Novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.11.027

A series of novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents were designed, synthesized and characterized by IR, NMR and HRMS spectra. The biological evaluation in vitro revealed that some of the target compounds exerted good anti

Full text of DOI:10.1016/j.ejmech.2017.11.027

Accepted Manuscript
Novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents: Design,
synthesis and biological evaluation
Han-Bo Liu, Wei-Wei Gao, Vijai Kumar Reddy Tangadanchu, Cheng-He Zhou, Rong-
Xia Geng
PII:
S0223-5234(17)30922-4
10.1016/j.ejmech.2017.11.027
EJMECH 9902
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2017
Revised Date: 26 October 2017
Accepted Date: 8 November 2017
Please cite this article as: H.-B. Liu, W.-W. Gao, V.K.R. Tangadanchu, C.-H. Zhou, R.-X. Geng, Novel
aminopyrimidinyl benzimidazoles as potentially antimicrobial agents: Design, synthesis and biological
evaluation, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.027.
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Graphical Abstract
Novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents: Design, synthesis and
biological evaluation
†
*
*
Han-Bo Liu, Wei-Wei Gao, Vijai Kumar Reddy Tangadanchu , Cheng-He Zhou and Rong-Xia Geng
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality,
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China;
†
*
Postdoctoral researcher from CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
Corresponding Address:
Tel.: +86-23-68254967; fax: +86-23-68254967; E-mail: zhouch@swu.edu.cn (Cheng-He Zhou);
geng0712@swu.edu.cn (Rong-Xia Geng).
A series of novel aminopyrimidinyl benzimidazoles were synthesized and screened for their antimicrobial
activities. Molecular modeling and experimental investigation with DNA suggested the possible
antibacterial mechanism.

