Design, synthesis and anticancer activities of diaryl urea derivatives bearing N-acylhydrazone moiety

Article abstract of DOI:10.1248/cpb.c12-00234

A new series of diaryl urea derivatives bearing N-acylhydrazone moiety were designed and synthesized. All the target compounds were evaluated for their cytotoxic activities in vitro against human lung adenocarcinoma epithelial cell line (A549), human breast cancer cell line (MDA-MB-231) and human leukemia cell line (HL-60) by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Several compounds (1a, 1f and 1h) were further evaluated against human embryonic fibroblast, lung-derived cell line (WI38). The pharmacological results indicated that some compounds exhibited promising anticancer activities. In particular, compound 1f showed the most potent cytotoxicity against the tested three cell lines with IC₅₀ values of 0.41 μM, 0.24 μM and 0.23 μM, respectively.

Full text of DOI:10.1248/cpb.c12-00234

1046
Regular Article
Chem. Pharm. Bull. 60(8) 1046–1054 (2012)
Vol. 60, No. 8
Design, Synthesis and Anticancer Activities of Diaryl Urea Derivatives
Bearing N-Acylhydrazone Moiety
Bei Zhang,a Yanfang Zhao,a Xin Zhai,a Lihui Wang,b Jingyu Yang,b Zehui Tan,a and
,a
Ping Gong*
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Pharmaceutical
b
Engineering, Shenyang Pharmaceutical University; and School of Life Science and Biopharmaceutics, Shenyang
Pharmaceutical University; Shenyang, Liaoning 110016, P.R. China.
Received April 16, 2012; accepted May 18, 2012
A new series of diaryl urea derivatives bearing N-acylhydrazone moiety were designed and synthesized.
All the target compounds were evaluated for their cytotoxic activities in vitro against human lung adenocar-
cinoma epithelial cell line (A549), human breast cancer cell line (MDA-MB-231) and human leukemia cell
line (HL-60) by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Several
compounds (1a, 1f and 1h) were further evaluated against human embryonic fibroblast, lung-derived cell line
(WI38). The pharmacological results indicated that some compounds exhibited promising anticancer activi-
ties. In particular, compound 1f showed the most potent cytotoxicity against the tested three cell lines with
IC50 values of 0.41µM, 0.24µM and 0.23µM, respectively.
Key words diaryl urea; N-acylhydrazone; anticancer activity
During the past decade, a large number of multitargeted did the PAC-1 derivatives display antitumor activity in vitro,12)
inhibitors were reported to exhibit anticancer activities with 2-hydroxyl substitution was retained for the phenyl ring Ar2.
various mechanisms of action as distinct from those cytotoxic Four- and 5-benzoxyl groups were respectively introduced to
drugs and mono-targeted inhibitors.1–4) Some of them have the phenyl ring Ar2 to investigate the effect of the extension
been developed successfully such as sorafenib (Nexavar), of the hydrophobic region. Furthermore, the phenyl group at
sunitinib (Sutent) and lapatinib (Tykerb). Sorafenib (Fig. 1), Ar2 was replaced with a substituted chromenonyl group which
the first orally bioavailable multitargeted receptor tyrosine was often associated with a variety of biological activities to
kinase inhibitor, has a reasonable enzyme potency against explore whether such a replacement would bring a significant
Raf, vascular endothelial growth factor receptor (VEGFR) and rise in anticancer activity.
platelet derived growth factor receptor (PDGFR) and pathway
inhibition in cells (IC50 values of 40–1200nM depending on
Results and Discussion
Chemistry The synthetic route of target compounds
the cell line).5–9) It was approved by Food and Drug Admin-
istration (FDA) for the treatment of advanced renal cell car- 1a–i is described in Chart 1. The commercially available
cinoma in December 2005 and hepatocellular carcinoma in 4-aminobezonitrile reacted with triphosgene in dioxane to
November in 2007, respectively.10,11)
obtain 4-isocyanatobenzonitrile 3, which was treated with
PAC-1 (Fig. 1) is the first small molecular procaspase-3 different substituted anilines to get diaryl ureas 4a–h. 4a–h
activator that induces apoptotic death in tumor cell lines and were then turned into thioamides 5a–h under the condition
retards tumor growth in vivo.12,13) In an attempt to discover of magnesium chloride and sodium bisulfide in N,N-dimeth-
new anticancer agents with multi-targeted molecular mecha- ylformamide. Cyclization of 5a–h with 1,3-dichloroacetone
nisms, we combined the diaryl urea moiety from sorafenib in tetrahydrofuran readily afforded thiozoles 6a–h, which
and N-acylhydrazone, the pharmacophore of PAC-1, with a reacted with piperazine in ethanol by nucleophilic substitution
thiazolyl ring (structure 1) or an amide bond (structure 2) as to get 7a–h. Consequently, ethyl chloroacetate was added to a
the linker. Thus a series of diaryl urea derivatives bearing N- solution of 7a–h in ethanol to afford esters 8a–h, followed by
acylhydrazone moiety (Fig. 2) were designed and synthesized. hydrazinolysis to obtain acylhydrazines 9a–h. Finally, target
Various substituted ureido-linked phenyl (Ar1) and hydrazone- compounds 1a–i were prepared via respective condensation
linked phenyl (Ar2) groups were introduced to explore the in- of 9a–h with various benzaldehydes and benzyloxybenzalde-
fluence of electronic and steric effects on the anticancer activ- hydes 17 and 18.
ity. Since only with the hydroxyl group on the phenyl ring Ar2
The synthesis of target compounds 2a–i is described in
Fig. 1. Structures of Sorafenib and PAC-1
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: gongpinggp@126.com
© 2012 The Pharmaceutical Society of Japan

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Fig. 2. The General Structures of Target Compounds
Reactions and conditions: (a) triphosgene, dioxane, 80°C, 24h; (b) Ar1NH2, THF, r.t.; (c) MgCl2, NaSH, DMF, overnight; (d) 1,3-dichloroacetone, THF, 50°C, 7h; (e)
piperazine, ethanol, r.t., 2h; (f) ethyl chloroacetate, K2CO3, NaI, ethanol, 50°C, 2h; (g) 80% hydrazine hydrate, ethanol, 50°C, 48h; (h) i. Ar2CHO or 17 or 18, ethanol;
ii. HCl-ethanol.
