Regioselective synthesis of water-soluble monophosphate derivatives of combretastatin A-1

Article abstract of DOI:10.1021/np200104t

The natural products combretastatin A-4 (CA4) and combretastatin A-1 (CA1) are potent cancer vascular disrupting agents and inhibitors of tubulin assembly (IC₅₀ = 1-2 μM). The phosphorylated prodrugs CA4P and CA1P are undergoing human clinical

Full text of DOI:10.1021/np200104t

ARTICLE
pubs.acs.org/jnp
Regioselective Synthesis of Water-Soluble Monophosphate
Derivatives of Combretastatin A-1
Rajendra P. Tanpure,†,r Benson L. Nguyen,†,r Tracy E. Strecker,† Savannah Aguirre,† Suman Sharma,‡
David J. Chaplin,‡ Bronwyn G. Siim,‡ Ernest Hamel,§ John W. Lippert, III,^ George R. Pettit,||
Mary Lynn Trawick,† and Kevin G. Pinney*,†
†Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798-7348, United States
‡OXiGENE Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, California 94080, United States
§Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe,
Arizona 85287-1604, United States
S Supporting Information
b
ABSTRACT: The natural products combretastatin A-4 (CA4)
and combretastatin A-1 (CA1) are potent cancer vascular
disrupting agents and inhibitors of tubulin assembly (IC50
=
1ꢀ2 μM). The phosphorylated prodrugs CA4P and CA1P are
undergoing human clinical trials against cancer. CA1 is unique
due to its incorporation of a vicinal phenol, which has afforded
the opportunity to prepare both diphosphate and regioisomeric
monophosphate derivatives. Here, we describe the first syn-
thetic routes suitable for the regiospecific preparation of the
CA1-monophosphates CA1MPA (8a/b) and CA1MPB (4a/b).
The essentialregiochemistrynecessary to distinguish between the
two vicinal phenolic groups was accomplished with a tosyl protecting group strategy. Each of the four monophosphate analogues
(including Z and E isomers) demonstrated in vitro cytotoxicity against selected human cancer cell lines comparable to their
corresponding diphosphate congeners. Furthermore, Z-CA1MPA (8a) and Z-CA1MPB (4a) were inactive as inhibitors of tubulin
assembly (IC50 > 40 μM), as anticipated in this pure protein assay.
ombretastatin A-1 (CA1)1 and combretastatin A-4 (CA4)2ꢀ4
are both Z-stilbenoid naturalproducts originally isolated from
showed more consistent results against murine P388 leukemia in
vitro, and CA1 (Z), in preclinical development, showed higher
vascular disruption and antitumor activity than CA4 (Z).31 The
increased effectiveness of CA1 (Z), compared to CA4 (Z), may be
attributed to the presence of the second hydroxy substituent,
which facilitates formation of the highly reactive ortho quinone
analogue, obtainable through biological oxidation of the 1,2-diol
functionality present in CA1 (Z).33,34
Recent studies by Kirwan et al. have shown that the addi-
tional phosphate group present in CA1P, as compared to CA4P,
results in the formation of numerous metabolites of CA1,
several of which have been identified as monophosphates,
monoglucuronides, and a bis-glucuronide.35 Partial enzymatic
dephosphorylation of CA1P may lead to two regioisomeric
CA1-monophosphates [combretastatin A-1 monophosphate
A (CA1MPA) and combretastatin A-1 monophosphate B
(CA1MPB), Figure 1] in addition to formation of the active drug
C
the Africanbush willow tree, Combretum caffrum Kuntze (Combre-
taceae). CA1 and CA4 are remarkably potent against human
cancer cell lines (in vitro)5 and are strongly inhibitory against the
polymerization of tubulin into microtubules.6,7 Formulated as
phosphate prodrugs [(CA1P, OXi4503)8,9 and (CA4P, Zybrestat,
fosbretabulin)10] to increase aqueous solubility, these compounds
are currently under investigation in human clinical trials as antic-
ancer drugs.11ꢀ21 CA1P and CA4P fall into a relatively new
grouping of compounds collectively referred to as vascular dis-
rupting agents (VDAs).14,22ꢀ24 Mechanistically distinct from
antiangiogenic agents, VDAs are characterized by their ability to
selectively damage existing microvasculature25 feeding a tumor,
thus starving that tumor of oxygen and required nutrients.26ꢀ30
Interestingly, while CA4 (Z) is generally more potent than CA1
(Z) against human cancer cell lines (in vitro), the corresponding
prodrug CA4P (Z) is somewhat less active than CA1P (Z) in
certain in vivo preclinical tumor growth delay studies carried out in
severe combined immunodeficiency (SCID) mice.31,32 CA1 (Z)
Received: February 2, 2011
Published: June 30, 2011
r
Copyright
2011 American Chemical Society and
1568
dx.doi.org/10.1021/np200104t J. Nat. Prod. 2011, 74, 1568–1574
American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
reference compound. This procedure was based on the
standard sulforhodamine B (SRB) assay.38,39 The GI50 values
are shown in Table 1. The Z-series CA1-monophosphates
(4a and 8a) showed enhanced cytotoxicity compared to the
corresponding E-series regioisomers (4b and 8b). This re-
flects the increased cytotoxicity of Z-CA1 compared to E-CA1.
The Z-series CA1-monophosphates (4a and 8a) were evalu-
ated for their ability to inhibit tubulin assembly and were
found to be inactive (IC50 > 40 μM), which is consistent with
the results obtained with CA1P.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Dichloromethane, aceto-
nitrile, and tetrahydrofuran (THF) were used in their anhydrous
forms, as obtained from the chemical suppliers. Reactions were
performed under an inert atmosphere using nitrogen gas, unless
specified. Thin-layer chromatography (TLC) plates (precoated glass
plates with silica gel 60 F254, 0.25 mm thickness) were used to
monitor reactions. Purification of intermediates and products was
carried out with a flash purification system using silica gel (200ꢀ400
mesh, 60 Å) or RP-18 prepacked columns. Intermediates and
products synthesized were characterized on the basis of their 1H
NMR (500 MHz), 13C NMR (125 MHz), 31P NMR (202 MHz),
gHSQC, and gHMBC spectroscopic data. TMS was used as an
internal standard for spectra recorded in CDCl3. For spectra
Figure 1. Combretastatin A-1 (CA1) and combretastatin A-4 (CA4)
derivatives.
CA1.35 Each of the monophosphates (CA1MPA 8a(Z), 8b(E)
and CA1MPB 4a(Z), 4b(E)) has distinct chemical properties.36
Since the regioisomeric monophosphate salts are structurally
distinct from both CA1P and CA1, it is anticipated that they
may have different activity profiles in biological systems.
’ RESULTS AND DISCUSSION
1
The synthesis of the stilbenoid core of CA4 and CA1, along
with numerous derivatives, has been efficiently achieved using
the Wittig approach.4,32,33 The regiospecificity necessary for the
synthesis of the CA1-monophosphates (Scheme 1) was accom-
plished by distinguishing between the two vicinal phenolic
functional groups at C-20 and C-30 of stilbenes 1a/b and 5a/b
with a selective tosyl protecting group strategy.37 The phosphor-
ylation of monophenols 1a/b and 5a/b was achieved with
dibenzylphosphite as the reagent of choice due to its high
reactivity, which resulted in good yields. Deprotection of the
resultant benzyloxy phosphate esters 2a/b and 6a/b with TMSBr
followed by treatment with MeONa provided the corresponding
CA1-disodium phosphate salts 3a/b and 7a/b. Removal of the
tosyl group was achieved using NaOH (2 M) in a microwave
reactor at a moderate temperature (50 ꢀC) to afford CA1MPA
8a(Z), CA1MPA 8b(E), CA1MPB 4a(Z), or CA1MPB 8b(E).
