N-Mannich bases of benzimidazole as a potent antitubercular and antiprotozoal agents: Their synthesis and computational studies

Article abstract of DOI:10.1080/00397911.2020.1725057

This article dealing with the microwave assisted synthesis of N-Mannich bases of pyridine clubbed with two different benzimidazole cores with their micromolar biological potency. All the synthesized compounds were evaluated for their in-vitro antibacteria

Full text of DOI:10.1080/00397911.2020.1725057

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
N-Mannich bases of benzimidazole as a potent
antitubercular and antiprotozoal agents: Their
synthesis and computational studies
Vatsal M. Patel, Navin B. Patel, Manuel J. Chan-Bacab & Gildardo Rivera
To cite this article: Vatsal M. Patel, Navin B. Patel, Manuel J. Chan-Bacab & Gildardo Rivera
(2020): N-Mannich bases of benzimidazole as a potent antitubercular and antiprotozoal
agents: Their synthesis and computational studies, Synthetic Communications, DOI:
10.1080/00397911.2020.1725057
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https://doi.org/10.1080/00397911.2020.1725057
N-Mannich bases of benzimidazole as a potent
antitubercular and antiprotozoal agents: Their synthesis
and computational studies
Vatsal M. Patela , Navin B. Patelb, Manuel J. Chan-Bacabc, and Gildardo Riverad
aDepartment of Chemistry, Jamanaben Narottambhai Motiram Patel Science College, Surat, India;
c
bDepartment of Chemistry, Veer Narmad South Gujarat University, Surat, India; Departamento de
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Microbiologıa Ambiental y Biotecnologıa, Universidad Autonoma de Campeche, Campeche, Mexico;
d
ꢀ
ꢀ
Laboratorio de Biotecnologia Farmaceutica, Centro de Biotecnologıa Genomica, Instituto Politecnico
Nacional, Reynosa, Mexico
ABSTRACT
ARTICLE HISTORY
Received 9 December 2019
This article dealing with the microwave assisted synthesis of N-
Mannich bases of pyridine clubbed with two different benzimidazole
cores with their micromolar biological potency. All the synthesized
compounds were evaluated for their in-vitro antibacterial, antimyco-
bacterial and antiprotozoal potency. One of the final compound was
found to be most active against M. tuberculosis (MIC ¼ 3.125 mM) in
the primary screening. N-Mannich bases of benzimidazole with pyri-
dine-3-amine and 5-methyl-pyridine-2-amine showed potency
against L. mexicana and T. cruzi respectively with IC50 value 0.25 and
1.02 mg/mL. Compound 4a showed good binding energy in the
active pocket of receptor (PDB: 4cod) with ꢀ11.013 docking score.
The stability of docked complex was validated by performing
Molecular dynamics (MD) up to 20 ns simulation time. In silico ADME
parameters and toxicity predicted that the active compounds belong
to the Class IV GHS with LD50 value 1360 mg/kg and hence found to
be mildly toxic.
KEYWORDS
antichagasic; antitubercular;
benzimidazole; leishmanici-
dal; MD simulation;
molecular docking
GRAPHICAL ABSTRACT
CONTACT Vatsal M. Patel
patelvatsal2119@gmail.com, patelvatsal1904@gmail.com
Department of Chemistry,
Jamanaben Narottambhai Motiram Patel Science College, Surat, D C Patel Education Campus, Bharthana, Vesu, Surat,
395017 India; Navin B. Patel
University, Surat, India.
drnavinbpatel@gmail.com
Department of Chemistry, Veer Narmad South Gujarat
Supplemental data for this article can be accessed on the publisher’s website
ß 2020 Taylor & Francis Group, LLC

