Design, synthesis and evaluation of novel 2-thiophen-5-yl-3H-quinazolin-4-one analogues as inhibitors of transcription factors NF-k{cyrillic}B and AP-1 mediated transcriptional activation: Their possible utilization as anti-inflammatory and anti-cancer ag

Article abstract of DOI:10.1016/j.bmc.2010.01.007

In an attempt to discover novel inhibitors of NF-κB and AP-1 mediated transcriptional activation utilizing the concept of chemical lead based medicinal chemistry and bioisosterism a series of 2-(2,3-disubstituted-thiophen-5-yl)-3H-quinazolin-4-one analogs

Full text of DOI:10.1016/j.bmc.2010.01.007

Bioorganic & Medicinal Chemistry 18 (2010) 2796–2808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel 2-thiophen-5-yl-3H-quinazolin-4-one
analogues as inhibitors of transcription factors NF-rB and AP-1 mediated
transcriptional activation: Their possible utilization as anti-inflammatory
and anti-cancer agents
Rajan S. Giri a, , Hardik M. Thaker a, Tony Giordano b, Jill Williams b, Donna Rogers b, Kamala K. Vasu a,
,
*
Vasudevan Sudarsanam a
a B. V. Patel Pharmaceutical Education and Research Development Centre, S.G. Road, Thaltej, Ahmedabad 54, India
b Feist-Weiller Cancer Center, LSUHSCs, 1501 Kings Highway Shreveport, LA 71130, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to discover novel inhibitors of NF-
j
B and AP-1 mediated transcriptional activation utilizing
Received 10 November 2009
Revised 3 January 2010
Accepted 5 January 2010
Available online 11 January 2010
the concept of chemical lead based medicinal chemistry and bioisosterism a series of 2-(2,3-disubsti-
tuted-thiophen-5-yl)-3H-quinazolin-4-one analogs was designed. A facile and simple route for the syn-
thesis of the designed molecules was developed. Synthesized molecules were evaluated for their
activity as inhibitors towards NF-rB and AP-1 mediated transcriptional activation in a cell line report-
based assay. This series provides us with a substantial number of compounds inhibiting the activity of
NF-rB and/or AP-1 mediated transcriptional activation. These compounds also exhibit anti-inflammatory
and anti-cancer activity in in vivo models of inflammation and cancer. The 4-pyridyl group is found to be
Keywords:
Thiophene
Inhibitors of NF-jB and AP-1 mediated
transcriptional activation
Antiinflammatory agents
Transcription inhibitors
Anti-cancer agents
the most important pharmacophore on the third position of thiophene ring for inhibiting NF-jB and AP-1
mediated transcriptional activation. The relationships between the activities shown by these compounds
in the in vivo and in vitro models have been established by using FVB transgenic mice model. These
results suggest the suitability of the designed molecular framework as a potential scaffold for the design
of molecules with inhibitory activity towards NF-jB and AP-1 mediated transcriptional activation, which
may also exhibit anti-inflammatory and anti-cancer activity. This series of molecules warrants further
study to explore their potential as therapies for use in chronic inflammatory conditions and cancer.
Development of the synthetic protocol for the synthesis of this series of molecules, biological activities
and a structure–activity relationship (SAR) have been discussed herein.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Transcription factors are regulatory proteins which bind to spe-
cific DNA sequences in the gene promoter or enhancer regions to
The current treatment options for cancer and inflammatory dis-
eases are by and large unsatisfactory due to poor overall efficacy
and relatively significant side effect profiles.1 A complex network
of proteins involving multiple molecular pathways, for which a
great deal of redundancy exists, act as mediators of inflammation
and cancer.2 In view of the polygenic nature of these diseases, with
a number of pathways acting in parallel, it seems appropriate to
look for therapies acting on more than one pathway.3 The targeting
of transcription factors with small molecules represents one such
approach of modulating the activity of a number of proteins and
pathways simultaneously.4
activate or inhibit the transcription of genes.5 The synthesis of
new messenger RNA by the process of transcription is a major con-
trol point in the expression of many proteins. This is especially true
for cytokines, cell adhesion molecules, erosive enzymes and other
proteins that mediate inflammatory diseases and cancer. Nuclear
factor-
tion factors that orchestrate expression of many genes involved in
inflammation and cancer. NF- B controls the expression of virtu-
ally all significant pro-inflammatory chemokines/cytokines, many
angiogenic growth factors, TNF- , inducible nitric oxides synthase,
jB (NF-jB) and activating protein (AP-1) are key transcrip-
j
a
COX-2, ICAM-1, VCAM-1, E-selectin and pathways of cell prolifera-
tion and survival.6,7 Similarly AP-1 regulates the activity of
interleukin-2 (IL-2), IL-3 and granulocyte-macrophage colony
stimulating factor (GM-CSF), metalloproteinases, collagenase and
stromelysin.8 Many other pro-inflammatory genes are also known
* Corresponding author. Tel.: +91 079 27439375; fax: +91 07927450449.
E-mail addresses: kamkva@gmail.com, perd@perdcentre.com (K.K. Vasu).
Present address: Department of Medicinal Chemistry, University of Minnesota, 308
Harvard St. SE, MN 55445, United States.
to have binding sites for NF-jB and AP-1.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.007

R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
2797
The role of the immune system in cancer has also become better
understood. The tumor microenvironment is highly influenced by
inflammatory cells responsible for fostering tumor proliferation,
survival and migration.9 Thus, altering cytokine expression can
have a profound affect on cancer progression as demonstrated by
the success of Celgene’s immunomodulatory drugs, thalidomide
and revlimid. Therefore, the selective inhibition of a limited num-
quinazolin-4-one analogues 9 (Fig. 1). The monocyclic hetroaro-
metic ring system at the fifth position of thiophene ring was
replaced with
a bicyclic heteroarometic ring system in the
3-aryl-3H-quinazolin-4-one scaffold, which can be considered as
a bioisostere of the quinoline ring in SPC-839 and is a well known
anti-inflammatory scaffold. Herein, we report the synthesis and
evaluation of 2-thiophen-5-yl-3H-quinazolin-4-one analogues as
ber of key transcription factors like NF-
jB and AP-1 involved in the
inhibitors of transcription factors NF-jB and AP-1 mediated
inflammatory process may control a plethora of mediators of
inflammation and cancer. The dual targeting of these two tran-
scription factors may also overcome some of the redundancy that
is inherent to biological systems and possibly responsible for the
failure of the more specific, ‘magic bullet’ targeted therapies.10,11
The anti-inflammatory effect of steroids, retinoids and a variety
of anti-rheumatic drugs act through a mechanism that converges
on a limited number of transcription factors, most notably tran-
transcriptional activation. We also discuss their potential as anti-
inflammatory and anti-cancer agents.
2. Chemistry
In designing the synthetic protocol for 2-thiophen-5-yl-3H-qui-
nazolin-4-one analogues, we envisioned that 2-chloro methyl-3-
aryl-3H-quinazoline-4-one 2 and 1,3-di-morpholin-4-yl-2-substi-
tuted-propenethione intermediate 5 may represent interesting
starting materials for synthesis of designed compounds 9 (Scheme
1). A chemical interaction between 2 and 5 may result into the
S-alkylation of the sulfur group of 5 by the active methylene group
(–CH2–) of 2, yielding intermediate 1. It can be observed from the
structure of 1 that it is chemically not likely to be very stable,
therefore an intramolecular cyclization reaction followed by stable
aromatic ring formation would be the favored synthetic route
(Schemes 1 and 2).
Mindful of this consideration we initiated to synthesize inter-
mediates 2 and 5. The synthesis of 2-chloromethyl-3-aryl-3H-qui-
nazolin-4-one 2 was carried out using a modified Niementowski
synthesis, beginning with the chloro-acetylation of anthranilic acid
4 to yield the corresponding 2-(2-chloro-acetyl amino) benzoic
acid 3. Acid 3 was then treated with trichlorophosphate (PCl3) to
convert into an acid chloride which was immediately treated with
substituted aniline in situ to replace the chloro group of the acid
chloride with substituted aniline. This was then refluxed in toluene
to generate the cyclized product 2.21,22 2-Aryl-1,3-dimorpholin-4-
scription factors NF-
the compounds that exert their anti-inflammatory effects by inhib-
iting the activation of NF- B and AP-1 may cause much milder gas-
j
B and AP-1.11 Importantly, it is believed that
j
trointestinal mucosal damage and other side-effects associated
with long term NSAID and glucocorticoid use.12 Based on these
observations, it appears that inhibition of NF-jB and/or AP-1 tran-
scriptional activation may represent a potential and safe target in
the development of novel agents to treat inflammatory diseases
and cancer.13,14
Quinazoline scaffolds and their bioisosteres are well known as a
privileged scaffold in drug discovery. During their search for small
molecule inhibitors of NF-jB and AP-1, Palanki and his coworkers
have reported quinazoline derivatives which have a thiophene ring
attached at the second position of the quinozoline (SPC-839,
Fig. 1).15–17
Further, tri-substituted thiophenes have been reported by us as
inhibitors of the transcription factors, NF-jB and AP-1, and as anti-
inflammatory agents.18–20 The SAR study in our earlier work sug-
gested that a morpholine at the second position, with aryl and het-
eroaryl substitutions at the third and fifth position of thiophene
scaffold resulted in an advantageous pharmacophore for designing
yl-propenethiones
5 were prepared using Willgerodt–Kindler
reaction, where appropriate acetophenones 7 were reacted with
elemental sulfur and morpholine 8 to yield the phenylthioacetic
acid morpholide intermediates 6.23 Phenylthioacetic acid morpho-
lide was reacted with morpholine and triethylorthoformate (as a
one carbon donor), to yield 2-aryl-1,3-dimorpholin-4-yl-propen-
ethiones 424 (Scheme 2). In the final step, intermediate 2 was
better inhibitors of NF-j
B and AP-1.18–20 In the process of engi-
neering better inhibitors of transcriptional activation of NF-rB
and AP-1 utilizing the concept of bioisosterism and chemical lead
based medicinal chemistry, we delineated the gross framework
for the design of several new compounds as 2-thiophen-5-yl-3H-
Figure 1. Template based approach for the designing of novel 2-thiophene-5-yl-3H-quinazoline-4-one analogues as NF-jB and AP-1 inhibitors.

