Discovery of potent and selective rhodanine type IKKβ inhibitors by hit-to-lead strategy

Article abstract of DOI:10.1016/j.bmcl.2012.06.088

Regulation of NF-κB activation through the inhibition of IKKβ has been identified as a promising target for the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop novel IKKβ inhibitors, we performed high throughput screening toward around 8000 library compounds, and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound to obtain potent and selective IKKβ inhibitors. Throughout hit-to-lead studies, we have discovered optimized compounds which possess blocking effect toward NF-κB activation and TNFα production in cell as well as inhibition activity against IKKβ. Among them, compound 3q showed the potent inhibitory activity against IKKβ, and excellent selectivity over other kinases such as p38α, p38β, JNK1, JNK2, and JNK3 as well as IKKα.

Full text of DOI:10.1016/j.bmcl.2012.06.088

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5668–5674
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and selective rhodanine type IKKb inhibitors by
hit-to-lead strategy
Hyeseung Song c, Yun Suk Lee c, Eun Joo Roh b, Jae Hong Seo a, Kwang-Seok Oh d, Byung Ho Lee d,
Hogyu Han c, Kye Jung Shin a,
⇑
a Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu,
Bucheon, Gyeonggi-do 420-743, Republic of Korea
b Life/Health Division, Korea Institute of Science and Technology, 5 Wolsong-gil, Seongbuk-gu, Seoul 136-791, Republic of Korea
c Department of Chemistry, Korea University, Seongbuk-gu, Seoul 136-713, Republic of Korea
d Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Regulation of NF-jB activation through the inhibition of IKKb has been identified as a promising target for
Received 25 May 2012
Revised 26 June 2012
Accepted 27 June 2012
Available online 15 July 2012
the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop
novel IKKb inhibitors, we performed high throughput screening toward around 8000 library compounds,
and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound
to obtain potent and selective IKKb inhibitors. Throughout hit-to-lead studies, we have discovered opti-
mized compounds which possess blocking effect toward NF-
well as inhibition activity against IKKb. Among them, compound 3q showed the potent inhibitory activity
against IKKb, and excellent selectivity over other kinases such as p38 , p38b, JNK1, JNK2, and JNK3 as
well as IKK
jB activation and TNFa production in cell as
Keywords:
IKKb inhibitor
a
NF-jB
a
.
TNFa
Ó 2012 Elsevier Ltd. All rights reserved.
Reumatoid athritis
Hit-to-lead
NF-
role in the regulation of a variety of genes associated with inflam-
mation, anti-apoptosis, and proliferation.1 NF-
B exists as an inac-
tive form bound with inhibitory B (I B) protein in the cytoplasm.
Upon stimulation by proinflammatory cytokine, I B is phosphory-
lated, polyubiquitinated, and degraded through the S26 proteasome
pathway, thus NF- B sets free from I B and translocates to the nu-
cleus where it binds to
B site and activates target genes.2 Phos-
phorylation of I B is catalyzed by I B kinase (IKK) complex
protein which is composed of two catalytic subunits, IKK (IKK1)
and IKKb (IKK2), and a regulatory subunit, IKK
(NEMO).3 IKK
and IKKb are both serine–threonine kinase, but IKK is considered
to regulate the duration of NF- B response with prolonged expres-
sion of proinflammatory cytokines observed in IKK -deficient
cells.4 In this reason, development of selective inhibitors of IKKb
j
B is a multifunctional transcription factor and plays central
hit compound (1) was selected from druggable and transformative
points of view. Selected compound 1 has rhodanine ring as a core
structure with diethylaminoalkoxyphenyl moiety in western part
and phenylmethylene moiety in eastern part. In the present study,
we replaced western and eastern parts of parent compound (1)
with various substituents to obtain the maximum inhibitory
activity against IKKb. Compound 3q was found to have more than
100-folds increased inhibitory activity compared to hit compound,
j
j
j
j
j
j
j
j
j
and to show excellent selectivity over other kinases such as IKK
a,
a
p38 , p38b, JNK1, JNK2, and JNK3. Also NF- B activation and TNFa
a
j
c
a
production were efficiently blocked by compound 3q in cell-based
assay. All these findings suggest that optimized compound 3q has
high possibility as a potential candidate for treatment of disease
a
j
a
associated with NF-jB activation such as rheumatoid arthritis,
chronic obstructive pulmonary disease (COPD), and cancer.
