Antineoplastic agents. 548. Synthesis of iodo- and diiodocombstatin phosphate prodrugs

Article abstract of DOI:10.1021/np200797x

Toward the objective of designing a structurally modified analogue of the combretastatin A-4 phosphate prodrug (1b) with the potential for increased specificity toward thyroid carcinoma, synthesis of a series of iodocombstatin phosphate (11a-h) and diiodocombstatin phosphate prodrugs (12a-h) has been accomplished. The diiodo series was obtained via 8a and 9c from condensation of 4 and 6, and the iodo sequence involved a parallel pathway. Both series of iodocombstatins were found to display significant to powerful inhibition of the growth of a panel of human cancer cell lines and of the murine P388 lymphocytic leukemia cell line. Of the diiodo series, 12a was also found to markedly inhibit growth of pediatric neuroblastoma, and monoiodocombstatin 9a strongly inhibited HUVEC growth. Overall, the strongest activity was found against the breast, CNS, leukemia, lung, and prostate cancer cell lines and the least activity against the pancreas and colon lines. Parallel biological investigations of tubulin interaction, antiangiogenesis, and antimicrobial effects were also conducted.

Full text of DOI:10.1021/np200797x

Article
pubs.acs.org/jnp
Antineoplastic Agents. 548. Synthesis of Iodo- and Diiodocombstatin
Phosphate Prodrugs
George R. Pettit,*,† Heidi J. Rosenberg,† Rachel Dixon,† John C. Knight,† Ernest Hamel,‡
Jean-Charles Chapuis,† Robin K. Pettit,† Fiona Hogan,† Brandy Sumner,† Kenneth B. Ain,§
and Brindi Trickey-Platt†
†Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604,
United States
‡Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702-1201, United States
§Division of Endocrinology & Molecular Medicine, Department of Internal Medicine, Room MN524, University of Kentucky Medical
Center, 800 Rose Street, Lexington, Kentucky 40536-0298, United States
ABSTRACT: Toward the objective of designing a structurally
modified analogue of the combretastatin A-4 phosphate prodrug
(1b) with the potential for increased specificity toward thyroid
carcinoma, synthesis of a series of iodocombstatin phosphate (11a−
h) and diiodocombstatin phosphate prodrugs (12a−h) has been
accomplished. The diiodo series was obtained via 8a and 9c from
condensation of 4 and 6, and the iodo sequence involved a parallel pathway. Both series of iodocombstatins were found to
display significant to powerful inhibition of the growth of a panel of human cancer cell lines and of the murine P388 lymphocytic
leukemia cell line. Of the diiodo series, 12a was also found to markedly inhibit growth of pediatric neuroblastoma, and
monoiodocombstatin 9a strongly inhibited HUVEC growth. Overall, the strongest activity was found against the breast, CNS,
leukemia, lung, and prostate cancer cell lines and the least activity against the pancreas and colon lines. Parallel biological
investigations of tubulin interaction, antiangiogenesis, and antimicrobial effects were also conducted.
rom our initial discoveries over 30 years ago of the
combretastatins, which are constituents of the African tree
cancer. In addition, excess production of the pituitary hormone
thyroid-stimulating hormone (TSH), important in the
regulation of thyroid gland growth and function, may be
involved in the etiology of thyroid cancer. Previously used
clinical treatments5c,d for thyroid cancer include surgery,
suppression of TSH, 131I-radiotherapy,5e and anticancer drugs.
Thyroid cancer is the fastest increasing (240% increase in the
past 30 years) cancer type in both men and women in the
United States; most of the increase in incidence of papillary
carcinomas is probably due to earlier detection,5f but the need
for new and effective anticancer drugs, especially for the more
aggressive carcinomas, is clear. Early in the human cancer phase
I clinical trials of CA4 (1a), as the sodium phosphate prodrug
1b (CA4P, Fosbretabulin, Zybrestat), evidence for activity
against otherwise refractory anaplastic thyroid carcinoma was
observed, and this was further supported in a phase II clinical
trial.2b,6,7 The pioneering clinical studies of Remick and
colleagues2b,7a have led via phase II clinical trials of CA4P
against anaplastic thyroid carcinoma to the current phase III
level.5b
F
Combretum caffrum (Combretaceae), knowledge of their
chemistry, biology, and medical potential has continued to
advance.2 Preclinical and clinical developments over the last 12
years have been rapidly accelerating, especially for combretas-
tatin A-4 (CA4, 1a), in the form of the phosphate prodrug
CA4P (1b), and combretastatin A-1 (CA1, 1c) as promising
cancer vascular-disrupting and ophthalmology drugs.2,3 In turn,
these encouraging developments have stimulated a variety of
efforts devoted to synthesis and biological evaluation of
combretastatin structural modifications, related primarily to
CA4 (1a) and CA1 (1c). Recent reports include SAR studies
that provide varying levels of cancer cell growth inhibition.4
For 2011, the estimated incidence of thyroid cancer in the
United States is at about 48 000, including 1740 deaths.5a Most
thyroid cancers are well differentiated papillary (about 80%)
and follicular (about 14%) carcinomas. Both types of tumor
cells are believed to be derived from follicular epithelial cells
that produce thyroid hormone. Of the remaining thyroid
malignancies, about 4% are medullary carcinoma (neuro-
endocrine) and about 2% are the exceptionally aggressive
anaplastic carcinoma (median survival of 4−5 months and a
near-100% lethal outcome).2b,5b Significantly, the incidence of
both follicular and anaplastic carcinomas is increased in
populations residing in areas of iodine deficiency. Radiation
exposure represents the most general risk factor for thyroid
The most active thyroid hormone components are
triiodothyronine (2a) and its tetraiodo derivative thyroxine
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: September 30, 2011
Published: February 10, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Article
(2b). Both are derived from 3,5-diiodotyrosine, with release
controlled by TSH. Consequently, the focus of the present
investigation was evaluation of iodo modifications of CA4P
(1b), with the ultimate goal of obtaining enhanced concen-
tration of drug in the thyroid carcinoma tissue. Of the
continuing CA4 (1a) structural modification studies,4 those
involving halogen substitutions have been primarily concerned
with fluoro, chloro, bromo, and iodo derivatives.4a,8 Our
objective here was the synthesis and initial evaluation of the 3-
iodo- and 3,5-diiodostilbene CA4 modifications, herein
designated iodocombstatin (3a) and diiodocombstatin (3b),
and of their phosphate prodrug modifications.
