rathi, S.; Aedo, S. J.; Abrenica, M. V.; Tse-Dinh, Y. C. Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region. Nucleic Acids Res. 2008, 36, 4788-4796. 47. Kakkar, R.; Suruchi; Grover, R. Theoretical study of molecular recognition by Hoechst 33258 derivatives. J. Biomol. Struct. Dyn. 2005, 23, 37-47. 48. Chen, S. J.; Wang, J. C. Identification of active site residues in Escherichia coli DNA topoisomerase I. J. Biol. Chem. 1998, 273, 6050-6056. 49. Wolber, G.; Dornhofer, A. A.; Langer, T. Efficient overlay of small organic molecules using 3D pharmacophores. J. Comput. Aided Mol. Des 2006, 20, 773-788. 50. Perdih, A.; Wolber, G.; Solmajer, T. Molecular dynamics simulation and linear interaction energy study of D-Glu-based inhibitors of the MurD ligase. J. Comput. Aided Mol. Des 2013, 27, 723-738. 51. Clinical Laboratory and Standards Institut. Methods for Dilution Suceptible Test for Bacteria that grow Aerobically 8th ed.; Approved Standard M07-A8; Clinical Laboratory and Standards Institut: Wayne, PA, USA 2009.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

54

Page 54 of 73

52. Annamalai, T.; Dani, N.; Cheng, B.; Tse-Dinh, Y. C. Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol. BMC. Biochem. 2009, 10, 18. 53. Reimer, L. G.; Stratton, C. W.; Reller, L. B. Minimum inhibitory and bactericidal concentrations of 44 antimicrobial agents against three standard control strains in broth with and without human serum. Antimicrob. Agents Chemother. 1981, 19, 1050-1055. 54. Domanico, P. L.; Tse-Dinh, Y. C. Mechanistic studies on E. coli DNA topoisomerase I: divalent ion effects. J. Inorg. Biochem. 1991, 42, 87-96. 55. Castelli, S.; Vieira, S.; D'Annessa, I.; Katkar, P.; Musso, L.; Dallavalle, S.; Desideri, A. A derivative of the natural compound kakuol affects DNA relaxation of topoisomerase IB inhibiting the cleavage reaction. Arch. Biochem. Biophys. 2013, 530, 7-12. 56. Mancini, G.; D'Annessa, I.; Coletta, A.; Chillemi, G.; Pommier, Y.; Cushman, M.; Desideri, A. Binding of an indenoisoquinoline to the topoisomerase-DNA complex induces reduction of linker mobility and strengthening of protein-DNA interaction. PLoS. One. 2012, 7, e51354. 57. Sandeep, G.; Nagasree, K. P.; Hanisha, M.; Kumar, M. M. AUDocker LE: A GUI for virtual screening with AUTODOCK Vina. BMC. Res. Notes 2011, 4, 445. 58. Kaplan, W.; Littlejohn, T. G. Swiss-PDB Viewer (Deep View). Brief. Bioinform. 2001, 2, 195-197. 59. Wolber, G.; Langer, T. LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters. J. Chem. Inf. Model. 2005, 45, 160-169.


Page 55 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55

60. Reyes, N.; Aggen, J. B.; Kostrub, C. F. In vivo efficacy of the novel aminoglycoside ACHN-490 in murine infection models. Antimicrob. Agents Chemother. 2011, 55, 17281733. 61. Xiao, X. Y.; Hunt, D. K.; Zhou, J.; Clark, R. B.; Dunwoody, N.; Fyfe, C.; Grossman, T. H.; O'Brien, W. J.; Plamondon, L.; Ronn, M.; Sun, C.; Zhang, W. Y.; Sutcliffe, J. A. Fluorocyclines. 1. 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: a potent, broad spectrum antibacterial agent. J. Med. Chem. 2012, 55, 597-605. 62. Andes, D.; Craig, W.; Nielsen, L. A.; Kristensen, H. H. In vivo pharmacodynamic characterization of a novel plectasin antibiotic, NZ2114, in a murine infection model. Antimicrob. Agents Chemother. 2009, 53, 3003-3009. 63. Zuluaga, A. F.; Salazar, B. E.; Rodriguez, C. A.; Zapata, A. X.; Agudelo, M.; Vesga, O. Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases. BMC. Infect. Dis. 2006, 6, 55. 64. Reyes, N.; Aggen, J. B.; Kostrub, C. F. In vivo efficacy of the novel aminoglycoside ACHN-490 in murine infection models. Antimicrob. Agents Chemother. 2011, 55, 17281733.


