Synthesis and p38 Inhibitory Activity of Some Novel Substituted N,N′-Diarylurea Derivatives

We have identified a novel series of substituted N,N′-diarylurea p38α inhibitors. The inhibitory activity of the target compounds against the enzyme p38α, MAPKAPK2 in BHK cells, TNF-α release in LPS-stimulated THP-1 cells and p38α binding experiments were tested. Among these compounds, 25a inhibited the p38α enzyme with an IC50 value of 0.47 nM and a KD value of 1.54 × 10−8 and appears to be the most promising one in the series.


Introduction
The mitogen-activated protein kinase p38 (p38 MAPK) belongs to the serine/threonine kinase family and plays a major role in the regulation, biosynthesis and actions of key proinflammatory mediators like tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), p53, and mitogen-activated protein-kinase-activated protein kinase 2 (MAPKAPK2), etc. [1]. According to the literature, there are four known distinct subunits of p38 MAPK (p38α, β, γ and δ). Previous investigations have indicated that p38α plays an important role in serious diseases such as chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), psoriasis, and Crohn's disease [2,3]. In recent years, it has been shown that other diseases such as cancer, neuropathic pain, periodontal diseases and lung inflammation caused by acute gas may also be treated and prevented using p38α inhibitors [3][4][5][6][7][8]. However, although some select candidates are in clinical trials, including losmapimod in phase III for treatment of acute coronary syndrome, LY2228820 in phase II for epithelial ovarian cancer and PH-797804 in phase III for treatment of COPD [9][10][11][12], no p38α inhibitors are currently available on the market. Therefore, there is continuing need to identify novel bioavailable small molecule inhibitors of p38α.
Based on their mode of action, p38α inhibitors can be divided into two categories: ATP-competitive inhibitors (e.g., SB-203580) and non-competitive inhibitors (e.g., BIRB-796) that trap the p38α in an Asp-Phe-Gly-out (DFG-out) conformation (shown in Figure 1) [13][14][15]. ATP-competitive inhibitors use the ATP binding pocket and inhibit the kinase by directly competing with the binding of ATP [16]. Non-competitive inhibitors typically make use of the ATP binding site, but they also form unique hydrogen bonding and hydrophobic interactions with p38α. The non-competitive compounds inhibit p38α by maintaining a kinase conformation that is not suitable for ATP binding [17]. Compared with ATP competitive inhibitors, non-competitive inhibitors do not compete directly with the binding of ATP, which could provide a kinetic advantage. In addition, the amino acid residues of the allosteric pocket BIRB-796 is a classical allosteric p38 inhibitor with a N,N′-diarylurea scaffold. According to the X-ray single crystal structure of p38α/BIRB-796, BIRB-796 matched very well with the DFG-out conformation by forming several hydrogen bonding and hydrophobic interactions [19]. TNF-α secretion assays showed that this material inhibits LPS-induced TNF-α production with IC50 values of 18 nM and 780 nM for THP-1 cells in human whole blood and in mice in vivo, respectively (by 84%, 30 mg/kg p.o.) [20]. The major clinical problem of BIRB-796 is its hepatotoxicity, and the literature has suggested that a naphthalene epoxide intermediate might be the cause. Therefore, some structure modification and structure-activity relationship (SAR) investigations of BIRB-796 were carried out in order to discover novel p38α inhibitors.
According to the SAR of BIRB-796, the "urea linkage" occupied a hydrogen-bonding site and is important to maintain the activity [21]. Thus, this "urea linkage" is preserved in our compounds. The naphthyl ring of BIRB-796 pushes quite deeply into the hydrophobic gatekeeper pocket and hydrophobic interactions play a key role in ensuring high activity and selectivity for p38α inhibitors [22]. In order to increase the activity and selectivity and reduce or even eliminate the hepatotoxicity, the naphthalene ring was replaced with other aromatic hydrophobic scaffolds, such as phenyl, benzyl or chromene moieties, which allowed for synthetic flexibility and these structural modifications are also the correct size for the small gatekeeper. According to the SAR information for kinase inhibitors, a variety of functional groups are well tolerated by this adenine binding site, and introducing some rigid groups in the site could improve p38α inhibitory activity according to the literature [23,24]. We therefore attempted to either replace this morpholinoethoxy moiety with an indazole moiety or replace the morpholinoethoxy ring with a naphthalenol with the overall aim of increasing the binding affinity. BIRB-796 also has a unique allosteric pocket that is created when the activation loop adopts the "DFG-out" conformation. Two selectivity sites could be discovered in the allosteric pocket: one is occupied by the t-butyl group, and the other by the p-tolyl group. It has been reported that the t-butyl group offers a unique interaction with p38α that guarantees the inhibitory activity and kinase selectivity [21]. According to the above information, the t-butyl group was retained in our compounds, and we tried to replace the 4-tolyl group with other substituted phenyls to investigate the SAR surrounding the phenyl ring unit. According to the information mentioned above, we now report the design and synthesis of some series of new urea derivatives that were then evaluated for their inhibitory activities against MAPKAPK2, TNF-α, and p38α.

