Synthesis and antitumor activity of novel nitrogen mustard-linked chalcones

Article abstract of DOI:10.1002/ardp.201200443

A series of nitrogen mustard-linked chalcones were synthesized and evaluated for their antitumor activity in vitro against the K562 and HepG2 cell lines. The aldol condensation of [N,N-bis(chloroethyl)-3-amino]-acetophenone (2) with aromatic aldehydes afforded the nitrogen mustard-linked chalcones. Among the analogs tested, compounds 5e and 5k exhibited significant anti-proliferation activities against K562 cells with IC₅₀ values of 2.55 and 0.61 μM, respectively, which revealed higher cell toxicity than the standard drugs cisplatin (IC₅₀ > 200 μM) and adriamycin (IC₅₀ = 14.88 μM). The methoxyl and N,N-dimethyl groups on the B-ring of the chalcone frame enhanced the inhibitory activities against both the K562 and HepG2 cell lines. The structure-activity relationship study indicated that the inhibitory activities significantly varied with the position(s) and species of the substituted group(s). A series of nitrogen mustard-linked chalcones were prepared and evaluated for their antitumor activities against the K562 and HepG2 cell lines. The structure-activity relationship study indicated that the methoxyl and N,N-dimethyl groups on the B-ring of the chalcone frame may enhance the inhibitory activities of the chalcones. Copyright

Full text of DOI:10.1002/ardp.201200443

292
Arch. Pharm. Chem. Life Sci. 2013, 346, 292–299
Full Paper
Synthesis and Antitumor Activity of Novel Nitrogen
Mustard-Linked Chalcones
Xianwen Fang, Bingqin Yang, Zhao Cheng, Meipan Yang, Na Su, Lizhen Zhou, and Jia Zhou
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education,
Department of Chemistry and Materials Science, Northwest University, Xi’an, P. R. China
A series of nitrogen mustard-linked chalcones were synthesized and evaluated for their antitumor
activity in vitro against the K562 and HepG2 cell lines. The aldol condensation of [N,N-bis(chloroethyl)-3-
amino]-acetophenone (2) with aromatic aldehydes afforded the nitrogen mustard-linked chalcones.
Among the analogs tested, compounds 5e and 5k exhibited significant anti-proliferation activities
against K562 cells with IC50 values of 2.55 and 0.61 mM, respectively, which revealed higher cell
toxicity than the standard drugs cisplatin (IC50 > 200 mM) and adriamycin (IC50 ¼ 14.88 mM). The
methoxyl and N,N-dimethyl groups on the B-ring of the chalcone frame enhanced the inhibitory
activities against both the K562 and HepG2 cell lines. The structure–activity relationship study
indicated that the inhibitory activities significantly varied with the position(s) and species of the
substituted group(s).
Keywords: Anti-proliferation / Cell toxicity / Chalcones / Crystal structure / Nitrogen mustard
Received: November 23, 2012; Revised: December 25, 2012; Accepted: January 18, 2013
DOI 10.1002/ardp.201200443
Additional supporting information may be found in the online version of this article at the publisher’s
:
web-site.
Introduction
reports indicated that chalcones, as a kind of anticancer
agents [8, 9], exhibit potential cytotoxicity in the treatment
of some kinds of cancer neoplastic diseases. Wang et al.
[10] have shown that millepachine 1 (Fig. 2), which was
isolated from Millettia pachycarpa for the first time in their
group, exhibited potent apoptosis-inducing effects, making
them promising candidates for the treatment of cancer.
Nakamura et al. [11] have designed and synthesized fluori-
nated 3,4-dihydroxychalcone 2, which showed 5-lipoxy-
genase inhibition on rat basophilic leukemia-1 (RBL-1)
cells and inhibitory action on Fe3þ-ADP-induced NADPH-
dependent lipid peroxidation in rat liver microsomes. Xia
et al. [12] synthesized the novel 20-amino chalcone 3, which
had high activity towards the multi-drug resistant KB-VIN and
the ovarian 1A9 cell lines, with IC50 values of 0.30 and
0.35 mg/mL, respectively. In order to investigate and develop
efficient and broad-spectrum antitumor drugs, the structure
modification of chalcones has become a focus in related
research in the recent years.
Chalcones are a group of natural compounds belonging to
the flavonoid family and are found in the heartwood, bark,
leaves, fruits, and roots of a variety of trees and plants [1].
Chemically, with a common 1,3-diphenyl-2-propen-1-one
framework (Fig. 1), chalcones, as the main precursors in
the biosynthesis of flavonoids and as an important substruc-
ture of some natural compounds, have been known for over
a century. Up to date, many studies have shown that the
natural and synthetic chalcone derivatives show a diverse
array of biological activities, among which are antioxidant
[2], antimalarial [3], anti-inflammatory [4], antileishmanial
[5], antibacterial, and antifungal activities [6, 7]. Recent
Correspondence: Bingqin Yang, Key Laboratory of Synthetic and Natural
Functional Molecule Chemistry of the Ministry of Education, Department
of Chemistry and Materials Science, Northwest University, Xi’an 710069,
P. R. China.
E-mail: fxw325@126.com; yangbq@nwu.edu.cn
Fax: þ86 29 88302217
Bifunctional alkylating agents, particularly nitrogen mustards,
are an important class of anticancer drugs [13, 14]. With the
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Arch. Pharm. Chem. Life Sci. 2013, 346, 292–299
Antitumor Activity of Nitrogen Mustard-Linked Chalcones
293
strand cross-linking with DNA. Like other bifunctional
alkylating agents, they are active against both resting and
rapidly dividing tumor cells [23].
