Synthesis and biological evaluation of 1-(2-aminophenyl)-3-arylurea derivatives as potential EphA2 and HDAC dual inhibitors

Article abstract of DOI:10.1248/cpb.c16-00154

A series of 1-(2-aminophenyl)-3-arylurea novel derivatives were synthesized and evaluated against Ephrin type-A receptor 2 (EphA2) and histone deacetylases (HDACs) kinase. Most of the compounds exhibited inhibitory activity against EphA2 and HDAC. The antiproliferative activities were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (thiazolyl blue, tetrazolium blue) against the human cancer cell lines HCT116, K562 and MCF7. Compounds 5a and b showed the most potent inhibitory activity against EphA2 and HDAC. However, compound 5b exhibited higher potency against HCT116 (IC₅₀=5.29 μM) and MCF7 (IC₅₀=7.42 μM). 1-(2-Aminophenyl)-3-arylurea analogues may serve as new EphA2-HDAC dual inhibitors.

Full text of DOI:10.1248/cpb.c16-00154

1136
Chem. Pharm. Bull. 64, 1136–1141 (2016)
Vol. 64, No. 8
Regular Article
Synthesis and Biological Evaluation of 1-(2-Aminophenyl)-3-arylurea
Derivatives as Potential EphA2 and HDAC Dual Inhibitors
,a
Yong Zhu,a Ting Ran,b Xin Chen,a Jiaqi Niu,a Shuang Zhao,a Tao Lu,a,c and Weifang Tang*
a Department of Organic Chemistry, China Pharmaceutical University; Nanjing 210009, P. R. China: b Laboratory of
Molecular Design and Drug Discovery, School of Sciences, China Pharmaceutical University; Nanjing 210009, P. R.
c
China: and State Key Laboratory of Natural Medicines, China Pharmaceutical University; Nanjing 210009, P. R.
China.
Received February 17, 2016; accepted April 6, 2016
A series of 1-(2-aminophenyl)-3-arylurea novel derivatives were synthesized and evaluated against Eph-
rin type-A receptor 2 (EphA2) and histone deacetylases (HDACs) kinase. Most of the compounds exhibited
inhibitory activity against EphA2 and HDAC. The antiproliferative activities were evaluated by 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (thiazolyl blue, tetrazolium blue) against the
human cancer cell lines HCT116, K562 and MCF7. Compounds 5a and b showed the most potent inhibi-
tory activity against EphA2 and HDAC. However, compound 5b exhibited higher potency against HCT116
(IC50=5.29µM) and MCF7 (IC50=7.42 µM). 1-(2-Aminophenyl)-3-arylurea analogues may serve as new
EphA2–HDAC dual inhibitors.
Key words Ephrin type-A receptor 2 inhibitor; histone deacetylase inhibitor; synthesis; antiproliferative
activity; dock
Mechanism-based targeted therapies as a treatment for tu- sion.14) Histone acetyltransferases (HATs) catalyze the acety-
mors has been proclaimed as one of the major breakthroughs lation of lysine residues on histone and non-histone proteins.
in human cancer research in the past several decades. How- The reverse reaction is catalyzed by histone deacetylases
ever, the clinical effectiveness of such treatment is gener- (HDACs), thus promoting a more closed chromatin structure
ally unsustainable in the long run and relapses are almost where transcription is repressed.15,16) HDAC inhibitors have
inevitable after the treatment.1) One explanation is that, the been applied to treatment of human cancers.17,18) Zolinza (vori-
core hallmark capabilities of cancer are regulated by partially nostat, SAHA) was approved by the Food and Drug Adminis-
redundant signaling molecules. As such, a targeted therapeu- tration (FDA) for the treatment of advanced cutaneous T-cell
tic agent inhibiting only one key molecule in a tumor may lymphoma (CTCL).19) A benzamide HDAC inhibitor, MS275,
not completely shut off the core hallmark capability, allow- is reported to be a selective HDAC inhibitor targeting class
ing some cancer cells to survive with residual function.1) To I HDACs, which is a moderately potent HDAC inhibitor in
address this problem, the medicine will have to incorporate, phase II clinical trial.20) Several small molecule HDAC inhibi-
within a single molecule, elements that simultaneously tackle tors have entered clinical trials for the treatment of a variety
multiple targets for cancer therapy.2)
of haematological and solid tumors.21) Preclinical data with
The erythropoietin-producing hepatocellular (Eph) recep- numerous cancer cell lines has shown synergistic and additive
tor tyrosine kinases (RTKs) has been recognized increasingly effects when combining HDAC inhibitors with various anti-
as key regulators of both normal development and disease tumor therapies, suggesting HDAC might be an ideal target
including organ development, tissue remodeling, neuronal for combination.22)
signaling, vascular development and tumor progression.3–7)
HDAC inhibitors have been shown to synergize with other
To date, Eph receptor tyrosine kinases are classified in the agents, including RTK inhibitors, to suppress proliferation
EphA (EphA1–10) and EphB (EphB1–6) subclasses based on and induce apoptosis in tumor cells.23,24) This evidence sug-
sequence homology and binding affinity for ephrins.8) One gests that simultaneous EphA2 activation and HDAC in-
family member in particular, the EphA2 was identified from hibition could be a promising approach in cancer therapy.
