Synthesis and evaluation of (Z)-2,3-diphenylacrylonitrile analogs as anti-cancer and anti-microbial agents

Article abstract of DOI:10.1016/j.ejmech.2013.08.031

In the present study, a series of (Z)-2,3-diphenylacrylonitrile analogs were synthesized and then evaluated in terms of their cytotoxic activities against four human cancer cell lines, e.g. lung cancer (A549), ovarian cancer (SK-OV-3), skin cancer (SK-MEL-2), and colon cancer (HCT15), as well as anti-microbial activities against three microbes, e.g. Staphylococcus aureus, Salmonella typhi, and Aspergillus niger. The title compounds were synthesized by Knoevenagel condensation reaction of benzyl cyanide or p-nitrobenzyl cyanide with substituted benzaldehydes in good yields. Most of the compounds exhibited significant suppressive activities against the growth of all cancer cell lines. Compound 3c was most active in inhibiting the growth of A549, SK-OV-3, SK-MEL-2, and HCT15 cells lines with IC₅₀ values of 0.57, 0.14, 0.65, and 0.34 mg/mL, respectively, followed by compounds 3f, 3i, and 3h. Compound 3c exhibited 2.4 times greater cytotoxic activity against HCT15 cells, whereas it showed similar potency against SK-OV-3 cells to that of the standard anti-cancer agent doxorubicin. Structure-activity relationship study revealed that electron-donating groups at the para-position of phenyl ring B were more favorable for improved cytotoxic activity, whereas the presence of electron-withdrawing groups was unfavorable compare to unsubstituted acrylonitrile. An optimal electron density on phenyl ring A of (Z)-2,3-diphenylacrylonitrile analogs was crucial for their cytotoxic activities against human cancer cell lines used in the present study. Qualitative structure-cytotoxic activity relationships were studied using physicochemical parameters; a good correlation between calculated polar surface area (PSA), a lipophobic parameter, and cytotoxic activity was found. Moreover, all compounds showed significant anti-bacterial activities against S. typhi, whereas compound 3k showed potent inhibition against both S. aureus and S. typhi bacterial strains.

Full text of DOI:10.1016/j.ejmech.2013.08.031

Accepted Manuscript
Synthesis and evaluation of (Z)-2,3-diphenylacrylonitrile analogues as anti-cancer
and anti-microbial agents
Mohammad Sayed Alam, Young-Joo Nam, Dong-Ung Lee
PII:
S0223-5234(13)00547-3
10.1016/j.ejmech.2013.08.031
EJMECH 6377
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2013
Revised Date: 25 August 2013
Accepted Date: 31 August 2013
Please cite this article as: M.S. Alam, Y.-J. Nam, D.-U. Lee, Synthesis and evaluation of (Z)-2,3-
diphenylacrylonitrile analogues as anti-cancer and anti-microbial agents, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.08.031.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis and evaluation of (Z)-2,3-diphenylacrylonitrile analogues as anti-
cancer and anti-microbial agents
a
b
b,
Mohammad Sayed Alam , Young-Joo Nam , Dong-Ung Lee *
a
Department of Chemistry, Jagannath Univeristy, Dhaka 1100, Bangladesh
b
Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea
*
Corresponding author. Tel.:+82 54 770 2224; fax: +82 54 742 9833.
E-mail address: dulee@dongguk.ac.kr (D.U. Lee)

ACCEPTED MANUSCRIPT
Abstract
In the present study, a series of (Z)-2,3-diphenylacrylonitrile analogues were synthesized and
then evaluated in terms of their cytotoxic activities against four human cancer cell lines, e.g.
lung cancer (A549), ovarian cancer (SK-OV-3), skin cancer (SK-MEL-2), and colon cancer
(HCT15), as well as anti-microbial activities against three microbes, e.g. Staphylococcus
aureus, Salmonella typhi, and Aspergillus niger. The title compounds were synthesized by
Knoevenagel condensation reaction of benzyl cyanide or p-nitrobenzyl cyanide with
substituted benzaldehydes in good yields. Most of the compounds exhibited significant
suppressive activities against the growth of all cancer cell lines. Compound 3c was most
active in inhibiting the growth of A549, SK-OV-3, SK-MEL-2, and HCT15 cells lines with
IC50 values of 0.57, 0.14, 0.65, and 0.34 mg/mL, respectively, followed by compounds 3f, 3i,
and 3h. Compound 3c exhibited 2.4 times greater cytotoxic activity against HCT15 cells,
whereas it showed similar potency against SK-OV-3 cells to that of the standard anti-cancer
agent doxorubicin. Structure-activity relationship study revealed that electron-donating
groups at the para-position of phenyl ring B were more favorable for improved cytotoxic
activity, whereas the presence of electron-withdrawing groups was unfavorable compare to
unsubstituted acrylonitrile. An optimal electron density on phenyl ring A of (Z)-2,3-
diphenylacrylonitrile analogues was crucial for their cytotoxic activities against human
cancer cell lines used in the present study. Qualitative structure-cytotoxic activity
relationships were studied using physicochemical parameters; a good correlation between
calculated polar surface area (PSA), a lipophobic parameter, and cytotoxic activity was found.
Moreover, all compounds showed significant anti-bacterial activities against S. typhi, whereas
compound 3k showed potent inhibition against both S. aureus and S. typhi bacterial strains.
Keywords: acrylonitriles; cytotoxicity; antimicrobial activity; human cancer cell line