Scheme 1
RESULTS AND DISCUSSION
■
The initial intermediates were synthesized by Wittig reaction of
compound 4 with 3-iodo-4,5-dimethoxybenzaldehyde (5) and
with 3,5-diiodo-4-methoxybenzaldehyde (6) to provide iodos-
tilbenes 7 and 8, respectively, as Z/E mixtures (Scheme 1). The
isomers were separated by silica gel column chromatography
(CC), and subsequent deprotection with tetrabutylammonium
fluoride afforded the corresponding phenols 9a−d. Iodocomb-
statins 9a and 9c were further characterized as their 3-acetyl
derivatives 9e and 9f. The Z-isomers 9a and 9c were treated
with dibenzylphosphite and CCl4 in the presence of
diisopropylethylamine and N,N-dimethylaminopyridine
(DMAP) to provide bisbenzylphosphates 10a and 10b, from
which the benzyl ester protecting groups were cleaved with
bromotrimethylsilane.3b,8b,9 Subsequent treatment of the
resulting phosphoric acids with the appropriate cation
precursor readily afforded prodrugs 11a−h and 12a−h. In
this sequence, the debenzylation step proved to be the most
challenging: in order to minimize conversion of the Z- to E-
geometry, presumably due to available bromonium ion, cooling
(0 °C) was required, as well as an aqueous sodium thiosulfate
treatment. Once the potassium salts of the prodrugs were in
hand, a selection of alkali metal, ammonium, and amino acid
salts of the phosphates were synthesized by ion exchange
chromatography to complete the 11a−h and 12a−h prodrug
series.
aqueous solubility (Table 1) than the iodostilbene precursors
(9a−d). Compound 12a was also tested against three pediatric
neuroblastoma cell lines (GI50 in μg/mL: SK-N-SH, 0.0075;
SK-N-AS, 0.002; IMR-32, 0.081).
Against two cancer cell lines long believed to be human
anaplastic thyroid carcinoma cells (KAT-4 and SW1736), we
had earlier evaluated the potential for increased specificity by
the new iodocombstatins. Diiodocombstatin 9c proved to be
more inhibitory than iodocombstatin (9a, Table 2), but both
strongly inhibited growth of the ATC line KAT-4. However,
phenols 9a and 9c were 100× less active against the SW1736
line. The potassium phosphates 11a and 12a as expected
proved to be less inhibitory (10×). While growth of the KAT-4
cells was significantly reduced by phosphates 11a and 12a, the
SW1736 cells proved to be resistant. It has since been reported
that KAT-4 is one of 12 redundant cell lines of 40 presumptive
human thyroid cancer cell lines tested for cross-contamination
and is likely not of thyroid origin.11a Thus the KAT-4 cancer
cell line data, while interesting, are not directly significant with
respect to thyroid cancer but are a further indication of the
cancer cell growth inhibitory activity of these compounds.
The ability of the iodostilbenes to inhibit cancer cell growth
was examined and compared to the activities of combretastatin
A-3 (3c),10 fluorcombstatin (3d),8b and combretastatin A-4
(1a) (Table 1). The iodostilbene phosphate salts all retained
strong activity and, as expected, demonstrated markedly better
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Table 1. Human Cancer Cell Line Growth Inhibition (GI50, μg/mL) and Murine P388 Lymphocytic Leukemia Inhibitory
Activities (ED50, μg/mL)
a
compound solubility (mg/mL) leukemia P388 pancreas BXPC-3 breast MCF-7 CNS SF-268 lung-NSC NCI-H460 colon KM20L2 prostate DU-145
1a
0.0003
0.0004
0.26
0.39
0.23
2.3
<0.001
0.036
0.0083
0.0016
0.00018
0.0036
0.55
0.0006
0.029
0.19
0.061
0.034
1.2
0.0008
0.0072
0.0043
0.0019
0.00018
0.0021
0.27
1b
3c
0.49
3d
0.0020
0.0020
0.0028
0.189
0.745
0.048
0.038
2.7
0.0027
0.00022
0.0027
0.18
0.0032
0.00029
0.0034
0.21
>1
9a
0.328
0.15
1.7
9c
9b
9d
>10
3.0
0.94
3.3
3.4
>10
0.41
>1
>1
0.24
5.8
11a
11b
11c
11d
11e
11f
11g
11h
12a
12b
12c
12d
12e
12f
12g
12h
14
2
0.0021
0.0020
0.017
0.381
0.469
0.490
0.21
0.32
0.16
0.26
0.37
0.44
0.0064
0.018
0.0038
0.0047
0.0065
0.0044
0.035
0.0048
0.050
0.066
0.051
0.066
0.070
0.10
0.0057
0.018
0.0040
0.0037
0.0044
0.0033
0.0097
0.0043
0.053
0.051
0.050
0.054
0.041
0.054
0.086
0.040
0.0043
0.017
0.0039
0.0036
0.0036
0.0031
0.0034
0.0040
0.046
0.327
0.050
0.033
0.025
0.030
0.033
0.025
0.0038
0.011
≥2.4
0.0043
0.0026
0.0029
0.0021
0.0030
0.0047
0.028
0.0032
0.0026
0.0026
0.0022
0.0029
0.0034
0.030
≥4
≥2
0.51
0.32
0.59
0.40
22
2
>1
>1
>1
>1
>1
>1
>1
>1
0.242
≥4
0.021
0.37
0.35
0.33
0.36
0.37
0.33
0.032
0.014
0.028
0.011
0.025
0.011
0.023
0.017
0.22
0.026
0.026
0.047
0.94
0.021
a
Solubility values represent solution in 1 mL of H2O at 25 °C.
Table 2. Human Anaplastic Thyroid Carcinoma Cell Line
Inhibition Values (GI50, μg/mL)
11a (Figure 1F) showed a slightly larger reduction in the size of
the cords as compared to 12a (Figure 1G). Such inhibitory
activity against the HUVECs is of considerable interest since
endothelial cells play a central role in the angiogenic process.
Moreover, combretastatin derivatives have been shown to be
involved in vascular targeting therapy by destroying the existing
vasculatures of a tumor, specifically staunching blood flow and
inhibiting tumor growth through the disruption of the tubulin
cytoskeleton of endothelial cells, which leads to thrombosis of
the vasculature (see Table 5).12
The mono (9a) and diiodo (9b) analogues of CA4 (1a) and
their sodium phosphate derivatives (11b and 12b, respectively)
were also evaluated and compared to 1a for inhibitory effects
on tubulin (experiments were performed contemporaneously,
Table 5). Similar IC50 values were obtained for the non-
phosphorylated compounds, with 9a appearing to be somewhat
less inhibitory than 1a, and 9c more active than 1a. The two
phosphorylated derivatives 11b and 12b had little or no
inhibitory effect on tubulin assembly, as had been observed
previously with CA4P (1b).
Combretastatin A-4 (1a) is a potent inhibitor of the binding
of [3H]colchicine to tubulin. In our standard assay with 1.0 μM
tubulin and 5.0 μM [3H]colchicine, the binding of the
radiolabel to the protein has routinely been 95−100% inhibited
by 5.0 μM 1a and 75−85% inhibited when 1a was present in
the reaction mixture at 1.0 μM, equimolar with tubulin.13 In the
current experiments, this finding was obtained again (Table 5).
