Design, synthesis and biological evaluation of novel substituted N,N′-diaryl ureas as potent p38 inhibitors

Article abstract of DOI:10.3390/molecules200916604

A novel series of substituted N,N′-diaryl ureas that act as p38α inhibitors have been designed and synthesized based on two key residues (Gly110 and Thr106) that are different in p38α MAPK than in other kinases. Preliminary biological evaluation indicated

Full text of DOI:10.3390/molecules200916604

Molecules 2015, 20, 16604-16619; doi:10.3390/molecules200916604
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis and Biological Evaluation of Novel Substituted
N,N′-Diaryl ureas as Potent p38 Inhibitors
Dianxi Zhu 1, Xingzhou Li 2, Wu Zhong 2,* and Dongmei Zhao 1,*
1
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; E-Mail: zhudianxi@126.com
2
Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China; E-Mail: xingzhou1970@gmail.com
* Authors to whom correspondence should be addressed; E-Mails: zhongwu@bmi.ac.cn (W.Z.);
dongmeiz-67@163.com (D.Z.); Tel.: +86-135-0133-4184 (W.Z.); +86-24-2398-6413 (D.Z.).
Academic Editor: James W. Leahy
Received: 26 August 2015 / Accepted: 9 September 2015 / Published: 11 September 2015
Abstract: A novel series of substituted N,N′-diaryl ureas that act as p38α inhibitors have been
designed and synthesized based on two key residues (Gly110 and Thr106) that are different
in p38α MAPK than in other kinases. Preliminary biological evaluation indicated that most
compounds possessed good p38α inhibitory potencies. Among these compounds, 9g appeared
to be the most powerful and is the main compound that we will study in the future.
Keywords: kinase inhibitor; p38 inhibitors; p38 MAPK; TNF-α; glycine flip
1. Introduction
p38α mitogen-activated protein (MAP) kinase belongs to the serine/threonine family of kinases and
plays an important role in regulating the production of inflammatory cytokines, including tumor necrosis
factor-alpha (TNF-α) and interleukin-1b (IL-1β) [1]. In its activated state, p38α phosphorylates a range
of intracellular protein substrates that post-transcriptionally regulate the biosynthesis of TNF-α and
IL-1β. Excessive levels of TNF-α and IL-1β are thought to be responsible for the progression of many
inflammatory diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease [2,3]. To
date, many research programs have focused on the inhibition of TNF-α production, antagonism of TNF-α.
Therefore, p38α mitogen-activated protein (MAP) kinase has attracted considerable attention as a

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molecular target for the treatment of these conditions. Furthermore, the development of p38α MAP
kinase inhibitors into anti-inflammatory drugs was obstructed by their severe liver toxicity and lack of
kinase selectivity, as SB203580 and BIRB-796 were found to interact with the hepatic cytochrome P450
enzymes involved in drug metabolism [4–7]. Therefore searching for potent selective p38α inhibitors
has become a research focus.
Members of the MAP kinase family share similar sequence and conserved structural motifs, and are
all activated by dual phosphorylation of conserved threonine and tyrosine residues in the activation
loop. A reasonable strategy to improve the activity and selectivity of p38α MAPK inhibitors is to take
advantage of the key residues that are different between p38α MAPK and other kinases. Sequence
alignment of human MAP kinases and related kinases showed that Thr106 and Gly110 are unique in
p38α and β (Figure 1a), and these two residues are both responsible for the p38α selectivity of the
inhibitors pyridinylimidazole, triazolopyridylbenzamides and disubstituted dibenzosuberones (Figure 1b).
The high selectivity of p38α inhibitors has been attributed to the presence of Thr106 (a small gatekeeper)
in the p38α ATP-binding site (residues 100–118, which cover hydrophobic region I, linker region and
hydrophobic region II [8]. However, MAP kinases other than p38β have either a methionine or a
glutamine residue in this position which prevents the binding of phenyl ring inhibitors to hydrophobic
region I [9–12]. Gly110 is a residue that is specific to p38α, β and γ isoforms, and larger residues that are
present in other MAP kinases would make the peptide chain rotation much more difficult, explaining the
high activity and selectivity of most compounds that are able to form a hydrogen bond with Gly110 [8].
Therefore, the special amino acids (Gly110/Thr106) were considered as the key factor of our p38α
inhibitor design.
Figure 1. (a) Sequence alignment of human MAP kinases (residues 100–118) and related
kinase: both Thr106 (red) and Gly110 (blue) are unique in p38α/β, and allow for close
interactions of the proteins with the p38α inhibitors [13]; (b) Schematic of the binding mode
of 2-(2,4-difluoro-phenylamino)-7-(2R,3-dihydroxy-propoxy)-10,11-dihydro-dibenzo[a,d]
cyclohepten-5-one (Skepinone-L, PDB ID 3QUE) with p38α MAP kinase [14].
p38α inhibitors can be classified into two types based on their mode of action: ATP-competitive
p38α inhibitors (e.g., SB203580), and non-competitive inhibitors (e.g., BIRB-796) (Figure 2). Like

