Regio-and stereospecific synthesis of mono-β-d-glucuronic acid derivatives of combretastatin A-1

Article abstract of DOI:10.1021/np100108e

Synthetic routes have been established for the preparation of regio-and stereoisomerically pure samples of the mono-β-d-glucuronic acid derivatives of combretastatin A-1, referred to as CA1G1 (5a) and CA1G2 (6a). Judicious choice of protecting groups for the catechol ring was required for the regiospecific introduction of the glucuronic acid moiety. The tosyl group proved advantageous in this regard. The two monoglucuronic acid analogues demonstrate low cytotoxicity (compared to CA1, 2) against selected human cancer cell lines, with CA1G1 being slightly more potent than CA1G2.

Full text of DOI:10.1021/np100108e

J. Nat. Prod. 2010, 73, 1093–1101
1093
Regio- and Stereospecific Synthesis of Mono-ꢀ-D-Glucuronic Acid Derivatives of Combretastatin
A-1
Rajendra P. Tanpure,† Tracy E. Strecker,† David J. Chaplin,‡ Bronwyn G. Siim,‡ Mary Lynn Trawick,† and Kevin G. Pinney*,†
Department of Chemistry and Biochemistry, Baylor UniVersity, One Bear Place #97348, Waco, Texas 76798-7348, and OXiGENE Inc.,
Magdalen Centre, Robert Robinson AVenue, The Oxford Science Park, Oxford, OX4 4GA, U.K.
ReceiVed February 16, 2010
Synthetic routes have been established for the preparation of regio- and stereoisomerically pure samples of the mono-
ꢀ-D-glucuronic acid derivatives of combretastatin A-1, referred to as CA1G1 (5a) and CA1G2 (6a). Judicious choice
of protecting groups for the catechol ring was required for the regiospecific introduction of the glucuronic acid moiety.
The tosyl group proved advantageous in this regard. The two monoglucuronic acid analogues demonstrate low cytotoxicity
(compared to CA1, 2) against selected human cancer cell lines, with CA1G1 being slightly more potent than CA1G2.
The members of the combretastatin family of natural products,1
isolated from the African bush willow tree, Combretum caffrum
Kuntze (Combretaceae), are exemplary in terms of both their
relative simplicity in chemical structure and their pronounced
biochemical and biological activity. Two Z-(cis)-stilbenoid com-
pounds from this family (Figure 1), combretastatin A-4 (CA4, 1)2,3
and combretastatin A-1 (CA1, 2),4 are potent inhibitors of tubulin
assembly (IC50 ) 1.0 and 1.1 µM, respectively)5 and are highly
cytotoxic toward selected human cancer cell lines (for example,
CA4, GI50 ) 0.001 µM, and CA1, GI50 ) 0.013 µM against DU-
145 prostate cancer cells).6 The corresponding phosphate prodrugs
(Figure 1), combretastatin A-4 phosphate (3; CA4P, Zybrestat,
fosbretabulin)7-10 and combretastatin A-1 phosphate (4; CA1P,
Oxi4503),11-13 are both in human clinical trials as vascular
disrupting agents (VDAs). VDAs represent a relatively new and
rapidly emerging field of anticancer therapy.14,15 These compounds
are characterized by their ability to interfere with the dynamics of
Figure 1. Combretastatin A-1 (CA1) and combretastatin A-4 (CA4)
derivatives.
the tubulin-microtubule protein system in the endothelial cells
lining the microvessels that feed tumors. This results in a cascade
of cell signaling events involving activation of RhoA and subse-
corresponding glucuronide derivative (CA4G), 7a (Figure 1), which
quently RhoA kinase, leading to morphological changes in the
is rapidly cleared from the human body.23 Enzymatic conjugation
endothelial cells and eventual microvessel occlusion, thus starving
the tumor of necessary nutrients and oxygen.16-18 In addition,
resulting in glucuronidation is a major mechanism to convert
exogenous compounds to less active and more water-soluble
disruption of vascular endothelial (VE) cadherins leads to further
microvessel permeability and damage.19
metabolites for excretion. Each of the CA1 monoglucuronides
(CA1G1, 5a,b; CA1G2, 6a,b) (Figure 1) has distinct chemical and
biological properties. Initial clinical data from an ongoing phase I
study indicates that CA1G1(5a) is structurally stable and has a long
CA4 (1) and CA1 (2) are almost identical structurally, with the
only difference being an additional hydroxy substituent at the C-2′
position of CA1. The enhanced effectiveness of CA1P (4) in
delaying growth of tumor xenografts in mice is thought to be related
to a combination of the vascular disrupting effect along with a
distinct secondary mechanism of biological activity involving
formation of the highly reactive ortho quinone analogue, obtainable
half-life in human plasma, while CA1G2 (6a) has a much shorter
plasma half-life and appears less stable owing to ortho-phenolic-
mediated isomerization from the Z- to the corresponding E-isomer.24
Accordingly, it became necessary to develop robust, regio- and
stereospecific synthetic routes for both of the CA1 monoglucu-
ronides (CA1G1, 5a; CA1G2, 6a) in order to have sufficient
quantities of these compounds available for further biological
through biological oxidation of the 1,2-diol functionality present
in CA1.20,21 Owing to the additional hydroxy group, the oxidation/
reduction properties of CA1 are different from those of CA4,
evaluation in support of the ongoing human clinical development
rendering it susceptible to oxidative metabolism in the liver by
of CA1P.
hepatic enzymes and to formation of free radical species, hence
The monoglucuronides of CA1 have been previously prepared,
implicating it in the formation of at least 13 additional metabolites,
in an unpublished study, through an enzymatic route.24 While this
in comparison to those formed from CA4.20 Some of these CA1P
enzymatic route yielded relatively pure samples of CA1G1 (5a)
and CA1G2 (6a), it did not allow unequivocal regioisomeric
assignment. In addition, the enzymatic route is cost prohibitive for
metabolites have been identified as monophosphates, monoglucu-
ronides, and bisglucuronides (8).22 Pharmacokinetic data have
indicated that CA4P (3) once converted to CA4 (1), most likely by
use as a scale-up procedure. To the best of our knowledge, the
nonspecific phosphatase enzymes, is further metabolized to its
regio- and stereospecific chemical synthesis of the monoglucuronic
acid derivatives of CA1 reported herein represents the first available
* To whom correspondence should be addressed. Tel: (254) 710-4117.
synthetic route to these important glucuronide analogues. This work
Fax: (254) 710-4272. E-mail: Kevin_Pinney@baylor.edu.
† Baylor University.
‡ OXiGENE Inc.
was based, in part, on the previous synthesis of CA4G (7a) by
Pettit and co-workers.25
10.1021/np100108e  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/24/2010

1094 Journal of Natural Products, 2010, Vol. 73, No. 6
Tanpure et al.
Scheme 1. Selective Protection of OH-2′ and OH-3′ of
Combretastatin A-1 (CA1)
column chromatography (Scheme 2). Subsequent sonication of
stilbenes 24-26 in the presence of KF and catalytic HBr success-
fully displaced the DPS or TBS group, while leaving intact the
sulfonate ester at the C-2′ position, producing phenols 28 and 29
(Scheme 2). Single-crystal X-ray diffraction provided structural
confirmation for compounds 22, 25a, and 27a.34 Treatment of
bromotriacetylglucuronate 31 with phenol 28 or 29 in the presence
of Cs2CO3 successfully displaced bromide at the anomeric carbon,
providing stilbene-triacetylglucuronates 32a and 33a (Scheme 2).
The coupling constant (32a J ) 7.6 Hz, 33a J ) 7.7 Hz) in the 1H
NMR spectrum for the hydrogen [32a (δH 4.86), 33a (δH 5.13)] on
the anomeric carbon confirmed that the bromide displacement
proceeded in the anticipated fashion to afford the requisite
ꢀ-linkage.35,36 Hydrolysis of acetyl and sulfonyl esters of stilbene-
glucuronates 32a (R1 ) tosyl) and 33a (R1 ) mesyl) in the presence
of NaOH in a microwave oven (sealed vessel) readily provided
monoglucuronide CA1G2 6a (Scheme 2).
The successful synthesis of CA1G1 relied on the protecting group
differentiation present in stilbene 27a. Selective cleavage of the
isopropyl group upon treatment with a variety of Lewis acids (AlCl3,
BCl3, and TiCl4) was accompanied by undesired Z to E partial
isomerization. The binary salt [TMAH][Al2Cl7], which is described
as an acidic ionic liquid, has been utilized for the selective
deprotection of alkyl-aryl ethers.37 Treatment of 27a with ionic
liquid [TMAH][Al2Cl7] resulted in selective cleavage of the
isopropyl group to generate stilbene phenol 30a. Reaction of phenol
30a with bromotriacetylglucuronate 31 in the presence of Cs2CO3
provided stilbene-glucuronate 34a. The coupling constant (J ) 7.8
It is instructive to note that glucoside derivatives of CA1 are
also known. Isolation of combretastatin A-1 2′-O-ꢀ-D-glucoside (9)
from Combretum krausii26 as well as from Combretum erythro-
phyllum27 and its synthesis have been reported.28 The glucoside
analogue combretastatin A-1 3′-O-ꢀ-D-glucoside (10) has not been
reported as an isolated natural product; however, its synthesis has
been achieved.28 Both 9 and 10 have been described to possess
cytotoxicity against B16F10 cancer cell lines, and analogue 10 is
reportedly 5 times more active compared to 9.28 In addition,
glucoside analogues 9 and 10 demonstrate antimitotic activity by
microtubule disassembly.28 The synthesis of combretastatin A-4
3′-O-ꢀ-D-glucoside (11) has also been reported.25
1
Hz) in the H NMR spectrum for the hydrogen (δH 5.17) on the
anomeric carbon confirmed that the bromide displacement pro-
ceeded in the anticipated fashion to afford the requisite ꢀ-linkage.
Treatment of 34a with NaOH in a microwave oven (sealed vessel)
readily hydrolyzed acetyl, methyl, and p-tosyl ester functionalities
to provide CA1G1 (5a).
Results and Discussion
The Koenigs-Knorr methodology29 was adopted to synthesize
the mono-ꢀ-D-glucuronic acid derivatives of combretastatin A-1
(CA1) using methyl R-D-(1-deoxy-1-bromo,2,3,4-triacetyl)glucur-
onate (31) as the glucuronide donor and CA1 as the glucuronide
acceptor. Substitution of the bromide at the anomeric center of the
glucuronide donor, by a phenolate of an appropriately protected
CA1 analogue (28-30), achieved the required stereochemistry.30,31
While yields for the glucuronidation step were only modest,
attempts to increase yield were not successful, presumably due to
the sterically crowded phenolic moiety and instability of 31 at room
temperature. Syntheses of CA1 and its various derivatives have
been efficiently achieved using the Wittig approach.4,32,33 In order
to achieve the required regiospecificity in the synthesis of the CA1
monoglucuronides, it was important to distinguish between the two
vicinal phenolic functional groups at C-2 and C-3 of the corre-
sponding pre-Wittig aldehyde 13 with a strategically chosen
selective protecting group. This selectivity was achieved by treating
aldehyde 13 with Hunig’s base (DIPEA) followed by a stoichio-
metric amount of DPSCl or TBSCl. In each case, silylation occurred
exclusively at the C-3 position, leaving the C-2 phenol intact, to
provide 14 or 15, respectively (Scheme 1). Selective protection of
the C-2 phenol of aldehyde 13 was achieved upon treatment with
a stoichiometric amount of p-TsCl to form sulfonyl ester 16
(Scheme 1). Interestingly, the reaction of a stoichiometric amount
of 2-bromopropane with aldehyde 13 in the presence of K2CO3
provided the C-2 monoisopropyl ether 18 as the major product in
a mixture of mono- and di-isopropyl ethers (Scheme 1), which were
readily separated by column chromatography. The yield of this
reaction was improved by utilizing a sealed vessel in a microwave
oven (Scheme 1).
