Antineoplastic agents. 445. Synthesis and evaluation of structural modifications of (Z)- and (E)-combretastatin A-4

Article abstract of DOI:10.1021/jm0205797

A series of cis- and trans-stilbenes related to combretastatin A-4 (1a), with a variety of substituents at the 3′-position of the aryl B-ring, were synthesized and evaluated for inhibitory activity employing six human cancer cell lines (NCI-H460 lung carc

Full text of DOI:10.1021/jm0205797

J. Med. Chem. 2005, 48, 4087-4099
4087
Antineoplastic Agents. 445. Synthesis and Evaluation of Structural
Modifications of (Z)- and (E)-Combretastatin A-41
George R. Pettit,*,† Monte R. Rhodes,† Delbert L. Herald,† Ernest Hamel,‡ Jean M. Schmidt,† and
Robin K. Pettit†
Cancer Research Institute and Department of Chemistry, Arizona State University, P.O. Box 872404,
Tempe, Arizona 85287-2404, and Screening Technologies Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702-1201
Received December 27, 2002
A series of cis- and trans-stilbenes related to combretastatin A-4 (1a), with a variety of
substituents at the 3′-position of the aryl B-ring, were synthesized and evaluated for inhibitory
activity employing six human cancer cell lines (NCI-H460 lung carcinoma, BXPC-3 pancreas,
SK-N-SH neuroblastoma, SW1736 thyroid, DU-145 prostate, and FADU pharynx-squamous
sarcoma) as well as the P-388 murine lymphocyte leukemia cell line. Several of the cis-stilbene
derivatives were significantly inhibitory against all cell lines used, with potencies comparable
to that of the parent 1a. All were potent inhibitors of tubulin polymerization. The corresponding
trans-stilbenes had little or no activity as tubulin polymerization inhibitors and were relatively
inactive against the seven cancer cell lines. In terms of inhibition of both cancer cell growth
and tubulin polymerization, the dimethylamino and bromo cis-stilbenes were the most potent
of the new derivatives, the latter having biological activity approaching that of 1a. As part of
the present study, the X-ray crystal structure of the 3′-O-phosphate of combretastatin A-4 (1b)
was successfully elucidated. Compound 1b has been termed the “combretastatin A-4 prodrug”,
and it is currently undergoing clinical trials for the treatment of human cancer patients.
Introduction
Tubulin is one of the most useful and strategic
molecular targets for anticancer drugs.2 Ligands such
as combretastatin A-4 (1a) that bind in the colchicine3
(2) site of tubulin and inhibit cancer cell proliferation
include certain other cis-stilbenes4 (but not their trans
isomers, for example, resveratrol 35), as well as podo-
phyllotoxin6 (4), steganacin7 (5), and some of their
synthetic analogues. The most promising of these
compounds thus far is combretastatin A-44 (1a), which
has been shown to cause mitotic arrest4,8 in murine
leukemia cells and human ovarian and colon cancer cell
lines, exhibits activity against multi-drug-resistant
(MDR) cancer cell lines8a and, most importantly, has
demonstrated powerful cancer antiangiogenesis proper-
ties.9 Currently, the sodium combretastatin A-4 3′-
phosphate4b,c prodrug (1b) is undergoing human cancer
clinical trials and shows significant promise.4m,n
The relative molecular simplicity of combretastatin
A-4 (1a) suggests a number of practical approaches to
the design of new antineoplastic agents, and such efforts
in recent years have been devoted to detailed studies of
the structure-activity relationships (SAR) of variously
substituted stilbenes2a,10 as well as water-soluble
prodrugs.4b,c,9a From these investigations, we noticed
that (a) the (Z)-configuration of the olefin bridge is
essential for biological activity and (b) a substituent at
the 3′-position of the B-ring was almost always neces-
sary for significant cytotoxic activity. The present report
summarizes the synthesis of a selection of 3′-halide,
-amine, and -O-alkylamine derivatives as potential
cancer cell growth inhibitors and evaluation of these
compounds for inhibition of tubulin polymerization and
antimicrobial activity.
* To whom correspondence should be addressed. Tel.: (480) 965-
3351. Fax: (480) 965-8558. E-mail: bpettit@asu.edu.
† Arizona State University.
Wittig reaction4b,11 between phosphonium bromides
6a12 or 6b4d with the aryl aldehydes 7a-e in tetrahy-
‡ National Cancer Institute.
10.1021/jm0205797 CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/24/2005

4088 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Pettit et al.
drofuran with n-butyllithium (sodium hydride may also
be used) to generate the ylide, followed by separation
using flash column chromatography, led to the corre-
sponding cis-stilbenes 8a-e and trans-stilbenes 9a-e.
Next, we completed a synthesis of the 3′-amino deriva-
tive 8f of combretastatin A-4 by reducing the 3′-nitro
precursor 8a. While this synthesis seemed simple upon
initial inspection, the presence of the ethylene bridge
made selective reduction difficult. Initial attempts with
aqueous Na2S2O4/acetone,13 Fe-ammonium chloride,14
NaBH4/CuSO4 in ethanol,15 and hydrazine/montmoril-
lonite16 in refluxing ethanol, while all superficially
promising, failed to give the 3′-amine 8f. In all in-
stances, a complex mixture of products and/or isomer-
ization of the product to the undesired (E)-olefin re-
sulted. Turning our attention to several newer methodolo-
gies involving organometallics, we first tried sonication
of the 3′-nitrostilbene with anhydrous NiCl2,17 but no
reaction product was detected. Attempting a similar
reaction utilizing NiCl2 hexahydrate in combination
with sodium borohydride18 gave the 3′-amino derivative
in yields greater than 50% following purification. Al-
though this is a new route to amine 8f, the compound
has since been reported in the literature by several other
groups using two different methods.10e,19 Nevertheless,
with such an efficient route in hand, the path has been
opened to other potentially useful structural modifica-
tions of combretastatin A-4. Comparison of the biological
activity of the 3′-amine 8f with that of the 3′-amine
derivatives already reported showed a good correlation.
yields, whereas the trans isomers were barely detect-
able. Consequently, the trans-stilbenes 9a-c were
prepared by the Wittig-Horner reaction employing
phosphonate ester 1020 with aryl aldehydes 7a-c and
15-crown-5 as a cocatalyst.21 Under these conditions the
trans isomers were obtained exclusively. The 3′,4-
dihydroxystilbenes 11b and 12b were prepared from the
corresponding O-silyloxylated stilbenes 8e and 9e by
deprotection using tetra-n-butylammonium fluoride in
tetrahydrofuran. The 3′-hydroxypropyloxy derivatives
11a and 12a were prepared by an analogous route from
8d and 9d.
Preparation of the 3′-side-chain tertiary amine de-
rivatives was accomplished by use of two separate
approaches. The first involved synthesis of 2-hydroxy-
ethoxy derivative 13 by reaction of combretastatin A-4
(1a) with ethylene carbonate and base. Treatment of 13
By use of the chemistry described above, the cis
isomers were the major products, and the trans isomers
were isolated as the minor products. From aryl alde-
hydes 7a-c, the cis isomers were obtained in high
with methanesulfonyl chloride gave 14b, and subse-
quent reaction of 14b with the selected amine in
acetonitrile containing triethylamine22 gave products

Structural Modifications of Combretastatin A-4
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4089
15a,c-g. However, owing to the number of steps and
less than desirable yields, most of these amines were
made via a second route: reaction of 1a itself with the
appropriate aminoethyl halide and sodium hydride23
afforded amines 15a,c-f,h,j in yields of 70% or greater.
singlets in the 300-MHz NMR spectrum. Consequently,
those isomers were assigned relative to their corre-
sponding geometric isomer by 500-MHz NMR spectros-
copy, which gave distinct couplets with characteristic
coupling constants, and by 2D NMR experiments.
Further study of the 3′-O-phosphate sodium salt4b,c
(1b) with respect to its crystal structure proved to be
an especially useful avenue of the present investigation.
A crystal of prodrug 1b suitable for X-ray analysis
(Figure 1) was grown from a mixture of water and
acetone (1:2, v/v). On the basis of the X-ray crystal
structure combined with the combustion analysis for
sodium and results of a Karl Fisher water analysis, it
appears that the crystalline combretastatin A-4 prodrug
specimen selected for crystal structure determination
corresponded on average to one sodium atom per
molecule. Presumably the disodium, monosodium, and
acid phosphates exist in equilibrium, and the relative
amounts are a function of pH. For example, at pH near
9.5, the disodium salt should predominate, while at pH
4.5 the monosodium salt is the most stable form, and
at pH <4 the phosphoric acid should be present.
The X-ray crystal structure of combretastatin A-4
prodrug (1b) also suggests that the conformation of this
stilbene is not planar. The crystal structure reveals that
the planes of the two phenyl rings are inclined to each
other, suggesting a low-energy conformation that may
be the one involved in binding at the tubulin receptor
site. After our original isolation and initial structure
determination of combretastatin A-4, we, and more
recently others,25 completed X-ray crystal structure
elucidations of this natural product and found the same
nonplanarity.8d The planar conformation of phosphate
1b would be a high-energy species owing to a nonbonded
interaction between the protons of the two aromatic
rings.
The effects on cancer cell growth and tubulin polym-
erization of the 4 trans- and 20 cis-stilbenes have been
summarized in Tables 1 and 2, respectively. Previous
studies involving combretastatin A-4 (1a) have estab-
lished that the positions of the methoxy groups in both
the A- and B-rings determine the magnitude of cyto-
toxicity and antitumor activity.10f,j Replacement of the
4-methoxy group of the A-ring with a hydroxyl group
(11b and 12b) resulted in a significant reduction (about
100 times) in cancer cell growth inhibition with the cis
isomer, whereas the trans isomer maintained activity
comparable with that of trans-combretastatin A-4.4a,d
The ability of 12b to inhibit tubulin polymerization was
greater than that of the parent trans-stilbene, but we
were unable to evaluate the cis isomer 11b since it was
unstable and isomerized to trans-stilbene 12b in 1 day
at room temperature. This may also account for the
reduced cytotoxicity observed with 11b.
Compound 15i was prepared from the ethoxy chloride
analogue (14a) of 1a, and methylation of 15a gave 15b.
Treatment of 1a with N,N-(2-chloroethyl)aminocarbonyl
chloride in dry pyridine over 54 h gave the 3′-glycine
mustard 16 in 72% yield. The cis or trans geometries
Replacement of the B-ring 3′-hydroxyl group of cis-
stilbene 1a with nitro, dimethylamino, or bromo groups
(compounds 8a-c) resulted in products with a decreased
potency relative to the parent stilbene (1a), albeit still
cytotoxic (ED50 < 10-1 µM). These results suggest that
the location of the four methoxy groups and the presence
of a substituent at the 3′-position of the cis-stilbene are
important features for pronounced inhibition of cancer
cell growth.
1
were established by their characteristic H NMR cou-
pling constants for the olefinic protons, consistent with
this class of compounds of approximately 12 Hz for the
cis and 16-17 Hz for the trans isomers.24,4d In some
cases (8a,b and 9d), the two olefinic protons gave

4090 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Pettit et al.
Figure 1. X-ray crystal structure of combretastatin A-4. Atoms are depicted as 50% thermal probability ellipsoids, and the
dashed lines depict bonds to alternate disordered sites.
a
a
Table 1. Human Cancer Cell Line (GI50
) and Murine P-388 Lymphocytic Leukemia Inhibitory Activity (ED50) of Combretastatins
A-4 and A-4 Prodrug, and Synthetic Modifications
compd
(µg/mL)
leukemia
P-388
pancreas
BXPC-3
neuroblast
SK-N-SH
thyroid
SW1736
lung-NSC
NCI-H460
prostate
DU-145
pharynx
FADU
1a
0.0026
0.0029
2.4
>0.1
0.23
0.029
0.015
0.007
0.00043
>10.0
-
0.34
0.26
>4.0
>10.0
5.0
0.00026
0.00025
0.014
0.010
0.002
0.00023
7.3
0.00026
0.00061
0.0067
0.80
0.034
0.00080
2.8
0.00056
0.00035
0.0038
0.4
0.00076
0.00072
0.047
0.11
0.027
0.00033
>10.0
-
0.00065
0.00045
0.047
0.090
0.0058
0.00053
3.8
1b
8a
8b
0.9
8c
0.16
0.033
0.00033
>0.0
-
8f
>0.010
>10.0
9a
b
9b
-
-
-
-
9c
3.08
0.63
0.40
0.18
0.19
8.1
10.8
0.38
3.4
0.36
0.46
0.43
0.17
4.5
6.3
0.32
3.4
0.59
0.38
0.33
3.4
0.35
3.4
0.40
4.1
0.56
1.5
11.4
0.63
0.23
2.3
3.2
0.069
4.6
0.41
0.47
0.24
1.1
0.49
4.2
0.53
2.9
0.0030
0.67
0.54
0.15
11a
11b
12a
12b
13
0.37
0.25
0.74
6.8
11.6
>10.0
0.28
4.7
>10.0
4.9
4.49
5.5
0.195
2.9
0.045
2.7
2.7
0.028
2.0
0.14
14a
14b
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
16
3.7
2.87
0.523
1.94
0.255
0.421
0.348
0.634
1.60
0.0232
19.0
0.75
0.074
3.0
1.0
0.42
>10.0
37.2
>10.0
2.0
>10.0
2.9
0.21
0.28
1.5
0.64
0.10
1.1
0.34
0.36
3.3
0.22
1.6
0.22
2.8
0.0064
0.31
0.22
0.14
0.60
5.1
0.34
3.3
0.97
>10.0
4.9
0.41
>10.0
2.3
3.4
0.0033
0.56
0.82
1.01
2.8
1.5
2.3
0.68
0.37
0.32
0.31
0.72
a GI50 (HTCL) and ED50 (P-388) refer to the drug dosages required to inhibit tumor cell growth by 50%. There is no mathematical
difference between the two values which are both calculated identically; the only difference is historical usage. b Not tested owing to
instability of the compound.
Among the structural features considered to be im-
portant in such structurally related anticancer agents
as tamoxifen, clomiphene, and nafoxidine26 is the amine
side chain. This substituent has been reported to be an
essential contributor to the anticancer properties of both
tamoxifen and trioxifene.27 Consequently, as an exten-
sion of our SAR efforts with combretastatin A-4, a
selection of derivatives that include amine-containing
(15a,c-j and 16) side chains, as well as quaternary
salt28 15b, were prepared from the parent stilbene (1a).
The result was a series of substances that are only
weakly active against the cancer cell lines employed.
Consistent with earlier observations,4a,d all of the
trans-stilbenes (9a,c, 12a,b) were less potent than their
corresponding cis isomers. Compound 9c showed mod-
erate cytotoxicity (1.0 × 10-1 µM range) in several of
the cell lines tested, while the other trans-stilbenes
proved to be less active.
The principal mechanism of action of the com-
bretastatins has been shown to involve microtubules.29
Accordingly, the newly synthesized compounds were
evaluated for inhibitory effects on tubulin assembly in
comparison with combretastatin A-4 (1a) and its trans
isomer, when sufficient sample was available (Table 2).

