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

European Journal of Medicinal Chemistry 123 (2016) 544e556

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Optimization and biological evaluation of aminopyrimidine-based IkB kinase b inhibitors with potent anti-inflammatory effects Yongje Shin a, b, 1, Sang Min Lim b, c, 1, Hong Hua Yan d, 1, Sungwoo Jung a, b, 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 Center for Catalytic Hydrocarbon Functionalizations, Institute of Basic Science (IBS), Daejeon 34141, South Korea 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 b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 April 2016 Received in revised form 28 July 2016 Accepted 31 July 2016 Available online 1 August 2016

Targeting IkB kinase b (IKKb) can be a promising strategy in the development of a therapeutic treatment of inflammatory diseases because IKKb is well-recognized as a key mediator of the NF-kB signaling pathway. In this study, we have successfully developed a structure-activity relationship (SAR) profile of the aminopyrimidine-based IKKb 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 cytokines (IL-1a, IL-6, and TNF-a). The inhibitory effects of 8e on the phosphorylation in the NF-kB pathway further supported that the suppression of the NF-kB signaling pathway induced the anti-inflammatory effect in LPS-stimulated Raw 264.7 cells. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: IKKb Inhibitor Structure-activity relationship NO reduction Anti-inflammatory effect

1. Introduction Inflammation has been known as an immune response of body tissues caused by a wide variety of stimuli such as injury or infection. The Inflammatory reaction is characterized by the local rapid expression of pro-inflammatory cytokines, chemokines and adhesion molecules [1]. These are mainly regulated by nuclear factor kappa-B (NF-kB) protein, which is a ubiquitously expressed transcription factor [2,3]. Whereas NF-kB is normally maintained in an inactive state in the cytoplasm, NF-kB is activated at the inflammatory sites by several pro-inflammatory stimuli, such as tumor necrosis factor alpha (TNF-a), interleukin-1 (IL-1), and lipopolysaccharide (LPS) [4]. The activation of NF-kB results in its translocation from the cytosol to the nucleus, which promotes transcription of diverse pro-inflammatory cytokines (IL-1, IL-6, and TNF-a), chemokines (IL-8), and adhesion molecules (VCAM-1,

* Corresponding author. Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea. ** Corresponding author. E-mail addresses: [email protected] (S.-S. Hong), [email protected] (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.

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 inflammatory process. Recent studies have demonstrated the critical role of NF-kB in the inflammatory process in several animal models. Moreover, it was reported that NF-kB is highly activated in a variety of human inflammatory diseases such as rheumatoid arthritis (RA), atherosclerosis, multiple sclerosis, and inflammatory bowel disease [5]. Therefore, numerous strategies suppressing the activation of NF-kB have been pursued to develop therapeutics for inflammatory diseases [6]. Among them, targeting IkB kinase b (IKKb) has appeared to be one of the most promising strategies because IKKb has been well-recognized as a key mediator of the NF-kB signaling pathway [7,8]. In a resting state, IkB (inhibitor of kappa-B) binds to NF-kB, keeping it in the cytosol. Once the NF-kB signaling pathway is stimulated, IKKb phosphorylates IkB triggering ubiquitination and subsequent degradation of IkB, which renders NF-kB free to translocate into the nucleus and to induce the transcription of various genes responsible for inflammation and cell survival [9,10]. 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

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inflammatory diseases [11e21]. Recently, we disclosed a series of aminopyrimidine-based potent IKKb inhibitors through a virtual screening of a large chemical library with improved scoring functions [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 optimization of a series of aminopyrimidine-based IKKb inhibitors and demonstrate the biological evaluation of these compounds in terms of their anti-inflammatory effects. 2. Results and discussion 2.1. Discovery of IKKb inhibitors Our efforts to develop potent IKKb 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 IKKb complex with the aminopyrimidine 1 (Fig. 1b). Molecular simulation of 2-anilino-4-phenylpyrimidine with a crystal structure of IKKb (PDB ID: 4KIK) revealed that it forms bidentate hydrogen bonding with Cys 99 in the hinge region (Fig. 1b). This series was attractive for developing potent IKKb inhibitors because an aminopyrimidine scaffold can be easily diversified at the 2- and 4positions of the pyrimidine core via a convergent synthetic approach for the rapid elucidation of the structure-activity relationship (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 4positions (A and C rings) of pyrimidine through structure-based de novo design and compared the calculated binding free energies of the derivatives with respect to the IKKb kinase. We

