The design and synthesis of novel orally active inhibitors of AP-1 and NF-κB mediated transcriptional activation. SAR of in vitro and in vivo studies

Article abstract of DOI:10.1016/j.bmcl.2003.08.047

We have developed novel orally active quinazoline analogues as inhibitors of AP-1 and NF-κB mediated transcriptional activation. Among the derivatives prepared, 1-[2-(2-thienyl)quinazolin-4-ylamino]-3-methyl-3- pyrroline-2,5-dione (10) showed significant activity in an adjuvant-induced arthritis rat model by reducing the swelling by 65% in the non-injected foot. The synthesis, structure-activity relationship, and in vivo activity are described.

Full text of DOI:10.1016/j.bmcl.2003.08.047

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4077–4080
The Design and Synthesis of Novel Orally Active Inhibitors of
AP-1 and NF-ꢀB Mediated Transcriptional Activation.
SAR of In Vitro and In Vivo Studies
Moorthy S. S. Palanki,a,* Paul E. Erdman,a Minghuan Ren,a Mark Suto,a
Brydon L. Bennett,a Anthony Manning,a Lynn Ransone,a Cheryl Spooner,a Sonal Desai,a
Arnie Ow,a Ryuichi Totsuka,b Peter Tsaob and Wataru Toriumib
aCelgene, 4550 Towne Centre Court, San Diego, CA 92121, USA
bTanabe Seiyaku Co. Ltd., 16-89 Kashima, 3-Chome, Yodogawa-ku, Osaka 532-8505, Japan
Received 17 March 2003; accepted 18 August 2003
Abstract—We have developed novel orally active quinazoline analogues as inhibitors of AP-1 and NF-kB mediated transcriptional
activation. Among the derivatives prepared, 1-[2-(2-thienyl)quinazolin-4-ylamino]-3-methyl-3-pyrroline-2,5-dione (10) showed sig-
nificant activity in an adjuvant-induced arthritis rat model by reducing the swelling by 65% in the non-injected foot. The synthesis,
structure–activity relationship, and in vivo activity are described.
# 2003 Elsevier Ltd. All rights reserved.
There is abundant evidence that T-lymphocytes (T-cells)
orchestrate both the initiation and propagation of
immune responses through the secretion of protein
mediators termed cytokines and chemokines.1 These
cytokines and chemokines play very important roles in a
number of inflammatory diseases.2 Transcription fac-
tors are regulators of inducible gene expression.3 In
activated T cells, transcription factors such as the acti-
vator protein-1 (AP-1) regulate interleukin-2 (IL-2),
IL-3, and granulocyte-macrophage colony stimulating
factor (GM-CSF), while the nuclear factor-kB (NF-kB),
is essential for the transcriptional regulation of the
proinflammatory cytokines IL-1, IL-6, IL-8 and tumor
necrosis factor-a (TNFa).4,5 Based on these observa-
tions, it appears that inhibition of AP-1 and/or NF-kB
transcriptional activation in T cells may represent an
attractive target in the development of novel antiin-
flammatory drugs. Our goal was to develop inhibitors of
both AP-1 and NF-kB mediated transcriptional activa-
tion as novel therapeutic agents for treating inflammatory
mediated conditions.
no oral activity in rat PK studies and poor Caco-2 per-
meability. We reasoned that the carboxylate group in 1
was responsible for the lack of oral activity. Several
analogues with carboxylate bioisosteres on the 5-posi-
tion of pyrimidine ring of 1 resulted in the loss of activ-
ity.6 In order to develop potent inhibitors without a
carboxylate ester function, a newclass of inhibitors, 2,
was designed by introducing a fused phenyl ring on the
pyrimidine ring and placing a methoxy group at the
5-position (Fig. 1) in 2. The synthesis, structure activity
relationship, and in vivo activity in an animal model are
discussed below.
The synthesis of the quinazoline compounds (Scheme 1)
started from the appropriately substituted 2-aminophe-
nyl carboxylic acids (3).8 The treatment of the above
acids with an acid chloride followed by acetic anhydride
resulted in 4.9 The lactone 4 was converted to a quin-
azoline 5 by heating with ammonium acetate at 220 ꢀC
in a steel bomb.10 The hydroxy group at the 4-position
in 5 was converted to a chloro group by treating with
phosphorus oxychloride at 110 ꢀC.7 The chlorine at the
4-position was replaced with hydrazine.7 The hydrazine
analogue was heated at reflux with citraconic anhydride
to give 6 in good yield.7 All these compounds were
evaluated in Jurkat T-cells stably transfected with either
AP-1 binding site or a NF-kB binding site promoter–
reporter sequence.11
Earlier we reported the identification of a novel com-
pound, 1, that inhibited both AP-1 and NF-kB mediated
transcriptional activation.6,7 However, compound 1 had
*Corresponding author. Corresponding author. Tel.: +1-858-558-
7500; fax: +1-858-795-4719; e-mail: mpalanki@celgene.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.047

