2-Chloro-4-(trifluoromethyl)pyrimidine-5-N-(3',5'- bis(trifluoromethyl)phenyl)carboxamide: A potent inhibitor of NF-κB- and AP- 1-mediated gene expression identified using solution-phase combinatorial chemistry

Article abstract of DOI:10.1021/jm970671g

Described is the identification of a novel series of compounds that blocks the activation of two key transcription factors, AP-1 and NF-κB. These transcription factors regulate the expression of several critical proinflammatory proteins and cytokines and represent attractive targets for drug discovery. Through the use of high throughput screening and solution- phase parallel synthesis, inhibitors of both NF-κB and AP-1 were identified. In subsequent testing, these compounds were also shown to block both IL-2 and IL-8 levels in the same cells. One of the most potent compounds in this series, 28, was active in several animal models of inflammation and immunosuppression, thus validating the importance of AP-1 and NF-κB as potential therapeutic targets. The synthesis and preliminary structure- activity relationships of these compounds is addressed.

Full text of DOI:10.1021/jm970671g

413
J . Med. Chem. 1998, 41, 413-419
2-Ch lor o-4-(tr iflu or om eth yl)p yr im id in e-5-N-(3′,5′-bis(tr iflu or om eth yl)p h en yl)-
ca r boxa m id e: A P oten t In h ibitor of NF -KB- a n d AP -1-Med ia ted Gen e Exp r ession
Id en tified Usin g Solu tion -P h a se Com bin a tor ia l Ch em istr y
Robert W. Sullivan, Colin G. Bigam, Paul E. Erdman, Moorthy S. S. Palanki, David W. Anderson,
Mark E. Goldman, Lynn J . Ransone, and Mark J . Suto*
Departments of Medicinal Chemistry and Pharmacology, Signal Pharmaceuticals, Inc., 5555 Oberlin Drive,
San Diego, California 92121
Received October 3, 1997
Described is the identification of a novel series of compounds that blocks the activation of two
key transcription factors, AP-1 and NF-κB. These transcription factors regulate the expression
of several critical proinflammatory proteins and cytokines and represent attractive targets for
drug discovery. Through the use of high throughput screening and solution-phase parallel
synthesis, inhibitors of both NF-κB and AP-1 were identified. In subsequent testing, these
compounds were also shown to block both IL-2 and IL-8 levels in the same cells. One of the
most potent compounds in this series, 28, was active in several animal models of inflammation
and immunosuppression, thus validating the importance of AP-1 and NF-κB as potential
therapeutic targets. The synthesis and preliminary structure-activity relationships of these
compounds is addressed.
In tr od u ction
In certain autoimmune diseases and chronic inflam-
matory states, the continuous activation of T-cells leads
to a self-perpetuating destruction of normal tissues or
organs.1 This activation initiates a cascade of events
that results in the overproduction of certain transcrip-
tion factors and proinflammatory cytokines.2,3 Tran-
scription factors are a family of proteins that act as
molecular switches and regulate several cellular events,
including gene expression, cytokine production, and the
synthesis of additional cellular regulators.4
tory effect on the production of IL-2 and IL-8 levels in
Two transcription factors in particular, nuclear fac-
stimulated cells and was active in an animal model of
tor-κ binding (NF-κB) and activator protein-1 (AP-1),
inflammation.11 Through the use of solution-phase
control the production of many cytokines and related
parallel chemistry and targeted synthesis, a 10-fold
proteins elevated in immunoinflammatory diseases.5-7
more potent derivative, 28, was identified. This paper
These include interleukin-1 (IL-1), interleukin-2 (IL-2),
describes the use of these techniques and the resulting
interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor
structure-activity relationships of this series of com-
necrosis factor-R (TNFR). NF-κB plays a significant role
pounds.
in the regulation of IL-8 transcription during exposure
of cells to external stimuli, while AP-1 regulates both
IL-2 and TNF-R production during T-cell activation.
Ch em istr y
Therefore, modulation of either one or both of these
transcription factors should lead to suppression of
cytokine levels and, thus, represent attractive targets
for the prevention of immunoinflammatory diseases.8
The compounds listed in Table 1 were prepared as
illustrated in Schemes 1 and 2. The 2-amino derivatives
(2-8) were synthesized by stirring 1 with an appropri-
ate amine in THF. The 2-hydroxy and the 2-methoxy
derivatives (9 and 10) were prepared by treatment of 1
with sodium hydroxide or sodium methoxide, respec-
tively. Reduction of 1 with Pd/C/MgO in EtOH/H2O (2/
1) provided 11. The N-methylamide 12 was prepared
by alkylation of the sodium salt of 1 with MeI. Alky-
lation of 1 with benzyl bromide was unsuccessful; thus,
the corresponding N-benzyl derivative 13 was prepared
using the corresponding N-benzylaniline.12
To date, no known antiinflammatory or autoimmune
drugs have been specifically developed clinically as
inhibitors of NF-κB and/or AP-1. Herein, we report the
identification of a series of novel inhibitors of both NF-
κB and AP-1 transcriptional activation and the subse-
quent validation of these transcription factors as drug
discovery targets. Using automated high-throughput
assays with stably transfected human J urkat T-cells,
we identified a compound that inhibited both NF-κB and
The preparation of 160 amides of general structure
15 was done using solution-phase combinatorial tech-
niques.13 A series of 160 commercially available alkyl-
AP-1 transcriptional activation (1, IC
) 0.3-0.5
50
µM).9,10 In addition, compound 1 had a similar inhibi-
S0022-2623(97)00671-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/24/1998

414 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Ta ble 1. In Vitro Evaluation of Substituted Pyrimidines
Sullivan et al.
Sch em e 1
IC50 (µM)
mp
yield
no.
