Development of 2-(4-oxoquinazolin-3(4 H)-yl)acetamide derivatives as novel enoyl-acyl carrier protein reductase (InhA) inhibitors for the treatment of tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2014.09.028

InhA, the enoyl acyl carrier protein reductase of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel anti-tubercular agents. Twenty eight 2-(4-oxoquinazolin-3(4H)-yl)acetamide derivatives were synthesized and evaluated for their in vitro MTB InhA inhibition. Compounds were further evaluated for their in vitro activity against drug sensitive and resistant MTB strains and cytotoxicity against RAW 264.7 cell line. Compounds were docked at the active site of InhA to understand their binding mode and differential scanning fluorimetry was performed to ascertain their protein interaction and stability.

Full text of DOI:10.1016/j.ejmech.2014.09.028

Accepted Manuscript
Development of 2-(4-oxoquinazolin-3(4H)-yl)acetamide derivatives as novel enoyl-
acyl carrier protein reductase (InhA) inhibitors for the treatment of tuberculosis
Ganesh S. Pedgaonkar, Jonnalagadda Padma Sridevi, Variam Ullas Jeankumar,
Shalini Saxena, Parthiban Brindha Devi, Janupally Renuka, Perumal Yogeeswari,
Dharmarajan Sriram, Chair Professor
PII:
S0223-5234(14)00846-0
10.1016/j.ejmech.2014.09.028
EJMECH 7334
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2014
Revised Date: 5 August 2014
Accepted Date: 8 September 2014
Please cite this article as: G.S Pedgaonkar, J.P. Sridevi, V.U. Jeankumar, S. Saxena, P.B. Devi, J.
Renuka, P. Yogeeswari, .D. Sriram, Development of 2-(4-oxoquinazolin-3(4H)-yl)acetamide derivatives
as novel enoyl-acyl carrier protein reductase (InhA) inhibitors for the treatment of tuberculosis, European
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.09.028.
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ACCEPTED MANUSCRIPT
SHORT COMMUNICATION
Development of 2-(4-oxoquinazolin-3(4H)-yl)acetamide derivatives as novel
enoyl-acyl carrier protein reductase (InhA) inhibitors for the treatment of
tuberculosis
Ganesh S Pedgaonkar, Jonnalagadda Padma Sridevi, Variam Ullas Jeankumar,
Shalini Saxena, Parthiban Brindha Devi, Janupally Renuka,
*
Perumal Yogeeswari, Dharmarajan Sriram
Department of Pharmacy, Birla Institute of Technology & Science - Pilani, Hyderabad
Campus, Jawahar Nagar, Hyderabad – 500078, India.
Abstract
InhA, the enoyl acyl carrier protein reductase of Mycobacterium tuberculosis (MTB) is an
attractive target for developing novel anti-tubercular agents. Twenty eight 2-(4-
oxoquinazolin-3(4H)-yl)acetamide derivatives were synthesized and evaluated for their in
vitro MTB InhA inhibition. Compounds were further evaluated for their in vitro activity
against drug sensitive and resistant MTB strains and cytotoxicity against RAW 264.7 cell
line. Compounds were docked at the active site of InhA to understand their binding mode and
differential scanning fluorimetry was performed to ascertain their protein interaction and
stability.
Key words: Tuberculosis, Enoyl acyl carrier protein reductase, InhA, Cytotoxicity
Corresponding Author*
D. Sriram
Chair Professor, Department of Pharmacy,
Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Jawaharnagar, Hyderabad- 500 078.
INDIA
Telephone: +91-40663030506
Fax: +91-4066303998
Email: dsriram@hyderabad.bits-pilani.ac.in
1

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1
. Introduction
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis
MTB) in humans, whose global burden remains enormous. The TB epidemic is complicated
by the emergence of multidrug resistant-TB (MDR-TB) and extremely drug resistant-TB
XDR-TB) strains. Worldwide in 2012, 3.6% of newly diagnosed TB cases and 20% of those
(
(
previously treated for TB were MDR-TB cases. At least one case of XDR-TB had been
reported by 92 countries by the end of 2012. Also on average, an estimated 9.6% of MDR-TB
cases have XDR-TB [1]. Mycolic acids are the core constituents essential for the integrity of
mycobacterial cell wall. Among various enzymes involved in the biosynthesis of mycolic
acids, NADH-dependent enoyl-acyl carrier protein reductase (InhA) (E.C.1.3.1.9) encoded by
inhA gene is key layer which catalyses reduction of long-chain trans-2-enoyl-ACP in the type
II fatty acid biosynthesis pathway [2]. Studies have validated InhA as the primary target for
anti-tubercular drugs such as isoniazid (INH) and ethionamide [3]. As a prodrug, INH must
first get activated by KatG, a catalase-peroxidase enzyme to form an active INH-NAD adduct
which then functions as potent inhibitor of InhA [4]. Clinical studies demonstrated that most
INH-resistant strains arise from KatG-associated mutations [5]. Consequently, inhibitors
targeting InhA directly without prerequisite for activation would be promising candidates for
development of new anti-TB agents. Several series of direct InhA inhibitors have been
reported, such as pyrazoles [6], indole-5-amides [6], alkyl diphenyl ethers [7], pyrrolidine
carboxamides [8], arylamides [9] and imidazopiperidine [10] derivatives. We previously
reported design of novel leads of MTB InhA inhibitors [11]. In the course of our studies on
one of the lead 2-(4-oxoquinazolin-3(4H)-yl)-N-(thiazol-2-yl)acetamide (UPS 17) (Fig. 1),
we synthesized twenty eight derivatives and report herein their biological activities.
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2. Results and discussion
2.1. Design and Chemistry
In our earlier work we generated and validated quantitative pharmacophore models using
known set of reported MTB InhA inhibitors. The validated pharmacophore model was then
used as a query to screen an in-house/commercial database of 400,000 compounds and
retrieved 25,000 hits. These hits were further ranked based on its shape and feature similarity
with potent MTB InhA inhibitor using rapid overlay of chemical structures (ROCS) and
subsequent hits were subjected for docking. Based on the pharmacophore, ROCS model and
docking interactions, 32 compounds were selected and assayed for InhA inhibitory activity
[
11]. One of the lead 2-(4-oxoquinazolin-3(4H)-yl)-N-(thiazol-2-yl)acetamide (UPS 17) (Fig.
) showed 31% inhibition of MTB InhA at 10µM. The quest for more potent molecules
1
encouraged us to utilize these moderately active hits (shown in Fig. 1) as a structural
framework to construct a library for developing a strong structure-activity relationship (SAR)
profile and also to understand the ideal site for introducing chemical diversity. Thus, a library
of 28 molecules utilizing this lead as a template were designed using a combination of
molecular docking and medicinal chemistry strategies, and is depicted in Table 1. Based on
the input from protein-ligand interactions observed on docking of lead molecules
(compounds 1, 2 & 3), the quinazolin-4(3H)-one core was decided to be retained in the initial
th
SAR explorations. However, the introduction of chloro group at 6 position and a methyl tail
nd
at 2 position of the quinazolin-4(3H)-one core, were found to be favorable in improving the
interaction with the active site residues, and hence were attempted in synthesis. Various aryl
and heteroaryl substituents (as shown in Table 1) were also attempted as the right hand core
in the hit expansion step to introduce structural diversification.
The target molecules were achieved by using a simple and straight forward strategy as
depicted in Schemes 1 & 2. Construction of the target molecule began by condensing
3

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commercially available and relatively cheap anthranilamide with formamidine acetate in
refluxing ethanol as similarly reported by Li et al. [12]. Though the condensation reaction of
unsubstituted and 5-chloro-substituted anthranilamide with formamidine acetate went
smoothly yielding the desired quinazolin-4(3H)-one in very good yield, our initial effort to
nd
introduce a methyl tail at the 2 position of the quinazolin-4(3H)-one core by condensing the
appropriate anthranilamide with acetamidine hydrochloride gave the required product in very
low yield. Hence, an alternate strategy by condensing corresponding anthranilic acid with
acetic anhydride followed by dehydration with formamide as previously demonstrated by
Ning et al. [13] was followed to achieve the desired 2-methylquinazolin-4(3H)-one in very
good yield. Acetic acid linker was then introduced to the quinazolin-4(3H)-one core by
2 3
alkylation with methyl bromoacetate (BrCH COOCH ) followed by subsequent hydrolysis
with lithium hydroxide. The final library was then assembled by coupling the so formed acid
with various substituted amines to the give the target compounds. Though the reaction
progressed smoothly for aromatic amines in good yield, a similar conversion was not
observed in the case of heterocyclic amines especially with 2-amino-5-nitrothiazole, 2-
aminobenzothiazole and 2-amino-6-nitrobenzothiazole, probably due to their low
nucleophillicity and solubility issues. Hence, an alternative strategy was designed as depicted
in Scheme 3, wherein the corresponding amine was first treated with chloroacetyl chloride in
presence of triethylamine (TEA) as base to give the corresponding 2-chloro-N-
(
aryl/heteroaryl)acetamide. The so formed 2-chloro-N-(aryl/heteroaryl)acetamide was then
introduced into the desired quinazolin-4(3H)-one unit by alkylation using cesium carbonate
Cs CO ) as base at 60°C.
.2 InhA enzyme inhibition studies and SAR. Recombinant MTB InhA was expressed in E.
(
2
3
2
coli and subsequently purified according to a previously reported procedure [14]. The
synthetic compounds were evaluated in vitro for their percentage inhibition of MTB InhA at
4

