Synthesis and cytotoxicity of gossypol related compounds

Article abstract of DOI:10.1016/S0223-5234(00)00165-3

Gossypol, gossypolone, reduced gossypol and new Schiff's bases of racemic gossypol and gossypolone were extracted or synthesized. Their cytotoxic activities on KB human cancer cells were determined. Gossypolone and the ethylamine derivative of gossypolone

Full text of DOI:10.1016/S0223-5234(00)00165-3

Eur. J. Med. Chem. 35 (2000) 805−813
2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001653/FLA
©
Original article
Synthesis and cytotoxicity of gossypol related compounds
a
a
a
a
b
Vi-Thuy Dao , Christiane Gaspard , Michel Mayer , Georges H. Werner , Suong N. Nguyen ,
a*
Robert J. Michelot
a
Institut de chimie des substances naturelles, CNRS, 91198 Gif sur Yvette, France
b
Department of Organic Chemistry, University of Ho Chi Minh City, 227 Nguyen Van Cu, Quan 5, Ho Chi Minh City, Viet Nam
Received 28 December 1999; revised 7 March 2000; accepted 9 March 2000
Abstract – Gossypol, gossypolone, reduced gossypol and new Schiff’s bases of racemic gossypol and gossypolone were extracted or
synthesized. Their cytotoxic activities on KB human cancer cells were determined. Gossypolone and the ethylamine derivative of gossypolone
were the most active compounds (IC50 in the micromolar range in both cases). The cytotoxicity of gossypol and gossypolone was increased
when the tests were performed in the absence of serum and decreased when catalase as well as mannitol were added to the culture medium.
©
2000 Éditions scientifiques et médicales Elsevier SAS
gossypol / gossypolone / Schiff’s bases / reduced gossypol / oxygen radical scavengers / cytotoxicity
1
. Introduction
limited because the phenolic hydroxyl groups in the 1 and
′ (peri) positions complicate the chemistry of the func-
tional group at the 8 and 8′ positions [1].
1
Gossypol [1,1′,6,6′,7,7′-hexahydroxy-5,5′diisopropyl-
,3′dimethyl- [2,2′] binaphthalenyl-[8,8′] dicarbaldehyde]
3
Nevertheless, the reaction of gossypol with primary
amines, giving gossypol imines or Schiff’s bases has been
extensively developed and, in the meantime, the broad
biological activity of gossypol has been explained by its
easy coupling to lysine side chains of proteins [1] and to
enzymes involved in the cellular growth process [19].
Gossypol imines have been investigated for their
antitumour or inhibitory effects and some structure–ac-
tivity comparisons have already been proposed pointing
out the importance of the substituting group [8, 20].
More recently, the metabolic fate of gossypol was
reinvestigated in view of the toxicity of gossypolone. As
a polyphenol, gossypol has been cited as a promoter of
free radical formation in biological media and part of its
cytotoxicity has been attributed also to its transformation
into gossypolone which represents the major metabolite
of gossypol in vivo [21, 22].
In the present paper, we describe the synthesis of
several original derivatives of gossypol and gossypolone
and their cytotoxic activities on the KB human cancer cell
line. Furthermore, to get some insight into the role played
by oxidation in cytotoxicity, we investigated the activity
is a polyphenolic dialdehyde (figure 1), a naturally
occurring yellow pigment found in high concentrations in
the pigment glands of the cotton plant gossypium [1].
Extensive information is available concerning the toxi-
cology and pharmacokinetics of this compound since it
has already been administered to humans as a male oral
contraceptive [2].
Gossypol and some of its derivatives also display
several potentially useful biological activities: antitrypa-
nosomal [3], antimalarial [4–6], antitumour [7–9] and
inhibition of enveloped viruses [10–12] including HIV
[13–18].
While being marginally toxic, gossypol represents a
potential starting point for the development of antiviral or
antitumour drugs.
Numerous studies suggest that modifications to gossy-
pol’s functional aldehyde groups could reduce its host
toxicity while retaining its therapeutic effects [15, 16].
The availability of stable derivatives of gossypol, in
which only the aldehyde groups are modified, has been
*
Correspondence and reprints: michelot@icsn.cnrs-gif.fr

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V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
Figure 1. Structures of gossypol derivatives and IC50 on KB cells (µM).
of gossypol or gossypolone on the growth of these cells in
the presence of radical scavengers.
These considerations prompted us to synthesize new
gossypol derivatives; Schiff’s bases of gossypol; reduced
gossypol; Schiff’s bases of gossypolone and to compare
their cytotoxicity.
2
. Chemistry
The protected tetra and hexamethoxy ethers of gossy-
pol have also been synthesized to evaluate the toxicity
associated with the phenolic groups in gossypol.
For structure–activity comparison we reduced the al-
dehyde groups into alcohol using a selective reducing
agent.
The toxicity of gossypol has been associated by many
authors with the presence of the aldehyde groups which
can easily bind nucleophilic targets of biological impor-
tance.

V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
807
Figure 2.
