Synthesis of substituted fluorobenzimidazoles as inhibitors of 5-lipoxygenase and soluble epoxide hydrolase for anti-inflammatory activity

Article abstract of DOI:10.1002/ardp.201800030

A new series of 4-((5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)methyl)-benzoic acids 4a–o and 2-(5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)-2-methylpropanoic acids 8a–e were synthesized, and their inhibitory potencies against soluble epoxide hydrolase (sEH) and 5-lipoxygenase (5-LOX) were investigated. These molecules were designed based on the combination of 5-LOX and sEH pharmacophores, resulting in hybrid analogs with potent sEH and 5-LOX inhibitory activity. Compound 4g showed remarkable activity with IC₅₀ values of less than 1 μM (0.9 μM) against 5-LOX, while compound 4k displayed promising activity against sEH with IC₅₀ ≤ 1 μM (0.7 μM). These compounds were evaluated for their in vivo potential using the carrageenan-induced rat paw edema assay. Based on the obtained results, the structure–activity relationship was established and a correlation between the activities was observed. Compounds 4f, 4g, 4k, 4n, and 8e showed potent anti-inflammatory activity and significant inhibition of edema (64.13, 67.39, 66.30, 65.21, and 58.69%, respectively) at a dose of 100 mg/kg, comparable to the standard drug ibuprofen (70.65%) at 3 h.

Full text of DOI:10.1002/ardp.201800030

Received: 14 February 2018
Revised: 8 April 2018
Accepted: 11 April 2018
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DOI: 10.1002/ardp.201800030
FULL PAPER
Synthesis of substituted fluorobenzimidazoles as
inhibitors of 5-lipoxygenase and soluble epoxide
hydrolase for anti-inflammatory activity
B. Nandha1
Sureshbabu A. Ramareddy2
Hazra Kuntal3
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1 Department of Pharmaceutical Chemistry,
Vivekananda College of Pharmacy, Rajiv
Gandhi University of Health Sciences,
Bangalore, India
Abstract
A new series of 4-((5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)methyl)-
benzoic acids 4a–o and 2-(5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)-2-
methylpropanoic acids 8a–e were synthesized, and their inhibitory potencies against
soluble epoxide hydrolase (sEH) and 5-lipoxygenase (5-LOX) were investigated. These
molecules were designed based on the combination of 5-LOX and sEH pharmaco-
phores, resulting in hybrid analogs with potent sEH and 5-LOX inhibitory activity.
Compound 4g showed remarkable activity with IC50 values of less than 1 μM (0.9 μM)
against 5-LOX, while compound 4k displayed promising activity against sEH with
IC50 ≤ 1 μM (0.7 μM). These compounds were evaluated for their in vivo potential using
the carrageenan-induced rat paw edema assay. Based on the obtained results, the
structure–activity relationship was established and a correlation between the activities
was observed. Compounds 4f, 4g, 4k, 4n, and 8e showed potent anti-inflammatory
activity and significant inhibition of edema (64.13, 67.39, 66.30, 65.21, and 58.69%,
respectively) at a dose of 100 mg/kg, comparable to the standard drug ibuprofen
(70.65%) at 3 h.
2 Department of Pharmaceutical Chemistry,
KLE University's College of Pharmacy,
Bangalore, India
3 Department of Pharmaceutical Chemistry,
Bharat Technology, Howrah, Banitabla, India
Correspondence
B. Nandha, Department of Pharmaceutical
Chemistry, Vivekananda College of Pharmacy,
Rajiv Gandhi University of Health Sciences,
Dr. Rajkumar road, Rajajinagar 2nd stage,
Bangalore 560055, Karnataka, India.
Email: pharmnandha@gmail.com
K E Y W O R D S
5-lipoxygenase, anti-inflammatory activity, fluorobenzimidazole, rat paw edema, soluble
epoxide hydrolase
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1
INTRODUCTION
of COX, LOX, and soluble epoxide hydrolase (sEH). Among the three
isoforms of LOXs, the enzyme primarily responsible for the role in the
Lipoxygenase (LOX), cyclooxygenase (COX), and cytochrome P450
(CYP 450) epoxygenase are the key enzymes within the arachidonic
acid (AA) cascade, where they play vital roles in the synthesis of
prostaglandins (PGs), leukotrienes (LTs), and undesirable pro-
inflammatory mediators leading to inflammation and pain.[1,2] Various
inflammatory diseases are associated with the excessive formation of
LTs and PGs. In the past three decades, significant effort has been
directed toward the understanding of numerous biological roles of
mediators of inflammation and beneficial effects of selective inhibiton
inflammatory process was found to be 5-lipoxygenase (5-LOX), due to
its key role in LTs biosynthesis.[3,4] COX enzymes exist in two isoforms,
cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2).[5] In
contrast to the constitutive COX-1, the inducible COX-2 is expressed
in many tissues and the prostaglandins produced by COX-2 play a
major role in inflammatory reactions, and are responsible for the
characteristic inflammatory symptoms.[6] The preferential binding of
selective inhibitors to the COX-2 enzyme over COX-1 enzyme
prevents the side effects associated with the COX-1 inhibition.[7]
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Arch Pharm Chem Life Sci. 2018;ARDP201800030.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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The enzyme sEH is a novel target for pharmacologic therapies of
inflammation as it is actively involved in the conversion of EETs
(epoxyeicosatrienoic acids) to DHETs (dihydroxyeicosatrienoic acids),
and EETs are key anti-inflammatory mediators that are generated by
the CYP 450 enzymes by catalyzing the epoxidation of AA.[8] There is
recent evidence that when EETs are stabilized by sEH inhibitors, they
have dramatically increased anti-inflammatory properties due to the
rise in localized endogenous EETs levels.[9,10] Efficacies of selective
inhibitors of sEH in acute animal models of inflammatory disease have
been demonstrated to produce beneficial effects, as they were found
to possess marked anti-inflammatory activity.[11,12] Furthermore, it
was demonstrated using a xylene-induced ear edema model in mice
that sEH inhibition reduces inflammation in vivo.[13] Accordingly, any
agent that inhibits sEH is expected to be of considerable therapeutic
value for the treatment of conditions associated with local or systemic
inflammation. Dual inhibitors able to block equally the COX/LOX,
COX/sEH, or LOX/sEH enzymatic pathways offer a better alternate
approach in designing a new drug with a superior anti-inflammatory
profile with fewer side effects than targeting a single enzyme
or pathway inhibition.[14–17] Nimesulide, sorafenib, dCP2PU (1-(3,
while the benzoic acid motif is supposed to inhibit sEH. Both the
primary and secondary pharmacophore share the common acidic
imide linkage present in the bezimidazole ring (Figure 2). While
evidence from in vitro studies does not prove in vivo biological activity,
these do provide a rationale and support for the possible use of these
novel compounds to suppress inflammation in vivo. The approach of
dual inhibition of sEH and 5-LOX has been proposed as a more
promising initiative for the development of safer drugs in inflamma-
tory disorders. Accordingly, sEH and 5-LOX inhibition was evaluated
and furthermore the compounds were screened for anti-inflamma-
tory activity by carrageenan-induced rat paw edema method. Keeping
in view the benefits of DMLs, an attempt was made to combine the
structural characteristics of some potent sEH and 5-LOX inhibitors.
Association of such pharmacophoric groups has led to some novel
sEH/5-LOX dual inhibitors with in vitro and in vivo anti-inflammatory
activity.
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
4-dichlorophenyl)-3-(4-phenoxyphenyl)urea),
t-AUCB
(trans-4-
[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid), t-TUCB
(trans-4-[4-(3-trifluoromethoxyphenyl-1-ureido)-cyclohexyloxy]-ben-
zoic acid), and RWJ-63556 are some of the aryl ether analogs with
potential in vitro and in vivo anti-inflammatory activity,[18–21] as
depicted in Figure 1. Benzimidazole analog 2 inhibits sEH with IC50
value of 0.6 μM and compound 3 inhibited 5-LOX and sEH with IC50
values of 36 and 3.5 μM, respectively.[22,23] Recently synthesis and
structure–activity relationship (SAR) of some halogenated phenyl
ether triazole-based compounds as potent dual mPGES-1 and 5-LOX
inhibitors were reported, and biological evaluation of selected
compounds from the data set has disclosed three new potential
anti-inflammatory drugs.[24,25] Benzimidazole scaffolds and their
bioisosteres are well known as a privileged scaffold in drug discovery.
Benzimidazole analogues have been reported as effective anti-
inflammatory agents with COX-2 and 5-LOX inhibiting action.[26–32]
Several non-urea derivatives and aminoheterocycles as amide mimics
were reported as potent sEH inhibitors, and to our knowledge very few
papers have been published on the synthesis and in vitro evaluation of
benzimidazoles as sEH inhibitors.[33–36]
As illustrated in Schemes 1 and 2, designed molecules 4a–o and 8a–e
were prepared via a two-step process involving S-alkylation of
substituted-2-mercaptobenzimidazoles 1a–o followed by hydrolysis
of the resulting ester. The substituted-2-mercaptobenzimidazoles
1a–o required for the synthesis of esters 3a–o were prepared as
described in the earlier report.[40] The esters 3a–o were obtained by
S-alkylation of substituted-2-mercaptobenzimidazoles 1a–o with
methyl 4-(bromomethyl)benzoate 2 in the presence of potassium
carbonate in dry DMF at room temperature. The crude esters 3a–o
so obtained were used for the next step without further purification.
Hydrolysis of the ester group in compounds 3a–o under alkaline
condition followed by acidification yielded the desired benzoic acids
4a–o (Scheme 1).
A similar synthetic protocol was adopted for the synthesis of 2-(6-
(substituted)-5-fluoro-1H-benzo[d]imidazol-2-ylthio)-2-methylpropa-
noic acids 8a–e under same conditions as depicted in Scheme 2. The
esters 7a–e obtained by SN2 reaction of substituted-2-mercaptoben-
zimidazoles 5a–e with ethyl-2-bromo-2-methylpropionate 6 were
subjected to alkaline hydrolysis to afford the propanoic acids 8a–e.
The structures of the newly synthesized substituted fluorobenz
imidazoles were characterized by FTIR, 1H NMR, 13C NMR, and mass
spectroscopy. 1H NMR spectral analysis of compounds 4a–o showed a
singletat4.59–4.65 ppm, due tothepresence ofthemethylenic─CH2─
proton and another singlet at 12.22–12.98 ppm corresponding to one
proton of COOH group. IR spectrum characteristic bands were
observed at 3398–3425 cm−1 and 2910–2969 cm−1, assignable to
stretching vibrations of the NH and benzylic S-CH2 respectively. While