Characterization of Compounds 5a and 5b Using 1H
NMR, 13C NMR, NOE, and HMBC Data. For CA1G1 (5a),
1
the doublet in the H NMR spectrum at δH 4.85 with a coupling
constant of JG1,G2 ) 7.8 Hz confirmed the stereochemistry of the
glucuronic acid moiety with a ꢀ-acetal linkage at its anomeric
carbon (δC 105.8). The regioselective incorporation of the glucu-
ronide at the C-2′ phenol was determined with the long-range
HMBC correlation between the proton H-G1 (δH 4.85) on the
anomeric carbon and C-2′ (δC 143.7) of CA1G1 (5a). In addition,
observation of an NOE between H-G1 (δH 4.85) and H-1′a (δH
6.89) in the 1D NOESY spectrum confirmed the regioselectivity
of the acetal linkage between C-2′ and C-G1 of the glucuronic acid
CA1G1. The presence of the carboxylic acid functionality was
substantiated by a long-range correlation between H-G5 (δH 3.96)
and C-G6 (δC 169.1). The J1a,1′a value of 12.0 Hz for 5a confirmed
the Z stereochemistry of the stilbene bridge, while the J1a,1′a value
of 17.0 Hz attested to the E stereochemistry for 5b. In a similar
1
manner, CA1G2 (6a and 6b) were characterized using H NMR,
13C NMR, NOE, and HMBC data (see Supporting Information).
Characterization of Compounds 6a and 6b Using 1H
NMR, 13C NMR, NOE, and HMBC Data. For CA1G2 (6a),
1
the doublet in the H NMR spectrum at δH 4.56 with a coupling
constant of JG1,G2 ) 7.5 Hz confirmed the stereochemistry of the
glucuronic acid moiety with a ꢀ-ether linkage at its anomeric carbon
(δC 106.1). The regioselective incorporation of the glucuronide at
the C-3′ phenol was determined with the long-range HMBC
correlation between the proton H-G1 (δH 4.56) on the anomeric
carbon and C-3′ (δC 135.2) of CA1G2. In addition, observation of
an NOE between H-G1 (δH 4.56) and H-4′OCH3 (δH 3.73) in the
1D NOESY spectrum confirmed the regioselectivity of the ether
linkage between C-3′ and C-G1 of glucuronic acid CA1G2. The
presence of the carboxylic acid functionality was confirmed by a
long-range correlation between H-G5 (δH 4.56) and C-G6 (δC
Treatment of aldehydes 14-18 with Hunig’s base followed by
the appropriate silyl halide or sulfonyl halide formed aldehydes
19-22, respectively (Scheme 1). A Wittig reaction between salt
23 and the accompanying aldehyde (19-22) provided a mixture
of E and Z stilbenes 24-27, which were readily separated by

Synthesis of DeriVatiVes of Combretastatin A-1
Journal of Natural Products, 2010, Vol. 73, No. 6 1095
Scheme 2. Synthesis of Combretastatin A-1-O-ꢀ-D-Glucuronic Acids (CA1G1 and CA1G2)
purification system using silica gel (200-400 mesh, 60 Å) or RP-18
Table 1. Cytotoxicity against Human Cancer Cell Lines
DU-145, SK-OV-3, and NCI-H460
prepacked columns. Intermediates and products synthesized were
characterized on the basis of their 1H NMR (500 MHz), 13C NMR (125
MHz), gDQCOSY, NOESY1d, gHSQC, and gHMBC spectroscopic
data. 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). HRMS were obtained using electron impact (EI)
ionization or electrospray ionization (ESI) or (+ve or -ve) atmospheric
pressure chemical ionization (APCI)/atmospheric pressure photoion-
ization (APPI) techniques. Purity of the final compounds was further
analyzed at 25 °C using a HPLC system with a diode-array detector (λ
) 190-400 nm), a Zorbax XDB-C18 HPLC column (4.6 mm ×150
mm, 5 µm), and a Zorbax reliance cartridge guard-column (eluents,
solvent A, 0.025% ammonium trifluoroacetate in water; solvent B,
acetonitrile; gradient, 90% A/10% B f 40% A/60% B over 0 to 18
min; flow rate 1.0 mL/min; injection volume 20 µL; monitored at
wavelengths λ 254, 280, and 300 nm).
GI50 (µM) SRB assaya
compound
DU-145
SK-OV-3
NCI-H460
CA1 (Z)
5a (Z)
5b (E)
6a (Z)
6b (E)
0.013b
4.4
>88
3.3
nac
2.3
30
2.1
>85
0.046b
3.0
26
1.8
26
>98
a Average of n g 3 independent determinations. b Ref 6. c Not
available.
172.8). The J1a,1′a value of 12.0 Hz for 6a substantiated the Z
stereochemistry of the stilbene bridge, while the J1a,1′a value of 17.0
Hz confirmed the E stereochemistry for 6b.
Biological Evaluation. The cytotoxicity of the glucuronides
CA1G1 (5a,b) and CA1G2 (6a,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 reference
compound. This procedure was based on the standard sulfor-
hodamine B (SRB) assay.33,38,39 The GI50 values are shown in Table
1. A comparison of the CA1 glucuronide analogues (5a,b and 6a,b)
showed that the Z-isomers (5a and 6a) were more potent against
all three cancer cell lines; however, all of the glucuronide analogues
were found to be substantially less active than CA1.
(Z)/(E)-(3,4,5-Trimethoxy)-{2′-(p-toluenesulfonyloxy)-3′-[(tert-
butyldiphenylsilyl)oxy]}stilbene (24a/24b). n-BuLi (2.5 M in hexanes,
2.0 mL, 5.0 mmol) was added dropwise to a well-stirred solution of
Wittig salt 23 (1.6 g, 3.0 mmol) in THF (60 mL, anhydrous) at 0 °C.
The reaction mixture was then warmed to rt, stirred for 15 min, and
then cooled to -78 °C. A solution of aldehyde 19 (1.4 g, 2.5 mmol) in
THF (5 mL, anhydrous) was added dropwise to the reaction mixture,
and the reaction was stirred until the temperature gradually warmed to
room temperature. The reaction was quenched by careful addition of
H2O (25 mL) and extracted with Et2O (3 × 50 mL). The combined
organic phase was washed with brine, dried over MgSO4, filtered, and
evaporated under reduced pressure. Flash chromatography of the crude
product using a prepacked 100 g silica column [eluents: solvent A,
EtOAc; solvent B, hexanes; gradient, 10% A/90% B over 3.18 min (1
CV), 10% A/90% B f 50% A/50% B over 33.0 min (10 CV), 50%
A/50% B over 6.36 min (2 CV); flow rate 40.0 mL/min; monitored at
λ 254 and 280 nm] afforded the Z-isomer 24a (1.17 g, 1.6 mmol, 64%
yield) as an off-white solid and the E-isomer 24b (0.0460 g, 0.0063
mmol, 2.5% yield) as an off-white solid.
Experimental Section
General Experimental Procedures. Methylene chloride (CH2Cl2),
acetone, and tetrahydrofuran (THF) were used in their anhydrous form
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

1096 Journal of Natural Products, 2010, Vol. 73, No. 6
Tanpure et al.
Z-Isomer 24a: mp 92-94 °C; 1H NMR (CDCl3, 500 MHz) δ 7.83
(2H, d, J ) 8.3 Hz, H-2′′, H-6′′), 7.68 [4H, d, J ) 8.0 Hz, Ph(H-2,
H-6)], 7.36 [2H, m, Ph(H-4)], 7.31 [4H, m, Ph(H-3, H-5)], 7.21 (2H,
d, J ) 8.5 Hz, H-3′′, H-5′′), 6.66 (1H, d, J ) 8.6 Hz, H-6′), 6.44 (2H,
s, H-2, H-6), 6.25 (1H, d, J ) 8.6 Hz, H-5′), 6.10 (1H, d, J ) 12.0 Hz,
H-1′a), 6.04 (1H, d, J ) 12.0 Hz, H-1a), 3.83 (3H, s, OCH3-4), 3.68
(6H, s, OCH3-3, -5), 2.77 (3H, s, OCH3-4′), 2.37 (3H, s, CH3-4′′), 1.06
[9H, s, -C(CH3)3]; 13C NMR (CDCl3, 125 MHz) δ 152.7 (C, C-3,
C-5), 149.9 (C, C-4′), 144.8 (C, C-4′′), 139.34 (C, C-3′), 139.28 (C,
C-2′), 137.1 (C, C-4), 134.6 [C, Ph(C-1)], 134.5 (C, C-1′′), 134.4 [CH,
Ph(C-2, C-6)], 132.2 (C, C-1), 129.9 (CH, C-1a), 129.6 (CH, C-3′′,
C-5′′), 129.0 [CH, Ph(C-4)], 128.5 (CH, C-2′′, C-6′′), 127.1 [CH, Ph(C-
3, C-5)], 124.9 (C, C-1′), 124.6 (CH, C-1′a), 121.8 (CH, C-6′), 109.5
(CH, C-5′), 106.2 (CH, C-2, C-6), 60.9 (CH3, OCH3-4), 56.0 (CH3,
OCH3-3, -5), 54.0 (CH3, OCH3-4′), 26.4 [CH3, -C(CH3)3], 21.6 (CH3,
CH3-4′′), 20.0 [C, -C(CH3)3]; HRMS m/z 725.2596 [M + 1]+ (calcd
for C41H45O8SSi+, 725.2599); anal. C 67.78, H 6.05%, calcd for
C41H44O8SSi, C 67.93, H 6.12%.
