Design, synthesis and characterization of novel 2-(2,4-disubstituted-thiazole-5-yl)-3-aryl-3H-quinazoline-4-one derivatives as inhibitors of NF-κB and AP-1 mediated transcription activation and as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2008.10.031

A series of 2-(2,4-disubstituted-thiazole-5-yl)-3-aryl-3H-quinazoline-4-one derivatives were designed and synthesized. Synthesized molecules were further evaluated for their inhibitory activity towards transcription factors NF-κB and AP-1 mediated transcr

Full text of DOI:10.1016/j.ejmech.2008.10.031

European Journal of Medicinal Chemistry 44 (2009) 2184–2189
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and characterization of novel 2-(2,4-disubstituted-thiazole-5-
yl)-3-aryl-3H-quinazoline-4-one derivatives as inhibitors of NF- B and AP-1
mediated transcription activation and as potential anti-inflammatory agents
k
,
b
c
c
c
Rajan S. Giri a , Hardik M. Thaker , Tony Giordano , Jill Williams , Donna Rogers ,
*
Vasudevan Sudersanam b, Kamala K. Vasu b
a Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street, SE, MN 55445, USA
b B. V. Patel Pharmaceutical Education and Research Development Centre, Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad 380 054, Gujarat, India
c Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center at Shreveport, 1501 Kings Highway Shreveport, LA 71130, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-(2,4-disubstituted-thiazole-5-yl)-3-aryl-3H-quinazoline-4-one derivatives were designed
Received 24 September 2008
Received in revised form
28 October 2008
Accepted 29 October 2008
Available online 5 November 2008
and synthesized. Synthesized molecules were further evaluated for their inhibitory activity towards
transcription factors NF-
as well as for their anti-inflammatory activity in in vivo model of acute inflammation. This series provides
us with selective and dual inhibitors of NF- B and AP-1 mediated transcriptional activation which also
exhibit significant efficacy in in vivo model of inflammation. Two of the compounds 9m and 9o turned
out to be the most promising dual inhibitors of NF- B and AP-1 mediated transcriptional activation with
an IC50 of 3.3 M) and 9p (IC50 ¼ 5.5 M) emerged as selective inhibitors of
M for both. 9n (IC50 ¼ 5.5
NF- M) were found to be
M) and 9d (IC50 ¼ 5.5
kB and AP-1 mediated transcriptional activation in a cell line based in vitro assay
k
k
Keywords:
Thiazole
Inhibitors of NF-kB and AP-1 mediated
transcriptional activation
Anti-inflammatory agents and transcription
inhibitors
m
m
m
k
B mediated transcriptional activation and 9c (IC50 ¼ 5.5
m
m
more selective inhibitor of AP-1 mediated transcriptional activity. Though the relationship between the
activities shown by these compounds in in vivo and in vitro model is still to be established, these results
suggest the suitability of the designed molecular framework as a potential anti-inflammatory molecular
framework which also exhibits the inhibitory activity towards NF-kB and AP-1 mediated transcriptional
activation. This will be worth studying further to explore its complete potential particularly in chronic
inflammatory conditions. The structure activity relationship (SAR) of this series has been discussed
herein.
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
and activate or inhibit transcription of genes [2]. Transcription
factor nuclear factor-
key transcription factors that orchestrate expression of many genes
involved in inflammatory conditions. NF- B regulates the tran-
scription of genes including TNF- , interleukin-1 (IL-1), IL-2, IL-6,
adhesion molecules and chemokines, among others [3,4]. Similarly,
(AP-1) regulates the production of the cytokines TNF- , IL-1, IL-2,
granulocyte-macrophage colony stimulating factor (GM-CSF) and
matrix metalloproteases (MMP) [5]. Therefore, the selective inhi-
bition of transcription factors like NF-kB and AP-1 involved in
inflammatory diseases may thus control a plethora of mediators of
inflammation [6]. This may also overcome some of the redundancy
that is inherent to biological systems and possibly responsible for
the failure of single-targeted therapy [7,8]. It is also reported that
compounds which shows their anti-inflammatory effect through
kB (NF-kB) and the activating factor (AP-1) are
Inflammation remains a common and, all too often poorly
controlled clinical problem which can be life threatening in
extreme form of allergy, autoimmune diseases and rejection of
transplanted organs. The treatment options available for inflam-
matory diseases are unsatisfactory and complicated due to their
lack of efficacy and adverse effect profile. Due to the failure of single
targeted therapy, and in view of the polygenic nature of inflam-
matory diseases which involves a number of pathways acting in
series and in parallel it seemed appropriate to look for candidates
acting on more than one pathway involved in inflammatory
conditions [1].
k
a
a
Transcription factors are regulatory proteins which bind to
specific DNA sequences in the gene promoter or enhancer regions
inhibiting the activation NF-kB and AP-1 does not cause gastroin-
testinal mucosal damage and other side effects associated with
long term NSAIDs and glucocorticoid use [11]. Based on these
observations, it appears that inhibition of NF-kB and/or AP-1
* Corresponding author. Tel.: þ1 318 655 3063.
