Discovery of imidazo[1,2-b]pyridazine derivatives as IKKβ inhibitors. Part 1: Hit-to-lead study and structure-activity relationship

Article abstract of DOI:10.1016/j.bmcl.2010.07.026

Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKβ inhibitors. By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKβ inhibitory activity and TNFα inhibitory activity in TH

Full text of DOI:10.1016/j.bmcl.2010.07.026

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5113–5118
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of imidazo[1,2-b]pyridazine derivatives as IKKb inhibitors.
Part 1: Hit-to-lead study and structure–activity relationship
a
a
a
a
Hiroki Shimizu a, , Shinji Tanaka , Tadashi Toki , Isao Yasumatsu , Toshihiko Akimoto ,
*
Kaoru Morishita a, Tomonori Yamasaki a, Takanori Yasukochi b, 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:
Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKb inhibitors.
By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKb inhibi-
Received 22 May 2010
Revised 5 July 2010
Accepted 7 July 2010
Available online 13 July 2010
tory activity and TNF
a inhibitory activity in THP-1 cell increased. Also, these compounds showed high
kinase selectivity. The structure–activity relationship was revealed and the interaction model of imi-
dazo[1,2-b]pyridazine compounds with IKKb was constructed.
Ó 2010 Published by Elsevier Ltd.
Nuclear factor
j
B (NF-
j
B) is the transcription factor that has a
B plays a number of
(IKK
(RelA/p50).9,10 RelA/p50 exists in an inactive complex associated
with I B. The degradation of I B as a result of phosphorylation
by IKK complex and subsequent K48-linked polyubiquitination
causes RelA/p50 to be released from I B. The RelA/p50 promotes
a/IKKb/NEMO) plays an important role in activating NF-jB
crucial part in the immune system.1,2 NF-
j
important roles such as immune-response, inflammation, cell
proliferation and survival/cell death by regulating the expression
j
j
of
(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
variety of genes including pro-inflammatory cytokines
j
a
, IL-1, IL-6), chemokines, anti-apoptotic proteins, adhen-
transcription of genes of pro-inflammatory cytokines and other
inducible proteins in nucleus. Of the IKK components, IKKb is
j
essential in phosphorylation of I
j
B. It is anticipated that a potent
matoid arthritis. It is observed that NF-
jB is highly active in the
area of inflammation.3–5,8
Cl
Cl
H
There are some signal transduction cascades for the activation
a
N
Cl
N
of NF-
j
B.6d,9 In the classical (canonical) pathway, IKK complex
N
N
N
N
N
Br
Br
2
3
Cl
Cl
H
6 N
Cl
Cl
Cl
Cl
N
H
N
H
N
N
N
1
d
b, c
3
N
N
N
N
N
N
H
N
R1
CH3
O
N
H3C
N
OH
R2
O
O
4
Compound 1
1, 5a~5z
IKKβ: IC50 1.1 μM
CDK2: IC50 >100 μM
GSK3β: IC50 >100 μM
Scheme 1. Syntheses of 1 and modification of terminal substituent in the 3-
position of imidazo[1,2-b]pyridazines 5a–5z.24 Reagents and conditions: (a) 3,4-
dichlorobenzylamine, 140 °C, 81%; (b) 4-ethoxycarbonylphenylboronic acid,
PdCl2(dppf)ꢀCH2Cl2, K3PO4, dppf, dioxane, 80 °C, 49%; (c) 1 N NaOH aq, MeOH,
reflux, 36%; (d) amine (R1R2NH), HBTU, Et3N, DMF then TFA, CH2Cl2 (to cleave the
Boc group that protects primary amine to give 5a, 5e, 5i–5m, 5s; and to cleave the
2,4-dimethoxybenzyl group that protects primary amine to give 5z) 19–69%. Yields
are not optimized.
Figure 1. HTS-hit compound 1.
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8609.