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10 µM concentration by utilizing a commonly used method [14]. The results are shown in
Table 1. Out of the 28 compounds tested, 16 compounds exhibited InhA enzyme inhibition
IC50 less than 10µM. Dose-response curves were plotted for the potential inhibitors (12 and
25) using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) by taking log
concentrations on the X-axis and response as % inhibition on the Y-axis as shown in Fig. 2.
To support the SAR, we carried out docking studies for all the synthesized compounds and
compared with lead compound UPS 17. The crystal structure of MTB InhA complexed with
reference inhibitor 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide
(PDB ID: 2H7M with a resolution of 1.62Å) was downloaded and docking was carried out
using Glide, version 5.7, Schrodinger, LLC (New York, NY, 2012). Analysis of the crystal
structure of 2H7M revealed that the reference inhibitor in the InhA active site formed
hydrogen bonding network with Tyr158, and the NAD+ cofactor that probably served as the
key feature to govern the orientation of the compound within the active site. Dual hydrogen
bonding network involved the oxygen atom on the pyrrolidine carbonyl group, InhA catalytic
residue Tyr158, and the NAD+. This hydrogen bonding network seemed to be conserved
among all the InhA-inhibitor complexes identified so far. We also analyzed the pi-stacking
interactions at the active site cavity where some pi-stacking interactions were observed
between the quinazoline nucleus and, Phe97 and Phe149 amino acid residues. All compounds
showed pi-stacking interactions but were week as compared to observed hydrogen bonding
interactions. The reference inhibitor was re-docked with the active site residues of the InhA
to validate the active site cavity. The ligand exhibited good Glide score of -8.02 kcal/mol and
was found in the vicinity of amino acids Tyr158, Phe149, Met199, Ile215, Pro156, Leu218,
Met155, Ala211, Ile202, Met103, Leu207, Ala157, Met161, Phe97, Met98, Gly96, Gly104
and Lys165. Re-docking results showed that the compound exhibited similar interactions as
that of the original crystal structure and showed RMSD of 0.87 Å suggesting a high docking
5

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reliability of the docking method in terms of reproducing the experimentally observed
binding mode. The superimposition of Glide docked conformation of co-crystal with the
original crystal pose of 2H7M is shown in Fig. 3.
With the aim of getting insights into the structural basis of activity, lead compound UPS 17
was analysed in more detail. Docking of the lead compound 2-(4-oxo-3,4-dihydroquinazolin-
3-yl)-N-(1,3-thiazol-2-yl)acetamide (UPS17) within the active site of the InhA protein is
illustrated in Fig. 4. The predicted bound conformation of the lead compound UPS17 showed
that the oxygen atom of carbonyl group of the acetamide linker formed a hydrogen bonding
network with the side chain of the Tyr158. The quinazolin-4(3H)-one ring was surrounded by
hydrophobic amino acids such as Phe149, Pro193, Leu218, Ile215, Ala211, Pro156, Ala157,
Met103 and Met199. The thiazole ring was enclosed by hydrophobic amino acids such as
Met98, Phe97 and Met161. The compound was very well fit in the active site cavity with
docking score of -6.75 kcal/mol. The effect of various aryl/heteroaryl substitutions at the R
2
position was explored. As shown in Table 1, all the compounds showed % inhibition in the
range of 88.12 to 33.70% at 10 ꢀM concentration against MTB InhA which was better than
the lead compound UPS17 (31% at 10 ꢀM).
The first subset of compounds (7, 9, 11, 13, 29, 33 and 37), with various aryl/heteroaryl
groups at R
2
position through an acetamide linker is shown in Table 1. The phenyl group
substituent at the R position (compound 7) displayed good InhA inhibitory activity.
2
nd
th
Compound containing substitution at 2 and 5 position of the phenyl ring (2-chloro-5-
trifluoromethyl)-2-phenyl group) (compound 9) provided moderate InhA inhibitory activity,
whereas introduction of 2-furylmethyl group at R position (compound 11) slightly reduced
(
2
InhA inhibitory activity. The compounds substituted with heteroaryl groups such as 2-
benzothiazoyl and 5-nitro-2-thiazolyl group (compound 33 and 29 respectively) at R2
position resulted in satisfactory in vitro InhA inhibitory activity. The 6-nitro-2-benzothiazolyl
6

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group substituent at the R
2
position (compound 37) displayed highest activity with IC50 of
2
5.12 ꢀM whereas 2-thiophenylmethyl ring at the R position (compound 13) led to less
potency compared to other compounds from this subset. The in vitro InhA inhibitory activity
of compounds from this subset was in the range of 72.18 to 33.70% at 10 ꢀM concentrations
and the docking score was found in the range of -7.61 to -7.27 kcal/mol suggesting that these
compounds were well fit in the active site of the InhA enzyme. Docking analysis of
compound 37 at the active site revealed hydrogen bonding interaction with the hydroxyl
ribose group of the NAD+ whereas interaction with Tyr158 was found missing (Fig. 5). The
6-nitro-2-benzothiazolyl ring was buried into the hydrophobic pocket and was found to make
contacts with Tyr158, Ile215, Met155, Leu218, Pro156, Ala157, Met103 and. Apart from
th
hydrophobic interactions the nitro group present at 6 position on benzothiazole ring made
polar contact with Gln214. On the other hand, compound 13 was found to be oriented in a
similar manner to that of lead UPS17 retaining hydrogen bonding with Tyr158, but failed to
demonstrate any interaction with hydroxyl ribose group of the NAD+ which was earlier
demonstrated to be crucial for activity and specificity observed at the enzyme level and thus
may have resulted in reduced activity (Fig. 6).
In the second subset of compounds (8, 10, 12, 14, 30, 34 and 38), effect of chloro group
1
substituent at R position on the quinazolin-4(3H)-one ring was investigated to understand the
effect of similar aryl/heteroaryl substituents at R position. The SAR study revealed that,
2
hydrophobic chloro group at R position increased hydrophobicity of the compounds and
1
hydrophobic interactions with the active site which was crucial for inhibition of the enzyme.
This subset of compounds displayed better potency than the previous subset. Here, all the
compounds showed satisfactory in vitro InhA inhibitory activity in the range of 82.43 to
45.62% at 10 ꢀM concentration. The compounds 10, 14 and 8 with 2-chloro-5-
(trifluoromethyl)-2-phenyl, 2-thiophenylmethyl and phenyl groups respectively as R2
7

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substituents showed good InhA enzyme inhibition activity (IC50 of 4.56, 7.12, and 7.78 µM
respectively). It was observed that substitution with heteroaryl groups such as 2-
benzothiazolyl, 6-nitro-2-benzothiazolyl and 5-nitro-2-thiozolyl group at R
2
position
(respectively 34, 38 and 30) was somewhat less favored than the other substitutions at the R2
position. Compound 12 with 2-furylmethyl ring as R substituent exhibited the most
2
promising inhibitory potency with IC50 of 3.12 µM whereas compound 38 with 6-nitro-2-
2
benzothiazolyl group at R position showed lesser potency compared to other compounds
from this subset. The ligand binding analysis of compound 12 revealed two hydrogen
bonding interactions by oxygen atom of carbonyl group of the acetamide linker with the
enzyme, first with side chain of Tyr158 and another with the ribose hydroxyl group of the
NAD+, correlating with the protein–inhibitor complex interactions outlined in InhA crystal
structure. The compound was very well fit in to the active site cavity of the protein with
docking score of -7.86 kcal/mol and the 6-chloroquinazolin-4(3H)-one ring was surrounded
by hydrophobic amino acids residues such as Phe149, Leu218, Ile215, Pro156, Met103,
Ala211, Ala157, Ile202 and Leu207. The 2-furylmethyl ring was enclosed by hydrophobic
amino acids like Met161, Phe97 and Met98 (Fig. 7). Also, the orientation of compound 12
was found to be similar to that of crystal ligand exhibiting a very good fitness in the active
site pocket making this compound more potent. The ligand binding analysis of one of the less
active derivative from this subset (compound 38) demonstrated that it was well inserted into
the hydrophobic cavity but failed to produce any hydrogen bonding interactions with the
protein which explained its reduced activity (Fig. 8).
The third subset of compounds (20, 22, 24, 26, 31, 35 and 39) consisted of methyl substituent
3 2
at R position on the quinazolin-4(3H)-one ring and R was substituted with various
aryl/heteroaryl groups same as that of the above two subsets as shown in Table 1. Here, all
the 7 compounds exhibited inhibitory potency in the range of 70.12 to 34.12%. Compounds
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2
24 and 39 with 2-furylmethyl and 6-nitro-2-benzothiazolyl groups respectively at R position,
showed good InhA inhibition potency with IC50 of 6.86 and 6.16 ꢀM respectively, whereas
compounds with phenyl and 2-chloro-5-(trifluoromethyl)-2-phenyl groups at R position
compounds 20 and 22 respectively), showed moderate IC50 of 8.92 and 9.06 ꢀM. It was
interesting to note that compound substituted with 2-thiophenemethyl ring as R substituent
2
(
2
(compound 26) exhibited the most promising inhibitory potency (IC50 of 6.01 µM) whereas
compound 31 substituted with 5-nitro-2-thiozolyl group at R position showed less potency
2
compared to other compounds from this subset. Closer analysis of compound 26 in the
binding site revealed hydrogen bonding interactions with side chain of Tyr158 as well as
ribose hydroxyl group of the NAD+ similar to other active compounds. The compound was
well placed in the active site of the protein and further stabilized by the hydrophobic
interaction with Phe149, Leu218, Met155, Pro156, Ala157, Ile215, Met199 and Met103 (Fig.
9
). One of the less active derivatives from this subset, compound 31 showed low docking
score of -5.46 kcal/mol as compared to other active compounds and though the compound
was well inserted into the active site pocket of the protein, it was not involved in any
hydrogen bonding interactions resulting in reduced activity (Fig. 10).
In the fourth subset of compounds (21, 23, 25, 27, 32, 36 and 40) effect of chloro group at R
1
position and methyl group at R position on the quinazolin-4(3H)-one ring was investigated
3
to understand the effect of same aryl/heteroaryl substituent’s at R position. In this subset, all
2
the compounds exhibited good inhibitory potency in the range of 88.12 to 45.68%.
3
Compounds substituted with 2-furylmethyl and 2-thiophenylmethyl group at R position (25
and 27) displayed good inhibitory potency (IC50 of 4.52 and 6.76 ꢀM respectively). It was
noted that heteroaryl 5-nitro-2-thiazolyl, 6-nitro-2-benzothiazolyl and 2-benzothiazolyl group
substitutions (compounds 32, 40 and 36) were less favoured than other substituents at R2
position. Compound 21 where the phenyl ring was substituted at R position emerged as most
2
9

