Density functional theory molecular modeling, chemical synthesis, and antimicrobial behaviour of selected benzimidazole derivatives

Article abstract of DOI:10.1016/j.molstruc.2016.10.066

Eco-friendly, one-pot, solvent-free synthesis of biologically active 2-substituted benzimidazoles is presented and discussed herein. Novel N-Mannich bases are synthesized from benzimidazoles, secondary amines and formaldehyde, and their structures are confirmed by ¹H nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and elemental analysis. All benzimidazole derivatives are evaluated by qualitative and quantitative methods against 9 bacterial strains. The largest microbicide and anti-biofilm effect is observed for the 2-(1-hydroxyethyl)-compounds. Density functional theory (DFT) modeling of the molecular structure and frontier molecular orbitals, i.e. highest occupied molecular orbital and lowest unoccupied molecular orbital (HOMO/LUMO), is accomplished by using the GAMESS 2012 software. Antimicrobial activity is correlated with the electronic parameters (chemical hardness, electronic chemical potential, global electrophilicity index), Mullikan atomic charges and geometric parameters of the benzimidazole compounds. The planarity of the compound, symmetry of the molecule, and the presence of a nucleophilic group, are advantages for a high antimicrobial activity. Finally, we briefly show that further accurate processing of such compounds into thin films and hybrid structures, e.g. by laser ablation matrix-assisted pulsed laser evaporation and/or laser-induced forward transfer, may indeed provide simple and environmental friendly, state-of-the-art solutions for antimicrobial coatings.

Full text of DOI:10.1016/j.molstruc.2016.10.066

Accepted Manuscript
Density functional theory molecular modeling, chemical synthesis, and antimicrobial
behaviour of selected benzimidazole derivatives
Maria Marinescu, Diana Gabriela Tudorache, George Iuliu Marton, Christina-Marie
Zalaru, Marcela Popa, Mariana-Carmen Chifiriuc, Cristina-Elena Stavarache, Catalin
Constantinescu
PII:
S0022-2860(16)31123-1
10.1016/j.molstruc.2016.10.066
MOLSTR 23067
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 20 June 2016
Revised Date: 31 August 2016
Accepted Date: 20 October 2016
Please cite this article as: M. Marinescu, D.G. Tudorache, G.I. Marton, C.-M. Zalaru, M. Popa, M.-C.
Chifiriuc, C.-E. Stavarache, C. Constantinescu, Density functional theory molecular modeling, chemical
synthesis, and antimicrobial behaviour of selected benzimidazole derivatives, Journal of Molecular
Structure (2016), doi: 10.1016/j.molstruc.2016.10.066.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Density functional theory molecular modeling, chemical synthesis,
and antimicrobial behaviour of selected benzimidazole derivatives
∗
1
1
2
1
Maria Marinescu , Diana Gabriela Tudorache , George Iuliu Marton , Christina-Marie Zalaru ,
3,4
3,4
5
6,7
Marcela Popa , Mariana-Carmen Chifiriuc , Cristina-Elena Stavarache , Catalin Constantinescu
1
UB - University of Bucharest, Faculty of Chemistry, Department of Organic Chemistry,
Biochemistry and Catalysis, 90-92 Şoseaua Panduri, Sector 5, RO-050663 Bucharest, Romania
2
UPB - University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science,
1
-7 Polizu bd., RO-011061 Bucharest, Romania
3
UB - University of Bucharest, Faculty of Biology, Department of Botanic-Microbiology,
-3 Aleea Portocalilor, RO-60101 Bucharest, Romania;
1
4
ICUB - Research Institute of the University of Bucharest,
1-95 Splaiul Independentei, RO-050095 Bucharest, Romania
9
5
Institute of Organic Chemistry “C.D. Nenitzescu” of the Romanian Academy,
02B Splaiul Independentei, RO-060023, Bucharest, Romania
2
6
INFLPR - National Institute for Laser, Plasma and Radiation Physics, Department of Lasers,
09 Atomistilor St., RO-077125 Magurele (Bucharest), Romania
4
7
University of Limoges, CNRS, ENSCI, SPCTS - UMR 7315, F-87000 Limoges, France
Abstract
Eco-friendly, one-pot, solvent-free synthesis of biologically active 2-substituted benzimidazoles is presented and discussed
herein. Novel N-Mannich bases are synthesized from benzimidazoles, secondary amines and formaldehyde, and their
1
structures are confirmed by H nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and elemental
analysis. All benzimidazole derivatives are evaluated by qualitative and quantitative methods against 9 bacterial strains.
The largest microbicide and anti-biofilm effect is observed for the 2-(1-hydroxyethyl)-compounds. Density functional
theory (DFT) modeling of the molecular structure and frontier molecular orbitals, i.e. highest occupied molecular orbital
and lowest unoccupied molecular orbital (HOMO / LUMO), is accomplished by using the GAMESS 2012 software.
Antimicrobial activity is correlated with the electronic parameters (chemical hardness, electronic chemical potential,
global electrophilicity index), Mullikan atomic charges and geometric parameters of the benzimidazole compounds. The
planarity of the compound, symmetry of the molecule, and the presence of a nucleophilic group, are advantages for a high
antimicrobial activity. Finally, we briefly show that further accurate processing of such compounds into thin films and
hybrid structures, e.g. by laser ablation matrix-assisted pulsed laser evaporation and/or laser-induced forward transfer,
may indeed provide simple and environmental friendly, state-of-the-art solutions for antimicrobial coatings.
Keywords: 2-substituted benzimidazoles; antimicrobial activity; spectral analysis; disc diffusion, minimal inhibitory
concentration (MIC), biofilm, laser processing.
*
Corresponding authors: maria.marinescu@chimie.unibuc.ro, catalin.constantinescu@inflpr.ro, catalin.constantinescu@unilim.fr
1

