Synthesis and anti-cancer activity evaluation of new dimethoxylated chalcone and flavanone analogs

Article abstract of DOI:10.1002/ardp.201400215

A novel series of chalcones and flavanones discriminated by the presence of a 3,4-dimethoxyphenyl moiety in their structures were synthesized as anti-cancer agents. The cytotoxicity evaluation of the analogs against the MCF-7, MDA-MB-231 (human breast can

Full text of DOI:10.1002/ardp.201400215

Arch. Pharm. Chem. Life Sci. 2014, 347, 853–860
853
Full Paper
Synthesis and Anti-Cancer Activity Evaluation of New
Dimethoxylated Chalcone and Flavanone Analogs
Shima H. M. E. Ketabforoosh1, Asma Kheirollahi2, Maliheh Safavi3, Nasim Esmati4, Sussan
K. Ardestani2, Saeed Emami5, Loghman Firoozpour4, Abbas Shafiee6, and Alireza Foroumadi4,6
1
Faculty of Pharmacy, Lorestan University of Medical Sciences, Khorramabad, Iran
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran
Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
2
3
4
5
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
6
Tehran University of Medical Sciences, Tehran, Iran
A novel series of chalcones and flavanones discriminated by the presence of a 3,4-dimethoxyphenyl
moiety in their structures were synthesized as anti-cancer agents. The cytotoxicity evaluation of the
analogs against the MCF-7, MDA-MB-231 (human breast cancer), and SK-N-MC (human neuroblastoma)
cell lines demonstrated that the introduction of a halogen on the 3,4-dimethoxyphenyl part of both
series and the attachment of a pyrrolidinylethoxy group on the C-7 position of the flavanone derivatives
increased their activity. Indeed, 3-halogenated chalcones (1c and 1d) were more potent than the
standard drug etoposide against all tested cell lines. Fluorescence microscopy and flow cytometry
analyses confirmed that the anti-cancer effect of the most potent compounds 1c and 1d occurs via
apoptosis induction.
Keywords: Apoptosis / Anti-cancer agents / Chalcone / Cytotoxicity / Flavanone
Received: May 26, 2014; Revised: July 10, 2014; Accepted: July 11, 2014
DOI 10.1002/ardp.201400215
Introduction
Plants are remarkable source of anticancer drugs and lead
compounds. Among natural products, flavonoids are one of
the most important groups [5]. More than 4000 flavonoids
have been found in plants [6]. Dietary flavonoids usually
exist in nuts, fruits, vegetables, spices, and seeds [5, 6] and
exhibit wide spectrum of biological activities such as anti-
inflammatory, antioxidant, and antiproliferation [7–9].
Structure of flavonoids includes phenyl benzopyrone
(C6-C3-C6 skeleton) or 1,3-diarylpropane skeleton in which
arrangement of skeleton depends on degree of oxidation
[7, 10]. This large family is classified into flavons, flavonols,
flavanones, isoflavones, flavanes, chalcones, and anthocyani-
dins [11, 12]. Chalcones are bicyclic flavonoids that consist of
two aromatic rings that are linked by an a,b-unsaturated
ketone system [13]. They display several pharmacological
activities such as antibacterial, antifungal, antileishmanial,
and antitumor [14–17]. Recently, many derivatives of
chalcones were synthesized for evaluation of their cytotoxic
activity. According to the results of various studies, chalcones,
Cancer is characterized by uncontrolled growth of abnormal
cells in the body [1]. It is a serious cause of mortality in the
present society [2]. Although there are outstanding develop-
ments in diagnostic methods and therapeutic approaches,
cancer is one of the most frequent clinical problems
worldwide [3]. Nowadays, chemotherapy is one of the notable
healing approaches. Since most of the anti-cancer agents have
toxic effects on the normal cells, chemotherapy leads to
various adverse reactions. Therefore, designing safe and
effective new anti-cancer drugs is a necessary issue in the
medicinal chemistry [4].
Correspondence: Dr. Alireza Foroumadi, Department of Medicinal
Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran 14174, Iran.
E-mail: aforoumadi@yahoo.com
Fax: þ98 21 66461178
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S. H. M. E. Ketabforoosh et al.
