Synthesis, biological evaluation and molecular modelling insights of 2-arylquinazoline benzamide derivatives as anti-tubercular agents

Article abstract of DOI:10.1016/j.molstruc.2020.128493

New 2-arylquinazoline benzamide derivatives were synthesized and screened against H37RV strain, compounds displayed specific and potent anti-mycobacterial activity against Mycobacterium tuberculosis. 9a, 9c, 9d, 9e, 9f, 9h, 13b, 17d and 17e exhibited selective and good inhibitory activity against Mycobacterium tuberculosis with the MIC values range of 4–32 μg/mL. Molecular modelling studies also supports that the active molecules can fit well in the binding pocket of GlmU with good ligand-protein interactions, strong binding energies and satisfactory ADMET properties results (obeys the Lipinski rule of 5). Comprehensively, the studies recommended that these new quinazoline derivatives have the potential to be further develop as prospective anti-mycobacterial leads.

Full text of DOI:10.1016/j.molstruc.2020.128493

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Synthesis, biological evaluation and molecular modelling insights of 2-arylquinazoline
benzamide derivatives as anti-tubercular agents
Satyaveni Malasala, Md Naiyaz Ahmad, Jitendra Gour, Manjulika Shukla, Grace Kaul,
Abdul Akhir, Srikanth Gatadi, Y.V. Madhavi, Sidharth Chopra, Srinivas Nanduri
PII:
S0022-2860(20)30818-8
DOI:
https://doi.org/10.1016/j.molstruc.2020.128493
MOLSTR 128493
Reference:
To appear in: Journal of Molecular Structure
Received Date: 5 February 2020
Revised Date: 18 May 2020
Accepted Date: 19 May 2020
Please cite this article as: S. Malasala, M.N. Ahmad, J. Gour, M. Shukla, G. Kaul, A. Akhir, S. Gatadi,
Y.V. Madhavi, S. Chopra, S. Nanduri, Synthesis, biological evaluation and molecular modelling insights
of 2-arylquinazoline benzamide derivatives as anti-tubercular agents, Journal of Molecular Structure
(2020), doi: https://doi.org/10.1016/j.molstruc.2020.128493.
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Synthesis, biological evaluation and molecular modelling insights of 2-
arylquinazoline benzamide derivatives as anti-tubercular agents
Satyaveni Malasalaa, Md. Naiyaz Ahmadb, Jitendra Goura, Manjulika Shuklab, Grace Kaulb,
Abdul Akhirb, Srikanth Gatadia, Y.V Madhavia, Sidharth Choprab*, Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India

Synthesis, biological evaluation and molecular modelling insights of 2-
arylquinazoline benzamide derivatives as anti-tubercular agents
Satyaveni Malasalaa, Md. Naiyaz Ahmadb, Jitendra Goura, Manjulika Shuklab, Grace Kaulb,
Abdul Akhirb, Srikanth Gatadia, Y.V Madhavia, Sidharth Choprab*, Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
Corresponding author: Dr. Srinivas Nanduri, E-mail: nandurisrini92@gmail.com
Co-corresponding author: Dr. Sidharth Chopra: skchopra007@gmail.com
Abstract: New 2-arylquinazoline benzamide derivatives were synthesised and screened
against H37RV strain, compounds displayed specific and potent anti-mycobacterial activity
against Mycobacterium tuberculosis. 9a, 9c, 9d, 9e, 9f, 9h, 13b, 17d and 17e exhibited
selective and good inhibitory activity against Mycobacterium tuberculosis with the MIC
values range of 4-32 µg/mL. Molecular modelling studies also supports that the active
molecules can fit well in the binding pocket of GlmU with good ligand-protein interactions,
strong binding energies and satisfactory ADMET properties results (obeys the Lipinski rule
of 5). Comprehensively, the studies recommended that these new quinazoline derivatives
have the potential to be further develop as prospective anti-mycobacterial leads.
Keywords: 2-arylquinazoline, tuberculosis, minimum inhibitory concentration (MIC),
molecular modelling.

1.0 Introduction:
Increased global prevalence and rise in multi-drug resistance status of various infectious
disease like tuberculosis fuelled the search for novel antimycobacterial agents (Figure 1) [1]
acting through different mechanisms to overcome resistance. Tuberculosis is a transmissible
disease caused by Mycobacterium tuberculosis (Mtb) and recognized to have a high mortality
rate globally [2]. According to WHO reports, 10 million cases were reported in 2017
globally, with India having the highest incidence burden [3]. Emergence of drug-resistant TB
(DR-TB) is often accompanied with chronic immunosuppressive conditions like HIV and
diabetes, thus severely limiting current treatment options. Clinical approval of Bedaquiline
(A, Fig. 1) [5] after a gap of 40 years has kindled hope for discovering new drugs by
consideration of different heterocycles as anti-mycobacterial agents [1]. Quinazoline is one of
the versatile scaffold of medicinal importance possessing diverse pharmacological properties
such as anti-tubercular [6], antibacterial [7], anticonvulsant [8], anti-HIV [9], antifungal [9],
anti-inflammatory, analgesic [10] and anticancer [11] activities. The present work focuses on
the exploration of 2-aryl quinazolines as potential antimycobacterial agents. A survey of
literature suggests that very few reports exist on quinazoline based compounds as
antimycobacterial agents. Pyrazinamide (B, Fig. 1) is the front line therapy used to treat
tuberculosis. For active tuberculosis, it is often used with other first line drugs like
Rifampicin, Isoniazid and Ethambutol [5]. Macozinone (C, Fig. 1), a promising fused
heterocyclic derivative was identified against drug-susceptible (DS) and drug-resistant (DR)
Mtb strains which led to the exploration of different fused heterocycles as antimycobacterials
[12]. Wang et al. reported 4-(aminopyrazolyl)-substituted quinazolines (D, Fig. 1) to possess
inhibitory activity against protein kinases (PknA & PknB) of Mycobacterium tuberculosis
[13]. Tran et al. reported 4-aminoquinazolines (E & F, Fig. 1), as potent inhibitors of N-
acetylglucosamine-1-phosphate uridyltransferase (GlmU) of M. tuberculosis [14]. Our
continued interest in exploration of the potential biological applications of Quinazolines led
to the synthesis of 4-anilino-2-phenylquinazolines with various substitution pattern.

Fig. 1. Approved TB-drugs and Quinazoline derivatives as anti-mycobacterial agents.
2.0 Results and discussion:
2.1 Chemistry
A series of new 2-arylquinazoline derivatives were synthesized as described in Schemes 1 &
2. 2-aminobenzamide 1 was condensed with different substituted aryl aldehydes 2a-c for
about 12-24 h in presence of dimethylsulphoxide to afford 2-arylquinazolinones 3a-c [15].
These derivatives were treated with phosphorus (V) oxychloride and N, N-diethyl aniline to
provide 2-aryl 4-chloroquinazoline derivatives 4a-c [15]. The chlorinated derivatives 4a-c
undergo nucleophillic substitution with substituted nitroanilines (5&10) to obtain N-(4/3-
nitrophenyl)-2-phenylquinazolin-4-amine 6a-c. Further, the nitro derivatives were reduced to
amine functionality by using iron powder and calcium chloride in presence of ethanol, to
afford N1-(2-phenylquinazolin-4-yl)benzene-1,4-diamine 7a-c & 11. Finally, coupling of
amines with diverse carboxylic acids 8a-i & 12a-f afforded the corresponding amide
derivatives 9a-m and 13a-f in moderate to excellent yields. Chlorinated derivative 4a was
also treated with 4-aminobenzoic acid 14 to yield 4-((2-phenylquinazolin-4-yl) amino)
benzoic acid 15 which was further subjected to amide bond formation with different
substituted amines 16a-f to afford the corresponding amide derivatives 17a-f in moderate to
excellent yields.
Scheme 1. Synthesis of N-(4-((2-phenylquinazolin-4/3-yl) amino) phenyl) benzamide
derivatives.

