Bisphenolic compounds that enhance cell cation transport are found in commercial phenol red

Article abstract of DOI:10.1021/jm960300k

We have isolated two bisphenolic compounds (4 and 5) that have a marked effect on K⁺ and Na⁺ concentrations in human cells from commercial preparations of the pH indicator dye phenol red (phenolsulfonphthalein). We used a bioassay to

Full text of DOI:10.1021/jm960300k

J . Med. Chem. 1996, 39, 4897-4904
4897
Bisp h en olic Com p ou n d s Th a t En h a n ce Cell Ca tion Tr a n sp or t Ar e F ou n d in
Com m er cia l P h en ol Red
Philip R. Kym,† Kim L. Hummert,† Anna G. Nilsson,† Martin Lubin,‡ and J ohn A. Katzenellenbogen*,†
Department of Chemistry, University of Illinois, Urbana, Illinois 61801, and Department of Microbiology,
Dartmouth Medical School, Hanover, New Hampshire 03755
Received April 22, 1996X
We have isolated two bisphenolic compounds (4 and 5) that have a marked effect on K+ and
Na+ concentrations in human cells from commercial preparations of the pH indicator dye phenol
red (phenolsulfonphthalein). We used a bioassay to identify active chromatographic fractions
from the lipophilic impurities present in phenol red, and we determined the structure of two
active components (4 and 5) by 1H and 13C NMR and mass spectrometry. When added to human
fibroblasts in serum-free medium, the bisphenol fluorene derivative 9,9-bis(4′-hydroxyphenyl)-
3-hydroxyfluorene (5) produced a rapid loss of K+ and a gain of Na+, at low concentrations,
with an EC50 between 30 and 60 ng/mL (80-160 nM). The 2- and 4-hydroxy isomers of the
fluorene 5 (i.e., compounds 6 and 7), prepared by synthesis, had similar activity, although
compound 6 was somewhat less potent. The bisphenol xanthene derivative 9,9-bis(4′-
hydroxyphenyl)xanthene (4) elicited a similar biological response but was less potent than 5-7;
it also had a strong effect on cell adhesion, causing release of cells from the plastic substrate
at concentrations as low as 2-5 µg/mL (5.5-14 µΜ). The structures of xanthene (4) and fluorene
(5) bisphenols have been confirmed by synthesis from xanthone and hydroxyfluorenone,
respectively, by Friedel-Crafts alkylation with phenol. In the latter case, the desired
3-hydroxyfluorene isomer was formed in situ by rearrangement of the 1-hydroxy isomer.
In tr od u ction
Phenol red (phenolsulfonphthalein, 1) has been used
for almost a century to evaluate many biological proc-
esses, including measurement of renal tubular function,1
estimation of kidney blood flow,2 and measurement of
lipid and hydrogen peroxide formation.3 Its most far-
reaching and common use, however, is as a pH indicator
in tissue culture media. Commercially, phenol red is
prepared in one step by the acid-catalyzed Friedel-
Crafts reaction of phenol with 2-sulfobenzoic acid,
2-sulfobenzoic acid cyclic anhydride, or saccharin at
elevated temperatures.4 Excess phenol functions as
solvent, and yields are good. Most commercial prepara-
tions of phenol red are sold at a purity of 90% or 95%
“dye content”, with the remaining 5-10% of material
consisting of lipophilic impurities.
F igu r e 1. Structures of phenol red (1), estrogenic lipophilic
impurity 2, and oxidized estrogenic impurity 3.
Some time ago, commercial preparations of phenol red
were shown to have weak estrogenic activity in several
estrogen-dependent human breast cancer-derived cell
lines (MCF-7,5 T47D,6 ZR-75-17). Although the estro-
genic activity observed with phenol red preparations
was initially attributed to the phenolsulfonphthalein
dye itself,5-7 later studies showed it to be due to a
lipophilic impurity present in the phenol red prepara-
tion.8 This impurity was isolated and characterized as
the phenyl sulfonate ester of the reduced form of phenol
red (2) which is found together with its oxidized
congener (3) (Figure 1).9 Phenol red preparations have
also been identified as the source of cytotoxicity in
MCF-7 breast cancer cells under alkaline (pH > 7.4)
conditions,10 as an inhibitor of a thromboxane A2/
prostaglandin H2 receptor agonist,11 and as an inhibitor
of renal epithelial cell growth.12
Recently, one of us (M. Lubin) found that impurities
in samples of several commercial preparations of phenol
red caused rapid loss of cell K+ and gain of Na+ (with a
reversal of the normally high K+/Na+ ratio) in human
MCF-7, EJ bladder carcinoma, and fibroblast (HF) cells,
when cells were incubated in serum-free medium.13 This
action on ion transport was effectively inhibited by
albumin or serum, which presumably absorbed most of
the impurities. Moreover, an ether extract of phenol
red was active in the bioassay, but the residual ex-
tracted phenol red was not. The ion channel blockers
quinine and verapamil partially inhibited the action of
the impurities. Interestingly, the effects observed with
phenol red in human MCF-7, EJ , and HF cells were not
apparent in the rodent NIH-3T3 or CHO-K1 cell lines.
* Address correspondence to: Prof. J ohn Katzenellenbogen, Depart-
ment of Chemistry, University of Illinois, Box 37 Roger Adams
Laboratory, 600 S. Mathews Ave., Urbana, IL 61801. E-mail:
jkatzene@uiuc.edu.
† University of Illinois.
‡ Dartmouth Medical School.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
S0022-2623(96)00300-7 CCC: $12.00 © 1996 American Chemical Society

4898 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Kym et al.