Structural Modifications of Combretastatin A-4
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4091
Table 2. Effect of Newly Synthesized Combretastatin
examined. cis-Stilbenes with bulkier substituents at
position C-3′ were invariably much less active as inhibi-
tors of assembly, and in most cases (14a,b, 15a,c-g,
16) showed minimal or no inhibitory activity at 40 µM,
the highest concentration evaluated. Nevertheless, the
hydroxypropyl derivative 11a and the imidazolylethyl
derivative 15i had IC50 values (6.5 and 7.6 µM, respec-
tively) substoichiometric to the tubulin concentration of
10 µM, while the pyrrolloethyl derivative 15h was
weakly inhibitory, with an IC50 value of 20 µM. The
activity of the latter two compounds is somewhat
surprising, but it suggests that the aromatic substituent
overcomes the presumably steric effect that interferes
with the stilbene-tubulin interaction implied by the
inactivity of 14a,b, 15a,c-g, and 16. Curiously, the
better tubulin inhibitor 15i had little cytotoxicity, while
the less effective 15h was quite cytotoxic in four of the
seven cell lines.
Analogues on Tubulin Polymerizationa
compd
IC50 (µM) ( SD
combretastatin A-4 (1a)
1.2 ( 0.03
33 ( 7
2.6 ( 0.5
1.1 ( 0.02
31 ( 1
trans-combretastatin A-4
8a
8c
9c
11a
12b
13
6.5 ( 0.03
14 ( 4
2.8 ( 0.4
>40
>40
>40
>40
>40
>40
>40
>40
20 ( 6
7.6 ( 0.2
>40
14a
14b
15a
15c
15d
15e
15f
15g
15h
15i
16
a Reaction mixtures contained 0.8 M monosodium glutamate
(pH 6.6, with HCl in 2 M stock solution), 10 µM (1.0 mg/mL)
purified tubulin, 4% (v/v) dimethyl sulfoxide (drug solvent), and
varying concentrations of compounds being evaluated. Reaction
mixtures were incubated for 15 min at 37 °C and then chilled on
ice. GTP (0.4 mM) was added, and reaction mixtures were
transferred to cuvettes held at 0 °C. The temperature was raised
to 30 °C, and tubulin assembly was followed for 20 min by
turbidimetry at 350 nm in Beckman DU7400/7500 diode array
spectrophotometers. IC50 values were determined by interpolation
between experimental values. A minimum of two determinations
was performed with each compound. SD, standard deviation.
Two of the new trans-stilbenes were also evaluated
for antitubulin activity. The bromo derivative 9c had
weak activity that was essentially identical to that of
trans-combretastatin A-4 (IC50 values of 31 and 33 µM,
respectively). Somewhat surprisingly, the C-4 demethyl
analogue of trans-combretasatain A-4, compound 12b,
was over twice as active as the parent compound, with
an IC50 value of 14 µM. This could be the result of some
isomerization to the cis isomer, since rapid isomerization
of the latter to 12b had precluded its evaluation for
inhibition of tubulin assembly (see above).
In these studies we evaluated the ability of these
compounds to inhibit the extent of assembly of 10 µM
tubulin following an incubation of 30 °C for 20 min.
In previous work the amino derivative 8f was shown
to have inhibitory activity identical to that of com-
bretastatin A-4 (1a).30 In the present study, the bromo
analogue 8c also was as active as 1a (IC50 values of 1.1
and 1.2 µM, respectively), while the nitro derivative 8a
and the hydroxyethyl derivative 13 were about half as
active (IC50 values of 2.6 and 2.8 µM, respectively). This
contrasts with the cytotoxicity evaluation presented in
Table 1, where only 8f had activity against cells
comparable to the activity of 1a, and 8a,c and 13,
although similar to each other in overall activity, were
substantially less active than 1a in most cell lines
Combretastatin A-4, combretastatin A-4 prodrug, and
several prodrug modifications were previously shown to
have marginal antimicrobial activity.4b Of the com-
bretastatin A-4-related stilbenes available for antimi-
crobial evaluation, compounds 11b, 12b, and 15b,c,d,i
had the most activity (Table 3). Compound 15c was the
most potent, with a minimum inhibitory concentration
(MIC) of <6.25 µg/disk for Micrococcus luteus. Com-
pound 12b had MICs of 6.25-12.5 µg/disk for Crypto-
coccus neoformans and 12.5-25 µg/disk for Neisseria
gonorrhoeae, while compound 11b exhibited a MIC of
12.5-25 µg/disk against N. gonorrhoeae. Compounds
15b,d,i had MICs of 12.5-25 µg/disk for M. luteus.
Table 3. Antimicrobial Activities [MIC (µg/disk)] of the Combretastatin A-4-Related Stilbenes in the Disk Diffusion Assay
Candida Cryptococcus Staphylococcus Streptococcus Enterococcus Micrococcus Stenotrophomonas Escherichia Enterobacter Neisseria
compd albicans neoformans
aureus
pneumoniae
faecalis
luteus
maltophilia
coli
cloacae
gonorrhoeae
8a
*
*
*
*
*
*
*
*
*
*
*
*
*
NT
*
*
*
*
*
*
*
*
*
*
*
*
NT
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NT
*
*
8c
*
*
*
*
9a
9c
11a
11b
*
*
*
*
*
*
*
*
*
*
*
*
*
NT
*
NT
NT
*
NT
NT
*
50-100
*
NT
*
12.5-25
12b 50-100 6.25-12.5
13
*
*
*
12.5-25
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NT
*
NT
*
NT
*
50-100
14a
14b
15a
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
15b 25-50
25-50
50-100
*
12.5-25
*
*
*
15c
15d
15e
15f
15g
15h
15i
16
*
*
*
*
*
*
*
*
25-50
NT
NT
NT
NT
*
50-100
<6.25
NT
NT
NT
NT
*
NT
NT
NT
NT
*
50-100
50-100
*
*
*
*
*
*
*
12.5-25
*
*
*
*
*
*
*
*
*
*
50-100
*
*
50-100
*
*
*
*
*
*
*
12.5-25
*
*
*
*
*
*

4092 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Pettit et al.
mixture, and two phases separated. The aqueous phase was
washed with ether (3 × 200 mL), and the ethereal solution
was added to the THF layer from the reaction mixture. The
combined organic phase was washed with water (3 × 200 mL)
and dried. Removal of solvent in vacuo yielded a crude residue,
which was subjected to flash chromatography (silica gel; eluent
5% EtOAc in hexane) to afford the pure isomeric stilbenes.
3′-Nitro-3,4,4′,5-tetramethoxy-(Z)-stilbene (8a). Follow-
ing flash column chromatography (eluent hexanes-ethyl
acetate, 5:1), the product was obtained as a yellow solid that
recrystallized from ethyl acetate-hexane as a pale yellow
powder (2.2 g, 66%): mp 127-127.5 °C; IR (KBr) νmax 2960
(aromatic) 2937, 2827, 1577, 1527, 1502 (assym. NO2) 1464,
1413, 1365, 1288 (symm. NO2), 1236, 1126, 1004, 856 (C-N),
781 cm-1; EIMS m/z (relative intensity) 345 (M+, 100), 330
(65), 315, 283, 270, 254, 240, 211, 195, 181, 165, 152, 139; 1H
NMR (500 MHz) δ 3.71 (s, 6H, 2 × OCH3), 3.84 (s, 3H, OCH3),
3.93 (s, 3H, OCH3), 6.44 (d, J ) 12.0 Hz, 1H, H-1a′), 6.46 (s,
2H, H-2, H-6), 6.58 (d, J ) 12.1 Hz, 1H, H-1a), 6.93 (d, J )
8.1 Hz, 1H, H-5′), 7.42 (dd, J ) 8.1, 2.1 Hz, 1H, H-6′), 7.79 (d,
J ) 2.1 Hz, 1H, H-2′); 13C NMR (75 MHz) δ 56.95, 56.59, 60.99,
105.85, 113.10, 125.99, 126.87, 129.74, 131.32, 131.82, 134.64,
137.72, 139.48, 151.72, 153.2. Anal. Calcd for C18H19NO6: C,
62.60; H, 5.55; N, 4.06. Found: C, 62.74; H, 5.63; N, 3.92.
Experimental Section
Tetrahydrofuran (THF) was freshly distilled from sodium/
benzoketal, dimethylformamide (DMF) from CaH2, and tri-
ethylamine (TEA) from KOH. All other solvents were redis-
tilled prior to use. Air-sensitive reactions were carried out
under argon. Ether refers to diethyl ether. Organic extracts
of aqueous solutions were dried over MgSO4 unless specified
otherwise.
The following compounds were prepared according to lit-
erature procedures: combretastatin A-4,4d 1-(2-chloroethyl)-
1H-pyrrole,31 1-(2-chloroethyl)-1H-imidazole hydrochloride,32
2-(2-chloroethyl)pyridine,33 3-(2-chloroethyl)pyridine hydro-
chloride,34 2-diethylaminoethyl chloride hydrochloride,35 4-meth-
oxy-3-nitrobenzaldehyde,36 [(3,4,5-trimethoxyphenyl)methyl]-
phosphoric acid diethyl ether,20 4-[(tert-butyldimethylsilyl)oxy]-
3,5-dimethoxybenzaldehyde,10j 3-(3-hydroxypropoxy)-4-meth-
oxybenzaldehyde,37 [3-(tert-butyldimethylsilyl)oxy]-4-methoxy-
benzyltriphenylphosphonium bromide,38 3,4,5-trimethoxy-
benzyltriphenylphosphonium bromide,12 N-(chloroformyl)bis-
(2-chloroethyl)amine,39 and 3-dimethylamino-4-methoxyben-
zaldehyde (7).40 All other chemicals were purchased from either
Acros Organics or Sigma-Aldrich Chemical Co.
Reaction progress and purity of products were checked by
analytical TLC using Analtech (GHLF) silica-coated glass
plates, with visualization by UV254 light, iodine, 0.3% ninhy-
drin in (97:3) n-butanol-HOAc, or 5% anisaldehyde in 95:5:1
ethanol-acetic acid-H2SO4. Separation of products was
achieved by flash column chromatography on fine silica (EM
Sciences, 230-400 mesh ASTM).
Melting points were determined in a capillary tube using
an Electrothermal apparatus (Model IA9200) and are uncor-
rected. The 1H and 13C NMR spectra were determined with a
Varian Gemini 300-MHz spectrometer, unless otherwise noted,
with TMS as an internal standard in CDCl3. The IR spectra
were recorded by employing a Nicolet FTIR model MX-1
spectrophotometer, and the EIMS data were measured with
a MAT 312 mass spectrometer. Microanalyses were performed
at Galbraith Laboratories. X-ray crystal structure analysis was
performed on a Bruker SMART 6000 diffractometer.
3′-Dimethylamino-3,4,4′,5-tetramethoxy-(Z)-stilbene
(8b). Flash column chromatography (eluent hexanes-EtOAc,
5:1) gave the product as a viscous, yellow oil (200 mg, 37%),
which was found to be unstable at room temperature: Rf 0.30
(hexanes-ethyl acetate, 5:1); EIMS m/z (relative intensity) 343
(45), 315 (5), 300 (15), 100 (100); IR (neat) νmax 2850, 2770,
1
1572, 1500, 1450, 1241, 1120, 1010, 870 cm-1; H NMR (500
MHz) δ 2.93 (s, 6H, N(CH3)2), 3.70 (s, 6H, 2 × OCH3), 3.81 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 6.46 (d, J ) 12.0 Hz, 1H, H-1a′),
6.49 (d, J ) 12.0 Hz, 1H, H-1a), 6.50 (s, 2H, H-2, H-6), 6.77
(d, J ) 8.1 Hz, 1H, H-5′), 6.86 (dd, J ) 8.1, 1.6 Hz, 1H, H-6′),
7.23 (d, J ) 1.8 Hz, 1H, H-2′).
3′-Bromo-3,4,4′,5-tetramethoxy-(Z)-stilbene (8c). Re-
crystallization from ethyl acetate-hexane, following purifica-
tion by flash column chromatography, gave prismatic crystals
(4.4 g, 40%): mp 100-101 °C; Rf 0.30 (hexanes-ethyl acetate,
3-[(tert-Butyldimethylsilyloxy)propoxy]-4-methoxy-
benzaldehyde (7d). To a stirred solution of 3-(3-hydroxypro-
poxy)-4-methoxybenzaldehyde37 (1.00 g, 4.8 mmol) in dry DMF
(20 mL) was added imidazole (0.82 g, 12 mmol, 2.5 equiv),
followed by tert-butyldimethylsilyl chloride (0.87 g, 5.76 mmol,
1.2 equiv). The reaction mixture was stirred at room temper-
ature for 4 h, and water (20 mL) was added, followed by
another 10 min of stirring. Ether (25 mL) was added, followed
by saturated sodium bicarbonate solution (25 mL), and stirring
was continued for another 15 min. The ethereal phase was
separated, and the aqueous phase was washed with ether (3
× 25 mL). The combined ether extract was washed with brine
(50 mL) and water (50 mL), dried, and filtered, and solvent
was removed in vacuo. Purification by flash chromatography
gave a clear viscous oil (1.4 g, 98%): Rf 0.38 (hexanes-EtOAc,
9:1); IR (KBr vmax 2850, 2695, 1250, 1110, 1010, 870 cm-1
;
EIMS m/z 379, (M+, 100), 363 (M - CH3)+; 1H NMR δ 3.71 (s,
6H, 2 × OCH3), 3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.42
(d, J ) 12.0 Hz, 1H, H-1a′), 6.47 (d, J ) 12.0 Hz, 1H, H-1a),
6.51 (s, 2H, H-2, H-6), 6.78 (d, J ) 8.7 Hz, 1H, H-5′), 7.19 (dd,
J ) 8.7, 2.4 Hz, 1H, H-6′), 7.55 (d, J ) 2.4 Hz, 1H, H-2′). Anal.
Calcd for C18H19BrO4: C, 57.01; H, 5.05; Br, 21.07. Found: C,
56.99; H, 5.1; Br, 21.00.
3′-[(tert-Butyldimethylsilyloxy)propoxy]-3,4,4′,5-tet-
ramethoxy-(Z)-stilbene (8d). Following purification by flash
chromatography, the product was isolated as a clear oil
(65%): Rf 0.45 (hexanes-ethyl acetate, 7:3); IR (neat) νmax
3001, 2978, 2901, 1745, 1545, 1432, 1211, 1189, 1010, 854
cm-1; EIMS m/z (relative intensity) 488 (M+, 100), 473, 431,
1
9:1); IR (neat) νmax 2955, 2856, cm-1; H NMR δ 0.03 (s, 6H,
1
398, 373, 358, 343, 163, 73; H NMR δ 0.03 (s, 6H, Si(CH3)2),
0.87 (s, 9H, C(CH3)3), 2.05 (q, 2H, CH2CH2CH2-), 3.69 (s, 6H,
2 × OCH3), 3.81 (t, J ) 6.0 Hz, 2H, CH2Si), 3.83 (s, 3H, OCH3),
3.95 (s, 3H, OCH3), 4.20 (t, J ) 6.0 Hz, 2H, OCH2), 6.43 (d, J
) 12.3 Hz, 1H, H-1a′), 6.50 (d, J ) 12.3 Hz, 1H, H-1a), 6.52
(s, 2H, H-2, H-6), 6.76 (d, J ) 8.7 Hz, 1H, H-5′), 7.44 (dd, J )
8.7, 1.5 Hz, 1H, H-6′), 7.47 (d, J ) 1.5 Hz, 1H, H-2′).
Si(CH3)2), 0.87 (s, 9H, C(CH3)3), 2.05 (qn, J ) 7.5, 2 Hz, 2H,
CH2CH2CH2), 3.81 (t, J ) 6.0 Hz, 2H, CH2OSi), 3.93 (s, 3H,
OCH3), 4.17 (t, J ) 6.0 Hz, 2H, OCH2CH2CH2), 6.86 (d, 1H,
H-5), 7.20 (dd, J ) 9.0, 2.4 Hz, 1H, H-6), 7.32 (d, J ) 2.1 Hz,
1H, H-2), 9.79 (s, 1H, CHO); 13C NMR (75 MHz) δ 0.00, 23.74,
31.35, 37.61, 61.53, 64.87, 71.16, 116.04, 131.89, 135.54,
154.56, 160.28, 196.37. Anal. Calcd for C17H28O4Si: C, 62.9;
H, 8.7. Found: C, 63.07; H, 8.76.
3′,4-Di[(tert-butyldimethylsilyl)oxy)]-3,4′,5-trimethoxy-
(Z)-stilbene (8e). Wittig coupling of phosphonium salt 6b with
TBDMS aldehyde 7e and separation (eluent hexanes-ethyl
acetate, 50:1) led to a clear oil (1.3 g, 36%): EIMS m/z 530
(M+, 70), 473 (75), 458 (45), 386 (100); 1H NMR δ 0.086 (s, 6H,
Si(CH3)2), 0.12 (s, 6H, Si(CH3)2), 0.95 (s, 9H, C(CH3)3), 1.00 (s,
9H, C(CH3)3), 3.62 (s, 6H, 2 × OCH3), 3.77 (s, 3H, 4′-OCH3),
6.41 (d, J ) 12.0 Hz, 1H, H-1a′), 6.48 (d, J ) 12.0 Hz, 1H,
H-1a), 6.48 (s, 2H, H-2, H-6), 6.71 (d, J ) 8.4 Hz, 1H, H-5′),
6.83 (m, 1H, H-6′), 7.01 (d, J ) 2.0 Hz, 1H, H-2′). Anal. Calcd
for C29H46O5Si2: C, 65.6; H, 8.7. Found: C, 65.98; H, 9.05.
General Procedure for the Preparation of (Z)-Stil-
benes 8a-d. A homogeneous suspension of phosphonium
bromide 6a (15.1 g, 28.9 mmol) in THF (900 mL) under argon
was cooled and retained at -23 °C for 1 h. Butyllithium (11.6
mL, 28.9 mmol of a 2.5 M solution in hexanes) was added
dropwise via syringe, and the resultant blood-red solution was
stirred at -23 °C for 60 min, at which time the aldehyde (28.9
mmol) in 100 mL of dry THF was added (dropwise) from an
addition funnel. Stirring was continued at -23 °C for 3 h and
at room temperature for 18 h. By this time, the red color had
completely disappeared. Ice-water (300 mL) was added to the
3′-Amino-3,4,4′,5-tetramethoxy-(Z-)stilbene (8f). Nitro-
stilbene 8a (50 mg, 0.8 mmol) and nickel(II) chloride hexahy-