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dihydrochalcones, bis-chalcones, or hybrids of chalcone
with coumarin exhibited anti-proliferative activity against
different cancer cell lines especially breast and prostate
cancers [1, 5, 18–20]. Rao et al. [2] demonstrated that chalcones
with methoxy groups on aromatic rings have potent anti-
proliferative effects on tumor cell line without cytotoxicity on
normall cells.
Besides chalcones, current reports are focused on antitu-
mor activity of flavanones. Min et al. synthesized flavanone
analogs with cytotoxic activity on leukemia and lung
carcinoma cells [21]. Derivatives consist of methoxy and
halogen groups on aromatic rings exhibiting inhibitory effect
on proliferation of human breast cancer cell lines [8, 22].
Furthermore, it was illustrated that the halogenated flava-
nones can induce apoptosis [7, 8].
The structures of compounds were confirmed by FT-IR and
1H NMR spectroscopy. In the 1H NMR spectra of chalcones, the
signal of phenolic hydroxyl group appeared at 12.77–
13.53 ppm. Also, the protons of olefinic group resonanced
at 7.81–7.94 and 7.46–7.59 ppm as two doublets. Coupling
constant for olefinic protons was about 15.2–16.0 Hz in accord
to trans-configuration for chalcone derivatives. In the IR
spectra, the carbonyl stretching vibration band of chalcones
1a–f appeared at 1631–1687 cmꢀ1. The observed band at
3420–3443 cmꢀ1 was related to the phenolic OH of chalcones
1a–f. In the 1H NMR of flavanones 2a–e, there were three peaks
approximately at 5.4, 3.1, and 2.9 ppm. The pattern of these
peaks was characteristic of protons located at the C2 and C3
–
position of chroman ring [7]. The band due to the C O
–
stretching of flavanones 2a–e occurred at 1673–1696 cmꢀ1
.
According to the available data, we designed and synthe-
sized a novel series of chalcones and flavanones discriminated
by presence of 3,4-dimethoxyphenyl moiety in their struc-
tures to find new potential anti-cancer agents. The cytotoxici-
ty of compounds was determined by MTT assay. Finally,
assessment of apoptosis induction of more potent compounds
was performed.
Biology
The cytotoxic activity of synthesized compounds was
evaluated against three human cancer cell lines, including
MCF-7, MDA-MB-231, and SK-N-MC by MTT assay. The
inhibition percentages of compounds were assessed by the
formula [(absorbance of control cells ꢀ absorbance of treated
cells)/absorbance of control cells] ꢁ 100. The concentration
causing 50% cell growth inhibition (IC50) was calculated from
concentration–response curves by regression analysis.
Results and discussion
Chemistry
Table 1 shows the IC50 value of synthesized compounds
against three human cancer cell lines. All compounds with
the exception of 1e and 2b showed good activity against SK-N-
MC cells (IC50 values ꢂ 16.8 mM). Compounds 1a–d and 2c
were more potent than standard drug etoposide against these
cells. Also, the cytotoxic activity of compounds 2d and 2e
against SK-N-MC was equal to that of etoposide. In the case of
Chalcones 1a–f were obtained from Claisen–Schmidt conden-
sation of an appropriate acetophenone with various benzal-
dehydes in the presence of sodium hydroxide. The second
series of compounds (flavanones) 2a–e were prepared by
cyclizing of chalcone derivatives 1 in refluxing ethanol and in
the presence of sodium acetate (Scheme 1) [7].
Scheme 1. Synthesis of chalcone and flavanone derivatives.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 853–860
Synthesis and Anti-Cancer Activity of New Flavanone Analogs
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Table 1. Results of MTT assay (IC50, mM) of compounds 1a–f and 2a–e against three human cancer cell lines.