Scheme 2. Synthesis of N-phenyl-4-((2-phenylquinazolin-4-yl) amino) benzamide derivatives
O
O
R3
N
OH
H
Cl
NH2
HN
HN
R3
N
4-Aminobenzoic acid (14)
16a-f
N
N
2-Propanol, 85 oC
12 h, 75 %
N
HATU, DIPEA, N2
DMF, 0 °C-rt, 10-12 h
70-81 %
N
N
4a
17a-f
15
17a
: R3 = 3,5-dimethoxyphenyl
17b: R3 = 3,4,5-trimethoxyphenyl
17c
: R3 = 3-chloro-4-fluorophenyl
17d: R3 =ethyl alanate
17e
: R3 = ethyl leucinate
17f: R3 = 4-chlorobenzyloxy
2.2 In vitro anti-mycobacterial activity
All the newly synthesized 2-aryl quinazoline benzamide derivatives were assessed for their
anti-mycobacterial activity against M. tuberculosis H37Rv ATCC 27294 (Mtb) using
Rifampicin and Isoniazid as reference compounds. The compounds exhibited interesting
inhibitory activity in a range of MIC 4-64 µg/mL. The results are tabulated in Table 1. In all

these compounds, the C2-position is maintained as phenyl or substituted phenyls. The
compounds exhibited good to moderate activity when the C2 position is phenyl, however,
when it is changed to 3,4-dimethoxyphenyl (9i - 9j) or 4-cyanophenyl (9k - 9m) the
compounds exhibited loss of activity. Among the synthesized compounds, when the C2-
position was substituted with phenyl and C4 was substituted with various differently
substituted anilino amines at para position compounds 9a-9m were obtained. When the
substituents on the anilino amines R2 were varied by different electron withdrawing groups as
in 5-bromo-2-chlorophenyl 9e, 4-cyanophenyl 9f, 4-N,N-dimethyl amino phenyl 9d, 4-
iodophenyl 9h and 5-chlorothiophenyl 9c derivatives, the compounds exhibited potent
inhibitory activities against Mtb H37Rv (MIC ranging from 4-16 µg/mL). When R2 groups
are substituted with bulky group like 2-naphthyl 9a, the compound has also shown good
inhibition against Mtb H37Rv (MIC 16 µg/mL). However, the compound with triazolophenyl
9b as R2 was found to be inactive. Further, change of Phenyl at C2-position with 3,4-
dimethoxyphenyl (9i and 9j) and 4-cyanophenyl (9k to 9m) resulted in reduction in activity.
Table 1. MIC (µg/mL) of 2-arylquinazoline based amide derivatives 9a-m, 13a-f & 17a-f.
M. tuberculosis
H37Rv
S.no Compound
R1
R2/ R3
ATCC 27294
MIC (µg/mL)
1.
9a
16

2.
>64
9b
3.
4.
9c
16
8
9d
5.
9e
4
6.
7.
8.
9f
9g
9h
4
>64
16
9.
>64
9i
10.
11.
>64
>64
9j
9k

12.
>64
9l
13.
14.
15.
>64
64
9m
13a
13b
32
16.
17.
13c
13d
64
64
18.
19.
>64
>64
13e
13f
20.
21.
>64
>64
17a
17b

22.
23.
>64
17c
17d
16
24.
25.
17e
17f
32
>64
26.
27.
-
-
-
-
0.03
0.06
Isoniazid
Rifampicin
Further, when we changed the substitution on anilino amines from para to meta position, as in
5-chloro-2-thiophenyl 13a, 5-bromo-2-chlorophenyl 13b, 4-cyanophenyl 13c and 4-
iodophenyl 13d, the derivatives showed moderate to good inhibitory activity (MIC 16-64
µg/mL). In further modification, when compounds 17a-f with reverse amide substitution
were tested, they exhibited encouraging results (17d and 17e showed MIC of 16 & 32 µg/mL
respectively). In order to assess the broad biological profile of the compounds, the derivatives
were also evaluated against ESKAP pathogen panel, the compounds were found to be
inactive (supporting information). It was inferred that the synthesized molecules have shown
selective inhibition particularly against Mycobacterium tuberculosis. The broad Structure
Activity Relationships (SAR) derived is presented in Fig. 2.

Fig. 2. Structure Activity Relationship (SAR) of new 2-aryl quinazoline derivatives.
3.0 Molecular modelling studies:
As structurally related molecules are reported to exhibit inhibitory activity against Mtb GlmU
[16], the newly synthesized molecules were docked against M. tuberculosis GlmU crystal
structure to determine their inhibitory potential. The 3D crystal co-ordinates of M.
tuberculosis GlmU were retrieved from the protein data bank (PDB ID: 4K6R). Molecular
docking studies were performed for the selected bio-active molecules (9a, 9c, 9d, 9e, 9f, 9h,
13b, 17d and 17e) of 2-arylquinazoline benzamide derivatives and the best orientations of 9e
and 9f in to the active-site of GlmU protein is represented in Fig. 2. The compound 9e forms
two hydrogen bonding interactions, in which initial one was found at a distance of 2.99 Å,
where C=O of 9e act as an acceptor and NH of Gly15 as a donor and the additional hydrogen
bonding interaction was found at a distance of 2.89 Å between aromatic hydrogen of 9e as a
donor and C=O of Glu207 as an acceptor. Besides, five aromatic hydrogen bonding
interactions were observed with Leu12, Gln83, Glu166 and Glu207. Further, π-cation
interaction was found between nitrogen of Arg19 and Lys26 with aromatic ring of 9e.
Hydrogen bonding interactions of the other potent compound 9f was found at a distance of
2.62 Å in which C=O of 9f acts as an acceptor and NH of Gly88 as a donor. The

supplementary hydrogen bonding interaction was found at a distance of 2.76 Å, where C=O
of 9f acts as an acceptor and NH of Leu87 as a donor and 2 aromatic hydrogen bonding
interactions were observed with Gln83 and Asp114. Further, π-cation interaction was found
between nitrogen of Arg19 and Lys26 with aromatic ring of 9f, whereas the co-crystal has
shown 2 hydrogen bonding interactions with Gln83, i.e. (i) C=O of Gln83 at a distance of
2.04 Å and (ii) the C=O of Gln83 at a distance of 2.76 Å. In our studies we observed that all
the active molecules have shown interactions with the key amino acid residues. Further, we
observed that all of the active molecules were fitted well in the active binding pocket and
interacted with all the amino residues related to the co-crystal.
Fig. 3 a) Binding pose of 9e and 9f (ball and stick representation) ligand interactions in the
active pocket of binding pocket of GlmU (PDB ID: 4K6R). The gold colour lines represent

hydrogen bond interactions, the cyan colour lines imply the aromatic hydrogen bond
interactions and green line indicates cation-arene interactions. b) Super imposition of 9a, 9d,
9e and 9f at the binding pocket (binding site indicated in the rainbow colour) of GlmU (PDB
ID: 4K6R).
Table 2. Binding energy and ligand interactions of docked molecules (9a, 9c, 9d, 9e, 9f, 9h,
13b, 17d and 17e).
Ligand Interactions
Binding
Ligand
id
Energy
S.no
π -cation/
H-Bond
Hydrophobic
(Kcal/mol)
π-π stacking
Arg19,
Lys26
Gln83, Leu87,
Gly88
Leu12, Ala14, Pro16,Pro86, Leu87,
Ala92, Tyr150, Ala182, Val238
1.
2.
3.
-51.036
-37.440
-45.570
9a
9c
9d
(π-cation)
Tyr209
(π-π)
Ala14, Pro16, Pro86,Leu87,
Leu87, Gly88
Gln83, Gly88,
Ala182, Tyr209, Leu210, Val238
Arg19
Leu12, Ala14, Pro16, Pro86, Leu87,
Ala92, Tyr150, Tyr209
Asp114,Glu166,
Glu207
(π-cation)
Gln83, Gly88,
Arg19,
Lys26
Leu12, Ala13, Ala14, Pro16, Val27,
Pro86,Leu87, Ala92
Ser112, Glu166,
4.
5.
-43.442
-49.529
9e
9f
Glu207
(π-cation)
Arg19
(π-π)
Gln83, Leu87,
Gly88, Asp114
Leu12, Ala14, Pro16, Pro86,Leu87,
Ala92, Tyr150
Tyr209
(π-π)
Ala14, Pro16, Ala182, Pro86,
Leu87, Tyr150, Tyr209, Val238
6.
7.
-51.165
-54.488
Leu87, Gly88
Arg19
9h
Leu12, Ala13, Ala14, Pro16, Val27,
Pro86, Ala92
-
13b

