Anti-inflammatory effect and inhibition of nitric oxide production by targeting COXs and iNOS enzymes with the 1,2-diphenylbenzimidazole pharmacophore

Article abstract of DOI:10.1016/j.bmc.2020.115427

Being the base of several non-communicable diseases, including cancer, inflammation is a complex process generated by tissue damage or change in the body homeostatic state. Currently, the therapeutic treatment for chronic inflammation related diseases is

Full text of DOI:10.1016/j.bmc.2020.115427

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-inflammatory effect and inhibition of nitric oxide production by
targeting COXs and iNOS enzymes with the 1,2-diphenylbenzimidazole
pharmacophore
Mónica I. García-Arandaa, Jazmin E. Gonzalez-Padillac, Carlos Z. Gómez-Castrod,
Yolanda M. Gómez-Gómeza, Martha C. Rosales-Hernándezc, Efrén V. García-Báeza,
⁎
⁎
Marina O. Franco-Hernándeza, José L. Castrejón-Floresb, , Itzia I. Padilla-Martíneza,
a Laboratorio de Química Supramolecular y Nanociencias, Unidad Profesional Interdisciplinaria de Biotecnología del Instituto Politécnico Nacional, Av. Acueducto s/n
Barrio la Laguna Ticomán, Ciudad de México 07340, Mexico City, Mexico
b Laboratorio de Cultivo Celular y Biología Molecular, Unidad Profesional Interdisciplinaria de Biotecnología del Instituto Politécnico Nacional, Av. Acueducto s/n Barrio la
Laguna Ticomán, Ciudad de México 07340, Mexico City, Mexico
c Laboratorio de Biofísica y Biocatálisis, Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina del Instituto Politécnico Nacional, Plan de San Luis y
Díaz Mirón s/n, Casco de Santo Tomás, 11340 Mexico City, Mexico
d CONACyT – Universidad Autónoma del Estado de Hidalgo, km 4.5 Carretera Pachuca-Tulancingo, Col. Carboneras, Mineral de la Reforma, Hidalgo 42184, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Being the base of several non-communicable diseases, including cancer, inflammation is a complex process
generated by tissue damage or change in the body homeostatic state. Currently, the therapeutic treatment for
chronic inflammation related diseases is based on the use of selective cyclooxygenase II enzyme, COX-2, in-
hibitors or Coxibs, which have recently regained attention giving their preventive role in colon cancer. Thus, the
discovery of new molecules that selectively inhibit COX-2 and other inflammatory mediators is a current
challenge in the medicinal chemistry field. 1-Phenylbenzimidazoles have shown potential COX inhibitory ac-
tivity, because they can reproduce the interaction profile of known COX inhibitors. Therefore, in the present
investigation a series of 1,2-diphenylbenzimidazoles (DPBI) with different aromatic substitutions in the para
position were synthesized and their interaction with COX-2 and nitric oxide synthase, iNOS, was determined in
silico, in vitro and in vivo. Compound 2-(4-bromophenyl)-1-(4-nitrophenyl)-1H-benzo[d]imidazole showed the
best inhibition towards COX-2, while compounds N-(4-(2-(4-bromophenyl)-1H-benzo[d]imidazol-1-yl)phenyl)
acetamide and N-(4-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide diminished the produc-
tion of NO in vitro. Additionally, they had a significant anti-inflammatory activity in vivo when given orally.
Benzimidazole
Anti-inflammatory
COX-inhibition
iNOS inhibition
Interaction profile of COX-2
1. Introduction.
acid inhibition and mucosal production regulated by PGs.
At the beginning of the new century, the development of selective
Inflammation is the base of several non-communicable diseases,
which kills 41 million people each year representing the 71% of all the
global deaths according with the World Health Organization.1 Several
drugs targeting different receptors are currently used as anti-in-
flammatory agents, and can be classified as steroidal, non-steroidal
drugs (NSAIDs) or agent modifiers of the diseases, which includes
biological drugs such as antibodies. Among them, NSAIDs are widely
and commonly used. They inhibit, non-selectively, the action of the
cyclooxygenases (COX-1 and COX-2) enzymes decreasing the produc-
tion of prostaglandins (PGs), although their chronic use correlates with
the presence of gastric ulcers as a result of the dysregulation in gastric
COX-2 inhibitors opened a new horizon for the treatment of chronic
inflammation associated diseases making a breakthrough in the control
of inflammation.2 COX-2 is highly expressed during the inflammatory
response, although recent reports have shown that it is also con-
stitutively expressed in other organs.3 Unfortunately, COX-2 inhibitors
were not free of adverse effects and their prolonged use, more than five
years, resulted in myocardial infarction in a few patients leading to the
withdrawn of certain coxibs and the restriction in their use. However,
recent clinical studies have confronted previous findings demonstrating
their safety.4 A renewed interest in the use of coxibs has arisen, because
it has been shown that their use together with other anti-carcinogenic
⁎ Corresponding authors.
E-mail addresses: jlcastrejon@ipn.mx (J.L. Castrejón-Flores), ipadillamar@ipn.mx (I.I. Padilla-Martínez).
https://doi.org/10.1016/j.bmc.2020.115427
Received 17 December 2019; Received in revised form 2 March 2020; Accepted 5 March 2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:MónicaI.García-Aranda,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2020.115427

M.I. García-Aranda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
drugs might help to prevent colon cancer.5 Nonetheless, the COX-2
inhibition effect and its relationship with cancer prevention needs to be
further investigated into detail.
The 1H NMR data of the series of compounds 2, 3 and 4 showed four
signals, for each of the protons of the benzofused ring, and four more
for the eight protons of the p-substituted C2-Ph and N1-Ph rings. Their
chemical shifts were the expected in agreement with the electro-donor
(ED) or electro-withdrawing (EW) nature of the substituents. For the
acetylated compounds 4, the characteristic signal for the NH and Me
groups were also observed at low and high frequencies, respectively.
Finally, 13C NMR data confirmed the structures. The chemical shifts of
C2 are almost independent of the nature of the substituents appearing
in the 149.8–152.4 range, whereas the δ of the ipso C13 and C19 de-
pend on the EW or ED nature of the substituents, as the most relevant
features. Furthermore, the stretching bands (cm−1) of the nitrile, acid
and amide groups were clearly observed in the IR spectra of compounds
A vast amount of research has been focused exclusively in the in-
hibition of COX-1 or −2 as modifiers agents of the inflammatory pro-
cess, and although it has been proved very effective, this process is a
complex phenomenon with several cells and effector molecules in-
volved. For example, nitric oxide (NO) is a small but potent pro-in-
flammatory molecule secreted during inflammation that causes vaso-
dilation and cellular migration, and at higher concentrations, it
downregulates adhesion molecules and induce apoptosis of in-
flammatory cells. Inducible nitric oxide synthase or iNOS, a target for
drug designing, is expressed by macrophages and transforms L-arginine
into NO and L-citrulline.6,7
2a (νCN
=
2225 cm−1), 2f (νCO
=
1707 cm−1
) and 4
The benzimidazole ring (BI), a benzene and imidazole fused struc-
ture, is a widely used pharmacophore with a myriad of clinical appli-
cations including diseases with an inflammatory etiology.8 Substituted-
BI structures are not structurally related to NSAIDs or selective COX-2
inhibitors, although they have shown a similar inhibition profile than
these commercial drugs given their capacity to bind to similar sites and
mimic interaction patterns than NSAIDs.9 Pioneering work with 2-
substituted-BI, -benzothiazole and -benzoxazole heterocycles showed
that the BI ring had a moderate selective inhibition against human COX-
2 when compared with the other heterocycles.10 Moreover, a series of
(νCO = 1707–1667 cm−1, νNH = 3465–3251 cm−1).