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1. Introduction
With the occurrence of multi-drug, extended-drug, and pan-drug resistance phenotypes,
bacterial resistance to antibiotics is one of the most important global health problem, justifying the
necessity of identifying innovative, radical antimicrobial agents [1]. The antibiotic resistance problem
is amplified by the ability of bacteria to grow in a sessile, adherent state, developing microbial
communities called biofilms, that exhibit a phenotypic resistance, i.e. tolerance, rendering them to
1,000-fold more resistant to antibiotic treatments with respect to their planktonic counterparts [2, 3].
Benzimidazole is a bi-cyclic aromatic organic compound, consisting of the fusion of benzene
and imidazole. Together with its derivatives, they represent an important class of bioactive molecules
of biological and pharmaceutical interest [4], mainly used as ligands for transition metal complexes.
To better exemplify, the most prominent natural benzimidazole compound is N-ribosyl-
dimethylbenzimidazole, which serves as an axial ligand for cobalt in vitamin B . They are important
1
2
not only as structural units in natural compounds and synthetic pharmaceuticals, but have also
elicited considerable theoretical interest. In recent years, reports on compounds which contain the
benzimidazole moiety have increased. Various pharmaceutical applications of such compounds
include anticancer [5-10], antihypertensive [11, 12], anti-inflammatory [13, 14], antimicrobial [15-17],
antiviral [18, 19] and lipid modulating [20, 21] activities. Some benzimidazole compounds (e.g.
astemizole) have demonstrated anti-prion activity, with a view to possible treatment for Creutzfeldt–
Jakob disease, while other compounds (e.g. albendazole) are currently used as medication for the
treatment of a variety of parasitic worm infestations. Some benzimidazole compounds also exhibit
fungicidal properties, mainly due to their binding to the fungal microtubules and stopping hyphal
growth. They are valuable agents in Alzheimer disease [22], and are also used as psychoactive drugs
[
23]. Furthermore, benzimidazoles are mentioned as anticoagulants [24] and antidiabetic agents [25].
In this work, we present and discuss results on the eco-friendly synthesis of 2-substituted
benzimidazoles and new Mannich bases (Figure 1), density functional theory (DFT) study for these
structures, antimicrobial activity screening, followed by a brief discussion on their potential use as
antimicrobial coatings accomplished by thin film laser processing. The antimicrobial activity of the
compounds was assessed on Gram-negative (Escherichia coli ATCC 8739, Klebsiella pneumoniae 40,
Enterobacter cloacae 56, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii complex
230) and Gram-positive (Staphylococcus aureus ATCC 6538, Stapylococus saprophyticus ATCC 15305,
Enterococcus faecium 17, Enterococus faecalis ATCC 29212) bacterial strains.
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2. Experimental section
2.1 Materials and devices
All chemicals and reagents (provided by Sigma-Aldrich) were used without any further
purification. Elemental analysis was performed with a “multi EA 4000” device from “Analytik Jena”.
Fourier transform infrared (FTIR) spectra have been acquired by using a “Vertex 70-Bruker”
1
spectrophotometer, in KBr pellets. The H nuclear magnetic resonance (NMR) spectra have been
recorded on a “Varian Inova-400” (400 MHz, 2 RF channels) in deuterated dimethyl sulfoxide (DMSO-
1
d
6
). The chemical shifts (δ, ppm, TMS internal standard) by H NMR and the FTIR spectra have all been
recorded at room temperature.
2.2 Synthesis of benzimidazole compounds
A mixture of organic acid (40 mmol) and o-phenylenediamine (40 mmol), thoroughly grounded
with a pestle in a mortar at room temperature until liquefied, was subsequently heated at 140°C for
.5 - 2 h. The progress of the reaction was monitored by thin-layer chromatography (TLC). After
1
cooling, the resulting mass was washed with water, filtered, and the final product was re-crystallized.
The synthesized 2-benzimidazoles were then characterized and confirmed by comparing their physical
data with those of literature known compounds, as follows.
2.2a Synthesis 2-substituted benzimidazoles
(1A) (S)-1-(1H-Benzo[d]imidazole-2-yl)ethanol, colourless solid. Yield 80%; m.p. 177-178°C,
−1
similar to [26] (m.p. 176-178°C) IR (cm , KBr): 3356 (aromatic –NH bending), 3364 (OH stretching),
3070 (aromatic C-H stretching), 1505 (C-N stretching), 1458 (-C=C stretching), 1315 (-C-N stretching);
1
H-RMN: δ 7,58 (d, 2H), 7,26 (t, 2H), 1,72 (d, 3H, CH ), 5,21 (q, 1H), 2.3 (brs, 1H, NH), 2,28 (s, 1H, OH).
3
Elemental analysis (%) found for C H N O: C, 66.57; H, 6.25; N, 17.29; O, 9.89. calcd.: C, 66.65; H,
9
10 2
6.21; N, 17.27; O, 9.86%.
(
2B) 2-Phenyl-1H-benzo[d]imidazole, white solid. Yield 75%; m.p. 291-293°C, similar to [27]
−
1
(
(
m.p. 291-293°C); IR (cm , KBr): 3352 (aromatic –NH bending), 3044 (aromatic C-H stretching), 1622
alkenyl C=C stretch), 1587, 1537 (aromatic ring stretch) 1458 (-C=C stretch), 1410 (vinyl C-H in-plane
1
bend), 1312, 1290 (vinylidene C-H in-plane bend); H-RMN: δ 12.91 (brs, 1H, NH), 8.13 (d, 2H, J = 7.0
10 2
Hz), 7.51–7.43 (m, 5H), 7.19 (brs, 2 H). Elemental analysis (%) found for C13H N : C, 80.35; H, 5.22; N,
14.41. Calcd: C, 80.41; H, 5.17; N, 14.42%).
3

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(
3B) 2-o-Tolil-1H-benzo[d]imidazole, colourless solid. Yield 72%; m.p. 220-221°C, similar to [28]
−
1
(
m.p. 220-222°C); IR (cm , KBr): 3355 (aromatic –NH bending), 3087, 3062 (aromatic C-H stretching),
2925 (alkyl stretch), 1620 (alkenyl C=C stretch), 1614, 1505 (aromatic ring C-C stretch) 1458 (-C=C
1
stretch), 1410 (vinyl C-H in-plane bend), 1086, 1035 (in plane C-H bending), 738 (C-H bending); H-
NMR: δ 13.08 (br s, 1H), 7.82 - 7.75 (m, 3H), 7.60 - 7.58 (m, 1H), 7.55 - 7.43 (m, 4H), 2.56 (s, 3H).
Elemental analysis (%) found for C H N : C, 80.72; H, 5.72; N, 13.55. Calcd: C, 80.77; H, 5.76; N,
1
4 12 2
13.46%).
(
4B) 1-(1H-benzo[d]imidazole-2-yl)ethanone, synthesized according to the procedure
−1
described in [29]. Brown crystals, Yield (90%). IR (cm , KBr): 3356 (aromatic –NH bending), 3070
(
aromatic C-H stretching), 1705 (C=O stretch), 1612, 1505 (aromatic ring C-C stretch), 1458 (-C=C
1
stretching), 1315 (-C-N stretching); H-RMN: δ 2,83 (s, 3H), 7,58 (t, 2H), 7,91 (s, 1H), 7,54 (s, 1H), 7,26
(
s, 2H), 10,25 (s, 1H, NH). Elemental analysis (%) found for C H N O: C, 67.41; H, 5.03; N, 17.52; O,
9 8 2
10.04. Calcd: C, 67.50; H, 5.00; N, 17.50; O, 10.00%).
2
.2b Synthesis of the Mannich bases
A Mannich base is a beta-amino-ketone, formed by the reaction of an amine, formaldehyde
or another aldehyde) and a carboxilic acid. Here, the Mannich bases were prepared in a solution of 2-
(
substituted benzimidazole (0.005 mol) in 10 ml ethanol, 0.005 mol of secondary amine and 0.005 mol
of formaldehyde. The reaction mixtures were stirred at room temperature for 2h and then refluxed
for an additional 3h. After cooling, the products were filtered, dried, and re-crystallized from
dimethylformamide (DMF).
(
1M) Dimethyl(2-o-tolyl-1H-benzo[d]imidazole-1-yl)methanamine. Pale yellow (crystals). TLC:
−1
R (ethyl acetate : methanol : chloroform 4:3:3 v/v): 0.65; Yield 65%; m.p. 175-176°C. IR (cm , KBr):
f
2
990, 1450, 1439 methyl CHasym; 1485 CH bending; 1210 aromatic tertiary amine; 1028m skeletal C-C
2
vibrations; 1583 vs C=C aromatic stretch; 2876 methylene CH asym/sym stretch; 766 orto-phenylene.
1
H-RMN: δ 7,65 (dd, 2H, J = 2, 7Hz), 7,35 (d, 1H, J = 8Hz), 7,23 (m, 2H), 7,11 (m, 3H), 4,80 (s, 2H, CH
,27 (s, 6H, CH ). Elemental analysis (%) found for C17 : C, 76.91; H, 7.22; N, 15.86. Calcd for
C H N : C, 76.95; H, 7.21; N, 15.83%).
2
),
2
3
19 3
H N
1
3 10 2
(
2M) 1-(1-((Diethylamino)methyl)-1H-benzo[d]imidazol-2-yl)ethanol. Pale yellow (semisolid).
−
1
TLC: R (ethyl acetate : methanol : chloroform 4:3:3 v/v): 0.72; Yield 75%; IR (cm , KBr): 3200-3400
f
normal ‘‘polymeric’’ OH stretch, 3080 (aromatic C-H stretch), 2904, 2822 methyne C-H stretch, 1457
4

