One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2014.05.089

A series of cinnamylideneacetophenones were synthesized via a modified Claisen-Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue assay. Derivatives 17 and 18 bearing a 2-nitro group on the B rin

Full text of DOI:10.1016/j.bmcl.2014.05.089

Accepted Manuscript
One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxic-
ity in breast cancer cells
David J. Weldon, Marilyn D. Saulsbury, Joshua Goh, Leah Rowland, Petreena
Campbell, Laijia Robinson, Calvin Miller, Joshua Christian, Louisa Amis, Nia
Taylor, Willie Davis Jr., Stanley L. Evans, Eileen Brantley
PII:
S0960-894X(14)00598-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.089
BMCL 21703
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
11 April 2014
22 May 2014
23 May 2014
Please cite this article as: Weldon, D.J., Saulsbury, M.D., Goh, J., Rowland, L., Campbell, P., Robinson, L., Miller,
C., Christian, J., Amis, L., Taylor, N., Davis, W. Jr., Evans, S.L., Brantley, E., One-pot synthesis of
cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells, Bioorganic & Medicinal
Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.089
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One-pot synthesis of cinnamylideneacetophenones and
their in vitro cytotoxicity in breast cancer cells.
David J. Weldon, Marilyn D. Saulsbury, Joshua
Goh, Leah Rowland, Petreena Campbell, Laijia
Robinson, Calvin Miller, Joshua Christian,
Louisa Amis, Nia Taylor, Willie Davis Jr.,
Stanley L. Evans and Eileen Brantley

Bioorganic & Medicinal Chemistry Letters journal homepage:
www.elsevier.com
One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in
breast cancer cells.
David J. Weldona, Marilyn D. Saulsburyb, Joshua Goha, Leah Rowlandc, Petreena Campbellc, Laijia Robinsonc,d, Calvin Millera,
Joshua Christiana, Louisa Amisc, Nia Taylorc, Willie Davis Jr.a,c, Stanley L. Evanse,# and Eileen Brantleya,c,f*
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of cinnamylideneacetophenones were synthesized via a modified Claisen-
Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer
cells using the Alamar Blueassay. Derivatives 17 and 18 bearing a 2-nitro group
on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC50 = 71 and
1.9 nM) respectively. Derivative 17 also displayed sub-micromolar (IC50 = 780 nM)
cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly
less cytotoxicity than the chemotherapeutic doxorubicin in non-tumorigenic MCF-
10A cells. This study provides evidence supporting the continued development of
nitro-substituted cinnamylideneacetophenones as small molecules to treat breast
cancer.
Keywords: breast cancer; estrogen receptor;
chalcone derivatives; leinamycin;
cytotoxicity
2014 Elsevier Ltd. All rights reserved.
aDepartment of Pharmaceutical and Administrative Sciences, School of Pharmacy, Loma Linda University, Loma Linda, CA, US
bDepartment of Pharmaceutical Sciences, Hampton University, Hampton, VA, US
cDepartment of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA, US
dDepartment of Chemistry, Geology and Physics, School of Mathematics, Science & Technology, Elizabeth City State
University, Elizabeth City, NC, US
eDepartment of Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN, US
fDepartment of Chemistry, University of California, Riverside, California, 92521, USA
#Deceased
____________________________________
*Corresponding author. Tel.: +1 909 558 7703; fax: +1 909 558 4035. Email addresses: ebrantley@llu.edu, brantley71@yahoo.com (E.