Chart 1. Synthesis of Compounds 1a–i
Chart 2. 4-Nitroaniline reacted with triphosgene in dioxane from benzene-1,3-diol via a two-step reaction pathway with
to afford 1-isocyanato-4-nitrobenzene 10, which was treated ethyl acetoacetate and urotropine in sequence.
with corresponding substituted anilines to give diaryl ureas
Biological Evaluation The cytotoxicity of target com-
11a–d. Reduction of compounds 11a–d in the presence of pounds 1a–i and 2a–i were evaluated against human lung
iron powder and ammonium chloride in 95% ethanol gave adenocarcinoma epithelial cell line (A549), human breast
ureidoanilines 12a–d, followed by reaction with chloroacetyl cancer cell line (MDA-MB-231) and human leukemia cell line
chloride to afford chloroactetamides 13a–d, which underwent (HL-60) with sorafenib and PAC-1 as the positive controls.
nucleophilic attack by piperazine to give 14a–d. According to Three compounds (1a, 1f and 1h) were further evaluated
the same procedures for the preparation of 1a–i, the desired against human embryonic fibroblast, lung-derived cell line
compounds 2a–i were obtained via respective condensation of (WI38) in vitro. The results expressed as IC50 values were
16a–d with various benzaldehydes, benzyloxybenzaldehyde 17 summarized in Table 1.
and chromenealdehyde 20.
As listed in Table 1, most of the target compounds exhibited
As shown in Chart 3, benzyloxybenzaldehydes 17 and 18 moderate to strong activities to the tested cell lines. Generally,
were prepared from 2,5-dihydroxybenzaldehyde and 2,4-dihy- compounds 1a–i with thiazolyl linker were more potent than
droxybenzaldehyde respectively through reactions with benzyl compounds 2a–i with amide. It suggested that the heterocy-
chloride in acetonitrile in the presence of sodium bicarbonate clic ring bridging the two pharmacophores is essential for the
and potassium iodide. Chromenealdehyde 20 was synthesized activity, and replacement with an acyclic linker may cause a

1048
Vol. 60, No. 8
Reactions and conditions: (a) triphosgene, dioxane, 80°C, 24h; (b) Ar1NH2, THF, r.t.; (c) Fe, NH4Cl, HOAc, ethanol, 80°C, 5h; (d) chloroacetyl chloride, TEA,
CH2Cl2, 0°C, r.t., 5h; (e) piperazine, ethanol, r.t., 6h; (f) ethyl chloroacetate, K2CO3, NaI, ethanol, 50°C, 2h; (g) 80% hydrazine hydrate, ethanol, 50°C, 48h; (h) i.
Ar2CHO or 17 or 20, ethanol; ii. HCl-ethanol.
Chart 2. Synthesis of Compounds 2a–i
compounds 1h and 1i with the benzyloxyl group at the 4-posi-
tion of the phenyl ring did not show a similar result. These
findings indicated that the aryl substituent at the 5-position of
Ar2 would significantly enhance the cytotoxicity.
To investigate the effect of a heterocylic ring for Ar2, we
replaced the phenyl group with a substituted chromenonyl
group to obtained compounds 2h and 2i. Unfortunately, these
compounds exhibited even lower inhibition activities than
the unfavored compounds 2a–g. It suggested that the phenyl
group substituted at Ar2 is critical for the activity.
Compounds 1a, 1f and 1h exerted weaker cytotoxicity
against WI38 (the normal cell line) than the three cancer cell
lines. The most promising compound 1f possessed selective
indexes (IC50 normal cell/IC50 cancer cell) of 11, 19 and 20 for
A549, MDA-MB-231 and HL-60 cell lines, respectively.
Reactions and conditions: (a) benzyl chloride NaHCO3, CH3CN, r.f., 30h; (b)
benzyl chloride, NaHCO3, CH3CN, reflux, 30h; (c) ethyl acetoacetate, H2SO4,
10°C, 4h; (d) urotropine, HOAc, 115°C, 4h.
Chart 3. Synthesis of Intermediates 17, 18 and 20
Conclusion
dramatical decrease in the activity, or even a complete loss.
In summary, a new series of diaryl urea derivatives bearing
Compounds 1f, 1g and 1i with trifluoromethoxyl or tri- N-acylhydrazone moiety were designed and synthesized. All
fluoromethyl group at the ortho-position and dichloro group the compounds were screened for their cytotoxicity against
at the meta- and para-positions of phenyl ring Ar1 repectively A549, MDA-MB-231 and HL-60 cell lines. The pharmaco-
showed more potent cytotoxicity than compound 1d with no logical results indicated that some of the prepared compounds
substituent and 1h with only chloro group at the meta-posi- showed promising activities. Compound 1f showed the most
tion. Compounds 2e and 2g with trifluoromethyl or more elec- potent cytotoxicity against A549, MDA-MB-231 and HL-60
tron-withdrawing groups exhibited comparable cytotoxicity cell lines with IC50 values of 0.41µM, 0.24µM and 0.23µM,
to sorafenib and PAC-1, while compound 2f with only chloro repectively, which were 3.2- to 19.8-fold higher than sorafenib
group at the meta-position of phenyl Ar1 showed no activity and PAC-1. Moreover, compound 1f exhibited the most selec-
against A549 and MDA-MB-231 cell lines. These findings tive cytotoxicity against the three cancer cell lines when com-
suggested that the electron-withdrawing groups on the phenyl pared with WI38 cell line. The preliminary strucuture–activ-
ring Ar1 are favorable to the activity.
ity relationships (SARs) showed that the thiazolyl linker and
Compounds with alkyl groups or no substituents at the the phenyl group of Ar2 are favorable for the activity. More-
phenyl ring Ar2, e.g. compounds 1c and 1a, exhibited com- over, electron-withdrawing groups on Ar1 and the benzyloxy
parable activities to sorafenib and PAC-1. Compounds 1f and group at the 5-position of phenyl ring Ar2 could enhance the
1g with the benzyloxyl group at the 5-position of the phenyl activity. These investigations prompt us to carry on further
ring showed a remarkable increase in the activity, especially studies on their mechanisms of action.
compound 1f displayed the most potent activity with IC50 val-
ues of 0.41µM, 0.24µM and 0.23µM, respectively, which were
Experimental
3.2- to 19.8-fold higher than sorafenib and PAC-1. However,
Chemistry Melting points were obtained on a Büchi

August 2012
1049
Table 1. Structures and Cytotoxicity of Compounds 1a–i and 2a–i against A549, MDA-MB-231, HL-60 and WI38 Cell Lines
IC50 (µM)
Compd.