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active compound among the entire series with InhA inhibitory IC50 of 3.12 ꢀM which was
well supported by a very good interaction profile in docking study as well with a glide
docking score of -8.06 kcal/mol. The in silico analysis of the most active compound 21
displayed two hydrogen bonding interactions, one with the side chain of Tyr158 and another
with ribose hydroxyl group of the NAD+ with similar orientation to that of crystal ligand
which was considered to be crucial for bioactivity making this compound more potent (Fig.
11). Further the 6-chloro-2-methylquinazolin-4(3H)-one ring was oriented deeply into the
hydrophobic pocket and found to be stabilized by hydrophobic interactions with the Pro193,
Ile215, Leu218, Pro156, Ala211, Ile202, Met103, Leu207, Ala157, and Met199. The ligand
binding analysis of one of the less active compound 32 showed its association with two
+
hydrogen bonding interactions with NAD , but it was disorientated in the active site of
protein along with low docking energy of -6.05 kcal/mol (Fig. 12).
From the docking results, presence of of hydrogen bonds with side chain of Tyr158 and
hydroxyl group of NAD+ along with hydrophobic interactions with the active site were
predicted to be the most crucial factors affecting the inhibitory potency of these compounds.
An analysis of enzyme inhibition activity revealed the following trend at R
= chloro group, R = methyl group) > (R = chloro group, R = H atom) > (R
2
= methyl group) > (R = R = H atom). In case of substitution at R position, substitution
1
and R
3
positions.
(R
1
3
1
3
1
= H atom,
R
3
1
3
with 2-furylmethyl and 2-thiophenylmethyl group was more favored than aryl/heteroaryl
groups such as 6-nitro-2-benzothiazolyl, 2-chloro-5-(trifluoromethyl)-2-phenyl, phenyl, 2-
benzothiazolyl and 5-nitro-2-thiazolyl group.
2.3. In vitro MTB activity studies. All the synthesized compounds were also screened in vitro
for their activity against MTB H37Rv (ATCC27294) using agar dilution method [15] with
drug concentrations from 50 µg/mL to 0.78 µg/mL in duplicates. The minimum inhibitory
concentration (MIC) was determined for each compound as the minimum concentration of
1
0

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compound required to completely inhibit the bacterial growth. Isoniazid, ethambutol,
rifampicin and ofloxacin were used as reference compounds for comparison. MIC values of
the synthesized compounds along with the standard drug for comparison are presented in
Table 1. All synthesized compounds showed activity against MTB with MIC ranging from
3.74 to 84.08 µM. Six compounds (8, 10, 12, 21, 25 and 37) inhibited MTB with MIC of <5
µM. Compound 21 which showed IC50 of 3.12±0.46µM in the MTB InhA enzyme inhibition
activity, showed MTB MIC of 4.76 µM. In general compounds which contained (sub)-
nitrothiazolyl, -benzothiazolyl and -nitrobenzothiazolyl showed higher MIC as compared to
other compounds in this series. Some of the compounds failed to exhibit inhibition of MTB
despite good potency in the enzyme inhibition study, and this could be a major challenge in
TB drug discovery. Among other causes, efflux mechanisms contributed in a major way to
intrinsic resistance to drugs [16]. Efflux pumps of MTB belong to the ABC (ATP-binding
cassette) transporters and major facilitator superfamily (MFS) proteins [17], or antibiotic-
modifying and -degrading enzymes [18], and SMR (small multidrug resistance) family.
Efflux-mediated drug resistance in MTB could be due to one or more efflux pumps working
alone or in coordination. This was possible because of the redundancy of their functions,
which might overlap extensively [19].
In the current work, we repeated the MTB screening of the synthesized compounds in
presence of reported efflux pump inhibitor piperine (8 µg/mL) [20]. In most of the cases MIC
decreased 2 to 4 fold when compared to absence of an efflux pump inhibitor. Compound 21
showed MTB MIC of 2.38 µM in the presence of piperine and the MTB InhA IC50 was well
correlated. Encouraged by the promising in vitro anti-tubercular activity, the more potent
analogues were further evaluated for their in vitro potency against the clinical isolate of
XDR-TB (strain resistant to Isoniazid, Rifampicin, Ofloxacin and Kanamycin). All the
1
1

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compounds displayed mycobacterial kill against the XDR strain and six compounds (9, 11,
21, 25-26 and 37) showed XDR-TB MIC of <20 µM.
2.4. In vitro cytotoxicity studies. The synthesized compounds were further examined for
toxicity in a RAW 264.7 cell line (mouse leukemic monocyte macrophage) at concentrations
0 and 100 µM. After 72 h of exposure, viability of the cells was assessed on the basis of
5
cellular conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
into a formazan product using the Promega Cell Titer 96 non-radioactive cell proliferation
assay [21]. Percentage inhibitions are reported in Table 1. The most active MTB InhA
inhibitor (compound 21) showed no toxicity (19.85 and 28.68% inhibition at 50 and 100 µM
drug concentrations respectively).
2.5 Biophysical characterization. One of the active compounds from this series was further
investigated using a biophysical technique, differential scanning fluorimetry (DSF). The
ability of the compounds to stabilize the catalytic domain of the InhA protein was assessed
utilizing the DSF technique by which the thermal stability of the catalytic domain of InhA
native protein and of the protein bound with the ligand was measured [22]. Complex with
compound 33 was heated stepwise from 25 to 95°C in steps of 0.1°C rise in the presence of a
fluorescent dye (sypro orange), whose fluorescence increased as it interacted with
hydrophobic residues of the InhA protein. As the protein was denatured, the amino acid
residues became exposed to the dye. A right side positive shift of T in comparison to native
m
protein meant higher stabilization of the protein-ligand complex, which was a consequence of
the inhibitor binding. In our study, compound 33 showed significant positive T
m
shift of
2.6°C confirming the stability of the protein-ligand complex as shown in Fig. 13 which
depicts the curves obtained in the DSF experiment for MTB InhA protein (pink) and protein-
compound 33 complex (green).
3. Conclusion
1
2

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In present work, we report the synthesis and screening results of twenty eight substituted 2-
(4-oxoquinazolin-3(4H)-yl)acetamide derivatives against MTB InhA as well as drug sensitive
and resistant MTB strains. All the synthesized compounds showed better InhA inhibition as
compared to lead molecule, and compound 21 emerged as the most active compound
exhibiting 88.12% inhibition of InhA at 10 µM with an IC50 of 3.12 µM. It inhibited drug
sensitive MTB with MIC of 4.76 µM and was non-cytotoxic at 100µM.
4
4
4
. Experimental Data
.1. Chemistry
.1.1. General procedure for the synthesis of quinazolin-4(3H)-ones (4a-b). To a refluxing
solution of corresponding anthranilamide (1mmol) in ethanol (30mL) was added
formamidine acetate (2mmol). The solution was refluxed for about 6h (monitored by TLC &
LCMS for completion), and solvent evaporated under reduced pressure. The residue was
further diluted with water (30mL) and ethyl acetate (50mL) and the layers separated. The
organic layer was dried over anhydrous sodium sulphate and evaporated under reduced
pressure. The residue was purified by silica gel column chromatography using hexane: ethyl
acetate as eluent to give the corresponding quinazolin-4(3H)-one (4a-b) in good yield.
4.1.1.1. Quinazolin-4(3H)-one (4a). The compound was synthesized according to the general
procedure using anthranilamide (3a) (2.0g, 14.68mmol) and formamidine acetate (3.05g,
1
2
9.37mmol) to afford 4a (1.95g, 90.7%) as white solid. M.p: 152-154°C. H NMR (DMSO-
13
d ): δ 10.34 (s, 1H), 8.13 – 7.72 (m, 5H). C NMR (DMSO-d ): δ 161.1, 148.4, 146.0,
6
H
6
C
+
1
6
4
8 6 2
34.1, 128.2, 125.5, 125.2, 120.9. ESI-MS m/z 147 (M+H) . Anal. Calcd. for C H N O: C,
5.75; H, 4.14; N, 19.17; Found: C, 65.71; H, 4.12; N, 19.19.
.1.1.2. 6-Chloroquinazolin-4(3H)-one (4b). The compound was synthesized according to
the general procedure using 2-amino-5-chlorobenzamide (3b) (2.0g, 11.72mmol) and
formamidine acetate (2.44g, 23.44mmol) to afford 4a (1.87g, 88.6%) as off white solid. M.p:
1
3