Structural Modifications of Combretastatin A-4
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4093
drate (1.6 mmol, 2 equiv) were dissolved in dry methanol (5
mL). Sodium borohydride (0.12 g, 3.2 mmol, 4 equiv) was added
(in portions with stirring under cooling) over the course of 30
min. The reaction mixture was then warmed to room temper-
ature and stirred for 30 min. The solvent was removed (in
vacuo), the resulting black, granular solid residue was redis-
solved in methanol, and the mixture was filtered through a
1-in. pad of silica gel. The filtrate (200 mL) was reduced in
volume, adsorbed onto silica gel, and separated by flash column
chromatography (hexanes-ethyl acetate, 5:1) to give the
product as a pale yellow gum (25 mg, 63%) that was stored in
an amber vial below 0 °C: Rf 0.38 (hexanes-ethyl acetate, 2:1);
IR (neat) νmax 3472 (N-H stretch), 3371 (N-H stretch), 3001
(aromatic C-H stretch), 2937, 2835, 1614, 1579 (aromatic Cd
C stretch), 1512, 1454, 1327, 1284 (C-N stretch), 1234, 1126,
1006, 875 cm-1; EIMS m/z (relative intensity) 315 (M+, 100),
MHz) δ 56.50, 60.85, 63.33, 103.58, 122.95, 123.93, 125.07,
129.34, 131.80, 132.32, 132.63, 133.48, 134.62, 152.09, 153.31.
Anal. Calcd for C18H19NO6: C, 62.6; H, 5.55; N, 4.05. Found:
C, 61.6; H, 5.49; N, 3.88.
3′-Dimethylamino-3,4,4′,5-tetramethoxy-(E)-stilbene
(9b). Following purification by flash column chromatography,
the resultant pale yellow oil (0.5 g, 29%) was stored at low
temperature owing to thermal and UV instability: Rf 0.26
(hexanes-ethyl acetate, 4:1); EIMS m/z 343 (M+), 328 (M+
-
CH3), 315, 270, 181 (bases); IR (neat) νmax 2930, 2850, 2780,
1
1735, 1580, 1126, 1000 cm-1; H NMR (400 MHz) δ 2.90 (s,
6H, N(CH3)2), 3.90 (s, 3H, OCH3), 3.91 (s, 6H, 2 × OCH3), 3.98
(s, 3H, OCH3), 6.73 (s, 2H, H-2, H-6), 6.96 (d, J ) 17.0 Hz,
1H, H-1a′), 7.01 (d, J ) 17.1 Hz, 1H, H-1a), 7.10 (d, J ) 8.5
Hz, 1H, H-5′), 7.67 (dd, J ) 8.6, 2.2 Hz, 1H, H-6′), 8.03 (d, J )
2.4 Hz, 1H, H-2′); 13C NMR (100 MHz) δ 40.6 (N(CH3)2), 56.00,
60.98, 64.01, 103.66, 122.90, 124.01, 125.21, 129.66, 132.00,
132.56, 132.72, 133.59, 134.83, 152.11, 151.6 (C-3′).
3′-Bromo-3,4,4′,5-tetramethoxy-(E)-stilbene (9c). After
purification by column chromatography, the solid was recrys-
tallized from ethanol as needles (0.56 g, 65%): mp 142-144
°C; Rf 0.23 (hexanes-ethyl acetate, 9:1); EIMS m/z (relative
intensity) 379 (M+, 100), 363, 302, 256, 241, 225, 211, 198; 1H
NMR δ 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.91 (s, 6H, 2
× OCH3), 6.71 (s, 2H, H-1a′, H-1a), 6.88 (d, J ) 8.4 Hz, 1H,
H-5′), 6.89 (s, 2H, H-2, H-6), 7.38 (dd, J ) 8.7, 2.1 Hz, 1H,
H-6′), 7.89 (d, J ) 2.2 Hz, 1H, H-2′); 13C NMR (75 MHz) δ
56.06, 56.27, 60.93, 103.43, 111.89, 126.187, 126.75, 127.84,
130.84, 131.44, 132.89, 137.88, 153.36. Anal. Calcd for C18H19-
BrO4: C, 57.01; H, 5.05; Br, 21.07. Found: C, 56.34; H, 5.04;
Br, 21.00.
General Method for the Preparation of Stilbenes 11a,b
and 12a,b. A solution of tetra-n-butylammonium fluoride in
THF (1.0 M, 2 mL, 2 mmol) was added to a solution of the
stilbene (8d,e; 9d,e, 1 mmol) in THF (5 mL), and the mixture
was stirred at room temperature. After 30 min, ice (1 g) was
added, followed by ether (10 mL). The organic layer was
washed with water (2 × 10 mL), dried, and filtered. Evapora-
tion of the solvent and separation of the crude product by flash
chromatography (40% ethyl acetate in hexanes) afforded the
product.
1
300 (M - NH)+, 285, 254, 240, 225, 152; H NMR δ 3.70 (s,
6H, 2 × OCH3), 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.37
(d, J ) 12.6 Hz, 1H, H-1a′), 6.46 (d, J ) 12.1 Hz, 1H, H-1a),
6.55 (s, 2H, H-2, H-6), 6.68 (s, 2H, H-5′, H-6′), 6.70 (d, J ) 1.2
Hz, 1H, H-2′); 13C NMR (75 MHz) δ 55.49, 55.90, 60.90, 106.04,
110.06, 115.23, 119.50, 128.36, 129.99, 132.96, 135.73, 137.03,
146.61, 152.79; HRFABMS (M)+ calcd for C18H21NO4, 315.1471,
found 315.1465.
3′-[(tert-Butyldimethylsilyl)propoxy]-3,4,4′,5-tetra-
methoxy-(E)-stilbene (9d). The olefin recrystallized from
ethyl acetate-heptane as colorless needles (35%): mp 116-
117 °C; Rf 0.36 (hexanes-ethyl acetate, 7:3); IR (KBr) νmax
2998, 2875, 1725, 1654, 1432, 1211, 1178, 1020, 854 cm-1
;
EIMS m/z (relative intensity) 488 (M+, 100), 473, 431, 398,
373, 358, 343, 163; 1H NMR (500 MHz) δ 0.087 (s, 6H, Si-
(CH3)2), 1.01 (s, 9H, C(CH3)3), 2.10 (q, 2H, CH2CH2CH2), 3.70
(s, 3H, OCH3), 3.80 (t, J ) 6.0 Hz, 2H, -CH2-Si), 3.81 (s, 6H,
2 × OCH3), 3.91 (s, 3H, OCH3), 4.18 (t, J ) 6.0 Hz, 2H, OCH2),
6.56 (d, J ) 16.3 Hz, 1H, H-1a′), 6.58 (s, 2H, H-2, H-6), 6.62
(d, J ) 16.4 Hz, 1H, H-1a), 7.01 (d, J ) 8.7 Hz, 1H, H-5′), 7.36
(s, 1H, H-6′), 7.50 (d, J ) 1.2 Hz, 1H, H-2′). Anal. Calcd for
C27H40O6Si: C, 66.36; H, 8.25. Found: C, 66.35; H, 8.45.
3′,4-Di((tert-butyldimethylsilyl)oxy)-3,4′,5-trimethoxy-
(E)-stilbene (9e). Following the Wittig reaction and purifica-
tion as described above (see 8e), the solid product recrystallized
from ethyl acetate-hexane as prisms (1.4 g, 37%): mp 85-86
°C; EIMS m/z (relative intensity) 530 (M+, 70), 473 (75), 458
(45), 386 (100); 1H NMR δ 0.139 (s, 6H, Si(CH3)2), 0.181 (s,
6H, Si(CH3)2), 1.02 (s, 9H, C(CH3)3), 1.16 (s, 9H, C(CH3)3), 3.82
(s, 3H, 4′-OCH3), 3.84 (s, 6H, 2 × OCH3), 6.69 (s, 2H, H-2, H-6),
6.80 (d, J ) 16.5 Hz, 1H, H-1a′), 6.85 (s, 1H, H-5′), 6.88 (d, J
) 16.5 Hz, 1H, H-1a), 7.02 (s, 1H, H-6′), 7.05 (d, J ) 1.8 Hz,
1H, H-2′). Anal. Calcd for C29H46O5Si2: C, 65.6; H, 8.7.
Found: C, 65.68; H, 9.00.
General Procedure for Preparation of (E)-Stilbenes
(9a-c). A solution of the aldehyde (5 mmol) and the diethyl
phosphonate (10, 5 mmol) in dry THF (10 mL) was added to a
stirred slurry of sodium hydride (NaH) (5 mmol) in THF (10
mL) containing 15-crown-5 (0.15 mmol) at 0 °C (under argon).
A rapid evolution of H2 was observed, and the solution
developed a gelatinous, orange/red appearance. Upon comple-
tion of addition (ca. 15 min), the reaction mixture was warmed
to room temperature and stirred for 12 h. The mixture was
poured into water (15 mL), transferred to a separatory funnel,
and extracted with ether (3 × 20 mL). The combined extract
was washed with 10% NaHSO3 (2 × 10 mL) and saturated
NaCl (2 × 10 mL) and dried. Evaporation of solvent followed
by flash chromatographic separation gave the desired product.
3′-O-(3′′-Hydroxypropyl)-combretastatin A-4 (11a).
Deprotection as described above, followed by recrystallization
from ethyl acetate-hexane, afforded colorless needles (0.98 g,
89%): mp 48-50 °C; Rf 0.25 (hexanes-ethyl acetate, 3:2); IR
(neat) νmax 3533 (OH), 2999, 2937, 2837, 1736, 1579, 1126,
1004, 869 (cis CdC) cm-1; EIMS m/z (relative intensity) 374
1
(M+, 100), 359 (35), 316 (5), 301 (10), 241 (5); H NMR δ 1.88
(q, 2H, CH2CH2CH2), 2.84 (bs, 1H, -OH), 3.63 (s, 6H, 2 ×
OCH3), 3.70 (t, J ) 6.0 Hz, 2H, ArOCH2), 3.74 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 3.87 (t, J ) 6.0 Hz, 2H, CH2CH2OH), 6.37
(d, J ) 12.0 Hz, 1H, H-1a′), 6.42 (d, J ) 12.0 Hz, 1H, H-1a),
6.45 (s, 2H, H-2, H-6), 6.68 (d, J ) 8.1 Hz, 1H, H-5′), 6.76 (dd,
J ) 8.1, 1.5 Hz, 1H, H-6′), 6.80 (d, J ) 1.5 Hz, 1H, H-2′); 13C
NMR (75 MHz) δ 31.69, 55.92, 60.84, 67.84, 105.96, 111.08,
113.59, 122.37, 128.79, 129.69, 129.80, 133.09, 136.99, 147.58,
148.59, 152.97. Anal. Calcd for C21H26O6: C, 62.9; H, 8.7.
Found: C, 63.07; H, 8.76.
3′,4-Dihydroxy-3,4′,5-trimethoxy-(Z)-stilbene (11b).
Deprotection as described above yielded a clear oil that
solidified upon standing. Recrystallization from ethyl acetate-
hexane afforded pale yellow needles (0.15 g, 80%): mp 104-
106 °C; Rf 0.38 (hexane-acetone, 3:2); EIMS m/z (relative
intensity) 302 (M+, 100), 287, 269, 256, 227, 213, 199, 181; IR
(neat) νmax 3005 (OH), 2998 (aromatic), 2920, 2840, 1641, 1573,
1500, 1454, 1325, 1242, 1121, 870 (cis CdC) cm-1; 1H NMR δ
3.65 (s, 6H, 2 × OCH3), 3.75 (s, 3H, 4′-OCH3), 4.98 (bs, 2H, 2
× OH), 6.51 (d, J ) 12.1 Hz, 1H, H-1a′), 6.58 (d, J ) 12.0 Hz,
1H, H-1a), 6.59 (s, 2H, H-2, H-6), 6.71 (d, J ) 8.3 Hz, 1H, H-5′),
6.80 (dd, J ) 8.4, 2.1 Hz, 1H, H-6′), 6.83 (d, J ) 1.8 Hz, 1H,
H-2′); 13C NMR (75 MHz) δ 56.01 (OCH3), 56.11 (4′-OCH3),
105.20 (C-2, C-6), 111.52 (C-5′), 117.08 (C-2′), 121.22 (C-6′),
129.01 (C-1a′), 129.51 (C-1a), 130.81 (C-1′), 132.48 (C-1), 137.54
3′-Nitro-3,4,4′,5-tetramethoxy-(E)-stilbene (9a). Follow-
ing purification by column chromatography, the resultant
yellow solid recrystallized slowly from ethyl acetate-hexane
as pale yellow needles (1.0 g, 64%): mp 159-160 °C; Rf 0.20
(hexanes-ethyl acetate, 3:1); EIMS m/z 345 (M+), 330, 315,
270, 254, 240, 195, 181 (base); 1H NMR δ 3.89 (s, 3H, OCH3),
3.92 (s, 6H, 2 × OCH3), 3.99 (s, 3H, OCH3), 6.73 (s, 2H, H-2,
H-6), 6.93 (d, J ) 16.5 Hz, 1H, H-1a′), 7.00 (d, J ) 16.5 Hz,
1H, H-1a), 7.09 (d, J ) 8.7 Hz, 1H, H-5′), 7.65 (dd, J ) 8.7, 2.1
Hz, 1H, H-6′), 8.01 (d, J ) 2.4 Hz, 1H, H-2′); 13C NMR (75

