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

Article abstract of DOI:10.1016/S0960-894X(02)00517-6

Several analogues of ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-4-(trifluoromethyl)pyrimidine- 5-carboxylate (1) were synthesized and tested as inhibitors of AP-1 and NF-κB mediated transcriptional activation in Jurkat T cells. From our SAR work, ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))-N-methylamino]-4-(trifluoromethyl)- pyrimidine-5-carboxylate was identified as a novel and potent inhibitor.

Full text of DOI:10.1016/S0960-894X(02)00517-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2573–2577
Structure–Activity Relationship Studies of Ethyl 2-[(3-Methyl-
2,5-dioxo(3-pyrrolinyl))amino]-4-(trifluoromethyl)pyrimidine-
5-carboxylate: An Inhibitor of AP-1 and
NF-ꢀB Mediated Gene Expression
Moorthy S. S. Palanki,* Leah M. Gayo-Fung, Graziella I. Shevlin, Paul Erdman,
Mark Sato, Mark Goldman, Lynn J. Ransone and Cheryl Spooner
Celgene Signal Research Division, 5555 Oberlin Drive, San Diego, CA 92121, USA
Received 22 March 2002; accepted 21 June 2002
Abstract—Several analoguesof ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-4-(trifluoromethyl)pyrimidine-5-carboxylate ( 1)
were synthesized and tested as inhibitors of AP-1 and NF-kB mediated transcriptional activation in Jurkat T cells. From our SAR
work, ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))-N-methylamino]-4-(trifluoromethyl)-pyrimidine-5-carboxylate wasidentified asa
novel and potent inhibitor. # 2002 Elsevier Science Ltd. All rights reserved.
T-lymphocytes(T-cell) orchetsrate both the initiation
and propagation of various immune responses through
the secretion of protein mediators termed cytokines.1
These cytokines play a significant role in a number of
inflammatory diseases such as asthma, psoriasis, rheu-
matoid arthritis, and transplant rejection.2 Several
studies have shown that T-cell driven immune responses
appear to overreact in these disease states.3,4 In acti-
Figure 1.
vated T cells, transcription factors such as the activator
protein-1 (AP-1), regulate IL-2 and matrix metallo-
proteinases production, while the nuclear factor-kB
(NF-kB), is essential for the transcriptional regulation
of the proinflammatory cytokinesIL-1, IL-6, IL-8, and
TNF-a.5 Based on these findings, it appears that inhibi-
tion of AP-1 and NF-kB transcriptional activation in T
cellsmay represent an attractive target in the develop-
ment of novel antiinflammatory drugs.6
mediated transcriptional activation.9 We screened our
diversity library using automated high-throughput
assays with stably transfected human Jurkat T-cells and
identified a novel compound (1) that inhibited both AP-1
and NF-kB mediated transcriptional activation
(IC50=2 mM). Compound 1 did not block basal tran-
scription driven by the b-actin promoter. In addition, 1
had an inhibitory effect on the production of IL-2 and
IL-8 levelsin a stimulated Jurkat T-cells. Our goal was
to improve the potency of 1 by exploring different sub-
stituents around the 2, 4, and 5-positions of the pyr-
imidine ring. This paper describes the synthesis and
structure-activity relationship of this series of novel
compounds(Fig. 1).
Very few compoundsare known to inhibit both the AP-1
and NF-kB mediated transcriptional activation.7
A
pyrrole analogue, PNU156804, wasshown to block the
activation of both AP-1 and NF-kB transcription fac-
tors.8 Macrolide antibiotic such as Erythromycin at
therapeutic concentrationsinhibited AP-1 and NF- kB
Scheme 1 shows the synthesis of various analogues of 1
that contain different groupsat the 4- and 5-position of
*Corresponding author. Tel.: +1-858-558-7500; fax: +1-858-623-
0870; e-mail: mpalanki@signalpharm.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00517-6

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M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2573–2577
with phosphorus oxychloride.11 The chlorine wasfirts
displaced with hydrazine and then converted to a
3-methyl-2, 5-dioxo(3-pyrrolinyl) or citraconamido
group by heating at reflux with citraconic anhydride.