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νC=C aromatic stretch, 1352, 1341 methyne C-H bend, 1314, 1223 aromatic tertiary amine, 1271
1
primary OH in plan bend, 743 primary OH out-of-plane bend. H-RMN: δ 7,80 (d, 1H, J = 2 Hz), 7,53 (s,
1
H), 7.34 (t, 1H, J = 3 Hz), 7.20 (t, 1H, J = 3Hz), 5.07 (q, CH, J = 2Hz), 4.83 (dd, CH , J = 3, 14 Hz), 2.65
2
(
doublet sextet, 2 CH , J = 1, 12 Hz), 2.20 (s, 1H from OH), 1.80 (d, 3H, CH , J = 2Hz), 1.11 (t, 6H, 2 CH ,
2 3 3
J = 2Hz). Elemental analysis (%) found for C H N O: C, 72.05; H, 9.03; N, 12.04; O, 6.88, calcd.: C,
1
4 12 2
72.10; H, 9.01; N, 12.01; O, 6.87%.
2.3 Antimicrobial activity assays
2.3a Qualitative screening of the antimicrobial activity
8
-1
Standardized bacterial suspensions with a density of 1.5 -3 x 10 CFU mL (corresponding to
the 0.5 McFarland nephelometric standard) were obtained from 15-18h fresh bacterial cultures
developed on solid media. The compounds were suspended in DMSO to prepare a stock solution of
-1
1
0 mg /mL concentration. The antimicrobial activity was tested on Mueller–Hinton Agar (MHA)
medium. The qualitative screening was performed by an adapted disc diffusion method [30].
2.3b Quantitative assay of the antimicrobial activity
The quantitative assay of the antimicrobial activity was performed by liquid medium
microdilution method in 96 multi-well plates. Two-fold serial dilutions of the compounds solutions
-1
(
ranging between 1000 μg and 4 μg mL ) were performed in a 200 μL volume of broth, and each was
well seeded with 50 μL microbial inoculum. Bacterial culture positive controls (wells containing
culture medium seeded with the microbial inoculum) as well negative sterility controls (containing
only culture medium) were used. The influence of the DMSO solvent was also quantified in a series of
wells containing DMSO, diluted accordingly with the dilution scheme used for the tested compounds.
o
The multi-well plates were incubated for 24h at 37 C, and the minimal inhibitory concentration (MIC)
values were considered as the lowest concentration of the tested compound that inhibited the visible
growth of the microbial overnight cultures, as compared to the positive control, correlated with a
decreased value of the absorbance read at 600 nm (by using an “Apollo LB 911” ELISA reader).
2.3c Quantitative assay of the anti-biofilm activity
In order to evaluate the influence of the obtained compounds upon the colonization ability of
microbial strains to the inert substratum, a microtiter method was employed. The multi-well plates
5

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used for the MIC assay were emptied and washed three times with phosphate buffered saline. The
biofilm formed on the plastic wells wall. After it was fixed for 5 min with cold methanol and coloured
for 15 min by violet crystal solution, it was re-suspended in a 33% acetic acid solution. The minimal
biofilm eradication concentration (MBEC) values were considered at the lowest concentration of the
tested compound that inhibited the development of biofilm on the plate wells, as revealed by the
decreased values of the optical density of the coloured solution at 490 nm, and as compared to that
of the positive control [31].
2.4 Computational and modeling details
A quantum mechanical modeling method was implemented for each benzimidazole
compound, using the GAMESS 2012 software [32], in order to assess their structural parameters. The
modeling was performed on a computer cluster consisting of 12 nodes and 96 cores running on Linux
CeontOS. The results were visualized using wxMacMolPlt [33]. The molecular geometries of the
benzimidazoles were optimized by using DFT at M11/ktzvp level of theory. Tuhlar's M11 [34] is a
modern range-separated hybrid functional that provides better results compared to the traditional
B3LYP functional class of approximations to the exchange–correlation energy in DFT. Also, the basis
set that we used is a more recent one (Karlsruhe valence triple zeta basis with a set of single
polarization), introduced by Prof. Ahlrichs [35, 36]. The parameters used for geometry optimization
are the default ones used in GAMESS, and the geometry used during the calculations is described by
"
natural internal coordinates" generated by the software.
3. Results and discussion
3.1 Synthesis of benzimidazoles and benzimidazole Mannich bases
Three 2-substituted-benzimidazoles have been synthesized using an eco-friendly, one-pot,
solvent-free reaction, from 1,2-phenylenediamine and the corresponding carboxylic acid: (S)-lactic,
benzoic, and o-toluic acid, respectively. For the lactic acid (liquid), the reactants were mixed
thoroughly for a given period of time, instead of grinding. It should be noted that, by the eco-friendly
method presented herein, better yields have been obtained in comparison to those reported in
literature by other classical methods [37]. 1-(1H-Benzo[d]imidazole-2-yl)ethanone (4B) was obtained
by classic oxidation of (S)-1-(1H-benzo[d]imidazole-2-yl)ethanol with potassium dichromate and acetic
acid. As it was previously mentioned, the new Mannich bases were synthesized by stirring of the 2-
6

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substituted benzimidazoles, the secondary amine, and the formaldehyde, in a molar ratio of 1:1:3.
The two methylene protons from the -CH - group in the second Mannich base (2M) are diastereotopic
2
and, as a consequence, they appear as a doublet in doublets at 4.83 ppm. Also, the protons from -
CH - ethylene groups are diastereotopic, and a doublet of sextets at 2.65 ppm can be observed.
2
Antimicrobial properties of all benzimidazole derivatives have been screened, as they are presented
and discussed in the following sections.
3.2 Antimicrobial activity
The antimicrobial activity of these compounds have been assessed against a range of
planktonic and adherent bacterial strains, including all “ESKAPE” pathogens, which are exhibiting
epidemiologically important resistance mechanisms, namely Escherichia coli (E. coli), Staphylococcus
aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii),
Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecalis (E. faecalis) [38]. In the qualitative
assay, we have quantified the growth inhibition zones, induced after the deposition of 10 µl of the
DMSO stock solution (10 mg/ml concentration) over the microbial culture (Table 1). From the tested
compounds, the most active proved to be 3B, active against five of the 9 tested strains, mostly Gram-
positive ones, followed by 2B and 1B, each of them active against four tested microbial strains.
Compound 2B exhibited the largest spectrum of activity, including Gram positive cocci (E. faecium),
Pseudomonadaceae (P. aeruginosa, A. baumannii) and Enterobacteriaceae (E. coli), while compound
1
B antimicrobial spectrum included only Gram-negative strains. Compound 2M was active against the
two Enterococcus sp. strains and the E. cloacae strain, while compounds 4B, 1M exhibited a much
lower and narrower antimicrobial activity, against two or one of the tested Gram positive cocci
strains. The quantitative assay of the microbicidal activity for the tested compounds confirmed the
results obtained in the qualitative assay only for the compound 2B, for which the same spectrum of
activity has been recorded, with MIC values ranging from 0.5 to 0.125 mg/ml (Table 2). Compound 3B
exhibited moderate antimicrobial activity (MIC of 0.25-0.5 mg/ml) against the members of
Pseudomonadaceae family and very good MIC (0.031 mg/ml) against E. coli. Compound 1B exhibited
very low MIC values against S. saprophyticus (0.002 mg/ml), P. aeruginosa (0.008 mg/ml) and K.
pneumoniae (0.031 mg/ml). Compound 2M was active against the two Enterococcus sp. strains, with
MIC values ranging from 0.008 tp 0.031 mg/ml and the K. pneumoniae strain (0.031 mg/ml). The
investigation of the anti-biofilm activity of the obtained compounds revealed a different behaviour as
7

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compared to their microbicidal properties (Table 3). Compound 1B proved to be the most effective
anti-biofilm agent, inhibiting the biofilm development of strains belonging to all three bacterial
groups, i.e. Gram positive cocci, Pseudomonadaceae and Enterobacteriaceae, the lowest MBEC value
(
0. 008 mg/ml) being recorded against E. faecium. The second most active anti-biofilm compound was
M, also exhibiting low MBEC against the ability to form biofilms by three strains belonging to all
2
three tested bacterial groups. Compound 2B exhibited anti-biofilm activity against E. faecium and
Pseudomonadaceae strains, with very good anti-P. aeruginosa activity. Compound 4B exhibited very
good activity against E. faecium with MBEC of 0.008 mg/mL. It is to be noticed that two of the most
feared, biofilm forming opportunistic and nosocomial pathogens, i.e. E. faecium and P. aeruginosa,
proved to be very susceptible to at least 4 of the tested compounds, proving their utility for the
development of novel anti-biofilm agents.
3.3 Electronic and structural properties
The molecular geometries of the benzimidazole compounds were optimized using DFT at
M11/ktzvp level [34-36]. The parameters of the optimized geometries of the compounds were
subsequently determined. Relatively small molecules belonging to the group of symmetry C1, with
lengths between 8.935 Å and 10.775 Å can be seen in Table 4. The smallest molecule is 1B, which has
the best anti-biofilm activity. Mannich base 1M has a total energy of -823.309 Hartree (Ha), thus it is
the most stable of all compounds, which correlates very well with its poor antimicrobial activity.
3.3a Electronic parameters of the benzimidazole compounds
The following parameters of the benzimidazole compounds have been obtained by using DFT
at M11/ktzvp level of theory, i.e. EHOMO (energy of high occupied molecular orbital), ELUMO (energy of
low unnoccupied molecular orbital), Egap (EHOMO- ELUMO), η (chemical hardness), µ (electronic chemical
potential), ω (global electrophilicity index), are shown in Table 5. These were used to appreciate
chemical reactivity indices of benzimidazole compounds.
Chattaraj et al. [39] noted that the larger the HOMO–LUMO energy gap is, the harder and
more stable/less reactive the molecule reveals to be. This means that the order of the decreasing the
reactivity from the Egap value is:
2
B > 3B > 4B > 1M > 1B > 2M.
8

