Inhibitors of NF-kappaB and AP-1 gene expression: SAR studies on the pyrimidine portion of 2-chloro-4-trifluoromethylpyrimidine-5-[N-(3', 5'-bis(trifluoromethyl)phenyl)carboxamide].

Article abstract of DOI:10.1021/jm0001626

We investigated the structure-activity relationship studies of N-[3, 5-bis(trifluoromethyl)phenyl][2-chloro-4-(trifluoromethyl)pyrimidin-5 -yl]carboxamide (1), an inhibitor of transcription mediated by both NF-kappaB and AP-1 transcription factors, with the goal of improving its potential oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski's rule of 5, and were examined for potential gastrointestinal permeability using the intestinal epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity. However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed comparable in vitro activity and improved Caco-2 permeability compared to 1.

Full text of DOI:10.1021/jm0001626

J . Med. Chem. 2000, 43, 3995-4004
3995
In h ibitor s of NF -KB a n d AP -1 Gen e Exp r ession : SAR Stu d ies on th e P yr im id in e
P or tion of 2-Ch lor o-4-tr iflu or om eth ylp 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]
Moorthy S. S. Palanki,* Paul E. Erdman, Leah M. Gayo-Fung, Graziella I. Shevlin, Robert W. Sullivan,
Mark J . Suto, Mark E. Goldman, Lynn J . Ransone, Brydon L. Bennett, and Anthony M. Manning
Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, California 92121
Received April 7, 2000
We investigated the structure-activity relationship studies of N-[3,5-bis(trifluoromethyl)phenyl]-
[2-chloro-4-(trifluoromethyl)pyrimidin-5-yl]carboxamide (1), an inhibitor of transcription medi-
ated by both NF-κB and AP-1 transcription factors, with the goal of improving its potential
oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski’s
rule of 5, and were examined for potential gastrointestinal permeability using the intestinal
epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of
the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a
fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity.
However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl
group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a
substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If
it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed
comparable in vitro activity and improved Caco-2 permeability compared to 1.
Ch a r t 1
In tr od u ction
The production of proinflammatory cytokines, as well
as a number of other cellular regulators, is controlled
by a family of proteins known as transcription factors
(TFs). These TFs, when activated, bind to a specific
region of DNA and upregulate gene expression. The
activation of these TFs is caused by a variety of external
signals including physiological stress, infectious agents,
and other bioregulatory molecules. Two transcription
factors, nuclear factor-κB (NF-κB) and activator pro-
tein-1 (AP-1), play an important role in mediating
inflammatory and immune responses.2 AP-1 is impor-
tant for the regulation of interleukin-2 (IL-2) production,
T-cell activation, and production of matrix metallopro-
teinases, while NF-κB is essential for the regulation of
interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6
(IL-6), interleukin-8 (IL-8), and tumor necrosis factor-R
(TNF-R).3 Therefore, the inhibition or modulation of
AP-1 and/or NF-κB activation represents an approach
for the identification of novel immunoinflammatory
agents.4,5
Previously, N-[3,5-bis(trifluoromethyl)phenyl][2-chloro-
4-(trifluoromethyl)pyrimidin-5-yl]carboxamide (1; Chart
1) was reported as an inhibitor of AP-1 and NF-κB
activation.6,7 The compound was active in several ani-
mal models of inflammation and immunosuppression
(ip, 10-20 mg/kg).5 However, the compound exhibited
poor oral activity, consistent with poor permeability in
the gastrointestinal cell line, Caco-2 (apparent perme-
ability coefficient (Papp) ) 11 ( 4 × 10-7 cm/s; Table 4).
Using Caco-2 cells as a measure of potential oral
bioavailability,8 and J urkat T cells as an in vitro
measure of efficacy, we sought to further define the key
structure-activity parameters (SAR) for this class of
compounds and identify analogues of 1 with comparable
in vitro activity and improved Caco-2 permeability.
Herein, we describe the preparation of a series of
pyrimidines modified at the 2-, 4-, 5-, and 6-positions
and the parallel synthesis of amide derivatives using a
solution-phase parallel synthesis technique. In addition,
we examined several related ring systems such as
pyridazine, pyrazine, and benzene rings in the place of
the pyrimidine ring. We identified compounds that
retained inhibitory activity against AP-1- and NF-κB-
mediated transcriptional activation while displaying
improved Caco-2 permeability.
Ch em istr y
Libraries of pyrimidine-, pyridazine-, pyrazine-, and
benzenecarboxamides were designed and prepared to
explore SARs and to identify compounds with compa-
rable in vitro activity and enhanced Caco-2 perme-
ability. The preparation of the libraries involved two
components: (1) synthesis of the appropriately substi-
* To whom correspondence should be addressed. Phone: 858-558-
7500. Fax: 858-623-0870. E-mail: mpalanki@signalpharm.com.
10.1021/jm0001626 CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/19/2000

3996 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Palanki et al.
Sch em e 1a
Sch em e 3a
a
Reagents: (a) urea, MeOH, HCl; (b) POCl3; (c) NaOH (for R
) CH3) or LiOH (for R ) CF3); (d) (COCl)2, CH2Cl2, rt, 30 min.
Ch a r t 2
if the pyrmidine ring was essential for the biological
activity observed and if other ring systems would have
improved potency and Caco-2 permeability. The use of
parallel synthesis enabled rapid preparation and iden-
tification of optimum amide substituents for each aryl
and heteroaryl investigated.
a
Reagents: (a) Mg(OCOCH2CO2Et)2, CDI, THF; (b) CH(OEt)3;
(c) urea or NH2(CR2)dNH, ∆, NaOEt; (d) POCl3; (e) aq NaOH-
THF; (g) (COCl)2, DMF, CH2Cl2; (h) Me3N, THF; (i) KF, DMF-
H2O; (j) KCN, DMF-H2O.
Sch em e 2a
Illustrated in Scheme 1 is synthesis of the pyrimidine
acid chlorides 38-46 and 51-54 used for the 2,4,5-
substituted pyrimidine libraries. The 2,4-disubstituted
pyrimidine-5-carbonyl chlorides were prepared from
various â-keto esters 4-10 or from diethyl propane-1,3-
dioate (11). The â-keto esters 4-9 and diethyl propane-
1,3-dioate (11) were commercially available; however,
thienoyl acetoacetate (10) required synthesis and was
prepared by reaction of 2-thiophenecarboxylic acid (3)
with carbonyldiimidazole and ethylmagnesium acetate.9
Treatment of the esters 4-11 with triethyl orthoformate
provided the corresponding ethoxymethylene intermedi-
ates 12-19.10 The pyrimidines 20-30 were subse-
quently prepared by treating the ethoxymethylene
derivatives with either urea or the appropriately sub-
stituted amidines such as acetamidine or trifluoro-
methylacetamidine in the presence of NaOEt.11,12
Treatment of the 2-hydroxypyrimidines 21, 23, 25,
and 26 with POCl3,13 followed by hydrolysis of the esters
with 1 N NaOH in THF provided the corresponding
2-chloro-5-carboxylic acids 32, 34, 36, and 37, respec-
tively. In the case of 20, 22, and 24, the compounds were
hydrolyzed to the corresponding acids 31, 33, and 35
respectively, without POCl3 treatment. Conversion of
the acids 31-37 to the acid chlorides 38-44 was
accomplished using either oxalyl chloride in CH2Cl2 (in
the case of 2-chloro acids) or phosphorus oxychloride (in
the case of 2-hydroxy acids). The 2-fluoro-5-carbonyl
a
Reagents: (a) DMF, POCl3; (b) NH2C(SMe)dNH; (c) NH2-
C(R2)dNH; (d) KOH; (e) NaOH, THF-H2O; (f) POCl3; (g) (COCl)2,
CH2Cl2.
tuted aryl or heteroaryl acid chlorides and (2) reaction
of the acid chlorides with various commercially available
amines using solution-phase parallel synthesis tech-
niques. For the pyrimidine libraries, a variety of 2,4,5-,
2,5-, and 2,4,6-substituted pyrimidine acid chlorides
(Schemes 1-3) were prepared in order to determine the
optimum pyrimidine substituents and substitution pat-
tern. Various pyridazine, pyrazine, and benzene acid
chlorides (Chart 2) were also synthesized to determine

Inhibitors of NF-κB and AP-1 Gene Expression
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3997
chloride 45 and the 2-cyano-5-carbonyl chloride 46 were
prepared from the corresponding 2-chloro-5-carboxylic
acid 32 by reaction with Me3N in THF, followed by KF
or KCN in DMF/H2O, respectively,14 and finally with
oxalyl chloride in CH2Cl2.
The 2-methyl- and 2-trifluoromethylpyrimidine acid
chlorides 51-54 were prepared in a similar fashion. The
pyrimidine esters 27-30 were first hydrolyzed to the
corresponding carboxylic acids 47-50 using NaOH. The
acids 48-50 were then converted to the acid chlorides
52-54 using oxalyl chloride. For the 4-chloropyrimidine
acid chloride 51, the 4-hydroxypyrimidine 47 was re-
fluxed in POCl3.
Amberlyst 21, which is a basic ion-exchange resin. The
Amberlyst 21 functioned as a solid-phase basic scaven-
ger and adsorbed any HCl produced in the reaction,
allowing the limited amount of amine present to react.
