Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 3: Exploration of effective compounds in arthritis models

Article abstract of DOI:10.1016/j.bmcl.2011.05.115

We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine potent IKKβ inhibitory activity, TNFα inhibitory

Full text of DOI:10.1016/j.bmcl.2011.05.115

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4550–4555
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of imidazo[1,2-b]pyridazines as IKKb inhibitors. Part 3: Exploration
of effective compounds in arthritis models
Hiroki Shimizu a, , Tomonori Yamasaki a , Yoshiyuki Yoneda a, Fumihito Muro a, Tomoaki Hamada a,
⇑
Takanori Yasukochi b, Shinji Tanaka a, Tadashi Toki a, Mika Yokoyama a, Kaoru Morishita a, Shin Iimura a
a R&D Division, Daiichi Sankyo Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Research Department II, Daiichi Sankyo RD Associe Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation
in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine
potent IKKb inhibitory activity, TNFa inhibitory activity in vivo and excellent pharmacokinetics. The com-
pound we have acquired, which had both potent activities and good pharmacokinetic profiles based on
improved physicochemical properties, demonstrated efficacy on collagen-induced arthritis models in
mice and rats.
Received 30 March 2011
Revised 27 May 2011
Accepted 31 May 2011
Available online 6 June 2011
Keywords:
Inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
IKK
TNF
Imidazo[1,2-b]pyridazine
Collagen-induced arthritis
Pharmacokinetic
Nuclear factor-
j
B (NF-
j
B) is a transcription factor that has a
companies and research institutes have tried to develop IKKb inhib-
itors.13 We also continued our effort to acquire orally active small
molecule IKKb inhibitors.14,15 We have confirmed the potency of
imidazo[1,2-b]pyridazine derivatives as IKKb inhibitors as we have
reported compounds 1 and 2 showed potent IKKb inhibitory activ-
ity (Fig. 1).14 Furthermore, compounds 3 and 4 exhibited strong
crucial part in the immune system.1,2 NF-
jB plays a number of
important roles such as immune response, inflammation, cell pro-
liferation, survival and cell death by regulating the expression of a
variety of genes of proteins including pro-inflammatory cytokines
(e.g., TNF
sion molecules, osteoclastogenesis-related factors and inducible
proteins.3–7 NF-
B is implicated in the pathogenesis of multiple
inflammatory diseases and autoimmune diseases including rheu-
a, IL-1, IL-6), chemokines, anti-apoptotic proteins, adhe-
j
H
H
matoid arthritis. It is observed that NF-
jB is highly active in the
N
N
site of inflammation.3–5,8
N
N
N
N
N
N
There are some signal transduction cascades for the activation
of NF-
B.6d,9 In the classical (canonical) pathway, known as one
of the major pathways, IKK complex (IKK /IKKb/NEMO) plays an
important role in activating NF-
B (RelA/p50).9,10 RelA/p50 exists
as an inactive complex associated with I B. The phosphorylation
of I B by the IKK complex and subsequent K48-linked polyubiqui-
tination lead to the degradation of I B. The released RelA/p50
j
H
N
H
N
a
j
CH3
O
N
O
N
H
j
H3C
2
1
j
j
H
N
H
N
promotes transcription of genes of pro-inflammatory cytokines
and other inducible proteins in nucleus.
N
N
Cl
N
N
N
N
Of the IKK components, IKKb is essential in the phosphorylation
CH3
of IjB. It is anticipated that a potent IKKb inhibitor could be a prom-
F
F
ising anti-inflammatory agent.2a,11,12 A number of pharmaceutical
N
N
O
O
N
H
N
H
3
4
⇑
Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8609.
E-mail address: shimizu.hiroki.hu@daiichisankyo.co.jp (H. Shimizu).
Figure 1. Potent IKKb inhibitors with an imidazo[1,2-b]pyridazine scaffold.14,15
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.115

H. Shimizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4550–4555
4551
TNF
a
inhibitory activity in mice.15 In the following step, it becomes
the masking effect of amide NH moiety by the formation of weak
internal hydrogen bonding between fluorine and NH of benzamide
and/or the weakening of crystalinity.16,18 However, there was con-
cern that the clearance of 5d and 5e seemed to be higher as ob-
served in the decrease of plasma concentration of 5d and 5e
compared with 1 and 5a at 90 min after oral administration. To re-
duce the clearance, we tried to change the terminal amine sub-
structures such as pyrrolidin-2-ylmethyl derivatives 5f and 5g.15
important to find compounds that show efficacy in arthritis models
to develop an anti-inflammatory agent.
To resolve the issue, we need to explore the compounds which
combine potent IKKb inhibitory activity, TNF
in vivo and good pharmacokinetic properties. We consider that
compounds 3 and 4, which exhibited TNF inhibitory activity in
vivo, did not necessarily show satisfactory plasma concentration
for continuously administrated studies.15 As a result, we decided
to further explore and modify compounds 1 and 2 with the goal
to increase plasma concentrations and functional activity to help
improve efficacy in arthritis models.
To begin with, we planned to modify the benzamide moiety in
the 3-position of imidazo[1,2-b]pyridazine. It was assumed that
the benzamide moiety affects the pharmacokinetic properties.16
The results are summarized in Table 1. Compounds 5d and 5e,
a inhibitory activity
a
We found that 5g showed potent IKKb inhibitory activity, TNF
a
inhibitory activity and a higher level of plasma concentration
(MDCK Papp: 5g = 4.2 ꢀ 10ꢁ6 cm/s). The 2- or 3-methyl benzamide
group such as 5b or 5c led to the decrease in IKKb inhibitory activ-
ities. IKKb inhibitory activity of 5b decreased 10-fold than that of
5c. It is considered that the methyl group interfered with the pla-
narity of phenyl moiety and the imidazo[1,2-b]pyridazine scaffold.
