Antineoplastic agents. 509. Synthesis of fluorcombstatin phosphate and related 3-halostilbenes

Article abstract of DOI:10.1021/np058038i

The present SAR study of combretastatin A-3 (3a) focused on replacement of the 3-hydroxyl group by a series of halogens. That approach with Z-stilbenes resulted in greatly enhanced (> 10-100-fold) cancer cell growth inhibition against a panel of human cancer cell lines and the murine P388 lymphocytic leukemia cell line. Synthesis of the 3-fluoro-Z-stilbene designated fluorcombstatin (11a) and its potassium 3′-O-phosphate derivative (16c) by the route 7 → 8a → 11a → 14 → 16c illustrates the general synthetic pathway. The 3′-O-phosphoric acid ester (15) of 3-bromo-Z-stilbene 13a was also converted to representative cation salts to evaluate the potential for improved aqueous solubility, and the potassium salt (16 mg/mL in water) proved most useful. The fluoro (11a), chloro (12a), and bromo (13a) halocombstatins were nearly equivalent to combretastatin A-4 (1a) as inhibitors of tubulin polymerization and of the binding of colchicine to tubulin. The tubulin binding in cell-free systems was also retained in human umbilical vein endothelial cells. All three halocombstatins retained the powerful human cancer cell line inhibitory activity of combretastatin A-4 (1a) and proved superior to combretastatin A-3 (3a). In addition, the halocombstatins targeted Gram-positive bacteria and Cryptococcus neoformans.

Full text of DOI:10.1021/np058038i

1450
J. Nat. Prod. 2005, 68, 1450-1458
Antineoplastic Agents. 509. Synthesis of Fluorcombstatin Phosphate and
Related 3-Halostilbenes|,1
George R. Pettit,*,† Mathew D. Minardi,† Heidi J. Rosenberg,† Ernest Hamel,‡ Michael C. Bibby,§
Sandie W. Martin,§ M. Katherine Jung, Robin K. Pettit,† Timothy J. Cuthbertson,† and Jean-Charles Chapuis†
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 872404, Tempe,
Arizona 85287-2404, Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, University of
Bradford, Cancer Research Unit, West Yorkshire BD7 1DP, United Kingdom, and Science Applications International Corp.,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702
Received March 14, 2005
The present SAR study of combretastatin A-3 (3a) focused on replacement of the 3-hydroxyl group by a
series of halogens. That approach with Z-stilbenes resulted in greatly enhanced (>10-100-fold) cancer
cell growth inhibition against a panel of human cancer cell lines and the murine P388 lymphocytic
leukemia cell line. Synthesis of the 3-fluoro-Z-stilbene designated fluorcombstatin (11a) and its potassium
3′-O-phosphate derivative (16c) by the route 7 f 8a f 11a f 14 f 16c illustrates the general synthetic
pathway. The 3′-O-phosphoric acid ester (15) of 3-bromo-Z-stilbene 13a was also converted to representa-
tive cation salts to evaluate the potential for improved aqueous solubility, and the potassium salt (16
mg/mL in water) proved most useful. The fluoro (11a), chloro (12a), and bromo (13a) halocombstatins
were nearly equivalent to combretastatin A-4 (1a) as inhibitors of tubulin polymerization and of the
binding of colchicine to tubulin. The tubulin binding in cell-free systems was also retained in human
umbilical vein endothelial cells. All three halocombstatins retained the powerful human cancer cell line
inhibitory activity of combretastatin A-4 (1a) and proved superior to combretastatin A-3 (3a). In addition,
the halocombstatins targeted Gram-positive bacteria and Cryptococcus neoformans.
The African Bush Willow Combretum caffrum (Combre-
taceae) has proved to be a very important source of new
cancer cell growth inhibitory constituents that we discov-
ered beginning in 1979 and named combretastatins.2 The
most potent cancer cell line inhibitor among a series of cis-
stilbene constituents (Figure 1) was combretastatin A-4
(1a, CA4), and its sodium phosphate (CA4P) derivative (1b)
was advanced to Phase I human cancer clinical trials in
1998.2 Overall results continue to be promising, and human
cancer Phase II and combination (Phase 1b) trials with
CA4P (1b) are currently underway. Antivascular,3 antian-
giogenesis,4 and general antimetastatic5 activities of CA4P,
as well as its synergistic utility in combination5f with other
anticancerdrugs,radioimmunotherapy,6a andhyperthermia,6b
are all areas of active cancer research interest.
Figure 1.
After the discovery of CA4, we7 and others8 have been
conducting SAR investigations of this relatively simple but
and transport in vivo, we also synthesized phosphate ester
biologically fascinating molecule. In addition, we have been
derivatives of the 3-fluoro-10 and 3-bromostilbenes.11
(2a),9a,b A-2,7a and A-3 (3a)9c to new antineoplastic agents.
Results and Discussion
extending SAR insights derived from combretastatins A-1
Now we report a series of structural modifications of
Preparation of the stilbenes was accomplished as shown
in Scheme 1 and Figure 2. Phosphonium bromide 7 was
prepared as previously described,9c and condensation with
the appropriate halobenzaldehyde10-12 using n-butyllithium
in THF led to silyl-protected stilbenes 8-10. Subsequent
deprotection (Scheme 1) with tetrabutylammonium fluoride
afforded 3-halostilbenes 11-13. The Z-isomers 11a and
13a were phosphorylated9 using dibenzyl phosphite, diiso-
propylethylamine, N,N-(dimethylamino)pyridine, and car-
bon tetrachloride in acetonitrile to provide bisbenzyl phos-
phates 14 and 15. Debenzylation of the phosphate esters
(14 and 15) was achieved using trimethylsilybromide9b,c
followed by the corresponding base to produce phosphates
16 and 17. Compounds 18 and 19 were used to synthesize
combretastatin A-3 (3a) and its phosphate prodrug (3b) by
replacing the 3-hydroxy group with halogens and with
primary focus on the fluoro substituent. Since the phos-
phate ester derivative of combretastatins A-1-A-4 proved
to be an effective method for increasing aqueous solubility
|
Dedicated to the memory of Professor Thomas A. Connors (1934-Feb
4, 2002), a remarkably effective advocate of new anticancer drug discovery
and development. Please refer to Newell et al. (Br. J. Cancer 2003, 89, 437-
454).
* To whom correspondence should be addressed. Tel: 480-965-3351.
Fax: 480-965-8558.
† Arizona State University.
‡ STB, NCI, Frederick.
§ University of Bradford.
SAIC, NIH, Frederick.
10.1021/np058038i CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/12/2005

Synthesis of Fluorcombstatin Phosphate
Journal of Natural Products, 2005, Vol. 68, No. 10 1451
Scheme 1
Figure 2.
equimolar to the tubulin concentration, binding of the
radiolabeled ligand was inhibited by 75-89%. Interest-
ingly, the lowest and highest inhibitory effects were
observed with stilbenes 11a and 13a, the two compounds
that displayed the greatest inhibitory effects in the polym-
erization assay.
In an earlier study,13 combretastatin A-3 (3a, with a C-3
hydroxyl instead of a methoxyl or halogen) was found to
be about half as active as combretastatin A-4 (1a) as an
inhibitor of tubulin assembly, about one-fifth as active as
an inhibitor of colchicine binding to tubulin, and about one-
seventh as active as an inhibitor of cancer cell growth. A
related finding was that elimination of the C-3 substituent
entirely, by replacing it with a hydrogen atom, resulted in
about a 7-fold reduction in inhibitory effect on polymeri-
zation and complete loss of cytotoxic activity.14 Thus, the
optimal activity observed with combretastatin A-4 (1a) and
the three halocombstatins suggests a C-3 substituent of
some size is necessary where the fluorine atom may
represent a minimum and a C-3 methoxy group is quite
favorable. Therefore, it seems unlikely that the predomi-
nant effect of the substituent results from direct enhance-
ment of the interaction of ligand with protein. The A-ring
substituents most likely cause the active cis-stilbenes to
assume with greater probability a conformation that favors
the drug-tubulin interaction.