One should note that gossypol exhibits atropisomerism
due to hindered rotation about the binaphthyl bond. Since
the gossypol used in this work was racemic and none of
the derivatives were resolved, the differences in cytotoxic
properties of their (+) and (–) isomers were not deter-
mined.
Schiff’s bases were synthesized by reacting gossypol
(compounds 6–18) or gossypolone (compounds 19–20) to
the amines in a mixture of ether/isopropanol under mild
conditions (25–45 °C, argon). After 5–30 min the product
precipitated and could be recrystallized except for the
methylamine derivative which was insoluble in the com-
mon solvents and which was prepared from a solution in
dimethylformamide. NMR studies of these compounds in
deuteriochloroform revealed the enamine structure for
compounds 7, 10, 12, 16 and 17.
2.1. Coupling with amines – Schiff’s bases of gossypol
Many Schiff’s bases resulting from the coupling to
amines of racemic gossypol or its enantiomers have
already been described [8, 20, 23, 24]. Biological activi-
ties of the compounds are dependent both on the structure
of the Schiff’s base and also on the hydrophobicity of the
substituting chain [20].
2
.2. Reduction of gossypol
For the reduction of gossypol derivatives, different
approaches have been proposed. Reduction of gossypol
hexamethoxy derivative by hydrogen and platinum in
acetic acid was attempted for the first time in 1930 by
Adams et al. [27]. They obtained the deoxygossypol
tetramethyl ether. A direct reduction of gossypol with
The high reactivity of the aldehyde groups in gossypol
or in gossypolone enables the rapid coupling of nucleo-
philes followed by a possible dehydration of an interme-
diate carbinol (figure 2). The molecular structure of the
Schiff’s bases depends on the equilibrium between the
imino and the enamine form and the enamine proton has
been used as a probe for the NMR quantitation of the
equilibrium [25]. Since NMR studies are often performed
in aprotic solvents, the position of the observed equilib-
rium is not representative of the equilibrium in a physi-
ological medium [26]. Thus NMR data could scarcely
correlate with the biological activities, and the sharp
structure–activity relationships observed for the antitu-
mour activity [8] do not reflect the position of the
enamine–imine equilibrium observed in aprotic solvents.
LiAlH followed by acetylation of the reaction mixture
4
led to deoxygossypol tetraacetate and methylapogossypol
hexaacetate [28].
More recently, Deck et al. reduced gossypol 1,1′
dimethyl ether with NaBH and they obtained the corre-
4
sponding alcohol [29].
Using NaBH CN, a smooth reducing agent, selective
3
of the aldehyde group [30], we were able to obtain
directly the alcohol from gossypol itself. The alcohol was
purified by HPLC.

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V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
3. Results and discussion
gossypol and no toxicity for gossypolone on the SK-
mel-28 cell line [22].
The following new derivatives of gossypol have been
Our data confirm that the cytotoxicity of gossypol
derivatives depends on the availability of the phenolic
oxygen atoms (4 and 5 are inactive) and also on the bulk
and structure of the radical substituent in a Schiff’s base.
In addition, the oxidation state of gossypol seems to play
an important role since gossypolone and gossypolone
Schiff’s bases are more active than the reduced parent
compound.
This last fact prompted us to evaluate the idea that
gossypolone, which is a major metabolite of gossypol,
could be, to a large extent, the toxic agent for the cells.
Following this hypothesis, co-incubation of antioxidants
or radical scavengers should reduce the toxicity of
gossypol and less that of gossypolone.
For these experiments it was necessary to eliminate
any compound known to bind gossypol or protect it from
oxidation in the culture medium [32]. Furthermore, in
view of the potential in vivo antitumour activities of
gossypol and its derivatives, it was of interest to deter-
mine whether or not the various constituents of serum had
an influence on their cytotoxic activities in vitro.
Table I shows that when gossypol and gossypolone are
tested for their cytotoxic effects on KB cell monolayers
maintained either in serum-free medium or in medium
containing 10% foetal calf serum, the IC50 values of the
compounds are 5–10 times lower in the serum-free
medium. In addition, the toxicity of gossypol is more
affected by the presence of serum than that of gossy-
polone.
synthesized and characterized: gossypolol 3 and imines
of gossypolone 19 and 20. Imines of gossypol 6–18 have
been fully characterized (MS, NMR) and gossypol tet-
ramethoxy ether 4 has been attributed a structure which is
different from that previously proposed [1], considering
its reactivity and NMR data.
The cytotoxic activities of compounds 1–20 are re-
ported in figure 1. We found the ethylamine derivative 7
more toxic than other Schiff’s bases of gossypol against
KB cells (IC50 5.6 µM), immediately followed by 8, 9
and 11 (6.5, 6.6 and 6.7 µM, respectively).
Liang et al. [8] have determined the antiproliferative
activities of 1, 2, 8, 9, 10 on MCF7, MCF7/adr malignant
cells and HBL-100 immortalized human mammary epi-
thelial cells. They concluded that, among numerous other
Schiff’s bases of gossypol, the isopropyl amine derivative
9
, was the most active.