Hopp and Bunker have also recently reported that
lipophilic impurities in phenol red cause a reversal of
cell K+/Na+ ratio in human fibroblasts and COS-7 and
Hs68 cell lines.14 A change in the intracellular K+ and
Na+ of the magnitude observed by Lubin, and by Hopp
and Bunker, would be expected to have an effect on
multiple cellular functions. Furthermore, the observa-
tion that crude phenol red preparations were active
suggested that the responsible components may be very
potent, since it is likely that these components make
up less than 1% of the total material present.
In this report, we describe the isolation and structure
determination of compounds in the lipophilic extract of
commercial phenol red that display ion transport activ-
ity or toxicity. We used reversed-phase HPLC to
fractionate the impurities, and guided by a bioassay, we
were able to identify those fractions with maximum
activity and isolate two active substances. We deter-
mined their structures by spectroscopy, confirmed them
by synthesis, and tested them, and certain analogs, in
cell culture assays.
Resu lts a n d Discu ssion
Isola tion a n d Id en tifica tion of Com p on en ts Th a t
Ca u se Loss of Cell K+. Our strategy for identifying
components of phenol red preparations that possess
biological activity involved four steps: (1) solvent par-
titioning of the lipophilic impurities of phenol red, (2)
reversed-phase HPLC analysis of the lipophilic impuri-
ties, (3) systematic bioassay of crude fractions to identify
active components, and (4) final purification and iden-
tification of active components.
Extraction of a 5.42 g portion of phenol red mono-
sodium salt (Aldrich) with diethyl ether (3 × 50 mL)
provided 0.127 g (2.4%) of a reddish brown oil. Analyti-
cal reversed-phase HPLC of this material revealed the
presence of about 20 different lipophilic components;
most of the mass (about 90%) was present in six major
peaks (Figure 2, panel A). The eluent containing the
major lipophilic impurity (tR ) 11 min) was deep red;
this component had previously been identified as the
oxidized estrogenic impurity 3.9 The estrogenic im-
purity 2 had previously been identified as the minor
peak at tR ) 13 min.8 The identity of these peaks was
confirmed by coinjection of authentic samples.
We initially tested the effect of the crude lipophilic
impurities on intracellular K+ and Na+ in HF cells,
using the procedure previously described.13 We found
that incubation of the crude material from the ether
extract changed the intracellular ratio of K+/Na+ from
7.4 to 0.68 (Table 1) (typical ratios observed in healthy
cells range from about 3.5 to 10).13 Encouraged by this
preliminary result, we proceeded with preparative
reversed-phase HPLC fractionation of the crude ether
extract. Since the resolution of the preparative scale
separation was poorer than the analytical scale separa-
tion (cf., Figures 2 and 3), we collected only 10 fractions
(fractions I-X, Figure 3) and assayed them for their
effect on the K+ and Na+ content of HF cells.
F igu r e 2. Panel A: Reversed-phase analytical HPLC of the
ether extract of phenol red sodium salt (C-18 silica gel column;
1 mL/min eluting with isocratic 50:50 water:acetonitrile; UV
absorbance monitored at 254 nm). Panel B: Alignment of
characterized, pure components from the ether extract of
phenol red with the HPLC trace from the crude lipophilic
impurities; the starred peaks in panel A correspond to the pure
components in panel B. HPLC conditions were identical with
those described for panel A.
may have been present in these tail fractions. Fraction
VII also demonstrated weak activity in the bioassay, as
the observed ratio of K+/Na+ was 1.7. The more
pronounced effect of fraction VII, however, was its
apparent cytotoxicity. After 1 h of incubation with
fraction VII, most of the fibroblasts were partly re-
tracted, and a few of the cells had detached themselves
from the culture dish. Since the effect of fraction VII
on ion content was less than that of fraction II, its toxic
effect on adhesion is probably not due to its effect on
ion distribution.
Reevaluation of fraction VII by analytical reversed-
phase HPLC revealed only two components, present in
a ratio of about 9:1. We were able to isolate 4.4 mg of
the major component in pure form by silica gel flash
chromatography (50% hexanes/EtOAc). Injection of
fraction II onto the analytical column also revealed only
one major component. We repeated the ether extraction
on 25 g of the same lot of phenol red (Aldrich) and
isolated 0.418 g of lipophilic impurities. Initial purifica-
tion of the ether extract was accomplished by normal
phase silica gel chromatography (gradient elution from
80% hexanes in EtOAc to 100% EtOAc). A portion of
the fraction that contained the components of fraction
We found that two of the collected fractions possessed
significant biological activity (Table 1). Fraction II
caused a dramatic effect on the K+/Na+ ratio, dropping
it to 0.30, almost completely reversing the value typi-
cally observed in healthy cells. Fractions III and IV
showed a weak effect, possibly attributable to small
amounts of the potent component from fraction II that

Bisphenolic Compounds in Commercial Phenol Red
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4899
ion at m/ z 366, which at high resolution gave a mass
at 366.1256, calcd 366.1256, indicating a composition
of C25H18O3. The most prominent fragment was 273
(M+ - 93), indicative of loss of a phenol group.
The 500 MHz J -correlated 2D 1H NMR spectrum
established the presence of a 1,4-disubstituted aromatic
four-spin (AA′XX′) system (two doublets at δ 6.73 and
6.58, J ) 8.8 Hz, equivalent to four protons each). The
remaining eight protons were nested in a 1,2-disubsti-
tuted aromatic four-spin (ABCD) system, indicated by
the two sets of doublet of doublet of doublets (δ 7.21,
6.99) and two sets of doublet of doublets (δ 7.07, 6.93),
each set equivalent to four protons. The only other
signal was a singlet (equivalent to two protons) at δ
8.22.
