Exploring electronic structure, and substituent effect of some biologically active benzimidazole derivatives: Experimental insights and DFT calculations

Article abstract of DOI:10.1016/j.molstruc.2020.128996

A series of (1H-benzimidazol-2-ylmethyl)-N-(4-phenyl)amine derivatives incorporating different electron-donating and withdrawing groups (X = 4[sbnd]OCH₃ (1), 4[sbnd]CH₃ (2), H (3), 4[sbnd]Cl (4), and 4[sbnd]Br (5)) on the para-position of the phenyl substituent was prepared, characterized and screened for their potential antimicrobial activity against some microbes. The substituent effect on the spectroscopic data (vibrational modes and NMR resonances) is well established by fitting with the Hammett constant. The unsubstituted derivative 4 exhibited comparable activity (MIC = 0.26 μM) to the standard tetracycline (MIC = 0.18 μM) against Staphylococcus aureus. Introduction of a substituent to the phenyl ring led to diminishing of the antibacterial activity. The substituent effect on the electron structure of 1–5 was investigated by TDDFT calculations. The acid dissociation constants of the ionizable NH group correlate well with Kubota's σ^? parameter (R² = 0.9196). The solvatochromism behavior of 1–5, in solvents of different polarity and hydrogen-bond tendency, was investigated by linear solvation–energy relationship equation analysis. Correlation between various quantum chemical descriptors, and antibacterial activity was carried out to verify a structural activity relation for this series of benzimidazole derivatives.

Full text of DOI:10.1016/j.molstruc.2020.128996

Journal Pre-proof
Exploring electronic structure, and substituent effect of some
biologically active benzimidazole derivatives: Experimental insights
and DFT calculations
Ola R. Shehab , Ahmed M. Mansour
PII:
S0022-2860(20)31319-3
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128996
MOLSTR 128996
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Journal of Molecular Structure
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Accepted date:
1 July 2020
27 July 2020
28 July 2020
Please cite this article as: Ola R. Shehab , Ahmed M. Mansour , Exploring electronic struc-
ture, and substituent effect of some biologically active benzimidazole derivatives: Ex-
perimental insights and DFT calculations, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.128996
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Highlights:

Acid dissociation constants fit well with Hammett constant.

Introduction of a substituent reduces the toxicity compared to the unsubstituted
one.


Solvatochromism behaviour is mainly attributed to solvent dipolarity/polarizability.
Structural activity relationship is recognized by correlating toxicity to total energy.
1

Exploring electronic structure, and substituent effect of some biologically active
benzimidazole derivatives: Experimental insights and DFT calculations
Ola R. Shehab,a and Ahmed M. Mansoura*
Chemistry Department, Faculty of Science, Cairo University, Gamaa Street, Giza 12613, Egypt.
Corresponding author: mansour@sci.cu.edu.eg, inorganic_am@yahoo.com, Tel: +2 01220689432
Abstract
A series of (1H-benzimidazol-2-ylmethyl)-N-(4-phenyl)amine derivatives incorporating
different electron-donating and withdrawing groups (X = 4-OCH3 (1), 4-CH3 (2), H (3), 4-Cl (4),
and 4-Br (5)) on the para-position of the phenyl ring was prepared, characterized and
screened for their potential antimicrobial activity against some microbes. The substituent
effect on the spectroscopic data (vibrational modes and NMR resonances) is well established
by fitting with the Hammett constant. The unsubstituted derivative 4 exhibited comparable
activity (MIC = 0.26 μM) to the standard tetracycline (MIC = 0.18 μM) against Staphylococcus
aureus. Introduction of a substituent to the phenyl ring led to diminishing of the antibacterial
activity. The substituent effect on the electron structure of 1–5 was investigated by TDDFT
calculations. The acid dissociation constants of the ionizable NH group correlate well with
Kubota’s σ- parameter (R2 = 0.9196). The solvatochromism behavior of 1–5, in solvents of
different polarity and hydrogen-bond tendency, was investigated by linear solvation–energy
relationship equation analysis. Correlation between various quantum chemical descriptors,
and antibacterial activity was carried out to verify a structural activity relation for this series
of benzimidazole derivatives.
Keywords: Hammett constant; Spectrophotometry; Acid dissociation constant; TDDFT
2