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envisioned that adequately located substituents and chemical groups on the aniline group (A ring) and the phenyl ring (C ring) could achieve additional molecular interactions with IKKb, providing more potent IKKb 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 IKKb-aminopyrimidine 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 was generated with a reasonable potency against IKKb (IC50 ¼ 1.52 mM). Introduction of an o-amino group on the phenoxy 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 investigated 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 pocket region and has close contact with IKKb in the back-pocket. The amino group of the phenoxy ring maintains a key hydrogenbonding 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). 2.2. Exploration of the solvent exposed region Considering the tolerance within the IKKb active site, compound 3k is a promising scaffold from which more potent IKKb inhibitors 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

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.

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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 substituent 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

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. The resulting compounds in which various substituents were incorporated into the solvent exposed region, were tested to measure the potency of their inhibition of IKKb. Compound 3a was prepared as the control to investigate the role of the substituents (3a, IC50 ¼ 11.8 mM). The first series of compounds, which contain electron donating substituents (e.g., methyl, methoxy and dimethylamino groups) at the para position, showed decreased potencies as shown in Table 2(3b-3d). The enzyme potency of the derivatives was also decreased when a chloro group was introduced (3e). We observed that the potency was slightly increased when a cyano (3f, IC50 ¼ 6.2 mM) or anilide group (3i, IC50 ¼ 4.2 mM) were installed at this position. The incorporation of a sulfonyl group enhanced the potency (3j, IC50 ¼ 2.7 mM). Moving the substituent 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 IKKb. For example, a comparison of the IC50 value of 3k (IC50 ¼ 97 nM) with that of 3m (IC50 ¼ 1.6 mM) clearly reveals that substitutions at the meta position resulted in a substantial loss of the inhibitory activity against IKKb, indicating that the orientation of the sulfonamide group is

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critical in sustaining potency. In addition, the use of a bicyclic aniline (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 mono-alkylated sulfonamide (3r, IC50 ¼ 1.1 mM) or a sulfonyl moiety (3j, IC50 ¼ 2.7 mM) in the A ring, implying the importance of the free-NH2 in this region for IKKb inhibition. It appears that the 4sulfonamide group of 3k can form robust hydrogen bonding interactions 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 inhibitors, suggesting that this group serves as a critical factor for potent activity against IKKb.

Table 1 Structure-activity relationship of derivatives 3a-3s against IKKb.

Compd

R

X

IC50 (mM)

Compd

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

12.0 ± 4.3

3ma

NH2

1.6 ± 0.7

3d

N(CH3)2

NH2

28.7 ± 12.8

3n

NH2

4.6 ± 1.7

3e

Cl

NH2

34.1 ± 14.2

3o

NO2

0.034 ± 0.011

3f

CN

NH2

6.2 ± 1.7

3p

NO2

>20

3g

NH2

8.1 ± 1.9

3q

NO2

>20

3h

NH2

14.2 ± 6.4

3r

NO2

1.1 ± 0.3

3i

NH2

4.2 ± 2.2

3s

NO2

0.32 ± 0.21

3j

NH2

2.7 ± 0.8

a

Sulfonamide group was placed at the meta position.

R

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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 inhibition 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 process, 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. 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 simulations 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 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). 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 cellbased 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 compounds possess potent IKKb inhibition; derivatives bearing aminoethoxy 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 strategy has served well without compromising the IKKb inhibitory activity. For example, both chloride (8d) and anilino derivatives (8e) exhibited good potencies (8d, IC50 ¼ 19 nM and 8e, 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

Fig. 3. Predicted binding mode of compound 8a with IKKb (PDB ID: 4KIK).

Table 3 Structure-activity relationship.

IC50 (nM)

In silico solubility

LogDa

3k

97 ± 45

5.98

3.29

8a

65 ± 18

4.79

1.39

8b

79 ± 32

4.75

e

8c

138 ± 57

5.42

e

Compd

R

Y

8d

Cl

19 ± 6

5.47

e

8e

NH2

41 ± 24

4.39

e

8f

CN

58 ± 31

4.68

3.81

8g

NH2

53 ± 28

3.69

0.58

8h

CN

111 ± 61

3.79

e

8i

m-CN

190 ± 63

3.81

1.49

a

Experimental logD values were generated at pH 7.4.

