Synthesis of fluorescent and photoaffinity-labeled derivatives of bisphenol A and their inhibitory activity toward hypoxic expression of erythropoietin

Article abstract of DOI:10.1016/j.bmcl.2007.07.007

Bisphenol A derivatives, possessing a fluorescent dye and a photo-reactive group, were synthesized from bisphenol A, and the inhibitory activity of the derivatives was evaluated against hypoxic response. The synthesized derivatives were found to inhibit t

Full text of DOI:10.1016/j.bmcl.2007.07.007

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5121–5124
Synthesis of fluorescent and photoaffinity-labeled derivatives
of bisphenol A and their inhibitory activity toward hypoxic
expression of erythropoietin
Nobuhiro Maezawa,a Hiroshi Tsuchikawa,a Shigeo Katsumura,a,*
Tomoko Kubob and Susumu Imaokab,*
aDepartment of Chemistry and Open Research Center on Organic Tool Molecules, School of Science and Technology,
Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan
bDepartment of Bioscience and Nanobiotechnology Research Center, School of Science and Technology,
Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan
Received 7 May 2007; revised 30 June 2007; accepted 4 July 2007
Available online 23 July 2007
Abstract—Bisphenol A derivatives, possessing a fluorescent dye and a photo-reactive group, were synthesized from bisphenol A, and
the inhibitory activity of the derivatives was evaluated against hypoxic response. The synthesized derivatives were found to inhibit
the hypoxic expression of erythropoietin in Hep3B cells as well as bisphenol A.
Ó 2007 Elsevier Ltd. All rights reserved.
Bisphenol A (BpA) is widely used as a major source of
polycarbonate and epoxy resins in industry and is well
known as one of the common environmental endocrine
disruptors. This molecule is a xenoestrogen and affects
the reproductive functions of animals.1 In addition, as
non-estrogenic effects, BpA was reported to induce
developmental abnormalities in the neuronal systems
of humans and animals.2 The use of BpA, however,
has not been restricted because of the lack of develop-
ment of its alternatives. Thus, the elucidation of its
mode of action and molecular mechanism is strongly
desired. It is also important to pursue the behavior of
BpA in the environment, cells, and organizations. Under
these situations, the methods for detection and measure-
ment of BpA have broadly been studied, and some
methods have been reported; for example, the use of a
specific monoclonal antibody against BpA3 and the
use of isotope-labeled BpA for detection.4 However,
the detection and the quantification of BpA are not nec-
essarily facile, because these methods often require a
special facility and instruments. In this paper, we
describe the synthesis and evaluation of BpA derivatives
possessing a fluorescent dye in order to detect BpA more
simply and efficiently. In addition, we synthesized a BpA
derivative possessing a photo-reacting group, namely a
photoaffinity group. Furthermore, we report the synthe-
sis of a valuable BpA derivative having both fluores-
cence and photoaffinity groups. These are expected to
be useful tool molecules to elucidate the BpA behavior
based on the evaluation of their inhibitory activity
toward the hypoxic expression of erythropoietin.
In the previous study, we found that BpA inhibited the
hypoxic expression of the erythropoietin (EPO) gene,
which is known as a hematopoietic cytokine. In the struc-
ture–activity relationship study of BpA, we reported that
the blocking of two phenol groups in BpA did not change
the inhibitory effect on the induction of EPO, but the
inhibition completely disappeared with the removal of
the two central methyl groups.5 Taking into consider-
ation these previous results, we designed and synthesized
three kinds of labeled compounds as shown in Figure 1,
which have labels at the terminal of a C4 or C2 chain as a
linker extended from the hydroxyl group of phenol. One
of these compounds contains the 4-nitrobenzo-2-oxa-
1,3-diazole (NBD) group as a fluorescence-labeled
group6 (1, 2), and the second one contains the 3-(4-alk-
oxyphenyl)-3-trifluoromethyldiazirine group (3), which
Keywords: Bisphenol A; Fluorescence; Photoaffinity-label; Synthesis;
Inhibition.
*
Corresponding authors. Tel.: +81 79 565 8314; fax: +81 79 565 9077
(S.K.); tel.: +81 79 565 7673; fax: +81 79 565 9077 (S.I.); e-mail
addresses: katsumura@ksc.kwansei.ac.jp; imaoka@ksc.kwansei.ac.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.007