2798
R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
Scheme 1. Retro analysis of the designed molecule.
Scheme 2. Synthesis of designed compounds.
treated in acetonitrile with intermediate 5 in acetonitrile at 75–
80 °C for 2–4 h to yield the desired 2-thiophen-5-yl-3H-quinazo-
lin-4-one analogues.
nucleophilic attack may take place due to the initial tautomeriza-
tion of the quinazolinone double bond to generate an enamine like
intermediate; the quinazolinone nitrogen then serves as an elec-
tron source for the nucleophilic attack. The driving force for the
thiophene ring formation is the elimination of the morpholine 8
residue and the formation of the stable five membered aromatic
heterocycle. The probable mechanism has been summarized in
Scheme 3.
In this synthesis, a functionalized 2-aryl-1,3-dimorpholin-4-yl-
propenethiones 5 derivative provided three ring carbon (C-2, C-3,
C-4) atoms and the heteroatom (S-1) of the resultant thiophene
ring; the remaining carbon atom (C-5) was supplied by the meth-
ylene group of the 2-chlormethyl group at the second position of
the quinazolinone ring 2. The mechanism involves the S-alkylation
of the –SH group of 2-aryl-1,3-dimorpholin-4-yl-propenethione 5
by –CH2– of chloromethyl at the second position of quinazolinone
ring followed by the intramolecular cyclization through the nucle-
ophilic attack of the active methylene group. Presumably this
3. Results and discussion
The focus of our approach in this work was to optimize the sub-
stitutions on the phenyl ring at third (R2) position of the quinazoli-

R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
2799
Scheme 3. Plausible mechanism for the synthesis of designed compounds.
none ring and on the third position of the thiophene ring (R1) to
generate maximum inhibition of NF- B and/or AP-1 mediated
transcriptional activation. The selection of substituents at both
3.1.1. Structure–activity relationship
j
Analysis of the data generated from this study suggests that:
(1) the activity of the designed molecular framework is depen-
dent upon the substituents at the R1 and R2 positions and/or their
combination. (2) Compounds which have –SO2CH3 group at R1
position were found to be more active for the biological activities
discussed in this paper than compounds which have –H, –Cl,
–SCH3 and –NHCOCH3 at R1 position (Table 1). These results sug-
gest that electron withdrawing hydrophilic substitutes (e.g.,
–SO2CH3) are more desirable at the R1 position rather than electron
withdrawing (e.g., –Cl), electron donating (e.g., –SCH3) lipophilic
substituents or electronically neutral hydrophilic substitutes
(e.g., –NHCOCH3) for achieving the desired activity. (3) The com-
pounds substituted with –Cl on R2 positions yielded the least ac-
tive series of compounds in this study (except in combination
with 4-pyridyl at the 3rd position of thiophene ring). The pres-
ence of –Cl at R2 position is detrimental even where R1 is
–SO2CH3 (9bf, Table 1). Which suggests that electron withdraw-
ing groups with lipophilic characteristics like –Cl may not be a
ideal substitution on R2 position to get the good activity from
the designed molecular framework (9bc–9bf and 9bi, Table 1)
and presence of this kind of substitution may also eliminate po-
tency of the –SO2CH3 group at R1 position. (4) The compounds
substituted with –CH3 group at R2 position were shown to exhibit
mild to very weak activity in both the assays tested (9ag, 9ah,
9aj, 9al–9ao, 9aq–9as, 9au, 9av, Table 1). These results suggest
that substituents with electron donating and lipophilic character-
istics like –CH3 may not be the ideal substituents on R2 position
for the desired activity but presence of this group does not elim-
inate the potency of the –SO2CH3 group at R1 position (9ai and
9at, Table 1). (5) The compounds substituted with electron donat-
ing and very low steric value like –OCH3 at R2 position were
found to be more active in the designed series of compounds than
compounds having electron donating and lipophilic characteris-
tics like –CH3 (9ax, 9ay, 9ba Table 1). (6) Compounds which have
–COCH3 group at R2 position were found to be active for the bio-
logical activities discussed in this paper (9bj and 9bk, Table 1).
Combination of –COCH3 group at R2 position with –H and –Cl
groups at R1 proved to be detrimental for the activity of designed
molecular framework. (7) Combination of –SO2CH3 group at R1
position with –COCH3 groups at R2 position was not found to
be beneficial as compared to the other combination (Table 1).
(8) 4-Pyridyl group at the third position of the thiophene ring
positions were mainly guided by lipophilicity and electronic con-
siderations as defined by the
way that they fall within the
r
r, p
–
p
Craig plot, chosen in such a
quadrant of the Craig plot.25
One substituent was chosen for the R1 position from every quad-
rant (i.e., –CH3 from first, Cl from second, –COCH3 from third and
–OCH3 from fourth quadrant) to generate a structure–activity pro-
file of the compounds. Similarly, one substituent was chosen from
every quadrant (i.e., –SCH3 from first, –Cl from second, –SO2CH3
from third and –NHCOCH3 from fourth quadrant) for the R2
position.
3.1. Inhibition of NF-rB and AP-1 mediated gene transcription
The ability of the synthesized 2-thiophen-5-yl-3H-quinazolin-
4-one analogues to modulate NF-jB and AP-1 mediated transcrip-
tional activation was evaluated using a luciferase reporter assay in
human embryonic kidney (HEK-293) cells with a stably transfected
NF-
mycin selection, a chromosomal integration of a luciferase reporter
construct regulated by multiple copies of the NF- B and AP-1 re-
sponse elements. For induction of transcription and to test the bio-
logical response of the promoters, the HEK/NF- B cell line was
stimulated with TNF- (1 ng/ l) and the HEK/AP-1 cell line was
stimulated with PMA (1 ng/ l) in the presence or absence of test
jB-luc and AP-1-luc gene. These cells maintain, through hygro-
j
j
a
l
l
compounds. All of the 37 compounds synthesized were evaluated
for their activities. A reasonable number of compounds have been
identified exhibiting moderate to excellent inhibitory activity to-
wards NF-
this cell based in vitro assay at10
pounds showing more than 50% inhibition at 10
j
B and/or AP-1 mediated transcriptional activation in
M concentration (Table 1). Com-
M were taken
l
l
forward for in vivo testing in carrageenan induced rat paw edema
model of inflammation and were found to exhibit a significant pro-
tective activity in this model at 50 mg/kg, po in comparison to the
existing anti-inflammatory drug ibuprofen (Table 2). The most
promising compounds from the in vitro screening were tested
in vitro for dose response from 10 nM to 10
was found to be the most promising compound from the series
with IC50 value of 10 M and 5 M for NF-rB and AP-1, respectively
lM. Compound 9bk
l
l
(Table 2). Compound 9bk was also found to be the most active
compound in the in vivo anti-inflammatory activity (Table 2).

2800
R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
Table 1
3.1.2. 9bk Decreased LPS-stimulated NF-
transgenic FVB mice
In our in vitro assay we have used human embryonic kidney cell
line, (HEK293) stably expressed with a plasmid containing a mini-
jB activity by 43% in
Evaluation of synthesized compounds in in vitro assays for their inhibitory activities
towards NFkB and AP-1 mediated transcriptional activation
mal promoter and tandem copies of the NF-jB transcriptional
element (50-AGTTGAGGGGACTTTCCCAGGC-30) or the AP-1 tran-
scriptional element (TGACTAA), which regulates the expression of
the luciferase reporter gene. The read out of our in vitro screening re-
sults suggest that our compounds are inhibiting the activation of NF-
rB and AP-1 mediated transcriptional process. Therefore to see if our
compounds inhibit the activation of NF-rB directly in vivo we tested
the activity of 9bk to inhibit the activation of NF-rB in vivo using
transgenic FVB mice consisting of a minimal promoter and tandem
No.
Entrya
R1
R2
X
% Inhibition in vitro (10 lM)
NF-
j
B binding sites in all cells were used as in vivo ‘reporters’ of
NF-
j
B
AP-1
NFj
B activity. Three transgenic mice which express an NF- B-luci-
j
1
2
3
4
5
6
7
8
9
9aa
9ab
9ac
9ad
9ae
9af
9ag
9ah
9ai
–H
–Cl
–NHCOH3
–SO2CH3
–SCH3
—
–H
–Cl
–SO2CH3
–SCH3
—
–H
–H
–H
–H
–H
–H
C
C
C
C
C
N
C
C
C
C
N
C
C
C
C
N
C
C
C
C
C
N
C
C
N
C
C
N
C
C
C
C
N
C
C
C
N
0
0
7
9
0
30
23
1
51
15
53
28
35
20
11
42
0
15
4
55
22
26
0
55
55
20
60
80
0
0
0
0
68
0
11
0
26
28
0
firase reporter gene in all tissues were taken per group. Mice injected
with luciferin only (not stimulated with LPS) were shown to have
very low endogenous levels of NF-
(Fig. 2A). All mice were confirmed to glow by injection of 60
(Sigma) 3 h prior to injection of luciferin and imaging on the IVIS 100
system (Xenogen) (Fig. 2B). These results suggest that stimulation
with LPS treatment is able to activate the NF-
ment group was treated for four days with 50 mg/kg of 9bk daily
once and on the fourth day all three mice were injected with 60
LPS and after 3 h all mice were injected with luciferin and were im-
aged on the IVIS 100 system (Xenogen) (Fig. 2C). Compound 9bk
treated mice (Fig. 2C) had a significantly decreased level of biolumi-
nescence compared to the control (maximally) stimulated mice
j
B activity limited to the thymus
g LPS
l
38
27
32
52
13
0
10
13
48
14
35
17
27
35
51
16
29
0
33
33
0
10
35
23
14
17
7
2-CH3
2-CH3
2-CH3
2-CH3
2-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
2-OCH3
2-OCH3
2-OCH3
3-OCH3
4-OCH3
2-Cl
jB in vivo. The treat-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
9aj
9ak
9al
l
g
–Cl
9am
9an
9ao
9ap
9aq
9ar
9as
9at
9au
9av
9aw
9ax
9ay
9az
9ba
9bb
9bc
9bd
9be
9bf
9bg
9bh
9bi
–NHCOCH3
–SO2CH3
–SCH3
—
–H
–Cl
–NHCOCH3
–SO2CH3
–SCH3
—
–H
–SO2CH3
—
–SO2CH3
–SO2CH3
—
–H
–Cl
–NHCOCH3
–SO2CH3
—
–H
–Cl
(Fig. 2B), indicating that 9bk is inhibiting activation of NF-jB di-
rectly in vivo.
Inflammation and cancer have been shown to have a close
association and hence anti-inflammatory drugs of steroidal as
well as non-steroidal nature have been proven to decrease the
risk of developing various malignancies including colon,26 lung,27
prostate and breast cancers.28 Under this concept, the targets NF-
rB and AP-1 have been widely described as very important links
between the inflammatory mechanism and cancer and as a result
this association has been studied in depth in various aspects of
cancer.28–30 Based on this understanding, the most active com-
pound from this study 9bk was tested for its ability to inhibit
proliferation or cause apoptosis on various cancer cell lines,
which are used as models for different types of cancers. Com-
pound 9bk was shown to inhibit the growth of LNCap and PC-3
(prostate cancer cell lines) and TCCSUP (bladder cancer cell line).
Compound 9bk was also found to inhibit tumor growth and tu-
mor angiogenesis in a various human tumor xenograft models.
In a cytokine profiling assay 9bk was found to alter the expres-
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
32
25
7
31
84
4-COCH3
4-COCH3
4-COCH3
4-COCH3
0
46
66
9bj
9bk
–SO2CH3
—
a
Synthesized compounds were characterized by their IR, NMR and LC–MS
(Section 5). SPC-839, reduced luciferase activity by ꢀ40% in the NF-rB and AP-1 cell
lines at 1.0
M.20
sion of cytokines like TNF-a, MCP-3, MCP-4, BMP-4, BMP-6, RAN-
l
TES, MCP-1, GCP-1 and various anti-apoptotic genes involved in
several type of cancer progression.31 These results suggest that
9bk is potential inhibitor of NF-rB and AP-1 mediated transcrip-
tional activation which may be utilized to treat inflammatory dis-
eases and cancer.
A new molecular scaffold 2-(2,3-disubstituted-thiophen-5-yl)-
3H-quinazolin-4-one, has been developed and can be considered
as a potential molecular scaffold for designing of novel inhibitors
of NF-rB and AP-1 mediated transcriptional activation, with po-
tential for use as anti-inflammatory and anti-cancer therapies.
This was developed by utilizing the molecular scaffold generated
through a chemical lead based medicinal chemistry approach
(Fig. 1) and incorporating the morpholine, aryl and heteroaryl
groups on thiophene ring, which was an already established
pharmacophore as an inhibitor of NF-rB and AP-1 mediated
transcriptional activation and as an anti-inflammatory
pharmacophore.
emerged as the most important pharmacophore for inhibiting
NF- B and AP-1 mediated transcriptional activation, as all the
j
compounds (except 9av, which was found to be least active in 4-pyr-
idyl series) in this series were found to have highly significant activ-
ity in both the assays (Table 1). (9) To study the effect of –COCH3
group at the R2 position in combination with the 4-pyridyl group
at the third position of the thiophene ring, compound 9bk was syn-
thesized and was found to be the most promising dual inhibitor of
NF-jB and AP-1 mediated transcriptional activation.
These results suggest that the electron withdrawing hydrophilic
substituents like –COCH3 at R2 position in combination with lipo-
philic cyclic ring containing weakly basic nitrogen may be the ideal
feature for obtaining the dual inhibitory activity towards NF-jB
and AP-1 mediated transcriptional activation from the designed
molecular framework.