Ar1
Ar2
A549
3.88
MDA-MB-231
4.65
HL-60
2.40
WI38
11.67
1a
1b
1c
2.40
1.49
1.27
0.90
0.33
2.11
NDa)
NDa)
1d
1e
1f
1.67
2.30
0.41
0.45
0.83
0.74
0.59
0.24
0.30
0.52
0.55
1.03
0.23
0.35
1.18
NDa)
NDa)
4.53
NDa)
6.91
1g
1h
1i
0.78
8.97
0.47
2.39
0.80
4.24
NDa)
NDa)
2a
2b
1.34
0.49
1.29
NDa)
2c
52.0
10.7
1.18
1.07
2.44
4.42
NDa)
NDa)
2d
2e
2f
3.16
1.48
2.66
1.70
NDa)
NDa)
>100
>100

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Vol. 60, No. 8
Table 1. Structures and Cytotoxicity of Compounds 1a–i and 2a–i against A549, MDA-MB-231, HL-60 and WI38 Cell Lines
IC50 (µM)
Compd.
Ar1
Ar2
A549
4.02
MDA-MB-231
1.92
HL-60
1.82
WI38
NDa)
2g
2h
2i
18.0
>100
5.54
NDa)
NDa)
>100
8.92
3.08
Sorafenib
PAC-1
1.30
2.81
2.70
2.04
NDa)
4.56
10.8
6.63
a) ND: not determined.
Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, 77.5%. ESI-MS m/z: 272.0 (M+H)+.
Switzerland) and were uncorrected. Mass spectra (MS) were 1-(4-Cyanophenyl)-3-(3,4-dichlorophenyl)urea (4h): Yield
taken in electrospray ionization (ESI) mode on Agilent 1100 82.5%. ESI-MS m/z: 306.0 (M+H)+.
LC-MS (Agilent, Palo Alto, CA, U.S.A.). Proton (1H) nuclear
General Procedure for the Preparation of 4-(3-Substi-
magnetic resonance spectroscopy was performed using Bruker tuted phenylureido)benzothioamides (5a–h) To a solution
ARX-300, 300MHz spectrometers (Bruker Bioscience, Bil- of benzonitrile (4a–h) (0.086mol) in N,N-dimethylformamide
lerica, MA, U.S.A.) with tetramethylsilane (TMS) as an inter- (250mL) was added magnesium chloride (22g, 0.109mol) and
nal standard. Unless otherwise noted, all the materials were sodium bisulfide (12.2g, 0.218mol). The reaction mixture was
obtained from commercial available sources and were used stirred at room temperature overnight and then was added
without further purification.
to 1L water, acidified to pH 4 with dilute hydrochloride and
4-Isocyanatobenzonitrile (3) 4-Aminobezonitrile (100g, filtered. The collected solid was washed with water until the
0.847mol) was treated with excess hydrochloride-ethanol filtrate became neutral and dried to obtain 5a–h (47.6–59.2%).
and the resulted solution was evaporated to dryness. The
hydrochloride salt obtained was then dissolved in dioxane Chloromethylthiazol-2-yl)phenyl)-3-substituted
General Procedure for the Preparation of 1-(4-(4-
Phenyl-
(200mL) and added dropwise to a solution of triphosgene ureas (6a–h) Benzothioamide (5a–h) (0.083mol) was dis-
(125g, 0.423mol) in dioxane (200mL). The reaction mixture solved in THF (300mL) and heated to 50°C. To the stirred so-
was heated to 50°C and stirred for 24h. The resulting mixture lution was added 1,3-dichloroacetone (10.5g, 0.083mol). The
was concentrated in vacuo and distilled under reduced pres- reaction mixture was stirred for 7h. The resulting mixture
sure to give 3 (103g, 84.4%) as colorless oil. bp: 130–131°C was evaporated in vacuo to remove most of the solvent, cooled
(15mmHg).
and filtered off. The residue was suspended in 1L water and
General Procedure for the Preparation of 1-(4-Cyano- the suspension was stirred and basified to pH 8 with saturated
phenyl)-3-substituted Phenylureas (4a–h) To a solution potassium carbonate solution. The precipitates was filtered,
of 3 (16g, 0.111mol) in tetrahydrofuran (100mL) was added washed with water and dried to obtain 6a–h (74.2–80.0%).
the corresponding substituted aniline (0.111mol) slowly. The
General Procedure for the Preparation of 1-Substituted
resulted mixture was stirred at room temperature and the re- Phenyl-3-(4-(4-(piperazin-1-ylmethyl)thiazol-2-yl)phenyl)-
action was monitored by TLC. The reaction mixture was con- ureas (7a–h) To a solution of piperazine (64g, 0.748mol)
centrated in vacuo. the product that precipitated was filtered in ethanol was added urea (6a–h) in portions. The reaction
and dried to obtain 4a–h.
mixture was stirred at room temperature for 2h. The resulting
(4a): mixture was evaporated in vacuo to remove most of the sol-
vent and poured into 1.5L water. The white precipitates was
1-(4-Cyanophenyl)-3-(2-trifluoromethylphenyl)urea
Yield 85.8%. ESI-MS m/z: 306.1 (M+H)+.
1-(4-Cyanophenyl)-3-(3,5-dichlorophenyl)urea (4b): Yield filtered, washed with water and dried to obtain 7a–h.
83.4%. ESI-MS m/z: 306.1 (M+H)+.
1-(4-(4-(Piperazin-1-ylmethyl)thiazol-2-yl)phenyl)-3-(2-
1-(4-Cyanophenyl)-3-(3-trifluoromethoxyphenyl)urea (4c): trifluoromethylphenyl)urea (7a): Yield 58.2%. ESI-MS m/z:
Yield 79.7%. ESI-MS m/z: 322.2 (M+H)+.
1-(4-Cyanophenyl)-3-phenylurea (4d): Yield 87.1%. ESI-MS
m/z: 238.1 (M+H)+.
462.2 (M+H)+.
1-(3,5-Dichlorophenyl)-3-(4-(4-(piperazin-1-ylmethyl)-
thiazol-2-yl)phenyl)urea (7b): Yield 55.4%. ESI-MS m/z: 462.2
1-(2-Chloro-4-trifluoromethylphenyl)-3-(4-cyanophenyl)urea (M+H)+.
(4e): Yield 85.3%. ESI-MS m/z: 340.0 (M+H)+.
1-(4-(4-(Piperazin-1-ylmethyl)thiazol-2-yl)phenyl)-3-(3-
1-(4-Cyanophenyl)-3-(2-trifluoromethoxyphenyl)urea (4f): trifluoromethoxyphenyl)urea (7c): Yield 63.7%. ESI-MS m/z:
Yield 80.4%. ESI-MS m/z: 322.1 (M+H)+.
478.3 (M+H)+.
1-Phenyl-3-(4-(4-(piperazin-1-ylmethyl)thiazol-2-yl)phenyl)-
1-(3-Chlorophenyl)-3-(4-cyanophenyl)urea
(4g):
Yield

August 2012
1051
urea (7d): Yield 55.9%. ESI-MS m/z: 394.2 (M+H)+.
under reduced pressure to give 10 (70g, 58.9%) as yellow
1-(2-Chloro-4-trifluoromethylphenyl)-3-(4-(4-(piperazin-1- solid. bp: 168–170°C (15mmHg).
ylmethyl)thiazol-2-yl)phenyl)urea (7e): Yield 51.8%. ESI-MS
General
Procedure
for
the
Preparation
of
m/z: 496.1 (M+H)+.