In the same reason, 3-pyridyl derivative 5j was more potent than
2-pyridyl derivative 5k in cell-free inhibitory assay. Compounds
5i and 5j showed lower inhibitory activities in mouse whole blood
cell assay for IKKb inhibitory activities because of lower permeabil-
ity as shown in the PAMPA values in Table 1. The substitution of
benzamide to five-membered heteroaryl carbamoyl moieties such
as 5l and 5m reduced IKKb inhibitory activities. It is considered
that the difference of angle from 1,4-phenyl, which is larger in
the 2,5-furyl moiety than in the 2,5-thienyl moiety, causes IKKb
which have 2-fluorobenzamide moiety, showed greater TNF
a
inhibitory activity in vivo. Improvement was considered to be
caused by the enhancement of oral absorption, which was
indicated as the improvement of values in the permeability test
with Madin–Darby canine kidney (MDCK) cells (Papp: 5d = 6.2,
5e = 5.6 compared with 1 = 2.1, 5a = 2.3 (ꢀ10ꢁ6 cm/s)).17 The
improvement of permeability is possibly due to the enhancement
of hydrophobicity as seen in the distribution coefficient values,
Table 1
The modification of the 3-position of imidazo[1,2-b]pyridazine.
X
X
X
X
X
X
X
CH3
X
F
CH3
Cl
c
h
e
d
i
Ar:
a
f
b
g
X
H
H
H
N
H
N
H
H
N
N
N
N
O
O
O
N
O
O
S
O
*
*
*
*
*
*
*
N
N
N
X
3
H
N
H
N
N
Ar
H
N
H
N
H
*
*
O
O
N
O
O
O
*
5a-m
F
CH3
R1:
k
l
j
*
*
*
*
m
N
N
N
H
N
CH3
H
Compds
Ar
R1
IKKba IC50
M)
TNF
IC50
a
productionb
M)
Inhibition of
Plasma leveld at
Microsomal stabilitye
(%) mice/rats
Log Df
(pH 7.4)
PAMPAgPe (ꢀ10ꢁ6 cm/s)
(
l
(
l
TNFa
(%) in micec
30 mg/kg p.o. (
l
g/ml)
(pH 7.4)
1
a
a
j
l
0.20
0.055
0.016
0.017
0.071
20
0.80
6.8
0.17
1.2
0.64
–
10
0.39
0.0075
0.16
0.45
0.44
–
71/–h
77/–
33/–
28/–
75/–
–/–
2.5
1.8
3.4
5.0
2.6
–
>50
0.84
>50
39
>50
–
2
No inhibition
52
61
2.0
–
–
60
61
No inhibition
37
–
–
–
–
–
–
3i
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
a
b
c
d
d
d
d
e
f
k
j
j
j
k
l
m
l
j
k
k
j
1.9
–
–
–/–
–
–
0.30
0.14
0.064
0.027
0.41
0.20
0.14
0.68
2.0
1.2
1.0
1.3
0.26
4.1
4.0
3.7
–
0.11
0.29
0.14
0.38
–
–
–
–
–
79/-
57/-
85/-
87/46
92/-
100/-
100/-
–/–
2.7
2.8
1.8
3.1
2.2
2.0
2.0
–
>50
>50
19
>50
14
14
11
–
5j
5k
5l
f
g
h
i
–
–
–/–
–/–
–
–
–
–
5m
j
19
–
a
b
c
d
e
f
The method is described in Ref. 14.
Mouse whole blood cell. This method is described in Ref. 15.
The inhibition ratio of TNF at the oral dose of 30 mg/kg in mice. This method is described in Ref. 15.
Plasma concentration of test compounds at 90 min after oral administration. This method is described in Ref. 15.
The method is described in Ref. 19.
The method is described in Ref. 20.
Parallel artificial membrane permeability assay. This method is described in Ref. 21.
Not tested.
Structure, see Figure 1.
a
g
h
i

4552
H. Shimizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4550–4555
Table 2
The optimization of the 6-position of imidazo[1,2-b]pyridazine.
H
6
N
R2
F
N
N
N
F
H
N
N
O
H
Compds
R2
IKKba
IC50
TNF
a
Inhibition of TNF
a
Plasma leveld at
30 mg/kg
p.o. (lg/ml)
Microsomal stabilitye
(%) mice/rats
Log Df
(pH 7.4)
PAMPAg Pe
(ꢀ10ꢁ6 cm/s)
(pH 7.4)
(
lM)
productionb
(%) in micec
IC50
(l
M)
6a
6b
6c
6d
6e
6f
6g
6h
6i
n-Propyl
n-Butyl
n-Pentyl
Cyclobutyl
Cyclopentyl
–(CH2)2SCH3
–(CH2)3SCH3
–(CH2)3F
0.064
0.019
0.026
0.017
0.016
0.097
0.061
0.12
0.61
0.47
0.71
0.23
0.31
1.6
1.2
1.5
1.2
0.71
26
45
0.54
0.60
0.093
1.4
0.66
–
0.15
0.48
0.42
0.11
69/–h
56/–
21/–
54/42
65/–
44/–
3.1
3.5
4.0
3.3
3.6
2.8
3.0
2.5
2.8
3.3
>50
>50
>50
>50
>50
>50
40
33
>50
37
No inhibition
56
14
–
No inhibition
13/–
32
57
18
85/47
80/18
44/–
–(CH2)4F
–(CH2)5F
0.043
0.029
6j
a-hSee notes of Table 1.
inhibitory activities to decrease more in 5m. The 2-chlorobenzam-
ide 5h, instead of 2-fluorobenzamide 5f, was not adequate.