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Title page
Title:
Novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents: Design, synthesis and
biological evaluation
Author Names and Affiliations:
†
*
*
Han-Bo Liu, Wei-Wei Gao, Vijai Kumar Reddy Tangadanchu , Cheng-He Zhou and Rong-Xia Geng
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality,
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China;
†
*
Postdoctoral researcher from CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
Corresponding Address:
Tel.: +86-23-68254967; fax: +86-23-68254967; E-mail: zhouch@swu.edu.cn (Cheng-He Zhou);
geng0712@swu.edu.cn (Rong-Xia Geng).
Abstract:
A series of novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents were designed,
synthesized and characterized by IR, NMR and HRMS spectra. The biological evaluation in vitro revealed
that some of the target compounds exerted good antibacterial and antifungal activity in comparison with the
reference drugs. Noticeably, compound 7d could effectively inhibit the growth of A. flavus, E. coli DH52
and MRSA with MIC values of 1, 1 and 8 µg/mL, respectively. Further studies revealed that pyrimidine
derivative 7d could exhibit bactericidal mode of action against both Gram positive (S. aureus and MRSA)
and Gram negative (P. aeruginosa) bacteria. The active molecule 7d showed low cell toxicity and did not
obviously trigger the development of resistance in bacteria even after 16 passages. Furthermore, compound
7d was able to beneficially regulate reactive oxygen species (ROS) generation for an excellent safety
profile. Molecular docking study revealed that compound 7d could bind with DNA gyrase by the formation
of hydrogen bonds. The preliminary exploration for antimicrobial mechanism disclosed that compound 7d
could effectively intercalate into calf thymus DNA to form a steady supramolecular complex, which might
further block DNA replication to exert the powerful bioactivities. The binding investigation of compound
7d with human serum albumins (HSA) revealed that this molecule could be effectively transported by
HSA.
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Keywords:
Aminopyrimidine; Benzimidazole; Antimicrobial; DNA; HSA
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1
. Introduction
Pyrimidine is an important six-membered aromatic framework containing two nitrogen atoms, and
widely present in a range of bioactive agents. The unique structural feature of pyrimidine ring enables its
derivatives to readily interact with biological macromolecules like enzymes, receptors, deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA) [1]. Thus, the pyrimidine fragment has been prevalently employed
in the design of new drugs and a lot of pyrimidine-based drugs have been successfully developed and
extensively used in clinic [2]. The amino modified pyrimidines are a special type of pyrimidine derivatives
in which the amino fragment as an electron-rich center can improve the lipophilicity and modulate the
electron density of the electron-deficient pyrimidine ring, and have drawn much attention in medicinal
chemistry in very recent years. Especially in antimicrobial aspect,
a
great number of
aminopyrimidine-based derivatives have been reported to possess efficient antibacterial activities by
inhibiting dihydrofolate reductase (DHFR), a validated therapeutic target that could catalyze the reduction
of dihydrofolate to tetrahydrofolate [3]. Inhibition of the folate cycle could lead to interruption of the
supply of thymidine, and further bring about inhibition of DNA biosynthesis and the death of bacterial cells
[4]. Noticeably, aminopyrimidine-based trimethoprim and iclaprim as bacterial DHFR inhibitors have been
successfully applied in clinic to treat a variety of infectious diseases [5]. However, the appearance of
drug-resistant strains among community acquired infections and the rapidly global spread of resistant
clinical isolates make many clinical drugs decrease therapeutic effects or totally lose activities [6], and
force an immense need to exploit new class of anti-infective agents with strong activity, low toxicity and
distinct modes of action from currently clinical drugs.
It is well-known that benzimidazoles are structural isosteres to purines that are essential substrates for the
biosynthesis of nucleic acids and proteins inside the bacterial cell wall. The purine-like structure enables
benzimidazole derivatives to obstruct the biosynthesis of nucleic acids and proteins by competing with
purines, ultimately leading to the death of the bacterial cell [7]. Additionally, benzimidazole derivatives
have been reported to efficiently inhibit bacterial topoisomerases [8]. These suggested that benzimidazole
derivatives should have potential antimicrobial activities with distinct action mechanisms from the
well-known antimicrobial agents, which would be helpful for benzimidazole-based compounds to reduce
the probability of bacteria to develop resistance.
Much work has been directed towards the development of benzimidazole compounds as medicinal
agents, and an increasing number of benzimidazole compounds have displayed great potency to treat
microbial infections [9]. Benzimidazolyl quinolone ZL showed significant inhibition against all the tested
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strains including multidrug resistant organisms, and it also effectively inhibited the formation of biofilm
and dispersed the established bacterial biofilms [10]. Berberine-benzimidazole PJ was found to be a
potentially antibacterial molecule, especially towards Methicillin-Resistant Staphylococcus aureus (MRSA),
Escherichia coli and Salmonella typhi with low MIC values of 2−8 µg/mL, and the antibacterial
mechanism was experimentally proven to be mainly in relation with its dual action modes to block bacterial
DNA replication [11]. All the above observations revealed that the development of benzimidazoles as novel
antimicrobial agents may be served as a promising strategy to exploit new anti-infective agents with better
activity and less bacterial resistance (Fig. 1).
Fig. 1
In view of this situation and as an extension of our studies on the development of antimicrobial azoles
[12], it is of great interest for us to combine the aminopyrimidine fragment and benzimidazole ring to
generate a new structural type of potentially antimicrobial agents. Much literature evidenced that the alkyl
chain could exert large effect on biological potency by regulating the lipid-water partition coefficient and
binding affinity to target enzymes [13]. Rationally, various alkyl chains with different lengths were
incorporated into benzimidazole nucleus to investigate the effects on antimicrobial activities. The phenyl
moieties bearing fluoro, chloro and methyl groups were also considered because they could usually affect
the pharmacological properties by modulating lipophilicity and cell membrane permeability, thus
influencing the rate of absorption and transport of drugs in vivo [14]. An in-depth binding analysis between
bioactive small molecules and DNA or human serum albumins (HSA, the most important and abundant
macromolecule proteins in the circulatory system) may not only beneficially provide useful information for
the absorption, transportation, distribution, metabolism, excretion properties, and possible mechanism of
agents, but also be constructive for the design, modification, and screening of drug molecules [15]. In view
of the above observations, it is reasonable for us to investigate the antimicrobial mechanism with DNA and
the further transportation behavior of the highly active compound by HSA to preliminarily evaluate the
pharmacokinetic and transportation properties by fluorescence and UV–vis absorption spectroscopy on
molecular level.
Fig. 2
2. Results and discussion
2.1. Chemistry
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The target aminopyrimidinyl benzimidazoles were prepared from commercial o-phenylene diamine and
-hydroxypropanoic acid. The synthetic route was outlined in Scheme 1. o-Phenylene diamine was reacted
2
with 2-hydroxypropanoic acid via cyclization to produce intermediate 2 in an excellent yield of 91.2%. The
hydroxyl oxidation of compound in the presence of potassium dichromate provided
-acetylbenzimidazole 3. Benzimidazolyl chalcones 4a−d were generated from compound 3 with
2
2
substituted aromatic aldehydes involving Claisen-Schmidt condensation, and further treated with a series of
alkyl bromides to give N-alkyl benzimidazolyl chalcones 5a−p in desirable yields ranging from 61% to
77%. Benzimidazolyl pyrimidines 7a−d and N-alkyl derivatives 6a−p were smoothly obtained via
cyclization of chalcones (4a−d and 5a−p) with guanidine hydrochloride in the presence of sodium hydride.
Scheme 1
2.2. Biological Activity
All the prepared benzimidazolyl chalcones and pyrimidine derivatives were evaluated for their
antimicrobial activities in vitro against four Gram positive bacteria (Staphylococcus aureus ATCC25923,
Methicillin-Resistant Staphylococcus aureus N315, Bacillus subtilis and Micrococcus luteus ATCC4698),
six Gram negative bacteria (Bacillus proteus ATCC13315, Escherichia coli DH52, Pseudomonas
aeruginosa, Bacillus typhi, Escherichia coli JM109 and Shigella dysenteriae) and five fungi (Candida
albicans ATCC76615, Candida mycoderma, Candida utilis, Saccharomyces cerevisia and Aspergillus
flavus) using the standard two folds serial dilution method in 96-well micro-test plates recommended by the
National Committee for Clinical Laboratory Standards (NCCLS) [16]. Minimal inhibitory concentration
(MIC, µg/mL) was defined as the lowest concentration of new compounds that completely inhibited the
growth of bacteria. Currently available antimicrobial drugs chloromycin, norfloxacin and fluconazole were
used as the positive controls. To investigate whether this new type of pyrimidine compounds are
bactericidal or bacteriostatic in nature, the minimal bactericidal concentration (MBC) values of some target
compounds against Gram positive strain (S. aureus and MRSA) and Gram negative strain (P. aeruginosa)
were determined and subsequently compared to respective MIC values. According to NCCLS, a MBC/MIC
ratio of 1 to 2 is considered indicative of bactericidal behavior. Contrastively, a MBC/MIC ratio ≥8 is
determined as being indicative of bacteriostatic behavior [17].
2.2.1. Antibacterial activity
The antibacterial results in vitro (Table 1 and Table 2) revealed that most of the prepared compounds
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could effectively inhibit the growth of some tested strains. Excitingly, some of them were even more active
than the reference drugs. The benzimidazole intermediates 4a–d and 5a–p, as shown in Table 1, also
displayed moderate to good activities against all the tested bacterial strains in comparison with the
reference drugs. Particularly, 4-methylphenyl chalcone 4d and 4-fluorophenyl derivative 5a with a butyl
group gave strong inhibition against most of the tested bacterial strains. Compound 4d showed better
antimicrobial efficiencies with MIC values of 4 µg/mL against B. proteus, P. aeruginosa and E. coil JM109
than the corresponding reference drug chloromycin. It was noteworthy that compound 5a, with an excellent
MIC value of 0.5 µg/mL against P. aeruginosa, was 32- and 2-fold more potent than chloromycin and
norfloxacin, respectively. For the Gram positive S. aureus, MRSA and B. subtilis strains, derivatives 4d
and 5a (MICs = 4−16 µg/mL) displayed stronger inhibitory activity than chloromycin (MICs = 8−32
µg/mL). Expectantly, most of the target aminopyrimidines 6a−p and 7a−d showed higher inhibitory
potency than their chalcone precursors, especially towards Gram negative bacteria, suggesting that the
conversion of α,β-unsaturated ketone moiety into aminopyrimidine framework was conducive to exerting
the biological activity.
Table 2 showed the significant effects of the types of substituents on the benzene ring on biological
activity. Among compounds 7a−d, 4-fluorophenyl derivative 7a demonstrated effective anti-S. aureus
activity with a MIC value of 2 µg/mL, which was 4-fold more potent than the reference drug chloromycin
and norfloxacin (MIC = 8 µg/mL). Meanwhile, both 2,4-dichlorophenyl pyrimidine 7c and 4-methylphenyl
derivative 7d showed low MIC values of 8 µg/mL against S. aureus, equipotent to chloromycin. Notably, B.
subtilis was more sensitive to target compounds 7a (MIC = 2 µg/mL), 7c (MIC = 8 µg/mL) and 7d (MIC =
8
µg/mL) than chloromycin (MIC = 32 µg/mL). Besides, derivative 7d could not only inhibit the growth of
B. proteus (MIC = 8 µg/mL), M. luteus (MIC = 8 µg/mL) and S. aureus (MIC = 8 µg/mL), with equivalent
or superior activities to chloromycin, but also show inhibitory potency against Gram negative bacteria
including B. proteus, E. coil DH52, P. aeruginosa, B. typhi, and E. coil JM109 at low concentrations (MICs
=
1−4 µg/mL). These indicated that pyrimidine 7d should have the potency as a lead compound for the
further development of more effectively board-spectrum antimicrobial agents. Excitedly, compound 7c
gave the best antibacterial activity against B. proteus with a MIC value of 1 µg/mL, which was 32-fold
more potent than the reference drug chloromycin and 4-fold more active than norfloxacin. However,
compound 7b bearing 4-chlorophenyl group displayed fairly poor antibacterial activities with MIC values
in the range of 8–512 µg/mL in comparison with other substituted counterparts. These suggested that the
substituents in benzene skeleton should have an important effect on the antibacterial activity.
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The effects of alkyl chains in benzimidazole nucleus were also considered. Unfortunately, the
introduction of alkyl groups into benzimidazole nucleus resulted in less potent against the tested pathogens
(Table 1) and they contributed to the antibacterial activities in the order of butyl > heptyl > hexyl > octyl.
Among the alkyl derivatives 6a−p, butyl compound 6m with a 4-methylphenyl moiety gave
broad-spectrum and better antibacterial activity in comparison with other alkyl substituted ones, especially
towards Gram positive bacteria with MIC values ranging from 8 to 16 µg/mL. Butyl derivative 6a
containing a 4-fluorophenyl group could efficiently inhibit the growth of B. subtilis (MIC = 1 µg/mL),
superior to chloromycin (MIC = 32 µg/mL) and norfloxacin (MIC = 2 µg/mL). Meanwhile, B. subtilis was
also more sensitive to target compounds 6c, 6g and 6h with MIC values of 2 µg/mL than chloromycin.
Hexyl derivative 6f bearing 4-chlorophenyl moiety showed strong anti-M. luteus and anti-E. coli DH52
activities with low MIC values of 2 µg/mL, being 4 and 8 times more potent than chloromycin, respectively.
Derivative 6m with a butyl group exerted broad-spectrum antimicrobial activity against all the tested Gram
positive bacteria (MICs = 8−16 µg/mL), comparable to chloromycin. However, the replacement of butyl
group by heptyl moiety led to compound 6o, which gave stronger anti-Gram negative bacteria activities
than chloromycin against E. coli DH52 (MIC = 4 µg/mL), B. typhi (MIC = 4 µg/mL), P. aeruginosa (MIC
=
2 µg/mL) and S. dysenteriae (MIC = 4 µg/mL), but displayed poor activity against Gram positive bacteria.
Besides, 2,4-dichlorophenyl derivatives 6i−l displayed overall moderate to weak bioactivities, and only
strong anti-B. proteus activity (MIC = 4 µg/mL) was observed for octyl compound 6l, which was more
active than chloromycin (MIC = 32 µg/mL) and equivalent to norfloxacin (MIC = 4 µg/mL).
Table 1
Table 2
2.2.2. Antifungal activity
The antifungal results (Table 3 and Table 4) revealed that most of the target aminopyrimidinyl
benzimidazoles displayed a similar tendency and intensity to their antibacterial activities. Notably, C. utilis
was more sensitive to target compounds 6a−h, 6l−n and 7b−d (MICs = 2–8 µg/mL) than fluconazole (MIC
=
16 µg/mL). Derivatives 6a−p and 7a−d were also more significantly beneficial than compounds 4a−d
and 5a−p in antifungal activities, which indicated that pyrimidines were positive to antimicrobial activities.
Noticeably, non-alkyl substituted benzimidazole derivatives 7c and 7d exerted excellent and broad
spectrum activity in this series, with MIC values between 1 and 8 µg/mL. Pyrimidine derivatives 7b−d
gave much better anti-A. flavus efficiency than fluconazole at the concentrations of 1–8 µg/mL, particularly
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compound 7d (MIC = 1 µg/mL), which was even 256-fold more potent than fluconazole (MIC = 256
µg/mL). The less acidic NH group in benzimidazole motif was observed to be favorable for biological
potency probably due to its participation in formation of hydrogen bonds, which might play an important
role in exerting biological activity.
Table 3
Table 4
2.2.3. Minimal bactericidal concentration (MBC)
As depicted in Table 5, most of the selected compounds displayed bactericidal mode of action against the
tested microorganisms. Among them, compounds 7a, 7c and 7d, with a corresponding MBC/MIC ratio of 1,
were highly bactericidal against S. aureus (MBC = 2, 8, 8 µg/mL, respectively). Towards MRSA strain,
derivatives 6f, 6m, 6o and 7d exerted identical MBC and MIC values (MBC/MIC ratio = 1), being
indicative of desirable bacteriostatic behavior. Meanwhile, most of the compounds showed moderate to
weak bactericidal activity against the Gram negative P. aeruginosa strain with the exception of compound
6o, which showed the highest bactericidal potency (MBC = 4 µg/mL). However, compound 6e showed
8-fold difference between MIC and MBC against P. aeruginosa, indicating a bacteriostatic behavior. It was
worthy to note that pyrimidine derivatives 7c and 7d exhibited bactericidal mode of action against both
Gram positive and Gram negative bacteria (MBC/MIC ratio ≤ 2). These implied that this new type of
pyrimidine compounds had effective bactericidal potentiality against the tested strains, especially for Gram
positive bacteria.
Table 5
2.3. Development of resistance to compound 7d
An important attribute of potentially antimicrobial agents is that the development of resistance to their
action is not readily attained [18]. To evaluate the probability of these pyrimidine molecules to elicit
resistance in bacteria, the drug resistance studies of compound 7d against MRSA were carried out. The
standard strain of MRSA was exposed to compound 7d from the MIC for sustained passages and new MIC
values were determined every 24 h after propagation of MRSA cultures with fresh media and serially
diluted concentrations of the tested compound 7d. To make comparative analysis, parallel cultures were
exposed to 2-fold dilutions with the reference drugs chloromycin and norfloxacin as positive controls. The
experiments were repeated for 16 days. As shown in Fig. 3, the susceptibility of MRSA bacteria to
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compound 7d remained nearly unchanged even after 16 passages. By contrast, MRSA quickly developed
resistance to chloromycin and norfloxacin after several passages indicating that compound 7d did not easily
induce drug resistance in MRSA as clinical drugs did.
Fig. 3
2.4. Cell toxicity of compound 7d
The most bioactive compound 7d was further examined for cytotoxic properties against human
hepatocyte LO2 cells using the colorimetric cell proliferation MTT assay. As shown in Fig. 4, compound
7d showed relative low toxicity to LO2 cells at high concentrations.
Fig. 4
2.5. Reactive oxygen species
ROS are metabolic byproducts of aerobic respiration and are responsible for maintaining redox
homeostasis in cells. The relation between antimicrobial mechanism and cell toxicity has not been fully
elucidated, but it is thought to involve the photo-excitation of endogenous porphyrin molecules, a process
which could generate ROS [19]. However, oxidative damaging effects of excessive ROS on
biomacromolecules, such as DNA, proteins, and lipids, through nonspecific rapid reactions could cause
toxicity to cells, thus leading to cell injury or death [20]. ROS level was investigated using DCFH-DA as a
fluorescence probe and imaged by fluorescence microscope. As shown in Fig. 5, target compound 7d
showed much lower ROS generation than H O at concentration of 6.25, 12.5, 25, 50 µM, respectively.
2
2
Therefore, the newly synthesized compound 7d could possess an excellent safety profile by regulating ROS
generation.
Fig. 5
2.6. Molecular docking
To rationalize the observed antibacterial activity of aminopyrimidinyl benzimidazole 7d, a flexible
ligand receptor docking investigation was undertaken. The crystal structure data (gyrase−DNA complex)
was obtained from the protein data bank (PDB code: 2XCS), which was representative target to investigate
the antibacterial mechanism [21]. The interaction of compound 7d with gyrase−DNA receptor was shown
in Fig. 6. The docking result of interaction between compound 7d and gyrase−DNA complex might
rationalize the possible antibacterial mechanism. The NH group of benzimidazole ring was in close vicinity
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to the residue ASP1083 of gyrase through hydrogen bonds with distance of 1.6 and 1.9 Å, respectively.
Compound 7d could also form hydrogen bonds with the residue ASP1083 through hydrogen atom of NH2
group, which revealed that NH and NH groups played important role in biological activity. Besides,
2
electrostatic interactions existed between the aromatic fragment of compound 7d and residues ARG1122,
MET1121, ALA1120, TYR1087 and ASP1083 in gyrase. These cooperative binding might be favorable to
stabilize the compound−enzyme−DNA complex, which might be responsible for the good inhibitory
efficacy of compound 7d against the tested strains.
Fig. 6
2.7. Molecular electrostatic potential (MEP) surface
The molecular electrostatic potential (MEP) surface gives an indication of the charged surface area of the
compounds and the orientation of drug candidates for their activities. Therefore, MEP maps can be applied
to investigate the key structural features for the target compounds such as steric, electrostatic interactions
and hydrogen donor/acceptor properties [22]. In an attempt to understand the lower antimicrobial activity
of compound 6p and higher antimicrobial activity of 7d, MEPs were performed to examine the differences
in electrostatic binding characteristic of the surface of the molecules at the DFT-B3LYP level of theory for
the neutral state. As shown in Table 6, aminopyrimidinyl benzimidazole 7d had positive charge regions (in
blue) mainly located on the NH and NH groups, therefore the regions were more capable for interactions
2
with the electronically surroundings, which were also in agreement with the binding mode obtained from
above docking study. In the alkylated analogue 6p, the positive charge regions of NH group were buried in
2
the structure and only partially available for interaction, and the electropositively charged zones of the NH2
group in compound 6p were also decreased as a result of the introduction of octyl group at the N-1 position
of the benzimidazole ring. These results might indicate the capability of hydrogen bond formation in the
regions of aminopyrimidine and the benzimidazole ring, which could reveal the differences in electrostatic
binding characteristics between compounds 6p and 7d. This explanation was also consistent with the
obtained antimicrobial results for the series of benzimidazole analogues where there was a significant loss
of potency for alkylated analogue 6p, compared to non-alkylated counterpart 7d.
Table 6
2.8. Interactions with calf thymus DNA
DNA is a potent drug target due to its essentially biological effect through the presence of multiple sites
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for drug interaction which is exploited for rational design and construction of newly and efficiently
antibacterial drugs [23]. Calf thymus DNA is always selected as the DNA model because of its medical
importance, low cost and ready availability properties [24]. Therefore, the in vitro binding studies between
biologically active compound 7d and calf thymus DNA on molecular level were carried out by fluorescence
and UV-vis absorption spectroscopy.
2.8.1. Absorption spectra of DNA in the presence of compound 7d
With a fixed concentration of DNA, UV-vis absorption spectra were recorded with the increasing amount
of compound 7d. As shown in Fig. 7, the maximum absorption peak of DNA at 260 nm exhibited
proportional increase accompanied by slightly red shift with the increasing concentration of compound 7d.
Meanwhile the absorption value of simply sum of free DNA and compound 7d was a little greater than the
measured value of 7d–DNA complex (inset of Fig. 7), indicating that a weak hypochromic effect existed
between DNA and compound 7d. Furthermore, the intercalation of the aromatic chromophore of compound
7d into the helix and the strong overlap of π-π* states of the large π-conjugated system with the electronic
states of DNA bases were consistent with the observed spectral changes [25].
Fig. 7
Based on the variations in the absorption spectra of DNA upon binding to compound 7d, equation (1) [26]
can be utilized to calculate the binding constant (K):
0
A
ξC
ξC
1
=
+
×
0
A− A
ξD−C − ξC ξD−C − ξC K[Q]
(1)
0
where A and A represent the absorbance of DNA in the absence and presence of compound 7d at 260 nm,
ξC and ξD–C are the absorption coefficients of compound 7d and 7d–DNA complex respectively. The plot
0
0
of A /(A–A ) versus 1/[compound 7d] was constructed by using the absorption titration data and linear
4
fitting (Fig. 8), yielding the binding constant, K = 1.54 × 10 L/mol, R = 0.999, SD = 0.136 (R is the
correlation coefficient. SD is standard deviation).
Fig. 8
2.8.2. Absorption spectra of AO interaction with DNA
To further explore the action mode of molecule 7d and calf thymus DNA, acridine orange (AO), a
cationic dye whose binding action with DNA has been sufficiently demonstrated to be intercalative mode
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[27], was employed as a spectral probe in the present work. Fig. 9 displayed the absorption spectra of a
competitive binding between AO and 7d with DNA. With the increasing concentration of compound 7d, a
remarkable fluorescence decrease in DNA–AO system was observed around 540 nm, suggesting that
compound 7d could compete with AO for the intercalation sites in DNA. The experimental results showed
that the mode of compound 7d binding to DNA was intercalation.
Fig. 9
2.8.3. Iodide quenching experiments
Steady-state emission quenching experiments may provide further information about the binding of
compound 7d with DNA. In the presence of negatively charged anionic quencher, if a molecule is protected
from being quenched as a result of repelling action between anionic quencher and anionic sugar phosphate
backbone of DNA, the binding mode of the small molecule with DNA will be intercalation [28]. In this
work, potassium iodide (KI) fluorescence quenching curves of compound 7d in the absence and presence of
DNA were given in Fig. S1 & S2, which indicated that the addition of KI resulted in extensive quenching
of the fluorescence intensity. The quenching constant can be deduced from Stern-Volmer equation [29]:
F0
=
1+ K [Q]
SV
F
(2)
In this equation F and F represent the fluorescence intensity of compound 7d/7d−DNA system in the
0
absence and presence of the quencher KI, respectively. KSV (L/mol) is the Stern–Volmer quenching constant,
and [Q] is the concentration of KI. The Stern–Volmer equation was applied to determine KSV by linear
regression of a plot of F /F versus [Q] (Fig. 10). The observed quenching constant K was 39.87 L/mol in
0
SV
the presence of DNA, which was lower than that of 46.23 L/mol in the absence of DNA. The reduction of
the fluorescence quenching of compound 7d by anionic quencher in presence of DNA, reconfirmed that
compound 7d interacted with calf thymus DNA by intercalative mode.
Fig. 10
2.9. Interactions of compound 7d with HSA
HSA is an important extracellular protein with extensive distribution in the circulatory system, which
accounts for 60−65% of the total plasma protein concentration. The enormous ligand binding capability of
HSA enables the most important carrier protein to transport various exogenous and endogenous molecules,
thus significantly affecting absorption, distribution, and metabolism of numerous therapeutic drugs and
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other bioactive agents [30], which is not only beneficial for the understanding of pharmacology and
pharmacodynamics behaviors of drugs, but also instructive for the design of drug molecules. Herein, the
binding between HSA and the highly active molecule 7d was investigated by UV-vis and fluorescence
spectroscopy on the molecular level to preliminarily study the absorption, distribution, and metabolism.
2.9.1. UV-vis absorption spectral study
UV-vis absorption measurement is a convenient and applicable technique for exploring the structural
change of protein and the formation of complex. In our binding experiment, UV-vis absorption
spectroscopy was adopted to evaluate the binding behaviors between compound 7d and HSA. As shown in
Fig. 11, the absorption peak at 278 nm was observed, and the peak intensity increased with the addition of
compound 7d. The enhancement of absorbance intensity evidenced the binding interaction of compound 7d
to HSA. However, the maximum absorption wavelength remained unchanged, implying that the
interactions of compound 7d and HSA were noncovalent interactions. This result can be explained by the
occurrence of π–π stacking between the aromatic rings of compound 7d and tryptophan (Trp-214), tyrosine
(Tyr-411) and phenylalanine (Phe) residues, which possess conjugated π-electrons and are located in the
binding cavity of HSA [31].
Fig. 11
2.6.2. Fluorescence quenching mechanism
Fluorescence spectroscopy is also an effective method to study the interactions of small molecules with
HSA. The fluorescence intensity of Trp-214 may change when HSA interacts with other small molecules,
which can be reflected in the fluorescence spectra of HSA in the UV region. The effect of compound 7d on
the fluorescence intensity to HSA at 298 K was shown in Fig. 12. It was obvious that HSA had a strong
fluorescence emission with a peak at 352 nm owing to the single Trp-214 residue. The intensity of this
characteristic broad emission band decreased steadily with the increased concentrations of compound 7d,
but the maximum emission wavelength of HSA remained unchanged. This suggested that Trp-214 did not
undergo any change in polarity, and hence compound 7d was likely to interact with HSA via the
hydrophobic region located in HSA. The fluorescence quenching data can be analyzed by the equation (2).
The Stern–Volmer plots of HSA in the presence of compound 7d at different concentrations and
temperatures were shown in Fig. S3.
Fig. 12
13