Brantley)

two aromatic rings joined by a three-carbon α,β-unsaturated
carbonyl system.15 The stilbene configuration within the α,β-
unsaturated carbonyl system has been used as a combinatorial
starting point for novel medicinal entities and may also
contribute to their antitumor properties. Stilbene derivatives
include tamoxifen and the naturally occurring flavonoid
genistein.16
We speculate that leinamycin’s α,β,γ,δ−unsaturated carbonyl
group, as highlighted in Figure 1, may also contribute to its
anticancer activity. As a result, we designed, synthesized and
evaluated a series of cinnamylideneacetophenones, or (E,E)-1,5-
Despite current advances in therapy, breast cancer causes
nearly 40,000 deaths each year in the US.1 As a result, it is
evident that more effective therapy is needed to treat these
malignancies. Historically, breast cancer has been classified
broadly as endocrine receptor (i.e. estrogen receptor or
progesterone receptor) positive or negative. This stratification
frequently serves as a guide for targeted therapy. Breast cancer
that is estrogen receptor positive (ER+) and progesterone
receptor positive (PR+) is frequently treated with hormone-based
therapeutic agents such as the anti-estrogen tamoxifen
(Nolvadex®) or aromatase inhibitors such as anastrazole
(Arimidex®).2 Despite their effectiveness, tamoxifen can cause
thromboembolisms and endometrial cancer; while, anastrazole
therapy increases the risk of arthralgia and bone fracture. In
addition, there is a risk of hormone therapy-related resistance.3-5
More recently, breast cancer has included a classification for
tumors that overexpress human epidermal growth factor receptor
2 (HER2) irrespective of ER/PR status. Such patients are
frequently candidates for a targeted monoclonal antibody
trastuzumab (Herceptin®). However, only about 25% of HER2
positive tumors respond to trastuzumab and a substantial
percentage of these tumors acquire resistance to this agent.
Interestingly, those that use this agent are at risk for developing
cardiotoxicity.6
Figure 1. The development of cinnamylideneacetophenones from
common features of leinamycin and chalcone skeleton.
diarylpenta-2,4-dien-1-ones, with aryl substitution similar to
published chalcones to determine if this portion of the molecule
also contributes to their anticancer activity (Figure 1). Recent
studies
have
extensively
characterized
certain
cinnamylideneacetophenones though little is known about their
potential to display anticancer activity.17
Tumors that lack appreciable expression of ER, PR and HER2
are denoted as ‘triple negative’ and are unresponsive to hormone-
based or targeted therapeutic agents. As a result, patients bearing
such tumors are limited to chemotherapy protocols with cytotoxic
(non-specific) agents such as doxorubicin (Adriamycin®,
DOXIL®), cyclophosphamide (Cytoxan®), and paclitaxel
(Taxol®, Abraxane®), which are associated with severe adverse
effects. Specifically doxorubicin can cause cardiotoxicity and
secondary malignancies.7 Considering the risk of hormone
therapy-related resistance, the ineffectiveness of endocrine
therapy in triple negative breast cancer and the severe side effects
of nonspecific chemotherapeutic agents, a need persists for novel
small molecules with the potential to effectively manage different
breast cancer subtypes.
Using a Claisen-Schmidt condensation reaction, we
initially synthesized a library of chalcones. We are reporting the
two derivatives (4 and 5) with the most biological activity.
Scheme 1. Synthesis of chalcone derivatives 4 and 5. (i) NaOH, H2O/EtOH
(50% v/v), rt, 24 h, 74-82%.
There have been a number of small molecules isolated and
synthesized in hopes of discovering the next pharmacophore to
serve as a template for effective agents to treat cancer,
particularly breast cancer. Leinamycin, a naturally occurring
antibiotic containing extended conjugation shows antitumor
activity and is reported to cleave DNA.8 Leinamycin exhibits
potent anticancer activity both in cancer cell lines and in human
tumor xenografts.9 At least a portion of leinamycin’s anticancer
activity resides within the disulfide bond of the 1,2-dithiolan-3,1-
Chalcone 4 has a tri-methoxy phenyl substitution on the A-ring
and chalcone 5 bears a 2-nitro group on the B-ring (Scheme 1).