6.46 (2H, s, H-2, H-6), 3.88 (6H, s, OCH3-3, -5), 3.87 (3H, s, OCH3-
4), 3.82 (3H, s, OCH3-4′), 2.16 (3H, s, CH3-4′′), 1.01 [9H, s, -C(CH3)3],
0.13 [6H, s, -Si(CH3)2]; 13C NMR (CDCl3, 125 MHz) δ 153.1 (C,
C-3, C-5), 151.6 (C, C-4′), 145.1 (C, C-4′′), 140.1 (C, C-2′), 139.4 (C,
C-3′), 137.7 (C, C-4), 133.9 (C, C-1′′), 133.1 (C, C-1), 129.5 (CH,
C-3′′, C-5′′), 128.4 (CH, C-2′′, C-6′′), 128.0 (CH, C-1′a), 125.0 (C,
C-1′), 122.1 (CH, C-1a), 117.3 (CH, C-6′), 110.2 (CH, C-5′), 103.5
(CH, C-2, C-6), 60.9 (CH3, OCH3-4), 56.1 (CH3, OCH3-3, -5), 55.4
(CH3, OCH3-4′), 25.8 [CH3, C(CH3)3-3′], 21.4 (CH3, CH3-4′′), 18.7 [C,
-C(CH3)3], -4.4 [CH3, -Si(CH3)2]; HRMS m/z 601.2286 [M + 1]+
(calcd for C31H41O8SSi+, 601.2286); anal. C 62.05, H 6.76%, calcd
for C31H40O8SSi, C 61.97, H 6.71%.
(Z)-(3,4,5-Trimethoxy)-(2′-(methylsulfonyloxy)-3′-[(tert-butyl-
diphenylsilyl)oxy])stilbene (26a). n-BuLi (2.5 M in hexanes, 3.10 mL,
7.75 mmol) was added dropwise to a solution of Wittig salt 23 (2.10
g, 4.01 mmol) in THF (50 mL, anhydrous) at 0 °C. The reaction mixture
was then warmed to rt, stirred for 15 min, and then cooled to -78 °C.
A solution of aldehyde 21 (1.23 g, 2.54 mmol) in THF (15 mL,
anhydrous) was added dropwise to the reaction mixture, and the reaction
was stirred until the temperature gradually warmed to rt. The reaction
was quenched by careful addition of H2O (25 mL) and extracted with
Et2O (3 × 50 mL). The combined organic phase was washed with brine,
dried over MgSO4, filtered, and evaporated under reduced pressure.
Flash chromatography of the crude product using a prepacked 50 g
silica column [eluents: solvent A, EtOAc; solvent B, hexanes; gradient,
10% A/90% B over 1.39 min (1 CV), 10% A/90% B f 50% A/50%
B over 16.3 min (10 CV), 50% A/50% B over 3.18 min (2 CV); flow
rate 40.0 mL/min; monitored at λ 254 and 280 nm] afforded the
Z-isomer 26a (1.28 g, 1.97 mmol, 78% yield) as an off-white solid:
1
E-Isomer 24b: mp 179-181 °C; H NMR (CDCl3, 500 MHz) δ
7.76 (2H, d, J ) 8.3 Hz, H-2′′, H-6′′), 7.70 [4H, dd, J ) 8.0 Hz, 1.5
Hz, Ph(H-2, H-6)], 7.35 [2H, m, Ph(H-4)], 7.32 [4H, m, Ph(H-3, H-5)],
7.06 (2H, d, J ) 8.0 Hz, H-3′′, H-5′′), 7.02 (1H, d, J ) 8.7 Hz, H-6′),
6.64 (1H, d, J ) 16.2 Hz, H-1′a), 6.58 (1H, d, J ) 16.2 Hz, H-1a),
6.44 (1H, d, J ) 8.5 Hz, H-5′), 6.43 (2H, s, H-2, H-6), 3.88 (6H, s,
OCH3-3, -5), 3.87 (3H, s, OCH3-4), 2.82 (3H, s, OCH3-4′), 2.12 (3H,
s, CH3-4′′), 1.11 [9H, s, -C(CH3)3]; 13C NMR (CDCl3, 125 MHz) δ
153.1 (C, C-3, C-5), 150.1 (C, C-4′), 145.2 (C, C-4′′), 139.5 (C, C-3′),
139.0 (C, C-2′), 137.7 (C, C-4), 134.5 [CH, Ph(C-2, C-6)], 134.3 [C,
Ph(C-1)], 133.8 (C, C-1′′), 133.1 (C, C-1), 129.5 (CH, C-3′′, C-5′′),
129.0 [CH, Ph(C-4)], 128.5 (CH, C-2′′, C-6′′), 127.8 (CH, C-1a), 127.1
[CH, Ph(C-3, C-5)], 124.7 (C, C-1′), 122.2 (CH, C-1′a), 117.1 (CH,
C-6′), 110.1 (CH, C-5′), 103.5 (CH, C-2, C-6), 60.9 (CH3, OCH3-4),
56.1 (CH3, OCH3-3, -5), 54.0 (CH3, OCH3-4′), 26.4 [CH3, -C(CH3)3],
21.4 (CH3, CH3-4′′), 20.0 [C, -C(CH3)3]; HRMS m/z 725.2596 [M +
1]+ (calcd for C41H45O8SSi+, 725.2599).
1
mp 70-72 °C; H NMR (CDCl3, 500 MHz) δ 7.70 [4H, m, Ph(H-2,
H-6)], 7.35 [2H, m, Ph(H-4)], 7.33 [4H, m, Ph(H-3, H-5)], 6.73 (1H,
d, J ) 8.6 Hz, H-6′), 6.69 (1H, d, J ) 12.0 Hz, H-1′a), 6.61 (1H, d, J
) 12.0 Hz, H-1a), 6.52 (2H, s, H-2, H-6), 6.33 (1H, d, J ) 8.6 Hz,
H-5′), 3.82 (3H, s, OCH3-4), 3.67 (6H, s, OCH3-3, -5), 3.25 (3H, s,
-OSO2CH3), 2.85 (3H, s, OCH3-4′), 1.07 [9H, s, -C(CH3)3]; 13C NMR
(CDCl3, 125 MHz) δ 152.8 (C, C-3, C-5), 149.9 (C, C-4′), 139.0 (C,
C-2′), 138.7 (C, C-3′), 137.3 (C, C-4), 134.4 [CH, Ph(C-2, C-6)], 134.2
[C, Ph(C-1)], 132.0 (C, C-1), 131.5 (CH, C-1a), 129.2 [CH, Ph(C-4)],
127.3 [CH, Ph(C-3, C-5)], 125.4 (C, C-1′), 125.1 (CH, C-1′a), 122.4
(CH, C-6′), 109.7 (CH, C-5′), 106.3 (CH, C-2, C-6), 60.9 (CH3, OCH3-
4), 55.9 (CH3, OCH3-3, -5), 54.1 (CH3, OCH3-4′), 40.1 (CH3,
-OSO2CH3), 26.6 [CH3, -C(CH3)3], 20.0 [C, -C(CH3)3]; HRMS m/z
649.2285 [M + 1]+ (calcd for C35H41O8SSi+, 649.2286); anal. C 64.61,
H 6.28%, calcd for C35H40O8SSi, C 64.79, H 6.21%.
(Z)/(E)-(3,4,5-Trimethoxy)-(2′-(p-toluenesulfonyloxy)-3′-[(tert-bu-
tyldimethylsilyl)oxy])stilbene (25a/25b). n-BuLi (2.5 M in hexanes,
6.0 mL, 15.0 mmol) was added dropwise to a well-stirred solution of
Wittig salt 23 (7.85 g, 15.0 mmol) in THF (250 mL, anhydrous) at 0
°C. The reaction mixture was then warmed to rt, stirred for 15 min,
and cooled to -78 °C. Aldehyde 20 (3.70 g, 8.47 mmol) in THF (30
mL, anhydrous) was added dropwise to the reaction mixture and stirred
until the temperature gradually warmed to rt. The reaction was quenched
by careful addition of H2O (100 mL) and extracted with Et2O (3 ×
200 mL). The combined organic phase was washed with brine, dried
over MgSO4, filtered, and evaporated under reduced pressure. Flash
chromatography of the crude product using a prepacked 100 g silica
column [eluents: solvent A, EtOAc; solvent B, hexanes; gradient, 10%
A/90% B over 3.18 min (1 CV), 10% A/90% B f 50% A/50% B over
33.0 min (10 CV), 50% A/50% B over 6.36 min (2 CV); flow rate
40.0 mL/min; monitored at λ 254 and 280 nm] afforded the Z-isomer
25a (4.44 g, 7.39 mmol, 87% yield) as an off-white solid and the
E-isomer 25b (0.293 g, 0.487 mmol, 6% yield) as an off-white solid.
(Z)/(E)-(3,4,5-Trimethoxy)-(2′-isopropyloxy-3′-[p-toluenesulfony-
loxy])stilbene (27a/27b). n-BuLi (2.5 M in hexanes, 22.0 mL, 55.0
mmol) was added dropwise to a well-stirred solution of Wittig salt 4
(25.85 g, 49.39 mmol) in THF (250 mL, anhydrous) at 0 °C. The
reaction mixture was then warmed to rt, stirred for 30 min, and then
cooled to -78 °C. A solution of aldehyde 21 (15.00 g, 41.16 mmol) in
THF (30 mL, anhydrous) was added dropwise to the reaction mixture,
and the reaction was stirred until the temperature gradually warmed to
rt. The reaction was quenched by careful addition of H2O (100 mL)
and extracted with Et2O (3 × 200 mL). The combined organic phase
was washed with brine, dried over MgSO4, filtered, and evaporated
under reduced pressure. Flash chromatography of the crude product
using a prepacked 100 g silica column [eluents: solvent A, EtOAc;
solvent B, hexanes; gradient, 20% A/80% B over 3.18 min (1 CV),
20% A/80% B f 70% A/30% B over 33.0 min (10 CV), 70% A/30%
B over 6.36 min (2 CV); flow rate 40.0 mL/min; monitored at λ 254
and 280 nm] afforded the Z-isomer 27a (17.68 g, 33.44 mmol, 81%
yield) as a white solid and the E-isomer 27b (2.620 g, 4.956 mmol,
12% yield) as an off-white solid.
1
Z-Isomer 25a: mp 139-141 °C; H NMR (CDCl3, 500 MHz) δ
7.80 (2H, d, J ) 8.3 Hz, H-2′′, H-6′′), 7.24 (2H, d, J ) 8.4 Hz, H-3′′,
H-5′′), 6.76 (1H, d, J ) 8.6 Hz, H-6′), 6.60 (1H, d, J ) 8.6 Hz, H-5′),
6.44 (2H, s, H-2, H-6), 6.16 (2H, s, H-1a, H-1′a), 3.82 (3H, s, OCH3-
4), 3.75 (3H, s, OCH3-4′), 3.66 (6H, s, OCH3-3, -5), 2.38 (3H, s, CH3-
4′′), 0.95 [9H, s, -C(CH3)3], 0.04 [6H, s, -Si(CH3)2]; 13C NMR
(CDCl3, 125 MHz) δ 152.6 (C, C-3, C-5), 151.3 (C, C-4′), 144.7 (C,
C-4′′), 140.2 (C, C-2′), 139.1 (C, C-3′), 137.0 (C, C-4), 134.6 (C, C-1′′),
132.2 (C, C-1), 130.4 (CH, C-1a), 129.5 (CH, C-3′′, C-5′′), 128.4 (CH,
C-2′′, C-6′′), 125.3 (C, C-1′), 124.7 (CH, C-1′a), 122.1 (CH, C-6′),
109.5 (CH, C-5′), 106.1 (CH, C-2, C-6), 60.8 (CH3, OCH3-4), 55.9
(CH3, OCH3-3, -5), 55.4 (CH3, OCH3-4′), 25.7 [CH3, -C(CH3)3], 21.6
(CH3, CH3-4′′), 18.6 [C, -C(CH3)3], -4.5 [CH3, -Si(CH3)2]; HRMS
m/z 600.2223 [M]+ (calcd for C31H40O8SSi+, 600.2208); anal. C 61.77,
H 6.74%, calcd for C31H40O8SSi, C 61.97, H 6.71%. Single-crystal
X-ray diffraction further confirmed the Z-configuration of 25a.34 X-ray
CCDC #736463.