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silico calculations with Discovery Studio (8e: 4.39, 8g: 3.69). We also examined the significance of the para-substituents on the C4aryl 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 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 inhibitors that are anticipated to possess more favorable physicochemical properties. 2.4. Mechanistic studies 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 enzymatic 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 excessively generated from inducible nitric oxide synthase (iNOS) during inflammation [26]. Because activated NF-kB is responsible for inducing iNOS, the degree of NO reduction was measured to identify promising IKKb inhibitors having good cellular activities [5,9]. The RAW 264.7 cells were incubated with three different concentrations (1, 2, and 5 mM) of compounds for 6 h and treated with 100 ng/mL LPS for 18 h. The generation of NO was compared to the negative control. In general, derivatives incorporating the sulfonamide group on the A ring were slightly superior to the corresponding 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

Fig. 4. Calculated binding mode of 8e in the ATP-binding site of IKKb (PDB ID: 4KIK).

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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 investigated 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 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 dosedependent manner (Fig. 6). 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 264.7 cells were pre-treated with 8e (5 mM) for 6 h, and then, cells were stimulated by adding 100 ng/mL LPS for an additional 18 h, which promoted the release of pro-inflammatory cytokines (IL-1a, IL-6, and TNF-a) as well as other inflammatory mediators. We observed that treatment with 8e (5 mM) strongly inhibited the expression of pro-inflammatory cytokines (IL-1a, IL-6, and TNF-a). In addition, the expressions of other inflammation-related proteins including SiCAM-1, TIMP-1, and I-TAC were substantially suppressed by 8e (Fig. 7). We next analyzed the effect of 8e on proinflammatory cytokines by treating Raw 264.7 cells with various concentrations of 8e, revealing that the expressions of IL-6 and TNF-a decreased upon treating macrophages with 8e in a dosedependent manner (Fig. 8). 2.4.3. Effects on the NF-kB signaling pathway To ensure that the anti-inflammatory effect of 8e can be attributed to the inhibition of the NF-kB signaling pathway, the inhibitory effect of 8e on the phosphorylation of crucial signaling proteins in the NF-kB 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 IKKb and IkBa were measured by Western blotting using b-actin as a loading control. We observed that treatment of 8e gradually decreased the LPS-induced phosphorylation of both IKKb and IkBa in a dose dependent manner (Fig. 9a). Moreover, the IKKb-mediated phosphorylation of p65 of NF-kB was suppressed by 8e in a dosedependent manner (Fig. 9b). Next, immunofluorescence staining with confocal microscopy was performed to investigate the effect of 8e on the translocation of NF-kB from the cytoplasm to the nucleus (Fig. 10). After treating Raw 264.7 cells with 8e and LPS, fixed cells were treated with the pNF-kB antibody as well as a rhodamine-B-isothiocyanate-based secondary antibody. The fixed cells were stained with the fluorescent DNA-binding agent, 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Consistent with the expectations, upon stimulation of macrophages with LPS, p65 of NF-kB was mainly translocated to the nucleus. In addition, we observed that treatment with 8e (5 mM) significantly inhibited the LPS-stimulated nuclear translocation of p65 of NF-kB in Raw 264.7 cells, further supporting the conclusion that the suppression of the NF-kB signaling pathway induced the anti-inflammatory effect of 8e in LPS-stimulated Raw 264.7 cells.

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Fig. 5. The NO reduction results of our representative IKKb inhibitors.

resulted in the enhanced cellular potency in terms of the antiinflammatory effects. Representative compounds exhibited strong effect in NO reduction by inhibiting the synthesis of iNOS protein, and strongly inhibited the expression of pro-inflammatory cytokines (IL-1a, IL-6, and TNF-a). Moreover, the inhibitory effects of 8e on the phosphorylation in the NF-kB pathway supported the conclusion that the suppression of the NF-kB signaling pathway induced the anti-inflammatory effect.

Fig. 6. Dose-dependent inhibition of NOS induction by 8e.

4. Experimental section 4.1. Cells

3. Conclusion In conclusion, we extensively modified the structure of a hit compound to optimize a series of aminopyrimidine-based IKKb inhibitors utilizing a structural-based drug design strategy. 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 replaces the aromatic D ring with acyclic hydrophilic groups has served well without compromising the IKKb inhibitory activity. The introduction of simple water-solubilizing groups at this position

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). 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

Fig. 7. Reduction of cytokine expression by 8e.