Sonication of the plates provided sufficient agitation of
all the reactants. Following complete reaction of each
amine, a small amount of water was added to the wells
to tranform any unreacted acid chloride into the corre-
sponding carboxylic acid. The Amberlyst 21 present
would also adsorb the carboxylic acid onto the resin. The
products were easily isolated by transferring the organic
layer of each well into tared test tubes followed by
concentration of the solutions.
Prior to testing, the purity of each compound within
a library (80 compounds constituted one library) was
determined using thin-layer chromatography (TLC). By
TLC, all of the compounds were clean products with no
residual starting materials present. Fifteen to twenty
compounds from each library were randomly selected
and analyzed by high-performance liquid chromatogra-
phy (HPLC) and electron-impact mass spectroscopy
(EIMS) for purity and structural verification, respec-
tively. Molecular weights of the randomly selected
compounds were confirmed, and all compounds had
>90% purity.
Scheme 2 illustrates the synthesis of 2-substituted
pyrimidine-5-carbonyl chlorides 61, 64, and 65. Reaction
of potassium ethyl malonate with POCl3 and DMF
provided ethyl 3-(dimethylamino)-2-formylprop-2-enoate
(56),15 which was treated with S-methylthiopseudourea,
trifluoroacetamidine, and benzamidine to give the py-
rimidines 57,16 58,17 and 59,18 respectively. The hy-
drolysis of 2-methylthiopyrimidine carboxylate (57) with
NaOH was slow and resulted in incomplete reaction.
Hydrolysis using KOH, however, was efficient and
provided the pyrimidinecarboxylic acid 60, which was
then refluxed in POCl3 to give the 2-chloropyrimidine
acid chloride 61. The 2-trifluoromethyl- and 2-phenyl-
pyrimidine acid chlorides (64 and 65) were prepared
from the corresponding pyrimidine esters (58 and 59,
respectively) as described above.
The synthesis of two 2,4-disubstituted pyrimidine-6-
carbonyl chlorides 72 and 73 is illustrated in Scheme
3. Methyl and trifluoromethyl â-keto esters (66 and 67)
were treated with urea under acidic conditions to
provide the 2-hydroxy-4-methylpyrimidine-6-carboxy-
late and the 2-hydroxy-4-trifluoromethylpyrimidine-6-
carboxylate, respectively (68 and 69).19 These com-
pounds were hydrolyzed to the corresponding carboxylic
acids 70 and 71, treated with POCl3, and then reacted
with oxalyl chloride to provide 2-chloro-4-methylpyri-
midine-5-carbonyl chloride (72) and 2-chloro-4-trifluo-
romethylpyrimidine-5-carbonyl chloride (73). Yields from
this two-step procedure were higher than yields ob-
tained from the one-pot procedure described earlier.
In addition to preparing a variety of 2,4,5-, 2,5-, and
2,4,6-substituted pyrimidines, various pyrimidine ring
isosteres, such as pyridazine, pyrazine, and benzene,
were also synthesized. 2,4-Dichloropyrimidine-5-carbo-
nyl chloride (74),10 2,4-dichloropyrimidine-6-carbonyl
chloride (75),10 3-chloro-5-trifluoromethylpyridazine-6-
carbonyl chloride (76),20 3-chloropyridazine-6-carbonyl
chloride (77),21 2-chloropyrazine-5-carbonyl chloride
(78),22 and 4-chloro-2-trifluoromethylbenzoyl chloride
(79),23 shown in Chart 2, were synthesized using
literature procedures referenced herein.
The amide sublibraries were prepared by treating the
aryl and heteroaryl acid chlorides (38-46, 51-54, 61,
64, 65, 72-79) with a variety of amines and anilines
using solution-phase parallel synthesis.7,24,25 In a mi-
crotiter format, each acid chloride described above was
reacted with 160 amines and anilines using two plates
(80 compounds/plate). The synthesis involved adding
1.05 equiv of each acid chloride to 80 wells of a
microtiter plate with each well containing one amine
or aniline in EtOAc. Also present in each well was
Biology
High-throughput screening and followup studies were
performed using three distinct cell lines. J urkat T-cells
stably transfected with either an NF-κB-dependent, an
AP-1-dependent, or the â-actin26 promoter driving lu-
ciferase were pretreated for 0.5 h with compounds
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 centrifuga-
tion for determination of luciferase activity. The results
are expressed as IC50 values (Tables 1-3) where the IC50
value is defined as the concentration of compound
required to reduce luciferase activity to 50% of control
values.
Compounds that were active in the cell-based assay
were studied for their Caco-2 permeability.27 Com-
pounds were dissolved in DMSO, then added to the
apical layer, and monitored in the basolateral layer. The
concentrations of the compound on the basolateral side
were measured using HPLC equipped with a YMC C-18
column at 254 nm. The concentrations were calculated
by comparing to standard curves. The results of the
permeability coefficient of the compounds are sum-
marized in Table 4.
Resu lts a n d Discu ssion
The goal of the current study was to further define
the optimum substituents at the 2-, 4-, and 6-positions
of the pyrimidine ring, as well as the importance of the
pyrimidine ring itself, in an effort to identify analogues
of 1 with comparable potency and improved Caco-2
permeability. Through the use of solution-phase parallel
synthesis, we were able to determine the optimum
amide functionality for each new pyrimidine synthe-
sized. This study also resulted in the identification of
the optimum positioning of the substituents on the
pyrimidine ring (2, 4, 5 vs 2, 4, 6).

3998 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Palanki et al.
Ta ble 4. Caco-2 Permeability of Active Compounds
Ta ble 1. Inhibition of AP-1- and NF-κB-Mediated
Transcriptional Activation in J urakat Cells
Caco-2 permeability,
×10-7 cm/s
follows
Lipinski’s
rule?
IC50, µM
no.
R4
R2
(n ) 3; mean ( SD)
no.
R4
R2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
CH3
CF3
CN
CF3
CF3
Ph
AP-1
NF-kB
1
80
81
82
83
84
85
86
87
88
CF3
H
CH3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
11 ( 4
61 ( 7
62 ( 6
58 ( 9
31 ( 6
9 ( 3
31 ( 6
9 ( 7
21 ( 3
19 ( 6
yes
yes
yes
yes
yes
yes
yes
no
1
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
CF3
H
CH3
0.05
0.05
0.3
0.05
0.4
0.5
0.8
0.6
0.4
9.8
0.4
0.3
0.02
0.3
0.5
0.6
0.2
0.4
8
CH3CH2
CH3CH2CH2
CF3CF2
Cl
CH3CH2
CH3CH2CH2
CF3CF2
Cl
Ph
Ph
2′-thienyl
CF3
yes
yes
2′-thienyl
CF3
CF3
CF3
CF3
Cl
0.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
derivatives (>50% at the 0.3 µg/mL dose) were then run
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 against the â-actin control at the highest dose
tested (3.3 µg/mL; data not shown).
H
H
CH3
CF3
CF3
CH3
Tables 1-3 contain the IC50 values for a select group
of 3′,5′-bis(trifluoromethyl)aniline pyrimidine deriva-
tives. Originally, the compounds were prepared as part
of the libraries. The library was composed of individual,
pure compounds (>90%) in 5 ( 2 mg. This quantity of
material is enough to test a compound at three different
doses in three assays (AP-1, NF-κB, and â-actin assays).
However, the individual, discrete compounds shown in
Table 1 were also prepared in large quantities since each
of the compounds listed in Table 1 was the most potent
member of the sublibrary. The large-scale synthesis of
the pure (>99%) potent compounds enabled us to fully
characterize them using various analytical methods and
evaluate them in various assays.
Ta ble 2. Inhibition of AP-1- and NF-κB-Mediated
Transcriptional Activation in J urakat Cells
IC50, µM
no.
R4
R2
AP-1
NF-kB
97
98
99
CF3
CH3
Cl
Cl
Cl
Cl
>10
>10
> 10
>10
>10
>10
4-P osition Der iva tives. Examination of the various
substituents in the 4-position in which the 2-position
was held constant (chloro) indicated that small lipophilic
groups were best tolerated (80-87). However, a sub-
stituent other than a hydrogen was needed as indicated
by the decrease in activity as seen with the unsubsti-
tuted derivative (1 vs 80). Comparing the 4-chloro
derivative (85) to the 4-CF3 (1), we observed a loss in
activity even though these substituents are of compa-
rable size and have similar properties. An interesting
observation was the difference in activity between the
4-phenyl (86) and 4-thienyl (87) derivatives. Thiophene
had been reported as an isostere for a phenyl group.
However, in this series the phenyl derivative was
considerably more active.
Ta ble 3. Inhibition of AP-1- and NF-κB-Mediated
Transcriptional Activation in J urakat Cells
IC50, µM
no.
R4
X
Y
Z
AP-1
NF-kB
100
101
102
103
CF3
CF3
H
CH
CH
CH
N
CH
N
N
CH
N
N
>30
10
>30
2-P osition Der iva tives. Examination of the 2-posi-
tion reveals that the chloro substituent is optimal and
absolutely required. Reversing the 2- and 4-substituents
(1 vs 92) resulted in a loss of activity. In addition, any
modification to the 2-position (CH3, CF3, CN, Ph)
resulted in inactive derivatives (89-91). Only the
2-fluoro compound had inhibitory activity (1 vs 88).