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V. M. PATEL ET AL.
Introduction
The parasitic disease leishmaniasis and chagas which are caused by the protozoa
Leishmania mexicana and protist Trypanosoma cruzi are of the most neglected proto-
zoal diseases. The choice for the antiparasitic treatment for this particular class is also
limited. The available drugs to treat the particular condition of leishmaniasis are limited
to miltefosine, pentavalent antimony, paromomycin, imiquimod, etc. and these pharma-
ceuticals display low efficacy, serious toxic side effects including patient intolerance, and
the induction toward resistant strains opens the door for medicinal chemists to discover
of new safe and effective drugs for leishmaniasis treatment.[1] Anti-chagasic drugs are
also confined to particular azole or nitro derivative class viz., benznidazole or nifurti-
mox. Researchers around the world continue to search for new efficient compounds in
the treatment of this disease. In the continuation of our research[2] to find the better
leishmanicidal and trypanocidal agent, another class of azole is synthesized to get more
potent results is described in this article. According to the latest global tuberculosis
report-2019 released by the World Health Organization (WHO), tuberculosis (TB)
remains the single most lethal infectious disease. As per the report, the cases of exten-
sively drug-resistant (XDR) TB are increased globally by 6 million.[3] The development
for the search of new promising anti-TB agents results in emerging of newer hybrid
molecules of class diarylquinolines, oxazolidinones, nitroimidazoles, etc.[4] Isoniazid
(INH) is one of the most effective and widely used drugs for the treatment of TB.
InhA, the enoyl-ACP reductase in Mycobacterium tuberculosis is found to be an appeal-
ing target for the development of novel drugs against TB.[5] In our previous work, we
report the inhibitor that directly targets InhA which resulted in the development of
newer series of triazole[6] and imidazolinone[7] that are in-vitro micromolar inhibitors
of M. tuberculosis H37Rv.
The biological profile of benzimidazole derivatives occupies an important position in
medicinal chemistry.[8] Benzimidazole has been first synthesized by Hoebrecker in 1872
after that different synthetic routes have been developed to synthesize varied benzimida-
zole analogous by conventional[9–11] and non-conventional[12–15] environmentally
benign approach. After the successful evaluation of the very first benzimidazole scaffold
in 1944 as an antifungal agent, the numbers of research papers have been published
dealing with the various pharmacological importances of benzimidazoles. This versatile
heterocyclic core was found to possess bioactivity as analgesic (Bezitramide), antiviral
(Enviroxime), antiulcer (Omeprazole, Rabeprazole), anti-neoplastic (Nocodazole), anti-
fungal (Ketoconazole, Fluconazole), etc. The presence of benzimidazole nucleus in
numerous categories of therapeutic agents[16] such as antimicrobial,[17] antivirals,[18]
anti-parasites,[19] anticancer,[20] proton pump inhibitors,[21] immunomodulators,[22] hor-
mone modulators,[23] CNS stimulants,[24] etc. has made it an indispensable anchor for
development of new therapeutic agents. On the other hand, Mannich base derivatives of
benzimidazole attain great attention in the development of the newer pharmacologically
important candidate. Mannich bases of benzimidazole derivative were reported for anti-
microbial,[25] antitubercular,[26] analgesic and anti-inflammatory activity,[25] anti-
cancer,[27] antiviral,[28] etc.
In view of the preceding facts, we design newer hybrid candidates that combine ben-
zimidazole moiety with pyridine via N-Mannich reaction[29–31] with the aim to produce

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R =
a
b
e
c
d
Scheme 1. Synthesis of N-Mannich base derivatives (3a-e) & (4a-e).
promising antitubercular and antiprotozoal agents. The substitution pattern was care-
fully chosen to confer different electronic environments to the molecules. All the syn-
thesized molecules were spectrally characterized and subjected to in-vitro biological
evaluation. Furthermore, all synthesized compounds were in silico evaluated for ADME
properties, toxicity profile and were docked against an enoyl-acyl carrier protein(ACP)-
reductase of M. tuberculosis (PDB ID: 4COD)[32] and ACP reducatse of S. aureus (PDB
ID: 4NZ9).[33] Molecular dynamics (MD) simulations was performed for up to 20 ns
simulation time to investigate ligand-protein complex stability.[34]
Result and discussion
Chemistry
The synthetic route of the N-((2-(pyridin-4-yl)1H-benzo[d]imidazol-1-yl)methyl)-substi-
tuted-aminopyridine (3a–e) and N-((2-(3-(trifluoromethyl)styryl)-1H-benzo[d]imidazol-
1-yl)methyl)-substituted-aminopyridine (4a–e) is outlined in Scheme 1. The compound
1 and 2 on reaction with substituted-amino-pyridine and 37% formaldehyde afforded
(3a–e) & (4a–e) via microwave induced synthetic approach. The key intermediate 2-
(pyridin-4-yl)-1H-benzo[d]imidazole (1) and 2-(3-(trifluoromethyl)styryl)-1H-benzo[d]i-
midazole (2) were obtained by the reaction of o-phenylenediamine with isonicotinic
acid and 3-trifluoromethyl cinnamic acid respectively. We have carried out the MW
synthesis under the Q-proM modified microwave synthesis system. The structures of

