Optimization and biological evaluation of aminopyrimidine-based IκB kinase β inhibitors with potent anti-inflammatory effects

Article abstract of DOI:10.1016/j.ejmech.2016.07.075

Targeting IκB kinase β (IKKβ) can be a promising strategy in the development of a therapeutic treatment of inflammatory diseases because IKKβ is well-recognized as a key mediator of the NF-κB signaling pathway. In this study, we have successfully develope

Full text of DOI:10.1016/j.ejmech.2016.07.075

European Journal of Medicinal Chemistry 123 (2016) 544e556
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Optimization and biological evaluation of aminopyrimidine-based I
kinase inhibitors with potent anti-inflammatory effects
kB
b
,
b
,
,
c
,
,
, b
Yongje Shin a 1, Sang Min Lim b 1, Hong Hua Yan d 1, Sungwoo Jung a
,
Zhenghuan Fang d, Kyung Hee Jung d, Soon-Sun Hong d **, Sungwoo Hong b
,
, a, *
a Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea
b Center for Catalytic Hydrocarbon Functionalizations, Institute of Basic Science (IBS), Daejeon 34141, South Korea
c Korea Institute of Science and Technology (KIST), Seoul 02792, South Korea
d Department of Biomedical Sciences, College of Medicine, Inha University, Incheon 400-712, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Targeting I
of inflammatory diseases because IKK
pathway. In this study, we have successfully developed a structure-activity relationship (SAR) profile of
the aminopyrimidine-based IKK inhibitors through the structure-based design strategy to improve the
physicochemical properties and cellular activity in terms of the anti-inflammatory effects. Representative
compounds exhibited desirable activity in nitric oxide (NO) reduction by inhibiting the synthesis of
inducible nitric oxide synthase (iNOS), and strongly inhibited the expression of pro-inflammatory cy-
k
B kinase
b
(IKK
b
) can be a promising strategy in the development of a therapeutic treatment
Received 1 April 2016
Received in revised form
28 July 2016
Accepted 31 July 2016
Available online 1 August 2016
b
is well-recognized as a key mediator of the NF- B signaling
k
b
Keywords:
tokines (IL-1
a, IL-6, and TNF-
a
). The inhibitory effects of 8e on the phosphorylation in the NF-
k
B pathway
IKKb
further supported that the suppression of the NF-
effect in LPS-stimulated Raw 264.7 cells.
kB signaling pathway induced the anti-inflammatory
Inhibitor
Structure-activity relationship
NO reduction
© 2016 Elsevier Masson SAS. All rights reserved.
Anti-inflammatory effect
1. Introduction
ICAM-1, E-selectin) [5]. Furthermore, activated NF-kB induces the
generation of nitric oxide synthase (NOS) and cyclooxygenase-2
(COX-2), both of which are important regulators of the inflamma-
tory process. Recent studies have demonstrated the critical role of
Inflammation has been known as an immune response of body
tissues caused by a wide variety of stimuli such as injury or infec-
tion. The Inflammatory reaction is characterized by the local rapid
expression of pro-inflammatory cytokines, chemokines and adhe-
sion molecules [1]. These are mainly regulated by nuclear factor
NF-
k
B in the inflammatory process in several animal models.
B is highly activated in a variety
Moreover, it was reported that NF-
k
of human inflammatory diseases such as rheumatoid arthritis (RA),
atherosclerosis, multiple sclerosis, and inflammatory bowel disease
[5]. Therefore, numerous strategies suppressing the activation of
kappa-B (NF-
scription factor [2,3]. Whereas NF-
inactive state in the cytoplasm, NF-
matory sites by several pro-inflammatory stimuli, such as tumor
necrosis factor alpha (TNF- ), interleukin-1 (IL-1), and lipopoly-
saccharide (LPS) [4]. The activation of NF- B results in its trans-
k
B) protein, which is a ubiquitously expressed tran-
B is normally maintained in an
B is activated at the inflam-
k
k
NF-
diseases [6]. Among them, targeting I
appeared to be one of the most promising strategies because IKK
has been well-recognized as a key mediator of the NF- B signaling
pathway [7,8]. In a resting state, I B (inhibitor of kappa-B) binds to
NF- B, keeping it in the cytosol. Once the NF- B signaling pathway
is stimulated, IKK phosphorylates I B triggering ubiquitination
and subsequent degradation of I B, which renders NF- B free to
k
B have been pursued to develop therapeutics for inflammatory
k
B kinase (IKK ) has
b
b
a
b
k
k
location from the cytosol to the nucleus, which promotes
transcription of diverse pro-inflammatory cytokines (IL-1, IL-6, and
k
k
k
TNF-a
), chemokines (IL-8), and adhesion molecules (VCAM-1,
b
k
k
k
translocate into the nucleus and to induce the transcription of
various genes responsible for inflammation and cell survival [9,10].
* Corresponding author. Department of Chemistry, Korea Advanced Institute of
Science and Technology (KAIST), Daejeon 34141, South Korea.
** Corresponding author.
As a result, potent small molecule IKKb inhibitors have been
developed through intensive studies and drug discovery programs
as therapeutic options for the treatment of cancer and
E-mail addresses: hongs@inha.ac.kr (S.-S. Hong), hongorg@kaist.ac.kr (S. Hong).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.07.075
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
545
inflammatory diseases [11e21]. Recently, we disclosed a series of
aminopyrimidine-based potent IKK inhibitors through a virtual
envisioned that adequately located substituents and chemical
groups on the aniline group (A ring) and the phenyl ring (C ring)
b
screening of a large chemical library with improved scoring func-
tions [22]. In the present study, we extensively modified the
structure of a hit compound using a structure-based drug design
strategy to improve its physicochemical properties and cellular
activity. Herein, we report our studies on the design and optimi-
could achieve additional molecular interactions with IKK
providing more potent IKK inhibitors from the hit. In the first
step, a variety of aminopyrimidines were generated with the
LigBuilder program [23] based on the structure of the IKK -ami-
nopyrimidine complex obtained from docking simulations. By
installing a phenoxy group on the C4 aryl group (C ring) and a
sulfonamide group on the C2 aniline ring (A ring), compound 2
b,
b
b
zation of a series of aminopyrimidine-based IKKb inhibitors and
demonstrate the biological evaluation of these compounds in terms
of their anti-inflammatory effects.
was generated with
a
reasonable potency against IKKb
(IC50 ¼ 1.52 M). Introduction of an o-amino group on the phe-
m
noxy ring (D ring) dramatically improved the potency (3k,
IC50 ¼ 98 nM). To obtain structural insight into the inhibiton of 3k,
its binding modes in the ATP-binding site were further investi-
gated in a comparative fashion. Fig. 2 shows the lowest energy
conformation of compound 3k (calculated with the Discovery
Studio software), which defines the binding mode of 3k at the ATP
biding site. The phenoxy ring appears to point toward the binding
2. Results and discussion
2.1. Discovery of IKK
b inhibitors
Our efforts to develop potent IKK
b
inhibitors commenced with
de novo design to identify promising molecular scaffolds, which
led us to discover 2-anilino-4-phenylpyrimidine (1) as a hit
compound (Fig. 1a). To obtain structural insight into the inhibitory
mechanism of the identified inhibitor 1, we carried out molecular
docking studies and subsequent structural analysis of an IKK
complex with the aminopyrimidine 1 (Fig. 1b). Molecular simu-
pocket region and has close contact with IKKb in the back-pocket.
The amino group of the phenoxy ring maintains a key hydrogen-
bonding interaction with GLY27. Therefore, the phenoxy ring (D
ring) was thoroughly investigated by incorporating a variety of
substituents. To find lead-level inhibitors that are potent at both
enzymatic and cellular levels, we next planned to focus our design
attempts on the investigation of the solvent exposure region (A
ring region).
b
lation of 2-anilino-4-phenylpyrimidine with a crystal structure of
IKK
bonding with Cys 99 in the hinge region (Fig. 1b). This series was
attractive for developing potent IKK inhibitors because an ami-
b (PDB ID: 4KIK) revealed that it forms bidentate hydrogen
b
nopyrimidine scaffold can be easily diversified at the 2- and 4-
positions of the pyrimidine core via a convergent synthetic
approach for the rapid elucidation of the structure-activity rela-
tionship (SAR). For these reasons, chemical derivatization of hit 1
was initiated for further optimization by in-depth structural
modification. We observed that there is additional space near the
4-phenyl group (red circle) surrounded by several polar residues
such as Gly 27, Lys 44, Glu 149, Asn 150, and Asp 166. Additionally,
the 2-anilino group is pointing toward the solvent-exposed region
near Asp 103 and Lys 106 (Fig. 1b). In an initial effort to enhance
the binding affinity, we explored the space at the 2- and 4-
positions (A and C rings) of pyrimidine through structure-based
de novo design and compared the calculated binding free en-
2.2. Exploration of the solvent exposed region
Considering the tolerance within the IKK
b
active site, compound
inhibitors
3k is a promising scaffold from which more potent IKK
b
can be derivatized. Because the 2-amino group was the optimal
substituents on the D ring, we tentatively fixed this group in this
region. Therefore, the next round of analogues was focused on the
incorporation of a variety of substituents on the A ring by exploring
the solvent exposed region observed in the molecular simulation.
