Synthesis and evaluation of new quinazoline-benzimidazole hybrids as potent anti-microbial agents against multidrug resistant Staphylococcus aureus and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2020.112996

Owing to the rapid rise in antibiotic resistance, infectious diseases have become serious threat to public health. There is an urgent need to develop new antimicrobial agents with diverse chemical structures and novel mechanisms of action to overcome the resistance. In recent years, Quinazoline-benzimidazole hybrids have emerged as a new class of antimicrobial agents active against S. aureus and M. tuberculosis. In the current study, we designed and synthesized fifteen new Quinazoline-benzimidazole hybrids and evaluated them for their antimicrobial activity against S. aureus ATCC 29213 and M. tuberculosis H37Rv. These studies led to the identification of nine potent antibacterial agents 8a, 8b, 8c, 8d, 8f, 8g, 8h, 8i and 10c with MICs in the range of 4–64 μg/mL. Further, these selected compounds were found to possess potent antibacterial potential against a panel of drug-resistant clinical isolates which include methicillin and vancomycin-resistant S. aureus. The selected compounds were found to be less toxic to Vero cells (CC₅₀ = 40-≥200 μg/mL) and demonstrated a favourable selectivity index. Based on the encouraging results obtained these new benzimidazol-2-yl quinazoline derivatives have emerged as promising antimicrobial agents for the treatment of MDR- S. aureus and Mycobacterial infections.

Full text of DOI:10.1016/j.ejmech.2020.112996

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Synthesis and evaluation of new Quinazoline-benzimidazole hybrids as potent
anti-microbial agents against multidrug resistant Staphylococcus aureus and
Mycobacterium tuberculosis
Satyaveni Malasala, Md. Naiyaz Ahmad, Ravikumar Akunuri, Manjulika Shukla,
Grace Kaul, Arunava Dasgupta, Y.V. Madhavi, Sidharth Chopra, Srinivas Nanduri
PII:
S0223-5234(20)30968-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112996
EJMECH 112996
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 August 2020
Revised Date: 12 October 2020
Accepted Date: 2 November 2020
Please cite this article as: S. Malasala, M.N. Ahmad, R. Akunuri, M. Shukla, G. Kaul, A. Dasgupta, Y.V
Madhavi, S. Chopra, S. Nanduri, Synthesis and evaluation of new Quinazoline-benzimidazole hybrids
as potent anti-microbial agents against multidrug resistant Staphylococcus aureus and Mycobacterium
tuberculosis, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112996.
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Synthesis and evaluation of new Quinazoline-benzimidazole hybrids as
potent anti-microbial agents against multidrug resistant Staphylococcus
aureus and Mycobacterium tuberculosis
Satyaveni Malasalaa, Md. Naiyaz Ahmadb, c, Ravikumar Akunuria, Manjulika Shuklab,
Grace Kaulb, c, Arunava Dasguptab,c, Y.V Madhavia, Sidharth Choprab,c*, Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
cAcSIR, Ghaziabad, Sector 19, Kamla Nehru Nagar, Ghaziabad-201002, Uttar Pradesh, India
Graphical Abstract:

Synthesis and evaluation of new Quinazoline-benzimidazole hybrids as
potent anti-microbial agents against multidrug resistant Staphylococcus
aureus and Mycobacterium tuberculosis
Satyaveni Malasalaa, Md. Naiyaz Ahmadb,c, Ravikumar Akunuria, Manjulika Shuklab,
Grace Kaulb, c, Arunava Dasguptab,c, Y.V Madhavia, Sidharth Choprab,c*, Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
cAcSIR, Ghaziabad, Sector 19, Kamla Nehru Nagar, Ghaziabad-201002, Uttar Pradesh, India
Corresponding author: Dr. Srinivas Nanduri, E-mail: nandurisrini92@gmail.com
Co-corresponding author: Dr. Sidharth Chopra: skchopra007@gmail.com
Abstract: Owing to the rapid rise in antibiotic resistance, infectious diseases have become
serious threat to public health. There is an urgent need to develop new antimicrobial agents
with diverse chemical structures and novel mechanisms of action to overcome the resistance.
In recent years, Quinazoline-benzimidazole hybrids have emerged as a new class of
antimicrobial agents active against S. aureus and M. tuberculosis. In the current study, we
designed and synthesized fifteen new Quinazoline-benzimidazole hybrids and evaluated them
for their antimicrobial activity against S. aureus ATCC 29213 and M. tuberculosis H37Rv.
These studies led to the identification of nine potent antibacterial agents 8a, 8b, 8c, 8d, 8f,
8g, 8h, 8i and 10c with MICs in the range of 4-64 µg/mL. Further, these selected compounds
were found to possess potent antibacterial potential against a panel of drug-resistant clinical
isolates which include methicillin and vancomycin-resistant S. aureus. The selected
compounds were found to be less toxic to Vero cells (CC50 = 40-≥200 µg/mL) and
demonstrated a favourable selectivity index. Based on the encouraging results obtained these
new benzimidazol-2-yl quinazoline derivatives have emerged as promising antimicrobial
agents for the treatment of MDR- S. aureus and Mycobacterial infections.
Keywords: Quinazoline, Benzimidazole, anti-bacterial agents, MRSA, Tuberculosis,
cytotoxicity, resistance.