56


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 73

Figure 1. N N H

N N

N H OH

Hoechst 33258 N N

N

N

N

N H

N H Hoechst 33342

Figure 2.


OC 2H 5

Page 57 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.


57


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


58

Page 58 of 73

Page 59 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4

.


59


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5.


60

Page 60 of 73

Page 61 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6.


61


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1.

Scheme 2.


62

Page 62 of 73

Page 63 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

63


Scheme 3. NH2

R3 NH2

N R2

R1

N H

OHC

N N

9a-9c

5a-5j (a)

N N R1

N H

N

N R3

N H

R2

10a-j, 11a-i, 12a-e 10a 10b 10c 10d 10e 10f 10g 10h 10i 10j

R1= ethyl, R2 = OH, R3 = H R1 = propyl, R2 = OH, R3 = H R1 = N,N-dimethylaminoethyl, R2 = OH, R3 = H R1 = N-trif luoroacetylaminoethyl, R2 = OH, R3 = H R1 = phenyl, R2 = OH, R3 = H R1 = 2-cyanophenyl, R2 = OH, R3 = H R1 = 4-fluorophenyl, R2 = OH, R3 = H R1 = 4-pyridyl, R2 = OH, R3 = H R1 = 2-pyrimdyl, R2 = OH, R3 = H R1 = 4-aminophenyl, R2= OH, R3 = H

12a 12b 12c 12d 12e

R1 = ethyl, R2 = OEt, R3 = H R1 = propyl, R2 =OEt, R3 = H R1 = N,N-dimethylaminoethyl, R2 = OEt, R3= H R1 = N-trif luoroacetylaminoethyl, R2 = OEt, R3 = H R1 = 4-pyridyl, R2= OEt, R3 = H

11a R1 = ethyl, R2= OMe, R3 = OMe 11b R1 = propyl, R2 = OMe, R3 = OMe 11c R1 = N,N-dimethylaminoethyl, R2 = OMe, R3 = OMe 11d R1 = phenyl, R2 = OMe, R3 = OMe 11e R1 = 2-cyanophenyl, R2 = OMe, R3 = OMe 11f R1 = 4-f luorophenyl, R2 = OMe, R3 = OMe 11g R1 = 4-pyridyl, R2 = OMe, R3 = OMe 11h R1 = 2-pyrimdyl, R2 = OMe, R3 = OMe 11i R1 = 4-aminophenyl, R2 = OMe, R3 = OMe


64


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 64 of 73

Table 1: Susceptibility of Standard E. coli Strains against Bisbenzimidazole Analogs and their Topoisomerase I Inhibitory Activity (IC50) Compounds

MIC50 (µg/mL) DH5α ATCC 25922

MIC90 (µg/mL) DH5α ATCC 25922

MBC (µg/mL) DH5α ATCC 25922

IC50 (µM)

64

>128

120.9

−

128

−

5.8

>128

>128

−

−

−

−

20.8

64

>128

−

−

−

−

8

>128

>128

−

−

−

−

45.8

>128

>128

−

−

−

−

60.3

>128

>128

−

−

−

−

20.4

>128

>128

−

−

−

−

20.32

>128

>128

−

−

−

−

>50

10a

10b

10c

10d

10e

10f

10g

10h

Contd.