human epithelial cDNA library and widespread expression Here, compound N-(4-(3-(2-aminophenyl)ureido)phenyl)-4-
in epithelial cells.9) An oncogenic role for EphA2 has been methylbenzamide (5e) was disclosed to inhibit EphA2 and
suggested due to its overexpression in breast, prostate, lung HDAC based on the virtual screening. Then we designed a se-
cancer as well as malignant melanoma.10) The level of EphA2 ries of 1-(2-aminophenyl)-3-arylurea derivatives and a simple
expression on tumor cells correlates with the degree of malig- and convenient route was applied for synthesis of compounds
nant progression and poor prognosis.11) The possibility of tar- 5a–h (Chart 1). In addition, biological assay showed some
geting EphA2/ephrin therapeutically may be the most straight- compounds exhibited potential antiproliferative activity across
forward in the context of inhibiting Eph/ephrins signaling in a range of human cancer cell lines.
the vasculature as a means of preventing tumor angiogenesis.7)
Currently only a few small molecule EphA2 inhibitors have
Results and Discussion
Chemistry The substituted aromatic acid was treated with
been reported in the literature.12,13)
The reversible acetylation of lysine residues in histone tails thionyl chloride, affording the corresponding acyl chloride 1.
plays a critical role in transcriptional activation and repres- The reaction was conducted using 1–2 drops of N,N-dimethyl-
*To whom correspondence should be addressed. e-mail: twf@cpu.edu.cn
© 2016 The Pharmaceutical Society of Japan

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1137
Reagents and conditions: (a) SOCl2, DMF, reflux, 4h; (b) 4-nitroaniline, TEA, 0°C; (c) Fe, NH4Cl, C2H5OH, H2O, reflux, 6h; (d) 1-isocyanato-2-nitrobenzene, toluene,
reflux, 3h; (e) Fe, HOAc, 95% C2H5OH, reflux, 6h.
Chart 1
formamide (DMF) as the catalyst under anhydrous conditions.
After the thionyl chloride was evaporated, the crude product
was used for the next step without purification. Then the acyl
Table 1. The Structures and Inhibitory Activities of Compounds 5a–h,
Dasatinib and MS-275 against EphA2 and HDACs
chloride 1 was reacted with 4-nitroaniline in dry dichloro-
methane in the presence of anhydrous triethylamine at 0°C to
afford the corresponding amide 2. Reduction with iron powder
in water/ethanol gave desired amine 3. Compound 3 reacted
with 1-isocyanato-2-nitrobenzene to afford the urea 4. The
IC50 (µM)
target compounds 5a–h were synthesized by reduction of the
corresponding urea 4 with iron powder in 95% ethanol.
Pharmacology To study biological activity, compounds
5a–h were evaluated against EphA2 kinase activity assays
with Dasatinib as positive control and HDACs enzyme assay
with MS-275 as positive control. EphA2 was expressed from
CHO-CK cell-line and HDACs were prepared from HeLa cell
extracts. Compounds 5a–h were initially screened at final
concentrations of 10.0, 1.0 and 0.1µM and an enzymatic assay
measuring total HDACs activity. IC50s of compounds 5a–h
and the reference compounds Dasatinib and MS-275 were
calculated according to inhibitory rates, the in vitro data for
enzymatic inhibition are listed in Table 1.
The data in Table 1 demonstrated that compounds 5a–h
exhibited the inhibitory activity against EphA2 and HDACs,
especially compound 5a (IC50=0.68µM against EphA2,
IC50=0.94µM against HDACs) and 5b (IC50=0.57 µM against
EphA2, IC50=1.52 µM against HDACs). Compounds 5b–d,
h relatively exhibited higher inhibitory activities than that
of substituted aryl compounds against EphA2. These results
suggest that methoxy groups at benzene ring are favorable
for EphA2 inhibition. The activity of compound 5c against
EphA2 was found to be less potent than 5b, d and h. This
means that the methoxy group at 2-position is unfavorable for
EphA2 inhibition. The inhibitory activity against EphA2 of
compound 5a was close to that of compounds 5b and d, so
it means that the pyridine is an important group for EphA2
inhibition.