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V. M. PATEL ET AL.
Table 1. Antiprotozoal, antibacterial, antifungal and antimycobacterial activity.
Antiprotozoal activity
Antibacterial activity
Antifungal activity Anti tubercular activity
MIC mg/mL
IC50 mg/mL
L. mexicana
MNYC/BZ/
62/M379
T. cruzi
MHOM/MX/
94/NINOA
E. coli
MTCC 442
S. aureus
MTCC 96
C. albicans
MTCC 227
M. tuberculosis
Compound No.
H37Rv
1
2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
>50
>50
>50
2.43
>50
0.41
4.59
>50
0.25
9.60
7.04
>50
0.55
–
>50
>50
>50
>50
>50
1.02
>50
>50
1.46
>50
>50
>50
–
200
200
100
100
250
125
200
200
250
125
62.5
250
–
250
62.5
250
100
500
250
125
25
62.5
100
12.5
150
–
500
500
500
1000
500
500
500
250
500
1000
100
1000
–
100
100
500
150
250
125
250
3.125
62.5
150
6.25
125
–
Miltefosine
Benznidazole
Ciprofloxacin
Nystatin
Isoniazid
2.90
–
–
–
25
–
–
–
50
–
–
–
–
100
–
–
–
–
0.2
–
–
–
–
compounds were established on the basis of their spectral data. IR spectrum of final
compounds showed band at cmꢀ1 3390 for N-H, 3055 for aromatic C-H str., 2868 for
1
methylene C-H str., 1430 for methylene C-H bend., and 1608 for C ¼ N. H NMR spec-
trum of (3a–e) showed singlet at 8.60 for NH which disappeared on D2O exchange and
1
doublet at 5.02 for CH2 proton whereas, H NMR (400 MHz, DMSO-d6, d ppm) spec-
trum of (4a–e) showed signal at 12.69 for NH which disappeared on D2O exchange,
doublet at 7.74 and 7.36 for corresponding styryl CH with coupling constant value16 Hz
confirming the trans isomer and doublet at 5.63 for CH2 proton. 13C NMR (100 MHz,
DMSO-d6, d ppm) spectra of final compounds showed at 61.41 for CH2, 150.47, 150.05
for C ¼ N, 136.69 to 106.79 for corresponding aromatic carbons and 112.73 for styryl
carbon. Moreover, the mass spectrum of these compounds agrees with the molecular
weight of the proposed structure. The data generated from all these spectral techniques
confirming the formation of a final compounds.
Biology
In-vitro antiprotozoal activity
Compounds 1, 2, 3a–e and 4a–e were tested in-vitro for their potency as antiprotozoal
agents against Leishmania mexicana and Trypanosoma cruzi. Biological assays results on
two protozoans parasitic tested are summarized in Table 1. The comparison was made
among new benzo[d]imidazole derivatives and the antiprotozoal drugs of choice: milte-
fosine and benznidazole on L. mexicana and T. cruzi, respectively. From series 3a–e,
compounds 3b, 3d and 3e, showed a biological activity against L. mexicana. SAR ana-
lysis, showed that a change in the nitrogen atom position drastically affect the biological
activity, compounds 3a and 3c, have a null leishmanicidal activity (IC50 >50 mg/mL),

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however, compound 3b, showed the higher activity (IC50 ¼ 2.43 mg/mL). After the
incorporation of an electron donating (methyl) group at para-position on the pyridine
ring enhanced the biological activity, compound 3d showed a similar IC50 value
(0.41 mg/mL) that miltefosine (IC50 ¼ 0.55 mg/mL), however a phenoxy group at ortho-
position on the pyridine ring reduce the effect ten times (IC50 ¼ 4.59 mg/mL). From ser-
ies 4a–e, compounds 4b, 4c and 4d showed also a leishmanicidal activity; in this case,
compound 4b had the best biological effect (IC50 ¼ 0.25 mg/mL), two times more than
miltefosine. SAR analysis showed that the position of the nitrogen atom on the pyridine
ring is a key factor in the biological activity. After, substitutions at para- and ortho-
position on the pyridine ring reduce drastically the leishmanicidal effect (IC50 >50 mg/
mL). A comparison between analogous compounds such as 3a and 4a shown that sub-
stitution of pyridine ring at 2-position on benzimidazole ring with a 3-trifluoromethyl-
styryl not affect the biological activity.
The analysis of trypanocidal activity from series 3a–e shown that para-substitution
with electron-donating group affects the biological effects drastically, compound 3d
obtained an IC50 value (1.02 mg/mL) better than benznidazole (2.90 mg/mL). In series
4a–e, the biological behavior was different; here the position of nitrogen atom in the
pyridine ring is the more important factor in the trypanocidal activity. Compound 4b
showed an IC50 value of 1.46 mg/mL, which is better than reference drug.
In-vitro antimicrobial activity
The minimum inhibitory concentrations (MIC) for antimicrobial potency of 1, 2, 3a–e
and 4a–e were screened against two different bacterial strain and one fungal strain. The
susceptibility of the organisms was determined by the broth microdilution method and
compared with standard drugs; ciprofloxacin and nystatin for antibacterial and antifun-
gal activity respectively. The results of this activity are summarized in Table 1. Initially,
compounds 1 and 2showed moderate to poor activity on both bacterial and fungal
strains. Therefore, we had incorporated common amine agents via N-Mannich reaction
in compounds 1 and 2 to enhance the antimicrobial potency. The results show that
there has been no significant improvement for antimicrobial activity while incorporating
different pyridine moieties into compound 1. Interestingly, incorporation of those same
pyridine moieties to compound 2 has the positive antimicrobial effect. Firstly we had
incorporated simple amino pyridine with different in nitrogen position. 4a found to
possess higher MIC values than control drug. An introduction of electron releasing
group (-CH3) on amino pyridine reagent enhances the antimicrobial potency when it
has been incorporated with compound 2. The compound 4d exhibited excellent activity
(MIC ¼ 12.5 lM) against gram-positive S. aureus that compare to control antibacterial
drug. Rests of the compounds were poor to moderately active. None of the compounds
displayed significant potency against gram-negative E. coli. Similar trend has been
observed for the minimum fungicidal concentration against C. albicans. The incorpor-
ation of similar amine component to compound 1 exhibited no interesting results, but
with compound 2 some of the derivatives showed relatively significant result as antifun-
gal. Compound 4d showed potency with MIC value 100 lM against C. albicans, similar
to that of reference drug nystatin. Rests of the compounds showed higher MIC values
against the tested fungi species and hence found to be poorly active or non-active.