1. Introduction
Alarming rise of difficult to treat multi drug resistant bacterial infections has emerged as one
of the formidable challenges of 21st century [1-5]. The World Health Organization (WHO)
has categorised 12 of the pathogens as high-risk priority II pathogens which require
immediate attention [6,7]. In particular, S. aureus is known to cause a wide range of diseases,
from skin infections to serious illnesses like pneumonia, meningitis, bacteraemia and sepsis
[8-10]. Among the various resistant strains, methicillin resistant (MRSA) and vancomycin
resistant (VRSA) strains are known to be sources of spread of community infections. Multi
Drug Resistant Tuberculosis (MDR-TB) caused due to Mycobacterium tuberculosis (Mtb)
has emerged as another major life-threatening disease. A number of studies indicated that the
anti-microbial resistance (AMR) has attained frightening levels globally [11-13]. Around
750,000 deaths are reported annually due to AMR and the toll is likely to rise to 10 million
by 2050 [14]. Hence, search for newer treatments for multi drug resistant microbial infections
have been the focus of the scientific community in recent years.
Quinazoline is a remarkable scaffold of medicinal importance possessing promising
pharmacological activities like antitubercular [16], antibacterial [17], anticonvulsant [18],
anti-HIV, antifungal [19], anti-inflammatory, analgesic [20] and anticancer [21] activities.
Bouley et al. [22] reported 4(3H)-quinazolinones as orally bioavailable antibacterial agents I.
Our group also reported 3-phenylquinazolin-4(3H)-ones and related derivatives II [23] and 2-
phenylquinazoline derivatives III as potent anti-MDR-S. aureus agents and as anti-
mycobacterial agents [24]. Benzimidazole scaffold has attracted wide attention as an
interesting heterocyclic in medicinal chemistry owing to its presence as a core scaffold in a
number of drugs such as albendazole, mebendazole, candesartan, astemizole etc. [25].
Several compounds possessing benzimidazole as a core are reported to possess diverse
therapeutic properties such as antibacterial, antiulcer, antiviral, antihypertensive, anticancer,
antifungal, anti-inflammatory and anticoagulant properties [26]. Dokla et al. [27] reported
1,2-disubstituted benzimidazole scaffold IV to possess potent antibacterial activity against E.
coli. Mustafa et al. [28] reported the benzimidazole-acrylonitrile hybrids V as anti-
mycobacterial agents. p-aminobenzoic acid (PABA) serves as a bioactive linker because of
its occurrence in many human pathogens. p-Aminosalicylic acid VI [29], a derivative of
PABA is used to treat active drug resistant tuberculosis together with other anti-tubercular
medications.

Br
O
O
NH Cl
O
N
OH
OH
O
N
HN
N
N
N
OH
N
CN
III
MIC: 4 g/mL M. tuberculosis
II
I
MIC: 0.03 g/mL S. aureus
MIC: 64 g/mL M. tuberculosis
MIC: 0.03 g/mL S. aureus
O
OH
OH
N
N
CN
N
O
N
H
N
N
HN S
O
NH2
VI
IV
V
MIC: 2 g/mL E. coli
MIC: 0.78 g/mL M. tuberculosis
p-aminosalicylic acid
Second line anti-tb drug
Figure 1. Reported anti-bacterial agents.
Based on the key role being played by quinazoline, benzimidazole and p-aminobenzoic acid
moieties in a number of anti-microbial agents, we designed and synthesized a number of new
quinazoline-benzimidazole hybrids with p-aminobenzoic acid as linker.
R2
N
N
H
HN
N
N
R1
Figure 2. Designed quinazolines-benzimidazole hybrids.

2. Results and Discussion:
2.1 Chemistry:
A series of new benzimidazolo-2-arylquinazoline derivatives were synthesized as described
in Scheme 1. 2-aminobenzamide 1 was condensed with different substituted aryl aldehydes
2a-d for 12-24 h in presence of dimethylsulphoxide to afford 2-arylquinazolinones 3a-d [30].
These derivatives were treated with phosphorus (V) oxychloride and N, N-diethyl aniline to
provide 2-aryl 4-chloroquinazoline derivatives 4a-d [30]. The chlorinated derivatives 4a-d
undergo nucleophillic substitution with p-aminobenzoic acid 5 to obtain 4-[(2-
phenylquinazolin-4-yl)amino]benzoic acid derivatives 6a-c. Further, the 4-[(2-
phenylquinazolin-4-yl)amino]benzoic acid derivatives 6a-c were condensed with substituted
o
o-phenylenediamines 7a-e in presence of polyphosphoric acid at 180 C to afford the
corresponding benzimidazole derivatives 8a-j in moderate to excellent yields.
Scheme 1. Synthesis of N-(4-(1H-benzoimidazol-2-yl)aryl)-2-arylquinazolin-4-amine
derivatives.
4-[(2-phenylquinazolin-4-yl) amino] benzoic acid derivatives 6a-c were also coupled with
different substituted 2-aryl-benzoimidazol-6-amines 9a-d to afford the corresponding
benzimidazoloamide derivatives 10a-d in excellent yields (Scheme-2).

Scheme 2. Synthesis of 4-((2-phenylquinazolin-4-yl)amino)-N-(2-(aryl)-1H-benzoimidazol-
6-yl)benzamide derivatives.
2.2 In vitro antibacterial activity
2.2.1 Antibiotic susceptibility testing against E. coli, S. aureus, K. pneuminiae, A. baumannii,
and P. aeruginosa pathogen panel.
The newly synthesized benzimidazol-2-yl quinazoline derivatives were subjected to antibiotic
susceptibility testing against E. coli, S. aureus, K. pneuminiae, A. baumannii, and P.
aeruginosa pathogen panel. The minimum inhibitory concentrations (MICs) against gram
positive and gram-negative bacterial strains viz., E. coli, S. aureus, K. pneuminiae, A.
baumannii, and P. aeruginosa pathogen panel were determined by using broth microdilution
assay. In addition, the synthesized compounds were also evaluated against M. tuberculosis
(ATCC 27294), M. abscessus (ATCC 19977), M. fortuitum (ATCC 6841) and M. chelonae
(ATCC 35752). The results are tabulated in Tables 1 & 2. As per our previous studies [24],
2-phenylquinazolines are reported to possess potent anti-bacterial and anti-mycobacterial
activities. Based on this observation and also the promising antimicrobial activities of
benzimidazole, we explored a number of 2-aryl-quinazolines with benzimidazole hybrids
containing a number of electron withdrawing and electron donating substituents. We studied
the effect of 3,4-dimethoxyphenyl and 4-chlorophenyl as aryl substituents at C-2 of
quinazolines. Compounds with 4-nitro, 4-bromo, 4-chloro and 4-fluoro substituents on
benzimidazole moiety were studied. Based on this, a total of 15 new quinazoline-
benzimidazole hybrids were synthesized and evaluated for their anti-bacterial and anti-
mycobacterial activities.