R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
2801
Table 2
Evaluation of most promising compounds in in vitro assays for their anti-inflammatory activity in carrageenin induced rat paw edema model of acute inflammation
% Protectiona in in vivo (50 mg/kg)
b
Entry
R1
R2
X
IC50
(lM)
NF-kB
AP-1
9ak
9ax
9ay
9ba
9bb
9bg
9bk
—
2-CH3
2-OCH3
2-OCH3
4-OCH3
2-Cl
N
C
N
C
N
N
N
7.1
9.2
12.2
26
10.3
11.4
10
nac
nac
nac
nac
nac
nac
5
51
63
42
53
43
52
81
–SO2CH3
—
–SO2CH3
—
—
—
4-Cl
4-COCH3
a
Oral administration for all test compounds for in vivo anti-inflammatory activity. For the standard drug ibuprofen, dose and % protection were 50 mg/kg, 52%,
respectively.
b
IC50 values for inhibition of luciferase production in pNF-
j
B-luc HEK and pNF-AP-1-luc HEK cells following TNF-
na, not applicable (only compounds showing more than 50% inhibition in initial screening were subjected to six point dose response study. 9ai and 9at have shown
promising activity in primary screening but failed to show good dose response).
a and PMA activation, respectively.
c
Figure 2. Compound 9bk decreased LPS-stimulated NF-
tissues. (A) Mice injected with luciferin only were shown to have very low endogenous levels of NF-
injection of 60 g LPS (Sigma) 3 h prior to injection of luciferin and imaging on the IVIS 100 system (Xenogen). (C) Mice were given 50 mg/kg 9bk by oral gavage for 4
j
B activity by 43% in luciferase reporter mice. Transgenic mice expressed an NFjB-luciferase reporter gene in all
jB activity limited to the thymus. (B) All mice were confirmed to glow by
l
consecutive days prior to LPS treatment, luciferin injection, and imaged as above. Representative images from similar exposures with the same color scale are shown. (D)
Compound 9bk (LS-122) treated mice had a significantly decreased level of bioluminescence compared to the control (maximally) stimulated mice, indicating that 9bk is
inhibiting NF-jB directly in vivo.
activity. These results suggest the suitability of the designed molec-
ular framework for use in the development of anti-inflammatory
and anti-cancer therapies.
4. Conclusion
In an attempt to discover novel inhibitors of NF-jB and AP-1
mediated transcriptional activationfor use as potent anti-inflamma-
tory and/or anti-cancer agents, a series of 2-(2,3-disubstituted-
thiophen-5-yl)-3H-quinazolin-4-one analogs were designed,
synthesized and evaluated for its biological activities. A facile and
simple route for the synthesis of the designed compounds was
developed. This series eventually turned out to be very promising
such that a substantial number of compounds which inhibit the acti-
vation of NF-jB and AP-1 mediated transcriptional activation were
generated. These inhibitors also demonstrated significant in vivo
efficacy as anti-inflammatory agents. Compound 9bk, the most ac-
tive dual inhibitor of NF-jB and AP-1 mediated transcriptional acti-
vation from the entire series, was also found to demonstrate
potential anti-cancer activity in vitro as well as in an in vivo model
of cancer. The relationship between the activities shown by these
compounds in the in vivo and in vitro models has been establish
by using transgenic FVB mice model. Therefore, the activity of 9bk
5. Experimental
Melting points (mp) were determined on a Toshniwal melt-
ing point apparatus using open glass capillary tubes and repre-
sent the uncorrected values. Proton NMR spectra were measured
on a Bruker spectrometer using the specified solvents at RSIC
Chandigarh. LC–MS analyses were done on a Perkin Elmer Ap-
plied Bio-Sciences API-165 in the analytical laboratory of B. V.
Patel PERD Centre, Ahmedabad, India using electron spray ioni-
zation (EI) in positive mode. Elemental analysis was carried out
at RSIC, IIT Powai on Perkin Elmer 2400 CHN elemental ana-
lyzer. All the reactions were monitored using thin layer chroma-
tography (TLC) using glass plate coated with silica gel G or
GF254. TLC plates were developed in iodine and toluene/acetoni-
trile (7:3) was taken as mobile phase, unless mentioned
otherwise.
in in vivo model may be atleast in part is due to its NF-jB inhibitory

