Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives as potent antibacterial agents effective against multidrug resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bioorg.2018.11.007

Treatment of nosocomial and community acquired Staphylococcus aureus infections has become more challenging due to the egression of multi-drug resistance. This has spurred the need for rapid development of new therapeutic agents which can effectively negate the resistance mechanisms. In our current work, several new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives were synthesized and examined for their antimicrobial activity against ESKAP pathogen panel and pathogenic mycobacteria. In the primary screening, compounds 4a, 4b, 6′a, 6′b, 6′h, 6′i and 6′j were found to demonstrate selective and potent inhibitory activity against Staphylococcus aureus (MICs = 0.25–0.5 μg/mL). When tested against Vero cells, all the compounds were found to be non toxic possessing favourable selectivity index (SI > 10), which encouraged us for carrying out further studies. Compound 6′a (SI > 40) was tested against a number of multiple clinical strains of multi-drug resistant S. aureus and was found to exhibit potent activity, irrespective of the resistant status of the strain. Besides, compound 6′a also exhibited concentration dependent bactericidal activity and synergized with the FDA approved drugs tested. The interesting results obtained suggest the potential utility of the newly synthesized compounds for treatment of multidrug resistant S. aureus infections.

Full text of DOI:10.1016/j.bioorg.2018.11.007

Accepted Manuscript
Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and
benzamide derivatives as potent antibacterial agents effective against multidrug
resistant Staphylococcus aureus
Srikanth Gatadi, Jitendra Gour, Manjulika Shukla, Grace Kaul, Swetarka das,
Arunava Dasgupta, Y.V. Madhavi, Sidharth Chopra, Srinivas Nanduri
PII:
S0045-2068(18)30966-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.007
YBIOO 2615
Reference:
Bioorganic Chemistry
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Please cite this article as: S. Gatadi, J. Gour, M. Shukla, G. Kaul, S. das, A. Dasgupta, Y.V. Madhavi, S. Chopra,
S. Nanduri, Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives as
potent antibacterial agents effective against multidrug resistant Staphylococcus aureus, Bioorganic Chemistry
(2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.007
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Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide
derivatives as potent antibacterial agents effective against multidrug resistant
Staphylococcus aureus
Srikanth Gatadi a, Jitendra Gour a, Manjulika Shukla b, Grace Kaul b, Swetarka das b, Arunava
Dasgupta b, Y.V. Madhavi a, Sidharth Chopra b,*, Srinivas Nanduri a,*
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
Abstract:
Treatment of nosocomial and community acquired Staphylococcus aureus infections has become
more challenging due to the egression of multi-drug resistance. This has spurred the need for
rapid development of new therapeutic agents which can effectively negate the resistance
mechanisms. In our current work, several new 4-oxoquinazolin-3(4H)-yl)benzoic acid and
benzamide derivatives were synthesized and examined for their antimicrobial activity against
ESKAP pathogens and pathogenic mycobacteria. In the primary screening, compounds 4a, 4b,
6'a, 6'b, 6'h, 6'i, 6'j were found to demonstrate selective and potent inhibitory activity against
Staphylococcus aureus (MICs = 0.25-0.5 µg/mL). When tested against Vero cells, all the
compounds were found to be less toxic possessing favourable selectivity index (SI >10), which
encouraged us for carrying out further studies. Compound 6'a (SI >40) was tested against a
number of multiple clinical strains of multi-drug resistant S. aureus and was found to exhibit
potent activity, irrespective of the resistant status of the strain. Besides, compound 6’a also
exhibited concentration dependent bactericidal activity and synergized with the FDA approved
drugs tested. The interesting results obtained suggest the potential utility of the newly
synthesized compounds for treatment of multidrug-resistant S. aureus infections.
Keywords: Drug resistance, antibacterial agents, Methicillin-resistant and Vancomycin resistant
Staphylococcus aureus, Minimum Inhibitory concentration, Vero cells.

Corresponding author. Dr. Srinivas Nanduri, Tel: +91-40-23073740; Fax: +91-40-23073751;

E-mail: nandurisrini92@gmail.com; Dr. Sidharth Chopra, Tel.: +91-522-2771940; Fax: +91-
522-2771941; Email: skchopra007@gmail.com
1

1. Introduction
Multi-drug resistant Staphylococcus aureus (MDR-SA) infections are a significant health crisis
globally and have spurred a need for rapid development of new classes of therapeutic agents
with a potential to circumvent the drug resistant mechanisms [1]. MDR-SA exhibits resistance to
methicillin, β-lactams, glycopeptides, fluoroquinolones, macrolides, oxazolidinones and
carbapenems [2-7]. Sensible use and development of new, effective antibiotics is a vital step to
palliate the complications associated with MDR-SA infections.
In the quest for new antibacterials, we found quinazolinones to be a privileged structure in
modern medicinal chemistry, possessing wide range of biological properties like antibacterial,
antifungal, anticonvulsant, anti-inflammatory, anti-HIV, anticancer and analgesic activities [8-
18]. Exploration of quinazolinones has gained enormous interest and proven promise with the
identification of potent antimicrobial agents. Saravanan et al. [19] and Zayed et al. [20] reported
quinazolinones 1 & 2 respectively as potent antibacterial agents. Desai et al. [21] reported the
antibacterial properties of 2-styryl-3,4-dihydroquinazolin-6-yl-(1,3,5-triazin-2-yl)-3-methylurea
derivatives 3. Recently, Bouley et al. [22] reported 4(3H)-quinazolinones 4 as potent
antibacterial agents with good in vivo activity. Jadhavar et al. [23] also reported 2-styryl
quinazolinones 5 (Figure 1) as potent anti-mycobacterial agents. In the present study, we have
designed a number of 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives
(Figure 2) and evaluated them for their antimicrobial properties against ESKAP pathogens and
pathogenic mycobacteria.
<Insert Figure 1 here>
<Insert Figure 2 here>
2.0 Results and Discussion
2.1 Chemistry
The newly designed compounds were synthesized by following the synthetic schemes outlined
in Schemes 1 & 2. Substituted anthranilic acids 1, 1’ were cyclised to the corresponding
benzoxazinone intermediates 2, 2' by refluxing in acetic anhydride. To a solution of 2, 2' in
glacial acetic acid, 3-amino benzoic acid was added and the reaction mixture was refluxed for 5
h to afford 3-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzoic acid as intermediates 3, 3’. To obtain
the amide derivatives 4’, the intermediate 3’ was reacted with various amines by using N,N-
2