The antimicrobial screening results suggested that the synthesized compounds exhibited
selective inhibitory activity against S. aureus. Compounds 8d, 8h and 8i exhibited equipotent
inhibitory activity with MIC of 4 µg/mL. Compounds 8b and 8c have shown anti-bacterial
inhibition with MIC of 8 µg/mL. Compounds 8g, 10a, 10b and 11 have exhibited moderate
inhibition with MIC of 16-64 µg/mL. The remaining compounds are found to be inactive
against E. coli, K. pneuminiae, A. baumannii, and P. aeruginosa pathogens. With phenyl
group at C-2 position, the substitutions on benzimidazole were altered such as 4-nitro 8a, 4-
chloro 8b, unsubstituted 8c, 4-fluoro 8d and 4-bromo 8e. While, the compound 8a did not
show any inhibitory activity against S. aureus, the compounds 8b, 8c and 8d exhibited good
inhibitory activity against S. aureus with MIC 8, 8 and 4 µg/mL respectively. Further, with
3,4-dimethoxyphenyl at C-2 position compounds 8f with 4-nitro and 8g with 4-chloro
substitutions on benzimidazole were synthesized. While the compound 8g inhibited S. aureus
with MIC value 16 µg/mL, the compound 8f did not show any inhibitory activity. With 4-
chlorophenyl at C-2 position, compounds 8h, 8i and 8j were synthesized. While, the
compounds 8h and 8i were found to possess potent inhibitory activity against S. aureus with
MIC: 4 µg/mL, the compound 8j was found to be inactive. Next, we focused our attention on
the modification of the carboxyl moiety at para position of the C-4 phenyl by modifying to
various amides by coupling with substituted benzimidazoloamines as shown in Scheme 2.
Thus, the compounds 10a, 10b, 10c and 10d were synthesized. The synthesized compounds
were found to exhibit moderate inhibition against S. aureus with MIC values of 32->64
µg/mL. Compound 11 was synthesized by direct substitution of 2-trifluoromethyl
benzimidazolo-5-amine at C-4. The compound 11 was found to be inactive.
The synthesized compounds were tested for their anti-mycobacterial potential with four
different mycobacterial strains. The inhibitory screening results are tabulated in Table 2.
Compounds 8a-e showed moderate inhibitory activity against Mtb (MIC = 8–>64 µg/mL).
The compound 8a exhibited inhibitory activity against M. tuberculosis with MIC value of 8
µg/mL. The compound 8b showed moderate activity against M. tuberculosis, M. fortuitum
and M. chelonae. The compounds 8c and 8d did not show any inhibitory activity against M.
tuberculosis. Interestingly, compound 8f exhibited good inhibitory activity against M.
tuberculosis (H37Rv) with MIC value of 8 µg/mL. The compound 8f also showed good
inhibition against M. fortuitum (ATCC 6841) with the MIC value of 4 µg/mL and M.
chelonae (ATCC 35752) with the MIC value of 8 µg/mL. Compound 8g exhibited moderate
activity against M. tuberculosis with MIC value of 32 µg/mL. Compound 10c exhibited good

inhibition activity with MIC 16 µg/mL against M. tuberculosis. The compounds 10a and 10d
were found to be moderately active and compound 11 showed moderate inhibition with the
MIC 64 µg/mL.
Table 1. MIC values (µg/mL) of the tested compounds against gram positive and gram
negative pathogen panel.
S. No
Sample
code
E. coli
ATCC
25922
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
S. aureus
ATCC
29213
>64
8
K. pneumoniae A. baumannii
P. aeruginosa
ATCC 27853
BAA 1705
BAA 1605
1
2
3
4
5
6
7
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
10a
10b
10c
8
4
>64
>64
16
4
4
>64
32
64
9
10
11
12
13
14
15
16
>64
>64
64
10d
11
Levofloxacin
0.125
0.015
Table 2. MIC values (µg/mL) of the tested compounds against Mtb and other mycobacterium
species.
Mtb H37Rv
ATCC 27294
M. abscessus
M. fortuitum
ATCC 6841
>64
M. chelonae
S.No
Sample code
ATCC 19977
>64
>64
>64
>64
NT
ATCC 35752
>64
32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
10a
10b
10c
10d
11
8
32
>64
>64
NT
8
32
>64
>64
NT
4
>64
NT
NT
NT
>64
>64
>64
>64
>64
NT
NT
>64
32
NT
8
>64
NT
>64
>64
NT
NT
NT
>64
>64
>64
>64
>64
NT
32
NT
NT
NT
64
>64
16
64
64
0.03
0.06
1
NT
NT
>64
>64
>64
>64
>64
NT
NT
NT
NT
0.03
Isoniazid
Rifampicin
Streptomycin
Ethambutol
Levofloxacin
NT
NT
NT
1
NT
NT
0.03
1
NT
*NT=not tested

The Structure-Activity Relationships derived are explained in Figure 3.
Figure 3. Structure-Activity Relationship of new quinazoline-benzimidazole hybrids.
2.2.2 Cytotoxicity against Vero cells:
The selected compounds were further subjected to cytotoxicity assay against Vero cells
(African green monkey kidney cell line, ATCC CCL-81) using MTT assay. The CC50 (50%
cytotoxic concentration) was defined as the reduction of cell viability to 50% by compound.
Doxorubicin and Levofloxacin were used as a reference standard and each experiment was
performed in triplicate. The cytotoxicity data demonstrates that 8a, 8b, 8c, 8d, 8f, 8g, 8h, 8i
and 10c were nontoxic to Vero cells (CC50 = 40->100 µg/mL) and displayed favourable
selectivity index. The results are tabulated in Tables 3.
Table 3. Cytotoxicity profile against Vero cells and Selectivity Index (SI).
S. aureus
ATCC 29213 (µg/mL)
CC50
Selective index
(CC50/MIC)
12.5
S. No
Sample code
1.
2.
3.
4.
5.
6.
7.
8.
9.
100
100
80
8a
8b
8c
8d
8f
8g
8h
8i
8
8
8
4
8
16
4
4
12.5
10
10
12.5
>6.25
12.5
12.5
40
100
>100
50
50
>100
>6.25
10c
16

2.2.3 Determination of MIC against MDR-S. aureus including VRSA
To further determine their potential against multiple strains of MDR S. aureus, 8b, 8c, 8h and
8i were tested against various well defined and characterized clinical isolates of MSSA,
MRSA and VRSA as per standard CLSI guidelines. The results are summarised in Table 4.
The compounds 8b, 8c, 8h and 8i were found to be active against various resistant strains.
From the close examination of the results, it can be inferred that 8b, 8c, 8h and 8i displayed
potent antibacterial activity with MIC 4–16 µg/mL against a number of clinical strains of
MSSA, MRSA and VRSA. The obtained results suggest that compounds 8b, 8c, 8h and 8i
are less resistant to all the MRSA and VRSA isolates with MIC 4–16 µg/mL in comparison
with Methicillin. Levofloxacin and Meropenam. The compounds 8b, 8c, 8h and 8i were also
found to be active against VRSA clinical isolates with MIC 4–16 µg/mL.
Table 4. MIC of a compound 8b, 8c, 8h and 8i against MRSA and VRSA strains.

