Design and synthesis of a gossypol derivative with improved antitumor activities

Article abstract of DOI:10.1002/ardp.200800185

A novel chemical process has been devised for the synthesis of a new derivative of gossypol, 6,7,6',7'-tetrahydroxy-5,5'-diisopropyl-3,3'-dimethyl- [2,2']binaphthalenyl-1,4,1',4'-tetraone (Apogossypolone). This new process has only four steps, with a shorter synthesis span, a simple purification process, and improved yield and quality. The structure of apogossypolone was characterized by ¹H-nuclear magnetic resonance, ¹³C- nuclear magnetic resonance, mass spectroscopy, infrared spectroscopy, and elemental analysis. Cell-cytotoxicity assay demonstrates that apogossypolone is three- to six-fold more potent than the parent compound, (-)-gossypol, in inhibiting the human prostate tumor cell lines PC-3 and DU-145 as well as the human breast cancer cell line MDA-MB-231. The colony-formation assay with DU-145 cells showed that apogossypolone inhibited more than 70% of colony formation at 1 μM, whereas (-)-gossypol at 10 μM only inhibited less than 50% of colony formation. The results indicate that apogossypolone exerts strong antitumor activities in human prostate and breast cancer cells, and thus represents a promising cancer therapeutic.

Full text of DOI:10.1002/ardp.200800185

Arch. Pharm. Chem. Life Sci. 2009, 342, 223–229
Y. Zhan et al.
223
Full Paper
Design and Synthesis of a Gossypol Derivative with Improved
Antitumor Activities
Yonghua Zhan1, Guangfeng Jia1, Daocheng Wu1,*, Yiqing Xu2, and Liang Xu2
1 Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and
Technology, Xi'an Jiaotong University, Xi'an, China
2 Department of Radiation Oncology, Division of Cancer Biology, University of Michigan, Ann Arbor, MI, USA
A novel chemical process has been devised for the synthesis of a new derivative of gossypol,
6,7,69,79-tetrahydroxy-5,59-diisopropyl-3,39-dimethyl-[2,2']binaphthalenyl-1,4,19,49-tetraone
(Apo-
gossypolone). This new process has only four steps, with a shorter synthesis span, a simple puri-
fication process, and improved yield and quality. The structure of apogossypolone was character-
1
ized by H-nuclear magnetic resonance, 13C-nuclear magnetic resonance, mass spectroscopy,
infrared spectroscopy, and elemental analysis. Cell-cytotoxicity assay demonstrates that apogos-
sypolone is three- to six-fold more potent than the parent compound, (–)-gossypol, in inhibiting
the human prostate tumor cell lines PC-3 and DU-145 as well as the human breast cancer cell
line MDA-MB-231. The colony-formation assay with DU-145 cells showed that apogossypolone
inhibited more than 70% of colony formation at 1 lM, whereas (–)-gossypol at 10 lM only inhib-
ited less than 50% of colony formation. The results indicate that apogossypolone exerts strong
antitumor activities in human prostate and breast cancer cells, and thus represents a promising
cancer therapeutic.
Keywords: Antitumor activity / Apogossypolone / Assay / Gossypol / Synthesis /
Received: October 14, 2008; accepted: November 14, 2008
DOI 10.1002/ardp.200800185
Supporting Information for this article is available from the author or on the WWW under
http://www.wiley-vch.de/contents/jc_2019/2009/2008-00185_s.pdf
lignancy characteristics [5–7]. Its anticancer activities
have been attributed to its binding affinity to Bcl-2, Bcl-
xL, and Mcl-1 proteins-rational therapeutic targets
because of their roles in regulating the pathway which
leads to apoptosis of tumor cells [8–12]. However, it is not
widely used clinically because it is very unstable, toxic,
and insoluble.
Thus, extensive attempts to chemically modify gossy-
pol to obtain therapeutic antitumor agents with greater
potency and less toxicity have been performed. More
than 50 new analogues of gossypol have been synthesized
to date [13–16]. Unfortunately, none of them has been
used in the clinic as an antitumor agent because of unde-
sirable side effects, insolubility and a lack of selectivity
against tumor cells. Therefore, (–)-gossypol is still the
world's only orally available small-molecule inhibitor of
Bcl-2, Bcl-xL and Mcl-1 proteins that has advanced into
clinical trials [17].