ACCEPTED MANUSCRIPT
The different mechanisms of quenching are usually classified as either dynamic quenching or static
quenching, which can be distinguished by their different dependence on temperature. For dynamic
quenching, higher temperatures result in faster diffusion and larger amounts of collisional quenching,
consequently, the quenching constant increases with increasing temperature, but the reverse effect will be
observed for static quenching [32]. The value of KSV for the interaction of compound 7d with HSA at
different temperatures was shown in Table 7. The results disclosed that the KSV values decreased with
increasing temperature, which suggested that the quenching mechanism of HSA by compound 7d was not
initiated by dynamic quenching but by static quenching.
Table 7
2.9.3. Binding constant and site
For a static quenching process, the data can be described by the Modified Stern−Volmer equation [33]:
F0
1
1
1
=
+
∆
F
fa Ka
[
Q
]
fa
(3)
∆
F is the difference in fluorescence intensity in the absence and presence of compound 7d at concentration
[
Q], f is the fraction of accessible fluorescence, and K is the effective quenching constant for the
a
a
accessible fluorophores, which are analogous to associative binding constants for the quencher-acceptor
-1
system. The dependence of F /∆F on the reciprocal value of quencher concentration [Q] is linear with the
0
-1
-1
slope equaling to the value of (f K ) . The value of f is fixed on the ordinate. The constant K is a quotient
a
a
a
a
-1
-1
of the ordinate fa and the slope (f K ) . As shown in Table 8, the decreasing trend of K with increasing
a
a
a
temperature was in accordance with K ’s dependence on temperature as discussed above, which further
SV
confirmed that the fluorescence quenching of HSA was static quenching.
Table 8
Fig. 13
When small molecules bind independently to a set of equivalent sites on a macromolecule, the apparent
binding constant (K ) and the number of binding sites (n) can be obtained from the double logarithm
b
regression curve of log[(F −F)−1] versus log[Q] according to the following equation [34]:
0
log
[
(F / F) −1
]
= log K + nlog
[Q
]
0
b
(4)
Fig. S4 and Table 7 showed a linear temperature dependence of log[(F −F)−1] versus log[Q] plots, the
0
14

ACCEPTED MANUSCRIPT
slopes and intercepts of which were equal to n and logK values, respectively. The calculated K values
b
b
suggested that a moderate affinity existed between compound 7d and HSA, and the values of K decreased
b
with the increasing temperature indicating that the capacity of compound 7d binding to HSA was reduced.
The increasing temperature resulted in the increasing diffusion coefficient and the reduction of stability of
the 7d−HSA complex. The high linear correlation coefficients at different temperatures indicated the
assumptions underlying the derivation of equation 4 were valid. The values of n were approximately equal
to 1 suggesting that there was a single class of binding sites on HSA for compound 7d
.
2
.9.4. Binding mode and thermodynamic parameters
The major forces involved in protein-ligand interactions include electrostatic interactions, hydrogen
bonds, van der Waals interactions, and hydrophobic interactions [35]. The thermodynamic parameters, free
energy change ( G), enthalpy change ( H), and entropy change ( S) of the binding reaction can provide
evidence for clarifying interaction modes. Assuming that the enthalpy ( H) does not change significantly
∆
∆
∆
∆
over the temperature range studied, it can be regarded as a constant, and then the values of
∆H, ∆S, and ∆G
are estimated from the van’t Hoff equation [36]:
∆
H
∆S
ln K = −
+
RT
R
(5)
where K is analogous to the associative binding constants at the corresponding temperature and R is the gas
constant. In order to explain the binding model between compound 7d and HSA, the thermodynamic
parameters were calculated from the van’t Hoff plots. The ∆H was estimated from the slope of the van’t
Hoff relationship (Fig. 14). The free energy change was then calculated from the following equation [37]:
∆
G = ∆H −T∆S
(6)
Fig. 14
The thermodynamic parameters were obtained in Table 9, the negative charge of
binding of compound 7d to HSA was a spontaneous process, and the positive S value was frequently
regarded as evidence for a hydrophobic interaction. The negative value of H suggested that the binding
∆G indicated that the
∆
∆
process was predominately enthalpy driven and by means of hydrogen binding interactions [38]. Therefore,
both hydrophobic interactions and hydrogen bonds played a vital role in the binding of compound 7d to
HSA and seemed to favor the stability of the 7d−HSA complex.
Table 9
15

ACCEPTED MANUSCRIPT
2
.9.5. Synchronous fluorescence spectroscope
Synchronous fluorescence is a popular technique to study the microenvironment of aromatic amino acid
residues, particularly tyrosine (Tyr) and tryptophan (Trp). The synchronous fluorescence spectrum is
obtained by simultaneously scanning the excitation and emission monochromators [39]. When the
wavelength differences (∆λ) of excitation and emission are fixed at 15 and 60 nm, respectively, the
spectrum will provide the characteristic information of Tyr and Trp residues in a protein, respectively. It
was clear that the maximum emission wavelength of Tyr residue remained unaltered in presence of
compound 7d (Fig. S5), suggesting that the polarity around this residue was retained. However, compared
with Tyr residue, a stronger fluorescence quenching of Trp residue with concomitant blue shift was
observed on the addition of compound 7d, as a result of modified microenvironment around Trp residue
(
Fig. 15).
Fig. 15
3. Conclusion
In conclusion, a series of aminopyrimidinyl benzimidazoles were successfully synthesized by a
1
13
convenient and efficient procedure. Their structures were confirmed by H NMR, C NMR, IR and HRMS
spectra. The in vitro antimicrobial evaluation revealed that some of the target compounds exhibited good
bioactivities against the tested strains, comparable or even superior to reference drugs. Particularly,
compound 7d could effectively inhibit the growth of all the tested bacterial and fungal strains, especially
against A. flavus, E. coli DH52 and MRSA with MIC values of 1, 1 and 8 µg/mL, respectively. Further
studies revealed that pyrimidine derivative 7d could exhibit bactericidal mode of action against both Gram
positive (S. aureus and MRSA) and Gram negative (P. aeruginosa) bacteria. In addition, it also displayed
low toxicity against normal human hepatocyte LO2 cells and did not trigger the development of resistance
in bacteria. Compound 7d also beneficially regulated ROS generation for an excellent safety profile.
Further molecular docking study indicated that compound 7d could bind with DNA gyrase by the formation
of hydrogen bonds. The preliminary exploration for antimicrobial mechanism revealed that compound 7d
could effectively intercalate into calf thymus DNA to form a steady supramolecular complex, which might
further block DNA replication to exert the powerful bioactivities. The transportation behavior of HSA to the
highly active compound 7d was evaluated and revealed that the association of benzimidazole derivative 7d
with HSA was spontaneous and the hydrophobic interactions and hydrogen bonds played important roles in
the transportation of compound 7d. The calculated parameters indicated that compound 7d could be
16