The concept of antiangiogenesis as a therapeutic ap-
proach is now robustly pursued as a promising novel
antitumor strategy.15 Previous investigations have also
shown that combretastatin A-4 disrupts the microtubules
of human umbilical vein endothelial cells (HUVECs) in
culture.16a These studies confirmed that the tubulin-
binding properties shown in cell-free systems are retained
when the compound enters cells and that tubulin binding
is a significant component of the biological activity. In the
present study, the ability of stilbenes 11a and 12a to
disrupt microtubules in HUVECs was determined. HU-
VECs were isolated according to the method of Jaffe et al.16b
Loss of cellular microtubules and disruption of cell division
(binucleated cells) were clearly seen following treatment
of HUVECs with halocomstatins 11a and 12a for 30 min
at a concentration of 1 µM (see Figure 4). Stilbenes 11a
and 12a caused marked cell rounding and depolymerization
of tubulin under similar conditions (scoring ) ++ at 30
min), but stilbene 12a appeared slightly more potent (Table
3). The tubulin in HUVECs exposed to 1 µM 12a for g1 h
appeared mainly depolymerized with only a few microtu-
the nitro- and aminostilbenes 20a-h, for purposes of
comparison (Figure 2).
Compared to the related combretastatins, the new Z-
halostilbenes all showed very strong inhibition of cancer
cell growth (Table 1). Their cancer cell growth inhibitory
properties were stronger than those of combretastatin A-3
(3a) and equivalent to combretastatin A-4 (1a). The nitro-
and aminostilbenes (20c-h) proved to be generally less
active. The strong inhibitory activities of stilbenes 11a and
13a were retained upon conversion to phosphate salts and
displayed markedly better aqueous solubility than their
phenol precursors. As expected,9 all of the E geometrical
isomers evaluated proved to be much less effective as
inhibitors of cancer cell growth.
Because of their potent cancer cell line inhibitory activ-
ity, the three halocombstatins (11a, 12a, and 13a) were
compared to combretastatin A-4 (1a) with respect to
inhibitory effects on tubulin polymerization and on the
binding of [3H]colchicine to tubulin (Table 2). These experi-
ments demonstrated that all were essentially identical in
their apparent interactions with tubulin. The three halo-
combstatins inhibited the polymerization reaction with IC50
values of 1.5-1.6 µM, versus an IC50 value of 1.8 µM for
combretastatin A-4 (1a). The minor differences were within
experimental error, as indicated by the standard devia-
tions. Similarly, all three cis-stilbenes were highly potent
inhibitors of the colchicine binding assay. When present
at a concentration one-fifth that of [3H]colchicine but

1452 Journal of Natural Products, 2005, Vol. 68, No. 10
Pettit et al.
Table 1. Human Cancer Cell Line Growth Inhibition (GI50 µg/mL) and Murine P388 Lymphocytic Leukemia Inhibitory Activity
(ED50 µg/mL) of Combretastatin A-1 (2a), A-3 (3a), A-4 (1a), Fluorcombstatin (11a), and Synthetic Modifications Including Phosphate
Salts
leukemia
P388
pancreas
BXPC-3
breast
MCF-7
CNS
SF268
lung-NSC
NCI-H460
colon
KM20L2
prostate
DU-145
compound
1a
0.0003
0.0004
0.251
0.39
<0.001
0.0006
0.029
0.74
0.061
0.034
0.061
0.53
1.2
0.0008
1b
0.036
2a
4.4
1.5
0.17
2b
<0.01
0.26
0.024
0.49
0.036
0.0083
0.052
0.038
0.19
0.034
0.0043
0.048
0.0019
0.18
3a
2.3
3b
0.305
2.8
0.92
0.45
<0.0032
0.18
0.00026
0.034
0.00038
0.15
3.5
11a
11b
12a
12b
13a
13b
16a
16b
16c
16d
16e
16f
16g
16h
17a
17b
17c
17d
17e
17f
1g
<0.0020
0.253
0.745
2.2
<0.0027
0.051
<0.0016
0.35
>1
0.53
0.27
1.4
0.0024
0.027
0.32
0.59
0.64
1.6
0.0026
0.041
<0.00090
0.048
0.00025
0.038
0.00082
0.13
0.0021
0.0174
0.0298
0.019
0.00046
0.14
0.00050
0.18
0.59
1.2
0.59
0.490
0.548
0.73
0.39
>1
0.0044
0.0038
0.0040
0.0079
0.011
0.0051
0.0040
0.0039
0.0087
0.0075
0.0073
0.0027
0.017
0.0094
0.0039
0.0043
0.0050
0.022
0.0034
0.0032
0.034
0.0028
0.0026
0.0029
0.0066
0.041
0.0068
0.0039
0.0047
2.9
1.5
0.0036
0.0043
0.0037
0.0046
0.011
0.0037
0.0018
0.019
0.0046
0.0066
0.0028
0.0079
0.042
0.0063
0.0045
0.0049
2.8
>1
0.0074
0.015
>1
>1
0.0040
0.030
>1
0.0060
0.0020
0.031
0.0041
0.0039
0.0035
0.0044
0.043
>1
0.014
0.033
0.99
0.093
0.13
0.20
0.15
0.56
<0.001
0.074
0.17
>10
2.5
0.062
>1
0.015
<0.01
<0.01
<0.01
<0.01
<0.01
0.288
0.0034
0.0030
0.0032
0.0064
0.023
0.23
0.11
0.24
0.48
2.6
0.0022
0.0045
0.0049
3.2
0.0022
0.0053
0.0067
4.1
0.37
0.27
0.45
>10
1.8
<0.01
<0.01
2.22
0.402
0.224
17h
17i
20c
20d
20e
20f
20g
20h
0.49
0.62
0.31
0.25
0.39
0.36
1.1
0.56
0.41
1.2
0.0174
0.0289
>10
0.37
1.0
0.060
0.040
0.030
0.035
0.24
0.39
1.3
0.035
0.012
0.34
<0.010
0.37
0.0022
0.59
0.39
0.053
0.27
0.030
0.0360
<0.010
0.076
0.014
0.044
Table 2. Inhibition of Tubulin Polymerization and Binding of
from commercial sources (Acros Organics, Sigma-Aldrich Co.,
Alfa Aesar, City Chemicals, or Lancaster Synthesis, Inc.).
Solvent extracts of aqueous solutions were dried over anhy-
drous MgSO4. Gravity column chromatography was performed
using silica gel from VWR Scientific (70-230 mesh) or from
E. Merck (230-400). Analtech silica gel GHLF plates were
employed for TLC.
Melting points were determined with an electrochemical
digital melting point apparatus and are uncorrected. NMR
spectra were recorded employing Varian Gemini 300 or Varian
Unity 400 instruments. Chemical shifts are reported in ppm
downfield from tetramethylsilane as an internal standard in
CDCl3 or where noted in D2O. High-resolution mass spectra
were obtained with a Kratos Ms-50 instrument (Midwest
Center for Mass Spectroscopy, University of Nebraska-
Lincoln) or with a JEOL LCmate instrument (ASU). Elemental
analyses were determined by Galbraith Laboratories, Inc.,
Knoxville, TN.
Heteronuclear couplings were seen in both the 1H NMR and
13C NMR in the compounds containing fluorine. In the 13C
NMR, some couplings were easy to distinguish, while others
were more difficult. When the couplings were difficult to
distinguish, all signals are noted in the data.
General Procedure for Synthesis of Dimethoxyha-
lobenzaldehydes. 3-Fluoro-4,5-dimethoxybenzaldehyde
(4). To a stirred solution prepared from 100 mL of DMF and
5-fluorovanillin (lit.10 1.0 g, 5.88 mmol) was added portionwise
over 15 min sodium hydride (60% in mineral oil, 282 mg, 7
mmol). After 15 min, iodomethane (1.5 mL, 24 mmol) was
added, and stirring continued for 16 h at RT. The reaction was
terminated by the addition of H2O, the mixture was extracted
with hexane (3 × 100 mL), and solvents were removed in
vacuo. Purification by flash chromatography on a column of
silica gel using hexane-ethyl acetate (4:1) as eluent afforded
a colorless solid (1 g, 93%): mp 51-53 °C (lit.11 mp 52-53 °C);
[3H]Colchicine to Tubulin by Halocombstatins
inhibition of
polymerization
IC50 (µM) ( SD
inhibition of colchicine
binding
% inhibition ( SD
compound
1a
1.8 ( 0.2
1.5 ( 0.2
1.6 ( 0.2
1.5 ( 0.2
81 ( 3
75 ( 6
85 ( 4
89 ( 2
11a
12a
13a
bules apparent (scoring ) +++, Table 3). In another series
of experiments, fluorcombstatin (11a) was further evalu-
ated against HUVECs in vitro. These cells showed signifi-
cant sensitivity to the fluorcombstatin (11a): ED50 0.00025
µg/mL. Cord lengths as well as junction numbers were
markedly reduced at both 0.01 and 0.001 µg/mL (Figure
3B and 3C) compared to untreated controls (Figure 3A).
Such activity against endothelial cells is of the utmost
interest, as these cells play a central role in the angiogenic
process.