Hit compound 1 has two distinct substituents on the rhodanine
nucleus, diethylaminopropanoxyphenyl and 4-hydroxyphenylm-
ethylene group (Fig. 1). In our preliminary fragment analysis study
of hit compound 1, we found that deletion of the aminoalkyl group
at the western part resulted in a complete loss of activity. It sug-
gests that the aminoalkyl group is essential for keeping inhibitory
activity against IKKb. On the basis of fragment study of 1, we first
tried to investigate the substituent effect of eastern part fixing the
western part with diethylaminopropanoxyphenyl group. We
over IKKa is advantageous to anti-inflammatory and autoimmune
disease such as rheumatoid arthritis.5
As a program of searching compounds for showing an inhibitory
activity on IKKb in our in-house library, high-throughput screening
(HTS) is employed for the generation of novel hit structure. In this
process, we have identified several hit compounds. Among them, a
⇑
Corresponding author. Tel.: +82 2 2164 4060; fax: +82 2 2164 4059.
E-mail address: kyejung@catholic.ac.kr (K.J. Shin).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.088

H. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5668–5674
5669
cell-based assay. In bulky aromatic analogues, phenoxyphenyl
(2m–2p) and 5-phenylfuranyl (2q, 2r) were found to be useful
replacement for the eastern part. Notably, carboxamido substituted
phenoxyphenyl and phenylfuranyl analogues except o-carboxami-
dophenoxyphenyl (2n) provided a considerable improvement in
enzymatic activities compared to hit compound, and also blocked
N
O
S
N
S
western part
O
NF-jB activation and TNFa production in cell-based assay. How-
OH
ever, they were still inferior to reference compound11 in enzymatic
and cell-based activities.
In order to obtain the optimized compound, structure activity
relationship of the western part was investigated. With the fixed
carboxamidophenoxyphenyl and carboxamidophenylfuranyl in
eastern part, we next focused our efforts on the structural modifi-
cation and activity evaluation for a variety of aminoalkoxyphenyl
derivatives in western part. We have diversified diethylamino
group to other amino group such as pyrrolidine, morpholine, and
piperazine, and we also gave variety to carbon chain length ranging
from 2 to 5. We made this series of compounds as the same man-
ners with Scheme 1, and the method for preparation of 3a–s was
depicted in Scheme 2.
The inhibitory activities of various aminoalkoxyphenyl deriv-
ativs in western part of rhodanine ring are summarized in Table
2. The results showed that the IKKb inhibitory activities were
mainly influenced by amino groups and carbon length between
nitrogen and oxygen in western part, and the position of carboxa-
mido substituent in eastern part. As the length of carbon chain
goes to increase, proportional positive effect was observed in IKKb
inhibitory activity (3s > 3r > 3q). Especially 3s was as potent as ref-
erence compound. However, 3s and 3r which has 4 and 5 carbons
as a linker, respectively, did not show inhibitory activity against
eastern part
1: % inhibition at 10µM; 56%, IC50; 23.6 µM
Figure 1. Hit structure and its screening data.