In addition to characterization of the intermediates and final
recorded in D2O: δ H 4.80. All the chemical shifts are expressed
in ppm (δ), coupling constants (J) are presented in Hz, and peak
patterns are reported as broad (br), singlet (s), doublet (d), triplet
(t), quartet (q), septet (sept), and multiplet (m). HRESIMS were
obtained using (+ve or ꢀve) electrospray ionization (ESI) techni-
ques. Purity of the final compounds was further analyzed at 25 ꢀC
using an Agilent 1200 HPLC system with a diode-array detector (λ =
190ꢀ400 nm), a Zorbax XDB-C18 HPLC column (4.6 mm ꢁ 150 mm,
5 μm), and a Zorbax reliance cartridge guard-column; eluents, solvent A,
0.1% TFA in H2, solvent B, 0.08% TFA in acetonitrileꢀH2 (80:20 (v/v)
ratio); gradient, 80% A/20% B over 0 to 5 min; 80% A/20% B f 5% A/
95% B over 5 to 35 min; 5% A/95% B over 35 to 45 min; post-time
15 min; flow rate 1.0 mL/min; injection volume 20 μL; monitored at
wavelengths of 254, 264, 280, and 300 nm.
(Z)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[(para-toluene-
sulfonyl)oxy]-300-[([bis[(benzyl)oxy]]phosphoryl)oxy]-400-
methoxyphenyl]ethene (2a). Phenol 1a37 (0.120 g, 0.247 mmol)
was dissolved in acetonitrile (10 mL) and cooled to ꢀ10 ꢀC. CCl4
(0.20 mL, 2.1 mmol) was added, and the reaction mixture was stirred for
5 min. Diisopropylethylamine (0.40 mL, 2.3 mmol) and DMAP (0.065 g,
0.532 mmol) were added, and the reaction mixture was stirred for an
additional 10 min. Dibenzylphosphite (0.30 mL, 1.4 mmol) was added
slowly to the reaction mixture, which was then stirred for 45 min and
monitored by TLC. On completion, KH2PO4 (25 mL) was added, and the
reaction mixture was allowed to return to ambient temperature. H2O
(25 mL) was added to the reaction mixture, and the organic phase was
separated. The aqueous phase was extracted with EtOAc (3 ꢁ 10 mL), and
the combined organic phases were dried with Na2SO4, filtered, and
concentrated under reduced pressure. Flash chromatography of the crude
product using a prepacked 10 g silica column [eluents; solvent A, EtOAc,
solvent B, hexanes; gradient, 40% A/60% B over 1.15 min (1 CV), 40% A/
60% B f 10% A/90% B over 12.30 min (10 CV), 10% A/90% B over 3.07
min (2.5 CV); flow rate 12.0 mL/min; monitored at 254 and 280 nm]
afforded 2a (0.12 g, 0.59 mmol, 67%) as a yellow oil: 1HNMR(CDCl3, 500
MHz) δ 7.77 (2H, d, J = 8.4 Hz, H-200, H-600), 7.40ꢀ7.30 (10H, m, Ph),
7.16 (2H, d, J = 8.4 Hz, H-300, H-500), 7.00 (1H, d, J = 8.8 Hz, H-60), 6.71
(1H, d, J = 8.8 Hz, H-50), 6.47 (2H, s, H-2, H-6), 6.36 (1H, d, J = 16.2 Hz,
H-1a), 6.30 (1H, d, J = 16.2 Hz, H-10a), 5.12 (4H, m, OCH2Ph), 3.81 (3H,
1
compounds by H and 13C NMR and HRMS data, the phos-
phorus-containing compounds were further characterized by
their 31P NMR data. Compounds 4a, 4b, 8a, and 8b each showed
a singlet in their respective 31P NMR spectrum at ca. ꢀ6.2 ppm, a
characteristic of phosphoric acid triesters. On conversion of
these phosphoric acid triesters to their respective sodium salts,
each of them showed a downfield shift of ∼9 ppm in their
31P NMR spectrum. HPLC studies were carried out on each
of the Z-monophosphates (4a, 8a) to provide further confirma-
tion of their chemical purity. Under these HPLC conditions
neither dephosphorylation, Z/E isomerization, nor intra-
molecular phosphate migration was observed (see Supporting
Information).
’ BIOLOGICAL EVALUATION
The cytotoxicity of the four CA1-monophosphates CA1MPA
(8a,b) and CA1MPB (4a,b) was evaluated using a panel of
three human cancer cell lines, prostate (DU-145), ovarian
(SK-OV-3), and lung (NCI-H460), with doxorubicin as a
1569
dx.doi.org/10.1021/np200104t |J. Nat. Prod. 2011, 74, 1568–1574

Journal of Natural Products
ARTICLE
Scheme 1. Synthesis of Z/E-CA1-20-monophosphates and Z/E-CA1-30-monophosphates
Table 1. Cytotoxicity of CA1-monophosphate Analogues and Related Compounds against Human Cancer Cell Lines SK-OV-3,
NCI-H460, and DU-145 and Inhibition of Tubulin Polymerization
GI50 (μM) SRB assaya
inhibition of tubulin polymerization IC50 (μM)
compd
SK-OV-3
NCI-H460
DU-145
CA1 (Z)b
CA1 (E)d
8a (Z)
0.0384 ( 0.0242
2.27
0.0153 ( 0.0158
1.32
0.0326 ( 0.0173
2.14
1.9c
11 ( 0.9
>40
nde
0.00164 ( 0.000700
0.465 ( 0.0597
0.00260 ( 0.0000403
0.681 ( 0.177
0.00103
0.00356 ( 0.000267
1.55 ( 1.30
0.00277 ( 0.000884
2.13 ( 1.37
8b (E)
4a (Z)
0.0334 ( 0.00206
0.336 ( 0.0856
0.0133
0.0155 ( 0.00836
0.520 ( 0.302
0.00287
>40
nde
>40g
4b (E)
CA1P (Z)f
a Average of n g 3 independent determinations. b See ref 7 for additional data. This batch of CA1 was synthesized by the current authors using the
method found in ref 1. c See ref 40. d See ref 41. e nd = not determined. f See ref 42. g See ref 43 for additional data.
s, OCH3-4), 3.75 (3H, s, OCH3-40), 3.68 (6H, s, OCH3-3, -5), 2.33 (3H, s,
CH3-400); 13C NMR (CDCl3, 125 MHz) δ 152.8 (C, C-3, C-5), 151.5 (C,
C-40), 145.2 (C, C-400), 140.4 (C, C-20), 137.2 (C, C-4), 135.9 (C, Ph),
134.2 (C, C-30), 133.7 (C, C-100), 132.0 (C, C-1), 131.5 (CH, C-1a), 129.6
(CH, C-300, C-500), 128.5 (CH, C-200, C-600), 128.4 (4CH, Ph), 128.3 (2CH,
Ph), 127.7 (4CH, Ph), 127.0 (CH, C-60), 126.0 (C, C-10), 123.9 (CH,
C-10a), 110.8 (CH, C-50), 106.1 (CH, C-2, C-6), 69.7 (2CH2, OCH2Ph),
60.9 (CH3, OCH3-4), 56.5 (CH3, OCH3-40), 56.0 (CH3, OCH3-3, -5), 21.6
(CH3, CH3-400); 31P NMR (CDCl3, 202 MHz) δ ꢀ6.16; HRESIMS m/z
746.2017 [M + 1]+ (calcd for C39H40O11PS+, 746.2024).