We found racemic gossypolone 2 to be more toxic
2.4 µM) than gossypol and any gossypol Schiff’s base.
(
Interestingly enough, the Schiff’s base of gossypolone 19
was the most toxic derivative (0.8 µM), whereas gossy-
polol 3 was almost inactive as well as methoxy-protected
gossypol derivatives 4 and 5.
Our results on KB cells parallel the results obtained by
Liang et al. [8] on the antiproliferative effects of racemic
gossypolone and Schiff’s bases of gossypol. From the
other reports in the literature it is difficult to draw firm
conclusions since cytotoxicity was measured using dif-
ferent methods and on different cell lines. For instance,
Shelley et al. reported recently that racemic gossypol and
racemic gossypolone displayed IC50 values ranging from
Among the possible explanations for the inhibitory
effect of serum on the activities of both compounds, we
examined the role which free radical scavengers, known
to be present in serum, could exert. To that effect,
2
0–30 µM [31]. Blackstaffe et al. reported IC50 values in
an MTT viability assay of 22 µM for gossypol and
gossypolone on the SK-mel-19 cell line and 22 µM for
catalase, mannitol, glutathione and vitamin
(α-tocopherol) were added to KB cell cultures at non-
E
Table I. Effect of foetal calf serum on the cytotoxic activities of gossypol and gossypolone.
Medium containing 10 % FCS
Gossypol or gossypolone concentration (µM)
10
60
85
5
50
68
2.5
30
60
1
0
42
0.5
0
21
%
%
cell destruction with gossypol
cell destruction with gossypolone
IC50 gossypol = 5 µM; IC50 gossypolone = 1.2 µM
Medium without FCS
Gossypol or gossypolone concentration (µM)
10
100
91
5
100
80
2.5
91
74
1
86
68
0.5
75
58
%
%
cell destruction with gossypol
cell destruction with gossypolone
IC50 of gossypol or gossypolone < 0.5 µM

V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
809
Table II. Effect of free radical scavengers on the cytotoxic
were determined at the Service de Spectrométrie de
Masse de l’Institut de Chimie des Substances Naturelles
(ICSN) on an AEI MS50 spectrometer or an MS80 RT
activities of gossypol and gossypolone.
Treatment
Cell mortality
after 48 h
1
13
Kratos spectrometer. H-, C-NMR spectra were re-
incubation (%)
corded in CDCl or DMSO-d on a Bruker AM300
3
6
Gossypol 0.5 µM
63
31
37
57
49
27
20
72
spectrometer. HPLC analysis was performed on a Waters
HPLC 600E, with a reversed-phase Alltima C-18, 5 µm
column (4.6 mm i.d. × 250 mm) by isocratic elution (I):
Gossypol 0.5 µM + catalase (150 µg/mL)
Gossypol 0.5 µM + mannitol (25 mM)
Gossypol 0.5 µM + glutathione (5 mM)
Gossypolone 0.5 µM
Gossypolone 0.5 µM + catalase (150 µg/mL)
Gossypolone 0.5 µM + mannitol (25 mM)
Gossypolone 0.5 µM + glutathione (5 mM)
1
1
0% A, 90% B; (II): 20% A, 80% B the flow rate was
mL/min; (III): 2% A, 98% B the flow rate was 2 mL/
min at 50 ± 1 °C, with the solvent system: A: 95% water,
% CH CN, 0.1% TFA; B: 100% CH CN, 0.1% TFA.
5
3
3
The detection wavelength was 254 nm.
4
.1.1. Synthesis of 6,7,6′,7′-
tetrahydroxy-5,5′-diisopropyl-3,3′-dimethyl-
,4,1′,4′-tetraoxo-1,4,1′,4′-tetrahydro-[2,2′]-
cytotoxic concentrations and at the same time as gossypol
and gossypolone. Table II shows that, under such condi-
tions, catalase and mannitol (both known to interact with
H O formation) did exert a significant inhibitory effect
1
binaphthalenyl-8,8′-dicarbaldehyde (gossypolone) 2
Following the method described by Haas et al. [34]. A
solution of 1.5 g (2.59 mmol) of 1 in 75 mL of acetone
and 150 mL of acetic acid was heated on a steam bath
2
2
on the cytotoxic activities of gossypol and gossypolone.
Glutathione had no effect and results with α-tocopherol
were inconsistent. Again, the toxicity of gossypol remains
higher than the toxicity of gossypolone in the presence of
radical scavengers, suggesting that serum actually pro-
tects the cells from gossypol not only by quenching free
radicals.
A preliminary clinical study of gossypol in advanced
human cancer has been performed by Stein et al. [33].
This was essentially a phase I study in which 34 patients
were given escalating doses of oral gossypol. No major
adverse effects occurred and there was no evidence of
haematological or biochemical disturbances, the dose-
limiting toxicity being emesis. There was no evidence
either of tumour regression, but in our opinion these
negative results do not rule out the possibility that
gossypol derivatives endowed with higher in vitro cyto-
toxicity than that of gossypol itself and administered by
various routes in combination with other anticancer
agents could prove to play a useful role in the therapy of
some cancer states.
