High content screening of diverse compound libraries identifies potent modulators of tubulin dynamics

Article abstract of DOI:10.1021/ml5000564

Tubulin modulating agents such as the taxanes are among the most effective antimitotic cancer drugs, although resistance and toxicity present significant problems in their clinical use. However, most tubulin modulators are derived from complex natural products, which can make modification of their structure to address these problems difficult. Here, we report the discovery of new antimitotic compounds with simple structures that can be rapidly synthesized, through the phenotypic screening of a diverse compound library for the induction of mitotic arrest. We first identified a compound, which induced mitotic arrest in human cells at submicromolar concentrations. Its simple structure enabled rapid exploration of activity, defining a biphenylacetamide moiety required for activity, A family of analogues was synthesized, yielding optimized compounds that caused mitotic arrest and cell death in the low nanomolar range, comparable to clinically used antimitotic agents. These compounds can be synthesized in 1-3 steps and good yields. We show that one such compound targets tubulin, partially inhibiting colchicine but not vinblastine binding, suggesting that it acts allosterically to the known colchicine-binding site. Thus, our results exemplify the use of phenotypic screening to identify novel antimitotic compounds from diverse chemical libraries and characterize a family of biphenylacetamides (biphenabulins) that show promise for further development.

Full text of DOI:10.1021/ml5000564

Letter
pubs.acs.org/acsmedchemlett
High Content Screening of Diverse Compound Libraries Identifies
Potent Modulators of Tubulin Dynamics
Luca Laraia,†,‡ Jamie Stokes,†,‡ Amy Emery,‡ Grahame J. McKenzie,‡ Ashok R. Venkitaraman,*,‡
and David R. Spring*,†
†Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.
‡MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Biomedical Campus, Cambridge CB2 0XZ, U.K.
S
* Supporting Information
ABSTRACT: Tubulin modulating agents such as the taxanes are
among the most effective antimitotic cancer drugs, although
resistance and toxicity present significant problems in their
clinical use. However, most tubulin modulators are derived from
complex natural products, which can make modification of their
structure to address these problems difficult. Here, we report the
discovery of new antimitotic compounds with simple structures
that can be rapidly synthesized, through the phenotypic screening
of a diverse compound library for the induction of mitotic arrest.
We first identified a compound, which induced mitotic arrest in
human cells at submicromolar concentrations. Its simple structure
enabled rapid exploration of activity, defining a biphenylaceta-
mide moiety required for activity, A family of analogues was
synthesized, yielding optimized compounds that caused mitotic
arrest and cell death in the low nanomolar range, comparable to clinically used antimitotic agents. These compounds can be
synthesized in 1−3 steps and good yields. We show that one such compound targets tubulin, partially inhibiting colchicine but
not vinblastine binding, suggesting that it acts allosterically to the known colchicine-binding site. Thus, our results exemplify the
use of phenotypic screening to identify novel antimitotic compounds from diverse chemical libraries and characterize a family of
biphenylacetamides (biphenabulins) that show promise for further development.
KEYWORDS: Phenotypic screening, antimitotics, tubulin, colchicine, allosteric
henotypic screening involves testing small molecules in
approved clinically.13,14 All approved compounds target
tubulin,15−17 and many unfortunately suffer from adminis-
tration and/or resistance problems.18 Additionally, as current
clinical compounds are mostly complex natural products or
derivatives thereof, modification to improve properties can be
limited to a few functional handles. Despite this, impressive
progress has been made in the total synthesis and derivatization
of tubulin-targeted antimitotics.19−21 New antimitotic com-
pounds could offer an alternative to existing therapies, and
several groups have dedicated significant efforts in this area,
utilizing natural product inspired compound collections,22−24 as
well as more traditional screening approaches.25−28 Our own
efforts in this area resulted in the identification of dosabulin, a
novel tubulin inhibitor from a DOS library of 35 compounds.29
In this work, we describe the evaluation of several compound
libraries produced by DOS and other methods, for the
induction of mitotic arrest. We identify one compound that
causes significant mitotic arrest followed by cancer cell death.