The de novo design studies identified a variety of functional groups
to introduce on the aminopyrimidine core at this position. The
aminopyrimidine scaffold can be easily equipped with a variety of
groups by standard synthetic chemistry, allowing in-depth
ergies of the derivatives with respect to the IKK
b kinase. We
Fig. 1. (a) De novo hit scaffold (b) Design of scaffolds as IKKb inhibitors and opportunities for modification in the schematic active site of IKKb.

546
Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
Fig. 2. Predicted binding mode of compound 3k with IKKb (PDB ID: 4KIK).
structural modification for rapid exploration of the SAR profile.
With this agenda, a diverse set of substituents were investigated to
expand the structure-activity relationship by incorporating a sub-
stituent into the A ring. Scheme 1 illustrates the general synthetic
route for the preparation of compounds 3a-3s. Suzuki coupling
between 2,4-dichloropyrimidine (4) and 4-hydroxyphenylboronic
acid provided 4-(2-chloropyrimidin-4-yl)phenol (5) as a common
synthetic intermediate. To increase structural diversity at C2, a
.
Reagents and conditions: (a) Pd(dppf)Cl2 CH2Cl2, 4-hydroxyphenylboronic acid, Na2CO3, EtOH/toluene,
50 °C, 3 h; (b) aniline, cat. HCl, EtOH, 150 °C, 30 min, microwave; (c) 1-fluoro-2-nitrobenzene, K2CO3,
DMSO, 130 °C, 2 h; (d) Fe, NH4Cl, EtOH/H2O, 85 °C, 30 min.
Scheme 1. Preparation of 2-anilino-4-phenylpyrimidine derivatives of 3a.

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
547
variety of anilines were connected to C2 through amination under
acidic conditions. Next, nucleophilic aromatic substitution
employing 1-fluoro-2-nitrobenzene furnished 7a-7n and 3o-3s
equipped with four aryl rings. Final reduction of the nitro group
smoothly provided the target compounds 3a-3n.
critical in sustaining potency. In addition, the use of a bicyclic an-
iline (6-aminobenzothiazole) also led to reduced activity (3n,
IC50 ¼ 4.6
mM). With the sulfonamide group on the A ring, we tested
another polar nitro-substituent on the D ring instead of the amino
group, which increased the potency approximately 3-fold (3o,
IC50 ¼ 34 nM). Of particular significance is the observation that a
marked reduction in potency was observed by the incorporation of
The resulting compounds in which various substituents were
incorporated into the solvent exposed region, were tested to
measure the potency of their inhibition of IKK
prepared as the control to investigate the role of the substituents
M). The first series of compounds, which contain
electron donating substituents (e.g., methyl, methoxy and dime-
thylamino groups) at the para position, showed decreased po-
tencies as shown in Table 2(3b-3d). The enzyme potency of the
derivatives was also decreased when a chloro group was intro-
duced (3e). We observed that the potency was slightly increased
b
. Compound 3a was
mono-alkylated sulfonamide (3r, IC50 ¼ 1.1
(3j, IC50 ¼ 2.7 M) in the A ring, implying the importance of the
free-NH2 in this region for IKK inhibition. It appears that the 4-
mM) or a sulfonyl moiety
m
(3a, IC50 ¼ 11.8
m
b
sulfonamide group of 3k can form robust hydrogen bonding in-
teractions with Asp 103 and Lys 106. In addition, the amino group
on the D ring of 3k can form a hydrogen bond with Gly 27, and the
nitro group of 3o can have molecular interactions with Lys 147 and
Asp 166, which might explain the exceptional potency of these
compounds. Several alternative substituents to the sulfonamide
such as reversed sulfonamide (3s) and carbamate (3p and 3q) were
investigated, and the IC50 values of the resulting compounds were
determined. However, no compound bearing the sulfonamide
surrogates offered any advantage relative to the corresponding
sulfonamide compounds (Table 1). Cumulatively, derivatives
bearing the sulfonamide group appeared to be the superior in-
hibitors, suggesting that this group serves as a critical factor for
when a cyano (3f, IC50 ¼ 6.2
were installed at this position. The incorporation of a sulfonyl group
M). Moving the substituent
mM) or anilide group (3i, IC50 ¼ 4.2
mM)
enhanced the potency (3j, IC50 ¼ 2.7
m
from the para- to the meta-position of the A ring led to a decrease in
the inhibitory activity, probably due to the disruption of key
hydrogen bonds with Asp 103 and Lys 106 in IKK
comparison of the IC50 value of 3k (IC50 ¼ 97 nM) with that of 3m
(IC50 ¼ 1.6 M) clearly reveals that substitutions at the meta posi-
tion resulted in a substantial loss of the inhibitory activity against
IKK , indicating that the orientation of the sulfonamide group is
b. For example, a
m
potent activity against IKKb.
b
Table 1
Structure-activity relationship of derivatives 3a-3s against IKKb.
Compd
R
X
IC50
(
m
M)
Compd
R
X
IC50 (mM)
3a
H
NH2
11.8 2.5
3k
NH2
0.097 0.013
3b
CH3
NH2
>50
3l
NH2
1.1 0.4
3c
OCH3
NH2
NH2
12.0 4.3
3ma
3n
NH2
NH2
1.6 0.7
4.6 1.7
3d
N(CH3)2
28.7 12.8
3e
3f
Cl
NH2
NH2
34.1 14.2
6.2 1.7
3o
3p
NO2
NO2
0.034 0.011
CN
>20
3g
3h
3i
NH2
NH2
NH2
8.1 1.9
14.2 6.4
4.2 2.2
3q
3r
3s
NO2
NO2
NO2
>20
1.1 0.3
0.32 0.21
3j
NH2
2.7 0.8
a
Sulfonamide group was placed at the meta position.

548
Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
Table 2
In silico calculation results of our representative IKKb inhibitors.a
Compd
ADMET solubility
AlogP
Molecular solubility
MW
3k
3o
8a
ꢀ5.98
ꢀ6.41
ꢀ4.79
3.588
4.229
1.929
ꢀ6.724
ꢀ6.809
ꢀ4.811
433.48
463.47
385.44
a
Molecular solubility and AlogP were calculated employing Discovery studio.
2.3. Optimization for improving the cellular activity
Although this series of compounds present potent IKKb inhibi-
tion at the enzyme level, they are regarded as poor lead compounds
for drug discovery due to the poor solubility. Since the calculated
solubility values of this series are generally very low (e.g., 3k: -5.98,
3o: -6.41 from calculation with Discovery Studio), we decided to
make some structural modifications to enhance the solubility. It
was recently reported that the number of aromatic rings is highly
associated with solubility [24]. To expedite the optimization pro-
cess, a “drug-likeness” filter [25] was applied to identify molecules
with more suitable physicochemical properties for drug candidates.
When in silico solubility values of the synthesized compounds
were calculated from Discovery Studio, these values were within
the range of ꢀ7 and ꢀ6. Therefore, the modification of the aryl D
ring appears to be essential for enhancing the in silico solubility
values. For example, it was envisaged that the designed compound
8a bearing an aminoethanol substituent at the para position of the
C ring possesses more favorable physicochemical properties than
3k based on the calculated solubility values as shown in Table 2.
Due to the relationship between the number of aromatic rings and
solubility, we planned to remove the aryl D ring to facilitate cellular
uptake through increased solubility.
Fig. 3. Predicted binding mode of compound 8a with IKKb (PDB ID: 4KIK).
Table 3
Structure-activity relationship.
Recognizing the possibility that the aryl D ring of this series
might confer poor solubility, we examined the influence of the D
ring on the enzymatic potency by carrying out molecular simula-
tions of 8a. Although removing the D ring of 3k may be perceived as
a potential disadvantage by losing a hydrogen bonding interaction
with Gly 27, the excellent predicted fit in the binding site led us to
explore a variety of substituents. In addition, we noted that the
proper functional groups of the C ring, such as an aminoethoxy
group could be allocated to generate further stabilizing interactions
Compd
R
Y
IC50 (nM) In silico solubility LogDa
3k
97 45
ꢀ5.98
3.29
8a
65 18
ꢀ4.79
ꢀ1.39
through the formation of a hydrogen bond with Asp 166 in IKKb.
With the binding mode set in this manner, the aromatic D ring
appeared to be unnecessary for producing an inhibitory activity of
IKKb (Fig. 3).