ACCEPTED MANUSCRIPT
1
13
1
d
92-194°C. H NMR (DMSO-d
6
): δ
H
10.41 (s, 1H), 8.34 – 7.48 (m, 4H). C NMR (DMSO-
-
6
): δ
C
161.3, 146.6, 144.5, 135.1, 134.7, 129.1, 128.8, 122.5. ESI-MS m/z 179 (M-H) . Anal.
Calcd. for C
.1.2. General procedure for the synthesis of methyl 2-(4-oxoquinazolin-3(4H)-yl)acetates
5a-b). To a stirred mixture of corresponding quinazolin-4(3H)-one (4a-b) (1mmol) and
8 5 2
H ClN O: C, 53.21; H, 2.79; N, 15.51; Found: C, 53.25; H, 2.77; N, 15.49.
4
(
methyl bromoacetate (1.2mmol) in N,N-dimethylformamide (DMF) (15mL) was added
anhydrous cesium carbonate (1.5mmol) at room temperature (rt). The mixture was heated at
60°C for about 3h (monitored by TLC & LCMS for completion). The reaction mixture was
filtered through celite and concentrated under reduced pressure. The residue was partitioned
between ethyl acetate (20mL) and water (15mL), and the organic layer dried over anhydrous
sodium sulphate, and concentrated under reduced pressure. The crude product was purified
by silica gel column chromatography using hexane: ethyl acetate as eluent to give the
corresponding methyl 2-(4-oxoquinazolin-3(4H)-yl)acetate (5a-b).
4.1.2.1. Methyl 2-(4-oxoquinazolin-3(4H)-yl)acetate (5a). The compound was synthesized
according to the general procedure using quinazolin-4(3H)-one (4a) (1.5g, 10.26mmol) and
methyl bromoacetate (1.88g, 12.31mmol) to afford 5a (1.84g, 82.4%) as white solid. M.p:
1
13
2
22-224°C. H NMR (DMSO-d
6
): δ
H
8.27 – 7.61 (m, 5H), 4.42 (s, 2H), 3.58 (s, 3H).
C
NMR (DMSO-d
6
): δ
C
168.2, 161.4, 148.0, 147.3, 133.1, 127.5, 126.8, 126.5, 120.9, 56.3,
+
4
6.2. ESI-MS m/z 219 (M+H) . Anal. Calcd. for C H N O : C, 60.55; H, 4.62; N, 12.84;
11
10
2
3
Found: C, 60.51; H, 4.64; N, 12.82.
.1.2.2. Methyl 2-(6-chloro-4-oxoquinazolin-3(4H)-yl)acetate (5b). The compound was
synthesized according to the general procedure using 6-chloroquinazolin-4(3H)-one (4b)
4
(1.5g, 8.30mmol) and methyl bromoacetate (1.52g, 9.96mmol) to afford 5b (1.68g, 80.1%) as
1
white solid. M.p: 263-265°C. H NMR (DMSO-d ): δ 8.28 (s, 1H), 7.96 – 7.46 (m, 3H),
6
H
1
3
4
.49 (s, 2H), 3.60 (s, 3H). C NMR (DMSO-d ): δ 168.1, 161.5, 147.4, 146.5, 133.6, 133.1,
6
C
1
4

ACCEPTED MANUSCRIPT
-
1
5
4
27.9, 127.6, 122.1, 51.5, 46.2. ESI-MS m/z 251 (M-H) . Anal. Calcd. for C11
H
9
ClN
2
O
3
: C,
2.29; H, 3.59; N, 11.09; Found: C, 52.31; H, 3.61; N, 11.06.
.1.3. General procedure for the synthesis of 2-(4-oxoquinazolin-3(4H)-yl)acetic acids (6a-
b). To a stirred mixture of corresponding methyl 2-(4-oxoquinazolin-3(4H)-yl)acetate (5a-b)
(
1mmol) in tetrahydrofuran (THF) (6mL) at rt was added drop wise lithium hydroxide
2mmol) dissolved in water (2mL). The reaction mixture was slowly warmed to rt and stirred
(
for 3h (monitored by TLC & LCMS for completion), and solvent evaporated under reduced
pressure. The residue was diluted with water (10mL) and ethyl acetate (10mL), and the layers
separated. The aqueous layer was acidified with 2N hydrochloric acid and further extracted
with dichloromethane (10mL). The organic layer was dried over anhydrous sodium sulphate
and evaporated under reduced pressure. The crude product was further purified by silica gel
column chromatography using hexane: ethyl acetate as eluent to give the corresponding 2-(4-
oxoquinazolin-3(4H)-yl)acetic acid (6a-b).
4.1.3.1. 2-(4-Oxoquinazolin-3(4H)-yl)acetic acid (6a). The compound was synthesized
according to the general procedure using methyl 2-(4-oxoquinazolin-3(4H)-yl)acetate (5a)
1
(
1.5g, 6.87mmol) to afford 6a (1.1g, 78.7%) as white solid. M.p: 261-263°C. H NMR
1
3
(
DMSO-d
69.4, 161.7, 148.4, 147.6, 133.5, 127.4, 126.9, 126.7, 120.3, 49.9. ESI-MS m/z 203 (M-H) .
Anal. Calcd. for C H N O : C, 58.82; H, 3.95; N, 13.72; Found: C, 58.79; H, 3.94; N, 13.69.
6 H 6 C
): δ 11.23 (s, 1H), 8.67 – 7.51 (m, 5H), 4.64 (s, 2H). C NMR (DMSO-d ): δ
-
1
1
0
8
2
3
4.1.3.2. 2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)acetic acid (6b). The compound was
synthesized according to the general procedure using methyl 2-(6-chloro-4-oxoquinazolin-
3
2
(4H)-yl)acetate (5b) (1.5g, 5.93mmol) to afford 6b (1.1g, 78.1%) as white solid. M.p: 249-
1
13
6 H
51°C. H NMR (DMSO-d ): δ 11.17 (s, 1H), 8.61 – 7.41 (m, 4H), 4.58 (s, 2H). C NMR
(
DMSO-d ): δ 169.2, 161.4, 147.7, 146.1, 133.3, 133.0, 127.7, 127.5, 122.5, 49.7. ESI-MS
6 C
1
5

ACCEPTED MANUSCRIPT
-
7 2 3
m/z 237 (M-H) . Anal. Calcd. for C10H ClN O : C, 50.33; H, 2.96; N, 11.74; Found: C,
50.29; H, 2.95; N, 11.72.
4.1.4. General procedure for the synthesis of substituted 2-(4-oxoquinazolin-3(4H)-
yl)acetamides (7-14). To a solution of corresponding amine (1.1mmol) in dry
dichloromethane (DCM) (4mL) was added TEA (2mmol) and corresponding 2-(4-
oxoquinazolin-3(4H)-yl)acetic acid (6a-b) (1mmol) at 0°C. Propylphosphonic anhydride
solution (T3P) (≥50 wt. % in ethyl acetate; 2mmol) was then added drop wise to the reaction
mixture and was stirred at rt for 6h (monitored by TLC & LCMS for completion). The
reaction mixture was then washed with water (4mL), brine (4mL). The organic layer was
dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude
product was further purified by silica gel column chromatography using hexane: ethyl acetate
as eluent to give the corresponding 2-(4-oxoquinazolin-3(4H)-yl)acetamides (7-14).
4.1.4.1. 2-(4-Oxoquinazolin-3(4H)-yl)-N-phenylacetamide (7). The compound was
synthesized according to the general procedure using aniline (0.05g, 0.53mmol) and 2-(4-
oxoquinazolin-3(4H)-yl)acetic acid (6a) (0.1g, 0.48mmol) to afford 7 (0.098g, 72.2%) as pale
1
yellow solid. M.p: 241-243°C. H NMR (DMSO-d
6
): δ
H
10.45 (s, 1H), 8.37 – 7.04 (m, 10H),
1
3
4
.86 (s, 2H). C NMR (DMSO-d
6
): δ
C
161.3, 161.7, 148.5, 147.6, 138.7, 133.6, 128.8, 128.2,
+
1
27.5, 126.9, 126.4, 121.7, 121.0, 50.2. ESI-MS m/z 280 (M+H) . Anal. Calcd. for
C H N O : C, 68.81; H, 4.69; N, 15.05; Found: C, 68.78; H, 4.67; N, 15.02.
16
13
3
2
4.1.4.2. 2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N-phenylacetamide (8). The compound was
synthesized according to the general procedure using aniline (0.042g, 0.46mmol) and 2-(6-
chloro-4-oxoquinazolin-3(4H)-yl)acetic acid (6b) (0.1g, 0.41mmol) to afford 8 (0.096g,
1
7
3.1%) as off white solid. M.p: 258-260°C. H NMR (DMSO-d
6
): δ
H
10.32 (s, 1H), 8.57 –
13
7
.15 (m, 9H), 5.01 (s, 2H). C NMR (DMSO-d ): δ 168.6, 161.8, 147.7, 146.5, 138.7,
6
C
1
6