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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 ethlenediamine dihydrochloride, 14% glacial acetic acid) (Promega, Medison, WI, USA). 4.3. Western blotting Total protein was extracted with lysis buffer containing 1% Igepal CA-630, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol,

2 mM EDTA, and the following protease and phosphatase inhibitor: aprotinin (10 mg/ml), leupeptin (10 mg/ml; ICN Biomedicals, AsseRelegem, Belgium), phenylmethylsulfonyl fluoride (1.72 mM), NaF (100 mM), NaVO3 (500 mM), and Na4P2O7 (500 mg/mL; SigmaAldrich). Protein was separated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The nitrocellulose membranes were immunostained with the appropriate primary

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antibodies followed by the secondary antibodies conjugated to horseradish peroxidase. Antibody binding was detected with an enhanced chemiluminescence reagent (Amersham Biosciences). Antibodies against p-IKK-b (Tyr199) IKK-b, p-IKBa and (Ser32/36) and IKBa were obtained from Cell signaling (Boston, MA, USA). iNOS and b-actin were purchased from Santa Cruze (Texas, USA). 4.4. Cytokine Array Mouse Cytokine Array Panel A was purchased from R&D Systems. We treated Raw 264.7 cells with 8e (5 mM) for 6 h. And then 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 membranes 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. 4.5. p-NF-kB p65 ELISA 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 5 mM) for 6 h p-NF-kB p65 in cytoplasm was using ELISA Kit (Cell Signaling Technology, Inc., Beverly, MA, USA), according to the manufacturer's instructions. 4.6. Cytokine assay (IL-6 and TNF-a) 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 proinflammatory cytokine assays using a mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems), according to the manufacturer's instructions. 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  C. The fixed cells were washed and blocked in blocking buffer for 1 h at room temperature and then incubated overnight with p-NF-kB antibody (1:50; Santa Cruz Biotechnology, Inc.) at 4  C. 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). 4.8. Preparation of Compounds General Procedure I: A reaction mixture of 2-Chloro-4-[4-(2nitrophenoxy)phenyl]pyrimidine (150 mg, 0.46 mmol), Aniline (42 mg, 0.46 mmol) and catalytic amount of 1 N HCl (a few drops) in Ethanol (5 mL) was heated for 2 h at 160  C under microwave irradiation. After cooling and evaporation of the mixture, the resulting solid was washed with methanol (20 mL) to give 4-[4-(2Nitrophenoxy)phenyl]-N-phenyl-pyrimidin-2-amine (7a) (89 mg,

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  C 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  C. 1H NMR (500 MHz, DMSO-d6) d 9.63 (s, 1H), 8.49 (d, J ¼ 5.2 Hz, 1H), 8.15 (d, 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-2amine (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  C. 1H NMR (500 MHz, DMSO-d6) 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) d 163.4, 160.6, 160.5, 159.2, 141.3, 141.0, 138.5, 130.9, 130.5, 129.3, 129.0, 126.2, 121.6, 119.4, 116.9, 116.8, 116.5, 107.5, 20.8. HRMS (ESIþ) m/z calcd for C23H21N4O [M þ H]þ, 369.1710; found, 369.1711. 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  C. 1H NMR (500 MHz, DMSO-d6) 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, 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). 13 C 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. 4.8.3. N4-{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}-N1,N1dimethyl-benzene-1,4-diamine (3d) Compound 3d was prepared (7 mg, 12% overall yield of 2 steps) according to GP I from N4,N4-Dimethylbenzene-1,4-diamine instead of Aniline as a starting material. mp 70e75  C. 1H NMR (500 MHz, DMSO-d6) d 9.25 (s, 1H), 8.41 (d, 1H), 8.12 (d, 2H), 7.60 (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) 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  C. 1H NMR (500 MHz, DMSO-d6) 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). 13C NMR (150 MHz, DMSO-d6) d 163.1, 160.2, 159.9, 158.8, 140.8, 140.5, 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

