Synthesis of 1,2,3-triazole linked 4(3H)-Quinazolinones as potent antibacterial agents against multidrug-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.ejmech.2018.08.070

Methicillin and vancomycin resistant Staphylococcus aureus infections are an emerging global health concern leading to increasing morbidity and mortality. Continuous increase in drug resistance has underlined the need for discovery and development of new antibacterial agents acting via novel mechanisms to overcome this pressing issue. In this context, a number of 1,2,3-triazole linked 4(3H)-quinazolinone derivatives were designed and synthesized as potent antibacterial agents. When evaluated against ESKAP pathogen panel, compounds 7a, 7b, 7c, 7e, 7f, 7g, 7h, 7i, 9a, 9c, 9d and 9e exhibited significantly selective inhibitory activities towards Staphylococcus aureus (MIC = 0.5–4 μg/mL). To understand and confirm the specificity of these compounds, the compounds 7a and 9a were tested against E. coli and A. baumannii in combination with sub-lethal concentrations of Polymyxin B nonapeptide (PMBN) and were found to be inactive. This clearly indicated that these compounds possess specific and potent activity towards S. aureus and are inactive against gram-negative pathogens. Encouragingly, the compounds were also found to be non toxic to Vero cells and displayed favourable selectivity index (SI = 40 to 80). Furthermore, 7a and 9a were found to possess potent inhibitory activity when tested against multidrug resistant S. aureus including strains resistant to vancomycin (MIC values 0.5–32 μg/mL), indicating that the compounds are able to escape current drug-resistance mechanisms. With the potent anti-bacterial activity exhibited the new series of 1,2,3-triazole linked 4(3H)-quinazolinones have emerged as promising candidates for treating multidrug resistant Staphylococcus aureus infections.

Full text of DOI:10.1016/j.ejmech.2018.08.070

Accepted Manuscript
Synthesis of 1,2,3-triazole linked 4(3H)-Quinazolinones as potent antibacterial agents
against multidrug-resistant Staphylococcus aureus
Srikanth Gatadi, Jitendra Gour, Manjulika Shukla, Grace Kaul, Swetarka Das,
Arunava Dasgupta, Satyaveni Malasala, Ramya Sri Borra, Y.V. Madhavi, Sidharth
Chopra, Srinivas Nanduri
PII:
S0223-5234(18)30742-6
10.1016/j.ejmech.2018.08.070
EJMECH 10681
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 May 2018
Revised Date: 13 August 2018
Accepted Date: 25 August 2018
Please cite this article as: S. Gatadi, J. Gour, M. Shukla, G. Kaul, S. Das, A. Dasgupta, S. Malasala,
R.S. Borra, Y.V. Madhavi, S. Chopra, S. Nanduri, Synthesis of 1,2,3-triazole linked 4(3H)-
Quinazolinones as potent antibacterial agents against multidrug-resistant Staphylococcus aureus,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.070.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis of 1,2,3-triazole linked 4(3H)-Quinazolinones as potent antibacterial
agents against multidrug-resistant Staphylococcus aureus
Srikanth Gatadi,a Jitendra Gour,a Manjulika Shukla,b Grace Kaul,b Swetarka Das,b Arunava
Dasgupta,b Satyaveni Malasala,a Ramya Sri Borra,a 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

ACCEPTED MANUSCRIPT
Synthesis of 1,2,3-triazole linked 4(3H)-Quinazolinones as potent antibacterial agents
against multidrug-resistant Staphylococcus aureus
Srikanth Gatadi,a Jitendra Gour,a Manjulika Shukla,b Grace Kaul,b Swetarka Das,b Arunava
Dasgupta,b Satyaveni Malasala,a Ramya Sri Borra,a 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
Abstract:- Methicillin and vancomycin resistant Staphylococcus aureus infections are an
emerging global health concern leading to increasing morbidity and mortality. Continuous
increase in drug resistance has underlined the need for discovery and development of new
antibacterial agents acting via novel mechanisms to overcome this pressing issue. In this context,
a number of 1,2,3-triazole linked 4(3H)-quinazolinone derivatives were designed and
synthesized as potent antibacterial agents. When evaluated against ESKAP pathogen panel,
compounds 7a, 7b, 7c, 7e, 7f, 7g, 7h, 7i, 9a, 9c, 9d and 9e exhibited significantly selective
inhibitory activities towards Staphylococcus aureus (MIC - 0.5–4 µg/mL). To understand and
confirm the specificity of these compounds, the compounds 7a and 9a were tested against E. coli
and A. baumannii in combination with sub-lethal concentrations of Polymyxin B nonapeptide
(PMBN) and were found to be inactive. This clearly indicated that these compounds possess
specific and potent activity towards S. aureus and are inactive against gram-negative pathogens.
Encouragingly, the compounds were also found to be non toxic to Vero cells and displayed
favourable selectivity index (SI = 40 to 80). Furthermore, 7a and 9a were found to possess
potent inhibitory activity when tested against multidrug-resistant S. aureus including strains
resistant to vancomycin (MIC values - 0.5–32 µg/mL), indicating that the compounds are able to
escape current drug-resistance mechanisms. With the potent anti-bacterial activity exhibited the
new series of 1,2,3-triazole linked 4(3H)-quinazolinones have emerged as promising candidates
for treating multidrug resistant Staphylococcus aureus infections.
Keywords: Methicillin-resistant Staphylococcus aureus, drug resistance, 1,2,3-triazole,
quinazolinones, Vero cells, cytotoxicity.
1

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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
1.0. Introduction
Continuous emergence of multidrug resistant Staphylococcus aureus infections has created a
critical and unmet medical need for the development of new classes of antibacterial agents for
their treatment [1]. β-lactam antibiotics have been the preferred choice for the treatment of S.
aureus infections for several years. However, these chemotherapeutics are found to be
inadequate due to the rise of methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant Staphylococcus aureus (VRSA). The multidrug resistant Staphylococcus
aureus strains have shown resistance to a number of antibiotics such as methicillin, oxacillin,
nafcillin and carbapenems. Of the drug resistant S. aureus strains VRSA have been the most
difficult bacterial strains to treat [2]. At present, vancomycin [3], daptomycin [4], orally active
linezolid [5] or ceftaroline and ceftobiprole [6] are used for treatment of MRSA infections.
However, resistance to all the available antibiotics has been on rise [7]. Hence, discovery of new
antibacterial agents to overcome the various resistance mechanisms is a crucial step to address
the multidrug resistant Staphylococcus aureus infections.
In the quest for new antibacterial agents, several heterocyclic scaffolds have been explored.
Among them, quinazolinones have emerged as a privileged structure [8] possessing a wide range
of biological properties viz., antibacterial, antifungal, anti-inflammatory anticonvulsant,
anticancer, anti-HIV, and analgesic activities [9–15]. Utilization of quinazolinone core for the
synthesis of new antibacterial agents has recently gained importance. Xioping et al. [16]
reported 2-methyl quinazolinones 1 with good antibacterial activity. Saravanan et al. [17] and
Suresh et al. [18] reported the antimicrobial properties of quinazolinone compounds 2 and 3
respectively. Jadhavar et al. [19] reported 2-styryl quinazolinones 4 as potent antimycobacterial
agents. Bouley et al. [20–21] reported 4(3H)-quinazolinones 5 and related compounds as potent
PBP1 and PBP2a inhibitors of MRSA with promising in vivo activity (Figure 1).
<Insert Figure 1 here>
Several 1,2,3˗triazole containing structures have been investigated as interesting therapeutic
agents against several targets possessing wide spectrum of biological properties [22] including
antibacterial activity. In addition to the interesting pharmacological properties, triazole moiety is
2

