Synthesis and biological evaluation of novel bisbenzimidazoles as Escherichia coli topoisomerase IA inhibitors and potential antibacterial agents

Article abstract of DOI:10.1021/jm5003028

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 analogues as Escherichia 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 (IC ₅₀ = 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 up to 100 μM. 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 × 10⁶ M^-1 and -10.84 kcal mol^-1 from 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.

Full text of DOI:10.1021/jm5003028

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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
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Synthesis and Biological Evaluation of Novel
Bisbenzimidazoles as Escherichia coli
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Topoisomerase IA Inhibitors and Potential
Antibacterial Agents
†
χ
†χ
†χ
†
††
Hemlata Nimesh, Souvik Sur, Devapriya Sinha, Pooja Yadav, Prachi Anand, Priyanka
§
§
† ,ҡ
Bajaj, Jugsharan S. Virdi, Vibha Tandon *
†
††
Department of Chemistry, University of Delhi, Delhiꢀ110 007, India. Department of Chemistry
& Biochemistry, CUNYꢀHunter College, New York, NY 10065 USA.
§
Department of Microbiology, University of Delhi, Delhiꢀ110 021, India.
ҡ
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi – 110 067.
χ
First, second and third authors have equal contributions.
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KEYWORDS
Bisbenzimidazoles, Piperazine, E. coli topoisomerase, Docking, Antibacterial agent
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ABSTRACT
Novel bisbenzimidazole inhibitors of bacterial type IA topoisomerase are of interest for the
development of new antibacterial agents that are impacted by targetꢀmediated cross resistance
with fluoroquinolones. The present study demonstrates the successful synthesis and evaluation of
bisbenzimidazole analogs as E. coli topoisomerase IA inhibitors. 5ꢀ(4ꢀPropylpiperazinꢀ1ꢀyl)ꢀ2ꢀ
[2’ꢀ(4ꢀethoxyphenyl)ꢀ5’ꢀbenzimidazolyl]benzimidazole (12b) showed significant relaxation
inhibition activity against EcTopo 1A (IC = 2 ± 0.005 ꢁM) and a tendency to chelate metal ion.
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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
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ꢀ1
ꢀ 1
with EcTopo 1A was observed 6.8 x 10 M and ꢀ10.84 Kcal mol from 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
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strategy in the development of new therapies. DNA topoisomerases are ubiquitous enzymes
which utilize an active site tyrosine residue for nucleophilic attack on DNA phosphodiester
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linkage resulting in formation of covalent proteinꢀDNA intermediate. 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,
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which trigger cell death. 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
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exploited by various antibacterial agents.
Topoisomerase I targeting poisons may be
particularly effective when the bacterial pathogen is responding to host defense, or in the
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presence of other antibiotics that induce the bacterial stress response. 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
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and cleaved DNA.
Recently anziac acid, an aqueous Lichen Depside obtained from
hypotrachyma species was identified as a potent Bacillus subtillis and E. coli topoisomerase I
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(EcTopo 1A) inhibitor. TseꢀDinh 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
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topoisomerase IA.
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
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fluoroquinolones as they have different mechanism of inhibition. But the major lead compound
of Aventis program was (3R,4R)ꢀ4ꢀ[(S)ꢀ3ꢀ(3ꢀfluoroꢀ6ꢀmethoxyꢀquinolinꢀ4ꢀyl)ꢀ3ꢀhydroxyꢀ
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propyl]ꢀ1ꢀ[2ꢀ(thiophenꢀ2ꢀylsulfanyl)ꢀethyl]ꢀpiperidineꢀ3ꢀcarboxylic acid (NXLꢀ101) having
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quinoline nucleus had to be discontinued due to QT interval prolongation.
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 crossꢀresistance between NBTIs and
fluroquinolones and paved the way for the developement of novel non fluoroquinolone
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inhibitors. 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
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against these drugs.
In continuation of above efforts novel class of substituted 4,5’ꢀbithiazoles and 5ꢀ(2ꢀ
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hydroxybenzylidene) rhodanine were observed as potent DNA gyrase inhibitor.
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
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S. pneumoniae taking homologous structure EcTopo 1A as model in consideration.
Our group has reported a bisbenzimidazole, 2ꢀ(3,4ꢀdimethoxyphenyl)ꢀ5ꢀ[5ꢀ(4
methylpiperazinꢀ1ꢀyl)ꢀ1Hꢀbenzimidazolꢀ2ꢀyl]ꢀ1Hꢀbenzimidazole (DMA), a synthetic analogue
of Hoechst 33342, less cytotoxic which does not affect cell viability in mammalian cells up to a
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concentration of 100 µM, inhibits growth of E. coli cultures even at a concentration of 2 µM
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when grown in coꢀculture with human cells.
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,
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Human Topoisomerase IIα (HuTopoIIα) and E. coli gyrase enzyme. The parent analogue
Hoechst 33258 & 33342 (Figure 1) were found to be mutagenic, clastogenic and cytotoxic to
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human cells.
but DMA appeared to be a safer and effective molecule. 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
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AT rich sequences of DNA. 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
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analogues, Hoechst 33342 and 33258, known hTop1 inhibitors. It was imperative to confirm
safety index of 11b, 11gꢀh, 12aꢀc 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).
