An amino-benzosuberene analogue that inhibits tubulin assembly and demonstrates remarkable cytotoxicity

Article abstract of DOI:10.1039/c2md00318j

The recent discovery of a small-molecule benzosuberene-based phenol that demonstrates remarkable picomolar cytotoxicity against selected human cancer cell lines and strongly inhibits tubulin polymerization (1-2 μM) inspired the design and synthesis of a variety of new, structurally diverse benzosuberene derivatives. An efficient synthetic route to functionalized benzosuberenes was developed. This methodology utilized a Wittig reaction, followed by a selective alkene reduction and ring-closing cyclization to form the core benzosuberone structure. This synthetic route facilitated the preparation of a 4-nitro-1-(3′,4′,5′-trimethoxyphenyl) benzosuberene derivative and its corresponding 4-amino analogue in good yield. The 4-amino analogue was a strong inhibitor of tubulin polymerization (1.2 μM), demonstrated enhanced cytotoxicity against the human cancer cell lines examined (GI₅₀ = 33 pM against SK-OV-3 ovarian cancer, for example), and exhibited a concentration dependent disruption of a pre-established capillary-like network of tubules formed from human umbilical vein endothelial cells.

Full text of DOI:10.1039/c2md00318j

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CONCISE ARTICLE
An amino-benzosuberene analogue that inhibits tubulin assembly and
demonstrates remarkable cytotoxicity†
Rajendra P. Tanpure,‡a Clinton S. George,‡a Madhavi Sriram,‡a Tracy E. Strecker,a Justin K. Tidmore,a
b
Ernest Hamel, Amanda K. Charlton-Sevcik, David J. Chaplin, Mary Lynn Trawick and Kevin G. Pinney
a
c
a
a
*
Received 26th December 2011, Accepted 10th February 2012
DOI: 10.1039/c2md00318j
The recent discovery of a small-molecule benzosuberene-based phenol that demonstrates remarkable
picomolar cytotoxicity against selected human cancer cell lines and strongly inhibits tubulin
polymerization (1–2 mM) inspired the design and synthesis of a variety of new, structurally diverse
benzosuberene derivatives. An efficient synthetic route to functionalized benzosuberenes was
developed. This methodology utilized a Wittig reaction, followed by a selective alkene reduction and
ring-closing cyclization to form the core benzosuberone structure. This synthetic route facilitated the
preparation of a 4-nitro-1-(30,40,50-trimethoxyphenyl) benzosuberene derivative and its corresponding
4-amino analogue in good yield. The 4-amino analogue was a strong inhibitor of tubulin
polymerization (1.2 mM), demonstrated enhanced cytotoxicity against the human cancer cell lines
examined (GI50 ¼ 33 pM against SK-OV-3 ovarian cancer, for example), and exhibited a concentration
dependent disruption of a pre-established capillary-like network of tubules formed from human
umbilical vein endothelial cells.
a dihydronaphthalene analogue 2 (discovered previously in our
laboratory) that is strongly cytotoxic and inhibitory against
tubulin assembly.1,8 In addition to its antiproliferative mecha-
nism of action, both CA4 and compound 2 damage tumor
vasculature.8,9 Preliminary studies with the benzosuberene
phenol 1, in appropriate prodrug form, also suggest vascular
damage as one component of its overall mechanism of action as
an antitumor agent.2 This makes it likely that the benzosuberene
amino derivative 3 would function through a similar mechanistic
pathway.
Introduction
The discovery of small-molecule anticancer agents that demon-
strate nanomolar to subnanomolar cytotoxicity against human
cancer cell lines is noteworthy, and such compounds have the
potential to become drug candidates through an appropriately
focused development program. The discovery of compounds that
demonstrate picomolar cytotoxicity is even more exciting. In
previous studies,1–3 we identified a functionalized benzosuberene-
based phenol 1 as one such compound (Fig. 1). Its structure is
reminiscent of both colchicine4,5 and combretastatin A-4 (CA4),6
which are natural products that are potent inhibitors of tubulin
assembly.7 Both compounds interact with tubulin at a small-
molecule binding site named the colchicine site (Fig. 1).
