Synthesis and structure-activity relationships of novel IKK-β inhibitors. Part 3: Orally active anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2004.05.041

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. Modification of a novel IKK-β inhibitor 1 (IKK-β IC ₅₀=1500nM, Cell IC₅₀=8000nM) at the 4-p

Full text of DOI:10.1016/j.bmcl.2004.05.041

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4019–4022
Synthesis and structure–activity relationships of novel IKK-b
inhibitors. Part 3: Orally active anti-inflammatory agents
Toshiki Murata,a,* Mitsuyuki Shimada,a Sachiko Sakakibara,a Takashi Yoshino,a
Tsutomu Masuda,a Takuya Shintani,a Hiroki Sato,a Yuji Koriyama,a
Keiko Fukushima,a Noriko Nunami,a Megumi Yamauchi,a
Kinji Fuchikami,b Hiroshi Komura,b Akihiko Watanabe,b Karl B. Ziegelbauer,b
Kevin B. Baconb 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 30 April 2004; revised 17 May 2004; accepted 18 May 2004
Available online
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. Modification of a novel IKK-b inhibitor 1 (IKK-b IC50 ¼ 1500 nM, Cell IC50 ¼ 8000 nM) at the
4-phenyl ring and 6-phenol group on the pyridine core ring resulted in a marked increased in biological activities. An optimized
compound, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile, exhibited excellent in vitro
profiles (IKK-b IC50 ¼ 8.5 nM, Cell IC50 ¼ 60 nM) and a strong oral efficacy in in vivo anti-inflammatory assays (significant effects
at 1 mg/kg, po in arachidonic acid-induced ear edema model in mice).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
the pyridine ring with an aliphatic group incorporating a
primary amine resulted in a marked increase of the
IjBkinase 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-
jB) in response to various inflammatory stimuli.2 NF-
jBis an inducible transcription factor that is thought to
be a pivotal target for drugs for cancer and chronic
inflammatory diseases. Herein, we report the synthesis
and the structure–activity relationship (SAR) study of a
novel class of small molecule IKK-b inhibitors.
H
O
N
O
O
H2N
CN
CN
OH
OH
N
NH2
N
NH2
1
2
IC50 = 1500 nM (IKK-
IC50 = 8000 nM (cell)
)
IC50 = 60 nM (IKK- )
IC50 = 8000 nM (cell)
β
β
In the preceding communications,3;4 we described the
SAR study of a novel IKK-b inhibitor 1 identified via
screening of the Bayer compound library (Fig. 1). First
we found that various functional groups were tolerated
on the 4-phenyl ring without losing activity.3 Moreover,
replacing the substituted phenyl ring at the 4-position of
H
N
NH
CN
NH2
CN
OH
OH
N
N
NH2
3a
4a
β
IC50 = 25 nM (IKK- )
IC50 = 300 nM (cell)
IC50 = 300 nM (IKK- )
IC50 = 400 nM (cell)
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 compound 1 and optimized compounds.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.041

4020
T. Murata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4019–4022
IKK-b inhibitory activity, albeit with no improvement
in the cellular activity (TNFa-induced RANTES pro-
duction in A549 cells), as exemplified by compound 2
derived from a phenylalanine derivative.4 In order to
improve the cellular activity, the 4-aminoalkyl group
was further optimized, and 4-piperidin-3-ylpyridine
analog 3a was identified as a potent IKK-b inhibitor
with excellent cellular activities.4 However, the bio-
availability of compound 3a in mice and rats was less
than satisfactory. Thus, we continued the modification
of compound 3a to discover highly potent and orally
active IKK-b inhibitors with anti-inflammatory effect.
3. Results and discussion
In our initial SAR study, the in vitro activity was
improved readily by the modification of the substituent
at the 4-position on the pyridine core ring. In order to
identify more potent and orally active IKK-b inhibitors,
our synthetic strategy was next shifted to optimization
of the ortho-phenol group of the compound 3a, which
would be readily implemented using commercially
available substituted 20-hydroxyacetophenone deriva-
tives by the usual synthetic procedure. In addition, since
the initial SAR study indicated that the phenol
hydroxide was an essential moiety for activity, we
focused on the optimization of the substituents on the
ortho-phenol.
2. Chemistry5
Table 1 describes the SAR of 4-piperidin-3ylpyridine
analogs 3a–r for optimization of the ortho-phenol
group. Addition of a methyl substituent at the 30- or 60-
position (3b,e) to the parent compound 3a resulted in a
drastic loss of activity. Although the addition of a
methoxy substituent at the 30- or 40-position (3c–d) also
resulted in a loss of activity, the IKK-b inhibitory and
cellular activities were maintained when the methoxy
substituent was introduced at the 60-position (3h).