Introduction
Gossypol is a yellow polyphenolic compound found in
pigment glands distributed throughout the cotton plant
(Gossypium sp.). It has been associated with a wide range
of biological and medicinal activities, including antifer-
tility [1], antimalarial [2], antitumor [3], and antiviral
effects [4]. Therefore, it is widely used in the medical and
pharmaceutical field. Recently, a number of research
reports suggested that gossypol is a promising novel anti-
cancer drug, especially (–)-gossypol, which has received
considerable attention because of its unique antima-
Correspondence: Daocheng Wu, Key Laboratory of Biomedical Informa-
tion Engineering of Education Ministry, School of Life Science and Tech-
nology, Xi'an Jiaotong University, Xi'an, 710049, China.
E-mail: wudaocheng@mail.xjtu.edu.cn
Fax: +86 29 82663941
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Arch. Pharm. Chem. Life Sci. 2009, 342, 223–229
Figure 1. Structures of (–)-gossypol ((–)-G) and gossypol.
Figure 2. Structures of gossypolic acid and gossypolonic acid.
Although (–)-gossypol represents a potentially interest- charged at physiological conditions (pH = 7.4), and this
ing lead compound, it can be further optimized for better may prevent them from entering cells. Both compounds,
efficacy of therapy and reduced toxicity as a new drug tar- in fact, were found, in spite of their excellent binding
geting Bcl-2, Bcl-xL and Mcl-1 proteins [18, 19]. Both (–)- affinities to Bcl-2, to be poor inhibitors of cell growth in
gossypol ((–)-G) and gossypol contain two reactive alde- PC-3 cells with IC50 values greater than 10 lM.
hyde groups in their structures (Fig. 1). These two reac-
To overcome the poor cell permeability of gossypolic
tive groups can combine with lysine residues of proteins and gossypolonic acid, we designed and synthesized apo-
to form Schiff's bases, which have been assumed to cause gossypolone, in which the two aldehyde groups of gossy-
the toxicity of gossypol in animals and humans. This tox- pol are completely removed. Computational docking of
icity, albeit mild, greatly limits the maximum dose of (–)- these compounds, followed by lengthy molecular
gossypol that can be given to patients. In addition, the dynamic simulation, revealed that the three hydroxyl
compound can combine with other proteins when it groups on the left naphthyl ring in apogossypolone form
enters the bodies of animals and humans, causing reduc- several highly optimized hydrogen bonds with Arg 139
tion of therapeutic efficacy and enhancement of toxicity. in Bcl-2, compensating for the removal of the aldehyde
In cancer clinical trials, gossypol could not be adminis- group in (–)-gossypol [28, 29]. Apogossypolone was deter-
trated i.v. and has a maximum tolerated dose (MTD) of mined to have Ki values of 35 nM. The binding curve for
40 mg daily when given orally [20]. It is expected that apogossypolone to Bcl-2 is shown in Fig. 3. The Ki value
removal of the aldehyde groups will significantly reduce for apogossypolone to Bcl-xL was determined to be
gossypol's toxicity in humans. Moreover, although the 660 € 40 nM, similar to that of (–)-gossypol. Hence, apo-
binding affinity of (–)-gossypol to Bcl-2 is fairly high (Ki = gossypolone represents a new and more potent small
237 nM) and it has been one of the first Bcl-2 inhibitors molecular inhibitor of Bcl-2/Bcl-xL.
reported, its binding affinity to Bcl-2 protein is signifi-
In view of the available studies on the structure-activity
cantly less than that of Bad 25-mer BH3 peptide (Ki = relationship and in continuation of our ongoing efforts
6.9 nM) in binding assay [21–24]. Therefore, despite the on the design and development of structurally modified
fact that (-)-gossypol represents a potentially interesting gossypol, here, we describe a novel chemical synthesis
lead compound, it is necessary to find novel and safer process of 6,7,69,79-tetrahydroxy-5,59-diisopropyl-3,3'-
derivatives of gossypol with greatly improved binding dimethyl-[2,29] binaphthalenyl-1,4,19,49-tetraone (Apogos-
affinity to Bcl-2 protein, provided that these new ana- sypolone) with improved yield, and quality of its antitu-
logues have comparable or better cellular activity in can- mor activities.
cer cells with high levels of endogenous Bcl-2 protein, as
well as good selectivity in normal and cancer cells with
low levels of Bcl-2 protein.