1
Z-Isomer 27a: mp 153-155 °C; H NMR (CDCl3, 500 MHz) δ
7.85 (2H, d, J ) 8.5 Hz, H-2′′, H-6′′), 7.32 (2H, d, J ) 8.5 Hz, H-3′′,
H-5′′), 7.08 (1H, d, J ) 8.5 Hz, H-6′), 6.55 (1H, d, J ) 12.5 Hz, H-1′a),
6.53 (1H, d, J ) 8.5 Hz, H-5′), 6.47 (1H, d, J ) 12.0 Hz, H-1a), 6.44
(2H, s, H-2, H-6), 4.37 [1H, sept, J ) 6.5 Hz, -CH(CH3)2], 3.83 (3H,
s, OCH3-4), 3.69 (3H, s, OCH3-4′), 3.67 (6H, s, OCH3-3, -5), 2.46 (3H,
s, CH3-4′′), 1.06 [6H, d, J ) 6.5 Hz, -CH(CH3)2]; 13C NMR (CDCl3,
125 MHz) δ 152.9 (C, C-4′), 152.8 (C, C-3, C-5), 150.0 (C, C-2′),
144.6 (C, C-4′′), 137.1 (C, C-4), 135.1 (C, C-1′′), 133.3 (C, C-3′), 132.5
(C, C-1), 129.9 (CH, C-1a), 129.2 (CH, C-3′′, C-5′′), 128.6 (CH, C-6′),
128.5 (CH, C-2′′, C-6′′), 125.4 (CH, C-1′a), 125.3 (C, C-1′), 106.8
1
E-Isomer 25b: mp 114-116 °C; H NMR (CDCl3, 500 MHz) δ
7.71 (2H, d, J ) 8.3 Hz, H-2′′, H-6′′), 7.13 (1H, d, J ) 8.8 Hz, H-6′),
7.08 (2H, d, J ) 8.0 Hz, H-3′′, H-5′′), 6.78 (1H, d, J ) 8.8 Hz, H-5′),
6.67 (1H, d, J ) 16.2 Hz, H-1′a), 6.62 (1H, d, J ) 16.1 Hz, H-1a),

Synthesis of DeriVatiVes of Combretastatin A-1
Journal of Natural Products, 2010, Vol. 73, No. 6 1097
(CH, C-5′), 106.0 (CH, C-2, C-6), 76.0 [CH, -CH(CH3)2], 60.9 (CH3,
OCH3-4), 56.0 (CH3, OCH3-4′), 55.8 (CH3, OCH3-3, -5), 22.1 [CH3,
-CH(CH3)2], 21.6 (CH3, CH3-4′′); HRMS m/z 528.1817 [M]+ (calcd
for C28H32O8S+, 528.1812); anal. C 63.63, H 6.16%, calcd for
C28H32O8S, C 63.62, H 6.10%. Single-crystal X-ray diffraction further
confirmed the Z-configuration of 27a.34 X-ray CCDC #736601.
4.5 mmol) in DMF (25 mL, anhydrous) was stirred for a few minutes.
HBr (0.30 mL, 48% solution in water) was added to the reaction
mixture, which was subjected to sonication in a water bath at rt. The
reaction mixture was monitored by TLC to completion. After 60 min,
water was added to the reaction mixture followed by extraction with
Et2O (2 × 50 mL). The combined organic layer was washed with brine,
dried over MgSO4, filtered, and evaporated under reduced pressure.
Flash chromatography of the crude product 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 75% A/25%
B over 13.12 min (10 CV), 75% A/25% B over 2.38 min (2 CV); flow
rate 25.0 mL/min; monitored at λ 254 and 280 nm] afforded phenol
28b (0.14 g, 0.29 mmol, 82% yield) as a pink solid: mp 175-177 °C;
1H NMR (CDCl3, 500 MHz) δ 7.84 (2H, d, J ) 8.4 Hz, H-2′′, H-6′′),
7.18 (2H, d, J ) 9.1 Hz, H-3′′, H-5′′), 7.11 (1H, d, J ) 8.6 Hz, H-6′),
6.85 (1H, d, J ) 16.2 Hz, H-1′a), 6.81 (1H, d, J ) 8.6 Hz, H-5′), 6.72
(1H, d, J ) 16.2 Hz, H-1a), 6.55 (2H, s, H-2, H-6), 5.83 (1H, b, OH-
3′), 3.93 (3H, s, OCH3-4′), 3.89 (6H, s, OCH3-3, -5), 3.88 (3H, s, OCH3-
4), 2.29 (3H, s, CH3-4′′); 13C NMR (CDCl3, 125 MHz) δ 153.2 (C,
C-3, C-5), 147.6 (C, C-4′), 145.4 (C, C-4′′), 139.5 (C, C-3′), 137.9 (C,
C-4), 135.2 (C, C-2′), 133.3 (C, C-1′′), 133.0 (C, C-1), 129.6 (CH,
C-3′′, C-5′′), 129.0 (CH, C-1a), 128.5 (CH, C-2′′, C-6′′), 125.5 (C,
C-1′), 121.7 (CH, C-1′a), 116.2 (CH, C-6′), 109.7 (CH, C-5′), 103.7
(CH, C-2, C-6), 60.9 (CH3, OCH3-4), 56.4 (CH3, OCH3-4′), 56.1 (CH3,
OCH3-3, -5), 21.6 (CH3, CH3-4′′); HRMS m/z 486.1346 [M]+ (calcd
for C25H26O8S+, 486.1343); anal. C 61.69, H 5.39%, calcd for
C25H26O8S, C 61.72, H 5.39%.
1
E-Isomer 27b: mp 161-163 °C; H NMR (CDCl3, 500 MHz) δ
7.89 (2H, d, J ) 8.4 Hz, H-2′′, H-6′′), 7.45 (2H, d, J ) 8.8 Hz, H-6′),
7.35 (1H, d, J ) 8.0 Hz, H-3′′, H-5′′), 7.23 (1H, d, J ) 16.5 Hz, H-1′a),
6.89 (1H, d, J ) 16.4 Hz, H-1a), 6.72 (2H, s, H-2, H-6), 6.68 (1H, d,
J ) 8.8 Hz, H-5′), 4.31 [1H, sept, J ) 6.2 Hz, -CH(CH3)2], 3.90 (6H,
s, OCH3-3, -5), 3.87 (3H, s, OCH3-4), 3.67 (3H, s, OCH3-4′), 2.47 (3H,
s, CH3-4′′), 1.14 [6H, d, J ) 6.2 Hz, -CH(CH3)2]; 13C NMR (CDCl3,
125 MHz) δ 153.4 (C, C-3, C-5), 152.7 (C, C-4′), 149.7 (C, C-2′),
144.6 (C, C-4′′), 137.8 (C, C-4), 134.9 (C, C-1′′), 133.5 (C, C-1), 133.3
(C, C-3′), 129.2 (CH, C-3′′, C-5′′), 128.6 (CH, C-2′′, C-6′′), 127.8 (CH,
C-1a), 125.8 (C, C-1′), 124.0 (CH, C-6′), 122.8 (CH, C-1′a), 107.6
(CH, C-5′), 103.4 (CH, C-2, C-6), 76.9 [CH, -CH(CH3)2], 61.0 (CH3,
OCH3-4), 56.1 (CH3, OCH3-3, -5), 55.9 (CH3, OCH3-4′), 22.2 [CH3,
-CH(CH3)2], 21.7 (CH3, CH3-4′′); HRMS m/z 529.1891 [M + 1]+
(calcd for C28H33O8S+, 529.1891); anal. C 63.68, H 6.29%, calcd for
C28H32O8S, C 63.62, H 6.10%.
[TMAH][Al2Cl7]. 37 To a suspension of AlCl3 (13.52 g, 101.4 mmol)
in 100 mL of CH2Cl2 cooled to 0 °C was added with stirring
trimethylammonium chloride [TMAH] (4.85 g, 50.67 mmol). The
reaction mixture was allowed to warm to ambient temperature and
stirred for 2 h. This clear yellow solution of the ionic liquid was used
as such for the deprotection of isopropyl ether of CA-1.
(Z)-(3,4,5-Trimethoxy)-(2′-[methanesulfonyloxy]-3′-hydroxy)stil-
bene (29a). To a solution of 26a (1.05 g, 1.62 mmol) in THF (25 mL,
anhydrous) was added TBAF (1.70 mL, 1.0 M solution in THF), and
the solution was stirred for a few minutes. The reaction mixture was
monitored by TLC to completion. After 60 min, water (10 mL) was
added to the reaction mixture followed by extraction with Et2O (2 ×
50 mL). The combined organic layer was washed with brine, dried
over MgSO4, filtered, and evaporated under reduced pressure. Flash
chromatography of the crude product using a prepacked 25 g silica
column [eluents: solvent A, EtOAc; solvent B, hexanes; gradient, 20%
A/80% B over 3.18 min (1 CV), 20% A/80% B f 70% A/30% B over
33.0 min (10 CV), 70% A/30% B over 6.36 min (2 CV); flow rate
25.0 mL/min; monitored at λ 254 and 280 nm] afforded phenol 29a
(Z)- (3,4,5-Trimethoxy)-(2′-[p-toluenesulfonyloxy]-3′-hydroxy)stil-
bene (28a). A mixture of stilbene 24a (1.26 g, 1.74 mmol) and KF
(0.151 g, 2.60 mmol) in DMF (20 mL, anhydrous) was stirred for a
few minutes. HBr (0.100 mL, 48% solution in water) was added to the
reaction mixture, and the reaction mixture was sonicated in a water
bath at rt and monitored by TLC to completion. After 60 min, water
was added to the reaction mixture followed by extraction with Et2O (2
× 100 mL). The combined organic layer was washed with brine, dried
over MgSO4, filtered, and evaporated under reduced pressure. Flash
chromatography of the crude product using a prepacked 100 g silica
column [eluents: solvent A, EtOAc; solvent B, hexanes; gradient, 20%
A/80% B over 3.18 min (1 CV), 20% A/80% B f 75% A/25% B over
33.0 min (10 CV), 75% A/25% B over 6.36 min (2 CV); flow rate
40.0 mL/min; monitored at λ 254 and 280 nm] afforded phenol 28a
(0.760 g, 1.56 mmol, 90% yield) as a white solid.