The simplicity of the 1H NMR spectrum (a 1,4-
disubstituted four-spin system, a 1,2-disubstituted four-
spin system, and a singlet), coupled with the elemental
composition (C18H25O3) available from high-resolution
mass spectrometry, suggested that this compound pos-
sesses a plane of symmetry. This symmetry was further
supported by the 13C NMR spectrum, which revealed
the presence of only 11 unique carbon centers.
F igu r e 3. Crude partitioning of the lipophilic impurities of
phenol red by preparative reversed-phase HPLC (C-18 silica
gel column; 3 mL/min with water:acetonitrile gradient starting
from 50:50 (first 20 min), increasing to 20:80 over 10 min, and
maintaining 20:80 for 20 min; UV absorbance monitored at
254 nm).
The 1H-13C chemical shift correlation spectrum
showed the presence of five quaternary carbons and six
carbon centers that correlated with proton resonances.
The five quaternary carbons are assigned following
Ta ble 1. Effect of Crude HPLC Fractions from Lipophilic
Impurities in Phenol Red on Ion Content of HF Cellsa
additive
control
purified phenol red
crude impurities
K+
Na+
K+/Na+
literature precedent15 and comparison with the 13C
35
34
17
35
10
28
30
38
27
35
36
35
4.7
4.4
25
4.9
33
7.4
7.7
0.68
7.1
0.30
2.0
3.0
7.6
1.7
8.0
8.8
6.6
13
NMR spectrum for 5, the two hydroxy-substituted
C
4′
carbons being the most downfield at δ 156.2 followed
by the oxygen-substituted C4a carbons in the xanthene
ring system at δ 152.6, the C1′ carbons that are para to
hydroxyl substitution at δ 136.7, the remaining two
quaternary C1a carbons of the xanthene system at δ
131.3, and finally the only aliphatic carbon (δ 53.0, C9)
at the tetrahedral center of the molecule.
HPLC Fr I
II
III
IV
V
VII
VIII
IX
X
14
10
5.0
16
4.4
4.1
5.3
The combination of the J -correlated 2D 1H NMR
a
1
spectrum and the H-13C-correlated spectrum enabled
Each of the nine fractions displayed in Figure 3 was dried,
taken up in equal volumes of ethanol, and then added to DMEM
without phenol red or serum and applied to cell cultures. Final
solvent concentration was 0.1%. Purified phenol red was at 15
µg/mL, and crude impurities were at 2.7 µg/mL. Final concentra-
tions of each HPLC fraction, in µg/mL: I, 4.5; II, 6.3; III, 7.5; IV,
10.1; V, 4.2; VII, 24.3; VIII, 3.5; IX, 0.4; X, 0.4. After 45 min at 37
°C in CO2/air, cells were processed for analysis. Values shown
are 108 × mol of K+ or Na+ per dish, assayed as described
previously.13
us to make definitive assignments for the remaining
protons and carbons. The C3′ carbons in the 1,4-
disubstituted aromatic ring systems are the most upfield
aromatic resonances at δ 114.3, and the protons at-
tached to these carbons (H3′, δ 6.58, ortho to the
hydroxyl) are the most upfield-shifted proton signals.
These protons are coupled to the protons meta to the
hydroxyl groups (H2′, δ 6.73), which are attached to the
C2′ carbons at δ 130.9. In the 1,2-disubstituted aromatic
ring system, the most upfield doublet is assigned as
protons H1, δ 6.93, which are attached to the C1 carbons
(δ 130.1). These protons are most strongly correlated
with the doublet of doublets at δ 6.99 (H2), which are
attached to the C2 carbons (δ 122.5). These protons are
coupled to the most downfield protons in the 1,2-
disubstituted system (H3, δ 7.21, meta to the oxygen
substitution), which are correlated with the C3 carbons
(δ 127.4). The final correlation in this four-spin system
is between H3 (δ 7.21) and H4 (δ 7.07); the H4 protons
are correlated with the C4 carbons (δ 116.0).
II was subjected to preparative reversed-phase HPLC
(50% CH3CN:H2O, isocratic). From these chromato-
graphic runs, 2.4 mg of the major component of fraction
II was isolated.
The pure major components of fractions II and VII
were reevaluated by bioassay for K+ and Na+. We found
that these components produced a more pronounced
effect on K+/Na+ ratios than had been previously
observed for the crude fractions; therefore, we were
confident that they were the principal source of the
biological activity. The traces from pure samples of
fractions II and VII, as well as pure samples of the
estrogenic compound 2 and its oxidized form 3, are
aligned with the peaks to which they correspond in the
analytical reversed-phase HPLC trace of the crude
lipophilic impurities of phenol red (Figure 2).
The corroboration of NMR spectroscopy and mass
spectrometry led us to assign the structure of the major
component of fraction VII as 9,9-bis(4′-hydroxyphenyl)-
xanthene (4). The final proof of this structure was
provided by the synthesis of 4 from xanthone and phenol
(vide infra).
Str u ctu r e Elu cid a tion of th e Ma jor Com p on en t
in F r a ction VII. Electron-impact (EI, 70 eV) and field-
desorption (17 µA) mass spectrometry of the purified
material from fraction VII gave an intense molecular
Str u ctu r e Elu cid a tion of th e Ma jor Com p on en t
of F r a ction II. Electron-impact (70 eV) and field-

4900 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Kym et al.
