Synthesis and evaluation of new quinazolin-4(3H)-one derivatives as potent antibacterial agents against multidrug resistant Staphylococcus aureus and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2019.04.067

Staphylococcus aureus and Mycobacterium tuberculosis are major causative agents responsible for serious nosocomial and community-acquired infections impacting healthcare systems globally. Over several decades, these pathogens have developed resistance to multiple antibiotics significantly affecting morbidity and mortality. Thus, these recalcitrant pathogens are amongst the most formidable microbial pathogens for which international healthcare agencies have mandated active identification and development of new antibacterial agents for chemotherapeutic intervention. In our present work, a series of new quinazolin-4(3H)-one derivatives were designed, synthesized and evaluated for their antibacterial activity against ESKAP pathogens and pathogenic mycobacteria. The experiments revealed that 4'c, 4'e, 4'f and 4'h displayed selective and potent inhibitory activity against Staphylococcus aureus with MIC values ranging from 0.03-0.25 μg/mL. Furthermore, compounds 4'c and 4'e were found to be benign to Vero cells (CC₅₀ = >5 μg/mL) and displayed promising selectivity index (SI) > 167 and > 83.4 respectively. Additionally, 4'c and 4'e demonstrated equipotent MIC against multiple drug-resistant strains of S. aureus including VRSA, concentration dependent bactericidal activity against S. aureus and synergized with FDA approved drugs. Moreover, compound 4′c exhibited more potent activity in reducing the biofilm and exhibited a PAE of ～2 h at 10X MIC which is comparable to levofloxacin and vancomycin. In vivo efficacy of 4'c in murine neutropenic thigh infection model revealed that 4'c caused a similar reduction in cfu as vancomycin. Gratifyingly, compounds 4d, 4e, 9a, 9b, 14a, 4'e and 4'f also exhibited anti-mycobacterial activity with MIC values in the range of 2-16 μg/mL. In addition, the compounds were found to be less toxic to Vero cells (CC₅₀ = 12.5->100 μg/mL), thus displaying a favourable selectivity index. The interesting results obtained here suggest the potential utilization of these new quinazolin-4(3H)-one derivatives as promising antibacterial agents for treating MDR-Staphylococcal and mycobacterial infections.

Full text of DOI:10.1016/j.ejmech.2019.04.067

Accepted Manuscript
Synthesis and evaluation of new quinazolin-4(3H)-one derivatives as potent
antibacterial agents against multidrug resistant Staphylococcus aureus and
Mycobacterium tuberculosis
Srikanth Gatadi, Jitendra Gour, Manjulika Shukla, Grace Kaul, Arunava Dasgupta,
Y.V. Madhavi, Sidharth Chopra, Srinivas Nanduri
PII:
S0223-5234(19)30395-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.067
EJMECH 11302
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 February 2019
Revised Date: 25 April 2019
Accepted Date: 25 April 2019
Please cite this article as: S. Gatadi, J. Gour, M. Shukla, G. Kaul, A. Dasgupta, Y.V. Madhavi, S.
Chopra, S. Nanduri, Synthesis and evaluation of new quinazolin-4(3H)-one derivatives as potent
antibacterial agents against multidrug resistant Staphylococcus aureus and Mycobacterium tuberculosis,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.067.
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ACCEPTED MANUSCRIPT
Synthesis and evaluation of new Quinazolin-4(3H)-one derivatives as potent antibacterial
agents against multidrug resistant Staphylococcus aureus and Mycobacterium tuberculosis
a,c
a
b,c
b,c
b
Srikanth Gatadi, Jitendra Gour, Manjulika Shukla, Grace Kaul, Arunava Dasgupta,
a
b
a
Y.V. Madhavi, Sidharth Chopra, *, Srinivas Nanduri, *,
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500037, India
b
Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
c: These authors contributed equally
Graphical abstract:
1

ACCEPTED MANUSCRIPT
Synthesis and evaluation of new Quinazolin-4(3H)-one derivatives as potent antibacterial
agents against multidrug resistant Staphylococcus aureus and Mycobacterium tuberculosis
a,c
a
b,c
b,c
b
Srikanth Gatadi, Jitendra Gour, Manjulika Shukla, Grace Kaul, Arunava Dasgupta,
a
b
a
Y.V. Madhavi, Sidharth Chopra, *, Srinivas Nanduri, *,
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500037, India
b
Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
c: These authors contributed equally
Abstract
Staphylococcus aureus and Mycobacterium tuberculosis are major causative agents responsible
for serious nosocomial and community-acquired infections impacting healthcare systems
globally. Over several decades, these pathogens have developed resistance to multiple antibiotics
significantly affecting morbidity and mortality. Thus, these recalcitrant pathogens are amongst
the most formidable microbial pathogens for which international healthcare agencies have
mandated active identification and development of new antibacterial agents for
chemotherapeutic intervention. In our present work, a series of new quinazolin-4(3H)-one
derivatives were designed, synthesized and evaluated for their antibacterial activity against
ESKAP pathogens and pathogenic mycobacteria. The experiments revealed that 4’c, 4’e, 4’f and
4’h displayed selective and potent inhibitory activity against Staphylococcus aureus with MIC
values ranging from 0.03-0.25 µg/mL. Furthermore, compounds 4’c and 4’e were found to be
benign to Vero cells (CC = >5 µg/mL) and displayed promising selectivity index (SI) >167 and
5
0
>83.4 respectively. Additionally, 4’c and 4’e demonstrated equipotent MIC against multiple
drug-resistant strains of S. aureus including VRSA, concentration dependent bactericidal activity
against S. aureus and synergized with FDA approved drugs. Moreover, compound 4'c exhibited
more potent activity in reducing the biofilm and exhibited a PAE of ~2 h at 10X MIC which is
comparable to levofloxacin and vancomycin. In vivo efficacy of 4’c in murine neutropenic thigh
infection model revealed that 4’c caused a similar reduction in cfu as vancomycin. Gratifyingly,
compounds 4d, 4e, 9a, 9b, 14a, 4’e and 4’f also exhibited anti-mycobacterial activity with MIC
values in the range of 2-16 µg/mL. In addition, the compounds were found to be less toxic to
1

ACCEPTED MANUSCRIPT
Vero cells (CC = 12.5->100 µg/mL), thus displaying a favourable selectivity index. The
5
0
interesting results obtained here suggest the potential utilization of these new quinazolin-4(3H)-
one derivatives as promising antibacterial agents for treating MDR-Staphylococcal and
mycobacterial infections.
Keywords: Quinazolinones, Minimum Inhibitory Concentration, Multidrug-resistant
Staphylococcus aureus (MDR-S. aureus), 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
2

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1. Introduction
Staphylococcus aureus and Mycobacterium tuberculosis are ubiquitous dreadful pathogens
responsible for major human suffering worldwide [1]. Although causing inherently treatable
infections, due to rapid acquiring of multidrug-resistance (MDR), their treatment has impacted
healthcare systems significantly [2]. In view of the serious crisis, MRSA was recently
categorised as high risk Priority II pathogen by World Health Organization (WHO), one among
the 12 listed pathogens which imperils human health along with mycobacteria [3]. The incessant
rise in drug-resistance exhibited by these pathogens as well as number of nosocomial and
community infections caused by these pathogens in recent years has compelled the need to
identify and develop new antibacterials for treating infection under hospital and community
settings [4-11].
In our search of various chemical classes for developing new antibacterials, we found
quinazolinone to be a promising heterocycle which could be considered as a privileged structure
in the arena of drug design and development, due to its wide spectrum of biological properties
including anti-cancer, anti-bacterial, anti-HIV, anti-fungal, anti-inflammatory, analgesic and
anti-convulsant activities and also has broad scope for structural diversity [12-19].
Potential anti-bacterial properties of various quinazolin-4(3H)-one derivatives have been cited in
the literature. Xioping et al. [20] and Zayed et al. [21] reported the anti-bacterial properties of 2-
methylquinazolinone compounds I and II respectively (Figure 1). Saravanan et al. [22] reported
the antimicrobial nature of quinazolinone compound III. Nandy et al. [23] and Jadhavar et al.
[
24] also reported the potent anti-mycobacterial activity of Mannich bases of 2-methyl-3H-
quinazolin-4-ones IV and 2-styrylquinazolinones V respectively (Figure 1). Desai et al.[25]
reported the antibacterial properties of series of new 2-styryl-3,4-dihydroquinazolin-6-yl)-1,3,5-
triazin-2-yl)-3-methylurea derivative VI (Figure 1). Bouley et al. [26] reported 4(3H)-
quinazolinones as potential orally bioavailable antibacterial agent VII. Recently, our group has
reported 3-phenylquinazolin-4(3H)-ones VIII, 4-oxoquinazolin-3(4H)-yl)benzoic acid and
benzamide derivatives as potent anti-MDR-S. aureus agents [27].
<Insert Figure 1 here>
Many 1,2,3˗triazole containing molecules have been explored as interesting therapeutic agents
against several biological targets while expressing wide range of biological properties [28]
including antimicrobial activity. In addition to the promising pharmacological properties,
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triazole heterocycle is also known to have better hydrogen bond forming ability and metabolic
stability. Antibiotics cefatrizine (β-lactam antibiotic) IX and tazobactam [29] (β-lactamase
inhibitor) X contain 1,2,3-triazole as a key structural feature [30]. We have recently reported
1,2,3-triazole linked 4(3H)˗quinazolinones XI as potent antibacterial agent [31]. (Figure 2).
<Insert Figure 2 here>
1.1 Rationale
Quinazolinone core is considered to be a promising heterocyclic structure in the area of drug
design and development, due to its broad spectrum of biological properties including
antibacterial activity. In addition, the 1,2,3-triazole heterocycle is found to exhibit good
biological properties along with better hydrogen bond forming ability and metabolic stability.
We hypothesized that hybridizing the structural features of quinazolinone and 1,2,3-triazole
moieties could potentially have advantageous effect on the antibacterial activity. In the present
study, we designed and synthesized a number of new quinazolin-4(3H)-one derivatives and
tested them for their antibacterial and antimycobacterial properties (Figure 3).
<Insert Figure 3 here>
2
2
.0 Results and Discussion
.1 Chemistry
A series of newly designed quinazolin-4(3H)-one derivatives were synthesized as described in
Schemes 1, 2 & 3. Substituted anthranilic acids were converted into the substituted
benzoxazinone intermediate by treating with acetic anhydride under reflux conditions for 2 h.
Further, substituted benzoxazinone intermediate was dissolved in glacial acetic acid on gentle
heating followed by addition of substituted aniline, and the contents were refluxed for 5 h to give
an 2-methylquinazolin-4(3H)-one derivatives (Schemes 1, 2 & 3).
6
or 7-bromo substituted 2-methyl quinazolin-4(3H)-one 3 reacted with substituted boronic acids
under Suzuki conditions to afford 6 or 7- phenyl-2-methylquinazolin-4(3H)-ones 4. Besides, 6-
hydroxy-2-methyl-3-phenylquinazolin-4(3H)-one 7 or 7-amino-2-methyl-3-phenylquinazolin-
4(3H)-one 13 intermediates was subjected to O or N-propargylation by using propargyl bromide
under inert conditions to afford corresponding 6-O- or 7-N-propargylated quinazolin-4(3H)-one
derivatives 8 or 14. Treatment of the derivatives 8 and 14 with various substituted azides under
click conditions afforded the corresponding 1,2,3-triazole derivatives 9 and 15 respectively.
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Intermediates 4 were reacted with benzaldehyde or 4-cyano benzaldehyde in glacial acetic acid
under reflux conditions for overnight to afford the new 2-styrylquinazolin-4(3H)-one derivatives
as final compounds in moderate to excellent yields (Scheme 1, 2 & 3).
To the intermediate 2-methyl-7-nitro-4H-benzo[d][1,3]oxazin-4-one 11 dissolved in glacial
acetic acid, aniline was added and the reaction mixture was refluxed for 5 h to give 2-methyl-7-
nitro-3-phenylquinazolin-4(3H)-one as intermediate 12. To obtain the amine 13, the nitro group
of the intermediate 12 was reduced by using tin(II) chloride dihydrate under reflux for 6 h in
ethanol. The amine 13 was allowed to undergo azidation reaction by using sodium nitrite and
sodium azide in 2N HCl to afford azide intermediate 16. Finally, preparation of the designed
compounds 17 and 18 were accomplished by reacting the azide intermediate 16 with various
substituted phenylacetylenes or phenoxymethylacetylene by using Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction. Finally, treatment of 17 with 4-cyanobenzaldehyde in glacial
acetic acid afforded the 2-styrylquinazolin-4(3H)-one compounds 4’u-4’v as final products in
moderate to excellent yields (Schemes 2 & 3). Structures of all the newly synthesized
1
13
compounds were confirmed by H NMR, C NMR and HRMS (ESI) spectroscopic techniques.
Representative compounds were also analysed for purity by using HPLC (Agilent Infinity-2
1260 series).
<Insert Scheme 1 here>
<Insert Scheme 2 here>
<Insert Scheme 3 here>
<Insert Table 1 here>
2.2. In vitro antibacterial activity
2.2.1. Antibiotic Susceptibility Testing against ESKAP pathogen panel
A series of new quinazolin-4(3H)-one derivatives were evaluated for their antibacterial activity
against ESKAP pathogen panel by using broth microdilution assay. Minimum Inhibitory
Concentration (MIC) was determined as per Clinical Laboratory Standards Institute (CLSI)
guidelines [32]. The concentration of new quinazolin-4(3H)-one derivatives used is in the range
of 64-0.03 µg/mL and Levofloxacin, isoniazid and refampicin were used as reference standards.
The results are listed in Table 2.
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<Insert Table 2 here>
A perusal of the antimicrobial screening results suggested that the compounds 4a-4f (Scheme 1)
with substituted phenyl group at C-6 or C-7 with methyl group at C-2 position exhibited poor
inhibitory activity against the ESKAP pathogen panel (MIC = 32–>64 µg/mL). However, the
compounds 4d and 4e exhibited modest inhibitory activity against M. tuberculosis H R .
3
7
v
Compounds 9a-9g (Scheme 1) with 1,2,3-triazole linked to 3-N-phenyl or 3-N-benzyl 2-methyl
quinazolin-4(3H)-one via ether linker at C-6 position did not exhibit activity against the ESKAP
pathogen panel. However, the compounds 9a and 9b exhibited modest inhibitory activity against
M. tuberculosis H R (MIC = 16 µg/mL). Replacement of phenyl with benzyl moiety (9f-9g) at
37
v
N-3 on ring A was found to be detrimental on antimycobacterial activity. Interestingly,
compound 14a with prop-2-yn-1-ylamino group at C-7 position of 2-methyl-3-phenyl
quinazolin-4(3H)-one exhibited good inhibitory activity against M. tuberculosis H R (MIC =
3
7 v
4
µg/mL). However, 14a did not display activity against ESKAP pathogen panel. Replacement of
prop-2-yn-1-yl group (14a) with substituted 1-phenyl-1H-1,2,3-triazol-4-yl-methyl at C-7 as in
5a-15c did not exhibit any activity against both Staphylococcus aureus and mycobacteria.
In our continued efforts, we introduced 4-phenyl-1H-1,2,3-triazole (compounds 17a–17d) or 4-
phenoxymethyl)-1H-1,2,3-triazole (compounds 18a–18c) at C-7 position of quinazolin-4(3H)-
1
(
one on ring B. These compounds did not exhibit inhibitory activity against S. aureus and
mycobacteria (MIC = >64 µg/mL). Moreover, even after replacement of methyl group at C-2
with styryl moiety (compounds 4’u & 4’v), they did not exhibit antibacterial activity against S.
aureus and mycobacteria (MIC = >64 µg/mL). These broad SAR observations are summarised
in Figure 4.
<Insert Table 3 here>
Significantly, (scheme 3) compound 4’a with methyl acetate group, 4’b with tert-butyl phenyl at
C-3 position with C-2 styryl moiety (with p-C H -CN group) on ring A was found to be inactive
6
4
against ESKAP pathogen panel. However, compound 4’c, with 3-hydroxy-4-carboxy phenyl
group at N-3 with styryl moiety (with p-C H -CN group) displayed selective and highly potent
6
4
inhibitory activity against S. aureus (MIC = 0.03 µg/mL). Interestingly, compound 4’d having
-carboxy-4-hydroxy phenyl group at N-3 with 4-cyanostyryl moiety at C-2 did not exhibit
2
activity (MIC = >64 µg/mL). Compound 4’e having hydroxy at meta position on the N-phenyl
with fluoro group at C-5 exhibited selective and potent inhibitory activity against S. aureus
(MIC = 0.0625 µg/mL). This prompted us to investigate a number of fluoro quinazolinones with
4-cyanostyryl moiety at C-2 position by varying substituents on 3-N-phenyl at meta position.
6