4094 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Pettit et al.
(C-4), 145.21 (C-4′), 145.67 (C-4′), 146.02 (C-3′), 152.67 (C-3,
C-5). Anal. Calcd for C17H18O5: C, 67.5; H, 6.0. Found: C, 67.2;
H, 6.12.
followed by purification by flash chromatography (eluent
hexanes-ethyl acetate, 2:1), gave the product as a colorless
oil (0.16 g, 65%): Rf 0.63 (hexanes-ethyl acetate, 2:1); EIMS
m/z (relative intensity) 378 (M+, 100), 363 (58), 328 (3), 313
(5), 284 (7), 269 (7), 252 (10), 241 (13), 225 (10), 210 (8), 197
(12), 155 (12), 115 (12), 64 (20), 27 (22); 1H NMR δ 3.67 (s,
6H, 2 × OCH3), 3.68 (t, J ) 6.0 Hz, 2H, -CH2CH2Cl), 3.80 (s,
3H, OCH3), 3.81 (s, 3H, OCH3), 4.04 (t, J ) 6.0 Hz, 2H, OCH2-
CH2Cl), 6.42 (d, J ) 12.6 Hz, 1H, H-1a′), 6.46 (d, J ) 12.6 Hz,
1H, H-1a), 6.47 (s, 2H, H-2, H-6), 6.76 (d, J ) 8.7 Hz, 1H, H-5′),
6.81 (d, J ) 2.4 Hz, 1H, H-2′), 6.88 (dd, J ) 8.7, 2.1 Hz, 1H,
H-6′); 13C NMR (75 MHz) δ 41.42 (CH2Cl), 55.89 (3 × OCH3),
60.75 (4-OCH3), 68.96 (-OCH2CH2), 105.80 (C-2), 111.65 (C-
5′), 114.83 (C-2′), 123.03 (C-6′), 128.97 (C-1a′), 129.26 (C-1a),
129.83 (C-1′), 132.73 (C-1), 137.00 (C-4), 146.90 (C-4′), 148.82
(C-3′), 152.85 (C-5,3).
3′-O-(3′′-Hydroxypropyl)-3,4,4′,5-tetramethoxy-(E)-stil-
bene (12a). Deprotection by the general procedure gave a
colorless solid that was crystallized from ethanol-pentane as
colorless prisms (0.12 g, 90%): mp 121-123 °C; Rf 0.20
(hexanes-ethyl acetate, 3:12); EIMS m/z (relative intensity)
374 (M+, 83), 359 (20), 316 (8), 301 (10), 241 (7); IR (KBr) νmax
3501 (OH), 2989 (aromatic), 2934, 1585, 1501, 1460, 1450,
1
1404, 1323, 1270, 1262, 1253, 1125 cm-1; H NMR δ 1.81 (q,
2H, CH2CH2CH2OH), 2.75 (bs, 1H, OH), 3.71 (t, J ) 6.5 Hz,
2H, ArOCH2), 3.85 (s, 3H, OCH3), 3.88 (t, J ) 6.5 Hz, 2H, CH2-
OH), 3.91 (s, 3H, OCH3), 3.92 (s, 6H, 2 × OCH3), 6.72 (s, 2H,
H-2, H-6), 6.85 (d, J ) 8.5 Hz, 1H, H-5′), 6.92 (d, J ) 16.1 Hz,
1H, H-1a′), 6.93 (d, J ) 16.3 Hz, 1H, H-1a), 7.01 (dd, J ) 8.5,
2.0 Hz, 1H, H-6′), 7.23 (d, J ) 2.3 Hz, 1H, H-2′); 13C NMR (75
MHz) δ 32.10 (CH2), 56.01 (OCH3), 60.66 (4-OCH3), 60.84 (CH2-
OH), 67.84 (ArOCH2-), 105.96 (C-6, C-2′), 111.08 (C-2′), 113.59
(C-5′), 122.37 (C-6′), 128.79 (C-1a′), 129.69 (C-1a), 129.80 (C-
1′), 133.09 (C-1), 136.99 (C-4), 147.58 (C-4′), 148.59 (C-3′),
152.97 (C-3, C-5). Anal. Calcd for C21H26O6: C, 62.9; H, 8.7.
Found: 62.5; H, 8.5.
Note: Ether 14a proved to be thermally unstable and
decomposed readily at room temperature. Although it is stable
in the refrigerator under an inert atmosphere for approxi-
mately 1 day, it should be used immediately following puri-
fication.
3′-O-[2′′-(Methanesulfonyl)ethyl]-combretastatin A-4
(14b). Freshly distilled triethylamine (0.791 mL, 5.65 mmol,
4 equiv) was slowly added (dropwise) to a stirring solution of
alcohol 13 (0.51 g, 1.4 mmol) and methanesulfonyl chloride
(0.44 mL, 5.65 mmol, 4 equiv) in THF (10 mL) at 0 °C. After
0.5 h, water (10 mL) was added to the reaction mixture, and
the aqueous phase was removed and extracted with ethyl
acetate (3 × 10 mL). The combined organic extract was dried,
filtered, and concentrated in vacuo, resulting in a pale yellow
oil. This was subjected to flash column chromatography (eluent
hexane-acetone, 2:1) to yield the product as a clear oil (0.66
g, 95%): EIMS m/z (relative intensity) 438 (M+, 100), 423 (38),
327 (6), 269 (4), 241 (6), 197 (4), 155 (4), 123 (25), 79 (28); IR
(neat) νmax 2937 (aromatic), 2839, 1736, 1579, 1512, 1356 (S(d
O)2 stretch), 1242 (C-O-C), 1177 (asymm. S(dO)2 stretch),
3′,4-Dihydroxy-3,4′,5-trimethoxy-(E)-stilbene (12b).
Deprotection followed by recrystallization from ethyl acetate-
hexane gave colorless opalescent flakes (0.11 g, 98%): mp 150-
152 °C; Rf 0.40 (hexane-acetone, 3:2); EIMS m/z 302 (M+, 100),
287, 269, 256, 181; IR (KBr) νmax 3010 (OH), 2998, 1560, 1500,
1
1463, 1271, 1010 cm1; H NMR δ 3.81 (s, 3H, OCH3), 3.92 (s,
6H, 2 × OCH3), 5.03 (bs, 1H, OH), 6.68 (s, 2H, H-2, H-6), 6.71
(d, J ) 8.4 Hz, 1H, H-5′), 6.86 (d, J ) 16.0 Hz, 1H, H-1a′),
6.88 (d, J ) 16.2 Hz, 1H, H-1a), 7.10 (dd, J ) 8.5, 1.2 Hz, 1H,
H-6′), 7.18 (d, J ) 1.2 Hz, 1H, H-2′); 13C NMR (75 MHz) δ
55.98 (OCH3), 56.10 (OCH3), 60.83 (4-OCH3), 103.41 (C-2, C-6),
110.61 (C-5′), 111.62 (C-2′), 120.01 (C-6′), 126.99 (C-1a′), 127.81
(C-1a), 130.90 (C-1′), 133.31 (C-1), 137.59 (C-4), 145.75 (C-4′),
146.52 (C-3′), 153.32 (C-3, C-5). Anal. Calcd for C17H18O5: C,
67.5; H, 6.0. Found: C, 66.90; H, 6.05.
1
1128, 925 cm-1; H NMR δ 3.10 (s, 3H, SO2CH3), 3.70 (s, 6H,
2 × OCH3), 3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.05 (t, J
) 4.5 Hz, 2H, CH2OSO2), 4.49 (t, J ) 4.5 Hz, 2H, ArOCH2-),
6.46 (s, 2H, H-1a′, H-1a), 6.51 (s, 2H, H-2, H-6), 6.76 (d, J )
8.4 Hz, 1H, H-5′), 6.79 (d, J ) 1.8 Hz, 1H, H-2′), 6.90 (dd, J )
8.3, 1.8 Hz, 1H, H-6′); 13C NMR (75 MHz) δ 37.59, 55.82, 55.97,
60.84, 66.95, 68.40, 106.00, 111.70, 114.81, 123.33, 129.15,
129.31, 129.93, 132.88, 137.18, 146.93, 148.90, 152.99. Anal.
Calcd for C21H26O8S: C, 57.5; H, 6.0. Found: C, 56.8; H, 5.7.
3′-O-(2′′-Hydroxyethyl)-combretastatin A-4 (13). A mix-
ture of the phenol 1a (4.0 g, 12.6 mmol), ethylene carbonate
(3.7 g, 42 mmol, 3.3 equiv), and potassium carbonate (5.22 g,
37.8 mmol, 3 equiv) in anhydrous DMF (15 mL) was heated
for 24 h at 100 °C under argon, whereupon TLC analysis
showed that the reaction was complete. After cooling, water
(25 mL) was added, followed by dichloromethane (30 mL). The
organic layer was removed, washed with 3 N NaOH (3 × 25
mL) and brine (25 mL), dried, filtered, and evaporated under
reduced pressure. Separation by flash column chromatography
(hexane-acetone, 2:1) gave a colorless oil that solidified upon
cooling. Recrystallization from ethyl acetate-hexane gave
colorless crystals (4.1 g, 90%): mp 89 °C; Rf 0.24 (hexane-
acetone, 2:1); EIMS m/z (relative intensity) 360 (100), 345 (46),
301 (4), 268 (2), 241 (4), 181 (3), 142 (4), 115 (4); IR (neat) νmax
General Procedures for the Preparation of Com-
bretastatin A-4 3′-Derivatives 15a,c-j. Method A. 3′-O-
[2′′-(N-Pyrrolidinyl)ethyl]-combretastatin A-4 (15d). To
a stirred solution of phenol 1a (0.5 g, 1.6 mmol) in DMF (10
mL) under argon was added NaH (0.38 g of a 60% dispersion,
9.5 mmol, 6 equiv). The mixture was stirred at room temper-
ature for 30 min, by which point H2 evolution had ceased. The
reaction mixture was heated to 90 °C (oil bath), and 1-(2-
chloroethyl)pyrrolidine hydrochloride (0.61 g, 3.6 mmol, 2.25
equiv) was added in portions over 30 min. The reaction mixture
was stirred at 90 °C for another 12 h, whereupon TLC analysis
(hexanes-ethyl acetate, 3:2) showed the reaction to be com-
plete. The mixture was cooled to ambient temperature, and
isopropyl alcohol (3 mL) was added to destroy excess NaH.
The slurry was partitioned between water (50 mL) and ether
(50 mL), and the water was extracted with ether (3 × 20 mL).
The combined ethereal extract was dried (Na2SO4), filtered,
and concentrated in vacuo to yield a brown oil. Chromato-
graphic separation (hexanes-ethyl acetate-triethylamine, 6:4:
1) gave the product as a pale yellow oil (0.60 g, 90%). The oily
product was dissolved in ether and cooled in an ice bath, and
1.0 equiv of 1.0 N HCl in ether was added dropwise until a
precipitate formed. The solvent was removed in vacuo, and
the colorless solid was recrystallized (3×) from 2-propanol-
ether to give the hydrochloride salt as colorless needles: mp
131-132 °C; Rf 0.47 (hexanes-ethyl acetate-triethylamine,
6:4:1); IR (neat) νmax 2957, 2833, 2783, 1676, 1579, 1512, 1462,
1327, 1238, 1128, 1028, 869, 776 cm-1; EIMS m/z 413 (M+),
3418, 2937, 1641, 1512, 1413, 1238, 1188, 1016, 868, 775 cm-1
;
1H NMR δ 2.44 (bs, 1H, OH), 3.70 (s, 6H, 2 × OCH3), 3.83 (t,
J ) 3.9 Hz, 2H, CH2OH), 3.84 (s, 3H, 4-OCH3), 3.85 (s, 3H,
4′-OCH3), 3.93 (t, J ) 3.9 Hz, 2H, CH2CH2), 6.43 (d, J ) 12.0
Hz, 1H, H-1a′), 6.49 (d, J ) 12.0 Hz, 1H, H-1a), 6.51 (s, 2H,
H-2, H-6), 6.78 (d, J ) 8.4 Hz, 1H, H-5′), 6.88 (dd, J ) 8.4, 2.1
Hz, 1H, H-6′), 6.92 (d, J ) 2.1 Hz, 1H, H-2′); 13C NMR (75
MHz) δ 55.89 (OCH3), 56.00 (OCH3), 60.89 (4-OCH3), 61.09
(CH2CH2), 71.19 (CH2CH2), 106.01 (C-2), 160.28, 196.37. Anal.
Calcd for C20H24O6: C, 66.65; H, 6.71. Found: C, 66.63; H,
6.78.
3′-O-(2′′-Chloroethyl)-combretastatin A-4 (14a). A 60%
dispersion of NaH in mineral oil (25 mg, 0.63 mmol) was added
in portions to a stirred solution of phenol 1a (0.20 g, 0.63 mmol)
in dry DMF (10 mL). The mixture was stirred for 30 min and
cooled to 0 °C before the addition of 2-chloroethyl methane-
sulfonate (120 mg, 0.76 mmol, 1.2 equiv). Stirring was
continued at 65 °C for 2 h, whereupon the reaction mixture
was poured into water and extracted with ethyl acetate (3 ×
20 mL). Drying, filtration, and removal of solvent in vacuo,