10
1.7
4.0
1.7
4.0
H
CH
N
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

Inhibitors of NF-κB and AP-1 Gene Expression
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3999
Ch a r t 3
2,4,5- vs 2,4,6-P osition P yr im id in es. To investigate
the importance of the positioning of the carboxamide
group with relation to the substituents at the 2- and
4-positions, we prepared the corresponding 2-chloro-4-
substituted-6-carboxamide libraries as well as the in-
dividual 2-chloro-4-methyl, 4-trifluoromethyl, and 4-chlo-
ro derivatives 97-99. The preparation of this library
was critical since 2,4,6-substituted pyrimidines could
have a different SAR profile than the 2,4,5-substituted
compounds. Of interest was the benzyl derivatives
prepared as part of the library, since the extended chain
would in theory allow for a similar orientation to the
parent carboxamide 1. Upon evaluation of the com-
pounds, it was found that none had potency in our
assays. This was surprising due to the similarity in
structures and indicated that a very critical interaction
or binding was occurring.
opportunity for identifying a compound with the desired
characteristics. The substitution of the trifluoromethyl
group with a methyl or phenyl group still resulted in
submicromolar inhibitors of NF-κB and AP-1 activation.
The methyl analogue 81 was comparable in activity in
the whole-cell assay.
Oth er Heter ocycles. Having established that only
2,4,5-substituted pyrimidines were active, we next
turned our attention to the criticality of the pyrimidine
ring. Table 3 contains several other ring systems with
the various bis(trifluoromemthyl) derivatives examined
(100-103). First, the 2-chloro-4-trifluorobenzamide 100
was prepared and found to be completely inactive. Next,
several pyrimidine isomers were examined including the
1,2- and 1,4-positional isomers (pyridazines 101, 102
and pyrazine 103). The results for the pyridazine ring
system were somewhat different in that the 5-hydrogen
compound 102 was more potent than the 5-trifluoro-
methyl compound 101. Compound 101 was 300-fold less
potent than 1 (10 µM vs 30 nM). However, 102 was
comparable in potency to the corresponding unsubsti-
tuted pyrimidine 80. The 1,4-isomer 103 had compa-
rable potency to 102, (4 vs 1.7 µM), indicating that a
heterocyclic ring is critical for activity.
Ca co-2 P er m ea bility. Compounds 1 and 80-88
were tested in the Caco-2 assay (gastrointestinal cell
line). Compound 1 exhibited poor permeability in the
Caco-2 (apparent permeability coefficient (Papp) ) 11 (
4 × 10-7 cm/s; Table 4). The corresponding nor-trifluo-
romethyl analogue 80 and 4-methyl analogue 81 showed
increased Caco-2 permeability. The 4-ethyl analogue
also exhibited increased permeability. The 4-pentafluo-
roethyl analogue 84, 4-chloro analogue 85, 4-phenyl
analogue 86, and 4-(2′-thienyl) analogue 87 all exhibited
low Caco-2 permeability. The 2-fluoro-4-trifluorometh-
ylpyrimidine analogue 88 also exhibited low Caco-2
permeability.
The pyridazine analogue was less active than the
pyrimidine analogue (101 vs 1). A similar trend was
observed with the pyrazine analogues (103 vs 1). Several
attempts to prepare the corresponding pyrazine ana-
logue of 1 maintaining the trifluoromethyl substituent
were unsuccessful. The introduction of a phenyl ring in
place of the pyrimidine ring resulted in a loss of activity
(100 vs 1). The orientation of groups on the pyrimidine
ring was more crucial to activity, and the heterocyclic
variation resulted in a less dramatic effect on activity.
Among the compounds synthesized, 81 was comparable
to 1 in activity in the in vitro assay. Table 4 shows the
list of active compounds that were tested in the Caco-2
assay.28 We also performed calculations on these com-
pounds using Lipinski’s rule of 5.29 All the compounds
except compound 86 followed Lipinski’s rule. Com-
pounds 80-82 showed superior Caco-2 permeability
(Table 4) than 1. Thus we identified a compound that
is comparable in activity to 1 with superior Caco-2
permeability and potential oral bioavailability.
Exp er im en ta l Section
Melting points (uncorrected) were obtained on a Mel-Temp-
II using capillary tubes. Proton nuclear magnetic resonance
spectra were obtained on a Varian Gemini 2000 spectrometer
operating at 300 MHz for proton with tetramethylsilane as
an internal standard, and chemical shifts are reported on the
δ scale. Infrared spectra were obtained on a Nicolet Impact
400D spectrometer. Electron impact mass spectra (EIMS) were
Con clu sion s
obtained on
a Hewlett-Packard 5890 Series II plus gas
As described previously, compound 1 was a potent
inhibitor of NF-κB and AP-1 activation. The compound
was active in several animal models of inflammation
but only when administered ip. The major focus of the
work described here was the identification of the
optimum combination of 2-, 4-, and 5-position substit-
uents that would provide a compound with improved
potential oral bioavailability. To this end, approximately
3200 analogues of 1 were prepared and evaluated in
vitro. The results of the SAR studies are summarized
in Chart 3. The most active derivatives all contained a
chloro substituent at the 2-position of the pyrimidine
ring. Examination of a variety of substituents at the
4-position indicated that this may provide a better
chromatography coupled to a Hewlett-Packard 5972 mass
selective detector. Electrospray ionization (ESMS) was ob-
tained on a Hewlett-Packard 1100 MSD at Mass Consortium
in San Diego, CA. Flash chromatography was performed using
silica gel 60 (230-400 mesh). High-performance liquid chro-
matography was performed on a Rainin Dynamax system
equipped with a YMC C-18 column using acetonitrile contain-
ing 35% water and 0.1% CF3COOH. Elemental analyses were
performed at Desert Analytics, Tucson, AZ, and were within
0.4% of the calculated values. All reactions requiring anhy-
drous conditions and/or an inert atmosphere were performed
under positive nitrogen atmosphere. Tetrahydrofuran was
distilled from sodium benzophenone ketyl. All other solvents
and reagents were used as received from commercial suppliers.
Eth yl Eth oxym eth ylen e-4,4,4-tr iflu or oacetoacetate (12).
A solution of 4,4,4-trifluoroacetoacetate (46 g, 0.25 mol),

4000 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Palanki et al.
triethyl orthoformate (74 g, 0.50 mol), and Ac2O (77 g, 0.75
mol) was heated under N2 at 120 °C for 2 h and 140 °C for 5
h. The solution was concentrated to an oil and distilled to
obtain the title compound (58.6 g, 98%; bulb-to-bulb distilla-
tion, 80-90 °C, 1.5 mm/Hg): 1H NMR (CDCl3) δ 7.80 (d, 1H),
4.3 (m, 4H), 1.3 (m, 6H); IR (neat) 2992, 1741, 1623, 1590,
1202, 1019 cm-1; EIMS m/z 240 (M+).
1711, 1583, 1470, 1375, 1277, 1215, 1107, 1022 cm-1; EIMS
m/z 182 (M+).
E t h yl
4-et h yl-2-h yd r oxyp yr im id in e-5-ca r b oxyla t e
1
(24): yield 81%; mp 215-217 °C; H NMR (DMSO-d6) δ 8.47
(s, 1H), 8.30 (s, 1H), 4.09 (q, J ) 7.2 Hz, 2H), 2.69 (q, J ) 7.2
Hz, 2H), 1.22 (t, J ) 7.2 Hz, 3H), 1.04 (t, J ) 7.2 Hz, 3H); IR
2981, 1716, 1579, 1497, 1367, 1275, 1205, 800 cm-1; EIMS m/z
196 (M+).
Eth yl Ur eid om eth ylen ep en ta flu or op r op ion yla ceta te
(13). A solution of ethyl pentafluoropropionylacetate (5 g, 21
mmol), urea (1.3 g, 21 mmol), and triethyl orthoformate (3.2
g, 21 mmol) was heated at reflux for 3 h. The reaction mixture
was cooled, and the solid was collected by filteration. The solid
was washed with water, ether, and dried to give the title
E t h yl 2-h yd r oxy-4-p r op ylp yr im id in e-5-ca r b oxyla t e
1
(25): yield 72%; mp 185-186 °C; H NMR (DMSO-d6) δ 8.75
(bs, 1H), 8.04 (s, 1H), 4.21 (q, J ) 6.8 Hz, 2H), 2.85 (t, J ) 8.0
Hz, 2H), 1.59 (m, 2H), 1.26 (t, J ) 6.8 Hz, 3H), 0.90 (t, J ) 8.0
Hz, 3H); IR (neat) 2962, 1747, 1687, 1475, 1275 cm-1; EIMS
m/z 209 (M - H+).
1
compound (4.4 g, 68%) as a white solid: mp 160-162 °C; H
NMR (DMSO-d6) δ 9.90 (s, 1H), 8.4 (s, 1H), 7.5 (d, 2H), 4.1 (q,
J ) 7.2 & 6.9, 2H), 1.19 (t, J ) 7.2 Hz, 3H); IR (KBr) 3088,
2910, 1749, 1670, 1402, 1173, 1099 cm-1; EIMS m/z 258 ((M
- EtOH)+).
