Discovery of Quinazolin-4(3 H)-ones as NLRP3 Inflammasome Inhibitors: Computational Design, Metal-Free Synthesis, and in Vitro Biological Evaluation

Article abstract of DOI:10.1021/acs.joc.9b00138

NLRP3 inflammasome is an important therapeutic target for a number of human diseases. Herein, computationally designed series of quinazolin-4(3H)-ones were synthesized using iodine-catalyzed coupling of arylalkynes (or styrenes) with O-aminobenzamides. The key event in this transformation involves the oxidative cleavage of the C-C triple/double bond and the release of formaldehyde. The reaction relies on the C-N bond formation along with the C-C bond cleavage under metal-free conditions. The nitro-substituted quinazolin-4(3H)-one 2k inhibited NLRP3 inflammasome (IC₅₀ 5 μM) via the suppression of IL-1β release from ATP-stimulated J774A.1 cells.

Full text of DOI:10.1021/acs.joc.9b00138

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Article
Discovery of Quinazolin-4(3H)-ones as NLRP3 Inflammasome Inhibitors:
Computational Design, Metal-free Synthesis and In-vitro Biological Evaluation
Mohd Abdullaha, Shabber Mohammed, Mehboob Ali, Ajay
Kumar, Ram A. Vishwakarma, and Sandip B. Bharate
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Mar 2019
Downloaded from http://pubs.acs.org on March 21, 2019
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Discovery of Quinazolin-4(3H)-ones as NLRP3
Inflammasome Inhibitors: Computational Design, Metal-
free Synthesis and In-vitro Biological Evaluation
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Mohd Abdullaha,†‡ Shabber Mohammed,†‡ Mehboob Ali,‡‖ Ajay Kumar,‡‖
Ram A. Vishwakarma,†‡ Sandip B. Bharate†‡*
†Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180001, India
‡Academy of Scientific & Innovative Research, CSIR-Indian Institute of Integrative Medicine,
Canal Road, Jammu-180001, India
‖PKPD Toxicology & Formulation Division, CSIR - Indian Institute of Integrative Medicine,
Canal Road, Jammu-180001, India
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O
O
Iodine (20 mol%)
R
C
Ph
150
125
100
75
NH
Ph
R-CHO
+
NH2
NH2
O2, DMSO, H2O
110 oC, 12 h
OR
N
R
8
Ph
Quinazolin-4(3H)-ones
9
Tyr 168
Leu 171
Lys 238
***
Thr 169
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***
25
O
Met 239
Arg 237
0
Tyr 381
NH
N
Trp 416
2k
NO2
Lys 232
InhibitsNLRP3
inflammasome
(IC50 = 5 µM)
Thr 233
ABSTRACT.
NLRP3 inflammasome is an important therapeutic target for number of human diseases. Herein,
a computationally-designed series of quinazolin-4(3H)-ones were synthesized using iodine-
catalyzed coupling of arylalkynes (or styrenes) with O-aminobenzamides. Key event in this
transformation involves oxidative-cleavage of C–C triple bond and the release of formaldehyde.
Reaction relies on the C-N bond formation along with C-C bond cleavage under metal-free
conditions. The nitro-substituted quinazolin-4(3H)-one 2k inhibited NLRP3-inflammasome (IC50
5 µM) via suppression of IL-1β release from ATP-stimulated J774A.1 cells.
KEYWORDS.
NLRP3-inflammasome; Homology model; quinazolin-4(3H)-ones; C-C triple bond;
aminobenzamides; alkynes; Alzheimer's disease.
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INTRODUCTION
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The NLRP3 inflammasome play an important role in the pathogenesis of wide range of human
diseases including Alzheimer's disease,1 diabetic encephalopathy,2 rheumatoid arthritis,3 chronic
liver disease,4 gout,5 cryopyrin-associated periodic syndrome (CAPS), etc.6 The NLRP3 protein
belongs to the family of nucleotide-binding and oligomerization domain-like receptors (NLRs).
In addition to the NLRP3 protein, the NLRP3 inflammasome also contains adapter protein
apoptosis-associated speck-like protein (ASC) and procaspase-1. The interaction between these
three proteins tightly regulates the inflammasome function in order to ensure immune activity.7
The active caspase-1 released from NLRP3 inflammasome converts pro-IL-1β to its mature form
IL-1β, which is a potent pro-inflammatory cytokine involved in the process of inflammation.
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In recent years, number of small molecules have been identified as inhibitors of NLRP3
inflammasome.8 The inhibition of this complex has been achieved by small molecules, via
inhibiting ATP-sensitive potassium channels (e.g. glyburide),9 inhibiting the activation of
caspase-1 (e.g. parthenolide),10 inhibiting NLRP3 ATPase activity (e.g. parthenolide, Bay 11-
7082,10 acrylamide derivative11), inhibiting the release of IL-1β (e.g. MCC95012) and via
inhibition of ATP receptor P2X7 (e.g. AZD9056) (Figure 1).13 The β-estradiol14 has been
reported to inhibit levels of IL-1β, caspase-1 as well as P2X7 receptor.14 OLT1177, 3-
(methylsulfonyl)propanenitrile, inhibits the IL-1β release as well as caspase-1 activity, and has
been advanced to the clinical trials in patients with knee osteoarthritis. Majority of the reported
NLRP3 inflammasome inhibitors are sulphonyl derivatives and thus identification of new
chemotype (non-sulphonyl) would be an important addition to NLRP3 inhibitors. The structure-
based drug design approach provides an opportunity to come up with the new chemotype;
therefore herein we employed computational approach to design quinazolin-4(3H)-ones as a new
class of NLRP3 inflammasome inhibitors.
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O
O
O
O
S
S
OMe O
N
N
CN
H
H
H
O
S
N
HN
N
H
O
O
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O
OH
Cl
MCC950
Glyburide
BAY11-7082
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O
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OH
NH2
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O
O
H
O
O
N
S
CN
H
H
H
Cl
HO
Acrylamide derivative
OLT1177
-Estradiol
Figure 1. Structures of representative NLRP3 inflammasome inhibitors
Because of the biological importance of quinazolin-4(3H)-ones,15 it has been explored
extensively by synthetic chemists.16 The condensation of O-aminobenzamides with aldehydes in
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20
the presence of oxidants such as CuCl2, DDQ,18 MnO2, and KMnO4 is one of the most
elegant approach for its synthesis. In recent years, numerous protocols involving sp3 carbon
synthons such as benzyl halides,21 benzyl alcohols,22 and benzyl amines23 have been reported. Pd
catalyzed condensation with aryl halides via carbon insertion from CO24 or isocyanide25 are also
reported. Other reported coupling partners include aryl carboxylic acid,26 aryl acid chloride,27
methylarenes.28 Cheng and Cui’s group29 reported TFA mediated synthesis of 2-arylquinazolin-
4(3H)-ones via condensation of O-aminobenzamides with ketoalkynes. This transformation
involved TFA mediated C-C triple bond cleavage of ketoalkynes producing 2-arylquinazolin-
4(3H)-ones along with acetophenone as a byproduct. However, to the best of our knowledge, the
synthesis of 2-arylquinazolin-4(3H)-ones directly from terminal alkynes and styrenes has never
been reported. Furthermore, the use of terminal alkynes / styrenes has several advantages over
ketoalkynes, in terms of cost and commercial availability. In continuation to our interest in
synthesis of medicinally important heterocycles,30 and to validate computationally designed new
NLRP3 inflammasome inhibitors, herein we have synthesized a series of 2-arylquinazolin-
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4(3H)-ones via coupling of O-aminobenzamides with terminal aryl alkynes (and also with
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styrenes) under metal-free condition using molecular oxygen as an oxidant (Figure 2).