dimethyl amino pyridine (DMAP), N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide
hydrochloride (EDC) in dichloromethane and allowed to react at room temperature for 12-14 h.
Further intermediate 3 or 4’ were reacted with benzaldehyde or 4-chloro or 4-cyano
benzaldehyde in glacial acetic acid or N,N-Dimethylformamide dimethylacetal (DMF-DMA) in
N,N-dimethyl formamide (DMF) under reflux conditions overnight (18-20 h) to afford the final
compounds in moderate to excellent yields. The structures of the newly synthesized compounds
1
were confirmed by H NMR, 13C NMR (given in supplementary data B) and HRMS (ESI)
spectroscopic techniques.
<Insert Scheme 1 here>
<Insert Scheme 2 here>
2.2 In vitro antibacterial activity
2.2.1 Antibiotic Susceptibility Testing against ESKAP panel of bacteria
All the newly synthesised 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives
were evaluated for their antibacterial activity against ESKAP panel of pathogens. Antibiotic
susceptibility testing was performed by determining minimum inhibitory concentration (MIC)
according to the CLSI guidelines [24]. The newly synthesized derivatives were tested in the
range of 64-0.03 μg/mL with Levofloxacin as a control and the results are given in Table 1. In
order to find their spectrum of activity, these compounds were also tested against pathogenic M.
tuberculosis H37Rv strain, wherein all the compounds were found to be inactive (Supplementary
data A).
Compound 4a with chloro at C-5 position and carboxy group at meta position on the N-phenyl
with styryl moiety (with p-C6H4-CN group) at C-2 exhibited selective activity against S. aureus
(MIC = 8 µg/mL). Compound 4b having C-CH3 at C-8 position carboxy group at meta position
on the N-phenyl and with styryl moiety (p-C6H4-CN group) at C-2 displayed potent activity
against S. aureus with MIC of 2 µg/mL (Scheme 1). This observation prompted us to investigate
a number of quinazolinones with 4-cyano styryl moiety at C-2 position by varying R1, R2, R3 &
3

X. The corresponding aza derivatives 4c-4e (pyrido pyramidinones) with carboxy group at meta
position on the N-phenyl and styryl moiety (with p-C6H4-R2 group) at C-2 were found to be
devoid of activity. Besides, compound 4f with 2-carboxy group and compound 4g with 2-
carboxy-4, 5-dimethoxy group on the N-Phenyl moiety were found to be inactive against
ESKAP pathogen panel. Based on the potent activity shown by the 4-oxoquinazolin-3(4H)-
yl)benzoic acid derivatives, a number of corresponding amide derivatives were prepared and
evaluated for their antibacterial activity and some of the highlights are discussed below.
Compounds 4’ and 5’ with methyl and (E)-N,N-dimethylprop-1-en-1-amine group at C-2
respectively and cyclopropyl amide at meta position on the N-phenyl moiety were found to be
inactive. However, when the C-2 methyl group is replaced with styryl moiety (with p-C6H4-CN
group) as in 6’b, the compound exhibited selective and potent inhibitory activity against S.
aureus (MIC = 0.5 µg/mL), suggesting the usefulness of styryl moiety (with p-C6H4-CN group).
Replacement of cyclopropyl amide with cyclohexyl (6’c), 4-fluoro phenyl (6’d), 3-pyridyl (6’e),
thiazolyl (6’f), 5-methylisoxazolyl (6’g) amides led to loss of activity. Interestingly, compound
6’a with isopropyl amide was found to be the most potent compound with a MIC of 0.25 µg/mL.
Gratifyingly, when secondary amides were replaced with tertiary amides at C-3’ of N-phenyl as
in compound 6’h (pyrrolidinyl), 6’i (piperidinyl), 6’j (morpholinyl), the compounds were found
to exhibit potent and selective activity against S. aureus with MIC values 4-8 µg/mL (Figure 3
& 4).
<Insert Figure 3 here>
<Insert Figure 4 here>
<Insert Table 1 here>
4

2.2 Cytotoxicity Assay against Vero cells
The compounds 4a, 4b, 6’a, 6’b, 6’h, 6’i and 6’j were tested for cytotoxicity against Vero cells
using the MTT assay [25]. CC50 is defined as the lowest concentration of compound which leads
to a 50% reduction in cell viability with doxorubicin as positive control. A perusal of results
(Table 2) indicate that 4a, 4b, 6’a, 6’b, 6’h, 6’i and 6’j are non toxic to Vero cells and exhibited
a selectivity index (SI) of (>1.25->40).
<Insert Table 2 here>
Among the tested compounds, 6'a was found to be promising with potent activity against S.
aureus and also possessing favourable selectivity index (SI >40), which encouraged us to carry
further studies.
2.3 Determination of MIC against MDR-SA including VRSA
To determine the spectrum of activity of 6'a against multiple strains of MDR-SA, it was tested
against various well defined and characterized clinical strains of MRSA and VRSA. The results
are summarised in Table 3. Levofloxacin, Meropenem and Vancomycin were used as reference
standards. From the examination of the results, it can be inferred that the compound 6’a
exhibited potent antibacterial activity with MIC = <0.125–0.5 µg/mL against various clinical
strains of MRSA and VRSA. Thus, 6’a exhibits equipotent activity against MDR-SA
irrespective of its drug-resistance status.
<Insert Table 3 here>
3. Time Kill kinetics of 6’a
The bactericidal activity was assessed by the time-kill method. S. aureus ATCC 29213 cells
were diluted up to ~105 CFU/mL and treated with compound for concentrations corresponding
to 1X and 10X of MIC of 6’a and vancomycin in MHB in triplicate and incubated at 37 °C.
0.1mL samples were collected after time intervals of 0, 1, 6 and 24 h, serially diluted in PBS and
plated on TSA followed by incubation at 37 °C for 18-20 h. Kill curves were constructed by
counting the colonies from plates and plotting the CFU/mL of surviving bacteria at each time
point in the presence and absence of compound (Figure 5). 6’a exhibits concentration dependent
bactericidal activity.
<Insert Figure 5 here>
5