E t h yl 2-h yd r oxy-4-p h en ylp yr im id in e-5-ca r b oxyla t e
(26): yield 65%; mp >260 °C dec; 1H NMR (DMSO-d6) δ 1.00
(t, 3H, J ) 7.2 Hz), 4.02 (q, 2H, J ) 6.9 Hz), 7.46 (m, 5H),
8.05 (s, 1H), 10.30 (s, 1H); IR (KBr) 2981, 1713, 1587, 1547,
1261, 1105 cm-1; EIMS m/z 243 (M - H+).
Eth yl Ur eid om eth ylen ea cetoa ceta te (14). A mixture of
ethyl acetoacetate (200 g, 1.54 mmol), urea (105 g, 1.54 mmol),
and triethyl orthoformate (228 g, 1.54 mmol) was heated at
140 °C under N2 for 22 h. The reaction mixture was cooled,
and filtered. The solid was washed with Et2O (3 × 100 mL),
water (3 × 150 mL), and dried under vacuum to provide 156
g (51%) of the title compound: mp 173-174 °C; 1H NMR
(DMSO-d6) δ 11.42 (d, J ) 12.9 Hz, 1H), 8.46 (d, J ) 12.6 Hz,
1H), 7.72 (s, 1H), 7.26 (s, 1H), 4.17 (q, J ) 7.2 Hz, 2H), 2.42
(s, 3H), 1.27 (t, 3H); IR (KBr) 3418, 2995, 1709, 1655, 1558,
1466, 1391, 1234, 1062 cm-1; EIMS m/z 200 (M+).
Eth yl 4-Hyd r oxy-2-tr iflu or om eth ylp yr im id in e-5-ca r -
boxyla te (27). A solution of diethyl ethoxymethylenemalonate
(35.0 g, 162 mmol), trifluoroacetamidine (18 g, 162 mmol) and
NaOEt (11.0 g, 162 mmol) in EtOH (200 mL) was heated at
reflux under N2 for 6 h. The reaction mixture was concentrated
under reduced pressure and treated with H2O (48 mL). The
solid was filtered and washed with Et2O (300 mL) and H2O
(200 mL), and dried under vacuum to give the title compound
1
(21 g, 50%): mp > 220 °C (turned brown); H NMR (DMSO-
d6) δ 8.38 (s, 1H), 4.16 (q, J ) 7.2 & 6.9 Hz, 2H), 1.25 (q, J )
7.2 Hz, 3H); IR (KBr) 3230, 2932, 1689, 1657, 1471, 1376, 1280,
1206,1062 cm-1; EIMS m/z 236 (M+).
Eth yl u r eid om eth ylen ep r op ion oyla ceta te (15): yield
1
37%; mp 148-150 °C; H NMR (CDCl3) δ 10.2 (dd, J ) 12.6
Hz, 1H), 8.39 (d, J ) 12.6 Hz, 1H), 7.67 (bs, 1H), 7.29 (s, 1H),
4.17 (m, 2H), 2.80 (q, J ) 7.2 Hz, 2H), 1.25 (t, J ) 7.2 Hz,
3H), 0.96 (t, J ) 6.0 Hz, 3H); IR (KBr) 3386, 2995, 1713, 1643,
1552, 1230 cm-1; EIMS m/z 214 (M+).
Eth yl u r eidom eth ylen ebu ton oylacetate (16): yield 42%;
mp 153-154 °C; 1H NMR (CDCl3) δ 10.45 (d, J ) 12 Hz, 1H),
8.56 (dd, J ) 12 Hz, 1H), 4.27 (m, 2H), 2.96 (t, J ) 7.2 Hz,
2H), 2.78 (t, J ) 7.2 Hz, 2H0, 2.55 (s, 2H), 1.35 (m, 3H), 0.94
(q, J ) 7.2 Hz, 3H); IR (neat) 3396, 3209, 2950, 1703, 1643,
1552, 1221, 1147, 766 cm-1; EIMS m/z 228 (M+).
Eth yl 4-m eth yl-2-tr iflu or om eth ylp yr im id in e-5-ca r box-
yla te (28): yield 55%; H NMR (CDCl3) δ 9.26 (s, 1H), 4.51
1
(q, J ) 6.9 Hz, 2H), 2.82 (s, 3H), 1.46 (t, J ) 7.2 Hz, 3H); IR
(KBr) 2863, 1748, 1652, 1593, 1487, 1238 cm-1; EIMS m/z 234
(M+).
Eth yl 2-m eth yl-4-tr iflu or om eth ylp yr im id in e-5-ca r box-
yla te (29): yield 58%; 1H NMR (CDCl3) δ 9.13 (s, 1H), 4.46 (q,
J ) 7.2 Hz, 2H), 2.90 (s, 3H), 1.38 (t, J ) 7.1 Hz, 3H); IR (KBr)
2932, 1758, 1668, 1620, 1418, 1381, 1275, 1179 cm-1; EIMS
m/z 234 (M+).
Eth yl u r eid om eth ylen eben zoyla ceta te (17): yield 55%;
mp 124-126 °C; 1H NMR (CDCl3) δ 11.43 (d, J ) 12.9 Hz,
1H), 8.44 (d, J ) 12.7 Hz, 1H) 7.72 (s, 1H), 7.3 (m, 6H), 4.17
(q, J ) 7.2 Hz, 2H), 1.27 (t, J ) 7.1 Hz, 3H); IR (KBr) 3411,
2994, 1711, 1644, 1551, 1472 cm-1; EIMS m/z 246 (M+).
Eth yl Ur eid om eth ylen eth ien oyla ceta te (18) a n d Di-
eth yl Tr iflu or oa ceta m id om eth ylen em a lon a te (19). These
compound were ring-closed to 20 and 27, respectively, under
the reaction conditions.
Eth yl 2-h yd r oxy-4-(2′-th ien yl)p yr im id in e-5-ca r boxy-
la te (20): yield 51%; mp >220 °C; 1H NMR (CDCl3) δ 8.55 (s,
1H), 7.82 (dd, J ) 1.2, 3.9 Hz, 1H), 7.59 (dd, 1H, 0.9, 5.1 Hz,
1H), 7.11 (m, 1H), 5.25 (bs, 1H), 4.31 (q, J ) 7.2 Hz, 2H), 1.33
(t, J ) 6.9 Hz, 3H), IR (neat) 2995, 1714, 1647, 1605, 1409,
1290 cm-1; ESMS m/z 251 (M + H+).
Eth yl 2-h yd r oxy-4-tr iflu or om eth ylp yr im id in e-5-ca r -
boxyla te (21): yield 45%; 1H NMR (DMSO-d6) δ 9.89 (s, 1H),
8.41 (bs, 1H), 4.0 (q, J ) 7.2 Hz, 2H), 1.2 (t, J ) 7.1 Hz, 3H);
EIMS m/z 236 (M+).
Eth yl 2,4-Bis(tr iflu or om eth yl)p yr im id in e-5-ca r boxy-
la te (30). A solution of ethyl ethoxymethylene-4,4,4-trifluo-
roacetoacetate (15 g, 62.5 mmol), and trifluoroacetamidine
(12.6 g, 112.5 mmol) in EtOH (50 mL) was heated at reflux
for 24 h under N2. The reaction mixture was cooled to room
temperature and concentrated to an oil under reduced pres-
sure. Flash chromatography (SiO2, 20% ethyl acetate in
hexane) afforded the title compound as an oil (7.0 g, 39%): 1H
NMR (CDCl3) δ 9.37 (s, 1H), 3.70 (q, J ) 7.2 & 6.9 Hz, 2H),
1.27 (t, J ) 7.2 Hz, 3H); IR (neat) 2978, 2864, 1732, 1216, 1159
cm-1; EIMS m/z 287 (M+).
2-Hyd r oxy-4-(2′-th ien yl)p yr im id in e-5-ca r boxylic a cid
1
(31): yield 89%; mp > 200 °C; H NMR (DMSO-d6) 8.37 (s,
1H), 7.81 (m. 2H), 7.15 (m, 1H); IR (KBr) 2994, 1630, 1605,
1420, 1400, 1215 cm-1; ESMS m/z 221 (M - H+). Anal.
(C9H6N2O3S) C, H, N.
2-Ch lor o-4-tr iflu or om eth ylpyr im idin e-5-car boxylic acid
(32): mp 158 °C; 1H NMR (DMSO-d6) δ 12.4 (bs, 1H), 9.36 (s,
1H); ESMS m/z 225 (M - H+).
Eth yl 2-h yd r oxy-4-p en ta flu or oeth ylp yr im id in e-5-ca r -
boxyla te (22): yield 66%; mp 171 °C; 1H NMR (DMSO-d6) δ
8.56 (s, 1H), 4.15 (q, 2H), 1.23 (t, 3H); IR 2968, 1730, 1653,
1420, 1179, 1221 cm-1; EIMS m/z 258 (M+).
