Benzimidazole scaffold based hybrid molecules for various inflammatory targets: Synthesis and evaluation

Article abstract of DOI:10.1016/j.bioorg.2018.05.014

Designing of hybrid drugs with specific multitarget profile is a promising line of attack against inflammation. In light of this, a series of benzimidazole scaffold based hybrid molecules were designed by integrating benzimidazoles (containing pharmacophoric elements for COXs and LOXs inhibitors) with phthalimide subunit of thalidomide (pharmacophore element for TNF-α inhibitor) under one construct via molecular hybridization strategy. The designed molecules were synthesized and evaluated for their inhibitory activity against COXs (COX-1, COX-2), LOXs (5-LOX, 15-LOX) enzymes as well as TNF-α inhibitory effect. The results revealed that, compounds (3a–l) obtained showed inhibition in submicromolar range against COXs and LOXs targets whereas milder inhibitory activity was obtained against lipopolysaccharides (LPS)-induced TNF-α secretion by murine macrophage-like cells (RAW264.7). Within this class of compounds, 3j emerged as having alluring multiple inhibitory effects on set of COX-1/2 and 5-/15-LOX enzymes (COX-1 IC₅₀ = 9.85 μM; COX-2 IC₅₀ = 1.00 μM; SI = 9.85; 5-LOX IC₅₀ = 0.32 μM; 15-LOX IC₅₀ = 1.02 μM) in conjunction with a good anti-inflammatory and analgesic activities. Additionally, compound 3j showed gastrointestinal safety with reduced lipid peroxidation. Docking results of compound 3j with COX-2 and 5-LOX were also consistent with the in vivo anti-inflammatory results.

Full text of DOI:10.1016/j.bioorg.2018.05.014

Accepted Manuscript
Benzimidazole scaffold based hybrid molecules for various inflammatory tar-
gets: Synthesis and Evaluation
Gaganpreet Kaur, Om Silakari
PII:
S0045-2068(18)30215-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.014
YBIOO 2367
Reference:
Bioorganic Chemistry
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Accepted Date:
5 March 2018
16 May 2018
16 May 2018
Please cite this article as: G. Kaur, O. Silakari, Benzimidazole scaffold based hybrid molecules for various
inflammatory targets: Synthesis and Evaluation, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.05.014
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Benzimidazole scaffold based hybrid molecules for various
inflammatory targets: Synthesis and Evaluation.
Gaganpreet Kaur and Om Silakari*
*Corresponding Author- Molecular Modeling Lab (MML), Department of Pharmaceutical
Sciences and Drug Research, Punjabi University, Patiala. Punjab 147002, India.
E-mail: omsilakari@gmail.com, Telephone: +919501542696
1

ABSTRACT
Designing of hybrid drugs with specific multitarget profile is a promising line of attack
against inflammation. In light of this, a series of benzimidazole scaffold based hybrid
molecules were designed by integrating benzimidazoles (containing pharmacophoric
elements for COXs and LOXs inhibitors) with phthalimide subunit of thalidomide
(pharmacophore element for TNF-α inhibitor) under one construct via molecular
hybridization strategy. The designed molecules were synthesized and evaluated for their
inhibitory activity against COXs (COX-1, COX-2), LOXs (5-LOX, 15-LOX) enzymes as
well as TNF-α inhibitory effect. The results revealed that, compounds (3a-l) obtained showed
inhibition in submicromolar range against COXs and LOXs targets whereas milder inhibitory
activity was obtained against lipopolysaccharides (LPS)-induced TNF-α secretion by murine
macrophage-like cells (RAW264.7). Within this class of compounds, 3j emerged as having
alluring multiple inhibitory effects on set of COX-1/2 and 5-/15-LOX enzymes (COX-1
IC50=9.85µM; COX-2 IC50=1.00µM; SI=9.85; 5-LOX IC50=0.32µM; 15-LOX IC50=1.02µM)
in conjunction with a good anti-inflammatory and analgesic activities. Additionally,
compound 3j showed gastrointestinal safety with reduced lipid peroxidation. Docking results
of compound 3j with COX-2 and 5-LOX were also consistent with the in vivo anti-
inflammatory results.
Keywords: Anti-inflammatory, Benzimidazole scaffold, COXs, Hybrid drugs, LOXs,
Multitarget profile, TNF-α
2

1. Introduction
Despite years of studies and irrespective of long passage of anti-inflammatory drugs, efficacy
and safety of the drugs- a set of extremely important fundamental issues remains unresolved
[1]. The growing realization that inflammation is a complex, multifactorial and crucial in
many diseases opens up a whole new avenues for treatment [2]. Current research emphasizes
designing of hybrid drugs with a specific multi-target profile as the promising line of attack
against inflammation [3].
Demand for hybrid drugs has driven the pursuit of structures possessing multitargeted
aptitude. It is well documented that designing of novel drugs on “privileged scaffold” is one
of the successful directions in drug discovery. “Privileged scaffold” offered an optimal source
of core structure and presented remarkable capability of binding across multitudes of
therapeutically relevant biological targets [4]. Among the privileged scaffolds engaged in
drug designing, focus on benzimidozole nucleus is noteworthy. Above and beyond synthetic
versatility, benzimidazole scaffold endows its derivatives with diverse portfolio as of
inflammation targeted drugs by virtue of its inherent affinity against various inflammation
related targets. This inspiring biological background has raised lots of concerns on their
suitability as a viable scaffold for design of multitargeted anti-inflammatories [5]. On the
other hand thalidomide is another significant drug molecule noticeable for its well-known
inherent TNFα-inhibitor property liable for its distinguishing immunomodulatory and anti-
inflammatory properties. Structure activity studies, analogs and metabolites of thalidomide
now clearly disclosed that phthalimide subunit (pharmacophore) of thalidomide is essential
for its distinctive pharmacological functions whereas its glutarimide portion (toxicophore)
facilitates binding to the human cereblon gene (component of an E3 ubiquitin ligase
3

complex), which is a primary target protein responsible for thalidomide-mediated
teratogenicity [3,6].
Following the emerging trend of hybrid drugs and encouraged by biological background of
benzimidazole and thalidomide, the possibility of designing novel hybrid molecules was
therefore explored using privileged benzimidazole as a core scaffold, which combines, under
one construct, pharmacophoric elements that characterize well-known classes of inhibitors of
cyclooxygenase isoenzymes (COXs), lipoxygenase isoenzymes (LOXs) and tumor necrosis
factor-alpha (TNF-α) via molecular hybridization drug design strategy. A preview of the
overall design strategy illustrated in Figure 1. The pharmacophoric element needed for
cyclooxygenase and lipoxygenase inhibitors where they shares the same “privileged”
benzimidazole scaffold as putative binding motif integrated with phthalimide subunit of
thalidomide as pharmacophoric group for TNF-α inhibition. It was envisaged that such a
benzimidazole scaffold based hybrid molecules might therefore, endowed with an efficient
and safer anti-inflammatory action.
The relevant set of targets selected in this study, cyclooxygenases (COX- 1, COX-2), TNF-
α, and lipoxygenases (5-LOX, 15-LOX) were chosen for variety of reason. The first and
foremost, is all of them shares inflammation as the therapeutic niche. Secondly, cross-talks
(or communications) amongst inflammatory pathways may lead to strong pharmacodynamics
synergy between the elected targets. COXs and TNF-α were carefully selected set of primary
targets. The COXs are the principally involved and overly explored best known targets of
anti-inflammatory drugs to date [7], while TNF-α is a central key player in the initiation of
multiple inflammatory cascades [8]. Moreover because of suggested role of lipoxygenase in
the mechanism and safety of anti-inflammatory drugs [9] they are intentionally being elected
as key inflammatory target for the present study.
2. Results and discussion
4