ACCEPTED MANUSCRIPT
effectively stored and carried by HSA.
4. Experimental
4
.1. General methods
Melting points were recorded on X–6 melting point apparatus and uncorrected. TLC analysis was done
using pre-coated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer
-1
1
13
(
Bio-Rad, Cambridge, MA, USA) using KBr pellets in the 400–4000 cm range. H NMR and C NMR
spectra were recorded on a Bruker AV 600 spectrometer using TMS as an internal standard. The following
abbreviations were used to designate aryl groups: Bim = benzimidazolyl, Pyr = pyrimidinyl, Ph = phenyl.
The chemical shifts were reported in parts per million (ppm), the coupling constants (J) were expressed in
hertz (Hz) and signals were described as singlet (s), doublet (d), triplet (t) as well as multiplet (m). The
high-resolution mass spectra (HRMS) were recorded on an IonSpec FT-ICR mass spectrometer with ESI
resource. All fluorescence spectra were recorded on F-7000 spectrofluorimeter (Hitachi, Tokyo, Japan)
equipped with 1.0 cm quartz cells. The UV spectrum was recorded at room temperature on a TU-2450
spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells.
Calf thymus DNA, HSA and AO were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tris, NaCl,
HCl were analytical purity. Sample masses were weighed on a microbalance with a resolution of 0.1 mg.
All chemicals and solvents were commercially available and were used without further purification
4
.1.1. Synthesis of 1-(1H-benzo[d]imidazol-2-yl)ethan-1-ol (2) and 1-(1H-benzo[d]imidazol-2-yl)ethan-1-
one (
3
)
The intermediates
.1.2. Synthesis of (E)-1-(1H-benzo[d]imidazol-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one (4a
To a solution of 2-acetylbenzimidazole (0.800 g, 5.00 mmol) in ethanol (50 mL) were added 60%
2 and 3 were prepared according to the previously reported methods [40].
4
)
3
o
aqueous potassium hydroxide (15 mL) and 4-fluorobenzaldehyde (0.620 g, 5 mmol) at 0 C with stirring.
The reaction mixture was stirred at room temperature until complete consumption of starting material
(monitored by TLC, petroleum ether/chloroform, 1/3, V/V). The crushed ice and diluted hydrochloric acid
solution were added for total precipitation of the yellow solid (0.846 g), which were obtained by vacuum
o
filtration and then recrystallized in ethanol. Yield: 63.6%; mp: 226–227 C (Table 10); IR (KBr)
ν
: 3262
(Bim-NH), 3065, 3033 (aromatic C−H), 1659 (C=O), 1587, 1511 (aromatic frame), 1430, 1331, 1222, 1091,
-1
1
8
30, 739 cm ; mp: 226−228; H NMR (600 MHz, DMSO-d ) δ: 13.51 (s, 1H, Bim-NH), 8.10 (d, J = 16.1
6
17

ACCEPTED MANUSCRIPT
Hz, 1H, 4-FPh-CH=CH), 8.00 (d, J = 16.1 Hz, 1H, 4-FPh-CH=CH), 7.98–7.96 (m, 2H, 4-FPh-3,5-H), 7.89
(d, J = 6.9 Hz, 1H, Bim-4-H), 7.62 (d, J = 6.9 Hz, 1H, Bim-7-H), 7.42 (d, J = 5.8 Hz, 1H, Bim-6-H), 7.33
1
3
(
m, 3H, 4-FPh-3,5-H, Bim-5-H) ppm; C NMR (151 MHz, DMSO-d )
δ: 181.4, 165.0, 163.3, 149.4, 143.5,
6
1
35.3, 131.8, 131.8, 131.5, 131.5, 126.2, 123.6, 122.0, 121.6, 116.7, 116.6, 113.4 ppm.
4
.1.3. Synthesis of (E)-1-(1H-benzo[d]imidazol-2-yl)-3-(4-chlorophenyl)prop-2-en-1-one (4b)
Compound 4b (1.148 g) was obtained as yellow solid according to general procedure described for 4a
starting from 2-acetylbenzimidazole (0.800 g, 5.00 mmol) and 4-chlorobenzaldehyde (0.700 g, 5.00
mmol). Yield: 81.4%; mp: 231–232 C; IR (KBr) : 3276 (Bim-NH), 3059, 3013 (aromatic C−H), 1660
3
o
ν
-1 1
(
C=O), 1599, 1510, 1487 (aromatic frame), 1327, 1212, 1080, 977, 819, 740 cm ; H NMR (600 MHz,
DMSO-d6) δ: 13.52 (s, 1H, Bim-NH), 8.14 (d, J = 16.1 Hz, 1H, 4-ClPh-CH=CH), 7.98 (d, J = 16.1 Hz, 1H,
4
8
=
-ClPh-CH=CH), 7.92 (d, J = 8.4 Hz, 2H, 4-ClPh-3,5-H), 7.89 (d, J = 8.1 Hz, 1H, Bim-4-H), 7.61 (d, J =
.1 Hz, 1H, Bim-7-H), 7.56 (d, J = 8.4 Hz, 2H, 4-ClPh-2,6-H), 7.43 (t, J = 7.5 Hz, 1H, Bim-6-H), 7.34 (d, J
1
3
7.3 Hz, 1H, Bim-6-H) ppm; C NMR (151 MHz, DMSO-d ) δ: 181.4, 149.4, 143.6, 143.2, 136.1, 135.3,
6
1
33.8, 131.1, 129.6, 126.3, 123.7, 122.8, 121.7, 113.4 ppm.
.1.4. Synthesis of (E)-1-(1H-benzo[d]imidazol-2-yl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (4c
Compound 4c (1.166 g) was obtained as yellow solid according to general procedure described for 4a
4
)
starting from 2-acetylbenzimidazole
3
(0.800 g, 5.00 mmol) and 2,4-dichlorobenzaldehyde (0.875 g, 3.00
o
mmol). Yield: 73.8%; mp: 248–249 C; IR (KBr)
ν
: 3278 (Bim-NH), 3057 (aromatic C−H), 1661 (C=O),
-1 1
1
588, 1515, 1468 (aromatic frame), 1427, 1329, 1213, 1090, 978, 738 cm ; H NMR (600 MHz, DMSO-d )
6
δ
: 13.56 (s, 1H, Bim-NH), 8.17 (s, 2H, 2,4-Cl Ph-CH=CH), 8.15 (d, J = 8.5 Hz, 1H, 2,4-Cl Ph-3-H), 7.88
2
2
(
d, J = 8.1 Hz, 1H, Bim-4-H), 7.77 (d, J = 2.0 Hz, 1H, 2,4-Cl Ph-6-H), 7.61 (d, J = 8.1 Hz, 1H, Bim-7-H),
2
7
.54 (dd, J = 8.5, 2.0 Hz, 1H, 2,4-Cl Ph-5-H), 7.43 (t, J = 7.5 Hz, 1H, Bim-6-H), 7.35 (t, J = 7.5 Hz, 1H,
2
1
3
Bim-5-H) ppm; C NMR (151 MHz, DMSO-d )
δ: 181.1, 149.1, 143.5, 138.0, 136.5, 135.8, 135.4, 131.5,
6
1
30.2, 130.1, 128.7, 126.4, 125.4, 123.8, 121.7, 113.4 ppm.
4
.1.5. Synthesis of (E)-1-(1H-benzo[d]imidazol-2-yl)-3-(p-tolyl)prop-2-en-1-one (4d)
Compound 4d (0.917 g) was obtained as yellow solid according to general procedure described for 4a
starting from 2-acetylbenzimidazole (0.800 g, 5.00 mmol) and 4-methylbenzaldehyde (0.600 g, 5.00
3
o
mmol). Yield: 69.5%; mp: 215–216 C; IR (KBr)
ν: 3264 (Bim-NH), 3064, 3025 (aromatic C−H), 2919
-1
(
aliphatic C−H), 1658 (C=O), 1590, 1513, 1487 (aromatic frame), 1427, 1362, 1216, 1088, 980, 741 cm ;
18

ACCEPTED MANUSCRIPT
1
H NMR (600 MHz, DMSO-d6) δ: 13.53 (s, 1H, Bim-NH), 8.09 (d, J = 16.1 Hz, 1H, 4-CH Ph-CH=CH),
3
7
.97 (d, J = 16.0 Hz, 1H, 4-CH Ph-CH=CH), 7.78 (d, J = 7.5 Hz, 4H, Bim-4,7-H, 4-CH Ph-2,6-H), 7.39 (s,
3 3
1
3
2
H, Bim-5,6-H), 7.32 (d, J = 7.4 Hz, 2H, 4-CH Ph-3,5-H), 2.37 (s, 3H, 4-CH Ph-CH ) ppm; C NMR (151
3 3 3
MHz, DMSO-d6) δ: 181.44, 149.55, 144.79, 141.78, 132.13, 130.25, 129.41, 121.11, 21.58 ppm.
4
.1.6. Synthesis of (E)-1-(1-butyl-1H-benzo[d]imidazol-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one (5a)
To a stirred suspension of potassium carbonate (0.553 g, 4.00 mmol) in acetonitrile (60 mL) was added
o
compound 4a (0.532 g, 2.00 mmol). The mixture was stirred at 70 C for 3 h and then cooled to room
o
temperature. 1-Bromobutane (0.361 g, 2.20 mmol) was added and stirred under 70 C. After the reaction
was completed (monitored by TLC, petroleum ether/chloroform, 1/1, V/V), the solvent was evaporated and
the residue was treated with water (40 mL) and extracted with chloroform (3 × 35 mL). The combined
organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography (eluent, petroleum ether/chloroform,
o
3
3
/1−2/1, V/V) to afford the pure compound 5a as yellow solid. Yield: 74.6%; mp: 75–76 C; IR (KBr)
ν
:
060, 3039 (aromatic C–H), 2957, 2932, 2964 (aliphatic C–H), 1659 (C=O), 1588, 1506, 1475 (aromatic
-1
1
frame), 1331, 1224, 1047, 828, 738 cm ; H NMR (600 MHz, CDCl )
δ: 8.21 (d, J = 16.0 Hz, 1H,
3
4
-FPh-CH=CH), 7.93 (d, J = 8.2 Hz, 1H, Bim-4-H), 7.86 (d, J = 16.0 Hz, 1H, 4-FPh-CH=CH), 7.72 (dd, J
=
8.4, 5.5 Hz, 2H, 4-FPh-3,5-H), 7.46 (d, J = 8.0 Hz, 1H, Bim-7-H), 7.43 (t, J = 7.5 Hz, 1H, Bim-6-H),
7
.37 (t, J = 7.4 Hz, 1H, Bim-5-H), 7.09 (t, J = 8.5 Hz, 2H, 4-FPh-2,6-H), 4.67 (t, J = 7.5 Hz, 2H,
Bim-NCH ), 1.89–1.82 (m, 2H, CH CH CH ), 1.46–1.38 (m, 2H, CH CH ), 0.97 (t, J = 7.4 Hz, 3H,
2
2
2
3
2
3
1
3
CH CH ) ppm; C NMR (151 MHz, CDCl3) δ: 182.5, 165.1, 163.4, 146.8, 143.0, 141.9, 136.6, 131.1,
2
3
1
30.9, 130.9, 125.9, 123.7, 123.1, 121.9, 116.1, 116.0, 110.8, 45.5, 32.5, 20.2, 13.8 ppm; HRMS (ESI)
+
calcd. for C H FN O [M+H] , 323.1560; found, 323.1558.
2
0
19
2
4
.1.7. Synthesis of (E)-3-(4-fluorophenyl)-1-(1-hexyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (5b)
Compound 5b (0.566 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4a (0.798 g, 3.00 mmol) and 1-bromohexane (0.541 g, 3.30 mmol). Yield: 53.8%; mp: 74–75
ºC; IR (KBr) : 3057, 3035 (aromatic C–H), 2958, 2937, 2966 (aliphatic C–H), 1658 (C=O), 1589, 1506,
ν
-
1 1
1
1
7
475 (aromatic frame), 1331, 1224, 1047, 828, 738 cm ; H NMR (600 MHz, CDCl ) δ: 8.21 (d, 1H, J =
3
6.0 Hz, 4-FPh-CH=CH), 7.93 (d, 1H, J = 8.1 Hz, Bim-4-H), 7.86 (d, 1H, J = 16.0 Hz, 4-FPh-CH=CH),
.75–7.71 (m, 2H, Bim-6,7-H), 7.48–7.42 (m, 2H, 4-FPh-3,5-H), 7.38 (t, J = 7.3 Hz, 1H, Bim-5-H), 7.10 (t,
J = 8.4 Hz, 2H, 4-FPh-2,6-H), 4.67 (t, 2H, J = 7.5 Hz, Bim-NCH ), 1.90–1.84 (m, 2H, CH (CH ) CH ),
2
2
2 3
3
19