Ala14, Gly15,
Gln83
His58
Leu12, Ala13, Ala14, Pro16, Pro86,
Ala92
8.
-61.058
17d
17e
(π-π)
Leu12, Ala13, Ala14, Pro16, Val27,
Pro86, Leu87, Ala92
9.
-37.289
-55.550
Lys26
Gln83
-
-
Ala14, Pro16, Leu82, Pro86, Leu87,
Ala92, Tyr209
Co-
10.
crystal
3.1 Prime MM/GBSA binding energy calculations
In our studies, we observed that all the active compounds have shown greater binding
energies compared to the co-crystal binding energy, calculated by using MM/GBSA tool. The
objective of MM/GBSA and docking studies is to explain the potential of the synthesized
compounds as inhibitors of GlmU. In our studies, we observed that the synthesized
compounds have shown good ligand interactions and binding energies at the binding pocket
of GlmU as shown in Table 2. The active molecules displayed binding energies ranging from
-37.289 to -61.058 kcal/mol in comparison to the co-crystal binding energy -55.550 kcal/mol.
Finally, some of the identified ligands confirmed better binding energy compared to the co-
crystal, suggesting that the synthesized compounds fit well for inhibiting Mtb GlmU.
3.2 ADMET properties
ADMET properties of the synthesized compounds were calculated using Qikprop program
(Qikprop, version 6.5, Schrödinger, LLC, New York, NY, 2014). As can be seen below, the
partition coefficient (QPlogPo/w), hydrogen bond donors (donor HB), hydrogen bond
acceptors (acceptor HB), molecular weight (mol. Wt.) and percent human oral absorption
exhibited satisfactory results. The compounds also followed Lipinski rule of five. ADMET
properties for Isoniazid and Rifampicin were also calculated and compared with the results of
the synthesized compaunds. The predicted results are shown in Table 3.
Table 3. ADMET properties
Recomme
nded
values
Isoniaz
id
Rifampi
cin
Descriptors
9a
9c
9d
9e
9f
9h
13b
17d
17e

Molecular
weight
130.0–
725.0
466.5
41
456.9
48
459.5
49
529.8
22
441.4
91
542.3
78
529.8
22
440.5
01
482.5
81
137.14
1
820.978
5.739
Dipole
moment
1.0–12.5
7.028
3.836
9.957
7.522
8.358
4.489
5.767
6.668
2.112
3.32
827.2
28
775.1
72
843.6
19
810.6
57
804.9
04
845.7
94
762.7
09
786.5
64
868.9
52
329.90
9
Total SASA
300–1000
983.65
No. of
rotatable
bonds
0–15
5
5
6
5
6
5
5
8
9
2
24
Donor HB
0.0–6.0
2
5
2
5
2
6
2
5
2
2
5
2
5
1
6
1.25
6.25
3
5
Acceptor
HB
2.0–20.0
6.5
4.5
18.65
QP
Polarizabilit
y
56.64
2
50.37
5
55.51
8
53.88
4
52.68
2
55.21
5
52.18
3
49.94
8
55.44
2
13.0–70.0
2.0–6.5
14.107
71.616
QP logP
o/w
6.606
6.074
6.142
6.715
5.052
-1.4
6.488
6.478
5.444
6.403
-1.06
-0.646
-0.844
3.187
−3.0 and
1.2
QP log BB
-0.586 -0.371 -0.696 -0.221
-0.846 -0.186 -1.144
-2.113
Human
Oral
Absorption
1–3
1
100
1
1
100
1
1
100
1
1
100
2
1
91.88
1
1
1
100
2
1
100
1
1
100
1
2
66.83
0
1
56.518
2
Percent
Human
Oral
> 80% is
high
92.48
8
Absorption
Rule of
Five
violations
< 25% is
low
2
4.0 Conclusion
In conclusion, a series of new 2-arylquinazoline benzamide derivatives were synthesized and
evaluated against Mtb H37Rv strains. Compounds 9e and 9f exhibited selective and potent
anti-mycobacterial activity with MIC value 4 µg/mL. Compounds 9a, 9c, 9d, 9h and 17d also
showed anti-mycobacterial activity with MIC values in the range of 8-16 µg/mL. Molecular
modelling studies conducted suggested that the synthesized compounds act as inhibitors of
Mtb GlmU and bind well in the active binding pocket. Mtb GlmU is an important therapeutic
target for Mtb. The compounds also showed good ligand-protein interactions, strong binding
energies and satisfactory ADMET properties results (obeys the Lipinski rule of 5). With the
encouraging results obtained the newly synthesized 2-arylquinazoline benzamide derivatives
have shown the potential for further development as promising anti-mycobacterial agents.

5.0 Experimental Section:
5.1 General Methods. All the reagents and solvents were obtained from commercial
suppliers and were used without further purification. Analytical thin layer chromatography
(TLC) was performed on MERCK pre-coated silica gel 60-F254 (0.5 mm) aluminum plates.
Visualization of the spots on TLC plates was achieved by UV light. 1H and 13C NMR spectra
were recorded on Bruker 500 MHz by making a solution of samples in the DMSO-d6 as
1
solvent using tetramethylsilane (TMS) as the internal standard. Chemical shifts for H and
13C are reported in parts per million (ppm) downfield from tetra methyl silane. Spin
multiplicities are described as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet), and m (multiplet). Coupling constant (J) values are reported in hertz (Hz). HRMS
were determined with Agilent QTOF mass spectrometer 6540 series instrument. Wherever
required, column chromatography was performed using silica gel (60-120). The reactions
wherever anhydrous conditions required are carried under nitrogen positive pressure using
freshly distilled solvents. All evaporation of solvents was carried out under reduced pressure
using rotary evaporator below 45 oC. Melting points were determined with an electro thermal
digital melting point apparatus IA9100 and are uncorrected. The names of all the compounds
given in the experimental section were taken from ChemBioDraw Ultra, Version 12.0.
5.1.1 Preparation of intermediates 3(a-c), 4(a-c), 6(a-c), 11 and 15.
Intermediates 3(a-c), 4(a-c), 6(a-c), 11 and 15 (Scheme 1 & 2) were prepared according to
the procedures described in the previously reported methods [15].
5.1.2 General Experimental Procedure for the Synthesis of piperazine amide derivatives (9a-
m, 13a-f and 16a-f);
To the mixture of substituted benzoic acids (8a-i and 12a-f 1 mmol) and HATU (1 mmol),
DMF (3mL) was added slowly under nitrogen atmosphere. The reaction mixture was then
stirred for 20 minutes at 0 oC, followed by the addition of substituted 2-arylquinazoline (7a-c,
11 and 15, 1 mmol). The reaction mixture was stirred for 20 minutes at room temperature,
followed by the addition of DIPEA. Upon completion of the reaction as monitored by TLC,
crushed ice was added to the reaction mixture. The resulting solid was then subjected to
vacuum filtration; excess of water was used to wash off the insoluble solids to obtain crude
powder which was purified using column chromatography (elution with hexane/EtOAc =
7:3). The pure products were collected as white colour solids in good yields.

5.1.2.1 N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)-2-naphthamide (9a)
Off-white solid; yield 80 %; mp:160–164 °C; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.51 (s, 1H), 9.93 (s, 1H), 8.64 (s, 1H), 8.60 (d, J =
8.3 Hz, 1H), 8.48 (d, J = 6.4 Hz, 2H), 8.12 (d, J = 6.9 Hz, 1H), 8.09 (s, 2H), 8.04 (d, J = 7.1
Hz, 1H), 7.99-7.94 (m, 4H), 7.89 (d, J = 3.8 Hz, 2H), 7.69-7.61 (m, 3H), 7.53 (t, J = 7.2 Hz,
3H); 13C NMR (125 MHz, DMSO- d6): δ 165.8, 159.5, 158.3, 150.9, 138.9, 135.6, 135.5,
134.7, 133.6, 132.8, 132.6, 130.7, 129.4, 128.9, 128.6, 128.5, 128.4, 128.1, 127.3, 126.3,
124.9, 123.5, 123.1, 121.0, 114.5; HRMS (ESI): m/z calculated for C31H22N4O 467.1872
found 467.1896 [M+H]+.
5.1.2.2
1-phenyl-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)-1H-1,2,3-triazole-4-
carboxamide (9b)
Off-white solid; yield 76 %; mp:163–168 °C; FT-IR (cm−1): 3325, 3059, 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.64 (s, 1H), 9.91 (s, 1H), 9.49 (d, J = 17.0 Hz, 1H),
8.59 (d, J = 8.4 Hz, 1H), 8.49-8.89 (m, 2H), 8.04 (d, J = 7.6 Hz, 2H), 7.97 (s, 3H), 7.88 (d, J
= 3.8 Hz, 2H), 7.68-7.62 (m, 3H), 7.57-50 (m, 5H); 13C NMR (125 MHz, DMSO- d6): δ
159.5, 158.4, 158.3, 150.9, 144.3, 138.8, 136.7, 135.7, 134.8, 133.6, 130.7, 130.4, 129.7,
128.8, 128.5, 128.3, 126.3, 125.9, 123.5, 123.1, 121.1, 121.0, 114.4; HRMS (ESI): m/z
calculated for C29H21N7O 484.1886 found 484.1904 [M+H]+.
5.1.2.3 5-chloro-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)thiophene-2-carboxamide (9c)
White solid; yield 78 %; mp:161–166 °C; FT-IR (cm−1): 3326, 3042, 1656, 1585, 1040, 770;
1H NMR (500 MHz, DMSO-d6): δ 10.40 (s, 1H), 10.02 (s, 1H), 8.58 (d, J = 8.3 Hz, 3H),
7.99-7.87 (m, 7H), 7.82 (s, 2H), 7.69 (s, 2H), 7.30 (s, 1H); 13C NMR (125 MHz, DMSO- d6):
δ 159.5, 159.1, 158.3, 150.9, 139.8, 138.8, 135.8, 134.5, 134.2, 133.6, 130.7, 129.4, 128.8,
128.7, 128.5, 128.4, 126.4, 123.5, 123.2, 121.1, 114.5; HRMS (ESI): m/z calculated for
C25H17ClN4OS 457.0890 found 457.0924 [M+H] +.
5.1.2.4 4-(dimethylamino)-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (9d)
White solid; yield 73 %; mp:163–167 °C; FT-IR (cm−1): 3325, 3059, 1650, 1585, 770, 710;
1H NMR (500 MHz, DMSO-d6): δ 9.91 (d, J = 9.6 Hz, 1H), 8.83 (s, 1H), 8.74 (d, J = 5.8 Hz,
1H), 8.56 (t, J = 6.4 Hz, 1H), 8.47 (d, J = 5.9 Hz, 1H), 8.33 (s, 1H), 8.19–7.98 (m, 2H), 8.00–

