Discovery of novel and selective IKK-β serine-threonine protein kinase inhibitors. Part 1

Article abstract of DOI:10.1016/S0960-894X(02)01046-6

IκB kinase β (IKK-β) is a serine-threonine protein kinase critically involved in the activation of the transcription factor Nuclear Factor kappa B (NF-κB) in response to various inflammatory stimuli. We have identified a small molecule inhibitor of IKK-β. Optimization of the lead compound resulted in improvements in both in vitro and in vivo potency, and provided IKK-β inhibitors exhibiting potent activity in an acute cytokine release model (LPS-induced TNFα).

Full text of DOI:10.1016/S0960-894X(02)01046-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 913–918
Discovery of Novel and Selective IKK-ꢀ Serine-Threonine
Protein Kinase Inhibitors. Part 1
Toshiki Murata,a,* Mitsuyuki Shimada,a Sachiko Sakakibara,a Takashi Yoshino,a
Hiroshi Kadono,a Tsutomu Masuda,a Makoto Shimazaki,a Takuya Shintani,a
Kinji Fuchikami,b Katsuya Sakai,b Hisayo Inbe,b Keisuke Takeshita,b Toshiro Niki,b
Masaomi Umeda,b Kevin B. Bacon,b Karl B. Ziegelbauerb and Timothy B. Lowingera
aDepartment of Chemistry, Research Center Kyoto, Bayer Yakuhin, Ltd., Kizu, Soraku, Kyoto 619-0216, Japan
bDepartment of Biology, Research Center Kyoto, Bayer Yakuhin, Ltd., Kizu, Soraku, Kyoto 619-0216, Japan
Received 22 October 2002; accepted 9 December 2002
Abstract—IkBkinase b (IKK-b) is a serine-threonine protein kinase critically involved in the activation of the transcription factor
Nuclear Factor kappa B(NF- kB) in response to various inflammatory stimuli. We have identified a small molecule inhibitor of
IKK-b. Optimization of the lead compound resulted in improvements in both in vitro and in vivo potency, and provided IKK-b
inhibitors exhibiting potent activity in an acute cytokine release model (LPS-induced TNFa).
# 2003 Elsevier Science Ltd. All rights reserved.
IkBkinase b (IKK-b) is a 756 amino acid-containing
serine-threonine protein kinase.1 As part of the IKK-
complex, IKK-b is critically involved in the activation
of the transcription factor Nuclear Factor kappa B
(NF-kB) in response to various inflammatory stimuli
including Tumor Necrosis Factor a (TNFa), Interleukin
1b (IL-1b) and lipopolysaccharide (LPS). In addition to
IKK-b, the IKK-complex contains IKK-a, IKK-g, NIK
and various other known and unknown proteins. NF-
kBis an inducible transcription factor that is thought to
be a pivotal target for drugs for cancer and chronic
inflammatory diseases.2
A number of reports on IKK-b inhibitors exist. The
non-specific protein kinase inhibitors, quercetin and
staurosporine,5 as well as well-known anti-inflammatory
agents such as aspirin6 and cyclopentenone prosta-
glandins7 are known to inhibit IKK-b kinase. In our
assays the natural protein kinase inhibitor staurosporine
inhibited IKK-b with an IC50 of 0.25 mM, and more
potently inhibited IKK-a with an IC50 of 0.05 mM.
More recently, it has been reported that a novel IkB
kinase (IKK) inhibitor, PS-1145, specifically blocks
TNFa-induced NF-kBactivation, resulting in a sup-
pression of the release of various cytokines both in vitro
and in vivo.8 These data suggest that specific inhibition
of IKK-b will result in a strong in vivo anti-inflammatory
and immuno-modulatory effect.
A catalytically inactive mutant of IKK-b has been
shown to inhibit activation of NF-kBby TNF a, IL-1b,
LPS, and anti-CD3/anti-CD28 stimulation.3 This pro-
vides further evidence for the involvement of IKK-b in
these pathways leading to NF-kBactivation. In addi-
tion, embryonic fibroblast cells isolated from IKK-b-
deficient mice show defects in TNFa- and IL-1-induced
degradation of IkB. In contrast, in cells derived from
IKK-a-deficient mice, pro-inflammatory cytokine-
induced IkBdegradation is not inhibited, suggesting
that IKK-b controls the NF-kBactivation rather than
IKK-a.4
From high-throughput screening of the Bayer com-
pound library, the 2-amino-3-cyano-4-aryl-6-(2-
hydroxy-phenyl)pyridine analogue 1 was identified as a
potent IKK-b inhibitor.