In the past several years, designs and modifications of the
structure of nitrogen mustards to increase their cytotoxicity,
especially for their specificity toward tumor cells, have been
in the focus of interest of many biochemists [24–26]. But,
to the best of our knowledge, there have so far been no
literature reports regarding nitrogen mustard-containing
chalcone derivatives in which the N,N-bis(2-chloroethyl)-
amine pharmacophore was linked to the chalcone frame-
work. In our present study, we designed and synthesized a
series of nitrogen mustard-linked chalcone derivatives, and
their anticancer activity against two cancer cell lines was
evaluated in vitro, with the aim of searching new potent and
selective anticancer agents. We also elucidated the influence
of the electronic effect of different substituents on the
chalcone framework on the cytotoxicity of the chalcones.
Figure 1. The basic framework of chalcones.
N,N-bis(2-chloroethyl)amine pharmacophore, nitrogen mus-
tards are believed to exert their biological activity through
interstrand cross-linking in the major groove of DNA
(Scheme 1) [13, 15], and this linkage represents the highest
toxicity of all alkylation events. However, due to their high
reactivity and the fact that they have no particular affinity
for alkylating DNA, a high proportion of those nitrogen
mustards drugs can easily react with other cellular com-
ponents such as proteins [16], thiols, or genes [17] before
reaching the DNA. And this disadvantage produced many
unwanted side effects such as bone marrow toxicity [18],
leading to the loss of the drugs’ therapeutic activity against
malignancy. Also, a significant amount of the drug may reach Results and discussion
the DNA with only one arm of the mustard intact, forming
the monoadducts that are considered to be more genotoxic
rather than toxic [19] (Scheme 1). Drugs of this class, such
as mechlorethamine [20], melphalan [21], and chlorambucil
[22], are widely used in cancer treatment, and their cyto-
toxicity appears to be related to the extent of their inter-
Synthesis
The newly synthesized compounds are nitrogen mustards
linked to substituted chalcones. In order to investigate the
structure–activity relationship (SAR) of the chalcone-nitro-
gen mustard conjugates, we introduced several different
Figure 2. Structures of some reported chal-
cones with antitumor potency.
Scheme 1. Chemical structures of nitrogen mustards and formation of DNA-mustard cross-links.
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X. Fang et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 292–299
substituents into the B-ring of the chalcone framework. The
novel nitrogen mustard-linked chalcone derivatives 5a–5k
were obtained through the synthetic route shown in
Scheme 2. First, 3-nitroacetophenone was reduced by
Fe/NH4Cl–CH3COOH in ethanol, giving 3-aminoacetophenone
1 [27]. Hydroxyethylation of 1 with 2-chloroethanol in water
and in the presence of CaCO3 at 1208C for 7 h afforded 3-[N,N-
bis(2-hydroxyethyl)-amino]-acetophenone 2 [28]. In order to
obtain the intermediate 3-[N,N-bis(2-hydroxyethyl)-amino]-
acetophenone 3, we first attempted the method of direct
chlorination by treating the diol 2 with SOCl2 in CH2Cl2 or
by treating with POCl3 [29, 30]. Unfortunately, this did not
give the required chlorinated compound. So we adopted the
indirect chlorination method: Tosylation of diol 2 followed
by displacement with NaCl in DMF [31] gave the intermediate
4. Compounds 5a–k were obtained by a base-catalyzed
Claisen–Schmidt condensation [32] of 3 with substituted
aromatic aldehydes. All of the synthetic compounds gave
satisfactory analytical and spectroscopic data, which were
in full accordance with their depicted structures. The IR and
1H NMR spectra of compounds 1--4 and 5a--k are given in
Supporting Information Figures S1--S30.
the full-matrix least squares based on F2 using the SHELXTL-
97 program. All non-hydrogen atoms were refined anisotropi-
cally and hydrogen atoms were generated geometrically. The
crystal data and structural refinement parameters of 3 and 5a
are summarized in Supporting Information Table S1. The two
molecular structures with the atom numbering scheme are
shown in Fig. 3.