The substituents on the secondary amine at the 2-posi-
tion wasintroduced either by treating 5 with an alkyl
halide and a base or an acid chloride or an isocyanate or
a sulphonyl chloride or a chloroformate. The chloro
intermediate 5 wastreated with ammonia to give a
2-amino analogue 17.
The synthesis of 5-substituted ketones and ethers is
shown in Scheme 2. An appropriately substituted
b-ketoester was cyclized with S-methylisothiourea under
basic conditions to give pyrimidine intermediate 7. The
ester group was hydrolyzed, converted to acid chloride
using oxalyl chloride and then treated with a Grignard
reagent to give a keto analogue. The S-methyl group
wasoxidized to a uslphone, 8. Treatment of the sul-
phone with hydrazine followed by citraconic anhydride
resulted in compounds 45, 46, 48, and 53. An alkyl
group wasintroduced on the secondary amine by treat-
ment with an appropriate alkyl halide and sodium
hydride in THF to give compound 66. The synthesis of
ether analogue (71) started with the ester 7, which was
hydrolyzed and reduced to an alcohol using mixed
anhydride and sodium borohydride. The intermediate
alcohol wasmethylated uisng iodomethane. The S-Me
group wasthen converted to a citraconamido group as
described earlier to give 71.
Scheme 1. (a) Urea, CH(OEt)3, reflux, overnight; (b) NaOEt, EtOH,
rt, overnight; (c) POCl3, reflux, N2, 3 h; (d) hydrazine, THF, rt, 5 min;
(e) citraconic anhydride, chloroform, reflux, 3 h; (f) R1Br, NaH, THF,
rt, overnight, (for R1=alkyl) or RCOCl, pyridine, rt, overnight (for
R1=RCO) or RSO2Cl, pyridine, rt, overnight, (for R1=RSO2) or
RNCO, THF, rt, overnight, (for R1=RNHCO) or ROCOCl, pyridine,
rt, overnight (for R’=ROCO); (g) NH3, THF, 0 ꢀC to rt, 6 h.
Scheme 3 illustrates the introduction of various groups
such as amides, ethers, and ester bioisosteres at the
5-position of the pyrimidine ring. A carboxylic acid in
compound 7 wastreated with acetone oxime and
n-butylithium, followed by acid to give an isoxazole, 10.
The S-Me group in 10 wasconverted to a citracona-
mido group, asdescribed above, to give compound, 54.
The carboxylic acid in 11 wasconverted to a cyano
group by treating 11 with oxalyl chloride, followed by
the pyrimidine ring. An appropriately substituted
b-keto ester was heated at reflux with urea and triethyl
orthoformate to give 3. A base promoted cyclization of
the intermediate 3 resulted in a pyrimidine ring 4.10 The
hydroxy group in 4 wasconverted to a chloro by treating
Scheme 2. (a) S-methylisothiourea, NaOEt, EtOH, reflux, overnight; (b) NaOH, THF-methanol-water, rt, overnight; (c) (i) (COCl)2, DCM, rt, 1 h;
(ii) RMgBr, THF, À78 ꢀC, 2 h; (iii) m-CPBA; DCM, rt, overnight; (d) (i) Hydrazine, THF, rt, 5 min; (ii) citraconic anhydride, chloroform, reflux,
overnight; (e) R’Br, NaH, THF, rt, overnight (R’=alkyl); (f) (i) Isobutylchloroformate; N-methyl morpholine (NMM), sodium borohydride, water,
rt, 1 h, (ii) methyl iodide, silver (II) oxide, acetonitrile, rt, overnight, (iii) m-CPBA; DCM; rt; overnight.

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2573–2577
2575
Scheme 4. (a) (i) ClCH2COCH3, (ii) acetamide, BF3–Et2O, xylenes;
(b) (i) m-CPBA, (ii) hydrazine, THF, rt, 5 min; (iii) citraconic anhy-
dride, chloroform, reflux, overnight; (c) (i) CH3CONHNH2, POCl3,
reflux, overnight; (d) (i) (COCl)2, (ii) NH2CH2CH2OH; (iii) SOCl2,
EtOAc–CHCl3.
