Design, synthesis, and biological evaluation of lipophilically modified bisphenol Z derivatives

Article abstract of DOI:10.1111/cbdd.13531

In the present study, a small library of bisphenol Z (BPZ) derivatives was synthesized and investigated for anti-proliferative effects in cultured breast and glioblastoma cell lines. Synthesized BPZ derivatives varied in molecular size, polarity, and lipo

Full text of DOI:10.1111/cbdd.13531

DR RICHARD W DUDLEY (Orcid ID : 0000-0001-5020-371X)
Article type : Research Letter
Lea M. Stitzlein, Christopher R.T. Stang, Laura R. Inbody, P.S.S. Rao, Ryan A. Schneider,
Richard W. Dudley*
Design, synthesis, and biological evaluation of lipophilically modified bisphenol Z
derivatives
The University of Findlay, College of Pharmacy, 1000 North Main Street, Findlay, OH
45840
*Corresponding author email: dudley@findlay.edu Phone: 419-434-5387
ORCID : 0000-0001-5020-371X
Conflict of Interest
No conflict of interest exists for any of the authors.
Abstract
In the present study, a small library of bisphenol Z (BPZ) derivatives was synthesized
and investigated for anti-proliferative effects in cultured breast and glioblastoma cell lines.
Synthesized BPZ derivatives varied in molecular size, polarity, and lipophilicity. Of the 8
derivatives tested, compounds 4 and 6, both of which displayed the highest degree of
lipophilicity, were most active at inducing cell death as determined by the XTT assay. Cell
membranes were interrogated using trypan blue staining and were shown to remain intact
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13531
This article is protected by copyright. All rights reserved.

during treatments with 4 and 6. Activation of caspase enzymes (3 and/or 7) was noted to
occur following treatment with compound 4. Polar BPZ derivatives, those with a substituted
amine or alcohol, were devoid of any inhibitory or proliferative effects. The remaining
derivatives seem to lack sufficient lipophilicity to execute an overt toxic effect. Our results
suggest that increasing the lipophilic character of BPZ enhances the cytotoxic effects.
1. Introduction
Bisphenols, including bisphenol A (BPA), are a class of diverse molecules bearing
phenolic residues joined by varied linkers. Bisphenol A is the most famous bisphenol
derivative as it has been used extensively in industry, primarily in polymerization reactions to
form plastics. The synthesis of BPA is relatively straight-forward and can be accomplished
by an electrophilic aromatic substitution reaction using acetone and phenol under acid
catalysis (Fig. 1) (Dermer, 1983).
Over the past decade, negative effects due to BPA exposure have been reported
showing that BPA possess endocrine modulatory effects (de Andrade et al., 2017; Gassman,
2017; Izzotti et al., 2010; Jenkins, Betancourt, Wang, & Lamartiniere, 2012; Lamartiniere,
Jenkins, Betancourt, Wang, & Russo, 2011; Lee et al., 2014; Matsumoto, Adachi, & Suzuki,
2005; Rochester & Bolden, 2015). The published data surrounding this effect in exposed
populations has led to the withdrawal of BPA from numerous commercial products including
plastic water bottles, infant formula bottles, as well as plastic storage containers for fear of
BPA leaching into foodstuff. The endocrine modulatory effects of BPA may be a result of the
molecule’s structural resemblance to estradiol. Research has shown that BPA can act as an
estrogen receptor agonist or antagonist and has the capacity to elicit numerous “estrogen-
like” effects in cultured cells (Gassman, 2017; Izzotti et al., 2010; Suzuki, Ide, & Ishida,
2001; Washington, Hubert, Jones, & Gray, 2001).
This article is protected by copyright. All rights reserved.