Based upon the mechanism of (–)-gossypol's binding
Results and discussion
affinity to Bcl-2/Bcl-xL, it has been proposed that one aldy- Chemistry
hyde group interacts with a conserved Arginine residue We have adopted a concise synthetic route for the prepa-
in Bcl-2/Bcl-xL, while the other aldehyde group is exposed ration of apogossypolone. The target compound 5 was
to solvent [25–27]. To maintain this interaction with Arg synthesized as shown in Scheme 1. Generally, the two
residue in Bcl-2/Bcl-xL, we designed and synthesized two aldehyde groups of gossypol may be removed through
new analogues, gossypolic acid and gossypolonic acid heating in a basic solvent. In addition, gossypol may also
(Fig. 2). These two compounds were determined to have be decarbonylated by reaction with HSCH2CH2SH in the
Ki values of 90 nM and 150 nM, respectively, and thus presence of BF3 / Et2O. Here, gossypol was decarbonylated
were slightly more potent than (–)-gossypol. However, the by heating in concentrated aqueous sodium hydroxide
two acid groups in these compounds are negatively using a steam bath. The whole reaction process was not
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Arch. Pharm. Chem. Life Sci. 2009, 342, 223–229
Gossypol Derivative with Improved Antitumor Activities
225
to shield the hydroxy group in compound 2; this is
important because of its instability. There were many
chemical groups we could select, such as alkanoyl, aral-
kanoyl, benzoyl, methyl, and alkyl groups. Here, we
selected the alkanoyl group to protect compound 2 by
treatment with anhydride in pyridine at high tempera-
tures. After adding the same equivalent of water, we
easily obtained the crude product ester 3 easily. It is note-
worthy that the crude compound was purified only by
treating it with an equivalent volume of boiling ethyl
acetate and petroleum ether, resulting in a 98% yield of
pure ester 3. In addition to these steps, it was important
to select a suitable solvent because it directly affected the
reaction condition and the yield. Fortunately, we
obtained compound 3 by selecting a suitable reagent
anhydride and the solvent pyridine, thereby having to
heat it for only several minutes at the defined tempera-
tures throughout the whole process.
Figure 3. Binding curve for apogossypolone to Bcl-2.
only easy to operate but also lasted only 30 minutes
because of the use of the NaOH or KOH instead of other
bases. Then, the reaction mixture was poured onto ice
containing concentrated sulfuric acid. The resulting pre-
cipitate was extracted with ether and further purified
through hot methanol and water, first obtaining com-
pound 2. After optimization, the yield was significantly
increased from 90% to 98% by canceling the purification
process without affecting the last result. After compound
2 was obtained, we looked for a suitable protection group
With the ester 3 in hand, the selection of an oxidation
agent was critical in order to get compound 4. Based on
the structure of compound 3, a powerful oxidation
reagent was needed in order to successfully complete the
structure modification. With our knowledge of the oxida-
tion reagents, the use of periodic acid with chromium
oxide was obvious for its superior oxidation character.
However, at last we selected the Kiliani's solution which
is a mixture of H2SO4, H2O, Cr2O3 in defined proportions.
We subjected the ester 3 to seven equivalents Kiliani's sol-
Scheme 1. Synthesis of the target compound 5.
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Y. Zhan et al.
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Table 1. Structures and physico-chemical data of compounds 2–5.
Compound
M.p.
(8C)
Reaction time
(min)
Yield
(%)
Formula
M.W.
2
3
4
5
215–217
289–291
228–230
217–219
30
10
10
98
90
48
98
C28H30O6
C40H42O12
C36H34O12
C28H26O8
462.52
714.74
658.63
490.49
120
Figure 4. MTT-assay results for com-
pound 5.
ution at 958C in acetic acid for only five minutes, fol- 2.2 Biological evaluation
lowed by addition of 50 equivalents of water, obtaining To test the anticancer activity of compound 5 obtained
the crude product ester 4. The resulting crude product with the new synthesis method, the MTT-based cell cyto-
was further purified from hot methanol aided by ultra- toxicity assay and colony-formation assay were carried
sound to reach crystallization. The target compound 5 out. Figure 4 shows the MTT assay results. Compound 5
was obtained by removal of the protection groups of ester has IC50 values 1.0, 2.0, and 0.3 lM, three- to sixfold more
4 by reacting it with ten equivalents sodium carbonate in potent than (–)-gossypol which has IC50 of 6.1, 6.0, and
dioxane at 808C for 2 h. The product was then acidified 1.5 lM in human prostate cancer cell lines, PC-3 and DU-
and isolated by extraction with ether. After purification 145, and human breast cancer cell line MDA-MB-231,
from the hot methanol and water with the aid of ultra- respectively. The colony formation assay was conducted
sound. Compound 5 was obtained with a significant yield in DU-145 cells. Figure 5 shows that apogossypolone
of 98% (Table 1). As can be seen from the whole synthesis potently inhibited the colony formation of DU-145 cells.