Chemistry
The synthetic methods used to prepare our target compounds are summarized in Schemes 1-6. Firstly, compounds 2a-2i were synthesized using substituted phenylhydrazines 1a-1i and pivaloylacetonitrile in the presence of diluted hydrochloric acid. Treating 2a-2i with 2,2,2-trichloro-ethyl carbonochloridate in THF furnished intermediates 3a-3i (Scheme 1), while the intermediates 7a-7b were synthesized from substituted 2-fluorobenzonitriles 4a-4b (Scheme 1). The intermediates 5a-5b were prepared from 4a-4b and 4-amino-3-methylphenol in the presence of K2CO3. Intermediates 5a-5b were cyclized with isoamyl nitrite in the presence of potassium acetate and acetic anhydride to generate 6a-6b. Finally, intermediates 7a-7b were synthesized from 6a-6b using LiAlH4 in THF at room temperature or H2 and Pd/C. BIRB-796 is a classical allosteric p38 inhibitor with a N,N 1 -diarylurea scaffold. According to the X-ray single crystal structure of p38α/BIRB-796, BIRB-796 matched very well with the DFG-out conformation by forming several hydrogen bonding and hydrophobic interactions [19]. TNF-α secretion assays showed that this material inhibits LPS-induced TNF-α production with IC 50 values of 18 nM and 780 nM for THP-1 cells in human whole blood and in mice in vivo, respectively (by 84%, 30 mg/kg p.o.) [20]. The major clinical problem of BIRB-796 is its hepatotoxicity, and the literature has suggested that a naphthalene epoxide intermediate might be the cause. Therefore, some structure modification and structure-activity relationship (SAR) investigations of BIRB-796 were carried out in order to discover novel p38α inhibitors.
According to the SAR of BIRB-796, the "urea linkage" occupied a hydrogen-bonding site and is important to maintain the activity [21]. Thus, this "urea linkage" is preserved in our compounds. The naphthyl ring of BIRB-796 pushes quite deeply into the hydrophobic gatekeeper pocket and hydrophobic interactions play a key role in ensuring high activity and selectivity for p38α inhibitors [22]. In order to increase the activity and selectivity and reduce or even eliminate the hepatotoxicity, the naphthalene ring was replaced with other aromatic hydrophobic scaffolds, such as phenyl, benzyl or chromene moieties, which allowed for synthetic flexibility and these structural modifications are also the correct size for the small gatekeeper. According to the SAR information for kinase inhibitors, a variety of functional groups are well tolerated by this adenine binding site, and introducing some rigid groups in the site could improve p38α inhibitory activity according to the literature [23,24]. We therefore attempted to either replace this morpholinoethoxy moiety with an indazole moiety or replace the morpholinoethoxy ring with a naphthalenol with the overall aim of increasing the binding affinity. BIRB-796 also has a unique allosteric pocket that is created when the activation loop adopts the "DFG-out" conformation. Two selectivity sites could be discovered in the allosteric pocket: one is occupied by the t-butyl group, and the other by the p-tolyl group. It has been reported that the t-butyl group offers a unique interaction with p38α that guarantees the inhibitory activity and kinase selectivity [21]. According to the above information, the t-butyl group was retained in our compounds, and we tried to replace the 4-tolyl group with other substituted phenyls to investigate the SAR surrounding the phenyl ring unit. According to the information mentioned above, we now report the design and synthesis of some series of new urea derivatives that were then evaluated for their inhibitory activities against MAPKAPK2, TNF-α, and p38α.
Compound 10 was cyclized with isoamyl nitrite in the presence of potassium acetate and acetic anhydride to generate 11. The intermediate 12 was synthesized by reduction with SnCl 2 in EtOH or by catalytic hydrogenation under catalysis of Pd/C followed by (Boc) 2 O protection to obtain the compound 13. The products of interest 25a-25k, 26a-26f, 27a-27e, 28a-28i, 29a-29i, 30a-30d were obtained in quite good yields by the reaction of 3a-3i with the intermediates 7, 14, 16, 18, 21 and 24 in the presence of DIEA in DMSO via a microwave-assisted method under the following conditions: temperature: 55-100 °C; power: 60-100 W; pressure: 8.0 bar; time: 10-20 min [27,28]. We chose the microwave-assisted pathway as a new method because it is easier and quicker and because yields are improved to a certain degree. The products of interest 25a-25k, 26a-26f, 27a-27e, 28a-28i, 29a-29i, 30a-30d were obtained in quite good yields by the reaction of 3a-3i with the intermediates 7, 14, 16, 18, 21 and 24 in the presence of DIEA in DMSO via a microwave-assisted method under the following conditions: temperature: 55-100˝C; power: 60-100 W; pressure: 8.0 bar; time: 10-20 min [27,28]. We chose the microwave-assisted pathway as a new method because it is easier and quicker and because yields are improved to a certain degree.

