Design, synthesis and antimicrobial evaluation of novel benzimidazole-incorporated sulfonamide analogues

Article abstract of DOI:10.1016/j.ejmech.2017.04.077

A novel series of benzimidazole-incorporated sulfonamide analogues were designed and synthesized with an effort to overcome the increasing antibiotic resistance. Compound 5c gave potent activities against Gram-positive bacteria and fungi, and 2,4-dichlorobenzyl derivative 5g showed good activities against Gram-negative bacteria. Both of these two active molecules 5c and 5g could effectively intercalate into calf thymus DNA to form compound?DNA complex respectively, which might block DNA replication to exert their powerful antimicrobial activity. Molecular docking experiments suggested that compounds 5c and 5g could insert into base-pairs of DNA hexamer duplex by the formation of hydrogen bonds with guanine of DNA. The transportation behavior of these highly active compounds by human serum albumin (HSA) demonstrated that the electrostatic interactions played major roles in the strong association of active compounds with HSA, and which was also confirmed by the full geometry calculation optimizations.

Full text of DOI:10.1016/j.ejmech.2017.04.077

Accepted Manuscript
Design, synthesis and antimicrobial evaluation of novel benzimidazole-incorporated
sulfonamide analogues
Hui-Zhen Zhang, Shi-Chao He, Yan-Jun Peng, Hai-Juan Zhang, Lavanya Gopala,
Vijai Kumar Reddy Tangadanchu, Lin-Ling Gan, Cheng-He Zhou
PII:
S0223-5234(17)30359-8
10.1016/j.ejmech.2017.04.077
EJMECH 9428
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 December 2016
Revised Date: 24 April 2017
Accepted Date: 30 April 2017
Please cite this article as: H.-Z. Zhang, S.-C. He, Y.-J. Peng, H.-J. Zhang, L. Gopala, V.K.R.
Tangadanchu, L.-L. Gan, C.-H. Zhou, Design, synthesis and antimicrobial evaluation of novel
benzimidazole-incorporated sulfonamide analogues, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.04.077.
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Graphical Abstract
Design, synthesis and antimicrobial evaluation of novel benzimidazole-incorporated sulfonamide analogues
a,b
a
a
b
a
Hui-Zhen Zhang , Shi-Chao He , Yan-Jun Peng , Hai-Juan Zhang , Lavanya Gopala , Vijai Kumar Reddy
a,
c
,*
a,*
Tangadanchu ‡, Lin-Ling Gan , Cheng-He Zhou
a
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China;
b
Shandong Provincial Engineering Technology Research Center for Lunan Chinese Herbal Medicine, School of
Pharmacy, Linyi University, Linyi 276000, China
;
c
School of Pharmacy, Chongqing Medical and Pharmaceutical College, Chongqing 401331, China.
A novel series of benzimidazole-incorporated sulfonamide analogues were developed and screened for their
antimicrobial activities. Interaction with calf thymus DNA and molecular docking were investigated.
Transportation behavior with HSA was explored and molecular electrostatic potentiality was studied by full
geometry optimizations.
NHCOCH
3
O
S
O
N
N
R
5
5
c, R = 4-F
g, R = 2,4-2Cl
5
g-DNA hexamer duplex
5
c-DNA hexamer duplex

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Title page
Title:
Design, synthesis and antimicrobial evaluation of novel benzimidazole-incorporated sulfonamide analogues
Author Names and Affiliations:
a,b,#
a,#
a
b
a,
Hui-Zhen Zhang , Shi-Chao He , Yan-Jun Peng , Hai-Juan Zhang , Lavanya Gopala †, Vijai Kumar
a,
c,
*
a,*
Reddy Tangadanchu ‡, Lin-Ling Gan , Cheng-He Zhou
a
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China;
b
School of Pharmacy, Linyi University, Linyi 276000, China
;
c
School of Pharmacy, Chongqing Medical and Pharmaceutical College, Chongqing 401331, China.
#
These two authors contributed equally to this work
†
‡
*
Postdoctoral fellow from Sri Venkateswara University, India
Postdoctoral researcher from CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
Corresponding Address:
Prof. Cheng-He Zhou, School of Chemistry and Chemical Engineering, Southwest University, Chongqing
00715, China
4
Tel.: +86-23-68254967, +86-23-68254165; Fax: +86-23-68254967
E-mail: zhouch@swu.edu.cn (Cheng-He Zhou)
Dr. Lin-Ling Gan, School of Pharmacy, Chongqing Medical and Pharmaceutical College, Chongqing
401331, China
E-mail: ganlinling2012@163.com (Lin-Ling Gan)
Abstract:
A novel series of benzimidazole-incorporated sulfonamide analogues were designed and synthesized with
an effort to overcome the increasing antibiotic resistance. Compound 5c gave potent activities against
Gram-positive bacteria and fungi, and 2,4-dichlorobenzyl derivative 5g showed good activities against
Gram-negative bacteria. Both of these two active molecules 5c and 5g could effectively intercalate into calf
thymus DNA to form compound−DNA complex respectively, which might block DNA replication to exert
their powerful antimicrobial activity. Molecular docking experiments suggested that compounds 5c and 5g
could insert into base-pairs of DNA hexamer duplex by the formation of hydrogen bonds with guanine of
1

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DNA. The transportation behavior of these highly active compounds by human serum albumin (HSA)
demonstrated that the electrostatic interactions played major roles in the strong association of active
compounds with HSA, and which was also confirmed by the full geometry calculation optimizations.
Keywords:
Benzimidazole; Sulfonamide analogues; Antibacterial; Antifungal; Calf thymus DNA; HSA
2