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1
. Introduction
A number of acrylonitrile analogues with a wide range of interesting biological activities
have been reported. For example, Tagmose and co-workers have described the synthesis and
biological evaluation of 3,3-diamino-sulfonylacrylonitriles as novel inhibitors of glucose-
induced insulin secretion from beta cells [1]. Carta and co-workers have reported the
synthesis of 3-aryl-2-(1H-benzotriazol-1-yl)acrylonitrile analogues along with their anti-
bacterial, anti-fungal, anti-mycobacterial, anti-retroviral, and anti-tumour activities [2-4].
Further, Sączewski et al. [5, 6] have reported the synthesis, cytotoxic and antibacterial
activities of novel 2,3- and 2,6-disubstituted heteroarylacrylonitriles. Recently, Hranjec and
co-workers reported the synthesis and in vitro antitumor evaluation of benzimidazolyl 2,3-
disubstituted acrylonitriles [7]. More recently, Zificsak and co-workers reported the synthesis
of sulfonyl acrylonitrile derivatives and their biological evaluation as novel inhibitors of
peritoneal carcinomatosis [8].
Resveratrol (Fig. 1), a naturally occurring hydroxylated stilbene analogue, which has
been found in many medicinal plants, grape skin, peanuts, and red wine [9], shows potent
chemopreventive activity [10] by exerting anti-proliferative and pro-apoptotic effects in
human cancer cells [11]. Recently, a good number of stilbene analogues has been isolated as
well as synthesized from natural sources, displaying a wide range of interesting biological
activities [12-16]. Polyhydroxy stilbenes possesses strong anti-oxidative [17] and anti-
inflammatory [18] activities that lead to chemotherapeutic properties such as anti-cancer-
promoting activity. Resveratrol, a polyhydroxylated stilbene analogue, exerts its anti-cancer
activity by triggering the synthesis of endogenous ceramide [19,20], a bioactive sphingolipid
[
21,22]. Ceramide is a promising pharmacological target when either major apoptotic
pathway is disrupted or resistance to DNA damage elevated. Moreover, drugs that trigger
ceramide, while highly effective in malignant cells, are less toxic to normal cells and tissues

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[
23]. Resveratrol and its analogue have been reported as apoptosis-inducing agents, aryl
hydrocarbon receptor (AhR) modulators, and human cytochrome P450 (CYP) inhibitors,
specifically as selective inhibitors of the isoform CYP1B1 [24-26]. Tyrphostins (tyrosine
phosporylation inhibitors), hydroxylated styrenes (Fig. 1), are used as potential protein
tyrosine kinase inhibitors [27]. Protein tyrosine kinase plays an important role in normal cell
division and abnormal cell proliferation, its enhanced activity is considered to be related with
proliferative diseases such as cancer. In addition, several kinds of tyrphostin derivatives such
as substituted quinazoline-tyrphostins [28] and modified phenolic tyrphostins [29] exerted
anticancer properties against human cancer cell lines.
Due to our ongoing interest in identifying novel biologically active molecules, we have
designed and synthesized (Z)-2,3-diphlenylacrylonitriles, which are structurally similar to
resveratrol, a trans-stilbene analogue and tyrphostins. The syntheses of (Z)-2,3-
diphenylacrylonitrile analogues were carried out by a simple but efficient Knoevenagel
condensation reaction of benzyl cyanide or substituted benzyl cyanide with various
substituted benzaldehydes in ethanol. Structures of the new compounds were elucidated by
1
IR, H-NMR, and elemental analyses. We investigated the in vitro cytotoxic activities of
these new compounds against four culture cell lines, e.g. A549 (human lung cancer), SK-OV-
3
(human ovarian cancer), SK-MEL-2 (human skin cancer), and HCT15 (human colon
cancer). Physicochemical calculations were also carried out in order to determine the
relationship between the electronic properties and cytotoxic activities of (Z)-2,3-
diphenylacrylonitrile analogues. Furthermore, in vitro anti-microbial activities were screened
against two bacterial strains and one fungal strain, e.g. Staphylococcus aureus, Salmonella
typhi, and Aspergillus niger.
2
. Results and Discussion
2
.1. Chemistry

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The desired compounds 3a-r were synthesized according to a convenient one-step
reaction depicted in Scheme 1, by Knoevenagel condensation of the appropriate
benzylcyanide or p-nitrobenzylcyanide with the suitable substituted benzaldehyde in ethanol,
using sodium ethoxide as basic catalyst. All compounds except 3a, 3b, and 3i are new. The
1
structures of the compounds were assigned by IR, H NMR, and elemental analyses. In the IR
spectra of the compounds, characteristic –C≡N stretching absorption bands appeared around
-1
2
190–2230 cm . Although two geometric isomers were obtained in the Knoevenagel
condensation, we observed that in all cases, the Z-isomer was the only product supported by
the proton NMR spectra. From the literature survey, it has been found that the vinylic-H of E-
isomers exhibits a down-field shift (δ 8.81–7.65) compared to that of Z-isomers (δ 7.60–6.03)
1
[
3,4,30]. In the H NMR spectra, the vinylic proton (–CH=C–) of all compounds appeared at
6
.96-7.52 ppm as a broad singlet equivalent to one proton, and at higher field compared to
that of E-isomer, which supported the Z-configuration of all compounds. The aromatic
protons were assigned in the usual manner, according to their substitution pattern. The
methoxy protons of compounds 3b and 3k appeared as singlets at 3.87 and 3.89 ppm,
respectively. The methyl protons of compounds 3i and 3r were observed as singlets at 2.41
and 2.42 ppm, respectively, equivalent to three protons each. The N,N′-dimethyl protons of
compounds 3c and 3l were observed as singlets at 2.91 and 2.89 ppm, respectively,
equivalent to six protons each.
2
.2 . Cytotoxic activity
The cytotoxic activities of (Z)-2,3-diphenylacrylonitrile analogues (3a-r) were evaluated
by an in vitro assay performed on four human cancer cell lines, e.g. lung cancer (A549),
ovarian cancer (SK-OV-3), skin cancer (SK-MEL-2), and colon cancer (HCT15). Their
cytotoxic activities were evaluated by measuring the inhibition of net cell growth, as
measured as a percentage of the control samples, after incubation for 48 h with the test