In light of these observations, and our exploration for new
molecules with anti-inflammatory activity that embody these
structural characteristics encouraged us to design and synthesize
new aryl ether substituted fluorobenzimidazoles for examination as
inhibitors of sEH and 5-LOX for anti-inflammatory activity. Current
research focuses on the design of multi-target ligands interfering with
AA cascade. We chose the strategy of designed multiple ligands
(DMLs) involving dual inhibition of 5-LOX and sEH. The main design
strategy of DMLs comprises the linking of two selective pharmaco-
phores leading to only one active compound,[37–39] targeting various
steps of the AA cascade. Benzimidazole core (central linker) was
combined with benzoic acid moiety on one side and aryl ether linkage
on the other. The latter is responsible for inhibitory activity on 5-LOX,
C
O stretching bands of carboxylic acid appeared from 1689 to
1710 cm−1, the OH stretching bands was observed from 3200 to
3300 cm−1 in the IR spectra of the compounds 4a–o. The 13C NMR
results showed that the compounds presented the expected carbonyl
signal at δ 166–170 ppm and methylene signal at δ 33–35 ppm.

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FIGURE 1 Chemical structures of some representative 5-LOX and sEH inhibitors reported in the literature and of the synthesized
substituted fluorobenzimidazoles (4a–o and 8a–e) as anti-inflammatory agents
On the other hand, IR spectra of compounds with 2-methyl
propanoic acid head 8a–e exhibited the absorption band at around
1700–1735 cm−1 (CO stretch) and 3210–3250 cm−1 (OH stretch),
indicating the presence of carboxyl group. The 1H and 13C NMR
spectra further support the presence of acid group. The 1H NMR
spectrum displayed COOH proton signal as a singlet at 12.88–
13.59 ppm, while carbonyl carbon appeared at δ 169–175 ppm in the
FIGURE 2 5-LOX and sEH dual inhibitor pharmacophore model

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NANDHA ET AL.
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SCHEME 1 Synthesis of compounds 4a–o
13C NMR spectrum. IR spectra showed bands at 2914–2969 cm−1
,
2.2 Biological evaluation
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attributed to a methyl group. The presence of strong bands at 3406–
3425 cm−1 was clear indication of NH stretching. The 1H NMR
spectrum revealed the presence of signals for methyl groups
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2.2.1 In vitro soybean lipoxygenase and sEH
inhibition
resonating between
δ 1.53 and 1.60 ppm as singlets. In the
13C NMR spectrum, the two carbons of the methyl groups were
visible at δ 26–27 ppm, while the quaternary carbon attached to the
methyl groups was observed at δ 50–53 ppm. The mass spectra were
in agreement with the suggested structures of all the synthesized
compounds.
Test compounds and inhibitors were screened for potency in vitro
against recombinant human sEH and soybean lipoxygenase. The
concentration of the test compounds and inhibitors that reduces the
enzyme activity by 50% was designated as IC50. The test compounds
were assayed for soybean lipoxygenase and sEH inhibitory activity in

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SCHEME 2 Synthesis of compounds 8a–e
triplicates, and the IC50 values are reported as the average of the
obtained results with at least two points in the linear region of the
curve on either side of the IC50 (Table 1).
ring with electron withdrawing group at 4-position of the aromatic ring
was important for 5-LOX inhibitory activity. The in vitro 5-LOX
inhibitory activity of phenyl ether analog 4o (IC50 = 14 μM) was
comparable to the compounds 4c and 4d with IC50 values of 10 and
17 μM. Compound 4a, with a chlorine atom at 6-position of the
benzimidazole ring, was found to be least active of the series with IC50
value of 63 μM.
RWJ-63556 which is structurally related to nimesulide and its
analogues is an acidic anti-inflammatory drug, and was reported as a
potent orally active 5-LOX inhibitor. Acidic nature of RWJ-63556 is
attributed to the presence of a sulfonanilide group. As depicted in
Figure 3, our strategy was based on incorporating the two key
pharmacophores of RWJ-63556 in the benzimidazole ring. Primary
pharmacophore in RWJ-63556 is a diaryl ether function, while the
secondary pharmacophore is an acidic NH group. We have
investigated new molecules designed by retaining these two
pharmacophores in the benzimidazole ring for 5-LOX inhibitory
activity.
As discussed in Section 1, there are several excellent lead
structures under investigation as sEH inhibitors. An effort toward
designing novel synthetic inhibitors of sEH based on reported active
lead compounds resulted in a series of fluorobenzimidazoles 4a–o.
Inhibition potency of the described compounds ranges from 0.7 to
100 μM against recombinant human sEH. Compared to the
unsubstituted benzimidazole compound 4a (IC50 = 41 μM), the
inhibition potencies increased dramatically when 6-chlorine in
4a was replaced with aryl ethers resulting in compounds 4b
(IC50 = 14 μM), 4c (IC50 = 2.7 μM), 4d (IC50 = 12 μM), 4e
(IC50 = 10 μM), 4f (IC50 = 7 μM), and 4g (IC50 = 4 μM). Analogues 4f
and 4g with electron withdrawing Cl and F showed a dramatic
increase in potency over other aryl ethers 4b–e. Likewise, replace-
ment of the 6-chlorine atom in 4a with aromatic azoles yielded 4j and
4k as potent inhibitors of human sEH. Compound 4k with triazole ring
was found to be a potent inhibitor of sEH (IC50 = 0.7 μM), with
activity better than derivative 4j (IC50 = 1.8 μM) with isosterically
replaced imidazole ring. Introduction of pyrrolidine and piperidine
ring in the 6-position of the benzimidazole ring, resulted in
This compound displayed significant anti-inflammatory activity in
a canine model of local inflammation, and served as lead compound for
the synthesis of compounds 4b–g and 4l–o, retaining the 5-LOX
pharmacophores present in RWJ-63556. The synthesized compounds
4a–o were tested for their ability to inhibit 5-LOX. Among the tested
compounds, aryl ether analogs 4f and 4g with electron withdrawing Cl
(IC50 = 3 μM) and F (IC50 = 0.9 μM) at 4-position of the 6-phenyl ring
were found to exhibit significant 5-LOX inhibitory activity. The other
aryl ether analogs 4b (IC50 = 27 μM), 4c (IC50 = 10 μM), 4d
(IC50 = 17 μM), and 4e (IC50 = 21 μM) also exhibited good, although
not the best 5-LOX inhibitory action. It was found that introduction of
heterocyclic amines like pyrrolidine 4h (IC50 = 89 μM) and piperidine 4i
(IC50 = 77 μM) proved to be detrimental for activity. However,
introduction of aromatic azoles like imidazole 4j (IC50 = 32 μM) and
triazole 4k (IC50 = 35 μM) exhibited moderate activity. These results
illustrated that aryl ether function at 6-position of the benzimidazole
compounds 4h (IC50 = 77 μM) and 4i (IC50 = 100 μM), with
a
significant drop in potency compared to the other derivatives.
Interestingly, the unsubstituted phenyl ether analog 4o
(IC50 = 25 μM) was the least active.