We compared 9a and 9c with 1a as inhibitors of colchicine
binding in the standard reaction conditions. While 9a was
identical to 1a in its inhibitory effect, the diiodo 9c was more
potent. Virtually complete inhibition of colchicine binding
occurred with 9c at 1 μM, making this compound the most
potent inhibitor of colchicine binding we have yet examined.
compound
KAT-4
0.089
SW1736
9a
2.2
1.2
9c
0.039−0.063
0.37−0.43
0.38−0.44
11a
12a
>10
>10
Antiangiogenesis is now actively pursued as a promising
antitumor strategy.11b Iodo- (9a) and diiodocombstatin (9b),
as well as phosphate prodrugs 11a,b and 12a,b, were evaluated
against human umbilical vein endothelial cells (HUVEC) in
vitro (Table 3). These cells showed significant sensitivity to the
Table 3. Human Umbilical Vein Endothelial Cell (HUVEC)
Inhibition Values (GI50, μg/mL)
compound
HUVEC
9a
0.000040
0.00028
0.00025
0.00035
0.0049
9c
11a
11b
12a
12b
0.051
new compounds. Iodocombstatin (9a) was the most active,
with an ED50 value of 4 × 10−5 μg/mL, followed by
diiodocombstatin (9c). A similar pattern was observed with
phosphates 11a and 12a. With both the mono- and
diiodocombstatins, 9a and 9c, cord lengths as well as junction
numbers were markedly reduced at 0.001 μg/mL (see Table 4
and Figure 1B,D) but were similar to the untreated control
(Figure 1A) at 0.0001 μg/mL (Figure 1C,E). At 0.001 μg/mL,
387
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Journal of Natural Products
Article
Table 4. Length of Cords Formed, Number of Junctions, and
Relative Percent Growth of Cells
EXPERIMENTAL SECTION
■
General Experimental Methods. All solvents were redistilled,
and ether refers to diethyl ether. Isovanillin, 3-iodo-4,5-dimethox-
ybenzaldehyde, anhydrous CH2Cl3, anhydrous toluene, tetrabutylam-
monium fluoride [1.0 M solution in tetrahydrofuran (THF)], dibenzyl
phosphate, 4-dimethylaminopyridine, CCl4, Dowex-50W (HCR-W2),
and L-tryptophan methyl ester hydrochloride were obtained from
Sigma-Aldrich Chemical Co. 3,5-Diiodo-4-hydroxybenzaldehyde was
obtained from Lancaster Synthesis Inc., and diisopropylethylamine
from Avocado (Alfa Aesar, Ward Hill, MA, USA). The remaining
reagents were purchased from Acros Organics (Fisher Scientific,
Pittsburgh, PA, USA). All reactions were performed under an argon
atmosphere, protected from bright light to avoid the potential for
photochemical side reactions. The reactions and products were
monitored by TLC using Analtech silica gel GHLF Uniplates
visualized under long-wave and short-wave UV irradiation and stained
by dipping into phosphomolybdic acid in EtOH followed by heating.
All organic extracts of aqueous solutions were dried over anhydrous
magnesium sulfate. Where appropriate, the crude products were
separated by column chromatography, using flash (230−400 mesh
ASTM) silica gel from E. Merck.
drug conc (μg/
lengths of
junction
rel %
c
a
b
mL)
cords
no.
growth
9a
0.01
−
+
−
+
14
14
18
90
0.001
0.0001
0.00001
++(+)
++(+)
9c
0.01
−
+
−
(+)
4
8
0.001
0.0001
0.00001
+++
+++
84
87
11a
11b
12a
12b
0.01
1
10
77
95
0.001
++
++(+)
+++
0.0001
0.00001
+++
+++
+++
0.1
−
−
+
−
−
+
7
14
Melting points were measured with an electrothermal digital
0.01
melting point apparatus and are uncorrected. All H and 13C NMR
1
0.001
0.0001
5
spectra were obtained using Varian Gemini 300 MHz or Varian Unity
400 or 500 MHz instruments with CDCl3 (TMS internal reference) as
solvent unless otherwise noted. The 31P NMR spectra were obtained
in CDCl3 or D2O solution with 85% H3PO4 as an external standard
employing a Varian Unity 400 or 500 MHz instrument. Elemental
analyses were performed by Galbraith Laboratories, Inc. Mass spectra
were obtained in our institute with a JEOL LCmate instrument.
3,5-Diiodo-4-methoxybenzaldehyde (6). A solution of 3,5-diiodo-
4-hydroxybenzaldehyde (5 g, 13.37 mmol) in anhydrous DMF (50
mL) was cooled to 0 °C, and sodium hydride (0.64 g, 16 mmol, 60%
dispersion in mineral oil) was slowly added. Iodomethane was then
added, and stirring was continued at rt in the dark for 19 h. The
reaction was terminated by the addition of H2O (50 mL), and the
mixture was extracted with EtOAc−hexane (1:1, 3 × 50 mL). The
combined organic extract was filtered and concentrated in vacuo. The
residue was separated by CC on silica gel using EtOAc−hexane (1:9)
as eluent. The product was a colorless solid (4.2 g, 80%) that
104
0.1
−
++(+)
−
++(+)
15
33
88
96
0.01
0.001
0.0001
+++
+++
1
−
+
−
(+)
−7
−2
0.1
0.01
0.001
++(+)
++(+)
>100
>100
a
Lengths of cords: −, no cords; +, small; +(+), 25% control; ++, 50%
b
control; ++(+), 75% control; +++, same as control. Number of
junctions: −, no junctions; +, few; +(+), 25% control; ++, 50%
c
control; +(+), 75% control; +++, same as control. Relative to control.
crystallized from hexane: mp 121−123 °C (lit.15 mp 124 °C); H
1
Combretastatin A-4 (1a) and the prodrug CA4P (1b) were
previously shown to have marginal antimicrobial activity
(minimum inhibitory concentration [MIC] = 25−100 μg/
disk) against the Gram positive opportunist Micrococcus luteus
and the Gram negative pathogen Neisseria gonorrhoeae.14 The
new CA4 analogues 9a,c, as well as 9b, and phosphate salts
11a−h and 12a−h were tested against a panel of bacteria and
fungi. Phenols 9a and 9c were active against M. luteus (MIC:
2−4 and 4−16 μg/mL, respectively), and the diiodocombstatin
phosphates 12a−12h were very active against N. gonorrhoeae
(MIC in μg/mL: 12a, <0.5−4; 12b, 32−64; 12c, <0.5−2; 12d,
<0.5; 12e, <0.5; 12f, <0.5−1; 12g, <0.5; 12h, <0.5−2).
Iodocombstatins 11d, 11g, and 11h were also active against N.
gonorrheae (MIC in μg/mL: 11d, 16−32; 11g, 4−8; 11h, 32−
64), and iodocombstatin 9a had marginal activity against the
pathogenic yeast Cryptococcus neoformans (MIC: 64 μg/mL).
From all evidence now in hand, diiodocombstatin (9c)
appears especially suitable for further preclinical development.
The very powerful biological properties of the 3-iodo- and
especially the 3,5-diodocombstatins may be the result, in part,
of increased halogen−protein bonding, as suggested by Ley and
colleagues.4a 3,5-Diiodocombstatin (9c) and derived prodrugs
offer promising candidates for further preclinical development.
