An efficient synthetic strategy for obtaining 4-methoxy carbon isotope labeled combretastatin a-4 phosphate and other Z-combretastatins

Article abstract of DOI:10.1021/np9004486

Human cancer and other clinical trials under development employing combretastatin A-4 phosphate (1b, CA4P) should benefit from the availability of a [¹¹C]-labeled derivative for positron emission tomography (PET). In order to obtain a suitable

Full text of DOI:10.1021/np9004486

J. Nat. Prod. 2010, 73, 399–403
399
An Efficient Synthetic Strategy for Obtaining 4-Methoxy Carbon Isotope Labeled
Combretastatin A-4 Phosphate and Other Z-Combretastatins1⊥
George R. Pettit,*,† Mathew D. Minardi,† Fiona Hogan,† and Pat M. Price‡
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State UniVersity, P.O. Box 871604, Tempe, Arizona 85287-1604,
and UniVersity of Manchester, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, United Kingdom
ReceiVed July 24, 2009
Human cancer and other clinical trials under development employing combretastatin A-4 phosphate (1b, CA4P) should
benefit from the availability of a [11C]-labeled derivative for positron emission tomography (PET). In order to obtain a
suitable precursor for addition of a [11C]methyl group at the penultimate step, several new synthetic pathways to CA4P
were evaluated. Geometrical isomerization (Z to E) proved to be a challenge, but it was overcome by development of
a new CA4P synthesis suitable for 4-methoxy isotope labeling.
In 1987, we reported1 the isolation, structure, and synthesis of
combretastatin A-1 (1c, CA1) from the subtropical tree Combretum
caffrum (Eckl. and Zeyh.) Kuntze (Combretaceae)1b collected in
southern Africa, and two years later combretastatin A-4 (1a, CA4)
from the same source was isolated and synthesized.2 Subsequently,
both CA1 and CA4 were converted to phosphate prodrugs (1d,
CA1P,3 and 1b, CA4P4) and developed5 to human cancer clinical
trials.6 CA1P and CA4P are presently in phase I/II and III cancer
trials, respectively, and CA4P is also in phase II human macular
degeneration (leading cause of blindness)7 clinical trials. The
potential of CA4P in treating other eye diseases such as diabetic
retinopathy7d and retinoblastoma7b,e is also being developed.
Presently, CA4P (aka Zybrestat) followed by CA1P is the lead
among cancer vasculature disrupting drugs.8a-e A large number
of other potentially important recent observations concerning
medical applications of the leading combretastatins include evidence
that CA4P is antiangiogenic,8f increases aberrant organization of
metaphase chromosomes in non-small cell lung cancer cells,8g
inhibits gastric cancer cell metastasis,8h and improves glucose
Results and Discussion
tolerance in diabetic mice, which in turn suggests a possible new
approach to treatment of type 2 diabetes.8i The latter evidence
provides another mechanistic parallel to resveratrol (2)8j and
suggests many other avenues for research from cancer prevention8h
to longevity.8k
When we began this research in 2001, the knowledge of CA4 in
vivo metabolism left some uncertainties as to whether [11C]-CA4
was an appropriate target. That has in the interim been resolved.
Very recently, it has been confirmed that CA4 is transformed in
rat and human liver fractions to a glucuronide metabolite.11 In the
rat, with an approximate therapeutic dose, CA4 is metabolized to
both the glucuronide and a previously undetected sulfate derivative
at the 4′-phenol position.11 One or both of these relatively long-
lived (compared to the 20.1 min half-life of [11C]) modifications
would be useful for measurement of radiation (dosimetry) in the
study of absorption, distribution, metabolism, and elimination
(pharmacokinetics) of CA4P as part of current human trials. In turn,
that would allow a better knowledge of the distribution of CA4
between neoplastic and normal tissue as well as the relative binding
potential/effective blockade vs dose. Those early objectives required
a synthesis of CA4P that would allow a short reaction path and
high-yield penultimate step for obtaining [11C]-CA4P.