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Interestingly, the compound 4’f with propargyl group and 4’h with acetylene moiety at meta
position on the N-phenyl displayed potent antibacterial activity with MIC values 0.5 and 0.25
µg/mL respectively. Intriguingly, compound 4’e and 4’f exhibited inhibitory activity against M.
tuberculosis H R (MIC 8 and 2 µg/mL respectively). However, compound 4’g and 4’i with
3
7
v
allyloxy and nitrile group at meta position of the N-Phenyl moiety was found to be inactive
MICs = 32 - >64 µg/mL) against both S. aureus and mycobacteria. Compound 4’j with
(
naphthalen-1-yl substitution, compounds 4’k and 4’l with 4-fluoro or 3,4-dichloro benzyl
substitution, compound 4’m with 4-(trifluoromethyl)phenyl amino substitution, compound 4’n
with (indol-3-yl)ethyl substitution, compound 4’o with pyridin-3-yl substitution at N-3 position
were found to be inactive (MIC = ≥64 µg/mL). Replacement of X=C with X=N in compounds
4’z-4’aj did not display any activity except compound 4’ad with thiol (-SH) group at para
position of the N-Phenyl moiety which exhibited MIC = 2 µg/mL against S. aureus. However,
all the aza variants of quinazolin-4(3H)-one did not display activity against mycobacteria (MIC
=
>64 µg/mL). Compounds 4’p & 4’q with methyl acetate group at N-3 with prop-2-yn-1-yloxy
or phenyl-1H-1,2,3-triazol-4-yl)methoxy group at C-6 also did not exhibit antibacterial activity.
Compounds 4’r (phenyl at C-6), 4’s (prop-2-yn-1-ylamino group at C-7), 4’t (phenyl at C-7),
4’u (4-phenyl-1H-1,2,3-triazole group at C-7), 4’v ((4-(4-methoxyphenyl)-1H-1,2,3-triazole
group at C-7)) with styryl moiety at C-2 position on ring A of quinazolin-4(3H)-one did not
exhibit activity against both S. aureus and mycobacteria. Similarly, compounds 4’w, 4’x & 4’y
with benzyl group at N-3 and prop-2-yn-1-yloxy group at C-6 were found to be inactive. The
broad Structure Activity Relationships derived are summarised in Figure 4 & 5.
<Insert Figure 4 here>
<Insert Figure 5 here>
2.2.1.1. Brief comparison of results
All the newly synthesized compounds exhibited promising antibacterial activity similar to the
compounds reported by various research groups. However, in contrast to 2-methylquinazolin-
4(3H)-one compounds reported by Xioping et al. [20], Zayed et al. [21], Saravanan et al. [22],
Nandy et al. [23] and Desai et al.[25], compounds 4d and 4e with methyl at C-2 and substituted
phenyl group at C-7 position exhibited inhibitory activity against M. tuberculosis H R . In
3
7
v
addition, compounds 9a-9b with 1,2,3-triazole linked to 3-N-phenyl or 3-N-benzyl 2-methyl
quinazolin-4(3H)-one via ether linker at C-6 position exhibited modest inhibitory activity against
M. tuberculosis H R (MIC = 16 µg/mL). Interestingly, compound 14a with prop-2-yn-1-
3
7
v
7

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ylamino group at C-7 position of 2-methyl-3-phenyl quinazolin-4(3H)-one showed potent
activity against M. tuberculosis H R (MIC = 4 µg/mL) However, they did not show activity
3
7
v
.
against the ESKAP pathogen panel. Unlike 2-styrylquinazolin-4(3H)-ones reported by Jadhavar
et al. [24] and Bouley et al. [26], compounds 4’e and 4’f with C-2-styryl moiety (p-C H -CN
6
4
group) exhibited good inhibitory activity against M. tuberculosis H R with MIC values 8 and 2
3
7
v
respectively. In comparison with our recent reports on quinazolinones [27a][27b], replacement
of 3-(prop-2-yn-1-yloxy) phenyl (MIC = 0.25 µg/mL), 3-allyloxy phenyl (MIC = 0.125 µg/mL),
3
-ethynylphenyl (MIC = 0.5 µg/mL), 3-cyanophenyl (MIC = 0.5 µg/mL) group with 3-hydroxy-
-carboxy phenyl group (4’c) at N-3 position with styryl moiety (with p-C H -CN group)
4
6
4
displayed selective and highly potent inhibitory activity against S. aureus (MIC = 0.03 µg/mL).
Furthermore, compound 4’c exhibited 8 and 16 fold potent activity than the secondary amides N-
isopropylbenzamide (MIC = 0.25 µg/mL) and N-cyclopropylbenzamide (MIC = 0. 5 µg/mL)
quinazolinone derivatives reported by Gatadi et al. [27a]. Recently, Gatadi et al. [27b] also
reported the compound having hydroxy at meta position on the N-phenyl with chloro group at C-
5, which did not exhibit activity against S. aureus (MIC = ≥64 µg/mL). Interestingly, the
compound having chloro group, when replaced with fluoro group at C-5 position (4’e) exhibited
selective and potent activity against S. aureus (MIC = 0.0625 µg/mL). Additionally, the 5-fluoro
compounds 4’f with prop-2-yn-1-yloxy group and 4’h with acetylene moiety at meta position on
the N-phenyl displayed potent antibacterial activity against S. aureus with MIC values of 0.5 and
0.25 µg/mL respectively. Intriguingly, compound 4’e and 4’f also exhibited inhibitory activity
against M. tuberculosis H R (MICs 8 and 2 µg/mL respectively). Replacement of X=C with
3
7
v
X=N in compound 4’ad with 4-mercaptophenyl group at N-3 position (MIC = 2 µg/mL)
exhibited potent inhibitory activity against S. aureus than the compounds reported by Gatadi et
al. [27a]. Compounds 4’c and 4’e exhibited 8-16 fold more potent activity than the compounds
with 3-chlorophenyl, 3-toluoyl and 3-fluorophenyl at N-3 position against Staphylococcus
aureus (MIC = 0.5 µg/mL). Moreover, compounds 4’c and 4’e also exhibited 8-16 fold more
potent activity than compounds with benzyl (Bz, 3-Br-Bz, 4-OCH -Bz) groups on the 1,2,3-
3
triazole moiety (MIC = 0.5 µg/mL) against Staphylococcus aureus as reported in the recent
literature [31].
2.3. Cytotoxicity against Vero cells
To examine the effect of the active compounds 4’c, 4’e, 4’f and 4’h on mammalian cells, a
cytotoxicity assay was performed against Vero cells (African green monkey kidney cell line,
8

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ATCC CCL-81) using the MTT assay. The 50% cytotoxic concentration (CC ) is defined as the
50
lowest concentration of compound which leads to a 50% reduction in cell viability. Doxorubicin
was used as a reference standard and each experiment was performed in triplicate. A perusal of
cytotoxicity data suggests that 4’c, 4’e, 4’f and 4’h are non toxic to Vero cells (CC = >5–>10
5
0
µg/mL) and displayed a promising Selectivity Index (SI) of (>10–>167). In a similar vein, active
compounds 4d, 4e, 9a, 9b, 14a, 4’e and 4’f against mycobacteria were found to be non toxic to
Vero cells (CC = 12.5->100 µg/mL) and exhibited favourable selectivity index. The results are
5
0
tabulated in Table 4 & Table 5.
<Insert Table 4 here>
<Insert Table 5 here>
2.4. Determination of MIC against MDR-S. aureus including VRSA
To determine their range of activity against multiple strains of MDR-S. aureus, 4’c and 4’e were
examined against various well defined and characterized clinical isolates of MRSA and VRSA
as per standard CLSI guidelines [32]. The results are summarised in Table 6. Levofloxacin,
meropenem and vancomycin were used as reference standards. From the close examination of
the results, it can be inferred that 4’c and 4’e displayed potent antibacterial activity with MIC
ranging from <0.125–0.5 µg/mL against various clinical strains of MRSA and VRSA.
<Insert Table 6 here>
2.5. Time kill kinetics of 4’c and 4’e
The bactericidal activity was determined by the time-kill method. S. aureus ATCC 29213 cells
5
were diluted up to ~10 cfu/mL and treated with compound for concentrations corresponding to
1
X and 10X of MIC of 4’c, 4’e and vancomycin in MHB in triplicate and incubated at 37 °C.
00 µL samples were collected after time intervals of 0h, 1h, 6h and 24h and serially diluted in
1
Phosphate Buffered Saline (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 stipulated time point in the presence and absence of compound
(Figure 6).
<Insert Figure 6 here>
2.6. Determination of synergy with FDA approved drugs
9