To further optimize 5g, we explored the substituent located at
the 6-position of imidazo[1,2-b]pyridazine. The results are summa-
rized in Table 2. The compounds with cycloalkyl moieties such as
6d and 6e were more potent than n-alkyl (6a–6c) or substituted al-
vehicle control = 8.0 3.2, mean SD) when the compound was or-
ally administrated once a day for 14 days after the second chal-
lenge of collagen.
Due to the more favorable pharmacokinetic and protein binding
properties of 6d we decided conducted a collagen-induced arthritis
model in rats. Compound 6d significantly reduced paw swelling in
a dose-dependent manner (Fig. 2).27 The paw volume decreased
58% at the 24th day after the first immunization when the com-
pound was orally administrated at the dose of 100 mg/kg once a
day for 18 days after the second challenge of collagen. It was ob-
served that 6d delayed onset, relieved symptoms of paw swelling
and suppressed bone destruction. Even though 6d possesses lower
functional activity in the rat whole blood cell assay (mouse:
kyl moieties (6f–6j) in IKKb inhibitory activity and/or TNFa inhib-
itory activity in mouse whole blood cell assay. The plasma
concentrations were moderate in most compounds, and we could
see the tendency for plasma levels to become lower as the sizes
of 6-substituents become larger. The same tendency can be seen
in microsomal stability values.
We discovered that compound 6d possesses an excellent profile
with potent in vitro and in vivo inhibitory activities and higher
plasma concentrations based on good physicochemical properties
(MDCK Papp: 6d = 7.0 ꢀ 10ꢁ6 cm/s).22 Pharmacokinetic properties
of 5g and 6d in mice and rats are displayed in Table 3. The pharma-
cokinetic parameters of 6d showed remarkable improvement com-
pared with that of 5g in AUC, Cmax and clearance. It is considered
that the improvement of permeability by the covering of the polar
secondary amine in the 6-position of imidazo[1,2-b]pyridazine by
the more bulky cyclobutyl group would increase the plasma level.
The difference can be seen between the species. The difference of
the plasma protein binding ratio between mice and rats could be
considered as one of the reasons for the difference in species. On
the basis of these results, we selected 6d for further evaluation in
rodent arthritis models.
IC50 = 0.23 lM vs rat: IC50 = 1.7 l
M)28 the superior pharmacoki-
netic and physical properties led to efficacy.
The conversion of the benzamide moiety and the combination
with terminal amine units in the 3-position of imidazo[1,2-b]pyr-
idazine was conducted as shown in Schemes 1 and 2. We prepared
the benzamide parts as arylboronic acid (pinacol ester) or aryltin
derivatives 8a–8i, which are commercially available or are pre-
pared by the known procedures as indicated in a previous re-
ports.29 Palladium catalyzed coupling reactions with bromide 7,15
followed by hydrolysis of esters as appropriate gave carboxylic acid
10a–10i (Scheme 1). The condensation reaction of 10a–10i with
amine units R1NH2 and subsequent deprotection when needed
lead to compounds 5a–5m (Scheme 2).
Synthesis of analogs containing 2-fluoro-N-{[(2S,4R)-4-fluoro-
pyrrolidin-2-yl]methyl}benzamide group (6a–6j) at the 3-position
of imidazo[1,2-b]pyridazine is described in Schemes 3 and 4. The
carboxylic acids 14a–14j were prepared from 1214 by way of nucle-
ophilic substitution, conversion and/or protection of the functional
We evaluated 6d for the anti-inflammatory effect in a collagen-
induced arthritis model in mice. Compound 6d showed efficacy by
the reduction of paw swelling at the oral dose of 100 mg/kg.26 The
clinical scores were reduced by 67% (100 mg/kg of 6d = 2.6 2.3,
Table 3
Pharmacokinetic properties of 5g and 6d in mice and rats at the oral dose of 30 mg/kg.
Compd
AUC0–24 h
(l
g h/ml)
MRT (h)
CL/F (ml/min/kg)
Vdss/F (L/kg)
T1/2(h)
Cmax
(
lg/ml)
Tmax (h)
Protein bindinga
Mouseb
Ratc
5g
6d
5g
6d
0.888
1.86
23.1
95.3
2.3
2.0
4.9
5.1
563
269
22.2
5.46
78
32
6.5
1.7
0.89
0.90
2.8
0.26
0.75
4.1
1.0
1.0
4.0
2.8
90
94
98
99
2.4
16
a
b
c
The methods are described in Ref. 23.
Male BALB/c mouse, 30 mg/kg, p.o. This method is described in Ref. 24.
Female Wistar–Lewis rat, 30 mg/kg, p.o. This method is described in Ref. 25.

H. Shimizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4550–4555
4553
R3
N
H
N
(c or d)
a or b
N
N
N
N
10a-i
N
N
Ar
Ar
H
H
N
N
R1
R1
O
O
11a (Ar: a, R1: k, R3: Boc)
11b = 5b
5a-5m
11c = 5c
11d (Ar: d, R1: j, R3: Boc)
11e (Ar: d, R1: k, R3: Boc)
11f (Ar: d, R1: l, R3, R5: Boc)
11g (Ar: d, R1: m, R3, R5: Boc)
11h (Ar: e, R1: l, R3, R5: Boc)
11i (Ar: f, R1: j, R3: Boc)
11j (Ar: f, R1: k, R3: Boc)
11k (Ar: g, R1: k, R3: Boc)
11l = 5l
Figure 2. Effect of compound 6d on the collagen-induced arthritis model in rats.