NMR (CDCl3, 300 MHz) δ 3.93 (s, 3 H), 8.27 (s, 2 H), 9.81 (s, 1 H);
13C NMR (CDCl3, 75 MHz) δ 60.35, 90.72, 140.65, 140.71, 187.51,
187.56; anal. C 24.86, H 1.58%, calcd for C8H6I2O2, C 24.77, H 1.56%.
3-Iodo-4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-Z-stilbene
(7a) and 3-Iodo-4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-E-
stilbene (7b). Method A. Phosphonium bromide 410 (3.67 g,
5.13 mmol) was dissolved in DCM at 0 °C. Sodium hydride
(60% dispersion in mineral oil, 0.41 g, 10.2 mmol) was added,
and the mixture turned orange. Next, 3-iodo-4,5-dimethox-
ybenzaldehyde (5, 1 g, 3.42 mmol) was added, and stirring was
continued for 21 h. The reaction was terminated by addition of
H2O (50 mL), and the mixture was extracted with DCM (3 ×
50 mL). The organic phase was filtered and concentrated. The
oil obtained was subjected to flash chromatography on silica gel
and eluted with 0−3% EtOAc in hexane to afford Z-stilbene 7a
(0.86 g, 39%), which crystallized as a colorless solid from
hexane: mp 122−124 °C; 1H NMR (CDCl3, 300 MHz) δ 1.07
(s, 9 H), 3.45 (s, 3 H), 3.55 (s, 3 H), 3.79 (s, 3 H), 6.21 (d, 1
H, J = 12 Hz), 6.31 (d, 1 H, J = 12 Hz), 6.59 (d, 1 H, J = 7.8
Hz), 6.72 (s, 2 H), 6.77 (dd, 1 H, J = 7.8, 1.5 Hz), 7.19 (d, 1 H,
J = 1.8 Hz), 7.40−7.20 (m, 6 H), 7.64 (d, 4H, J = 7.5 Hz); 13C
NMR (CDCl3, 75 MHz) δ 19.68, 26.62, 55.05, 55.56, 60.33,
91.94, 111.72, 113.09, 120.78, 122.43, 126.73, 127.33, 129.32,
130.28, 130.93, 133.54, 135.17, 144.70, 149.82, 151.82; HRMS
m/z 651.1474 [M + H]+ (calcd for C33H36IO4Si, 651.1428);
anal. C, 60.79; H, 5.67%, calcd for C33H35IO4Si, C 60.92, H
5.45%.
388
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Figure 1. Treated human umbilical vein endothelial cells. Morphological appearance of the cords in HUVEC cells: (A) untreated control; (B) after
exposure to 9a (0.001 μg/mL for 24 h); (C) after exposure to 9a (0.0001 μg/mL for 24 h); (D) after exposure to 9c (0.001 μg/mL for 24 h); (E)
after exposure to 9c (0.0001 μg/mL for 24 h); (F) after exposure to 11a (0.001 μg μg/mL for 24 h); (G) after exposure to 12a (0.001 μg/mL for 24
h).
In a parallel series of Wittig reactions evaluating a variety of solvents
(CH2Cl2, EtOAc, toluene, and CHCl3), temperature (−70 to 0 °C to
rt), time (2.5−9.25 h), and base (NaH, KOH/18-crown-6),
production of the Z-isomer ranged from 42% to 52% yields, as
judged by NMR analyses.
Further elution gave E-stilbene 7b (0.96 g, 43%), which crystallized
from hexane as a colorless solid: mp 98−99 °C; 1H NMR (CDCl3, 300
MHz) δ 1.14 (s, 9 H), 3.55 (s, 3 H), 3.82 (s, 3 H), 3.82 (s, 3 H), 3.89
(s, 3 H), 6.43 (d, 1 H, J = 15.9 Hz), 6.71−6.76 (m, 2 H), 6.86−6.95
(m, 3 H), 7.33−7.42 (m, 6 H); 13C NMR (CDCl3, 100 MHz) δ 19.81,
26.67, 55.28, 60.50, 92.65, 110.22, 112.11, 117.70, 120.56, 124.58,
127.52, 128.45, 128.68, 129.60, 129.77, 133.64, 135.38, 135.82, 145.18,
148.13, 150.56, 152.49; HRMS m/z 651.1400 [M + H]+ (calcd for
C33H36IO4Si, 651.1428); anal. C 60.88, H 5.63%, calcd for
C33H35IO4Si, C 60.92, H 5.42%.
Table 5. Inhibitory Effects of Iodocombstatins on Tubulin
Assembly and on Colchicine Binding to Tubulin in
Comparison with Those of Combretastatin A-4 (1a)
inhibition of colchicine binding to
tubulin
inhibition of tubulin
assembly
1.0 μM
compound
10 μM
compound
IC50 (μM) SD
% inhibition SD
1a
2.1 0.03
81
78
5
99 0.9
a
1b
>40
9a
2.5 0.04
1.5 0.3
>40
8
100 0.8
100 0.3
9c
95 0.8
11b
12b
Method B. Butyllithium (4.5 mL, 11.3 mmol) was added to a stirred
and cooled (−70 °C) suspension of phosphonium bromide 4 in dry
THF (100 mL). The solution was stirred for 30 min at −70 °C and
then for 6 h at rt. Water (50 mL) was added, and the reaction mixture
>40
a
Data from previous evaluations, not repeated in current studies.
389
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was extracted with EtOAc (3 × 100 mL). The organic phase was
filtered and concentrated to an oil, which was subjected to flash
chromatography on silica and eluted with 0−3% EtOAc in hexane to
afford Z-stilbene 7a (1.4 g, 21%) as a colorless solid: mp 122−124 °C.
3,5-Diiodo-4,4′-dimethoxy-3′-O-tert-butyldiphenylsilyl-Z-stilbene
(8a) and 3,5-Diiodo-4,4′-dimethoxy-3′-O-tert-butyldiphenylsilyl-E-
stilbene (8b). Method A. To a solution of phosphonium
bromide 410 (2.77 g, 3.87 mmol) in DCM at 0 °C was added
sodium hydride (60% dispersion in mineral oil, 0.31 g, 7.7
mmol), and the mixture turned orange. After addition of
aldehyde 6 (1.0 g, 2.57 mmol), stirring was continued for 7.5 h.
The reaction was terminated by addition of H2O (50 mL) and
extracted with DCM (3 × 50 mL). The organic extract was
filtered and concentrated. The oily residue was flash chromato-
graphed on silica gel using hexane as eluent to give an isomeric
mixture of 8a and 8b (71% yield, 1.35 g). Further elution gave
mmol) as described above for the synthesis of Z-isomer 9a. The oily
mixture was separated by CC with 2:1 hexane−EtOAc as eluent to
1
provide Z-isomer 9c as an oil (0.45 g, 49%): H NMR (CDCl3, 300
MHz) δ 3.85 (s, 3 H), 3.89 (s, 3 H), 5.54 (s, 1 H), 6.26 (d, 1 H, J = 12
Hz), 6.49 (d, 1 H, J = 12 Hz), 6.74 (s, 2H), 6.82 (s, 1 H), 7.67 (s, 2
H); 13C NMR (CDCl3, 125 MHz) δ 55.98, 60.73, 89.98, 110.46,
114.87, 120.98, 125.08, 129.47, 131.57, 137.37, 139.96, 145.42, 146.21,
157.50; HRMS m/z 508.9111 [M + H]+ (calcd for C16H15I2O3,
508.9113); anal. C 37.80, H 2.83%, calcd for C16H14I2O3, C 37.82, H
2.78%.