4. Determination of synergy with FDA approved drugs
The checkerboard method was used to determine synergy between 6’a and various antibiotics
linezolid, meropenem, ceftriaxone and vancomycin for the treatment of staphylococcal
infections. As can be seen in Table 4, compound 6’a synergized with the FDA approved drugs
tested, thus exhibiting great potential to be a part of multi-drug regimen.
<Insert Table 4 here>
5. Conclusion
In conclusion, a series of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives
were synthesised and evaluated against ESKAP panel of bacteria. Compounds 4a, 4b, 6’a, 6’b,
6’h, 6’i and 6’j displayed selective and potent antibacterial activity against Staphylococcus
aureus. They were found to be non toxic to Vero cells (CC50 = >5->20 µg/mL) with good
selectivity index. In further studies, compound 6’a displayed potent inhibitory activity when
screened against clinical MRSA and VRSA isolates. Besides compound 6’a exhibited
concentration dependent bactericidal activity and synergized with the FDA approved drugs
tested. With the promising results obtained, the synthesized new 4-oxoquinazolin-3(4H)-
yl)benzoic acid and benzamide derivatives present potential for further development as anti-
staphylococcal leads.
6. Materials & Experimental methods
All the chemicals, reagents and starting materials were procured from commercial providers.
The thorough monitoring of reactions were performed by thin layer chromatography (TLC-
MERCK pre-coated silica gel 60-F254 aluminium plates) under UV light. Melting points were
1
13
checked using Stuart® SMP30 apparatus and are uncorrected. H and C NMR were taken on
Bruker Avance 500 MHz spectrometer using tetramethylsilane (TMS) as the internal standard
1
and chemical shifts are reported in ppm. Chemical shifts are referenced to TMS (δ 0.00 for H
13
1
13
NMR and C NMR), DMSO˗d6 (δ 2.50 for H NMR and 39.5 for C NMR) or CDCl3 (δ 7.26
for 1H NMR and 77.16 for 13C NMR) or combination of DMSO-d6 and. CDCl3, in which CDCl3
was used as an internal reference. Spin multiplicities are reported as s (singlet), brs (broad
singlet), d (doublet), dd (double doublet), t (triplet) and m (multiplet). Coupling constant (J)
values are reported in hertz (Hz). HRMS were determined with Agilent QTOF mass
spectrometer 6540 series instrument and were performed in the ESI techniques at 70 eV.
Column chromatography was performed using silica gel 60–120 or 100–200 mesh.
6

Intermediates 2, 3 (Scheme 1) were prepared according to the procedures described in literature
[22].
6.1. General reaction procedure for the synthesis of (E)-3-(4-oxo-2-styrylquinazolin-3(4H)-
yl)benzoic acid and its corresponding aza derivatives 4a–4g.
Substituted 3-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzoic acid (3, 2 mmol) was dissolved in 15
mL of glacial acetic acid, to which 4-substituted benzaldehyde (2 mmol) was added. The
reaction was refluxed for 18–20 h, and monitered by using TLC. After completion of reaction,
the reaction mixture was allowed to cool at room temperature and then 5mL volume of water
was added to give crude precipitate. The resulting crude precipitate was filtered and washed with
water followed by cold methanol and hexane to obtain a (E)-3-phenyl-2-styrylquinazolin-4(3H)-
ones and its corresponding aza derivatives 4a–4g as pure yellow to pale yellow solids in 68–83
1
13
% yields. All the newly synthesized compounds were characterized by H NMR, C NMR and
HRMS (ESI).
6.1.1. (E)-3-(5-chloro-2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (4a):Yellow
solid, Yield 74%; mp: 191-193 °C, 1H NMR (500 MHz, DMSO–d6) δ 13.25 (s, 1H), 8.17–8.12
(m, 1H), 8.09–8.06 (m, 1H), 7.90 (d, J = 15.5 Hz, 1H), 7.82–7.73 (m, 5H), 7.71 (dd, J = 8.2, 1.1
Hz, 1H), 7.57–7.52 (m, 3H), 6.40 (d, J = 15.6 Hz, 1H) ppm; 13C NMR (125 MHz, DMSO–d6) δ
167.00, 159.77, 151.85, 150.16, 139.51, 137.95, 137.41, 135.03, 133.93, 133.40, 133.27, 132.88,
130.63, 130.55, 130.53, 129.74, 128.76, 127.39, 123.48, 119.01, 118.17, 112.18 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C24H15ClN3O3 428.0802; found 428.0808.
6.1.2. (E)-3-(2-(4-cyanostyryl)-8-methyl-4-oxoquinazolin-3(4H)-yl)benzoic acid (4b): Yellow
solid, Yield 81%; mp: 193–195 °C. 1H NMR (500 MHz, DMSO–d6) δ 13.13 (s, 1H), 8.17–8.13
(m, 1H), 8.05–8.03 (m, 1H), 7.99–7.93 (m, 2H), 7.81–7.77 (m, 2H), 7.77–7.71 (m, 3H), 7.60–
7.56 (m, 2H), 7.44 (t, J = 7.6 Hz, 1H), 6.49 (d, J = 15.5 Hz, 1H), 2.70 (s, 3H) ppm; 13C NMR
(125 MHz, DMSO–d6) δ 166.99, 161.96, 149.92, 145.99, 139.78, 137.52, 137.48, 136.11,
135.61, 133.86, 133.25, 132.85, 130.56, 130.52, 130.41, 128.70, 127.02, 124.59, 124.06, 121.11,
119.04, 111.97, 17.49 ppm; HRMS (ESI): m/z calcd for [M+H]+ C25H18N3O3 408.1348; found
408.1352.
6.1.3. (E)-3-(4-oxo-2-styrylpyrido[2,3-d]pyrimidin-3(4H)-yl)benzoic acid (4c):Yellow solid,
1
Yield 83%; mp: 229–231 °C. H NMR (500 MHz, DMSO–d6) δ 13.29 (s, 1H), 9.02 (d, J = 2.6
Hz, 1H), 8.51 (d, J = 6.9 Hz, 1H), 8.16 (d, J = 6.7 Hz, 1H), 8.09 (s, 1H), 8.02 (d, J = 15.4 Hz,
7