1-(4-Nitrophenyl)-3-substituted phenylureas (11a–d) To
1-(4-(4-(Piperazin-1-ylmethyl)thiazol-2-yl)phenyl)-3-(2- a solution of 10 (40g, 0.244mol) in tetrahydrofuran (500mL)
trifluoromethoxyphenyl)urea (7f): Yield 58.4%. ESI-MS m/z: was added the corresponding substituted aniline (0.244mol)
478.1 (M+H)+.
slowly. The resulted mixture was stirred at room temperature
1-(3-Chlorophenyl)-3-(4-(4-(piperazin-1-ylmethyl)thiazol-2- and the reaction was monitored by TLC. The reaction mixture
yl)phenyl)urea (7g): Yield 50.7%. ESI-MS m/z: 428.1 (M+H)+. was concentrated in vacuo and filtered. The solid was dried to
1-(3,4-Dichlorophenyl)-3-(4-(4-(piperazin-1-ylmethyl)- obtain 11a–d.
thiazol-2-yl)phenyl)urea (7h): Yield 53.0%. ESI-MS m/z: 462.0
1-(4-Nitrophenyl)-3-(3-(trifluoromethyl)phenyl)urea
(11a):
(M+H)+.
Yield 75.7%. ESI-MS m/z: 326.2 (M+H)+.
General Procedure for the Preparation of Ethyl 2-
1-(3-Chlorophenyl)-3-(4-nitrophenyl)urea
(11b):
Yield
(4-((2-(4-(3-Substituted phenylureido)phenyl)thiazol-4-yl)- 80.1%. ESI-MS m/z: 292.1 (M+H)+.
methyl)piperazin-1-yl)acetates (8a–h) To a solution of N-
substituted piperazine (7a–h) (0.063mol) in ethanol (300mL) urea (11c): Yield 81.4%. ESI-MS m/z: 360.0 (M+H)+.
was added potassium carbonate (5.2g, 0.038mol), ethyl 1-(3-Chloro-4-(trifluoromethyl)phenyl)-3-(4-nitrophenyl)-
chloroacetate (7.7g, 0.063mol) and sodium iodide (cat.). The urea (11d): Yield 72.4%. ESI-MS m/z: 360.0 (M+H)+.
reaction mixture was heated to 50°C and stirred for 2h. The General Procedure for the Preparation of 1-(4-Amino-
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-nitrophenyl)-
reaction mixture was concentrated in vacuo and cooled. The phenyl)-3-substituted phenylureas (12a–d) To a stirred
product precipitated was filtered off, washed with ethanol and solution of ureidonitrobenzene (11a–d) (0.185mol) in 95%
water, and dried to obtain 8a–h (87.5–92.1%).
ethanol (500mL) was added glacial acetic acid (5mL), am-
General Procedure for the Preparation of 1-Substituted monium chloride (15g, 0.280mol) and iron powder (62g,
Phenyl-3-(4-(4-((4-(2-hydrazinyl-2-oxoethyl)piperazin-1- 1.107mol). The mixture was heated to 80°C and stirred
yl)methyl)thiazol-2-yl)phenyl)ureas (9a–h) To a solution of for 5h. The reaction mixture was filtered hot. The filtrate
ester (8a–h) (0.044mol) in ethanol (250mL) was added 80% was concentrated in vacuo and poured into water (500mL).
hydrazine hydrate (27.7g, 0.443mol). The reaction mixture The precipitates were filtered off and dried to afford 12a–d
was heated to 50°C and stirred for 48h. The reaction mixture (93.1–98.2%).
was evaporated to remove most of the solvent. The residue
General Procedure for the Preparation of 2-Chloro-
was filtered off, washed with water, and dried to obtain 9a–h. N-(4-(3-substituted phenylureido)phenyl)acetamides (13a–
1-(4-(4-((4-(2-Hydrazinyl-2-oxoethyl)piperazin-1-yl)methyl)- d) A solution of amine (12a–d) (0.183mol) and triethylamine
thiazol-2-yl)phenyl)-3-(2-trifluoromethylphenyl)urea
(9a): (28g, 0.277mol) in dichloromethane (500mL) was cooled
Yield 89.8%. ESI-MS m/z: 534.2 (M+H)+.
below 0°C in an ice-salt bath and chloroacetyl chloride (25g,
1-(3,5-Dichlorophenyl)-3-(4-(4-((4-(2-hydrazinyl-2- 0.221mol) was added dropwise. The ice-salt bath was removed
oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)phenyl)urea (9b): and the resulting mixture was stirred at room temperature for
Yield 88.0%. ESI-MS m/z: 534.2 (M+H)+.
5h. The precipitates were filtered off, washed with dichloro-
1-(4-(4-((4-(2-Hydrazinyl-2-oxoethyl)piperazin-1-yl)methyl)- methane and dried to afford 13a–d (88.6–91.2%).
thiazol-2-yl)phenyl)-3-(3-trifluoromethoxyphenyl)urea
(9c):
General Procedure for the Preparation of 2-(Piperazin-
1-yl)-N-(4-(3-substituted phenylureido)phenyl)acetamides
Yield 92.6%. ESI-MS m/z: 550.2 (M+H)+.
1-(4-(4-((4-(2-Hydrazinyl-2-oxoethyl)piperazin-1-yl)methyl)- (14a–d) To a stirred solution of piperazine (194g, 1mol)
thiazol-2-yl)phenyl)-3-phenylurea (9d): Yield 88.7%. ESI-MS in ethanol (500mL) was added chloroactetamide (13a–d)
m/z: 466.2 (M+H)+.
(0.500mol) in portions. The reaction mixture was stirred at
1-(2-Chloro-4-trifluoromethylphenyl)-3-(4-(4-((4-(2- room temperature for 6h. The resulting mixture was evaporat-
hydrazinyl-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)- ed in vacuo to remove the solvent and then poured into water
phenyl)urea (9e): Yield 85.2%. ESI-MS m/z: 568.1 (M+H)+.
(500mL). The precipitates were filtered off, washed with water
1-(4-(4-((4-(2-Hydrazinyl-2-oxoethyl)piperazin-1-yl)methyl)- and dried to give 14a–d.
thiazol-2-yl)phenyl)-3-(2-(trifluoromethoxy)phenyl)urea (9f):
2-(Piperazin-1-yl)-N-(4-(3-(3-trifluoromethyl)phenylureido)-
phenyl)acetamide (14a): Yield 68.9%. ESI-MS m/z: 422.2
Yield 90.4%. ESI-MS m/z: 550.3 (M+H)+.
