Novel N-phenyl dichloroacetamide derivatives as anticancer reagents: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2010.06.032

A current study shows that sodium dichloroacetate (DCA) can induce cancer cell apoptosis and inhibit tumor growth, but its cytotoxic activity is low (IC₅₀ > 1000 μM for A549). In this paper, a variety of DCA derivatives were synthesized, and their cytotoxic activities were evaluated. The result showed that the N-phenyl-2,2-dichloroacetamide analogues had satisfactory potencies. Among them, N-(3-iodophenyl)-2,2-dichloroacetamide (3e), an optimized lead compound, has an IC₅₀ against A549 as low as 4.76 μM. Furthermore, it can induce cancer cell apoptosis and has a low toxicity in mice (LD₅₀ = 1117 mg/kg). N-phenyl-2,2-dichloroacetamide analogues has higher cytotoxic activity and N-(3-iodophenyl)-2,2-dichloroacetamide (3e) is an optimized lead compound.

Full text of DOI:10.1016/j.ejmech.2010.06.032

European Journal of Medicinal Chemistry 45 (2010) 4300e4306
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel N-phenyl dichloroacetamide derivatives as anticancer reagents:
Design, synthesis and biological evaluation
,
b
a
c
Yongchong Yang a, Peihua Shang a, Changmei Cheng a , Dongchun Wang , Ping Yang , Feng Zhang ,
*
Tianwen Li a, Aijun Lu b, Yufen Zhao a
a Key laboratory of Bioorganic Phosphorus and Chemical Biology, The Ministry of Education, Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
b Jiangsu Simcere Pharmaceutical Research Institute and Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18 XuanWu Avenue,
XuanWu District, Nanjing 210042, PR China
c Department of Pharmacology, Taishan medical college, Taian, 271016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A current study shows that sodium dichloroacetate (DCA) can induce cancer cell apoptosis and inhibit
tumor growth, but its cytotoxic activity is low (IC50 > 1000 mM for A549). In this paper, a variety of DCA
derivatives were synthesized, and their cytotoxic activities were evaluated. The result showed that the
N-phenyl-2,2-dichloroacetamide analogues had satisfactory potencies. Among them, N-(3-iodophenyl)-
2,2-dichloroacetamide (3e), an optimized lead compound, has an IC50 against A549 as low as 4.76 mM.
Received 20 April 2010
Received in revised form
21 June 2010
Accepted 24 June 2010
Available online 30 June 2010
Furthermore, it can induce cancer cell apoptosis and has a low toxicity in mice (LD50 ¼ 1117 mg/kg).
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Sodium dichloroacetate (DCA)
Cytotoxic activity
Apoptosis
N-phenyl dichloroacetamide
1. Introduction
channel Kv1.5 is increased. DCA upregulates Kv1.5 through an
NFAT1-dependent mechanism, in which DCA reverses the metab-
In 1973, Whitehouse et al. discovered that sodium dichlor-
oacetate (DCA, Fig. 1) could activate mitochondrial pyruvate
dehydrogenase [1,2]. Since then, DCA has been studied as an
inhibitor of pyruvate dehydrogenase kinase [2e8] (PDK) with effi-
cacies on treating diabetes [9e11], ischemia [12], endotoxic shock
[13] acute hepatitis [14], cardiac insufficiency [3], lactic acidosis
[15e19] and so on. Clinical studies [16,18,19] have shown that DCA
is a lactate-lowering drug which can be employed for treating
various diseases caused by acquired and congenital lactic acidosis,
with an oral dose of 25e100 mg/kg body weight.
Recently, another great success has been reported [20,21]. DCA
can induce cancer cell apoptosis, decrease proliferation and inhibit
tumor growth without apparent toxicity [20]. In DCA-treated
cancer cells, mitochondrial PDK is inhibited, mitochondrial
membrane potential is decreased and the expression of the Kþ
olism of cancer cells from glycolysis to glucose oxidation, increases
mitochondrial H2O2, activates Kv channels, depolarizes mitochon-
drial membrane and decreases mitochondrial membrane potential
(MMP), which opens mitochondrial transition pores (MTP). In the
meantime, cytochrome C and AIF are released from the mito-
chondria to the cytoplasm, activating caspases and inducing
apoptosis [20]. Very recent reports have showed that DCA could
also induce apoptosis in endometrial cancer cells [22] and help to
radiate prostate cancer cells [23] in vitro by a similar mechanism.
This news is so encouraging [20e24] that many dying cancer
patients opted for the unapproved drug [24] because they had no
time to wait for a long-term clinical study. Embarrassingly, the
patients had to adopt the oral dosage for lactic acidosis because so
far there is no clinical study for cancer. However, compared to other
anticancer drugs in the market, the dosage is very high. Our study
also discloses that DCA’s cytotoxic activity is indeed not ideal, and
the value of IC50 for A549 cancer cell is more than 1000
Therefore it is necessary to modify DCA to enhance its cytotoxic
activity.
In this paper, a variety of DCA derivatives have been designed,
synthesized, and biologically evaluated. We found that a potential
compound N-3-iodophenyl-2,2-dichloroacetamide (3e) possesses
mM.
Abbreviations: DCA, sodium dichloroacetate; SAR, structure-activity relation-
ship; IC50, 50% inhibitory concentration; MTP, mitochondrial transition pores;
MMP, mitochondrial membrane potential; AIF, apoptosis-inducing factor; NFAT1,
nuclear factor of activated T cells 1.
* Corresponding author. Tel./fax: þ86 10 62784642.
E-mail address: chengcm@mail.tsinghua.edu.cn (C. Cheng).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.032

Y. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 4300e4306
4301
increasing potency. Optimization of the aniline ring has been
accomplished by systematically varying the substituents at ortho-,
meta-, and para-positions. These studies showed that the cytotoxic
activities of N-phenyl-2,2-dichloroacetamide analogues were
completely dependent on the characters and positions of the
substituents on the benzene ring (Table 1). Comparing to meta- and
para-positions, substitution at ortho-positions obviously had
a lower potency. For example, the IC50 of 2d was lower than that of
3d and 4d. It was proposed that the ortho-substituent was close to
the pharmacophore (2,2-dichloroacetyl) and may block the inter-
action between the pharmacophore and the receptor. The meta-
substituted structures had the best potencies, followed by the ones
with para-substituents. For example, the IC50 of N-(3-iodo-
O
Cl
O- Na+
Cl
sodium dichloroacetate(DCA)
Fig. 1. Structure of sodium dichloroacetate (DCA).
high activity, low toxicity and could be used as a candidate
compound for preclinical study.