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other kinase inhibitors, first generation p38α MAPK inhibitors like the pyridinyl-imidazole (SB203580)
target the ATP binding site of the kinase in its active conformation. Type II inhibitors typically use the
ATP binding site, but they also exploit unique hydrogen bonding and hydrophobic interactions made
possible by the DFG residues of the activation loop being folded away from the conformation required
for ATP phosphate transfer [15]. The N,N′-diaryl urea compounds (BIRB796) inhibit p38α by stabilizing
a conformation of the kinase that is incompatible with ATP binding [16]. The X-ray co-crystal structure
of human p38α MAP kinase and BIRB-796 shows that conserved residues Asp168-Phe169-Gly170
(DFG) move to a new position during binding [17–20]. In the new conformation (DFG-out), ATP
binding to p38α MAP kinase is inhibited. The non-competitive inhibitors target the unique DFG-out
inactive kinase conformation, they are likely to possess greater cellular potency and altered selectivity
relative to their ATP-competitive counterparts. Ususlly tape II inhibitors have a better kinase selectivity,
because the allosteric site could provide another handle for tuning kinase selectivity [15,21]. Therefore,
in this context, we focused on the discovery of novel non-competitive p38α inhibitors using BIRB-796
as a lead that the compounds could form a hydrogen bond with Gly110. In BIRB-796, the ethoxy
morpholine group forms hydrogen bond interactions with the residue of Met109, for our compounds,
we attempted to replace the ethoxy morpholine group with much more rigid benzo [d] thiazol-2-amine
group so as to form a hydrogen bond with Gly110. Furthermore, we replaced the naphthalene group of
BIRB-796 with a benzyl or 5-fluorobenzyl group, which generated synthetic flexibility for structural
modification of these compounds and maintained connection with the small gatekeeper. We hypothesized
that structural modification of the inhibitors based on the key residues that are different between p38α
MAPK and related kinases would generate highly potent p38α inhibitors.
Figure 2. Chemical structures of SB-203580 and BIRB-796.
2. Results and Discussion
Based on the above hypothesis, a novel series of N,N′-diaryl urea p38α inhibitors were designed
and synthesized, with the aim of developing compounds with tight interactions with residues Thr106
and Gly110. The synthesis of target compounds 8a–8c and 9a–9j is outlined in Scheme 1. The key
intermediates 7a–7b were prepared from substituted 2-fluorobenzonitrile 4a–4b. Treating 4a–4b with
4-aminophenol generated the intermediates 5a–5b. Compounds 5a–5b were cyclized with KSCN and
Br2 in the presence of acid to generate thiazoles 6a–6b [22]. Intermediate 7a–7b were synthesized by
reduction with LiAlH4 in THF at room temperature or by catalytic hydrogenations under the catalysis
of Pd/C [23]. For the pyrazole-benzyl moieties, substituted phenylhydrazines 1a–1b were cyclized

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with pivaloylacetonitrile in the presence of diluted hydrochloric acid to provide compounds 2a–2b [24],
and 2 was treated with 2,2,2-trichloroethyl carbonochloridate to generate the intermediates 3a–3b. Urea
formation was achieved by coupling the corresponding benzyl amine 7a–7b with carbamate 3a–3b [25], to
generate urea compounds 8a–8c [26]. The target compounds 9a–9j were synthesized by substitution of
8 with isocyanate or methyl carbonochloridate in dichloromethane at room temperature [27].
Reactions and conditions: (a) ethanol, pivaloylacetonitrile, HCl, 80 °C, 6–8 h, 86.4%; (b) THF, 2,2,2-trichloroethyl
carbonochloridate, NaHCO3, 0 °C, 12 h, 85.3%; (c) DMSO, 4-aminophenol, K2CO3, 90 °C, 4 h, 71.1%;
(d) AcOH, KSCN, Br2, 0–10 °C, 8 h, 62.5%; (e) THF, LiAlH4, 65 °C, 30–40 min, 73.7%; (f) DMSO, DIEA,
85 °C, 1 h, 53.5%; (g) DCM, RT, 1–2 h, 88.7%.
Scheme 1. Synthesis of substituted N,N′-diaryl ureas derivatives.
Our compounds were profiled for their p38α inhibitory activities and ability to inhibit TNF-α release
in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) [28,29]. The
results are presented in Table 1.Then the p38α kinase selectivity assay was continued and the results
are presented in Table 2. Nearly all our compounds showed good activities, and good kinase selectivity
with p38α/β showed in Table 2 which indicating that this strategy is successful. However, owing to the
limited number of compounds, our knowledge of the structure-activity relationship is limited. Overall,
R1 and R2 substituents have little impact on the enzyme’s activity, but TNF-α inhibition is reduced
when R2 is a nitro group compared to when R2 is a different group, as in compounds 9a, 9c, 8a and 8b.
The polarity of compounds with a nitro group at the R2 position is higher, but transmittance through
the cell membrane may be lower than for compounds with a methyl group at the R2 position; therefore,
the TNF-α inhibition activities of 8a and 9a in PBMC cells were lower than those of the other
compounds. Secondly, the compounds with little substituents in the R3 position that also have the
ability to form hydrogen bonds showed good activities (compounds 8a, 8b, 9d and 9g). Hydrogen
bonding of the compounds with p38α may have been impacted by large groups at the R3 position (9h, 9i).
Finally, the activities of compounds 9h, 9i, and 9j were lower than those of 9f, 9b and 9a. This is
probably because compounds 9h, 9i, and 9j contained a fluorine atom, which may affect their ability to
interact with the small gatekeeper of the p38α protein. However, the fluorine atom at the R1 position
may increase kinase selectivity with p38α/β (9d, 9g), and with a fluorine atom at the R1 position may
allow the compounds to have lower hepatotoxicity, as the benzene ring in this position has been reported
to be metabolized by CYP and biotransformed into ring epoxides, which are highly toxic metabolites [30].