(
60–70 °C) during the addition of 112.5 mL of a 10%
aqueous solution of ferric chloride hexahydrate (42 mmol)
and for several minutes longer. The solution was cooled
and 200 mL of water was added to precipitate a dark
iron-containing compound which was removed and treated
with a mixture of ether and aqueous 20% sulfuric acid.
The liberated phenol was taken into the ether layer which
was washed with water and dried (Na SO ), and the ether
2
4
was evaporated. The residue was precipitated from
toluene/hexane to yield 0.6–0.7 g (42–49%). HPLC:
1
K′(II): 2.75; EI-MS: m/z: 553 (M + Li); H-NMR-CDCl :
3
1
4
.46 (d, 12H, 2 HC–(CH ) ), 2.07 (s, 6H, 2 Ar–CH ),
3 2 3
.14 (m, 2H, 2 HC–(CH ) ), 6.59 (s, 2H, 2 OH at 6,6′
3
2
positions), 10.60 (s, 2H, 2 CHO), 13.03 (s, 2H, 2 OH at
13
7
1
,7′ positions); C-NMR-CDCl :198.65 (CHO), 186.70,
3
84.60 (2 C=O at 1 and 4 positions).
4
5
1
.1.2. Synthesis of 8,8′-bis-hydroxymethyl-
,5′-diisopropyl-3,3′-dimethyl-[2,2′]-binaphthalenyl-
,6,7,1′,6′,7′-hexaol (gossypolol or gossylic alcohol) 3
A solution of 1 g (1.73 mmol) of 1 in 20 mL of
Further studies are underway to synthesize new deriva-
tives of gossypol and particularly of gossypol enanti-
omers which could, as pointed out by many authors [22,
methanol and 108 mg (1.73 mmol) of NaBH CN and
3
3
1], lead to interesting antitumour drugs.
2
N HCl/methanol was added dropwise with stirring to
maintain the pH of the solution near 3–4. After ca. 5 h the
methanol was evaporated at reduced pressure and the
residue was taken up in 20 mL of water, saturated with
sodium chloride and extracted with three 15 mL portions
of ether, the combined extracts were dried (Na SO ) and
evaporated to give a residue (1 g), a small amount of the
gossypol-acetic acid was detected in the residue by
HPLC. To eliminate the gossypol-acetic acid, we used
4
. Experimental protocols
4
.1. Chemistry
2
4
Reagents and solvents used for the reactions were
purchased from Fluka. Gossypol-acetic acid 1 was ex-
tracted and purified following Carruth [21]. Mass spectra

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V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
Girard’s T-reagent: 500 mg of the residue and 100 mg of
Girard’s T-reagent were dissolved in 50 mL of alcohol
containing 10% acetic acid, the solution was heated for
Ar–CH ), 3.31–3.46 (m, 6H, 2 O–HC–OCH ), 3.83–4.19
3 3
(m, 14H, 2 HC–(CH ) , 4 OCH at 6,6′,7,7′ positions),
3
2
3
6.95–7.06 (m, 2H, 2 O–HC–OAr), 7.48 (d, 2H, 2 Ar-H).
3
5
0 min at 60 °C. Then, the reaction mixture was cooled,
0 mL of water was added, and solution was extracted
4
.1.5. General method for the synthesis of gossypol
with three 15 mL portions of ether. The organic phase
was washed with water and dried over Na SO , the ether
was removed in vacuo to yield 75 mg (15% yield) HPLC:
and gossypolone Schiff’s bases: 6–18 and 19, 20
2
4
Two hundred milligrams (0.35 mmol) of 1 [(0.37 mmol)
of 2] was dissolved in a solution of 5 mL ether and 2 mL
of isopropanol and stirred at room temperature. The
amine (1.4 mmol) was added and heated for 5 min at
1
k′(II): 3.04; EI-MS: m/z 545 (M + Na); H-NMR-CDCl :
3
1
.5 (m, 2 HC–(CH ) ), 2.2 (6H: 2 Ar–CH ), 3.9 (2H:
3 2 3
–1
HC–(CH ) ), 5.6 (4H: CH OH); IR 3 517 cm (alcohol).
3
2
2
4
5 °C and the mixture was allowed to stand, under argon,
for 2 h at room temperature. The precipitate was filtered
off, washed with hexane and vacuum dried to give the
Schiff’s base as a yellow solid.
4
2
2
.1.3. Synthesis of 5,5′-diisopropyl-
,4,2′,4′-tetramethoxy-7,7′-dimethyl-
H,2′H-[8,8′]bi[naphtho[1,8-bc]furanyl]-3,3′-diol 4
One gram (1.73 mmol) of 1 in a mixture solution of
mL of absolute methyl alcohol and 5 mL of dimethyl
6: 5,5′-diisopropyl-3,3′-dimethyl-8,8′-bis-methylimino-
methyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol. The pre-
cipitate, 180.4 mg (yield 95%) was obtained from a
solution of methylamine in dimethylformamide. It was
purified by repeated precipitations from dimethylforma-
mide solutions The very low solubility of this compound
in chloroform did not allow NMR analysis or mass
spectrometry.