Further elution led to the E-stilbene 9d (0.46 g, 50% yield) as a
1
colorless solid, which crystallized from hexane: mp 127−129 °C; H
NMR (CDCl3, 300 MHz) δ 3.86 (s, 3 H), 3.91 (s, 3 H), 5.62 (s, 1 H),
6.71 (d, 1 H, J = 16.5 Hz), 6.83 (d, 1 H, J = 8.1 Hz), 6.90 (d, 1 H, J =
17.1 Hz), 6.95 (d, 1 H, J = 8.4 Hz), 7.10 (d, 1 H, J = 2.4 Hz), 7.85 (s, 2
H, H-2); 13C NMR (CDCl3, 75 MHz) δ 55.51, 60.30, 90.17, 100.17,
100.21, 110.18, 111.35, 119.17, 122.56, 129.63, 129.81, 136.82, 137.19,
146.34, 157.25; HRMS m/z 508.9119 [M + H]+ (calcd for
C16H15I2O3, 508.9113); anal. C 38.01, H 2.91%, calcd for
C16H14I2O3, C 37.82, H 2.78%.
1
the E-isomer 8b (0.10 g, 5%) as a colorless oil: H NMR
(CDCl3, 300 MHz) δ 1.14 (s, 9 H), 3.56 (s, 3 H), 3.84 (s, 3 H),
6.33(d, 1 H, J = 15.9 Hz), 6.72 (d, 1 H, J = 8.4 Hz), 6.73 (d, 1
H, J = 15.9 Hz), 6.72 (d, 1 H, J = 8.4 Hz, ArH), 6.85 (d, 1H, J =
2.1 Hz), 6.92 (dd, 1 H, J = 1.8 Hz and J 8.4 Hz), 7.34−7.46 (m,
6 H), 7.72−7.75 (m, 6 H); 13C NMR (CDCl3, 100 MHz) δ
19.82, 26.69, 55.30, 60.77, 90.59, 112.09, 117.73, 120.83,
122.47, 127.55, 129.38, 129.65, 129.99, 133.58, 135.40, 137.15,
137.73, 145.22, 150.84, 157.55; HRMS m/z 747.0442 [M +
H]+ (calcd for C32H33I2O3Si, 747.0289).
Method B. Butyllithium (0.6 mL, 1.47 mmol) was added to a stirred
and cooled (−10 °C) suspension of phosphonium bromide 4 (1.01 g,
1.4 mmol) in dry THF (80 mL). The orange-red solution was stirred
for 10 min at rt. Aldehyde 6 (0.50 g, 1.33 mmol) was added, and the
reaction mixture color changed from red to yellow. Stirring was
continued at rt for 10 min before addition of ice water (100 mL) and
extraction of the mixture with EtOAc (3 × 100 mL). The organic
phase was washed with water (100 mL), filtered, and concentrated.
The resulting oil was partially separated by flash chromatography on
silica gel using hexane−EtOAc (100:1) as eluent to give an isomeric
mixture in a ratio of approximately 1:1.9 (Z:E, 0.90 g, 90%).
3-Iodo-4,4′,5-trimethoxy-3′-acetyl-Z-stilbene (9e). Phenol 9a
(0.45 g) was dissolved in pyridine (3 mL)−acetic anhydride (170
μL), and the mixture was stirred for 2 h. The mixture was concentrated
under reduced pressure from toluene (3 × 10 mL). The residue was
diluted with EtOAc (30 mL), and the solution was washed successively
with H2O (10 mL) and NaHCO3 (10% aq sol, 10 mL), filtered, and
concentrated. Flash chromatography on silica using 1:24 EtOAc−
hexane yielded acetate 9e (0.20 g, 41%) as a colorless solid that
1
crystallized from hexane: mp 121−122 °C; H NMR (CDCl3, 300
MHz) δ 2.29 (s, 3 H), 3.83 (s, 3 H), 3.85 (s, 3 H), 6.29 (d, 1 H, J = 12
Hz), 6.48 (d, 1 H, J = 12 Hz), 6.85 (d, 1 H, J = 8.7 Hz), 6.93 (d, 1 H, J
= 2.43), 7.06 (d, 1 H, J = 1.5 Hz), 7.09 (d, 1 H, J = 2.4 Hz), 7.67 (s, 2
H); 13C NMR (CDCl3, 125 MHz) δ 20.66, 55.94, 60.72, 90.11,
112.16, 123.25, 125.41, 127.47, 128.85, 130.64, 137.10, 139.54, 139.89,
150.69, 157.67, 168.79; HRMS m/z 582.9482 [M + CH3OH]+ (calcd
for C19H20I2O5, 582.9479); anal. C 39.30, H 3.13%, calcd for
C18H16I2O4, C 39.30, H 2.93%
3-Iodo-4,4′,5-trimethoxy-3′-hydroxy-Z-stilbene (9a). To a solu-
tion of silyl ether 7a (1.30 g, 1.99 mmol) in THF was added
tetrabutylammonium fluoride (2.2 mL, 2.2 mmol). The mixture was
stirred under Ar in the dark for 10 min, and the reaction was
terminated by the addition of H2O (5 mL). The product was extracted
with EtOAc (3 × 15 mL), and the organic phase was filtered and
concentrated. The crude product was separated by silica gel CC using
1:4 EtOAc−hexane to give stilbene 9a (0.70 g, 85%) as a colorless
solid: mp 92−94 °C; IR (film) νmax 3543, 3011, 2937, 2841, 1510,
1273, 1001, 908, 732 cm−1; 1H NMR (CDCl3, 300 MHz) δ 3.61 (s, 3
H), 3.81 (s, 3 H), 3.84 (s, 3 H), 6.32 (d, 1 H, J = 12 Hz), 6.34 (s, 1 H),
6.56 (d, 1 H, J = 12 Hz), 6.75 (s, 1 H), 6.83 (d, 1 H, J = 1.8 Hz), 6.85
(s, 3 H), 7.25 (d, 1 H, J = 1.5 Hz); 13C NMR (75 MHz, CDCl3) δ
55.56, 55.82, 60.33, 91.78, 110.50, 113.11, 115.00, 120.91, 126.96,
129.94, 130.28, 135.93 145.29, 146.10, 147.67, 151.79; HRMS m/z
413.0250 [M + H]+ (calcd for C17H18IO4, 413.0259); anal. C 49.38, H
4.24%, calcd for C17H17IO4, C 49.53, H 4.16%.