The lead compound 1 shows potent inhibitory activity
against IKK-b (IC50=1.5 mM) and excellent selectivity
versus other kinases such as IKK-a, Syk and MKK4
(mitogen-activated protein kinase kinase 4) (IC50 values
>20 mM). Furthermore, this lead compound inhibits
NF-kB-dependent expression of several reporter genes,
chemokines, cytokines and IgE production in various
functional cellular assays (Table 1). These results suggest
*Corresponding author. Tel.: +81-774-75-2483; fax: +81-774-75-
2510; e-mail: toshiki.murata.tm@bayer.co.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01046-6

914
T. Murata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 913–918
Table 1. Inhibitory activities of the lead compound 1 in various
kinase and cellular assays
Scheme 1. (a) 30% NaOH, EtOH, rt; (b) malononitrile, ammo-
nium acetate, EtOH, reflux, 24 h, 51% (3, R=4-NO2), 11% (4,
R=4-N(CH3)2).14
Cells/Cell Line
Stimulus
Read-Out
IC50, mMa
hydroxyphenyl)pyridine analogues 3 and 4 by a reaction
of substituted 1,3-diaryl-2-propen-1-ones 2 with mal-
ononitrile in the presence of ammonium acetate
(Scheme 1).11 Although the o-hydroxyacetophenone
was utilized for the pyridine contraction reaction with-
out protection of the phenolic hydroxyl group, protec-
tion became necessary for further modifications of the
substituents on the aromatic group at the 4-position of
the pyridine.
A549
Jurkat T-cell
HEK293
Mouse B-cells
Human PBMCs
TNFa
anti-CD3/anti-CD28
TNFa
RANTES
IL-2
NF-kB-Luciferase
IgE
8
15
8
0.35
10
LPS/IL-4
LPS
TNFa
aValues are means of more than three experiments.
that compound 1 is a lead structure for specific inhibi-
tors of the IKK-complex activated by various inflam-
matory stimuli and it can be therefore pharmacologically
confirmed that IKK-b is a key component of the signal
transduction pathway in those physiological responses.
When protected 20-hydroxyacetophenones were used as
starting materials, the 2-amino-3-cyano-4,6-diarylpyr-
idine core structures were simply constructed using a
one-pot coupling reaction of four components, aceto-
phenone, benzaldehyde, malononitrile and ammonium
acetate, following literature precedent12 (Scheme 2 and
3). The corresponding pyridone analogue 7 was pre-
pared by the similar one-pot reaction using ethyl
cyanoacetate instead of malononitrile, and was subse-
quently utilized to synthesize the 2-diethylaminopyridine
analogue 9. The benzylether was found to be one of the
most suitable protecting groups of the 20-hydroxy-
acetophenones, enabling the construction of the 2-amino-
pyridine core structures in good yield, as exemplified by
the synthesis of the pyridine analogue 10 (Scheme 3).13
Herein, we report the synthesis, structure–activity
relationships (SAR) and characterization of a series of
2-amino-3-cyano-4-aryl-6-(2-hydroxyphenyl)pyridines.
Chemistry9
Synthesis of 2-amino-3-cyano-4,6-diarylpyridines has
been reported in several publications.10 Manna et al.
actually synthesized the 2-amino-3-cyano-4-aryl-6-(2-
Scheme 2. (a) Malononitrile, ammonium acetate, 1,4-dioxane, reflux, 18%; (b) H2, 10% Pd-C, AcOEt, rt, 87%; (c) 5-oxotetrahydro-2-fur-
ancarbonyl chloride, pyridine, acetonitrile, 0 ꢀC, rt, 77%; (d) n-Bu4NF, THF, 0 ꢀC, 93%; (e) NaOH, water, methanol, rt, 60%; (f) ethyl cyanoace-
tate, ammonium acetate, toluene, reflux, 19%; (g) (CF3SO2)2O, pyridine, 0 ꢀC, 81%; (h) diethylamine, DMSO, 40 ꢀC, 88%; (i) H2, 10% Pd-C,
methanol, THF, rt, 45%; (j) 3-(1-piperidinyl)propanoyl chloride, pyridine, THF, 0 ꢀC, rt, 69%.