Introduction
The parasitic and antiviral activities of benzimidazole and its derivatives received
considerable attention because of their widely applications in the pharmaceutical
applications. The benzimidazole ring system is essential for displaying chemical functionality
in some biologically active compounds. Some of its derivatives have powerful biological
activities as antiviral, antitumor [1], antihistaminic, antimicrobial [2] and anthelminthic
agents [3]. To date, some chloroaryloxyalkyl derivatives showed great bactericidal activity
against Staphylococcus aureus A 15091 and Salmonella Typhi O-901 [4]. Commercial
benzimidazole based drugs such as Thiabendazole and Albendazole are using as antibacterial
agents against both gram-positive and gram-negative microbes [5,6]. Benzimidazole
derivatives act as bioisosteres for purine, which is vital for bacterial cell wall, nucleic acid,
and protein biosynthesis [7]. Besides, benzimidazole based drugs in clinical use have an
established safety and efficacy profiles [6]. In addition, benzimidazole as its 5,6-dimethyl
derivative is present in vitamin B12 and some related biomolecules [8].
Benzimidazole compounds have been broadly investigated in industrial processes as
corrosion inhibitors for metal and alloy surfaces [9]. Also, benzimidazoles can be used in
liquid crystals [10], OLED’s [11], and switch’s devices [12]. These various applications of
benzimidazole compounds have attracted many experimentalists and theorists to explore
the spectroscopic and structural properties of benzimidazole, and some of its derivatives. In
the present contribution, a series of (1H-benzimidazol-2-ylmethyl)-N-(4-phenyl) amine
derivatives (Scheme 1) functionalized with different electron-donating and withdrawing
groups (X = 4-OCH3 (1), 4-CH3 (2), H (3), 4-Cl (4), and 4-Br (5)) was reported. The substituent
3

effect on the photophysical properties and antibacterial activity was investigated with the
aid of quantum chemical calculations. The acid dissociation constants of the ionizable groups
were determined spectrophotometrically and correlated with Hammett constant.
Solvatochromism behavior of the compounds was investigated by multi-parameter equation
namely linear solvation–energy relationship. Finally, a relation between the antibacterial
activity and various quantum chemical descriptor was investigated.
Experimental section
Materials and Instruments
2-chloromethyl benzimidazole [13] and (1H-benzimidazol-2-ylmethyl)-N-(4-phenyl)
amine derivatives (X = 4-OCH3 (1) [14], 4-CH3 (2) [14], H (3) [15], 4-Cl (4) [16] and 4-Br (5)
[17]) were prepared by following the previously methods. Characterization of 1–5 was done
1
by ATIR and H NMR. ATIR spectra were recorded in the solid state on a Nicolet 380 FT-IR
1
spectrometer equipped with a smart iFTR accessory. H NMR analysis was done with
Brucker-Avance 200 MHz spectrometer at room temperature. The purity of the previously
published compounds was checked by elemental analysis, which were determined by
Elementar Vario MICRO cube CHN analyzernor an EA 3000 elemental analyser from HEKtech,
Germany [1; C15H15N3O C 71.13, H 5.97, N16.59: found C 71.06, H 5.92, N 16.58. 2; C15H15N3 C
75.92, H 6.37, N 17.71, found C 75.85, H 6.32, N 17.70. 3; C14H13N3 C 75.31, H 5.87, N 18.82:
found C 75.18, H 5.84, N 18.64. 4; C14H13ClN3 C 65.25, H 4.69, N 16.30: found C 65.25, H 4.69,
N 16.30. 5; C14H13BrN3 C 55.65, H 4.00, N 13.91: found C 55.43, H 3.94, N 13.82]. Electronic
absorption spectra were measured on an Agilent 8453 diode array spectrophotometer.
4