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1. Introduction
The antibiotic resistance which was accelerated by the use and misuse of antimicrobial drugs has been a
major global challenge for public health. During the past decades, a dramatic increase in human-pathogenic
bacteria worldwide was seen due to resistance to one or multiple antibiotics. More and more infections
caused by resistant microorganisms fail to respond to conventional treatment, and in some cases, even last
resort antibiotics have lost their power. Recently, the World Health Organization titled a recent world health
day as “Combat drug resistance: no action today means no cure tomorrow”, and triggered an increase in
research activity, and several promising strategies have been developed to restore treatment options against
infections by resistant bacterial pathogens [1].
Sulfonamides as artificial antifolic agents have been widely used for the prevention and cure of bacterial
infections in biological systems and recently have evoked high favor in biology and medicine because of
their diverse pharmacological activities [2] including antibacterial [3], antifungal [4], antiviral [5],
antitumor [6], anti-inflammatory [7], and carbonic anhydrase inhibitors [8]. As the analogues of
aminobenzoic acid, sulfonamides could compete with it to availably prevent the synthesis of nucleic acids
and proteins, and then inhibit the growth of various microorganisms. Furthermore, sulfonamide compounds
have attracted increasing research in supramolecular chemistry because they could combine the features of
different fragments through the coordination of phenylamino and sulfonyl amino groups [9]. Particularly,
Ag-sulfadiazine has been significantly employed in burn therapy, which is better than the free ligand or
AgNO . Up to now, numerous sulfonamides bearing aromatic heterocycles such as isoxazole, thiazole,
3
pyridazine and pyrimidine have been successfully developed and used in clinic like sulfadiazine,
sulfachlorpyridazine, sulfathiazole and sulfisoxazole with excellent antimicrobial activities. This has
stimulated considerable efforts towards the synthesis and development of completely new structural
sulfonamide derivatives with excellent activity, broad spectrum and low toxicity.
Our previous efforts have identified that the introduction of aromatic heterocycles such as imidazole [10],
triazole [11], tetrazole [12], thiazole [13] and benzene-fused azoles like benzimidazole [14] and
benzotriazole [15] into target molecules could highly improve the antimicrobial activities. 1,2,3-Triazole
sulfonamide WXL-1 containing 2,4-difluorobenzyl group was almost 32-fold more potent than precursor
sulfonamide against Pseudomonas aeruginosa and Shigella dysenteriae [16]. The sulfonamide ZHZ−1
bearing 2-methyl-5-nitroimidazole fragment displayed almost equivalent anti-P. aeruginosa efficiency to
Chloromycin (MIC = 16 µg/mL) (Fig. 1), and further research found that this compound could effectively
intercalate into calf thymus DNA to form compound−DNA complex which might block DNA replication to
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exert their powerful antimicrobial activities. Moreover, the transportation behavior of this compound by
human serum albumin (HSA) showed that electrostatic interactions played major roles in the strong
association of compound ZHZ-1 and HSA [17].
Fig. 1
In view of such stimulating properties and as an extension of our studies on the development of
sulfonamide azoles, the methylene moiety as the bioisostere of amino group was introduced into the
sulfonamide fragment with the aim to investigate the effect on antimicrobial activity [18]. Benzimidazole
ring as the fused heterocycle of benzene and imidazole was structurally similar to purine, and its derivatives
could compete with purine to inhibit the synthesis of nucleic acids and proteins inside the bacterial cell wall,
and then kill the bacterial strains or inhibit their growth [19]. Additionally, fluoro- and chloro-substituted
benzyl groups were introduced into the target molecules in order to improve the pharmacological properties
by enhancing the rate of absorption and the transportation of drugs in vivo [20].
The newly synthesized sulfonamide analogues were screened for their antibacterial and antifungal
activities in vitro. DNA as one of the most important targets has attracted growing interest in investigating
the interaction of small molecules with DNA to explore the possible antimicrobial action mechanism [21].
The interactions of most active compounds with calf thymus DNA were evaluated by UV-vis absorption
spectroscopy on a molecular level to explore their probable antimicrobial mechanisms. Moreover,
molecular docking studies were employed to reconfirm the interaction behaviors between the prepared
compounds and DNA hexamer duplex [22]. Additionally, the transportation behavior of the highly active
molecules by human serum albumin (HSA) was investigated by fluorescence spectroscopy to preliminarily
study their absorption, distribution and metabolism [23].
2. Results and discussion
2.1. Chemistry
The target benzimidazole-incorporated sulfonamide analogues were prepared from commercial
acetanilide and chlorosulfonic acid. Their synthetic routes were outlined in Scheme 1. Acetanilide was
reacted with chlorosulfonic acid to produce N-protected sulfonyl chloride 2, and then further treated by
sodium sulfite and sodium bicarbonate to give p-acetylamino benzenesulfinic acid sodium salt 3. The latter
was subsequently reacted with chloromethyl benzimidazole to afford benzimidazole-incorporated
sulfonamide analogue 4. The N-alkylation of benzimidazole ring of compound 4 with halobenzyl halides in
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acetonitrile at 70 ºC using potassium carbonate as base respectively produced the target aralkyl
benzimidazole sulfonamide analogues 5a−h in 52.1−73.7% yields which showed that the substituents
exhibited effect on the formation of target compounds to some extent. Generally, the chlorobenzyl halides
gave higher yields than fluorobenzyl ones. In this series, it was found that the strong electron-withdrawing
3-F group gave the lowest yield (52.1%), while 3-Cl substituted one provided the highest yield of 73.7%.
Moreover, compound 4 was also reacted with a series of alkyl bromides to produce compounds 6a−i with a
large difference in yields of 24.2−71.2%. Furthermore, the N-alkylation of compound 4 with carbazole
alkyl bromide was successfully performed to give carbazole alkylated benzimidazole sulfonamide analogue
7
in yield of 72.0%. Generally, it was thought that the presence of protons on the nitrogen atom of the
sulfonamide skeleton was favorable for the bioactivity, and thus compounds 5−7 were further transformed
into the deprotected sulfonamide analogues 8−10 in ethanol in the presence of sodium hydroxide in order to
explore their effect on the bioactivities.
Scheme 1
2.2. Analysis of spectra
1
13
All the new compounds were characterized by IR, H NMR, C NMR, MS and HRMS spectra. Their
spectral analyses were consistent with the assigned structures and listed in the experimental section. The
+
mass spectra for benzimidazole compounds gave a major fragment of [M+H] according to their molecular
formula.
2.2.1. IR spectra
In IR spectra, all the synthesized benzimidazole sulfonamide analogues 4−7 gave broad absorption in
-1
3
369−3352 cm which indicated the presence of NH group of amide moiety, whereas two broad absorption
-1
between 3332−3307 and 3209−3185 cm were attributed to the NH group in compounds 8−10. The
characteristic C=N bands of benzimidazole ring in all benzimidazole derivatives appeared in the region
-1
between 1694 and 1639 cm . All the other absorption bands were also observed at the expected regions.
1
2
.2.2. H NMR spectra
1
In H NMR spectra, compounds 4−7 gave singlets at 2.12–2.10 ppm assigned to the CH protons linked
3
to the amide moiety. The singlets at 5.19–5.07 ppm of compounds 5−7 were assigned to the CH protons
2
linked to benzimidazole ring, while the N-CH protons of alkyl chain in compound 6 gave lower shift
2
signals at 4.35–4.21 ppm. The substitution of alkyl group by halobenzyl moiety to yield compound 5 led to
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downfield shifts of N-CH protons (5.63–5.56 ppm), which were higher than those of CH protons attached
2
2
C2-benzimidazole ring because of the strong electron-withdrawing ability of halophenyl moieties and
nitrogen atom in benzimidazole ring. The corresponding deprotected compounds 8 and 9 gave upfield shifts
of SO -CH protons down to 4.90–4.87 and 5.03–4.88 ppm due to the absence of acetyl group respectively.
2
2
Moreover, the peaks for the 3,5-H protons in the sulfonamide analogue ring in the protected compounds
−7 appeared at δ 7.76–7.67 ppm, whereas deprotection led to an upfield shift to 6.61–6.58 ppm. In
4
addition, all the other aromatic and aliphatic protons appeared at the appropriate chemical shifts and
integral values.
1
3
2
.2.3. C NMR spectra
1
3
The C NMR spectral analyses were in accordance with the assigned structures. No large differences
13
were found in the C NMR chemical shifts for the carbonyl carbon in compounds 5–7 (δ 169.7–169.6
ppm). The signals for the phenyl 3,5-C in the sulfonamide analogue skeletons in derivatives 5–7 were
observed at 119.0–118.9 ppm, respectively. It was noticeable that the deprotection of these compounds to
13
compounds 8–10 resulted in upfield C shifts (5.9–5.8 ppm) of these two carbons because of the absence
of the strong electron-withdrawing acetyl group. The signals at 56.3–54.5 ppm in compounds 4–10 were
assigned to the methylene carbon which was connected with sulfonyl group. For compounds 5 and 8, the
methylene carbon linked to the benzimidazole ring was appeared at 50.2–45.1 ppm. All the other carbons
13
gave C peaks at the expected regions.
2.3. Biological Activity
The in vitro antimicrobial screening for all the synthesized compounds was evaluated against four
Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Methicillin-resistant Staphylococcus aureus
N315 (MRSA), Micrococcus luteus ATCC 4698 and Bacillus subtilis ATCC 21216), four Gram-negative
bacteria (Escherichia coli ATCC 8099, Pseudomonas aeruginosa ATCC 27853, Bacillus typhi and Bacillus
proteus ATCC 13315) and five fungi (Candida albicans ATCC 76615, Candida mycoderma, Candida utilis,
Saccharomyces cerevisia and Aspergillus flavus) using two fold serial dilution technique recommended by
National Committee for Clinical Laboratory Standards (NCCLS) with the positive control of clinically
antimicrobial drugs Chloromycin, Norfloxacin and Fluconazole [24]. The values of ClogP, a partition
coefficient as a kind of measurement for hydrophobicity/lipophilicity, were calculated using ChemDraw
Ultra 10.0 software integrated with Cambridge Software (Cambridge Soft Corporation). The antibacterial
and antifungal data as well as ClogP values were depicted in Tables 1 and 2.
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2.3.1. Antibacterial activity
The in vitro antibacterial screening demonstrated that some of the newly synthesized compounds could
effectively inhibit the growth of all the tested microorganisms and exhibit broad antimicrobial spectrum
and potent antibacterial activity. Compound 4 exhibited moderate to good activity against the tested
bacteria (MIC = 32−128 µg/mL) in comparison with clinical drugs.
Table 1 displayed the significant effects of the substituents on the benzene ring on the biological activity.
Noticeably, in this series of halobenzyl benzimidazole sulfonamide analogues, compound 5c with
4
4
-fluorobenzyl group gave the best activities against the tested Gram-positive bacteria with MIC values of
–64 µg/mL. Particularly, its anti-S. aureus (MIC = 4 µg/mL) activity was 2-fold more potent to clinical
Chloromycin and Norfloxacin, and for B. subtilis strains, it also showed two times more active than
Chloromycin (MIC = 16 µg/mL). Especially, this compound displayed equivalent inhibition activity against
MRSA to Chloromycin (MIC = 16 µg/mL). The replacement of 4-fluorobenzyl moiety by
2,4-dichlorobenzyl group, which yielded compound 5g, resulted in good bioactivities against
Gram-negative bacteria with MIC values ranging from 4 to 32 µg/mL. Compound 5g showed eight times
higher activity (MIC = 4 µg/mL) than Chloromycin against B. typhi. However, the substitution of
4-fluorobenzyl fragment in compound 5c by 3,4-dichlorobenzyl group which gave derivative 5h was not
beneficial for the antibacterial activities.
In comparison with halobenzyl compounds 5a−h, most of alkyl derivatives 6a−i exerted relatively lower
activities in inhibiting the growth of the tested strains. Compound 6c with pentyl group gave good anti-P.
aeruginosa activity with MIC value of 4 µg/mL which was 4-fold more potent than Chloromycin. The
replacement of pentyl chain by heptyl group, which yielded compound 6e, resulted in strong activity
towards the tested S. aureus strains (MIC = 8 µg/mL). Additionally, when the alkyl substituents were
extended to decyl, dodecyl and octadecyl groups, compounds 6f−i gave relatively weaker inhibitory activity.
These results suggested that the length of alkyl chain possessed remarkable effects on biological activities.
The short alkyl chains with poor lipophilicity or long alkyl chains with poor hydrophilicity in these
compounds might make them unfavorable for being delivered to the binding sites.
Some of the deprotected halobenzyl benzimidazole sulfonamide analogues 8a−h exerted relatively better
activity than the corresponding protected ones to some extent in inhibiting the growth of the tested bacteria.
Specially, compound 8f with 4-chlorobenzyl moiety was 32-fold or 16-fold more potent than its precursor
against B. subtilis or M. luteus strains (MIC = 8 and 8 µg/mL, respectively). 2,4-Dichlorobenzyl
benzimidazole sulfonamide analogue 8g displayed equivalent bioactivity against S. aureus to Chloromycin
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and Norfloxacin with MIC value of 8 µg/mL. However, the deprotection of alkyl compounds 6a−i, which
yielded compounds 9a−i, led to weak antibacterial activities.
Much research has reported that the introduction of carbazole heterocycle is helpful to improve
antimicrobial potency [18]. In our work, it was found that the carbazole incorporated sulfonamide analogue
7
possessed considerable potentiality with MIC value of 16 µg/mL. Its corresponding deprotected
compound 10 gave moderate activity against B. subtilis and B. typhi strains.
Table 1
2.3.2. Antifungal activity
The antifungal evaluation in vitro displayed that some prepared benzimidazole sulfonamide analogues
exhibited good bioactivities against the tested fungal strains. Compound 4 showed moderate to good
antifungal activities with MIC values ranging from 16 to 128 µg/mL.
In the series of sulfonamide analogues 5a−h, compound 5c bearing 4-fluorobenzyl group exerted the
relatively best activities in inhibiting the growth of all the tested fungal strains. The replacement of
4-fluorobenzyl moiety by 2-chlorobenzyl fragment, which yielded compound 5d, resulted in good activity
against Fluconazole-insensitive A. flavus (MIC = 8 µg/mL). Moreover, 3,4-dichlorobenzyl substituted
compound 5g gave MIC value of 16 µg/mL against A. flavus, which was 16 times more active than
reference drug.
The length of aliphatic chain exhibited obvious effects on antifungal activity. The suitable length of alkyl
chain to exert the best antifungal efficacy was observed to be (CH ) moiety, and the propyl derivative 6b
2
2
generally gave better activity in contrast with other alkyl derivatives with shorter or longer chain length. It
displayed comparable inhibitory activity against S. cerevisiae to reference (MIC = 16 µg/mL). Compound
6f containing octyl group was more effective than the reference at 8 µg/mL concentration. Additionally, the
deprotected compound 9b also gave good activity in inhibiting the growth of the tested fungal with MIC
values ranging from 4 to 64 µg/mL.
The deprotected compound 8a bearing 2-fluorobenzyl fragment exhibited good activities against C.
mycoderma, C. utilis and S. cerevisiae with MIC values of 8, 4 and 8 µg/mL, respectively. Moreover, its
anti-A. flavus activity was 8-fold more potent than the reference drug (MIC = 256 µg/mL). Notably,
compound 8g with 2,4-dichlorobenzyl moiety showed much stronger activities against C. utilis and A.
flavus (MIC = 2 and 4 µg/mL) than Fluconazole (MIC = 8 and 256 µg/mL).
Table 2
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2.3.3. Effect of ClogP values on antimicrobial activity
Hydrophobic/lipophilic properties possessed an important role in exerting biological activity [25]. The
ClogP values as one of the most important factors have been extensively employed to predict the
bioactivity of target molecules. The calculated liposome/water partition coefficients (ClogP) for all newly
prepared compounds were shown in Table 1. The results demonstrated that compounds with lower values
of ClogP showed better antimicrobial activities, and these compounds possessed comparable ClogP values
to the reference drugs with equivalent potency. As shown in Table 1, the ClogP values of compounds 6a–i
generally increased with the increasing length of alkyl groups, and the enhancement of the antimicrobial
activities was observed in compounds 6a–e, but the bioactivities were decreased in compounds 6f–i. These
might be explained by the possibility that higher lipophilic compounds were unfavorable for being
delivered to the binding sites in organism, and manifested the significant role of suitable lipophilicity in
drug design.
2.4. Interactions with calf thymus DNA
DNA is the informational molecule encoding the genetic instructions and has been widely researched for
the advisable design and development of efficient drugs. Calf thymus DNA has always been employed as a
model because of its biological importance and commercial available properties. The binding behavior of
compound 5c (exerting good inhibition against Gram-positive bacteria and fungal strains) and 5g
(displaying good inhibition against Gram-negative bacteria strains) with calf thymus DNA was studied to
explore the possible antimicrobial mechanism of action on a molecular level in vitro with neutral red (NR)
dye as a spectral probe using UV-vis spectroscopic methods [26].
2.4.1. Absorption spectra of DNA in the presence of compounds 5c and 5g
The absorption spectroscopy as one of the most important techniques is extensively employed in
DNA-binding studies. Generally, it is considered that hypochromism and hyperchromism are vital spectral
features to distinguish changes in the DNA double-helical structure. As reported, hyperchromism was
generated from the breakage of the DNA duplex secondary structure, while hypochromism was originated
from the stabilization of the DNA duplex by either an intercalation binding mode or the electrostatic effects
of small molecules [27]. The observed hypochromism intensively recommended a close proximity of the
aromatic chromophore to the DNA bases, which might be due to the strong interaction between the
electronic states of intercalating chromophore and that of the DNA base.
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With a fixed concentration of DNA, the UV-vis absorption spectra were recorded with an increasing
amount of compounds 5c and 5g. As shown in Fig. 2 and Fig. 3, the UV-vis spectra showed that the
maximum absorption of DNA (at 260 nm) displayed a proportional increase with increase in the
concentration of compounds 5c and 5g. Meanwhile, the absorption value for the measured values of the
5c–DNA or 5g–DNA complex was slightly greater than the simply sum of free DNA and free compound 5c
or 5g, which was observed in the inset of Fig. 2 and Fig. 3. These indicated a weak hyperchromic effect
existed between DNA and compound 5c or 5g. These demonstrated that a weak hypochromic effect existed
between DNA and compound 5c or 5g. Moreover, the intercalation of the aromatic chromophore of
compound 5c or 5g into the helix and the strong overlap of π-π* states in the large π-conjugated system
with the electronic states of DNA bases were consistent with the observed spectral changes [28].
Fig. 2
Fig. 3
On the basis of the variations in the absorption spectra of DNA upon binding to 5c or 5g, equation 1 can
be utilized to calculate the binding constant (K).
(1)
0
0
The plot of A /(A-A ) versus 1/[compound 5c(or 5g)] is constructed by using the absorption titration data
and linear fitting (Supporting Information: Fig. S1 or Fig. S2), yielding the binding constant, K = 1.26 ×
4
4
1
0 or 1.52 × 10 L/mol, R = 0.999 or 0.999, SD = 0.04 or 0.09 respectively (R is the correlation
coefficient. SD is standard deviation).
2.4.2. Absorption spectra of NR interaction with DNA
Neutral Red (NR) as a planar phenazine dye is structurally similar to other planar dyes acridine, thiazine
and xanthene. It has been displayed that the binding of NR with DNA is intercalation binding type.
Therefore, NR was used as a spectral probe to investigate the binding mode of 5c or 5g with DNA in this
work.
The absorption spectra of the NR dye upon the addition of DNA were showed in Fig. S3 (Supporting
Information). It was apparent that the absorption peak of the NR at around 460 nm gave gradual decrease
with the increasing concentration of DNA, and a new band at around 530 nm developed. This was because
of the formation of the new DNA–NR complex. An isosbestic point at 504 nm provided evidence of
10