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M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4077–4080
We have reported pyrimidine based analogues as inhib-
itors of AP-1 and NF-kB mediated transcriptional acti-
vation.6,7 Based on our earlier studies, we selected three
substituents to study at the 2-position of the quinazoline
ring. We examined the effect of a 2-(20-thienyl) (7), a
phenyl (8) and a trifluoromethyl (9) substituion at the 2-
position of the quinazoline ring (Table 1). Among the
three groups examined, the compound with the 2-(20-
thienyl) group (7) was the most potent. We selected the
2-(20-thienyl) and 2-trifluoromethyl substituted analo-
gues for further lead optimization.
N-morpholine group (19) at the 7-position resulted in a
10-fold drop in potency compared to the 7-methoxy
analogue (12). However, a compound with an N,N-
dimethylamino group at the 7-position (20) had com-
parable potency to the 7-methoxy analogue (12) and was
5-fold less potent than 10. The hydrochloride salts of
both the morpholino compound (19) and the dimethyla-
mino analogue (20) had better water solubility than 10.
A similar trend was observed with the trifluoromethyl
series (Table 3). A 5-methoxy analogue (21) and a
6-methoxy analogue (22) were 10-fold more potent than
the unsubstituted analogue (9). However both 21 and 22
were approximately 10-fold less active than 10 and 11,
respectively. The substitution of an alkyl group at the
5-position resulted in a 40-fold drop in potency. How-
ever, the substitution of SMe at the 6-position resulted
only in a 3-fold drop in potency. The introduction of
methylsulfoxide (25) and methylsulfone (26) at the
6-position resulted in a 30-fold and a 300-fold drop in
potency, respectively. An hydroxy group at the 6-posi-
tion (27) in the place of OMe (22) resulted in very small
A series of 2-thienyl compounds was synthesized as
shown in Scheme 1. The initial focus was on the intro-
duction of a methoxy group on the phenyl position of
the quinazoline molecule (Table 2). Compounds with a
methoxy group at the 5-position (10) and a methoxy at
the 6-position (11) had comparable potency. However, a
methoxy group at the 7-position (12) or the 8-position
(13) resulted in the loss of 2- to 4-fold in potency. The
introduction of two (14) and three methoxy (15) groups
resulted in compounds that were 10-fold less potent
than 10. The introduction of electron withdrawing
atoms such as fluorine at the 5-position (16) or chlorine
at the 6-position (17) resulted in a 4- to 5-fold loss in
potency, compared to 10. The introduction of an
Table 1. Inhibition of AP-1 and NF-kB mediated transcriptional
activation in Jurkat T-cells
No
R2
IC50, mM
7
8
9
(20Thienyl)
Phenyl
CF3
0.06
0.1
0.1
Table 2. Inhibition of AP-1 and NF-kB mediated transcriptional
activation in Jurkat T-cells
Figure 1.
No.
R0
IC50, mM
10
11
12
13
14
15
16
17
18
19
20
5-OMe
6-OMe
7-OMe
0.008
0.003
0.02
0.05
0.05
0.04
0.05
0.02
0.02
0.2
8-OMe
6,7-di-OMe
6,7,8-tri-OMe
5-F
6-Cl
5-Me
7-(N-morphinoyl)
7-(NMe2)
Scheme 1. (a) R2COCl, THF, rt, 2 h; (b) (CH3CO)2O, reflux, 1 h, 82–
99% from 3; (c) CH3COONH4, 220 ꢀC, steel bomb, 7 h, 81–97%;
(d) POCl3 (10 equiv), reflux, 3 h, 84–94%; (e) hydrazine (2.2 equiv),
THF, rt, 1 h; (f) citraconic anhydride (1.1 equiv), chloroform, Dean-
Stark receiver, reflux, 12 h, 82–98% combined yield for steps e and f.
0.04