Compounds 3 and 5c crystallized in different space groups,
P21/n and P-1. XRD studies on compound 3 showed that the two
benzene rings derived from the p-toluenesulfonyl unit are
planar, exist on both sides of the benzene ring plane [C11,
C12, C13, C14, C15, C16, C17] and are almost parallel with it
(Fig. 2). The dihedral angles between the three benzene rings
are 5.278, 11.228, and 5.918, respectively. There are similarities
in the bond lengths of S(1)–O(3) and S(2)–O(5), with distances of
˚
1.573(2) and 1.570(2) A, respectively, because of the existence of
the same electronegativity and space volume. In the crystal
lattice, there are several weak hydrogen bond intramolecular
interactions (Supporting Information Table S2) and the crystal
packing is further stabilized by these hydrogen bonds.
XRD studies were also carried out for the title compound
5c. The two benzene rings are almost co-planar, with dihedral
angles of 4.9(42)8. The bond lengths and angles of the benzene
rings are quite unequivalent, which may be the result of the
different space volumes and electronegativities of the sub-
stituents in each ring. There are several weak intramolecular
hydrogen bonds (Supporting Information Table S3).
Crystallographic structures of compounds 3 and 5c
Suitable crystals of compounds 3 and 5c for X-ray diffraction
(XRD) were grown by the solvent evaporation method. The
diffraction intensity data were collected on a Bruker Smart
Apex II CCD diffractometer with graphite-monochromated
Mo Ka radiation (k ¼ 0.71073) at 296(2) K. Empirical absorp-
tion corrections were applied using the SADABS program.
The structures were solved by direct methods and refined by
Biological activity tests
The cytotoxicities of the compounds were evaluated against
the human cancer cell lines HepG2 (liver cells) and K562
OTs
OH
O
O
O
O
NO2
NH2
N
N
c
a
b
OH
OTs
1
2
3
Cl
Cl
Cl
O
O
N
N
e
d
R
Cl
CHO
R
_
4
5a k
5a: R= 2-N(CH2CH2Cl)2 5b: R= 2,4-Cl2 5c: R= p-NO2
5d: R= p-Br
5h: R= o-Cl
5e: R= p-OCH3
5i: R= p-F
5f: R= 2-F
5j: R= p-Cl
5g: R= H
5k: R= p-N(CH3)2
Reagents and conditions: (a) Fe powder, NH4Cl, CH3COOH, ethanol, reflux, 2 h; (b) 2-chloroethanol, CaCO3,
H2O, 120°C, 7 h; (c) TsCl, Et3N, THF, CH2Cl2, r.t., 26 h; (d) DMF, LiCl, 110°C, 30 min; (e) substituted aromatic
aldehyde, ethanol, 2.5 M NaOH, r.t., 4–6 h.
Scheme 2. Synthesis of nitrogen mustard-linked chalcone derivatives 5a–5k.
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Antitumor Activity of Nitrogen Mustard-Linked Chalcones
295
Figure 3. ORTEP diagrams for compounds 3 and 5c.
(human leukemia cells) by the colorimetric MTT assay [33,
34]. Although their binding sites might not be identical with
those of the series of nitrogen mustard-linked chalcone
derivatives, the anticancer drugs cisplatin (DDP) and adria-
mycin (ADM) were used as reference compounds because of
their well-established clinical application in cancer. The
results are presented in Table 1, which illustrates the con-
centrations required to inhibit cell growth by 50% (IC50
values); a low IC50 is desired and implies cytotoxicity or
anti-proliferation activity at low drug concentrations [35, 36].
The anti-proliferation activity results for the K562 cells
treated with each compound indicate that compound 5k
(4-N(CH3)2) showed the highest activity with an IC50 value
of 0.61 mM. The compounds displaying a potent anti-
proliferation effect on K562 cells were 5a (4-N(CH2CH2Cl)2),
5b (2,4-di-Cl), 5c (4-NO2), 5d (4-Br), 5e (4-OCH3), 5f (2-F), and 5g,
with IC50 values of 6.53, 10.39, 10.82, 13.43, 2.55, 4.22, and
14.75 mM, respectively. They all revealed higher activity
than the standard drug adriamycin (IC50 ¼ 14.88 mM).
Compounds 5h (2-Cl), 5i (4-F), and 5j (4-Cl) were as active as
the control drug (adriamycin) or less active than it. All the
newly synthesized chalcones displayed much higher potency
than the other standard drug, cisplatin (IC50 > 200 mM).
As we can see, replacement of the 4-H of the B-ring with
substituents such as N,N-dimethyl (5k), nitro (5c), a bromine
atom (5d), methoxyl (5e), and the N,N-dichloroethyl group
(5a) displayed different degrees of increase in their anti-
proliferation activity against K562 cells. In contrast, groups
as fluorine and chlorine atoms introduced at the para-
position of the B-ring may decrease the cytotoxicity on
the selected cell lines. Turning to the effects of an electron-
releasing group at the para-position, we found that replace-
ment of electron-withdrawing groups with electron-donating
groups (5a, 5k) causes an increase in cytotoxicity on the
K562 cell line. Moreover, comparison of the cytotoxicities
of 5b, 5h and 5j, 5f and 5i, respectively, led to the impression
that a fluorine or chlorine atom at the ortho-position of the
B-ring, instead of the same atom at the para-position, may
enhance the cytotoxicity on the K562 cell line.