Compound 2c crystallized in the triclinic form, space group P-1
(No.2) with two molecules in the unit cell. The molecular structure of
compound 2c, shown in Figure 1(a), is similar to the bromine derivative
2b, reported elsewhere.13 The C2-Ph and N1-Ph rings are out of the
mean plane formed by the BI-heterocycle by 19.95(4)° and 58.08(3)°,
respectively, and the torsion angle between the two planes is 68.14(4)°.
The strong EW effect of the p-NO2 group, in the N1-Ph ring, is re-
sponsible of most of the non covalent dispersive interactions observed
in the crystal network, such as CeH∙∙∙A (A = π, N, O) and ONO∙∙∙ π in
addition to the Cl∙∙∙ π interaction that rise the supramolecular structure
(Figure 1(b) and Table S1 (Supporting Information)). The ab initio
MKS15 calculated charges for the N1-Ph hydrogens were in 1.50 to 1.80
range, in agreement with their involvement in CeH∙∙∙A dispersive in-
teractions (Table S2). Theoretical calculations underscore the im-
portance of enhanced electrostatic stabilization in the non-covalent
interactions in the crystal structure.
5-substitued-1-phenylsulfonyl-2-methylbenzimidazole
compounds
showed moderate anti-inflammatory effect when tested in a rat edema
in vivo assay, but their effect over COX-2 inhibition was not estab-
lished.11 More recently, 2-coumarin-BI series were evaluated as anti-
inflammatory compounds in vivo demonstrating a good inhibitory effect
in the rat paw edema model, where the withdrawing groups in the
aromatic ring seem to benefit the anti-inflammatory effect.12 Ad-
ditionally, the benzimidazole heterocycle, particularly 5-chloro-1,3-di-
hydro-2H-benzimidazol-2-one, showed a potent NO inhibition, which
varies upon the different substitutions, although the effect of a double
phenyl substitution to the core benzimidazole ring and their effect to-
wards iNOS has not been assessed.
2.2. Docking computational analysis.
2.2.1. Predicted COX-1 and −2 affinity of the DPBI-substituted
compounds.
Therefore, following previous findings published by our group de-
monstrating the affinity of 1-phenylbenzimidazole derivatives towards
COXs enzymes,9 the synthesis of a novel family of 1,2-diphenylbenzi-
midazole (DPBI) substituted compounds was achieved with the aim to
profiling them as selective COXs and iNOS inhibitors by in vitro and in
vivo assays. The results demonstrate that the DPBI substitution with
strong and weak electro withdrawing groups not only resulted in a
significant inhibition towards COX-2, but also in an important anti-in-
flammatory effect in vivo, while the acetamide substitution diminished
the production of NO. Moreover, 2-(4-bromophenyl)-1-(4-nitrophenyl)-
1H-benzo[d]imidazole reduced inflammation when administrated or-
ally in rats.
From the molecular docking calculations an estimated free energy of
binding (ΔG°b) was obtained for the set of DPBIs and the control
compounds in both COX-1 and COX-2 models. The number of binding
modes (BM) found inside the COX binding site, the lowest free energy of
binding (ΔG°b), and the selectivity indices for COX-2 are listed in
Table 1. These results show that the estimated activity of the tested
compounds is comparable to the control inhibitors, while in some cases
the affinity of the DPBIs is even higher than that calculated for both
ibuprofen (Ibp) and etoricoxib (Etox). According to docking calcula-
tions, it was observed that all compounds would be more active for
COX-2 than for COX-1 with free energies of binding ranging between
−8.94 to −10.73 kcal/mol and −6.87 to −9.18 kcal/mol, respec-
tively. From the calculated affinities, the COX-2 selectivity index
(ΔG°b,COX-2/ΔG°b,COX-1) is higher in the series of compounds 2, with the
p-NO2 substitution at the N1-Ph ring, than in the analogous compounds
of the series 3 and 4.
2. Results and discussion.
2.1. Route of synthesis of the DPBI substituted compounds.
The synthetic pathways employed for the synthesis of the inter-
mediate and final compounds are depicted in Scheme 1. In the initial
step, the synthesis of compounds 1a-e was carried out by the con-
densation reaction between 1,2-phenylenediamine and the corre-
sponding aromatic aldehyde with excellent yields (90–97%). In a
second step, the N1 position of the compounds was substituted ac-
complishing an aromatic nucleophilic substitution reaction, using 1-
fluoro-4-nitrobenzene as nucleophile in basic media, to obtain com-
pounds 2a-e13 with moderate to good yields (52–82%). The set of
compounds 3 was obtained through the reduction of the NO2 group to
NH2, with Sn in acidic medium,14 which were further acetylated to
generate the series of compounds 4, in both cases with high yields
(90–95%).
2.2.2. COX binding modes of DPBI substituted compounds.
The calculated COX-inhibitor complexes, indicate that all com-
pounds bind to COX-1 through a single binding mode or BM-I, see
Figure 2(a). In this conformation, the inhibitors simultaneously occupy
three relevant pockets within the COX binding site, i.e. the stereo-
selective pocket close to the entrance of the COX binding site, the long
hydrophobic COX channel, and the side pocket. This last is associated to
the binding of Coxibs, while the other two bind all the classic NSAIDs as
well. By comparing the binding pose of the studied inhibitors with the
crystallographic structure Ibp, it can be observed that the benzo-fused
ring of the BI occupies the same space as the chiral isopropyl moiety of
Ibp, interacting closely with residues Leu359, Val316, and Tyr355. The
longest axis of the compounds, i.e. the BI-C2-Ph moiety, was aligned
2

M.I. García-Aranda, et al.
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Scheme 1. Chemical route for the synthesis of 1,2-diphenilbenzimidazole (DPBI) substituted compounds defined as 2a-f, 3b,c,g and 4b,c,h.
Figure 1. (a) Ortep of compound 2c at 50% probability level. (b) Two-dimensional supramolecular architecture in the (0 8–5) plane.
along the COX channel placing the functional group in close contact
with Met522, Trp387 and Tyr385 residues, at the top of the hydro-
phobic channel. The N1-Ph moiety of the model compounds was, in
most cases, able to bind the side pocket, reaching residues such as
His90, Gln192, Phe518, and Ile523; this last residue is known to shrink
the access to the side channel in COX-1, whereas, its substitution to Val
in COX-2 allows the access to the side pocket for many Coxibs.16
It is important to note that the above described BM-I was actually
unfavorable for the compound series 2 in COX-1, because several non-
COX binding modes were actually more stable than those found within
the COX binding site for this series (data not shown). For series 3, the
N1-Ph rings binds to the side pocket of COX-1 but with low affinity. The
reduced calculated affinity of all compounds for COX-1 is due to the
inaccessibility of the BM-I to the COX-1 side pocket, however the
NHCOCH3 substitution at the N1-Ph ring in compound series 4 was able
to access to the side pocket in COX-1 with increased affinity, see
Table 1.