E-mail address: shimizu.hiroki.hu@daiichisankyo.co.jp (H. Shimizu).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.07.026

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H. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5113–5118
Table 1
Table 1 (continued)
IKKb inhibition and TNFa production inhibition assay results for terminal substituent
R1
R2
in the 3-position, compounds 1 and 5a–5z
IKKb inhibitiona
TNF
IC50
a
(
inhibitionb
lM)
Compds
N
*
*
IC50
(
lM)
Cl
Cl
H
N
5t
1.4
>30
12
N
N
N
N
N
N
5u
>30
N CH3
OH
*
*
R1
R2
N
H
5v
5w
5x
>30
>30
>30
NT
NT
NT
N
O
O
*
*
*
*
N
H
CH3
R1
R2
IKKb inhibitiona
TNF
IC50
a
(
inhibitionb
lM)
Compds
N
*
N
H
OH
IC50
1.1
(l
M)
CH3
N
H
5y
5z
27
27
N
1
>30
*
*
N
H
CH3
>30
1.7
NH2
NH2
N
N
H
a
5a
5b
2.8
2.4
The method for measuring cell-free IKKb inhibitory activity is described in Ref.
25.
b
The method for measuring the inhibitory activity of TNFa release by THP-1 cell
is described in Ref. 27.
*
*
0.59
>30
N
H
c
Not tested.
N
N
5c
2.4
>30
NTc
N
H
IKKb inhibitor could be a promising anti-inflammatory agent by
the inhibition of the NF-
B pathway.2a,11,12
O
j
5d
>30
*
*
N
H
Recently, biological agents such as anti-TNF antibodies, soluble
TNF receptor and anti-IL-6 receptor antibody are well used for the
treatment of inflammatory disorders and autoimmune disorders
such as rheumatoid arthritis.13,14 However, these biological agents
have some limitation in price, a limited administration route and
induction of autoantibody. Therefore, it is necessary to develop an
orally available small molecule which can decrease the production
of pro-inflammatory cytokines.12a,14 A number of pharmaceutical
companies have made efforts to develop an IKKb inhibitor.11,15–23
Here, we report on the research to acquire the compounds that show
strong IKKb inhibitory activity to develop an anti-inflammatory
agent.
N
H
NH2
CH3
5e
5f
4.0
2.4
11
4.7
>30
6.5
*
*
N
H
N
CH3
NH2
N
H
5g
H
N
*
*
5h
5i
>30
>30
4.0
N
H
N
H
0.32
N
H
To acquire small molecules that demonstrate IKKb inhibitory
activity, we performed high-throughput screening (HTS) and dis-
covered a series of potent compounds, which commonly have the
imidazo[1,2-b]pyridazine scaffold. Among these compounds, com-
*
*
*
N
H
5j
10
14
5.6
17
N
H
N
H
pound 1 showed IC50 1.1
Moreover, this compound showed good kinase selectivity over
CDK2 (IC50 >100 M) and GSK3b (IC50 >100 M). We adopted 1
lM of IKKb inhibitory activity (Fig. 1).
5k
5l
>30
6.5
NH
l
l
NH
N
H
as a HTS-hit compound and tried to improve the potency.
As the first step in the modification of compound 1, the terminal
amine moiety in the 3-position of imidazo[1,2-b]pyridazine was
replaced with another amine or polar substituent to demonstrate
the structure–activity relationship of this substructure.
The general synthetic route for compounds 1 and 5a–5z are
shown in Scheme 1. 3-Bromo-6-chloroimidazo[1,2-b]pyridazine 2
was prepared by a known procedure as indicated in a previous re-
port.28 Nucleophilic substitution to compound 2 gave amine 3. Su-
zuki-Miyaura coupling to 3, followed by hydrolysis of ethyl ester
gave carboxylic acid 4. By the condensation of various amines with
compound 4, compounds 1 and 5a–5z were synthesized.