Previous research on BPA and other bisphenol analogs have shown that across all
derivatives, a proliferative effect is induced at micromolar concentrations in cell lines which
express estrogen receptors (Matsumoto et al., 2005; Mesnage et al., 2017). Importantly, as the
concentration of bisphenol derivative was increased, cell viability was shown to decrease.
One interesting observation from these studies is that of three bisphenols (BPA, BPF, and
BPZ), the more lipophilic the derivative, the greater the cytotoxic effect at higher
concentrations (BPF < BPA < BPZ) (Mesnage et al., 2017). The results from this study
showed that bisphenol Z (BPZ) displayed the greatest degree of cytotoxicity among the
bisphenol analogs tested and may be in part due to its enhanced lipophilic character relative
to BPA or BPF (CLogP of 5, 3.6, and 2.8 respectively). This same study demonstrated that
the addition of a true estrogen receptor antagonist, ICI 182,780, decreased the proliferative
effects of bisphenol analogs. The addition of an antagonist and the removal of proliferative
effects strongly indicated that BPZ may be acting as an agonist of estrogen receptors.
In addition to their estrogenic effects, BPA and its derivatives have been shown to
modulate PPAR-γ signaling and can also induce caspase-dependent apoptotic pathways in
cell culture (Fehlberg, Gregel, Goke, & Goke, 2003; Terasaka, Kadoma, Sakagami, &
Fujisawa, 2005). Numerous bisphenol derivatives have been synthesized as BPA
substitutes and unfortunately also possess similar endocrine disrupting effects. Simple
alterations in the linker between the two phenolic rings is insufficient to prevent hormone
receptor activation as seen with bisphenol S and bisphenol F (Rochester & Bolden, 2015).
BPZ can be synthesized in a similar manner to BPA by substituting
cyclohexanone for acetone (Fig. 2) (Gregor, 2012).
This article is protected by copyright. All rights reserved.

2. Methods and Materials
Given the dichotomy in BPZ activity, this study focused on the design, synthesis and
evaluation of both polar and non-polar enhancing modification made to BPZ and their effect
on proliferation in breast (MCF-7) and glioblastoma (A-172) cell lines. The present small
library focused attention on the 4-position of the cyclohexanone ring used as the
phenol linker. Compounds were synthesized in one or two steps with reasonable
yields. Synthesized compounds were then screened against the aforementioned cell lines as
both culture lines express estrogen receptors and are sensitive to estrogen receptor antagonists
(Bardon, Vignon, Derocq, & Rochefort, 1984; He, Liu, Yang, & Yuan, 2015).
Bisphenol Z derivatives were synthesized from phenol and the appropriate
cyclohexanone derivative under acidic conditions. Analog 1 was synthesized as shown in
Scheme 1a using 4-aminocyclohexanone (1 mmol) dissolved in concentrated HCl followed
by the addition of 2.5 mmol phenol and stirred at room temperature overnight. Product was
precipitated with ice cold water and filtered. Additional drying in an oven at 100°C removed
excess phenol. This synthetic procedure was also used for the preparation of analogs 2, 5, 6,
and 8. For the synthesis of derivatives 4 and 7 (Scheme 1b), analog 1 was dissolved in dry
DMF and treated with triethylamine followed by acylation using hexanoyl chloride or
palmitoyl chloride at room temperature overnight. Purification of analogs 4 and 7 was done
using a 1000 Micron preparative plate with dichloromethane: ethyl acetate (90: 10) as the
mobile phase. All compounds were characterized using 1H NMR and high-resolution mass
spectrometry. Details on characterization are provided in the supporting information.
The in vitro cytotoxicity of BPZ and synthesized derivatives was measured in A-172
cells and MCF-7 cells using the XTT assay. Briefly, cells were seeded at a concentration of
5,000 cells per well and allowed to equilibrate overnight. The following day, BPZ derivatives
were applied in concentrations ranging from 1 to 100 µmol and cells were incubated for 24
This article is protected by copyright. All rights reserved.