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known to have better metabolic stability and hydrogen bond forming ability. Interestingly, β-
lactam antibiotic cefatrizine 6 and the β-lactamase inhibitor tazobactam 7 [23] contain 1,2,3-
triazole as a structural feature [24] (Figure 2).
<Insert Figure 2 here>
We hypothesized that combining the structural features of quinazolinone and triazole moieties
could potentially have beneficial effect on the antibacterial activity. In the current study, we
synthesized a number of 1,2,3˗triazole linked 4(3H)˗quinazolinones 8, 9, 10 and 11 (Figure 3)
and examined them for their antibacterial properties.
<Insert Figure 3 here>
2.0 Results and Discussion
2.1 Chemistry
All the newly designed quinazolinones are synthesized as described in Schemes 1 & 2.
Substituted anthranilic acids 1 were converted into their corresponding benzoxazinone
intermediate 2 by refluxing in acetic anhydride. To the intermediate 2 in glacial acetic acid, 3 or
4-hydroxy aniline were added and the reaction mixture was refluxed for 5 h to give 3-(3 or 4-
hydroxyphenyl)-2-methyl quinazolinone 3 as key intermediates. Further, the intermediate 3 was
subjected to O-propargylation by using propargylbromide to afford 3-(3 or 4-O-propargyl
phenyl) quinazolinone derivatives 4. Treatment of the derivatives 4 with various substituted
azides under click conditions gave the corresponding triazole derivatives. Treatment of these
triazole derivatives with 4-cyano or 4-chlorobenzaldehyde in acetic acid under reflux condition
overnight afforded the compounds (Scheme 1) in moderate to excellent yields.
<Insert Scheme 1 here>
In the second scheme, to the intermediate 2 dissolved in glacial acetic acid, 3-nitro aniline 10
was added and the reaction mixture was refluxed for 5 h to give 2-methyl-3-(3-nitrophenyl)
quinazolinone as intermediate 11. To obtain the amine 12, the nitro group of the intermediate 11
was reduced in the presence of tin(II) chloride under reflux in ethanol. The amine 12 was
allowed to undergo azidation reaction by using sodium nitrite and sodium azide in dilute HCl to
afford azide intermediate 13. Finally, preparation of the designed compounds 15 and 19 were
accomplished by reacting the azide intermediate 13 with various substituted phenylacetylenes or
phenoxymethylacetylene by employing Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
3

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reaction. Finally, treatment of 15 and 19 with 4-chloro or 4-cyanobenzaldehyde in glacial acetic
acid yielded the compounds 17 and 21 respectively (Scheme 2).
<Insert Scheme 2 here>
13
Structures of all the newly synthesized compounds were confirmed by 1H NMR, C NMR and
HRMS (ESI) spectroscopic techniques.
2.2 In vitro antibacterial activity
2.2.1 Antibiotic susceptibility testing against ESKAP panel of bacteria.
All the newly synthesised 1,2,3-triazole linked 4(3H)-quinazolinones were evaluated for their
antibacterial activity against ESKAP pathogen panel. Minimum Inhibitory Concentration (MIC)
was determined by performing antibiotic susceptibility testing on the newly synthesized
compounds in accordance with the standard CLSI guidelines [25]. The concentration of the
newly synthesized compounds used is in the range of 64-0.03 µg/mL. Levofloxacin was used as
a reference compound. The results are given in Table 1. In order to determine their spectrum,
these compounds were also tested against M. tuberculosis H37Rv where only 6f and 6k
exhibited moderate activity (Supplementary data).
<Insert Table 1 here>
A perusal of the screening results indicated that the compounds 6a to 6n (Scheme 1) with
methyl group at C-2 position did not show any inhibitory activity against the ESKAP panel.
However, when the C-2 methyl group is converted into the corresponding 4-cyanostyryl moiety
as in 7a, 7b, 7c, 7e, 7f, 7g, 7h the compounds exhibited potent and selective activity against
Staphylococcus aureus (MIC - 0.5–4 µg/mL). Among these compounds, 7a, 7c and 7 g with 3-
chloro phenyl, 3-toluoyl and 3-fluoro phenyl at N-3 exhibited potent activity with MIC value of
0.5 µg/mL. However, compound 7e with 3-carboxy phenyl exhibited reduced potency (MIC - 4
µg/mL). Similarly, replacement of 4-cyanostyryl with 4-chlorostyryl moiety at C-2 as in 7i was
found to exhibit reduced activity (MIC - 4 µg/mL). Surprisingly, compound 7d with styryl
moiety at C-2 and methyl group at C-8 did not exhibit any inhibitory activity. Compounds with
substituted benzyl groups on the triazole moiety 9a (Bz), 9c (3-Br-Bz) exhibited selective and
potent activity against Staphylococcus aureus (MIC - 0.5 µg/mL). However compound 9b (4-Br-
Bz) resulted in 32 fold decrease in activity and the compound 9e (4-NO2-Bz) with electron
withdrawing group resulted in 4 fold reduction in activity against Staphylococcus aureus (MIC -
4

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2 µg/mL). Interestingly, compound 9d (4-OCH3-Bz) with electron donating methoxy group
restored potent activity against Staphylococcus aureus (MIC - 0.5 µg/mL). Compound 9'a with
ether link changed to C'4 position did not show activity. In our continued efforts, we introduced
4-phenyl-1H-1,2,3-triazole or 4-(phenoxymethyl)-1H-1,2,3-triazole directly at meta position of
3-N-phenyl of 2-methyl-quinazolin-4(3H)-one on ring B (compounds 15 & 19), but they did not
show antibacterial activity against S. aureus (MIC >64 µg/mL). Moreover, even after
replacement of methyl group at C-2 with 4-cyanostyryl moiety (compounds 17&21), they did
not show activity against S. aureus (MIC >64 µg/mL). These broad SAR observations are
summarised in Figure 4.
<Insert Figure 4 here>
To rule out the possibility of outer membrane permeability as a factor for inactivity of the
compounds against gram-negative pathogens, we determined the MIC of 7a and 9a in
combination with sub-lethal concentrations of Polymyxin B nonapeptide (PMBN) against E. coli
ATCC 25922 and A. baumannii BAA-1605 [26]. As can be seen in Table 2, 7a and 9a were not
active in the presence of 10 µg/mL PMBN, while appreciable difference could be seen for
Rifampicin, thus indicating the tested compound’s specificity towards S. aureus.
<Insert Table 2 here>
2.3 Cytotoxicity assay against Vero cells
The active compounds 7a, 7b, 7c and 9a were tested for their cytotoxicity against Vero cells
using the MTT assay. 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. The results indicated that the compounds 7a, 7b, 7c and 9a are non
toxic to vero cells (CC50 = 20–40 µg/mL) and exhibited selectivity index (SI) of 40 to 80, which
is an acceptable starting point for drug discovery program. The results are tabulated in Table 3.
<Insert Table 3 here>
2.4 Determination of MIC against MDR-S. aureus including VRSA
To determine their spectrum of activity against multiple strains of MDR-S. aureus, the MIC
assay was performed on compounds 7a and 9a against various well defined and characterized
clinical strains of MRSA and VRSA in accordance with standard CLSI guidelines [25]. The
5

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results are summarised in Table 4. Levofloxacin, meropenem and vancomycin were used as
reference standards. From examination of the results, it can be inferred that the compound 7a
and 9a exhibited potent antibacterial activity with MIC values in the range of 0.5˗2 µg/mL
against various clinical strains of MDR-S. aureus. The only exception is VRS1 (MIC - 32
µg/mL), which is also resistant to Meropenem, Vancomycin and a number of other β-lactam
antibiotics.
<Insert Table 4 here>
3.0. Conclusion
In conclusion, a series of newly designed 1,2,3-triazole linked 4(3H)-quinazolinone derivatives
have been synthesised and evaluated against ESKAP panel of bacteria. Compounds 7a, 7b, 7c,
7e, 7f, 7g, 7h, 7i, 9a, 9c, 9d and 9e displayed selective and potent antibacterial activity against
Staphylococcus aureus. The compounds 7a and 9a were found to be inactive against E. coli and
A. baumannii when tested in combination with sub-lethal concentrations of Polymyxin B
nonapeptide (PMBN), confirming their specificity for S. aureus and ruled out the outer
membrane permeability as a factor for the compound's inactivity against gram-negative
pathogens. Additionally, the compounds were found to be non toxic to Vero cells (CC50 = 20 to
40 µg/mL) with a good selectivity index (SI = 40 to 80). 7a and 9a also displayed potent
inhibitory activity against several clinical MRSA and VRSA isolates indicating that the
compounds are able to overcome the existing drug-resistance mechanisms. Taken together, these
compounds exhibit potential to be further developed as narrow spectrum anti-staphylococcal
leads with potential to evade drug resistance.
4.0. Materials & methods
All the chemicals, reagents and starting materials were procured from commercial providers and
were used as such. The monitoring of reactions were performed by TLC-MERCK pre-coated
1
silica gel 60-F254 (0.5 mm) aluminium plates under UV light. H and 13C NMR spectra were
obtained on Bruker Avance 500 MHz spectrometer using tetramethylsilane (TMS) as the internal
standard and chemical shifts are reported in ppm. Chemical shifts are referenced to TMS (δ 0.00
1
13
1
13
for H NMR and C NMR), CDCl3 (δ 7.26 for H NMR and 72.6 C NMR) or DMSO˗d6 (δ
1
2.50 for H NMR and 39.5 for 13C NMR). Spin multiplicities are reported as s (singlet), brs
(broad singlet), d (doublet), dd (double doublet), t (triplet) and m (multiplet). Coupling constant
6