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samples following the SRB (sulforhodamine-B) method. All activities were compared with
doxorubicin as a positive control. As observed in Table 1, compound 3c showed the highest
cytotoxic activity against all cancer cell lines, followed by compounds 3f, 3i, and 3h.
Compound 3c exhibited 2.4 times greater cytotoxic activity against HCT15 cancer cells (IC50
-1
0
.34 µg mL ), whereas it showed similar potency against SK-OV-3 cancel cells (IC50 0.14 µg
-1
-1
mL ) to that of the standard anti-cancer agent doxorubicin (IC 0.814 and 0.092 µg mL ,
5
0
respectively). However, this compound displayed lower activity against other two cancer cell
lines, A549 and SK-MEL-2.
Structure-activity relationship study revealed that the nature of the substituted group on
the phenyl ring of (Z)-2,3-diphenylacrylonitriles plays an important role in the cytotoxic
activity of the compound. Compound 3a, which possessed two unsubstituted phenyl rings,
-1
showed greater cytotoxicity against SK-MEL-2 cells (IC50 38.45 mg mL ) than against A549
-1
-1
-1
(
IC50 43.76 mg mL ), SK-OV-3 (IC50 47.39 mg mL ), and HCT15 (IC50 68.98 mg mL )
cells. Introduction of a N,N-dimethylamino group, a stronger electron-donating group, at the
para-position of phenyl ring B resulted in compound 3c, which exhibited 76, 339, 59, and
-1
-1
2
03 times greater activities against A549 (IC50 0.57 mg mL ), SK-OV-3 (IC50 0.14 mg mL ),
-1
-1
SK-MEL-2 (IC50 0.65 mg mL ), and HCT15 (IC50 0.34 mg mL ) cells, respectively,
compared with those of 3a. Further, introduction of a nitro group, a stronger electron-
withdrawing group, at the para-position of phenyl ring A or B resulted in compounds 3e and
3
j, respectively. The former compound showed similar cytotoxicities against SK-MEL-2 and
SK-OV-3 cancer cells along with increased cytotoxicity against A549 and HCT15 cells
compared with those of 3a, whereas the latter compound exhibited increased activities
against all cancer cell lines. Introduction of a nitro group at the ortho-position instead of the
para-position of phenyl ring B of 3a yielded compound 3d, which showed similar
cytotoxicity as 3a. On the other hand, compound 3d showed significantly increased activities

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against A549, SK-MEL-2, and HCT15 cells as well as similar activity against SK-OV-3 cells
compared with those of a para-nitro analogue, compound 3e. Introduction of a methyl group,
a weak electron-donating group, at the para-position of phenyl ring B resulted in compound
3
i, which exhibited 24, 81, 23, and 45 times greater activities against A549 (IC50 1.82 mg
-1
-1
-1
mL ), SK-OV-3 (IC50 0.58 mg mL ), SK-MEL-2 (IC50 1.63 mg mL ), and HCT15 (IC50
-1
1
.51 mg mL ) cells, respectively, compared with those of 3a. On the other hand, it showed
2
.5-4.5 times lower cytotoxicities against all cancer cell lines compared to those of 3c, a N,N-
dimethylamino analogue. Increasing the electron-donating capacity of the methyl group by
replacement with a methoxy group (compound 3b) led to a substantial (14-25 times) loss in
activity, although compound 3b showed significant increased cytotoxicities (1.7-3.2 times)
against all cell lines compared with those of 3a, an unsubstituted acrylonitrile. Compounds
with halogen substituents (3f and 3h) at the para-position of phenyl ring B, which showed
weak electron-withdrawing capacity but also electron-donating ability due to the resonance
effect, displayed significantly improved cytotoxicities against all cell lines compared to those
of 3a. Though the methoxy group has greater electron donating capacity than chlorine atom,
compound 3b, a para-methoxy analogue showed less cytotoxicity against all cancer cell lines
compared to that of 3f, a para-chloro analogue. Compound 3f, with chlorine substituted at
the para-position of phenyl ring B, showed 24, 237, 25, and 57 times greater cytotoxicities
-
1
-1
against A549 (IC 1.81 mg mL ), SK-OV-3 (IC 0.20 mg mL ), SK-MEL-2 (IC 1.53 mg
5
0
50
50
-1
-1
mL ), and HCT15 (IC50 1.22 mg mL ) cells, respectively, compared with those of 3a.
Compound 3h, with bromine substituted at the para-position of phenyl ring B, showed
reduced activities (9-28 times) against all cancer cell lines compared with those of compound
3
f. Introduction of a chlorine atom at the ortho-position instead of para-position of phenyl
ring B of 3a yielded compound 3g, which resulted in improved cytotoxicity compared with
that of 3a but dramatically reduced activities against all cancer cell lines compared with those

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of compound 3f. On the other hand, introduction of a nitro group, a highly polar group, at the
para-position of phenyl ring A of compounds 3b-i yielded compounds 3k-r. All of these
compounds (3k-m, 3o, 3q, and 3r) showed significantly reduced activities against all cancer
cell lines except for compounds 3n and 3p. Compound 3n showed significantly improved
activities against A549, SK-OV-3, and HCT15 cell lines but reduced activity against SK-
MEL-2 cells, whereas compound 3p exhibited significantly improved activities against A549,
SK-OV-3, and SK-MEL-2 cell lines but reduced activity against HCT15 cells compared to
their corresponding compounds 3e and 3g, respectively. The above structure-activity
relationships led us to hypothesize that an optimum electron density on phenyl ring B of (Z)-
2
,3-diphenylacrylonitriles may be closely related to maximum cytotoxic activity against the
cell lines used in the present study. Further structure-activity studies with versatile analogues
are needed to clearly elucidate the role of structure on the cytotoxicity of (Z)-2,3-
diphenylacrylonitriles as well as to identify their molecular targets.
2
.3 . Computational Studies
The physicochemical properties of a molecule play an important role in determining
molecular reactivity in a biological response [31]. As biological systems are furnished with a
number of heterogeneous phases, e.g. water, serum protein, lipid particles etc., drug transport
processes and drug-receptor interactions are essentially physicochemical. Polar surface area
(PSA) or lipophilicity is recognized as a meaningful parameter in structure-activity
relationship studies and has become the single most informative and successful
physicochemical property in medicinal chemistry [32]. Nowadays, it is recognized as a major
experimental and theoretical tool in drug design.
To explain the qualitative structure-anti-cancer activity relationships (QSAR) of (Z)-2,3-
diphenylacrylonitriles (3a-r), physicochemical calculations were carried out using
molinspiration cheminformatics software. Physiochemical parameters of some selected (Z)-