5
sulfate was stirred at room temperature under argon.
When the solid was completely dissolved, 7 mL of 10%
methyl alcoholic potassium hydroxide was added drop-
wise and the mixture was allowed to stand for 24 h at
room temperature. The solution turned a deep brown
colour and a precipitate of potassium sulfate formed, the
excess dimethyl sulfate was decomposed with 10% aque-
ous sodium hydroxide solution. The white flocculent
precipitate that formed was filtered, washed with cool
methanol and dried to yield 0.85 g (85.5% yield). HPLC:
7
:
8,8′-bis-ethyliminomethyl-5,5′-diisopropyl-3,3′-
dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol: see
general method for synthesis. Obtained 190 mg (yield
+
9
6%) HPLC: k′(III): 0.71, EI-MS: m/z 573 (MH );
1
+
1
H-NMR-CDCl :1.37 (t, 6H, 2 HC=N–CH –CH ), 1.52
k′(I): 2.83; EI-MS: m/z: 575 (MH ); H-NMR-CDCl :
3
2
3
3
(
d, 12H, 2 HC-(CH ) ), 2.12 (s, 6H, 2 Ar–CH ), 3.52 (m,
H, 2 HC=N–CH –CH ), 3.74 (m, 2H, 2 HC–(CH ) ),
2 3 3 2
.59 (s, 2H, 2 Ar–H), 9.69 (d, 2H, 2 HC=N–R); 13.34 (br,
H, 2 OH at 7,7′ positions).
1
3
4
.52 (d, 12H, 2 HC–(CH ) ), 2.29 (s, 6H, 2 Ar–CH ),
.83 (m, 2H, 2 HC–(CH ) ), 3.28 (s, 6H, 2 O–HC–OCH ),
3 2 3
3
2
3
4
7
2
3
2
3
.19 (s, 6H, 2 OCH at positions 6,6′), 6.28 (s, 2H, 2 OH
3
at 7,7′ positions), 7.1 (s, 2H, 2 O–HC–OAr), 7.42 (s, 2H,
Ar–H). These data, compared to NMR data from Haas
2
8: 5,5′-diisopropyl-3,3′-dimethyl-8,8′-bis-propylimino-
methyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol: see gen-
et al. [34], are more consistent with a methoxy group in
positions 6,6′ than in position 7,7′ as previously proposed
by Adams et al. [1].
eral method for synthesis. Obtained 191 mg (yield 92%)
+
1
HPLC: k′(III): 1.09; EI-MS: m/z 601 (MH ); H-NMR-
CDCl : 1.02 (t, 6H, 2 HC=N–CH –CH –CH ), 1.54
3
2
2
3
(
d, 12H, 2 HC–(CH ) ), 1.73 (m, 4H, 2 HC=N–
4
2
2
.1.4. Synthesis of 5,5′-diisopropyl-
3 2
CH –CH –CH ), 2.13 (s, 6H, 2 Ar–CH ), 3.46 (t, 4H, 2
HC=N–CH –CH –CH ), 3.74 (m, 2H, 2 HC–(CH ) ),
7
2
,3,4,2′,3′,4′-hexamethoxy-7,7′-dimethyl-
H,2′H-[8,8′]bi[naphtho[1,8-bc]furanyl] 5
Two grams (3.46 mmol) of 1 in a solution of 20 mL of
2
2
3
3
2
2
3
3 2
.60 (s, 2H, 2 Ar–H), 9.65 (d, 2H, 2 HC=N–R); 13.27 (br,
H, 2 OH at 7,7′ positions).
9: 5,5′-diisopropyl-8,8′-bis-(isopropylimino-methyl)-
absolute methyl alcohol and 20 mL of dimethyl sulfate
was stirred at room temperature under argon. When
solution was complete, a solution of 30 mL 10% methyl
alcoholic potassium hydroxide was added dropwise and
the mixture was allowed to stir for 48 h at room tempera-
ture. Excess dimethyl sulfate was decomposed with 10%
aqueous sodium hydroxide solution. The white precipi-
tate that formed was filtered, washed with cool methanol
and dried: 1.6 g (76.9% yield). HPLC: k′(I): 5.31 and
3,3′-dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol:
see general method for synthesis. Obtained 201 mg (yield
+
96%). HPLC: k′(III): 1.04; EI-MS: m/z 601 (MH );
1
H-NMR-CDCl : 1.39 (t, 6H, 2 HC=N–CH–(CH ) ),
3
3 2
1.55 (d, 12H, 2 HC–(CH ) ), 2.13 (s, 6H, 2 Ar–CH ),
3.46 (t, 4H, 2 HC=N–CH –CH –CH ), 3.73 (m, 4H, 2
HC=N–CH–(CH ) ) and 2 HC–(CH ) ), 7.61 (s, 2H, 2
Ar–H), 9.72 (d, 2H, 2 HC=N–R); 13.28 (br, 2H, 2 OH at
7,7′ positions).
