Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acrylonitriles: in vitro anticancer activity against MCF-7, PC-3 and A2780 cancer cell lines

Article abstract of DOI:10.1007/s11164-016-2562-3

A series of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acrylonitrile (2–9) were designed and synthesized to develop new cancer drugs. The structures of synthesized compounds 2–9 were described by using melting point, mass (MALDI-TOF-MS), FT-IR, elemental analysis, ¹H, ¹³C, ¹³C-APT and 2D NMR spectroscopy. The in vitro anticancer activities of 2–9 against human breast cancer (MCF-7), human prostate cancer (PC-3) and human ovarian cancer cells (A2780) were investigated by [3-(4,5-dimethylthiazol)-2-yl]-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assay method. Additionally, the LogIC₅₀ values of these compounds on A2780, MCF-7 and PC-3 cell lines were calculated by using inhibition % values by the GraphPad Prism 6 program on a computer. The results indicated that these compounds have high anticancer activity against MCF-7, PC-3 and A2780 cell lines (especially A2780 cell lines, p?

Full text of DOI:10.1007/s11164-016-2562-3

Res Chem Intermed
DOI 10.1007/s11164-016-2562-3
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-
phenyl)acrylonitriles: in vitro anticancer activity
against MCF-7, PC-3 and A2780 cancer cell lines
1
Furkan Ozen Suat Tekin2 Kenan Koran1
¨
•
•
•
Su¨leyman Sandal2 Ahmet Orhan Gorgulu
¨ ¨1
•
¨
Received: 5 February 2016 / Accepted: 26 April 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A series of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acryloni-
trile (2–9) were designed and synthesized to develop new cancer drugs. The
structures of synthesized compounds 2–9 were described by using melting point,
1
mass (MALDI-TOF-MS), FT-IR, elemental analysis, H, 13C, 13C-APT and 2D
NMR spectroscopy. The in vitro anticancer activities of 2–9 against human breast
cancer (MCF-7), human prostate cancer (PC-3) and human ovarian cancer cells
(A2780) were investigated by [3-(4,5-dimethylthiazol)-2-yl]-2,5-diphenyl-2H-te-
trazolium bromide] (MTT) assay method. Additionally, the LogIC50 values of these
compounds on A2780, MCF-7 and PC-3 cell lines were calculated by using inhi-
bition % values by the GraphPad Prism 6 program on a computer. The results
indicated that these compounds have high anticancer activity against MCF-7, PC-3
and A2780 cell lines (especially A2780 cell lines, p \ 0.05).
Keywords Phenylacrylonitrile derivatives Á Human ovarian cancer cell Á MCF-7 Á
Anticancer evaluation Á PC-3
Introduction
Cancer, also known as a malignant neoplasm or tumor, is a group of diseases
involving abnormal cell growth in any part of the body. Irregular cell growth can
begin almost anywhere in the body of humans, which consists of trillions of cells.
Various cancer types have been identified such as stomach, breast, prostate and
ovarian. Breast and ovarian cancers are the most common malignancy among
&
Kenan Koran
kkoran@firat.edu.tr
1
2
Department of Chemistry, Faculty of Science, Firat University, 23119 Elazig, Turkey
Department of Physiology, Faculty of Medicine, Inonu University, 44280 Malatya, Turkey
123