2.1 Design rationale
The scientific rationale behind the design concept is the “synergistic benefit” that could be
achieved when COXs and LOXs inhibitory activities combined with the inhibition of TNF-α
in alleviating inflammation. Support for this concept provided by findings evolving around
the use of NSAIDs and COX-2 selective inhibitors. First is, NSAIDs and coxibs
preferentially decreased prostaglandin (PG) compared with thromboxane (TX) synthesis, and
this imbalance may create a persistent “rebound effect” on the proinflammatory signaling
hence increases the TNF-alpha production, an effect that is paradoxical and certainly
undesired for anti-inflammatory drugs [10]. Second is, selective inhibition of cyclooxygenase
(COX) pathway may also lead to a shunt of the arachidonic metabolism towards the other
untargeted lipoxygenase (LOX) pathway thus increasing the formation of proinflammatory
and gastrotoxic leukotriene’s [11].
A possible reason of these effects is redundancy and robustness in many biological networks
and pathways in which cells often find way to compensate for protein whose activity is
affected by a drug [12]. Moreover an ideal drug may be one whose efficacy is based not on
inhibition of a single target but rather on rebalancing of several proteins or events that
contributed to etiology, pathogenesis and progression of diseases. The described effects
support the need for paradigm shift in drug discovery from single disease target inhibitor
towards multi-target concept, which might favors improved safety and efficacy. It is
therefore seems reasonable that a balanced approach targeting selective inhibition of COX-2
combined with inhibition of LOXs and TNF-α could deliver superior therapeutic benefits in
treatment of inflammation. As of yet no such hybrid molecules holding anti-inflammatory
profile indicated in the literature.
2.2 Chemistry
5

The synthetic pathway for the target hybrid compounds is illustrated in Scheme 1(A). Scheme
1(A) describes the facile cyclisation reaction of 4-nitrobenzene-1,2-diamine with cyanogen
bromide in water to give desired 5-nitro-1H-benzimidazol-2-amine scaffold 1 [13], which
was transformed into corresponding 2-(5-nitro-1H-benzimidazol-2-yl)-1H-isoindole-1,3(2H)-
dione an intermediate hybridized structure 2 by means of phthaloylation reaction using o-
phthaloyl dichloride in the presence of pyridine [14]. The synthesis of target hybrid
molecules was achieved by cross coupling reactions. Among the available synthetic
approaches, we opted to begin with the N-arylation reactions driven by potassium
carbonate/N,N-dimethyformamide system in situ which led to the coupling of free (NH) site
in benzimidazole scaffold of intermediate hybridized structure 2 with different aryl halides as
their electrophilic coupling partner leading to formation of target hybrid compounds (3a-l)
[15]. Approximate presentation of what may happen depicted in Scheme 1(B). In Scheme
1(B), reaction (I) is deprotonation which involves generation of anionic nucleophile by
reaction of sodium carbonate and free (NH) site of benzimidazole in N,N-dimethylformamide
as solvent. In order to balance charge, the in situ generated anionic nucleophile further
interact with floating potassium ion and converted into the corresponding potassium salts.
While reaction (II) is expected to be classic SN2 substitution in Scheme 2.
2.3 Biological evaluation
2.3.1 In vitro COX-1 and COX-2 inhibitory assay
Target compounds 3a-l were tested for COX-1 and COX-2 inhibition at five concentrations
(0.01, 0.1, 1, 10 and100µM) to determine the concentration produced 50% inhibition of
COX-1 and COX-2 enzymes (IC50 values) and their selectivity indices (SI= IC50COX-1/
IC50COX-2) using indomethacin and celecoxib as reference drugs. The results recorded in
Table 1 indicated that these compounds exhibit broad range of COX-1/2 activities (COX-2
IC50=1.0 to >100µM range; COX-1 IC50=7.0 to >100µM range). Since both isoforms (COX-1
6

and COX-2) are structurally quite similar (>50% sequence homology) their three-
dimensional structures are almost superimposable and they also share common binding
partner(substrate), accordingly results further explicitly indicated that some compounds are
more active on COX-1 and others are on COX-2. Among them, methoxy, phenethyl,
isopropyl, and nitro substituted compounds (3b, 3f, 3h, and 3k) provided much weaker COX
inhibition (COX-1 IC50= >15µM; COX-2 IC50= >100µM). However for the methyl
substituted compounds, COX-2 potency order was 3d > 3e >3c, it appeared that m- position
of the methyl (3d) at peripheral phenyl group were more favourable than o- and p- positions.
Compound 3c, which provided a potent COX-1 inhibition (COX-1 IC50= 7.76µM) among
series, on the other hand did not inhibit COX-2 at concentration of 10µM. Comparison of 3h
(COX-1 IC50= 17µM; COX-2 IC50=>100µM) with that of 3i (COX-1 IC50= 15.99µM; COX-2
IC50=16.07µM) revealed that presence of p-position of tert-butyl group provided an optimal
combination of in vitro COX-1/COX-2 inhibitory effects hence seems to be more favourable
than isopropyl group. Compounds 3j displayed significant COX-2 inhibitory activity (COX-2
IC50 = 1.00 µM; COX-1 IC50 = 9.85 µM), and it was 4-fold more potent compared to
reference drug indomethacin (COX-2 IC50 = 4.02 µM) although less potent than celecoxib
(COX-2 IC50 = 0.04 µM). It appeared that introduction of phenyl substituent at the m-position
of peripheral phenyl ring (3j) was most favourable for the cyclooxygenase inhibition. It is
notable that substituting a phenylethyl group (3f) for a 3-phenylbenzyl (3j) resulted lesser
potent inhibitor of COX-2(IC50=29.73 µM). In terms of selectivity index (SI), compound 3j
showed modest COX-2 selectivity (SI=9.85) than the reference drug celecoxib although put
on show good selectivity when compared to its structural analogue indomethacin, an indole
based NSAIDs (SI=0.08) as shown in Table 1.
2.3.2 In vitro 5-LOX and 15-LOX inhibitory assay
7

Target compounds 3a-l were tested for human 5-LOX and 15-LOX inhibition at five
concentrations (0.01, 0.1, 1, 10 and 100µM) to determine the concentration produced 50%
inhibition of 5-LOX and 15-LOX enzymes (IC50 values) using reductive inhibitor
nordihydroguiarectic acid (NDGA) as reference drug. The results reported in Table 1
indicated that all the target compounds had more potential in inhibitory human 5-LOX (IC50
value range from 0.32µM to >5µM) compared to 15-LOX (IC50 value range from 1.02 to
>5µM). The 5-LOX inhibitory potency was of the order 3j > 3a > 3e >3b >3h > 3d > 3k > 3f
>3l >3g >3c> 3i.Within subgroup of methoxy substituted compounds lipoxygenase potency
order was 3a>3b. Incorporation of isopropyl group (3g) resulted in potent 15-LOX inhibition
(15-LOX IC50= 1.76 µM) although it was less potent than reference drug NDGA. As
indicated, compound 3j possessed the most potent LOX inhibitory activity and the moderate
subtype selectivity for 5-LOX (3-fold selectivity versus 15-LOX). Compound 3j was 1.5 fold
more potent inhibitor of 5-LOX than the reference drug NDGA (5-LOX IC50= 0.52µM). It
appeared that presence of the biphenyl ring (3j) was most favourable for the dual 5/15-
lipoxygenase inhibition. Also compounds 3h and 3j (15-LOX IC50= 1.05 µM and 1.02 µM
respectively) were found to be slightly more potent than that of NDGA (15-LOX
IC50=1.10µM) suggesting that these compounds could act as antioxidants. Additionally,
compound 3j exhibited significant dual COX/LOX inhibition over the reference drugs.
2.3.3 In vitro TNF-α inhibitory assay
Target compounds 3a-l were tested against LPS-induced TNF-α secretion in mouse
RAW264.7 macrophages at different concentrations to determine the concentration produced
50% inhibition of TNF-alpha (IC50 values) using dexamethasone as reference drug. The
results of biological activity of all target compound have been presented in Table 1. It was
observed that a majority of the target compounds of this series demonstrated weak inhibitory
activities as compared to the standard dexamethasone while few of them (3f, 3g) found to
8