389.1164; found, 389.1162. 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  C. 1H NMR (500 MHz, DMSO-d6) 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) d 163.8, 160.8, 160.0, 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 C23H18N5O [M þ Na]þ, 380.1506; found, 380.1509. 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  C. 1H NMR (500 MHz, DMSO-d6) d 10.09 (s, 1H), 8.55 (d, J ¼ 5.2 Hz, 1H), 8.16 (d, J ¼ 8.7 Hz, 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, DMSO-d6) d 163.3, 160.3, 159.7, 158.9, 145.3, 140.8, 140.5, 130.1, 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. 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  C. 1H NMR (500 MHz, DMSO-d6) 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, 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 167.6, 163.2, 160.2, 159.9, 158.9, 143.4, 140.8, 140.5, 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. 4.8.8. N-{4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl} amino}phenyl}acetamide (3i) 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  C. 1H NMR (500 MHz, 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 (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, 116.45, 116.37, 116.04, 107.04, 23.85. HRMS (ESIþ) m/z calcd for C24H21N5NaO2 [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  C. 1H NMR (500 MHz, DMSOd6) 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) d 163.3, 160.3, 159.6, 158.9, 145.4, 140.8, 140.5, 132.2, 130.0, 128.8,

553

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. 4.8.10. 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  C. 1H NMR (500 MHz, DMSO-d6) d 10.07 (s, 1H), 8.56 (d, J ¼ 5.3 Hz, 1H), 8.17 (d, J ¼ 8.8 Hz, 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, 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. 4.8.11. 4-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino}N-thiazol-2-yl-benzenesulfonamide (3l) Compound 3l was prepared (7 mg, 10% overall yield of 2 steps) according to GP I from 4-Amino-N-thiazol-2-yl-benzenesulfonamide instead of Aniline as a starting material. mp 110e115  C. 1H NMR (500 MHz, DMSO-d6) d 12.62 (br, 1H), 10.06 (s, 1H), 8.56 (d, 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) 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, 116.07, 112.42, 108.34, 107.91. HRMS (ESIþ) m/z calcd for C25H20N6NaO3S2 [M þ Na]þ, 539.0931; found, 539.0944. 4.8.12. 3-{{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}amino} benzenesulfonamide (3m) 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  C. 1H NMR (500 MHz, DMSO-d6) d 10.00 (s, 1H), 8.70 (s, 1H), 8.55 (d, 1H), 8.23 (d, 2H), 7.85 (d, 1H), 7.49 (m, 1H), 7.43 (m, 2H), 7.33 (s, 2H), 7.02e6.97 (m, 3H), 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 C22H19N5NaO3S [M þ Na]þ, 456.1101; found, 456.1137. 4.8.13. N-{4-[4-(2-Aminophenoxy)phenyl]pyrimidin-2-yl}-1,3benzothiazol-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  C. 1H NMR (500 MHz, CDCl3) 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) d 163.7, 160.6, 160.4, 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. 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 204e206  C. 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, 1H), 7.20 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d 162.9, 159.8,

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159.2, 158.6, 148.1, 143.7, 141.7, 136.1, 135.4, 132.1, 129.2, 126.7, 125.9, 125.3, 122.4, 118.1, 117.9, 108.6. HRMS (ESIþ) m/z calcd for C22H17N5NaO5S [M þ Na]þ, 486.0843; found, 486.0876. 4.8.15. Methyl N-{4-{{4-[4-(2-nitrophenoxy)phenyl]pyrimidin-2yl}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  C. 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 (150 MHz, DMSO-d6) d 163.1, 160.6, 159.4, 158.7, 154.5, 148.7, 142.0, 135.9, 135.7, 133.5, 132.9, 129.4, 126.2, 125.6, 122.6, 120.0, 119.2, 118.6, 107.7, 51.9. HRMS (ESIþ) m/z calcd for C24H19N5NaO5 [M þ H]þ, 480.1278; found, 480.1291. 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  C. 1H NMR (500 MHz, DMSO-d6) d 9.54 (s, 1H), 9.45 (br, 1H), 8.50 (d, 1H), 8.20 (d, 2H), 8.12 (d, 1H), 7.76 (m, 1H), 7.69 (d, 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, 148.7, 142.0, 135.8, 135.7, 133.6, 133.0, 129.4, 126.2, 125.6, 122.6, 120.0, 119.1, 118.6, 107.7, 60.4, 15.0. HRMS (ESIþ) m/z calcd for C25H21N5NaO5 [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) 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 148e152  C. 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, 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. 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 194e196  C. 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 (m, 1H), 7.37e7.32 (m, 2H), 7.19 (m, 4H), 2.92 (s, 3H). 13C NMR (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. General Procedure II: A solution of 2,4-Dichloropyrimidine (2.00 g, 13.42 mmol), 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan2-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  C 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 concentrated in vacuo. After removal of the solvent, the residue was