replaced phenylmethylene moiety with other aryl groups involving
heteroaromatics for the eastern part, and examined their enzyme
inhibitory activities against IKKb and suppression abilities of NF-
j
B activation and TNFa production in cell-based assay. The synthe-
sis of this series of compounds was described in Scheme 1. Alkyl-
ation of 4-nitrophenol with dibromopropane gave corresponding
monoalkyl product, which was reacted with diethylamine in the
presence of K2CO3 to afford 4-(diethylaminopropanoxy)-1-nitro-
benzene (5). Reduction of nitro compound 5 by catalytic hydroge-
nation over Pd/C in methanol provided aniline 6. Aniline 6 was
converted to rhodanine 7 by reaction with bis(carboxymethyl)tri-
thiocarbonate in ethanol and water (4:3),6 which was condensed
with various aryl aldehydes in the presence of sodium acetate in
acetic acid to afford compounds 2a–r.7
Based on the aromatic nature of size and physicochemical prop-
erties, a series of 18 compounds were synthesized and their biolog-
ical activities were evaluated. Inhibitory enzymatic activity of IKKb
was measured by TR-FRET (time-resolved fluorescence resonance
NF-jB activation and TNFa production in cell-based assay. These
results seemed to be caused by poor cell permeability of com-
pounds 3s and 3r. In the case of 2 and 3 carbon length linker, com-
pound with 3 carbons linker (2n, 2o and 2p) was moderately
superior to the corresponding compounds with 2 carbons linker
(3a, 3b and 3c) in IKKb inhibitory activities. These results indicate
that optimal carbon length is three carbons apart from oxygen to
nitrogen in western part.
As we can see in Table 2, structural modification of amino group
has considerably affected the potency of inhibitors. We have re-
placed diethylamino group with cyclic secondary amino groups
such as pyrrolidine, piperidine, morpholine, and 4-methylpipera-
zine. In analogues of pyrrolidine and piperidine with p-CONH2 sub-
stituent in eastern part (3f, 3i), their inhibitory activities were
equipotent to parent diethylamino compound (2p). The most
noticeable improvement effect resulted from the introduction of
4-methylpiperazine moiety, which led to around 100-folds in-
energy transfer) method on IMAPÒ platform,8 and NF-
j
B report
gene assay was performed to evaluate inhibitory ability of synthe-
sized compounds toward NF-
B activation.9 Additionally, inhibi-
j
tion study of TNF
a production was carried out with the spleen
cell of collagen induced arthritis (CIA) mouse model.10
Various aromatic analogues such as simple monocyclic aromat-
ics, heteroaromatics, and bulky bicyclic and phenoxyphenyl aro-
matics were introduced to the eastern part. Table 1 shows
representative enzymatic and subsequent cell-based assay results
from the analogues of eastern part. Simple aromatic and heteroar-
omatic compounds possessing either electron donating or electron
withdrawing group did not show any inhibition activity compared
to p-hydroxyphenyl ring of hit compound. Only 2b compound
which has 3,4-dichloro substituent was improved in activity com-
pared to hit compound, but did not display subsequent activity in
HO
c
a, b
N
O
N
O
NO2
NO2
NH2
6
5
4
d
e
N
O
N
O
S
S
S
N
N
S
O
O
R
7
2a~r
Scheme 1. Reagents and conditions: (a) Dibromopropane, K2CO3, CH3CN, 80 °C, 78%; (b) diethylamine, K2CO3, CH3CN, 80 °C, 53%; (c) Pd/C (10%), H2, MeOH, 98%; (d)
bis(carboxymethyl)trithiocarbonate, EtOH/H2O (4:3), reflux, 52%; (e) aldehydes, NaOAc, AcOH, reflux, 57–88%.

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H. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5668–5674
Table 1
IKKb inhibitory activities of rhodanine derivatives 2a–2r
N
O
S
N
S
O
R1
Compd.