1H), 7.82–7.73 (m, 2H), 7.55 (dd, J = 7.6, 4.6 Hz, 1H), 7.39 (d, J = 17.3 Hz, 5H), 6.35 (d, J =
13
15.4 Hz, 1H) ppm; C NMR (125 MHz, DMSO–d6) δ 166.97, 162.56, 157.82, 156.76, 154.62,
141.08, 137.42, 136.44, 135.03, 133.84, 132.91, 130.70, 130.64, 130.59, 130.39, 129.55, 128.31,
122.56, 119.99, 116.45 ppm; HRMS (ESI): m/z calcd for [M+H]+ C22H16N3O3 370.1191; found
370.1209.
6.1.4. (E)-3-(2-(4-chlorostyryl)-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)benzoicacid (4d):Yellow
solid, Yield 73%; mp: 220–222 °C. 1H NMR (500 MHz, DMSO–d6) δ 13.30 (s, 1H), 9.02 (dd, J
= 4.5, 1.9 Hz, 1H), 8.53–8.49 (m, 1H), 8.18–8.11 (m, 1H), 8.08 (s, 1H), 8.00 (d, J = 15.4 Hz,
1H), 7.81–7.72 (m, 2H), 7.55 (dd, J = 7.8, 4.6 Hz, 1H), 7.47–7.43 (m, 4H), 6.37 (d, J = 15.5 Hz,
1H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 166.97, 162.54, 157.76, 156.77, 154.48, 139.66,
137.31, 136.45, 135.09, 133.95, 133.83, 132.90, 130.74, 130.59, 130.40, 130.03, 129.58, 122.65,
120.75, 116.49 ppm; HRMS (ESI): m/z calcd for [M+H]+ C22H15ClN3O3 404.0802; found
404.0815.
6.1.5. (E)-3-(2-(4-cyanostyryl)-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)benzoic acid (4e):Yellow
solid, Yield 68%; mp: 242–244 °C. 1H NMR (500 MHz, DMSO–d6) δ 9.03 (dd, J = 4.5, 1.8 Hz,
1H), 8.52 (dd, J = 7.8, 1.8 Hz, 1H), 8.19–8.11 (m, 1H), 8.09 (s, 1H), 8.04 (d, J = 15.5 Hz, 1H),
7.82 (d, J = 8.2 Hz, 2H), 7.79–7.71 (m, 2H), 7.64 (d, J = 8.3 Hz, 2H), 7.60–7.53 (m, 1H), 6.52
13
(d, J = 15.5 Hz, 1H) ppm; C NMR (125 MHz, DMSO–d6) δ 172.49, 166.96, 162.51, 157.69,
156.84, 154.20, 139.51, 138.93, 137.16, 136.51, 133.81, 133.35, 132.89, 130.60, 130.41, 129.00,
123.53, 122.93, 119.05, 116.71, 112.32 ppm; HRMS (ESI): m/z calcd for [M+H]+ C23H15N4O3
395.1144; found 395.1151.
6.1.6. (E)-2-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (4f):Yellow solid, Yield
78%; mp: 154–156 °C; 1H NMR (500 MHz, DMSO–d6) δ 8.20–8.11 (m, 2H), 8.95–8.87 (m,
2H), 7.87–7.77 (m, 4H), 7.76–7.71 (m, 1H), 7.62–7.53 (m, 4H), 6.46 (d, J = 15.6 Hz, 1H) ppm;
13C NMR (125 MHz, DMSO–d6) δ 165.05, 160.67, 150.41, 146.75, 138.66, 136.37, 135.99,
134.27, 133.19, 132.24, 131.16, 130.10, 129.33, 128.79, 127.60, 126.71, 126.29, 125.86, 122.63,
120.11, 117.99, 110.98 ppm; HRMS (ESI): m/z calcd for [M+H]+ C24H16N3O3 394.1191; found
394.1194.
6.1.7.(E)-2-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-4,5-dimethoxybenzoic
acid
(4g):
1
Yellow solid, Yield 71%; mp: 185–187 °C;. H NMR (500 MHz, DMSO–d6) δ 8.14 (d, J = 7.8
Hz, 1H), 7.96–7.85 (m, 2H), 7.84–7.76 (m, 3H), 7.60 (d, J = 8.2 Hz, 2H), 7.55 (t, J = 7.5 Hz,
1H), 7.16–7.10 (m, 2H), 7.00–6.96 (m, 1H), 6.59 (d, J = 15.6 Hz, 1H), 3.86 (s, 3H), 3.76 (s, 3H)
8

13
ppm; C NMR (125 MHz, DMSO–d6) δ 161.79, 151.86, 149.82, 149.64, 147.68, 139.94,
137.09, 135.21, 133.34, 129.74, 128.65, 127.75, 127.36, 126.97, 124.14, 121.47, 121.30, 119.07,
113.05, 112.35, 111.98, 56.29, 56.18 ppm; HRMS (ESI): m/z calcd for [M+H]+ C26H20N3O5
454.1403; found 454.1406.
Intermediates 2’, 3’ (Scheme 2) were prepared according to the procedures described in
literature [22].
6.2. General reaction procedure for the synthesis of 2-methyl-4-oxoquinazolin-3(4H)-
yl)benzamide derivatives 4’ .
To a mixture of the amine (2 mmol) (Scheme 2) and N,N-dimethyl amino pyridine (DMAP) (2.6
mmol) in dichloromethane was added the 3-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzoic acid
(3’, 2 mmol), followed by N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(EDC) (2.7 mmol.), and was allowed to stir 12-14 h at room temperature. After completion of
reaction, the reaction mixture was extracted with ethylacetate and the organic layer was washed
with 10% aq citric acid, water, sat. aq NaHCO3, and brine. The organic layer was dried with
sodium sulphate (Na2SO4) and concentrated in vacuum to afford the amides 4’ as pure white
products in good yields without further purification.
6.2.1. N-cyclopropyl-3-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzamide (4’):White solid, Yield
1
61%; mp: 140–142 °C. H NMR (500 MHz, CDCl3+DMSO-d6) δ 8.11 (d, J = 7.6 Hz, 1H),
7.90–7.86 (m, 1H), 7.75–7.69 (m, 1H), 7.67–7.65 (m, 1H), 7.59 (d, J = 8.1Hz, 1H), 7.52 (t, J =
7.7 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.31(d, J = 7.7 Hz, 1H), 6.90 (brs, 1H), 2.89–2.79 (m, 1H),
2.17 (s, 3H), 0.79–0.72 (m, 2H), 0.57–0.49 (m, 2H) ppm; 13C NMR (125 MHz, CDCl3+DMSO-
d6) δ 171.96, 166.68, 158.87, 152.23, 142.49, 141.25, 139.45, 135.57, 134.57, 133.29, 133.14,
132.04, 131.67, 131.45, 125.36, 29.18, 28.00, 10.82, 10.78 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C19H18N3O2 320.1399; found 320.1402.
6.3. General reaction procedure for the synthesis of (E)-N-cyclopropyl-3-(2-(2-
(dimethylamino)vinyl)-4-oxoquinazolin-3(4H)-yl)benzamide 5’, or (E)-3-(2-(4-cyanostyryl)-4-
oxoquinazolin-3(4H)-yl-benzamide derivatives 6’a–6’j.
Compound (4’, 2 mmol) was reacted with 4-cyano benzaldehyde (2 mmol) in acetic acid or N,N-
Dimethylformamide dimethyl acetal (2 mmol) in N, N-dimethyl formamide (DMF) under reflux
conditions (18-20 h). The reaction was monitered by using TLC. After completion of reaction,
5mL volume of water was added to the cooled reaction mixture. The resulting crude precipitate
9