ACCEPTED MANUSCRIPT
1
3
1
.41–1.37 (m, 2H, CH (CH ) CH ), 1.32 (s, 4H, (CH ) CH ), 0.88 (t, 3H, J = 6.6 Hz, CH CH ) ppm; C
2
2 2
3
2 2
3
2
3
NMR (151 MHz, CDCl3)
δ: 182.6, 165.1, 163.4, 146.9, 143.0, 141.9, 136.6, 130.9, 130.9, 125.9, 123.7,
1
23.1, 121.9, 116.1, 116.0, 110.8, 45.7, 31.4, 30.4, 26.6, 22.6, 13.9 ppm; HRMS (ESI) calcd. for
+
C H FN O [M+H] , 351.1873; found, 351.1871.
22
23
2
4
.1.8. Synthesis of (E)-3-(4-fluorophenyl)-1-(1-heptyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (5c)
Compound 5c (1.032 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4a (1.331 g, 5.00 mmol) and 1-bromoheptane (0.985 g, 5.50 mmol). Yield: 60.0%; mp: 62–63
ºC; IR (KBr) : 3058, 3040 (aromatic C–H), 2958, 2931, 2966 (aliphatic C–H), 1659 (C=O), 1588, 1506,
475 (aromatic frame), 1331, 1224, 1047, 828, 738 cm ; H NMR (600 MHz, CDCl )
ν
-1 1
1
δ
: 8.21 (d, J = 16.0
Hz, 1H, 4-FPh-CH=CH), 7.93 (d, J = 8.2 Hz, 1H, Bim-4-H), 7.87 (d, J = 16.0 Hz, 1H, 4-FPh-CH=CH),
.74 (dd, J = 8.4, 5.4 Hz, 2H, 4-FPh-3,5-H), 7.47 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.44 (t, J = 7.9 Hz, 1H,
3
7
Bim-6-H), 7.38 (t, J = 8.0 Hz, 1H, Bim-5-H), 7.11 (t, J = 8.5 Hz, 2H, 4-FPh-2,6-H), 4.68 (t, J = 7.5 Hz, 2H,
Bim-NCH ), 1.91–1.84 (m, 2H, CH (CH ) CH ), 1.43–1.32 (m, 4H, (CH ) CH CH CH ), 1.30–1.27 (m,
2
2
2 4
3
2 2
2
2
3
1
3
4
1
H, (CH ) CH ), 0.87 (t, J = 6.9 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl3)
δ: 182.6, 165.1,
2
2
3
2
3
63.4, 146.8, 143.1, 141.9, 136.6, 131.2, 131.1, 130.9, 130.8, 125.9, 123.7, 123.1, 121.9, 116.1, 116.0,
1
10.8, 77.2, 77.0, 76.8, 45.7, 31.7, 30.5, 28.9, 26.9, 22.5, 14.0 ppm; HRMS (ESI) calcd. for C H FN O
2
3
25
2
+
[
M+H] , 365.2029; found, 365.2026.
4
.1.9. Synthesis of (E)-3-(4-fluorophenyl)-1-(1-octyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (5d
)
Compound 5d (0.925 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4a (1.331 g, 5.00 mmol) and 1-bromooctane (1.056 g, 5.50 mmol). Yield: 51.2%; mp: 67–68
ºC; IR (KBr) : 3061, 3038 (aromatic C–H), 2956, 2931, 2964 (aliphatic C–H), 1659 (C=O), 1588, 1506,
475 (aromatic frame), 1331, 1224, 1047, 828, 738 cm ; H NMR (600 MHz, CDCl )
ν
-1 1
1
δ
: 8.21 (d, J = 16.0
Hz, 1H, 4-FPh-CH=CH), 7.93 (d, J = 8.2 Hz, 1H, Bim-4-H), 7.87 (d, J = 16.0 Hz, 1H, 4-FPh-CH=CH),
.74 (dd, J = 8.2, 5.6 Hz, 2H, 4-FPh-3,5-H), 7.47 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.44 (t, J = 7.5 Hz, 1H,
3
7
Bim-6-H), 7.38 (t, J = 7.5 Hz, 1H, Bim-5-H), 7.11 (t, J = 8.5 Hz, 2H, 4-FPh-3,5-H), 4.68 (t, J = 7.5 Hz, 2H,
Bim-NCH ), 1.91–1.83 (m, 2H, CH (CH ) CH ), 1.44–1.37 (m, 2H, CH (CH ) CH ), 1.37–1.31 (m, 2H,
2
2
2 5
3
2
2 4
3
1
3
CH (CH ) CH ), 1.31–1.21 (m, 6H, (CH ) CH ), 0.86 (t, J = 6.8 Hz, 3H, CH CH ) ppm; C NMR (151
2
2 3
3
2 3
3
2
3
MHz, CDCl3)
δ: 182.6, 165.1, 163.4, 146.9, 143.1, 141.9, 136.6, 130.9, 130.9, 125.9, 123.7, 123.1, 121.9,
116.1, 116.0, 110.8, 45.7, 31.7, 30.8, 30.5, 29.2, 29.2, 26.9, 22.6, 14.0 ppm; HRMS (ESI) calcd. for
+
C H FN O [M+H] , 379.2186; found, 379.2184.
24
27
2
20

ACCEPTED MANUSCRIPT
4
.1.10. Synthesis of E)-1-(1-butyl-1H-benzo[d]imidazol-2-yl)-3-(4-chlorophenyl)prop-2-en-1-one (5e)
Compound 5e (0.417 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4b (0.564 g, 2.00 mmol) and 1-bromobutane (0.301 g, 2.20 mmol). Yield: 61.7%; mp:
01–102 ºC; IR (KBr) : 3060 (aromatic C−H), 2949, 2868 (aliphatic C−H), 1666 (C=O), 1603, 1483, 1456
aromatic frame), 1405, 1327, 1172, 1044, 826, 742 cm ; H NMR (600 MHz, CDCl )
1
ν
-1 1
(
δ: 8.25 (d, J = 16.0
3
Hz, 1H, 4-ClPh-CH=CH), 7.93 (d, J = 8.9 Hz, 1H, Bim-4-H), 7.84 (d, J = 16.0 Hz, 1H, 4-ClPh-CH=CH),
7
7
.67 (d, J = 8.4 Hz, 2H, 4-ClPh-3,5-H), 7.47 (d, J = 8.2 Hz, 1H, Bim-7-H), 7.45–7.42 (m, 1H, Bim-6-H),
.39 (m, 3H, 4-ClPh-2,6-H, Bim-5-H), 4.68 (t, J = 7.4 Hz, 2H, Bim-NCH ), 1.89–1.80 (m, 2H,
2
1
3
CH CH CH ), 1.47–1.38 (m, 2H, CH CH ), 0.97 (t, J = 7.4 Hz, 3H, CH CH ) ppm; C NMR (151 MHz,
2
2
3
2
3
2
3
CDCl3)
δ: 182.5, 146.8, 142.9, 141.9, 136.7, 136.6, 133.4, 130.1, 129.2, 126.0, 123.8, 121.9, 110.8, 50.7,
+
3
0.8, 20.1, 13.7 ppm; HRMS (ESI) calcd. for C H ClN O [M+H] , 339.1264; found, 339.1263.
2
0
19
2
4
.1.11. Synthesis of (E)-3-(4-chlorophenyl)-1-(1-hexyl-1H-benzo[d]imidazol-2-yl) prop-2-en-1-one (5f)
Compound 5f (0.478 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4b (0.564 g, 2.00 mmol) and 1-bromohexane (0.361 g, 2.20 mmol). Yield: 65.2%; mp: 84–85
ºC; IR (KBr) : 3058, 3023 (aromatic C−H), 2994, 2868 (aliphatic C−H), 1665 (C=O), 1603, 1483, 1456
ν
-
1 1
(
aromatic frame), 1405, 1327, 1172, 1044, 826, 742 cm ; H NMR (600 MHz, CDCl ) δ: 8.11 (d, 1H, J =
3
1
7
4
6.0 Hz, 4-ClPh-CH=CH), 7.80 (d, 1H, J = 8.1 Hz, Bim-4-H), 7.68 (d, 1H, J = 16.0 Hz, 4-ClPh-CH=CH),
.49 (d, 2H, J = 7.2 Hz, 4-ClPh-3,5-H), 7.31–7.21 (m, 3H, Bim-5,6,7-H), 7.21 (d, J = 7.2 Hz, 2H,
-ClPh-2,6-H), 4.50 (t, 2H, J = 7.4 Hz, Bim-NCH ), 1.75–1.69 (m, 2H, CH (CH ) CH ), 1.30–1.23 (m, 2H,
2
2
2 3
3
1
3
CH (CH ) CH ), 1.22–1.15 (m, 4H, (CH ) CH ), 0.75 (t, 3H, J = 6.8 Hz, CH CH ) ppm; C NMR (151
2
2 2
3
2 2
3
2
3
MHz, CDCl3)
δ: 182.4, 146.8, 142.6, 142.0, 136.6, 136.6, 133.4, 130.0, 129.1, 125.9, 123.8, 123.7, 122.0,
+
1
10.8, 45.7, 31.4, 30.4, 26.6, 22.5, 14.0 ppm; HRMS (ESI) calcd. for C H ClN O [M+H] , 367.1577;
2
2
23
2
found, 367.1576.
4
.1.12. Synthesis of (E)-3-(4-chlorophenyl)-1-(1-heptyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (5g)
Compound 5g (0.450 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4b (0.564 g, 2.00 mmol) and 1-bromoheptane (0.394 g, 2.20 mmol). Yield: 59.2%; mp: 71–72
ºC; IR (KBr) : 3058, 3023 (aromatic C−H), 2996, 2924, 2852 (aliphatic C−H), 1668 (C=O), 1610, 1587,
482 (aromatic frame), 1329, 1052, 975, 823, 748 cm ; H NMR (600 MHz, CDCl )
ν
-1 1
1
δ: 8.24 (d, J = 16.0
3
Hz, 1H, 4-ClPh-CH=CH), 7.92 (d, J = 8.1 Hz, 1H, Bim-4-H), 7.83 (d, J = 16.0 Hz, 1H, 4-ClPh-CH=CH),
21