R1
Enzyme assay
Cell-based assay
B (10 M)
% Inhibition of IKKb (10
l
M)
IKKb IC50
(l
M)
% Inhibition of NF-
j
l
TNFa IC50 (lM)
OCH3
2a
2b
—
—
—
—
OH
Cl
61.8
3.4
9.3
—
—
—
—
—
Cl
Cl
2c
Cl
2d
2e
2f
—
—
—
—
—
—
—
—
—
—
N
28.1
—
N
NH
2g
2h
—
—
—
—
—
—
—
N
H
23.4
2i
2j
52.6
3.2
14.1
—
—
—
—
—
N
N
2k
—
—
—
—
O
NH2
2l
15.5
36.5
—
—
—
O
Cl
2m
15.6
71.4
13
O
2n
4.8
—
—
—
O
O
NH2
NH2
NH2
2o
2p
84.9
95.7
7.3
2.6
39.3
46.6
10
8
O
O
O
O

H. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5668–5674
5671
Table 1 (continued)
Compd.
R1
Enzyme assay
Cell-based assay
B (10 M)
% Inhibition of IKKb (10
l
M)
IKKb IC50
(l
M)
% Inhibition of NF-
j
l
TNFa IC50 (lM)
O
O
2q
87.1
5.7
59.1
—
NH2
O
O
2r
77.2
88.7
3.3
57.4
73.9
—
NH2
Reference compound
0.17
1.8
R1
O
X
O
HO
( )n
a
b, c
( )n
NO2
NO2
NH2
9a: n=2, R1=NEt2
8a: n=2, X=Br
8b: n=3, X=Br
8c: n=4, X=Br
8d: n=5, X=I
4
9b: n=3, R1=pyrrolidinyl
9c: n=3, R1=piperidinyl
9d: n=3, R1=morpholidinyl
9e: n=3, R1=4-methylpiperazinyl
9f: n=4, R1=4-methylpiperazinyl
9g: n=5, R1=4-methylpiperazinyl
R1
O
R1
O
( )n
( )n
S
S
d
e
N
N
S
S
O
O
R2
10a: n=2, R1=NEt2
3a~s
10b: n=3, R1=pyrrolidinyl
10c: n=3, R1=piperidinyl
10d: n=3, R1=morpholidinyl
10e: n=3, R1=4-methylpiperazinyl
10f: n=4, R1=4-methylpiperazinyl
10g: n=5, R1=4-methylpiperazinyl
Scheme 2. Reagents and conditions: (a) Dihaloalkane, K2CO3, CH3CN, 80 °C, 64–82%; (b) amines, K2CO3, CH3CN, 80 °C, 48–76%; (c) Pd/C (10%), H2, MeOH, 90–99%; (d)
bis(carboxymethyl)trithiocarbonate, EtOH/H2O (4:3), reflux, 33–64%; (e) aldehydes, NaOAc, AcOH, reflux, 62–86%.
crease in enzymatic inhibitory activity compared to hit compound
(1). Interestingly, replacement of diethylamino group with mor-
pholine ring resulted in the complete loss of inhibitory activity.
Considering the biological effects on the position of carboxa-
mido substituent in eastern part, there was a declining tendency
of inhibitory activities as an order of para > meta > ortho whatever
aminoalkoxy substituents were attached in western part. One
exception was observed in morpholine substituent (3i–3n), in this
case every compounds having morpholine moiety were inactive
against IKKb.
and JNK3 as well as IKKa. It is noteworthy that IKKa/IKKb IC50 ratio
of 3f, 3p and 3q is over 20 compared with reference compound.
In summary, from high throughput screening technology, we
have identified a hit compound 1 which has rhodanine nucleus as
a core structure. Starting from this hit compound, structural modi-
fication was carried out in eastern and western part of rhodanine
nucleus in order to obtain potent and selective IKKb inhibitors.