R2
R5
R6
(°C)
(%) NF-κB AP-1
1
2
3
4
5
6
7
8
9
Cl
N(CH3)2
NH2
nBuNH
aniline
benzylamine
cyclohexylamine
piperidyl
OH
H
H
H
H
H
H
H
H
H
H
H
A
A
A
A
A
A
A
A
A
A
A
A
A
B
C
D
E
183-184 92
163-164 80
0.50 0.50
>10
>10
>10
>10
>10
>10
5.0
>10
>10
>10
>10
>10
>10
4.0
>250
60
162-163 41
228-229 91
202-203 87
198-199 86
186-187 58
dec 160
68
>10
>10
>10
>10
>10
>10
10 OCH3
11
172-173 87
188-189 53
124-125 20
102-104 25
H
12 Cl
13 Cl
19 Cl
20 Cl
21 Cl
22 Cl
23 Cl
24 Cl
25 Cl
26 Cl
27 Cl
28 Cl
CH3
Benzyl
2.30 3.0
2.70 2.70
H
H
H
H
H
H
H
H
H
H
96-97
61
>10
>10
>10
>10
>10
>10
180-181 62
248-249 27
172-173 85
135-136 55
190-191 35
141-143 35
170-171 75
200-201 40
165-166 75
0.38 0.49
1.20 1.60
0.80 0.50
2.30 2.80
F
G
H
I
J
K
4.0
0.9
3.80
0.9
0.05 0.05
a
Key: A ) 3,5-dichlorophenyl; B ) butyl; C ) phenyl; D ) 2,6-
dimethylphenyl; E ) 4-(trifluoromethyl)phenyl; F ) 3,5-dimeth-
oxyphenyl; G ) 2,6-dichloro-4-pyrimidine; H ) 5-methyl-2-
thiophene; I ) 3-methyl-5-isoxazole; J ) 3,4,5-trichlorophenyl; K
) 3,5-bis(trifluoromethyl)phenyl.
Sch em e 2
amines, anilines, and heterocyclic amines was selected
for inclusion (see the Supporting Information) in this
study. The compounds were prepared in a microtiter
format 80 compounds at a time. The procedure involved
sonicating EtOAc solutions of the amines and a slight
excess of the pyrimidine acid chloride 14 in the presence
of Amberlyst A-21 ion-exchange resin in a 96-well plate.
A small amount of H2O was added to each well to
“quench” the excess acid chloride. The organic layer
from each well was transferred to individually tared test
tubes (Zymark BenchMate) and concentrated. The
samples were then solvated (DMSO) for high through-
put screening at a concentration of 5 mg/mL. TLC
analysis was done on the entire plate, and HPLC
analysis was performed on 15 random samples. All
samples were run in a three-point dose response analy-
sis in the cell-based transcription assays. Yields (based
upon tared weight) ranged from 60 to 90%, and purities
were greater than 85%.
T-cells stably transfected with either an NF-κB binding
site, an AP-1 binding site, or the â-actin promoter
driving luciferase were pretreated for 0.5 h with com-
pounds dissolved in 0.2% DMSO/H2O. The cells were
then stimulated with phorbol 12-myristate-13-acetate
(PMA) and phytohemagglutin (PHA) and incubated for
an additional 5 h. The cells were harvested by cen-
trifugation for determination of luciferase activity. The
results are expressed as IC50 values where the IC50
value is defined as the concentration of compound
required to reduce luciferase activity to 50% of control
values.
Cytokine determinations (IL-2 and IL-8) were per-
formed by ELISA using commercially available kits.14
The production IL-2 and IL-8 was determined in super-
natants collected in the above luciferase studies. The
results are plotted as percent of control (DMSO treated
cells).
The 3-(trifluoromethyl)-5-carbonylanilines were pre-
pared starting with the commercially available 3-nitro-
5-(trifluoromethyl)benzoic acid (16) as shown in Scheme
3. Treatment of 16 with oxalyl chloride followed by the
appropriate alcohol or amine provided the desired ester
or amide. Reduction with Pd/C and reaction with 14
provided the compounds listed in Table 2.
Resu lts a n d Discu ssion
The compounds synthesized in library format were
evaluated in a three-point dose response analysis (3.3,
0.3, and 0.03 µg/mL) in all three assays, and active
derivatives (>50% at the 0.3 µg/mL dose) were then run
Biology
High throughput screening and follow-up studies
were performed using three distinct cell lines. J urkat

NF-κB- and AP-1-Mediated Gene Expression Inhibition
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 415
Ta ble 2. In Vitro Evaluation of Substituted Pyrimidine Ester
and Amides
allowed for an immediate assessment of the key func-
tional groups needed at the R5 position. Dramatic
changes in activity were seen with the different sub-
stituents attached to the amide nitrogen. To validate
this approach to lead optimization, we also synthesized
individually several key derivatives in the series and
compared their activity to the analogues prepared in the
library.16 Clearly, a substituted aromatic group is
essential since the n-butyl derivative 19 and the het-
erocyclic amides are less active (24-26).
The most active compounds were substituted aniline
derivatives. A substituent on the ring is needed at
either the 3-, 4-, or 5-position. The simple aniline
derivative 20 was inactive. Compound 20 was synthe-
sized as part of the library and then prepared individu-
ally to verify the unexpected lack of activity. Several
other structure-activity relationships were observed.
First, aniline derivatives substituted at the 3- or 4-posi-
tion with small electron-withdrawing groups were the
most potent (CF3 > Cl > F > CH3). Large bulky groups
tended to decrease activity, and substituents at the 2-
and/or 6-position resulted in a loss of activity (inactive
at highest concentration tested). Increasing the dis-
tance between the amide nitrogen and the aromatic
rings (benzyl derivatives) also resulted in inactive
compounds. The results indicated that a small electron-
withdrawing group was required at either the 3- or
4-position with the best activity found with substituents
at both the 3- and 5-positions or the 3- and 4-positions.
Trisubstituted compounds such as the 3′,4′,5′-trichloro
derivative 27 offered no advantage over the 3,5-
substituted compound (27, IC50 ) 0.9 vs 1, IC50 ) 0.3
µM).
IC50 (µM)
mp
(°C)
yield
(%)
no.
R
NF-κB
AP-1
29a
29b
29c
29d
29e
29f
29g
29h
NH2
218-219
243-244
67-71
49-53
103-105
oil
55
21
68
71
72
35
58
55
5.00
>10
0.28
0.17
0.62
1.80
1.80
4.60
3.00
>10
0.15
0.12
0.66
1.20
1.20
3.00
NHBu
OC2H5
OC4H9
OC6H13
OC8H17
O-cyclohexyl
OCH2PO(OEt)2
59-60
125-126
Sch em e 3
On the basis of the results obtained from the solution-
phase libraries, we synthesized the 3,5-bis(trifluoro-
methyl)aniline derivative 28 (SP100030). This com-
pound was the most potent inhibitor identified with an
IC50 value of 50-100 nM in the cell-based assays (NF-
κB and AP-1). Additional 3-(trifluoromethyl)-5-carbonyl
derivatives were prepared in an effort to improve the
solubility of these compounds and to further define the
structure-activity relationships at the 5-position (Table
2). In summary, the ester derivatives were more active
than the corresponding amides (29b vs 29d ), and the
activity of the esters was optimal with a four carbon
chain 29d . However, all of the derivatives were less
active than 28.
in a six-point dose-response analysis. Compounds in
Table 1 were run immediately in a six-point dose-
response analysis. None of the compounds discussed
had activity on the â-actin control at the highest dose
tested (3.3 mg/mL, data not shown).