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R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
5.1. Synthesis of 2-(2-chloro-acetyl amino) benzoic acid 321,22
procedure described for 2a to afford the title compound as off light
yellow solid. Yield: 32.2%, mp 180–182 °C, Rf: 0.62, LC–MS (m/z):
301 (M+1).
Anthranilic acid 4 (50.0 g, 0.37 mol) was taken into dichloro-
methane and equimolar amount of triethylamine was added, and
then reaction was cooled to 0 °C. While maintaining this tempera-
ture chloro acetyl chloride (41.1 g, 0.37 mol) was added to it in
15 min. Reaction was stirred at room temperature for 4 h. White
precipitates were filtered and washed with ample amount of water
and dried. The crude compound was crystallized with hot water to
get (55.0 g, 64.5%) of 2-(2-cholo-acetylamino) benzoic acid as a
white solid. mp 183–185 °C, Rf: 0.40, LC–MS (m/z): 214 (M+1).
5.2.7. 2-Chloro methyl-3-(4-methoxy-phenyl)-3H-quinazoline-
4-one (2g)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 4-methoxy aniline (11.4 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound as a light
yellow solid.Yield: 34.0%, mp 154–155 °C, Rf: 0.57, LC–MS (m/z):
301 (M+1).
5.2. Synthesis of 2-chloromethyl quinazoline-4-one derivatives
5.2.8. 2-Chloro methyl-3-(2-chloro-phenyl)-3H-quinazoline-4-
one (2h)
5.2.1. Synthesize of 2-chloro methyl-3-(phenyl)-3H-
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 2-choloro aniline (12.0 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound as a light
yellow solid. Yield: 26.4%, mp 145–146 °C, Rf: 0.61, LC–MS (m/z):
305 (M+1).
quinazoline-4-one (2a)22
20.0 g (0.09 mol) of 2-(2-chloro-acetyl amino) benzoic acid 3
and equimolar amount of aniline (8.70 g, 0.09 mol) were taken in
toluene and PCl3 (18 mL) was added into it. Temperature was
raised to reflux the reaction and maintained till TLC showed com-
pletion of reaction. Then the reaction was cooled to room temper-
ature and solvent was evaporated in vacuo. To this residue water
was added and reaction mixture was neutralized with the addition
of sodium bicarbonate and extracted with chloroform (4 ꢁ 50 mL).
The organic layers were combined and washed with water
(2 ꢁ 30 mL). The organic layer was then dried over Na2SO4 evapo-
rated in vacuo. Resulting crude was crystallized in methanol to get
2-chloromethyl-3-phenyl-3H-quinazoline-4-one (7.20 g, 28.0%) as
a white solid. mp 152–153 °C, Rf: 0.48, LC–MS (m/z): 271 (M+1).
5.2.9. 2-Chloro methyl-3-(3-chloro-phenyl)-3H-quinazoline-4-
one (2i)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 3-choloro aniline (12.0 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound a light yel-
low solid. Yield: 27.4%, mp 202–205 °C, Rf: 0.59, LC–MS (m/z): 305
(M+1).
5.2.10. Chloro methyl-3-(4-chloro-phenyl)-3H-quinazoline-4-
one (2j)
5.2.2. 2-Chloro methyl-3-(2-methyl-phenyl)-3H-quinazoline-4-
one (2b)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 3-choloro aniline (12.0 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound a light yel-
low solid. Yield: 26.4%, mp 234–235 °C, Rf: 0.62, LC–MS (m/z): 305
(M+1).
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 2-methyl aniline (9.80 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound as an off
white solid. Yield: 29.0%, mp 205–206 °C, Rf: 0.54, LC–MS (m/z):
285 (M+1).
5.2.11. 2-Chloro methyl-3-(p-acetyl phenyl)-3H-quinazoline-4-
one (2k)
5.2.3. 2-Chloro methyl-3-(3-methyl-phenyl)-3H-quinazoline-4-
one (2c)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 4-amino acetophenone (12.7 g, 0.09 mol) using the
same procedure described for 2a to afford the title compound a
light yellow solid. Yield: 36.6%, mp 180–181 °C, Rf: 0.54, LC–MS
(m/z): 313 (M+1).
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 3-methyl aniline (9.80 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound as an off
white solid. Yield: 32.5%, mp 183–185 °C, Rf: 0.53, LC–MS (m/z):
285 (M+1).
5.3. Synthesis of phenylthioacetic acid morpholide
intermediates (6)23
5.2.4. 2-Chloro methyl-3-(4-methyl-phenyl)-3H-quinazoline-4-
one (2d)
5.3.1. 1-Morpholin-4-yl-2-phenyl-ethanethione (6a)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 4-methyl aniline (9.80 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound as an off
white solid. Yield: 36.0%, mp 265–266 °C, Rf: 0.64, LC–MS (m/z):
285 (M+1).
A 100 mL round-bottomed flask was charged with 24.0 g
(0.20 mol) of acetophenone, 1.0 g of p-toluenesulfonic acid mono-
hydrate, 36.0 g (0.41 mol) of morpholine, and 6.40 g (0.20 mol) of
sulfur. The flask was equipped with a reflux condenser and was
heated to reflux for 3 h. The resulting reddish brown solution
was poured into 100 mL of stirred hot methanol. The resulting pre-
cipitates were filtered and washed twice with ice-cold methanol
(0–5 °C, 50 mL). The crude was re-crystallized by methanol and
water mixture to afford desire compounds (21.9 g) as a yellow so-
lid. Yield: 50%, mp 78–79 °C, LC–MS (m/z): 222 (M+1).
5.2.5. 2-Chloro methyl-3-(2-methoxy-phenyl)-3H-quinazoline-
4-one (2e)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20.0 g, 0.09 mol) was
reacted with 2-methoxy aniline (11.4 g, 0.09 mol) using the same
procedure described for 2a to afford the title compound as a light
yellow solid. Yield: 31.4%, mp 193–194 °C, Rf: 0.62, LC–MS (m/z):
301 (M+1).
5.3.2. 2-(4-Chloro-phenyl)-1-morpholin-4-yl-ethanethione
(6b)
1-(4-Chloro-phenyl)-ethanone (10.0 g, 0.07 mol) was reacted
with morpholine (0.13 mol) using the same procedure described
for 6a to afford the title compound (10.0 g) as a yellow solid. Yield:
60.15%, mp 175–177 °C, LC–MS (m/z): 256, 258 (M+1, M+2).
5.2.6. Chloro methyl-3-(3-methoxy-phenyl)-3H-quinazoline-4-
one (2f)
2-(2-Chloro-acetyl amino) benzoic acid 3 (20 g, 0.094 mol) was
reacted with 3-methoxy aniline (11.4 g, 0.094 mol) using the same

R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
2803
5.3.3. N-[4-(2-Morpholin-4-yl-2-thioxo-ethyl)-phenyl]-
acetamide (6c)
N-(4-Acetyl-phenyl)-acetamide (10.0 g, 0.06 mol) was reacted
with morpholine (0.13 mol) using the same procedure described
for 6a to afford the title compound (8.0 g) as a yellow solid. Yield:
50.72%, mp 210–212 °C, LC–MS (m/z): 279 (M+1).
(M+1). 1H NMR(300 MHz, DMSO-d6), d 3.05 (s, 3H), 3.29–3.62 (m,
8H), 4.15–4.44 (m, 8H), 6.5 (s, 1H), 7.27–7.49 (m, 2H), 7.65–7.76
(m, 2H).
5.4.5. 3-(4-Morpholino)-2-(4-methylmercapto-phenyl)-
thioacrylic acid morpholide (5e)
2-(4-Methylsulfanyl-phenyl)-1-morpholin-4-yl-ethanethione
6e (10.0 g, 0.04 mol) was reacted with morpholine and triethylor-
thoformate using the same procedure described for 5a to afford
title compound (5.8 g) as a yellow solid. Yield: 42%, mp 164–
165 °C, M.F.: C18H24N2O2S2, LC–MS (m/z): 365 (M+1).
5.3.4. 2-(4-Methanesulfonyl-phenyl)-1-morpholin-4-yl-
ethanethione (6d)
1-(4-Methanesulfonyl-phenyl)-ethanone (10.0 g, 0.051 mol)
was reacted with morpholine (0.13 mol) using the same procedure
described for 6a to afford the title compound (9.0 g) as a yellow so-
lid. Yield: 60%, mp 198–199 °C, LC–MS (m/z): 300 (M+1).
5.4.6. 1,3-Di-morpholin-4-yl-2-pyridin-4-yl-propenthione (5f)
1-Morpholin-4-yl-2-pyridine-4-yl-ethanethione
6f
(5.0 g,
5.3.5. 2-(4-Methylsulfanyl-phenyl)-1-morpholin-4-yl-
ethanethione (6e)
1-(4-Methylsulfanyl-phenyl)-ethanone (10.0 g, 0.08 mol) was
reacted with morpholine (0.13 mol) using the same procedure de-
scribed for 6a to afford the title compound (8.0 g) as a yellow solid.
Yield: 48.39%, mp 158–160 °C, LC–MS (m/z): 268 (M+1).
0.02 mol) was reacted with morpholine and triethylorthoformate
using the same procedure described for 5a to afford title com-
pound (3.0 g) as a yellow solid. Yield: 39.68%, mp 122–125 °C,
Molecular Formula C16H21N3O2S, LC–MS (m/z): 320 (M+1), 1H
NMR(300 MHz, DMSO-d6), d 3.19–3.59 (m, 8H), 3.7–4.2 (m, 8H),
6.09 (s, 1H), 7.01–7.32 (m, 4H).
5.3.6. 1-Morpholin-4-yl-2-pyridine-4-yl-ethanethione (6f)
1-Pyridine-4-yl-ethanone (10.0 g, 0.08 mol) was reacted with
morpholine (0.13 mol) using the same procedure described for 6a
to afford the title compound (7.9 g) as a yellow solid. Yield:
47.4%, mp 162–163 °C, LC–MS (m/z): 223 (M+1).
5.5. Synthesis of 2-thiophen-5-yl-3H-quinazolin-4-one
analogues
5.5.1. 2-(5-Morpholin-4-yl-4-phenyl-thiophene-2-yl)-3-phenyl-
3H-quinazolin-4-one (9aa)
1,3-Di-morpholin-4-yl-2-phenyl-propenethione
5a
(1.0 g,
5.4. 2-Aryl-1,3-dimorpholin-4-yl-propenethiones derivatives
(4)
0.003 mol) was taken in 5 mL of acetonitrile and heated to
78 °C. At this temperature 2-chloro methyl-3-(phenyl)-3H-qui-
nazoline-4-one 2a (0.86 g, 0.003 mol) in 5 mL acetonitrile was
added into it and this reaction mixture was heated at this tem-
perature for 6–8 h. Then it was cooled to rt and solvent was re-
moved in vacuo. The solid residue obtained was crystallized
using methanol to afford title compound as yellow solid. Yield
58.0%, mp 218–220 °C, Rf: 0.53, IR (KBr, cmꢂ1); 3021,
1691,1539, 1308, 1281, 758, Molcular Formula C28H23N3O2S, LC–
MS (m/z): 466 (M+1), 1H NMR (300 MHz, CDCl3) d 2.96–3.0 (m,
4H), 3.72–3.74 (m, 4H), 5.96 (s, 1H), 7.22–7.78 (m, 13H), 8.27–
8.30 (m, 1H), Elemental Anal. Calcd: C, 72.23; H, 4.98; N, 9.03.
Found: C, 72.22; H, 4.97; N, 8.45.
5.4.1. 1, 3-Di-morpholin-4-yl-2-phenyl-propenethione (5a)
A mixture of 8.0 g of phenyl thioacetic acid morpholide 6a, 8.0 g
of morpholine and 20.0 g of triethylorthoformate was refluxed for
8 hours in an oil bath. Excess of triethylorthoformate was removed
from reaction in vacuo. The residue was dissolved in mixture of
warm chloroform: methanol (10 mL: 50 mL) and then cooled to
0 °C. The product separated as fine yellow crystals. The product
was filtered, washed with methanol and dried to afford 6.1 g
(56.0%). mp 115 °C, M.F.: C17H22N2O2S, LC–MS (m/z): 319 (M+1).
5.4.2. 2-(4-Chloro-phenyl)-1,3-di-morpholin-4-yl-
propenethione (5b)
5.5.2. 2-[4-(4-Chloro-phenyl)-5-morpholin-4-yl-thiophen-2-
yl]-3-phenyl-3H-quinazolin-4-one (9ab)
2-(4-Chloro-phenyl)-1-morpholin-4-yl-ethanethione 6b (5.0 g,
0.02 mol) was reacted with morpholine and triethylorthoformate
using the same procedure described for 5a to afford title com-
pound (5.40 g) as a yellow solid. Yield: 72%, mp 134–135 °C,
M.F.: C17H21N2O2SCl, LC–MS (m/z): 353, 354 (M+1, M+2).
The title compound was prepared from 2-(4-chloro-phenyl)-
1,3-di-morpholin-4-yl-propenethione 5b (1.1 g, 0.003 mol) and 2-
chloro methyl-3-(phenyl)-3H-quinazoline-4-one 2a (0.81 g,
0.003 mol) using the same procedure described for 9aa to afford ti-
tle compound as a yellow solid. Yield 51.0%, mp 229–230 °C, Rf:
0.52, IR (KBr, cmꢂ1); 3010, 1691, 1538, 1309, 1282, 758, Molcular
Formula C28H22N3O2SCl, LC–MS (m/z): 500 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.93–2.96 (m, 4H), 3.70–3.73 (m, 4H), 5.91 (s,
1H), 7.20–7.79 (m, 12H), 8.25–8.28 (m, 1H).
5.4.3. N-{4-[1-(Morpholine-4-carbothioyl)-2-morpholin-4-yl-
vinyl]-phenyl}-acetamide (5c)
N-[4-(2-Morpholin-4-yl-2-thioxo-ethyl)-phenyl]-acetamide 6c
(5.0 g, 0.02 mol) was reacted with morpholine and triethylortho-
formate using the same procedure described for 5a to afford title
compound (4.0 g) as a yellow solid. Yield: 59.26%, mp 145–
147 °C, Molecular Formula C19H25N3O3S, LC–MS (m/z): 376
(M+1), 1H NMR (300 MHz, DMSO-d6), d 2.02 (s, 3H), 3.19–3.38
(m, 8H), 4.19–4.50 (m, 8H), 4.67 (s, 1H), 6.36 (s, 1H), 7.20–7.34
(m, 2H), 7.70–7.80 (m, 2H).
5.5.3. N-(4-(2-Morpholino-5-(4-oxo-3-phenyl-3,4-
dihydroquinazolin-2-yl)thiophen-3-yl)phenyl)acetamide (9ac)
The title compound was prepared from N-{4-[1-(Morpholine-4-
carbothioyl)-2-morpholin-4-yl-vinyl]-phenyl}-acetamide 5c (1.0
gm, 0.003 mol) and 2-chloro methyl-3-(phenyl)-3H-quinazoline-
4-one 2a (0.70 g, 0.003 mol) using the same procedure described
for 9aa to afford title compound as a yellow solid. Yield 57.0%,
mp >275 °C, Rf: 0.34, IR (KBr, cmꢂ1): 3186, 3066, 1689, 1535,
1314, 1281, 758, Molcular Formula C30H26N4O3S, LC–MS (m/z):
523 (M+1), 1H NMR (300 MHz, CDCl3) d 2.18 (s, 3H), 2.94–2.97
(m, 4H), 3.70–3.73 (m, 4H), 5.94 (s, 1H), 7.22–7.78 (m, 13H),
8.27–8.3 (m, 1H).
5.4.4. 2-(4-Methanesulfonyl-phenyl)-1,3-di-morpholin-4-yl-
propenethione (5d)
2-(4-Methanesulfonyl-phenyl)-1-morpholin-4-yl-ethanethione
6d (5.0 g, 0.013 mol) was reacted with morpholine and triethylor-
thoformate using the same procedure described for 5a to afford
title compound (5.0 g) as
a yellow solid. Yield: 75.5%, mp
167–169 °C, Molecular Formula C18H24N2O4S2, LC–MS (m/z): 397