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(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. Melting
points were taken using Stuart® SMP30 apparatus.
Intermediates 2, 3 and 4 (Scheme 1) were prepared according to the procedures described in
literature [20][24].
4.1. General reaction procedure for the synthesis of 2-methyl-3-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)quinazolin-4(3H)-one derivatives 6a-6n.
Compound (4, 2 mmol) (scheme 1) and azide (5 or 8, 2 mmol) were suspended in 10mL of a 1:1
tert-BuOH/H2O mixture. To the heterogeneous mixture, sodium ascorbate (10 mol%), copper
(II) sulfate pentahydrate (2 mol%) were added and the reaction mixture was allowed to stir for
12-14 h to give crude white or pale yellow precipitates, which were filtered, washed with water,
followed by hexane and finally purified by using column chromatography to obtain pure solid
compounds 6a-6n in 68-85% yields. All the newly synthesized compounds were characterized
by 1H NMR, 13C NMR and HRMS (ESI).
4.1.1.
3-(3-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-methylquinazolin-
1
4(3H)-one (6a): White solid, Yield 83%; mp: 190–192 °C; H NMR (500 MHz, DMSO–d6) δ
9.05 (s, 1H), 8.13 (dd, J = 7.9, 1.4 Hz, 1H), 8.07 (t, J = 2.0 Hz, 1H), 7.96–7.92 (m, 1H), 7.87–
7.83 (m, 1H), 7.69–7.63 (m, 2H), 7.59–7.54 (m, 1H), 7.55–7.49 (m, 2H), 7.27 (t, J = 2.0 Hz,
1H), 7.23 (dd, J = 8.3, 2.4 Hz, 1H), 7.07 (dd, J = 8.0, 0.7 Hz, 1H), 5.30 (m, 2H), 2.18 (s, 3H)
ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.76, 159.09, 154.87, 147.80, 144.08, 139.40,
138.05, 135.08, 134.69, 132.14, 130.91, 129.11, 127.12, 126.90, 126.78, 123.88, 121.46, 120.98,
120.46, 119.28, 116.20, 115.46, 61.53, 24.31 ppm; HRMS (ESI): m/z calcd for [M+H]+
C24H19ClN5O2 444.1227; found 444.1236.
4.1.2.
3-(3-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-methylquinazolin-
1
4(3H)-one (6b): Yellow solid, Yield 84%; mp: 187–189 °C; H NMR (500 MHz, DMSO–d6) δ
9.00 (s, 1H), 8.12 (m, 1H), 7.98–7.94 (m, 2H), 7.87–7.82 (m, 1H), 7.72–7.68 (m, 2H), 7.67 (m,
1H), 7.55–7.51 (m, 1H), 7.49 (m, 1H), 7.27–7.24 (m, 1H), 7.24–7.20 (m, 1H), 7.08–7.04 (m,
1H), 5.29 (m, 2H), 2.17 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.73, 159.16, 154.85,
147.82, 144.12, 139.42, 135.85, 135.06, 133.58, 130.88, 130.37, 127.12, 126.90, 126.78, 123.74,
7

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122.39, 121.47, 121.00, 116.17, 115.50, 61.63, 24.28 ppm; HRMS (ESI): m/z calcd for [M+H]+
C24H19ClN5O2 444.1227; found 444.1234.
4.1.3.
3-(3-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-
1
methylquinazolin-4(3H)-one (6c): White solid, Yield 78%; mp: 202–204 °C; H NMR (500
MHz, CDCl3) δ 8.29 (dd, J = 8.0, 1.3 Hz, 1H), 8.11 (s, 1H), 7.94 (dd, J = 6.2, 2.7 Hz, 1H), 7.81
–7.76 (m, 1H), 7.70 (m, 2H), 7.50 (m, 2H), 7.32 (t, J = 8.6 Hz, 1H), 7.16 (m, 1H), 6.96 (t, J = 2.2
Hz, 1H), 6.93 – 6.89 (m, 1H), 5.35 (m, 2H), 2.31 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6)
δ 161.76, 159.09, 156.44, 154.86, 147.80, 144.09, 139.40, 135.08, 134.01, 130.91, 127.11,
126.91, 126.78, 124.06, 123.01, 121.51, 121.47, 120.98, 118.75, 118.57, 116.17, 115.46, 61.53,
24.31 ppm; HRMS (ESI): m/z calcd for [M+H]+ C24H18ClFN5O2 462.1133; found 462.1136.
4.1.4. 2-methyl-3-(3-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)quinazolin-4(3H)-one
(6d): Off white solid, Yield 85%; mp: 178–180 °C; 1H NMR (500 MHz, DMSO–d6) δ 8.94 (s,
1H), 8.13 (dd, J = 7.9, 1.1 Hz, 1H), 7.88–7.84 (m, 1H), 7.75 (s, 1H), 7.69 (m, 2H), 7.56–7.46 (m,
3H), 7.32 (d, J = 7.6 Hz, 1H), 7.29–7.25 (m, 1H), 7.23 (m, 1H), 7.06 (m, 1H), 5.29 (m, 2H), 2.42
(s, 3H), 2.19(s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.63, 159.19, 155.18, 147.37,
143.86, 140.16, 139.28, 137.01, 135.15, 130.90, 130.18, 129.86, 127.01, 126.81, 123.57, 121.40,
121.10, 120.93, 117.77, 116.25, 115.47, 61.67, 24.16, 21.38 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C25H22N5O2 424.1773; found 424.1777.
4.1.5.
3-(3-((1-(3-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-methylquinazolin-
4(3H)-one (6e): white solid, Yield 76%; mp: 212–214 °C; 1H NMR (500 MHz, DMSO–d6) δ
10.04 (s, 1H), 8.89 (s, 1H), 8.12 (dd, J = 8.0, 1.3 Hz, 1H), 7.86–7.82 (m, 1H), 7.67 (m, 1H),
7.54–7.47 (m, 2H), 7.41–7.36 (m, 1H), 7.32–7.28 (m, 2H), 7.27–7.24 (m, 1H), 7.24–7.20 (m,
13
1H), 7.07–7.03 (m, 1H), 6.91–6.87 (m, 1H), 5.32–5.22 (m, 2H), 2.17 (s, 3H) ppm; C NMR
(125 MHz, DMSO–d6) δ 161.72, 159.18, 158.97, 154.85, 147.83, 143.80, 139.42, 138.02,
135.05, 131.25, 130.86, 127.12, 126.88, 126.78, 123.58, 121.43, 121.01, 116.23, 116.18, 115.48,
111.02, 107.62, 61.63, 24.28 ppm; HRMS (ESI): m/z calcd for [M+H]+ C24H20N5O3 426.1566;
found 426.1571.
4.1.6.
3-(3-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-methylquinazolin-
1
4(3H)-one (6f): White solid, Yield 78%; mp: 264–266 °C; H NMR (500 MHz, DMSO–d6) δ
9.97 (s, 1H), 8.79 (s, 1H), 8.12 (dd, J = 8.0, 1.3 Hz, 1H), 7.88–7.82 (m, 1H), 7.67 (m, 3H), 7.56–
7.47 (m, 2H), 7.26 (t, J = 2.0 Hz, 1H), 7.22 (dd, J = 8.3, 2.2 Hz, 1H), 7.06 (m, 1H), 6.97–6.92
(m, 2H), 5.26 (m, 2H), 2.17 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.74, 159.19,
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158.31, 154.88, 147.80, 143.52, 139.40, 135.07, 130.87, 129.16, 127.11, 126.90, 126.76, 123.56,
122.55, 121.37, 120.98, 116.53, 116.11, 115.42, 61.63, 24.30 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C24H20N5O3 426.1566; found 426.1570.
4.1.7.
3-(3-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-methylquinazolin-
4(3H)-one (6g): White solid, Yield 74%; mp: 164–166 °C; 1H NMR (500 MHz, DMSO–d6) δ
8.86 (s, 1H), 8.12 (dd, J = 8.0, 1.3 Hz, 1H), 7.87–7.78 (m, 3H), 7.67 (d, J = 8.2 Hz, 1H), 7.55–
7.47 (m, 2H), 7.26 (m, 1H), 7.24–7.19 (m, 1H), 7.15 (m, 2H), 7.08–7.03 (m, 1H), 5.27 (m, 2H),
13
3.84 (s, 3H), 2.17 (s, 3H) ppm; C NMR (125 MHz, DMSO–d6) δ 161.73, 159.86, 159.21,
154.86, 147.82, 143.69, 139.42, 135.05, 130.86, 130.47, 127.12, 126.89, 126.77, 123.60, 122.37,
121.41, 121.00, 116.16, 115.48, 115.40, 61.70, 56.07, 24.28 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C25H22N5O3 440.1722; found 440.1729.
4.1.8.
3-(4-((3-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzoic acid (6h): White solid, Yield 77%; mp: 233–235 °C; 1H NMR (500 MHz, DMSO–d6)
δ 13.43 (s, 1H), 9.10 (s, 1H), 8.44–8.40 (m, 1H), 8.21–8.16 (m, 1H), 8.12 (m, 1H), 8.05 (m, 1H),
7.87–7.81 (m, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.55–7.47 (m, 2H), 7.29–
7.25 (m, 1H), 7.23 (m, 1H), 7.06 (m, 1H), 5.30 (m, 2H), 2.18 (s, 3H) ppm; 13C NMR (125 MHz,
DMSO–d6) δ 166.76, 161.73, 159.16, 154.85, 147.82, 144.15, 139.42, 137.22, 135.05, 133.10,
130.90, 130.87, 129.80, 127.12, 126.89, 126.80, 124.77, 123.84, 121.47, 121.04, 121.01, 116.21,
115.52, 61.62, 24.29 ppm; HRMS (ESI): m/z calcd for [M+H]+ C25H20N5O4 454.1515; found
454.1520.
4.1.9.
4-(4-((3-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-
yl)benzoic acid (6i): White solid, Yield 69%; mp: 229–231 °C; 1H NMR (500 MHz, DMSO–d6)
δ 13.26 (s, 1H), 9.09 (s, 1H), 8.20–8.03 (m, 5H), 7.88–7.81 (m, 1H), 7.71–7.63 (m, 1H), 7.56 –
7.47 (m, 2H), 7.29–7.19 (m, 2H), 7.09–7.03 (m, 1H), 5.31 (m, 2H), 2.17 (s, 3H) ppm; 13C NMR
(125 MHz, DMSO–d6) δ 166.84, 161.73, 159.16, 154.85, 147.82, 144.30, 139.94, 139.43,
135.05, 131.56, 131.26, 130.88, 127.13, 126.89, 126.77, 123.76, 121.49, 121.00, 120.41, 116.17,
115.52, 61.62, 24.29 ppm; HRMS (ESI): m/z calcd for [M+H]+ C25H20N5O4 454.1515; found
454.1519.
4.1.10.
yl)methoxy)phenyl)quinazolin-4(3H)-one (6j): Off white solid, Yield 71%; mp: 200–202 °C; 1H
NMR (500 MHz, DMSO–d ) δ 9.11 (s, 1H), 8.54 (d, J = 2.2 Hz, 1H), 8.21 (dd, J = 8.4, 2.2 Hz,
2-methyl-3-(3-((1-(4-methyl-3-nitrophenyl)-1H-1,2,3-triazol-4-
6
1H), 8.11 (dd, J = 7.9, 1.2 Hz, 1H), 7.87–7.82 (m, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.1
9