13
7.84 (m, 4H), 7.81–7.43 (m, 4H), 6.99-6.71 (m, 3H), 3.13 (s, 3H), 3.07–2.97 (m, 3H); C
NMR (125 MHz, DMSO- d6): δ 166.5, 136.5, 131.9, 131.6, 131.1, 131.0, 130.9, 130.7, 130.5,
130.2, 130.1, 128.7, 128.6, 128.4, 128.1, 127.9, 127.8, 126.8, 126.4, 122.5, 115.2, 52.8;
HRMS (ESI): m/z calculated for C29H25N5O 460.2137 found 460.2160 [M+H] +.
5.1.2.5 5-bromo-2-chloro-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (9e)
Light brown solid; yield 78 %; mp:160–165 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 780,
710, 680, 605; 1H NMR (500 MHz, DMSO-d6): δ 10.63 (s, 1H), 9.92 (s, 1H), 8.59 (d, J = 8.4
Hz, 1H), 8.48-8.44 (m, 2H), 7.96 (d, J = 8.9 Hz, 2H), 7.91-7.85 (m, 2H), 7.81 (d, J = 8.9 Hz,
13
2H), 7.74 (dd, J = 8.6, 2.4 Hz, 1H), 7.66-7.62 (m, 1H), 7.58–7.49 (m, 5H); C NMR (125
MHz, DMSO- d6): δ 163.6, 159.5, 158.3, 150.9, 139.2, 138.8, 135.7, 135.0, 134.2, 133.6,
132.2, 131.9, 130.7, 129.9, 128.9, 128.5, 128.3, 126.4, 123.5, 123.2, 120.5, 120.2, 114.5;
HRMS (ESI): m/z calculated for C27H18BrClN4O 529.0431 found 531.0446 [M+2] +.
5.1.2.6 4-cyano-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (9f)
Light yellow solid; yield 78 %; mp:159–163 °C; FT-IR (cm−1): 3325, 3060, 2230 1650, 1585,
780, 710; 1H NMR (500 MHz, DMSO-d6): δ 10.55 (s, 1H), 9.93 (s, 1H), 8.59 (d, J = 8.3 Hz,
1H), 8.46 (dd, J = 7.6, 1.8 Hz, 2H), 8.16 (d, J = 8.3 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 7.97 (d,
13
J = 8.9 Hz, 2H), 7.92–7.87 (m, 4H), 7.66-7.62 (m, 1H), 7.53 (d, J = 7.1 Hz, 3H); C NMR
(125 MHz, DMSO- d6): δ 164.4, 159.5, 158.3, 139.5, 138.7, 135.8, 135.0, 133.7, 132.9,
130.7, 129.0, 128.9, 128.8, 128.5, 128.3, 126.4, 123.4, 123.1, 121.0, 118.8, 114.4, 114.2;
HRMS (ESI): m/z calculated for C28H19N5O 442.1668 found 442.1694 [M+1] +.
5.1.2.7 6-bromo-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)picolinamide (9g)
Brown solid; yield 72 %; mp:161–165 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 780, 710,
680; 1H NMR (500 MHz, DMSO-d6): δ 10.56 (s, 1H), 9.92 (s, 1H), 8.97 (s, 1H), 8.59 (d, J =
8.2 Hz, 1H), 8.46 (d, J = 6.2 Hz, 2H), 8.29 (d, J = 6.3 Hz, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.87
(t, J = 6.0 Hz, 5H), 7.65–7.60 (m, 1H), 7.52 (d, J = 6.8 Hz, 3H); 13C NMR (125 MHz,
DMSO- d6): δ 163.2, 159.5, 158.3, 150.9, 150.1, 144.6, 139.0, 138.8, 135.9, 134.9, 133.6,
130.7, 128.8, 128.6, 128.5, 128.4, 128.3, 126.3, 123.4, 123.1, 121.0, 114.4 ; HRMS (ESI):
m/z calculated for C26H18BrN5O 495.0695 found 497.0646 [M+2] +.
5.1.2.8 4-iodo-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (9h)

White solid; yield 79 %; mp:163–168 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 780, 710,
680; 1H NMR (500 MHz, DMSO-d6): δ 10.36 (s, 1H), 9.90 (s, 1H), 8.59 (d, J = 8.2 Hz, 1H),
8.47 (d, J = 6.0 Hz, 2H), 7.95 (d, J = 8.6 Hz, 4H), 7.90–7.85 (m, 4H), 7.81 (d, J = 8.2 Hz,
13
2H), 7.65-761 (m, 1H), 7.52 (t, J = 6.4 Hz, 3H); C NMR (125 MHz, DMSO- d6): δ 159.4,
158.2, 151.1, 141.8, 138.7, 133.8, 130.8, 129.0, 128.9, 128.7, 128.6, 128.4, 128.2, 127.4,
126.6, 124.9, 123.5, 122.7, 122.3, 114.6, 114.0.; HRMS (ESI): m/z calculated for C27H19IN4O
543.0682 found 543.0710 [M+H] +.
5.1.2.9 5-chloro-N-(4-((2-(3,4-dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)thiophene-2-
carboxamide (9i)
Yellow solid; yield 80 %; mp:158–162 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 1245, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 11.82 (s, 1H), 10.13 (s, 1H), 8.20–7.97 (m, 4H), 7.83
(s, 2H), 7.69 (s, 1H), 7.51 (s, 1H), 7.39 (s, 1H), 6.73 (s, 2H), 6.56 (s, 1H), 5.00 (s, 1H), 3.96–
13
3.79 (m, 6H); C NMR (125 MHz, DMSO- d6): δ 163.6, 163.4, 161.6, 159.2, 152.3, 149.6,
146.0, 139.8, 134.8, 132.8, 132.3, 128.7, 128.1, 127.7, 126.6, 126.2, 122.7, 121.2, 118.8,
115.0, 114.1, 106.3, 99.0, 56.5, 56.0; HRMS (ESI): m/z calculated for C27H21ClN4O3S
517.1101 found 517.1125 [M+H] +.
5.1.2.10
5-bromo-2-chloro-N-(4-((2-(3,4-dimethoxyphenyl)quinazolin-4-
yl)amino)phenyl)benzamide (9j)
Yellow solid; yield 75 %; mp:158–162 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 1245, 780,
710, 605; 1H NMR (500 MHz, DMSO-d6): δ 11.80 (s, 1H), 9.95 (s, 1H), 8.13 (d, J = 6.7 Hz,
1H), 7.84–7.76 (m, 3H), 7.69 (d, J = 7.5 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.29 (d, J = 8.6
Hz, 2H), 7.23 (d, J = 4.0 Hz, 1H), 6.77–6.66 (m, 2H), 6.55 (d, J = 8.7 Hz, 2H), 4.98 (s, 1H),
3.90 (d, J = 8.3 Hz, 3H), 3.88 (d, J = 11.9 Hz, 3H); 13C NMR (125 MHz, DMSO- d6): δ
163.4, 161.6, 159.2, 158.5, 152.3, 149.6, 146.0, 140.3, 134.8, 133.5, 132.3, 129.2, 128.6,
128.5, 127.7, 127.5, 126.6, 126.2, 122.8, 121.5, 121.2, 115.1, 114.1, 106.3, 99.0, 56.5, 56.0;
HRMS (ESI): m/z calculated for C29H22BrClN4O3 589. 0642 found 591. 0622 [M+2] +.
5.1.2.11 4-cyano-N-(4-((2-(4-cyanophenyl)quinazolin-4-yl)amino)phenyl)benzamide (9k)
Light yellow solid; yield 74 %; mp:157–161 °C; FT-IR (cm−1): 3325, 3060, 2230 1650, 1585,
780, 710; 1H NMR (500 MHz, DMSO-d6): δ 10.57 (s, 1H), 10.03 (s, 1H), 8.60 (dd, J = 13.3,
8.4 Hz, 3H), 8.16 (d, J = 8.1 Hz, 2H), 8.06 (d, J = 8.1 Hz, 2H), 8.00 (d, J = 8.2 Hz, 2H),