Spectrophotometric measurements
Compounds 1–5 were weighed and dissolved in anhydrous ethyl alcohol to prepare
stock solutions with a concentration of 10-3 M. The solvatochromism behavior was
investigated by recording the absorption electronic spectra of the compounds in different
solvents (DMSO, DMF, acetonitrile, ethyl alcohol, 1,4-dioxane, and cyclohexane). This
achieved by transferring 0.2 mL from the stock solution into 10 mL measuring flask and
diluting up the solution to the mark with the solvent.
The acid dissociation constants of 1–5 were determined spectrophotometrically. To 100
mL measuring flask charged with 2 mL of the stock solution of the benzimidazole derivative
and 23 mL ethyl alcohol, water was added until the mark. The resulting solutions were made
strongly acidic (pH = 2) by adding few drops from hydrochloric acid (0.2 M). Then the
solution was partially neutralized with sodium hydroxide solution (0.2 M). The pH values of
the partially neutralized or basic solutions were measured and the corresponding electronic
absorption spectra were recorded. The acid dissociation constants were determined by half-
height [18] and the limiting absorbance [19] methods. The rationale of using HCl/NaOH
rather than buffer solution is eliminating the spectral changes corresponding to the
absorption of the buffer components in the same range of the measurements.
Computational details
In the ground state, geometry optimizations of the benzimidazole derivatives were carried
out at B3LYP [20]/6-311++G(d,p) and M06-2X [21]/def2TZVP [22] level of theories using
Gaussian03 program package [23]. Visualization of the local minimum structures was carried
out with the aid of Gauss view [24]. Time dependent density functional (TDDFT) calculations
5

were carried out by B3LYP/6-311++G(d,p) method using the continuum polarizable model to
add the solvent effect. Frontier molecular orbitals and molecular electrostatic potential maps
were obtained at B3LYP/6-311++G(d,p) level of theory. The quantum chemical descriptors
were calculated according to Koopman’s theorem [25] as follows: the ionization energy (I.E.)
and electro-affinity (E.A.) were deduced from the energies of HOMO and LUMO (I.E. = –
EHOMO and E.A. = –ELUMO), Mulliken electronegativity is equal (I.E.+E.A.)/2, hardness is
approximately ((I.E.–E.A.)/2) [26] and softness is the reciprocal of the hardness. The
electrophilicity index (ω) [27], a measure of the lowering of the energy as electron flow
between the donor and acceptor, is equal to –(electronegativity)2/hardness.
Results and discussion
Correlation with Hammett constant
The derivatives of (1H-benzimidazol-2-ylmethyl)-N-(4-phenyl)amine (Scheme 1) were
prepared by heating to reflux a mixture of 2-(chloromethyl)benzimidazole, sodium iodide
and substituted anilines (X = 4-OCH3 (1) [14], 4-CH3 (2) [14], H (3) [15], 4-Cl (4) [16] and 4-Br
(5) [17]) in anhydrous ethanol for 12 h. These derivatives were chosen to investigate the
influence of the character of the substituent on the electronic structure and photophysical
properties of the investigated compounds. The structures of the previously published
1
benzimidazoles were characterized by AT IR (Figure S1) and H NMR spectroscopy (Figure
S2). The AT IR spectra of the benzimidazole derivatives show an intense ν(NH) mode of the
aniline residue in the range of 3429–3449 cm-1. Correlation between the stretching ν(NH)
mode of 1–5 and Hammett (σ) [28], Brown’s (σ+) [29], and Kubota’s (σ-) [30] parameters was
investigated. The plot of the ν(NH) mode (except for 3) with Hammett constant is fit (R2 =
6