Substitution of a CA4P A-ring methyl group with a [11C]methyl
seemed to offer the fewest difficulties10b and would also utilize
some of the synthetic procedures we previously developed for
synthesizing combretastatin A-3, the 3-desmethyl derivative of
CA4.12 When an approach based on the 3-hydroxy-4,5-dimethoxy-
phenyl A-ring of combretastatin A-3 was discontinued owing to
geometrical isomerization problems at the stilbene stage, attention
was next focused on a 3,5-dimethoxy-4-hydroxyphenyl A-ring
precursor that would lead to phenol 3 and allow methylation without
cis/trans isomerism. The first such attempt to synthesize phenol 3
By 2000, positron emission tomography (PET) was already well
established as a noninvasive technique for biomedical imaging, and
its potential for necessary applications in preclinical and clinical
research employing the lead combretastatins, particularly CA4P,
was clearly evident. To follow is both a brief outline of the
radiolabeling rationale and a practical synthetic route to employ as
a model for later introduction of a [11C]-isotope into CA4/CA4P.
A [11C]methyl group was selected as the most efficient method,
which required completion of an appropriate new synthesis of CA4
and now remains a most practical option. Meanwhile, a variety of
other new syntheses of CA49a,b and its analogues9c-g have been
reported, as well as syntheses of CA1P radiolabeled with a
[14C]methyl group in high specific activity10a and [11C]methyl
derivatives10b of resveratrol (2), augmented by other structural
modifications8j of this increasingly important naturally occurring
stilbene.8h
⊥ Dedicated to the late Dr. John W. Daly of NIDDK, NIH, Bethesda,
Maryland, and to the late Dr. Richard E. Moore of the University of Hawaii
at Manoa for their pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: 480 965-3351.
Fax: 480 965-2747. E-mail: bpettit@asu.edu.
† Arizona State University.
‡ University of Manchester.
10.1021/np9004486  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/22/2009

400 Journal of Natural Products, 2010, Vol. 73, No. 3
Pettit et al.
Scheme 1
Scheme 3
Scheme 2
the basic conditions required for a Wittig condensation, it had to
be on the A-ring aldehyde unit rather than on the phosphonium
salt moiety (ring B). The new A-ring was prepared by the
acetylation of 3,5-dimethoxy-4-hydroxybenzaldehyde to give 12
(Scheme 3). The Wittig olefin reaction was accomplished by
treatment of phosphonium salt 1312b with n-butyllithium followed
by the addition of aldehyde 12 to produce stilbene 14 (16%).
Desilylation with tetrabutylammonium fluoride (TBAF) afforded
3′-phenol 15 (84%). The phenol group was phosphorylated4,12b,13
with dibenzylphosphite to produce phosphate ester 16 (68%), and
deacetylation by treatment with 5% potassium carbonate in
methanol afforded 4-phenol 3, albeit in moderate yields (30%; 2.4%
overall yield from 13) owing to partial isomerization to the
corresponding trans-stilbene.
Once the necessary precursor of combretastatin A-4 prodrug was
in hand, a suitable methylation procedure was needed that could
be easily employed to produce [11C]combretastatin A-4 phosphate
prodrug. A selection of procedures for the methylation of 4-phenol
3 were attempted using CH3I, as this would be a choice methylating
agent for producing a [11C]-methylated derivative. However, all
attempts to methylate the phenol (3) with CH3I resulted in cis/
trans isomerization.10a Therefore, a new approach that would avoid
isomerization by way of direct conversion of the silyl ether to a
methyl ether was examined.
In a new route to 11 (Scheme 4), Wittig salt 7 was treated with
NaH and aldehyde 17, which was prepared directly from isovanillin
(8a) in 85% yield. Desilylation of the product, stilbene 11, with
concurrent methylation that could be used to efficiently obtain
[11C]combretastatin A-4 was first explored by modification of an
existing procedure, in which a TBDMS ether can be converted into
a benzyl ether by use of KF and benzyl bromide in tetrahydrofuran.