3
2
3
2
2
3
3
2
3 2
+
1
5
.85; EI-MS: m/z: 603 (MH ); H-NMR-CDCl3
(300 MHz) 1.54 (d, 12H, 2 HC–(CH ) ), 2.32 (m, 6H, 2
3
2

V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
811
1
0: 8,8′-bis-butyliminomethyl-5,5′-diisopropyl-3,3′-
CH –(CH ) –CH ), δ 1.55 (d, 12H, 2 HC–(CH ) ), δ
2 2 2 3 3 2
dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol: see
1.73 (m, 4H, 2 HC=N–CH –CH –(CH ) –CH ), 2.13 (s,
2 2 2 3 3
general method for synthesis. Obtained 196 mg (yield
6H,
2
Ar–CH3),
δ
3.49 (t, 4H,
2
3 2
HC=N–
9
0%) HPLC: k′(III): 1.58; EI-MS: m/z 635 (M + Li), 629
CH –(CH ) –CH ), 3.74 (m, 2H, 2 HC–(CH ) ), 7.61 (s,
2
2 4
3
+ 1
(MH ); H-NMR-CDCl : 0.98 (t, 6H, 2 HC=N–
2H, 2 Ar–H), δ 9.64 (s, 2H, 2 HC=N–R); 13.31 (br, 2H,
2 OH at 7,7′ positions).
3
CH –CH –CH –CH ), 1.43 (m, 4H,
2
HC=N–
2
2
2
3
CH –CH –CH –CH ),1.53 (d, 12H, 2 HC–(CH ) ),1.72
15: 8,8′-bis-heptyliminomethyl-5,5′-diisopropyl-3,3′-
dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol: see
general method for synthesis. Obtained 176 mg (yield
2
2
2
3
3 2
(
m, 4H, 2 HC=N–CH –CH –CH –CH ), 2.13 (s, 6H, 2
2 2 2 3
Ar–CH ), 3.49 (t, 4H, 2 HC=N–CH –CH –CH –CH ), δ
3
2
2
2
3
+
3
.74 (m, 2H, 2 HC–(CH ) ), 7.61 (s, 2H, 2 Ar–H), 9.65
71%) HPLC: k′(III): 5.23; EI-MS: m/z 713 (MH );
3
2
1
(s, 2H, 2 HC=N–R), 13.35 (br, 2H, 2 OH at 7,7′
H-NMR-CDCl : 0.88 (t, 6H, 2 HC=N–(CH ) –CH ),
3
2 6
3
positions).
1.31 (m, 18H, 2 HC=N–(CH ) –CH –CH , 2 HC=N–
2
5
2
3
1
1: 8,8′-bis-(sec-butylimino-methyl)-5,5′-diisopropyl-
(CH ) –CH –CH –CH , 2 HC=N–(CH ) –CH –(CH ) –
CH , 2 HC=N–(CH ) –CH –(CH ) –CH ), 1.54 (d, 12H,
3 2 2 2 2 3 3
2 4 2 2 3 2 3 2 2 2
3
,3′-dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol:
see general method for synthesis. Obtained 196 mg
yield 90%) HPLC: k′(III): 1.51; EI-MS:m/z 635 (M +
2
HC–(CH ) ), 1.71 (m, 4H,
2
HC=N–CH2–
3
2
(
CH –(CH ) –CH ), 2.13 (s, 6H, 2 Ar–CH ), 3.49 (t, 4H,
2 HC=N–CH –(CH ) –CH ), 3.74 (m, 2H, 2 HC–(CH ) ),
2
2 4
3
3
+
1
Li), 629 (MH ); H-NMR-CDCl : 0.98–1.38 (m, 12H, 2
3
2 2 5 3 3 2
CH –CH–(CH ) ), 1.54 (d, 12H, 2 CH–(CH ) ), 1.67
7.61 (s, 2H, 2 Ar–H), 9.65 (s, 2H, 2 HC=N–R);13.34 (br,
2H, 2 OH at 7,7′ positions).
2
3 2
3 2
(
m, 4H, 2 CH –CH–(CH ) ), 2.12 (s, 6H, 2 Ar–CH ),
2 3 2 3
3
.44 (m, 2H, 2 CH –CH–(CH ) ) 3.76 (m, 2H, 2
16: 5,5′-diisopropyl-3,3′-dimethyl-8,8′-bis[(2-phenyl-
propylimino)-methyl]-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-
hexaol: see general method for synthesis. Obtained
241 mg (yield 92%) HPLC: k′(III): 1.00, 1.57; EI-MS:
2
3 2
CH–(CH ) ), 5.58 (s, 2H, 2 OH at 1,1′ positions), 7.61
3
2
(
(
s, 2H, 2 Ar–H), 8.03 (s, 2H, 2 OH at 6,6′ positions), 9.7
2s, 2H, 2 HC=N–R), 13.3 (br, 2H, 2 OH at 7,7′
+
1
positions).
