Novel heterocycle-substituted pyrimidines as inhibitors of NF-κB transcription regulation related to TNF-α cytokine release

Article abstract of DOI:10.1016/j.bmcl.2007.11.064

Novel heterocyclic ring-substituted pyrimidines have been designed as inhibitors of glycogen synthase kinase-3β (GSK-3β) from the modification of known inhibitors. Several potent inhibitors exhibiting nanomolar activities were discovered against GSK-3β kinase as well as in an NF-κB reporter gene assay. Based on the results from in vitro TNF-α release inhibition and in vivo endotoxima, these inhibitors are expected to be useful candidates for treatment of inflammation-related diseases.

Full text of DOI:10.1016/j.bmcl.2007.11.064

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 653–656
Novel heterocycle-substituted pyrimidines as inhibitors of
NF-jB transcription regulation related to TNF-a cytokine release
Hyung-Ho Ha, Jee Seon Kim and B. Moon Kim*
Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea
Received 9 August 2007; revised 29 October 2007; accepted 17 November 2007
Available online 22 November 2007
Abstract—Novel heterocyclic ring-substituted pyrimidines have been designed as inhibitors of glycogen synthase kinase-3b (GSK-
3b) from the modification of known inhibitors. Several potent inhibitors exhibiting nanomolar activities were discovered against
GSK-3b kinase as well as in an NF-jB reporter gene assay. Based on the results from in vitro TNF-a release inhibition and
in vivo endotoxima, these inhibitors are expected to be useful candidates for treatment of inflammation-related diseases.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The transcription nuclear factor jB (NF-jB)1 is a pivotal
regulator of the inducible expression of key pro-inflam-
matory mediators. Activated NF-jB has been observed
in several debilitating inflammatory disorders including
rheumatoid arthritis (RA)2 and osteoarthritis. In acti-
vated T cells, transcription factors such as the activator
protein-1 (AP-1)3 regulate IL-2 and matrix metallopro-
teinase production, while the NF-jB is essential for the
transcriptional regulation of the pro-inflammatory cyto-
kines4 IL-1, IL-6, IL-8, and TNF-a. Though the mecha-
nism for the inhibition of NF-jB is well established,1
complications which depend on the signal inducing mole-
cules, cell types, and target genes are still waiting to be
deciphered. However, the IjB protein family members
are the most promising classical target of inhibition for
NF-jB. The IjB kinase (IKK) complex is the primary
mediator of IjB phosphorylation and is activated by var-
ious stimuli including cytokines such as tumor necrosis
factor alpha (TNF-a) and interleukin 1 (IL-1).
activity. However, NF-jB DNA binding and luciferase
reporter assay activity were reduced in the GSK-3b null
cells, which is consistent with the report that GSK-3b is
able to affect NF-jB function downstream of release
from IjBb. Recently, a number of reports have emerged
describing molecules that inhibit GSK-3b targeting
mostly diabetes and Alzheimer disease.7
However, a recent publication has demonstrated that
TNF-induced NF-jB reporter gene transcription was
also suppressed in GSK-3b gene-deleted cells.8,9 GSK-
3b was also reported to inhibit the systemic inflamma-
tion of in vivo endotoxic model induced by LPS,10 while
another study has demonstrated that inhibition of GSK-
3b potentiated TNF-induced expression of IL-6 and
MCP-1 by 2–6-fold.11 Generally TNF-a contributes to
the regulation of the body’s response to infection and
cellular stress. Chronic and excessive production of
TNF-a are believed to underlie the progression of many
autoimmune diseases such as rheumatoid arthritis (RA),
Chrohn’s disease, and psoriasis.12
The glycogen synthase kinase-3b (GSK-3b)5 is a critical
factor in the regulation of a wide variety of signaling
proteins and transcription factors, including cyclin D1,
c-Jun, NF-ATc, and b-catenin, among others. Numer-
ous reports have implicated GSK-3b in the control of
various signaling pathways that activate NF-jB, includ-
ing regulation of NF-jB1/p105 stability6 as well as IKK
GlaxoSmithKline researchers reported pyrazolo[3,4-
b]pyridines represented by compound 1 as potent inhib-
itors of GSK-3 and they showed very promising kinase
inhibitor activities (Fig. 1).13 Based upon our prelimin-
ary modeling studies of the GlaxoSmithKline structures,
two possible structural modifications were envisioned,
namely structures A and B in Figure 1.
Keywords: Glycogen synthase kinase 3b; Nuclear factor jB; Anti-inf-
lammation; TNF-a cytokine; Heterocyclic fused ring.
In structure A, the 6-position of the pyridine ring ap-
peared to have some room for extra binding.14 Fixing
*
Corresponding author. Tel.: +82 2 880 6644; fax: +82 2 872 7505;
e-mail: kimbm@snu.ac.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.064