inactive even at higher tested concentration(>500 µM). However, single compound 3j among
the series, in particular, showed IC50 value of 461µM against TNF-alpha inhibitory activity.
2.3.4 In vivo assay
2.3.4.1 Carragennan-induced paw edema model
Carrageenan test is greatly sensitive to clinically useful steroidal and nonsteroidal anti-
inflammatory drugs and has been widely accepted as a useful model to measure anti-
inflammatory drugs. Carrageenan injection generated intense inflammation (edema as being
the principal symptom) which peaked between 3-5 hours and is attributed to release of
inflammation related mediators (e.g. prostaglandins, leukotrienes, cytokines and nitric oxide),
which is the moment when its maximum effect is demonstrated and the moment when the
anti-inflammatory effect of the test product is best observed. The pharmacological results
listed in Table 2(see also Table 2S, supplementary data for detail) and illustrated in Fig. 2
represents the mean changes in paw edema volume mL±SD of animals pretreated with the
reference drugs and test compound 3j after 3h and 5h from the induction of inflammation,
together with the percent inhibition of induced rat paw edema by the test compound (percent
anti-inflammatory activity). Statistical differences of control, reference and test groups were
carried out using F test (ANOVA) followed by post hoc test. The screened results revealed
that, the strong inhibition of edema was observed after 3 h. The tested compound 3j showed
significant anti-inflammatory activity (% edema reduction=81.88) comparable to that of
celecoxib (87.59% edema reduction) whereas higher than indomethacin (79.41% edema
reduction). The significant inhibition of edema of 3j coupled with its marked in vitro
inhibitory effects against cyclooxygenases, lipoxygenases and TNF-α suggested an obvious
mechanism of anti-inflammatory action.
2.3.4.2 Analgesic activity
9

Sodium chloride-induced writhings was significantly reduced in rats receiving the test
compound 3j, indicating peripheral analgesic effect. The analgesic activity of the compounds
was done at the same dose as used for anti-inflammatory activity. The positive controls,
indomethacin and celecoxib, inhibited the writhing response by 76.28% and 78.57%
respectively whereas the analgesic effect of 3j was 77.14% at 3hr post drug administration
(Table 3).
2.3.4.3 Gastric ulcerogenic activity
The compound (3j), which was screened for analgesic activity, further screened for their
acute ulcerogenic risk. In the indomethacin induced ulcerated control group, the severity
index was 5.33±0.47 which was significantly reduced by the newly synthesized compound 3j
as shown in Table 3. Compound 3j showed severity index of 1.16, which is less than one-
fourth of the value of indomethacin hints at that compound 3j is relatively selective for COX-
2.
2.3.4.4 Lipid peroxidation
Compound 3j showing significantly reduced gastric mucosal lesions is also reported to show
reduced gastric MDA content, an index of lipid peroxidation-mediated tissue damage.
Therefore, by determining the MDA content it can be make certain that the compound is
actually lesser toxic to gastric mucosa. The lipid peroxidation was measured as nmoles of
MDA/100mg of tissue. Animals treated with indomethacin exhibited 2.89, whereas control
group showed 1.09 and the groups treated with synthesized compounds showed lipid
peroxidation content of 1.17(Table 3), suggesting that newly synthesized compound 3j results
in relatively lesser gastric mucosal injury.
2.4 Structure-activity correlation
Careful inspection of structures of the tested compounds revealed that central core
(benzimidazole scaffold) might contribute largely to the interaction with the amino acid
10

residues of the enzyme’s active site and is mainly responsible for projecting the
pharmacophoric elements in correct orientation for the efficient binding. In general
substantial part of the compounds obtained showed dual inhibition of both COX-1/2 and
5/15-LOX enzymes whereas weak inhibitory activity was obtained against LPS-induced
TNF-α secretion. The in vitro data acquired for this class of compounds further indicated that
COX and LOX inhibition can be manipulated by varying the substituents attached at N-1
position (D framework, see Fig.1) of target compounds but virtually on the TNF-α inhibitory
activity no significant impact was observed. It is also notable that replacement of benzyl
group in the D framework of the target compound (see Fig. 1 and Table 1) by phenylethyl
group as represented by 3f provided weak COX-2 inhibition and moderate COX-2 selectivity
(COX-2 IC50=29.73µM; SI=15.2) while retaining 5- and 15-LOX inhibitory activity and led
to complete loss in TNF-α inhibitory activity. Individual compound 3j showed significant
dual COX/LOX inhibition (COX-1 IC50=9.85µM, COX-2 IC50=1.00µM; SI=9.85; 5-LOX
IC50 = 0.32 µM; 15-LOX IC50 = 1.02 µM) along with milder TNF-α inhibitory activity for
which the presence of 3-phenylbenzyl group at the N-1 position, electron withdrawing group
i.e. nitro substituent at C-5 position of benzimidazole scaffold observed to be beneficial
features clearly.
2.5 Anti-inflammatory docking study
All the designed compounds were docked into the active sites of COX-1, COX-2, 5-LOX and
15-LOX. Results of molecular docking analysis indicate that all the designed and
subsequently synthesized compounds especially 3j, were having equally good binding
affinity towards COX-2, 5-LOX and 15-LOX, while 3c showed significant binding affinity in
COX-1. The binding energy of docked compounds towards COX-2 was found to be between
−24.56 and −31.94 kcal/mol, in COX-1, it ranged from −20.73 and −47.99 kcal/mol, in 5-
LOX, the binding energy ranged between −52.96 and −56.77 kcal/mol and in 15-LOX, it was
11

found between -25.63 and 57.68 kcal/mol. The most potent compound 3j revealed by
biological evaluation showed good binding energy among the docked compounds with the
score of −31.94 kcal/mol towards COX-2, −56.77 kcal/mol towards 5-LOX and 57.68
kcal/mol towards 15-LOX. This binding energy score reflects the affinity of 3j towards both
COX-2 and 5-LOX. While the most potent compound against COX-1, 3c showed good
binding energy among the docked compounds with the score of −44.02 kcal/mol towards
COX-1
Analysis of docked complex of 3j and COX-2 proved that the nitro group of 3j plays a
crucial role in COX-2 inhibition by interacting with both Phe-518 and Arg-513 (Fig. 3).
Following the designing of the molecules, the biphenyl rings formed π-π interactions with
Tyr-355 and Arg-120, respectively. While, analysis of docked complex of 3j and 5-LOX
showed significance of nitro group for 5-LOX inhibition also. The nitro group formed
interactions with His-432 present in the active site of 5-LOX. Moreover, nitrogen of
benzimidazole nucleus forms hydrogen bonding with Gln-557. Complying with the π-π
interactions in COX-2, in 5-LOX also biphenyls formed π-π interaction with His-600 and an
additional π-π interaction formed between benzimidazole ring and His-367(Fig. 4). Similarly,
analysis of docked complex of 3j and 15-LOX showed several π-π interactions between fused
aryl ring of benzimidazole and Val-348, and between aryl ring of thalidomide and Val-116.
Additionally, the nitro group formed another π-π interactions with Tyr-348 present in the
active site of 15-LOX (Fig. 5). Finally, most significant COX-1 inhibitor, 3c showed
hydrogen bond between N of benzimidazole and Ser-510 (Fig. 6). Additionally, another
hydrogen bond interaction was observed between nitro group and Ile857. While the
thalidomide ring formed the hydrophobic interactions with the catalytic domain to improve
the overall binding affinity.
3. Conclusion
12