However, the corresponding 60-amino analog 3f had no
activity.
The 2-amino-6-aryl-3-cyano-4-piperidinylpyridine core
structures can be easily constructed using a one-pot
coupling reaction of four components, acetophenone,
N-Boc-formylpiperidine, malononitrile and ammonium
acetate, as exemplified in Schemes 1 and 2.
Boc
N
BnO
O
OH
O
a
b
CN
BnO
OBn
OH
N
OBn
Boc
NH2
Thus, we further concentrated on the optimization of
the alkoxy moiety at the 60-position of the ortho-phenol
group, as in 3g–r. The 60-hydroxy analog 3g exhibited
Boc
N
N
c
d
e
CN
CN
3r
OH
O
N
NH2
N
OH
NH2
Table 1. SAR of 4-piperidin-3-ylpyridine analogs 3a–r
OH
NH
Scheme 1. Reagents and conditions: (a) benzylbromide, K2CO3, NaI,
acetone, reflux, 18 h, 46% yield; (b) N-Boc-3-formylpiperidine, mal-
ononitrile, ammonium acetate, 1,4-dioxane, 120 ꢁC in a sealed vessel,
12 h, 37% yield; (c) 10% Pd–C, acetic acid, ethyl acetate, H2 at 3 atm,
rt, 2 days, 76% yield; (d) benzyl bromide, K2CO3, acetone, THF, rt,
2 days, 65% yield; (e) 2 N HCl in 1,4-dioxane, rt, 18 h, 88% yield.
CN
OH
3'
R
N
NH2
4'
6'
3a-r
5'
IC50 (nM)
Cellb
Compd
–R
IKK-ba
3a
3b
3c
3d
3e
3f
–H
25
1300
20,000
560
2400
7800
15
300
30-CH3
30-OCH3
40-OCH3
60-CH3
60-NH2
60-OH
7000
Nd
OH
O
O
O
O
a
b
O
O
2500
8000
>10,000
200
OH
OH
OMe
Boc
3g
3h
3i
H
N
N
60-OCH3
60-OCH2CH3
34
14
300
150
HCl
c
d
3j
60-O(CH2)2CH3
60-O(CH2)4CH3
60-O(CH2)6CH3
60-OCH(CH3)2
5
80
300
CN
NH2
CN
O
O
3k
3l
6
14
N
1500
800
N
NH2
3m
3n
3o
3p
3q
3r
81
5
O
OH
60-OCH2CH(CH3)2
150
80
OMe
4j
60-OCH2-cyclopropyl
60-OCH2-cyclobutyl
60-OCH2-cyclohexyl
60-OCH2-phenyl
3
4
26
80
900
Scheme 2. Reagents and conditions: (a) (bromomethyl)cyclopropane,
K2CO3, acetone, 50 ꢁC, 2 days, 83% yield; (b) 4-methoxybenzyl chlo-
ride, tetrabutylammonium iodide, acetone, reflux, 18 h, 89% yield; (c)
N-Boc-4-formylpiperidine, malononitrile, ammonium acetate, 1,4-
dioxane, 110 ꢁC, 3 h, 42% yield; (d) 2 N HCl in 1,4-dioxane, rt, 10 h,
quant.
9
300
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) 4019–4022
4021
Table 2. SAR of 4-piperidin-4-ylpyridine analogs 4a–l
somewhat better activity than the parent compound 3a.
In a series of compounds with a straight-chain alkoxy
moiety (3h–l), n-propoxy analog 3j demonstrated the
most potent activity. The longer alkoxy chain has a
beneficial effect on the IKK-b inhibitory activity but a
negative effect on cellular activity, as in 3k–l. While
introduction of a branch at the a-position on the ether
moiety (3m) led to a decrease of activity, the b-branch
compounds 3n–q exhibited greatly potent activity.
Among all such compounds, cyclopropyl and cyclobutyl
analogs (3o,p) were the most potent inhibitors. We also
found that various substituted benzylether moieties were
tolerated to maintain the potent activity, as exemplified
by compound 3r.