Compound 3q12 which possesses p-carboxamidophenoxyphenyl
moiety in eastern part and 4-methylpiperazinylpropoxyphenyl
group in western part was found as a potent inhibitor against IKKb,
There are lots of kinases in human biological system. To reduce
side effects possibly occurring on inhibiting other kinases, selectiv-
ity for target kinase is needed. In this reason, we performed kinase
and showed balanced inhibitory activity against NF-
and TNF production in cell-based assay. Moreover, compound
3q has excellent selectivity over other kinases such as IKK
p38 , p38b, JNK1, JNK2, and JNK3. Taken together, optimized com-
pound 3q has high possibility as a potential candidate for treatment
of disease associated with NF- B activation such as rheumatoid
jB activation
a
a,
panel test against IKK
a
and other kinases such as p38
a
, p38b, JNK1,
a
JNK2 and JNK3 with selected compounds (3f, 3p and 3q) which
possessed good inhibitory activities in enzymatic and cell-based
assay, and the results are summarized in Table 3. Selected com-
j
arthritis, chronic obstructive pulmonary disease (COPD), and
pounds showed excellent selectivity over p38a, p38b, JNK1, JNK2,
cancer.

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H. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5668–5674
Table 2
IKKb inhibitory activities of rhodanine derivatives 3a–3s
R1
O
S
n
N
S
O
R2
Compd
n. R1
R2
Enzyme assay
% Inhibition of IKKb
Cell-based assay
% Inhibition of NF- TNF
M)
IKKb IC50
M)
jB
a IC50
(10
lM)
(
l
(10
lM)
(l
3a
2
NEt2
1.6
—
—
—
—
O
O
O
O
O
O
NH2
NH2
NH2
3b
3c
2
2
NEt2
NEt2
17.1
62.6
57.0
58.5
7
8
O
O
8.7
—
O
O
N
3d
3
21.3
—
—
NH2
N
N
NH2
NH2
3e
3f
3
3
57.3
85.3
6.3
1.5
57.0
58.5
7
5
O
O
O
O
O
N
3g
3
18.6
—
—
—
NH2
N
N
NH2
NH2
3h
3i
3
3
83.0
81.2
6.3
4.2
42.1
48.2
30
30
O
O
O
O
O
O
O
N
3j
3
3
—
—
—
—
—
—
—
NH2
O
N
NH2
3k
48.8
O

H. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5668–5674
5673
O
O
O
O
N
N
N
NH2
3l
3
—
—
—
—
O
O
O
3m
3n
3
3
2.1
—
—
—
—
—
—
—
NH2
O
O
NH2
N
N
N
3o
3
52.7
8.5
42.4
12
O
O
O
NH2
N
N
N
NH2
NH2
NH2
NH2
3p
3q
3r
3
3
4
5
86.8
90.1
90.4
1.5
64.7
65.4
—
5
2
4
O
O
N
N
0.35
0.23
O
O
O
O
O
N
N
3s
89.3
88.7
0.16
0.17
—
—
Reference
73.9
1.8
compound
4. Strnad, J.; Burke, J. R. Trends Pharmacol. Sci. 2007, 28, 142.
5. (a) Brown, K. D.; Claudio, E.; Siebenlist, U. Arthritis Res. Ther. 2008, 10, 212; (b)
Coish, P. D. G.; Wickens, P. L.; Lowinger, T. B. Trends Opin. Ther. Patents 2006, 16,
1.
Table 3
Selectivity data of 3f, 3p and 3q over other kinases (IC50 values in
l
M)
6. Basak, S.; Shih, V. F. S.; Hoffmann, A. Mol. Cell. Biol. 2008, 28, 3139.
7. (a) Potin, D.; Launay, M.; Nicolai, E.; Fabreguette, M.; Malabre, P.; Caussade, F.;
Besse, D.; Skala, S.; Stetsko, D. K.; Todderud, G.; Beno, B. R.; Cheney, D. L.;
Chang, C. J.; Sheriff, S.; Hollenbaugh, D. L.; Barrish, J. C.; Iwanowicz, E. J.;
Suchard, S. J.; Dhar, T. G. Bioorg. Med. Chem. Lett. 2005, 15, 1161; (b) Khodair, A.
I. J. Heterocycl. Chem. 2002, 39, 1153; (c) Xing, C.; Wang, L.; Tang, X. H.; Sham, Y.
Y. Bioorg. Med. Chem. 2007, 15, 2167.
Compd.