Structural Modifications of Combretastatin A-4
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4095
382, 316, 241, 211, 165, 115, 98, 84 (base); 1H NMR δ 1.78 (m,
4H, pyrrolidine 3,4-H), 2.58 (m, 4H, pyrrolidine 2,5-H), 2.86
(t, J ) 6.6 Hz, 2H), 3.34 (s, 3H, OCH3), 3.69 (s, 6H, 2 × OCH3),
3.83 (s, 3H, OCH3), 3.98 (t, J ) 6.6 Hz, 2H), 6.43 (d, J ) 12.0
Hz, 1H, H-1a′), 6.48 (d, J ) 12.0 Hz, 1H, H-1a), 6.51 (s, 2H,
H-2, H-6), 6.76 (d, J ) 8.7 Hz, 1H, H-5′), 6.86 (dd, J ) 4.0 Hz,
1.8, 1H, H-6′), 6.88 (bd, J ) 1.8 Hz, 1H, H-2′). Anal. Calcd for
C24H32ClNO5: C, 64.06; H, 7.17; N, 3.11. Found: C, 64.00; H,
7.17; N, 3.00.
119-120 °C; Rf 0.29 (hexanes-ethyl acetate-triethylamine, 6:4:
1); EIMS m/z (relative intensity) 429 (M+, 22), 398 (1), 316
(2), 114 (32), 100 (100); IR (neat) νmax 2999, 2953, 2835, 1579,
1
1512, 1454, 1327, 1126, 1026, 858 cm-1; H NMR δ 2.53 (t, J
) 4.5 Hz, 4H, R-CH2), 2.74 (t, J ) 6.0 Hz, 2H, OCH2CH2N),
3.70 (s, 6H, 2 × OCH3), 3.71 (t, J ) 4.5 Hz, 4H, â-CH2), 3.83
(s, 3H, 4′-OCH3), 3.84 (s, 3H, 4-OCH3), 3.95 (t, J ) 6.0 Hz,
2H, OCH2CH2), 6.44 (d, J ) 12 Hz, 1H, H-1a′), 6.49 (d, J ) 12
Hz, 1H, H-1a), 6.52 (s, 2H, H-2, H-6), 6.77 (d, J ) 8.1 Hz, 1H,
H-5′), 6.86 (dd, J ) 8.1 Hz, 1.5, 1H, H-6′), 6.89 (d, J ) 1.5 Hz,
1H, H-2′); 13C NMR (75 MHz) δ 53.55 (R-CH2), 54.00 (OCH3),
55.95 (OCH3), 57.39 (OCH2CH2N), 60.89 (4-OCH3), 66.61
(-OCH2CH2N), 66.90 (â-CH2), 106.00 (C-2), 111.44 (C-5′),
114.19 (C-2′), 122.45 (C-6′), 128.89 (C-1a′), 129.62 (C-1a),
129.93 (C-1′), 132.90 (C-1), 137.15 (C-4), 147.68 (C-4′), 148.79
(C-3′), 152.97 (C-5,3). Anal. Calcd for C24H32ClNO6: C, 61.86;
H, 6.92; N, 3.01. Found: C, 61.96; H, 7.67; N, 3.00.
3′-O-[2′′-(N,N-Dimethylamino)ethyl]-combretastatin A-4
(15a). Ether formation via method A gave a pale yellow oil
(0.52 g, 85%) following flash column chromatography (hex-
anes-ethyl acetate-triethylamine, 6:4:1). The hydrochloride
salt was prepared as described and crystallized from 2-pro-
panol-ether as colorless needles: mp 125-126 °C; Rf 0.27
(hexanes-ethyl acetate-triethylamine, 6:4:1); EIMS m/z (rela-
tive intensity) 387 (M+, 30), 356 (M+ - Me2, 1), 316 (1), 241
(1), 195 (1), 165 (2), 126 (1), 98 (2), 86 (2), 72 (28), 58 (100); IR
(neat) νmax 2939, 2831, 2771, 1579, 1512, 1462, 1427, 1327,
3′-O-[2′′-(N-Pyrrollyl)ethyl]-combretastatin A-4 (15h).
Use of method A led to 0.43 g (67% yield) of amine 15h as a
deep yellow-brown gum. Subsequent conversion to the hy-
drochloride salt yielded a solid that crystallized from ethanol-
ether to afford colorless needles: mp 155-157 °C; Rf 0.25
(acetone-hexane, 3:1); EIMS m/z (relative intensity) 409 (M+,
25), 329 (11), 316 (5), 100 (100); IR (neat) νmax 2870, 2710, 1580,
1
1238, 1128, 1028, 869 cm-1; H NMR δ 2.30 (s, 6H, N(CH3)2),
2.70 (t, 2H, J ) 6.0 Hz, CH2CH2N), 3.70 (s, 6H, 3,5-OCH3),
3.83 (s, 3H, 4-OCH3), 3.84 (s, 3H, 4′-OCH3), 3.92 (t, 2H, J )
6.0 Hz, OCH2CH2), 6.44 (d, 1H, J ) 12.0 Hz, H-1a′), 6.50 (d,
1H, J ) 12.0 Hz, H-1a), 6.52 (s, 2H, H-2, H-6), 6.77 (d, 1H, J
) 7.5 Hz, H-5′), 6.86 (s, 1H, H-6′), 6.89 (d, 1H, J ) 1.8 Hz,
H-2′). Anal. Calcd for C22H30ClNO5: C, 62.33; H, 7.13; N, 3.30.
Found: C, 62.14; H, 7.08; N, 3.23.
1
1512, 1455, 1327, 1127, 1050, 825 cm-1; H NMR δ 3.12 (t, J
) 6.6 Hz, 2H, CH2CH2N), 3.70 (s, 6H, 2 × OCH3), 3.84 (s, 3H,
4′-OCH3), 3.85 (s, 3H, 4-OCH3), 3.95 (t, J ) 6.6 Hz, 2H, OCH2-
CH2), 6.38 (m, 3J ) 3.5, 2.6, 1.3 Hz, 2H, $$4,4′-CH), 6.45 (d, J
) 12.0 Hz, 1H, H-1a′), 6.49 (d, J ) 12.1 Hz, 1H, H-1a), 6.51
(s, 2H, H-2, H-6), 6.76 (d, J ) 8.1 Hz, 1H, H-5′), 6.86 (dd, J )
8.1, 1.5 Hz, 1H, H-6′), 6.89 (d, J ) 1.5 Hz, 1H, H-2′), 6.94 (m,
3J ) 3.5, 2.6, 1.2 Hz, 2H, R,R′-CH). Anal. Calcd for C24H28-
ClNO5: C, 64.64; H, 6.33; N, 3.14. Found: C, 64.22; H, 6.30;
N, 3.11.
3′-O-[2′′-(N,N-Diethylamino)ethyl]-combretastatin A-4
(15c). Use of method A led to a pale yellow oil (0.26 g, 98%)
following purification by flash column chromatography (hex-
anes-ethyl acetate-triethylamine, 6:4:1). The hydrochloride
salt (prepared as described above, see 15d) crystallized from
2-propanol-ether as colorless needles: mp 113-114 °C; Rf 0.60
(hexanes-ethyl acetate-triethylamine, 6:4:1); EIMS m/z (rela-
tive intensity) 415 (M+, 40), 400 (M+ - CH3, 7), 384 (12), 342
(3), 316 (10), 100 (40), 86 (100); IR (neat) νmax 2966, 2833, 2771,
3′-[2′′-(2′′Pyridyl)ethy]-combretastatin A-4 (15j). Method
A led to a pale yellow-brown oil (0.26 g, 39%), following
purification by flash column chromatography (hexanes-ethyl
acetate-triethylamine, 4:4:2). The hydrochloride salt (pre-
pared as described above, see 15d) crystallized from 2-pro-
panol-ether as colorless needles: mp 178-180 °C; Rf 0.30;
EIMS m/z (relative intensity) 421 (M+, 10), 316 (11), 100 (100);
1579, 1512, 1483, 1427, 1327, 1238, 1128, 1026, 871, 763 cm-1
;
1H NMR δ 1.01 (t, J ) 7.2 Hz, 6H, N(CH2CH3)2), 2.56 (q, J )
7.2 Hz, 4H, N(CH2CH3)2), 2.83 (t, J ) 6.6 Hz, 2H, CH2N), 3.68
(s, 6H, 3, 5-OCH3), 3.82 (s, 3H, 4-OCH3), 3.83 (s, 3H, 4′-OCH3),
3.90 (t, J ) 6.6 Hz, 2H, OCH2), 6.42 (d, J ) 12.0 Hz, 1H, H-1a′),
6.49 (d, J ) 12.0 Hz, 1H, H-1a), 6.51 (s, 2H, H-2, H-6), 6.75
(d, J ) 8.7 Hz, 1H, H-5′), 6.85 (dd, J ) 4.2, 1.8 Hz, 1H, H-6′),
6.87 (d, J ) 1.8 Hz, 1H, H-2′); 13C NMR (75 MHz) δ 11.78
(N(CH2CH3)2), 47.74 (N(CH2CH3)2), 51.50 (CH2N), 55.83 (3 ×
OCH3), 60.81 (4-OCH3), 67.20 (OCH2CH2), 106.02 (C-2, C-6),
111.35 (C-5′), 113.66 (C-2′), 122.00 (C-6′), 128.91 (C-1a′), 129.72
(C-1a), 130.01 (C-1′), 132.86 (C-1), 137.23 (C-4), 148.01 (C-4′),
148.70 (C-3′), 152.99 (C-3, C-5). Anal. Calcd for C24H34C1NO5:
C, 63.78; H, 7.58; N, 3.10. Found: C, 63.47; H, 7.94; N, 3.19.
1
IR (neat) νmax 2980 cm-1; H NMR δ 3.16 (t, J ) 6.0 Hz, 2H,
CH2CH2), 3.70 (s, 6H, 2 × OCH3), 3.83 (s, 6H, 2 × OCH3), 4.08
(t, J ) 6.1 Hz, 2H, OCH2CH2), 6.46 (d, J ) 12.2 Hz, 1H, H-1a′),
6.48 (d, J ) 12.1 Hz, 1H, H-1a), 6.50 (s, 2H, H-2, H-6), 6.76
(d, J ) 8.0 Hz, 1H, H-5′), 6.88 (dd, J ) 8.1, 2.1 Hz, 1H, H-6′),
6.90 (d, J ) 2.1 Hz, 1H, H-2′), 7.19 (d, J ) 7.6 Hz, 1H, â-CH),
7.34 (m, 1H, δ-CH), 7.79 (m, 1H, (-CH), 8.66 (d, J ) 5.5 Hz,
1H, -CH). Anal. Calcd for C25H28ClNO5; C, 65.57; H, 6.16; N,
3.06. Found: C, 65.76; H, 6.30; N, 3.30.
Method B. 3′-O-[2′′-(N-Methylpiperazinyl)ethyl]-com-
bretastatin A-4 (15g). Both N-methylpiperazine (10 mL) and
triethylamine (10 mL) were added to a solution of methane-
sufonate 14b (0.66 g) in 15 mL of dry acetonitrile. The solution
was stirred (under argon) for 48 h at room temperature, and
the solvent was then evaporated to yield a brown oil that was
dissolved in ether (25 mL). After extraction with 10% hydro-
chloric acid (3 × 10 mL), the pH of the combined aqueous
extract was raised to 11 with 20% sodium hydroxide, and the
solution was extracted with ether (3 × 10 mL). The combined
ethereal extract was washed with water (10 mL), dried, and
concentrated (reduced pressure) to yield, following chroma-
tography (hexanes-ethyl acetate-triethylamine, 6:4:1 as elu-
ent), a yellow oil (0.51 g, 76%). The dihydrochloride salt was
prepared as described above as a colorless solid, which
recrystallized from 2-propanol-ether as colorless needles: mp
190-191 °C; Rf 0.28 (hexanes-ethyl acetate-triethylamine, 6:4:
1); EIMS m/z (relative intensity) 442 (M+, 10) 316 (2), 114 (25),
100 (100); IR (neat) νmax 2940, 2880, 1580, 1510, 1325, 1128,
3′-O-[2′′-(N-Piperidinyl)ethyl]-combretastatin A-4 (15e).
Method A led to 0.53 g (78% yield) of amine 15e. The
hydrochloride salt crystallized from 2-propanol-ether as color-
less needles: mp 144-145 °C; Rf 0.49 (hexanes-ethyl acetate-
triethylamine, 6:4:1); EIMS m/z (relative intensity) 427 (M+,
20), 396 (7), 316 (5), 112 (20), 98 (100); IR (neat) νmax 3474,
3030, 3003, 2938, 2837, 1579, 1512, 1454, 1327, 1280, 1128,
1
1010, 889 cm-1; H NMR δ 1.43 (m, 2H, γ-CH2), 1.56 (m, 4H,
â-CH2), 2.44 (t, J ) 4.8 Hz, 4H, R-CH2), 2.71 (t, J ) 6.6 Hz,
2H, OCH2CH2N), 3.69 (s, 6H, 2 × OCH3), 3.83 (s, 6H, 2 ×
OCH3), 3.96 (t, J ) 6.6 Hz, 2H, OCH2CH2N), 6.43 (d, J ) 12.0
Hz, 1H, H-1a′), 6.50 (d, J ) 12.0 Hz, 1H, H-1a), 6.51 (s, 2H,
H-2, H-6), 6.76 (dd, J ) 6.6, 2.1 Hz, 1H, H-5′), 6.86 (dd, J )
6.6, 1.8 Hz, 2H, H-6′, H-2′); 13C NMR (75 MHz) δ 24.14 (γ-
CH2), 25.88 (â-CH2), 54.92 (OCH3), 55.85 (OCH3), 57.59 (R-
CH2), 60.79 (4-OCH3), 66.65 (OCH2CH2), 106.06 (C-2), 111.44
(C-5′), 114.00 (C-2′), 122.08 (C-6′), 128.89 (C-1a′), 129.67 (C-
1a), 130.00 (C-1′), 132.83 (C-1), 137.20 (C-4), 147.92 (C-4′),
148.72 (C-3′), 152.94 (C-5,3). Anal. Calcd for C25H34C1NO5: C,
64.71; H, 7.39; N, 3.02. Found: C, 65.00; H, 7.86; N, 3.00.
1
1010, 869 cm-1; H NMR δ 2.28 (s, 3H, N-CH3), 2.45 (m, 4H,
R-CH2), 2.57 (m, 4H, â-CH2), 2.77 (t, J ) 6.0 Hz, 2H,
OCH2CH2N), 3.70 (s, 6H, 2 × OCH3), 3.79 (t, J ) 6.0 Hz, 2H,
OCH2CH2N), 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.43 (d,
J ) 12.0 Hz, 1H, H-1a′), 6.50 (d, J ) 12.0 Hz, 1H, H-1a), 6.52
3′-O-[2′′-(N-Morpholino)ethoxy]-combretastatin A-4
(15f). Use of method A led to the hydrochloride, which
crystallized from 2-propanol-ether as colorless needles: mp