Sch em e 1. Synthesis of Bisphenols 4-7
desorption (17 µA) mass spectrometry of the purified
material from fraction II gave an intense molecular ion
at m/ z 366, which at high resolution gave a mass at
366.1254, calcd 366.1256, indicating a composition of
C25H18O3. This material was identical in mass and
composition with the previously identified fraction VII.
Prominent fragments included m/ z 348 (M+ - 18) and
273 (M+ - 93), indicating the possible loss of a hydroxyl
group and a phenol group, respectively.
The 500 MHz J -correlated 2D 1H NMR spectrum
established the presence of a 1,4-disubstituted aromatic
four-spin (AA′XX′) system (two doublets at δ 6.95 and
6.53, J ) 8.7 Hz, four protons each). One 1,2-disubsti-
tuted aromatic four-spin (ABCD) system equivalent to
four protons is indicated by the two sets of doublets (δ
7.65, 7.31) and the two sets of doublet of doublets (δ
7.24, 7.16). The only remaining correlated spin system
is characteristic of a 1,2,4-trisubstituted aromatic spin
system indicated by two doublets (δ 7.12, 7.10) and a
doublet of doublets (δ 6.62). The two singlets at δ 8.28
and 8.06 correspond to the hydroxyl protons OH3 and
OH4′, respectively.
The 1H-13C chemical shift correlation spectrum
showed the presence of eight quaternary carbons and
nine carbon centers correlated with proton resonances.
The quaternary carbons were tentatively assigned fol-
lowing literature precedent15 and by comparison with
the 13C NMR spectrum for 4. The hydroxyl-substituted
13C3 carbon is the most downfield signal at δ 158.1
followed by the two hydroxyl-substituted C4′ centers at
δ 157.0 and the C8a carbon of the fluorene system at δ
154.0. The other quaternary carbons of the fluorene
system (C1a, C4a, and C5a) appear at δ 143.9, 141.9, and
141.0, respectively. The C1′ carbons para to the hy-
droxyl substituent in the bisphenol system show up at
δ 138.0, while the aliphatic carbon at the tetrahedral
center of the molecule appears at δ 64.2.
proton (H4, δ 7.12) which correlates to C4 at δ 106.9.
This proton experiences both the shielding effect of the
ortho hydroxyl group and the deshielding effect of the
edge-on interaction with the second aromatic ring of the
fluorene ring system; these opposing electronic influ-
ences effectively cancel each other out.
The combination of the J -correlated 2D 1H NMR
1
spectrum and the H-13C correlated spectrum enabled
us to make definitive assignments for the remaining
protons and carbons. The H3′ protons in the 1,4-
disubstituted aromatic ring system are the most upfield
proton resonances at δ 6.53 and are attached to the C3′
carbons (δ 115.2). These protons are coupled to the
protons meta to the hydroxyl group (H2′, δ 6.95), which
are attached to the C2′ carbons at δ 129.6. In the 1,2-
disubstituted aromatic ring system, the most downfield
doublet is assigned as proton H5 (δ 7.65), which is
attached to the C5 carbon (δ 120.3). This proton is ortho
coupled to the doublet of doublets centered at δ 7.24
(H6), which correlates to the C6 carbon (δ 127.4). The
doublet at δ 7.31 is assigned to the proton H8, ortho to
the quaternary center, which is attached to the C8
carbon at δ 126.7. This proton is coupled to the proton
resonance H7 (δ 7.16) which is attached to the C7 carbon
at δ 127.7. The protons that are meta (H7) and para
(H6) to the quaternary center are also coupled. In the
1,2,4-trisubstituted aromatic ring system, the protons
that are ortho (H1, δ 7.10) and meta (H2, δ 6.62) to the
quaternary center are ortho coupled to each other. The
proton that is meta to the quaternary center (H2) is
shifted upfield by the resonance-donating effect of the
ortho hydroxyl group at C3 of the fluorene ring system.
The H1 proton correlates to the C1 carbon at δ 127.2
and the H2 proton is attached to the C2 carbon at δ
115.4. The H2 proton is meta coupled to the remaining
From the combined data from NMR spectroscopy and
mass spectrometry, we assign the major component of
fraction II from the HPLC separation as 9,9-bis(4′-
hydroxyphenyl)-3-hydroxyfluorene (5). Final proof of
structure for this compound was provided by synthesis
from 3-hydroxyfluorenone and phenol (vide infra).
Syn th esis of Bisp h en ols 4 a n d 5. The bisphenol
xanthene derivative 4 was available in one step from
commercially available phenol and xanthone (8) (Scheme
1). Lewis acid-catalyzed phenol addition to the xan-
thone carbonyl was effected by stirring xanthone (8)
neat in an excess of phenol with 3 equiv of aluminum
chloride. After 4 h at 140 °C, no products that coeluted
with the major component of fraction VII isolated by
HPLC methods from phenol red had been formed. The
reaction mixture was allowed to stir for an additional
12 h at 180 °C, at which point a new spot that coeluted
with the major component of fraction VII was observed.
The bis-para-substituted phenol derivative 4 was the
major product isolated (30%) from the reaction mixture.
We suspect that the products resulting from alkylation
ortho to the hydroxyl group are also formed under these
forcing conditions. The synthetic material 4 was found
to be identical with the material isolated from phenol
red, by
1H NMR, mass spectrometry, and HPLC com-
parisons. Bisphenol 4 has been reported once previously
in the literature;16 it was used as the repeating unit of

Bisphenolic Compounds in Commercial Phenol Red
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4901
Sch em e 2. Synthesis of 3-Hydroxyfluorenone (15)
novel polycarbonate and polyester polymers with large
cross-planar substituents.