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DNA–NR complex formation.
2.4.3. Absorption spectra of competitive interaction of compound 5c or 5g and NR with DNA
As showed in Fig. 4 or Fig. 5, the competitive binding between NR and 5c (or 5g) with DNA was
observed in the absorption spectra. With the increasing concentration of compound 5c or 5g, an apparent
intensity increase was observed around 275 nm. Compared with the absorption around 275 nm of
NR–DNA complex, the absorbance at the same wavelength exhibited the reverse process (inset of Fig. 4 or
Fig. 5). These various spectral changes were consistent with the intercalation of compound 5c or 5g into
DNA by substituting for NR in the DNA–NR complex.
Fig. 4
Fig. 5
As depicted above, although compounds 5c and 5g displayed different bioactivities, both of them could
intercalate into calf thymus DNA to form compound−DNA complex which might block DNA replication to
exert their powerful antimicrobial activities.
2.5. Molecular docking of compound 5c or 5g with DNA hexamer duplex
Molecular docking study as a useful method is widely employed to investigate the binding modes of
small molecules to DNA. Up to now, the full-length 3D structure of calf thymus DNA is not available in
Protein Data Bank (PDB). Since calf thymus DNA is B-DNA, the CT-DNA sequence d(CGATCG) (PDB
2
code: 3FT6) was chosen as receptor model. In our research, molecular docking study was performed
between compound 5c or 5g and DNA hexamer duplex to understand the binding model. The docking
−
1
−1
mode with the lowest binding free energy (
−4.01 kJ·mol for 5c or −4.26 kJ·mol for 5g) is shown in Fig.
6
and Fig. 7 or Fig. 8 and Fig. 9. The results demonstrated that the hydrogen atom connected to the nitrogen
atom and oxygen atom in the sulfonyl group of compounds 5c and 5g formed two hydrogen bonds with the
guanine of DNA, thus preventing the formation of hydrogen bond between cytosine in DNA. This kind of
interaction resulted in decreased stability of DNA, and therefore inhibited its physiological function. All
these suggested that the simulation results were in accordance with the above spectral experiment results.
Fig. 6
Fig. 7
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Fig. 8
Fig. 9
2.6. Interactions of compound 5c or 5g with HSA
2.6.1. UV-vis absorption spectral study
UV-vis absorption measurement as operational method is applicable to explore the structural change of
protein and to identify the complex formation. In our binding experiment, UV-vis absorption spectroscopic
method was employed to evaluate the binding behaviors between compound 5c or 5g and HSA. As shown
in Fig. S4 or Fig. S5 (Supporting Information), the absorption peak observed at 278 nm was attributed to
the aromatic rings in Tryptophan (Trp-214), Tyrosine (Tyr-411) and Phenylalanine (Phe) residues in HSA.
With the addition of compound 5c or 5g, the peak intensity increased, indicating that compound 5c or 5g
could interact with HSA and the peptide strands of HSA were extended [29].
2.6.2. Fluorescence quenching mechanism
Fluorescence spectroscopy is a favorable method to investigate the interactions of small molecules with
HSA. The fluorescence intensity of Trp-214 may change when HSA interacts with other small molecules,
which could be reflected in the fluorescence spectra of HSA in the UV region [30]. The effect of compound
5c or 5g on the fluorescence intensity to HSA at 293 K was shown in Fig. 11 or Fig. 12. It was obvious that
HSA had a strong fluorescence emission with a peak at 348 nm owing to the single Try-214 residue. The
intensity of this characteristic broad emission band regularly decreased with the increased concentrations of
compound 5c or 5g. In Fig. 10 or Fig. 11, the black line showed the only emission spectrum of compound
5c or 5g, which indicated that compound 5c or 5g did not possess significant fluorescence features, and
therefore the effect of compound 5c or 5g on fluorescence of HSA would be negligible at the excitation
wavelength (295 nm) [31].
Fig. 10
Fig. 11
The fluorescence quenching data can be analyzed by the well-known Stern-Volmer equation [32]:
F0
=
1+ K [Q] =1+ Kqτ0[Q]
(2)
SV
F
The Stern-Volmer plots of HSA in the presence of compound 5c or 5g at different concentrations and
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temperatures could be calculated and were showed in Fig. S6 or Fig. S7 (Supporting Information).
The values of KSV and K for the interaction of compound 5c or 5g with HSA at different temperatures
q
were showed in Table 3. The KSV values were inversely correlated with the temperature, which indicated
that the fluorescence quenching of HSA might be initiated by the formation of compound–HSA complex
1
2
-1
rather than dynamic collisions. The K values obtained at different temperatures were in 10 L/mol s
q
(Table 3), which far exceeded the diffusion controlled rate constants of various quenchers with a
1
0
-1
biopolymer (2.0 × 10 L/mol s ), and indicated that the quenching was not initiated by the dynamic
diffusion process but occurred in the statically formation of compound–HSA complex [31].
Table 3
2.6.3. Binding constant and site
For a static quenching process, the data could be described by the Modified Stern-Volmer equation [33]:
F0
1
1
1
(3)
=
+
∆F
f K [Q] fa
a a
The modified Stern-Volmer plots were showed in Fig. S8 or Fig. S9 (Supporting Information) and the
calculated results were depicted in Table 4.
Table 4
When small molecules bind to a set of equivalent sites on a macromolecule, the equilibrium binding
constants and the numbers of binding sites can also be calculated according to the Scatchard equation [34]:
r
=
nK − rK
b b
Df
(4)
The Scatchard plots were shown in Fig. S10 or Fig. S11 (Supporting Information) and the K and n were
b
listed in Table 4.
The modified Stern-Volmer and Scatchard plots for the compound–HSA system at different temperatures
were given in Table 4. The decreased trend of K and K with increased temperatures was in accordance
a
b
with K ’s depended on temperatures. The value of the binding site n was approximately 1, which showed
SV
one high affinity binding site, was present in the interaction of compound 5c or 5g with HSA. The results
also showed that the binding constants were moderate and the effects of temperatures were not significant,
thus both compounds 5c and 5g might be stored and carried by this protein.
2.6.4. Binding mode and thermodynamic parameters
13

ACCEPTED MANUSCRIPT
Generally, there are four types of non-covalent interactions including hydrogen bonds, van der Waals
forces, electrostatic interactions and hydrophobic bonds, which play substantial roles in small molecules
binding to proteins [35]. The thermodynamic parameters enthalpy (∆H) and entropy (∆S) change of binding
reaction are the main evidence for confirming the interactions between small molecules and protein. If the
∆H does not vary significantly over the studied temperatures range, then its value and ∆S can be evaluated
from the van’t Hoff equation:
∆
H
∆S
ln K = −
+
RT
R
(5)
In order to explain the binding model between compound 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. 12 or Fig. 13). The free energy change (∆G) was then calculated from the following equation:
G = ∆H −T∆S
∆
(6)
Fig. 12
Fig. 13
The values of ∆H, ∆G and ∆S were summarized in Table 5. The negative values of free energy ∆G of the
interaction between compound 5c or 5g and HSA suggested that the binding process was spontaneous, and
the negative values of ∆H indicated that the binding was mainly enthalpy-driven and involved an
exothermic reaction, the ∆S was unfavorable for it. A positive ∆S value is frequently taken as a typical
evidence for hydrophobic interaction, which was consistent with the above discussion. Therefore, ∆H < 0
and ∆S > 0 obtained in this case indicated that the electrostatic interactions played an important role in the
binding of compound 5c or 5g to HSA [36].
Table 5
The above experiments displayed that compounds 5c and 5g could effectively interact with HSA, thereby
causing hypochromic effect of ultraviolet spectroscopy. When the electronic transfer occurred between
compound 5c or 5g and HSA, it caused energy transfer without radiation, and therefore resulted in the
quenching of fluorescence spectrum. Further molecular electrostatic potentiality for the compounds 5c and
5g was investigated by full geometry optimizations of the studied systems which was performed by using
the B3LYP functional with 6-31G* basis set. Calculation presented in this work was carried out by the
GAUSSIAN 09 program package. The results manifested that the nucleophilic effect of carbonyl and
sulfonyl groups (Fig. 14, red region) in compound 5c or 5g might induce an electrostatic effect with
14