E-mail address: girix007@umn.edu (R.S. Giri).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.031

R.S. Giri et al. / European Journal of Medicinal Chemistry 44 (2009) 2184–2189
2185
transcriptional activation may represent an attractive target in the
development of novel anti-inflammatory agents [9,10].
SPC-839 (Fig. 1) reported by Palanki and his coworkers is one of
the very few compounds which demonstrate their anti-inflamma-
toryactivity by inhibiting the activation of NF-kB and AP-1 mediated
transcriptional activation [12–14]. Taking SPC-839 as a chemical
lead [15] and by using the concept of bioisosterism [16], we have
designed a novel scaffold (2-(2,4-disubstituted-thiazole-5-yl)-3-
aryl-3H-quinazolin-4-one (9, Fig.1)) for its evaluation as inhibitor of
H
N
N
R2
R1
b
HN
a
N
N
NH2
Cl
S
R2
C
N
1
3
R2
2
H
N
HN
N
NH2
c
R1
HCl
NH2
H2N
S
5
4
NF-
report the synthesis of various analogs of designed scaffold and their
evaluation as inhibitors of NF- B and AP-1 mediated transcriptional
kB and/or AP-1 mediated transcriptional activation. Herein, we
O
O
k
O
activation and as anti-inflammatory agents. Thus the structure
activity relationship (SAR) of this series has been discussed.
OH
OH
e
d
N
R3
NH
NH2
N
67
2. Chemistry
Cl
8
O
3, f
5, f
The synthesis of designed compounds is outlined in Scheme 1.
The thiourea derivatives (3) were prepared by treating different
amidines (2) with suitable isothiocyanates in toluene or hexane. 1-
amidino-3-substituted thiourea derivatives (5) were prepared by
treating guanidine hydrochloride (4) with suitable isothiocyanates
in aqueous basic media [17]. Amidines (2) were prepared from
commercially available acetonitrile or benzonitrile (1) [18]. The
synthesis of 2-chloromethyl-3-aryl-3H-quinazoline-4-one (8) was
furnished by using modified Niementowski synthesis, where we
began with chloro-acetylation of antranilic acid (6) to get corre-
sponding 2-(2-choroacetyl amino) benzoic acid (7). Acid (7) was
then treated with trichlorophosphate (PCl3) to convert into acid
chloride which was immediately treated with substituted aniline
insitu to substitute chloro group of acid chloride with anilines. This
was then refluxed in toluene to get cyclised product (8) [19–21]. In
the final step, we treated thiourea derivatives (3 and 5) in acetoni-
trile with 2-chloromethyl-3-aryl-3H-quinazolin-4-one derivatives
(8) in acetonitrile at 75–80 ꢀC for 2–4 h to furnished desired 2-
(2-alkylamino/arylamino-4-methyl/amino/phenyl-thiazole-5-yl)-3-
aryl-3H-quinazolin-4-one derivatives (9) [22,23]. The synthesized
compounds were characterized by their 1H NMR chemical shift,
LC-MS (m/e), and elemental analysis.
In this synthesis, functionalized thiourea derivatives 3 and 5
provide two ring carbon atoms and both the heteroatom of resul-
tant thiazole ring; the remaining carbon atom (C-5) was supplied
by the methylene group of 2-chlormethyl group at the second
position of quinazolinone ring.
The mechanism involves the S-alkylation of the –SH group of
amidine by –CH2– of chloromethyl at the second position of qui-
nazolinone ring followed by the intramolecular cyclization by the
nucleophilic attack of active methylene group. Presumably this
nucleophilic attack may take place due to initial tautomerization of
quinazolinone double bond to generate an enamine like interme-
diate; the quinazolinone nitrogen then serves as an electron source
O
R1= methyl, phenyl, p-chloro phenyl,
p-CH3 phenyl
4
N
3
R2= -CH3, Ph, -NH2
R3= -H, -Cl, -CH3,
R3
NH
2
1
N
-OCH3. -COCH3
S
5
1
9a-z
2
4
N
3
R2
R1
Scheme 1. Reactions and conditions: a) AlCl3, diethyl amine, 120–140 ꢀC, b) R1-NCS,
toluene or hexane 0–5 ꢀC, c) R1-NCS, NaOH, 0–5 ꢀC, d) Chloroacetylchloride (ClAcCl),
TEA, 0–5 ꢀC for 30 min then on RT of 3 h, e) Arylamine, PCl3, toluene, 110 ꢀC, 5–6 h, f)
Acetonitrile, 80 ꢀC 2–4 h.
for the nucleophilic attack. The driving force for the thiazole
formation is the elimination of the diethyl amine residue in case of
intermediate 3 (in case of intermediate 5 ammonia will be liber-
ated) and the formation of the stable five membered aromatic
heterocyclic ring. The mechanism is summarized in Scheme 2.
3. Results and discussion
All of the 26 compounds synthesized were evaluated for their
activities. A substantial number of compounds have been identified
exhibiting moderate to excellent inhibitory activity towards NF-
and/or AP-1 mediated transcriptional activation in a cell line based
in vitro assay at 10 M concentration. IC50 was determined for the
compounds showing more than 50% inhibition at 10 M (Table 2).