Page 65 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

65


>128

>128

−

−

−

−

>50

128

>128

−

−

−

−

4.7

8

16

1.4

14.8

16

32

12.32

2

8

3.2

12.5

4

16

3.0

128

128

−

−

−

−

3.0

>128

>128

−

−

−

−

3.8

>128

>128

−

−

−

−

3.76

>128

>128

−

−

−

−

>50

0.5

2.00

1.8

3.7

1

4

6.2

10i

10j

11a

11b

11c

11d

11e

11f

11g

Contd.


66


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 66 of 73

2.00

32

3.6

49

4

64

6.2

>128

>128

−

−

−

−

20.3

0.5

1.78

0.7

3.1

1

4

5.8

0.2

0.2

0.8

3.3

0.2

0.4

2.0

1

8

14.5

26.6

2

16

9.4

16

10.4

22.5

20.8

32

32

50.8

>128

>128

−

−

−

−

>50

11h

11i

12a

12b

12c

12d

12e


Page 67 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

67


Table2. Susceptibility of clinical E. coli Strains against Potent Bisbenzimidazole Analogs E. coli

11b

11g

11h

12a

12b

12c

strain

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

R

2

3.4

4

1

1.6

2

16

30.1

32

4

7.2

8

2

3.1

4

32

57.2

64

118R

2

3.2

4

1

1.7

2

4

7.4

8

1

1.4

2

1

1.7

2

8

15.2

16

85R

8

15.2

16

2

3.6

4

32

55.8

64

4

6.9

8

1

1.7

2

16

28.6

32

360R

8

15.2

16

1

1.7

2

>128

-

-

3

58.1

64

1

1.6

2

64

120.8

128

451R

72

4

7.6

8

2

3.6

4

16

28.8

32

2

3.6

4

1

1.7

2

32

55.3

64

R

4

7.5

8

1

3.6

4

16

30.2

32

4

7.4

8

2

3.4

4

16

28.8

32

81R

8

14.5

16

2

3.1

4

16

29.7

32

4

6.5

8

1

1.6

2

32

60.2

64

132R

64

118

128

2

3.7

4

64

122

128

4

7.2

8

2

3.5

4

64

122.6

128

151R

8

15.1

16

1

1.7

2

16

30.4

32

4

6.8

8

1

1.7

2

32

61.6

64

401R

4

7.2

8

1

1.6

2

16

29

32

2

3.7

4

1

1.7

2

16

30.4

32

555R

16

29.8

32

16

58.6

64

32

59.4

64

8

13.9

16

4

6.8

8

32

60.4

64

59

R

R

R

R

R, resistant; E. coli 72 resistant to CIP; 118 resistant to AMP, TMP, NAL; E. coli 85 resistant to CHL, AMP, KAN, TMP, CIP, NAL; E. coli 360 resistant to GEN, KAN, TET, NAL; E. coli 451R resistant to AMP, TET, GEN; E. coli 59R resistant to CHL, AMP, TMP, CIP, NAL; E. coli 81R resistant to AMP, KAN, TMP, TET, CIP, NAL; E. coli 132R resistant to CHL, AMP, KAN, TMP, CIP, NAL; E. coli 151R resistant to CHL, AMP, KAN, TMP, CIP, NAL; E. coli 401R resistant to AMP, CHL, CIP, GEN, TET; E. coli 555R resistant to AMP, CIP, GEN, TET MIC and MBC were done in triplicate and average of the three experiments are depicted. AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; NAL, Nalidixic acid; TMP, trimethoprim; TET, tetracycline;


68


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 68 of 73

Table 3: Susceptibility of Water Borne E. coli Strains against Potent Bisbenzimidazole Analogs E. coli strain