ACCEPTED MANUSCRIPT
Hz, 1H), 7.55–7.48 (m, 2H), 7.27 (t, J = 2.1 Hz, 1H), 7.25–7.20 (m, 1H), 7.08–7.04 (m, 1H),
5.31 (m, 2H), 2.59 (s, 3H), 2.18 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.68, 159.14,
155.00, 149.80, 147.61, 144.28, 139.36, 135.52, 135.10, 134.83, 133.45, 130.90, 126.97, 126.94,
126.79, 124.82, 123.91, 121.49, 120.97, 116.30, 116.22, 115.51, 61.61, 24.24, 19.54 ppm;
HRMS (ESI): m/z calcd for [M+H]+ C25H21N6O4 469.1624; found 469.1629.
4.1.11.
2-methyl-3-(3-((1-(2-methyl-4-nitrophenyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)quinazolin-4(3H)-one (6k): Pale yellow solid, Yield 73%; mp: 198–200 °C;
1H NMR (500 MHz, DMSO–d6) δ 8.81 (s, 1H), 8.41 (d, J = 2.4 Hz, 1H), 8.26 (dd, J = 8.7, 2.5
Hz, 1H), 8.11 (dd, J = 7.9, 1.3 Hz, 1H), 7.86–7.82 (m, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.66 (d, J =
8.0 Hz, 1H), 7.54–7.49 (m, 2H), 7.25 (m, 2H), 7.06 (m, 1H), 5.32 (m, 2H), 2.34 (s, 3H), 2.18 (s,
3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.71, 159.21, 154.85, 148.09, 147.81, 143.43,
141.29, 139.44, 135.59, 135.05, 130.87, 127.75, 127.12, 126.95, 126.93, 126.89, 126.77, 122.70,
121.50, 120.99, 116.20, 115.48, 61.58, 24.29, 18.31 ppm; HRMS (ESI): m/z calcd for [M+H]+
C25H21N6O4 469.1624; found 469.1631.
4.1.12.
2-methyl-3-(3-((1-(3,4,5-trimethylphenyl)-1H-1,2,3-triazol-4-
1
yl)methoxy)phenyl)quinazolin-4(3H)-one (6l): White solid, Yield 77%; mp: 244–246 °C; H
NMR (500 MHz, DMSO–d6) δ 8.47 (s, 1H), 8.10 (dd, J = 7.9, 1.3 Hz, 1H), 7.86–7.81 (m, 1H),
7.68–7.63 (m, 1H), 7.54–7.47 (m, 2H), 7.25–7.21 (m, 2H), 7.09 (m, 2H), 7.05 (m, 1H), 5.30 (m,
2H), 2.33 (s, 3H), 2.16 (s, 3H), 1.86 (s, 6H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.67,
159.25, 154.85, 147.81, 142.96, 140.02, 139.40, 135.03, 134.94, 133.79, 130.81, 129.35, 127.15,
127.08, 126.87, 126.76, 121.44, 120.99, 116.29, 115.59, 61.84, 24.26, 21.09, 17.28 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C27H26N5O2 452.2086; found 452.2107.
4.1.13.
2-methyl-3-(3-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-
1
yl)methoxy)phenyl)quinazolin-4(3H)-one (6m): White solid, Yield 68%; mp: 184–186 °C; H
NMR (500 MHz, DMSO–d6) δ 8.99 (s, 1H), 8.11 (dd, J = 7.9, 1.3 Hz, 1H), 7.84 (m, 1H), 7.67
(d, J = 8.1 Hz, 1H), 7.51 (m, 2H), 7.27–7.20 (m, 4H), 7.08–7.04 (m, 1H), 5.30 (m, 2H), 3.88 (s,
6H), 3.72 (s, 3H), 2.17 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.72, 159.20, 154.85,
154.02, 147.82, 143.82, 139.43, 138.01, 135.05, 132.90, 130.88, 127.12, 126.88, 126.75, 123.77,
121.46, 120.97, 116.08, 115.51, 98.78, 61.74, 60.65, 56.81, 24.29 ppm; HRMS (ESI): m/z calcd
for [M+H]+ C27H26N5O5 500.1934; found 500.1940.
4.1.14.
3-(3-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,8-
1
dimethylquinazolin-4(3H)-one (6n): White solid, Yield 72%; mp: 223–225 °C; H NMR (500
10

