Design and synthesis of novel enhanced water soluble hydroxyethyl analogs of combretastatin A-4

Article abstract of DOI:10.1016/j.bmcl.2011.01.136

Thirteen hydroxyethyl- analogs of combretastatin A-4 (CA-4) that contain the 1-(1′-hydroxyethyl)-1-(3″,4″,5″-trimethoxyphenyl)-2- (substituted phenyl)ethene framework were synthesized. Molecular modeling studies at the DFT level showed that compound 3j ad

Full text of DOI:10.1016/j.bmcl.2011.01.136

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2087–2091
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel enhanced water soluble hydroxyethyl analogs
of combretastatin A-4
Megan Lee a, Olivia Brockway a, Armaan Dandavati a, Samuel Tzou a, Robert Sjoholm a, Alexis Nickols b,
Balaji Babu a, Sameer Chavda a, Vijay Satam a, Rachel M. Hartley c, Cara Westbrook c, Susan L. Mooberry c,d
,
Gregory Fraley b, Moses Lee a,
⇑
a Department of Chemistry, Division of Natural and Applied Sciences, Hope College, Holland, MI 49422, USA
b Department of Biology, Division of Natural and Applied Sciences, Hope College, Holland, MI 49422, USA
c Department of Pharmacology, University of Texas Health Science Center, San Antonia, TX 78229, USA
d Department of Medicine, University of Texas Health Science Center, San Antonia, TX 78229, USA
a r t i c l e i n f o
a b s t r a c t
Thirteen hydroxyethyl- analogs of combretastatin A-4 (CA-4) that contain the 1-(10-hydroxyethyl)-1-
(300,400,500-trimethoxyphenyl)-2-(substituted phenyl)ethene framework were synthesized. Molecular
modeling studies at the DFT level showed that compound 3j adopts a ‘twisted’ conformation mimicking
CA-4. The cytotoxicity of the novel compounds against the growth of murine B16 melanoma and L1210
lymphoma cells in culture was measured using an MTT assay. Three analogs 3f, 3h, and 3j were active. Of
these, 3j, which has the same substituents as CA-4 and IC50 values of 16.1 and 4.1 lM against B16 and
L1210 cells, respectively, was selected for further biological evaluation. The activity of 3j was verified
by the NCI 60 cell line screen. Compound 3j causes microtubule depolymerization in A-10 cells with
Article history:
Received 8 January 2011
Accepted 31 January 2011
Available online 3 February 2011
Keywords:
Combretastatins
Cytotoxicity
Lymphoma
Melanoma
Solubility
an EC50 of 21.2
lM. Analog 3j, which has excellent water solubility of 479 lM, had antitumor activity
in a syngeneic L1210 murine model.
Ó 2011 Elsevier Ltd. All rights reserved.
Tubulin
Microtubules
Combretastatin A-4 (1a, CA-4), as shown in Figure 1 is a natural
product and vascular disrupting agent (VDA), first isolated by G.R.
Pettit et al. in 1989.1 It was isolated from the bark of the African
Willow Tree, Combretum caffrum. VDAs including CA-4 are effective
antitumor agents that are capable of rapidly and selectively disrupt-
ing the abnormal tumor vasculature resulting in vascular collapse
and subsequent tumor necrosis.2 Combretastatin A-4 binds to tubu-
lin within the colchicine binding site, altering endothelial cell struc-
ture and causing vascular permeability and rapid destruction of
tumor vasculature.3 However, CA-4’s possibilities as a clinical anti-
tumor agent is hindered by its low bioavailability and poor aqueous
solubility.4 These limitations have led to the development of many
water-soluble derivatives and analogs, the most important of which
include a phosphate containing pro-drug (1b, CA-4P) as shown in
Figure 1.5 Other promising analogs include AC-7739 (1c), the pro-
drug 1d and A105972, 1e.6a These derivatives of CA-4 have high
potency and increased aqueous solubility as compared to CA-4.
Several combretastatin analogs are advancing in clinical trials.6
Other examples of cytotoxic analogs of CA-4 that have been
synthesized in the author’s laboratory include pyrazole,7 pyrazo-
line,8 cyclohexenone,9 oxadiazoline (1f)10 2-thioxo-pyrimidine,11
and acetyl-combretastatin analogs.12 The X-ray crystallography
structure and molecular modeling studies revealed that
1a, R=OH, Combretastatin A4 (CA-4)
-2
1b, R= O-PO3
H3CO
H3CO
R
1c, R=NH2
1d, R=
H
N
CH(NH2)CH2OH
Ac
OCH3
OCH3
O
Ac
N
N
N
O
N
H3CO
OCH3
OCH3
NH2
O
H3CO
H3CO
OCH3
H3CO
OCH3
1e, A-105972
CH3
1f
CH3
1'
O
HO
1
2
5''
H3CO
H3CO
OH
OCH3
A
H3CO
B
OH
3''
4''
OCH3
H3CO
OCH3
OCH3
2j
3j
⇑
Corresponding author.
E-mail address: lee@hope.edu (M. Lee).
Figure 1. Structures of combretastatin A-4 derivatives 1a–d, acetyl-analog 2j, and
the novel hyroxyethyl analog 3j.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.136