P
cellular or in vivo models directly, without any target bias.1
It has recently seen a resurgence, with the obervation that most
first-in-class drugs with novel mechanisms of action originate
from such approaches.2 Historically, the most successful drugs
have been identified in this way. Phenotypic screening is
particularly suitable when diverse compound collections are
available. These are predicted to display a broader range of
biological activity, which can be identified more efficiently when
a range of targets can elicit the same phenotype when
modulated. Diverse chemical libraries are often produced by
diversity-oriented synthesis (DOS).3−6 This approach aims to
build complex diverse small molecule libraries in an efficient
manner. The combination of DOS with phenotypic screening
has been successful in identifying antibacterial, antimalarial, and
anticancer compounds, validating this approach.7−9
Our group has recently produced several distinct libraries
using DOS and other more focused approaches,10−12 and their
potential to modulate biological systems has not been
evaluated. Recently, we have described an optimized
phenotypic high content screen (HCS) to identify compounds
that cause mitotic arrest. Causing mitotic arrest is a validated
anticancer mechanism, and several mitotic inhibitors are
Received: February 6, 2014
Accepted: February 24, 2014
Published: February 24, 2014
© 2014 American Chemical Society
598
dx.doi.org/10.1021/ml5000564 | ACS Med. Chem. Lett. 2014, 5, 598−603

ACS Medicinal Chemistry Letters
Letter
We obtain structure−activity relationships (SARs) to discover
more potent analogues, and the mechanism of action is
identified as perturbation of tubulin dynamics by binding to a
site that causes partial displacement of colchicine from tubulin.
These compounds, termed biphenabulins, are structurally
simple and can be assembled in just 2−3 synthetic steps,
providing a potential future alternative to currently used
microtubule targeting agents (MTAs) and promising molecules
for further optimization and biological evaluation.
Scheme 1. Synthesis of Antimitotic Biphenylacetamides and
Analogues
Using an optimized assay for identifying molecules that cause
mitotic arrest,11,30 a library of over 400 compounds was
screened. The library was composed of recently reported DOS
efforts focusing on macrocycles,10−12 as well as more focused
compound sets from methodology or medicinal chemistry
efforts in our group (Supplementary Figure S1). The assay is
undertaken in U2OS osteosarcoma cells and involves staining
for phosphorylated histone H3 (pH3), a mitotic marker,
following 20 h treatment with the test compounds. Cells are
visualized with Hoechst and imaged with the Cellomics
Arrayscan, a high content microscope. An automated algorithm
then calculates the percentage of cells arrested in mitosis. The
primary screen consisted of screening all compounds at a single
concentration of 50 μM in triplicate.29
From the primary screen one compound (1), which caused
mitotic arrest in 40−50% of all cells at 50 μM, was identified
(Figure 1). This hit was reconfirmed and showed a clear dose-
of Suzuki couplings with commercially available boronic acids
were performed with the para-bromoacetamide intermediates
4a and 4b. Finally, amide couplings on building block 6
delivered p-amido substituted analogues 7a−b in moderate
yield.
The importance of the biphenylacetate moiety was initially
investigated. This was replaced with a series of aromatic and
benzylic groups (Table 1, compounds 2a−f). Interestingly, only
the naphthyl acetate group retained some antimitotic activity,
albeit at higher concentrations than 1, while all other analogues
were inactive. Having established that the biphenylacetate
group was important for activity, we began exploring the amine
substituents (Table 1, 3a−j). All compounds with the exception
of the benzylamine-substituted 3a were less active than 1.
Compound 3a exhibited a 2-fold increase in activity, which
could be attributed to the increase in lipophilicity. Methylation
on the benzylic carbon (3b and 3c) was not tolerated, with the
(S)-enantiomer 3c being completely inactive, while the (R)-
enantiomer was an order of magnitude less potent than 1.
Methylation on the amide nitrogen was also not tolerated (3d),
leading to a reduction in activity of almost 2 orders of
magnitude. Substitution at the para-position led to a small
reduction in activity (3e), while substitution with a bromide at
the meta-position led to complete loss of activity (3f). Pyridines
3g and 3h also showed a significant reduction in activity, while
substitution for a phenyl (3i) or aliphatic iso-propyl group (3j)
completely abolished activity.