Compound
R
EphA2
HDACs
0.94
5a
0.68
0.57
5b
5c
1.52
29.1
1.89
5d
5e
0.51
5.72
12.3
2.54
5f
8.20
9.33
15.5
6.43
5g
5h
0.79
18.5
Dasatinib
MS-275
0.43
Nt
Nt
As shown in Table 1, compounds 5a–h exhibited inhibitory
activity against HDACs. Compared with reference compound
MS-275, all the synthesized compounds inhibit HDACs activ-
ity with reduced potency. Only the activity of compound 5a
(IC50=0.94µM) against HDACs was close to that of MS-275
(IC50=0.83µM). It seems that the effect of pyridine substitut-
ing benzene ring can obviously affect EphA2 and HDACs
inhibitory activity. Compound 5b (IC50=1.52 µM) also had
0.83
Nt: Not tested.

1138
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
higher activity against HDACs than other substituted phenyl (hinge), of the ureido NH with the side chain T692 (hinge)
compound.
are reproduced. The 2-aminophenyl moiety is aligned with a
To test the anticancer activities of the synthesized com- hydrophobic pocket composed of I690, G663, M667 and L646.
pounds we evaluated antiproliferative activities of compounds 4-Methoxybenzene moiety is at the entrance of the active site.
5a–h against human colon carcinoma cell line (HCT116), As shown in Fig. 1B, the carbonyl and 2-aminophenyl NH are
erythroleukemic cell line (K562) and breast cancer cell line coordinated with the catalytic zinc ion. The hydrogen bonds
(MCF7) by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5- of 2-amino substituent with the backbone of A168, of the
diphenyltetrazolium bromide (MTT) colorimetric assay. The ureido NH with the carbonyl group of G140 may contribute
results were summarized in Table 2.
to the tight fit. The phenyl ring of the ureidophenyl moiety is
Compounds 5a, b, g and h exhibited potential effects stacked between P141, P198 and H170.
against HCT116, K562 and MCF7 in vitro (Table 2). In the
case of against K562, all synthesized compounds showed less
inhibitory activities than reference compounds. When tested
Conclusion
In summary, a series of 1-(2-aminophenyl)-3-arylurea de-
against HCT116 and MCF7, compound 5b displayed enhanced rivatives were synthesized efficiently as novel HDAC–EphA2
inhibition (IC50=5.29 µM against HCT116) as compared with dual inhibitors. As expected, these compounds exhibited
Dasatinib (IC50=5.50 µM against HCT116) and compounds distinct inhibitory activity against HDAC and EphA2 and
5a (IC50=15.38 µM against MCF7), 5b (IC50=7.42 µM against antiproliferative activities in vitro. Compounds 5a, b, g and
MCF7), 5d (IC50=7.55 µM against MCF7), 5g (IC50=10.81 µM h exhibited potential effects against HCT116, K562 and
against MCF7) and 5h (IC50=9.17 µM against MCF7) showed MCF7 in vitro. Especially compound 5b exhibited higher po-
a higher potency than MS-275 (IC50=16.69 µM against MCF7). tency against HCT116 than Dasatinib and against MCF7 than
Docking Studies of Compound 5b on EphA2 as Well MS275. According to the docking study, the binding mode
as on HDAC The binding mode of the representative com- was discussed. The results suggested that 1-(2-aminophenyl)-
pound 5b to the EphA2 and HDAC are shown in Fig. 1. It 3-phenylurea motif offered us some useful information to lead
was possible to dock compound 5b in an energetically favor- future anticancer design efforts targeting EphA2 and HDAC
able conformation into the model (Fig. 1A). The hydrogen proteins.
bonds of the benzoylamido NH with the backbone of M695
Experimental
Chemistry All chemicals were reagent grade and pur-
Table 2. The Antiproliferative Activities of Compounds 5a–h and Refer-
chased from commercial suppliers. Melting points were deter-
ence Compounds
mined in open capillaries on a WRS-1A digital melting point
apparatus (Shenguang). The NMR spectra were recorded on
a Bruker Avance 500 (Bruker), using tetramethylsilane, or
trimethylsilyl (TMS) as the internal standard. The chemical
shifts are reported in ppm (δ) and coupling constants (J) val-
ues are given in Hertz (Hz). The IR spectra were performed
on a FTIR-8400S (Shimadzu) in KBr pellets; the frequencies
are expressed in cm−1. Mass spectra were obtained from Agi-
lent 1100 LC/MSD (Agilent) or Q-tof micro MS (Micromass)
and the high-resolution (HR) electrospray ionization-time of
flight (ESI-TOF)-MS was recorded on Agilent 6224A (TOF)
LC/MS.