2. Chemistry
phenyl)-2,2-dichloroacetamide (3e) was 4.76
N-(4-iodophenyl)-2,2-dichloroacetamide (4e) was 12.54
m
M, and the IC50 of
Synthesis of N-phenyl-2,2-dichloroacetamide derivatives is
outlined in Scheme 1. Substituted aniline and 1.3 mol equiv
dichloroacetyl chloride reacted in dry toluene, affording the
product N-phenyl-2,2-dichloroacetamide derivatives after reflux-
ing about 1e5 h, with yields of 90e99%. 3l and 4h were oxygenated
[25] for 48 h by AcOH/30% H2O2 (v:v/2:1), generating 3m and 4i.
mM. To
further study the influence of substituted positions, we synthesized
compounds 5aed. Compared to 3c, these compounds possessed
another Cl atom in 2-, 3-, 4- or 6-position beside the 3-position. The
results showed that ortho-Cl reduced the activity (e.g., 5a, 5d),
while para-Cl (5b) and meta-Cl (5c) helped to improve the activity,
and meta-Cl (5c) was the best.
3. Pharmacology
Hence, we focused on the optimization of the meta-position.
A series of meta-substituent analogues (3aeq) have been synthe-
sized and studied. The compounds possessing electron-donating
substituents (e.g., 3a, 3i) had lower potencies, while those with
strong electron-withdrawing substituents (e.g., 3h, 3j, 3k) showed
higher activities. However, the ones with moderate electron-
withdrawing substituents (e.g., 3e) exhibited the best potencies,
with a 27-fold increase in activity compared to N-phenyl-2,2-
dichloroacetamide (1). On the other hand, the compounds
with hydrophobic substituents (e.g., 3dee, 3jem) enhanced the
activity, and, with hydrophilic groups, decreased or even lost the
activity (e.g., 3nep). In conclusion, the moderate electron-with-
drawing and hydrophobic substituents at the meta-position were
beneficial to the activity.
3.1. Cytotoxic activity and SAR study
The cytotoxicity of all the synthesized compounds was
measured in vitro by MTT assay [26,27] using human tumor cell
lines derived from gastric carcinoma (BGC-823), oral epidermoid
carcinoma (KB), nonsmall cell lung cancer (A549), and liver
carcinoma (BEL-7402). As DCA derivatives on the A549 cell line
generally had higher activities, the IC50 on A549 was selected as
the activity index involved in the SAR discussion. The cytotoxicity
of DCA derivatives against the four tumor cell lines will be
discussed later.
DCA is a metal-salt that probably cannot penetrate the bio-
membrane due to its low liposolubility. Accordingly, we designed
and synthesized a mass of DCA prodrugs (more than 60) to
enhance the liposolubility, including metal-salts, ammonium salts,
esters and amides (Figs. 2 and 3). Unexpectedly, most of the
compounds could not show better potencies, while N-phenyl-
On the whole, DCA derivatives presented powerful potencies in
inhibiting not only A549, but also BEL-7402 and KB. The values of
IC50 against BEL-7402, KB and A549 were almost 5- to 10-fold more
potent than against BGC-823. For example, the IC50 of N-(3-iodo-
phenyl)-2,2-dichloroacetamide (3e) against BEL-7402, KB and A549
2,2-dichloroacetamide (1) displayed a 10-fold increase in IC50
.
was about 5
m
M, and 58.83 ꢀ 23.68
mM against BGC-823. Therefore,
Subsequently, we synthesized a homologous series of N-phenyl-
2,2-dichloroacetamide (Table 1). Biological evaluation showed that
N-benzyl-2,2-dichloroacetamide and 2,2-dichloro-N-(naphthalen-
4-yl)acetamide had no improvement in potency, while substituent
N-phenyl-2,2-dichloroacetamides presented satisfactory potency.
Thereafter, we were acutely aware that dichloroacetyl was the
active group, and the aniline ring was the essential factor in
N-(3-iodophenyl)-2,2-dichloroacetamide (3e) had a potential value
for drug development.
3.2. Annexin V-FITC/propidium iodide analysis of apoptosis
In order to study the mode of cell death (apoptosis or necrosis),
compound 3e was used to induce A549 cells apoptosis by Annexin
V-FITC/PI assay (Fig. 4). In early apoptotic cells, membrane phos-
pholipid phosphatidylserine (PS) was translocated from the inner
to the outer leaflet of the plasma membrane, exposing PS to the
external cellular environment [28]. Annexin V, a Ca2þ-dependent
phospholipid binding protein with high affinity for PS, binded to
cells with exposed PS. With a combination of Annexin V and PI
staining, the early apoptosis could be determined. Fig. 4 shows that
the percentages of apoptosis (Q2 þ Q4) of A549 cells treated with
Cl
H
NH2
N
O
Cl
a
R
Cl
O
R
Cl
Cl
yield,90-99%
2a-f;3a-l,n-q;4a-h,,j-l;5a-d.
Cl
H
compound 3e in 10 mM, 15 mM, 30 mM for 24 h were 19.4%, 19.5%,
Cl
H
N
35.5% respectively, and when the time increased to 48 h, the
percentages were 25.4%, 31.3%, 46.2% respectively. Compared to the
control assay, compound 3e could obviously induce apoptosis, and
the apoptosis percentage of A549 cells (Q2 þ Q4) increased with
the concentration of 3e and treatment time. For example, the
N
Cl
b
Cl
SO2CF3
SCF3
O
O
3m
4i
3l
4h
apoptosis percentage of cells treated with 30
35.5% compared to 19.4% in 10 M, and 46.4% in 30
treated for 48 h was more than 35.5% at 24 h. The results revealed
mM of 3e for 24 h was
m
mM when
Scheme 1. General synthetic N-phenyl-2,2-dichloroacetamide derivatives. Reagents
and conditions: (a) toluene, reflux, 1e5 h; (b) AcOH/30% H2O2 (v:v/2:1), 35 ꢁC, 48 h.

