Synthesis and structure-activity relationships of novel IKK-β inhibitors. Part 2: Improvement of in vitro activity

Article abstract of DOI:10.1016/j.bmcl.2004.05.040

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as IκB kinase β (IKK-β) inhibitors. Substitution of an aminoalkyl group for the aromatic group at the 4-position on the core pyridine ring resulted i

Full text of DOI:10.1016/j.bmcl.2004.05.040

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4013–4017
Synthesis and structure–activity relationships of novel IKK-b
inhibitors. Part 2: Improvement of in vitro activity
Toshiki Murata,a,* Mitsuyuki Shimada,a Hiroshi Kadono,a Sachiko Sakakibara,a
Takashi Yoshino,a Tsutomu Masuda,a Makoto Shimazaki,a Takuya Shintani,a
Kinji Fuchikami,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 26 April 2004; revised 17 May 2004; accepted 18 May 2004
Available online 17 June 2004
Abstract—A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as IjB
kinase b (IKK-b) inhibitors. Substitution of an aminoalkyl group for the aromatic group at the 4-position on the core pyridine ring
resulted in a marked increase in both kinase enzyme and cellular potencies, and provided potent IKK-b inhibitors with IC50 values
of below 100 nM.
Ó 2004 Elsevier Ltd. All rights reserved.
Previously we described the identification of 2-amino-
3-cyano-4-aryl-6-(2-hydroxyphenyl)pyridines as small
molecule inhibitors of IjB kinase b (IKK-b).1 IKK-b is
a serine–threonine protein kinase, which is critically in-
volved in the activation of the transcription factor Nu-
clear Factor kappa B (NF-jB) in response to various
inflammatory stimuli.2 In general, inhibition of NF-jB
activation results in strong anti-inflammatory effects
similar or superior to those brought upon by steroids.
Therefore, such IKK-b inhibitors are of potential use in
disease indications such as asthma, atopic dermatitis,
allergic rhinitis, and rheumatoid arthritis.
(IC50 > 20 lM). Furthermore, compound 2 demonstrates
significant in vivo activity in an acute model of cytokine
release (LPS-induced TNFa production model in mice).
However, further improvement of the in vitro activity,
especially cellular activity, could not be accomplished by
modification of the substituents on the 4-phenyl ring.
Thus, our synthetic strategy shifted to replacing the
4-phenyl ring itself. Conveniently, the improved and
facile procedure for the pyridine core ring synthe-
sis offers ample possibilities for significant structural
modifications at the 4-position (Fig. 1).
Herein, we report the synthesis and SAR of a series of
2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine
analogs.
The initial structure–activity relationships (SAR) study1
of our first lead compound 1 indicated that various
functional groups were tolerated on the 4-phenyl ring
without losing activity whereas the ortho-phenol and the
2-aminopyridine were demonstrated to be essential
moieties for activity. Hence, the emphasis of our initial
synthetic efforts was focused on a modification of the
substituents on the 4-phenyl ring. The optimized analog
2 shows potent inhibitory activity against IKK-b
(IC50 ¼ 600 nM) and excellent selectivity versus other
kinases such as IKK-a (IC50 ¼ 20 lM), Syk and MKK4
H
N
H
N
O
N
O
O
O
CN
CN
OH
OH
N
NH2
N
NH2
1
2
IC50 = 1500 nM (IKK-β)
IC50 = 600 nM (IKK-β)
Keywords: IKK-b; NF-jB; Kinase inhibitor.
* Corresponding author. Tel.: +81-774-75-2483; fax: +81-774-75-2510;
e-mail: muratato@kcn.ne.jp
Figure 1. Initial lead compounds.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.040