Small-molecule anticancer agents that target solid-tumor
vasculature represent an important emerging area of research
significance.9,10 Solid tumors require nutrients and oxygen
provided by a network of vasculature, which has distinct
morphological differences compared with a corresponding
vascular network feeding normal healthy tissue.11 Tumor
vasculature is highly disorganized with abnormal bulges, blind
ends, and shunts.12 It is also characterized as leaky and discon-
tinuous. It is these physiological dissimilarities that collectively
offer a therapeutic advantage for the selective targeting and
disruption of tumor vasculature through small-molecule drug
intervention.
Compound
1
also bears structural
similarity
to
aDepartment of Chemistry and Biochemistry, Baylor University, One Bear
Place #97348, Waco, Texas, 76798-7348, USA. E-mail: Kevin_Pinney@
baylor.edu; Fax: +1 254 710 4272; Tel: +1 254 710 4117
bScreening Technologies Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute
at Frederick, National Institutes of Health, Frederick, Maryland, 21702,
USA
Tumor vasculature can be efficiently targeted through two
distinct mechanistic pathways.13 Angiogenic inhibiting agents
(AIAs) prevent new blood vessel growth.14 Vascular disrupting
agents (VDAs), on the other hand, damage existing tumor
vasculature through a series of cell-signaling pathways.15,16 One
important class of small-molecule VDAs bind to tubulin
cOXiGENE Inc., 701 Gateway Boulevard, Suite 210, South San Francisco,
California, 94080, USA
† Electronic supplementary information (ESI) available: Synthesis and
characterization of compounds 3, 4, 8–16; as well as the methodology
used in the tubulin assembly, colchicine binding, SRB assays, and
endothelial tube disruption assay. See DOI: 10.1039/c2md00318j
‡ Authors with equal contribution.
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Fig. 1 Representative small-molecule inhibitors of tubulin assembly.
heterodimers at the colchicine site and thereby potently inhibit
tubulin assembly. The colchicine site is located on the b-subunit
of the ab-tubulin heterodimer, near the interface between the two
subunits.17
butoxide, and 3-methoxy-2-nitrobenzaldehyde resulted in
compound 8 in good yield (Scheme 2, Route 1). A selective
reduction of the alkene moiety to afford compound 9 was ach-
ieved using a 10% Pd/C catalyst with 1,4-cyclohexadiene in
excess. Reduced analogue 9 underwent cyclization upon treat-
ment with Eaton’s reagent (7.7 wt% P2O5 in methanesulfonic
acid)32 to afford nitrobenzosuberone 10 (ref. 33) in good yield.
Alternatively, nitrobenzosuberone 10 was prepared by nitration
of benzosuberone 15. The regioisomeric mixture obtained (10,
37% and 12, 49%) was separated chromatographically (Scheme
2, Route 2).33 Dropwise addition of nitrobenzosuberone 10 to
3,4,5-trimethoxyphenyllithium (prepared in situ from the corre-
sponding aryl bromide) gave tertiary alcohol 11, which under-
went elimination upon exposure to acetic acid to generate
nitrobenzosuberene 4. The nitro group in compound 4 was
reduced to its corresponding amine 3 upon treatment with zinc
dust in acetic acid. Eaton’s reagent has been successfully
employed for cyclization to form tetralone (6,6-fused) and ben-
zosuberone (6,7-fused) ring systems,32,37,38 and we were encour-
aged by the efficient extension of this methodology to these
functionalized benzosuberene core structures.