ACCEPTED MANUSCRIPT
positive electricity of Lys199 in HSA. Therefore, compound 5c or 5g with HSA might interact by
electrostatic interactions [37]. This result was also evidenced by the value of enthalpy change (∆H) and
entropy change (∆S) from the van’t Hoff equation, which was accordant with the literature (when ∆H < 0
and ∆S > 0, the main force is electrostatic interaction).
Fig. 14
3. Conclusion
In conclusion, a novel series of benzimidazole-incorporated sulfonamide analogues have been
successfully prepared starting from commercially avaliable acetanilide. All the new compounds were
1
13
confirmed by H NMR, C NMR, IR, MS and HRMS spectra. Among these sulfonamide analogues,
compound 5c bearing 4-fluorobenzyl group gave potent activities against Gram-positive bacteria and
fungal strains (MIC = 4−64 µg/mL), and 2,4-dichlorobenzyl derivative 5g showed good activities against
Gram-negative bacteria with MIC values ranging from 4 to 32 µg/mL. These results manifested that
compounds 5c and 5g should be worthy to be further investigated as potential antimicrobial agents. Further
research demonstrated that the two active molecules 5c and 5g could effectively intercalate into calf thymus
DNA to form the compound−DNA complex, which might block DNA replication to exert their powerful
antimicrobial activity. Molecular docking experiments suggested that compounds 5c and 5g could
intercalate into base-pairs of DNA hexamer duplex by the formation of hydrogen bonds with guanine of
DNA. The binding research demonstrated that HSA could effectively store and carry compounds 5c and 5g
by electrostatic interactions, and which was also confirmed by the full geometry calculation optimizations.
All these results opened up a promising starting point to optimize the structures of sulfonamide
benzimidazoles as potent antimicrobial agents.
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, Ph = phenyl. The chemical shifts
were reported in parts per million (ppm), the coupling constants (J) were expressed in hertz (Hz) and
15

ACCEPTED MANUSCRIPT
signals were described as singlet (s), doublet (d), triplet (t) as well as multiplet (m). The mass spectra were
recorded on LCMS–2010A and HRMS. All chemicals and solvents were commercially available and were
used without further purification.
4.1.1. Synthesis of 4-acetamidobenzene-1-sulfonyl chloride (2)
4
-Acetamidobenzene-1-sulfonyl chloride 2 was prepared according to the literature procedure, starting
from acetaniline (5.002 g, 0.037 mol) and chlorosulfonic acid (14 mL). Yield: 81.6%; mp: 138–140 ºC.
literature mp: 142–144 ºC) [17].
(
4.1.2. Synthesis of sodium 4-acetamidobenzenesulfinate (3)
A mixture of compound 2 (4.343 g, 18.6 mmol), sodium sulfite (3.486 g, 27.7 mmol) and sodium
bicarbonate (2.322 g, 27.6 mmol) was stirred in water at 80 ºC. After the reaction was completed
monitored by TLC, eluent, methanol/ethyl acetate, 1/2, V/V), the system was washed by ethyl acetate, and
the water phase was collected and evaporated to afford compound 3 as white solid.
(
4.1.3. Synthesis of N-(4-((1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (4)
A suspension of compound 3 (3.959 g, 17.9 mmol), chloromethyl benzimidazole (2.992 g, 17.8 mmol)
and tetrabutylammonium iodide (0.010 g) was stirred in acetone (30 mL) at 50 ºC. After the reaction was
completed (monitored by TLC, eluent, acetone/petroleum ether, 1/1, V/V), the reaction system was filtered,
and the residue was collected and washed with water to give compound 4 as yellow solid. Yield: 60%; mp:
1
1
2
55−156 ºC; IR (KBr) ν: 3429 (Bim-NH), 3359 (N−H), 3024 (aromatic C−H), 3024 (CH ), 1681 (C=N),
2
-1 1
589, 1543 (aromatic frame), 737 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.10 (s, 3H, COCH ), 4.91 (s,
6
3
H, Bim-CH ), 7.20−7.19 (m, 2H, Bim-6,7-H), 7.53 (d, 2H, J = 6.0 Hz, Bim-5,8-H), 7.69 (d, 2H, J = 6.0
2
Hz, Ph-3,5-H), 7.75 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.39 (s, H, NHCOCH ), 12.54 (s, H, Bim-1-H) ppm;
3
13
C NMR (150 MHz, DMSO-d ) δ: 24.7, 54.6, 115.7, 117.1, 119.9, 123.0, 123.4, 129.0, 130.0, 137.9, 139.0,
6
145.8, 152.8, 169.8 ppm.
4.1.4. Synthesis of N-(4-((1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide
(5a)
A suspension of compound 4 (0.492 g, 1.49 mmol) and potassium carbonate (0.425 g, 3.07 mmol) was
stirred in acetonitrile (30 mL) at 50 ºC. After 0.5 h, 1-(chloromethyl)-2-fluorobenzene (0.342 g, 2.37 mmol)
was added and the reaction system was stirred at 70 ºC continuously. After the reaction was completed
16

ACCEPTED MANUSCRIPT
(monitored by TLC, eluent, acetone/petroleum ether, 1/1, V/V), the solvent was removed and the residue
was exacted with chloroform (3 × 20 mL), dried over anhydrous sodium sulfate and purified by silica gel
column chromatography (eluent, acetone/petroleum ether, 1/1, V/V) to afford compound 5a as yellow solid.
Yield: 54%; mp: 215−217 ºC; IR (KBr) ν: 3360 (N−H), 3037 (aromatic C−H), 2994 (CH ), 1687 (C=N),
2
-1 1
1
584, 1531 (aromatic frame), 739 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.14 (s,
6 3
2
H, Bim-CH ), 5.63 (s, 2H, 2-FPh-CH ), 6.93−6.90 (t, H, J = 9.0 Hz, 2-FPh-5-H), 7.11−7.09 (t, H, J = 6.0
2
2
Hz, 2-FPh-4-H), 7.24−7.16 (m, 4H, Bim-5,6,7,8-H), 7.35−7.32 (m, H, 2-FPh-6-H), 7.60−7.59 (m, H,
2-FPh-3-H), 7.74 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H,
1
3
NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 24.6, 49.1, 54.6, 111.4, 116.0, 119.0, 119.90, 122.7,
3
6
123.5, 123.7, 125.1, 129.5, 129.9, 130.3, 132.5, 135.6, 142.8, 144.0, 144.8, 160.6, 169.7 ppm; TOF-MS
+
+
(
m/z): 460 [M+Na] ; HRMS (TOF) calcd. for C H FN O SNa [M+Na] , 460.1102; found, 460.1105.
23 20 3 3
4.1.5. Synthesis of N-(4-((1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide
(5b)
Compound 5b was prepared according to the procedure described for compound 5a, starting from
compound 4 (0.210 g, 0.638), 1-(chloromethyl)-3-fluorobenzene (0.216 g, 1.505 mmol) and potassium
carbonate (0.187 g, 1.355 mmol). The pure product 5b was obtained as yellow solid. Yield: 52.1%; mp:
2
21−224 ºC; IR (KBr) ν: 3362 (N−H), 3038 (aromatic C−H), 2993 (CH ), 1689 (C=N), 1582, 1533
2
-1
1
(
aromatic frame), 736 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.16 (s, 2H,
6
3
Bim-CH ), 5.61 (s, 2H, 3-FPh-CH ), 6.98−6.94 (m, 2H, 3-FPh-2,6-H), 7.11−7.08 (m, H, 3-FPh-4-H),
2
2
7.20−7.18 (m, 2H, Bim-6,7-H), 7.30−7.29 (m, H, Bim-8-H), 7.37−7.33 (m, H, 3-FPh-5-H), 7.61−7.59 (m,
H, Bim-5-H), 7.76 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H,
1
3
NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 24.7, 47.1, 54.5, 111.6, 114.3, 114.8, 118.9, 119.9,
3
6
122.7, 123.4, 123.5, 130.0, 131.0, 132.6, 135.6, 139.9, 142.9, 144.0, 144.8, 162.8, 169.7 ppm; TOF-MS
+
+
(
m/z): 460 [M+Na] ; HRMS (TOF) calcd. for C H FN O SNa [M+Na] , 460.1102; found, 460.1101.
23 20 3 3
4.1.6. Synthesis of N-(4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide
(5c)
Compound 5c was prepared according to the procedure depicted for compound 5a, starting from
compound 4 (0.201 g, 0.610 mmol), 1-(chloromethyl)-4-fluorobenzene (0.176 g, 1.225 mmol) and
potassium carbonate (0.174 g, 1.260 mmol). The pure product 5c was obtained as yellow solid. Yield:
6
7.7%; mp: 199−201 ºC; IR (KBr) ν: 3364 (N−H), 3037 (aromatic C−H), 2995 (CH ), 1687 (C=N), 1585,
2
17

ACCEPTED MANUSCRIPT
-
1 1
1
534 (aromatic frame), 740 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.14 (s, 2H,
6
3
Bim-CH ), 5.56 (s, 2H, 4-FPh-CH ), 7.19−7.12 (m, 6H, 4-FPh-2,3,5,6-H, Bim-6,7-H), 7.30−7.29 (m, H,
2
2
Bim-8-H), 7.59−7.58 (m, H, Bim-5-H), 7.75 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz,
1
3
Ph-2,6-H), 10.40 (s, H, NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 24.7, 47.0, 54.6, 111.7, 115.9,
3
6
119.0, 119.9, 122.6, 123.4, 129.5, 130.0, 132.6, 133.1, 135.6, 142.9, 143.9, 144.8, 162.1, 169.7 ppm;
+
+
TOF-MS (m/z): 460 [M+Na] ; HRMS (TOF) calcd. for C H FN O SNa [M+Na] , 460.1102; found,
2
3
20
3
3
460.1104.
4.1.7. Synthesis of N-(4-((1-(2-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide
(5d)
Compound 5d was prepared according to the experimental procedure described for compound 5a,
starting from compound 4 (0.271 g, 0.824 mmol), 1-chloro-2-(chloromethyl)benzene (0.200 g, 1.242 mmol)
and potassium carbonate (0.180 g, 1.260 mmol). The pure product 5d was obtained as yellow solid. Yield:
6
1
6.7%; mp: 158−160 ºC; IR (KBr) ν: 3367 (N−H), 3039 (aromatic C−H), 2993 (CH ), 1689 (C=N), 1587,
2
-1 1
535 (aromatic frame), 738 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.11 (s, 2H,
6
3
Bim-CH ), 5.62 (s, 2H, 2-ClPh-CH ), 6.52 (d, H, J = 6.0 Hz, 2-ClPh-6-H), 7.22−7.17 (m, 4H,
2
2
Bim-5,6,7,8-H), 7.32−7.30 (t, H, J = 6.0 Hz, 2-ClPh-4-H), 7.53 (d, H, J = 6.0 Hz, 2-ClPh-5-H), 7.63 (d, H,
J = 6.0 Hz, 2-ClPh-3-H), 7.73 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H,
1
3
NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 24.7, 45.5, 54.7, 111.4, 119.0, 120.0, 122.8, 123.7,
3
6
127.9, 128.4, 129.8, 129.9, 130.0, 132.1, 132.4, 134.0, 135.6, 142.9, 144.2, 144.9, 169.7 ppm; TOF-MS
+
+
(
m/z): 477 [M+Na] ; HRMS (TOF) calcd. for C H ClN O SNa [M+Na] , 476.0806; found, 476.0805.
23 20 3 3
4.1.8. Synthesis of N-(4-((1-(3-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide
(5e)
Compound 5e was prepared according to the procedure described for compound 5a, starting from
compound 4 (0.239 g, 0.726 mmol), 1-chloro-3-(chloromethyl)benzene (0.176 g, 1.089 mmol) and
potassium carbonate (0.156 g, 1.130 mmol). The pure product 5e was obtained as yellow solid. Yield:
7
1
3.7%; mp: 205−207 ºC ; IR (KBr) ν: 3365 (N−H), 3039 (aromatic C−H), 2997 (CH ), 1686 (C=N), 1589,
2
-
1 1
536 (aromatic frame), 739 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.13 (s, 2H,
6
3
Bim-CH ), 5.64 (s, 2H, 3-ClPh-CH ), 6.48 (d, H, J = 6.0 Hz, 3-ClPh-6-H), 7.23−7.17 (m, 4H,
2
2
Bim-5,6,7,8-H), 7.39−7.33 (t, H, J = 6.0 Hz, 3-ClPh-5-H), 7.45 (d, H, J = 6.0 Hz, 3-ClPh-4-H), 7.57 (d, H,
J = 6.0 Hz, 3-ClPh-2-H), 7.74 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H,
18