process, we adopted the crystallization and recrystalliza- At 1 lM, apogossypolone inhibited more than 70% of col-
tion to purify the crude product in contrast to using flash ony formation compared with that of the solvent control,
silica gel column chromatograpy. In addition, the prod- whereas (–)-gossypol, at 10 lM, only inhibited less than
uct yield was also significantly improved by adopting 50% of colony formation. These data suggest that the apo-
ultrasound crystallization. The structures of the com- gossypolone we synthesized has strong antitumor activ-
pounds in Scheme 1 were confirmed by infrared spectro- ity. Regarding the antitumor mechanism of apogossypo-
1
scopy (IR), H-NMR, 13C-NMR, mass spectroscopy, and ele- lone, the likely mechanism is that apogossypolone binds
mental analysis.
to Bcl-2 (or Mcl-1, Bcl-xL, A1, Bcl-w) and prevents its associ-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 223–229
Gossypol Derivative with Improved Antitumor Activities
227
Figure 5. Inhibition of the colony formation of DU-145 cells by apogossypolone.
ation with BH3-only pro-apoptotic proteins, thus process, and improved quality. Our results differ from
unleashing the pro-apoptotic proteins to participate in another recent report [32]. In addition, it is noteworthy
the apoptotic response. Yet, it should be noted that the that we applied ultrasound crystallization, which
exact mechanism of action of apogossypolone is unclear improved the yield significantly. The MTT-based cell-cyto-
to date. Currently, apogossypolone has been reported to toxicity assay and colony-formation assay show that apo-
be a strong inhibitor of Bcl-2, Bcl-xL and Mcl-1 proteins gossypolone has significantly improved antitumor activ-
both in vitro and in vivo according to our earlier report ity in the prostate-cancer model compared to the parent
[31], as well as in a follicular small cleaved cell lymphoma compound (-)-gossypol. Since the anticancer mechanism
model, nasopharyngeal carcinoma xenografts, and is not clear, further research in this area is in progress in
human leukemia in a recent study [30, 33]. Our experi- our laboratories.
ment further proved that apogossypolone has antitumor
activity in the prostate cancer model.
Apogossypolone showed potent radiosensitization
Experimental
potential in the prostate cancer PC-3 xenograft model in
Synthesis
nude mice via oral administration, leading to complete
tumor regression in seven out of ten tumors in the com-
bination treatment group [31]. Besides these results, apo-
gossypolone is very stable with no decomposition
detected when it was stored at room temperature for sev-
eral weeks without the protection of nitrogen. Because
the major toxicity of gossypol in animals and humans
has been associated with its two reactive aldehyde
groups, removal of these aldehydes should significantly
reduce the toxicity. Our earlier studies have examined
the maximal tolerated dose (MTD) of apogossypolone and
(-)-gossypol using two different routes of administrations
oral and i.v. Via both routes of administration, the MTD
of apogossypolone was 240 mg/kg (orally) and 80 mg/kg
(i.v.), eight times better than (–)-gossypol, with MTD of
30 mg/kg and 10 mg/kg, respectively [31]. All the results
indicated that apogossypolone was superior to (–)-gossy-
pol, possibly making apogossypolone the most potent
gossypol derivative identified to date.
All reagents were purchased from commercial sources and were
used without further treatment, unless otherwise indicated.
Melting points were determined with a Mettler FP82 + FP80
apparatus (Mettler-Toledo, Greifensee, Switzerland) and have
not been corrected. The 1H-NMR and 13C-NMR spectroscopy were
recorded on a Bruker 400 UltrashieldTM (Bruker, Rheinstetten,
Germany), using TMS as the internal standard. IR spectroscopy
were obtained using a ThermoNicolet FT-IR Nexus spectropho-
tometer (Thermo Electron Corporation, Waltham, MA, USA)
with samples as KBr pellets. Elemental microanalyses were car-
ried out on vacuum-dried samples using an elemental analyzer
(LECO, CHN-900 Elemental Analyzer; LECO Corporation, St.