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RESULTS AND DISCUSSION
Chemistry
Synthetic approaches for the preparation of target bisbenzimidazole compounds and their
intermediates are depicted in Schemes 1ꢀ3. Total 24 bisbenzimidazoles, 10aꢀj, 11aꢀi and 12aꢀe
were synthesized by reacting two intermediates; oꢀphenylenediamine derivatives 5aꢀj and the
respective aldehydes 9aꢀc (Scheme 3) in the presence of sodium metabisulfite (Na S O ) as
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oxidizing agent in 40ꢀ60% yield using reported procedure The Na S O in water forms sodium
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bisulfite (NaHSO ), which forms an adduct with aldehyde, and reacts with diamine to form the
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product. The diamine intermediates 5aꢀj were prepared by nucleophilic substitution reaction of
5ꢀchloroꢀ2ꢀnitroacetanilide 1 with the Nꢀsubstituted piperazines 2aꢀj to provide 2ꢀnitroacetanilide
derivatives 3aꢀj in > 90% yields. Deacetylation of 3aꢀj was done using 10% sulphuric acid to
get 4aꢀj 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
5aꢀk in good yield (Scheme 1). 2ꢀArylꢀ5ꢀcyanoꢀ1Hꢀbenzimidazole 8aꢀc were synthesized in 75ꢀ
90% yield by reacting 4ꢀcyanoꢀ1,2ꢀphenylenediamine 6 with the appropriate substituted
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benzaldehydes 7aꢀc using reported (Scheme 2).
Catalytic reduction of 8aꢀc using NiꢀAl alloy
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in the presence of 75% formic acid in water, provided intermediates 9aꢀc in good yield (Scheme
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2).
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.
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The synthetic analogues of bisbenzimidazoles 11aꢀi and 12aꢀe have shown good antibacterial
potential in comparison to 10aꢀj 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 10aꢀj, with hydroxyl group at para position have less +R effect as compared to 11aꢀi
which have two methoxy group, whereas compounds 12aꢀe 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
11aꢀi have 3,4ꢀdisubstituted methoxy group, but methoxy group at 3ꢀposition have more stronger
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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
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interaction with DNA.
But, we observed through docking of bisbenzimidazoles in EcTopo
1Aꢀds 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 Nꢀ6 of imidazole ring, increasing the chances of protonation of nitrogen.
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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 Nꢀ6, 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.
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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 MIC values in the range
90
of 0.1ꢀ8µg/mL with the best results observed in case of 11g, 12a, and 12b ranging from 0.1ꢀ
4µg/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, 11gꢀh, 12aꢀc 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
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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, 11gꢀh, 12aꢀ
c) showed good inhibitory activity against all the strains tested with comparatively lower MIC ,
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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 0.1ꢀ8 µg/mL when screened against
standard, clinical and waterꢀborne E. coli strains.
Inhibition of DNA Relaxation Activity of EcTopo 1A by bisbenzimidazoles.
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Bisbenzimidazole derivatives were proven to inhibit EcTopo 1A significantly. 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
ꢁ
M) and DNA relaxation products were then resolved by gel electrophoresis on 1% agarose gel.
Out of the total 24 bisbenzimidazoles, compounds 10a, 10c, 10j, 11bꢀe, 11gꢀh, 12aꢀc showed
lower IC values in the range of 1ꢀ10 µM concentration (Table 1). Considering the results of
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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 2ꢁM 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
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centre connecting the anionic centres in the toprim and the anionic centre in the nucleic acids.
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Yuk Ching et. al. have shown Mg dependence on DNA cleavage patterns of Topoisomerase
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A. There are literature reports showing metal chelation activity of bisbenzimidazoles. In
view of above it becomes obligatory to perform metal dependent relaxation assay with EcTopo
1
A using 12b (Figure 3C). The probable reason behind this may be that higher concentration of
0
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Mg 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
2+
2
5mM concentration of Mg (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, 11gꢀh, 12aꢀc 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,
1
2b and 12c did not show any significant inhibition even at 100 µM concentration .
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DNA Binding Study using Fluorescence Spectrscopy. We performed fluorescence
7
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titrations of 12b with poly(AT) sequence and observed high binding constant (7 x 10 M ),
suggesting that 12b is a DNA binding molecule and have higher affinity with AT rich DNA
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(
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
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macromolecules.
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
6
ꢀ1
EcTopo 1A are collated in Table 4. The binding affinity value of 12b is 6.8 x 10 M for
ꢀ1
EcTopo 1A and binding free energy (ꢀG) was ꢀ10.84 kcal mol 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.
ꢀ1
A large enthalpy change (ꢀH) of ꢀ22.78 kcal mol and less entropy change (TꢀS) of ꢀ11.94 kcal
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mol 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.
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Docking and Molecular modeling studies with EcTopo 1AꢀdsDNA complex. Six
different docking experiments were carried out in order to understand mode of interaction of
bisbenzimidazoles with EcTopo 1AꢀdsDNA 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
bisbenzimidaozleꢀenzyme interaction. The threeꢀdimensional 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
ꢀ1
and ꢀ8.57 Kcal mol . 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 S2ꢀS3). 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
4
3
that acidic triad participate in metal coordination, which is important for religation of DNA.