0.9995) (Figure S3). A systematic shift to higher wavenumber was observed upon change of
the character of the substituent on the phenyl ring from the electron-donating to electron
1
withdrawing. The H NMR spectra of 1–5 (Figure S2) show the NH resonance of the aniline
residue in the range of δ = 5.89–6.52 ppm [14–17]. The NH resonances correlate best with
Kubota’s σ- parameter (R2 = 0.9869) (Figure S3) compared with R2 = 0.9586 and 0.9094 for σ
and σ+, respectively. We tried to investigate the long-range influence of the substituent by
correlating the NMR resonances of the methylene group with Hammett parameter. Plotting
1
of the –CH2 H NMR resonance as a function of σ, σ+ and σ- is fit with R2 = 0.8396, 0.8431 and
0.8101 designating stabilization of the partial or full charges compared to the inductive effect
of the conjugated framework.
Geometry optimization
Ground-state geometry of the benzimidazole derivatives 1–5 was executed using B3LYP
[20]/6-311++G(d,p) and M06-2X [21]/def2TZVP [22] methods. The optimized structures were
characterized as local minima via harmonic frequency analysis, recognized with the absence
of the imaginary modes. A view of the local minimum energy structures of the benzimidazole
derivatives is shown in Figure 1. The optimized bond lengths for the compounds 1–5 are
given in Table 1 along with the available X-ray crystallographic data for compound 5 (X = Br)
[17]. The root means square error in the bond lengths was calculated by comparing the data
of the optimized structures with the crystal data of compound 5 (X = Br). It is worthy mention
that the optimized bond lengths compare well with the crystal data of compound 5 and the
RMS values were found to be in the range of 0.0244–0.0260 and 0.0222–0.0231 for B3LYP/6-
311++G(d,p) and M06-2X/def2TZVP methods, respectively. Some of the calculated bond
7

lengths are slightly longer than the experimental finding except the benzimidazole C7–N15
and C7–N16, which are significantly longer by about 0.06 Å. This may be attributed that the
optimization process was carried out one single molecule, while packing state including intra-
and intermolecular interactions was considered in the solution of the crystal structure. It is
well-known that the benzimidazole moiety is involved in intermolecular H-bond via the
benzimidazole NH and pyridine-type nitrogen [17]. We explicit that the introduction of the
substituent in the para-position on the phenyl moiety may lead to a change in the charge
distribution of the molecule and consequently influence the electronic structure of the
compound. As shown in Table 1, there is a slightly change in the optimized C–C bond lengths
of the phenyl moiety in the vicinity of the para-substituent as well as C9–N17 bond by
changing the character of the substituent. In particular, by introduction of σ-withdrawing
substituent (Cl and Br), the calculated C–C bonds adjacent to the halogen atom become
shorter than the corresponding bonds in the unsubstituted derivative (3), while the
substitution with the electron-donating group leads to an elongation in the phenyl C–C
bonds.
It is essential to examine the variation of Mulliken charge of 1–5 with the character of
the substituent on the phenyl ring. Comparison of Mulliken charges of the phenyl moiety of
1–5 is shown in Figure 2. Indeed, since Cl and Br substituents have electron-withdrawing
character, the Mulliken charge on the bearing atom (C12) changes dramatically from
–0.35574e (X = H) to 0.397318e (X = Cl) and 0.051221e (X = Br). Compound 2 (X = CH3)
behaves similarly to the electron-withdrawing derivatives (4 and 5) with a positively charge
of 0.594653e on C12 atom because of the hyperconjugation effect. In 1–5, the C11 atom
8

shows an accumulation of the negative charge density except the para-methoxy derivative
(1). The benzimidazole derivatives show accumulation of the positive charge on the nitrogen
of the aniline residue in the following order: OCH3 (0.047326e) > Br (0.022857e) > H
(0.018639e) > CH3 (0.016136e) > Cl (0.004205e) that may be accounted for the planarity of
the whole molecule and the increasing of the dihedral angle (N17C8C7N15) on going from a
derivative with electron-donating character to that with electron withdrawing character.
Electronic structure
Time dependent density functional theory
The absorption spectra of 1–5 (Figure 3) show six electronic transitions in ethanol at
204, 222–225, 242–250, 274, 280 and 293–311 nm. The first and second transitions may be
assigned to the medium and low energies π–π* transitions within the phenyl moiety [31]. The
next three bands may be assigned to π–π* transitions of the heteroatomic groups inside the
benzimidazole moiety [32]. The two bands at 274 and 281 nm appear doublet because of a
tautomeric structure of the benzimidazole ring [33]. The transition in the range of 293–311
nm may be assigned to n–π* of the aniline residue. On comparing the electronic absorption
spectra of 1–5 with their parent benzimidazole (243, 272 and 278 nm) [34] and 2-methyl
benzimidazole (245, 274 and 280 nm) [33], it was found that the transitions of the
benzimidazole moiety remain intact.
To understand the observed electronic transitions and to get an insight into the
substituent effect on the absorption spectra of 1–5, time-dependent density functional
theory (TDDFT) calculations were carried out at B3LYP/6-311++G(d,p) level of theory using
the default PCM solvation model to introduce the solvent effect (ethanol). The first 30 singlet
9