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V. M. PATEL ET AL.
From SAR studies we may conclude that the presence of styryl functionality and the
electron releasing group on amine reagent plays an important role in obtaining better
antimicrobial and antifungal activity.
In-vitro antimycobacterial activity
Compounds 1, 2and all the newer Mannich derivatives (3a–e and 4a–e) were evaluated
to find better antimycobacterial agent against M. tuberculosis H37Rv. The evaluation
results are given in Table 1. Both the key intermediate 1 and 2 were found to possess
higher MIC value and hence found to be poorly active. As discussed previously, five dif-
ferent amine reagents were introduced to both key intermediates to acquire better
mycobactericidal candidate. In this studies, we have observed that the change in the
position of nitrogen atom in pyridine ring enhance the antimycobacterial potency.
Initially three different amino pyridines, viz., 2-amino pyridine, 3-amino pyridine and
4-amino pyridine were selected. Incorporation of these three amine reagents with 1
gives no significant potency. Those amine components while incorporated with 2
enhance the activity. The compound 4d displayed encouraging potency whereas 4a
found to possess better micromolar potency (MIC ¼ 3.125 lM) against M. tuberculosis
compared to control drug. The SAR studies revealed that change in amine component
i.e., pyridine-2-amine to pyridine-3-amine decreased the activity by twenty fold.
Moreover, introduction of methyl group decreased the activity by two folds. The pos-
ition of nitrogen atom in pyridine ring and styryl functionality concerned with benzimi-
dazole plays an important role for getting a better antitubercular agent.
Computational studies
In silico molecular docking studies
All the synthesized derivatives 1, 2, 3a–e and 4a–e were docked against two different
targets viz., enoyl-acyl-carrier-protein reductase of S. aureus (PDB ID: 4nz9) and enoyl-
acyl-carrier-protein reductase of M. tuberculosis (PDB ID: 4cod). The results were
described in the terms of docking score, XP GScore, glide evdw, glide ecoul, glide
energy and glide emodel in Tables 2 and 3 respectively. The glide docking score of all
the compounds with respect to 4nz9 were in the range from ꢀ11.792 to ꢀ4.563, where
Table 2. Docking scores of the compounds 1, 2, 3a–e and 4a–e with an enoyl-acyl-carrier-protein
reductase of S. aureus (PDB ID:4nz9).
Compound No.
docking score
XP GScore
glide evdw
glide ecoul
glide energy
glide emodel
1
2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
ꢀ6.015
ꢀ9.87
ꢀ6.021
ꢀ9.904
ꢀ6.463
ꢀ8.664
ꢀ4.573
ꢀ6.525
ꢀ7.781
ꢀ10.528
ꢀ9.83
ꢀ25.626
ꢀ32.055
ꢀ41.076
ꢀ35.872
ꢀ33.565
ꢀ37.268
ꢀ39.597
ꢀ44.011
ꢀ43.087
ꢀ35.511
ꢀ48.446
ꢀ41.051
ꢀ40.801
ꢀ0.759
ꢀ4.722
ꢀ6.236
ꢀ5.167
ꢀ5.416
ꢀ6.39
ꢀ26.385
ꢀ36.778
ꢀ47.311
ꢀ41.039
ꢀ38.981
ꢀ43.658
ꢀ42.51
ꢀ37.331
ꢀ51.7
ꢀ6.299
ꢀ8.617
ꢀ4.563
ꢀ6.146
ꢀ7.776
ꢀ10.528
ꢀ9.692
ꢀ8.601
ꢀ11.792
ꢀ7.034
ꢀ10.007
ꢀ65.547
ꢀ62.179
ꢀ60.383
ꢀ66.441
ꢀ61.869
ꢀ80.684
ꢀ64.128
ꢀ63.435
ꢀ75.542
ꢀ60.071
ꢀ74.439
ꢀ2.913
ꢀ5.869
2.692
ꢀ49.88
ꢀ40.394
ꢀ41.351
ꢀ54.213
ꢀ42.361
ꢀ48.711
ꢀ8.697
ꢀ12.249
ꢀ7.129
ꢀ10.007
ꢀ5.84
ꢀ5.767
ꢀ1.31
4nz9_molecule
ꢀ7.91