hours. The XTT proliferation indicator was added the following day after washing cells and
measured per manufacturer instructions on a BioTek Cytation 3 microplate reader.
To assess the mechanism of cytotoxicity for the lead compounds (4 and 6), the
luminescence-based Caspase-Glo® 3/7 (Promega) assay was conducted in MCF-7 and A-172
cells. Briefly, as per the manufacturer’s protocol, cells were plated at a density of 5,000 cells
per well and allowed to equilibrate overnight followed by next day treatment with
compounds 4 and 6 at 2 or 3 times the calculated IC50 values for 12 hours. Camptothecin and
cisplatin served as positive controls for the Caspase-Glo® 3/7 assay and provided
statistically significant increases in caspase 3/7 activities (data not shown). Luminescence
signal as a result of caspase 3 and/or 7 activation was measured on a BioTek Cytation 3 plate
reader. Activation of caspase 3 and/or 7 induced by compounds 4 and 6 was expressed in
terms of fold change relative to vehicle-treated cells. Statistical significance was determined
using GraphPad Prism and a p-value <0.05 represented statistically significant elevation in
caspase 3/7 activity (compared to vehicle-treated cells) by the treated compounds.
3. Results and Discussion
Of the analogs tested in the XTT assay, those which appear to have the highest degree
of lipophilic character, specifically compounds 4 and 6, were clearly the most active at
inhibiting cell growth in both the A-172 cell line and the MCF-7 cell line. The concentration
response curves for all tested derivatives are shown in Figure 3. The IC50 values for all tested
compounds were calculated and are presented in Table 1 along with their structural
modification to the 4-position and the respective CLogP values. Substituents made to the 4-
position on the cyclohexane ring of BPZ which were polar or of less lipophilic significance
failed to induce any detectable cytotoxic effect or inhibition of growth. Molecules
which failed to inhibit growth or induce cell death did not induce proliferation either. We
This article is protected by copyright. All rights reserved.

speculate that the derivatives devoid of activity are excluded from the cells as a consequence
of their polarity. Enhanced lipophilic character may contribute to activity by
allowing diffusion through membranes followed by an intracellular event which culminates
in cell death. Previous studies surrounding bisphenol analogs parallels ours in that as the
lipophilicity increases within the bisphenol scaffold, cytotoxic effects increase. The cause of
this enhanced cytotoxic effect could point to additional lipophilic interaction with
intracellular receptors including the estrogen receptor as a consequence of the substituent
coming off the 4-position of the cyclohexane ring. It will be important to determine the nature
of the interaction with the estrogen receptors or other receptors of interest.
The observed cell death occurring in A-172 cells and MCF-7 cells is not occurring
through a non-specific membrane damaging effect. Trypan blue, a vital dye which is
excluded from cells with intact membranes, was used to probe membranes in A-172 and
MCF-7 cells after a 6-hour treatment with 4 and 6 at twice the calculated IC50 concentration.
Relative to acetone-permeabilized controls which took up and retained trypan blue, no
significant dye retention was detected in either cell line post-treatment (Fig. 4). Furthermore,
preliminary mechanistic studies revealed that the cytotoxicity observed with compound 4
involves the possible activation of caspase-3 and -7. As shown in Table 2, compound 4
treatment led to a statistically significant (9-fold) increase in executioner caspase activity in
A-172 cells and a mild yet statistically significant activation of executioner caspase in MCF-7
treated cells. It is possible that the functional deficit of caspase 3 in MCF-7 cells contributed
to a much lower signal in the luminescence assay relative to the signal generated in A-172
cells (Jänicke, Sprengart, Wati, & Porter, 1998). Interestingly, our preliminary data did not
reveal the activation of caspases-3 or -7 by compound 6. The lack of significant caspase-3 or
-7 activation by compound 6 in this assay does not completely rule out apoptosis as a
mechanism of cell death, but may point to an alternative pathway to apoptosis. Studies have
This article is protected by copyright. All rights reserved.