4096 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Pettit et al.
(s, 2H, H-2, H-6), 6.77 (d, J ) 8.7 Hz, 1H, H-5′), 6.86 (s, 1H,
H-6′), 6.88 (d, J ) 2.1 Hz, 1H, H-2′). Anal. Calcd for C25H36-
Cl2N2O5 (di-HCl salt): C, 58.2; H, 7.1; N, 5.4. Found: C, 58.29;
H, 7.51; N, 5.15.
X-ray Crystal Structure of Sodium Combretastatin
A-4 Phosphate (1b). (a) X-ray Crystal Structure Deter-
mination. For sodium combretastatin A-4 phosphate prodrug
hydrate (1b), a thin plate (∼0.18 mm × 0.24 mm × 0.36 mm),
grown from an acetone-water solution, was mounted on the
tip of a glass fiber. Cell parameter measurements and data
collection were performed at 165 ( 1 K with a Bruker SMART
6000 diffractometer system using Mo KR radiation. A sphere
of reciprocal space was covered using the MULTIRUN tech-
nique.41 Thus, six sets of frames of data were collected with
0.30° steps in ω, and a last set of frames with 0.30° steps in
æ, such that 86.6% coverage of all unique reflections to a
resolution of 0.84 Å was accomplished.
Method
C.
3′O-[2′′-(1H-Imidazol-1-yl)ethyl-com-
bretastatin A-4 (15i). Sodium hydride (60% dispersion in
mineral oil, 18 mg, 0.45 mmol, 1.1 equiv) was added in portions
to a stirred solution of imidazole (28 mg, 0.41 mmol) in dry
DMF (4 mL). The mixture was stirred for 30 min, and
2-chloroethyl ether 14a (0.16 g, 0.41 mmol) was added. The
solution was heated for 6 h at 80-90 °C and then poured into
water. The aqueous phase was extracted with ethyl acetate
(3 × 15 mL), and the combined extract was dried and
concentrated. Separation by flash chromatography (eluent
acetone-hexane, 2:1) gave the product as a colorless oil that
was then treated with ethereal hydrogen chloride (see method
a) to give the hydrochloride salt, which crystallized from
2-propanol-ether as nearly colorless needles (0.12 g, 71%): mp
133-135 °C; Rf 0.35 (acetone-hexane, 2:1); EIMS m/z (relative
intensity) 410 (M+, 100), 395 (95), 379 (15), 363 (12), 349 (5),
312 (8), 300 (10), 241 (8), 205 (7), 128 (5), 115 (7), 110 (15), 96
(60), 82 (16), 68 (50), 55 (7), 42 (25); IR (neat) νmax 3396, 2937,
2839, 2700, 2609, 1581, 1512, 1454, 1327, 1236, 1126, 1062,
(b) Crystal data. C18H20NaO8P‚2H2O (hydrate), FW )
454.33, monoclinic, C2/c, a ) 61.421(12) Å, b ) 4.5533(9) Å, c
) 14.558(3) Å, â ) 100.44(3)°, V ) 4004.1(14) Å3, Z ) 8, Fc )
1.507 Mg/m3, µ(Mo KR) ) 0.214 mm-1, λ) 0.71073 Å, F(000)
) 1904.
A total of 2028 reflections were collected, of which 1503
reflections were independent reflections (Rint ) 0.0310). Sub-
sequent statistical analysis of the data set with the XPREP42
program indicated the space group was C2/c. Final cell
constants were determined from the set of the 1089 observed
(>2σ(I)) reflections which were used in structure solution and
refinement. An absorption correction was applied to the data
with SADABS.43 Structure determination and refinement was
readily accomplished with the direct-methods program SHELX-
TL.44 All non-hydrogen atom coordinates were located in a
routine run using default values for that program. Surpris-
ingly, the structure proved to be the monosodium phosphate
derivative, rather than the disodium salt initially anticipated.
In addition to the parent molecule of the A-4 monosodium salt,
two molecules of water were present in each asymmetric unit
of the cell. For subsequent refinement, the remaining H atom
coordinates were calculated at optimum positions. Consider-
able disorder was noted for the phosphate moiety, with two
equally occupied sites being noted for each of the phosphate
oxygen atoms (O95, O96, and O97), as well as the sodium
cation. In addition, one of the water molecules was disordered
over two sites. The final model for 1b is shown in Figure 1, in
which the alternate disordered sites are shown with dashed
lines. All non-hydrogen atoms were refined anisotropically
(with the exception of the disordered atoms) in a full-matrix
least-squares refinement procedure. The H atoms were in-
cluded; their Uiso thermal parameters were fixed at either 1.2
or 1.5 (depending on atom type), the value of the Uiso of the
atom to which they were attached, and forced to ride that
atom. The final standard residual R1 value for 1b was 0.0771
for observed data and 0.0941 for all data. The goodness-of-fit
on F 2 was 0.967. The corresponding Sheldrick R values were
wR2 ) 0.2028 and 0.2182, respectively. A final difference
Fourier map showed minimal residual electron density, the
largest difference peak and hole being 0.282 and -0.783 e/Å3,
respectively. Final bond distances and angles were all within
expected and acceptable limits.
1
1006, 825, 750 cm-1; H NMR δ 3.68 (s, 6H, 2 × OCH3), 3.83
(s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.02 (t, J ) 5.1 Hz, 2H,
OCH2CH2N), 4.25 (t, J ) 5.1 Hz, 2H, OCH2CH2), 6.46 (d, J )
12.0 Hz, 1H, H-1a′), 6.48 (d, J ) 12.0 Hz, 1H, H-1a), 6.50 (s,
2H, H-2, H-6), 6.73 (d, J ) 1.8 Hz, 1H, H-2′), 6.78 (d, J ) 8.4
Hz, 1H, H-5′), 6.91 (dd, J ) 8.4, 1.8 Hz, 1H, H-6′), 7.04 (d, J )
1.2 Hz, 2H, H-δ4, H-δ5), 7.60 (s, 1H, H-δ2); 13C NMR (75 MHz)
δ 46.27, 55.88 (3 × OCH3), 60.80 (4-OCH3), 68.33 (-OCH2-
CH2), 105.84 (C-2, C-6), 111.56 (C-5′), 114.38 (C-2′), 119.40 (C-
6′), 123.15 (C-δ4,δ5), 128.93 (C-1a′), 129.19 (C-δ2), 129.26 (C-
1a), 129.76 (C-1′), 132.81 (C-1), 137.57 (C-4), 146.89 (C-4′),
148.83 (C-3′), 152.90 (C-5,3). Anal. Calcd for C23H27ClN2O5: C,
61.81; H, 6.09; N, 6.27. Found: C, 61.93; H, 6.36; N, 6.00.
3′-O-[2′′-(Trimethylamino)ethyl]-combretastatin A-4
iodide (15b). Method A. A solution of dimethylamine 15a
(0.30 g, 0.77 mmol) in methyl iodide (1 mL) was stirred under
argon for 30 min at room temperature. The solution was
concentrated in vacuo to give a yellow gum that was dissolved
in boiling methanol. Cooling afforded crystals (46% yield) of
the iodide: mp 201-205 °C; identical spectroscopically with
the sample prepared by method B.
Method B. A solution of amine 15a (0.40 g, 1 mmol) in a
mixture of methyl iodide (4 mL) and dry methanol (10 mL)
was stirred in the dark for 16 h at room temperature and then
concentrated in vacuo to an oily residue that crystallized from
ethanol (25%): mp 202-204 °C; 1H NMR δ 1.90 (s, 9H, (CH3)3),
2.70 (t, J ) 6.0 Hz, 2H, CH2N), 3.60 (s, 6H, 2 × OCH3), 3.78
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.15 (t, J ) 6.0 Hz, 2H,
OCH2), 6.46 (d, J ) 12.0 Hz, 1H, H-1a′), 6.49 (d, J ) 12.0 Hz,
1H, H-1a), 6.51 (s, 2H, H-2, H-6), 6.78 (d, J ) 8.1 Hz, 1H, H-5′),
6.84 (dd, J ) 8.1, 1.2 Hz, 1H, H-6′), 6.90 (d, J ) 1.2 Hz, 1H,
H-2′); HRFABMS M+ calcd for C23H32INO5 529.1325, found
529.2318.
Disk Diffusion Susceptibility Testing. Antimicrobial
activity was assayed by the National Committee for Clinical
Laboratory Standards (NCCLS) disk susceptibility test.45
Compounds were reconstituted in a small volume of sterile
dimethyl sulfoxide immediately prior to susceptibility experi-
ments. Mueller-Hinton agar supplemented with 5% sheep
blood was used for Streptococcus pneumoniae, gonococcal
typing agar for Neisseria gonorrhoeae, and Mueller-Hinton
agar for all other bacteria. Candida albicans was tested on
Sabouraud Dextrose Agar and Cryptococcus neoformans on
Yeast Morphology agar. The MIC was defined as the lowest
drug concentration resulting in a clear zone of growth inhibi-
tion.
3′-O-[Bis(2-chloroethyl)carbamoyl]-combretastatin A-4
(16). A solution of N-(chloroformyl)-bis(2-chloroethyl)amine
(0.15 g, 0.76 mmol, 1.2 equiv) was added to a solution of phenol
1a (0.2 g, 0.63 mmol) in dry pyridine (5 mL) at 0 °C. The
mixture was stirred at room temperature for 4 h, then at 50-
60 °C for 12 h. The mixture was allowed to cool, and ice was
added. The product was extracted with ethyl acetate (3 × 15
mL). Drying of the combined extract, filtration, and solvent
removal followed by flash chromatography (hexanes-ethyl
acetate, 3:1) gave the hydrochloride salt as a clear gum (0.27
g, 88%): EIMS m/z (relative intensity) 483 (M+, 50), 316 (45);
IR (neat) νmax 1730 (CdO) cm-1 1H NMR δ 3.72 (t, J ) 4.2
;
Hz, 4H, 2 × CH2), 3.75 (t, J ) 4.2 Hz, 4H, 2 × CH2), 3.80 (s,
3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 6H, 2 × OCH3), 6.45
(s, 2H, H-1a, H-1a′), 6.53 (s, 2H, H-2, H-5), 6.83 (d, J ) 9.0
Hz, 1H, H-5′), 7.10 (dd, J ) 7.8, 2.1 Hz, 1H, H-6′), 7.14 (d, J )
2.1 Hz, 1H, H-2′). Anal. Calcd for C23H27Cl2NO6‚HCl: C, 53.0;
H, 5.4; N, 2.7. Found: C, 53.19; H, 5.6; N, 3.3.
Acknowledgment. We are pleased to thank the
following for the very necessary financial assistance:
Outstanding Investigator Grants CA44344-06-12 and
RO1CA90441-01 awarded by the Division of Cancer
Treatment and Diagnosis, National Cancer Institute,