MIC (µg/mL)
Meropenem
Strains
S. aureus
Antibiotics resistant to
None
Details about the strain
8b
8
8c
8
8h
8
8i
4
Levofloxacin
0.125
Vancomycin
1
Daptomycin
1
Methicillin
1
MSSA
ATCC
29213
0.0625
32
Methicillin, Ceftriaxone,
Meropenem, Gentamycin
and Linezolid
Methicillin, Ceftriaxone,
Meropenem, Gentamycin
and Linezolid
Resistant to tetracycline, contains mec subtype I
cassette, Large variety of virulence factors
NR 100
NR 119
8
8
32
8
8
4
4
0.25
16
2
2
1
1
>64
>64
Contains mec subtype IV cassette, G2576T mutation in
domain V in one or more 23 S rRNA genes
32
64
NR
10129
NR
10198
NR
10192
NR
10186
NR
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone,
Meropenem
Also called as TCH60
8
4
16
4
8
8
4
8
2
4
8
4
0.25
8
4
4
1
1
1
1
2
1
0.5
0.5
1
>64
>64
>64
>64
>64
32
USA100 Community acquired-MRSA, Contains mec
type II cassette, Negative for PVL virulence factor
Community acquired-MRSA, Contains mec type II
cassette, Negative for PVL virulence factor
USA 300 Community acquired-MRSA, Contains mec
type IV cassette, Positive for PVL virulence factor
Community acquired-MRSA, Contains mec type II
cassette, Negative for PVL virulence factor
Community acquired-MRSA, Contains mec type V
cassette, Positive for PVL virulence factor
MRSA
8
32
8
4
16
64
8
8
16
8
8
0.5
1
Methicillin, Ceftriaxone,
Meropenem
16
8
16
8
32
64
1
10193
NR
10194
Methicillin, Ceftriaxone
8
0.125
0.5
USA 600 Community acquired-MRSA
Contains mec type II cassette, Negative for PVL
virulence factor
NR
10191
Methicillin, Ceftriaxone,
Meropenem
16
4
16
8
8
4
8
8
8
4
2
4
32
32
64
64
64
16
2
1
>64
>64
>64
>64
Methicillin, Ceftriaxone,
Meropenem, Gentamycin,
Vancomycin, Teicoplanin
Methicillin, Ceftriaxone,
Meropenem, Vancomycin
and Teicoplanin
Methicillin, Ceftriaxone,
Meropenem, Vancomycin
and Teicoplanin
USA100 and contains mec (subtype II) and van A.,
Negative for van B, van C1, van C2, van D, van E, PVL
and arginine catabolic mobile element (ACME)
USA100 and contains mec (subtype II) and van A.
Negative for van B, van C1, van C2, van D, van E, PVL
and arginine catabolic mobile element (ACME)
VRS1
VRS4
>64
>64
>64
1
VRSA
8
8
>64
32
0.5
0.5
VRS12
NA*
8
16

2.3 ADMET properties
ADMET properties of the synthesized compounds were calculated using Qikprop program (Qikprop, version 6.5, Schrödinger, LLC, New York,
NY, 2014). As can be seen below, the partition coefficient (QPlogPo/w), hydrogen bond donors (donor HB), hydrogen bond acceptors (acceptor
HB), molecular weight (mol. Wt.) and percent human oral absorption exhibited satisfactory results. The compounds also followed Lipinski rule
of five. ADMET properties for Isoniazid and Rifampicin were also calculated and compared with the results of the synthesized compounds. The
predicted results are shown in Table 5.
Table 5. ADMET properties of 8a-j, 10a-d and 11.
Recommended
Descriptors
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
10a
10b
10c
10d
11
Levofloxacin
Isoniazid
Rifampicin
values
Molecular
weight
130.0–725.0
458.47
413.48
431.47
492.37
518.53
507.97
447.92
465.91
465.37
524.50
629.49
618.09
582.66
411.40
361.37
137.141
820.978
Dipole
moment
1.0–12.5
10.49
9.71
9.30
5.25
8.15
8.13
5.16
9.33
7.32
7.33
7.79
9.61
8.30
5.25
4.15
5.33
3.32
4.33
Total SASA
300–1000
969.54
899.24
789.81
819.49
773.04
909.35
721.15
687.16
829.909
1005.33
855.54
979.24
1021.81
1011.49
653.04
587.16
329.909
1018.33
No. of
rotatable
0–15
2
3
4
3
4
4
4
1
2
2
2
3
4
3
4
1
2
25
bonds
2
5
2
4
2
4
2
4
2
4
2
6.5
2
5.5
2
6.5
2
4
2
4
3
6.5
3
7.5
3
6.5
2
4
Donor HB
Acceptor HB
QP
Polarizability
QP logP o/w
QP log BB
Human Oral
Absorption
Percent
Human Oral
Absorption
Rule of Five
violations
0.0–6.0
2.0–20.0
3
6.5
0
7.25
3
4.5
6
20.35
13.0–70.0
44.20
44.42
46.05
46.08
48.52
55.81
52.87
36.38
56.80
68.01
54.20
41.42
40.05
56.01
38.52
36.38
13.80
68.019
2.0–6.5
−3.0 and 1.2
8.08
-1.01
7.66
-1.41
9.81
-0.513
7.54
-0.112
5.08
-0.940
7.82
-0.96
5.04
-0.137
8.39
-0.42
7.67
-0.87
5.89
-2.430
7.85
-1.39
5.16
-0.61
7.66
-1.01
7.87
-1.11
7.72
-1.07
-0.39
-0.42
-0.653
-0.87
2.893
-2.430
1
91.49
1
1
100
1
1
100
1
1
100
1
1
100
1
1
79.59
1
1
100
1
1
100
1
1
100
1
1
100
1
1–3
1
100
2
1
100
2
1
100
2
1
100
2
1
100
1
2
48.823
0
2
66.30
0
1
33.90
3
> 80% is high
< 25% is low