excited states were considered in the TDDFT calculations. The calculated spectra of 1–5,
assignment of the electronic transitions and their energies are given in (Figure S4) and (Table
S1). The absorption spectra were stimulated by GAUSSSUM software. Each excited state was
interpolated by a Gaussian convolution with the full-width at half-maximum (FWHM) of 3000
cm-1.
In the range of 185–350 nm, the calculated spectra of 1–5 shows three main transitions
at 206, 242–252 and 271–296 nm (Figure S4). The calculated spectra of 1–5 are comparable
and the only difference is the positions of the bands at 242–252 and 271–296 nm. The
calculated TDDFT data compare well with the experimental findings reflecting the suitability
of the applied method in predicting and understanding the electronic transitions of this size
of compounds. The compositions of selected frontier molecular orbitals, involved in the main
transitions of 1–5, are given in Figure 4 and Table S2–5. In general, the assignment of the
theoretical electronic transitions and the corresponding FMO give a cutoff proof about the
suggested nature of the transitions in the observed absorption spectra. Compound 2 (X =
CH3) has five main transitions at 204, 218, 246, 280 and 303 nm corresponding to HOMO–1→
LUMO+3, HOMO–1→LUMO+1, HOMO→LUMO+3, HOMO→LUMO+1 and HOMO→LUMO,
respectively. HOMO shows π(benzimidazole) character, while LUMO results from π* of the
phenyl moiety and thus the HOMO→LUMO transition at 303 nm may be considered as
charge transfer (CT) (Table S3). For 1 and 3, the lowest energy transition (HOMO→LUMO) at
317 and 294 nm has a nature of CT as 2, but the electron donation is from the phenyl moiety
to benzimidazole moiety (Table S2 & 4). The HOMO–LUMO gap of 1 is 0.22 eV lower than
that of 2.
10

The calculated spectrum of 4 (X = Cl) shows five main transitions at 206, 251, 258, 282
and 294 nm due to HOMO–3→LUMO, HOMO–1/HOMO–2→LUMO, HOMO→LUMO+2/
LUMO+3, HOMO→LUMO+1 and HOMO→LUMO, respectively. The transition at 294 nm
(Figure 4) has a ground-state composed of π(phenyl moiety)/CH2–NH, whereas the excited
state is of π*(benzimidazole/phenyl) forming CT/π–π*/n–π*. The transition at 282 nm has the
same nature as that at 294 nm. The data obtained (Table S5) for the bromo derivative 5
show a good match with 4 in the position of the transition and the compositions of FMO
(Figure 4), which are participated in the same electronic movement.
Electronic absorption spectra in different pH values
The electronic absorption spectra of 1–5 in 75% (v/v) aqueous-ethanolic mixture of
varying pH values were recorded to determine the ionization constants of the ionizable NH
groups. It was found that the mono positively species are formed by protonating the
benzimidazolic sp2 nitrogen and di-cation species by protonating the latter mention group in
addition to the NH of the aniline residue. A blue shift is observed during the formation of the
mono- and di-cation species, while a red shift is seen if the NH group(s) is ionized.
In strongly acidic medium (pH = 2), the bands of the unsubstituted derivative (X = H) at
242, 274 and 280 nm in neat ethanol are blue shifted to 236, 268 and 275 nm revealing the
formation of di-cation species. The positions of the new bands remain unchanged by
increasing the pH up to 4, but the intensities decrease with lowering [H+]. This may be taken
as a sign for renovation of the mono positively species. At pH = 5 (Figure 5), a shoulder grows
at 279 nm. This may be inferred that compound 3 remains in its neutral form. An isosbestic
point is shown at 213 nm in the pH range of 3–5 corresponding to presence of an acid-base
11