We previously reported that combretastatin A-3, but not
its sodium phosphate prodrug, inhibited growth of the
pathogenic fungus Cryptococcus neoformans.9c In the present
study, the halocombstatins were also subjected to antimi-
crobial evaluation. Susceptibility testing was performed by
the reference broth microdilution assay.17,18 The antimi-
crobial activities of the halocombstatins were very similar,
targeting Gram-positive bacteria and C. neoformans (Table
4). As with the sodium combretastatin A-3 prodrug,9c the
sodium phosphate derivative (16a) of fluorcombstatin (11a)
did not retain significant antimicrobial activity (Table 4).
Experimental Section
General Experimental Procedures. All solvents (ether
refers to diethyl ether) were redistilled, and reagents were

Synthesis of Fluorcombstatin Phosphate
Journal of Natural Products, 2005, Vol. 68, No. 10 1453
Figure 3. Morphological appearance of the cords in HUVEC cells.
HUVEC were plated on Matrigel for 24 h. Control cells (A) differenti-
ated into well-defined tube-like structures (note large amounts of cords
and junctions). Cells that were exposed to 11a (0.01 µg/mL) did not
form complete tube-like structures (B) compared to the control (see
marked reduction of cord lengths and junction numbers. Cells that
were exposed to 11a (0.001 µg/mL) formed a reduced number of such
structures (C).
1H NMR (300 MHz, CDCl3) δ 3.94 (s, 3H), 4.05 (s, 3H), 7.24
(s, 1H), 7.26 (s, 1H), 9.82 (s, 1H).
3-Chloro-4,5-dimethoxybenzaldehyde (5). The preceding
reaction was repeated with 5-chlorovanillin (10 g, 54 mmol)
to give the title compound, which was isolated as recorded in
the preceding experiment (4) to afford a colorless solid (10.4
g, 97%): mp 88-90 °C (lit.11 mp 87-89 °C); 1H NMR (300 MHz,
CDCl3) δ 3.95 (s, 3H), 3.96 (s, 3H), 7.36 (d, 1H, J ) 1.5 Hz),
7.50 (d, 1H, J ) 1.5 Hz), 9.85 (s, 1H).
3-Bromo-4,5-dimethoxybenzaldehyde (6). The experi-
ment was repeated with 5-bromovanillin (10 g, 43.3 mmol) as
described for aldehyde 4. Separation by flash chromatography
on a column of silica gel using hexane-ethyl acetate (9:1) as
eluent afforded a colorless solid (8 g, 75%): mp 64-65 °C; 1H
NMR (300 MHz, CDCl3) δ 3.94 (s, 3H), 3.95 (s, 3H), 7.39 (d,
1H, J ) 1.8 Hz), 7.65 (d, 1H, J ) 1.8 Hz), 9.85 (s, 1H).
General Procedure for the Stilbene Syntheses. 3-Fluoro-
4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-Z-stilbene (8a).
To a mixture of phosphonium salt 79c (4.7 g, 6.5 mmol) and
Figure 4. Morphological appearance of the microtubules in HU-
VECs: A, untreated control; B, after exposure to chlorocombstatin (12a,
1 µM for 30 min); C, after exposure to fluorcombstatin (11a, 1 µM for
30 min).

1454 Journal of Natural Products, 2005, Vol. 68, No. 10
Pettit et al.
Table 3. Assessment of Microtubule Damage in Primary
HUVECs Treated with 11a or 12a as Monolayer Culturesa
129.7, 133.6, 134.3, 135.4, 144.5, 145.2, 150.6, 153.7; anal. calcd
for C33H35Cl04Si.
3-Bromo-4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-
Z-stilbene (10a). Employment of 3-bromo-4,5-dimethoxybenz-
aldehyde (8 g, 33 mmol) using the procedure for 8a provided
the Z-isomer (10a, 4.2 g, 21%) as a clear oil: IR 2962, 1730,
1510, 1267, 908, 735, 650 cm-1; 1H NMR (300 MHz, CDCl3) δ
1.07 (s, 9H), 3.45 (s, 3H), 3.58 (s, 3H), 3.82 (s, 3H), 6.23 (d,
1H, J ) 12 Hz), 6.32 (d, 1H, J ) 12 Hz), 6.59 (d, 1H, J ) 8.1
Hz), 6.69-6.75 (m, 1H), 6.97 (d, 1H, J ) 1.5 Hz), 7.25-7.37
(m, 6H), 7.65 (dd, 4H, J ) 1.5 Hz, J ) 8.1 Hz); 13C NMR (100
MHz, CDCl3) δ 19.8, 26.7, 55.1, 55.8, 60.6, 94.4, 111.7, 112.0,
117.2, 120.8, 122.4, 125.2, 126.9, 127.3, 129.2, 129.4, 130.4,
133.5, 134.4, 135.2, 144.7, 145.2, 149.8, 152.9; HRMS calcd
for C33H35NaO4SiBr 79Br 625.1386 [M + Na]+, found 625.1364,
81Br 627.1365 [M + Na]+, found 627.1338.
Further elution of the chromatogram led to isolation of the
E-isomer 10b (8.1 g): IR 2934, 2859, 1710, 1510, 1275, 908,
732, 650 cm-1; 1H NMR (300 MHz, CDCl3) δ 1.14 (s, 9H), 3.54
(s, 3H), 3.84 (s, 3H), 3.88 (s, 3H),6.46 (d, 1H, J ) 12 Hz), 6.71
(d, 1H, J ) 8.1 Hz), 6.76 (d, 1H, J ) 12 Hz), 6.85 (d, 1H, J )
2.1 Hz), 6.87 (d, 1H, J ) 2.1 Hz), 6.94 (dd, 1H, J ) 8.4 Hz, J
) 2.4 Hz), 7.15 (d, 1H, J ) 2.4 Hz) 7.30-7.44 (m, 6H), 7.74
(dd, 4H, J ) 1.8 Hz, J ) 7.8 Hz); 13C NMR (75 MHz, CDCl3)
δ 19.8, 26.6, 55.2, 56.0, 60.6, 109.2, 109.8, 112.1, 117.7, 120.6,
122.6, 124.8, 127.5, 128.8, 129.6, 133.6, 134.9, 135.3, 145.2,
150.6, 153.6.
3-O-tert-Butyldimethylsilyl-4,4′,5-trimethoxy-3′-nitro-
Z- and E-stilbenes (20a,b). The reaction was conducted in
freshly distilled toluene (under argon) employing phosphonium
bromide19 19b (41 g, 80.86 mmol, mp 109-111 °C, prepared
in 95% yield, from benzyl alcohol19 19a and phosphorus
tribromide in CH2Cl2), aldehyde 18a9c (11.3 g, 38.12), and
sodium hydride (6.5 g, 162.5 mmol of 60% in mineral oil) at
ice-bath temperature for 8 h followed by 14 h at ambient
temperature. After initial isolation, the crude product was
separated by flash column chromatography on silica gel (4:1
hexane-ethyl acetate) to provide Z-stilbene 20a as a bright
yellow oil (13.1 g, 55%): 1H NMR (300 MHz) δ 0.08 (s, 6H),
0.94 (s, 9H), 3.70 (s, 3H), 3.77 (s, 3H), 3.92 (s, 3H), 6.34 (d,
0.5H, J ) 1.8 Hz), 6.42 (d, 1H, J ) 12 Hz), 6.43 (d, 0.5H, J )
2.1 Hz), 6.54 (d, 1H, J ) 12 Hz), 6.93 (d, 0.5H, J ) 8.7 Hz),
6.97 (d, 1H, J ) 8.7 Hz), 7.33 (dd, 1H, J ) 1.8 Hz, J ) 8.3 Hz),
7.41 (dd, 0.5H, J ) 2.1 Hz, J ) 8.9 Hz), 7.65 (d, 0.5H, J ) 2.1
Hz), 7.75 (d, 0.5H, J ) 2.1 Hz); HRMS calcd for C23H32NO6Si
446.1998 [M + H]+, found 446.2385.