Joseph, MI, USA) and were in an acceptable range of € 0.4% for all
compounds. Mass spectra were recorded using a Hewlett-Pack-
ard MSD 5973N spectrometer (GC 6890plus/DIP; Agilent Tech-
nologies, Inc., Santa Clara, CA, USA).
(+)-5,59-Diisopropyl-3,39-dimethyl-[2,29]binaphthalenyl-
1,6,7,19,69,79-hexaol 2
Gossypol acetic acid (5.0 g, 8.7 mmol) was heated in 40% aqueous
sodium hydroxide (30 mL) at 958C for 30 min, then the reaction
mixture was poured onto ice-containing 98% concentrated sul-
furic acid. The resulting mixture was extracted with ether, the
combined extracts were washed with water, and concentrated
to yield crude 2. Recrystallization from methanol and water
afforded pure product 2 (4.9 g, 98%). Brown solide; m.p.: 215–
Conclusions
In conclusion, we have designed a novel synthesis
method for apogossypolone with only four steps, with a
shorter synthesis span, lower cost, a simple purification
1
2178C. H-NMR (300 MHz, CDCl3) d: 1.54 (d, J = 6.56 Hz, 12H, 12-
CH3, 129-CH3, 13-CH3, 139-CH3), 2.14 (s, 6H, 14-CH3, 149-CH3), 3.52
(m, 2H, 11-CH), 5.0 (s, 6H, 1-C-OH, 19-C-OH, 6-C-OH, 69-C -OH, 7-C-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 223–229
OH, 79-C-OH), 7.26 (s, 2H, 4-C-H, 49-C-H); 13C-NMR (75 MHz, MeOD)
d: 147.5, 116.9, 130.6, 113.1, 146.7, 145.7, 100.2, 118.8, 127.0,
18.0, 25.2, 16.8. IR (KBr, cm–1): 3427, 2872, 1615, 1594,1269. Anal.
Calcd. (%) for C28H30O6: C, 72.65; H, 6.49. Found: C, 72.64; H, 6.50.
Antitumor-activity assay
Cell culture and reagents
Prostate cancer cell lines used were obtained from the American
Type Culture Collection (Manassas, VA, USA). Cells were rou-
tinely maintained in an improved minimal essential medium
(Biofluids, Rockville, MD, USA) with 10% fetal bovine serum and
2 mM L-glutamine. Cultures were maintained in a humidified
incubator at 378C and 5% CO2. Apogssypolne was dissolved in
DMSO at 20 mM as the stock solution.
Acetic acid 1,7,19,69,79-pentaacetoxy-5,59-diisopropyl-3,39-
dimethyl-[2,29]binaphthalenyl-6-yl ester 3
Acetic anhydride (9 mL, 96.3 mmol) was added to the solution of
compound 2 in pyridine (20 mL). The reaction mixture was
heated at 1258C for 10 min and then allowed to cool and stand
at room temperature for 30 min. Water (250 mL) was added to
the reaction mixture and crystals of compound 3 formed. Recrys-
tallization from boiling ethyl acetate and petroleum ether
afforded pure product 3 (4.25 g, 90%). Pale yellow solid; m.p.:
289-2918C. 1H-NMR (300 MHz, CDCl3) d: 1.49 (d, J = 6.56 Hz, 12H,
12-CH3, 129-CH3, 13-CH3, 139'-CH3), 2.31-2.39 (m, 6H, 14-CH3, 149-
CH3), 3.82 (s, 2H, 11-CH), 7.98 (s, 2H, 4-C-H, 49-C-H), 7.26 (s, 2H, 8-C-
H, 89-C-H), 2.04 (s, 16-CH3, 169-CH3, 18-CH3, 189-CH3, 20-CH3, 209-CH3);
13C-NMR (75 MHz, CDCl3) d: 141.2, 126.9, 135.7, 122.7, 124.5,
144.9, 113.7, 131.1, 168.6, 27.5, 21.6, 20.7. IR (KBr, cm–1): 3444,
2960, 1770, 1592,1608. Anal. Calcd. (%) for C40H42O12: C, 67.16; H,
5.88. Found: C, 67.17; H, 5.87.
Growth / cytotoxicity assay
Cell-growth inhibition by Apogossypolone was determined by
the MTT-based cytotoxicity assay using Cell Proliferation
Reagent WST-1 (Roche) according to the manufacturer's instruc-
tion. Briefly, cancer cells (5000 cells/well) were plated in 96-well
culture plates, and various concentrations of apogossypolone
were added to the cells in triplicates. Four days later, WST-1 was
added to each well and incubated for 1.5 h at 378C. Absorbance
was measured with a plate reader at 450 nm with correction at
650 nm. The results are expressed as the % of absorbance of
treated wells versus that of vehicle control. IC50, the drug concen-
tration giving 50% growth inhibition was calculated via sigmoid
curve fitting using GraphPad Prism 5.0 (GraphPad, Inc.).
