Antineoplastic agents. 579. Synthesis and cancer cell growth evaluation of E-stilstatin 3: A resveratrol structural modification

Article abstract of DOI:10.1021/np9002146

As an extension of our earlier structure/activity investigation of resveratrol (1a) cancer cell growth inhibitory activity compared to the structurally related stilbene combretastatin series (e.g., 2a), an efficient synthesis of E-stilstatin 3 (3a) and its phosphate prodrug 3b was completed. The trans-stilbene 3a was obtained using a convergent synthesis employing a Wittig reaction with phosphonium bromide 9 as the key reaction step. Deprotection of the Z-silyl ether 13 gave E-stilstatin 3 (3a) as the exclusive product. The structure and stereochemistry of 3a was confirmed by X-ray crystal structure determination.

Full text of DOI:10.1021/np9002146

J. Nat. Prod. 2009, 72, 1637–1642
1637
Antineoplastic Agents. 579. Synthesis and Cancer Cell Growth Evaluation of E-Stilstatin 3: A
Resveratrol Structural Modification⊥
George R. Pettit,* Noeleen Melody, Andrew Thornhill, John C. Knight, Thomas L. Groy, and Cherry L. Herald
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State UniVersity, P.O. Box 871604,
Tempe, Arizona 85287-1604
ReceiVed April 9, 2009
As an extension of our earlier structure/activity investigation of resveratrol (1a) cancer cell growth inhibitory activity
compared to the structurally related stilbene combretastatin series (e.g., 2a), an efficient synthesis of E-stilstatin 3 (3a)
and its phosphate prodrug 3b was completed. The trans-stilbene 3a was obtained using a convergent synthesis employing
a Wittig reaction with phosphonium bromide 9 as the key reaction step. Deprotection of the Z-silyl ether 13 gave
E-stilstatin 3 (3a) as the exclusive product. The structure and stereochemistry of 3a was confirmed by X-ray crystal
structure determination.
The therapeutic potential of the E-stilbene resveratrol (1a),2a first
isolated from roots of Vertarum grandiflorum (white hellebore) in
1940, covers a wide range of diseases as noted in recent reviews.2b,c
Stilbene 1a has been reported to be an inhibitor of carcinogenesis
at multiple stages via its ability to inhibit cyclooxygenase3a,b and
is an anticancer agent with a role in antiangiogenesis2a,4 and in the
upregulation of phase II enzymes, which are involved in detoxifying
harmful metabolites and are therefore an indication of chemopre-
ventive activity.2a,5 More recently both in vitro and in vivo studies
showed that stilbene 1a induces cell cycle arrest and apoptosis in
tumor cells.6 The cardioprotective effects of resveratrol have been
found to include prevention of platelet aggregation in vitro,7
promotion of vasorelaxation,8,9 prevention of LDL oxidation,10 and
reduced formation of atherosclerotic plaques and restoration of flow-
mediated dilation in rabbits fed on a high-cholesterol diet.11
Resveratrol’s (1a) inhibition of cyclooxygenase enzymes3 led to
the discovery that, along with suppression of the apoptotic neuronal
cell death initiated by neuroinflammation typical of Parkinson’s
disease12a as well as other inflammatory responses,12b specific
immune responses were enhanced.13 Resveratrol has been suggested
to inhibit tissue death due to oxygen starvation during a heart attack
or stroke.14a,b Very importantly, resveratrol’s effects on longevity
have been of increasing interest.2c,15 In concert with these observa-
tions, stilbene 1a and the related Z-stilbene combretastatin A-4 (2a)
have been shown to improve fasting blood glucose levels and insulin
resistance as summarized below.
Scheme 1a
a Reagents and conditions: (a) piperidine/water, reflux, 48 h; (b) DIPEA,
DMF, TBDMSCl, rt, 18 h; (c) NaBH4, CH3CH2OH, 0 °C, rt, 2 h; (d) DCM,
PBr3, 0 °C, rt, 2 h; (e) toluene, PPh3, rt, 24 h.
Combretum caffrum.19 Among the combretastatins, the A-4 (2a,
CA4)19a has received major attention owing to its strong inhibition
of tubulin polymerization and selective targeting of tumor vascular
systems (cancer vascular disrupting), which cuts off the tumor blood
flow, leading to hemorrhagic necrosis as well as cancer antiangio-
genesis.20 After 10 years of advancing through phase I and II
clinical trials, the prodrug sodium combretastatin A-4 phosphate
(2b, CA4P) has now entered phase III human cancer clinical trials.
In addition, CA4P has advanced to phase II human clinical trials
against both myopic and wet macular degeneration (a leading cause
of blindness). Current results from the clinical trials have been very
encouraging and have stimulated a large number of important
SAR20a-c and broad-spectrum biological/medical studies.20a-c,21,22
Among the latter investigations are the recent reports that CA4P
strongly inhibits gastric tumor metastases by attenuating p-AKT
expression (facilitates migration of cancer cells to distant tissue
sites)21 and that both CA4 (2a) and resveratrol (1a) activate AMPK
(and downregulate gluconeogenic enzyme mRNA levels) in the
liver, which improved the fasting blood glucose levels in diabetic
db/db mice.22 The AMPK studies with CA4 also showed, as with
1a, that it activates PPAR transcriptional activity, which improved
the glucose tolerance in the diabetic mice.22 Overall these results,
with AMPK serving as a useful therapeutic target for controlling
type 2 diabetes and obesity, suggest another very important avenue
for future resveratrol (1a) and CA4 (and CA4P) new drug
research.22
An investigation of resveratrol metabolism in humans by Walle16
revealed that 1a was metabolized extensively in the body and that
the bulk of an intravenous initial dose was rapidly converted (∼30
min) to sulfate esters and other derivatives. Furthermore, when the
methylated analogue 1b was investigated, it was found to be more
active than the natural product in the majority of bioassays.17 Such
evidence raised the possibility that one (or more) of its many
metabolites is the biologically active agent, rather than resveratrol
itself, and has led to a number of investigations focused on the
synthesis of new structural modifications of resveratrol (1a),12b,18
including our earlier synthesis and biological study of sodium
resverastatin phosphate prodrug19j and stilstatins 1 and 2.1
For over 30 years, we have been exploring the chemistry and
the anticancer and other important biological properties of the
combretastatins that we isolated from the South African bush willow
⊥ Dedicated to the memory of Professor Georgy B. Elyakov (1929-2005)
for his pioneering contributions to the chemistry and biology of natural
products.
