Antibacterial activity and mechanism of action of the benzazole acrylonitrile-based compounds: In?vitro, spectroscopic, and docking studies

Article abstract of DOI:10.1016/j.ejmech.2017.05.010

A new series of pyrimidine derivatives 5, 9a-d and 12a-d was synthesized by an efficient procedure. The antibacterial activity of the new compounds was studied against four bacterial strains. Compound 5 was found to exhibit the highest potency, with?=?1.0?μg/ml, against both Escherichia coli and Pseudomonas aeruginosa when compared with amoxicillin (MIC?=?1.0–1.5?μg/mL). Transmission electron microscope results confirmed that activities against bacteria occurred via rupturing of the cell wall. Molecular modeling results suggested that compounds 5, 9a-d and 12a-d have the potential to irreversibly bind to the penicillin-binding protein (PBP) Ser62 residue in the active site and were able to overcome amoxicillin resistance in bacteria by inhibiting the β-lactamase enzyme. Docking studies showed that compounds 5, 9a-d and 12a-d inhibit the β-lactamase enzyme through covalent bonding with Ser70. The synergistic effect with amoxicillin was studied. The newly synthesized compounds reported in this study warrant further consideration as prospective antimicrobial agents.

Full text of DOI:10.1016/j.ejmech.2017.05.010

Accepted Manuscript
Antibacterial activity and mechanism of action of the benzazole acrylonitrile-based
compounds: In vitro, spectroscopic, and docking studies
Shaikha S. AlNeyadi, Alaa A. Salem, Mohammad A. Ghattas, Noor Atatreh, Ibrahim
M. Abdou
PII:
S0223-5234(17)30371-9
10.1016/j.ejmech.2017.05.010
EJMECH 9440
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 December 2016
Revised Date: 1 May 2017
Accepted Date: 2 May 2017
Please cite this article as: S.S. AlNeyadi, A.A. Salem, M.A. Ghattas, N. Atatreh, I.M. Abdou,
Antibacterial activity and mechanism of action of the benzazole acrylonitrile-based compounds: In vitro,
spectroscopic, and docking studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.010.
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ACCEPTED MANUSCRIPT
77 % yield
MIC = 1.0 µg/mL (E. coli)
The binding mode of compound 5 (ball and stick in orange) docked into the PBP binding site

ACCEPTED MANUSCRIPT
Antibacterial activity and mechanism of action of the benzazole
acrylonitrile-based compounds: in vitro, spectroscopic, and docking
studies
Shaikha S. AlNeyadia, Alaa A. Salema, Mohammad A. Ghattasb, Noor Atatrehb and Ibrahim M. Abdou*a
a
Department of Chemistry, College of Science, UAE University Al-Ain, 15551 UAE
b
College of Pharmacy, Al Ain University of Science and Technology, Al Ain, 64141 UAE
Abstract: A new series of pyrimidine derivatives 5, 9a-d and 12a-d was synthesized by an efficient procedure. The antibacterial activity
of the new compounds was studied against four bacterial strains. Compound 5 was found to exhibit the highest potency, with = 1.0 µg/mL,
against both Escherichia coli and Pseudomonas aeruginosa when compared with amoxicillin (MIC = 1.0–1.5 µg/mL). Transmission
electron microscope results confirmed that activities against bacteria occurred via rupturing of the cell wall. Molecular modeling results
suggested that compounds 5, 9a-d and 12a-d have the potential to irreversibly bind to the penicillin-binding protein (PBP) Ser62 residue in
the active site and were able to overcome amoxicillin resistance in bacteria by inhibiting the β-lactamase enzyme. Docking studies showed
that compounds 5, 9a-d and 12a-d inhibit the β-lactamase enzyme through covalent bonding with Ser70. The synergistic effect with
amoxicillin was studied. The newly synthesized compounds reported in this study warrant further consideration as prospective
antimicrobial agents.
Keywords: pyrimidine, synthesis, antibacterial, β-lactamase, PBP, docking.
*Corresponding author.
E-mail address: i.abdou@uaeu.ac.ae (Ibrahim M. Abdou)
1. Introduction
Microbial infections are becoming the most pressing issue for global health and the economy. Therefore, intensive
efforts are underway to develop new antimicrobial agents. The design of new compounds to deal with resistant bacteria has
become one of the most important areas of antibacterial research today, since the resistance of pathogenic bacteria to
available antimicrobial drugs is rapidly becoming a major problem worldwide. The discovery of novel and potent
antibacterial agents is the greatest challenge for today’s chemists and pharmacists to prevent the emergence of resistance to
existing drugs. Organisms such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci (VRE), and
Gram-negative bacilli are resistant to all antibiotics used in the treatment of infections [1]. Results of bioassays and
structure-activity relationship (SAR) investigations indicate that existing marketed drugs have limitations, and some have
toxic side effects. An understanding of the mechanism of resistance can play a major role in developing new agents that
might have better activity.
Benzimidazoles and benzothiazoles are important pharmacophores in medicinal chemistry owing to their
involvement as key components for various biological activities. Benzothiazoles and their derivatives exhibit antitumor [2],
antimicrobial [3], and antiviral [4] activities. Benzimidazoles have been found to display anticancer [5], antiviral [6], anti-
inflammatory [7], antimicrobial [8], antioxidant [9], and anticoagulant properties [10]. Extensive biochemical and
pharmacological studies have confirmed that benzimidazole and benzothiazole derivatives are effective against various
strains of microorganisms. The antibacterial activities of these compounds are due to structural similarity to purine, resulting
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in competitive inhibition of the synthesis of nucleic acids and proteins inside the bacterial cell wall [11]. Organic compounds
containing pyrimidine scaffold as a core unit are known to exhibit various biological and pharmaceutical actions such as
antiviral, antibacterial, antitumor, anti-inflammatory, and antifungal activities [12]. Considerable improvement in the
biological potential and discovery of many efficacious and safer drugs was observed when two or more pharmacophores
were combined together in one molecule. The hybrid molecules have the advantage of low toxicity and exhibit synergy in
the medicinal efficacy of the individual moieties [13].
The therapeutic importance of these classes of compounds inspired us towards the development of small molecules
as therapeutic agents; herein we report the design and synthesis of pyrimidine analogues containing benzimidazole and
benzothiazole moieties by a simple and efficient method to evaluate the antibacterial potencies of this class of novel
compounds. The synthesized compounds 5, 9a-d and 12a-d were characterized by their elemental analysis, IR, and 1D-NMR
spectral studies. Findings of biological activities indicate that some compounds possess potential antimicrobial activity.
2. Results and Discussion
2.1. Chemistry
The synthesis of new and potentially useful compounds 5, 9 and 12, in which a pyrimidine ring was linked to
the benzimidazole or benzothiazole moieties through an acrylonitrile bridge, was carried out by a simple and
efficient synthetic procedure (Schemes 1-3). The new pyrimidines were designed by introducing an α,β-
unsaturated nitrile as an active β-center to be targeted by nucleophilic residues in the enzyme’s active site, and
also to form additional H-bonds, resulting in new features which enhance the biological activities of the obtained
products 5, 9 and 12.
It is known that heteroaromatic nitrile derivatives undergo Knoevenagel condensation reactions with aryl
aldehydes to yield 3-aryl-2-hetarylacrylonitrile derivatives [14]. In the current study, we report the synthesis of a
new series of benzazole pyrimidine acrylonitriles 5, 9a-d and 12a-d. A condensation between an equimolar
amount of 2-benzazole acrylonitriles 4,11 and pyrimidine aldehydes 2,8 in the presence of a catalytic amount of
piperidine proceeded well and gave the final products 5, 9 and 12 in yields 77–89 %, with excellent purities
(Schemes 1-3).
Scheme 1. Synthesis of (E)-2-(benzo[d]thiazol-2-yl)-3-hetarylacrylonitriles 5: i = POCl3/DMF, NaHCO3 ii = CH2(CN)2,
EtOH, 25°C, 5 h iii = piperidine, 25°C, 15 min.
2

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Scheme 2. Synthesis of (E)-2-(benzo[d]thiazol-2-yl)-3-hetarylacrylonitriles 9: i = R-NH2, N,N-diisopropyl ethylamine
(DIPEA), EtOH, MW, 120°C, 150 W, 10 min ii = POCl3/DMF, NaHCO3 iii = piperidine, 25°C, 15 min.
CHO
H
H
N
NH2
NH2
CN
N
CN
ii
i
N
N
+
N
N
N
R
N
R
8
12a-d
R = (CH ) N-, (89%)
11
10
12a:
3
2
12b: R = C6H5CH2NH-, (81%)
12c: R = N-morpholino, (84%)
12d: R = p-Cl-C6H4NH-, (87%)
Scheme 3. Synthesis of (E)-2-(benzo[d]imidazol-2-yl)-3-hetarylacrylonitriles 12: i = ethyl cyanoacetate, EtOH, reflux, 20
min. ii = piperidine, 25°C, 15 min
It seems reasonable that the condition used in this work would promote the Knoevenagel reaction through
the active methylene of 4 or 11 as a precursor. The reaction begins by deprotonation of the activated methylene in
the presence of a catalytic amount of piperidine. The activated methylene attacks the carbonyl carbons of the
aldehydes in pyrimidines 2 or 8 to produce 5, 9, 12 (see Appendix A, Fig S1) [15].