8b
8c
79 32
ꢀ4.75
ꢀ5.42
e
e
Based on the observation, we hypothesized that the cellular
activities might be improved by replacing the aromatic D ring with
acyclic hydrophilic groups without significantly influencing the
enzyme inhibitory activities. To verify this assumption, a diverse set
of water-soluble moieties were employed in the region to enhance
the physicochemical properties, which is required for planned cell-
based assays. To build a variety of groups at the Y position (Table 3),
a systematic evaluation of the structural features necessary to
impart activity was performed. In general, this new series of com-
138 57
8d
8e
8f
Cl
19
6
ꢀ5.47
ꢀ4.39
ꢀ4.68
ꢀ3.69
ꢀ3.79
ꢀ3.81
e
NH2
CN
41 24
58 31
53 28
111 61
190 63
e
3.81
pounds possess potent IKKb inhibition; derivatives bearing ami-
noethoxy groups (8a-8c) showed IC50 values between 65 and
138 nM (Table 3). These results prompted us to investigate the
possibility of using simple functional groups. Intriguingly, this
8g
8h
8i
NH2
CN
ꢀ0.58
e
strategy has served well without compromising the IKK
activity. For example, both chloride (8d) and anilino derivatives
(8e) exhibited good potencies (8d, IC50 19 nM and 8e,
b inhibitory
m-CN
1.49
¼
IC50 ¼ 41 nM). The N-(2-hydroxyethyl)-sulfonamide group was well
tolerated (8g, IC50 ¼ 53 nM and 8h, IC50 ¼ 111 nM), and modest
increases in the molecular solubility values were obtained by in
a
Experimental logD values were generated at pH 7.4.

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
549
silico calculations with Discovery Studio (8e: ꢀ4.39, 8g: ꢀ3.69). We
also examined the significance of the para-substituents on the C4-
aryl ring and observed that the inhibitory activity was reduced
when the para-cyano group of compound 8h (IC50 ¼ 111 nM) was
moved to the meta-position (8i, IC50 ¼ 190 nM). The interactions of
aminoethanol derivatives 8a and 8g also moderately induced NO
reduction. The N-(2-hydroxyethyl)-sulfonamide derivatives 8b and
8i presented similar inhibitory patterns of NO production to those
of the corresponding sulfonamide series.
Because 8e displayed the best activity in both the enzymatic and
NO reduction cellular assays, compound 8e was further investi-
gated for its anti-inflammatory effects in RAW 264.7 cells. To ensure
that the NO reduction effect originated from the inhibition of the
synthesis of iNOS protein, the level of the iNOS protein was
8e in the ATP-binding site of IKKb are similar to those of 8a in that
the sulfonamide group maintains favorable hydrogen bonding with
the sidechain of Lys106 and Asp103 (Fig. 4). In addition, the NH2
moiety of 8e establishes an additional hydrogen bond with the
backbone aminocarbonyl oxygen of Gly22. As a consequence of the
process of design and synthesis, we were able to find potent in-
hibitors that are anticipated to possess more favorable physico-
chemical properties.
measured by Western blotting using b-actin as a loading control.
Raw 264.7 cells were treated with increasing concentrations of 8e
for 6 h before LPS stimulation, and we clearly observed that iNOS
was highly expressed upon treatment with LPS. Furthermore, the
induced synthesis of NOS by LPS was inhibited by 8e in a dose-
dependent manner (Fig. 6).
2.4. Mechanistic studies
2.4.2. Effects on the production of pro-inflammatory cytokines
In general, cell responses to inflammation include stimulation of
the production of a variety of cytokines, chemokines, adhesion
molecules, and inflammation mediators [5,9]. Because compound
8e successfully demonstrated the ability to suppress the generation
of NO in Raw 264.7 cells, we further investigated the effect of 8e on
the generation of the inflammation-related proteins. Raw
The IKKb signaling is transmitted through multiple pathways to
regulate cell inflammation by mediating a variety of inflammatory
responses [5,9]. The selected inhibitors displaying potent enzy-
matic and cellular activities were further investigated to determine
their anti-inflammatory effects in the RAW 264.7 cell line.
2.4.1. Effects on the generation of nitric oxide
Nitric oxide (NO) is a gaseous signaling molecule that is exces-
sively generated from inducible nitric oxide synthase (iNOS) during
264.7 cells were pre-treated with 8e (5
were stimulated by adding 100 ng/mL LPS for an additional 18 h,
which promoted the release of pro-inflammatory cytokines (IL-1
IL-6, and TNF- ) as well as other inflammatory mediators. We
observed that treatment with 8e (5 M) strongly inhibited the
expression of pro-inflammatory cytokines (IL-1 , IL-6, and TNF- ).
mM) for 6 h, and then, cells
a
,
inflammation [26]. Because activated NF-
inducing iNOS, the degree of NO reduction was measured to
identify promising IKK inhibitors having good cellular activities
[5,9]. The RAW 264.7 cells were incubated with three different
concentrations (1, 2, and 5 M) of compounds for 6 h and treated
kB is responsible for
a
m
b
a
a
In addition, the expressions of other inflammation-related proteins
including SiCAM-1, TIMP-1, and I-TAC were substantially sup-
pressed by 8e (Fig. 7). We next analyzed the effect of 8e on pro-
inflammatory cytokines by treating Raw 264.7 cells with various
concentrations of 8e, revealing that the expressions of IL-6 and
m
with 100 ng/mL LPS for 18 h. The generation of NO was compared to
the negative control. In general, derivatives incorporating the sul-
fonamide group on the A ring were slightly superior to the corre-
sponding N-(2-hydroxyethyl)-sulfonamide derivatives in terms of
the reduction of NO. In addition, molecular solubility appeared to
affect, in part, the degree of NO reduction as well. Compound 8d
possessing the lowest in silico molecular solubility did not show
any appreciable effect in NO reduction, whereas aniline derivative
8e showing the greatest in silico molecular solubility, substantially
suppressed the generation of NO after LPS activation (Fig. 5). The
TNF-a decreased upon treating macrophages with 8e in a dose-
dependent manner (Fig. 8).
2.4.3. Effects on the NF-
To ensure that the anti-inflammatory effect of 8e can be
attributed to the inhibition of the NF- B signaling pathway, the
inhibitory effect of 8e on the phosphorylation of crucial signaling
proteins in the NF- B pathway was assessed. Raw 264.7 cells were
treated with increasing concentrations of 8e for 6 h, 100 ng/mL LPS
was added for 30 min, and the phosphorylation levels of IKK and
were measured by Western blotting using -actin as a loading
control. We observed that treatment of 8e gradually decreased the
LPS-induced phosphorylation of both IKK and I in a dose
dependent manner (Fig. 9a). Moreover, the IKK -mediated phos-
phorylation of p65 of NF- B was suppressed by 8e in a dose-
dependent manner (Fig. 9b).
kB signaling pathway
k
k
b
IkB
a
b
b
kBa
b
k
Next, immunofluorescence staining with confocal microscopy
was performed to investigate the effect of 8e on the translocation of
NF-
Raw 264.7 cells with 8e and LPS, fixed cells were treated with the p-
NF- B antibody as well as a rhodamine-B-isothiocyanate-based
kB from the cytoplasm to the nucleus (Fig. 10). After treating
k
secondary antibody. The fixed cells were stained with the fluores-
cent DNA-binding agent, 4,6-diamidino-2-phenylindole (DAPI) to
visualize the nuclei. Consistent with the expectations, upon stim-
ulation of macrophages with LPS, p65 of NF-
located to the nucleus. In addition, we observed that treatment
with 8e (5 M) significantly inhibited the LPS-stimulated nuclear
translocation of p65 of NF- B in Raw 264.7 cells, further supporting
the conclusion that the suppression of the NF- B signaling pathway
kB was mainly trans-
m
k
k
induced the anti-inflammatory effect of 8e in LPS-stimulated Raw
Fig. 4. Calculated binding mode of 8e in the ATP-binding site of IKK
b
(PDB ID: 4KIK).
264.7 cells.

550
Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
Fig. 5. The NO reduction results of our representative IKKb inhibitors.
resulted in the enhanced cellular potency in terms of the anti-
inflammatory effects. Representative compounds exhibited strong
effect in NO reduction by inhibiting the synthesis of iNOS protein,
and strongly inhibited the expression of pro-inflammatory cyto-
kines (IL-1
on the phosphorylation in the NF-
a
, IL-6, and TNF-
a
). Moreover, the inhibitory effects of 8e
B pathway supported the
k
conclusion that the suppression of the NF-kB signaling pathway
induced the anti-inflammatory effect.
4. Experimental section
Fig. 6. Dose-dependent inhibition of NOS induction by 8e.
4.1. Cells
3. Conclusion
RAW 264.7 cell line was obtained from Korean Cell Line Bank
(Seoul, Korea). The cells were cultured in DMEM with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Grand
Island, NY, USA).