MAPKAPK2 Inhibitory Activity
In order to study p38α inhibition in a cell system, we investigated the ability of all compounds to inhibit the phosphorylation of mitogen-activated protein-kinase-activated protein kinase 2 (MAPKAPK2). Each compound was prepared at seven concentrations (1, 3, 10 and 30 nM, 0.1 and 0.3 µM), and the percentages of inhibition were calculated from the numbers of cells after treatment with the different compounds. The results are displayed in Table 1.

p38α Assay
To assess the p38α inhibition in an enzyme system, we selected the compounds that showed good inhibitory activity against MAPKAPK2 and monitored their ability to inhibit the phosphorylation of activation transcription factor (2ATF-2) in the enzyme system in an in vitro assay. The results are shown in Table 1.

Inhibition of TNF-α Production in THP-1 Cells
To assess the inhibition of TNF-α release from THP-1 cells after LPS stimulation, we chose the compounds that showed good inhibitory activity against the p38α enzyme and MAPKAPK2 and monitored their ability to inhibit TNF-α release from THP-1 cells after LPS stimulation. The results are also shown in Table 1.

Surface Plasmon Resonance (SPR)
To assess the binding ability between the compounds and p38α protein, we used SPR for kinase inhibitor interaction studies. The results are shown in Table 2 and Figure 2. To assess the p38α inhibition in an enzyme system, we selected the compounds that showed good inhibitory activity against MAPKAPK2 and monitored their ability to inhibit the phosphorylation of activation transcription factor (2ATF-2) in the enzyme system in an in vitro assay. The results are shown in Table 1.

Inhibition of TNF-α Production in THP-1 Cells
To assess the inhibition of TNF-α release from THP-1 cells after LPS stimulation, we chose the compounds that showed good inhibitory activity against the p38α enzyme and MAPKAPK2 and monitored their ability to inhibit TNF-α release from THP-1 cells after LPS stimulation. The results are also shown in Table 1.

Surface Plasmon Resonance (SPR)
To assess the binding ability between the compounds and p38α protein, we used SPR for kinase inhibitor interaction studies. The results are shown in Table 2 and Figure 2. Table 2. Detailed binding analysis of small molecule inhibitors with p38α mitogen-activated protein kinase.

Discussion
MAPKAPK2 is a well-known substrate of p38α. It forms an adduct with p38α, and when a stress signal is received, this protein complex is disrupted because of phosphorylation of p38α [29]. Thus, MAPKAPK2 could be used as an effector of p38α by phosphorylating further substrates. Certain compounds showed good activities, and compounds 25a-25k and 27a-27e were also profiled for their p38α inhibitory activity and ability to inhibit TNF-α release in the lipopolysaccharide (LPS)-stimulated human acute monocytic leukaemia cell line (THP-1). The activities and structures of the compounds revealed certain structure-activity relationship information. Overall, the R1 and R2 Concentration series (0.003-1 µM) of inhibitor with different color lines enzyme kinetic characteristics were determined by surface plasmon resonance on the inactive form of the kinase using a Biacore instrument.