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4077–4080
4079
Table 3. Inhibition of AP-1 and NF-kB mediated transcriptional
activation in Jurkat T-cells
animals treated with compound 10 (Fig. 2). However,
the rest of the compounds (9, 11, 20) caused modest
reduction in swelling. In conclusion, we have developed
an inhibitor of AP-1 and NF-kB mediated transcrip-
tional activation that showed efficacy in an animal dis-
ease model. This compound (10) showed significant
activity in the adjuvant-induced arthritis rat model by
reducing the swelling by 65% in the non-injected foot.
Further evaluation of 1014 is in progress.
References and Notes
No.
R0
IC50, mM
21
22
23
24
25
26
27
28
29
5-OMe
6-OMe
5-Me
0.01
0.03
0.4
0.08
1
1. Palanki, M. S. S.; Manning, A. M. Exp. Opin. Ther.
Patents 1999, 9, 27.
2. Lewis A. J. Emerging Drugs: The prospect for improved
medicines; Annual Executive Briefing, Ashley Publications
Ltd., 1996, 31.
3. Vossen, A. C. T. M.; Savelkoul, H. F. J. Mediat. Inflamm.
1994, 3, 403.
4. Manning, A. M.; Lewis, A. J. Rheumatoid Arthritis 1997, 1,
65.
6-SMe
6-SOMe
6-SO2Me
6-OH
7-(1-Piperidyl)
7-(NMe2)
9
0.1
0.2
0.02
5. Palanki, M. S. S. Curr. Med. Chem. 2002, 9, 219.
6. Palanki, M. S. S.; Erdman, P. E.; Manning, A. M.; Ow, A.;
Ransone, L. J.; Spooner, C.; Suto, C.; Suto, M. Bioorg. Med.
Chem. Lett. 2000, 10, 1645.
Table 4. Caco-2 permeability coefficient values
No.
Pc, cm/s
7. Palanki, M. S. S.; Gayo, L. M.; Shevlin, G. I.; Erdman, P.;
Suto, M.; Goldman, M.; Ransone, L. J.; Spooner, C. Bioorg.
Med. Chem. Lett. 2002, 12, 2573.
8. Palanki, M. S. S.; Suto, M. WO 9901441.
9. Afify, A. A.; El-Nagdy, S.; Sayed, M. A.; Mohey, I. Indian
J. Chem. 1988, 27B, 920.
Caffeine
4.4ꢂ0.1ꢁ10ꢃ5
1.41ꢂ0.4ꢁ10ꢃ5
1.62ꢂ0.2ꢁ10ꢃ6
1.41ꢂ0.6ꢁ10ꢃ6
1.49ꢂ0.3ꢁ10ꢃ6
1.68ꢂ0.3ꢁ10ꢃ6
10
11
20
21
29
10. Hauteville, M.; Ponchet, M.; Ricci, M.; Favre-Bonvin, J.
J. Heterocycl. Chem. 1988, 25, 715.
11. NF-ꢀB Assay: Human Jurkat T-cells stably transfected
with an NF-kB binding site (from the MHC promoter) fused
to a minimal SV-40 promoter driving luciferase were used in
these experiments.1 Cells were counted, resuspended in fresh
medium containing 10% serum-plus at a density of 1ꢁ106
cells/mL, and plated in 96-well round-bottom plates (200 mL
per well) 18 h prior to running the assays. Compounds dis-
solved in 0.2% DMSO/H2O at the appropriate concentrations
were then added to the microtiter plates containing the cells,
and the plates were incubated at 37 ꢀC for 0.5 h. To induce
transcriptional activation, 50 mg/mL of phorbol 12-myristate-
13-acetate (PMA) and 1 ug/mL of phytohemagglutin (PHA)
were added to each well, and the cells were incubated for an
additional 5 h at 37 ꢀC. The plates were centrifuged at 2200
rpm for 1 min at room temperature followed by the removal of
the media; 60 mL of cell lysis buffer was added to each well,
and cells were lysed 0.25 h; 40 mL of each cell lysate was
transferred to a black 96-well plate, and 50 mL of luciferase
substrate buffer was added. Luminescence was immediately
measured using a Packard TopCount. The results are expres-
sed as IC50 values where the IC50 value is defined as the con-
centration of compound required to reduce luciferase activity
to 50% of control values.
AP-1 Assay: The AP-1 assay was run as described above for
NF-kB except that the Jurkat T-cells were stably transfected
with a plasmid that contained an AP-1 binding site from the
collagenase promoter driving luciferase expression. In addi-
tion, the concentration of PMA and PHA were 5 mg/mL and 1
mg/mL, respectively.
12. Chong, S.; Dando, S. A.; Morrison, R. A. Pharm. Res.
1997, 14, 1835.
13. Female Lewis rats, 8 weeks of age, were purchased from
Charles River, Japan. After acclimatization, the rats were
randomly assigned into 12 groups (n=5 per group) according
Figure 2. Effects of compounds on footpad swelling in adjuvant
arthritic rats. Compounds were administered 30 mg/kg, po. Each
point represents the mean ꢂ S.E. of 5 animals per group. *, **: Sig-
nificantly different from the control, p<0.01, respectively.
drop in potency. Two analogues with a basic nitrogen at
the 7-position were prepared. The 7-(1-piperidyl) ana-
logue (28) was 20-fold less active than 21 but the
7-(N,N-dimethyl) analogue (29) was comparable in
potency to 21.
We selected a set of 5 compounds to study their Caco-2
permeability.12 As shown in Table 4, compound 10
showed acceptable permeability. However, the rest of the
analogues were less permeable than 10. We further exam-
ined these compounds in the adjuvant induced arthritis
rat-model.13 On day 21, the swelling on both injected foot
and non-injected foot pad went down significantly in the