Compounds
R
R0
SK-N-MC
MDA-MB-231
MCF-7
1a
1b
1c
1d
H
H
H
H
H
OCH3
Cl
2.5 ꢃ 0.8
4.7 ꢃ 2.3
1.5 ꢃ 0.2
1.1 ꢃ 0.6
6.9 ꢃ 2.6
9.8 ꢃ 1.3
6.2 ꢃ 0.8
1.3 ꢃ 0.3
6.1 ꢃ 2.1
6.5 ꢃ 0.7
1.2 ꢃ 0.6
1.0 ꢃ 0.6
Br
1e
1f
H
57.7 ꢃ 4.5
>100
>100
OCH3
11.9 ꢃ 1.4
>100
14.0 ꢃ 1.3
2a
2b
2c
H
H
H
H
OCH3
Cl
16.8 ꢃ 7.2
39.3 ꢃ 7.4
3.7 ꢃ 0.8
16.3 ꢃ 2.9
>100
11.5 ꢃ 1.8
10.6 ꢃ 6.4
14.5 ꢃ 0.7
9.7 ꢃ 3.9
2d
H
7.9 ꢃ 1.7
13.9 ꢃ 3.2
8.9 ꢃ 1.9
2e
OCH3
7.9 ꢃ 3.7
8.3 ꢃ 3.9
11.8 ꢃ 5.9
7.1 ꢃ 2.6
13.1 ꢃ 6.5
8.9 ꢃ 2.4
Etoposide
breast cancer cells (MDA-MB-231 and MCF-7), most compounds
displayed significant activity with IC50 values of 1.0–16.3 mM.
In general, bromochalcone 1d was the most potent compound
against all tested cell lines. It was five to eight times more
potent than etoposide.
The cytotoxic activity of chlorochalcone 1c and bromo-
chalcone 1d as the most potent compounds was evaluated on
non-tumoral cell line MRC-5. According to the results, the IC50
values of compounds 1c and 1d on human normal cell line
were >100 mM and 31 ꢃ 3.1 mM, respectively. Therefore, the
cytotoxicity of these compounds was apparently selective for
cancer cells, and compounds 1c and 1d had less cytotoxic
activity toward normal human cell line MRC-5.
Apoptosis induction by the selected compounds 1c and 1d
was evaluated using acridine orange/ethidium bromide
double staining technique. Analysis of the acridine orange/
ethidium bromide staining showed that the compounds 1c
and 1d induced apoptosis in MCF-7 cell line. As shown in
Fig. 1, the viable cells were observed green and the apoptotic
cells had orange particles in their nuclei. These results were
also established by the flow cytometry analyses of the
apoptotic cells. We used annexin V/7-AAD flow cytometric
analysis as a quantitative method of determining apoptosis.
According to the results, the cells undergo apoptosis after
treatment with IC50 concentrations of 1c and 1d. As shown in
Fig. 2, compounds 1c and 1d induced 17.9 and 17% apoptosis
in the MCF-7 cells, respectively. These percentages are
The tested compounds were structurally related to
chalcones and flavanones. The comparison of chalcone
derivatives with flavanone analogs indicated that
chalcones (1a–c) were more potent than corresponding
flavanone (2a–c) against all tested cancer cell lines. In
contrast, flavanone compounds containing pyrrolidinyl-
ethoxy moiety (2d and 2e) are more potent than related
chalcones analogs (1e and 1f). The observed IC50 values
revealed that the insertion of chlorine or bromine atom on
3,4-dimethoxyphenyl part of the molecules (both chalcones
and flavanones) improved the profile of cytotoxicity.
However, the introduction of third methoxy substituent
on the 3,4-dimethoxyphenyl moiety could not improve the
activity and even in some cases diminished the cytotoxicity.
Also, pyrrolidinylethoxy substituent on C-7 position of
flavanone derivatives was favorable for anti-proliferative
effect of these compounds.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 853–860
Figure 1. Acridine orange/ethidium bromide double staining of MCF-7 cells with characteristic symptoms of apoptosis: (a) DMSO 1% as
control, (b) etoposide as positive control, (c and d) cells treated with IC50 of compounds 1c and 1d, respectively, for 24 h. White arrow
indicates live cells and dashed arrow indicates apoptotic cells. The images of cells were taken with a fluorescence microscope at 400ꢁ.
implicated on the cells in the early stages of apoptosis.
Therefore, the evaluation of apoptosis in cells treated with the
test compounds confirmed the anti-proliferative activity.
These data confirmed compounds 1c and 1d induce apoptosis
in MCF-7 cell line. Also percentages of apoptosis for
compounds 1c and 1d are comparable with etoposide as
reference drug, which induced 18.24% apoptosis in MCF-7
cells.