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
4
4
with DNA form the previously determined structures of EcTopo 1A. The Glu9, Asp113,
Glu115 are responsible for DNA breakage and rejoining. Glu9, Asp111, Asp113 and Tyr319
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make a tetrad in the vicinity of DNA double helix. 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
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2
+
phosphates with the help of Mg 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
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46
and Mg ions near the religation site. 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 religationꢀstep in two ways, 1)
benzimidazoles might not be allowing the conformational change in EcTopo 1Aꢀds DNA
complex 2)benzimidazole being a positively charged molecule at physiological pH increase the
4
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positive electrostatic potential significantly and decrease the negative electrostatic potential
around the side chains of the acidic triad of Asp111, Asp113 and Glu115 responsible for binding
2
+
of Mg 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 1AꢀdsDNA complex. Hence, we can hypothesize
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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, 11gꢀh, 12aꢀc used for docking study bind in the similar pocket of
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EcTopo 1A , 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 Nꢀalkyl 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 Cꢀterminal 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, 11gꢀh, 12aꢀc were superimposed and
found (Figure S4) that all six ligands bind in the same pocket in Ec Topo IAꢀdsDNA, suggesting
uniformity of docking protocol. The pharmacophore was created using Ligand Scout application
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framework and concluded the detection of directed hydrogen bonding interactions, lipophilic
regions, three hydrophobic spheres, charge interactions and steric exclusions . Exclusion volume
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spheres were also included to account for the protein environment.
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Mouse Systemic Infection Model. Systemic infection model of Balb/c mice was
8
developed by injecting E. coli ATCC 25922 (0.5 X 10 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 postꢀinfection. Treatment of mice with
compound 12b led to the survival of the mice infected with E. coli ATCC 25922 with an ED of
5
0
5
mg/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
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showing low MIC and MBC against E. coli strains. Out of 24, six compounds 11b, 11gꢀh, 12aꢀc
showed significant inhibition of EcTopo 1A with low MIC , MIC and MBC values against
50
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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 1AꢀdsDNA complex revealed that bisbenzimidazoles used in this study
are positively charged molecules which significantly increased positive electrostatic potential at
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the active site in EcTopo 1A enzyme affecting the Mg affinity of the ternary complex and
2
+
inhibited DNA religation, this fact was further proven by Mg 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
1
13
uncorrected. The H (300/400/500 MHz) and C NMR (300/400 MHz) spectra were recorded in
CDCl , MeOD and DMSOꢀd as the solvent on an ECXꢀ400P, Jeol/Bruker NMR spectrometers
3
6
using CDCl MeOD and DMSOꢀd as an internal standard. Chemical shifts are reported in ppm
3
,
6
1
by assigning the CDCl DMSOꢀd , and MeOD resonance in the H NMR spectrum as 7.26, 2.50
3
,
6
1
3
and 3.31 ppm and in the 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
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Accurate Mass QꢀTOF LC/MS mass spectrometer and IR spectra were taken on a PerkinꢀElmer,
FTꢀIR 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 (Fꢀ254, 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 ODSꢀAQ 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
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(
e.g. Et O, EtOAc, CHCl , MeOH, EtOH, CH CN, DMF, hexane, toluene, etc.) were purchased
2 3 3
from commercial sources and used as stated.
General Procedure for the Synthesis of 2ꢀNitroacetanilide Derivatives (3aꢀj):
2
4
Synthesis of 2ꢀnitroacetanilide derivatives was done using the reported procedure. To a stirred
solution of 5ꢀchloroꢀ2ꢀnitroacetanilide (4.65 mmol) in DMSO (10ml), respective Nꢀsubstituted
piperazine (1.5 equiv) and triethylamine (1.0 equiv) were added. The resulting reaction mixture was
heated at 120ºC for 2ꢀ4 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
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over silica gel (60ꢀ120 mesh size) using ethylacetate: petroleum ether as mobile phase in quantitative
yield of the title compound.
5ꢀ(4ꢀEthylꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3a). Yellow solid; (95% yield, 1.29g); mp
0
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09−110 ºC. H NMR (400 MHz, CDCl ) δ ppm 1.13 (t, J = 7.2 Hz, 3H), 2.28 (s, 3H), 2.46 (q, J
3
=
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 =
1
3
9
2
2
1
6
.6 Hz, 1H), 8.32 (d, J = 3.0 Hz, 1H), 11.01 (bs, 1H). C NMR (100 MHz, CDCl ) δ ppm 11.9,
3
–1
6.0, 41.0, 46.6, 52.2, 102.61, 107.7, 126.0, 128.5, 137.9, 155.5, 169.7. FTIR (KBr,cm ): 3317.49,
971.10, 2818.73, 1700.08, 1612.64, 1576.40, 1540.74, 1492.36, 1451.13, 1384.29, 1312.15,
242.00, 1187.89, 1157.70, 1127.48, 1103.13, 1022.22, 978.24, 856.82, 806.79, 750.43, 717.53,
