Development of a new benzophenone-diketopiperazine-type potent antimicrotubule agent possessing a 2-pyridine structure

Article abstract of DOI:10.1021/ml5001883

A new benzophenone-diketopiperazine-type potent antimicrotubule agent was developed by modifying the structure of the clinical candidate plinabulin (1). Although the right-hand imidazole ring with a branched alkyl chain at the 5-position in 1 was critical for the potency of the antimicrotubule activity, we successfully substituted this moiety with a simpler 2-pyridyl structure by converting the left-hand ring from a phenyl to a benzophenone structure without decreasing the potency. The resultant compound 6b (KPU-300) exhibited a potent cytotoxicity, with an IC₅₀value of 7.0 nM against HT-29 cells, by strongly binding to tubulin (K_d= 1.3 μM) and inducing microtubule depolymerization.

Full text of DOI:10.1021/ml5001883

Letter
pubs.acs.org/acsmedchemlett
Development of a New Benzophenone−Diketopiperazine-Type
Potent Antimicrotubule Agent Possessing a 2‑Pyridine Structure
†
†
†
‡
†
§,∥
Yoshiki Hayashi, Haruka Takeno, Takumi Chinen, Kyohei Muguruma, Kohei Okuyama,
†
†
§
‡
Akihiro Taguchi, Kentaro Takayama, Fumika Yakushiji, Masahiko Miura, Takeo Usui,
,
†
and Yoshio Hayashi*
†
Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
‡
§
∥
Section of Oral Radiation Oncology, Department of Oral Health Science, and Section of Maxillofacial Surgery, Department of
Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo 113-8549, Japan
*
S Supporting Information
ABSTRACT: A new benzophenone−diketopiperazine-type potent antimicrotubule agent
was developed by modifying the structure of the clinical candidate plinabulin (1). Although
the right-hand imidazole ring with a branched alkyl chain at the 5-position in 1 was critical
for the potency of the antimicrotubule activity, we successfully substituted this moiety with a
simpler 2-pyridyl structure by converting the left-hand ring from a phenyl to a
benzophenone structure without decreasing the potency. The resultant compound 6b
(
KPU-300) exhibited a potent cytotoxicity, with an IC value of 7.0 nM against HT-29 cells,
50
by strongly binding to tubulin (K = 1.3 μM) and inducing microtubule depolymerization.
d
KEYWORDS: Cyclic dipeptide, diketopiperazine, antimicrotubule agent, anticancer
icrotubules are tubular polymers formed by the
have yet been developed using colchicine site-binding
M
polymerization of α- and β-tubulin heterodimers and
compounds, probably due to the strong in vivo toxicity of
these compounds or the difficulties associated with demon-
strating superiority over existing drugs that bind to other
1
are major cytoskeletal components in eukaryotic cells. The
dynamic and mutual interconversion of tubulins and micro-
tubules is responsible for a variety of cellular processes involved
in maintaining the cellular structure, providing an intracellular
rail-like transport platform for the motor proteins kinesin and
dynein, and enabling cell division through spindle formation1
during mitosis, which pulls apart the eukaryotic chromosomes.
Because of these important roles, microtubules are attractive
17−19
sites.
Colchicine site binders (Figure 1) were recently recognized
20
as vascular disrupting agents (VDAs) that damage tumor-
induced immature vascular endothelial cells, resulting in the
selective disruption of the angiogenic blood vessels that are
formed in and around solid tumor tissues. Clinical trials have
been conducted in which these vascular disrupting compounds
2
,3
molecular targets in cancer chemotherapy. Several anti-
microtubule agents that interfere with the dynamic functions of
microtubules have been developed as clinical drugs for the
treatment of various cancers.
and dosetaxel), which can stabilize the polymerized states of
microtubules, and the vinca alkaloids (vincristine, vinblastine,
and vindesine), which can depolymerize microtubules to form
α/β-tubulin heterodimers.
Antimicrotubule agents generally recognize one of three
specific major drug binding sites on β-tubulin.
21
were tested as anticancer drugs. Because vascular disruption
prevents oxygen and nutrition supplies from reaching solid
tumor tissues, VDA can induce tumor regression.