All obtained compounds 5, 9 and 12 were fully characterized using elemental analysis, FT-IR, and NMR
1
spectroscopic techniques. Both H- and 13C-NMR data were consistent with the presence of pyrimidine,
benzothiazole, and benzimidazole moieties. The 1H-NMR data of all derivatives revealed a single olefinic proton,
consistent with the formation of a single isomer, which was assigned to be the thermodynamically more stable E
configuration [16]. The obtained E-isomer is stabilized by the presence of hydrogen bonding between the olefinic
proton and the imidazole nitrogen, in addition to the steric repulsions between the aromatic and heteroaromatic
groups in the Z-conformer [17],[18].
The IR spectra of compounds 9a-d showed characteristic absorption bands at 2010–2232 cm-1 and 1565–
1614 cm-1 corresponding to the CN and C=C functions, respectively. In addition, it confirmed the absence of both
1
the carbonyl group and the aldehyde proton (CHO) in the final products. The H-NMR spectra for compounds
9a-d showed that the olefinic protons were observed at very low fields within a range δ = 8.17–8.87 ppm due to
the highly deshielded protons in the E-form. This is one of the driving forces for greater stability and planarity for
the compounds 9a-d [19]. The 1H-NMR spectra also demonstrated the absence of a sharp aldehyde-proton singlet
at δ = 9.90 ppm, confirming the formation of the targeted products 9a-d, while the pyrimidine protons (H-4,6)
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13
appeared within a range δ = 8.52–9.44 ppm. Further C-NMR evidence confirming the formation of a carbon-
carbon double bond is the disappearance of the signal at δ = 22.8 ppm, corresponding to the methylene CH2
carbon and the appearance of the new olefinic signal. In the 13C-spectra, the olefinic carbons resonated in the
range of δ = 147.1–162.9 ppm, and the signal appeared at δ = 112.2–117.4 ppm assigned to the CN group, while
the pyrimidine C4 and C6 appeared at δ = 152.8–164.5 ppm. The presence of aromatic C-H, aliphatic C-H, CN,
and C=C signals at 3053, 2968, 2210, and 1582 cm-1, respectively, in the IR spectrum of compound 9c, indicated
the formation of the desired product. Furthermore, its 1H-NMR spectrum recorded a singlet peak at δ = 8.51 ppm,
attributable to the olefinic proton, as well as retaining the morpholine ring protons appearing at δ =3.67–3.68 ppm
and δ =3.83–3.84 ppm. The H4 and H6 of pyrimidine ring resonated as a singlet at δ =8.94 ppm. Benzimidazole-2-
acetonitrile 11 is the second convenient precursor used [20],[21] in our study. Benzimidazole-2-acetonitrile 11
was allowed to react with pyrimidine aldehydes 8 in the presence of catalytic amounts of piperidine to afford (E)-
2-(benzo[d]imidazol-2-yl)-3-arylacrylonitriles 12a-d at yields of 81–89 % (Scheme 2). Structures of the obtained
acrylonitrile derivatives 12a-d were established on the basis of their elemental analysis along with their
compatible spectral data. The FT-IR spectra of compounds 12b showed characteristic absorption bands at
2207 cm-1, 1604 cm-1, and 3439 cm-1 corresponding to the CN, C=C and NH- groups, respectively. The 1H-NMR
spectrum indicated the presence of a low-field olefinic proton in the region of δ = 8.46–8.88 ppm accounting for
the formation of target compound 12b. In addition, the docking study and energy minimization supported the
formation of the E-isomer, with a potential energy of 11.83 kcal/mol compared with 12.66 kcal/mol for the Z-
isomer.
2.2. Biological Studies
2.2.1. Antimicrobial activity
Biological activities of compounds containing pyrimidine rings have stimulated considerable interest in
exploring the synthesis of new and potentially useful compounds. Novel derivatives 5, 9a-d and 12a-d were
evaluated for their in-vitro antibacterial activity against two Gram-positive bacterial strains, namely,
Staphylococcus aureus (ATCC-25923) and Bacillus subtilis (ATCC 6633); and two Gram-negative bacterial
strains, namely, Escherichia coli (ATCC-25922) and Pseudomonas aeruginosa (ATCC-27853) using the well
diffusion method. Amoxicillin was used as the reference standard. The results of screening derivatives 5, 9a-d
and 12a-d are summarized in Table 1. Among the series, two compounds, 5 and 12d, exhibited excellent
antibacterial activity against both Gram-positive and Gram-negative bacteria with a MIC ranging from 1 to
13 µg/mL. Compounds 9a, 9d, 12b, and 12a exhibited moderate antibacterial activity against the tested
organisms (MIC = 11.1 to 25 µg/mL). However, all other compounds in the series were found to have good
activity against both Gram-positive and Gram-negative bacteria, as compared to amoxicillin. The in-vitro
antibacterial screening of compounds 5, 9a-d against E. coli showed that compound 5 (MIC = 1.0 µg/mL)
exhibited slightly higher antibacterial activity than the control (amoxicillin, MIC = 1.5 µg/mL), whereas
compound 12d had an MIC value of 9.0 µg/mL. The remaining compounds showed good activity against E. coli
with MIC values between 11.1 and 50 µg/mL, compared with amoxicillin 1.5 µg/mL. Compounds 5, 9 and 12
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were found to inhibit S. aureus at MIC values between 8.0 and 35.8 µg/mL. In addition, compounds 5 and 12d
exhibited good antimicrobial activities, compared with amoxicillin (MIC = 1.5 µg/mL), against Gram-positive
Bacillus subtilis bacteria, with MIC ranging between 8.0-13.0 µg/mL (Table 1), (Fig. 1). Therefore, the biological
results obtained for the new pyrimidine analogues 5, 9d, and 12d showed promising antibacterial activity against
Gram-positive and Gram-negative bacteria (Table 1). A brief investigation of the structure-activity relationship
(SAR) revealed that halogen substitution at the pyrimidine ring contributed to better antibacterial activity.
Furthermore, the presence of amino groups at positions 2- and 4- of the pyrimidine ring improved the
antibacterial properties. It has been reported that fusing two biodynamic heteroaryl systems can result in the
formation of a new scaffold with significant biological activity [22]. In addition, the linkage of pyrimidine to
benzothiazole or benzimidazole was found to enhance the antibacterial activities of the final products. Results
presented in Table 1 indicate that all the tested compounds exhibited greater activity against Gram-positive and
Gram-negative bacteria strains than amoxicillin, while the benzimidazole analogues exhibited more potent
activity than the benzothiazole derivatives.
Table 1
Agar well diffusion method – zone of inhibition and MIC (µg/ml) of pyrimidines 5, 9a–d and 12a–d.
Compounds
Zone of inhibition (mm)
MIC (µg/ml)
E. coli
P. aeruginosa
S. aureus
B. subtilis
E. coli
P. aeruginosa S. aureus
B. subtilis
47
28
32
40
1.0
1.0
8.0
8.0
5
9a
9b
9c
52
50
40
32
35
30
35
35
32
45
39
39
50
35.8
18.3
35.8
35.8
18.3
27.8
50
18.3
50
18.3
35.8
CN
S
N
N
N
N
O
9d
40
30
30
35
11.1
11.1
11.1
20
12a
12b
12c
50
45
40
30
30
35
30
35
35
39
40
39
14.3
18
11.1
18
11.1
18
20
25
27.8
27.8
27.8
27.8
12d
40
21
28
24
32
25
40
14
9.0
1.5
9.0
1
9.0
1.5
13
Amoxicillin
1.5
5

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60
50
40
30
20
10
0
b
E.coli
a
E.coli
140
120
100
80
P.aeruginosa
S. aureus
B.subtilis
P.aeruginosa
S. aureus
B.subtilis
60
40
20
0
Compounds
Compounds
Fig. 1. a) A plot of the minimum inhibitory concentration (µg/ml) for four strains of bacteria; b) Zone of inhibition areas of
the new pyrimidines 5, 9 and 12 compared with amoxicillin.
2.2.2. Investigation of in-vitro amoxicillin combined with compound 5 or 12d
Antibiotic combinations are used to enhance antibacterial efficacy and to prevent the development of
resistance. Antibiotic combinations are frequently used in order to obtain broad-spectrum effects in the treatment
of serious infections such as septicemia and endocarditis, and also to produce an in-vivo effect against strains
which are defined as resistant to the known inhibiting or fatal dose of one antibiotic [23].
Certain combinations of β-lactam antibiotics exhibit synergistic antibacterial effects against various
strains. Amoxicillin is similar to penicillin in its bactericidal action against susceptible bacteria during the stage
of active multiplication. It acts through the inhibition of cell wall biosynthesis, leading to the death of the
bacterium. Its usefulness is limited by its susceptibility to β-lactamase enzyme hydrolysis produced by the
organism [24].
The synergistic effects of combinations such as (amoxicillin + 5) and (amoxicillin + 12d) were studied.
Equal volumes of amoxicillin (MIC) with 5 (MIC), and 12d with amoxicillin were mixed. A 1.0 µg/ml
concentration of amoxicillin was mixed with a 13.0 µg/ml concentration of 12d and made up to a range of
dilutions (100, 80, 60, 40, 10, and 5 %). Thereby, the 100 % dilution will contain amoxicillin 1.0 µg/ml and 12d
MIC 13.0 µg/ml, and 10 % will contain amoxicillin 0.1 µg/ml and 12d 1.3 µg/ml (Tables 2, 3 and Fig. 2).
6

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Table 2
MIC for amoxicillin (MIC) with 5 and 12d (MIC).