equilibrium between mono-cation and neutral species. Between pH = 6–12, the bands
located at 236, 268 and 275 nm (pH < 6) are red shifted to 241, 273 and 279 nm,
respectively, due to ionization of the NH groups.
The para-substituted compounds (1, 2 and 5) behaved similarly to the substituted
compound 3 and 4 [16]. Unlike 4, there is no clear isosbestic point in the spectra of 5 (Figure
S5) overall the pH range and this may be the non-planarity of the p-Br derivative as indicated
by its crystal data [17] and quantum chemical calculations. At pH = 2, a shoulder appears at
219 nm and vanishes with decreasing [H+], which may be accounted for deprotonation of the
NH group of the aniline residue. The bands located at 249, 273 and 279 nm have the same
behavior as those in the unsubstituted compound 3 under the same experimental condition.
One clear isosbestic point is observed in the electronic absorption spectra of 1 (Figure S5)
and 2 (Figure S5) at 212 nm corresponding to the existence of an acid-base equilibrium
between the mono-positively and neutral species. For 1 and 2, the bands located at 243, 273
and 280 nm in ethanol have the same trend of variation with the pH as the para bromo
derivative.
The plots of absorbance versus pH values, taken at certain wavelength, for compounds
1–5 were used to determine the acid ionization constants using the well-known methods;
half-height [18] and limiting absorbance [19] methods. The values of the acid ionization
constants are given in Table 2. In general, four ionization constants were determined for 1–5.
The first pKa value in the pH range of 2.34–2.65 is accounted for deprotonation of the
protonated NH group of the aniline residue, while the second value at 4.32–5.33 may be
assigned to deprotonation of the protonated benzimidazole sp2 nitrogen [35]. The third pKa
12

value in the range of 7.96–9.14 may be due to the ionization of the aniline NH group (Figure
S6). The ionization of the latter group is directly influenced by the character of the
substituent on the phenyl ring as the electron-donating group decreases the acidity of NH
group, while that of the electro withdrawing group nature increases the acidity of the group.
The pKa value of the aniline residue NH group correlates well with Kubota’s σ- parameter (R2
= 0.9196) compared with R2 = 0.8861 and 0.7716 for σ and σ+, respectively. The fourth pKa
value at 10.66–11.42 may be accounted for the ionization of benzimidazolic NH group.
Electronic absorption spectra of 1–5 in different solvents
The electronic absorption spectra of 1–5 were recorded in solvents of different polarity
and H-bond tendency [DMSO, DMF and 1,4-dioxane (H–bond acceptor), acetonitrile (H-bond
donor) and ethyl alcohol (amphoteric H-bond acceptor-donor)] as well as cyclohexane
(Figure 6 and Figure S7). The bands at 274 and 281 nm blue shift slightly on going from the
highly polar solvents e.g. DMSO to a non-polar solvent e.g. cyclohexane. In ethyl alcohol, a
solvent of highly H-bond tendency, the latter bands are blue shifted as the benzimidazole
derivatives are acting as proton acceptors at the benzimidazolic sp2 nitrogen. Compared to
cyclohexane, the bands of 1–5 exhibit a red shift in DMSO and DMF (Figure 6). To
authenticate whether the band shift is corresponding to a change in the solvation energy or
pure dielectric effect, the wavenumbers of the electronic transitions were plotted against
(D–1)/(D+1), where D is the dielectric constant of the solvent. The plots of all the bands are
not linear indicating that the dielectric constant is not the only determining variable and the
so-called specific solute-solvent interaction may be the key managing factor in determining
the band position.
13

