An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site

Abstract

Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding site is one of the most important pockets for potential tubulin polymerization destabilizers. Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. A large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers. CBSIs are believed to act by a common mechanism via binding to the colchicine site on tubulin. The present review is a synopsis of compounds that have been reported in the past decade that have provided an increase in our understanding of the actions of CBSIs.

Full text

An Overview of Tubulin Inhibitors That Interact with the
Colchicine Binding Site
Yan Lu, Jianjun Chen, Min Xiao, Wei Li, and Duane D. Miller
Department of Pharmaceutical Sciences, Health Science Center, University of Tennessee, 847
Monroe Ave, Room 327, Memphis, TN 38163, USA
Duane D. Miller: dmiller@uthsc.edu
Abstract
Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding
site is one of the most important pockets for potential tubulin polymerization destabilizers.
Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin
assembly and suppressing microtubule formation. A large number of molecules interacting with
the colchicine binding site have been designed and synthesized with significant structural
diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic
properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers.
CBSIs are believed to act by a common mechanism
via
binding to the colchicine site on tubulin.
The present review is a synopsis of compounds that have been reported in the past decade that
have provided an increase in our understanding of the actions of CBSIs.
Keywords
antimitotic; cancer; colchicine; multidrug resistance; tubulin polymerization inhibitor
INTRODUCTION
Drugs that disrupt microtubule/tubulin dynamics are used widely in cancer chemotherapy.
The vast majority of these molecules act by binding to the protein tubulin, an α, β-
heterodimer that forms the core of the microtubule. Microtubule targeting agents (MTA) are
also named antimitotic agents which perturb not only mitosis but also arrest cells during
interphase. MTAs are known to interact with tubulin through at least four binding sites: the
laulimalide, taxane/epothilone, vinca alkaloid, and colchicine sites (Fig. 1). Similar to
paclitaxel, Laulimalide can promote the tubulin-microtubule assembly, but binds to a
different site on the microtubules (1). Taxanes, including paclitaxel and docetaxel, bind to
polymerized microtubules at the inner surface of the β subunit, and are widely used in the
treatment of lung, breast, ovarian and bladder cancers. Taxanes promote tubulin
stabilization, thereby interfering with tubulin dynamics. Vinca alkaloids, including
vinblastine, vincristine, and vinorelbine, promote depolymerization of microtubules. They
generally bind with high affinity to one or a few tubulin molecules at the tip of microtubules
but do not copolymerize into microtubules. Indeed, vinblastine prevents self-association of
© Springer Science+Business Media, LLC 2012
Correspondence to: Duane D. Miller, dmiller@uthsc.edu.
DISCLOSURES
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of
Health. Additional supports were provided by the Van Vleet Endowed Professorship.
NIH Public Access
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. 2012 November ; 29(11): 2943–2971. doi:10.1007/s11095-012-0828-z.
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tubulin by interacting at the interface between two αβ–tubulin heterodimers (2). The fourth
group of microtubule interfering agents is represented by colchicine, which also induces
microtubule depolymerization. In contrast to agents binding to the other three sites,
colchicine binds with high affinity to tubulin that can become copolymerized into
microtubules. Colchicine binding to β-tubulin results in curved tubulin dimer and prevents it
to adopt a straight structure, due to a steric clash between colchicine and α-tubulin, which
inhibits microtubule assembly (3).
Given the success of the taxanes and vinca alkaloids, which have established tubulin as a
valid target in cancer therapy, research efforts have been focused on developing colchicine-
like compounds for cancer treatment.
COLCHICINE AS A MEDICINE
Colchicine was extracted from the poisonous meadow saffron
Colchicum autumnale L.
and
was the first tubulin destabilizing agent. It has been used for many years as an unapproved
drug to treat gout, familial mediterranean fever, pericarditis and Behçet’s disease. In 2009,
U.S. Food and Drug Administration (FDA) approved colchicine as a monotherapy drug to
treat familial mediterranean fever and acute gout flares. Colchicine can effectively inhibits
mitosis. Since cancer cells undergo mitosis at a significantly increased rate, this means that
cancer cells are more susceptible to colchicine poisoning than are normal cells. Therefore
colchicine is also being investigated as an anti-cancer drug. However, the therapeutic value
of colchicine against cancer is restrained by its low therapeutic index. Its toxicity includes
neutropenia, gastrointestinal upset, bone marrow damage and anemia.
Although colchicine is not used as an anticancer agent, there have been multiple efforts to
clinically develop colchicine binding site agents (CBSI). As microtubules are important
regulators of endothelial cell biology, one advantage of mechanism of actions of CBSIs is
targeting the tumor vasculature. CBSIs can prevent new blood vessels formation by
outgrowth from preexisting ones (angiogenesis inhibitors) or destroy the existing tumor
vasculature (vascular disrupting agents, VDA). The targeting of tumor blood vessels
introduces a therapeutically promising application for these compounds. Another favorable
factor is that most of these drugs have no multidrug resistance (MDR) issues. The major
limitation of using microtubule-targeting agents clinically is innate and acquired drug
resistance. The most common form of clinical resistance is overexpression of the MDR1
gene, which encodes the P-glycoprotein (Pgp) drug efflux pump. This membrane-associated
ATP-binding cassette (ABC) transporter is overexpressed in many tumor cell lines,
including tissues of the liver, kidney, and gastrointestinal tract. Over-expression of Pgp
decreases intracellular drug levels, consequently limiting drug cytotoxicity. In addition,
over-expression of Pgp is associated with poor response to microtubule-targeted agents
including taxanes and vinca alkaloids and subsequent treatment failure.
Besides over-expression of ABC transporters, other significant mechanism of resistance
including mutantions in tubulin and overexpression of βIII -tubulin isoform. Among eight
identified β-tubulin isotypes in human, overexpression of class III β-tubulin is an indicator
of resistance to tubulin targeting agents such as paclitaxel and vinorelbine. While the
efficacies of some CBSIs such as colchicines and 2-methoxyestradiol were not affected by
the expression pattern of β-tubulin (4,5). The clinical development of a microtubule-
targeting agent that circumvents both of these drug resistance mechanisms could have
advantages for patients with drug resistant tumors.
To overcome these resistance problems, many research efforts have concentrated on
developing CBSIs, although to date no agent that binds within the colchicine binding site is
approved for use against cancer. Many CBSIs have been identified as potential anticancer
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agents for clinical studies due to their ability to overcome Pgp/β-III tubulin mediated drug
resistance and their antiangiogenic or antivascular actions. Most CBSIs have small
molecular weight with chemically modifiable structures, thus they afford adequate space for
chemical modification to improve pharmacokinetic (PK) properties, efficacy and reduce
toxicity.
The current review searched the PubMed using key words “colchicine, tubulin and cancer”.
A total of 441 references were found from 1971 to 2010 and showed the trend of increasing
interests every decade (Fig. 2) in CBSIs for treatment of cancer. In this article, we focus on
CBSI agents in current clinical trials and recently published research papers after 2000 (Fig.
2, total 296 publications) with high citation rates.
NATURAL PRODUCTS AND CBSI IN CLINICAL TRIALS
Many of the CBSIs are based on natural products such as colchicinoids and combretastatins,
while others are synthetic compounds such as ABT-751. In this section, we will discuss the
well-known CBSIs and recent progress of CBSIs in clinical trials (Fig. 3).
Colchicine (1) and ZD6126 (2)—A number of clinical trials have been done on
colchicine (1) for treatment of various diseases including cancer. However, the clinical use
in treatment of cancer was hampered by its significant toxicity. ZD6126 (2) is a water-
soluble phosphate prodrug of
N-
acetylcolchinol structurally very similar to colchicine with
potential antiangiogenesis and antineoplastic activities (6,7). ZD6126 was developed by
AstraZeneca for the treatment of metastatic colorectal cancer. However the study was
terminated at phase II due to apparent cardiotoxicity at pharmacological doses.
CA-4 and its Analogs (3–7)—Combretastatins are a class of stilbenoid phenols isolated
from
Combretum caffrum
. Combretastatin A-4 (CA-4, 3) is the most potent naturally
occurring combretastatin known in regards to both tubulin binding ability and cytotoxicity.
CA-4P (Zybrestat, fosbretabulin, and its salt fosbretabulin disodium, 3P) is the prodrug of
CA-4 developed by OxiGene. Currently it is being evaluated in clinical trials as a treatment
for solid tumors.
In vivo
, it is dephosphorylated to its active metabolite CA-4. Several phase
II studies using CA-4P have finished or are ongoing for different type of cancers including
anaplastic thyroid cancer, non-small cell lung cancer, relapsed ovarian cancer,
etc.
(8).
Oxi4503 (4) is combretastatin A-1 diphosphate (CA-1P) targeting tumor vasculature. It is a
phosphorylated CA-4 analog developed by OxiGene for the treatment of solid tumors. A
phase I study is currently recruiting participants to determine the safety and maximum
tolerated dose of OXi4503 in patients with relapsed and refractory acute myelogenous
leukemia and myelodis-plastic syndrome.
AVE8062 (ombrabulin, 5) is another CA-4 analog which exerts its anticancer activity
through disrupting the blood vessel formation in tumors. Compared with CA-4, it has
improved water solubility and is orally available. AVE8062 has enhanced antitumor activity
and decreased toxicity in a murine Colon 26 carcinoma model. It is also effective against a
number of cancer cells that are resistant to taxanes (9). In a phase I study, the combination of
AVE8062 with docetaxel was well tolerated. A phase III study is currently ongoing for
advanced cancer treatment (10).
Phenstatin (6) is also a CA-4 analog with the double bond of CA-4 being replaced by a
carbonyl group. Phenstatin showed strong cytotoxicity and antitubulin activity similar to
CA-4, but it is more stable compare with CA-4 which is known to be unstable
In vivo
due to
the transformation from the active
cis-
configuration to the more stable but inactive
trans-
configuration (11). CC-5079 (7) belongs to 1,1-diarylethenes analogs of CA-4, which is
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called isocombretastatins A. CC-5079 is a dual inhibitor of tubulin polymerization and
phosphodiesterase-4 (PDE4) activity. It showed antiangiogenic and antitumor activities.
CC-5079 can arrest cell cycle in G2/M phase, increase phosphorylation of G2/M checkpoint
proteins, and induce apoptosis (12).
Podophyllotoxin (8), otherwise known as podofilox, is a non-alkaloid toxic lignan extracted
from the roots and rhizomes of
Podophyllum
species. In 1890, Kiirsten isolated crystalline
podophyllotoxin (13). Podophyllotoxin competitively inhibits the binding of colchicine. It
binds to tubulin more rapidly than does colchicine. The utilization of 8 as a lead in
anticancer drug design has resulted in useful cancer fighting drugs such asetoposide,
teniposide, and etoposide phosphate (13).
Steganacin (9), a new lignan lactone from the alcoholic extract of
Steganataenia araliacea
Hochest
, has significant anti-tumor activity
in vivo
against P388 leukemia in mice and
in
vitro
against cells derived from a human carcinoma of the nasopharynx (KB) (14). It was
found that 9 prevented the formation of the spindle that forms prior to the first cleavage.
This suggested that steganacin, like other spindle poisons such as colchicine and
podophyllotoxin, exerts its antimitotic activity through an effect on spindle microtubules
(14).
Nocodazole (10) is a natural product which has been shown to have antimitotic and
antitumor activity. The action of this agent is readily reversible and relatively rapid. Like 8
and 9, this agent exerts its effect in cells by interfering with the polymerization of
microtubules. However, the full therapeutic efficacy of this agent is limited owing to the
development of various side effects in patients, including bone marrow suppression,
neutropenia, leukopenia and anemia (15). This agent is now often used as a lead compound
to discover novel CBSIs or as a reference compound to study cell mitosis.
Curacin A (11), originally purified as a major lipid component from a strain of the cyano
bacterium
Lyngbya majuscula
isolated in Curaçao, is a potent inhibitor of cell growth and
mitosis. It binds rapidly and tightly at the colchicine site of tubulin. A recurring structural
theme in the colchicine binding site agents has been at least one and generally two aromatic
domains (16), while 11, as a potent colchicine binding site antimitotic agent, is a major
exception to this structural generalization in that it has no aromatic residue. Poor water-
solubility and lack of chemical stability prevent the clinical development of curacin A, but
synthetic analogs with improved bioavailability may provide new promises.
2-Methoxyestradiol (2-ME, 12) is an endogenous estrogen metabolite, formed by hepatic
cytochrome P450 2-hydroxylation of β-estradiol and 2-
O
-methylation
via
catechol
O-
methyltranseferase. This metabolite has attracted interest because of its potent inhibition of
tumor vasculature and tumor cell growth. Because solid tumor growth is dependent on
angiogenesis, the potent antiangiogenic activity and tubulin polymerization inhibition of 2-
ME
in vivo
are of potential therapeutic value and have warranted further investigation in
clinical trials. A recent clinical study indicated that the main adverse effects of 2-ME
included fatigue, nausea, diarrhea, neuropathy, edema, and dyspnea (17). Studies have
shown that 2-ME is metabolized by conjugation at positions 3 and 17 and oxidation at
position 17. The conjugated forms of 2-ME are inactive, and oxidation to 2-methoxyestrone
results in 10- to 100-fold loss in activity
in vitro
(18). In order to make metabolically stable
analogs with improved anti-tubulin properties, ENMD-1198 (13) was generated
via
chemical modification at 3 and 17 position. This agent also binds to the colchicine binding
site in tubulin, induces G2/M cell cycle arrest and apoptosis, and reduces hypoxia-inducible
factor (HIF)-1α levels. Studies also showed that ENMD-1198 was very potent at inhibiting
endothelial cell proliferation, motility, migration, and morphogenesis. In addition,
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ENMD-1198 induced a significant decrease in vascular endothelial growth factor receptor
(VEGFR)-2 protein expression in endothelial cells. Furthermore, ENMD-1198 is able to
disrupt vascular structures very quickly (19).
ABT-751 (E7010, 14) is an orally bioavailable tubulin-binding agent that is currently in a
phase II clinical trial for cancer treatment. It is a novel sulfonamide antimitotic that binds to
the colchicine site on β-tubulin that leads to a block in the cell cycle at the G2/M phase,
resulting in cellular apoptosis. ABT-751 was investigated in a recent phase I clinical trial to
assess its PK profile and safety (20). The maximum tolerated dose for the daily schedule
was 250 mg/day. Dose-limiting toxicities included abdominal pain, constipation, and
fatigue. ABT-751 was absorbed after oral administration with an overall mean Tmax of about
2 h. The PK properties of ABT-751 were dose-proportional and time independent. ABT-751
metabolism occurred primarily by glucuronidation and sulfation.
T138067 (15) was first reported by Shan
et al.
in 1999 as a novel antimitotic agent (21). This
compound has been shown to covalently bind to Cys239 on β-tubulin isoforms 1, 2, and 4
by way of a nucleophilic aromatic substitution reaction (21). The covalent modification of
β-tubulin prevents the polymerization of the α, β-tubulin dimers into microtubules. This
leads to cell cycle arrest at the G2/M phase followed by apoptosis (21). T138067 is effective
against a variety of tumors, including those that express the MDR phenotype (IC50=11–165
nM) (21). A phase II clinical trial showed that treatment with T138067 was tolerable with
moderate hematologic and gastrointestinal toxicity. Neurotoxicity, an expected side effect,
was minimal.
BNC-105P (16) was developed by Bionomics (Australia) as a low-molecular-weight VDA
for treatment of cancers. BNC-105P is a phosphorylated prodrug which rapidly transforms
to the active form BNC-105 by nonspecific endogenous phosphatases in plasma and on
endothelial cells (22). BNC-105 exhibits selectivity (81 fold) for growth factor activated
endothelial cells compared to quiescent human umbilical vein endothelial cells (HUVECs).
A phase I study has been completed and the drug was shown to be generally well tolerated.
A phase II study for BNC105P in combination with Everolimus for progressive metastatic
clear cell renal cell carcinoma is currently recruiting participants (23).
Indibulin (D-24851, ZIO-301, 17) is an orally active anti-mitotic drug that is effective
against various human tumor cell lines and xenografts, including taxane resistant tumors. In
preclinical studies indibulin lacks neurotoxicity which is largely associated with other
tubulin binding drugs. The antitumor activity against MDR cancers, the lack of
neurotoxicity, and the oral dosing make indibulin a promising candidate for further
development as an anticancer drug. Indibulin was reported not to overlap with the colchicine
site, and it was shown to partially compete for binding with “colchicine” site binders (40%
inhibition) (24).
In vivo
, oral application of indibulin showed a remarkable efficacy in the
Yoshida AH13 rat sarcoma model without systemic toxicity being observed. Indibulin not
only inhibits growth of tumor cell lines with different resistance phenotypes including
MDR1 and multi-drug resistance-associated protein (MRP), but also retains its antitumor
activity against cancer cell lines with resistance to cisplatin, the topoisomerase-I-inhibitor
SN-38, and the thymidylate synthase inhibitors 5-FU and raltitrexed. Although indibulin
also alters microtubule function, no neurotoxic effects on rats was seen at curative doses
compared to paclitaxel and vincristine treatment groups.