An attempt to apply this method using KF and CH3I to convert the
TBDPS ether directly to a methyl ether did not succeed. However,
the approach was successful when TBAF and CH3I were used and
led to combretastatin A-4 3′-dibenzylphosphate (18). That result
completed a very useful synthetic approach that can now be
employed to obtain 4-methoxy isotope-labeled combretastatins and
related stilbenes.
began with silyl protection of 3,5-dimethoxy-4-hydroxybenzalde-
hyde using tert-butyldiphenylsilyl chloride (TBDPSCl), which gave
a 71% yield of silyl ether 4 (Scheme 1). The benzaldehyde (4)
was reduced with NaBH4 to afford benzyl alcohol 5 (94%), which
was then converted to benzyl bromide 6, and subsequent treatment
with triphenylphosphine led to Wittig salt 7 (62%).
Synthesis of the B-ring was begun (Scheme 2) by protection of
isovanillin (8a) with the tert-butyldimethylsilyl (TBDMS) group
(8b, 84%) as previously reported.12a The Wittig coupling of the
A- and B-rings was accomplished by the treatment of phosphonium
bromide 7 with n-butyllithium followed by addition of aldehyde
8b to afford stilbene 9 in 25% yield. Disilyl ether 9 was then
selectively deprotected with pyridinium p-toluenesulfonate (PPTS)
in ethanol to afford 3′-phenol 10 in 65% yield. Stilbene 10 was
phosphorylated with dibenzylphosphite to afford the phosphate ester
(11, 38%).4,12b,13 Subsequently, the desilylation of phosphorylated
stilbene 11 to obtain 4-phenol 3 was attempted, but all efforts failed
owing to concomitant cleavage of the phosphate ester.
Since removal of the silyl protecting group was causing difficulty,
a different protecting group such as acetate was needed in order to
eventually obtain phenol 3. Because an acetate group is labile under

4-Methoxy Carbon Isotope Labeled Combretastatin
Journal of Natural Products, 2010, Vol. 73, No. 3 401
4-(tert-Butyldiphenylsilyloxy)-3,5-dimethoxybenzyltriphenylphos-
phonium Bromide (7). Benzyl bromide 6 (5.5 g) was dissolved in
toluene (200 mL), and triphenylphosphine (3.5 g, 13 mmol) was added.
The solution was heated at reflux for 1 h, cooled to rt, and allowed to
stir an additional 2 h. The resulting precipitate was collected and
triturated with ether to afford bromide 7 (5.5 g) as a colorless,
Scheme 4
1
amorphous solid (62% over 2 steps): mp 152-153 °C (CH3OH); H
NMR (CD3OD, 300 MHz) δ 1.061 (s, 9H), 3.11 (s, 6H), 4.88 (d, JPCH
) 14.4 Hz, 2H), 6.08 (s, 1H), 6.09 (s, 1H), 7.28 (m, 6H), 7.65 (m,
20H), 7.85 (m, 4H); 13C NMR (CD3OD, 75 MHz) δ 20.83, 27.28, 30.98,
31.60, 55.55, 109.14, 109.22, 118.54, 119.67, 120.35, 120.48, 128.11,
129.20, 129.90, 130.55, 131.14, 131.30, 134.95, 135.44, 135.57, 136.35,
152.35, 152.40; anal. C 68.62%, H 6.11%, calcd for C43H44BrO3PSi,
C 69.07%, H 5.93%.
4-(tert-Butyldimethylsilyloxy)-3-methoxybenzaldehyde (8b). A
solution of isovanillin (8a, 20 g, 131 mmol) in DCM (400 mL) and
DIPEA (25 mL, 145 mmol) was stirred for 10 min, and TBDMSCl
(22 g, 145 mmol) was added. After stirring 16 h, the reaction was
terminated by the addition of water (50 mL). The organic phase was
separated and washed with NaOH (5%, 50 mL) followed by brine.