The results shown in Table 1 clearly indicate the
importance of the chlorine atom at the 2-position of the
pyrimidine ring. Any substitution at this position
besides chlorine resulted in a complete loss of activity.15
Derivatizations of the amide nitrogen as the N-methyl
(12) or N-benzyl (13) analogues resulted in a 10-fold loss
of activity, and these compounds were more active
against the â-actin control.
The ability of compound 28 to block the production
of IL-2 and IL-8 in J urkat T-cells was also examined.
As expected, both IL-2 and IL-8 were inhibited at the
same concentrations as seen in the luciferase assay (IC50
≈ 0.03 µM). In addition, the inhibitory activity seen
with this compound was specific to T-cells. Subsequent
studies were conducted examining the ability of 28 to
block induced cytokine production in monocytes, epi-
thelial cells, fibroblasts, osteoblasts, or endothelial cells.
Surprisingly, this compound had no activity in the other
cell lines examined, although studies indicated that the
compound was able to cross the cell membranes.17 In
vivo studies were conducted with 28 and the compound
was active i.p. in a dose-dependent manner in several
animal models of inflammation and immunosuppres-
sion.10
The power of the combinatorial techniques described
was demonstrated by the synthesis and evaluation of
160 analogues of 1 in a 2-week period. These results

416 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sullivan et al.
2-(N-Cycloh exyla m in o)-4-(t r iflu or om et h yl)-5-N-(3′,5′-
d ich lor op h en yl)p yr im id in eca r boxa m id e (7): 86% yield;
mp 198-199 °C; H NMR (500 MHz, acetone-d6) δ 9.82 (bs,
1H), 8.75 (s, 1H), 7.80 (s, 2H), 7.24 (s, 1H), 3.91 (bs, 1H), 2.81
(m, 2H), 1.80 (m, 2H), 1.65 (m, 6H); IR (KBr) 1583, 1523, 1306
cm-1. Anal. (C18H16Cl2F3N4O): C, H, N.
Con clu sion s
1
Compound 28 (SP100030) represents one of the first
inhibitors of NF-κB and AP-1 transcriptional activation
specifically identified from high-throughput screening
and solution-phase parallel synthesis. The exact mech-
anism of action of 28 is under investigation and will be
reported shortly.18 However, the compound has dem-
onstrated activity in several animal models,10 but its
low aqueous solubility and high lipophilicity likely
account for the lack of oral activity in the animal models
tested. The activity of this series of compounds suggests
that inhibitors of AP-1 and NF-κB may be useful as
novel immunoinflammatory agents. Additional studies
focused on further defining the structure-activity re-
lationships of the pyrimidine ring as well as increasing
the solubility of this novel class of compounds are in
progress.
2-N-P ip er id yl-4-(t r iflu or om et h yl)-5-N-(3′,5′-d ich lor o-
p h en yl)p yr im id in eca r boxa m id e (8): 58% yield; mp 186-
1
187 °C; H NMR (500 MHz, acetone-d6) δ 9.85 (bs, 1H), 8.80
(s, 1H), 7.80 (s, 2H), 7.24 (d, J ) 1.5 Hz, 1H), 3.90 (s, 4H),
1.74 (m, 2H), 1.60 (m, 4H); IR (KBr) 3267, 1583, 1531, 1267
cm-1. Anal. (C17H15F3Cl2N4O): C, H, N.
2-Hyd r oxy-4-(tr iflu or om eth yl)-5-N-(3′,5′-d ich lor op h en -
yl)p yr im id in eca r boxa m id e (9). A mixture of 1 (370 mg,
1.0 mmol) in THF (10 mL) and aqueous NaOH (1 M, 10 mL,
10 mmol) was stirred for 16 h at room temperature. The
mixture was acidified with HCl (1 N) and extracted with
EtOAc (3 × 30 mL). The organic layers were combined,
washed with brine, and dried over Na2SO4. The solvent was
removed, and the resulting crude material was purified by
column chromatography (SiO2, 2/1 hexanes/EtOAc) to provide
240 mg (68%) of 9 as a white solid: mp dec >160 °C; 1H NMR
(500 MHz, acetone-d6) δ 8.55 (s, 1H), 8.18 (bs, 1H), 7.77 (s,
1H), 7.67 (s, 2H), 2.82 (s, 1H); IR (KBr) 3390, 1611, 1508 1216
cm-1. Anal. (C12H6Cl2F3N3O2): C, H, N.
Exp er im en ta l Section
Starting materials were obtained from commercial sources
and used without purification. Silica gel (E. Merck, 60-230
mesh) was used for flash column chromatography, and silica
gel plates (E. Merck) were used for thin-layer chromatography.
All NMR spectra were recorded on a Varian Gemini 300 or a
Bruker AM-500 spectrometer, and shifts are reported in parts
per million relative to internal tetramethylsilane. Melting
points were determined on a Mel Temp II and are uncorrected.
IR spectra were recorded with a Nicolet Impact 400d spectro-
photometer; mass spectra were obtained on a Hewlett-Packard
5890 Series II gas chromatogram with a Hewlett-Packard 5972
mass selective detector. Combustion elemental analyses were
performed by Desert Analytics Laboratory, Tucson, AZ, and
found values were within 0.4% of the theoretical values (unless
otherwise indicated).
2-Meth oxy-4-(tr iflu or om eth yl)-5-N-(3′,5′-d ich lor op h en -
yl)p yr im id in eca r boxa m id e (10). A mixture of 1 (100 mg,
0.27 mmol) in MeOH (15 mL) and NaOMe (50 mg, 0.92 mmol)
was stirred for 2.5 h under an atmosphere of N2. The reaction
was acidified with HCl (1 N) and extracted with EtOAc (3 ×
30 mL). The organic layers were combined, washed with brine,
and dried over Na2SO4. The organic layer was concentrated,
and the resulting crude material was purified by column
chromatography (SiO2, 10/1 hexanes/EtOAc) to provide 84 mg
1
(87%) of 10 as a white solid: mp 172-173 °C; H NMR (500
MHz, acetone-d6) δ 10.1 (bs, 1H), 9.17 (s, 1H), 7.79 (s, 2H),
7.29 (s, 1H), 4.10 (s, 3H); IR (KBr) 3242, 1655, 1494, 1391 cm-1
Anal. (C13H8Cl2F3N3O2): C, H, N.