IC50 (µM)
Compound
HCT116
K562
MCF7
5a
5b
32.17
5.29
8.36
9.17
15.38
7.42
5c
>100
9.31
>100
>100
65.42
>100
26.54
68.13
0.022
0.989
>100
7.55
>100
>100
10.81
5d
5e
85.21
>100
19.87
6.36
5f
5g
5h
9.17
3.90
Dasatinib
MS-275
5.50
General Synthesis of N-(4-Nitrophenyl)arylamide (2)
The substituted aromatic acid (1.0mmol), DMF (0.05mL)
0.76
16.69
Fig. 1. Docking of Compound 5b at EphA2 (A) and HDAC (B)
Compound 5b was shown in light gray. EphA2 and HDAC is represented by ribbons with the interacting residues represented as sticks. Hydrogen-bonding interactions
and coordination with zinc ion are drawn as dashed lines.

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1139
and SOCl2 (6.0mL) were mixed and stirred at reflux for 4h,
then cooled and evaporated to give reactive acyl chloride (4e)
(1), respectively. A solution of acyl chloride 1 (1.0mmol) in
4-Methyl-N-(4-(3-(2-nitrophenyl)ureido)phenyl)benzamide
Yellow powder, yield: 60%; mp: 249–251°C; 1H-NMR
CH2Cl2 (5.0mL) was added dropwise to the solution of 4-ni- (DMSO-d6, 500MHz): δ, ppm: 10.15 (s, 1H), 9.93 (s, 1H),
troaniline (1.0mmol) and triethylamine (0.5mL) in 20mL 9.82 (s, 1H), 8.34–8.31 (m, 1H), 8.12–8.09 (m, 1H), 7.75–7.67
CH2Cl2 at 0°C. After stirring overnight at room temperature, (m, 3H), 7.55 (d, J=7.9Hz, 2H), 7.43–7.40 (m, 1H), 7.24–7.16
the reaction mixture was then poured in excess of diluted (m, 4H), 2.53 (s, 3H); HR-ESI-MS m/z: 413.1232 (Calcd for
NaOH and extracted with CH2Cl2. The extraction liquid was C19H17N5O2Na [M+Na]+: 413.1220).
purified by a flash chromatography with petroleum ether–
EtOAc (10:1, v/v) to give N-(4-nitrophenyl)arylamide as a (4f)
yellow powder.
3-Methyl-N-(4-(3-(2-nitrophenyl)ureido)phenyl)benzamide
Yellow powder, yield: 58%; mp: 235–238°C; 1H-NMR
General Synthesis of N-(4-Aminophenyl)arylamide (3)
(DMSO-d6, 500MHz): δ, ppm: 10.17 (s, 1H), 9.94 (s, 1H),
To a solution of 2 (1.0mmol) in H2O (5mL) were added 9.84 (s, 1H), 8.33–8.29 (m, 1H), 8.10–8.07 (m, 1H), 7.81–7.70
NH4Cl (0.1mmol), iron powder (3.0mmol) and EtOH (0.5mL). (m, 3H), 7.56 (d, J=8.0Hz, 2H), 7.41–7.38 (m, 1H), 7.26–7.18
The mixture was stirred at reflux for 6h, and filtered. The (m, 4H), 2.53 (s, 3H); HR-ESI-MS m/z: 413.1239 (Calcd for
solution was extracted with EtOAc, and the organic layer C19H17N5O2Na [M+Na]+: 413.1220).