* To whom correspondence should be addressed. Tel: (480) 965-3351.
Fax: (480) 965-2747. E-mail: bpettit@asu.edu.
10.1021/np9002146 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/31/2009

1638 Journal of Natural Products, 2009, Vol. 72, No. 9
Pettit et al.
Scheme 2a
Table 1. Human Cancer Cell Growth Inhibition Results (GI50,
µg/mL)
cell line
pancreas breast
CNS
lung-NSC colon
prostate
structure BXPC-3 MCF-7 SF-268 NCI-H460 KM20L2 DU-145
1a19j
1b19j
1c19j
1d19j
2a19j
2c19g
3a
3.3
0.35
15.5
0.0034
0.39
3.9
>10
>10
5.0
3.9
4.1
0.56
5.0
0.0044
>0.01
3.6
0.62
13.2
0.0028
0.00060
0.038
>10
>10
4.9
13.1
22.0
3.5
1.5
10.2
0.0054
0.00080
0.037
>10
>10
6.3
a Reagents and conditions: (a) DCM, BCl3, 2 h, rt, BCl3, rt, 24 h; (b) DMF,
DIPEA, rt, TBDMS-Cl.
14.8
0.34
Because of the ever-increasing biological relationships of certain
combretastatins to resveratrol (1a), we have continued our structure/
activity investigations19g,j,23 and now report the synthesis of a new
structural modification of 1a, the polyhydroxylated trans-stilbene
designated stilstatin 3 (3a), and its phosphate prodrug (3b). Stilstatin
3 was evaluated first as a cancer cell growth inhibitor, and the data
were compared with those of the structurally similar 1a and 1c,
methylated derivatives 1b and 1d, combretastatin A4 isomers and
salts 2a-d, and the structural modification 4.
5.5 >10
>10 >10
>10
>10
3b
423
geometrical instability of related Z-isomers previously. For example,
the Z-isomer of stilbene 423 could be isolated but evaded biological
investigations owing to isomerization to the E-stilbene 4 over 1
day (room temperature). E-Stilstatin 3 (3a) recrystallized from
benzene-chloroform, and following initial characterization the
geometry and overall structure were confirmed by an X-ray crystal
structure determination (Figure 1).
Photochemical isomerization24 of 3a was also investigated as a
route to the Z-isomer, which was needed for biological comparison
and evaluation. For the E f Z conversion, Z-stilstatin 3 (3a) in
benzene was stirred with benzil and irradiated with a 254 nm UV
lamp for 29 h. The products were separated by column chroma-
tography to afford only unreacted E-isomer. Further attempts at
photochemical isomerization of the E-isomer in acetonitrile with
fluorenone as the sensitizer25 were also unsuccessful. Ultraviolet
irradiation was conducted using a standard laboratory UV lamp at
365 nm (and at >280 nm) and also using a medium-pressure UV
lamp under an argon atmosphere (photochemical cabinet) with the
exclusion of light for 4 h. In the latter experiments, the light pink
solution of 3a turned dark orange over time, but examination by
NMR did not reveal the Z-isomer as a product in any of these
experiments.
Next 3a was phosphorylated with dibenzylphosphite in the
presence of CCl419j,26 to give triphosphate 15. The phosphoric acid
intermediate was obtained by deprotection of 15 with TMS-Br at
rt and was isolated following extraction of the reaction mixture
with water. The sodium prodrug was obtained when the aqueous
fraction was concentrated to a small volume and eluted through a
column containing Dowex 50X8-200 ion-exchange resin (sodium
form). The UV-active fractions were combined and freeze-dried
to yield a cream solid, which was reprecipitated from a solution of
ethanol-water at 0 °C overnight to yield sodium phosphate 3b as
a colorless crystalline powder in quantitative yield.
Stilstatin 3a and its phosphate prodrug (3b) were found to exhibit
less cancer cell growth inhibitory activity than resveratrol (1a),
resveratrol analogue 1b, and E-stilbene 4 against a panel of human
cancer cell lines. Stilstatin 3a showed moderate cytotoxicity (GI50
5.5 µg/mL) against the breast MCF-7 cell line. A comparison of
cancer cell line data for the resveratrol synthetic modifications 1b,19j
4,23 and 3a showed a decrease in activity with successive addition
of hydroxyl groups to the B-ring of the E-stilbene system. Our
previous studies have shown the E-isomer of combretastatin A-419g
(2c) to be in some cases 100 times less active in various cancer
cell lines than the remarkably potent cis-isomer 2a. These results
are consistent with general observations to date that the E-stilbenes
are less active as cancer cell growth inhibitors compared to their
Z-counterparts. The Z-isomer of resveratrol (1c) was the exception
to this general rule and showed slightly less activity than the
E-isomer (1a). However the methylated Z-isomer (1d) proved to
be more active than its E-isomer (1b). Future investigation of
stilstatin 3a and the other resveratrol structural modifications noted
herein as sirtuin activators along with the combretastatin series may
provide further SAR insights.