We screened these chalcones using the Alamar Blue assay to
compare their cytotoxicity levels with the unsubstituted trans-
chalcone (6), which was purchased from Sigma-Aldrich® (CAS
Number 614-47-1, Catalog Number 136123). We initially
evaluated the anticancer activity of 4 and 5 in comparison to 6
and later to their corresponding cinnamylideneacetophenones to
determine the impact of extending the conjugation on
cytotoxicity in MCF-7 ER+ and MDA-MB-468 ER- breast
cancer cells. We also evaluated their potential cytotoxicity in a
oxide heterocycle molecule which readily promotes reactive
10
oxygen species formation and DNA damage.8,
Although a
significant amount of antitumor activity may exist in this portion
of the molecule, it may not account for all of its antitumor
actions.
Another class of anti-cancer compounds that have been
extensively synthesized are chalcones (1,3-diaryl-2-propen-1-
Table 1. Biological activity of chalcones 4-6 produced via Scheme 1a
Compound MDA-MB-468
MCF-7
IC50 (M)
4.8 (1.99-11.8)
MCF-10A
IC50 (M)
0.032 (0.004-0.28)
number
IC50 (M)
ones), flavonoid small molecules that possess
a broad
4
2.05 (1.27-3.32)
pharmacological profile including antioxidant, anti-inflammatory
and anticancer actions.11 In addition, these naturally occurring
compounds disrupt mitosis and inhibit tubulin polymerization.12
While the unsubstituted chalcone 2 does not occur naturally,
5
6
> 50
>50
3.75 (1.17-11.9)
> 50
4.2 (0.9-17.8)
3.15 (1.63-6.07)
aAll tests were performed in replicate (n = 5) and repeated at least twice.
Numbers in parentheses represent 95% confidence intervals.
polyhydroxylated chalcones and flavonoids exist in a myriad of
14
edible plants.13,
Structurally, chalcones are characterized by

non-tumorigenic breast epithelial cell line (MCF-10A).
Chalcone 4 demonstrated activity in the MDA-MB-468 cell
line in the micromolar range while chalcones 5 and 6 were
inactive. Both chalcones 4 and 5 displayed activity in MCF-7
cells while chalcone 6 was inactive in this cell line. All three
chalcones displayed substantial toxicity in the MCF-10A non-
cancerous cell line to suggest their high potential for toxicity in
non-target cells (Table 1).
cancer cells. However, pronounced cytotoxicity was detected in
MCF-10A cells following exposure to 10 as compared to 9.
Surprisingly, the presence of a single methoxy group in the 4-
position on the A-ring seen in 11 resulted in complete loss of
cytotoxic activity. The presence of a fluorine group in the 4-
position on the A-ring as seen in 12 resulted in a loss of
cytotoxicity in MDA-MB-468 cells yet substantially increased
Following the construction of the chalcone analogues, we
designed
cinnamylideneacetophenones using a one-pot modified Claisen-
Schmidt condensation reaction. The synthesis of
and
synthesized
a
library
of
cinnamylideneacetophenones 9-18 was accomplished by reacting
trans-cinnamaldehydes containing varying functional groups
with either acetophenone or 2-nitro acetophenone in the
presence of aqueous NaOH and ethanol (Scheme 2). This library
combines the common aryl groups of reported chalcones
including those of 4 and 5, with the extended conjugation and
increased carbon length seen in the aforementioned portion of
leinamycin
(Table
2
and
Figure
2).
The
cinnamylidenenacetophenones were evaluated for cytotoxicity
against MDA-MB-468 and MCF-7 cells and the non-tumorigenic
Scheme 2. Construction of target molecules 9-18. (i) NaOH, H2O/EtOH
(50% v/v), rt, 24h, 65-83%.