(Z)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[(para-toluene-
sulfonyl)oxy]-300-[(disodium)phosphate]-400-methoxyphe-
nyl]ethene (3a). Dibenzylphosphate 2a (0.31 g, 0.41 mmol) was
dissolved in acetonitrile (25 mL) cooled to ꢀ10 ꢀC. Freshly distilled
TMSBr (0.27 mL, 2.1 mmol) was added, and the reaction mixture was
stirred for 3 h at ꢀ10 ꢀC. Next the reaction mixture was added dropwise
to a suspension of NaOMe (0.111 g, 2.06 mmol) in MeOH (10 mL)
cooled to ꢀ10 ꢀC. The reaction mixture was stirred for 3 h and then
allowed to slowly return to ambient temperature. On completion, MeOH
was removed in vacuo. Flash chromatographic separation of the crude
product using a prepacked 30 g RP-18 silica column [eluents, solvent A,
H2O, solvent B, acetonitrile; gradient, 100% A/0% B over 0 to 1.19 min
(1 CV), 100% A/0% B f 60% A/40% B over 18.28 min (14 CV), 0% A/
100% B over 3.57 min (3 CV); flow rate 25.0 mL/min; monitored at 254
and 280 nm] afforded sodium phosphate 3a (0.13 g, 52%) as an off-white
solid: 1H NMR (CD3OD, 500 MHz) δ 7.93 (2H, d, J = 8.1 Hz, H-200,
H-600), 7.28 (2H, d, J = 8.1 Hz, H-300, H-500), 6.78 (1H, d, J = 8.6 Hz, H-60),
6.75 (1H, dd, J = 8.6 Hz, H-50), 6.53 (2H, s, H-2, H-6), 6.04 (1H, d, J =
12.2 Hz, H-10a), 6.01 (1H, d, J = 12.2 Hz, H-1a), 3.86 (3H, s, OCH3-40),
3.74 (3H, s, OCH3-4), 3.64 (6H, s, OCH3-3, -5), 2.38 (3H, s, CH3-400);
13C NMR (CD3OD, 125 MHz) δ 154.9 (C, C-40), 154.1 (C, C-3, C-5),
146.8 (C, C-400), 143.4 (C, C-20), 140.5 (C, C-30), 138.4 (C, C-4), 135.7
(C, C-100), 133.9 (C, C-1), 131.5 (CH, C-1a), 130.8 (CH, C-300, C-500),
129.8 (CH, C-200, C-600), 126.1 (CH, C-10a), 124.8 (CH, C-60), 124.5 (C,
C-10), 112.2 (CH, C-50), 107.9 (CH, C-2, C-6), 61.3 (CH3, OCH3-4),
57.2 (CH3, OCH3-40), 56.6 (CH3, OCH3-3, -5), 21.8 (CH3, CH3-400); 31P
1570
dx.doi.org/10.1021/np200104t |J. Nat. Prod. 2011, 74, 1568–1574

Journal of Natural Products
ARTICLE
NMR (CD3OD, 122 MHz) δ 2.73; HRESIMS m/z 611.0723 [M + 1]+
(calcd for C25H26Na2O11PS+, 611.0723).
that point the mixture was added to a suspension of NaOMe (0.45 g, 8.3
mmol) in MeOH (25 mL) cooled to ꢀ10 ꢀC. The mixture was then
stirred for 2 h and allowed to slowly return to ambient temperature. On
completion, MeOH was evaporated at 50 ꢀC in vacuo, and flash
chromatographic separation of the crude product using a prepacked
12 g RP-18 silica column [eluents, solvent A, H2O, solvent B, acetonitrile;
gradient, 100% A/0% B over 1.15 min (1 CV), 100% A/0% B f 45% A/
55% B over 17.30 min (14 CV), 0% A/100% B over 3.45 min (3 CV);
flow rate 12.0 mL/min; monitored at 254 and 280 nm] led to sodium
phosphate 7a (0.43 g, 0.71 mmol, 88%): 1H NMR (D2O, 500 MHz) δ
7.71 (2H, d, J = 8.4 Hz, H-200, -600), 7.39 (2H, d, J = 8.1 Hz, H-300, -500),
7.01 (1H, d, J = 8.7 Hz, H-60), 7.00 (1H, d, J = 12.1 Hz, H-10a), 6.60 (1H,
d, J = 12.1 Hz, H-1a), 6.59 (1H, d, J = 8.8 Hz, H-2, -6), 6.49 (2H, s, H-50),
3.73 (3H, s, OCH3-4), 3.70 (6H, s, OCH3-3, -5), 3.29 (3H, s, OCH3-40),
2.41 (3H, s, CH3-400); 13C NMR (D2O, 125 MHz) δ 151.9 (C, C-3,
C-5), 151.3 (C, C-40), 146.6 (C, C-20), 146.5 (C, C-400), 135.6 (C, C-4),
133.7 (C, C-1), 131.8 (C, C-100), 131.5 (C, C-30), 129.5 (CH, C-1a),
129.5 (CH, C-300, C-500), 128.8 (CH, C-60), 128.2 (CH, C-200, C-600),
127.2 (CH, C-10a), 125.3 (C, C-10), 106.9 (CH, C-50), 106.6 (CH, C-2,
C-6), 60.8 (CH3, OCH3-4), 55.8 (CH3, OCH3-3, -5), 55.5 (CH3,
OCH3-40), 20.7 (CH3, CH3-400); 31P NMR (D2O, 202 MHz) δ
ꢀ3.19; HRESIMS m/z 611.0716 [M + 1]+ (calcd for C25H26-
Na2O11PS+, 611.0723).