+
87.76. HRMS (ESI): m/z calcd for C H N O [M + H] 293.1613 obsd 293.1618.
1
4
20
4
3
5ꢀ(4ꢀAllylꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3b). Yellow solid; (98% yield, 1.38g); mp
1
1
43.5−144.5 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 2.14 (s, 3H), 2.45−2.48 (m, 4H), 2.98
6
(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 =
13
2.2, 9.52 Hz, 1H), 7.67 (s, 1H), 7.97 (d, J = 9.52 Hz, 1H), 10.43 (s, 1H). C NMR (100 MHz,
DMSO−d ) 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,
6
–
1
1
69.0. FTIR (KBr,cm ): 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 C H N O [M + H] 305.1613 obsd 305.1615.
1
4
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4
3
5ꢀ(4ꢀ(2ꢀNꢀTrifluoroacetylaminoethyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3c). Yellow
1
solid; (93% yield, 1.74%); mp 181.9−182.9 ºC. H NMR (400 MHz, CDCl ) δ ppm 2.27 (s, 3H),
3
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 =
13
1
0.72 Hz, 1H), 8.33 (d, J = 2.68 Hz, 1H), 10.97 (bs, 1H). C NMR (100 MHz, CDCl ) δ ppm
3
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5.9, 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,
–
1
169.6. FTIR (KBr,cm ): 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,
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+
1
100.84, 976.01, 876.25, 725.11. HRMS (ESI): m/z calcd for C H F N O [M + H] 404.1545
1
6
20
3
5
4
obsd 404.1553.
5
ꢀ[4ꢀ(2ꢀ(N, NꢀDimethylaminoethyl)ꢀ1ꢀpiperazinyl]ꢀ2ꢀnitroacetanilide (3d). Yellow solid;
1
(
80% yield, 1.24g); mp 167.2−168.7 ºC. H NMR (400 MHz, CDCl ) δ ppm 1.73 (s, 6H), 2.27 (s,
3
4
1
4
H), 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,
13
H), 8.32 (d, J = 2.68 Hz, 1H), 11.01 (bs, 1H). C NMR (100 MHz, CDCl ) δ ppm 26.4, 41.3,
3
–1
6.0, 48.1, 102.9, 108.0, 126.3, 128.9, 138.3, 156.2, 170.1. FTIR (KBr,cm ): 3439.34, 3294.69,
3159.61, 2836.94, 1618.69, 1567.04, 1420.31, 1384.63, 1250.59, 1114.24, 1002.01, 935.15,
+
7
89.70, 805.56, 438.24. HRMS (ESI): m/z calcd for C H N O [M + H] 336.2035 obsd
1
6
25
5
3
3
36.2032.
5ꢀ(4ꢀPhenylꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3e). Orangish yellow solid; (90% yield,
1
1
.42g); mp 151.6−152.3ºC. H NMR (500 MHz, CDCl ) δ ppm 2.28 (s, 3H), 3.33−3.34 (m, 4H),
3
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, CDCl ) δ ppm 26.0, 40.9, 46.6, 48.8, 102.8, 107.8, 116.2, 120.4, 126.2, 128.6,
3
–
1
1
29.3, 137.9, 150.6, 155.4, 169.7. FTIR (KBr,cm ): 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,
1
029.40, 977.17, 925.14, 850.54, 801.50, 769.34, 750.14, 695.13, 652.62, 537.39. HRMS (ESI):
+
m/z calcd for C H N O [M + H] 341.1613 obsd 341.1606.
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ꢀ(4ꢀ(2ꢀCyanophenyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3f). Yellow solid; (94% yield,
1
1
.59g); mp 175.9−176.6 ºC. H NMR (400 MHz, CDCl ) δ ppm 2.36 (s, 1H), 3.31−3.34 (m,
3
4
H), 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
0
1
2
3
4
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6
7
8
9
0
1
2
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8
9
0
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8
9
0
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9
0
=
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),
13
8.36 (d, J = 2.92 Hz, 1H), 10.95 (br s, 1H). C NMR (100 MHz, CDCl ) δ ppm 25.9, 46.8, 50.9,
3
1
02.9, 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.
–
1
FTIR (KBr,cm ): 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,
+
9
80.07, 928.33, 751.87. HRMS (ESI): m/z calcd for C H N O [M + H] 366.1566 obsd
1
9
19
5
3
3
66.1556.
5ꢀ(4ꢀ(4ꢀFluorophenyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3g). Yellow solid; (92% yield,
1
1
.53g); mp 130.2−132.5 ºC. H NMR (500 MHz, CDCl ) δ ppm 2.29 (s, 3H), 3.22−3.24 (m, 4H),
3
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
13
Hz, 1H), 8.38 (d, J = 3.0 Hz, 1H), 11.01 (br s, 1H). C NMR (100 MHz, CDCl ) δ ppm 25.9,
3
4
1
1
9
6.7, 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,
–
1
69.6. FTIR (KBr,cm ): 3308.42, 2925.51, 2852.50, 1701.49, 1615.19, 1579.55, 1543.74,
510.30, 1454.43, 1376.22, 1309.63, 1276.54, 1228.64, 1182.62, 1088.11, 1035.07, 975.98,
23.05, 830.81, 751.85, 714.78, 579.02, 528.74. HRMS (ESI): m/z calcd for C H FN O [M +
1
8
19
4
3
+
H] 359.1519 obsd 359.1511.