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2
,3-diphenylacrylonitrile analogues are listed in Table 2. The lipophilic character of a
molecule depends on two important factors, i.e. hydrophobicity and polarity, which help the
molecule to cross or irreversibly damage the cellular membrane. Figure 2 shows the
molecular lipophilicity potential (MLP) map and polar surface areas (PSAs) of selected (Z)-
2
,3-diphenylacrylonitrile analogues. In the present study, compound 3c was the most active,
followed by compounds 3f, 3i, and 3h with PSAs of 27.03, 23.792, 23.792, and 23.792,
respectively. Although a small number of compounds were used in the present study, a good
correlation was observed in which cytotoxic activity decreased with increasing PSA (Fig. 3).
It is well known that PSA, i.e. polarity, is correlated with the lipophilicity of a molecule,
2
which is an important factor for biological activity. The correlation coefficients (r ) between
the PSAs and inhibitory potencies of selected (Z)-2,3-diphenylacrylonitrile analogues against
A549, human SK-OV-3, SK-MEL-2, and HCT15 cells were found to be 0.78 (n=11), 0.83
(
n=10), 0.78 (n=11), and 0.79 (n=13), respectively. The above correlations should be treated
with caution since there were three exceptions, e.g. PSAs of moderately active compounds 3a
23.792) and 3g (23.792) were the same as highly active compound 3f (23.792), whereas PSA
(
of active compound 3p (69.616) was as high as similarly active compound 3h (23.792).
Therefore, maps of PSA and MLP were compared for compounds 3a, 3f, 3g, 3h, and 3p (Fig.
2
). It was found that polarity and lipophilicity were different for all molecules. On the other
hand, compounds 3a, 3f, and 3g showed similar PSAs, whereas compound 3f showed a
greater lipophilic area compared to that of compound 3a or 3g. Similarly, although the PSA
of compound 3p was higher than that of compound 3h, their MLP maps were almost the
same. The design and synthesis of additional (Z)-2,3-diphenylacrylonitrile analogues
containing lipophilic and hydrophilic substituents are in progress in order to corroborate our
findings.
2
.4 . Anti-microbial activity

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The newly synthesized (Z)-2,3-diphenylacrylonitrile analogues (3a-r) were evaluated for
their in vitro anti-bacterial activities against two bacterial strains, e.g. Staphylococcus aureus
(Gram-positive) and Salmonella typhi (Gram-negative), as well as their anti-fungal activities
against Aspergillus niger by the disc diffusion method. Results of the in vitro evaluation of
anti-microbial activity are reported in Table 3. Inhibition zones of synthesized compounds
-1
were measured at doses of 100 mg disc , and azithromycin as a positive control was
-1
evaluated at a dose of 25 mg disc . As presented in Table 3, all compounds inhibited the
growth of S. typhi, and compounds 3k-n resisted both S. aureus and S. typhi. None of the
-1
compounds were significantly active against A. niger at a concentration of 100 mg disc .
Among all of them, compound 3k showed the highest activities against both S. aureus and S.
typhi, followed by compound 3l. Compounds 3c, 3f, 3i, 3k, 3p, and 3r showed high activities
against S. typhi, whereas compounds 3b, 3d, 3e, 3g, 3h, 3j, 3m-o, and 3q exhibited moderate
-1
activities against the same bacterial strain. At the same concentration, i.e. 100 mg disc ,
compounds 3k-n demonstrated greater activities against S. aureus compared to S. typhi. From
this study, (Z)-2,3-diphenylacrylonitriles appeared to be more active against Gram-negative
bacteria compared to Gram-positive bacteria.
Structure-activity relationships may be explained briefly as follows: introduction of an
2
electron-withdrawing group (NO ), a highly polar group, into the para-position of ring A of
compounds 3a-i yielded compounds 3j-r, which showed slightly increased bactericidal
activities against S. typhi in the case of compounds 3j-l, 3p, and 3r, reduced activities in the
case of compounds 3m, 3o, and 3q, and no change in the case of compound 3n. Introduction
2
of this same polar group (NO ) into the para-position of ring A of compounds 3b-e yielded
compounds 3k-n, which showed good anti-bacterial activities against S. aureus, whereas
compounds 3a-i, and their para-nitro analogues (3j and 3o-r) were inactive. Introduction of
2 3 3
electron-donating groups (halogen, OMe, NMe , and CH ) at the R position resulted in

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increased activity (cf., 3b, 3c, 3f, 3i, 3k, and 3l), whereas the presence of an electron-
withdrawing group (NO ) caused a reduction in activity (cf., 3d, 3e, 3m, and 3n).
2
3
. Conclusion
The present study reports the synthesis of novel (Z)-2,3-diphenylacrylonitrile
analogues as well as their biological evaluation as cytotoxic and anti-bacterial agents. Using
the Knoevenagel condensation reaction, 18 compounds were prepared and their cytotoxicities
against four cancer cell lines, e.g. lung cancer (A549), ovarian cancer (SK-OV-3), skin cancer
(SK-MEL-2), and colon cancer (HCT15), as well as anti-microbial activities against three
microbes, e.g. S. aureus, S. typhi, and A. niger, evaluated. Compound 3c showed the highest
cytotoxic activities against all cancer cell lines, followed by compounds 3f, 3i, and 3h
containing N,N-dimethylamine, chloro, methyl, and bromo substituents at the para-position
of the phenyl ring A, respectively. Compound 3c exhibited 2.4 times greater cytotoxic
activity against HCT15 cells, whereas it showed similar potency against SK-OV-3 cells to
that of the standard anti-cancer agent doxorubicin. Introduction of electron-donating groups
at the para-position of the phenyl ring B was favorable for improved cytotoxic activity,
whereas the presence of an electron-withdrawing group was unfavorable compare to
unsubstituted acrylonitrile. Physicochemical calculations indicate that the cytotoxicities of
(Z)-2,3-diphenylacrylonitriles correlated well with the calculated PSA and MLP. Most of the
compounds showed significant bactericidal activities against S. typhi, whereas compound 3k
showed potent activity against both S. aureus and S. typhi. The strong cytotoxicities of
compounds 3c, 3f, 3i, and 3h combined with our computational results will be helpful in
synthesizing a large library of (Z)-2,3-diphenylacrylonitrile analogues for extensive anti-
cancer study as well as for the development of a more appropriate drug candidate.
4
. Experimental