Seven days after the first immunization, the second immunization was performed
and each dose of compound 6d was orally administrated once a day. Data are
expressed as mean SD. Statistical significance was determined using Dunnett’s
test. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 compared with the vehicle control group.
11m (Ar: i, R1: j, R3: Boc)
F
l
j
CH3
k
R1:
*
*
m
*
*
N
N
groups and Suzuki–Miyaura coupling reaction (Scheme 3). Com-
pounds 6a–6j were synthesized by the condensation of 14a–14j
with tert-butyl (2S,4R) 2-(aminomethyl)-4-fluoropyrrolidinecarb-
oxylate, followed by the cleavage of protective groups (Scheme 4).
In conclusion, we successfully optimized the substructures at
the 3- and 6-positions of imidazo[1,2-b]pyridazine derivatives to
N
N
CH3
R5
R5
Scheme 2. Condensation reaction with terminal amine units. Reagents and
conditions: (a) R1NH2, EDCꢂHCl, HOBt, Et3N, CH2Cl2, 90% for 11a, 81% for 11d, 83%
for 11e, 88% for 11f, 95% for 11g, quant. for 11h, for 11i–11k, 69% for 11m; (b)
R1NH2, DMT-MM, DMF, 94% for 5b, 94% for 5c, 57% for 5l; (c) TFA, CH2Cl2, 73% for 5f,
87% for 5g, 67% 2 steps for 5i, 68% 2 steps for 5j, 59% 2 steps for 5k, 69% for 5m; (d)
4 N HCl in 1,4-dioxane, 86% for 5a, 79% for 5d, 59% for 5e, 19% for 5h.
combine potent IKKb inhibitory activity, TNFa inhibitory activity,
good pharmacokinetics. During the optimization process it became
clear that the 2-fluorobenzamide improved permeability of the
compounds while the (2S,4R)-4-fluoropyrrolidin-2-ylmethyl group
improved potency and improved physicochemical properties as
demonstrated by compound 5g. Subsequent modifications of
compound 5g led to the development of 6d by replacing the
R3
N
R3
N
a, b, c or d
N
O
N
N
Ar
OR4
X
+
N
N
N
Ar
OR4
Br
O
7 (R3: H or Boc)
8a (Ar: a, X: n, R4: Me)
8b (Ar: b, X: o, R4: Me)
8c (Ar: c, X: o, R4: Me)
8d (Ar: d, X: n, R4: H)
8e (Ar: e, X: o, R4: Me)
8f (Ar: f, X: Br, R4: Me)
8g (Ar: g, X: o, R4: Me)
8h (Ar: h, X: n, R4: H)
8i (Ar: i, X: o, R4: Me)
9a (Ar: a, R3: Boc, R4: Me)
9b (Ar: b, R3: H, R4: Me)
9c (Ar: c, R3: H, R4: Me)
9d = 10d
9e (Ar: e, R3: Boc, R4: Me)
9f (Ar: f, R3: Boc, R4: Me)
9g (Ar: g, R3: Boc, R4: Me)
9h = 10h
9i (Ar:i, R3: Boc, R4: Me)
R3
N
(e or f)
N
N
N
HO
B
HO
X:
n
o
*
Ar
OH
O
O
B
10a (Ar: a, R3: Boc)
10b (Ar: b, R3: H)
10c (Ar: c, R3: H)
10d (Ar: d, R3: Boc)
10e (Ar: e, R3: Boc)
10f (Ar: f, R3: Boc)
10g (Ar: g, R3: Boc)
10h (Ar:h, R3: H)
10i (Ar: i, R3: Boc)
*
O
Scheme 1. Preparation of arylcarboxylic acid derivatives. Reagents and conditions: (a) 8, PdCl2(dppf)ꢂCH2Cl2, K3PO4ꢂnH2O, 1,4-dioxane–H2O (9:1), 80–95 °C, 93% for 9a, 75%
for 9b, 46% for 9c, quant. for 9e, 62% for 9g, 97% for 9i; (b) 8d, PdCl2(PPh3)2, 2 M K2CO3 aq., 1,4-dioxane, 90 °C, 81% for 10d; (c) 8f, hexamethylditin, Pd2(dba)3, P(t-Bu)3, 1,4-
dioxane, 90 °C, 18% for 9f; (d) 8h, Pd(PPh3)4, Cs2CO3, DMF–EtOH–H2O (7:2:3), microwave irradiation 130 °C, 5 min, 66% for 10h; (e) 1 N NaOH aq., MeOH, 82% for 10b, 82% for
10c, 51% for 10f, 88% for 10g, 90% for 10i; (f) 1 N NaOH aq, THF–MeOH (4:1), 97% for 10a, 66% for 10e. Boc: tert-butoxycarbonyl.