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TABLE 1 In vitro screening of the synthesized compounds (4a–o, 8a,
and 8e) for 5-LOX and sEH inhibitory activity
Having established the role of aryl ether linkage for 5-LOX
inhibition and heteroaromatic azole linkage for sEH inhibition at 6-
position, hybrid analog 4n was synthesized. The molecular hybrid 4n
(IC50 = 30 μM) with aryl ether linkage at 6-position bearing an
imidazole ring at 4-position of the phenyl ring has significantly
enhanced the 5-LOX inhibitory activity as compared to diether analog
4l (IC50 = 45 μM) and compound 4m (IC50 = 49 μM) with methylene-
dioxyphenyl moiety. On the other hand, N-phenoxyimidazole substi-
tuted compound 4n (IC50 = 3.1 μM) was more active compared to
diether 4l (IC50 = 17 μM) and methylenedioxyphenyl containing
benzimidazole molecule 4m (IC50 = 19 μM), that are well tolerated
with moderate sEH inhibitory activity. The novel hybrid compound 4n
demonstrated an in vitro 10-fold increase in potency against sEH
relative to 5-LOX inhibition.
5-LOX inhibitory activity
(IC50d, μM)
sEH inhibitory activity
(IC50d, μM)
Compoundª
4a
63
27
10
17
21
3
41
4b
14
4c
2.7
12
4d
4e
10
4f
7
4g
0.9
89
77
32
35
45
49
30
14
19
5
4
4h
77
4i
100
1.8
0.7
17
4j
In continuation of these studies, and in order to gain more
insight on the SAR, compounds with an aliphatic carboxylic head
were synthesized and evaluated for in vitro anti-inflammatory
activity. Among the compounds containing the propanoic acid
group, 8a and 8e were the selected candidates for in vitro anti-
inflammatory activity. Replacement of benzoic acid head with
aliphatic propanoic acid function resulted in a significant decrease
in sEH inhibition (IC50 = >100 μM), while the 5-LOX inhibitory
activity for the compounds 8a (IC50 = 19 μM) and 8e (IC50 = 5 μM)
was retained. Importantly, this observation suggested that aryl ether
linkage at 6-position plays an important role in determining the anti-
inflammatory activities of these analogs. The study also suggested
that the aryl ether linkage is required for 5-LOX inhibiton, while
benzoic acid part is essential for the sEH inhibitory activity.
We found that the compound with fluorophenyl ether linkage at
6-position 8e appeared to be the most potent 5-LOX inhibitor
among the propanoic acid derivatives synthesized.
4k
4l
4m
4n
19
3.1
25
4o
8a
>100
>100
2 nM
–
8e
t-AUCBb
NDGAc
–
12.5
aDetailed structures are shown in Schemes 1 and 2.
bA fluorescent-based assay was performed and t-AUCB used as a positive
control was found to have an IC50 value of 2 nM.[44]
cNordihydroguaiaretic acid (NDGA) was used as positive control for 5-LOX
inhibitory activity by UV absorbance-based enzyme assay.
dFor each compound, the percent inhibition was determined in triplicates.
Synthesized compounds and standard drugs in bold - showing significant inhibition.
FIGURE 3 Rationale for the design and synthesis of the substituted fluorobenzimidazoles (4a–o and 8a–e) as anti-inflammatory agents, and
the importance of an acidic NH group in the representative anti-inflammatory agents and synthesized compounds for 5-LOX inhibitory
activity

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2.2.2 In vivo inhibition of carrageenan-induced paw
inhibition of 64.13 and 67.39% comparable to the standard drug
ibuprofen. The result shows that an electron-withdrawing group
increases the anti-inflammatory activity of the compound. Another
feature observed during the study is that the analogues 4j and 4k with
imidazole and 1,2,4-triazole at C-6 on the phenyl ring of fluorobenzi-
midazole show better anti-inflammatory properties (59.78 and 66.30%)
than the analogues 4h and 4i substituted with alicyclic amines (48.91
and 47.82%). Surprisingly, the hybrid analogue 4n showed 65.21%
inhibition comparable to ibuprofen at 3 h. The SAR was not very
discernable for compounds 4e and 4m that were equipotent with
55.43% inhibition, while compound 4l with 53.26% inhibition displayed
activity similar to 4b with 52.17% inhibition.
edema
All the newly synthesized fluorobenzimidazoles 4a–o were screened for
in vivo anti-inflammatory activity by employing carrageenan-induced rat
paw edema method and the results are summarized in Tables 2 and 3.
Most of the test compounds showed appreciable inhibition of the
edema size in comparison with standard drug ibuprofen. The tested
compounds showed anti-inflammatory activity ranging from 45.65 to
67.39%, whereas the reference drug ibuprofen showed 70.65%
inhibition after 3 h. The in vivo anti-inflammatory activity data revealed
that the substitution pattern at 6-position of the benzimidazole ring
seemedtohavedifferentinfluenceontheinhibitionofinflammation, i.e.,
paw edema. The unsubstituted fluorobenzimidazole counterpart 4a was
synthesized to see its effect on activity profile, and was found to have
the least inhibitory effect with percent inhibition of 45.65%. Among the
aryl ether derivatives, compounds 4b–e with alkyl or aryl substituents
showed good anti-inflammatory activity with a percent inhibition of
52.17, 56.52, 57.60, and 55.43%, respectively. In the ether series, the
phenyl ether analog 4o was least active with 50.00% inhibition. On the
other hand, a considerable increase in activity was observed in
compounds 4f and 4g containing chlorine and fluorine with a percent
On the other hand, compounds 8a and 8e with propanoic acid
function were well tolerated andshowedgood anti-inflammatoryactivity
(53.26 and 58.69%). In this series 8e emerged as the potent analogue.
SAR study reveals that imidazole and triazole substituted
derivatives (4j and 4k), and compounds with F and Cl bearing ether
linkage at C-6 on the phenyl ring of fluorobenzimidazole (4f and 4g),
were the most active of all the compounds and showed significant
inhibitory activity both in vitro and in vivo. Clear correlation between in
vitro activity for 5-LOX and sEH inhibition, and in vivo activity for
inhibition of edema (%) by test compounds, has been established.
TABLE 2 Anti-inflammatory activity of the tested compounds using acute carrageenan-induced paw edema in rats at 100 mg/kg body weight
Mean paw edema volume in mL SEMa,b
Groups
I
Treatment
Dose (mg/kg) orally
1 h
2 h
3 h
4 h
Control
Ibuprofen
4a
1% CMCc
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.34 0.004
0.23 0.009
0.26 0.013
0.27 0.008
0.24 0.013
0.30 0.009
0.30 0.009
0.27 0.008
0.25 0.011
0.23 0.009
0.28 0.004
0.29 0.011
0.25 0.011
0.27 0.008
0.28 0.004
0.24 0.013
0.31 0.002
0.25 0.007
0.24 0.003
0.70 0.006
0.36 0.008
0.47 0.022
0.45 0.004
0.42 0.006
0.52 0.022
0.54 0.011
0.48 0.013
0.40 0.022
0.38 0.011
0.49 0.006
0.50 0.005
0.42 0.008
0.41 0.004
0.48 0.013
0.44 0.003
0.53 0.009
0.41 0.003
0.40 0.022
0.92 0.003
0.27 0.006
0.50 0.011
0.44 0.006
0.40 0.013
0.39 0.011
0.41 0.009
0.33 0.008
0.30 0.022
0.47 0.006
0.48 0.011
0.37 0.004
0.31 0.010
0.43 0.006
0.41 0.009
0.32 0.011
0.46 0.007
0.43 0.008
0.38 0.006
0.95 0.006
0.38 0.003
0.53 0.022
0.52 0.006
0.58 0.013
0.45 0.013
0.43 0.022
0.40 0.008
0.49 0.011
0.50 0.006
0.52 0.006
0.55 0.011
0.47 0.010
0.51 0.006
0.45 0.013
0.48 0.004
0.46 0.005
0.57 0.009
0.49 0.011
II
III
IV
4b
V
4c
VI
4d
VII
VIII
IX
4e
4f
4g
X
4h
XI
4i
XII
XIII
XIV
XV
XVI
XVII
XVIII
XIX
4j
4k
4l
4m
4n
4o
8a
8e
aSEM denotes the standard error of the mean.
bAll data are significantly different from control (p < 0.001).
cCarboxymethyl cellulose.
Synthesized compounds and standard drugs in bold - showing significant inhibition.