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4
.1. General
Melting points were determined on an X-5 melting point apparatus (Yuxiagyiqi, Gongyi City
Yuxiang Instruments Co., Ltd., China) and are uncorrected. IR spectra were obtained with an
FTIR-8430S (Shimadzu, Japan) using KBr discs. NMR spectra were recorded on a Unity
INOVA 500 MHz NMR (Varian, USA) or an AM-400MH (Bruker, USA) spectrometer using
3 6
CDCl or acetone-d with TMS as an internal standard. Elemental analyses (C, H, N) were
performed on a Perkin–Elmer 2400 II CHN elemental analyzer.
4
.2. General procedure for preparation of 2,3-diphenylacrylonitrile analogues (3a-r)
Benzyl cyanide or substituted benzyl cyanides (2.0 mmol) were added to substituted
benzaldehydes (2.5 mmol) in ethanol (15-20 mL), after which the mixture was stirred at room
temperature for 10-15 min. To this solution, sodium ethoxide (0.7 g, 10 mmol) in the same
solvent (10 mL) was added dropwise with constant stirring, and the reaction mixture was
vigorously stirred for 20-72 h to complete the reaction, which was monitored by TLC. After
removal of the solid materials and solvent, crude products were purified by silica gel column
chromatography (eluent: mixtures of dichloromethane and n-hexane from 1:1 to 7:3
gradient).
4
.2.1. (Z)-2,3-Diphenylacrylonitrile (3a) [33]
o
-1
1
Yield 96 %, mp 85-86 C (white soilds). IR (KBr) νmax: 2218 cm (CN). H
NMR spectrum: (400 MHz; CDCl
3
) δ (ppm): 7.41 (m, 6H, Ar), 7.52 (brs, 1H, =CH),
.66 (m, 2H, Ar), 7.87 (m, 2H, Ar). Anal. Calcd for C15 11N C 87.77; H 5.40; N
.82%. Found C 87.70; H 5.38; N 6.87%.
7
6
H
4
.2.2. (Z)-3-(4-Methoxyphenyl)-2-phenylacrylonitrile (3b) [34]
o
-1
1
Yield 81%, mp 93-94 C (yellow solids). IR (KBr) νmax: 2208 cm (CN). H NMR
3 3
spectrum: (400 MHz; CDCl ) δ (ppm): 3.87 (s, 3H, OCH ), 6.98 (m, 2H, Ar), 7.39 (m, 3H,

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Ar), 7.16 (brs, 1H, =CH), 7.65 (m, 2H, Ar), 7.88 (m, 2H, Ar). Anal. Calcd for C16
H
13NO C
8
1.68; H 5.57; N 5.95%. Found C 81.57; H 5.52; N 6.02%.
4
.2.3. (Z)-3-(4-(Dimethylamino)phenyl)-2-phenylacrylonitrile (3c)
ο
-1
1
Yield 94 %, mp 101-102 C (pale yellow solids). IR (KBr) ν : 2210 cm (CN). H
max
NMR spectrum: (400 MHz; CDCl
m, 3H, Ar), 7.49 (brs, 1H, =CH), 7.67 (m, 2H, Ar), 7.85 (m, 2H, Ar). Anal. Calcd for
C 82.22; H 6.49; N 11.28%. Found C 82.17; H 6.45; N 11.36%.
3 3 2
) δ (ppm): 2.91 (s, 6H, -N(CH ) ), 7.01 (m, 2H, Ar), 7.38
(
17 16 2
C H N
4
.2.4. (Z)-3-(2-Nitrophenyl)-2-phenylacrylonitrile (3d)
ο
-1
1
Yield 95 %, mp 106-107 C (white solids). IR (KBr) νmax: 2218 cm (CN). H NMR
spectrum: (400 MHz; CDCl
3
) δ (ppm): 7.36 (m, 3H, Ar), 7.58 (m, 2H, Ar), 6.99 (brs, 1H,
C 71.99; H 4.03; N
=
CH), 7.83 (m,2H, Ar), 7.94 (m, 1H, Ar). Anal. Calcd for C15H N O
10 2 2
1
1.19%. Found C 71.91; H 3.98; N 11.26%.
4
.2.5. (Z)-3-(4-Nitrophenyl)-2-phenylacrylonitrile (3e)
ο
-1
1
Yield 94 %, .p 110-112 C (yellow solids). IR (KBr) νmax: 2220 cm (CN). H NMR
spectrum: (400 MHz; acetone-d
6
) δ (ppm): 7.41 (m, 3H, Ar), 7.08 (brs, 1H, =CH), 7.68 (m,
2
1
H, Ar), 7.81 (m, 2H, Ar), 7.96 (m, 2H, Ar). Anal. Calcd for C15 C 71.99; H 4.03; N
10 2 2
H N O
1.19%. Found C 71.93; H 3.97; N 11.25%.
4
.2.6. (Z)-3-(4-Chlorophenyl)-2-phenylacrylonitrile (3f)
ο
-1
1
Yield 96 %, mp 103-104 C (white solids). IR (KBr) νmax: 2205 cm (CN). H NMR
spectrum: (400 MHz; CDCl ) δ (ppm): 7.11 (m, 2H, Ar), 7.42 (m, 3H, Ar), 7.51 (brs, 1H,
3
=
CH), 7.67 (m, 2H, Ar), 7.86 (m, 2H, Ar). Anal. Calcd for C15
H10ClN C 75.16; H 4.21; N
5
.84%. Found C 75.11; H 4.18; N 5.89%.