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H.-H. Ha et al. / Bioorg. Med. Chem. Lett. 18 (2008) 653–656
Table 1. Inhibition of pyrazolopyridine analogues against GSK-3b,
and NF-jB and AP-1 reporter gene assay results for compounds 2–6
H
N
N
F
O
O
R1
F
N
HN
HN
F
O
R2
N
N
HN
X
N
H
N
N
H
N
N
Y
N
F
N
N
X, Y = N, C
Structures B
R
IC50 = 7nM
N
H
N
N
1
Structures A
N
R
Figure 1. New inhibitor design based upon the GlaxoSmithKline
inhibitor 1 against GSK-3.
Com-
pound
R
H
GSK-3b,
NF-jB,
AP-1b,
a
a
a
IC50 (lM) IC50 (lM) IC50 (lM)
cyclopropanecarboxamide for 3-amino group and a
small fluoride group at the 5 position, we focused on
the diversification at the 6-position of the bicyclic ring
with piperazine derivatives. Derivatives of the structure
A were prepared as outlined in Scheme 1.
2
3
4
5
6
na
na
na
na
2-CH3OPh
10.9 ( 0.5) na
13.1 ( 1.2) na
19.3 ( 4.6) na
9.4 ( 0.8) na
2-CH3OPhC(O) na
2,4-F2-PhC(O) na
2-CF3-PhS(O2) na
a Values are means of three experiments, standard deviation is given in
parentheses (na, not active).
b In vitro therapeutic index (IC50 cytotoxicity/IC50 complement
inhibition).
In Table 1 in vitro activity results from this modification
are listed. All five compounds tested were found to be
inactive for GSK-3b kinase and also for the reporter
gene assay of NF-jB, and AP-1 showed moderate and
unsatisfactory results, respectively.
N
O
O
From the above results, it was not possible to draw any
conclusion on the relationship between GSK-3b and
NF-jB transcription. We then turned our attention to
targeting GSK-3 inhibition using structure B.
O
a
b
R
R
N
N
R
N
N
7
8
9
Diversity in the structure B was given in three parts; R1,
R2, and the heteroatom (X and/or Y) in the bicyclic ring.
Compounds 12–16 were prepared through the sequence
of reactions shown in Scheme 2. Pyrazolopyridine ring
formation and aminopyrimidine synthesis, two key syn-
thetic reactions in this scheme, proceeded smoothly.
NH
NH2
c
R1
R1
d
N
H
11
NH2
10
H
N
12: R1=H, R2=H
R1
N
The R2 group was fixed as H or phenyl as shown in
Scheme 2. However, for R1, broader diversity was intro-
duced and from this primary SAR study three aniline
derivatives were selected. In Scheme 3 a reaction se-
quence is depicted for the construction of pyrolopyri-
dine or indole core structures. Methoxyethyl group15
was introduced for the enhancement of physicochemical
property of the inhibitors which often exhibit high lipo-
philicity (Table 2).
13: R1=3-Methoxy, R2=H
14: R1=2-Me-5-Cl, R2=H
15: R1=H, R2=Phenyl
N
R2
16: R1=3-Methoxy, R2=Phenyl
N
N
Scheme 2. A general synthetic process for structure B. Reagents and
conditions: (a) 1-aminopyridinium iodide, KOH, H2O, rt, 60–70%; (b)
DMF–DMA, DMF, 130 ꢁC, 65–75%; (c) conc nitric acid, 50%
cyanamide, ethanol, 90 ꢁC, 82%; (d) K2CO3, 2-methoxyethanol,
120 ꢁC, 40–50%.
NH2
F
CN
Cl
F
F
CN
Cl
N
a
b
N
H
N
N
N
N
N
O
Cl
N
N
PG
PG
HN
PG=BOC, 2-MeOPh
2: R=H
O
HN
F
3: R=2-CH3OPh
4: R=2-CH3OPhC(O)
5: R=2,4-F2-PhC(O)
6: R=2-CF3-PhS(O2)
F
e
c, d
N
N
N
H
N
N
N
H
N
N
N
R
HN
Scheme 1. A general synthetic scheme for structure A. Reagents and conditions: (a) piperazine, Et3N, CH3CN, refluxed 2 h, 94–96%; (b) hydrazine
hydrate, 2-methoxyethanol, refluxed 6 h, 82–86%; (c) cyclopropanecarbonyl chloride, pyridine, refluxed 4 h, 80–84%; (d) 4 N-HCl in dioxane, rt 5 h,
96%; (e) RCOCl or RSO3Cl, Et3N, CH2Cl2, 82–86%.