3. Conclusion:
Rapid rise in multidrug resistance is one of the alarming threats to public health worldwide.
In order to alleviate the situation, there is an urgent need to develop new antimicrobial drugs
with novel mechanisms of action. In this work, we designed and synthesized a library of 15
new Quinazoline-Benzimidazole hybrids and evaluated them against a panel of gram positive
and gram negative bacterial strains consisting of against E. coli, S. aureus, K. pneuminiae, A.
baumannii, and P. aeruginosa, M. tuberculosis H37Rv and other mycobacterial strains.
Among the studied compounds, 8a, 8b, 8c, 8d, 8f, 8g, 8h, 8i and 10c exhibited good to
moderate inhibitory activity against S. aureus and different mycobacterium species. The
compounds were found to be non-toxic to Vero cells (CC50 40->100 µg/mL) with favourable
selectivity index (SI <10->25). Further, the compounds 8b, 8c, 8h and 8i were found to
display potent inhibitory activity against clinical MRSA and VRSA isolates. In a separate
study, the compounds 8a, 8f and 10c were found to exhibit potent anti-mycobacterial activity
with MIC values in the range of 8-16 µg/mL. The compounds were also found to be less
toxic to Vero cells (CC50 ≥100 µg/mL) and exhibited promising selectivity index. Based on
the above studies, the newly synthesized quinazoline-benzimidazole hybrids were found to
possess promising anti-bacterial and anti-mycobacterial activities and possess the potential
for further development.
4. Experimental section:
4.1 General Methods. All the reagents and solvents were obtained from commercial
suppliers and were used without further purification. Analytical thin layer chromatography
(TLC) was performed on MERCK pre-coated silica gel 60-F254 (0.5 mm) aluminum plates.
Visualization of the spots on TLC plates was achieved by UV light. 1H and 13C NMR spectra
were recorded on Bruker 500 MHz by making a solution of samples in the DMSO-d6 as
1
solvent using tetramethyl silane (TMS) as the internal standard. Chemical shifts for H and
13C are reported in parts per million (ppm) downfield from tetra methyl silane. Spin
multiplicities are described as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet), and m (multiplet). Coupling constant (J) values are reported in hertz (Hz). HRMS
were determined with Agilent QTOF mass spectrometer 6540 series instrument. Wherever
required, column chromatography was performed using silica gel (60-120). The reactions are
carried under nitrogen positive pressure using freshly distilled solvents wherever anhydrous
conditions required. All evaporation of solvents was carried out under reduced pressure using

rotary evaporator below 45 . Melting points were determined with an electro thermal digital
melting point apparatus IA9100 and are uncorrected. The names of all the compounds given
in the experimental section were taken from ChemBioDraw Ultra, Version 12.0.
General Experimental Procedure for the Synthesis of 8a-j, 10a-d and 11.
Procedure for 8a-j: To the mixture of 6 (1 mmol), substituted o-phenylenediamines (7a-f, 1
mmol) was added to the RBF, then polyphospheric acid (5gm) was added to the reaction
o
mixture and stir the contents at 180 C for 4-6 h. The reaction mixture was stirred for 20
minutes at room temperature, followed by the addition of ice cold water. Then neutralise the
reaction mixture by using the aqueous solution of NaOH. After neutralization of the reaction,
the suspension was extracted with ethyl acetate (3x5.0 mL), washed with 1:1 mixture of
brine. The combined organic extracts were dried over anhydrous sodium sulphate. After
removal of the solvent under reduced pressure, the crude product was purified by using
column chromatography, EtOAc:Hexane (2:8) as eluent on silica gel to afford the pure
products.
Procedure for 10a-d: To the mixture of 6 (1 mmol) and HATU (1mmol), DMF (3mL) was
added slowly under nitrogen atmosphere. The reaction mixture was then stirred for 20
o
minutes at 0 C, followed by the addition of substituted benzimidazole amines (9a-d,
1mmol). The reaction mixture was stirred for 20 minutes at room temperature, followed by
the addition of DIPEA. Upon completion of the reaction as monitored by TLC, crushed ice
was added to the reaction mixture. The resulting solid was then subjected to vacuum
filtration, excess of water was used to wash off the insoluble solids to obtain crude powder
which was purified using column chromatography (elution with hexane/EtOAc = 3:7). The
pure products were collected as white color solids in good yields.
4.1.1 N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)-2-phenylquinazolin-4-amine (8a)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 13.57 (s, 1H), 10.14 (s, 1H), 8.65 (d, J = 8.3 Hz, 1H),
8.59-8.42 (m, 3H), 8.34 (d, J = 8.7 Hz, 2H), 8.29 (d, J = 8.7 Hz, 2H), 8.15 (d, J = 8.2 Hz,
1H), 7.93 (d, J = 3.2 Hz, 2H), 7.76 (s, 1H), 7.69-7.61 (m, 1H), 7.60–7.53 (m, 3H); 13C NMR
(125 MHz, DMSO- d6): δ 159.4, 158.2, 151.0, 143.0, 142.5, 138.6, 133.9, 130.9, 129.0,
128.9, 128.7, 128.4, 128.2, 127.9, 126.6, 126.3, 124.1, 123.5, 122.2, 118.4, 114.6; HRMS
(ESI): m/z calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.

4.1.2 N-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-2-phenylquinazolin-4-amine (8b)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 13.07 (d, J = 14.8 Hz, 1H), 10.10 (s, 1H), 8.64 (d, J =
8.3 Hz, 1H), 8.58–8.44 (m, 2H), 8.33–8.19 (m, 4H), 7.99–7.87 (m, 2H), 7.74–7.64 (m, 2H),
7.55 (t, J = 7.2 Hz, 4H), 7.24 (t, J = 9.1 Hz, 1H); 13C NMR (125 MHz, DMSO- d6): δ 159.4,
158.2, 151.0, 141.7, 138.7, 133.8, 130.8, 129.0, 128.9, 128.7, 128.4, 128.2, 127.4, 126.6,
124.9, 123.5, 122.8, 122.3, 121.8, 118.4, 114.6, 114.0, 112.8; HRMS (ESI): m/z calculated
for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.13 N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-phenylquinazolin-4-amine (8c)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
1
710; H NMR (500 MHz, DMSO-d6): δ 13.83 (s, 1H), 10.53-10.46 (m, 1H), 10.14 (s, 1H),
8.66 (d, J = 5.8 Hz, 1H), 8.53 (s, 2H), 8.40 (s, 1H), 8.25 (s, 2H), 8.18 (s, 2H), 7.93 (s, 2H),
13
7.79 (d, J = 33.7 Hz, 2H), 7.68 (s, 1H), 7.56 (s, 3H); C NMR (125 MHz, DMSO- d6): δ
159.4, 158.2, 151.2, 151.0, 141.6, 138.7, 133.8, 130.9, 128.9, 128.7, 128.4, 128.2, 127.3,
126.6, 123.5, 122.3, 117.4, 114.6, 114.0, 109.5; HRMS (ESI): m/z calculated for C31H22N4O
467.1872 found 467.1896 [M+H]+.
4.1.4 N-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-2-phenylquinazolin-4-amine (8d)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 12.69 (s, 1H), 8.78–8.14 (m, 3H), 8.14–8.02 (m, 2H),
7.98-7.85 (m, 2H), 7.85–7.67 (m, 3H), 7.75–7.17 (m, 6H); 13C NMR (125 MHz, DMSO- d6):
δ 164.6, 158.4, 157.9, 150.6, 143.1, 141.0, 135.4, 135.2, 134.9, 133.9, 132.9, 131.4, 129.1,
128.9, 128.7, 127.1, 123.5, 123.3, 120.2, 119.3, 118.0, 114.6, 112.9; HRMS (ESI): m/z
calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.1.5 N-(4-(5-bromo-1H-benzo[d]imidazol-2-yl)phenyl)-2-phenylquinazolin-4-amine (8e)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.60 (s, 1H), 9.98 (d, J = 8.9 Hz, 1H), 8.76–8.51 (m,
3H), 8.02 (t, J = 9.3 Hz, 2H), 7.98–7.85 (m, 4H), 7.82 (d, J = 8.9 Hz, 2H), 7.78 (d, J = 8.6
13
Hz, 1H), 7.75 (t, J = 4.7 Hz, 1H), 7.70–7.66 (m, 1H), 7.53 (d, J = 8.6, 1H); C NMR (125
MHz, DMSO- d6): δ 165.5, 159.4, 158.2, 151.0, 143.0, 138.6, 133.9, 130.9, 130.5, 129.6,