In other words, the formation of H-bond between the benzimidazole derivatives 1–5
and the solvent molecules leads to such solvolysis, which overcome the influence of the
dielectric constant. One of the most impressive and most popular quantitative treatments of
the solvent effects using a multi-parameter equation is linear solvation–energy relationship
(LSER).
(
)
(
)
Where
and
are the positions of the absorption bands in a given
solvent and reference solvent (e.g. cyclohexane), respectively. The π* scale is an indicator of
solvent dipolarity/polarizability [36], α is an index of the hydrogen donor ability of the
solvent [37] and β is a scale describing the strength of the hydrogen acceptor of a given
solvent [38]. The a, b and s coefficients determine the relative susceptibilities of the
wavenumber value to the designated solvent property scales. Therefore, the solute-solvent
interaction can be separated into nonspecific contacts expressed by so-called polarity,
polarizability solvent functions and specific interactions as explained by the means of α and
β. The results of the multiple regressions for the band around 242 nm are given Table 3. The
negative sign of coefficients ''s'' reveals a bathochromic shift of the band around 242 nm
with increasing the basicity of H-bond acceptor of the solvent and solvent
dipolarity/polarizability. The negative sign of ''a'' shows that a bathochromic shift with
increasing the solvent H-bond donor acidity. The positive sign of "b" indicates a
hypsochromic shift with increasing the solvent H-bond acceptor basicity and solvent polarity.
The percentage contributions of the solvatochromic parameters in Table 3 confirm that most
of the solvatochromism is mainly owing to solvent dipolarity/polarizability.
14

Antimicrobial activity and structural activity relationship (SAR)
In general, benzimidazole containing compounds are extremely effective compounds
with respect to both their microbe inhibitory activity and their satisfactory selectivity ratio. It
is expected that the character of the para-substituent on the phenyl ring of 1–5 is
responsible for the variation in the toxicity of this series of benzimidazole compounds.
Compounds 1–5 were previously screened [14–17] for their potential antibacterial activity
against gram-positive (Bacillus subtilis, Streptococcus faecalis and Staphylococcus aureus)
and gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and Neisseria
Gonorrhoeae) (Figure 7). In classifying the antibacterial activity as gram-positive or gram-
negative, it would usually be expected that a much greater number of biologically active
compounds would be toxic against gram-positive than gram-negative bacteria. However, in
this contribution, compounds 1–5 are active against both types of the microbes revealing
broad spectrum properties. The toxicity of 1–5 is affected by the character of the para-
substituent that is may be attributed to electronic factor and lipophilicity value, a key
parameter controlling their entry inside the cells. For Staphylococcus aureus, the
antibacterial activity of 1–5 was found to be in the following order tetracycline > 3 >>> 4 > 2
> (1, 5). It is worthy mention that compound 3 (MIC = 0.26 μM) [15] has comparable toxicity
to tetracycline (MIC = 0.18 μM) against Staphylococcus aureus. Besides, compound 3 exhibits
higher toxicity to the other microbes than the other derivatives.
Frontier molecular orbitals may provide some information about the way by which the
biologically active species interact with the components of the cell. One of the goals of SAR is
to correlate the antibacterial activity of a series of compounds with a given quantum
15

chemical descriptor based on density functional theory calculations. Various descriptors such
as EHOMO, ELUMO, ΔE, dipole moment, softness (S), hardness (η), Mulliken electronegativity (χ),
and electrophilicity index (ω) were calculated according to Koopman’s theorem [25] and
presented in Table 4. The strength of the molecule to donate or accept electron can be
determined by EHOMO and ELUMO, respectively. The chemical reactivity of the molecule and the
chemical hardness-softness properties can be measured by ΔE. Unfortunately, we did not
find a good correlation, with a good regression value, between the antibacterial activity of 1–
5 and the previously mentioned quantum chemical descriptor except the total energy of the
molecule. As shown in Table 4, benzimidazole derivative 3 has the lowest calculated total
energy value among the investigated compounds at B3LYP/6-311++G(d,p) level of theory.
Molecular electrostatic potential map is an interesting quantum chemical descriptor
usually used to explore the sites for hydrogen-bond interactions, electrophilic attack as well
as nucleophilic reactions [13]. The MEP maps of 1–5 were obtained at B3LYP/6-311++G(d,p)
level of theory. As shown in Figure 8, a strongly positively area is contained on the acidic
benzimidazolic sp3 group in the all the derivatives, while area of a weakly positively charge is
established over the methylene group. In the contrast, the halide and methoxy substituents
represent areas of a weakly negative charge (greenish yellow). In addition, a slightly negative
charge is contained on the phenyl moieties of 1–3. It seems that in particular, the phenyl ring
in the case of the unsubstituted compound carrys higher negatively MEP value than the
other derivatives that may be contributed to its higher bacteriostatic activity.
Conclusion
16