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It can be seen that for compounds 1B and 2M, the data correlate poorly. The stability and the
reactivity of an organic compound are correlated with the chemical hardness, which may be
calculated by using the following equation [40]:
ELUMO − EHOMO
η
=
2
From the calculated values of the chemical hardness (η) of the studied benzimidazoles and
Mannich bases, one can see that compound 2B should be the most reactive, because it is less hard of
all, followed by 3B, 4B, 1M, and 1B, 2M should be the least reactive. Again, one could say that for
compound 1B data do not fit all. Parr defined the negative of the electronegativity as the electronic
chemical potential of a molecule, determined by the equation:
EHOMO + ELUMO
µ
=
2
It is known that the greater the electronic chemical potential (µ) is, the less stable or more reactive
the compound reveals to be. In Table 4, one can see that the decreasing order of the compounds
reactivity is as follows:
1
B > 1M > 2M > 3B > 2B > 4B.
Compound 1M correlates the worst in this case, but for the first time we find a parameter that
correlates the activity of compound 1B. Parr introduced the global electrophilicity index using the
electronic chemical potential and chemical hardness [41]:
2
µ
ω
=
2η
This index measures the stability of one molecule after it accepts an electronic charge from the
surrounding environment. In Table 5, one can notice a decreasing order for the
ω parameter, which
means an increasing order for the stability of the molecules:
1B > 2M > 1M> 3B > 2B > 4B.
Compound 1B is the strongest nucleophile, while 4B is the strongest electrophile. We observe a good
correlation for the antimicrobial activity for all compounds, except for the 1M compound. An
explanation for their poor activity with respect to the geometrical parameters will be provided.
9

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3.3b Atomic charges and geometric parameters of the benzimidazole compounds
The nucleophilicity and electrophilicity concepts are related with the atomic charges on the
atoms. The Mulliken charges on the atoms were calculated using DFT at M11/ktzvp level of theory
(
Table 6). The numbering of the atoms in the compounds discussed herein is shown in Figure 2. The
calculated geometric parameters for all compounds are summarized in Table 7 and Table 8. To the
best of our knowledge, there is no experimental data on the geometric structure of these
benzimidazoles to be found in literature. The following observations can be made relative to atomic
charges and the antimicrobial activity of the discussed benzimidazoles.
Charges of -0.435 and -0.454 can be observed on the oxygen atom in 1B and the 2M molecule,
respectively, which suggests that this is the most reactive atom for a substitution reaction. However,
the base 2M is less reactive than the benzimidazole 1B. A possible explanation for this behaviour is
the stereochemistry of the 1B, which can be seen from the data for the bond lengths, bond and
dihedral angles (Table 7 and Table 8). Therefore, 1B has a slight deviation from co-planarity due to the
-
OH and -CH groups. However, these are almost symmetric and are therefore compensated. About
3
5
° deviation from co-planarity, meaning (α-β)/2, is finally determined in the 1B compound. In the case
of 2M, the same angle is 30.5°, a big deviation from co-planarity due to the presence of nitrogen
substituents. In addition, the substituents to nitrogen are unsymmetrically located, i.e. the angles γ
and δ, and a deviation of about 44 degrees was determined, (γ-δ)/2.
2
Compound 4B has a charge of -0,354 on oxygen atom (hybridized sp ), not available to be
involved in substitution. It also exhibits minimum nucleophilicity and, as a consequence, reduced
reactivity. However, in this case the stereochemistry of the compound is very advantageous, because
the entire molecule is planar, as (α-β)/2 is 0,05 degrees. These facts explain the reactivity of the 4B
against about 22% of strains.
Benzimidazole 2B is perfectly planar. This fact explains their good reactivity against ~44% of
the strains, although a chemical substituent with high charge is not present in this molecule.
Benzimidazole 3B has a tolyl ring rotated with 28.761°, a deviation from co-planarity of the
entire molecule; however, the -CH substituent has a high charge, i.e. -0.685, which possibly functions
3
as a strong nucleophile, as it can be seen from the large spectra of strains in this case.
The Mannich base compound 1B has a tolyl ring rotated with 66.348°, which is double
compared with benzimidazole 3B, and also a big deviation from the co-planarity of the N-substituents,
10

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i.e. (θ-τ)/2, meaning 73.14°. Such a stereochemical structure doesn’t favour antimicrobial activity of
the 1M, in concordance with the qualitative and quantitative determinations.
3.3c Chemical shifts
Finally, in order to find and compare the chemical shifts of the protons in our new compounds,
we used data from literature [42, 43]. In NMR spectroscopy, the chemical shift is the resonant
frequency of a nucleus relative to a standard in a magnetic field, while the position and number of
chemical shifts are diagnostic of the structure of a molecule. Two scenarios have been employed. For
3
the hydrogen atoms bound to sp carbon atoms, the following equation was used:
δXYZC-H = (0.23 + σ + σ + σ ) ppm,
X
Y
Z
where: σ , σ σ are the increments for the substituents of the calculated hydrogen atom. Likewise,
X
Y,
Z
2
for the aromatic hydrogen atoms bound to sp carbon atoms, the following equation was used:
δAr-H = (7.27 + σortho + σmeta + σpara) ppm,
where: σortho, σmeta,
σpara are the increments for the substituents in the ortho, meta, and para
positions, respectively. For the Mannich bases (1M and 2M), the determined chemical shifts (in ppm)
are presented in black colour while the calculated ones (also in ppm) are in blue colour (Figure 3). In
both cases, one can observe a reasonable agreement between the determined and the calculated
chemical shifts, hence confirming our approach, except for the circumstances in which the values for
the special increment are not available and a difference can be observed between the two values (e.g.
the case of the -CH group).
3
To summarize, we show that the electronic parameters of the benzimidazole compounds are
in accordance with their antimicrobial activity. Such results fit with previously reported data for the
coumarin compounds [44]. Further accurate processing of such compounds into thin films and hybrid
structures, e.g. by laser ablation matrix-assisted pulsed laser evaporation (MAPLE) and/or laser-
induced forward transfer (LIFT) [45-52] could provide simple and environmental friendly, state-of-the-
art solutions for antimicrobial applications. It was shown that the antimicrobial activity of lysozyme
[
46, 49-52] and lactoferrin [53-55] is preserved after laser processing, as thin films. Therefore,
11

ACCEPTED MANUSCRIPT
embedded and/or multi-layered structures [55], patterned coatings and blends [56-59] can be further
imagined, in which novel structures can be designed and produced to further enhance, adjust and
control such microbicidal / anti-biofilm properties.
4. Conclusion
Six benzimidazole compounds, including new Mannich bases, have been synthesized,
characterized, presented and discussed herein. Density functional theory analysis of the molecular
structure, together with the HOMO-LUMO orbitals, has been employed by using the GAMESS 2012
software. It has been found that the electronic and structural parameters of the benzimidazole
compounds are well correlated to their antimicrobial activity. The tested compounds exhibited
different microbicidal and anti-biofilm features, being active against a wide spectrum of Gram positive
and Gram negative bacterial strains both in planktonic and adherent state, endorsing their potential
in developing novel anti-microbial and anti-biofilm agents. The microbicidal activity of the compounds
is linked to the presence of the nucleophilic groups within the molecule, such as -OH or -CH3.
Therefore, the largest microbicidal and anti-biofilm effect is noticed for the OH-compounds, in both
types, i.e. benzimidazole and Mannich bases. The planarity and the symmetry of the molecule also
represent strong support in their antimicrobial activity. Finally, we briefly show that further accurate
processing of such compound into thin films and hybrid structures, e.g. by laser ablation matrix-
assisted pulsed laser evaporation and/or laser-induced forward transfer, and may provide simple and
environmental friendly, state-of-the-art solutions for antimicrobial coating applications.
References
[
1] A.P. Magiorakos, A. Srinivasan, R.B. Carey, Y. Carmeli, M.E. Falagas, C.G. Giske, S. Harbarth, J.F.
Hindler, G. Kahlmeter, B. Olsson-Liljequist, D.L. Paterson, L.B. Rice, J. Stelling, M.J. Struelens, A.
Vatopoulos, J.T. Weber, D.L. Monnet, “Multidrug-resistant, extensively drug-resistant and pandrug-
resistant bacteria: an international expert proposal for interim standard definitions for acquired
resistance”, Clin. Microbiol. Infect. 18 (2011) 268-281; http://dx.doi.org/10.1111/j.1469-
0691.2011.03570.x
[
2] F. Sun, F. Qu, Y. Ling, P. Mao, P. Xia, H. Chen, D. Zhou, “Biofilm-associated infections: antibiotic
resistance and novel therapeutic strategies”, Future Microbiology 8 (2013) 877-886;
http://dx.doi.org/10.2217/fmb.13.58
12