Figure 1. Data of the phenotypic screen for mitotic arrest. Each
compound was screened at 50 μM in U2OS cells, which were stained
with Hoechst and a phospho-histone H3 specific antibody. The
structure and associated data for hit compound 1 are overlaid. Data
shown as the mean of a single experiment conducted in triplicate;
nocodazole was used at 200 nM.
dependent effect, with an EC50 for mitotic arrest of 0.51 μM.
This was particularly significant as the structure of the molecule
was very simple and thus amenable to optimization. With this
result in hand, we sought to investigate the mode of action of 1
and determine the SARs. To synthesize analogues, several
similar strategies relying on amide and Suzuki couplings were
employed (Scheme 1). Commercial carboxylic acids were
subjected to amide coupling conditions with furfurylamine to
explore right-hand side substitutions. All couplings proceeded
in moderate to good yields (2a−f, 45−69%) using T3P and
DIPEA in ethyl acetate. Using similar amide coupling
conditions, 10 analogues were synthesized in moderate to
excellent yield (3a−j, 32−98%) from biphenyl acetate and
commercial amines. In most cases, column chromatography
was not required to purify the final compounds. To further
investigate SARs on the right-hand side of the molecule, a range
It must be noted that because the SAR was obtained from a
phenotypic screen, factors such as cell permeability and
metabolic stability may affect activity. To address this issue,
physiochemical properties were calculated for all molecules
599
dx.doi.org/10.1021/ml5000564 | ACS Med. Chem. Lett. 2014, 5, 598−603

ACS Medicinal Chemistry Letters
Letter
Table 1. Synthesis and Biological Evaluation of
Biphenylacetamides with Differing Substituents on the Left-
and Right-Hand Sides
Table 2. Effect of Substitution on the Right-Hand Side Aryl
Ring on the Induction of Mitotic Arrest and Growth
a
Inhibition in U2OS Cells
mitotic index
(MI)
growth
inhibition
GI50 (μM)
mitotic index (MI)
a
Code
-R1
-R2
EC50 (μM)
code
R
Ar
EC50 (μM)
1
2-furfuryl
2-furfuryl
2-furfuryl
2-furfuryl
2-furfuryl
2-furfuryl
2-furfuryl
benzyl
4-phenylbenzyl
2- naphthylmethyl
2-phenylbenzene
4-benzoylphenyl
2-benzofuran
0.51
12.9
>40
>40
>40
>40
>40
0.25
4.83
>40
29.9
1.90
>40
16.6
9.70
>40
>40
1
2-furfuryl phenyl
0.51
0.15
2a
2b
2c
2d
2e
2f
5a
5b
5c
2-furfuryl 2-acetylphenyl
12.9
N/D
0.120
0.100
2-furfuryl 3-acetamido phenyl
0.490
0.340
2-furfuryl 3-fluoro-4-methyl
phenyl
3-quinoline
5d
5e
5f
2-furfuryl 3,4,5-trimethoxyl
phenyl
>40
N/D
0.603
0.120
biphenyl
2-furfuryl 3,5-dimethyl-4-
methoxy phenyl
1.17
N/D
3a
3b
3c
3d
3e
3f
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
4-phenylbenzyl
(R)-N-(1-phenyl ethyl)
(S)-N-(1-phenyl ethyl)
N-(Me)-N-(Bn)
p-methoxybenzyl
m-bromobenzyl
4-pyridyl
2-furfuryl 4-trifluoromethoxy
phenyl
5g
5h
2-furfuryl 4-acetyl phenyl
0.076
0.073
0.024
0.024
2-furfuryl 4-benzoate methyl
ester
3g
3h
3i
5i
5j
5k
5l
2-furfuryl 4-cyanophenyl
2-furfuryl benzodioxole
0.526
0.144
0.028
0.081
0.111
0.031
0.011
0.027
2-pyridyl
phenyl
benzyl
benzyl
4-acetyl phenyl
3j
iso-butyl
4-benzoate methyl
ester
a
MI EC50 data is the mean of at least two independent experiments
conducted in triplicate.