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Table 3. Docking scores of 1, 2, 3a–e and 4a–e with an enoyl-[acyl-carrier-protein] reductase of M.
tuberculosis (PDB ID: 4cod).
Compound No.
docking score
XP GScore
glide evdw
glide ecoul
glide energy
glide emodel
1
2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
ꢀ7.769
ꢀ7.664
ꢀ3.331
ꢀ6.288
ꢀ5.139
ꢀ7.626
ꢀ5.416
ꢀ11.013
ꢀ10.154
ꢀ5.352
ꢀ10.679
ꢀ6.168
ꢀ11.160
ꢀ8.523
ꢀ7.775
ꢀ7.698
ꢀ4.172
ꢀ6.335
ꢀ5.235
ꢀ7.631
ꢀ5.86
ꢀ27.871
ꢀ28.765
ꢀ36.83
ꢀ4.185
ꢀ5.450
ꢀ2.48
ꢀ32.056
ꢀ34.214
ꢀ39.31
ꢀ42.749
ꢀ45.298
ꢀ55.639
ꢀ52.324
ꢀ58.608
ꢀ81.452
ꢀ58.410
ꢀ95.986
ꢀ59.458
ꢀ59.915
ꢀ68.454
ꢀ64.492
ꢀ112.71
ꢀ99.810
ꢀ41.374
ꢀ34.301
ꢀ52.113
ꢀ41.966
ꢀ55.204
ꢀ42.31
ꢀ2.119
ꢀ0.594
ꢀ2.464
ꢀ3.246
ꢀ4.873
ꢀ4.402
ꢀ1.427
ꢀ1.119
ꢀ3.086
ꢀ11.75
ꢀ21.305
ꢀ43.493
ꢀ34.895
ꢀ54.577
ꢀ45.212
ꢀ60.077
ꢀ46.711
ꢀ43.517
ꢀ46.456
ꢀ48.085
ꢀ73.454
ꢀ71.604
ꢀ11.266
ꢀ11.016
ꢀ6.510
ꢀ10.817
ꢀ7.302
ꢀ11.160
ꢀ8.523
ꢀ42.089
ꢀ45.337
ꢀ45.001
ꢀ61.695
ꢀ50.299
4cod_molecule1
4cod_molecule2
compound 4d showed very good binding energy in the active pocket of receptor with
ꢀ11.792 docking score (ꢀ12.249 XP Gscore), showed most potent as well in wet lab
result against S. aueus (MIC ¼ 12.5 mM). It was found to interact with amino acid resi-
dues Tyr157 and Tyr147 through p–p stacking and found the potential to inhibit Enoyl
ACP reductase of S. aureus. Figure 1a shows the fit of compound 4d into the active site
of the receptor and the 2D plots of ligand interaction map are shown in Figure 1b for
the same. The same compound also displayed good binding energy in the active pocket
of receptor Enoyl ACP reductase of M. tuberculosis with ꢀ10.154 docking score, showed
moderate potency with in-vitro antimycobacterial potency against M. tuberculosis with
MIC 6.25 mM. Whereas compound 4a interacts within the active pocket of the receptor
with ꢀ11.013 docking score (ꢀ11.266 XP Gscore). On the basis of activity data and
docking result 4a and 4d both had potential to inhibit an Enoyl ACP reductase of
M. tuberculosis. The binding interactions of both of these compounds are almost similar
as both compounds bind with the amino acid residue through hydrogen bonding and
p–p stacking with. For compound 4a, hydrogen bonding and p–p stacking interactions
were observed with Ala198 (Ala198 C ¼ O … … HN of ligand) and Phe97 along with
other hydrophobic interactions with Phe149, Ala157, Tyr158, Met161, Met199, Ala201,
Ile202 and Leu207 of Enoyl ACP reductase. These interactions have been observed for
the co-crystallized ligand of PDB 4cod. Hence this protein-ligand complex has been
selected for further studies. Figures 2a and 3a shows the fit of 4a and 4d into the active
site of the receptor. The 2D plot of protein-ligand interaction diagram for the same was
figured out in 2b and 3b respectively.
MD Simulation studies
The stability of protein-ligand complex formed by molecular modeling was then vali-
dated by implementing a MD simulation using DESMOND. The production run for the
complex was carried out for 20 ns. The Simulation Quality Analysis was performed and
an analysis of the total energy, potential energy, temperature, pressure, and volume over
the length of the simulation was done. The analysis includes the average value, the
standard deviation, and the slope of a linear fit to the values of the property as a func-
tion of time. All the values indicated a stable MD simulation production run (Fig. 4).

8
V. M. PATEL ET AL.
Figure 1. (a) 3D presentation of ligand 4d interaction into the active site of long enoyl-acyl-carrier-
protein reductase of S. aureus (4NZ9). (b) 2D presentation of ligand 4d interacting with amino
acid residues.
To explore the dynamic stability of the complex and to ensure the rationality of the
sampling method, RMSD from the structure in frame 0 was analyzed on a structure in
1001 frames. The plots (Figs. 5–7) showed no significant increase in deviation until the
completion of 20 ns. The ligand RMSD value was also monitored and was found to be