was washed with saturated brine and dried over MgSO4. The
solvent was removed under reduced pressure. The residue (4g)
was purified by a flash chromatography with petroleum ether–
4-Chloro-N-(4-(3-(2-nitrophenyl)ureido)phenyl)benzamide
Yellow powder, yield: 51%; mp: 285–287°C; 1H-NMR
EtOAc (2:1, v/v) to give N-(4-aminophenyl)arylamide as a (DMSO-d6, 500MHz): δ, ppm: 10.14 (s, 1H), 9.95 (s, 1H), 9.84
white powder. (s, 1H), 8.32–8.29 (m, 1H), 8.10–8.07 (m, 1H), 7.86–7.83 (m,
General Synthesis of N-(4-(3-(2-Nitrophenyl)ureido)phenyl)- 2H), 7.71–7.68 (m, 1H), 7.56–7.43 (m, 4H), 7.41–7.38 (m, 1H),
arylamide (4) 7.17 (d, J=8.0Hz, 2H); HR-ESI-MS m/z: 433.0692 (Calcd for
1-Isocyanato-2-nitrobenzene (1.0mmol) was added to a so- C19H17N5O2Na [M+Na]+: 433.0674).
lution of 3 (1.0mmol) in dry toluene (10mL). The mixture was
3,4,5-Trimethoxy-N-(4-(3-(2-nitrophenyl)ureido)phenyl)-
refluxed for 3h. The solvent was removed under reduced pres- benzamide (4h)
sure, and the mixture was washed with CH2Cl2. The product
Yellow powder, yield: 52%; mp: 232–234°C; 1H-NMR
was recrystallized from tetrahydrofuran (THF) as a yellow (DMSO-d6, 500MHz): δ, ppm: 1H-NMR (DMSO-d6,
solid.
N-(4-(3-(2-Nitrophenyl)ureido)phenyl)nicotinamide (4a)
500MHz): δ, ppm: 10.32 (s, 1H), 9.93 (s, 1H), 9.83 (s, 1H),
8.33–8.29 (m, 1H), 8.12–8.09 (m, 1H), 7.71–7.68 (m, 1H), 7.54
Yellow powder, yield: 57%; mp: 263–266°C; 1H-NMR (d, J=8.0Hz, 2H), 7.42–7.38 (m, 1H), 7.25–7.18 (m, 4H), 3.88
(dimethyl sulfoxide (DMSO)-d6, 500MHz): δ, ppm: 10.42 (s, (s, 6H), 3.79 (s, 3H); HR-ESI-MS m/z: 489.1393 (Calcd for
1H), 9.92 (s, 1H), 9.83 (s, 1H), 8.78 (s, 1H), 8.32–8.29 (m, C19H17N5O2Na [M+Na]+: 489.1381).
2H), 8.13–8.10 (m, 1H), 7.70–7.62 (m, 2H), 7.54–7.40 (m, 3H),
General Synthesis of N-(4-(3-(2-Aminophenyl)ureido)-
7.36 (d, J=8.0 HZ, 1H), 7.30–2.18 (m, 2H); HR-ESI-MS m/z: phenyl)arylamide (5)
400.1023 (Calcd for C19H17N5O2Na [M+Na]+: 400.1016).
To a solution of 4 (1.0mmol) in 95% EtOH (5mL) was
4-Methoxy-N-(4-(3-(2-nitrophenyl)ureido)phenyl)benzamide added iron powder (3.0mmol) and HOAc (0.1mL). The mix-
(4b)
ture was stirred at reflux for 6h, and filtered. The solvent was
Yellow powder, yield: 58%; mp: 276–278°C; 1H-NMR removed under reduced pressure, and the residue was purified
(DMSO-d6, 500MHz): δ, ppm: 10.14 (s, 1H), 9.93 (s, 1H), by a flash chromatography with CH2Cl2–MeOH (10:1, v/v) to
9.82 (s, 1H), 8.36–8.32 (m, 1H), 8.15–8.13 (m, 1H), 7.87 (d, give N-(4-(3-(2-aminophenyl)ureido)phenyl)arylamide.
J=8.0Hz, 2H), 7.72–7.68 (m, 1H), 7.56 (d, J=8.0Hz, 2H),
7.42–7.39 (m, 1H), 7.21–7.10 (m, 4H), 3.94 (s, 3H); HR-ESI-MS
m/z: 429.1185 (Calcd for C19H17N5O2Na [M+Na]+: 429.1169).