ACCEPTED MANUSCRIPT
[
3] J. Davies, D. Davies, “Origins and evolution of antibiotic resistance”, Microbiol. Mol. Biol. Rev. 74
(
2010) 417-433; http://dx.doi.org/10.1128/MMBR.00016-10
[
4] H. Thakuria, G. Das, “An expeditious one-pot solvent free synthesis of benzimidazole derivatives”,
ARKIVOK 2008 (2008) 321-328; ISSN: 1551-7012
[
5] T. Liu, C. Sun, X. Xing, L. Jing, R. Tan, Y. Luo, W. Huang, H. Song, Z. Li, Y. Zhao, “Synthesis and
evaluation of 2-[2-(phenylthiomethyl)-1H-benzo[d] imidazol-1-yl)acetohydrazide derivatives as
antitumor agents”, Bioorg. Med. Chem. Lett. 22 (2012) 3122–3125;
http://dx.doi.org/10.1016/j.bmcl.2012.03.061
[
6] X.J. Wang, N.Y. Chu, Q.H. Wang, C. Liu, C.G. Jiang, X.Y. Wang, T. Ikejima, M.S. Cheng, “Newly
synthesized bis-benzimidazole derivatives exerting anti-tumor activity through induction of apoptosis
and autophagy”, Bioorg. Med. Chem. Lett. 22 (2012) 6297–6300;
http://dx.doi.org/10.1016/j.bmcl.2012.06.102
[
7] C. Guo, A. Linton, M. Jalaie, S. Kephart, M. Ornelas, M. Pairish, S. Greasley, P. Richardson, K.
Maegley, M. Hickey, J. Li, X. Wu, X. Ji, Z. Xie, “Discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-
pyrido[1,2-a]pyrimidin-4-ones as novel PKM2 activators”, Bioorg. Med. Chem. Lett. 23 (2013) 3358–
3363; http://dx.doi.org/10.1016/j.bmcl.2013.03.090
[
8] Y.K. Yoon, M.A. Ali, A.C. Wei, A.N. Shirazi, K. Parang. T.S. Choon, “Benzimidazoles as new scaffold
of sirtuin inhibitors: Green synthesis, in vitro studies, molecular docking analysis and evaluation of
their anti-cancer properties”, Eur. J. Med. Chem 83 (2014) 448–454;
http://dx.doi.org/10.1016/j.ejmech.2014.06.060
[
9] S. Nakao, M. Mabuchi, T. Shimizu, Y. Itoh, Y. Takeuchi, M. Ueda, H. Mizuno, N. Shigi, I. Ohshio, K.
Jinguji, Y. Ueda, M. Yamamoto, T. Furukawa, S. Aoki, K. Tsujikawa, A. Tanaka, “Design and synthesis of
prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors as anti-prostate cancer drugs”, Bioorg. Med.
Chem. Lett. 24 (2014) 1071–1074; http://dx.doi.org/10.1016/j.bmcl.2014.01.008
[
10] M. Azam, A.A. Khan, S.I. Al-Resayes, M.S. Islam, A.K. Saxena, S. Dwivedi, J. Musarrat, A.
Trzesowska-Kruszynska, R. Kruszynski, “Synthesis and characterization of 2-substituted
benzimidazoles and their evaluation as anticancer agent”, Spectrochim. Acta A. 142 (2015) 286–291;
http://dx.doi.org/10.1016/j.saa.2015.01.106
[
11] S. Estrada-Soto, R. Villalobos-Molina, F. Aguirre-Crespo, J. Vergara-Galicia, H. Moreno-Díaz, M.
Torres-Piedra, G. Navarrete-Vázquez, “Relaxant activity of 2-(substituted phenyl)-1H-benzimidazoles
on isolated rat aortic rings: Design and synthesis of 5-nitro derivatives”, Life Sci. 79 (2006) 430–435;
http://dx.doi.org/10.1016/j.lfs.2006.01.019
[
“
12] A. Bali, Y. Bansal, Y. Sugumaran, J.S. Saggu, P. Balakumar, G. Kaur, G. Bansal, A. Sharma, M. Singh,
Design, synthesis, and evaluation of novelly substituted benzimidazole compounds as angiotensin II
receptor antagonists”, Bioorg. Med. Chem. Lett. 15 (2005) 3962–3965;
http://dx.doi.org/10.1016/j.bmcl.2005.05.054
13

ACCEPTED MANUSCRIPT
[
13] K.C.S. Achar, K.M. Hosamani, H.R. Seetharamareddy, “In-vivo analgesic and anti-inflammatory
activities of newly synthesized benzimidazole derivatives”, Eur. J. Med. Chem. 45 (2010) 2048–2054;
http://dx.doi.org/10.1016/j.ejmech.2010.01.029
[
14] A.O.H. El-Nezhawy, A.R. Biuomy, F.S. Hassan, A.K. Ismaiel, H.A. Omar, “Design, synthesis and
pharmacological evaluation of omeprazole-like agents with anti-inflammatory activity”, Bioorg. Med.
Chem. 21 (2013) 1661–1670; http://dx.doi.org/10.1016/j.bmc.2013.01.070
[
“
15] P.J. Chen, A. Yang, Y.F. Gu, X.S. Zhang, K.P. Shao, D.Q. Xue, P. He, T.F. Jiang, Q.R. Zhang, H.M. Liu,
Synthesis, in vitro antimicrobial and cytotoxic activities of novel pyrimidine–benzimidazol
combinations”, Bioorg. Med. Chem. Lett. 24 (2014) 2741–2743;
http://dx.doi.org/10.1016/j.bmcl.2014.04.037
[
16] B. Zhou, B. Li, W. Yi, X. Bu, L. Ma, “Synthesis, antioxidant, and antimicrobial evaluation of some 2-
arylbenzimidazole derivatives”, Bioorg. Med. Chem. Lett. 23 (2013) 3759–3763;
http://dx.doi.org/10.1016/j.bmcl.2013.05.004
[
17] F.A.S. Alasmary, A.M. Snelling, M.E. Zain, A.M. Alafeefy, A.S. Awaad, N. Karodia, “Synthesis and
Evaluation of Selected Benzimidazole Derivatives as Potential Antimicrobial Agents”, Molecules 20
(
2015) 15206-15223; http://dx.doi.org/10.3390/molecules200815206
[
18] J.R. Burgeson, A.L. Moore, D.N. Gharaibeh, R.A. Larson, N.R. Cerruti, S.M. Amberg, D.E. Hruby, D.
Dai, “Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from
benzimidazole and related heterocycles”, Bioorg. Med. Chem. Lett. 23 (2013) 750–756;
http://dx.doi.org/10.1016/j.bmcl.2012.11.093
[
19] Y.M. Shaker, M.A. Omar, K. Mahmoud, S.M. Elhallouty, W.M. El-Senousy, M.M. Ali, A. Mahmoud,
A.H. Abdel-Halim, S.M. Soliman, H.I. El Diwani, “Synthesis, in vitro and in vivo antitumor and antiviral
activity of novel 1-substituted benzimidazole derivatives”, J. Enzyme. Inhib. Med. Chem. 30 (2015)
826-845; http://dx.doi.org/10.3109/14756366.2014.979344
[
20] H.G.F. Richter, G.M. Benson, D. Blum, E. Chaput, S. Feng, C. Gardes, U. Grether, P. Hartman, B.
Kuhn, R.E. Martin, J.M. Plancher, M.G. Rudolph, F. Schuler, S. Taylor, K.H. Bleicher, “Discovery of novel
and orally active FXR agonists for the potential treatment of dyslipidemia & diabetes”, Bioorg. Med.
Chem. Lett. 21 (2011) 191–194; http://dx.doi.org/10.1016/j.bmcl.2010.11.039
[
21] Y. Qian, K. Conde-Knape, S.D. Erickson, F. Falcioni, P. Gillespie, I. Hakimi, F. Mennona, Y. Ren, H.
Salari, S.S. So, J.W. Tilley, “Potent MCH-1 receptor antagonists from cis-1,4-diaminocyclohexane-
derived indane analogs”, Bioorg. Med. Chem. Lett. 23 (2013) 4216–4220;
http://dx.doi.org/10.1016/j.bmcl.2013.05.017
[
22] K. Matsumura, M. Ono, M. Yoshimura, H. Kimura, H. Watanabe, Y. Okamoto, M. Ihara, R.
Takahashi, H. Saji, “Synthesis and biological evaluation of novel styryl benzimidazole derivatives as
probes for imaging of neurofibrillary tangles in Alzheimer’s disease”, Bioorg. Med. Chem. 21 (2013)
3356–3362; http://dx.doi.org/10.1016/j.bmc.2013.02.054
14