ACCEPTED MANUSCRIPT
MHz, DMSO–d6) δ 9.03 (s, 1H), 8.05 (t, J = 1.9 Hz, 1H), 7.98–7.90 (m, 2H), 7.70 (m, 1H), 7.64
(t, J = 8.1 Hz, 1H), 7.60–7.55 (m, 1H), 7.50 (t, J = 8.1 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.26–
7.20 (m, 2H), 7.05 (m, 1H), 5.30 (s, 2H), 2.57 (s, 3H), 2.20 (s, 3H) ppm; 13C NMR (125 MHz,
DMSO–d6) δ 161.95, 159.03, 153.67, 146.24, 144.11, 139.53, 138.08, 135.36, 135.22, 134.70,
132.11, 130.86, 129.08, 126.36, 124.46, 123.83, 121.46, 120.83, 120.47, 119.27, 116.16, 115.52,
61.61, 24.64, 17.52 ppm; HRMS (ESI): m/z calcd for [M+H]+ C25H21ClN5O2 458.1384; found
458.1390.
4.2. General reaction procedure for the synthesis of (E)-4-(2-(4-oxo-3-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile derivatives 7a-7i, 9a-9e.
Compound (6 or 6’, 1 mmol) was dissolved in glacial acetic acid, to which 4-chloro or 4-
cyanobenzaldehyde (1 mmol) was added and the homogeneous mixture was allowed to reflux
overnight (16-18 h). The reaction was monitored by using TLC. After completion of reaction,
the reaction mixture was allowed to cool to room temperature and then 10mL water was added
to give crude precipitates, which were filtered under vacuum, washed with water, methanol and
finally washed with hexane to afford a pure yellow to pale yellow crystal products 7a-7i, 9a-9e
1
13
in 50–78% yields . All the newly synthesized compounds were characterized by H NMR, C
NMR and HRMS (ESI).
4.2.1.
(E)-4-(2-(3-(3-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (7a): Yellow solid, Yield 78%; mp: 274–276 °C; H
NMR (500 MHz, DMSO–d6) δ 9.00 (s, 1H), 8.19 (dd, J = 7.9, 1.2 Hz, 1H), 8.14 (dd, J = 6.4, 2.7
Hz, 1H), 7.94–7.87 (m, 3H), 7.82 (m, 1H), 7.76 (m, 2H), 7.73–7.68 (m, 1H), 7.65 (t, J = 9.0 Hz,
1H), 7.62–7.57 (m, 1H), 7.56–7.52 (m, 1H), 7.50 (m, 2H), 7.33–7.30 (m, 1H), 7.29–7.25 (m,
1H), 7.09–7.06 (m, 1H), 6.49 (d, J = 15.6 Hz, 1H), 5.33 (s, 2H) ppm; 13C NMR (125 MHz,
DMSO–d6) δ 161.61, 159.03, 151.30, 147.68, 144.00, 139.74, 138.29, 137.25, 135.72, 135.37,
133.52, 133.26, 131.07, 130.37, 130.30, 128.53, 127.79, 127.52, 126.98, 123.78, 123.65, 122.37,
122.16, 121.95, 121.27, 119.03, 116.84, 115.82, 111.99, 61.48 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C32H22ClN6O2 557.1493; found 557.1498.
4.2.2.
(E)-4-(2-(3-(3-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
dihydroquinazolin-2-yl)vinyl)benzonitrile (7b): Yellow solid, Yield 76%; mp: 278–280 °C; 1H
NMR (500 MHz, DMSO–d6) δ 8.99 (s, 1H), 8.17 (dd, J = 7.9, 1.1 Hz, 1H), 7.94–7.87 (m, 4H),
7.81 (d, J = 8.0 Hz, 1H), 7.77 (m, 2H), 7.67–7.62 (m, 2H), 7.61–7.56 (m, 1H), 7.54 (m, 3H),
7.32 (t, J = 2.0 Hz, 1H), 7.28 (dd, J = 8.4, 2.1 Hz, 1H), 7.08 (m, 1H), 6.51 (d, J = 15.6 Hz, 1H),
11

ACCEPTED MANUSCRIPT
5.32 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.61, 159.03, 151.30, 147.68, 144.00,
139.74, 138.29, 137.25, 135.72, 135.37, 133.52, 133.26, 131.07, 130.30, 128.53, 127.79, 127.52,
126.98, 123.78, 123.65, 122.16, 121.95, 121.27, 119.03, 116.84, 115.82, 111.99, 61.48 ppm;
HRMS (ESI): m/z calcd for [M+H]+ C32H22ClN6O2 557.1493; found 557.1499.
4.2.3.
(E)-4-(2-(4-oxo-3-(3-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (7c): Yellow solid, Yield 65%; mp: 264–266 °C; H
NMR (500 MHz, CDCl3) δ 8.31 (d, J = 7.8 Hz, 1H), 8.10 (s, 1H), 8.00 (m, 1H), 7.89–7.79 (m,
2H), 7.60–7.45 (m, 7H), 7.44–7.35 (m, 3H), 7.31–7.19 (m, 1H), 7.05 (s, 1H), 6.94 (m, 1H), 6.51
(d, J = 15.2 Hz, 1H), 5.35 (s, 2H), 2.45 (s, 3H) ppm; 13C NMR (125 MHz, CDCl3) δ 161.31,
159.36, 151.82, 150.56, 149.73, 143.83, 140.13, 139.09, 139.06, 137.11, 136.77, 135.34, 132.58,
131.14, 129.81, 129.61, 128.55, 127.74, 127.39, 126.37, 121.53, 121.37, 121.20, 120.60, 118.37,
117.55, 116.95, 115.49, 113.12, 62.23, 21.44 ppm; HRMS (ESI): m/z calcd for [M+H]+
C33H25N6O2 537.2039; found 537.2049.
4.2.4.
(E)-4-(2-(3-(3-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-8-methyl-4-
oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (7d): Yellow solid, Yield 66%; mp: 271– 273
°C; 1H NMR (500 MHz, DMSO–d6) δ 9.03 (s, 1H), 8.01 (dd, J = 7.8, 0.6 Hz, 1H), δ 7.97 (t, J =
2.0 Hz, 1H), 7.94 (d, J = 15.5 Hz, 1H). 7.88–7.84 (m, 1H), 7.76 (m, 3H), 7.60 (t, J = 8.0 Hz,
1H), 7.57–7.52 (m, 2H), 7.52–7.48 (m, 2H), 7.46 (t, J = 7.6 Hz, 1H), 7.32–7.29 (m, 1H), 7.29–
7.26 (m, 1H), 7.06 (m, 1H), 6.50 (d, J = 15.6 Hz, 1H), 5.32 (s, 2H), 2.71 (s, 3H) ppm; 13C NMR
(125 MHz, TFA-d) δ 162.81, 161.03, 159.84, 149.67, 142.75, 142.30, 139.42, 139.29, 137.63,
137.12, 136.50, 135.44, 135.06, 134.59, 133.68, 133.19, 131.51, 131.29, 129.09, 127.49, 124.15,
123.85, 121.45, 120.72, 119.64, 118.19, 117.55, 117.36, 116.33, 115.11, 61.20 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C33H24ClN6O2 571.1649; found 571.1654.
4.2.5.
(E)-3-(4-((3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)phenoxy)methyl)-1H-1,2,3-
triazol-1-yl)benzoic acid (7e): Yellow solid, Yield 59%; mp: 281– 283 °C; 1H NMR (500 MHz,
DMSO–d6) δ 13.49 (s, 1H), 9.16 (s, 1H), 8.42–8.40 (m, 1H), 8.26–8.20 (m, 1H), 8.19–8.14 (m,
1H), 8.10–8.06 (m, 1H), 8.00–7.93 (m, 2H), 7.89–8.84 (m, 1H), 7.83–7.74 (m, 3H), 7.68–7.51
(m, 4H), 7.43–7.37 (m, 1H), 7.37–7.31 (m, 1H), 7.16–7.09 (m, 1H), 6.56 (d, J = 15.6 Hz, 1H),
13
5.38 (s, 2H) ppm; C NMR (125 MHz, DMSO–d6) δ 166.72, 161.58, 159.06, 151.31, 147.71,
144.03, 139.75, 138.31, 137.25, 137.12, 135.33, 133.23, 133.09, 131.04, 130.79, 129.72, 128.51,
127.80, 127.48, 127.00, 124.51, 123.82, 123.74, 121.96, 121.31, 120.85, 118.99, 116.93, 115.87,
112.00, 61.54 ppm; HRMS (ESI): m/z calcd for [M+H]+ C33H23N6O4 567.1781; found 567.1783.
12