Table 1. IC50a) (mM) values of the target compounds for K562 and
HepG2 cells.
Compound
K562
HepG2
In HepG2 cells, the results followed a different trend:
Compound 5e was a good substrate for inhibition of these
cells, providing the lowest IC50 value (5.19 mM) among the
newly prepared chalcones. However, replacement of 4-H of 5g
with the electron-withdrawing groups mentioned before
resulted in an increase in cytotoxicity on HepG2 cells to a
minor degree. When comparing the cytotoxicities of com-
pounds with groups as the fluorine or chlorine atom at the
ortho- and para-positions of the B-ring, we found that only
fluorine in the ortho-position caused a minor increase in anti-
proliferation activity, while the chlorine atom had the oppo-
site effect. Compounds 5i, 5j, 5f, and 5k showed moderate
to good cytotoxicity on the HepG2 cell line, with IC50 values
of 16.06, 15,60, 15.22, and 8.54 mM, respectively, and the
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
ADM
DDP
6.53
10.39
10.82
13.43
2.55
34.98
44.24
24.97
41.66
5.19
15.22
26.50
18.97
16.06
15.60
8.53
4.22
14.75
15.06
16.73
16.57
0.61
14.88
Nb)
4.19
11.31
a)
IC50 values are the mean of three experiments in replicate of at
least three independent experiments.
b)
N means inactive (IC50 values > 200 mM).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 292–299
AXIMA-CFRTM plus MALDI-TOF mass spectrometer. Column flash
chromatography was carried out on Merck silica gel (250–400
mesh ASTM). Thin-layer chromatography (TLC) was performed on
silica gel GF254. The solvents and chemicals used for synthesis
were commercially available and were used without further
purification unless otherwise noted.
cytotoxicities on HepG2 cells of these compounds were close to
that of the standard drug cisplatin (IC50 ¼ 11.31 mM), but
higher than that of adriamycin (IC50 ¼ 4.19 mM). More impres-
sively, the chalcones 5e and 5k with the methoxyl and N,N-
dimethyl group at the para-position of the B-ring, respectively,
showed high potency in cytotoxicity on both the K562 and
HepG2 cell lines, suggesting that these kinds of compounds
are of value in the future as drugs for chemotherapy.
3-Aminoacetophenone (1)
Iron powder (3.9 g, 69 mmol) was activated by refluxing it with a
mixture of 10 mL distilled water, ethanol (40 mL), glacial acetic
acid (1 mL), and NH4Cl (0.2 g, 4 mmol) for 0.5 h under vigorous
stirring at 808C; then, ethanol (10 mL) and 3-nitroacetophenone
(1.65 g, 10 mmol) were added to the mixture within 10 min.
After the mixture was heated slowly until reflux under vigorous
stirring for 2 h, the hot reaction mixture was filtered and the
residue was washed with ethanol for three times. After evapor-
ation of the alcohol, the residue was diluted with water and
extracted with CHCl3/CH3OH 2:1 for three times; the extract was
dried over anhydrous Na2SO4 and the solvent was evaporated at
reduced pressure. The crude was purified on a flash column
(CH3CO2Et/petroleum ether 2:1 as eluent) to give 3-aminoaceto-
phenone 1. Yield: 84% of white solid, mp 96–978C; 1H NMR
(400 MHz, CDCl3): d 7.32 (dd, 1H, J ¼ 7.6, 0.8 Hz, ArH), 7.24
(t, 2H, J ¼ 7.8 Hz, ArH), 6.89–6.84 (m, 1H, ArH), 3.90 (s, 2H,
–NH2), 2.55 (s, 3H, –CH3); IR (KBr) n (cmꢀ1): 3467, 3369, 3222,
1668, 1628, 1597, 1490, 1457, 1356, 1324, 1236, 777, 683.
These results are consistent with previous reports on SAR
studies on chalcones derivatives, which showed that even a
subtle modification of the chalcone chemical structure may
result in significant changes in their biological potency [37].
Taking into account the cytotoxicity data of this study on
both the K562 and HepG2 cell lines, all the tested chalcones
present moderate to good toxicity, which may indicate that
this kind of compounds is worthy of further investigation.
Conclusion
In this paper, we reported on the synthesis, characterization,
and antitumor activities of nitrogen mustard-linked chal-
cones analogs. The crystal structures of the intermediate 3
and the title compound 5a were obtained, and structural
analysis revealed that compounds 3 and 5a are monoclinic
with the space group P21/c and triclinic with the space group
P-1, respectively. The SAR results revealed that the inhibition
activities of this kind of compounds are dependent on the
substituents on the B-ring; the structure modification indi-
cated that a methoxyl or an N,N-dimethyl group at the para-
position of the B-ring may increase the inhibitory activity.
According to the MTT assay, most of these derivatives had
micromolar-level potency against the tested cell lines.