(Z)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[hydroxy]-300-[(di-
sodium)phosphate]-400-methoxyphenyl]ethene (4a). A solu-
tion of sulfonate ester 3a (0.089 g, 0.146 mmol) and NaOH (3 mL, 2M)
inMeOH(3 mL) ina 5 mLmicrowavesafe sealedvialwas heatedto50ꢀC
for 30 min. Reversed-phase TLC (30:70 acetonitrileꢀH2O) was used to
monitor the reaction. On completion, aqueous solvents were evaporated
under reduced pressure. The crude product was subjected to flash
chromatography using a prepacked 30 g RP-18 silica column [eluents,
solvent A, H2O, solvent B, acetonitrile; gradient, 100% A/0% B over 1.19
min (1 CV), 100% A/0% B f 60% A/40% B over 13.12 min (10 CV), 0%
A/100% B over 3.57 min (2 CV); flow rate 25.0 mL/min; monitored at
254 and 280 nm], affording sodium phosphate 4a (0.063 g, 0.14 mmol,
95%) as an off-white solid: 1H NMR (D2O, 500 MHz) δ 6.82 (1H, d, J =
8.6 Hz, H-60), 6.69 (1H, d, J = 12 Hz, H-10a), 6.65 (2H, s, H-2, H-6), 6.60
(1H, d, J = 12 Hz, H-1a), 6.53 (1H, d, J = 8.6 Hz, H-50), 3.83 (3H, s,
OCH3-40), 3.75 (3H, s, OCH3-4), 3.69 (6H, s, OCH3-3, -5); 13C NMR
(D2O, 125 MHz) δ 152.0 (C, C-3, C-5), 151.6 (C, C-40), 147.3 (C, C-20),
135.6 (C, C-4), 133.7 (C, C-1), 131.3 (C, C-30), 129.5 (CH, C-1a), 126.3
(CH, C-10a), 123.8 (CH, C-60), 119.7 (C, C-10), 106.4 (CH, C-2, C-6),
104.4 (CH, C-50), 60.8 (CH3, OCH3-4), 56.0 (CH3, OCH3-40), 55.8
(CH3, OCH3-3, -5); 31P NMR (D2O, 122 MHz) δ 3.68; HRESIMS m/z
457.0633 [M + H]+ (calcd for C18H20Na2O9P+, 457.0635).
(Z)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[(disodium)phos-
phate]-300-[hydroxy]-400-methoxyphenyl]ethene (8a). Sulfo-
nate ester 7a (0.107 g, 0.175 mmol) was dissolved in MeOH (3 mL)
in a 5 mL microwave safe vial with a stir bar. To this solution NaOH
(2 mL, 2 M) was added, the vial was capped, and the reaction mixture
was prestirred for 5 min. The reaction mixture was heated at 50 ꢀC in a
microwave reactor for 30 min. Temperatures higher than 50 ꢀC may lead
to isomerization of the compound. On completion, the solvents were
evaporated in vacuo and the crude product was subjected to flash
chromatographic separation using a prepacked 30 g RP-18 silica column
[eluents, solvent A, H2O, solvent B, acetonitrile; gradient, 100% A/0% B
over 0 to 1.19 min (1 CV), 100% A/0% B f 60% A/40% B over 13.12
min (10 CV), 60% A/40% B over 3.57 min (3 CV); flow rate 25.0 mL/
min; monitored at 254 and 280 nm], affording 8a (0.046 g, 0.100 mmol,
58%): 1H NMR (500 MHz, D2O) δ 6.82 (1H, d, J = 12 Hz, H-10a), 6.67
(1H, d, J = 8.7 Hz, H-60), 6.66 (2H, s, H-2, -6), 6.64 (1H, d, J = 8.7 Hz,
H-50), 6.62 (1H, d, J = 12 Hz, H-1a), 3.83 (3H, s, OCH3-40), 3.76 (3H, s,
OCH3-4), 3.69 (6H, s, OCH3-3, -5); 13C NMR (125 MHz, D2O)
δ 151.9 (C, C-3, C-5), 148.6 (C, C-40), 140.4 (C, C-20), 138.5 (C, C-30),
135.5 (C, C-4), 133.9 (C, C-1), 129.1 (CH, C-1a), 127.2 (CH, C-10a),
124.2 (CH, C-10), 120.2 (C, C-60), 107.3 (CH, C-50), 106.5 (CH, C-2,
C-6), 60.9 (CH3, OCH3-4),56.0(CH3, OCH3-40), 55.8(CH3, OCH3-3, -5);
31P NMR (D2O, 202 MHz) δ 3.21. HRESIMS m/z 457.0632 [M + H]+
(calcd for C18H20Na2O9P+, 457.0635).
(E)-1-[30,40,50-Trimethoxy]-2-[200-[(benzyl)oxy]]phosphoryl)-
oxy]-300-[(para-toluenesulfonyl)oxy]-400-methoxyphenyl]ethene
(6b). Phenol 5b37 (0.47 g, 0.96 mmol) was dissolved in acetonitrile (10 mL)
cooled to ꢀ10 ꢀC, CCl4 (1 mL, 10 mmol) was added, and the reaction
mixture was stirred for 5 min. Diisopropylethylamine (0.3 mL, 1.7 mmol)
and DMAP (0.1 g, 0.8 mmol) were added, and the reaction mixture was
stirred for an additional 10 min. Dibenzylphosphite (0.25 mL, 1.1 mmol)
was added slowly to the mixture, and stirring continued for 45 min
(monitored by TLC). On completion, saturated KH2PO4 solution
(50 mL) was added, and the mixture was allowed to return to ambient
temperature. H2O (50 mL) was added, and the organic phase was
separated. The aqueous phase was extracted with EtOAc (3 ꢁ 10 mL),
and the combinedorganicphase was dried overNa2SO4. The solution was
filtered and concentrated under reduced pressure. Flash chromatographic
separation of the crude product was performed using a prepacked 100 g
silica column [eluents; solvent A, EtOAc, solvent B, hexanes; gradient,
40%A/60%B over1.15min(1 CV), 40% A/60%Bf 54% A/46%B over
(Z)-1-[30,40,50-Trimethoxy]-2-[200-[(benzyl)oxy]]phosphoryl)-
oxy]-300-[(para-toluenesulfonyl)oxy]-400-methoxyphenyl]ethene
(6a). Phenol 5a37 (0.77 g, 1.8 mmol) was dissolved in acetonitrile (15 mL)
cooled to ꢀ10 ꢀC. CCl4 (2.00 mL, 20.7 mmol) was added, and the reaction
mixture was stirred for 5 min. Diisopropylethylamine (0.7 mL, 4 mmol) and
DMAP (0.151 g, 1.23 mmol) were added, and the reaction mixture was
stirred for an additional 10 min. Dibenzylphosphite (0.50 mL, 2.3 mmol) was
added slowly to the reaction mixture, which was then stirred for 1 h and
monitored by TLC. On completion, saturated KH2PO4 solution (25 mL)
was added, and the reaction mixture was allowed to return to ambient
temperature. H2O (25 mL) was added to the reaction mixture, and the
organic phase was separated. The aqueous phase was extracted with EtOAc
(2 ꢁ 20 mL), and the combined organic phases were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude product was
subjected to flash chromatography using a prepacked 100 g silica column
[eluents, solvent A, EtOAc, solvent B, hexanes; gradient, 30% A/70% B over
3.18 min (1 CV), 30% A/70% B f 80% A/20% B over 33.00 min (10 CV),
80% A/20% B over 6.36 min (2 CV); flow rate 40.0 mL/min; monitored at
254 and 280 nm], affording 6a (0.91 g, 1.2 mmol, 78%) as a yellow oil: 1H
NMR (CDCl3, 500 MHz) δ7.75 (2H, d, J=8.4Hz, H-200, H-600), 7.35ꢀ7.25
(10H, m, Ph), 7.21 (2H, d, J = 8.4 Hz, H-300, H-500), 7.04 (1H, d, J = 8.8 Hz,
H-60), 6.63 (1H, d, J = 11.9 Hz, H-10a), 6.62 (1H, d, J = 8.8 Hz, H-50), 6.51
(1H, d, J = 11.9 Hz, H-1a), 6.42 (2H, s, H-2, H-6), 5.06 (4H, m, OCH2Ph),
3.82 (3H, s, OCH3-4), 3.64 (6H, s, OCH3-3, -5), 3.60(3H, s, OCH3-40), 2.36
(3H, s, CH3-400);13CNMR(CDCl3,125MHz) δ152.8 (C, C-3, C-5), 152.7
(C, C-40), 144.9 (C, C-400), 142.8 (C, C-20), 137.2 (C, C-4), 135.7 (2C, Ph-
C1), 134.3 (C, C-100), 132.1 (C, C-1a), 132.0 (C, C-1), 131.6 (C, C-30),
129.3 (CH, C-300, -500), 129.0 (CH, C-60), 128.5 (CH, C-200, C-600), 128.43
(4CH, Ph), 128.36 (2CH, Ph), 127.8 (4CH, Ph), 124.7 (C, C-10), 124.3
(CH, C-10a), 109.1 (CH, C-50), 106.2 (CH, C-2, C-6), 69.9 (2CH2,
OCH2Ph), 60.9 (CH3, OCH3-4), 56.1 (CH3, OCH3-40), 55.9 (CH3,
OCH3-3, -5), 21.6 (CH3, CH3-400); 31P NMR (CDCl3, 202 MHz) δ
ꢀ6.16; HRESIMS m/z 747.2024 [M]+ (calcd for C39H40O11PS+,
747.2023); anal. C 62.92, H 5.29%, calcd for C39H39O11PS, C 62.73,
H 5.26%.