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  C under microwave irradiation. After cooling and evaporation of the mixture, the resulting solid was washed with Methanol (50 mL) to give 4-{[4-(4Hydroxyphenyl)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 (2hydroxyethyl)carbamate (68 mL, 0.44 mmol) in THF (3 mL) was added DIAD (87 mL, 0.44 mmol) at 0  C. After 10 min, the mixture 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  C and the resulting solid was washed with excess MeOH to afford the product 8a (71 mg, 58% yield). mp 265e267  C. 1H NMR (500 MHz, DMSOd6) d 10.15 (s, 1H), 8.57 (d, J ¼ 4.8 Hz, 1H), 8.34 (br, 3H), 8.20 (d, 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), 3.24 (br, 2H). 13C NMR (100 MHz, DMSO-d6) d 164.1, 160.6, 158.7, 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. 4.8.19. 4-{{4-{4-[2-(Dimethylamino)ethoxy]phenyl}pyrimidin-2yl}amino}benzenesulfonamide hydrochloride (8b) 4-{[4-(4-Hydroxyphenyl)pyrimidin-2-yl]amino}benzenesulfonamide (6k) was prepared from GP II and it was used as a starting material to synthesize the desired compound 8b. To a stirred solution of 4-{[4-(4-hydroxyphenyl)pyrimidin-2-yl]amino}benzenesulfonamide (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-ChloroN,N-dimethylethanamine hydrochloride (63 mg, 0.438 mmol) at 0  C. After 10 min, the mixture was heated for 2 h at 90  C. The 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 110e114  C. 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 (100 MHz, DMSO-d6) d 163.8, 160.1, 159.0, 158.0, 143.4, 136.4, 129.2, 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. 4.8.20. Tert-Butyl N-{2-{4-[2-(4-sulfamoylanilino)pyrimidin-4-yl] phenoxy}ethyl}carbamate (8c) Compound 8c was prepared (80 mg, 3 steps- 64% overall yield) according to GP II without final deprotection of the Boc group. mp 179e182  C. 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). 13C NMR (150 MHz, DMSO-d6) d 163.8, 161.3, 160.1, 159.2, 156.2, 144.3, 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;

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found, 508.1623. 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  C 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 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 2-Chloro-4-(4chlorophenyl)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  C 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, 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 (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 C16H13ClN4NaO2S [M þ Na]þ, 383.0340; found, 383.0343. 4.8.21. 4-{[4-(4-Aminophenyl)pyrimidin-2-yl]amino} benzenesulfonamide (8e) 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-dioxaborolan2-yl)aniline instead of 4-Chlorophenylboronic acid as a starting material. Also, the reaction condition of Suzuki Coupling was changed from Na2CO3 at 90  C to NaHCO3 at 80  C. mp 301e304  C. 1 H 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, 135.7, 128.5, 126.6, 123.0, 117.6, 113.5, 107.1. HRMS (ESIþ) m/z calcd for C16H15N5NaO2S [M þ Na]þ, 364.0839; found, 364.0829. 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 4Chlorophenylboronic acid as a starting material. mp 278e281  C. 1H NMR (500 MHz, DMSO-d6) d 10.22 (s, 1H), 8.71 (d, 1H), 8.36 (d, 2H), 8.06 (d, 2H), 7.98 (d, 2H), 7.78 (d, 2H), 7.63 (d, 1H), 7.20 (s, 2H). 13C NMR (150 MHz, DMSO-d6) d 162.3, 160.2, 143.9, 141.1, 136.8, 133.4, 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-(2hydroxyethyl)benzenesulfonamide (8g) 4-(2-Chloropyrimidin-4-yl)aniline was used as a starting material for the synthesis of compound 8g. A solution of 4-(2Chloropyrimidin-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  C 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 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 N-[4-(2Chloropyrimidin-4-yl)phenyl]acetamide (138 mg, 77% yield). A mixture of this intermediate (29 mg, 0.12 mmol), 4-Amino-N-(2-