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The checkerboard method was used to determine the interaction between 4’c, 4’e and various
FDA approved antibiotics including meropenem, linezolid, ceftriaxone and vancomycin which
are typically used for the treatment of staphylococcal infections (Table 7 & 8). As can be seen,
4’c and 4’e synergized with drugs tested, thus displaying great potential to be a part of multi-
drug regimen.
<
Insert Table 7 here>
Insert Table 8 here>
<
2.7. Determination of Post-Antibiotic effect of 4'c
The post-antibiotic effect of 4'c was determined with vancomycin and levofloxacin as controls.
As can be seen from Table 9, 4'c exhibits a PAE of ~2 h at 10X MIC which is comparable to
levofloxacin and vancomycin.
<Insert Table 9 here>
2.8. 4'c eradicates S. aureus biofilm
It has been repeatedly demonstrated that bacteria under different stresses make biofilm in order
to protect themselves, which often leads to prolonging therapeutic intervention and increasing
drug-resistance. Incidentally, most of the approved drugs have very limited activity against
pathogens in biofilms owing to an altered physiological state, thus it is imperative to determine
the antibiofilm activity of molecules under development. In this context, 4'c at 10X MIC
exhibited potent anti-biofilm activity as it reduced biofilm by 14.1% as compared to untreated.
On the other hand, similar reduction in biofilm was achieved at 10X MIC of levofloxacin
(30.6% reduction) while vancomycin only reduced by 7.7%. Thus, 4'c is more potent in reducing
the biofilm as compared to vancomycin (Figure 7).
<Insert Figure 7 here>
3. In vivo animal efficacy of 4’c
The in vivo efficacy of 4’c was tested in murine neutropenic thigh infection model of S. aureus
infection. This model has been utilized extensively for evaluating the in vivo efficacy of several
molecules against S. aureus. Briefly, the thigh of neutropenic mice was infected with S. aureus,
followed by two IP injections of 4’c at 25 and 50 mg/Kg while vancomycin was injected at 25
mg/Kg. Saline and vancomycin treated mice groups were used as a negative and positive control
1
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respectively. As can be seen in Figure 8, mice infected with S. aureus were treated with
vancomycin (25 mg/Kg) and 4’c (25 and 50 mg/Kg) and the treatment caused significantly equal
reduction at 25 mg/Kg (P <0.05). When dosed at 50 mg/Kg, 4’c reduced significantly more cfu
(P<0.005) than vancomycin, thus displaying a concentration dependent reduction in CFU. Taken
together, 4’c is as potently capable of causing reduction in cfu as is vancomycin.
<Insert Figure 8 here>
4. Conclusion
In conclusion, a series of new quinazolin-4(3H)-one derivatives have been synthesised and
evaluated against ESKAP pathogen panel and pathogenic mycobacteria. Compounds 4’c, 4’e,
4’f and 4’h displayed selective and potent antibacterial activity against Staphylococcus aureus
and were found to be non toxic to Vero cells (CC = >5–>10 µg/mL) with a good selectivity
50
index (SI = >10–>167). 4’c and 4’e displayed potent inhibitory activity when screened against
clinical MRSA and VRSA isolates. Gratifyingly, compounds 4d, 4e, 9a, 9b, 14a, 4’e and 4’f
also exhibited antimycobacterial activity with MIC values in the range of 2-16 µg/mL. Besides,
the compounds were found to be less toxic to Vero cells (CC = 12.5->100 µg/mL) and
5
0
exhibited favourable selectivity index. Compounds 4’c and 4’e also exhibited concentration
dependent bactericidal activity and synergized with the FDA approved drugs tested. Moreover,
compound 4'c exhibited more potent activity in reducing the biofilm and exhibited a PAE of ~2
h at 10X MIC which is comparable to levofloxacin and vancomycin. In vivo efficacy of 4’c in
the murine neutropenic thigh infection model, revealed that the compound 4’c was equipotently
capable of causing reduction in cfu as compared to vancomycin. All together, these new
compounds display antibacterial potential to be further modified and developed as
antimycobacterial and anti-multidrug resistant S. aureus leads.
5. Materials and 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
13
silica gel 60-F254 (0.5 mm) aluminium plates under UV light. H and C 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), DMSO˗d (δ 2.50 for H NMR and 39.7 for C NMR) or CDCl (δ
6
3
1
13
7
.26 for H NMR and 77.00 or 77.16 for C NMR) or combination of CDCl and DMSO-d in
3 6,
1
1

ACCEPTED MANUSCRIPT
1
3
which DMSO-d was used as an internal reference or TFA-d (δ 164.2 and δ 116.6 for C NMR).
6
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. HPLC purity were determined with Agilent Infinity-2
1260 series instrument (UV–Vis diode-array detector) and were performed at 258-268 nm.
Column chromatography was performed using silica gel 60–120 or 100–200 mesh. Melting
points were taken using Stuart® SMP30 apparatus.
Intermediates 2, 3, 6, 7 and 8 (Scheme 1) were prepared according to the procedures described
in literature [26,27].
5.1. General reaction procedure for the synthesis of substituted 6 or 7-phenyl-2-methyl-3-
phenylquinazolin-4(3H)-one derivatives 4a-4f.
To a stirred solution of 6 or 7-bromo-2-methyl-3-phenylquinazolin-4(3H)-ones (3, 2 mmol)
(
scheme 1), in a schlenk tube, Cs CO (2 mmol), substituted phenylacetylenes or
2 3
phenoxymethyl acetylenes (2 mmol), Pd-(PPh ) (5 mol%), followed by LiCl (2 mol%) were
3
4
added. A septa was placed on the schlenk tube which then was purged with N for 5 min. The
2
solvents 1:1 (5 mL of toluene and 5 mL of ethanol) were added, and the test tube was put in an
oil bath previously set at 100 °C and allowed to stir for 12-14 h. The reaction was monitored by
TLC. After completion of reaction, it was extracted with ethylacetate and washed with NH Cl
4
solution, water and brine and dried by using sodium sulfate (Na SO ). The combined organic
2
4
extracts were added directly to a plug of silica gel to remove the catalyst, thus yielding the crude
product. Further purification was accomplished via column chromatography, yielding a pure
1
13
white solids 4a-4f. All the newly synthesized compounds were characterized by H NMR, C
1
NMR and HRMS (ESI). H NMR signals was also determined for some representative
compounds. The synthesis of the final compounds 4a-4f was followed by general procedure
given above.
5.1.1. 2-methyl-3,6-diphenylquinazolin-4(3H)-one (4a): White solid, Yield 71 %; mp: 133–135
1
°
C. H NMR (500 MHz, CDCl ) δ 8.49 (d, J = 2.1 Hz, 1He), 8.02 (dd, J = 8.5, 2.2 Hz, 1Hi),
3
7.79–7.75 (m, 1Hj), 7.71–7.67 (m, 2Hf), 7.61–7.55 (m, 2Hg), 7.55–7.50 (m, 1Hh), 7.49–7.45
1
3
(
m, 2Hb), 7.41–7.35 (m, 1Hd), 7.31–7.27 (m, 2Hc), 2.27 (s, 3Ha) ppm; C NMR (125 MHz,
CDCl ) δ 162.3, 154.3, 146.5, 139.6, 137.8, 133.6, 130.0, 129.4, 129.0, 128.0, 127.9, 127.2,
3
1
2

ACCEPTED MANUSCRIPT
+
1
3
27.2, 124.9, 121.0, 24.4 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 313.1341; found
21 17 2
13.1344.
5.1.2. 3-(3-chlorophenyl)-6-(4-fluorophenyl)-2-methylquinazolin-4(3H)-one (4b): White solid,
1
Yield 80 %; mp: 165–167 °C. H NMR (500 MHz, DMSO-d ) δ 8.27 (d, J = 2.0 Hz, 1Hf), 8.14
6
(dd, J = 8.4, 2.1 Hz, 1Hb), 7.84–7.78 (m, 2Hij), 7.76–7.72 (m, 1Hd), 7.71 (s, 1He), 7.62 (d, J =
13
4
.4 Hz, 2Hg), 7.53–7.47 (m, 1Hc), 7.35–7.31 (m, 2Hh), 2.17 (s, 3Ha) ppm; C NMR (125 MHz,
DMSO-d ) δ 162.6 (d, J = 244.8 Hz), 161.7, 154.6, 147.0, 139.7, 137.6, 135.8 (d, J = 2,7 Hz),
6
1
2
3
34.1, 133.6, 131.6, 129.7, 129.3 (d, J = 8.3 Hz), 129.2, 128.0, 128.0, 124.0, 121.2, 116.5 (d, J =
+
1.5 Hz, 2H), 24.5 ppm; HRMS (ESI): m/z calcd for [M+H] C H FClN O 365.0857; found
2
1
15
2
65.0860.
5.1.3. 6-(4-methoxyphenyl)-2-methyl-3-phenylquinazolin-4(3H)-one (4c): white solid, Yield 68
1
%
; mp: 111–113 °C. H NMR (500 MHz, CDCl ) δ 8.43 (d, J = 2.1Hz, 1He), 8.00 (dd, J = 8.5,
3
2
7
.2 Hz, 1Hi), 7.84–7.78 (m, 1Hj), 7.65–7.61 (m, 2Hf), 7.60–7.56 (m, 2Hc), 7.56–7.52 (m, 1Hd),
1
3
.32–7.27 (m, 2Hb), 7.03–6.99 (m, 2Hg), 3.87 (s, 3Hh), 2.32 (s, 3Ha) ppm; C NMR (125
MHz, CDCl ) δ 162.2, 159.6, 154.2, 139.4, 137.6, 134.7, 133.3, 132.0, 130.0, 129.4, 128.3,
3
+
128.0, 126.9, 124.2, 120.9, 114.5, 55.4, 24.1 ppm; HRMS (ESI): m/z calcd for [M+H]
C H N O 343.1446; found 343.1450.
22
19
2
2
5.1.4. 3-(3-chlorophenyl)-2-methyl-7-phenylquinazolin-4(3H)-one (4d): White solid, Yield 85
1
%
; mp: 145–147 °C. H NMR (500 MHz, CDCl ) δ 8.33–8.30 (m, 1H), 7.95–7.92 (m, 1H),
3
7
1
1
.76–7.70 (m, 3H), 7.54–7.48 (m, 4H), 7.47–7.42 (m, 1H), 7.34–7.32 (s, 1H), 7.23–7.18 (m,
1
3
H), 2.27(s, 3H) ppm; C NMR (125 MHz, CDCl ) δ 161.8, 154.3, 147.8, 147.4, 145.8, 139.5,
3
38.7, 135.7, 131.0, 129.8, 129.1, 128.6, 127.6, 127.4, 126.5, 126.0, 124.7, 119.2, 24.3 ppm;
+
HRMS (ESI): m/z calcd for [M+H] C H ClN O 347.0951; found 347.0954.
21
16
2
5.1.5. 3-(3-chlorophenyl)-7-(4-fluorophenyl)-2-methylquinazolin-4(3H)-one (4e): White solid,
1
Yield 76 %; mp: 140–142 °C H NMR (500 MHz, DMSO-d ) δ 8.14 (d, J = 8.3 Hz, 1H), 7.95–
6
7.87 (m, 3H), 7.90 (dd, J = 8.6, 5.5 Hz, 1H), 7.72–7.69 (m, 1H), 7.64–7.59 (m, 2H), 7.52–7.46
13
(
m, 1H), 7.40–7.32 (m, 2H), 2.17 (s, 3H) ppm; C NMR (125 MHz, DMSO-d ) δ 163.04 (d, J =
6
246.2 Hz), 161.5, 154.9, 148.3, 145.5, 139.6, 135.6 (d, J = 3.1 Hz), 134.1, 131.6, 129.8 (d, J =
8.4 Hz), 129.6, 129.2, 128.0, 127.5, 125.4, 124.6, 119.7, 116.5 (d, J = 21.5 Hz), 24.4 ppm;
+
HRMS (ESI): m/z calcd for [M+H] C H FClN O 365.0857; found 365.0859.
21
15
2
1
3

ACCEPTED MANUSCRIPT
5.1.6. 3-(3-chlorophenyl)-7-(4-methoxyphenyl)-2-methylquinazolin-4(3H)-one (4f): White solid,
1
Yield 78 %; mp: 155–157 °C. H NMR (500 MHz, CDCl ) δ 8.28 (d, J = 8.3 Hz, 1H), 7.91 (s,
3
1H), 7.73–7.69 (m, 1H), 7.69–7.65 (m, 2H), 7.54–7.50 (m, 2H), 7.33 (s, 1H), 7.23–7.18 (m, 1H),
1
3
7
.06–7.01 (m, 2H), 3.88 (s, 3H), 2.31 (s, 3H) ppm; C NMR (125 MHz, CDCl ) δ 161.5, 160.3,
3
154.7, 147.5, 138.4, 135.7, 131.6, 131.1, 129.9, 128.6, 127.6, 127.1, 126.5, 125.8, 123.3, 123.3,
+
1
18.5, 114.6, 55.4, 24.0 ppm; HRMS (ESI): m/z calcd for [M+H] C H ClN O 377.1057;
22
18
2
2
found 377.1058.
Compound 14a (Scheme 1) were prepared according to the procedures described in literature
26,27].
[
5.2. General reaction procedure for the synthesis of 6-((1H-1,2,3-triazol-4-yl)methoxy)-2-
methylquinazolin-4(3H)-one derivatives 9a-9g or 7-(((1H-1,2,3-triazol-4-yl)methyl)amino)-2-
methyl-3-phenylquinazolin-4(3H)-one derivatives 15a-15c.
Compound (8 or 14, 2 mmol) (scheme 1 & 2) and phenyl or benzyl azides (2 mmol) were
suspended in 10 mL of a 1:1 tert-BuOH/H O mixture. To the heterogeneous mixture, sodium
2
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 9a-9g in 61-77% yields and 15a-15c in 51-56%
1
13
yields. All the newly synthesized compounds were characterized by H NMR, C NMR and
HRMS (ESI). The synthesis of the final compounds 9a-9g and 15a-15c was followed by general
procedure given above.
5
4
.2.1.
6-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-methyl-3-phenylquinazolin-
1
(3H)-one (9a): White solid, Yield 72 %; mp: 177–179 °C H NMR (500 MHz, CDCl ) δ 8.12
3
(s, 1Hg), 7.81–7.78 (m, 1Hk), 7.76 (d, J = 2.7 Hz, 1He), 7.67–7.62 (m, 2Hhj), 7.59–7.53 (m,
2
3
1
2
Hb), 7.53–7.49 (m, 1Hm), 7.49–7.40 (m, 3Hci), 7.31–7.26 (m, 2Hld), 5.36 (s, 2Hf), 2.23 (s,
1
3
Ha) ppm; C NMR (125 MHz, DMSO–d ) δ 161.5, 156.6, 152.7, 144.3, 142.6, 138.5, 138.0,
6
34.7, 132.1, 130.1, 129.4, 129.1, 128.9, 128.9, 124.8, 123.6, 121.7, 120.4, 119.2, 108.4, 62.1,
+
4.3 ppm; HRMS (ESI): m/z calcd for [M+H] C H ClN O 444.1227; found 444.1229.
2
4
19
5
2
5
4
9
.2.2.
6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-methyl-3-phenylquinazolin-
1
(3H)-one (9b): White solid, Yield 74 %; mp: 171–173 °C. H NMR (500 MHz, DMSO–d ) δ
6
.01 (s, 1Hg), 7.99–7.94 (m, 2Hh), 7.71–7.66 (m, 3Hie), 7.66–7.63 (m, 1Hk), 7.61–7.56 (m,
1
4