1-(3-Chlorophenyl)-3-(4-(4-((4-(2-hydrazinyl-2-oxoethyl)- (M+H)+.
piperazin-1-yl)methyl)thiazol-2-yl)phenyl)urea (9g): Yield
N-(4-(3-(3-Chlorophenyl)ureido)phenyl)-2-(piperazin-1-yl)-
87.5%. ESI-MS m/z: 500.2 (M+H)+.
acetamide (14b): Yield 72.1%. ESI-MS m/z: 388.1 (M+H)+.
N-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-
1-(3,4-Dichlorophenyl)-3-(4-(4-((4-(2-hydrazinyl-2-
oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)phenyl)urea (9h): phenyl)-2-(piperazin-1-yl)acetamide (14c): Yield 65.4%. ESI-
Yield 86.7%. ESI-MS m/z: 534.1 (M+H)+.
1-Isocyanato-4-nitrobenzene (10) 4-Nitroaniline (100g,
MS m/z: 456.1 (M+H)+.
N-(4-(3-(3-Chloro-4-(trifluoromethyl)phenyl)ureido)-
0.725mol) was treated with excess hydrochloride-ethanol and phenyl)-2-(piperazin-1-yl)acetamide (14d): Yield 69.6%. ESI-
evaporated to dryness. The hydrochloride salt obtained was MS m/z: 456.1 (M+H)+.
then dissolved in dioxane (200mL) and added dropwise to a
solution of triphosgene (108g, 0.362mol) in dioxane (200mL). (2-Oxo-2-((4-(3-substituted
General Procedure for the Preparation of Ethyl 2-(4-
phenylureido)phenyl)amino)-
The reaction mixture was heated to 50°C and stirred for 24h. ethyl)piperazin-1-yl)acetates (15a–d) To a solution of N-
The resulting mixture was concentrated in vacuo and distilled substituted piperazine (14a–d) (0.157mol) in ethanol (500mL)

1052
Vol. 60, No. 8
was added sodium dicarbonate (20g, 0.238mol) and ethyl dryness to afford 20 (2.2g, 38.0%) as white solid. ESI-MS
chloroacetate (21g, 0.171mol). The reaction mixture was heat- m/z: 203.0 (M−H)−.
ed to 65°C and stirred for 20h. The mixture was concentrated
General Procedure for the Preparation of Target Com-
to about 200mL in vacuo and cooled to room temperature. pounds 1a–i and 2a–i To a solution of acethydrazide (9a–h,
The precipitates were filtered off and dried to afford 15a–d 16a–d) (0.002mol) in ethanol (10mL) was added appropriate
(82.3–86.4%).
benzaldehyde or the prepared aromatic aldehyde (17, 18, 20).
General Procedure for the Preparation of 2-(4-(2- The reaction mixture was stirred and refluxed for 2h. The
Hydrazinyl-2-oxoethyl)piperazin-1-yl)-N-(4-(3-substituted reaction mixture was cooled and precipitates were collected
phenylureido)phenyl)acetamides (16a–d) To a solution of by filtration to obtain the crude product, which was then puri-
ester (15a–d) (0.059mol) in ethanol (300mL) was added 80% fied by flash column chromatography. The pure product was
hydrazine hydrate (37g, 0.592mol). The reaction mixture was dissolved in chloroform. To the solution was added excessive
heated to 50°C and stirred for 48h. The precipitates were fil- hydrochloride-ethanol and stirred for 1h. Ether was added to
tered off and dried to afford 16a–d.
the mixture above. The precipitates were filtered off and dried
2-(4-(2-Hydrazinyl-2-oxoethyl)piperazin-1-yl)-N-(4-(3-(3- to afford 1a–i and 2a–i.
(trifluoromethyl)phenyl)ureido)phenyl)acetamide (16a): Yield
81.2%. ESI-MS m/z: 494.2 (M+H)+.
1-(4-(4-((4-(2-(2-(2-Hydroxybenzylidene)hydrazinyl)-2-
oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)phenyl)-3-(2-
N-(4-(3-(3-Chlorophenyl)ureido)phenyl)-2-(4-(2- trifluoromethylphenyl)urea Dihydrochloride (1a): Yield 55.8%.
hydrazinyl-2-oxoethyl)piperazin-1-yl)acetamide (16b): Yield mp 186–188°C. ESI-MS m/z: 638.2 (M+H)+. 1H-NMR
85.4%. ESI-MS m/z: 460.2 (M+H)+.
(300MHz, DMSO-d6) δ: 11.96 (s, 1H), 10.15 (s, 1H), 8.42–8.37
N-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)- (m, 2H), 7.95–7.88 (m, 5H), 7.77–7.64 (m, 5H), 7.34–7.24 (m,
phenyl)-2-(4-(2-hydrazinyl-2-oxoethyl)piperazin-1-yl)- 2H), 7.01–6.84 (m, 2H), 4.58 (s, 1H), 4.48 (s, 2H), 3.81 (s, 1H),
acetamide (16c): Yield 77.5%. ESI-MS m/z: 528.2 (M+H)+.
N-(4-(3-(3-Chloro-4-(trifluoromethyl)phenyl)ureido)-
3.66 (brs, 8H).
1-(3,5-Dichlorophenyl)-3-(4-(4-((4-(2-(2-(2-hydroxybenzyli-
phenyl)-2-(4-(2-hydrazinyl-2-oxoethyl)piperazin-1-yl)- dene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-
yl)phenyl)urea Dihydrochloride (1b): Yield 53.3%. mp
acetamide (16d): Yield 81.1%. ESI-MS m/z: 528.2 (M+H)+.
1
5-Benzyloxy-2-hydroxybenzaldehyde (17) To a solution 230–232°C. ESI-MS m/z: 638.3 (M+H)+. H-NMR (300MHz,
of 2,5-dihydroxybenzaldehyde (50g, 0.362mol) in acetonitrile DMSO-d6) δ: 11.98 (s, 1H), 10.15–10.01 (m, 3H), 8.38 (s,
(500mL) was added benzyl chloride (60g, 0.471mol), sodium 1H), 7.94 (d, J=8.6Hz, 3H), 7.76 (d, J=7.8Hz, 1H), 7.63 (d,
bicarbonate (35g, 0.413mol) and potassium iodide (cat.). The J=8.6Hz, 2H), 7.54 (s, 2H), 7.29–7.23 (m, 1H), 7.16 (s, 1H),
reaction mixture was heated to reflux and stirred for 30h. 6.95–6.84 (m, 2H), 4.63 (s, 1H), 4.53 (s, 2H), 3.99 (s, 1H), 3.64
The mixture was poured into water (500mL). The precipitates (brs, 8H).
were filtered off, dried, recrystallized from methanol to give
17 (46.5g, 56.3%) as white solid. ESI-MS m/z: 227.1 (M−H)−. hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)-
4-Benzyloxy-2-hydroxybenzaldehyde (18) To a solution phenyl)-3-(3-trifluoromethoxyphenyl)urea Dihydrochloride
1-(4-(4-((4-(2-(2-(3,5-Di-tert-butyl-2-hydroxybenzylidene)-
of 2,4-dihydroxybenzaldehyde (50g, 0.362mol) in acetonitrile (1c): Yield 59.7%. mp 221–222°C. ESI-MS m/z: 766.2 (M+H)+.