The solvent was removed to provide an orange-brown oil, which was
separated by vacuum distillation to afford a colorless oil (29.3 g, 84%):
Experimental Section
General Experimental Procedures. Abbreviations: TBDMS, tert-
butyldimethylsilyl; TBDPS, tert-butyldiphenylsilyl; DIPEA, diisopro-
pylethylamine; LAH, lithium aluminum hydride; DMAP, dimethylami-
nopyridine; TBAF, tetrabutylammonium fluoride; DMF, N,N-
dimethylformamide; THF, tetrahydrofuran; DCM, dichloromethane;
TLC, thin-layer chromatography. All solvents (ether refers to diethyl
ether) used in chemical reactions were redistilled and dried. Other
reagents were purchased from Sigma-Aldrich Chemical Co., Lancaster-
Clariant, or Fisher-ACROS Chemical Co. Solvent extracts of aqueous
solutions were dried over anhydrous magnesium sulfate unless otherwise
noted. Gravity column chromatography (CC) was performed using silica
gel (70-230 mesh) from VWR Scientific. All melting points were
determined with an Electrochemical digital melting point apparatus,
model IA 9200, and are uncorrected. NMR spectra were recorded using
a Varian Gemini 300 or a Varian Unity 400 instrument. Chemical shifts
are reported in ppm (δ) downfield from tetramethylsilane as an internal
standard. High-resolution FAB mass spectra were obtained on a Kratos
MS-50 (Midwest Center for Mass Spectrometry, University of
Nebraska-Lincoln). Elemental analyses were obtained from Galbraith
Laboratories, Inc., Knoxville, TN.
4-(tert-Butyldiphenylsilyloxy)-3,5-dimethoxybenzaldehyde (4). To
a solution of imidazole (7.5 g, 109 mmol), 3,5-dimethoxy-4-hydroxy-
benzaldehyde (10 g, 55 mmol), and DMF (100 mL) was added
TBDPSC12 (16 mL, 60 mmol). After stirring 16 h, the reaction was
terminated by the addition of water and extracted with hexane-EtOAc
(1:1, 3 × 75 mL). The extract was washed with brine, and following
removal of solvent, the residue was separated by flash chromatography
(9:1, hexane-EtOAc) to afford aldehyde 4 as a clear oil (16.25 g, 71%):
bp 202-204 °C (0.01 mmHg); 1H NMR (CDCl3, 300 MHz) δ 1.12 (s,
9H), 3.51 (s, 6H), 6.98 (s, 2H), 7.36 (m, 6H), 7.68 (d, J ) 8.1 Hz,
4H), 9.76 (s, 1H); 13C NMR (CDCl3, 75 MHz) δ 26.62, 55.23, 106.51,
127.15, 127.66, 129.32, 129.55, 133.82, 134.78, 134.97, 140.43, 151.33,
191.01; anal. C 71.17%, H 6.90%, calcd for C25H28O4Si, C 71.39%, H
6.71%.
1
bp 140-142 °C (0.01 mmHg): H NMR (CD3OD, 300 MHz) δ 0.13
(s, 6H), 0.96 (s, 9H), 3.84 (s, 3H), 6.91 (d, J ) 8.1 Hz, 1H), 7.33 (d,
J ) 2.4 Hz, 1H), 7.43 (dd, J ) 2.1, 8.1 Hz, 1H), 9.77 (s, 1H).