Discussion
MAPKAPK2 is a well-known substrate of p38α. It forms an adduct with p38α, and when a stress signal is received, this protein complex is disrupted because of phosphorylation of p38α [29]. Thus, MAPKAPK2 could be used as an effector of p38α by phosphorylating further substrates. Certain compounds showed good activities, and compounds 25a-25k and 27a-27e were also profiled for their p38α inhibitory activity and ability to inhibit TNF-α release in the lipopolysaccharide (LPS)-stimulated human acute monocytic leukaemia cell line (THP-1). The activities and structures of the compounds revealed certain structure-activity relationship information. Overall, the R 1 and R 2 substituents have little effect on the activity of compounds, but the inhibitory activity was reduced when R 1 was substituted with a sulphamoyl group compared with R 1 substituted with another group, as in compounds 25a-25k, 26a-26f. The polarity of compounds with a sulphamoyl group at the R 1 position is higher, therefore, the activities of 25k and 26c were lower than those of the other compounds owing to their low transport through the cell membrane. Second, the activities of compounds 25i, 25j, 27d and 27e were lower than those of 25h, 25a, 27b and 27a, probably because the fluorine atom can affect the ability to interact with the small gatekeeper of the p38α protein.
In a comparison of the activity of compounds 25a-25k and BIRB-796 with those of 26a-26f, 29a-29i and 28a-28i, 29a-29i and 28a-28i in particular showed almost no inhibitory activity. These results demonstrate that the naphthyl ring of BIRB-796 is important for maintaining the inhibitory activity. We consider that the length of the naphthyl ring and chromene affects the anti-p38α activity of the corresponding derivatives. However, the activity of chromene urea compounds was poor compared with that of the corresponding benzylurea analogues or BIRB-796, which indicates that the chromene ring could not replace the naphthyl ring without reducing activity. Perhaps the chromene ring does not offer a correct interaction with the p38α protein. Although the structures of compounds 25a-25k and 29a-29i are similar, compounds 29a-29i showed almost no inhibitory activity compared with compounds 25a-25k, and similar activity as 28a-28i. We consider that the phenyl ring substituents in 28a-28i and 29a-29i cannot replace the hydrophobic interactions of the naphthyl ring. A docking model of the p38α/25a complex was built using Discovery Studio (PDB code: 1KV2) and is shown in Figure 3. Compound 25a fitted very well in the small gatekeeper position (Thr106) in Figure 3, while the 29a-linked phenyl ring system could not insert into hydrophobic region I and provide a complementary interaction surface. According to the previous SAR discussion, compounds 30a-30d with a directly linked benzyl ring system should show good activity, but the results indicated that compounds 30a-30d also showed poor activity, similar to compounds 28a-28i. In analysing the structure activity relationship of all compounds, we considered that 30a-30d could not bind with the p38α protein in the correct configuration like BIRB-796 because of the flexibility of the molecules (ethoxymorpholine and benzyl ring), while the rigidity of the indazole and 1,2,3,4-tetrahydronaphthalene moieties in compounds 25a-25k and 27a-27e ensured that the active configuration was the same as in BIRB-796. In summary, compounds substituted with a rigid indazole instead of a morpholinoethoxy could show good p38α inhibitory activity performance. The indazole also offers a hydrophobic interaction with p38α. The benzyl ring system instead of a naphthyl ring effectively occupied the small gatekeeper (Thr106) by forming hydrophobic interactions with the hydrophobic region I of the p38α protein, therefore, it was observed that compounds 25a-25k, and especially 25a, stood out due to their high activity. substituents have little effect on the activity of compounds, but the inhibitory activity was reduced when R1 was substituted with a sulphamoyl group compared with R1 substituted with another group, as in compounds 25a-25k, 26a-26f. The polarity of compounds with a sulphamoyl group at the R1 position is higher, therefore, the activities of 25k and 26c were lower than those of the other compounds owing to their low transport through the cell membrane. Second, the activities of compounds 25i, 25j, 27d and 27e were lower than those of 25h, 25a, 27b and 27a, probably because the fluorine atom can affect the ability to interact with the small gatekeeper of the p38α protein.
In a comparison of the activity of compounds 25a-25k and BIRB-796 with those of 26a-26f, 29a-29i and 28a-28i, 29a-29i and 28a-28i in particular showed almost no inhibitory activity. These results demonstrate that the naphthyl ring of BIRB-796 is important for maintaining the inhibitory activity. We consider that the length of the naphthyl ring and chromene affects the anti-p38α activity of the corresponding derivatives. However, the activity of chromene urea compounds was poor compared with that of the corresponding benzylurea analogues or BIRB-796, which indicates that the chromene ring could not replace the naphthyl ring without reducing activity. Perhaps the chromene ring does not offer a correct interaction with the p38α protein. Although the structures of compounds 25a-25k and 29a-29i are similar, compounds 29a-29i showed almost no inhibitory activity compared with compounds 25a-25k, and similar activity as 28a-28i. We consider that the phenyl ring substituents in 28a-28i and 29a-29i cannot replace the hydrophobic interactions of the naphthyl ring. A docking model of the p38α/25a complex was built using Discovery Studio (PDB code: 1KV2) and is shown in Figure 3. Compound 25a fitted very well in the small gatekeeper position (Thr106) in Figure 3, while the 29a-linked phenyl ring system could not insert into hydrophobic region I and provide a complementary interaction surface. According to the previous SAR discussion, compounds 30a-30d with a directly linked benzyl ring system should show good activity, but the results indicated that compounds 30a-30d also showed poor activity, similar to compounds 28a-28i. In analysing the structure activity relationship of all compounds, we considered that 30a-30d could not bind with the p38α protein in the correct configuration like BIRB-796 because of the flexibility of the molecules (ethoxymorpholine and benzyl ring), while the rigidity of the indazole and 1,2,3,4-tetrahydronaphthalene moieties in compounds 25a-25k and 27a-27e ensured that the active configuration was the same as in BIRB-796. In summary, compounds substituted with a rigid indazole instead of a morpholinoethoxy could show good p38α inhibitory activity performance. The indazole also offers a hydrophobic interaction with p38α. The benzyl ring system instead of a naphthyl ring effectively occupied the small gatekeeper (Thr106) by forming hydrophobic interactions with the hydrophobic region I of the p38α protein, therefore, it was observed that compounds 25a-25k, and especially 25a, stood out due to their high activity.  According to the information in Figure 3, the structure of 25a permits key interactions with p38α which are similar to those of BIRB-796. In addition, the important amino acids (Thr106, Lys53 and Met109) that bind with p38α also preserve the activity. First, the nitrogen atom of the indazole in 25a directly interacts with Met109 of p38α the same as pexmetinib, which could explain the previously discussed high activity of 25a. In addition, the p38α inhibitory activity of 25a was better than that of pexmetinib, which suggests that the unsubstituted indazole offers better binding ability with p38α [30][31][32][33][34]. Second, 25a occupied the small gatekeeper (Thr106) region by forming hydrophobic interactions with the hydrophobic region I of the p38α protein. (Figure 3). Finally, the π-cation interaction between the pyrazole ring and the side chains of Lys53 also plays a role on the activity. The above data also could explain why the binding capacity of 25a (KD = 1.54ˆ10´8) was better than that of SB203580. The binding affinity of compound 25a is lower than literature value of the reference BIRB-796, however it is more potent than BIRB-796 in other assays. We consider that SPR is a rough binding assay method, as the data could only explain binding capacity with the protein instead of binding capacity with the active site.

