Novel inhibitors of AP-1 and NF-κB mediated gene expression: Structure-activity relationship studies of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)pyrimidine-5-carb oxylate

Article abstract of DOI:10.1016/S0960-894X(00)00312-7

In an effort to identify novel inhibitors of AP-1 and NF-κB mediated transcriptional activation, several analogues of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)pyrimidine-5-carb oxylate (1) were synthesized and tested in two in vitro assays. The 2-(2'-thienyl) substituted compound (11) was identified as the most potent in this series. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00312-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1645±1648
Novel Inhibitors of AP-1 and NF-ꢀB Mediated Gene Expression:
Structure±Activity Relationship Studies of Ethyl 4-[(3-Methyl-
2,5-Dioxo(3-pyrrolinyl))amino]-2-(tri¯uoromethyl)pyrimidine-
5-carboxylate
Moorthy S. S. Palanki,* Paul E. Erdman, Anthony M. Manning, Arnold Ow,
Lynn J. Ransone, Cheryl Spooner, Carla Suto and Mark Suto
Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, USA
Received 16 March 2000; accepted 12 May 2000
AbstractÐIn an e€ort to identify novel inhibitors of AP-1 and NF-kB mediated transcriptional activation, several analogues of
ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(tri¯uoromethyl)pyrimidine-5-carboxylate (1) were synthesized and tested in
two in vitro assays. The 2-(20-thienyl) substituted compound (11) was identi®ed as the most potent in this series. # 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
e€ect on the production of IL-2 and IL-8 levels in stimu-
lated cells. Our goal was to improve potency by exploring
di€erent substituents around the pyrimidine ring. We
introduced various groups on the pyrimidine ring at 2, 4
and 6-positions of 1. This paper describes the synthesis
and the structure±activity relationship of this series of
compounds.
There is now abundant evidence that T-lymphocytes
orchestrate both the initiation and propagation of
immune responses through the secretion of protein med-
iators termed cytokines.1 These cytokines play a very
important role in a number of in¯ammatory diseases.2,3
In allergies and autoimmune diseasesÐsuch as asthma,
psoriasis, rheumatoid arthritis, and transplant rejectionÐ
T-cell driven immune responses appear to overreact. In
activated T cells, transcription factors such as the activator
protein-1 (AP-1), regulate IL-2 production and produc-
tion of matrix metalloproteinases, while the nuclear fac-
tor-kB (NF-kB), is essential for the transcriptional
regulation of the proin¯ammatory cytokines IL-1, IL-6,
IL-8, and TNFa.4 Based on these observations, it appears
that inhibition of AP-1 and NF-kB transcriptional acti-
vation in T cells may represent an attractive target in the
development of novel antiin¯ammatory drugs (Fig. 1).5
Chemistry
The synthesis of 2- and 4-substituted analogues is shown
in Scheme 1. An appropriately substituted amidine was
cyclized with diethyl 2-(ethoxymethylene)propane-1,3-
dioate in ethanol and sodium ethoxide to give 4. The
amidines that are not available commercially were pre-
pared either from the corresponding nitrile or acid as
shown.6 The hydroxy group in 4 was converted to a chloro
group7 and then treated with hydrazine or methylhy-
drazine to give the appropriate intermediate, 5. Treatment
of the hydrazine intermediates (5) with appropriately
substituted maleic anhydride resulted in the ®nal products
(6±44). The treatment of 10 with acetic anhydride
resulted in 38. Table 1 shows the list of compounds that
were prepared with modi®cations at R1, R2, R3, and R4.
Using automated high-throughput assays with stably
transfected human jurkat T-cells, we identi®ed a com-
pound (1) from a diversity library that inhibited both AP-1
and NF-kB mediated transcriptional activation (IC50=1
mM) without blocking basal transcription driven by the
b-actin promoter. In addition, 1 had a similar inhibitory
We also examined the importance of the carboxylate
group at the 5-position. Several ester bioisosteres were
introduced in place of the ethyl ester of 11, which were
*Corresponding author. Tel.: +1-858-558-7500; fax: +1-858-623-
0870. e-mail: mpalanki@signalpharm.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00312-7