4302
Y. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 4300e4306
R
CHCl COO
CHCl2COO
H3N C COOMe
M
CHCl2COO
CHCl2COO B
CHCl COO
H3N R
2
N
2
H
Fig. 2. Structure of DCA salt derivatives. Explanation: I, M ¼ metallic ion; II, N ¼ natural nucleoside and derivatives; III, B ¼ base in nucleoside and derivatives; IV, R ¼ the R
substituting group in natural amino acid; V, R0 ¼ alkyl.
a good time-dependent and dose-dependent relationship of 3e
induced A549 cell apoptosis.
The general procedure for the preparation of N-phenyl-2,2-
dichloroacetamide derivatives was as follows: a mixture of
aniline or substituted aniline (3 mmol) and dichloroacetyl chlo-
ride (3.9 mmol, 1.3 equiv) in dry toluene (20 mL) was stirred
under reflux in a round-bottom flask. The progress of the reac-
tion was monitored by TLC (petroleum ether/ethyl acetate 1:1
to 4:1). After 1e5 h, the solvent and excess dichloroacetyl
chloride was allowed to evaporate under vacuum. The residual
powder was N-phenyl-2,2-dichloroacetamide derivatives, with
a 90e99% yield.
3.3. Acute toxicity
The median lethal dose (LD50) in Kunming mice for the
compounds with high cytotoxic activities (3c, 3e) were evaluated
by intragastric administration for 14 days. The LD50 values of 3c and
3e were 4549 mg/kg and 1117 mg/kg respectively. The results
indicate that the two compounds exhibit low toxicity and are well
tolerated by experimental animals.
5.2. General procedure for the synthesis of 3m and 4i
4. Conclusion
NHCOCHCl2
More than 100 DCA derivatives were designed, synthesized,
cytotoxic activity screened and SAR studied. The results show that
N-phenyl-2,2-dichloroacetamide analogues have satisfactory
potencies, and compound 3e has the highest potency. An apoptosis
and acute toxicity study on 3e showed that it could induce cancer
cell apoptosis and had lower toxicity (LD50 for mice as 1117 mg/kg).
Therefore, it was significant that 3e would be used as a candidate
compound for preclinical research. At the same time, we have
offered a novel strategy to modify the classic small molecule drug
(DCA), while there has been no report about DCA derivatives since
its bioactivity was discovered.
SO2CF3
5.2.1. N-(3-(trifluoromethylsulfonyl)phenyl)-2,2-dichloroacetamide 3m
AcOH 20 mL, 30% H2O2 10 mL and N-(3-(trifluoromethylthio)-
phenyl)-2,2-dichloroacetamide (3l) 0.496 g (1.6 mmol) were added
in a round-bottom flask, and the mixture was stirred at 35 ꢁC for
48 h. The progress of the reaction was monitored by TLC (petroleum
ether/ethyl acetate 4:1). When the reaction finished, the solvent
was evaporated under vacuum. The residue was dissolved in 20 mL
CH2Cl2 and washed with saturated aqueous NaHCO3 (2 ꢃ 20 mL)
and saturated brines (20 mL). The organic phases were dried with
Na2SO4 and concentrated. The residue was purified by column
chromatography (petroleum ether/ethyl acetate 8:1) to afford the
product. Yield: 209 mg (38%), Light yellow solid, mp 98e100 ꢁC; Rf
(petroleum ether/ethyl acetate 4:1): 0.50. 1H NMR (300 MHz,
CHLOROFORM-d) 8.34 (s, 1H), 8.19 (s, 1H), 8.17 (d, J ¼ 8.9, 1H),
7.88 (d, J ¼ 7.6, 1H), 7.72 (dd, J ¼ 7.6, 8.9, 1H), 6.08 (s, 1H);
13C NMR (75 MHz, CHLOROFORM-d)
162.79, 138.36, 132.27,
131.15,128.25,127.45, 122.02, 119.84 (q, J ¼ 325.8), 66.62; ESI-MS:
287.0 (M ꢄ H)ꢄ; HR-MS calculated for C9H6Cl2F3NO3S (M ꢄ H)ꢄ
333.9325, found 333.9331.
5. Experimental protocols
5.1. Chemistry
Toluene was distilled over calcium hydride, and other reagents,
including aniline or substituted aniline and dichloroacetyl chloride,
were obtained from commercial suppliers and used without further
purification. Proton and carbon magnetic resonance spectra (1H
NMR and 13C NMR, JOEL JNM-ECA) were recorded with tetrame-
thylsilane or solvent resonance as the internal standard (1H NMR:
TMS at 0.00 ppm, CDCl3 at 7.26 ppm, DMSO-d6 at 2.50 ppm; 13C
NMR: CDCl3 at 77.23 ppm, DMSO-d6 at 39.51 ppm). Mass spectra
were recorded with a Bruker ESQUIRE-LC ion trap spectrometer
equipped with a gas nebulizer probe, capable of analyzing ions up
to m/z 6000. HR-MS were obtained using a Bruker Fourier Trans-
form Ion Cyclotron Resonance Mass Spectrometer. TLC analyses
were carried out on silica gel F254, and the spots were examined
with UV light. All final products had a purity of ꢂ95%. The purity of
final products was determined by HPLC (LabTech). Method: Dia-
d
d
NHCOCHCl2
SO2CF3
monsil C18 column (4.6 mm ꢃ 250 mm, 5
mm); mobile phase:
5.2.2. N-(4-(trifluoromethylsulfonyl)-phenyl)-2,2-
dichloroacetamide 4i
methanol (90%), H2O (10%); flow rate ¼ 1.0 mL/min;
l
¼ 254 nm.
Procedure for synthesis of 4i followed 3m’s steps, N-(4-(tri-
fluoromethylthio)-phenyl)-2,2-dichloroacetamide (4h) 0.668 g (2.2
mmol), Yield: 226 mg (31%), light yellow solid, mp 137e139 ꢁC; Rf
(petroleum ether/ethyl acetate 4:1): 0.50. 1H NMR (300 MHz,
O
O
O
O
R
Cl
Cl
Cl
Cl
B
N
N
H
C
COOMe
N
H
R
H
Cl
Cl
Cl
Cl
CHLOROFORM-d)
d
8.36 (s, 1H), 8.07 (d, J ¼ 8.9, 2H), 7.91 (d, J ¼ 8.9,
2H), 6.08 (s, 1H); 13C NMR (75 MHz, CHLOROFORM-d)
d
162.67,
144.23, 132.55, 126.74, 120.69, 119.91 (q, J ¼ 326.6), 66.62; ESI-MS:
335.4 (M ꢄ H)ꢄ; HR-MS calculated for C9H6Cl2F3NO3S (M ꢄ H)ꢄ
333.9325, found 333.9335.