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hydroxyethyl)benzenesulfonamide (25 mg, 0.12 mmol) and catalytic amount of 1 N HCl (a few drops) in n-Butanol (2 mL) was heated for 2 h at 180  C 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  C. 1H NMR (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, DMSOd6) 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-(2hydroxyethyl)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 4Chlorophenylboronic acid as a starting material of the first step and 4-Amino-N-(2-hydroxyethyl)benzenesulfonamide instead of 4Aminobenzenesulfonamide as a starting material of the second step. mp 271e274  C. 1H NMR (500 MHz, DMSO-d6) d 10.28 (s, 1H), 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 (150 MHz, DMSO-d6) d 162.4, 160.3, 160.2, 144.4, 141.1, 133.4, 132.8, 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. 4.8.25. 4-{[4-(3-Cyanophenyl)pyrimidin-2-yl]amino}-N-(2hydroxyethyl)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 4Chlorophenylboronic acid as a starting material of the first step and 4-Amino-N-(2-hydroxyethyl)benzenesulfonamide instead of 4Aminobenzenesulfonamide as a starting material of the second step. mp 239e241  C. 1H NMR (500 MHz, DMSO-d6) d 10.25 (s, 1H), 8.71 (d, 1H), 8.63 (m, 1H), 8.54 (m, 1H), 8.06e8.01 (m, 3H), 7.80 (t, 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, 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 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. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2016.07.075. References [1] Z. Zhang, B. Rigas, NF-kappaB, inflammation and pancreatic carcinogenesis: NF-kappaB as a chemoprevention target (review), Int. J. Oncol. 29 (2006) 185e192. [2] C. Nakanishi, M. Toi, Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs, Nat. Rev. Cancer 5 (2005) 297e309. [3] N.D. Perkins, Integrating cell-signalling pathways with NF-kappaB and IKK function, Nat. Rev. Mol. Cell Biol. 8 (2007) 49e62. [4] K. Ziegelbauer, F. Gantner, N.W. Lukacs, A. Berlin, K. Fuchikami, T. Niki, K. Sakai, H. Inbe, K. Takeshita, M. Ishimori, H. Komura, T. Murata, T. Lowinger,

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[5] [6] [7]

[8]

[9] [10] [11]

[12]

[13]

[14]

[15]

Y. Shin et al. / European Journal of Medicinal Chemistry 123 (2016) 544e556 K.B. Bacon, A selective novel low-molecular-weight inhibitor of IkappaB kinase-beta (IKK-beta) prevents pulmonary inflammation and shows broad anti-inflammatory activity, Br. J. Pharmacol. 145 (2005) 178e192. P.P. Tak, G.S. Firestein, NF-kappaB: a key role in inflammatory diseases, J. Clin. Invest. 107 (2001) 7e11. M. Karin, Y. Yamamoto, Q.M. Wang, The IKK NF-kappa B system: a treasure trove for drug development, Nat. Rev. Drug Discov. 3 (2004) 17e26. P. Bamborough, J.F. Callahan, J.A. Christopher, J.K. Kerns, J. Liddle, D.D. Miller, M.A. Morse, W.L. Rumsey, R. Williamson, Progress towards the development of anti-inflammatory inhibitors of IKKbeta, Curr. Top. Med. Chem. 9 (2009) 623e639. J. Suzuki, M. Ogawa, S. Muto, A. Itai, M. Isobe, Y. Hirata, R. Nagai, Novel IkB kinase inhibitors for treatment of nuclear factor-kB-related diseases, Expert. Opin. Investig. Drugs 20 (2011) 395e405. T. Lawrence, The nuclear factor NF-kappaB pathway in inflammation, Cold Spring Harb, Perspect. Biol. 1 (2009) a001651. M.S. Hayden, S. Ghosh, Shared principles in NF-kappa B signaling, Cell 132 (2008) 344e362. J. Liddle, P. Bamborough, M.D. Barker, S. Campos, C.W. Chung, R.P. Cousins, P. Faulder, M.L. Heathcote, H. Hobbs, D.S. Holmes, C. Ioannou, C. RamirezMolina, M.A. Morse, R. Osborn, J.J. Payne, J.M. Pritchard, W.L. Rumsey, D.T. Tape, G. Vicentini, C. Whitworth, R.A. Williamson, 4-Phenyl-7-azaindoles as potent, selective and bioavailable IKK2 inhibitors demonstrating good in vivo efficacy, Bioorg. Med. Chem. Lett. 22 (2012) 5222e5226. X. Qiu, Y. Du, B. Lou, Y. Zuo, W. Shao, Y. Huo, J. Huang, Y. Yu, B. Zhou, J. Du, H. Fu, X. Bu, Synthesis and identification of new 4-arylidene curcumin analogues as potential anticancer agents targeting nuclear factor-kappaB signaling pathway, J. Med. Chem. 53 (2010) 8260e8273. A.L. Crombie, F.W. Sum, D.W. Powell, D.W. Hopper, N. Torres, D.M. Berger, Y. Zhang, M. Gavriil, T.M. Sadler, K. Arndt, Synthesis and biological evaluation of tricyclic anilinopyrimidines as IKKbeta inhibitors, Bioorg. Med. Chem. Lett. 20 (2010) 3821e3825. J.A. Christopher, P. Bamborough, C. Alder, A. Campbell, G.J. Cutler, K. Down, A.M. Hamadi, A.M. Jolly, J.K. Kerns, F.S. Lucas, G.W. Mellor, D.D. Miller, M.A. Morse, K.D. Pancholi, W. Rumsey, Y.E. Solanke, R. Williamson, Discovery of 6-aryl-7-alkoxyisoquinoline inhibitors of IkappaB kinase-beta (IKK-beta), J. Med. Chem. 52 (2009) 3098e3102. P. Lorenzo, R. Alvarez, M.A. Ortiz, S. Alvarez, F.J. Piedrafita, A.R. de Lera, Inhibition of IkappaB kinase-beta and anticancer activities of novel chalcone