¨
F. Ozen et al.
women in many countries; on the other hand, prostate cancer is the most widespread
malignancy and age-related cause of cancer deaths among males worldwide [1–4].
Several treatment methods have been developed for such cancers like chemotherapy
and radiation therapy. Although these therapy methods are the most valid to cope
with cancer, they possess many drawbacks such as a decrease in susceptibility to
infection and production of blood cells, skin, mouth and gum problems,
inflammation of the lining of the digestive tract, hair loss, etc. Cancer drugs which
are used in chemotherapy are distributed evenly within the body of a patient and
cannot distinguish the cancer cells from healthy ones which produces several side
effects [5, 6]. Drug resistance in the treatment of cancer is a cause that hinders
achievement. Thus, augmentations of the effectiveness of existing drugs or
designing novel drugs are being tried. For this reason, the development of potential
anticancer drugs is important. Recently, to reduce the negative effects of
chemotherapeutic drugs and to increase productivity, the development of new
anticancer drugs such as acrylonitrile analogues [7–10], coumarin derivatives [11–
13], phosphazene compounds [14–16], chalcone compounds [17–19], some
pyrazoline derivatives [20, 21], and metal complexes [22–24] has been reported.
The anticancer and antibacterial properties of heteroaryl-acrylonitrile analogues
have been reported in the literature [7–10]. But no studies were found about the
anticancer activities of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acryloni-
trile derivatives.
In the present study, we aimed to design and synthesize phenylacrylonitrile
compounds 2–9 in order to determine the anticancer activities against three different
types of human cancer cells (MCF-7, PC-3, A2780). For this reason, 2-(2,3,4-
trimethoxyphenyl)-1-(substituted-phenyl)acrylonitrile compounds 2–9 were
obtained by using the Knoevenagel condensations protocol [25–27]. The structures
of these compounds 2–9 were described by using melting point, mass (MALDI-
TOF-MS), FT-IR, elemental analysis, 1H, 13C, 13C-APT and 2D NMR spectroscopy.
And their possible anticancer properties were investigated against onCF-7, PC-3 and
A2780 cell lines by using the MTT assay method. The logIC50 values were
determined. Our results indicate that these phenylacrylonitrile compounds displayed
strong anticancer activity towards MCF-7, PC-3 and A2780 cell lines.
Experimental
Reagents and equipment
2,3,4-Trimethoxybenzaldehyde, 3-methylphenylacetonitrile, 4-methylphenylacetoni-
trile, 3-(trifluoromethyl)phenylacetonitrile, 4-(trifluoromethyl)phenylacetonitrile, 3,4-
(methylenedioxy)phenylacetonitrile,
3,5-bis(trifluoromethyl)phenylacetonitrile,
3-chlorophenylacetonitrile, 4-chlorophenylacetonitrile, ethyl alcohol and NaOH were
purchased from Sigma-Aldrich (USA). The PC-3, MCF-7 and A2780 cancer cell lines
were supplied from the American Type Culture Collection. Trypsin, calf serum,
streptomycin and penicillin were supplied by Hyclone (Waltham, USA).
123

Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-…
FT-IR and mass results were recorded on a Perkin Elmer spectrum one FT-IR and
on a Bruker Daltonics microflex mass spectrometer, respectively. Positive ion and
linear mode MALDI TOF-MS spectra of phenylacrylonitrile derivatives were
recorded on a MALDI matrix [in 1,8,9-anthracenetriol (20 mg/mL Tetrahydrofu-
ran)] using a nitrogen laser accumulating 50 laser shots. 1D and 2D NMR analysis
were recorded using a Bruker DPX-400 spectrometer at ambient temperature with
SiMe4 as an internal standard. CDCl3-d was used as solvent for the NMR studies.
Elemental analysis was carried out by a CHNS-932 (LECO) apparatus. The melting
points of 2–9 were determined using a SHIMADZU DSC-50 thermobalance (10 °C/
min).
General methods for 2-(2,3,4-trimethoxyphenyl)-1-(substituted-
phenyl)acrylonitrile derivatives (2–9)
Absolute ethyl alcohol (100 mL), substituted benzylcyanide (5.4 mmol), and 2,3,4-
trimethoxybenzaldehyde (5.4 mmol) were added to three-necked reaction flask with
a mechanical stirrer. The reaction mixture was stirred for 0.5 h at 70 °C. Then, a
sodium hydroxide (20 %) solution was added dropwise until a precipitate formed.
The reaction was cooled to room temperature and the mixture was poured into ice-
water. The residue was filtered and washed with hot water to pH 7. The crude
product was dried under vacuum and then recrystallized in ethanol [25–27].
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(3-methylphenyl)acrylonitrile (2)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 0.67 g (5.4 mmol) 3-methyl-
benzylcyanide, yellow crystalline solid, m.p. 79–80 °C, yield: 90 %. FT-IR (KBr)
cm-1 = 3059, 3094 mC–H(Ar), 2831, 2935 mC–H(Aliph.), 2214 mC:N, 1498, 1583, 1601
1
m
C=C. H NMR (400 MHz, CDCl3): d (ppm) = 2.45 (s, 3H, H19), 3.92 (s, 3H, H9),
3.96 (s, 3H, H8), 3.97 (s, 3H, H7), 6.83 (s, 1H, H4), 7.21 (d, 1H, H16), 7.38 (t, 1H,
H17), 7.50–7.48 (m, 2H, H14 and H18), 7.86 (s, 1H, H10), 8.05 (d, 1H, H5). 13C NMR
(400 MHz, CDCl3): d (ppm) = 21.5 (C19), 56.1 (C9), 60.9 (C8), 61.8 (C7), 107.4
(C4), 109.1 (C11), 118.6 (C12), 121.0 (C6), 123.0 (C18), 123.4 (C5), 126.69 (C14),
128.8 (C17), 129.6 (C16), 134.8 (C13), 136.5 (C15), 138.8 (C10), 141.9 (C2), 153.1
(C1), 155.8 (C3). MALDI-MS: m/z calc. 309.36; found: 309.66 [M]?. Anal. calcd.
for C19H19NO3: C, 73.77; H, 6.19; N, 4.53. Found: C, 73.46; H, 5.9; N, 4.19 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(4-methylphenyl)acrylonitrile (3)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 0.67 g (5.4 mmol) 4-methyl-
benzylcyanide, yellow crystalline solid, m.p. 153–154 °C, yield: 88 %. FT-IR (KBr)
cm-1 = 3010, 3038 mC–H(Ar), 2835, 2935 mC–H(Aliph.), 2212 mC:N, 1461, 1510, 1587
1
m
C=C. H NMR (400 MHz, CDCl3): d (ppm) = 2.42 (s, 3H, H17), 3.92 (s, 3H, H9),
3.95 (s, 3H, H8), 3.96 (s, 3H, H7), 6.82 (s, 1H, H4), 7.28 (d, 2H, H15), 7.61 (t, 2H,
H14), 7.83 (s, 1H, H10), 8.05 (d, 1H, H5). 13C NMR (400 MHz, CDCl3): d
(ppm) = 21.2 (C19), 56.1 (C9), 60.9 (C8), 61.8 (C7), 107.4 (C4), 109.9 (C11), 118.6
(C12), 121.0 (C6), 123.3 (C5), 125.8 (C14, C18), 129.6 (C15, C17), 132.1 (C13), 135.17
123