EPC2407 (Crolibulin, 18), MPI-0441138 (19), and MPC-6827 (Azixa, Verubulin,
20)—The 4-aryl-
4H
-chromenes, which were developed by EpiCept Corp. in California,
inhibit tubulin polymerization and induce apoptosis. Through structure-activity relationship
(SAR) studies of the 4-aryl-
4H
-chromenes, the anticancer drug candidate EPC2407 with
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potent vascular disrupting activity and
in vivo
efficacy has been identified (25) and is
currently in phase II clinical trial for the treatment of anaplastic thyroid cancer.
MPI-0441138 (19) is the lead compound for MPC-6827 (20) discovered by EpiCept and
identified as a highly active apoptosis inducer (EC50 for caspase activation of 2 nM) and as a
potent inhibitor of cell proliferation (GI50 of 2 nM) in T47D cells (26). This compound
inhibits tubulin polymerization and growth of Pgp overexpressing cells, and shows efficacy
in the MX-1 human breast and PC-3 prostate cancer mouse models (26). A recent phase I
study indicated that MPC-6827 was well tolerated at the recommended dose. The most
common adverse events were nausea, fatigue, flushing, and hyperglycemia (27). However,
recent news (http://www.biopharmcatalyst.com/2011/09/myrxhalts-phase-2b-azixa-pphm-
pipeline-update-scmp/) released in Sep. 2011 reported that Myrexis, which has exclusive
rights to MPC-6827 from EpiCept discontinued development of MPC-6827 due to
“disproportionate investment of time and resources relative to its likelihood of technical and
regulatory success.”
CYT997 (21) is originally discovered as a structurally novel, orally active microtubule
targeting agent. It is now in phase II clinical trials for the treatment of selected cancers.
CYT997 inhibits tubulin polymerization by binding at the colchicine binding site of tubulin.
CYT997 blocks the cell cycle at the G2/M phase, and western blot analysis indicates an
increase in phosphorylated Bcl-2, along with increased expression of cyclin B1 (28). This
compound also possesses favorable PK properties and is orally active in different tumor
models, including paclitaxel resistant cancer (28). CYT997 exhibits vascular disrupting
activity
in vitro
by effects on the permeability of human umbilical vein endothelial cell
monolayers, as well as
in vivo
on tumor blood flow (28).
MN-029 (denibulin, 22) is a novel benzoimidazole carbamate that reversibly inhibits
microtubule assembly, resulting in disruption of the cytoskeleton of tumor vascular
endothelial cells. MN-029 was found to demonstrate striking antivascular effects in tumors,
leading to the induction of necrosis and a consequential rapid loss of clonogenic neoplastic
cells. This VDA also was successfully incorporated into conventional cisplatin or radiation
therapy treatments (29). A recent phase I clinical study of MN-029 in patients with advanced
solid tumors showed that MN-029 was generally well tolerated and showed decrease in
tumor vascular parameters (30). The most common toxicities of MN-029 included nausea,
dose related vomiting, diarrhea, fatigue, headache, and anorexia. No significant
myelotoxicity, stomatitis or alopecia was observed in clinical (30).
CI-980 ((S)-(−)-NSC 613862, 23) is one of a novel class of 1, 2-dihydropyrido [
3, 4-b
]
pyrazines that inhibits tubulin polymerization presumably by interacting with the colchicine
binding site of tubulin. The (
R
)-(+)-isomer NSC 613863 showed potency in several
biological assays. However, the
S
-isomer is the more potent inhibitor on tubulin
polymerization and cell proliferation (31). CI-980 treated cells accumulate in the M-phase of
the cell cycle and subsequently die. In sensitive tumor models, the potency for this agent is
similar to that of vincristine, but the spectrum of antitumor activity is wider. CI-980 shows
activity against a variety of cancer cells
in vitro
, including leukemia, melanoma, sarcoma,
mammary adenocarcinoma, and colon adenocarcinomas. CI-980 is currently in a phase II
clinical trial (32). Neurotoxicity is the biggest problem for this agent. It can cause a
significant but reversible decline in recent memory functioning. So careful monitoring of
cognitive function in patients receiving this agent should be performed if dose or schedule
parameters are changed.
CP248 (24) and CP461 (25) are derivatives of Exisulind (Aptosyn, inhibitor of enzyme
cyclic guanosine monophosphate phosphodiesterase (cGMP-PDE)). Tubulin polymerization
is believed to be their target. Both CP248 and CP461 cause growth inhibition and apoptosis
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in several cancer cell lines. There are at least two modes of inhibiting tumor cells identified
for CP248. One is its inhibition of the cGMP-specific PDE2 and PDE5 and activate a
protein kinase G mediated signaling pathway that triggers apoptosis. The other is its ability
to bind to tubulin, inhibit its polymerization, and cause cells to be arrested in mitosis (33).
CP461 is a member of a class of novel proapoptotic drugs that inhibit cyclic GMP
phosphodiesterases specifically but not cyclooxygenase-1 or -2. It was in a phase I study for
the treatment of patients with advanced melanoma. CP-461 inhibits the growth of a broad
range of human tumor cell lines
in vitro
at micromolar concentrations. It selectively induces
apoptosis in cancer cells but not normal cells (34).
TN16 (26) is a tenuazonic acid derivative exhibiting anti-tumor effects
in vitro
and
in vivo
by inhibiting microtubule assembly and produces M phase arrest. TN16 has a structure
distinct from the representative microtubule inhibitor colchicine, and yet it inhibits
microtubule assembly, and prevents the stabilization of microtubules (35).
COMPUTER MODELING STUDIES OF CBSI
Computer-aided drug design methodologies have been increasingly applied to drug
development and have already provided some useful directions in the design and discovery
of anticancer drugs. Along with the increased publications of crystal structures recently, an
increasing number of molecular modeling studies on tubulin have been reported.
A study published in 2000 by Hamel
et al.
(36) is considered the first report of structure-
based approach for the colchicine binding site agents. The authors tried to identify two
potential colchicinoids binding sites on tubulin with the aids of biochemical and molecular
modeling techniques. The colchicine binding site was identified by Ravelli
et al.
in 2004 by
the determination of a 3.5 Å X-ray structure of α, β-tubulin complexed with N-deacetyl-N-
(2-mercaptoacetyl) colchicine (DAMA-colchicine) (3). Experimental data showed that
colchicine binds to β-tubulin at its interface with α-tubulin, resulting in inhibition of tubulin
polymerization. Colchicine and podophyllotoxin bind to β-tubulin at its interface with α-
tubulin with a similar orientation. An X-ray diffraction study demonstrated that the
trimethoxyphenyl (TMP) groups of both DAMA-colchicine and podophyllotoxin are located
in the β-tubulin structure in the vicinity of the amino acid residue Cysβ241 (note: In some
publications (36) this residue is numbered as Cysβ239). The width of the colchicine binding
site is approximately 4–5 Å, and the volume of this site is confined in β-tubulin by helix 7
(H7) containing Cysβ241, loop 7 (T7) and helix 8 (H8).
In recent years, several molecules structurally distinct from colchicine have been
crystallized in the colchicine binding site. These X-ray structures show a new and interesting
binding mode of tubulin in complex with CBSIs (Table I). ABT-751 (E7010, 14) interacts
with the colchicine binding site (37). The methoxy and pyridine groups of ABT-751
superimpose with the colchicine C and A rings, respectively; the sulfonamide bridge
overlaps with the B ring. ABT-751 interacts with β–strand S6
via
a hydrogen bond between
Tyrβ202 and the phenolic group. ABT-751 is more deeply buried than colchicine in β-
tubulin pocket and does not interact with the α-subunit. TN16 (26, Fig. 3) (37) competes
with colchicine for tubulin binding. It is even more deeply buried in the β monomer than
ABT-751. The tubulin residues involved in binding belong to β strands S4, S5, and S6 (van
der Waals contact with Thrβ239, Valβ238, Tyrβ202, Gluβ200, and Pheβ169). T138067 (15,
Fig. 3) is a bi-aryl molecule sharing with ABT-751 a sulfonamide linker. T138067-tubulin
complex showed a dual binding mode with a covalent component (37). T138067 occupies a
site largely overlapping with that of colchicine while another mode is covalently linked to
Cysβ241. In the latter case, only pentafluorophenyl ring A bound covalently to residue
Cysβ241 of β-tubulin. When T138067 is not covalently linked to the protein, it interacts
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primarily with strand S9 (Leuβ313– Argβ320) and loop T7 (Alaβ250–Pheβ244), that border
the colchicine binding site on the α-tubulin side. CI-980 (23, Fig. 3) and NSC-613863 are
enantiomers, which are also buried deeply into β-tubulin than colchicine (38). They show no
interactions with the α subunit. The molecular partially overlapped with the A ring of
colchicine. Gluβ200 has a hydrogen bonding interaction with the amino group of the
pyridine. The carbamate substituent is embedded in a hydrophobic pocket though van der
Waals contacting with residues of Thrβ239, Tyrβ202, Asnβ167, Glnβ136, and Ileβ4.
In order to rationalize their key common interactions at the colchicines binding site, many
pharmacophore models have been reported. Gussio
et al.
(39) employed docking studies and
molecular dynamics simulations to construct binding models for a set of structurally diverse
CBSIs, using the α, β-tubulin: DAMA-colchicine X-ray structure as the template.
Examination of the binding models of a set of structurally diverse CBSIs revealed common
pharmacophore groups for the CBSIs and extended the understanding of interactions at the
colchicine binding site. According to the report, the common pharmacophore of ligands of
the colchicine binding site contains the following seven pharmacophoric points (Fig. 4):
three hydrogen bond acceptors (A1, A2, and A3), one hydrogen bond donor (D1), two
hydrophobic centers (H1 and H2), and one planar group (R1). Hydrogen bond acceptor A1
is in contact with Valα179, A2 is in contact with Cysβ239, and A3 establishes one contact
mainly with Alaβ248, Aspβ249, and Leuβ250. Hydrogen bond donor D1 interacts with
Thrα177. Drug activity requires one hydrogen bond acceptor, two hydrophobic centers, and
a planar group.
The above docking results provide explanations for many known and new CBSIs on their
structure-binding interactions for the colchicine binding site. The fact that none of the
known structures of CBSIs contains all seven pharmacophore groups suggests that the
binding affinity of each chemotype can be improved by appropriate chemical modifications.
The binding models and pharmacophore may provide useful insights for rational structure-
based drug design.
The accuracy of computer models reported are growing, especially when relatively rapid
calculations such as molecular docking and pharmacophore queries are connected with more
advanced methodologies such as molecular dynamics. But there are still many challenges for
researchers in this research field. The fact that all the available crystal structures of tubulin
in complex with a CBSI have low resolution (3.5~4.0 Å) should not be overlooked (Table I).
This can explain the poor performance of the scoring system and provide justification for
why molecular dynamics is necessary to improve accuracy of model. Another point to keep
in mind is that the various isoforms of tubulin and their specific roles. The tubulin
superfamily includes α-, β-, γ-, δ-, ε-, ζ-, and η-tubulin (40). Different isotypes for both α
and β subunits are present in human cells. β-tubulin represents the main binding domain for
CBSI and includes at least eight isotypes that are expressed in different human tissue/organs.
The discovery of novel CBSIs that target specific isoforms selectively could have a
remarkable impact and molecular modeling could prove to be a very helpful tool in this
research area.
REPORTED CBSI IN PRECLINICAL STUDIES
Along with the rapid development of colchicine binding site inhibitors in the last decade,
especially with the elucidations of several tubulin colchicine pocket-ligand binding crystal
structures, more structures of CBSIs were reported and many of them showed excellent
potency and drug-like properties for their preclinical applications. We now summarize
several classes of CBSIs with diverse chemical structures.
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Combretastatin A-4 and its Analogs
Combretastatin A-4 was shown to exhibit potent antiangiogenic and antitumor activities.
However, poor solubility of the drug impinged its clinical development and required the
preparation of more soluble derivatives such as CA-4P phosphate sodium salt (3P, Fig. 3)
and the amino acid hydrochloride salt (5). In addition, the activity of CA-4 is hampered by a
short biological half-life (41,42) and isomerization of the active
cis
-olefinic conformation
into the corresponding inactive
trans-
analogs under the influence of heat, light, and protic
media (43,44). It could therefore be hypothesized that analogs that retain the potency and
efficacy of CA-4, but that have a different pharmacokinetic profile might be useful. To
overcome these problems, new analogs of CA-4 have been synthesized and developed in the
recent years.
The Ethenyl Bridge of the Stilbene Moiety (Fig. 5)—The olefinic bridge of CA-4
represents a weak point for metabolic stability. Sulfonate analogs of CA-4 have been
prepared by Gwaltney
et al.
(45) (
i.e.,
27). Compound 27 competitively binds with
colchicine and CA-4 for the colchicine binding site in tubulin and is potent inhibitor of
tubulin polymerization and cell proliferation. Importantly, this compound also inhibits the
proliferation of Pgp over-expressing cancer cells, which are resistant to many other
antitumor agents. Recently, Fortin
et al.
also reported that sulfonate and sulfonamide
moieties are bioisosteres of ethenyl bridge (PIB-SO: 28, PIB-SA: 29) (46,47). Quantitative
structure-activity relationships (QSAR) of 28 and 29 derivatives were established using
comparative molecular similarity indices and comparative molecular field analyses
(CoMSIA and CoMFA). Chick chorioallantoic membrane tumor assays show that active
PIB-SO and PIB-SA analogs efficiently block angiogenesis and tumor growth at similar
levels as CA-4 and exhibit low toxicity on the chick embryos. Interestingly, the SAR studies
of PIB-SO suggest that the phenylimidazolidin-2-one moiety was utilized to mimic the TMP
(A ring) moiety in CA-4, which is commonly found in the design of potent antimicrotubule
agents and described as a key structural element for the binding of antimitotics to the
colchicine binding site. Also, the other report from Simoni
et al.
(48) demonstrated that
benzo[
b
]thiophene (30, 31) or benzofuran (32) could replace TMP in CA-4 structure and
keep antiproliferative and inhibition of tubulin polymerization activity. Compounds 30 and
31 have a binding affinity to colchicine site five times stronger than CA-4.
A number of heterocyclic ring bridging CA-4 analogs have been prepared to restrict the
cis-
configuration and provide optimal conformational geometry for interaction with the
colchicine binding site. Introducing the heterocycles in place of the double bond can prevent
the isomerization of the double bond from
cis-
to
trans
-, and may improve the drug-like
properties. In 1998, Ohsumi
et al.
reported using five-membered heterocycle rings such as
pyrazole, tetrazole, and thiazole as
cis
-restricted bridges in combretastatin analogs which
showed potent antitubulin activity and cytotoxicity (49). Inspired by this strategy, more
CA-4 analogs were prepared and their activity was evaluated. Since 2000, imidazole (33-35)
(50), pyrazole (36) (50), 1,3-oxazole (37) (50), 2(
5H
)-furanone (38) (51), cyclopentenone
(39, 40) (52), oxazolone (41, 42) (53), 4-arylcoumarin (43) (54,55), furazan (44) (56),
triazoles (45) (57), 4,5-dihydroisoxazole (46) (58), 2,3-dihydrothiophene (47) (59),
azetidinone (48-52) (35,60), 2-aminothiazole (53-55) (61), and tetrazole (56-58) (62) have
been synthesized and appeared to elicit their tumor cytoxicity in a fashion similar to
combretastatin. Some compounds were found to be slightly more potent than combretastatin
itself (for example, 2-aminothiazole and tetrazole bridged compounds). Incorporation of an
N
-methyl group into the bridging imidazole ring (35) improved PK profiles (larger oral
AUC, longer half-life, and higher bioavailability). It is also the first CA-4 analog showing
potent antitumor activity
in vivo
orally (50). Another series of rigid analogs of CA-4, which
contain the 2-amino thiazole ring system in place of the ethylene bridge present in CA-4
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were reported recently (61). Compounds (53-55) with different A rings displayed
antiproliferative activity at picomolar concentrations against all tested cancer cell lines as
well as different drug resistant cell lines. Compound 53 was the most potent inhibitor of
tubulin polymerization and one of the most potent inhibitors of colchicine binding
(IC50=0.44 μM for assembly, 88% inhibition of the binding of [
3H
]-colchicine). Compound
54 induced apoptosis and this was partially dependent on caspase activation. In 2012, a new
series of tetrazole analogs with 3, 5-dihalophenyl rings (57, 58) (62) were reported and that
it appearts that a dihalogen substitution can consistently increase potency by up to 5-fold
when compared to the TMP ring compound 56 on HUVECs and a range of cancer cells.
Similar studies show that a halogen substituted phenyl A ring could replace TMP and gave a
new vision for further modifying CA-4 (3) aryl moiety (
i.e.
single halogen substituted
compounds 59 and 60 are more active on CA-4 resistant HT-29 cells at picomolar range
inhibition) (63). Tron
et al.