adamantyl arotinoids, J. Med. Chem. 51 (2008) 5431e5440. [16] A.H. Bingham, R.J. Davenport, R. Fosbeary, L. Gowers, R.L. Knight, C. Lowe, D.A. Owen, D.M. Parry, W.R. Pitt, Synthesis and structure-activity relationship of aminopyrimidine IKK2 inhibitors, Bioorg. Med. Chem. Lett. 18 (2008) 3622e3627. [17] R. Waelchli, B. Bollbuck, C. Bruns, T. Buhl, J. Eder, R. Feifel, R. Hersperger, P. Janser, L. Revesz, H.G. Zerwes, A. Schlapbach, Design and preparation of 2benzamido-pyrimidines as inhibitors of IKK, Bioorg. Med. Chem. Lett. 16 (2006) 108e112. [18] M.S. Palanki, P.E. Erdman, M. Ren, M. Suto, B.L. Bennett, A. Manning, L. Ransone, C. Spooner, S. Desai, A. Ow, R. Totsuka, P. Tsao, W. Toriumi, The design and synthesis of novel orally active inhibitors of AP-1 and NF-kappaB mediated transcriptional activation, SAR of in vitro and in vivo studies, Bioorg, Med. Chem. Lett. 13 (2003) 4077e4080. [19] N. Kishore, C. Sommers, S. Mathialagan, J. Guzova, M. Yao, S. Hauser, K. Huynh, S. Bonar, C. Mielke, L. Albee, R. Weier, M. Graneto, C. Hanau, T. Perry, C.S. Tripp, A selective IKK-2 inhibitor blocks NF-kappa B-dependent gene expression in interleukin-1 beta-stimulated synovial fibroblasts, J. Biol. Chem. 278 (2003) 32861e32871. [20] C. Frelin, V. Imbert, E. Griessinger, A. Loubat, M. Dreano, J.F. Peyron, AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNFalpha in Jurkat leukemic cells, Oncogene 22 (2003) 8187e8194. [21] J.R. Burke, M.A. Pattoli, K.R. Gregor, P.J. Brassil, J.F. MacMaster, K.W. McIntyre, X. Yang, V.S. Iotzova, W. Clarke, J. Strnad, Y. Qiu, F.C. Zusi, BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice, J. Biol. Chem. 278 (2003) 1450e1456. [22] H. Park, Y. Shin, H. Choe, S. Hong, Computational design and discovery of nanomolar inhibitors of IkappaB kinase beta, J. Am. Chem. Soc. 137 (2015) 337e348. [23] R.X. Wang, Y. Gao, L.H. Lai, LigBuilder: a multi-purpose program for structurebased drug design, J. Mol. Model 6 (2000) 498e516. [24] T.J. Ritchie, S.J.F. Macdonald, The impact of aromatic ring count on compound developability e are too many aromatic rings a liability in drug design, Drug Discov. Today 14 (2009) 1011e1020. [25] O. Ursu, A. Rayan, A. Goldblum, T.I. Oprea, Understanding drug-likeness, WIREs. Comput. Mol. Sci. 1 (2011) 760e781. [26] J. MacMicking, Q.W. Xie, C. Nathan, Nitric oxide and macrophage function, Annu. Rev. Immunol. 15 (1997) 323e350.