General Procedures
All reagents and solvents were used as received from commercial sources. 1 H-and 13 C-NMR spectra were recorded at 400 MHz and 100 MHz, respectively, on a JNM-ECA-400 instrument (JEOL, Tokyo, Japan) in CDCl 3 or DMSO-d 6 , respectively. Proton and carbon chemical shifts are expressed in parts per million (ppm) relative to internal tetramethylsilane (TMS), and coupling constants (J) are expressed in Hertz (Hz). The splitting pattern abbreviations are described as follows: multiplicity (s: singlet, d: doublet, dd: double doublet, ddd: double double doublet, dm: double multiplet, ds: double single, dt: double triplet, t: triplet, td triple doublet, tm, triple multiplet, tt: triple triplet, q: quartet, quint: quintuplet, m: multiplet, br: broad). The electronic information of compounds are available in the Supplementary Materials. Infrared (IR) spectra were recorded with a Perkin-Elmer 1330 infrared spectrophotometer (PerkinElmer, Waltham, MA, USA) using KBr as solid matrix. Low-resolution mass spectra were obtained using a 3000LC/MS instrument (API, Rockwell, MD, USA) equipped with an ESI source or an 620BTOF LC/MS equipped with an ESI source (Agilent Technologies, Santa Clara, CA, USA). Purities of the compounds were established by analytical HPLC, which was carried out on an Agilent HPLC system. HPLC analysis conditions were as follows: Diamonsil C18 column (250ˆ4.6 mm, 5 µm); flow rate, 1.0 mL¨min´1; DAD detection at 245 nm; mobile phase: 60% acetonitrile in water.

General Method for the Synthesis of 2a-2i
A solution of the corresponding substituted phenylhydrazine hydrochloride (1 equiv.) and pivaloylacetonitrile (1.2 equiv.) was stirred overnight at 80˝C in ethanol. The solution was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO 4 ), filtered, and evaporated to dryness.

General Method for the Synthesis of 3a-3i
A solution of the corresponding 2a-2i (1 equiv.) and 2,2,2-trichloroethyl carbonochloridate (1.1 equiv.) in THF was stirred 6 h at 0˝C, then the mixture was filtered and extracted three times with ethyl acetate. The solution was dried with MgSO 4 and concentrated to dryness.