(500mL) was added benzyl chloride (60g, 0.471mol), sodium 1H-NMR (300MHz, DMSO-d6) δ: 12.12 (s, 1H), 11.45 (s,
bicarbonate (35g, 0.413mol) and potassium iodide (cat.). The 1H), 9.10 (s, 1H), 9.08 (s, 1H), 8.49 (s, 1H), 7.86 (d, J=8.6Hz,
reaction mixture was heated to reflux and stirred for 30h. 2H), 7.72 (s, 1H), 7.59 (d, J=8.6Hz, 2H), 7.44–7.39 (m, 2H),
The mixture was poured into water (500mL). The precipitates 7.33–7.30 (m, 2H), 7.18 (s, 1H), 6.96 (d, J=7.7Hz, 1H), 3.67 (s,
were filtered off, dried, recrystallized from methanol to give 2H), 3.32 (s, 1H), 3.14 (s, 2H), 2.56 (s, 8H), 1.39 (s, 9H), 1.27
18 (43.6g, 52.8%) as white solid. ESI-MS m/z: 227.1 (M−H)−. (s, 9H)
7-Hydroxy-4-methyl-2H-chromen-2-one
(19) Sulfuric
1-(4-(4-((4-(2-(2-(5-Benzyloxy-2-hydroxybenzylidene)-
acid (500mL) was cooled below 10°C in an ice-salt bath, and hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)-
to the cooled acid was slowly added a solution of benzene-1,3- phenyl)-3-phenylurea Dihydrochloride (1d): Yield 62.5%. mp
1
diol (55g, 0.500mol) in ethyl acetoacetate (65g, 0.500mol). 202–204°C. ESI-MS m/z: 676.3 (M+H)+. H-NMR (300MHz,
The reaction mixture was stirred for 4h. The resulting mix- DMSO-d6) δ: 11.94 (s, 1H), 9.71 (d, J=13.4Hz, 1H), 9.42 (d,
ture was poured onto cracked ice and stirred. The precipitates J=12.7Hz, 1H), 8.32 (s, 1H), 7.90 (s, 4H), 7.63 (d, J=8.1Hz,
were filtered off, washed with water and dried to afford 19 3H), 7.49–7.38 (m, 7H), 7.35–7.26 (m, 4H), 6.99 (d, J=7.1Hz,
(67.0g, 76.1%) as white solid. ESI-MS m/z: 175.1 (M−H)−.
2H), 6.87 (d, J=8.8Hz, 1H), 5.05 (s, 2H), 4.56 (s, 1H), 4.47 (s,
7-Hydroxy-4-methyl-2-oxo-2H-chromene-6-carbalde- 2H), 3.78 (s, 1H), 3.59 (brs, 8H).
hyde (20) A suspension of urotropine (20g, 0.143mol) in
1-(4-(4-((4-(2-(2-(5-Benzyloxy-2-hydroxybenzylidene)hydra-
glacial acetic acid (80mL) was heated to 40°C and stirred zinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)phenyl)-3-
to form a clear solution. To this solution was added 19 (5g, (2-chloro-4-(trifluoromethyl)phenyl)urea Dihydrochloride (1e):
0.028mol) in portions. The reaction mixture was stirred for Yield 52.3%. mp 214–215°C. ESI-MS m/z: 778.4 (M+H)+.
20min and then heated to 115°C and stirred for another 2h. 1H-NMR (300MHz, DMSO-d6) δ: 11.96 (s, 1H), 10.62 (d,
Finally the reaction mixture was cooled to 95°C, 30% sulfuric J=14.0Hz, 1H), 9.75 (s, 1H), 9.02 (d, J=7.0Hz, 1H), 8.62
acid (15mL) was added and the reaction continued for 1.5h. (s, 1H), 8.51 (s, 1H), 8.33 (s, 1H), 7.96–7.93 (m, 3H), 7.72
The resulting mixture was evaporated to dryness and the dry (d, J=8.2Hz, 2H), 7.68 (s, 1H), 7.44–7.32 (m, 7H), 6.99 (dd,
residue obtained was extracted with ether. The organic layer J1=2.8Hz, J2=8.9Hz, 1H), 6.88 (d, J=8.9Hz, 1H), 5.05 (s,
was then washed with saturated sodium bicarbonate solu- 2H), 4.58 (s, 1H), 4.50 (s, 2H), 3.84 (s, 1H), 3.68 (brs, 8H).
tion, dried over anhydrous sodium sulfate and evaporated to
1-(4-(4-((4-(2-(2-(5-Benzyloxy-2-hydroxybenzylidene)-

August 2012
1053
hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)- 7.32–7.23 (m, 3H), 7.02–6.99 (m, 1H), 6.93–6.88 (m, 2H), 3.33
phenyl)-3-(2-trifluoromethoxyphenyl)urea Dihydrochloride (s, 1H), 3.14 (s, 1H), 3.12 (s, 2H), 2.59 (s, 8H).
(1f): Yield 54.1%. mp 225–226°C. ESI-MS m/z: 760.3
N-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-
(M+H)+. 1H-NMR (300MHz, DMSO-d6) δ: 11.98 (s, 1H), phenyl)-2-(4-(2-(2-(2-hydroxybenzylidene)hydrazinyl)-2-
10.06 (d, J=11.7Hz, 1H), 9.98 (d, J=10.9Hz, 1H), 8.51 (s, 1H), oxoethyl)piperazin-1-yl)acetamide Dihydrochloride (2d): Yield
8.34 (s, 1H), 7.96–7.91 (m, 4H), 7.71 (s, 1H), 7.64 (d, J=8.6Hz, 51.4%. mp 260–261°C. ESI-MS m/z: 632.1 (M+H)+. H-NMR
1
1H), 7.46–7.31 (m, 8H), 7.22 (d, J=2.9Hz, 1H), 7.01–6.87 (300MHz, DMSO-d6) δ: 11.99 (s, 1H), 10.69 (s, 1H), 10.06 (s,
(m, 3H), 5.06 (s, 2H), 4.62 (s, 1H), 4.53 (s, 2H), 3.94 (s, 1H), 1H), 9.53 (s, 1H), 8.40 (s, 1H), 8.11 (s, 1H), 7.77 (d, J=7.3Hz,
3.72–3.51 (m, 8H).