Continued elution of the chromatographic column led to the
isolation of E-stilbene 20b as a yellow oil (1.2 g, 5%): 1H NMR
(300 MHz) δ 0.09 (6H, s), 0.94 (s, 9H), 3.70 (s, 3H), 3.77 (s,
3H), 3.92 (s, 3H), 6.34 (d, 1H, J ) 1.8 Hz), 6.42 (d, 1H, J ) 12
Hz), 6.43 (d, 1H, J ) 1.8 Hz), 6.54 (d, 1H, J ) 12 Hz), 6.92 (d,
1H, J ) 8.7 Hz), 7.41 (dd, 1H, J ) 2.1 Hz, J ) 8.9 Hz), 7.74 (d,
1H, J ) 2.1 Hz); HRMS calcd for C23H32NO6Si 446.19989 [M
+ H]+, found 446.20599.
concentration
stilbene
(µM)
t ) 30 min t ) 1 h t ) 2 h t ) 4 h
11a
0
1
10
0
1
10
normal
++
+++
normal
++
normal
++
normal normal
++
++
+++
+++
+++
12a
normal
normal normal
++/+++ +++
+++
+++
+++
+++
+++
a Key for scoring of HUVECS: Normal: endothelial cells have
normal microtubules. +: Slight depolymerization of tubulin visible;
slight rounding of cells; many strands of microtubules still visible.
++: Marked cell rounding and microtubule disruption; depolym-
erization of microtubules apparent although some microtubules
still visible. +++: Microtubules mainly depolymerized; occasional
microtubules apparent; cells show marked rounding with trans-
parent edges; rigid, fixed appearance.
tetrahydrofuran (25 mL, cooled to -78 °C) was added n-BuLi
(2.6 mL, 2.5 M in hexane, 6.5 mmol, over 5 min). After 1 h,
3-fluoro-4,5-dimethoxybenzaldehyde (1 g, 5.4 mmol) in tet-
rahydrofuran (10 mL) was added over 30 min. The mixture
was allowed to warm to RT and stirring continued for 16 h.
The reaction was terminated by the addition of H2O (50 mL),
the product was extracted with ethyl acetate, solvents were
removed in vacuo, and the residue obtained was subjected to
flash column chromatography on silica gel using hexane-ethyl
acetate (9:1) as eluent to afford Z-stilbene 8a (1 g, 34%) as a
clear oil: 1H NMR (300 MHz, CDCl3) δ 1.07 (s, 9H), 3.46 (s,
3H), 3.65 (s, 3H), 3.90 (d, 3H, JHF ) 1.2 Hz), 6.24 (d, 1H, J )
12 Hz), 6.33 (d, 1H, J ) 12 Hz), 6.52-6.61 (m, 2H), 6.72-6.76
(m, 3H), 7.26-7.40 (m, 6H), 7.64-7.67 (m, 4H); 13C NMR (125
MHz, CDCl3) δ 19.7, 26.6, 55.1, 56.0, 61.4 (d, JFC ) 3.5 Hz),
108.3, 108.3, 109.5 (d, JFC ) 20 Hz, ortho C), 111.7, 120.8,
122.4, 127.3, 127.4, 127.4, 127.7, 129.3, 129.5, 129.6, 130.3,
132.6 (d, JFC ) 9 Hz, meta C), 133.5, 134.7, 135.2, 135.8 (d,
JFC ) 14 Hz, ortho C), 144.7, 149.8, 152.9, 152.9, 155.5 (d, JFC
) 242 Hz, ipso C); HRMS (calcd for C33H36FO4Si) [M + H]+
543.2368, found 543.2372; anal. calcd for C33H35FO4Si, C, H.
Further elution gave the E-isomer 8b as a colorless solid,
which was crystallized from hexane (1.2 g, 41%): 1H NMR (300
MHz, CDCl3) δ 1.14 (s, 9H), 3.56 (s, 3H), 3.91 (s, 3H), 3.93 (d,
JHF ) 1.2 Hz), 6.45 (d, 1H, J ) 15.9 Hz), 6.70-6.78 (m, 4H),
6.87 (d, 1H, J ) 2.1 Hz), 6.93 (d, 1H, J ) 2.14 Hz), 6.97 (d,
1H, J ) 2.1 Hz), 7.34-7.45 (m, 6H), 7.73-7.77 (m, 4H); 13C
NMR (75 MHz, CDCl3) δ 19.7, 26.6, 55.2, 56.1, 61.3, 105.4,
106.4, 106.7, 112.6, 117.6, 120.6, 125.3, 127.5, 128.5, 129.6,
139.7, 133.1, 133.3, 133.6, 135.3, 145.1, 150.5, 153.5; HRMS
calcd for C33H36FO4Si 543.2368 [M + H]+, found 543.2392;
anal. calcd for C33H35FO4Si, C, H.
3-Chloro-4,4′,5-trimethoxy-3′-O-tert-butyldiphenylsilyl-
Z-stilbene (9a). The experimental procedure noted above (8a)
was repeated with 3-chloro-4,5-dimethoxybenzaldehyde (2.8
g, 14 mmol) to yield the Z-isomer (1.6 g, 21%) as a clear oil:
1H NMR (300 MHz, CDCl3) δ 1.07 (s, 9H), 3.46 (s, 3H), 3.60
(s, 3H), 3.84 (s, 3H), 6.24 (d, 1H, J ) 12 Hz), 6.34 (d, 1H, J )
12 Hz), 6.59 (d, 1H, J ) 7.5 Hz), 6.66 (s, 1H), 6.73 (s, 1H),
6.73 (d, 1H, J ) 9 Hz) 6.81 (s, 1H), 7.26-7.38 (m, 6H), 7.65
(dd, 4H, J ) 6.67 Hz, J ) 1.2 Hz); 13C NMR (125 MHz, CDCl3)
δ 19.8, 26.7, 55.1, 55.8, 60.7, 111.3, 111.7, 120.9, 122.4, 122.5,
127.1, 127.4, 127.8, 129.3, 129.5, 130.5, 133.6, 133.8, 135.3,
144.2, 144.7, 149.9, 153.1; HRMS calcd for C33H36ClO4Si 37Cl
561.2042 [M + H]+, found 561.2449, 35Cl 559.2071 [M + H]+,
found 559.1996; anal. calcd for C33H35Cl04Si.
3,3′-Dinitro-4,4′,5-trimethoxy-Z-stilbene (20g). The pre-
ceding Wittig reaction (see 20a) was carried out with 3-nitro-
4,5-dimethoxybenzaldehyde 18b20 (0.5 g, 2.37 mmol), phos-
phonium salt 19b19 (1.2 g, 2.36 mmol), sodium hydride (0.3 g,
7.5 mmol, 60% in mineral oil), and toluene (200 mL). Purifica-
tion by flash chromatography on silica gel using hexane-ethyl
acetate (1:1) as eluent afforded dinitrostilbene 20g as an off-
1
white powder (0.47 g, 55%): mp 114-115 °C; H NMR (300
MHz) δ 3.72 (s, 3H), 3.95 (s, 3H), 3.98 (s, 3H), 6.57 (d, 2H, J
) 3.0 Hz), 6.95 (d, 1H, J ) 1.5 Hz), 6.98 (d, 1H, J ) 9.0 Hz),
7.20 (d, 1H, J ) 1.8 Hz), 7.40 (dd, 1H, J ) 2.0 Hz, J ) 6.2 Hz),
7.75 (d, 1H, J ) 2.1 Hz); HRMS calcd for C17H17N2O7 361.10365
[M + H]+, found 361.10358.
Continued elution of the chromatographic column led to the
isolation of E-stilbene 9b in 4.9 g, 62% yield as a clear oil: 1H
NMR (300 MHz, CDCl3) δ 1.14 (s, 9H), 3.56 (s, 3H), 3.86 (s,
3H), 3.90 (s, 3H), 6.44 (d, 1H, J ) 16.5 Hz), 6.74 (d, 1H, J )
16.5 Hz), 6.74 (s, 1H), 6.82 (d, 1H, J ) 1.5 Hz), 6.87 (d, 1H, J
) 1.8 Hz), 6.94 (dd, 1H, J ) 8.1 Hz, J ) 2.1 Hz), 6.99 (d, 1H,
J ) 1.5 Hz), 7.34-7.42 (m, 6H), 7.72-7.76 (m, 4H); 13C NMR
(125 MHz, CDCl3) δ 19.8, 26.7, 55.3, 55.1, 60.8, 108.4, 112.1,
117.7, 119.8, 120.6, 125.0, 127.5, 128.4, 128.8, 128.8, 129.6,
General Procedure for Cleavage of the Silyl Ether
Protecting Group. 3-Fluoro-4,4′,5-trimethoxy-3′-hydroxy-
Z-stilbene (11a, Fluorcombstatin). A solution prepared
from Z-isomer 8a (2.4 g, 4.4 mmol), tetrahydrofuran (50 mL),
and 1 M tetrabutylammonium fluoride (4.5 mL, 4.5 mmol) was
stirred for 3 h. The reaction was terminated by the addition
of H2O (50 mL), the mixture was extracted with ethyl acetate,
and solvents were removed in vacuo. Separation by flash

Synthesis of Fluorcombstatin Phosphate
Table 4. Antimicrobial Activities of Fluorcombstatin (11a) and Related Compoundsa
range of minimum inhibitory concentration (µg/mL)
Journal of Natural Products, 2005, Vol. 68, No. 10 1455
microorganism
11a
11b
12a
13a
13b
16a
Cryptococcus neoformans ATCC 90112
Candida albicans ATCC 90028
Staphylococcus aureus ATCC 29213
Streptococcus pneumoniae ATCC 6303
Enterococcus faecalis ATCC 29212
Micrococcus luteus Presque Isle 456
Escherichia coli ATCC 25922
Enterobacter cloacae ATCC 13047
Stenotrophomonas maltophilia ATCC 13637
Neisseria gonorrhoeae ATCC 49226
64
64
64
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
32-64
*
*
64
64
*
32-64
*
*
*
32
*
*
*
16
*
32-64
16-32
32-64
*
*
*
*
*
*
*
*
*
*
*
*
*
32
8-16
32-64
16
a * ) no inhibition at 64 µg/mL.