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R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
5.5.4. 2-[4-(4-Methanesulfonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-phenyl-3H-quinazolin-4-one (9ad)
5.5.9. 2-[4-(4-Methanesulfonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-o-tolyl-3H-quinazolin-4-one (9ai)
The title compound was prepared from 2-(4-methanesul-
fonyl-phenyl)-1, 3-di-morpholin-4-yl-propenethione 5d (1.0 g,
0.003 mol) and 2-chloro methyl-3-(phenyl)-3H-quinazoline-4-one
2a (0.68 g, 0.003 mol) using the same procedure described for 9aa
to afford title compound as a yellow solid. Yield 58.4%, mp
>275 °C, Rf: 0.53, IR (KBr, cmꢂ1): 3212, 3036, 1681, 1608, 1448,
1326, 1275, 770, Molcular Formula C29H25N3O4S2, LC–MS (m/z):
544 (M+1), 1H-NMR (300 MHz, CDCl3) d 2.94–2.98 (m, 4H), 3.12 (s,
3H), 3.78–3.80 (m, 4H), 5.91 (s, 1H), 7.23–7.89 (m, 12H), 8.31–8.33
(m, 1H).
The title compound was prepared from 2-(4-methanesulfo-
nyl-phenyl)-1,3-di-morpholin-4-yl-propenethione 5d (1.0 g,
0.003 mol) and 2-chloro methyl-3-(2-methyl-phenyl)-3H-quinazo-
line-4-one (2b) (0.74 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid. Yield
56.0%, mp 255–258 °C, Rf: 0.42, IR (KBr, cmꢂ1): 3218, 3047, 1692,
1610, 1451, 1320, 1279, 771, Molcular Formula C30H27N3O4S2,
LC–MS (m/z): 558 (M+1), 1H NMR (300 MHz, CDCl3) d 2.1–2.2
(s, 3H), 2.97–3.0 (m, 4H), 3.1 (s, 3H) , 3.75–3.78 (m, 4H), 5.93
(s, 1H), 7.28–7.88 (m, 11H), 8.29–8.32 (m, 1H).
5.5.5. 2-[4-(4-Methylsulfanyl-phenyl)-5-morpholin-4-yl-
thiphen-2-yl]-3-phenyl-3H-quinazolin-4-one (9ae)
5.5.10. 2-[4-(4-Methylsulfanyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-o-tolyl-3H-quinazolin-4-one (9aj)
The title compound was prepared from 3-(4-morpholino)-2-(4-
methylmercapto-phenyl)-thioacrylic acid morpholide 5e (0.98 g,
0.003 mol) and 2-chloro methyl-3-(phenyl)-3H-quinazoline-4-
one 2a (0.73 g, 0.003 mol) using the same procedure described
for 9aa to afford title compound as a yellow solid. Yield 46%, mp
218–220 °C, Rf: 0.53, IR (KBr, cmꢂ1): 3042, 1688, 1438, 1281, 762,
Molcular Formula C29H25N3O2S2, LC–MS (m/z): 526 (M+1), 1H
NMR (300 MHz, CDCl3) d 2.49 (s, 3H), 2.94–2.97 (m, 4H), 3.70–
3.73 (m, 4H), 5.89 (s, 1H), 7.07–7.78 (m, 12H), 8.27–8.3 (m, 1H).
The title compound was prepared from 3-(4-morpholino)-2-(4-
methylmercapto-phenyl)-thioacrylic acid morpholide 5e (1.0 g,
0.003 mol) and 2-chloro methyl-3-(2-methyl-phenyl)-3H-quinazo-
line-4-one 2b (0.78 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid.
Yield 56.0%, mp 255–258 °C, Rf: 0.42, IR (KBr, cmꢂ1): 3042,
1688, 1441, 1281, 761, Molcular Formula C30H27N3O4S2, LC–MS
(m/z): 526 (M+1), 1H NMR (300 MHz, CDCl3) d [ppm]: 2.12 (s,
3H), 2.32 (s ,3H), 2.95–2.98 (m, 4H), 3.71–3.74 (m, 4H), 5.91 (s,
1H), 7.08–7.76 (m, 11H), 8.26–8.29 (m, 1H).
5.5.6. 2-(5-Morpholin-4-yl-4-pyridin-4-yl-thiophen-2-yl)-3-
phenyl-3H-quinazolin-4-one (9af)
5.5.11. 2-(5-Morpholin-4-yl-4-pyridin-4-yl-thiophen-2-yl)-3-o-
tolyl-3H-quinazolin-4-one (9ak)
(Z)-1,3-Di-morpholin-4-yl-2-pyridin-4-yl-propenethione
5f(1.0 g, 0.003 mol) was taken in dimethylformamide and 2-chlo-
romethyl-3-phenyl-3H-quinazoline-4-one 2a (0.84 g, 0.003 mol)
in dimethylformamide was added into it at rt and reaction mass
was stirred on room temperature for 24 h and then was poured
into ice-cold water and stirred well for 10 min and then filtered
to remove water. The residue was dried and recrystallised with
methanol to get title compound as a yellow solid. Yield: 38.0%,
mp 230–231 °C, Rf: 0.34, IR (KBr, cmꢂ1): 3053, 2908, 1689,1543,
1307,1281,758, 710, Molcular Formula C27H22N4O2S2, LC–MS (m/
z): 467 (M+1), 1H NMR (300 MHz, CDCl3) d 2.94–2.97 (m, 4H),
3.72–3.75 (m, 4H), 5.92 (s, 1H), 7.21–7.80 (m, 10H), 8.26–8.28
(m, 1H), 8.42–8.48 (m, 2H).
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(2-methyl-phenyl)-3H-quinazoline-4-one 2b (1.0 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 39.0%, mp 208–210 °C, Rf:
0.38, IR (KBr, cmꢂ1): 3021, 2911, 1686,1538, 1305,1240,750, Mol-
cular Formula C28H24N4O2S, LC–MS (m/z): 481(M+1), 1H NMR
(300 MHz, CDCl3) d 2.14 (s,3H), 2.95–2.98 (m, 4H), 3.73–3.76 (m,
4H), 5.9 (s, 1H), 7.2–7.8 (m, 9H), 8.27–8.30 (dd, 1H, J = 8.7,
1.2 Hz), 8.30–8.44 (m, 2H).
5.5.12. 2-[4-(4-Chloro-phenyl)-5-morpholin-4-yl-thiophen-2-
yl]-3-m-tolyl-3H-quinazolin-4-one (9al)
5.5.7. 2-(5-Morpholin-4-yl-4-phenyl-thiophene-2-yl)-3-o-tolyl-
3H-quinazolin-4-one (9ag)
The title compound was prepared from 2-(4-chloro-phenyl)-
1,3-di-morpholin-4-yl-propenethione 5b (1.0 g, 0.003 mol) and 2-
chloro methyl-3-(3-methyl-phenyl)-3H-quinazoline-4-one (2c)
(0.78 g, 0.003 mol) using the same procedure described for 9aa to
afford title compound as a yellow solid. Yield 56.0%, mp 228–
230 °C, Rf: 0.57, IR (KBr, cmꢂ1): 3040, 1680, 1445, 1285, 759, Mol-
cular Formula C29H24N3O2SCl, LC–MS (m/z): 514.5 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.42 (s, 3H), 2.93–2.96 (m, 4H), 3.71–3.74 (m,
4H), 5.91 (s, 1H), 7.16–7.87 (m, 11H), 8.26–8.28 (d, 1H).
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-phenyl-propenethione 5a (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(2-methyl-phenyl)-3H-quinazoline-4-one 2b (0.9 g,
0.003 mol) using the same procedure described for 9aa to afford ti-
tle compound as a yellow solid. Yield 54.0%, mp 187–188 °C, Rf:
0.60, IR (KBr, cmꢂ1): 3045, 1687, 1445, 1282, 790, 750, Molecular
Formula C29H25N3O2S, LC–MS (m/z): 480 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.15 (s, 3H), 2.95–2.98 (m, 4H), 3.70–3.73 (m,
4H), 5.97 (s, 1H), 7.17–7.76 (m, 12H), 8.26–8.28 (d, 1H).
5.5.13. N-{4-[2-Morpholin-4-yl-5-(4-oxo-3-m-tolyl-3–4-
dihidro-quinazolin-2-yl)-thiophe-ne-3-yl]-phenyl}-acetamide
(9am)
5.5.8. 2-[4-(4-Chloro-phenyl)-5-morpholin-4-yl-thiophen-2-
yl]-3-o-tolyl-3H-quinazolin-4-one (9ah)
The title compound was prepared from N-{4-[1-(morpholine-4-
The title compound was prepared from 2-(4-chloro-phenyl)-
1,3-di-morpholin-4-yl-propenethione 5b (1.1 g, 0.003 mol) and 2-
chloro methyl-3-(2-methyl-phenyl)-3H-quinazoline-4-one 2b
(0.9 g, 0.003 mol) using the same procedure described for 9aa to
afford title compound as a yellow solid. Yield 53.0%, mp 228–
230 °C, Rf: 0.62, IR (KBr, cmꢂ1): 3044, 1670, 1440, 1275, 769, Mol-
cular Formula C29H24N3O2SCl, LC–MS (m/z): 514 (M+1), 1H NMR
(300 MHz, CDCl3) d [ppm] = 2.42 (s, 3H), 2.93–2.96 (m, 4H), 3.71–
3.74 (m, 4H), 5.91 (s, 1H), 7.16–7.87 (m, 11H), 8.26–8.28 (d, 1H).
carbothioyl)-2-morpholin-4-yl-vinyl]-phenyl}-acetamide
5c
(0.98 g, 0.003 mol) and 2-Chloro methyl-3-(3-methyl-phenyl)-3H-
quinazoline-4-one 2c (0.74 g, 0.003 mol) using the same procedure
described for 9aa to afford title compound as yellow solid. Yield
52%, mp >275 °C, Rf: 0.61, IR (KBr, cmꢂ1): 3216, 1682, 1455,
1275, 763, Molcular Formula C31H28N4O3S , LC–MS (m/z): 537.5
(M+1), 1H NMR (300 MHz, CDCl3) d 2.18 (s, 3H), 2.42 (s, 3H),
2.94–2.97 (m, 4H), 3.71–3.74 (m, 4H), 5.94 (s, 1H), 7.12–7.84 (m,
12H), 8.24–8.27 (d, 1H).