In conclusion, we extensively modified the structure of a hit
compound to optimize a series of aminopyrimidine-based IKK
inhibitors utilizing structural-based drug design strategy.
b
a
Through an in-depth evaluation of the structural features at the C2
and C4 positions along with modulating the D aryl ring of the
original hit compound, we have successfully explored the structure
activity relationships of the series to improve its physicochemical
properties and cellular activity. Intriguingly, the strategy that re-
places the aromatic D ring with acyclic hydrophilic groups has
4.2. Nitrite assay
The cells (1 ꢁ 105 cells/ml) were cultured in 48-well plates. After
incubating for 24 h, compounds were pre-treated for 6 h and then
100 ng/ml of LPS was added for another 18 h. NO synthesis was
measured by assaying the culture medium for nitrite, which is the
stable reaction product of nitric oxide with molecular oxygen, using
served well without compromising the IKKb inhibitory activity. The
introduction of simple water-solubilizing groups at this position
Fig. 7. Reduction of cytokine expression by 8e.

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
551
Fig. 8. Dose-dependent reduction of IL-6 and TNF-a by 8e.
Fig. 9. Dose-dependent inhibition of the NF-kB signaling pathway.
Fig. 10. Inhibition of the translocation of NF-kB from the cytosol to the nucleus.
Griess reagent (0.5% sulfanilic acid, 0.002% N-1-naphtyl ethlene-
diamine dihydrochloride, 14% glacial acetic acid) (Promega, Medi-
son, WI, USA).
2
m
M EDTA, and the following protease and phosphatase inhibitor:
aprotinin (10 mg/ml), leupeptin (10 mg/ml; ICN Biomedicals, Asse-
Relegem, Belgium), phenylmethylsulfonyl fluoride (1.72 M), NaF
(100 M), NaVO3 (500 M), and Na4P2O7 (500 mg/mL; Sigma-
m
m
m
Aldrich). Protein was separated by sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and then trans-
ferred onto nitrocellulose membranes. The nitrocellulose mem-
branes were immunostained with the appropriate primary
4.3. Western blotting
Total protein was extracted with lysis buffer containing 1% Ige-
pal CA-630, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol,

552
Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
antibodies followed by the secondary antibodies conjugated to
horseradish peroxidase. Antibody binding was detected with an
enhanced chemiluminescence reagent (Amersham Biosciences).
51% yield) as an intermediate. To a solution of 7a (60 mg,
0.16 mmol) in EtOH (6 mL) were added Fe (57 mg, 1.02 mmol) and
NH4Cl (7 mg, 0.13 mmol) in H2O (2 mL). The mixture was stirred
vigorously for 30 min at 85 ꢂC and monitored by TLC. After cooling
to room temperature, the mixture was diluted with EtOAc (50 mL),
and washed with brine (30 mL x 2). The combined organic phases
were dried over anhydrous MgSO4, filtered and concentrated in
vacuo. After removal of the solvent, the residue was purified by
flash chromatography (ethylacetate/hexanes) on silica gel to give
the desired product 3a (41 mg, 74% yield). mp 137e139 ꢂC. 1H NMR
Antibodies against p-IKK-
IKB were obtained from Cell signaling (Boston, MA, USA). iNOS
and -actin were purchased from Santa Cruze (Texas, USA).
b b, p-IKBa
(Tyr199) IKK- and (Ser32/36) and
a
b
4.4. Cytokine Array
Mouse Cytokine Array Panel A was purchased from R&D Sys-
tems. We treated Raw 264.7 cells with 8e (5
m
M) for 6 h. And then
(500 MHz, DMSO-d6)
d
9.63 (s, 1H), 8.49 (d, J ¼ 5.2 Hz, 1H), 8.15 (d,
the cells were stimulated with LPS (100 ng/ml) for another 18 h.
After blocking membranes with blocking buffer, membranes were
incubated with lysates extracting from Raw 264.7 cells which were
mixed with antibody cocktail overnight. The next day, the mem-
branes were washed with washing buffer for 3 times, incubated
with Streptavidin-HRP detection antibody and then detected with
an enhanced Chemi reagent A and Chemi reagent B. To analyze the
inflammation-related proteins, the relative expression of
inflammatory-proteins was determined following quantification of
scanned images by Image J.
J ¼ 8.8 Hz, 2H), 7.84 (d, J ¼ 7.8 Hz, 2H), 7.33e7.29 (m, 3H), 7.02 (d,
J ¼ 8.8 Hz, 2H), 6.98e6.93 (m, 2H), 6.91e6.85 (m, 2H), 6.60 (m, 1H),
4.98 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
d 163.1, 160.1, 160.1,
158.8, 140.8, 140.6, 140.5, 130.5, 128.6, 128.5, 125.7, 121.3, 121.2,
118.8,116.5,116.4,116.1,107.3. HRMS (ESIþ) m/z calcd for C22H19N4O
[M þ H]þ, 355.1553; found, 355.1552.
4.8.1. 4-[4-(2-Aminophenoxy)phenyl]-N-(p-tolyl)pyrimidin-2-
amine (3b)
Compound 3b was prepared (23 mg, 2 steps- 33% overall yield)
according to GP I from 4-Methylaniline instead of Aniline as a
starting material. mp 66e70 ꢂC. 1H NMR (500 MHz, DMSO-d6)
4.5. p-NF-kB p65 ELISA
d
9.51 (s, 1H), 8.47 (d, 1H), 8.13 (d, 2H), 7.70 (d, 2H), 7.29 (d, 1H), 7.11
(d, 2H), 7.01 (m, 3H), 6.90e6.84 (m, 2H), 6.59 (m, 1H), 4.97 (s, 2H),
2.25 (s, 3H). 13C NMR (150 MHz, DMSO-d6)
163.4, 160.6, 160.5,
Raw 264.7 cells were incubated with 100 ng/ml of LPS for
30 min after the exposure to various concentrations of 8e (1, 2 and
d
5
mM) for 6 h p-NF-
kB p65 in cytoplasm was using ELISA Kit (Cell
159.2, 141.3, 141.0, 138.5, 130.9, 130.5, 129.3, 129.0, 126.2, 121.6,
Signaling Technology, Inc., Beverly, MA, USA), according to the
manufacturer's instructions.
119.4, 116.9, 116.8, 116.5, 107.5, 20.8. HRMS (ESIþ) m/z calcd for
C
23H21N4O [M þ H]þ, 369.1710; found, 369.1711.
4.6. Cytokine assay (IL-6 and TNF-
a
)
4.8.2. 4-[4-(2-Aminophenoxy)phenyl]-N-(4-methoxyphenyl)
pyrimidin-2-amine (3c)
Compound 3c was prepared (45 mg, 2 steps- 33% overall yield)
according to GP I from 4-Methoxyaniline instead of Aniline as a
starting material. mp 126e128 ꢂC. 1H NMR (500 MHz, DMSO-d6)
Raw 264.7 cells were plated onto 24 well (4 ꢁ 105 cells/well)
overnight for the attachment. 8e (1, 2 and 5 mM) was pretreated
with Raw 264.7 cells for 6 h before adding 100 ng/ml of LPS for 18 h.
Cell-free supernatants were subsequently employed to pro-
inflammatory cytokine assays using
immunosorbent assay (ELISA) kit (R&D Systems), according to the
manufacturer's instructions.
d
9.43 (s, 1H), 8.44 (d, J ¼ 5.2 Hz, 1H), 8.12 (d, J ¼ 8.8 Hz, 2H), 7.71 (d,
a
mouse enzyme-linked
J ¼ 8.9 Hz, 2H), 7.26 (d, J ¼ 5.2 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.98
(m, 1H), 6.91e6.84 (m, 4H), 6.59 (m, 1H), 4.97 (s, 2H), 3.72 (s, 3H).
13C NMR (100 MHz, DMSO-d6)
d 163.0, 160.3, 160.0, 158.7, 154.1,
140.8,140.5,133.8,130.6,128.6,125.7, 121.2, 120.6,116.5,116.3, 116.1,
113.7, 106.8, 55.2. HRMS (ESIþ) m/z calcd for C23H21N4O2 [M þ H]þ,
385.1659; found, 385.1670.
4.7. Immunofluorescence
Raw 264.7 cells were pretreated with 5 mM of 8e for 6 h before
adding 100 ng/ml of LPS for 30 min. Washed with PBS twice and
fixed in an acetone: methanol solution (1:1) for 10 min at ꢀ20 ꢂC.
The fixed cells were washed and blocked in blocking buffer for 1 h
4.8.3. N4-{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}-N1,N1-
dimethyl-benzene-1,4-diamine (3d)
Compound 3d was prepared (7 mg, 12% overall yield of 2 steps)
at room temperature and then incubated overnight with p-NF-
k
B
according to GP
instead of Aniline as a starting material. mp 70e75 ꢂC. 1H NMR
(500 MHz, DMSO-d6) 9.25 (s, 1H), 8.41 (d, 1H), 8.12 (d, 2H), 7.60 (d,
I from N4,N4-Dimethylbenzene-1,4-diamine
antibody (1:50; Santa Cruz Biotechnology, Inc.) at 4 ꢂC. Before
incubating with mouse RITC secondary antibody (1:100, Dianova,
Germany) for 1 h at room temperature, the fixed cells were washed
with PBS for several times. The fixed cells were also stained with
4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. After
washing with PBS twice, the slides were covered with DABCO
(Sigma-Aldrich, St. Louis, MO) and then viewed with a confocal
laser scanning microscope (Olympus, Tokyo, Japan).
d
2H), 7.22 (d, 1H), 7.01e6.96 (m, 3H), 6.90e6.83 (m, 2H), 6.73 (d, 2H),
6.59 (m,1H), 4.97 (s, 2H), 2.84 (s, 6H). 13C NMR (150 MHz, DMSO-d6)
d
163.3, 160.8, 160.4, 159.1, 146.6, 141.3, 141.0, 131.1, 131.1, 128.9,
126.1, 121.6, 121.1, 116.9, 116.7, 116.5, 113.4, 106.7, 41.2. HRMS (ESIþ)
m/z calcd for C24H23N5NaO [M þ Na]þ, 420.1795; found, 420.1794.