Fig. 3. Structure of DCA amide and ester derivatives. Explanation: VI, N ¼ natural
nucleoside and derivatives; VII, B ¼ base in nucleoside and derivatives; VIII, R ¼ the R
substituting group in natural amino acid; IX, R0 ¼ alkyl.

Y. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 4300e4306
4303
Table 1
Structure and in vitro activity data for N-phenyl-2,2-dichloroacetamide analogues.
Cl
Cl
Cl
R
Cl
Cl
H
N
H
N
H
N
H
N
H
N
Cl
R
R
Cl
Cl
Cl
Cl
Cl
O
O
O
O
O
R
1
2a-f
3a-q
4a-l
5a-d
Entry
R
IC50
(
m
M ꢀ error)a
BGC-823
KB
A549
BEL-7402
DCA
1
1051 ꢀ 360
1424 ꢀ 148
1011 ꢀ 300
130 ꢀ 44
860 ꢀ 271
eb
e
e
e
e
e
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
Me
F
Cl
Br
NO2
NHCOCHCl2
Me
F
Cl
Br
I
CN
C^CH
NO2
e
e
e
e
e
87.53 ꢀ 25.25
50.15 ꢀ 3.54
36.99 ꢀ 25.33
197.56 ꢀ 58.18
130.94 ꢀ 6.16
41.05 ꢀ 12.37
e
e
e
e
67.87 ꢀ 20.46
38.17 ꢀ 28.59
194.1 ꢀ 24.83
e
e
253.30 ꢀ 96.92
e
e
e
e
e
e
e
e
e
e
14.78 ꢀ 4.75
7.80 ꢀ 5.01
4.76 ꢀ 2.81
66.53 ꢀ 8.90
11.22 ꢀ 7.08
18.04 ꢀ 11.28
25.78 ꢀ 9.97
12.18 ꢀ 10.25
15.05 ꢀ 5.38
14.07 ꢀ 4.67
6.53 ꢀ 1.52
e
44.45 ꢀ 28.8
7.72 ꢀ 8.52
5.39 ꢀ 4.30
48.35 ꢀ 15.12
4.74 ꢀ 3.99
15.74 ꢀ 10.45
54.80 ꢀ 6.13
5.97 ꢀ 1.77
10.28 ꢀ 4.44
6.54 ꢀ 3.44
9.01 ꢀ 2.57
e
95.30 ꢀ 22.81
8.18 ꢀ 7.66
5.25 ꢀ 3.25
60.88 ꢀ 10.02
6.26 ꢀ 2.17
44.71 ꢀ 23.14
57.69 ꢀ 33.59
8.86 ꢀ 1.28
7.41 ꢀ 2.17
4.84 ꢀ 1.85
20.55 ꢀ 18.95
e
58.83 ꢀ 23.68
e
171.82 ꢀ 41.22
122.55 ꢀ 43.90
OMe
CF3
179.37
142.54
142.14
122.88
OCF3
SCF3
SO2CF3
COOMe
NHCOCHCl2
OCOCHCl2
SO2Ph(m-NHCOCHCl2)
Me
F
Cl
Br
I
NO2
OMe
SCF3
SO2CF3
COOMe
NHCOCHCl2
OCOCHCl2
2Cl
63.98 ꢀ 13.39
e
e
e
e
e
e
e
e
e
198.35
e
23.96 ꢀ 15.08
e
5.89 ꢀ 2.87
48.92 ꢀ 4.29
68.41 ꢀ 22.11
20.37 ꢀ 15.32
13.74 ꢀ 7.00
12.54 ꢀ 3.78
34.42 ꢀ 32.80
80.88 ꢀ 41.11
10.56 ꢀ 9.73
8.50 ꢀ 4.51
e
15.88
e
e
e
e
e
60.83 ꢀ 12.29
21.70 ꢀ 7.51
66.17 ꢀ 77.54
60.57 ꢀ 23.16
277.69 ꢀ 100.14
2.08 ꢀ 1.12
9.71 ꢀ 3.36
e
49.55 ꢀ 16.51
9.77 ꢀ 4.84
11.92 ꢀ 6.04
29.50 ꢀ 16.72
192.18 ꢀ 120.64
3.61 ꢀ 3.72
72.82 ꢀ 3.20
e
e
221.90 ꢀ 77.50
190.00 ꢀ 78.44
e
203.35 ꢀ 164.98
146.50
e
e
e
e
e
e
e
e
e
5a
5b
5c
5d
e
216.98 ꢀ 180.78
7.05 ꢀ 3.87
4.53 ꢀ 1.65
11.53 ꢀ 7.30
e
180.45 ꢀ 0.63
7.37 ꢀ 7.06
4.65 ꢀ 3.15
18.16 ꢀ 14.00
4Cl
5Cl
6Cl
176.90 ꢀ 102.43
60.05 ꢀ 67.14
e
5.09 ꢀ 0.51
4.40 ꢀ 0.50
12.38 ꢀ 7.34
a
IC50 is 50% inhibitory concentration.
‘e’ means IC50 could not be determined (IC50 > 400 mM).
b
5.2.3. N-phenyl-2,2-dichloroacetamide 1
White solid, mp 121e122 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
8.11 (s, 1H), 7.56 (d, J ¼ 8.4, 2H), 7.39 (t, J ¼ 7.2, 2H), 7.21 (t, J ¼
MHz, CHLOROFORM-d)
d
161.87, 153.05 (d, J ¼ 242.3), 126.11 (d, J ¼
7.7), 124.99, 124.95, 121.83, 115.33 (d, J ¼ 18.9), 66.75; ESI-MS: 220.0
d)
d
(M ꢄ H)ꢄ.
6.9, 1H), 6.04 (s, 1H); 13C NMR (75 MHz, CHLOROFORM-d)
d 162.14,
136.39, 129.39, 125.91, 120.56, 67.05; ESI-MS: 205.6 (M þ H)þ.