T. Murata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 913–918
915
The straightforward synthetic method could be adopted
to solid-phase synthesis using an O-tethered o-hydroxy-
acetophenone on solid support instead of the protected
o-hydroxyacetophenone (Scheme 4). This combinatorial
chemistry method provided the target compounds in
good yield (>80%) and high purity (>80%, ELSD),
and was used to generate approximately 1000 com-
pounds for the optimization of the substituents on the
phenyl group at the 4-position of the pyridine. All the
potent compounds were purified by column chromato-
graphy or re-synthesized using solution phase chemistry
to confirm the biological activity of the pure com-
pounds.
amine, are tolerated at this position, suggesting the
hypothesis that this portion of the molecule may interact
with the hydrophilic pocket of the enzyme.
The functionality on the phenyl group appears to affect
cellular activity significantly. Introduction of a basic
amino moiety on the side chain of the phenyl group
(26 and 28) improved cellular activity. However, the
carboxylic acid analogues (6, 18–20, 22–23) exhibited
either no or weak cellular activity, albeit with potent
IKK-b inhibitory activity, suggesting poor cell mem-
brane permeability of these derivatives. While dis-
placement of substituents from the 30 to the 40-position
on the phenyl group did not significantly impact IKK-
b inhibitory activity, such modifications resulted in a
complete loss of cellular activity (6 vs 22, 26 vs 27 and
29 vs 30).
Results and Discussion
Recombinant human IKK-b was used to measure
kinase activity in vitro.15 To test cellular efficacy of
IKK-b inhibition, an ELISA assay measuring TNFa-
induced RANTES production was employed.16
In contrast to the diversity of substitution tolerated on
the phenyl group at the 4-position, modification of the
phenol group at the 6-position appears to be rather
restrictive (Table 3). Removal of the phenolic hydroxide
(31) results in a complete loss of activity. Change of the
substitution position (32 and 40) or replacement of the
hydroxy group with a methoxy group also yielded inac-
tive compounds, suggesting that the 20-phenol group is
necessary for activity. Interestingly, the 20-phenol ana-
logue 39 incorporating a methyl group on the 5-position
of the pyridine ring exhibited no inhibitory activity. The
common characteristic of the inactive compounds was
found to be a lack of hydrogen-bonding interaction
between the phenolic hydroxide and the pyridine nitro-
gen atom, which could typically be observed via the
chemical shift of the phenolic hydroxide proton in the
1H NMR spectrum.17 The hydrogen-bonding interac-
tion of the 5-methyl analogue 39 appears to be inter-
rupted by the steric collision of the methyl group with
the hydrogen atom at the 60-position of the phenol
We initially synthesized para-nitro (3) and para-dime-
thylamine (4) analogues to assess their IKK-b inhibitory
activity because these compounds have been reported to
exhibit anti-inflammatory activity in animal models
(paw edema tests using carrageenin).11 These com-
pounds showed no inhibitory activity in our in vitro
assays (Table 2).
The phenyl group at the 4-position of the pyridine can
accommodate a variety of substituents (Table 2). Since
the lead compound 1 is sparingly soluble in an aqueous
solution, our initial optimization efforts focused on the
introduction of hydrophilic substituents onto this
phenyl group with the aim to improve the solubility as
well as the inhibitory activity. Various hydrophilic
functionalities, such as alcohol, carboxylic acid and
Scheme 3. (a) Malononitrile, ammonium acetate, toluene, reflux, 63%; (b) HNR1R2, DMF, rt, 60 ꢀC, 92%; (c) Fe powder, ammonium chloride,
water, ethanol, reflux, 75%; (d) 3-(1-piperidinyl)propanoyl chloride, pyridine, rt, 32%; (e) H2, 10% Pd-C, AcOH, AcOEt, rt, 86%.
ꢀ
.
Scheme 4. (a) Wang resin, malononitrile, ammonium acetate, 1,4-dioxane, 80 C; (b) SnCl2 2H2O, DMF, rt; (c) chloroacetyl chloride, rt; (d) piper-
idine, diisopropylethylamine, DMF; (e) 50% TFA/CH2Cl2, 86% overall yield, 96% purity (ELSD).

916
T. Murata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 913–918
group. Thus, the attainment of a geometry allowing for
the internal hydrogen bond appears to play a significant
role in achieving IKK-b inhibitory activity.
10-fold more potent than the parent compound 1 in the
cellular assay (Table 4).