3,5-Diiodo-4,4′-dimethoxy-3′-acetyl-Z-stilbene (9f). To a solution
of phenol 9c (0.1 g, 0.24 mmol) in 3 mL of anhydrous pyridine was
added acetic anhydride (50 μL, 0.51 mmol) and a catalytic amount of
DMAP. The mixture was stirred for 90 min, and the reaction was
terminated by the addition of CH3OH (5 mL). Toluene was added,
and the solution was concentrated under reduced pressure prior to
flash chromatography on silica gel. EtOAc−hexane (1:9) as eluent
provided a colorless solid (0.1 mg, 91%) that crystallized from hexane:
1
mp 103−104 °C; H NMR (CDCl3, 300 MHz) δ 2.27 (s, 3 H), 3.61
(s, 3 H), 3.81 (s, 6 H), 6.38 (d, 1 H, J = 12 Hz), 6.48 (d, 1 H, J = 12
Hz), 6.77 (d, 1 H, J = 1.8 Hz), 6.83 (d, 1 H, J = 8.4 Hz), 6.96 (d, 1 H, J
= 1.5 Hz), 7.09 (dd, 1 H, J = 8.4 Hz, J = 2.4 Hz), 7.26 (s, 1 H); 13C
NMR (CDCl3, 125 MHz) δ 20.61, 55.67, 55.93, 60.44, 92.07, 112.07,
112.92, 123.17, 127.63, 127.74, 129.39, 129.65, 103.97, 134.96, 139.49,
147.99, 150.39, 152.05, 168.81; HRMS m/z 455.0356 [M + H]+ (calcd
for C19H20IO5, 455.0355); anal. C 49.67, H 4.18%, calcd for
C19H19IO5, C 50.24, H, 4.22%.
3-Iodo-4,4′,5-trimethoxy-3′-hydroxy-E-stilbene (9b). The E-iso-
mer 9b (0.29 g, 98%) was obtained from silyl ether 7b (0.46 g, 0.7
mmol) as described above for the synthesis of the Z-isomer 9a.
Separation by CC (7:3 hexane−EtOAc as eluent) gave E-isomer 9b
(0.29 g, 98%) as a colorless solid: mp 111−113 °C; 1H NMR (CDCl3,
300 MHz) δ 3.84 (s, 3 H), 3.87 (s, 3 H), 3.88 (s, 3 H), 5.85 (bs, 1 H),
6.77 (d, 1 H, J = 16.5 Hz), 6.89 (d, 1 H, J = 16.5 Hz), 6.82 (s, 1 H),
6.96 (s, 1 H), 6.93 (d, 1 H, J = 2.4 Hz), 7.11 (d, 1 H, J = 1.5 Hz), 7.46
(d, 1 H, J = 1.5 Hz); 13C NMR (CDCl3, 75 MHz) δ 55.85, 60.41,
92.56, 110.40, 110.63, 111.77, 119.28, 124.97, 128.36, 128.70, 130.15,
135.71, 145.71, 146.56, 148.11, 152.44: HRMS m/z 413.0250 [M +
H]+ (calcd for C17H18IO4, 413.0257); anal. C 49.38, H 4.24%, calcd for
C17H17IO4, C 49.53, H 4.16%.
Dibenzyl 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate
(10a). A solution of Z-stilbene 9a (0.68 g, 1.64 mmol) in acetonitrile
(7 mL) was cooled to −10 °C before the addition of CCl4 (1.6 mL,
16.4 mmol), and the mixture was stirred for 10 min at −10 °C in the
dark. Diisopropylamine (0.57 mL, 3.28 mmol) and DMAP (20 mg,
cat.) were added in rapid succession. After 1 min, dibenzylphosphite
(0.44 mL, 1.96 mmol) was added, and the mixture was stirred for 20
min at −10 °C. The reaction was terminated by the addition of 0.5 M
KH2PO4 (7 mL) and extracted with EtOAc (3 × 15 mL). The organic
phase was filtered and concentrated. The oily residue was separated by
CC using 4:1 hexane−EtOAc as eluent to yield 0.94 g (86%) of a pure
1
oil: bp (dec) 274 °C (0.01 mmHg); H NMR (CDCl3, 300 MHz) δ
3.51 (s, 3 H), 3.65 (s, 3 H), 3.72 (s, 3 H), 5.04 (s, 2 H), 5.06 (s, 2 H),
6.36 (d, 1 H, J = 9 Hz), 6.42 (d, 1 H, J = 9 Hz), 6.77 (d, 1 H, J = 1.2
Hz), 6.89 (d, 1 H, J = 6 Hz), 7.02 (d, 1 H, J = 6 Hz), 7.01 (s, 1 H),
7.19 (d, 1 H, J = 1.2 Hz), 7.28−7.35 (m, 10 H); 13C NMR (CDCl3, 75
3,5-Diiodo-4,4′-dimethoxy-3′-hydroxy-Z-stilbene (9c) and 3,5-
Diiodo-4,4′-dimethoxy-3′-hydroxy-E-stilbene (9d). Stilbenes 9c and
9d were obtained from a mixture of silyl ethers 8a and 8b (1.35 g, 1.81
390
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MHz) δ 56.20, 56.50, 60.73, 71.18, 71.24, 92.73, 113.72, 114.23,
122.58, 122.61, 128.10, 128.93, 129.50, 129.56, 130.18, 130.85, 130.86,
131.87, 136.26, 136.66, 136.73, 140.32, 140.39, 149.12, 151.21, 151.25,
153.26; HRMS m/z 673.0808 [M + H]+ (calcd for C31H31IO7P,
673.0852); anal. C 55.37, H 4.64%, calcd for C31H30IO7P, C 55.37, H
4.50%.
(d, 1 H, J = 12 Hz), 6.50 (d, 1 H, J = 12 Hz), 6.73 (s, 2 H), 6.82 (s, 1
H), 7.18 (s, 1 H), 7.20 (s, 1 H).
Piperidine 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate
1
(11e): colorless oil; H NMR (D2O, 300 MHz) δ 1.51 (m, 4 H),
1.62 (m, 8 H), 3.00 (t, 8 H, J = 6 Hz), 3.51 (s, 3 H), 3.63 (s, 3 H), 3.67
(s, 3 H), 6.34 (d, 1 H, J = 12.6 Hz), 6.51 (d, 1 H, J = 12.6 Hz), 6.72 (s,
2 H), 6.83 (s, 1 H), 7.21 (s, 1 H).
Glycine-OMe 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phos-
phate (11f): colorless solid; mp 74−78 °C; 1H NMR (D2O, 300
MHz) δ 3.48 (s, 3 H), 3.61 (s, 3 H), 3.67 (s, 3 H), 3.68 (s, 3 H), 3.76
(s, 2 H), 6.30 (d, 1 H, J = 12 Hz), 6.46 (d, 1 H, J = 12 Hz), 6.69−6.77
(m, 3 H), 7.10 (s, 1 H), 7.16 (s, 1 H).