.
Gen er a l Syn th esis for th e 2-Am in o Der iva tives (3-7).
2-(N,N-Dim eth yla m in o)-4-(tr iflu or om eth yl)-5-N-(3′,5′-d i-
ch lor op h en yl)p yr im id in eca r boxa m id e (2). To a solution
of 1 (100 mg, 0.270 mmol) in THF was added gaseous N,N-
dimethylamine. The mixture was stirred at room temperature
under an atmosphere of N2 for 3 h. The reaction was
concentrated, and the resulting oil was purified by column
chromatography (SiO2, 12/1 hexanes/EtOAc), providing 82 mg
(80%) of 2 as a white solid: mp 163-164 °C; 1H NMR (500
MHz, acetone-d6) δ 9.89 (bs, 1H), 8.82 (s, 1H), 7.80 (s, 2H),
7.24 (s, 1H), 3.27 (s, 6H); IR (KBr) 3383, 1652, 1585 cm-1. Anal.
(C14H11Cl2F3N4O): C, H, N.
2-Am in o-4-(tr iflu or om eth yl)-5-N-(3′,5′-dich lor oph en yl)-
p yr im id in eca r boxa m id e (3): 58% yield; mp >250 °C; 1H
NMR (300 MHz, CDCl3) δ 10.85 (s, 1H), 8.74 (s, 1H), 7.75 (s,
2H), 7.72 (s, 2H), 7.60 (s, 1H); IR (KBr) 3378, 1661, 1590, 1419
cm-1. Anal. (C12H7Cl2F3N4O): C, H, N.
4-(Tr iflu or om et h yl)-5-N-(3′,5′-d ich lor op h en yl)p yr im -
id in eca r boxa m id e (11). To a solution of 1 (100 mg, 0.270
mmol) in EtOH/H2O (2/1, 2.5 mL) were added Pd/C (5%, 10
mg) and MgO (24 mg, 0.59 mmol). The mixture was stirred
under an atmosphere of H2 for 2.5 h, filtered through Celite,
and concentrated. The resulting crude material was purified
by column chromatography (SiO2, 10/1 hexanes/EtOAc), pro-
viding 48 mg (53%) of 11 as a white solid: mp 189-190 °C;
1H NMR (300 MHz, DMSO-d6) δ 11.2 (s, 1H), 9.60 (s, 1H), 9.40
(s, 1H), 7.70 (s, 2H), 7.40 (s, 1H); IR (KBr) 3262, 1650, 1585,
1151 cm-1. Anal. (C12H6Cl2F3N3O): C, H, N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-m eth yl-N-(3′,5′-dich lo-
r op h en yl)p yr im id in eca r boxa m id e (12). To a mixture of
NaH (21 mg, 0.525 mmol) in DMF (20 mL) under N2 was added
a solution of 1 (86 mg, 0.23 mmol) in DMF (5 mL). This was
stirred for 0.3 h, MeI (0.10 mL, 1.61 mmol) was added, and
stirring continued for an additional for 2 h. The solution was
acidified with 1 N HCl and concentrated. The resulting oil
was dissolved in EtOAc, extracted with HCl (1 N, 2 × 20 mL),
and washed with brine. The organic layer was dried over
MgSO4, filtered, and concentrated. The resulting oil was
purified by column chromatography (SiO2, 8/2 hexanes/EtOAc)
to provide a solid that was recrystallized from EtOH/H2O to
provide 20 mg (22%) of 12 as a white solid: mp 124-125 °C;
1H NMR (300 MHz, CDCl3) δ 8.53 (s, 1H), 7.30 (s, 1H), 6.97
2-(N-Bu tyla m in o)-4-(tr iflu or om eth yl)-5-N-(3′,5′-d ich lo-
r op h en yl)p yr im id in eca r b oxa m id e (4): 41% yield; mp
1
162-163 °C; H NMR (500 MHz, acetone-d6) δ 9.87 (bs, 1H),
8.81 (s, 1H), 8.71 (s, 1H), 7.80 (s, 2H), 7.24 (s, 1H), 3.50 (m,
2H), 1.62 (m, 2H), 1.41 (m, 2H), 0.93 (m, 3H); IR (KBr) 3281,
1583, 1529, 1199 cm-1. Anal. (C16H15Cl2F3N4O‚0.5H2O): C,
H, N.
2-(N-P h en ylam in o)-4-(tr iflu or om eth yl)-5-N-(3′,5′-dich lo-
r op h en yl)p yr im id in eca r boxa m id e (5): 91% yield; mp 228-
229 °C; 1H NMR (500 MHz, acetone-d6) δ 8.99 (s, 1H), 7.86 (s,
2H), 7.82 (s, 2H), 7.39 (m, 2H), 7.26 (s, 1H), 7.12 (m, 1H) 2.10
(s, 2H), 3.46 (s, 3H); IR (KBr) 1669, 1577, 1337 1171 cm-1
Anal. (C13H7Cl3F3N3O): C, H, N.
.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-ben zyl-N-(3′,5′-dich lo-
r op h en yl)p yr im id in eca r b oxa m id e (13). A mixture of
benzaldehyde (1.04 g, 9.40 mmol), 3,5-dichloroaniline (1.71 g,
10.6 mmol), acetic acid (0.20 mL), and methanol (100 mL was
cooled to 0 °C. A solution of NaBH3CN (1 M, 28.0 mL, 28.0
mmol) was added via a syringe pump over 0.3 h. The solution
was stirred at 0 °C for 0.5 h and allowed to warm to room
temperature, and stirring continued for 18 h. The mixture
was acidified with HCl (1 N) and then concentrated. The
(bs, 2H); IR (KBr) 3237, 1579, 1517, 1145 cm-1. Anal. (C18
-
H
11Cl2F3N4O): C, H, N.
2-(N-Ben zylam in o)-4-(tr iflu or om eth yl)-5-N-(3′,5′-dich lo-
r op h en yl)p yr im id in eca r boxa m id e (6): 87% yield; mp 202-
1
203 °C; H NMR (500 MHz, acetone-d6) δ 9.85 (bs, 1H), 8.90
(s, 1H), 7.95 (m, 1H), 7.79 (s, 2H), 7.40 (m, 2H), 7.32 (m, 2H)
7.24 (m, 2H), 4.73 (m, 2H); IR (KBr) 3272, 1583, 1145 cm-1
Anal. (C19H13Cl2F3N4O): C, H, N.