ACCEPTED MANUSCRIPT
7
.65 (d, J = 8.3 Hz, 2H, 4-ClPh-3,5-H), 7.47–7.42 (m, 2H, Bim-6,7-H), 7.37 (m, 3H, 4-ClPh-2,6-H,
Bim-5-H), 4.65 (t, J = 7.5 Hz, 2H, Bim-NCH ), 1.92–1.81 (m, 2H, CH (CH ) CH ), 1.41–1.32 (m, 4H,
2
2
2 4
3
1
3
(
CH ) CH CH CH ), 1.30–1.23 (m, 4H, (CH ) CH ), 0.87 (t, J = 6.9 Hz, 3H, CH CH ) ppm; C NMR
2 2 2 2 3 2 2 3 2 3
(151 MHz, CDCl3)
δ: 182.46, 146.79, 142.73, 141.94, 136.64, 136.63, 133.37, 130.05, 129.16, 125.92,
1
23.82, 123.72, 121.95, 110.80, 45.70, 31.70, 30.45, 28.93, 26.88, 22.54, 14.01 ppm; HRMS (ESI) calcd.
+
for C H ClN O [M+H] , 381.1734; found, 381.1732.
23
25
2
4
.1.13. Synthesis of (E)-3-(4-chlorophenyl)-1-(1-octyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (5h)
Compound 5h (0.631 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4b (0.564 g, 2.00 mmol) and 1-bromooctane (0.423 g, 2.20 mmol). Yield: 80.0%; mp: 82–83
ºC; IR (KBr) : 3055, 3031 (aromatic C−H), 2959, 2868 (aliphatic C−H), 1664 (C=O), 1603, 1483, 1456
ν
-
1 1
(
aromatic frame), 1405, 1327, 1172, 1044, 826, 742 cm ; H NMR (600 MHz, CDCl ) δ: 8.26 (d, 1H, J =
3
1
7
6.0 Hz, 4-ClPh-CH=CH), 7.93 (d, 1H, J = 9.8 Hz, Bim-4-H), 7.85 (d, 1H, J = 15.9 Hz, 4-ClPh-CH=CH),
.67 (m, 2H, 4-ClPh-3,5-H), 7.45 (m, 2H, Bim-6,7-H), 7.40 (m, 3H, 4-ClPh-2,6-H, Bim-5-H), 4.67 (t, 2H, J
=
7.5 Hz, Bim-NCH ), 1.90–1.83 (m, 2H, CH (CH ) CH ), 1.42–1.31 (m, 4H, (CH ) CH CH CH CH ),
2 2 2 5 3 2 2 2 2 2 3
1
3
1
1
3
3
.29–1.21 (m, 6H, (CH ) CH ), 0.86 (t, J = 6.0 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl3) δ:
2 3 3 2 3
82.5, 146.8, 142.8, 141.9, 136.7, 136.6, 133.4, 130.1, 129.2, 126.0, 123.8, 121.9, 110.8, 45.7, 31.7, 30.8,
+
0.4, 29.2, 29.1, 26.9, 22.6, 14.0 ppm; HRMS (ESI) calcd. for C H ClN O [M+H] , 395.1890; found,
2
4
27
2
95.1892.
.1.14. Synthesis of (E)-1-(1-butyl-1H-benzo[d]imidazol-2-yl)-3-(2,4-dichlorophenyl)prop-2-en-1- one (5i
4
)
Compound 5i (0.538 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4c (0.633 g, 2.00 mmol) and 1-bromobutane (0.302 g, 2.20 mmol). Yield: 72.3%; mp:
10–111 ºC; IR (KBr) : 3068, 3009 (aromatic C−H), 2956, 2843 (aliphatic C−H), 1668 (C=O), 1603, 1472
aromatic frame), 1434, 1306, 1161, 1037, 745 cm ; H NMR (600 MHz, CDCl )
1
ν
-1 1
(
δ: 8.28 (d, J = 16.1 Hz,
3
1
7
H, 2,4-Cl Ph-CH=CH), 8.25 (d, J = 15.9 Hz, 1H, 2,4-Cl Ph-CH=CH), 7.92 (d, J = 8.1 Hz, 1H, Bim-4-H),
2
2
.89 (d, J = 8.5 Hz, 1H, 2,4-Cl Ph-5-H), 7.47 (m, 3H, Bim-6,7-H, 2,4-Cl Ph-3-H), 7.38 (t, J = 7.4 Hz, 1H,
2
2
Bim-5-H), 7.30 (d, J = 8.4 Hz, 1H, 2,4-Cl Ph-6-H), 4.69 (t, J = 7.4 Hz, 1H, Bim-NCH ), 1.88 (m, 2H,
2
2
1
3
CH CH CH ), 1.44 (m, 2H, CH CH ), 0.98 (t, J = 7.3 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl )
2
2
3
2
3
2
3
3
δ: 182.1, 146.6, 141.9, 138.4, 136.7, 136.4, 131.6, 130.0, 128.9, 127.5, 126.1, 125.8, 123.8, 122.0, 110.9,
+
4
5.5, 30.8, 20.2, 13.7 ppm; HRMS (ESI) calcd. for C H Cl N O [M+H] , 373.0874; found, 373.0875.
2
0
18
2
2
22

ACCEPTED MANUSCRIPT
4
.1.15. Synthesis of (E)-3-(2,4-dichlorophenyl)-1-(1-hexyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1- one (5j)
Compound 5j (0.631 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4c (0.633 g, 2.00 mmol) and 1-bromohexane (0.361 g, 2.20 mmol). Yield: 71.7%; mp: 60–61
ºC; IR (KBr) : 3065, 3008 (aromatic C−H), 2932, 2869 (aliphatic C−H), 1669 (C=O), 1603, 1472
ν
-1 1
(
aromatic frame), 1434, 1306, 1161, 1037, 745 cm ; H NMR (600 MHz, CDCl )
δ: 8.26 (d, J = 16.0 Hz,
3
1
7
1
2
H, 2,4-Cl Ph-CH=CH), 8.23 (d, J = 16.1 Hz, 1H, 2,4-Cl Ph-CH=CH), 7.91 (d, J = 8.2 Hz, 1H, Bim-4-H),
2
2
.87 (d, J = 8.5 Hz, 1H, 2,4-Cl Ph-5-H), 7.47–7.42 (m, 3H, Bim-6,7-H, 2,4-Cl Ph-3-H), 7.37 (t, J = 7.4 Hz,
2
2
H, Bim-5-H), 7.30–7.26 (m, 1H, 2,4-Cl Ph-6-H), 4.68–4.64 (t, J = 7.8 Hz, 2H, Bim-NCH ), 1.90–1.84 (m,
2
2
H, CH (CH ) CH ), 1.44–1.37 (m, 2H, CH CH CH CH ), 1.36–1.29 (m, 4H, (CH ) CH ), 0.88 (t, J = 7.0
2
2 3
3
2
2
2
3
2 2
3
1
3
Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl3) δ: 182.1, 146.6, 141.9, 138.4, 136.7, 136.7, 136.4,
2
3
1
31.6, 130.0, 128.8, 127.5, 126.1, 125.8, 123.8, 122.0, 110.8, 45.7, 31.4, 30.4, 26.6, 22.5, 13.9 ppm; HRMS
+
(
ESI) calcd. for C H Cl N O [M+H] , 401.1187; found, 401.1186.
2
2
22
2
2
4
.1.16. Synthesis of (E)-3-(2,4-dichlorophenyl)-1-(1-heptyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1-one (5k)
Compound 5k (0.689 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4c (0.633 g, 2.00 mmol) and 1-bromoheptane (0.394 g, 2.20 mmol). Yield: 83.2%; mp: 61–62
ºC; IR (KBr) : 3062, 3004 (aromatic C−H), 2918, 2844 (aliphatic C−H), 1667 (C=O), 1603, 1472
ν
-1 1
(
aromatic frame), 1434, 1306, 1161, 1037, 745 cm ; H NMR (600 MHz, CDCl )
δ: 8.21 (d, J = 16.0 Hz,
3
1
7
7
H, 2,4-Cl Ph-CH=CH), 8.17 (d, J = 16.0 Hz, 1H, 2,4-Cl Ph-CH=CH), 7.88 (d, J = 8.1 Hz, 1H, Bim-4-H),
2
2
.78 (d, J = 8.5 Hz, 1H, 2,4-Cl Ph-5-H), 7.40–7.37 (m, 2H, Bim-6,7-H), 7.36 (s, 1H, 2,4-Cl Ph-3-H),
2
2
.34–730 (m, 1H, Bim-5-H), 7.19 (dd, J = 8.4, 1.8 Hz, 1H, 2,4-Cl Ph-6-H), 4.59 (t, J = 7.8 Hz, 2H,
2
Bim-NCH ), 1.86–1.80 (m, 2H, CH (CH ) CH ), 1.40–1.30 (m, 4H, (CH ) CH CH CH ), 1.30–1.23 (m,
2
2
2 4
3
2 2
2
2
3
1
3
4
1
3
4
H, (CH ) CH ), 0.86 (t, J = 7.0 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl3) δ: 181.9, 146.5,
2 2 3 2 3
41.9, 138.1, 136.6, 136.6, 136.3, 131.5, 129.9, 128.7, 127.4, 126.0, 125.7, 123.7, 122.0, 110.7, 45.7, 31.7,
+
0.4, 28.9, 26.9, 22.6, 14.0 ppm; HRMS (ESI) calcd. for C H Cl N O [M+H] , 415.1344; found,
2
3
24
2
2
15.1345.
.1.17. Synthesis of (E)-3-(2,4-dichlorophenyl)-1-(1-octyl-1H-benzo[d]imidazol-2-yl)prop-2-en-1- one (5l
4
)
Compound 5l (0.598 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4c (0.633 g, 2.00 mmol) and 1-bromooctane (0.423 g, 2.20 mmol). Yield: 69.8%; mp: 65–66
ºC; IR (KBr) : 3063, 3005 (aromatic C−H), 2922, 2853 (aliphatic C−H), 1666 (C=O), 1603, 1472
ν
23