7.96–7.87 (m, 6H), 7.69 (d, J = 4.2 Hz, 1H); 13C NMR (125 MHz, DMSO- d6): δ 164.4,
158.5, 157.9, 150.6, 143.1, 139.4, 135.5, 135.2, 133.9, 132.9, 128.9, 128.8, 128.8, 128.7,
127.1, 123.5, 123.3, 121.1, 119.3, 118.8, 114.6, 114.2, 112.9; HRMS (ESI): m/z calculated
for C29H18N6O 467.1620 found 467.1648 [M+H] +.
5.1.2.12
5-chloro-N-(4-((2-(4-cyanophenyl)quinazolin-4-yl)amino)phenyl)thiophene-2-
carboxamide (9l)
Yellow solid; yield 77 %; mp:159–163 °C; FT-IR (cm−1): 3325, 3060, 2230 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.64 (s, 1H), 10.02 (s, 1H), 8.63–8.55 (m, 3H), 8.01
(d, J = 8.4 Hz, 2H), 7.99-7.89 (m, Hz, 4H), 7.82 (d, J = 8.9 Hz, 2H), 7.78 (d, J = 8.6 Hz, 1H),
7.75 (d, J = 2.5 Hz, 1H), 7.57-7.51 (m, 1H); 13C NMR (125 MHz, DMSO- d6): δ 164.4,
158.5, 157.9, 150.6, 143.1, 139.4, 135.5, 135.2, 133.9, 132.9, 128.9, 128.9, 128.8, 128.7,
127.1, 123.5, 123.3, 121.1, 119.3, 118.8, 114.6, 114.2, 112.9; HRMS (ESI): m/z calculated
for C26H16ClN5OS 482.0842 found 482.0870 [M+H] +.
5.1.2.13
5-bromo-2-chloro-N-(4-((2-(4-cyanophenyl)quinazolin-4-
yl)amino)phenyl)benzamide (9m)
Yellow solid; yield 72%; mp:159–163 °C; FT-IR (cm−1): 3325, 3060, 2230 1650, 1585, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 10.64 (s, 1H), 10.02 (s, 1H), 8.65-8.61 (m, 3H), 8.01
(d, J = 8.4 Hz, 2H), 7.92 (t, J = 5.9 Hz, 4H), 7.82 (d, J = 8.9 Hz, 2H), 7.78 (d, J = 8.6 Hz,
13
1H), 7.75 (d, J = 2.5 Hz, 1H), 7.69-7.66 (m, 1H), 7.55-7.51 (m, 1H); C NMR (125 MHz,
DMSO- d6): δ 164.6, 158.4, 157.9, 150.6, 143.1, 141.0, 135.4, 135.2, 134.9, 133.9, 132.9,
131.4, 129.1, 128.9, 128.7, 127.1, 123.5, 123.3, 120.5, 120.2, 119.3, 118.0, 114.6, 112.9;
HRMS (ESI): m/z calculated for C28H17BrClN5O 554.0383 found 556.0401 [M+2] +.
5.1.2.14
5-chloro-N-(3-((2-phenylquinazolin-4-yl)amino)phenyl)thiophene-2-carboxamide
(13a)
White solid; yield 81 %; mp:160–164 °C; FT-IR (cm−1): 3326, 3042, 1656, 1585, 1040, 770;
1H NMR (500 MHz, DMSO-d6): δ 10.40 (s, 1H), 10.02 (s, 1H), 8.69–8.45 (m, 3H), 8.06–7.78
13
(m, 9H), 7.72–7.47 (m, 2H), 7.30 (s, 1H); C NMR (125 MHz, DMSO- d6): δ 164.6, 158.4,
157.9, 150.6, 143.1, 141.0, 135.4, 135.3, 134.9, 133.9, 132.9, 131.4, 129.1, 128.9, 128.7,
127.1, 123.5, 123.3, 120.2, 119.3, 118.0, 114.6, 112.9; HRMS (ESI): m/z calculated for
C25H17ClN4OS 457.0890 found 457.0924 [M+H] +.

5.1.2.15 5-bromo-2-chloro-N-(3-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (13b)
Brown solid; yield 75 %; mp:159–163 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 780, 710,
1
680, 605; H NMR (500 MHz, DMSO-d6): δ 10.63 (s, 1H), 9.99-9.91 (m, 1H), 8.58 (t, J =
10.8 Hz, 1H), 8.49-8.41 (m, 2H), 7.99-7.89 (m, 2H), 7.89-7.82 (m, 3H), 7.81 (d, J = 8.9 Hz,
2H), 7.79-7.89 (m, 1H), 7.65-7.61 (m, 1H), 7.58–7.50 (m, 4H); 13C NMR (125 MHz, DMSO-
d6): δ 162.6, 160.3, 152.8, 149.2, 139.7, 137.3, 135.1, 133.8, 131.7, 129.4, 127.8, 127.5,
127.1, 126.4, 124.4, 124.1, 123.5, 123.1, 122.3, 121.5, 120.4, 120.2, 117.1, 112.9, 104.6;
HRMS (ESI): m/z calculated for C27H18BrClN4O 529.0431 found 531.0446 [M+2] +.
5.1.2.16 4-cyano-N-(3-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (13c)
Light yellow solid; yield 76 %; mp:159–163 °C; FT-IR (cm−1): 3325, 3060, 2230 1650, 1585,
780, 710; 1H NMR (500 MHz, DMSO-d6): δ 10.56 (s, 1H), 9.92 (s, 1H), 8.97 (s, 1H), 8.59 (d,
J = 8.2 Hz, 1H), 8.46 (d, J = 6.2 Hz, 2H), 8.33–8.23 (m, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.89-
13
7.78 (m, 5H), 7.75–7.32 (m, 5H); C NMR (125 MHz, DMSO- d6): δ 164.4, 158.5, 157.9,
150.6, 143.1, 139.4, 135.5, 135.2, 133.9, 132.9, 128.9, 128.8, 128.7, 128.4, 127.1, 123.5,
123.3, 121.2, 121.1, 119.3, 118.8, 114.6, 114.2, 112.9; HRMS (ESI): m/z calculated for
C28H19N5O 442.1668 found 442.1694 [M+H] +.
5.1.2.17 4-iodo-N-(3-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (13d)
White solid; yield 82 %; mp:163–168 °C; FT-IR (cm−1): 3325, 3060, 1650, 1585, 780, 710,
680; 1H NMR (500 MHz, DMSO-d6): δ 10.36 (s, 1H), 9.90 (s, 1H), 8.59 (d, J = 8.2 Hz, 1H),
8.53–8.43 (m, 2H), 7.99-7.93 (m, 4H), 7.92–7.84 (m, 4H), 7.81 (d, J = 8.2 Hz, 2H), 7.67–
13
7.50 (m, 4H); C NMR (125 MHz, DMSO- d6): δ 163.2, 159.5, 158.3, 150.9, 150.1, 144.6,
139.1, 139.0, 138.8, 135.9, 134.9, 133.6, 130.7, 128.8, 128.7, 128.6, 128.4, 128.3, 126.3,
123.4, 123.1, 121.0, 114.4; HRMS (ESI): m/z calculated for C27H19IN4O 543.0682 found
543.0710 [M+H] +.
5.1.2.18 4-(dimethylamino)-N-(3-((2-phenylquinazolin-4-yl)amino)phenyl)benzamide (13e)
White solid; yield 79%; mp:158–162 °C; FT-IR (cm−1): 3325, 3059, 1650, 1585, 770, 710;
1H NMR (500 MHz, DMSO-d6): δ 9.91 (d, 1H), 8.93–8.51 (m, 3H), 8.47 (d, J = 5.9 Hz, 1H),
8.09-8.01 (m, 2H), 7.97–7.76 (m, 5H), 7.76–7.58 (m, 2H), 7.52 (s, 2H), 7.15 (s, 1H), 6.92–
6.88 (m, 1H), 6.79-7.68 (m, 1H), 3.13 (s, 3H), 3.08–2.96 (m, 3H); 13C NMR (125 MHz,