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Previous work (Cheng & Cui's group)
O
O
O
O
9
NH
Ph
TFA (3 equiv)
NH2
NH2
+
Ph
Ph
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N2, Toluene,
90 oC, 16 h
Ph
N
This work
O
O
Iodine (20 mol%)
R
C
Ph
NH
Ph
R-CHO
+
NH2
NH2
O2, DMSO, H2O
110 oC, 12 h
OR
N
R
Ph
Figure 2. Synthesis of quinazolin-4(3H)-ones via coupling of O-
aminobenzamide with aryl alkynes/ styrenes
The homology model based design followed by synthesis and biological evaluation for
suppression of IL-1β release has resulted in identification of quinazolin-4(3H)-one as a new class
of NLRP3 inflammasome inhibitors. The computational design, new synthetic strategy and
biological evaluation of the series of quinazolin-4(3H)-ones is presented in this paper.
RESULTS AND DISCUSSION
Building Homology Model and Design of NLRP3 Inflammasome Inhibitors. The lack of
crystal structure of any molecular target always adds challenges to medicinal chemists to design
new class of modulators. Here, we performed the protein structure alignment of NACHT domain
of the human NLRP3 protein (residues 220–536, Entry Id: Q96P20, Entry Name:
NLRP3_HUMAN) with the resolved structure of NLRC4 (PDB ID: 4KXF).31 The homology
model was developed by multiple-thread alignments, as described by Zhang's research group
through a web server (I-TASSER).32 The prepared model was validated by studying the
interaction of ATP and a known NLRP3 inflammasome inhibitor, MCC950. The ATP binding
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site of the built protein contains a highly conserved residue Lys 232. The docking studies were
performed by preparing a grid around this residue. The docking of ATP at this site has shown
that its phosphate groups display several key H-bonding interactions with Lys 232, Arg 237, Thr
233 and His 522 residues. In addition, the Arg 237 residue have shown H-bonding with ATP
sugar moiety and Trp 416 residue display π-π stacking with adenine base (Figure 3B).
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The structure of β-estradiol could be mimicked via 2-aryl quinazolin-4(3H)-ones, as shown in
Figure 3C. The molecular docking of 2-aryl quinazolin-4(3H)-one scaffold at the ATP binding
site, has indicated that this scaffold occupies this cavity, and display several similar interactions
like ATP, and known NLPR3 inflammasome inhibitors, MCC950 and β-estradiol. One of the
key interaction (π-π stacking with Trp 416) of adenine moiety of ATP was found to be
maintained by this new structural motif. Interestingly, this interaction was found to be common
among known inflammasome inhibitors e.g. MCC950, acrylamide derivative [2-(2-
chlorobenzyl)-N-(4-sulfamoylphenethyl)acrylamide].11
Thr 169
(A)
(B)
Lys 238
Tyr 168
Met 239
Tyr 381
Trp 416
Arg 237
Lys 232
Thr 233
His 522
(C)
R
NH2
N
Ar
OH
N
N
O
N
H
HN
N
OH
HO
P
HO
H
H
O
O
O
O
O
O
HO
P
P
HO
O
HO
OH
-Estradiol
2-aryl quinazolin-4(3H)-one
ATP
Figure 3. The homology model of NLRP3 inflammasome and design of
its new inhibitors. A. homology model; B. binding site of the model
showing interactions of ATP; C. designed "2-aryl quinazolin-4(3H)-
one" scaffold.
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Synthesis. Our synthetic efforts were initiated by examining the reaction of O-
aminobenzamide 1a with phenylacetylene 4a using iodine as a catalyst and DMSO as a solvent
by varying catalyst loading, temperature, reaction time and oxidant (Table 1). Reaction at room
temperature produced desired 2-aryl quinazolin-4(3H)-one 2a; however only in traces (entry 1).
Under heating condition, in an open air atmosphere, the product 2a was obtained in 28% yield
(entry 2). When the molecular oxygen was employed, the yield of 2a was improved up to 49%
(entry 4). Further, on increasing the reaction temperature up to 140 °C, it has not positively
impacted on the product yield (entry 5). On the use of iodine in stoichiometric equivalents, the
2a yield was slightly improved, however along with the formation of iodo-byproduct 3a (entries
6 and 7). Next, in order to reduce the formation of side product 3a, reaction was then performed
using catalytic amounts of I2 and longer reaction time. These efforts have led to the identification
of a optimized reaction condition, wherein, catalyst loading of 20 mol% I2 using molecular
oxygen at 110 °C for 16 h, produced 72% yield of desired product 2a (entry 10). On lowering the
reaction temperature to 80 °C, there was significant decrease in the product yield (2a, 20%; not
shown in Table 1). Further investigation of other solvents such as DMF, toluene, ACN (entries
11-13), has shown that none of them are superior to DMSO, although all of them supported the
progress of this reaction. Further, on addition of 1 equivalent of water to the optimized condition
of entry 10, the desired product was formed in 70% yield, only after 12 h reaction time (entry
14). This condition was used for synthesis of quinazolin-4(3H)-one molecular library. Under this
optimized condition, the desired quinazolin-4(3H)-one 2a was obtained in equivalent yield, even
after replacing the substrate phenylacetylene 4a with styrene 5a.
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Table 1. Optimization of the Reaction Conditionsa
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Entry Iodine
(mol%)
Solvent Oxidant temp Time % yieldb
(o C) (h)
2a
traces
28
18
49
48
53
53
47
62
72
25
30
20
70
3a
10
11
12
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14
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16
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1
10
10
10
10
10
150
100
70
50
20
20
20
20
20
DMSO air
DMSO air
DMSO O2
DMSO O2
DMSO O2
DMSO O2
DMSO O2
DMSO O2
DMSO O2
DMSO O2
rt
10
10
10
10
10
10
10
10
16
16
16
16
16
12
0
2
110
rt
0
3
0
4
110
140
110
110
110
110
110
110
110
110
110
0
5
0
6
22
20
20
13
traces
0
7
8
9
10
11c
12c
13c
DMF
Toluene O2
ACN O2
DMSO O2
O2
0
0
14d
traces
a
O-aminobenzamide (1.0 mmol), phenylacetylene (1.2 mmol) and I2 in DMSO
b
c
stirred at a specified temperature; isolated yield; in case of entries 11-13, no
reaction occurred when atmospheric air was used as oxidant; d addition of 1 equiv
H2O.
Using optimized reaction conditions, a series of 2-aryl quinazolin-4(3H)-ones were
synthesized by varying aryl alkynes (Scheme 1). Phenylacetylenes substituted with various
EDGs such as alkyls, OMe participated well in this reaction producing desired products in good
yields (examples 2b-f). Phenylacetylenes substituted with EWGs such as F, Br, CF3, CN, and
NO2 also produced corresponding quinazolin-4(3H)-ones in good yields (examples 2g-k).
Further, the acetylenes with fused bicyclics and biphenyl rings (examples 2m and 2n) and
heterocycles (example 2o) also participated well in this reaction; however in the case of 9-
ethynylanthracene, the desired product was obtained only in traces (example 2p).33
Unfortunately, aliphatic alkynes were not suitable for this kind of transformations under our
optimized conditions. Furthermore, the reaction of 1a with dimethyl acetylenedicarboxylate was
also not successful.
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O
O
R
I2, O2 balloon
DMSO, H2O, 110 o
NH
N
NH2
+
Ar
C
NH2
1a
R
Ar = Ph, bi-Ph,heterocycle
Ar
2
4
O
N
O
O
O
NH
NH
NH
N
NH
N
N
2d
(72%)
2a
2b
2c
(68%)
(70%)
O
(72%)
O
9
O
10
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O
NH
NH
NH
NH
N
N
N
N
2g
(73%)
O
2e
2i
(75%)
O
2f
F
(80%)
O
2h
2l
(62%)
O
O
Br
NH
NH
N
NH
NH
N
N
N
2j
(50%)
O
(68%)
2k
(70%)
O
(55 %)
O
CN
NO2
CF3
N
O
NH
N
NH
NH
NH
N
N
N
N
2o
2p
(traces)
2n
(53%)
(72%)
2m
(70%)
Ph
Scheme 1. Scope of the reaction for synthesis of various quinazolin-4(3H)-ones
2a-p. Reagents and conditions: O-aminobenzamide (1a, 1.0 equiv.),
phenylacetylene (4, 1.2 equiv.), 20 mol% I2, DMSO, H2O (1 equiv.) 110 °C, O2
atmosphere, 12 h, 50-75%.