m/z 753 (MH ); H-NMR-CDCl : 1.4 (d, 6H, 2
3
1
2: 8,8′-bis-(tert-butylimino-methyl)-5,5′-diisopropyl-
CH –CH(CH )–C H ), 1.53 (d, 12H, 2 HC–(CH ) ), 2.1
2
3
6
5
3 2
3
,3′-dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol:
(s, 6H, 2 Ar–CH ), 3.08 (m, 2H, 2 CH –CH(CH )–C H ),
3 2 3 6 5
see general method for synthesis. Obtained 190 mg
3.67 (m, 6H, 2 CH –CH(CH )–C H and 2 HC–(CH ) ),
2 3 6 5 3 2
(
yield 87%) HPLC: k′(III): 0.77, 1.42; EI-MS: m/z 635
M + Li), 629 (MH ); H-NMR-CDCl : 1.27 (s,
5.51 (br, 2H, 2 OH at 1,1′ positions), 7.31 (m, 10H, 2
CH –CH(CH )–C H ), 7.59 (s, 2H, 2 Ar–H), 7.99 (br,
+
1
(
3
2
3
6 5
C–(CH ) ), 1.53 (d, 12H, 2 CH–(CH ) ), 2.13 (s, 6H, 2
2H, 2 OH at 6,6′ positions), 9.5 (d, 2H, 2 HC=N–R),
13.32 (br, 2H, 2 OH at 7,7′ positions).
3
3
3 2
Ar–CH ), 3.74 (m, 2 CH–(CH ) ), 7.62 (s, 2H, 2 Ar–H),
3
3 2
8
.02 (br, 2H, 2 OH at 6,6′ positions), 9.84 (2s, 2H, 2
HC=N–R), 13.43 (br, 2H, 2 OH at 7,7′ positions).
3: 5,5′-diisopropyl-3,3′-dimethyl-8,8′-bis-pentyl-
17: 2-({1,6,7,1′,6′,7′-hexahydroxy-5,5′-diisopropyl-8′-
[(1-methoxycarbonyl-2-phenyl-ethylimino)-methyl]-3,3′-
dimethyl-[2,2′]binaphthalenyl-8-ylmethylene}-amino)-3-
phenyl-propionic acid methyl ester: see general method
for synthesis. Obtained 220 mg (yield 84%). EI-MS: m/z
1
iminomethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol:
see general method for synthesis. Obtained 190 mg (yield
+
1
8
3%). HPLC: k′(III): 2.31; EI-MS: m/z 663 (M + Li), 657
753 (MH ); H-NMR-CDCl3: 1.53 (d, 12H,
2
+
1
(MH ); H-NMR-CDCl : 0.91 (t, 6H, 2 HC=N–
HC–(CH ) ), 2.07 (s, 6H, 2 Ar–CH ), 3.17–3.32 (m, 4H,
3
3 2
3
(
2
2
CH ) –CH ), 1.34 (m, 8H, 2 HC=N–(CH ) –CH –CH ,
2 CH(COOCH )CH –C H ), 3.72 (m, 2H,2 HC–(CH ) ),
3.77 (s, 6H, 2 CH(COOCH )CH –C H ), 4.3 (m, 2H, 2
3 2 6 5
2
4
3
2 3
2
3
3 2 6 5 3 2
HC=N–(CH ) –CH –CH –CH ), 1.53 (d, 12H,
2
2
2
2
3
HC–(CH ) ),
δ
1.71 (m, 4H,
2
HC=N–
CH(COOCH )CH –C H ), 5.37 (br, 2H, 2 OH at 1,1′
3
2
3 2 6 5
CH –CH –(CH ) –CH ), δ 2.13 (s, 6H, 2 Ar–CH ), δ
positions), 7.23 (m, 10H, 2 CH(COOCH )CH –C H ),
3 2 6 5
2
2
2 2
3
3
3
2
2
.49 (t, 4H, 2 HC=N–CH –(CH ) –CH ), δ 3.74 (m, 2H,
7.55 (s, 2H, 2 Ar–H), 7.94 (s, 2H, 2 OH at 6,6′ positions),
9.27–9.39 (d, 2H, 2 HC=N–R), 13.36 (br, 2H, 2 OH at
7,7′ positions).
2
2 3
3
HC–(CH ) ), δ 7.61 (s, 2H, 2 Ar–H), 9.64 (s, 2H,
3
2
HC=N–R); 13.31 (br, 2H, 2 OH at 7,7′ positions).