R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
2805
5.5.14. 2-[4-(4-Methanesulfonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-m-tolyl-3H-quinazolin-4-one (9an)
The title compound was prepared from 2-(4-methanesulfonyl-
5.5.19. N-{4-[2-Morpholin-4-yl-5-(4-oxo-3-p-tolyl-3-,4-dihidro-
quinazolin-2-yl-thiophene-3-yl]-phenyl}-acetamide (9as)
The title compound was prepared from N-{4-[1-(morpholine-4-
phenyl)-1,3-di-morpholin-4-yl-propenethione
5d
(1.0 g,
carbothioyl)-2-morpholin-4-yl-vinyl]-phenyl}-acetamide
5c
0.003 mol) and 2-chloro methyl-3-(3-methyl-phenyl)-3H-quinazo-
line-4-one 2c (0.71 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid. Yield
54.5%, mp 208–210 °C, Rf: 0.62, IR (KBr, cmꢂ1): 3066, 1687, 1456,
1302, 1280, 760, Molcular Formula C30H27N3O4S2, LC–MS (m/z):
558 (M+1), 1H NMR (300 MHz, CDCl3) d 2.18 (s, 3H), 2.93–2.99
(m, 4H), 3.71–3.75 (m, 4H), 5.99 (s, 1H), 7.10–7.74 (m, 12H),
8.26–8.28 (d, 1H).
(0.97 g, 0.003 mol) and 2-chloro methyl-3-(4-methyl-phenyl)-3H-
quinazoline-4-one 2d (0.73 g, 0.003 mol) using the same procedure
described for 9aa to afford title compound as a yellow solid.
Yield: 40.0%, mp >275 °C, Rf: 0.61, IR (KBr, cmꢂ1): 3265, 3052,
2895, 1685, 1600, 1456, 1275, 760, Molcular Formula C31H28N4O3S,
LC–MS (m/z): 537.52 (M+1), 1H NMR (300 MHz, CDCl3) d 2.18 (s,
3H), 2.48 (s, 3H), 2.94–2.97 (m, 4H), 3.71–3.74 (m, 4H), 5.89 (s,
1H), 7.16–7.78 (m, 12H), 8.25–8.27 (d, 1H).
5.5.15. 2-[4-(4-Methylsulfanyl-phenyl)-5-morpholin-4-yl-
thiphen-2-yl]-3-m-tolyl-3H-quinazolin-4-one (9ao)
5.5.20. 2-[4-(4-Methanesulfonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-p-tolyl-3H-quinazolin-4-one (9at)
The title compound was prepared from 3-(4-morpholino)-2-(4-
methylmercapto-phenyl)-thioacrylic acid morpholide 5e (1.1 g,
0.003 mol) and 2-chloro methyl-3-(3-methyl-phenyl)-3H-quinazo-
line-4-one 2c (0.88 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid. Yield
43%, mp 220–221 °C, Rf: 0.59, IR (KBr, cmꢂ1): 3010, 1685, 1439,
1276, 762, Molcular Formula C30H27N3O2S2, LC–MS (m/z): 526.5
(M+1), 1H NMR (300 MHz, CDCl3) d 2.33 (s, 3H), 2.43 (s, 3H),
2.95–2.98 (m, 4H), 3.71–3.74 (m, 4H), 5.97 (s, 1H), 7.08–7.78 (m,
12H), 8.25–8.27 (d, 1H).
The title compound was prepared from 2-(4-methanesulfonyl-
phenyl)-1, 3-di-morpholin-4-yl-propenethione 5d (1.0 g,
0.003 mol) and 2-chloro methyl-3-(4-methyl-phenyl)-3H-quinazo-
line-4-one 2d (0.71 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid. Yield
57.0%, mp 265–266 °C, Rf: 0.57, IR (KBr, cmꢂ1): 3066, 1680, 1610,
1438, 1309, 1281, 765, Molcular Formula C30H27N3O4S2, LC–MS
(m/z): 558 (M+1), 1H NMR (300 MHz, CDCl3) d 2.51 (s, 3H), 2.96–
2.99 (m, 4H), 3.08 (s, 3H) , 3.74–3.77 (m, 4H), 5.91 (s, 1H), 7.26–
7.86 (m, 11H), 8.27–8.29 (m, 1H).
5.5.16. 2-(5-Morpholin-4-yl-4-pyridin-4-yl-thiophen-2-yl)-3-
m-tolyl-3H-quinazolin-4-one (9ap)
5.5.21. 2-[4-(4-Methylsulfanyl-phenyl)-5-morpholin-4-yl-
thiphen-2-yl]-3-p-tolyl-3H-quinazolin-4-one (9au)
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(3-methyl-phenyl)-3H-quinazoline-4-one 2c (0.88 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 36%, mp 230–232 °C, Rf:
0.57, IR (KBr, cmꢂ1): 3047, 2929, 1691,1534, 1324,1252,710, Mol-
cular Formula C28H24N4O2S, LC–MS (m/z): 481 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.43 (s, 3H), 2.95–2.98 (m, 4H), 3.73–3.76 (m,
4H), 5.96 (s, 1H), 7.17–7.77 (m, 9H), 8.26–8.29 (dd, 1H, J = 8.1,
1.2 Hz), 8.43–8.48 (m, 2H).
The title compound was prepared from 3-(4-morpholino)-2-(4-
methylmercapto-phenyl)-thioacrylic acid morpholide 5e (1.0 g,
0.003 mol) and 2-chloro methyl-3-(4-methyl-phenyl)-3H-quinazo-
line-4-one 2d (0.71 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid. Yield
42.0%, mp 238–240 °C, Rf 0.59, IR (KBr, cmꢂ1): 3025, 2897, 1684,
1598, 1444, 1287, 790, Molcular Formula C30H27N3O2S2, LC–MS
(m/z): 527 (M+1), 1H NMR (300 MHz, CDCl3) d 2.13 (s, 3H), 2.30
(s, 3H), 2.93–2.987(m, 4H), 3.71–3.75 (m, 4H), 5.98 (s, 1H), 7.18–
7.76 (m, 11H), 8.26–8.28 (m, 1H).
5.5.17. 2-(5-Morpholin-4-yl-4-phenyl-thiophene-2-yl)-3-p-
tolyl-3H-quinazolin-4-one (9aq)
5.5.22. 2-(5-Morpholin-4-yl-4-pyridin-4-yl-thiophen-2-yl)-3-p-
tolyl-3H-quinazolin-4-one (9av)
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-phenyl-propenethione 5a (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(4-methyl-phenyl)-3H-quinazoline-4-one 2d (0.9 g,
0.003 mol) using the same procedure described for 9aa to afford ti-
tle compound as a yellow solid. Yield 54.0%, mp 207–209 °C, Rf:
0.63, IR (KBr, cmꢂ1): 3054, 1682, 1441, 1284, 770, Molcular For-
mula C29H25N3O2S, LC–MS (m/z): 481 (M+1), 1H NMR (300 MHz,
CDCl3) d 2.48 (s, 3H), 2.95–2.98 (m, 4H), 3.71–3.74 (m, 4H), 5.99
(s, 1H), 7.15–7.74 (m, 12H), 8.25–8.27 (d, 1H).
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(4-methyl-phenyl)-3H-quinazoline-4-one 2d (0.88 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 39.0%, mp 220–221 °C, Rf:
0.52, IR (KBr, cmꢂ1): 3011, 2897, 1679, 1540, 1321, 1270, 750, Mol-
cular Formula C28H24N4O2S, LC–MS (m/z): 481 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.25 (s, 3H), 2.95–2.97 (m, 4H), 3.73–3.76 (m,
4H), 5.91 (s, 1H), 7.25–7.76 (m, 9H), 8.26–8.28 (m, 1H), 8.44–
8.51 (m, 2H).
5.5.18. 2-[4-(4-Chloro-phenyl)-5-morpholin-4-yl-thiophen-2-
yl]-3-p-tolyl-3H-quinazolin-4-one (9ar)
5.5.23. 3-(2-Methoxy-phenyl)-2-(5-morpholin-4-yl-4-phenyl-
thiophen-2-yl)-3H-quinazolin-4-one (9aw)
The title compound was prepared from 2-(4-chloro-phenyl)-
1,3-di-morpholin-4-yl-propenethione 5b (1.1 g, 0.003 mol) and 2-
chloro methyl-3-(4-methyl-phenyl)-3H-quinazoline-4-one 2d
(0.9 g, 0.003 mol) using the same procedure described for 9aa to
afford title compound as a yellow solid. Yield 51.0%, mp 228–
230 °C, Rf: 0.63, IR (KBr, cmꢂ1): 3035, 2965, 1685, 1435, 1275,
786, Molcular Formula C29H24N3O2SCl, LC–MS (m/z): 514.5 (M+1),
1H NMR (300 MHz, CDCl3) d 2.14 (s, 3H), 2.93–2.96 (m, 4H),
3.71–3.75 (m, 4H), 5.98 (s, 1H), 7.17–7.76 (m, 11H), 8.26–8.28 (d,
1H).
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-phenyl-propenethione 5a (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(2-methoxy-phenyl)-3H-quinazoline-4-one 2e (0.96 g,
0.003 mol) using the same procedure described for 9aa to afford ti-
tle compound as a yellow solid. Yield 43.0%, mp 152–154 °C, Rf:
0.56, IR (KBr, cmꢂ1): 3016, 2952, 1680, 1453, 1278, 761, Molcular
Formula C29H25N3O3S, LC–MS (m/z): 496.5 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.93–2.94 (m, 4H), 3.70–3.72 (m, 7H), 6.21 (s,
1H), 7.04–7.77 (m, 12H), 8.23–8.28 (d, 1H).