S. No Dilution (%)
B. infantis
(105 cells/ml)
E. coli
(105 cells/ml)
A+ 12d
S. aureus
(105 cells/ml)
P. aeruginosa
(105 cells/ml)
A+ 5
N
A+ 12d A+ 5
A+ 5 A+ 12d A+ 5 A+ 12d
1
2
3
4
5
6
7
8
100
N
N
N
N
N
G
G
N
N
N
N
N
N
N
G
G
N
N
N
N
N
N
G
G
N
N
N
N
N
N
G
G
N
N
N
N
N
N
G
G
N
N
N
N
N
N
G
G
N
80
N
N
N
N
N
G
G
N
60
40
N
N
10
N
05
G
+ve control
-ve control
G
N
Note: A: amoxicillin; N: no growth; G: bacterial growth; +ve control: bacteria with only nutrient broth (to check if bacteria
grown or not); -ve control: sterile nutrient broth (to check contamination).
Table 3
Activities of compounds 5 and 12d (MICs) and the combination of amoxicillin with 5 and amoxicillin with 12d against
bacteria strains.
Bacteria
Amoxicillin
5 MIC
(µg/ml)
1.0
12d MIC
(µg/ml)
9.0
MIC (µg/ml) of
MIC (µg/ml) of
MIC (µg/ml)
amoxicillin + 5
amoxicillin + 12d
B. infantis
E. coli
1.5
1.5
1.5
1.0
0.1
0.1
0.8
0.8
0.9
0.9
0.9
1.3
1.0
9.0
S. aureus
8.0
9.0
P. aeruginosa
8.0
13.0
14
B. infantis
12
10
8
E. coli
S. aureus
P. aeruginosa
6
4
2
0
Amoxicillin
5
12d
Amoxicillin + 5 Amoxicillin + 12d
After combination
Before combination
Fig. 2. Activities of pyrimidine acrylonitrile derivatives 5, 12d, and amoxicillin (MICs) before and after combination.
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Synergistic effects can be clearly observed from Table 3. We can see that in the case of E. coli, promising
antibacterial activity is obtained for the amoxicillin combinations, whereas for the amoxicillin alone less activity
is observed.
Synergy has been defined as requiring a fourfold reduction in the MIC of both antibiotics in combination,
compared with each one being used alone, measuring the fractional inhibitory concentration index (FICI). The
FICI was calculated for each combination (5 + amoxicillin and 12d + amoxicillin) using the following formula
[25]:
ꢊ
ꢓ
ꢊ
ꢓ
ꢃꢁꢂꢄ ꢅꢆꢇꢈꢉ ꢋꢌꢍꢎꢏꢐꢑꢒꢏꢌꢐ
ꢃꢁꢂꢖꢗꢘꢙꢚꢛꢚꢜꢜꢚꢝ ꢂꢌꢍꢎꢏꢐꢑꢒꢏꢌꢐ
ꢀꢁꢂꢁ =
+
ꢊ
ꢓ
ꢃꢁꢂꢄ ꢅꢆꢇꢈꢉ ꢑꢔꢌꢐꢕ
ꢃꢁꢂꢖꢗꢘꢙꢚꢛꢚꢜꢜꢚꢝ ꢊꢑꢔꢌꢐꢕꢓ
The FICI was interpreted as follows: synergistic, FICI ≤ 0.5; additive, 0.5 < FICI ≤ 1; indifferent, 1 < FICI ≤ 2;
antagonistic, FICI > 2.
The FICI values for the two combinations of (5 + amoxicillin) and (12d + amoxicillin) were 0.2, indicating
synergy. The synergy between 5 and 12d and amoxicillin has been attributed to the increase in the uptake of
compounds 5 and 12d. The compounds 5 and 12d are resistant to hydrolysis by the β-lactamase enzyme
elaborated by the strain of organism being tested. Compounds 5 and 12d seem to act not only as antibacterial
agents, but also may act as β-lactamase inhibitors, preventing bacterial degradation of amoxicillin. Thus, the
synergistic effect on the MIC of the mixture is more effective than applying amoxicillin alone. The combination
also allows lower doses of both amoxicillin and compounds 5 or 12d to be used, helping to reduce toxicity,
broadening the empiric coverage provided by the two antimicrobial agents (with different spectra of activity), and
preventing or delaying the emergence of resistance during antimicrobial therapy.
2.3. Docking Study
2.3.1. Penicillin-binding protein
The novel pyrimidine acrylonitrile derivatives have an α,β-unsaturated nitrile that can be easily attacked
by a nucleophile. Similar acrylonitrile-based compounds showed time-dependent inhibition for the botulinum
neurotoxin serotype A metalloprotease enzyme [26], suggesting the formation of a covalent bond between its
acrylonitrile group and an allosteric site cysteine residue. Hence, it might be sensible to suggest that our
compounds can also inhibit the target enzyme in an irreversible manner (Fig. 3).
One of the most well-known bacterial targets for irreversible inhibitors is the penicillin-binding protein
(PBP), which could be a target for our benzazole acrylonitriles. To study whether that is theoretically possible,
our compounds were covalently docked into the E. coli PBP active pocket. The resultant docking affinities
showed favorable binding modes for all docked compounds (Table 4), where all ligands had scores between -3.0
and -4.0 kcal/mol. Compound 5 achieved the lowest docking score amongst all synthesized compounds, which
was in line with the experimental antimicrobial results.
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The reference compound, amoxicillin, used in the experiment was also docked into the PBP active site and
obtained a minimum energy of -6.2 kcal/mol (Table 4). In contrast to the in silico results obtained, the pyrimidine
acrylonitrile derivatives 5, 9 and 12 showed better in-vitro antimicrobial activity than amoxicillin. This can be
explained by the poor correlation between predicted binding energies and the corresponding experimental
affinities scoring functions [27].
The docked binding mode of 5 shows how the ligand (nitrile-activated) double bond formed a covalent
interaction with the nucleophilic residue in the PBP active site Ser62 (Fig. 4). As for the non-covalent
interactions stabilizing the 5-PBP complex, the cyano group nitrogen seems to be involved in two possible
electrostatic interactions with the side chains of Asn308 and Ser360. The pyrimidine nitrogen’s presence in
compound 5 allowed the formation of additional hydrogen bond with the hydroxyl group of Thr418, whilst the
aromatic amine side chain allowed further hydrogen bond with the backbone amide of Ser420. Moreover, the
benzothiazole ring was docked into a hydrophobic pocket, creating a π-π stacking interaction with the aromatic
ring of Phe160 and multiple van der Waals interactions with the side chains of Leu359 and Leu421, providing
further stabilization for the ligand-receptor complex (Fig. 4).
Overall, the newly synthesized compounds 5, 9 and 12 appear to have the major features required to enter
the PBP catalytic pocket and irreversibly occupy it. The formation of the covalent complex suggested that the
enzyme would be permanently damaged, resulting in cell wall synthesis inhibition. Indeed, further work is
required to confirm that the PBP enzyme is a target for our novel antibacterial agents.
Fig. 3. The proposed mechanism of PBP inhibition by the acrylonitrile-based compounds.
Fig. 4. The binding mode of compound 5 (ball and stick in orange) docked into the PBP binding site (surrounding residues
shown as gray sticks). The nucleophilic residue is labeled in red. The picture was generated by MOE [28]. Hydrogen
bonding is shown as yellow dotted lines with distances in angstrom.
9

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Table 4
Docking affinities obtained from covalent docking of acrylonitrile-based antibacterial agents into the E. coli PBP active site
and from experimentally testing them against E. coli.
Compound No.
Docking affinity
(kcal/mol)
-4.0
5
-3.3
12d
Amoxicillin
-6.2
2.3.2. β-Lactamase enzyme
It was found that a similar class of compounds showed time-dependent inhibition of the botulinum
neurotoxin serotype A metalloprotease enzyme through the formation of a covalent interaction between their
acrylonitrile group and an allosteric site cysteine residue [26]. To further investigate the action of the newly
synthesized benzazole derivatives as antimicrobial active compounds, an experiment was designed to study the
possible mode of interaction with the β-lactamase enzyme (Fig. 5). Consequently, a theoretical study was
undertaken to evaluate the possibility of a covalent interaction with the E. coli β-lactamase active pocket. The
docking affinity results showed stable binding energies for all docked compounds ranging between -4.2 and -6.3
kcal/mol (Table 5).
The ligand 5 appears to fit in the β-lactamase pocket very nicely, explaining how this ligand was able to
obtain a minimum binding energy of -5.9 kcal/mol (Fig. 6). This was probably attributed to the hydrogen bonding
formed between the 5 cyanide and the Ser130 side chain; positioning the nitrile-activated double bond to form a
covalent bond with the Ser70 hydroxyl group. Additionally, the 2-amino group of the pyrimidine ring formed a
hydrogen bond with the hydroxyl group of Ser235. Thus, these compounds can irreversibly bind the β-lactamase
enzyme. Further in-vitro testing is required, however, to confirm that Ser70 is the site of reaction for compounds
5, 9 and 12.
Fig. 5. The proposed mechanism of inhibition of the β-lactamase enzyme by 5.
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Fig. 6. The binding mode of 5 (ball and stick in orange) docked into the β-lactamase pocket (surrounding residues shown as
gray sticks). The nucleophilic residue is labeled in red. The picture was generated by MOE [28]. Hydrogen bonding is shown
as yellow dotted lines with distance in angstrom.
Table 5
Docking affinities obtained from covalent docking of compounds 5, 9 and 12 into the E. coli β-lactamase active site.
Compound No.