3′-(tert-Butyldimethylsilyloxy)-4-(tert-butyldiphenylsilyloxy)-3,4′,5-
trimethoxy-(Z)-stilbene (9). Phosphonium bromide 7 (5 g, 6.7 mmol)
was dissolved in THF (100 mL) and cooled to -78 °C. n-BuLi (2.5 M
in hexane, 2.7 mL, 6.7 mmol) was added, followed after 1 h by aldehyde
8b (1.95 g, 7.4 mmol) over 5 min. After stirring for 16 h, the reaction
was terminated by the addition of water, and the mixture was extracted
with EtOAc. The solvent was removed from the organic phase, and
the residue was separated by CC to afford a clear oil (9, 0.5 g, 25%):
1
bp (dec) 94-96 °C (0.01 mmHg); H NMR (CD3OD, 300 MHz) δ
0.15 (s, 6H), 1.02 (s, 9H), 1.15 (s, 9H), 3.32 (s, 6H), 3.79 (s, 3H), 6.41
(s, 2H), 6.97 (d, J ) 7.8 Hz, IH), 6.82 (s, 1H), 6.85 (s, 1H), 7.04 (s,
1H), 7.37 (m, 10H), 7.75 (m, 4H); 13C NMR (CD3OD, 75 MHz) δ
-4.81, 18.31, 20.05, 25.64, 26.73, 30.27, 30.79, 34.16, 54.92, 55.38,
100.61, 105.79, 111.56, 121.45, 122.56, 125.46, 126.91, 128.57, 128.96,
129.16, 129.73, 130.36, 133.47, 134.55, 135.12, 144.57, 149.95, 150.47;
anal. C 71.19%, H 7.74%, calcd for C39H50O5Si2, C 71.52%, H 7.69%.
4-(tert-Butyldiphenylsilyloxy)-3′-hydroxy-3,4′,5-trimethoxy-(Z)-
stilbene (10). Stilbene 9 (0.34 g, 0.5 mmol) and PPTS (12 mg, 0.05
mmol) were dissolved in ethanol (25 mL). The mixture was heated to
60 °C and allowed to stir for 100 h. Ethanol was removed, and the
residue was separated by CC (9:1 hexane-EtOAc) to yield stilbene
10 (0.18 g, 65%): mp 86-87 °C; 1H NMR (CD3OD, 300 MHz) δ 1.089
(s, 9H), 3.28 (s, 6H), 3.84 (s, 3H), 5.46 (s, 1H), 6.36 (s, 2H), 6.65 (d,
J ) 8.7 Hz, 1H), 6.72 (dd, J ) 1.8, 8.4 Hz, 1H), 7.33 (m, 10H), 7.70
(m, 4H); 13C NMR (CD3OD, 75 MHz) δ 19.99, 26.67, 29.10, 30.76,
54.94, 55.79, 105.78, 110.21, 115.06, 120.93, 126.88, 128.44, 128.96,
129.24, 129.68, 130.69, 133.46, 134.39, 135.12, 145.14, 145.60, 150.50;
anal. C 72.98%, H 6.53%, calcd for C33H36O5Si, C 73.30%, H 6.71%.
3′-O-Bis(benzyl)phosphoryl-4-(tert-butyldiphenylsilyloxy)-3,4′,5-
trimethoxy-(Z)-stilbene (11). From Phenol 10. Method A. To a
solution of 3′-phenol 10 (0.18 g, 0.33 mmol) in acetonitrile (10 mL)
that was cooled to -10 °C was added CCl4 (0.32 mL, 3.3 mmol). The
mixture was stirred for 10 min before the addition of DIPEA (0.12
mL, 0.68 mmol) and DMAP (4 mg, 0.033 mmol), and after 1 min
dibenzylphosphite (0.11 mL, 0.49 mmol)4 was added (dropwise over
5 min). The reaction mixture was stirred for 3 h at -10 °C, treated
with KH2PO4 (20 mL, 0.5 M), stirred for 10 min, and then extracted
with EtOAc. Removal of solvent and separation by CC (1:1
hexane-EtOAc) afforded phosphate 11 (0.10 g, 38%): bp (dec) 148
°C (0.01 mmHg); 1H NMR (CDCl3, 300 MHz) δ 3.64 (s, 6H), 3.76 (s,
3H), 5.11 (s, 2H), 5.14 (s, 2H), 6.44 (d, J ) 2.1 Hz, 2H), 6.50 (s, 2H),
6.77 (d, J ) 8.4 Hz, 1H), 7.06 (d, J ) 8.1 Hz, 1H), 7.12 (m, 1H), 7.32
(s, 10H); anal. C 70.22%, H 6.31%, calcd for C47H49O8PSi, C 70.48%,
H 6.17%.