.

NF-κB- and AP-1-Mediated Gene Expression Inhibition
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 417
resulting oil was partitioned between EtOAc and H2O and
basified with NaOH (1 N) until pH 9. The combined organic
layers were washed with brine, dried over MgSO4, filtered, and
concentrated. The crude material was purified by column
chromatography (SiO2, 9/1 hexanes/EtOAc) to provide 1.61 g
(64%) of N-(3,5-dichlorophenyl)benzylamine:9 1H NMR (300
MHz, CDCl3) δ 7.31 (m, 5H), 6.66 (t, J ) 1.8 Hz, 1H), 6.45 (d,
J ) 1.8 Hz, 2H), 4.24 (s, 1H), 4.13 (s, 1H).
N-Bu tyl-3-am in o-5-(tr iflu or om eth yl)ben zylam ide (18b):
1H NMR (300 MHz, CDCl3) δ 7.26 (s, 1H), 7.23 (s, 1H), 6.99
(s, 1H), 6.10 (bs, 1H), 4.02 (bs, 2H), 3.46 (m, 2H), 1.61 (m, 2H),
1.43 (m, 2H), 0.96 (t, J ) 7.2 Hz, 3H). GC-MS 11.42 min
(m/e 260, 99).
Eth yl 3-a m in o-5-(tr iflu or om eth yl)ben zoa te (18c): 1H
NMR (300 MHz, CDCl3) δ 7.65 (s, 1H), 7.49 (s, 1H), 7.05 (s,
1H), 4.39 (q, J ) 7.2 Hz, 2H), 1.40 (t, J ) 7.5 Hz, 3H); GC-
MS 8.66 min (m/e 233, 96).
Bu tyl 3-a m in o-5-(tr iflu or om eth yl)ben zoa te (18d ): 1H
NMR (300 MHz, CDCl3) δ 7.64 (s, 1H), 7.48 (s, 1H), 7.05 (s,
1H), 4.32 (t, J ) 6.6 Hz, 2H), 1.75 (m, 2H), 1.48 (m, 2H), 1.01
(t, J ) 7.2 Hz, 3H); GC-MS 9.79 min (m/e 261, 100).
Hexyl 3-a m in o-5-(tr iflu or om eth yl)ben zoa te (18e): 1H
NMR (300 MHz, CDCl3) δ 7.64 (s, 1H), 7.49 (s, 1H), 7.05 (s,
1H), 4.31 (t, J ) 6.9 Hz, 2H), 2.0-1.2 (m, 8H), 0.91 (t, J ) 6.6
Hz, 3H); GC-MS 10.97 min (m/e 289, 100).
To a mixture of Amberlyst A-21 resin (1 g) and N-(3,5-
dichlorophenyl)benzylamine (0.204 g, 0.761 mmol) in EtOAc
(15 mL) was added a solution of 2-chloro-4-(trifluoromethyl)-
5-pyrimidine acid chloride (0.241 g, 0.984 mmol) in EtOAc (2.0
mL). The mixture was stirred for 1 h, H2O (0.2 mL) was added,
and stirring was continued for an additional 0.25 h. The
organic layer was decanted and concentrated. The resulting
oil was purified by column chromatography (SiO2, 9/1 hexanes/
EtOAc) to provide 52 mg (15%) of 13 as a clear oil: 1H NMR
(300 MHz, CDCl3) δ 8.54 (s, 1H), 7.35 (m, 4H), 7.23 (m, 2H),
Oct yl 3-a m in o-5-(t r iflu or om et h yl)b en zoa t e (18f): 1H
NMR (300 MHz, CDCl3) δ 7.64 (s, 1H), 7.48 (s, 1H), 7.05 (s,
1H), 4.31 (t, J ) 6.6 Hz, 2H), 4.00 (bs, 2H), 1.8-1.2 (m, 12H),
0.88 (t, J ) 6.9 Hz, 3H); GC-MS 11.87 min (m/e 317, 99).
Cycloh exyl 3-am in o-5-(tr iflu or om eth yl)ben zoate (18g):
1H NMR (300 MHz, CDCl3) δ 7.65 (s, 1H), 7.50 (s, 1H), 7.08
(s, 1H), 5.00 (m, 1H), 2.0-1.3 (m, 10H); GC-MS 11.23 min
(m/e 287, 98).
6.77 (m, 2H), 5.06 (s, 2H); IR (KBr) 3064, 1667, 1562 cm-1
Anal. (C19H11Cl3F3N3O): C, H, N.
.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
17a -h . 3-Nitr o-5-(tr iflu or om eth yl)ben zyla m id e (17a ). To
a solution of 16 (1.0 g, 4.2 mmol) in CH2Cl2 (50 mL) were added
oxalyl chloride (1.45 mL, 18.8 mmol) and DMF (2 drops). The
solution was stirred for 18 h under an atmosphere of N2 and
concentrated, and the resulting oil was dissolved in THF (20
mL). The solution was added to NH4OH (2.2 mL) in THF (40
mL) and stirred for 18 h. The reaction was concentrated, and
the residue was partitioned between EtOAc and H2O. The
organic layer was washed with H2O (2×), extracted with
NaHCO3 (2 × 40 mL), washed with brine, dried over MgSO4,
filtered, and concentrated. The crude solid was purified by
column chromatography (SiO2, 1/1 hexanes/EtOAc) to provide
0.912 g (92% yield) of 17a as a white solid: 1H NMR (300 MHz,
CDCl3) δ 8.81 (s, 1H), 8.63 (s, 1H), 8.42 (s, 1H) 6.20 (bs, 2H);
GC-MS 10.33 min (m/e 234, 100).
N-Bu tyl-3-n itr o-5-(tr iflu or om eth yl)ben zyla m id e (17b):
1H NMR (300 MHz, CDCl3) δ 8.71 (s, 1H), 8.62 (s, 1H), 8.42
(s, 1H), 6.34 (bs, 1H), 3.52 (m, 2H), 1.66 (m, 2H), 1.45 (m, 2H),
0.98 (t, J ) 6.6 Hz, 3H); GC-MS 10.86 min (m/e 290, 99).