The BMs found for COX-2 included the BM-I described in COX-1
only in the case of para-substituted compounds with Br or Cl at the C2-
Ph ring, i.e., compound series b and c, along with compound 2e, un-
substituted at the same ring, see Figure 2(b). The fact that the rest of the
compounds with different functional groups were unable to reproduce
the BM-I in COX-2 reflects the selectivity of this isoform for halogen
atoms at the top of the hydrophobic channel, a feature that can be
exploited to achieve selectivity. Nevertheless, all the tested compounds
showed alternative binding modes in COX-2 besides the one shared
3

M.I. García-Aranda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Table 1
the docking calculations. The LRCD scores were ranked for each DPBI,
COX isoform and BM; Table S4 lists the combination of crystallographic
complex and BM that produced the lowest LRCD score for each com-
pound. Thus, the best match for each compound determined the BM
considered to be the most representative among the distribution ob-
served in COX-2 (highlighted in Table S3) as an alternative criterion to
the ΔG°b.
Docking results for Ibuprofen, Etoricoxib and DPBIs 2a-f, 3b,c,g and 4b,c,h
with COX-1 and COX-2.
Compound Number of COX
ΔG°b (kcal/mol)
Selectivity index
binding modes (BM)
COX-1
COX-2
COX-1
COX-2
ΔG°b,COX-2
ΔG°b,COX-1
/
Kd,COX-2
/
Kd,COX-1
It is worth noting that the best matches found in the data base of
reference complexes included Celecoxib complexes with either COX-1
or COX-2 as well as complexes of Indomethacin derivatives known to
occupy the side pocket of COX-1.18 The followed knowledge-based
criteria suggest that compound series 2, would bind COX-2 pre-
ferentially through BM-III or BM-IV, whereas series 3 and 4 would bind
through BM-II and BM-V, respectively. These matches generally point to
the lowest energy BMs, except for compound series 4, for this series the
energy criteria select BM-I whereas the knowledge-based criteria points
to BM-V. This discrepancy may be solved by considering the in vitro
assessment on COX enzymes presented later; compound series 4 showed
selective COX-1 inhibition which may be explained by a change in the
binding mode in both isoforms. As already mentioned, the calculations
showed that this series are active for COX-1 due to the capability of the
N1-Ph ring and its acetamide group to interact with the side pocket of
this isoform. Therefore, a change in the binding mode from BM-I to BM-
V would explain the observed activity since BM-V in COX-2 does not
keep the key interactions observed in COX-1. Thus, these results suggest
that the specificity of the binding mode in each of the compound series
is a key factor to explain the inhibitory profile and selectivity of the
studied DPBI compounds.
2a
1
1
1
1
0
0
2
1
1
1
1
1
1
2
2
4
2
2
3
3
3
4
1
4
3
2
1
3
−7.29
−7.42
−7.26
−6.87
NA
−10.65
−9.87
−9.85
−10.17
−9.48
−9.84
−9.24
−9.17
−8.94
−10.73
−10.69
−10.26
−7.16
−10.47
1.46
1.33
1.36
1.48
NA
0.0034
0.0160
0.0126
0.0038
NA
2b
2c
2d
2e
2f
NA
NA
NA
3b
−8.33
−8.01
−7.02
−9.18
−8.97
−9.09
−8.31
−7.57
1.11
1.14
1.27
1.17
1.19
1.13
0.86
1.38
0.2153
0.1412
0.0391
0.0731
0.0549
0.1388
6.9658
0.0075
3c
3g
4b
4c
4h
Ibuprofen
Etoricoxib
NA = Not Available
with COX-1 as predicted from previous computational calculations by
the research group.9 Relative to N1-phenylbenzimidazoles, the addition
of the second substituting phenyl ring at the BI center increases se-
lectivity and reduces the number of binding modes. The alternative
binding modes in COX-2 occupy the three COX pockets already men-
tioned but flipping each of the three vertices of the molecules. Five out
of six possible binding modes (I-V) were observed in the calculations,
see Figure 2(b-f). Considering the energetics together with a knowl-
edge-based criteria, a selection of the most plausible BM for each
complex was performed as specified in Table S3 and described in the
next section.
2.3. Pharmacological evaluation of the DPBI substituted compounds.
2.3.1. Evaluation of the cytotoxic activity in vitro.
Before any functional assay, the effect of all compounds over the
viability of the macrophage cell line RAW 264.7 was determined by the
change in the metabolic activity, as a readout of viability, using the
MTT assay. The compounds of the series 3, (p-NH2 group in the N1-Ph)
were the less cytotoxic, maintaining 80% viability or more, at the
highest concentration tested (500 μM), above this concentration most
compounds were not soluble. In contrast, the series of compounds 2 (p-
NO2 group in the N1-Ph) were more cytotoxic. In fact, compound 2d,
with a double p-NO2 substitution, exhibited the highest cytotoxicity,
reducing viability to less than half at 500 μM. The high toxicity seen
with the NO2 substituted benzimidazoles has been positively related
with in vitro toxicity in tumoral cells lines as a result of the inhibition of
2.2.3. Interaction profile of DPBI relative to known inhibitor complexes.
To rule out alternative BMs the Ligand-Receptor Contact Distance
(LRCD) parameter was used as a measure of similarity in the interaction
profile of enzyme-inhibitor complexes.9 The strategy consisted in se-
lecting the BM that maximized the similarity in the pattern of interac-
tions relative to know inhibitors co-crystallized with the COX enzymes.
Briefly, we performed a search in the PDB finding 74 crystallographic
COX-inhibitor complexes (24 for COX-1 and 50 for COX-2) for which
the LRCD was calculated against each of the calculated complexes in
Figure 2. Binding modes found for DPBI-substituted in COX-1 and COX-2. (a) Complexes of compounds in binding mode I with COX-1. (b)-(f) Complexes of
compounds in binding modes I-V in COX-2, respectively. See Table S3 for a list of the specific molecules in each binding mode.
4

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100
98
96
94
92
90
88
86
84
82
80
2
3
4
6
7
8
10
11
12
25e
2f
4b
21a
2b
3b
39g
4c
4h
2c
2d
3c
Figure 3. Cytotoxic activity in vitro of the DPBI-substituted compounds over murine macrophages at pharmacological relevant concentrations. Results are present as
the mean standard deviation of a triplicate of three independent experiments at concentration of 50 (solid bars) and 100 (grid bars) μM.
the PARPs enzymes.17 Moreover, NO2 substituted compounds can
transform to the nitroso reactive compound, formed during NO2 re-
duction, a powerful electrophile that reacts with proteins and DNA.18,19
After evaluating the cytotoxicity, the in vitro therapeutic con-
centration, defined as the concentration at which the viability remains
at 90%, was established, see Figure 3. Almost all compounds, but 2d,
kept viability up to 90% at 100 μM, so further experiments were exe-
cuted using this maximal concentration or below.
Table 2
Selective Inhibitory activity and IC50 concentration of DPBIs against COX-1 and
COX-2 enzymes.
Compounds
% of Enzyme Inhibition
Selectivity
SI
IC50 μM
COX-2
COX-2
COX-1
COX-1
2a
34.3
50.8
23.3
47.7
42.7
32.8
17.6
44.8
45.9
28.4
23.8
27.0
62.6
53.7
1.1
1.1
0.6
1.0
0.3
0.7
0.8
3.3
0.8
1.0
1.6
4.4
0.8
0.5
29.0
18.3
17.6
29.3
40.6
31.0
62.3
39.0
29.7
48.7
47.4
59.5
23.4
56.6
0.2
0.4
0.8
0.7
0.5
1.4
0.7
0.4
1.1
0.8
1.0
0.2
1.3
1.2
1.63
2.77
1.37
1.63
1.05
1.06
0.11
1.15
1.55
0.58
0.51
0.53
2.68
0.96
–
–
2b
102
–
–
2c
–
2d
–
–
2e
162
–
175
–
2.3.2. In vitro inhibition of COX-1 and COX-2 enzymes.
2f
The COXs inhibition assay was performed with all the compounds
including commercial drugs at a concentration of 100 μM for direct
comparison. Ibp, Etox and the kit selective inhibitor compounds, SC-
560 and DUP-697, were used as selective inhibitors for COX-1 and −2.