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Although some trends were observed, to thoroughly analyze the relationship between structure and
activity, more compounds of this group will be synthesized in the future.
Table 1. Structure and biological activity of compounds 8a–8b, 9a–9j.
Compound No. R1
R2
—
R3
—
—
H
p38α IC50 (nM) a TNF-α IC50 (nM) b
BIRB-796
SB203580
8a
—
—
H
13.74 ± 2.01
13.89 ± 1.17
2.878 ± 0.01
1.108 ± 0.20
n.d. c
—
800 ± 13.15
31.60 ± 5.27
8.94 ± 1.02
NO2
Methyl
8b
F
H
9a
9b
9c
9d
9e
9f
H
H
H
H
H
H
NO2
1.309 ± 0.09
6.165 ± 0.07
1.023 ± 0.09
1.501 ± 0.24
3.288 ± 0.25
4.499 ± 0.04
33.16 ± 1.17
62.11 ± 5.01
18.32 ± 4.20
8.86 ± 3.32
90.21 ± 6.12
56.44 ± 4.09
Methyl
Methyl
Methyl
Methyl
Methyl
9g
9h
9i
F
F
F
Methyl
Methyl
Methyl
0.844 ± 1.04
12.38 ± 0.18
7.538 ± 1.99
6.22 ± 2.07
995.97 ± 9.11
106.62 ± 4.10
9j
F
Methyl
4.277 ± 1.14
52.82 ± 8.03
a
p38α MAP kinase activity was assessed based on the rate of phosphorylation of ATF-2 (activation
transcription factor 2) in an in vitro assay; b LPS-induced TNF-α production assay in human peripheral blood
mononuclear cell (PBMC); c n.d.: not determined.
Table 2. Kinase selectivity assay of selected compounds (9d, 9g).
IC50 (nM)
Compound No.
p38α
p38β
p38γ
p38δ
9d
9g
1.501 ± 0.20 17.54 ± 1.25 191 ± 7.20 230.9 ± 7.15
2.844 ± 0.65 20.07 ± 1.40 424.3 ± 5.75 948.4 ± 8.35
To illustrate the structure-activity relationship of 9g and p38α protein, a docking model of the
p38α/compound 9g complex was built using Discovery studio (PDB code: 1KV2), and the results are
shown in Figure 3. The docking model of 9g with p38α protein reveals a hydrogen bond formed by
one hydrogen of the urea group and the carboxylate oxygen of Glu71 and a hydrogen bond formed by
oxygen of the urea group and N-H of Asp168. The two structures of 9g and BIRB-796 overlap each

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other, and the key interactions in the p38α active site, consistent with previous reports, are highlighted
(Figure 3a). In addition, the two primary differences (Gly110/Thr106) between p38α MAPK and other
kinases could improve the selectivity of a p38α MAPK inhibitor. Firstly oxygen atoms in 9g directly
interact with the N-H of Met109 and Gly110 (Figure 3a), which can explain the high activity and
selectivity of 9g discussed previously, and perhaps compounds that bind the glycine-flipped form of
p38α MAPK dramatically lose their potency when glycine is replaced with another amino acid, which
disables the flip. Secondly, 9g has a directly linked aromatic ring system and makes good use of the
small gatekeeper (Thr106) by forming a tight complementary surface with hydrophobic region I of the
enzyme (Figure 3b).
Figure 3. (a) A docking model of the p38α/9g complex was built on the basis of the
p38α/BIRB796 structure (PDB code: 1KV2) [31] using the program Discovery Studio;
(b) Binding mode of compound 9g.
3. Experimental Section
3.1. General Synthetic Information and Synthesis Procedures
13
All reagents and solvents were used as received from commercial sources. 1H-NMR and C-NMR
spectra were recorded at 400 MHz and 100 MHz on a JNM-ECA-400 instrument in CDCl3 or DMSO-d6,
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 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). Low-resolution
mass spectra were obtained using an API 3000 LC/MS with an ESI source or an Agilent 620B TOF
LC/MS with an ESI source.

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3.2. Chemistry
3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-amine (2a). A solution of 4-tolyllhydrazine hydrochloride (5.20 g,
33 mmol) and pentylacyl acetonitrile (3.75 g, 30 mmol) in 0.4 M ethanolic solution of HCl (100 mL)
was heated under reflux during 8 h. After cooling to room temperature, 1 M NaOH was added to the
mixture until the pH reached 10–11. The mixture was partitioned between water and ethyl acetate. The
water phase was extracted twice with dichloromethane. The organic phases were combined and washed
with water and brine, then dried with Na2SO4. Evaporation of the solvent in vacuo afforded the crude
product, which was subjected to recrystallization from ethyl acetate and petroleum ether to produce
1
compound 2a as a white solid (5.88 g,). Yield: 86.4%. H-NMR (CDCl3, 400 MHz), δ: 7.40 (d, 2H),
7.25 (d, 2H), 5.50 (s, 1H), 4.72 (brs, 2H), 2.37 (s, 3H), 1.32 (s, 9H). ESI-MS (+Q, m/z): 230 [M + H]+,
252 [M + Na]+.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)carbamate (3a). The 2a (0.80 g, 3.5 mmol)
was put in a 100 mL three-necked bottle and then dissolved in 30 mL THF . The bottle was cooled to
0 °C and then put 2.9 g NaHCO3 into the bottle while stirring. Then drip the 2,2,2-trichloroethyl
carbonochloridate into the bottle, keep the temp for 30 min, then 0 °C for 12 h. The mixture was filtered
and then extracted with ethyl acetate for 3 times. Dry the solution with sodium sulfate, concentrating
then separate the production with column chromatograph. Yield: 85.2%. 1H-NMR (CDCl3, 400 MHz),
δ: 9.10 (s, 1H), δ: 7.40 (d, 2H), 7.25 (d, 2H), 5.56 (s, 1H), 4.79 (s, 2H), 2.36 (s, 3H), 1.30 (s, 9H). ESI-MS
(+Q, m/z): 404 [M + H]+, 426 [M + Na]+.
2-(4-Aminophenoxy)-5-fluorobenzonitrile (5a). The 4-aminophenol (5 g, 45.87 mmol) was put in a 250 mL
three-necked bottle and then dissolved in 100 mL DMSO .The bottle was heated to 90 °C and put 19 g
K2CO3 in to the bottle while stirring. Then drip the 4a (7.76 g, 55.0 mmol) into the bottle and heated to
90 °C for 4 h. The mixture was put into 200 mL water and the water phase was extracted twice with
ethyl acetate. The organic phases were combined and washed with water and brine, dried with Na2SO4
and the solvent removed under vacuum to yield the crude product, which was purified by column
chromatography petroleum ether/ethyl acetate (5:1) to give compound 5a as a yellow solid. Yield: 71.1%.
1H-NMR (CDCl3, 400 MHz), δ: 7.49 (m, 1H), 7.29 (m, 1H), 7.15 (d, 1H), 6.79 (d, 2H), 6.70 (d, 2H),
6.17 (s, 2H). ESI-MS (+Q, m/z): 229 [M + H]+, 251 [M + Na]+.
2-((2-Aminobenzo[d]thiazol-6-yl)oxy)-5-fluorobenzonitrile (6a). To a solution of compound 5a (3 g,
13.2 mmol) in AcOH 20 mL was added KSCN (1.92 g, 19.8 mmol). The mixture was cooled to 0 °C
and a solution of Br2 (13.2 mmol) in AcOH (6 mL) was added. The mixture was then stirred at room
temperature for 8 h. The mixture was poured into water, basified with NH4OH (aq), and extracted with
EtOAc. The organic layer was washed with water and brine and dried over Na2SO4. The compound 6a
was purified by column chromatography petroleum ether/ethyl acetate (1:1) to give a yellow solid.
Yield: 62.5%. 1H-NMR (CDCl3, 400 MHz), δ: 7.88 (d, 1H), 7.67 (m, 1H), 7.50 (m, 1H), 7.30 (m, 1H),
7.18 (d, 1H), 7.15 (m, 1H), 7.01 (d, 2H). ESI-MS (+Q, m/z): 286 [M + H]+, 308 [M + Na]+.
6-(2-(Aminomethyl)-4-fluorophenoxy)benzo[d]thiazol-2-amine (7a). To 15mL of anhydrous THF was
added 0.35 g (9 mmol) LiAlH4 in a 50 mL bottle, the bottle was kept in ice bath. Take 0.85 g (3 mmol)