MCF-10A cells as seen in Table 3. When comparing 4 and 10, 5
and 18, and 6 and 9, we detected a distinct decrease in
cytotoxicity in MCF-10A cells when the conjugation was
extended from the unsaturated ketone to the
Figure 2. The library of cinnamylideneacetophenones synthesized for
biological evaluation.
cytotoxicity in MCF-7 cells as compared to 9. Unlike 10,
fluorine substitution in the 4-position on the A-ring in 12 resulted
in no toxicity in the MCF-10A cells. The presence of chlorine in
the 4-position on the A-ring resulted in a derivative (13) that was
completely inactive. 3,4-dichloro substitution on the A-ring
resulted in derivative (14) which demonstrated cytotoxicity in the
MDA-MB-468 and MCF-7 breast cancer cells although
substantial cytotoxicity was also detected in the MCF-10A cells.
Substitution of a nitro group in the 4-position of the A ring
resulted in an inactive derivative 15 while insertion of a napthyl
group in the A-ring resulted in derivative 16 with activity in
MDA-MB-468 cells but no activity in MCF-7 or MCF-10A cells.
Since chalcone 5 exhibited some activity in MCF-7 cells with
an IC50 of 3.75 M though inactive in MDA-MB-468 cells, we
synthesized two cinnamylideneacetophenones with a 2-nitro
group on the B ring. We found that derivative 17 exhibited potent
nanomolar activity in both the MDA-MB-468 and MCF-7 breast
cancer cell lines though cytotoxicity was apparent in MCF-10A
cells unlike 9. Derivative 18 exhibited low micromolar activity
in MDA-MB-468 cells and extremely potent activity in the
nanomolar range in MCF-7 cells. In addition, there was no
appreciable cytotoxicity detected in MCF-10A cells following
exposure to 18. None of the cinnamylideneacetophenones were
as cytotoxic to the MCF-10A cells as the reference
chemotherapeutic doxorubicin. Additionally, 18 displayed more
potent anticancer activity in MCF-7 cells than doxorubicin
(Table 3).
Table 2. Cinnamylideneacetophenones 9-18 produced via Scheme 2a
Compound
number
9
R1
R2
Ph
3,4,5-trimethoxyphenyl
4-methoxyphenyl
4-fluorophenyl
4-chlorophenyl
3,4-dichlorophenyl
4-nitrophenyl
Naphthyl
Ph
10
11
12
13
14
15
16
17
18
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Furyl
2-nitrophenyl
2-nitrophenyl
Ph
unsaturated ketone. Though extending the conjugation
appeared to not appreciably impact breast cancer cell viability for
the unsubstituted or trimethoxy substituted chalcone (6 vs. 9 or 4
vs. 10), such extension did improve in vitro cytotoxicity for both
breast cancer cell lines for the 2-nitro-substituted chalcone (5 vs.
18).
With respect to the unsubstituted cinnamylideneacetophenone
9, the substitution of trimethoxy phenyl for the unsubstituted
phenyl as the A-ring (10) resulted in a substantial enhancement in
cytotoxicity in both the MDA-MB-468 and the MCF-7 breast
One key assessment into the suitability of a potential agent for
the treatment of breast cancer is how selective the agent is in
exhibiting cytotoxicity. Ideally, an agent will exhibit cytotoxicity
only in malignant cells while sparing non-tumorigenic cells. We

evaluated the extent of selective cytotoxicity for each of the
compounds as well as doxorubicin by dividing the IC50 for the
non-tumorigenic MCF-10A cells by the IC50 values determined
for both breast cancer cell lines (Table 4). Among the chalcones,
5 showed 2.7-fold greater toxicity in MCF-7 cells as compared to
the MCF-10A cells. Otherwise, the chalcones were actually
more cytotoxic in MCF-10A cells than the breast cancer cells.
This finding strengthened our rationale for synthesizing
and sub-micromolar activity in the cancerous cell lines along
with the high SI of derivative 18 make it the most promising
candidate of all the cinnamylideneacetophenones for future
studies and derivation. The next most promising derivative 17
only differs from 18 in that ring A consists of a furan heterocycle
rather than a phenyl group.