As expected, the SC-560 and DUP-697, compounds showed a potent
inhibition, 94% and 84%, for COX-1 and −2, respectively, validating
the assay. Ibp and Etox did cause selective inhibition of 54% and 63%
over COX-1 and −2, respectively, and the selectivity index (SI), the
ratio between COX-2 and COX-1 activities, was obtained for Etox
(SI = 2.68) and Ibp (SI = 0.96). Although the computational studies
showed a preference interaction of all the compound with COX-2, some
of the compounds had a strong inhibition towards COX-1, which was
higher than Ibp, see Table 2. The in vitro assays validate the docking
findings demonstrating that compounds of series 2 had the strongest
affinity towards COX-2. Among them, compound 2b and 2d, showed
the highest percentages of inhibition towards COX-2, 51% and 48%,
respectively. Compound 2b had the best in vitro inhibition, 51%, which
is lower than the inhibition seen with Etox, 62%. Although having the
best inhibitory parameters including a high affinity towards COX-2 in
silico, the compounds in series 2 exhibited a medium to low potency
towards the therapeutic target. Importantly, compound 2b was less
selective towards COX-1 which resulted in a slightly higher selectivity
index (SI = 2.77) than Etox (Table 2). The anti-inflammatory activity of
a C2-Ph substitution in the BI ring, particularly, the 2-(1H-benzo[d]
imidazole-2-yl)-N-(2,3-dimethylphenyl)benzamine was previously as-
3b
–
–
3c
193
173
–
224
–
3g
4b
–
4c
–
–
4h
–
–
Etoricoxib
Ibuprofen
–
–
–
–
Cyclooxygenases 1 and 2 = COX-1, COX-2, SI = Selectivity Index (COX-2/
COX-1), IC50 = Half maximal inhibitory concentration.
demonstrating the relevance of this substitution over the C2-Ph ana-
logues. Additionally, the halogenated para substitution at the N1-Ph
synergized the inhibition by interacting directly with another relevant
COX binding site, such as the long hydrophobic COX channel.
2.3.3. Inhibition of NO production.
The capacity of the compounds to inhibit the production of nitric
oxide was assessed in murine macrophages activated with LPS at
therapeutic concentrations. Thus, the cells were incubated at 50 μM and
100 μM to determine the potency and dose dependent effect of the
tested compounds, see Table 3. Interestingly, most compounds showed
better inhibition at the lowest concentration, suggesting dose depen-
dent effect of the molecules over the pharmacological target. Com-
pound 2b, which showed the best inhibition over COX-2, was not the
best in reducing NO production, suggesting that this result is not di-
rectly related with the decrease of COX-2 activity. On the contrary,
compounds 4b and 4c reduced the NO production at the levels observed
with the commercial drugs. Structurally, compounds 4b and 4c, had
both an acetamide group in the para position and a para halogenated at
N1-Ph and C2-Ph, respectively.
sessed showing
a
weak selective inhibition towards COX-2
(SI = 0.651).20 Thus, the N1-Ph substitution in the BI is a decisive
factor not only strengthening COX-2 inhibition, but also reducing the
specificity for COX-1, in the tested compounds. According with the
binding modes studies, the N1-Ph substitution will bind to the side
pocket,
a particular structural feature of the COX-2 enzyme,
5

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Table 3
3 h after the injection of carrageenan. Ibp (100 mg/kg) and Etox
(10 mg/kg) reduced edema formation by 42% and 35%, respectively.
Importantly, compound 2b and 2d, the best COX-2 inhibitors, exhibited
better reduction in the edema formation, 42%, which is comparable
with the effect of the commercial drugs, see Figure 5. The rest of the
compounds showed a reduction in edema formation, but it was smaller
than the positive controls. The results in edema reduction are similar to
those reported with a series of 2-methylamino-1H-benzimidazole deri-
vatives particularly with the N-(1H-benzimidazol-2-ylmethyl)-3-chlor-
oaniline.23
In vitro inhibition of NO production of the DPBIs compounds in macrophages
stimulated with LPS.
Compounds
% Nitric Oxide inhibition
50 μM
100 μM
2a
24.4
46.5
53.2
22.4
38.1
32.6
62.5
24.9
42.5
77.3
75.9
10.1
79.8
82.1
8.9
6.7
5.7
9.6
6.2
7.8
7.3
7.5
6.9
5.3
3.7
4.4
1.9
1.9
46.0
45.8
53.9
33.8
44.0
46.2
74.2
52.2
44.1
83.6
86.4
15.3
87.3
83.7
6.8
5.8
6.4
6.8
4.5
7.6
2.8
5.2
7.3
6.1
1.9
7.0
1.4
2.8
2b
2c
2d
2e
2f
3b
3. Conclusions.
3c
3g
Benzimidazoles are important pharmacophores with a myriad of
clinical applications. Although 1,2-diphenylbenzimidazole compounds
are not structurally related with conventional anti-inflammatory drugs,
including NSAIDs or Coxibs, the in-silico studies herein reported de-
monstrated that some of the synthetized compounds bind to similar
sites that the Coxibs. The N1-Ph substitution increased the selectivity
towards COX-2 by producing a differential binding mode in each iso-
form. Moreover, the different para substitutions at the 1,2-diphe-
nilbenzimidazole structure allowed to obtain a handful of selective
active compounds to different pharmacological targets. The para-NO2
and para-Br at N1-Ph and C2-Ph substitutions, respectively, favour the
inhibition towards COX-2, as seen with the 2-(4-bromophenyl)-1-(4-
nitrophenyl)-1H-benzo[d]imidazole compound. On the other hand, by
keeping the halogenated groups in the C2-Ph para position but func-
tionalizing the N1-Ph para position with an acetamide group, a new
series of compounds were generated capable to inhibit the secretion of
NO as seen with N-(4-(2-(4-bromophenyl)-1H-benzo[d]imidazol-1-yl)
phenyl)acetamide and N-(4-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-
1-yl)phenyl)acetamide. Importantly, the docking studies towards COX-
2 correctly predicted those compounds with the best anti-inflammatory
activity in the in vitro and in vivo assays, although some chemical
modification towards the 1,2-DPBIs are necessary to increase the po-
tency of the synthetized compounds. In summary, the functionalization
of the 1,2-diphenylbenzimidazole scaffold represents a unique oppor-
tunity for the synthesis of diverse compounds which target different
anti-inflammatory pathways that might lead to the development of new
anti-inflammatory drugs.