shown that the apoptotic cell death pathway can be achieved despite no net change or an
actual reduction in caspase-3 or -7 activation (Peng et al., 2009; Sagar et al., 2014). In lieu of
these previous reports we would like to investigate the possible involvement of alternate
pathways of cell death in compound 6 treated cells.
We have synthesized a small library of BPZ derivatives with variable lipophilic
character. Those with the highest degree of lipophilicity exerted an observable decline in cell
proliferation with IC50 values in the 30 µmol range while those bearing hydrophilic or minor
lipophilic character relative to BPZ were essentially devoid of activity. Additional derivatives
are being synthesized which present lipophilicity in a compact manner including
an adamantane derivative, planar derivatives including a naphthoyl and piperonyl derivative,
and a longer chain fatty acylated derivative using stearoyl chloride. This will help determine
if overall lipophilicity or the presentation of the lipophilic group governs activity. One
additional aspect to consider is cellular localization after treatment. To determine where BPZ
derivatives may accumulate post-treatment, we plan to synthesize a fluorescent derivative
using 1 and dansyl chloride to allow for fluorescence microscopy.
It is important to note limitations to our study. The synthetic library overall is
comparatively small yet represents a range of molecules bearing extremes in terms of
lipophilic character. The synthesis and testing of additional hydrophobic and hydrophilic
analogs, as suggested previously, will help to elucidate the overall impact of a bisphenol’s
lipophilic character on cytotoxic activity. We are in the process of identifying a mechanism to
account for the observed cytotoxicity. We have ruled out loss of membrane integrity as a
mechanism and are exploring numerous potential causes for cell death. Moving forward,
Western blotting for activated caspase enzymes may provide insight for determining a
possible mechanism to help explain the observed cell death after treatment with compound
This article is protected by copyright. All rights reserved.

4 or 6. It will also be important to investigate the role of poly-ADP ribose polymerase
(PARP) as a potential mechanism for cytotoxicity.
Figure Legends
Figure 1. Acid-catalyzed synthesis of BPA from phenol and acetone.
Figure 2. Chemical structure of bisphenol Z.
Figure 3. Concentration response curves of A-172 and MCF-7 cell lines following 24-hour
incubation with indicated compounds. Results are presented as percent of vehicle control
(DMSO). Data points are representative of mean SEM of three independent experiments
ran in quadruplicate.
Figure 4. Effect of compounds 4 and 6 on membrane integrity as measured by trypan blue
dye exclusion. Membranes were still intact following 6-hour incubation with compounds 4
and 6 in the A-172 and MCF-7 cells lines. Data points are representative of mean SEM of
three independent experiments ran in quadruplicate. Fifteen-minute incubation with ice-cold
acetone was used as a positive control. * denotes significance between DMSO and indicated
treatment group at p < 0.05 (One-Way ANOVA, Dunnett’s multiple comparison test).
Scheme 1. Synthesis of aliphatic and acylated bisphenol Z derivatives. (1a) A one-step
reaction was used as displayed to synthesize the aliphatic substituted BPZ derivates. (1b) A
two-step reaction was used as displayed to synthesize acylated BPZ derivatives via initial
synthesis of 4-amino bisphenol Z followed by fatty acid acylation.
Table 1. Bisphenol Z derivatives and respective IC50 values. Compounds 1 and 8 did not
display activity at 100 µM and were not evaluated using complete response curves. The IC50
values represents the mean SEM of three independent experiments ran in quadruplicate.
CLogP values were calculated using ChemDraw Ultra.
Table 2. Influence of compound 4 and 6 on caspase activation. Compound 4 and 6 were
applied to cells at 2 to 3 times the calculated IC50 value and incubated for 12 hours. Data
represent the fold increase of caspase activation relative to untreated control as determined by
This article is protected by copyright. All rights reserved.