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T. Murata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4013–4017
The 2-amino-3-cyano-4,6-diarylpyridine core structures
can be simply constructed using a one-pot coupling
reaction of four components, acetophenone, benzalde-
hyde, malononitrile, and ammonium acetate. Although
only aromatic aldehydes were exemplified as the starting
aldehyde in the reported procedure,3 we found that var-
ious aliphatic aldehydes could also be used in the pyridine
construction reaction using traditional and combinato-
rial chemistry approaches,4 as shown in Scheme 1.
We next focused our attention on the synthesis and SAR
of the 4-aminoalkyl analogs prepared from N-Boc-pro-
tected glycinal and b-alaninal derivatives. Table 1 de-
scribes the SAR of these compounds. While compounds
3a–c prepared from phenylalaninal derivatives exhibit
potent IKK-b inhibitory activity with IC50 values below
100 nM, compounds 3e–g derived from aliphatic amino
acids also maintain moderate activity, suggesting that
the aromatic ring is not so important for activity. Sim-
ilar results are also observed with the 4-aminoethyl-
pyridine analogs 4a–c. The simple aminoethyl analog 4a
is more potent than the phenyl-substituted analog 4c.
The IKK-b inhibitory activity could be improved by
introduction of the primary aminoalkyl moiety at the 4-
position, and these analogs are more potent than the
natural protein kinase inhibitor staurosporine5 in our
assay. Nevertheless, no significant improvement in
cellular potency (TNFa-induced RANTES production)
was achieved. We next sought to investigate the SAR
of 4-aminoalkylpyridine analogs using not only
amino acid moieties but also other aminoalkyl
aldehydes as the starting material for the pyridine core
synthesis.
In our initial efforts to replace the substituted phenyl
ring with an aliphatic group for the R2 moiety, com-
pound 3, which incorporates a primary amine was found
to be 25-fold more potent than the lead compound 1 in
the IKK-b biochemical assay. Compound 3 was syn-
thesized starting from commercially available N-Boc-
phenylalaninal, as shown in Scheme 2. Although opti-
cally pure N-Boc-phenylalaninal was also used as the
starting material, the desired chiral analog was not ob-
tained because racemization of the asymmetric center
readily occurred during the pyridine ring synthesis. For
the sake of simplicity, all the analogs were evaluated as
racemic mixtures.
Table 1. SAR of 4-aminoalkylpyridine analogs
R2
R1
O
O
R1
CN
R
AcONH4
CN
CN
O
R2
+
+
H2N
R
CHO
H2N
OH
N
NH2
CN
CN
OH
N
NH2
N
NH2
Scheme 1. Four component coupling reaction to construct the pyr-
idine core ring. R1 ¼ phenol protecting group (benzyl, p-methoxy-
benzyl, methoxyethoxy-methyl, tert-butyldimethylsilyl), or Wang
resin. R2 ¼ aryl or alkyl group.
3a-g
4a-c
Compds
–R
IKK-ba
IC50 (nM)
RANTESb
IC50 (nM)
1
2
1500
600
8000
7000
3a
3b
3c
3d
60
80
8000
5000
H
O
N
a
+
H
N
O
O
O
CN
O
BnO
OMe
O
CHO
N
NH2
40
5000
F
HCl
H2N
730
15,000
H
b
c
O
N
3e
3f
–CH3
150
480
240
70
4000
15,000
5000
O
CN
CN
n-Butyl
iso-Propyl
–H
HO
OH
3g
4a
4b
N
NH2
N
NH2
800
3000
–CH3
470
3a
IC50 = 60 nM (IKK- β)
4c
110
250
2000
Staurosporine
Not done
Scheme 2. Reagents and conditions: (a) malononitrile, ammonium
acetate, toluene, 120 °C in a sealed vessel, 3 h, 49% yield; (b) 10% Pd–
C, ethyl acetate, THF, H2 at 3 atm, rt, 24 h, 89% yield; (c) 2.5 N HCl in
1,4-dioxane, rt, 3 h, quant.
a Enzyme inhibition assay using recombinant human IKK-b.
b ELISA assay measuring TNFa-induced RANTES production in
A549 cells.