4b
4c
4h
Etoricoxib
Ibuprofen
Results are represented as the mean
three independent experiments.
standard deviation of a triplicate of
Most synthetic NO inhibitors are competitive inhibitors of L-arginine
which is the natural substrate of the inducible nitric oxide synthase
enzyme (iNOS). The acetamide group, present in compounds of the
series 4, has been highlighted as potent iNOS inhibitor. This group in
the C5 position of indole derivatives, resulted highly specific showing
only inhibition towards iNOS, but not against the constitutively ex-
pressed neural and endothelial forms. These compounds interact di-
rectly with the active site of iNOS blocking the enzymatic reaction that
leads to NO production.21 In order to enlighten the mechanism of NO
inhibition in the presence of the 1,2-DPBIs in vitro, docking calculations
of the best inhibitory compounds, 4b and 4c, were performed on h-
iNOS. The resulting free energy of binding (ΔG°b
= -8.9 and
−9.0 kcal mol−1) and dissociation constant values (Kd = 0.284 and
0.249 μM), calculated for 4b and 4c, respectively, are of the same order
of magnitude than potent h-iNOS inhibitors.22 The interaction profiles
of 4b and 4c, in h-iNOS active site, are depicted in Figure 4. Both
compounds are close to the amino acid residues Asp382, Arg388,
Gln263, Pro350, Val352, Trp372, Tye373, Glu377. However, com-
pound 4c is hydrogen bonded with Asp382 and Arg388, whereas
compound 4c is forming hydrophobic interactions with these two
amino acids.
4. Experimental.
2.3.4. Evaluation of the anti-inflammatory activity in vivo
4.1. Instrumental and chemicals.
The anti-inflammatory effect of all the compounds (100 mg/kg) was
tested using the in vivo carrageenan paw animal model, where maximal
edema formation, 75% more than the saline solution group, was seen
The uncorrected melting points were measured in open capillary
tubes in an Electrothermal apparatus IA 9100. 1H (300.01 MHz) and
Figure 4. Interaction profiles of compounds 4b (left) and 4c (right) in the h-iNOS active site.
6

M.I. García-Aranda, et al.
50
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
45
40
35
30
25
20
15
10
5
0
Etox
Ibp
2a
2b
2c
2d
2e
2f
3b
3c
3g
4b
4c
4h
Figure 5. Anti-inflammatory activity of DPBI-substituted compounds in vivo orally administered in a rat paw edema model using a dose of 100 mg/kg. Results are
present as the mean standard deviation of a triplicate of five independent experiments.
13C NMR (75.46 MHz) spectra were recorded on a Varian Mercury-300
spectrometer, except compound 4h which was recorded on a Bruker
(400 MHz 1H, 75 MHz 13C), at 20 °C using CDCl3 as solvent, unless
otherwise is specified, and tetramethylsilane (TMS) as internal re-
ference; chemical shift values (δ) are in parts per million (ppm) and
coupling constants (nJ values) are in Hertz (Hz). IR spectra were ob-
tained neat with a 3100 FT–IR Excalibur Series spectrophotometer. All
chromatographic purifications were performed with silica gel (200
–300 mesh), whereas all TLC developments were performed on silica
gel coated (Merck 60 F254) aluminium foils. Yields reported are iso-
lated yields of the materials. To indicate the multiplicity of the signals,
the abbreviations are used: s, for a simple signal; d, for a double signal;
dd, for a double of doubles signal; t, for a triple signal; q, for a quad-
ruple signal; m, for a multiple signals, or different combinations of the
above.
phenylenediamine (2 mmol), aryl aldehyde (2 mmol) and sodium me-
tabisulfite (2.02 mmol) in 5 mL of DMSO were heated at 140 °C for 2 h.
After the completion of the reaction, cold water (50 mL) was added.
The resulting precipitate was filtered, washed with plenty of water and
air dried. Their spectroscopic properties were in agreement with re-
ported values: compounds 1a, 1b, 1c, 1e28–29 and 1d30 see SI.
Compounds 2b and 2e are known, the first was prepared as re-
ported13 and the second following the methodology herein described,
the data agrees with reported values31, see SI.
4-(1-(4-nitrophenyl)-1H-benzo[d]imidazol-2-yl)benzonitrile (2a).
In a 100 mL round bottom flask were placed 0.645 g (2.94 mmol) of 4-
(1H-benzo[d]imidazol-2-yl)benzonitrile (1a), 0.406 g (2.94 mmol) of
anhydrous K2CO3 and 20 mL of DMF. The flask, equipped with a con-
denser, was heated to 70 °C during 15 min and then 0.35 mL (0.457 g,
3.24 mmol) of 1-fluoro-4-nitrobenzene were added. The reaction mix-
ture was heated to 120 °C, during 48 h under magnetic stirring, left to
cool to RT and poured into 50 mL of cold distilled water acidified with
10 drops of concentrated HCl. The resulting solid was filtered, washed
with distilled water and dried at RT to obtain 0.650 g of 2a. The raw
product was purified by column chromatography (Hexane/AcOEt
80:20) to obtain 0.40 g (1.47 mmol) of a yellow solid in 50% yield (m.p.
X-Ray structure determination. General crystallographic data for
compound 2c has been deposited in the Cambridge Crystallographic
Data Centre (CCDC) as supplementary publication number 1963116.
Crystal data, collection and refinement parameters are listed in Table
S5. Single crystal X-ray diffractions data was collected on an Agilent
Super Nova (dual source). The cell refinement and data reduction were
carried out with the CrysAlisPro software. The structure was solved by
direct methods using the SHELXS-97 program24 of the WINGX
package.25 The final refinement was performed by full-matrix least-
squares methods using the SHELX97 program.24 The H atoms on C were
geometrically positioned and treated as riding atoms with: C e H
0.93 Å, Uiso(H) = 1.2 eq(C) for carbon atoms. Platon26 and Mercury27
were used to prepare the material for publication.
198–200 °C). 1H NMR: δ 8.40 (m AÁ, 2H, H-12,14), 7.91 (dd, 1H, H-4,
4
3J = 7.0, J = 1.0), 7.64 (s, 4H, H-17,18,20,21), 7.50 (m XX, 2H, H-
́
11,15), 7.42 (dt, 1H, H-5, 3J = 7.5, 4J = 2.0), 7.36 (dt, 1H, H-6,
3J = 7.5, 4J = 2.0), 7.30 (dd, 1H, H-7, 3J = 7.0, 4J = 2.0 Hz). 13C
NMR: δ 149.8 (C2), 147.4 (C13), 143.1 (C16), 141.9 (C10), 136.4 (C9),
133.6 (C8), 132.4 (C18,20), 130.0 (C17,21), 127.9 (C11,15), 125.7
(C12,14), 124.9 (C6), 124.3 (C5), 120.8 (C4), 118.0 (C22), 113.6 (C19),
110.1 (C7). IR (ν, cm−1): 2225 (C^N), 1584 (C]C Ar), 1516, 1346
(NO2), 839, 748 (CeH Ar out of plane). ESI: [M + H]+ 341.1029
found, 341.1039 calcd.
Mass analysis was performed by Electro Spray Ionization in a High-
Resolution Mass spectrometry Bunker micrOTQF–QII instrument
(Brunker Daltonik GmbH, Bremen, Germany).
2-(4-chlorophenyl)-1-(4-nitrophenyl)-1H-benzo[d]imidazole (2c).
Prepared and purified as described for 2a, from 2-(4-chlorophenyl)-1H-
benzo[d]imidazole (1c) (0.694 g, 2.42 mmol), 1-fluoro-4-nitrobenzene
(0.399 g, 2.83 mmol) and K2CO3 (0.351 g, 2.54 mmol) as a yellow solid
in 75% yield (m.p. 166– 167 °C). 1H NMR: δ 8.38 (m, 2H, H-12,14),
7.88 (d, 1H, H-4, 3J = 8.0), 7.48 (m, 2H, H-11,15), 7.45 (m, 2H, H-
18,20), 7.38 (m, 1H, H-5), 7.33 (m, 2H, H-6,7), 7.32 (m, 2H, H-17,21).