From our spectroscopic work, we suspected that the
biologically active material in fraction II was a bis(4-
substituted phenol) adduct of a hydroxyfluorenone
derivative. The J -correlated 2D 1H NMR spectrum
suggested a 1,2,4-trisubstituted ring system, thus re-
quiring attachment of the hydroxyl group at either the
C2 or C3 carbons of the fluorene ring system. The
position of the hydroxyl group could be unambiguously
defined by comparison with authentic bisphenol deriva-
tives of 3-hydroxyfluorenone (5) and 2-hydroxyfluo-
renone (6). Our first synthetic target was 6 because
2-hydroxyfluorenone (9) is commercially available.
Stirring 2-hydroxyfluorenone (9) in excess phenol and
aluminum chloride (3 equiv) for 2 h at 80 °C provided
the bisphenol 6 in high yield (89%) (Scheme 1). Spec-
troscopic analysis of this compound (1H and 13C NMR)
showed it to be different from the material isolated from
phenol red. The most notable difference in the 1H NMR
was the presence of two upfield-shifted signals (protons
ortho to hydroxy substitution) and one downfield-shifted
signal (proton in the deshielding region of the second
aromatic ring of the fluorenone ring system) in the 1,2,4-
trisubstituted aromatic ring system. Although bis-
phenol 6 was not the same compound as isolated in
fraction II from phenol red, it was very similar, and it
was evaluated in the bioassay and for relative binding
affinity for the estrogen receptor.
Ta ble 2. Effect of Various Concentrations of Compounds 4-7
on Ion Content of HF Cellsa
expt no.
I
additive
none
5
5
5
5
conctn
K+
30
Na+ K+/Na+
0 ng/mL
8.0
8.0
3.8
3.8
15.6 ng/mL 30
31.0 ng/mL 27
62.5 ng/mL 10
125 ng/mL
125 ng/mL
12
29
5.2 38
2.3
0.34
0.14
0.91
1.3
4.1
1.8
5 + quinine
5 + verapamil 125 ng/mL
20
22
16
10
19
27
20
41
34
24
43
II
none
4
4
none
4
4 + quinine
0 µg/mL
2.4 µg/mL
4.8 µg/mL
0 µg/mL
0.89
5.7
III
7.6
4.8 µg/mL
4.8 µg/mL
2.5 29
27
0.09
2.3
3.4
In this series, we also prepared the bisphenol deriva-
tive (7) of 4-hydroxyfluorenone (10) (Scheme 1). Stirring
commercially available 4-hydroxyfluorenone (10) with
an excess of phenol and aluminum chloride (3.0 equiv)
at 80 °C for 2 h resulted in facile formation of the
bisphenol derivative 7 (85%). As expected, this material
was also different from the major component in fraction
II from phenol red. Our attempt at forming the bis-
phenol derivative of 1-hydroxyfluorenone (11), however,
led to a surprising finding (Scheme 1). The starting
material, 1-hydroxyfluorenone (11), remained unreacted
at 80 and 120 °C. Increasing the temperature to 160
°C resulted in formation of a bisphenol product. Isola-
tion of this compound revealed it to be a mixture of two
isomers (about 80:20), of which the major isomer was
identical with the active component (5) in fraction II of
phenol red (1H NMR, HPLC). Thus, under the reaction
conditions, it appears that the 1-hydroxyfluorene system
has rearranged to the 3-hydroxy system.
12
11
4 + verapamil 4.8 µg/mL
none
6
6
6
6
6 + verapamil 3.0 µg/mL
none
7
7
7
37
29
IV
0 µg/mL
0.03 µg/mL 28
0.1 µg/mL
0.3 µg/mL
3.0 µg/mL
4.5
3.5
8.7 24
3.8 35
5.5 39
15 21
4.5
7.1
11
6.4
8.0
0.36
0.11
0.14
0.71
6.7
4.2
2.2
0.29
V
0 ng/mL
30
30
24
10 ng/mL
30 ng/mL
100 ng/mL
6.7 23
a
At the start of each experiment, medium was changed to
DMEM without phenol red or serum, with additions as indicated.
After 45 min (experiments I, II, IV, and IV), or 30 min (experiment
III), at 37 °C in CO2/air, cells were processed for analysis. Values
shown are 108 × mol of K+ or Na+ per dish, assayed as described
previously.13 When present, quinine was at 150 µM, verapamil
at 100 µM.
3-hydroxyfluorenone (15) and phenol (Scheme 2). Syn-
thesis of the 3-hydroxy derivative required 3-hydroxy-
fluorenone (15), which was prepared by Suzuki cou-
pling17 of the aryl bromide 12 with boronic acid 13,
followed by methanesulfonic acid-catalyzed ring closure
and methyl ether deprotection with boron trifluoride-
dimethyl sulfide complex (Scheme 2). Aluminum chlo-
ride-catalyzed Friedel-Crafts reaction of 15 with phenol
provided bisphenol 5 in 81% yield. We have not found
any previous reports of bisphenol 5 in the literature.
Ion Tr a n sp or t a n d Estr ogen Recep tor Bin d in g
Activity of Bisp h en ols 4 a n d 5 a n d Rela ted Com -
p ou n d s. The synthetic bisphenol derivatives 4 and 5
were tested by measuring changes in K+ and Na+ in
HF cells. The fluorene derivative 5 was active at quite
low concentrations and produced 50% of the maximum
effect between 30 and 60 ng/mL (80-160 nM) (Table
2).