4.8.4. 4-[4-(2-Aminophenoxy)phenyl]-N-(4-chlorophenyl)
pyrimidin-2-amine (3e)
4.8. Preparation of Compounds
Compound 3e was prepared (29 mg, 38% overall yield of 2 steps)
according to GP I from 4-Chloroaniline instead of Aniline as a
starting material. mp 64e68 ꢂC. 1H NMR (500 MHz, DMSO-d6)
General Procedure I: A reaction mixture of 2-Chloro-4-[4-(2-
nitrophenoxy)phenyl]pyrimidine (150 mg, 0.46 mmol), Aniline
(42
m
g, 0.46 mmol) and catalytic amount of 1 N HCl (a few drops) in
d
9.80 (d, 1H), 8.52 (t, 1H), 8.15 (m, 2H), 7.88 (m, 2H), 7.36 (m, 3H),
7.04e6.97 (m, 3H), 6.92e6.84 (m, 2H), 6.60 (m, 1H), 4.98 (d, 2H). 13
NMR (150 MHz, DMSO-d6) 163.1, 160.2, 159.9, 158.8, 140.8, 140.5,
Ethanol (5 mL) was heated for 2 h at 160 ꢂC under microwave
irradiation. After cooling and evaporation of the mixture, the
resulting solid was washed with methanol (20 mL) to give 4-[4-(2-
Nitrophenoxy)phenyl]-N-phenyl-pyrimidin-2-amine (7a) (89 mg,
C
d
139.6, 130.3, 128.7, 128.4, 125.8, 124.7, 121.2, 120.2, 116.4, 116.3,
116.0, 107.7. HRMS (ESIþ) m/z calcd for C22H18ClN4O [M þ H]þ,

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
553
389.1164; found, 389.1162.
128.1, 125.8, 121.2, 118.0, 116.5, 116.4, 116.1, 108.6, 44.0. HRMS (ESIþ)
m/z calcd for C23H20N4NaO3S [M þ Na]þ, 455.1148; found, 455.1168.
4.8.5. 4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino}
benzonitrile (3f)
Compound 3f was prepared (9 mg, 16% overall yield of 2 steps)
according to GP I from 4-Aminobenzonitrile instead of Aniline as a
starting material. mp 210e214 ꢂC. 1H NMR (500 MHz, DMSO-d6)
4.8.10. 4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino}
benzenesulfonamide (3k)
Compound 3k was prepared (86 mg, 52% overall yield of 2 steps)
according to GP I from 4-Aminobenzenesulfonamide instead of
Aniline as a starting material. mp 152e155 ꢂC. 1H NMR (500 MHz,
d
10.22 (s, 1H), 8.59 (d, 1H), 8.17 (d, 2H), 8.04 (d, 2H), 7.75 (d, 2H),
7.48 (d, 1H), 7.04e6.97 (m, 3H), 6.91e6.84 (m, 2H), 6.60 (m, 1H),
4.98 (s, 2H). 13C NMR (150 MHz, DMSO-d6)
163.8, 160.8, 160.0,
DMSO-d6)
d
10.07 (s, 1H), 8.56 (d, J ¼ 5.3 Hz, 1H), 8.17 (d, J ¼ 8.8 Hz,
d
2H), 8.01 (d, J ¼ 8.8 Hz, 2H), 7.78 (d, J ¼ 8.8 Hz, 2H), 7.43 (d,
J ¼ 5.3 Hz, 1H), 7.19 (s, 2H), 7.03 (d, J ¼ 8.8, 2H), 6.99 (m, 1H),
6.91e6.85 (m, 2H), 6.60 (m, 1H), 4.98 (s, 2H). 13C NMR (100 MHz,
159.3,145.5,141.3,140.9,133.5,130.4,129.2,126.3,121.7,120.1,118.8,
116.9, 116.8, 116.5, 109.2, 102.7. HRMS (ESIþ) m/z calcd for
C
23H18N5O [M þ Na]þ, 380.1506; found, 380.1509.
DMSO-d6) d 163.3, 160.3, 159.8, 158.9, 143.8, 140.8, 140.5, 136.1,
130.2,128.8,126.7, 125.8, 121.2, 117.9, 116.5,116.4, 116.1, 108.4. HRMS
(ESIþ) m/z calcd for C22H19N5NaO3S [M þ Na]þ, 456.1101; found,
456.1121.
4.8.6. 1-{4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}
amino}phenyl}ethanone (3g)
Compound 3g was prepared (51 mg, 35% overall yield of 2 steps)
according to GP I from 1-(4-Aminophenyl)ethanone instead of
Aniline as a starting material. mp 87e89 ꢂC. 1H NMR (500 MHz,
4.8.11. 4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino}-
N-thiazol-2-yl-benzenesulfonamide (3l)
DMSO-d6)
d
10.09 (s, 1H), 8.55 (d, J ¼ 5.2 Hz, 1H), 8.16 (d, J ¼ 8.7 Hz,
Compound 3l was prepared (7 mg, 10% overall yield of 2 steps)
according to GP I from 4-Amino-N-thiazol-2-yl-benzenesulfona-
mide instead of Aniline as a starting material. mp 110e115 ꢂC. 1H
2H), 7.98 (d, J ¼ 8.7 Hz, 2H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.42 (d,
J ¼ 5.2 Hz, 1H), 7.01 (d, J ¼ 8.7 Hz, 2H), 6.97 (m, 1H), 6.89e6.83 (m,
2H), 6.58 (m, 1H), 4.96 (s, 2H), 2.49 (s, 3H). 13C NMR (100 MHz,
NMR (500 MHz, DMSO-d6)
d 12.62 (br, 1H), 10.06 (s, 1H), 8.56 (d,
DMSO-d6)
d
163.3, 160.3, 159.7, 158.9, 145.3, 140.8, 140.5, 130.1,
1H), 8.17 (d, 2H), 7.98 (d, 2H), 7.74 (d, 2H), 7.44 (d, 1H), 7.23 (d, 1H),
7.03 (d, 2H), 6.99 (m, 1H), 6.91e6.85 (m, 2H), 6.80 (d, 1H), 6.61 (m,
1H), 5.04 (br, 1H), 4.78 (br, 1H). 13C NMR (100 MHz, DMSO-d6)
129.7, 129.5, 128.8, 125.8, 121.2, 117.5, 116.5, 116.4, 116.1, 108.4, 26.3.
HRMS (ESIþ) m/z calcd for C24H21N4O2 [M þ H]þ, 397.1659; found,
397.1673.
d
168.51, 163.26, 160.24, 159.69, 158.87, 144.03, 140.81, 140.49,
133.99, 130.11, 128.79, 126.90, 125.76, 121.18, 117.78, 116.47, 116.43,
4.8.7. 4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino}
benzamide (3h)
Compound 3h was prepared (36 mg, 21% overall yield of 2 steps)
according to GP I from 4-Aminobenzamide instead of Aniline as a
starting material. mp 155e157 ꢂC. 1H NMR (500 MHz, DMSO-d6)
116.07, 112.42, 108.34, 107.91. HRMS (ESIþ) m/z calcd for
C
25H20N6NaO3S2 [M þ Na]þ, 539.0931; found, 539.0944.
4.8.12. 3-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino}
benzenesulfonamide (3m)
d
9.93 (s, 1H), 8.55 (d, J ¼ 5.1 Hz, 1H), 8.16 (d, J ¼ 8.5 Hz, 2H), 7.91 (d,
Compound 3m was prepared (4.5 mg, 9% overall yield of 2 steps)
according to GP I from 3-Aminobenzenesulfonamide instead of
Aniline as a starting material. mp 156e159 ꢂC. 1H NMR (500 MHz,
J ¼ 8.5 Hz, 2H), 7.85 (d, J ¼ 8.6 Hz, 2H), 7.80 (br, 1H), 7.40 (d,
J ¼ 5.1 Hz, 1H), 7.16 (br, 1H), 7.02 (d, J ¼ 8.6, 2H), 6.98 (m, 1H),
6.91e6.84 (m, 2H), 6.60 (m, 1H), 4.98 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) d 10.00 (s, 1H), 8.70 (s, 1H), 8.55 (d, 1H), 8.23 (d, 2H), 7.85
DMSO-d6)
d
167.6, 163.2, 160.2, 159.9, 158.9, 143.4, 140.8, 140.5,
(d, 1H), 7.49 (m, 1H), 7.43 (m, 2H), 7.33 (s, 2H), 7.02e6.97 (m, 3H),
130.3, 128.7, 128.3, 126.6, 125.8, 121.2, 117.5, 116.5, 116.4, 116.1, 108.0.