5ꢀ(4ꢀ(4ꢀPyridyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3h). Yellow solid; (80% yield, 1.27g);
1
mp 179.2−180.9 ºC. H NMR (400 MHz, CDCl ) δ ppm 2.28 (s, 3H), 3.56−3.59 (m, 4H),
3
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
13
=
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). C NMR
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(
100 MHz, CDCl ) δ ppm 26.0, 44.9, 45.7, 102.5, 107.3, 107.8, 126.5, 128.6, 137.8, 149.5,
3
–
1
1
1
7
54.2, 154.8, 169.7. FTIR (KBr,cm ): 3325.90, 2924.84, 2851.54, 1742.65, 1700.42, 1612.90,
578.34, 1539.44, 1460.45, 1316.47, 1279.83, 1221.32, 1186.01, 1086.14, 1029.76, 808.43,
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+
48.59, 540.79. HRMS (ESI): m/z calcd for C H N O [M + H] 342.1566 obsd 342.1568.
1
7
19
5
3
5
ꢀ(4ꢀ(2ꢀPyrimidyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3i). Yellow solid; (94% yield,
1
1
.49g); mp 226.2−227.2 ºC. H NMR (300 MHz, CDCl ) δ ppm 2.29 (s, 3H), 3.60−3.63 (m, 4H),
3
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, CDCl ) δ ppm 26.0, 42.9, 46.3, 102.6, 107.6, 110.5, 126.2, 128.6, 137.9,
3
–
1
1
55.4, 157.8, 161.3, 169.6. FTIR (KBr,cm ): 3432.23, 3318.76, 3134.83, 2906.68, 2855.88,
1
701.16, 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 C H N O [M + H] 343.1518
1
6
18
6
3
obsd 343.1519.
5ꢀ(4ꢀ(4ꢀNitrophenyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroacetanilide (3j). Yellow solid; (97% yield,
1
1.79g); mp 196.2−197.1 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 2.16 (s, 3H), 3.64−3.67 (m,
6
4
H), 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 =
13
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). C NMR (75
MHz, CDCl ) 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,
3
–1
1
53.8, 154.7, 169.7. FTIR (KBr,cm ): 3312.85, 2922.88, 2856.09, 1694.87, 1596.26, 1542.52,
492.05, 1461.41, 1386.67, 1301.24, 1216.61, 1186.73, 1113.72, 1030.10, 994.55, 976.54,
1
921.92, 851.15, 817.51, 751.84, 691.51, 583.73, 519.96. HRMS (ESI): m/z calcd for
+
C H N O [M + H] 386.1464 obsd 386.1482.
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General Procedure for the Synthesis of 2ꢀNitroaniline Derivatives (4a−j). Synthesis
of 2ꢀnitroaniline derivatives was done using the reported procedure. Acetylated compound 3a−j
was taken in round bottom flask and 10% H SO solution was added to it (10mL/g). The
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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.
5ꢀ(4ꢀEthylꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4a). Yellow solid; (98% yield, 0.83g); mp
1
1
22.9−123.6 ºC. H NMR (400 MHz, CDCl ) δ ppm 1.12 (t, J = 7.04 Hz, 3H), 2.46 (q, J = 7.92
3
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
13
(
dd, J = 2.68, 9.4 Hz, 1H), 8.01 (d, J = 10.08 Hz, 1H). C NMR (75 MHz, CDCl ) δ ppm 10.9,
3
–
1
4
5.6, 51.0, 51.2, 97.1, 104.3, 122.9, 126.6, 147.2, 154.3. FTIR (KBr,cm ): 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,
+
4
60.54. HRMS (ESI): m/z calcd for C H N O [M + H] 251.1508 obsd 251.1507.
12 18
4
2
5ꢀ(4ꢀAllylꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4b). Yellow solid; (97% yield, 0.83g); mp
1
8
3.6−85.4 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 2.43−2.45 (m, 4H), 2.97 (d, J = 6.58 Hz,
6
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
13
(dd, J = 2.2, 9.52 Hz, 1H), 7.26 (s, 2H), 7.80 (d, J = 9.56 Hz, 1H). C NMR (100 MHz,
DMSO−d ) 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
6
–
1
(
KBr,cm ): 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,
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8
11.87, 744.56, 644.54. HRMS (ESI): m/z calcd for C H N O [M + H] 263.1508 obsd
12 16 4 2
2
63.1508.
5ꢀ(4ꢀ(2ꢀNꢀTrifluoroacetylaminoethyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4c). Yellow solid;
10
11
12
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15
16
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18
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26
27
28
29
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(
94% yield, 0.84g); mp 135.6−137.2 ºC. H NMR (400 MHz, CDCl ) δ ppm 2.54−2.63 (m, 6H),
3
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
13
(
dd, J = 2.68, 10.08 Hz, 1H), 6.96 (br s, 1H), 8.00 (d, J = 9.44 Hz, 1H). C NMR (100 MHz,
CDCl ) 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,
3
–
1
1
57.7. FTIR (KBr,cm ): 3439.77, 3338.93, 2851.31, 1619.58, 1598.95, 1563.63, 1509.96,
1
477.41, 1385.65, 1322.11, 1221.20, 1111.93, 991.67, 804.96, 742.79, 534.26. HRMS (ESI):
+
m/z calcd for C H F N O [M + H] 362.1440 obsd 362.1435.