E t h yl 2-H yd r oxy-4-m et h ylp yr im id in e-5-ca r b oxyla t e
(23). A solution of ethyl ureidomethylene acetoacetate (50 g,
250 mmol), NaOEt (22.1 g, 325 mmol) in EtOH (500 mL) was
stirred at room temperature under N2 for 3 days. The white
precipitate was collected by filtration and washed with EtOH
and dried under reduced pressure to yield 45 g (88%) of the
title compound as the sodium salt: mp >220 °C dec; 1H NMR
(DMSO-d6) δ 12.22 (bs, 1H), 8.75 (s, 1H), 4.25 (q, J ) 7.2 &
6.7 Hz, 2H), 1.31 (t, J ) 7.2 Hz, 3H); IR (KBr) 2646, 1755,
2-H yd r oxy-4-p en t a flu or oet h ylp yr im id in e-5-ca r b oxy-
lic a cid (33): yield 98%; mp >210 °C dec; H NMR (DMSO-
d6) δ 9.9 (bs, 1H), 8.43 (s, 1H); IR (KBr) 3232; 2964, 1732, 1657,
1427, 1223, 1176 cm-1; ESMS m/z 257 (M - H+). Anal.
(C7H3F5N2O3‚1.25H2O) C, H, N.
2-Ch lor o-4-m eth ylp yr im id in e-5-ca r boxylic Acid (34).
A solution of ethyl 2-chloro-4-methylpyrimidine-5-carboxylate
(1.0 g, 5 mmol), NaOH (0.24 g, 6 mmol), and H2O (30 mL) was
stirred at room temperature for 3 h. The solution was acidified
to below pH ) 1 with 6 N HCl and cooled to 0 °C. The white
solid was filtered, and dried to give 0.67 g (78%) of the title
compound: 1H NMR (DMSO-d6) δ 9.01 (s, 1H), 2.75 (s, 3H);
13C NMR (DMSO-d6) δ 171.95, 165.33, 161.59, 161.47, 123.33,
1

Inhibitors of NF-κB and AP-1 Gene Expression
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4001
23.80; IR (KBr) 3230, 2991, 1732, 1578, 1514, 1423, 1275, 1174
cm-1; ESMS m/z 172 (M+).
5-carboxylic acid (2.80 g, 12.4 mmol) in dry THF (50 mL) at 0
°C was stirred with Me3N (gas, 5 min). A preceipitate was
immediately formed. The solid was filtered (3.40 g, 97%), and
washed with dry THF to give 2-trimethylammonium 4-tri-
fluoromethylpyrimidine-5-carboxylic acid chloride: mp 120-
121 °C dec; 1H NMR (DMSO-d6) δ 10.82 (bs, 1H), 9.20 (2, 1H),
4-E t h yl-2-h yd r oxyp yr im id in e-5-ca r b oxylic a cid (35):
1
yield 87%; mp >200 °C; H NMR (CDCl3) δ 9.11 (s, 1H), 8.12
(s, 1H), 3.24 (q, J ) 7.5 Hz, 2H), 1.32 (t, J ) 7.5 Hz, 3H); ESMS
m/z 186 (M - H+).
3.62 (s, 9H); IR (KBr) 3510, 2994, 1705, 1507, 1196, 990 cm-1
;
2-Ch lor o-4-p r op ylp yr im id in e-5-ca r b oxylic Acid (36).
Ethyl 2-chloro-4-propylpyrimidine-5-carboxylate (25) was hy-
drolyzed with NaOH to give the title compound (88%): mp
201 °C, 1H NMR (CDCl3) δ 9.18 (s, 1H), 3.19 (t, J ) 6.9 Hz,
2H), 1.79 (m, 2H), 1.05 (t, J ) 7.0 Hz, 3H); ESMS m/z 199 (M
- H+).
ESMS m/z 285 (M+). A solution of 2-trimethylammonium
chloride 4-trifluoromethylpyrimidine-5-carboxylic acid (75 mg,
0.26 mmol) and potassium fluoride (20 mg, 0.34 mmol) in
DMF-water (50-50, 3 mL) was stirred for 0.5 h. The reaction
mixture was acidified with 1 mL of 1 N HCl and extracted
with EtOAc. The organic layer was concentrated to give
2-fluoro-4-trifluoromethylpyrimidine-5-carboxylic acid (40 mg,
73%): 1H NMR (DMSO-d6) δ 9.41 (s, 1H); IR (KBr) 3448, 2995,
1757, 1656, 1455, 1172, 995 cm-1; ESMS m/z 210 (M+). A
solution of 2-fluoro-4-trifluoromethylpyrimidine-5-carboxylic
acid (40 mg, 0.19 mmol), oxalyl chloride (0.2 mL), and DMF
(1 drop) was stirred for 1 h at room temperature. The reaction
mixture was concentrated to give the title compound (40 mg,
93%): 1H NMR (CDCl3) δ 9.42 (s, 1H); IR (neat) 2945, 1786,
1581, 1450, 1412, 1169, 945 cm-1; EIMS m/z 228 (M+).
2-Ch lor o-4-p h en ylp yr im id in e-5-ca r boxylic Acid (37).
A solution of ethyl 2-hydroxy-4-phenylpyrimidine-5-carboxy-
late (2.0 g, 8.1 mmol) and POCl3 was heated at reflux for 1 h
under N2 atmosphere. The reaction mixture was poured on
crushed ice, and extracted with EtOAc (4 × 50 mL). The EtOAc
layers were combined and concentrated. Flash chromatography
of the residue afforded ethyl 2-chloro-4-phenylpyrimidine-5-
carboxylate (1.5 g, 72%): mp 45-47 °C; 1H NMR (CDCl3) δ
8.96 (s, 1H), 7.6-7.4 (m, 5H), 4.25 (t, 2H, J ) 6.9 Hz), 1.14 (t,
3H, J ) 7.2 Hz); IR (KBr) 2998, 1703, 1645, 1560, 1213 cm-1
;
EIMS m/z 262 (M+). A solution of ethyl 2-chloro-4-phenyl-
pyrimidine-5-carboxylate (1.5 g, 5.7 mmol) and NaOH (0.34
g, 8.58 mmol) in 20% THF in water (50 mL) was stirred at
room temperature for 2 h. The reaction mixture was made
acidic with 1 N HCl and EtOAc. The EtOAc layer was removed
under reduced pressure to give the title compound (0.71 g,
53%): mp 108-110 °C; 1H NMR (DMSO-d6) δ 9.05 (s, 1H),
7.55 (m, 5H); IR (KBr) 2983, 1724, 1556, 1400, 1295, 1192
cm-1; ESMS m/z 222 (M+).
2-Cyan o-4-tr iflu or om eth ylpyr im idin e-5-car bon yl Ch lo-
r id e (46). A solution of 2-trimethylammonium chloride-4-
trifluoromethylpyrimidine-5-carboxylic acid (3.62 g, 12.7 mmol)
and KCN (0.988 g, 15.2 mmol) in DMF (36.5 mL) and H2O
(18.3 mL) was stirred at room temperature under nitrogen for
0.25 h. The reaction mixture was concentrated under reduced
pressure and dissolved in EtOAc (400 mL). The EtOAc layer
was washed with H2O (4 × 100 mL), brine (100 mL), and dried
(Na2SO4). The EtOAc layer was filtered, and concentrated
under reduce pressure to yield the title compound (2.03 g,
74%): mp 148-149 °C dec; 1H NMR (DMSO-d6) δ 9.56 (s); IR
2-Ch lor o-4-tr iflu or om eth ylpyr im idin e-5-car bon yl Ch lo-
r id e (39). The material was identical to the commercially
obtained sample.30
(KBr) 3331, 2996, 2226, 1705, 1663, 1588, 1296, 1142 cm-1
;
ESMS m/z 216 (M - 1+). A solution of 2-cyano-4-trifluoro-
methylpyrimidine-5-carboxylic acid (2.0 g, 9.2 mmol), oxalyl
chloride (1.4 g, 11 mmol) and DMF (4 drops) in CH2Cl2 (46
mL) was stirred at room temperature under N2 for 40 min.
The reaction mixture was concentrated under reduced pres-
sure, and distilled (bulb-to-bulb, 100 °C, 1.5 mm/Hg) to give
the title compound as a clear liquid (1.8 g, 82%): 1H NMR
(CDCl3) δ 9.49 (s); 13C NMR (CDCl3) δ 162.25, 160.07, 153.25
(q, J ) 39 Hz), 145.88, 129.48, 118.72 (q, J ) 276 Hz), 113.78;
IR (neat) 3066, 2985, 2254, 1786, 1564, 1435, 1379, 1178, 908
cm-1; EIMS m/z 235 (M+).
2-Ch lor o-4-p e n t a flu or oe t h ylp yr im id in e -5-ca r b on yl
Ch lor id e (40). The reaction mixture was concentrated under
reduced pressure and distilled (bulb-to-bulb, 80-85 °C, 1 mm/
Hg) to give the title compound (1.2 g, 35%): 1H NMR (DMSO-
d6) δ 9.18 (s, 1H); IR (neat) 2895, 1794, 1566, 1227, 1005 cm-1
;
EIMS m/ z 294 (M+).