H
N
CN
R
N
NH2
OH
4a-l
IC50 (nM)
IKK-ba Cellb
Compd
–R
4a
4b
4c
4d
4e
4f
–H
–OH
300
270
120
24
400
500
200
70
–OCH2CH3
–O(CH2)2CH3
–O(CH2)3CH3
–O(CH2)4CH3
–O(CH2)5CH3
–O(CH2)6CH3
15
20
80
The 4-piperidin-3-ylpyridine analogs with an alkoxy
moiety at the 60-position have potent in vitro activity,
but do have poor pharmacokinetic profiles probably due
to the intrinsic physicochemical instability. The 4-pip-
eridin-3-ylpyridine analogs were prepared as the
hydrochloric acid salts, which were stable in the solid
state. However, the compounds were unstable under
basic conditions and even in neutral buffer solution. For
instance, compound 3r was rapidly decomposed at 37 ꢁC
in a pH 7.4 isotonic buffer solution, and only 25% of the
compound remained after 24 h. One of the major deg-
radation products was a cyclized compound produced
by a nucleophilic attack of the piperidine nitrogen onto
the nitrile group, as exemplified in Scheme 3. Thus, we
found that the combination of 4-piperidin-3-yl moiety
with 3-nitrile moiety on the pyridine ring would cause
physicochemical instability.
200
300
400
80
4g
4h
4i
25
50
–OCH2CH(CH3)2
–OCH2-cyclopropyl
–OCH2-cyclobutyl
–OCH2-phenyl
15
4j
8.5
40
40
4k
4l
12
110
1500
a Enzyme inhibition assay using recombinant human IKK-b.
b ELISA assay measuring TNFa-induced RANTES production in
A549 cells.
The cyclopropyl analog 4j (IKK-b IC50 ¼ 8.5 nM)
moderately inhibits IKK-a with an IC50 of 250 nM but
exhibits good selectivity towards other kinases, such as
IKK3, Syk and MKK4 (IC50 > 20,000 nM). Moreover,
compound 4j demonstrates quite potent activity in var-
ious cellular assays, as shown in Table 3. Importantly,
compound 4j inhibited NF-jB-dependent reporter gene
Next, we focused our attention on the synthesis and
SAR of the 4-piperidin-4-ylpyridine analogs, because
compound 4a (Fig. 1) has only moderate IKK-b inhib-
itory activity but potent cellular activity comparable to
the corresponding 4-piperidin-3-yl analog 3a. In addi-
tion, no degradation of the 4-piperidine analogs was
observed even under basic conditions because the reac-
tion of the 4-piperidine nitrogen with the 3-nitrile moiety
would be geometrically unfavourable. Table 2 describes
the SAR of the 4-piperidin-4-ylpyridine analogs 4a–l,
which displays the similar tendency with that of the 4-
piperidin-3-yl analogs 3a–r shown in Table 1. Intro-
duction of an alkoxy substituent at the 60-position of the
phenol (4b–l) resulted in an increase of activity relative
to the parent compound 4a. In this series, cyclopropyl
analog 4j and cyclobutyl analog 4k exhibit the most
potent IKK-b inhibitory and cellular activities.
Table 3. Inhibitory activities of compound 4j in various kinase and
cellular assays
H
N
CN
O
NH2
N
OH
4j
IC50 = 8.5 nM
IC50 = 250 nM
(IKK-β)
(IKK-α)
IC50 > 20000 nM
(IKK3, Syk and MKK4)
Cell
Stimulus
Read-out
IC50 (nM)a
A549
HUVECS
HEK293
TNFa
TNFa
TNFa
PMA/Ca
PMA/Ca
PMA
LPS
RANTES
VCAM-1
40
30
NF-jBtransactivation
NF-jBtransactivation
130
147
Jurkat T-cell
Jurkat T-cell
MRC5
NH
NF-AT transactivation >10,000
>10,000
50
N
AP-1 transactivation
TNFa
N
huPBMCs
huPBMCs
huPBMCs
O
O
NH
NH2
96b
45b
46b
pH 7.4 buffer
37 °C
LPS
LPS
IL-1b
IL-6
Proliferation
N
NH2
N
OH
3r
OH
Mouse B-cells LPS
a Values are means of more than three experiments.
Scheme 3. Possible mechanism for degradation of 4-piperidin-3-yl-
pyridine analogs.
b Results were obtained from MDS pharmacology services
(www.mdsps.com).

4022
T. Murata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4019–4022
onto the ortho-phenol group resulted in a marked
0.100
0.075
0.050
0.025
0.000
-0.025
Vehicle
4j
4j
4j
0.3 mg / kg, p.o.
improvement of activity in IKK-b and cellular assays.