These compounds were also found to exhibit a significant protec-
tive activity in the carrageenan induced rat paw edema model (in
vivo, 50 mg/kg, p.o.) in comparison to the existing anti-inflamma-
tory drug ibuprofen (Table 1).
On the basis of the biological activity exhibited by the designed
series we can classify this set of compounds into three classes. (1)
kB
m
m
Compounds selectively efficacious as inhibitors of NF-kB mediated
transcriptional activation and also showing good in vivo activity
like, compounds 9l, 9n and 9p (Table 1). (2) Compounds selectively
efficacious as inhibitors of AP-1 mediated transcriptional activation
and also showing in vivo activity, like compounds 9c, 9d and 9g
(Table 1). (3) Compounds that are dual inhibitors of both NF-kB and
AP-1 mediated transcriptional activation and are also active in in
vivo model of acute inflammation like 9m and 9o (Table 1).
Our findings suggest that selectivity of the compound for these
transcriptional factors is primarily dependent on the combination
of substitutions at the 2nd position of the thiazole (R1), 4th position
of the thiazole (R2) and on the phenyl ring at the 3rd (R3) position of
the quinazolinone ring.
For selectivity towards inhibition of NF-kB mediated transcrip-
tional activation, both arylamino and methylamino have shown
comparable selectivity at the 2nd position of thiazole, while
Fig. 1. Chemical lead based approaches for the discovery of novel quinazolinone
derivatives as inhibitors of NF-kB and AP-1 mediated transcriptional activation.

2186
R.S. Giri et al. / European Journal of Medicinal Chemistry 44 (2009) 2184–2189
Cl
R
H
R
N
R1
R1
HN
R1
R1
S
N
S
N
1
SH
S
N
2
N
R
R2
N
N
C
3
R2
R2
N
R2
C
C
4
N
R2
N
N
N
R
H
3
N
S
R
O
N
R2
N
N
R1
R3
R=
N
H
R
S
5
9
Scheme 2. Plausible mechanism for the synthesis of designed compounds.
electron withdrawing groups like –Cl and –COCH3 are more
favorable on the para position of phenyl ring at the third position of
the quinazolinone ring.
For selectivity towards inhibition of AP-1 mediated transcrip-
tional activation, both arylamino and methylamino have shown
comparable selectivity at the 2nd position of thiazole. However,
electron-releasing groups like –CH3 on ortho position of the phenyl
ring at the third position of quinazolinone ring are more desirable.
Table 1
Evaluation of synthesized compounds for their inhibitory activity towards NF-
and AP-1 mediated transcriptional activation and as anti-inflammatory agents.
kB
O
N
R3
Dual inhibitory activity towards NF-kB and AP-1 mediated
S
N
NH
transcriptional activation was accomplished when R1 was methyl
and p-Cl phenyl in combination with p-Cl substitution at the phenyl
ring at the 3rd position of quinazolinone. Change in the position of
–Cl group from the para position (9o) to the meta position (9l) of
the phenyl ring at 3rd position of the quinazolinone ring decreases
the potency and increases the selectivity of the compounds towards
R1
N
R2
R3
Compounds
R1
R2
% Protectiona in
vivo (50 mg/kg)
% Inhibition in
vitro (10 M)
m
NF-kB
AP-1
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
9u
9v
9w
9x
Ph
p-Cl Ph
–CH3
Ph
p-Cl Ph
–CH3
p-Cl Ph
p-Cl Ph
p-Cl Ph
p-Cl Ph
Ph
p-Cl Ph
–CH3
Ph
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–NH2
–NH2
–Ph
–H
–H
12
4
25
0
16
0
inhibition of NF-kB mediated transcriptional activation.
These results also suggest that the presence of electron with-
drawing or releasing group on phenyl ring at 3rd position of qui-
nazolinone is very important for the biological activities discussed
above as absence of these groups at this position make the
2-CH3
2-CH3
2-CH3
3-CH3
3-CH3
4-CH3
2-OCH3
4-OCH3
2-Cl
2-Cl
4-Cl
4-Cl
4-Cl
4-COCH3
4-COCH3
4-COCH3
4-Cl
4-COCH3
–H
2-CH3
2-Cl
4-Cl
35
42
45
n.d
n.d
37
27
5
n.d
58
71
72
38
42
n.d
13
37
53
n.d
51
61
n.d
n.d
23
52
16
21
n.d
10
2
12
n.d
0
67
60
n.d
20
39
2
n.d
0
21
21
82
9
84
0
7
2
0
Table 2
6
IC50 value determined for compounds showing more than 50% inhibition in in vitro
assay.