(64) synthesized rigid analogs of CA-4 (61-64) in two steps
exploiting a regioselective Suzuki coupling. Compound 63 displayed low nanomolar
cytotoxicity (IC50=9.4 nM) and proved to have no
cis-trans
isomerization and a slower
phase II glucuronidation compared to CA-4.
In contrast, some
cis
-restricted bridging CA-4 analogs were observed with reduced activity
(Fig. 6). Three-membered cyclopropyl ring (65) (65) and anti-epoxide (66) (66) retained a
certain antiproliferative activity. Substituted five membered rings such as 3-aminopyrazole
(67) (67), 1,2,3-triazole (68) (68), imidazol-2-one (69) (69) showed decreased activity. Six-
membered pyridine (70) (58), cyclohexenone (71) (70), 1,2,3,4-tetrahydro-2-
thioxopyrimidine (72) (71) were also used to replace the olefinic moiety. A 3,5-pyrazoline
(73) (72) analog was also prepared, but it showed reduced cytotoxicity.
Overall, it can be concluded that certain five-membered ring systems seem to be the best
option for chemical modifications of CA-4 analogs. It is difficult to say if the decrease in
activity, observed in some cases, is due to steric interactions or an incorrect orientation of
the two phenyl rings in the binding site. In some cases, the two phenyl rings should have 1,
2-substituents to maximize the potency; while 1, 3- relationships (73) give a strong
reduction in potency. Moreover, the presence of an aromatic character does not seem to be
necessary (27-29, 46-52 and 64). An interesting investigation to rigidify the
cis-
conformation of CA-4 is to synthesize a
para
-cyclophane derivative. Although the idea of a
macrocyclic analog of CA-4 is quite attractive, the resulting compounds (
e.g.
, 74, 75) did
not lead to biological activity on tubulin, precluding their anticancer applications (73).
Indole, Quinolone and Thiophene-Based CBSIs
Many natural products such as alkaloids contain indole group showed a variety of biological
activities. The indole ring is a structural component of a large amount of antimitotic
compounds. A series of microtubule inhibitors and anti-cancer drugs bearing indole nucleus
are reviewed with their enhanced cytotoxic activity.
Arylthioindole (ATI, 76-81, Fig. 7) analogs, which possess a 3-(3,4,5-trimethoxyphenyl)thio
moiety at the 2-position of the indole ring were effective tubulin assembly inhibitors (74,75).
Sulfur bridging linker ATI along with the corresponding methylene (77) and ketone (78)
compounds were potent tubulin assembly inhibitors reported by Silvestri
et al.
(76). Sulfur
derivatives were superior or equivalent to the ketones for growth inhibition of MCF-7 breast
cancer cells, while the methylene derivatives were substantially less effective. These
compounds inhibited the growth of MCF-7, HEK, M14, and U937 cells with IC50 values in
the 13–220 nM range. All three linked (S 76, CH2 77, CO 78) analogs bearing either 5-Br or
5-OMe and a 2-COOMe of the indole were found to be potent inhibitors in both tubulin
polymerization and MCF-7 cell growth assays, with potencies comparable to that of CA-4.
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Further investigation of new ATIs by replacing the 2-ester in 76 with 5-membered
heterocyclic rings (79-81) improved their biological profile. New ATI agents were active in
the Pgp-overexpressing and human transformed cell lines with improved solubility. They
triggered caspase-3 activation and induced p53-independent apoptosis, differently from the
classical apoptotic response induced by DNA damage that requires functional p53. The
sulfur bridging ATI 79 showed satisfactory metabolic stability and PK properties (77).
Molecular modeling simulation from the most active to the least active analogs in ATI series
confirmed the importance of TMP by its interaction with Cysβ241. The key role of H-bond
between the indole nitrogen atom and Thrα179 amide group was also confirmed by
molecular dynamics simulation (77).
2-Aroylindoles with 5-methoxy-
1H
-2-indolyl-phenylmethanone (D-64131, 82) were
discovered as tubulin inhibitors by high throughput screening from synthetic 2-aroylindole
derivatives (78). D-64131 arrests tumor cells in G2/M phase, interferes with the colchicine
binding site of tubulin, but does not affect β-tubulin GTPase activity. It is also cytotoxic
toward MDR/MRP resistant cell lines, depicts antiangiogenic activity and shows oral
bioavailability with marked
in vivo
antitumor activity in the human MEXF 989 melanoma
xenograft model.
BPR0L075 (83) is a 3-indolyl-phenylmethanone with antimitotic activity in human cancer
cells. It exerts potent antitumor and antimitotic activities through the inhibition of tubulin
polymerization by binding to tubulin at the colchicine binding site. BPR0L075 showed
in
vitro
anticancer activity against a variety of human tumor cell lines including glioblastoma,
breast, gastric, leukemia, liver, and colorectal cancer cells. Furthermore, phosphorylated
Bcl-2, perturbed mitochondrial membrane potential, and activation of the caspase-3 cascade
may be involved in BPR0L075-induced apoptosis. Notably, BPR0L075 can overcome
Pgp170/MDR and MRP mediated multidrug-resistant to vincristine, paclitaxel, and
colchicine. Moreover, BPR0L075 shows potent activity against the growth of xenograft
tumors at
i.v.
doses of 50 mg/kg in nude mice (79).
N
-Heterocyclic indolyl glyoxylamide BPR0C261 (84) is an analog of indibulin (17) and
possesses
in vitro/in vivo
anticancer activities (80,81). BPR0C261 destabilizes microtubules
and blocks cell cycle transition specifically at G2/M phase. Moreover, apoptosis induction in
the cancer cells is another underlying mechanism for the anticancer effects of BPR0C261.
Colchicine binding assay indicated BPR0C261 at both 5 and 20 μM competitively binds to
tubulin and strongly interferes with the colchicine binding to tubulin (80). In addition,
BPR0C261 concentration-dependently inhibited the proliferation and migration of HUVECs
with an IC50 value of 1.6 nM and disrupted the endothelial capillary-like 2D tube formations
of HUVEC. Given orally, BPR0C261 suppressed angiogenesis in a mouse model. It was
found orally absorbable in mice and showed a good oral bioavailability (
F
=43%) in dogs.
Moreover, the combination of BPR0C261 plus cisplatin synergistically prolonged the
lifespans of mice inoculated with murine leukemia cells.
1-Aroylindole, 1-aroylindoline, 1-aroyl-1,2,3,4-tetrahydroquinoline, and 2-, 3-, 4-, 5-, 6-, 7-,
8-aroylquinolines were synthesized and evaluated for anticancer activity as CA-4 derivatives
(82,83). Among these substituents, 1-aroylindoles with C4-amino (85) and C4-hydroxy (86)
substituents exhibited antitubulin activity superior or comparable to that of colchicine and
CA-4 with IC50 values of 0.9 and 0.6 μM, respectively. They also showed antiproliferative
activity with an IC50 range of 0.3–5.4 nM in a set of human cancer cell lines. In an
aroylquinoline series, only 2- and 6-aroylquinoline showed potency against five cancer cell
lines. 5-Amino-6-methoxy-2-aroylquinoline 87 has the most potent antiproliferative activity
(IC50=0.2 to 0.4 nM) against various human cancer cell lines including a MDR-resistant
cancer cell line KB-vin10. Compound 87 exhibited more potent inhibition of tubulin
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polymerization (IC50=1.6 μM) than CA-4 (IC50= 2.1 μM) and showed strong binding
property to the colchicine binding site on microtubules. A continued modification of the
linkers (-O-, -NH-, -S-, -SO2-, and direct bond) between TMP and 5-amino-6-
methoxyquinoline identified active oxygen and sulfur atom linked analogs (88-90) (84).
Although less active than parent compound 87, the most potent compounds, sulfide 89 and
sulfone 90, still showed low nanomolar inhibition against cancer growth and revealed that
the linkage has a capacity for various bridging groups. Moreover, Compound 87
demonstrated the ability to overcome the efflux protein (MDR/Pgp or MRP) mediated drug-
resistance in human cancer cell lines (KB-Vin 10, KB-S15, and KB-7D) with IC50 values
ranging from 2.4 to 2.8 nM.
Florent’s group explored the possibility of replacing the usual TMP ring present in a large
majority of CA-4 analogs with a trimethoxyindole ring (91) to obtain substituted 2-
aroylindoles through a palladium-catalyzed domino reaction (85). Compound 91 only
displayed fair activity (IC50= 4 μM). However, Liou
et al.
(86,87) successfully identified
potent 1-benzyl-4, 5, 6-trimethoxyindole (92 and 93, mean IC50=26 and 27 nM,
respectively), 1-indolylindole (94) and 1-quinolinylindole (95) as a new class of colchicine
binding site microtubule destabilizing agents. 4, 6- or 5, 6-Dimethoxyindole analogs showed
dramatically reduced bio-activity indicating that the trimethoxyindole moiety in the 1-
benzylindoles series is critical for activity. Compounds 94 and 95 exhibited anti-
proliferative activity with IC50 values ranging from 11 to 49 nM in a diverse set of human
cancer cell lines, including MDR1-expressing cervical carcinoma cell line KB-VIN10.
These two compounds also demonstrated potent tubulin polymerization inhibitory activity
with IC50 values of 1.7 and 2.7 μM, respectively. Molecular modeling and docking studies
showed A-ring of 94 and CA-4 are located in a similar area and have hydrophobic
interactions with Cys241, Leu248, Ala250 and Leu255. The Brings of both compounds also
occupied the same pocket and have hydrophobic interactions with Asn258, Met259, Lys352
and Val181.
Hu
et al.
(88) discovered 2-(2-amino-5-(1-ethyl-
1H
-indol-5-yl)pyrimidin-4-yl)phenol (97)
from 2-(2-amino-5-(4-(ethyl (methyl)amino)phenyl)pyrimidin-4-yl)phenol (96, IC50 range
from 90 to 550 nM) by forming a 5-indolyl substitution
via
cyclization of
N
-methyl to
phenyl ring. Compound 97 displays activity as an inhibitor of tubulin polymerization (IC50
=0.79 μM, 3.39 fold more active than colchicine IC50=2.68 μM), and it possesses the ability
to arrest cells at the G2/M phase of the cell cycle and antiproliferative activities against
several tumor cell lines with IC50 values ranging from 16 to 62 nM.
El-Nakkady
et al.
(89) reported introduction of hydrazides at the 3 position of 2-
phenylindole. The most potent compound 98 exhibited an IC50 value of 1.6 nM, being more
active than vincristine (IC50=2.0 nM). Structures of compound 98 docked in the colchicine
binding site of tubulin showed a hydrogen bond between the indole NH and Asnα101 in the
colchicine binding site of tubulin, suggesting that these phenolic indoles might act through
inhibition of tubulin.
T115 (99) is a recently reported N-substituted 1, 2, 4-triazole based colchicine binding site
tubulin inhibitor. It has potent and selective inhibitory effects against several cancer cell
lines and their corresponding drug resistance cells. T115 is devoid of the instability issue of
stilbene-like CA-4 analogs by introducing the triazole ring system as a bridge to retain the
cis
-configuration which is the biologically active form. Acute toxicity studies showed T115
was well-tolerated
in vivo
with a maximum tolerated dose of 400 mg/kg and showed no
cytotoxicity against normal fibroblasts cell lines. T115 significantly inhibited tumor growth
(
i.p.
) in mouse xenograft HT-29 and PC-3 models (90).
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Compound 100 was originally designed by Kelly’s group (91) for a nuclear hormone
receptor program but it exhibited potent inhibition of mitosis at the G2/M stage and proved
to be a colchicine binding site tubulin polymerization inhibitor. The 4-indolyl group at the 1-
position of phenyl was found to be critical for potency. Compounds with NH/C=O linkers or
without the linker at the 3-position all showed good activities against multiple cell lines (as
low as 0.01 μM). Replacement of the phenolic hydroxyl group in 100, which suffered from
rapid glucuronidation, with a sulfonylamide gave a potent compound LP-261 (101) with
significantly improved oral bioavailability in rat PK studies (
F
=24%
vs.
80%).
NSC 664171 (102) is a quinolinone derivative that has demonstrated strong cytotoxic effects
with GI50 values in the nanomolar or subnanomolar range in many different tumor cell lines
such as lung, ovary, prostate, breast cancers. It is also a potent inhibitor of tubulin
polymerization with activity comparable to those of the other well-known antimitotic natural
products such as colchicine, podophyllotoxin, and CA-4 (92).
CHM-1 (103) was discovered
via
SAR studies of a series of 2-phenyl-4-quinolones as a new
class of antimitotic antitumor agents. It showed potent cytotoxicity with an average log GI50
value of −6.47 (log of the concentration that reduced cell growth by 50%) in the National
Cancer Institute (NCI)’s 60 human tumor cell line (93). This compound was also a potent
inhibitor of tubulin polymerization with an IC50 value of 0.85 μM. Most importantly, it
demonstrated good
in vivo
activity against the OVCAR-3 ovarian cell line, prolonging the
life span of mice bearing the tumor by 130%.
S9 (104) (94), a hybrid molecule of α-methylene-γ-lactones and 2-phenyl indoles derived
from PI3K inhibitor wortmannin, is a multi-inhibitor simultaneously targeting both PI3K-
Akt-mTOR pathway and the microtubule cytoskeleton. S9 down-regulated phosphorylation
of Akt, mTOR, p70S6K and 4EBP1 through stimulation of EGF in Rh30 cells. It inhibited
the polymerization of tubulin by binding to the colchicine binding site and cause M phase
arrest. Dual mechanism of PI3k-Akt-mTOR signaling and tubulin inhibiting contributes to
cytotoxicity observed
in vitro
against a panel of tumor cells including MDR tumor cells and
in vivo
antitumor activity in human tumor xenografted mice models.
A series of thiophene derivatives (105, 106) have been synthesized and were found to be
potent inhibitors of tubulin polymerization (61,95). Compounds 105 and 106 inhibit cancer
cell growth at subnanomolar concentrations and interact strongly with tubulin by binding to
the colchicine site. Flow cytometry of these compounds had cellular effects typical of agents
that bind to tubulin, causing accumulation of cells in the G2/M phase of the cell cycle and a
substantial increase in the number of apoptotic cells.
Dodd’s group reported a series of fused indole ring compounds (107-109) (96,97), which
contain a seven-member amide ring. It showed similar effect with colchicine in potency and
inhibiting tubulin polymerization, by the interruption of cancer cell growth at the G2/M
stage. The ability of these compounds to promote apoptosis in the cancer cells studied was
also clearly demonstrated. Compound 108 was more effective than colchicine in causing
tumor volume regression after 7 days of treatment in a chick chorioallantoic membrane
model of transplanted human glioma (U87) tumor growth. The research group also
synthesized rigid analogs of these compounds by fusing another five-member ring between
the phenyl and seven member ring, but this led to a loss of activity (97).
Chalcone Compounds
Antitubulin activity was found in chalcone compounds which bears an aromatic ketone and
an enone as the central core. Chalcones are precursors of flavonoids and scaffolds for a
variety of important biological compounds. They are abundant in edible plants and display
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biological activities, including anti-cancer, anti-inflammatory, anti-tubercular, and anti-
fungal,
etc.
Their biological properties are largely due to the α, β-unsaturated ketone moiety.
Modifications on the two aromatic rings remains an area of pharmacological interest in the
screening of active chalcones, such as MDL-27048 (110, Fig. 8) and JAI-51 (111) (98). The
effects of MDL-27048 on microtubules are similar to those of colchicine or combretastatin
analogs. It represents a new type of antitubulin agent, which could prove to be valuable as
an experimental inhibitor in the study of microtubules and microtubule-mediated functions.
A series of aryl- and aroyl-substituted chalcone analogs of the tubulin binding agent CA-4
(3) were prepared by Flynn’s group (99). All compounds were assessed as inhibitors of
tubulin polymerization, but only E-31 (112) had activity similar to that of CA-4 (2.5
vs.
2.0
μM). However, compound E-31 did not exhibit antiproliferative activity against the MCF-7
cell line.
Zhu’s group recently reported a series of novel antitubulin polymerization inhibitors
containing the chalcone skeleton and a sulfonamide moiety (113) (100) or containing the
resveratrol skeleton and chalcone moiety (114) (101). Compound 113 showed the most
potent inhibitory activity with an IC50 value of 0.8 μg/mL and antitubulin polymerization
activity with an IC50 of 2.4 μg/mL in the cinnamic acyl sulfonamide derivatives. Among the
resveratrol derivatives, compound 114 showed the most potent inhibitory activity. It
inhibited the growth of a number of cancer cell lines with IC50 values ranging from 0.1 to
1.4 μg/mL. It also inhibited the polymerization of tubulin with an IC50 of 2.6 μg/mL.
Computational docking analysis of the binding conformation of compound 113 and 114 in
the colchicine binding site demonstrated that interactions with the protein residues in tubulin
led to the antiproliferative activity.