N-(4-(3-(2-Aminophenyl)ureido)phenyl)nicotinamide (5a)
White powder, yield: 84%; mp: 239–243°C; 1H-NMR
(DMSO-d6, 500MHz): δ, ppm: 10.34 (s, 1H), 9.10 (d, J=2.0Hz,
2-Methoxy-N-(4-(3-(2-nitrophenyl)ureido)phenyl)benzamide 1H), 8.76–8.74 (m, 2H), 8.28 (d, J=8.1Hz, 1H), 7.70–7.65 (m,
(4c)
3H), 7.58–7.54 (m, 1H), 7.44 (d, J=8.9Hz, 2H), 7.34 (dd,
Yellow powder, yield: 55%; mp: 276–278°C; 1H-NMR J=7.9Hz and 1.4Hz, 1H), 6.84 (dt, J=8.5Hz and 1.5Hz, 1H),
(DMSO-d6, 500MHz): δ, ppm: 10.09 (s, 1H), 9.90 (s, 1H), 6.74 (dd, J=7.8Hz and 1.4Hz, 1H), 6.58 (dt, J=7.8Hz and
9.78 (s, 1H), 8.33–8.30 (m, 1H), 8.10–8.07 (m, 1H), 7.72–7.68 1.5Hz, 1H), 4.78 (s, 2H); 13C-NMR (DMSO-d6, 125MHz):
(m, 1H), 7.60–7.45 (m, 4H), 7.43–7.40 (m, 1H), 7.21–7.10 δ, ppm: 168.1, 157.9, 151.5, 149.1, 148.2, 138.3, 137.8, 135.3,
(m, 4H), 3.95 (s, 3H); HR-ESI-MS m/z: 429.1177 (Calcd for 134.6, 129.7, 129.1, 128.9, 127.8, 126.5, 121.2, 119.4, 117.6;
C19H17N5O2Na [M+Na]+: 429.1169).
HR-ESI-MS m/z: 370.1297 (Calcd for C19H17N5O2Na [M+Na]+:
3-Methoxy-N-(4-(3-(2-nitrophenyl)ureido)phenyl)benzamide 370.1274); IR (KBr, cm−1): 3298, 1643, 1572, 1510, 1312, 1232,
(4d)
Yellow powder, yield: 49%; mp: 225–228°C; 1H-NMR
835, 746, 710, 660.
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-4-methoxy-
(DMSO-d6, 500MHz): δ, ppm: 10.22 (s, 1H), 9.91 (s, 1H), 9.80 benzamide (5b)
(s, 1H), 8.35–8.32 (m, 1H), 8.14–8.10 (m, 1H), 7.74–7.70 (m,
White powder, yield: 85%; mp: 230–232°C; 1H-NMR
3H), 7.57–7.53 (m, 2H), 7.42–7.40 (m, 1H), 7.19–7.05 (m, 3H), (DMSO-d6, 500MHz): δ, ppm: 10.02 (s, 1H), 8.83 (s, 1H),
6.75–6.72 (m, 1H), 3.95 (s, 3H); HR-ESI-MS m/z: 429.1182 7.97–7.67 (m, 4H), 7.85 (s, 1H), 7.43–6.81 (m, 7H), 6.65 (s,
(Calcd for C19H17N5O2Na [M+Na]+: 429.1169).
1H), 4.03 (s, 3H); 13C-NMR (DMSO-d6, 125MHz): δ, ppm:
170.4, 169.1, 157.7, 155.5, 139.7, 138.5, 134.4, 129.5, 129.0,

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Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
128.1, 125.8, 125.0, 123.9, 122.8, 118.4, 60.5; HR-ESI-MS m/z: (DMSO-d6, 500MHz): δ, ppm: 10.19 (s, 1H), 8.96 (s, 1H), 7.98
399.1461 (Calcd for C21H20N4O3Na [M+Na]+: 399.1428); IR (d, J=8.5Hz, 2H), 7.94 (s, 1H), 7.64 (d, J=8.5Hz, 2H), 7.59
(KBr, cm−1): 3348, 1639, 1547, 1510, 1398, 1250, 837, 756, 675, (d, J=8.5Hz, 2H), 7.43 (d, J=8.5Hz, 2H), 7.36 (d, J=9.0Hz,
572.
1H), 6.83 (t, J=8.0Hz, 1H), 6.73 (d, J=6.5Hz, 1H), 6.57 (t,
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-2-methoxybenz- J=7.5Hz, 1H), 4.80 (s, 2H); 13C-NMR (DMSO-d6, 125MHz):
amide (5c)
δ, ppm: 170.9, 157.9, 155.0, 144.4, 139.9, 138.7, 135.4, 135.1,
White powder, yield: 80%; mp: 202–206°C; 1H-NMR 133.5, 131.2, 130.1, 127.2, 126.5, 124.1, 123.9, 118.6; HR-
(DMSO-d6, 500MHz): δ, ppm: 9.97 (s, 1H), 8.74 (s, 1H), 7.71 ESI-MS m/z: 403.0968 (Calcd for C20H17ClN4O2Na [M+Na]+:
(s, 1H), 7.66 (dd, J=7.5Hz and 1.5Hz, 1H), 7.62 (d, J=9.0Hz, 403.0932); IR (KBr, cm−1): 3425, 2920, 1645, 1533, 1462, 1313,
2H), 7.49 (dt, J=8.5Hz and 1.5Hz, 1H), 7.40 (d, J=9.0Hz, 1018, 837, 750, 648.