Apoptosis induction of compound 1c and 1d was assessed on
MCF-7 cell line. According to acridine orange/ethidium
bromide staining and flow-cytometric data, compounds 1c
and 1d induce apoptosis in MCF-7 cell line.
Experimental
Chemistry
Chemicals and all solvents used in this study were purchased from
Merck AG and Aldrich Chemicals. Melting points were determined
on a Kofler hot stage apparatus. The IR spectra were obtained on a
Shimadzu 470 spectrophotometer (potassium bromide disks).
1H NMR spectra were recorded using Bruker 400 spectrometer
and chemical shifts (d) are reported in parts per million (ppm)
relative to tetramethylsilane (TMS) as an internal standard.
Elemental analyses were carried out on a Costec rapid elemental
analyzer model 4010 (Costec, Germany) for C and H, and the
results are within ꢃ0.4% of the theoretical values. The chalcones
1a–f and flavanone derivatives 2a–e were synthesized starting
from appropriate 2-hydroxyacetophenone and dimethoxy-
benzaldehyde as outlined in Scheme 1. 3-Chloro-4,5-dimethoxy-
benzaldehyde, 3-bromo-4,5-dimethoxybenzaldehyde, and 1-(2-hydroxy-
4-(2-tetrahydro-1H-1-pyrrolylethoxy)phenyl)-1-ethanone were pre-
pared according to the literature methods [23, 24].
Conclusion
In summary, to find new potential anti-cancer agents, we
synthesized a novel series of chalcones and related flavanones
discriminated by the presence of 3,4-dimethoxyphenyl moiety
in their structures. The limited structure–activity relation-
ships study demonstrated that the introduction of halogen on
the 3,4-dimethoxyphenyl part of both series, and attachment
of pyrrolidinylethoxy group on the C-7 position of flavanone
derivatives increased cytotoxic activity. Cytotoxicity evalua-
tion revealed that 30-halogenated chalcones (1c and 1d) were
more potent than etoposide and 7-(pyrrolylethoxy)flavanones
2e and 2d were as potent as etoposide. Moreover, cytotoxic
activity of compounds 1c and 1d on human normal cell line
(MRC-5) was significantly less than cancer cell lines. Thus,
the anti-proliferative effect of these analogs was selective.
General procedure for the synthesis of chalcones 1a–f
To
a solution of substituted acetophenone (1 mmol) and
appropriate 3,4-dimethoxybenzaldehyde (1 mmol) in ethanol
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Arch. Pharm. Chem. Life Sci. 2014, 347, 853–860
Synthesis and Anti-Cancer Activity of New Flavanone Analogs
857
Figure 2. Apoptosis analysis of MCF-7 cells treated with compounds 1c and 1d by annexin V-PE/7-AAD double staining. (a) MCF-7 cells
treated with DMSO 1% (negative control) or with IC50 values of etoposide (positive control) and compounds 1c and 1d. Fluorescence
intensity of annexin V-PE was shifted to the right in cells treated with compounds 1c and 1d. (b and c) Percentage apoptotic cells (%PE-
positive cells) was calculated. Exposure of MCF-7 cells to IC50 concentrations of etoposide, 1c and 1d induced apoptosis in 18.24, 17.97, and
17.05% of the cells, respectively.
(20 mL), 50% NaOH solution (4 mL) was added. Color of mixture
changed and precipitate formed. Then, the volume of solution
was increased up to 100 mL and it was stirred at room
temperature over night. The precipitate was filtered and
suspended in water. The suspension was acidified by 37% HCl
and pH of solution was 3. The product was extracted with ethyl
acetate three times and the organic layer was washed with water
and brine, and then dried (Na2SO4). After evaporation of solvent,
the residue was crystallized from ethanol or methanol to give
compounds 1a–f.