The present study shows that the synthesized benzimidazole scaffold based hybrids
represents promising multitargeting potential by influencing various inflammation-related
targets. E.g., COX-1, COX-2, 5-LOX, 15-LOX and TNF-α. Compound 3j showed potent
dual inhibition of both COX-2 and 5-LOX enzymes was also screened for its in vivo anti-
inflammatory activity, analgesic (algesia being the main symptom of inflammation) and
ulcerogenic and lipid peroxidation activities. The results revealed that it showed significant
decrease in carrageenan-induced paw edema in conjunction with good analgesic activity.
Besides, compound 3j offered gastrointestinal safety with reduced lipid peroxide profile.
Biological results was also consistent with the docking studies in the active sites of the target
enzymes (COX-2 and 5-LOX). Accordingly results of the study concluded that observed
combination of COX-2 selectivity and 5-LOX inhibition in 3j may be responsible for the
efficient anti-inflammatory action and high gastrointestinal safety and thus supports the
prevailing hypothesis.
4. Experimental section
4.1 Chemistry
All chemicals and solvents required for synthesis were procured commercially from various
suppliers (Sigma, Merk and Loba) and were of LR grade, used without any purification. The
solvents were dehydrated according to the standard methods. The synthesis was carried out
using steam bath, magnetic stirrer and hot plate (Perfit), and solvents were recovered using
rotary vacuum evaporator (Perfit). The completion of each reaction was monitored by thin
layer chromatography (DC-Alufolien (20x20 cm) Kieselgel 60 F254 chromato plates) using
hexane: ethylacetate (7:3 v/v) and chloroform: methanol (9.6:0.4 v/v) as a TLC development
solvent system and visualized in UV chamber (Perfit) at short as well as long wavelengths.
Impure compounds and intermediates were purified on silica columns from appropriate
solvent. Compounds were purified by silica columns (100-200mesh) or recrystallization
13

technique. The melting point were recorded in open glass capillaries with electrical melting
1
point apparatus and uncorrected. H-NMR spectra were recorded on a Bruker Avance II 400
MHz NMR spectrometer using DMSO-d6 (or in CDCl3) as solvent and tetramethylsilane
(TMS) as an internal standard. Proton chemical shifts are expressed in parts per million
13
(ppm). C NMR spectra were recorded on same spectrometer operating at 400 MHz. IR
spectra were measured with Perkin Elmer RZX FT-IR and Bruker (Alpha E) FT-IR
spectrometer. IR peaks were recorder at cm-1 scale. Mass spectra were recorded on Waters Q-
TOF micro MS spectrometer at positive ionization mode (ESI+). The MS peaks were
recorded as m/z ratio and corrected, using which candidate structures, their relative
abundances were assessed.
Compounds 1[13], 2 [14] and 3a-l [15] were prepared according to reported procedures (see
the Table 1S (Supplementary data) for the physicochemical properties and TLC data of
compounds 3a-l).
4.1.1. Synthesis of 5-Nitro-1H-benzimidazol-2-amine (1)
A suspension of 4-nitro-o-phenylenediamine (1.4g, 9.1mmol) in a solution of BrCN (0.97g,
9.2mmol) in water (30ml) was heated under reflux for 7hr, cooled and neutralized with 25%
NH4OH to pH 10-11. The formed precipitate was then filtered, washed with water, air-dried
and recrystallized from hot water. The title compound was obtained as an oranges yellow
º
1
shiny crystals. Yeild=1.28g (91.4%); mp. 245-247 C. H-NMR (400 MHz, DMSO-d6, δ
ppm): 8.10 (d, 1H, J=2.2Hz, ArH), 7.90 (dd, 1H, J = 8.70, 2.2 Hz, ArH), 7.20 (d, 1H, J = 8.7
Hz, ArH), 6.90 (s, 2H, NH2). 13C-NMR (400 MHz, DMSO-d6, δ ppm): 158.91, 139.92,
136.67, 116.50, 111.16, 106.16. IR (KBr, cm-1): 3514, 3457 (N-H stretching), 3092 (C-H
aromatic stretching), 1658(C=N ring stretching), 1587, 1507 (skeletal bands), 1421
(asymmetric N=O stretching), 1471 (N-H scissoring), 1336 (symmetric N=O stretching), 876
14

(N-H wagging). MS (+ESI-QTOF): m/z calculated for C7H6N4O2 [M+H]+ 179.14, found
179.1(100%).
4.1.2. Synthesis of 2-(5-Nitro-1H-benzo[d]imidazol-2-yl)isoindoline-1,3-dione (2)
5-Nitro-1H-benzimidazole-2-amine (5.11g, 0.028mmole) is dissolved in 35ml of anhydrous
pyridine, and phthaloyl chloride (5.81g, 0.034mmole) is added causing immediate
precipitation. On mixing and heating under reflux for 1.5h, all dissolves. After cooling,
addition of water (42ml) yielded the desired product as a shiny light yellow powder which
º
can used in the next step without further purification. Yield=4.25g(83.1%); mp. 266-268 C.
1H-NMR (400 MHz, DMSO-d6, δ ppm): 8.57 (d, 1H, J=2.16Hz, H-4), 8.19 (dd, 1H, J = 8.90,
2.4 Hz, H-6), 8.09 (m, 2H, H-1’, H-4’), 7.97 (m, 2H, H-2’, H-3’), 7.80(d, 1H, J=9.40Hz, H-
7). 13C-NMR (400 MHz, DMSO-d6, δ ppm): 168.85, 165.60, 153.66, 123.17, 123.31, 115.20.
IR (KBr, cm-1): 3386 (N-H stretching), 3106 (C-H aromatic stretching), 1793, 1728 (N-C=O
imide stretching), 1626 (C=N ring stretching), 1423(asymmetric O-N=O stretching),
1344(symmetric O-N=O stretching), 1599, 1474 (skeletal bands). MS (+ESI-QTOF): m/z
calculated for C15H8N4O4 [M+H]+ 309.24, found 309.2(25.0%).
4.1.3 General procedure for the synthesis of target compounds (3a-l): N-Arylation of free
(NH) site in benzimidazole scaffold of intermediate hybridized structure 2.
The target compounds were prepared following the literature method. Briefly, mixture of
intermediate hybridized structure 2 (1 equiv) and potassium carbonate (2 equiv) in N,N-
dimethylformamide (10mL/mmol) was treated in one portion with the appropriate aryl halide
(2 equiv). The reaction mixture was heated under reflux. The reactions usually completed
within 2–4ꢀh (monitored by TLC). After the completion of reaction (as evidenced by TLC),
reaction mixture cooled to room temperature, and partitioned between ethyl acetate and
water. The organic layer was washed with water and brine, dried over sodium sulphate, and
concentrated in vacuo. The residue was subjected to silica gel chromatography to give the
15