Eth yl 3-n itr o-5-(tr iflu or om eth yl)ben zoa te (17c): 1H
NMR (300 MHz, CDCl3) δ 9.04 (s, 1H), 8.67 (s, 1H), 8.63 (s,
1H), 4.51 (q, J ) 7.2 Hz, 2H), 1.46 (t, J ) 6.9 Hz, 3H); GC-
MS 8.41 min (m/e 263, 100).
Bu tyl 3-n itr o-5-(tr iflu or om eth yl)ben zoa te (17d ): 1H
NMR (300 MHz, CDCl3) δ 9.03 (s, 1H), 8.67 (s, 1H), 8.61 (s,
1H), 4.44 (t, J ) 6.6 Hz, 2H), 1.82 (m, 2H), 1.50 (m, 2H), 1.01
(t, J ) 7.2 Hz, 3H); GC-MS 9.08 min (m/e 292, 100).
Hexyl 3-n itr o-5-(tr iflu or om eth yl)ben zoa te (17e): 1H
NMR (300 MHz, CDCl3) δ 9.04 (s, 1H), 8.68 (s, 1H), 8.62 (s,
1H), 4.43 (t, J ) 6.9 Hz, 2H), 1.82 (m, 2H), 1.46 (m, 6H), 0.94
(t, J ) 7.2 Hz, 3H); GC-MS 10.17 min (m/e 320, 98).
Octyl 3-n itr o-5-(tr iflu or om eth yl)ben zoa te (17f): 1H
NMR (300 MHz, CDCl3) δ 9.04 (s, 1H), 8.68 (s, 1H), 8.62 (s,
1H), 4.43 (t, J ) 6.9 Hz, 2H), 1.86 (m, 2H), 1.6-1.2 (m, 10H),
0.87 (m, 3H); GC-MS 11.24 min (m/e 348, 98).
Cycloh exyl 3-n itr o-5-(tr iflu or om eth yl)ben zoa te (17g):
1H NMR (300 MHz, CDCl3) δ 9.03 (s, 1H), 8.67 (m, 1H), 8.61
(m, 1H), 5.10 (m, 1H), 2.0-1.3 (m, 10 H); GC 10.50 min (98).
Dieth yl p h osp h a te m eth yl-3-n itr o-5-(tr iflu or om eth yl)-
ben zoa te (17h ): 1H NMR (300 MHz, CDCl3) δ 9.06 (s, 1H),
8.71 (s, 1H), 8.63 (s, 1H), 4.74 (d, J ) 8.7 Hz, 2H), 4.26 (m,
4H), 1.38 (t, J ) 6.9 Hz, 6H).
Dieth yl p h osp h a te m eth yl-3-a m in o-5-(tr iflu or om eth -
yl)ben zoa te (18h ): 1H NMR (300 MHz, CDCl3) δ 7.63 (s, 1H),
7.49 (s, 1H), 7.08 (s, 1H), 4.63 (d, J ) 8.4 Hz, 2H), 4.23 (m,
6H), 1.36 (t, J ) 7.2 Hz, 6H); GC-MS 12.6 min (m/e 355 M+).
Gen er a l P r oced u r e for Syn th esis of th e P yr im id in e
Am id e Der iva tives. 2-Ch lor o-4-(tr iflu or om eth yl)-5-N-
bu tylp yr im id in eca r boxa m id e (19). To a 20 mL round-
bottom flask were added Amberlyst A-21 ion-exchange resin
(0.2 g), n-butylamine (0.068 g, 0.932 mmol), and EtOAc (10
mL). A solution of 2-chloro-4-(trifluoromethyl)-5-pyrimidine
acid chloride (0.240 g, 0.981 mmol) in EtOAc (0.5 mL) was
added to the round-bottom flask and the mixture stirred for
0.3 h. Water (0.2 mL) was added and stirring continued for 5
min. The reaction was filtered, dried over MgSO4, and filtered
and the solvent removed under reduced pressure. The result-
ing crude solid was recrystallized from EtOH/H2O, providing
1
109 mg (61%) of 19 as a white solid: mp 96-97 °C; H NMR
(500 MHz, CDCl3) δ 8.90 (s, 1H), 5.90 (bs, 1H), 3.46 (q, J )
6.75 Hz, 2H), 1.58 (m, 2H), 1.42 (m, 2H), 0.97 (t, J ) 7.2 Hz,
3H); IR (KBr) 3268, 1645, 1501 cm-1. Anal. (C10H11ClF3N3-
O): C, H, N.
2-Ch lor o-4-(t r iflu or om et h yl)-5-N-p h en ylp yr im id in e-
ca r boxa m id e (20): 62% yield; mp 180-181 °C; 1H NMR (300
MHz, CDCl3) δ 9.04 (s, 1H), 7.56 (d, J ) 7.5 Hz, 2H), 7.49 (bs,
1H), 7.41 (t, J ) 7.5 Hz, 2H), 7.25 (t, J ) 7.5 Hz, 1H); IR (KBr)
3185, 1650, 1326, 1172 cm-1. Anal. (C12H7ClF3N3O): C, H,
N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-(2′,6′-dim eth ylph en yl)-
p yr im id in eca r boxa m id e (21): 27% yield; mp 248-249 °C;
1H NMR (300 MHz, CDCl3) δ 9.99 (s, 1H), 9.06 (s, 1H), 7.13
(s, 3H), 2.31 (s, 6H); IR (KBr) 3235, 1661, 1353, 1155 cm-1
Anal. (C14H11ClF3N3O): C, H, N.
.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[4′-(tr iflu or om eth yl)-
p h en yl]p yr im id in eca r boxa m id e (22): 74% yield; mp 172-
1
173 °C; H NMR (300 MHz, CDCl3) δ 11.05 (s, 1H), 9.27 (s,
1H), 7.84 (d, J ) 8.6 Hz, 2H), 7.64 (d, J ) 8.6 Hz, 2H); IR
(KBR) 3290, 1667, 1535, 1337 cm-1
0.5H2O): C, H, N.
. Anal. (C13H6ClF6N3O‚
2-Ch lor o-4-(t r iflu or om e t h yl)-5-N -(3′,5′-d im e t h oxy-
p h en yl)p yr im id in eca r boxa m id e (23): 55% yield; mp 135-
136 °C; 1H NMR (300 MHz, CDCl3) δ 9.01 (s, 1H), 7.56 (s, 1H),
6.77 (s, 1H), 6.76 (s, 1H), 6.34 (t, J ) 1.5 Hz, 1H), 3.81 (s, 6H);
IR (KBR) 3290, 1661, 1606, 1568 cm-1. Anal. (C14H11ClF3N3-
O3): C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
18a -h . 3-Am in o-5-(tr iflu or om eth yl)ben zyla m id e (18a ).