6
2-furfuryl 4-benzoic acid
>20
N/D
0.021
7a
2-furfuryl 4-(methyl
carbamoyl)
0.051
synthesized in this series using Schrodinger’s QikProp
application (Supplementary Table 1).31 Their cell permeability,
clogP, and solubility were not predicted to vary greatly between
the series, suggesting that phenotypic SAR may be a viable
surrogate in the absence of on-target activity. In summary, even
small substitutions in the left-hand portion of the molecules
had a very big impact on activity. This suggests that it is not
possible to obtain large increases in activity from changes in this
region.
phenyl
7b
2-furfuryl 4-(isobutyl
carbamoyl)
0.101
0.046
phenyl
a
MI EC50 and GI50 values are the mean of at least two independent
experiments conducted in triplicate. N/D = not determined.
in activity was observed. Both the ketone 5g and ester 5h
displayed very high levels of activity in the mitotic index assay.
The carbonyl functionality was essential for maximal levels of
activity, as the para-nitrile 5i and benzodioxole 5j both
displayed lower acitivities.
To investigate SAR on the right-hand side of the molecule,
the para-bromoacetamide intermediates 4a and 4b, were
synthesized in one step using standard amide coupling
conditions. Following this, a range of Suzuki couplings with
commercially available boronic acids were performed to explore
different substitution patterns at this position. The substrates
selected were predominantly aromatics with substituents of
differing sizes and electronic properties attached to different
positions. Twelve final compounds (5a−l) were screened for
their ability to induce mitotic arrest in U2OS cells (Table 2).
A compound with an ortho-substituent (5a) was considerably
less active than the original hit, while the activity of those
containing a meta-substituent varied depending on the other
substituents present. Compounds 5b and 5c both displayed
similar activity to 1, whereas the trimethoxy-substituted 5d was
completely inactive. Reducing the size and polarity of the
substituents flanking the para-methoxy group with two methyl
groups restored an active compound (5e), though this was still
2-fold less potent than 1. The trifluoromethoxy analogue (5f)
displayed potent cytotoxicity; however, mitotic arrest was not
measurable. This suggests that 5f is acting by a different
mechanism. The trifluoromethoxy functional group is poten-
tially reactive and thus was not pursued further. When a para-
substituent bearing a carbonyl was introduced, a 7-fold increase
Having established the substituents that dictated activity in
the left and right portions of the molecule, these were
combined to observe whether an additive effect existed. For the
left-hand side, the benzyl substituent was selected, while for the
right-hand side the para-acetyl (5k) and para-methylester (5l)
groups were chosen. Both compounds showed an approximate
2-fold increase in activity compared to the furan containing
analogues 5g and 5h, consistent with the increase observed
from 1 to 3a. Compound 5k was particularly active at inducing
mitotic arrest and growth inhibition, with values in the low
nanomolar range (Table 2 and Supplementary Figure S2).
However, this compound was poorly soluble even in organic
solvents at concentrations higher than 1 μM, which was
predicted to cause problems at later stages of biological
evaluation. At this stage it was also observed that the activity of
5l decreased over time. This could be attributed to a slow
hydrolysis of the ester upon storage of the compound in
DMSO. To overcome these problems, analogues with
improved stability and solubility were sought. It was
hypothesized that an analogue with an amide substituent at
the para-position could improve stability. Returning to the
600
dx.doi.org/10.1021/ml5000564 | ACS Med. Chem. Lett. 2014, 5, 598−603

ACS Medicinal Chemistry Letters
Letter
Figure 2. Target identification of the biphenylacetamide antimitotic compounds. (a) Confocal microscopy images of U2OS cells stained with DAPI
(blue) and an α-tubulin specific antibody (green). Cells treated with 5k and biphenabulin (7a) showed a significantly disrupted tubulin network,
suggesting that this may be their molecular target; scale bar, 18 μm. (b) Representative tubulin polymerization assay with compounds from the
biphenylacetamide series. Tubulin (3 mg/mL) was incubated with test compounds and GTP at 37 °C for 1 h. All active compounds from the mitotic
index assay were also inhibitors of tubulin polymerization, whereas 3f, the inactive analogue, was not. All compounds were tested at 10 μM. (c) A
fluorescence polarization (FP) assay using FL-BODIPY-vinblastine (2 μM) as a tracer and tubulin (2 μM) showed that biphenabulin did not
interfere with vinblastine binding to tubulin; data shown as mean s.e.m. for an experiment conducted in triplicate (ex/em wavelengths 485/520
nm). (d) Fluorescence intensity (FI) assay data of biphenabulin inhibiting tubulin at the colchicine site. Colchicine (3 μM), tubulin (3 μM), and
biphenabulin were incubated for 1 h at 37 °C. While a dose-dependent decrease in FI was observed for this compound, it did not reach the same
level of inhibition as the positive control nocodazole. This could suggest that biphenabulin is a tubulin inhibitor that acts near or allosteric to the
colchicine site; data shown as mean s.e.m. for an experiment conducted in triplicate (ex/em wavelengths 365/435 nm).