HRMS (ESIþ) m/z calcd for C23H19N5NaO2 [M þ Na]þ, 420.1431;
found, 420.1436.
6.91e6.85 (m, 2H), 6.61 (m, 1H), 4.98 (s, 2H). 13C NMR (100 MHz,
DMSO-d6)
d 163.06, 160.25, 159.89, 159.00, 144.51, 141.08, 140.83,
140.52, 130.10, 129.13, 128.88, 125.80, 121.54, 121.21, 118.25, 116.52,
116.35, 116.11, 115.57, 107.88. HRMS (ESIþ) m/z calcd for
4.8.8. N-{4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}
amino}phenyl}acetamide (3i)
C
22H19N5NaO3S [M þ Na]þ, 456.1101; found, 456.1137.
Compound 3i was prepared (11 mg, 15% overall yield of 2 steps)
according to GP I from N-(4-Aminophenyl)acetamide instead of
Aniline as a starting material. mp 121e125 ꢂC. 1H NMR (500 MHz,
4.8.13. N-{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}-1,3-
benzothiazol-6-amine (3n)
Compound 3n was prepared (23 mg, 38% overall yield of 2 steps)
according to GP I from 1,3-Benzothiazol-6-amine instead of Aniline
as a starting material. mp 137e141 ꢂC. 1H NMR (500 MHz, CDCl3)
DMSO-d6)
d 9.82 (s, 1H), 9.53 (s, 1H), 8.47 (d, 1H), 8.13 (d, 2H), 7.71
(d, 2H), 7.49 (d, 2H), 7.30 (d, 1H), 7.01 (d, 2H), 6.99 (m, 1H),
6.90e6.83 (m, 2H), 6.59 (m, 1H), 4.97 (s, 2H), 2.02 (s, 3H). 13C NMR
d
8.87 (s, 1H), 8.66 (br, 1H), 8.47 (br, 1H), 8.05 (m, 3H), 7.70 (s, 1H),
7.55 (d, 1H), 7.15 (m, 1H), 7.08 (m, 3H), 6.97e6.87 (m, 2H), 6.78 (m,
1H), 3.82 (s, 2H). 13C NMR (150 MHz, DMSO-d6)
163.7, 160.6, 160.4,
(100 MHz, DMSO-d6) d 167.75,163.04,160.14,160.03,158.74,140.78,
140.56, 135.95, 133.32, 130.51, 128.59, 125.69, 121.11, 119.48, 119.28,
d
116.45, 116.37, 116.04, 107.04, 23.85. HRMS (ESIþ) m/z calcd for
159.2, 153.7, 148.4, 141.3, 140.9, 139.1, 134.7, 130.8, 129.1, 126.2, 123.1,
121.6, 119.4, 116.9, 116.8, 116.5, 110.8, 108.3. HRMS (ESIþ) m/z calcd
for C23H18N5OS [M þ H]þ, 412.1227; found, 412.1221.
C
24H21N5NaO2 [M þ Na]þ, 434.1587; found, 434.1602.
4.8.9. 4-[4-(2-Aminophenoxy)phenyl]-N-[4-(methylsulfonyl)
phenyl]pyrimidin-2-amine (3j)
Compound 3j was prepared (51 mg, 19% overall yield of 2 steps)
according to GP I from 4-(Methylsulfonyl)aniline instead of Aniline
as a starting material. mp 100e104 ꢂC. 1H NMR (500 MHz, DMSO-
4.8.14. 4-{{4-[4-(2-Nitrophenoxy)phenyl]pyrimidin-2-yl}amino}
benzenesulfonamide (3o)
Compound 3o was prepared (243 mg, 57% yield) according to GP
I from 4-Aminobenzenesulfonamide instead of Aniline as a starting
material without final reduction of the nitro group. mp
d6)
d
10.21 (s, 1H), 8.58 (d, J ¼ 5.2 Hz, 1H), 8.18 (d, J ¼ 8.6 Hz, 2H),
8.10 (d, J ¼ 8.7 Hz, 2H), 7.86 (d, J ¼ 8.7 Hz, 2H), 7.46 (d, J ¼ 5.2 Hz,
1H), 7.03 (d, J ¼ 8.6 Hz, 2H), 6.99 (m, 1H), 6.91e6.85 (m, 2H), 6.60
(m, 1H), 4.98 (s, 2H), 3.15 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
204e206 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 10.11 (s, 1H), 8.60 (d,
J ¼ 2.6 Hz, 1H), 8.24 (d, J ¼ 7.0 Hz, 2H), 8.12 (d, J ¼ 7.6 Hz, 1H), 8.00
(d, J ¼ 7.2 Hz, 2H), 7.79e7.75 (m, 3H), 7.48e7.45 (m, 2H), 7.34 (m,
d
163.3, 160.3, 159.6, 158.9, 145.4, 140.8, 140.5, 132.2, 130.0, 128.8,
1H), 7.20 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
d 162.9, 159.8,

554
Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
159.2, 158.6, 148.1, 143.7, 141.7, 136.1, 135.4, 132.1, 129.2, 126.7, 125.9,
purified by flash chromatography (Ethylacetate/Hexanes) on silica
gel to give 4-(2-Chloropyrimidin-4-yl)phenol (5) (1.08 g, 78% yield).
A mixture of 5 (1.08 g, 5.23 mmol), 4-Aminobenzenesulfonamide
(900 mg, 5.23 mmol) and catalytic amount of 1 N HCl (30 drops)
in Ethanol (13 mL) was heated for 2 h at 160 ꢂC under microwave
irradiation. After cooling and evaporation of the mixture, the
resulting solid was washed with Methanol (50 mL) to give 4-{[4-(4-
Hydroxyphenyl)pyrimidin-2-yl]amino}benzenesulfonamide (6k)
as a key intermediate (1.77 g, 99% yield). To a stirred solution of 4-
{[4-(4-Hydroxyphenyl)pyrimidin-2-yl]amino}benzenesulfonamide
(100 mg, 0.29 mmol), PPh3 (115 mg, 0.44 mmol) and tert-Butyl (2-
125.3, 122.4, 118.1, 117.9, 108.6. HRMS (ESIþ) m/z calcd for
C
22H17N5NaO5S [M þ Na]þ, 486.0843; found, 486.0876.
4.8.15. Methyl N-{4-{{4-[4-(2-nitrophenoxy)phenyl]pyrimidin-2-
yl}amino}phenyl}carbamate (3p)
Compound 3p was prepared (2 mg, 4% yield) according to GP I
from methyl N-(4-Aminophenyl)carbamate instead of Aniline as a
starting material without final reduction of the nitro group. mp
168e172 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 9.55 (s, 1H), 9.49 (br,
1H), 8.51 (d, 1H), 8.21 (d, 2H), 8.13 (d, 1H), 7.76 (m, 1H), 7.70 (d, 2H),
7.46 (m, 1H), 7.38e7.33 (m, 4H), 7.21 (d, 2H), 3.65 (s, 3H). 13C NMR
hydroxyethyl)carbamate (68
mL, 0.44 mmol) in THF (3 mL) was
(150 MHz, DMSO-d6)
d
163.1, 160.6, 159.4, 158.7, 154.5, 148.7, 142.0,
added DIAD (87
m
L, 0.44 mmol) at 0 ꢂC. After 10 min, the mixture
135.9, 135.7, 133.5, 132.9, 129.4, 126.2, 125.6, 122.6, 120.0, 119.2,
was stirred for 48 h at room temperature. The resulting solution
was monitored by TLC and was diluted with EtOAc (30 mL). The
organic layer was washed with water and brine, dried over MgSO4,
and concentrated in vacuo. After removal of the solvent, the residue
was purified by flash chromatography (Ethylacetate/Hexanes) on
silica gel to give crude product. A mixture of crude intermediate
and 1.25 M HCl in MeOH (2 mL) was stirred for 1 h at 50 ꢂC and the
resulting solid was washed with excess MeOH to afford the product
8a (71 mg, 58% yield). mp 265e267 ꢂC. 1H NMR (500 MHz, DMSO-
118.6, 107.7, 51.9. HRMS (ESIþ) m/z calcd for
[M þ H]þ, 480.1278; found, 480.1291.