Docking Affinity
(kcal/mol)
-5.9
5
-4.4
9a
-4.4
9b
-4.5
9c
-4.9
9d
-4.4
12a
12b
12c
12d
-4.6
-4.2
-6.3
2.4. Biophysical studies
To confirm the results obtained from the in-vitro antimicrobial and docking studies, the interactions
between the benzazole-pyrimidines 5 and 12d and the β-lactamase enzyme were investigated using UV-vis
absorption, fluorescence, circular dichroism, 1HNMR, and SEM techniques.
2.4.1. UV-vis study
UV-vis absorption technique is a simple operational method to validate the structural change in an enzyme
and to identify complex formation. In the binding experiment, UV-vis absorption spectroscopy was adopted to
evaluate the binding behavior between compound 5 and the β-lactamase enzyme. The band at 280 nm was
attributed to the n-σ*, while the band at 330 nm appeared to be a combination of n-π* and π-π* transitions. The
11

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β-lactamase enzyme band at 275 nm clearly interfered with the absorption band of compound 5 at 280 nm. The
intensity of the band at 280 nm increased, probably because of overlapping with the β-lactamase enzyme band at
275 nm. In contrast, with the addition of compound 5, the peak intensity of the band at 330 nm increased,
indicating that compound 5 interacted with the β-lactamase enzyme (see Appendix A, Fig. S2).
The UV-spectra of compound 12d showed two absorption bands at λ = 287 and 336 nm (see Appendix A,
Fig. S3). The band at λ = 287 nm was attributed to the n-σ*, while the band at λ = 336 nm is a combination of n-
π* and π-π* transitions. The β-lactamase enzyme demonstrated one absorption band at λ = 275 nm and clearly
interfered with the absorption band at λ = 287 nm of 12d. Therefore, the absorbance at λ = 287 nm increased
upon the addition of the β-lactamase enzyme. The intensity of the band at λ = 336 nm also increased upon the
addition of the enzyme which might indicate an interaction between the enzyme and the ligand.
2.4.2. Fluorescence study
Fluorescence spectroscopy was the second method used to confirm the interaction between the active
benzazole-pyrimidines 5 and 12d and the β-lactamase enzyme in phosphate buffer, pH 7.4. The fluorescence
spectrum of compound 5, using excitation λmax of 280 nm, showed an emission band at λ = 443 nm attributed to
the n-π* singlet-singlet transition. Upon sequential additions of the β-lactamase enzyme, this band had a slight
red shift (~2 nm) with an increased intensity, indicating that compound 5 interacted with the β-lactamase enzyme.
The same result was observed when compound 12d was used in titration with β-lactamase (see Appendix A, Fig.
S4).
2.4.3. Circular dichroism (CD) study
Circular dichroism (CD) is a spectroscopic technique where the absorption of molecules is measured over a
range of wavelengths. CD spectroscopy is used extensively to study chiral molecules of all types and sizes. A
primary use is to analyze the secondary structure and the conformation of macromolecules, such as protein
secondary structures, which are found to be more sensitive to the environment. Structures change on interaction
with other molecules and can be detected and studied using CD spectroscopy [29]. We conducted CD titration
experiments to get more information on the interactions between compounds 5, 9 and 12 with β-lactamase and
conformation changes upon interaction. The CD spectrum of the β-lactamase enzyme in phosphate buffer at pH
7.4 showed a negative band centered at 220 nm and one positive band at 270 nm. This finding is consistent with
previously reported works [30] (see Appendix A, Fig. S5).
Additions of compound 5 to the β-lactamase enzyme resulted in CD spectral changes. The intensities of
the band centered at 220 nm became more positive upon the addition of compound 5, while after extensive
addition of 5, the negative band at 220 nm blue-shifted slightly and gradually transformed into a positive band
centered at 215 nm. The transformation of the negative band at 220 nm into the positive one at 215 nm indicates a
change in the β-lactamase enzyme conformation upon interaction with 5. The same results were obtained when
12d was used, as can been seen in Appendix A, Fig. S6.
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2.4.4. NMR studies: Complexation of β-lactamase with benzazole-pyrimidine 12d
The docking studies demonstrated that the nitrile-activated double bond in compound 12d was able to
form a covalent bond with the Ser70 residue in the β-lactamase pocket. NMR was used to confirm the formation
of the covalent bond between the benzazole-pyrimidine 12d and the β-lactamase active site.
The complexation behavior of the synthesized benzazole-pyrimidine 12d with β-lactamase enzyme was
investigated as induced chemical shifts of the benzazole-pyrimidine protons. Upon addition of β-lactamase
enzyme, no significant changes occurred in the chemical shifts of the benzazole-pyrimidine protons. After 60 min,
the chemical shifts of the benzazole-pyrimidine protons of 12d were observed in the upfield region. The ∆δ
calculated suggested that β-lactamase enzyme preferentially encapsulates the benzazole-pyrimidine 12d moiety.
The inclusion of compound 12d into the β-lactamase enzyme active site led to a significant shift of the
pyrimidine H-4 and H-6 to a higher field of 0.95 ppm. The disappearance of the olefinic proton of 12d at δ = 7.78
ppm and the appearance of a new signal resonance at δ = 5.28 ppm integrated to one proton and assigned for the
methine proton confirmed the formation of a new covalent bond in the β-lactamase pocket (see Appendix A, Fig.
S7).
2.4.5. Morphological changes of E. coli bacteria upon the addition of compound 5
The morphological effect observed using TEM provided evidence for interactions with, and disruption of,
the cell wall. For instance, PBP located in the cytoplasmic membrane, may bind to compound 5 and changes the
cell morphology which could result in PBP-compound 5 interaction. Additionally, untreated cells must be studied
by electron microscopy as controls (untreated bacteria) to confirm observed changes in the bacterial cells exposed
to compound 5.
TEM analysis of unstained bacteria showed normal external morphological features of the bacterial strain.
E. coli cells diluted in phosphate buffer exhibited many filaments, such as flagella and fimbriae (see Appendix A
Fig. S8a–c). The fimbriae measured approximately 7.00 nm wide, and up to 900 nm long. In contrast, after
treatment with compound 5 for 2 h the bacterial cells appeared to be seriously damaged (see Appendix A, Fig.
S8d–i). The cells demonstrated unusual morphology of being cracked and ruptured. Electron-dense particles or
precipitates were also observed around damaged bacterial cells. Damaged cells showed either localized or
complete separation of the cell membrane from the cell wall. The cellular degradation was also accompanied by
electron-translucent cytoplasm and cellular disruption in the damaged cells. These findings support the previously
mentioned theory that compounds 5, 9 and 12 could exert their antibacterial activity via inhibiting penicillin-
binding protein, which is essential for bacterial cell wall synthesis.
3. Conclusion
A new series of nine benzimidazole and benzothiazole pyrimidines 5, 9 and 12 were synthesized via an
environmentally friendly and efficient procedure, and the products were then screened for their antimicrobial
activities. Among the newly synthesized derivatives, compound 5 exhibited the highest antibacterial activities,
which were comparable to those of the test control, amoxicillin. The bactericidal activity of these compounds was
proposed, by docking, to result from the irreversible inhibition of PBP, which is important for cell wall synthesis.
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In line with the in silico data, TEM studies showed that these compounds can cause bacterial cell wall rupture.
Furthermore, these compounds exhibited the potential to overcome bacterial resistance by demonstrating
significant synergism when combined with amoxicillin. Docking studies showed that these compounds have the
ability to covalently bind the β-lactamase enzyme via forming an irreversible interaction with the key amino acid
residue Ser62. The results obtained were confirmed using 1H-NMR, UV, fluorescence, and TEM techniques.
Therefore, these compounds open new avenues for the development of anti-bacterial therapeutic agents
for the treatment of infectious diseases. In addition, these results give an insight into structure-activity
relationships, which are tremendously important for the design of further new antimicrobial compounds.
4. Experimental
4.1. Chemistry
All reagents and chemicals were purchased from Sigma-Aldrich and used without further purification. Thin-
layer chromatography (TLC) was performed on silica gel glass plates (Silica gel, 60 F254, Fluka) and the spots
were visualized under a UV lamp. Column chromatography was performed on a Kieselgel S (silica gel S, 0.063–
0.1 mm). The melting points were recorded on a Gallenkamp apparatus and are uncorrected. Infrared spectra
1
were measured using KBr pellets on a Thermo Nicolet model 470 FT-IR spectrophotometer. H-NMR spectra
were recorded on Varian, 400 MHz instruments by using DMSO-d6 and CDCl3 solutions and tetramethylsilane
(TMS) as an internal reference. The element analysis was performed on a Euro Vector EA 3000. Fluorescence
measurements were conducted inside quartz cells using a Cary Eclipse model-3 spectrofluorometer equipped
with a high-intensity Xenon flash lamp and 1.0 cm path length (Varian, Austria). Absorption measurements were
carried out using an Agilent 8453 spectrophotometer supported by 1.0 cm quartz cells (Austria). CD
measurements were made using a Jasco J-815 spectrometer (Jasco, USA). A detailed description of the
morphology was obtained by transmission electron microscopy (TEM; CM10-Phillips Amsterdam, Netherlands).