furan or an alternative heterocycle on the left-hand side was
predicted to improve solubility. The precursor for such
compounds (6) was synthesized by Suzuki coupling and
subjected to amide coupling conditions using a very small and a
sterically hindered amine to assess what size substituent could
be tolerated at this position (Table 2).
Both compounds synthesized retained very good levels of
antimitotic and growth inhibitory activity, with 7a being the
most active (Table 2 and Supporting Information Figure S2).
This suggests that a larger amide substituent is less tolerated
than a simple methyl amide, though the difference in activity
was not large. The precursor containing the carboxylic acid (6)
was completely inactive, which may be the result of a predicted
lack of cell permeability (Supplementary Table S1).
Having obtained compounds with low nanomolar activity for
mitotic arrest and growth inhibition, the identification of their
targets was the next goal. Confocal microscopy was used for in-
depth analysis of the phenotype resulting from compound
treatment. The DNA dye 4′,6-diamidino-2-phenylindole
(DAPI) and an α-tubulin specific antibody were selected to
image mitotic arrest. Compounds 5k and 7a were selected for
analysis, as these were the most potent and stable compounds
identified. Images of the interphase cells showed a disrupted
tubulin network, which suggested that tubulin itself could be
the target of these molecules (Figure 2a).
Having identified a potential target of the series, several
compounds were selected for testing in a tubulin polymer-
ization assay.22 Four compounds with differing potencies in the
cell-based mitotic index assay were selected to give a
representative spread in activity (Figure 2b). All compounds
that were active in the cell-based mitotic index assay were also
inhibitors of tubulin polymerization, whereas the inactive
compound 3f showed no activity in this assay. Interestingly,
the order of activity in the mitotic index assay was also reflected
in the tubulin polymerization assay, with 7a being the most
potent at reducing polymerization followed by 1 and then 5i
(Supplementary Table S2). Compound 7a was therefore
termed biphenabulin, for biphenyl tubulin inhibitor. Nocoda-
zole was significantly more potent at reducing the tubulin
polymerization rate than the biphenylacetamides despite having
similar potencies in the MI assay (EC50 = 50 nM for
nocodazole and 30−70 nM for the biphenylacetamides). This
could suggest a differing mechanism of inhibition of polymer-
ization for the biphenylacetamide compounds. To investigate
601
dx.doi.org/10.1021/ml5000564 | ACS Med. Chem. Lett. 2014, 5, 598−603

ACS Medicinal Chemistry Letters
Letter
their potential binding site(s), biphenabulin was selected for
further analysis as it displayed good antimitotic activity,
solubility, and stability. Several binding sites on tubulin have
been reported for inhibitors of tubulin polymerization of which
the vinca alkaloid and the colchicine ones are the best
studied.32,33 For other small molecules such as noscapine, the
binding site has not yet been identified.23 The Vinca alkaloid
and colchicine sites were selected for analysis, as we had
recently optimized assays to study their engagement.29
Biphenabulin was initially tested in a fluorescence polarization
assay monitoring the interaction of vinblastine with tubulin
(Figure 2c). The data clearly showed that this compound was
not able to inhibit the binding of fluorescent vinblastine to
tubulin, while the positive control (unlabeled vinblastine) was.