2H), 7.34 (dd, J=8.0Hz and 1.0Hz, 1H), 7.17 (d, J=8.5Hz,
1H), 7.06 (t, J=7.5Hz, 1H), 6.84 (t, J=7.5Hz, 1H), 6.73 (dd, benzamide (5h)
J=7.5Hz and 1.0Hz, 1H), 6.57 (dt, J=7.5Hz, 1H), 4.76 (s,
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-3,4,5-trimethoxy-
White powder, yield: 78%; mp: 197–200°C; 1H-NMR
2H), 3.91 (s, 3H); 13C-NMR (DMSO-d6, 125MHz): δ, ppm: (DMSO-d6, 500MHz): δ, ppm: 10.00 (s, 1H), 8.77 (s, 1H),
169.8, 162.5, 158.1, 155.5, 139.1, 138.5, 138.0, 133.6, 130.7, 7.77 (s, 1H), 7.61 (d, J=9.0Hz, 2H), 7.43 (d, J=9.0Hz,
130.2, 129.2, 126.0, 125.6, 125.0, 123.3, 122.4, 119.2, 115.6, 2H), 7.36–7.35 (m, 1H), 7.3 (s, 2H), 6.84 (dt, J=8.5Hz and
58.8; HR-ESI-MS m/z: 399.1455 (Calcd for C21H20N4O3Na 1.5Hz, 1H), 6.74 (dd, J=8.0Hz and 1.5Hz, 1H), 6.56 (dt,
[M+Na]+: 399.1428); IR (KBr, cm−1): 3352, 1647, 1553, 1514, J=7.5Hz and 1.0Hz, 1H), 4.77 (s, 2H), 3.87 (s, 6H), 3.73 (s,
1402, 1244, 1022, 837, 752, 667.
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-3-methoxybenz- 158.1, 155.1, 148.8, 140.6, 138.7, 130.6, 130.1, 129.5, 127.8,
3H); 13C-NMR (DMSO-d6, 125MHz): δ, ppm: 171.4, 159.2,
amide (5d)
126.8, 123.6, 123.0, 119.4, 111.3, 65.2, 60.6; HR-ESI-MS m/z:
White powder, yield: 80%; mp: 213–216°C; 1H-NMR 459.1647 (Calcd for C23H24N4O5Na [M+Na]+: 459.1639); IR
(DMSO-d6, 500MHz): δ, ppm: 10.12 (s, 1H), 8.74 (s, 1H), (KBr, cm−1): 3354, 1738, 1645, 1551, 1514, 1483, 1307, 742,
7.70–7.64 (m, 3H), 7.53 (d, J=7.7Hz, 1H), 7.48–7.41 (m, 4H), 667, 521.
7.34 (d, J=6.9Hz, 1H), 7.14 (dd, J=8.2Hz and 2.4Hz, 1H),
EphA2 Inhibition Assay The inhibitory activities of
6.84 (dt, J=8.0Hz and 1.4Hz, 1H), 6.74 (d, J=6.5Hz, 1H), the compounds to EphA2 were determined by using a time
6.56 (t, J=8.0Hz, 1H), 4.79 (s, 2H), 3.84 (s, 3H); 13C-NMR resolved fluorescence resonance energy transfer (TR-FRET)
(DMSO-d6, 125MHz): δ, ppm: 171.1, 165.7, 158.0, 154.9, 139.8, assay. To evaluate inhibitory activity of compounds, the
139.0, 137.4, 135.1, 130.5, 129.8, 127.4, 126.5, 124.8, 124.1, ATP concentration (3µM) was adjusted to equal Km and the
123.0, 120.8, 116.5, 115.6, 59.4; HR-ESI-MS m/z: 399.1449 concentration of EphA2 (0.02µg/mL) was used at an EC50
(Calcd for C21H20N4O3Na [M+Na]+: 399.1428); IR (KBr, value. TK-substrate-biotin (0.5µM) and SEB (20nM) were used
cm−1): 3348, 1651, 1553, 1514, 1310, 1240, 831, 752, 667, 523.