IKKb IKK
a
p38a
p38b JNK1 JNK2 JNK3
3f
3p
3q
1.5
1.5
>30
>30
>30
>30
>30
—
>30
>30
>30
—
>30
>30
>30
—
>30
>30
>30
—
>30
>30
>30
—
0.38 >30
Reference compound 0.17
0.64
8. (a) Oh, K.-S.; Lee, S.; Choi, J. K.; Lee, B. H. Comb. Chem. High Throughput Screen.
2010, 13, 790; (b) Nagarajan, S.; Doddareddy, M. R.; Choo, H.; Cho, Y. S.; Oh, K.-
S.; Lee, B. H.; Pae, A. N. Bioorg. Med. Chem. 2009, 17, 2759.
Acknowledgments
9. For NF-
Luc reporter plasmid DNA using calcium phosphate precipitation methods.
Cells were pre-treated with 10 M of inhibitors for 1 h and further incubated
jB report gene assay, HeLa cells were transfected with 1 lg of NF-jB-
This work was financially supported by Research Fund 2011 of
The Catholic University of Korea and Development of Bio-oriented
Technology Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
Technology.
l
with TPA (50 ng/mL) for 8 h. Luciferase activity was determined and
normalized to the protein content of each extract. Results are presented as
relative luciferase unit (RLU) per mg compared to non-treated cell (n = 3).
10. Eight weeks after the primary type II collagen immunization, the mouse
spleens were collected for cell preparation and washed twice with PBS. The
spleens were minced and the red blood cells were lysed with 0.83%
ammonium chloride. The cells were filtered through
a cell strainer and
centrifuged at 1300 rpm at 4 °C for 5 min. The cell pellets were re-suspended
in RPMI 1640 medium and plated in 24-well plates (Corning, NY, USA) at a
concentration of 1 Â 106 cells/well. Isolated splenocytes were cultured with
References and notes
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inhibitors for 72 h. The amounts of TNF
a in the culture supernatants were
measured by ELISA. Antibodies directed against mouse TNF
biotinylated anti-mouse TNF were used as the capture and detection
antibodies, respectively. Alkaline phosphatase (Sigma) was used for the
chromogenic reaction. The amounts of TNF present in the test samples were
determined from standard curves constructed with serial dilutions of
recombinant murine TNF (R&D Systems). The absorbance was determined
with an ELISA microplate reader at 405 nm.
a and against
a
a
a

5674
H. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5668–5674
11. Murata, T.; Shimada, M.; Kadono, H.; Sakakibara, S.; Yoshino, T.; Masuda, T.;
Shimazaki, M.; Shintani, T.; Fuchikami, K.; Bacon, K. B.; Ziegelbauer, K. B.;
Lowinger, T. B. Bioorg. Med. Chem. Lett. 2004, 14, 4013.
12. 2HCl salt form of 3q: 1H NMR (400 MHz, DMSO-d6) d 2.17–2.18 (m, 2H), 2.81
(bs, 3H), 3.53–3.60 (m, 10H), 4.13 (t, J = 5.7 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H), 7.16
(d, J = 8.8 Hz, 2H), 7.21 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 9.0 Hz, 2H), 7.36 (bs, 1H),
7.74 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.8 Hz, 2H), 7.98 (bs, 1H), 11.55 (bs, 2H); 13
H
NMR (100 MHz, DMSO-d6) d 23.9, 42.5, 48.8, 50.2, 53.6, 65.6, 115.4, 118.6,
118.9, 119.3, 119.7, 122.3, 128.2, 128.9, 130.3, 130.7, 132.4, 133.4, 158.2, 158.8,
159.2, 167.5, 194.5: HRMS Calcd for C31H32N4O4S2 (M+H)+ 589.1937. Found
589.1986.
NH
CN
OH
N
NH2