A-ring dihalogenation increases the cellular activity of combretastatin-templated tetrazoles

Article abstract of DOI:10.1021/ml200149g

The combretastatins have been investigated for their antimitotic and antivascular properties, and it is widely postulated that a 3,4,5-trimethoxyaryl A-ring is essential to maintain potent activity. We have synthesized new tetrazole analogues (32a€"34), demonstrating that 3,5-dihalogenation can consistently increase potency by up to 5-fold when compared to the equivalent trimethoxy compound on human umbilical vein endothelial cells (HUVECs) and a range of cancer cells. Moreover, this increased potency offsets that lost by installing the tetrazole bridge into combretastatin A-4 (1), giving crystalline, soluble compounds that have low nanomolar activity, arrest cells in G₂/M phase, and retain microtubule inhibitory activity. Molecular modeling has shown that optimized packing within the binding site resulting in increased Coulombic interaction may be responsible for this improved activity.

Full text of DOI:10.1021/ml200149g

Letter
pubs.acs.org/acsmedchemlett
A-Ring Dihalogenation Increases the Cellular Activity of
Combretastatin-Templated Tetrazoles
Thomas M. Beale,† Daniel M. Allwood,† Andreas Bender,‡ Peter J. Bond,‡ James D. Brenton,§
D. Stephen Charnock-Jones,∥ Steven V. Ley,† Rebecca M. Myers,*,† James W. Shearman,† Jill Temple,§
Jessica Unger,§ Ciorsdaidh A. Watts,† and Jian Xian§
†Department of Chemistry, Lensfield Road, University of Cambridge, Cambridge CB2 1EW, U.K.
‡Unilever Centre for Molecular Science Informatics, Lensfield Road, University of Cambridge, Cambridge CB2 1EW, U.K.
§Functional Genomics of Ovarian Cancer Laboratory, Cancer Research U.K., Cambridge Research Institute, Li Ka Shing Centre,
Robinson Way, Cambridge CB2 0RE, U.K.
∥Department of Obstetrics and Gynaecology, University of Cambridge, and National Institute for Health Research, Cambridge
Comprehensive Biomedical Research Centre, Cambridge CB2 0SW, U.K.
S
* Supporting Information
ABSTRACT: The combretastatins have been investigated for their
antimitotic and antivascular properties, and it is widely postulated that a
3,4,5-trimethoxyaryl A-ring is essential to maintain potent activity. We have
synthesized new tetrazole analogues (32−34), demonstrating that 3,5-
dihalogenation can consistently increase potency by up to 5-fold when
compared to the equivalent trimethoxy compound on human umbilical vein
endothelial cells (HUVECs) and a range of cancer cells. Moreover, this
increased potency offsets that lost by installing the tetrazole bridge into
combretastatin A-4 (1), giving crystalline, soluble compounds that have low
nanomolar activity, arrest cells in G2/M phase, and retain microtubule in-
hibitory activity. Molecular modeling has shown that optimized packing
within the binding site resulting in increased Coulombic interaction may be
responsible for this improved activity.