1
(0.599 g, 1.46 mmol, 90% yield) as a white solid: mp 55-58 °C; H
NMR (CDCl3, 500 MHz) δ 6.73 (1H, d, J ) 8.6 Hz, H-6′), 6.64 (1H,
d, J ) 8.6 Hz, H-5′), 6.63 (1H, d, J ) 12.1 Hz, H-1a), 6.60 (1H, d, J
) 12.0 Hz, H-1′a), 6.48 (2H, s, H-2, H-6), 3.88 (3H, s, OCH3-4′), 3.81
(3H, s, OCH3-4), 3.65 (6H, s, OCH3-3, -5), 3.37 (3H, s, -OSO2CH3);
13C NMR (CDCl3, 125 MHz) δ 152.8 (C, C-3, C-5), 146.8 (C, C-4′),
138.7 (C, C-3′), 137.2 (C, C-4), 135.3 (C, C-2′), 132.05 (C, C-1), 132.12
(CH, C-1a), 126.2 (C, C-1′), 124.1 (CH, C-1′a), 120.9 (CH, C-6′), 108.9
(CH, C-5′), 106.2 (CH, C-2, C-6), 60.8 (CH3, OCH3-4), 56.5 (CH3,
OCH3-4′), 55.9 (CH3, OCH3-3, -5), 40.0 (CH3, -OSO2CH3); HRMS
m/z 411.1108 [M + 1]+ (calcd for C19H23O8S+, 411.1108); anal. C
55.37, H 5.67%, calcd for C19H22O8S, C 55.60, H 5.40%.
(Z)-(3,4,5-Trimethoxy)-(2′-[p-toluenesulfonyloxy]-3′-hydroxy)stil-
bene (28a). A mixture of stilbene 25a (1.22 g, 2.03 mmol) and KF
(0.420 g, 7.23 mmol) in DMF (20 mL, anhydrous) was stirred for a
few minutes. HBr (0.3 mL, 48% solution in water) was added to the
reaction mixture, sonicated in a water bath at rt, and monitored by
TLC to completion. After 60 min, water was added to the reaction
mixture followed by extraction with Et2O (2 × 100 mL). The combined
organic layer was washed with brine, dried over MgSO4, filtered, and
evaporated under reduced pressure. Flash chromatography of the crude
product using a prepacked 100 g silica column [eluents: solvent A,
EtOAc; solvent B, hexanes; gradient, 20% A/80% B over 3.18 min (1
CV), 20% A/80% B f 75% A/25% B over 33.0 min (10 CV), 75%
A/25% B over 6.36 min (2 CV); flow rate 40.0 mL/min; monitored at
λ 254 and 280 nm] afforded phenol 28a (0.921 g, 1.89 mmol, 93%
yield) as a white solid: mp 137-138 °C; 1H NMR (CDCl3, 500 MHz)
δ 7.91 (2H, d, J ) 8.4 Hz, H-2′′, H-6′′), 7.29 (2H, d, J ) 8.0 Hz,
H-3′′, H-5′′), 6.71 (1H, d, J ) 8.6 Hz, H-6′), 6.62 (1H, d, J ) 8.6 Hz,
H-5′), 6.43 (2H, s, H-2, H-6), 6.36 (1H, d, J ) 12.0 Hz, H-1a), 6.32
(1H, d, J ) 12.0 Hz, H-1′a), 5.89 (1H, b, OH-3′), 3.86 (3H, s, OCH3-
4′), 3.82 (3H, s, OCH3-4), 3.67 (6H, s, OCH3-3, -5), 2.42 (3H, s, CH3-
4′′); 13C NMR (CDCl3, 125 MHz) δ 152.7 (C, C-3, C-5), 147.4 (C,
C-4′), 145.3 (C, C-4′′), 139.4 (C, C-3′), 137.2 (C, C-4), 135.4 (C, C-2′),
133.5 (C, C-1′′), 132.1 (C, C-1), 131.3 (CH, C-1a), 129.5 (CH, C-3′′,
C-5′′), 128.5 (CH, C-2′′, C-6′′), 125.7 (C, C-1′), 124.2 (CH, C-1′a),
120.8 (CH, C-6′), 109.2 (CH, C-5′), 106.2 (CH, C-2, C-6), 60.9 (CH3,
OCH3-4), 56.4 (CH3, OCH3-4′), 55.9 (CH3, OCH3-3, -5), 21.7 (CH3,
CH3-4′′); HRMS m/z 487.1421 [M + 1]+ (calcd for C25H27O8S+,
487.1421); anal. C 61.75, H 5.57%, calcd for C25H26O8S, C 61.72, H
5.39%.
(Z)-(3,4,5-Trimethoxy)-(2′-hydroxy-3′-[p-toluenesulfonyloxy])stil-
bene (30a). To a solution of 27a (3.990 g, 7.546 mmol) in CH2Cl2
(100 mL) cooled to 0 °C was added dropwise ionic liquid (46.00 mL,
23.32 mmol, 0.507 M [TMAH][Al2Cl7] solution in CH2Cl2) while
stirring the solution. The reaction was monitored by TLC. After the
reaction was complete (typically 5-10 min), ice cold water was added
to quench the reaction. The mixture was stirred vigorously for 2 min
and the organic layer was separated. The aqueous layer was extracted
with CH2Cl2 (2 × 100 mL), and the combined organic phase was
washed with brine, dried over MgSO4, filtered, and evaporated under
reduced pressure. Flash chromatography of the crude product using a
prepacked 100 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 40% A/60% B over 3.18 min (1 CV), 40% A/60%
B f 80% A/20% B over 33.0 min (10 CV), 80% A/20% B over 6.36
min (2 CV); flow rate 40.0 mL/min; monitored at λ 254 and 280 nm]
afforded phenol 30a (2.99 g, 6.145 mmol, 81%) as an off-white solid:
mp 137-138 °C; 1H NMR (CDCl3, 500 MHz) δ 7.85 (2H, d, J ) 8.3
Hz, H-2′′, H-6′′), 7.34 (2H, d, J ) 8.4 Hz, H-3′′, H-5′′), 7.03 (1H, d,
J ) 8.8 Hz, H-6′), 6.67 (1H, d, J ) 8.8 Hz, H-5′), 6.56 (1H, d, J )
11.8 Hz, H-1a), 6.43 (2H, s, H-2, H-6), 6.35 (1H, d, J ) 11.8 Hz,
H-1′a), 3.81 (3H, s, OCH3-4), 3.66 (6H, s, OCH3-3, -5), 3.66 (3H, s,
OCH3-4′), 2.46 (3H, s, CH3-4′′); 13C NMR (CDCl3, 125 MHz) δ 152.8
(E)-(3,4,5-Trimethoxy)-(2′-[p-toluenesulfonyloxy]-3′-hydroxy)stil-
bene (28b). A mixture of 25b (0.21 g, 0.35 mmol) and KF (0.26 g,

1098 Journal of Natural Products, 2010, Vol. 73, No. 6
Tanpure et al.
(C, C-3, C-5), 152.3 (C, C-4′), 144.9 (C, C-4′′), 143.0 (C, C-2′), 137.2
(C, C-4), 134.5 (C, C-1′′), 132.4 (CH, C-1a), 131.9 (C, C-1), 131.8 (C,
C-3′), 129.4 (CH, C-3′′, C-5′′), 128.6 (CH, C-6′), 128.4 (CH, C-2′′,
C-6′′), 125.1 (C, C-1′), 123.5 (CH, C-1′a), 109.8 (CH, C-5′), 106.2
(CH, C-2, C-6), 60.8 (CH3, OCH3-4), 56.1 (CH3, OCH3-4′), 55.9 (CH3,
OCH3-3, -5), 21.7 (CH3, CH3-4′′); HRMS m/z 486.1349 [M]+ (calcd
for C25H26O8S+, 486.1343); anal. C 61.46, H 5.35%, calcd for
C25H26O8S, C 61.72, H 5.39%.
2035); anal. C 56.74, H 5.41, S 3.82%, calcd for C38H42O17S, C 56.85,
H 5.27, S 3.99%.
(E)-3(S),4(S),5(R)-Triacetoxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-2′-[p-toluenesulfonyloxy]-3′-stilbenyloxy)]tetrahydropyran-2(S)-
carboxylic Acid Methyl Ester (32b). A mixture of phenol 28b (0.114
g, 0.234 mmol) and Cs2CO3 (0.450 g, 1.38 mmol) in acetone (10 mL,
anhydrous) was stirred for 10 min under N2. Bromide 31 (0.301 g,
0.758 mmol) was added to this reaction mixture, which was subse-
quently stirred for 24 h. The suspension formed was filtered through a
Buchner funnel, and the solid was washed with acetone and ether. The
organic solvents were evaporated under reduced pressure to afford a
viscous dark liquid. Flash chromatography of the crude product using
a prepacked 25 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 40% A/60% B over 1.19 min (1 CV), 40% A/60%
B f 70% A/30% B over 13.12 min (10 CV), 70% A/30% B over 2.38
min (2 CV); flow rate 25.0 mL/min; monitored at λ 254 and 280 nm]
afforded the glucuronate 32b (0.120 g, 0.150 mmol, 64% yield) as a
pink-white solid: mp 118-121 °C; 1H NMR (CDCl3, 500 MHz) δ 7.93
(2H, d, J ) 8.2 Hz, H-2′′, H-6′′), 7.45 (1H, d, J ) 8.9 Hz, H-6′), 7.42
(2H, d, J ) 8.4 Hz, H-3′′, H-5′′), 7.31 (1H, d, J ) 16.2 Hz, H-1′a),
6.90 (1H, d, J ) 16.2 Hz, H-1a), 6.84 (1H, d, J ) 8.9 Hz, H-5′), 6.75
(2H, s, H-2, H-6), 5.11 (1H, t, J ) 9.4 Hz, H-G3), 4.93 (1H, t, J ) 9.8
Hz, H-G4), 4.92 (1H, d, J ) 7.7 Hz, H-G1), 4.34 (1H, dd, J ) 7.7, 9.2
Hz, H-G2), 3.91 (6H, s, OCH3-3, -5), 3.86 (3H, s, OCH3-4), 3.84 (3H,
s, OCH3-4′), 3.78 (1H, d, J ) 10.0 Hz, H-G5), 3.73 (3H, s, COOCH3-
G6), 2.49 (3H, s, CH3-4′′), 2.03 (6H, s, OCOCH3-G2, -G3), 2.00 (3H,
s, OCOCH3-G4); 13C NMR (CDCl3, 125 MHz) δ 170.0 (C, OCOCH3-
G3), 169.4 (C, OCOCH3-G4), 169.1 (C, OCOCH3-G2), 166.6 (C,
COOCH3-G6), 153.3 (C, C-3, C-5), 151.9 (C, C-4′), 144.9 (C, C-4′′),
142.1 (C, C-2′), 138.0 (C, C-4), 137.6 (C, C-3′), 134.7 (C, C-1′′), 133.1
(C, C-1), 129.6 (CH, C-3′′, C-5′′), 129.3 (CH, C-1a), 128.7 (CH, C-2′′,
C-6′′), 126.1 (C, C-1′), 121.5 (CH, C-1′a), 121.4 (CH, C-6′), 110.9
(CH, C-5′), 103.7 (CH, C-2, C-6), 100.3 (CH, C-G1), 72.2 (CH, C-G5),
72.0 (CH, C-G3), 71.3 (CH, C-G2), 68.8 (CH, C-G4), 60.9 (CH3, OCH3-
4), 56.2 (CH3, OCH3-4′), 56.1 (CH3, OCH3-3, -5), 52.7 (CH3, COOCH3-
G6), 21.5 (CH3, CH3-4′′), 20.6 (CH3, OCOCH3-G2, -G3), 20.5 (CH3,
OCOCH3-G4); HRMS m/z 803.2212 [M + 1]+ (calcd for C38H43O17S+,
803.2216); anal. C 57.04, H 5.32%, calcd for C38H42O17S, C 56.85, H
5.27%.