T. Murata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4013–4017
4015
As a result, 4-(3-piperidinyl)pyridine analog 5a was
Boc
N
N
Boc
NH
OH
found to have a desirable in vitro biological profile
(IKK-b IC50 ¼ 25 nM). Figure 2 describes the SAR of
the piperidine analogs. Change of the substitution po-
sition (5b and 5c), aromatization (5d) or N-methylation
(5e) of the piperidine nitrogen result in a decrease of
activity, suggesting that the piperidine NH is necessary
for high in vitro potency. However, the 4-piperidine
analog 5b shows cellular activity comparable to the 3-
piperidine analog 5a. Although the N-methylation of the
2-amino moiety (5f) leads to a drastic loss in activity, the
N-acetylation (5g) maintains moderate activity. Re-
moval of the phenolic hydroxide (5h) results in a com-
plete loss of activity. The same result has been observed
in initial SAR of the lead compound 1 and ascribed to
the hydrogen-bonding interaction between the phenolic
hydroxide and the pyridine nitrogen atom, which is
hypothesized to retain an important conformation for
potent IKK-b inhibitory activity. Compound 5i, incor-
porating a fluorine atom at the 5-position of the pyridine
core, maintains moderate activity, suggesting that the
fluorine atom does not significantly interrupt the
hydrogen bonding.
N
a, b
c
d
OMe
O
CHO
Boc
O
Boc
N
N
e
f
CN
CN
5a
O
OH
N
NH2
N
NH2
6
Scheme 3. Reagents and conditions: (a) di-tert-butyl dicarbonate, 2 N
NaOH, 1,4-dioxane, rt, 12 h, 88% yield; (b) N,O-dimethylhydroxyl-
amine hydrochloride, PyBOP, Et3N, CH2Cl2, rt, 12 h, quant.; (c)
LiAlH4, THF, )15 °C, 30 min, then 1 N KHSO4 aq, quant.; (d) 20-
benzyloxyacetophenone, malononitrile, ammonium acetate, toluene,
150 °C in a sealed vessel, 2 h, 34% yield; (e) 10% Pd–C, ethyl acetate,
THF, H2 at 3 atm, rt, 24 h, 89% yield; (f) 0.5 N HCl in 1,4-dioxane, rt,
12 h, 53% yield.
In this piperidine series, compound 5a was found to be
the most promising. Compound 5a exhibits good
450 nM) and MKK4 (IC50 > 20 lM), and excellent
activity in various cellular assays such as TNFa-
selectivity versus other kinases such as IKK-a (IC50
¼
induced RANTES production in A549 cell (IC50
300 nM), anti-CD3/anti-CD28-induced IL-2 produc-
¼
tion in Jurkat T-cell (IC50 ¼ 600 nM) and LPS-
induced TNFa production in human PBMC (IC50
400 nM).
¼
H
N
NH
NH
The synthesis of compound 5a is depicted in Scheme 3.
The key step of piperidine analogs 5a–i is the one-pot
coupling reaction of four components. The starting
aldehyde, N-Boc-3-formylpiperidine, was prepared by a
reduction of the corresponding Weinreb amide using
LiAlH4. The syntheses of the N-methylated compounds
(5e and 5f) and the N-acetyl compound 5g were con-
ducted starting from the key intermediate 6,6 as described
in Scheme 4. The methyl group on the piperidine nitrogen
was introduced by a reductive alkylation of the piperidine
nitrogen using formalin and NaBH3CN to provide
compound 5e. The synthesis of the 2-alkylaminopyridine
analog 5f was achieved by a Sandmeyer reaction using
CuBr2 and tert-butyl nitrite followed by a nucleophilic
attack of methylamine onto 2-bromopyridine. Reaction
of compound 6 with acetyl chloride in pyridine gave the
diacetylated analog, which was then treated with dilute
ammonium hydroxide solution to cleave an acetyl bond
to provide mono-acetyl analog 5g. The fluoropyridine
analog 5i was synthesized from 2-fluoroacetophenone as
the starting material for the pyridine synthesis, as shown
in Scheme 5.
CN
CN
CN
NH2
OH
OH
OH
N
NH2
N
NH2
N
5a
25 nM (IKK-β)
300 nM (RANTES)
5b
5c
530 nM (IKK-β)
6000 nM (RANTES)
300 nM (IKK-β)
400 nM (RANTES)
CH3
N
N
NH
CN
OH
CN
OH
CN
OH
N
NH2
N
NH2
N
NH
CH3
5d
5e
5f
6200 nM (IKK-β)
230 nM (IKK-β)
1000 nM (RANTES)
6500 nM (IKK-β)
20 µM (RANTES)
NH
NH
CN
NH
CN
OH
CN
F
HO
In summary, replacement of the substituted phenyl with
an aminoalkyl moiety at the 4-position of the pyridine
core ring resulted in a marked increase in in vitro
activity. The optimized piperidine analog 5a exhibited
not only selective and potent inhibition of IKK-b kinase
but also reasonably potent activity in a variety of rele-
vant cellular assays.
N
NH
N
NH2
N
NH2
O
5g
330 nM (IKK-β)
1500 nM (RANTES)
5h
>20 µM (IKK- β)
5i
230 nM (IKK-β)
2000 nM (RANTES)
Figure 2. SAR of piperidine analogs (IC50 values).