A series of dihalogenated chalcones and structurally related dienones have been synthesized
and showed fair cytotoxic activities (IC50 in low micromolar range) toward individual
cancer cell lines. Most of this series of compounds are tubulin polymerization inhibitors.
However, one dienone derivative (115) was found unexpectedly to stabilize tubulin similar
to docetaxel. This is the first reported chalcone derivative with microtubule-stabilizing
activity.
Sulfonanilides Compounds
The sulfonamides have been in clinical use for yearsdue to their biological activities such as
antibacterial, antidiabetic, antithyroid, antihypertensive, or antiviral activities. Recently,
many structurally novel sulfonamide derivatives have shown substantial antitumor activity.
Several CBSIs containing the sulfonamide group were used in clinical studies such as
ABT-751 (14, Fig. 3) and T138067 (15). Sulfonamide analog ELR510444 (116, Fig. 9)
(102) has potent microtubule disrupting activity
via
direct interaction with tubulin at the
colchicine binding site. ELR510444 potently inhibited cell proliferation with an IC50 value
of 9.0 nM in MDA-MB-435 cells and did not serve as a substrate for the Pgp drug
transporter and it retains activity in class III βtubulin overexpressing cell lines, suggesting
that it circumvents both clinically relevant mechanisms of drug resistance to this class of
agents. ELR510444 also shows efficacy in the MDA-MB-231 xenograft model with at least
a 2-fold therapeutic window. Studies in tumor endothelial cells show that ELR510444 has
potential antivascular effects. ELR510444 also leads to caspase-3/7 activation and
subsequent apoptosis with cellular EC50 values of 50–100 nM. The compound induces an
initial cellular arrest in G2/M and a significant tubulin depolymerizing effect, followed by
an increase in a sub-G1 (apoptotic) population after 24 h.
J30 (117) is an orally active sulfonamide CBSI discovered by Liou’s group (103,104) and
shows strong antiproliferative activity with IC50 values ranging from 8.6 to 11.1 nM against
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human tumor cell lines, as well as the ability to overcome drug resistance. J30
depolymerizes microtubules in the KB cell line, resulting in an accumulation of G2/M phase
cells. Further studies indicate that J30 causes cell cycle arrest, as assessed by flow analyses
and the appearance of MPM-2 (a specific mitotic marker), and is associated with up-
regulation of cyclin B1, phosphorylation of Cdc25C, and dephosphorylation of Cdc2. J30
also causes Bcl-2 phosphorylation, cytochrome C translocation, and activation of the
caspase-9 and caspase-3 cascades, suggesting it mediated apoptotic signaling pathway that
depends on caspases and mitochondria. J30 given orally inhibits tumor growth in NOD/scid
mice bearing human oral, gastric, and drug-resistant tumor xenografts.
2-Methoxyestradiol and its Derivatives
2-Methoxyestradiol (2-ME, 12, Fig. 3) is the main metabolite of the hormone β–estradiol
and is a weak competitive inhibitor of colchicine binding to tubulin. 2-ME and its analog
ENMD-1198 (13, Fig. 3) are under clinical studies as promising anticancer agents with dual
activity against cancer cell proliferation and angiogenesis (17,19). Cushman and colleagues
have focused on changing substituents at the 2- and 6-positions (105) of 2-methoxyestradiol
to increase cytotoxicity and tubulin polymerization inhibition, and their efforts have
produced 2-ethoxyestradiol (118, Fig. 10), which was 17-fold more cytotoxic than 2-ME in
the whole panel of tumor cell lines in MGM value (Mean graph midpoint for growth
inhibition of all human cancer cell lines tested, 76 nM
vs.
1.3 μM). Furthermore, molecules
carrying an NOH moiety in the 6-position with either CH3CH2O or CF3CH2O in the 2-
position (119) were also superior to 2-ME in all three parameters (MGM=79 and 66 nM). A
2-ethoxyestradiol derivative with an additional ketone moiety in position 6 (120) was still
highly potent (MGM=130 nM). Later modifications at the 2-position by the same research
group resulted in some other potent compounds, although not superior to 2-ME, such as 2-
(
E
-3′-hydroxy-1′-propenyl) estradiol (121, MGM=1.1 μM) and 2-(1′-propynyl) estradiol
(122, MGM=1.7 μM) (106). Additionally, the 17-position contributes significantly to the
anticancer activity and pharmacokinetic profile of 2-ME. Oxidation of the 17-OH of
estradiol to estrone is one of the main routes for metabolic deactivation and steroid
clearance. 2-Ethoxy-17-methylene analog of 2-ME (123, MGM=0.79 μM) showed greater
tubulin polymerization inhibition and cytotoxicity than 2-ME and contained moieties that
are expected to inhibit deactivating metabolic processes (107). Agoston and colleagues
investigated whether substituents at the neighboring 16-position may inhibit this process
(108). Larger substituents tended to result in lower cytotoxicity, but a few 16-position
modified compounds including 124 were comparable to 2-ME in assays with HUVEC and
MDA-MD-231 cells. However, all these agents did not address the major problem of low
oral bioavailability. If sulfamate is introduced on the hydroxyl groups of the 3 and 17
positions (125 and 126), (109,110) the sulfamoylated compounds retained the ability to bind
to the colchicine site of tubulin, conferred superior biological activity and demonstrated
resistance to metabolism with a high bioavailability (85%). Most importantly, superior
biological activity
in vivo
was found after oral application of bis-sulfamate (125) compared
to 2-ME at a daily dose of 20 mg/kg in mouse xenograft models of MDA-MB-435 and
MCF-7 cancer cells. Similar results were obtained in the MCF-7 model for a 17-
cyanomethyl analog (126). Given orally, 125 and 126 were also superior to 2-ME in an
assay testing the inhibition of blood vessel growth, and demonstrated a superior ability to
inhibit neovascularization. These agents work as inhibitors of tubulin polymerization and
inhibit the binding of radiolabeled colchicine to tubulin. The potential hydrogen bonding of
the terminal NH2 group of the sulfamate is not essential based on SAR studies of this series
of compounds. As illustrated by compounds 127 and 128 (111), when the NH2 is replaced
by a CH3 along with a 2-ethyl substituents, A 4–11 fold enhancement of anti-proliferative
activity was observed.
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Analogs of Natural Products
Natural products are excellent sources for drug discovery and development. A large amount
of anticancer drugs are either natural products or derivatives from natural sources including
plants, animals and microorganisms. Colchicinoids and combretastatins are isolated from
plants and are well-studied tubulin binding agents. Structure modification of natural
products which bind to the colchicine site are continually being performed and have
successfully generated plenty of synthesized derivatives.
Desmosdumotins and Analogs—Desmosdumotins AC (129-131, Fig. 11) are isolated
from the root bark of
Desmos dumosus
. These natural flavonoid and chalcone scaffolds
represent promising new lead structures for further new analog development as potential
antitumor agents. Lee’s group synthesized derivatives of desmosdumotin B and C with
improved anticancer activity and selectivity against Pgp overexpressing multidrug resistant
cell lines (112). Triethyl analogs with 4′-alkyl desmosdumotin B (130) derivatives 132 and
133 showed significant selectivity with ED50 values of 0.03 and 0.025 μg/mL, respectively,
against KB-VIN cells (MDR) with ratios of >460- and 320-fold compared with that of KB
(non-MDR) cells (113). Further modifications on B-ring systems with bicyclic or tricyclic
aromatic rings identified derivative 134 with a benzo[
b
]thiophenyl B-ring, which was highly
active with GI50 values of 0.06–0.16 μM over a panel of cancer cell lines including Pgp
expressed MDR cells. Furthermore, 134 inhibited tubulin assembly
in vitro
with an IC50
value of 2.0 μM and colchicine binding by 78% as well as cellular microtubule
polymerization and spindle formation, which confirmed they are a new class of CBSIs
(114). Modifications of desmosdumotin C (131) led to 3,5,5-tripropyl-4′-bromo analog 135,
which possessed the most potent activity against A549, HCT-8, 1A9, PC-3, KB and KB-
VIN cells with ED50 values of 0.87–2.25 μg/mL (1.8–2.6 μM). A MeO- or PrO- group at
C-4 was generally preferred over other alkyl ether groups. Oligonucleotide microarray
studies showed that 135 may modulate spindle assembly checkpoint and chromosome
separation and arrest cells mainly in the G2/M phase (112,115).
Centaureidin (136, Fig. 11) is an
O-
methylated flavonol isolated from plants like
tanacetum
microphyllum, brickellia veronicaefolia, bidens pilosa
and
polymnia fruticosa
. Centaureidin
inhibits tubulin polymerization and competes with the binding of [
3H
]-colchicine to tubulin.
It can induce mitotic figure formation in whole cells at cytotoxic concentrations.
Centaureidin is the first known example of a flavone with anti-mitotic activity (116).
Noscapine (137, Fig. 11), an anti-cough opium alkaloid, binds to tubulin, alters its
conformation, affects microtubule assembly, and causes apoptosis in many cell types. Oral
administration of noscapine has potent antitumor activity against solid murine lymphoid
tumors and human breast and bladder tumors implanted in nude mice. Amino-noscapine was
designed and chemically synthesized following the guidance of a linear interaction energy
(LIE) method with a surface generalized Born (SGB) continuum solvation model. The
amino noscapine derivative 138 has higher tubulin binding activity with the binding pocket
of tubulin involved in three hydrogen bonds and they are distinct compared to noscapine
which involved only one hydrogen bond. The LIE–SGB model constitutes the first evidence
that this class of compounds binds to the colchicine binding site. Amino noscapine has
overall much stronger anti-tumor activity than noscapine against the NCI-60 cancer cells
panel (117).
Polygamain (139, Fig. 11): Lignan polygamain was recently isolated as the microtubule-
active constituent from the crude extract of the Mountain torchwood,
Amyris madrensis
(118). Polygamain has structural similarities to podophyllotoxin (8, Fig. 3) and has potent
inhibition against a wide panel of cancer cell lines (average IC50 is 52.7 nM). It inhibits the
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tubulin assembly and interacts within the colchicine binding site. Molecular modeling
suggests that the benzodioxole group of polygamain occupies the same pocket as the TMP
group of podophyllotoxin, but has distinct interactions within the hydrophobic pocket.
Polygamain circumvents two common mechanisms of drug resistance for microtubule
targeting agents, the expression of Pgp pump and the class βIII isotype of tubulin.
Diketopiperazine—Cyclopeptides are known to exhibit biological activities ranging from
cell-cycle inhibition to specific enzyme-activity modulation. The smallest cyclopeptides
studied for their potential therapeutic effects are diketopiperazines. Tryprostatin A and B
(140 and 141, Fig. 11), diketopiperazines isolated from the fermentation broth of
Aspergillus
fumigatus
by Osada and co-workers in 1995, hold great anti-cancer promise (119).
Tryprostatin A reverses resistance against mitoxantrone in various breast cancer resistance
protein-expressing human cancer cells accompanied by a selective inhibition of the ATP-
dependent drug transport activity of breast cancer resistance protein (120). Phenylahistin
(NPI-2350, 142), a metabolite isolated from
Aspergillus ustus
, is another fungal
isoprenylated diketopiperazine, composed of the phenylalanine and dehydrohistidine. The
fungus produces phenylahistin as a racemic mixture, but only the (-)-enantiomer proved to
be cytotoxic (121). Interestingly, (Z)-dehydrophenylahistin (143), in which chirality is lost
by dehydrogenation of the phenylalanine moiety, has also been reported as an antimitotic
agent being 1,000 times more active than (-)-phenylahistin toward the first cleavage of sea
urchin embryo (122). 143 also showed significant activity against human cancer cells lines,
with IC50 values in the nanomolar range. The isoprenyl group attached to the imidazole ring
was also indicated to be important for activity. A series of analogs of dehydrophenylahistin
was synthesized, resulting in the identification of plinabulin (NPI-2358, 144). Plinabulin is
a
tert
-butyl analog of phenylahistin with a colchicine-like tubulin depolymerization activity
and a potent microtubule-targeting diketopiperazine derivative with IC50 values in the low
nanomolar range. Compound 144 is equally active against MDR tumor cell lines and is now
under phase II clinical trials as a vascular disrupting anti-cancer drug. To develop more
potent anti-microtubule and cytotoxic derivatives based on the dehydrophenylahistin
skeleton, Hayashi’s group performed further SAR study on the
tert
-butyl and the phenyl
groups of 144, and evaluated their cytotoxic and tubulin-binding activities (123).
Compounds 145 with a 2, 5-difluorophenyl and 146 with a benzophenone in place of the
phenyl group had both vascular disrupting and cytotoxic activities (5- and 10-times more
potent than that of CA-4, respectively). Compounds 145 and 146 exhibited a lowest
effective concentration of 2 nM and 1 nM for microtubule depolymerization, respectively.
Curacin A—The marine natural product curacin A (11, Fig. 3) is a potent inhibitor of
cellmitosis, binding quickly at the colchicine binding site of tubulin. The lipid structure of
curacin A differs greatly from that of colchicine and other CBSIs. Analog studies of curacin
A were made to simplify the structure and increase water solubility and chemical stability.
However, minor changes in the lipid chain or fragments of curacin A can lead to inactive
derivatives: opening of the thiazoline ring and longer chains or acyl groups as oxygen
substituents on C13 are not tolerated. The C7-C10 diene segment of curacin A is sensitive to
modification. Replacement of cyclopropyl ring with a
tert-
butyl group (147-150, Fig. 11)
leads to a>2-fold decrease in activity. The oxazoline and oxazole analogs 147 and 148 lack
any appreciable biological efficacy (124). Compound 151, with an ethylenedioxy bridge at
C13 causing loss of chirality, is equivalent to curacin A in potency. Classic TMP group
introductions provide effective replacements for the labile cyclopropyl thiazoline moiety in
analog 152. Further introducing the oxime ether linker to replace the
(Z
)-C3-C4 alkene
moiety lead to discovery of analog 153, which is found to be more potent than curacin A in
inhibiting the assembly of purified tubulin and shows more chemical stability in the
presence of plasma (125).
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Agents Covalently Binding to the Tubulin Colchicine Binding Site (Fig. 12)
While most of CBSIs reversibly bind to the tubulin pocket, design and synthesis of
irreversible CBSIs has aroused interest since covalent bond formation could circumvent the
resistance caused by the mutations of tubulin residues and trigger microtubule disruption. A
subset of irreversible CBSIs was reported, which formed covalent bonds with tubulin amino
acid residues, generally cysteines. Alkylation of Cysβ239 caused loss of tubulin’s ability to
polymerize. As previously mentioned aryl-pentafluorosulfonamaide T138067 (15, Fig. 3), as
well as its analog T113242 (154, Fig. 12) (126), covalently alkylate tubulin Cysβ239 and
induce microtubule depolymerization. Colchicinoids 2-chloroacetyl-2-
demethylthiocolchicine (2CTC, 155) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC,
156) resemble colchicine in binding to tubulin and react covalently with β–tubulin, forming
adducts with Cysβ239 and Cysβ354 (127).
A screening program aimed at the discovery of new antimicrotubule agents yielded
RPR112378 (ottelione A, 157), a natural inhibitor of tubulin polymerization, first isolated
from the fresh water plant
Ottelia alismoides
. RPR112378 is an efficient inhibitor of tubulin
polymerization (IC50 =1.2 μM) and a highly cytotoxic compound with an IC50 of 0.02 nM
against KB cell growth. The cytotoxicity of RPR112378 is probably caused by an addition
reaction of ethylenic bond with sulfhydryl of cysteine residues (128).
t
BCEU (158), a derivative of N-aryl-N′-2-chloroethylurea (CEU), was reported to react
exclusively with Cysβ239. Further modification of CEU by introducing a branched
R
-alkyl
chain (159, 160) led to enhanced cytotoxicity (including cell line with mutations in tubulin)
and rapid alkylation of β-tubulin in cells. It is speculated that the active site of the β-tubulin
is stereo selective and the
R
-isomer of the branched chloroethyl group allowed a sterically
favored orientation of the alkylating moiety, promoting the approach of the chlorine atom
toward the sulfhydryl of Cysβ239 residue. However, subsequent work using mass
spectrometry has identified that the Gluβ198, which is adjacent to the colchicine binding site
behind the two potent nucleophilic residues, Cysβ239 and Cysβ354, has been shown to
covalently react with CEU (129,130). None of the cysteine residues of β-tubulin was linked
to the alkylating agent. This result is contrary to the previous hypothesis that the reacting
amino acids in tubulins would be mainly the cysteine residues.
Other CBSI: Screened and Synthesized
Anthracenone—Antimitotic anthracenone-based molecules (161-168, Fig. 13) that inhibit
tubulin polymerization have been reported (131). The majority of these compounds possess
an unsaturated bond between the anthracenone and the terminal aromatic ring. The
modifications of the linker between the anthracenone and the terminal phenyl ring were
performed with the benzylidene C=C double bond (161, 162), chalcone (165), oxime (166)
and a C=N bond (167, 168). The active anthracenone analogs inhibit the growth of various
tumor cell lines in the G2/M phase with a cell cycle dependent manner by interacting with
tubulin at the colchicine binding site.