3-(3,4-Dimethoxyphenyl)-1-(2-hydroxyphenyl)prop-2-en-
1-one (1a)
1
Yield 90%; m.p. 100–102°C; H NMR (CDCl3) d: 12.98 (s, 1H, OH),
7.94 (d, 1H hydroxyphenyl, J ¼ 7.6 Hz), 7.9 (d, 1H, vinyl, J ¼ 15.2
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Arch. Pharm. Chem. Life Sci. 2014, 347, 853–860
Hz), 7.53 (m, 2H, vinyl and hydroxyphenyl), 7.27 (m, 1H
hydroxyphenyl), 7.17 (s, 1H phenyl), 7.03 (d, 1H phenyl, J ¼ 8.8 Hz),
6.96 (m, 2H, phenyl), 3.96 (s, 3H, OCH3), 3.94 (s, 3H, OCH3); IR (KBr,
The mixture was refluxed for 48 h. It was poured in cold water
and the precipitate was extracted with ethyl acetate. Organic
phase was dried (Na2SO4) and the solvent was removed. The
residue was crystallized from methanol or ethanol to give
corresponding flavanone.
cmꢀ1) nmax: 3443 (OH), 1631 (C O). Anal. calcd. for C17H16O4: C,
–
–
71.62; H, 5.67. Found: C, 71.51; H, 5.72.
1-(2-Hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-
2-(3,4-Dimethoxyphenyl)-2,3-dihydrochromen-4-one (2a)
Yield 57%; m.p. 118–120°C; 1H NMR (CDCl3) d: 7.95 (dd, 1H, J ¼ 8.0
and 1.6 Hz, H5 chroman), 7.51 (td, 1H, J ¼ 8.0 and 2.0 Hz, H7
en-1-one (1b)
1
Yield 82%; m.p. 132–133°C; H NMR (CDCl3) d: 12.82 (s, 1H, OH),
0
0
chroman), 7.07 (m, 4H, H6, H8 chroman and H2 , H6 ), 6.95 (d, 1H,
7.95 (d, 1H hydroxyphenyl, J ¼ 8.0 Hz), 7.88 (d, 1H, vinyl,
J ¼ 16.0 Hz), 7.56 (m, 2H, vinyl and hydroxyphenyl), 7.06 (d, 1H
hydroxyphenyl, J ¼ 8.0 Hz), 6.89 (s, 1H phenyl), 6.16 (s, 1H phenyl),
3.94 (s, 3H, OCH3), 3.93 (s, 6H, OCH3); IR (KBr, cmꢀ1) nmax: 3438
–
0
J ¼ 8.0 Hz, H5 ), 5.45 (dd, 1H, J ¼ 15.2 and 2.8 Hz, H2 chroman), 3.93
(s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.16 (t, 1H, J ¼ 15.2 Hz, H3
chroman), 2.9 (dd, 1H, J ¼ 15.2 and 2.8 Hz, H3 chroman); IR (KBr,
cmꢀ1) nmax: 1696 (C O). Anal. calcd. for C17H16O4: C, 71.62; H,
–
–
(OH), 1637 (C O). Anal. calcd. for C18H18O5: C, 68.78; H, 5.77.
–
5.67. Found: C, 71.87; H, 5.97.
Found: C, 68.93; H, 5.94.
2-(3,4,5-Trimethoxyphenyl)-2,3-dihydrochromen-4-one
3-(3-Chloro-4,5-dimethoxyphenyl)-1-(2-hydroxyphenyl)-
(2b)
prop-2-en-1-one (1c)
Yield 54%; m.p. 123–127°C; 1H NMR (CDCl3) d: 7.95 (d, 1H,
J ¼ 7.2 Hz, H5 chroman), 7.53 (m, 2H, H6 and H7 chroman), 7.35 (s,
1
Yield 36%; m.p. 113–116°C; H NMR (CDCl3) d: 12.77 (s, 1H, OH),
7.94 (d, 1H, hydroxyphenyl, J ¼ 8.0 Hz), 7.81 (d, 1H, vinyl,
J ¼ 13.6 Hz), 7.59 (m, 2H, vinyl and hydroxyphenyl), 7.35 (s, 1H,
phenyl), 7.06 (m, 3H, hydroxyphenyl and phenyl), 3.95 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3); IR (KBr, cmꢀ1) nmax: 3429 (OH), 1687
–
0
0
1H, H2 ), 7.09 (d, 1H, J ¼ 7.2 Hz, H8 chroman), 6.71 (s, 1H, H6 ), 5.43
(d, 1H, J ¼ 15.6 Hz, H2 chroman), 3.9 (s, 6H, OCH3), 3.14 (t, 1H,
J ¼ 15.6 Hz, H3 chroman), 2.91 (d, 1H, J ¼ 15.6 Hz, H3 chroman); IR
(KBr, cmꢀ1) nmax: 1688 (C O). Anal. calcd. for C18H18O5: C, 68.78;
–
–
(C O). Anal. calcd. for C17H15ClO4: C, 64.06; H, 4.74. Found: C,
–
H, 5.77. Found: C, 68.88; H, 5.54.