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N. Maezawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5121–5124
group of the mono-MOM ether of BpA by the Mitsun-
obu reaction using diisopropylazodicarboxylate (DIAD)
with mono-protected butanediol,8 followed by the re-
moval of the TIPS group to produce alcohol 6 in 70%
yield. The terminal hydroxyl group was converted into
azide by reaction with diphenylphosphorylazide
(DPPA), and primary amine 7 was obtained by treat-
ment of the resulting azide with triphenylphosphine.
Introduction of the NBD group was achieved by a
nucleophilic substitution reaction of 7 with 7-chloro-4-
nitrobenzo-2-oxa-1,3-diazole (NBDCl) to provide com-
pound 2 in 52% yield over three steps.9 The subsequent
deprotection of the MOM group under acidic condition
gave fluorescence-labeled BpA 1 in 82% yield.10
Next is the preparation of photoaffinity-labeled BpA
derivative 3 (Scheme 2). The synthesis was commenced
with 3-(4-hydroxyphenyl)-3-trifluoromethyl diazirine 8,
which was prepared according to the reported proce-
dure.11 The hydroxy group of phenol 8 was directly con-
nected with BpA derivative 6 using our own method
utilizing the Mitsunobu reaction to quantitatively pro-
duce 3.12 Thus, we realized the synthesis of both fluores-
cence-labeled and photoaffinity-labeled BpA derivatives.
The third derivative of labeled BpA was both fluorescent
and photoaffinity-double-labeled BpA derivative 4. The
hydroxy group of phenol 8 was reacted with mono-pro-
tected butanediol by our method utilizing the Mitsun-
obu reaction, followed by treatment with TBAF to
give the corresponding alcohol 9 in good yield. In this
case, a C4 chain was also adopted as a linker part.
Unfortunately, the direct coupling between photo-
Figure 1. The structure of BpA and the derivatives.
can produce the corresponding carbene accompanied by
liberating nitrogen by photoirradiation at 360 nm and
can form a covalent bond with the neighboring func-
tion.7 The third one contains both fluorescent and photo-
affinity groups (4, 5). The length of the linker was
considered to avoid the interaction with the central
methyl groups of BpA.
The synthesis was started from BpA as illustrated in
Scheme 1. The C4 linker was introduced to the hydroxyl
Scheme 1. Reagents and conditions: (a) MOMCl, NaH, DMF, rt, 1 h,
43%; (b) TIPSO(CH2)4OH, PPh3, DIAD, THF, 3 h; (c) TBAF, THF,
rt, 3 h, 70% (two steps); (d) (PhO)2P(O)N3, PPh3, DIAD, THF, 3 h; (e)
PPh3, H2O, 60 °C, 3 h; (f) NBDCl, Et3N, rt, 30 min, 52% (three steps);
(g) 2 M HCl, AcOH, rt, 3 days, 82%.
Scheme 2. Reagents and conditions: (a) TIPSO (CH2)4OH, PPh3,
DIAD, THF, 1.5 h; (b) TBAF, THF, rt, 3 h; (c) TsCl, Et3N, DMAP,
CH2Cl2, rt, 2 h, 69% (three steps); (d) 1, NaH, DMF, rt, 5 min, 64%.

N. Maezawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5121–5124
5123
affinity-labeled 9 and fluorescence-labeled 1 under the
Mitsunobu conditions did not proceed,12 and both com-
pounds were recovered. The primary hydroxy group was
then converted into the corresponding tosylate, which
reacted with the sodium phenoxide of 1 to produce the
desired 4 in 44% yield from 8.13 Thus, we succeeded in
the preparation of double-labeled BpA derivative 4.
We also synthesized another double-labeled BpA deriv-
ative 5 which contained a C4 chain as the linker part by
the same method.14
We then evaluated the biological activities of
fluorescence-labeled derivative 1, MOM ether 2, photo-
affinity-labeled derivative 3, and fluorescence-photo-
affinity-double-labeled derivatives
4 and 5. After
addition of BpA or these derivatives to Hep3B cells
under hypoxia, respectively, we measured the expression
of EPO by RT-PCR (Figs. 2 and 3).15 EPO was strongly
induced under hypoxia, and the addition of BpA com-
pletely suppressed EPO induction.5 By addition of com-
pound 1, the expected inhibition effect on EPO
expression was observed. Interestingly, the MOM ether
2 showed stronger inhibitory activity than 1, and the
magnitude of the suppression was comparable to that
of BpA. Moreover, the double-labeled compound 4 also
revealed the inhibitory activity almost equal to that of
BpA, and compound 5 showed less inhibitory activity
than compound 4 because compound 5 needed higher
concentration (300 lM) than compound 4 (200 lM) to
reveal the similar inhibitory effect. On the other hand,
photoaffinity-labeled compound 3 did not show the
inhibitory activity. These results strongly suggest that
Figure 3. Inhibition of EPO induction under hypoxia by BpA and
BpA derivatives (4 and 5). The conditions are same in the legend of
Figure 2 except the concentration of the derivative 5 (300 lM). Nor,
normoxia; Hyp, hypoxia; Hyp + BpA, addition of bisphenol A under
hypoxia; Hyp + 5, addition of derivative 5 under hypoxia; and
Hyp + 4, addition of derivative 4 under hypoxia.
the synthesized BpA derivatives 2 and 4 would be recog-
nized in cells as well as BpA. Thus, they are considered to
be simpler and more practical tool molecules for the
detection of BpA. Furthermore, they are expected to
identify the target molecule of BpA by photoaffinity
labeling and to disclose the behavior and distribution
of BpA in cells.
In conclusion, we synthesized the BpA derivatives which
possess a fluorescent dye and a photo-reactive group. To
the best of our knowledge, these molecules are the first
examples which possess fluorescence and affinity labels
in the BpA molecule. Furthermore, we observed that
these derivatives (1, 2, 4, and 5) inhibited the hypoxic
expression of EPO in Hep3B cells as well as BpA, and
hence they are expected to be powerful tool molecules
to elucidate BpA behavior.
Acknowledgments
This work was partially supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan. This study
was also supported by a Matching Fund Subsidy for a
Private University.
Figure 2. Inhibition of EPO induction under hypoxia by BpA and
BpA derivatives (1–3). Hep3B cells were cultured for 6 h under
hypoxia in the presence of BpA (200 lM), the derivative 1 (200 lM),
derivative 2 (200 lM), and derivative 3 (200 lM). These chemicals
were dissolved in DMSO. Expression of the EPO gene was detected by
RT-PCR (reverse transcription-polymerase chain reaction). b-Actin
was used as a control. Bands of amplified DNA fragments on agarose
gel were quantified by NIH Image. Values in the graph are means of
two different samples and are expressed as ratios of EPO mRNA and
b-actin mRNA. Control value is set at 1.0. Nor, normoxia; Hyp,
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Hyp + 3, addition of derivative 3 under hypoxia; Hyp + 1, addition
of derivative 1 under hypoxia; and Hyp + 2, addition of derivative 2
under hypoxia.