KEYWORDS: Combretastatin, tetrazole, dihalogenation, human umbilical vein endothelial cell, ovarian cancer,
vascular disrupting agent
ombretastatin A-4 (CA-4) (1) isolated from the African
that 3,5-dibromo- (7) and 3,5-diiodo- (8) substitution of the A-
1
Cwillow tree, Combretum caffrum, binds to the colchicine ring maintained, and even improved upon, the activity of 1 in
binding site on β-tubulin.2 At nanomolar concentrations, it
human umbilical vein endothelial cells (HUVECs) and ovarian
cancer cells (SK-OV-3 and paclitaxel-resistant SK-OV-3TR)
stabilizes the dynamics of cellular microtubules, arresting the
cell cycle in metaphase and triggering apoptosis.3 It is also a
potent vascular disrupting agent (VDA). It is this property that
has made 1 an attractive lead for the development of new
anticancer therapies, since in vivo it readily cuts off blood supply
to tumors.4,5 Such is the interest in 1 that several structurally
related VDAs are in clinical trials: the phosphate prodrug of 1
(Zybrestat, 2), the phosphate prodrug of combretastatin A-1
(3), OXi4503 (4), and a serine prodrug (Ombrabulin, 5) of a
3-amino CA-4 analogue (6) (Figure 1).6−8 The syntheses and
biological activities of combretastatin analogues have been
reviewed, and what emerges from this literature is an intriguing
correlation.9−12 Almost without exception, the shared structural
feature present in combretastatin analogues is a 3,4,5-trime-
thoxyaryl ring (A-ring), which is widely accepted to be essential
for biological activity. However, we and others have recently
reported that this is not necessarily the case (Figure 1).13−17 In
our recent work, we modified the A-ring of 1 and discovered
(Figure 1).13
Cell cycle analysis of SK-OV-3 cells treated with 5 nM 7 and
8 indicated that a higher percentage of cells were arrested at the
G2/M phase: 52% and 84%, respectively, when compared to
cells treated with 1 (31%).13 These encouraging results led us
to look at additional chemical improvements that could be
made. We envisaged replacing the double bond with a tetrazole
to mitigate the solubility problems of the cis-stilbenes in
aqueous media and address the tendency of the most potent
compound in the series (8) to isomerize in solution.18 The few
reports of tetrazole-containing VDAs have structures based on
the template of 1. For instance, direct replacement of the
double bond in 6 with a tetrazole ring resulted in a compound
Received: June 22, 2011
Accepted: January 19, 2012
Published: January 19, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Tetrazoles 32−34
Figure 1. Combretastatin A-4 (1) and A-1 (3) and analogues in
clinical trials. Dihalogenated A-ring analogues 7 and 8.13
with nanomolar activity (IC50 = 7.2 nM) on colon 26 cells that
maintained antitubulin activity (IC50 = 2 μM).19 Replacement
of the double bond in chalcone analogues with a tetrazole
resulted in compounds with low micromolar activity (IC50
=
4.1 μM) on neuroblastoma SH-SY5Y cells.20
Our general approach for synthesis of tetrazoles 32−34 was
to perform an amide coupling reaction between A-ring anilines
and a B-ring acid derivative. Conversion of the resulting amides
into thioamides and tetrazole formation using trimethylsilyl
azide in the presence of mercury(II) acetate were envisaged as
key steps toward the desired compounds (Scheme 1).
3,5-Dibromo-4-methoxy aniline (12) was obtained from 9 in
77% yield via acetyl protection of amine 9, phenol methylation,
and subsequent deacetylation. Iodination of 4-nitrophenol (13)
using iodine monochloride formed in situ from sodium chlorite
and sodium iodide gave 14.21 Treatment of 14 with dimethyl
sulfate and potassium carbonate in refluxing acetone gave 15,
which was reduced using tin chloride in ethyl acetate and
ethanol to give 3,5-diiodo-4-methoxyaniline (16) in 79% yield.
Benzoic acid 19, its ethyl carbonic anhydride derivative 20, or
its acid fluoride 21 were investigated as B-ring coupling partners.
Acid 19 was obtained from tert-butyldimethylsilyl (TBDMS)
protection of 17 in 39% yield or from aldehyde 18 via a two-
step sequence of silyl protection and then aldehyde oxidation
with copper(I) in 82% yield; 19 was transformed into 20 using
ethyl chloroformate, or alternatively into 21 using cyanuric
fluoride. After screening various coupling conditions, the
optimal procedure for the synthesis of 23 was found to be
diisopropylcarbodiimide (DIC) and N-hydroxybenzotriazole
(HOBt). For 24 and 25 it was found that treatment of anilines
12 and 16 with sodium hydride followed by 20 gave the best
results. Amides 23−25 were converted into their correspond-
ing thioamides 26−28 using Lawesson’s reagent. These were
then treated with mercury(II) acetate in the presence of
trimethylsilyl azide (TMSN3) to give 29−31 through a one-pot
desulfuration and cyclization reaction. After silyl protecting
group removal, tetrazoles 32−34 were obtained in 48%, 30%,
and 20% overall yield, respectively.