In summary, the substituent effect on the phenyl ring of (1H-benzimidazol-2-ylmethyl)-N-(4-
phenyl)amine derivatives (X = 4-OCH3 (1), 4-CH3 (2), H (3), 4-Cl (4), and 4-Br (5)) on the
photophysical properties and antibacterial activity was investigated. The vibrational
stretching mode and NMR resonance of the NH group of the aniline residue correlated well
with Hammett parameter. The RMS values of the optimized bond lengths obtained at two
different level of theories, with respect to X-ray crystallographic data, were found to be in
the range of 0.0244–0.0260 and 0.0222–0.0231 for B3LYP/6-311++G(d,p) and M06-
2X/def2TZVP methods, respectively, reflecting the appropriate of this calculation method for
this size of compounds. The substituent effect on the electronic structure and the
understanding of the electronic transitions were performed with the aid of TDDFT
calculations. The acid dissociation constants of the ionizable and protonated groups were
determined spectrophotometrically. The values of the acid dissociation constants correlate
well with Kubota’s σ- parameter (R2 = 0.9196). The effect of the solvents on the electronic
transitions was investigated. The percentage contributions of the solvatochromic
parameters, calculated by linear solvation–energy relationship equation, confirmed that
most of the solvatochromism is mainly due to solvent dipolarity/polarizability. The
antibacterial activity of the benzimidazole derivatives was found to be reduced upon the
introduction of the substituent in the para-position of the aniline moiety. Structural activity
relationship was investigated by correlating the antibacterial activity with some quantum
chemical descriptors such as total energy, EHOMO, ELUMO, hardness, softness, energy gap and
electronegativity. It is worthy mention that the compound with a lower energy is the highly
active derivatives.
17

AUTHOR CONTRIBUTION
Ola Shehab carried out all the experimental work and Ahmed Mansour performed the DFT
calculations. The manuscript was written by Ola Shehab and revised by both authors.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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Figure captions:
Scheme 1 Synthesis of (1H-benzimidazol-2-ylmethyl)-N-(4-phenyl)amine derivatives (1–5).
Figure 1 Local minimum structures of benzimidazole derivatives 1–5 calculated at B3LYP/6-
311++G(d,p).
Figure 2 Comparison of Mulliken charges for the benzimidazole derivatives 1–5 (X = OCH3,
CH3, H, Cl and Br) calculated at B3LYP/6-311++G(d,p) level of theory.
Figure 3 Electronic absorption spectra of 1–5 in ethanol.
22

Figure 4 Selected TDDFT calculated electronic transitions and Frontiers molecular orbitals of
4 obtained at B3LYP/6-311++G(d,p) level of theory.
Figure 5 Electronic absorption spectra of 3 in solutions of varying pH value.
Figure 6 Electronic absorption spectra of 1 in solvents of different polarity and hydrogen-
bond tendency.
Figure 7 Representative and comparison of the previously reported antibacterial data [13–
16] of 1–5 and the standard tetracycline against gram-positive (B. subtilis, S. aureus, and S.
faecalis) and gram-negative bacteria (P. aeruginosa, E. coli, and N. Gonorrhea). DMSO was
used as a control and its inhibition zone was withdrawing from that of each case.
Figure 8 Molecular electrostatic potential energy maps of 1–5 obtained at B3LYP/6-
311++G(d,p) level of theory. The electron density isosurface is 0.004 a.u.
23

Scheme 1
Figure 1
24

Figure 2
Figure 3
25

Figure 4
Figure 5
26

Figure 6
Figure 7
27

Figure 8
28

GRAPHICAL ABSTRACT
29