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M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4077–4080
to treatment received. Rats in 11 groups were induced with
arthritis by injecting 100 mg of heat-inactivated Mycobacter-
ium butyricum (Difco, USA) in 100 mL of liquid paraffin in the
right hind paw(Day 0), and the rest group was used as a
healthy control group. Compound 9, 10, 11 and 20 were sus-
pended in 0.5% carboxymethylcellulose solution containing
0.1% Tween80 and saline for 30 mg/kg, po administration.
Dexamethsone 21-phosphate (DEX, a antiinflammatory ster-
oid from Sigma), a positive reference compound, was sus-
pended in 0.5% carboxymethylcellulose solution and
administered to rats at a dose level of 0.1 mg/kg, po All
compounds and vehicles were administered to arthritic rats
once daily for 21 days. The degree of arthritis was determined
by measuring hind-pawvolume using a plethysmometer (TK-
lO5, Unicom, Tokyo) on the Day 0 (immediately before adju-
vant injection), 3, 7, 10, 14, 17, and 21.
Statistical Analysis: All values are means+SEM unless
otherwise stated. Statistically significance was determined by
the Tukey-Kamer procedure using SuperANOVA software
(Abacus Concepts, USA). The level of significance was
p<0.05.
14. 1-[2-(2-thienyl)quinazolin-4-ylamino]-3-methyl-3-pyrroline-
2,5-dione (10). Mp 199--200 ꢀC. 1H NMR (300 MHz, CDCl3) d
9.40 (s, 1H), 7.82 (dd, J=1.5 and 3.9 Hz, 1H), 7.56 (t, J=8.1
Hz, 1H), 7.42 (m, 2H), 7.07 (dd, J=3.6 and 1.2 Hz, 1H), 6.62
(m, 2H), 3.83 (m, 3H), 2.26 (d, J=1.8 Hz, 3H); IR (KBr,
cmꢃ1) 3099, 2942, 1734, 1576, 1500, 1315, 1259, 1086, 731;
EIMS m/z 366 (M+);