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compound 6a and dissolve in 15 mL anhydrous THF. Drip the solution into the LiAlH4 suspension
then stirred at 65 °C for 30–40 min. The mixture was added ethanol till no bubble generates.
Then filter and concentrate the suspension. The product was separated by column chromatograph
1
(dichloromethane/methanol 100:2), Yield: 73.7%. H-NMR (CDCl3, 400 MHz), δ: 8.70 (s, 1H), 7.85
(d, 1H), 7.65 (m, 1H), 7.48 (m, 1H), 7.30 (m, 1H), 7.18 (d, 1H), 7.15 (m, 1H), 7.01 (d, 2H), 4.36
(d, 1H). ESI-MS (+Q, m/z): 290 [M + H]+, 312 [M + Na]+.
1-(2-((2-Aminobenzo[d]thiazol-6-yl)oxy)-5-fluorobenzyl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea
(8b). The 6-(2-(aminomethyl)-4-fluorophenoxy)benzo[d]thiazol-2-amine 7a (1 g, 3.5 mmol) was put in
a 100 mL three-necked bottle and then dissolved in 50 mL DMSO. Then drip the 3a (1.7 g, 4.2 mmol)
and 1 mL Et3N into the bottle and heated to 85 °C for 1 h. The mixture was put into 200 mL water and
the water phase was extracted twice with ethyl acetate. The organic phases were combined and washed
with water and brine, dried with Na2SO4 and the solvent removed under vacuum to yield the crude
product, which was purified by column chromatography petroleum ether/ethyl acetate (5:1) to give
1
compound 8b as a yellow solid. Yield: 53.5%. H-NMR (DMSO, 400 MHz), δ: 8.58 (s, 1H), 8.32
(d, 2H), 7.82 (d, 2H), 7.41 (s, 2H), 7.25 (m, 3H), 7.06 (m, 2H), 6.98 (m, 1H), 6.85 (m, 1H), 6.32 (s, 1H),
4.29 (d, 2H), 1.27 (s, 9H). ESI-MS (+Q, m/z): 545 [M + H]+, 567 [M + Na]+. 145.1–147.2 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-ethylureido)benzo[d]thiazol-6-yl)oxy)-5-
fluorobenzyl)urea (9g). The 8b (1 g, 1.8 mmol) was put in a 100 mL three-necked bottle and then
dissolved in 50 mL DCM . Then drip the ethyl isocyanate (0.4 g, 5.4 mmol) into the bottle, and the
mixture was stirred at room temperature for 2 h. The mixture was put into 100 mL water and the water
phase was extracted twice with DCM. The organic phases were combined and washed with water and
brine, dried with Na2SO4 and the solvent removed under vacuum to yield the crude product, which
was purified by column chromatography petroleum ether/ethyl acetate (3:1) to give compound 9g as
a white solid. Yield: 88.7%. 1H-NMR (DMSO, 400 MHz), δ: 10.64 (s, 1H), 8.28 (s, 1H), 7.48 (d, 2H),
7.35 (d, 2H), 7.31 (s, 2H), 7.25 (m, 2H), 7.06 (m, 2H), 6.98 (m, 1H), 6.85 (m, 1H), 6.22 (s, 1H), 4.29
13
(d, 2H), 3.16 (m, 2H), 2.49 (s, 3H), 1.27 (s, 9H), 1.06 (m, 3H). C-NMR (100 MHz, DMSO d6), δ:
160.90 (1C, thiazole), 159.89 (1C, pyrazole), 157.51 (1C, CO), 155.05 (1C, CO), 154.19 (1C,
pyrazole), 153.16, 150.91, 138.02, 136.99, 136.80, 133.71, 133.35, 130.05, 124.56, 121.03, 120.74,
117.33, 115.63, 115.38, 115.06, 111.04 (18C, Ar-C), 96.22 (1C, pyrazole), 38.54 (1C, N-CH2), 34.80
(1C, N-CH2CH3), 32.49 (1C, CH3-C), 30.72 (3C, C-CH3), 21.09 (1C, Ar-CH3), 15.67 (1C, CH2-CH3).
ESI-MS (+Q, m/z): 616 [M + H]+, 638 [M + Na]+. mp 197.7–199.9 °C.
3-(Tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-amine (2b). The title compound was obtained similarly
1
to 2a. H-NMR (CDCl3, 400 MHz), δ: 8.40 (d, 2H), 8.15 (d, 2H), 5.50 (s, 1H), 4.72 (brs, 2H), 1.32
(s, 9H). ESI-MS (+Q, m/z): 261 [M + H]+, 283 [M + Na]+.
2,2,2-Trichloroethyl (3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)carbamate (3b). The title compound
was obtained similarly to 3a. 1H-NMR (CDCl3, 400 MHz), δ: 9.10 (s, 1H), 8.40 (d, 2H), 8.15 (d, 2H),
5.56 (s, 1H), 4.79 (s, 2H), 1.30 (s, 9H). ESI-MS (+Q, m/z): 435 [M + H]+, 457 [M + Na]+.