13C NMR: δ 150.8 (C2), 146.9 (C13), 142.9 (C16), 142.0 (C10), 136.1
(C9), 135.9 (C8), 130.5 (C18,20), 128.8 (C17,21), 127.7 (C11,15),
4.2. Chemical synthesis of DPBI-substituted compounds.
All reagents and solvents were purchased from commercial sup-
pliers and were dried and purified when necessary by standard tech-
niques. Compounds 1a-e are known but they are not commercially
available. Therefore, they were synthesized following the methodology
described elsewhere with modifications.13 The general procedure for
the synthesis of 1a-1e is briefly described:
a mixture of o-
7

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Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
127.4 (C19), 125.2 (C12,14), 124.1 (C6), 123.7 (C5), 120.2 (C4), 109.7
(C7). IR (ν, cm−1): 1588 (C]C Ar), 1514, 1348 (NO2), 834, 771, 746
(CeH Ar out of plane). ESI: [M + H]+ 350.0693 found, 350.0697
calcd.
4,4′-(1H-benzo[d]imidazole-1,2-diyl)dianiline (3g). Synthesized
and purified as described for 3b, starting from 1.50 g (4.17 mmol) of 2d
and 1.98 g (16.6 mmol) of metallic tin to obtain 0.662 g (2.20 mmol,
53%) of a brown powder, d.p. = 249 °C. 1H NMR (DMSO‑d6): δ 7.60 (d,
1H, H-4, 3J = 8.0), 7.25 (d, 2H, H-11,15, 3J = 8.0), 7.17 (t, 1H, H-5,
3J = 8.0), 7.11 (t, 1H, H-6, 3J = 7.0), 7.00 (d, 1H, H-7, 3J = 7.0), 6.97
(d, 2H, H-17,21, 3J = 8.0), 6.66 (d, 2H, H-18,20, 3J = 8.0), 6.45 (d,
2H, H-12,14, 3J = 8.0), 5.47, 5.45 (s, 4H, NH2). 13C NMR (DMSO‑d6): δ
153.0 (C2), 150.0 (C13), 149.0 (C19), 142.6 (C9), 137.9 (C8), 130.1
(C17,21), 128.1 (C11,15), 124.8 (C10), 122.0 (C5,6), 118.3 (C4), 116.9
(C16), 114.5 (C12,14), 113.1 (C18,20), 110.1 (C7). IR (ν, cm−1): 3423,
3385, 3186 (NeH), 1608 (NH2), 1520 (C]C Ar), 1275 (CeN), 831, 749
(CeH Ar out of plane). (ESI): [M + H]+ 301.1452 found, 301.1454
calcd.
1,2-bis(4-nitrophenyl)-1H-benzo[d]imidazole (2d). Prepared and
purified as described for 2a, from 2-(4-nitrophenyl)-1H-benzo[d]imi-
dazole (2.00 g, 8.37 mmol), 1-fluoro-4-nitrobenzene (1d) (1.42 g,
10.04 mmol) and K2CO3 (1.157 g, 8.37 mmol) as a yellow solid in 71%
yield (m.p. 234–236 °C). 1H NMR: δ 8.10 (d, 2H, H-12,14, 3J = 8.9),
7.92 (d, 2H, H-18,20, 3J = 8.8), 7.57 (m, 1H, H-4), 7.48 (d, 2H, H-
11,15, 3J = 8.9), 7.45 (d, 2H, H-17,21, 3J = 8.8), 7.08 (m, 3H, H-
5,6,7). 13C NMR: δ 149.8 (C2), 147.8 (C19), 147.1 (C13), 142.7 (C16),
141.5 (C9), 136.5 (C10), 135.5 (C8), 131.7 (C6), 130.7 (C18, 20), 128.8
(C17,21), 125.5 (C12,14), 124.6 (C5), 123.7 (C11,15), 120.1 (C4),
110.8 (C7). IR (ν, cm−1): 1588 (C]C Ar), 1508, 1343 (NO2), 854, 740,
702 (CeH Ar out of plane). ESI: [M + H]+ 361.0972 found, 361.0938
calcd.
N-(4-(2-(4-bromophenyl)-1H-benzo[d]imidazol-1-yl)phenyl)acet-
amide (4b). In a 100 mL round bottom flask, immersed in an ice-water
bath, were placed 1.00 g (2.75 mmol) of compound 3b, 40 mL of THF
and 1 mL of TEA. Under vigorous stirring, 1.4 mL of acetyl chloride
were added, the reaction mixture was left under stirring for 2 h to reach
RT. The solvent was evaporated and the remaining solid was washed
with distilled water and dried at RT to obtain 0.900 g (1.99 mmol, 90%)
of a beige solid m.p. = 153–156 °C. 1H NMR: δ 10 (s, 1H, NH), 7.86 (d,
1H, H-4, 3J = 8.0), 7.68 (d, 2H, H-11,15, 3J = 8.8), 7.60 (b, 1H, H-7),
7.43 (s, 4H, H-17,18,20,21), 7.34 (dt, 1H, H-5, 3J = 7.5, 4J = 2.0), 7.28
(dt, 1H, H-6, 3J = 7.5, 4J = 2.0), 7.26 (d, 1H, H-12,14, 3J = 8.0), 7.25
(d, 1H, H-7, 3J = 8.0), 2.22 (s, 3H, CH3). 13C NMR: δ 168.5 (C]O),
151.2 (C2), 142.7 (C16), 138.4 (C13), 137.3 (C9), 132.2 (C8), 131.6
(C18), 130.8 (C17), 128.7 (C10), 127.9 (C11,15), 124.2 (C19), 123.7
(C6), 123.3 (C5), 120.8 (C12,14), 119.8 (C4), 110.5 (C7), 24.7 (CH3).
IR (ν, cm−1): 3465 (NeH), 1667 (C]O), 1594 (C]C Ar), 1264 (CeN),
821, 746 (CeH Ar out of plane). (ESI): [M + H]+ 406.0588 found,
406.0556 calcd.
4-(1-(4-nitrophenyl)-1H-benzo[d]imidazol-2-yl)benzoate (2f). In
100 mL bottom flask, equipped with condenser, were refluxed 0.500 g
(1.47 mmol) of 2a and 60 mL of concentrated HCl (37%) during 9 h.
After cooling to RT the solution was alkalinized to pH = 5 with NaOH
solution (60%). The resulting solid was washed with distilled water and
dried at RT to obtain 0.30 g (0.84 mmol, 57% yield) of a pale yellow
powder, d.p. = 258 °C. 1H NMR: δ 8.40 (d, 2H, H-12,14, J = 8.9 Hz),
7.92 (d, 2H, H-18,20, 3J = 8.2), 7.84 (m, 1H, H-4), 7.73 (d, 2H, H-
17,21, 3J = 8.9), 7.57 (d, 2H, H-11,15, 3J = 8.3), 7.36 (m, 3H, H-
5,6,7). 13C NMR: δ 167.2 (C]O), 151.4 (C2), 147.4 (C13), 143.2 (C16),
142.2 (C10), 136.8 (C9), 133.5 (C8), 132.5 (C19), 129.9 (C18,20),
129.8 (C17,21), 129.1 (C11,15), 125.8 (C12,14), 124.6 (C6), 123.9
(C5), 120.3 (C4), 111.0 (C7). IR (ν, cm−1): 3410, 2780, 2608 (OeH),
1707 (C]O), 1526, 1348 (NO2), 1275 (CeO), 861, 788, 761 (CeH Ar
out of plane). (ESI): [M + H]+ 360.0965 found, 360.0985 calcd.