desired target compound. The compounds prepared according to this procedure are as
follows:
4.1.3.1 2-(1-(3-Methoxybenzyl)-5-nitro-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3a)
Compound 3a was prepared from 2 and 3-methoxybenzyl chloride as a brownish yellow
1
powder. Yield= (0.95g) 65.5%; mp. 242-244ºC. H-NMR (400 MHz, DMSO-d6, δ ppm):
7.96 (d, 1H, J=2.16Hz, H-4), 7.82 (dd, 1H, J=8.68, 2.20 Hz, H-6), 7.23-7.19 (m, 2H, H-1’, H-
4’), 7.09 (s, 2H, H-2’, H-3’), 6.82-6.79(m, 3H, H-1’’, H-3’’and H-4’’), 6.73(d, 1H, J=7.68,
H-5’’), 5.31 (s, 2H, benzylic protons, CH2). 3.62(s, 3H, OCH3). 13C-NMR (400 MHz,
DMSO-d6, δ ppm): 158.66, 157.71, 142.85, 141.92, 139.49, 128.48, 128.38, 128.18, 114.72,
114.03, 109.50, 107.58, 55.03, 44.49. IR (KBr, cm-1): 3108 (C-H aromatics stretching), 2837,
2737(C-H stretching), 1773, 1723 (N-C=O imide stretching), 1658(C=N ring stretching),
1431 (asymmetric O-N=O stretching), 1600, 1553, 1462 (skeletal bands), 1398 (symmetric
O-N=O stretching), 1254, 1036 (C-O-C stretching). MS (+ESI-QTOF): m/z calculated for
C23H16N4O5 [M+H]+ 429.39, found 429.3(20.0%).
4.1.3.2 2-(1-(4-Methoxybenzyl)-5-nitro-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3b)
Compound 3b was prepared from 2 and 4-methoxybenzyl chloride as a brownish yellow
powder. Yield= (0.92) 63.4%; mp. 231-233ºC. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 7.94
(d, 1H, J=2.24Hz, H-4), 7.82 (dd, 1H, J=8.68, 2.20 Hz, H-6), 7.24-7.22 (m, 1H, H-7), 7.19-
7.17 (m, 3H, H-1’, H-4’, H-2’), 7.09(s, 2H, H-1’’, H-5’’), 6.89-6.85(m, 2H, H-2’’, H-4’’),
13
5.30 (s, 2H, benzylic protons, CH2). 3.70(s, 3H, OCH3). C-NMR (400 MHz, DMSO-d6, δ
ppm): 159.37, 157.78, 142.84, 142.01, 139.56, 137.83, 129.85, 118.88, 114.80, 113.10,
112.51, 109.86, 107.56, 55.00, 44.91. IR (KBr, cm-1): 3018 (C-H aromatics stretching), 2837,
2738(C-H stretching), 1779 (N-C=O imide stretching), 1663(C=N ring stretching), 1430
(asymmetric O-N=O stretching), 1618, 1600, 1480 (skeletal bands), 1329 (symmetric O-N=O
16

stretching), 1256, 1069 (C-O-C stretching). MS (+ESI-QTOF): m/z calculated for
C23H16N4O5 [M+H]+ 429.397, found 429.3(10.0%).
4.1.3.3 2-(1-(2-Methylbenzyl)-5-nitro-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3c)
Compound 3c was prepared from 2 and 2-methylbenzyl chloride as dark brown powder.
1
Yield= (0.98) 68.2%; mp. 126-128ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 8.00 (d, 1H,
J=2.2Hz, H-4), 7.80 (dd, 1H, J=8.7, 2.2 Hz, H-6), 7.25-7.23 (m, 1H, H-7), 7.18-7.12 (m, 1H,
H-1’), 7.09-7.10(m, 3H, H-2’, H-3’, H-2’’), 7.04(t, 1H, J=7.2, H-3’’), 6.27(d, 1H, J=7.2, H-
5’’), 5.36 (s, 2H, benzylic protons, CH2). 2.37(s, 3H, CH3). 13C-NMR (400 MHz, DMSO-d6,
δ ppm): 158.06, 142.90, 142.03, 139.80, 135.49, 134.02, 132.23, 127.04, 125.95, 123.97,
114.75, 109.57, 107.56, 43.48, 18.63. IR (KBr, cm-1): 3065 (C-H aromatics stretching), 2762
(C-H stretching), 1661 (N-C=O imide stretching), 1477 (asymmetric O-N=O stretching),
1616(C=N ring stretching), 1549, 1408 (skeletal bands), 1321 (symmetric O-N=O
stretching). MS (+ESI-QTOF): m/z calculated for C23H16N4O5 [MH+Li]+ 419.39, found
419.3(80.0%).
4.1.3.4 2-(1-(3-Methylbenzyl)-5-nitro-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3d)
Compound 3d was prepared from 2 and 3-methylbenzyl chloride as brown powder. Yield=
1
(0.98g) 67.5%; mp. 145-147ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 7.96 (d, 1H,
J=2.24Hz, H-4), 7.84 (dd, 1H, J=8.68, 2.24 Hz, H-6), 7.26-7.14 (m, 3H, H-7, H-1’, H-4’),
7.15 (s, 2H, H-2’, H-3’), 7.09(d, 1H, J=7.60, H-3’’), 7.03(s, 1H, H-1’’), 6.97(d, 1H, J=7.68,
H-5’’), 5.03 (s, 2H, benzylic protons, CH2). 2.25(s, 3H, CH3). 13C-NMR (400 MHz, DMSO-
d6, δ ppm): 157.80, 142.84, 141.98, 139.58, 137.83, 136.83, 128.58, 128.26, 127.42, 127.30,
123.94(2C), 114.80, 109.54, 107.53, 44.97, 20.98. IR (KBr, cm-1): 3063 (C-H aromatics
stretching), 2837, 2738(C-H stretching), 1776, 1727 (N-C=O imide stretching), 1660(C=N
ring stretching), 1450 (asymmetric O-N=O stretching), 1603, 1555, 1479 (skeletal bands),
17

1321 (symmetric O-N=O stretching). MS (+ESI-QTOF): m/z calculated for C23H16N4O5
[M+K]+ 451.29, found 451.0.
4.1.3.5 2-(1-(4-Methylbenzyl)-5-nitro-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3e)
Compound 3e was prepared from 2 and 4-methylbenzyl chloride as brownish orange powder.
1
Yield= (1.00g) 68.9%; mp. 135-137ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 7.94 (d, 1H,
J=2.24Hz, H-4), 7.83 (dd, 1H, J=8.68, 2.24 Hz, H-6), 7.40 (s, 1H, H-7), 7.26-7.20 (m, 2H, H-
1’, H-4’), 7.16-7.09(m, 6H, H-2’, H-3’, H-1’’, H-3’’, H-4’’, H-5’’), 5.30 (s, 2H, benzylic
13
protons, CH2). 2.20(s, 3H, CH3). C-NMR (400 MHz, DMSO-d6, δ ppm):157.78, 142.83,
141.95, 139.53, 133.24, 129.22, 129.18, 126.95, 126.86, 114.76, 109.51, 107.56, 44.78,
20.60. IR (KBr, cm-1): 3091 (C-H aromatics stretching), 2737(C-H stretching), 1777, 1721
(N-C=O imide stretching), 1661(C=N ring stretching), 1452 (asymmetric O-N=O stretching),
1600, 1553, 1480 (skeletal bands), 1324 (symmetric O-N=O stretching). MS (+ESI-QTOF):
m/z calculated for C23H16N4O5 [M+H]+ 413.39, found 413.0(0.9%).
4.1.3.6 2-(5-Nitro-1-phenethyl-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3f)
Compound 3f was prepared from 2 and 2-phenylethylbenzyl chloride as brown powder.
1
Yield= (0.95g) 65.5%; mp. 258-260ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 8.00 (d, 1H,
J=2.16 Hz, H-4), 7.77 (dd, 1H, J=8.64, 2.20 Hz, H-6), 7.24-7.14 (m, 7H, H-7, H-1’, H-2’, H-
3’, H-4’, H-2’’, H-4’’), 7.09-7.07 (m, 1H, H-3’’), 6.98(s, 2H, H-1’’, H-5’’), 4.30 (t, 2H,
13
J=7.68Hz, CH2, H-6’’, H-7’’). 2.97(t, 2H, J=7.64, CH2, H-8’’, H-9’’). C-NMR (400 MHz,
DMSO-d6, δ ppm): 158.74, 157.37, 149.33, 142.53, 141.70, 139.42, 137.73, 137.63, 133.64,
128.87, 128.12, 128.07, 126.34, 126.29, 117.53, 114.40, 113.18, 109.50, 106.76, 103.49,
43.29, 34.14. IR (KBr, cm-1): 3068 (C-H aromatics stretching), 2743(C-H stretching), 1768
(N-C=O imide stretching), 1665(C=N ring stretching), 1479(C-H methylene bending), 1461
(asymmetric O-N=O stretching), 1598, 1554, 1400(skeletal bands), 1329 (symmetric O-N=O
stretching). MS (+ESI-QTOF): m/z calculated for C23H16N4O5 [M+K]+ 450.39, found 450.2.
18