ACCEPTED MANUSCRIPT
-1 1
(
aromatic frame), 1434, 1306, 1161, 1037, 745 cm ; H NMR (600 MHz, CDCl )
δ: 8.11 (d, J = 16.0 Hz,
3
1
7
7
1
H, 2,4-Cl Ph-CH=CH), 8.07 (d, J = 16.0 Hz, 1H, 2,4-Cl Ph-CH=CH), 7.77 (d, J = 8.1 Hz, 1H, Bim-4-H),
2
2
.69 (d, J = 8.5 Hz, 1H, 2,4-Cl Ph-5-H), 7.31–7.27 (m, 2H, Bim-6,7-H), 7.26 (s, 1H, 2,4-Cl Ph-5-H),
2
2
.24–7.19 (m, 1H, Bim-5-H), 7.10 (d, J = 8.4 Hz, 1H, 2,4-Cl Ph-5-H), 4.49 (t, J = 7.6 Hz, 2H, Bim-NCH ),
2
2
.77–1.69 (m, 2H, CH (CH ) CH ), 1.29–1.23 (m, 2H, CH (CH ) CH ), 1.23–1.18 (m, 2H,
2
2 5
3
2
2 4
3
1
3
CH (CH ) CH ), 1.17–1.10 (m, 6H, (CH ) CH ), 0.74 (t, J = 6.9 Hz, 3H, CH CH ) ppm; C NMR (151
2
2 3
3
2 3
3
2
3
MHz, CDCl3)
δ
181.9, 146.6, 141.9, 138.1, 136.7, 136.6, 136.4, 131.6, 129.9, 128.8, 127.4, 126.0, 125.8,
+
1
23.7, 122. 0, 110.8, 45.7, 31.8, 30.4, 29.2, 29.2, 26.9, 22.6, 14.0 ppm; MS (m/z): 429.3 [M+H] ; HRMS
+
(
ESI) calcd. for C H Cl N O [M+H] , 429.1500; found, 429.1501.
2
4
26
2
2
4
.1.18. Synthesis of (E)-1-(1-butyl-1H-benzo[d]imidazol-2-yl)-3-(p-tolyl)prop-2-en-1-one (5m)
Compound 5m (0.241 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4d (0.528 g, 2.00 mmol) and 1-bromobutane (0.301 g, 2.20 mmol). Yield: 37.9%; mp: 73–74
ºC; IR (KBr) : 3061, 3025 (aromatic C−H), 2927, 2860 (aliphatic C−H), 1663 (C=O), 1592, 1511, 1474,
451 (aromatic frame), 1328, 1162, 1050, 980, 753 cm ; H NMR (600 MHz, CDCl )
ν
-1 1
1
δ: 8.27 (d, J = 16.0
3
Hz, 1H, 4-CH Ph-CH=CH), 7.96 (d, J = 8.1 Hz, 1H, Bim-4-H), 7.93 (d, J = 16.0 Hz, 1H,
3
4
-CH Ph-CH=CH), 7.68 (d, J = 7.8 Hz, 2H, 4-CH Ph-2,6-H), 7.50 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.46 (t, J
3 3
=
7.5 Hz, 1H, Bim-6-H), 7.40 (t, J = 7.5 Hz, 1H, Bim-5-H), 7.26 (d, J = 7.7 Hz, 2H, 4-CH Ph-3,5-H), 4.72
3
(
t, J = 7.4 Hz, 2H, Bim-NCH ), 2.42 (s, 3H, 4-CH Ph-CH ), 1.93–1.85 (m, 2H, CH CH CH ), 1.51–1.41
2
3
3
2
2
3
1
3
(
m, 2H, CH CH ), 1.00 (t, J = 7.3 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl )
δ: 182.8, 147.1,
2
3
2
3
3
1
1
44.6, 141.9, 141.3, 136.6, 132.2, 129.6, 129.0, 125.7, 123.6, 122.4, 121.9, 110.8, 45.5, 32.5, 21.5, 20.2,
+
3.7 ppm; HRMS (ESI) calcd. for C H N O [M+H] , 319.1810; found, 319.1811.
2
1
22
2
4
.1.19. Synthesis of (E)-1-(1-hexyl-1H-benzo[d]imidazol-2-yl)-3-(p-tolyl)prop-2-en-1-one (5n)
Compound 5n (0.400 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4d (0.528 g, 2.00 mmol) and 1-bromohexane (0.361 g, 2.20 mmol). Yield: 57.8%; mp: 79–80
ºC; IR (KBr) : 3056, 3024 (aromatic C−H), 2924, 2856 (aliphatic C−H), 1660 (C=O), 1592, 1511, 1474,
451 (aromatic frame), 1328, 1162, 1050, 980, 753 cm ; H NMR (600 MHz, CDCl )
ν
-1 1
1
δ: 8.12 (d, J = 16.0
3
Hz, 1H, 4-CH Ph-CH=CH), 7.82 (d, J = 8.1 Hz, 1H, Bim-4-H), 7.78 (d, J = 16.0 Hz, 1H,
3
4
-CH Ph-CH=CH), 7.51 (d, J = 7.9 Hz, 2H, 4-CH Ph-2,6-H), 7.32 (d, J = 8.0 Hz, 1H, Bim-7-H), 7.29 (t, J
3 3
=
7.4 Hz, 1H, Bim-6-H), 7.24 (t, J = 7.4 Hz, 1H, Bim-5-H), 7.08 (d, J = 7.8 Hz, 2H, 4-CH Ph-3,5-H), 4.52
3
(
t, J = 7.6 Hz, 2H, Bim-NCH ), 2.25 (s, 3H, 4-CH Ph-CH ), 1.78–1.70 (m, 2H, CH (CH ) CH ), 1.32–1.25
2 3 3 2 2 3 3
24

ACCEPTED MANUSCRIPT
1
3
(
m, 2H, CH (CH ) CH ), 1.24–1.15 (m, 4H, (CH ) CH ), 0.76 (t, J = 6.8 Hz, 3H, CH CH ) ppm; C NMR
2 2 2 3 2 2 3 2 3
(151 MHz, CDCl3)
δ: 182.8, 147.1, 144.5, 142.0, 141.3, 136.6, 132.2, 129.6, 129.0, 125.7, 123.6, 122.3,
+
1
3
21.9, 110.8, 45.7, 31.5, 30.4, 26.6, 22.5, 21.5, 14.0 ppm; HRMS (ESI) calcd. for C H N O [M+H] ,
2
3
26
2
47.2123; found, 347.2125.
.1.20. Synthesis of (E)-1-(1-heptyl-1H-benzo[d]imidazol-2-yl)-3-(p-tolyl)prop-2-en-1-one (5o
4
)
Compound 5o (0.486 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4d (0.528 g, 2.00 mmol) and 1-bromoheptane (0.394 g, 2.20 mmol). Yield: 67.4%; mp: 50–51
ºC; IR (KBr) : 3054, 3027 (aromatic C−H), 2934, 2858 (aliphatic C−H), 1659 (C=O), 1592, 1511, 1474,
ν
-
1 1
1
1
7
7
1
451 (aromatic frame), 1328, 1162, 1050, 980, 753 cm ; H NMR (600 MHz, CDCl )
δ
: 8.24 (d, J = 16 Hz,
3
H, 4-CH Ph-CH=CH), 7.93 (d, J = 8.2 Hz, 1H, Bim-4-H), 7.89 (d, J = 16.0 Hz, 1H, 4-CH Ph-CH=CH),
3
3
.62 (d, J = 7.9 Hz, 2H, 4-CH Ph-2,6-2H), 7.45–7.38 (m, 2H, Bim-6,7-H), 7.37–7.32 (m, 1H, Bim-5-H),
3
.18 (t, J = 8.1 Hz, 2H, 4-CH Ph-3,5-H), 4.63 (t, J = 7.2 Hz, 2H, Bim-NCH ), 2.35 (s, 3H, 4-CH Ph-CH ),
3
2
3
3
.89–1.81 (m, 2H, CH (CH ) CH ), 1.41–1.31 (m, 4H, (CH ) CH CH CH ), 1.29–1.21 (m, 4H,
2
2 4
3
2 2
2
2
3
1
3
(
CH ) CH ), 0.86 (t, J = 6.2 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl3) δ: 182.8, 147.1, 144.5,
2 2 3 2 3
1
2
42.0, 141.3, 136.6, 132.2, 129.6, 129.0, 125.7, 123.6, 122.3, 121.9, 110.7, 45.7, 31.7, 30.5, 29.0, 26.9,
+
2.6, 21.5, 14.0 ppm; HRMS (ESI) calcd. for C H N O [M+H] , 361.2280; found, 361.2283.
2
4
28
2
4
.1.21. Synthesis of (E)-1-(1-octyl-1H-benzo[d]imidazol-2-yl)-3-(p-tolyl)prop-2-en-1-one (5p)
Compound 5p (0.360 g) was obtained as yellow solid according to general procedure described for 5a
starting from 4d (0.528 g, 2.00 mmol) and 1-bromooctane (0.423 g, 2.20 mmol). Yield: 48.1%; mp: 50–51
ºC; IR (KBr) : 3053, 3021 (aromatic C−H), 2944, 2866 (aliphatic C−H), 1659 (C=O), 1592, 1511, 1474,
451 (aromatic frame), 1328, 1162, 1050, 980, 753 cm ; H NMR (600 MHz, CDCl )
ν
-1 1
1
δ: 8.13 (d, J = 16.0
3
Hz, 1H, 4-CH Ph-CH=CH), 7.81 (d, J = 8.1 Hz, 1H, Bim-4-H), 7.78 (d, J = 16.1 Hz, 1H,
3
4
7
-CH Ph-CH=CH), 7.52 (d, J = 8.1 Hz, 2H, 4-CH Ph-2,6-H), 7.35–7.28 (m, 2H, Bim-6,7-H), 7.24 (t, J =
3 3
.5 Hz, 1H, Bim-5-H), 7.09 (d, J = 7.8 Hz, 2H, 4-CH Ph-3,5-H), 4.53 (t, J = 7.6 Hz, 2H, Bim-NCH ), 2.25
3
2
(
s, 3H, 4-CH Ph-CH ), 1.78–1.70 (m, 2H, CH (CH ) CH ), 1.31–1.19 (m, 4H, (CH ) CH CH CH CH ),
3 3 2 2 5 3 2 2 2 2 2 3
1
3
1
1
3
3
.18–1.10 (m, 6H, (CH ) CH ), 0.75 (t, J = 6.3 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, CDCl3) δ:
2 3 3 2 3
82.8, 147.1, 144.5, 142.0, 141.3, 136.6, 132.2, 129.6, 129.0, 125.7, 123.6, 122.3, 121.9, 110.8, 45.7, 31.8,
+
0.5, 29.3, 29.2, 26.9, 22.6, 21.5, 14.0 ppm; HRMS (ESI) calcd. for C H N O [M+H] , 375.2436; found,
2
5
30
2
75.2437.
25

ACCEPTED MANUSCRIPT
4
.1.22. Synthesis of 4-(1-butyl-1H-benzo[d]imidazol-2-yl)-6-(4-fluorophenyl)pyrimidin-2-amine (6a)
To a stirred solution of sodium hydride (0.096 g, 4 mmol) and guanidine hydrochloride (0.191 g, 2.00
o
mmol) in DMF (5 mL) was added gradually 5a (0.483 g, 1.50 mmol) at 0 C, and the reaction mixture was
o
stirred for 1h at room temperature and then heated at 100 C for 12 h. After the completion of reaction
(
monitored by TLC, chloroform/methanol, 15/1, V/V), the reaction mixture was poured into water and the
obtained precipitate was filtered and purified by column chromatography (chloroform/methanol, 10/1, V/V)
to afford target compound 6a. Yield: 49.1%; mp: 199–200 ºC; IR (KBr) : 3365, 3308 (NH ), 3155
ν
2
(aromatic C−H), 2960, 2929, 2868 (aliphatic C−H), 1640, 1566, 1505, 1465 (aromatic frame), 1411, 1330,
-
1 1
1
7
226, 1012, 829, 750 cm ; H NMR (600 MHz, DMSO-d )
δ
: 8.24 (dd, J = 8.5, 5.7 Hz, 2H, 4-FPh-3,5-H),
6
.94 (s, 1H, Pyr-H), 7.78 (d, J = 8.0 Hz, 1H, Bim-4-H), 7.70 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.38 (d, J = 8.7
Hz, 2H, 4-FPh-2,6-H), 7.36 (m, 1H, Bim-6-H), 7.31 (t, J = 7.5 Hz, 1H, Bim-5-H), 6.98 (s, 2H, Pyr-NH2),
.96 (t, J = 7.2 Hz, 2H, Bim-NCH ), 1.78–1.72 (m, 2H, CH CH CH ), 1.34–1.27 (m, 2H, CH CH ), 0.86 (t,
4
2
2
2
3
2
3
1
3
J = 7.4 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 164.3, 163.9, 159.5, 148.5, 142.5, 137.0,
2
3
6
1
33.9, 133.9, 129.7, 129.7, 124.2, 123.0, 120.3, 116.3, 116.1, 111.6, 105.0, 45.0, 32.6, 19.8, 14.1 ppm;
+
HRMS (ESI) calcd. for C H FN [M+H] , 362.1781; found, 362.1783.
2
1
20
5
4
.1.23. Synthesis of 4-(4-fluorophenyl)-6-(1-hexyl-1H-benzo[d]imidazol-2-yl)pyrimidin-2-amine (6b)
Compound 6b (0.365 g) was obtained as yellow solid according to general procedure described for 6a
starting from 5b (0.525 g, 1.50 mmol) and guanidine hydrochloride (0.191 g, 2.00 mmol). Yield: 62.5%;
mp: 175–176 ºC; IR (KBr) : 3317, 3190 (NH ), 3057 (aromatic C−H), 2956, 2927, 2855 (aliphatic C−H),
ν
2
-1
1
1
640, 1566, 1507, 1467 (aromatic frame), 1411, 1331, 1229, 1156, 829, 750 cm ; H NMR (600 MHz,
DMSO-d6) δ: 8.24 (dd, J = 8.4, 5.7 Hz, 2H, 4-FPh-3,5-H), 7.92 (s, 1H, Pyr-H), 7.77 (d, J = 8.0 Hz, 1H,
Bim-4-H), 7.70 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.37 (t, J = 8.2 Hz, 3H, 4-FPh-2,6-H, Bim-6-H), 7.30 (t, J =
7
.5 Hz, 1H, Bim-5-H), 6.97 (s, 2H, Pyr-NH ), 4.95 (t, J = 7.1 Hz, 2H, Bim-NCH ), 1.79–1.71 (m, 2H,
2
2
1
3
CH (CH ) CH ), 1.29–1.14 (m, 6H, (CH ) CH ), 0.78 (t, J = 6.9 Hz, 3H, CH CH ) ppm; C NMR (151
2
2 3
3
2 3
3
2
3
MHz, DMSO-d6)
δ: 165.1, 164.3, 163.9, 163.4, 159.5, 148.5, 142.5, 137.0, 133.9, 129.7, 129.6, 124.1,
1
23.0, 120.3, 116.2, 116.1, 111.6, 105.0, 45.1, 31.3, 30.4, 26.1, 22.5, 14.2 ppm; HRMS (ESI) calcd. for
+
C H FN [M+H] , 390.2094; found, 390.2093.
23
24
5
4
.1.24. Synthesis of 4-(4-fluorophenyl)-6-(1-heptyl-1H-benzo[d]imidazol-2-yl)pyrimidin-2-amine (6c
)
Compound 6c (0.441 g) was obtained as yellow solid according to general procedure described for 6a
26