60
93
79
96
71
15
0
0
12
0
27
24
0
4
0
O
p-Cl Ph
–CH3
–Ph
N
R3
S
N
p-Cl Ph
p-Cl Ph
p-Cl Ph
p-Cl Ph
p-Cl Ph
p-Cl Ph
p-CH3 Ph
–CH3
NH
R1
N
2
R2
27
32
0
–Ph
–Ph
–Ph
–Ph
–Ph
–
Compounds
R1
R2
R3
IC50 (in vitro)
NF-kB
AP-1
0
9c
9d
9l
9m
9n
9o
9p
–CH3
Ph
p-Cl Ph
–CH3
Ph
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
2-CH3
2-CH3
2-Cl
4-Cl
4-Cl
n.d
n.d
5.5
3.3
5.5
3.3
5.5
5.5
5.5
n.d
3.3
n.d
3.3
n.d
m
m
M
M
9y
9z
4-COCH3
4-COCH3
–
15
31
–
p-Cl Ph
–
mM
mM
mM
mM
mM
Ibuprofen
–
m
m
M
M
n.d ¼ Not determined.
a
Oral administration for all test compounds for in vivo anti-inflammatory activity,
p < 0.05, Student’s unpaired t-test versus controls. For the standard drug ibuprofen,
dose and % protection were 50 mg/kg, 52% respectively.
p-Cl Ph
–CH3
4-Cl
4-COCH3

R.S. Giri et al. / European Journal of Medicinal Chemistry 44 (2009) 2184–2189
2187
compounds only mildly active or virtually inactive in all the bio-
logical assays mentioned (9a, 9b and 9u, Table 1).
Replacement of –CH3 group at the 4th position of the thiazole
ring (9o) with an –NH2 (9s and 9t) group or phenyl group (9u–9z)
was found to be detrimental for the inhibitory activity towards NF-
5.1. General synthesis of 2-(2-alkylamino/arylamino-4-alkyl/
phenyl/amino-thiazole-5-yl)-3-aryl-3H-quinazolin-4-one
derivatives
The general procedure for synthesis of designed compounds (9)
is as follows. 0.01 mole of thiourea derivatives (3) or 1-amidino-3-
substituted thiourea derivatives (5) in 5 ml of acetonitrile
was added to a stirred solution of 0.01 mole of the respective
2-chloromethyl-3-aryl-3H-quinazoline-4-one (8) in 5 ml of aceto-
nitrile at 75–80 ꢀC. This reaction mixture was stirred at this
temperature for 2–4 h and then cooled to room temperature. The
compound that precipitated was filtered and recrystalized in
methanol to obtain solid crystals. In case the solid did not
separate from the reaction mixture, the completion of the
reaction was confirmed on TLC, the solvent was evaporated
completely and chilled water was added to it and triturated till the
solid separated.
k
B and AP-1 mediated transcriptional activation. However, in both
cases where an –NH2 group was introduced instead of a –CH3 group
at the 4th position of the thiazole ring, the compounds retained
their anti-inflammatory activity in in vivo system (9s and 9t, Table
1). In most cases in vivo and in vitro results exhibited by designed
series are reasonably comparable. A notable exception is the in vivo
result of 9o which shows less inhibition compared to the other hits
even though it has emerged as the most potent dual inhibitor of NF-
k
B and AP-1 mediated transcriptional activation. This suggests that
the designed compounds may also interfere with some other
pathways involved in the inflammatory cascade. The experiments
for concluding the actual molecular aspect of the anti-inflammatory
activity of the compounds are currently underway where we are
trying to see the relationship between the activities shown by these
compounds in in vivo and in vitro models.
5.2. Characterization of synthesized compounds
We have hereby generated a novel molecular framework,
2-(2,4-disubstituted-thiazole-5-yl)–3-aryl-3H-quinazoline-4-one
by utilizing the chemical lead based approach and concept of bio-
isosterism. Compounds based on this framework have found to be
5.2.1. 2-(4-Methyl-2-phenylamino-thiazol-5-yl)-3-phenyl-3H-
quinazolin-4-one (9a)
Yield: 72.36%, M.P.: 210–12 ꢀC, Rf: 0.51, M.F.: C24H18N4OS, LC-MS
active as an inhibitor of NF-
k
B and AP-1 mediated transcriptional
(m/e): 411 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.27 (s,
d
activation (in vitro) and, also shown anti-inflammatory activity in
acute model of inflammation (in vivo). These results may open up
new avenues in designing candidates acting on more than one rate-
limiting step along the inflammatory cascade.
3H), 7.21–7.90 (m, 13H), 8.11–8.14 (d, 1H, Jortho7.6 Hz), 10.32 (s, 1H),
Anal. Calcd. For. C24H18N4OS: C, 70.22; H, 4.42; N, 13.65, Found: C,
70.19; H, 4.51; N, 13.67.
5.2.2. 2-[2-(4-Cholro-phenylamino)-4-methyl-thiazol-5-yl]-3-
phenyl-3H-quinazoline-4-one (9b)
4. Conclusion
Yield: 39%, M.P.: 230–2 ꢀC, Rf: 0.32, M.F.: C24H17N4OSCl, LC-MS
(m/e): 445 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.25 (s,
d
In an attempt to discover novel inhibitors of NF-kB and AP-1
3H), 7.23–7.87 (m, 12H), 8.10–8.15 (d, 1H, Jortho7.2 Hz), 10.35 (s, 1H),
Anal. Calcd. For. C24H17N4OSCl: C, 64.79; H, 3.85; N, 12.59, Found: C,
64.81; H, 3.86; N, 12.61.
mediated transcriptional activation and potent anti-inflammatory
agents, a series of 2-(2,4-disubstituted-thiazole-5-yl)-3-aryl-3H-
quinazoline-4-one derivatives were designed, synthesized and
evaluated for its biological activity. This series eventually turned
out to be very promising as it provides us with selective and dual
5.2.3. 2-(4-Methyl-2-methylamino-thiazol-5-yl)-3-o-tolyl-3H-
quinazolin-4-one (9c)
inhibitors of NF-kB and AP-1 mediated transcriptional activation.