4
−6
These are taxanes (paclitaxel
4
,5
In our efforts to develop a potent microtubule depolymeriza-
tion agent based on an original skeleton that can provide a
pharmacological profile distinct from that of the known
colchicine site binders, we focused on a natural cyclic dipeptide
6
1
2−16
1
−3
(diketopiperazine, DKP), phenylahistin,
which was
The taxanes
isolated from Aspergillus ustus in 1997. This compound has a
and vinca alkaloids bind specifically to taxane and vinca sites,
respectively. The third binding site is a colchicine site, to which
colchicine and many other natural products, such as
Received: May 9, 2014
Accepted: July 23, 2014
7
,8
9,10
podophyllotoxin, combretastatin A-4 (CA-4),
stegana-
1
1
12−16
cine, and phenylahistin
can bind. No anticancer drugs
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ml5001883 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Benzophenone−DKP Derivatives
Figure 1. Structures of natural colchicine-site binders, plinabulin, and
its representative derivatives.
a
Reagents and conditions: (a) t-BuOK, t-BuOH, DMF, −15 °C, 2.5 h,
6
4%; (b) 28% NH OH, DMF, rt, 1 h, 53%; (c) Cs CO , degassed
4
2
3
DMF, 110 °C, 2 h, 14−86%.
distinctive hydrophilic chemical structure, displays moderate
colchicine-like microtubule depolymerization activity, and has
been used to develop a DKP-type antimicrotubule clinical
compound 10 for biological evaluation. Next, the intermediate
9 was used for the second Aldol condensation with a variety of
aromatic aldehydes 11 and 12a−12e in the presence of Cs CO
22
candidate (phase II), plinabulin (1, Figure 1). Agent 1
exhibited a potent cytotoxicity, with an IC50 value of 10 nM
level against a variety of human cancer cell lines in addition to
vascular disrupting activity. A further structure−activity
relationship (SAR) study of agent 1 was used to identify a
more potent derivative 3 (KPU-105) having a benzoyl group at
2
3
in degassed DMF under an Ar atmosphere. These aldehydes
were purchased commercially, except for 12e, which was
prepared by the reduction of the 2-cyanopyrimidine with
DIBAL-H and was used directly without purification. All
derivatives used for the biological evaluation were purified using
silica-gel column chromatography to obtain with the desired
purity (>95%), as characterized by a reversed-phase HPLC
analysis monitored at 230 nm. The chemical structures of all
derivatives were characterized by electrospray ionization time-
of-flight mass spectrometry (ESI-TOF-MS) and IR and NMR
spectroscopy (see the Supporting Information).
22
the C-3 position of the phenyl ring in agent 1. The
cytotoxicity against HT-29 (human colon adenocarcinoma)
cells and the binding dissociation constant of this benzophe-
none derivative 3 to porcine tubulin were 1.4 and 65 nM,
respectively. Its derivative 4, bearing a fluorine atom
substitution at the 4-position of the additional benzoyl group,
showed a more potent cytotoxicity (IC50 = 0.5 nM) and a
Cytotoxicities of the synthesized derivatives were evaluated
against human HT-29 colorectal cancer cells. In a previous
study, we observed that branched alkyl chains, such as the tert-
butyl group at the 5-position of the imidazole ring, were critical
23
similar binding dissociation constant (K = 72 nM).
d
The potent activities of the derivatives 3 and 4 suggested that
the benzophenone moiety (the left-hand unit of the structure)
was important for tubulin recognition. Further structural
modifications of these derivatives were, therefore, attractive
for the development of a potent DKP-derivative composed of a
new pharmacophore. In the present study, we focused on the
imidazole moiety (right-hand unit) at the opposite site of the
benzophenone moiety in the derivative 3 to synthesize a series
of new derivatives. The cytotoxicities of the compounds in this
series were evaluated to discover a new DKP pharmacophore
that displayed good antimicrotubule activity.