Chromene—4-Aryl-4
H
-chromenes (169, Fig. 13) are identified as apoptosis-inducing
agents, possessing vascular disrupting activity through a cell-based apoptosis screening
assay (25,132). One chromene lead compound EPC2407 (crolibulin, 18, Fig. 3) has been
used in clinical trials. The 4-aryl-4
H
-chromenes inhibit tubulin polymerization and bind at
or close to the binding site of colchicine. The cells treated with these agents undergo a G2/M
arrest prior to caspase activation. They are also active in the multidrug resistant MES-SA/
DX5 tumor cells and are highly active as single agents (subnanomolar range potency) and in
combination with other anticancer agents such as cisplatin in several tumor xenograft
models. SAR studies of the 4-aryl group with 5-, 6-, 7-, 8-substituents and fused 7,8-
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chromene rings were carried out and led to the discovery of a 7-methyl-4H-pyrrolo[
2,3-
H
]chromene analog with low nanomolar potency (GI50=0.3 nM against T47D colon cancer
cells) (133).
The small-molecule fluorenone UA62784 (170, Fig. 13) was first reported by Henderson
et
al.
in 2009 (134) from a high throughput cytotoxicity screening that selectively targeted
DPC4-deleted pancreatic cancer cells. UA62784 shows cytotoxic in the nanomolar range,
causes G2/M arrest, induces apoptosis, and prevents the formation of a functional bipolar
spindle during mitosis by inhibiting the microtubule-associated ATPase activity of the
CENP-E kinesin-like protein, but it shows no effect on tubulin polymerization. Continued
research by Abrieu
et al.
(135) on this molecule revealed an alternative mechanism of
antitumor action that showed UA62784 interacts with tubulin at or near the colchicine
binding site, not
via
inhibiting ATPase activity of kinesin CENP-E. Immunofluorescence
and live cell imaging indicate that UA62784 perturbs the mitotic spindles. It also shows
additive effects with some known microtubule-depolymerizing drugs including vinca
alkaloids, colchicine, or nocodazole, but not paclitaxel.
JG-03-14 (171, Fig. 13) (136,137), a
tetra
-substituted brominated pyrrole, has been shown to
have broad cytotoxic and antiproliferative effects against cancer cells
in vitro
(IC50=36–80
nM, including drug-resistant cell line) and
in vivo
activity due to its ability to potently bind
at or near the colchicine binding site on tubulin. JG-03-14 can cause dose-dependent loss of
cellular microtubules and it can phosphorylate Bcl-2, arrest cells in the G2/M phase and it is
a poor substrate of Pgp (137). JG-03-14 has direct effects on endothelial cells that could be
indicative of therapeutically useful anti-vascular actions. Molecular modeling studies have
indicated that while the dimethoxyphenyl group of JG-03-14 occupies a space similar to that
of the TMP group of colchicine and interacts with Cysβ241, the
tetra-
substituted pyrrole
group interacted with both α- and β-tubulin in space not shared with colchicine. The side
chain ethoxy oxygen forms a bifurcated H-bond interaction with the NH2 of Asnα101. This
may suggest significant differences as compared to colchicine in the mechanism of binding
to tubulin.
MT119 (172, Fig. 13) is a planar-structured compound, which was optimized as a new CBSI
from a combinatorial library (138). MT119 inhibit tubulin polymerization significantly both
in tumor cells and in cell-free systems, which is followed by the disruption of mitotic
spindle assembly. It arrests different tumor cells at the G2/M phase, and inhibits the
proliferation of ten tested tumor cells with IC50s ranging from 0.06 μM to 0.53 μM. MT119
is also cytotoxic to cancer cells resistant to vincristine, adriamycin or mitoxantrone.
NSC 676693 (173, Fig. 13) is a novel antimitotic compound based on the
arylthienopyrrolizinone molecular skeleton. It has strong anticancer activity in human cancer
cells with IC50 in the nanomolar range and it interacts with tubulin in the micromolar range.
Based on the structure of NSC 676693, a series of it analogs have been synthesized and the
best compound shows 10-fold improvement of potency compared with NSC 676693 (139).
Indanocine (174, Fig. 13) is a synthetic indanone that was first identified by the NCI’s
Developmental Therapeutics Program as an antiproliferative agent. It is a tubulin inhibitor as
well as an apoptosis inducer in certain cancers. The activity of indanocine on multidrug-
resistant cancer cells indicates that indanocine could be a potential lead compound for the
development of chemotherapeutic strategies for drug-resistant cancers (140).
IRC-083927 (175, Fig. 13) is an orally available synthetic imidazole derivative, which was
developed by ISPEN, and inhibits tubulin polymerization by binding to the colchicine site. It
shows highly potent antiproliferative activity on human tumor cell lines including taxane,
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vinca alkaloid, or epothilone resistant cells due to the presence of efflux pumps (Pgp, MRP)
and/or mutated tubulin (141). IRC-083927 displayed cell cycle arrest in G2/M phase in
tumor cells and inhibited endothelial cell proliferation
in vitro
and vessel formation in the
low nanomolar range supporting an antiangiogenic action. Furthermore, the oral
administration of IRC-083927 in athymic mouse models showed a significant
in vivo
antitumor activity without apparent toxicity (141). The antitumor effect induced by
IRC-083927 supports its potential for the treatment of advanced cancers, particularly those
resistant to current clinically available drugs.
XRP44X (176, Fig. 13) is a pyrazole derivative reported by Wasylyk
et al
. Itintergrates two
anticancer mechanisms: antimitotic and inhibition of MAPK/ERK signaling pathway. It
binds to the colchicine binding site of tubulinand depolymerizes the microtubules, thus
affects the morphology of the actin cytoskeleton. It also inhibits fibroblast growth factor 2
(FGF-2)–and phosphorylation by the Ras-Erk signaling upstream from Ras (142). XRP44X
also inhibits the growth of transformed cells in culture and angiogenesis in an
ex vivo
assay
of endothelial cell sprouting (142).
KRIBB3 (177, Fig. 13) is a novel microtubule inhibitor with an isoxazole moiety in the
structure. It can induce mitotic arrest and apoptosis in human cancer cells. It was first
developed by Korea Research Institute of Bioscience and Biotechnology. KRIBB3 exerts
significant antitumor activity against a variety of cancer cells such as colon, prostate, breast,
and lung by inhibiting tubulin polymerization. KRIBB3 selectively arrests cell cycle at the
mitotic phase.
In vivo
, KRIBB3 decreased tumor volume by 49.5% (50 mg/kg) and 70.3%
(100 mg/kg) compared to control mice (143).
A-105972 (178) and A-204197 (179, Fig. 13) are both oxadiazoline analogs identified by
Abbott Laboratories from a high throughput screening of 60,000 compounds. The two drugs
have the exact same scaffold and the only difference is a functional group in the aromatic
ring. They are potential anticancer agents and were undergoing preclinical development by
Abbott Laboratories (144). A-105972 inhibited the growth of several tumor cell lines,
including breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, as
well as multidrug-resistant cells with IC50 in between 20 and 200 nM depending on specific
cell types, but its utility
in vivo
was limited by a short half-life. A-105972 and A-204197
interact with tubulin and induce apoptosis and Bcl-2 phosphorylation. A-204197 has shown
high potency
in vitro
(IC50 36–48 nM) and is especially effective against MDR cancer cell
lines. These results indicate A-204197 is a promising antimitotic agent that has potential for
treating neoplastic diseases with great efficacy (145).
A-289099 (180, Fig. 13) is an indolyloxazoline derivative with antimitotic activity
developed also by Abbott Laboratories. It was discovered as a result of structural
optimizations of the lead compound A-105972 (146). A-289099 exerts its anticancer activity
by inhibition of tubulin polymerization and by binding at the colchicine binding site.
A-289099 has high anti-proliferative activity in a number of cancer cells with EC50 values
ranging from 5.1 to 12.8 nM. A-289099 is orally active in a syngeneic M5076 murine
reticulum sarcoma flank tumor model. Pharmacokinetic study showed that the
bioavailability of A-289099 in monkey is 18.6%. The half-life ranges from 1.1 h to 3.3 h
depending on species (146,147).
2-Indolinones (181-183, Fig. 13), a series of methylene-bridged heterocycles discovered by
Andreani
et al.
(148,149) have similar inhibitory effects on colchicine binding site and
tubulin polymerization. These compounds arrest the cells in the G2/M phase of the cell
cycle, and are associated with activation of apoptosis, disrupt the mitochondrial membrane
potential, and increase ROS production and Bax translocation into mitochondria. The
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apoptosis in the HT-29 cells is accompanied by caspase activation and phosphatidylserine
externalization. Interestingly, the most potent compounds 181 and 183, which show more
potent mean GI50 (40–70 nM in NCI screening) than vincristine (GI50=200 nM), strongly
inhibit the activation of the kinase Akt associated with cell survival and proliferation.
CBSI with Improved Solubility and PK Profiles
A major problem with CBSIs is their limited aqueous solubility that substantially reduces
absorption of a drug. Problems with the delivery of drugs to the tumor occur also when the
active agent has a high molecular weight which limits tissue penetration. Various strategies
and structural modifying approaches have been investigated to solve this problem.
Prodrugs have been successfully used to increase aqueous solubility. For example,
phosphate prodrugs are typically converted to the parent molecule
via
non-specific alkaline
or acidic phosphatases. This approach has allowed intravenous administration of phosphate
prodrugs to overcome otherwise bleak pharmacokinetic profiles. In particular, CA-4 has
recently benefited from a phosphate prodrug administration (3P). Similarly, Cushman’s
group also addressed the issue of low water solubility by producing water soluble 2-ME
derivatives by coupling phosphate to the hydroxyl groups in positions 3 and 17 (150). The 3-
phosphate (184, Fig. 14) exhibited similar properties as compared with the parental drug 2-
ME. As expected, in rats the 3- phosphate was metabolized rapidly (within one hour) to the
active drug 2-ME. However, the oral administration of the 3-phosphate did not enhance 2-
ME plasma levels in rats relative to 2-ME feeding. But the enhanced water solubility
facilitates intravenous application. Similarly, hydrophilic monosodium phosphate prodrug
(185) of CHM-1 (103, Fig. 7) was prepared, and possesses improved pharmacological
effects over CHM-1, and readily converts to its parent molecule during both
i.v.
and
p.o.
administration, and shows antitumor activity in the SKOV-3 xenograft mouse model.
Another strategy to increase aqueous solubility of CA-4 is replacing the C-3 hydroxyl group
in ring B with an amine substituent as an HCl salt (AC-7739, 186, Fig. 14). The
L
-serine
amide HCl salt (AC-7700, 187) has also been prepared. Both compounds showed enhanced
aqueous solubility over CA-4 and were efficacious
in vivo
(49). On the basis of these
results, a variety of amide prodrugs of irreversible tubulin inhibitor T138067 (15) were
prepared. T138067 has the ability to penetrate the blood brain barrier (BBB). While
compounds that have the ability to penetrate the BBB are promising candidates for treating
brain tumors, it brought the main dose-limiting side effect of central nervous system (CNS)
toxicity. Amides 188-191 showed no detectable or extremely small amounts of crossing the
BBB. The
in vivo
efficacy of amide 191 approached that of T138067 and was better
tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor
xenografts (151).
A PEG-based polymeric nanomedicine prodrug (192, Fig. 14) of colchicine was synthesized
to increase its aqueous solubility, reduce systemic toxicity and to enhance its therapeutic
window in a recent study (152). Cell viability studies with human umbilical vein endothelial
cells demonstrated the degradation kinetics of the prodrug accordingly. It was observed that
distinct vascular disruption and consequent tumor necrosisin the prodrug treatment but not
for free colchicine at an equivalent dose by
i.v.
treatment in the B16F10 melanoma-bearing
mouse model. Furthermore, a five-times-higher dose of the prodrug is well tolerated with
reduced toxicity. The polymeric conjugated prodrug is another potential strategy to improve
solubility/efficacy/toxicity compared with the parent drug, and it appears to be a promising
approach for the application of CBSIs in cancer therapy.
Liposome based drug delivery possesses advantages including their prolonged circulation
kinetics, passive targeting properties, and the ability to encapsulate both hydrophobic and
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hydrophilic drugs. However, colchicinoids are difficult to retain in liposomes due to their
physicochemical properties. Two hydrolysable PEGylated derivatives (153) of colchicine
were developed for encapsulation into the aqueous core of long-circulating liposomes with
glycolic acid linker (193), and a lactic acid linker (194, Fig. 14). Hydrolysis studies at 37°C
and pH 7.4 showed that 193 possessed a relatively rapid half-life (5.4 h) whereas 194
hydrolyzed much slower (T1/2 =217 h). The parent drug, colchicine, was released rapidly
after encapsulation into liposomes, whereas both PEGylated colchicine prodrugs were
efficiently retained and released only after cleavage of the PEG-linker. Unlike colchicine,
these PEGylated colchicine-derived prodrugs are retained within the aqueous interior after
encapsulation into liposomes, and the release of colchicine can be controlled by using
different biodegradable linkers.
Chemical Structure Modifications—Our groups (154) discovered a series of 4-
substituted methoxybenzoyl-aryl-thiazoles (SMART, 195, Fig. 15) as a result of structural
modifications of the lead compounds 2-arylthiazolidine-4-carboxylic acid amides (ATCAA).
The antiproliferative activity of the SMART agents against melanoma and prostate cancer
cells has been improved from micromolar to low nanomolar range as compared to the early
ATCAA series. Preliminary mechanism of action studies indicated that the SMART
compounds exert their anticancer activity through inhibition of tubulin polymerization
via
the colchicine binding site, overcome the Pgp mediated MDR and show lower neurotoxicity
as compared to vinblastine (155). In order to improve the poor aqueous solubility of
SMART, it was formulated in polyethylene-
b-
poly(
D,L
-lactide) (PEG-PLA) micelles. The
solubility of SMART was increased by more than 1.1×105 fold (156). In a continuing effort
at chemical structure modifications to improve the poor aqueous solubility and
bioavailability, an imidazole B ring analog (ABI, 196) and a phenyl-aminothiazole (PAT,
197) template have been designed in which an amino linkage was inserted between the “A”
and “B” rings of compound 195. The most potent ABI analogs possess low nanomolar
activity and have substantially improved aqueous solubility by 50-fold (157). The PAT
analogs maintained nanomolar range potency against cancer cell lines
via
inhibiting tubulin
polymerization and markedly improved solubility and bioavailability (R=Fluoro,
F
=21%)
compared with the SMART template (
F
=3.3%).
Lee’s group (158,159) developed a series of acetylated (198) and methylpyrazoline (199,
Fig. 15) analogs of CA-4 with the intention to improve solubility of the CA-4 analogs.
Compound 199 has aqueous solubility of 372 μM, which is slightly higher than that for
CA-4 (350 μM), but they also lost the potency.
Gangjee
et al.
(160) reported water soluble colchicine binding site inhibitor and microtubule
depolymerizing agents (200-201, Fig. 15) that inhibited the growth of cancer cells with GI50
values in the nanomolar range. This molecule contains a chiral center and both the
R
and
S
enantiomers cause a G2/M cell cycle arrest and circumvent tumor resistance due to
overexpression of Pgp and βIII tubulin. The
S
isomer (200) is a single digit nanomolar
inhibitor in 51 cancer cell lines and is 10- to 88-fold more potent against most of the cell
lines than either the racemate or the
R
isomer (201).
CONCLUSION
Tubulin targeting agents have played a key role in cancer treatment since the approval of
vincristine by FDA in 1963, and research in this field maintains active. Among the different
types of anti-tubulin agents, only CBSIs have not yet reached the commercial phase.
Literature searching shows hundreds of potential CBSIs have been synthesized and tested
with the hope to find a better clinical drug for cancer therapy. A large number of structurally
diverse CBSI molecules display their anticancer activity through their abilities to arrest cell
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cycle and to kill cancer cells
via
both mitotic and apoptotic pathways, thus hold great
promise for new family of anticancer drugs. Over the last decade, the potential of CBSIs to
act as selective VDAs and overcome efflux pump/mutant tubulin/βIII tubulin
overexpression mediated multidrug resistance has been recognized. A number of agents
have been examined in clinical studies, and their capacity to inhibit growth of the tumor
vasculature and resistant tumor has been confirmed. The current drawbacks in developing
this series of agents as anticancer drugs exist in several areas including narrow therapeutic
windows and lack of oral bioavailability. The side effects such as neural toxicity,
cardiovascular and thromboembolic events remain major concerns and their therapeutic
efficacy as single agents has been disappointing to this point. However, combination
strategies of established anticancer drugs with CBSIs suggest that targeting the vascular
tissue may be a profitablet direction for the further development of CBSIs as anti-cancer
drugs. Furthermore, many CBSIs have limited clinical application due to their poor aqueous
solubility, inadequate metabolic stability, and unsatisfactory pharmacokinetic profiles.