5.2.6. N-(2-chlorophenyl)-2,2-dichloroacetamide 2c
White solid, mp 115e116 ꢁC, 1H NMR (300 MHz, CHLORO-
5.2.4. N-(2-methylphenyl)-2,2-dichloroacetamide 2a
FORM-d)
1.4, 1H), 7.33 (td, J ¼ 7.9, 1.4, 1H), 7.14 (td, J ¼ 7.9, 1.4, 1H), 6.07
(s, 1H); 13C NMR (75 MHz, CHLOROFORM-d)
161.84, 133.34,
d
8.82 (s, 1H), 8.32 (dd, J ¼ 8.3, 1.4, 1H), 7.43 (dd, J ¼ 8.0,
White solid, mp 140e141 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
d)
d
8.09 (s, 1H), 7.77 (d, J ¼ 7.9, 1H), 7.25 (t, J ¼ 7.6, 1H), 7.23 (d, J ¼
d
7.9, 1H), 7.15 (t, J ¼ 7.2, 1H), 6.06 (s, 1H), 2.32 (s, 3H); 13C NMR (75
129.46, 128.10, 126.20, 123.98, 121.58, 67.08; ESI-MS: 238.0
MHz, CHLOROFORM-d)
d
162.18, 134.15, 130.91, 130.10, 127.16,
(M ꢄ H)ꢄ.
126.62, 123.14, 67.21, 17.59; ESI-MS: 216.0 (M ꢄ H)ꢄ.
5.2.7. N-(2-bromophenyl)-2,2-dichloroacetamide 2d
5.2.5. N-(2-fluorophenyl)-2,2-dichloroacetamide 2b
White solid, mp 113e115 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
White solid, mp 108e109 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
d)
d
8.82 (s, 1H), 8.29 (dd, J ¼ 8.2, 1.4, 1H), 7.59 (dd, J ¼ 8.0, 1.4, 1H),
d)
d
8.41 (s, 1H), 8.26 (m, 1H), 7.16 (m, 3H), 6.06 (s, 1H); 13C NMR (75
7.37 (t, J ¼ 7.2, 1H), 7.07 (t, J ¼ 7.0, 1H), 6.07 (s, 1H); 13C NMR (75

4304
Y. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 4300e4306
Fig. 4. Flow cytometric analysis of phosphoatidylserine externalization (Annexin V binding) and cell membrane integrity (PI staining). A549 cells were treated with compound 3e
at 10, 15 and 30 M for 24 and 48 h. Early apoptotic cells were presented in the Q4 quadrant, and late apoptotic cells were presented in the Q2 quadrant.
m
MHz, CHLOROFORM-d)
d
161.92, 134.45, 132.70, 128.74, 126.67,
5.2.14. N-(3-iodophenyl)-2,2-dichloroacetamide 3e
121.87, 114.43, 67.14; ESI-MS: 281.8(M ꢄ H)ꢄ.
White solid, mp 116e117 ꢁC, 1H NMR (300 MHz, DMSO-d6)
d
10.74 (s, 1H), 8.07 (s, 1H), 7.56 (d, J ¼ 8.2, 1H), 7.52 (d, J ¼ 8.0, 1H),
5.2.8. N-(2-nitrophenyl)-2,2-dichloroacetamide 2e
7.18 (t, J ¼ 8.0, 1H), 6.58 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
Yellow solid, mp 79e81 ꢁC, 1H NMR (300 MHz, DMSO-d6)
d
11.01
d 161.87, 138.91, 133.18, 130.96, 127.99, 119.09, 94.58, 67.16; ESI-MS:
(s, 1H), 8.04 (d, J ¼ 8.1, 1H), 7.78 (t, J ¼ 7.6, 1H), 7.69 (d, J ¼ 7.7, 1H),
328 (M ꢄ H)ꢄ.
7.49 (t, J ¼ 7.7, 1H), 6.78 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
162.87, 143.26, 134.84, 130.25, 127.30, 126.53, 125.75, 67.25; ESI-
5.2.15. N-(3-cyanophenyl)-2,2-dichloroacetamide 3f
MS: 248.0(M ꢄ H)ꢄ.
Yellow solid, mp 149e150 ꢁC, 1H NMR (300 MHz, CHLORO-
FORM-d)
d
8.27 (s,1H), 7.99 (s,1H), 7.87e7.70 (m,1H), 7.60e7.42 (m,
162.28, 138.43,
5.2.9. N-(2-(2,2-dichloroacetamido)phenyl)-2,2-dichloroacetamide 2f
White solid, mp 214e216 ꢁC, 1H NMR (300 MHz, DMSO-d6)
2H), 6.07 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
130.45, 128.19, 124.48, 122.60, 118.39, 111.91, 67.13; ESI-MS:
d
10.08 (s, 2H), 7.51 (dd, J ¼ 5.9, 3.6, 2H), 7.32 (dd, J ¼ 6.0, 3.5, 2H),
227.2 (M ꢄ H)ꢄ.
6.65 (s, 2H); 13C NMR (75 MHz, DMSO-d6)
d 162.54, 130.02, 126.69,
125.61, 67.02; ESI-MS: 328.9(M ꢄ H)ꢄ.
5.2.16. N-(3-ethynylphenyl)-2,2-dichloroacetamide 3g
Yellow solid, mp 111e113 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
5.2.10. N-(3-methylphenyl)-2,2-dichloroacetamide 3a
d)
d
8.11 (s, 1H), 7.69 (s, 1H), 7.58 (d, J ¼ 3.5, 1H), 7.35e7.31 (m, 2H),
White solid, mp 105e106 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
6.04 (s, 1H), 3.11 (s, 1H); 13C NMR (75 MHz, CHLOROFORM-d)
d)
d
8.06 (s, 1H), 7.39 (s, 1H), 7.35 (d, J ¼ 8.1, 1H), 7.26 (t, J ¼ 7.7, 1H),
d 162.45, 136.39, 129.57, 129.39, 124.12, 123.31, 121.16, 82.83, 78.34,
7.02 (d, J ¼ 7.4, 1H), 6.03 (s, 1H), 2.37 (s, 3H); 13C NMR (75 MHz,
66.91; ESI-MS: 226.2 (M ꢄ H)ꢄ.
CHLOROFORM-d)
d 162.05, 139.44, 136.30, 129.20, 126.71, 121.13,
117.61, 67.10, 21.6; ESI-MS: 216.0 (M ꢄ H)ꢄ.