22
for generating potent activity in agent 1. By contrast, the
methyl derivative 2 (Figure 1), possessing a plinabulin skeleton,
showed a dramatically reduced activity (IC = 339 nM, Table
50
22
1). The imidazole moiety bearing an appropriate blanched
alkyl group was, therefore, indispensable in the right-hand unit
for tubulin recognition by the DKP-type antimicrotubule agents
derived from natural DKP, (−)-phenylahistin. The introduction
of a methyl group into the imidazole ring of the benzophenone
derivative 3 yielded the corresponding derivative 5, which,
however, retained its potent cytotoxicity (IC50 = 15 nM).
Although this cytotoxicity was a factor of 10.7 less than that of
the corresponding benzophenone derivative 3 bearing a tert-
butyl group, the activity was nearly equal to the value obtained
from agent 1. This result suggested that the quaternary carbon
(i.e., tert-butyl group) was not essential for the potent activity of
the benzophenone−DKP derivatives, probably due to the
strong interactions between the tubulin molecules and the
benzophenone moiety. Complete removal of the imidazole
As shown in Scheme 1, the new benzophenone−DKP
derivatives 5 and 6a−6e were synthesized via a tandem Aldol
22
condensation with two aldehydes onto the DKP ring. First,
the benzophenone−DKP intermediate 9 was synthesized by
the first Aldol condensation between 3-benzoylbenzaldehyde 7
and N,N′-diacetylpiperazine-2,5-dione 8 in the presence of t-
BuOK and t-BuOH in DMF under an Ar atmosphere. The
intermediate 9 was hydrolyzed in the presence of an excess
amount of 28% NH OH to afford a deacetylated reference
4
B
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ACS Medicinal Chemistry Letters
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Table 1. Cytotoxicities of the Plinabulin (1) Derivatives
against HT-29 Cells
Table 2. Cytotoxicities of the Benzophenone−DKP
Derivatives against HT-29 Cells
a
The values represent the average from two independent dose
b
response curves. The values represent the mean ± SD from at least
three independent dose response curves.
pyridine ring in derivative 6b could form a hydrogen bond to
the DKP ring, thereby retaining the pseudoplanar tricyclic
structure critical for eliciting potent antimicrotubule activity
among the DKP-type compounds. The observed lower activity
of the phenyl derivative 6a was attributed to a lack of planarity
due to repulsion between the DKP and phenyl rings. This
conformation would have precluded hydrogen bond formation
with the DKP ring. However, the introduction of a nitrogen
atom at the other sites on the pyridine ring reduced the activity.
The complete loss of activity in the 3- and 4-pyridyl derivatives
a
The values represent the mean ± SD from at least three independent
dose response curves, except for compounds 9 and 10, the IC50 values
of which were only measured once because their activities were quite
low. The IC50 values are reported in ref 22. The IC50 values are
reported in ref 23.
b
c
6c and 6d was most likely due to the unfavorable effects of the
nitrogen atom, in addition to preventing hydrogen bond
formation. The significant activity of the 2-pyrimidyl derivative
moiety from the benzophenone derivative 3 yielded the
derivative 10, which showed almost no cytotoxicity and its
N-acetylated derivative 9 showed a dramatically reduced
activity with an IC50 value on the micromolar level. Therefore,
some side-chain structure at the right-hand part would be
required to exhibit the activity. This knowledge suggests a new
route to converting the right-hand imidazole moiety into other
units while retaining the potent activity.
6e (IC50 = 75.3 nM) suggested that hydrogen bond formation
between the DKP and pyrimidine rings somewhat restored the
negative effects of the other unfavorable nitrogen atom on the
pyrimidine ring. These results suggested that the hydrogen
bond was necessary for maintaining the pseudoplanar tricyclic
structure, which was critical to the antimicrotubule activity of
the DKP-type compounds.
Encouraged by these findings, we attempted to modify the
critical imidazole moiety of derivative 3 to form other
heteroaromatic structures. A pseudoplanar tricyclic structure
formed by hydrogen bonding between the DKP-amide NH
group and the imidazole nitrogen is thought to be critical for
An X-ray crystal analysis of the potent derivative 6b, as
shown in Figure 2, revealed that the 2-pyridyl and DKP rings
retained their planarity, with a dihedral angle of only 0.99°. The
DKP−amide NH and 2-pyridyl nitrogen atoms were proximally
positioned to permit hydrogen bonding. The formation of
12−16,22
the potent activity;
therefore, the position of the
15
hydrogen bonds was also supported by the NMR analysis.