Similarly, the substrate scope was investigated with various substituted styrenes under
optimized reaction conditions. The corresponding quinazolin-4(3H)-ones were obtained in good
yield by varying the substitution on styrene precursor (Scheme 2). Styrenes substituted with
EDGs (2f) as well as EWGs (2g, 2h, 2k, 2q, 2r, 2s, 2t) were well tolerated in this reaction.
Biphenyl styrene (e.g. 2n) as well as heterocyclic styrenes (e.g. 2o, 2u) also produced
corresponding quinazolin-4(3H)-ones in good yields.
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O
O
R
NH
I2,O2 Balloon
NH2
Ar
R
DMSO, H2O, 110 o
C
+
N
NH2
Ar
Ar = Ph, bi-Ph, heterocycle
R = EDG, EWG
1a
5
2
O
N
O
O
O
NH
NH
NH
N
NH
N
N
2g
2a
(72%)
(74%)
O
2f
2h
(70%)
(78%)
O
OMe
Br
Cl
9
F
10
11
12
13
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O
O
NH
NH
NH
NH
N
N
N
N
N
2k
2n
(73%)
(74%)
O
NO2
2o (50%)
2q
(74%)
O
Ph
O
O
NH
NH
NH Cl
NH
Br
Cl
N
N
N
N
2r
(60%)
2s
2t
(63%)
(50%)
2u
(53%)
N
Scheme 2. Scope of the reaction for synthesis of various quinazolin-4(3H)-ones
from O-aminobenzamide and styrenes. Reagents and conditions: O-
aminobenzamide (1a, 1.0 equiv.), styrenes (5, 1.2 equiv.), 20 mol% I2, DMSO,
H2O (1 equiv.) 110 °C, O2 atmosphere, 12 h, 50-78%.
To further explore the scope of this reaction, various substituted 2-aminobenzamides were
employed under optimal conditions. A series of halo groups on benzamide substrate such as
chloro and iodo were well tolerated (examples 2v-2y). In the case of 2-amino-N-ethylbenzamide,
the desired quinazolin-4(3H)-one 2z was not obtained. Next, we employed this protocol for
preparation of pyrazolo[4,3-d]pyrimidin-7(6H)-one, which is another medicinally important
highly privileged scaffold.34 Using this protocol, pyrazolo[4,3-d]pyrimidin-7(6H)-one 6a was
prepared in 50% yield (Scheme 3). Interestingly, the internal alkyne as well as internal alkene
also participated in this reaction, producing corresponding quinazolin-4(3H)-ones in 27-61%
yields (Scheme 4).
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O
O
R
Ar
4
R1
I2, O2 balloon
DMSO, H2O, 110 o
Ar = Ph, substituted-Ph
Ar1 = Pyrazole, substituted-Ph
NH
Ar1
N
Ar1
H
+
C
NH2
N
Ar
R
1
2, 6
5
R = EDG, EWG; R1 = H, alkyl
6
7
8
9
O
O
O
N
I
I
Cl
NH
NH
NH
N
N
2x
6a
2w
OMe
(65%)
NO2
(76%)
2v
(72%)
O
OMe
10
11
12
13
14
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60
O
H
O
N
N
N
NH
I
N
NH
N
N
2y
2z
(0%)
(72%)
(50%)
Scheme 3. Scope of the reaction for synthesis quinazolin-4(3H)-ones
and pyrazolo[4,3-d]pyrimidin-7(6H)-ones 6. Reagents and
2
conditions: O-aminobenzamide (1, 1.0 equiv.), phenylacetylenes (4,
1.2 equiv.), 20 mol% I2, DMSO, H2O, 110 °C, O2 atmosphere, 12 h,
50-76%.
O
O
NH
NH
+
Ph
N
N
2a
(30%)
2f
(27%)
O
Ph
OMe
OMe
+
8b
O
Ph
Ph
NH
Ph
(57%)
O
+
7a
Ph
NH2
N
2a
NH2
1a
+
Ph
8a
NH
N
Ph
2a
(61%)
Scheme 4. Scope of the reaction for synthesis quinazolin-4(3H)-ones 2
using internal alkynes/ alkenes. Reagents and conditions: O-
aminobenzamide (1, 1.0 equiv.), alkyne 7a/ stilbene 8a-b (1.2 equiv.), 20
mol% I2, DMSO, H2O, 110 °C, O2 atmosphere, 12 h, 27-61%.
To gain mechanistic insights, a few control experiments were performed (Scheme 5A-E). In
the presence of molecular oxygen, the reaction of α-iodophenylketone 9 with O-aminobenzamide
1a produced 2-benzoyl quinazolin-4(3H)-one 10a in good yield, both with I2 (20 mol%) and
without I2 (Scheme 5A). These results indicated that α-iodophenylketone is very sensitive to
DMSO-I2 system which immediately forms phenylglyoxal 11 and leads to the formation of 2-
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benzoyl product 10a. Second control experiment was the condensation of phenylglyoxal 11 (1
4
5
o
mmol) and O-aminobenzamide 1a (1 mmol) under molecular oxygen at 110 C with catalytic
6
7
8
9
amount of I2. In this case also, we obtained 2-benzoyl product 10a as a major product. However,
in case of acetophenone 12 (1 mmol) using same reaction conditions, the desired quinazolin-
4(3H)-one 2a was obtained as a major product. In fourth experiment (Scheme 5D), reaction of
O-aminobenzamide 1a (1 mmol) with formaldehyde (3 mmol) in presence of molecular oxygen
with 20 mol% I2, produced quinazolin-4(3H)-one 13 (m/z 146) which we also observed as a
byproduct during synthesis of all quinazolin-4(3H)-ones (based on MS analysis of reaction
mixture; see Figure S1 of Supporting Information). Similarly, when O-aminobenzamide 1a (1
mmol) was reacted with acetaldehyde (3 mmol) under optimized reaction conditions, the
quinazolin-4(3H)-one 2a was obtained (Scheme 5E).
10
11
12
13
14
15
16
17
18
19
20
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22
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25
26
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49
50
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52
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57
58
59
60
O
2a
(80%)
,
NH
H
O
S
+
PhCHO
14
I2 (20 mol%),
DMSO, O2
110 oC, 4 h
M
D
N
,
)
h
%
l
o
4
13
(55%)
,
m
o
C
O
0
H
2
0
(
C
1
H
1
I 2
,
+
O 2
(E)
O
O
(D)
(A)
I
+
O
NH
Ph
9
Ph
NH2
N
I2, DMSO, O2
10a
110 oC, 5 h
O
NH2
1a
with I2 (20 mol%): 84%
without I2: 82%
I
2
(
(B)
1
D
0
1
M
0
S
o
C
m
(C)
1
O
+
O
o
l
,
%
O
H
)
,
+
,
2
3
Ph
h
O
12
O
I2 (10 mol%),
11
Ph
DMSO, O2
110 oC, 5 h
10a
(75%)
2a
(50%)
Scheme 5. Control experiments.