To a solution of 17a (0.550 g, 2.35 mmol) in EtOH (25 mL)
was added 5% Pd/C (30 mg). The mixture was flushed with
H2 four times and then stirred under an atmosphere of H2 for
18 h. The mixture was filtered through Celite and concen-
trated. The resulting crude material 18a (0.425 g, 89% yield)
was used without further purification: 1H NMR (300 MHz,
acetone-d6) δ 7.51(bs, 1H), 7.45 (s, 1H), 7.40 (s, 1H), 7.10 (s,
1H), 6.70 (bs, 1H), 5.30 (bs, 2H); GC-MS 10.53 min (m/e 204,
98).
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[4-(2′,6′-d ich lor op yr i-
d in o)]p yr im id in eca r boxa m id e (24): 40% yield; mp 189-
190 °C; 1H NMR (300 MHz, CDCl3) δ 9.02 (s, 1H), 8.10 (s, 1H),
7.59 (s, 2H); IR (KBr) 1711, 1579, 1210, 1167 cm-1. Anal. (C11
H4Cl3F3N4O): C, H, N.
-

418 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sullivan et al.
2-Ch lor o-4-(t r iflu or om et h yl)-5-N-[2′-(5′′-m et h ylt h io-
p h en eyl)]p yr im id in eca r boxa m id e (25): 21% yield; mp
4H), 1.37 (t, J ) 7.2 Hz, 6); IR (KBr) 1739, 1573, 1365 cm-1
.
Anal. (C19H17ClF6N3O6P): C, H, N.
1
142-143 °C; H NMR (300 MHz, CDCl3) δ 9.02 (s, 1H), 8.22
Solu tion -P h a se Libr a r y P r ep a r a tion . Amberlyst A-21
ion-exchange resin (5-10 beads) was placed into 80 wells of a
1 mL deep well microtiter plate (rows 1 and 12 open). The 80
individual amines as shown in Table 2 (100 µL, 22.4 µmol in
EtOAc) were added to the individual wells and diluted with
additional EtOAc (0.2 mL). Then to each well was added a
solution of 2-chloro-4-(trifluoromethyl)-5-pyrimidine acid chlo-
ride (100 µL, 24.6 µmol) in EtOAc. The wells were capped,
and the plate was sonicated in a desk top sonicator (Branson
1210) for 0.25 h. To each well was added H2O (30 µL), and
the plate was sonicated for an additional 0.1 h. The organic
layer (280 µL) of each well was transferred into a tared test
tube and concentrated to provide 80 individual compounds.
Each compound was analyzed by TLC (1/1 hexanes/EtOAc),
and 15 random samples were analyzed by HPLC. The test
tubes were then solvated at 5 mg/mL and submitted for
biological testing.
NF -KB Assa y. Human J urkat T-Cells stably transfected
with an NF-κB binding site (from the MHC promoter) fused
to a minimal SV-40 promoter driving luciferase were used in
these experiments.19 Cells were counted, resuspended in fresh
medium containing 10% Serum-Plus at a density of 1 × 106
cells/mL, and plated in 96-well round-bottom plates (200 µL
per well) 18 h prior to running the assays.
Compounds dissolved in 0.2% DMSO/H2O at the appropriate
concentrations (3.3, 0.33, and 0.03 µg/mL for initial evaluation
of libraries) 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 ng/mL of phorbol 12-
myristate-13-acetate (PMA) and 1 µg/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 removal
of the media; 60 µL of cell lysis buffer was added to each well,
and cells were lysed 0.25 h; 40 µL of each cell lysate was
transferred to a black 96-well plate, and 50 µL of luciferase
substrate buffer was added. Luminescence was immediately
measured using a Packard TopCount. The results are ex-
pressed as IC50 values where the IC50 value is defined as the
concentration of compound required to reduce luciferase activ-
ity to 50% of control values.
(s, 1H), 6.63 (m, 1H), 6.56 (m, 1H), 2.46 (s, 3H); IR (KBr) 3246,
1651, 1563, 1326 cm-1. Anal. (C11H7ClF3N3OS): C, H, N.
2-Ch lor o-4-(t r iflu or om et h yl)-5-N-[5′-(3′′-m et h ylisoxa -
zolyl)]p yr im id in eca r boxa m id e (26): 75% yield; mp 170-
171 °C; 1H NMR (300 MHz, acetone-d6) δ 9.42 (s, 1H), 6.74 (s,
1H), 2.27 (s, 3H); IR (KBr) 1704, 1562, 1209 cm-1. Anal. (C10
H6ClF3N4O2): C, H, N.
-
2-Ch lor o-4-(t r iflu or om e t h yl)-5-N -(3′,4′,5′-t r ich lor o-
p h en yl)p yr im id in eca r boxa m id e (27): 40% yield; mp 200-
1
201 °C; H NMR (300 MHz, CDCl3) δ 10.48 (bs, 1H), 8.91 (s,
1H), 7.85 (s, 2H), 7.59 (s, 2H); IR (KBr) 1706, 1561, 1203 cm-1
Anal. (C12H4Cl4F3N3O): C, H, N.
.
2-Ch lor o-4-(t r iflu or om et h yl)-5-N-[3′,5′-b is(t r iflu or o-
m eth yl)p h en yl]p yr im id in eca r boxa m id e (28): 75% yield;
mp 166-167 °C; 1H NMR (300 MHz, CDCl3) δ 9.05 (s, 1H),
8.10 (s, 2H), 7.86 (s, 1H), 7.76 (s, 1H); 13C NMR (300 MHz,
acetone-d6) δ 162.9, 141.2, 133.4, 132.9, 128.2, 126.3, 122.8,
122.7, 121.0, 118.7; IR (KBr) 3292, 1668, 1517, 1380 cm-1
Anal. (C14H5ClF9N3O): C, H, N.