The results of the modification of the terminal amine moiety are
described in Table 1. The structure–activity relationship in the
3-position was revealed as follows. For the distance between
amide and amino nitrogen, the compounds with three atom dis-
tances showed higher potency (1, 5a, 5b) than those with four
atom distances (5e, 5f). The (S)-2-pyrrolidinylmethyl moiety 5i
NH
5m
5n
18
4.4
NT
*
*
N
H
>30
N
H
N
N
*
*
N
H
5o
5p
>30
>30
>30
NT
N
H
N
N
*
N
H
5q
>30
NT
CH3
N
*
*
5r
5s
2.1
8.9
15
N
CH3
CH3
increased potency (IKKb IC50 0.32
compound has cyclic alkylamine unit, compounds 5j–5n
decreased in potency. When the amine was piperidine 5c,
lM). However, even when the
N
>30
NH2
a

H. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5113–5118
5115
R3
N
R3
N
R4
R4
Cl
Cl
a, b
c
N
d, e
N
N
N
N
N
N
N
N
N
N
N
Br
H
N
OH
OH
O
N
H
O
O
6
2
8a~8p
7a~7p
Scheme 2. Modification of substituents in the 6-position of imidazo[1,2-b]pyridazine.24 Reagents and conditions: (a) 4-methoxycarbonylphenylboronic acid,
PdCl2(dppf)ꢀCH2Cl2, K3PO4, dppf, dioxane, 80 °C, 44%; (b) LiOHꢀH2O, THF-H2O (3:1), 86%; (c) amine (R3R4NH), 140 °C; (d) tert-Butyl (2S)-2-(aminomethyl)-1-pyrrolidine-
carboxylate, HBTU, Et3N, DMF; (e) TFA, CH2Cl2, 8–29% 3 steps (to cleave the Boc group that protects the pyrrolidine moiety and the 2,4-dimethoxybenzyl group that protects
primary amine to give 8a). Yields are not optimized.
morpholine 5d, 2-amino-2,2-dimethethyl 5g, aniline 5h or pyri-
dines 5o–5q, the inhibitory activity decreased. By these results it
is revealed that the substructure around the terminal amine influ-
ences the affinity to IKKb. The N,N,-disubstituted amide such as the
amide of N,N,N0-trimethyldimethylamine 5r and 4-(pyrrolidin-
1-yl)-1-piperidine 5t remained active, but the amide of azetidine
5s and piperazine 5u had little activity. In the case of replacing
the amine moiety to the neutral groups 5v–5y or the removal of
substituent 5z, the potency decreased.
After the exploration of the 3-position, we introduced various
structure to the 6-position of imidazo[1,2-b]pyridazine. The modi-
fied compounds of the 6-position were synthesized as in Scheme 2.
3-Bromo-6-chloroimidazo[1,2-b]pyridazine 2 was converted to
carboxylic acid 6 by Suzuki-Miyaura coupling reaction, followed
by hydrolysis of methyl ester. To compound 6, nucleophilic
substitution by various amines afforded compounds 7a–7p. The
condensation reaction of carboxylic acids 7a–7p with tert-butyl
(2S)-2-(aminomethyl)-1-pyrrolidinecarboxylate, followed by the
cleavage of protective groups gave 8a–8p.
the size of the substituent, an adjacent alkyl moiety to the amine
moiety that is not bulky, such as the cyclopropylmethyl group
was suitable. Other moieties such as isopropyl 8c, cyclopropyl
8d, benzyl 8f, 8g and cyclohexyl 8m were less favored (Table 2).
After determining the potent cyclopropylmethyl group in the 6-
position, we revisited the terminal amine moiety in the 3-position
again to further optimize and investigate the detailed structure–
activity relationship.
The compounds 9a–9k were synthesized as shown in Scheme 3.
Compounds 9a–9i were formed by the condensation reaction of
carboxylic acid 7e and the amine moiety.29 Compound 9j was syn-
thesized by reductive amination of acetaldehyde to 8e. Compound
9k was synthesized in a different manner to avoid methylation of
the 6-NH moiety of imidazo[1,2-b]pyridazine. By the condensation
of carboxylic acid 6 with tert-butyl (2S)-2-(aminomethyl)-1-pyrr-
olidinecarboxylate, amide 10 was formed. The cleavage of the
Boc group of 10 and subsequent methylation of pyrrolidine with
formaldehyde gave N-methylpyrrolidine 11. The nucleophilic sub-
stitution of 11 by cyclopropylmethylamine afforded 9k.