C24H19N5NaO5
4.8.16. Ethyl N-{4-{{4-[4-(2-nitrophenoxy)phenyl]pyrimidin-2-yl}
amino}phenyl}carbamate (3q)
Compound 3q was prepared (30 mg, 64% yield) according to GP I
from Ethyl N-(4-Aminophenyl)carbamate instead of Aniline as a
starting material without final reduction of the nitro group. mp
179e181 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d
9.54 (s, 1H), 9.45 (br,
d6)
d
10.15 (s, 1H), 8.57 (d, J ¼ 4.8 Hz, 1H), 8.34 (br, 3H), 8.20 (d,
1H), 8.50 (d, 1H), 8.20 (d, 2H), 8.12 (d, 1H), 7.76 (m, 1H), 7.69 (d, 2H),
J ¼ 8.0 Hz, 2H), 7.99 (d, J ¼ 7.2 Hz, 2H), 7.77 (d, J ¼ 7.8 Hz, 2H), 7.49
(d, J ¼ 4.7 Hz, 1H), 7.25 (br, 2H), 7.17 (d, J ¼ 7.7 Hz, 2H), 4.30 (br, 2H),
7.45 (m, 1H), 7.38e7.32 (m, 4H), 7.21 (d, 2H), 4.10 (m, 2H), 1.24 (t,
3H). 13C NMR (150 MHz, DMSO-d6)
d
163.1, 160.6, 159.4, 158.7, 154.1,
3.24 (br, 2H). 13C NMR (100 MHz, DMSO-d6)
d 164.1, 160.6, 158.7,
148.7, 142.0, 135.8, 135.7, 133.6, 133.0, 129.4, 126.2, 125.6, 122.6,
157.5, 143.3, 136.6, 129.1, 129.0, 126.7, 118.3, 115.2, 108.2, 64.6, 38.2.
HRMS (ESIþ) m/z calcd for C18H20N5O3S [M þ H]þ, 386.1281; found,
386.1293.
120.0, 119.1, 118.6, 107.7, 60.4, 15.0. HRMS (ESIþ) m/z calcd for
C
25H21N5NaO5 [M þ Na]þ, 494.1435; found, 494.1454.
4.8.17. N-Methyl-4-{{4-[4-(2-nitrophenoxy)phenyl]pyrimidin-2-yl}
amino}benzenesulfonamide (3r)
4.8.19. 4-{{4-{4-[2-(Dimethylamino)ethoxy]phenyl}pyrimidin-2-
yl}amino}benzenesulfonamide hydrochloride (8b)
Compound 3r was prepared (92 mg, 42% yield) according to GP I
from 4-Amino-N-methyl-benzenesulfonamide instead of Aniline as
a starting material without final reduction of the nitro group. mp
4-{[4-(4-Hydroxyphenyl)pyrimidin-2-yl]amino}benzenesulfo-
namide (6k) was prepared from GP II and it was used as a starting
material to synthesize the desired compound 8b. To a stirred so-
lution of 4-{[4-(4-hydroxyphenyl)pyrimidin-2-yl]amino}benzene-
sulfonamide (100 mg, 0.292 mmol) and K2CO3 (202 mg, 1.46 mmol)
in DMF (3 mL) were added KI (14 mg, 0.074 mmol) and 2-Chloro-
N,N-dimethylethanamine hydrochloride (63 mg, 0.438 mmol) at
148e152 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 10.17 (s, 1H), 8.61 (d,
J ¼ 5.0 Hz, 1H), 8.25 (d, J ¼ 8.6 Hz, 2H), 8.12 (d, J ¼ 7.0 Hz, 1H), 8.06
(d, J ¼ 8.6 Hz, 2H), 7.78e7.72 (m, 3H), 7.50e7.44 (m, 2H), 7.34 (d,
J ¼ 8.0 Hz, 1H), 7.24e7.20 (m, 3H), 2.40 (d, J ¼ 4.9 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6)
d
162.9, 159.7, 159.1, 158.6, 148.1, 144.3, 141.7,
0
ꢂC. After 10 min, the mixture was heated for 2 h at 90 ꢂC. The
135.3,132.1, 130.8,129.2,127.8, 125.8, 125.3, 122.4, 118.1, 118.0, 108.7,
28.7. HRMS (ESIþ) m/z calcd for C23H19N5NaO5S [M þ Na]þ,
500.0999; found, 500.1026.
resulting solution was cooled to room temperature and was diluted
with Dichloromethane (30 mL). The organic layer was washed with
saturated NH4Cl solution and brine, dried over MgSO4, and
concentrated in vacuo. The reaction mixture was treated with
1.25 M HCl (2 mL) in MeOH and the resulting solid was washed
with excess MeOH to afford the product 8b (21 mg, 16% yield). mp
4.8.18. N-{4-{{4-[4-(2-Nitrophenoxy)phenyl]pyrimidin-2-yl}
amino}phenyl}methanesulfonamide (3s)
Compound 3s was prepared (24 mg, 51% yield) according to GP I
from N-(4-Aminophenyl)methanesulfonamide instead of Aniline as
a starting material without final reduction of the nitro group. mp
110e114 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 10.69 (br, 1H), 10.10 (s,
1H), 8.57 (d, J ¼ 5.2 Hz, 1H), 8.20 (d, J ¼ 8.5 Hz, 2H), 7.99 (d,
J ¼ 8.6 Hz, 2H), 7.76 (d, J ¼ 8.6 Hz, 2H), 7.48 (d, J ¼ 5.1 Hz, 1H),
7.20e7.18 (m, 4H), 4.47 (br, 2H), 3.54 (br, 2H), 2.85 (s, 6H). 13C NMR
194e196 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 9.67 (s, 1H), 9.44 (s,
1H), 8.52 (d, 1H), 8.21 (d, 2H), 8.12 (d, 1H), 7.80e7.75 (m, 3H), 7.45
(100 MHz, DMSO-d6) d 163.8, 160.1, 159.0, 158.0, 143.4, 136.4, 129.2,
(m, 1H), 7.37e7.32 (m, 2H), 7.19 (m, 4H), 2.92 (s, 3H). 13C NMR
128.9, 126.6, 118.1, 115.2, 108.2, 62.6, 55.0, 42.7. HRMS (ESIþ) m/z
calcd for C20H24N5O3S except for HCl [M þ H]þ, 414.1594; found,
414.1609.
(100 MHz, DMSO-d6) d 162.74, 160.10, 159.03, 158.36, 148.20, 141.61,
137.52, 135.33, 132.42, 131.66, 129.06, 125.82, 125.21, 122.26, 121.96,
119.67, 118.13, 107.58. HRMS (ESIþ) m/z calcd for C23H19N5NaO5S
[M þ Na]þ, 500.0999; found, 500.1035.
4.8.20. Tert-Butyl N-{2-{4-[2-(4-sulfamoylanilino)pyrimidin-4-yl]
phenoxy}ethyl}carbamate (8c)
General Procedure II: A solution of 2,4-Dichloropyrimidine
(2.00 g, 13.42 mmol), 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-
2-yl)phenol (1.48 g, 6.71 mmol), 2 N Na2CO3 (10 mL) and Pd(dppf)
Cl$2CH2Cl2 (1.64 g, 2.01 mmol) in Toluene/EtOH (v/v ¼ 9/1, 160 mL)
was heated to 50 ꢂC for 3 h and monitored by TLC. After cooling to
room temperature, the mixture was poured into water (300 mL),
and extracted with EtOAc (500 mL x 2). The combined organic
phases were dried over anhydrous MgSO4, filtered and concen-
trated in vacuo. After removal of the solvent, the residue was
Compound 8c was prepared (80 mg, 3 steps- 64% overall yield)
according to GP II without final deprotection of the Boc group. mp
179e182 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d
10.04 (s, 1H), 8.55 (d,
1H), 8.16 (d, 2H), 8.00 (d, 2H), 7.76 (d, 2H), 7.45 (d, 1H), 7.19 (s, 2H),
7.11 (d, 2H), 7.06 (m, 1H), 4.06 (m, 2H), 3.33 (br, 2H), 1.39 (s, 9H). 13
NMR (150 MHz, DMSO-d6) 163.8, 161.3, 160.1, 159.2, 156.2, 144.3,
C
d
136.4, 129.2, 129.1, 127.0, 118.2, 115.3, 108.6, 78.3, 67.1, 40.5, 28.7.