General Method for the Synthesis of 5a-5b
A solution of the corresponding 4a-4b (1 equiv.) and 4-amino-3-methylphenol (10 mmol) was stirred 2 h at 90˝C in the presence of K 2 CO 3 (4.14g 30 mmol) in DMSO (50 mL). Then the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO 4 ), filtered and evaporated to dryness. The product was separated by column chromatography using PE/EA (3:1) as eluent.

General Method for the Synthesis of 6a-6b
A solution of the corresponding 5a-5b (1 equiv.) and isoamyl nitrite (1.5 equiv.) was stirred 8 h at 90˝C in the presence of potassium acetate (0.98 g, 10 mmol) and acetic anhydride (1.75 g, 15 mmol) in toluene (50 mL). The solution was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO 4 ), filtered, and evaporated to dryness. The product was separated by column chromatography using PE/EA (8:1) as eluent.

General Method for the Synthesis of 7a-7b
A solution of the corresponding 6a or 6b (1 equiv.) and LiAlH 4 (3 equiv.) in THF was stirred 1 h at 65˝C. Then the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO 4 ), filtered and evaporated to dryness. Products were purified by column chromatography using DCM/MeOH (50:1) as eluent.

Synthesis of 14
4-Fluoro-1-nitro-2-(prop-2-yn-1-yloxy)benzene (9). Compound 8 (6.7 g, 40 mmol) was dissolved in DMF (60 mL), and K 2 CO 3 (8.3 g, 60 mmol) was added while stirring vigorously. 3-Bromoprop-1-yne (50 mmol, 5.9 g) was then added dropwise while the mixture was on ice, and the reaction was allowed to proceed at room temperature for 12 h. The mixture was poured into ice water and kept in fridge for a day. The yellow solid 9 that appeared was filtered off and dried (yield 97%). IR  (10). To DMF (40 mL) compound 9 (1.95 g, 10 mmol) and K 2 CO 3 (4.0 g, 30 mmol) were added and the mixture was stirred at room temperature for 4 h, followed by the addition of 4-amino-3-methylphenol (1.23 g, 10 mmol). The suspension formed was stirred at 100˝C for 3 h. The reaction mixture was cooled, and poured into ice water (100 mL), then the product was briefly placed in a fridge whereby a white solid appeared. The product was recovered by suction filtration and the filter cake was dried to give 2.55 g (90%) of  (11). Compound 10 (0.8 g, 2.7 mmol) and acetic anhydride (1.14 g) were added to dry toluene (50 mL), followed by potassium acetate (0.3 g). The suspension was stirred at 80˝C and then isopentyl nitrite (0.6 g, 5 mmol) was added. The reaction was continued for 12 h, then the suspension was cooled down and filtered. Product  (12). Compound 11 (2.0 g, 6 mmol) was placed in a 100 mL flask and then dissolved in ethyl acetate (40 mL). Ethanol (40 mL) was then added, followed by SnCl 2 (4.5 g, 21 mmol). The mixture was heated to reflux for 6 h. Next saturated NaHCO 3 solution was added until bubbles ceased to come out, and the mixture was filtered. The filtrate was extracted with ethyl acetate and then dried and then separated by column chromatography using DCM/MeOH (100:1) as eluent, yield (54%). IR (  2-((5-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)benzonitrile (17a). 6-Hydroxy-1-tetralone (5 g, 30.86 mmol) was placed in a 250 mL three-necked flask and dissolved in DMSO (100 mL). The flask was heated to 90˝C, and K 2 CO 3 (12.78 g) was added under stirring. Compound 4a (4.48 g, 37.03 mmol) was added dropwise into the flask and the reaction mixture was heated to 90˝C for 4 h. The mixture was poured into water (200 mL), and the water phase was extracted twice with ethyl acetate. The organic phases were combined, washed with water and brine, dried with Na 2 SO 4 , and the solvent removed under vacuum to yield the crude product, which was purified by column chromatography using petroleum ether/ethyl acetate (5:1) as eluent to give compound 17a as a yellow solid (75.3%). IR ( (21). Compound 20 (2.52 g, 10 mmol) was added to EtOH (50 mL) in a 100-mL flask, followed by SnCl 2¨2 H 2 O (11.28 g (50 mmol) and the mixture was heated at 80˝C for 2 h. The mixture was then poured into 10% aqueous NaOH solution (200 mL), and the solid was filtered. The water phase was extracted twice with ethyl acetate, and the organic phases were combined and washed with water and brine and dried with Na 2 SO 4 . The crude product was obtained after concentration; yield 60%. IR 2-((1H-Indazol-5-yl)oxy)aniline (24). Compound 23 (2.97 g, 10 mmol) was added to EtOH (50 mL) in a 100-mL flask, followed by SnCl 2¨2 H 2 O (11.30 g, 50 mmol) and the mixture was reacted at 80˝C for 2 h. The mixture was poured into 10% aqueous NaOH solution (200 mL), and the solid formed was filtered. The water phase was extracted twice with ethyl acetate, and the organic phases were combined, washed with water and brine, and dried with Na 2 SO 4 . A yellow solid was collected after concentration; yield 57%. IR