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4
.2.7. (Z)-3-(2-Chlorophenyl)-2-phenylacrylonitrile (3g)
ο
-1
1
Yield 92 %, mp 97-98 C (white solids). IR (KBr) νmax: 2230 cm (CN). H NMR
spectrum: (400 MHz; CDCl ) δ (ppm): 7.19 (m, 3H, ArH), 7.48 (m, 3H, ArH), 7.29 (brs, 1H,
3
=
CH), 7.79 (m, 3H, ArH). Anal. Calcd for C15H10ClN: C 75.16, H 4.21, N 5.84. Found: C
7
5.10, H 4.19, N 5.90.
4
.2.8. (Z)-3-(4-Bromophenyl)-2-phenylacrylonitrile (3h)
ο
-1
1
Yield 95 %, mp 89-90 C (white solids). IR (KBr) νmax: 2190 cm (CN). H NMR
spectrum: (400 MHz; CDCl
3
) δ (ppm): 7.07 (m, 2H, Ar), 7.34 (m, 3H, Ar), 7.47 (brs, 1H,
10BrN C 63.40; H 3.55; N
=
CH), 7.58 (m, 2H, Ar), 7.69 (m, 2H, Ar). Anal. Calcd for C15
H
4
.93%. Found C 63.37; H 3.51; N 4.99%.
4
.2.9. (Z)-2-Phenyl-3-p-tolylacrylonitrile (3i) [30]
o
-1
1
Yield 85%, mp 61-62 C (white solids). IR (KBr) νmax: 2213 cm (CN). H NMR
spectrum: (400 MHz; CDCl
3
) δ (ppm): 2.41 (s, 3H, CH
3
), 7.27 (m, 2H, Ar), 7.41 (m, 3H, Ar),
7
5
.50 (brs, 1H, =CH), 7.67 (m, 2H, Ar), 7.80 (m, 2H, Ar). Anal. Calcd for C H N C 87.64; H
16
13
.98; N 6.39%. Found C 87.53; H 5.86; N 6.42%.
4
.2.10. (Z)-2-(4-Nitrophenyl)-3-phenylacrylonitrile (3j)
ο
-1
1
Yield 91 %, mp 143-142 C (red solids). IR (KBr) νmax: 2220 cm (CN). H NMR
spectrum: (400 MHz; CDCl
CH), 7.79 (m, 2H, Ar), 7.94 (m, 2H, Ar). Anal. Calcd for C15
1.19%. Found C 71.95; H 4.00; N 11.23%.
3
) δ (ppm): 7.31 (m, 3H, Ar), 7.51 (m, 2H, Ar), 7.11 (brs, 1H,
=
10 2 2
H N O
C 71.99; H 4.03; N
1
4
.2.11. (Z)-3-(4-Methoxyphenyl)-2-(4-nitrophenyl)acrylonitrile (3k)

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ο
-1
1
Yield 92 %, mp 109-110 C (pale red solids). IR (KBr) νmax: 2201 cm (CN). H
NMR spectrum: (400 MHz; CDCl
H, Ar), 7.08 (brs, 1H, =CH), 7.79 (m, 2H, Ar), 7.91 (m, 2H, Ar). Anal. Calcd for
C 68.56; H 4.32; N 9.99%. Found C 68.49; H 4.29; N 10.02%.
3 3
) δ (ppm): 3.89 (s, 3H, OCH ), 6.97 (m, 2H, Ar), 7.41 (m,
2
16 12 2 3
C H N O
4
.2.12. (Z)-3-(4-(Dimethylamino)phenyl)-2-(4-nitrophenyl)acrylonitrile (3l)
ο
-1
1
Yield 89 %, mp 114-115 C (deep red solids). IR (KBr) νmax: 2208 cm (CN).
H
NMR spectrum: (400 MHz; CDCl
m, 2H, Ar), 7.48 (brs, 1H, =CH), 7.71 (m, 2H, Ar), 7.89 (m, 2H, Ar); Anal. Calcd for
C 69.61; H 5.15; N 14.33%. Found C 69.58; H 5.11; N 14.37%.
3 3 2
) δ (ppm): 2.89 (s, 6H, -N(CH ) ), 6.89 (m, 2H, Ar), 7.33
(
17 15 3 2
C H N O
4
.2.13. (Z)-3-(2-Nitrophenyl)-2-(4-nitrophenyl)acrylonitrile (3m)
-1
1
Yield 92 %, mp 161-162 (yellowish red solids). IR (KBr) νmax: 2222 cm (CN). H
NMR spectrum: (400 MHz; CDCl ) δ (ppm): 7.46 (m, 1H, Ar), 6.96 (brs, 1H, =CH), 7.91 (m,
H, Ar), 8.07 (m, 3H, Ar). Anal. Calcd for C15 C 61.02; H 3.07; N 14.23%. Found C
0.96; H 3.02; N 14.27%.
3
4
6
9 3 4
H N O
4
.2.14. (Z)-2,3-bis(4-Nitrophenyl)acrylonitrile (3n)
ο
-1
1
Yield 94 %, mp 156-157 C (pale red solids). IR (KBr) νmax: 2230 cm (CN).
H
NMR spectrum: (400 MHz; CDCl ) δ (ppm): 6.96 (brs, 1H, =CH), 7.89 (m, 4H, Ar), 8.02 (m,
3
4
H, Ar); Anal. Calcd for C15
4.28%.
9 3 4
H N O C 61.02; H 3.07; N 14.23%. Found C 60.98; H 3.03; N
1
4
.2.15. (Z)-3-(4-Chlorophenyl)-2-(4-nitrophenyl)acrylonitrile (3o)
ο
-1
Yield 87 %, mp 139-140 C (yellowish red solids). IR (KBr) νmax: 2199 cm (CN).
1
3
H NMR spectrum: (400 MHz; CDCl ) δ (ppm): 7.39 (m, 2H, Ar), 7.52 (m, 2H, Ar), 7.19