About the structure–activity relationship in the 6-position of
imidazo[1,2-b]pyridazine, we found that the secondary amine with
an appropriate size of non-polar substituents was favored (8e,
methyl group 8b was too small). Primary or tertiary amine in the
6-position decreased potency (8a, 8n–8p), and the polar substitu-
ent near the NH moiety was not favored (8h, 8j, 8k). Regarding
The compounds with a terminal substructure in the 3-position
were N,N-dimethylamine 9a, N,N-diethylamine 9b, N,N,N0-trimeth-
ylethylenediamine 9c and the aminoethyl derivatives 9d and 9e,
which produced IKKb inhibitory activities that were about IC50
0.07–0.3
lM. The compound with (S)-2-piperidinylmethyl 9i
showed strong IKKb inhibitory activity almost the same as 8e.
H
N
H
N
H
N
H
N
N
N
N
N
a (, b)
c
N
N
N
N
N
N
N
N
R5
R6
H
N
H
N
OH
N
O
N
O
N
H
O
O
7e
9a~9i
8e
9j
H
Cl
Cl
N
Cl
N
N
g
N
N
e, f
d
N
N
N
N
N
N
N
N
H
N
H
N
H
N
OH
O
N
O
N
CH3
O
O
N
CH3
6
11
O
9k
10
O
Scheme 3. Modification of the terminal unit in the 3-position of imidazo[1,2-a]pyridazine.24 Reagents and conditions: (a) amine (R5R6NH), DMT-MM, DMF; (b) TFA, 20–60% 2
steps. Yields are not optimized; (c) CH3CHO, NaBH(OAc)3, DCE, 45%; (d) tert-Butyl (2S)-2-(aminomethyl)-1-pyrrolidinecarboxylate, DMT-MM, DMF, 78%; (e) TFA; (f) 37%
HCHO in water, NaBH3CN, AcOH, MeOH, 84% 2 steps; (g) cyclopropylmethylamine, 120 °C, 49%.

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H. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5113–5118
Table 2
Table 3
IKKb inhibition and TNF
a
production inhibition assay results for the substituent in
IKKb inhibition and TNFa production inhibition assay results for the modification of
the 6-position, compounds 8a–8p
terminal substituent in the 3-position, compounds 9a–9k
R3
N
H
N
R4
N
N
N
N
N
N
R5
R6
H
N
N
O
O
N
H
R5
N
IKKb inhibitiona
TNF
IC50
a
(
inhibitionb
lM)
R3
R4
Compd
IKKb inhibitiona
IC50
TNF
IC50
a
(
inhibitionb
lM)
R6
IC50
(
lM)
*
Compds
N
(l
M)
*
H
N
CH3
NH2
N
9a
9b
0.20
0.69
1.2
8a
14
NTc
29
*
*
CH3
H
N
8b
8c
1.3
H
N
CH3
*
*
0.079
0.33
N
*
*
H
N
0.27
0.20
1.7
3.2
CH3
N
CH3
H
N
9c
2.4
N
8d
8e
CH3
*
*
H
N
H
N
0.055
0.33
0.69
3.7
9d
9e
0.31
1.5
2.1
NH2
*
*
H
N
H
N
CH3
8f
N
0.073
*
CH3
H
N
8g
0.19
2.7
NTc
Cl
*
*
*
9f
5.1
N
N
*
*
*
H
N
8h
8i
3.7
>30
>30
OH
OH
N
9g
9h
9i
0.16
0.10
2.1
1.6
1.5
H
N
N
0.55
H
O
N
H
N
N
8j
11
>30
>30
H
N
*
*
H
H
N
H
N
H
N
0.047
N
H
*
*
8k
3.0
N
H
N
H
N
N
9j
0.14
0.11
0.49
1.3
8l
0.38
0.53
>30
>30
N
N
*
*
H
N
H
N
OH
8m
*
9k
N
CH3
CH3
N
8n
7.1
20
CH3
a
*
*
*
Values are the means of at least two experiments.
The method for measuring cell-free IKKb inhibitory activity is described in Ref.
b
8o
8p
>30
>30
NT
NT
N
N
O
25.
c
Not tested.