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TABLE 3 Inhibition of edema (%) by standard drug and test compounds (4a–o, 8a, and 8e)
Inhibition of edema (%)
Treatment
Dose (mg/kg) orally
1 h
2 h
3 h
4 h
Ibuprofen
4a
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
32.35
23.52
20.58
29.41
11.76
11.76
20.58
26.47
32.35
17.64
14.70
26.47
20.58
17.64
29.41
08.82
26.47
29.41
48.57
32.85
35.71
40.00
25.71
22.85
31.42
42.85
45.71
30.00
28.57
40.00
41.42
31.42
37.14
24.28
41.42
42.85
70.65
45.65
52.17
56.52
57.60
55.43
64.13
67.39
48.91
47.82
59.78
66.30
53.26
55.43
65.21
50.00
53.26
58.69
60.00
44.21
45.26
38.94
52.63
54.73
57.89
48.42
47.36
45.26
42.10
50.52
46.31
52.63
49.47
51.57
40.00
48.42
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
8a
8e
Synthesized compounds and standard drugs in bold - showing significant inhibition.
|
3
CONCLUSION
shown by compounds 4f, 4g, 4k, 4n, and 8e comparable to the standard
drug ibuprofen clearly indicates that these compounds can serve as lead
molecules for further development as potent anti-inflammatory agents.
Further optimization of the lead compounds for their anti-inflammatory
activities is actively underway in our laboratory.
A series of novel 4-((5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-
ylthio)methyl)benzoic acids 4a–o and 2-(5-fluoro-6-(substituted)-1H-
benzo[d]imidazol-2-ylthio)-2-methylpropanoic acids 8a–e were synthe-
sizedandexaminedforactivityagainstsEH and5-LOX. Thein vitro assays
undertakeninthisstudyshowedthatthecompoundsexhibitedmoderate
to good activity in sEH and 5-LOX inhibition. The benzimidazole scaffold
with aryl ether and azole linkage at 6-position has been shown to be
effective inhibitor of 5-LOXandsEH, and theinhibitiondisplayed were all
in micromolar range. The most active analogue against sEH was 4k, and
compound 4g emergedasthepromisingbenzimidazolederivativeagainst
5-LOX. From the assays undertaken in this study, it is clear that the
incorporationof fluorine intothemolecule has had asignificant impact on
the inhibition of sEH and 5-LOX. However, when compared to the
heterocyclic amine substituted derivative, the aryl ether analog reduced
the IC50 value. The azole substituted compounds emerged as the potent
sEH inhibitors, whilethearyletheranalogsexhibitedmoderatetogood5-
LOX inhibition. It was interesting that the sEH inhibitory activity of the
phenylimidazole hybrid analog 4n was better than the 5-LOX inhibitory
action. A substantial reduction in sEH inhibitory activity was observed
in case of substituted fluorobenzimidazoles with propanoic acid head
(8a and 8e), leading to the assumption that selective 5-LOX inhibition
might be contributing to their anti-inflammatory properties. To rational-
ize the in vitro anti-inflammatory activity, the compounds were further
evaluated in vivo for their anti-inflammatory activity by the carrageenan-
induced rat paw edema assay. In conclusion, the significant inhibition
|
4
EXPERIMENTAL
|
4.1 Chemistry
|
4.1.1 General
All chemicals were purchased from commercial sources and were used
without further purification. Solvents were purified and dried by
standard methods. Melting points (uncorrected) were determined in
open glass capillaries. The IR spectra were recorded on a Shimadzu-
5400 FT-IR spectrometer in KBr pellets and band positions are reported
in wave numbers (cm−1). 1H and 13C NMR were measured in CDCl3 or
DMSO-d6 on a Bruker Avance-400 MHZ spectrometer. Chemical shifts
are expressed in δ (ppm) relative to the internal standard and coupling
constants J in Hz. Electrospray ionization mass spectra (ESI-MS) were
recorded on a LCMS 3200 triple quad (Q Trap) and LCMS 3000 API
SCIEX mass spectrometer. Microanalyses were performed on a Perkin-
Elmer 2400 CHN element analyzer. All compounds were within 0.4%
of the theoretical values. Analytical thin-layer chromatography (TLC)
was performedon0.2 mmsilicagelprecoatedaluminumsheets (60F254
,
Merck) and UV light was used for visualization at 254 nm.

NANDHA ET AL.
9 of 13
|
The NMR and mass spectra and the InChI codes of the
4-((6-(4-Phenylphenoxy)-5-fluoro-1H-benzo[d]imidazol-2-
investigated compounds together with some biological activity data
are provided as Supporting Information.
ylthio)methyl)benzoic acid (4c)
White solid; yield 69%; mp 183–185°C; IR (KBr cm−1): 3400 (NH str),
3220 (OH str), 3090 (Ar CH str), 2965 (CH2 str), 1703 (Ar CO str), 1605


(C N str), 1514, 1485, 1473 (C C ring str), 1352 (Ar C-N str), 1218
(Ar-O-Ar, C-O-C str), 1151 (C-F str); 1H NMR (DMSO-d6): δ 4.61 (s, 2H,
CH2), 6.98–7.01 (d, 2H, J = 12.0 Hz, ArH), 7.29–7.63 (m, 11H, ArH),
7.86–7.88 (d, 2H, J = 8.0 Hz, ArH), 12.86 (br.s, 2H, NH and COOH);
13C NMR (DMSO-d6): δ 34.75 (S-CH2), 116.27, 126.35, 127.00,
128.11, 128.78, 128.84, 129.40, 130.91, 134.62, 137.58, 139.48,
142.14, 143.37, 148.60, 148.87, 149.40, 151.21, 151.78, 157.78 (Ar-
C), 167.79 (CO); MS (ESI) m/z: 470.1 (M−1). Anal. calcd. for
|
4.1.2 General procedure for the synthesis of 4-((5-
fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acids (4a–o)
A
mixture of 5-fluoro-6-(substituted)-1H-benzo[d]imidazole-2-
thiol 1a–o (2 mmol), methyl 4-(bromomethyl)benzoate 2 (2 mmol),
and K2CO3 (4 mmol) in dry DMF (10 mL) was stirred at room
temperature for 5 h in a nitrogen-flushed flask (completion of the
reaction was monitored by TLC). After completion of reaction, the
reaction mixture was treated with water and extracted with ethyl
acetate (3 × 50 mL). The combined organic layer was washed with
water (50 mL), brine (50 mL), dried over anhydrous Na2SO4, and
filtered. The organic solvent was evaporated under vacuum to
afford the crude esters 3a–o that were used for the next step
without further purification. The ester was subjected to alkaline
hydrolysis by refluxing with 50% KOH in ethanol for 3 h. After
removal of alcohol under vacuum, water was added to the residue
with stirring and acidified with 2 N HCl to pH 4. The precipitated
product was separated by filtration at the pump, followed by water
wash and dried. The crude residue was purified by recrystallization
from ethanol to afford 4a–o.
C
27H19FN2O3S: C, 68.93; H, 4.07; N, 5.95. Found: C, 68.79; H,
3.98; N, 5.90.
4-((5-Fluoro-6-(p-tolyloxy)-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acid (4d)
White solid; yield 53%; mp 155–157°C; IR (KBr cm−1): 3408 (NH str),
3218 (OH str), 3105, 3050 (Ar CH str), 2916 (CH2 str), 2865 (CH3 str),