2806
R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
5.5.24. 2-[4-(4-Methanesulphonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-(2-methoxy-phenyl)-3H-quinazolin-4-one
(9ax)
5.5.29. 3-(4-Chloro-phenyl)-2-(5-morpholin-4-yl-4-phenyl-
thiophen-2-yl)-3H-quinazolin-4-one (9bc)
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-phenyl-propenethione 5a (1.0 g, 0.003 mol) and 2-chloro
The title compound was prepared from 2-(4-methanesulfonyl-
phenyl)-1,3-di-morpholin-4-yl-propenethione 5d (1.0 g, 0.003 mol)
and 2-chloro methyl-3-(2-methoxy-phenyl)-3H-quinazoline-4-
one 2e (0.75 g, 0.003 mol) using the same procedure described
for 9aa to afford title compound as a yellow solid. Yield 55.0%,
mp 222–223 °C, Rf: 0.62, IR (KBr, cmꢂ1): 3056, 1682, 1611, 1434,
1314, 1278, 761, Molcular Formula C30H27N3O5S2, LC–MS (m/z):
574 (M+1), 1H NMR (300 MHz, CDCl3) d 2.89–2.97 (m, 4H), 3.2 (s,
3H) , 3.72–3.78 (m, 4H), 3.91 (s, 3H), 5.93 (s, 1H), 7.27–7.89 (m,
11H), 8.28–8.30 (m, 1H).
methyl-3-(4-chloro-phenyl)-3H-quinazoline-4-one
2j
(0.97 g,
0.003 mol) using the same procedure described for 9aa to afford
title compound as a yellow solid. Yield 54.0%, mp 223–224 °C,
Rf: 0.68, IR (KBr, cmꢂ1): 3054, 1682, 1600, 1441, 1285, 772, Molcu-
lar Formula C28H22ClN3O2S, LC–MS (m/z): 500 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.93–2.98 (m, 4H), 3.71–3.75 (m, 4H), 5.96
(s, 1H), 7.12–7.72 (m, 12H), 8.22–8.25 (d, 1H).
5.5.30. 3-(4-Chloro-phenyl)-2-[4-(4-chloro-phenyl)-5-
morpholin-4-yl-thiophen-2-yl]-3H-quinazolin-4-one (9bd)
The title compound was prepared from 2-(4-chloro-phenyl)-
1,3-di-morpholin-4-yl-propenethione 5b (1.1 g, 0.003 mol) and 2-
5.5.25. 3-(2-Methoxy-phenyl)-2-(5-morpholin-4-yl-4-pyridin-
4-yl-thiophen-2-yl)-3H-quinazolin-4-one (9ay)
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(2-methoxy-phenyl)-3H-quinazoline-4-one 2e (0.93 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 41.0%, mp 204–205 °C, Rf:
0.58, Molcular Formula C28H24N4O3S, LC–MS (m/z): 497 (M+1), 1H
NMR (300 MHz, CDCl3) d 2.94–2.98 (m, 4H), 3.71–3.77 (m, 4H),
3.92 (s, 3H), 5.91 (s, 1H), 7.26–7.79 (m, 9H), 8.27–8.29 (m, 1H),
8.42–8.48 (m, 2H).
chloro
methyl-3-(4-chloro-phenyl)-3H-quinazoline-4-one
2j
(0.97 g, 0.003 mol) using the same procedure described for 9aa to
afford title compound as a yellow solid. Yield 55.0%, mp 240–
243 °C, Rf: 0.68, IR (KBr, cmꢂ1): 3032, 2967, 1682, 1432, 1276,
790, Molcular Formula C28H21Cl2N3O2S, LC–MS (m/z): 514.5
(M+1), 1H NMR (300 MHz, CDCl3) d 2.91–2.95 (m, 4H), 3.70–3.74
(m, 4H), 5.99 (s, 1H), 7.21–7.77 (m, 11H), 8.29–8.31 (d, 1H).
5.5.31. N-(4-{5-[3-(4-Chloro-phenyl)-4-oxo-3,4-dihydro-
quinazolin-2-yl]-2-morpholin-4-yl-thiophene-3-yl}-acetamide
(9be)
5.5.26. 2-[4-(4-Methanesulphonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-(3-methoxy-phenyl)-3H-quinazolin-4-one
(9az)
The title compound was prepared from N-{4-[1-(morpholine-
The title compound was prepared from 2-(4-methanesulfonyl-
phenyl)-1,3-di-morpholin-4-yl-propenethione 5d (1.0 g, 0.003 mol)
2-chloro methyl-3-(3-methoxy-phenyl)-3H-quinazoline-4-one 2f
(0.75 g, 0.003 mol) using the same procedure described for 9aa to
afford title compound as a yellow solid. Yield 57.0%, mp 203–
205 °C, Rf: 0.62, IR (KBr, cmꢂ1): 3059, 1680, 1606, 1438, 1313,
1279, 763, Molcular Formula C30H27N3O5S2, LC–MS (m/z): 574
(M+1), 1H NMR (300 MHz, CDCl3) d 2.90–2.98 (m, 4H), 3.25 (s,
3H) , 3.74–3.79 (m, 4H), 3.93 (s, 3H), 5.91 (s, 1H), 7.26–7.90 (m,
11H), 8.27–8.31 (m, 1H).
4-carbothioyl)-2-morpholin-4-yl-vinyl]-phenyl}-acetamide
5c
(1.2 g, 0.003 mol) and 2-chloro methyl-3-(4-chloro-phenyl)-3H-
quinazoline-4-one 2j (0.97 g, 0.003 mol) using the same proce-
dure described for 9aa to afford title compound as a yellow solid.
Yield 54.0%, mp >275 °C, Rf: 0.29, IR (KBr, cmꢂ1): 3245, 3042,
1690, 1610, 1445, 1280, 761, Molcular Formula C30H25ClN4O3 S,
LC–MS (m/z): 557 (M+1),1H NMR (300 MHz, CDCl3) d 2.18 (s,
3H), 2.94–2.98 (m, 4H), 3.71–3.74 (m, 4H), 5.99 (s, 1H), 7.25–
8.26 (m, 13H).
5.5.32. 2-[4-(4-Methanesulphonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-(4-chloro-phenyl)-3H quinazolin-4-one (9bf)
The title compound was prepared from 2-(4-methanesulfonyl-
phenyl)-1,3-di-morpholin-4-yl-propenethione 5d (1.3 g, 0.003 mol)
and 2-chloro methyl-3-(4-chloro-phenyl)-3H-quinazoline-4-one
2j (0.97 g, 0.003 mol) using the same procedure described for 9aa
to afford title compound as a yellow solid. Yield 57.0%, mp
>275 °C, Rf: 0.51, , IR (KBr, cmꢂ1): 3021, 1684, 1435, 1311, 1281,
760, Molcular Formula C29H24N3O4SCl, LC–MS (m/z): 578 (M+1),
1H NMR (300 MHz, DMSO-d6), d 2.98–3.04 (m, 4H), 3.10 (s, 3H),
3.76–3.79 (m, 4H), 6.04 (s, 1H), 7.28–7.92 (m, 11H), 8.26–8.29
(dd,1H, J = 9.3, 1.2 Hz), Elemental Anal. Calcd: C, 60.25; H, 4.18;
N, 7.27. Found: C, 60.26; H, 4.09; N, 7.26.
5.5.27. 2-[4-(4-Methanesulphonyl-phenyl)-5-morpholin-4-yl-
thiophen-2-yl]-3-(4-methoxy-phenyl)-3H-quinazolin-4-one
(9ba)
The title compound was prepared from 2-(4-methanesulfonyl-
phenyl)-1,3-di-morpholin-4-yl-propenethione 5d (1.0 g, 0.003 mol)
and 2-chloro methyl-3-(4-methoxy-phenyl)-3H-quinazoline-4-
one 2g (0.75 g, 0.003 mol) using the same procedure described
for 9aa to afford title compound as a yellow solid. Yield: 56.0%,
mp 240–243 °C, Rf: 0.49, IR (KBr, cmꢂ1): 3053, 2935, 1680, 1452,
1320, 1275, 765, Molcular Formula C30H27N3O5S, LC–MS (m/z):
574 (M+1), 1H NMR (300 MHz, CDCl3) d 2.97–3.0 (m, 4H), 3.1 (s,
3H), 3.76–3.79 (m, 4H), 3.93 (s, 3H), 6.1(s, 1H), 7.1–7.8 (m, 11H),
8.27–8.30 (dd, 1H, J = 9.0, 1.0 Hz).
5.5.33. 3-(4-Chloro-phenyl)-2-(5-morpholin-4-yl-4-pyridin-4-
yl-thiophen-2-yl)-3H-quinazolin-4-one (9bg)
5.5.28. 3-(2-Chloro-phenyl)-2-(5-morpholin-4-yl-4-pyridin-4-
yl-thiophen-2-yl)-3H-quinazolin-4-one (9bb)
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(4-chloro-phenyl)-3H-quinazoline-4-one 2j (0.94 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 39.0%, mp 195–197 °C , Rf:
0.57, Molcular Formula C27H21ClN4O2S, LC–MS (m/z): 501 (M+1),
1H NMR (300 MHz, CDCl3) d 2.94–2.98 (m, 4H), 3.73–3.76 (m,
4H), 6.12 (s, 1H), 7.26–7.78 (m, 9H), 8.25–8.28 (m, 1H), 8.45–
8.46 (m, 2H).
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(2-chloro-phenyl)-3H-quinazoline-4-one 2h (0.94 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 39%, mp 179–180 °C, Rf:
0.58, Molecular Formula C27H21ClN4O2S, LC–MS (m/z): 501 (M+1),
1H NMR (300 MHz, CDCl3) d 2.92–2.99 (m, 4H), 3.71–3.75 (m,
4H), 6.11 (s, 1H), 7.27–7.80 (m, 9H), 8.24–8.29 (m, 1H), 8.43–
8.46 (m, 2H).