The 4-piperidin-3-ylpyridine analogs 3 tend to be more
potent than 4-piperidin-4-ylpyridine analogs 4 but have
poor bioavailability due to the physicochemical insta-
bility. Thus, we have focused on the optimization of
4-piperidin-4-ylpyridine analogs 4. As a result, com-
pound 4j has been identified as a highly potent inhibitor
of IKK-b, which is orally bioavailable in mice and rats
and demonstrates significant in vivo activity in anti-
inflammatory models (arachidonic acid-induced mouse
ear edema model).
1 mg / kg, p.o.
3 mg / kg, p.o.
Dex. 0.3 mg / kg, p.o.
###
##
**
**
**
**
**
**
0
3
6
Time after treatment (h)
Figure 2. Oral efficacy of compound 4j (blue) and dexamethasone (red)
in arachidonic acid-induced mouse ear edema model:7 Statistical dif-
ferences between vehicle control and compound 4j groups were ana-
lyzed using one-way ANOVA and Dunnett’s method (**: P < 0:01,
***: P < 0:001). For dexamethasone, data were analyzed by Student’s
t-test (##, ###: P < 0:01, P < 0:001). Dex: dexamethasone.
References and notes
1. (a) Mercurio, F.; Zhu, H.; Murray, B. W.; Shevchenko, A.;
Bennett, B. L.; Jian; Wu Li; 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.
2. For recent reviews, see: (a) Ghosh, S.; Karin, M. Cell 2002,
109(Suppl: S81); (b) Senftleben, U.; Karin, M. Crit. Care
Med. 2002, 30, S18.
3. 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.
activation in TNFa -activated HEK293 cells and PMA/
calcium ionophore-activated Jurkat T cells. On the other
hand, compound 4j failed to inhibit PMA-induced AP-1
activation in MRC-5 cells and PMA/calcium ionophore-
induced NF-AT dependent reporter gene transcription
in Jurkat cells even at concentrations exceeding 10 lM.
These results indicate that compound 4j selectively
interferes with the NF-jBsignaling cascade by inhibi-
tion of IKK-b in living cells.
4. Murata, T.; Shimada, M.; Kadono, H.; Sakakibara, S.;
Yoshino, T.; Masuda, T.; Shimazaki, M.; Shintani, T.;
Fuchikami, K.; Bacon, K. B.; Ziegelbauer, K. B.; Lowin-
ger, T. B. Bioorg. Med. Chem. Lett. 2004, 14, preceding
paper in this issue. doi:10.1016/j.bmcl.2004.05.040.
5. 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.; Fuchikami, K.; Komura,
H.; Lowinger, T. B. WO 0224679, 2002; Chem. Abstr. 2002,
136, 279345.
6. (a) D’Acquisto, F.; Iuvone, T.; Rombola, L.; Sautebin, L.;
Di Rosa, M.; Carnuccio, R. FEBS Lett. 1997, 418, 175; (b)
Camandola, S.; Leonarduzzi, G.; Musso, T.; Varesio, L.;
Carini, R.; Scavazza, A.; Chiarpotto, E.; Baeuerle, P. A.;
Poli, G. Biochem. Biophys. Res. Commun. 1996, 229, 643;
(c) Puignero, V.; Queralt, J. Inflammation 1997, 21, 431.
7. In vivo arachidonic acid-induced ear edema in mice: ear
edema was induced by topical application of arachidonic
acid (500 lg/ear). Compound 4j, dexamethasone and vehi-
cle (10% cremophor in saline) were given po 60 min before
the arachidonic acid application. Ear thickness was mea-
sured at 0, 1, 3 and 6 h after the arachidonic acid
application. Each column indicates the mean and SEM of
5 mice.
Compound 4j has reasonable aqueous solubility
(0.12 mg/mL in pH 7.4 isotonic buffer) and excellent
Caco-2 permeability (Papp 62.3 · 10ꢀ7 cm/s), and dem-
onstrates orally bioavailability in mice (BA: 16%) and
rats (BA: 60%). The favourable bioavailability of com-
pound 4j in rats is likely due to its low clearance (0.33
L/h/kg).
Figure 2 describes oral efficacy of compound 4j and
dexamethasone in an acute inflammation model. The
arachidonic acid-induced ear edema mechanistically
depends on COX-2 expression, which is regulated by
NF-jBactivation. 6 Thus, this model is considered to be
a suitable model of inflammation for the evaluation of
IKK-b inhibitors. In this model, compound 4j exhibited
oral efficacy at 1 mg/kg in a dose-dependent manner.
The effect was slightly stronger than that of dexameth-
asone (0.3 mg/kg).
In summary, we have investigated the SAR for IKK-b
inhibition of 4-piperidinylnicotinonitrile analogs, 3a and
4a. Introduction of an alkoxy moiety at the 60-position