Results and Discussion
The experimental procedures19 developed for our prior syntheses
of combretastatins19i were modified to give E-stilstatin 3. Scheme
1 outlines the synthesis of the necessary phosphonium bromide,
where intermediates 6 and 7 were prepared by modifications of
prior methods. Silyl ether 7 was reduced to alcohol 8 followed by
bromination and phosphorylation to give bromide 9, the A-ring
precursor of 3a. Synthesis of the B-ring counterpart was begun as
follows (Scheme 2). Commercially available aldehyde 10 was
selectively demethylated with BCl3 as previously described19i to
furnish diphenol 11. Subsequent protection19d with TBDMS-Cl gave
access to aldehyde 12 in very good overall yields. A Wittig reaction
sequence (Scheme 3) employing phosphonium bromide 9 and
aldehyde 12 led to stilbene 3a.
Treatment of 9 with n-butyllithium at -15 °C afforded the ylide,
and addition of aldehyde 12 gave the silyl ethers 13 and 14. The
cis/Z-isomer was isolated (by fractional crystallization from ethanol)
from the reaction product in 65% yield as a colorless crystalline
compound. Further separation of the reaction products (now rich
in trans/E) by recrystallization from CH3OH-CH2Cl2 gave the E
silyl ether as needles in ∼10% yield, confirmed by NMR spectro-
scopic analysis. A number of attempts at desilylation of Z-isomer
13 using tetrabutylammonium fluoride (TBAF) did not yield the Z
product but instead E-stilstatin 3 (3a). We have encountered the

A ResVeratrol Structural Modification
Journal of Natural Products, 2009, Vol. 72, No. 9 1639
Scheme 3a
a Reagents and conditions: (a) n-BuLi, THF, -15 °C; (b) THF, -10 °C, TBAF (1 M in THF); (c) CCl4, -10 °C, acetonitrile; (d) DIPEA, DMAP, dibenzyl
phosphite; (e) TMS-Br, DCM, rt; (f) ion exchange, Dowex 50X8-200 (sodium form).
Figure 1. X-ray crystal structure of E-stilstatin 3 (3a).
Experimental Section
benzaldehyde (5). Additional NMR and IR data are reported: 74% yield;
mp 113-115 °C [lit.27 mp 113-114 °C]; Rf 0.20 in 1:1 hexanes-EtOAc;
General Experimental Procedures. All solvents and reagents were
obtained from Sigma-Aldrich (Milwaukee, WI) and Acros Organics
(Fischer Scientific, Pittsburgh, PA). Ether refers to diethyl ether, and
Ar refers to argon gas. All starting reagents were used as purchased
unless otherwise stated. Reactions were monitored by TLC using
Analtech silica gel GHLF uniplates visualized under long- and short-
wave UV irradiation along with staining with phosphomolybdic acid/
heat, iodine, or KMnO4. Solvent extracts were dried over anhydrous
magnesium sulfate unless otherwise stated. Where appropriate, the crude
products were separated by flash column chromatography on silica gel
(230-400 mesh ASTM) from E. Merck. The photochemical experi-
ments were conducted with a standard laboratory UV lamp at 365 and
254 nm and with a medium-pressure mercury lamp at >280 nm in a
photochemical cabinet under argon.
Melting points are uncorrected and were determined employing an
electrothermal 9100 apparatus. The 1H and 13C NMR spectra were
recorded employing Varian Gemini 300, Varian Unity 400, or Varian
Unity 500 instruments using a deuterated solvent and were referenced
to either TMS or the solvent. The reported figures are given as ppm.
Phosphorus NMR was referenced against a standard of 85% H3PO4.
HRMS data were recorded with a Jeol LCmate mass spectrometer and
Jeol GCmate. Elemental analyses were determined by Galbraith
Laboratories, Inc., Knoxville, TN.
1
IR (neat) νmax 3231, 1671, 1607, 1513, 1464, 1330, 1113 cm-1; H
NMR (CDCl3, 300 MHz) δ 3.97 (6H, s, 2 × OCH3), 6.12 (1H, brs,
OH), 7.15 (2H, s, 2 × ArH), 9.82 (1H, s, CHO); 13C NMR (CDCl3,
100 MHz) δ 56.41, 106.65, 128.30, 140.81, 147.31, 190.72; HREIMS
m/z 182.0508 (calcd for C9H10O4, 182.0579).
4-(tert-Butyldimethylsilyloxy)-3,5-dimethoxybenzaldehyde (7). DI-
PEA (4.47 g, 34.6 mmol, 6.02 mL) was added to a stirred solution of
4-hydroxy-3,5-dimethoxybenzaldehyde 6 (4.1 g, 22.47 mmol) and tert-
butyldimethylsilyl chloride (4.06 g, 26.96 mmol) in anhydrous DMF
(100 mL). After the mixture was stirred at room temperature for 18 h,
water (100 mL) was added and the reaction mixture extracted with
EtOAc (3 × 100 mL). The combined organic phase was washed with
water (2 × 50 mL) and dried, and the solvent was removed in vacuo.
Separation was achieved by column chromatography, eluting with 1:1
hexanes-EtOAc, to give the title compound in quantitative yield (6.65
g) as an off-white solid: mp 78-79 °C [lit.28 mp 69-71 °C]; Rf 0.50
in 1:1 EtOAc-hexanes; IR (neat) νmax 2932, 1690, 1503, 1331, 1126
cm-1; 1H NMR (CDCl3, 300 MHz) δ 0.08 (6H, s, (CH3)2Si), 0.93 (9H,
t
s, Bu), 3.77 (6H, s, 2 × OCH3), 7.01 (2H, s, 2 × ArH), 9.72 (1H, s,
CHO); 13C NMR(CDCl3, 100 MHz) δ -4.63, 18.68, 25.58, 55.66,
106.59, 129.28, 140.53, 151.86, 190.88; HRMS APCI+ m/z 297.1519
[M + H]+ (calcd for C15H25O4Si, 297.1522).
4-(tert-Butyldimethylsilyloxy)-3,5-dimethoxybenzyl Alcohol (8).