ACCEPTED MANUSCRIPT
1
3
NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 34.6, 49.9, 54.7, 111.4, 119.0, 120.0, 122.8, 123.3,
3
6
123.4, 128.4, 129.7, 129.9, 130.0, 132.2, 132.5, 133.9, 135.6, 142.9, 144.4, 144.9, 169.7 ppm; TOF-MS
+
+
(
m/z): 477 [M+Na] ; HRMS (TOF) calcd. for C H ClN O SNa [M+Na] , 476.0806; found, 476.0803.
23 20 3 3
4.1.9. Synthesis of N-(4-((1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide
(5f)
Compound 5f was prepared according to the procedure described for compound 5a, starting from
compound 4 (0.287 g, 0.872 mmol), 1-chloro-4-(chloromethyl)benzene (0.254 g, 1.570 mmol) and
potassium carbonate (0.182 g, 1.319 mmol). The pure product 5f was obtained as yellow solid. Yield:
6
1
8.1%; mp: 208−210 ºC ; IR (KBr) ν: 3366 (N−H), 3038 (aromatic C−H), 2996 (CH ), 1687 (C=N), 1588,
2
-
1 1
537 (aromatic frame), 738 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.15 (s, 2H,
6
3
Bim-CH ), 5.59 (s, 2H, 4-ClPh-CH ), 7.14 (d, 2H, J = 6.0 Hz, 4-ClPh-2,6-H), 7.21−7.17 (m, 2H,
2
2
Bim-6,7-H), 7.29−7.27 (m, H, Bim-8-H), 7.36 (d, 2H, J = 6.0 Hz, 4-ClPh-3,5-H), 7.60−7.59 (m, H,
Bim-5-H), 7.75 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH3)
1
3
ppm; C NMR (150 MHz, DMSO-d ) δ: 24.7, 47.0, 54.6, 111.7, 119.0, 119.8, 122.7, 123.5, 129.0, 129.3,
6
+
1
30.0, 132.5, 132.6, 135.5, 135.9, 142.8, 143.9, 144.8, 169.7 ppm; TOF-MS (m/z): 477 [M+Na] ; HRMS
+
(
TOF) calcd. for C H ClN O SNa [M+Na] , 476.0806; found, 476.0809.
2
3
20
3
3
4.1.10. Synthesis of N-(4-((1-(2,4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)
acetamide (5g)
Compound 5g was prepared according to the procedure described for compound 5a, starting from
compound 4 (0.247 g, 0.751 mmol), 2,4-dichloro-1-(chloromethyl)benzene (0.185 g, 0.931 mmol) and
potassium carbonate (0.207 g, 1.500 mmol). The pure product 5g was obtained as white solid. Yield: 59.4%;
mp: 166−168 ºC ; IR (KBr) ν: 3363 (N−H), 3039 (aromatic C−H), 2993 (CH ), 1689 (C=N), 1585, 1531
2
-
1
1
(
aromatic frame), 740 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.12 (s, 3H, COCH ), 5.15 (s, 2H,
6
3
Bim-CH ), 5.61 (s, 2H, 2,4-Cl Ph-CH ), 6.49 (d, H, J = 6.0 Hz, 2,4-Cl Ph-6-H), 7.28−7.21 (m, 4H,
2
2
2
2
Bim-5,6,7,8-H), 7.64 (d, H, J = 6.0 Hz, 2,4-Cl Ph-5-H), 7.69 (s, H, 2,4-Cl Ph-3-H), 7.73 (d, 2H, J = 6.0 Hz,
2
2
1
3
Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150 MHz,
3
DMSO-d ) δ: 24.7, 45.2, 54.7, 111.4, 119.0, 120.1, 122.9, 123.8, 128.1, 129.4, 129.6, 129.9, 132.4, 133.1,
6
+
1
33.3, 133.4, 135.5, 142.9, 144.3, 144.9, 169.7 ppm; ESI-MS (m/z): 511 [M+Na] ; HRMS (ESI) calcd. for
+
C H Cl N O SNa [M+Na] , 510.0416; found, 510.0425.
23
19
2
3
3
19

ACCEPTED MANUSCRIPT
4.1.11. Synthesis of N-(4-((1-(3,4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)
acetamide (5h)
Compound 5h was prepared according to the procedure described for compound 5a, starting from
compound 4 (0.180 g, 0.547 mmol), 3,4-dichloro-1-(chloromethyl)benzene (0.157 g, 0.803 mmol) and
potassium carbonate (0.114 g, 0.826 mmol). The pure product 5h was obtained as yellow solid. Yield:
61.0%; mp: 167−169 ºC ; IR (KBr) ν: 3365 (N−H), 3038 (aromatic C−H), 2995 (CH ), 1687 (C=N), 1585,
2
-
1 1
1
535 (aromatic frame), 741 cm ; H NMR (600 MHz, DMSO-d ) δ: 2.11 (s, 3H, COCH ), 5.19 (s, 2H,
6
3
Bim-CH ), 5.61 (s, 2H, 3,4-Cl Ph-CH ), 7.04 (d, H, J = 6.0 Hz, 3,4-Cl Ph-6-H), 7.29−7.19 (m, 4H,
2
2
2
2
Bim-5,6,7,8-H), 7.44 (s, H, 3,4-Cl Ph-2-H), 7.57 (d, H, J = 6.0 Hz, 3,4-Cl Ph-5-H), 7.75 (d, 2H, J = 6.0 Hz,
2
2
1
3
Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150 MHz,
3
DMSO-d ) δ: 24.7, 46.6, 54.5, 111.6, 118.9, 119.9, 122.8, 123.6, 127.7, 129.5, 129.9, 130.6, 131.2, 131.7,
6
+
1
32.6, 135.4, 138.2, 142.9, 144.0, 144.8, 169.6 ppm; ESI-MS (m/z): 511 [M+Na] ; HRMS (ESI) calcd. for
+
C H Cl N O SNa [M+Na] , 510.0416; found, 510.0424.
23
19
2
3
3
4.1.12. Synthesis of N-(4-((1-ethyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6a)
Pure compound 6a was obtained in process of synthesizing compound 5a as yellow oil. Yield: 52.8%;
mp: >250 ºC; IR (KBr) ν: 3360 (N−H), 3041 (aromatic C−H), 2990 (CH ), 1691 (C=N), 1587, 1500
2
-
1 1
(
aromatic frame), 738 cm ; H NMR (600 MHz, DMSO-d ) δ: 1.34−1.32 (t, 3H, J = 6.0 Hz, CH CH ),
6
2
3
2
.10 (s, 3H, COCH ), 4.35−4.31 (m, 2H, CH CH ), 5.10 (s, 2H, Bim-CH ), 7.21−7.19 (t, H, J = 6.0 Hz,
3 2 3 2
Bim-6-H), 7.29−7.26 (t, H, J = 9.0 Hz, Bim-7-H), 7.59−7.55 (m, 2H, Bim-5,8-H), 7.72 (d, 2H, J = 6.0 Hz,
1
3
Ph-3,5-H), 7.77 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150 MHz,
3
DMSO-d ) δ: 15.2, 24.7, 39.2, 54.6, 111.2, 118.9, 119.7, 122.4, 123.2, 129.9, 132.6, 135.2, 142.8, 143.2,
6
+
+
1
44.8, 169.6 ppm; ESI-MS (m/z): 358 [M+H] ; HRMS (ESI) calcd. for C H N O S [M+H] , 358.1220;
18 20 3 3
found, 358.1223.
4.1.13. Synthesis of N-(4-((1-propyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6b)
Pure compound 6b was obtained in process of synthesizing compound 5a as yellow solid. Yield: 51.2%;
mp: 116−118 ºC; IR (KBr) ν: 3362 (N−H), 3037 (aromatic C−H), 2991 (CH ), 1687 (C=N), 1589, 1508
2
-1 1
(
aromatic frame), 739 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.88−0.86 (t, 3H, J = 6.0 Hz, CH CH CH ),
6 2 2 3
1.77−1.71 (m, 2H, CH CH CH ), 2.10 (s, 3H, COCH ), 4.24−4.21 (t, 2H, J = 9.0 Hz, CH CH CH ), 5.13 (s,
2 2 3 3 2 2 3
2
H, Bim-CH ), 7.24−7.22 (t, H, J = 6.0 Hz, Bim-6-H), 7.30−7.28 (t, H, J = 6.0 Hz, Bim-7-H), 7.58 (d, H, J
2
20

ACCEPTED MANUSCRIPT
=
6.0 Hz, Bim-8-H), 7.63 (d, H, J = 6.0 Hz, Bim-5-H), 7.71 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.77 (d, 2H, J =
1
3
6
5
3
.0 Hz, Ph-2,6-H), 10.44 (s, H, NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 15.2, 22.1, 24.6, 39.2,
3
6
4.5, 111.2, 119.0, 119.7, 122.4, 123.2, 129.9, 132.6, 135.2, 142.8, 143.2, 144.7, 169.6 ppm; ESI-MS (m/z):
+
+
72 [M+H] ; HRMS (ESI) calcd. for C H N O S [M+H] , 372.1376; found, 372.1385.
1
9
22
3
3
4.1.14. Synthesis of N-(4-((1-pentyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6c)
Pure compound 6c was obtained in process of synthesizing compound 5a as yellow solid. Yield: 28.1%;
mp: 94−96 ºC; IR (KBr) ν: 3359 (N−H), 3030 (aromatic C−H), 2994 (CH ), 1680 (C=N), 1589, 1491
2
-1 1
(
aromatic frame), 741 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.86−0.84 (t, 3H, J = 6.0 Hz, (CH ) CH ),
6 2 4 3
1
4
.31−1.26 (m, 4H, (CH ) (CH ) CH ), 1.72−1.67 (m, 2H, CH CH (CH ) CH ), 2.10 (s, 3H, COCH ),
2 2 2 2 3 2 2 2 2 3 3
.24−4.21 (t, 2H, J = 9.0 Hz, CH (CH ) CH ), 5.08 (s, 2H, Bim-CH ), 7.20−7.18 (t, H, J = 6.0 Hz,
2
2 3
3
2
Bim-6-H), 7.28−7.25 (t, H, J = 9.0 Hz, Bim-7-H), 7.57−7.55 (t, 2H, J = 6.0 Hz, Bim-5,8-H), 7.71 (d, 2H, J
1
3
=
6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.40 (s, H, NHCOCH ) ppm; C NMR (150
3
MHz, DMSO-d ) δ: 14.3, 22.3, 24.6, 29.2, 29.4, 44.2, 54.7, 111.3, 118.9, 119.7, 122.4, 123.2, 129.9, 132.6,
6
+
1
35.5, 142.6, 143.5, 144.8, 169.7 ppm; ESI-MS (m/z): 401 [M+H] ; HRMS (ESI) calcd. for C H N O S
2
1
26
3
3
+
[
M+H] , 400.1685; found, 400.1693.
4.1.15. Synthesis of N-(4-((1-hexyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6d)
Pure compound 6d was obtained in process of synthesizing compound 5a as yellow oil. Yield: 47.5%;
mp: 108−110 ºC; IR (KBr) ν: 3352 (N−H), 3037 (aromatic C−H), 2999 (CH ), 1687 (C=N), 1589, 1493
2
-1 1
(
aromatic frame), 738 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.86−0.84 (t, 3H, J = 6.0 Hz, (CH ) CH ),
6 2 5 3
1
4
.30−1.25 (m, 6H, (CH ) (CH ) CH ), 1.70−1.65 (m, 2H, CH CH (CH ) CH ), 2.10 (s, 3H, COCH ),
2 2 2 3 3 2 2 2 3 3 3
.24−4.21 (t, 2H, J = 9.0 Hz, CH (CH ) CH ), 5.08 (s, 2H, Bim-CH ), 7.21−7.18 (t, H, J = 9.0 Hz,
2
2 4
3
2
Bim-6-H), 7.28−7.25 (t, H, J = 9.0 Hz, Bim-7-H), 7.57−7.55 (t, 2H, J = 6.0 Hz, Bim-5,8-H), 7.71 (d, 2H, J
1
3
=
6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150
3
MHz, DMSO-d ) δ: 14.3, 22.5, 24.6, 26.3, 29.6, 31.3, 44.2, 54.6, 111.3, 118.9, 119.6, 122.4, 123.2, 129.9,
6
+
1
32.6, 135.5, 142.6, 143.4, 144.8, 169.6 ppm; ESI-MS (m/z): 415 [M+H] ; HRMS (ESI) calcd. for
+
C H N O S [M+H] , 414.1846; found, 414.1855.
22
28
3
3
4.1.16. Synthesis of N-(4-((1-heptyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6e)
Compound 6e was obtained as yellow solid. Yield: 24.2%; mp: 102−103 ºC; IR (KBr) ν: 3354 (N−H),
-1
1
3
032 (aromatic C−H), 2994 (CH ), 1685 (C=N), 1592, 1498 (aromatic frame), 738 cm ; H NMR (600
2
21