1H), 7.65–7.56 (m, 5H), 7.45 (d, J=8.9Hz, 2H), 7.33–7.24 (m,
1-(4-(4-((4-(2-(2-(5-Benzyloxy-2-hydroxybenzylidene)- 1H), 6.98–6.93 (m, 1H), 6.91–6.85 (m, 1H), 4.62 (s, 1H), 4.12
hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)- (s, 2H), 3.95 (s, 1H), 3.64 (brs, 8H).
phenyl)-3-(2-trifluoromethylphenyl)urea Dihydrochloride (1g):
2-(4-(2-(2-(5-(Benzyloxy)-2-hydroxybenzylidene)-
Yield 53.7%. mp 241–244°C. ESI-MS m/z: 744.4 (M+H)+. hydrazinyl)-2-oxoethyl)piperazin-1-yl)-N-(4-(3-(3-
1H-NMR (300MHz, DMSO-d6) δ: 12.00 (s, 1H), 10.14 (d, (trifluoromethyl)phenyl)ureido)phenyl)acetamide Dihydrochlo-
J=11.9Hz, 1H), 10.02 (d, J=10.1Hz, 1H), 8.51 (s, 1H), 8.34 (s, ride (2e): Yield 58.3%. mp 210–212°C. ESI-MS m/z: 704.3
1H), 8.02–7.92 (m, 4H), 7.67 (s, 1H), 7.63 (d, J=8.7Hz, 2H), (M+H)+. 1H-NMR (300MHz, DMSO-d6) δ: 11.40 (s, 1H),
7.53 (t, J=7.7Hz, 1H), 7.44–7.30 (m, 7H), 6.99 (dd, J1=3.0Hz, 10.65 (s, 1H), 9.58 (s, 1H), 8.99 (s, 1H), 8.71 (s, 1H), 8.48 (s,
J2=8.9Hz, 1H), 6.89 (d, J=8.8Hz, 1H), 5.06 (s, 2H), 4.64 (s, 1H), 8.01 (s, 1H), 7.55–7.50 (m, 4H), 7.47–7.28 (m, 9H), 7.15
1H), 4.56 (s, 2H), 4.03 (s, 1H), 3.75–3.58 (m, 8H).
(d, J=2.9Hz, 1H), 6.98 (dd, J1=3.0Hz, J2=8.9Hz, 1H), 6.84
1-(4-(4-((4-(2-(2-(4-Benzyloxy-2-hydroxybenzylidene)- (d, J=8.8Hz, 1H), 5.05 (s, 2H), 3.30 (s, 1H), 3.12 (s, 3H), 2.59
hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)- (s, 8H).
phenyl)-3-(3-chlorophenyl)urea Dihydrochloride (1h): Yield
2-(4-(2-(2-(5-(Benzyloxy)-2-hydroxybenzylidene)hydra-
54.1%. mp 213–215°C. ESI-MS m/z: 710.2 (M+H)+. H-NMR zinyl)-2-oxoethyl)piperazin-1-yl)-N-(4-(3-(3-chlorophenyl)-
(300MHz, DMSO-d6) δ: 11.78 (s, 1H), 9.65 (d, J=10.8Hz, ureido)phenyl)acetamide Dihydrochloride (2f): Yield 53.1%.
1H), 9.59 (d, J=12.4Hz, 1H), 8.26 (s, 1H), 7.91 (d, J=8.7Hz, mp 203–205°C. ESI-MS m/z: 670.4 (M+H)+. 1H-NMR
2H), 7.86 (d, J=9.0Hz, 1H), 7.71 (s, 1H), 7.66–7.61 (m, 3H), (300MHz, DMSO-d6) δ: 11.40 (s, 1H), 10.65 (s, 1H), 9.58
7.45–7.40 (m, 5H), 7.37–7.33 (m, 2H), 7.31 (d, J=5.2Hz, 2H), (s, 1H), 8.99 (s, 1H), 8.71 (s, 1H), 8.48 (s, 1H), 8.01 (s, 1H),
7.04–7.02 (m, 1H), 6.57–6.55 (m, 2H), 5.12 (s, 1H), 5.09 (s, 7.55–7.50 (m, 4H), 7.47–7.28 (m, 9H), 7.15 (d, J=2.9Hz, 1H),
1
1H), 4.48 (s, 1H), 4.43 (s, 2H), 3.46 (brs, 9H).
1-(4-(4-((4-(2-(2-(4-Benzyloxy-2-hydroxybenzylidene)hydra- 5.05 (s, 2H), 3.30 (s, 1H), 3.12 (s, 3H), 2.59 (s, 8H).
zinyl)-2-oxoethyl)piperazin-1-yl)methyl)thiazol-2-yl)phenyl)-3- 2-(4-(2-(2-(5-(Benzyloxy)-2-hydroxybenzylidene)hydra-
(3,4-dichlorophenyl)urea Dihydrochloride (1i): Yield 50.4%. zinyl)-2-oxoethyl)piperazin-1-yl)-N-(4-(3-(4-chloro-3-(tri-
mP 205–207°C. ESI-MS m/z: 744.1 (M+H)+. 1H-NMR fluoromethyl)phenyl)ureido)phenyl)acetamide
Dihydrochlo-
6.98 (dd, J1=3.0Hz, J2=8.9Hz, 1H), 6.84 (d, J=8.8Hz, 1H),
(300MHz, DMSO-d6) δ: 11.77 (s, 1H), 9.65 (brs, 2H), 8.25 ride (2g): Yield 47.8%. mp 180–182°C. ESI-MS m/z: 738.4
(s, 1H), 7.93–7.88 (m, 5H), 7.62 (d, J=8.6Hz, 3H), 7.53 (d, (M+H)+. 1H-NMR (300MHz, DMSO-d6) δ: 11.43 (s, 1H),
J=8.8Hz, 1H), 7.45–7.33 (m, 6H), 6.61–6.54 (m, 2H), 5.12 (s, 10.66 (s, 1H), 9.60 (s, 1H), 9.11 (s, 1H), 8.77 (s, 1H), 8.48 (s,
1H), 5.09 (s, 1H), 4.43 (brs, 3H), 3.45 (brs, 9H).