Structural Modifications of Combretastatin A-4
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4097
Chem. 1987, 65, 2390-2396. (k) Pettit, G. R.; Singh, S. B.; Cragg,
G. M. Synthesis of Natural (-)-Combretastatin. J. Org. Chem.
1985, 50, 3404-3406. (l) Pettit, G. R.; Cragg, G. M.; Herald, D.
L.; Schmidt, J. M.; Lohavanijaya, P. Isolation and Structure of
Combretastatin. Can. J. Chem. 1982, 60, 1374-1376. (m) Nabha,
S. M.; Mohammad, R. M.; Wall, N. R.; Dutcher, J. A.; Salkini,
B. M.; Pettit, G. R.; Al-Katib, A. M. Evaluation of Combretastatin
A-4 Prodrug in a Non-Hodgkin’s Lymphoma Xenograft Model:
Preclinical Efficacy. Anti-Cancer Drugs 2001, 12, 57-63. (n)
Nabha, S. M.; Wall, N. R.; Mohammad, R. M.; Pettit, G. R.; Al-
Katib, A. M. Effects of Combretastatin A-4 Prodrug against a
Panel of Malignant Human â-Lymphoid Cell Lines. Anti-Cancer
Drugs 2000, 11, 385-392.
DHHS; the Arizona Disease Control Research Commis-
sion; the Robert B. Dalton Endowment Fund; the Caitlin
Robb Foundation; Gary L. and Diane R. Tooker; Lottie
Flugel; the Eagles Art Ehrmann Cancer Fund; and the
Ladies Auxiliary to the Veterans of Foreign Wars. For
other helpful assistance we thank Drs. Fiona Hogan and
Michael Williams.
Supporting Information Available: X-ray data for com-
bretastatin A-4 phosphate prodrug. This material is available
free of charge via the Internet at http://pubs.acs.org.
(5) (a) Mannila, E.; Talvitie, A.; Kolehmainen, E. Antileukemic
Compounds Derived from Stilbenes in Picea abies Bark. Phy-
tochemistry 1993, 33, 813-816. (b) Schmitt, E.; Lehmann, L.;
Metzler, M.; Stopper, H. Hormonal and Genotoxic Activity of
Resveratrol. Toxicol. Lett. 2002, 136, 133-142. (c) Matsuoka,
A.; Takeshita, K.; Furuta, A.; Ozaki, M.; Fukuhara, K.; Miyata,
N. The 4′-Hydroxy Group is Responsible for the in vitro Cyto-
genetic Activity of Resveratrol. Mutat. Res. 2002, 521, 29-35.
(d) Arichi, H.; Kimura, Y.; Okuda, H.; Baba, H.; Kozawa, K.;
Arichi, S. Effects of Stilbene Components of the Roots of
Polygonum cuspidatum SIEB ET ZUCC on Lipid-metabolism.
Chem. Pharm. Bull. 1982, 30, 1766-1770. (e) Ingham, J. L.
3,5,4′-Trihydroxystilbene as a Phytoalexin from Groundnuts
(Arachis hypogeae). Phytochemistry 1976, 15, 1791-1793. (f)
Pettit, G. R.; Grealish, M. P.; Jung, M. K.; Hamel, E.; Pettit, R.
K.; Chapuis, J.-C.; Schmidt, J. M. Antineoplastic Agents 465.
Structural Modification of Resveratrol: Sodium Resverastatin
Phosphate. J. Med. Chem. 2002, 45, 2534-2542.
(6) (a) Gordaliza, M.; Castro, M. A.; del Corral, J. M. M.; Lopez-
Vazquez, M. L.; Garcia, P. A.; San Feliciano, A.; Garavia-
Gravalos, M. D.; Broughton, H. Preparation and Cytotoxicity of
Podophyllotoxin Derivatives Lacking the Lactone Ring. Tetra-
hedron 1997, 53, 15742-15760. (b) Utsugi, T.; Shibata, J.;
Sugimoto, Y.; Aoyagi, K.; Wierzba, K.; Kobunai, T.; Terada, T.;
Oh-hara, T.; Tsuruo, T.; Yamada, Y. Antitumor Activity of a
Novel Podophyllotoxin Derivative (TOP-53) against Lung Cancer
and Lung Metastatic Cancer. Cancer Res. 1996, 56, 2809-2814.
(c) Kamal, A.; Atchison, K.; Daneshtalab, M.; Micetich, R. G.
Synthesis of Podophyllotoxin Congeners as Potential DNA
Topoisomerase II Inhibitors. Anti-Cancer Drug Des. 1995, 10,
545-554. (d) Lee, K.-H.; Imakura, Y.; Haruna, M.; Beeers, S.
A.; Thurston, L. S.; Dai, H.-J.; Chen, C.-H.; Liu, S.-Y.; Cheng,
Y.-C.; Antitumor Agents. 107. New Cytotoxic 4-Alkylamino
Analogs of 4′-Demethyl-Epipodophyllotoxin as Inhibitors of
Human DNA Topoisomerase II. J. Nat. Prod. 1989, 52, 606-
613.
(7) (a) Wickdramaratne, D. B. M.; Pengsuparp, T.; Mar, W.; Chai,
H.-B.; Chagwedera, T. E.; Beecher, C. W. W.; Farnsworth, N.
R.; Kinghorn, A. D.; Pezzuto, J. M.; Cordell, G. A. Novel
Antimitotic Dibenzocyclo-octadiene Lignan Constituents of the
Stem Bark of Steganotaenia araliacea. J. Nat. Prod. 1993, 56,
2083-2090. (b) Tomioka, K.; Ishiguro, T.; Mizuguchi, H.; Ko-
meshima, N.; Koga, K.; Tsukagoshi, S.; Tsuruo, T.; Tashiro, T.;
Tanida, S.; Kishi, T. Absolute Structure-Cytotoxic Activity
Relationships of Steganacin Congeners and Analogues. J. Med.
Chem. 1991, 34, 54-57. (c) Schiff, P. B.; Kende, A. S.; Horwitz,
S. B. An Inhibitor of Hela Cell Growth and Microtubule
Assembly in vitro. Biochem. Biophys. Res. Commun. 1978, 85,
737-746. (d) Wang, R. W.-J.; Rebhun, L. I.; Kupchan, S. M.
Antimitotic and Antitubulin Activity of the Tumor Inhibitor
Steganacin. Cancer Res. 1977, 37, 3071-3079.
(8) (a) McGown, A. T.; Fox, B. W. Differential Cytotoxicity of
Combretastatins A-1 and A-4 in Two Daunorubicin-Resistant
P388 Cell Lines. Cancer Chemother. Pharmacol. 1990, 26, 79-
81. (b) Jiang, J. B.; Hesson, D. P.; Dusak, B. A.; Dexter, D. L.;
Kang, G. L.; Hamel, E. Synthesis/Biological Evaluation of
2-Styrylquinazolin-4(3H)-ones, a New Class of Antimitotic An-
ticancer Agents Which Inhibit Tubulin Polymerization. J. Med.
Chem. 1990, 33, 1721-1728. (c) Lin, C. M.; Ho, H. H.; Pettit, G.
R.; Hamel, E. Antimitotic Natural Products Combretastatin A-4
and Combretastatin A-2: Studies on the Mechanism of Their
Inhibition of the Binding of Colchicine to Tubulin. Biochemistry
1989, 28, 6984-6991. (d) McGown, A. T.; Fox, B. W. Structural
and Biochemical comparison of the Antimitotic Agents Colchi-
cine, Combretastatin A-4 and Amphethinile. Anti-Cancer Drug
Des. 1989, 3, 249-254.
References
(1) For contribution no. 444, refer to: Pettit, G. R.; Lippert, J. W.,
III; Herald, D. L. A Pinacol Rearrangement/Oxidation Synthetic
Route to Hydroxyphenstatin. J. Org. Chem. 2000, 65, 7438-
7444.
(2) (a) Pinney, K. G.; Bounds, A. D.; Dingeman, K. M.; Mocharla,
V. P.; Pettit, G. R.; Bai, R.; Hamel, E. A New Anti-tubulin Agent
Containing the Benzo(b)thiophene Ring System. Bioorg. Med.
Chem. Lett. 1999, 9, 1081-1086. (b) Miller, T. A.; Bulman, A.
L.; Thompson, C. D.; Macdonald, T. L. The Synthesis and
Evaluation of Temperature Sensitive Tubulin Toxins. Bioorg.
Med. Chem. Lett. 1999, 9, 407-412. (c) Jordan, A.; Hadfield, J.
A.; Lawrence, N. J.; McGown, A. T. Tubulin as a Target for
Anticancer Drugs: Agents Which Interact with the Mitotic
Spindle. Med. Res. Rev. 1998, 18, 259-296. (d) Chaudhuri, A.
R.; Seetharamalu, P.; Schwarz, P. M.; Hausheer, F. H.; Luduen˜a,
R. F. The Interaction of the B-ring of Colchicine with R-Tubu-
lin: A Novel Footprinting Approach. J. Mol. Biol. 2000, 303,
679-692. (e) ter Haar, E.; Rosenkranz, H. S.; Hamel, E.; Day,
B. W. Computational and Molecular Modeling Evaluation of the
Structural Basis for Tubulin Polymerization Inhibition by
Colchicine Site Agents. Bioorg. Med. Chem. 1996, 4, 1659-1671.
(3) (a) DeVincenzo, R.; Scambia, G.; Ferlini, C.; Distefano, M.;
Filippini, P.; Riva, A.; Bombardelli, E.; Pocar, D.; Gelmi, M. L.;
Benedetti-Panici, P.; Mancuso, S. Antiproliferative Activity of
Colchicine Analogues on MDR-Positive and MDR-Negative
Human Cancer Cell Lines. Anti-Cancer Drug Des. 1998, 13, 19-
33. (b) Shi, Q.; Verdier-Pinard, P.; Brossi, A.; Hamel, E.; McPhail,
A. T.; Lee, K.-H. Antitumor Agents. 172. Synthesis and Biological
Evaluation of Novel Deacetamidothiocolchicine-7-ols and Ester
Analogs as Antitubulin Agents. J. Med. Chem. 1997, 40, 961-
966. (c) Ducray, P.; Lebeau, L.; Mioskowski, C. Synthesis of Lipid
Derivatives of Colchicine. Helv. Chim. Acta 1996, 79, 2346-2352.
(d) Boye, O.; Brossi, A. Tropolonic Colchicum Alkaloids and Allo
Congeners. In The Alkaloids; Brossi, A., Ed.; Academic Press:
New York, 1992; Vol. 41, pp 125-176. (e) Muzaffar, A.; Brossi,
A.; Lin, C. M.; Hamel, E. Antitubulin Effects of Derivatives of
3′-Dimethylthiocolchine, Methylthio Ethers of Natural Colchi-
cinoids, and Thioketones Derived from Thiocolchicine. Compari-
son with Colchicinoids. J. Med. Chem. 1990, 33, 567-571.
(4) (a) Pettit, G. R.; Toki, B. E.; Herald, D. L.; Boyd, M. R.; Hamel,
E.; Pettit, R. K.; Chapuis, J. C. Antineoplastic Agents 410.
Asymmetric Hydroxylation of trans-Combretastatin A-4. J. Med.
Chem. 1999, 42, 1459-1465. (b) Pettit, G. R.; Rhodes, M. R.;
Herald, D. L.; Chaplin, D. J.; Stratford, M. R. L.; Hamel, E.;
Pettit, R. K.; Chapuis, J.-C.; Oliva, D. Antineoplastic Agents 393.
Synthesis of the trans-isomer of Combretastatin A-4 Prodrug.
Anti-Cancer Drug Des. 1998, 13, 981-993. (c) Pettit, G. R.;
Rhodes, M. R. Antineoplastic Agents 389. New Syntheses of the
Combretastatin A-4 Prodrug. Anti-Cancer Drug Des. 1998, 13,
183-191. (d) Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.;
Pettit, R. K.; Schmidt, J. M.; Hogan, F. Antineoplastic Agents.
291. Isolation and Synthesis of Combretastatins A-4, A-5 and
A-6. J. Med. Chem. 1995, 38, 1666-1672. (e) Pettit, G. R.; Singh,
S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D.
Isolation and Structure of the Strong Cell Growth and Tubulin
Inhibitor Combretastatin A-4. Experientia 1989, 45, 209-211.
(f) Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.; Schmidt,
J. M.; Pettit, G. R.; Hamel, E. Interactions of Tubulin with Potent
Natural and Synthetic Analogs of the Antimitotic Agent Com-
bretastatin: A Structure-Activity Study. Mol. Pharmacol. 1988,
34, 200-208. (g) Pettit, G. R.; Singh, S. B.; Schmidt, J. M.; Niven,
M. L.; Hamel, E.; Lin, C. M. Isolation, Structure, Synthesis and
Antimitotic Properties of Combretastatins B-3 and B-4 from
Combretum caffrum. J. Nat. Prod. 1988, 51, 517-527. (h) Pettit,
G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.; Schmidt, J. M.
Isolation, Structure and Synthesis of Combretastatins A-1 and
B-1, Potent New Inhibitors of Microtubule Assembly, Derived
from Combretum caffrum. J. Nat. Prod. 1987, 50, 119-131. (i)
Pettit, G. R.; Cragg, G. M.; Singh, S. B. Antineoplastic Agents
122. Constituents of Combretum caffrum. J. Nat. Prod. 1987,
50, 386-391. (j) Pettit, G. R.; Singh, S. B. Isolation, Structure,
and Synthesis of Combretastatin A-2, A-3, and B-2. Can. J.
(9) (a) Chaplin, D. J.; Pettit, G. R.; Hill, S. A. Anti-Vascular
Approaches to Solid Tumour Therapy: Evaluation of Combre-
tastatin A4 Phosphate. Anticancer Res. 1999, 19, 189-196. (b)
Dark, G. G.; Hill, S. A.; Prise, V. E.; Tozer, G. M.; Pettit, G. R.;
Chaplin, D. J. Combretastatin A-4, an Agent That Displays
Potent and Selective Toxicity toward Tumor Vasculature. Cancer
Res. 1997, 57, 1829-1834.