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F. Ozen et al.
(C10), 138.9 (C16), 141.9 (C2), 153.1 (C1), 155.7 (C3). MALDI-MS: m/z calc.
309.36; found: 310.27 [M ? H]?. Anal. calcd. for C19H19NO3: C, 73.77; H, 6.19;
N, 4.53. Found: C, 73.53; H, 6.02; N, 4.32 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(3-
(trifluoromethyl)phenyl)acrylonitrile (4)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 1.0 g (5.4 mmol) 3-(trifluo-
romethyl)benzylcyanide, yellow crystalline solid, m.p. 72–73 °C, yield: 40 %. FT-
IR (KBr) cm-1 = 3010, 3038 mC–H(Ar), 2835, 2935 mC–H(Aliph.), 2212 mC:N, 1461,
1
1496, 1582, 1600 mC=C. H NMR (400 MHz, CDCl3): d (ppm) = 3.92 (s, 3H, H9),
3.97 (s, 3H, H8), 4.0 (s, 3H, H7), 6.84 (d, 1H, H4), 7.62 (t, 1H, H16), 7.67 (m, 1H,
H17), 7.89 (d, 1H, H18), 7.93 (s, 2H, H14), 8.08 (d, 1H, H5). 13C NMR (400 MHz,
CDCl3): d (ppm) = 56.1 (C9), 60.9 (C8), 61.9 (C7), 107.4 (C4), 108.3 (C11), 118.1
(C12), 120.3 (C6), 122.6 (C14), 123.5 (C5), 125.3 (C16), 129.1 (C18), 129.5 (C17),
131.4 (C15), 131.7 (C19), 135.9 (C13), 138.2 (C10), 141.9 (C2), 153.4 (C1), 156.4
(C3). MALDI-MS: m/z calc. 363.33; found: 364.27 [M ? H]?. Anal. calcd. for
C19H16F3NO3: C, 62.81; H, 4.44; N, 3.86. Found: C, 62.71; H, 4.26; N, 3.67 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(4-
(trifluoromethyl)phenyl)acrylonitrile (5)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 1.0 g (5.4 mmol) 4-(trifluo-
romethyl)benzylcyanide, yellow crystalline solid, m.p. 101–102 °C, yield: 50 %.
FT-IR (KBr) cm-1 = 3005, 3030 mC–H(Ar), 2940, 2967 mC–H(Aliph.), 2209 mC:N
,
1461, 1497, 1588, 1615 mC=C. 1H NMR (400 MHz, CDCl3): d (ppm) = 3.92 (s, 3H,
H9), 3.98 (s, 3H, H8), 3.99 (s, 3H, H7), 6.85 (d, 1H, H4), 7.74 (d, 2H, H14), 7.82 (d,
2H, H15), 7.97 (s, 1H, H10), 8.11 (d, 1H, H5). 13C NMR (400 MHz, CDCl3): d
(ppm) = 56.1 (C9), 60.9 (C8), 61.9 (C7), 107.5 (C4), 108.2 (C11), 118.1 (C12), 120.3
(C6), 123.5 (C5), 125.9 (C13), 126.1 (C14, C18), 130.4 (C16), 130.7 (C19), 138.4 (C10),
138.5 (C15, C17), 141.9 (C2), 153.4 (C1), 156.5 (C3). MALDI-MS: m/z calc. 363.33;
found: 364.27 [M ? H]?. Anal. calcd. for C19H16F3NO3: C, 62.81; H, 4.44; N, 3.86.
Found: C, 62.73; H, 4.25; N, 3.75 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(3,4-
(methylenedioxy)phenyl)acrylonitrile (6)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 0.87 g (5.4 mmol) 3,4-
(methylenedioxy)benzylcyanide, pale yellow crystalline solid, m.p. 125–126 °C,
yield: 80 %. FT-IR (KBr) cm-1 = 3015, 3043 mC–H(Ar), 2895, 2939 mC–H(Aliph.)
,
1
2211 mC:N, 1413, 1495, 1590 mC=C. H NMR (400 MHz, CDCl3): d (ppm) = 3.92
(s, 3H, H9), 3.95 (s, 3H, H8), 3.97 (s, 3H, H7), 6.09 (s, 2H, H19), 6.81 (d, 1H, H4),
6.90 (d, 1H, H17), 7.16 (s, 1H, H14), 7.22 (s, 1H, H10), 7.71 (s, 1H, H5), 8.01 (d, 1H,
H18). 13C NMR (400 MHz, CDCl3): d (ppm) = 56.1 (C9), 60.9 (C8), 56.1 (C7), 61.8
(C7), 101.6 (C19), 105.9 (C14), 107.4 (C4), 108.6 (C17), 109.6 (C11), 118.62 (C12),
120.5 (C5), 120.9 (C6), 123.2 (C18), 129.2 (C13), 135.2 (C10), 141.9 (C2), 148.3
123

Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-…
(C16), 148.4 (C15), 153.0 (C1), 155.7 (C3). MALDI-MS: m/z calc. 339.34; found:
339.41 [M]?. Anal. calcd. for C19H17NO5: C, 67.25; H, 5.05; N, 4.13. Found: C,
67.03; H, 4.98; N, 3.99 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(3,5-
bis(trifluoromethyl)phenyl)acrylonitrile (7)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 0.87 g (5.4 mmol) 3,5-
bis(trifluoromethyl)benzylcyanide, pale yellow crystalline solid, m.p. 124–125 °C,
yield: 88 %. FT-IR (KBr) cm-1 = 3000, 3040 mC–H(Ar), 2923, 2950 mC–H(Aliph.)
,
2211 mC:N, 1413, 1465, 1500, 1578 mC=C.
1H NMR (400 MHz, CDCl3): d
(ppm) = 3.92 (s, 3H, H9), 3.99 (s, 3H, H8), 4.02 (s, 3H, H7), 6.85 (d, 1H, H4), 7.90
(s, 1H, H10), 7.99 (d, 1H, H5), 7.22 (s, 1H, H10), 7.71 (s, 1H, H5), 8.09 (s, 3H, H14,
H16, H18). 13C NMR (400 MHz, CDCl3): d (ppm) = 56.2 (C9), 60.9 (C8), 61.9 (C7),
106.7 (C4), 107.5 (C11), 117.6 (C12), 119.8 (C6), 121.6 (C20), 122.1 (C19), 123.7
(C5), 124.3 (C16), 125.8 (C14, C18), 132.4 (C17), 132.7 (C15), 137.3 (C13), 139.8
(C10), 141.8 (C2), 153.6 (C1), 157.1 (C3). MALDI-MS: m/z calc. 431.33; found:
431.48 [M]?. Anal. calcd. for C20H15F6NO3: C, 55.69; H, 3.51; N, 3.25. Found: C,
55.39; H, 3.31; N, 3.07 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(3-(chlorophenyl)acrylonitrile (8)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 0.82 g (5.4 mmol)
3-chlorobenzylcyanide, yellow crystalline solid, m.p. 98–99 °C, yield: 85 %. FT-
IR (KBr) cm-1 = 3000, 3054 mC–H(Ar), 2829, 2938 mC–H(Aliph.), 2211 mC:N, 1497,
1
1581, 1596, 1626 mC=C. H NMR (400 MHz, CDCl3): d (ppm) = 3.98 (s, 3H, H9),
3.96 (s, 3H, H8), 3.92 (s, 3H, H7), 6.83 (d, 1H, H4), 7.38–7.40 (m, 2H, H15, H17),
7.58(d, 1H, H16), 7.68 (s, 1H, H14), 7.88 (s, 1H, H10), 8.07 (d, 1H, H5). 13C NMR
(400 MHz, CDCl3): d (ppm) = 61.9 (C7), 60.9 (C8), 56.1 (C9), 106.1 (C11), 107.4
(C4), 108.3 (C11), 118.2 (C12), 120.4 (C6), 123.5 (C5), 124.1 (C18), 125.9 (C14),
128.7 (C16), 130.2 (C17), 135.1 (C13), 136.7 (C15), 141.9 (C2), 143.1 (C2), 153.3
(C1), 156.3 (C3). MALDI-MS: m/z calc. 329.77; found: 329.35 [M]?. Anal. calcd.
for C18H16ClNO3: C, 65.56; H, 4.89; N, 4.25. Found: C, 65.49; H, 4.63; N, 4.11 %.
Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(4-(chlorophenyl)acrylonitrile (9)
1.06 g (5.4 mmol) 2,3,4-trimethoxybenzaldehyde (1), 0.82 g (5.4 mmol)
4-chlorobenzylcyanide, yellow crystalline solid, m.p. 125–126 °C, yield: 78 %.
FT-IR (KBr) cm-1 = 3065, 3098 mC–H(Ar), 2974, 2939 mC–H(Aliph.), 2211 mC:N
,
1501, 1518, 1581, 1609 mC=C. 1H NMR (400 MHz, CDCl3): d (ppm) = 3.91 (s, 3H,
H9), 3.96 (s, 3H, H8), 3.97 (s, 3H, H7), 6.82 (d, 1H, H4), 7.41 (d, 2H, H15), 7.63 (d,
2H, H14), 7.85 (s, 1H, H10), 8.05 (d, 1H, H5). 13C NMR (400 MHz, CDCl3): d
(ppm) = 61.8 (C7), 60.9 (C8), 56.1 (C9), 107.5 (C4), 108.6 (C11), 118.2 (C12), 120.6
(C6), 123.4 (C5), 127.1 (C15, C17), 129.2 (C14, C18), 133.5 (C13), 134.7 (C16), 136.9
(C10), 141.9 (C2), 153.3 (C1), 156.2 (C3). MALDI-MS: m/z calc. 329.77; found:
123