1H), 8.11 (s, 1H), 7.61–7.54 (m, 4H), 7.46–7.32 (m, 7H), 7.15
2-(4-(2-(2-(2-Hydroxybenzylidene)hydrazinyl)-2-oxoethyl)- (d, J=2.9Hz, 1H), 6.98 (dd, J1=2.9Hz, J2=8.8Hz, 1H), 6.84
piperazin-1-yl)-N-(4-(3-(3-(trifluoromethyl)phenyl)ureido)- (d, J=8.8Hz, 1H), 5.05 (s, 2H), 3.32 (s, 1H), 3.12 (s, 3H), 2.58
phenyl)acetamide Dihydrochloride (2a): Yield 45.1%. mp (s, 8H).
1
245–248°C. ESI-MS m/z: 598.4 (M+H)+. H-NMR (300MHz,
2-(4-(2-(2-((7-Hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)-
DMSO-d6) δ: 11.98 (s, 1H), 10.59 (s, 1H), 10.17 (s, 1H), 9.77 (s, methylene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)-N-(4-(3-(3-
1H), 9.42 (s, 1H), 8.39 (s, 1H), 8.01 (s, 1H), 7.71 (d, J=6.5Hz, (trifluoromethyl)phenyl)ureido)phenyl)acetamide Dihydrochlo-
1H), 7.58–7.53 (m, 4H), 7.50–7.43 (m, 3H), 7.33–7.25 (m, 2H), ride (2h): Yield 54.2%. mp 261–263°C. ESI-MS m/z: 680.3
6.97–6.85 (m, 2H), 4.59 (s, 1H), 4.04 (s, 2H), 3.86 (s, 1H), 3.54 (M+H)+. 1H-NMR (300MHz, DMSO-d6) δ: 12.53 (s, 1H),
(s, 8H).
10.64 (s, 1H), 9.71 (d, J=9.0Hz, 1H), 9.39 (d, J=7.4Hz, 1H),
2-(4-(2-(2-(3,5-Di-tert-butyl-2-hydroxybenzylidene)hydra- 9.36 (s, 1H), 9.01 (s, 1H), 8.00 (s, 1H), 7.75 (d, J=8.9Hz, 1H),
zinyl)-2-oxoethyl)piperazin-1-yl)-N-(4-(3-(3-(trifluoromethyl)- 7.59–7.50 (m, 4H), 7.45 (d, J=8.9Hz, 2H), 7.29 (d, J=7.4Hz,
phenyl)ureido)phenyl)acetamide Dihydrochloride (2b): Yield 1H), 6.97 (d, J=8.9Hz, 1H), 6.28 (s, 1H), 4.52 (s, 1H), 4.12 (s,
58.9%. mp 275–277°C. ESI-MS m/z: 710.3 (M+H)+. H-NMR 2H), 3.77 (s, 1H), 3.55–3.27 (m, 8H), 2.42 (s, 3H).
1
(300MHz, DMSO-d6) δ: 11.99 (s, 1H), 10.56 (s, 1H), 9.66 (s,
N-(4-(3-(3-Chloro-4-(trifluoromethyl)phenyl)ureido)-
1H), 9.32 (s, 1H), 8.51 (s, 1H), 8.00 (s, 1H), 7.59–7.43 (m, 6H), phenyl)-2-(4-(2-(2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-6-
7.32–7.24 (m, 3H), 4.06 (s, 4H), 3.82 (s, 4H), 3.46 (s, 6H), 1.40 yl)methylene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)acetamide
(s, 9H), 1.28 (s, 9H).
Dihydrochloride (2i): Yield 49.0%. mp 249–251°C. ESI-MS
N-(4-(3-(3-Chlorophenyl)ureido)phenyl)-2-(4-(2-(2-(2- m/z: 714.0 (M+H)+. 1H-NMR (300MHz, DMSO-d6) δ:
hydroxybenzylidene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)- 12.47 (s, 1H), 10.61 (s, 1H), 9.85 (d, J=8.9Hz, 1H), 9.37 (d,
acetamide Dihydrochloride (2c): Yield 57.2%. mp 259–261°C. J=6.1Hz, 1H), 9.01 (s, 1H), 8.63 (s, 1H), 8.11 (s, 1H), 7.74 (d,
ESI-MS m/z: 564.2 (M+H)+. H-NMR (300MHz, DMSO-d6) J=8.9Hz, 1H), 7.61 (s, 2H), 7.55 (d, J=8.9Hz, 2H), 7.44 (d,
1
δ: 11.43 (s, 1H), 11.21 (s, 1H), 9.60 (s, 1H), 8.84 (s, 1H), 8.68 J=8.9Hz, 2H), 6.97 (d, J=8.9Hz, 1H), 6.27 (s, 1H), 4.49 (s,
(s, 1H), 8.53 (s, 1H), 7.71 (s, 1H), 7.54 (d, J=8.9Hz, 2H), 1H), 4.10 (s, 2H), 3.74 (s, 1H), 3.52–3.24 (m, 8H), 2.42 (s, 3H).
7.48 (dd, J1=1.7Hz, J2=7.2Hz, 1H), 7.38 (d, J=8.9Hz, 2H),
Cytotoxicity The cytotoxicity of compounds 1a–i and

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Vol. 60, No. 8
2) Traxler P., Allegrini P. R., Brandt R., Brueggen J., Cozens R., Fab-
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2a–i were evaluated against A549, MDA-MB-231 and HL-60
cell lines, and 1a, 1f and 1h were evaluated against WI38 cell
line by the MTT method in vitro, with sorafenib and PAC-1
as the positive controls. The cancer cell lines and the normal
cell line were cultured in minimum essential medium (MEM)
supplement with 10% fetal bovine serum (FBS).
Approximately 4×103 cells, suspend in MEM medium, were
plated onto each well of a 96-well plate and incubated in 5%
CO2 at 37°C for 24h. The tested compounds at indicated final
concentrations were added to the culture medium and the cell
cultures were continued for 72h. Fresh MTT was added to
each well at a terminal concentration of 5µg/mL and incubat-
ed with cells at 37°C for 4h. The formazan crystals were dis-
solved in 100µL dimethyl sulfoxide (DMSO) per well, and the
absorbency at 492nm (for absorbance of MTT formazan) and
630nm (for the reference wavelength) was measured with the
enzyme-linked immunosorbent assay (ELISA) reader. All of
the compounds were tested twice in the cell lines. The results
expressed as IC50 (inhibitory concentration of 50%) were the
averages of two determinations and were calculated by using
the Bacus Laboratories Inc. Slide Scanner (Bliss) software.
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Acknowledgements This work was supported by the Na-
tional High Technology Research and Development Program
(“863” Program) of China (No. 2012AA020305) and National
Natural Science Foundation of China (No. 21002065 and No.
81102470).
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