for EphA2 kinase reaction. The total reaction volume was
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-4-methylbenzamide 10 µL and test compounds were preincubated with enzyme
(5e)
for 10min before adding peptide substrate and ATP. Kinase
White powder, yield: 86%; mp: 202–204°C; 1H-NMR reactions were conducted for 30min at room temperature in
(DMSO-d6, 500MHz): δ, ppm: 10.02 (s, 1H), 8.68 (s, 1H), standard 384 well plates and then 10µL of detection mixture
7.87–7.86 (m, 2H), 7.67 (s, 1H), 7.65 (d, J=9.5Hz, 2H), 7.40 including 10mM ethylenediaminetetraacetic acid (EDTA) and
(d, J=9.5Hz, 2H), 7.33 (m, 3H), 6.84 (dt, J=9.0Hz and 1µM europium-tagged antibody was added to the reaction
1.5Hz, 1H), 6.74 (dd, J=8.0Hz and 1.5Hz, 1H), 6.58 (dt, plates 1h before reading the plates. Following the addition
J=7.5Hz and 1.5Hz, 1H), 4.75 (s, 2H), 2.50 (s, 3H); 13C-NMR of reagents for detection, the TR-FRET signal was measured
(DMSO-d6, 125MHz): δ, ppm: 168.5, 157.8, 154.5, 146.5, using an EnVision multi-label reader. The instrument settings
140.6, 137.9, 136.0, 133.7, 132.0, 130.4, 129.6, 127.3, 126.3, used were 340nm for excitation and 615nm and 665nm for
123.5, 123.0, 118.7, 27.7; HR-ESI-MS m/z: 383.1519 (Calcd for emission with a 100µs delay time. The TR-FRET counts are
C21H20N4O2Na [M+Na]+: 383.1478); IR (KBr, cm−1): 3325, expressed as ratio F665/F615nm×104, where F665 and F615nm
1649, 1553, 1516, 1310, 1242, 752, 669, 627, 521.
are fluorescence counts at 665 and 615nm for fluorescein and
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-3-methylbenzamide europium, respectively. IC50 was calculated by a non-linear
(5f)
White powder, yield: 72%; mp: 222–226°C; 1H-NMR
regression using Prism version 5.01.
HDAC Inhibition Assay In vitro HDAC inhibition assays
(DMSO-d6, 500MHz): δ, ppm: 10.10 (s, 1H), 8.73 (s, 1H), were conducted as described in reference.25) Briefly, 10µL of
7.76–7.71 (m, 3H), 7.66–7.65 (m, 2H), 7.43–7.38 (m, 4H), 7.34 HeLa nuclear extract was mixed with tested compound (50µL).
(dd, J=7.5Hz and 1.0Hz, 1H), 6.84 (dt, J=8.0Hz and 1.5Hz), Five minutes later, fluorogenic substrate tert-butoxycarbonyl
6.74 (dd, J=7.5Hz and 1.0Hz, 1H), 6.58 (dt, J=7.5Hz and (Boc)-Lys (acetyl)-7-amino-4-methyl-coumarin (AMC) (40µL)
1.0Hz, 1H), 4.77 (s, 2H), 2.40 (s, 3H); 13C-NMR (DMSO-d6, was added, and the mixture was incubated at 37°C for 30min
125MHz): δ, ppm: 168.0, 157.2, 154.2, 144.6, 140.2, 139.4, and then stopped by addition of 100µL of developer contain-
138.7, 137.1, 136.5, 134.7, 129.9, 129.5, 128.5, 127.4, 126.1, ing trypsin and p-toluenesulfonic acid (TSA). After incubation
123.8, 123.3, 118.9, 26.9; HR-ESI-MS m/z: 383.1506 (Calcd at 37°C for 20min, fluorescence intensity was measured using
for C21H20N4O2Na [M+Na]+: 383.1478); IR (KBr, cm−1): 3281, a microplate reader at excitation and emission wavelengths of
1645, 1556, 1510, 1402, 1231, 744, 694, 625, 523.
390 and 460nm, respectively. The inhibition ratios were calcu-
N-(4-(3-(2-Aminophenyl)ureido)phenyl)-4-chlorobenzamide lated from the fluorescence intensity readings of tested wells
(5g)
relative to those of control wells. Experiment with triplicate
White powder, yield: 79%; mp: 289–291°C; 1H-NMR data were performed. The IC50 values were calculated using a

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1141
regression analysis of the concentration/inhibition data.
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Acknowledgments This study was supported by the
National Natural Science Foundation of China (Grant No.
81202410), Ph.D. Programs Foundation of Ministry of Educa-
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Conflict of Interest The authors declare no conflict of
interest.