ACCEPTED MANUSCRIPT
MHz, DMSO-d ) δ: 0.86−0.83 (t, 3H, J = 9.0 Hz, (CH ) CH ), 1.27−1.23 (m, 8H, (CH ) (CH ) CH ),
6
2 6
3
2 2
2 4
3
1
.70−1.66 (m, 2H, CH CH (CH ) CH ), 2.10 (s, 3H, COCH ), 4.23−4.21 (t, 2H, J = 6.0 Hz,
2 2 2 4 3 3
CH (CH ) CH ), 5.08 (s, 2H, Bim-CH ), 7.21−7.18 (t, H, J = 9.0 Hz, Bim-6-H), 7.27−7.25 (t, H, J = 6.0 Hz,
2
2 5
3
2
Bim-7-H), 7.57−7.55 (t, 2H, J = 6.0 Hz, Bim-5,8-H), 7.70 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J =
1
3
6
2
1
4
.0 Hz, Ph-2,6-H), 10.40 (s, H, NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 14.4, 22.5, 24.6, 26.6,
3
6
8.8, 29.7, 31.6, 44.1, 54.6, 111.3, 118.9, 119.7, 122.4, 123.2, 129.9, 132.6, 135.5, 142.7, 143.4, 144.8,
+
+
69.6 ppm; ESI-MS (m/z): 429 [M+H] ; HRMS (ESI) calcd. for C H N O S [M+H] , 428.2002; found,
2
3
30
3
3
28.2010.
4.1.17. Synthesis of N-(4-((1-octyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6f)
Pure compound 6f was obtained in process of synthesizing compound 5a as yellow solid. Yield: 71.2%;
mp: 97–99 ºC; IR (KBr) ν: 3357 (N−H), 3038 (aromatic C−H), 2996 (CH ), 1685 (C=N), 1590, 1498
2
-1 1
(
aromatic frame), 739 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.86−0.83 (t, 3H, J = 9.0 Hz, (CH ) CH ),
6 2 7 3
1
4
.27−1.23 (m, 10H, (CH ) (CH ) CH ), 1.69−1.66 (m, 2H, CH CH (CH ) CH ), 2.10 (s, 3H, COCH ),
2 2 2 5 3 2 2 2 5 3 3
.23−4.21 (t, 2H, J = 6.0 Hz, CH (CH ) CH ), 5.08 (s, 2H, Bim-CH ), 7.21−7.18 (t, H, J = 9.0 Hz,
2
2 6
3
2
Bim-6-H), 7.28−7.25 (t, H, J = 9.0 Hz, Bim-7-H), 7.57−7.55 (t, 2H, J = 6.0 Hz, Bim-5,8-H), 7.70 (d, 2H, J
1
3
=
6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150
3
MHz, DMSO-d ) δ: 14.4, 22.5, 24.6, 26.6, 29.0, 29.1, 29.7, 31.7, 44.1, 54.6, 111.3, 118.9, 119.7, 122.4,
6
+
1
23.2, 129.9, 132.6, 135.5, 142.7, 143.4, 144.8, 169.6 ppm; ESI-MS (m/z): 443 [M+H] ; HRMS (ESI)
+
calcd. for C H N O S [M+H] , 442.2159; found, 442.2168.
2
4
32
3
3
4.1.18. Synthesis of N-(4-((1-decyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6g)
Compound 6g was synthesized according to the experimental procedure reported for compound 5a,
starting from compound 4 (0.407 g, 1.237 mmol), 1-bromononane (0.410 g, 1.856 mmol) and potassium
carbonate (0.256 g, 1.856 mmol). The crude product 6g was obtained as yellow solid. Yield: 47.7%; mp:
7
8–80 ºC; IR (KBr) ν: 3352 (N−H), 3034 (aromatic C−H), 2996 (CH ), 1687 (C=N), 1590, 1505 (aromatic
2
-1 1
frame), 741 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.86−0.84 (t, 3H, J = 6.0 Hz, (CH ) CH ), 1.26−1.22
6
2 9
3
(
m, 14H, (CH ) (CH ) CH ), 1.70−1.66 (m, 2H, CH CH (CH ) CH ), 2.10 (s, 3H, COCH ), 4.23−4.21 (t,
2 2 2 7 3 2 2 2 7 3 3
2
H, J = 6.0 Hz, CH (CH ) CH ), 5.08 (s, 2H, Bim-CH ), 7.21−7.18 (t, H, J = 9.0 Hz, Bim-6-H), 7.27−7.25
2 2 8 3 2
(t, H, J = 6.0 Hz, Bim-7-H), 7.57−7.55 (t, 2H, J = 6.0 Hz, Bim-5,8-H), 7.70 (d, 2H, J = 6.0 Hz, Ph-3,5-H),
1
3
7
2
.76 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 14.4,
3 6
2.5, 24.6, 26.6, 29.1, 29.4, 29.4, 29.6, 31.7, 44.1, 54.7, 111.3, 118.9, 119.7, 122.4, 123.2, 129.9, 132.7,
22

ACCEPTED MANUSCRIPT
+
1
35.6, 142.7, 143.4, 144.8, 169.6 ppm; ESI-MS (m/z): 471 [M+H] ; HRMS (ESI) calcd. for C H N O S
2
6
36
3
3
+
[
M+H] , 470.2472; found, 470.2476.
4.1.19. Synthesis of N-(4-((1-dodecyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6h)
Prepared according to the general procedure described for compound 5a, starting from compound 4
0.363 g, 1.103 mmol), 1-bromododecane (0.412 g, 1.655 mmol) and potassium carbonate (0.228 g, 1.655
mmol), the pure compound 6h (0.243 g) was obtained as yellow solid. Yield: 44.3%; mp: 135–137 ºC; IR
KBr) ν: 3357 (N−H), 3038 (aromatic C−H), 2991 (CH ), 1687 (C=N), 1590, 1508 (aromatic frame), 736
(
(
2
-
1 1
cm ; H NMR (600 MHz, DMSO-d ) δ: 0.86−0.83 (t, 3H, J = 9.0 Hz, (CH ) CH ), 1.26−1.22 (m, 18H,
6
2 11
3
(
CH ) (CH ) CH ), 1.69−1.67 (m, 2H, CH CH (CH ) CH ), 2.10 (s, 3H, COCH ), 4.24−4.21 (t, 2H, J = 9.0
2 2 2 9 3 2 2 2 9 3 3
Hz, CH (CH ) CH ), 5.08 (s, 2H, Bim-CH ), 7.21−7.18 (t, H, J = 9.0 Hz, Bim-6-H), 7.27−7.25 (t, H, J =
2
2 10
3
2
6
6
2
1
.0 Hz, Bim-7-H), 7.56 (d, 2H, J = 6.0 Hz, Bim-5,8-H), 7.71 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J =
1
3
.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 14.4, 22.5, 24.6, 26.6,
3
6
9.1, 29.2, 29.4, 29.4, 29.5, 29.5, 29.7, 31.8, 44.2, 54.7, 111.3, 118.9, 119.7, 122.4, 123.2, 129.9, 132.7,
+
35.6, 142.7, 143.4, 144.8, 169.6 ppm; ESI-MS (m/z): 499 [M+H] ; HRMS (ESI) calcd. for C H N O S
2
8
40
3
3
+
[
M+H] , 498.2785; found, 498.2793.
4.1.20. Synthesis of N-(4-((1-octadecyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)phenyl)acetamide (6i)
Compound 6i was synthesized according to the experimental procedure reported for compound 5a,
starting from compound 4 (0.350 g, 1.064 mmol), 1-bromooctadecane (0.495 g, 1.486 mmol) and
potassium carbonate (0.220 g, 1.596 mmol). The crude product was obtained and purified via silica gel
column chromatography (eluent, acetone/petroleum ether, 1/1, V/V) to give pure compound 6i (0.328 g) as
yellow solid. Yield: 53.0%; mp: 143–145 ºC; IR (KBr) ν: 3356 (N−H), 3034 (aromatic C−H), 2997 (CH2),
-1 1
1
689 (C=N), 1593, 1501 (aromatic frame), 738 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.85−0.83 (t, 3H, J
6
=
6.0 Hz, (CH ) CH ), 1.26−1.22 (m, 30H, (CH ) (CH ) CH ), 1.69−1.66 (m, 2H, CH CH (CH ) CH ),
2 17 3 2 2 2 15 3 2 2 2 15 3
2
.10 (s, 3H, COCH ), 4.23−4.21 (t, 2H, J = 6.0 Hz, CH (CH ) CH ), 5.07 (s, 2H, Bim-CH ), 7.20−7.17 (t,
3 2 2 16 3 2
H, J = 9.0 Hz, Bim-6-H), 7.26−7.24 (t, H, J = 6.0 Hz, Bim-7-H), 7.56 (d, 2H, J = 6.0 Hz, Bim-5,8-H), 7.70
1
3
(
d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 10.41 (s, H, NHCOCH ) ppm; C NMR
3
(
150 MHz, DMSO-d ) δ: 14.3, 22.5, 24.6, 26.6, 29.1, 29.2, 29.4, 29.4, 29.5, 29.5, 29.6, 31.8, 44.1, 54.7,
6
111.2, 118.9, 119.7, 122.3, 123.1, 129.9, 132.7, 135.6, 142.8, 143.4, 144.8, 169.6 ppm; ESI-MS (m/z): 583
+
+
[
M+H] ; HRMS (ESI) calcd. for C H N O S [M+H] , 582.3724; found, 582.3727.
34 52 3 3
23