The analogues synthesized as part of this study were
evaluated in Jurkat T-cellsstably transfected with pro-
moter-reporter gene constructs driven by either an AP-1
binding site or a NF-kB binding site.15,16 All the com-
pounds were tested in both assays. The IC50 valuesare
shown in Table 1. Since all the compounds had similar
IC50 valuesin both AP-1 and NF- kB assays, average
IC50 values in both assays are shown in Table 1.
Scheme 3. (a) (i) Acetone oxime, n-BuLi, THF; (ii) H2SO4, THF, (b)
(i) m-CPBA, DCM, rt, overnight, (ii) Hydrazine, THF, rt, 5 min; (iii)
citraconic anhydride, chloroform, reflux, overnight; (c) (i) (COCl)2,
DCM, rt, 2 h, (ii) NH3 (gas), THF, rt, overnight, (iii) SOCl2; DMF; (e)
(i) NaN3; NH4Cl, DMF, (ii) MeI, K2CO3; (g) (i) COCl2, DCM, 1 h, rt,
(ii) YH, THF, 1 h, rt.
ammonia and then finally with thionyl chloride. The
N-methylated tetrazole wasobtained by treating the
cyano analogue (12) with sodium azide and ammonium
chloride followed by methylation using iodomethane. The
citraconamido group wasintroduced at the 2-position of
compound 12, asdescribed earlier, to give 57. Similarly,
the carboxylic acid analogue 42 wassynthesized from 11
asshown. The carboxylic acid of 11 wasalso converted to
an ester or amide via an acid chloride to give intermediate
13. A citraconamido group wasintroduced at the 2-posi-
tion, asdescribed above, to give 43–46, 48, 49, and 53.
Compound 1 was active in both the cell-based assays
(IC50=2 mM). Initial SAR studies were focused on
modifying 3-methyl-2,5-dioxo(3-pyrrolinyl)amino or
citraconamido group. The removal of a methyl group
(16), or the introduction of a phenyl group in the place
of methyl (18) or the introduction of a 2nd methyl
group on the citracanamido ring (19) resulted in very
little change in activity. However, when the citracona-
mido ring wasremoved, compound 17 had much
weaker potency. Next, we examined the importance of
NH at the 2-position. The hydrogen was replaced with
alkyl groups( 20 and 21), substituted carbonyl groups
(22 and 23), a urea group (24), and a carbamate group
(25). All these compounds showed 2- to 6-fold
improvement in potency. The importance of a tri-
fluoromethyl group at the 4-position was also examined.
Removal of the trifluoromethyl group (26) resulted in a
6-fold loss in potency. Substitution of the tri-
fluoromethyl with a methyl (27) resulted in a compound
with comparable potency. However, groupsusch as
ethyl- (28) and pentafluoroethyl- (29) substituted com-
pounds resulted in 5- to 10-fold increase in potency.
Other bulky groupssuch asphenyl ( 30) and benzyl (31)
did not improve the potency, and methoxymethyl (32)
The synthesis of additional ester bioisosteres is illu-
strated in Scheme 4. Treatment of the carboxylic acid
analogue 11 with chloroacetone, followed by acetamide
in BF3-etherate provided the methyl oxazole 14. The
intermediate 14 wasconverted to 56, 58, and 59 as
described previously.12 Oxadiazole analogues( 55) were
synthesized by treating 11 with N-acetylhydrazide and
phosphorus oxychloride, followed by hydrazine and
then citraconic anhydride.13 The carboxylic acid in 11
wasconverted to a dihydro oxazole analogue ( 15) by
treating with oxalyl chloride, followed by aminoethanol.
The intermediate 15 wasconverted to the citraconamide
14
analogue 47 asdescribed before.

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M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2573–2577
Table 1. Inhibition of AP-1 and NF-kB-mediated transcriptional activation in Jurkat cells
No.
R2
R4
R5
IC50, ꢁM
No.