Compounds 5e and 5k showed the most potent inhibition,
with IC50 values of 2.55 and 5.19 mM for K562 cells and 0.61
and 8.53 mM for HepG2 cells, respectively, revealing much
higher inhibitory potency than the standard drug cisplatin
and similar inhibition capacity with regard to adriamycin.
The results of this study show that this kind of compounds
may be considered as potential anticancer agents, and fur-
ther studies need to be conducted to reach a better under-
standing of the signaling mechanisms of the subject
compounds, and the relevant study is underway.
[N,N-Bis(2-hydroethyl)-3-amino]-acetophenone (2)
A mixture of 3-aminoacetophenone (6.4 g, 47.5 mmol), 2-chloro-
ethanol (16 mL, 240 mmol), and CaCO3 (6.5 g, 65 mmol) in water
(60 mL) was heated under reflux with vigorous stirring for 7 h.
After hot filtering, the unreacted CaCO3 was washed with a few
portions of hot water; then, the filtrate was extracted with
CH2Cl2 (40 mL ꢁ 3) dried over anhydrous MgSO4 and concen-
trated under reduced pressure to afford a yellow oil. The residue
was further purified by SiO2 flash column chromatography
(CH3CO2Et/petroleum ether 2:1 as eluent) to give the required
diol 2. Yield: 68% of faint yellow liquid; 1H NMR (400 MHz,
CDCl3): d 7.20–7.24 (m, 3H, ArH), 6.85–6.87 (m, 1H, ArH), 4.74
(s, 2H, –OH), 3.77 (s, 4H, –OCH2–), 3.53–3.54 (d, 4H, J ¼ 4.8 Hz,
–NCH2–), 2.49 (s, 3H, –CH3); IR (KBr) n (cmꢀ1): 3378, 2933, 2880,
1673, 1597, 1494, 1444, 1357, 1267, 1178, 1010, 779, 688; HRMS
(ESI) C12H17NO3 [MþNa]þ: 246.1101, found: 246.1105.
N,N-Bis[2-(tosyloxyethyl)-3-amino]-acetophenone (3)
A solution of diol 2 (5.8 g, 26 mmol) in dry THF/CH2Cl2 (208 mL,
THF/CH2Cl2 1:3) was cooled at 08C, and Et3N (10.27 mL,
77.35 mmol) was added, followed by tosylchloride (16.65 g,
87 mmol). The mixture was stirred at r.t. for 26 h, diluted with
CH2Cl2 and washed many times with aqueous NaHCO3 and brine.
After drying with anhydrous Na2SO4, the solvent was evaporated
under reduced pressure. The residue was chromatographed on
silica gel (CH3CO2Et/petroleum ether 1:5 as eluent) to give the
required ditosylate. Yield: 46% of faint yellow solid, mp 87–898C;
1H NMR (400 MHz, CDCl3): d 7.68 (d, 4H, J ¼ 8.2 Hz, ArH), 7.28–
7.22 (m, 6H, ArH), 7.03 (d, 1H, J ¼ 1.0 Hz, ArH), 6.68 (dd, 1H,
J ¼ 7.9, 2.0 Hz, ArH), 4.11 (t, 4H, J ¼ 5.7 Hz, –OCH2–), 3.60 (t, 4H,
J ¼ 5.7 Hz, –NCH2–), 2.55 (s, 3H, –CH3), 2.39 (s, 6H, –PhCH3);
Experimental
Chemistry
Melting points are uncorrected and were taken on a XT-4 micro
melting point apparatus. 1H NMR spectra were recorded on an
INOVA-400 NMR spectrometer and chemical shifts are reported
in parts per million (ppm, d) relative to tetramethylsilane as
internal standard. The infrared spectra were recorded on an
EQUINOX-55 FTIR spectrometer using KBr pellets and values
are expressed in cmꢀ1. ESI-MS data were recorded on an
IR (KBr)
n
(cmꢀ1): 3426, 2959, 2923, 1678, 1589, 1515,
1356, 1173, 1178, 1092, 812, 764; HRMS (ESI) calcd. for
C26H29NO7S2 [MþNa]þ: 554.1278, found: 554.1270.
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Antitumor Activity of Nitrogen Mustard-Linked Chalcones
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IR (KBr) n (cmꢀ1): 2923, 2855, 1664, 1600, 1563, 1507, 1452,
1340, 1249, 833, 789, 744; HRMS (ESI) calcd. for C19H18Cl2N2O3
[MþNa]þ: 415.0587, found: 415.0611.