2-Ch lor o-4-m et h ylp yr im id in e-5-ca r b on yl
Ch lor id e
(41).31 A solution of ethyl 2-hydroxy-4-methylpyrimidine-5-
carboxylate (5 g, 27.5 mmol) and POCl3 (84 g, 550 mmol) was
heated at reflux under N2 for 1 h. The reaction mixture was
cooled to room temperature and concentrated under reduced
pressure. The oil was dissolved in CHCl3 (600 mL) and washed
with H2O (3 × 100 mL). The aqueous layers were combined
and extracted with CHCl3 (100 mL). The CHCl3 layers were
combined, dried (Na2SO4), filtered through a silica plug,
concentrated under reduced pressure. The residue was distilled
(bulb-to-bulb, 110-115 °C, 0.5 mm/Hg) to yield 1.5 g (27%) of
ethyl 2-chloro-4-methylpyrimidine-5-carboxylate: 1H NMR
(CDCl3) δ 9.04 (s, 1H), 4.42 (q, J ) 7.2 & 6.9 Hz, 2H), 2.85 (s,
3H), 1.43 (t, J ) 7.2 Hz, 3H); 13C NMR (CDCl3) δ 172.32,
163.84, 162.78, 161.17, 122.32, 62.03, 24.34, 14.17; IR (neat)
4-H yd r oxy-2-t r iflu or om et h ylp yr im id in e-5-ca r b oxyl-
ic Acid (47). A solution of ethyl 2-trifluoromethyl-4-hydroxy-
pyrimidine-5-carboxylate (5.00 g, 19.4 mmol), NaOH (0.932 g,
23.3 mmol), and H2O (20 mL) was stirred at 60 °C for 15 h.
The reaction mixture was acidified to pH ) 1 using 6 N HCl
and concentrated under reduced pressure to one-half its
volume. The solid was filtered and dried under reduced
pressure to give the title compound (2.1 g, 53%): mp >198 °C
dec; 1H NMR (DMSO-d6) δ 8.83; 13C NMR (DMSO-d6) δ 171.43,
16613, 159.19, 157.25 (q, J ) 6.4 Hz,), 119.36 (q, J ) 275 Hz),
113.15 IR 3336, 2922, 1716, 1567, 1344, 1291, 1222, 1163 cm-1
;
3001, 1743, 1570, 1541, 1448, 1362, 1284, 1132, 1072 cm-1
;
ESMS m/z 207 (M - 1+).
EIMS m/z 200 (M+). Ethyl 2-chloro-4-methylpyrimidine-5-
carboxylate was hydrolyzed to an acid using aqueous 1 N
NaOH and THF. The reaction mixture was acidified to give
2-chloro-4-methylpyrimidine-5-carboxylic acid. A solution of
2-chloro-4-methylpyrimidine-5-carboxylic acid (0.81 g, 4.7
mmol), oxalyl chloride (0.89 g, 7.05 mmol), and DMF (1 drop)
in CH2Cl2 (23 mL) was stirred at room temperature under N2
for 4 h. The reaction mixture was concentrated and distilled
(bulb-to-bulb, 100 °C, 1.3 mm/Hg) to give 0.548 g (61%) of the
title compound as a clear liquid: 1H NMR (CDCl3) δ, 9.02 (s,
1H), 2.74 (s, 3H); IR 2987, 1747, 1578, 1535, 1455, 1370, 1200
cm-1; EIMS m/z 191 (M+).
4-Meth yl-2-tr iflu or om eth ylpyr im idin e-5-car boxylic acid
(48): yield 85%; mp >210 °C dec; 1H NMR (DMSO-d6) δ 9.27
(s, 1H), 2.81 (s, 3H), IR (KBr) 3375, 3065, 2863, 1748, 1652,
1487, 1238, 1153 cm-1; FABMS m/z 207 ((M + 1)+).
2-Meth yl-4-tr iflu or om eth ylpyr im idin e-5-car boxylic acid
(49): yield 82%; mp >211 °C dec; 1H NMR (DMSO-d6) δ 9.22
(s, 1H), 2.79 (s, 3H); IR (KBr) 3416, 2980, 1721, 1578, 1546,
1285, 1099 cm-1; FABMS m/z 207 ((M + 1)+).
2,4-Bis(tr iflu or om eth yl)p yr im id in e-5-ca r boxylic Acid
(50). A solution of ethyl 2,4-bis(trifluoromethyl)pyrimidine-5-
carboxylate (5.0 g, 17 mmol) and NaOH (0.72 g, 18 mmol) in
EtOH (20 mL) and H2O (50 mL) was stirred at room temper-
ature for 1 h. The solution was acidified at °C to pH ) 1 using
concentrated HCl. The resulting solid was filtered and dried
under reduced pressure to give the title compound (1.5 g,
2-Ch lor o-4-p h en ylp yr im id in e-5-ca r b on yl
ch lor id e
1
(44): yield 71%; mp 42 °C; H NMR (CDCl3) δ 9.04 (s, 1H),
7.55 (m, 5H); IR (KBr) 2976, 1722, 1562, 1528, 1407, 1190
cm-1; EIMS m/z 252 (M+).
1
2-Flu or o-4-tr iflu or om eth ylpyr im idin e-5-car bon yl Ch lo-
r id e (45). A solution of 2-chloro-4-trifluoromethylpyrimidine-
25%): mp 59 °C; H NMR (DMSO-d6) δ 9.62; IR (KBr) 2968,
1734, 1455, 1250, 1157 cm-1; EIMS m/z 260 (M+).

4002 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Palanki et al.
2-Tr iflu or om eth yl-4-ch lor opyr im idin e-5-car bon yl Ch lo-
r id e (51). A solution of 2-trifluoromethyl-4-hydroxypyrimidine-
5-carboxylic acid (2.2 g, 10.6 mmol), POCl3 (32 g, 212 mmol)
and SOCl2 (25 g, 212 mmol) was heated at reflux for 4 days.
The reaction mixture was concentrated under reduced pres-
sure and distilled (bulb-to-bulb, 90-95 °C, 1.5 mm/Hg) to
provide the title compound (2.1 g, 81%): 1H NMR (CDCl3) δ
9.45 (s); IR (neat) 2998, 1774, 1645, 1566, 1535, 1443, 1362,
1163, 897 cm-1; EIMS m/z 244 (M+).
EtOAc in hexanes): mp 53-55 °C; 1H NMR (CDCl3) δ 7.81 (s,
1H), 4.49 (q, 2H), 2.65 (S, 3H), 1.44 (t, 3H); IR (KBr) 2987,
1743, 1574, 1529, 1263, 905 cm-1; EIMS m/z 200 (M+). Starting
from ethyl 2-chloro-4-methylpyrimidine-6-carboxylate (5.1 g,
25 mmol) and NaOH (1.0 g, 25 mmol), in 20% THF in water
(125 mL) 2-chloro-4-methylpyrimidine-6-carboxylic acid was
obtained as a white solid (1.7 g, 84% yield): mp 172-173 °C;
1H NMR (DMSO-d6) δ 7.95 (s, 1H), 2.58 (s, 3H); IR (KBr) 3230,
2991, 1732, 1577, 1514, 1423, 1275, 1174, 1083 cm-1; EIMS
m/z 173 (M+).
4-Meth yl-2-tr iflu or om eth ylpyr im idin e-5-car bon yl ch lo-
r id e (52): acid chloride was distilled (70-72 °C at 0.5 mm/
2-Ch lor op yr im id in e -5-N -[3′,5′-b is(t r iflu or om e t h yl)-
p h en yl]ca r boxa m id e (80). This compound was prepared in
96% yield (HPLC) pure in a combinatorial well and screened
in the whole-cell assay for IC50 values.
1
Hg; yield 89%); H NMR (CDCl3) δ 9.53 (s, 1H), 2.93 (s, 3H);
IR (neat) 2937, 1780, 1726, 1581, 1398, 1209, 1151, 930 cm-1
;
EIMS m/z 224 (M+).
2-Meth yl-4-tr iflu or om eth ylpyr im idin e-5-car bon yl ch lo-
r id e (53): yield 66%; bp 90 °C at 1.5 mm/Hg; 1H NMR (CDCl3)
δ 9.32 (s, 1H), 2.93 (s, 3H); IR 2989, 1788, 1680, 1576, 1211,
843 cm-1; EIMS m/z 224 (M+).
2,4-Bis(tr iflu or om eth yl)p yr im id in e-5-ca r bon yl ch lo-
r id e (54): yield 89%; bp 105 °C at 1.5 mm/Hg; 1H NMR
(CDCl3) δ 9.12; IR (neat) 2981, 2889, 1738, 1325, 1161, 1052
cm-1; EIMS m/z 278 (M+).
2-Ch lor o-4-m et h ylp yr im id in e-5-N-[3′,5′-b is(t r iflu or o-
m eth yl)p h en yl]p yr im id in e-5-ca r boxa m id e (81). A solu-
tion of 2-chloro-4-methylpyrimidine-5-carbonyl chloride (100
mg, 0.53 mmol), 3,5-bis(trifluoromethyl)aniline (121 mg, 0.53
mmol) and Amberlyst A-21 (100 mg) in EtOAc (5.3 mL) was
stirred at room temperature for 1 h. The solution was filtered,
concentrated, and purified by preparative centrifugal thin-
layer chromatography (4 mm SiO2, 10% EtOAc in hexane) to
afford the title compound (170 mg, 84%): mp 156-157 °C; 1H
NMR (acetone-d6) δ 10.34 (bs, 1H), 8.92 (s, 1H), 8.39 (s 2H),
7.23 (s, 1H), 2.72 (s 3H); IR (KBr) 3275, 3080, 1660, 1570, 1535,
1381, 1176, 1136, 893 cm-1; EIMS m/z 383 (M+). Anal. (C14H8-
ClF6N3O) C, H, N.