Molecules 2015, 20
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1
2-(4-Aminophenoxy)benzonitrile (5b). The title compound was obtained similarly to 5a. H-NMR
(CDCl3, 400 MHz), δ: 7.69 (m, 1H), 7.49 (m, 1H), 7.29 (m, 1H), 7.15 (d, 1H), 6.79 (d, 2H), 6.70 (d, 2H),
6.17 (s, 2H). ESI-MS (+Q, m/z): 211 [M + H]+, 233 [M + Na]+.
2-((2-Aminobenzo[d]thiazol-6-yl)oxy)benzonitrile (6b). The title compound was obtained similarly to 6a.
1H-NMR (CDCl3, 400 MHz), δ: 7.88 (d, 1H), 7.67 (m, 2H), 7.50 (m, 1H), 7.30 (m, 1H), 7.18 (d, 1H),
7.15 (m, 1H), 7.01 (d, 2H). ESI-MS (+Q, m/z): 268 [M + H]+, 290 [M + Na]+.
6-(2-(Aminomethyl)phenoxy)benzo[d]thiazol-2-amine (7b). The title compound was obtained similarly
1
to 7a. H-NMR (CDCl3, 400 MHz), δ: 8.70 (s, 1H), 7.85 (d, 1H), 7.65 (m, 1H), 7.48 (m, 2H), 7.28
(m, 1H), 7.18 (d, 1H), 7.15 (m, 1H), 6.98 (d, 2H), 4.34 (d, 1H). ESI-MS (+Q, m/z): 272 [M + H]+,
294 [M + Na]+.
1-(2-((2-Aminobenzo[d]thiazol-6-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)urea
(8a). The title compound was obtained similarly to 8b. 1H-NMR (DMSO, 400 MHz), δ: 8.58 (s, 1H), 8.32
(d, 2H), 7.82 (d, 2H), 7.41 (s, 2H), 7.25 (m, 4H), 7.06 (m, 2H), 6.99 (m, 1H), 6.87 (m, 1H), 6.32 (s, 1H),
4.29 (d, 2H), 1.27 (s, 9H). ESI-MS (+Q, m/z): 558 [M + H]+, 580 [M + Na]+. mp 135.2–137.9 °C.
Methyl(6-(2-((3-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)ureido)methyl)phenoxy)benzo[d]thiazol-
2-yl)carbamate (9a). The 8a (1 g, 1.8 mmol) was put in a 100 mL three-necked bottle and then dissolved
in 50 mL DCM . Then drip the methyl chloroformate (0.5 g, 5.4 mmol) into the bottle, and the mixture
was stirred at room temperature for 2 h. The mixture was put into 100 mL water and the water phase
was extracted twice with DCM. The organic phases were combined and washed with water and brine,
dried with Na2SO4 and the solvent removed under vacuum to yield the crude product, which was purified
by column chromatography petroleum ether/ethyl acetate (3:1) to give compound 9a as a white solid.
1
Yield: 85.6%. H-NMR (DMSO, 400 MHz), δ: 12.06 (s, 1H), 8.88 (s, 1H), 8.38 (d, 2H), 7.85 (d, 2H),
7.61 (s, 2H), 7.55 (m, 2H), 7.26 (m, 2H), 7.08 (m, 1H), 7.01 (m, 1H), 6.32 (s, 1H), 4.29 (d, 2H), 3.86
(s, 3H), 1.27 (s, 9H). ESI-MS (+Q, m/z): 616 [M + H]+, 638 [M + Na]+. mp 190.1–193.1 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-(tert-butyl)ureido)benzo[d]thiazol-6-yl)oxy)
benzyl)urea (9b). The title compound was obtained similarly to 9g. 1H-NMR (DMSO, 400 MHz), δ: 10.26
(s, 1H), 8.22 (s, 1H), 7.58 (d, 2H), 7.49 (d, 2H), 7.29 (m, 6H), 7.15 (m, 1H), 7.06 (m, 1H), 7.01 (m, 1H),
6.91 (m, 1H), 6.82 (s, 1H), 6.22 (s, 1H), 4.29 (d, 2H), 2.36 (s, 3H), 1.32 (s, 9H), 1.27 (s, 9H). 13C-NMR
(100 MHz, DMSOe d6), δ: 160.42 (1C, thiazole), 159.06 (1C, pyrazole), 154.87 (1C, CO), 154.35 (1C,
CO), 152.64 (1C, pyrazole), 152.24, 145.41, 137.74, 136.50, 136.24, 132.78, 130.27, 129.60, 129.05,
128.53, 124.22, 123.35, 120.54, 117.91, 117.41, 111.18 (18C, Ar-C), 95.05 (1C, pyrazole), 50.14 (N-C),
38.28 (1C,N-CH2 ), 32.00 (1C, CH3-C), 30.25 (3C, C-CH3), 28.74 (3C, C-CH3), 20.64 (1C, Ar-CH3).
ESI-MS (+Q, m/z): 626 [M + H]+, 648 [M + Na]+. mp 200.1–201.2 °C.
Methyl(6-(2-((3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)methyl)phenoxy)benzo[d]thiazol-2-yl)
carbamate (9c). The title compound was obtained similarly to 9a. 1H-NMR (DMSO, 400 MHz), δ:
12.06 (s, 1H), 8.25 (s, 1H), 7.68 (d, 2H), 7.55 (d, 2H), 7.48 (s, 2H), 7.35 (m, 2H), 7.26 (m, 2H), 7.08
(m, 1H), 7.01 (m, 1H), 6.22 (s, 1H), 4.31 (d, 2H), 3.76 (s, 3H), 2.35 (s, 3H), 1.24 (s, 9H). 13C-NMR