chromatography with 1:4 ethyl acetate-hexane as eluent
provided Z-stilbene 11a (1.12 g, 83%) as a colorless solid, which
was recrystallized from ethyl acetate-hexane: mp 93-94 °C;
1H NMR (300 MHz, CDCl3) δ 3.67 (s, 3H), 3.87 (s, 3H), 3.90
(d, 1H, JHF ) 0.9 Hz), 5.30 (bs, 1H), 6.35 (d, 1H, J ) 12 Hz),
6.48 (d, 1H, J ) 12 Hz), 6.61-6.66 (m, 2H), 6.72 (d, 1H, J )
8.4 Hz), 6.75-6.84 (m, 2H), 6.86 (d, 1H, J ) 1.5 Hz); 13C NMR
(75 MHz, CDCl3) δ 55.9, 56.0, 61.4 (d, JCF ) 3.5 Hz), 108.2,
109.4, 109.6, 110.33, 114.8, 120.9, 127.7, 127.7, 130.0, 130.1,
132.4, 132.5, 135.8, 135.9, 145.1, 145.8, 152.8, 152.9, 154.2,
156.6; 19F NMR (CDCl3) δ -11.32 (d, J ) 12.8 Hz, 1F); HRMS
calcd for C17H18FO4 305.1189 [M + H]+, found 305.1187; anal.
calcd for C17H17FO4 C, H.
3-Fluoro-4,4′,5-trimethoxy-3′-hydroxy-E-stilbene (11b).
Cleavage of silyl ester 8b (150 mg, 0.27 mmol) was performed
as described for the synthesis of 11a. Separation by flash
chromatography on silica using ethyl acetate-hexane (3:7)
gave the title compound (75 mg, 88%). Recrystallization from
hexane gave a colorless solid: mp 86-87 °C; 1H NMR (300
MHz, CDCl3) δ 3.91 (s, 3H), 3.92 (s, 3H), 3.93 (d, JHF ) 0.9
Hz), 5.75 (bs, 1H), 6.77-6.98 (m, 6H), 6.86 (d, 1H, J ) 1.8
Hz); 13C NMR (75 MHz, CDCl3) δ 55.9, 56.2, 61.4, 100.6, 105.7,
106.5, 106.8, 107.5, 110.6, 111.8, 119.3, 125.8, 127.6, 128.6,
129.5, 130.6, 133.1, 133.2, 134.7, 136.4, 145.8, 146.6, 153.6,
156.0 (d, JCF ) 242 Hz, ipso C); anal. calcd for C17H17FO4 C,
67.10%; H, 5.63%, found C, 66.66%; H, 6.51%.
3-Chloro-4,4′,5-trimethoxy-3′-hydroxy-Z-stilbene (12a).
Deprotection of silyl ester 9a (1.5 g, 2.7 mmol) was conducted
as summarized for the synthesis of 11a. Separation by flash
chromatography on silica using ethyl acetate-hexane (3:7)
gave the title compound (754 mg, 89%). Recrystallization from
hexane gave a white solid: mp 105-106 °C; 1H NMR (500
MHz, CDCl3) δ 3.65 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 5.52 (s,
1H), 6.36 (d, 1H, J ) 12 Hz), 6.50 (d, 1H, J ) 12 Hz), 6.72-
6.76 (m, 3H), 6.88 (dd, 2H, J ) 1.5 Hz, J ) 6 Hz); 13C NMR
(75 MHz, CDCl3) δ 55.9, 55.9, 60.8, 110.4, 111.4, 114.9, 121.0,
122.5, 127.6, 127.9, 130.1, 130.4, 133.7, 144.4, 145.3, 145.9,
153.2; anal. calcd for C17H17ClO4 C, H.
908, 732 cm-1; 1H NMR (300 MHz, CDCl3) δ 3.63 (s, 3H) 3.84
(s, 3H), 3.86 (s, 3H), 6.34 (d, 1H, J ) 12 Hz), 6.49 (d, 1H, J )
12 Hz), 6.73 (d, 1H, J ) 8.4 Hz), 6.77 (d, 1H, J ) 8.7, 1.8 Hz),
6.79 (d, 1H, J ) 1.8 Hz), 6.86 (d, 1H, J ) 1.5 Hz), 7.04 (d, 1H,
J ) 1.5 Hz); 13C NMR (75 MHz, CDCl3) δ 55.8, 55.9, 60.6,
110.4, 112.1, 115.0, 117.2, 121.0, 125.3, 127.3, 130.1, 130.4,
134.4, 145.3, 146.0, 153.0; HRMS calcd for C17H17O481Br
366.0290 [M + H]+, found 366.0287; anal. calcd for C17H17-
BrO4 C, H.
3-Bromo-4,4′,5-trimethoxy-3′-hydroxy-E-stilbene (13b).
By the same procedure used to obtain phenol 13a, silyl ester
10b was converted to E phenol 13b and isolated by flash
column chromatography on silica gel with ethyl acetate-
hexane (3:7) as eluent to provide the title compound (0.14 g,
81%). Recrystallization from hexane gave a colorless solid: mp
1
152-154 °C; H NMR (300 MHz, CDCl3) δ 3.86 (s, 3H), 3.89
(s, 3H), 3.90 (s, 3H), 6.80 (d, 1H, J ) 15.9 Hz), 6.82 (d, 1H, J
) 8.4 Hz), 6.94-6.97 (m, 1H), 6.88 (s, 1H), 7.11 (d, 1H, J )
1.8 Hz), 7.25 (d, 1H, J ) 1.5 Hz), 7.28 (d, 1H, J ) 15.9 Hz);
13C NMR (75 MHz, CDCl3) δ 56.0, 56.1, 60.6, 109.3, 110.6, 11.7,
117.8, 119.3, 122.6, 125.3, 128.8, 130.5, 134.8, 145.6, 145.7,
146.5, 153.5; anal. calcd for C17H17BrO4 C, H.
3-Hydroxy-4,4′,5-trimethoxy-3′-nitro-Z-stilbene (20c).
Cleavage of the silyl ester 20a (0.15 g, 70.34 mmol) was
performed as noted for the synthesis of phenol 11a to provide
phenol 20c (0.11 g, 99%) as a yellow oil: 1H NMR (300 MHz,
CDCl3) δ 3.69 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 5.70 (s, 1H),
6.35 (s, 1H), 6.41 (d, 1H, J ) 12.5 Hz), 6.49 (d, 1H, J ) 1.5
Hz), 6.53 (d, 1H, J ) 12.5 Hz), 6.92 (d, 1H, J ) 8.5 Hz), 7.40
(dd, 1H, J ) 2.1 Hz, J ) 9 Hz) and 7.72 (d, 1H, J ) 2.1 Hz);
HRMS calcd for C17H18NO6 332.1134 [M + H]+, found 332.1108;
anal. calcd for C17H17NO6 C, H, N.
3-Hydroxy-4,4′,5-trimethoxy-3′-nitro-E-stilbene (20d).
The preceding cleavage reaction was repeated using 20b (175
mg, 0.39 mmol), and following separation by flash column
chromatography on silica gel, phenol 20d (76 mg, 66%) was
obtained as a yellow oil: mp 123-124 °C; FABMS calcd for
C17H17NO6 331.1056, found m/z 331 [M]+.
3-Chloro-4,4′,5-trimethoxy-3′-hydroxy-E-stilbene (12b).