DMSO- d6): δ 166.5, 136.5, 131.9, 131.8, 131.6, 131.1, 131.0, 130.9, 130.7, 130.5, 130.2,
130.1, 128.9, 128.7, 128.6, 128.4, 128.1, 127.9, 127.8, 126.8, 126.4, 122.5, 115.2, 52.8;
HRMS (ESI): m/z calculated for C29H25N5O 460.2137 found 460.2160 [M+H] +.
5.1.2.19 6-bromo-N-(3-((2-phenylquinazolin-4-yl)amino)phenyl)-2-naphthamide (13f)
Light brown solid; Yield 81 %; mp: 163-168 oC; FT-IR (cm−1): 3325, 3062, 1650, 1585, 780,
1
710; H NMR (500 MHz, DMSO-d6): δ 10.58 (s, 1H), 9.97 (s, 1H), 8.71-8.61 (m, 4H), 8.55
(s, 1H), 8.32 (s, 1H), 8.15–8.09 (m, 3H), 8.03 (d, J = 5.9 Hz, 2H), 7.90 (s, 2H), 7.75 (d, J =
13
15.6 Hz, 2H), 7.64 (s, 1H), 7.49 (s, 3H); C NMR (125 MHz, DMSO- d6): δ 165.8, 159.5,
158.3, 150.9, 138.9, 135.6, 135.5, 134.7, 134.7, 133.6, 132.8, 132.8, 132.6, 130.7, 129.7,
129.4, 128.9, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 127.3, 126.3, 124.9, 123.5, 123.1,
121.0, 114.5; HRMS (ESI): m/z calculated for C31H21BrN4O 545.0977 found 542.0966
[M+2] +.
5.1.2.20 N-(3,5-dimethoxyphenyl)-4-((2-phenylquinazolin-4-yl)amino)benzamide (17a)
o
White solid; Yield 78 %; mp: 165-169 C; FT-IR (cm−1): 3320, 3053, 1645, 1582, 780, 710;
1H NMR (500 MHz, DMSO-d6): δ 10.14 (s, 1H), 10.10 (s, 1H), 8.65 (d, J = 8.2 Hz, 1H), 8.51
(d, J = 6.8 Hz, 2H), 8.22 (d, J = 8.4 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2H), 7.93 (s, 2H), 7.68 (s,
13
1H), 7.56 (d, J = 7.3 Hz, 3H), 7.15 (s, 2H), 6.28 (s, 1H), 3.77 (s, 6H); C NMR (125 MHz,
DMSO- d6): δ 165.4, 160.8, 159.4, 158.2, 151.1, 143.1, 141.5, 138.6, 130.9, 129.6, 129.0,
128.8, 128.7, 128.4, 126.6, 123.6, 121.3, 114.6, 99.0, 96.1, 55.6; HRMS (ESI): m/z
calculated for C29H24N4O3 477.1927 found 477.1960 [M+H] +.
5.1.2.21 4-((2-phenylquinazolin-4-yl)amino)-N-(3,4,5-trimethoxyphenyl)benzamide (17b)
o
White solid; Yield 77 %; mp: 163-167 C; FT-IR (cm−1): 3320, 3053, 1645, 1582, 780, 710;
1H NMR (500 MHz, DMSO-d6): δ 10.48 (s, 1H), 10.13 (s, 1H), 8.63 (d, J = 8.3 Hz, 1H), 8.30
(s, 1H), 8.26 (d, J = 8.8 Hz, 2H), 8.18-8.10 (m, 3H), 7.93 (t, J = 7.0 Hz, 2H), 7.86 (s, 2H),
7.73–7.52 (m, 3H), 7.47 (d, J = 7.7 Hz, 1H), 3.94 (s, 6H), 3.77 (s, 3H); 13C NMR (125 MHz,
DMSO- d6): δ 165.7, 158.8, 158.0, 153.3, 152.7, 151.1, 143.4, 140.6, 140.2, 133.9, 130.3,
129.2, 128.7, 128.6, 126.5, 124.2, 123.5, 121.5, 116.8, 114.4, 105.6, 60.6, 56.2; HRMS (ESI):
m/z calculated for C30H26N4O4 507.2032 found 407.2060 [M+H] +.
5.1.2.22 N-(3-chloro-4-fluorophenyl)-4-((2-phenylquinazolin-4-yl)amino)benzamide (17c)

o
White solid; Yield 70 %; mp: 157-161 C; FT-IR (cm−1): 3320, 3053, 1645, 1582, 780, 710;
1H NMR (500 MHz, DMSO-d6): δ 10.37 (s, 1H), 10.14 (s, 1H), 8.64 (t, J = 8.2 Hz, 1H), 8.50
(t, J = 7.5 Hz, 2H), 8.39–8.18 (m, 2H), 8.18–8.00 (m, 3H), 7.93 (d, J = 2.8 Hz, 2H), 7.90–
7.76 (m, 1H), 7.75–7.66 (m, 1H), 7.55 (t, J = 9.1 Hz, 3H), 7.44 (t, J = 9.1 Hz, 1H); 13C NMR
(125 MHz, DMSO- d6): δ 162.7, 159.3, 158.3, 152.8, 135.0, 134.1, 133.2, 131.8, 131.1,
129.0, 128.4, 128.2, 128.1, 127.8, 127.0, 126.8, 126.3, 124.1, 123.6, 122.5, 122.4, 121.4,
114.5; HRMS (ESI): m/z calculated for C27H18ClFN4O 469.1231 found 469.1250 [M+H] +.
5.1.2.23 Ethyl 3-(4-((2-phenylquinazolin-4-yl)amino)benzamido)propanoate (17d)
o
White solid; Yield 80 %; mp: 155-159 C; FT-IR (cm−1): 3320, 3053, 1645, 1582, 780, 710;
1H NMR (500 MHz, DMSO-d6): δ 10.04 (s, 1H), 8.62 (d, J = 8.1 Hz, 1H), 8.58–8.39 (m,
3H), 8.13 (d, J = 8.0 Hz, 2H), 8.03–7.85 (m, 4H), 7.66 (s, 1H), 7.54 (s, 3H), 4.10 (d, J = 7.0
Hz, 2H), 3.54 (d, J = 5.6 Hz, 2H), 2.62 (s, 2H), 1.22-1.17 (m, 3H); 13C NMR (125 MHz,
DMSO- d6): δ 171.8, 166.3, 159.4, 158.2, 151.0, 142.6, 138.6, 133.9, 130.8, 129.3, 128.9,
128.6, 128.3, 128.2, 126.6, 123.5, 121.4, 114.5, 60.4, 36.0, 34.3, 14.5; HRMS (ESI): m/z
calculated for C26H24N4O3 441.1927 found 441.1960 [M+H] +.
5.1.2.24 Ethyl (4-((2-phenylquinazolin-4-yl)amino)benzoyl)leucinate (17e)
o
White solid; Yield 79 %; mp: 152-156 C; FT-IR (cm−1): 3320, 3053, 1645, 1582, 780, 710;
1H NMR (500 MHz, DMSO-d6): δ 10.07 (d, J = 12.4 Hz, 1H), 8.69-8.60 (m, 2H), 8.49 (d, J =
6.8 Hz, 1H), 8.16 (d, J = 8.1 Hz, 2H), 8.07–7.96 (m, 2H), 7.91 (s, 2H), 7.84 (s, 1H), 7.66 (s,
2H), 4.55 (s, 1H), 3.88-3.81 (m, 2H), 3.68 (s, 1H), 3.48 (s, 3H), 1.86-1.65 (m, 3H), 1.13-0.93
(m, 6H); 13C NMR (125 MHz, DMSO- d6): δ 173.7, 166.5, 159.4, 158.2, 153.3, 151.0, 142.9,
138.6, 133.9, 130.8, 128.9, 128.6, 128.3, 126.6, 123.5, 121.6, 121.3, 114.5, 56.2, 52.3, 51.4,
24.9, 23.3, 21.6; HRMS (ESI): m/z calculated for C29H30N4O3 483.2396 found 483.2426
[M+H] +.
5.1.2.25 N-((4-chlorobenzyl)oxy)-4-((2-phenylquinazolin-4-yl)amino)benzamide (17f)
o
White solid; Yield 8 %; mp: 152-156 C; FT-IR (cm−1): 3320, 3053, 1645, 1582, 1550, 780,
710; 1H NMR (500 MHz, DMSO-d6): δ 11.75 (s, 1H), 10.06 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H),
8.50-8.46 (m, 2H), 8.14 (d, J = 8.7 Hz, 2H), 7.96-7.91 (m, 4H), 7.69–7.64 (m, 1H), 7.55 (t, J
= 6.1 Hz, 3H), 7.50 (d, J = 6.9 Hz, 2H), 7.45-7.37 (m, 3H), 4.97 (s, 2H); 13C NMR (125
MHz, DMSO- d6): δ 159.4, 158.2, 151.1, 143.0, 138.6, 136.5, 133.9, 130.9, 129.3, 128.9,