Future new CBSIs will concentrate on the design of metabolically stable analogs and
pharmacokinetic optimization will focus on how to improve their oral bioavailability as well
as to increase their potency. With the new insights into the design of new agents, there is
hope that CBSIs will move from basic research to clinical practice.
Acknowledgments
Research reported in this publication was supported by the National Cancer Institute of the National Institutes of
Health under Award Number R01CA148706.
ABBREVIATIONS
2-ME 2-methoxyestradiol
ABC ATP binding cassette
BS binding site
CA-4 combretastatin A-4
CBSI colchicine binding site inhibitors
cGMP cyclic guanosine monophosphate
CNS central nervous system
CoMFA comparative molecular field analyses
CoMSIA comparative molecular similarity indices analyses
DAMA-colchicine N-deacetyl-N-(2-mercaptoacetyl) colchicine
FDA Food and Drug Administration
FGF fibroblast growth factor
HIF hypoxia-inducible factor
HUVEC human umbilical vein endothelial cell
LIE linear interaction energy
MDR multidrug resistance
MRP multidrug resistance-associated protein
MTA microtubule targeting agent
NCI National Cancer Institute
Lu et al. Page 23
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PDE phosphodiesterase
Pgp P-glycoprotein
PK pharmacokinetic
QSAR quantitative structure-activity relationships
SAR structure-activity relationship
SGB surface-generalized Born
TMP trimethoxyphenyl
TNF-αtumor necrosis factor-α
VDA vascular-disrupting agent
VEGFR vascular endothelial growth factor receptor
References
1. Pryor DE, O’Brate A, Bilcer G, Diaz JF, Wang Y, Kabaki M, et al. The microtubule stabilizing
agent laulimalide does not bind in the taxoid site, kills cells resistant to paclitaxel and epothilones,
and may not require its epoxide moiety for activity. Biochemistry. 2002; 41:9109–15. [PubMed:
12119025]
2. Gigant B, Wang C, Ravelli RB, Roussi F, Steinmetz MO, Curmi PA, et al. Structural basis for the
regulation of tubulin by vinblastine. Nature. 2005; 435:519–22. [PubMed: 15917812]
3. Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, et al. Insight into tubulin
regulation from a complex with colchicine and a stathmin-like domain. Nature. 2004; 428:198–202.
[PubMed: 15014504]
4. Seveand P, Dumontet C. Is class III beta-tubulin a predictive factor in patients receiving tubulin-
binding agents? The Lancet Oncology. 2008; 9:168–75. [PubMed: 18237851]
5. Stengel C, Newman SP, Leese MP, Potter BV, Reed MJ, Purohit A. Class III beta-tubulin
expression and
in vitro
resistance to microtubule targeting agents. Br J Cancer. 2010; 102:316–24.
[PubMed: 20029418]
6. Goto H, Yano S, Zhang H, Matsumori Y, Ogawa H, Blakey DC, et al. Activity of a new vascular
targeting agent, ZD6126, in pulmonary metastases by human lung adenocarcinoma in nude mice.
Cancer Res. 2002; 62:3711–5. [PubMed: 12097279]
7. Lippert JW 3rd. Vascular disrupting agents. Bioorg Med Chem. 2007; 15:605–15. [PubMed:
17070061]
8. Rustin GJ, Shreeves G, Nathan PD, Gaya A, Ganesan TS, Wang D, et al. A Phase Ib trial of CA4P
(combretastatin A-4 phosphate), carboplatin, and paclitaxel in patients with advanced cancer. Br J
Cancer. 2010; 102:1355–60. [PubMed: 20389300]
9. Kim TJ, Ravoori M, Landen CN, Kamat AA, Han LY, Lu C, et al. Antitumor and antivascular
effects of AVE8062 in ovarian carcinoma. Cancer Res. 2007; 67:9337–45. [PubMed: 17909042]
10. http://clinicaltrials.gov/ct2/results?term=AVE8062.
11. Pettit GR, Toki B, Herald DL, Verdier-Pinard P, Boyd MR, Hamel E, et al. Antineoplastic agents.
379. Synthesis of phenstatin phosphate. J Med Chem. 1998; 41:1688–95. [PubMed: 9572894]
12. Zhang LH, Wu L, Raymon HK, Chen RS, Corral L, Shirley MA, et al. The synthetic compound
CC-5079 is a potent inhibitor of tubulin polymerization and tumor necrosis factor-alpha
production with antitumor activity. Cancer Res. 2006; 66:951–9. [PubMed: 16424030]
13. Bohlinand L, Rosen B. Podophyllotoxin derivatives: drug discovery and development. Drug
Discov Today. 1996; 1:343–51.
14. Kupchan SM, Britton RW, Ziegler MF, Gilmore CJ, Restivo RJ, Bryan RF. Steganacin and
steganangin, novel antileukemic lignan lactones from Steganotaenia araliacea. J Am Chem Soc.
1973; 95:1335–6. [PubMed: 4687687]
Lu et al. Page 24
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

15. Attia SM. Molecular cytogenetic evaluation of the mechanism of genotoxic potential of amsacrine
and nocodazole in mouse bone marrow cells. Journal of Applied Toxicology: JAT. 201110.1002/
jat.1753
16. Hamel E. Antimitotic natural products and their interactions with tubulin. Medicinal Research
Reviews. 1996; 16:207–31. [PubMed: 8656780]
17. Matei D, Schilder J, Sutton G, Perkins S, Breen T, Quon C, et al. Activity of 2 methoxyestradiol
(Panzem NCD) in advanced, platinum-resistant ovarian cancer and primary peritoneal
carcinomatosis: a Hoosier Oncology Group trial. Gynecol Oncol. 2009; 115:90–6. [PubMed:
19577796]
18. LaVallee TM, Burke PA, Swartz GM, Hamel E, Agoston GE, Shah J, et al. Significant antitumor
activity
in vivo
following treatment with the microtubule agent ENMD-1198. Mol Cancer Ther.
2008; 7:1472–82. [PubMed: 18566218]
19. Pasquier E, Sinnappan S, Munoz MA, Kavallaris M. ENMD-1198, a new analogue of 2-
methoxyestradiol, displays both anti-angiogenic and vascular-disrupting properties. Mol Cancer
Ther. 2010; 9:1408–18. [PubMed: 20442304]
20. Hande KR, Hagey A, Berlin J, Cai Y, Meek K, Kobayashi H, et al. The pharmacokinetics and
safety of ABT-751, a novel, orally bioavailable sulfonamide antimitotic agent: results of a phase 1
study. Clin Cancer Res. 2006; 12:2834–40. [PubMed: 16675578]
21. Shan B, Medina JC, Santha E, Frankmoelle WP, Chou TC, Learned RM, et al. Selective, covalent
modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with
in vivo
efficacy against multidrug-resistant tumors. Proc Natl Acad Sci U S A. 1999; 96:5686–91.
[PubMed: 10318945]
22. Patterson DM, Rustin GJS, Serradell N, Rosa E, Bolos J. Combretastatin A-4 phosphate. Drugs of
the Future. 2007; 32:1025–32.
23. Rischin D, Bibby DC, Chong G, Kremmidiotis G, Leske AF, Matthews CA, et al. Clinical,
pharmacodynamic, and pharmacokinetic evaluation of BNC105P: a phase I trial of a novel
vascular disrupting agent and inhibitor of cancer cell proliferation. Clin Cancer Res. 2011;
17:5152–60. [PubMed: 21690571]
24. Bacher G, Nickel B, Emig P, Vanhoefer U, Seeber S, Shandra A, et al. D-24851, a novel synthetic
microtubule inhibitor, exerts curative antitumoral activity
in vivo
, shows efficacy toward
multidrug-resistant tumor cells, and lacks neurotoxicity. Cancer Res. 2001; 61:392–9. [PubMed:
11196193]
25. Gourdeau H, Leblond L, Hamelin B, Desputeau C, Dong K, Kianicka I, et al. Antivascular and
antitumor evaluation of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes, a
novel series of anticancer agents. Mol Cancer Ther. 2004; 3:1375–84. [PubMed: 15542776]
26. Sirisoma N, Kasibhatla S, Pervin A, Zhang H, Jiang S, Willardsen JA, et al. Discovery of 2-chloro-
N-(4-methoxy-phenyl)-N-methylquinazolin-4-amine (EP128265, MPI-0441138) as a potent
inducer of apoptosis with high
in vivo
activity. J Med Chem. 2008; 51:4771–9. [PubMed:
18651728]
27. Tsimberidou AM, Akerley W, Schabel MC, Hong DS, Uehara C, Chhabra A, et al. Phase I clinical
trial of MPC-6827 (Azixa), a microtubule destabilizing agent, in patients with advanced cancer.
Mol Cancer Ther. 2010; 9:3410–9. [PubMed: 21159616]
28. Burns CJ, Fantino E, Phillips ID, Su S, Harte MF, Bukczynska PE, et al. CYT997: a novel orally
active tubulin polymerization inhibitor with potent cytotoxic and vascular disrupting activity
in
vitro
and
in vivo
. Mol Cancer Ther. 2009; 8:3036–45. [PubMed: 19887548]
29. Shiand W, Siemann DW. Preclinical studies of the novel vascular disrupting agent MN-029.
Anticancer Res. 2005; 25:3899–904. [PubMed: 16309177]
30. Ricart AD, Ashton EA, Cooney MM, Sarantopoulos J, Brell JM, Feldman MA, et al. A phase I
study of MN-029 (denibulin), a novel vascular-disrupting agent, in patients with advanced solid
tumors. Cancer Chemotherapy and Pharmacology. 2011; 68:959–70. [PubMed: 21305290]
31. de Ines C, Leynadier D, Barasoain I, Peyrot V, Garcia P, Briand C, et al. Inhibition of microtubules
and cell cycle arrest by a new 1-deaza-7,8-dihydropteridine antitumor drug, CI 980, and by its
chiral isomer, NSC 613863. Cancer Res. 1994; 54:75–84. [PubMed: 8261466]
Lu et al. Page 25
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

32. Thomas JP, Moore T, Kraut EH, Balcerzak SP, Galloway S, Vandre DD. A phase II study of
CI-980 in previously untreated extensive small cell lung cancer: an Ohio State University phase II
research consortium study. Cancer Investigation. 2002; 20:192–8. [PubMed: 11901539]
33. Yoon JT, Palazzo AF, Xiao D, Delohery TM, Warburton PE, Bruce JN, et al. CP248, a derivative
of exisulind, causes growth inhibition, mitotic arrest, and abnormalities in microtubule
polymerization in glioma cells. Mol Cancer Ther. 2002; 1:393–404. [PubMed: 12477052]
34. Sun W, Stevenson JP, Gallo JM, Redlinger M, Haller D, Algazy K, et al. Phase I and
pharmacokinetic trial of the proapoptotic sulindac analog CP-461 in patients with advanced
cancer. Clin Cancer Res. 2002; 8:3100–4. [PubMed: 12374677]
35. Tripodi F, Pagliarin R, Fumagalli G, Bigi A, Fusi P, Orsini F, et al. Synthesis and biological
evaluation of 1,4-Diaryl-2-azetidinones as specific anticancer agents: activation of adenosine
monophosphate activated protein kinase and induction of apoptosis. J Med Chem. 2012; 55:2112–
24. [PubMed: 22329561]
36. Bai R, Covell DG, Pei XF, Ewell JB, Nguyen NY, Brossi A, et al. Mapping the binding site of
colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts
predominantly with cysteine 239 and secondarily with cysteine 354. J Biol Chem. 2000;
275:40443–52. [PubMed: 11005811]
37. Dorleans A, Gigant B, Ravelli RB, Mailliet P, Mikol V, Knossow M. Variations in the colchicine-
binding domain provide insight into the structural switch of tubulin. Proc Natl Acad Sci U S A.
2009; 106:13775–9. [PubMed: 19666559]
38. Barbier P, Dorleans A, Devred F, Sanz L, Allegro D, Alfonso C, et al. Stathmin and interfacial
microtubule inhibitors recognize a naturally curved conformation of tubulin dimers. J Biol Chem.
2010; 285:31672–81. [PubMed: 20675373]
39. Nguyen TL, McGrath C, Hermone AR, Burnett JC, Zaharevitz DW, Day BW, et al. A common
pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach. J
Med Chem. 2005; 48:6107–16. [PubMed: 16162011]
40. Dutcher SK. The tubulin fraternity: alpha to eta. Curr Opin Cell Biol. 2001; 13:49–54. [PubMed:
11163133]
41. Simoni D, Romagnoli R, Baruchello R, Rondanin R, Rizzi M, Pavani MG, et al. Novel
combretastatin analogues endowed with antitumor activity. J Med Chem. 2006; 49:3143–52.
[PubMed: 16722633]
42. Stevenson JP, Rosen M, Sun W, Gallagher M, Haller DG, Vaughn D, et al. Phase I trial of the
antivascular agent combretastatin A4 phosphate on a 5-day schedule to patients with cancer:
magnetic resonance imaging evidence for altered tumor blood flow. Journal of Clinical Oncology:
Official Journal of the American Society of Clinical Oncology. 2003; 21:4428–38. [PubMed:
14645433]
43. Tron GC, Pirali T, Sorba G, Pagliai F, Busacca S, Genazzani AA. Medicinal chemistry of
combretastatin A4: present and future directions. J Med Chem. 2006; 49:3033–44. [PubMed:
16722619]
44. Aprile S, Del Grosso E, Tron GC, Grosa G.
In vitro
metabolism study of combretastatin A-4 in rat
and human liver microsomes. Drug Metabolism and Disposition: the Biological Fate of Chemicals.
2007; 35:2252–61. [PubMed: 17890446]
45. Gwaltney SL 2nd, Imade HM, Barr KJ, Li Q, Gehrke L, Credo RB, et al. Novel sulfonate
analogues of combretastatin A-4: potent antimitotic agents. Bioorganic & Medicinal Chemistry
Letters. 2001; 11:871–4. [PubMed: 11294380]
46. Fortin S, Wei L, Moreau E, Lacroix J, Cote MF, Petitclerc E, et al. synthesis, biological evaluation,
and structure-activity relationships of substituted phenyl 4-(2-oxoimidazolidin-1-
yl)benzenesulfonates as new tubulin inhibitors mimicking combretastatin A-4. J Med Chem. 2011;
54:4559–80. [PubMed: 21604746]
47. Fortin S, Wei L, Moreau E, Lacroix J, Cote MF, Petitclerc E, et al. Substituted phenyl 4-(2-
oxoimidazolidin-1-yl)benzenesulfonamides as antimitotics. Antiproliferative, antiangiogenic and
antitumoral activity, and quantitative structure-activity relationships. European Journal of
Medicinal Chemistry. 2011; 46:5327–42. [PubMed: 21920638]
Lu et al. Page 26
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

48. Simoni D, Romagnoli R, Baruchello R, Rondanin R, Grisolia G, Eleopra M, et al. Novel A-ring
and B-ring modified combretastatin A-4 (CA-4) analogues endowed with interesting cytotoxic
activity. J Med Chem. 2008; 51:6211–5. [PubMed: 18783207]
49. Ohsumi K, Hatanaka T, Fujita K, Nakagawa R, Fukuda Y, Nihei Y, et al. Syntheses and antitumor
activity of cis-restricted combretastatins: 5-membered heterocyclic analogues. Bioorganic &
Medicinal Chemistry Letters. 1998; 8:3153–8. [PubMed: 9873694]
50. Wang L, Woods KW, Li Q, Barr KJ, McCroskey RW, Hannick SM, et al. Potent, orally active
heterocycle-based combretastatin A-4 analogues: synthesis, structure-activity relationship,
pharmacokinetics, and
in vivo
antitumor activity evaluation. J Med Chem. 2002; 45:1697–711.
[PubMed: 11931625]
51. Kim Y, Nam NH, You YJ, Ahn BZ. Synthesis and cytotoxicity of 3,4-diaryl-2(5H)-furanones.
Bioorganic & Medicinal Chemistry Letters. 2002; 12:719–22. [PubMed: 11844709]
52. Nam NH, Kim Y, You YJ, Hong DH, Kim HM, Ahn BZ. Synthesis and anti-tumor activity of
novel combretastatins: combretocyclopentenones and related analogues. Bioorganic & Medicinal
Chemistry Letters. 2002; 12:1955–8. [PubMed: 12113817]
53. Nam NH, Kim Y, You YJ, Hong DH, Kim HM, Ahn BZ. Combretoxazolones: synthesis,
cytotoxicity and antitumor activity. Bioorganic & Medicinal Chemistry Letters. 2001; 11:3073–6.
[PubMed: 11714613]
54. Bailly C, Bal C, Barbier P, Combes S, Finet JP, Hildebrand MP, et al. Synthesis and biological
evaluation of 4-arylcoumarin analogues of combretastatins. J Med Chem. 2003; 46:5437–44.