5124
N. Maezawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5121–5124
calcd for C37H37F3N6O6 [M+Na]+ 741.2624, found
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14. Data for 5: 1H NMR (CDCl3, 400 MHz) d 8.51 (d,
J = 8.8 Hz, 1H), 7.16 (AB-q, J = 8.5 Hz, 2H), 7.15 (AB-q,
J = 9.0 Hz, 2H), 7.13 (AB-q, J = 8.5 Hz, 2H), 6.83 (AB-q,
J = 8.8 Hz, 2H), 6.83 (AB-q, J = 8.5 Hz, 2H), 6.82 (AB-q,
J = 8.5 Hz, 2H), 6.55 (br s, 1H), 6.27 (d, J = 8.8 Hz, 1H),
4.28–4.31 (m, 6H), 3.89 (dt, J = 4.9, 5.0 Hz, 2H), 1.63 (s,
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J = 8.8 Hz, 2H), 6.44 (br s, 1H), 6.19 (d, J = 8.4 Hz, 1H),
5.15 (s, 2H), 4.64 (t, J = 5.6 Hz, 2H), 3.60 (t, J = 6.3 Hz,
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15. The detailed experimental conditions are as follows:
Hep3B cells were cultured in DMEM containing 10%
FCS, and the FCS concentration was reduced to 0.1% at
24 h before the treatment with chemicals. For hypoxic
treatment, the cells were incubated in 5% O2, 5% CO2, and
90% N2 balanced with a modulator incubator chamber
(Napco 7101, Winchester, VA) or were incubated in a
sealed 2.5-L box with an Anero Pack for cells. Hep3B cells
were incubated for 6 h under hypoxia in the presence of
BpA and derivatives (200–300 lM). Total RNA was
extracted from Hep3B cells and a reaction mixture
containing 1 lg of RNA and 200 U of reverse transcrip-
tase was reacted according to the condition as follows:
incubation for 10 min at 25 °C and 60 min at 42 °C,
followed by heating for 10 min at 70 °C to stop the
reaction. Polymerase chain reaction (PCR) was performed
using a reaction mixture containing 10 pmol of each
primer, 1.5 U of Ampli Taq, and 100 ng of cDNA
according to the following protocol: 10 min at 96 °C and
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72 °C. Primers for b-actin were 50-CAAGAGATGGCC
ACGGCTGCT-30 (sense) and 50-TCCTTCTGCATCC
TGTCGGCA-30 (antisense). Primers for EPO were 50-G
CCAGAGGAACTGTCCAGAG-30 (sense) and 50-TTC
TCCAGGTCATCCTGTCC-30 (antisense). The cycle
number is within the linear range of amplification. Bands
of amplified DNA fragments on agarose gel were quan-
tified by NIH Image.
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J = 8.4 Hz, 1H), 7.15 (AB-q, J = 7.4 Hz, 2H), 7.09 (AB-q,
J = 8.8 Hz, 2H), 6.82 (AB-q, J = 8.8 Hz, 2H), 6.73 (AB-q,
J = 8.5 Hz, 2H), 6.47 (br s, 1H), 6.18 (d, J = 8.4 Hz, 1H),
4.04 (t, J = 5.6 Hz, 2H), 3.51–3.60 (m, 2H), 3.47 (s, 3H),
1.94–2.07 (m, 2H), 1.63 (s, 6H); ESI HRMS m/z calcd for
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J = 8.5 Hz, 1H), 7.16 (AB-q, J = 9.0 Hz, 2H), 7.12 (AB-q,
J = 9.0 Hz, 4H), 6.88 (AB-q, J = 8.5 Hz, 2H), 6.82 (AB-q,
J = 8.5 Hz, 2H), 6.78 (AB-q, J = 8.5 Hz, 2H), 6.19 (br s,
1H), 6.18 (d, J = 8.5 Hz, 1H), 3.98–4.06 (m, 6H), 3.57–3.62
(m, 2H), 1.93–2.04 (m, 8H), 1.64 (s, 6H); ESI HRMS m/z