ACCEPTED MANUSCRIPT
[
23] K.L. Erb, S.B. Ravula, J. Yu, S.Z. Kord, W.J. Moree, R.E. Petroski, J. Wen, S. Malany, S.R.J. Hoare, A.
Madan, P.D. Crowe, G. Beaton, “The discovery and structure–activity relationships of 2-(piperidin-3-
yl)-1H-benzimidazoles as selective, CNS penetrating H1-antihistamines for insomnia”, Bioorg. Med.
Chem. Lett. 20 (2010) 2916–2919; http://dx.doi.org/10.1016/j.bmcl.2010.03.027
[
24] A. Kolesnikov, R. Rai, W.B. Young, J. Mordenti, L. Liu, S. Torkelson, D.W. Shrader, E.M. Leahy, H.
Hu, E. Gjerstad, J. Janc, B.A. Katz, A.P. Sprengeler, “Factor VIIa inhibitors: Improved pharmacokinetic
parameters”, Bioorg. Med. Chem. Lett. 16 (2006) 2243–2246;
http://dx.doi.org/10.1016/j.bmcl.2006.01.037
[
25] M. Ishikawa, K. Nonoshita, Y. Ogino, Y. Nagae, D. Tsukahara, H. Hosaka, H. Maruki, S. Ohyama, R.
Yoshimoto, K. Sasaki, Y. Nagata, J. Eiki, T. Nishimura, “Discovery of novel 2-(pyridine-2-yl)-1H-
benzimidazole derivatives as potent glucokinase activators”, Bioorg. Med. Chem. Lett. 19 (2009)
4450–4454; http://dx.doi.org/10.1016/j.bmcl.2009.05.038
[
26] K.G. Rao, D. Chakraborty, “Synthesis, Characterization and Antibacterial Evaluation of Some
Potent 2-Substituted Benzimidazole Analogues”, Int. J. Pharm. Sci. Drug Res. 6 (2014) 67-69; ISSN:
975-248X
0
[
27] V.A. Sontakke, S. Ghosh, P.P. Lawande, B.A. Chopade, V.S. Shinde, “A Simple, Efficient Synthesis
of 2-Aryl Benzimidazoles Using Silica Supported Periodic Acid Catalyst and Evaluation of Anticancer
Activity”, ISRN Organic Chemistry 2013 (2013) 453682; http://dx.doi.org/10.1155/2013/453682
[
28] R. Srinivasulu, K.R. Kumar, P.V.V. Satyanayarana, “Facile and Efficient Method for Synthesis of
Benzimidazole Derivatives Catalyzed by Zinc Triflate”, Green & Sust. Chem. 4 (2014) 33-37;
http://dx.doi.org/10.4236/gsc.2014.41006
[
4
29] P.K. Kumar, P.K. Dubey, “Studies on preparation of 2-Acetylbenzimidazole”, Der Pharma Chemica
(2012) 1292-1295; ISSN: 0975-413X
[
30] C.S. Ciobanu, S.L. Iconaru , M.C. Chifiriuc , A. Costescu, P.L. Coustumer, D. Predoi, “Synthesis and
Antimicrobial Activity of Silver-Doped Hydroxyapatite Nanoparticles”, BioMed. Res. Int. 2013 (2013)
16218; http://dx.doi.org/10.1155/2013/916218
9
[
31] C. Araniciu, L. Marutescu, S. Oniga, O. Oniga, M.C. Chifiriuc, M. Palage, “Evaluation of the
antimicrobial and anti-biofilm activity of some 4,2 and 5,2 bisthiazoles derivatives”, Dig. J. Nanomater.
Biostruct. 9 (2014) 123-131;
[
32] M.W. Schmidt , K.K. Baldridge , J.A. Boatz , S.T. Elbert , M.S. Gordon , J.H. Jensen , S. Koseki, N.
Matsunaga, K.A. Nguyen, S. Su , T.L. Windus , M. Dupuis, J.A. Montgomery, “General Atomic and
Molecular Electronic Structure System”, J. Comput. Chem. 14 (1993) 1347-1363;
http://dx.doi.org/10.1002/jcc.540141112
[
33] B.M. Bode, M.S. Gordon, “Macmolplt: a graphical user interface for GAMESS”, J. Mol. Graphics
Mod. 16 (1998) 133–138; http://dx.doi.org/10.1016/S1093-3263(99)00002-9
15

ACCEPTED MANUSCRIPT
[
34] R. Peverati, D.G. Truhlar, “Improving the Accuracy of Hybrid Meta-GGA Density Functionals by
Range Separation”, J. Phys. Chem. Lett. 2 (2011) 2810–2817; http://dx.doi.org/10.1021/jz201170d
[
35] A. Schaefer, H. Horn, R. Ahlrichs, “Fully optimized contracted Gaussian basis sets for atoms Li to
Kr”, J. Chem. Phys. 97 (1992) 2571-2577; http://dx.doi.org.ezproxy.unilim.fr/10.1063/1.463096
[
36] A. Schaefer, H. Horn, R. Ahlrichs, “Fully optimized contracted Gaussian basis sets of triple zeta
valence quality for atoms Li to Kr”, J. Chem. Phys. 100 (1994) 5829;
http://dx.doi.org.ezproxy.unilim.fr/10.1063/1.467146
[
37] S.S. Panda, R. Malik, S.C. Jain, “Synthetic Approaches to 2-Arylbenzimidazoles: A Review”, Curr.
Org. Chem. 16 (2012) 1905-1919; http://dx.doi.org/10.2174/138527212802651232
[
38] C. Araniciu, S. Oniga, O. Oniga, M. Palage, M.C. Chifiriuc, L. Marutescu, “Antimicrobial and anti-
pathogenic activity evaluation of some 2-(Trimethoxyphenyl)-4-AR1-5-R2-thiazoles”, Farmacia 63
(
2015) 40-45;
[
39] P.K. Chattaraj, B. Maiti, “HSAB Principle Applied to the Time Evolution of Chemical Reactions”, J.
Am. Chem. Soc. 125 (2003) 2705–2710; http://dx.doi.org/10.1021/ja0276063
[
40] J.W. Peters, W.N. Lanzilotta, B.J. Lemon, L.C. Seefeldt, “X-ray crystal structure of the Fe-only
hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution”, Science 282 (1998)
853-1858; http://dx.doi.org/10.1126/science.282.5395.1853 (Corrections and Clarifications in:
Science 283 (1999) 33, http://dx.doi.org/10.1126/science.283.5398.33h)
1
[
41] R.G. Parr, R.G. Pearson, “Absolute hardness: companion parameter to absolute
electronegativity”, J. Am. Chem. Soc. 105 (1983) 7512–7516; http://dx.doi.org/10.1021/ja00364a005
1
13
[
5
42] M. Balci, in: “Basic H and C-NMR spectroscopy”; (2005) Elsevier Science. ISBN: 978-0-444-
1811-8.
[
43] T.D.W. Claridge, in: “High-Resolution NMR Techniques in Organic Chemistry”, 3nd Edition; (2016)
Elsevier Science. ISBN: 978-0-080-99986-9.
[
44] S. Špirtovic-Halilovic, M. Salihovic, H. Dzudzevic-Čancar, S. Trifunovic, S. Roca, D. Softic, D.
Zavrsnik, “DFT study and microbiology of some coumarin-based compounds containing a chalcone
moiety”, J. Serb. Chem. Soc. 79 (2014) 435–443; http://dx.doi.org/10.2298/JSC130628077S
[
45] A. Luches, A. Piqué, L.V. Zhigilei (eds.), special issue on “Matrix-assisted pulsed laser evaporation:
fundamentals and applications”, Applied Physics A: Materials Science & Processing 105 (2011) 517–
71; ISSN: 0947-8396 (Print) / 1432-0630 (Online)
6
[
46] T.E. Itina, L.V. Zhigilei, B.J. Garrison, “Matrix-assisted pulsed laser evaporation of polymeric
materials: a molecular dynamics study”, Nuclear Instruments and Methods in Physics Research Section
B: Beam Interactions with Materials and Atoms 180 (2001) 238–244;
http://dx.doi.org/10.1016/S0168-583X(01)00423-2
16