ACCEPTED MANUSCRIPT
+
1
35.4, 133.0, 129.0, 128.1, 127.8, 127.6, 122.4, 121.7, 50.3. ESI-MS m/z 314 (M+H) . Anal.
Calcd. for C16 : C, 61.25; H, 3.86; N, 13.39; Found: C, 61.21; H, 3.88; N, 13.42.
.1.4.3. N-(2-Chloro-5-(trifluoromethyl)phenyl)-2-(4-oxoquinazolin-3(4H)-yl)acetamide
9). The compound was synthesized according to the general procedure using 2-chloro-5-
H
3 2
12ClN O
4
(
trifluoromethylaniline (0.1g, 0.53mmol) and 2-(4-oxoquinazolin-3(4H)-yl)acetic acid (6a)
1
(
0.1g, 0.48mmol) to afford 9 (0.13g, 70.6%) as white solid. M.p: 260-262°C. H NMR
13
(
DMSO-d
6 H 6 C
): δ 10.24 (s, 1H), 8.61 – 7.27 (m, 8H), 5.04 (s, 2H). C NMR (DMSO-d ): δ
1
1
5
4
68.7, 161.8, 148.4, 147.7, 137.7, 133.5, 129.5, 129.3, 127.6, 126.8, 126.6, 126.1, 124.2,
-
22.2, 120.0, 118.9, 50.7. ESI-MS m/z 380 (M-H) . Anal. Calcd. for C H ClF N O : C,
17
11
3
3
2
3.49; H, 2.90; N, 11.01; Found: C, 53.52; H, 2.92; N, 11.02.
.1.4.4. 2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N-(2-chloro-5-
trifluoromethyl)phenyl)acetamide (10). The compound was synthesized according to the
(
general procedure using 2-chloro-5-trifluoromethylaniline (0.089g, 0.46mmol) and 2-(6-
chloro-4-oxoquinazolin-3(4H)-yl)acetic acid (6b) (0.1g, 0.41mmol) to afford 10 (0.13g,
1
7
4.5%) as white solid. M.p: 218-220°C. H NMR (DMSO-d ): δ 10.34 (s, 1H), 8.43 – 7.55
6
H
13
(
m, 7H), 5.06 (s, 2H). C NMR (DMSO-d
6 C
): δ 168.8, 161.6, 147.8, 146.4, 137.7, 133.6,
133.0, 129.5, 129.3, 127.8, 127.6, 126.1, 124.2, 122.3, 122.0, 118.9, 50.6. ESI-MS m/z 415
-
(
2 3 3 2
M-H) . Anal. Calcd. for C17H10Cl F N O : C, 49.06; H, 2.42; N, 10.10; Found: C, 49.09; H,
2.43; N, 10.12.
4.1.4.5. N-(Furan-2-yl-methyl)-2-(4-oxoquinazolin-3(4H)-yl)acetamide
(11).
The
compound was synthesized according to the general procedure using furfurylamine (0.051g,
0.53mmol) and 2-(4-oxoquinazolin-3(4H)-yl)acetic acid (6a) (0.1g, 0.48mmol) to afford 11
1
(
0.097g, 70.6%) as pale yellow solid. M.p: 187-189°C. H NMR (DMSO-d
6
): δ
H
8.83 (s, 1H),
1
3
8
1
.42 – 6.27 (m, 8H), 4.35 (s, 2H), 4.29 (s, 2H). C NMR (DMSO-d ): δ 171.2, 161.4, 148.4,
6 C
47.7, 145.9, 142.3, 133.5, 127.6, 126.8, 126.5, 120.9, 110.7, 110.5, 50.4, 37.3. ESI-MS m/z
1
7

ACCEPTED MANUSCRIPT
+
2
4
4
84 (M+H) . Anal. Calcd. for C15
13 3 3
H N O : C, 63.60; H, 4.63; N, 14.83; Found: C, 63.57; H,
.65; N, 14.81.
.1.4.6. 2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N-(furan-2-ylmethyl)acetamide (12). The
compound was synthesized according to the general procedure using furfurylamine (0.044g,
0.46mmol) and 2-(6-chloro-4-oxoquinazolin-3(4H)-yl)acetic acid (6b) (0.1g, 0.41mmol) to
1
afford 12 (0.10g, 76.2%) as white solid. M.p: 223-225°C. H NMR (DMSO-d
6
): δ
H
8.86 (s,
13
1
H), 8.48 – 6.31 (m, 7H), 4.87 (s, 2H), 4.32 (s, 2H). C NMR (DMSO-d
6
): δ
C
171.2, 161.7,
147.7, 146.2, 145.9, 142.2, 133.6, 133.0, 127.6, 127.4, 122.3, 110.5, 110.3, 50.1, 36.9. ESI-
+
MS m/z 318 (M+H) . Anal. Calcd. for C H ClN O : C, 56.70; H, 3.81; N, 13.23; Found: C,
1
5
12
3
3
56.74; H, 3.83; N, 13.20.
4.1.4.7. 2-(4-Oxoquinazolin-3(4H)-yl)-N-(thiophen-2-ylmethyl)acetamide (13). The
compound was synthesized according to the general procedure using 2-
thiophenemethylamine (0.059g, 0.53mmol) and 2-(4-oxoquinazolin-3(4H)-yl)acetic acid (6a)
1
(
(
(
0.1g, 0.48mmol) to afford 13 (0.10g, 71.4%) as white solid. M.p: 234-236°C. H NMR
13
DMSO-d ): δ 8.97 (s, 1H), 8.52 – 6.98 (m, 8H), 4.74 (s, 2H), 4.40 (s, 2H). C NMR
6
H
DMSO-d
6
): δ
C
171.2, 161.4, 148.3, 147.7, 140.5, 133.6, 127.4, 127.1, 126.8, 126.5, 126.3,
+
1
25.4, 121.0, 51.1, 43.2. ESI-MS m/z 300 (M+H) . Anal. Calcd. for C15
H
13
N
3
O
2
S: C, 60.18;
H, 4.38; N, 14.04; Found: C, 60.21; H, 4.37; N, 14.06.
.1.4.8. 2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N-(thiophen-2-ylmethyl)acetamide (14).
4
The compound was synthesized according to the general procedure using 2-
thiophenemethylamine (0.052g, 0.46mmol) and 2-(6-chloro-4-oxoquinazolin-3(4H)-yl)acetic
1
acid (6b) (0.1g, 0.41mmol) to afford 14 (0.11g, 78.6%) as white solid. M.p: 212-214°C. H
13
NMR (DMSO-d
6 H
): δ 8.95 (s, 1H), 8.54 – 6.95 (m, 7H), 4.89 (s, 2H), 4.42 (s, 2H). C NMR
(
DMSO-d ): δ 171.2, 161.5, 147.7, 146.2, 140.8, 133.6, 133.1, 127.6, 127.4, 127.1, 126.8,
6
C
1
8

ACCEPTED MANUSCRIPT
+
1
5
4
25.3, 122.1, 51.2, 42.9. ESI-MS m/z 334 (M+H) . Anal. Calcd. for C15
H12ClN
3
O
2
S: C,
3.97; H, 3.62; N, 12.59; Found: C, 54.00; H, 3.64; N, 12.62.
.1.5. General procedure for the synthesis of 2-methyl-4H-benzo[d][1,3]oxazin-4-ones
(16a-b). A solution of corresponding anthranilic acid (1mmol) in acetic anhydride (15mL)
was heated to reflux for 1h (monitored by TLC & LCMS for completion), quenched with ice
and concentrated under reduced pressure. The residue was partitioned between ethyl acetate
(20mL) and water (20mL), and organic layer was dried over anhydrous sodium sulphate,
filtered, and concentrated under reduced pressure. The crude product was purified by silica
gel column chromatography using hexane: ethyl acetate as eluent to give the corresponding
2-methyl-4H-benzo[d][1,3]oxazin-4-one (16a-b).
4.1.5.1. 2-Methyl-4H-benzo[d][1,3]oxazin-4-one (16a). The compound was synthesized
according to the general procedure using anthranilic acid (2.0g, 14.58mmol) to afford 16a
1
(
1.84g, 78.4%) as white solid. M.p: 117-119°C. H NMR (DMSO-d
6
): δ
H
8.19 – 7.58 (m,
1
3
4
H), 1.21 (s, 3H). C NMR (DMSO-d ): δ 163.9, 160.1, 144.6, 135.4, 128.3, 126.7, 124.3,
6 C
+
1
14.2, 25.5. ESI-MS m/z 162 (M+H) . Anal. Calcd. for C H NO : C, 67.07; H, 4.38; N, 8.69;
9 7 2
Found: C, 67.10; H, 4.40; N, 8.66.
4.1.5.2. 6-Chloro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (16b). The compound was
synthesized according to the general procedure using 2-amino-5-chlorobenzoic acid (2.0g,
1
1
1.65mmol) to afford 16b (1.74g, 76.7%) as white solid. M.p: 158-160°C. H NMR (DMSO-
1
3
d ): δ 8.07 – 7.52 (m, 3H), 1.19 (s, 3H). C NMR (DMSO-d ): δ 173.1, 161.8, 154.2,
6
H
6
C
-
1
31.1, 128.0, 121.2, 39.6, 16.6, 8.8. ESI-MS m/z 194 (M-H) . Anal. Calcd. for C
C, 55.26; H, 3.09; N, 7.16; Found: C, 55.30; H, 3.07; N, 7.13.
.1.6. General procedure for the synthesis of 2-methylquinazolin-4(3H)-ones (17a-b). A
9 6 2
H ClNO :
4
solution of corresponding 2-methyl-4H-benzo[d][1,3]oxazin-4-one (16a-b) (1mmol) in
formamide (15mL) was heated to 130°C for 7h (monitored by TLC & LCMS for
1
9

ACCEPTED MANUSCRIPT
completion). Upon cooling, the mixture got solidified, which was further washed with water,
and recrystallized from ethanol to give 2-methylquinazolin-4(3H)-one (17a-b).
4.1.6.1. 2-Methylquinazolin-4(3H)-one (17a). The compound was synthesized according to
the general procedure using 2-methyl-4H-benzo[d][1,3]oxazin-4-one (16a) (1.6g, 9.98mmol)
1
to afford 17a (1.31g, 82.8%) as white solid. M.p: 247-249°C. H NMR (DMSO-d ): δ 12.19
6
H
1
3
(
s, 1H), 8.08 – 7.42 (m, 4H), 2.34 (s, 3H). C NMR (DMSO-d
6
): δ
C
161.4, 155.0, 147.2,
O:
+
1
34.2, 127.9, 125.7, 125.3, 120.6, 21.2. ESI-MS m/z 161 (M+H) . Anal. Calcd. for C
9
H
8
N
2
C, 67.49; H, 5.03; N, 17.49; Found: C, 67.52; H, 5.01; N, 17.47.
.1.6.2. 6-Chloro-2-methylquinazolin-4(3H)-one (17b). The compound was synthesized
4
according to the general procedure using 6-chloro-2-methyl-4H-benzo[d][1,3]oxazin-4-one
1
(
16b) (1.6g, 8.22mmol) to afford 17b (1.31g, 82.7%) as white solid. M.p: 251-253°C. H
13
NMR (DMSO-d
6
): δ
H
10.07 (s, 1H), 8.01 – 7.39 (m, 3H), 2.45 (s, 3H). C NMR (DMSO-d
6
):
-
δ
C
161.1, 155.0, 145.7, 134.2, 130.8, 128.3, 126.9, 121.2, 22.4. ESI-MS m/z 193 (M-H) .
Anal. Calcd. for C H ClN O: C, 55.54; H, 3.63; N, 14.39; Found: C, 55.51; H, 3.64; N,
9
7
2
14.36.
4.1.7. General procedure for the synthesis of methyl 2-(2-methyl-4-oxoquinazolin-3(4H)-
yl)acetates (18a-b). To a stirred mixture of corresponding 2-methylquinazolin-4(3H)-ones
17a-b) (1mmol) and methyl bromoacetate (1.2mmol) in DMF (15 mL) at rt was added
(
cesium carbonate (1.5mmol). The mixture was heated to 60°C for about 3h (monitored by
TLC & LCMS for completion). The reaction mixture was filtered through celite and
concentrated under reduced pressure. The residue was partitioned between ethyl acetate
(20mL) and water (15mL) and the organic layer dried over anhydrous sodium sulphate,
filtered, and concentrated under reduced pressure. The crude product was purified by silica
gel column chromatography using hexane: ethyl acetate as eluent to give the corresponding
methyl 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetate (18a-b).
2
0