A solution of benzaldehyde 7 (6.65 g, 22.47 mmol) in ethanol (30 mL)
4-Hydroxy-3,5-dimethoxybenzaldehyde (6). Preparation of com-
pound 6 was repeated as originally described27 from 3,4,5-trimethoxy-

1640 Journal of Natural Products, 2009, Vol. 72, No. 9
Pettit et al.
was stirred for 10 min at 0 °C. NaBH4 (1.1 g, 29.21 mmol) was added,
and the reaction mixture was stirred at 0 °C for 3 h. Solid NaHCO3
was added until effervescing stopped. The solution was filtered and
the solvent removed in vacuo. Separation was realized by filtration
through a 2 in. silica gel plug, eluting with 1:1 hexanes-EtOAc, to
give the alcohol as a colorless solid in 83% (5.54 g) yield: mp 117-118
°C; Rf 0.24 in 7:3 hexanes-EtOAc; IR (neat) νmax 3295, 2932, 2858,
overall yield (4.3 g) of 13. The remaining mother liquor was
concentrated to a yellow oil, which was dissolved in CH3OH and
cooled to provide crystals (0.77 g). Examination by 1H NMR showed
this product to be a mixture of Z/E-isomers (13/14, 0.77 g) in a
ratio of 1:3, respectively. Recrystallization of this mixture (0.1 g)
from CH2Cl2-CH3OH gave the E-stilbene 14 derivative as needles
(0.074 g).
Z-13: mp 110-111 °C, Rf 0.73 in 95:5 hexanes-EtOAc; 1H NMR
(CDCl3, 300 MHz) δ 0.08 (6H, s, 2 × (CH3)2Si), 0.102 (6H, s, 2 ×
(CH3)2Si), 0.171 (6H, s, 2 × (CH3)2Si), 0.980 (18H, s, 2 × (tBu)),
1.01 (9H, s, tBu), 3.59 (6H, s, 2 × OCH3), 3.708 (3H, s, OCH3), 6.29
(1H, d, J ) 8.7 Hz, H-5′), 6.34 (1H, d, J ) 12.3 Hz, -CHdCH-),
6.56 (2H, s, H-2, H-6), 6.87 (1H, d, J ) 8.4 Hz, H-6′); 13C NMR
(CDCl3, 125 MHz) δ -4.40, -3.7, -3.0, 18.8, 19.0, 26.0, 26.4, 26.6,
55.2, 55.8, 104.3, 106.3, 122.5, 123.7, 126.5, 128.3, 130.1, 133.6, 137.0,
146.4, 151.3, 151.7; HRMS-APCI m/z 661.3750 [M + H]+ (calcd for
C35H61O6Si3, 661.3776); anal. C 63.96%, H 9.24%, calcd for
C35H60O6Si3, C 63.59% H 9.15%.
1
1591, 1511, 1245, 1134, 906 cm-1; H NMR (CDCl3, 300 MHz) δ
0.00 (6H, s, (CH3)2Si), 0.89 (9H, s, tBu), 1.80 (1H, brs, OH), 3.67 (6H,
s, 2 × OCH3), 4.46 (2H, s, CH2OH), 6.42 (2H, s, 2 × ArH); 13C NMR
(CDCl3, 100 MHz) δ -4.66, 25.79, 55.73, 65.80, 104.10, 133.37,
151.68; HRMS APCI+ m/z 281.1574 [M + H - H2O]+ (calcd for
C15H25O3Si, 281.1573).
4-(tert-Butyldimethylsilyloxy)-3,5-dimethoxybenzyltriphenylphos-
phonium Bromide (9). To a cooled (0 °C) and stirred solution of
alcohol 8 (5.54 g, 18.6 mmol) in anhydrous CH2Cl2 (50 mL) was added
a solution of PBr3 (6.04 g, 22.32 mmol, 2.10 mL) in anhydrous CH2Cl2
(25 mL) dropwise at 0 °C. After stirring at 0 °C for 10 min, the reaction
mixture was poured onto ice-cold saturated NaHCO3 solution (100 mL).
The separated organic phase was washed with water (20 mL) and dried,
and the solvent was removed in vacuo to furnish the bromide
intermediate. The oily residue was dissolved in toluene (70 mL), and
a solution of PPh3 (4.87 g, 18.6 mmol) in toluene (20 mL) was added.
The reaction mixture was stirred at room temperature for 10 min.
Toluene (∼50 mL) was removed in vacuo, and the reaction mixture
was allowed to cool over 24 h. The white precipitate was collected by
filtration and recrystallized from ethanol to afford the phosphonium
salt in 68% yield (7.89 g) as a colorless solid: mp 246-248 °C; Rf
0.50 in 1:9 CH3OH-CH2Cl2; IR (neat) νmax 3050, 2950, 1511, 1463,
E-14: mp 118-119 °C, Rf 0.54 in 95:5 hexanes-EtOAc; 1H NMR
(CDCl3, 500 MHz) δ 0.11 (6H, s, 2 × (CH3)2Si), 0.13 (6H, s, 2 ×
t
(CH3)2Si), 0.14 (6H, s, 2 × (CH3)2Si), 0.99 (9H, s, Bu), 1.02 (9H, s,
t
tBu), 1.08 (9H, s, Bu), 3.79 (3H, s, OCH3), 3.81 (6H, s, 2 × OCH3),
6.54 (1H, d, J ) 8.5 Hz, H-5′), 6.68 (2H, s, H-2, H-6), 6.78 (1H, d, J
) 16.5 Hz, -CHdCH-), 7.18 (1H, d, J ) 8.5 Hz, H-6′), 7.24 (1H, d,
J ) 16.5 Hz, -CHdCH-); HRMS-APCI+ m/z 661.3782 [M + H]+
(calcd for C35H61O6Si3, 661.3776); anal. C 63.52%, H 9.18%, calcd
for C35H60O6Si3, C 63.59% H 9.15%.