In order to investigate the pharmacological profile of
these potent IKK-b inhibitors, we tested their activity in
an acute model of cytokine release (LPS-induced TNFa
production in mice),18 which is considered to be one of
the most mechanism-relevant models for the evaluation
of IKK-b inhibition (Table 4). The initial lead com-
pound 1 exhibits in vivo activity only when adminis-
tered ip (intraperitoneally) but lacks oral efficacy due to
low oral bioavailability. On the other hand, the more
hydrophilic analogues, such as the carboxylic acid ana-
logue 6 and the basic amino analogues (13, 26),
demonstrate potent oral activity as well as high potency
when administered ip The piperidyl analogue 26 inhibits
LPS-induced TNFa production with an ED50 of 0.03
mg/kg and 2 mg/kg after intraperitoneal and oral
administration, respectively.
Addition of a substituent at the 50-position on the 20-
phenol group usually resulted in a loss of cellular activity,
despite maintaining IKK-b inhibitory activity (34–36).
The introduction of a methyl group at the 60-position
gave an inactive compound 37, in which the
methyl group possibly interrupts the hydrogen-bonding
interaction.
The 2-aminopyridine ring also appears to be important
for inhibitory activity (Table 3). Replacement of the
amino moiety by a hydroxy moiety (41) leads to a loss
of activity, as does substitution of the amino moiety by
a diethylamino group (9). The acetylamide analogue 42
showed moderate activity.
Introduction of an alkylamine group at the 40-position
on the phenyl ring was tolerated for IKK-b inhibition,
and the pyrrolidine analogue 16 was shown to be
The compound 26 identified by the optimization efforts
of this compound class maintains excellent selectivity
versus other kinases such as IKK-a (IC50=20 mM), Syk
and MKK4 (IC50>20 mM).
Table 2. Modification of the C-4 phenyl group
In summary, screening of the Bayer compound library
resulted in the identification of a novel class of IKK-b
inhibitors. Optimization of the lead compound 1
through a combined combinatorial and medicinal
chemistry effort resulted in improvements in both in
vitro and in vivo potency. As a result, compound 26 has
been identified as a selective and potent inhibitor of the
IKK-b which is orally bioavailable in mice, and which
Compd
R
IC50 (mM)
IKK-b15
RANTES16
Table 3. Modification of the C-6 phenyl group
3
4
40-
40-
30-
20-
30-
40-
30-
NO2
N(CH3)2
NH2
CO2Na
CO2Na
CO2Na
>20
>20
20
>50
>50
>50
>50
>50
>50
>50
17
18
19
20
21
0.7
2.5
0.7
1.4
1
6
30-
1.5
0.9
8
30-
20
Compd
R1
R2
R3
IC50 (mM)15,16
22
23
40-
30-
0.5
1.4
>50
>50
IKK-b RANTES
6
–OH
–H
–H
–OCH3
–OH
–OH
–OH
–OH
–OH
–OH
–H
–H
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
0.9
>20
>20
>20
20
>50
>50
>50
>50
>50
>50
>50
>50
>50
24
25
16
30-
30-
30-
0.6
0.6
3
15
35
15
31
32
33
34
35
36
37
38
39
40–OH
–H
50–F
1.7
5.5
5.3
50–Cl
50–OCH3
60–CH3
60–OCH3
–H
>20
2
>20
26
27
30-
40-
0.6
1.0
7
>50
26
40
41
9
–OH
–H
–OH
–OH
–OH
–H
30–OH
–H
–H
–H
–NH2
–NH2
–OH
0.6
7
10
>50
nd
>20
>20
>20
15
28
29
30
20-
30-
40-
0.5
1.8
0.6
6
15
>50
–NEt2
–NHCOCH3
42
30

T. Murata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 913–918
917
Table 4. Modification of the C-4 phenyl group
K. B.; Fuchikami, K.; Umeda, M.; Komura, H.; Yoshida, N.
WO 0244153, 2002; Chem Abstr. 2002, 137, 20298.
10. For the first report, see: Sakurai, A.; Midorikawa, H. Bull.
Chem. Soc. Jpn. 1968, 41, 430.
11. (a) Manna, F.; Chimenti, F.; Bolasco, A.; Filippelli, A.;
Palla, A.; Filippelli, W.; Lampa, E.; Mercantini, R. Eur. J.
Med. Chem. 1992, 27, 627. (b) Manna, F.; Chimenti, F.;
Bolasco, A.; Bizzarri, B.; Filippelli, W.; Filippelli, A.;
Gagliardi, L. Eur. J. Med. Chem. 1999, 34, 245.