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(brs, 1H, =CH), 7.81 (m, 2H, Ar), 7.97 (m, 2H, Ar). Anal. Calcd for C15
H
9
ClN
2
O
2
C 63.28;
H 3.19; N 9.84%. Found C 63.24; H 3.16; N 9.87%.
4
.2.16. (Z)-3-(2-Chlorophenyl)-2-(4-nitrophenyl)acrylonitrile (3p)
ο
-1
Yield 92 %, mp 124-126 C (yellowish red solids). IR (KBr) ν : 2226 cm (CN).
max
1
3
H NMR spectrum: (400 MHz; CDCl ) δ (ppm): 7.11 (m, 2H, Ar), 7.46 (m, 2H, Ar), 7.13
(brs, 1H, =CH), 7.78 (m, 2H, Ar), 8.03 (m, 2H, Ar); Anal. Calcd for C15
9
H ClN
2
O
2
C 63.28; H
3
.19; N 9.84%. Found C 63.25; H 3.15; N 9.88%.
4
.2.17. (Z)-3-(4-Bromophenyl)-2-(4-nitrophenyl)acrylonitrile (3q)
ο
-1
Yield 95 %, mp 128-130 C (yellowish red solids). IR (KBr) νmax: 2220 cm (CN).
1
3
H NMR spectrum: (400 MHz; CDCl ) δ (ppm): 7.43 (m, 2H, Ar), 7.15 (brs, 1H, =CH), 7.64
(
m, 2H, Ar), 7.82 (m, 2H, Ar), 7.99 (m, 2H, Ar); Anal. Calcd for C H BrN O C 54.74; H
1
5
9
2
2
2
.76; N 8.51%. Found C 54.69; H 2.71; N 8.55%.
4
.2.18. (Z)-2-(4-Nitrophenyl)-3-p-tolylacrylonitrile (3r)
ο
-1
1
Yield 91 %, mp 13-132 C (pale yellow solids). IR IR (KBr) νmax: 2215 cm (CN). H
NMR spectrum: (400 MHz; CDCl
H, Ar), 7.09 (brs, 1H, =CH), 7.87 (m, 2H, Ar), 8.01 (m, 2H, Ar). Anal. Calcd for
C H N O C 72.72; H 4.58; N 10.60%. Found C 72.68; H 4.55; N 10.66%.
3 3
) δ (ppm): 2.42 (s, 3H, CH ), 7.17 (m, 2H, Ar), 7.40 (m,
2
16
12
2
2
4
4
.3. Biological assay
.3.1. Cytotoxicity Assay
Cytotoxicity after treatment of tumor cells with the test materials was determined
using the SRB (sulforhodamine-B) method, currently adopted in the NCI’s in vitro anti-
cancer drug screening [35], i.e. the inhibition rate of cell proliferation was estimated after
continuous exposure to the test materials for 48 h. All samples were tested in triplicate, and

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the mean IC50 values (mg/mL) (concentration of compound resulting in 50% inhibition of cell
proliferation) and S.E.M. were calculated.
4
.3.2. Anti-bacterial Screening
A previously described filter paper disc diffusion method [36] against two strains was
used for determination of the in vitro anti-bacterial effects of all samples. Briefly, nutrient
agar (NA) media (Difco laboratories, Lawrence, KS) was used as a basal medium for test
bacteria. These agar media were inoculated with 0.2 mL of the 24-h liquid cultures containing
the microorganisms. The sample discs were placed gently on pre-inoculated agar plates and
o
then incubated aerobically at 37 C for 24 h. Discs with only DMSO were used as a control,
and azithromycin was used as a positive control. Inhibitory activity was measured (in mm) as
the diameter of the observed inhibition zones. These evaluations were performed in triplicate
-1
for each compound at a concentration of 100 µg disc .
4
.3.3. Anti-fungal Screening
Using the standard disc diffusion method [36], all samples were tested in vitro for
their anti-fungal properties against Aspergillus niger. Briefly, potato dextrose agar (Difco)
o
was used as a basal medium for testing of fungi. Sterilized melted PDA medium (~45 C) was
poured into a Petri dish (90 mm) and solidified. Prepared discs of samples were placed gently
on solidified agar plates and freshly seeded with the test organisms using sterile forceps.
Discs with DMSO and azithromycin were used as negative and positive controls, respectively.
o
Plates were incubated at 30±1 C for 72 h. DMSO was used as a solvent for preparation of
desired solutions of the test samples.
4
.4. Computational Methods
The molecular geometries of the (Z)-2,3-diphenylacrylonitrile analogues were built with a
standard bond length and angles using the ChemBio3D ultra Ver. 12 molecular modeling

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program (CambridgeSoft Corporation, Cambridge, MA 02140 USA). The energy was
minimized by the semi-empirical molecular orbital PM3 method [37]. Physicochemical
properties were calculated using molinspiration cheminformatics software (Molinspiration
Cheminformatics, SK 90026 Slovensky Grob, SR). The method for calculation of clogP was
developed by Molinspiration (miLogP2.2 - 2005) based on group contributions and
correction factors by fitting calculated logP with experimental logP for a training set more
than twelve thousand, mostly drug-like molecules. Molecular polar surface area (PSA) was
calculated based on the methodology published by Ertl et al. [38] as a sum of fragment
contributions. The maps of molecular lipophilicity potential (MLP) and polar surface area
(PSA) were viewed in Molinspiration Galaxy 3D Structure Generator (ver. 2010.02 beta)
using an optimized structure generated by the semi-empirical molecular orbital PM3 method.
Acknowledgments
Dr. Seen Ae Chae at Korea Basic Science Institute (Daegu) is acknowledged for the NMR
data.
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Table 1
In vitro cytotoxicity data of (Z)-2,3-diphenylacrylonitrile analogues (3a-r) against selected
human cancer cell lines.
CN
R1
A
B
R3
H
R2
-
1 a
IC (µg mL )
5
0
Comp
R1
H
R2
H
R3
b
c
d
e
A549
SK-OV-3
SK-MEL-2
HCT15
3a
3b
3c
3d
3e
H
43.76
25.51
0.57
47.39
14.93
0.14
38.45
25.17
0.65
68.98
23.16
0.34
H
H
H
H
OMe
NMe
H
2
H
NO
2
44.45
37.76
1.81
48.59
48.97
0.20
38.95
35.16
1.53
70.12
44.85
1.22
H
H
H
NO
Cl
H
2
3
f
H
3
3
g
h
H
Cl
31.56
18.19
1.82
18.63
5.57
25.53
14.46
1.63
33.76
13.98
1.51
H
H
H
Br
3
i
j
H
CH
H
3
0.58
3
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
34.10
35.65
77.59
72.35
32.95
82.96
17.86
89.75
75.51
0.011
39.24
34.05
78.97
74.90
35.85
85.30
7.27
33.95
32.14
75.85
52.65
37.50
83.43
16.14
69.76
59.98
0.009
40.12
69.12
81.12
3
k
H
OMe
3
l
H
NMe
H
2
3
m
NO
2
>100.0
39.86
83.06
88.90
92.65
>100.0
0.814
3
n
o
p
q
H
H
NO
2
3
Cl
3
3
Cl
H
H
H
Br
>100.0
71.98
0.092
3
r
CH
3
Doxorubicin
a
IC50 values were obtained using a dose response curve by nonlinear regression using a
b
c
d
curve fitting program, OriginPro 7.5. human lung cancer, human ovarian cancer, human
e
skin cancer, and human colon cancer.