was filtered and washed with water followed by cold methanol and hexane to obtain a
quinazolin-3(4H)-yl)benzamide derivatives 5’, 6’a–6’j derivatives as pure yellow to pale yellow
solids in 50–80% yields. All the newly synthesized compounds were characterized by 1H NMR,
13C NMR and HRMS (ESI).
6.3.1.
(E)-N-cyclopropyl-3-(2-(2-(dimethylamino)vinyl)-4-oxoquinazolin-3(4H)-yl)benzamide
1
(5’): White solid, Yield 50%; mp: 156–158 °C. H NMR (500 MHz, DMSO–d6) δ 8.52 (s, 1H),
8.03–7.91 (m, 2H), 7.87 (d, J = 12.1 Hz, 1H), 7.75 (s, 1H), 7.71–7.61 (m, 2H), 7.52–7.39 (m,
2H), 7.25–7.17 (m, 1H), 3.97 (d, J = 12.0 Hz, 1H), 2.82–2.78 (m, 7H), 0.64 (m, 4H) ppm; 13C
NMR (125 MHz, DMSO–d6) δ 166.68, 162.24, 155.43, 150.59, 149.55, 138.48, 136.06, 134.84,
132.10, 130.01, 128.00, 127.94, 126.72, 125.86, 123.41, 118.89, 86.70, 23.61, 6.18, 6.04 ppm;
HRMS (ESI): m/z calcd for [M+H]+ C22H23N4O2 375.1821; found 375.1828.
6.3.2. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-isopropylbenzamide (6’a): Yellow
solid, Yield 58%; mp: 188–189 °C. 1H NMR (500 MHz, DMSO–d6) δ 8.34 (d, J = 7.6 Hz, 1H),
8.18–8.14 (m, 1H), 8.09–8.05 (m, 1H), 7.98–7.88 (m, 3H), 7.84–7.77 (m, 3H), 7.70 (t, J = 7.8
Hz, 1H), 7.67–7.63 (m, 1H), 7.61–7.53 (m, 3H), 6.50 (d, J = 15.6 Hz, 1H), 4.13–4.09 (m, 1H),
1.19–1.15 (m, 6H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 164.58, 161.69, 151.26, 147.67,
139.77, 137.51, 137.16, 136.65, 135.44, 133.31, 131.99, 130.11, 128.70, 128.26, 127.86, 127.58,
126.96, 123.89, 121.16, 119.03, 112.10, 41.69, 22.77, 22.72 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C27H23N4O2 435.1821; found 435.1827.
6.3.3. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-cyclopropylbenzamide (6’b): Pale
yellow solid, Yield 68%; mp: 167–169 °C. 1H NMR (500 MHz, DMSO–d6) δ 8.59 (s, 1H), 8.15
(d, J = 7.7 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.99–7.87 (m, 3H), 7.83–7.77 (m, 3H), 7.72–7.68
(m, 1H), 7.66–7.62 (m, 1H), 7.60–7.56 (m, 3H), 6.48 (d, J = 15.6 Hz, 1H), 2.88–2.84 (m, 1H),
0.72–0.68 (m, 2H), 0.60–0.56 (m, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 166.82, 161.72,
151.25, 147.64, 139.73, 137.49, 137.17, 136.20, 135.49, 133.32, 132.15, 130.23, 128.71, 128.60,
128.22, 127.85, 127.63, 126.96, 123.85, 121.10, 119.06, 112.07, 23.59, 6.23, 6.06 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C27H21N4O2 433.1664; found 433.1667.
6.3.4. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-cyclohexylbenzamide (6’c): Yellow
solid, Yield 62%; mp: 190-192 °C. 1H NMR (500 MHz, DMSO–d6) δ 8.33 (d, J = 7.9 Hz, 1H),
8.16 (dd, J = 7.9, 1.2 Hz, 1H), 8.08–8.03 (m, 1H), 7.96–7.90 (m, 3H), 7.84–7.80 (m, 3H), 7.70
(t, J = 7.8 Hz, 1H), 7.66–7.62 (m, 1H), 7.61–7.57 (m, 3H), 6.51 (d, J = 15.6 Hz, 1H), 3.85–3.69
10

(m, 1H), 1.89–1.77 (m, 2H), 1.77–1.67 (m, 2H), 1.64–1.55 (m, 1H), 1.38–1.20 (m, 4H), 1.19–
1.04 (m, 1H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 164.58, 161.71, 151.24, 147.65, 139.73,
137.49, 137.14, 136.63, 135.45, 133.31, 132.00, 130.12, 128.77, 128.70, 128.31, 127.85, 127.59,
126.96, 123.86, 121.13, 119.05, 112.07, 49.02, 32.87, 32.82, 25.72, 25.38 ppm; HRMS (ESI):
m/z calcd for [M+H]+ C30H27N4O2 475.2134; found 475.2140.
6.3.5. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-(4-fluorophenyl)benzamide (6’d):
1
Yellow solid, Yield 70%; mp: 245–247 °C. H NMR (500 MHz, DMSO–d6) δ 10.43 (s, 1H),
8.20–8.16 (m, 2H), 8.10–8.06 (m, 1H), 7.98–7.89 (m, 2H), 7.85–7.76 (m, 6H), 7.75–7.71 (m,
13
1H), 7.63–7.56 (m, 3H), 7.25–7.17 (m, 2H), 6.55 (d, J = 15.6 Hz, 1H) ppm; C NMR (125
MHz, DMSO–d6) δ 164.69, 161.74, 158.89 (d, J1 = 240.5 Hz), 151.26, 147.67, 139.75,
C-F
137.54, 137.36, 136.54, 135.70 (d, J4 = 2.6 Hz), 135.48, 133.31, 132.71, 130.37, 129.09,
C-F
128.78, 128.74, 127.87, 127.62, 126.98, 123.90, 122.83 (d, J3 = 7.9 Hz), 121.16, 119.06,
C-F
115.72 (d, J2 = 22.2 Hz), 112.09 ppm; HRMS (ESI): m/z calcd for [M+H]+ C30H20FN4O2
C-F
487.1570; found 487.1576.
6.3.6. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-(pyridin-3-yl)benzamide (6’e):
Yellow solid, Yield 69%; mp: 280–282 °C. 1H NMR (500 MHz, DMSO–d6) δ 10.59 (s, 1H),
8.92 (d, J = 2.4 Hz, 1H), 8.33 (dd, J = 4.7, 1.4 Hz, 1H), 8.23–8.15 (m, 3H), 8.10 (t, J = 1.7 Hz,
1H), 7.98–7.90 (m, 2H), 7.86–7.78 (m, 4H), 7.77–7.73 (m, 1H), 7.66–7.56 (m, 3H), 7.41 (dd, J =
8.3, 4.7 Hz, 1H), 6.55 (d, J = 15.6 Hz, 1H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 165.22,
161.76, 151.26, 147.67, 145.28, 142.55, 139.75, 137.58, 137.41, 136.12, 136.04, 135.51, 133.30,
132.98, 130.46, 129.20, 128.88, 128.75, 128.02, 127.88, 127.65, 126.98, 124.07, 123.90, 121.14,
119.05, 112.09 ppm; HRMS (ESI): m/z calcd for [M+H]+ C29H20N5O2 470.1617; found
470.1624.
6.3.7. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-(thiazol-2-yl)benzamide (6’f):
1
Yellow solid, Yield 64%; mp: 220–222 °C. H NMR (500 MHz, DMSO–d6) δ 12.77 (s, 1H),
8.30–8.26 (m, 1H), 8.23–2.20 (m, 1H), 8.17 (dd, J = 7.9, 0.9 Hz, 1H), 7.97–7.89 (m, 2H), 7.86–
7.73 (m, 5H), 7.65–7.54 (m, 4H), 7.30 (s, 1H), 6.58 (d, J = 15.6 Hz, 1H) ppm; 13C NMR (125
MHz, DMSO–d6) δ 164.48, 161.72, 151.29, 147.72, 139.82, 137.49, 137.45, 135.46, 133.52,
133.30, 133.42, 130.54, 129.51, 129.35, 128.78, 127.89, 127.60, 126.99, 124.07, 121.19, 119.82,
119.04, 114.54, 112.71, 112.08 ppm; HRMS (ESI): m/z calcd for [M+H]+ C27H18N5O2S
476.1181; found 476.1182.
11