ACCEPTED MANUSCRIPT
1
3
2
Hb), 7.56–7.50 (m, 2Hc), 7.46–7.42 (m, 2Hjd), 5.37 (s, 2Hf), 2.10 (s, 3Ha) ppm; C NMR (125
MHz, DMSO–d ) δ 161.5, 156.7, 152.7, 144.3, 142.6, 138.5, 135.8, 133.6, 130.4, 130.1, 129.4,
6
128.9, 128.9, 124.8, 123.5, 122.3, 121.7, 108.4, 62.1, 24.3 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H ClN O 444.1227; found 444.1231.
24
19
5
2
5.2.3.
2-methyl-3-phenyl-6-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-
1
yl)methoxy)quinazolin-4(3H)-one (9c): White solid, Yield 66 %; mp: 178–180 °C, H NMR (500
MHz, CDCl ) δ 8.09 (s, 1Hg), 7.81 (d, J = 2.8 Hz, 1He), 7.75–7.70 (m, 1Hk), 7.63–7.57 (m,
3
2Hb), 7.56–7.52 (m, 1Hj), 7.51–7.47 (dd, J = 8.9, 2.9 Hz, 1Hd), 7.31–7.27 (m, 2Hc), 6.97 (s,
1
3
2
Hh), 5.39 (s, 2Hf), 3.95 (s, 6Hi), 3.91 (s, 3Hi’), 2.29 (s, 3Ha) ppm; C NMR (125 MHz,
CDCl ) δ 161.7, 157.0, 153.9, 152.9, 143.9, 141.3, 138.5, 137.4, 132.8, 130.1, 129.5, 128.1,
3
127.9, 125.0, 121.6, 121.4, 108.1, 98.7, 62.3, 61.1, 56.5, 23.8 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H N O 500.1934; found 500.1941.
27
26
5
5
5.2.4.
2-methyl-7-(4-(((2-methyl-4-oxo-3-phenyl-3,4-dihydroquinazolin-6-yl)oxy)methyl)-1H-
1,2,3-triazol-1-yl)-3-phenylquinazolin-4(3H)-one (9d): White solid, Yield 61 %; mp: 255–257
1
°
C H NMR (500 MHz, DMSO–d ) δ 9.25 (s, 1H), 8.28 (d, J = 8.6 Hz, 1H), 8.23 (d, J = 2.1 Hz,
6
1H), 8.13 (dd, J = 8.6, 2.1 Hz, 1H), 7.71 (d, J = 2.9 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 7.62–7.52
1
3
(
m, 7H), 7.52–7.47 (m, 2H), 7.47–7.43 (m, 2H), 5.41 (s, 2H), 2.16 (s, 3H), 2.11 (s, 3H) ppm; C
NMR (125 MHz, DMSO–d ) δ 161.5, 161.1, 156.7, 156.7, 152.7, 149.0, 144.6, 142.6, 141.2,
6
1
1
5
38.5, 138.1, 130.1, 130.0, 129.6, 129.4, 129.2, 129.0, 128.9, 128.9, 124.8, 123.7, 121.7, 120.6,
+
18.2, 116.9, 108.5, 62.1, 24.6, 24.3 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O
3
3
26
7
3
68.2097; found 568.2102.
5
4
8
7
1
1
1
.2.5.
6-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-methyl-3-phenylquinazolin-
1
(3H)-one (9e): White solid, Yield 71 %; mp: 155–157 °C. H NMR (500 MHz, DMSO–d ) δ
6
.32 (s, 1H), 8.13–8.09 (m, 1H), 7.88–7.82 (m, 1H), 7.69–7.65 (m, 1H), 7.61–7.56 (m, 2H),
.55–7.50 (m, 1H), 7.50–7.46 (m, 1H), 7.31–7.26 (m, 2H), 7.20–7.15 (m, 2H), 7.05–7.01 (m,
1
3
H), 5.61 (s, 2H), 5.23–5.14 (m, 2H), 2.14 (s, 3H) ppm; C NMR (125 MHz, DMSO–d ) δ
6
61.7, 159.2, 154.9, 147.8, 143.2, 139.4, 135.8, 135.1, 132.2, 130.8, 130.7, 127.1, 126.9, 126.8,
25.4, 121.9, 121.3, 121.0, 116.0, 115.4, 61.7, 52.6, 24.3 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H BrN O 502.0878; found 502.0880.
25
21
5
2
5
4
9
.2.6.
3-benzyl-6-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-methylquinazolin-
1
(3H)-one (9f): White solid, Yield 70 %; mp: 240–242 °C. H NMR (500 MHz, DMSO–d ) δ
6
.06 (s, 1Hh), 8.08 (s, 1Hf), 7.97–7.93 (m, 1Hl), 7.75 (d, J = 2.8 Hz, 1Hi), 7.66–7.59 (m, 2Hkn),
1
5

ACCEPTED MANUSCRIPT
7
7
1
1
.59–7.56 (m, 1Hj), 7.55–7.50 (m, 1Hm), 7.40–7.32 (m, 2Hc), 7.31–7.25 (m, 1Hd), 7.20 (d, J =
13
.6 Hz, 2Hb), 5.39 (s, 4Hge), 2.47 (s, 3Ha) ppm; C NMR (125 MHz, DMSO–d ) δ 161.7,
6
56.7, 153.4, 144.3, 142.5, 138.1, 137.0, 134.7, 132.1, 129.3, 129.1, 129.0, 127.8, 126.8, 124.9,
+
23.6, 121.1, 120.5, 119.3, 108.4, 62.1, 46.9, 23.2 ppm; HRMS (ESI): m/z calcd for [M+H]
C H ClN O 458.1384; found 458.1384.
25
21
5
2
5
4
9
1
.2.7.
3-benzyl-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-methylquinazolin-
1
(3H)-one (9g): White solid, Yield 74 %; mp: 235–237 °C. H NMR (500 MHz, DMSO–d ) δ
6
.01 (s, 1Hh), 8.02–7.93 (m, 2Hi), 7.75 (d, J = 2.8 Hz, 1Hf), 7.71–7.66 (m, 2Hj), 7.63–7.58 (m,
Hl), 7.52 (dd, J = 8.9, 2.9 Hz, 1Hd), 7.39–7.32 (m, 2Hc), 7.31–7.25 (m, 1Hk), 7.20 (d, J = 7.4
1
3
Hz, 2Hd), 5.39 (s, 4Heg), 2.47 (s, 3Ha) ppm; C NMR (125 MHz, DMSO–d ) δ 161.7, 156.7,
6
1
1
4
53.4, 144.3, 142.5, 137.0, 135.8, 133.6, 130.3, 129.3, 128.9, 127.8, 126.8, 124.9, 123.5, 122.3,
+
21.1, 108.3, 62.1, 46.9, 23.2 ppm; HRMS (ESI): m/z calcd for [M+H] C H ClN O
25
21
5
2
58.1384; found 458.1387.
5.2.8. 2-methyl-3-phenyl-7-(prop-2-yn-1-ylamino)quinazolin-4(3H)-one (14a): White solid,
1
Yield 77 %; mp: 155–157 °C. H NMR (500 MHz, DMSO–d ) δ 7.80 (d, J = 8.7 Hz, 1He),
6
7.57–7.51 (m, 2Hb), 7.50–7.46 (m, 1Hd), 7.40–7.35 (m, 2Hc), 7.00 (t, J = 5.9 Hz, 1Hi), 6.81
(dd, J = 8.7, 2.3 Hz, 1Hf), 6.69–6.67 (m, 1Hj), 4.01 (dd, J = 5.9, 2.3 Hz, 2Hg), 3.15 (t, J = 2.3
13
Hz, 1Hh), 2.06 (s, 3Ha). C NMR (125 MHz, DMSO–d ) δ 161.3, 154.6, 153.4, 149.9, 138.7,
6
129.9, 129.1, 127.7, 114.9, 110.4, 105.5, 81.6, 73.9, 49.1, 32.2, 24.5 ppm; HRMS (ESI): m/z
+
calcd for [M+H] C H N O 290.1293; found 290.1295.
1
8
16
3
5.2.9.
7-(((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2-methyl-3-
1
phenylquinazolin-4(3H ) (15a): White solid, Yield 53 %; mp: 122-124 °C. H NMR (500 MHz,
DMSO-d ) δ 8.83 (s, 1Hi), 8.27–8.22 (m, 1He), 8.01–7.95 (m, 1Hf), 7.78 (d, J = 8.7 Hz, 1Hj),
6
7.68 (t, J = 9.0 Hz, 1Hk), 7.57–7.51 (m, 2Hb), 7.51–7.46 (m, 1Hd), 7.38–7.33 (m, 2Hc), 7.26 (t,
J = 5.7 Hz, 1Hg), 6.87 (dd, J = 8.8, 2.1 Hz, 1Hm), 6.67 (d, J = 2.0 Hz, 1Hl), 4.53 (d, J = 5.6 Hz,
1
3
2
Hh), 2.04 (s, 3Ha) ppm; C NMR (125 MHz, DMSO-d ) δ 160.9, 157.9, 155.9, 154.1, 153.4,
6
149.6, 146.3, 138.3, 133.7, 129.5, 128.7, 127.3, 122.2, 121.7, 120.9, 120.8 (d, J = 7.8 Hz), 118.2
+
(d, J = 22.7 Hz), 114.5, 109.7, 104.4, 38.1, 24.0 ppm; HRMS (ESI): m/z calcd for [M+H]
C H ClFN O 461.1293; found 461.1294.
24
19
6
5.2.10.
2-methyl-7-(((1-(3-methyl-4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-3-
1
phenylquinazolin-4(3H)-one (15b): White solid, Yield 56 %; mp: 176-178 °C. H NMR (500
MHz, DMSO-d ) δ 8.61 (s, 1Hi), 8.40 (d, J = 2.3 Hz, 1Hk), 8.25 (dd, J = 8.7, 2.5 Hz, 1Hj), 7.79–
6
1
6