1700 (Ar CO str), 1608 (C N str), 1550, 1520, 1430 (CC ring str), 1360
(Ar C-N str), 1218 (Ar-O-Ar, C-O-C str), 1160 (C-F str); 1H NMR
(DMSO-d6): δ 2.24 (s, 3H, CH3), 4.59 (s, 2H, CH2), 6.81–6.83 (d, 2H,
J = 8.0 Hz, ArH), 7.11–7.13 (d, 2H, J = 8.0 Hz, ArH), 7.47–7.53 (m, 4H,
ArH), 7.84–7.86 (d, 2H, J = 8.0 Hz, ArH), 8.24 (s, 1H, NH), 12.78 (br.s,
1H, COOH); 13C NMR (DMSO-d6): δ 20.08 (CH3), 34.78 (S-CH2),
98.03, 98.93, 103.12, 115.58, 116.08, 128.75, 129.39, 130.12,
131.57, 140.03, 149.30, 150.92, 151.65, 155.83 (Ar-C), 166.03
(CO); MS (ESI) m/z: 408.3 (M−1). Anal. calcd. for C22H17FN2O3S: C,
64.70; H, 4.19; N, 6.85. Found: C, 64.55; H, 4.11; N, 6.75.
4-((6-Chloro-5-fluoro-1H-benzo[d]imidazol-2-ylthio)methyl)-
benzoic acid (4a)
Pale beige solid; yield 57%; mp 195–197°C; IR (KBr cm−1): 3420 (NH
str), 3200 (OH str), 3080 (Ar CH str), 2950 (CH2 str), 1700 (Ar CO str),


1615 (C N str), 1500, 1455 (C C ring str), 1350 (Ar C-N str), 1154
(C-F str), 1020 (C-Cl str); 1H NMR (DMSO-d6): δ 4.61 (s, 2H, CH2),
7.53–7.63 (m, 4H, ArH), 7.85–7.86 (d, 2H, ArH), 12.87 (br.s, 2H, NH
and COOH); 13C NMR (DMSO-d6): δ 34.89 (S-CH2), 99.42, 99.86,
114.20, 115.26, 128.78, 129.40, 139.78, 140.40, 150.85, 161.86 (Ar-
C), 166.93 (CO); MS (ESI) m/z: 336.1 (M−1). Anal. calcd. for
4-((6-(4-tert-Butylphenoxy)-5-fluoro-1H-benzo[d]imidazol-2-
ylthio)methyl)benzoic acid (4e)
White solid; yield 61%; mp 166–168°C; IR (KBr cm−1): 3420 (NH str),
3205 (OH str), 3070 (Ar CH str), 2960 (CH2 str), 2882 (CH3 str), 1690


(ArCOstr),1605(C Nstr),1515,1475(C Cringstr),1350(ArC-Nstr),
1225 (Ar-O-Ar, C-O-C str), 1175 (C-F str); 1H NMR (DMSO-d6): δ 1.25 (s,
9H, 3CH3), 4.61 (s, 2H, CH2), 6.83–6.93 (d, 2H, ArH), 7.33–7.38 (m, 2H,
ArH), 7.41–7.43 (d, 1H, J = 8.0 Hz, ArH), 7.46–7.49 (d, 1H, J = 12.0 Hz,
ArH), 7.54–7.56 (d, 2H, J = 8.0 Hz, ArH), 7.85–7.87 (d, 2H, J = 8.0 Hz,
ArH), 7.90 (s, 1H, NH), 12.64 (s, 1H, COOH); 13C NMR (DMSO-d6): δ
31.23 ((CH3)3), 33.92 (C(CH3)3), 34.78 (S-CH2), 115.64, 116.87, 126.49,
126.81, 127.04, 128.97, 129.27, 129.47, 129.64, 129.75, 138.38,
138.53, 142.73, 143.87, 144.98, 149.46, 150.86, 151.84, 155.68 (Ar-C),
166.96 (CO); MS (ESI) m/z: 450.2 (M−1). Anal. calcd. for C25H23FN2O3S:
C, 66.65; H, 5.14; N, 6.21. Found: C, 66.51; H, 5.03; N, 6.14.
C
15H10ClFN2O2S: C, 53.50; H, 2.99; N, 8.31. Found: C, 53.41; H,
2.92; N, 8.39.
4-((5-Fluoro-6-naphthalen-2-yloxy)-1H-benzo[d]imidazol-2-
ylthio)methyl)benzoic acid (4b)
White solid; yield 63%; mp 177–179°C; IR (KBr cm−1): 3398 (NH str),
3207 (OH str), 3039 (Ar CH str), 2969 (CH2 str), 1691 (Ar CO str), 1610


(C N str), 1597, 1577, 1473 (C C str), 1354 (Ar C-N str), 1225 (Ar-O-
Ar, C-O-C str), 1159 (C-F str); 1H NMR (DMSO-d6): δ 4.62 (s, 2H, CH2),
6.70–6.71 (d, 1H, ArH), 7.32–7.37 (m, 1H, ArH), 7.42 (s, 1H, ArH),
7.53–7.65 (m, 7H, ArH), 7.85–7.91 (m, 2H, ArH), 7.97 (s, 1H, NH),
8.25–8.28 (d, 1H, J = 12.0 Hz, ArH), 12.70 (br.s, 1H, COOH); 13C NMR
(DMSO-d6): δ 35.27 (S-CH2), 109.89, 121.76, 122.89, 125.24, 126.47,
126.61, 127.34, 127.55, 128.23, 129.48, 129.77, 129.97, 130.25,
134.85, 138.97, 143.28, 151.53, 154.22 (Ar-C), 167.46 (CO); MS (ESI)
m/z: 444.2 (M−1). Anal. calcd. for C25H17FN2O3S: C, 67.56; H, 3.85; N,
6.30. Found: C, 67.41; H, 3.72; N, 6.25.
4-((6-(4-Chlorophenoxy)-5-fluoro-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acid (4f)
White solid; yield 64%; mp 159–160°C; IR (KBr cm−1): 3410 (NH str),
3225 (OH str), 3090 (Ar CH str), 2925 (CH2 str), 1697 (Ar CO str), 1620


(C N str), 1485, 1473 (C C ring str), 1354 (Ar C-N str), 1220 (Ar-O-
Ar, C-O-C str), 1151 (C-F str), 1080 (C-Cl str); 1H NMR (DMSO-d6): δ
4.60 (s, 2H, CH2), 6.42 (m, 2H, ArH), 6.93–6.94 (d, 2H, ArH), 7.35–7.37

10 of 13
NANDHA ET AL.
|
(m, 2H, J = 8.0 Hz, ArH), 7.51 (s, 2H, ArH), 7.85 (s, 2H, ArH), 8.29 (s, 1H,
NH), 12.88 (br.s, 1H, COOH); 13C NMR (DMSO-d6): δ 36.35 (S-CH2),
101.87, 102.12, 103.27, 107.12, 109.19, 110.64, 112.19, 112.51,
114.65, 117.71, 126.43, 128.95, 129.66, 137.87, 138.01, 147.09,
147.98, 149.51, 151.13, 151.89, 156.89 (Ar-C), 169.87 (CO); MS (ESI)
m/z: 428.0 (M−1). Anal. calcd. for C21H14ClFN2O3S: C, 58.82; H, 3.29;
N, 6.52. Found: C, 58.68; H, 3.20; N, 6.49.
1H NMR (DMSO-d6): δ 4.63 (s, 2H, CH2), 7.30–7.55 (m, 3H, ArH), 7.75
(br.s, 1H, ArH), 7.89–7.91 (d, 2H, J = 8.0 Hz, ArH), 8.09–8.13 (d, 2H,
imidazole), 8.77 (s, 1H, imidazole), 12.83 (br.s, 2H, NH and COOH); MS
(ESI) m/z: 368.0 (M−1). Anal. calcd. for C18H13FN4O2S: C, 58.70; H,
3.55; N, 15.20. Found: C, 58.52; H, 3.41; N, 15.15.
4-((5-Fluoro-6-(1H-1,2,4-triazol-1-yl)-1H-benzo[d]imidazol-2-
ylthio)methyl)benzoic acid (4k)
4-((5-Fluoro-6-(4-fluorophenoxy)-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acid (4g)
Brown solid; yield 67%; mp 227–229°C; IR (KBr cm−1): 3413 (NH str),
3210 (OH str), 3092 (Ar CH str), 2915 (CH2 str), 1690 (Ar CO str), 1615
White solid; yield 58%; mp 150–152°C; IR (KBr cm−1): 3422 (NH str), 3210
(C N str), 1511, 1450 (C C ring str), 1350 (Ar C-N str), 1145 (C-F str);
1H NMR (DMSO-d6): δ 4.65 (s, 2H, CH2), 7.54–7.61 (m, 3H, ArH),
7.75–7.76 (d, 1H, ArH), 7.85–7.87 (d, 2H, J = 8.0 Hz, ArH), 8.24 (s, 1H,
triazole), 8.93 (s, 1H, triazole), 12.98 (br.s, 2H, NH and COOH); 13C NMR
(DMSO-d6): δ 34.88 (S-CH2), 116.20, 126.31, 126.86, 127.99, 128.61,
128.71, 129.39, 134.65, 139.56, 148.40, 151.15, 152.25, 157.78 (Ar-C),
167.70 (CO); MS (ESI) m/z: 369.2 (M+1). Anal. calcd. for C17H12FN5O2S:
C, 55.29; H, 3.27; N, 18.95. Found: C, 55.20; H, 3.18; N, 18.86.