[PubMed: 14640552]
55. Combes S, Barbier P, Douillard S, McLeer-Florin A, Bourgarel-Rey V, Pierson JT, et al. Synthesis
and biological evaluation of 4-arylcoumarin analogues of combretastatins. Part 2. J Med Chem.
2011; 54:3153–62. [PubMed: 21488686]
56. Tron GC, Pagliai F, Del Grosso E, Genazzani AA, Sorba G. Synthesis and cytotoxic evaluation of
combretafurazans. J Med Chem. 2005; 48:3260–8. [PubMed: 15857132]
57. Pati HN, Wicks M, Holt HL, Leblanc R, Weisbruch P, Forrest L, et al. Synthesis and biological
evaluation of cis-combretastatin analogs and their novel 1,2,3-triazole derivatives. Heterocyclic
Commun. 2005; 11:117–20.
58. Simoni D, Grisolia G, Giannini G, Roberti M, Rondanin R, Piccagli L, et al. Heterocyclic and
phenyl double-bond-locked combretastatin analogues possessing potent apoptosis-inducing
activity in HL60 and in MDR cell lines. J Med Chem. 2005; 48:723–36. [PubMed: 15689156]
59. Flynn BL, Flynn GP, Hamel E, Jung MK. The synthesis and tubulin binding activity of thiophene-
based analogues of combretastatin A-4. Bioorganic & Medicinal Chemistry Letters. 2001;
11:2341–3. [PubMed: 11527727]
60. O’Boyle NM, Carr M, Greene LM, Bergin O, Nathwani SM, McCabe T, et al. Synthesis and
evaluation of azetidinone analogues of combretastatin A-4 as tubulin targeting agents. J Med
Chem. 2010; 53:8569–84. [PubMed: 21080725]
61. Romagnoli R, Baraldi PG, Brancale A, Ricci A, Hamel E, Bortolozzi R, et al. Convergent
synthesis and biological evaluation of 2-amino-4-(3′,4′,5′-trimethoxyphenyl)-5-aryl thiazoles as
microtubule targeting agents. J Med Chem. 2011; 54:5144–53. [PubMed: 21663319]
62. Beale TM, Allwood DM, Bender A, Bond PJ, Brenton JD, Charnock-Jones DS, et al. Xian A-ring
dihalogenation increases the cellular activity of combretastatin-templated tetrazoles. ACS
Medicinal Chemistry Letters: Ahead of Print. 2012
63. Schobert R, Biersack B, Dietrich A, Effenberger K, Knauer S, Mueller T. 4-(3-Halo/amino-4,5-
dimethoxyphenyl)-5-aryloxazoles and -N-methylimidazoles that are cytotoxic against
combretastatin A resistant tumor cells and vascular disrupting in a cisplatin resistant germ cell
tumor model. J Med Chem. 2010; 53:6595–602. [PubMed: 20731355]
64. Theeramunkong S, Caldarelli A, Massarotti A, Aprile S, Caprioglio D, Zaninetti R, et al.
Regioselective Suzuki coupling of dihaloheteroaromatic compounds as a rapid strategy to
synthesize potent rigid combretastatin analogues. J Med Chem. 2011; 54:4977–86. [PubMed:
21696175]
Lu et al. Page 27
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

65. Furst R, Zupko I, Berenyi A, Ecker GF, Rinner U. Synthesis and antitumor-evaluation of
cyclopropyl-containing combretastatin analogs. Bioorganic & Medicinal Chemistry Letters. 2009;
19:6948–51. [PubMed: 19879758]
66. Hadfield JA, Gaukroger K, Hirst N, Weston AP, Lawrence NJ, McGown AT. Synthesis and
evaluation of double bond substituted combretastatins. European Journal of Medicinal Chemistry.
2005; 40:529–41. [PubMed: 15922837]
67. Liu T, Cui R, Chen J, Zhang J, He Q, Yang B, et al. 4,5-Diaryl-3-aminopyrazole derivatives as
analogs of Combretastatin A-4: synthesis and biological evaluation. Arch Pharm. 2011; 344:279–
86.
68. Akselsen OW, Odlo K, Cheng JJ, Maccari G, Botta M, Hansen TV. Synthesis, biological
evaluation and molecular modeling of 1,2,3-triazole analogs of combretastatin A-1. Bioorg Med
Chem. 2012; 20:234–42. [PubMed: 22137934]
69. Xue N, Yang X, Wu R, Chen J, He Q, Yang B, et al. Synthesis and biological evaluation of
imidazol-2-one derivatives as potential antitumor agents. Bioorg Med Chem. 2008; 16:2550–7.
[PubMed: 18226907]
70. Ruprich J, Prout A, Dickson J, Younglove B, Nolan L, Baxi K, et al. Design, synthesis and
biological testing of cyclohexenone derivatives of combretastatin-A4. Letters in Drug Design &
Discovery. 2007; 4:144–8.
71. Lee L, Davis R, Vanderham J, Hills P, Mackay H, Brown T, et al. 1,2,3,4-tetrahydro-2-
thioxopyrimidine analogs of combretastatin-A4. European Journal of Medicinal Chemistry. 2008;
43:2011–5. [PubMed: 18226429]
72. Johnson M, Younglove B, Lee L, LeBlanc R, Holt H Jr, Hills P, et al. Design, synthesis, and
biological testing of pyrazoline derivatives of combretastatin-A4. Bioorganic & Medicinal
Chemistry Letters. 2007; 17:5897–901. [PubMed: 17827004]
73. Mateo C, Perez-Melero C, Pelaez R, Medarde M. Stilbenophane analogues of deoxycombretastatin
A-4. J Org Chem. 2005; 70:6544–7. [PubMed: 16050729]
74. De Martino G, Edler MC, La Regina G, Coluccia A, Barbera MC, Barrow D, et al. New
arylthioindoles: potent inhibitors of tubulin polymerization. 2. Structure-activity relationships and
molecular modeling studies. J Med Chem. 2006; 49:947–54. [PubMed: 16451061]
75. De Martino G, La Regina G, Coluccia A, Edler MC, Barbera MC, Brancale A, et al.
Arylthioindoles, potent inhibitors of tubulin polymerization. J Med Chem. 2004; 47:6120–3.
[PubMed: 15566282]
76. La Regina G, Sarkar T, Bai R, Edler MC, Saletti R, Coluccia A, et al. New arylthioindoles and
related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth
inhibition, and molecular modeling studies. J Med Chem. 2009; 52:7512–27. [PubMed: 19601594]
77. La Regina G, Bai R, Rensen W, Coluccia A, Piscitelli F, Gatti V, et al. Design and synthesis of 2-
Heterocyclyl-3-arylthio-1H-indoles as potent tubulin polymerization and cell growth inhibitors
with improved metabolic stability. J Med Chem. 2011; 54:8394–406. [PubMed: 22044164]
78. Mahboobi S, Pongratz H, Hufsky H, Hockemeyer J, Frieser M, Lyssenko A, et al. Synthetic 2-
aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents. J Med
Chem. 2001; 44:4535–53. [PubMed: 11741473]
79. Kuo CC, Hsieh HP, Pan WY, Chen CP, Liou JP, Lee SJ, et al. BPR0L075, a novel synthetic indole
compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity
in
vivo
. Cancer Res. 2004; 64:4621–8. [PubMed: 15231674]
80. Hu CB, Chen CP, Yeh TK, Song JS, Chang CY, Chuu JJ, et al. BPR0C261 is a novel orally active
antitumor agent with antimitotic and anti-angiogenic activities. Cancer Science. 2011; 102:182–
91. [PubMed: 21040217]
81. Li WT, Hwang DR, Chen CP, Shen CW, Huang CL, Chen TW, et al. Synthesis and biological
evaluation of N-heterocyclic indolyl glyoxylamides as orally active anticancer agents. J Med
Chem. 2003; 46:1706–15. [PubMed: 12699388]
82. Nien CY, Chen YC, Kuo CC, Hsieh HP, Chang CY, Wu JS, et al. 5-Amino-2-aroylquinolines as
highly potent tubulin polymerization inhibitors. J Med Chem. 2010; 53:2309–13. [PubMed:
20148562]
Lu et al. Page 28
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

83. Liou JP, Wu ZY, Kuo CC, Chang CY, Lu PY, Chen CM, et al. Discovery of 4-amino and 4-
hydroxy-1-aroylindoles as potent tubulin polymerization inhibitors. J Med Chem. 2008; 51:4351–
5. [PubMed: 18588280]
84. Lee HY, Chang JY, Nien CY, Kuo CC, Shih KH, Wu CH, et al. 5-Amino-2-aroylquinolines as
highly potent tubulin polymerization inhibitors. Part 2. The impact of bridging groups at position
C-2. J Med Chem. 2011; 54:8517–25. [PubMed: 22060033]
85. Arthuis M, Pontikis R, Chabot GG, Quentin L, Scherman D, Florent JC. Domino approach to 2-
aroyltrimethoxyindoles as novel heterocyclic combretastatin A4 analogues. European Journal of
Medicinal Chemistry. 2011; 46:95–100. [PubMed: 21112130]
86. Lai MJ, Chang JY, Lee HY, Kuo CC, Lin MH, Hsieh HP, et al. Synthesis and biological evaluation
of 1-(4′-Indolyl and 6′-Quinolinyl) indoles as a new class of potent anticancer agents. European
Journal of Medicinal Chemistry. 2011; 46:3623–9. [PubMed: 21641700]
87. Lai MJ, Kuo CC, Yeh TK, Hsieh HP, Chen LT, Pan WY, et al. Synthesis and structure-activity
relationships of 1-benzyl-4,5,6-trimethoxyindoles as a novel class of potent antimitotic agents.
ChemMedChem. 2009; 4:588–93. [PubMed: 19266513]
88. Xie F, Zhao H, Li D, Chen H, Quan H, Shi X, et al. Synthesis and biological evaluation of 2,4,5-
substituted pyrimidines as a new class of tubulin polymerization inhibitors. J Med Chem. 2011;
54:3200–5. [PubMed: 21480626]
89. El-Nakkady SS, Hanna MM, Roaiah HM, Ghannam IA. Synthesis, molecular docking study and
antitumor activity of novel 2-phenylindole derivatives. European Journal of Medicinal Chemistry.
2012; 47:387–98. [PubMed: 22119129]
90. Arora S, Wang XI, Keenan SM, Andaya C, Zhang Q, Peng Y, et al. Novel microtubule
polymerization inhibitor with potent anti-proliferative and antitumor activity. Cancer Res. 2009;
69:1910–5. [PubMed: 19223556]
91. Shetty RS, Lee Y, Liu B, Husain A, Joseph RW, Lu Y, et al. Synthesis and pharmacological
evaluation of N-(3-(1H-indol-4-yl)-5-(2-methoxyisonicotinoyl)phenyl)methanesulfonamide
(LP-261), a potent antimitotic agent. J Med Chem. 2011; 54:179–200. [PubMed: 21126027]
92. Li L, Wang HK, Kuo SC, Wu TS, Mauger A, Lin CM, et al. Antitumor agents. 155. Synthesis and
biological evaluation of 3′,6,7-substituted 2-phenyl-4-quinolones as antimicrotubule agents. J Med
Chem. 1994; 37:3400–7. [PubMed: 7932568]
93. Li L, Wang HK, Kuo SC, Wu TS, Lednicer D, Lin CM, et al. Antitumor agents. 150. 2′,3′,4′,5′,
5,6,7-substituted 2-phenyl-4-quinolones and related compounds: their synthesis, cytotoxicity, and
inhibition of tubulin polymerization. J Med Chem. 1994; 37:1126–35. [PubMed: 8164254]
94. Zhang C, Yang N, Yang CH, Ding HS, Luo C, Zhang Y, et al. S9, a novel anticancer agent, exerts
its anti-proliferative activity by interfering with both PI3K-Akt-mTOR signaling and microtubule
cytoskeleton. PLoS One. 2009; 4:e4881. [PubMed: 19293927]
95. Romagnoli R, Baraldi PG, Carrion MD, Lopez Cara C, Preti D, Fruttarolo F, et al. Synthesis and
biological evaluation of 2- and 3-aminobenzo[b]thiophene derivatives as antimitotic agents and
inhibitors of tubulin polymerization. J Med Chem. 2007; 50:2273–7. [PubMed: 17419607]
96. Keller L, Beaumont S, Liu JM, Thoret S, Bignon JS, Wdzieczak-Bakala J, et al. New C5-alkylated
indolobenzazepinones acting as inhibitors of tubulin polymerization: cytotoxic and antitumor
activities. J Med Chem. 2008; 51:3414–21. [PubMed: 18503262]
97. Pons V, Beaumont S, Dau METH, Iorga BI, Dodd RH. Rigid analogues of antimitotic
indolobenzazepinones: new insights into tubulin binding via molecular modeling. ACS Medicinal
Chemistry Letters. 2011; 2:565–570.
98. Boumendjel A, McLeer-Florin A, Champelovier P, Allegro D, Muhammad D, Souard F, et al. A
novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-
gp and BCRP in
in-vitro
and
in-vivo
glioblastoma models. BMC Cancer. 2009; 9:242. [PubMed:
19619277]
99. Kerr DJ, Hamel E, Jung MK, Flynn BL. The concise synthesis of chalcone, indanone and indenone
analogues of combretastatin A4. Bioorg Med Chem. 2007; 15:3290–8. [PubMed: 17360188]
100. Luo Y, Qiu KM, Lu X, Liu K, Fu J, Zhu HL. Synthesis, biological evaluation, and molecular
modeling of cinnamic acyl sulfonamide derivatives as novel antitubulin agents. Bioorg Med
Chem. 2011; 19:4730–8. [PubMed: 21783370]
Lu et al. Page 29
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

101. Ruan BF, Lu X, Tang JF, Wei Y, Wang XL, Zhang YB, et al. Synthesis, biological evaluation,
and molecular docking studies of resveratrol derivatives possessing chalcone moiety as potential
antitubulin agents. Bioorg Med Chem. 2011; 19:2688–95. [PubMed: 21440448]
102. Risinger AL, Westbrook CD, Encinas A, Mulbaier M, Schultes CM, Wawro S, et al. ELR510444,
a novel microtubule disruptor with multiple mechanisms of action. J Pharmacol Exp Ther. 2011;
336:652–60. [PubMed: 21148249]
103. Chang JY, Hsieh HP, Chang CY, Hsu KS, Chiang YF, Chen CM, et al. 7-Aroyl-aminoindoline-1-
sulfonamides as a novel class of potent antitubulin agents. J Med Chem. 2006; 49:6656–9.
[PubMed: 17154496]
104. Liou JP, Hsu KS, Kuo CC, Chang CY, Chang JY. A novel oral indoline-sulfonamide agent, N-[1-
(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-isonicotinamide (J30), exhibits potent
activity against human cancer cells
in vitro
and
in vivo
through the disruption of microtubule. J
Pharmacol Exp Ther. 2007; 323:398–405. [PubMed: 17660383]
105. Cushman M, He HM, Katzenellenbogen JA, Varma RK, Hamel E, Lin CM, et al. Synthesis of
analogs of 2-methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and
cancer cell growth. J Med Chem. 1997; 40:2323–34. [PubMed: 9240348]
106. Cushman M, Mohanakrishnan AK, Hollingshead M, Hamel E. The effect of exchanging various
substituents at the 2-position of 2-methoxyestradiol on cytotoxicity in human cancer cell cultures
and inhibition of tubulin polymerization. J Med Chem. 2002; 45:4748–54. [PubMed: 12361402]
107. Edsall AB, Mohanakrishnan AK, Yang D, Fanwick PE, Hamel E, Hanson AD, et al. Effects of
altering the electronics of 2-methoxyestradiol on cell proliferation, on cytotoxicity in human
cancer cell cultures, and on tubulin polymerization. J Med Chem. 2004; 47:5126–39. [PubMed:
15456256]
108. Agoston GE, Shah JH, Lavallee TM, Zhan X, Pribluda VS, Treston AM. Synthesis and structure-
activity relationships of 16-modified analogs of 2-methoxyestradiol. Bioorg Med Chem. 2007;
15:7524–37. [PubMed: 17910916]
109. Chander SK, Foster PA, Leese MP, Newman SP, Potter BV, Purohit A, et al.
In vivo
inhibition of
angiogenesis by sulphamoylated derivatives of 2-methoxyoestradiol. Br J Cancer. 2007;
96:1368–76. [PubMed: 17426705]
110. Ireson CR, Chander SK, Purohit A, Perera S, Newman SP, Parish D, et al. Pharmacokinetics and
efficacy of 2-methoxyoestradiol and 2-methoxyoestradiol-bis-sulphamate
in vivo
in rodents. Br J
Cancer. 2004; 90:932–7. [PubMed: 14970876]
111. Jourdan F, Leese MP, Dohle W, Hamel E, Ferrandis E, Newman SP, et al. Synthesis, antitubulin,
and antiproliferative SAR of analogues of 2-methoxyestradiol-3,17-O,O-bis-sulfamate. J Med
Chem. 2010; 53:2942–51. [PubMed: 20225862]
112. Nakagawa-Goto K, Chen TH, Peng CY, Bastow KF, Wu JH, Lee KH. Antitumor agents 259.
Design, syntheses, and structure-activity relationship study of desmosdumotin C analogs. J Med
Chem. 2007; 50:3354–8. [PubMed: 17569518]
113. Nakagawa-Goto K, Bastow KF, Chen TH, Morris-Natschke SL, Lee KH. Antitumor agents 260.
New desmosdumotin B analogues with improved
in vitro
anticancer activity. J Med Chem. 2008;
51:3297–303. [PubMed: 18473435]
114. Nakagawa-Goto K, Wu PC, Lai CY, Hamel E, Zhu H, Zhang L, et al. Antitumor agents. 284.
New desmosdumotin B analogues with bicyclic B-ring as cytotoxic and antitubulin agents. J Med
Chem. 2011; 54:1244–55. [PubMed: 21284385]
115. Nakagawa-Goto K, Wu PC, Bastow KF, Yang SC, Yu SL, Chen HY, et al. Antitumor agents 283.
Further elaboration of desmosdumotin C analogs as potent antitumor agents: activation of spindle
assembly checkpoint as possible mode of action. Bioorg Med Chem. 2011; 19:1816–22.