Accordingly, we proposed a plausible reaction mechanism for formation of 2a from 1a and 4a
as depicted in Figure 4. We propose two possible pathways. It is well known that under oxidative
conditions, phenylacetylenes gets oxidized to α-iodophenylketone 935 and acetophenones 12.36 In
first path (Figure 4), phenylacetylene 4a in the presence of I2/DMSO gets converted to α-
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iodophenylketone 9 which predominantly led to the formation of 10a (Scheme 5B). In another
pathway, phenylacetylene 4a in the presence of I2/DMSO gets converted to acetophenone 12 and
facilitating the formation of intermediate III via formation of Schiff’s base II. Intermediate III
undergoes iodination to produce IV. The intermediate III gets converted to V via Kornblum type
oxidation in the presence of DMSO.37 Finally, the intermediate V undergoes HCHO elimination
to produce iminium intermediate VI, which on deprotonation produces 2a. Interestingly, a mass
of m/z 146 for byproduct 13 [quinazolin-4(3H)-one] was observed when reaction mixture was
monitored by ESI-MS. This clearly indicated that HCHO must be forming in-situ which leads to
formation of 13 as a minor byproduct. Similarly, when internal alkyne 7a (Scheme 4) was used,
still the product 2a was formed, via similar reaction mechanism. In this case, it is presumed that
benzaldehyde 14 gets released which further on coupling with 1a, produces same product 2a
(control experiment E, Scheme 5). This was also evidenced in case of stilbenes 8a and 8b
(Scheme 4). The formation of 2-arylquinazolin-4(3H)-ones 2a via condensation of O-
aminobenzamide 1a and styrene 5a and stilbenes 8a-b also occurs via iodine mediated
conversion of styrene/ stilbene to corresponding acetophenone.38
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O
S
M
D
/
I2
O
Ar
4a
I2
Ph
Ar
O
N
O
12
C
.
t
I2/DMSO
NH
Ph
p
H3C
x
O
E
I
O
NH2
NH2
2a
Ar
O
O2
O
I
9
1a
B
.
NH2
t
p
Expt. A
x
E
N
O
NH
Ph
II
Ph
1a
Expt. B
+
10a
H
Ar
N
H
S
11
O
+
VI
O
+
HCHO
13
S
O
1a
Expt. D
NH
S
I2
N
H
Ph
O
III
HI
H
O
NH
S
O
I
S
NH
N
H
O
Ph
V
N
Ph
H
IV
I
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Figure 4. Plausible mechanism (control experiments from Scheme 5 are
indicated here, in green colour) for formation of quinazolin-4(3H)-ones
2a via condensation of O-aminobenzamide 1a with phenylacetylene 4a.
3
4
5
6
7
8
9
Inhibition of NLRP3 Inflammasome. For NLRP3 inhibition assay, mouse macrophages
(J774A.1 cells) after priming with LPS were treated with 10 µM of test compound and were
challenged with ATP (3 mM) for 30 min as a second signal. Activation of NLRP3 is culminated
with the cleavage of pro-IL-1β into its activated form IL-1β. Compounds synthesized herein, and
few quinazolin-4(3H)-ones prepared earlier 2aa-2ag30b (in all, total 30 compounds; chemical
structures of 2aa-2ag are shown in Figure S2 of Supporting Information) were tested in this
assay. The supernatant of vehicle/ test compound treated cells was collected and analyzed for IL-
1β levels using ELISA. The results are shown in Table 2. Four compounds viz. 2f, 2i, 2k and 2y
displayed significant suppression of IL-1β release in culture supernatant. Compound 2k was the
most active with 40% suppression of IL-1β release into the media. Further, in order to determine
the IC50 value of compound 2k, J774A.1 cells were treated with different concentrations (0.1- 10
µM) of quinazolin-4(3H)-one 2k. A mild inhibitory effect was observed even at 1.25 µM;
however, it showed maximum inhibition at a concentration of 10 µM. The IC50 of 2k was found
to be 5 µM.
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Table 2. In-vitro inhibition of IL-1β release in J774A.1 cells by quinazolin-
4(3H)-ones.ab
O
O
% suppression
Glide Score
H
N
NH
NH
R2
of IL-1β release (ATP-binding
N
R1
N
N
at 10 µM
site of NLRP3
6a
2a-2ag
inflammasome)
[mean SD]
Entry
R1 R2
LPS + ATP + 2a
LPS + ATP + 2f
LPS + ATP + 2i
LPS + ATP + 2j
LPS + ATP + 2k
LPS + ATP + 2r
H
H
H
H
H
H
Ph
12.7 ± 0.15
6.60
8.50
8.42
7.66
6.64
6.63
Ph (4-OMe) 28.8 ± 0.29
Ph (4-CF3)
Ph (4-CN)
Ph (4-NO2)
Ph (3-Br)
21.3 ± 0.10
13.9 ± 0.01
39.8 ± 0.11
16.1 ± 0.24
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LPS + ATP + 2t
H
Ph (3-Cl)
12.9 ± 0.12
6.90
7.17
8.05
-
LPS + ATP + 2y 6-I Ph
LPS + + ATP + MCC950c
LPS + DMSO + ATPd
29.3 ± 0.12
105.8 ± 0.01
0
aAll compounds were dissolved in DMSO. J774A.1 cells after priming with
LPS, were treated with compounds (10 µM) or vehicle (DMSO). Cells were
then treated with ATP and the secretion of IL-1β in culture supernatants was
measured by ELISA. bcompounds 2b-2e, 2g, 2h, 2l-2q, 2s, 2u-2x, 2aa-2ag, and
6a showed  10% inhibition of IL-1β release at 10 µM, and thus these
compounds are not listed in this table. cMCC950 was used as a positive control
in this assay. dATP treated cells without any test compound acts as a "control
group".
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Upon activation of NLRP3 inflammasome, the caspase-1 gets released which converts the
pro IL-1β into its mature form IL-1β which is highly potent pro-inflammatory cytokine.
Therefore, next we sought to investigate the effect of quinazolin-4(3H)-one 2k on the expression
of mature IL-1β in ATP-stimulated J774A.1 cells via western-blot analysis. As shown in Figure
5, the compound 2k displayed dose-dependent decrease in the levels of cleaved IL-1β in the
culture supernatant (p < 0.005). This study has further validated the results (Table 2) obtained in
NLRP3 inflammasome activation assay.
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A.
MCC950 (100 nM)
-
-
-
-
-
-
-
+
-
-
+
+
+
-
+
+
-
10
+
+
-
-
-
-
2k, µM
ATP(3mM)
LPS (1 µg/ml)
5
+
+
2.5 1.25 0.625
+
+
+
+
+
+
6
7
8
9
Supernatant
Lysate
Cleaved IL-1β
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pro IL-1β
β -actin
B.
150
125
100
75
50
25
0
***
***
***
***
***
***
Figure 5. Western-blot analysis showing the effect of quinazolin-4(3H)-one
2k on the expression of pro IL-1β and cleaved IL-1β in J774A.1 cells
(statistical analysis was performed by comparing the test compound treated
groups with "LPS+ATP" group. p value *** < 0.005).
To predict the putative binding mode of the quinazolin-4(3H)-one 2k, the docking was
performed with the developed homology model of NLRP3 inflammasome. The 3D-interaction
map of 2k is depicted in Figure 6B. The quinazolin-4(3H)-one ring displayed π- π interactions
with Trp 416 residue, whereas the 3-phenyl ring showed π-π interactions with Tyr 381 residue of
the active site. The nitro-functionality displayed additional cation-π (with Leu 171) and H-
bonding interactions (with Thr 168, Thr 169). The known NLRP3 inflammasome inhibitor
MCC950 strongly occupied the ATP binding cavity (glide score = 8.05) and displayed three π-
π interactions and a H-bonding (via terminal OH group) with Trp 416 residue (Figure 6A). The
other two active quinazolin-4(3H)-ones 2f and 2i were also found to occupy the ATP binding site
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of NLRP3 inflammasome, and displayed interactions with Trp 416 and Tyr 381 residues (Figure
S3 of Supporting Information).
6
7
8
(B)
(A)
Lys 238
Leu 171
Thr 169
Tyr 168
Lys 238
Leu 171
Thr 169
9
10
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Tyr 168
Tyr 381
Met 239
Tyr 381
Met 239
Arg 237
Trp 416
Arg 237
Trp 416
Lys 232
Lys 232
Thr 233
Thr 233
Figure 6. Interactions of MCC950 (A) and quinazolin-4(3H)-one 2k (B) with ATP binding site of NLRP3 inflammasome. The
light blue dotted lines indicate - interactions, dark blue as H-bonding and green-dotted lines as cation-π interactions.