the Caspase®Glo 3/7 assay. Compound 6 failed to influence caspase-3 or -7 activity in both
cell lines tested. The values represents the fold increase SEM of quadruplicate
treatments. Statistical significance was determined using one-way ANOVA
and Dunnett’s multiple comparison test. A p-value <0.05, denoted with an * represented
statistically significant elevation in caspase-3/7 activity compared to vehicle treated cells.
References
Bardon, S., Vignon, F., Derocq, D., & Rochefort, H. (1984). The antiproliferative effect of
tamoxifen in breast cancer cells: mediation by the estrogen receptor. Mol Cell
Endocrinol, 35(2-3), 89-96.
de Andrade, A. L. C., Soares, P. R. L., da Silva, S., da Silva, M. C. G., Santos, T. P., Cadena,
M. R. S., . . . Cadena, P. G. (2017). Evaluation of the toxic effect of endocrine
disruptor Bisphenol A (BPA) in the acute and chronic toxicity tests with Pomacea
lineata gastropod. Comp Biochem Physiol C Toxicol Pharmacol, 197, 1-7.
doi:10.1016/j.cbpc.2017.04.002
Dermer, O. C. (1983). Bisphenol-A, in J.J. McKelta (ed.). Encyclopedia of Chemical
Processing and Design 4(Marcel Dekker, New York), 406-430.
Fehlberg, S., Gregel, C. M., Goke, A., & Goke, R. (2003). Bisphenol A diglycidyl ether-
induced apoptosis involves Bax/Bid-dependent mitochondrial release of apoptosis-
inducing factor (AIF), cytochrome c and Smac/DIABLO. Br J Pharmacol, 139(3),
495-500. doi:10.1038/sj.bjp.0705275
Gassman, N. R. (2017). Induction of oxidative stress by bisphenol A and its pleiotropic
effects. Environ Mol Mutagen, 58(2), 60-71. doi:10.1002/em.22072
Gregor, R. W. (2012). Synthesis of Bisphenol Z: An Organic Chemistry Experiment. Journal
of Chemical Education, 89(5), 669-671. doi:10.1021/ed200293k
He, W., Liu, R., Yang, S. H., & Yuan, F. (2015). Chemotherapeutic effect of tamoxifen on
temozolomide-resistant gliomas. Anticancer Drugs, 26(3), 293-300.
doi:10.1097/CAD.0000000000000197
Izzotti, A., Longobardi, M., Cartiglia, C., D'Agostini, F., Kanitz, S., & De Flora, S. (2010).
Pharmacological modulation of genome and proteome alterations in mice treated with
the endocrine disruptor bisphenol A. Curr Cancer Drug Targets, 10(2), 147-154.
Jänicke, R. U., Sprengart, M. L., Wati, M. R., & Porter, A. G. (1998). Caspase-3 is required
for DNA fragmentation and morphological changes associated with apoptosis. J Biol
Chem, 273(16), 9357-9360.
Jenkins, S., Betancourt, A. M., Wang, J., & Lamartiniere, C. A. (2012). Endocrine-active
chemicals in mammary cancer causation and prevention. J Steroid Biochem Mol Biol,
129(3-5), 191-200. doi:10.1016/j.jsbmb.2011.06.003
Lamartiniere, C. A., Jenkins, S., Betancourt, A. M., Wang, J., & Russo, J. (2011). Exposure
to the Endocrine Disruptor Bisphenol A Alters Susceptibility for Mammary Cancer.
Horm Mol Biol Clin Investig, 5(2), 45-52. doi:10.1515/HMBCI.2010.075
Lee, H. S., Park, E. J., Oh, J. H., Moon, G., Hwang, M. S., Kim, S. Y., . . . Hong, J. H.
(2014). Bisphenol A exerts estrogenic effects by modulating CDK1/2 and p38 MAP
kinase activity. Biosci Biotechnol Biochem, 78(8), 1371-1375.
doi:10.1080/09168451.2014.921557
Matsumoto, H., Adachi, S., & Suzuki, Y. (2005). Bisphenol A in ambient air particulates
responsible for the proliferation of MCF-7 human breast cancer cells and Its
This article is protected by copyright. All rights reserved.