(OH str), 3070 (Ar CH str), 2910 (CH2 str), 1692 (Ar CO str), 1610 (C Nstr),
1545, 1500, 1475 (CC ring str), 1350 (Ar C-N str), 1200 (Ar-O-Ar, C-O-C
str), 1152 (C-F str); 1H NMR (DMSO-d6):
δ 4.59 (s, 2H, CH2),
6.92–6.95 (d, 2H, J = 12.0 Hz, ArH), 7.06–7.08 (d, 2H, J = 8.0 Hz, ArH),
7.10–7.11 (m, 2H, ArH), 7.51–7.53 (d, 2H, J = 8.0 Hz, ArH), 7.87–7.89 (d,
2H, J = 8.0 Hz, ArH), 8.17 (s, 1H, NH), 12.64 (s, 1H, COOH); 13C NMR
(DMSO-d6): δ34.80 (S-CH2), 99.46, 99.89, 108.30, 115.89, 116.26, 123.25,
128.73, 129.39, 133.35, 138.55, 149.15, 150.25, 158.33, 163.25 (Ar-C),
168.73 (CO); MS (ESI) m/z:412.3(M−1). Anal. calcd. for C21H14F2N2O3S: C,
61.17; H, 3.42; N, 6.79. Found: C, 61.02; H, 3.29; N, 6.69.
4-((5-Fluoro-6-(4-phenoxyphenoxy)-1H-benzo[d]imidazol-2-
ylthio)methyl)benzoic acid (4l)
White solid; yield 62%; mp 149–151°C; IR (KBr) cm−1: 3425 (NH str),
4-((5-Fluoro-6-(pyrrolidin-1-yl)-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acid (4h)
Off-whitesolid;yield 68%;mp122–124°C; IR(KBr cm−1):3420 (NH str),
3222 (OH str), 3082, 3066, 3039 (Ar CH str), 2929, 2854 (CH2 str), 1710


(Ar CO str), 1639 (C Nstr), 1593,1487, 1467(C Cringstr),1354(ArC-
N str), 1222 (Ar-O-Ar, C-O-C str), 1165 (C-F str); 1H NMR (DMSO-d6): δ
4.60 (s, 2H, CH2), 6.98–7.00 (d, 2H, J = 8.0 Hz, ArH), 7.28–7.32 (d, 2H,
ArH), 7.39–7.43 (m, 3H, ArH), 7.50–7.53 (d, 2H, J = 12.0 Hz, ArH), 7.57–
7.60 (m, 4H, ArH), 7.87–7.89 (d, 2H, J = 8.0 Hz, ArH), 8.23 (s, 1H, NH),
12.77 (br.s, 1H, COOH); 13C NMR (DMSO-d6): δ 33.91 (S-CH2), 98.30,
98.54, 103.18, 116.76, 126.40, 127.10, 128.09, 128.20, 128.68, 128.77,
128.88, 135.05, 138.09, 138.23, 139.39, 149.36, 151.75, 157.27 (Ar-C),
169.55 (CO); MS (ESI) m/z: 486.1 (M−1). Anal. calcd. for C27H19FN2O4S:
C, 66.65; H, 3.93; N, 5.75. Found: C, 66.46; H, 3.81; N, 5.69.
3223 (OH str), 3000 (Ar CH str), 2925 (CH2 str), 2880 (pyrrolidine CH2


str), 1692 (Ar CO str), 1625 (C N str), 1550, 1525, 1510 (C C ring str),
1226 (R C-N str), 1173 (C-F str); 1H NMR (CDCl3): δ 1.55–1.62 (m, 4H,
pyrrolidine), 2.52–2.71 (m, 4H, pyrrolidine), 4.47 (s, 2H, CH2), 5.87–5.90
(d, 2H, J = 12.0 Hz, ArH), 6.80–6.82 (d, 1H, J = 8.0 Hz, ArH), 6.89–6.91
(d, 1H, J = 8.0 Hz, ArH), 7.01 (s, 1H, NH), 7.06 (d, 2H, ArH), 12.81 (s, 1H,
COOH); MS (ESI) m/z: 371.1 (M+1). Anal. calcd. for C19H18FN3O2S: C,
61.45; H, 4.88; N, 11.30. Found: C, 61.29; H, 4.74; N, 11.19.
4-((5-Fluoro-6-(piperidin-1-yl)-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acid (4i)
Off-white solid; yield 59%; mp 135–137°C; IR (KBr cm−1): 3420 (NH str),
4-((6-(Benzo[d][1,3]dioxol-5-yloxy)-5-fluoro-1H-benzo[d]-
imidazol-2-ylthio)methyl)benzoic acid (4m)
White solid; yield 70%; mp 140–142°C; IR (KBr) cm−1: 3387 (NH str),
3211 (OH str), 3080 (Ar CH str), 2920 (CH2 str), 2860 (piperidine CH2 str),
3215 (OH str), 3072, 3039 (Ar CH str), 2939 (CH2 str), 2847 (─OCH2O─




1699 (Ar CO str), 1609 (C N str), 1547, 1507, 1476 (C C ring str), 1228
(R C-N str), 1175 (C-F str); 1H NMR (CDCl3): δ 1.52 (m, 6H, piperidine),
2.92–2.96 (m, 4H, piperidine), 4.61 (s, 2H, CH2), 7.40–7.42 (d, 1H,
J = 8.0 Hz, ArH), 7.53–7.58 (m, 3H, ArH), 7.86–7.88 (d, 2H, J = 8.0 Hz,
ArH), 8.22 (s, 1H, NH), 12.57 (s, 1H, COOH); 13C NMR (CDCl3): δ 25.99
(CH2), 29.40 (CH2), 34.99 (S-CH2), 49.19 (CH2), 115.78, 116.40, 131.30,
131.89, 132.99, 133.65, 134.55, 135.59, 136.55, 152.55, 161.75 (Ar-C),
169.99(CO); MS (ESI) m/z: 385.1(M+1). Anal. calcd. for C20H20FN3O2S:C,
62.33; H, 5.23; N, 10.89. Found: C, 62.17; H, 5.17; N, 10.77.
str), 1705 (Ar CO str), 1625 (C N str), 1580, 1550, 1470 (C C ring str),
1320 (Ar C-N str), 1222 (Ar-O-Ar, C-O-C str), 1170 (C-F str); 1H NMR
(DMSO-d6): δ 4.95 (s, 2H, CH2), 5.87 (s, 2H, ─OCH2O─), 7.09–7.13 (d,
2H, ArH), 7.16–7.21 (d, 2H, ArH), 7.28–7.30 (d, 1H, J = 8.0 Hz, ArH),
7.98–8.08 (m, 4H, ArH), 8.29(s, 1H, NH), 12.59 (s, 1H, COOH); 13C NMR
(DMSO-d6): δ 35.60 (S-CH2), 99.89, 100.35 (OCH2O), 101.98, 103.61,
108.72, 109.32, 114.66, 114.73, 114.90, 122.81, 128.42, 129.12,
131.53, 132.04, 136.35, 143.50, 148.64, 153.42 (Ar-C), 169.22 (CO);
MS (ESI) m/z: 438.3 (M−1). Anal. calcd. for C22H15FN2O5S: C, 60.27; H,
3.44; N, 6.38. Found: C, 60.04; H, 3.30; N, 6.29.
4-((5-Fluoro-6-(1H-imidazol-1-yl)-1H-benzo[d]imidazol-2-ylthio)-
methyl)benzoic acid (4j)
Brown solid; yield 65%; mp 212–214°C; IR (KBr cm−1): 3410 (NH str),
4-((6-(4-(1H-Imidazol-1-yl)phenoxy)-5-fluoro-1H-benzo[d]-
imidazol-2-ylthio)methyl)benzoic acid (4n)
3222 (OH str), 3050 (Ar CH str), 2910 (CH2 str), 1689 (Ar CO str), 1606
Pale brown solid; yield 73%; mp 203–205°C; IR (KBr) cm−1: 3462 (NH

(C N str), 1500, 1550 (CC ring str), 1340 (Ar C-N str), 1150 (C-F str);
str), 3208 (OH str), 3091, 3072 (Ar CH str), 2940 (CH2 str), 1719 (Ar CO