General Method for the Synthesis of 25a-25k
A solution of the corresponding 3a-3i (1.1 equiv.) and 7a-7b (1 equiv.) was stirred for 2 h at 90˝C in the presence of Et 3 N (1 mL) in DMSO (50 mL). The mixture was poured into ice water, and the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO 4 ), filtered and evaporated to dryness. The product was separated by column chromatography using DCM/MeOH (100:1) as an eluent.

General Method for the Synthesis of 26a-26f
A solution of the corresponding 3a-3i (1.1 equiv.) and 14 (1 equiv.) was stirred 2 h at 90˝C in the presence of Et 3 N (1 mL) in DMSO (50 mL). The mixture was poured into ice water, and the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO 4 ), filtered, and evaporated to dryness. The product was separated by column chromatography using DCM/MeOH (150:1) as eluent.
1-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (26a). From 3a (4.03 g, 10 mmol), and 14 (2.79 g, 10 mmol), after work-up and purification the title compound   The GFP-MAPKAPk2_BHK cell model can differentially express the MAPKAPK2 molecule and GFP information system so the activity can be derived from the contrast of the compound BIRB-796 and the obtained compounds. GFP-MAPKAPk2_BHK cells were cultured in 96-well plates (2.0ˆ10 4 /100 µL/well) containing 10% heat-inactivated foetal bovine serum (FBS) and 1 mg/mL G418 at 37˝C in humidified air containing 5% CO 2 for 18-24 h. The cells were washed with nutrient solution (100 µL/well) and changed to the compound liquid (100 µL/well). Experiments were set up with the control, and the compound was repeated at each concentration in three wells. The cells were cultured in 96-well plates at 37˝C in humidified air containing 5% CO 2 for 90 min. The cells were washed with the dye solution (20 µL/well), and the dyed cells were held for 1 hour at room temperature in the dye liquid. Analysis of nuclear translocation and calculation of the ability of the compounds to inhibit nuclear translocation was performed using the IN Cell Analyzer 1000 Nuclear Trafficking Analysis Module System (GE, Madison, WI, USA). The cultured cells were treated with the obtained compounds in seven concentration grads (1, 3, 10 and 30 nM, 0.1 and 0.3 µM). The inhibitory percentage can be calculated using the amounts of cells after the disposition of the different compounds.

IC 50 Determination of Inhibition of TNF-α Release from THP-1 Cells after LPS Stimulation
THP-1 cells were cultured in RPMI medium 1640 with 10% (v/v) foetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-mercaptoethanol at 37˝C and 5% CO 2 . LPS stock was prepared at 1 mg/mL by adding 1 mL sterile endotoxin-free water to 1 mg LPS powder. The LPS stock was aliquoted and stored at´20˝C. Test medium: RPMI Medium 1640 with 0.5% (v/v) foetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol. An amount of 100 K THP-1 cells were seeded into a 96-well cell culture plate in 100 µL of test medium. The compounds were diluted at 1:3.5 in DMSO over 10 points (9 + 0) with a starting concentration of 333 µM. An amount of 3.6 µL of diluted compound was transferred to 96.4 µL test medium, with a starting concentration of 12 µM. A total of 10 µL compound was added to the plate with cells, and the cells were incubated for 1 hour at 37˝C. An amount of 10 µL/well LPS was added to stimulate the cells. The final LPS concentration was 300 ng/mL, and the DMSO concentration was 0.3%. Final compound concentrations: 1000.00, 285.71, 81.63, 23.32, 6.67, 1.90, 0.54, 0.16, 0.04 and 0 nM. Two replicates were used for each dose. The plate was incubated for 5 hours at 37˝C. The TNF-α standard curve samples were prepared with final concentrations of 2500, 1250, 625, 312, 156, 78, 39, 19.5, 9.75 and 0 pg/mL. Three replicates were used for each dose. At the 5th hour, the TNF-α in the supernatants and standard curve samples was determined using the HTRF kit. The cells were centrifuged at 2000 rpm for 5 min, and 10 µL supernatant was added to a ProxiPlate-384 Plus assay plate (PerkinElmer). Amounts of 5 µL Anti-TNF-α-Cryptate (Invitrogen, Carlsbad, CA, USA) and 5 µL Anti-TNFα-XL665 (Invitrogen) were added, and the plate was incubated at RT for 15 h. The plate was read on an Envision instrument (PerkinElmer) and the IC 50 was calculated.