ACCEPTED MANUSCRIPT
4.1.7.1. Methyl 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetate (18a). The compound was
synthesized according to the general procedure using 2-methylquinazolin-4(3H)-ones (17a)
(1.3g, 8.11mmol) and methyl bromoacetate (1.48g, 9.73mmol) to afford 18a (1.47g, 78.4%)
1
as white solid. M.p: 235-237°C. H NMR (DMSO-d ): δ 8.01 – 7.59 (m, 4H), 4.47 (s, 2H),
6
H
1
3
3
.63 (s, 3H), 2.52 (s, 3H). C NMR (DMSO-d ): δ 168.1, 161.4, 154.7, 147.1, 133.5, 127.5,
6 C
+
1
12 2 3
26.6, 126.3, 120.9, 51.8, 43.4, 22.2. ESI-MS m/z 233 (M+H) . Anal. Calcd. for C12H N O :
C, 62.06; H, 5.21; N, 12.06; Found: C, 62.09; H, 5.19; N, 12.09.
.1.7.2. Methyl 2-(6-chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)acetate (18b). The
4
compound was synthesized according to the general procedure using 6-chloro-2-
methylquinazolin-4(3H)-one (17b) (1.3g, 6.67mmol) and methyl bromoacetate (1.22g,
1
8
d
1
2
4
4
.01mmol) to afford 18b (1.36g, 76.8%) as white solid. M.p: 267-269°C. H NMR (DMSO-
13
6
H 6 C
): δ 7.89 – 7.41 (m, 3H), 4.49 (s, 2H), 3.62 (s, 3H), 2.53 (s, 3H). C NMR (DMSO-d ): δ
68.3, 161.4, 154.5, 145.2, 133.4, 133.1, 127.7, 127.4, 122.3, 51.3, 43.2, 22.4. ESI-MS m/z
-
65 (M-H) . Anal. Calcd. for C H ClN O : C, 54.05; H, 4.16; N, 10.50; Found: C, 54.08; H,
12
11
2
3
.15; N, 10.53.
.1.8. General procedure for the synthesis of 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetic
acids (19a-b). To a stirred solution of corresponding methyl 2-(2-methyl-4-oxoquinazolin-
(4H)-yl)acetate (18a-b) (1mmol) in THF (6mL) at rt was added drop wise lithium
3
hydroxide (2mmol) dissolved in water (2mL). The reaction mixture was stirred at room
temperature for 3h (monitored by TLC & LCMS for completion), and solvent evaporated
under reduced pressure. The residue was diluted with water (10mL) and ethyl acetate (10mL)
and the layers separated. The aqueous layer was acidified with 2N hydrochloric acid and
further extracted with DCM (10mL). The organic layer was dried over anhydrous sodium
sulphate and evaporated under reduced pressure. The crude product was further purified by
2
1

ACCEPTED MANUSCRIPT
silica gel column chromatography using hexane: ethyl acetate as eluent to give the
corresponding 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19a-b).
4.1.8.1. 2-(2-Methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19a). The compound was
synthesized according to the general procedure using methyl 2-(2-methyl-4-oxoquinazolin-
3
1
8
1
(4H)-yl)acetate (18a) (1.3g, 5.59mmol) and lithium hydroxide (0.26g, 11.18mmol) to afford
1
9a (0.95g, 78.6%) as white solid. M.p: 244-246°C. H NMR (DMSO-d
6
): δ
H
10.96 (s, 1H),
13
.06 – 7.38 (m, 4H), 4.49 (s, 2H), 2.52 (s, 3H). C NMR (DMSO-d
6
): δ
C
168.9, 161.7, 154.6,
-
47.1, 133.6, 127.6, 126.8, 126.6, 121.0, 47.9, 22.4. ESI-MS m/z 217 (M-H) . Anal. Calcd.
for C H N O : C, 60.55; H, 4.62; N, 12.84; Found: C, 60.58; H, 4.60; N, 12.87.
11
10
2
3
4.1.8.2. 2-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19b). The compound
was synthesized according to the general procedure using methyl 2-(6-chloro-2-methyl-4-
oxoquinazolin-3(4H)-yl)acetate (18b) (1.3g, 4.87mmol) and lithium hydroxide (0.23g,
1
9
.74mmol) to afford 19b (0.91g, 74.5%) as white solid. M.p: 261-263°C. H NMR (DMSO-
1
3
d ): δ 11.02 (s, 1H), 8.03 – 7.52 (m, 3H), 4.47 (s, 2H), 2.47 (s, 3H). C NMR (DMSO-d ):
6
H
6
δC 169.1, 161.8, 154.8, 145.3, 133.8, 133.1, 127.8, 127.5, 122.5, 48.4, 22.3. ESI-MS m/z 251
-
(
M-H) . Anal. Calcd. for C11
H
9
ClN
2
O
3
: C, 52.29; H, 3.59; N, 11.09; Found: C, 52.32; H,
3
4
3
.61; N, 11.12.
.1.9. General procedure for the synthesis of substituted 2-(2-methyl-4-oxoquinazolin-
(4H)-yl)acetamides (20-27). To a solution of corresponding amine (1.1mmol) in dry DCM
(
4mL) was added TEA (2mmol) and corresponding 2-(2-methyl-4-oxoquinazolin-3(4H)-
yl)acetic acid (19a-b) (1mmol) at 0°C. Propylphosphonic anhydride solution (T3P) (≥50 wt.
in ethyl acetate; 2mmol) was then added drop wise at 0°C, and the reaction mixture was
%
slowly warmed to rt and stirred for 6h (monitored by TLC & LCMS for completion). The
reaction mixture was then washed with water (4mL) and brine (4mL). The organic layer was
dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude
2
2

ACCEPTED MANUSCRIPT
product was further purified by silica gel column chromatography using hexane: ethyl acetate
as eluent to give the corresponding 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamides (20-
27).
4.1.9.1. 2-(2-Methyl-4-oxoquinazolin-3(4H)-yl)-N-phenylacetamide (20). The compound
was synthesized according to the general procedure using aniline (0.046g, 0.50mmol) and 2-
(2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19a) (0.1g, 0.45mmol) to afford 20 (0.11g,
1
7
2.4%) as white solid. M.p: 261-263°C. H NMR (DMSO-d
6
): δ
): δ
38.5, 133.6, 129.1, 128.1, 127.5, 126.8, 126.6, 121.8, 120.7, 49.1, 22.3. ESI-MS m/z 294
H
10.41 (s, 1H), 8.35 – 7.11
1
3
(
m, 9H), 4.72 (s, 2H), 2.49 (s, 3H). C NMR (DMSO-d
6
C
168.8, 161.6, 154.8, 147.1,
1
+
(
M+H) . Anal. Calcd. for C H N O : C, 69.61; H, 5.15; N, 14.33; Found: C, 69.64; H, 5.13;
17 15 3 2
N, 14.30.
4.1.9.2. 2-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)-N-phenylacetamide (21). The
compound was synthesized according to the general procedure using aniline (0.040g,
0.43mmol) and 2-(6-chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19b) (0.1g,
1
0
.39mmol) to afford 21 (0.09g, 71.2%) as off white solid. M.p: 260-262°C. H NMR
1
3
(
DMSO-d
6
6
): δ
): δ
H
10.38 (s, 1H), 8.33 – 7.13 (m, 8H), 4.75 (s, 2H), 2.58 (s, 3H). C NMR
(DMSO-d
C
168.7, 161.9, 154.7, 145.2, 138.7, 133.6, 133.1, 129.1, 128.2, 127.8, 127.6,
+
1
22.0, 121.5, 49.0, 22.0. ESI-MS m/z 328 (M+H) . Anal. Calcd. for C17
H14ClN
3
O
2
: C, 62.30;
H, 4.31; N, 12.82; Found: C, 62.34; H, 4.29; N, 12.84.
.1.9.3. N-(2-Chloro-5-(trifluoromethyl)phenyl)-2-(2-methyl-4-oxoquinazolin-3(4H)-
yl)acetamide (22). The compound was synthesized according to the general procedure using
4
2
3
2
-chloro-5-trifluoromethylaniline (0.097g, 0.50mmol) and 2-(2-methyl-4-oxoquinazolin-
(4H)-yl)acetic acid (19a) (0.1g, 0.45mmol) to afford 22 (0.13g, 74.5%) as white solid. M.p:
1
19-221°C. H NMR (DMSO-d ): δ 10.26 (s, 1H), 8.60 – 7.22 (m, 7H), 5.03 (s, 2H), 2.50
6
H
13
(
s, 3H). C NMR (DMSO-d ): δ 168.6, 161.5, 154.6, 147.2, 137.7, 133.6, 129.4, 129.2,
6 C
2
3