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(100 MHz, DMSOe d6), δ: 160.85 (1C, thiazole), 159.62 (1C, pyrazole), 155.12 (1C, CO), 154.91 (1C,
CO), 154.58 (1C, pyrazole), 153.28, 145.98, 138.31, 137.02, 136.67, 133.38, 130.87, 130.09, 129.53,
129.02, 124.73, 123.97, 121.71, 118.57, 118.21, 111.73 (18C, Ar-C), 95.74 (1C, pyrazole), 53.50 (1C,
O-CH3), 38.71 (1C, N-CH2), 32.49 (1C, CH3-C), 30.70 (3C, C-CH3), 21.13 (1C, Ar-CH3). ESI-MS
(+Q, m/z): 585 [M + H]+, 607 [M + Na]+. mp 183.1–185.7 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-ethylureido)benzo[d]thiazol-6-yl)oxy)benzyl)
1
urea (9d). The title compound was obtained similarly to 9g. H-NMR (DMSO, 400 MHz), δ: 10.66
(s, 1H), 8.22 (s, 1H), 7.68 (d, 2H), 7.55 (d, 2H), 7.48 (s, 2H), 7.41 (m, 2H), 7.35 (m, 1H), 7.30 (m, 1H),
7.29 (m, 1H), 7.26 (m, 1H), 7.23 (s, 1H), 6.22 (s, 1H), 4.31 (d, 2H), 3.23 (m, 2H), 2.35 (s, 3H), 1.24
(s, 9H), 0.96 (m, 3H). ESI-MS (+Q, m/z): 598 [M + H]+, 620 [M + Na]+. mp 186.5–188.1 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-(2-chloroethyl)ureido)benzo[d]thiazol-6-yl)oxy)
benzyl)urea (9e). The title compound was obtained similarly to 9g. 1H-NMR (DMSO, 400 MHz), δ: 10.86
(s, 1H), 8.26 (s, 1H), 7.58 (d, 2H), 7.51 (d, 2H), 7.45 (s, 2H), 7.41 (m, 2H), 7.35 (m, 1H), 7.30 (m, 1H),
7.29 (m, 1H), 7.26 (m, 1H), 7.23 (s, 1H), 6.22 (s, 1H), 4.31 (d, 2H), 3.73 (m, 2H), 3.63 (m, 2H), 2.35
(s, 3H), 1.24 (s, 9H). ESI-MS (+Q, m/z): 632 [M + H]+, 654 [M + Na]+. mp 192.5–195.1 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-cyclohexylureido)benzo[d]thiazol-6-yl)oxy)
1
benzyl)urea (9f). The title compound was obtained similarly to 9f. H-NMR (DMSO, 400 MHz), δ:
10.39 (s, 1H), 8.23 (s, 1H), 7.61 (d, 2H), 7.53 (d, 2H), 7.46 (s, 2H), 7.40 (m, 2H), 7.35 (m, 1H), 7.30
(m, 1H), 7.29 (m, 1H), 7.26 (m, 1H), 7.23 (s, 1H), 6.22 (s, 1H), 4.29 (d, 2H), 3.43 (m, 1H), 2.34 (s, 3H),
13
1.86 (m, 2H), 1.66 (m, 4H), 1.24 (s, 9H), 1.02 (m, 4H). C-NMR (100 MHz, DMSOe d6), δ: 160.44
(1C, thiazole), 159.32 (1C, pyrazole), 154.87 (1C, CO), 154.39 (1C, CO), 152.94 (1C, pyrazole),
152.30, 145.47, 137.76, 136.52, 136.27, 132.86, 130.29, 129.62, 129.03, 128.51, 124.22, 123.37, 120.56,
117.93, 117.43, 111.24 (18C, Ar-C), 95.10 (1C, pyrazole), 48.14 (1C, N-CH), 38.28 (1C, N-CH2),
33.42 (1C, cyclohexane), 32.63 (1C, cyclohexane) 32.02 (1C, CH3-C), 30.25 (3C, C-CH3), 25.14 (1C,
cyclohexane), 24.53 (1C, cyclohexane), 24.28 (1C, cyclohexane), 20.64(1C, Ar-CH3). ESI-MS (+Q,
m/z): 652 [M + H]+, 674 [M + Na]+. mp 171.3–173.3 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-cyclohexylureido)benzo[d]thiazol-6-yl)oxy)-
5-fluorobenzyl)urea (9h). The title compound was obtained similarly to 9g. 1H-NMR (DMSO, 400 MHz),
δ: 10.38 (s, 1H), 8.23 (s, 1H), 7.61 (d, 2H), 7.53 (d, 2H), 7.46 (s, 2H), 7.40 (m, 2H), 7.35 (m, 1H), 7.30
(m, 3H), 6.22 (s, 1H), 4.29 (d, 2H), 3.53 (m, 1H), 2.34 (s, 3H), 1.88 (m, 2H), 1.68 (m, 4H), 1.24 (s, 9H),
0.91 (m, 4H). δ: 160.41 (1C, thiazole), 159.43 (1C, pyrazole), 157.11 (1C, CO), 154.55 (1C, CO), 153.79
(1C, pyrazole), 152.96, 150.41, 137.51, 136.42, 136.20, 133.23, 129.95, 129.55, 124.00, 120.52, 120.14,
116.83, 115.11, 114.78, 114.09, 110.54 (18C, Ar-C), 95.72 (1C, pyrazole), 48.13(1C, N-CH), 38.24
(1C, N-CH2), 33.39 (1C, cyclohexane), 32.60 (1C, cyclohexane) 32.00 (1C, CH3-C), 30.22 (3C, C-CH3),
25.11 (1C, cyclohexane), 24.50 (1C, cyclohexane), 24.25 (1C, cyclohexane), 20.61(1C, Ar-CH3). ESI-MS
(+Q, m/z): 670 [M + H]+, 692 [M + Na]+. mp 170.2–172.3 °C.
1-(3-(Tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((2-(3-(tert-butyl)ureido)benzo[d]thiazol-6-yl)oxy)-
5-fluorobenzyl)urea (9i). The title compound was obtained similarly to 9g. 1H-NMR (DMSO, 400 MHz), δ:

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10.25 (s, 1H), 8.28 (s, 1H), 7.48 (d, 2H), 7.42 (d, 2H), 7.29 (m, 6H), 7.15 (m, 1H), 7.06 (m, 1H), 7.01
(m, 1H), 6.91 (m, 1H), 6.82 (s, 1H), 6.22 (s, 1H), 4.29 (d, 2H), 2.36 (s, 3H), 1.30 (s, 9H), 1.26 (s, 9H).
ESI-MS (+Q, m/z): 644 [M + H]+, 666 [M + Na]+. mp 164.7–166.9 °C.
Methyl(6-(2-((3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)methyl)-4-fluorophenoxy)benzo[d]
1
thiazol-2-yl)carbamate (9j). The title compound was obtained similarly to 9a. H-NMR (DMSO,
400 MHz), δ: 12.05(s, 1H), 8.24 (s, 1H), 7.68 (d, 2H), 7.55 (d, 2H), 7.48 (s, 2H), 7.35 (m, 2H), 7.26 (m,
1H), 7.08 (m, 1H), 7.01 (m, 1H), 6.22 (s, 1H), 4.31 (d, 2H), 3.73 (s, 3H), 2.35 (s, 3H), 1.23 (s, 9H).
13C-NMR (100 MHz, DMSOe d6), δ: 160.29 (1C, thiazole), 159.12 (1C, pyrazole), 157.16 (1C, CO),
154.76 (1C, CO), 154.51 (1C, pyrazole), 153.23, 150.18, 146.42, 145.37, 141.73, 137.84, 136.68, 135.95,
132.92, 129.58, 127.39, 124.13, 121.25, 117.17, 114.88, 1110.57 (18C, Ar-C), 96.23 (1C, pyrazole),
53.02 (1C, O-CH3), 37.98 (1C, N-CH2), 32.01 (1C, CH3-C), 30.16 (3C, C-CH3), 20.64 (1C, Ar-CH3).
ESI-MS (+Q, m/z): 603 [M + H]+, 625 [M + Na]+. mp 154.8–157.1 °C.
3.3. Pharmacology
3.3.1. In Vitro Pharmacological Activity. IC50 Determi Nation of Inhibition of TNF-α Release from
Isolated Human Peripheral Blood Mononuclear Cells (PBMCs) after LPS Stimulation
Peripheral venous blood from healthy, nonmedicated donors was collected using
ethylenediaminetetraacetic acid (EDTA) as the anticoagulant. For PBMC preparation, samples of
blood were diluted 1:1 with sterile phosphate buffered saline and then separated using SepMate tubes
(No. 15450) with 15 mL lymphoprep (No. 07851), centrifuged at 1200× g for 30 min. Buffy coat cells
were removed into PBS, centrifuged at 200× g for 10 min, and resuspended in PBMC assay buffer. A
differential white cell count was performed, and PBMCs were diluted to 10,000 lymphocytes per mL
in PBMC assay buffer. Test compounds were dissolved in DMSO and diluted in PBMC assay buffer to
cover an appropriate concentration range. Samples of test compound solution or vehicle (20 μL) were
added into 96-well tissue culture treated plates (Corning, Shanghai, China), and PBMC (160 μL) added
to each well. The assay mixtures were incubated at 37 °C for 1 h in a humidified incubator containing
an atmosphere of air supplemented with 5% CO2 before adding LPS (10 ng/mL). Plates were returned
to the incubator for a further 18 h and then centrifuged before recovery of samples of supernatant. TNF-α
in the samples was determined using an enzyme-linked immunosorbent assay (ELISA) (ebioscience
No. 88-7346). Dose response curves were constructed from which IC50 values were calculated. At least
n = 2 determinations were made from a single donor of PBMCs.
3.3.2. p38α MAP Kinase Activity was Assessed Based on the Rate of Phosphorylation of ATF-2
(Activation Transcription Factor 2) in an in Vitro Assay
1× kinase reaction buffer composition: 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl2
and 1 mM EGTA. Titration MAPK14/p38α at 90 μM ATP: Prepare MAPK14/p38α in 1× kinase buffer
with concentration at 500 ng/mL. Perform two-fold serial dilution using 1× kinase buffer from
500 ng/mL, 16 dose points. Add 5 μL of the serial diluted MAPK14/p38α into the 384-well plate in
triplicate. Prepare 1 mL of 0.8 μM substrate GFP-ATF2 (19-96) and 180 μM ATP in 1× kinase reaction