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Figure 2. (a) 3D presentation of protein-ligand interactions of 4a into the active site of an enoyl-
[acyl-carrier-protein] reductase of M. tuberculosis (4COD). (b) 2D presentation of ligand 4a interacting
with amino acid residues.
stabilized during the production run. All the findings strongly support the high stability
of the complex formed.
The descriptive analyses of RMSD of the compounds in 20 ns MD simulation from
the zero frames have been given in Table 4. The maximum RMSD fitted to the target

10
V. M. PATEL ET AL.
Figure 3. (a) 3D presentation of protein-ligand interactions of 4d into the active site of an enoyl-
[acyl-carrier-protein] reductase of M. tuberculosis (4COD). (b) 2D presentation of ligand 4d interacting
with amino acid residues.
ligand in the active pocket of Enoyl ACP reductase complex is 1.376 Å and the standard
deviation for ligand 4a complex with Enoyl ACP reductase is 0.287. Similarly, the mean
RMSD value for C-alpha of protein was 2.104 Å and the standard deviation being 0.252
which showed the stability ligand and protein. The plot indicates the RMSF values for

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Figure 4. Simulation Quality Analysis.
Figure 5. Protein RMDS for Backbone, Side chain and Heavy atoms.
the C-alpha atom of the residues (Fig. 8). The beginning and the end values are high as
they indicate the N-terminal and C-terminal residues. The other residues have reliable
RMSF values below 2 Å.
The interaction of ligand 4a with protein was monitored throughout the simulation
(Figs. 9 and 10). The stability of the hydrogen bonding network predicted by Glide XP
docking method was examined by monitoring the percentage occurrence of predicted
hydrogen bonds during the simulation time. The presence of some prominent hydrogen
bonds between the inhibitor and Enoyl ACP protein reductase with modest to high fre-
quencies has been observed during the analyses of the MD trajectories of input inhibi-
tors. The molecular docking for ligand 4a predicted one hydrogen bond for 4a/Enoyl
ACP reductase complex by GlideXP. However, this hydrogen bond (Ala198 C¼O—HN
of ligand), was observed only during 10% of the MD trajectory. For 52% of the simula-
tion time, a new hydrogen bond was observed with Gln 100 (Gln100 O¼C–NH—N of

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V. M. PATEL ET AL.
Figure 6. Protein RMDS for Ca atoms.
Figure 7. Ligand RMDS.
Table 4. RMSD values to compare the docked conformation.
Mean (Å)
Median (Å)
Standard Deviation
Range (Å)
RMSD_C-alpha
RMSD_Backbone
RMSD_All residues
RMSD_ligand
2.104
2.138
2.935
1.376
2.158
2.193
3.014
1.439
0.252
0.247
0.290
0.287
0.000, 2.517
0.000, 2.540
0.000, 3.366
0.000, 1.939
ligand) as shown in the simulation interaction diagram (Fig. 11). Several strong hydro-
phobic interactions with Met103 have also been observed during the course of the simu-
lation. Some hydrophobic interactions were also observed with Phe97, Phe149, Ala157,
Tyr158, Met161, Met199, Ala201, Ile202 and Leu207, which were short-lived.
Additionally, water bridges have been observed with Met 98, Gln 100 and Ala198 of for
short period during the production run. Figure 10 represents a timeline of the interac-
tions and contacts in terms of H-bonds, hydrophobic interactions, ionic interactions

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SYNTHETIC COMMUNICATIONSV
13
Figure 8. RMSF values for the C-aplha atom of the residues.
Figure 9. Histogram plot for protein-ligand contacts.
and water bridges. The top panel shows the total number of specific contacts the pro-
tein makes with the ligand over the course of the trajectory. The bottom panel shows
which residues interact with the ligand in each trajectory frame. Some residues make
more than one specific contact with the ligand, which is represented by a darker shade
of orange, according to the scale to the right of the plot.
In silico ADMET studies. We have calculated In silico physicochemical properties and
logarithm of partition coefficient to determine lipophilicity (Log Po/w) to forecasting
Lipinski’s, Ghose, Veber and Egan drug-likeness (Table 5) of final compounds using

14
V. M. PATEL ET AL.
Figure 10. A timeline representation of the interactions and contacts (H-bonds, Hydrophobic, Ionic,
Water bridges).
Figure 11. A schematic diagram of detailed ligand atom interactions with the protein residue at the
end of simulation.