R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
2807
5.5.34. 3-(4-Acetyl-phenyl)-2-(5-morpholin-4-yl-4-phenyl-
thiophen-2-yl)-3H-quinazolin-4-one (9bh)
tion, the reduction in the paw volume compared to vehicle control
was measured plethysmometrically. All experimental protocols
were reviewed and accepted by the Institutional Animal House
Ethics Committee (IAEC) prior to the initiation of the experiment.
All the final compounds were tested simultaneously with the
vehicle control to evaluate their anti-inflammatory activities. The
percentage protection at 50 mg/kg dose was calculated according
to the following formula:
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-phenyl-propenethione 5a (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(p-acetyl phenyl)-3H-quinazoline-4-one 2k (1.0 g,
0.003 mol) using the same procedure described for 9aa to afford ti-
tle compound as a yellow solid. Yield 52.0%, mp >275 °C, Rf: 0.47, IR
(KBr, cmꢂ1): 3015, 2894, 1685, 1452, 1284, 761, Molcular Formula
C30H25N3O3S, LC–MS (m/z): 558 (M+1), 1H NMR (300 MHz, CDCl3) d
2.67 (s, 3H), 2.94–2.97(m, 4H), 3.70–3.73 (m, 4H), 5.98 (s, 1H),
7.17–8.17 (m, 12H), 8.2–8.3 (m, 1H).
% Protection ¼ ½ðControl ꢂ TestÞ=Controlꢃ ꢁ 100
6.2. In vitro cell line based assays for NF-jB and AP-1
5.5.35. 3-(4-Acetyl-phenyl)-2-[4-(4-chloro-phenyl)-5-
morpholin-4-yl-thiophen-2-yl]-3H-quinazolin-4-one (9bi)
The title compound was prepared from 2-(4-chloro-phenyl)-
1,3-di-morpholin-4-yl-propenethione 5b (1.1 g, 0.003 mol) and 2-
chloro methyl-3-(p-acetyl phenyl)-3H-quinazoline-4-one 2k
(0.96 g, 0.003 mol) using the same procedure described for 9aa to
afford title compound a yellow solid. Yield: 50.0%, mp 222–4 °C,
Rf: 0.45, M.F.:C30H24N3O3SCl, LC–MS (m/z): 542 (M+1), 1H NMR
(300 MHz, CDCl3) d 2.68 (s, 3H), 2.87–2.93 (m, 4H), 3.65–3.71 (m,
4H), 5.89 (s, 1H), 7.20–7.80 (m, 11H), 8.21–8.24 (m, 1H).
The human embryonic kidney cell line, (HEK293), from
Panomics, Inc, Freemont, CA, was used for the in vitro cell based
screening for inhibition of NF-
jB and AP-1 activity. These cells
stably expressed a plasmid containing a minimal promoter and
tandem copies of the NF-j
B transcriptional element (50-AGTT-
GAGGGGACTTTCCCAGGC-30) or the AP-1 transcriptional element
(TGACTAA), which regulates the expression of the luciferase re-
porter gene. Cells were plated at 5 ꢁ 104 cells /well in 96 well
plates at 37 °C in 5% CO2 for 24 h. Test compounds were dis-
solved in 0.1% DMSO, added at desired concentrations (10 lM–
1 nM) to replicate wells, and incubated at 37 °C in 5% CO2 for
an additional 24 h. For induction of transcription and to test
5.5.36. 3-(4-Acetyl-phenyl)-2-[4-(4-methanesulfonyl-phenyl)-
5-morpholin-4-yl-thiophen-2-yl]-3H-quinazolin-4-one (9bj)
The title compound was prepared from 2-(4-methanesulfonyl-
the biological response of the promoters, the HEK/NF-
was stimulated with TNF- (1 ng/ l) and the HEK/AP-1 cell line
was stimulated with PMA (1 ng/ l) and the cells were incubated
jB cell line
phenyl)-1,3-di-morpholin-4-yl-propenethione
5d
(1.0 g,
a
l
0.003 mol) and 2-chloro methyl-3-(p-acetyl phenyl)-3H-quinazo-
line-4-one 2k (0.80 g, 0.003 mol) using the same procedure de-
scribed for 9aa to afford title compound as a yellow solid. Yield
54.0%, mp 239–240 °C, Rf: 0.51, IR (KBr, cmꢂ1): 3024, 2913, 1680,
1440, 1308, 1276, 664, Molcular Formula C31H27N3O5S2, LC–MS
(m/z): 586 (M+1), 1H-NMR (300 MHz, CDCl3) d 2.71 (s, 3H), 2.95–
2.98 (m, 4H), 3.08 (m, 3H), 3.75–3.77 (m, 4H), 6.11 (s, 1H), 7.28–
8.2 (m, 11H), 8.27–8.30 (dd, 1H, J = 9.0, 1.2 Hz).
l
at 37 °C in 5% CO2 for 20–24 h. After incubation, cell lysis buffer
containing the luciferase substrate (Bright-Glo Luciferase Assay
System, Promega) was added to each well. Luminescence was
immediately measured using the Rosys Anthos Lucy II or Victor
III (Perkin Elmer) luminometer. The luminescence of each test
compound was reported as relative light units (RLU) by taking
the mean of the replicate wells and normalizing them to the
maximal control values. In separate assays, viability of the cells
incubated with similar concentrations of compound was analyzed
to confirm that changes in RLU were not due to cytotoxicity (data
not shown).
5.5.37. 3-(4-Acetyl-phenyl)-2-(5-morpholin-4-yl-4-pyridin-4-
yl-thiophen-2-yl)-3H-quinazolin-4-one (9bk)
The title compound was prepared from 1,3-di-morpholin-4-yl-
2-pyridin-4-yl-propenthione 5f (1.0 g, 0.003 mol) and 2-chloro
methyl-3-(p-acetyl phenyl)-3H-quinazoline-4-one 2k (0.97 g,
0.003 mol) using the same procedure described for 9af to afford ti-
tle compound as a yellow solid. Yield: 42.0%, mp 241–242 °C, Rf:
0.53, Molcular Formula C29H24N4O3S, LC–MS (m/z): 509 (M+1),
1H-NMR (300 MHz, CDCl3) d 2.69 (s, 3H), 2.92–2.95 (m, 4H),
2.71–2.75(m, 4H), 6.12 (s, 1H), 7.19–8.16 (m, 9H), 8.28–8.48 (m,
1H), 8.43–8.45 (m, 2H), Elemental Anal. Calcd: C, 68.49; H, 4.76;
N, 11.02. Found: C, 68.44; H, 4.64; N, 11.54.
6.3. In vivo regulation of transgenic model for NF-jB
Transgenic FVB mice carrying a transgene consisting of a mini-
mal promoter and tandem NF B binding sites in all cells were used
as in vivo ‘reporters’ of NF B activity. Three groups of 8–10 week
j
j
old mice (n = 3) were used including (1) untreated control, (2)
LPS treated, or (3) mice treated with a daily dose (50 mg/kg) of
9bk for 3 days before LPS stimulation. On the 4th day, mice in
6. Biology
groups 2 and 3 were treated with an intraperitoneal (60
lg/mouse)
dose of bacterial lipopolysaccharide (from E. coli 0111:B4) in 100
l
l
6.1. In vivo acute model of inflammation
sterile saline. The mice were transferred to the LSUHSC-S Small
Animal in vivo Imaging Core for bioluminescence imaging (IVIS
100; Xenogen, Inc.). Approximately 4 h after LPS inoculation, mice
were anesthetized with 150 mg/kg ketamine and 5 mg/kg xylazine
in 100 ll sterile saline and injected with 100 ll ip 15 mg/kg lucif-
erin in sterile saline, and imaged approximately 5–10 min later
The carrageenan induced rat paw edema test was used for the
determination of in vivo anti-inflammatory activity.32 Sprague-
Dawley rats (bred in the animal colony at the B. V. Patel PERD, Cen-
tre, Ahmedabad) of either sex, in the weight range of 150–250 g
were used for the study. Animals were fasted for 18 h prior to
the experiment. Each group designating a single compound was
tested against a control group receiving only the vehicle treatment
(n = 4/group). All compounds were administered orally (50 mg/kg
body weight) in 0.2% agar suspension prepared fresh prior to use.
One hour post dosing, 0.1 ml of 1% carrageenan solution in normal
saline (ripened for 7 days) was injected in the subplantar region of
the right hind paw of each rat. After (3 h) the carrageenan injec-
using the IVIS imager. Representative images are included showing
endogenous NF-
jB activity limited to the thymus in unstimulated
control mice, maximally stimulated NF-
j
B activity after LPS inoc-
ulation, and mice treated with 9bk. The percentage change in bio-
luminescence was determined by comparing the mean
luminescence minus the background activity in the thymus be-
tween maximal LPS stimulation and 9bk + LPS treatment
(p = 0.03).

2808
R. S. Giri et al. / Bioorg. Med. Chem. 18 (2010) 2796–2808
12. Tegeder, I.; Niederberger, E.; Israr, E.; Guhring, H.; Bruce, K.; Euchenhofer, C.;
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R.S.G. thanks to RSIC Mumbai and Chandigarh for recording
NMR and elemental analysis. Support from the Feist-Weiller Can-
cer Center and an IC grant provided by Industrial Commissionerate
of Gujarat for carrying out this work is acknowledged. We thank
Professor Harish Padh and Professor C. J. Shisoo, Directors, B.V. Pa-
tel PERD centre, for their support towards this project.
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