ACCEPTED MANUSCRIPT
7.74 (m, 2Hb), 7.56–7.52 (m, 2Hc), 7.50–7.46 (m, 1Hd), 7.39–7.33 (m, 2Hem), 7.22 (t, J = 5.6
Hz, 1Hg), 6.90–6.86 (m, 1Hf), 6.73 (d, J = 2.1 Hz, 1Hn), 4.55 (d, J = 5.6 Hz, 2Hh), 2.35 (s,
1
3
3
1
3
4
Hl), 2.05 (s, 3Ha) ppm; C NMR (125 MHz, DMSO-d ) δ 161.3, 154.5, 153.9, 150.0, 148.0,
6
45.8, 141.4, 138.7, 135.5, 129.9, 129.1, 127.7, 127.6, 126.9, 125.3, 122.7, 114.8, 110.1, 105.0,
+
8.4, 24.4, 18.4 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O
468.1784; found
25
22
7
3
68.1784.
5
4
.2.11.
2-methyl-3-phenyl-7-(((1-(3,4,5-trimethylphenyl)-1H-1,2,3-triazol-
1
yl)methyl)amino)quinazolin-4(3H)-one (15c): White solid, Yield 51 %; mp: 126-128 °C. H
NMR (500 MHz, DMSO-d ) δ 8.25 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.55–7.52 (m, 2H), 7.51–
6
7
6
.49 (m, 1H), 7.39–7.36 (m, 2H), 7.18 (t, J = 5.5 Hz, 1H), 7.09 (s, 2H), 6.90–6.86 (m, 1H), 6.74–
13
.71 (m, 1H), 4.53 (d, J = 5.5 Hz, 2H), 2.33 (s, 3H), 2.05 (s, 3H), 1.88 (s, 6H) ppm; C NMR
(
125 MHz, DMSO-d ) δ 161.3, 154.4, 154.0, 150.0, 145.0, 139.9, 138.7, 134.9, 133.9, 129.9,
6
129.3, 129.1, 127.7, 125.6, 114.7, 110.0, 106.9, 105.0, 38.6, 24.4, 21.1, 17.3 ppm; HRMS (ESI):
+
m/z calcd for [M+H] C H N O 451.2246; found 451.2250.
2
7
27
6
5.3. General reaction procedure for the synthesis of 2-methyl-3-phenyl-7-(1H-1,2,3-triazol-1-
yl)quinazolin-4(3H)-one derivatives 17a-17d, 18a-18c.
7-azido-2-methyl-3-phenylquinazolin-4(3H)-one (16, 2 mmol) (scheme 2) and substituted
phenylacetylenes or phenoxymethyl acetylenes (2 mmol) were mixed in a 10 mL volume of 1:1
tert-BuOH/H O mixture. To the heterogeneous mixture, sodium ascorbate (10 mol%), followed
2
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 17a-17d, 18a-18c in 58-82% yields. All the newly synthesized
1
13
compounds were characterized by H NMR, C NMR and HRMS (ESI). The synthesis of the
final compounds 17a-17d, 18a-18c was followed by general procedure given above.
5.3.1. 2-methyl-3-phenyl-7-(4-phenyl-1H-1,2,3-triazol-1-yl)quinazolin-4(3H)-one (17a): White
1
solid, Yield 78 %; mp: 210–212 °C. H NMR (500 MHz, DMSO–d ) δ 9.58 (s, 1Hg), 8.31 (d, J
6
=
8.6 Hz, 1He), 8.28–8.25 (m, 1Hf), 8.19–8.14 (m, 1Hj), 8.01–7.96 (m, 2Hh), 7.63–7.57 (m,
1
3
2
Hb), 7.57–7.47 (m, 5Hcik), 7.44–7.39 (m, 1Hd), 2.17 (s, 3Ha) ppm; C NMR (125 MHz,
DMSO–d ) δ 161.2, 156.7, 149.0, 148.2, 141.3, 138.1, 130.4, 130.1, 129.6, 129.5, 129.2, 128.9,
6
+
128.9, 125.9, 120.5, 120.3, 118.0, 116.7, 24.7 ppm; HRMS (ESI): m/z calcd for [M+H]
C H N O 380.1511; found 380.1516.
23
18
5
1
7

ACCEPTED MANUSCRIPT
5.3.2.
7-(4-(4-bromophenyl)-1H-1,2,3-triazol-1-yl)-2-methyl-3-phenylquinazolin-4(3H)-one
1
(
17b): White solid, Yield 82 %; mp: 204–206 °C; H NMR (500 MHz, DMSO–d ) δ 9.65 (s,
6
1
7
2
1
Hg), 8.33 (d, J = 8.6 Hz, 1He), 8.26 (d, J = 2.1 Hz, 1Hf), 8.16 (dd, J = 8.6, 2.2 Hz, 1Hj), 7.95–
.91 (m, 2Hh), 7.77–7.73 (m, 2Hi), 7.63–7.58 (m, 2Hb), 7.58–7.52 (m, 1Hd), 7.52–7.48 (m,
1
3
Hc), 2.18 (s, 3Ha) ppm; C NMR (125 MHz, DMSO–d ) δ 161.1, 156.8, 149.0, 147.1, 141.2,
6
38.1, 132.5, 130.1, 129.7, 129.6, 129.3, 128.9, 127.8, 122.0, 120.7, 120.6, 118.0, 116.7, 24.8
+
ppm; HRMS (ESI): m/z calcd for [M+H] C H BrN O 458.0616; found 458.0619.
2
3
17
5
5.3.3. 2-methyl-3-phenyl-7-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)quinazolin-4(3H)-one (17c): White
1
solid, Yield 77 %; mp: 200–202 °C; H NMR (500 MHz, CDCl ) δ 8.46 (d, J = 8.6 Hz, 1H),
3
8.33 (s, 1H), 8.09 (s, 1H), 8.06–8.03 (m, 1H), 7.87–7.83 (m, 2H), 7.65–7.59 (m, 2H), 7.59–7.54
13
(
m, 1H), 7.35–7.30 (m, 4H), 2.44 (s, 3H), 2.31 (s, 3H) ppm; C NMR (125 MHz, CDCl ) δ
3
1
1
3
61.4, 156.1, 149.0, 148.7, 141.5, 138.7, 137.4, 130.2, 129.7, 129.6, 129.4, 127.9, 127.0, 125.9,
+
22.4, 120.4, 118.3, 117.0, 24.5, 21.4 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O
2
4
20
5
94.1668; found 394.1670.
5.3.4.
7-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-methyl-3-phenylquinazolin-4(3H)-one
1
(
17d): White solid, Yield 69 %; mp: 198–200 °C; H NMR (500 MHz, CDCl ) δ 8.46 (d, J = 8.5
3
Hz, 1H), 8.28 (s, 1H), 8.08 (s, 1H), 8.06–8.02 (m, 1H), 7.9 1–7.86 (m, 2H), 7.65–7.59 (m, 2H),
13
7.59–7.53 (m, 1H), 7.34–7.30 (m, 2H), 7.07–7.02 (m, 2H), 3.89 (s, 3H), 2.30 (s, 3H) ppm;
C
NMR (125 MHz, CDCl ) δ 161.4, 160.0, 156.1, 148.7, 141.5, 137.4, 132.2, 130.2, 129.6, 129.4,
3
127.9, 127.3, 122.5, 120.4, 118.3, 116.9, 116.5, 114.4, 55.4, 24.6 ppm; HRMS (ESI): m/z calcd
+
for [M+H] C H N O 410.1617; found 410.1619.
2
4
20
5
2
5.3.5.
2-methyl-7-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)-3-phenylquinazolin-4(3H)-one
1
(
18a): White solid, Yield 58 %; mp: 203–205 °C; H NMR (500 MHz, CDCl ) δ 8.45 (d, J = 8.5
3
Hz, 1He), 8.25 (s, 1Hg), 8.08 (s, 1Hl), 8.04–8.00 (m, 1Hf), 7.65–7.59 (m, 2Hj), 7.59–7.55 (m,
1Hd), 7.38–7.33 (m, 2Hb), 7.33–7.30 (m, 2Hc), 7.09–7.05 (m, 2Hi), 7.05–7.01(m, 1Hk), 5.36
13
(
s, 2Hh), 2.33 (s, 3Ha) ppm; C NMR (125 MHz, CDCl ) δ 161.2, 158.1, 156.7, 148.1, 141.4,
3
1
2
37.1, 130.2, 129.7, 129.5, 127.9, 121.5, 121.1, 120.5, 118.6, 116.8, 114.8, 113.3, 111.3, 61.8,
+
4.3 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 410.1617; found 410.1621.
2
4
20
5
2
5.3.6.
3-(3-chlorophenyl)-7-(4-((3,5-dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-2-
1
methylquinazolin-4(3H)-one (18b): White solid, Yield 65 %; mp: 255–257 °C. H NMR (500
MHz, CDCl ) δ 8.43 (d, J = 8.6 Hz, 1Hf), 8.22 (s, 1Hh), 8.13–8.09 (m, 1Hb), 8.07–8.02 (m,
3
1Hg), 7.61–7.54 (m, 2Hed), 7.38–7.36 (m, 1Hm), 7.27–7.23 (m, 1Hc), 6.70–6.66 (m, 3Hjl), 5.33
1
8

ACCEPTED MANUSCRIPT
1
3
(
s, 2Hi), 2.40 (s, 3Ha), 2.33 (s, 6Hk) ppm; C NMR (125 MHz, CDCl ) δ 160.8, 158.1, 156.2,
3
1
1
4
47.6, 146.1, 141.6, 139.5, 138.0, 135.9, 131.2, 130.2, 129.5, 128.4, 126.4, 123.3, 120.7, 120.1,
+
18.8, 116.5, 112.5, 61.8, 24.2, 21.5 ppm; HRMS (ESI): m/z calcd for [M+H] C H ClN O
2
6
23
5
2
72.1540; found 472.1544.
5.3.7.
3-(3-chlorophenyl)-2-methyl-7-(4-((4-(tert-pentyl)phenoxy)methyl)-1H-1,2,3-triazol-1-
1
yl)quinazolin-4(3H)-one (18c): White solid, Yield 65 %; mp: 255–257 °C. H NMR (500 MHz,
DMSO–d ) δ 9.24 (s, 1H), 8.30 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 1.8 Hz, 1H), 8.15 (dd, J = 8.6,
6
1
7
7
1
6
5
.9 Hz, 1H), 7.75–7.71 (m, 1H), 7.66–7.60 (m, 2H), 7.56–7.50 (m, 1H), 7.31–7.24 (m, 2H),
.05–6.99 (m, 2H), 5.25 (s, 2H), 2.19 (s, 3H), 1.59 (q, J = 7.4 Hz, 2H), 1.23 (s, 6H), 0.62 (t, J =
13
.4 Hz, 3H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.1, 156.3, 156.1, 148.9, 145.1, 141.8,
6
41.3, 139.4, 134.2, 131.7, 129.8, 129.2, 129.1, 128.0, 127.3, 123.5, 120.5, 118.2, 116.9, 114.7,
+
1.4, 37.4, 36.7, 28.9, 24.6, 9.5 ppm; HRMS (ESI): m/z calcd for [M+H] C H ClN O
2
9
29
5
2
14.2010; found 514.1975.
.4. General reaction procedure for the synthesis of (E)-2-styrylquinazolin-4(3H)-one
5
derivatives and (E)-2-styrylpyrido[2,3-d]pyrimidin-4(3H)-one 4’ (4’a-4’z) and (4’aa-4’aj).
Compound (3’, 1 mmol) (scheme 3) was dissolved in glacial acetic acid on gentle heating, to
which benzaldehyde 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 10 mL 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 4’ (4’a-4’z) and (4’aa-4’aj) in 48–78% yields. All the newly synthesized
1
13
compounds were characterized by H NMR, C NMR and HRMS (ESI). Some representative
potent compounds were also analysed for purity, which showed >95 %, by using Agilent
Infinity-2 1260 series instrument (UV–Vis diode-array detector) at 258–268 nm. The synthesis
of the final compounds 4’ (4’a-4’z) and (4’aa-4’aj) was followed by general procedure given
above.
5.4.1. (E)-methyl 2-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)acetate (4’a): Yellow solid,
1
Yield 52 %; mp: 144–146 °C. H NMR (500 MHz, DMSO–d + 2-3 drops of CDCl ) δ 8.16 (dd,
6
3
J = 8.0, 1.1 Hz, 1Hg), 7.99 (d, J = 15.3 Hz, 1Hc), 7.97–7.94 (m, 2Hdf), 7.85–7.80 (m, 1He),
7.81–7.77 (m, 2Hj), 7.75–7.72 (m, 1Hh), 7.53–7.48 (m, 2Hi), 5.20 (s, 2Hb), 3.76 (s, 3Ha) ppm;
1
3
C NMR (125 MHz, DMSO–d + CDCl ) δ 168.8, 161.6, 151.8, 147.4, 139.9, 139.4, 134.9,
6
3
1
9