ACCEPTED MANUSCRIPT
-
1
27.4, 126.8, 126.5, 126.1, 124.3, 122.2, 120.9, 118.9, 48.3, 22.4. ESI-MS m/z 394 (M-H) .
Anal. Calcd. for C18 : C, 54.63; H, 3.31; N, 10.62; Found: C, 54.66; H, 3.29; N,
H
3 3 2
13ClF N O
10.59.
4.1.9.4.
2-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)-N-(2-chloro-5-
(trifluoromethyl)phenyl)acetamide (23). The compound was synthesized according to the
general procedure using 2-chloro-5-trifluoromethylaniline (0.084g, 0.43mmol) and 2-(6-
chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19b) (0.1g, 0.39mmol) to afford 23
1
(
0.11g, 70.3%) as white solid. M.p: 237-239°C. H NMR (DMSO-d
6
): δ
H
10.35 (s, 1H), 8.64
1
3
–
1
4
9
4
7.19 (m, 6H), 5.01 (s, 2H), 2.52 (s, 3H). C NMR (DMSO-d ): δ 168.7, 161.7, 154.7,
6 C
45.2, 137.8, 133.6, 133.1, 129.4, 129.1, 127.7, 127.4, 126.0, 124.3, 122.3, 122.1, 118.9,
-
8.2, 21.9. ESI-MS m/z 429 (M-H) . Anal. Calcd. for C18
H12Cl F
2 3
N
3
2
O : C, 50.25; H, 2.81; N,
.77; Found: C, 50.28; H, 2.84; N, 9.75.
.1.9.5. N-(Furan-2-ylmethyl)-2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide (24). The
compound was synthesized according to the general procedure using furfurylamine (0.048g,
0.50mmol) and 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19a) (0.1g, 0.45mmol) to
1
afford 24 (0.097g, 71.2%) as white solid. M.p: 216-218°C. H NMR (DMSO-d
6
): δ
H
8.81 (s,
): δ
13
1
H), 8.09 – 6.27 (m, 7H), 4.79 (s, 2H), 4.31 (s, 2H), 2.47 (s, 3H). C NMR (DMSO-d
6
C
171.6, 163.8, 154.7, 146.3, 144.9, 141.5, 133.6, 127.5, 126.9, 126.7, 121.0, 110.9, 110.5,
+
4
7.1, 37.1, 22.5. ESI-MS m/z 298 (M+H) . Anal. Calcd. for C H N O : C, 64.64; H, 5.09;
16
15
3
3
N, 14.13; Found: C, 64.68; H, 5.06; N, 14.16.
4.1.9.6. 2-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)-N-(furan-2-ylmethyl)acetamide
(
(
(
(
25). The compound was synthesized according to the general procedure using furfurylamine
0.041g, 0.43mmol) and 2-(6-chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (19b)
1
0.1g, 0.39mmol) to afford 25 (0.091g, 69.8%) as white solid. M.p: 234-236°C. H NMR
1
3
DMSO-d ): δ 8.85 (s, 1H), 8.01 – 6.21 (m, 6H), 4.92 (s, 2H), 4.34 (s, 2H), 2.51 (s, 3H). C
6
H
2
4

ACCEPTED MANUSCRIPT
NMR (DMSO-d
6
): δ
C
171.1, 161.6, 154.3, 145.7, 145.1, 142.3, 133.6, 133.0, 127.9, 127.6,
+
1
22.3, 110.7, 110.4, 48.1, 37.4, 22.3. ESI-MS m/z 332 (M+H) . Anal. Calcd. for
C
16
H
14ClN
3
O
3
: C, 57.93; H, 4.25; N, 12.67; Found: C, 57.97; H, 4.22; N, 12.64.
4.1.9.7. 2-(2-Methyl-4-oxoquinazolin-3(4H)-yl)-N-(thiophen-2-ylmethyl)acetamide (26).
The compound was synthesized according to the general procedure using 2-
thiophenemethylamine (0.056g, 0.50mmol) and 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetic
1
acid (19a) (0.1g, 0.45mmol) to afford 26 (0.10g, 70.6%) as white solid. M.p: 243-245°C. H
NMR (DMSO-d
6
): δ
H
8.34 (s, 1H), 8.05 – 6.94 (m, 7H), 4.83 (s, 2H), 4.33 (s, 2H), 2.49 (s,
1
3
3
1
H). C NMR (DMSO-d ): δ 171.1, 161.5, 154.5, 147.1, 140.8, 133.5, 127.4, 127.1, 126.8,
6
C
+
26.6, 126.3, 125.4, 121.0, 48.6, 43.1, 22.3. ESI-MS m/z 314 (M+H) . Anal. Calcd. for
C
16
H
15
N
3
O
2
S: C, 61.32; H, 4.82; N, 13.41; Found: C, 61.36; H, 4.84; N, 13.39.
4.1.9.8.
2-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)-N-(thiophen-2-
ylmethyl)acetamide (27). The compound was synthesized according to the general procedure
using 2-thiophenemethylamine (0.048g, 0.43mmol) and 2-(6-chloro-2-methyl-4-
oxoquinazolin-3(4H)-yl)acetic acid (19b) (0.1g, 0.39mmol) to afford 27 (0.095g, 69.3%) as
1
white solid. M.p: 222-224°C. H NMR (DMSO-d
6
): δ
H
8.95 (s, 1H), 8.03 – 6.95 (m, 6H),
): δ 166.3, 160.2, 155.9, 145.8,
13
4
.79 (s, 2H), 4.48 (s, 2H), 2.48 (s, 3H). C NMR (DMSO-d
6
C
141.8, 134.5, 130.4, 128.8, 126.6, 125.5, 125.19, 125.11, 120.9, 46.1, 37.3, 22.7. ESI-MS m/z
+
3
48 (M+H) . Anal. Calcd. for C H ClN O S: C, 55.25; H, 4.06; N, 12.08; Found: C, 55.28;
16
14
3
2
H, 4.04; N, 12.11.
4.1.10. General procedure for the synthesis of 2-chloro-N-(aryl/heteroaryl)acetamides
(28a-c). Chloroacetyl chloride (2mmol) was added drop wise to a well stirred suspension of
corresponding amine (1mmol) and TEA (3mmol) in DMF (15mL) at 0°C. The reaction
mixture was then stirred at rt for about 3h (monitored by TLC & LCMS for completion), and
the solvent evaporated under reduced pressure. The residue was further diluted with water
2
5

ACCEPTED MANUSCRIPT
(20mL) and ethyl acetate (30mL), and the layers separated. The aqueous layer was re-
extracted with ethyl acetate (2×30mL) and the combined organic layer was washed with brine
20mL), dried over anhydrous sodium sulphate and evaporated under reduced pressure. The
(
residue was purified by silica column chromatography using hexane: ethyl acetate as eluent
to give corresponding 2-chloro-N-(aryl/heteroaryl)acetamides (28a-c).
4.1.10.1. 2-Chloro-N-(5-nitrothiazol-2-yl)acetamide (28a). The compound was synthesized
according to the general procedure using chloroacetyl chloride (1.55g, 13.77mmol) and 5-
nitrothiazol-2-amine (1.0g, 6.88mmol) to afford 28a (1.03g, 67.7%) as pale yellow solid.
1
13
M.p: 252-254°C. H NMR (DMSO-d ): δ 9.94 (s, 1H), 8.34 (s, 1H), 4.45 (s, 2H). C NMR
6
H
-
(
DMSO-d ): δ 169.4, 166.3, 148.1, 138.6, 43.1. ESI-MS m/z 220 (M-H) . Anal. Calcd. for
6
C
C
5
H
4
ClN
3
O
3
S: C, 27.10; H, 1.82; N, 18.96; Found: C, 27.14; H, 1.79; N, 18.99.
4.1.10.2. N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide (28b). The compound was synthesized
according to the general procedure using chloroacetyl chloride (1.50g, 13.31mmol) and
benzo[d]thiazol-2-amine (1.0g, 6.65mmol) to afford 28b (0.92g, 66.2%) as pale yellow solid.
1
M.p: 276-278°C. H NMR (DMSO-d ): δ 11.78 (s, 1H), 8.21 – 7.63 (m, 4H), 4.39 (s, 2H).
6
H
1
3
C NMR (DMSO-d
6
): δ
C
173.7, 168.8, 153.4, 130.9, 125.5, 124.7, 121.5, 118.4, 42.9. ESI-
ClN OS: C, 47.69; H, 3.11; N, 12.36; Found: C,
-
MS m/z 225 (M-H) . Anal. Calcd. for C
9
H
7
2
47.72; H, 3.13; N, 12.39.
4.1.10.3. 2-Chloro-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (28c). The compound was
synthesized according to the general procedure using chloroacetyl chloride (1.15g,
1
6
1
1
3
0.24mmol) and 6-nitrobenzo[d]thiazol-2-amine (1.0g, 5.12mmol) to afford 28c (0.92g,
1
6.2%) as pale yellow solid. M.p: 273-275°C. H NMR (DMSO-d
6
): δ
H
12.62 (s 1H), 9.11 (s,
13
H), 8.34 – 7.97 (m, 2H), 4.55 (s, 2H). C NMR (DMSO-d
6
): δ
C
173.9, 168.5, 159.6, 144.8,
-
31.6, 121.5, 119.2, 117.1, 43.0. ESI-MS m/z 270 (M-H) . Anal. Calcd. for C H ClN O S: C,
9
6
3
3
9.79; H, 2.23; N, 15.47; Found: C, 39.76; H, 2.25; N, 15.50.
2
6