Removal of the silyl group from 9b (5.6 g, 6.0 mmol) was as
described for the synthesis of 11a. Purification by flash
chromatography on silica using ethyl acetate-hexane (3:7)
gave the title compound (1.62 g, 79%). Recrystallization from
hexane led to a colorless solid: mp 138-140 °C; 1H NMR (300
MHz, CDCl3) δ 3.87 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H), 5.69 (bs,
1H), 6.79 (d, 1H, J ) 15.9 Hz), 6.81 (d, 1H, J ) 8.4 Hz), 6.84-
6.96 (m, 3H), 7.10 (dd, J ) 1.8 Hz, J ) 8.4 Hz); 13C NMR (75
MHz, CDCl3) δ 55.9, 56.1, 60.7, 108.7, 110.6, 111.8, 119.4,
119.8, 125.5, 128.4, 128.8, 130.6, 134.2, 144.6, 145.8, 146.6,
153.8; HRMS calcd for C17H17ClO4 35Cl 321.0894 [M + H]+,
found 321.0893; anal. calcd for C17H17ClO4 C, H.
3-Bromo-4,4′,5-trimethoxy-3′-hydroxy-Z-stilbene (13a).
The silyl ester cleavage reaction for 10a (4 g, 6.6 mmol) was
completed as described for the synthesis of phenol 11a.
Isolation by flash column chromatography on silica gel using
ethyl acetate-hexane (1:4) gave 13a (2.22 g, 92%). Recrystal-
lization from hexane afforded a colorless solid: mp 108-109
°C; IR 3539, 3441, 3011, 2939, 2839, 1554, 1510, 1273, 1047,
3-Hydroxy-4,4′,5-trimethoxy-3′-amino-Z-stilbene (20e)
and Hydrochloride (20f). Platinum-on-carbon (5%, 0.5 g)
was added to a solution of Z-stilbene 20c (1.2 g, 3.62 mmol)
and ammonium formate (1.0 g, 15.85 mmol) in MeOH (5 mL).
The mixture was heated under reflux for 30 min and filtered,
and the residue was fractionated on a column of silica gel in
7:3 hexane-ethyl acetate to yield amine 20e (0.94 g, 86%) as
an amorphous yellow solid: 1H NMR (400 MHz, CDCl3) δ 3.59
(s, 3H), 3.75 (s, 3H), 3.81 (s, 3H), 6.25 (d, 1H, J ) 12 Hz), 6.35
(d, 1H, J ) 12 Hz), 6.38 (d, 1H, J ) 2 Hz), 6.47 (d, 1H, J ) 1.6
Hz), 6.60 (s, 1H), 6.61 (s, 1H), 6.62 (s, 1H); 13C NMR (75 MHz
w/APT) 55.5n, 55.6n, 61.0n, 104.9n, 108.8n, 110.1n, 115.4n,
119.5n, 128.2n, 130.0p, 130.0n, 133.5p, 134.5p, 135.7p, 146.6p,
148.9p, 151.8p; FABMS calcd for C17H19NO4 301.13, found, m/z
301 [M]+. A solution of amine 20e (1.0 g, 3.32 mmol) in ether
(5 mL) was treated with ethereal hydrogen chloride (1 M, 5
mL), and the product was collected as a pale yellow solid (20f,
1.14 g, 99%): mp 153-156 °C; 1H NMR (500 MHz, D2O) δ 3.48
(s, 3H), 3.62 (s, 3H), 3.76 (s, 3H), 6.29 (s, 1H), 6.32 (s, 1H),
6.36 (d, 1H, J ) 12 Hz), 6.42 (d, 1H, J ) 12 Hz), 6.89 (d, 1H,

1456 Journal of Natural Products, 2005, Vol. 68, No. 10
Pettit et al.
J ) 8.5 Hz), 7.09 (s, 1H), 7.13 (s, 1H); 13C NMR (75 MHz, D2O,
w/APT) 55.9p, 56.3p, 61.0p, 105.6p, 110.0p, 112.4p, 118.9n,
124.0p, 128.7p, 129.9p, 130.2n, 131.0p, 133.6n, 135.8n, 149.0n,
151.7n, 152.7n; HRMS calcd for C17H20NO4 302.1392 [M + H]+,
found 302.1381.
3,3′-Diamino-4,4′,5-trimethoxy-Z-stilbene (20h). To dini-
trostilbene 20g (0.11 g, 0.31 mmol) in acetic acid (25 mL) was
added zinc powder (4 g, 61.2 mmol). After 1 h the mixture was
filtered through Celite and the solvent was removed in vacuo
to yield diamine 20h as a yellow oil (72 mg, 75%): 1H NMR
(300 MHz) δ 3.66 (s, 3H), 3.73 (br s, 4H), 3.81 (s, 3H), 3.83 (s,
3H), 6.32 (s, 1H), 6.35 (d, 1H, J ) 2.1 Hz), 6.35 (dd, 2H, J )
12.0 Hz, J ) 13.2 Hz), 6.67 (s, 1H), 6.68 (s, 1H), 6.71 (s, 1H);
HRMS calcd for C17H21N2O3 301.1552 [M + H]+, found
301.1639; anal. calcd for C17H20N2O3 C, H.
was collected and washed with ether to provide sodium salt
1
16a (0.61 g, 76%): colorless solid; mp 200-202 °C; H NMR
(300 MHz, D2O) δ 3.52 (s, 3H), 3.67 (s, 3H), 3.68 (s, 3H), 6.52
(d, 1H, J ) 12 Hz), 6.71 (d, 1H, J ) 12 Hz), 6.72 (s, 1H), 6.78
(s, 1H), 6.79 (s, 1H), 7.03 (s, 1H), 7.16 (s, 1H).
Lithium 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-O-
1
phosphate (16b): colorless solid; mp 255-274 °C; H NMR
(300 MHz, D2O) δ 3.55 (s, 3H), 3.68 (s, 3H), 3.72 (s, 3H), 6.36
(d, 1H, J ) 12 Hz), 6.51 (d, 1H, J ) 12 Hz), 6.62-6.65 (m,
2H), 6.69-6.76 (m, 2H), 7.22 (s, 1H).
Potassium 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-O-
1
phosphate (16c): colorless solid; mp 192-210 °C; H NMR
(300 MHz, D2O) δ 3.56 (s, 3H), 3.69 (s, 3H), 3.74 (s, 3H), 6.37
(d, 1H, J ) 12 Hz), 6.51 (d, 1H, J ) 12 Hz), 6.62-6.78 (m,
4H), 7.24 (s, 1H).
Dibenzyl 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (14). A solution of phenol 11a (1.1 g, 3.6 mmol),
acetonitrile (20 mL), and 3.5 mL (36 mmol) of CCl4 was cooled
to -10 °C and stirred for 10 min. Then diisopropyl ethylamine
(1.3 mL, 7.4 mmol) and DMAP (44 mg, 0.36 mmol) were added.
After 1 min, dibenzyl phosphite (1.2 mL, 5.4 mmol) was added
(over 5 min), and the mixture stirred for an additional 3 h at
-10 °C. The reaction was terminated by the addition of 0.5 M
KH2PO4, the mixture was extracted with ethyl acetate, sol-
vents were removed in vacuo, and the product was isolated
by flash chromatography (1:1 elution with ethyl acetate-
hexane) to yield phosphate 14 (1.5 g, 74%): bp dec 280 °C (0.01
mmHg); 1H NMR (300 MHz, CDCl3) δ 3.65 (s, 3H), 3.77 (s,
3H), 3.87 (d, 3H, JHF ) 1.2 Hz), 5.12 (s, 2H), 5.14 (s, 2H), 6.38
(d, 1H, J ) 12 Hz), 6.43 (d, 1H, J ) 12 Hz), 6.57 (s, 1H) 6.62
(dd, 1H, J ) 1.5 Hz, J ) 11.5 Hz), 6.78 (d, 1H, J ) 8.5 Hz),
Sodium 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (17a). To a solution of dibenzyl phosphate 15 (0.28
g, 0.45 mmol) in dry CH2Cl2 (10 mL) was added trimethylsi-
lylbromide (125 µL, 0.95 mmol). The reaction mixture was
stirred for 30 min under argon, and the reaction was termi-
nated by the addition of MeOH (20 mL). Following removal of
solvents (in vacuo), the phosphoric acid was dissolved in EtOH
(10 mL), and NaOMe (49 mg, 0.9 mmol) was added to the
solution. After the reaction mixture was stirred for 30 min,
the precipitate was collected and washed with ether to provide
sodium salt 17a (0.17 g) as a colorless solid: mp 196-197 °C;
1H NMR (300 MHz, D2O) δ 3.53 (s, 3H), 3.68 (s, 3H), 3.70 (s,
3H), 6.37 (d, 1H, J ) 12 Hz), 6.52 (d, 1H, J ) 12 Hz), 6.75 (s,
1H), 6.77 (s, 1H), 6.79 (s, 1H), 7.01 (s, 1H), 7.15 (s, 1H).