Colony-formation assay
Acetic acid 6,69,79-triacetoxy-5,59-diisopropyl-3,39-
dimethyl-1,4,19,49- tetraoxo-1,4,19,49-tetrahydro-[2,29]
binaphthalenyl-7-yl ester 4
The colony-formation assay was conducted in DU-145 cells. Two
hundred cells were plated in each well of a six-well plate, and
24 h later, (–)-gossypol and apogossypolone with appropriate
doses were added. After 5 days of incubation, 0.5 mL serum was
supplemented to each well. The colonies were stained with crys-
tal violet on day 14 and the colonies with over 50 cells were
counted.
Kiliani's solution (25 mL) was added to a solution of compound 3
in acetic acid (200 mL) and the reaction mixture was stirred at
958C for 10 min. Ice water (350 mL) was added to quench the
reaction and the yellow amorphous compound 4 was formed.
Recrystallization from hot methanol afforded pure compound 4
1
This study was supported in part by the National Nature Science Foun-
dation of China (Nos.30570494, 30772658 and 30710403089) (to D. Wu)
and USA Department of Defense, Prostate Cancer Research Program
(Nos. W81XWH-04-1-0215 and W81XWH-06-1-0010) (to L. Xu). We
thank Ms. Wenhua Tang and Yang Meng for technical support.
(1.7 g, 48%). Yellow solid; m.p.: 228-2308C. H-NMR (300 MHz,
CDCl3) d: 7.26 (s, 2H, 8-C-H, 89-C-H), 3.82 (s, 2H, 11-CH), 1.36 (m,
12H, 12-CH3, 129-CH3, 13-CH3, 139-CH3), 1.98-2.02 (m, 6H, 14-CH3,
149-CH3), 2.31 (s, 16-CH3, 169-CH3, 18-CH3, 189-CH3); 13C-NMR
(75 MHz, CDCl3) d: 184.2, 146.7, 144.7, 165.2, 166.7, 120.8, 122.6,
125.0, 18.7, 25.6, 13.1, 179.2, 19.7. IR ( KBr, cm–1): 3462, 2960,
1770, 1662, 1373, 1200. Anal. Calcd. (%) for C36H34O12: C, 65.59; H,
5.16. Found: C, 65.60; H, 5.17.
The author have declared no conflict of interest.
6,7,69,79-Tetrahydroxy-5,59-diisopropyl-3,39-dimethyl-
[2,29]binaphthalenyl-1,4,19,49-tetraone 5
References
A 20% solution of sodium carbonate (7 mL) was added to a solu-
tion of compound 4 (0.5 g, 0.9 mmol) in dioxane (10 mL) and the
reaction mixture was stirred at 808C for 2 h. After cooling, 4 M
HCl was added to the solution and the pH was adjusted to 4.
Ether was added, and the aqueous phase was extracted three
times with ether. The combined extracts were dried and yielded
the crude compound 5. Recrystallization from methanol and
water afforded pure compound 5 (0.45 g, 98%). Brick red solid;
m.p.: 217–2198C. 1H-NMR (300 MHz, MeOD) d: 7.26 (s, 2H, 8-C-H,
89-C-H ), 3.27–3.29 (m, 2H, 11-CH), 1.42 (d, J = 6.56 Hz, 12H, 12-
CH3, 129-CH3, 13-CH3, 139-CH3), 5.0 (s, 4H, 6-C-OH, 69-C-OH, 7-C-OH,
79-C-OH), 7.26 (s, 2H, 8-C-H, 89-C-H), 1.95 (s, 6H, 14-CH3, 149-CH3);
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135.2, 17.1, 24.8, 11.6. IR (KBr, cm–1): 3447, 2970, 1770, 1662,
1592. Anal. Calcd. (%) for C28H26O8: C, 68.50; H, 5.30. Found: C,
68.51; H, 5.29. MS (m/z): 489.1 [M]–, 491.0 [M]+.
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