ACCEPTED MANUSCRIPT
4.1.11. General procedure for the synthesis of substituted 2-(4-oxoquinazolin-3(4H)-
yl)acetamides
(29-40).
To
a
solution
of
corresponding
2-chloro-N-
(aryl/heteroaryl)acetamides (28a-c) (1.2mmol) in DMF (3mL) was added anhydrous cesium
carbonate (2mmol) and corresponding quinazolin-4(3H)-one (4a-b, 17a-b) (1mmol). The
reaction mixture was heated at 60°C for 3h (monitored by TLC & LCMS for completion).
The residue was further diluted with water (10mL) and ethyl acetate (20mL) and the layers
separated. The aqueous layer was re-extracted with ethyl acetate (2x15mL) and the combined
organic layer was washed with brine (15mL), dried over anhydrous sodium sulphate and
evaporated under reduced pressure and the residue was purified by silica column
chromatography using hexane: ethyl acetate as eluent to give the desired substituted 2-(4-
oxoquinazolin-3(4H)-yl)acetamides (29-40).
4.1.11.1.
N-(5-Nitrothiazol-2-yl)-2-(4-oxoquinazolin-3(4H)-yl)acetamide
(29).
The
compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (28a) (0.16g, 0.75mmol) and quinazolin-4(3H)-one (4a) (0.1g,
1
0
.68mmol) to afford 29 (0.15g, 68.6%) as pale yellow solid. M.p: 223-225°C. H NMR
13
(
6 H 6 C
DMSO-d ): δ 12.15 (s, 1H), 8.61 – 7.57 (m, 6H), 4.51 (s, 2H). C NMR (DMSO-d ): δ
168.6, 162.8, 161.5, 148.1, 147.6, 147.4, 135.4, 133.2, 127.4, 126.8, 126.5, 120.9, 50.8. ESI-
-
MS m/z 332 (M-H) . Anal. Calcd. for C13
H
9
N
5
O
4
S: C, 47.13; H, 2.74; N, 21.14; Found: C,
47.17; H, 2.72; N, 21.16.
4.1.11.2. 2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (30). The
compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (28a) (0.134g, 0.60mmol) and 6-chloroquinazolin-4(3H)-one
1
(
4b) (0.1g, 0.55mmol) to afford 30 (0.14g, 70.1%) as pale yellow solid. M.p: 198-200°C. H
13
NMR (DMSO-d ): δ 12.17 (s, 1H), 8.64 – 7.49 (m, 5H), 4.48 (s, 2H). C NMR (DMSO-d ):
6
H
6
δC 168.6, 162.8, 161.5, 147.7, 147.4, 146.4, 135.4, 133.7, 133.1, 127.8, 127.5, 122.3, 51.0.
2
7

ACCEPTED MANUSCRIPT
-
8 5 4
ESI-MS m/z 364 (M-H) . Anal. Calcd. for C13H ClN O S: C, 42.69; H, 2.20; N, 19.15;
Found: C, 42.65; H, 2.22; N, 19.17.
4.1.11.3. 2-(2-Methyl-4-oxoquinazolin-3(4H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (31). The
compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (28a) (0.152g, 0.68mmol) and 2-methylquinazolin-4(3H)-one
1
(
17a) (0.1g, 0.62mmol) to afford 31 (0.15g, 73.4%) as pale yellow solid. M.p: 226-228°C. H
13
6 H
NMR (DMSO-d ): δ 12.21 (s, 1H), 8.60 – 7.61 (m, 5H), 4.47 (s, 2H), 2.49 (s, 3H). C NMR
(
DMSO-d
6
): δ
C
168.7, 162.9, 161.7, 154.6, 147.4, 147.1, 135.4, 133.2, 127.5, 126.7, 126.5,
-
1
20.9, 48.2, 22.2. ESI-MS m/z 344 (M-H) . Anal. Calcd. for C H N O S: C, 48.69; H, 3.21;
14
11
5
4
N, 20.28; Found: C, 48.70; H, 3.23; N, 20.25.
.1.11.4. 2-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)-N-(5-nitrothiazol-2-yl)acetamide
32). The compound was synthesized according to the general procedure using 2-chloro-N-(5-
nitrothiazol-2-yl)acetamide (28a) (0.125g, 0.56mmol) and 6-chloro-2-methylquinazolin-
4
(
4
2
(3H)-one (17b) (0.1g, 0.51mmol) to afford 32 (0.14g, 72.7%) as pale yellow solid. M.p:
1
17-219°C. H NMR (DMSO-d ): δ 12.23 (s, 1H), 8.62 – 7.43 (m, 4H), 4.46 (s, 2H), 2.52
6
H
1
3
(
s, 3H). C NMR (DMSO-d
6 C
): δ 168.6, 162.7, 161.4, 154.5, 147.3, 144.8, 135.2, 133.7
-
1
33.1, 127.7, 127.4, 122.0, 48.2, 22.3. ESI-MS m/z 378 (M-H) . Anal. Calcd. for
S: C, 44.28; H, 2.65; N, 18.44; Found: C, 44.31; H, 2.67; N, 18.42.
.1.11.5. N-(Benzo[d]thiazol-2-yl)-2-(4-oxoquinazolin-3(4H)-yl)acetamide (33). The
14 5 4
C H10ClN O
4
compound was synthesized according to the general procedure using N-(benzo[d]thiazol-2-
yl)-2-chloroacetamide (28b) (0.17g, 0.75mmol) and quinazolin-4(3H)-one (4a) (0.1g,
1
0
.68mmol) to afford 33 (0.15g, 67.8%) as pale yellow solid. M.p: 196-198°C. H NMR
13
(
DMSO-d
6 H
): δ 10.67 (s, 1H), 8.63 (s, 1H), 8.21 – 7.56 (m, 8H), 4.74 (s, 2H). C NMR
(
DMSO-d ): δ 174.6, 168.7, 161.5, 153.4, 148.5, 147.7, 133.6, 131.0, 127.4, 126.7, 126.4,
6
C
2
8

ACCEPTED MANUSCRIPT
+
1
25.2, 124.6, 122.0, 120.9, 118.4, 50.7. ESI-MS m/z 337 (M+H) . Anal. Calcd. for
S: C, 60.70; H, 3.60; N, 16.66; Found: C, 60.73; H, 3.61; N, 16.64.
.1.11.6. N-(Benzo[d]thiazol-2-yl)-2-(6-chloro-4-oxoquinazolin-3(4H)-yl)acetamide (34).
The compound was synthesized according to the general procedure using N-(benzo[d]thiazol-
17 12 4 2
C H N O
4
2-yl)-2-chloroacetamide (28b) (0.136g, 0.60mmol) and 6-chloroquinazolin-4(3H)-one (4b)
1
(
(
(
0.1g, 0.55mmol) to afford 34 (0.15g, 73.5%) as pale yellow solid. M.p: 209-211°C. H NMR
1
3
DMSO-d
6
6
): δ
): δ
H
10.68 (s, 1H), 8.61 (s, 1H), 8.19 – 7.41 (m, 7H), 4.71 (s, 2H). C NMR
DMSO-d
C
174.4, 168.6, 161.6, 153.1, 147.7, 146.5, 133.4, 133.1, 130.9, 127.7, 127.5,
+
1
25.4, 124.6, 122.3, 121.9, 118.6, 50.4. ESI-MS m/z 371 (M+H) . Anal. Calcd. for
C H ClN O S: C, 55.06; H, 2.99; N, 15.11; Found: C, 55.10; H, 3.00; N, 15.13.
17
11
4
2
4.1.11.7. N-(Benzo[d]thiazol-2-yl)-2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide (35).
The compound was synthesized according to the general procedure using N-(benzo[d]thiazol-
2-yl)-2-chloroacetamide (28b) (0.154g, 0.68mmol) and 2-methylquinazolin-4(3H)-one (17a)
1
(
(
(
0.1g, 0.62mmol) to afford 35 (0.14g, 68.7%) as pale yellow solid. M.p: 206-208°C. H NMR
1
3
DMSO-d ): δ 10.61 (s, 1H), 8.22 – 7.49 (m, 8H), 4.41 (s, 2H), 2.53 (s, 3H). C NMR
6
H
DMSO-d
6
): δ
C
174.7, 168.7, 161.4, 154.5, 153.3, 147.2, 133.6, 131.0, 127.3, 126.7, 126.4,
+
1
25.1, 124.8, 121.9, 121.0, 118.5, 48.2, 22.1. ESI-MS m/z 351 (M+H) . Anal. Calcd. for
C
18
H
14
N
4
O
2
S: C, 61.70; H, 4.03; N, 15.99; Found: C, 61.73; H, 4.01; N, 15.97.
4.1.11.8.
N-(Benzo[d]thiazol-2-yl)-2-(6-chloro-2-methyl-4-oxoquinazolin-3(4H)-
yl)acetamide (36). The compound was synthesized according to the general procedure using
N-(benzo[d]thiazol-2-yl)-2-chloroacetamide (28b) (0.126g, 0.56mmol) and 6-chloro-2-
methylquinazolin-4(3H)-one (17b) (0.1g, 0.51mmol) to afford 36 (0.13g, 69.7%) as pale
1
yellow solid. M.p: 237-239°C. H NMR (DMSO-d
6
): δ
H
10.62 (s, 1H), 8.19 – 7.54 (m, 7H),
1
3
4
.47 (s, 2H), 2.57 (s, 3H). C NMR (DMSO-d ): δ 174.5, 168.5, 161.3, 154.7, 153.3, 145.2,
6 C
133.6, 133.1, 130.9, 127.6, 127.4, 125.3, 124.4, 122.6, 122.0, 118.6, 48.3, 22.4. ESI-MS m/z
2
9