4.1.3.7 2-(5-Nitro-1-(4-vinylbenzyl)-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3g)
Compound 3g was prepared from 2 and 4-vinyllbenzyl chloride as brownish orange powder.
1
Yield= (1.03g) 71.0%; mp. 179-181ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 7.97 (d, 1H,
J=2.08Hz, H-4), 7.82 (dd, 1H, J=8.68, 2.00Hz, H-6), 7.42-7.35 (m, 3H, H-7, H-1’, H-4’),
3
7.21-7.16 (m, 3H, H-2’, H-3’, H-2’’), 7.10(s, 2H, H-1’’, H-5’’), 6.67 (dd, 1H, Jtrans=17.60,
3Jcis=10.92 Hz, vinylic proton, H-1’’’), 5.76(d, 1H, J=17.64, vinylic proton, H-2’’’), 5.36(s,
2H, benzylic protons, CH2), 5.23(d, 1H, J=10.96, vinylic proton, H-3’’’). 13C-NMR (400
MHz, DMSO-d6, δ ppm): 157.73, 142.85, 142.06, 139.44, 136.43, 135.97, 135.73, 127.10,
127.02, 126.51, 126.25, 114.67, 114.21, 113.49, 107.27, 103.75, 103.75, 44.88. IR (KBr, cm-
1): 3086 (C-H aromatics stretching), 2743(C-H stretching), 1777 (N-C=O imide stretching),
1660(C=N ring stretching), 1629 (C=C vinylnic stretching), 1454 (asymmetric O-N=O
stretching), 1600, 1553, 1479(skeletal bands), 1325 (symmetric O-N=O stretching), 994, 918
(C-H out of plane bending). MS (+ESI-QTOF): m/z calculated for C24H16N4O4 [M+H]+
425.40, found 425.0(4.2%).
4.1.3.8
2-(1-(4-Isopropylbenzyl)-5-nitro-1H-benzo[d]imidazol-2-yl)isoindoline-1,3-dione
(3h)
Compound 3h was prepared from 2 and 4-isopropylbenzyl chloride as yellow powder. Yield=
1
(1.00g) 68.9%; mp. 265-267ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 8.00 (d, 1H, J=2.2
Hz, H-4), 7.82 (dd, 1H, J=8.6, 2.2 Hz, H-6), 7.20-7.10 (m, 7H, H-7, H-1’, H-4’, H-2’, H-3’,
H-2’’, H-4’’), 6.96 (s, 2H, H-1’’, H-5’’), 5.30 (s, 2H, benzylic protons, CH2), 2.80 (sep, 1H,
J=6.90 Hz, isopropyl methine proton), 1.20(d, 6H, J=6.88Hz, isopropyl methyl protons). 13C-
NMR (400 MHz, DMSO-d6, δ ppm): 157.62, 147.74, 142.72, 142.08, 139.34, 133.16,
126.59, 126.51, 126.34, 114.65, 109.83, 106.94, 44.98, 33.12, 23.64. IR (KBr, cm-1): 3090
(C-H aromatics stretching), 2965, 2870(C-H stretching), 1765 (N-C=O imide stretching),
1660 (C=N ring stretching), 1431 (asymmetric O-N=O stretching), 1322, 1301 (C-H gem-
19

dimethyl bending), 1617, 1550 (skeletal bands). MS (+ESI-QTOF): m/z calculated for
C25H20N4O4 [M+H]+ 441.44, found 441.0(3.3%).
4.1.3.9 2-(1-(4-(tert-Butyl) benzyl)-5-nitro-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione
(3i)
Compound 3i was prepared from 2 and 4-tert-butyl benzyl chloride as dark yellow powder.
Yield= (1.01g) 70.0%; mp. 189-191ºC. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 7.95 (d, 1H,
J=2.20 Hz, H-4), 7.82 (dd, 1H, J=8.68, 2.2 Hz, H-6), 7.33-7.31 (m, 3H, H-1’, H-4’, H-2’’),
7.23(d, 1H, J=8.68, H-7), 7.11-7.13 (m, 2H, H-2’, H-3’), 7.05 (s, 2H, H-1’’, H-5’’), 5.30 (s,
2H, benzylic protons, CH2), 1.20(s, 9H, tert-butyl). 13C-NMR (400 MHz, DMSO-d6, δ ppm):
157.74, 149.91, 142.85, 142.02, 140.56, 139.51, 133.16, 126.52, 126.44, 125.26, 114.65,
109.61, 107.29, 44.73, 34.11, 31.00. IR (KBr, cm-1): 3092 (C-H aromatics stretching), 2959,
2743(C-H stretching), 1773 (N-C=O imide stretching), 1661(C=N ring stretching), 1431
(asymmetric O-N=O stretching), 1383, 1336 (C-H tert-butyl bending), 1601, 1553, 1458
(skeletal bands). MS (+ESI-QTOF): m/z calculated for C26H22N4O4 [M+H]+ 455.45, found
455.0(4.2%).
4.1.3.10 2-(1-([1,1'-Biphenyl]-3-ylmethyl)-5-nitro-1H-benzo[d]imidazol-2-yl)isoindoline-1,3-
dione (3j)
Compound 3j was prepared from 2 and 3-phenylbenzyl chloride as yellow brownish powder.
1
Yield= (1.02g) 70.3%; mp. 247-249ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 7.98 (d, 1H,
J=2.2 Hz, H-4), 7.84 (dd, 1H, J=8.68, 2.2 Hz, H-6), 7.57-7.52 (m, 5H, H-7, H-1’, H-4’, H-2’,
H-3’), 7.45-7.39 (m, 4H, H-1’’, H-3’’, H-4’’, H-5’’), 7.37-7.34 (m, 2H, H-1’’’, H-5’’’), 7.30-
7.27 (m, 1H, H-3’’’), 7.15-7.13(m, 2H, H-2’’’, H-4’’’), 5.50 (s, 2H, benzylic protons, CH2).
13C-NMR (400 MHz, DMSO-d6, δ ppm):157.66, 142.85, 142.12, 140.63, 139.81, 139.46,
136.84, 129.13, 128.75, 127.42, 126.55, 125.86, 125.71, 125.62, 125.49, 114.72, 109.14,
107.27, 45.17. IR (KBr, cm-1): 3063 (C-H aromatics stretching), 2739(C-H stretching), 1778
20