R2
R4
R5
IC50, mM
34
35
36
37
38
39
40
41
42
43
44
45
46
"
"
"
"
"
"
"
"
"
"
"
"
"
2-Thienyl
3-Thienyl
2-(5-Methylthienyl)
2-(5-Chlorothienyl)
2-Benzo[b]thienyl
2-Thiazolyl
Cyclopropyl
CF3
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2-tBu
CO2H
CONH2
CONMe2
COMe
COPh
0.14
0.20
0.045
0.52
0.8
2.2
1.5
0.21
30
30
30
4.4
0.098
1
CF3
CO2Et
2
CF3
CF3
CF3
CF3
16
17
CF3
CF3
CO2Et
CO2Et
1.6
30
CF3
NH2
47
"
CF3
6.4
18
19
CF3
CF3
CO2Et
CO2Et
3.7
3.9
48
49
50
51
52
53
"
"
"
"
"
"
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
COPh
COCH2CH2CH3
Cyclopropyl ester
CH2OH
0.65
0.45
0.12
4.9
1.1
7.7
CN
COCH3
54
"
CH2CH3
10
20
21
CF3
CF3
CO2Et
CO2Et
0.3
55
56
57
"
"
"
CH2CH3
CH2CH3
CH2CH3
10
0.83
10
0.45
22
23
CF3
CO2Et
CO2Et
0.41
0.83
CF3
58
"
CH2CH3
2.8
59
60
"
"
2-Thienyl
2-Thienyl
0.76
0.05
24
CF3
CO2Et
0.59
CO2-tBu
61
62
CH2CH2CH3
CO2Et
0.43
0.45
25
26
CF3
CO2Et
CO2Et
0.38
CF2CF3
CO2Et
H
13
63
64
65
66
67
68
69
70
71
"
"
"
"
"
"
"
"
"
CH2CH3
2-Benzo[b]thienyl
2-Thiazolyl
CH2CH3
CF2CF3
2-Thienyl
3-Thienyl
2-(5-Methylthienyl)
CH2CH3
CO2Et
CO2Et
CO2Et
COPh
CO2Et
CO2Et
CO2Et
CO2Et
CH2OCH3
0.035
0.13
1.7
0.41
0.35
0.094
0.12
0.35
0.63
27
28
29
30
31
32
33
"
"
"
"
"
"
"
CH3
CH2CH3
CF2CF3
Ph
CH2Ph
CH2OCH3
2-Furanyl
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
1.9
0.4
0.2
1.2
3.8
6.0
1

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2573–2577
2577
resulted in decreased potency. The introduction of a
2-thienyl group (34) resulted in more than 10-fold
improvement in potency. The introduction of
2-(5-methylthienyl) group (36) resulted in a compound
with more than 40-fold improvement in potency. The
significance of the ethyl carboxylate at the 5-position of
the ring wasalso examined. The introduction of a bulky
alkyl group such as t-butyl group (41) in the place of
ethyl resulted in 10-fold increase in potency. However,
groupssuch asa carboxylic acid ( 42), carboxamide (43),
and N,N-dimethyl carboxamide (44) all resulted in the
loss of potency. While a methyl ketone (45) at the
5-position resulted in small improvement in potency, a
phenyl ketone (46) analogue was20-fold more active.
We also examined several bioisosteres for the replace-
ment of ethyl carboxylate. The introduction of an oxa-
zoline (47), isoxazole (54) oxadiazole (55), tetrazole (57),
phenyloxazole (58) all resulted in compounds with less
potency than 1. However, the methyloxazoles( 56 and
59) resulted in compounds with improved potency.
Several groupsat the 4-poitsion ( 62–71) were also
explored while keeping the N-methyl citraconamide
group at the 2-position. The analogues 62 through 71
had submicromolar potency with the 4-ethyl-substituted
analogue 63 being the most potent compound in the
series, having an IC50 value of 35 nM.
Figure 2.
groupsat the 2-, 4- and 5-poistion of the pyrimidine
ring. Asa result of our SAR effort, we identified potent
dual inhibitorsof AP-1 and NF- kB mediated tran-
scription with inhibitors (Fig. 2, 36, and 63) having an
IC50 value of 0.045 and 0.035 mM respectively, in the
Jurkat T cell assays. Evaluation of compound 63 and
other compoundsdecsribed herein in variousin vivo
studiesisthe next step in defining the beneficial effect of
inhibiting both AP-1 and NF-kB mediated transcrip-
tional activation.