ACCEPTED MANUSCRIPT
132.7, 129.1, 127.6, 127.1, 126.7, 122.9, 120.2, 118.8, 112.3, 52.7, 45.1 ppm; HRMS (ESI):
+
m/z calcd for [M+H] C H N O 346.1191; found 346.1194.
2
0
16
3
3
5.4.2.
(E)-4-(2-(3-(4-(tert-butyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile
1
(
4’b): Yellow solid, Yield 60 %; mp: 211-213 °C. H NMR (500 MHz, DMSO–d ) δ 8.15 (d, J =
6
7.9 Hz, 1H), 7.92–7.85 (m, 2H), 7.84–7.76 (m, 3H), 7.64–7.60 (m, 2H), 7.57 (t, J = 7.5 Hz, 1H),
1
3
7
.54–7.50 (m, 2H), 7.41–7.35 (m, 2H), 6.46 (d, J = 15.6 Hz, 1H), 1.38 (s, 9H) ppm; C NMR
(
125 MHz, DMSO–d ) δ 161.7, 152.2, 151.6, 147.7, 139.9, 137.0, 135.3, 134.6, 133.4, 128.9,
6
128.5, 127.8, 127.5, 127.0, 126.9, 124.6, 124.2, 121.2, 119.0, 112.0, 35.1, 31.6 ppm; HRMS
+
(
ESI): m/z calcd for [M+H] C H N O 406.1919; found 406.1922.
2
7
24
3
5.4.3. (E)-4-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-2-hydroxybenzoic acid (4’c): Yellow
1
solid, Yield 48 %; mp: 138-140 °C. H NMR (500 MHz, DMSO–d ) δ 9.93 (s, 1Hb), 8.18–8.13
6
(m, 1Hf), 7.95–7.87 (m, 2Hkd), 7.86–7.81 (m, 2Hm), 7.79 (d, J = 8.1 Hz, 1Hh), 7.61–7.53 (m,
2Hl), 7.44–7.36 (m, 1Hg), 7.01–6.96 (m, 1Hi), 6.89–6.83 (d, J = 8.4 Hz, 2Hea), 6.55 (d, J = 15.6
1
3
13
Hz, 1Hj) ppm; C NMR (125 MHz, DMSO–d ) δ C NMR (125 MHz, DMSO–d ) δ 161.5,
6
6
158.8, 151.3, 147.7, 139.9, 138.2, 137.1, 135.3, 133.4, 130.9, 129.1, 128.6, 127.8, 127.4, 127.0,
+
124.0, 121.3, 119.8, 119.1, 116.9, 116.4, 112.0 ppm; HRMS (ESI): m/z calcd for [M+H]
C H N O 410.1141; found 410.1139; HPLC purity 96.3%.
24
16
3
4
5.4.4. (E)-2-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl)-5-hydroxybenzoic acid (4’d): Yellow
1
solid, Yield 52 %; mp: 258–260 °C. H NMR (500 MHz, DMSO–d ) δ 12.93 (s, 1H), 10.28 (s,
6
1H), 8.13–8.10 (m, 1H), 7.91–7.86 (m, 2H), 7.84–7.77 (m, 3H), 7.64–7.58 (m, 2H), 7.56–7.50
1
3
(
m, 2H), 7.35–7.30 (m, 1H), 7.18–7.13 (m, 1H), 6.54 (d, J = 15.6 Hz, 1H) ppm; C NMR (125
MHz, DMSO–d ) δ 166.0, 161.9, 158.4, 152.1, 147.8, 139.9, 137.2, 135.2, 133.3, 132.2, 128.7,
6
128.5, 128.0, 127.7, 127.2, 126.9, 124.0, 121.2, 120.6, 119.1, 118.4, 112.0 ppm; HRMS (ESI):
+
m/z calcd for [M+H] C H N O 410.1141; found 410.1145.
2
4
16
3
4
5.4.5.
(E)-4-(2-(5-fluoro-3-(3-hydroxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’e): Yellow solid, Yield 49 %; mp: 188-190 °C. H NMR (500 MHz,
DMSO–d ) δ 9.93 (s, 1Hb), 7.91 (d, J = 15.6 Hz, 1Hj), 7.88–7.79 (m, 3Hgl), 7.61–7.55 (m,
6
3Hfk), 7.45–7.35 (m, 1Hh), 7.33–7.37 (m, 1Hd), 7.00–6.93 (m, 1He), 6.91–6.81 (m, 2Hac), 6.49
1
3
(
d, J = 15.6 Hz, 1Hi) ppm; C NMR (125 MHz, DMSO–d ) δ 160.6 (d, J = 263.4 Hz), 158.3,
6
1
58.0 (d, J = 4.0 Hz), 151.8, 149.3, 139.2, 137.2 (d, J = 17.0 Hz), 135.3 (d, J = 10.7 Hz), 132.9,
30.4, 128.1, 123.4 (d, J = 3.6 Hz), 123.2, 119.3, 118.6, 116.4, 115.9, 113.2 (d, J = 20.5 Hz),
1
2
0

ACCEPTED MANUSCRIPT
+
1
11.7, 110.3, 110.3 ppm; HRMS (ESI): m/z calcd for [M+H] C H FN O 384.1148; found
2
3
15
3
2
384.1153; HPLC purity 98.3 %.
5.4.6.
(E)-4-(2-(5-fluoro-4-oxo-3-(3-(prop-2-yn-1-yloxy)phenyl)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’f): Yellow solid, Yield 51 %; mp: 220–222 °C. H NMR (500 MHz,
DMSO–d ) δ 7.91 (d, J = 15.6 Hz, 1H), 7.89–7.79 (m, 3H), 7.62–7.59 (m, 1H), 7.58–7.51 (m,
6
3
4
1
1
1
H), 7.34–7.28 (m, 1H), 7.23–7.16 (m, 2H), 7.11–7.07 (m, 1H), 6.46 (d, J = 15.6 Hz, 1H), 4.89–
1
3
.84 (m, 2H), 3.55 (m, 1H) ppm; C NMR (125 MHz, DMSO–d ) δ 160.7 (d, J = 263.4 Hz),
6
58.1, 151.9, 149.4, 139.3, 137.6, 137.5, 135.5 (d, J = 11.0 Hz), 133.3, 133.0, 130.6, 130.0,
28.3, 123.5 (d, J = 3.7 Hz), 123.2, 122.0, 118.6, 116.0 (d, J = 2.9 Hz), 113.3 (d, J = 20.5 Hz),
+
11.8, 110.4 (d, J = 5.3 Hz), 78.9, 78.5, 55.9 ppm; HRMS (ESI): m/z calcd for [M+H]
C H FN O 422.1305; found 422.1309; HPLC purity 95.9 %.
26
17
3
2
5.4.7.
(E)-4-(2-(3-(3-(allyloxy)phenyl)-5-fluoro-4-oxo-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’g): Yellow solid, Yield 56 %; mp: 200–202 °C. H NMR (500 MHz,
DMSO–d ) δ 7.91 (d, J = 15.6 Hz, 1Hl), 7.89–7.80 (m, 3Hnh), 7.63–7.54 (m, 3Hmj), 7.51 (t, J =
6
8.1 Hz, 1Hi), 7.34–7.27 (m, 1Hf), 7.19–7.12 (m, 2Hga), 7.06–7.01 (m, 1He), 6.47 (d, J = 15.6
Hz, 1Hk), 6.09–5.96 (m, 1Hc), 5.42 –5.35 (m, 1Hd), 5.26–5.19 (m, 1Hd’), 4.65–4.58 (m, 2Hb)
13
ppm; C NMR (125 MHz, DMSO–d ) δ 162.2, 160.1, 159.5, 158.5 (d, J = 4.2 Hz), 152.3,
6
149.8, 139.7, 137.9 (d, J = 23.0 Hz), 135.9 (d, J = 10.5 Hz), 133.8, 133.4, 130.9, 128.7, 123.9 (d,
J = 3.7 Hz), 123.7, 121.7, 119.0, 118.2, 116.4, 116.0, 113.7 (d, J = 20.7 Hz), 112.2, 110.8, 110.8,
+
6
8.9 ppm; HRMS (ESI): m/z calcd for [M+H] C H FN O 424.1461; found 424.1466.
2
6
19
3
2
5.4.8.
(E)-4-(2-(3-(3-ethynylphenyl)-5-fluoro-4-oxo-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’h): Yellow solid, Yield 69 %; mp: 171-173 °C. H NMR (500 MHz,
DMSO–d ) δ 7.91 (d, J = 15.6 Hz, 1H), 7.89–7.84 (m, 1H), 7.83–7.79 (m, 2H), 7.71–7.67 (m,
6
2H), 7.64 (t, J = 7.8 Hz, 1H), 7.62–7.57 (m, 3H), 7.57–7.53 (m, 1H), 7.33–7.27 (m, 1H), 6.43 (d,
1
3
J = 15.6 Hz, 1H), 4.36 (s, 1H) ppm; C NMR (125 MHz, DMSO–d ) δ 162.1, 160.0, 158.6,
6
152.1, 149.7, 139.6, 138.0, 137.2, 136.0, 135.9, 133.3, 133.2, 132.8, 130.6, 130.3, 128.7, 123.9,
123.6, 123.5, 119.0, 113.8, 113.7, 112.2, 110.7, 110.7, 82.9, 82.7 ppm; HRMS (ESI): m/z calcd
+
for [M+H] C H FN O 392.1199; found 392.1202; HPLC purity 98.8 %.
2
5
15
3
5.4.9. (E)-3-(2-(4-cyanostyryl)-5-fluoro-4-oxoquinazolin-3(4H)-yl)benzonitrile (4’i): Yellow
1
solid, Yield 70 %; mp: 180-182 °C. H NMR (500 MHz, DMSO–d ) δ 8.12-8.06 (m, 2H), 7.93
6
(d, J = 15.5 Hz, 1H), 7.90–7.86 (m, 2H), 7.85 (d, J = 7.8 Hz, 1H), 7.82–7.77 (m, 2H), 7.67–7.63
13
(
m, 2H), 7.62 (d, J = 8.2 Hz, 1H), 7.35–7.27 (m, 1H), 6.47 (d, J = 15.5 Hz, 1H) ppm; C NMR
2
1

ACCEPTED MANUSCRIPT
(
125 MHz, DMSO–d ) δ 162.1, 160.0, 158.6, 152.0, 149.6, 139.5, 138.4, 137.7, 136.2, 136.1,
6
1
1
3
34.8, 133.8, 133.6, 133.2, 131.5, 129.0, 123.9, 123.9, 123.4, 119.0, 118.5, 114.0, 113.8, 113.2,
+
12.2, 110.6, 110.5 ppm; HRMS (ESI): m/z calcd for [M+H] C H FN O 393.1151; found
24
14
4
93.1156.
5.4.10. (E)-4-(2-(3-(naphthalen-1-yl)-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (4’j):
1
Yellow solid, Yield 74 %; mp: 199-201 °C; H NMR (500 MHz, CDCl ) δ 8.34 (m, 1H), 8.08 (d,
3
J = 8.3 Hz, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 15.5 Hz, 1H), 7.90–7.84 (m, 2H), 7.69–
7
1
1
1
4
.63 (m, 1H), 7.60–7.52 (m, 2H), 7.51–7.45 (m, 5H), 7.26–7.18 (d, J = 8.3 Hz, 2H), 6.28 (d, J =
13
5.5 Hz, 1H) ppm; C NMR (125 MHz, CDCl ) δ 162.1, 151.5, 147.7, 139.4, 137.9, 136.0,
3
34.5, 133.2, 132.4, 130.4, 130.1, 128.8, 128.1, 128.0, 127.6, 127.4, 127.3, 127.2, 127.0, 125.7,
+
22.9, 121.9, 121.1, 118.4, 112.5 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O
27
18
3
00.1450; found 400.1453.
5.4.11. (E)-4-(2-(3-(4-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (4’k):
1
Yellow solid, Yield 71 %; mp: 238–240 °C. H NMR (500 MHz, DMSO–d ) δ 8.22–8.18 (m,
6
1
7
1
1
Hd), 7.95–7.85 (m, 6Hkefgi), 7.77–7.72 (m, 1Hh), 7.60–7.52 (m, 2Hj), 7.38–7.31(m, 2Ha),
1
3
.18–7.11(m, 2Hb), 5.65 (s, 2Hc) ppm; C NMR (125 MHz, DMSO–d ) δ 162.8, 161.9, 160.8,
6
52.0, 147.5, 140.0, 138.7, 135.2, 133.8, 133.2, 129.3 (d, J = 8.3 Hz), 129.1, 127.7, 127.4, 127.1,
23.9, 120.6, 119.1, 116.0 (d, J = 21.5 Hz), 112.1, 45.1 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H FN O 382.1355; found 382.1359.
24
17
3
5.4.12.
(E)-4-(2-(3-(3,4-dichlorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile
1
(
4’l): Yellow solid, Yield 66 %; mp: 231–233 °C. H NMR (500 MHz, DMSO–d ) δ 8.21–8.18
6
(m, 1H), 7.98–7.85 (m, 6H), 7.78–7.74 (m, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.60–7.50 (m, 3H),
1
3
7
1
1
.22 (dd, J = 8.4, 2.0 Hz, 1H), 5.64 (s, 2H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.9,
6
51.9, 147.5, 140.0, 139.0, 138.8, 135.3, 133.2, 131.7, 131.4, 130.5, 129.5, 129.2, 127.8, 127.5,
+
27.4, 127.1, 123.7, 120.6, 119.1, 112.1, 45.0 ppm; HRMS (ESI): m/z calcd for [M+H]
C H Cl N O 432.0670; found 432.0694.
24
16
2
3
5.4.13.
(E)-4-(2-(4-oxo-3-((4-(trifluoromethyl)phenyl)amino)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’m): Yellow solid, Yield 63 %; mp: 200-202 °C. H NMR (500 MHz,
DMSO–d ) δ 9.77 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 15.8 Hz, 1H), 7.92 (t, J = 7.4
6
1
3
Hz, 1H), 7.87–7.78 (m, 5H), 7.61–7.48 (m, 4H), 6.90 (d, J = 8.3 Hz, 2H) ppm; C NMR (125
MHz, DMSO–d ) δ 160.4, 153.8, 151.0, 147.1, 139.8, 139.1, 135.6, 133.3, 129.0, 128.1, 127.5,
6
2
2