One can formulate a reasonable mechanism for such
a rearrangement: Under forcing reaction conditions, one
molecule of phenol attacks the carbonyl group to form
the tetrahedral intermediate, which in this C1-OH
system is sterically too crowded to allow attack of the
second molecule of phenol. Instead, the C1a carbon is
protonated to form a dienone, which fragments at the
C9-C1a bond, allowing for rotation around the C4a-C5a
bond of a biphenyl intermediate. Subsequent ring
closure by attack of C9 on the carbon that was originally
C4 results in the monophenol adduct of 3-hydroxy-
fluorenone. Phenol attack on this less sterically crowded
intermediate affords the bisphenol derivative of 3-hy-
droxyfluorenone.
Confirmation of the structure of the biologically active
component isolated in fraction II from reversed-phase
HPLC and synthesized by rearrangement from 1-hy-
droxyfluorenone (11) was attained by its synthesis from
The bisphenol xanthene derivative 4 displayed partial
activity in the assay for cell K+ and Na+ content. The

4902 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Kym et al.
Sch em e 3. Mechanisms by which Phenol Red (1) and Compounds 4 and 5 Might Be Formed
concentration that produced 50% of the maximum effect
was between 2 and 5 µg/mL (5.5-14 µM). This com-
pound is more interesting, however, because of its
cytotoxicity. At concentrations not much greater than
that which reduced cell K+ by about 50% of control
values, the bisphenol xanthene 4 caused cells to be
released from the plastic substrate. This effect on cell
adhesion made it impossible to obtain satisfactory dose-
response data for 4, as the affected cells were washed
away during processing of the dishes (data not shown).
Compound 6 was active in the bioassay at 0.1 µg/mL
(280 nM) and 7 at 30 ng/mL (80 nM).
As described in previous reports on crude phenol
red,13,14 quinine and verapamil partially inhibit the
action of 5 and 4 on ion content of human fibroblasts.
Verapamil also partially inhibited the action of both 6
and 7 on cell K+ and Na+ (Table 2 and data not shown).
Neither compound caused cells to detach from the
plastic substrate. Hence, we think it likely that com-
pounds 5-7, and possibly 4, all act on ion channels.
Both quinine and verapamil have been well studied by
others: These agents inhibit the activity of several types
of ion channels, including K+ and Ca2+ channels.18,19
We have also tested bisphenols 4-7 for their competi-
tive binding affinities for the estrogen receptor (ER).20
We found them to exhibit weak, competitive binding for
the ER; the highest affinity compound, bisphenol 4, had
a relative binding affinity20 (RBA) of 0.03% (where
estradiol is 100%). The relative binding affinities of
bisphenols 5-7 are 0.006%, 0.007%, and 0.010%, re-
spectively. The RBA of phenol red prior to extraction
is 0.010%, and the RBA of the crude ether extract of
phenol red is 0.160%. The increased RBA value for the
crude ether extract reflects the presence of the high-
affinity bisphenol sulfonate 2 (50% relative to estradiol)
which has previously been identified as a lipophilic
impurity in phenol red.9 Compound 2 showed no
significant effect on cell K+ and Na+ content, when
tested on MCF-7 cells at 15 µg/mL (35 µM) (data not
shown).
intriguing to speculate on the mechanism of formation
of bisphenols 4 and 5 during the synthesis of phenol red.
At the elevated temperatures required to effect the
Friedel-Crafts reaction between phenol and 2-sulfo-
benzoic acid cyclic anhydride (16), the alkylation can
occur at either the para (major) or the ortho (minor)
positions of phenol (Scheme 3). Bisalkylation at the
para position followed by subsequent elimination of
water results in formation of phenol red (1) as the major
product. Formation of 3-hydroxyfluorenone 15, the
precursor to bisphenol 5, might be occurring from
sulfonic acid 17 by electrocyclic Nazarov ring closure,21
driven by the ultimate elimination of the sulfonic acid
as sulfur dioxide and water from the initial cyclization
product. Nazarov rearrangements are not precedented
in the fluorenone system.21 Nevertheless, a Nazarov-
like electrocyclic ring closure might occur from a bis-
phenol species formed by the reaction of 17 with phenol.
The xanthone (8) precursor to 4 could be formed in a
similar manner from 16 by initial attack of phenol at
the ortho position to provide 18 (Scheme 3). Hydroxyl
attack at the sulfonic acid-substituted aromatic carbon,
followed by elimination of sulfonic acid as sulfur dioxide
and water, would form xanthone (8). Ring closure could
also occur after a molecule of phenol has added to 18,
as was discussed as an alternate mechanism for forma-
tion of the fluorene ring system (vide supra).
Con clu sion
Two bisphenol derivatives with biological activity
have been isolated from the lipophilic impurities present
in a commercial preparation of phenol red; their struc-
tures have been determined by corroboration of 1H and
13C NMR methods and mass spectrometry and con-
firmed by synthesis. The bisphenol fluorene derivative
5 has been found to exhibit a rapid effect on the
intracellular content of K+ and Na+ ions in human
fibroblasts. The concentration that produces 50% of the
maximum loss of K+ and gain of Na+ lies between 30
and 60 ng/mL (80-160 nM). The 2- and 4-hydroxy
isomers of the fluorene compound, compounds 6 and 7,
prepared by synthesis are also active, 0.1 µg/mL (280
P u ta tive Mech a n ism of F or m a tion of Bisp h en ols
4 a n d 5 Du r in g th e Syn th esis of P h en ol Red . It is

Bisphenolic Compounds in Commercial Phenol Red
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4903
substituted bisphenol 4 (1.37 g, 30%) as a white solid (mp 230-
nM) and 30 ng/mL (80 nM), respectively. The bisphenol
xanthene derivative 4 elicits a similar biological re-
sponse but is less potent, and it also exhibits a marked
effect on cell adhesion, causing release of cells from the
plastic substrate at concentrations as low as 2 µg/mL.