OH
a
The method for measuring cell-free IKKb inhibitory activity is described in Ref. 25.
The method for measuring the inhibitory activity of TNFa release by THP-1 cell
bulky, such as cyclopropylmethyl, the compound was suitable.
For the terminal amine in the 3-position, the secondary amines
such as (S)-2-pyrrolidinylmethyl carbamoyl and (S)-2-piperidi-
nylmethyl carbamoyl units showed strong IKKb inhibitory activity.
In this position, some modifications such as alkylation of the amine
moiety of pyrrolidine or the 2-aminoethyl moiety are allowed, but
IKKb inhibitory activities decreased.
b
is described in Ref. 27.
c
Not tested.
The pyrrolidine N-ethylated compound 9j improved the activity in
cell assay for cell-free assay due to improved permeability.
Regarding the comparison between enantiomers, it was clearly
determined that one enantiomer was superior to another (the
(S)-isomer 8e compared with the (R)-isomer 9h, the (R)-isomer
9g compared with the (S)-isomer 9f) (Table 3).
In regards to the structure–activity relationship as a whole,
when the non-polar substituent that is adjacent to the amine moi-
ety in the 6-position of imidazo[1,2-b]pyridazine template is not
The weakness of TNFa inhibitory activities in cellular assay for
cell-free IKKb inhibitory activities might be explained by permeabil-
ity. In some compounds which showed potent activity in
cell-free assay, the values of parallel artificial membrane permeabil-
ity assays (PAMPA) were low (compound 5i: <2.0 ꢁ 10ꢂ6, 8e:
<2.0 ꢁ 10ꢂ6, 8g: <2.0 ꢁ 10ꢂ6, 9i: 2.3 ꢁ 10ꢂ6 cm/s Papp at pH 7.4).
These properties are thought to be the cause of decreased activity

H. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5113–5118
5117
Table 4
Kinase selectivity assay results for representative compounds
Compds
IKKb
IKK
IC50
a
(
CDK2
IC50
PDK1
GSK3b
IC50
JNK3
P38
IC50
a
(
IC50
(
l
M)
l
M)a
(
l
M)a
IC50
(l
M)a
(
l
M)a
IC50
(
l
M)a
l
M)a
1
5b
5i
8e
8g
9a
9b
9e
1.1
0.59
0.32
0.055
0.19
0.20
NTb
4.6
2.2
40
>100
>100
95
>100
>100
>100
NT
NT
>100
>100
>100
>100
>100
>100
NT
NT
90
NT
>100
>100
>100
>100
>100
NT
>100
>100
>100
>100
>100
NT
>100
>100
78
>100
NT
>4
>100
>100
>100
0.079
0.073
NT
NT
NT
NT
NT
a
Regarding the methods, in Ref. 26.
Not tested.
b
in THP-1 cells with poor ability to penetrate the cell membrane.
These compounds commonly have substructure of cyclic secondary
amine moiety in the 3-position. In the compoundswhich avoid these
substructures showed improved cellular activities for cell-free as-
says (compound 9a: >50 ꢁ 10ꢂ6, 9j: 23 ꢁ 10ꢂ6 Papp at pH 7.4).
About the kinase selectivity, each compound showed good
selectivity over some Ser/Thr kinases (Table 4). Compounds 8e
and 9a increased the inhibitory activity of IKKb, while decreasing
homology model.30 From the structure–activity relationship that
we have revealed, (S)-2-pyrrolidinylmethyl carbamoyl moiety in
the terminal of the 3-position had an important role for high affin-
ity. However, we could not find any effective interactions in this
part. Instead, there was steric hindrance between the pyrrolidine
moiety and the internal surface of IKKb, mainly the part of the salt
bridge among Lys44, Glu61 of helix C and Asp166 of the activation
loop (Fig. 2a).