Following this, biphenabulin was also assayed for its ability to
inhibit tubulin at the colchicine site, using a fluorescence
intensity (FI) assay previously described.29 A dose-dependent
decrease in colchicine FI was seen with biphenabulin, although
the decrease did not occur to the same extent as the positive
control nocodazole (Figure 2d). This corresponded to a
maximal inhibition of approximately 45% for biphenabulin,
while nocodazole reached 85−90% inhibition. We would expect
a molecule that bound in the same site as colchicine to
completely displace it and thus reduce fluorescence, which
could suggest that biphenabulin is a tubulin inhibitor that acts
allosterically to the colchicine site, resulting in a reduction of
the ability of tubulin to bind colchicine.
AUTHOR INFORMATION
Corresponding Authors
*(A.R.V.) E-mail: arv22@cam.ac.uk.
*(D.R.S.) E-mail: spring@ch.cam.ac.uk.
Author Contributions
The manuscript was written by L.L with contributions from all
authors. All authors have given approval to the final version of
the manuscript.
■
Funding
We thank Cancer Research U.K. (to L.L., A.R.V., and D.R.S.)
and the U.K. Medical Research Council (to J.S., A.E., G.J.M.,
and A.R.V.) for support.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
DOS, diversity-oriented synthesis; HCS, high content screen-
ing; SAR, structure−activity relationship
■
REFERENCES
■
(1) O’Connor, C. J.; Laraia, L.; Spring, D. R. Chemical genetics.
Chem. Soc. Rev. 2011, 40, 4332−4345.
(2) Swinney, D. C.; Anthony, J. How were new medicines
discovered? Nat. Rev. Drug. Discovery 2011, 10, 507−519.
(3) Schreiber, S. L. Target-oriented and diversity-oriented organic
synthesis in drug discovery. Science 2000, 287, 1964−1969.
(4) Galloway, W. R. J. D.; Isidro-Llobet, A.; Spring, D. R. Diversity-
oriented synthesis as a tool for the discovery of novel biologically
active small molecules. Nat. Commun. 2010, 1, 80.
In summary, a high content screen of a small diverse library
for induction of mitotic arrest was implemented. One
compound was identified that arrested cells in mitosis at
submicromolar concentration. Expedient synthesis of analogues
resulted in the rapid exploration of SAR. After exploration of
the SAR and having obtained compounds with low nanomolar
activity for mitotic arrest and growth inhibition, the
identification of their targets was the next goal.
(5) O’Connor, C. J.; Beckmann, H. S. G.; Spring, D. R. Diversity-
oriented synthesis: producing chemical tools for dissecting biology.
Chem. Soc. Rev. 2012, 41, 4444−4456.
(6) Tan, D. S. Diversity-oriented synthesis: exploring the
intersections between chemistry and biology. Nat. Chem. Biol. 2005,
1, 74−84.
(7) Thomas, G. L.; Spandl, R. J.; Glansdorp, F. G.; Welch, M.;
Bender, A.; Cockfield, J.; Lindsay, J. A.; Bryant, C.; Brown, D. F. J.;
Loiseleur, O.; Rudyk, H.; Ladlow, M.; Spring, D. R. Anti-MRSA agent
discovery using diversity-oriented synthesis. Angew. Chem., Int. Ed.
2008, 47, 2808−2812.
(8) Heidebrecht, R. W.; Mulrooney, C.; Austin, C. P.; Barker, R. H.;
Beaudoin, J. A.; Cheng, K. C.-C.; Comer, E.; Dandapani, S.; Dick, J.;
Duvall, J. R.; Ekland, E. H.; Fidock, D. A.; Fitzgerald, M. E.; Foley, M.;
Guha, R.; Hinkson, P.; Kramer, M.; Lukens, A. K.; Masi, D.;
Marcaurelle, L. A.; Su, X.-Z.; Thomas, C. J.; Weiwer, M.; Wiegand, R.