E-Stilstatin 3 (3a). To a cooled (-10 °C) solution of 2′,3′,4-tris(tert-
butyldimethylsilyloxy)-3,4′,5-trimethoxy-Z-stilstatin 3 (13, 6.1 g, 9.23
mmol) in anhydrous THF (30 mL) was added TBAF (11.1 mmol, 11
mL, 1.0 M solution in THF). The reaction mixture was stirred at -10
°C for 50 min. Ice-cold 6 N HCl (2 mL) was added, and the mixture
was extracted with EtOAc (4 × 50 mL). The organic phase was dried
and the solvent removed in vacuo to afford a brown oil (3.8 g).
Purification was realized by column chromatography (eluting with 1:1
hexanes-EtOAc), leading to a pink crystalline powder (1.2 g) that was
recrystallized from benzene-chloroform to yield 3a as colorless
crystals: mp 110-111 °C; 1H NMR (CDCl3, 300 MHz) δ 3.88 (3H, s,
OCH3), 3.91 (6H, s, 2 × OCH3), 5.39 (1H, s, OH), 5.51 (1H, s, OH),
5.61 (1H, s, OH), 6.47 (d, J ) 3 Hz, H-5′), 6.73 (2H, s, H-2, H-6),
7.04-6.98 (2H, m, -CHdCH-, H-6′), 7.15 (1H, d, J ) 15.9 Hz,
CHdCH); 13C NMR (CDCl3, 100 MHz) δ 56.2, 56.3 (2C), 103.1, 103.2,
117.5, 118.6, 121.4, 128.1, 128.3 (2C), 129.8, 132.3, 134.4, 141.9,
146.1, 147.2; anal. C 68.93%, H 6.11%, calcd for C17H18O6 ·C6H6, C
69.64%, H 6.05%.
X-ray Crystal Structure of 3a. (a) Data Collection. A small amber
crystal block (0.22 mm × 0.19 mm × 0.18 mm) of 3a grown from a
benzene-chloroform solution was mounted on the end of a thin glass
fiber using Apiezon type N grease and optically centered. Cell parameter
measurements and data collection were performed at ambient temper-
ature (298 ( 1 K) with a Bruker SMART APEX diffractometer using
Mo KR (0.71073 Å) radiation. A sphere of reciprocal space was
collected using the Multi-Run scheme available in the Bruker SMART29
data collection package. Three sets of frames were obtained, with each
set using 0.30°/frame steps to cover 182° in ω at fixed ꢀ values of 0°,
120°, and 240°, respectively. Data were integrated and reduced using
Bruker SAINT,29 resulting in 100% coverage of all unique reflections
to a resolution of 0.84 Å with an average redundancy of 4.51.
(b) Crystal Data: C17H18O6 ·(C6H6)1.5, fw ) 435.48, monoclinic,
P21/c, a ) 6.839(2) Å, b ) 16.920(5) Å, c ) 19.998(6) Å, ꢁ )
94.559(6)°, V ) 2306.8(12) Å3, Z ) 4, Fc ) 1.254 Mg m-3, µ(Mo
KR) ) 0.089 mm-1, λ ) 0.71073 Å, F(000) ) 924.
1
1438, 1338, 1249, 1125 cm-1; H NMR (CDCl3, 300 MHz) δ 0.00
(6H, s, (CH3)2Si), 0.89 (9H, s, tBu), 3.37 (6H, s, 2 × OCH3), 5.24 (2H,
d, J ) 13.8 Hz, CH2P), 6.30 (2H, d, J ) 2.4 Hz, 2 × ArH), 7.53-7.68
(15H, m, 3 × Ph); 31P NMR (CDCl3, 162 MHz) δ 24.06; HRMS-
APCI+ m/z 543.2513 [M - Br]+ (calcd for C33H40O3PSi, 543.2484).
2,3-Dihydroxy-4-methoxybenzaldehyde (11). Preparation of com-
pound 11 was repeated as originally described19i from 2,3,4-trimethoxy-
benzaldehyde (10). Colorless solid, 87% yield: mp 115-116 °C [lit.19i
mp 115-116 °C]; Rf 0.22 in 1:1 hexanes-EtOAc; IR (neat) νmax 3365,
1
2944, 1645, 1507, 1453, 1277, 1104, 1025 cm-1; H NMR (CDCl3,
300 MHz) δ 3.97 (3H, s, OCH3), 6.62 (1H, d, J ) 14.5 Hz, ArH), 7.12
(1H, d, J ) 13.5 Hz, ArH), 9.74 (1H, s, CHO); 13C NMR (CDCl3, 100
MHz) δ 56.58, 103.86, 116.32, 126.28, 133.25, 149.29, 153.23, 195.38;
HRMS-APCI+ m/z 169.0554 [M + H]+ (calcd for C8H9O4, 169.0500).
2,3-Bis(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (12).
Preparation of compound 12 was repeated as originally described19c
from benzaldehyde 11. Additional 13C NMR data are provided here.
Colorless solid, 84% yield: mp 75-76 °C [lit.19c mp 74.5-76 °C]; Rf
0.76 in 1:1 hexanes-EtOAc; IR (neat) νmax 2932, 2859, 1683, 1585,
1
1455, 1297, 1099 cm-1; H NMR (CDCl3, 300 MHz) δ 0.00 (12H, s,
2 × (CH3)2Si), 0.85 (9H, s, tBu), 0.89 (9H, s, tBu), 3.70 (3H, s, OCH3),
6.49 (1H, d, J ) 14.5 Hz, ArH), 7.36 (1H, d, J ) 15.0 Hz, ArH),
10.08 (1H, s, CHO); 13C NMR (CDCl3, 100 MHz) δ -3.92, -3.84,
18.55, 18.74, 26.02, 26.17, 55.17, 105.40, 115.21, 120.75, 121.37,
123.36, 136.80, 150.99, 157.53, 189.26; HRMS APCI+ m/z 397.2234
[M + H]+ (calcd for C20H37O4Si2, 397.2230).