H.-H. Ha et al. / Bioorg. Med. Chem. Lett. 18 (2008) 653–656
655
O
O
N
O
A
b
a
N
H
N
A
A
H
18
17
19
H
N
N
N
R1
O
N
d
c
N
N
O
A
A
21: A=N, R1=H
22: A=N, R1=3-Methoxy
23: A=C, R1=H
O
Figure 2. In silico surface model of the GSK-3 ATP-binding pocket
from PDB database with compound 21.
20
24: A=C, R1=3-Methoxy
Scheme 3. A general synthetic process for compounds 21–24. Reagents
and conditions: (a) AlCl3, AcCl, CH2Cl2, rt, 76–85%; (b) 2-bromoethyl
methyl ether, K2CO3, DMF, 50 ꢁC, 80–85%; (c) DMF–DMA, DMF,
130 ꢁC, 75–80%; (d) guanidine, t-BuOK, THF, 80 ꢁC, 50–55%.
compounds 21–24. The R1 substitution did not
appear to contribute much toward the activity except
for the compounds 13 and 14, where a pronounced dif-
ference toward GSK-3b was observed. However, the
phenyl substitution at R2 showed a dramatic increase
in activity. It appears that a major contribution for
the activity enhancement was from the aromatic nitro-
gen. To obtain selective compounds for inflammation
related mainly with NF-jB, three compounds were
selected and tested for the inhibition19 of TNF-a pro-
duction using (LPS)-stimulated human monocytic
cells (THP-1) by ELISA and the data are shown in
Table 3.
The main difference in structures A and B is the amino-
pyrimidine moiety. Generally this structural motif has
been well known for kinase inhibitor activity. A few
groups tried to combine this fragment and the pyrazol-
o-pyridine moiety to increase activity and selectivity for
kinase and antiviral inhibition.16 Binding mode of the
inhibitors featuring the two fragments combined was
predicted through in silico modeling as depicted in Fig-
ure 2.17 According to the modeling studies of com-
pound 21, phenylaminopyrimidine group appears to
have a unique interaction with Tyr134 and Val135 in
the hydrophobic pocket. The binding appears to be
optimal with the pyrimidyl-substituted bicyclic ring.
The result of inhibition against GSK-3 could be ana-
lyzed in two aspects, namely, the core structure and
peripheral substitution. Pyrolo-pyridines 12–16 showed
better activities not only in the GSK-3b kinase assay18
but also in the NF-jB reporter gene assay compared to
Table 3. In vitro functional assay for the inhibition of TNF-a release
in THP-1 cell line
Compound:
3
12
22
a
TNF-a, IC50 (lM)
na
0.14 ( 0.02)
0.99 ( 0.25)
a Values are means of three experiments, standard deviation is given in
parentheses (na, not active).
Table 2. Results of the inhibition of aminopyrimidine analogues for GSK-3b, NF-jB, and AP-1 reporter gene assay for compounds 12–24
H
N
N
R1
N
R2
X
N
Y
R3
a
a
a
Compound
R1
R2
R3
X,Y
GSK-3b, IC50 (lM)
NF-jBb, IC50 (lM)
AP-1c, IC50 (lM)
12
13
14
15
16
21
22
23
24
Ph
H
H
H
Ph
Ph
H
H
H
H
H
N,C
N,C
N,C
N,C
N,C
C,N
C,N
C,C
C,C
0.15 ( 0.02)
0.10 ( 0.02)
10.60 ( 1.08)
0.06 ( 0.01)
0.03 ( 0.14)
1.05 ( 0.08)
2.20 ( 0.07)
0.32 ( 0.13)
0.14 ( 0.18)
0.6 ( 0.05)
1.0 ( 0.08)
4.3 ( 0.3)
1.7 ( 0.1)
1.9 ( 0.1)
5.6 ( 0.6)
1.4 ( 0.1)
10.4 ( 1.1)
7.8 ( 0.5)
0.9 ( 0.03)
1.8 ( 0.08)
4.9 ( 0.7)
0.7 ( 0.2)
1.6 ( 0.3)
11.4 ( 1.5)
na
3-CH3OPh
2-CH3, 5-ClPh
Ph
H
H
H
3-CH3OPh
Ph
H
C2H4OMe
C2H4OMe
C2H4OMe
C2H4OMe
3-CH3OPh
Ph
3-CH3OPh
na
na
a Values are means of three experiments, standard deviation is given in parentheses (na, not active).
b Inhibition of NF-jB mediated transcriptional activation in A-549 cells.
c Inhibition of AP-1 mediated transcriptional activation in Jurkat cells.