Tryptophan-OMe 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phos-
phate (11g): colorless solid; mp 108−112 °C; 1H NMR (DMSO, 300
MHz) δ 3.19 (d, 2 H, J = 6.3 Hz), 3.56 (s, 3 H), 3.61 (s, 3 H), 3.66 (s,
3 H), 3.70 (s, 3 H), 4.09 (t, 1 H, J = 6 Hz), 6.35 (d, 1 H, J = 12 Hz),
6.47 (d, 1 H, J = 12 Hz), 6.81−6.85 (m, 2 H), 6.98 (t, 1 H, J = 7.2 Hz),
7.07 (t, 1 H, J = 8.1 Hz), 7.18 (s, 1 H), 7.22 (s, 1 H), 7.34 (d, 1 H, J =
8.1 Hz), 7.40 (s, 1 H), 7.46 (d, 1 H, J = 7.2 Hz).
Tris(3-iodo-4,4′,5-trimethoxy-Z-stilbene) 3′-O-phosphate (11h):
colorless solid; mp 75−81 °C; 1H NMR (DMSO, 300 MHz) δ 3.42 (s,
9 H), 3.57 (s, 3 H), 3.67 (s, 3 H), 3.70 (s, 3 H), 6.35 (d, 1 H, J = 12
Hz), 6.48 (d, 1 H, J = 12 Hz), 6.76 (d, 1 H, J = 8.4 Hz), 6.81 (d, 1 H, J
= 8.7 Hz), 6.92 (s, 1 H), 7.22 (s, 1 H), 7.42 (s, 1 H).
Dibenzyl 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate
(10b). Dibenzyl phosphate 10b (0.38 g, 55% yield) was obtained
from 9c (0.46 g, 0.91 mmol), by the method described above for the
synthesis of monoiodide 10a, as a colorless oil: bp (dec) 220 °C; (0.01
mmHg); 1H NMR (CDCl3, 300 MHz) δ 3.78 (s, 3 H), 3.81 (s, 6 H),
5.13 (s, 2 H), 5.16 (s, 2 H), 6.28 (d, 1 H, J = 12 Hz), 6.42 (d, 1 H, J =
12 Hz), 6.78 (d, 1 H, J = 9 Hz), 7.00 (d, 1 H, J = 8.7 Hz), 7.07 (s, 1
H), 7.33 (s, 10 H), 7.64 (s, 2 H); 13C NMR (CDCl3, 100 MHz) δ
55.96, 60.71, 69.83, 69.89, 90.15, 112.40, 122.23, 122.26, 125.60,
126.20, 126.21, 127.93, 128.49, 128.55, 130.66, 137.12, 139.92, 157.68;
31P NMR (CDCl3,162 MHz) δ −5.51; HRMS m/z 768.9699 [M +
H]+ (calcd for C30H28I2O6P, 768.9713).
General Procedures for Syntheses of the Phosphoric Acids
and Derivatives. Method A. Each of the metal cation
phosphate salts was obtained by the procedure outlined
below for preparing the potassium salt 11a, except for the
metal counterions introduced by treatment of the phosphoric
acid using either LiOH or NaOMe.
Potassium 3,5-diiodo-4,4′dimethoxy-Z-stilbene 3′-O-phosphate
(12a). Phosphate 12a (0.20 g, 80%) was obtained from ester 10b
(0.29 g, 0.38 mmol) as described above for the synthesis of 11a, except
that the aqueous phase was extracted with butyl alcohol (3 × 25 mL)
because the phosphoric acid was sparingly soluble in EtOAc and
DCM. The potassium salt was a colorless solid: mp 210−215 °C
(dec); 1H NMR (D2O, 300 MHz) δ 3.69 (s, 6 H), 6.27 (d, 1 H, J = 12
Hz), 6.49 (d, 1 H, J = 12 Hz), 6.64 (s, 2 H), 7.20 (s, 1 H), 7.62 (s, 2
H); 31P NMR (D2O, 162 MHz) δ 0.973.
Method B. Dowex-50W (2 g, HCR-W2) was placed in a column
and washed successively with CH3OH (50 mL), 1 N HCl (until pH
1), water (until pH 7), base/amine/amino acid (until pH 7−14), and
H2O (until pH 7). The column was recycled. The potassium salt 11a
or 12a (about 25 mg) was dissolved in deionized H2O (1 mL) and
applied to a Dowex-50W (HCR-W2) resin column (bearing the
appropriate amine or amino acid methyl ester), eluted with
approximately 40 mL of H2O. The eluent was concentrated by
freeze-drying to give the required cation derivative.
Method C. Amino Acid Methyl Esters. The amino acid methyl ester
hydrochloride was neutralized in CH3OH solution by addition of
potassium carbonate. Ether was added to precipitate the potassium
chloride, and the solution was filtered and concentrated. The amino
acid methyl ester residue was then applied to the Dowex-50W (HCR-
W2) resin column as described in method B.
Sodium 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate
1
(12b): colorless solid; mp 215−234 °C (dec); H NMR (D2O, 300
MHz) δ 3.69 (s, 3 H), 3.72 (s, 3 H), 6.29 (d, 1 H, J = 12 Hz), 6.49 (d,
1 H, J = 12 Hz), 6.69 (s, 2 H), 7.20 (s, 1 H), 7.64 (s, 2 H).
Lithium 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate
(12c): colorless solid; 250−270 °C (dec); 1H NMR (D2O, 300
MHz) δ 3.68 (s, 3 H), 3.71 (s, 3 H), 6.28 (d, 1 H, J = 12 Hz), 6.49 (d,
1 H, J = 12 Hz), 6.68 (s, 2 H), 7.19 (s, 1 H), 7.64 (s, 2 H); 31P NMR
(D2O, 162 MHz) δ 0.96.
Potassium 3-Iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate
(11a). Trimethylbromosilane (277 μL, 1.8 mmol) was added to a
cooled (0 °C) solution of benzyl phosphate 10a in DCM (40 mL),
and the mixture was stirred for 90 min before addition of sodium
thiosulfate (10% aq, 10 mL). After an additional 1 min of stirring, the
phases were separated and the aqueous phase was extracted
successively with DCM (20 mL) and EtOAc (2 × 20 mL). The
combined organic extracts were filtered and concentrated to afford the
phosphoric acid intermediate as a clear oil. After drying (high vacuum)
for 1 h, the oil was dissolved in CH3OH (10 mL) with cooling to 0 °C,
KOH (1.8 mL, 1 N solution in CH3OH) was added, and the mixture
was stirred for 20 min. The precipitate was collected and triturated
with ether to afford the potassium salt as a colorless solid: mp 197−
Morpholine 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phos-
phate (12d): colorless, waxy solid; mp 75−80 °C; 1H NMR
(DMSO, 300 MHz) δ 2.96−2.99 (m, 8 H), 3.74−3.77 (m, 8 H),
3.82 (s, 3 H), 3.83 (s, 3 H), 6.43 (d, 1 H, J = 12.5 Hz), 6.60 (d, 1 H, J
= 12.5 Hz), 6.86 (d, 1 H, J = 8.2 Hz), 6.93 (d, 1 H, J = 8.2 Hz), 7.49 (s,
1 H), 7.78 (s, 2 H).
Piperdine 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phosphate
(12e): colorless oil; 1H NMR (DMSO, 300 MHz) δ 1.51 (br s, 12 H),
2.79−2.81 (m, 8 H), 3.70 (s, 3 H), 3.72 (s, 3 H), 6.31 (d, 1 H, J = 12
Hz), 6.49 (d, 1 H, J = 12 Hz), 6.73 (d, 1 H, J = 8.4 Hz), 6.80 (d, 1 H, J
= 8.4 Hz), 7.40 (s, 1 H), 7.61 (s, 1 H).