concentration changes over 6 months. Arch Environ Contam Toxicol, 48(4), 459-466.
doi:10.1007/s00244-003-0243-x
Mesnage, R., Phedonos, A., Arno, M., Balu, S., Corton, J. C., & Antoniou, M. N. (2017).
Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives
Activate Estrogen Receptor Alpha in Human Breast Cancer Cells. Toxicol Sci, 158(2),
431-443. doi:10.1093/toxsci/kfx101
Peng, K. W., Wang, H., Qin, Z., Wijewickrama, G. T., Lu, M., Wang, Z., . . . Thatcher, G. R.
(2009). Selective estrogen receptor modulator delivery of quinone warheads to DNA
triggering apoptosis in breast cancer cells. ACS Chem Biol, 4(12), 1039-1049.
doi:10.1021/cb9001848
Rochester, J. R., & Bolden, A. L. (2015). Bisphenol S and F: A Systematic Review and
Comparison of the Hormonal Activity of Bisphenol A Substitutes. Environ Health
Perspect, 123(7), 643-650. doi:10.1289/ehp.1408989
Sagar, S., Esau, L., Moosa, B., Khashab, N. M., Bajic, V. B., & Kaur, M. (2014).
Cytotoxicity and apoptosis induced by a plumbagin derivative in estrogen positive
MCF-7 breast cancer cells. Anticancer Agents Med Chem, 14(1), 170-180.
Suzuki, T., Ide, K., & Ishida, M. (2001). Response of MCF-7 human breast cancer cells to
some binary mixtures of oestrogenic compounds in-vitro. J Pharm Pharmacol,
53(11), 1549-1554.
Terasaka, H., Kadoma, Y., Sakagami, H., & Fujisawa, S. (2005). Cytotoxicity and apoptosis-
inducing activity of bisphenol A and hydroquinone in HL-60 cells. Anticancer Res,
25(3B), 2241-2247.
Washington, W., Hubert, L., Jones, D., & Gray, W. G. (2001). Bisphenol a binds to the low-
affinity estrogen binding site. In Vitr Mol Toxicol, 14(1), 43-51.
doi:10.1089/109793301316882531
This article is protected by copyright. All rights reserved.

Compound -R
CLogP
A-172
MCF-7
IC50 (µM)
IC50 (µM)
67 1.5
-
-
4.9
2.9
5.5
9.9
60 2.0
-
BPZ
-NH2
-CH3
1
2
4
70 2.0
27 0.9
62 1.6
53 2.8
6
48 1.8
35 1.1
47 5.9
60 1.9
33 1.7
112 3.6
5
6
7
6.4
4.7
-OH
2.9
-
-
8
Table 1. Bisphenol Z derivatives and respective IC50 values. Compounds 1 and 8 did not
display activity at 100 µM and were not evaluated using complete response curves. The IC50
values represents the mean SEM of three independent experiments ran in quadruplicate.
CLogP values were calculated using ChemDraw Ultra.
This article is protected by copyright. All rights reserved.

Treatment
A-172
MCF-7
9.34 1.54*
-
1.23 0.04*
-
4
6
Table 2. Influence of compound 4 and 6 on caspase activation. Compound 4 and 6 were
applied to cells at 2 to 3 times the calculated IC50 value and incubated for 12 hours. Data
represent the fold increase of caspase activation relative to untreated control as determined by
the Caspase®Glo 3/7 assay. Compound 6 failed to influence caspase-3 or -7 activity in both
cell lines tested. The values represents the fold increase SEM of quadruplicate treatments.
Statistical significance was determined using one-way ANOVA and Dunnett’s multiple
comparison test. A p-value <0.05, denoted with an * represented statistically significant
elevation in caspase-3/7 activity compared to vehicle treated cells.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.