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H.-H. Ha et al. / Bioorg. Med. Chem. Lett. 18 (2008) 653–656
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17. The molecular modeling was performed with the Maestro
software package from Schrodinger. The ligand was
manually docked into the active site. Structural co-
ordinates for the GSK-3 structure were taken from: Bax,
B.; Carter, P. S.; Lewis, C.; Guy, A. R.; Bridges, A.;
Tanner, R.; Pettman, G.; Mannix, C.; Culbert, A. A.;
Brown, M. J. B.; Smith, D. G.; Reith, A. D. Structure
2001, 9, 1143.
18. GSK-3b was assayed in 96-well microtiter plates at a final
concentration of 20 nM in 100 mL Hepes at pH 7.2
containing 10 mM MgCl2, 0.1 mg/mL bovine serum albu-
min, 1 mM dithiothreitol, 0.3 mg/mL heparin, 2.8 M
peptide substrate (Biotin-Ahx-AAAKRREILSRRP-
S(PO3)YR-amide), 2.5 M ATP, and 0.2 Ci/well
[33P]ATP. After 40 min, the reaction was stopped by
addition of 100 mM EDTA and 1.0 mM ATP, solution in
100 mM Hepes followed by a solution of streptavidin
coated SPA beads (Amersham) in PBS to give a final
concentration of 0.25 mg of beads per assay well. The
plates were counted on a Packard TopCount NXT
microplate counter.
Figure 3. In vivo efficacy of compound 22 for serum TNF-a level after
LPS treatment.
Not surprisingly, compound 3 was not active in the
functional assay; however, compounds 12 and 22 exhib-
ited good activities. Based on this result, it was clear that
controlling cytokine levels involves disruption of the sig-
nal transduction pathway leading to their release from
stimulated inflammatory cells by way of GSK-3b kinase
and NF-jB transcription (Fig. 3).
Compound 22 was examined for inhibition20 of TNF-a
release in Balb/C mice challenged with LPS. It inhibited
TNF-a level dramatically at 300 mg/kg dose compared
to a reference compound,21 p38 Map kinase inhibitor
BIRB796.22 Further in vivo efficacy study on this series
of compounds is in progress.
In conclusion, we report a series of fused heterocyclic
GSK-3 inhibitors showing nanomolar potency against
GSK-3b, and submicromolar activities toward NF-jB
and LPS-stimulated human monocytic cells (THP-1).
From this result, in vitro TNF-a release inhibition and
in vivo sepsis model could be used to identify promising
candidates for treatment of inflammation related
diseases.
Acknowledgment
We are grateful to Choongwae Pharmaceuticals for run-
ning the reporter gene assay and TNF-a release assay.
19. Human monocytic cell line (THP-1) TNF-a release assay;
THP-1 cells were seeded into 96-well plates at 1 · 106 cells/
mL (200 L/well) in medium containing FCS (1%) and
incubated overnight. Following pretreatment with com-
pounds for 1 h, the cells were incubated with LPS (20 g/
mL) for a further 24 h. TNF-a release was measured in the
supernatants by sandwich ELISA. IC50 values shown from
repeat experiments are means (n = 3).
References and notes
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