[N,N-Bis(2-chloroxyethyl)-3-amino]-acetophenone (4)
A solution of ditosylate 3 (2.5 g, 4.7 mmol) in a minimum
volume of dry DMF was added to a LiCl solution (0.8 g,
18.8 mmol) also in DMF, and the resulting mixture was heated
at 1108C for 30 min. The mixture was diluted with water and
extracted with CH2Cl2 for three times. The organic phase was
washed with water and dried with anhydrous Na2SO4. The
solvent was evaporated under reduced pressure to give a
yellow residue, which was then chromatographed on silica gel
(CH3CO2Et/petroleum ether 1:15 as eluent) to give compound 4.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(4-bromophenyl)prop-2-en-1-one (5d)
Yield: 78% of yellow solid, mp 76–788C; 1H NMR (400 MHz,
CDCl3): d 7.72 (d, 1H, J ¼ 15.7 Hz, H17), 7.51 (dt, 4H, J ¼ 15.8,
9.0 Hz, H16 and ArH), 7.42–7.35 (m, 2H, ArH), 7.35–7.27 (m, 2H,
ArH), 6.95–6.89 (m, 1H, ArH), 3.83–3.77 (m, 4H, ClCH2–), 3.66
(dd, 4H, J ¼ 15.7, 6.9 Hz, –NCH2–); IR (KBr) n (cmꢀ1): 2959, 1674,
1595, 1562, 1489, 1440, 1452, 1351, 1277, 833, 777, 727; HRMS
(ESI) calcd. for C19H18Cl2BrNO [MþNa]þ: 447.9841, found:
447.9846.
1
Yield: 68% of light green solid, mp 55–578C; H NMR (400 MHz,
CDCl3): d 7.37–7.30 (m, 2H, ArH), 7.27 (s, 1H, ArH), 6.93–6.87
(m, 1H, ArH), 3.78 (t, 4H, J ¼ 6.8 Hz, ClCH2–), 3.65 (t, 4H,
J ¼ 6.8 Hz, –NCH2–), 2.58 (s, 3H, –CH3). IR (KBr) n (cmꢀ1): 3084,
2962, 2923, 1670, 1593, 1557, 1519, 1399, 1358, 1164, 814, 747;
HRMS (ESI) calcd. for C12H15Cl2NO [MþNa]þ: 282.0423, found:
282.0449.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(4-methoxyphenyl)prop-2-en-1-one (5e)
Yield: 82% of yellow solid, mp 123–1248C; 1H NMR (400 MHz,
CDCl3) d 7.77 (d, 1H, J ¼ 15.6 Hz, H17), 7.59 (d, 2H, J ¼ 8.5 Hz,
ArH), 7.36 (dd, 4H, J ¼ 18.8, 14.2 Hz, H16 and ArH), 6.93 (d, 3H,
J ¼ 8.6 Hz, ArH), 3.84 (s, 3H, –OCH3), 3.82–3.77 (m, 4H, ClCH2–),
3.66 (t, 4H, J ¼ 6.3 Hz, –NCH2–); IR (KBr) n (cmꢀ1): 2924,
2850, 1651, 1594, 1563, 1506, 1428, 1260, 826, 785, 724;
HRMS (ESI) calcd. for C20H21Cl2NO2 [MþNa]þ: 400.0842, found:
400.0862.
General procedure for the preparation of compounds 5a–k
To a solution of [N,N-bis(2-chloroxyethyl)-3-amino]-acetophenone
(3 mmol) and substituted aromatic aldehydes (3 mmol) in etha-
nol (9 mL), a solution of 2.5 M sodium hydroxide (1 mL) was
added slowly within 10 min, in an ice bath. After stirring for
4–6 h at room temperature, the formed precipitate was left. The
mixture was extracted with CH2Cl2 (10 mL ꢁ 3) and dried over
MgSO4. After the solvent was evaporated under reduced pressure,
the crude product was obtained; then, the crude product was
further purified by SiO2 flash column chromatography. The
yield, melting point, and spectral date of each compound were
collected as described below.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(2-fluorophenyl)prop-2-en-1-one (5f)
Yield: 85% of yellow solid, mp 85–868C; 1H NMR (400 MHz,
CDCl3): d 7.89 (d, 1H, J ¼ 15.9 Hz, H17), 7.63 (dd, 2H, J ¼ 19.3,
11.6 Hz, H16 and ArH), 7.44–7.29 (m, 4H, ArH), 7.23–7.09
(m, 2H, ArH), 6.93 (d, 1H, J ¼ 2.8 Hz, ArH), 3.81 (t, 4H,
J ¼ 6.6 Hz, ClCH2–), 3.68 (t, 4H, J ¼ 6.5 Hz, –NCH2–); IR (KBr)
n (cmꢀ1): 2922, 2578, 1654, 1599, 1564, 1491, 1450, 1352,
1219, 840, 745, 695; HRMS (ESI) calcd. for C19H18Cl2FNO
[MþNa]þ: 388.0642, found: 388.0665.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
{4-[N,N-bis(2-chloroxyethyl)phenyl]}prop-2-en-1-one (5a)
Yield: 84% of deep yellow solid, mp 124–1268C; 1H NMR
(400 MHz, CDCl3): d 7.75 (d, 1H, J ¼ 15.5 Hz, H17), 7.56 (d, 2H,
J ¼ 8.5 Hz, ArH), 7.42–7.27 (m, 4H, ArH and H17), 6.90 (d, 1H,
J ¼ 3.2 Hz, ArH), 6.70 (d, 2H, J ¼ 8.6 Hz, ArH), 3.81 (d, 8H,
J ¼ 4.0 Hz, ClCH20–), 3.67 (d, 8H, J ¼ 3.3 Hz, –NCH2–); IR (KBr)
n (cmꢀ1): 2923, 2855, 1648, 1570, 1519, 1443, 1351, 1164, 816,
769; HRMS (ESI) calcd. for C23H26Cl4N2O [MþNa]þ: 511.0664,
found: 511.0679.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
phenylprop-2-en-1-one (5g)
Yield: 85% of yellow sticky liquid; 1H NMR (400 MHz, CDCl3):
1H NMR (400 MHz, CDCl3): d 7.89 (d, 1H, J ¼ 15.9 Hz, H17),
7.63 (dd, 2H, J ¼ 19.3, 11.6 Hz, H16 and ArH), 7.44–7.29
(m, 4H, ArH), 7.23–7.09 (m, 2H, ArH), 6.93 (d, J ¼ 2.8 Hz,
1H, ArH), 3.81 (t, 4H, J ¼ 6.6 Hz, ClCH2–), 3.68 (t, 4H,
J ¼ 6.5 Hz, –NCH2–); IR (KBr) n (cmꢀ1): 3028, 2958, 2924, 1658,
1596, 1491, 1446, 1351, 1292, 1214, 756, 687; HRMS (ESI) calcd.