(Z)-1-[30,40,50-Trimethoxy]-2-[200-[(disodium)phosphate]-
300-[(para-toluenesulfonyl)oxy]-400-methoxyphenyl]ethene
(7a). Compound 6a (0.60 g, 0.80 mmol) was dissolved in acetonitrile
(12 mL) cooled to ꢀ10 ꢀC. Freshly distilled TMSBr (0.3 mL, 2.3 mmol)
was added dropwise, and the mixture was stirred for 1 h at ꢀ10 ꢀC. At
1571
dx.doi.org/10.1021/np200104t |J. Nat. Prod. 2011, 74, 1568–1574

Journal of Natural Products
ARTICLE
3.22 min (2.7 CV), 54% A/46% B f100% A/0% B over 11.15 min
(9 CV), 100% A/0% B over 3.07 min (2.5 CV); flow rate 12.0 mL/min;
monitored at 254 and 280 nm] and yielded a pure yellow oil, 6b (0.55 g,
0.74 mmol, 77%): 1H NMR (CDCl3, 500 MHz) δ 7.84 (2H, d, J = 8.4 Hz,
H-200, H-600), 7.50 (1H, d, J = 9.0 Hz, H-60), 7.34 (1H, d, J = 16.3 Hz,
H-10a), 7.28 (2H, d, J = 8.4 Hz, H-300, H-500), 7.26ꢀ7.22 (10H, m, Ph),
6.88 (1H, d, J = 16.3 Hz, H-1a), 6.76 (1H, dd, J = 9.0, 0.8 Hz, H-50), 6.67
(2H, s, H-2, H-6), 5.08 (4H, m, OCH2Ph), 3.86 (3H, s, OCH3-4), 3.76
(6H, s, OCH3-3, -5), 3.58 (3H, s, OCH3-40), 2.41 (3H, s, CH3-400); 13C
NMR (CDCl3, 125 MHz) δ 153.3 (C, C-3, C-5), 152.6 (C, C-40), 144.9
(C,0C0 -400), 142.4 (C, C-20), 137.9 (C, C-4), 135.5 (2C, Ph), 134.2 (C,
C-1 ), 133.0 (C, C-1), 131.5 (C, C-30), 129.5 (CH, C-1a), 129.3 (CH,
C-300, C-500), 128.6 (CH, C-200, C-600), 128.42 (4CH, Ph), 128.40 (2CH,
Ph), 127.9 (4CH, Ph), 124.5 (C, C-10), 124.2 (CH, C-60), 121.5 (CH,
C-10a), 109.6 (CH, C-50), 103.6 (CH, C-2, C-6), 70.0 (2CH2, OCH2Ph),
60.9 (CH3, OCH3-4), 56.0 (CH3, OCH3-3, -5), 55.9 (CH3, OCH3-40),
21.6 (CH3, CH3-400); 31P NMR (CDCl3, 202 MHz) δ ꢀ5.84; HRESIMS
m/z 747.2019 [M]+ (calcd for C39H40O11PS+, 747. 2023); anal. C 62.46,
H 5.23%, calcd for C39H39O11PS, C 62.73, H 5.26%.
δ7.46 (1H, d, J= 16.55 Hz, H-10a), 7.25 (1H, d, J=8.75Hz, H-60), 7.07 (1H,
d, J=16.55Hz,H-1a),7.03(2H,s,H-2,H-6),6.86(1H,d,J=8.75Hz, H-50),
3.94 (6H, s, OCH3-3, -5), 3.89(3H, s, OCH3-40), 3.81 (3H, s, OCH3-4); 13C
NMR (D2O, 125 MHz) δ 152.6 (C, C-3, C-5), 148.9 (C, C-40), 140.2 (C,
C-20), 138.3 (C, C-30), 136.1 (C, C-4), 134.6 (C, C-1), 127.5 (CH, C-1a),
124.2 (CH, C-10), 123.9 (C, C-10a), 116.5 (CH, C-60), 107.9 (CH, C-50),
104.1 (CH, C-2, C-6), 60.9 (CH3, OCH3-4), 56.1 (CH3, OCH3-3, -5), 56.0
(CH3, OCH3-40); 31P NMR (D2O, 202 MHz) δ 3.54; HRESIMS m/z
457.0632 [M + H]+ (calcd for C18H20Na2O9P+, 457.0635).
(E)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[(para-toluene-
sulfonyl)oxy]-300-[([bis[(benzyl)oxy]]phosphoryl)oxy]-400-
methoxyphenyl]ethene (2b). To a solution of phenol 1b37 (0.499 g,
1.03 mmol) in acetonitrile (10 mL) cooled to ꢀ10 ꢀC was added CCl4
(0.20 mL, 2.07 mmol), and the reaction mixture was stirred for 5 min.
Diisopropylethylamine (0.7 mL, 4.0 mmol) and DMAP (0.06 g, 0.49
mmol) were added, and the mixture was stirred for an additional 10 min.
Next, dibenzylphosphite (0.5 mL, 2.26 mmol) was slowly added to the
mixture. After stirring for 45 min (monitored by TLC), KH2PO4 (10 mL)
was added, and the mixture was allowed to return to ambient temperature.