231 °C, lit.16 mp 241 °C). Anal. (C25H18O3) C, H.
9,9-Bis(4′-h yd r oxyp h en yl)-3-h yd r oxyflu or en e
(5).
Meth od A: To a solution of 1-hydroxyfluorenone (11) (0.115
g, 0.586 mmol) stirring neat in phenol (3.0 g, 31.8 mmol) at
60 °C was added aluminum chloride (0.234 g, 1.76 mmol). The
reaction mixture was heated to 160 °C and stirred for 4 h. The
reaction mixture was allowed to cool to 80 °C, and excess
phenol was removed in vacuo. Flash chromatography (silica
gel, 4:1 hexanes:EtOAc to 2:1 hexanes:EtOAc) afforded the bis-
para-substituted bisphenol 5 (0.205 g, 35%) as the major
product. Reversed-phase HPLC (50% H2O, 50% CH3CN)
afforded 5 as a white solid.
The bisphenols would be expected to have their full
effect when cells are in protein-free media, causing a
fall in the steep gradients of monovalent ions that
normally exist across the plasma membrane. These
gradients, with intracellular K+ often about 20 times
higher than extracellular K, and with the reverse for
Na, are necessary for cell function.24 When cell K+
decreases, intracellular processes that depend on K+
may be compromised. The list of K-dependent processes
is long and includes protein synthesis, as has been
known for a long time, the recently reported stabiliza-
tion of chaperone-ADP binding, and suppression of
interleukin-1â maturation.25-30
The effect of these bisphenols on ion gradients could
be masked in part if the Na+/K+ ATPase (the Na+
pump), which continually removes excess intracellular
Na, were active. Since they do deplete cells of K, even
in the presence of an active Na+ pump, the increase in
the rates of K+ efflux and Na+ influx must be substan-
tial. In fact, as Hopp et al. have shown, even though
Na+ pump activity is enhanced by phenol red impurities,
it fails to prevent a sharp fall in K+ and Na+ gradients.14
Meth od B: To a solution of 3-hydroxyfluorenone (19) (0.089
g, 0.45 mmol) stirring neat in phenol (1.30 g, 13.8 mmol) at
60 °C was added aluminum chloride (0.191 g, 1.43 mmol). The
reaction mixture was heated to 80 °C and allowed to stir for 2
h. Flash chromatography (4:1 hexanes:EtOAc to 2:1 hexanes:
EtOAc) afforded bisphenol 5 (0.133 g, 81%) as the major
product. Recrystallization in ether-hexanes gave 5 as a white
crystalline solid (mp 248-250 °C). HRMS (EI) calcd for
C
25H18O3 366.1256, found 366.1254.
9,9-Bis(4′-h yd r oxyp h en yl)-2-h yd r oxyflu or en e (6). To
a solution of 2-hydroxyfluorenone (9) (0.202 g, 1.03 mmol)
stirring neat in phenol (3.0 g, 31.8 mmol) at 60 °C was added
aluminum chloride (0.442 g, 3.09 mmol). The reaction mixture
was heated to 80 °C, stirred for 2 h, and allowed to cool to
room temperature. Flash chromatography (silica gel, 4:1
hexanes:EtOAc to 2:1 hexanes:EtOAc) afforded 6 (0.339 g,
89%) as a white solid (mp 264-266 °C). HRMS (EI) calcd for
C
25H18O3 366.1256, found 366.1262.
9,9-Bis(4′-h yd r oxyp h en yl)-4-h yd r oxyflu or en e (7). To
The bisphenols are therefore quite efficacious. Po-
tency, with an EC50 as low as 80-160 nM, is comparable
to the potency of other inhibitors known to cause K+
and Na+ gradients to drop. The EC50 for ouabain, for
example, an inhibitor of the Na+ pump, is 20-30 nM
in human cells.31 The EC50 for amphotericin B, in
mouse sarcoma-180 cells, is about 2 µM.26 The calcium
ionophore ionomycin, with an EC50 about 1 µM in EJ
human bladder carcinoma cells, also causes marked loss
of cell K.13 The agent of ciguatera poisoning, maitotoxin,
produced by a marine dinoflagellate, is extraordinarily
potent; in addition to activating calcium influx, maito-
toxin causes collapse of K+ and Na+ gradients in mouse
BC3H1 cells at only 3 nM.32
a solution of 4-hydroxyfluorenone (10) (0.090 g, 0.46 mmol)
stirring neat in phenol (1.29 g, 13.7 mmol) at 60 °C was added
aluminum chloride (0.183 g, 1.37 mmol). The reaction mixture
was heated to 80 °C, stirred for 2 h, and allowed to cool to
room temperature. Flash chromatography (silica gel, 4:1
hexanes:EtOAc to 2:1 hexanes:EtOAc) afforded 7 (85%) as a
white solid (mp 261-263 °C). Anal. (C25H18O3) C, H.
Eth yl 2-(3′-Meth oxyp h en yl)ben zoa te (14). Ethyl 2-bro-
mobenzoate (12) (2.50 g, 10.9 mmol) and tetrakistriphenylphos-
phinepalladium (0.43 g, 0.32 mmol) were dissolved in 115 mL
of 1,2-dimethoxyethane. A 2 M aqueous solution of Na2CO3
(10.9 mL) was added via syringe. A solution of the boronic
acid 13 (1.84 g, 12.0 mmol) in 10 mL of DME was subsequently
added. The reaction mixture was heated to 100 °C and allowed
to stir for 12 h. It was then cooled, diluted with water,
extracted with ethyl acetate, and washed with saturated
aqueous NaHCO3 and brine. The organic layer was dried over
MgSO4, and the solvent was evaporated under reduced pres-
sure to give a brown oil. Flash chromatography (8:1 hexanes:
EtOAc to 1:1 hexanes:EtOAc) afforded 1.06 g (38%) of the
biaryl compound 14 as a colorless oil. Anal. (C16H16O3) C, H.