1
198 °C (dec); H NMR (D2O, 300 MHz) δ 3.51 (s, 3 H), 3.64 (s, 3
Glycine-OMe 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-phos-
1
phate (12f): colorless solid; mp 90−97 °C; H NMR (DMSO, 300
H), 3,71 (s, 3 H), 6.33 (d, 1 H, J = 12 Hz), 6.51 (d, 1 H, J = 12 Hz),
6.70 (s, 2 H), 6.84 (s, 1 H), 7.22 (s, 2 H); 31P NMR (D2O, 162 MHz)
δ 0.94.
MHz) δ 3.61 (s, 4 H), 3.68 (s, 6 H), 3.70 (s, 3 H), 3.72 (s, 3 H), 6.31
(d, 1 H, J = 12 Hz), 6.49 (d, 1 H, J = 12 Hz), 6.72 (d, 1 H, J = 9.6 Hz),
6.80 (d, 1 H, J = 8.1 Hz), 7.37 (s, 1 H), 7.67 (s, 1 H).
Sodium 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate
1
Tryptophan-OMe 3,5-diiodo-4,4′-dimethoxy-Z-stilbene 3′-O-
phosphate (12g): colorless solid; mp 125−130 °C; 1H NMR
(DMSO, 300 MHz) δ 3.34 (d, 1 H, J = 6.5 Hz), 3.36 (d, 1 H, J =
6.5 Hz), 3.66 (s, 3 H), 3.70 (s, 3 H), 3.72 (s, 3 H), 4.32 (t, 1 H, J = 6.5
Hz), 6.31 (d, 1 H, J = 12 Hz), 6.48 (d, 1 H, J = 12 Hz), 6.78−6.81 (m,
2 H), 7.01 (s, 1 H), 7.05 (t, 1 H, J = 7 Hz), 7.13 (t, 1 H, J = 7 Hz),
7.39 (d, 1 H, J = 7.5 Hz), 7.47 (d, 1 H, J = 8 Hz), 7.60 (s, 1 H).
Tris 3,5-diiodo-4,4′dimethoxy-Z-stilbene 3′-O-phosphate (12h):
colorless solid; mp 115−120 °C; 1H NMR (DMSO, 300 MHz) δ 3.34
(s, 18 H), 3.69 (s, 3 H), 3.71 (s, 3 H), 6.30 (d, 1 H, J = 12 Hz), 6.47
(d, 1 H, J = 12 Hz), 6.70 (d, 1 H, J = 8.1 Hz), 6.78 (d, 1 H, J = 8.1
Hz), 7.37 (s, 1 H), 7.67 (s, 2 H).
(11b): colorless solid; mp 194−195 °C (dec); H NMR (D2O, 300
MHz) δ 3.50 (s, 3 H), 3.67 (s, 3 H), 3.68 (s, 3 H), 6.50 (d, 1 H, J = 12
Hz), 6.70 (d, 1 H, J = 12 Hz), 6.72 (s, 1 H), 6.77 (s, 1 H), 6.79 (s, 1
H), 7.01 (s, 1 H), 7.13 (s, 1 H).
Lithium 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate
1
(11c): colorless solid; mp 245−275 °C (dec); H NMR (D2O, 400
MHz) δ 3.50 (s, 3 H), 3.62 (s, 3 H), 3.66 (s, 3 H), 6.33 (d, 1 H, J = 12
Hz), 6.49 (d, 1 H, J = 12 Hz), 6.70 (s, 2 H), 6.83 (s, 1 H), 7.20 (s, 1
H), 7.22 (s, 1 H).
Morpholine 3-iodo-4,4′,5-trimethoxy-Z-stilbene 3′-O-phosphate
(11d): colorless oil; 1H NMR (D2O, 300 MHz) δ 3.11−3.15 (m, 8 H),
3.50 (s, 3 H), 3.63 (s, 3 H), 3.68 (s, 3 H), 3.77−3.81 (m, 8 H), 6.33
391
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Article
Tubulin Evaluation Procedures. The methods used to measure
inhibition both of tubulin assembly16 and of [3H]colchicine binding to
tubulin13 have been described in detail elsewhere. In brief, the
assembly reaction was evaluated using purified tubulin at 10 μM in 0.8
M monosodium glutamate (pH 6.6 with HCl), varying compound
concentrations, 0.4 mM GTP, and 4% (v/v) dimethyl sulfoxide
(compound solvent). Extent of assembly after 20 min at 30 °C was
measured. In the colchicine binding assay, the tubulin concentration
was 1.0 μM, the [3H]colchicine concentration was 5.0 μM, and
compound concentrations were as indicated. Additional reaction
components are detailed in ref 13. Incubation was for 10 min at 37 °C,
and the tubulin−[3H] colchicine complex was separated from
unbound colchicine by retention of the complex by DEAE-cellulose
filters.
ACKNOWLEDGMENTS
■
We appreciate the financial support provided by grants RO1
CA90441-01-04, 2R56-CA 09441-06A1, and 5R01 CA 090441-
07 awarded by the Division of Cancer Treatment and
Diagnosis, National Cancer Institute, DHHS; the Arizona
Disease Control Research Commission; the Robert S. Dalton
Endowment Fund; Dr. Alec D. Keith; the J. W. Kieckhefer
Foundation; the Margaret T. Morris Foundation; the Caitlin
Robb Foundation; and Dr. J. C. Budzinski. For other helpful
assistance, we thank Drs. J. M. Schmidt, M. D. Minardi, and M.
P. Grealish, as well as C. Weber, M. J. Dodson, F. Craciunescu,
and L. Williams.
Cancer Cell Line Procedures. Inhibition of human cancer cell
growth was assessed using the National Cancer Institute's standard
sulforhodamine B assay as previously described.17 Briefly, cells in a 5%
fetal bovine serum/RPMI1640 medium were inoculated in 96-well
plates and incubated for 24 h. Serial dilutions of the compounds were
then added. After 48 h, the plates were fixed with trichloroacetic acid,
stained with sulforhodamine B, and read with an automated microplate
reader. A growth inhibition of 50% (GI50 or the drug concentration
causing a 50% reduction in the net protein increase) was calculated
from optical density data with Immunosoft software.
DEDICATION
■
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick, Mary-
land, for his pioneering work on the development of natural
product anticancer agents.
REFERENCES
■
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AUTHOR INFORMATION
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@
asu.edu.
■
Notes
́
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Hamze, A.; Provot, O.; Peyrat, J.-F.; De Losada, J. R.; Liu, J.-M.;
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