ACCEPTED MANUSCRIPT
4.1.21. Synthesis of N-(4-((1-(5-(9H-carbazol-9-yl)pentyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)
phenyl)acetamide (7)
Prepared the same way as the general procedure described for compound 5a, starting from compound 4
(0.176 g, 0.535 mmol), 9-(5-bromopentyl)-9H-carbazole (0.342 g, 1.082 mmol) and potassium carbonate
(0.152 g, 1.101 mmol), pure compound 7 (0.216 g) was synthesized as white solid. Yield: 72.0%; mp:
1
18–120 ºC; IR (KBr) ν: 3369 (N−H), 3042 (aromatic C−H), 2984 (CH ), 1694 (C=N), 1591, 1525
2
-1 1
(
aromatic frame), 741 cm ; H NMR (600 MHz, DMSO-d ) δ: 1.46−1.32 (m, 2H, carbazole-(CH ) CH ),
6 2 2 2
1
4
2
3
8
2
1
.75−1.71 (m, 2H, carbazole-CH CH ), 1.82−1.78 (m, 2H, carbazole-(CH ) CH ), 2.10 (s, 3H, COCH ),
2 2 2 3 2 3
.20−4.18 (t, H, J = 6.0 Hz, carbazole-CH ), 4.39−4.36 (t, H, J = 9.0 Hz, carbazole-(CH ) CH ), 5.00 (s,
2
2 4
2
H, Bim-CH ), 7.24−7.18 (m, 4H, Bim-5,6,7,8-H), 7.48−7.42 (m, 3H, carbazole-4,5,10-H), 7.57−7.53 (m,
2
H, carbazole-3,11,12-H), 7.67 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.76 (d, 2H, J = 6.0 Hz, Ph-2,6-H),
1
3
.15−8.13 (d, 2H, carbazole-6,9-H), 10.39 (s, H, NHCOCH ) ppm; C NMR (150 MHz, DMSO-d ) δ: 24.5,
3
6
4.7, 28.7, 29.5, 42.7, 44.1, 54.6, 109.7, 111.3, 119.0, 119.1, 119.7, 120.7, 122.3, 122.6, 123.2, 126.1, 129.9,
+
32.6, 135.6, 140.5, 142.7, 143.4, 144.8, 169.6 ppm; ESI-MS (m/z): 566 [M+H] ; HRMS (ESI) calcd. for
+
C H N O S [M+H] , 565.2268; found, 565.2271.
33
33
4
3
4.1.22. Synthesis of 4-((1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8a)
To a solution of compound 5a (0.300 g, 0.685 mmol) in ethanol 15 mL was added 0.4 mL 2 mol/L
sodium hydroxide solution. The mixture was refluxed for 10 h (monitored by TLC, eluent,
acetone/petroleum ether, 1/1, V/V). After cooling to the room temperature, the solvent was removed in
vacuo to give the deprotected compound 8a as yellow solid. Yield: 97.4%; mp: 205–207 ºC; IR (KBr) ν:
-1
3
318, 3207 (N−H), 3058 (aromatic C−H), 2988 (CH ), 1647 (C=N), 1587, 1506 (aromatic frame), 739 cm ;
2
1
H NMR (600 MHz, DMSO-d ) δ: 4.92 (s, 2H, Bim-CH ), 5.57 (s, 2H, 2-FPh-CH ), 6.21 (s, 2H, NH ),
6
2
2
2
6
.60 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 6.91−6.89 (t, H, J = 6.0 Hz, 2-FPh-5-H), 7.10−7.08 (t, H, J = 6.0 Hz,
-FPh-4-H), 7.24−7.19 (m, 4H, Bim-5,6,7,8-H), 7.34−7.31 (m, H, 2-FPh-6-H), 7.37 (d, 2H, J = 6.0 Hz,
2
1
3
Ph-2,6-H), 7.62−7.61 (m, H, 2-FPh-3-H) ppm; C NMR (150 MHz, DMSO-d ) δ: 49.1, 55.2, 111.3, 113.1,
6
116.0, 119.9, 122.6, 123.4, 123.6, 123.8, 125.1, 129.5, 130.3, 130.5, 135.6, 142.9, 144.5, 154.6, 160.4 ppm;
+
+
ESI-MS (m/z): 418 [M+Na] ; HRMS (ESI) calcd. for C H FN O SNa [M+Na] , 418.0996; found,
2
1
18
3
2
418.1005.
4.1.23. Synthesis of 4-((1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8b)
Compound 8b was prepared according to the experimental procedure described for compound 8a,
24

ACCEPTED MANUSCRIPT
starting from compound 5b (0.095 g, 0.217 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The
pure product 8b was obtained as yellow solid. Yield: 95.3%; mp: 212–214 ºC; IR (KBr) ν: 3314, 3209
-1 1
(
N−H), 3061 (aromatic C−H), 2993 (CH ), 1649 (C=N), 1588, 1501 (aromatic frame), 738 cm ; H NMR
2
(
600 MHz, DMSO-d ) δ: 4.95 (s, 2H, Bim-CH ), 5.57 (s, 2H, 3-FPh-CH ), 6.21 (s, 2H, NH ), 6.61 (d, 2H, J
6
2
2
2
=
6.0 Hz, Ph-3,5-H), 6.97−6.92 (m, 2H, 3-FPh-2,6-H), 7.11−7.08 (m, H, 3-FPh-4-H), 7.20−7.18 (m, 2H,
Bim-6,7-H), 7.31−7.30 (m, H, Bim-8-H), 7.37−7.33 (m, H, 3-FPh-5-H), 7.39 (d, 2H, J = 6.0 Hz, Ph-2,6-H),
1
3
7
.63−7.61 (m, H, Bim-5-H), 10.41 (s, H, NHCOCH ) ppm; C NMR (600 MHz, DMSO-d ) δ: 47.1, 55.2,
3
6
1
1
4
11.5, 113.1, 114.3, 114.9, 119.8, 122.6, 123.4, 123.8, 130.5, 131.1, 135.6, 139.9, 139.9, 142.9, 144.5,
+
+
54.6, 162.8 ppm; ESI-MS (m/z): 418 [M+Na] ; HRMS (ESI) calcd. for C H FN O SNa [M+Na] ,
2
1
18
3
2
18.0996; found, 418.1004.
4.1.24. Synthesis of 4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8c)
Compound 8c was prepared according to the procedure depicted for compound 8a, starting from
compound 5c (0.140 g, 0.320 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The pure product 5h
was obtained as yellow solid. Yield: 95.2%; mp: 224–226 ºC; IR (KBr) ν: 3307, 3200 (N−H), 3069
-1 1
(
aromatic C−H), 2997 (CH ), 1654 (C=N), 1594, 1507 (aromatic frame), 740 cm ; H NMR (600 MHz,
2
DMSO-d ) δ: 4.93 (s, 2H, Bim-CH ), 5.52 (s, 2H, 4-FPh-CH ), 6.21 (s, 2H, NH ), 6.60 (d, 2H, J = 6.0 Hz,
6
2
2
2
Ph-3,5-H), 7.19−7.12 (m, 6H, 4-FPh-2,3,5,6-H, Bim-6,7-H), 7.31−7.30 (m, H, Bim-8-H), 7.39 (d, 2H, J =
1
3
6
.0 Hz, Ph-2,6-H), 7.61−7.60 (m, H, Bim-5-H) ppm; C NMR (150 MHz, DMSO-d ) δ: 46.9, 55.3, 111.6,
6
113.1, 115.9, 119.8, 122.6, 123.3, 123.8, 129.5, 130.6, 133.2, 135.6, 143.0, 144.4, 154.6, 162.0 ppm;
+
+
ESI-MS (m/z): 418 [M+Na] ; HRMS (ESI) calcd. for C H FN O SNa [M+Na] , 418.0996; found,
2
1
18
3
2
418.1000.
4.1.25. Synthesis of 4-((1-(2-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8d)
Compound 8d was prepared according to the experimental procedure reported for compound 8a, starting
from compound 5d (0.148 g, 0.325 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The pure
product 5i was obtained as yellow solid. Yield: 97.5%; mp: 184–186 ºC; IR (KBr) ν: 3311, 3207 (N−H),
-1
1
3
062 (aromatic C−H), 2989 (CH ), 1659 (C=N), 1588, 1501 (aromatic frame), 741 cm ; H NMR (600
2
MHz, DMSO-d ) δ: 4.88 (s, 2H, Bim-CH ), 5.56 (s, 2H, 2-ClPh-CH ), 6.22 (s, 2H, NH ), 6.50 (d, H, J =
6
2
2
2
6.0 Hz, 2-ClPh-6-H), 6.60 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.24−7.17 (m, 4H, Bim-5,6,7,8-H), 7.32−7.29 (t,
H, J = 6.0 Hz, 2-ClPh-4-H), 7.35 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 7.53 (d, H, J = 6.0 Hz, 2-ClPh-5-H), 7.65
1
3
(
d, H, J = 6.0 Hz, 2-ClPh-3-H) ppm; C NMR (150 MHz, DMSO-d ) δ: 45.3, 56.3, 111.3, 113.1, 120.0,
6
25

ACCEPTED MANUSCRIPT
122.7, 123.5, 123.6, 128.0, 128.4, 129.8, 130.0, 130.5, 132.1, 134.1, 135.7, 142.9, 144.7, 154.6 ppm;
+
+
ESI-MS (m/z): 435 [M+Na] ; HRMS (ESI) calcd. for C H ClN O SNa [M+Na] , 434.0700; found,
2
1
18
3
2
434.0712.
4.1.26. Synthesis of 4-((1-(3-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8e)
Pure compound 8e was obtained in process of synthesizing compound 8a as yellow solid. Yield: 93.7%;
mp: 237–239 ºC; IR (KBr) ν: 3314, 3202 (N−H), 3067 (aromatic C−H), 2993 (CH ), 1657 (C=N), 1585,
2
-
1 1
1
3
7
503 (aromatic frame), 740 cm ; H NMR (600 MHz, DMSO-d ) δ: 4.92 (s, 2H, Bim-CH ), 5.59 (s, 2H,
6
2
-ClPh-CH ), 6.21 (s, 2H, NH ), 6.61 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.07 (d, H, J = 6.0 Hz, 3-ClPh-6-H),
2
2
.24−7.18 (m, 4H, Bim-5,6,7,8-H), 7.29−7.26 (t, H, J = 9.0 Hz, 3-ClPh-5-H), 7.35 (d, 2H, J = 6.0 Hz,
1
3
Ph-2,6-H), 7.51 (d, H, J = 6.0 Hz, 3-ClPh-4-H), 7.52 (d, H, J = 6.0 Hz, 3-ClPh-2-H) ppm; C NMR (150
MHz, DMSO-d ) δ: 50.2, 56.3, 111.2, 113.3, 119.5, 122.8, 123.7, 123.8, 127.8, 128.3, 129.7, 130.1, 130.4,
6
+
1
32.0, 134.2, 135.8, 142.9, 144.8, 154.7 ppm; ESI-MS (m/z): 435 [M+Na] ; HRMS (ESI) calcd. for
+
C H ClN O SNa [M+Na] , 434.0700; found, 434.0712.
21
18
3
2
4.1.27. Synthesis of 4-((1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8f)
Compound 8f was prepared according to the experimental procedure described for compound 8a,
starting from compound 5f (0.150 g, 0.329 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The
pure product 8f was obtained as yellow solid. Yield: 90.4%; mp: 223–225 ºC; IR (KBr) ν: 3319, 3206
-1 1
(
N−H), 3064 (aromatic C−H), 2989 (CH ), 1654 (C=N), 1587, 1505 (aromatic frame), 739 cm ; H NMR
2
(
600 MHz, DMSO-d ) δ: 4.93 (s, 2H, Bim-CH ), 5.54 (s, 2H, 4-ClPh-CH ), 6.21 (s, 2H, NH ), 6.61 (d, 2H,
6
2
2
2
J = 6.0 Hz, Ph-3,5-H), 7.13 (d, 2H, J = 6.0 Hz, 4-ClPh-2,6-H), 7.20−7.16 (m, 2H, Bim-6,7-H), 7.30−7.28
m, H, Bim-8-H), 7.36 (d, 2H, J = 6.0 Hz, 4-ClPh-3,5-H), 7.38 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 7.62−7.61 (m,
(
13
H, Bim-5-H) ppm; C NMR (150 MHz, DMSO-d ) δ: 47.0, 55.2, 111.6, 113.1, 119.8, 122.6, 123.4, 123.7,
6
+
1
29.0, 129.3, 130.6, 132.7, 135.6, 136.0, 143.0, 144.5, 154.6 ppm; ESI-MS (m/z): 413 [M+H] ; HRMS
+
(
ESI) calcd. for C H ClN O S [M+H] , 412.0881; found, 412.0885.
2
1
19
3
2
4.1.28. Synthesis of 4-((1-(2,4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8g)
Compound 8g was prepared according to the experimental procedure depicted for compound 8a, starting
from compound 5g (0.198 g, 0.405 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The pure
product 8g was obtained as yellow solid. Yield: 93.4%; mp: 216–218 ºC; IR (KBr) ν: 3329, 3201 (N−H),
-1
1
3
066 (aromatic C−H), 2993 (CH ), 1639 (C=N), 1593, 1500 (aromatic frame), 736 cm ; H NMR (600
2
26