6.3.8. (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-N-(5-methylisoxazol-3-yl)benzamide
1
(6’g): Yellow solid, Yield 66%; mp: 240–242 °C. H NMR (500 MHz, DMSO–d6) δ 11.45 (s,
1H), 8.21–8.18 (m, 1H), 8.17 (dd, J = 7.9, 1.3 Hz, 1H), 8.14–8.11 (m, 1H), 7.96–7.89 (m, 2H),
7.84–7.79 (m, 3H), 7.77–7.71 (m, 2H), 7.63–7.56 (m, 3H), 6.77 (s, 1H), 6.56 (d, J = 15.6 Hz,
1H), 2.42 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 170.03, 164.80, 161.71, 158.98,
151.28, 147.71, 139.81, 137.48, 137.37, 135.44, 135.16, 133.29, 130.44, 129.42, 129.25, 128.76,
127.88, 127.58, 126.98, 126.77, 124.05, 121.18, 119.04, 112.09, 97.41, 12.60 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C28H20N5O3 474.1566; found 474.1568.
6.3.9.
(E)-4-(2-(4-oxo-3-(3-(pyrrolidine-1-carbonyl)phenyl)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (6’h): Yellow solid, Yield 51%; mp: 258–260 °C. H NMR (500 MHz,
CDCl3) δ 8.30 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.89–7.81 (m, 2H), 7.75 (d, J = 7.7
Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.62–7.58 (m, 2H), 7.55–7.51 (m, 2H), 7.45–7.41 (m, 2H),
7.38–7.34 (m, 1H), 6.49 (d, J = 15.5 Hz, 1H), 3.71–3.59 (m, 2H), 3.53–3.43 (m, 2H), 2.01–1.92
(m, 2H), 1.92–1.80 (m, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 193.42, 167.56, 161.61,
151.47, 147.70, 139.79, 138.89, 137.20, 136.89, 135.41, 133.31, 130.78, 129.83, 128.62, 128.07,
127.83, 127.55, 127.01, 124.26, 121.24, 119.06, 112.02, 49.38, 46.36, 26.17, 24.34 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C28H23N4O2 447.1821; found 447.1826.
6.3.10.
(E)-4-(2-(4-oxo-3-(3-(piperidine-1-carbonyl)phenyl)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (6’i): Yellow solid, Yield 65%; mp: 280–282 °C. H NMR (500 MHz,
CDCl3) δ 8.32–8.28 (m, 1H), 7.99 (d, J = 15.5 Hz, 1H), 7.85–7.81 (m, 2H), 7.67–7.58 (m, 4H),
7.55–7.50 (m, 1H), 7.45–7.41 (m, 2H), 7.40–37 (m,1H), 7.37–7.33 (m, 1H), 6.50 (d, J = 15.5
Hz, 1H), 3.86–3.73 (m, 1H), 368–3.55 (m, 1H), 3.50–3.32 (m, 2H), 1.58–1.54 (m, 6H) ppm; 13C
NMR (125 MHz, DMSO–d6) δ 168.14, 161.62, 151.38, 147.69, 139.79, 138.23, 137.16, 137.11,
135.40, 133.32, 130.50, 130.39, 128.53, 128.06, 127.82, 127.61, 127.55, 127.00, 124.12, 121.23,
119.04, 112.04, 48.43, 42.77, 26.11, 25.63, 24.36 ppm; HRMS (ESI): m/z calcd for [M+H]+
C29H25N4O2 461.1977; found 461.1982.
6.3.11.
(E)-4-(2-(3-(3-(morpholine-4-carbonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (6’j): Yellow solid, Yield 62%; mp: 298–300 °C. H NMR (500 MHz,
DMSO–d6) δ 8.18–8.14 (m, 1H), 7.93–7.89 (m, 1H), 7.87 (d, J = 15.6 Hz, 1H), 7.84–7.78 (m,
3H), 7.71 (t, J = 7.8 Hz, 1H), 7.64–7.58 (m, 4H), 7.58–7.52 (m, 2H), 6.52 (d, J = 15.6 Hz, 1H),
3.67–3.51 (m, 4H), 3.43–3.22 (m, 4H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 168.36, 161.59,
151.41, 147.68, 139.74, 137.23, 137.20, 135.41, 133.33, 130.78, 130.53, 128.72, 128.61, 128.53,
12

128.04, 127.83, 127.55, 127.01, 124.19, 121.21, 119.04, 112.06, 66.32, 60.24, 21.23, 14.55 ppm;
HRMS (ESI): m/z calcd for [M+H]+ C28H23N4O3 463.1770; found 463.1767.
7. Bacterial strains and media
The ESKAP panel of bacteria consisted of Escherichia coli (ATCC 25922), Staphylococcus
aureus (ATCC 29213), Klebsiella pneumoniae (BAA-1705), Acinetobacter baumannii (BAA-
1605) and Pseudomonas aeruginosa (ATCC 27853). NRS100, NRS199, NRS129, NRS186,
NRS191, NRS192, NRS193, NRS194, NRS198 were the MRSA strains while VRS1, VRS4,
VRS12 were the 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).
Molecular details of Multi Drug Resistant (MDR) strains
Molecular details of strains
MDR-Strains
Community acquired-MRSA
positive for the Panton-Valentine leucocidin (PVL) virulence
NR 10194
factor
Contains staphylococcal chromosome cassette mec type V
Community acquired-MRSA
positive for the Panton-Valentine leucocidin (PVL) virulence
factor
NR 10186
NR 10193
NR 10191
Contains staphylococcal chromosome cassette mec type IV
Pulse-field gel electrophoresis (PFGE) typed as USA 300
Community acquired-MRSA
Negative for the Panton-Valentine leucocidin (PVL) virulence
factor
Contains staphylococcal chromosome cassette mec type II
Community acquired-MRSA
Pulse-field gel electrophoresis (PFGE) typed as USA 600
Contains staphylococcal chromosome cassette mec type II
Negative for the Panton-Valentine leucocidin (PVL) virulence
factor
MRSA
13