2-Ch lor o-4-e t h ylp yr im id in e -5-N -[3′,5′-b is(t r iflu or o-
m eth yl)p h en yl]ca r boxa m id e (82): mp 160-161 °C; 1H
NMR (CDCl3) δ 8.70 (s, 1H), 8.15 (s, 2H), 7.19 (s, 1H), 3.04 (t,
2H), 1.38 (q, 3H); IR (KBr) 3296, 2984, 1702, 1566, 1380, 1189,
1274, 887; EIMS m/z 397 (M+). Anal. (C15H10ClF6N3O) C, H,
N.
Eth yl 2-Tr iflu or om eth ylpyr im idin e-5-car boxylate (58).
A solution of R-carboethoxy-â-dimethylaminoacraldehyde16 (10
g, 58 mmol) and trifluoromethylacetamidine (16 g, 146 mmol)
in EtOH (200 mL) was heated at reflux for 3 h. The reaction
mixture was cooled and concentrated under reduced pressure
to an oil. Flash chromatography (SiO2, 20% EtOAc-hexanes)
afforded 5.4 g (44%) of the title compound as a white solid:
mp 65 °C; 1H NMR (CDCl3) δ 9.43 (s, 2H), 4.51 (q, 2H, J ) 6.9
Hz), 1.46 (t, 3H, J ) 7.2 Hz); IR (KBr) 3022, 1722, 1566, 1352,
1196, 1151 cm-1; EIMS m/z 220 (M+).
Eth yl 2-P h en ylp yr im id in e-5-ca r boxyla te (59). A solu-
tion of ethyl 3-N,N-dimethylamino-2-formylacrylate (4.0 g, 23
mmol), benzamidine hydrochloride (4.0 g, 26 mmol) and Na
(0.65 g, 28 mmol) in EtOH (40 mL) was heated at reflux under
N2 for 1 h. The solution was filtered and concentrated. The
residue was dissolved in EtOAc, washed with dilute HCl and
H2O. The EtOAc layer was dried (Na2SO4), filtered, and
concentrated to give the title compound (4.0 g, 75%): mp >220
°C dec; 1H NMR (DMSO-d6) δ 9.28 (s, 2H), 8.46 (m, 2H), 7.56
(m, 2H), 4.37 (q, J ) 7.1 Hz, 2H), 1.35 (t, J ) 7.2 Hz, 3H); IR
(KBr) 2987, 1724, 1657, 1431, 1181, 1219 cm-1; EIMS m/z 228
(M+).
2-Ch lor o-4-n -p r op ylp yr im id in e-5-N-[3′,5′-b is(t r iflu o-
1
r om eth yl)p h en yl]ca r boxa m id e (83): mp 152-153 °C; H
NMR (CDCl3) δ 8.71 (s, 1H), 8.15 (s, 2H), 7.93 (s, 1H), 7.73 (s,
1H), 2.98 (m, 2H), 1.83 (m, 2H), 1.0 (m, 3H); IR (KBr) 3279,
2969, 1662, 1569, 1529, 1380, 1275, 1179. 1135 cm-1; EIMS
m/z 411 (M+). Anal. (C16H12ClF6N3O) C, H, N.
2-Ch lor o-4-p en ta flu or oeth ylp yr im id in e-5-N-[3′,5′-bis-
(t r iflu or om et h yl)p h en yl]ca r b oxa m id e (84). This com-
pound was prepared in 92% yield (HPLC) pure in a combina-
torial well and screened in the whole-cell assay for IC50
values: EIMS m/z 487 (M+).
2-Ch lor op yr im id in e-5-ca r bon yl Ch lor id e (61). The pure
product was obtained from the reaction mixture by bulb-to-
bulb distillation under reduced pressure (100 °C, 1.5 mm/Hg;
yield 69%): 1H NMR (CDCl3) δ 9.25 (s); IR (neat) 2992, 1757,
1583, 1408, 1239, 1070, 586 cm-1; EIMS m/z 177 (M+).
2-Tr iflu or om et h ylp yr im id in e-5-ca r boxylic a cid (62):
yield 87%; mp 169-170 °C; 1H NMR (CDCl3) δ 9.42; IR (KBr)
3514, 2897, 1724, 1586, 1342, 1203, 1105 cm-1; EIMS m/z 192
(M+). Anal. (C6H3F3N2O2) C, H, N.
2,4-Dich lor op yr im id in e-5-N-[3′,5′-bis(tr iflu or om eth yl)-
ph en yl]car boxam ide (85): mp 104-105 °C; 1H NMR (acetone-
d6) δ 10.47 (bs, 1H), 9.01 (s 1H), 8.36 (s, 2H), 7.74 (s 1H); IR
(KBr) 3101, 1672, 1562, 1381, 1279, 1134, 887 cm-1; EIMS m/z
403 (M+). Anal. (C13H5Cl2F9N3O) C, H, N.
2-Ch lor o-4-p h en ylp yr im id in e-5-N-[3′,5′-b is(t r iflu or o-
m eth yl)p h en yl]ca r boxa m id e (86): mp 154-155 °C; 1H
NMR (DMSO-d6) δ 11.40 (s, 1H), 9.11 (s, 1H), 8.21 (s, 2H),
7.5-7.9 (m, 5H); EIMS m/z 445 (M+). Anal. (C19H8ClF6N3O)
C, H, N.
2-P h en ylp yr im id in e-5-ca r boxylic Acid (63). The title
compound was prepared (350 mg, 80%) from ethyl 2-phenyl-
pyrimidine-5-carboxylate in a manner similar to the synthesis
of 2,4-bis(trifluoromethyl)pyrimidine-5-carboxylic acid: mp >
2-F lu or o-4-t r iflu or om et h ylp yr im id in e-5-N-[3′,5′-b is-
(tr iflu or om eth yl)p h en yl]ca r boxa m id e (88): mp 133-135
°C; 1H NMR (CDCl3) δ 11.58 (s, 1H), 9.54 (s, 1H), 8.31 (s, 2H),
7.94 (s, 1H); IR (KBr) 3296, 2995, 1674, 1585, 1281, 1130, 950
cm-1; EIMS m/z 421 (M+). Anal. (C14H5F10N3O) C, H, N.
2-Met h yl-4-t r iflu or om et h ylp yr im id in e-5-N-[3′,5′-b is-
(tr iflu or om eth yl)p h en yl]ca r boxa m id e (89): mp 147-148
1
220 °C dec; H NMR (DMSO-d6) δ 9.29 (s, 2H), 8.47 (m, 2H),
7.58 (m, 3H); IR (KBr) 3400, 2968, 1693, 1580, 1410, 1294
cm-1; ESMS m/z 199 (M - H+).
2-Tr iflu or om et h ylp yr im id in e-5-ca r b on yl
ch lor id e
(64): yield 66%; mp 37-39 °C; 1H NMR (CDCl3) δ 9.4; IR 2897,
1724, 1586, 1342, 1203, 1105 cm-1; EIMS m/z 210 (M+).
2-P h en ylp yr im id in e-5-ca r bon yl Ch lor id e (65). The title
compound was prepared from 2-phenylpyrimidine-5-carboxylic
acid in a manner similar to the synthesis of 2-chloro-4-
methylpyrimidine-5-carbonyl chloride: yield 91%; mp 135 °C;
1H NMR (CDCl3) δ 9.39 (s, 1H), 8.57 (m, 2H), 7.56 (m, 3H); IR
(neat) 2875, 1682, 1585, 1410, 1298 cm-1; EIMS m/z 218 (M+).
2-Ch lor o-4-m eth ylp yr im id in e-6-ca r boxylic Acid (70).
Starting from ethyl 2-hydroxypyrimidine-6-carboxylate-urea
complex (25.0 g, 103 mmol) and POCl3 (238 g, 1.5 mol), ethyl
2-chloro-4-methylpyrimidine-6-carboxylate was obtained as a
white solid (5.1 g, 25%, flash chromatography, SiO2, 15%
°C; H NMR (CDCl3) δ 8.94 (s, 1H), 7.85 (s, 1H), 7.82 (s, 2H),
1
6.40 (bs, 1H); IR (KBr) 3267, 3082, 1655, 1560, 1284, 1136,
899 cm-1. Anal. (C15H8F6N3O) C, H, N.
2-Cya n o-4-tr iflu or om eth ylp yr im id in e-5-N-[3′,5′-bis(tr i-
flu or om eth yl)p h en yl]ca r boxa m id e (91): mp 146-147 °C;
1H NMR (acetone-d6) δ 10.69 (bs, 1H), 9.71 (s, 1H), 8.37 (s,
2H), 7.89 (2, 1H); IR (KBr) 3271, 2997, 1672, 1572, 1471, 1140,
893 cm-1; EIMS m/z 428 (M+). Anal. (C15H5F9N4O) C, H, N.
4-Ch lor o-2-t r iflu or om et h ylp yr im id in e-5-N-[3′,5′-b is-
(tr iflu or om eth yl)p h en yl]ca r boxa m id e (92): mp 165-166
°C; 1H NMR (CDCl3) δ 10.53 (s, 1H), 9.37 (s, 1H), 8.41 (s, 2H),
7.85 (s, 1H); EIMS m/z 437 (M+). Anal. (C14H5ClF9N3O) C, H,
N.