4.2. Amination
Compounds 7a-d obtained by the reaction between 2-chloropyrimidine 6 (1.0 mmol, 0.148g) with the
appropriate amines (1.0 mmol) in ethanol (2-3 ml) at 0°C in the presence of N,N-diisopropyl ethylamine (DIPEA,
1.1 mmol) under microwave irradiation for 10 min. The progress of reaction was monitored by TLC. Ethyl
acetate (10.0 ml) was added to the reaction mixture and the pH was adjusted to 7-7.5 using HCl (6.0 M). The
mixture was washed with saturated aqueous solution of NaHCO3. The organic layer was dried over anhydrous
MgSO4. The excess solvent was removed under reduced pressure and the obtained residue was further purified by
column chromatography using ethyl acetate-hexane (1:1) to afford the final products in good yields.
1
4.2.1. N,N-dimethylpyrimidin-2-amine (7a). H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 3.18 (s, 6H, CH3), 6.85
(m,1H, H5-pyrimidine), 8.70 (s, 2H, H4,6-pyrimidine); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 37.6 (CH3),
117.4 (C5-pyrimidine), 159.8 (C4,6-pyrimidine), 161.9 (C2-pyrimidine).
14

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1
4.2.2. N-benzylpyrimidin-2-amine (7b). H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 4.56 (d, 2H, Ph-CH2, J = 4.0
Hz), 6.68 (m,1H, H5-pyrimidine), 7.21-7.28 (m, 5H, aromatic), 8.69 (s, 2H, H4,6-pyrimidine), 8.82 (s, 1H, NH,
exchanges with D2O); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 44.3 (CH2, benzylamine), 119.4 (C5-
pyrimidine), 127.0, 127.3, 128.5, 138.7 (aromatic), 159.2 (C4,6-pyrimidine), 160.3 (C2-pyrimidine).
4.2.3. 4-(pyrimidin-2-yl)morpholine (7c). 1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 3.62-3.64 (m, 4H,
morpholine), 3.84-3.85 (m, 4H, morpholine), 6.70 (m,1H, H5-pyrimidine), 8.72 (s, 2H, H4,6-pyrimidine); 13C-
NMR [DMSO-d6, 100 MHz]: (δ, ppm) 45.1 (morpholine), 66.6 (morpholine), 119.0 (C5-pyrimidine), 159.2
(C4,6-pyrimidine), 160.1 (C2-pyrimidine).
1
4.2.4. N-(4-chlorophenyl)pyrimidin-2-amine (7d). H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 6.71 (m,1H, H5-
pyrimidine), 7.53-7.55 (d, 2H, p-chlorophenyl, J = 8.0 Hz ), 8.34 (s, 1H, NH, exchanges with D2O), 8.40-8.41 (d,
13
2H, p-chlorophenyl, J = 8.0Hz), 9.23 (s, 2H, H4,6-pyrimidine); C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 125.2
(C5-pyrimidine),129.4, 129.9, 134.8, 137.5 (aromatic), 158.1 (C4,6-pyrimidine), 162.3 (C2-pyrimidine).
4.3. General synthetic procedure for formulation of pyrimidines 2 and 8
Vilsmeier reagent was prepared by mixing ice-cold dry DMF (50 ml) and POCl3 (30.0 mmol, 2.8 ml). The
mixture was stirred for 15 min at º25C. To the previous mixture, aminopyrimidines (10.0 mmol) in dry DMF
(5.0 ml) were added over a period of 15 min at 0–5 °C. The reaction mixture was stirred for 24 h at º25C. The
mixture was then added to cold, saturated aq. K2CO3 and extracted with diethyl ether. The organic layer was
washed with water, dried over anhydrous Na2SO4, and evaporated under reduced pressure to afford the crude
product, which was purified in a silica gel column chromatography using hexane/ethyl acetate (9:1) as an eluent
to give the title compounds 2 and 8
1
4.3.1. 2,4-Diaminopyrimidine-5-carbaldehyde (2). Brown powder, yield 59 %; H-NMR [DMSO-d6, 400 MHz]:
(δ, ppm) 7.10 (d, 2H, NH2, exchange with D2O), 7.52 (d, 2H, NH2, exchange with D2O), 8.32 (s, 1H, H6-
pyrimidine), 9.45 (CHO); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 106.2 (C5-pyrimidine), 162.7 (C4-
pyrimidine), 164.4 (C6-pyrimidine), 167.0 (C2-pyrimidine), 189.5 (CHO).
4.3.2. 2-(N,N-Dimethylamino)pyrimidine-5-carbaldehyde (8a). Pale yellow crystals, yield 62 %; 1H-NMR
13
[DMSO-d6, 400 MHz]: (δ, ppm) 3.19 (s, 6H, CH3), 8.73 (s, 2H, H4,6-pyrimidine), 9.71 (s, 1H, CHO); C-NMR
[DMSO-d6, 100 MHz]: (δ, ppm) 37.4 (CH3), 119.4 (C5-pyrimidine), 160.7 (C4,6-pyrimidine), 162.8 (C2-
pyrimidine), 189.2 (CHO).
4.3.3. 2-(N-Benzylamino)pyrimidine-5-carbaldehyde (8b). Yellow powder, yield 70 %; 1H-NMR [DMSO-d6, 400
MHz]: (δ, ppm) 4.57 (d, 2H, Ph-CH2, J = 4.0 Hz), 7.20–7.28 (m, 5H, aromatic), 8.71 (s, 2H, H4,6-pyrimidine),
8.84 (s, 1H, NH, exchanges with D2O), 9.68 (CHO); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 44.5 (CH2,
15

ACCEPTED MANUSCRIPT
benzylamine), 120.7 (C5-pyrimidine), 127.3, 127.5, 128.8, 139.5 (aromatic), 160.8 (C4,6-pyrimidine), 163.8 (C2-
pyrimidine), 189.1 (CHO).
1
4.3.4. 2-(N-Morpholino)pyrimidine-5-carbaldehyde (8c). Pale yellow powder, yield 60 %; H-NMR [DMSO-d6,
400 MHz]: (δ, ppm) 3.62–3.64 (m, 4H, morpholine), 3.84–3.85 (m, 4H, morpholine), 8.77 (s, 2H, H4,6-
pyrimidine), 9.73 (s, 1H, CHO); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 44.5 (morpholine), 66.3 (morpholine),
120.1 (C5-pyrimidine), 160.9 (C4,6-pyrimidine), 162.1 (C2-pyrimidine), 189.2 (CHO).
1
4.3.5. 2-(4
′
-Chlorobenzylamino)pyrimidine-5-carbaldehyde (8d). Pale yellow powder, yield 61 %; H-NMR
[DMSO-d6, 400 MHz]: (δ, ppm) 7.58–7.60 (d, 2H, p-chlorophenyl, J = 8.0 Hz), 8.35 (s, 1H, NH, exchanges with
D2O), 8.41–8.43 (d, 2H, p-chlorophenyl, J = 8.0Hz), 9.28 (s, 2H, H4,6-pyrimidine), 10.10 (CHO); 13C-NMR
[DMSO-d6, 100 MHz]: (δ, ppm) 127.3 (C5-pyrimidine), 129.6, 130.8, 135.4, 137.6 (aromatic), 159.3 (C4,6-
pyrimidine), 165.6 (C2-pyrimidine), 191.3 (CHO).
4.4. Synthesis of benzothiazole-pyrimidines
4.4.1. 2-(Benzo[d]thiazol-2′-yl)acetonitrile (4). A mixture of 2-aminothiophenol 3 (0.01 mol, 1.1 ml) and
malononitrile (0.01 mol, 0.64 ml) in ethanol (10 ml) was stirred at º25C for 5 h. The yellow solid was collected
1
by filtration and recrystallized from ethanol to give light brown crystals; yield 86 %; mp 102 °C; H-NMR
[DMSO-d6, 400 MHz]: (δ, ppm) 4.72 (s, 2H, CH2), 7.45–7.52 (m, 2H, H5,6-benzothiazole), 8.00–8.10 (m, 2H,
H4,7-benzothiazole); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 22.8 (CH2), 117.5 (CN), 122.8 (C7-
benzothiazole), 123.1 (C4-benzothiazole), 126.1 (C5-benzothiazole), 127.0 (C6-benzothiazole), 135.5 (C3′a-
benzothiazole), 152.7 (C3′b-benzothiazole), 161.0 (C2-benzothiazole).
4.4.2. General synthetic procedure of (E)-2-(Benzo[d]thiazol-2′-yl)-3-arylacrylonitriles 5 and 9a–d
A mixture of 2-(benzo[d] thiazol-2′-yl)acetonitrile 4 (1.0 mmol, 0.17 g) and pyrimidine aldehyde 2 or 8
(1.0 mmol) was stirred at º25C for 10–15 min in ethanol (10 ml) which contained piperidine (2.0 mmol, 0.2 ml).
The reaction was monitored by TLC. The formed solid was filtrated and washed with hexane (2 × 10 ml) to give
the corresponding compounds 5 and 9a–d.