Community acquired-MRSA
Pulse-field gel electrophoresis (PFGE) typed not as USA100-1100
Contains staphylococcal chromosome cassette mec type II
Negative for the Panton-Valentine leucocidin (PVL) virulence
factor
NR 10192
Community acquired-MRSA
Pulse-field gel electrophoresis (PFGE) typed as USA100
Contains staphylococcal chromosome cassette mec type II
Negative for the Panton-Valentine leucocidin (PVL) virulence
factor
NR 10198
NR 100
Resistant to tetracycline
Positive for mec (subtype I)
Large variety of virulence factors
Also called as TCH60
G2576T mutation in domain V in one or more 23 S rRNA genes
Positive for mec (subtype IV)
Positive for 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)
NA*
NR 10129
NR 119
VRS1
VRSA
VRS4
VRS12
NA*- Not Available
7.1. Antibiotic susceptibility testing against ESKAP pathogen panel
Antibiotic susceptibility testing was performed on the newly synthesized compounds by
calculating the Minimum Inhibitory Concentration (MIC) according to standard CLSI guidelines
[24]. 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
(MHB) [26]. 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 64-0.03μg/mL. Controls i.e., cells
alone and media alone (without compound+cells) and drug levofloxacin used as a reference
standard in the whole experiment. 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 carried independently three times using duplicate samples each time.
7.2. Antibiotic susceptibility testing against pathogenic mycobacteria
Antimycobacterial susceptibility testing was performed on newly synthesized compounds (given
in Supplementary data), by using broth microdilution assay [27][28]. 10 mg/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 [29]
14

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. The newly synthesized compounds were tested from 64–0.5 mg/L in two-fold serial
diluted fashion with 2.5 μL of each concentration added per well of a 96-well round bottom
microtiter 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 newly
synthesized 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.
7.3. Cell Cytotoxicity Assay
The active newly synthesized compounds were screened for their cell toxicity against Vero cell
using the MTT assay [30]. ~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 an 5% CO2 atmosphere. After the incubation was over, 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 optical density
(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.
7.4. Time Kill Study
The bactericidal activity was assessed by the time-kill method [31]. S. aureus ATCC 29213 cells
were diluted up to ~105 CFU/ml and treated with compound for concentrations corresponding to
1X and 10X of MIC of 6’a and vancomycin in MHB in triplicate and incubated at 37 °C. 100 μL
samples were collected after time intervals of 0 h, 1 h, 6 h and 24 h and serially diluted in PBS
and plated on TSA followed by incubation at 37 °C for 18-20 h. Kill curves were constructed by
counting the colonies from plates and plotting the CFU/mL of surviving bacteria at each time
point in the presence and absence of compound.
7.5. Synergy screen
Checkerboard method was used to determine synergy between compound and the antibiotics
included in the study that included linezolid, meropenem, ceftriaxone and vancomycin against a
15

panel of MRSA and VRSA strains [27]. According to the recommendations of CLSI, serial two-
fold dilutions of each drug to at least double the MIC were freshly prepared prior to testing. The
compounds was serially diluted along the ordinate ranged from 0.03 to 4 µg/mL while the
antibiotics were serially diluted as shown along the abscissa ranged from 0.03 to 64 µg/mL /ml
in 96 well microtiter plate. An inoculum equal to 105 or 106 CFU/mL was prepared from each
MRSA and VRSA isolate in MHB. Each microtiter well was inoculated with 100 μL of a
bacterial inoculum of 105 or 106 CFU/mL, and plates were incubated at 37 °C for 24 hours under
aerobic conditions. According to the CLSI guidelines for broth microdilution, the MIC was
defined as the lowest concentration of antibiotic that completely inhibited the growth of the
organism as detected with the naked eye. The ΣFICs (fractional inhibitory concentrations) were
calculated as follows: ΣFIC = FIC A + FIC B, where FIC A is the MIC of drug A in the
combination/MIC of drug A alone, and FIC B is the MIC of drug B in the combination/MIC of
drug B alone. The combination is considered synergistic when the ΣFIC is ≤0.5, indifferent
when the ΣFIC is >0.5 to 4, and antagonistic when the ΣFIC is >4.
Acknowledgements
G. S. conveys warm regards to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, for the
award of NIPER fellowship. M.S. would like to thank UGC for her fellowship while G.K.
thanks DST for her fellowship. This study was supported by the DST grant from Department of
Science and Technology, Govt. of India to S.C. and S.N. (EMR/2017/000220). The following
reagents were provided by the Network on Antimicrobial Resistance in S. aureus (NARSA) for
distribution by BEI Resources, NIAID, NIH: NR 119, NR 10129, NR 10198, NR 10192, NR
10191, NR 10193, NR 10186, NR 10194, VRS1, VRS4 and VRS12.
Competing financial interests: The authors declare no competing financial interests.
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Table, Figures and Schemes
Table 1. MIC values (µg/mL) of the tested compounds against ESKAP panel of bacteria
Table 2. Cytotoxicity profile against Vero cells and SI
Table 3. MIC of 6’a against MRSA and VRSA strains
Table 4. Synergy screen data of compound 6’a
Figure 1. Structure of some literature reported bioactive quinazolinone compounds having anti-
bacterial and anti-mycobacterial activity
Figure 2: Newly designed quinazolinone derivatives
Figure 3. Structure Activity Relationship (SAR) of 4-oxoquinazolin-3(4H)-yl)benzoic acid and
benzamide derivatives
Figure 4. 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives selectively active
against Staphylococcus aureus
21

Figure 5. Time kill kinetics of compound 6’a
Scheme 1. Synthesis of 4-oxoquinazolin-3(4H)-yl)benzoic acid derivatives 4a-4g: Reagents and
conditions: (a) Ac2O, reflux, 2 h (b) R1-NH2, AcOH, reflux, 5 h (c) 4-R2-PhCHO, AcOH, reflux, 18-20 h,
83-68%.
Scheme 2. Synthesis of 4-oxoquinazolin-3(4H)-yl)benzamide derivatives 5’, 6’a-6’j: Reagents
and conditions: (a) Ac2O, reflux, 2 h (b) 3-amino benzoic acid, AcOH, reflux, 5 h (c) R1-NH2 or
R1R2NH, EDC.HCl, DMAP, DCM, rt., 12-14 h (d) 4-cyanobenzaldehyde, AcOH, reflux, 18 h (e) DMF-
DMA, DMF, reflux, 18-20 h, 50-70%.
Table 1. MIC values (µg/mL) of the tested compounds against ESKAP panel of bacteria.
S. aureus
ATCC
29213
8
E. coli
ATCC
25922
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
K.
A.
Compound
P. aeruginosa
ATCC 27853
pneumoniae baumannii
BAA-1705 BAA-1605
>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
>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
>64
>64
>64
>64
>64
0.5
4a
4b
4c
4d
4e
4f
4g
3’
4’
5’
6’a
6’b
6’c
6’d
6’e
6’f
2
>64
>64
>64
>64
>64
>64
>64
>64
0.25
0.5
32
>64
64
>64
64
>64
>64
>64
>64
>64
0.015
6’g
6’h
6’i
4
4
8
6’j
Levofloxacin
0.125
Table 2. Cytotoxicity profile against Vero cells and SI
Selectivity
Index
(CC50/MIC)
S. aureus
Compound
ATCC 29213
CC50
MIC (µg/mL) (µg/mL)
8
>10
>1.25
4a
22