4-(2-(4-bromophenyl)-1H-benzo[d]imidazol-1-yl)aniline (3b). In a
100 mL round bottom flask were placed 60 mL of concentrated HCl and
warmed to 60 °C in a water bath, 0.500 g (1.27 mmol) of raw 2b were
added, after 15 min, until complete dissolution, followed by 0.319 g
(2.69 mmol) of metallic tin in small portions. The mixture was allowed
to react during 2 h, cooled to RT and filtered. The filtrate was cooled
into an ice-water bath and alkalinized to pH 12 with NaOH solution
N-(4-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)phenyl)acet-
amide (4c). Synthesized and purified as described for 4b, starting from
1.50 g (4.69 mmol) of 3c and 1.5 mL of acetyl chloride to obtain 1.38 g
(3.81 mmol, 92%) of a beige powder, m.p. = 154–156 °C. 1H NMR
(DMSO‑d6): δ 9.94 (s, 1H, NH), 7.42 (d, 3H, H-4,11,15, 3J = 8.6), 7.18
(d, 2H, H-18,20, 3J = 8.6), 7.09 (d, 1H, H-17,21, 3J = 8.6), 6.99 (d,
2H, H-12,14, 3J = 8.7), 6.94 (t, 1H, H-5, 3J = 7.4), 6.91 (t, 1H, H-6,
3J = 7.0), 6.83 (m, 1H, H-7), 1.72 (s, 3H, CH3). 13C NMR (DMSO‑d6): δ
168.8 (C]O), 150.7 (C2), 142.1 (C9), 139.8 (C13), 137.2 (C10), 134.5
(C8), 131.9 (C16), 130.8 (C18,22), 130.5 (C19), 128.6 (C17,21), 128.0
(C11,15), 123.6 (C5), 123.0 (C6), 120.0 (C12,14), 119.3 (C4), 110.5
(C7), 24.2 (CH3). IR (ν, cm−1): 3316 (NeH), 1667 (C]O), 1594 (C]C
Ar), 1264 (CeN), 841, 750 (CeH Ar out of plane). (ESI): [M + H]+
362.1046 found, 362.1061 calcd.
(60%). The resulting precipitate was washed to obtain 0.250
g
(0.686 mmol) of raw 3b. Further purification was performed by column
chromatography (Hexane/AcOEt 4:6) to obtain 0.228 g (0.624 mmol,
49%) of a beige powder m.p. = 224–225 °C. 1H NMR: δ 7.86 (d, 1H, H-
4, 3J = 7.0), 7.51 (d, 2H, H-18,20, 3J = 8.5), 7.44 (d, 2H, H-17,21,
3J = 8.5), 7.27 (m, 3H, H-5,6,7), 7.06 (d, 2H, H-11,15, 3J = 8.4), 6.76
(d, 2H, H-12,14, 3J = 8.4), 3.93 (s, 2H, NH2). 13C NMR: δ 151.5 (C2),
147.1 (C13), 142.9 (C16), 137.9 (C9), 131.7 (C18,20), 131.0 (C17,21),
129.3 (C8), 128.5 (C11,15), 127.2 (C10), 124.1 (C19), 123.5 (C6),
123.1 (C5), 119.9 (C4), 115.9 (C12,14), 110.9 (C7). IR (ν, cm−1): 3383,
3316, 3188 (NeH), 1640, 1608 (NH2), 1520 (C]C Ar), 1280, 1260
(CeN), 831, 742 (CeH Ar out of plane). (ESI): [M(81Br) + H]+
366.0415 found, 366.045 calcd.
N,N'-((1H-benzo[d]imidazole-1,2-diyl)bis(4,1-phenylene))diaceta-
mide (4h). Synthesized and purified as described for 4b, starting from
1.50 g (5.00 mmol) of 3 g and 1.5 mL of acetyl chloride to obtain
0.920 g (2.39 mmol, 48%) of a beige powder, d.p. = 284 °C. 1H NMR
(DMSO‑d6): δ 10.45 (s, 2H, NH), 7.91 (d, 1H, H-4, 3J = 8.0), 7.84 (d,
2H, H-11,15, 3J = 8.0), 7.69 (d, 2H, H-17,21, 3J = 8.0), 7.54 (d, 2H, H-
18,20, 3J = 8.0), 7.50 (d, 2H, H-12,14, 3J = 9.0), 7.56 (t, 2H, H-5,6,
3J = 7.0), 7.37 (d, 1H, H-7, 3J = 7.0), 2.1, 2.0 (s, 6H, CH3). 13C NMR
(DMSO‑d6): δ 169.5 (C]O), 150.1 (C2), 143.3 (C19), 141.5 (C13),
135.5 (C10), 132.9 (C9), 131.4 (C8), 128.6 (C11,15), 128.3 (C12,14),
117.8 (C16), 128.6 (C17,21), 128.0 (C18), 123.6 (C6), 122.9 (C5),
119.3 (C4), 110.7 (C7), 24.1 (C24,27). IR (ν, cm−1): 3251 (NeH), 1707
(C]O), 1594 (C]C Ar), 1250 (CeN), 844, 755 (CeH Ar out of plane).
(ESI): [M + H]+ 385.1655 found, 385.1665 calcd.
4-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)aniline
(3c).
Synthesized and purified as described for 3b, starting from 0.500 g
(1.43 mmol) of 2c and 0.319 g (2.69 mmol) of metallic tin to obtain
0.237 g (0.740 mmol, 52%) of a beige powder, m.p. = 215–217 °C. 1
H
NMR: δ 7.85 (d, 1H, H-4, 3J = 7.9), 7.57 (d, 2H, H-18,20, 3J = 8.6),
7.31 (dd, 2H, H-17,21, J = 7.8, J = 1.4), 7.27 (t, 1H, H-5, 3J = 1.9),
7.24 (m, 1H, H-6), 7.21 (d, 1H, H-7, 3J = 7.8), 7.05 (d, 2H, H-11,15,
3J = 8.6 Hz), 6.74 (d, 2H, H-12,14, 3J = 8.7), 3.93 (s, 2H, NH2). 13C
NMR: δ 151.5 (C2), 147.1 (C13), 142.9 (C10), 137.9 (C9), 135.6 (C8),
130.8 (C18,20), 128.8 (C16), 128.7 (C17,21), 128.5 (C11,15), 127.2
(C19), 123.5 (C6), 123.1 (C5), 119.8 (C4), 115.9 (C12,14), 110.9 (C7).
IR (ν, cm−1): 3383, 3304, 3188 (NeH), 1640, 1608 (NH2), 1514 (C]C
Ar), 1280, 1260 (CeN), 834, 743 (CeH Ar out of plane). (ESI):
[M + H]+ 320.0952 found, 320.0955 calcd.
4.3. Modelling and in silico studies.
The affinities towards COX-1, COX-2 and h-iNOS enzymes of the set
of DPBIs were studied by theoretical means using the Autodock 4.2
program suit to perform molecular docking calculations.32 The
8

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crystallographic structure of each enzyme was obtained from the Pro-
tein Data Bank (PDB Ids: 1EQG, COX-1; 4PH9, COX-2; and 3EJ8, h-
iNOS).33,34
10 μL of the ADHP and AA were added, and fluorescence was measured
after 5 min for both COX-1 and −2 in a plate fluorometer using an
excitation/emission light of 530–540/585–595 nm wavelengths.