2′,3′,4-Tris(tert-butyldimethylsilyloxy)-3,4′,5-trimethoxy-4-Z- and
-E-stilbenes (13, 14). n-Butyllithium (13.7 mmol, 5.5 mL, 2.5 M
solution in hexane) was added (dropwise) to a stirred and cooled (-20
°C) suspension of triphenylphosphonium bromide 9 (6.2 g, 9.95 mmol)
in anhydrous THF (200 mL). The red reaction solution was stirred for
15 min at -15 °C, before addition of benzaldehyde 12 (4.0 g, 10.1
mmol). Stirring was continued at -15 °C for the duration of the
experiment. TLC analysis (49:1 hexanes-EtOAc) showed complete
conversion to product after 1 h, and the reaction was terminated with
the addition of iced water. The reaction mixture was extracted with
EtOAc (300 mL), the organic layer was washed with water (100 mL)
and dried, and the solvent was removed (in vacuo) to yield a yellow
residue. The crude product in ethanol (50 mL) was sonicated to effect
solution. The resulting white precipitate was collected and dried to yield
a colorless crystalline powder (3.58 g). The ethanol mother liquor was
cooled in an ice bath to yield crystals (0.72 g) that had the same Rf
value by TLC (95:5 hexanes-EtOAc) as the precipitate. Upon
(c) Structure Solution and Refinement. A total of 18 374 reflections
were measured, of which 4068 were independent (Rint ) 0.0296) and
2864 were considered observed (Iobs > 2σ(I)). Final unit cell parameters
were determined by least-squares fit of 6237 reflections covering the
resolution range of 8.601 to 0.773 Å. An absorption correction was
applied using SADABS29 on the full data set. Statistical analysis with
the XPREP program in SHELXTL29 indicated that the space group
was P21/c. Routine direct methods structure solution was performed
with SHELXTL, which produced all non-hydrogen atom coordinates
on the single 3a molecule in the asymmetric unit. Upon initial
refinement with SHELXTL, the remaining solvent in the asymmetric
unit became easily discernible in the Fourier difference map. After full
isotropic refinement, the thermal parameters for all non-hydrogen atoms
1
examination by H NMR, both precipitate and crystals were found to
be the Z-stilstatin 3 derivative 13 and were combined to give 65%

A ResVeratrol Structural Modification
Journal of Natural Products, 2009, Vol. 72, No. 9 1641
References and Notes
were then allowed to refine anisotropically. Subsequently, hydrogen
atoms were added at idealized positions and bond distances with their
isotropic thermal parameters fixed at 1.5 times the Uiso values of their
bonding partners for the methyl and hydroxyl hydrogens and at 1.2
times the Uiso values of their bonding partners for all others. The model
was then allowed to refine with hydrogens constrained as atoms riding
on their bonding partners. This resulted in a final standard residual R1
value of 0.0528 for observed data and 0.0731 for all data. Goodness
of fit on F2 was 1.022, and the weighted residual on F2, wR2, was 0.1432
for observed data and 0.1614 for all data. The final Fourier difference
map showed minimal electron density, with the largest difference peak
and hole having values of 0.218 electrons Å-3 and -0.165 electrons
Å-3, respectively. Final bond distances and angles were all within
expected and acceptable limits for the 3a molecule. However, the
average phenyl C-C distance in the included solvent (1.35 Å) was
shorter than typical values (1.39 Å), probably due to the large thermal
motion.30
(1) Part 579 in the Antineoplastic Agents series. For part 578, refer to:
Pettit, G. R.; Thornhill, A.; Melody, N.; Knight, J. C. J. Nat. Prod.
2009, 72, 380–388.
(2) (a) Takaoka, M. J. J. Fac. Sci. Hokkaido Imp. UniV. 1940, 3, 1–16.
(b) Gescher, A. J. Planta Med. 2008, 74, 1651–1655. (c) Orallo, F.
Curr. Med. Chem. 2008, 15, 1887–1898. (d) Saiko, P.; Szakmary, A.;
Jaeger, W.; Szekeres, T. Mutat. Res. 2008, 658, 68–94. (e) Baur, J. A.;
Sinclair, D. A. Nat. ReV. Drug DiscoVery 2006, 5, 493–506.
(3) (a) Handler, N.; Brunhofer, G.; Studenik, C.; Leisser, K.; Jaeger, W.;
Parth, S.; Erker, T. Bioorg. Med. Chem. 2007, 15, 6109–6118. (b)
Jang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.;
Beecher, C. W. W.; Fong, H. H. S.; Farnsworth, N. R.; Kinghorn,
A. D.; Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. Science 1997, 275,
218–220.
(4) Tseng, S. H.; Lin, S. M.; Chen, J. C.; Su, Y. H.; Huang, H. Y.;
Chen, C. K.; Lin, Y. P.; Chen, Y. Clin. Cancer Res. 2004, 10, 2190–
2202.
(5) Hebbar, V.; Shen, G.; Hu, R.; Kim, B. R.; Chen, C.; Korytko, P. J.;
Crowell, J. A.; Levine, B. S.; Kong, A.-N. T. Life Sci. 2005, 76, 2299–
2314.
2′,3′,4-O-Tri[bis(benzyl)phosphoryl]-3,4′,5-trimethoxy-E-stil-
bene (15). A solution of E-stilstatin 3 (3a, 1.5 g, 4.7 mmol) in CCl3
(7.5 mL) and acetonitrile (8 mL) was cooled to -10 °C. DIPEA (6
mL) and DMAP (0.19 g) were added, and the solution was stirred for
several minutes prior to addition of dibenzylphosphite (6.4 g). After
1 h the reaction had not gone to completion (by TLC), and an additional
5 mL of dibenzylphosphite was added to the brown solution. The
reaction mixture was stirred at rt for a further 60 min. Next, KH2PO4
(0.5 M, 200 mL) was added and the mixture stirred for 10 min before
extraction with ethyl acetate (3 × 100 mL). The solution was dried
(MgSO4) and the solvent removed in vacuo to yield a brown oil.