ACCEPTED MANUSCRIPT
MHz, DMSO-d ) δ: 4.91 (s, 2H, Bim-CH ), 5.55 (s, 2H, 2,4-Cl Ph-CH ), 6.21 (s, 2H, NH ), 6.47 (d, H, J =
6
2
2
2
2
6
.0 Hz, 2,4-Cl Ph-6-H), 6.60 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.29−7.18 (m, 4H, Bim-5,6,7,8-H), 7.34 (d, 2H,
2
1
3
J = 6.0 Hz, Ph-2,6-H), 7.65 (d, H, J = 6.0 Hz, 2,4-Cl Ph-5-H), 7.70 (s, H, 2,4-Cl Ph-3-H) ppm; C NMR
2
2
(
150 MHz, DMSO-d ) δ: 45.1, 55.2, 111.3, 113.1, 120.0, 122.8, 123.5, 123.6, 128.1, 129.4, 129.6, 130.5,
6
+
1
33.0, 133.3, 133.4, 135.5, 142.9, 144.8, 154.6 ppm; ESI-MS (m/z): 447 [M+H] ; HRMS (ESI) calcd. for
+
C H Cl N O S [M+H] , 446.0491; found, 446.0500.
21
18
2
3
2
4.1.29. Synthesis of 4-((1-(3,4-dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (8h)
Compound 8h was prepared according to the experimental procedure reported for compound 8a, starting
from compound 5h (0.140 g, 0.287 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The pure
product 8h was obtained as yellow solid. Yield: 94.7%; mp: 235–237 ºC; IR (KBr) ν: 3324, 3205 (N−H),
-1
1
3
061 (aromatic C−H), 2988 (CH ), 1642 (C=N), 1594, 1503 (aromatic frame), 740 cm ; H NMR (600
2
MHz, DMSO-d ) δ: 5.03 (s, 2H, Bim-CH ), 5.62 (s, 2H, 3,4-Cl Ph-CH ), 6.25 (s, 2H, NH ), 6.66 (d, 2H, J
6
2
2
2
2
=
6.0 Hz, Ph-3,5-H), 7.09 (d, H, J = 6.0 Hz, 3,4-Cl Ph-6-H), 7.26−7.23 (m, 2H, Bim-6,7-H), 7.34−7.33 (m,
2
H, Bim-8-H), 7.44 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 7.49 (s, H, 3,4-Cl Ph-2-H), 7.62 (d, H, J = 6.0 Hz,
2
1
3
3
,4-Cl Ph-5-H), 7.68−7.66 (m, H, Bim-5-H) ppm; C NMR (150 MHz, DMSO-d ) δ: 46.6, 56.1, 111.5,
2
6
113.1, 119.9, 122.7, 123.5, 123.8, 127.7, 129.5, 130.5, 130.6, 131.2, 131.7, 135.5, 138.2, 143.0, 144.6,
+
+
1
54.5 ppm; ESI-MS (m/z): 469 [M+Na] ; HRMS (ESI) calcd. for C H Cl N O SNa [M+Na] , 468.0311;
21 17 2 3 2
found, 468.0314.
4.1.30. Synthesis of 4-((1-ethyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (9a)
Prepared according to the general procedure described for 8a, starting from compound 6a (0.135 g, 0.378
mmol) and 0.2 mL 2 mol/L sodium hydroxide solution, pure compound 9a was obtained as yellow solid.
Yield: 90.8%; mp: 232–234 ºC; IR (KBr) ν: 3332, 3208 (N−H), 3061 (aromatic C−H), 2988 (CH ), 1675
2
-1 1
(
C=N), 1589, 1493 (aromatic frame), 740 cm ; H NMR (600 MHz, DMSO-d ) δ: 1.32−1.30 (t, 3H, J = 6.0
6
Hz, CH CH ), 4.30−4.27 (m, 2H, CH CH ), 4.90 (s, 2H, Bim-CH ), 6.19 (s, 2H, NH ), 6.60 (d, 2H, J = 6.0
2
3
2
3
2
2
Hz, Ph-3,5-H), 7.21−7.19 (t, H, J = 6.0 Hz, Bim-6-H), 7.27−7.25 (t, H, J = 6.0 Hz, Bim-7-H), 7.35 (d, 2H,
1
3
J = 6.0 Hz, Ph-2,6-H), 7.58−7.56 (t, 2H, J = 6.0 Hz, Bim-5,8-H) ppm; C NMR (150 MHz, DMSO-d ) δ:
6
15.2, 39.1, 55.2, 111.1, 113.1, 119.7, 122.3, 123.1, 123.9, 130.5, 135.2, 143.0, 143.8, 154.5 ppm; ESI-MS
+
+
(
m/z): 316 [M+H] ; HRMS (ESI) calcd. for C H N O S [M+H] , 316.1114; found, 316.1121.
16 18 3 2
4.1.31. Synthesis of 4-((1-propyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (9b)
27

ACCEPTED MANUSCRIPT
Prepared according to the general procedure described for 8a, starting from compound 6b (0.122 g,
.329 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution, pure compound 9b was obtained as yellow
0
solid. Yield: 97.2%; mp: 237–239 ºC; IR (KBr) ν: 3327, 3205 (N−H), 3051 (aromatic C−H), 2993 (CH2),
-1 1
1
684 (C=N), 1595, 1487 (aromatic frame), 737 cm ; H NMR (600 MHz, DMSO-d ) δ: 0.87−0.84 (t, 3H, J
6
=
9.0 Hz, CH CH CH ), 1.75−1.69 (m, 2H, CH CH CH ), 4.17−4.15 (t, 2H, J = 6.0 Hz, CH CH CH ),
2 2 3 2 2 3 2 2 3
4
.89 (s, 2H, Bim-CH ), 6.19 (s, 2H, NH ), 6.58 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.20−7.18 (t, H, J = 6.0 Hz,
2 2
Bim-6-H), 7.26−7.24 (t, H, J = 6.0 Hz, Bim-7-H), 7.33 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 7.58 (d, 2H, J = 6.0
1
3
Hz, Bim-5,8-H) ppm; C NMR (150 MHz, DMSO-d ) δ: 11.4, 22.9, 45.5, 55.3, 111.3, 113.1, 119.7, 122.2,
6
+
1
23.0, 123.9, 130.5, 135.6, 142.8, 144.1, 154.5 ppm; ESI-MS (m/z): 330 [M+H] ; HRMS (ESI) calcd. for
+
C H N O S [M+H] , 330.1271; found, 330.1277.
17
20
3
2
4.1.32. Synthesis of 4-((1-pentyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (9c)
Compound 9c was prepared according to the procedure depicted for compound 8a, starting from
compound 6c (0.042 g, 0.105 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The product 9c
(0.033 g) was obtained as yellow solid. Yield: 89.1%; mp: 233–235 ºC; IR (KBr) ν: 3316, 3200 (N−H),
-1
1
3
046 (aromatic C−H), 2987 (CH ), 1680 (C=N), 1587, 1494 (aromatic frame), 741 cm ; H NMR (600
2
MHz, DMSO-d ) δ: 0.86−0.84 (t, 3H, J = 6.0 Hz, (CH ) CH ), 1.31−1.24 (m, 4H, (CH ) (CH ) CH ),
6
2 4
3
2 2
2 2
3
1
.72−1.67 (m, 2H, CH CH (CH ) CH ), 4.19−4.16 (t, 2H, J = 9.0 Hz, CH (CH ) CH ), 4.88 (s, 2H,
2 2 2 2 3 2 2 3 3
Bim-CH ), 6.18 (s, 2H, NH ), 6.59 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.20−7.18 (t, H, J = 6.0 Hz, Bim-6-H),
2
2
7.27−7.24 (t, H, J = 9.0 Hz, Bim-7-H), 7.33 (d, 2H, J = 6.0 Hz, Ph-2,6-H), 7.58−7.54 (m, 2H, Bim-5,8-H)
1
3
ppm; C NMR (150 MHz, DMSO-d ) δ: 14.3, 22.3, 28.8, 29.3, 44.0, 55.3, 111.2, 113.1, 119.7, 122.3,
6
+
1
23.0, 123.8, 130.5, 135.6, 142.8, 144.0, 154.5 ppm; ESI-MS (m/z): 358 [M+H] ; HRMS (ESI) calcd. for
+
C H N O S [M+H] , 358.1584; found, 358.1587.
19
24
3
2
4.1.33. Synthesis of 4-((1-hexyl-1H-benzo[d]imidazol-2-yl)methylsulfonyl)aniline (9d)
Compound 9d was prepared according to the procedure depicted for compound 8a, starting from
compound 6d (0.134 g, 0.324 mmol) and 0.2 mL 2 mol/L sodium hydroxide solution. The product 9d
(0.108 g) was obtained as yellow solid. Yield: 94.2%; mp: 238–240 ºC; IR (KBr) ν: 3319, 3201 (N−H),
-1
1
3
042 (aromatic C−H), 2986 (CH ), 1672 (C=N), 1583, 1494 (aromatic frame), 740 cm ; H NMR (600
2
MHz, DMSO-d ) δ: 0.86−0.84 (t, 3H, J = 6.0 Hz, (CH ) CH ), 1.26−1.25 (m, 6H, (CH ) (CH ) CH ),
6
2 5
3
2 2
2 3
3
1
.70−1.66 (m, 2H, CH CH (CH ) CH ), 4.19−4.16 (t, 2H, J = 9.0 Hz, CH (CH ) CH ), 4.88 (s, 2H,
2 2 2 3 3 2 2 4 3
Bim-CH ), 6.20 (s, 2H, NH ), 6.59 (d, 2H, J = 6.0 Hz, Ph-3,5-H), 7.20−7.18 (t, H, J = 6.0 Hz, Bim-6-H),
2
2
28