4-tert-(Butyldiphenylsilyloxy)-3,5-dimethoxybenzyl Alcohol (5).
Benzaldehyde 4 (5.3 g, 12.6 mmol) was dissolved in ethanol (100 mL),
and NaBH4 (0.6 g, 15.1 mmol) was added. After stirring 3 h, the reaction
mixture was acidified with dilute HC1 (1 N) and extracted with EtOAc.
After solvent removal in vacuo, the resulting oil was purified by CC
(7:3 hexane-EtOAc) to afford alcohol 5 (5.01 g, 94%) as a clear oil:
bp 218 °C (0.01 mmHg); IR νmax 1134, 1242, 1340, 1427, 1462, 1512,
1593, 2859, 2936, 3420 cm-1; H NMR (CDCl3, 300 MHz) δ 1.091
1
(s, 9H), 1.99 (bs, 1H), 3.41 (s, 6H), 4.48 (s, 2H), 6.39 (s, 2H), 7.31
(m, 6H), 7.69 (d, J ) 7.8 Hz, 4H); 13C NMR (CDCl3, 75 MHz) δ
20.05, 26.52, 26.73, 55.13, 65.51, 103.77, 126.96, 127.61, 129.01,
133.36, 133.62, 134.55, 134.76, 135.12, 150.86; anal. C 71.22%, H
7.08%, calcd for C25H30O4Si, C 71.05%, H 7.16%.
4-(tert-Butyldiphenylsilyloxy)-3,5-dimethoxybenzyl Bromide (6).
To a solution of benzyl alcohol 5 (5 g, 12 mmol) in DCM (100 mL)
was added PBr3 (1 M in DCM, 6 mL, 6 mmol). After stirring 18 h, the
reaction was terminated by the addition of NaHCO3 (10%, 25 mL),
and the aqueous phase was extracted with DCM. The combined extract
was washed with cold water. Removal of solvent yielded a brown oil
(6) that was not further purified.
4-Acetoxy-3,5-dimethoxybenzaldehyde (12). A solution prepared
from 3,5-dimethoxy-4-hydroxybenzaldehyde (5 g, 27 mmol) and DMAP
(0.34 g, 2.7 mmol) in pyridine (50 mL) and acetic anhydride (8 mL,
81 mmol) was stirred for 16 h. Reaction was terminated by the addition
of water (100 mL) followed by extraction with EtOAc, washing of the

402 Journal of Natural Products, 2010, Vol. 73, No. 3
Pettit et al.
organic phase with HC1 (1 N), and removal of solvent. The residue
was purified by CC (7:3 hexane-EtOAc) to give 12 (2.4 g) as a
colorless solid that crystallized from EtOAc-hexane: mp 116-117 °C;
1H NMR (CD3OD) δ 2.36 (s, 3H), 3.90 (s, 6H), 7.15 (s, 2H), 9.91 (s,
1H).
was cooled to 0 °C before the addition of aldehyde 17 (4.13 g, 10
mmol). Stirring was continued at 0 °C for 6 h and at rt for 16 h before
the addition of water (100 mL) and extraction with EtOAc. After
removal of solvent (in vacuo) from the organic phase, the oil was
separated by CC (7:3 hexane-EtOAc), affording stilbene 11 (2.1 g,
25%) as a colorless oil.