NANDHA ET AL.
11 of 13
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

str), 1647 (C N str), 1600, 1485 (C C ring str), 1323 (Ar C-N str),
1230 (Ar-O-Ar, C-O-C str), 1161 (C-F str); 1H NMR (DMSO-d6): δ 4.60
(s, 2H, CH2), 7.14–7.17 (dd, 4H, J = 12.0 Hz, ArH), 7.40–7.46 (m, 3H,
ArH), 7.80–7.83 (d, 2H, J = 12.0 Hz, ArH), 8.07–8.09 (dd, 4H,
J = 8.0 Hz, ArH), 8.31 (s, 1H, NH), 12.22 (s, 1H, COOH); 13C NMR
(DMSO-d6): δ 32.00 (SCH2), 104.87, 110.20, 112.39, 117.22, 117.60,
118.86, 119.83, 122.79, 122.87, 130.17, 132.35, 136.14, 149.44,
151.00, 153.76, 153.91, 156.94, 157.27, 157.54, 159.86 (Ar-C),
170.00 (CO); MS (ESI) m/z: 460.3 (M−1). Anal. calcd. for
C24H17FN4O3S: C, 62.60; H, 3.72; N, 12.16. Found: C, 62.39; H,
3.64; N, 12.05.
423.3 (M+1). Anal. calcd. for C23H19FN2O3S: C, 65.39; H, 4.53; N, 6.62.
Found: C, 65.15; H, 4.47; N, 6.51.
2-(5-Fluoro-6-(naphthalen-2-yloxy)-1H-benzo[d]imidazol-2-
ylthio)-2-methylpropanoic acid (8b)
Off-white solid;yield65%; mp145–147°C; IR(KBr) cm−1: 3421(NHstr),
3221 (OH str), 3064 (Ar CH str), 2926, 2966 (CH3 str), 1700 (R CO str),


1629 (C N str), 1597, 1577, 1473 (C C ring str), 1356 (Ar C-N str),
1230(Ar-O-Ar, C-O-Cstr), 1159(C-Fstr);1H NMR(DMSO-d6):δ 1.54(s,
6H, (CH3)2), 6.72–6.74 (d, 1H, J = 8.0 Hz, ArH), 7.29–7.31 (d, 1H,
J = 8.0 Hz, ArH), 7.35–7.39 (t, 1H, J = 8.0 Hz, ArH), 7.53–7.65 (m, 5H,
ArH), 7.98(s, 1H, NH), 8.25–8.27(d, 1H, J = 8.0 Hz, ArH), 13.59(br.s, 1H,
COOH); 13C NMR (DMSO-d6): δ 26.75 (CH3)2, 52.93 (C(CH3)2), 109.67,
113.36, 121.24, 122.44, 124.78, 125.98, 126.10, 126.80, 127.71,
134.34, 138.88, 146.08, 147.11, 149.00, 151.65, 153.61 (Ar-C), 174.38
(CO); MS (ESI) m/z: 397.0 (M+1). Anal. calcd. for C21H17FN2O3S: C,
63.62; H, 4.32; N, 7.06. Found: C, 63.41; H, 4.22; N, 6.97.
4-((5-Fluoro-6-phenoxy-1H-benzo[d]imidazol-2-ylthio)methyl)-
benzoic acid (4o)
White solid; yield 66%; mp 145–147°C; IR (KBr) cm−1: 3408 (NH str),
3300 (OH str), 3050 (Ar CH str), 2925, 2855 (CH2 str), 1705 (Ar CO str),

1637 (CN str), 1629, 1512 (C C ring str), 1350 (Ar C-N str), 1220 (Ar-
O-Ar, C-O-C str), 1178 (C-F str); 1H NMR (DMSO-d6): δ 4.60 (s, 2H,
CH2), 6.87–6.89 (d, 1H, J = 7.2 Hz, ArH), 6.93–6.95 (m, 3H, ArH), 7.09–
7.17 (m, 4H, ArH), 7.32–7.36 (m, 2H, ArH), 12.57 (br.s, 2H, NH and
COOH); 13C NMR (DMSO-d6): δ 32.07 (SCH2), 102.85, 109.61,
114.28, 115.63, 125.79, 128.09, 128.20, 128.97, 129.91, 132.84,
140.90, 142.98, 143.61, 146.11, 151.95, 153.71 (Ar-C), 166.30 (CO);
MS (ESI) m/z: 394.3 (M−1). Anal. calcd. for C21H15FN2O3S: C, 63.95; H,
3.83; N, 7.09. Found: C, 63.71; H, 3.69; N, 7.01.
2-(6-(4-tert-Butylphenoxy)-5-fluoro-1H-benzo[d]imidazol-2-
ylthio)-2-methylpropanoic acid (8c)
Off-white solid; yield 59%; mp 132–135°C; IR (KBr) cm−1: 3406 (Ar NH
str), 3240 (OH str), 3060 (Ar CH str), 2929, 2964 (CH3 str in 2-methyl
propanoic acid), 2885 (CH3 str in tertiary butyl group), 1706 (R CO str),


1597 (C N str), 1508, 1473 (C C ring str), 1354 (Ar C-N str), 1224
(Ar-O-Ar, C-O-C str), 1178 (C-F str); 1H NMR (DMSO-d6): δ 1.25 (s, 9H,
(CH3)3), 1.54 (s, 6H, (CH3)2), 6.84–6.86 (d, 2H, J = 8.0 Hz, ArH), 7.21–
7.23 (d, 1H, J = 8.0 Hz, ArH), 7.33–7.35 (d, 2H, J = 8.0 Hz, ArH), 7.47–
7.49 (d, 1H, J = 8.0 Hz, ArH), 8.70 (s, 1H, NH), 13.00 (br.s, 1H, COOH);
13C NMR (DMSO-d6): δ 26.74 (CH3)2, 31.22 (CH3)3, 33.91 C(CH3)3,
53.08 (C(CH3)2), 102.37, 115.86, 126.49, 128.83, 129.83, 138.84,
138.99, 145.05, 148.63, 149.53, 151.91, 155.58 (Ar-C), 174.57 (CO);
MS (ESI) m/z: 403.3 (M+1). Anal. calcd. for C21H23FN2O3S: C, 62.66; H,
5.76; N, 6.95. Found: C, 62.43; H, 5.63; N, 6.87.
|
4.1.3 General procedure for the synthesis of 2-(5-
fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)-
2-methylpropanoic acids (8a–e)
The following propanoic acids 8a–e were synthesized from appropri-
ate 5-fluoro-6-(substituted)-1H-benzo[d]imidazole-2-thiols 5a–e and
ethyl-2-bromo-2-methylpropionate 6 according to the procedure
described for benzoic acids 4a–e.
A mixture of 5-fluoro-6-(substituted)-1H-benzo[d]imidazole-2-thiol
5a–e (2 mmol), ethyl-2-bromo-2-methylpropionate 6 (2 mmol), and
K2CO3 (4 mmol) in dry DMF (10 mL) was stirred at room temperature
for 5 h in a nitrogen-flushed flask (completion of the reaction was
monitored by TLC). Further steps of work-up and purification were done
in a similar manner as described for benzoic acids 4a–o.
2-(6-(4-Chlorophenoxy)-5-fluoro-1H-benzo[d]imidazol-2-
ylthio)-2-methylpropanoic acid (8d)
Off-white solid;yield68%; mp115–117°C; IR(KBr) cm−1: 3425(NHstr),
3210 (OH str), 3050 (Ar CH str), 2920 (CH3 str), 1715 (R CO str), 1603


(C N str), 1510, 1475 (C C ring str), 1355 (Ar C-N str), 1220 (Ar-O-Ar,
C-O-C str), 1170 (C-F str), 1110 (C-Cl str); 1H NMR (DMSO-d6): δ 1.60
(s, 6H, (CH3)2), 6.95–6.97 (d, 2H, J = 8.0 Hz, ArH), 7.19 (s, 1H, ArH),
7.35–7.38 (d, 2H, J = 12.0 Hz, ArH), 7.52 (br.s, 1H, ArH), 8.27 (br.s, 1H,
NH), 12.88 (br.s, 1H, COOH); 13C NMR (DMSO-d6): δ 26.39 (CH3)2,
52.71 (C(CH3)2), 87.40, 95.69, 98.96, 104.98, 112.77, 117.77, 126.58,
128.34, 129.65, 147.93, 156.87, 163.25, 168.03 (Ar-C), 173.99 (CO);
MS (ESI) m/z: 381.5 (M+1). Anal. calcd. for C17H14ClFN2O3S: C, 53.61;
H, 3.70; N, 7.35. Found: C, 53.42; H, 3.61; N, 7.24.
2-(6-(4-Phenylphenoxy)-5-fluoro-1H-benzo[d]imidazol-2-
ylthio)-2-methylpropanoic acid (8a)
Off-white solid; yield 62%; mp 166–169°C; IR (KBr) cm−1: 3421 (NH
str), 3250 (OH str), 3050 (Ar CH str), 2920 (CH3 str), 1717 (R CO str),