H2O (20 mL) was then added, and the organic phase was separated. The
aqueous phase was extracted with EtOAc (3 ꢁ 10 mL), the combined
organic phase was dried (Na2SO4), and the solution was filtered and
concentrated in vacuo. The crude product was separated by flash
chromatography using a prepacked 25 g silica column [eluents; solvent
A, EtOAc, solvent B, hexanes; gradient, 20% A/80% B over 1.19 min
(1 CV), 20% A/80% B f 70% A/30% B over 13.51 min (10.5 CV), 70%
A/30% B over 1.27 min (1.1 CV), 100% A/0% B over 3.57 min (3 CV);
flow rate 25.0mL/min; monitored at254 and 280 nm] to yield 2b(0.46 g,
0.61 mmol, 60%) as a yellow oil: 1H NMR (CDCl3, 500 MHz) δ 7.73
(2H, d, J = 8.1 Hz, H-200, H-600), 7.40 (1H, d, J = 9.0 Hz, H-60), 7.38ꢀ7.32
(10H, m, Ph), 7.06(2H, d, J =8.1 Hz, H-300, H-500), 6.90(1H, d, J = 9.0Hz,
H-50), 6.80 (1H, d, J = 16.2 Hz, H-10a), 6.71 (1H, d, J = 16.2 Hz, H-1a),
6.53 (2H, s, H-2, H-6), 5.21 (4H, m, OCH2Ph), 3.89 (6H, s, OCH3-3, -5),
3.88 (3H, s, OCH3-4), 3.80 (3H, s, OCH3-40), 2.16 (3H, s, CH3-400); 13C
NMR (CDCl3, 125 MHz): δ 153.2 (C, C-3, C-5), 151.7 (C, C-40), 145.6
(C, C-400), 140.1 (C, C-20), 137.9 (C, C-4), 136.0 (C, Ph), 134.5 (C, C-30),
133.2 (C, C-100), 132.8 (C, C-1), 129.6 (CH, C-300, C-500), 129.0 (CH,
C-1a), 128.6 (CH, C-200, C-600), 128.4(4CH, Ph), 128.3(2CH, Ph), 127.8
(4CH, Ph), 125.6 (C, C-10), 122.1 (CH, C-60), 121.4 (CH, C-10a), 111.5
(CH, C-50), 103.7 (CH, C-2, C-6), 69.8 (2CH2, OCH2Ph), 61.0 (CH3,
OCH3-4), 56.4 (CH3, OCH3-40), 56.1 (CH3, OCH3-3, -5), 21.5 (CH3,
CH3-400); 31P NMR (CDCl3, 202 MHz) δ ꢀ6.23; HRESIMS m/z
747.2020 [M + 1]+ (calcd for C39H40O11PS+, 747.2023); anal. C
62.71, H 5.31%, calcd for C39H39O11PS, C 62.73, H 5.26%.
(E)-1-[30,40,50-Trimethoxy]-2-[200-[(disodium)phosphate]-
300-[(para-toluenesulfonyl)oxy]-400-methoxyphenyl]ethene
(7b). Dibenzylphosphate 6b (0.32 g, 0.43 mmol) was dissolved in
acetonitrile (5 mL) cooled to ꢀ10 ꢀC. Freshly distilled TMSBr
(0.25 mL, 1.9 mmol) was added, and the reaction mixture was stirred
for 3 h at ꢀ10 ꢀC. The initial mixture was added dropwise to a
suspension of NaOMe (0.22 g, 4.1 mmol) in MeOH (15 mL) cooled to
ꢀ10 ꢀC. The reaction was stirred for 3 h and allowed to return to
ambient temperature. On completion, solvents were evaporated in
vacuo (<50 ꢀC to prevent isomerization), and the crude product was
subjected to flash chromatographic separation using a prepacked 30 g
RP-18 silica column [eluents, solvent A, H2O, solvent B, acetonitrile;
gradient, 100% A/0% B over 1.19 min (1 CV), 100% A/0% B f 61%
A/39% B over 12.52 min (9.7 CV), 61% A/39% B f 45% A/55% B
over 5.16 min (4 CV), 0% A/100% B over 3.57 min (3 CV); flow rate
25.0 mL/min; monitored at 254 and 280 nm], affording 7b (0.16 g, 2.6
mmol, 61%) as an off-white solid: 1H NMR (D2O, 500 MHz) δ 7.59
(2H, d, J = 8.4 Hz, H-200, H-600), 7.36 (1H, d, J = 16.3 Hz, H-10a), 7.28
(1H, d, J = 9.5 Hz, H-60), 7.22 (2H, d, J = 8.4 Hz, H-300, H-500), 6.69
(2H, s, H-2, H-6), 6.63 (1H, d, J = 16.5 Hz, H-1a), 6.58 (1H, dd, J = 8.5
Hz, H-50), 3.82 (6H, s, OCH3-3, -5), 3.70 (3H, s, OCH3-4), 3.33 (3H, s,
OCH3-40), 2.28 (3H, s, CH3-400); 13C NMR (D2O, 125 MHz) δ 152.3
(C, C-3, C-5), 151.6 (C, C-40), 146.4 (C, C-400), 144.5 (C, C-20), 136.1
(C, C-4), 134.0 (C, C-1), 131.6 (C, C-100), 131.0 (C, C-30), 129.5 (CH,
C-300, C-500), 128.3 (CH, C-200, C-600), 128.0 (CH, C-1a), 124.5 (C,
C-10), 124.3 (CH, C-60), 122.2 (CH, C-10a), 108.7 (CH, C-50), 103.7
(CH, C-2, C-6), 60.8 (CH3, OCH3-4), 55.9 (CH3, OCH3-3, -5), 55.6
(CH3, OCH3-40), 20.8 (CH3, CH3-400); 31P NMR (CDCl3, 202 MHz)
δ ꢀ4.25; HRESIMS m/z 611.0716 [M + H]+ (calcd for C25H26Na2-
O11PS+, 611.0723); anal. C 48.51, H 4.62%, calcd for C25H25Na2-
(E)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[(para-toluene-
sulfonyl)oxy]-300-[(disodium)phosphate]-400-methoxyphe-
nyl]ethene (3b). To a solution of dibenzylphosphate 2b (0.16 g,
0.21 mmol) in acetonitrile (12 mL cooled to ꢀ10 ꢀC) was added
freshly distilled TMSBr (0.15 mL, 1.14 mmol), and the mixture was
stirred for 3 h at ꢀ10 ꢀC. The solution was added dropwise to a
suspension of NaOMe (0.15 g, 2.78 mmol) in MeOH (10 mL) cooled
to ꢀ10 ꢀC. The mixture was stirred for 3 h and allowed to slowly return
to ambient temperature, and the solvent was evaporated in vacuo. The
crude product was separated by flash chromatography using a pre-
packed 30 g RP-18 silica column [eluents, solvent A, H2O, solvent B,
acetonitrile; gradient, 100% A/0% B over 1.19 min (1 CV), 100% A/
0% B f 45% A/55% B over 18.28 min (14 CV), 0% A/100% B over
3.57 min (3 CV); flow rate 25.0 mL/min; monitored at 254
and 280 nm] to afford 3b (0.042 g, 32%): 1H NMR (D2O, 500 MHz)
δ 7.52 (2H, d, J = 8.0 Hz, H-200, H-600), 7.08 (1H, d, J = 9.0 Hz, H-60),
6.91 (2H, d, J = 8.0 Hz, H-300, H-500), 6.84 (1H, d, J = 9.0 Hz, H-50), 6.42
(1H, d, J = 16.5 Hz, H-1a), 6.36 (1H, d, J = 16.5 Hz, H-10a), 6.34 (2H, s,
H-2, H-6), 3.82 (3H, s, OCH3-40), 3.73 (6H, s, OCH3-3, -5), 3.71 (3H,
s, OCH3-4), 1.90 (3H, s, CH3-400); 13C NMR (D2O, 125 MHz)
O11PS 0.5H2O, C 48.47, H 4.23%.