IS 54R WB31R

KP21R

NG3R

ISTR

IP18R

IP9R

KK45R

KK16R

MKNJR

11b

11g

11h

12a

12b

12c

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

MIC50

MIC90

MBC

8

14.8

16

1

1.7

2

32

61.6

64

4

7.1

8

0.5

1.6

2

32

60.6

64

8

15.2

16

2

3.7

4

16

30.2

32

4

7.2

8

0.5

1.7

2

16

29.4

32

8

14.8

16

2

3.6

4

32

60.5

64

4

6.8

8

0.5

3.8

4

32

61.4

64

7.6

8

1

1.8

2

16

30.6

32

4

7.7

8

0.5

1.7

2

8

15

16

8

14.9

16

0.5

0.8

1

16

30.8

32

4

3.7

8

0.5

1.7

2

32

60.6

64

4

7.6

8

2

3.7

4

16

30.8

32

4

7.6

8

1

3.4

4

16

30.6

32

8

15.4

16

2

3.6

4

128

-

-

4

7.2

8

1

3.1

4

64

118

128

4

7.7

8

2

3.7

4

16

30.2

32

4

7.5

8

0.5

1.7

2

16

29.4

32

2

3.7

4

1

1.7

2

16

30.4

32

1

1.8

2

0.5

1.8

2

16

27.6

32

4

7.6

8

2

3.7.

4

32

61.4

64

4

7.6

8

1

3.5

4

16

30.6

32

4

R, resistant; E. coli WB31R resistant to AMP, TET, TMP, PIP; E. coli KP21R resistant to AMP, ,TMP, CFZ,PIP, CFU. CFX, CFP CFT, E. coli NG3R resistant to CFZ, AMP, PIP, CFU, CFP, CFT, TET, CIP, AMP; E. coli MKNJR resistant to AMP,TMP,PIP; E. coli KK45R resistant to AMP, CHL,TET, TMP, CIP,; E. coli KK16R resistant to CFZ, AMP, PIP, CFU, CFP, CFT, TET, TMP; E. coli ISTR resistant to TET, TMP; E. coli IP18R resistant to AMP, TET. MIC and MBC were done in triplicate and average of the three experiments is depicted. AMP, ampicillin; CFZ, cefazolinl; PIP, piperacillin; CFU; cefuroxime; CFX, cefoxitine; CFP, cefepime; CFT, cefotaxime; AMP-CA, ampicillin+clavulanic acid. AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; KAN, kanamycin; TMP, trimethoprim; TET, tetracycline.Accession ID details: IS54;JX195708, WB31; JX195715, KP21; JX195712, NG3; JX195710, IST; JX195709, IP18; JX195706, IP9; JQ935969, KK45; JQ935973, KK16; JQ935972, MKNJ; JX195713


Page 69 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

69


Table 4: Thermodynamic Parameters of the 12b Binding to the EcTopo 1A in 20 mM TrisCl, 50 mM KCl, 10% Glycerol, 0.1mM DTT (pH 7.9) from ITCa Protein used

MgCl2 KCl (mM) (mM)

EcTopo 1A

10

50

Binding constant 6 -1 Ka x 10 (M ) 6.8 ± 0.46

∆H (Kcal/mol)

T∆S (Kcal/ mol)

∆G (Kcal/mol)

-22.78 ± 0.07

-11.94

-10.84

a

The data in this table are average of four determinations. The values of ∆G and T∆S were determined using the equations and ∆G =∆H-T∆S and T∆S = ∆H-∆G. All the ITC profiles were fit to a model of single binding sites. Uncertainties correspond to regression standard errors.

Table 5. In vivo Efficacy of Compound 12b S.No. Model Strain

12b

1

Septicemia

ATCC 25922

MIC50 (µg/mL) ED50 (mg/Kg)

0.2±0.024 5

2

Neutropenic ATCC thigh 25922

MIC50 (µg/mL)

0.2±0.024

Dose at 1 log reduction (mg/kg bw/day)

3 5

Dose at 3 log reduction (mg/kg bw/day)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