5.2.17. N-(3-nitrophenyl)-2,2-dichloroacetamide 3h
Yellow solid, mp 103e105 ꢁC, 1H NMR (300 MHz, DMSO-d6)
5.2.11. N-(3-fluorophenyl)-2,2-dichloroacetamide 3b
d
11.15 (s, 1H), 8.59 (t, J ¼ 2.1, 1H), 8.00 (dd, J ¼ 8.2, 2.3, 1H), 7.96 (dd,
White solid, mp 133e134 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
J ¼ 7.8, 1.6, 1H), 7.68 (t, J ¼ 8.2, 1H), 6.64 (s, 1H); 13C NMR (75 MHz,
d)
d
8.19 (s, 1H), 7.52 (s, 1H), 7.52 (dd, J ¼ 8.9, 8.9,1H), 7.08 (d, J ¼ 8.3,
DMSO-d6) d 162.40, 147.97, 138.75, 130.49, 125.83, 119.15, 114.05,
1H), 7.06 (d, J ¼ 8.9, 1H), 6.05 (s, 1H); 13C NMR (75 MHz,
67.15; ESI-MS: 246.8 (M ꢄ H)ꢄ.
CHLOROFORM-d)
d
162.14, 160.38 (d, J ¼ 244.5), 132.39, 122.49
(d, J ¼ 7.9), 116.20(d, J ¼ 22.2), 66.94; ESI-MS: 220.0 (M ꢄ H)ꢄ.
5.2.18. N-(3-methoxyphenyl)-2,2-dichloroacetamide 3i
White solid, mp 79e82 ꢁC, 1H NMR (300 MHz, CHLORO-
5.2.12. N-(3-chlorophenyl)-2,2-dichloroacetamide 3c
FORM-d)
d
8.12 (s, 1H), 7.34e7.21 (m, 2H), 7.04 (d, J ¼ 8.0, 1H),
White solid, mp 104e105 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
6.75 (dd, J ¼ 8.3, 2.4, 1H), 6.04 (s, 1H), 3.83 (s, 3H); 13C
d)
d
8.09 (s, 1H), 7.68 (s, 1H), 7.42 (d, J ¼ 8.0, 1H), 7.31 (t, J ¼ 8.0, 1H),
NMR (75 MHz, CHLOROFORM-d) d 162.51, 160.27, 137.49,
7.19 (d, J ¼ 7.7,1H), 6.04 (s,1H); 13C NMR (75 MHz, CHLOROFORM-d)
130.01, 112.92, 111.73, 106.48, 67.07, 55.45; ESI-MS:
d
162.02, 137.52, 135.18, 130.44, 126.01, 120.58, 118.42, 66.88; ESI-
256.0 (M þ Na)þ.
MS: 238.0 (M ꢄ H)ꢄ.
5.2.19. N-(3-(trifluoromethyl)-phenyl)-2,2-dichloroacetamide 3j
Pink solid, mp 80e82 ꢁC, 1H NMR (300 MHz, CHLOROFORM-d)
5.2.13. N-(3-bromophenyl)-2,2-dichloroacetamide 3d
White solid, mp 107e108 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
d
8.24 (s, 1H), 7.86 (s, 1H), 7.78 (d, J ¼ 7.7, 1H), 7.52 (t, J ¼ 7.9, 1H),
7.47 (d,
7.8, 1H), 6.06 (s, 1H); 13C NMR (75 MHz,
CHLOROFORM-d)
d)
d
8.11 (s, 1H), 7.81 (s, 1H), 7.48 (d, J ¼ 7.5, 1H), 7.34 (d, J ¼ 7.7, 1H),
J
¼
7.23 (t, J ¼ 7.9,1H), 6.04 (s,1H); 13C NMR (75 MHz, CHLOROFORM-d)
d
162.86, 136.92, 131.81 (q, J ¼ 32.7), 130.00,
d
162.10, 137.62, 130.69, 128.94, 123.46, 123.00, 118.99, 66.87;
123.90, 123.78 (q, J ¼ 270.3),122.60, 117.60, 66.82; ESI-MS:
ESI-MS: 281.7 (M ꢄ H)ꢄ.
271.3 (M ꢄ H)ꢄ.

Y. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 4300e4306
4305
5.2.20. N-(3-(trifluoromethoxy)-phenyl)-2,2-dichloroacetamide 3k
White solid, mp 76e77 ꢁC, 1H NMR (300 MHz, DMSO-d6)
10.93
5.2.29. N-(4-bromophenyl)-2,2-dichloroacetamide 4d
d
White solid, mp 148e150 ꢁC, 1H NMR (300 MHz, DMSO-d6)
10.76 (s, 1H), 7.57 (d, J ¼ 9.7, 4H), 6.58 (s, 1H); 13C NMR (75 MHz,
(s, 1H), 7.74 (s, 1H), 7.57 (d, J ¼ 8.3, 1H), 7.51 (t, J ¼ 8.0, 1H), 7.15
d
(d, J ¼ 7.1, 1H), 6.61 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
162.17,
DMSO-d6) d 162.37, 137.50, 132.37, 122.31, 117.03, 67.80; ESI-MS:
148.57, 139.29, 130.74, 120.08(q, J ¼ 254.6), 118.49, 116.67, 112.00,
67.20; ESI-MS: 287.3 (M ꢄ H)ꢄ; HR-MS calculated for C9H6Cl2F3NO2
(M ꢄ H)ꢄ 285.9655, found 285.9661.
281.8 (M ꢄ H)ꢄ.
5.2.30. N-(4-iodophenyl)-2,2-dichloroacetamide 4e
White solid, mp 173e175 ꢁC, 1H NMR (300 MHz, DMSO-d6)
5.2.21. N-(3-(trifluoromethylthio)-phenyl)-2,2-dichloroacetamide 3l
White solid, mp 88e89 ꢁC, 1H NMR (300 MHz, CHLOROFORM-d)
d
10.74 (s, 1H), 7.72(d, J ¼ 8.6, 2H), 7.44 (d, J ¼ 8.6, 2H), 6.58 (s, 1H);
13C NMR (75 MHz, DMSO-d6)
d 161.78, 137.66, 137.40, 121.93, 88.62,
d
8.19 (s, 1H), 7.87 (s, 1H), 7.75 (d, J ¼ 7.8, 1H), 7.50 (d, J ¼ 7.8, 1H),
67.25; ESI-MS: 327.9 (M ꢄ H)ꢄ.