4-Acetoxy-3′-(tert-butyldiphenylsilyloxy)-3,4′,5-trimethoxy-(Z)-
stilbene (14). Phosphonium bromide 13 (7.7 g, 1.07 mmol)12b in THF
(50 mL) was cooled to -78 °C, and n-BuLi (2.5 M in hexane, 3.9
mL) was added. The mixture was stirred for 1 h, aldehyde 12 (2 g, 8.9
mmol) was added in four portions (over 5 min), and the resulting
mixture was allowed to warm to rt and stirred an additional 3 h. Water
(100 mL) was added, and the mixture was extracted with EtOAc. The
extract was washed with brine and dried over sodium sulfate. After
removal of solvents, the resulting oil was separated by CC (9:1
hexane-EtOAc) to yield stilbene 14 (0.81 g, 16%): mp 81-82 °C
(hexane-acetone); 1H NMR (CD3OD, 300 MHz) δ 1.09 (s, 9H), 2.32
(s, 3H), 3.47 (s, 3H), 3.61 (s, 6H), 6.32 (d, J ) 1.2 Hz, 2H), 6.47 (s,
2H), 6.58 (d, J ) 8.1 Hz, 1H), 6.75 (d, J ) 2.4 Hz, 1H), 6.79 (dd, J
) 2.4, 8.1 Hz, 1H); anal. C 71.87%, H 6.42%, calcd for C35H38O6Si,
C 72.14%, H 6.57%.
4-Acetoxy-3′-hydroxy-3,4′,5-trimethoxy-(Z)-stilbene (15). To a
solution of stilbene 14 (0.81 g, 1.4 mmol) in THF (30 mL) was added
TBAF (1 M in THF, 1.5 mL), stirring was continued for 2 h, 50 mL
of 1 N HC1 was added, and the mixture was extracted with EtOAc.
The solvent was removed from the organic phase, and the residue was
separated by CC (7:3 hexane-EtOAc) to give stilbene 15 as a colorless
oil (0.40 g, 84%): bp (dec) 110-112 °C (0.01 mmHg) 1H NMR
(CD3OD, 300 MHz) δ 2.32 (s, 3H), 3.66 (s, 6H), 3.86 (s, 3H), 5.60 (s,
1H), 6.42 (d, J ) 12 Hz, 1H), 6.50 (d, J ) 12 Hz, 1H), 6.55 (s, 2H),
6.72 (d, J ) 8.1 Hz, 1H), 6.79 (dd, J ) 2.1, 8.1 Hz, 1H), 6.90 (d, J )
2.1 Hz, 1H); 13C NMR (CD3OD, 75 MHz) δ 20.53, 55.95, 56.02,
105.61, 110.27, 115.01, 121.04, 127.57, 128.74, 130.07, 130.26, 135.37,
145.15, 145.74, 151.64, 168.64; anal. C 66.33%, H 5.80%, calcd for
C19H20O6, C 66.27%, H 5.85%.
4-Acetoxy-3′-O-bis(benzyl)phosphoryl-3,4′,5-trimethoxy-(Z)-stil-
bene (16). To a solution of 3′-phenol 15 (0.55 g, 1.6 mmol) in
acetonitrile (10 mL) that was cooled to -10 °C was added CCl4 (1.5
mL, 15.8 mmol). The mixture was stirred for 10 min before the addition
of DIPEA (0.56 mL, 3.25 mmol) and DMAP (20 mg, 0.15 mmol).