References and Notes
From thiswork, it isevident that a single modification
at either the 2-, 4-, or 5-position of the pyrimidine can
significantly improve the potency of the initial hit, 1
(IC50=2 mM). N-methylation of the 2-amino group of
1, for example, improved potency over 6-fold (20,
IC50=0.3 mM). In another example, substitution of the
trifluoromethyl group at the 4-position of 1 with
2-(5-methylthienyl) improved potency 44-fold (36,
IC50=0.045 mM). These independent modifications
have significantly improved the potency of compound 1,
however, the potency resulting from a combination of
the structural modifications is not necessarily additive.
For example, N-methylation of the 2-amino group of
compound 36 doesnot further improve the potency of
36. To the contrary, the N-methylated analogue of 36 is
less potent than 36 (70, IC50=0.35 mM).
1. Palanki, M. S. S.; Manning, A. M. Exp. Opin. Ther.
Patents 1999, 9, 27.
2. O’Shea, J. J.; Ma, A.; Lipsky, P. Nature Reviews Immunol-
ogy 2002, 2, 37.
3. Manning, A. M. Drug Discovery Today 1996, 1, 151.
4. Lewis, A.J. Emerging Drugs: The Prospectfor Improved
Medicines; Annual Executive Briefing, Ashley Publications
Ltd., 1996; 31.
5. Vossen, A. C. T. M.; Savelkoul, H. F. J. Mediat. Inflamm.
1994, 3, 403.
6. Manning, A. M.; Lewis, A. J. Rheumatoid Arthritis 1997, 1,
65.
7. Palanki, M. S. S. CurrentMedicinal Chemistry 2002, 9, 219.
8. Mortellaro, A.; Songia, S.; Gnocchi, P.; Ferrari, M.; For-
nasiero, C.; D’Alessio, R.; Isetta, A.; Colotta, F.; Golay, J.
The J. of Immunology 1999, 162, 7102.
9. Desaki, M.; Takizawa, H.; Ohtoshi, T.; Kasama, T.;
Kobayashi, K.; Sunazuka, T.; Omura, S.; Yamamoto, K.; Ito,
K. Biochemical and Biophysical Res. Commun. 2000, 267, 124.
10. Schenone, P.; Sansebastiano, L.; Mosti, L. carboxylates. J.
Heterocycl. Chem. 1990, 27, 295.
11. Grohe, K.; Heitzer, H. Liebigs Ann. Chem. 1973, 1025.
12. Diana, G. D.; Oglesby, R. C.; Akullian, V.; Carabateas,
P. M.; Cutcliffe, D.; Mallamo, J. P.; Otto, M. J.; McKinlay,
M. A.; Maliski, E. G.; Michalec, S. J. J. Med. Chem. 1987, 30,
383.
13. Kotian, P.; Mascarella, W.; Abraham, P.; Lewin, A.;
Boja, J.; Kuhar, M.; Carroll, I. J. Med. Chem. 1996, 39,
2753.
14. 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.
15. Sullivan, R. W.; Bigam, C. G.; Erdman, P. E.; Palanki,
M. S. S.; Anderson, D. W.; Goldman, M.; Ransone, L. J.;
Suto, M. J. J. Med. Chem. 1998, 41, 413.
16. Palanki, M. S. S.; Erdman, P. E.; Gayo-Fung, L.; Shevlin,
G. I.; Sullivan, R. W.; Goldman, M. E.; Ransone, L. J.; Ben-
nett, B. L.; Manning, A. M.; Suto, M. J. J. Med. Chem. 2000,
43, 3995.
In other analogues, the potency resulting from a com-
bination of the structural modifications is cumulative.
Substitution of the trifluoromethyl group at the 4-posi-
tion of 1 with, for example, ethyl improved potency
5-fold (28, IC50=0.4 mM). In thiscase, methylation of
the 2-amino group of compound 28 further improved its
potency another 11-fold (63, IC50=0.035 mM). The
cumulative versus non-cumulative SAR may be due to
steric interactions between the 4-substituents and the
2-amino substituents, however, this is not yet well
understood and will be further studied.
In summary, a substituted pyrimidine compound, ethyl
2-[(3-methyl-2, 5-dioxo(3-pyrrolinyl))amino]-4-(trifluoro-
methyl)pyrimidine-5-carboxylate (1), wasidentified from
the screening of our in house diversity library. This
novel compound wasan inhibitor (IC 50=2 mM) of AP-1
and NF-kB mediated transcriptional activation in Jur-
kat T cells and was optimized by substituting various