1
4: 8,8′-bis-hexyliminomethyl-5,5′-diisopropyl-3,3′-
18: 8,8′-bis-dodecyliminomethyl-5,5′-diisopropyl-3,3′-
dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol: see
general method for synthesis. Obtained 270 mg (yield
dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol: see
general method for synthesis. Obtained 204 mg (yield
+
1
8
(
6%) HPLC: k′(III): 3.43; EI-MS: m/z 691 (M + Li), 684
91%) HPLC: k′(III): EI-MS: m/z 853 (M ); H-NMR-
CDCl : 0.88 (t, 6H, 2 HC=N–(CH ) –CH ), 1.27 (m,
+
1
M ); H-NMR-CDCl3: 0.87 (t, 6H,
2 HC=N–
3
2 11
3
(
2
CH ) –CH ), 1.28 (m, 12H, 2 HC=N–(CH ) –CH –CH ,
36H, 2 HC=N–(CH ) –(CH ) –CH ), 1.53 (d, 12H, 2
2
5
3
2 4
2
3
2 2 2 9 3
HC=N–(CH ) –CH –CH –CH , 2 HC=N–(CH ) –
HC–(CH3)2), 1.70 (m, 4H,
2
HC=N–CH2–
2
3
2
2
3
2 2

8
12
V.T. Dao et al. / Eur. J. Med. Chem. 35 (2000) 805–813
CH –(CH ) –CH ), 2.13 (s, 6H, 2 Ar–CH ), 3.48 (t, 4H,
the extracted dye at 540 nm, using a Uniskam-II micro-
plate reader (Labosystems, Life Sciences International).
For most of the compounds examined, final concentra-
tions tested ranged from 0.5–10 µM and each concentra-
tion was added to two wells per plastic plate. The IC50 of
each compound was calculated by plotting the percent of
cell death against the concentration of the test compound.
For the purpose of comparison, the cytotoxic activities
of two clinically useful anticancer agents, doxorubicin
and docetaxel, were determined using the same method as
described above. Doxorubicin was purchased as an in-
jectable solution for hospital use, docetaxel was a gift
from Rhône-Poulenc Rorer.
The possible implication of free radical formation in
the cytotoxic activities of gossypol and gossypolone was
examined by studying the effect of various scavengers on
these activities. Inasmuch as calf serum present in the
culture medium does contain endogenous scavengers of
free radicals, these tests were performed on cells main-
tained in serum-free medium.
KB cells were first grown as usual in the presence of
foetal calf serum, but after 24 hours incubation at 37 °C
adherent cell layers in the wells were washed with PBS
and the medium was changed to serum-free MEM.
Mannitol, catalase and α-tocopherol (all purchased from
Sigma) were added to the cultures at predetermined
non-toxic concentrations, at the same time as various
concentrations of gossypol and gossypolone. The percent
of cell destruction was determined after 48 h incubation
at 37 °C by the neutral red viability test as described
above.
2
2 9
3
3
2
HC=N–CH –(CH ) –CH ), δ 3.74 (m, 2H, 2
2 2 10 3
HC–(CH ) ), δ 7.61 (s, 2H, 2 Ar–H), 9.65 (s, 2H, 2
3
2
HC=N–R); 13.34 (br, 2H, 2 OH at 7,7′ positions).
1
9: 8,8′-bis-ethyliminomethyl-6,7,6′,7′-tetrahydroxy-
5
1
,5′-diisopropyl-3,3′-dimethyl-[2,2′]binaphthalenyl-1,4,
′,4′-tetraone: see general method for synthesis. Ob-
tained 182 mg (yield 82%) HPLC: k′(III): EI-MS: m/z
+
1
6
01 (MH ); H-NMR-CDCl : 1.27–1.44 (m, 18H, 2
3
HC–(CH ) , 2 HC=N–CH –CH ), 1.99 (s, 6H, 2
3
2
2
3
Ar–CH ), 3.69 (q, 4H, 2 HC=N–CH –CH ), 3.85 (m,
3
2
3
2
H, 2 HC–(CH ) ), δ 9.64 (d, 2H, 2 HC=N–R).
3 2
2
0:
methyl-8,8′-bis-pentyliminomethyl-[2,2′]binaphthalenyl-
,4,1′,4′-tetraone: see general method for synthesis. Ob-
tained 176 mg (yield 70%) HPLC: k′(III): EI-MS: m/z
6,7,6′,7′-tetrahydroxy-5,5′-diisopropyl-3,3′-di-
1
+
1
6
85 (MH );
H-NMR-CDCl3: 0.91 (t, 6H,
2
HC=N–CH –CH ), 1.35–1.46 (20H, 2 HC–(CH ) , 2
2
3
3 2
HC=N–(CH ) –(CH ) –CH ), 1.75 (m, 4H,
2
2
2
2 2
3
HC=N–CH –CH –(CH ) –CH ), 1.99 (s, 6H, 2 Ar–CH ),
2
2
2 2
3
3
3
.60 (t, 4H, 2 HC=N–CH –(CH ) –CH ), 3.87 (m, 2H, 2
2 2 3 3
HC–(CH ) ), 9.60 (d, 2H, 2 HC=N–R).
3
2
4.2. Biological studies
4.2.1. Cytotoxicity
The in vitro cytotoxic activities of all the various
gossypol derivatives were evaluated in KB cells (human
epidermoid carcinoma of the mouth), originally obtained
from the American Type Culture Collection.
The KB cell line was grown in MEM (minimal
essential medium, with Earle’s salt solution), purchased
at Seromed, containing 10% foetal calf serum, 2 mM
L-glutamine, 60 u/ml penicillin G and streptomycin sul-
fate and 40 µg/mL gentamycin. For the tests, KB cells
were grown as monolayers in Nunc 24-well plastic plates
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3