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M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2087–2091
CH3
Table 1
H
O
R1
Cytotoxicity of compounds 3a–m.
O
H3CO
H3CO
CH3 R5
R1
R2
R2
R3
H3CO
H3CO
piperidine,
benzoic Acid,
+
O
R4
toluene,
reflux 4h
R5
Compounds
L1210 IC50
(
lM)
B16 IC50 (lM)
OCH3
R3
R4
OCH3
2a-m
a, R1=R2=R3=R4=R5=H
NaBH4, CH3OH,
CeCl3.7 H2O
r.t. 24h
b, R1=R2=R4=R5=H; R3=CH3
c, R1=R2=R4=R5=H; R3=Cl
d, R1=R2=R4=R5=H; R3=Br
e, R1=R2=R4=R5=H; R3=NO2
GROUP A
3a
>100
33.5
>100
>100
>100
>100
>100
f, R1=R2=R4=R5=H; R3=OCH3
g, R1=R3=R4=R5=H; R2=NO2
h, R1=R4=R5=H; R2=R3=Cl
i, R1=R4=R5=H; R2=R3=OCH3
CH3
HO
R1
3b
3c
3d
3e
R2
R3
H3CO
H3CO
j, R1=R4=R5=H; R2=OH; R3=OCH3
k, R1=R4=R5=H; R2=NO2; R3=OCH3
l, R2=R3=R5=H; R1=R4=OCH3
R5
m, R1=R5=H; R2=R3=R4=OCH3
>100
>100
>100
R4
OCH3
3a-m
Scheme 1. Synthesis of the hydroxyethyl compounds 3a–m.
But the task of creating analogs with the potent cytotoxicity of
CA-4 and enhanced water solubility has proven much more chal-
lenging. For these reasons the related class of ‘‘hydroxyethyl’’ ana-
logs were synthesized (3j, Fig. 1), with the goal of further
increasing the aqueous solubility and potency of the acetyl analogs,
while retaining the optimal twisted conformation. Thirteen specific
analogs were synthesized and were divided into two groups shown
in Table 1. The compounds in Group A contain a mono substitution
on the B ring (3a–g), while compounds in Group B, contain a di- or
tri-substitution on the B ring (3h–m). The conformation of analog
3j was assessed by molecular modeling studies using MacSpartan.
The molecular structures were optimized by molecular mechanics
(MMFF) using a molecular equilibrium conformer procedure. The
equilibrium geometry was subsequently optimized using the Har-
tree–Fock (3-21 G) calculations, followed by a density-functional
calculation (B3LYP and 6-31 G). Results given in Figure 2, show
that compound 3j has a twisted conformation similar to CA-4 as
well as the acetyl-CA-4, 2j.
3f
3.9
17.5
3g
>100
>100
GROUP B
3h
3i
18.5
69
51.5
>100
3j
4.1
16.1
3k
67.5
>100
As described in Scheme 1, all 13 derivatives were synthesized in
a two-step process by the reaction of the appropriately substituted
benzaldehydes with 3,4,5-trimethoxy phenyl acetone14 in the
presence of benzoic acid and piperidine in refluxing toluene. This
process yielded the acetyl-combretastatins 2a–m,12b which were
reduced using NaBH4 and CeCl3ꢀ7H2O in methanol. The reaction
was stirred for 24 h. The hydroxyethyl analogs 3a–m were purified
using silica gel column chromatography with ethyl acetate and
hexane as eluents. The chemical yields were 25–80%. The struc-
tures of the compounds were confirmed by using IR, 1H-NMR
and mass spectral analysis. The z-configuration or cis-stilbene
framework of analogs 2a–m and 3a–m was confirmed by a single
3l
>100
51.5
>100
84
3m
IC50 values are given in
for 72 h
lM. The cells were continuously treated with compounds
compounds that lacked a ‘twisted geometry’ were inactive as
microtubule depolymerizers and had lower cytotoxicity.13
Among these classes of analogs the acetyl-combretastatins (2j,
Fig. 1), show great promise in both their potency and selectivity.
crystal X-ray structure of 2e.12b The chemical shift of the
c-vinylic
proton of 2a–m at the 7.50 ppm range12a also corroborated the cis
geometry.
Figure 2. Molecular models of CA-4 (1a) and novel analogs 2j and 3j determined from Hartree–Fock and density functional calculations.