.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-(am in ocar bon yl)ph en yl]pyr im idin ecar boxam ide (29a):
55% yield; mp 218-219 °C; 1H NMR (300 MHz, acetone-d6) δ
10.41 (bs, 1H), 9.45 (s, 1H), 8.42 (s, 1H), 8.38 (s,1H), 8.07 (s,
1H), 7.84 (bs, 1H), 6.67 (bs, 1H); IR (KBr) 1677, 1559, 1339,
1211 cm-1. Anal. (C14H7ClF6N4O2): C, H, N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-[(N-b u t yla m in o)ca r b on yl]p h en yl]p yr im id in eca r b ox-
a m id e (29b): 21% yield; mp 243-244 °C; 1H NMR (300 MHz,
acetone-d6) δ 10.38 (bs, 1H), 9.44 (s, 1H), 8.37 (s, 1H), 8.34 (s,
1H), 8.09 (bs, 1H), 7.99 (s, 1H), 3.42 (q, J ) 6.6 Hz, 2H), 1.60
(m, 2H), 1.40 (m, 2H), 0.93 (t, J ) 7.2 Hz, 3H); IR (KBr) 1652,
1586, 1360, 1211 cm-1. Anal. (C18H15ClF6N4O2): C, H, N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-(eth oxycar bon yl)ph en yl]pyr im idin ecar boxam ide (29c):
1
30% yield; mp 69-71 °C; H NMR (300 MHz, CDCl3) δ 9.06
(s, 1H), 8.30 (s, 1H), 8.25 (s, 1H), 8.17 (s, 1H), 7.94 (bs, 1H),
4.43 (q, J ) 6.9 Hz, 2H), 1.43 (t, J ) 6.9 Hz, 3H); IR (KBr)
1694, 1575, 1355, 1262 cm-1. Anal. (C16H10ClF6N3O3): C, H,
N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-(bu toxycar bon yl)ph en yl]pyr im idin ecar boxam ide (29d):
71% yield; mp 49-53 °C; H NMR (300 MHz, CDCl3) δ 9.12
AP -1 Assa y. The AP-1 assay was run as described above
for NF-κB except that the J urkat T-Cells were stably trans-
fected with a plasmid that contained an AP-1 binding site from
the collagenase promoter driving luciferase expression.19 In
addition, the concentrations of PMA and PHA were 5 ng/mL
and 1 µg/mL, respectively.
â-Act in Assa y. The â-actin assay was run as described
above for NF-κB except that the J urkat T-Cells were stably
transfected with a plasmid that contained the â-actin promoter
driving luciferase and the cells were not induced with PMA
and PHA.
In h ibition of Cytok in es. After centrifugation, superna-
tants from each well in the above luciferase experiments were
collected and stored at -20 °C until assay. Approximately 20-
50 µL aliquots were removed and cytokine levels determined
by ELISA (Biosource International, IL-2 (AP-1) and IL-8 (NF-
κB)). The results are expressed as IC50 values where the IC50
value is defined as the concentration of compound required to
reduce cytokine levels to 50% of control values.
1
(bs, 1H), 9.04 (s, 1H), 8.29 (m, 2H), 8.08 (s, 1H), 4.29 (t, J )
6.6 Hz, 2H), 1.76 (m, 2H), 1.46 (m, 2H), 0.98 (t, J ) 7.2 Hz,
3H); IR (KBr) 1708, 1574, 1267 cm-1. Anal. (C18H14ClF6N3-
O3‚0.5H2O): C, H, N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-[(h e x y lo x y )c a r b o n y l]p h e n y l]p y r im id in e c a r b o x a -
m id e (29e): 72% yield; mp 103-105 °C; 1H NMR (300 MHz,
CDCl3) δ 9.07 (s, 1H), 8.31 (s, 1H), 8.15 (s, 1H), 7.86 (bs, 1H),
4.38 (t, J ) 7.2 Hz, 2H), 1.8-0.8 (m, 11H); IR (KBr) 1704, 1560,
1350, 1206 cm-1. Anal. (C20H18ClF6N3O3): C, H, N.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-[(octyloxy)ca r bon yl]p h en yl]p yr im id in eca r boxa m id e
(29f): 35% yield; oil; 1H NMR (300 MHz, CDCl3) δ 9.05 (s,
1H), 8.73 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 8.07 (s, 1H), 4.22
(t, J ) 6.6 Hz, 2H), 1.76 (m, 2H), 1.31 (m, 10H), 0.86 (m, 3H);
IR (KBr) 3306, 1689, 1562, 1359 cm-1; HRMS calcd for C22H22
ClF6N3O3 526.1332, found 526.1339 (MH+).
-
Su p p or tin g In for m a tion Ava ila ble: Amine, aniline, and
heterocyclic amine structures for library and HTS data for
combinatorial plates (19 pages). Ordering information is given
on any current masthead page.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-[(cycloh exyloxy)ca r bon yl]p h en yl]p yr im id in eca r box-
1
a m id e (29g): 58% yield; mp 59-60 °C; H NMR (300 MHz,
CDCl3) δ 9.06 (s, 1H), 8.31 (s, 1H), 8.21 (s, 1H), 8.13 (m, 2H),
5.02 (m, 1H), 2.04-1.23 (m, 10H); IR (KBr) 3295, 1689, 1568,
1348 cm-1. Anal. (C20H16ClF6N3O3): C, H, N.
Refer en ces
(1) Manning, A. M.; Lewis, A. J . Transcription Factors, Rheumatoid
Arthritis and the Search for Improved Antirheumatic Agents.
Rheumatoid Arthritis 1997, 1, 65.
(2) Hill, C. S.; Treisman, R. Transcriptional Regulation by Extra-
cellular Signals: Mechanisms and Specificity. Cell 1995, 80, 199
and references therein.
2-Ch lor o-4-(tr iflu or om eth yl)-5-N-[3′-(tr iflu or om eth yl)-
5′-[[(d im eth yl p h osp h a te)m eth oxy]ca r bon yl]p h en yl]p y-
r im id in eca r boxa m id e (29h ): 55% yield; mp 125-126 °C;
1H NMR (300 MHz, CDCl3) δ 10.89 (s, 1H), 9.35 (s, 1H), 8.65
(s, 1H), 7.71 (s, 1H), 7.58 (s, 1H), 4.54 (d, J ) 8.1 Hz), 4.08 (m,

NF-κB- and AP-1-Mediated Gene Expression Inhibition
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 419
(3) Karin, M. Signal Transduction from the cell surface to the
nucleus through phosphorylation of transcription factors. Curr.
Biol. 1994, 6, 415.
(4) Manning, A. M. Transcription factors: A new frontier for drug
discovery. Drug Discovery Today 1996, 1, 151.
(11) Compound 1 was run in an adjuvanct arthritis model and was
found to be active at 25-30 mg/mg ip. Unpublished data, Signal
Pharmaceuticals.
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equiopotent.
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(18) Unpublished results, Signal Pharmaceuticals.
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