ACCEPTED MANUSCRIPT
4.2.6. (E)-4-(2-(3-(3-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (7f): Yellow solid, Yield 64%; mp: 231–233 °C; H
NMR (500 MHz, DMSO–d6) δ 8.84 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 7.95–7.86 (m, 2H), 7.85–
7.69 (m, 5H), 7.62–7.47 (m, 4H), 7.35–7.23 (m, 2H), 7.14–7.02 (m, 3H), 6.51 (d, J = 15.6 Hz,
1H), 5.30 (s, 2H), 3.83 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.58, 159.80, 159.14,
151.32, 147.71, 143.62, 139.79, 138.32, 137.27, 135.33, 133.27, 131.02, 130.40, 128.55, 127.80,
127.48, 126.98, 123.84, 123.46, 122.18, 121.92, 121.31, 119.03, 116.81, 115.91, 115.33, 112.00,
61.66, 56.04 ppm; HRMS (ESI): m/z calcd for [M+H]+ C33H25N6O3 553.1988; found 553.1991.
4.2.7.
(E)-4-(2-(3-(3-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (7g): Yellow solid, Yield 52%; mp: 260–262 °C; H
NMR (500 MHz, DMSO–d6) δ 9.02 (s, 1H), 8.20–8.12 (m, 1H), 7.96–7.88 (m, 2H), 7.87–7.72
(m, 5H), 7.67–7.49 (m, 5H), 7.41–7.31 (m, 2H), 7.30–7.26 (m, 1H), 7.11–7.05 (m, 1H), 6.51 (d,
J = 15.7 Hz, 1H), 5.33 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.60, 159.03, 151.31,
147.71, 144.02, 139.78, 138.31, 138.13, 137.25, 135.34, 133.26, 132.32, 132.24, 131.05, 128.54,
127.81, 126.96, 123.83, 123.76, 121.99, 121.31, 119.78, 119.01, 116.91, 116.40, 115.90, 112.00,
108.12, 107.91, 61.54 ppm; HRMS (ESI): m/z calcd for [M+H]+ C32H22FN6O2 541.1788; found
541.1792.
4.2.8.
(E)-4-(2-(4-oxo-3-(3-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (7h): Yellow solid, Yield
70%; mp: 288–290 °C; 1H NMR (500 MHz, DMSO–d6) δ 9.12 (s, 1H), 8.17 (dd, J = 7.9, 1.2 Hz,
1H), 8.13–8.08 (m, 2H), 7.99–7.94 (m, 2H), 7.94–7.88 (m, 2H), 7.83–7.79 (m, 1H), 7.77–7.72
(m, 2H), 7.62–7.52 (m, 2H), 7.52–7.48 (m, 2H), 7.35–7.27 (m, 2H), 7.11–7.06 (m, 1H), 6.50 (d,
J = 15.6 Hz, 1H), 5.34 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.59, 159.04, 151.30,
147.70, 144.26, 139.76, 138.32, 137.24, 135.34, 133.24, 131.05, 129.24 (d, J=32.6 Hz) 128.51,
127.80, 127.63 (d, J=3.8 Hz), 127.49, 126.98, 125.34, 123.80, 123.17, 122.01, 121.30, 121.16,
120.94, 118.97, 116.91, 115.86, 111.99, 61.52 ppm; HRMS (ESI): m/z calcd for [M+H]+
C33H22F3N6O2 591.1756; found 591.1753.
4.2.9.
(E)-2-(4-chlorostyryl)-3-(3-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-
1
yl)methoxy)phenyl)quinazolin-4(3H)-one (7i): Yellow solid, Yield 50%; mp: 218–220 °C; H
NMR (500 MHz, DMSO–d6) δ 9.02 (s, 1H), 8.16 (dd, J = 7.9, 1.1 Hz, 1H), 7.92–7.84 (m, 2H),
13

ACCEPTED MANUSCRIPT
7.84–7.74 (m, 3H), 7.69–7.60 (m, 1H), 7.59–5.51 (m, 2H), 7.40–7.34 (m, 4H), 7.33–7.20 (m,
3H), 7.09–7.03 (m, 1H), 6.38 (d, J = 15.6 Hz, 1H), 5.33 (s, 2H) ppm; 13C NMR (125 MHz,
DMSO–d6) δ 163.84, 161.89, 161.67, 159.04, 151.63, 147.83, 144.03, 138.46, 138.22, 138.14,
137.90, 135.28, 134.68, 134.22, 132.27(d, J=9.0Hz) 131.02, 129.53(d, J=9.9Hz), 127.69,
127.19, 126.92, 123.76, 121.99, 121.16(d, J=4.1Hz),, 116.80, 116.44, 115.93, 108.13, 107.92,
61.56 ppm; HRMS (ESI): m/z calcd for [M+H]+ C31H22FClN5O2 550.1446; found 550.1444.
4.2.10.
(E)-4-(2-(3-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (9a): Yellow solid, Yield 62%; mp: 254–256 °C; H
NMR (500 MHz, DMSO–d6) δ 8.31 (s, 1H), 8.16 (dd, J = 7.9, 1.1 Hz, 1H), 7.95–7.87 (m, 2H),
7.81 (m, 3H), 7.57 (m, 2H), 7.54–7.49 (m, 1H), 7.39–7.29 (m, 6H), 7.26–7.23 (m, 2H), 7.05 (m,
1H), 6.53 (d, J = 15.6 Hz, 1H), 5.58 (s, 2H), 5.19 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6)
δ 161.55, 159.32, 151.34, 147.67, 143.07, 139.84, 138.30, 137.26, 136.40, 135.35, 133.35,
130.96, 129.24, 128.65, 128.64, 128.43, 127.79, 127.48, 126.98, 125.37, 123.89, 121.83, 121.26,
119.09, 116.41, 115.97, 112.01, 61.79, 53.32 ppm; HRMS (ESI): m/z calcd for [M+H]+
C33H25N6O2 537.2039; found 537.2046.
4.2.11.
(E)-4-(2-(3-(3-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (9b): Yellow solid, Yield 64%; mp: 264–266 °C; H
NMR (500 MHz, DMSO–d6) δ 8.32 (s, 1H), 8.16 (dd, J = 7.9, 1.1 Hz, 1H), 7.94–7.88 (m, 2H),
7.83–7.78 (m, 3H), 7.60–7.54 (m, 5H), 7.52 (m, 1H), 7.29–7.23 (m, 4H), 7.06 (m, 1H), 6.53 (d, J
= 15.6 Hz, 1H), 5.58 (s, 2H), 5.20 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.55,
159.31, 151.33, 147.67, 143.14, 139.83, 138.30, 137.27, 135.79, 135.34, 133.34, 132.16, 130.96,
130.66, 128.65, 127.79, 127.48, 126.97, 125.42, 123.89, 121.91, 121.85, 121.26, 119.08, 116.44,
115.96, 112.02, 61.79, 52.57 ppm; HRMS (ESI): m/z calcd for [M+H]+ C33H24BrN6O2
615.1144; found 615.1144.
4.2.12.
(E)-4-(2-(3-(3-((1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (9c): Yellow solid, Yield 68%; mp: 259–261 °C; H
NMR (500 MHz, DMSO–d6) δ 8.35 (s, 1H), 8.20–8.14 (m, 1H), 7.97–7.87 (m, 2H), 7.85–7.78
(m, 3H), 7.63–7.49 (m, 6H), 7.38–7.28 (m, 2H), 7.27–7.22 (m, 2H), 7.09–7.03 (m, 1H), 6.54 (d,
13
J = 15.6 Hz, 1H), 5.61 (s, 2H), 5.21 (s, 2H) ppm; C NMR (125 MHz, DMSO–d6) δ 161.53,
159.34, 151.34, 147.69, 143.20, 139.87, 139.01, 138.32, 137.28, 135.32, 133.33, 131.53, 131.44,
131.21, 130.94, 128.65, 127.79, 127.54, 127.46, 126.98, 125.50, 123.94, 122.32, 121.89, 121.29,
14