ACCEPTED MANUSCRIPT
[
47] M. Nagel, T. Lippert, in chapter 5.4: “Laser-induced forward transfer for the fabrication of
devices”, from “Nanomaterials. Processing and characterization with lasers”, edited by Subhash
Chandra Singh, Haibo Zeng, Chunlei Guo, Weiping Cai; (2012) Wiley-VCH Verlag. ISBN: 978-3-527-
32715-7; http://dx.doi.org/10.1002/9783527646821.ch5
[
(
48] T. Lippert, in chapter 7: “UV Laser Ablation of Polymers: From Structuring to Thin Film Deposition”
pages 141-175), from “Laser-Surface Interactions for New Materials Production”, edited by A.
Miotello, P.M. Ossi (2010); Springer Berlin Heidelberg; ISBN: 978-3-642-03306-3 (Print) 978-3-642-
3307-0 (Online); http://dx.doi.org/10.1007/978-3-642-03307-0_7
0
[
49] A. Purice, J. Schou, N. Pryds, M. Filipescu, M. Dinescu, “Surface morphology of thin lysozyme films
produced by matrix-assisted pulsed laser evaporation (MAPLE)”, Applied Surface Science 254
(
2007)1244–1248; http://dx.doi.org/10.1016/j.apsusc.2007.07.181
[
50] A. Matei, S. Canulescu, C. Constantinescu, J. Schou, M. Dinescu, “A study on matrix assisted
pulsed evaporation (MAPLE) of organic materials”, AIP Conf. Proc. 1464 (2012) 256;
http://dx.doi.org/10.1063/1.4739879
[
51] C. Constantinescu, A. Matei, J. Schou, S. Canulescu, M. Dinescu, “Pulsed laser deposition of
lysozyme: the dependence on shot numbers and the angular distribution”, Applied Physics B: Lasers
and Optics 113 (2013) 367–371; http://dx.doi.org/10.1007/s00340-013-5485-3
[
52] M. Tabetah, A. Matei, C. Constantinescu, N.P. Mortensen, M. Dinescu, J. Schou, L.V. Zhigilei, “The
Minimum Amount of ‘Matrix’ Needed for Matrix-Assisted Pulsed Laser Deposition of Biomolecules”,
The Journal of Physical Chemistry B 118 (2014) 13290–13299;
http://dx.doi.org/10.1021/jp508284n
[
53] C. Constantinescu, A. Palla-Papavlu, A. Rotaru, P. Florian, F. Chelu, M. Icriverzi, A. Nedelcea, V.
Dinca, A. Roseanu, M. Dinescu, “Multifunctional thin films of lactoferrin for biochemical use deposited
by MAPLE technique”, Applied Surface Science 255 (2009) 5491–5495;
http://dx.doi.org/10.1016/j.apsusc.2008.07.163
[
54] V. Dinca, A. Palla Papavlu, C. Constantinescu, P. Florian, M. Icriverzi, F. Chelu, N. Scarisoreanu, M.
Filipescu, A. Moldovan, A. Roseanu, M. Dinescu, “Preliminary studies on biological compounds layers
and patterns for biomedical application”, Proceedings of the 6th European Symposium on Biomedical
Engineering, Chania, Crete, Greece (2008) PS.06;
[
“
55] V. Dinca, P.E. Florian, L.E. Sima, L. Rusen, C. Constantinescu, R.W. Evans, M. Dinescu, A. Roseanu,
MAPLE-based method to obtain biodegradable hybrid polymeric thin films with embedded
antitumoral agents”, Biomedical Microdevices 16 (2014) 11–21; http://dx.doi.org/10.1007/s10544-
13-9801-9
0
[
56] V. Dinca, P. Alloncle, P. Delaporte, V. Ion, L. Rusen, M. Filipescu, C. Mustaciosu, C. Luculescu, M.
Dinescu, “Excimer laser texturing of natural composite polymer surfaces for studying cell-to-substrate
17

ACCEPTED MANUSCRIPT
specific response”, Applied Surface Science 352 (2015) 82–90;
http://dx.doi.org/10.1016/j.apsusc.2015.02.141
[
/
57] C. Constantinescu, K.L.N. Deepak, P. Delaporte, O. Utéza, D. Grojo, “Arrays of metallic micro-
nano-structures by means of colloidal lithography and laser dewetting”, Applied Surface Science
(
2016); http://dx.doi.org/10.1016/j.apsusc.2015.10.073
[
58] I.A. Paun, A. Moldovan, C.R. Luculescu, M. Dinescu, “Antibacterial polymeric coatings grown by
matrix-assisted pulsed laser evaporation”, Applied Physics A: Materials Science and Processing 110
(
2013) 895-902; http://dx.doi.org/10.1007/s00339-012-7193-y
[
59] I.A. Paun, M. Zamfirescu, M. Mihailescu, C.R. Luculescu, C.C. Mustaciosu, I. Dorobantu, B. Calenic,
M. Dinescu, “Laser micro-patterning of biodegradable polymer blends for tissue engineering”, Journal
of Materials Science 50 (2015) 923–936; http://dx.doi.org/10.1007/s10853-014-8652-y
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List of tables and figures (table and figure captions):
Table 1. The antimicrobial activity of the tested compounds expressed semi-quantitatively, as the
absence of the microbial growth (-), slight decrease of the microbial growth (+/-), total inhibition of
microbial growth (+, ++).
-
1
Table 2. The MIC (µg mL ) values of the tested compounds against the tested microbial strains.
-1
Table 3. The MBEC (µg mL ) values of the tested compounds against the tested microbial strains.
Table 4. The symmetry group, length of molecule and total energy of the benzimidazoles.
Table 5. Chemical reactivity indices for compounds 1B-4B and 1M-2M.
Table 6. Mulliken atomic charges for compounds 1B-4B and 1M-2M.
Table 7. Bond lengths values of the discussed benzimidazoles.
Table 8. Bond and dihedral angles of the discussed benzimidazoles.
Figure 1. The optimized structures of benzimidazole compounds 1B-4B, 1M, 2M (black-C; grey-H; red-
O; blue-N).
Figure 2. Atom numbering, exemplified on Mannich bases 1M (like in 2B, 3B) and 2M (like in 1B, 4B).
Figure 3. The determined (in black) and calculated (in blue) chemical shifts for the Mannich base 1M
and 2M.
19

ACCEPTED MANUSCRIPT
Compound/Tested strain
S. aureus
1B
2B
+/-
+/-
+
3B
+
4B
-
1M
+/-
+/-
+
2M
+/-
+/-
+
+/-
+
S. saprophyticus
++
+
+
+
-
Gram positive cocci
E. faecium
+/-
+/-
+
E. faecalis
+/-
+
++
++
+/-
+/-
++
+/-
P. aeruginosa
A. baumannii
-
Gram negative bacilli
(
Pseudomonadaceae family)
++
+
+/-
-
+/-
+/-
K. pneumoniae
E. cloacae
E. coli
+
+/-
+
+/-
+/-
+
+/-
+/-
+/-
-
-
-
+/-
+/-
+/-
+/-
++
Gram negative baciili
(
Enterobacteriaceae family)
+/-
Table 1. The antimicrobial activity of the tested compounds expressed semiquantitatively, as the absence of the microbial growth (-),
slight decrese of the microbial growth (+/-), total inhibition of microbial growth (+, ++).