CONCLUSION
Herein we have identified quinazolin-4(3H)-one as a new class of NLRP3 inflammasome
inhibitors. The new method for synthesis of 2-aryl quinazolin-4(3H)-ones has also been
developed via condensation of O-aminobenzamide with aryl alkynes/ alkenes using molecular
oxygen as an oxidant. The quinazolin-4(3H)-one 2k inhibited NLRP3 inflammasome with IC50
value of 5 µM. The molecular modelling study has indicated that it occupies the ATP binding
site of the NLRP3 inflammasome. The promising NLRP3 inflammasome inhibitory activity of
this new non-sulphonyl chemotype opens up an opportunity to investigate it further for its lead
optimization and preclinical in-vivo validation.
EXPERIMENTAL SECTION
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General. 1H, C and DEPT NMR spectra were recorded on FT-NMR 500 and 400 MHz
instruments. Chemical data for protons are reported in parts per million (ppm) downfield from
tetramethylsilane and are referenced to the residual proton in the NMR solvent (CDCl3, 7.26
ppm; DMSO-d6, 2.50 ppm). Carbon nuclear magnetic resonance spectra (13C NMR) were
recorded at 125 MHz or 100 MHz: chemical data for carbons are reported in parts per million
(ppm, δ scale) downfield from referenced to the carbon resonance of the solvent (CDCl3, 77
ppm; DMSO-d6, 39.52 ppm). ESI-MS and HRMS spectra were recorded on Agilent 1100 LC-Q-
TOF and HRMS-6540-UHD machines. IR spectra were recorded on Perkin-Elmer IR
spectrophotometer. Melting points were recorded on digital melting point apparatus. The alkynes
4, 7a and styrenes 5, stilbenes 8a, 8b used herein, were commercially procured from Sigma-
Aldrich or TCI Chemicals.
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For NLRP3 inflammasome assay, J774A.1 cell line was obtained from ECACC through
Sigma-Aldrich. J774A.1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100U/ml penicillin and
100 mg/ml streptomycin (sigma). The reference standard MCC950 was purchased from Sigma-
Aldrich (MO, USA).
Building the Homology Model of NLRP3 Inflammasome. The protein structure alignment
of NACHT domain of the human NLRP3 protein (residues 220–536, Entry Id: Q96P20, Entry
Name: NLRP3_HUMAN) was done with the resolved structure of NLRC4 (PDB ID: 4KXF), by
multiple-thread alignments, as described by Zhang's research group through a web server (I-
TASSER).32 The obtained model was then subjected to the default protein preparation steps of
Glide module of Schrodinger molecular modelling software. For docking, the highly conserved
residue Lys 232 was considered and grid was prepared using 20 Ǻ distance from this residue.
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The developed model was validated by docking ATP and known inhibitors (MCC95012a, 2-(2-
4
5
chlorobenzyl)-N-(4-sulfamoylphenethyl)acrylamide11) of NLRP3 inflammasome.
6
7
8
9
Optimized Procedure for Preparation of 2-Aryl Quinazolin-4(3H)-ones (2) and
Pyrazolo[4,3-D]pyrimidin-7(6H)-ones (6). The reaction mixture of alkyne or alkene (4 or 5, 1.2
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o
equiv) and iodine (0.2 equiv) in DMSO and H2O (1 equiv) under molecular oxygen at 110 C
was stirred for 20 min. Then corresponding 2-aminobenzamides (1, 1 equiv.) or 4-amino-1-
methyl-3-propyl-1H-pyrazole-5-carboxamide (1 equiv) was added and the reaction mixture was
stirred further for 12 h. After completion of the reaction, water (20 mL) was added to the
reaction mixture. A saturated Na2S2O3 solution was then added until the brown color
disappeared. Product was extracted with EtOAc (3  10 mL). The organic layer was collected,
dried on anhydrous sodium sulphate and solvent was evaporated on rotary evaporator to get the
crude product. The crude product was purified by silica gel (#100-200) column chromatography
using 2 to 20% EtOAc: hexane to get pure quinazolin-4(3H)-ones. Most of the compounds were
purified without column chromatography. For this, the MeOH was added to the crude product
and the resulting suspension was filtered. Obtained residue was washed with MeOH (2-3 times)
and dried. To get good yield of the product, on addition of methanol, the product was allowed to
settle down for 2-3 days, which results in formation of a crystalline pure product.
2-Phenylquinazolin-4(3H)-one (2a).28a,39 White solid. yield: 70% (114 mg). m.p. 232-235 °C.
1H NMR (400 MHz, DMSO-d6, ppm): δ 11.57 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.26 - 8.24 (m,
2H), 7.86-7.80 (m, 2H), 7.60-7.50 (m, 3H), 7.52 (t, J = 7.2 Hz, 1H). C{1H} NMR (101 MHz,
13
CDCl3, ppm): δ 163.9, 151.9, 149.5, 134.9, 132.8, 131.6, 129.0, 127.9, 127.4, 126.8, 126.3,
120.8. IR (CHCl3): νmax 3436, 2926, 1666, 1481, 1297, 1144 cm-1. ESI-MS: m/z 223.08 [M+H]+.
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C14H11N2O 223.0871; Found 223.0869.
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2-(p-Tolyl)quinazolin-4(3H)-one (2b).28a,39 White solid. yield: 72% (124 mg). m.p. 239-242
°C. 1H NMR (400 MHz, DMSO-d6, ppm): δ 8.16 (d, J = 8.0 Hz, 1H), 8.11 ( d, J = 8.0 Hz, 2H),
7.84 (t, J = 8.2 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 8.2 Hz, 1H), 7.38 (d, J = 8.0 Hz,
2H), 2.40 (s, 3H). 13C{1H} NMR (101 MHz, DMSO-d6, ppm): δ 162.7, 152.7, 149.5, 149.3,
144.9, 135.1, 130.3, 129.6, 128.1, 127.9, 126.9, 126.3, 121.3, 21.5. IR (CHCl3): νmax 3436, 2918,
1667, 1600, 1486, 1241 cm-1. ESI-MS: m/z 237.10 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C15H13N2O 237.1022; Found 237.1029.
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2-(4-Ethylphenyl)quinazolin-4(3H)-one (2c).22a White solid. yield: 68% (125 mg). m.p. 205-
207 °C. 1H NMR (400 MHz, CDCl3, ppm): δ 8.27 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H),
7.83-7.81 (m, 1H), 7.52-7.49 (m, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.33 (m, 1H), 2.76 (q, J = 8.0,
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12.0 Hz, 2H), 1.29 (t, J = 8.0 Hz, 3H). C{1H} NMR (101 MHz, DMSO-d6, ppm): δ 162.8,
152.7, 149.3, 148.1, 135.1, 130.6, 128.5, 128.3, 127.9, 126.9, 126.3, 121.4, 28.5, 15.8. IR
(CHCl3): νmax 3436, 2920, 1669, 1612, 1482, 1240 cm-1. ESI-MS: m/z 251.11 [M+H]+. HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C16H15N2O 251.1179; Found 251.1201.
2-(4-Propylphenyl)quinazolin-4(3H)-one (2d). White solid. yield: 72% (139 mg). m.p. 200-
202 °C. 1H NMR (400 MHz, DMSO-d6, ppm): δ 12.50 (s, 1H), 8.16 - 8.11 (m, 3H), 7.84 (t, J =
8.2 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 2.65 (t, J
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= 7.5 Hz, 2H), 1.69 -1.62 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). C{1H} NMR (126 MHz, CDCl3,
ppm): δ 163.7, 151.8, 149.6, 147.0, 134.9, 130.1, 129.3, 127.9, 127.2, 126.5, 126.3, 120.8, 38.2,
24.4, 14.0. IR (CHCl3): νmax 3436, 2918, 1667, 1600, 1486, 1241 cm-1. ESI-MS: m/z 265.12
[M+H]+. HPLC purity: 92% (tR = 14.4 min).