the IKK
a
inhibitory activity compared with 5b and 5i. Potent com-
Considering this result and some studies about the conformation
of kinase and binding modes with ligands, we attempted to con-
struct a new model that would shift the conformation of the activa-
tion loop and the helix C in which the ligand crashed into. As one of
some ideas, we assumed that the conformational change such as
DLG-out occurs in the same way as with well-known DFG-out31
(Figs. 2b and 3). The binding mode is a result of the activation loop
which shifts to become an another form and amino acids on the acti-
vation loop which start to interact with imidazo[1,2-b]pyridazine
compounds. Leu167 on the activation loop causes a lipophilic
interaction with the cyclopropylmethyl moiety (cyclopropylmethyl
moiety was caught by Lue21, Val29 and Leu167). Asp166 on the
activation loop and Glu61 on helix C form multiple hydrogen bond-
ing interaction with the ammonium moiety of pyrrolidine in the
3-position of imidazo[1,2-b]pyridazine. The amide carbonyl moiety
in the 3-position interacts with the side chain of Lys44, which is sta-
bilized with the carbonyl moiety of the backbone of Asp166.
Imidazo[1,2-b]pyridazine scaffold interacts with the backbone of
Glu97 and Cys99 in the hinge region. These binding modes are
thought to be the key for strong inhibitory activity and high selectiv-
ity. We have indicated that the structure–activity relationship in the
3- and 6-position of imidazo[1,2-b]pyridazine follows the binding
pounds 8e and 9a showed more than 300-fold selectivity over IKK
and other Ser/Thr kinases.
To consider the characteristics of these imidazo[1,2-b]pyrida-
zine derivatives, we tried to construct an interaction model with
IKKb. As the X-ray structural data of IKKb has not been acquired,
we examined the docking study of compound 8e with the IKKb
a
Glu97
N
Helix C
Glu61
Hinge
O
H
N
O
O
Lys44
Cys99
O
+NH
N
H
H
H
N
N
O
NH3
N
N
N
O
H
O
O
H
N
Leu167
Asp166
HN
N
O
Val29
O
Activation loop
Leu21
Glu97
N
Helix C
Hinge
O
Cys99
H
N
O
Glu61
N
Lys44
H
N+H
H
H
O
N
O
N
H
O
N
N
O
H
O
N
NH3+
O
HN
O
Asp166
N
Val29
NH
Activation loop
O
Leu167
Leu21
Figure 2. 2D representations of predicted binding mode of compound 8e with IKKb.
The hydrogen bondings are represented as orange dotted lines. van der Waals
interactions are represented as yellow lines. (a) Pyrrolidine moiety crashes into the
internal surface composed of the salt bridge among Lys44, Glu61 and Asp166. (b)
Effective interactions are formed by the conformational shift of the activation loop
and helix C.31
Figure 3. Predicted binding mode of compound 8e with IKKb.32 View from the
N-lobe. For simplicity, only important residues are shown. The hydrogen bondings
are represented as yellow dotted lines.

5118
H. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5113–5118
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mode well. It is inferred that imidazo[1,2-b]pyridazine derivatives
could show a binding mode like the type-II inhibitor.32
In conclusion, we have developed potent IKKb inhibitory agents
with the imidazo[1,2-b]pyridazine scaffold from HTS-hit 1. The po-
tency was improved by the modification of the 3- and 6-position to
8e and 9i. These compounds showed good kinase selectivity over
IKKa and some Ser/Thr kinases. From the interaction model study,
it is assumed that appropriate interactions in the pyrrolidine moi-
ety in the 3-position are formed. The structure–activity relation-
ship we have revealed is explained in this model. Compound 8e
is an attractive lead compound and the predicted binding mode
is useful for further development of potent IKKb inhibitor. In the
proceeding paper, we would like to report further investigations
of the modification of 8e with the strategy based on this interac-
tion model.
24. All test compounds are purified by preparative HPLC and are acquired as formic
acid or hydrochloric acid salt.
25. IKKb kinase inhibition assay: Test compound at various concentrations or
jBa (180 nM final concentration) and [
33P]ATP
References and notes
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fetal bovine serum (FBS), 50 units/mL penicillin, 50 g/mL streptomycin) for 3–
a was performed at the Km of ATP for each recombinant
a
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l
g/mL) and
g/mL)
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a
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performed using Glide 4.0 (Schrödinger, LLC).