C.; Wirth, D.; Xia, M.; Yuan, J.; Zhao, J.; Palmer, M.; Munoz, B.;
Schreiber, S. Diversity-oriented synthesis yields a novel lead for the
treatment of malaria. ACS Med. Chem. Lett. 2012, 3, 112−117.
(9) Oh, S.; Nam, H. J.; Park, J.; Beak, S. H.; Park, S. B. Development
of a benzopyran-containing androgen receptor antagonist to treat
antiandrogen-resistant prostate cancer. ChemMedChem 2010, 5, 529−
533.
(10) Isidro-Llobet, A.; Murillo, T.; Bello, P.; Cilibrizzi, A.;
Hodgkinson, J. T.; Galloway, W. R. J. D.; Bender, A.; Welch, M.;
Spring, D. R. Diversity-oriented synthesis of macrocyclic peptidomi-
metics. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6793−6798.
(11) O’Connell, K. M. G.; Beckmann, H. S. G.; Laraia, L.; Horsley,
H. T.; Bender, A.; Venkitaraman, A. R.; Spring, D. R. A two-directional
strategy for the diversity-oriented synthesis of macrocyclic scaffolds.
Org. Biomol. Chem. 2012, 10, 7545−7551.
Using confocal microscopy, a disrupted tubulin network was
observed, and the mechanism of action was confirmed as
tubulin depolymerization. One of the most active compounds,
termed biphenabulin, was shown not to interfere with
vinblastine binding to tubulin, but to partially inhibit colchicine
binding. This suggests that it may be binding allosterically to
the colchicine site, as direct inhibitors of the colchicine site such
as nocodazole cause complete displacement of colchicine from
tubulin. The mode of action is very similar to dosabulin, which
we recently discovered. However, biphenabulin is 2 orders of
magnitude more potent and is synthesized in fewer (3
compared to 7) steps. Additionally, only 27 analogues were
required before molecules with a cellular potency in the low
nanomolar range (similar to clinically used drugs such as
vinblastine) were identified. Many anticancer drugs that target
tubulin display formulation and resistance problems; therefore,
new, simple modulators of tubulin dynamics amenable to rapid
modification would provide promising alternatives. We hope
that the molecules presented herein will serve as a starting
point or inspiration for future efforts in this area.
ASSOCIATED CONTENT
(12) Beckmann, H. S. G.; Nie, F.; Hagerman, C. E.; Johansson, H.;
Tan, Y. S.; Wilcke, D.; Spring, D. R. A strategy for the diversity-
oriented synthesis of macrocyclic scaffolds using multidimensional
coupling. Nat. Chem. 2013, 5, 861−867.
(13) Manchado, E.; Guillamot, M.; Malumbres, M. Killing cells by
targeting mitosis. Cell Death Differ. 2012, 19, 369−377.
■
S
* Supporting Information
Synthetic procedures and compound characterization as well as
biological assay protocols. This material is available free of
charge via the Internet at http://pubs.acs.org.
602
dx.doi.org/10.1021/ml5000564 | ACS Med. Chem. Lett. 2014, 5, 598−603

ACS Medicinal Chemistry Letters
Letter
(14) Chan, K. S.; Koh, C. G.; Li, H. Y. Mitosis-targeted anti-cancer
therapies: where they stand. Cell Death Dis. 2012, 3, e411.
(15) Dumontet, C.; Jordan, M. A. Microtubule-binding agents: a
dynamic field of cancer therapeutics. Nat. Rev. Drug Discovery 2010, 9,
790−803.
(16) Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer
drugs. Nat. Rev. Cancer 2004, 4, 253−265.
(17) Perez, E. A. Microtubule inhibitors: Differentiating tubulin-
inhibiting agents based on mechanisms of action, clinical activity, and
resistance. Mol. Cancer Ther. 2009, 8, 2086−2095.
(18) Kavallaris, M. Microtubules and resistance to tubulin-binding
agents. Nat. Rev. Cancer 2010, 10, 194−204.
(19) Smith, A. B.; Freeze, B. S.; Xian, M.; Hirose, T. Total synthesis
of (+)-discodermolide: a highly convergent fourth-generation
approach. Org. Lett. 2005, 7, 1825−1828.