(E)-(3,4,5-Trimethoxy)-(2′-hydroxy-3′-[p-toluenesulfonyloxy])stil-
bene (30b). To a solution of 27b (0.53 g, 1.0 mmol) in CH2Cl2 (25
mL) cooled to 0 °C was added dropwise ionic liquid (6.00 mL, 3.04
mmol, 0.517 M [TMAH][Al2Cl7] solution in CH2Cl2) while stirring
the solution. The reaction was monitored by TLC. After the reaction
was complete (typically 5-10 min), ice cold water was added to quench
the reaction. The mixture was stirred vigorously for 2 min, and the
organic layer was separated. The aqueous layer was extracted with
CH2Cl2 (2 × 25 mL), and the combined organic phase was washed
with brine, dried over MgSO4, filtered, and evaporated under reduced
pressure. Flash chromatography of the crude product using a prepacked
25 g silica column [eluents: solvent A, EtOAc; solvent B, hexanes;
gradient, 40% A/60% B over 1.19 min (1 CV), 40% A/60% B f 80%
A/20% B over 13.12 min (10 CV), 80% A/20% B over 2.38 min (2
CV); flow rate 25.0 mL/min; monitored at λ 254 and 280 nm] afforded
phenol 30b (0.392 g, 0.80 mmol, 81%) as an off-white solid: mp
137-138 °C; 1H NMR (CDCl3, 500 MHz) δ 7.85 (2H, d, J ) 8.2 Hz,
H-2′′, H-6′′), 7.36 (1H, d, J ) 8.8 Hz, H-6′), 7.33 (2H, d, J ) 8.0 Hz,
H-3′′, H-5′′), 7.26 (1H, d, J ) 16.4 Hz, H-1′a), 6.99 (1H, d, J ) 16.4
Hz, H-1a), 6.74 (2H, s, H-2, H-6), 6.37 (1H, d, J ) 8.8 Hz, H-5′), 3.91
(6H, s, OCH3-3, -5), 3.87 (3H, s, OCH3-4), 3.48 (3H, s, OCH3-4′),
2.46 (3H, s, CH3-4′′); 13C NMR (CDCl3, 125 MHz) δ 153.3 (C, C-3,
C-5), 151.8 (C, C-4′), 147.7 (C, C-2′), 145.9 (C, C-4′′), 137.7 (C, C-4),
133.6 (C, C-1), 131.5 (C, C-1′′), 129.4 (CH, C-3′′, C-5′′), 129.0 (CH,
C-2′′, C-6′′), 128.3 (CH, C-1a), 126.9 (C, C-3′), 125.0 (CH, C-6′), 122.0
(CH, C-1′a), 120.0 (C, C-1′), 103.9 (CH, C-5′), 103.5 (CH, C-2, C-6),
60.9 (CH3, OCH3-4), 56.1 (CH3, OCH3-3, -5), 55.5 (CH3, OCH3-4′),
21.7 (CH3, CH3-4′′); HRMS m/z 487.1418 [M + 1]+ (calcd for
C25H27O8S+, 487.1421).
(Z)-3(S),4(S),5(R)-Triacetoxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-2′-[p-toluenesulfonyloxy]-3′-stilbenyloxy)]tetrahydropyran-2(S)-
carboxylic Acid Methyl Ester (32a). A mixture of phenol 28a (0.762
g, 1.57 mmol) and Cs2CO3 (2.15 g, 6.60 mmol) in acetone (20 mL,
anhydrous) was stirred for 10 min under N2. Bromide 31 (1.21 g, 3.05
mmol) was added to this reaction mixture, which was subsequently
stirred for 24 h. The suspension formed was filtered through a Buchner
funnel, and the solid was washed with acetone and ether. The organic
solvents were evaporated under reduced pressure to afford a viscous
dark liquid. Flash chromatography of the crude product using a
prepacked 100 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 40% A/60% B over 3.18 min (1 CV), 40% A/60%
B f 70% A/30% B over 33.0 min (10 CV), 70% A/30% B over 6.36
min (2 CV); flow rate 40.0 mL/min; monitored at λ 254 and 280 nm]
afforded glucuronate 32a (0.450 g, 0.562 mmol, 36% yield) as an off-
(Z)-3(S),4(S),5(R)-Triacetoxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-2′-(methanesulfonyloxy)-3′-stilbenyloxy)]tetrahydropyran-2(S)-
carboxylic Acid Methyl Ester (33a). A mixture of phenol 29a (0.722
g, 1.76 mmol) and Cs2CO3 (1.55 g, 4.76 mmol) in acetone (20 mL,
anhydrous) was stirred for 10 min under N2. Bromide 31 (0.886 g,
2.23 mmol) was added to this reaction mixture, which was subsequently
stirred for 24 h. The suspension formed was filtered through a Buchner
funnel, and the solid was washed with acetone and ether. The organic
solvents were evaporated under reduced pressure to afford a viscous
dark liquid. Flash chromatography of the crude product using a
prepacked 50 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 40% A/60% B over 1.39 min (1 CV), 40% A/60%
B f 70% A/30% B over 16.3 min (10 CV), 70% A/30% B over 3.18
min (2 CV); flow rate 40.00 mL/min; monitored at λ 254 and 280 nm]
afforded glucuronate 33a (0.177 g, 0.244 mmol, 14% yield) as an off-
1
white solid: mp 95-97 °C; H NMR (CDCl3, 500 MHz) δ 7.93 (2H,
d, J ) 8.3 Hz, H-2′′, H-6′′), 7.45 (2H, d, J ) 8.0 Hz, H-3′′, H-5′′),
6.97 (1H, d, J ) 8.8 Hz, H-6′), 6.63 (1H, d, J ) 8.8 Hz, H-5′), 6.61
(1H, d, J ) 12.1 Hz, H-1a), 6.58 (1H, d, J ) 12.1 Hz, H-1′a), 6.52
(2H, s, H-2, H-6), 5.09 (1H, t, J ) 9.4 Hz, H-G3), 4.98 (1H, t, J ) 9.8
Hz, H-G4), 4.86 (1H, d, J ) 7.6 Hz, H-G1), 4.28 (1H, dd, J ) 7.6, 9.1
Hz, H-G2), 3.83 (3H, s, OCH3-4), 3.78 (1H, d, J ) 9.8 Hz, H-G5),
3.77 (3H, s, OCH3-4′), 3.75 (3H, s, COOCH3-G6), 3.68 (6H, s, OCH3-
3, -5), 2.54 (3H, s, CH3-4′′), 2.03 (3H, s, OCOCH3-G3), 2.02 (3H, s,
OCOCH3-G2), 2.00 (3H, s, OCOCH3-G4); 13C NMR (CDCl3, 125
MHz) δ 170.0 (C, OCOCH3-G3), 169.4 (C, OCOCH3-G4), 169.1 (C,
OCOCH3-G2), 166.6 (C, COOCH3-G6), 152.8 (C, C-3, C-5), 151.7
(C, C-4′), 144.8 (C, C-4′′), 142.6 (C, C-2′), 137.5 (C, C-3′), 137.1 (C,
C-4), 135.0 (C, C-1′′), 132.3 (C, C-1), 131.9 (CH, C-1a), 129.6 (CH,
C-3′′, C-5′′), 128.7 (CH, C-2′′, C-6′′), 126.7 (CH, C-6′), 126.5 (C, C-1′),
124.1 (CH, C-1′a), 110.2 (CH, C-5′), 106.3 (CH, C-2, C-6), 100.4 (CH,
C-G1), 72.3 (CH, C-G5), 72.1 (CH, C-G3), 71.4 (CH, C-G2), 68.8
(CH, C-G4), 60.9 (CH3, OCH3-4), 56.2 (CH3, OCH3-4′), 56.0 (CH3,
OCH3-3, -5), 52.8 (CH3, COOCH3-G6), 21.6 (CH3, CH3-4′′), 20.7 (CH3,
OCOCH3-G2/-G3), 20.6 (CH3, OCOCH3-G2/-G3), 20.5 (CH3, OCOCH3-
G4); HRMS m/z 825.2030 [M + Na]+ (calcd for C38H42O17SNa+, 825.
1
white solid: mp 86-88 °C; H NMR (CDCl3, 500 MHz) δ 6.96 (1H,
d, J ) 8.8 Hz, H-6′), 6.66 (1H, d, J ) 8.8 Hz, H-5′), 6.63 (1H, d, J )
11.9 Hz, H-1a), 6.57 (1H, d, J ) 11.9 Hz, H-1′a), 6.47 (2H, s, H-2,
H-6), 5.31 (2H, m, H-G3, H-G4), 5.22 (1H, m, H-G2), 5.13 (1H, d, J
) 7.7 Hz, H-G1), 4.01 (1H, m, H-G5), 3.82 (3H, s, OCH3-4′), 3.80
(3H, s, OCH3-4), 3.74 (3H, s, COOCH3-G6), 3.63 (6H, s, OCH3-3,
-5), 3.40 (3H, s, -OSO2CH3), 2.07 (3H, s, OCOCH3-G2), 2.03 (3H, s,
OCOCH3-G3), 2.02 (3H, s, OCOCH3-G4); 13C NMR (CDCl3, 125
MHz) δ 170.0 (C, OCOCH3-G3), 169.5 (C, OCOCH3-G2), 169.4 (C,
OCOCH3-G4), 166.7 (C, COOCH3-G6), 152.8 (C, C-3, C-5), 151.2
(C, C-4′), 141.7 (C, C-2′), 137.6 (C, C-3′), 137.1 (C, C-4), 132.1 (C,
C-1), 126.85 (CH, C-6′), 126.82 (C, C-1′), 123.9 (CH, C-1a, C-1′a),
110.4 (CH, C-5′), 106.2 (CH, C-2, C-6), 101.3 (CH, C-G1), 72.9 (CH,
C-G5), 71.9 (CH, C-G3), 71.3 (CH, C-G2), 69.3 (CH, C-G4), 60.9
(CH3, OCH3-4), 56.4 (CH3, OCH3-4′), 55.9 (CH3, OCH3-3, -5), 53.0
(CH3, COOCH3-G6), 40.2 (CH3, -OSO2CH3), 20.7 (CH3, OCOCH3-
G2), 20.6 (CH3, OCOCH3-G3), 20.5 (CH3, OCOCH3-G4); HRMS m/z
727.1897 [M + 1]+ (calcd for C32H39O17S+, 727.1902).