Lithium 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (17b): colorless solid; mp 265-268 °C (dec); 1H
NMR (300 MHz, D2O) δ 3.53 (s, 3H), 3.66 (s, 3H), 3.69 (s, 3H),
6.35 (d, 1H, J ) 12 Hz), 6.52 (d, 1H, J ) 12 Hz), 6.70 (s, 2H),
6.81 (d, 1H, J ) 1.5 Hz), 7.01 (d, 1H, J ) 1.5 Hz), 7.23 (s, 1H).
Potassium 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (17c): colorless solid; mp 230-233 °C (dec); 1H
NMR (300 MHz, D2O) δ 3.53 (s, 3H), 3.66 (s, 3H), 3.69 (s, 3H),
6.35 (d, 1H, J ) 12 Hz), 6.52 (d, 1H, J ) 12 Hz), 6.70 (s, 2H),
6.81 (d, 1H, J ) 1.5 Hz), 7.01 (d, 1H, J ) 1.5 Hz), 7.23 (s, 1H).
Cesium 3-bromo-4,4′,5-trimethoxy-phenyl-Z-stilbene
3′-O-phosphate (17d): colorless solid; mp 233-235 °C; 1H
NMR (300 MHz, DMSO) δ 3.51 (s, 3H), 3.62 (s, 3H), 3.65 (s,
3H), 6.38 (d, 1H, J ) 12 Hz), 6.50 (d, 1H, J ) 12 Hz), 6.71 (s,
1H), 6.83 (d, 1H, J ) 1.5 Hz), 7.03 (d, 2H, J ) 1.5 Hz), 7.23 (s,
1H).
7.03 (d, 1H, J ) 8.5 Hz), 7.12 (s, 1H), 7.14-7.32 (m, 10H); 13
C
NMR (100 MHz, CDCl3) δ 55.8, 61.4 (d, JCF ) 3 Hz), 69.7, 69.7,
108.1, 108.2, 109.5 (d, JCF ) 20.45 Hz, ortho), 112.3, 126.4,
126.4, 127.8, 128.4, 128.5, 129.3, 129.6, 129.6, 132.2 (d, JCF
9.1 Hz, meta C), 135.6, 135.7, 139.4, 139.5, 149.9, 149.9, 153.1,
153.1, 155.6 (d, JCF ) 243 Hz, ipso C); 31P NMR (162 MHz
CDCl3) δ -7.84; HRMS calcd for C31H30FO7P 565.1791 [M +
H]+, found 565.1812; anal. calcd for C31H30FO7P C, H.
Dibenzyl 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (15). Phosphorylation of 13a (1 g, 2.7 mmol) was
performed as described for the synthesis of phosphate 14.
Separation by flash column chromatography on silica using
ethyl acetate-hexane (1:1) as eluent gave phosphate 15 (1.6
1
g, 94%) as an oil: bp dec 271 °C (0.01 mmHg); H NMR (300
MHz, CDCl3) δ 3.59 (s, 3H), 3.76 (s, 3H), 3.79 (s, 3H), 5.11 (s,
2H), 5.13 (s, 2H), 6.37 (d, 1H, J ) 12 Hz), 6.43 (d, 1H, J ) 12
Hz), 6.72 (d, 1H, J ) 1.5 Hz), 6.78 (d, 1H, J ) 8.4 Hz), 7.01-
7.04 (m, 1H), 7.03 (d, 1H, J ) 2.4 Hz), 7.10 (d, 1H, J ) 1.8
Hz), 7.28-7.35 (m, 10H); 13C NMR (100 MHz, CDCl3) δ 55.8,
55.9, 60.5, 65.1, 69.7, 69.7, 111.8, 112.2, 117.2, 122.0, 122.0,
125.0, 126.3, 126.7, 127.3, 127.7, 127.8, 127.9, 128.3, 128.4,
129.3, 129.5, 129.5, 133.9, 135.4, 135.5, 139.3, 139.4, 145.3,
149.8, 149.8, 153.0; 31P NMR (162 MHz CDCl3) δ -7.84; HRMS
calcd for C31H31BrO7P79Br 625.0991 [M + H]+, found 625.0928,
81Br 627.0970 [M + H]+, found 627.09704; anal. calcd for
C31H30BrO7P C, H.
Rubidium 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
1
phosphate (17e): colorless solid; mp 204-206 °C; H NMR
(300 MHz, DMSO) δ 3.50 (s, 3H), 3.64 (s, 3H), 3.66 (s, 3H),
6.35 (d, 1H, J ) 12 Hz), 6.52 (d, 1H, J ) 12 Hz), 6.68 (s, 2H),
6.80 (d, 2H, J ) 1.5 Hz), 7.00 (d, 2H, J ) 1.5 Hz), 7.25 (s, 1H).
Calcium 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (17f): colorless solid; mp 245-248 °C (dec); 1H
NMR (300 MHz, DMSO) δ 3.53 (s, 3H), 3.69 (s, 3H), 3.70 (s,
3H), 6.33 (d, 1H, J ) 12 Hz), 6.50 (d, 1H, J ) 12 Hz), 6.71 (s,
2H), 6.81 (d, 2H, J ) 1.5 Hz), 7.99 (d, 2H, J ) 1.5 Hz), 7.23 (s,
1H).
Magnesium 3-bromo-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (17g): colorless solid; mp 280-285 °C (dec); 1H
NMR (300 MHz, DMSO) δ 3.50 (s, 3H) 3.60 (s, 3H), 3.65 (s,
3H), 6.33 (d, 1H, J ) 12 Hz), 6.50 (d, 1H, J ) 12 Hz), 6.68 (s,
2H), 6.79 (d, 2H, J ) 1.5 Hz), 7.00 (d, 2H, J ) 1.5 Hz), 7.21 (s,
1H).
General Procedure for Synthesis of the Phosphate
Cation Derivatives. Method A. Each of the metal cation
containing salts was obtained by the procedure outlined
directly below for preparing sodium salt 16a. The metal
counterions were introduced by treatment of the phosphoric
acid with either the corresponding hydroxide (potassium,
lithium) or acetate (magnesium).
Sodium 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-O-phos-
phate (16a). To a cooled solution of dibenzyl phosphate 14
(1.02 g, 1.8 mmol) in dry CH2Cl2 (40 mL) was added trimeth-
ylsilylbromide (530 µL, 3.9 mmol). The reaction mixture was
stirred at 0 °C for 2 h under argon, then terminated with 10%
sodium thiosulfate solution (8 mL). The organic layer was
separated and extracted with CH2Cl2 (3 × 40 mL). The
combined organic layers were dried, filtered, and concentrated.
The free phosphoric acid was dissolved in MeOH (5 mL), and
NaOMe (195 mg, 3.6 mmol) was added to the solution. After
the reaction mixture was stirred for 30 min, the precipitate
Method B. The potassium salt 16c (∼30 mg) was dissolved
in deionized H2O (1 mL) and applied to a Dowex-50w (HCR-
W2) resin column (amine or amino acid) and eluted with water.
The eluent was concentrated by freeze-drying to give the
required compound.
Morpholine 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′O-
1
phosphate (16d): colorless oil; H NMR (300 MHz, D2O) δ
3.10-3.14 (m, 8H), 3.54 (s, 3H), 3.67 (s, 3H), 3.71 (s, 3H), 3.76-
3.81 (m, 8H), 6.35 (d, 1H, J ) 12 Hz), 6.49 (d, 1H, J ) 12 Hz),
6.59-6.36 (m, 2H), 6.76-6.76 (m, 2H), 7.18 (s, 1H).
Piperidine 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-O-
phosphate (16e): waxy colorless solid; mp 55-57 °C; 1H
NMR (300 MHz, D2O) δ 1.47-1.59 (m, 12H), 2.94-3.03 (m,

Synthesis of Fluorcombstatin Phosphate
Journal of Natural Products, 2005, Vol. 68, No. 10 1457
8H), 3.51 (s, 3H), 3.64 (s, 3H), 3.67 (s, 3H), 6.30 (d, 1H, J ) 12
Hz), 6.48 (d, 1H, J ) 12 Hz), 6.56-6.72 (m, 4H), 7.20 (s, 1H).
Glycine-OMe 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-
Hoechst 33342 (Sigma, stock solution diluted with PBS to give
a final concentration of 1 µg/mL). Following the 30 min
incubation, the cover slips were again washed in PBS for 10
min and finally mounted using fluorescent mounting medium
(DAKO).