128.8, 128.7, 128.4, 128.2, 127.2, 126.6, 123.6, 121.5, 114.5, 77.5; HRMS (ESI): m/z
calculated for C28H21ClN4O2 481.1431 found 481.1456 [M+H] +.
5.2 Antibiotic susceptibility testing against ESKAP pathogen panel
Antibiotic susceptibility testing was carried out on the newly synthesized compounds by
determining the Minimum Inhibitory Concentration (MIC) with reference to the standard
CLSI guidelines [17, 18]. MIC is defined as the minimum concentration of compound at
which visible bacterial growth is inhibited. Bacterial cultures were grown in Mueller-Hinton
cation supplemented broth (CA-MHB). Optical density (OD600) of the cultures was measured,
followed by dilution for ~106 cfu/mL. This inoculum was added into a series of test wells in a
microtitre plate that contained various concentrations of compound under test ranging from
64-0.03 µg/mL. Controls i.e., cells alone and media alone (without compound+cells) and
levofloxacin used as a reference standard. Plates were incubated at 37 °C for 16-18 h
followed by observations of MIC values by the absence or presence of visible growth. For
each compound, MIC determinations were performed independently thrice using duplicate
samples each time.
5.3 Antibiotic susceptibility testing against pathogenic mycobacteria
Antimycobacterial susceptibility testing was carried out on the newly synthesized compounds
by using broth micro dilution assay [19]. 1g/100 mL stock solutions of test and control
compounds were prepared in DMSO and stored in −20 °C. Mycobacterial cultures were
inoculated in Middlebrook 7H9 enriched (Difco, Becton, NJ, USA) media supplemented with
10% ADC-Tween-80 (Bovine Serum Albumin, Dextrose, 0.2% glycerol and 0.05% Tween-
80) and OD600 of the cultures was measured, followed by dilution to achieve ~106 cfu/mL
[20]. The newly synthesized compounds were tested from 0.0064–0.00005 g/100 mL in two-
fold serial diluted fashion with 2.5 μL of each concentration added per well of a 96-well
round bottom microtitre plate. Later, 97.5 μL of bacterial suspension was added to each well
containing the test compound along with appropriate controls. Presto blue (Thermo Fisher,
USA) resazurin-based dye was used for the visualized identification of active compounds.
MIC of active compound was determined as lowest concentration of compound that inhibited
visible growth after incubation period. For each compound, MIC determinations were
replicated thrice using duplicate samples. The MIC plates were incubated at 37 °C for 7 days
for Mtb.

5.4 Molecular docking:
The crystal co-ordinates of Glmu were retrieved from the protein data bank (PDB ID: 4K6R).
The 3D structure of ligands was drawn on Maestro Molecule Builder of of Schrödinger suite
2015-4.9 version 10.4. The molecules were optimized using OPLS_2005 force field in
LigPrep 3.6 module of Schrödinger suite 2015-4.9. Docking procedure [21] was performed
according to the default settings implemented in maestro software, version 10.4 and the
ligands was docked into the binding site of GlmU (PDB ID: 4K6R).
5.4.1 Binding energy calculations for Active molecules with binding site of GlmU
The MM/GBSA (Molecular mechanics/generalized born surface area) analysis was used to
calculate ligand-binding energies based on docking complex, using the MM/GBSA
technology available in Prime module of Schrodinger software [22]. We performed binding
energy calculations of docked complexes of GlmU with the active molecules and the results
were depicted in Table 2.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
S. M is thankful to Department of Pharmaceuticals, Ministry of Chemicals & Fertilizers,
Govt. of India, for the award of NIPER fellowship. This study was supported by the DST
grant from Department of Science and Technology, Govt. of India to S.N and S.C.
(EMR/2017/000220).
References:
1. a) Yuan. T, Sampson. SN. Hit Generation in TB Drug Discovery: From Genome to
Granuloma. Chem. Rev. 2018; 118: 1887−1916. b) Poce. G, Cocozza. M, Consalvi. S,
Biava. M. SAR analysis of new anti-TB drugs currently in pre-clinical and clinical
development. Eur. J. Med. Chem. 2014; 86: 335-351. c) Ballell. L, Bates. HR, Young.
JR, Gomez. AD, Ruiz. AE, Barroso. V, Blanco. D, Crespo. B, Escribano. J, Lozano.
SR, Huss. S, Villarejo. AS, Plaza. JJ, Mendoza. A, Lopez. RM, Blanco. RM,
Lavandera. LJ, Herran. RE, Benito. FJ, Bustos. FJ, Barros. D, Castro. PJ, Cammack.

N. Fueling Open-Source Drug Discovery: 177 Small-Molecule Leads against
Tuberculosis. ChemMedChem. 2013; 8: 313-321. d) Kurz. GS, Furin. JJ, Bark. MC.
Drug Resistant Tuberculosis: Challenges and Progress. Infect Dis Clin North Am.
2016; 30: 509-522.
2. a) Palomino. CJ, Martin. A. Drug Resistance Mechanisms in Mycobacterium
tuberculosis. Antibiotics. 2014; 3: 317-340. b) Floyd. K, Glaziou. P, Zumla. A,
Raviglione. M. The global tuberculosis epidemic and progress in care, prevention, and
research: an overview in year 3 of the End TB era. Lancet Res Med. 2018; 6: 299-314.
c) Sandhu. KG. Tuberculosis: Current Situation, Challenges and Overview of its
Control Programs in India. J Glob Infect Dis. 2011; 3: 143-150.
3. https://www.who.int/en/news-room/fact-sheets/detail/tuberculosis.
4. a) Dartoisa. V, Barry. EC. A medicinal chemists guide to the unique difficulties of
lead optimization for tuberculosis. Bioorg. Med. Chem. Lett. 2013; 23: 4741-4750. b)
Chetty. S, Ramesh. M, Pillay. SA, Soliman. SEM. Recent advancements in the
development of anti-tuberculosis drugs. Bioorg. Med. Chem. Lett. 2017; 27: 370-386.
c) Makane. BV, Krishna. SV, Krishna. V, Shukla. Mahizhaveni. BM, Misra. S,
Chopra. S, Sriram. D, Dusthackeer. ANV, Rode. BH. Synthesis and evaluation of a-
aminoacyl amides as antitubercular agents effective on drug resistant tuberculosis.
Eur. J. Med. Chem. 2019; 164: 665-677.
5. Villemagne. B, Crauste. C, Flipo. M, Baulard. RA, Deprez. B, Willand. N.
Tuberculosis: The drug development pipeline at a glance, Eur. J. Med. Chem. 2012;
51: 1-16.
6. a) Kunes. J, Ant. AB, Pour. M, Waisser. K, Rek. MS, Janota. J. Quinazoline
derivatives with antitubercular activity. Il Farmaco. 2000; 55: 725-729. b) Srivastav.
MK, Shantakumar. SM. Design and Synthesis of Novel 2-Trichloromethyl-4-
Substituted Quinazoline Derivatives as Anti-tubercular Agents. Chem Sci Trans.
2013; 2: 1056-1062. c) Gatadi. S, Gour. J, Shukla. M, Kaul. G, Dasgupta. A,
Madhavi. VY, Chopra. S, Nanduri, S. Synthesis and evaluation of new quinazolin-
4(3H)-one derivatives as potent antibacterial agents against multidrug resistant
Staphylococcus aureus and Mycobacterium tuberculosis. Eur. J. Med. Chem. 2019;
175: 287-308. d) Asundaria. ST, Patel. NS, Patel. CK. Synthesis, characterization, and
antimicrobial studies of novel 1,3,4-thiadiazolium-5-thiolates. Med. Chem. Res. 2012;
21: 1199-1206.