12. Kambe, S.; Saito, K. Synthesis 1980, 366.
13. A typical procedure for the four component coupling to
construct the pyridine analogue (Synthesis of compound 10)—
A mixture of 2-benzyloxyacetophenone (5.0 g, 22.1 mmol),
40-chloro-30-nitrobenzaldehyde (8.2 g, 44.2 mmol), mal-
ononitrile (2.92 g, 44.2 mmol) and ammonium acetate (8.5 g,
110 mmol) in toluene (15 mL) was stirred at reflux for 3 h.
After cooling to rt, the mixture was diluted with ethyl ace-
tate and THF. The organic phase was washed twice with
water, dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was suspended in ethanol. The
precipitate was collected by filtration, washed with ethyl
acetate and dried under reduced pressure to give the desired
product 10 (6.4 g, 63%) as a white solid. 1H NMR
(500 MHz, DMSO-d6) d 5.16 (2H, s), 7.06 (2H, br s), 7.11
(2H, dd, J=7.5, 7.7 Hz), 7.26– 7.32 (4H, m), 7.38–7.47 (3H,
m), 7.69 (1H, dd, J=2.1, 8.4 Hz), 7.85 (1H, dd, J=1.7, 7.7
Hz), 7.90 (1H, d, J=8.4 Hz), 8.19 (1H, d, J=2.1 Hz).
14. The reaction procedures and chemical yields are cited
from the ref 11(a).
Compd
R
IC50 (mM)15, 16
IKK-b RANTES
ED50 (mg/kg)18
ipa
pob
1
6
26
12
13
—
—
–H
–NMe2
1.5
0.9
0.6
0.8
1.0
8
20
7
8
0.8
10
>30
1.3
<0.03
0.6
18.7
2
nd
0.8
10.1
43
2.5
5
nd
nd
aIntraperitoneal administration (30 min pretreatment).
bOral administration (60 min pretreatment).
demonstrates significant in vivo activity in an acute
model of cytokine release (LPS-induced TNFa).
15. IKK-b in vitro assay: Human IKK-b was cloned from
Quickclone cDNA library (CLONTECH) by polymerase
chain reaction and recombinant His-tagged IKK-b was
expressed in insect cells using by a baculovirus expression
vector system (Pharmingen). GST-IkBa (1-54) was expressed
in Escherichia coli BL21 (DE3). The sequences of all the con-
structed clones were verified by DNA sequencing.
Acknowledgements
We would like to thank Nagahiro Yoshida, Hiroshi
Komura, Florian Gantner and Klaus Urbahns for
helpful discussions.
Inhibition of IKK-b kinase activity was assayed in a 96-well
MTP format kinase assay. Compounds were dissolved in
DMSO (final 0.25%) and incubated with recombinant IKK-b
(final 0.6 mg/mL) and biotinylated-GST-IkBa (1-54) (final 0.2
mM) in kinase buffer b (20 mM Tris–HCl, pH 7.6, 20 mM
MgCl2, 20 mM b-glycerophosphate, 20 mM p-n-phenylphos-
phate, 1 mM EDTA, 20 mM creatine phosphate, 1 mM DTT,
1 mM Na3VO4, 0.1 mg/mL BSA and 0.4 mM phenyl-
methylsulfonyl fluoride; 50 mL/well) in U-bottomed 96-well
plate. Kinase reaction was started by addition of ATP (final 5
mM APT, 0.5 mCi/well [g-33P] ATP) and incubated at rt for 2
h. Kinase reactions were terminated by the addition of 150 mL
100 mM EDTA, 1 mg/mL BSA, and 150 mL of the sample
were transferred to streptavidin-coated, white MTP (Steffens
Biotechniche Analysen GmbH #08114E14.FWD) to capture
biotinylated substrates. After 1 h incubation, free radioactivity
was eliminated by washing the wells five times with 300 mL of
0.9% NaCl, 0.1% (w/v) Tween-20. The remaining radio-
activity was counted in 170 mL of MicroScint-PS scintillation
cocktail (Packard) using a TopCount scintillation counter.