Immunofluorescence microscopy using a confocal microscope
was then carried out to assess any disruption of the microtu-
bules in the cells. The cells were scored for tubulin disruption
(Table 3) and the results tabulated. Images to support the
scoring were also obtained.
Method B. In vitro Matrigel antiangiogenesis assays were
implemented according to the Developmental Therapeutics
Program NCI/NIH protocols.22 Matrigel, a basement mem-
brane matrix, was purchased from BD Biosciences. Growth
inhibition and cord formation assays were conducted using
HUVECs purchased from GlycoTech. HUVEC cells were grown
in EGM-2 medium (Cambrex).
Cord Formation Assay. An aliquot of Matrigel (60 µL) was
placed in each well of an ice-cold 96-well plate. The plates were
incubated 15 min at RT, followed by 30 min at 37 °C, to permit
the Matrigel to polymerize. Meanwhile, HUVECs were har-
vested and diluted to a concentration of 2 × 105 cells/mL, and
100 µL of the cell suspension was added to each well. A second
solution of 100 µL containing the compounds to be tested was
added next. After 24 h incubation, images were taken for each
concentration using an inverted Nikon Diaphot microscope and
D100 digital camera. Drug effect was assessed in comparison
to untreated controls by measuring the length of cords formed
and number of junctions.
1
O-phosphate (16f): colorless waxy solid; mp 88-92 °C; H
NMR (300 MHz, D2O) δ 3.53 (s, 2H), 3.68 (s, 2H), 3.71 (s, 3H),
3.76 (s, 3H), 6.35 (d, 1H, J ) 12 Hz), 6.49 (d, 1H, J ) 12 Hz),
6.58-6.62 (m, 2H), 6.72-6.81 (m, 2H), 7.13 (s, 1H).
Tryptophan-OMe 3-fluoro-4,4′,5-trimethoxy-Z-stilbene
3′-O-phosphate (16g): colorless solid; mp 75-80 °C; 1H NMR
(300 MHz, D2O) δ 3.24 (d, 2H, J ) 6 Hz), 3.53 (s, 3H), 3.61 (s,
3H), 3.67 (s, 3H), 3.72 (s, 3H), 4.11 (t, 1H, J ) 6 Hz), 6.35 (d,
1H, J ) 12 Hz), 6.60-6.64 (m, 2H), 7.04 (t, 1H, J ) 7.5 Hz),
7.09-7.14 (m, 2H), 7.20 (s, 1H), 7.38 (d, 1H, J ) 7.8 Hz), 7.46
(d, J ) 7.5 Hz).
‘Tris’ 3-fluoro-4,4′,5-trimethoxy-Z-stilbene 3′-O-phos-
phate (16h): colorless solid; mp 88-93 °C; 1H NMR (300
MHz, DMSO) δ 3.34 (s, 12H), 3.62 (s, 3H), 3.70 (s, 3H), 3.75
(s, 3H), 6.36 (d, 1H, J ) 12 Hz), 6.49 (d, 1H, J ) 12 Hz), 6.66-
6.71 (m, 1H), 6.75-6.77 (m, 2H), 6.81 (d, 1H, J ) 8.4 Hz), 7.42
(s, 1H).
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.21
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 Im-
munosoft software. Inhibition of the mouse leukemia P388 cells
was assessed in a 10% horse serum/Fisher medium solution
for 24 h, followed by a 48 h incubation with serial dilutions of
the compounds. Cell growth inhibition (ED50) was then calcu-
lated using a Z1 Beckman/Coulter particle counter.
Tubulin Evaluations. Tubulin polymerization was evalu-
ated by turbidimetry at 350 nm using Beckman DU7400/7500
spectrophotometers as described in detail elsewhere.20 Varying
concentrations of drug were preincubated with 10 µM (1.0 mg/
mL) purified tubulin,23 samples were chilled on ice, GTP (0.4
mM) was added, and polymerization was followed at 30 °C.
The parameter measured was extent of the reaction after 20
min. Colchicine binding was measured as described in detail
previously.24 Reaction mixtures contained 1.0 µM tubulin, 5.0
µM [3H]colchicine (from Dupont), and inhibitor at 1.0 µM.
Incubation was for 10 min at 37 °C.
Antiangiogenesis Evaluation. HUVEC Procedures.
Method A. Umbilical cords were obtained from the Maternity
Unit at Bradford Royal Infirmary following ethical approval
and consent from mothers. Isolated HUVECs were grown in
medium M199 (Sigma) supplemented with 20% human serum.
Authentication of cells was by staining for expression of the
endothelial cell marker CD31. Cover slips were initially coated
with 2% gelatin (Sigma) for 30 min and washed with Hanks
medium (Sigma) before being placed individually in 24-well
plates. Growth medium M199 (1 mL) was added, followed by
approximately 3 × 104 HUVECs to give a final volume of 1.5
mL in each well. The cells were incubated at 37 °C for 24 h
and checked microscopically before being treated with test
compounds.
Test compounds were dissolved in DMSO and added to
medium to give a final concentration of 1 µM, 10 µM, or 100
µM in each well. Duplicate plates were prepared for each
compound, and at time points of 30 min, 1, 2, and 4 h, the
appropriate set of cover slips was removed and fixed in ice-
cold methanol for at least 1 h. Untreated controls were treated
identically. The cover slips were then washed in PBS for 10
min before being incubated with 100 µL monoclonal anti-
human R-tubulin (mouse IgG1 isotype, Sigma) diluted 1:500
with PBS for 30 min. Following three 5 min washes with PBS,
100 µL of the secondary antibody TRITC (tetramethyl-
rhodamine isothiocyanate)-conjugated rabbit anti-mouse im-
munoglobulin (DAKO) was added to each cover slip at a
concentration of 1:50 in PBS. This and all subsequent staining
procedures were carried out in the dark. After a 30 min
incubation period, the cover slips were washed again in PBS
(three 5 min washes) before being stained for DNA using
The standard sulforhodamine B assay (see Cancer Cell Line
Procedures above) was used to evaluate results using HU-
VECs. The ED50 (drug concentration causing 50% inhibition)
was calculated from the plotted data.
Broth Microdilution Susceptibility Testing of Bac-
teria. The antibacterial activity of a subset of the halocomb-
statins was assessed by the NCCLS broth microdilution assay
(BMA).17 The halocombstatins were reconstituted in a small
volume of sterile DMSO and diluted in the appropriate media
prior to susceptibility experiments. Isolated colonies from
overnight cultures were suspended and diluted as recom-
mended to yield final inocula of approximately 5 × 105 CFU/
mL. Tests were performed in sterile 96-well plates containing
2-fold dilutions of the test compounds in gonococcal typing
broth (Neisseria), Mueller Hinton II (MHII) (cation-adjusted)
broth containing 3% lysed horse blood (Streptococcus), or MHII
broth (all other bacteria). One well was left drug-free (but
contained an equivalent volume of DMSO) for a turbidity
control. Plates were incubated without agitation at 37 °C with
5% CO2 (Neisseria) or at 35 °C (all other bacteria). MICs were
determined at 24 h. The MIC was defined as the lowest drug
concentration that inhibited all visible growth of the test
organism (optically clear).
Broth Microdilution Susceptibility Testing of Yeasts.
The halocombstatins were screened against yeasts by broth
microdilution assays (BMAs) according to the NCCLS.18
Isolated yeast colonies were suspended and diluted as recom-
mended to yield final inocula ranging from 0.5 to 2.5 × 103
CFU/mL. Tests were performed in sterile 96-well plates
containing 2-fold dilutions of the test compounds in 0.165 M
morpholinepropanesulfonic acid buffered RPMI 1640 medium
(pH 7.0). One well was left drug-free (but contained an
equivalent volume of DMSO) for a turbidity control. Plates
were incubated without agitation at 35 °C. MICs were deter-
mined at 48 h for Candida and at 72 h for Cryptococcus. The
MIC was defined as the lowest drug concentration that
inhibited all visible growth of the test organism (optically
clear).
Acknowledgment. The financial support was provided
with our appreciation by Outstanding Investigator Grant
CA44344-08-12 and Grant RO1 CA90441-01-05 awarded by
the Division of Cancer Treatment and Diagnosis, National
Cancer Institute, DHHS; the Arizona Disease Control Re-
search Commission; the Robert S. Dalton Endowment Fund;

1458 Journal of Natural Products, 2005, Vol. 68, No. 10
Pettit et al.
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Dr. Alec D. Keith; the J. W. Kieckhefer Foundation; the
Margaret T. Morris Foundation; the Caitlin Robb Foundation;
Gary L. and Diane R. Tooker; Polly J. Trautman; the Eagles
Art Ehrmann Cancer Fund; and the Ladies Auxiliary to the
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