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Table 2
Molinspiration calculations of molecular properties of compounds (3a-r).
MW
OH-NH
O-N
a
b
e
Comp.
cLogP
TPSA
nrotb
volume
c
d
(
g/mol)
interact
interact
3
3
3
3
3
a
b
c
d
e
205.260
235.286
248.329
250.257
250.257
239.705
239.705
284.156
219.287
250.257
280.283
293.326
295.254
295.254
284.702
284.702
329.153
264.284
3.785
3.842
3.887
3.516
3.744
4.463
4.235
4.594
4.234
3.744
3.801
3.846
3.475
3.703
4.422
4.194
4.553
4.193
23.792
33.026
27.03
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
4
4
1
1
1
1
4
5
5
7
7
4
4
4
4
2
3
3
3
3
2
2
2
2
3
4
4
4
4
3
3
3
3
199.728
225.274
245.634
223.063
223.063
213.264
213.264
217.614
216.289
223.063
248.608
268.968
246.397
246.397
236.598
236.598
240.948
239.624
69.616
69.616
23.792
23.792
23.792
23.792
69.616
78.85
3
f
3
3
g
h
3
3
i
j
3
k
3
l
72.854
115.44
115.44
69.616
69.616
69.616
69.616
3
m
3
n
o
p
q
3
3
3
3
r
a
Calculated octanol/water partition coefficient
Molecular polar surface area
Number of hydrogen-bond donors
Number of hydrogen-bond acceptors
Number of rotatable bonds
b
c
d
e

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Table 3
In vitro anti-microbial profiles of (Z)-2,3-diphenylacrylonitrile analogues (3a-r) in terms of
zone of inhibition.
CN
R1
A
B
R3
H
R2
Inhibition zone in mm
Comp
R
1
R
2
R
3
S. aureus
S. typhi
7 ± 0.5
10 ± 1.0
10 ± 1.0
8 ± 0.5
7 ± 0.5
A. niger
3
3
a
H
H
H
-
-
-
-
b
H
H
H
H
OMe
-
-
-
-
-
-
3
c
NMe
H
2
3
d
H
NO
2
3
e
H
H
H
NO
Cl
H
2
3
f
H
-
10 ± 1.0
-
3
3
g
H
Cl
-
-
8 ± 0.5
9 ± 0.5
-
-
h
H
H
H
Br
3
i
H
CH
H
3
-
-
11 ± 1.0
8 ± 0.5
-
-
3
j
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
18 ± 0.5
13 ± 1.0
11 ± 0.5
7 ± 0.5
7 ± 0.5
9 ± 0.5
10 ± 1.0
8 ± 0.5
10 ± 1.0
21 ± 1.0
-
3
k
H
OMe
12 ± 0.5
-
3
l
H
NMe
H
2
11 ± 0.5
-
3
m
NO
2
11 ± 1.0
-
3
n
o
p
q
H
H
NO
Cl
2
-
-
-
-
-
3
-
3
3
Cl
H
-
H
H
Br
-
3
r
CH
3
1
9 ± 1.0
18 ± 1.0
Azithromycin
Inhibitory activity is expressed as the diameter (in mm) of the observed inhibition
-1
zone. Data show means ± SD (n=3). Concentration of each compound is 100 µg disc ,
-1
whereas that of positive control is 25 µg disc .

ACCEPTED MANUSCRIPT
CN
NaOEt
R1
OHC
CN
R3
+
EtOH, r.t.
H
R1
R2
R3
R2
1
2
3a-r
Scheme 1. Synthesis of (Z)-2,3-diphenylacrylonitrile analogues.

ACCEPTED MANUSCRIPT
HO
HO
H
H
CN
R2
α
(
HO)n
R1
OH
β
R3
H
H
CN
H
(E)-Stilbene
(E)-Resveratrol
Tyrphostins
(Z)-Diphenylacrylonitriles
Figure 1. Structures of key molecules.

ACCEPTED MANUSCRIPT
A
B
C
D
E
F
Figure 2. Maps of lipophilicity potential (left) and polar surface area (right) of 3a (A), 3c (B),
3
f (C), 3g (D), 3h (E), and 3p (F) showing the most lipophilic area (pink color), intermediate
lipophilic area (green color), most hydrophobic area (blue color), nonpolar area (gray white
color), and polar area (red color).

ACCEPTED MANUSCRIPT
Figure 3. Correlation between polar surface area (PSA) and inhibitory potency in selected
(Z)-2,3-diphenylacrylonitrile analogues against (A) human lung cancer (A549), (B) human
ovarian cancer (SK-OV-3), (C) human skin cancer (SK-MEL-2), and (D) human colon
cancer (HCT15) cells.