In conclusion, the majority of the derivatives bore
substitutions on the A-ring yet the two most active derivatives
bore a 2-nitro group on the B-ring. Both of these agents
exhibited anticancer activity in the nanomolar to sub-micromolar
range yet exhibited substantially less cytotoxicity in the MCF-
10A cells. In fact the selectivity index for both 17 and 18
appears to be superior to that observed with the established
chemotherapeutic agent doxorubicin. Studies are underway to
further develop 17 and 18 as agents to treat breast cancer.
Table 3. Cinnamylideneacetophenones 9-18 produced via Scheme 2a
Compound MDA-MB-468
MCF-7
IC50 (M)
>50
MCF-10A
IC50 (M)
>50
number
IC50 (M)
9
31.9 (13.4-75.7)
10
11
12
13
14
15
16
17
18
6.35 (1.4-10.4) 2.13(0.24-18.4)
1.11(0.47-2.65)
>50
>50
>50
4.28 (0.13-1.4)
>50
>50
>50
>50
Acknowledgments
>50
15.6 (6.7-36.3)
>50
2.7 (0.5-14.6)
>50
1.37 (0.257-7.5)
>50
Research reported in this publication was supported in part by
the National Institute of Health Disparities and Minority Health
of the National Institutes of Health (NIH) under award
number P20MD006988, the National Cancer Institute of NIH
under award number R01 CA101864-09S1, the Elizabeth City
State University Support Program for Academic and Research
Enhancement (ESPARE) from the National Institute of General
Medical Sciences of NIH award number R25GM082808, Grants
to Promote Collaborative and Translational Research (Loma
Linda University) and from intramural funding from the Loma
Linda University School of Pharmacy Department of
Pharmaceutical and Administrative Sciences. The content is
solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health or
Loma Linda University.
8.429 (4.2-16.9)
>50
>50
0.78 (0.48-1.4) 0.071 (0.027-0.18)
9.11(1.5-56.4)
>50
1.37 (0.74-2.5) 0.0019 (0.0005-
0.0076)
doxorubicin 0.1 (0.015-0.67) 0.18 (0.06-0.53) 0.037 (0.026-0.54)
aAll tests were performed in replicate (n = 5). Numbers in parentheses
represent 95% confidence intervals.
derivatives with extended conjugation as opposed to additional
chalcones. We detected a greater than 11-fold selectivity for
MCF-7 cells following exposure to 12. We found nearly 6-fold
selectivity of 16 for MDA-MB-468 cells. Thus, fluorine
substitution on the phenyl group (ring A) and insertion of a
phenyl group for a naphthyl group in ring A led to derivatives
with promising activity and selectivity for MCF-7 and MDA-
MB-468 cells respectively.
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The selectivity indices for 17 in MDA-MB-468 and MCF-7
cells were 10- and 128-fold respectively. The greatest extent of
selective cytotoxicity was found when MDA-MB-468 and MCF-
7 cells were exposed to 18; a derivative that is more than 35
times more selective for MDA-MB-468 cells and more than
25,000 times more selective for MCF-7 cells. The micromolar
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cinnamylideneacetophenones
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MDA-MB-468
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0.004
SI
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18
doxorubicin
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19.
In vitro anticancer activity determination. The cytotoxic
activity of the cinnamylideneacetophenone derivatives was
determined using the Alamar Blue assay in accordance with a
previously published method.18. Stock solutions of the derivatives
were prepared in DMSO at a concentration of 100 mM. In this study
we used MCF-7 (estrogen receptor positive and MDA-MB-468
(estrogen receptor negative) breast cancer cells in comparison to the
non-tumorigenic MCF-10A cells. Briefly, cells were plated at their
appropriate densities in 96 well plates and allowed to attach for 24 h
before being exposed to varying concentrations of the derivatives for
72 h at 37C. The Alamar Blue dye was then added to each of the
wells for to initiate 4 h incubation. The plates were then placed in a
fluorescence microplate reader (BioTek FLx 800) to measure
fluorescence levels and the IC50 values determined using GraphPad
Prism 5.0.