7. a) Nepomuceno. MG, Chan. MK, Huynh.V, Martin. SK, Moore. TJ, Brien. ET, Pollo.
EAL, Sarabia. JF, Tadeus. C, Yao. Z, Anderson. ED, Ames. BJ, Shaw. TJ. Synthesis
and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein
FtsZ. ACS Med. Chem. Lett. 2015; 63: 308-312. b) Kung. PP, Casper. DM, Cook. LK,
Lingardo. WL, Risen. ML, Vickers. AT, Ranken. R, Blyn. BL, Wyatt. RJ, Cook. DP,
Ecker. JD. Structure−Activity Relationships of Novel 2-Substituted Quinazoline
Antibacterial Agents. J. Med. Chem. 1999; 42: 4705-4713. c) Gatadi. S, Gour. J,
Shukla. M, Kaul. G, Dasgupta. A, Madhavi. VY, Chopra. S, Nanduri, S. Synthesis
and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide
derivatives as potent antibacterial agents effective against multidrug resistant
Staphylococcus aureus. Bioorg. Chem. 2018; 83: 569-579.
8. Ugalea. GV, Bari. BS. Quinazolines: New horizons in anticonvulsant therapy. Eur. J.
Med. Chem. 2014; 80: 447-501.
9. a) Horn. VSK, Zhu. X, Pandharkar. T, Yang. S, Vesely. B, Vanaerschot. M, Dujardin.
CJ, Rijal. S, Kyle. ED, Wang. ZM, Werbovetz. AK, Manetsch. R. Antileishmanial
Activity of a Series of N2,N4-Disubstituted Quinazoline-2,4-diamines. J. Med. Chem.
2014; 57: 5141-5156. b) Hassanzadeh. F, Jafari. E, Hakimelahi. HG, Khajouei. RM,
Jalali. M, Khodarahmi. AG. Antibacterial, antifungal and cytotoxic evaluation of
some new quinazolinone derivatives. Res Pharm Sci. 2012; 7: 87-94. c) Modh. PR,
Clercq. DE, Pannecouque. C, Chikhalia. HK. Design, synthesis, antimicrobial activity
and anti-HIV activity evaluation of novel hybrid quinazoline–triazine derivatives. J
Enzyme Inhib Med Chem. 2013; 29: 1475-6374.
10. a) Farag. BD, Farag. AN, Esmat. A, Abuelezz. AS, Ibrahimd. SAE, El-Ellae. AD.
Synthesis, 3D pharmacophore, QSAR and docking studies of novel quinazoline
derivatives with nitric oxide release moiety as preferential COX-2 inhibitors. Med.
Chem. Commun. 2015; 6: 283-299. b) Alafeefya. M, Omar. KAA, DeebaKamal. AA,
TahirbNabila. EHE, Jaberc. AA. Synthesis, analgesic and anti-inflammatory
evaluation of some novel quinazoline derivatives. Eur. J. Med. Chem. 2010; 45: 4947-
4952. c) Smits. AR, Adami. M, Istyastono. PE, Zuiderveld. PO, Dam. EMC, Kanter.
JJF, Jongejan. A, Coruzzi. G, Leurs. R, Esch. PJI. Synthesis and QSAR of
Quinazoline Sulfonamides as Highly Potent Human Histamine H4 Receptor Inverse
Agonists. J. Med. Chem. 2010; 53: 2390-2400.

11. a) Hennequin. FL, Boyle. TF, Michael. J, Peter. W, Marsham. R, Kimbell. R,
Jackman. AL. Quinazoline Antifolates Thymidylate Synthase Inhibitors:ꢀ Lipophilic
Analogues with Modification to the C2-Methyl Substituent. J. Med. Chem. 1996; 39:
695-704. b) Krapf. KM, Gallus. J, Wiese. M. 4-Anilino-2-pyridylquinazolines and -
pyrimidines as highly potent and nontoxic inhibitors of breast cancer resistance
protein (ABCG2). J. Med. Chem. 2017; 60: 4474-4495. c) Chen. J, Sang. Z, Jiang. Y,
Yang. C, He. L. Design, synthesis, and biological evaluation of quinazoline
derivatives as dual HDAC1 and HDAC6 inhibitors for the treatment of cancer. Chem
Biol Drug Des. 2019; 93: 232-241.
12. a) Li. P, Wang. B, Zhang. X, Batt. MB, Besra. SG, Zhang. T, Ma. C, Zhang. D, Lin.
Z, Li. G, Huang. H, Lu. Y. Identification of novel benzothiopyranone compounds
against Mycobacterium tuberculosis through scaffold morphing from
benzothiazinones. Eur. J. Med. Chem. 2018; 160: 157-170. b) Yang. X, Wedajo. W,
Yamada. Y, Dahlroth. LS, Neo. JJ, Dick. T, Chui. KW. 1,3,5-triazaspiro [5.5] undeca-
2,4-dienes as selective Mycobacterium tuberculosis dihydrofolate reductase inhibitors
with potent whole cell activity. Eur. J. Med. Chem. 2018; 144: 262-276.
13. Wang. T, Bemis. G, Hanzelka. B, Zuccola. H, Wynn. M, Moody. C, Green. J, Locher.
C, Liu. A, Gao. H, Xu. Y, Wang. S, Wang. J, Bennani. Y, Thomson. J, Muh. U. Mtb
PKNA/PKNB dual inhibition provides selectivity advantages for inhibitor design to
minimize host kinase interactions. ACS Med. Chem. Lett. 2017; 8: 1224-1229.
14. Tran. AT, Wen. D, West. NP, Baker. EN, Britton. WJ, Payne. RJ. Inhibition studies
on Mycobacterium Tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase
(GlmU). Org. Biomol. Chem. 2013; 11: 8113-8126.
15. a) Kim. NY, Cheon. CH. Synthesis of quinazolinones from anthranilamides and
aldehydes via metal-free aerobic oxidation in DMSO. Tetrahedron Lett. 2014; 55:
2340-2344. b) Krapf. KM, Gallus. J, Spindler. A, Wiese. M. Synthesis and biological
evaluation of quinazoline derivatives- A SAR study of novel inhibitors of ABCG2.
Eur. J. Med. Chem. 2019; 161: 506-525.
16. Vithani, N., Bais, V., Prakash, B. GlmU (N-acetylglucosamine-1-phosphate
uridyltransferase) bound to three magnesium ions and ATP at the active site. Acta
Crystallogr.,Sect.F. 2014; 70: 703-708.

17. Wayne. AP. Methods for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically, approved standard, ninth ed., CLSI document M07-A9, Clinical and
Laboratory Standards Institute, 2012.
18. Jorgensen. HJ, Hindler.FJ, Reller. BL, Weinstein. PM. New consensus guidelines
from the clinical and laboratory standards institute for antimicrobial susceptibility
testing of infrequently isolated or fastidious bacteria. Clin. Infect. Dis. 2007; 44: 280-
286.
19. (a) Pandey. M, Singh. KA, Thakare. R, Talwar. S, Karaulia. P, Dasgupta. A, Chopra.
S, Pandey. KA. Diphenyleneiodonium chloride (DPIC) displays broad-spectrum
bactericidal activity. Sci. Rep. 2017; 7: 11521; (b) Wiegand. I, Hilpert. K, Hancock.
ER. Agar and broth dilution methods to determine the minimal inhibitory
concentration (MIC) of antimicrobial substances. Nat. Protoc. 2018; 3: 163-175.
20. Santiago. RB, Santiago. REC, Delgado. JEC, Contreras. LCJ, Hancock. ER, Pando.
HR. Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2,
E6 and CP26 against Mycobacterium tuberculosis, Int. J. Antimicrob. Agents. 2013;
41: 143-148.
21. Goud. SN, Ghouse. MS, Vishnu. J, Komal. D, Talla. V, Alvala. R, Pranay. J, Kumar.
J, Qureshid. AI, Alvala. M. Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1
mediated anticancer agents. Bioorg. Chem. 2019; 89: 103016.
22. Ye. J, Yang. X, Xu M, Chan. KP, Ma. C. Novel N-Substituted oseltamivir derivatives
as potent influenza neuraminidase inhibitors: Design, synthesis, biological evaluation,
ADME prediction and molecular docking studies. Eur. J. Med. Chem. 2019; 182:
111635.

Synthesis, biological evaluation and molecular modelling insights of 2-
arylquinazoline benzamide derivatives as anti-tubercular agents
Satyaveni Malasalaa, Md. Naiyaz Ahmadb, Jitendra Goura, Manjulika Shuklab, Grace Kaulb,
Abdul Akhirb, Srikanth Gatadia, Y.V Madhavia, Sidharth Choprab* and
Srinivas Nanduria*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, Telangana, India
bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow-226031, Uttar Pradesh, India
Highlights:
1. New 2-arylquinazoline aminobenzamide derivatives were synthesized successfully,
with simple reaction conditions.
2. A series of 25 molecules were screened against Mycobacterium tuberculosis H37RV
strain for the anti-mycobacterial activity.
3. 9a, 9c, 9d, 9e, 9f, 9h, 13b, 17d and 17e displayed potent and specific anti-
mycobacterial activity against Mycobacterium tuberculosis with MIC values in range
of 4-32 µg/mL.
4. 2 molecules (9e and 9f) identified as potent leads, with the minimum inhibitory
concentration of 4 µg/mL, respectively.