2
0.25
0.5
4
4
8
>20
>10
>5
>10
>10
>10
>10
>40
>10
>2.5
>2.5
>1.25
4b
6’a
6’b
6’h
6’i
6’j
Table 3. MIC of 6’a against MRSA and VRSA strains
Strains
Antibiotics resistant
to
MIC (µg/mL)
Levofloxaci Meropenem Vancomycin Methicillin
n
6’a
S.
None
<0.125 <0.5
<0.5
1
1
aureus
ATCC
29213
NR 119 Methicillin,
Ceftriaxone,
0.125 16
>64
1
32 - >64
Meropenem,
Gentamycin
and
Linezolid
NR
10129
Methicillin,
Ceftriaxone,
Meropenem
<0.125
<0.5
16
1
1
1
1
1
1
32 - >64
32 - >64
32 - >64
32 - >64
32 - >64
32 - >64
NR 100 Methicillin,
Ceftriaxone,
0.25-
<0.5
0.5
>64
32
Meropenem
NR
10198
Methicillin,
Ceftriaxone,
Meropenem
Methicillin,
Ceftriaxone,
Meropenem
Methicillin,
Ceftriaxone,
Meropenem
Methicillin,
Ceftriaxone,
Meropenem
Methicillin,
Ceftriaxone
Methicillin,
Ceftriaxone,
Meropenem
Methicillin,
Ceftriaxone,
Meropenem,
Gentamycin,
<0.125
32
NR
10192
<0.125
4 - 8
4 - 8
16 - 32
32
NR
10186
<0.125
4 - 8
NR
10193
<0.125
32
NR
10194
NR
10191
<0.125
<0.5
<0.5
>64
1
32 - >64
32 - >64
>64
<0.125
16 - 32
32
1
VRS1
0.25-
0.5
>64
>64
23

Vancomycin,
Teicoplanin
Methicillin,
Ceftriaxone,
Meropenem,
Vancomycin
Teicoplanin
Methicillin,
Ceftriaxone,
Meropenem,
Vancomycin
Teicoplanin
VRS4
0.125 >64
>64
>64
>64
>64
32 - >64
32 - >64
and
and
VRS12
0.125 - 32 - >64
0.25
Table 4. Synergy screen data of compound 6’a
S.
MIC of MIC of
aureus
ATCC
6’a in
the
drug in
the
Drug
29213 presence presence FIC A
MIC of drug of 6’a
(µg/mL) (µg/mL) (µg/mL)
FIC B
∑FIC= Inference
FIC A+
FIC B
A
B
0.25
8
1
0.25
2
0.25
0.5
0.125
0.015
1
6’a
Ceftazidime
Daptomycin
Gentamycin
Linezolid
Levofloxacin
Meropenem
Minocycline
Rifampicin
Vancomycin
0.007813 0.03125 0.03125 0.00390625 0.035156 Synergistic
0.007813 0.00195 0.03125
0.007813 0.00195 0.03125
0.007813 0.0075 0.03125
0.007813 0.0009 0.03125
0.007813 0.0009 0.03125
0.007813 0.0009 0.03125
0.007813 0.00003 0.03125
0.007813 0.0039 0.03125
0.00195
0.0078
0.00375
0.0036
0.0018
0.0072
0.002
0.0332 Synergistic
0.03905 Synergistic
0.035
Synergistic
0.03485 Synergistic
0.03305 Synergistic
0.03845 Synergistic
0.03325 Synergistic
0.03515 Synergistic
0.0039
24

Figure 1. Structure of some literature reported bioactive quinazolinone compounds having anti-
bacterial and anti-mycobacterial activity.
Figure 2: Newly designed quinazolinone derivatives
Figure 3. Structure Activity Relationship (SAR) of 4-oxoquinazolin-3(4H)-yl)benzoic acid and
benzamide derivatives
25

Figure 4. 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives selectively active
against Staphylococcus aureus
Figure 5. Time kill kinetics of a compound 6’a
26

Scheme 1. Synthesis of 4-oxoquinazolin-3(4H)-yl)benzoic acid derivatives 4a-4g: Reagents and
conditions: (a) Ac2O, reflux, 2 h (b) R1-NH2, AcOH, reflux, 5 h (c) 4-R2-PhCHO, AcOH, reflux, 18-20 h,
83-68%.
Scheme 2. Synthesis of 4-oxoquinazolin-3(4H)-yl)benzamide derivatives 5’, 6’a-6’j: Reagents
and conditions: (a) Ac2O, reflux, 2 h (b) 3-amino benzoic acid, AcOH, reflux, 5 h (c) R1-NH2 or
R1R2NH, EDC.HCl, DMAP, DCM, rt., 12-14 h (d) 4-cyanobenzaldehyde, AcOH, reflux, 18 h (e) DMF-
DMA, DMF, reflux, 18-20 h, 50-70%.
27

Graphical Abstract
Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide
derivatives as potent antibacterial agents effective against multidrug resistant
Staphylococcus aureus
Srikanth Gatadi a, Jitendra Gour a, Manjulika Shukla b, Grace Kaul b, Swetarka das b, Arunava
Dasgupta b, Y.V. Madhavi a, Sidharth Chopra b,*, Srinivas Nanduri a,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
28

Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide
derivatives as potent antibacterial agents effective against multidrug resistant
Staphylococcus aureus
Srikanth Gatadi a, Jitendra Gour a, Manjulika Shukla b, Grace Kaul b, Swetarka das b, Arunava
Dasgupta b, Y.V. Madhavi a, Sidharth Chopra b,*, Srinivas Nanduri a,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
Highlights:
1. New 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives synthesised and studied for
their anti-bacterial activity.
2. Compounds 4a, 4b, 6’a, 6’b, 6’h, 6’i and 6’j exhibited selective and potent activity against
Staphylococcus aureus and were also found to be non toxic.
3. Compound 6’a displayed potent inhibitory activity against clinical MRSA and VRSA isolates.
4. Compound 6’a exhibited concentration dependent bactericidal activity and also synergistic
effect with the tested drugs.
5. Structure Activity Relationships are summarized.
29