Yield: 30%, M.P.: 218–20 ꢀC, Rf: 0.63, M.F.: C20H18N4OS, LC-MS
All these inhibitors also demonstrated significant in vivo efficacy
compared to the known anti-inflammatory drug ibuprofen. Though
the relationship between the activities shown by these compounds
in in vivo and in vitro model is still to be established, these results
suggest the suitability of the designed molecular framework as
a potential anti-inflammatory lead. This will be worth studying
further to explore its complete potential particularly in chronic
inflammatory conditions. Additional characterization of these
compounds and related analogs will be reported in due course.
(m/e): 363 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.35 (s,
d
3H), 2.42 (s, 6H), 7.21–7.92 (m, 7H), 8.12–8.14 (d, 1H, Jortho7.4 Hz),
10.32 (s, 1H), Anal. Calcd. For. C20H18N4OS: C, 66.28; H, 5.01; N,
15.46, Found: C, 66.25; H, 4.98; N, 15.48.
5.2.4. 2-(4-Methyl-2-phenylamino-thiazol-5-yl)-3-o-tolyl-3H-
quinazolin-4-one (9d)
Yield: 49%, M.P.: 227–8 ꢀC, Rf: 0.64, M.F.: C25H20N4OS, LC-MS (m/
e): 425 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.18 (s,
3H), 2.28 (s, 3H), 6.98–7.76 (m, 12H), 8.13–8.15 (d, 1H, Jortho7.6 Hz),
10.21 (s, 1H), Anal. Calcd. For. C25H20N4OS: C, 70.73; H, 4.75; N,
13.20, Found: C, 70.69; H, 4.77; N, 13.27.
5. Experimental
Unless mentioned otherwise all the starting materials and
solvents were purchased from commercially available sources.
Melting points were determined on a Toshniwal melting point
apparatus using open glass capillary tubes and represent the
uncorrected values. Proton NMR spectra were measured on
a Bruker spectrometer using the specified solvents at RSIC Chan-
digarh. LC-MS analysis was done on a Perkin Elmer Applied Bio-
Sciences API-165 in the analytical laboratory of B. V. Patel PERD
Centre, Ahmedabad. Elemental analysis was carried out at RSIC, IIT
Powai on Perkin Elmer 2400 CHN elemental analyzer. All the
reactions were monitored using thin layer chromatography (TLC)
using glass plate coated with silica gel G or GF254. TLC plates were
developed in iodine and toluene:acetonitrile (7:3), taken as mobile
phase, unless mentioned otherwise.
5.2.5. 2-[2-(4-Chloro-phenylamino)-4-methyl-thiazol-5-yl]-3-o-
tolyl-3H-quinazolin-4-one (9e)
Yield: 54%, M.P.: >275 ꢀC, Rf: 0.63, M.F.: C25H19N4OSCl, LC-MS
(m/e): 459 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.19 (s,
d
3H), 2.26 (s, 3H), 7.08–7.75 (m, 11H), 8.11–8.12 (d, 1H, Jortho7.2 Hz),
10.27 (s, 1H), Anal. Calcd. For. C25H19N4OSCl: C, 65.42; H, 4.17; N,
12.21, Found: C, 65.39; H, 4.16; N, 12.22.
5.2.6. 2-(4-Methyl-2-methylamino-thiazol-5-yl)-3-m-tolyl-3H-
quinazolin-4-one (9f)
Yield: 30%, M.P.: >275 ꢀC, Rf: 0.49, M.F.: C20H18N4OS, LC-MS (m/
e): 363 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.18 (s,
6H), 2.28 (s, 3H), 6.98–7.76 (m, 7H), 8.13–8.15 (d, 1H, Jortho7.2 Hz),

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R.S. Giri et al. / European Journal of Medicinal Chemistry 44 (2009) 2184–2189
10.21 (s, 1H), Anal. Calcd. For. C20H18N4OS: C, 66.28; H, 5.01; N,
15.46, Found: C, 66.21; H, 4.93; N, 15.42.
5.2.15. 3-(4-Chloro-phenyl)-2-[2-(4-chloro-phenylamino)-4-
methyl-thiazol-5-yl]-3H-quinazolin-4-one (9o)
Yield: 35%, M.P.: 267–8 ꢀC, Rf: 0.48, M.F.: C24H16N4OSCl2, LC-MS
(m/e):480 (M þ 1), 1H NMR (300 MHz, CDCl3)
d
[ppm] ¼ 2.29 (s,
5.2.7. 2-[2-(4-Chloro-phenylamino)-4-methyl-thiazol-5-yl]-3-m-
tolyl-3H-quinazolin-4-one (9g)
3H), 7.29–7.9 (m, 11H), 8.12–8.15 (d, 1H, Jortho7.8 Hz), 10.33 (s, 1H),
Anal. Calcd. For. C24H16N4OSCl2: C, 60.13; H, 3.36; N, 11.69, Found: C,
60.26; H, 3.29; N 11.65.