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T. Murata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4013–4017
CH3
NH
N
b
c
CN
CN
5e
O
O
O
O
O
N
N
NH2
Boc
N
N
N
NH2
a
Boc
CN
N
N
e
d
f, g
6
CN
Br
5f
NH
CH3
h
Boc
Boc
N
N
O
i
j, k
5g
CN
O
CN
NH
O
N
N
O
Scheme 4. Reagents and conditions: (a) 2 N HCl in 1,4-dioxane, rt, 5 h, quant.; (b) formaldehyde, NaBH3CN, water, methanol, rt, 2 h, 74% yield;
(c) 10% Pd–C, ethyl acetate, THF, H2 at 3 atm, rt, 24 h, 50% yield; (d) CuBr2, tert-butyl nitrite, acetonitrile, 65 °C, 2 h, 45% yield; (e) methylamine
hydrochloride, Et3N, DMSO, 40 °C, 12 h, 58% yield; (f) 10% Pd–C, ethyl acetate, THF, H2 at 3 atm, rt, 24 h, 82% yield; (g) 2 N HCl in 1,4-dioxane, rt,
5 h, quant; (h) acetyl chloride, pyridine, rt, 5 h; (i) 1 N ammonium hydroxide, THF, rt, 1 h, 80 % yield in two steps; (j) 10% Pd–C, ethyl acetate, THF,
H2 at 3 atm, rt, 12 h, 31% yield; (k) 2 N HCl in 1,4-dioxane, rt, 5 h, quant.
OH
O
a, b
c
d
O
O
O
O
Br
F
Boc
Boc
N
N
e
f
F
CN
F
CN
5i
O
HO
N
NH2
N
NH2
Scheme 5. Reagents and conditions: (a) benzylbromide, K2CO3, acetone, reflux, 18 h, 88% yield; (b) Br2, ether, rt, 30 min, 92% yield; (c) tetrabutyl-
ammonium hydrogen difluoride, pyridine, THF, reflux, 22 h, 33% yield; (d) malononitrile, N-Boc-3-formylpiperidine, ammonium acetate, xylene,
120 °C, 3 h, 25% yield; (e) 10% Pd–C, ethyl acetate, THF, H2 at 3 atm, rt, 12 h, 34% yield; (f) 2 N HCl in 1,4-dioxane, rt, 5 h, quant.
4. Shintani, T.; Kadono, H.; Kikuchi, T.; Schubert, T.;
Shogase, Y.; Shimazaki, M. Tetrahedron Lett. 2003, 44,
6567.
References and notes
1. Murata, T.; Shimada, M.; Sakakibara, S.; Yoshino, T.;
Kadono, H.; Masuda, T.; Shimazaki, M.; Shintani, T.;
Fuchikami, K.; Sakai, K.; Inbe, H.; Takeshita, K.; Niki, T.;
Umeda, M.; Bacon, K. B.; Ziegelbauer, K. B.; Lowinger, T.
B. Bioorg. Med. Chem. Lett. 2003, 13, 913.
2. (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.;
Delhase, M.; Hayakawa, M.; Karin, M. Cell 1997, 91, 243.
3. Kambe, S.; Saito, K. Synthesis 1980, 366.
5. Peet, G. W.; Li, J. J. Biol. Chem. 1999, 274, 32655.
6. A typical procedure for the four component coupling to
construct the pyridine analog (synthesis of compound 6): A
mixture of 20-benzyloxyacetophenone (2.60 g, 11.5 mmol),
tert-butyl 3-formyl-1-piperidine carboxylate (4.90 g,
23.0 mmol), malononitrile (1.52 g, 23.0 mmol), ammonium
acetate (4.43 g, 57.5 mmol), and toluene (12 mL) in a sealed
vessel was stirred at 150 °C for 2 h. After cooled to room
temperature, the reaction mixture was partitioned between
ethyl acetate and water. The organic phase was separated,
washed with water and brine, dried over Na2SO4, filtered,

T. Murata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4013–4017
4017
and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (hexane/
ethyl acetate, 2/1) to give a yellow oil, which was then
recrystallized from ethanol to give the desired product as a
white solid (1.90 g, yield; 34% yield). For further details of
the syntheses, see: Murata, T.; Umeda, M.; Sakakibara, S.;
Yoshino, T.; Sato, H.; Masuda, T.; Koriyama, Y.; Shimada,
M.; Shintani, T.; Kadono, H.; Ziegelbauer, K. B.; Fuchi-
kami, K.; Komura, H.; Lowinger, T. B. WO 0224679, 2002;
Chem Abstr. 2002, 136, 279345.