4.4.2.1. (E)-2-(Benzo[d]thiazol-2′-yl)-3-(2″,4″-diaminopyrimidin-5″-yl)acrylonitrile (5). Yellow powder; yield
77 %; mp 280 °C; IR (KBr, cm-1): 3395, 3307 (br, NH2), 2101 (CN), 2733 (C-H aliphatic), 1565 (C=C); 1H-NMR
[DMSO-d6, 400 MHz]: (δ, ppm) 6.86–6.88 (bs, 2H, NH2, exchangeable with D2O), 7.47 (t, 1H, H5-benzothiazole,
J = 8.0 Hz), 7.55 (t, 1H, H6-benzothiazole, J = 8.0 Hz), 8.05 (d, 1H, H4-benzothiazole, J = 8.0 Hz), 8.11 (d, 1H,
H7-benzothiazole, J = 8.0 Hz), 8.30 (brs, 2H, NH2, exchangeable with D2O), 8.52 (s, 1H, H6-pyrimidine), 8.87
13
(olefinic H); C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 106.4 (C5-pyrimidine), 110.1 (C2′), 112.2 (CN), 122.3
(C7-benzothiazole), 133.3 (C4-benzothiazole), 139.9 (C5-benzothiazole), 139.9 (C6-benzothiazole), 153.1 (C3′a-
16

ACCEPTED MANUSCRIPT
benzothiazole), 160.0 (C6-pyrimidine), 161.5 (C3′b-benzothiazole), 162.9 (olefinic C), 164.5 (C4-pyrimidine),
164.8 (C2-benzothiazole), 167.1 (C2-pyrimidine); Anal. Calcd for C14H10N6S: C, 57.13; H, 3.42; N, 28.55; S,
10.89; Found: C, 57.18; H, 3.33; N, 28.63; S, 10.96; Anal. Calcd for C14H10N6S.2HBr: C, 36.86; H, 2.65; N,
18.42; S, 7.03; Found: C, 37.05; H, 2.45; N, 18.44; S, 7.05.
4.4.2.2. (E)-2-(Benzo[d]thiazol-2′-yl)-3-(2″-(dimethylamino)pyrimidin-5″-yl)acrylonitrile (9a). Yellow powder;
yield 86 %; mp 251 °C; IR (KBr, cm-1): 3408 (Ar-H stret), 2926 (aliphatic C-H stret.), 2209 (CN), 1607 (C=C);
1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 3.25 (s, 6H, 2CH3), 7.47–7.56 (m, 2H,H5,6 benzothiazole), 8.01 (d, 1H,
H4 benzothiazole, J = 8.0 Hz), 8.10 (d, 1H, H7 benzothiazole, J = 8.0 Hz), 8.17 (s, 1H, olefinic H), 9.02 (s, 2H,
H4,6 pyrimidine); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 37.4 (CH3), 100.5 (C2′), 115.3 (CN), 117.4 (C5-
pyrimidine), 122.7 (C7-benzothiazole), 123.2 (C4-benzothiazole), 126.3 (C5-benzothiazole), 127.4 (C6-
benzothiazole), 134.7 (C3`a-benzothiazole), 143.8 (C3`b-benzothiazole), 153.5 (olefinic C), 160.1 (C4,6-
pyrimidine), 161.7 (C2-benzothiazole), 163.7 (C2-pyrimidine); Anal. Calcd for C16H13N5S: C, 62.52; H, 4.26; N,
22.78; S, 10.43; Found: C, 62.57; H, 4.17; N, 22.86; S, 10.50; Anal. Calcd for C16H13N5S.HBr: C, 49.49; H, 3.63;
N, 18.04; S, 8.26; Found: C, 49.68; H, 3.43; N, 18.03; S, 8.27.
4.4.2.3. (E)-2-(Benzo[d]thiazol-2′-yl)-3-(2″-benzylaminopyrimidin-5″-yl)acrylonitrile (9b). Yellow powder; yield
78 %; mp 238 °C; IR (KBr, cm-1): 3458 (NH), 3228 (aromatic C-H- str.), 2985 (C-H aliphatic), 2211 (CN), 1614
1
(C=C); H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 4.62–4.64 (d, 2H, CH2, J = 8.0 Hz), 7.24–7.28 (m, 1H,
benzylamine), 7.34–7.35 (m, 4H, benzylamine), 7.48 (t, 1H, H5-benzothiazole, J = 8.0 Hz), 7.58 (t, 1H, H6-
benzothiazole, J = 8.0 Hz), 8.02 (d, 1H, H4-benzothiazole, J = 8.0 Hz), 8.13 (d, 1H, H7-benzothiazole), 8.17 (s,
13
1H, olefinic H), 8.92 (t, 1H, NH, exchangeable with D2O), 9.47 (s, 2H, H4,6-pyrimidine); C-NMR [DMSO-d6,
100 MHz]: (δ, ppm) 44.6 (CH2), 100.5 (C2′), 116.4 (CN), 117.4 (C5-pyrimidine), 122.8 (C7-benzothiazole),
123.2 (C4-benzothiazole), 126.4 (C5-benzothiazole), 127.3 (C6-benzothiazole), 127.6, 128.8, 134.5
(benzylamine), 139.6 (C3′a-benzothiazole), 143.8 (benzylamine), 153.4 (C3′b-benzothiazole), 160.6 (olefinic C),
160.9 (C4,6-pyrimidine),162.4 (C2-benzothiazole), 163.9 (C2-pyrimidine); Anal. Calcd for C21H15N5S: C, 68.27;
H, 4.09; N, 18.96; S, 8.68; Found: C, 68.32; H, 4.01; N, 19.01; S, 8.75; Anal. Calcd for C21H15N5S.HBr: C, 56.01;
H, 3.58; N, 15.55; S, 7.12; Found: C, 56.20; H, 3.38; N, 15.56; S, 7.14.
4.4.2.4. (E)-2-(Benzo[d]thiazol-2′-yl)-3-(2″-morpholinopyrimidin-5″-yl)acrylonitrile (9c). Yellow powder; yield
80 %; mp 248 °C; IR (KBr, cm-1): 3053 (aromatic C-H- str.), 2968 (CH-aliphatic), 2210 (CN), 1582 (C=C); 1H-
NMR [DMSO-d6, 400 MHz]: (δ,ppm) 3.67–3.68 (m, 4H, morpholine), 3.83–3.84 (m, 4H, morpholine), 6.54 (t,
1H, H5-benzothiazole, J = 8.0 Hz), 6.69 (d, 1H, H4-benzothiazole, J = 8.0 Hz), 6.94 (t, 1H, H6-benzothiazole, J =
13
8.0 Hz), 7.06 (d, 1H, H7-benzothiazole, J = 8.0 Hz), 8.51 (s, 1H, olefinic H), 8.94 (s, 2H, H4,6-pyrimidine); C-
NMR [DMSO-d6, 100 MHz]: (δ, ppm) 44.4, 66.4 (morpholine), 102.6 (C2′), 117.4 (CN), 119.5 (C5-pyrimidine),
124.8 (C7-benzothiazole), 125.4 (C4-benzothiazole), 128.4 (C5-benzothiazole), 129.5 (C6-benzothiazole), 136.8
(C3′a -benzothiazole), 145.9 (C3′b -benzothiazole), 155.7 (olefinic C), 162.3 (C4,6-pyrimidine), 163.8 (C2-
17

ACCEPTED MANUSCRIPT
benzothiazole), 165.9 (C2-pyrimidine); Anal. Calcd for C18H15N5OS: C, 61.87; H, 4.33; N, 20.04; S, 9.18; Found:
C, 61.92; H, 4.24; N, 20.12; S, 9.25; Anal. Calcd for C18H15N5OS.HBr: C, 50.24; H, 3.75; N, 16.27; S, 7.45;
Found: C, 50.43; H, 3.55; N, 16.28; S, 7.46.
4.4.2.5.
(E)-2-(Benzo[d]thiazol-2
′
-yl)-3-[2″-(p-chlorophenylamino)pyrimidin-5
″-yl]
acrylonitrile
(9d).
Crystallized from ethanol, orange powder; yield 83 %; mp 188 °C; IR (KBr, cm-1): 3445 (br, NH), 3269
(aromatic C-H- str.), 3050 (C-H aliphatic), 2232 (CN), 1572 (C=C); 1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm)
7.25 (d, 2H, p-chlorophenyl, J = 8.0 Hz), 7.40 (t, 1H, H5-benzothiazole, J = 8.0 Hz), 7.57 (d, 1H, H4-
benzothiazole, J = 8.0 Hz), 7.84 (t, 1H, H6- benzothiazole, J = 8.0 Hz), 8.03 (d, 1H, H7- benzothiazole, J = 8.0
Hz), 8.26 (s, 1H, NH, exchangeable with D2O), 8.44 (d, 2H, p-chlorophenyl, J = 7.8 Hz), 8.73 (s, 1H, olefinic H),
9.39 (s, 2H, H4,6-pyrimidine); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 113.2 (C2′), 117.4 (CN), 118.3 (C5-
pyrimidine), 119.2 (C7-benzothiazole), 126.9 (Ar-C, p-chlorophenyl), 127.8 (C4-benzothiazole), 130.6 (C5-
benzothiazole), 131.0 (C6-benzothiazole), 131.3 (Ar-C, p-chlorophenyl), 132.1 (Ar-C, p-chlorophenyl), 136.3
(C3a′-benzothiazole), 137.9 (Ar-C, p-chlorophenyl), 138.6 (C3b′-benzothiazole), 147.1 (olefinic-C), 152.8 (C4,6-
pyrimidine), 159.6 (C2-benzothiazole), 165.1 (C2-pyrimidine); Anal. Calcd for C20H12ClN5S: C, 61.62; H, 3.10;
N, 17.96; S, 8.22; Found: C, 61.67; H, 3.01; N, 18.03; S, 8.29; Anal. Calcd for C20H12ClN5S.HBr: C, 51.03; H,
2.78; N, 14.88; S, 6.81; Found: C, 51.22; H, 2.58; N, 15.01; S, 6.91.