Yield: 51%, M.P.: 236–8 ꢀC, Rf: 0.65, M.F.: C25H19N4OSCl, LC-MS
(m/e): 459 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.17 (s,
d
3H), 2.24 (s, 3H), 7.15–7.89 (m, 11H), 8.01–8.11 (d, 1H, Jortho 7.5 Hz),
10.5 (s, 1H), Anal. Calcd. For. C25H19N4OSCl: C, 65.42; H, 4.17; N,
12.21, Found: C, 65.36; H, 4.15; N, 12.19.
5.2.16. 3-(4-Acetyl-phenyl)-2-(4-methyl-2-methylamino-thiazol-5-
yl)-3H-quinazolin-4-one (9p)
Yield: 30%, M.P.: 245 ꢀC, Rf: 0.46, M.F.: C21H18N4O2S, LC-MS (m/
e): 391 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.38 (s,
5.2.8. 2-[2-(4-Chloro-phenylamino)-4-methyl-thiazol-5-yl]-3-p-
tolyl-3H-quinazolin-4-one (9h)
6H), 2.56 (s, 3H), 6.98–7.76 (m, 7H), 8.13–8.15 (d, 1H, Jortho8.0 Hz),
10.21 (s, 1H), Anal. Calcd. For. C21H18N4O2S: C, 64.60; H, 4.65; N,
14.35, Found: C, 64.61; H, 4.67; N, 14.32.
Yield: 55%, M.P.: >275 ꢀC, Rf: 0.61, M.F.: C25H19N4OSCl, LC-MS
(m/e): 459 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.3 (s,
3H), 2.47 (s, 3H), 7.21–7.86 (m, 11H), 8.11–8.14 (d, 1H, Jortho7.4 Hz),
10.30 (s, 1H), Anal. Calcd. For. C25H19N4OSCl: C, 65.42; H, 4.17; N,
12.21, Found: C, 65.40; H, 4.15; N, 12.17.
5.2.17. 3-(4-Acetyl-phenyl)-2-(4-methyl-2-phenylamino-thiazol-5-
yl)-3H-quinazolin-4-one (9q)
Yield: 48%, M.P.: 271–3 ꢀC, Rf: 0.51, M.F.: C26H20N4O2S, LC-MS
(m/e): 453.5 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.35
5.2.9. 2-[2-(4-Chloro-phenylamino)-4-methyl-thiazol-5-yl]-3-(2-
methoxy-phenyl)-3H-quinazolin-4-one (9i)
(s, 3H), 2.62 (s, 3H), 7.09–8.03 (m, 12H), 8.31–8.33 (dd, 1H, Jor-
tho8.6 Hz, Jmeta1.2), 9.87 (s, 1H), Anal. Calcd. For. C26H20N4O2S: C,
69.01; H, 4.45; N, 12.38, Found: C, 69.9; H, 4.48; N, 12.35.
Yield: 41%, M.P.: 225–6 ꢀC, Rf: 0.61, M.F.: C25H19N4O2SCl, LC-MS
(m/e): 475 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.34 (s,
d
3H), 3.75 (s, 3H) 6.95–7.88 (m, 11H), 8.11–8.14 (d, 1H, Jortho7.5 Hz),
10.33 (s, 1H), Anal. Calcd. For. C25H19N4O2SCl: C, 63.22; H, 4.03; N,
11.80, Found: C, 63.26; H, 4.10; N, 11.81.
5.2.18. 3-(4-Acetyl-phenyl)-2-[2-(4-chloro-phenylamino)-4-
methyl-thiazol-5-yl]-3H-quinazolin-4-one (9r)
Yield: 43%, M.P.: 209–10 ꢀC, Rf: 0.52, M.F.: C26H19N4O2SCl, LC-MS
(m/e): 488 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.32 (s,
d
5.2.10. 2-[2-(4-Chloro-phenylamino)-4-methyl-thiazol-5-yl]-3-(4-
methoxy-phenyl)-3H-quinazolin-4-one (9j)
3H), 2.61 (s, 3H), 7.27–8.0 (m, 11H), 8.13–8.16 (d, 1H, Jortho7.5 Hz),
10.35 (s, 1H), Anal. Calcd. For. C26H19N4O2SCl: C, 64.13; H, 3.93; N,
11.51, Found: C, 64.11; H, 3.92; N, 11.53.
Yield: 38%, M.P.: >275 ꢀC, Rf: 0.7, M.F.: C25H19N4O2SCl, LC-MS
(m/e): 475 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.28 (s,
d
3H), 3.71 (s, 3H), 6.92–7.92 (m, 11H), 8.11–8.14 (d, 1H, Jortho7.4 Hz),
10.33 (s, 1H), Anal. Calcd. For. C25H19N4O2SCl: C, 63.22; H, 4.03; N,
11.80, Found: C, 63.25; H, 4.07; N, 11.78.