7.44 (t, J ¼ 7.8,1H), 6.06 (s,1H); 13C NMR (75 MHz, CHLOROFORM-d)
d
162.68, 137.34, 133.47,130.36, 129.59 (q, J ¼ 308.5), 127.94, 125.67,
5.2.31. N-(4-nitrophenyl)-2,2-dichloroacetamide 4f
123.03, 66.83; ESI-MS: 302.2 (M ꢄ H)ꢄ; HR-MS calculated for
C9H6Cl2F3NOS (M ꢄ H)ꢄ 301.9427, found 301.9434.
Yellow solid, mp 130e132 ꢁC, 1H NMR (300 MHz, DMSO-d6)
d
11.24 (s, 1H), 8.29(d, J ¼ 9.2, 2H), 7.88 (d, J ¼ 9.2, 2H), 6.65 (s, 1H);
13C NMR (75 MHz, DMSO-d6)
d 162.46, 143.70, 143.26, 125.02,
5.2.22. N-(3-methoxycarbonylphenyl)-2,2-dichloroacetamide 3n
White solid, mp 108e110 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
119.78, 67.14; ESI-MS: 247.9 (M ꢄ H)ꢄ.
d)
d
8.54 (s, 1H), 8.13 (s, 1H), 7.92 (d, J ¼ 7.9, 1H), 7.87 (d, J ¼ 7.6, 1H),
5.2.32. N-(4-methoxyphenyl)-2,2-dichloroacetamide 4g
7.47 (t, J ¼ 7.8, 1H), 6.12 (s, 1H), 3.92 (s, 3H); 13C NMR (75 MHz,
White solid, mp 136e137 ꢁC, 1H NMR (300 MHz, CHLORO-
CHLOROFORM-d)
d
166.70, 162.54, 136.76, 131.15,129.52,126.81,
FORM-d)
d
8.07 (s, 1H), 7.46 (d, J ¼ 9.0, 2H), 6.90 (d, J ¼ 9.0, 2H),
125.12, 121.65, 66.91, 52.58; ESI-MS: 262 (M þ H)þ; HR-MS calcu-
6.04 (s, 1H), 3.81 (s, 3H); 13C NMR (75 MHz, CHLOROFORM-d)
lated for C10H9Cl2NO3 (M ꢄ H)ꢄ 259.9887, found 259.9893.
d
162.03, 157.61, 129.36, 122.39, 114.55, 67.06, 55.69; ESI-MS:
231.9 (M ꢄ H)ꢄ.
5.2.23. N-(3-(2,2-dichloroacetamido)phenyl)-2,2-
dichloroacetamide 3o
5.2.33. N-(4-(trifluoromethylthio)-phenyl)-2,2-dichloroacetamide 4h
White solid, mp 124e125 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
Brown solid, mp 199e201 ꢁC, 1H NMR (300 MHz, DMSO-d6)
d
10.76 (s, 2H), 8.03 (s,1H), 7.38 (d, J ¼ 1.2, 3H), 6.60 (s, 2H); 13C NMR
d)
CHLOROFORM-d)
d
8.20 (s, 1H), 7.91e7.49 (m, 4H), 6.05 (s, 1H); 13C NMR (75 MHz,
(75 MHz, DMSO-d6)
d
161.79, 138.12, 129.62, 115.98,111.16,67.29;
d
162.42, 138.96, 137.68, 129.60 (q, J ¼ 308.3),
ESI-MS: 329.0 (M ꢄ H)ꢄ.
121.07, 66.85; ESI-MS: 302.2 (M ꢄ H)ꢄ; HR-MS calculated for
C9H6Cl2F3NOS (M ꢄ H)ꢄ 301.9427, found 301.9434.
5.2.24. N-(3-(2,2-dichloroacetoxy)phenyl)-2,2-dichloroacetamide 3p
White solid, mp 156e158 ꢁC, 1H NMR (300 MHz, DMSO-d6)
5.2.34. N-(4-Methoxycarbonylphenyl)-2,2-dichloroacetamide 4j
White solid, mp 141e143 ꢁC, 1H NMR (300 MHz, DMSO-d6)
d
10.49 (s, 1H), 9.55 (s, 1H), 7.16 (s, 1H), 7.14 (t, J ¼ 8.3, 1H), 6.98 (d, J
¼ 7.9, 1H), 6.55 (dd, J ¼ 2.0, 8.3, 1H), 6.54 (s, 1H); 13C NMR (75 MHz,
d
10.98 (s, 1H), 7.98 (d, J ¼ 8.8, 2H), 7.75 (d, J ¼ 8.7, 2H), 6.62 (s, 1H),
DMSO-d6)
d
161.54, 157.81, 138.59, 129.78, 111.84, 110.47, 106.83,
3.84 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d 165.64, 162.15, 141.95,
67.40; ESI-MS: 330.4 (M ꢄ H)ꢄ; HR-MS calculated for C10H7Cl4NO3
130.43, 125.36, 119.35, 67.23, 52.00; ESI-MS: 261.7 (M þ H)þ;
HR-MS calculated for C10H9Cl2NO3 (M ꢄ H)ꢄ 259.9887, found
259.9891.
(M ꢄ H)ꢄ 327.9107, found 327.9113.