4554
H. Shimizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4550–4555
R3
N
R2
R3
N
R2 :
p
r
Cl
*
*
*
*
( )n
( )n
N
( )n
SCH3
R6
N
CH3
R2
e
N
1) a, b or c
2) d
N
N
N
N
N
F
N
q
s
( )n
Br
Br
OH
O
12
13a (R2: p, n = 1)
13b (R2: p, n = 2)
13c (R2: p, n = 3)
13d (R2: q, n = 1)
13e (R2: q, n = 2)
13f (R2: r, n = 1)
13g (R2: r, n = 2)
13h (R2: s, n = 1)
13i (R2: s, n = 2)
13j (R2: s, n = 3)
14a (R2: p, R3: Boc, n = 1)
14b (R2: p, R3: Boc, n = 2)
14c (R2: p, R3: Boc, n = 3)
14d (R2: q, R3: Boc, n = 1)
14e (R2: q, R3: H, n = 2)
14f (R2: r, R3: Boc, n = 1)
14g (R2: r, R3: Boc, n = 2)
14h (R2: s, R3: Boc, n = 1)
14i (R2: s, R3: Boc, n = 2)
14j (R2: s, R3: Boc, n = 3)
Scheme 3. Preparation of 2-fluorobenzoic acid derivatives. Reagents and conditions: (a) R2NH2, 120 °C for 13a (R3: H), for 13b (R3: H), for 13c (R3: H), 79% for 13e (R3: H); (b)
R2NH2, Et3N, NMP, 125–140 °C, 95% for 13d (R3: H), 79% for 13f (R3: H), 96% for 13g (R3: H); (c-1) R2NH2, NMP, 125 °C, 99% for 13h (R3: H, R6: OH), 96% for 13i (R3: H, R6: OH),
88% for 13j (R3: H, R6: OH); (c-2) PMBCl, NaH, DMF then CAN, MeOH–H2O (9:1), 56% for 13h (R3: PMB, R6: OH), 49% for 13i (R3: PMB, R6: OH), 45% for 13j (R3: PMB, R6: OH); (c-
3) MsCl, Et3N, CH2Cl2, 0°C, 88% for 13h (R3: PMB, R6: OMs), 82% for 13i (R3: PMB, R6: OMs), 48% for 13j (R3: PMB, R6: OMs); (c-4) TBAF, THF, 60 °C, for 13h (R3: PMB, R6: F), 83%
for 13i (R3: PMB, R6: F), 85% for 13j (R3: PMB, R6: F); (c-5) TFA, CH2Cl2, 79% 2 steps for 13h (R3: H, R6: F), 94% for 13i (R3: H, R6: F), 97% for 13j (R3: H, R6: F); (d) (Boc)2O, DMAP,
CH2Cl2, 60 °C, 60% 2 steps for 13a (R3: Boc), 88% 2 steps for 13b (R3: Boc), 86% 2 steps for 13c (R3: Boc), 80% for 13d (R3: Boc), No reaction. 13e (R3: H) was recovered, 89% 2
steps for 13f (R3: Boc), 94% for 13g (R3: Boc), 93% for 13h (R3: Boc, R6: F), quant. for 13i (R3: Boc, R6: F), 92% for 13j (R3: Boc, R6: F); (e) 4-Carboxy-3-fluorophenylboronic acid,
PdCl2(PPh3)2, 2 M K2CO3 aq., 1,4-dioxane 90 °C, 55% for 14a, 79% for 14b, 94% for 14c, 82% for 14d, 51% for 14e, quant. for 14f, 88% for 14g, quant. for 14h, 97% for 14i, 97% for
14j. PMB: p-methoxybenzyl, OMs: methanesulfonyloxy.
R3
N
References and notes
H
R2
N
R2
F
1. Sen, R.; Baltimore, D. Cell 1986, 47, 921.
2. (a) Li, Q.; Verma, I. M. Nat. Rev. Immunol. 2002, 2, 725; (b) Hayden, M. S.; Ghosh,
S. Cell 2008, 132, 344.
N
N
a or b
c
N
N
N
N
14a-j
F
F
3. Brown, K. D.; Claudio, E.; Siebenlist, U. Arthritis Res. Ther. 2008, 10, 1.
4. Viatour, P.; Merville, M. P.; Bours, V.; Chariot, A. Trends Biochem. Sci. 2005, 30, 43.
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Nature 2002, 420, 860; (c) Kim, H. J.; Hawke, N.; Baldwin, A. S. Cell Death Differ.
2006, 13, 738; (d) Perkins, N. D.; Gilmore, T. D. Cell Death Differ. 2006, 13, 759.
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Park, J.-M.; Lawrence, T.; Hsu, L.-C.; Cao, Y.; Schett, G.; Wagner, E. F.; Karin, M. J.
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Sci. STKE 2006, 357, re13.
10. (a) DiDonato, J. A.; Hayakawa, M.; Rothwarf, D. M.; Zandi, E.; Karin, M. Nature
1997, 388, 548; (b) Mercurio, F.; Zhu, H.; Murray, B. W.; Shevchenko, A.;
Bennett, B. L.; Li, J.; Young, D. B.; Barbosa, M.; Mann, M.; Manning, A.; Rao, A.