5.2.19. 2-[4-Amino-2-(4-chloro-phenylamino)-thiazol-5-yl]-3-(4-
chloro-phenyl)-3H-quinazolin-4-one (9s)
Yield: 34%, M.P.: 240–2 ꢀC,Rf:0.44, M.F.:C23H15N5OSCl2, LC-MS (m/
e): 481 (M þ 1),1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 7.23–7.76 (m,
d
5.2.11. 3-(2-Chloro-phenyl)-2-(4-methyl-2-phenylamino-thiazol-5-
yl)-3H-quinazolin-4-one (9k)
9H), 7.88–8.48(m, 5H),10.35 (s,1H). Anal. Calcd. For. C23H15N5OSCl2:C,
57.51; H, 3.15; N, 14.58, Found: C, 57.57; H, 3.1; N, 14.3.
Yield: 44%, M.P.: 228–30 ꢀC, Rf: 0.4, M.F.: C24H17N4O2SCl, LC-MS
(m/e): 446 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.48 (s,
d
5.2.20. 3-(4-Acetyl-phenyl)-2-[4-amino-2-(4-chloro-
phenylamino)-thiazol-5-yl]-3H-quinazolin-4-one (9t)
Yield: 32%, M.P.: 261–2 ꢀC, Rf: 0.56, M.F.: C25H18N5O2SCl, LC-MS
3H), 6.90–7.93 (m, 12H), 8.14–8.17 (d, 1H, Jortho 7.5 Hz), 10.2 (s, 1H),
Anal. Calcd. For. C24H17N4O2SCl: C, 64.79; H, 3.85; N, 12.59, Found:
C, 64.77; H, 3.84; N, 12.61.
(m/e): 488 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.67 (s,
d
3H), 7.21–7.70 (m, 9H), 7.91–8.44 (m, 5H), 10.32 (s, 1H), Anal. Calcd.
For. C25H18N5O2SCl: C, 61.54; H, 3.72; N, 14.35, Found: C, 61.52; H,
3.69; N, 14.37.
5.2.12. 3-(2-Chloro-phenyl)-2-[2-(4-chloro-phenylamino)-4-
methyl-thiazol-5-yl]-3H-quinazolin-4-one (9l)
Yield: 41%, M.P.: 237–8 ꢀC, Rf: 0.51, M.F.: C24H16N4OSCl, LC-MS
(m/e): 480.3 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.26
5.2.21. 2-[2-(4-Chloro-phenylamino)-4-phenyl-thiazol-5-yl]-3-
phenyl-3H-quinazolin-4-one (9u)
(s, 3H), 7.23–7.87 (m, 11H), 8.10–8.15 (d, 1H, Jortho7.2 Hz), 10.35 (s,
1H), Anal. Calcd. For. C24H16N4OSCl: C, 60.13; H, 3.36; N, 11.69,
Found: C, 60.11; H, 3.35; N, 11.71.
Yield: 71%, M.P.: >275 ꢀC, Rf: 0.68, M.F.: C29H19N4OSCl, LC-MS
(m/e): 507 (M þ 1), 1H NMR (400 MHz, CDCl3)
d
[ppm] ¼ 6.59–7.84
(m, 17H), 8.33–8.34 (dd, 1H, Jortho8.5 Hz, Jmeta1.1 Hz), 9.38 (s, 1H),
Anal. Calcd. For. C29H19N4OSCl: C, 68.70; H, 3.78; N, 11.05, Found: C,
68.65; H, 3.68; N, 11.14.
5.2.13. 3-(4-Chloro-phenyl)-2-(4-methyl-2-methylamino-thiazol-
5-yl)-3H-quinazolin-4-one (9m)
Yield: 41%, M.P.: 195–7 ꢀC, Rf: 0.61, M.F.: C19H15N4OSCl, LC-MS
(m/e): 384 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.33 (s,
d
5.2.22. 2-[2-(4-Chloro-phenylamino)-4-phenyl-thiazol-5-yl]-3-o-
tolyl-3H-quinazolin-4-one (9v)
3H), 2.82 (s, 3H), 7.17–7.82 (m, 7H), 8.28–8.30 (m, 1H), 9.37 (s, 1H),
Anal. Calcd. For. C19H15N4OSCl: C, 59.60; H, 3.95; N, 14.63, Found: C,
59.61; H, 3.97; N, 14.65.
Yield: 57%, M.P.: >275 ꢀC, Rf: 0.70, M.F.: C30H21N4OSCl, LC-MS
(m/e): 521 (M þ 1), 1H NMR (400 MHz, CDCl3)
[ppm] ¼ 2.1 (s, 3H),
d
6.92–7.87 (m,16H), 8.30–8.32 (dd,1H, Jortho7.8 Hz, Jmeta1.12 Hz), 9.65
(s, 1H), Anal. Calcd. For. C30H21N4OSCl: C, 69.16; H, 4.06; N, 10.75,
Found: C, 69.11; H, 3.98; N, 10.69.