1
4
18
3
5
3
5
ꢀ[4ꢀ(2ꢀ(N, NꢀDimethylaminoethyl)ꢀ1ꢀpiperazinyl]ꢀ2ꢀnitroaniline (4d). Yellow solid;
1
(
95% yield, 0.83g); mp 142.2−143.8 ºC. H NMR (400 MHz, CDCl ) δ ppm 2.25 (s, 6H),
3
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
13
Hz, 1H), 6.14 (br s, 2H), 6.25−6.28 (m, 1H), 7.99 (d, J = 9.52 Hz, 1H). C NMR (100 MHz,
CDCl ) 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):
3
+
m/z calcd for C H N O [M + H] 294.1930 obsd 294.1910.
1
4
23
5
2
5ꢀ(4ꢀPhenylꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4e). Yellow solid; (97% yield, 0.84g); mp
1
2
03.1−204.2 ºC. H NMR (500 MHz, CDCl ) δ ppm 3.30−3.35 (m, 4H), 3.51−3.54 (m, 4H), 5.99
3
(
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,
13
2H), 8.04 (d, J = 9.56 Hz, 1H). C NMR (100 MHz, DMSO−d ) δ ppm 46.1, 48.0, 97.5, 105.4,
6
–
1
1
15.7, 119.5, 123.0, 127.2, 129.1, 148.3, 150.4, 154.8. FTIR (KBr,cm ): 3574.22, 3479.85,
3
365.99, 2832.51, 1616.73, 1594.40, 1445.62, 1496.17, 1477.25, 1389.30, 1322.47, 1227.32,
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183.21, 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 C H N O [M + H] 299.1508 obsd 299.1500.
16 18
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5ꢀ(4ꢀ(2ꢀCyanophenyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4f). Yellow solid; (96% yield,
0
1
2
3
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9
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1
0
.84g); mp 175.7−177.7 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 3.26−3.28 (m, 4H),
6
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),
13
7
1
3
7
.84 (d, J = 9.52 Hz, 1H). C NMR (100 MHz, DMSO−d ): δ 46.4, 50.6, 97.9, 104.7, 105.4,
6
–
1
18.2, 119.1, 122.3, 123.2, 127.2, 134.3, 134.4, 148.3, 154.8, 154.9. FTIR (KBr, cm ): 3467.70,
348.83, 2832.04, 2217.05, 1618.07, 1478.22, 1320.28, 1223.38, 1167.10, 1111.43, 1028.78,
+
57.56. HRMS (ESI): m/z calcd for C H N O [M + H] 324.1460 obsd 324.1450.
1
7
17
5
2
5ꢀ(4ꢀ(4ꢀFluorophenyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4g). Yellow solid; (98% yield,
1
0.86g); mp 177.2−178.6 ºC. H NMR (400 MHz, CDCl ) δ ppm 3.19−3.21 (m, 4H), 3.49−3.51
3
(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),
13
6.96−7.00 (m, 2H), 8.01 (d, J = 9.84 Hz, 1H). C NMR (100 MHz, CDCl ) δ ppm 46.9, 50.0,
3
–
1
9
8.5, 105.7, 115.6, 115.8, 118.2, 118.3, 124.9, 128.3, 147.0, 147.7, 155.3. FTIR (KBr,cm ):
469.47, 3421.23, 3353.55, 3308.68, 3180.05, 2925.07, 2850.22, 2374.76, 2345.71, 1618.27,
3
1509.04, 1500.43, 1476.48, 1319.55, 1225.83, 1113.25, 1090.45, 1031.47, 966.79, 926.74,
+
8
18.19, 715.86. HRMS (ESI): m/z calcd for C H FN O [M + H] 317.1414 obsd 317.1404.
1
6
17
4
2
5ꢀ(4ꢀ(4ꢀPyridyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4h). Yellow solid; (95% yield, 0.83g); mp
1
177−180.9 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 3.54−3.57 (m, 4H), 3.67−3.70 (m, 4H),
6
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),
13
7
.85 (d, J = 10.24 Hz, 2H), 8.23 (s, 2H). C NMR (100 MHz, DMSO−d ) δ ppm 44.7, 44.8,
6
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6.7, 104.9, 123.0, 127.5, 148.4, 154.3. FTIR (KBr,cm ): 3445.37, 3335.57, 2918.76, 2851.54,
619.58, 1598.80, 1563.63, 1511.08, 1476.81, 1402.08, 1322.95, 122.06, 1113.36, 1029.66,
+
93.11, 937.70, 806.92, 743.71, 657.11. HRMS (ESI): m/z calcd for C H N O [M + H]
15
17
5
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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47
48
49
50
51
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53
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55
56
57
58
59
60
00.1460 obsd 300.1457.