(20) Ishikawa, H.; Colby, D. A.; Boger, D. L. Direct coupling of
catharanthine and vindoline to provide vinblastine: total synthesis of
(+)-and ent-(−)-vinblastine. J. Am. Chem. Soc. 2008, 130, 420−421.
(21) Ganem, B.; Franke, R. R. Paclitaxel from primary taxanes:
Perspective on creative invention in organozirconium chemistry. J. Org.
Chem. 2007, 72, 3981−3987.
(22) Voigt, T.; Gerding-Reimers, C.; Ngoc Tran, T. T.; Bergmann,
S.; Lachance, H.; Scholermann, B.; Brockmeyer, A.; Janning, P.;
̈
Ziegler, S.; Waldmann, H. A natural product inspired tetrahydropyran
collection yields mitosis modulators that synergistically target CSE1L
and tubulin. Angew. Chem., Int. Ed. 2013, 52, 410−414.
(23) Zimmermann, T. J.; Roy, S.; Martinez, N. E.; Ziegler, S.;
Hedberg, C.; Waldmann, H. Biology-oriented synthesis of a
tetrahydroisoquinoline-based compound collection targeting micro-
tubule polymerization. ChemBioChem 2013, 14, 295−300.
(24) Antonchick, A. P.; Gerding-Reimers, C.; Catarinella, M.;
Schuermann, M.; Preut, H.; Ziegler, S.; Rauh, D.; Waldmann, H.
Highly enantioselective synthesis and cellular evaluation of spiroox-
indoles inspired by natural products. Nat. Chem. 2010, 2, 735−740.
(25) Yang, W. S.; Shimada, K.; Delva, D.; Patel, M.; Ode, E.; Skouta,
R.; Stockwell, B. R. Identification of simple compounds with
microtubule-binding activity that inhibit cancer cell growth with high
potency. ACS Med. Chem. Lett. 2011, 3, 35−38.
(26) Tcherniuk, S.; Deshayes, S.; Sarli, V.; Divita, G.; Abrieu, A.
UA62784 is a cytotoxic inhibitor of microtubules, not CENP-E. Chem.
Biol. 2011, 18, 631−641.
(27) Yi, X.; Zhong, B.; Smith, K. M.; Geldenhuys, W. J.; Feng, Y.;
Pink, J. J.; Dowlati, A.; Xu, Y.; Zhou, A.; Su, B. Identification of a class
of novel tubulin inhibitors. J. Med. Chem. 2012, 55, 3425−3435.
(28) Chen, J.; Ahn, S.; Wang, J.; Lu, Y.; Dalton, J. T.; Miller, D. D.;
Li, W. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III)
analogues targeting tubulin polymerization as antiproliferative agents.
J. Med. Chem. 2012, 55, 7285−7289.
(29) Ibbeson, B. M.; Laraia, L.; Alza, E.; O’Connor, C. J.; Tan, Y. S.;
Davies, H. M. L.; McKenzie, G. J.; Venkitaraman, A. R.; Spring, D. R.
Diversity-oriented synthesis as a tool for identifying new modulators of
mitosis. Nat. Commun. 2014, 5, 3155.
(30) Zheng, S.; Aves, S. J.; Laraia, L.; Galloway, W. R. J. D.; Pike, K.
G.; Wu, W.; Spring, D. R. A concise total synthesis of
deoxyschizandrin and exploration of its antiproliferative effects and
those of structurally related derivatives. Chem.Eur. J. 2012, 18,
3193−3198.
(31) QikProp, version 3.8; Schrodinger, LLC.: New York, 2013.
̈
(32) Gigant, B.; Wang, C.; Ravelli, R. B. G.; Roussi, F.; Steinmetz, M.
O.; Curmi, P. A.; Sobel, A.; Knossow, M. Structural basis for the
regulation of tubulin by vinblastine. Nature 2005, 435, 519−522.
(33) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar,
S.; Sobel, A.; Knossow, M. Insight into tubulin regulation from a
complex with colchicine and a stathmin-like domain. Nature 2004,
428, 198−202.
603
dx.doi.org/10.1021/ml5000564 | ACS Med. Chem. Lett. 2014, 5, 598−603