Column chromatography (99:1 CH2Cl2-CH3OH) provided the desired
product as a yellow oil (1.29 g, 24.9% yield): Rf 0.25 (3:7
¨
(6) Garvin, S.; Ollinger, K.; Dabrosin, C. Cancer Lett. 2006, 231, 113–
122.
(7) Wang, Z.; Huang, Y.; Zou, J.; Cao, K.; Xu, Y.; Wu, J. M. Int. J. Mol.
Med. 2002, 9, 77–79.
(8) Naderali, E. K.; Doyle, P. J.; Williams, G. Clin. Sci. (London) 2000,
98, 537–543.
(9) Cruz, M.; Luksha, L.; Logman, H.; Poston, L.; Agewall, S.;
Kublickiene, K. Am. J. Physiol. Heart Circ. Physiol. 2006, 290,
H1969–H1975.
(10) Frankel, E. N.; Waterhouse, A. L.; Kinsella, J. E. Lancet 1993, 341,
1103–1104.
(11) Wang, Z.; Zou, J.; Cao, K.; Hsieh, T. C.; Huang, Y.; Wu, J. M. Int.
J. Mol. Med. 2005, 16, 533–540.
(12) (a) Bureau, G.; Longpre´, F.; Martinoli, M.-G. J. Neurosci. Res. 2008,
86, 403–410. (b) Chen, G.; Shan, W.; Wu, Y. L.; Ren, L. X.; Dong,
I. H.; Ji, Z. Z. Chem. Pharm. Bull. (Tokyo) 2005, 53, 1587–1590.
(13) Wu, S. L.; Yu, L.; Meng, K. W.; Ma, Z. H.; Pan, C. E. World J.
Gastroenterol. 2005, 11, 4745–4749.
(14) (a) Hung, L. M.; Su, M. J.; Chu, W. K.; Chiao, C. W.; Chan, W. F.;
Chen, J. K. Br. J. Pharmacol. 2002, 135, 1627–1633. (b) Hung, L. M.;
Chen, J. K.; Huang, S. S.; Lee, R. S.; Su, M. J. CardioVasc. Res.
2000, 47, 549–555.
(15) (a) Wood, J. G.; Rogina, B.; Lavu, S.; Howitz, K.; Helfand, S. L.;
Tatar, M.; Sinclair, D. Nature 2004, 430, 686–689. (b) Howitz, K. T.;
Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood,
J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L. L.; Scherer,
B.; Sinclair, D. A. Nature 2003, 425, 191–196.
1
hexanes-EtOAc); H NMR (CDCl3, 300 MHz) δ 3.66 (6H, s, 2 ×
OCH3), 3.79 (3H, s, OCH3), 5.02 (4H, m, 2 × CH2Ph), 5.18-5.27
(8H, m, CH2Ph), 6.64 (2H, s, Ar-H-2,6), 6.80-6.88 (m, 2H,
-CHdCH-), 7.12-7.14 (32 H, m, 6 × Ph, 2 × Ar-H); 13C NMR
(CDCl3, 125 MHz) δ 55.9 (2C), 56.4, 69.5 (2C), 69.8 (2C), 70.1, 70.2,
103.4 (2C), 109.9, 122.3, 122.6, 127.7-128.9, 134.8, 135.4, 135.5,
135.9, 136.0, 136.3, 151.8, 151.9, 152.0.
Sodium Stilstatin 3: 2′,3′,4-O-Triphosphate Prodrug (3b). To a
solution of benzyl phosphate 15 (4.5 g, 4.0 mmol) in anhydrous
dichloromethane (150 mL) was added bromotrimethylsilane (3.2 mL,
3.7 g, 24 mmol, 6 equiv) (dropwise), and the reaction mixture stirred
at rt for 30 min. The reaction was terminated with the addition of water
(100 mL). The phosphoric acid intermediate was not soluble in the
organic phase and was immediately converted to the sodium salt by
separation and concentration of the aqueous fraction to 30 mL and
elution through an ion-exchange column containing Dowex-50W resin
(sodium form). The fractions containing the salt were spotted onto TLC
plates, and the product-containing fractions were visualized using both
UV and I2 vapor. The pooled fractions were reduced to dryness via
freeze-drying for 24 h to yield a cream solid, which was recrystallized
from water-ethanol (ice bath at 0 °C) to yield an off-white powder
that was collected and dried (2.7 g, 96%): mp 220 °C (chars); 1H NMR
(D2O, 400 MHz) δ 3.82 (m, 9H, 3 × OCH3), 6.84 (1 H, d, J ) 8.8 Hz,
Ar-H), 6.9 (2H, s, Ar-H), 7.0 (1H, d, J ) 16.5 Hz, -CHdCH-),
7.4-7.44 (3H, m, Ar-H, -CHdCH-); 13C NMR (D2O, 100 MHz) δ
56.5, 104.1, 109.0, 120.8, 123.4, 127.7, 130.2, 130.3, 134.6, 134.9,
135.0, 144.0, 152.2, 152.3, 152.4.
(16) Walle, T.; Hsieh, F.; DeLegge, M. H.; Oatis, J. E., Jr.; Walle, U. K.
Drug Metab. Dispos. 2004, 32, 1377–1382.
(17) Cardile, V.; Chillemi, R.; Lombardo, L.; Sciuto, S.; Spatafora, C.;
Tringali, C. J. Biosci. 2007, 62, 189–195.