HRMS (ESIþ) m/z calcd for C23H27N5NaO5S [M þ Na]þ, 508.1625;

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556
555
found, 508.1623.
hydroxyethyl)benzenesulfonamide (25 mg, 0.12 mmol) and cata-
lytic amount of 1 N HCl (a few drops) in n-Butanol (2 mL) was
heated for 2 h at 180 ꢂC under microwave irradiation. After cooling
and evaporation of the mixture, the side product was removed by
washing with Methanol (10 mL) and the filtrate was purified by
flash chromatography (Dichloromethane/Methanol) on silica gel to
give the desired product 8g (5 mg,11% yield). mp 83e86 ꢂC. 1H NMR
General Procedure III: A solution of 2,4-Dichloropyrimidine
(575 mg, 3.86 mmol), 4-Chlorophenylboronic acid (600 mg,
3.86 mmol), 2 N Na2CO3 (3.86 mL, 7.72 mmol) and Pd(PPh3)4
(45 mg, 0.039 mmol) in Acetonitrile (13 mL) was heated to 90 ꢂC for
4 h and monitored by TLC. After cooling to room temperature, the
mixture was poured into water (30 mL), and extracted with EtOAc
(50 mL x 2). The combined organic phases were dried over anhy-
drous MgSO4, filtered and concentrated in vacuo. After removal of
the solvent, the residue was purified by flash chromatography
(Ethylacetate/Hexanes) on silica gel to give 2-Chloro-4-(4-
chlorophenyl)pyrimidine (669 mg, 77% yield). A mixture of the
intermediate (100 mg, 0.444 mmol), 4-Aminobenzenesulfonamide
(76 mg, 0.444 mmol) and catalytic amount of 1 N HCl (a few drops)
in Ethanol (2 mL) was heated for 2 h at 160 ꢂC under microwave
irradiation. After cooling and evaporation of the mixture, the
resulting solid was washed with Methanol (10 mL) to give the
product (8d) as white solid (105 mg, 66% yield). 1H NMR (500 MHz,
(500 MHz, DMSO-d6) d 9.93 (s,1H), 8.43 (d,1H), 8.03 (d, 2H), 7.93 (d,
2H), 7.72 (d, 2H), 7.35 (br, 1H), 7.30 (d, 1H), 6.67 (d, 2H), 5.79 (s, 2H),
4.67 (m, 1H), 3.36 (m, 2H), 2.78 (m, 2H). 13C NMR (150 MHz, DMSO-
d6) d 164.5, 160.0,158.3,152.9, 152.4,145.0,128.9,128.0,125.9,118.2,
113.9, 113.1, 60.3, 45.5. HRMS (ESIþ) m/z calcd for C18H19N5NaO3S
[M þ Na]þ, 408.1101; found, 408.1098.
4.8.24. 4-{[4-(4-Cyanophenyl)pyrimidin-2-yl]amino}-N-(2-
hydroxyethyl)benzenesulfonamide (8h)
Compound 8h was prepared (24 mg, 16% overall yield of 2 steps)
according to GP III from (4-Cyanophenyl)boronic acid instead of 4-
Chlorophenylboronic acid as a starting material of the first step and
4-Amino-N-(2-hydroxyethyl)benzenesulfonamide instead of 4-
Aminobenzenesulfonamide as a starting material of the second
DMSO-d6)
d
10.15 (s, 1H), 8.63 (d, J ¼ 5.1 Hz, 1H), 8.21 (d, J ¼ 8.4 Hz,
2H), 7.99 (d, J ¼ 8.7 Hz, 2H), 7.78 (d, J ¼ 8.6 Hz, 2H), 7.63 (d,
J ¼ 8.4 Hz, 2H), 7.52 (d, J ¼ 5.1 Hz, 1H), 7.21 (s, 2H). 13C NMR
step. mp 271e274 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 10.28 (s, 1H),
(100 MHz, DMSO-d6) d 162.7, 159.9, 159.5, 143.8, 136.3, 136.0, 135.4,
129.2, 128.9, 126.8, 118.1, 109.0. HRMS (ESIþ) m/z calcd for
8.72 (d, 1H), 8.38 (d, 2H), 8.07e8.01 (m, 4H), 7.75 (d, 2H), 7.64 (d,
1H), 7.40 (t, 1H), 4.67 (t, 1H), 3.37 (m, 2H), 2.78 (m, 2H). 13C NMR
C
16H13ClN4NaO2S [M þ Na]þ, 383.0340; found, 383.0343.
(150 MHz, DMSO-d6)
d 162.4, 160.3, 160.2, 144.4, 141.1, 133.4, 132.8,
4.8.21. 4-{[4-(4-Aminophenyl)pyrimidin-2-yl]amino}
benzenesulfonamide (8e)
128.3, 128.1, 119.0, 118.6, 113.7, 110.2, 60.4, 45.5. HRMS (ESIþ) m/z
calcd for C19H17N5NaO3S [M þ Na]þ, 418.0944; found, 418.0935.
Compound 8e was prepared (43 mg, 21% overall yield of 2 steps)
according to GP III from 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-
2-yl)aniline instead of 4-Chlorophenylboronic acid as a starting
material. Also, the reaction condition of Suzuki Coupling was
changed from Na2CO3 at 90 ꢂC to NaHCO3 at 80 ꢂC. mp 301e304 ꢂC.
4.8.25. 4-{[4-(3-Cyanophenyl)pyrimidin-2-yl]amino}-N-(2-
hydroxyethyl)benzenesulfonamide (8i)
Compound 8i was prepared (70 mg, 23% overall yield of 2 steps)
according to GP III from (3-Cyanophenyl)boronic acid instead of 4-
Chlorophenylboronic acid as a starting material of the first step and
4-Amino-N-(2-hydroxyethyl)benzenesulfonamide instead of 4-
Aminobenzenesulfonamide as a starting material of the second
1H NMR (500 MHz, DMSO-d6)
d
9.88 (s, 1H), 8.42 (d, J ¼ 5.3 Hz, 1H),
7.99 (d, J ¼ 7.6 Hz, 2H), 7.92 (d, J ¼ 7.5 Hz, 2H), 7.75 (d, J ¼ 7.7 Hz, 2H),
7.28 (d, J ¼ 5.1 Hz, 1H), 7.16 (s, 2H), 6.66 (d, J ¼ 7.5 Hz, 2H), 5.80 (br,
2H). 13C NMR (100 MHz, DMSO-d6)
d 164.0,159.6,158.0,152.0,144.1,
step. mp 239e241 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d 10.25 (s, 1H),
135.7, 128.5, 126.6, 123.0, 117.6, 113.5, 107.1. HRMS (ESIþ) m/z calcd
8.71 (d, 1H), 8.63 (m, 1H), 8.54 (m, 1H), 8.06e8.01 (m, 3H), 7.80 (t,
for C16H15N5NaO2S [M þ Na]þ, 364.0839; found, 364.0829.
1H), 7.75 (d, 2H), 7.66 (d, 1H), 7.40 (t, 1H), 4.66 (t, 1H), 3.37 (m, 2H),
2.78 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
d 162.2, 160.2, 160.1,
4.8.22. 4-{[4-(4-Cyanophenyl)pyrimidin-2-yl]amino}
benzenesulfonamide (8f)
Compound 8f was prepared (66 mg, 25% overall yield of 2 steps)
according to GP III from (4-Cyanophenyl)boronic acid instead of 4-
Chlorophenylboronic acid as a starting material. mp 278e281 ꢂC. 1H
144.4, 138.0, 134.8,132.8,132.0,131.1, 130.7, 128.0, 118.9, 118.6, 112.6,
109.7, 60.4, 45.5. HRMS (ESIþ) m/z calcd for C19H17N5NaO3S [M þ
Na]þ, 418.0944; found, 418.0942.
Acknowledgements
NMR (500 MHz, DMSO-d6)
8.06 (d, 2H), 7.98 (d, 2H), 7.78 (d, 2H), 7.63 (d, 1H), 7.20 (s, 2H). 13
NMR (150 MHz, DMSO-d6) 162.3, 160.2, 143.9, 141.1, 136.8, 133.4,
d 10.22 (s, 1H), 8.71 (d, 1H), 8.36 (d, 2H),
C
This research was supported by the Institute for Basic Science
(IBS-R010-G1), the gs2:National Research Foundation of Korea
grant (2015M3A9A8032178) and a grant (HI15C0554) from the
Korea Health Technology R&D Project, Ministry of Health and
Welfare, Korea.
d
128.7, 128.2, 127.1, 119.0, 118.5, 113.7, 110.0. HRMS (ESIþ) m/z calcd
for C17H13N5NaO2S [M þ Na]þ, 374.0682; found, 374.0667.
4.8.23. 4-{[4-(4-Aminophenyl)pyrimidin-2-yl]amino}-N-(2-
hydroxyethyl)benzenesulfonamide (8g)
Appendix A. Supplementary data
4-(2-Chloropyrimidin-4-yl)aniline was used as a starting ma-
terial for the synthesis of compound 8g. A solution of 4-(2-
Chloropyrimidin-4-yl)aniline (150 mg, 0.73 mmol), Acetyl acetate
(0.17 mL, 1.82 mmol), and TEA (0.92 mL, 0.039 mmol) in
Dichloromethane/DMF cosolvent (5 mL) was heated to 50 ꢂC for 6 h
and monitored by TLC. After cooling to room temperature, the
mixture was poured into water (30 mL), and extracted with EtOAc
(50 mL x 2). The combined organic phases were dried over anhy-
drous MgSO4, filtered and concentrated in vacuo. After removal of
the solvent, the residue was purified by flash chromatography
(Ethylacetate/Hexanes) on silica gel to give N-[4-(2-
Chloropyrimidin-4-yl)phenyl]acetamide (138 mg, 77% yield). A
mixture of this intermediate (29 mg, 0.12 mmol), 4-Amino-N-(2-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.07.075.
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