In the late 1970’s, the combretastatins were discovered by
Pettit and co-workers in the South African bush willow tree,
Combretum caffrum. Combretastatin A-1 (CA1)18–20 and com-
bretastatin A-4 (CA4)6 are two of the most potent compounds
isolated from C. caffrum, and both have pronounced biological
activity as VDAs and as inhibitors of tubulin polymerization. In
prodrug forms, both of these compounds, as well as the synthetic
combretastatin amino-analogue 7, are currently in human clin-
ical trials.21–25
Our original synthetic methodology towards the benzosuber-
ene molecular scaffold utilized a cyanogen azide ring expansion
reaction,1,26–28 and, while reliable, the yield for this overall
process was relatively low (Scheme 1). Accordingly, we devel-
oped a new, efficient synthetic strategy towards the functional-
ized six-seven fused ring system. This new method facilitated the
preparation of the nitro-benzosuberene analogue 4 and its cor-
responding amino derivative 3. These two new benzosuberene
analogues were evaluated for their ability to inhibit tubulin
assembly and for their cytotoxicity against three human cancer
cell lines.
Inhibition of tubulin assembly
The two new benzosuberene analogues 3 and 4 were found to be
potent inhibitors of tubulin assembly (IC50 ¼ 1.2 mM and
2.5 mM, respectively (Table 1)), comparable to CA1, CA4, and
the benzosuberene phenol 1. A binding assay utilizing tritium-
labeled colchicine indicated that amino-benzosuberene 3, at
a concentration of 5 mM, inhibited colchicine binding by 91%
(Table 1). At the lower concentration of 1 mM, amine 3 inhibited
colchicine binding by 65%. This is comparable with the activity
of CA4 (100% inhibition at 5 mM and 88% inhibition at 1 mM).
The nitro analogue 4 was less active as an inhibitor of colchicine
binding.
Results and discussion
Our improved synthetic approach to the target benzosuberene
analogues relied on a sequential Wittig reaction, selective
reduction, cyclization strategy to assemble the requisite 6,7-ring
fusion. Key features of this overall synthetic route include both
improved yields and fewer reaction steps. While other synthetic
approaches to this type of 6,7-fused ring system are known1,29,30
(including Friedel–Crafts cyclization31), the strategy that we
employed proved to be highly efficient. A Wittig reaction using 3-
(carboxypropyl)triphenylphosphonium bromide, potassium tert-
Scheme 1 Original synthetic route to the benzosuberenes.1–3
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Scheme 2 Improved ring-cyclization methodologies for the construction of benzosuberene-based compounds.
Table 1 Inhibition of tubulin polymerization and colchicine binding
Endothelial tube disruption
Inhibition of
colchicine bindinga
(%)
HUVECs, plated in medium supplemented with growth factors
onto Matrigelꢀ as a model for the extracellular matrix, form
a network of capillary-like tubules (Fig. 2, Control). The
disruption of these tubules by individual compounds provides an
in vitro assessment for vascular disrupting agents (VDAs). After
16 h, the endothelial tubule network was treated for 2 h with
varying concentrations of 3, 4, or CA4 (for comparison as
a positive control), or vehicle (medium containing 1% DMSO,
negative control). Representative photographs from a minimum
of three independent experiments are shown in Fig. 2. Tubule
disruption and cell rounding was observed for 3 at 0.10 mM, and
Inhibition of tubulin
polymerization IC50 (mM)
Compound
5 mM
1 mM
CA1
CA4
1
3
4
1.9b
ndc
100
nd
91
nd
88
nd
65
nd
1.2b d
,
1.7e
1.2
2.5
63
a
Reaction mixtures contained 1 mM tubulin, 5 mM [3H]colchicine, and
inhibitor as indicated. See ref. 34. nd ¼ not determined in this
b
c
e
study. d See ref. 35. See ref. 1.