Because verapamil and quinine partly inhibit the
effect of the bisphenols, they presumably interact with
ion channels. The full explanation of how the bisphe-
nols act, however, will require studies by patch-clamp
methods.
3-Hyd r oxy-9-flu or en on e (15). A solution of biaryl com-
pound 14 (0.756 g, 2.95 mmol) in 40 mL of methanesulfonic
acid (59.2 g, 616 mmol) was stirred and heated to 110 °C for
1 h. The resulting black mixture was poured slowly into
stirred ice water and then extracted with two portions of
diethyl ether. The combined organic layers were washed with
saturated NaHCO3 solution and water and then dried over
MgSO4. The solvent was evaporated under reduced pressure
to give a brown oil. Flash chromatography (8:1 hexanes:
EtOAc) yielded 0.378 g (61%) of the desired 3-methoxy-9-
fluorenone. To a solution of 3-methoxy-9-fluorenone (0.20 g,
0.94 mmol) in 10 mL of CH2Cl2 at 0 °C was added the boron
trifluoride-dimethyl sufide complex (4.9 mL, 47 mmol). Upon
addition of the complex, the reaction mixture turned from
yellow to dark red in color. It was allowed to warm to room
temperature and stir for 48 h. The reaction mixture was then
added to water and extracted with three portions of ethyl
acetate. The combined organic layers were dried over MgSO4,
and the solvent was evaporated under reduced pressure to a
brown solid. Flash chromatography (10:1 hexanes:EtOAc to
2:1 hexanes:EtOAc) yielded 0.067 g (37%) of the yellow solid
15 (mp 227-228 °C, lit.23 mp 228-229 °C). HRMS (EI) calcd
for C13H8O2 196.0524, found 196.0521.
Exp er im en ta l Section
Gen er a l. Reaction progress was monitored by analytical
thin-layer chromatography, using 0.25 mm silica gel glass-
backed plates with F-254 indicator (Merck). Flash chroma-
tography was performed with Woelm 32-63 µm silica gel
packing.22 Visualization was accomplished by phosphomolyb-
dic acid or UV illumination (254, 350 µM). Reversed-phase
HPLC was performed on a preparative Partisil M9 10 mm ×
50 cm ODS-2 column or an analytical MicroPak 4 mm × 30
cm C-18 silica gel column. Phenol red monosodium salt (Lot
#01601LF) was purchased from Aldrich Chemical Co. (Mil-
waukee, WI). Full spectroscopic characterization of com-
pounds 4-7, 18, and 19 is given in the Supporting Information.
Ch em ica l Syn th eses. 9,9-Bis(4′-h yd r oxyp h en yl)xa n -
th en e (4). To a solution of xanthone (8) (2.39 g, 12.2 mmol)
stirring neat in phenol (22.8 g, 242.4 mmol) at 60 °C was added
aluminum chloride (4.88 g, 36.6 mmol). The reaction mixture
was heated to 180 °C and stirred for 12 h. The reaction
mixture was allowed to cool to 80 °C, and excess phenol was
removed in vacuo. Flash chromatography (silica gel, 4:1
hexanes:EtOAc to 2:1 hexanes:EtOAc) afforded the bis-para-

4904 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Kym et al.
(12) Walsh-Reitz, M. M.; Toback, F. G. Phenol Red Inhibits Growth
of Renal Epithelial Cells. Am. J . Physiol. 1992, 262 (Renal Fluid
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Bioa ssa y. Human foreskin fibroblasts (HF) were grown in
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Grand Island, NY) with penicillin, streptomycin, neomycin,
and 10% fetal bovine serum (HyClone Laboratories, Logan,
UT) at 37 °C in CO2/air, pH 7.2-7.4. For all experiments,
DMEM without phenol red (Sigma Chemical, St. Louis, MO)
was freshly prepared from powder. Quinine and verapamil
were from Sigma. Stocks of dried HPLC fractions or of
compounds 4-7, prepared by synthesis, were dissolved in
either dimethyl sulfoxide or ethanol. Solvents in test solutions
were at 0.2% or less and had no activity.
For each experiment, values given in Tables 1 and 2 were
averaged from duplicate dishes; duplicates usually differed by
no more than 10-15%. Cell numbers per dish were from 5 ×
105 to 2 × 106. Medium was replaced with test solution by
four rapid rinses, with a final addition of 2.5 mL to each dish.
After incubation as described in Tables 1 and 2, each dish was
rapidly rinsed six times with iced 0.1 M MgCl2 or 0.15 MgSO4,
and the excess was removed by suction. After the dishes were
dry, 1.5 mL of the flame photometer diluent, lithium nitrate,
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Ack n ow led gm en t. We are grateful for support of
this research through a grant from the National Insti-
tutes of Health (PHS 5R37 DK15556 (J .A.K.)). We
thank Kathryn E. Carlson for estrogen receptor binding
affinity measurements.
Su p p or tin g In for m a tion Ava ila ble: Full spectroscopic
information on compounds 4-7, 14, and 15, 1D and 2D 1H
and 13C NMR spectra on compounds 4-6 and 19, and HPLC
traces on compounds 5 and 6 (14 pages). See any current
masthead page for ordering information.
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