4.5. Synthesis of benzimidazole-pyrimidines
4.5.1. 2-(1H-Benzo[d]imidazol-2′-yl)acetonitrile (11). A mixture of o-phenylenediamine 10 (0.01mol, 1.08 g)
and ethyl cyanoacetate (0.01 mol, 1.7g) were refluxed in ethanol (30 mL) for 20 min. After cooling, the solution
was extracted with ether (3 × 25 ml). The solvent was evaporated and the resultant solid product 11 was
o
1
recrystallized from ethanol as a brown crystals; yield 75 %; mp 212 C; H-NMR [DMSO-d6, 400 MHz]: (δ,
ppm) 4.36 (s, 2H, CH2), 5.17 (s, 1H, NH exchangeable with D2O), 6.35 (m, 2H, H5,6-benzimidazole), 6.47 (m, 2H,
H4,7-benzimidazole); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 22.8 (CH2), 117.5 (CN), 122.8 (C4-
benzimidazole), 123.1 (C7-benzimidazole), 126.1 (C5-benzimidazole), 127.0 (C6-benzimidazole), 135.5 (C3′-
benzimidazole), 152.7 (C2-benzimidazole).
4.5.2. General synthetic procedure of (E)-2-(Benzo[d]imidazol-2′-yl)-3-arylacrylonitriles 12a–d
A mixture of 2-(1H-benzo[d]imidazol-2′-yl)acetonitrile 11 (1.0 mmol, 0.16g) and pyrimidine aldehydes 8
(1.0 mmol) was stirred at º25Cfor 10–15 min in ethanol (10.0 ml) that contained piperidine (2.0 mmol, 0.2 ml).
The reactions were monitored by TLC, in order to verify the consumption of the precursors. The solid products
were isolated by filtration and washed with a mixture of hexane/ethanol (7:3) to give the corresponding
compounds 12a–d.
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4.5.2.1. (E)-2-(1H-Benzo[d]imidazol-2′-yl)-3-[2″-(dimethylamino)pyrimidin-5″-yl) acrylonitrile (12a). Orange
crystals; yield 89 %; mp 154 °C; IR (KBr, cm-1): 3439 (NH), 3307 (aromatic C-H-str.), 2860 (CH-aliphatic),
1
2395 (CN), 1616 (C=C); H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 3.21 (s, 6H, CH3), 5.17 (s, 1H, NH
exchangeable with D2O), 6.54 (t, 1H, H5-benzimidazole, J = 8.0 Hz), 6.68 (d, 1H, H6-benzimidazole, J = 8.0 Hz),
6.93 (t, 1H, H4- benzimidazole, J = 8.0 Hz), 7.05 (d, 1H, H7-benzimidazole, J = 8.0 Hz), 8.49 (s, 1H, olefinic H),
13
8.89 (s, 2H, H4,6-pyrimidine); C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 37.26 (CH3), 114.9 (C2′), 116.5 (C4,7-
benzimidazole), 117.1 (CN), 119.0 (C5-pyrimidine), 127.5 (C5,6-benzimidazole), 135.9 (C3′-benzimidazole),
144.1 (C2-benzimidazole), 152.5 (olefinic C), 158.7 (C4,6-pyrimidine), 162.3 (C2-pyrimidine); Anal. Calcd for
C16H14N6: C, 66.19; H, 4.86; N, 28.95; Found: C, 66.24; H, 4.77; N, 29.03; Anal. Calcd for C16H14N6.HBr: C,
51.77; H, 4.07; N, 22.64; Found: C, 51.96; H, 3.87; N, 22.66.
4.5.2.2. (E)-2-(1H-Benzo[d]imidazol-2′-yl)-3-[2″-(benzylamino)pyrimidin-5″-yl]acrylonitrile (12b). Light brown
powder; yield 81 %; mp 152 °C; IR (KBr, cm-1): 3471 (br, NH), 3367 (aromatic C-H-str.), 3226 (C-H aliphatic),
2207 (CN), 1614 (C=C); 1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 4.59 (d, 2H, CH2, J = 8Hz), 5.16 (s, 1H, NH
exchangeable with D2O), 6.53 (t, 1H, H5-benzimidazole, J = 8.0 Hz), 6.67 (d, 1H, H6-benzimidazole, J = 8.0 Hz),
6.93 (t, 1H, H4-benzimidazole, J = 8.0 Hz), 7.03 (d, 1H, H7- benzothiazole, J = 8.0 Hz), 7.22–7.25 (m, 1H,
benzylamine), 7.29–7.33 (m, 4H, benzylamine), 8.36 (t, 1H, NH, exchangeable with D2O), 8.46 (s, 1H, olefinic
H), 8.88 (s, 2H, H4,6-pyrimidine); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 44.5 (CH2), 114.9 (C2′), 116.5
(C4,7-benzimidazole), 117.2 (CN), 120.3 (C5-pyrimidine), 127.1 (C5,6-benzimidazole), 127.5-135.9 (Ar-C,
benzylamine), 140.2 (C3′-benzimidazole), 144.1 (C2- benzimidazole), 152.5 (olefinic C), 159.1 (C4,6-
pyrimidine), 163.1 ( C2-pyrimidine); Anal. Calcd for C21H16N6: C, 71.58; H, 4.58; N, 23.85; Found: C, 71.63; H,
4.49; N, 23.93; Anal. Calcd for C21H16N6.HBr: C, 58.21; H, 3.95; N, 19.40; Found: C, 58.41; H, 3.75; N, 19.39.
4.5.2.3. (E)-2-(1H-Benzo[d]imidazol-2′-yl)-3-(2″-morpholinopyrimidin-5″-yl)acrylonitrile (12c). Yellow powder;
yield 84 %; mp 176 °C; IR (KBr, cm-1): 3464 (br, NH), 3365 (aromatic C-H str.), 2967 (C-H aliphatic), 2331
1
(CN), 1604 (C=N); H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 3.67 (m, 4H, morpholine), 3.83 (m, 4H,
morpholine), 5.20 (s, 1H, NH exchangeable with D2O), 6.54 (t, 1H, H5-benzimidazole, J = 8.0 Hz), 6.69 (d, 1H,
H6-benzimidazole, J = 8.0 Hz), 6.94 (t, 1H, H4-benzimidazole, J = 8.0 Hz), 7.06 (d, 1H, H7-benzimidazole, J =
13
8.0 Hz), 8.51 (s, 1H, olefinic H), 8.93 (s, 2H, H4,6-pyrimidine); C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 44.4,
66.4 (morpholine), 114.9 (C2`), 116.5 (C4,7-benzimidazole), 117.1 (CN), 120.1 (C5-pyrimidine), 127.6 (C5,6-
benzimidazole), 135.7 (C3′-benzimidazole), 144.2 (C2-benzimidazole), 152.5 (olefinic C), 158.9 (C4,6-
pyrimidine), 161.7 (C2-pyrimidine); Anal. Calcd for C18H16N6O: C, 65.05; H, 4.85; N, 25.29; Found: C, 65.10; H,
4.76; N, 25.37; Anal. Calcd for C18H16N6O.HBr: C, 52.31; H, 4.15; N, 20.34; Found: C, 52.51; H, 3.95; N, 20.36.
4.5.2.4. (E)-2-(1H-Benzo[d]imidazol-2′-yl)-3-(2″-(p-chlorophenyl)amino)pyrimidin-5″-yl) acrylonitrile (12d).
Orange powder; yield 87 %; mp 258 °C; IR (KBr, cm-1): 3446 (br, NH), 3350 (aromatic C-H-str.), 3023 (C-H
aliphatic), 2260 (CN), 1606 (C=N); 1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 5.47 (s, 1H, NH exchangeable with
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D2O), 6.58 (t, 1H, H5-benzimidazole, J = 8.0 Hz), 6.74 (d, 1H, H6-benzimidazole, J = 8.0 Hz), 7.02 (t, 1H, H4-
benzimidazole, J = 8.0 Hz), 7.21(d, 1H, H7-benzimidazole, J = 8.0 Hz), 7.62 (d, 2H, p-Chlorophenyl, J = 7.8Hz),
7.98 (s,1H, NH, exchangeable with D2O), 8.46 (d, 2H, p-Chlorophenyl, J = 7.8Hz), 8.78 (s, 1H, olefinic H), 9.44
(s, 2H, H4,6-pyrimidine); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 115.4 (C2′), 116.3 (C4,7-benzimidazole),
117.3 (CN), 123.4 (C5-pyrimidine), 129.1(C5,6-benzimidazole) 129.4–135.9 (Ar-C, p-chlorophenyl), 136.7
(C3′-benzimidazole), 145.2 (C2-benzimidazole), 150.9 (olefinic C), 157.7 (C4,6-pyrimidine), 163.1 (C2-
pyrimidine); Anal. Calcd for C20H13ClN6: C, 64.43; H, 3.51; N, 22.54; Found: C, 64.48; H, 3.42; N, 22.62; Anal.
Calcd for C20H13ClN6.HBr: C, 52.94; H, 3.11; N, 18.52; Found: C, 53.13; H, 2.91; N, 18.51.
4.6. Salt formation
Compounds 5, 9a–d and 12a–d were dissolved in a minimal amount of methanol and treated with excess
hydrobromic acid to form the hydrobromide salt. The salt was collected, washed with diethyl ether, and dried.
4.7. Antibacterial activity
4.7.1. Medium
The antibacterial assay was performed using the well diffusion method for all compounds 5, 9a-d and
12a-d. Mueller Hinton agar (38.0 g of Mueller Hinton Agar in 1000 mL of distilled water) was prepared and
autoclaved. It was poured into the petri dishes and solidified at º25C. The bacterial strains (106 cells/ml) were
introduced in the plates using pipettes and hockey sticks to spread on the agar. For well diffusion, well were
made, and all the compounds were poured individually into the well (50 µl/well). It was then incubated at 37 °C
for 24–48 hours. The bacterial inhibition was determined by measuring the diameter of the inhibition zone (mm)
using a transparent scale. Antibiotic (amoxicillin 5.0 mg/ml) was then used as a positive control at 50 µl/well.