5.2.25. N-(3-(3-(2,2-dichloroacetamido)-phenylsulfonyl)phenyl)-
2,2-dichloroacetamid (3q)
5.2.35. N-(4-(2,2-dichloroacetamido)phenyl)-2,2-dichloroacetamide4k
White solid, mp 263e265 ꢁC, 1H NMR (300 MHz, DMSO-d6)
Light yellow solid, mp 205e206 ꢁC, 1H NMR (300 MHz, DMSO-
d6)
d
11.07 (s, 2H), 8.27 (s, 2H), 7.88 (d, J ¼ 8.0, 2H), 7.73 (d, J ¼ 7.9,
d
10.68 (s, 2H), 7.61 (s, 4H), 6.58 (s, 2H); 13C NMR (75 MHz, DMSO-
2H), 7.65 (t, J ¼ 7.9, 2H), 6.60 (s, 2H); 13C NMR (75 MHz, DMSO-d6)
d6)
d
161.58, 134.11, 120.44, 67.32; ESI-MS: 329.1 (M ꢄ H)ꢄ.
d
162.29, 141.47, 138.78, 130.85, 124.60, 123.24, 117.94, 67.17; ESI-
MS: 470.0 (M ꢄ H)ꢄ; HR-MS calculated for C16H12Cl4N2O4S (M ꢄ
5.2.36. N-(4-(2,2-dichloroacetoxy)phenyl)-2,2-dichloroacetamide 4l
White solid, mp 140e142 ꢁC, 1H NMR (300 MHz, DMSO-d6)
H)ꢄ 466.9199, found 466.9204.
d
10.38 (s, 1H), 9.39 (s, 1H), 7.38 (d, J ¼ 8.8, 2H), 6.75 (d, J ¼ 8.8, 2H),
5.2.26. N-(4-methylphenyl)-2,2-dichloroacetamide 4a
6.53 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d 161.16, 154.46, 129.06,
White solid, mp 159e160 ꢁC, 1H NMR (300 MHz, DMSO-d6)
121.60, 115.41, 67.46; ESI-MS: 330.4(M þ H)þ; HR-MS calculated for
d
10.54 (s, 1H), 7.48 (d, J ¼ 8.6, 2H), 7.18 (d, J ¼ 8.6, 2H), 6.57(s, 1H),
C10H7Cl4NO3 (M ꢄ H)ꢄ 327.9107, found 327.9112.
6.28 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d 161.78, 137.66, 137.40,
121.93, 88.62, 67.25; ESI-MS: 327.9 (M ꢄ H)ꢄ.
5.2.37. N-(2,3-dichloropheny)-2,2-dichloroacetamide 5a
White solid, mp 140e141 ꢁC, 1H NMR (300 MHz, DMSO-d6)
5.2.27. N-(4-fluorophenyl)-2,2-dichloroacetamide 4b
d
10.47 (s,1H), 7.62 (dd, J ¼ 8.0,1.3,1H), 7.56 (dd, J ¼ 8.1,1.4,1H), 7.42
White solid, mp 134e135 ꢁC, 1H NMR (300 MHz, CHLOROFORM-d)
(t, J ¼ 8.1, 1H), 6.81 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d 162.62,
d
8.10 (s, 1H), 7.53 (dd, J ¼ 8.8, 4.6, 2H), 7.08 (t, J ¼ 8.5, 2H), 6.04 (s, 1H); 13
C
135.36, 132.19, 128.27, 128.14, 126.44, 125.31, 66.66; ESI-MS:
NMR (75 MHz, CHLOROFORM-d)
d
162.07, 160.38 (d, J ¼ 243.8), 132.38,
271.5 (M ꢄ H)ꢄ.
122.52(d, J ¼ 8.3), 116.19(d, J ¼ 22.5), 66.93; ESI-MS: 220.9 (M ꢄ H)ꢄ.
5.2.38. N-(3,4-dichloropheny)-2,2-dichloroacetamide 5b
5.2.28. N-(4-chlorophenyl)-2,2-dichloroacetamide 4c
White solid, mp 148e149 ꢁC, 1H NMR (300 MHz, DMSO-d6)
10.95 (s, 1H), 7.95 (d, J ¼ 2.4, 1H), 7.64 (d, J ¼ 8.8, 1H), 7.54
White solid, mp 141e142 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
d
d)
d
8.11 (s, 1H), 7.52 (d, J ¼ 8.8, 2H), 7.35 (d, J ¼ 8.8, 2H), 6.04 (s, 1H);
(dd, J ¼ 8.8, 2.4, 1H), 6.60 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
13C NMR (75 MHz, CHLOROFORM-d)
d
162.10,134.95,131.15,129.48,
d 162.14, 137.65, 131.26, 130.92, 126.33, 121.11, 119.92, 67.12; ESI-MS:
121.80, 66.91; ESI-MS: 238.0 (M ꢄ H)ꢄ.
271.6 (M ꢄ H)ꢄ.

4306
Y. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 4300e4306
5.2.39. N-(3,5-dichloropheny)-2,2-dichloroacetamide 5c
doses of 633, 844, 1125, 1500, 2000 mg/kg. Changes in appearance,
behavior and mortality in mice were monitored for 14 days after
administration. LD50 values were calculated according to the
Kaerber method.
White solid, mp 145e146 ꢁC, 1H NMR (300 MHz, CHLOROFORM-
d)
d
8.11 (s, 1H), 7.54 (d, J ¼ 1.4, 2H), 7.20 (s, 1H), 6.03 (s, 1H); 13C
NMR (75 MHz, CHLOROFORM-d)
d 162.10, 138.19, 135.81, 125.94,
118.73, 66.72; ESI-MS: 295.9 (M þ Na)þ.
Acknowledgment
5.2.40. N-(2,5-dichloropheny)-2,2-dichloroacetamide 5d
White solid, mp 150e151 ꢁC, 1H NMR (300 MHz, DMSO-d6)
We would like to thank the National Natural Science Foundation
of China for financial support (No. 20972130, 20872078). We also
thank Dr. Wei Zhou for his help with writing this article.
d
10.42 (s, 1H), 7.80 (d, J ¼ 2.4, 1H), 7.60 (d, J ¼ 8.7, 1H), 7.38
(dd, J ¼ 8.6, 2.5, 1H), 6.83 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
162.72, 134.67, 131.78, 131.13, 127.32, 125.85, 125.60, 66.58; ESI-
MS: 271.5 (M ꢄ H)ꢄ.
Appendix. Supplementary data
5.3. Cell culture and cytotoxic activity assays
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.ejmech.2010.06.032.
Human gastric carcinoma cell line (BGC-823), human oral
epidermoid carcinoma cell line (KB), human nonsmall cell lung
cancer cell line (A549), and Human liver carcinoma cell Line (BEL-
7402), were obtained from ATCC. BGC-823, KB, and BEL-7402 cells
were cultured in RPMI 1640 supplemented with 10% fetal bovine
serum at 37 ꢁC in a humidified atmosphere containing 5% CO2. A549
cells were maintained in F12-k supplemented with 10% fetal bovine
serum at 37 ꢁC in a humidified atmosphere containing 5% CO2.
Cytotoxic activity was assessed by standard MTT assay. A
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