After 1 min, dibenzylphosphite (0.525 mL, 2.4 mmol) was added
(dropwise over 5 min). The reaction mixture was stirred an additional
3 h at -10 °C, treated with KH2PO4 (20 mL, 0.5 M), stirred for a
further 10 min, and then extracted with EtOAc. The solvent was
removed from the organic phase, and the residue was separated by CC
(3:2 hexane-EtOAC) to yield phosphate 16 (0.65 g, 68%): mp 82-83
°C; 1H NMR (CDCl3, 300 MHz) δ 3.64 (s, 6H), 3.76 (s, 3H), 5.11 (s,
2H), 5.14 (s, 2H), 6.44 (d, J ) 2.1 Hz, 2H), 6.50 (s, 2H), 6.77 (d, J )
8.4 Hz, 1H), 7.06 (d, J ) 8.1 Hz, 1H), 7.12 (m, 1H), 7.32 (s, 10H);
anal. C 66.82%, H 5.79%, calcd for C33H33O9P, C 66.57%, H 5.50%.
3′-O-Bis(benzyl)phosphoryl-4-hydroxy-3,4′,5-trimethoxy-(Z)-stil-
bene (3). To a solution of stilbene 16 (0.60 g, 0.1 mmol) in CH3OH
(10 mL) was added K2CO3 (5 mL, 5% in 1:1 CH3OH-H2O). After
stirring for 1 h, the reaction was terminated by the addition of HC1 (1
N, until pH ∼7) and extracted with EtOAc. Solvent was removed (in
vacuo) from the organic phase, and the residue was separated by CC
(3:2 hexane-EtOAc) to afford stilbene 3 as a yellow oil (0.18 g, 30%):
bp (dec) 53-55 °C (0.01 mm); 1H NMR (CDCl3, 300 MHz) δ 3.80 (s,
3H), 3.94 (s, 6H), 5.18 (s, 2H), 5.20 (s, 2H), 5.6 (bs, 1H), 6.70 (s, 2H),
6.81 (s, 1H), 6.88 (d, J ) 8.4 Hz, 1H), 7.21 (d, J ) 8.4 Hz, 1H), 7.33
(m, 10H); anal. C 65.93%, H 5.82%, calcd for C31H31O8P, C 66.19%,
H 5.55%.
3′-O-Bis(benzyl)phosphoryl-combretastatin A-4 (18). To a solution
of Na2S2O3 (20 mg, 0.125 mmol), TBAF (125 µL, 1 M in THF), and
CH3I (80 µL, 1.25 mmol) in THF (1 mL) was added stilbene 11 (0.10
g, 0.125 mmol), and the mixture was stirred for 1 h at rt. The reaction
was terminated by the addition of water (5 mL), and the mixture was
extracted with DCM. The solvent was removed (in vacuo) from the
organic phase, and the residue was separated by CC (3:2 hexane-EtOAc)
to yield stilbene 184 (51 mg, 74%): mp 73 °C; (lit4 mp 73 °C); H
1
NMR (CDCl3, 300 MHz) δ 3.67 (s, 6H), 3.77 (s, 3H), 3.80 (s, 3H),
5.12 (s, 2H), 5.14 (s, 2H), 6.40 (d, J ) 12 Hz, 1H), 6.45 (d, J ) 12
Hz, 1H), 6.48 (s, 2H), 6.78 (d, J ) 9 Hz, 1H), 7.06 (dd, J ) 1.8, 9 Hz,
1H), 7.15 (d, J ) 1.8 Hz, 1H), 7.30 (s, 10H).
Acknowledgment. Financial support we are pleased to record was
provided by Outstanding Investigator Grant CA44344-10-12 and RO1
CA90441-01-04, 2R56-CA 09441-06A1, and 5RO1 CA 090441-07
awarded by the Division of Cancer Treatment and Diagnosis, National
Cancer Institute, DHHS; the Arizona Biomedical Research Commission;
the Robert B. Dalton Endowment Fund; the Caitlin Robb Foundation;
Dr. John C. Budzinski; the Eagles Art Ehrmann Cancer Fund; and the
Ladies Auxiliary to the Veterans of Foreign Wars. For other helpful
assistance, we thank Drs. J. C. Knight and M. D. Williams (NSF Grant
CHE-9808678).
References and Notes
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1
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