a
Reagents and conditions: (a) Ac2O, AcOH, rt, 30 min, 98%; (b) MeI,
K2CO3, acetone, 120 °C, μW, 30 min, 83%; (c) 3 M HCl, 140 °C, μW,
10 min, 95%; (d) NaClO2, NaI, HCl, MeOH/H2O, 0 °C to rt, 18 h,
94%; (e) (MeO)2SO2, K2CO3, acetone, 65 °C, 18 h, 87%; (f) SnCl2,
EtOH/EtOAc, 70 °C, 2 h, 79%; (g) NaH, THF, 0 °C then TBDMSCl,
39%; (h) TBDMSCl, Et3N, DMF, rt, 16 h, 97%; (i) 5 mol % CuCl,
t-BuOOH, MeCN, 24 h, rt, 85%; (j) For 20, ethyl chloroformate,
NEt3, THF, 0 °C to rt, 1 h, 98%; (k) For 21, cyanuric fluoride, py,
THF, −20 to 0 °C, 2 h, 99%; (l) For 23, DIC, HOBt, 19, CH2Cl2,
DMF, 60 °C, μW, 30 min, 70%. For 24, NaH, THF, 0 °C, then 20,
THF, rt, 18 h 44%. For 25, NaH, THF, 0 °C, then 20, THF, rt, 18 h,
62%; (m) Lawesson’s reagent, THF. For 26, 17 h, 80 °C, 98%. For 27,
4 h, 70 °C, 80%. For 28, 4 h, 70 °C, 79%; (n) Hg(OAc)2, TMSN3,
THF. For 29, 0 °C, 3 h, 87%. For 30, 0 °C, 1 h, 97%. For 31, 0 °C, 3 h,
87%; (o) TBAF, THF, rt, 1 h; 32, 81%; 33, 80%; 34, 68%.
Using cancer cell lines and primary vascular endothelial cells,
we investigated the effects of 32−34 on cell growth and on the
cell cycle. We also observed their effects on isolated tubulin
polymerization. Sulforhodamine B (SRB) assays were per-
formed on six human carcinoma cell lines using three replicate
experiments with errors shown as 95% confidence intervals
(CI) (Table 1). In summary, 32−34 had low nanomolar
activity (IC50 = 0.5−24 nM) on all cell lines, excepting
combretastatin-resistant HT29 cells. These results were
comparable to those of the reference compound 1 on the
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ACS Medicinal Chemistry Letters
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Table 1. Growth Inhibition Effects of 1 and 32−34 on Human Carcinoma and Fibroblast Cell Lines
1 (CA-4)
32 (OMe)
33 (Br)
34 (I)
cell line
IC50/nM
95% CI
IC50/nM
95% CI
IC50/nM
95% CI
IC50/nM
95% CI
SK-OV-3
SK-OV-3TR
MCF-7
A549
1.7
4.2
3.0
7.9
2.9
3.3
538
2.8
0.9−2.5
3.3−5.1
2.0−4.0
7.1−8.8
1.8−4.1
1.1−5.4
9.5
11
8.9−10
8.4−13
13−29
17−28
8.1−9.8
21−28
−
6.1
5.6
5.5−6.6
2.0−9.1
4.6−9.5
16−27
2.5−3.3
7.9−8.5
−
2.6
1.4
1.9
2.8
0.5
2.4
513
2.2
1.7−3.4
0.2−5.9
1.0−2.9
2.4−3.3
0.4−0.7
1.8−3.1
452−574
0.0−7.2
21
7.0
22
21
PC3
9.0
24
2.9
DLD1
8.2
HT29
425−651
0.1−5.7
>500
12
>500
5.7
MRC5
8.7−15.8
4.5−6.9
same cell lines (IC50 = 1.7−7.9 nM) (Table 1). A general
increase in potency was observed across 32−34 on all cell lines.