Table 2 Cytotoxicity studies against human cancer cell lines NCI-H460,
DU-145, SK-OV-3
Cell line studies
GI50 (mM) SRB assaya
In addition, the compounds were evaluated for their cytotoxicity
against human non-small cell lung (NCI-H460), prostate (DU-
145), and ovarian (SK-OV-3) cancer cell lines. The amino-ben-
zosuberene analogue demonstrated remarkable cytotoxicity
against ovarian cancer with a GI50 value of 32.9 pM. In addition,
the compound was strongly cytotoxic against the non-small cell
lung and prostate cell lines. While somewhat less active than the
parent benzosuberene phenol 1, the amino derivative 3 was more
active against each cell line than the natural products CA4 and
CA1. The nitrobenzosuberene analogue 4 was significantly less
cytotoxic than any of the comparison compounds (Table 2).
Compound
NCI-H460
DU-145
SK-OV-3
CA1
CA4
1
3
4
16
0.015b
0.0028b
0.000028c
0.00469
0.216
0.033b
0.038b
0.00054b
0.0000032c
0.00111
0.211
0.00042b
0.00003c
0.0000329
0.152
ndd
0.00353
nd
Average of n $ 3 independent determinations. b For additional data see
a
ref. 36. See ref. 1. d nd ¼ not determined in this study.
c
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Fig. 2 HUVEC tube disruption. Effects of a 2 h treatment with (A) Cmpd. 3, (B) Cmpd. 4, and (C) CA4 on a pre-established network of capillary-like
tubules formed from HUVECS seeded onto Matrigelꢀ in a growth factor rich medium over a period of 16 h. CA4 was used as a positive control. All
experiments including the vehicle control contained a final concentration of 1% DMSO. These results are representative of the photographic record that
was obtained with a minimum of three independent experiments for each compound concentration and observing 9 fields per well (40ꢀ objective).
this effect was greatly increased at a concentration of 1 mM
(Fig. 2A). Compound 4 was much less active and demonstrated
no significant effects at concentrations of 0.01 or 0.10 mM. Only
minor disruption of tubules was observed at 1.0 mM compound
4, although a substantial number of cells were beginning to
round (Fig. 2B). In comparison, tubule disruption activity was
detected at 0.01 mM for CA4, and this activity was more
pronounced with increasing concentrations (Fig. 2C).
D2O). Obtaining a clear (to the naked eye) solution took several
days. A more robust solubilization strategy involved adding
DMSO (with vigorous vortexing and/or ultrasound to achieve
a clear solution) followed by the addition of water (to achieve
a favorable formulation for in vivo experiments in mice). The
amine salt 16 demonstrates cytotoxicity (in vitro) in the DU-145
prostate cancer cell line comparable to its parent amino-
benzosuberene analogue 3 (Table 1).
Hydrochloride salt formation
Conclusions
To facilitate planned in vivo experiments in mice, a hydrochloride
salt 16 was prepared synthetically from amine 3 in an effort to
obtain a more water-soluble derivative (Scheme 3). Although the
salt derivative 16 demonstrated limited solubility in water, it was
possible to obtain complete dissolution (NMR study used
approximately 3 mg of compound 16 in approximately 1.0 mL of
In summary, we have developed a sequential Wittig reaction,
selective reduction, Eaton’s reagent-mediated cyclization
strategy as an efficient synthetic route for the preparation of
functionalized benzosuberene derivatives. An amino-
benzosuberene analogue 3, prepared by this method, demon-
strated pronounced cytotoxicity against human cancer cell lines.
In addition, this compound was a potent inhibitor of tubulin
polymerization and effectively disrupted pre-established endo-
thelial tubes. In a collective sense, these data position compound
3 as an excellent candidate for further evaluation (through
additional biochemical/biological studies and in vivo tumor
imaging) as an anticancer, vascular disrupting agent.
Acknowledgements
The authors are grateful to the Cancer Prevention and Research
Institute of Texas (CPRIT (RP100406), to K.G.P. and M.L.T.),
Scheme 3 Synthesis of the HCl salt 16 of amine benzosuberene 3.
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