129.0, 128.8, 128.7, 128.4, 126.6, 123.6, 121.4, 121.3, 119.1, 118.5, 114.6; HRMS (ESI): m/z
calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.1.6 2-(3,4-dimethoxyphenyl)-N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)quinazolin-4-
amine (8f)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
1
710; H NMR (500 MHz, DMSO-d6): δ 13.63 (s, 1H), 10.09-10.03 (m, 1H), 8.61 (s, 1H),
8.49-8.41 (d, 1H), 8.32 (s, 3H), 8.14 (d, J = 16.2 Hz, 2H), 8.00 (s, 1H), 7.89-7.80 (m, 2H),
7.76 (s, 1H), 7.65-7.55 (m, 2H), 7.06-7.00 (m, 1H), 4.20–3.88 (m, 3H), 3.94–3.68 (m, 3H);
13C NMR (125 MHz, DMSO- d6): δ 163.4, 161.06, 159.2, 158.5, 152.3, 149.6, 146.0, 140.3,
134.8, 133.5, 132.3, 128.6, 128.5, 127.7, 127.5, 126.6, 126.2, 122.8, 121.2, 115.1, 114.1,
106.3, 99.0, 56.5, 56.0; HRMS (ESI): m/z calculated for C31H22N4O 467.1872 found
467.1896 [M+H]+.
4.1.7 N-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-2-(3,4-dimethoxyphenyl)quinazolin-
4-amine (8g)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
1
710; H NMR (500 MHz, DMSO-d6): δ 13.63 (s, 1H), 10.09-10.03 (m, 1H), 8.61 (s, 1H),
8.49-8.41 (d, 1H), 8.32 (s, 3H), 8.14 (d, J = 16.2 Hz, 2H), 8.00 (s, 1H), 7.89-7.80 (m, 2H),
7.76 (s, 1H), 7.65-7.55 (m, 2H), 7.06-7.00 (m, 1H), 4.20–3.88 (m, 3H), 3.94–3.68 (m, 3H);
13C NMR (125 MHz, DMSO- d6): δ 163.6, 163.4, 161.6, 159.2, 152.3, 149.6, 146.0, 139.8,
134.8, 132.8, 132.3, 128.7, 128.1, 127.7, 126.6, 126.2, 122.7, 121.2, 118.8, 115.0, 114.1,
113.8, 106.3, 99.0, 56.5, 56.0; HRMS (ESI): m/z calculated for C31H22N4O 467.1872 found
467.1896 [M+H]+.
4.1.8 N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-(4-chlorophenyl)quinazolin-4-amine (8h)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.57 (s, 1H), 10.03 (s, 1H), 8.60 (d, J = 8.4 Hz, 3H),
8.16 (d, J = 8.1 Hz, 2H), 8.06 (d, J = 8.1 Hz, 2H), 8.00 (d, J = 8.2 Hz, 2H), 7.94–7.88 (m,
13
5H), 7.70–7.66 (m, 1H); C NMR (125 MHz, DMSO- d6): δ 164.4, 159.5, 158.3, 139.5,
135.8, 135.0, 133.7, 132.9, 130.7, 128.9, 128.8, 128.3, 126.4, 123.4, 123.1, 121.0, 118.8,
114.4, 114.2; HRMS (ESI): m/z calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.

4.1.9
2-(4-chlorophenyl)-N-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)quinazolin-4-
amine (8i)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.56 (s, 1H), 9.92 (s, 1H), 8.97 (s, 1H), 8.59 (d, J =
8.2 Hz, 1H), 8.46 (d, J = 6.2 Hz, 2H), 8.33–8.24 (m, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.87 (t, J
13
= 6.0 Hz, 4H), 7.63 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 6.8 Hz, 3H); C NMR (125 MHz,
DMSO- d6): δ 159.5, 159.1, 158.3, 150.9, 139.8, 138.8, 135.8, 134.5, 134.2, 133.6, 130.7,
129.4, 128.8, 128.7, 128.5, 128.3, 126.3, 123.4, 123.1, 121.0, 114.4; HRMS (ESI): m/z
calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.1.10
N-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-2-(4-chlorophenyl)quinazolin-4-
amine (8j)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.36 (s, 1H), 9.90 (s, 1H), 8.59 (d, J = 8.2 Hz, 1H),
8.50–8.43 (m, 2H), 7.95 (d, J = 8.6 Hz, 4H), 7.88 (d, J = 3.7 Hz, 3H), 7.81 (d, J = 8.2 Hz,
13
2H), 7.62 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 7.1 Hz, 2H); C NMR (125 MHz, DMSO- d6): δ
162.4, 154.1, 149.2, 135.0, 133.6, 132.2, 130.8, 130.7, 128.8, 128.1, 127.9, 127.8, 127.5,
127.2, 126.8, 126.3, 125.7, 125.5, 125.3, 121.7; HRMS (ESI): m/z calculated for C31H22N4O
467.1872 found 467.1896 [M+H]+.
4.1.11 4-((2-phenylquinazolin-4-yl)amino)-N-(2-(trifluoromethyl)-1H-benzo[d]imidazol-5-
yl)benzamide (10a)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 11.38 (s, 1H), 11.33 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H),
8.31 (t, J = 7.5 Hz, 1H), 8.23–8.12 (m, 1H), 8.03–7.86 (m, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.59
(d, J = 7.7 Hz, 3H), 7.48 (t, J = 7.4 Hz, 1H), 7.36–7.28 (m, 1H), 7.19 (d, J = 7.6 Hz, 2H),
6.91 (t, J = 7.7 Hz, 2H), 6.76 (t, J = 7.4 Hz, 1H); 13C NMR (125 MHz, DMSO- d6): δ 163.6,
159.5, 158.3, 150.9, 139.1, 138.8, 135.7, 135.0, 134.2, 133.6, 133.4, 133.0, 132.1, 131.9,
130.7, 129.9, 128.9, 128.5, 128.3, 126.3, 123.4, 123.1, 120.5, 120.2, 114.4; HRMS (ESI): m/z
calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.1.12 N-(2-(4-bromo-2-fluorophenyl)-1H-benzo[d]imidazol-5-yl)-4-((2-phenylquinazolin-4-
yl)amino)benzamide (10b)

Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 12.78 (s, 1H), 10.13 (s, 1H), 8.66 (d, J = 8.2 Hz, 1H),
8.59–8.51 (m, 1H), 8.49 (d, J = 6.8 Hz, 1H), 8.44 (s, 1H), 8.20 (d, J = 8.5 Hz, 2H), 8.07 (d, J
= 8.6 Hz, 2H), 7.99 (d, J = 9.4 Hz, 2H), 7.89 (d, J = 6.9 Hz, 2H), 7.70 (d, J = 9.6 Hz, 2H),
13
7.64–7.34 (m, 6H); C NMR (125 MHz, DMSO- d6): δ 162.7, 159.3, 158.3, 152.8, 150.7,
149.1, 135.0, 134.1, 133.2, 131.8, 131.1, 129.0, 129.0, 128.4, 128.2, 128.1, 127.8, 127.5,
127.0, 126.8, 126.3, 124.1, 123.6, 122.4, 121.4, 114.5; HRMS (ESI): m/z calculated for
C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.1.13
N-(2-(8-chloroquinolin-3-yl)-1H-benzo[d]imidazol-5-yl)-4-((2-phenylquinazolin-4-
yl)amino)benzamide (10c)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 13.00 (s, 1H), 12.54 (s, 1H), 10.09 (s, 1H), 8.64 (d, J
= 8.2 Hz, 1H), 8.52 (d, J = 6.0 Hz, 2H), 8.41–8.05 (m, 4H), 7.93 (s, 2H), 7.83 (d, J = 7.9 Hz,
1H), 7.68 (s, 2H), 7.56 (d, J = 6.7 Hz, 3H), 7.50–7.26 (m, 2H), 7.09-7.00 (m, 2H), 6.67 (d, J
13
= 8.0 Hz, 1H), 5.62 (s, 1H); C NMR (125 MHz, DMSO- d6): δ 165.9, 163.6, 159.5, 158.3,
150.9, 139.2, 138.9, 138.8, 135.7, 135.5, 135.0, 134.2, 134.2, 133.7, 133.4, 133.0, 132.2,
131.9, 130.8, 130.7, 129.9, 128.9, 128.6, 128.3, 126.4, 123.4, 123.1, 120.5, 120.3, 120.2,
120.1, 114.4, 106.6. HRMS (ESI): m/z calculated for C31H22N4O 467.1872 found 467.1896
[M+H]+.
4.1.14
N-(2-(naphthalen-2-yl)-1H-benzo[d]imidazol-5-yl)-4-((2-phenylquinazolin-4-
yl)amino)benzamide (10d)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 12.54 (s, 1H), 10.23 (d, J = 9.6 Hz, 1H), 8.65 (d, J =
8.4 Hz, 1H), 8.52–8.50 (m, 1H), 8.30 (d, J = 7.1 Hz, 2H), 8.21–8.16 (m, 4H), 7.95 (t, J = 4.4
13
Hz, 2H), 7.86–7.84 (m, 1H), 7.80–7.65 (m, 4H), 7.61–7.52 (m, 8H), 7.40–7.35 (m, 1H); C
NMR (125 MHz, DMSO- d6): δ 167.1, 159.2, 157.9, 151.4, 151.2, 150.4, 149.0, 147.9, 146.8,
143.0, 142.6, 133.7, 133.1, 132.5, 131.2, 129.3, 128.9, 128.7, 128.5, 128.0, 127.8, 126.1,
124.0, 123.5, 122.2, 122.0, 121.6, 120.3, 118.3, 114.3, 112.2, 111.9, 111.5; HRMS (ESI): m/z
calculated for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.1.15
2-(thiophen-2-yl)-N-(2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)quinazolin-4-
amine (11)

Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 12.32 (s, 1H), 8.22–8.11 (m, 1H), 7.89–7.80 (m, 1H),
7.71 (d, J = 7.9 Hz, 1H), 7.67–7.39 (m, 2H), 7.40 (t, J = 1.9 Hz, 1H), 7.26 (t, J = 13.6 Hz,
13
1H), 7.17 (t, J = 7.8 Hz, 1H), 6.77 (d, J = 7.9, 1H), 5.36 (s, 2H); C NMR (125 MHz,
DMSO- d6): δ 167.5, 159.4, 158.2, 151.0, 144.1, 138.5, 134.0, 130.9, 130.5, 129.0, 128.6,
128.4, 128.2, 127.8, 127.8, 126.7, 125.5, 123.6, 121.4, 114.5; HRMS (ESI): m/z calculated
for C31H22N4O 467.1872 found 467.1896 [M+H]+.
4.2 Bacterial strains and media
The gram positive and gram-negative pathogen panel of bacteria consisted of Escherichia
coli (ATCC 25922), Klebsiella pneumoniae (BAA-1705), Acinetobacter baumannii
(BAA1605), Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC
29213). NRS199, NRS129, NRS186, NRS191, NRS192, NRS193, NRS194, NRS198 are
MRSA strains while VRS1, VRS4, VRS12 are VRSA strains. These strains were obtained
from BEI/NARSA/ATCC (Biodefense and Emerging Infections Research Resources
Repository/Network on Antimicrobial Resistance in Staphylococcus aureus/American Type
Culture Collection, USA) and routinely cultivated on Mueller-Hinton Agar (MHA). Prior to
the experiment, a single colony was picked from MHA plate, inoculated in Mueller-Hinton
cation supplemented broth II (CA-MHB) and incubated overnight at 37 °C with shaking for
18–24 h to get the starter culture.
M. tuberculosis H37Rv ATCC 27294 was cultured in Middlebrook 7H9 (Difco, Becton, NJ,
USA) media supplemented with 10% ADC (Bovine Serum Albumin, Dextrose, NaCl), 0.2%
glycerol and 0.05% Tween-80 (ADC-Tween-80).
4.2.1 Antibiotic susceptibility testing against pathogen panel consisting E. coli, S. aureus,
K. pneuminiae, A. baumannii, and P. aeruginosa.
Antibiotic susceptibility testing was carried out on the newly synthesized compounds by
determining the Minimum Inhibitory Concentration (MIC) with reference to the standard
CLSI guidelines [31, 32]. MIC is defined as the minimum concentration of compound at
which visible bacterial growth is inhibited. Bacterial cultures were grown in Mueller-Hinton
cation supplemented broth (CA-MHB). Optical density (OD600) of the cultures was measured,
followed by dilution for ~106 cfu/mL. This inoculum was added into a series of test wells in a
microtitre plate that contained various concentrations of compound under test ranging from