2-(4-(Tert-butyl)phenyl)quinazolin-4(3H)-one (2e).28a White solid. yield: 75% (153 mg). m.p.
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233-235 °C. H NMR (400 MHz, CDCl3, ppm): δ 10.85 (s, 1H), 8.34 (d, J = 7.7 Hz, 1H), 8.12
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(d, J = 8.0 Hz, 2H), 7.85-7.78 (m, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.52 (t, J = 8.0 Hz, 1H), 1.39 (s,
9H). 13C{1H} NMR (126 MHz, CDCl3, ppm): δ 163.1, 154.8, 151.1, 134.4, 129.3, 127.4, 126.5,
126.1, 125.9, 125.6, 120.3, 34.5, 30.7. IR (CHCl3): νmax 3436, 2918, 1667, 1600, 1486, 1241 cm-
1. ESI-MS: m/z 279.14 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C18H19N2O 279.1492;
Found 279.1491.
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2-(4-Methoxyphenyl)quinazolin-4(3H)-one (2f).28a,39 White solid. yield: 80% (148 mg). m.p.
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247-249 °C. H NMR (400 MHz, CDCl3, ppm): δ 8.27 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 8.0 Hz,
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2H), 7.79 (d, J = 4.0 Hz, 2H), 7.49 (m, 1H), 7.08 (d, J = 12.0 Hz, 2H), 3.91 (s, 3H). C{1H}
NMR (126 MHz, DMSO-d6 + CDCl3, ppm): δ 162.3, 161.8, 151.8, 148.9, 134.5, 129.4, 127.2,
126.1, 125.8, 124.7, 120.6, 114.0, 55.4. IR (CHCl3): νmax 3436, 2920, 1678, 1600, 1484, 1031
cm-1. ESI-MS: m/z 253.09 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C15H13N2O2
253.0972; Found 253.0967.
2-(4-Fluorophenyl)quinazolin-4(3H)-one (2g).28a,29 White solid. yield: 73% (128 mg). m.p.
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240-242 °C. H NMR (400 MHz, DMSO-d6, ppm): δ 12.58 (s, 1H), 8.26 (dd, J = 4.0, 8.0 Hz,
2H), 8.16 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0
Hz, 1H), 7.40 (t, J = 8.0 Hz, 2H). 13C{1H} NMR (126 MHz, DMSO-d6, ppm): δ 163.5 (d, 1JCF =
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103 Hz), 151.8, 149.1, 135.1, 130.9 (d, JCF = 8.8 Hz), 129.7, 127.9, 127.1, 126.3, 121.4, 116.2,
116.0. IR (CHCl3): νmax 3424, 2922, 2852, 2853, 1672, 1466, 1288, 1095 cm-1. ESI-MS: m/z
241.07 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C14H10FN2O 241.0772; Found
241.0782.
2-(4-Bromophenyl)quinazolin-4(3H)-one (2h).22d,40 White solid. yield: 62% (136 mg). m.p.
292-295 °C. 1H NMR (400 MHz, DMSO-d6, ppm): δ 12.66 (s, 1H), 8.21-8.16 (m, 3H), 7.88 (d, J
= 8.0 Hz, 1H), 7.80-7.78 (m, 3H), 7.58 (t, J = 7.3 Hz, 1H). 13C{1H} NMR (126 MHz, DMSO-d6,
ppm): δ 162.6, 151.9, 149.1, 135.2, 132.4, 132.1, 130.3, 128.0, 125.7, 121.5. IR (CHCl3): νmax
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3428, 2919, 1659, 1633, 1335, 1179 cm-1. ESI-MS: m/z 300.98 [M+H]+. HRMS (ESI-TOF) m/z:
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[M+H]+ Calcd for C14H1081BrN2O 302.9951; Found 302.9945.
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2-(4-(Trifluoromethyl)phenyl)quinazolin-4(3H)-one (2i).28a White solid. yield: 55% (117 mg).
m.p. 280-282 °C. 1H NMR (400 MHz, DMSO-d6, ppm) δ 12.77 (s, 1H), 8.37 (d, J = 8.1 Hz, 2H),
8.18 (d, J = 7.0 Hz, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.87 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.0 Hz,
1H), 7.57 (t, J = 7.2 Hz, 1H). 13C{1H} NMR (101 MHz, DMSO-d6, ppm) δ 162.6, 151.7, 148.9,
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137.1, 135.2, 131.8 (d, JCF = 32 Hz), 129.2, 128.1, 127.6, 126.4, 126.0 (d, JCF = 3 Hz), 125.8
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(d, JCF = 273 Hz), 121.7. IR (CHCl3): νmax 3428, 2919, 1659, 1633, 1335, 1179 cm-1. ESI-MS:
m/z 291.07 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C15H10F3N2O 291.0740; Found
291.0726.
2-(4-(Cyano)phenyl)quinazolin-4(3H)-one (2j).24,40 White solid. yield: 68% (123 mg). m.p.
280-282 °C. 1H NMR (400 MHz, DMSO-d6, ppm): δ 12.75 (s, 1H), 8.34 (t, J = 8.0 Hz, 2H), 8.20
(d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.95-7.78 (m, 3H), 7.58 (t, J = 8.0 Hz, 1H). IR
(CHCl3): νmax 3428, 2919, 1659, 1633, 1335, 1179 cm-1. ESI-MS: m/z 248.08 [M+H]+. HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C15H10N3O 248.0818; Found 248.0817.
2-(4-Nitrophenyl)quinazolin-4(3H)-one (2k).41 Yellow solid. yield: 70% (137 mg). m.p. >300
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°C. H NMR (400 MHz, DMSO-d6, ppm): δ 12.84 (s, 1H), 8.40 (m, 4H), 8.19 (d, J = 4.0 Hz,
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1H), 7.88 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H). C{1H} NMR
(101 MHz, DMSO-d6, ppm): δ 162.6, 151.1, 149.5, 148.8, 139.1, 136.3, 135.3, 129.8, 129.2,
127.9, 126.4, 124.1, 121.7. IR (CHCl3): νmax 3410, 2921, 1681, 1517, 1470, 1351, 1020 cm-1.
ESI-MS: m/z 266.05 [M-H]. HRMS (ESI-TOF) m/z: [M-H]- Calcd for C14H8N3O3 266.0571;
Found 266.0580.
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2-(4-(Dimethylamino)phenyl)quinazolin-4(3H)-one (2l).42 White solid. yield: 50% (97 mg).
m.p. > 300° C. 1H NMR (400 MHz, CDCl3, ppm): δ 9.98 (s, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.01
(d, J = 8.0 Hz, 2H), 7.76 (d, J = 4.0 Hz, 2H), 7.44-7.40 (m, 1H), 6.80 (d, J = 8.0 Hz, 2H), 3.08 (s,
6H). 13C{1H} NMR (126 MHz, CDCl3, ppm): δ 163.0, 152.6, 151.5, 150.0, 134.6, 128.1, 127.5,
126.4, 125.7, 120.4, 119.2, 111.7, 40.1. IR (CHCl3): νmax 3301, 2922, 1595, 1580, 1441, 1106,
1019 cm-1. ESI-MS: m/z 266.12 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C16H16N3O
266.1288; Found 266.1275.
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2-(Naphthalen-2-yl)quinazolin-4(3H)-one (2m).28a White solid. yield: 70% (140 mg). m.p. 247
- 249 °C. 1H NMR (500 MHz, DMSO-d6, ppm): δ 12.35 (s, 1H), 8.48 (d, J = 8.3 Hz, 1H), 8.29-
8.24 (q, J = 8.0, 12.0 Hz, 2H), 8.16-8.11 (q, J = 8.0, 12.0 Hz, 2H), 7.86 (t, J = 8.0 Hz, 1H), 7.69-
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7.65 (m, 5H). C{1H} NMR (126 MHz, DMSO-d6, ppm): δ 161.6, 150.5, 147.7, 135.2, 134.3,
133.8, 133.2, 131.7, 130.9, 129.3, 129.1, 128.9, 128.8, 127.2, 126.5, 125.3, 125.1, 123.4. IR
(CHCl3): νmax 3356, 2920, 1666, 1574, 1443, 1292, 1022 cm-1. ESI-MS: m/z 273.10 [M+H]+.