1646
M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1645±1648
was converted to N-aminocitraconamide as discussed
earlier to give 49.
The synthesis of oxazole analogue 51 started from the free
acid 47 (Scheme 3). The acid group was converted to an
ester using a chloroacetone and to methyl oxazole 50
using acetamide and BF3±Et2O.8 Compound 50 was con-
verted to 51 using a reaction sequence for the conversion
of 45 to 46. The carboxylic acid group in 47 was converted
to 2-methyl-1,3,4-oxadiazole.9 The oxadiazole inter-
mediate was converted to 52 using the reaction sequence
for the conversion of 45 to 46. The treatment of acid 47
with oxalyl chloride, followed by 2-aminoethanol, and
then thionyl chloride resulted in dihydro oxazole 53.
The citraconamido group was introduced on to 54 as
described earlier.
Figure 1.
synthesized as shown in Schemes 2 and 3. The synthesis
of 46 and 49 was initiated from the chloro derivative 44
(Scheme 2). The chlorine at the 4-position was replaced
with a benzyl thiol followed by conversion of the ester
to the isoxazole, 45.8
The S-benzyl group in 45 was oxidized with m-CPBA to
give a sulfone. The sulfone was treated with hydrazine
followed by citraconic anhydride to give 46. The ester
group in 44 was hydrolyzed and converted to cyano ana-
logue, 48. The treatment of 48 with sodium azide and
ammonium chloride in DMF resulted in a tetrazole ring.
The tetrazole ring was methylated and S-benzyl group
Results and Discussion
The analogues synthesized as part of this study were
evaluated in Jurkat T-cells stably transfected with pro-
moter-reporter gene constructs driven by either an AP-1
Scheme 1. (a) (i) (COCl)2, DMF; (ii) NH3, 3. SOCl2; (b) (i) HCl (gas), EtOH; (ii) NH3 (gas), EtOH; (c) Diethylethoxymethylene malonate, EtONa,
EtOH; (d) (i) POCl3; (ii) R4NHNH2; (e) Citraconic anhydride, CHCl3, Á.
Table 1. In vitro evaluation in Jurkat T-cells of compounds with R1 through R4 modi®cations on the pyrimidine ring. IC50 values for the both AP-1
and NF-kB mediated transcriptional activation were the same.
No.
R1
R2
R3
R4
IC50, mM
No.
R1
R2
R3
R4
IC50, mM
1
6
7
8
CF3
CH3
CH3CH2
t-Bu
SCH3
Ph
2-Thienyl
4-Pyridyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1
16
10
1
0.2
0.1
0.02
6
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
5-Methyl-2-thienyl
5-Chloro-2-thienyl
2-Benzo-thienyl
2-Furanoyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
0.2
0.3
4
0.2
1
2
0.3
7
15
1
10
2
4
1
9
Cyclopropyl
2-(Cyclo-hex-2-enyl-methyl
3,5-Dichloro-phenyl
Benzyl
10
11
12
13
14
15
16
17
18
19
20
21
23
24
25
2,6-Dichloro-4-pyridyl
3-Quinolinyl
2
6
8
Phenoxy
2-Phenyl-thio
2-Phenyl-sulfonyl
Phenyl
Phenyl
2-Thienyl
4-Fluoro-phenyl
CH3CH2
CF3
H
H
4-(2-Methyl-thiazolyl
1-(3, 5-Dimethyl-pyrazolyl
4-Methoxy-phenyl
3-Methoxy-phenyl
4-Fluoro-phenyl
4-Chloro-phenyl
4-Tri¯uoro-methyl-phenyl
3-Bromo-phenyl
3-Nitro-phenyl
3-Thienyl
10
0.9
5
0.7
0.3
0.4
0.7
7
CH3
Ac
CH3
Me
Me
H
3
10
10
20
10
CF3
CF3
H
H
H
H
0.1

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1645±1648
1647
Scheme 2. (a) (i) PhCH2SH, THF; (ii) Acetone oxime, n-BuLi, THF; (iii) H2SO4, THF; (b) (i) m-CPBA; (ii) NH2NH2; (iii) Citraconic anhydride,
CHCl3, re¯ux; (c) (i) PhCH2SH, THF; (ii) NaOH; (d) (i) (COCl)2; (ii) NH3 (gas); (iii) SOCl2, DMF; (e) (i) NaN3, NH4Cl, DMF; (ii) MeI, K2CO3;
(iii) m-CPBA; (iv) NH2NH2; (v) Citraconic anhydride, CHCl3, d.
Scheme 3. (a) (i) ClCH2COCH3; (ii) Acetamide, BF3-Et2O, xylenes; (b) (i) m-CPBA; (ii) NH2NH2; (iii) citraconic anhydride; (c) (i) CH3CONHNH2,
POCl3, d; (ii) m-CPBA; (iii) NH2NH2; (iv) Citraconic anhydride, CHCl3, d; (d) (i) (COCl)2; (ii) NH2CH2CH2OH; (iii) SOCl2, EtOAc-CHCl3.
binding site or a NF-kB binding site.10 All the compounds
were tested in both assays. The IC50 values for these com-
pounds are shown below. Since all the compounds had
similar IC50 values in the both AP-1 and NF-kB assays,
average values are shown (Table 1).
(8) group resulted in a compound with comparable
activity. The S-methyl (9) and phenyl (10) substituents
improved activity. The 2-thienyl (11) substituent resulted
in a 50-fold improved activity. The substituted and
unsubstituted heterocyclic rings (12±16) at 2-position
resulted in loss of activity. Any substitution on the phenyl
ring of 10 (compounds 17±24) resulted in decreased activ-
ity. Similarly, introduction of a group on the thienyl ring
of 11 (compounds 26±28) also resulted in decreased
activity. Introduction of a methyl group at R3 (42), a
Compound 1 had IC50 activity in both the cell based
assays at 1 mM. The substitution of a methyl (6) and ethyl
(7) in the place of tri¯uoromethyl group resulted in the
loss of activity. However, the introduction of a t-butyl

1648
M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1645±1648
Table 2. In vitro evaluation in Jurkat T-cells of compounds with
modi®cations of R5 of the pyrimidine ring. IC50 values for the both
AP-1 and NF-kB mediated transcriptional activation were the same.
tested. The bioisosteres 49, 51 and 53 were 10-fold less
active in the cell based assays. Analogues 46 and 52 were
15-fold less active and cyano analogue 48 was 250-fold
less active. Based on the above structural activity rela-
tionship studies, a thienyl group at 2-position ring, an N-
aminocitraconamido group at 4-position, and an ethyl
ester at 5-position of the pyrimidine ring 1 are optimum
substituents for the biological activity. To date we have
identi®ed a potent and novel inhibitor (11) of AP-1 and
NF-kB mediated transcriptional activation.
No
R5
IC50
,
No
R5
IC50,
mM
mM
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46
48
49
0.3
51
52
53
0.2
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phenyl group at R2 (43) or a hydrogen at R2 (44) resulted
in the decreased activity. Similarly, substitution of a
methyl group at R4 (37, 39, 40 and 41) resulted in the
decreased activity. The substitution of an acetyl group
at R4 (38) also resulted in decreased activity (Table 2).
Five ester bioisoster modi®cations were introduced in
place of the ester of 11. A cyano analogue (48) was also