5
ꢀ(4ꢀ(2ꢀPyrimidyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4i). Yellow solid; (96% yield, 0.84g); mp
1
1
68.5−166.8 ºC. H NMR (300 MHz, CDCl ) δ ppm 3.47−3.49 (m, 4H), 3.95−3.99 (m, 4H), 5.96
3
(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
13
(d, J = 9.6 Hz, 1H), 8.35 (d, J = 4.8 Hz, 2H). C NMR (100 MHz, CDCl ) δ ppm 25.6, 42.5, 45.8,
3
–
1
102.1, 107.3, 110.1, 128.1, 137.4, 155.0, 157.4, 169.2. FTIR (KBr,cm ): 3482.69, 3435.65,
3
368.12, 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,
68.14, 806.19, 790.68, 751.56, 656.89, 502.80. HRMS (ESI): m/z calcd for C H N O [M +
8
1
4
16
6
2
+
H] 301.1413 obsd 301.1401.
5ꢀ(4ꢀ(4ꢀNitrophenyl)ꢀ1ꢀpiperazinyl)ꢀ2ꢀnitroaniline (4j). Yellow solid; (95% yield, 0.84g); mp
1
1
82.3−184.5 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 3.55−3.57 (m, 4H), 3.60−3.68 (m, 4H),
6
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),
13
7.84 (d, J = 9.52 Hz, 1H), 8.08 (d, J = 9.52 Hz, 2H). C NMR (100 MHz, DMSO−d ) δ ppm
6
–1
4
5.3, 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 ):
3
467.91, 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 C H N O [M + H] 344.1359 obsd 344.1356.
1
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General Procedure for the Synthesis of 4ꢀ(4ꢀSubstitutedpiperazinyl)ꢀ1,2ꢀ
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
0
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0
temperature under 40 psi H pressure until TLC showed the disappearance of starting material
2
and reaction mixture becomes colorless. Reaction mixture was filtered through celite and filtrate
2
4
was used for next step without further purification and delay.
General Procedure for Preparation of 2ꢀArylꢀ5ꢀcyanobenzimidazoles (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 Na S O (0.5 equiv) in water
2
2
5
(
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
24
EtOAc/Pet ether as solid in 70−80% yield.
5ꢀCyanoꢀ2ꢀ(4ꢀhydroxyphenyl)benzimidazole (8a). White solid; (80% yield, 1.41g); mp.
1
1
90.6−193.2ºC. H NMR (400 MHz, DMSO−d ) δ ppm 6.94 (d, J = 8.72 Hz, 2H), 7.54 (d, J =
6
1
8
.4 Hz, 1H), 7.66 (bs, 1H), 8.04 (d, J = 8.4 Hz, 3H), 10.10 (s, 1H), 13.18 (bs, 1H). H NMR
(
400 MHz, DMSO−d + D O) δ ppm 6.93 (d, J = 8.72 Hz, 2H), 7.54 (dd, J = 1.32, 8.4 Hz, 1H),
6 2
13
7
.67 (d, J = 8.04 Hz, 1H), 7.98 (d, J = 8.72 Hz, 3H). C NMR (100 MHz, DMSO−d ) δ ppm
6
–
1
103.6, 116.0, 120.1, 120.3, 125.5, 128.9, 155.0, 160.0. FTIR (KBr, cm ): 3463.07, 3235.04,
2
924.36, 2851.54, 2588.23, 2228.91, 1610.10, 1497.78, 1466.59, 1432.01, 1313.71, 1269.93,
1
250.70, 1174.47, 1113.28, 966.64, 878.19, 841.98, 816.05, 749.57, 517.20, 439.55. HRMS
+
(ESI): m/z calcd for C H N O [M + H] 236.0820 obsd 236.0813.
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ꢀCyanoꢀ2ꢀ(4ꢀethoxyphenyl)benzimidazole (8b). White solid; (72% yield, 1.42g);
1
mp.223.1−224.8ºC. H NMR (400 MHz, DMSO−d ) δ ppm 1.36 (t, J = 6.56 Hz, 3H), 4.12 (q, J
6
1
=
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). H
10
11
12
13
14
15
16
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18
19
20
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22
23
24
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45
46
47
48
49
50
51
52
53
54
55
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58
59
60
NMR (400 MHz, DMSO−d + D O) δ ppm 1.34 (t, J = 7.32 Hz, 3H), 4.12 (q, J = 7.32 Hz, 2H),
6
2
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,
13
J = 8.76 Hz, 2H). C NMR (100 MHz, DMSO−d ) δ ppm 160.15, 155.29, 129.36, 125.96,
6
–
1
1
20.65, 120.26, 116.43, 103.92, 64.32, 14.74. FTIR (KBr, cm ): 3437.66, 2929.80, 2220.87,
+
1502.97, 1267.03, 1022.70, 813.60. HRMS (ESI): m/z calcd for C H N O [M + H] 264.1137
16
13
3
obsd 264.1129.
5ꢀCyanoꢀ2ꢀ(3,4ꢀdimethoxyphenyl)benzimidazole (8c). This compound was synthesized by
23
reported procedure. Off−white solid; (80% yield, 1.67g); mp 223.1−224.8ºC.
General Procedure for the preparation of 2ꢀArylꢀ5ꢀformyl benzimidazoles (9a−c).
NiꢀAl 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 Na SO and
2
4
filtered. The solvents were evaporated under reduced pressure. The residue was purified by
2
4
chromatography on silica gel (60−120 mesh size) in MeOH/EtOAc as solid in 65−74% yield.