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C18H13N2O 273.1022; Found 273.1040.
2-([1,1'-Biphenyl]-4-yl)quinazolin-4(3H)-one (2n).25 White solid. yield: 72% (157 mg).
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m.p. 266-269 °C. H NMR (400 MHz, DMSO-d6, ppm): δ 12.62 (s, 1H), 8.32 (d, J = 8.0 Hz,
2H), 8.19 (d, J = 8.0 Hz, 1H), 7.89-7.84 (m, 3H), 7.80-7.77 (m, 3H), 7.56-7.51 (m, 3H), 7.45 (m,
1H). 13C{1H} NMR (126 MHz, DMSO+10% CDCl3, ppm): δ 162.2, 151.9, 148.7, 142.8, 138.9,
134.6, 131.5, 129.0, 128.3, 127.4, 126.8, 126.7, 126.5, 125.8, 120.9. IR (CHCl3): νmax 3585,
2921, 1673, 1467, 1169 cm-1. ESI-MS: m/z 299.11 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C20H15N2O 299.1179; Found 299.1174.
2-(Pyridin-3-yl)quinazolin-4(3H)-one (2o).39 White solid. yield: 65% (90 mg). m.p. 270-
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273 °C. H NMR (400 MHz, DMSO-d6, ppm): δ 12.75 (s, 1H), 9.30 (s, 1H), 8.76 (d, J = 4.7 Hz,
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1H), 8.51 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.1
Hz, 1H), 7.62-7.51 (m, 2H). 13C{1H} NMR (101 MHz, DMSO-d6, ppm): δ 162.6, 152.3, 151.2,
149.0, 135.9, 135.2, 129.2, 128.1, 127.4, 126.4, 124.0, 121.6. IR (CHCl3): νmax 3585, 2921, 1673,
1467, 1169 cm-1. ESI-MS: m/z 224.07 [M+H]+. HPLC purity: 93% (tR = 5.6 min).
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2-(4-Chlorophenyl)quinazolin-4(3H)-one (2q).29,39 White solid. yield: 74% (140 mg).
m.p. 295-297 oC. 1H NMR (400 MHz, DMSO-d6, ppm) δ 12.59 (s, 1H), 8.18-8.11 (m, 3H), 7.83-
7.79 (m, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.59-7.43 (t, J = 8.0 Hz, 1H).
13C{1H} NMR (126 MHz, DMSO-d6, ppm) δ 162.7, 151.9, 149.0, 136.8, 135.2, 132.0, 130.1,
129.2, 127.9, 127.3, 126.3, 121.4. IR (CHCl3): Vmax 3584, 3393, 2922, 2851, 2346, 1705, 1674,
1346, 1017 cm-1. ESI-MS: m/z 257.0953 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C14H10ClN2O 257.0476; Found 257.0477.
2-(3-Bromophenyl)quinazolin-4(3H)-one (2r).43 White solid. yield: 60% (132 mg). m.p.
295-297 oC. 1H NMR (400 MHz, DMSO-d6, ppm) δ 12.63 (s, 1H), 8.38 (brs, 1H), 8.17 (t, J = 12
Hz, 2H), 7.86 (t, J = 4.0 Hz, 1H), 7.78 (t, J = 12 Hz, 2H), 7.57-7.50 (dd, J = 8.0, 20 Hz, 2H).
13C{1H} NMR (101 MHz, DMSO-d6, ppm) δ 162.6, 151.4, 148.9, 135.4, 135.2, 134.5, 131.2,
130.9, 128.1, 127.4, 127.3, 126.4, 122.4, 121.6. IR (CHCl3): max 3396, 2921, 2851, 2353, 1680,
1606 cm-1. ESI-MS: m/z 300.9 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C14H10BrN2O
300.9971; Found 300.9964.
2-(2-Chlorophenyl)quinazolin-4(3H)-one (2s).44 White solid. yield; 50% (95 mg). m.p.
196-198 oC. 1H NMR (400 MHz, DMSO-d6, ppm) δ 12.36 (s, 1H), 8.17 (d, J = 12 Hz, 2H), 8.09
(m, 1H), 7.78-7.76 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.45-7.43 (m, 1H), 7.07 (d, J = 8.0 Hz,
2H). 13C{1H} NMR (101 MHz, DMSO-d6, ppm) δ 161.9, 152.7, 149.1, 135.1, 134.3, 132.1,
132.0, 131.4, 130.1, 128.0, 127.7, 127.6, 126.3, 121.7. IR (CHCl3): max 3584, 3405, 2923, 2852,
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1686, 1117 cm-1. ESI-MS: m/z 257.0973 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C14H10ClN2O 257.0476; Found 257.0474; HPLC purity: 93% (tR = 10.8 min).
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2-(3-Chlorophenyl)quinazolin-4(3H)-one (2t).40 White solid. yield; 63% (120 mg). m.p.
252-254 oC. 1H NMR (400 MHz, DMSO-d6, ppm) δ 12.58 (s, 1H), δ 8.20 (dd, J = 5.3, 11.2 Hz,
1H), 8.11 (m, 1H), 7.86-7.79 (m, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.65-7.61 (m, 1H), 7.58-7.48 (m,
2H). 13C{1H} NMR (126 MHz, DMSO-d6, ppm) δ 162.6, 151.4, 149.1, 135.2, 133.9, 131.6,
131.0, 128.0, 127.4, 126.9, 126.4, 121.6. IR (CHCl3): νmax 3584, 3405, 2923, 2852, 1686, 1117
cm-1. ESI-MS: m/z 257.0973. HPLC purity: 98% (tR = 12.7 min).
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2-(Pyridin-4-yl)quinazolin-4(3H)-one (2u).40 white solid. yield: 53% (87 mg). m.p. 281-
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284 °C. H NMR (400 MHz, DMSO-d6, ppm) δ 8.80 (d, J = 5.8 Hz, 2H), 8.19 (d, J = 7.6 Hz,
1H), 8.13 (d, J = 6.0 Hz, 2H), 7.87 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.5
Hz, 1H). 13C{1H} NMR (101 MHz, DMSO-d6, ppm): 162.8, 151.4, 150.7, 148.8, 140.7, 135.2,
128.2, 127.8, 126.4, 122.1, 122.0. IR (CHCl3): νmax 3417, 2919, 1650, 1506, 1485, 1224, 1149
cm-1. ESI-MS: m/z 224.1 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C13H10IN3O
224.0818; Found 224.0796. HPLC purity: 99.8% (tR = 5.1 min).
6-Chloro-2-(4-methoxyphenyl)quinazolin-4(3H)-one (2v).39 White solid. yield: 72% (121 mg).
m.p. 286-288 °C. 1H NMR (400 MHz, DMSO-d6, ppm): δ 12.83 (s, 1H), 8.20-8.14 (m, 3H),
7.92 (dd, J = 4.0, 12.0 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.14 (d, J = 8.8 Hz, 2H), 3.90 (s, 3H).
IR (CHCl3): νmax 3418, 2924, 1682, 1651, 1460, 1263, 1127 cm-1. ESI-MS: m/z 287.05 [M+H]+.
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C15H12ClN2O2 287.0582; Found 287.0583.
6-Iodo-2-(4-methoxyphenyl)quinazolin-4(3H)-one (2w). White solid. yield: 76% (109 mg).