Science 1997, 278, 860; (c) Woronicz, J. D.; Gao, X.; Cao, Z.; Rothe, M.; Goeddel,
D. V. Science 1997, 278, 866.
F
H
H
N
N
N
O
N
O
H
O
O
15a (R2: p, R3: Boc, n = 1)
15b (R2: p, R3: Boc, n = 2)
15c (R2: p, R3: Boc, n = 3)
15d (R2: q, R3: Boc, n = 1)
15e (R2: q, R3: H, n = 2)
15f (R2: r, R3: Boc, n = 1)
15g (R2: r, R3: Boc, n = 2)
6a-6j
15h (R2: s, R3: Boc, R6: F, n = 1)
15i (R2: s, R3: Boc, R6: F, n = 2)
15j (R2: s, R3: Boc, R6: F, n = 3)
Scheme 4. Condensation reaction with terminal amine units. Reagents and
conditions: (a) tert-butyl (2S,4R)-2-(aminomethyl)-4-fluoropyrrolidinecarboxylate,
EDCꢂHCl, HOBt, Et3N, CH2Cl2, for 15a–15c, 79% for 15d, for 15e; (b) tert-butyl
(2S,4R)-2-(aminomethyl)-4-fluoropyrrolidinecarboxylate, DMT-MM, DMF, 88% for
15f, 85% for 15g, 89% for 15h, quant. for 15i, 99% for 15j; (c) TFA, CH2Cl2, 56% 2 steps
for 6a, 63% 2 steps for 6b, 59% 2 steps for 6c, 96% for 6d, 79% 2 steps for 6e, 45% for
6f, 61% for 6g, 84% for 6h, 81% for 6i, 45% for 6j.
11. Karin, M.; Yamamoto, Y.; Wang, Q. M. Nat. Rev. Drug Discov. 2004, 3, 17.
12. (a) Blake, S. M.; Swift, B. A. Curr. Opin. Pharmacol. 2004, 4, 276; (b) Burke, J. R.;
Strnad, J. Trends Pharmacol. Sci. 2007, 28, 142.
13. The latest IKKb inhibitors are reported in the following references and the
previous references are cited in Ref. 15. (a) Dyckman, A. J.; Langevine, C. M.;
Quesnelle, C.; Kempson, J.; Guo, J.; Gill, P.; Spergel, S. H.; Watterson, S. H.; Li, T.;
Nirschl, D. S.; Gillooly, K. M.; Pattoli, M. A.; McIntyre, K. W.; Chen, L.;
McKinnon, M.; Dodd, J. H.; Barrish, J. C.; Burke, J. R.; Pitts, W. J. Bioorg. Med.
Chem. Lett. 2011, 21, 383; (b) Cushing, T. D.; Baichwal, V.; Berry, K.; Billedeau,
R.; Bordunov, V.; Broka, C.; Cardozo, M.; Cheng, P.; Clark, D.; Dalrymple, S.;
DeGraffenreid, M.; Gill, A.; Hao, X.; Hawley, R. C.; He, X.; Jaen, J. C.; Labadie, S.
S.; Labelle, M.; Lehel, C.; Lu, P. P.; McIntosh, J.; Miao, S.; Parast, C.; Shin, Y.;
Sjogren, E. B.; Smith, M. L.; Talamas, F. X.; Tonn, G.; Walker, K. M.; Walker, N. P.;
Wesche, H.; Whitehead, C.; Wright, M.; Browner, M. F. Bioorg. Med. Chem. Lett.
2011, 21, 417; (c) Cushing, T. D.; Baichwal, V.; Berry, K.; Billedeau, R.;
Bordunov, V.; Broka, C.; Browner, M. F.; Cardozo, M.; Cheng, P.; Clark, D.;
Dalrymple, S.; DeGraffenreid, M.; Gill, A.; Hao, X.; Hawley, R. C.; He, X.; Labadie,
S. S.; Labelle, M.; Lehel, C.; Lu, P. P.; McIntosh, J.; Miao, S.; Parast, C.; Shin, Y.;
Sjogren, E. B.; Smith, M. L.; Talamas, F. X.; Tonn, G.; Walker, K. M.; Walker, N. P.;
Wesche, H.; Whitehead, C.; Wright, M.; Jaen, J. C. Bioorg. Med. Chem. Lett. 2011,
21, 423; (d) Noha, S. M.; Atanasov, A. G.; Schuster, D.; Markt, P.; Fakhrudin, N.;
Heiss, E. H.; Schrammel, O.; Rollinger, J. M.; Stuppner, H.; Dirsch, V. M.; Wolber,
G. Bioorg. Med. Chem. Lett. 2011, 21, 577; (e) Xie, J.; Poda, G. I.; Hu, Y.; Chen, N.
X.; Heier, R. F.; Wolfson, S. G.; Reding, M. T.; Lennon, P. J.; Kurumbail, R. G.;
Selness, S. R.; Li, X.; Kishore, N. N.; Sommers, C. D.; Christine, L.; Bonar, S. L.;
Venkatraman, N.; Mathialagan, S.; Brustkern, S. J.; Huang, H.-C. Bioorg. Med.
Chem. 2011, 19, 1242; (f) Takahashi, H.; Shinoyama, M.; Komine, T.; Nagao, M.;
Suzuki, S.; Tsuchida, H.; Katsuyama, K. Bioorg. Med. Chem. Lett. 2011, 21, 1758;
(g) Avila, C. M.; Lopes, A. B.; Gonçalves, A. S.; da Silva, L. L.; Romeiro, N. C.;
Miranda, A. L.; Sant’anna, C. M.; Barreiro, E. J.; Fraga, C. A. Eur. J. Med. Chem.
cyclopropylmethylamino moiety with cyclobutylamino moiety at
position 6 on the imidazo[1,2-b]pyridazine group. Compound 6d
showed potent functional activity, favorable physical properties,
good pharmacokinetics, and suitable plasma concentration level
that led to excellent efficacy in collagen-induced arthritis models.
Acknowledgments
We are grateful to the members of Drug Metabolism & Pharma-
cokinetics Research Laboratories for their measurement and vali-
dation of physicochemical data.