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F. Ozen et al.
329.34 [M]?. Anal. calcd. for C18H16ClNO3: C, 65.56; H, 4.89; N, 4.25. Found: C,
65.39; H, 4.69; N, 4.08 %.
In vitro antitumor evaluation
PC-3, MCF-7 and A2780 cell lines were preserved in DMEM (Dulbecco’s modified
Eagle’s medium) culture medium supplemented with 4500 mg/L glucose (10 %
heat-inactivated fetal bovine serum, L-glutamine (4 mM), 100 U/mL penicillin–
streptomycin) and addition of 10 mM non-essential amino acids for the culture of
A2780, PC-3 and MCF-7 cells. The anticancer activities of compounds 2–9 against
A2780, MCF-7 and PC-3 cell lines were examined by the MTT assay method,
which provides a simple way to detect living and growing cells without using
radioactivity. Briefly, 15 9 103 A2780, PC-3 and MCF-7 cell lines were plated in
triplicate in 96-well tissue culture plates, and treated with dimethyl sulfoxide (for
negative control or control group) and at 1, 5, 25, 50 and 100 lM concentrations of
compounds 2–9 in dimethyl sulfoxide. Then, A2780, PC-3 and MCF-7 cell lines
were kept for 24 h at 37 °C in a 5 % CO2 moistened incubator. After 24 h, MTT
(0.005 g/mL in phosphate buffer saline) was added to the cell culture and incubated
for 3 h. The formazan crystals formed during the interaction of active mitochondria
with MTT were dissolved in 100 mL (0.04 N) isopropyl alcohol and readings were
recorded on a microplate reader using a 570-nm filter. The relative cell viability (%)
was expressed as a percentage relative to the untreated control cells. Each value
represented an average of 10 measurements. All cellular results were obtained
against negative control cells [28–31].
Quantitative data are presented as mean standard deviation (SD). Normal
distribution was confirmed using the Kolmogorov–Smirnov test. Quantitative data
were analyzed using the Kruskal–Wallis H test following Mann–Whitney U test
with Bonferroni adjustment as a post hoc test. All p values \0.05 were considered
significant. All analyses were done by IBM SPSS Statistics 22.0 for Windows. The
logIC50 values (the half-maximal effective concentration) were determined by using
inhibition % values by the GraphPad Prism 6 program on a computer.
Results and discussion
Chemistry
In this work, 2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acrylonitrile com-
pounds 2–9 were prepared by the interaction of 2,3,4-trimethoxybenzaldehyde (1) with
substitute benzyl cyanide (3-methylbenzylcyanide, 4-methylbenzylcyanide, 3-(trifluo-
romethyl)benzylcyanide, 4-(trifluoromethyl)benzylcyanide, 3,4-(methylenedioxy)ben-
zylcyanide,
3,5-bis(trifluoromethyl)benzylcyanide,
3-chlorobenzylcyanide,
4-chlorobenzylcyanide) in the presence of ethyl alcohol and aqueous NaOH at 70 °C
[25–27]. The structures of compounds 2–9 have been described by MS, FT-IR,
microanalysis, 1D (1H and 13C-APT) and 2D (HETCOR) NMR spectroscopic methods.
The locations of characteristic peaks of primary, secondary and tertiary carbon atoms
123

Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-…
were determined by using 13C-APT NMR technique. The HETCOR NMR technique
was used for determination of –C–H carbon atoms. The process of the reactions and
structures is shown in Scheme 1.
The differential scanning calorimetry (DSC) spectra of compounds 2–9 showed a
single melting point (Fig. 1). Additionally, the molecular ion peaks of compounds
2–9 were obtained by MALDI TOF-MS analysis. The mass analyses of
phenylacrylonitriles were determined by the MALDI TOF-MS technique. The
molecular ion peak of each compound is given in the ‘‘Experimental’’ section. As an
example, the mass spectrum of compound 6 is given in Fig. 2.
The characteristic peaks in the 1D NMR and FT-IR spectra of 2–9 are given in
the ‘‘Experimental’’ section. The aldehyde carbonyl stretching vibrations were not
observed in the FT-IR spectra of 2–9. The –C:N peaks were shown between 2209
and 2214 cm-1 and aliphatic –C=C stretching vibrations were observed in the range
from 1578 to 1626 cm-1
.
1
The aldehyde carbonyl protons were not observed in the H-NMR spectrum of
phenyl acrylonitrile derivatives 2–9. The ratio of the protons integral height in the
spectra of the compounds 2–9 supports the proposed structures. The methoxy
protons for compounds 2–9 (7, 8 and 9 numbered protons in the Scheme 1) were
observed in the range from 3.92 to 4.02 ppm. The methyl protons for 2 and 3 in the
1H-NMR spectra were shown at 2.45 and 2.42, respectively. The methylene protons
and carbon peak at HETCOR NMR spectrum of 6 were observed at 6.09 and
101.61 ppm, respectively. The –C:N carbon peaks for compounds 2–9 were
N
CH3
CH3
CH3
O
O
CH3
O
CH3
O
O
CH3
O
CN
% 20 NaOH
EtOH
+
R3
O
R1
R2
R1
R2
( 2-9 )
( 1 )
R3
R3
-H
R1
-CH3
-H
R2
-H
2
3
4
5
6
7
CH3
8
CH3
8
-CH3
-H
-H
-H
-H
-H
CH
9
O
CH
CH
O
2
CH
3
3
3
3
7
9
7
2
O
O
O
O
-CF3
-H
3
4
1
6
3
4
1
6
CN
12
CN
12
11
14
17
11
14
13
-CF3
R1
R2
O
O
5
10
13
18
15
16
5
10
15
16
19
-bis(O2CH2)
18
17
( 2-9 )
-CF3
-Cl
-H
-H
-CF3
-H
( 6 )
R3
8
9
-H
-Cl
-H
Scheme 1 General presentations of compounds 2–9
123

¨
F. Ozen et al.
Fig. 1 The comparative melting points of compounds 2–9. These melting points were obtained by
differential scanning calorimetry using a SHIMADZU DSC thermo balance (10 °C/min)
Fig. 2 MALDI TOF-MS spectrum of compound 6
observed between 117.6 and 118.6 ppm. The 1H, 13C and HETCOR NMR spectra of
6 isaredepicted in Figs. 3, 4 and 5, respectively.
In vitro antitumor activity
The anticancer properties of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acry-
lonitrile derivatives were assessed in vitro using human prostate cancer cells (PC-3),
123

Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-…
H7
H8
H9
4.00
3.95
3.90
f1 (ppm)
H19
H10
H14
H17
H18
H4
H5
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Fig. 3 1H-NMR spectrum of compound 6. Compound 6 dissolved in deuterated chloroform (chloroform-
d) and obtained by using a Bruker (USA) DPX-400 spectrometer
0
1
2
3
4
H7, H8, H9
C7, C8, C9
5
H19, C19
6
H17, C17
H4, C4
H5, C5
7
8
9
H14, C14
H10, C10
H18, C18
150
140
130
120
110
100
90
80
70
60
50
40
30
Fig. 4 HETCOR (2D, 1H–13C coupling) NMR spectrum of compound 6. Compound 6 dissolved in
deuterated chloroform (chloroform-d) and obtained by using a Bruker (USA) DPX-400 spectrometer
123