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m.p. 220-222 °C. H NMR (400 MHz, DMSO-d6, ppm): δ 12.81 (s, 1H), 8.48 (d, J = 4.0 Hz,
1H), 8.17 (m, 3H), 7.57 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 12 Hz, 2H), 3.89 (s, 3H). 13C{1H} NMR
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(126 MHz, DMSO-d6, ppm): δ 185.6, 164.8, 160.4, 150.5, 146.9, 143.5, 134.8, 133.9, 126.9,
124.9, 114.5, 56.2; IR CHCl3): νmax 3418, 2920, 1658, 1457, 1313, 1167 cm-1. ESI-MS: m/z
378.99 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C15H12IN2O2 378.9938; Found
378.9928.
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6-Iodo-2-(4-nitrophenyl)quinazolin-4(3H)-one (2x). White solid. yield: 65% (97 mg). m.p. >
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260 °C. H NMR (400 MHz, DMSO-d6, ppm): δ 12.92 (s, 1H), 8.50 (d, J = 4.0 Hz, 1H), 8.48 -
8.38 (m, 4H), 8.20 (dd, J = 1.9, 8.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H). 13C{1H} NMR (126 MHz,
CDCl3 + DMSO-d6, ppm): δ 185.8, 159.9, 150.0, 148.2, 146.1, 143.2, 139.2, 134.3, 132.2, 130.5,
124.5, 123.1. IR (CHCl3): νmax 3426, 2918, 1666, 1519, 1342, 1160, 1016 cm-1. HRMS (ESI-
TOF) m/z: [M-H]- Calcd for C14H7IN3O3 391.9538; Found 391.9521.
6-Iodo-2-phenylquinazolin-4(3H)-one (2y). White solid. yield: 72% (95 mg). m.p. > 260 °C.
1H NMR (400 MHz, DMSO-d6, ppm): δ 12.87 (s, 1H), 8.49 (d, J = 4.0 Hz, 1H), 8.20-8.15 (m,
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3H), 7.76 (t, J = 7.4 Hz, 1H), 7.62-7.56 (q, J = 8.0, 16.0 Hz, 3H). C{1H} NMR (126 MHz,
DMSO-d6 + 10% CDCl3, ppm): δ 187.0, 143.1, 142.9, 134.3, 134.1, 133.9, 131.6, 130.8, 128.6,
128.5, 127.8, 124.5, 122.6, 94.2. IR (CHCl3): νmax 3397, 2921, 1662, 1591, 1285, 1173 cm-1.
ESI-MS: m/z 348.98 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C14H10IN2O 348.9832;
Found 348.9822.
1-Methyl-5-phenyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (6a).24 White
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solid. yield: 50% (73 mg). m.p.194-197 °C. H NMR (400 MHz, CDCl3, ppm): δ 10.91 (s, 1H),
8.11 (dd, J = 3.1, 6.5 Hz, 2H), 7.54 (d, J = 2.0 Hz, 3H), 4.30 (s, 3H), 2.98-2.92 (m, 2H), 1.91-
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1.85 (m, 2H), 1.04 (t, J = 7.3 Hz, 3H). C{1H} NMR (101 MHz, CDCl3, ppm): δ 155.3, 149.0,
146.4, 138.8, 132.4, 130.5, 128.3, 126.6, 124.8, 37.6, 27.2, 21.8, 13.5. IR (CHCl3): νmax 3440,
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2931, 1688, 1633, 1490, 1100 cm-1. ESI-MS: m/z 269.14 [M+H]+. HRMS (ESI-TOF) m/z:
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[M+H]+ Calcd for C15H17N4O 269.1397; Found 269.1389.
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2-Benzoylquinazolin-4(3H)-one (10a).45 White solid. yield: 84% (154 mg). m.p. 179-182 °C.
1H NMR (400 MHz, CDCl3, ppm): δ 10.18 (s, 1H), 8.52 (d, J = 8.0 Hz, 2H), 8.41 (d, J = 8.0 Hz,
1H), 7.95 (d, J = 8.0 Hz, 1H), 7.88 (t, J = 4.0 Hz, 1H), 7.72-7.64 (m, 2H), 7.58 (t, J = 7.8 Hz,
1H). 13C{1H} NMR (126 MHz, CDCl3, ppm): δ 185.6, 161.0, 147.9, 145.9, 134.9, 134.3, 134.0,
133.9, 131.8, 129.5, 129.4, 128.4, 126.9, 123.2. IR (CHCl3): νmax 3436, 2926, 1666, 1481, 1297,
1144 cm-1. ESI-MS: m/z 251.08 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C15H11N2O2
251.0815; Found 251.0812.
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Quinazolin-4(3H)-one (13).29,42 Light pink solid. yield: 55% (59 mg). m.p. 218-220 °C. H
NMR (400 MHz, DMSO-d6, ppm): δ 12.25 (s, 1H), 8.17-8.07 (m, 2H), 7.82 (t, J = 7.2 Hz, 1H),
7.67 (d, J = 8.1 Hz, 1H), 7.53 (t, J = 7.4 Hz, 1H). 13C{1H} NMR (101 MHz, DMSO-d6, ppm): δ
162.1, 148.4, 145.1, 134.6, 127.1, 126.5, 125.9, 122.4. IR (CHCl3): νmax 3417, 2921, 1675, 1517,
1453, 1110 cm-1. ESI-MS: m/z 147.04 [M+H]+. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C8H7N2O 147.0553; Found 147.0570.
NLRP3 Inflammasome Activation Assay. J774A.1 cells were seeded at a density of (0.4 ×
106 cells/mL) in 24 well plates. Cells were primed with LPS (1 µg/mL) for 5.5 h in DMEM with
10 % FBS and then treated with different compounds or vehicle (DMSO) in serum free media for
1 h. Followed by incubation with test compounds or vehicle, the cells were stimulated with ATP
(3 mM for 30 minutes). Supernatants were collected and analyzed for IL-1β levels by ELISA
(Invitrogen, Ebiosciences). IL-1β levels in the supernatants were observed through IL-1β ELISA
kit (Invitrogen, Ebioscience) according to manufacturer’s instruction. The absorbance for the
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cytokine was measured at 450 nm. The concentration of estimated cytokine was normalized with
the total protein amount extracted using NaCl-Triton X lysis buffer.
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Western-blot Analysis. Cell lysates were prepared in RIPA buffer (50 mM Tris-HCl [pH
7.5], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1% Nonidet P-40, and, 0.25% Na-deoxycholate)
with complete protease inhibitor cocktail (Sigma-Aldrich), 1 mM PMSF, 1 mM sodium ortho-
vanadate. The protein concentrations were determined by Bradford protein assay (Bio-Rad). The
cell supernatants were collected and concentrated by strata clear resin (Agilent). Proteins were
separated by SDS-PAGE gel electrophoresis and then transferred to PVDF membrane. The
PVDF membrane was blocked in TBST containing 5% skimmed milk for 2 h at room
temperature and kept for overnight incubation with IL-1β primary antibody at 4 °C. After
washing, incubation was done with secondary antibody at room temperature for 1-2 h and was
then developed by using ECL (Millipore). The level of IL-1β released in the supernatants was
measured through IL-1β ELISA kit (Invitrogen, Ebioscience) according to the manufacturer’s
instructions.
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Statistical Analysis. Data from biological experiments were analyzed using GraphPad Prism
(version 6). Student’s t tests (ANOVA) were performed to compare the groups (statistical
analysis.*p < 0.025; .**p < 0.01; .***p < 0.005)
ACKNOWLEDGEMENTS. Authors thanks analytical department IIIM for analytical support.
M. Abdullaha and M. Ali thanks UGC for research fellowship. S. Mohammed is thankful to
CSIR for research fellowship. The financial support from CSIR YSA grant (P90807) is gratefully
acknowledged.
AUTHOR INFORMATION.
Corresponding Author
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*Tel: +91-191-2586333; Tel: +91-191-2585006 (Extn. 345). E-mail: sbharate@iiim.ac.in,
sandipbharate@gmail.com (S.B.B.)
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ORCID: 0000-0001-6081-5787
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Notes
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The authors declare no competing financial interest.
SUPPORTING INFORMATION AVAILABLE: NMR spectra scans and HRMS/ HPLC
spectra scans for some compounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
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