We would like to express our sincere appreciation to
Dr. T. Washio and Dr. N. Masubuchi for conducting the pharmaco-
kinetic assays and their helpful discussions. We would also like to
thank Dr. K. Suzuki and Ms. A. Kameda for their support in pre-
paring the physicochemical data.

H. Shimizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4550–4555
4555
2011, 46, 1245; (h) Miller, D. D.; Bamborough, P.; Christopher, J. A.; Baldwin, I. 22. Compound 6d showed more than 50% inhibition against 6 kinases at 0.2
lM
R.; Champigny, A. C.; Cutler, G. J.; Kerns, J. K.; Longstaff, T.; Mellor, G. W.;
Morey, J. V.; Morse, M. A.; Nie, H.; Rumsey, W. L.; Taggart, J. T. Bioorg. Med.
Chem. Lett. 2011, 21, 2255; (i) Kim, S.; Jung, J.-K.; Lee, H.-S.; Kim, Y.; Kim, J.;
Choi, K.; Baek, D.-J.; Moon, B.; Oh, K.-S.; Lee, B.-H.; Shin, K.-J.; Pae, A.-N.; Nam,
G.; Roh, E.-J.; Cho, Y.-S.; Choo, H. Bioorg. Med. Chem. Lett. 2011, 21, 3002.
14. Shimizu, H.; Tanaka, S.; Toki, T.; Yasumatsu, I.; Akimoto, T.; Morishita, K.;
Yamasaki, T.; Yasukochi, T.; Iimura, S. Bioorg. Med. Chem. Lett. 2010, 20, 5113.
15. Shimizu, H.; Yasumatsu, I.; Hamada, T.; Yoneda, Y.; Yamasaki, T.; Tanaka, S.;
Toki, T.; Yokoyama, M.; Morishita, K.; Iimura, S. Bioorg. Med. Chem. Lett. 2011,
21, 904.
out of 250 kinases.
23. Protein binding studies.
To the 495
ll of serum samples of male BALB/c mice or female Wister–Lewis
rats, 5 l of test compound (10
l
l
g/ml, 20% acetonitrile aqueous solution) was
added and stirred weakly (final 100 ng/ml, n = 2). The mixture was shaken for
4 h at 37 °C in Rapid Equilibrium Dialysis Device.
To the 50
prepared in the same manner.
After addition of the 120 l of internal standard acetonitrile solution to the
samples, respectively and mixed, followed by centrifugation at 1800ꢀg for
5 min at 4 °C. The supernatants were filtered (0.45 m) and the concentrations
ll of the mixture, 50 ll of buffer was added. The blank serum was
l
16. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46(1–3), 3.
17. Madin–Darby canine kidney cell permeability.
l
of the test compounds were determined by an LC/MS/MS method.
24. Pharmacokinetic studies on mice.
The cell permeability of the selected compounds was determined with Madin–
Darby canine kidney (MDCK) cells. MDCK cells were maintained in Minimum
Female BALB/c mice (7 weeks of age, n = 3) were used. The fed mice were orally
administered the test compound at a dose of 30 mg/kg/10 ml suspended in
0.5% (w/v) methyl cellulose aqueous solution. Blood samples were collected at
0.5, 1, 2, 4, 8 and 24 h after administration. Blood samples (0.5 ml) were ice-
stored, followed by centrifugation at 1800ꢀg for 5 min at 4 °C. The plasma
fractions were subsequently stored in a ꢁ20 °C freezer until being analyzed.
The concentrations of the test compounds were determined by an LC/MS/MS
method. The plasma concentrations versus the time data were analyzed by
non-compartment approaches.
Essential Medium containing 10% (v/v) fetal bovine serum,
a penicillin–
streptomycin mixture and L-glutamine. For the transport assay, cells were
seeded into HTS 24-well transwells at 1.65 ꢀ 105 cells/ml, and grown for
6 days after seeding to allow the formation of a cell monolayer. Transport
buffer was prepared using NaHCO3 (final 0.35 g/l), D-glucose (final 3.5 g/l),
Hepes (final 10 mM), CaCl2 (final 0.14 g/l) and MgSO4 (final 0.098 g/l) in 10ꢀ
Hanks’ Balanced Salt Solution, and then adjusted to pH 6.0 or 7.4 with 1 M HCl
or 1 M NaOH. For each test compound, a 100
one of the compounds at a concentration of 10
was added to the apical (A) side of the monolayer. A 600
containing 4% (w/v) BSA in transport buffer (pH 7.4), which was re-adjusted to
pH 7.4, was added to the basolateral (B) side of the monolayer. Metoprolol was
used as a positive control. After 1 h of incubation at 37 °C, aliquots of the basal
solutions were analyzed on an LC/MS/MS system. For each compound, Eq. 1
was used to calculate the apparent permeability coefficient (Papp) from the LC/
l
l
l of dosing solution containing
M in transport buffer (pH 6.0)
l of blank solution
25. Pharmacokinetic studies on rats.
Female Wistar–Lewis rats (7–8 weeks of age, n = 3) were used. The fed rats
were orally administered the test compound at doses of 30 mg/kg/2 ml
suspended in 0.5% (w/v) methyl cellulose aqueous solution. Blood samples
(0.3 ml) were collected at 0.5, 1, 2, 4, 8 and 24 h after administration. These
analytical samples were stored at room temperature, followed by
centrifugation at 1800ꢀg for 5 min at 4 °C. The plasma fractions were
l
subsequently stored in
a
ꢁ20 °C freezer until being analyzed. The
MS/MS-determined concentration in the basolateral compartment (Cb,
lM)
concentrations of the test compounds were determined by an LC/MS/MS
method. The plasma concentrations versus the time data were analyzed by
non-compartment approaches.
and the initial 10 concentration in the donor compartment. In the
lM
following equation, 3600 s is the total time for the measurement of
compound flux, and 0.33 cm2 is the area of the transwell filter.