(18) Murias, M.; Ja¨ger, W.; Handler, N.; Erker, T.; Horvath, Z.; Szekeres,
T.; Nohl, H.; Gille, L. Biochem. Pharmacol. 2005, 69, 903–912.
(19) (a) Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J. M.;
Lohavanijaya, P. Can. J. Chem. 1982, 60, 1374–1376. (b) Pettit, G. R.;
Singh, S. B.; Cragg, G. M. J. Org. Chem. 1985, 50, 3404–3406. (c)
Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.; Schmidt, J. M. J.
Nat. Prod. 1987, 50, 119–131. (d) Pettit, G. R.; Singh, S. B. Can.
J. Chem. 1987, 65, 2390–2396. (e) Pettit, G. R.; Singh, S. B.; Hamel,
E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D. Experientia 1989,
45, 209–211. (f) Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.;
Pettit, R. K.; Schmidt, J. M.; Hogan, F. J. Med. Chem. 1995, 38, 1666–
1672. (g) Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Chaplin, D. J.;
Oliva, D. Anticancer Res. 1998, 13, 981–993. (h) Pettit, G. R.; Rhodes,
M. R. Anti-Cancer Drug Des. 1998, 13, 183–191. (i) Pettit, G. R.;
Lippert, J. W., III. Anti-Cancer Drug Des. 2000, 15, 203–216. (j) Pettit,
G. R.; Grealish, M. P.; Jung, M. K.; Hamel, E.; Pettit, R. K.; Chapuis,
J.-C.; Schmidt, J. M. J. Med. Chem. 2002, 45, 2534–2542.
(20) (a) Alloatti, D.; Giannini, G.; Cabri, W.; Lustrati, I.; Marzi, M.; Ciacci,
A.; Gallo, G.; Tinti, M. O.; Marcellini, M.; Riccioni, T.; Guglielmi,
M. B.; Carminati, P.; Pisano, C. J. Med. Chem. 2008, 51, 2708–2721.
(b) Zou, Y.; Xiao, C.-F.; Zhong, R.-Q.; Wei, W.; Huang, W.-M.; He,
S.-J. J. Chem. Res. 2008, 354–356. (c) Pinney, K. G.; Jelinek, C.;
Edvardsen, K.; Chaplin, D. J.; Pettit, G. R. In Anticancer Agents from
Natural Products; Cragg, G. M., Kingston, D. G. I., Newman, D. J.,
Eds.; Taylor and Francis: Boca Raton, FL, 2005; pp 23-46. (d) Cirla,
A.; Mann, J. Nat. Prod. Rep. 2003, 20, 558–564. (e) Pettit, G. R. In
Anticancer Agents: Frontiers in Cancer Chemotherapy; Ojima, I., Vite,
G. D., Altmann, K.-H., Eds.; American Chemical Society: Washington,
DC, 2001; pp 16-42.
Acknowledgment. We are pleased to acknowledge the very neces-
sary financial support provided by grants RO1 CA 90441-02-05, 2R56-
CA 09441-06A1, and 5RO1 CA 090441-07 from the Division of Cancer
Treatment and Diagnosis, NCI, DHHS; The Arizona Biomedical
Research Commission; Dr. Alec D. Keith; J. W. Kieckhefer Foundation;
Margaret T. Morris Foundation; the Robert B. Dalton Endowment Fund;
and Dr. William Crisp and Mrs. Anita Crisp. For other helpful assistance
we thank Drs. J.-C. Chapuis, F. Hogan, M. Hoard, and D. Doubek, as
well as M. Dodson.
Supporting Information Available: X-ray data for E-stilstatin 3
(3a). This material is available free of charge via the Internet at http://
pubs.acs.org.

1642 Journal of Natural Products, 2009, Vol. 72, No. 9
Pettit et al.
(21) Lin, H.-L.; Chiou, S.-H.; Wu, C.-W.; Lin, W.-B.; Chen, L.-H.; Yang,
Y.-P.; Tsai, M.-L.; Uen, Y.-H.; Liou, J.-P.; Chi, C.-W. J. Pharmacol.
Exp. Ther. 2007, 323, 365–373.
(22) (a) Zhang, F.; Sun, C.; Wu, J.; He, C.; Ge, X.; Huang, W.; Zou, Y.;
Chen, X.; Qi, W.; Zhai, Q. Pharmcol. Res. 2008, 57, 318–323. (b)
Sun, C.; Zhang, F.; Ge, X.; Yan, T.; Chen, X.; Shi, X.; Zhai, Q. Cell
Metabol. 2007, 6, 307–319.
(23) Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.; Schmidt, J. M.;
Pettit, R. K. J. Med. Chem. 2005, 48, 4087–4099.
(24) Pettit, G. R.; Anderson, C. R.; Herald, D. L.; Jung, M. K.; Lee, D. J.;
Hamel, E.; Pettit, R. K. J. Med. Chem. 2003, 46, 525–531.
(25) Hammond, G. S.; Saltiel, J.; Lamola, A. A.; Turro, N. J.; Bradshaw,
J. S.; Cowan, D. O.; Counsell, R. C.; Vogt, V.; Dalton, C. J. Am.
Chem. Soc. 1964, 86, 3197–3217.
(26) Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron Lett. 1996,
37, 771–774.
(27) Ren, X.; Chen, X.; Peng, K.; Xie, X.; Xia, Y.; Pan, X. Tetrahedron:
Asymmetry 2002, 13, 1799–1804.
(28) Snider, B. B.; Grabowski, J. F. Tetrahedron 2006, 62, 5171–5177.
(29) SMART (Version 5.632), SAINT (Version 6.45a), SHELXTL (Version
6.14), and SADABS (Version 2.10); Bruker AXS Inc.: Madison, WI,
2003.
(30) CCDC 736738 (3a) contains the supplementary crystallographic data
for 3a. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
NP9002146