A
nitrogen atom in the aromatic ring was considered in the
substitutions of the right-hand moiety. The introduction of a
simple 2-pyridyl ring in place of the alkylated imidazole ring
yielded the derivative 6b (KPU-300), which intriguingly
showed a sufficiently high potent cytotoxicity (IC50 = 7.0
nM), twice the activity of agent 1 and the benzophenone−
methyl derivative 5. This result successfully identified a new
pharmacophore comprising a DKP-type microtubule depoly-
merization agent. The cytotoxicities of the other pyridine
derivatives 6c and 6d bearing 3- and 4-pyridyl rings,
respectively, were lost, whereas the phenyl and 2-pyrimidyl
derivatives 6a and 6e showed reduced but significant activities
(
IC50 = 94.3 and 75.3 nM, respectively) (Table 2). These
2
4
results suggested that the nitrogen atom at the 2-position of the
Figure 2. ORTEP drawing of compound 6b.
C
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ACS Medicinal Chemistry Letters
Letter
downfield shift in the DKP amide NH peak was observed in
compounds 6b and 6e (δ 12.60 and 12.02 ppm, respectively) in
the presence of the o-nitrogen atom, although the downfield
shift was not observed in 6a, 6c, and 6d (around δ 10.63 ppm),
which did not include the relevant nitrogen atom (see the
Supporting Information). Two phenyl rings in the benzophe-
none moiety, which provided new interaction sites to the
tubulin molecule, were positioned in a different plane with a
dihedral angle of 48.2° resulting from the repulsive forces
between the two hydrogen atoms positioned proximally on
their respective rings. The bending geometry was important for
the potent activity. We previously reported that a derivative of
agent 1 with a planar fluorenone moiety, in which two phenyl
rings in the benzophenone were covalently connected,
23
displayed a dramatically reduced cytotoxicity.
The effects of the potent 2-pyridyl derivative 6b on
microtubule function were investigated by evaluating this
derivative for both tubulin binding and tubulin depolymeriza-
tion activities. As shown in Table 3, compound 6b had a
Table 3. Biological Activities of Plinabulin and 6b
a
cytotoxicity IC50 (nM)
inhibition of
tubulin
tubulin
binding K
polymerization
d
a
a
compd
(μM)
IC50 (μM)
HeLa
A549
1
1.0 ± 0.5
1.8 ± 0.3
20.6 ± 3.2
35.7 ± 25.1
Plinabulin
6b
1.3 ± 0.3
1.2 ± 0.1
5.4 ± 0.4
14.2 ± 1.2
a
The values represent the mean ± SD from at least three independent
Figure 3. Effect of agent 1 and derivative 6b on microtubules in HeLa
cell. HeLa cells were treated with DMSO, derivative 6b (7 and 30
nM), and agent 1 (20, 30, and 70 nM). α-Tubulin antibody was used
to visualize microtubules, and the DNA was visualized by DAPI
staining. Microtubules and DNA are depicted in green and blue,
respectively.
dose response curves.
tubulin binding dissociation constant (K =1.3 μM) similar to
d
that of agent 1 (1.0 μM). The IC50 value of 6b (1.2 μM) for
tubulin polymerization was slightly lower than that of 1 (1.8
μM). These results suggested that the new derivative 6b
interacted with tubulin in a fashion similar to that of other DKP
antimicrotubule derivatives. The cytotoxicity of the compounds
toward other cancer cell lines was tested using HeLa and A549
cells. The derivative 6b showed a higher cytotoxicity compared
to agent 1 against both cell lines (the cytotoxicities of 6b were
factors of 3.8 and 2.5 greater than the respective values
obtained from agent 1). These results suggested that the
cytotoxicity of 6b arose mainly from the antimicrotubule
activity. Meanwhile, the cytotoxic IC50 values of derivative 6b
toward primary human cell lines (human primary fibroblasts
NHSF46 and human peripheral blood mononuclear cells
mitotic spindle in the presence of any concentration tested. At
lower concentrations of the agent 1 (20 and 30 nM) and
derivative 6b (7 nM), multipolar spindle formation was
observed, whereas at higher concentrations the cells completely
lost the ability to form spindles. Abnormal mitosis is a well-
known signature of microtubule-depolymerized cells. The
results suggested that derivative 6b mainly affected the tubulins
and inhibited their polymerization in a manner similar to that of
agent 1.