Trimethoxy tetrazole 32 was consistently the least potent
compound, dibrominated 33 was intermediate in potency, and
diiodinated 34 was the most potent compound, in most cases
more potent than CA-4 (1).
which gives a measure of cell number in real-time using
impedance measurements (Table 2).
Table 2. Growth Inhibition Effects of 32−34 on HUVECs
HUVEC 24 h IC50/nM
isolate
The potency of tetrazoles 32−34 was unaffected by the
mechanism of resistance in a taxol-resistant cell line (SK-OV-
3TR), previously shown to be MDR1 overexpression.22 How-
ever, CA-4 (1) and tetrazoles 32−34 were affected by HT29
resistance, with potencies reduced by over 100-fold. The re-
sistance of this cell line is mediated by drug glucuronidation
and MRP efflux.23 However, it has been recently shown that
tumor drug resistance is not detrimental to combretastatin
efficacy in vivo due to the predomination of antivascular
effects.24 The tetrazoles 32−34 also exhibited the same profile
as 1 on MRC5 human fibroblasts but once again demonstrated
that diiodination of the tetrazole series allows greater potency.
Cell cycle analysis on SK-OV-3 cells following treatment with
32−34 at 5 nM showed that G2/M arrest was correlated with
increasing potency of inhibition of cell growth (Figure 2). The
compd
1
2
3
4
5
6
mean
1 (CA-4)
32 (OMe)
33 (Br)
34 (I)
1.9
10.0
6.0
2.2
−
2.2
9.9
8.1
2.2
2.3
21.4
2.9
−
2.2
13.6
6.6
−
−
3.2
20.4
8.8
2.8
6.5
7.1
1.9
1.8
2.6
2.4
The average IC50 values on HUVECs reprise the results
observed for the SK-OV-3 cell line; that is, potency increased
from the trimethoxy compound 32 (IC50 = 13.6 nM), to 33
(IC50 = 6.6 nM) then 34 (IC50 = 2.4 nM), comparable to 1
(IC50 = 2.2 nM). The absolute values mirror those obtained on
SK-OV-3 cells, suggesting 32−34 display no selectivity for
vascular cells over carcinoma cells.
The effect of 32−34 and 1 on tubulin polymerization was
also investigated. Compounds were tested at a concentration of
5 μM, and data is reported as the fold reduction in Vmax relative
to vehicle control averaged over four separate measurements
standard deviation. Change in Vmax was used, as it is the most
sensitive indicator of tubulin/drug interactions. Compound 32
(2.9 1.7) had very similar potency to 1 (3.0 0.4), with both
compounds able to reduce tubulin polymerization approx-
imately 3-fold relative to control. Compounds 33 and 34 were
less active, with 1.5
0.4 and 1.7
0.3 fold reduction in
tubulin polymerization, respectively. The induction of micro-
tubule depolymerization at this concentration suggests that
these tetrazole compounds retain ability to bind at the colchicine-
binding site on tubulin.
To further investigate the binding ability of 32−34 and to
propose a rationale for the added potency of dihalogenated
compounds 33 and 34 over 32, we performed molecular
modeling with tubulin (pdb: 1SA0)25 (Table 3). The final
interaction energies between protein and ligand consistently
predicted that 32−34 bind in a single orientation analogous to
that for 1 and colchicine, with the B-ring 3-hydroxyl picking up
a key hydrogen bond with the backbone carbonyl of Thr179
and an interaction with the side chain ε-amino group of Lys350,
as previously described for many combretastatin analogues.26 It
appears that in both 33 and 34 there is an additional interaction
between the B-ring 4-methoxy oxygen and Lys350 that is not
present for 32.