16. In vitro RANTES induction by TNFa in A549 cells: The
A549 human lung epithelium cell line (ATCC #CCL-885) was
maintained in Dulbecco’s modified Eagle’s medium (D-MEM,
Nikken Biomedical Institute) supplemented with 10% FCS
(Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin and 2
mM glutamine. A549 cells (4ꢁ104 cells in 80 mL/well) were
treated in a 96-well flat-bottom tissue culture plate for 1 h with
vehicle (0.1% DMSO) or test compounds. Then cells were sti-
mulated with 100 ng/mL TNF-a for 24 h. Concentration of
RANTES in the supernatants collected after 24 h was deter-
mined using a quantitative sandwich Fluorescent immu-
References and Notes
1. (a) Mercurio, F.; Zhu, H.; Murray, B. W.; Shevchenko, A.;
Bennett, B. L.; Jian, W. L.; Young, D. B.; Barbosa, M.;
Mann, M.; Manning, A.; Rao, A. Science 1997, 278, 860. (b)
Woronicz, J. D.; Gao, X.; Cao, Z.; Rothe, M.; Goeddel, D. V.
Science 1997, 278, 866. (c) Zandi, E.; Rothwarf, D. M.; Del-
hase, M.; Hayakawa, M.; Karin, M. Cell 1997, 91, 243.
2. Barnes, P. J.; Karin, M. N. Engl. J. Med. 1997, 336, 1066.
3. (a) O’Connell, M. A.; Bennett, B. L.; Mercurio, F.; Manning,
A. M.; Mackman, N. J. Biol. Chem. 1998, 273, 30410. (b) Harhay,
E. W.; Sun, S. C. J. Biol. Chem. 1998, 272, 25185. (c) Delhase, M.;
Hayakawa, M.; Chen, Y.; Karin, M. Science 1999, 284, 309.
4. Takeda, K.; Takeuchi, O.; Tsujimura, T.; Itami, S.; Adachi,
O.; Kawai, T.; Sanjo, H.; Yoshikawa, K.; Terada, N.; Akira,
S. Science 1999, 284, 313.
5. Peet, G. W.; Li, J. J. Biol. Chem. 1999, 274, 32655.
6. Yin, M.-J.; Yamamoto, Y.; Gaynor, R. B. Nature 1998,
396, 77.
7. Rossi, A.; Kapahi, P.; Natoli, G.; Takahashi, T.; Chen, Y.;
Karin, M.; Santoro, M. G. Nature 2000, 403, 103.
8. Hideshima, T.; Chauhan, D.; Richardson, P.; Mitsiades,
C.; Mitsiades, N.; Hayashi, T.; Munshi, N.; Dang, L.; Castro,
A.; Palombella, V.; Adams, J.; Anderson, K. C. J. Biol. Chem.
2002, 277, 16639.
9. For details of the syntheses, see: Murata, T.; Sakakibara,
S.; Yoshino, T.; Ikegami, Y.; Masuda, T.; Shimada, M.;
Shintani, T.; Shimazaki, M.; Lowinger, T.B.; Ziegelbauer,
noassay
recommendations (R&D Systems, Oxon, UK).
technique
following
the
manufacturer’s

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T. Murata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 913–918
18. In vivo TNFa induction by LPS in mice: Eight week old
BALB/c female mice were placed into two groups, a control
group and a treated group. A solution containing 200 mg/
mouse of LPS in 0.9% physiological saline was administered
by intraperitoneal (ip) injection into the control mice. Mice in
the treated group were first injected ip with test compounds 30
min prior to the LPS injection or po with test compounds
(10% cremophor/saline vehicle) 60 min prior to the LPS
injection. Under anesthesia with pentobarbital (80 mg/kg, ip),
blood was collected from the posterior venous cavity of the
treated and control mice at 90 min post-LPS injection into a
96-well plate containing 2% EDTA solution. The plasma was
separated by centrifugation at 1800 rpm for 10 min at 4 ꢀC and
then diluted with four times volumes of phosphate buffer sal-
ine (pH 7.4) containing 1% bovine serum albumin. TNFa
concentration in the sample was determined using an ELISA
kit (Pharmingen, San Diego, CA). The mean of TNFa level in
5 mice from each group was determined and the percent
reduction in TNFa levels was calculated.
17. The hydrogen-bonding interaction between the phenolic
hydroxide and the pyridine nitrogen atom was estimated by
the chemical shift of the phenolic hydroxide proton in the H
NMR spectrum, which was measured on a Brucker 500 MHz
spectrometer using DMSO-d6 as solvent and tetramethylsilane
(Me4Si) as an internal standard (0 ppm). When the structure is
suitable for the hydrogen-bonding, the chemical shift of this
proton is approximately 13 ppm. On the other hand, in the
absence of intramolecular hydrogen bonding, the chemical
.shift shifts upfield, toward 10 ppm.
1