3
(E)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[(disodium)phos-
phate]-300-[hydroxy]-400-methoxyphenyl]ethene (8b). Sulfonate
ester 7b (0.100 g, 0.164 mmol) was dissolved in MeOH (17 mL) in a
20mLmicrowavesafevial. TothissolutionwasaddedNaOH (3mL, 2M),
the vial was capped, and the reaction was prestirred for 2 min. The reaction
mixture was heated at 50 ꢀC in a microwave reactor for 30 min. As noted
above, temperatures higher than 50 ꢀC can lead to isomerization. On
completion, the solvents were evaporated in vacuo, and the crude product
was subjected to flash chromatographic separation using a prepacked 30 g
RP-18 silica column [eluents, solvent A, H2O, solvent B, acetonitrile;
gradient, 100% A/0% B over 1.19 min (1 CV), 100% A/0% B f 60% A/
40% B over 13.12 min (10 CV), 60% A/40% B over 3.57 min (3 CV); flow
rate 25.0 mL/min; monitored at 254 and 280 nm], providing sodium
phosphate 8b (0.063 g, 0.138 mmol, 84%): 1H NMR (D2O, 500 MHz)
1572
dx.doi.org/10.1021/np200104t |J. Nat. Prod. 2011, 74, 1568–1574

Journal of Natural Products
ARTICLE
δ 152.3 (C, C-40), 152.1 (C, C-3, C-5), 146.5 (C, C-400), 140.2 (C,
C-20), 136.1 (C, C-4), 135.7 (C, C-30), 133.5 (C, C-1), 131.8 (C,
C-100), 129.8 (CH, C-200, C-600), 128.3 (CH, C-1a), 127.9 (CH, C-300,
C-500), 124.2 (C, C-10), 121.4 (CH, C-60), 121.2 (CH, C-10a), 111.8
(CH, C-50), 103.7 (CH, C-2, C-6), 60.7 (CH3, OCH3-4), 56.1 (CH3,
OCH3-40), 55.8 (CH3, OCH3-3, -5), 20.5 (CH3, CH3-400); 31P NMR
(D2O, 202 MHz) δ ꢀ3.99; HRESIMS m/z 611.0713 [M + H]+ (calcd
for C25H26Na2O11PS+, 611.0723).
addition, portions of this project were partially supported by
Award Number 5R01CA140674 (to K.G.P. and M.L.T.) from
the National Cancer Institute. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Cancer Institute or the
National Institutes of Health. The authors also thank Dr. A.
Ramirez (Mass Spectrometry Core Facility, Baylor University)
for mass spectroscopic analysis, Dr. C. Moehnke for assistance
with NMR studies, and Dr. J. Karban and Dr. M. Nemec
(Director) for use of the shared Molecular Biosciences Center
at Baylor University.
(E)-1-[30,40,50-Trimethoxyphenyl]-2-[200-[hydroxy]-300-[(di-
sodium)phosphate]-400-methoxyphenyl]ethene (4b). A solution
of NaOH (3 mL, 2 M) was added to sulfonate ester 3b (0.050 g, 0.082
mmol) in MeOH (17 mL, in a 20 mL microwave safe vial with a stir bar).
The vial was capped and placed in the microwave. The solution was
prestirred for 2 min and then heated at 50 ꢀC in a microwave reactor for
30 min. (Caution: temperatures higher than 50 ꢀC may lead to iso-
merization.) Reversed-phase TLC (30:70 acetonitrileꢀH2O) was used to
monitor the reaction course. After the reaction was complete, the solvent
was evaporated in vacuo. The crude product was subjected to flash
chromatographic separation using a prepacked 30 g RP-18 silica column
[eluents, solvent A, H2O, solvent B, acetonitrile; gradient, 100% A/0% B
over 1.19 min (1 CV), 100% A/0% B f 60% A/40% B over 13.12 min
(10 CV), 0% A/100% B over 3.57 min (3 CV); flow rate 25.0 mL/min;
monitored at 254 and 280 nm] to yield 4b (0.035 g, 0.077 mmol, 94%): 1H
NMR (D2O, 500 MHz) δ7.36 (1H, d, J= 16.35 Hz, H-10a), 7.29 (1H, d, J=
8.75 Hz, H-60), 6.98 (1H, d, J = 16.35 Hz, H-1a), 6.86 (2H, s, H-2, H-6),
6.67 (1H, d, J = 8.65 Hz, H-50), 3.88 (3H, s, OCH3-40), 3.86 (6H, s, OCH3-
3, -5), 3.77 (3H, s, OCH3-4); 13C NMR (D2O, 125 MHz) δ 152.4 (C, C-3,
C-5), 151.9 (C, C-40), 147.6 (C, C-20), 135.8 (C, C-4), 134.7 (C, C-1),
131.3 (C, C-30), 126.5 (C, C-1a), 123.3 (CH, C-10a), 120.6 (CH, C-60),
119.6 (CH, C-10), 104.7 (CH, C-50), 103.4 (CH, C-2, C-6), 60.8 (CH3,
OCH3-4), 55.9 (CH3, OCH3-40), 55.8 (CH3, OCH3-3, -5); 31P NMR
(D2O, 202 MHz) δ ꢀ3.74; HRESIMS m/z 457.0631 [M + 1]+ (calcd for
C18H20Na2O9P+, 457.0635).
’ REFERENCES
(1) Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.; Schmidt, J. M.
J. Nat. Prod. 1987, 50, 119–131.
(2) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Experientia 1989, 45, 209–211.
(3) Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.; Schmidt,
J. M.; Pettit, G. R.; Hamel, E. Mol. Pharmacol. 1988, 34, 200–208.
(4) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989,
28, 6984–6991.
(5) NCI, Cancer Screening Data, DTP, 2010.
(6) Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.; Boyd,
M. R.; Hamel, E.; Pettit, R. K. J. Med. Chem. 1998, 41, 1688–1695.
(7) Pettit, G. R.; Grealish, M. P.; Herald, D. L.; Boyd, M. R.; Hamel,
E.; Pettit, R. K. J. Med. Chem. 2000, 43, 2731–2737.
(8) Pettit, G. R.; Lippert, J. W., III. Anti-Cancer Drug Des. 2000, 15,
203–216.
(9) Pettit, G. R.; Rhodes, M. R. Anti-Cancer Drug Des. 1998, 13,
183–191.
(10) Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma, R.;
Simpson, M. J.; Boyd, M. R.; Rener, G. A.; Bansal, N. Anti-Cancer Drug
Des. 1995, 10, 299–309.
(11) Lippert, J. W., III. Bioorg. Med. Chem. 2007, 15, 605–615.
(12) Patterson, D. M.; Rustin, G. J. Clin. Oncol. (R. Coll. Radiol.)
2007, 19, 443–456.
’ ASSOCIATED CONTENT
(13) Patterson, D. M.; Ross, P.; Koetz, B.; Saleem, A.; Stratford, M.;
Stirling, J.; Padhani, A.; Asselin, M.; Price, P.; Rustin, G. J. J. Clin. Oncol.
(Meet. Abstr.) 2007, 25, 14146.
(14) Siemann, D. W.; Chaplin, D. J.; Walicke, P. A. Expert Opin.
Investig. Drugs 2009, 18, 189–197.
S
Supporting Information. General experimental details
b
regarding tubulin and cytotoxicity assays, H NMR, 13C NMR,
gHSQC, gHMBC, HRESIMS, and HPLC. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
1
(15) Chaplin, D. J.; Pettit, G. R.; Hill, S. A. Anticancer Res. 1999,
19, 189–195.