Inhibitors of NF-κB and AP-1 Gene Expression
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4003
2-Tr iflu or om et h ylp yr im id in e-5-N-[3′,5′-b is(t r iflu or o-
m eth yl)p h en yl]ca r boxa m id e (93): mp 154 °C; 1H NMR
(CDCl3) δ 9.41 (s, 2H), 8.41 (2, 1H), 8.21 (s, 2H), 7.76 (s, 1H);
2981, 1655, 1592, 1301, 1211, 1011 cm-1. Anal. (C13H6F9N3O)
C, H, N.
2-P h en ylp yr im id in e-5-N-[3′,5′-b is(t r iflu or om et h yl)-
ph en yl]car boxam ide (94): mp 248-250 °C; 1H NMR (DMSO-
d6) δ 11.16 (s, 1H), 9.41 (s, 2H), 8.5 (m, 4H), 7.89 (s, 1H), 7.61
(m, 3H); EIMS m/z 411 (M+). Anal. (C19H11F6N3O) C, H, N.
4-Met h yl-2-t r iflu or om et h ylp yr im id in e-5-N-[3′,5′-b is-
(tr iflu or om eth yl)p h en yl]ca r boxa m id e (95): mp 152-153
°C; 1H NMR (CDCl3) δ 9.39 (s, 2H), 8.99 (s, 1H), 8.34 (bs, 1H),
8.18 (s, 1H), 2.96 (s, 3H); IR (KBr) 3256, 3055, 1705, 1660,
1290, 1153, 1122 cm-1; EIMS m/z 417 (M+). Anal. (C15H8F6N3O)
C, H, N.
these experiments.32 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/
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 mg/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 phorbol 12-
myristate-13-acetate (PMA) and 1 µg/mL 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 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.
2-Met h yl-4-t r iflu or om et h ylp yr im id in e-5-N-[3′,5′-b is-
(tr iflu or om eth yl)p h en yl]ca r boxa m id e (96): mp 147-148
°C; 1H NMR (CDCl3) δ 9.38 (s, 2H), 8.98 (s, 1H), 8.34 (bs, 1H),
8.17 (s, 1H), 2.94 (s, 3H); IR (KBr) 3244, 3021, 1701, 1665,
1294, 1153, 1123 cm-1; EIMS m/z 417 (M+). Anal. (C15H8F6N3O)
C, H, N.
AP -1 Assa y. The AP-1 assay was run as described above
for NF-kB 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.29 In
addition, the concentrations of PMA and PHA were 5 ng/mL
and 1 µg/mL, respectively.
Ca co-2 P er m ea bility Assa y. Caco-2 cells, strain HTB-37,
were acquired from ATCC and cultured in MEM media with
L-glutamine and Earles balanced salts (Hyclone, Logan, UT).33
Additional supplements included 20% heat-inactivated fetal
bovine serum (Hyclone, Logan, UT), 1 mM sodium pyruvate,
0.1 mM nonessential amino acids (GIBCO, Grand Island, NY),
and 50 U/mL penicillin/ 50 µg/mL streptomycin (Cellgro). Cells
were routinely passaged when 80% confluent as a 1:5 split
using 0.05% trypsin (Hyclone, Logan, UT). The 3-day Caco-2
culture was performed using the Biocoat Intestinal Epithelium
Differentiation Environment Kit (#30057 from Becton Dick-
inson, Bedford, MA) adhering to the manufacturers’ protocol.
2-Ch lor o-4-t r iflu or om et h ylp yr im id in e-6-N-[3′,5′-b is-
(t r iflu or om et h yl)p h en yl]ca r b oxa m id e (97). This com-
pound was prepared in 88% pure (HPLC) in a combinatorial
well and screened in the whole-cell assay for IC50 values:
ESMS m/z 437 (M+).
2-Ch lor o-4-m et h ylp yr im id in e-6-N-[3′,5′-b is(t r iflu or o-
m et h yl)p h en yl]ca r b oxa m id e (98). This compound was
prepared in 85% pure (HPLC) in a combinatorial well and
screened in the whole-cell assay for IC50 values: ESMS m/z
384 (M+).
2,4-Dich lor op yr im id in e-6-N-[3′,5′-bis(tr iflu or om eth yl)-
p h en yl]ca r boxa m id e (99): mp 168-169 °C; 1H NMR (CDCl3)
δ 10.78 (s, 1H), 8.49 (s, 1H), 7.99 (s, 2H), 7.60 (s, 1H); IR (KBr)
3001, 2867, 1735, 1535, 1279, 1134 cm-1; EIMS m/z 403 (M+).
Anal. (C13H5Cl2F6N3O) C, H, N.
3-Ch lor o-5-t r iflu or om et h ylp yr id a zin e-6-N-[3′,5′-b is-
(tr iflu or om eth yl)p h en yl]ca r boxa m id e (101): mp 122-123
°C; 1H NMR (CDCl3) δ 10.12 (s, 1H), 8.28 (s, 2H), 8.07 (s, 1H),
Briefly, cells were passaged and then resuspended at
a
7.73 (s, 1H); IR (KBr) 2995, 1702, 1601, 1450, 1280, 1120 cm-1
;
concentration of 0.4 × 106 cells/mL in basal seeding media
containing MITO-serum extender. Cells (0.5 mL) were added
to the precoated chamber inserts (200 000 cells/insert) and
cultured for 24 h. The media was then replaced with Entero-
Stim media containing MITO-serum extender and cultured for
an additional 48 h. The compound permeability assay was
performed at ambient room temperature and atmosphere.
Inserts were washed twice with PBS (+Mg, +Ca) by transfer-
ring the chamber insert to a well (basal chamber) containing
fresh PBS and washing the insert (apical chamber) with PBS.
The insert was placed in a well containing 1 mL of fresh PBS,
280 µL of compound solution (100 µM in PBS, 0.5% DMSO)
was added to the insert (apical chamber), and the plate was
incubated on a shaking table (90 rpm). After 45 min, the insert
was removed and the basal chamber PBS retained for analysis.
Assay viability was confirmed in parallel using [3H]PEG4000,
[3H]mannitol and [14C]caffeine (NEN, Boston MA). Typical
EIMS m/z 437 (M+). Anal. (C14H5ClF9N3O) C, H, N.
3-Ch lor op yr id a zin e -6-N -[3′,5′-b is(t r iflu or om e t h yl)-
p h en yl]ca r boxa m id e (102): mp 116-117 °C; 1H NMR
(CDCl3) δ 10.19 (s, 1H), 8.40 (d, J ) 9 Hz, 1H), 8.30 (s, 2H),
7.82 (d, J ) 9 Hz, 1H), 7.72 (s, 1H); IR (KBr) 2999, 1711, 1611,
1440, 1289, 1123 cm-1; EIMS m/z 369 (M+). Anal. (C13H5-
ClF6N3O) C, H, N.
3-Ch lor opyr azin e-6-N-[3′,5′-bis(tr iflu or om eth yl)ph en yl]-
ca r boxa m id e (103): mp 101-102 °C; 1H NMR (CDCl3) δ 9.76
(s, 1H), 9.30 (s, 1H), 8.62 (s, 1H), 8.28 (s, 2H), 7.70 (s, 1H); IR
(KBr) 3011, 1691, 1621, 1430, 1279, 1103 cm-1; EIMS m/z 369
(M+). Anal. (C13H5ClF6N3O) C, H, N.
Syn t h esis of a Lib r a r y. The synthesis involved adding
1.05 equiv of each acid chloride in ethyl acetate (100 µL) to 80
wells of a microtiter plate with each well containing 0.02 mmol
of one amine or aniline in EtOAc (100 µL) and Amberlyst 21
(100 mg), which is a basic ion-exchange resin. The plates were
covered and sonicated for 20 min. Water (50 µL) was added to
the wells and sonicated for an additional 10 min. Ethyl acetate
(1 mL) was added to each well and sonicated for 5 min. The
products were easily isolated by transferring the organic layer
(1 mL) of each well into tared test tubes followed by concentra-
tion of the solutions. The residue in each test tube was dried
under vacuum. The purity of each compound within a library
(80 compounds constituted one library) was determined using
TLC. By TLC, all of the compounds were clean with no residual
starting materials present. Fifteen to twenty compounds from
each library were randomly selected and analyzed by HPLC
and EIMS for purity and structural verification, respectively.
Molecular weights of the randomly selected compounds were
confirmed, and all compounds had >90% purity by HPLC.
NF -κB Assa y. Human J urkat 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
values for radiolabeled controls were 20 × 10-7, 55 × 10-7
,
and 320 × 10-7 cm/s, respectively. The compounds were
detected using HPLC on a Rainin Dynamax system equipped
with a YMC C-18 column using acetonitrile containing 35%
water and 0.1% CF3COOH
Ack n ow led gm en t. We thank Mr. Mark Rosen and
Ms. Palka Patel for synthesis of some of the intermedi-
ates and analysis libraries using HPLC, respectively.
Su p p or tin g In for m a tion Ava ila ble: List of amines that
were used in the combinatorial library. This material is
available free of charge via the Internet at http://pubs.acs.org.
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