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119.06, 116.43, 116.08, 112.04, 61.87, 52.50 ppm; HRMS (ESI): m/z calcd for [M+H]+
C33H24BrN6O2 615.1144; found 615.1147.
4.2.13. (E)-4-(2-(3-(3-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (9d): Yellow solid, Yield 71%; mp: 200–202 °C; H
NMR (500 MHz, DMSO–d6) δ 8.36 (s, 1H), 8.23–8.19 (m, 1H), 8.01–7.91 (m, 2H), 7.89–7.82
(m, 3H), 7.66–7.60 (m, 3H), 7.59–7.53 (m, 1H), 7.36–7.27 (m, 3H), 7.13–7.07 (m, 1H), 6.98–
6.92 (m, 2H), 6.92–6.87 (m, 1H), 6.59 (d, J = 15.6 Hz, 1H), 5.60 (s, 2H), 5.25 (s, 2H), 3.78 (s,
13
3H) ppm; C NMR (125 MHz, DMSO–d6) δ 161.53, 159.95, 159.35, 151.33, 147.69, 143.09,
139.86, 138.31, 137.81, 137.28, 135.30, 133.32, 130.94, 130.39, 128.64, 127.79, 127.45, 126.98,
125.35, 123.92, 121.86, 121.29, 120.48, 119.06, 116.43, 116.06, 114.23, 114.00, 112.03, 61.88,
55.58, 53.27 ppm; HRMS (ESI): m/z calcd for [M+H]+ C34H27N6O3 567.2144; found 567.2133.
4.2.14.
(E)-4-(2-(3-(3-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (9e): Yellow solid, Yield 66%; mp: 293–295 °C; H
NMR (500 MHz, DMSO–d6) δ 8.39 (s, 1H), 8.26–8.21 (m, 2H), 8.16 (dd, J = 7.9, 1.0 Hz, 1H),
7.95–7.88 (m, 2H), 7.84–7.78 (m, 3H), 7.62–7.48 (m, 6H), 7.28–7.22 (m, 2H), 7.09–7.04 (m,
1H), 6.53 (d, J = 15.6 Hz, 1H), 5.79 (s, 2H), 5.23 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6)
δ 161.54, 159.31, 151.34, 147.74, 147.69, 143.78, 143.30, 139.86, 138.33, 137.27, 135.33,
133.33, 130.96, 129.48, 128.64, 127.80, 127.47, 126.96, 125.74, 124.37, 123.93, 121.91, 121.29,
119.05, 116.53, 116.04, 112.04, 61.87, 52.43 ppm; HRMS (ESI): m/z calcd for [M+H]+
C33H24N7O4 582.1890; found 582.1898.
4.2.15.
(E)-4-(2-(3-(4-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-oxo-3,4-
dihydroquinazolin-2-yl)vinyl)benzonitrile (9’a): Compound 9’a was synthesized in a similar way
as procedure described in sec. 4.1 & 4.2. Yellow solid, Yield 60%; mp: 270–272 °C; 1H NMR
(500 MHz, DMSO–d6) δ 8.37 (s, 1H), 8.15 (dd, J = 7.8, 1.1 Hz, 1H), 7.93–7.87 (m, 2H), 7.84–
7.77 (m, 3H), 7.61–7.54 (m, 5H), 7.42–7.37 (m, 2H), 7.32–7.27 (m, 2H), 7.26–7.21 (m, 2H),
6.54 (d, J = 15.6 Hz, 1H), 5.64 (s, 2H), 5.25 (s, 2H) ppm; 13C NMR (125 MHz, DMSO–d6) δ
161.85, 158.72, 151.80, 147.68, 143.25, 139.85, 137.13, 135.88, 135.27, 133.36, 132.19, 130.67,
130.53, 129.97, 128.63, 127.77, 127.41, 126.99, 125.45, 124.06, 121.93, 121.27, 119.10, 116.10,
111.99, 61.84, 52.60 ppm; HRMS (ESI): m/z calcd for [M+H]+ C33H24BrN6O2 615.1144; found
615.1141.
15

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Intermediates 11, 12 and 13 (scheme 2) were synthesized in accordance with the procedures
described in literature [20][24].
4.3. General reaction procedure for the synthesis of 2-methyl-3-1H-1,2,3-triazol-1-
yl)phenyl)quinazolin-4(3H)-one derivatives 15a, 19a-19b.
3-(3-azidophenyl)-2-methylquinazolin-4(3H)-one (13, 2 mmol) (scheme 2) and acetylene (14 or
18, 2 mmol) were mixed in a 10mL volume of 1:1 tert-BuOH/H2O mixture. To the
heterogeneous mixture, sodium ascorbate (10 mol%), followed by copper(II)sulfate pentahydrate
(2 mol%) were added and the reaction mixture was allowed to stir for 12-14 h to give crude
white precipitates, which were filtered, washed with water, followed by hexane and finally
purified by using column chromatography to obtain as pure white solid compounds 15a, 19a-19b
1
13
in 84-90% yields. All the newly synthesized compounds were characterized by H NMR, C
NMR and HRMS (ESI).
4.3.1. 2-methyl-3-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)quinazolin-4(3H)-one (15a): White
solid, Yield 86%; mp: 166–168 °C; 1H NMR (500 MHz, CDCl3) δ 8.27 (dd, J = 8.0, 1.3 Hz, 1H),
8.25 (s, 1H), 7.98 (m, 1H), 7.91–7.88 (m 2H), 7.83–7.78 (m, 2H), 7.77–7.70 (m, 2H), 7.52 –
7.43 (m, 3H), 7.40–7.35 (m, 2H), 2.34 (s, 3H) ppm; 13C NMR (125 MHz, CDCl3) δ 162.11,
153.58, 148.76, 147.06, 139.06, 138.39, 135.01, 131.44, 129.89, 128.98, 128.64, 128.26, 127.08,
127.00, 126.80, 125.89, 121.04, 120.43, 120.36, 117.55, 24.32 ppm; HRMS (ESI): m/z calcd for
[M+H]+ C23H18N5O 380.1511; found 380.1516.
4.3.2.
2-methyl-3-(3-(4-((4-(tert-pentyl)phenoxy)methyl)-1H-1,2,3-triazol-1-
yl)phenyl)quinazolin-4(3H)-one (19a): White solid, Yield 84%; mp: 196–198 °C; 1H NMR (500
MHz, DMSO–d6) δ 8.98 (s, 1H), 8.19–8.08 (m, 3H), 7.90–7.85 (m, 1H), 7.82 (t, J = 8.0 Hz, 1H),
7.70 (d, J = 8.0 Hz, 1H), 7.65–7.62 (m, 1H), 7.57–7.52 (m, 1H), 7.25 (m, 2H), 6.99 (m, 2H),
5.22 (s, 2H), 2.22 (s, 3H), 1.58 (q, J = 7.4 Hz, 2H), 1.21 (s, 6H), 0.61 (t, J = 7.4 Hz, 3H) ppm;
13C NMR (125 MHz, DMSO–d6) δ 161.82, 156.16, 154.55, 147.79, 144.88, 141.80, 139.58,
137.91, 135.22, 131.59, 129.31, 127.26, 127.19, 127.03, 126.78, 123.25, 121.00, 120.96, 120.86,
114.61, 61.48, 37.40, 36.71, 28.88, 24.50, 9.52 ppm; HRMS (ESI): m/z calcd for [M+H]+
C29H30N5O2 480.2399; found 480.2402.
4.3.3. 3-(3-(4-((2,5-dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-methylquinazolin-
4(3H)-one(19b): White solid, Yield 90%; mp: 194–196 °C; 1H NMR (500 MHz, DMSO–d6) δ
8.96 (s, 1H), 8.16–8.11 (m, 3H), 7.90–7.85 (m, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.70 (m, 1H), 7.64
16

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(m, 1H), 7.57–7.52 (m, 1H), 7.03–7.00 (m, 1H), 6.99 (m,1H), 6.69 (m, 1H), 5.23 (s, 2H), 2.28 (s,
3H), 2.22 (s, 3H), 2.10 (s, 3H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.83, 156.46, 154.57,
147.78, 145.11, 139.58, 137.91, 136.66, 135.25, 131.60, 130.73, 129.32, 127.19, 127.05, 126.79,
123.28, 123.04, 121.70, 121.03, 120.94, 120.86, 113.19, 61.76, 24.50, 21.52, 16.16 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C26H24N5O2 438.1930; found 438.1936.
4.4. General reaction procedure for the synthesis of (E)-4-(2-(4-oxo-3-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile derivatives 17a-17b, 21a.
Compound (15 or 19, 1 mmol) was dissolved in glacial acetic acid, to which 4-cyano or 4-
chlorobenzaldehyde (1 mmol) was added and the reaction mixture was allowed to reflux for 16-
18 h (Scheme 2). The reaction was monitored by TLC. After completion of reaction, water was
added to afford crude precipitates, which were filtered, washed with water, followed by cold
methanol and hexane to afford a pure yellow to pale yellow products of (E)-3-(3-(4-phenyl-1H-
1,2,3-triazol-1-yl)phenyl)-2-styrylquinazolin-4(3H)-one derivatives 17a-17b or (E)-3-(3-(4-
(phenoxymethyl)-1H-1,2,3-triazol-1-yl)phenyl)-2-styrylquinazolin-4(3H)-one derivatives 21a in
71-83 % yields All the newly synthesized compounds were characterized by 1H NMR, 13C NMR
and HRMS (ESI).
4.4.1 (E)-2-(4-chlorostyryl)-3-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)quinazolin-4(3H)-one
(17a): Yellow solid, Yield 81%; mp: 246–248 °C; 1H NMR (500 MHz, DMSO–d6) δ 9.39 (s,
1H), 8.24–8.16 (m, 3H), 7.96–7.90 (m, 4H), 7.87 (t, J = 8.0 Hz, 1H), 7.83–7.80 (m, 1H), 7.64
(ddd, J = 7.9, 1.8, 0.9 Hz, 1H), 7.61–7.55 (m, 1H), 7.54–7.46 (m, 4H), 7.44–7.36 (m, 3H), 6.50
(d, J = 15.5 Hz, 1H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.77, 154.33, 151.51, 147.97,
147.82, 139.36, 138.68, 138.36, 138.03, 135.46, 134.79, 134.22, 131.71, 130.58, 129.90, 129.63,
129.51, 128.82, 127.78, 127.35, 126.96, 125.83, 121.14, 121.05, 120.95, 120.19 ppm; HRMS
(ESI): m/z calcd for [M+H]+ C30H21ClN5O 502.1434; found 502.1438.
4.4.2
(E)-4-(2-(4-oxo-3-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (17b): Yellow solid, Yield 83%; mp: 255–257 °C;. H NMR (500 MHz,
DMSO–d6) δ 9.40 (s, 1H), 8.26–8.16 (m, 3H), 7.99–7.91 (m, 4H), 7.90–7.82 (m, 2H), 7.80 (d, J
= 8.3 Hz, 2H), 7.68–7.63 (m, 3H), 7.61 (t, J = 7.5 Hz, 1H), 7.51 (m, 2H), 7.40 (t, J = 7.4 Hz,
1H), 6.65 (d, J = 15.5 Hz, 1H) ppm; 13C NMR (125 MHz, DMSO–d6) δ 161.71, 151.21, 147.96,
147.69, 139.75, 138.52, 138.01, 137.70, 135.53, 133.28, 131.73, 130.55, 129.62, 129.53, 128.87,
128.84, 127.89, 127.64, 127.00, 125.81, 123.86, 121.17, 120.98, 120.18, 119.06, 112.08 ppm;
HRMS (ESI): m/z calcd for [M+H]+ C31H21N6O 493.1777; found 493.1782.
17