70

Page 70 of 73

SCHEMES/FIGURES LEGENDS Scheme 1. Synthesis of different 4-(4-substitutedpiperazinyl)-1,2-phenylenediamine derivatives Scheme 2. Synthesis of different 2-aryl-5-formylbenzimidazoles Scheme 3. Synthesis of target bisbenzimidazoles Figure 1. Chemical Structures of Hoechst 33258 and 33342. Figure 2. Scientific approach of Present Study Figure 3. Inhibition of Relaxation Activity of EcTopo 1A in presence of different compounds 11b,11g, 11h, 12a, 12b,12c. FR (fully relaxed); PR: (partially relaxed); SC: (super coiled). (A) Ethidium bromide stained agarose gel showing lane 1: pHOT1 plasmid DNA (C), lane 2: relaxation of plasmid DNA by EcTopo 1A (CT), lane 3: plasmid DNA in presence of compound (CL), lane 4-10: Inhibition of relaxation of Plasmid DNA by Ec TopoIA in presence of 1,5,10,25,50,75 and 100µM of compounds respectively. (B) The % inhibition values as averages of results obtained from three independent experiments. Error bars denote the standard deviation. The graphical representation shows the data till 50 µM. (C) Ethidium bromide stained agarose gel showing influence of Mg2+ concentration on compound 12b in inhibiting EcTopo 1A. Lane 1-10: Inhibition of relaxation activity EcTopo 1A in presence of 5 µM compound 12b and 0, 1, 5, 10, 15, 20, 25, 30, 40, 50 mM of MgCl2 respectively. (D) Analysis of supercoiling of relaxed pHOT I plasmid with E. coli DNA gyrase in the presence of compounds. Ethidium bromide stained agarose gel showing lane 1: pHOT I relaxed plasmid DNA (C), lane 2: supercoiling of relaxed pHOT I plasmid DNA by E. coli DNA gyrase (CG), lane 3: relaxed pHOT I plasmid


Page 71 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


71

DNA in presence of compound, lane 4-10: supercoiling inhibition of relaxed pHOT I Plasmid DNA by E.coli DNA gyrase in presence of 1,5,10,25,50,75 and 100 µM of compounds respectively. (E) Inhibition of relaxation activity of hTop 1 in presence of different compounds. Ethidium bromide stained agarose gel showing lane 1: pHOT1 plasmid DNA lane 2: Relaxation of pHOT1 plasmid DNA by hTop 1 lane 3: plasmid DNA in presence of compound, lane 4-10 inhibition of relaxation of plasmid DNA by hTop 1 in presence of 1, 5, 10, 25, 50, 75 and 100 µM concentrations of compound respectively. Figure 4. (A) ITC titration of EcTopo 1A (1.0 x 10-5 M) with 12b (4 x 10-4 M) at 25°C in 20 mM Tris-Cl, 50 mM KCl, 10% Glycerol, 0.1mM DTT and 10 mM MgCl2(pH 7.9), (B) Binding isotherm derived from top panel. Figure 5. Docked pose of the ternary complex of 12b with E. coli topoisomerase-IA-dsDNA. (A) Docked structure of 12b with EcTopo 1A-dsDNA, (B) Inset docked pose showing interaction of 12b with Glu9, Pro11, Glu313, Arg321, His365; all the structures are coloured by element, and relevant structures are labeled. Dotted lines indicated interaction between 12b and residues whereas arrows indicate hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) sites. Figure 6. (A) Efficacy of compound 12b in mouse systemic infection model. Graphical representation of percentage survival versus drug dose (mg/kg body weight). (B) Efficacy of compound 12b in mouse neutropenic thigh model. Bar graph showing Log CFU/g Thigh versus drug dose. The standard deviations and errors were calculated.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

72

FOOTNOTE SCHEMES Scheme 1. Reagents and conditionsa: (a) (Et)3N, DMSO, 120 ºC; (b) 10% H2SO4, 80 ºC; (c) 10% Pd/C, EtOAc:MeOH (4:1). Scheme 2. Reagents and conditionsb: (a) Na2S2O5 in water, ethanol, reflux; (b) Ni-Al alloy, 75% HCOOH, 95 ºC. Scheme 3. Reagents and conditionsc: (a) Na2S2O5 in water, ethanol, reflux


Page 72 of 73

Page 73 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract


73