for C19H19Cl2NO [MþNa]þ: 370.0736, found: 370.0749.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(2,4-dichloro-phenyl)prop-2-en-1-one (5b)
Yield: 87% of yellow powdery solid, mp 109–1108C; 1H NMR
(400 MHz, CDCl3): 8.09 (d, 1H, J ¼ 15.7 Hz, H17), 7.68
(d, J ¼ 8.4 Hz, 1H, ArH), 7.54–7.27 (m, 6H, H16 and ArH), 6.94
(d, 1H, J ¼ 6.2 Hz, ArH), 3.89–3.77 (m, 4H, ClCH2–), 3.73–3.61
(m, 4H, –NCH2–); IR (KBr) n (cmꢀ1): 2922, 1656, 1582, 1457,
1357, 1312, 1250, 821, 774, 698; HRMS (ESI) calcd. for
C21H17Cl4NO [MþNa]þ: 463.9929, found: 463.9933.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(2-chlorophenyl)prop-2-en-1-one (5h)
Yield: 86% of yellow solid, mp 85–868C; 1H NMR (400 MHz,
CDCl3): d 8.16 (d, 1H, J ¼ 15.8 Hz, H17), 7.73 (s, 1H, ArH),
7.44 (d, 2H, J ¼ 15.7 Hz, H16 and ArH), 7.34 (d, 5H,
J ¼ 21.2 Hz, ArH), 6.93 (s, 1H, ArH), 3.81 (t, 4H, J ¼ 6.4 Hz,
ClCH2–), 3.68 (d, 4H, J ¼ 6.5 Hz, –NCH2–); IR (KBr) n (cmꢀ1):
3068, 2959, 2924, 1662, 1593, 1489, 1443, 1353, 1275, 1212,
1045, 756, 691; HRMS (ESI) calcd. for C19H18Cl3NO [MþNa]þ:
404.0346, found: 404.0359.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(4-nitrophenyl)prop-2-en-1-one (5c)
Yield: 89% of orange red solid, mp 140–1428C; 1H NMR
(400 MHz, CDCl3): d 8.28 (d, 2H, J ¼ 8.4 Hz, ArH), 7.80 (dd, 3H,
J ¼ 11.7, 7.5 Hz, ArH), 7.61 (d, 1H, J ¼ 15.7 Hz, H17), 7.38
(t, 3H, J ¼ 15.6 Hz, H17 and ArH), 6.97 (d, 1H, J ¼ 6.2 Hz, ArH),
3.89–3.79 (m, 4H, ClCH2–), 3.69 (t, 4H, J ¼ 6.3 Hz, –NCH2–);
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X. Fang et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 292–299
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(4-fluorophenyl)prop-2-en-1-one (5i)
Yield: 86% of pale yellow solid, mp 65–678C; 1H NMR (400 MHz,
CDCl3): d 7.74 (d, 1H, J ¼ 15.7 Hz, H16), 7.57 (d, 2H, J ¼ 8.4 Hz,
ArH), 7.47 (d, 2H, J ¼ 15.7 Hz, H17 and ArH), 7.39 (dd, 3H, J ¼ 7.3,
4.1 Hz, ArH), 7.32 (s, 1H, ArH), 6.94 (dd, 1H, J ¼ 5.9, 3.2 Hz, ArH),
3.82 (t, 4H, J ¼ 6.8 Hz, ClCH2–), 3.68 (t, 4H, J ¼ 6.8 Hz, –NCH2–);
IR (KBr) n (cmꢀ1): 2924, 2857, 1658, 1589, 1502, 1448, 1446, 1211,
825, 780, 735; HRMS (ESI) calcd. for C19H18Cl2FNO [MþNa]þ:
388.0642, found: 388.0665.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(4-chlorophenyl)prop-2-en-1-one (5j)
Yield: 92% of pale yellow solid, mp 103–1058C; 1H NMR (400 MHz,
CDCl3): d 7.76 (d, 1H, J ¼ 15.7 Hz, H17), 7.69–7.60 (m, 2H, H16 and
ArH), 7.49–7.30 (m, 4H, ArH), 7.11 (t, 2H, J ¼ 8.5 Hz, ArH), 6.93
(dd, 1H, J ¼ 5.7, 3.1 Hz, ArH), 3.82 (t, 4H, J ¼ 6.7 Hz, ClCH2–), 3.68
(t, 4H, J ¼ 6.7 Hz, –NCH2–); IR (KBr) n (cmꢀ1): 2923, 2855, 1654,
1596, 1560, 1486, 1391, 1276, 820, 769, 722; HRMS (ESI) calcd.