Figure 2. Cell cycle analysis for 32−34 at 5 nM on ovarian cancer cells
(SK-OV-3).
percentage of cells in G2/M increased from 25% for 32, to 40%
for 33, to a maximum of 89% for the diiodinated tetrazole 34.
These results compare to 43% of cells in G2/M for CA-4 (1)
at 5 nM. In all cases, the percentage of sub-G1 cells was less
than 5%.
We then explored whether 32−34 could have therapeu-
tic potential as VDAs. HUVECs are a model of tumor endo-
thelium and can be used to evaluate antivascular effects of
bioactive compounds. The results from treatment of HUVECs
with 32−34 were obtained using the xCELLigence system,
The key difference between the calculated binding of 32−34
is in the position of the A-ring in the binding pocket and
179
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ACS Medicinal Chemistry Letters
Letter
Table 3. Calculated Interaction Energies and Modeled
Binding to Tubulin for Compounds 32−34 and
Contribution from van der Waals and Coulombic
Interactions (32 in Green, 33 in Cyan, and 34 in Magenta)
AUTHOR INFORMATION
Corresponding Author
*Telephone: +44 (0) 1223 336334. Fax: +44 (0) 1223 336442.
E-mail: rmm32@cam.ac.uk.
■
Funding
T.M.B., J.W.S., D.M.A., and C.A.W. thank the CRUK Ph.D.
Training Programme for Medicinal Chemistry for funding.
P.J.B. and A.B. thank Unilever for funding. S.V.L. acknowledges
support from the BP Endowment.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Richard Farndale for use of the
xCELLigence facilities at the Department of Biochemistry at
the University of Cambridge. The computational work used the
Darwin Supercomputer at the University of Cambridge High
Performance Computing Service (http://www.hpc.cam.ac.uk/)
provided by Dell Inc., using Strategic Research, Infrastructure
Funding from the Higher Education Funding Council for England.
binding energy/(kcal mol−1)
compd
total
van der Waals
Coulombic
32 (OMe)
33 (Br)
34 (I)
−83.19
−83.38
−90.14
−50.90
−48.00
−49.55
−32.29
−35.38
−40.59
ABBREVIATIONS
■
HUVECs, human umbilical vein endothelial cells; CA-4,
combretastatin A-4; VDA, vascular disrupting agent; TBDMS,
tert-butyldimethylsilyl; DIC, diisopropylcarbodiimide; HOBt,
N-hydroxybenzotriazole; TMS, trimethylsilyl; TBAF, tetrabu-
tylammonium fluoride; SRB, sulforhodamine B; CI, confidence
intervals
especially in the location of the 4-methoxy group relative to
Cys239, which is the important hydrogen bonding interac-
tion for this part of the molecule. For 34 the S−H···O distance
is 1.9 Å, whereas for 32 and 33 this distance is longer, at 2.1
and 2.7 Å, respectively. This demonstrates an improved ability
to hydrogen bond at this position for 34, while 33 further
suffers from a loss of electrostatic interaction in the tetrazole
bridge region with the backbone amine of Ala248 that is
maintained by diiodinated 34. This is reinforced by the cal-
culated interaction energies, which show that our most potent
compound (34) is predicted to have the greatest energy of
interaction (Table 3). Surprisingly, given the large size and
polarizability of iodine, it is not a greater van der Waals
interaction that gives this added interaction energy but rather
that the optimal packing of 34 within the binding site results in
improved electrostatic and hydrogen bonding interactions with
protein residues.
Taken together, this data shows that substituting the double
bond of CA-4 (1) with a tetrazole reduces cellular potency by
5- to 10-fold but that introduction of bromine or iodine atoms
at the 3- and 5-positions of the A-ring can restore potency to
the level of CA−4 (1). The tetrazoles are crystalline and
chemically stable, which is not true for many stilbene-based
combretastatin analogues, bringing obvious advantages. Molec-
ular modeling shows that the enhanced activity of diiodinated
compound 34 may be due to optimized packing into the
colchicine binding site, resulting in greater electrostatic
interactions with tubulin.
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