ACCEPTED MANUSCRIPT
starting from 5c (0.546 g, 1.5 mmol) and guanidine hydrochloride (0.191 g, 2.00 mmol). Yield: 72.8%; mp:
1
1
76–177 ºC; IR (KBr)
ν: 3312, 3185 (NH ), 3057 (aromatic C−H), 2953, 2926, 2855 (aliphatic C−H), 1640,
2
-1 1
569, 1508, 1466 (aromatic frame), 1411, 1331, 1228, 1157, 832, 749 cm ; H NMR (600 MHz, DMSO-d )
6
δ
: 8.24 (dd, J = 8.4, 5.7 Hz, 2H, 4-FPh-3,5-H), 7.92 (s, 1H, Pyr-H), 7.77 (d, J = 8.0 Hz, 1H, Bim-4-H), 7.69
d, J = 8.1 Hz, 1H, Bim-7-H), 7.39-7.34 (m, 3H, 4-FPh-2,6-H, Bim-6-H), 7.30 (t, J = 7.5 Hz, 1H, Bim-5-H),
.96 (s, 2H, Pyr-NH ), 4.95 (t, J = 7.1 Hz, 2H, Bim-NCH ), 1.77–1.71 (m, 2H, CH (CH ) CH ), 1.27–1.21
(
6
2
2
2
2 4
3
(
m, 4H, (CH ) CH CH CH ), 1.20–1.16 (m, 2H, CH CH CH ), 1.15–1.10 (m, 2H, CH CH ), 0.77 (t, J =
2 2 2 2 3 2 2 3 2 3
1
3
7
1
2
.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d6) δ: 165.1, 164.3, 163.9, 163.4, 159.5, 148.6,
2 3
42.5, 137.0, 133.9, 129.7, 129.7, 124.1, 123.0, 120.3, 116.3, 116.1, 111.6, 105.0, 45.1, 31.7, 30.4, 28.8,
+
6.4, 22.4, 14.3 ppm; HRMS (ESI) calcd. for C H FN [M+H] , 404.2250; found, 404.2252.
2
4
26
5
4
.1.25. Synthesis of 4-(4-fluorophenyl)-6-(1-octyl-1H-benzo[d]imidazol-2-yl)pyrimidin-2-amine (6d)
Compound 6d (0.525 g) was obtained as yellow solid according to general procedure described for 6a
starting from 5d (0.567 g, 1.50 mmol) and guanidine hydrochloride (0.191 g, 2.00 mmol). Yield: 83.9%;
mp: 150–151 ºC; IR (KBr) : 3306, 3190 (NH ), 3068 (aromatic C−H), 2953, 2925, 2853 (aliphatic C−H),
ν
2
-1
1
1
638, 1570, 1508, 1466 (aromatic frame), 1412, 1332, 1230, 1158, 854, 748 cm ; H NMR (600 MHz,
DMSO-d6) δ: 8.24 (dd, J = 8.3, 5.7 Hz, 2H, 4-FPh-3,5-H), 7.92 (s, 1H, Pyr-H), 7.78 (d, J = 8.0 Hz, 1H,
Bim-4-H), 7.68 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.36 (t, 3H, 4-FPh-2,6-H, Bim-6-H), 7.30 (t, J = 7.5 Hz, 1H,
Bim-5-H), 6.97 (s, 2H, Pyr-NH ), 4.95 (t, J = 7.1 Hz, 2H, Bim-NCH ), 1.78–1.68 (m, 2H, CH (CH ) CH ),
2
2
2
2 5
3
1
.27–1.19 (m, 4H, (CH ) CH CH CH CH ), 1.19–1.15 (m, 2H, CH (CH ) CH ), 1.14–1.10 (m, 4H,
2 2 2 2 2 3 2 2 2 3
1
3
(
CH ) CH ), 0.77 (t, J = 7.0 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d6) δ: 165.1, 164.3,
2 2 3 2 3
1
1
4
63.9, 163.4, 159.5, 148.6, 142.5, 137.0, 133.9, 129.7, 129.7, 124.1, 123.0, 120.3, 116.2, 116.1, 111.6,
+
05.0, 45.1, 31.6, 30.3, 29.1, 29.0, 26.5, 22.5, 14.3 ppm; HRMS (ESI) calcd. for C H FN [M+H] ,
2
5
28
5
18.2407; found, 418.2406.
.1.26. Synthesis of 4-(1-butyl-1H-benzo[d]imidazol-2-yl)-6-(4-chlorophenyl)pyrimidin-2-amine (6e
4
)
Compound 6e (0.300 g) was obtained as yellow solid according to general procedure described for 6a
starting from 5e (0.507 g, 1.50 mmol) and guanidine hydrochloride (0.191 g, 2.00 mmol). Yield: 53.0%; mp:
1
1
86–187 ºC; IR (KBr)
ν: 3306, 3192 (NH ), 3057 (aromatic C−H), 2960, 2929, 2861 (aliphatic C−H), 1631,
2
-1 1
564, 1488, 1464 (aromatic frame), 1409, 1330, 1224, 1085, 834, 744 cm ; H NMR (600 MHz, DMSO-d )
6
δ: 8.20 (d, J = 8.5 Hz, 2H, 4-ClPh-3,5-H), 7.95 (s, 1H, Pyr-H), 7.78 (d, J = 8.0 Hz, 1H, Bim-4-H), 7.71 (d, J
=
8.1 Hz, 1H, Bim-7-H), 7.61 (d, J = 8.5 Hz, 2H, 4-ClPh-2,6-H), 7.37 (t, J = 7.6 Hz, 1H, Bim-6-H), 7.31 (t,
27

ACCEPTED MANUSCRIPT
J = 7.5 Hz, 1H, Bim-5-H), 7.02 (s, 2H, Pyr-NH ), 4.96 (t, J = 7.2 Hz, 2H, Bim-NCH ), 1.79–1.70 (m, 2H,
2
2
1
3
CH CH CH ), 1.34–1.70 (m, 2H, CH CH ), 0.86 (t, J = 7.4 Hz, 3H, CH CH ) ppm; C NMR (151 MHz,
2
2
3
2
3
2
3
DMSO-d6)
δ: 164.1, 163.9, 159.6, 148.4, 142.5, 137.0, 136.2, 136.0, 129.3, 129.1, 124.2, 123.0, 120.3,
+
1
11.6, 105.0, 45.1, 32.64, 19.8, 14.1 ppm; HRMS (ESI) calcd. for C H ClN [M+H] , 378.1485; found,
2
1
20
5
3
78.1486.
.1.27. Synthesis of 4-(4-chlorophenyl)-6-(1-hexyl-1H-benzo[d]imidazol-2-yl)pyrimidin-2-amine (6f
4
)
Compound 6f (0.399 g) was obtained as yellow solid according to general procedure described for 6a
starting from 5f (0.550 g, 1.50 mmol) and guanidine hydrochloride (0.191 g, 2.00 mmol). Yield: 65.7%; mp:
1
1
69–170 ºC; IR (KBr)
ν: 3314, 3187 (NH ), 3055 (aromatic C−H), 2953, 2925, 2855 (aliphatic C−H), 1640,
2
-1 1
548, 1493, 1466 (aromatic frame), 1410, 1330, 1227, 1091, 823, 747 cm ; H NMR (600 MHz, DMSO-d )
6
δ
: 8.19 (d, 2H, J = 8.4 Hz, 4-ClPh-3,5-H), 7.94 (s, 1H, Pyr-H), 7.77 (d, J = 8.0 Hz, 1H, Bim-4-H), 7.69 (d, J
8.1 Hz, 1H, Bim-7-H), 7.60 (d, J = 8.4 Hz, 2H, 4-ClPh-2,6-H), 7.36 (t, J = 7.6 Hz, 1H, Bim-6-H), 7.30 (t,
J = 7.5 Hz, 1H, Bim-5-H), 7.01 (s, 2H, Pyr-NH ), 4.95 (t, J = 7.1 Hz, 2H, Bim-NCH ), 1.77–1.70 (m, 2H,
=
2
2
1
3
CH (CH ) CH ), 1.29–1.13 (m, 6H, (CH ) CH ), 0.77 (t, J = 7.0 Hz, 3H, CH CH ) ppm; C NMR (151
2
2 3
3
2 3
3
2
3
MHz, DMSO-d6)
δ: 164.2, 163.9, 159.6, 148.5, 142.5, 137.0, 136.2, 136.0, 129.3, 129.1, 124.2, 123.1,
+
1
4
20.3, 111.6, 105.0, 45.1, 31.3, 30.4, 26.1, 22.5, 14.2 ppm; HRMS (ESI) calcd. for C H ClN [M+H] ,
2
3
24
5
06.1798; found, 406.1797.
.1.28. Synthesis of 4-(4-Chlorophenyl)-6-(1-heptyl-1H-benzo[d]imidazol-2-yl)pyrimidin-2-amine (6g
4
)
Compound 6g (0.427 g) was obtained as yellow solid according to general procedure described for 6a
starting from 5g (0.570 g, 1.50 mmol) and guanidine hydrochloride (0.191 g, 2.00 mmol). Yield: 67.9%;
mp: 177–178 ºC; IR (KBr) : 3277, 3156 (NH ), 3060, 3016 (aromatic C−H), 2925, 2853 (aliphatic C−H),
622, 1565, 1492, 1466 (aromatic frame), 1413, 1331, 1226, 1087, 1013, 857, 829, 749 cm ; H NMR
600 MHz, DMSO-d6) : 8.20 (d, J = 8.5 Hz, 2H, 4-ClPh-3,5-H), 7.94 (s, 1H, Pyr-H), 7.78 (d, J = 8.0 Hz,
H, Bim-4-H), 7.70 (d, J = 8.1 Hz, 1H, Bim-7-H), 7.60 (d, J = 8.4 Hz, 2H, 4-ClPh-2,6-H), 7.37 (t, J = 7.5
ν
2
-1
1
1
(
δ
1
Hz, 1H, Bim-6-H), 7.31 (t, J = 7.5 Hz, 1H, Bim-5-H), 7.02 (s, 2H, Pyr-NH ), 4.96 (t, J = 7.1 Hz, 2H,
2
Bim-NCH ), 1.77–1.71 (m, 2H, CH (CH ) CH ), 1.25–1.20 (m, 4H, (CH ) CH CH CH ), 1.20–1.15 (m,
2
2
2 4
3
2 2
2
2
3
1
3
2
H, CH CH CH ), 1.14–1.09 (m, 2H, CH CH ), 0.77 (t, J = 7.2 Hz, 3H, CH CH ) ppm; C NMR (151
2 2 3 2 3 2 3
MHz, DMSO-d6)
δ: 164.1, 163.9, 159.6, 148.5, 142.5, 137.0, 136.2, 136.0, 129.3, 129.1, 124.2, 123.0,
1
20.3, 111.6, 105.0, 45.1, 31.7, 30.4, 28.8, 26.4, 22.4, 14.3 ppm; HRMS (ESI) calcd. for C H ClN
2
4
26
5
+
[
M+H] , 420.1955; found, 420.1956.
28