Synthesis of DeriVatiVes of Combretastatin A-1
Journal of Natural Products, 2010, Vol. 73, No. 6 1099
(Z)-3(S),4(S),5(R)-Triacetoxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-3′-(p-toluenesulfonyloxy)-2′-stilbenyloxy)]tetrahydropyran-2(S)-
carboxylic Acid Methyl Ester (34a). A mixture of phenol 30a (3.500
g, 7.194 mmol), and Cs2CO3 (4.680 g, 14.36 mmol) in acetone (200
mL, anhydrous) was stirred for 10 min under N2. Bromoglucuronate
31 (5.710 g, 14.38 mmol) was added to this reaction mixture, which
was subsequently stirred for 24 h. The suspension was filtered through
a Buchner funnel, and the solid was washed with acetone (50 mL) and
ether (50 mL). The organic solvents were evaporated under reduced
pressure to afford a dark viscous liquid. Flash chromatography of the
crude product using a prepacked 100 g silica column [eluents: solvent
A, EtOAc; solvent B, hexanes; gradient, 80% A/20% B f 30% A/70%
B over 0 to 66 min (20 CV), 30% A/70% B over 9.54 min (2 CV);
flow rate 40.00 mL/min; monitored at λ 254 and 280 nm] afforded
CA1-glucuronate 34a (2.010 g, 2.510 mmol, 35% yield) as an off-
C38H42O17SNa+, 825.2035); anal. C 56.81, H 5.28, S 3.85%, calcd for
C38H42O17S, C 56.85, H 5.27, S 3.99%.
(Z)-3(S),4(S),5(R)-Trihydroxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-3′-hydroxy-2′-stilbenyloxy)]tetrahydropyran-2(S)-carboxylic Acid
(5a). A solution of CA1-glucuronate 34a (1.240 g, 1.584 mmol) and
NaOH (6 mL, 2 M solution) in methanol (15 mL) in a sealed vessel
was heated to 50 °C in a Biotage Initiator microwave for 30 min. The
reaction was monitored by TLC, and after completion the mixture was
transferred to a pear-shaped 200 mL flask, which was then cooled to
-20 °C. The reaction mixture was then neutralized with HCl (6 mL,
2 M solution) in MeOH (10 mL) and cooled to -20 °C while stirring
for 2 min. The aqueous solvents were evaporated under reduced pressure
at 40 °C, leaving behind Z-CA1G1 (5a). Flash chromatography of the
crude product using a prepacked 120 g RP-18 silica column [eluents:
solvent A, water; solvent B, acetonitrile; gradient, 90% A/10% B f
60% A/40% B over 0 to 31.80 min (10 CV), 40% A/60% B over 31.80
to 38.16 min (2 CV); flow rate 40.0 mL/min; monitored at λ 254 and
280 nm] afforded glucuronic acid (5a; 0.490 g, 9.637 mmol, 62%) as
1
white solid: mp 87-89 °C; H NMR (CDCl3, 500 MHz) δ 7.84 (2H,
d, J ) 8.3 Hz, H-2′′, H-6′′), 7.39 (2H, d, J ) 8.2 Hz, H-3′′, H-5′′),
7.03 (1H, d, J ) 8.8 Hz, H-6′), 6.60 (1H, d, J ) 12.0 Hz, H-1′a), 6.53
(1H, d, J ) 8.8 Hz, H-5′), 6.52 (1H, d, J ) 12.1 Hz, H-1a), 6.39 (2H,
s, H-2, H-6), 5.20 (1H, t, J ) 9.4 Hz, H-G3), 5.17 (1H, d, J ) 7.8 Hz,
H-G1), 5.12 (1H, t, J ) 9.8 Hz, H-G4), 4.85 (1H, dd, J ) 7.8, 9.3 Hz,
H-G2), 3.82 (3H, s, OCH3-4), 3.80 (1H, d, J ) 9.9 Hz, H-G5), 3.66
(6H, s, OCH3-3, -5), 3.60 (3H, s, COOCH3-G6), 3.52 (3H, s, OCH3-
4′), 2.50 (3H, s, CH3-4′′), 2.12 (3H, s, OCOCH3-G2), 2.02 (3H, s,
OCOCH3-G3), 1.98 (3H, s, OCOCH3-G4); 13C NMR (CDCl3, 125
MHz) δ 170.0 (C, OCOCH3-G3), 169.5 (C, OCOCH3-G2), 169.3 (C,
OCOCH3-G4), 166.5 (CH3, COOCH3-G6), 152.8 (C, C-3, C-5), 152.6
(C, C-4′), 147.1 (C, C-2′), 144.8 (C, C-4′′), 137.2 (C, C-4), 134.7 (C,
C-1′′), 132.3 (C, C-3′), 132.0 (C, C-1), 130.8 (CH, C-1a), 129.4 (CH,
C-3′′, C-5′′), 128.9 (CH, C-6′), 128.6 (CH, C-2′′, C-6′′), 125.1 (C, C-1′),
124.7 (CH, C-1′a), 108.4 (CH, C-5′), 106.1 (CH, C-2, C-6), 100.4 (CH,
C-G1), 72.6 (CH, C-G5), 72.2 (CH, C-G3), 71.2 (CH, C-G2), 69.2
(CH, C-G4), 60.9 (CH3, OCH3-4), 55.9 (CH3, OCH3-3, -5), 55.8 (CH3,
OCH3-4′), 52.7 (CH3, COOCH3-G6), 21.6 (CH3, CH3-4′′), 20.8 (CH3,
OCOCH3-G2), 20.6 (CH3, OCOCH3-G3), 20.5 (CH3, OCOCH3-G4);
HRMS (ESI+) m/z 825.2033 [M + Na]+ (calcd for C38H42O17SNa+,
825.2035); anal. C 57.10, H 5.39, S 4.12%, calcd for C38H42O17S, C
56.85, H 5.27, S 3.99%.
1
an off-white solid: mp 65-67 °C; H NMR (acetone-d6, 500 MHz) δ
8.15 (1H, b, OH-2′), 6.89 (1H, d, J ) 12.2 Hz, H-1′a), 6.71 (1H, d, J
) 8.6 Hz, H-6′), 6.69 (1H, d, J ) 8.6 Hz, H-5′), 6.55 (2H, s, H-2,
H-6), 6.44 (1H, d, J ) 12.2 Hz, H-1a), 4.85 (1H, d, J ) 7.8 Hz, H-G1),
3.96 (1H, d, J ) 9.8 Hz, H-G5), 3.80 (3H, s, OCH3-4′), 3.72 (1H, dd,
J ) 8.8, 9.6 Hz, H-G4), 3.70 (3H, s, OCH3-4), 3.66 (1H, dd, J ) 7.8,
9.1 Hz, H-G2), 3.64 (6H, s, OCH3-3, -5), 3.60 (1H, dd, J ) 8.8, 9.1
Hz, H-G3); 13C NMR (DMSO-d6, 125 MHz) δ 169.1 (C, COOH-G6),
153.0 (C, C-3, C-5), 148.4 (C, C-4′), 143.7 (C, C-2′), 139.9 (C, C-3′),
137.5 (C, C-4), 132.7 (C, C-1), 129.3 (CH, C-1a), 126.0 (CH, C-1′a),
124.4 (C, C-1′), 119.4 (CH, C-6′), 108.8 (CH, C-5′), 106.4 (CH, C-2,
C-6), 105.8 (CH, C-G1), 76.2 (CH, C-G3), 75.3 (CH, C-G5), 74.0 (CH,
C-G2), 71.7 (CH, C-G4), 59.6 (CH3, OCH3-4), 55.6 (CH3, OCH3-4′),
55.2 (CH3, OCH3-3, -5); HRMS (EI+) m/z 508.1580 [M]+ (calcd for
C24H28O12+, 508.1575); HRMS (ESI-) m/z 507.1525 [M - 1]- (calcd
for C24H27O12-, 507.1508).
(E)-3(S),4(S),5(R)-Trihydroxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-3′-hydroxy-2′-stilbenyloxy)]tetrahydropyran-2(S)-carboxylic Acid
(5b). A solution of glucuronate 34b (0.810 g, 1.01 mmol) and NaOH
(4 mL, 2 M solution) in methanol (16 mL) in a sealed vessel was heated
to 60 °C in a Biotage Initiator microwave for 30 min. The reaction
was monitored by TLC, and after completion, the reaction mixture was
cooled to -20 °C and treated with dropwise addition of HCl (4 mL, 2
M solution) in MeOH (20 mL) while stirring for 5 min. The neutralized
reaction mixture was then evaporated under reduced pressure to dryness
at 40 °C, leaving behind E-CA1G1 (5b). Flash chromatography of the
crude product using a prepacked 120 g RP-18 silica column [eluents:
solvent A, acetonitrile; solvent B, water; gradient, 10% A/90% B f
40% A/60% B over 33.0 min (10 CV), 40% A/60% B over 6.36 min
(2 CV); flow rate 40.0 mL/min; monitored at λ 254 and 280 nm]
afforded E-CA1G1 (5b; 0.448 g, 0.881 mmol, 87%) as an off-white
(E)-3(S),4(S),5(R)-Triacetoxy-6(S)-[(3,4,5-Trimethoxy)-(4′-meth-
oxy-3′-(p-toluenesulfonyloxy)-2′-stilbenyloxy)]tetrahydropyran-2(S)-
carboxylic Acid Methyl Ester (34b). A mixture of phenol 30b (0.650
g, 1.34 mmol) and Cs2CO3 (1.35 g, 4.14 mmol) in acetone (50 mL,
anhydrous) was stirred for 10 min under N2. Bromide 31 was added to
this reaction mixture, which was subsequently stirred for 24 h. The
suspension formed was filtered through a Buchner funnel, and the solid
was washed with acetone and ether. The organic solvents were
evaporated under reduced pressure to afford a viscous dark liquid. Flash
chromatography of the crude product 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
13.12 min (10 CV), 70% A/30% B over 2.38 min (2 CV); flow rate
25.0 mL/min; monitored at λ 254 and 280 nm] afforded the glucuronate
34b (0.604 g, 0.754 mmol, 56% yield) as an off-white solid: mp
108-110 °C; 1H NMR (CDCl3, 500 MHz) δ 7.81 (2H, d, J ) 8.3 Hz,
H-2′′, H-6′′), 7.46 (1H, d, J ) 8.8 Hz, H-6′), 7.36 (2H, d, J ) 8.0 Hz,
H-3′′, H-5′′), 7.32 (1H, d, J ) 16.4 Hz, H-1′a), 6.85 (1H, d, J ) 16.3
Hz, H-1a), 6.78 (2H, s, H-2, H-6), 6.66 (1H, d, J ) 8.9 Hz, H-5′), 5.27
(1H, sept, J ) 5.0 Hz, H-G3), 5.17 (1H, m, H-G1), 5.16 (1H, m, H-G2),
5.15 (1H, m, H-G4), 3.94 (6H, s, OCH3-3, -5), 3.92 (1H, d, J ) 10.0
Hz, H-G5), 3.88 (3H, s, OCH3-4), 3.57 (3H, s, COOCH3-G6), 3.43
(3H, s, OCH3-4′), 2.49 (3H, s, CH3-4′′), 2.08 (3H, s, OCOCH3-G2),
2.03 (3H, s, OCOCH3-G3), 2.00 (3H, s, OCOCH3-G4); 13C NMR
(CDCl3, 125 MHz) δ 169.9 (C, OCOCH3-G3), 169.7 (C, OCOCH3-
G2), 169.4 (C, OCOCH3-G4), 166.7 (C, OCOCH3-G5), 153.4 (C, C-3,
C-5), 152.3 (C, C-4′), 147.1 (C, C-2′), 144.9 (C, C-4′′), 137.9 (C, C-4),
134.3 (C, C-1′′), 133.4 (C, C-1), 131.8 (C, C-3′), 129.3 (CH, C-3′′,
C-5′′), 128.8 (CH, C-1a), 128.7 (CH, C-2′′, C-6′′), 125.8 (C, C-1′),
124.0 (CH, C-6′), 122.3 (CH, C-1′a), 108.8 (CH, C-5′), 103.8 (CH,
C-2, C-6), 100.9 (CH, C-G1), 72.6 (CH, C-G5), 72.0 (CH, C-G3), 71.3
(CH, C-G2), 69.4 (CH, C-G4), 61.0 (CH3, OCH3-4), 56.2 (CH3, OCH3-
3, -5), 55.6 (CH3, OCH3-4′), 52.7 (CH3, COOCH3-G6), 21.6 (CH3, CH3-
4′′), 20.8 (CH3, OCOCH3-G2), 20.6 (CH3, OCOCH3-G3), 20.4 (CH3,
1
solid: mp 106-108 °C; H NMR (acetone-d6, 500 MHz) δ 8.08 (1H,
b, OH-2′), 7.76 (1H, d, J ) 16.6 Hz, H-1′a), 7.23 (1H, d, J ) 8.7 Hz,
H-6′), 6.99 (1H, d, J ) 16.5 Hz, H-1a), 6.91 (2H, s, H-2, H-6), 6.85
(1H, d, J ) 8.8 Hz, H-5′), 4.84 (1H, d, J ) 7.9 Hz, H-G1), 4.04 (1H,
d, J ) 9.7 Hz, H-G5), 3.88 (6H, s, OCH3-3, -5), 3.84 (3H, s, OCH3-
4′), 3.76 (1H, m, H-G2), 3.75 (1H, m, H-G4), 3.74 (3H, s, OCH3-4),
3.66 (1H, m, H-G3); 13C NMR (acetone-d6, 125 MHz) δ 169.2 (C,
COOH-G6), 153.6 (C, C-3, C-5), 148.6 (C, C-4′), 143.4 (C, C-2′), 140.2
(C, C-3′), 137.9 (C, C-4), 133.8 (C, C-1), 127.6 (CH, C-1a), 124.6 (C,
C-1′), 122.4 (CH, C-1′a), 114.7 (CH, C-6′), 109.9 (CH, C-5′), 106.1
(CH, C-G1), 103.8 (CH, C-2, C-6), 76.2 (CH, C-G3), 75.2 (CH, C-G5),
74.2 (CH, C-G2), 71.8 (CH, C-G4), 59.7 (CH3, OCH3-4), 55.64 (CH3,
OCH3-4′), 55.61 (CH3, OCH3-3, -5); HRMS (ESI-) m/z 507.1497 [M
- 1]- (calcd for C24H27O16, 507.1508).