(N-C=O imide stretching), 1661(C=N stretching), 1445 (asymmetric O-N=O stretching),
1599, 1511, 1455 (skeletal bands), 1322 (symmetric O-N=O stretching). MS (+ESI-QTOF):
m/z calcd for C28H18N4O4 (Mꢀ+H)+ 475.46, found 475.1(3.3%).
4.1.3.11 2-(5-Nitro-1-(4-nitrobenzyl)-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3k)
Compound 3k was prepared from 2 and 4-nitrobenzyl chloride as brownish orange powder.
1
Yield= (0.84g) 57.9%; mp. 188-190ºC. H-NMR (400 MHz, DMSO-d6, δ ppm): 8.20-8.15
(m, 3H, H-2’’, H-4’’, H-7), 8.00(1H, d, J=2.16, H-4), 7.82 (dd, 1H, J=8.64, 2.20 Hz, H-6),
7.42-7.40 (m, 3H, H-1’, H-4’, H-2’), 7.21-7.18(m, 1H, H-3’), 7.11 (s, 2H, H-1’’, H-5’’), 5.53
13
(s, 2H, benzylic protons, CH2). C-NMR (400 MHz, DMSO-d6, δ ppm): 157.57, 146.85,
143.75, 142.88, 142.30, 139.20, 127.79, 123.57, 114.75, 109.88, 107.04, 44.61. IR (KBr, cm-
1): 3063 (C-H aromatics stretching), 2754(C-H stretching), 1664 (N-C=O imide stretching),
1475 (asymmetric O-N=O stretching), 1618 (C=N stretching) 1554, 1503 (skeletal bands),
1324 (symmetric O-N=O stretching). MS (+ESI-QTOF): m/z calculated for C22H13N5O6
[M+H]+ 444.36, found 444.1(2.5%).
4.1.3.12 2-(5-Nitro-1-(2-nitrobenzyl)-1H-benzo[d]imidazol-2-yl) isoindoline-1,3-dione (3l)
Compound 3l was prepared from 2 and 2-nitrobenzyl chloride as a yellowish brown powder.
Yield= (0.91g) 62.7%; mp. 210-212ºC. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 8.18-7.92(m,
2H, H-4, H-2’’), 7.84 (dd, 1H, J=8.64, 2.26Hz, H-6), 7.50-7.52(m, 1H, H-7), 7.37-7.31 (m,
3H, H-1’, H-4’, H-3’’), 7.21-7.16(m, 3H, H-2’, H-3’, H-4’’), 7.10(s, 1H, H-5’’), 5.53 (s, 2H,
13
benzylic protons, CH2). C-NMR (400 MHz, DMSO-d6, δ ppm): 158.46, 149.47, 139.25,
138.73, 133.59, 130.38, 130.24, 129.53, 125.58, 125.41, 121.95, 118.82, 113.57, 103.64,
44.24. IR (KBr, cm-1): 3070 (C-H aromatics stretching), 2754 (C-H stretching), 1664 (N-C=O
imide stretching), 1475 (asymmetric O-N=O stretching), 1618 (C=N stretching), 1554
(skeletal bands), 1324 (symmetric O-N=O stretching). MS (+ESI-QTOF): m/z calculated for
C22H13N5O6 (Mꢀ+K)+ 481.37, found 481.4.
21

4.2. Biological evaluation (Experiments in vitro)
4.2.1 Material and method
COX (ovine/human) inhibitor screening assay kit procured commercially from Cayman
suppliers. All materials, chemicals, drugs and reagents used in this experiments were
purchased from Sigma-Aldrich Co, (St Louis, MO, USA). For In vitro TNF-α inhibition
activity, Mouse TNF-alpha ELISA kit (Cat No: ELM-TNFα-1, Make: RayBiotech, USA)
procured commercially from suppliers. RAW (Mouse macrophages) cell line were procured
from National Centre for Cell Sciences (NCCS), Pune, India. Fetal Bovine Serum (FBS),
penicillin, MEM/DMEM, streptomycin, amphotericin B procured from Gibco, Hi-Media. For
lipoxygenase inhibitory activity, Human recombinant 5-LOX (Item no: 60402) procured from
Cayman suppliers, Lipoxidase (LOX) type 1-B from Glycine max (soybean) purchased from
Sigma-Aldrich Co, (St Louis, MO, USA). Boric acid, linoleic acid, NaOH, ethanol, NDGA,
Tris buffer, CaCl2, 0.1mM EDTA, ATP and DMSO were obtained from Sigma-Aldrich Co,
(St Louis, MO, USA).
4.2.2. In vitro COX-1 and COX-2 inhibitory assay
All the newly synthesized compounds were screened for their ability to inhibit COX-1 and
COX-2 enzymes. This was carried out using Cayman colorimetric COX (ovine) inhibitor
screening assay kit (Catalog No. 760111) supplied by Cayman chemicals, Ann Arbor, MI,
USA, according to reported method [16].
4.2.3. In vitro human recombinant 5-lipoxygenase (LOX) assay
All the newly synthesized compounds were screened for their ability to inhibit human
recombinant 5-LOX enzyme. Recombinant LOX activity assays were performed at room
temperature as reported previously. The newly generated conjugated diene (HETEs and
HpETEs) catalyzed by LOXs can be detected by the absorbance at 234 nm using an
ultraviolet visible spectrophotometer Hitachi U2900 (Tokyo, Japan) (assay buffer: 50mM
22

Tris-Buffer (pH=7.5) containing 2mM EDTA, 2mM CaCl2, 2mM ATP). All enzyme
reactions were carried out in 1 cm path length quartz cuvettes with a total volume of 2 mL,
and absorbance at 234 nm was continuously measured for 300s. To determine the inhibitor
activity, various concentrations of test compounds were used, and the reaction was started by
the introduction of 20mM linoleic acid [17].
4.2.4. In vitro 15-lipoxygenase assay procedure
The ability of the test compounds to inhibit human 15-LOX (IC50 value, µM) listed in Table
1. Lipoxidase (LOX) type 1-B from Glycine max (soybean), boric acid, linoleic acid, NaOH,
ethanol (96%) were used with no further purification. LOX inhibition activity was
determined spectrophotometrically by measuring the increase in absorbance at 234 nm for the
oxidation of linoleic acid. The reaction mixture contained (final concentration) the test
compounds, dissolved in DMSO at concentrations of 0.01–100 µM, or the solvent (control)
and linoleic acid 152 µM, in borate buffer (pH = 9.0). The reaction was started by adding a
lipoxygenase in amount of 500 units. Five different concentrations of each test compounds
were used for the inhibition activity experiments. The increase in absorbance was recorded
for 300 s under controlled temperature 25ºC [18].
4.2.5. In vitro TNF-alpha assay procedure
The ability of the test compounds to inhibit TNF-alpha (IC50 value, µM) listed in Table 1.
RAW cells seeded in to 6 well culture dishes at a cell population of 1.5 to 2x105 cells/ml in
DMEM with 10% FBS. After 24 h, the cells were treated with range of test concentrations of
test compounds along with 1μg/ml of lipopolysaccharide (LPS) and incubated at 37ºC with
5% CO2for 4 h. After incubation, the cell supernatant was collected, centrifuged, separated
and stored at -20º C till use. The TNF-alpha levels in the cell supernatants were estimated by
using standard ELISA kit specific for mouse (Mouse TNF-alpha ELISA, Cat No: ELM-
TNFα-1, Make: Ray Biotech, USA) as per the standard kit protocol. Based on the estimated
23