[PubMed: 21296579]
116. Beutler JA, Cardellina JH II, Lin CM, Hamel E, Cragg GM, Boyd MR. Centaureidin, a cytotoxic
flavone from Polymnia fruticosa, inhibits tubulin polymerization. Bioorganic & Medicinal
Chemistry Letters. 1993; 3:581–4.
117. Naik PK, Chatterji BP, Vangapandu SN, Aneja R, Chandra R, Kanteveri S, et al. Rational design,
synthesis and biological evaluations of amino-noscapine: a high affinity tubulin-binding
noscapinoid. J Comput Aided Mol Des. 2011; 25:443–54. [PubMed: 21544622]
Lu et al. Page 30
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

118. Hartley RM, Peng J, Fest GA, Dakshanamurthy S, Frantz DE, Brown ML, et al. Polygamain, a
new microtubule depolymerizing agent that occupies a unique pharmacophore in the colchicine
site. Molecular Pharmacology. 2011
119. Cui CB, Kakeya H, Okada G, Onose R, Ubukata M, Takahashi I, et al. Tryprostatins A and B,
novel mammalian cell cycle inhibitors produced by Aspergillus fumigatus. J Antibiot. 1995;
48:1382–4. [PubMed: 8557590]
120. Woehlecke H, Osada H, Herrmann A, Lage H. Reversal of breast cancer resistance protein-
mediated drug resistance by try-prostatin A. International Journal of Cancer Journal International
Du Cancer. 2003; 107:721–8. [PubMed: 14566821]
121. Kanoh K, Kohno S, Katada J, Takahashi J, Uno I. (-)-Phenyl-ahistin arrests cells in mitosis by
inhibiting tubulin polymerization. J Antibiot. 1999; 52:134–41. [PubMed: 10344567]
122. Kanzaki H, Yanagisawa S, Kanoh K, Nitoda T. A novel potent cell cycle inhibitor
dehydrophenylahistin–enzymatic synthesis and inhibitory activity toward sea urchin embryo. J
Antibiot. 2002; 55:1042–7. [PubMed: 12617513]
123. Yamazaki Y, Tanaka K, Nicholson B, Deyanat-Yazdi G, Potts B, Yoshida T, et al. Synthesis and
Structure-Activity Relationship Study of Antimicrotubule Agents Phenylahistin Derivatives with
a Didehydropiperazine-2,5-dione Structure. J Med Chem. 2012; 55:1056–71. [PubMed:
22185476]
124. Wipf P, Reeves JT, Balachandran R, Day BW. Synthesis and biological evaluation of structurally
highly modified analogues of the antimitotic natural product curacin A. J Med Chem. 2002;
45:1901–17. [PubMed: 11960501]
125. Wipf P, Reeves JT, Balachandran R, Giuliano KA, Hamel E, Day BW. Synthesis and Biological
Evaluation of a Focused Mixture Library of Analogues of the Antimitotic Marine Natural
Product Curacin A. J Am Chem Soc. 2000; 122:9391–5.
126. Ziegelbauer J, Shan B, Yager D, Larabell C, Hoffmann B, Tjian R. Transcription factor MIZ-1 is
regulated via microtubule association. Molecular Cell. 2001; 8:339–49. [PubMed: 11545736]
127. Bai R, Pei XF, Boye O, Getahun Z, Grover S, Bekisz J, et al. Identification of cysteine 354 of
beta-tubulin as part of the binding site for the A ring of colchicine. J Biol Chem. 1996;
271:12639–45. [PubMed: 8647876]
128. Combeau C, Provost J, Lancelin F, Tournoux Y, Prod’homme F, Herman F, et al. RPR112378
and RPR115781: two representatives of a new family of microtubule assembly inhibitors. Mol
Pharmacol. 2000; 57:553–63. [PubMed: 10692496]
129. Bouchon B, Chambon C, Mounetou E, Papon J, Miot-Noirault E, Gaudreault RC, et al.
Alkylation of beta-tubulin on Glu 198 by a microtubule disrupter. Mol Pharmacol. 2005;
68:1415–22. [PubMed: 16099845]
130. Fortin S, Bouchon B, Chambon C, Lacroix J, Moreau E, Chezal JM, et al. Characterization of the
covalent binding of N-phenyl-N′-(2-chloroethyl)ureas to {beta}-tubulin: importance of Glu198
in microtubule stability. J Pharmacol Exp Ther. 2011; 336:460–7. [PubMed: 20978170]
131. Prinz H, Schmidt P, Bohm KJ, Baasner S, Muller K, Gerlach M, et al. Phenylimino-10H-
anthracen-9-ones as novel antimicrotubule agents-synthesis, antiproliferative activity and
inhibition of tubulin polymerization. Bioorg Med Chem. 2011; 19:4183–91. [PubMed:
21705223]
132. Kasibhatla S, Gourdeau H, Meerovitch K, Drewe J, Reddy S, Qiu L, et al. Discovery and
mechanism of action of a novel series of apoptosis inducers with potential vascular targeting
activity. Mol Cancer Ther. 2004; 3:1365–74. [PubMed: 15542775]
133. Kemnitzer W, Drewe J, Jiang S, Zhang H, Crogan-Grundy C, Labreque D, et al. Discovery of 4-
aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high
throughput screening assay. 4. Structure-activity relationships of N-alkyl substituted pyrrole
fused at the 7,8-positions. J Med Chem. 2008; 51:417–23. [PubMed: 18197614]
134. Henderson MC, Shaw YJ, Wang H, Han H, Hurley LH, Flynn G, et al. UA62784, a novel
inhibitor of centromere protein E kinesin-like protein. Mol Cancer Ther. 2009; 8:36–44.
[PubMed: 19139111]
135. Tcherniuk S, Deshayes S, Sarli V, Divita G, Abrieu A. UA62784 Is a cytotoxic inhibitor of
microtubules, not CENP-E. Chem Biol. 2011; 18:631–41. [PubMed: 21609844]
Lu et al. Page 31
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

136. Dalyot-Herman N, Delgado-Lopez F, Gewirtz DA, Gupton JT, Schwartz EL. Interference with
endothelial cell function by JG-03-14, an agent that binds to the colchicine site on microtubules.
Biochem Pharmacol. 2009; 78:1167–77. [PubMed: 19576183]
137. Mooberry SL, Weiderhold KN, Dakshanamurthy S, Hamel E, Banner EJ, Kharlamova A, et al.
Identification and characterization of a new tubulin-binding tetrasubstituted brominated pyrrole.
Mol Pharmacol. 2007; 72:132–40. [PubMed: 17456786]
138. Zhang Z, Meng T, Yang N, Wang W, Xiong B, Chen Y, et al. MT119, a new planar-structured
compound, targets the colchicine site of tubulin arresting mitosis and inhibiting tumor cell
proliferation. International Journal of Cancer Journal International Du Cancer. 2011; 129:214–
24. [PubMed: 20830720]
139. Lisowski V, Leonce S, Kraus-Berthier L, Sopkovade Oliveira Santos J, Pierre A, Atassi G, et al.
Design, synthesis, and evaluation of novel thienopyrrolizinones as antitubulin agents. J Med
Chem. 2004; 47:1448–64. [PubMed: 14998333]
140. Leoni LM, Hamel E, Genini D, Shih H, Carrera CJ, Cottam HB, et al. Indanocine, a microtubule-
binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells. J Natl
Cancer Inst. 2000; 92:217–24. [PubMed: 10655438]
141. Liberatore AM, Coulomb H, Pons D, Dutruel O, Kasprzyk PG, Carlson M, et al. IRC-083927 is a
new tubulin binder that inhibits growth of human tumor cells resistant to standard tubulin-
binding agents. Mol Cancer Ther. 2008; 7:2426–34. [PubMed: 18723488]
142. Wasylyk C, Zheng H, Castell C, Debussche L, Multon MC, Wasylyk B. Inhibition of the Ras-Net
(Elk-3) pathway by a novel pyrazole that affects microtubules. Cancer Res. 2008; 68:1275–83.
[PubMed: 18316589]
143. Shin KD, Yoon YJ, Kang YR, Son KH, Kim HM, Kwon BM, et al. KRIBB3, a novel microtubule
inhibitor, induces mitotic arrest and apoptosis in human cancer cells. Biochem Pharmacol. 2008;
75:383–94. [PubMed: 17915194]
144. Szczepankiewicz BG, Liu G, Jae HS, Tasker AS, Gunawardana IW, von Geldern TW, et al. New
antimitotic agents with activity in multi-drug-resistant cell lines and
in vivo
efficacy in murine
tumor models. J Med Chem. 2001; 44:4416–30. [PubMed: 11728187]
145. Tahir SK, Han EK, Credo B, Jae HS, Pietenpol JA, Scatena CD, et al. A-204197, a new tubulin-
binding agent with antimitotic activity in tumor cell lines resistant to known microtubule
inhibitors. Cancer Res. 2001; 61:5480–5. [PubMed: 11454695]
146. Li Q, Woods KW, Claiborne A, Gwaltney SL 2nd, Barr KJ, Liu G, et al. Synthesis and biological
evaluation of 2-indolyloxazolines as a new class of tubulin polymerization inhibitors. Discovery
of A-289099 as an orally active antitumor agent. Bioorganic & Medicinal Chemistry Letters.
2002; 12:465–9. [PubMed: 11814821]
147. Tahir SK, Nukkala MA, Zielinski Mozny NA, Credo RB, Warner RB, Li Q, et al. Biological
activity of A-289099: an orally active tubulin-binding indolyloxazoline derivative. Mol Cancer
Ther. 2003; 2:227–33. [PubMed: 12657717]
148. Andreani A, Burnelli S, Granaiola M, Leoni A, Locatelli A, Morigi R, et al. Antitumor activity of
new substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-
b]thiadiazolylmethylene)-2-indolinones: selectivity against colon tumor cells and effect on cell
cycle-related events. J Med Chem. 2008; 51:7508–13. [PubMed: 19006285]
149. Andreani A, Granaiola M, Locatelli A, Morigi R, Rambaldi M, Varoli L, et al. Substituted 3-(5-
Imidazo[2,1-b]thiazolyl-methylene)-2-indolinones and analogues: synthesis, cytotoxic activity,
and study of the mechanism of action (1). J Med Chem. 2012
150. Edsall AB, Agoston GE, Treston AM, Plum SM, McClanahan RH, Lu TS, et al. Synthesis and
in
vivo
antitumor evaluation of 2-methoxyestradiol 3-phosphate, 17-phosphate, and 3,17-
diphosphate. J Med Chem. 2007; 50:6700–5. [PubMed: 18052315]
151. Rubenstein SM, Baichwal V, Beckmann H, Clark DL, Frankmoelle W, Roche D, et al.
Hydrophilic, prodrug analogues of T138067 are efficacious in controlling tumor growth
in vivo
and show a decreased ability to cross the blood brain barrier. J Med Chem. 2001; 44:3599–605.
[PubMed: 11606124]
152. Crielaard BJ, van der Wal S, Lammers T, Le HT, Hennink WE, Schiffelers RM, et al. A
polymeric colchicinoid prodrug with reduced toxicity and improved efficacy for vascular
Lu et al. Page 32
Pharm Res
. Author manuscript; available in PMC 2013 November 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

disruption in cancer therapy. International Journal of Nanomedicine. 2011; 6:2697–703.
[PubMed: 22114500]
153. Crielaard BJ, van der Wal S, Le HT, Bode AT, Lammers T, Hennink WE, et al. Liposomes as
carriers for colchicine-derived prodrugs: Vascular disrupting nanomedicines with tailorable drug
release kinetics. European Journal of Pharmaceutical Sciences: Official Journal of the European
Federation for Pharmaceutical Sciences. 2012; 45:429–35. [PubMed: 21907797]
154. Lu Y, Li CM, Wang Z, Ross CR 2nd, Chen J, Dalton JT, et al. Discovery of 4-substituted
methoxybenzoyl-aryl-thiazole as novel anticancer agents: synthesis, biological evaluation, and
structure-activity relationships. J Med Chem. 2009; 52:1701–11. [PubMed: 19243174]
155. Li CM, Wang Z, Lu Y, Ahn S, Narayanan R, Kearbey JD, et al. Biological activity of 4-
substituted methoxybenzoyl-aryl-thiazole: an active microtubule inhibitor. Cancer Res. 2011;
71:216–24. [PubMed: 21084278]
156. Li F, Lu Y, Li W, Miller DD, Mahato RI. Synthesis, formulation and
in vitro
evaluation of a
novel microtubule destabilizer, SMART-100. Journal of Controlled Release: Official Journal of
the Controlled Release Society. 2010; 143:151–8. [PubMed: 20060430]
157. Chen J, Wang Z, Li CM, Lu Y, Vaddady PK, Meibohm B, et al. Discovery of novel 2-aryl-4-
benzoyl-imidazoles targeting the colchicines binding site in tubulin as potential anticancer
agents. J Med Chem. 2010; 53:7414–27. [PubMed: 20919720]
158. Babu B, Lee M, Lee L, Strobel R, Brockway O, Nickols A, et al. Acetyl analogs of
combretastatin A-4: synthesis and biological studies. Bioorg Med Chem. 2011; 19:2359–67.
[PubMed: 21382720]
159. Lee M, Brockway O, Dandavati A, Tzou S, Sjoholm R, Satam V, et al. A novel class of trans-
methylpyrazoline analogs of combretastatins: synthesis and
in-vitro
biological testing. European
Journal of Medicinal Chemistry. 2011; 46:3099–104. [PubMed: 21524832]
160. Gangjee A, Zhao Y, Hamel E, Westbrook C, Mooberry SL. Synthesis and biological activities of
(R)- and (S)-N-(4-Methoxy-phenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimi din-4-
aminium chloride as potent cytotoxic antitubulin agents. J Med Chem. 2011; 54:6151–5.
[PubMed: 21786793]
Lu et al. Page 33
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Fig. 1.
Tubulin binding sites (BS) of microtubule targeting agents.
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Fig. 2.
Publications related to CBSIs over last decades from PubMed (data entry implementation
date until Dec 30th, 2011).
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Fig. 3.
Chemical structures of established drugs bound to colchicine binding site and CBSIs in
clinical trials (TMP: 3, 4, 5-trimethoxyphenyl).
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Fig. 4.
Interactions between the pharmacophoric points and the tubulin structure (based on Ref.
(60)).
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Fig. 5.
Chemical structures of chemical modified CA-4 analogs.
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Fig. 6.
Chemical structures of chemical modified CA-4 analogs (continued).
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Fig. 7.
Chemical structures of indole, quinolone and thiophene-based CBSIs.
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Fig. 8.
Chemical structures of chalcone analogs.
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Fig. 9.
Chemical structures of sulfonamide CBSIs.
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Fig. 10.
Chemical structures of 2-ME analogs.
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Fig. 11.
Chemical structures of CBSIs derivatives of natural products.
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Fig. 12.
Chemical structures of CBSIs covalently binding to tubulin colchicine binding site.
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Fig. 13.
Chemical structures of screened and synthesized CBSIs.
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Fig. 14.
Chemical structures of prodrugs of CBSIs.
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Fig. 15.
Chemical structures of CBSIs with improved solubility.
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Table I
Reported X-ray Structures of Tubulin in Complex with CBSIs
CBSIs, Compd ID PDB code Res (Å) Year(Ref)
Colchicine (1)1SA0 3.58 2004 (57)
Podophyllotoxin (8)1SA1 4.20 2004 (4)
ABT-751 (14)3HKC 3.80 2009 (58)
T138067 (15)3HKE 3.60 2009 (58)
TN16 (26)3HKD 3.70 2009 (58)
CI-980 (23,
S
-isomer) 3N2K 4.00 2010 (59)
NSC613863 (
R
-isomer of 23)3N2G 4.00 2010 (59)
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