(Z)-3(S),4(S),5(R)-Trihydroxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-2′-hydroxy-3′-stilbenyloxy)]tetrahydropyran-2(S)-carboxylic Acid
(6a). A solution of glucuronate 32a (0.20 g, 0.25 mmol) and NaOH (1
mL, 2 M solution) in MeOH (4 mL) in a sealed vessel was heated to
60 °C in a microwave for 15 min. The reaction was monitored by TLC,
and after completion, the reaction mixture was cooled to -20 °C and
treated with dropwise addition of HCl (1 mL, 2 M solution) in MeOH
(5 mL), while stirring for 5 min. The neutralized reaction mixture was
then evaporated under reduced pressure to dryness at 40 °C, leaving
behind Z-CA1G2 (6a). Flash chromatography of the crude product using
a prepacked 12 g RP-18 silica column [eluents: solvent A, acetonitrile;
OCOCH3-G4); HRMS m/z 825.2031 [M
+
Na]+ (calcd for

1100 Journal of Natural Products, 2010, Vol. 73, No. 6
Tanpure et al.
solvent B, water; gradient, 10% A/90% B over 1.15 min (1 CV), 10%
A/90% B f 40% A/60% B over 12.3 min (10 CV), 40% A/60% B
over 2.30 min (2 CV); flow rate 12.0 mL/min; monitored at λ 254 and
280 nm] afforded glucuronide 6a (0.095 g, 0.19 mmol, 75%) as an
off-white solid. Characterization of compound 6a is detailed following
an additional experimental procedure below.
for compounds 22, 25a, and 27a are displayed in the Supporting
Information. All data were processed using Bruker AXS SHELXTL
software, version 6.10.41 All hydrogen atoms were placed in idealized
positions, and all non-hydrogen atoms were refined anisotropically for
22, 25a, and 27a.
Biology. Effects on Tubulin Polymerization. Bovine brain tubulin
was purified as described previously.42 To evaluate the effect of
the compounds on tubulin assembly in vitro, varying concentrations
were preincubated with 10 µM tubulin (1.0 µg/mL) in glutamate
buffer at 30 °C and then cooled to 0 °C. After the addition of GTP,
the mixtures were transferred to 0 °C cuvettes in a recording
spectrophotometer and warmed to 30 °C, and the assembly of tubulin
was observed turbidimetrically.43 The IC50 was defined as the
compound concentration that inhibited the extent of assembly by
50% after a 20 min incubation.
(Z)-3(S),4(S),5(R)-Trihydroxy-6(S)-[(3,4,5-trimethoxy)-(4′-meth-
oxy-2′-hydroxy-3′-stilbenyloxy)]tetrahydropyran-2(S)-carboxylic Acid
(6a). A solution of glucuronate 33a (0.116 g, 0.160 mmol) and NaOH
(0.250 mL, 2 M solution) in MeOH (4 mL) in a sealed vessel was
heated to 50 °C in a microwave for 15 min. The reaction was monitored
by TLC, and after completion, the reaction mixture was cooled to -20
°C and treated with dropwise addition of HCl (0.250 mL, 2 M solution)
in MeOH (5 mL) while stirring for 5 min. The neutralized reaction
mixture was then evaporated under reduced pressure to dryness at 40
°C, leaving behind Z-CA1G2 (6a). Flash chromatography of the crude
product using a prepacked 12 g RP-18 silica column [eluents: solvent
A, acetonitrile; solvent B, water; gradient, 10% A/90% B over 1.15
min (1 CV), 10% A/90% B f 40% A/60% B over 12.3 min (10 CV),
40% A/60% B over 2.30 min (2 CV); flow rate 12.0 mL/min; monitored
at λ 254 and 280 nm] afforded glucuronide 6a (0.068 g, 0.134 mmol,
Cell Lines and Sulforhodamine B Assay. For details regarding
the cell lines and assay conditions see the Supporting Information.
Acknowledgment. The authors are grateful to OXiGENE, Inc.,
and the Welch Foundation (grant no. AA-1278 to K.G.P.) for their
generous financial support of this project, and to the NSF for
instrumentation awards (CHE-0420802 and CHE-0321214). The
authors also thank Mr. C. Carson for assisting with structural analysis
of selected compounds, Dr. A. Ramirez for mass spectral analysis,
and Dr. J. Karban and Dr. M. Nemec for use of the shared Molecular
Biosciences Center.
1
84%) as an off-white solid: mp 65-67 °C; H NMR (DMSO-d6, 500
MHz) δ 6.90 (1H, d, J ) 8.7 Hz, H-6′), 6.58 (2H, s, H-2, H-6), 6.54
(1H, d, J ) 12.2 Hz, H-1′a), 6.40 (1H, d, J ) 8.6 Hz, H-5′), 6.39 (1H,
d, J ) 12.3 Hz, H-1a), 4.56 (1H, d, J ) 7.4 Hz, H-G1), 3.73 (3H, s,
OCH3-4′), 3.63 (3H, s, OCH3-4), 3.59 (6H, s, OCH3-3, -5), 3.43 (1H,
d, J ) 8.9 Hz, H-G5), 3.37 (1H, t, J ) 7.5 Hz, H-G2), 3.26 (1H, t, J
) 7.5 Hz, H-G4), 3.26 (1H, t, J ) 8.6 Hz, H-G3); 13C NMR (DMSO-
d6, 125 MHz) δ 171.6 (C, s, COOH-G6), 152.4 (C, C-3, C-5), 152.2
(C, C-4′), 149.2 (C, C-2′), 136.5 (C, C-4), 134.3 (C, C-3′), 132.4 (C,
C-1), 128.2 (CH, C-1a), 125.2 (CH, C-1′a), 124.9 (CH, C-6′), 118.0
(C, C-1′), 105.9 (CH, C-2, C-6), 105.5 (CH, C-G1), 103.4 (CH, C-5′),
75.7 (CH, C-G3), 75.5 (CH, C-G5), 73.4 (CH, C-G2), 71.9 (CH, C-G4),
Supporting Information Available: Experimental details regarding
1
selected syntheses and the SRB assay, H NMR, 13C NMR, NOESY-
1D, gHSQC, gHMBC, HRMS, HPLC, and single-crystal X-ray
diffraction data. This material is available free of charge via the Internet
at http://pubs.acs.org.
References and Notes
60.0 (CH3, OCH3-4), 56.2 (CH3, OCH3-4′), 55.5 (CH3, OCH3-3, -5);
HRMS (ESI-) m/z 507.1524 [M - 1]- (calcd for C24H27O12
507.1508).
,
-
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using a prepacked 60 g RP-18 silica column [eluents: solvent A,
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J ) 8.8 Hz, H-6′), 7.29 (1H, d, J ) 16.5 Hz, H-1′a), 7.09 (1H, d, J )
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3.73 (1H, t, J ) 8.4 Hz, H-G4), 3.64 (2H, t, J ) 8.5 Hz, H-G2), 3.62
(2H, m, J ) 8.5 Hz, H-G3); 13C NMR (acetone-d6, 125 MHz) δ 170.2
(C, s, COOH-G6), 154.6 (C, C-3, C-5), 153.3 (C, C-4′), 149.7 (C, C-2′),
138.9 (C, C-4), 135.3 (C, C-1), 135.0 (C, C-3′), 128.3 (CH, C-1a),
123.8 (CH, C-6′), 123.6 (CH, C-1′a), 119.8 (C, C-1′), 107.0 (CH, C-G1),
105.1 (CH, C-5′), 104.6 (CH, C-2, C-6), 76.8 (CH, C-G3), 76.4 (CH,
C-G5), 74.8 (CH, C-G2), 72.6 (CH, C-G4), 60.7 (CH3, OCH3-4), 56.8
(CH3, OCH3-4′), 56.5 (CH3, OCH3-3, -5); HRMS (ESI-) m/z 507.1508
[M - 1]- (calcd for C24H27O12-, 507.1508).
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34
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NP100108E