5.2.14. 3-(4-Chloro-phenyl)-2-(4-methyl-2-phenylamino-thiazol-
5-yl)-3H-quinazolin-4-one (9n)
Yield: 51%, M.P.: 277–80 ꢀC, Rf: 0.84, M.F.: C24H17N4O2SCl, LC-MS
(m/e): 446 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
d
[ppm] ¼ 2.46 (s,
5.2.23. 3-(2-Chloro-phenyl)-2-[2-(4-chloro-phenylamino)-4-
phenyl-thiazol-5-yl]-3H-quinazolin-4-one (9w)
3H), 6.94–7.85 (m, 12H), 8.23–8.25 (dd, 1H, Jortho8.4 Hz, Jmeta1.0),
9.91 (s, 1H), Anal. Calcd. For. C24H17N4O2SCl: C, 64.79; H, 3.85; N,
12.59, Found: C, 64.75; H, 3.86; N, 12.57.
Yield: 51%, M.P.: 242–3 ꢀC, Rf: 0.77, M.F.: C29H18N4OSCl2, LC-MS
(m/e): 542 (M þ 1), 1H NMR (400 MHz, CDCl3)
d
[ppm] ¼ 7.41–8.21

R.S. Giri et al. / European Journal of Medicinal Chemistry 44 (2009) 2184–2189
2189
(m, 16H), 8.22–8.23 (m, 1H), 10.62 (s, 1H), Anal. Calcd. For.
C29H18N4OSCl2: C, 64.33; H, 3.35; N, 10.35, Found: C, 64.29; H, 3.38;
N, 10.26.
screening for NF-
either plasmids containing a minimal promoter and tandem copies
of the NF- B transcriptional element (5’-AGTTGAGGGGACTTTCC-
kB and AP-1 activity. These cells stably expressed
k
CAGGC-3’) or the AP-1 transcriptional element (TGACTAA) regu-
lating luciferase expression. Cells were plated at 5 ꢂ104 cells/well
in 96 well plates at 37 ꢀC in 5% CO2 for 24 h. Test compounds were
dissolved in 0.1% DMSO, added at desired concentrations (10 mM to
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 the bio-
5.2.24. 3-(4-Chloro-phenyl)-2-(4-phenyl-2-p-tolylamino-thiazol-
5-yl)-3H-quinazolin-4-one (9x)
Yield: 27%, M.P.: 225–7 ꢀC, Rf: 0.61, M.F.: C30H21N4OSCl, LC-MS
(m/e): 521 (M þ 1), 1H NMR (300 MHz, CDCl3)
d
[ppm] ¼ 2.3 (s, 3H),
6.46–6.48 (dd, 2H, Jortho7.2 Hz, Jmeta2.04 Hz), 7.0–7.29 (m, 11H),
7.51–7.55 (m, 1H), 7.77–7.82 (m, 2H), 8.28–8.30 (dd, 1H, Jortho8.9 Hz,
Jmeta1.0 Hz), 8.62 (s, 1H), Anal. Calcd. For. C30H21N4OSCl: C, 69.16; H,
4.06; N, 10.75, Found: C, 69.21; H, 3.99; N, 10.78.
logical activity of the promoters, the HEK/NF-
stimulated with TNF- (1 ng/ l) and the HEK/AP-1 cell line was
stimulated with PMA (1 ng/ l) and the cells were then incubated
kB cell line was
a
m
m
again at 37 ꢀC in 5% CO2 for 20–24 h. After incubation, the cell lysis
buffer containing luciferase substrate (Bright-Glo Luciferase Assay
System, Promega) was added to each well. Luminescence was
immediately measured using 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 that were not due to cytotoxicity (data not
shown).
5.2.25. 3-(4-Acetyl-phenyl)-2-(2-methylamino-4-phenyl-thiazol-5-
yl)-3H-quinazolin-4-one (9y)
Yield: 44%, M.P.: 239–40 ꢀC, Rf: 0.53, M.F.: C26H20N4O2S, LC-MS
(m/e): 453 (M þ 1), 1H NMR (300 MHz, CDCl3)
d
[ppm] ¼ 2.55 (s,
3H), 2.83 (s, 3H), 6.64–7.85 (m, 12H), 8.28–8.31 (dd, 1H, Jortho8.8 Hz,
Jmeta1.0), 9.38 (s, 1H), Anal. Calcd. For. C26H20N4O2S: C, 69.01; H,
4.45; N, 12.38, Found: C, 68.92; H, 4.41; N, 12.29.
5.2.26. 3-(4-Acetyl-phenyl)-2-[2-(4-chloro-phenylamino)-4-phenyl-
thiazol-5-yl]-3H-quinazolin-4-one (9z)
Yield: 44 %, M.P.: 218–20 ꢀC, Rf: 0.49, M.F.: C31H21N4O2SCl, LC-MS
(m/e): 549 (M þ 1), 1H NMR (300 MHz, DMSO-d6)
[ppm] ¼ 2.64 (s,
d
Acknowledgement
3H), 6.93–7.64 (m,16H), 8.2–8.3 (d,1H, Jortho7.4 Hz),10.54 (1H), Anal.
Calcd. For. C31H21N4O2SCl: C, 67.81; H, 3.86; N, 10.20; Found: C,
67.88; H, 3.82; N, 10.15.
The authors are thankful to the Industrial Commissionerate of
Gujarat for providing grant for carrying out this work.
5.3. Biology
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5.3.2. In vitro cell line based assays for NF-
kB and AP-1
The human embryonic kidney cell line, (HEK293), from Pano-
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