63.94; H, 4.87.
2-(3-Chloro-4,5-dimethoxyphenyl)-2,3-dihydrochromen-4-
3-(3-Bromo-4,5-dimethoxyphenyl)-1-(2-hydroxyphenyl)-
one (2c)
prop-2-en-1-one (1d)
Yield 10%; m.p. 120–123°C; 1H NMR (CDCl3) d: 7.94 (d, 1H,
J ¼ 7.6 Hz, H5 chroman), 7.55 (t, 1H, J ¼ 7.2 Hz, H7 chroman),
1
Yield 20%; m.p. 102–104°C; H NMR (CDCl3) d: 12.83 (s, 1H, OH),
7.94 (d, 1H, vinyl, J ¼ 15.6 Hz), 7.57 (m, 2H, vinyl and hydroxy-
phenyl), 7.09 (s, 1H phenyl), 7.05 (d, 1H phenyl, J ¼ 8.0 Hz), 6.97
(m, 2H, phenyl and hydroxyphenyl), 3.95 (s, 3H, OCH3), 3.92 (s, 3H,
0
0
7.11 (m, 3H, H6 and H8 chroman and H2 ), 6.97 (s, 1H, H6 ),
5.41 (d, 1H, J ¼ 13.2 Hz, H2 chroman), 3.91 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 3.09 (t, 1H, J ¼ 13.2 Hz, H3 chroman), 2.91
(d, 1H, J ¼ 13.2 Hz, H3 chroman); IR (KBr, cmꢀ1) nmax: 1687
–
OCH3); IR (KBr, cmꢀ1) nmax: 3431 (OH), 1639 (C O). Anal. calcd. for
–
–
C17H15BrO4: C, 56.22; H, 4.16. Found: C, 56.35; H, 4.08.
(C O). Anal. calcd. for C17H15ClO4: C, 64.06; H, 4.74. Found: C,
–
63.83; H, 4.64.
3-(3,4-Dimethoxyphenyl)-1-(2-hydroxy-4-(2-(pyrrolidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (1e)
2-(3,4-Dimethoxyphenyl)-7-(2-(pyrrolidin-1-yl)ethoxy)-2,3-
1
Yield 54%; m.p. 141–145°C; H NMR (CDCl3) d: 13.53 (s, 1H, OH),
dihydrochromen-4-one (2d)
7.88 (m, 2H, hydroxyphenyl and vinyl), 7.46 (d, 1H, vinyl,
J ¼ 15.2 Hz), 7.17 (s, 1H, phenyl), 6.93 (d, 1H, phenyl, J ¼ 8.0 Hz),
6.54 (m, 3H, phenyl and hydroxyphenyl), 4.29 (bs, 2H, alkane),
3.97 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.85 (bs, 2H, alkane), 1.65 (m,
Yield 23%; m.p. 132–135°C; 1H NMR (CDCl3) d: 7.9 (d, 1H,
0
0
J ¼ 8.4 Hz, H5 chroman), 7.01 (m, 2H, H2 and H6 ), 6.92 (d, 1H,
0
J ¼ 8.0 Hz, H5 ), 6.65 (d, 1H, J ¼ 8.4 Hz, H6 chroman), 6.5 (s, 1H, H8
chroman), 5.43 (d, 1H, J ¼ 15.0 Hz, H2 chroman), 4.25 (bs, 2H,
alkane), 3.93 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.82 (bs, 2H, alkane),
3.11 (t, 1H, J ¼ 15.0 Hz, H3 chroman), 2.85 (d, 1H, J ¼ 15.0 Hz, H3
chroman), 1.55 (m, 8H, pyrrolidine); IR (KBr, cmꢀ1) nmax: 1673
–
8H, pyrrolidine); IR (KBr, cmꢀ1) nmax: 3442 (OH), 1630 (C O). Anal.