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15

16
V. M. PATEL ET AL.
Table 6. In silico pharmacokinetics of 3a-e and 4a-e by SwissADME.
GI
BBB
Pgp
CYP1A2
CYP2C19
inhibitor
CYP2C9
CYP2D6
CYP3A4
log Kp
(cm/s)
Comp. No absorption permeant substrate inhibitor
inhibitor inhibitor inhibitor
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
High
High
High
High
High
High
High
High
High
Low
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
ꢀ6.00
ꢀ6.24
ꢀ6.24
ꢀ5.83
ꢀ5.48
ꢀ4.73
ꢀ4.96
ꢀ4.96
ꢀ4.55
ꢀ4.21
No
SwissADME. Screening of Lipinski’s Rule of Five and Veber filters showed that all the
synthesized compounds meet the criteria of drug-likeness assessment, however in Ghose
and Egan filter screening showed that compounds 3a–e meets all the criteria whereas
4a, 4b, 4c and 4d rejected with one violation. Pharmacokinetics for the same was eval-
uated by using SwissADME online web tool and results have been incorporated in
Table 6. All the compounds showed high gastrointestinal absorption except 4e.
Mannich derivative 3a–e exhibited BBB permeability, whereas 4a-e found to have no
permeability. A similar trend has been observed for p-glycoprotein substrate.
Cytochrome P450 (CYP) isoforms are responsible for metabolism of many drugs.
Inhibition of CYP isoforms results in decreased elimination and change in metabolic
pathways of their substrates, which is the major cause of adverse drug-drug interactions.
It is, therefore, essential to trace out the potential of the compounds for CYP inhibition.
The inhibitory ability (CYP3A4, CYP2D6, CYP2C19, CYP2C9 and CYP1A2) of the syn-
thesized compounds were summarized in Table 6. Most of all the newer Mannich deriv-
atives found the inhibitor of cytochrome P450 isoforms (CYP2C19 and CYP2D6).
Except for 4d, all the derivatives found the inhibitors of other cytochrome P450 iso-
forms (CYP1A2 and CYP2C9). 4d and 4e showed no ability to inhibit CYP3A4. The
values of Log Kp indicate the skin permeation for the respective inputs. The computa-
tional toxicity predictions can help to reduce the number of animal experiments and
save animal lives. From the wet lab and molecular modeling results, four compounds
viz., 3d, 4a, 4b and 4d have been selected for further screening to analyze their overall
drug score and toxicity risks. In slico toxicology predictions were evaluated by using
ProTox-II and results are given in Table 7. All the selected compounds had LD50 values
between 500–1360 mg/kgBodyWeight and belong to class IV Global Harmoni System
(GHS) indicating that it could be harmful if swallowed. As all the compounds belong to
class IV, it is predicted to found moderate to mild toxicity. Higher the value of LD50
displayed the lower its toxicity. 4a was of moderate toxicity with the LD50 value of
1360 mg/kgBW. From the results, it is predicted that all the selected compounds have
no hapatotoxicity and cytotoxicity. As the inputs showed inactive to organ toxicity, it
can be predicted that these inputs have no toxic effect on the liver. Moreover results
predicted to have mutagenic toxicity for all four inputs and active immunetoxicity
expect 3d.

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17
Table 7. In silico toxicology prediction of 3d, 4a, 4b and 4d by ProTox.
Toxicity end point
(Probability)
Organ toxicity
(Probability)
ꢂ
Oral toxicity prediction
Predicted Predicted
Comp. No LD50 mg/kg toxicity class Hepatotoxicity Carcinogenicity Immunotoxicity Mutagenicity Cytotoxicity
3d
4a
4b
4d
ꢂ
500
4
4
4
4
Inactive (0.71) Active (0.59)
Inactive (0.59) Active (0.58)
Inactive (0.59) Active (0.58)
Inactive (0.58) Active (0.58)
Inactive (0.9)
Active (0.96)
Active (0.96)
Active (0.96)
Active (0.65) Inactive (0.78)
Active (0.59) Inactive (0.81)
Active (0.59) Inactive (0.81)
Active (0.59) Inactive (0.80)
1360
1000
1000
Prediction accuracy 54.26 % for 3d, 4a, 4b and 4d.
Experimental
Reduce pressure distillation has been employed to remove and recover the solvents.
Melting points were determined by the open tube capillary method and are uncorrected.
The reaction was monitored by TLC plates (silica gel G) using different eluent system
and visualized under ultraviolet (UV) light, or iodine vapor. The IR spectra were
obtained on Thermo scientific Nicolet iS10 & Agilent Resolution Pro FT-IR spectrom-
1
eter (KBr pellets). The H-NMR spectra were recorded on a Bruker Avance II 400 MHz
while 13C NMR spectra 100 MHz spectrometers using Trimethysilane (TMS) as the
internal standard in deuterated dimethylsulfoxide (DMSO-d6) & deuterated chloroform
(CDCl3) solvents. Chemical shifts were reported in ppm units with the use of d scale.
The mass spectra were recorded by Waters, Q-TOF micromass (ESI-MS), SAIF,
Chandigarh. The microwave induced synthesis were carried out in a ‘‘QPro-M Modified
Microwave Synthesis System’’ manufactured by Questron Technologies Corporation,
Ontario L4Z 2E9 Canada, whereby microwaves are generated by magnetron at a fre-
quency of 2450 MHz having an output energy range of 100–500 W. The QPro-M
designed as multimode microwave cavity to perform faster Digestion (Open vessel),
evaporation, extraction and synthesis with ease.
3-Trifluromethyl-cinnamic acid required to synthesize the key intermediate (2) has
been synthesized as described in the literature.[35] Other than this, all the reagents or
starting materials used for the synthesis were taken directly and were used without fur-
ther purification and the solvents used were LR and AR grade. Full experimental detail
of the synthesis and characterization of intermediate (1) and (2) has been given in the
supporting information.
General procedure for synthesis of (3a–e) & (4a–e)
Benzo[d]imidazole (1) or (2) (1.0 mmol) was dissolved in methanol (10 vol). To this
solution, 0.19 mL 37% formaldehyde was added and stirred at 5–10 ꢁC for about 10 min.
Then a solution of substituted amino pyridine (1.0 equiv) in methanol (10 vol) was
added drop wise to resulting reaction mixture at the same temperature with vigorous
stirring for 1 h. The reaction mixture was then introduced to the microwave oven and
was irradiated for 8–9 min at 60–65 ꢁC (300 W) while monitoring the course of reaction
by TLC (using dichloromethane: methanol, 9:11). Then after reaction mass was kept
overnight at room temperature. The resulting solid was collected by filtration, washed
with cold petroleum ether (2 X 2.5 mL), dried and recrystallized from ethanol to
obtained pure (3a–e)
&
(4a–e). N-((2-(pyridin-4-yl)-1H-benzo[d]imidazol-1-