Both COX models were co-crystallized with the inhibitor ibuprofen,
considered together with etoricoxib as references in the calculations. A
re-docking calculation on the crystallographic complexes of Ibuprofen
with COX-1 and COX-2 was performed to validate the models. The
RMSD for the calculated complexes relative to PDB models 1EQG and
4PH9 were 0.883 and 0.803 Å, respectively. Figure S1 compares ex-
perimental and calculated docked structures of ibuprofen. The observed
RMSD below 0.9 Å confirm that the bioactive conformations are suffi-
ciently well reproduced. Thus, docking calculations on these models are
reliable.
4.4.2. Determination of the cytotoxicity of DPBI substituted compounds in
murine macrophages in vitro using the MTT assay.
Murine macrophages were growth in culture medium composed of
RPMI-1640 medium supplemented with 10% of inactivated fetal bovine
serum, 2 mmol/L HEPES buffer solution, 100 mM L-glutamine, 100 μg/
mL streptomycin, and 100 U/mL penicillin at standard culture conditions
37 °C and 5% CO2. Cells were counted using a hemocytometer, stained
with trypan blue (0.2%) and seeded at concentration of 4 x104 cells in
200 μL per well in a 96 well plate with ranging concentrations (0, 50,
100, 150, 200, 250, 300 and 500 μM) of all the compounds dissolved in
DMSO (0.2% final concentration) during 24 h. Then, the medium was
completely removed, and 100 μL of RPMI with the MTT reagent at a
concentration of 0.5 mg/mL was added to each well. The cells were in-
cubated at standard condition for 4 h, centrifuged at 1500 rpm and the
solution removed. In the final step, 100 μL of isopropyl alcohol was
added, mixed 5 min and read at 570 nm in a plate reader.
The structures of COXs and h-iNOs enzymes were cleaned up by
removing small molecules, the chain A of the homodimer including its
bounded heme prosthetic group were conserved for COXs enzymes. A
standard preparation procedure with the AutoDockTools program fol-
lowed, where polar hydrogen atoms were added, and Gasteiger atom
charges and solvation parameters were assigned. A grid made of
80 × 80 × 80 points with 0.375-Å spacing and centered at the COX
inhibitor binding site (i.e. at the center of mass of the crystallographic
ibuprofen molecules), defined the volume where the conformational
search was performed. Blind docking calculations were performed on h-
iNOS, using a 126 × 126 × 126 Å point grid with 0.375 Å spacing
centered on the protein. The enzyme-ligand complexes were visualized
using AutoDockTools,33 VMD,39 and Chimera v1.9.35
4.4.3. Nitric oxide inhibition of the DPBI substituted compounds in murine
macrophages activated with LPS
Macrophages were seeded at 4 x104 in 200 μL per well in a 96 well
plate in culture medium at standard conditions with all the compounds
at 50 and 100 μM, and in the presence or absence of LPS (100 ng/mL)
overnight. Then, 50 μL of each well was incubated with 50 μL of the
Griess reactive and read at 570 nm in a plate reader. The final nitric
oxide concentration was calculated using a NaNO2 standard curve at
known concentrations.
The protonation state of titratable residues was previously eval-
uated using the propka-3.1 program,36 particularly for the His90 re-
sidue located inside the COX binding site. Two variants of the neutral
form of this residue were used for the docking calculations. It was
verified that the same binding modes and energies were in general
obtained with both forms. The protonation at the imidazolic Nε of
His90 was selected to discuss the results.
4.4.4. Anti-inflammatory in vivo activity of DPBI substituted compounds in
the paw edema model.
The structure of the DPBIs was built and optimized at semiempirical
level of theory (AM1) using the Gaussian 09 program.37 The procedure
to prepare the ligands for docking calculations were also carried out
with AutoDockTools. Gasteiger atom charges and full flexibility of the
ligands was considered. The Lamarkian genetic algorithm was chosen
to perform 100 runs (conformational searches) for each ligand and COX
isoform. An exhaustive setup was selected to perform the calculation,
i.e. 150 individuals as initial population, 27,000 generations, and
2,500,000 energy evaluations. The 100 obtained complexes were
structurally clustered with a cutoff of 2 Å in the RMSD to select only the
lowest-energy conformation in each cluster for further analysis. The
free energy of binding (ΔG°b) of each complex considered in the ana-
lysis was estimated with the empirical scoring function of Autodock.38
Further structural analyses of the obtained complexes were performed
by calculating the ligand-receptor contact distance (LRCD) parameter to
compare the interaction profile of the studied compounds with the
profile of known COX-inhibitor complexes available in structural da-
tabases.9 This screening included 24 COX-1 and 50 COX-2 crystallized
complexes from the PDB and aimed to determine similarity with in-
teraction patterns of reference inhibitors
All experiments and protocols described in the present study were
conducted according to the Mexican Official Standard NOM-062-ZOO-
1999, Technical Specifications for Production, and Care and Use of
Laboratory Animals. Healthy male Wistar rats (250–300 g) were pro-
cured from the Animal Facility Laboratory at Institute of Neurobiology-
UNAM (S7). Animals were housed in polysulfonate cages in groups of 5
animals under controlled conditions of temperature 22
2 °C, relative
humidity, 55
5%, and photo-schedule (12 h light, and 12 h dark).
Animals had free access to food (Teklad Global Rodent Diet 2018S,
Harlan®) and purified water ad libitum.
Commercial drugs ibuprofen, etoricoxib and DPBI substituted
compounds were administered orally at 10 and 50 mg/kg, respectively,
to male Wistar rats (N = 5). One hour after the DPBI substituted
compounds were administered, the inflammatory response was induced
by injecting 0.1 mL of a 1% carrageenan solution in physiological saline
solution on the right paw and volume displacement was recorded at 0,
1, 2, 3, 4, 5 and 6 h after carrageenan administration with a ple-
thysmometer (PLM-01 plus, Orchid Scientific). Difference between the
left paw volume and the right paw volume gave the actual edema vo-
lumes. The percentage of paw edema inhibition was calculated as the
difference in the displaced volume obtained in the treated group and
the non-treated group divided by the non-treated group and multiplied
by 100 at the different times points.
4.4. In vitro and in vivo pharmacological evaluation.
4.4.1. COX-1 and COX-2 enzymatic assays.
COX-1 and −2 inhibition assays were performed according with the
manufacturer protocols (Cayman Chemicals). This assay measures the
formation of the highly fluorescent compound resorfin generated by the
reaction of ADPH (10-acetyl-3,7-dihidroxyphenoxazine) and PGG2
(hydroperoxy endoperoxide) which is formed by the oxidation of AA
(araquidonic acid) by COX. Thus, 10 μL of bovine COX-1 and human
recombinant COX-2 enzymes were incubated in buffer with the DPBI
substituted compounds (100 μM) or specific inhibitors (Dup-697 for
COX-2 and SC-560 for COX-1) during 15 and 5 min, respectively. Then,
Acknowledgements
Authors acknowledge the financial support from CONACYT (grant
255354) and the Secretaría de Investigación y Posgrado del Instituto
Politécnico Nacional (SIP-IPN). CZG-C thanks the CONACYT Research
fellowship 6286 in the project 222. MIG-A and JEG-P also thank
CONACYT for the scholarships 407918 and 264544. We also thank to
Dr. Susana Rojas-Lima and Dr. José Correa-Basurto for the access to the
X-ray and HR-Mass spectrometry instruments, respectively.
9

M.I. García-Aranda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
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