5
ꢀFormylꢀ2ꢀ(4ꢀhydroxyphenyl)benzimidazole (9a). Offꢀwhite solid, (74% yield,
1
0
.74g); mp 315.5−316.2 ºC. H NMR (400 MHz, DMSO−d ): δ 6.94 (d, J = 8.72 Hz, 2H), 7.69
6
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(
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,
1
1
H), 10.13 (s, 1H). H NMR (400 MHz, DMSO−d +D O) δ ppm 6.94 (d, J = 8.72 Hz, 2H), 7.69
6
2
1
3
(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). C
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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7
8
9
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1
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4
5
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7
8
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0
1
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3
4
5
6
7
8
9
0
NMR (100 MHz, DMSO−d6) δ ppm 115.9, 120.4, 123.0, 128.7, 130.9, 155.0, 159.9, 163.1,
–
1
192.5. FTIR (KBr, cm ): 3447.41, 2924.95, 2851.54, 1609.53, 1454.54, 1356.47, 1272.72,
+
1
019.15, 841.95, 774.82, 528.67, 430.76. HRMS (ESI): m/z calcd for C H N O [M + H]
14 10 2 2
2
39.0820 obsd 239.0813.
5
2
6
ꢀFormylꢀ2ꢀ(4ꢀethoxyphenyl)benzimidazole (9b). Offꢀwhite solid (70% yield, 0.70g); mp
1
66.8−268.6ºC. H NMR (400 MHz, DMSO−d6) δ ppm 1.36 (t, J = 6.84 Hz, 3H), 4.12 (q, J =
.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,
1
1H), 13.20 (bs, 1H). H NMR (400 MHz, DMSO−d + D O) δ ppm 1.35 (t, J = 6.84 Hz, 3H),
6
2
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 =
1
3
8
.68 Hz, 2H), 10.03 (s, 1H). C NMR (100 MHz, DMSO−d ) δ ppm 14.6, 63.4, 114.9, 121.7,
6
+
122.9, 128.5, 131.0, 160.5, 192.5. HRMS (ESI): m/z calcd for C H N O [M + H] 267.1133
16
14
2
2
obsd 267.1125.
5
ꢀFormylꢀ2ꢀ(3,4ꢀdimethoxyphenyl)benzimidazole (9c). This compound was synthesized by
23
reported procedure. Crystalline offꢀwhite compound; (65% yield, 0.65g); mp 232.6 − 235.4ºC.
General Procedure for Preparation of Bisbenzimidazole Derivatives (10aꢀj, 11aꢀi
and 12aꢀe). 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 Na S O (0.5 equiv) in water
2
2
5
(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
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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.
5ꢀ(4ꢀEthylpiperazinꢀ1ꢀyl)ꢀ2ꢀ[2’ꢀ(4ꢀhydroxyphenyl)ꢀ5’ꢀbenzimidazolyl]benzimidazole (10a).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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52
53
54
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56
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58
59
60
1
Orangish brown solid; (55% yield, 0.54g); mp 258.5−259.3 ºC. H NMR (400 MHz, DMSO−d )
6
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
13
Hz, 2H), 8.27−8.34 (m, 1H). C NMR (100 MHz, DMSO−d ) δ ppm 11.7, 49.8, 51.6, 52.4,
6
–
1
1
11.4, 115.82, 118.5, 120.4, 120.8, 124.2, 128.5, 147.6, 153.3, 159.5. FTIR (KBr, cm ):
3
113.70, 2823.08, 2361.39, 1610.72, 1443.69, 1240.25, 1173.15, 963.77, 817.12, 443.58. HRMS
+
(
ESI): m/z calcd for C H N O [M + H] 439.2246 obsd 439.2233.
2
6
26
6
5
ꢀ(4ꢀPropylpiperazinꢀ1ꢀyl)ꢀ2ꢀ[2’ꢀ(4ꢀhydroxyphenyl)ꢀ5’ꢀbenzimidazolyl]benzimidazole (10b).
1
Yellow colored solid; (52% yield, 0.50g); mp 238.5−239.1 ºC. H NMR (400 MHz, DMSO−d )
6
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
1
3
(
d, J = 8.24 Hz, 1H), 8.06 (d, J = 8.68 Hz, 2H), 8.22−8.28 (m, 1H). C NMR (100 MHz,
DMSO−d ) 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,
6
–
1
127.3, 128.4, 130.6, 136.4, 147.6, 151.6, 153.3, 156.8, 159.2, 159.5. FTIR (KBr, cm ): 3403.35,
2
930.07, 2705.51, 2372.79, 1605.83, 1466.67, 1305.37, 1257.42, 1189.98, 1036.66, 962.53,
+
8
10.63. HRMS (ESI): m/z calcd for C H N O [M + H] 453.2403 obsd 453.2373.
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6
5ꢀ(4ꢀ(2ꢀN,NꢀDimethylaminoethylpiperazinꢀ1ꢀyl))ꢀ2ꢀ[2’ꢀ(4ꢀhydroxyphenyl)ꢀ5’
benzimidazolyl]benzimidazole (10c). Brown crytalline solid; (56% yield, 0.51g); mp
1
2
08.5−209.3 ºC. H NMR (400 MHz, DMSO−d ) δ ppm 2.20 (s, 6H), 2.44−2.47 (m, 4H),
6
2
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