(
HMNC)) were 77 and 481 ± 73 nM, respectively, resulting
In conclusion, we designed and synthesized a series of
benzophenone−DKP antimicrotubule derivatives based on the
structure of derivative 3 and developed a potent 2-pyridyl
derivative 6b. This derivative, which possessed a pseudotricyclic
planar structure, as determined using X-ray crystal analysis,
displayed a strong cytotoxicity with IC50 values on the
nanomolar level against several human cancer cells. The
derivative 6b was found to bind strongly to tubulin and inhibit
tubulin polymerization. This function induced abnormal
mitotic spindle formation during metaphase in HeLa cells.
These results suggested that derivative 6b affected cancer cells
by obstructing the tubulin function. The 2-pyridine and
benzophenone moieties of the lead compound 6b may
potentially be modified toward the development of more
potent derivatives. Studies along these lines are in progress.
in being at least 10-fold less potent than that against HT-29
cells. This result suggested that the cancer cells were more
sensitive to derivative 6b than the normal cells.
In general, antimicrotubule agents such as colchicine and
CA-4 prevent the mitotic spindle formation. The effects of 6b
on the formation of the mitotic spindles were examined by
treating HeLa cells with agent 1 (as a positive control) and
derivative 6b over 6 h in concentrations of 20, 30, and 70 nM
(
agent 1) and 7 and 30 nM (derivative 6b). After treatment, the
microtubules and chromosomes were stained with an anti-α-
tubulin antibody and 4,6-diamidino-2-phenylindole (DAPI),
respectively. As shown in Figure 3, the untreated cells formed
dipolar mitotic spindles with condensed chromosomes aligned
at the metaphase plate during metaphase. By contrast, cells
treated with agent 1 and derivative 6b could not form a normal
D
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ACS Medicinal Chemistry Letters
Letter
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AUTHOR INFORMATION
■
*
3
(
Corresponding Author
(Yoshio H.) E-mail: yhayashi@toyaku.ac.jp. Tel: +81-42-676-
275. Fax: +81-42-676-3275.
Uno, I. Antitumor activity of phenylahistin in vitro and in vivo. Biosci.,
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Funding
Y. Synthesis and biological activities of phenylahistin derivatives.
This work was supported by Japan Society for the Promotion of
Science (JSPS), KAKENHI including a Grant-in-Aid for
Scientific Research (B) 23390029 and 23390427, and Platform
for Drug Discovery.
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Notes
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The authors declare no competing financial interest.
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(19) Pilat, M. J.; LoRusso, P. M. Vascular disrupting agents. J. Cell.
ACKNOWLEDGMENTS
■
Biochem. 2006, 99, 1021−1039.
The authors acknowledge Mr. H. Fukaya and Mr. Y. Shimura of
the Tokyo University of Pharmacy and Life Sciences for mass
spectra and crystallographic measurements. The authors thank
Mr. Yuki Shinozaki and Dr. Yuri Yamazaki, Tokyo University of
Pharmacy and Life Sciences, for technical assistance and for
useful discussion, respectively, and Professor Hiroyuki Yasui,
Kyoto Pharmaceutical University, for technical advice regarding
the binding dissociation constant.
(20) Daenen, L. G. M.; Roodhart, J. M. L.; Shaked, Y.; Voest, E. E.
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(22) Yamazaki, Y.; Tanaka, K.; Nicholson, B.; Deyanat-Yazdi, G.;
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Yasui, H.; Akamatsu, M.; Chinen, T.; Usui, T.; Shinozaki, Y.; Yakushiji,
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ABBREVIATIONS
■
VDA, vascular disrupting agent; DKP, diketopiperazine; SAR,
structure−activity relationship; CA-4, combretastatin A4;
DAPI, 4,6-diamidino-2-phenylindole
(23) Yamazaki, Y.; Sumikura, M.; Masuda, Y.; Hayashi, Y.; Yasui, H.;
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activity relationships of benzophenone-bearing diketopiperazine type
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