p38α MAP Kinase Activity Assessment Based on the Rate of Phosphorylation of ATF-2 (Activation Transcription Factor 2) in an in Vitro Assay
Kinase reaction buffer was composed of 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl 2 and 1 mM EGTA. Prepared MAPK14/p38α in 1ˆkinase buffer with concentration at 500 ng/mL. A 2-fold serial dilution was performed using 1ˆkinase buffer from 500 ng/mL using 16 dose points. The serially diluted MAPK14/p38α (5 µL) was added into the 384-well plate in triplicate and 0.8 µM substrate GFP-ATF2 (19-96) and 180 µM ATP in 1ˆkinase reaction buffer were prepared (1 mL). The reaction was started by adding 5 µL of the GFP-ATF2(19-96) substrate and ATP solution into each well of the assay plate. The final starting concentration of MAPK14/p38α was 250ng/ml, and the final GFP-ATF2 and ATP concentrations were 0.4 µM and 90 µM, respectively. The assay plate was sealed and incubated for 1 hour at room temperature (RT). Antibody solution (1 mL) was prepared from 20 mM EDTA and 4 nM Tb-antipATF2 (pThr71) antibody in TR-FRET dilution buffer. Antibody solution (10 µL) was added into each well of the assay plate and mixed gently. The final EDTA concentration was 10 mM, and the final Tb-antipATF2 (pThr71) concentration was 2 nM. The assay plate was sealed and incubated for 30 minutes at RT. Finally, the TR-FRET signal was readed on the Envision 2104 plate reader.
Inhibitor in 0.5% DMSO (2 µL/well) at 5-fold the final assay concentration was added into a 384-well assay plate. For the first inhibitor screening cycle, the final concentrations of inhibitors were 3333, 1111, 370, 123, 41, 13.7, 4.57, 1.52, 0.51, 0.17 and 0.056 nM (3-fold dilution, 11 dose points, two replicates for each dose). The inhibitor concentration was then adjusted according to the first cycle result. MAPK14/p38α (4 µL/well) was added to each well of the 384-well assay plate and incubated for 15 min at RT. To start the reaction, substrate GFP-ATF2 (19-96) and ATP (4 µL/well) were added into kinase reaction buffer. The TR-FRET signal was read on the Envision 2104 plate reader. The resulting TR-FRET emission ratio was plotted against the concentration of inhibitor, and the data fitted to a sigmoidal dose-response curve with a variable slope. The IC 50 concentration was calculated from the curve.

Binding Experiments
The Biacore instrument uses a technique based on surface plasmon resonance to follow the real-time binding of compounds to immobilized proteins on a carboxymethyl dextran surface in a liquid milieu. Compounds were stored as 10 mM stock solutions in 100% dimethyl sulfoxide (DMSO). All light-sensitive compounds were handled under yellow protective light. After dilution to 1 mM with 100% DMSO, samples were mixed with 1.03-to 1.05-fold concentrated assay buffer and DMSO to yield 10 µM compound in a final buffer composition of 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 , 1 mM MnCl 2 , and 3% or 5% DMSO (assay buffer), which was also used as the instrument running buffer and sample dilution buffer. ATP was dissolved directly in running buffer to make a 10 mM solution. The run was started with five start-up cycles in which running buffer was injected (instead of a sample) followed by sample injection cycles. Zero concentration samples were used as blanks. A typical analysis cycle consisted of a 60 s sample injection (30 µL/min), 120-200 s of buffer flow (dissociation phase), followed by a needle and tubing wash with 50% DMSO and buffer, and finally, a 30 s buffer injection to check for sample carry over. The flow cell temperature was 25˝C. Between sample series, a solvent correction cycle was run according to the instrument manual to adjust for referencing errors due to refractive index mismatches between running buffer and samples [35]. These data were used to calculate the KD for compounds.

Conclusions
In summary, we have designed and synthesized a novel series of substituted N,N 1 -diarylurea compounds as potential p38α inhibitors and showed that some compounds possessed good inhibitory potencies. In order to obtain these target compounds, we designed a number of routes to synthesize the targets. The target compounds were evaluated for the inhibitory activity against p38α, MAPKAPK2 in BHK cells, TNF-α release in LPS-stimulated THP-1 cells and p38α binding experiments. A promising overall profile led to the nomination of compound 25a as a p38α inhibitor development candidate. Further information related to the research and development of compound 25a will be reported in due course.