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4.4.3
(E)-4-(2-(4-oxo-3-(3-(4-((4-(tert-pentyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-
3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (21a): Yellow solid, Yield 71%; mp: 280–282 °C;
1H NMR (500 MHz, DMSO–d6) δ 8.99 (s, 1H), 8.18 (m, 3H), 8.02–7.87 (m, 2H), 7.86–7.76 (m,
4H), 7.66–7.56 (m, 4H), 7.24 (m, 2H), 6.98 (m, 2H), 6.62 (d, J = 15.5 Hz, 1H), 5.22 (s, 2H),
1.57 (q, 7.4 Hz, 2H), 1.20 (s, 6H), 0.61 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (125 MHz, DMSO–
d6) δ 161.68, 156.17, 151.22, 147.70, 144.93, 141.84, 139.77, 138.51, 137.95, 137.66, 135.49,
133.26, 131.66, 129.66, 128.86, 127.87, 127.60, 127.24, 126.99, 123.90, 123.19, 121.30, 121.20,
121.11, 119.05, 114.65, 112.08, 61.51, 37.41, 36.70, 28.88, 9.50 ppm; HRMS (ESI): m/z calcd
for [M+H]+ C37H33N6O2 593.2665; found 593.2670.
5.0. Bacterial strains and media
The ESKAP pathogens panel consisted of Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (BAA-1705), Acinetobacter baumannii (BAA-1605), 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 procured 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).
5.1. 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
[25][27]. 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). 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
18

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alone and media alone (without compound+cells) and levofloxacin used as a reference standard
in the 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.
5.2. Antibiotic susceptibility testing against pathogenic mycobacteria:
Antimycobacterial susceptibility testing was performed on selected newly synthesized
compounds listed in the Table 2, by using broth microdilution assay [28][29]. 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 [30] 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 compound was determined as lowest concentration of compound that inhibited visible
growth after incubation period. For each compound, MIC determinations were replicated three
times using duplicate samples. The MIC plates were incubated at 37 °C for 7 days for Mtb.
5.3. Cell cytotoxicity assay
The active newly synthesized compounds 7a, 7b, 7c, and 9a were screened for their cell toxicity
against Vero cell using the MTT assay [31]. ~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 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 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.
19

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Acknowledgements
G. S. conveys cordial thanks 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. thank
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. MIC (µg/mL) of 7a and 9a in presence of Polymyxin B nonapeptide (PMBN) against
gram-negative bacteria.
Table 3. Cytotoxicity profile against Vero cells and Selectivity index of selected compounds.
Table 4. MIC values (µg/mL) of the 7a and 9a against MRSA and VRSA strains
Figure 1. Structures of some of the literature cited quinazolinone antibacterial and
antimycobacterial agents.
Figure 2. Structures of 1,2,3˗triazole containing anti-bacterial agents.
Figure 3. Structures of the designed 1,2,3-triazole linked to the 3-N-phenyl quinazolinone core.
Figure 4: Effect of substitution on 3-N-phenyl quinazolinone core on antibacterial activity.
Scheme 1. Synthesis of 1,2,3-triazole linked to 4(3H)-quinazolinone derivatives via ether linker.
Scheme 2. Synthesis of 1,2,3-triazole linked 4(3H)-quinazolinone derivatives.
Table 1. MIC values (µg/mL) of the tested compounds against ESKAP panel of bacteria.
S. aureus
ATCC
29213
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
E. coli
ATCC
25922
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
K. pneumoniae
A. baumannii
BAA-1605
P. aeruginosa
ATCC 27853
Compound
BAA-1705
>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
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
7a
7b
7c
0.5
0.5
>64
4
7d
7e
25

ACCEPTED MANUSCRIPT
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.015
>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
0.5
7f
7g
7h
7i
9a
9b
9c
9d
9e
9’a
15a
17a
17b
19a
19b
21a
4
0.5
4
4
0.5
16
0.5
0.5
2
>64
>64
>64
>64
>64
>64
>64
0.125
Levofloxacin
Table 2. MIC (µg/mL) of 7a and 9a in presence of Polymyxin B nonapeptide (PMBN) against
gram-negative bacteria.
MIC (µg/mL)
Rifampicin
PMBN
7a
PMBN
9a
PMBN
Bacteria
E. coli
ATCC 25922
A. baumannii
BAA-1605
PMBN
>128
(-)
(+)
(-)
(+)
(-)
(+)
4
4
0.125
0.25
>64
>64
>64
>64
>64
>64
>64
>64
>128
Table 3. Cytotoxicity profile against Vero cells and Selectivity index of selected compounds.
S. aureus
ATCC 29213
CC50
(µg/mL)
Selectivity Index
(CC50/MIC)
Compound
MIC (µg/mL)
7a
7b
7c
9a
0.5
0.5
0.5
0.5
40
20
20
20
80
40
40
40
Table 4. MIC values (µg/mL) of the 7a and 9a against MRSA and VRSA strains.
Strains
Antibiotics resistant to
MIC (µg/mL)
Levofloxacin Meropenem Vancomycin
7a
9a
26

ACCEPTED MANUSCRIPT
S. aureus
ATCC
29213
None
0.5
1
0.5
4
1
2
NR 119
Methicillin, Ceftriaxone,
Meropenem, Gentamycin
and Linezolid
0.5
2
16
>64
NR
10129
NR
10198
NR
10192
NR
10186
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone,
Meropenem
0.5
0.5
0.5
0.5
2
2
1
0.5
32
8
16
1
2
2
2
>64
64
2
8
16
NR
10193
NR
10194
NR
10191
VRS1
Methicillin, Ceftriaxone,
Meropenem
Methicillin, Ceftriaxone
1
2
32
0.5
32
>64
4
2
0.5
1
1
1
Methicillin, Ceftriaxone,
Meropenem
1
>64
>64
1
Methicillin, Ceftriaxone,
Meropenem, Gentamycin,
Vancomycin, Teicoplanin
Methicillin, Ceftriaxone,
Meropenem, Vancomycin
and Teicoplanin
Methicillin, Ceftriaxone,
Meropenem, Vancomycin
and Teicoplanin
32
32
32
>64
VRS4
0.5
2
2
2
>64
32
>64
>64
>64
>64
VRS12
Figure 1. Structures of some of the literature cited quinazolinone antibacterial and
antimycobacterial agents.
27

ACCEPTED MANUSCRIPT
Figure 2. Structures of 1,2,3˗triazole containing anti-bacterial agents.
Figure 3. Structures of the designed 1,2,3-triazole linked to the 3-N-phenyl quinazolinone core.
28