ACCEPTED MANUSCRIPT
Compound/Tested strain
1B
>1
2B
>1
3B
>1
4B
>1
>1
>1
>1
>1
1M
>1
>1
>1
>1
>1
2M
>1
S. aureus
S. saprophyticus
0.002
>1
>1
>1
>1
Gram positive cocci
E. faecium
0.125
>1
>1
0.008
0.03125
>1
E. faecalis
>1
>1
P. aeruginosa
A. baumanii
0.008
0.25
0.25
Gram negative baciili
(
Pseudomonadaceae family)
>1
0.5
0.5
>1
>1
>1
K. pneumoniae
E. cloacae
E. coli
0.03125
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.03125
>1
Gram negative baciili
(
Enterobacteriaceae family)
>1
0.5
0.03125
>1
-
1
Table 2. The MIC (µg mL ) values of the tested compounds against the tested microbial strains.

ACCEPTED MANUSCRIPT
Compound/Tested strain
1B
>1
2B
>1
3B
>1
4B
>1
1M
>1
2M
>1
S. aureus
S. saprophyticus
0.002
0.002
>1
>1
>1
0.03125
0.008
>1
>1
>1
Gram positive cocci
E. faecium
E. faecalis
0.125
>1
>1
0.25
1
0.008
0.25
0.0625
>1
Gram negative baciili
P. aeruginosa
0.01562
0.3125
0.3125
>1
>1
(
Pseudomonadaceae family) A. baumanii
>1
0.5
0.25
>1
>1
>1
Gram negative baciili
K. pneumoniae
0.03125
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.03125
>1
(
Enterobacteriaceae family) E. coli
-
1
Table 3. The MBEC (µg mL ) values of the tested compounds against the tested microbial strains.

ACCEPTED MANUSCRIPT
Compound Symmetry Length (Å)
Etot (Ha)
1
2
3
4
B
B
B
B
C1
C1
C1
C1
C1
C1
8.935
10.823
10.733
8.974
-533.628
-610.780
-650.080
-532.427
-823.309
-785.457
1
M
M
10.775
10.322
2
Table 4. The symmetry group, length of molecule and total energy of the benzimidazoles.

ACCEPTED MANUSCRIPT
Benzimidazoles
Mannich bases
Energy (Hartree)
1B
2B
3B
4B
1M
2M
EHOMO
-0.322
0.048
-0.310
0.016
-0.311
0.023
-0.337
-0.001
-0.336
0.168
-0.169
0.085
-0.315
0.039
-0.354
0.177
-0.138
0.053
-0.325
0.046
ELUMO
Egap
-0.370
0.185
-0.326
0.163
-0.334
0.167
-0.371
0.1855
-0.1395
0.052
η
µ
-0.137
0.050
-0.152
0.070
-0.144
0.062
ω
Qualitative
44.44%
33.33%
44.44%
44.44%
44.44%
33.33%
55.55%
33.33%
22.22%
22.22% 11.11%
22.22%
33.33%
44.44%
Antimicrobial
activity
Quantitative (MIC)
-
-
Quantitative (MBEC)
22.22% 11.11%
Table 5. Chemical reactivity indices for compounds 1B-4B and 1M-2M.

ACCEPTED MANUSCRIPT
Mulliken charge
3B 4B
Atom
1
B
2B
1M
2M
C-1 (Benzimidazole)
C-2 (Benzimidazole)
C-3 (Benzimidazole)
C-4 (Benzimidazole)
C-5(Benzimidazole)
C-6 (Benzimidazole)
N-7 (Benzimidazole)
C-8 (Benzimidazole)
N-9 (Benzimidazole)
-0.132
-0.128 -0.135 -0.127 -0.131 0.132
-0.190 -0.185 -0.178 -0.185 -0.186
-0.072 -0.092 -0.095 -0.022 -0.077
-0.019 -0.007 0.000 -0.066 -0.039
-0.189
-0.089
0.013
0.010
-0.02
0.006 0.013 -0.035 0.023
-0.143
-0.140 -0.136 -0.140 -0.138 -0.135
-0.146 -0.406 -0.354 -0.295 -0.268
0.389 0.195 0.218 -0.002 0.267
-0.333 -0.325 -0.307 -0.271 -0.394
-0.341 -0.106 0.301 -0.046 -0.106
-0.389
0.167
-0.312
B1,M2
B2,B3,M1
, Ph
B4
C-10 (CH
O-11 (OH
C-11 (Ph)
C-12 (CH3
C-13 (Ph)
, CO )
-0.020
B1,M2
B4
, CO )
-0.435
-
-
-0.374
-
-0.454
-
-0.154 -0.039
-
-0.085
-
B1,B4,M2
B2,B3,M1
, Ph
)
-0.349
-0.130 -0.156 -0.552 -0.134 -0.421
-
-
-
-
-
-
-
-
-
-0.155 -0.161
-0.183 -0.117
-
-
-
-
-
-
-
-
-
-0.168
-0.067
-
-
B2,B3,M1
C-14’ (Ph
)
M1,M2)
2
C-14 (CH
N-15
-
-
-
-
-0.296 -0.408
-0.213 -0.203
C-15 (Ph)
-0.021 0.063
-0.060
-
B3,M1
M2
C-16 (CH -tol
, CH2
)
-
-
-
-
-0.685
-0.543 -0.240
3
C-17 (CH3)
C-18 (CH2)
C-19 (CH3)
-
-
-
-0.378 -0.408
-
-0.250
-0.426 -0.369
Ph: Phenyl; Tol: Tolyl
Table 6. Mulliken atomic charges for compounds 1B-4B and 1M-2M.

ACCEPTED MANUSCRIPT
Chemical
bond
Length (Å)
1B
4B
1M
2M
1.505
1.531
1.411
1.097
1.449
1.448
-
8
C -C
10
1.507
1.489
-
1
0
12
11
C -C
1.518
1.498
-
1
0
C -O
1.427
1.209
-
1
1
19
O -H
0.961
-
-
-
-
-
-
-
-
-
-
7
N -C
14
-
-
-
-
-
-
-
-
1.444
1
C -N
4
15
17
16
19
18
1.450
1
5
N -C
1.452
1
5
N -C
-
1.464
-
1
5
N -C
1.455
1
5
N -C
-
-
-
1.464
1.525
1.523
1
6
17
C -C
1
8
19
C -C
Table 7. Bond lengths values of the discussed benzimidazoles.

ACCEPTED MANUSCRIPT
Angle value
Parameter
1
B
2B
-
3B
-
4B
1M
2M
8 10 12
C C C
111.228
116.581
-
-
110.467
109.028
8 10 11
C C O
109.564
-
-
118.760
7 14 15
N C C
-
-
-
-
111.186 112.093
7
8 10 12
N C C C
148.807
-
-
0,390
-
77.533
99.982
161.069
-
9 8 10 12
N C C C (α)
33.184
-
-
0.435
-
7 8 10 11
N C C O (β)
23.312
-
-
0.335
-
7 8 10 15
N C C C
-
-
-
-
-
-
-
-
180.000 28.761
-
-
-
-
-
-
-
-
66.348
8 10 15 16
C C C C
-
-
-
-
-
-
-
1.832
1.673
-
7 14 15 17
N C N C (θ)
-
-
-
-
-
-
174.328
-
7
14 15 16
N C N C (γ)
-
160.232
-
7 14 15 19
N C N C (τ)
28.047
7 14 15 18
N C N C (δ)
-
-
-
72.499
79.587
160.545
1
4 15 16 17
C N C C
1
4 15 18 19
C N C C
Table 8. Bond and dihedral angles of the discussed benzimidazoles.

ACCEPTED MANUSCRIPT
3B
1B
2B
4B
1M
2M
Figure 1. The optimized structures of benzimidazole compounds 1B-4B, 1M, 2M (black-C; gray-H; red-O; blue-N).