1604 (C N str), 1514, 1485 (CC ring str), 1355 (Ar C-N str), 1222 (Ar-
O-Ar, C-O-C str), 1172 (C-F str); 1H NMR (DMSO-d6): δ 1.53 (s, 6H,
(CH3)2), 6.99–7.02 (d, 2H, J = 12.0 Hz, ArH), 7.29–7.35 (m, 2H, ArH),
7.41–7.44 (d, 2H, J = 12.0 Hz, ArH), 7.59–7.63 (m, 5H, ArH), 7.52 (s,
1H, NH), 13.55 (br.s, 1H, COOH); 13C NMR (DMSO-d6): δ 30.63 (CH3)2,
50.13 (C(CH3)2), 98.30, 98.54, 103.18, 116.76, 126.40, 127.10,
128.09, 128.20, 128.68, 128.77, 128.88, 135.05, 138.09, 138.23,
139.39, 149.36, 151.75, 157.27 (Ar-C), 169.55 (CO); MS (ESI) m/z:
2-(5-Fluoro-6-(4-fluorophenoxy)-1H-benzo[d]imidazol-2-ylthio)-
2-methylpropanoic acid (8e)
Off-white solid; yield 60%; mp 124–126°C; IR (KBr) cm−1: 3410 (NH
str), 3240 (OH str), 3066, 3101 (Ar CH str), 2969, 2914 (CH3 str), 1735

(R CO str), 1609 (CN str), 1505, 1475 (C C ring str), 1350 (Ar C-N str),

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NANDHA ET AL.
|
1200 (Ar-O-Ar, C-O-C str), 1151 (C-F str); 1H NMR (DMSO-d6): δ 1.54
(s, 3H, CH3), 1.60 (s, 3H, CH3), 6.96–6.99 (d, 2H, J = 12.0 Hz, ArH),
7.13–7.19 (m, 2H, ArH), 7.24–7.26 (d, 1H, J = 8.0 Hz, ArH), 7.47–7.50
(d, 1H, J = 12.0 Hz, ArH), 8.25 (br.s, 1H, NH), 12.92 (br.s, 1H, COOH);
13C NMR (DMSO-d6): δ 26.30 (CH3)2), 52.36 (C(CH3)2), 111.82,
117.84, 118.61, 126.13, 127.56, 129.00, 131.68, 135.61, 136.45,
149.35, 151.71, 154.18, 157.18 (Ar-C), 173.05 (CO); MS (ESI) m/z:
365.3 (M+1). Anal. calcd. for C17H14F2N2O3S: C, 56.04; H, 3.87; N,
7.68. Found: C, 55.85; H, 3.78; N, 7.61.
4.2.3 Determination of in vivo anti-inflammatory
|
acitivity[43]
The experimental protocol was reviewed and accepted by the
Institutional Animal Ethics Committee (IAEC) prior to the initiation
of the experiment (Proposal No. IAEC/NCP/50/10). Carrageenan-
induced rat paw edema was utilized as the model for acute
inflammation as previously described to carry out the evaluation of
test compounds for anti-inflammatory activity and is presented as the
percentage inhibition of induction of edema at the right hind paw.
Wister albino rats of either sex, in the weight range of 150–200 g were
used for the study. They were housed in groups of six animals in plastic
cages at 20–25°C and maintained on a standard pellet diet with free
access of water. Animals were fasted for 24 h before the experiment
and were marked with picric acid for individual animal identification.
Edema was induced in the right hind paw of male rats (150–200 g) by
the subplantar injection of 0.1 mL of a 1% carrageenan suspension in
saline. Six rats were used in each test group. Each group of animals
were orally dosed with the test compounds or drug free vehicle (1%
CMC) 1 h prior to carrageenan injection. Group I served as the control
group and received vehicle (1% CMC in water in a volume of 10 mL/
kg); group II received the reference standard ibuprofen at a dose of
100 mg/kg body weight. All the remaining groups received the test
compounds and were administered orally as suspensions in vehicle
(10 mL/kg) at the same dose orally. Paw volume was determined by
calculating the amount of mercury displaced after immersing the paw
to the level of the lateral malleolus. Foot volumes were measured using
the plethysmometer (model 7140, Ugo-Basile, Italy) at 0, 1, 2, 3, and
again 4 h after carrageenan injection, and the difference was
designated as edema volume. The percent inhibition of edema for
the treated groups was calculated by following formula:
|
4.2 Biological evaluation
|
4.2.1 Determination of in vitro IC50 for soybean
lipoxygenase inhibitory activity[41]
UV absorbance-based enzyme assay was adopted for the evaluation of
synthesized compounds for soybean lipoxygenase inhibitory activity.
The use ofsoybeanlipoxygenase enzyme asa modelfor the discoveryof
novel 5-LOX inhibitors is widely accepted because of its structural and
functional similarities with the mammalian LOXs. The principle involves
the measurement of absorbance at 234 nm to monitor the conversionof
sodium linoleate to 13-hydroperoxylinoleic acid, and commercially
purchased sample of nordihydroguaiaretic acid (NDGA) was used as the
standard inhibitor. Sodium linoleate (linoleic acid sodium salt), soybean
lipoxygenase, and NDGA were obtained from Sigma Chemical Co.
(Sigma–Aldrich, India). The enzymatic reaction was initiated by adding
the substrate (sodium linoleate) to the preincubated mixture of the test
compounds and NDGA with soybean lipoxygenase. The in vitro IC50
values of the target compounds dissolved in DMSO was determined by
incubating with fixed concentration of soybean lipoxygenase (0.2 mL of
enzyme solution at 1/3 × 10−4 w/v in saline) at 28°C for 5 min and later
followed by addition of sodium linoleate (0.1 mM).
% Inhibition ¼ 100 ½1 ꢀ Vt=Vcꢁ
|
4.2.2 Determination of in vitro IC50 for soluble
where Vt and Vc are the volumes of edema for the drug treated and
control groups, respectively. Compounds 4a–o, 8a, and 8e were
screened for anti-inflammatory activity and the results are expressed
as the mean SEM of six animals per group. Statistical evaluation was
performed using analysis of variance followed by t-test for sub-group
comparison and p < 0.001 was considered significant. Statistical
analysis was performed using STATISTICA 6.1 for Windows.
epoxide hydrolase inhibitory activity[42]
The in vitro IC50 values of the target compounds on human
recombinant sEH were carried out using the screening assay kit
(Cayman Chemicals-Item No. 10011671), according to the instructions
provided with it. The in vitro study utilized (3-phenyl-oxiranyl)-acetic
acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME) as
the specific substrate and a potent sEH inhibitor, trans-4-[4-(3-
adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) as the
reference standard. Solutions of the test compounds were freshly
prepared in DMSO and the IC50 values of inhibitors were determined
after carrying out assays at 10 different concentrations of each
compound. Recombinant human sEH was incubated with the test
compounds and t-AUCB before addition of the substrate (PHOME).
Incubated for 15 min at 25°C in 25 mM Bis-Tris/HCl buffer at pH 7.0
(total assay volume = 200 μL), and the activity was determined by
monitoring the appearance of 6-methoxy-2-naphthaldehyde (emits
fluorescence at 465 nm) by fluorescence detection with an excitation
wavelength of 330 nm and an emission wavelength of 465 nm.
ACKNOWLEDGMENTS
Authors would like to acknowledge the President, Vice-President and
Secretary, Janatha Education Society®, Vivekananda College of Pharmacy,
Bangalore, for providingthe necessary facilities for carrying out thisresearch
work. We are pleased to acknowledge the Indian Institute of Science,
Bangalore for providing spectral data of our synthesized compounds.
CONFLICT OF INTEREST
Authors declare that they have no conflict of interest.

NANDHA ET AL.
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How to cite this article: Nandha B, Ramareddy SA, Kuntal H.
Synthesis of substituted fluorobenzimidazoles as inhibitors of
5-lipoxygenase and soluble epoxide hydrolase for anti-
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