18
V. M. PATEL ET AL.
yl)methyl)pyridin-2-amine (3a) Yield 87%, m.p. 218–220 ꢁC, IR (KBr) ꢀ cmꢀ1: 3396
(N-H), 3055, 2936 (aromatic C-H), 2868 (methylene C-H, str.), 1606 (C¼N), 1430
1
(methylene C-H, bend.), 1350 (C-N); H NMR (400 MHz, DMSO-d6) d (ppm): 8.59 (t,
1H, NH, disappeared on D2O exchange), 8.32–7.71 (m, 4H, CH, aromatic; 4-pyridine),
7.46–7.08 (m, 8H, CH, aromatic; 2-pyridine & benzimidazole), 5.02 (d, 2H, CH2); 13C
NMR (100 MHz, DMSO-d6) d (ppm): (150.47; N–C¼N), (149.78, 135.19, 121.43; 4-pyri-
dine), (145.66, 140.75, 137.09, 123.67, 119.02; 2-pyridine), (122.58, 120.30, 118.53,
111.13, 106.79, 105.48; benzimidazole), 61.41 (CH2); ESI–MS: m/z calculated 301.13,
found [M þ H]þ 302.13; Anal.Calcd. for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found:
C, 71.65; H, 4.93; N, 23.13%.
Full experimental protocol to synthesize (3b–e) and (4a–e) with their characterization
data has been proved in the supporting information.
Conclusion
The present article deals with the synthesis of newer analogous of benz[d]imidazoles
which were synthesized by N-Mannich reaction using different pyridine amines by MW
induced synthetic approach. The synthetic pathways explain the edge of MW assisted
synthesis with the simplicity of procedure, increased reaction rate and escalate product
yields. These derivatives assessed for their antimicrobial, antituberculosis and antiproto-
zoal activity. Modification in key intermediate 1 and 2, impact the potency against dif-
ferent biological target. The electron releasing group on pyridine ring attached 2-
(pyridin-4-yl)-1H-benzo[d]imidazole showed potency against T.cruzi. The antimicrobial
and antifungal potency of 4d explains the key factor responsible for its potency is the
presence of electron realizing group on pyridine component along with the styryl func-
tionality in benz[d]imidazole. Hence, the modification of amine component with vari-
ous electron releasing and electron withdrawing substituents impact the biological
profile of derived motif against different biological target. 4d showed very good binding
energy in the active pocket of enoyl ACP reductase of S. aureus with ꢀ11.792 docking
score. It was also observed that the promising antimicrobials have proved to be encour-
aging antitubercular. The change in the position of nitrogen atom in pyridine ring
attached 2-(3-(trifluoromethyl)styryl)-1H-benzo[d]imidazole plays an important role to
attain better antimycobacterial activity as well as in-vitro leishmanicidal effect.
Compound 4a showed potency against M. tuberculosis H37Rv strain along with Glide
XP Gscore ꢀ11.266. Based on GlideXP docking simulations, one hydrogen bond with
Ala198 and one pi-pi inaction with Phe97 were predicted for the compound 4a to
Enoyl ACP reductase complex. However, this hydrogen bond was observed during 10%
of the MD trajectory and for 52% of the simulation time, a new interaction was
observed with Gln 100 (Gln100 O¼C–NH—N of ligand) displaying the stability of the
protein-ligand complex. In silico predicated toxicity results and calculated physicochemi-
cal properties, lipophilicity, pharmacokinetic parameters give the best choice for the
preparation of new derivatives with improved antitubercular and antiprotozoal activity
in the future.

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Acknowledgments
The authors are thankful to Veer Narmad South Gujarat University for providing necessary facili-
ties. The authors are thankful to Dr. Dhanji Rajani, Microcare Laboratory, Surat, for antimicro-
bial activity.
Disclosure statement
The authors declared no conflict of interest.
ORCID
Vatsal M. Patel
http://orcid.org/0000-0002-2286-1915
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