¨
F. Ozen et al.
C17
C5
C14
C4
C15
C16
C6
C11
C12
150 149 148 147
f1 (ppm)
120
115
110
105
f1 (ppm)
C14
C19
C17
C11
C5
C9
C8
C18
C10
C7
C3
C15,C16
C13
C1
C12
C2
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
f1 (ppm)
Fig. 5 13C-NMR spectrum of compound 6. Compound
(chloroform-d) and obtained by using a Bruker (USA) DPX-400 spectrometer
6 dissolved in deuterated chloroform
human ovarian cancer cells (A2780) and human breast cancer cells (MCF-7) at 1, 5,
25, 50 and 100 lM doses. Figure 6 shows the effects of compounds 2–9 on cell
viability measured at 24 h after exposure.
In the MCF-7 cell lines, compounds 2, 3, 4, 8 and 9 significantly reduced % cell
viability comparative to the control (p \ 0.05). This decrease usually showed at the
highest concentration tested (100 lM, p \ 0.05). When the structure activities of
the compounds 2–9 were investigated, the meta substituted compounds 2, 4 and 8
against MCF-7 cell lines were generally observed to be more active than the others.
Only the para substituted compounds 3 and 5 containing chloride and methyl
groups showed a similar effect to the meta substituted compounds. The calculated
logIC50 values for compounds are given in Table 1.
All the compounds 2–9 showed reduced % cell viability and were dose-
dependent (p \ 0.05) towards A2780 cell lines (p \ 0.05). All doses (1, 5, 25, 50
and 100 lM) of the compound 2 have good anticancer activity (p \ 0.01). At 5, 25,
50 and 100 lM doses of 6, 7 and 8 were found to be effective against A2780 cell
lines (p \ 0.05). When the structure activities of 2–9 were investigated, all the
compounds against A2780 cell lines were observed to be quite active. As a result,
compounds 2–9 exhibit significantly improved anticancer activity when compared
to the phenylacrylonitrile compounds given in the literature [10, 32–34].
Compound 4 did not show anticancer activity against PC-3 cell lines. At 25, 50
and 100 lM doses of the compounds 2, 3, 6 and 7 usually showed the highest
anticancer activity (p \ 0.05). Compound 5 only showed anticancer activity at the
123

Synthesis of 2-(2,3,4-trimethoxyphenyl)-1-(substituted-…
Fig. 6 The relative cell viability (%) of MCF-7, A2780 and PC-3 cells after a 24-h treatment with all the
compounds 2–9. The changes on the cell viability (%) caused by compounds 2–9 are compared with the
control data. Each data point is an average of 10 viabilities (*p \ 0.01)
Table 1 Evaluation of the
Compound
MCF-7
LogIC50 (lM)
PC-3
LogIC50 (lM)
A2780
LogIC50 (lM)
cytotoxicityand LogIC50 values
(lM), of phenylacrylonitrile
analogues 2–9 against a panel of
three cancer cell lines
2
3
4
5
6
7
8
9
2.15
2.21
2.11
2.54
2.82
3.02
2.13
2.14
1.91
1.68
NC
0.29
1.87
1.1
2.28
2.09
1.81
2.46
2.46
2.02
1.05
1.70
0.96
1.35
LogIC50 is the half-maximal
effective concentration of a drug
that reduces cell growth by
50 %
NC not converged
123

¨
F. Ozen et al.
highest concentration tested (100 lM, p \ 0.01). When the structure activities of
these compounds 2–9 were investigated, all the compounds we generally showed
anticancer activity similar to each other on PC-3 cell lines. The changes of the
functional groups (methyl, chlorine. etc.) in the structures of 2–9 did not change the
anticancer activities on PC-3 cells.
Conclusions
In conclusion, in the present study, we successfully reported the synthesis of
2-(2,3,4-trimethoxyphenyl)-1-(substituted-phenyl)acrylonitrile derivatives 2–9, and
their anticancer properties against three different human cancer cell lines (A2780,
PC-3 and MCF-7) were determined by using MTT assay. The structural
characterizations of compounds 2–9 were performed by FT-IR, elemental analysis,
melting point, mass, 1D and 2D NMR techniques. The results demonstrate that these
compounds have anticancer activity against three different human cancer cell lines.
Our results showed that A2780 cell lines are more effective than PC-3 and MCF-7
cell lines. Overall, compounds 2–9 may be candidates for anticancer drug
development in the future.
Acknowledgments This research was supported financially by The Scientific and Technological
Research Council of Turkey (TUBITAK) (Project Number: 110T652). The authors are grateful to the
Research Fund of the TUBITAK for their support.
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