4.7.2. Test microorganisms
Two Gram-positive bacteria, namely, Staphylococcus aureus (ATCC-25923) and Bacillus subtilis
(ATCC 6633) and two Gram-negative bacteria, namely, Escherichia coli (ATCC-25922) and Pseudomonas
aeruginosa (ATCC-27853) were used to determine antibacterial activity.
4.7.3. Minimum inhibitory concentration (MIC)
Compounds 5, 9a-d and 12a-d were serially diluted with autoclaved sterile distilled water and examined
from lower to higher dilution to find the MIC. Serially diluted compounds (100 µl) were added to sterile nutrient
broth (900 µl). A Control test-tube containing only medium (nutrient broth medium) was used to confirm the
sterility of the medium bacterial cultures. Bacterial suspension (10 µl) containing 105 cells/ml was inoculated into
all tubes. All of the test tubes were incubated at 37±1°C and observed for bacterial growth for 24 hours for MIC
determination. After incubation, for 24 hours, the test tube with no visible growth of the microorganism was
taken to represent the MIC value of the sample in µg/ml. Triplicates of each tested compound were performed,
and the average of the results was taken.
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4.7.4. Minimum inhibitory concentration (MIC) for combination of amoxicillin (MIC) with 5 and 12d
The minimal inhibitory concentration (MIC) of an antimicrobial compound is the lowest (i.e. minimum)
concentration of the antimicrobial compound that inhibits a given bacterial strain. Compounds (amoxicillin,
amoxicillin with 5 and 12d) were serially diluted with autoclaved sterile distilled water and examined from lower
to higher dilutions to find the MIC. Serially diluted compounds (100 µl) were added to sterile nutrient broth (900
µl). A Control test-tube containing only medium (nutrient broth medium) was used to confirm the sterility of the
medium bacterial cultures. Bacterial suspension (10 µl) containing 105 cells/ml was inoculated into all tubes. All
of the test tubes were incubated at 37±1°C and observed for bacterial growth for 24 hours for MIC determination.
After incubation, for 24 hours, the test tube with no visible growth of the microorganism was taken to represent
the MIC value of the sample in µg/ml.
4.7.5. Transmission electron microscopy analysis
Transmission electron microscopy (TEM) was used to evaluate the morphological changes in E. coli
after treatment with compound 5. Treated bacterial and control cells were processed by the same procedure for all
techniques. Upon incubation, the pellets obtained after centrifugation were fixed in Carnovsky’s fixative at º25C
for 3–4 hours with a mixture containing 2 % paraformaldehyde and 2.5% glutaraldehyde. The bacteria were then
washed three times with 0.1 M phosphate buffer, pH 7.2 for 5 minutes. The bacteria were then centrifuged at
10,000 rpm in an Eppendorf tube for 15 minutes. The bacterial pellet was resuspended in 1.0 ml of phosphate
buffer and then adsorbed on formvar carbon coated supports, Agar 200-mesh copper grids by floating the grids
on a drop of bacterial isolate. The bacteria on grids were stained by submerging the grids for 5 minutes in 12 %
(w/v) aqueous uranyl acetate, and were then rinsed with Milli-Q water three times. The grids were examined and
photographed under a Philips CM10 transmission electron microscope using an accelerating voltage of 80 kV.
Images were taken at different magnifications [31–33].
4.8. Docking studies using penicillin-binding protein receptor and β-lactamase enzyme
The E. coli penicillin-binding protein 4 (PBP4) crystal structure used in this docking study was
downloaded from the protein data bank [34] (PDB ID: 2EX8 [35]). The crystal structure of the E. coli β-
lactamase enzyme used in this docking study was downloaded from the protein data bank [34] (PDB ID: 1FQG
[36]). The crystal structure was checked by MOE using the protein preparation module [37], with a few
corrections to the structure. All water and ligand molecules were removed. It was then processed via the Protein
Preparation Wizard [38, 39] in the Maestro software [40], mainly to establish partial charges on each atom using
OPLS force field and to assign a protonation state on each ionizable group. The whole structure was relaxed via
conducting restrained minimization with an RMSD limit up to 0.3 Å. The binding pocket was identified with co-
crystallized ligand (penicillin G).
All ligands were created using MOE [28] which was also used to generate the dominant ionization state
for each ionizable functional group. Ligands were then prepared using the LigPrep module [41] in the Maestro
program [40] in order to give partial charges to ligand atoms and to generate a single low-energy conformation
for each ligand using the OPLS force field. Next, all ligands were covalently docked into the previously prepared
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binding site using the Covalent Docking module [42] in Maestro, with the thorough pose predication algorithm
employed for conformational sampling. Two main reaction types were set: β-lactam addition and conjugate
addition to nitrile-activated alkene, both of which were set to react with the Ser62 side chain. Pose refinement and
scoring were kept as default. Docked poses were scored by taking the average of two GlideScore [43, 44] values,
Glide docking score “pre-reacted” and a score-in-place “post-reacted” calculation, for the final docked pose [42].
GlideScore is an empirical scoring function that approximates the ligand-binding free energy. It has many terms,
including force field (electrostatic, van der Waals) contributions and terms rewarding or penalizing interactions
known to influence ligand binding [44].
4.9. Spectroscopic studies
4.9.1. Standard solutions
4.9.1.1. Phosphate buffer solution
A 0.01 M phosphate buffer solution (pH = 7.4) was prepared by dissolving appropriate amounts of H3PO4
and KOH in 1.0 L of deionized water. The pH was adjusted on glass electrode using the standard procedure for
buffer preparation.
4.9.1.2. β-Lactamase enzyme solution
β-lactamase enzyme solution (1 × 10-6 M) was prepared by dissolving an appropriate amount of the enzyme
in the phosphate buffer (pH 7.4). The stock solution was stored at 4 °C for 24 hours.
4.9.1.3. Pyrimidine acrylonitrile derivatives solutions
Stock solutions of compounds 5 and 12d were prepared by dissolving appropriate amounts of each
compound in the phosphate buffer (pH 7.4) to final concentrations of 6 × 10-5 M.
4.9.2. UV-Vis measurements
UV-vis absorption spectra were recorded using 6 × 10-5 M of compounds 5 and 12d and incremental
addition of enzyme solution (1 × 10-6 M, 10µl) in phosphate buffer, pH 7.4. The measurement ranged between
200 and 700 nm at the different enzyme-compound ratios and were recorded three minutes after incubation to
permit equilibrium between the species. Titration stopped at saturation, when no change in absorbance intensity
was observed.
4.9.3. Fluorescence measurements
Interactions of pyrimidine acrylonitrile derivatives 5 and 12d with β-lactamase enzyme were followed
fluorimetrically at 443 and 456 nm as λmax of emission using excitation λmax of 280 and 290 nm, respectively.
A fixed concentration of each compound was titrated with the incremental addition of enzyme (10 µl) in
phosphate buffer, pH 7.4 and the fluorescence measurements were performed keeping the excitation and emission
band slit width of 5–10 nm, after allowing three minutes’ equilibration for each enzyme addition, and scanned for
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fluorescence spectra in the range 300–600 nm. Titrations were stopped at saturation when no change in
fluorescence intensity was observed. A fluorescence-free quartz cell of 1 cm path length was used.
4.9.4. Circular dichroism measurements
Circular dichroism spectroscopy was used to confirm the results obtained by UV and fluorescence
spectroscopies. Interactions between pyrimidine acrylonitrile derivatives 5 and 12d with β-lactamase enzyme
were followed by adding successive amounts (6 × 10-5 M) to 1.0 mL of β-lactamase enzyme in phosphate buffer,
pH 7.4. The CD spectra were recorded at º25C in the wavelength range of 200–700 nm, at a speed of 50 nm/min
and bandwidth of 1 nm. Each spectrum was averaged from three successive accumulations. All CD spectra were
auto baseline-corrected against blank solutions. Before use, the optical chamber of the CD spectrometer was
deoxygenated with dry nitrogen and was held under nitrogen atmosphere during the measurements. UV-vis,
fluorescence and CD signals were corrected for dilution effects.
4.9.5. NMR studies of the interaction between β-lactamase and 12d
Compound 12d (10 mg) was dissolved in 0.3 ml of deuterium oxide (D2O, Merck) in an NMR tube, and the
1H-NMR spectrum was immediately recorded as a zero-time reference. The β-lactamase solution in 0.3 ml of 0.1
M phosphate buffer was added to the NMR tube, and the 1H-NMR spectrum was recorded at 37 °C after 60 min.
Acknowledgment
The authors wish to acknowledge the significant financial support of UAE University, Research Affairs
Sector (grant no. 31S030-1156-02-02-10).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://1drv.ms/b/s!Aoxf511qpqQthX9jJS6H0k92Mm1O
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Highlights
•
•
•
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Novel benzazole-pyrimidines with antimicrobial properties are synthesized.
Compounds 5 and 12d are highly active in both in vitro and molecular docking studies.
Physical studies of compounds 5, 12d with β–lactamase enzyme is described.
In-vitro study showed that a combination of the new analogues and Amoxicillin indicates
a synergistic effect.
•
Docking study and TEM suggest the membrane disruption action of 5.