64-4 µg/mL. Controls i.e., cells alone and media alone (without compound+cells) and
levofloxacin used as a reference standard. Plates were incubated at 37 °C for 16-18 h
followed by observations of MIC values by the absence or presence of visible growth. For
each compound, MIC determinations were performed independently thrice using duplicate
samples each time.
4.2.2 Antibiotic susceptibility testing against pathogenic mycobacteria
Antimycobacterial susceptibility testing was carried out on the newly synthesized compounds
by using broth micro dilution assay [33]. 1g/100 mL stock solutions of test and control
compounds were prepared in DMSO and stored in −20°C. Mycobacterial cultures were
inoculated in Middlebrook 7H9 enriched (Difco, Becton, NJ, USA) media supplemented with
10% ADC-Tween-80 (Bovine Serum Albumin, Dextrose, 0.2% glycerol and 0.05% Tween-
80) and OD600 of the cultures was measured, followed by dilution to achieve ~106 cfu/mL
[34]. The newly synthesized compounds were tested from 0.0064–0.00005 g/100 mL in two-
fold serial diluted fashion with 2.5 μL of each concentration added per well of a 96-well
round bottom micro titre plate. Later, 97.5 μL of bacterial suspension was added to each well
containing the test compound along with appropriate controls. Presto blue (Thermo Fisher,
USA) resazurin-based dye was used for the visualized identification of active compounds.
MIC of active compound was determined as lowest concentration of compound that inhibited
visible growth after incubation period. For each compound, MIC determinations were
replicated thrice using duplicate samples. The MIC plates were incubated at 37 °C for 7 days
for Mtb.
4.2.3 Cell Cytotoxicity Assay
The active newly synthesized compounds were screened for their cell toxicity against Vero
cells using MTT assay [35]. ~103 cells/well were seeded in 96 well plate and incubated at 37
°C with an 5% CO2 atmosphere. After 24 h, compound was added ranging from 100–5 mg/L
and incubated for 72 h at 37 °C with 5% CO2 atmosphere. After the incubation period, MTT
was added at 5 mg/L in each well, incubated at 37 °C for further 4 hours, residual medium
was discarded, 0.1 mL of DMSO was added to solubilise the formazan crystals and OD was
taken at 540 nm for the calculation of CC50. CC50 is defined as the lowest concentration of
compound which leads to a 50% reduction in cell viability. Doxorubicin was used as positive
control and each experiment was repeated in triplicate.

4.2.4 ADMET properties calculation
ADMET properties of the synthesized compounds were calculated using Qikprop program
(Qikprop, version 6.5, Schrödinger, LLC, New York, NY, 2014). As can be seen below, the
partition coefficient (QPlogPo/w), hydrogen bond donors (donor HB), hydrogen bond
acceptors (acceptor HB), molecular weight (mol. Wt.) and percent human oral absorption
exhibited satisfactory results. The compounds also followed Lipinski rule of five. ADMET
properties for Isoniazid and Rifampicin were also calculated and compared with the results of
the synthesized compounds.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
S. M is thankful to Department of Pharmaceuticals, Ministry of Chemicals & Fertilizers,
Govt. of India, for the award of NIPER fellowship. This study was supported by the DST
grant from Department of Science and Technology, Govt. of India to S.N and S.C.
(EMR/2017/000220).
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Synthesis and evaluation of new Quinazoline-benzimidazole hybrids as
potent anti-microbial agents against multidrug resistant Staphylococcus
aureus and Mycobacterium tuberculosis
Satyaveni Malasalaa, Md. Naiyaz Ahmadb, c, Ravikumar Akunuria, Manjulika Shuklab,
Grace Kaulb, c, Arunava Dasguptab, c, Y.V Madhavia, Sidharth Choprab,c*, Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
cAcSIR, Ghaziabad, Sector 19, Kamla Nehru Nagar, Ghaziabad-201002, Uttar Pradesh, India
Highlights:
1. Series of new quinazoline-benzimidazole hybrids were designed, synthesized and
evaluated for antibacterial activity against ESKAP pathogens and pathogenic
mycobacteria.
2. Compounds 8b, 8c, 8d, 8g, 8h, 8i and 10a displayed selective and potent inhibitory
activity against Staphylococcus aureus.
3. Compounds 8a, 8b, 8f, 8g, and 10c exhibited potent inhibitory activity against
different strains of Mycobacterium tuberculosis.
4. The selected compounds were found to show less cytotoxicity towards Vero cells with
favorable selectivity index.
5. The selected compounds were found to possess potent inhibitory activity against
multiple drug-resistant strains of S. aureus including VRSA.

Synthesis and evaluation of new Quinazoline-benzimidazole hybrids as potent
anti-microbial agents against multidrug resistant Staphylococcus aureus and
Mycobacterium tuberculosis
Satyaveni Malasalaa, Md. Naiyaz Ahmadb, Ravikumar Akunuria, Manjulika Shuklab,
Grace Kaulb, Arunava Dasguptab, Y.V Madhavia, Sidharth Choprab*, Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
Corresponding author: Dr. Srinivas Nanduri, E-mail: nandurisrini92@gmail.com
Co-corresponding author: Dr. Sidharth Chopra: skchopra007@gmail.com
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.
Conflicts of interest
The authors declare no conflicts of interest.