quantities of TNF-alpha against control group, percentage inhibition of TNF-alpha were
determined against control [19].
4.2.6 In vivo assay
4.2.6.1. Animals
Albino Wistar rats of either sex (150-330g) were obtained from Central Animal House,
Punjabi University, Patiala. All animals accessed food and water ad libitum and were housed
in 12h dark/light cycle in a controlled at room 23-25ºC. They were allowed to acclimatize to
the experiment room 2hr before the experiment. Experiments were carried out using
protocols approved by Institutional Animal Ethics Committee (registration number
107/GO/ReBi/99/CPSCEA) and care of animals carried out as per the Committee for the
Purpose of Control And Supervision of Experiments on Animals (CPSCEA). On the same set
of animals in vivo tests proposed to be carried out. In all tests, adequate considerations were
used to reduce discomfort or pain of animals.
4.2.6.2. Drugs
Indomethacin, Celecoxib, Carrageenan, Craboxymethylcellulose were purchased from
Sigma-Aldrich Chemicals Pvt. Limited, Bangalore, India. The novel compounds were
synthesized based on the previously described methods.
4.2.6.3. Preparation of samples for bioassay
Unless otherwise stated, the conditions are employed in all experiments. Test samples will be
given orally to test animals after suspending in 0.5% sodium carboxymethylcellulose. The
control group animals receives the same experimental handling as those of the test groups
except that the drug treatment is replaced with appropriate volumes of the dosing vehicle.
Both indomethacin and celecoxib in 0.5% sodium carboxymethyl cellulose were served as
standard drugs. The test compound (3j) administered on molar equivalent basis of reference
standards.
24

4.2.6.4. Anti-inflammatory assay
Compound 3j was evaluated for its in vivo anti-inflammatory activity applying the
carrageenan-induced paw edema model reported previously [20]. Rats were randomly
divided to five groups (n=6). The rats in group I (normal control) was kept as non-immunized
untreated. Rats that were given the vehicle (0.5% sodium carboxymethylcellulose) served as
carrageenan control group (group II).The other groups (group III, IV and V) were orally
administered the drugs—indomethacin and celecoxib as reference standards at a dose
0.05mmol/kg of body weight and the tested compound 3j (equimolar dosage to reference
standards). After 45 minutes, in all groups apart from the normal control, the rat paw oedema
induced by injected 100µl of carrageenan solution subcutaneously into sub plantar tissue of
the right hind paw. The paw volume were measured using plethysmometer in different
treatment groups, 3hr and 5hr following the carrageenan injection. The increase in paw
volume was calculated by subtracting the volumes before and 3hr and 5hr after the injection
of carrageenan. Edema was expressed as an increase in the volume of paw, and the
percentage of edema inhibition (or percent protection against inflammation) for each rat and
each rat was calculated according to the following equation:
% Inhibition= (Vt-V0) control-(Vt-V0) test compound/ (Vt-V0) control X 100
Where Vt is the mean volume of edema at specific time interval (after 3hr and 5hr) and V0 is
the mean volume of edema at zero time interval.
4.2.6.5. Analgesic assay
Compound 3j was evaluated for its in vivo analgesic activity using 4% sodium chloride
writhing (abdominal constriction) assay as described previously [21]. The analgesic activity
of the compounds was done at the same dose as used for anti-inflammatory activity. In this
model, at the time interval of 3hr after the oral administration of dosing vehicle (control),
reference standards (indomethacin and celecoxib) or test sample (3j) the writhing responses
25

were induced by intraperitoneal injection of 4% sodium chloride solution to groups of six rats
each. For each animal the total number of abdominal constrictions (writhes) will be counted
for the next 10 min, starting on the fifth minute after the 4% sodium chloride injection and is
expressed as writhing numbers. Percent inhibition of writhing was also calculated and
compared among control and drug-treated groups.
4.2.6.6. Gastric ulcerogenic activity
Compound 3j was further evaluated for acute gastric ulcerogenic risk evaluation using
indomethacin-induced gastric ulcer model [22]. This was done at three times higher dose in
comparison to the dose used for anti-inflammatory activity, i.e. 0.15mmol/kg of body weight
of indomethacin and the test compound were used. Each group had three animals which were
later scarificed according to CPSCEA guidelines. Their stomach is removed, opened along
the greater curvature, washed with saline, carefully examined and ulcers were scored
according to severity as follows:
Normal colored stomach-0, Red coloration-0.5, Spot ulcers-1.0, Hemorrhagic streaks-1.5,
Ulcers >3mm but <5mm-2.0, Ulcers>5mm-3.0
4.2.6.7. Lipid peroxidation assay
Lipid peroxidation studies were carried out according to the previously reported method
[23]. After scoring the gastric mucosa of animals for ulcerogenic effect of synthesized drugs,
the gastric mucosa of animals was scraped with two glass slides, weighed (100 mg), and
homogenized in 1.8 mL of 1.15% ice-cold potassium chloride (KCl) solution. The
homogenate was supplemented with 0.2 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL
of acetate buffer (pH 3.5), and 1.5 mL of 0.8% thiobarbituric acid (TBA). The mixture was
heated at 95°C for 60 min. The cooled reactants were shaken vigorously for 1 min and
centrifuged for 10 min at 4000 rpm after supplementing with 5 mL of a mixture of n-butanol
and pyridine (15:1 v/v). The supernatant organic layer was collected and absorbance was
26

measured at 532 nm on UV spectrophotometer. The results are expressed as nmoles of
malondialdehyde (MDA)/100 mg tissue, using extinction coefficient 1.56 × 105 per cm/M.
4.3 Anti-inflammatory docking study
Molecular docking analysis was performed using CDOCKER algorithm to find the binding
mode for synthesized molecules with target proteins. CDOCKER has been incorporated into
Discovery Studio 4.1 (Accelrys Software Inc., San Diego, CA) through the Dock Ligands
(CDOCKER) protocol. We extracted the crystal structure of COX-2, COX-1, 15-LOX and 5-
LOX (PDB ID: 6COX, 1PGF, 1IK3 and 3V99, respectively) from the RCSB Protein Data
Bank [http://www.rcsb.org/pdb]. In CDOCKER, random ligand conformations were
generated from the initial ligand structure through high-temperature molecular dynamics
followed by random rotations. Then, the random conformations were refined by grid-based
simulated annealing, which makes the results more accurate. The CDOCKER interaction
energy was then computed between the ligands and target proteins. The docking analysis
provided insights into the interactions between the synthesized molecules and target proteins,
which facilitated in mechanistic justification of the obtained biological results.
4.4. Statistical Analysis
Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San
Diego, CA). Data are expressed as mean and standard deviation± SD. Parametric test, one-
way analysis of variance (ANOVA) and the Tukey post hoc test were performed on the data
for intergroup comparisons besides two-way analysis of variance (ANOVA) followed by
Bonferroni post hoc test. Nonparametric tests, Kruskal-Wallis test and Mann–Whitney test
were used to compare ulcerogenicity in different groups. The nominal statistical significance
level was set at 0.05.
Conflict of interest
There are no conflicts to declare.
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Acknowledgements
The author(s) thank research group of Molecular Modelling Lab, Department of
Pharmaceutical and Drug Research, Punjabi University Patiala, for the helpful discussion and
suggestions during the redaction of this paper.
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