M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2087–2091
2089
Figure 3. NCI screen data (GI50 values) for compounds 3j against a panel of human cancer cell lines.
Table 2
Ratio of EC50 (microtubule depolymerization) to cytotoxicity (IC50, MDA-MB-435)
inhibit cell growth by 50%. The IC50 values for each compound in
B16 and L1210 cells are shown in Table 1.
Compound
EC50
(lM)
IC50
(l
M)
EC50/IC50
In this class of 13 derivatives, two compounds (3f and 3j) showed
1a10
1f10
2j12b
3j
0.003
0.5
1.8
0.007
0.14
0.18
0.88
2.3
3.6
10
modest cytotoxicity with IC50 values of 3.9 and 4.1
L1210 cell line, respectively, and 17.5 and 16.1 M for the B16 cell
line, respectively. Compounds 3b, 3i, 3k, and 3m showed low activ-
ity with IC50 values in the range of 33.5–67.5 M. The remaining
lM against the
l
21.2
24
l
analogs were inactive (IC50 >100 mM). It is worth noting that the
L1210 cells are more sensitive to these compounds than B16
cells.7–12 The nature of the differences in sensitivity might be related
to the higher proliferation rate of L1210. Overall, the cytotoxicity
results showed two clear trends. First, the results showed that
substitution in the 4-position (para) significantly enhanced
Each of the hydroxyethyl compounds 3a–m was evaluated for
activity against L1210 and B16 cells (murine lymphoma and mela-
noma respectively) using a 72-h continuous exposure MTT assay.15
The assays yielded IC50 values, the concentration required to