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buffer. Start the reaction by adding 5 μL of the substrate GFP-ATF2 (19-96) and ATP solution (prepared
at step iv.) into each well of the assay plate. Final starting concentration of MAPK14/p38α was 250 ng/mL;
final GFP-ATF2 and ATP concentration was 0.4 μM and 90μM. Seal the assay plate and incubate
for 1 h at room temperature (RT). Prepare 1 mL of antibody solution (20 mM EDTA and 4 nM
Tb-antipATF2 (pThr71) Antibody in TR-FRET dilution buffer. Add 10 μL of antibody solution into each
well of the assay plate and mix softly. Final EDTA concentration was 10 mM and final Tb-antipATF2
(pThr71) concentration was 2 nM. Seal the assay plate and incubate for 30 min at RT. Read TR-FRET
signal on Envision 2104 plate reader.
Determination of inhibitor IC50 value: Add 2 μL/well of inhibitor in 0.5% DMSO at 5-fold the final
assay concentration to the 384-well assay plate: For first cycle inhibitor screening, 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, 2 replicates for each dose). Adjust the inhibitor concentration according to the first cycle
result. Add 4 μL/well MAPK14/p38α to each well of the 384-well assay plate. Incubate for 15 min at
RT. To start the reaction, add 4 μL/well substrate GFP-ATF2 (19-96) and ATP in kinase reaction buffer.
(a) MAPK14/p38α final concentration: 1 ng/mL; (b) Substrate final concentration: 0.4 μM; (c) ATP final
concentration: 90 μM. Incubate for 1 h at RT. Add 10 μL/well of antibody solution. (a) EDTA final
concentration: 10 mM; (b) Antibody final concentration: 2 nM. Incubate for 30 min at RT. Read TR-FRET
signal on Envision 2104 plate reader.
Plot the resulting TR-FRET emission ratio against the concentration of inhibitor, and fit the data to
a sigmoidal dose-response curve with a variable slope. Calculate the IC50 concentration from the curve.
3.3.3. Kinase Selectivity Assay of Selected Compounds (9d, 9g)
1× kinase reaction buffer composition: 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl2 and
1 mM EGTA. Titration p38β/γ/δ: Prepare p38β/γ/δ in 1× kinase buffer with concentration at 2000 ng/mL.
Perform 3-fold serial dilution using 1×kinase buffer from 2000 ng/mL, 10 dose points. Add 5 μL of the
serial diluted p38β/γ/δ into the 384-well plate in two replicates. Prepare 2 × substrate GFP-ATF2 (19-96)
(0.6 μM/mL) and 10μMATP (p38 β), 6 μM ATP (p38γ/δ) in 1× kinase reaction buffer. Start the reaction
by adding 5 μL of the substrate GFP-ATF2 (19-96) and ATP solution (prepared at step iv.) into each
well of the assay plate. Final starting concentration of p38β/γ/δ was 1000 ng/mL; final GFP-ATF2
and ATP concentration was 0.3 μM and 5 μM ATP (p38β), 3 μM ATP (p38γ/δ). Sealthe assay plate
and incubate for 1 h at room temperature (RT). Prepare antibody solution (20 mM EDTA and 3 nM
Tb-antipATF2 (pThr71) Antibody in TR-FRET dilution buffer). Add 10 μL of antibody solution into
each well of the assay plate and mix softly. Final EDTA concentration was 10 mM and final Tb-antipATF2
(pThr71) concentration was 1.5 nM. Sealthe assay plate and incubate for 30 min at RT. Read TR-FRET
signal on Envision 2104 plate reader.
Determination of inhibitor IC50 value: Add 4μL/well p38β/γ/δ to each well of the 384-well assay
plate. Add 2 μL/well of inhibitor in 0.5% DMSO at 5-fold the final assay concentration to the 384-well
assay plate. For p38β, the final concentrations of inhibitors were 1111, 370, 123, 41, 13.7, 4.57, 1.52,
0.51, 0.17 and 0.056 nM (3 fold dilution, 10 dose points, 2 replicates for each dose). For p38γ/δ, the
final concentrations of inhibitors were 10,000, 3333, 1111, 370, 123, 41, 13.7, 4.57, 1.52 and 0.51 nM
(3 fold dilution, 10 dose points, 2 replicates for each dose). Incubate for 15 min at RT. To start the

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reaction, add 4 μL/well substrate GFP-ATF2 (19-96) and ATP in kinase reaction buffer. (a) p38β/γ/δ final
concentration: 50 ng/mL, 140 ng/mL, 25 ng/mL; (b) Substrate final concentration: 0.3 μM; (c) ATP final
concentration for p38β/γ/δ: 5 μM, 3 μM, 3 μM; (d) Final DMSO: 0.1%. Incubate for 1 h at RT. Add
10 μL/well of antibody solution. (a) EDTA final concentration: 10 mM; (b) Antibody final concentration:
1.5 nM. Incubate for 30 min at RT. Read TR-FRET signal on Envision 2104 plate reader.
Plot the resulting TR-FRET emission ratio against the concentration of inhibitor, and fit the data to
a sigmoidal dose-response curve with a variable slope. Calculate the IC50 concentration from the curve.
3.3.4. Docking (Discovery Studio)
Prepare Receptor: Firstly, download the PDB files (1KV2) at http://www.rcsb.org, add hydrogen
atom and electric charge after clearing the water of the protein. Secondly, define a active site using
BIRB-796 as a template.
Prepare Ligand: Draw the structure (9g) with chemdraw12.0 and minimize the molecule energy
using the function (generate conformations).
Molecular Docking: Run the docking and select the best conformation (Figure 3) and display
hydrogen bonds according to the docking results.
4. Conclusions
In summary, we have designed and synthesized a novel series of substituted N,N′-diaryl ureas
compounds that are p38α inhibitors. A wide variety of substituents can be tolerated, and most compounds
possessed good inhibitory potencies. However, owing to the limited number of compounds, we can
only provide a limited description of the structure-activity relationship. Among these compounds, 8b,
9d and especially 9g appeared to be the most potent ones, and will be the key compound that is studied
in the future. The activity results indicated that this novel substituted N,N′-diaryl ureas compounds
designed based on the two primary differences between p38αMAPK and other kinases could improve
the activity of p38αMAPK inhibitors and may serve as a novel chemotype for the development of
p38αMAPK inhibitors. Research on the p38 kinase biological actions of these compounds is ongoing,
and results will be reported in due course.
Acknowledgments
We gratefully acknowledge financial support from the program for innovative research team of
the ministry of education and program for Liaoning innovative research team at university and the
National Nature Science Foundation of China (Grant No. 81473139).
Author Contributions
Wu Zhong, Dongmei Zhao and Dianxi Zhu conceived and designed the experiments; Dianxi Zhu
performed the experiments; Xingzhou Li analyzed the data; Dianxi Zhu wrote the paper; All authors
read and approved the final manuscript.

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Conflicts of Interest
The authors declare no conflict of interest.
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