’ AUTHOR INFORMATION
(16) Mooney, C. J.; Nagaiah, G.; Fu, P.; Wasman, J. K.; Cooney,
M. M.; Savvides, P. S.; Bokar, J. A.; Dowlati, A.; Wang, D.; Agarwala,
S. S.; Flick, S. M.; Hartman, P. H.; Ortiz, J. D.; Lavertu, P. N.; Remick,
S. C. Thyroid 2009, 19, 233–240.
Corresponding Author
*Tel: +1 254 710 4117. Fax: +1 254 710 4272. E-mail:
Kevin_Pinney@baylor.edu.
(17) Akerley, W. L.; Schabel, M.; Morrell, G.; Horvath, E.; Yu, M.;
Johnsson, B.; Arbogast, K. J. Clin. Oncol. (Meet. Abstr.) 2007, 25, 14060.
(18) Nathan, P. D.; Judson, I.; Padhani, A.; Harris, A.; Carden, C. P.;
Smythe, J.; Collins, D.; Leach, M.; Walicke, P.; Rustin, G. J. J. Clin. Oncol.
(Meet. Abstr.) 2008, 26, 3550.
(19) www.Oxigene.com (accessed Aug 4, 2010).
(20) Dark, G. G.; Hill, S. A.; Prise, V. E.; Tozer, G. M.; Pettit, G. R.;
Chaplin, D. J. Cancer Res. 1997, 57, 1829–1834.
Present Addresses
^Albany Molecular Research Inc., Medicinal Chemistry, 26
Corporate Circle, PO Box 15098, Albany, New York 12212-
5098, United States
Author Contributions
rAuthors with equal contributions.
(21) Grosios, K.; Holwell, S. E.; McGown, A. T.; Pettit, G. R.; Bibby,
M. C. Br. J. Cancer 1999, 81, 1318–1327.
(22) Pinney, K. G. Molecular Recognition of the Colchicine Binding
Site as a Design Paradigm for the Discovery and Development of
Vascular Disrupting Agents; In Vascular-Targeted Therapies in Oncology;
John Wiley & Sons, Ltd: New York, 2006; pp 95ꢀ121.
(23) Pinney, K. G.; Jelinek, C.; Edvardsen, K.; Chaplin, D. J.; Pettit,
G. R. The Discovery and Development of the Combretastatins; Cragg,
G. R., Kingston, D. G. I., Newman, D. J., Eds.; In Anticancer Agents from
Natural Products; CRC Press/Taylor & Francis: Boca Raton, FL, 2005;
pp 23ꢀ46.
’ ACKNOWLEDGMENT
The authors are grateful to OXiGENE, Inc. (grants to K.G.P.
and M.L.T.), and the Welch Foundation (grant no. AA-1278 to
K.G.P.), and one of us (G.R.P.) is grateful for grants R01
CA90441-01 and SR01 CA090441-07-08 from The Division of
Cancer Treatment Diagnosis, NCI, DHHS, for their financial
support of this project, and to the NSF for funding the Varian
500 MHz NMR spectrometer (grant no. CHE-0420802). In
1573
dx.doi.org/10.1021/np200104t |J. Nat. Prod. 2011, 74, 1568–1574

Journal of Natural Products
ARTICLE
(24) Siemann, D. W.; Bibby, M. C.; Dark, G. G.; Dicker, A. P.;
Eskens, F. A.; Horsman, M. R.; Marme, D.; Lorusso, P. M. Clin. Cancer
Res. 2005, 11, 416–420.
(25) Vincent, L.; Kermani, P.; Young, L. M.; Cheng, J.; Zhang, F.;
Shido, K.; Lam, G.; Bompais-Vincent, H.; Zhu, Z.; Hicklin, D. J.; Bohlen,
P.; Chaplin, D. J.; May, C.; Rafii, S. J. Clin. Invest. 2005, 115, 2992–3006.
(26) Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253–265.
(27) Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat. Rev. Cancer
2005, 5, 423–435.
(28) Monk, K. A.; Siles, R.; Hadimani, M. B.; Mugabe, B. E.; Ackley,
J. F.; Studerus, S. W.; Edvardsen, K.; Trawick, M. L.; Garner, C. M.;
Rhodes, M. R.; Pettit, G. R.; Pinney, K. G. Bioorg. Med. Chem. 2006,
14, 3231–3244.
(29) Kanthou, C.; Tozer, G. M. Expert Opin. Ther. Targets 2007,
11, 1443–1457.
(30) Tozer, G. M.; Akerman, S.; Cross, N. A.; Barber, P. R.;
Bj€orndahl, M. A.; Greco, O.; Harris, S.; Hill, S. A.; Honess, D. J.; Ireson,
C. R.; Pettyjohn, K. L.; Prise, V. E.; ReyesAldasoro, C. C.; Ruhrberg, C.;
Shima, D. T.; Kanthou, C. Cancer Res. 2008, 68, 2301–2311.
(31) Hill, S. A.; Lonergan, S. J.; Denekamp, J.; Chaplin, D. J. Eur.
J. Cancer 1993, 29, 1320–1324.
(32) Hua, J.; Sheng, Y.; Pinney, K. G.; Garner, C. M.; Kane, R. R.;
Prezioso, J. A.; Pettit, G. R.; Chaplin, D. J.; Edvardsen, K. Anticancer Res.
2003, 23, 1433–1440.
(33) Folkes, L. K.; Christlieb, M.; Madej, E.; Stratford, M. R. L.;
Wardman, P. Chem. Res. Toxicol. 2007, 20, 1885–1894.
(34) Pettit, G. R.; Thornhill, A. J.; Moser, B. R.; Hogan, F. J. Nat.
Prod. 2008, 71, 1561–1563.
(35) Kirwan, I. G.; Loadman, P. M.; Swaine, D. J.; Anthoney, D. A.;
Pettit, G. R.; Lippert, J. W.; Shnyder, S. D.; Cooper, P. A.; Bibby, M. C.
Clin. Cancer Res. 2004, 10, 1446–1453.
(36) Stratford, M. R. L. J. Chromatogr. A 2008, 1181, 162–165.
(37) Tanpure, R. P.; Strecker, T. E.; Chaplin, D. J.; Siim, B. G.;
Trawick, M. L.; Pinney, K. G. J. Nat. Prod. 2010, 73, 1093–1101.
(38) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-
Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. J. Natl. Cancer Inst.
1991, 83, 757–766.
(39) Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112–1116.
(40) Monk, K. A.; Siles, R.; Hadimani, M. B.; Mugabe, B. E.; Ackley,
J. F.; Studerus, S. W.; Edvardsen, K.; Trawick, M. L.; Garner, C. M.;
Rhodes, M. R.; Pettit, G. R.; Pinney, K. G. Bioorg. Med. Chem. 2006, 14,
3231–3244.
(41) Batch prepared by Dr. Ming Zhou (Baylor University).
(42) Gift from OXiGENE, Inc.
(43) Chaplin, D. J.; Garner, C. M.; Kane, R. R.; Pinney, K. G.;
Prezioso, J. A.; Edvardsen, K. United States Patent 7384925, 2005.
1574
dx.doi.org/10.1021/np200104t |J. Nat. Prod. 2011, 74, 1568–1574