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M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2087–2091
cytotoxicity, especially for a methoxy group (3f) over its alkyl, halide,
or nitro counterpart. We hypothesize that this is related to the size
and electronic characteristics of the substituent. Second, the results
showed that substitution on the 3-position further affected the
potency of the compounds. Compound 3j, which contains a hydroxy
group on the 3-position and a methoxy group on the 4-position, is as
equally active as compound 3f. However, when the 3-position con-
tains a bulkier substituent, such as a methoxy (3i and 3m) or nitro
(3k) group, the activity dropped significantly. Based on these results,
analog 3j, which has a substitution pattern closest to CA-4, was
selectedforfurtherevaluationtoinvestigateitsmechanismofaction,
aqueous solubility, and activity in a murine tumor model.
The cytotoxicity of compound 3j, was further evaluated by the
NCI (National Cancer Institute). The NCI data provided the GI50 val-
ues of the compound against 60 human cancer cell lines.16 The data
show that this compound caused growth inhibition, with some
selectivity. The results are summarized in Figure 3. In a separate
study using a previously reported method,10 the cytotoxicity of
determined from the standard curve. The aqueous sample was
mixed for 6 h, centrifuged to pellet soluble material, and the super-
natant removed. The absorbance of the supernatant was measured
and the concentration of 3j calculated from the standard curve. Our
results show that compound 3j has a solubility of 479 20.0
This is substantially higher than the solubility of CA-4, which
was measured to be 350
M in this assay.6g,10
lM.
l
In conclusion, hydroxyethyl CA-4 compounds 3a–m are novel
analogs of combretastatin A-4. Several of the compounds were
cytotoxic against the growth of a number of cancer cells in vitro.
The mechanism of action of compound 3j is likely to be multifacto-
rial, as indicated by the lack of close linkage of the concentration
required to disrupt cellular microtubules and the IC50 concentra-
tion. The closest relative to CA-4, hydroxyethyl CA-4 3j demon-
strated antitumor activity in mice with no toxicity. In addition,
hydroxyethyl compounds have significant advantages, which in-
clude the ease of synthesis and enhanced aqueous solubility.
analog 3j was determined to have an IC50 value of 0.88
lM against
Acknowledgments
MDA-MB-435 human melanoma cells (Table 2), which was consis-
tent with the GI50 value of 0.2 mM.
The authors thank Conjura Pharmaceuticals, LLC (ML), and Hope
To gain insight into the mechanism of action of the hydroxy-
ethyl CA-4 analogs, the effect of compound 3j on interphase micro-
tubules was examined using A-10 cells. The EC50 values,
concentration required to cause 50% loss of interphase microtu-
bules, for compounds 1a, 1f, 2j, and 3j are shown in Table 2. The
results were further analyzed by calculating the ratio of EC50/IC50
(MDA-MB-435) for analogs 1f, 2j, and 3j, and the results are given
in Table 2. Compounds that give a ratio of 2:3, including 1a and 1f,
show tight linkage of the microtubule depolymerizing and cyto-
toxic effects consistent with microtubule mediated primary mech-
anism of action. Oxadiazoline analog 1f was previously confirmed
to cause microtubule depolymerization.10 With a ratio of EC50/IC50
of 10 for analog 2j, the result suggests that microtubule disruption
contributes to the mechanism of action, but the linkage is not as
tight as with 1a, suggesting the possibility of a secondary mecha-
nism of cytotoxicity.12b However, the hydroxyethyl analog, 3j,
which has an EC50/IC50 ratio of 24, strongly suggests a secondary
mechanism of action. This finding is not totally surprising, since
small changes in the structure of CA-4 analogs have been shown
to produce large changes in biological effects.12 In fact, some CA-
4 analogs are known to effect other biological targets, such as the
kinesin spindle protein (KSP)17 and DNA.18
College/Arnold & Mabel Beckman Scholars Program (RS) for
support. Support from the President’s Council Excellence award
is gratefully acknowledged (SLM).
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With the significant in vitro activity of compound 3j against
cancer cells in culture, the effects of compound 3j were preliminar-
ily tested in vivo using DBA2 female mice that were inoculated
with L1210 mouse lymphocytic leukemia cells on the right flank.19
The mice were treated via an intraperitoneal (ip) route once a day
with just the vehicle (control) or once a day with compound 3j at a
dose of 75 mg/kg starting on days 1, 3, 5, 7, 9, 11, 13, 15, and 17
after tumor inoculation. The volume of each injection was 100
l
L.20 On day 19, the tumor volume of 3j-treated mice was reduced
by 50% compared to the vehicle-treated control. It is worth noting
that in a separate in vivo experiment, administration of compound
3j at a dose of 75 mg/kg on days 1, 5, 9, 13, and 17 to healthy DBA2
female mice resulted in no visible toxicity as indicated by changes
in body weight, blood cell counts, and the color and texture of the
kidneys, liver, and spleen at the end of the experiment.
Since one of the goals of this work was to design water soluble
CA-4 analogs, the aqueous solubility of 3j was measured using a
modified version of the Multi Screen Solubility Filter Plate protocol
developed by Millipore.10 The maximum aqueous solubility for the
assay is 500 lM. A standard curve is developed for each compound
by diluting into an 80:20 aqueous buffer:acetonitrile mixture with
a final concentration of 5% DMSO (v/v). The concentration of the
compound soluble in aqueous buffer without acetonitrile was
17. (a) Cox, C.; Breslin, M.; Mariano, B.; Coleman, P.; Buser, C.; Walsh, E.; Hamilton,
K.; Huber, H.; Kohl, N.; Torrent, M.; Yan, Y.; Kuo, L.; Hartman, G. Bioorg. Med.

M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2087–2091
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Hoffman, W. F.; Tasber, E. S.; Coleman, P. J.; Buser, C. A.; Walsh, E. S.; Hamilton,
K.; Fernandes, C.; Schaber, M. D.; Lobell, R. B.; Tao, W.; South, V. J.; Yan, Y.; Kuo,
L.; Prueksaritanont, T.; Shu, C.; Torrent, M.; Heimbrook, D. C.; Kohl, N. E.;
Huber, H. E.; Hartman, G. D. Bioorg. Med. Chem. Lett. 2006, 16, 1775; (c)
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D.; Fernandes, C.; Lobell, R. B.; Tao, W.; South, V. J.; Yan, Y.; Kuo, L. C.;
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20. The stock drug solutions were prepared by first dissolving 45 mg of each
compound in a total of 450
glycol 400 and 150 L Tween 80. Each solution was then diluted to a final
L with a solution consisting of 1200 L 5% glucose in sterile
of DMSO to give stock solutions for each compound
lL of absolute ethanol, 900 lL of polyethylene
l
volume of 3000
water and 300
l
l
l
L
comprising of a 75 mg/kg dosage. Both solutions were prepared in glass vials
and stored at room temperature. Each 100
drug into the mice via an ip route.
lL injection delivers 1.5 mg of the
18. Gupton, J. T.; Burham, B. S.; Krumpe, K.; Du, K.; Sikorski, J. A.; Warren, A. E.;
Barnes, C. R.; Hall, I. H. Arch. Pharm. 2000, 333, 3.