4098 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Pettit et al.
(10) (a) Aleksandrzak, K.; McGown, A. T.; Hadfield, J. A. Antimitotic
Activity of Diaryl Compounds with Structural Features Resem-
bling Combretastatin A-4. Anti-Cancer Drugs 1998, 9, 545-550.
(b) Hatanaka, T.; Fujita, K.; Obsumi, K.; Nakagawa, R.; Fukuda,
Y.; Nihei, Y.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel B-Ring
Modified Combretastatin Analogues: Syntheses and Antine-
oplastic Activity. Bioorg. Med. Chem. Lett. 1998, 8, 3371-3374.
(c) Ohsumi, J.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda,
Y.; Hihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T.
Syntheses and Antitumor Activity of cis-Restricted Combret-
astatins: 5-membered Heterocyclic Analogues. Bioorg. Med.
Chem. Lett. 1998, 8, 3153-3158. (d) Medarde, M.; Ramos, A.;
Caballero, E.; Pela´ez-Lamamie´ de Clairac, R.; Lo´pez, L.; Gra´va-
los, D. G.; Feliciano, A. S. Synthesis and Antineoplastic Activity
of Combretastatin Analogues: Heterocombretastatins. Eur. J.
Med. Chem. 1998, 33, 71-77. (e) Ohsumi, K.; Nakagawa, R.;
Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei, Y.; Ohishi, K.;
Suga, Y.; Akiyama, Y.; Tsuji, T. Novel Combretastatin Analogues
Effective against Murine Solid Tumors: Design and Structure-
Activity Relationships. J. Med. Chem. 1998, 41, 3022-3032. (f)
Woods, J. A.; Hadfield, J. A.; Pettit, G. R.; Fox, B. W.; McGown,
A. T. The Interaction with Tubulin of a Series of Stilbenes Based
on Combretastatin A-4. Br. J. Cancer 1995, 71, 705-711. (g)
Gwaltney, S. L., II; Imade, H. M.; Barr, K. J.; Li, Q.; Gehrke,
L.; Credo, R. B.; Warner, R. B.; Lee, J. Y.; Kovar, P.; Wang, J.;
Nukkala, M. A.; Zielinski, N. A.; Frost, D.; Ng, S.-C.; Sham, H.
L. Novel Sulfonate Analogues of Combretastatin A-4: Potent
Antimitotic Agents. Bioorg. Med. Chem. Lett. 2001, 11, 871-
874. (h) Lawrence, N. J.; Rennison, D.; Woo, M.; McGown, A.
T.; Hadfield, J. A. Antimitotic and Cell Growth Inhibitory
Properties of Combretastatin A-4 Like Ethers. Bioorg. Med.
Chem. Lett. 2001, 11, 51-54. (i) Ohsumi, K.; Hatanaka, T.;
Nakagawa, R.; Fukuda, Y.; Morinaga, Y.; Suga, Y.; Nihei, Y.;
Ohishi, K.; Akiyama, Y.; Tsuji, T. Synthesis and Antitumor
Activities of Amino Acid Prodrugs of Amino-Combretastatins.
Anti-Cancer Drug Des. 1999, 14, 539-548. (j) Cushman, M.;
Nagarathnam, D.; Gopal, D.; He, H.-M.; Lin, C. M.; Hamel, E.
Synthesis and Evaluation of Analogues of (Z)-1-(4-Methoxyphe-
nyl)-2-(3,4,5-trimethoxyphenyl)ethene as Potential Cytotoxic and
Antimitotic Agents. J. Med. Chem. 1992, 35, 2293-2306.
(11) Vedejs, E.; Peterson, M. J. In Topics in Stereochemistry; Eliel,
E. L., Wilen, S. H., Eds.; John Wiley & Sons: New York, 1994;
Vol. 21, pp 1-125.
(23) Qian, X.; Abul-Hajj, Y. J. Synthesis and Biological Activity of
17â-Substituted Estradiol. J. Steroid Biochem. 1988, 28, 657-
664.
(24) Gu¨nther, H. In NMR Spectroscopy: Basic Principles, Concepts,
and Applications in Chemistry, 2nd ed.; Wiley: West Sussex,
England, 1995; pp 15-52.
(25) (a) Monaco, R. R.; Gardiner, W. C.; Kirschner, S. Semiempirical
Studies of Ring Twisting in cis-Stilbene and Related Biomol-
ecules. Int. J. Quantum Chem. 1999, 71, 57-62. (b) Nandy, P.;
Banerjee, S.; Gao, H.; Hui, M. B. V.; Lien, E. J. Quantitative
Structure-Activity Relationship Analysis of Combretastatins:
A Class of Novel Antimitotic Agents. Pharm. Res. 1991, 8, 776-
781.
(26) (a) Sato, M.; Rippy, M. K.; Bryant, H. U. Raloxifene, Tamoxifen,
Nafoxidine, or Estrogen Effects on Reproductive and Nonrepro-
ductive Tissues in Ovariectomized Rats. FASEB J. 1996, 10,
905-912. (b) Horwitz, K. B.; McGuire, W. L. Nuclear Mechanism
of Estrogen Action. Effect of Estradiol and Antiestrogens on
Estrogen Receptors and Nuclear Receptor Processing. J. Biol.
Chem. 1978, 253, 8185-8191.
(27) (a) Somjen, D.; Waisman, A.; Kaye, A. M. Tissue Selective Action
of Tamoxifen Methiodide, Raloxifene and Tamoxifen on Creatine
Kinase B Activity in vitro and in vivo. J. Steroid Biochem. Mol.
Biol. 1996, 59, 389-396. (b) Jordan, V. C.; Gosden, B. Impor-
tance of the Alkylaminoethoxy Side-Chain for the Estrogenic and
Antiestrogenic Actions of Tamoxifen and Trioxifene in the
Immature Rat Uterus. Mol. Cell. Endocrinol. 1982, 27, 291-
299. (c) Stauffer, S. R.; Huang, Y. R.; Aron, Z. D.; Coletta, C. J.;
Sun, J.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Tri-
arylpyrazoles with Basic Side Chains: Development of Pyrazole-
Based Estrogen Receptor Antagonists. Bioorg. Med. Chem. 2001,
9, 151-161.
(28) (a) Jarman, M.; Leung, O.-T.; Leclercq, G.; Devleeschouwer, N.;
Stoessel, S.; Coombes, R. C.; Skilton, R. A. Analogues of
Tamoxifen: The Role of the Basic Sidechain. Application of a
Whole-Cell Oestrogen-Receptor Binding Assay to N-Oxides and
Quaternary Salts. Anti-Cancer Drug Des. 1986, 1, 259-268. (b)
Foster, A. B.; McCague, R.; Seago, A.; Leclercq, G.; Stoessel, S.;
Roy, F. Modification of the Basic Side Chain in Tamoxifen:
Effects on Microsomal Metabolism and in vitro Biological Activ-
ity. Anti-Cancer Drug Des. 1986, 1, 245-257.
(29) Hamel, E. Antimitotic Natural Products and Their Interactions
with Tubulin. Med. Res. Rev. 1996, 16, 207-231.
(30) Pinney, K. G.; Mejia, M. P.; Villalobos, V. M.; Rosenquist, B. E.;
Pettit, G. R.; Verdier-Pinard, P.; Hamel, E. Synthesis and
Biological Evaluation of Aryl Azide Derivatives of Combretasta-
tin A-4 as Molecular Probes for Tubulin. Bioorg. Med. Chem.
Lett. 2000, 8, 2417-2425.
(31) Carpio, H.; Galeazzi, E.; Greenhouse, R.; Guzman, A.; Valarde,
E.; Antonio, Y.; Franco, F.; Leon, A.; Perez, V.; Salas, R.; Valdes,
D.; Ackrell, J.; Cho, D.; Gallegra, P.; Halpern, O.; Koehler, R.;
Maddox, M. L.; Muchowski, J. M.; Prince, A.; Tegg, D.; Thurber,
T. C.; Van Horn, A. R.; Wren, D. Synthesis of 1,2-Dihydro-3H-
pyrrolo[1,2-a]pyrrole-1-carboxylic Acids and Homologous Pyri-
dine and Azepine Analogues Thereof. Can. J. Chem. 1982, 60,
2295-2312.
(12) Asakawa, Y.; Tanikawa, K.; Aratani, T. New Substituted Biben-
zyls of Frullania brittoniae subsp. truncatifolia. Phytochemistry
1976, 15, 1057-1059.
(13) (a) Fieser, L. F. 1,2-Aminonaphthol Hydrochloride (2-Naphthol,
1-Amino-, Hydrochloride). Org. Synth. 1943, 35-39. (b) Fieser,
L. F. 1,4-Aminonaphthol Hydrochloride (1-Naphthol, 4-Amino-,
Hydrochloride). Org. Synth. 1943, Collect. Vol. 2, 39-42.
(14) Ramadas, K.; Srinivasan, N. Iron-Ammonium Chloridesa Con-
venient and Inexpensive Reductant. Synth. Commun. 1992, 22,
3189-3195.
(15) Yoo, S.; Lee, S.-L. Reduction of Organic Compounds with Sodium
Borohydride-Copper (II) Sulfate System. Synth. Lett. 1990,
419-420.
(16) Han, B. H.; Jang, D. G. Montmorillonite Catalyzed Reduction
of Nitroarenes with Hydrazine. Tetrahedron Lett. 1990, 31,
1181-1182.
(17) Li, H.; Zhang, R.; Wang, H.; Pan, Y.; Shi, Y. A Convenient and
Mild Synthetic Route to Aminoarenes by Reduction of Nitroare-
nes with Activated Nickel and Hydrazine Hydrate. Synth.
Commun. 1997, 27, 3047-3052.
(18) Nose, A.; Kudo, T. Reduction with Sodium Borohydride-Transi-
tion Metal Salt Systems. I. Reduction of Aromatic Nitro Com-
pounds with the Sodium Borohydride-Nickelous Chloride Sys-
tem. Chem. Pharm. Bull. 1981, 29, 1159-1161.
(19) (a) Pinney, K. L., personal communication. (b) Ohsumi, K.; Tsuji,
T.; Morinaga, Y.; Ohishi, K. Stilbene Derivatives and Pharma-
ceutical Compositions Containing Them. U.S. Patent No. 5,525,-
632, 1996; 7 pp. (c) Hatanaka, T.; Ohsumi, K.; Tsuji, T.; Nihei,
Y.; Nakagawa, R.; Ohishi, K. Stilbene Compounds and Phar-
maceutical Compositions Containing Them. U.S. Patent No. 5,-
674,906, 1997; 13 pp.
(20) Bachelor, F. W.; Loman, A. A.; Snowdon, L. R. Synthesis of
Pinosylvin and Related Hardwood Stilbenes. Can. J. Chem.
1970, 48, 1554-1557.
(21) (a) Baker, R.; Sims, R. J. A Convenient Modification of the
Wadsworth-Emmons Method of Olefin Formation Using a Crown
Ether. Synthesis 1981, 117. (b) Baker, R.; Sims, R. J. Synthesis
of Pallescensin-E: Use of a Crown Ether in the Wadsworth
Procedure for Olefin Formation. Tetrahedron Lett. 1981, 22,
161-162.
(22) Magarian, R. A.; Avor, K. S.; Overacre, L. B.; Kunchandy, J.;
Paxton, T. R. Synthesis and Biological Evaluation of Basic Side
Chain Derivatives of Analog II as Pure Antiestrogens and
Antitumor Agents. Anti-Cancer Drug Des. 1995, 10, 311-331.
(32) (a) Montginoul, C.; Richard, M. J.; Vigne, C.; Giral, L. Synthesis
of Aminoalkylbenzyltertiobutylketones. Ann. Pharm. Fr. 1985,
43, 249-255. (b) Bandi, A.; Sala, A.; Angelo Soresinetti, P.
Synthesis of New Imidazole Derivatives as Potential Inhibitors
of Thromboxane Synthetase. J. Heterocycl. Chem. 1990, 27, 215-
219.
(33) (a) Gump, W. S.; Nikawitz, E. J. U.S. Patent 2 533 243, 1950;
Chem. Abstr. 1950, 45, 4271c. (b) Bagley, J. R.; Thomas, S. A.;
Rudo, F. G.; Spencer, H. K.; Doorley, B. M.; Ossipov, M. H.;
Jerussi, T. P.; Benvenga, M. J.; Spaulding, T. New 1-(Hetero-
cyclylalkyl)-4-(propionanilido)-4-piperidinyl Methyl Ester and
Methylene Methyl Ether Analgesics. J. Med. Chem. 1991, 34,
827-841.
(34) Hawes, E. M.; Davis, H. L. Intramolecular Nucleophilic Cycliza-
tion of 3-Substituted Pyridylalkylamines onto the 2-Position of
the Pyridine Ring. J. Heterocycl. Chem. 1973, 10, 39-42.
(35) Gough, G. A. C.; King, H. Trypanocidal Action and Chemical
Constitution. Part VIII. Derivatives of â-Aminoethyl and γ-Ami-
nopropyl-arsinic Acids. J. Chem. Soc. 1928, 2426-2447.
(36) Johnson, T. B.; Bengls, R. Hydantoins: Synthesis of the Hy-
dantoin of 3-Amino-Tyrosine. J. Am. Chem. Soc. 1912, 34, 1054-
1061.
(37) Green, G. D.; Weinscherik, J. I., III; Mulvaney, J. E.; Hall, H.
K., Jr. Synthesis of Polyesters Containing a Nonrandomly Placed
Highly Polar Repeating Unit. Macromolecules 1987, 20, 722-
726.
(38) Singh, S. B.; Pettit, G. R. Isolation, Structure, and Synthesis of
Combretastatin C-1. J. Org. Chem. 1987, 54, 4105-4114.
(39) Mann, F. G. Hofmann’s Ethylene Bases. Synthesis of â,â′-
Diaminodiethylamine and of â,â′-Diaminodiethylmethylamine.
J. Chem. Soc. 1934, 461-466.

Structural Modifications of Combretastatin A-4
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4099
(40) Dave, M. A.; Kulkami, A. B. Synthesis of Dimethylamino
Aromatic Ketones and Aldehydes. II. Indian J. Chem. 1975, 13,
33-34.
crystal structures from diffraction data. This package includes,
among other, XPREP (an automatic space group determination
program), SHELXS (a structure solution program via Patterson
or direct methods), and SHELXL (structure refinement soft-
ware).
(41) SMART for Windows NT, v. 5.605; Bruker AXS Inc.: Madison,
WI, 2000.
(42) XPREP-The automatic space group determination program in
the SHELXTL (se ref 44).
(43) Blessing, R. H. Acta Crystallogr. 1995, A51, 33-38.
(44) SHELXTL-NT, Version 5.10; Bruker AXS Inc.: Madison, WI,
1997. An integrated suite of programs for the determination of
(45) National Committee for Clinical Laboratory Standards. Perfor-
mance standards for antimicrobial disk susceptibility tests;
Approved standard M2-A6; NCCLS: Wayne, PA, 1997.
JM0205797