–
–
calcd. for C23H27NO5: C, 69.5; H, 6.85. Found: C, 69.27; H, 6.7.
1-(2-Hydroxy-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-3-(3,4,5-
(C O). Anal. calcd. for C23H27NO5: C, 69.5; H, 6.85. Found: C, 69.78;
–
H, 6.98.
trimethoxyphenyl)prop-2-en-1-one (1f)
1
Yield 41%; m.p. 120–123°C; H NMR (CDCl3) d: 13.42 (s, 1H, OH),
7.87 (m, 2H, vinyl and hydroxyphenyl), 7.47 (d, 1H, vinyl,
J ¼ 15.6 Hz), 6.87 (s, 2H, phenyl), 6.54 (m, 2H, hydroxyphenyl), 4.26
(m, 2H, alkane), 3.94 (s, 3H, OCH3), 3.91 (s, 6H, OCH3), 3.87 (m, 2H,
alkane), 1.65 (bs, 4H, pyrrolidine); 1.47 (m, 4H, pyrrolidine);
7-(2-(Pyrrolidin-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-
2,3-dihydrochromen-4-one (2e)
Yield 20%; m.p. 75–77°C; 1H NMR (CDCl3) d: 7.9 (d, 1H,
0
0
J ¼ 8.8 Hz, H5 chroman), 6.69 (m, 3H, H2 and H6 and H6
chroman), 5.41 (d, 1H, J ¼ 14.5 Hz, H2 chroman), 4.27 (m, 2H,
alkane), 3.9 (s, 3H, OCH3), 3.87 (s, 6H, OCH3), 3.04 (t, 1H,
J ¼ 14.5 Hz, H3 chroman), 2.86 (d, 1H, J ¼ 14.5 Hz, H3 chroman),
1.54 (bs, 4H, pyrrolidine), 1.27 (bs, 4H, pyrrolidine); IR
IR (KBr, cmꢀ1) nmax: 3420 (OH), 1638 (C O). Anal. calcd. for
–
–
C24H29NO6: C, 67.3; H, 6.84. Found: C, 67.52; H, 6.79.
General procedure for the synthesis of flavanones 2a–e
A solution of an appropriate chalcone (1 mmol) in ethanol (10 mL)
was treated with sodium acetate (600 mg) and two drops of water.
(KBr, cmꢀ1) nmax: 1679 (C O). Anal. calcd. for C24H29NO6: C,
–
–
67.3; H, 6.84. Found: C, 67.54; H, 6.98.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 853–860
Synthesis and Anti-Cancer Activity of New Flavanone Analogs
859
Biology
Cell lines and cell culture
following treating the MCF-7 cells with and without compounds
1c, 1d, and etoposide at the concentration of their IC50, cells were
collected and washed twice with cold PBS and resuspended in the
binding buffer (100 mL of calcium buffer containing 10 mM
HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Then, the
cells were double stained with 5 mL of annexin V-PE and 5 mL of 7-
AAD solution. Finally, the samples were incubated for 15 min at
room temperature and then analyzed by flow cytometry.
Human cancer cell lines including MCF-7, MDA-MB-231, and SK-N-
MC were purchased from Pasteur Institute of Iran. The cells were
maintained in RPMI 1640 medium supplemented with 10% heat-
inactivated fetal calf serum (from Gibco BRL, UK) and 100 mg/mL
streptomycin and 100 U/mL penicillin at 37°C in a humidified
atmosphere with 5% CO2 in air.
This work was supported by grants from Tehran University of Medical
Sciences and Iran National Science Foundation (INSF).
MTT assay
The cytotoxicity evaluation of compounds 1a–f and 2a–e on three
human cancer cell lines including MCF-7 and MDA-MB-231
(human breast cancer cells), and SK-N-MC (human neuroblasto-
ma) was performed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) colorimetric assay [25]. Also,
the cytotoxic activity of compounds 1c and 1d were determined
on MRC-5 (human normal lung cell line).
The authors have declared no conflict of interest.
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