ACCEPTED MANUSCRIPT
127.1, 126.9, 126.2, 124.0, 121.9, 121.7, 121.3, 121.0, 119.1, 113.3, 112.2 ppm; HRMS (ESI):
+
m/z calcd for [M+H] C H F N O 433.1276; found 433.1283.
2
4
16
3
4
5.4.14. (E)-4-(2-(3-(2-(1H-indol-3-yl)ethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile
1
(
4’n): Yellow solid, Yield 55 %; mp: 229–231 °C. H NMR (500 MHz, DMSO–d ) δ 10.83 (s,
6
1
3
1
=
H), 8.20 (d, J = 7.8 Hz, 1H), 7.83 (t, J = 7.4 Hz, 1H), 7.75 (d, J = 7.4 Hz, 2H), 7.71–7.59 (m,
H), 7.54 (t, J = 7.4 Hz, 1H), 7.40–7.34 (m, 2H), 7.30 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 7.4 Hz,
H), 7.07 (m, 1H), 7.01 (t, J = 7.3 Hz, 1H), 6.96–6.89 (m, 1H), 4.49 (t, J = 5.8 Hz, 2H), 3.15 (t, J
1
3
6.3 Hz, 2H) ppm; C NMR (125 MHz, DMSO–d ) δ 160.5, 151.3, 146.4, 139.0, 136.7, 135.7,
6
133.8, 131.9, 127.7, 126.5, 126.5, 126.0, 125.8, 123.1, 122.7, 120.7, 119.6, 118.1, 118.1, 117.4,
+
1
11.0, 110.5, 109.7, 43.4, 23.6 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 417.1715;
2
7
21
4
found 417.1719.
5.4.15. (E)-4-(2-(4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (4’o):
1
Yellow solid, Yield 67 %; mp: 110–112 °C; H NMR (500 MHz, DMSO–d ) δ 8.79–8.75 (m,
6
1
7
1
1
1
3
Hb), 8.72–8.69 (m, 1He), 8.19–8.12 (m, 1Hh), 8.03–7.98 (m, 1Hf), 7.95–7.87 (m, 2Hgj), 7.85–
.78 (m, 3Hla), 7.68 (dd, J = 8.0, 4.8 Hz, 1Hd), 7.64 (d, J = 8.2 Hz, 2Hk), 7.59 (t, J = 7.5 Hz,
1
3
Hc), 6.52 (d, J = 15.5 Hz, 1Hi) ppm; C NMR (125 MHz, DMSO–d ) δ 161.8, 151.3, 150.7,
6
50.1, 147.7, 139.7, 137.7, 137.5, 135.6, 134.0, 133.3, 128.8, 127.9, 127.7, 127.0, 124.9, 123.9,
+
21.0, 119.1, 112.1 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 351.1246; found
2
2
15
4
51.1249.
5.4.16. (E)-methyl 2-(2-(4-cyanostyryl)-4-oxo-6-(prop-2-yn-1-yloxy)quinazolin-3(4H)-yl)acetate
1
1
(
4’p): Yellow solid, Yield 61 %; mp: 210–212 °C; H NMR (500 MHz, DMSO–d ) H NMR
6
(
500 MHz, DMSO–d ) δ 8.06–8.01 (m, 2H), 7.96–7.89 (m, 3H), 7.73 (d, J = 9.0 Hz, 1H), 7.62
6
(d, J = 2.9 Hz, 1H), 7.60–7.55 (m, 1H), 7.54–7.50 (m, 1H), 5.19 (s, 2H), 4.97 (d, J = 2.1 Hz,
1
3
2
H), 3.72 (s, 3H), 3.64 (t, J = 2.2 Hz, 1H) ppm; C NMR (125 MHz, DMSO–d ) δ 169.2, 161.3,
6
156.3, 150.2, 142.4, 140.2, 138.6, 133.1, 129.6, 129.3, 125.6, 123.2, 120.9, 111.9, 108.3, 79.3,
+
7
9.2, 56.5, 52.9, 45.5, 31.1 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 400.1297;
2
3
18
3
4
found 400.1300.
5.4.17.
(E)-methyl-2-(6-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-(4-
cyanostyryl)-4-oxoquinazolin-3(4H)-yl)acetate (4’q): Yellow solid, Yield 51 %; mp: 198–200
1
°
C; H NMR (500 MHz, DMSO–d ) δ 9.03 (s, 1H), 8.27–8.22 (m, 1H), 8.06–8.02 (m, 2H), 8.01–
6
7
2
.97 (m, 1H), 7.96–7.90 (m, 3H), 7.76–7.67 (m, 3H), 7.61–7.55 (m, 2H), 5.41 (s, 2H), 5.20 (s,
1
3
H), 3.73 (s, 3H) ppm; C NMR (125 MHz, DMSO–d ) δ 168.8, 160.9, 158.0, 156.8, 156.0,
6
2
3

ACCEPTED MANUSCRIPT
1
1
43.8, 141.9, 139.8, 138.1, 133.6 (d, J = 3.4 Hz), 132.7, 129.3, 128.9, 125.0, 123.5, 122.9, 122.6,
21.1 (d, J = 8.0 Hz), 120.9, 120.8, 120.6, 118.8, 118.3, 118.2, 111.5, 107.9, 61.7, 52.5, 45.1
+
ppm; HRMS (ESI): m/z calcd for [M+H] C H ClFN O 571.1297; found 571.1300.
2
9
21
6
4
5.4.18. (E)-6-(4-methoxyphenyl)-3-phenyl-2-styrylquinazolin-4(3H)-one (4’r): Yellow solid,
1
Yield 55 %; mp: 220–222 °C. H NMR (500 MHz, DMSO–d ) δ 8.29 (d, J = 2.0 Hz, 1H), 8.19–
6
8.15 (m, 1H), 7.90 (d, J = 15.5 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.77–7.71 (m, 2H), 7.67–7.57
(m, 3H), 7.52–7.46 (m, 2H), 7.40–7.30 (m, 5H), 7.12–7.04 (m, 2H), 6.33 (d, J = 15.6 Hz, 1H),
1
3
3
1
1
4
.82 (s, 3H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.8, 159.8, 151.5, 146.7, 139.1, 138.4,
6
37.5, 135.3, 133.3, 131.6, 130.3, 130.2, 129.7, 129.5, 129.4, 128.4, 128.4, 127.9, 123.3, 121.4,
+
20.5, 115.1, 55.7 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 431.1759; found
2
9
23
2
2
31.1765.
5.4.19.
(E)-4-(2-(4-oxo-3-phenyl-7-(prop-2-yn-1-ylamino)-3,4-dihydroquinazolin-
1
2
yl)vinyl)benzonitrile (4’s): Yellow solid, Yield 53 %; mp: 200–202 °C. H NMR (500 MHz,
DMSO–d ) δ 7.91–7.73 (m, 4H), 7.66–7.48 (m, 5H), 7.44–7.38 (d, J = 6.6 Hz, 2H), 7.10 (t,
6
1H), 6.90–6.85 (m, 1H), 6.79 (s, 1H), 6.43 (d, J = 15.5 Hz, 1H), 4.09–4.01 (m, 2H), 3.19 (s, 1H).
13
ppm; C NMR (125 MHz, DMSO–d ) δ 161.1, 153.6, 151.2, 149.7, 140.0, 137.6, 136.6, 133.3,
6
130.0, 129.6, 129.5, 128.5, 127.8, 124.3, 119.1, 115.8, 111.9, 110.8, 105.7, 81.7, 74.0, 32.2 ppm;
+
HRMS (ESI): m/z calcd for [M+H] C H N O 403.1559; found 403.1567.
26
19
4
5
6
1
2
1
1
.4.20. (E)-3-(3-chlorophenyl)-7-phenyl-2-styrylquinazolin-4(3H)-one (4’t): Yellow solid, Yield
1
0 %; mp: 219–221°C. H NMR (500 MHz, DMSO–d ) δ 8.20 (d, J = 8.3 Hz, 1He), 8.04 (d, J =
6
.6 Hz, 1Ha), 7.92–7.85 (m, 4Hjnl), 7.74–7.62 (m, 3Hfm), 7.60–7.53 (m, 4Hgh), 7.52–7.46 (m,
1
3
Hic), 7.42–7.36 (m, 2Hdb), 6.40 (d, J = 15.5 Hz, 1Hk) ppm; C NMR (125 MHz, DMSO–d ) δ
6
61.5, 151.8, 148.3, 146.8, 139.1, 138.7, 138.4, 134.5, 134.2, 132.5, 132.2, 131.8, 131.7, 130.0,
29.9, 129.7, 129.2, 128.5 127.7, 127.7, 125.9, 125.1, 123.6, 121.1, 119.9 ppm; HRMS (ESI):
+
m/z calcd for [M+H] C H ClN O 460.1216; found 460.1219.
2
9
19
3
5.4.21. (E)-4-(2-(4-oxo-3-phenyl-7-(4-phenyl-1H-1,2,3-triazol-1-yl)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’u): Yellow solid, Yield 58 %; mp: 225–227°C H NMR (500 MHz,
DMSO–d ) δ 9.64 (s, 1H), 8.39–8.32 (m, 2H), 8.22–8.17 (m, 1H), 7.99 (d, J = 7.4 Hz, 2H), 7.93
6
(d, J = 15.6 Hz, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.69–7.58 (m, 3H), 7.58–7.46 (m, 6H), 7.41 (t, J =
1
3
7
.1 Hz, 1H), 6.48 (d, J = 15.6 Hz, 1H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.0, 152.9,
6
148.9, 148.2, 141.4, 139.5, 137.9, 137.0, 133.4, 130.4, 130.2, 130.0, 129.8, 129.5, 129.4, 128.9,
2
4

ACCEPTED MANUSCRIPT
128.6, 125.9, 123.7, 120.7, 120.3, 119.0, 118.4, 117.1, 112.2 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H N O 493.1777; found 493.1780.
31
21
6
5.4.22.
(E)-4-(2-(7-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-4-oxo-3-phenyl-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (4’v): Yellow solid, Yield 53 %; mp: 229–231°C. H
NMR (500 MHz, DMSO–d ) δ 9.54 (s, 1H), 8.39–8.34 (m, 2H), 8.20 (dd, J = 8.7, 2.1 Hz, 1H),
6
7
2
1
1
.98–7.88 (m, 3H), 7.86–7.81 (m, 2H), 7.68–7.58 (m, 3H), 7.58–7.50 (m, 4H), 7.14–7.07 (m,
13
H), 6.50 (d, J = 15.6 Hz, 1H), 3.83 (s, 3H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.0,
6
59.9, 152.9, 151.2, 148.9, 148.2, 141.5, 139.6, 137.9, 137.0, 133.4, 130.2, 130.0, 129.4, 128.7,
27.3, 123.8, 122.9, 120.7, 119.3, 119.0, 118.4, 117.0, 115.0, 112.2, 55.7 ppm; HRMS (ESI):
+
m/z calcd for [M+H] C H N O 523.1882; found 523.1887.
3
2
23
6
2
5.4.23.
(E)-4-(2-(3-benzyl-4-oxo-6-(prop-2-yn-1-yloxy)-3,4-dihydroquinazolin-2-
1
yl)vinyl)benzonitrile (4’w): Yellow solid, Yield 62 %; mp: 171–173°C. H NMR (500 MHz,
DMSO–d ) δ 7.92–7.81 (m, 5H), 7.75–7.66 (m, 2H), 7.56–7.47 (m, 2H), 7.37–7.20 (m, 5H),
6
1
3
5
1
1
.67 (s, 2H), 4.99 (d, J = 1.9 Hz, 2H), 3.67 (t, 1H) ppm; C NMR (125 MHz, DMSO–d ) δ
6
61.6, 156.3, 150.2, 142.4, 140.2, 137.8, 137.7, 133.2, 129.6, 129.2, 128.9, 127.8, 127.0, 125.34,
23.9, 121.3, 119.2, 111.9, 108.5, 79.3, 79.3, 56.5, 45.8 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H N O 418.1555; found 418.1559.
27
20
3
2
5.4.24.
(E)-4-(2-(3-benzyl-6-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-
oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile (4’x): Yellow solid, Yield 64 %; mp: 229–231
1
°
C; H NMR (500 MHz, DMSO–d ) δ 9.04 (s, 1H), 8.28–8.24 (m, 1H), 8.03–7.97 (m, 1H), 7.91–
6
7
7
1
1
.84 (m, 4H), 7.84–7.78 (m, 2H), 7.76–7.66 (m, 2H), 7.60–7.50 (m, 2H), 7.35–7.30 (m, 2H),
13
.30–7.20 (m, 3H), 5.68 (s, 2H), 5.43 (s, 2H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.6,
6
57.2, 150.1, 144.2, 142.3, 140.2, 137.7 (d, J = 5.7 Hz), 134.0, 133.2, 129.7, 129.2, 129.0, 127.8,
27.0, 125.2, 125.0, 123.9, 123.0, 121.6, 121.5 (d, J = 8.2 Hz), 121.4, 121.2, 118.6 (d, J = 22.6
+
Hz), 111.9, 108.5, 62.1, 45.8 ppm; HRMS (ESI): m/z calcd for [M+H] C H ClFN O
33
23
6
2
589.1555; found 589.1563.
5.4.25.
(E)-4-(2-(3-benzyl-6-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-oxo-3,4-
1
dihydroquinazolin-2-yl)vinyl)benzonitrile (4’y): Yellow solid, Yield 68 %; mp: 200–202 °C. H
NMR (500 MHz, DMSO–d ) δ 9.08 (s, 1H), 8.08 (t, J = 2.0 Hz, 1H), 7.98–7.93 (m, 1H), 7.88–
6
7
7
5
.82 (m, 5H), 7.80 (d, J = 3.0 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.65 (t, J = 8.1 Hz, 1H), 7.61–
.56 (m, 2H), 7.53 (d, J = 15.4 Hz, 1H), 7.35–7.26 (m, 4H), 7.26–7.21 (m, 1H), 5.68 (s, 2H),
1
3
.42 (s, 2H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.6, 157.2, 150.1, 144.2, 142.3, 140.2,
6
2
5