for C19H18Cl3NO [MþNa]þ: 404.0346, found: 403.0364.
Figure 4. A 96-well plate showing the shaded wells in columns 2–
11 used for the experiments.
and the plate was then incubated at 378C for 24 h. A culture
medium-diluted solution (100 mL) of the test compounds in
DMSO (Grenier, Germany) was added into the wells in columns
2 (lowest) to 9 (highest), respectively, to obtain the required
quantitative concentrations of 256, 64, etc. down to 2 mg/mL;
the culture medium (100 mL) was also added to the wells
in columns 10 and 11, and the plate was incubated at 378C
for 72 h.
A modified procedure of Mosmann [38, 39] was used for the
MTT assays. An MTT solution in PBS (20 mL, 2.5 mg/mL) was
added to each well of the plate, which was then incubated for
4 h, by which time a purple precipitate of formazan formed at
the bottom of certain wells. The contents of each well were
carefully aspirated off to leave the formazan, and then 150 mL
DMSO was added and mixed thoroughly to dissolve the dark-blue
crystals. The plates were read on a SpectraMax 190 microplate
reader (Molecular Devices Inc., USA) at 490 nm within 1 h of
DMSO addition. The IC50 was defined as the compound concen-
tration required to inhibit cell proliferation by 50%. The exper-
iment was repeated three times.
(E)-30-[N,N-Bis(2-chloroxyethyl)-amino-phenyl]-1-
(4-dimethylaminophenyl)prop-2-en-1-one (5k)
Yield: 87% of orange-yellow solid, mp 105–1078C; 1H NMR
(400 MHz, CDCl3): d 7.78 (d, 1H, J ¼ 15.4 Hz, H17), 7.54 (d, 2H,
J ¼ 8.2 Hz, ArH), 7.34 (d, 4H, J ¼ 23.5 Hz, ArH and H16), 6.89
(s, 1H, ArH), 6.68 (d, 2H, J ¼ 8.1 Hz, ArH), 3.79 (d, 4H, J ¼ 6.1 Hz,
–CH2Cl), 3.67 (d, 4H, J ¼ 6.2 Hz, –NCH2–), 3.03 (s, 6H, –N(CH3)2);
IR (KBr) n (cmꢀ1): 2923, 2855, 1644, 1605, 1559, 1521, 1440, 1360,
1170, 810, 728; HRMS (ESI) calcd. for C21H24Cl2N2O [MþH]þ:
391.1338, found: 391.1349.
Cytotoxicity evaluation
All reagents were handled in a sterile fume hood. RPMI 1640
medium, fetal bovine serum (FBS) and HEPES were purchased
from Gibco; phosphate-buffered saline pH 7.4 (PBS) and trypsin–
EDTA (0.25% trypsin and 1 mM Na4(EDTA)) were obtained from
Sigma–Aldrich. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) was a Genview product. The growth medium
(RPMI 1640 medium with 10% FBS, 100 U/mL penicillin, and
100 mg/L streptomycin), HEPES, and MTT were stored at 48C,
while trypsin–EDTA and FBS were stored frozen at ꢀ208C and
thawed before use; PBS was stored at r.t.
The human cancer cell line HepG2 (liver cells) was obtained
from the Fourth Military Medical University; the K562 cell line
(human leukemia cells) was obtained from BeiJing DingGuo
ChangSheng Biotechnology Co., Ltd. Cells were cultured in 50-
cm2 culture flasks (Corning) using RPMI-1640 medium supple-
mented with 10% FBS, penicillin (100 IU/mL), and streptomycin
(100 mg/mL). The culture was maintained at 378C in an
atmosphere of 5% CO2 and 95% relative humidity. The cells
were transferred to a new flask every 2 days and treated with
trypsin–EDTA to detach them from the flask.
This work was supported by the National Natural Science Foundation of
China (No. 21172178). The authors are grateful to Prof. XiangDong Hu for
his encouragement and discussions on this research topic, and to Prof.
MingLi Peng and Ms. ZuHong Tian for assistance with the biological
activity test. We thank the ‘‘National Engineering Research Center for
Miniaturized Detection Systems’’ for providing biological services.
The authors have declared no conflict of interest.
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