26. Induction and assessment of collagen-induced arthritis in mice.
Female DBA/1 mice (7 weeks of age, n = 7) were immunized by an intradermal
injection at the base of the tail with 0.1 ml of emulsion containing 1.5 mg/ml of
bovine type II collagen in Freund’s complete adjuvant. Twenty-one days later,
mice were boosted by an intradermal injection at the abdomen with a total of
0.1 ml of same emulsion. Mice were treated with or without a test compound
from the day of the second injection. Clinical severity of inflammation was
scored periodically on a scale of 0–3 based on criteria as follows: 0, normal; 0.5,
swelling of one digit; 1, swelling of more than two digits or redness of paw; 2,
swelling of partial paw; 3, swelling of entire paw.
Pappð10ꢁ6 cm=sÞ ¼ ðCb ꢀ 600
l
lÞ=ð10
l
M ꢀ 3600 s ꢀ 0:33 cm2Þ
ð1Þ
18. Howard, J. A. K.; Hoy, V. J.; O’Hagan, D.; Smith, G. T. Tetrahedron 1996, 52,
12613.
19. Stability assay in mouse/rat liver microsome.
Test compound (final
1 lM) was incubated with male CD1 mouse liver
microsome/male IGS-SD rat liver microsome (final 0.1 mg/ml, n = 2) in sodium
phosphate buffer (pH 7.4) for 20 min at 37 °C. Assays were started by the
addition of NADPH at 37 °C and stopped by the addition of acetonitrile after
incubation for 30 min. The sample was ice-stored more than 30 min, followed
by centrifuged at 3500 rpm for 10 min at 4 °C. The supernatant was applied for
the analysis of supernatant by LC/MS/MS system. The remaining % of test
compound was calculated determined by the following equation:
Microsomal stability (% remaining) = {(Peak Area Ratio of test compound at
30 min)/(Peak Area Ratio of test compound at 0 min)} ꢀ 100
27. Induction and assessment of collagen-induced arthritis in rats.
Female Dark Agouti rats (9–10 weeks of age, n = 6) were immunized by an
intradermal injection at the back with a total of 0.2 ml of emulsion containing
250 lg/ml of bovine type II collagen in Freund’s incomplete adjuvant. Seven
days later, rats were boosted by an intradermal injection at the base of the tail
with a total of 0.1 ml of the same emulsion. Rats were treated with or without a
test compound from the day of the second injection. Hind paw volume was
measured periodically from induction of arthritis using a water displacement
plethysmometer. Paw swelling was expressed as the mean of the both hind
paw volumes of the change from the day before the boost.
20. Distribution coefficient.
Equal amounts of pH 7.4 phosphate buffered saline (PBS) and 1-octanol were
shaken and left for over 12 h. The upper layer (1-octanol) and lower layer (PBS)
were collected individually. Each compound was dissolved in 1-octanol or PBS
28. Inhibition assay of LPS-induced TNFa production by rat whole blood cell.
(200 lM). The same amount of either PBS or 1-octanol was added and the
Whole blood was collected from Lewis rat in heparinized tubes, and plated in a
96-well plate at 2 ꢀ 105 cells/well suspended in RPMI-1640 containing 10%
mixture was shaken vigorously for 30 min at room temperature. Then, both
phases were separated and assayed by HPLC or LC/MS. Log D7.4 was calculated
by the following equation: Log D7.4 = log(Peak area of compound in 1-octanol/
Peak area of compound in PBS)
fetal bovine serum, 25 mM HEPES, 50 U/ml penicillin and 50 lg/ml
streptomycin. Cells were pretreated with various concentrations of test
compounds just before stimulation with 1 g/ml LPS. After 4 h of incubation
at 37 °C in 5% CO2, the culture supernatants were harvested by centrifugation
(2000 rpm at 5 min, 4 °C). TNF concentration in the supernatants was
l
21. Membrane permeability assay.
The membrane permeability coefficients were assayed using PAMPA
a
Evolution™. The filter membrane of
phospholipids (GTI-0 lipid solution). At concentrations of 50
a
donor plate was coated with
M (containing
measured using ELISA kit according to the manufacturer’s instructions.
29. (a) Imanishi, M.; Tomishima, Y.; Itou, S.; Hamashima, H.; Nakajima, Y.;
Washizuka, K.; Sakurai, M.; Matsui, S.; Imamura, E.; Ueshima, K.; Yamamoto,
T.; Yamamoto, N.; Ishikawa, H.; Nakano, K.; Unami, N.; Hamada, K.;
Matsumura, Y.; Takamura, F.; Hattori, K. J. Med. Chem. 2008, 51, 1925; (b)
Ranbaxy Laboratories Limited, WO2009156966, 2009.; (c) Ishiyama, T.; Takagi,
J.; Yonekawa, Y.; Hartwig, J. F.; Miyaura, N. Adv. Synth. Catal. 2003, 345,
1103.
l
of 5% DMSO, pH 7.4), all the compounds were placed in a donor plate. The
acceptor plate was filled with Acceptor solution (ASB-7.4). The acceptor plate
was then stacked over the donor plate for
4
h
incubation at 25 °C. The
compound concentrations were assayed for both plates with
spectrometer. The effective permeability (Pe7.4) was calculated with PAMPA
Evolution Command Software.
a
UV