ACCEPTED MANUSCRIPT
1
1
38.0, 137.7, 137.7, 134.7, 133.2, 132.1, 129.7, 129.2, 129.1, 128.9, 127.8, 127.0, 125.2, 123.9,
23.7, 121.4, 120.5, 119.2, 119.2, 111.9, 108.5, 62.1, 45.8 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H ClN O 571.1649; found 571.1650.
33
24
6
2
5.4.26.
(E)-4-(2-(3-(3-hydroxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’z): Yellow solid, Yield 73 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.99 (s, 1Hb), 9.00 (dd, J = 4.6, 2.0 Hz, 1Hh), 8.49 (dd, J = 7.8, 2.0 Hz, 1Hf), 8.02
6
(d, J = 15.6 Hz, 1Hj), 7.83 (d, J = 8.4 Hz, 2Hl), 7.58 (d, J = 8.3 Hz, 2Hk), 7.55 (dd, J = 7.8, 4.6
Hz, 1Hj), 7.42 (t, J = 8.3 Hz, 1Hd), 7.06–6.94 (m, 1He), 6.95–6.84 (m, 2Hac), 6.55 (d, J = 15.6
1
3
Hz, 1Hi) ppm; C NMR (125 MHz, DMSO–d ) δ 162.2, 158.8, 157.6, 156.7, 154.2, 139.6,
6
138.6, 137.8, 136.5, 133.4, 131.0, 128.8, 123.5, 122.8, 119.7, 119.0, 117.1, 116.7, 116.3, 112.3
+
ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 367.1195; found 367.1198.
22
15
4
2
5.4.27.
(E)-4-(2-(3-(3-ethynylphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’aa): Yellow solid, Yield 70 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.03 (d, J = 2.6 Hz, 1H), 8.52 (d, J = 6.8 Hz, 1H), 8.04 (d, J = 15.5 Hz, 1H), 7.84
6
(d, J = 8.0 Hz, 2H), 7.71 (s, 2H), 7.65 (d, J = 7.1 Hz, 3H), 7.61–7.53 (m, 2H), 6.52 (d, J = 15.5
13
Hz, 1H), 4.37 (s, 1H) ppm; C NMR (125 MHz, DMSO–d ) δ 161.3, 156.6, 155.8, 153.1,
6
1
1
3
38.4, 137.8, 136.1, 135.4, 132.3, 132.2, 131.5, 129.6, 129.1, 127.9, 122.5, 122.4, 121.9, 118.0,
+
15.6, 111.3, 81.8, 81.8 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 375.1246; found
2
4
15
4
75.1253.
5.4.28.
(E)-3-(2-(4-cyanostyryl)-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)benzonitrile (4’ab):
1
Yellow solid, Yield 66 %; mp: 229–231 °C. H NMR (500 MHz, DMSO–d ) δ 9.03 (dd, J = 4.5,
6
2.0 Hz, 1Hg), 8.48 (dd, J = 7.8, 2.0 Hz, 1He), 8.15 (d, J = 1.7 Hz, 1Hf), 8.10 (dd, J = 10.9, 3.2
Hz, 1Hb), 8.03 (d, J = 15.4 Hz, 1Hi), 7.96–7.92 (m, 1Hd), 7.86 (t, J = 7.9 Hz, 1Ha), 7.82 (d, J =
8
1
1
.4 Hz, 2Hj), 7.68 (d, J = 8.4 Hz, 2Hk), 7.56 (dd, J = 7.8, 4.6 Hz, 1Hc), 6.55 (d, J = 15.5 Hz,
1
3
Hh) ppm; C NMR (125 MHz, DMSO–d ) δ 162.4, 157.6, 157.0, 154.0, 139.4, 139.3, 137.7,
6
36.5, 134.7, 133.9, 133.4, 133.3, 131.6, 129.2, 123.3, 123.0, 119.0, 118.5, 116.5, 113.2, 112.4
+
ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 376.1198; found 376.1200.
23
14
5
5.4.29.
(E)-4-(2-(3-(4-cyanophenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’ac): Yellow solid, Yield 63 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.04 (dd, J = 4.5, 1.9 Hz, 1H), 8.53 (dd, J = 7.8, 1.7 Hz, 1H), 8.13 (d, J = 8.3 Hz,
6
2
2
H), 8.05 (d, J = 15.5 Hz, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.80–7.74 (m, 2H), 7.70 (d, J = 8.2 Hz,
13
H), 7.58 (dd, J = 7.8, 4.6 Hz, 1H), 6.51 (d, J = 15.5 Hz, 1H) ppm; C NMR (125 MHz,
2
6

ACCEPTED MANUSCRIPT
DMSO–d ) δ 162.2, 157.6, 156.9, 153.7, 141.2, 139.4, 136.5, 134.4, 133.3, 130.8, 130.7, 129.2
6
+
123.2, 123.0, 119.0, 118.8, 116.5, 112.9, 112.4 ppm; HRMS (ESI): m/z calcd for [M+H]
C H N O 376.1198; found 376.1206.
23
14
5
5.4.30.
(E)-4-(2-(3-(4-mercaptophenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’ad ): Yellow solid, Yield 65 %; mp: 210–212°C. H NMR (500 MHz,
1
DMSO–d ) δ H NMR (500 MHz, DMSO) δ 9.02 (d, J = 2.2 Hz, 1H), 8.52 (d, J = 7.4 Hz, 1H),
6
7.96 (d, J = 15.5 Hz, 1H), 7.83–7.78 (m, 4H), 7.60–7.54 (m, 6H), 6.49 (d, J = 15.5 Hz, 1H) ppm;
1
3
C NMR (125 MHz, DMSO–d ) δ 162.4, 157.6, 156.9, 139.4, 138.9, 137.3, 136.5, 136.0, 133.3,
6
130.5, 128.9, 128.5, 128.3, 123.3, 122.9, 118.9, 116.6, 112.3 ppm; HRMS (ESI): m/z calcd for
+
[
M+H] C H N OS 383.0966; found 383.0970; HPLC purity 98.7 %.
22
15
4
5.4.31.
(E)-4-(2-(3-(4-(tert-butyl)phenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’ae): Yellow solid, Yield 78 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.02 (m, 1Hf), 8.52 (m, 1Hd), 8.00 (d, J = 15.6 Hz, 1Hh), 7.84 (d, J = 8.4 Hz, 2Hj),
6
7
9
1
.70–7.59 (m, 2Hi), 7.57 (m, 3Hae), 7.45–7.34 (m, 2Hb), 6.49 (d, J = 15.6 Hz, 1Hg), 1.38 (s,
13
Hc) ppm; C NMR (125 MHz, DMSO–d ) δ 162.4, 157.7, 156.8, 154.5, 152.5, 139.7, 138.5,
6
36.5, 134.2, 133.4, 128.8, 128.7, 127.0, 123.8, 122.9, 119.0, 116.6, 112.3, 35.1, 31.6 ppm;
+
HRMS (ESI): m/z calcd for [M+H] C H N O 407.1872; found 407.1880.
26
23
4
5.4.32.
(E)-4-(2-(3-(2,3-dimethylphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’af): Yellow solid, Yield 69 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.05 (dd, J = 4.6, 2.0 Hz, 1Hh), 8.54 (dd, J = 7.9, 2.0 Hz, 1Hf), 8.07 (d, J = 15.5
6
Hz, 1Hj), 7.84 (d, J = 8.4 Hz, 2Hl), 7.62–7.57 (m, 3Hkg), 7.41 (d, J = 7.4 Hz, 1He), 7.32 (t, J =
7.7 Hz, 1Hd), 7.25 (d, J = 7.5 Hz, 1Hc), 6.45 (d, J = 15.5 Hz, 1Hi), 2.37 (s, 3Hb), 1.95 (s, 3Ha)
13
ppm; C NMR (125 MHz, DMSO–d ) δ 161.9, 157.8, 157.0, 154.4, 139.5, 139.3, 138.9, 136.6,
6
135.7, 134.8, 133.4, 131.5, 128.9, 127.2, 126.8, 123.0, 122.8, 119.0, 116.5, 112.4, 20.5, 14.1
+
ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 379.1559; found 379.1561.
2
4
19
4
5.4.33.
(E)-4-(2-(3-(2,5-dimethylphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’ag): Yellow solid, Yield 71 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.05 (dd, J = 4.6, 2.0 Hz, 1H), 8.54 (dd, J = 7.9, 2.0 Hz, 1H), 8.09 (d, J = 15.5 Hz,
6
1
H), 7.84 (d, J = 8.4 Hz, 1H), 7.63–7.57 (m, 1H), 8.51–6.95 (m, 3H), 7.40 (d, J = 7.9 Hz, 1H),
.33 (dd, J = 7.8, 1.1 Hz, 1H), 7.25 (s, 1H), 6.47 (d, J = 15.5 Hz, 1H), 2.35 (s, 3H), 2.01 (s, 3H)
7
13
ppm; C NMR (125 MHz, DMSO–d ) δ 161.8, 157.7, 157.0, 154.2, 139.5, 137.5, 136.6, 135.6,
6
2
7

ACCEPTED MANUSCRIPT
133.4, 132.8, 131.5, 131.0, 129.5, 128.9, 123.0, 122.6, 119.0, 116.5, 112.4, 20.9, 17.0 ppm;
+
HRMS (ESI): m/z calcd for [M+H] C H N O 379.1559; found 379.1563.
24
19
4
5.4.34.
(E)-4-(2-(4-oxo-3-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’ah): Yellow solid, Yield 63 %; mp: 229–231 °C. H NMR (500 MHz,
DMSO–d ) δ 9.02 (s, 1H), 8.52 (d, J = 3.9 Hz, 1H), 8.06 (d, J = 15.3 Hz, 1H), 7.86 (d, J = 6.3
6
Hz, 2H), 7.67 (d, J = 6.0 Hz, 2H), 7.57 (s, 1H), 6.92 (s, 2H), 6.64 (d, J = 15.2 Hz, 1H), 3.78 (s,
1
3
3
1
H), 3.76 (s, 6H) ppm; C NMR (125 MHz, d-TFA) δ 156.8, 153.6, 150.1, 149.7, 149.6, 140.7,
40.5, 135.0, 132.5, 131.3, 124.6, 121.4, 120.7, 118.0, 117.2, 115.8, 114.9, 107.6, 63.0, 57.8
+
ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 441.1563; found 441.1565.
25
21
4
4
5.4.35.
(E)-4-(2-(3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-oxo-3,4-dihydropyrido[2,3-
1
d]pyrimidin-2-yl)vinyl)benzonitrile (4’ai): Yellow solid, Yield 77 %; mp: 229–231 °C. H NMR
(
500 MHz, DMSO–d ) δ 9.00 (dd, J = 4.5, 1.9 Hz, 1Hg), 8.50 (dd, J = 7.8, 1.8 Hz, 1He), 8.02 (d,
6
J = 15.5 Hz, 1Hi), 7.85 (d, J = 8.3 Hz, 2Hk), 7.66 (d, J = 8.3 Hz, 2Hj), 7.55 (dd, J = 7.8, 4.6 Hz,
1
1
1
1
4
Hc), 7.08 (dd, J = 12.9, 5.4 Hz, 2Hda), 6.94 (dd, J = 8.5, 2.4 Hz, 1Hf), 6.62 (d, J = 15.6 Hz,
13
Hh), 4.34 (d, J = 7.4 Hz, 4Hbb’) ppm; C NMR (125 MHz, DMSO–d ) δ 162.5, 157.6, 156.7,
6
54.7, 144.6, 144.3, 139.7, 138.6, 136.5, 133.4, 129.6, 128.9, 123.6, 122.8, 121.9, 119.1, 118.2,
+
16.6, 112.3, 64.6, 64.5 ppm; HRMS (ESI): m/z calcd for [M+H] C H N O 409.1300; found
2
4
17
4
3
09.1309.
5.4.36.
(E)-4-(2-(3-(4-fluorobenzyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-
1
yl)vinyl)benzonitrile (4’aj): Yellow solid, Yield 61 %; mp: 229–231°C H NMR (500 MHz,
DMSO–d ) δ 9.01 (s, 1H), 8.57 (d, J = 2.5 Hz, 1H), 8.12–7.98 (m, 2H), 7.93 (d, J = 13.6 Hz,
6
1
3
3
H), 7.59 (d, J = 15.1 Hz, 2H), 7.37 (s, 2H), 7.15 (s, 2H), 5.65 (s, 2H) ppm; C NMR (125
MHz, d-TFA) δ 166.0 (d, J = 248.1 Hz), 165.0, 164.8, 163.8, 163.7, 154.0, 150.8 (d, J = 26.4
Hz), 150.3, 141.6, 135.8, 133.4, 131.9, 130.8 (d, J = 8.5 Hz), 125.4, 122.1, 121.1, 119.1 (d, J =
+
2
3
2.5 Hz), 115.8, 50.6 ppm; HRMS (ESI): m/z calcd for [M+H] C H FN O 383.1308; found
2
3
16
4
83.1310.
6. Bacterial strains and media
The ESKAP panel of bacteria consisted of Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (BAA-1705), Acinetobacter baumannii (BAA1605), Pseudomonas aeruginosa
(ATCC 27853) and Staphylococcus aureus (ATCC 29213). NRS199, NRS129, NRS186,
NRS191, NRS192, NRS193, NRS194, NRS198 are MRSA strains while VRS1, VRS4, VRS12
2
8