Tubulin photoaffinity labeling study with a plinabulin chemical probe possessing a biotin tag at the oxazole

Article abstract of DOI:10.1016/j.bmc.2010.10.055

A new bioactive photoaffinity probe KPU-252-B1 (4) possessing a biotin tag on the oxazole ring of a potent plinabulin derivative KPU-244 (2) was synthesized via the Cu^I-catalyzed Huisgen's cycloaddition reaction to understand the precise binding mode of the diketopiperazine-based anti-microtubule agent plinabulin on tubulin. Probe 4 showed significant binding affinity toward tubulin and cytotoxicity against an HT-29 cells. A photoaffinity labeling study suggested that probe 4 specifically recognizes tubulin at a binding site that binds plinabulin or colchicine, most likely near or at the colchicine binding site, which is located at the interfacial region formed by α-and β-tubulin association. The results also demonstrated that probe 4 may serve as a useful plinabulin chemical probe to investigate the molecular mechanism by which anti-microtubule diketopiperazine derivatives operate.

Full text of DOI:10.1016/j.bmc.2010.10.055

Bioorganic & Medicinal Chemistry 19 (2011) 595–602
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tubulin photoaffinity labeling study with a plinabulin chemical probe
possessing a biotin tag at the oxazole
Yuri Yamazaki a, Yui Kido a, Koushi Hidaka b, Hiroyuki Yasui c, Yoshiaki Kiso b, Fumika Yakushiji a,
Yoshio Hayashi a,
⇑
a Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
b Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
c Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new bioactive photoaffinity probe KPU-252-B1 (4) possessing a biotin tag on the oxazole ring of a
potent plinabulin derivative KPU-244 (2) was synthesized via the CuI-catalyzed Huisgen’s cycloaddition
reaction to understand the precise binding mode of the diketopiperazine-based anti-microtubule agent
plinabulin on tubulin. Probe 4 showed significant binding affinity toward tubulin and cytotoxicity against
an HT-29 cells. A photoaffinity labeling study suggested that probe 4 specifically recognizes tubulin at a
binding site that binds plinabulin or colchicine, most likely near or at the colchicine binding site, which is
Received 25 September 2010
Revised 23 October 2010
Accepted 26 October 2010
Available online 31 October 2010
Keywords:
located at the interfacial region formed by
a-and b-tubulin association. The results also demonstrated
Anti-cancer agent
Diketopiperazine
Phenylahistin
that probe 4 may serve as a useful plinabulin chemical probe to investigate the molecular mechanism
by which anti-microtubule diketopiperazine derivatives operate.
Ó 2010 Elsevier Ltd. All rights reserved.
Tubulin
Vascular disrupting agent
1. Introduction
‘Plinabulin’ (NPI-2358/KPU-2, 1, IC50 of 15 nM against HT-29
cells, Fig. 1), designed and synthesized previously by us from a nat-
Microtubules, which are key components of the cytoskeleton,
are noncovalent polymers of - and b-tubulin heterodimers (each
ural diketopiperazine phenylahistin (PLH, halimide),5–9 is a potent
VDA.10 Plinabulin is currently being evaluated in Phase II clinical
trials in four countries, including the United States. Although PLH
exhibits colchicine-like tubulin depolymerizing activity, the chem-
ical structure of plinabulin is different from natural colchicine
binding molecules: it has a relatively hydrophilic diketopiperazine
(DKP) skeleton. Computer-assisted molecular models of plinabulin
and colchicine are not superimposable.11 Moreover, several biolog-
ical assays have shown that plinabilin displays properties that are
distinct from those of colchicine.10 Hence, it was attractive to ex-
plore the precise binding mode and microtubule depolymerization
mechanism of our dehydroDKP-type inhibitor, plinabulin.
a
with a molecular mass of about 50 kDa) that are assembled in a fil-
amentous tube-shaped structure essential to all eukaryotic cells.1
The cytoskeletal system of eukaryotic cells is an attractive target
for anti-cancer chemotherapeutic agents.2,3 Microtubule targeting
agents are generally classified into two main groups. One group,
the microtubule-stabilizing agents, stimulates microtubule poly-
merization and includes paclitaxel, dosetaxel, and epothilones.
The other group comprises microtubule depolymerizing agents.
These compounds inhibit microtubule polymerization and include
the Vinka alkaloids, colchicine and combretastatins.2 In particular,
colchicine and combretastatin, which recognize the colchicine
binding site on tubulin, induce tumor vascular collapse by rapid
depolymerization of microtubules in highly proliferating tumor
selective vascular endothelial cells.2 These agents are designated
‘vascular disrupting agents’ (VDA) and form a promising new class
of anti-cancer drugs which offers the attractive possibility of
inducing responses in all solid tumor types in combination
therapies.4
In the previous study,11,12 we demonstrated that plinabulin 1
directly interacted to tubulin with the Kd value of 1.1 lM in the
binding assay based on the intrinsic fluorescence quenching of por-
cine tubulin. To understand the binding mode of plinabulin on
tubulin, we performed a photoaffinity labeling study13 using
biotin-tagged photoaffinity probes derived from a benzophenone-
containing potent derivative KPU-244 (2, IC50 of 4 nM against
HT-29 cells, Fig. 1). We previously synthesized several photo-
affinity probes possessing a biotin tag at the 40-position of the
benzophenone moiety of compound 2.11,12 Photoaffinity labeling
studies of these probes and molecular dynamics (MD) simulations
suggested that plinabulin derivatives interact with the boundary
⇑
Corresponding author. Tel./fax: +81 42 676 3275.
E-mail address: yhayashi@toyaku.ac.jp (Y. Hayashi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.055

596
Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
O
O
O
H
N
O
O
O
O
N
H
HN
O
HN
HN
NH
N
O
NH
NH
N
NH
N
O
O
O
O
O
H
N
H
N
H
Plinabulin (1)
KPU-244 (2)
KPU-244-B2 (3)
NH
S
H
O
H3CO
H3CO
CH3
O
NH
NH
H
HN
H
O
H3CO
H
N
H
N
O
OCH3
N
H
S
O
O
Colchicine
N
N
N
O
O
O
HN
HN
NH
H
O
H
O
NH
N
H
H
N
N
N
H
S
O
O
O
O
KPU-252-B1 (4)
5
Figure 1. Structures of colchicine, plinabulin (1, NPI-2358/KPU-2), KPU-244 (2), KPU-244-B2 (3), a new photoaffinity probe KPU-252-B1 (4), and control compound 5.
region between the
the colchicine binding pocket but are not located inside the colchi-
cine binding site. MD simulations indicated the presence of an
interfacial pocket formed by
a
- and b-tubulin units at sites that surround
probe 4. After biological evaluation of the probe 4, tubulin photo-
affinity labeling was performed with or without photoirradiation
to investigate the binding mode of the probe. The results indicated
that probe 4 was a good chemical probe and behaved similarly to
our previous probes.
a- and b-tubulin that could accept a
biotin tag positioned at the oxazole ring of compound 2, which is
located at the opposite side of the benzophenone moiety. Based
on these findings, we designed a new plinabulin chemical probe
4 containing a biotin tag at the tert-butyl group on the oxazole ring
of compound 2. If the MD simulation were accurate, the newly de-
signed probe 4 would function in a manner similar to that of our
previous probes. In the synthesis of the new probe 4, we coupled
the biotin tag to the plinabulin derivative using the CuI-catalyzed
Huisgen’s 1,3-dipolar cycloaddition reactions developed by Sharp-
less and co-workers (click chemistry).14,15 According to the synthesis
shown in Scheme 3, we synthesized the plinabulin derivative 17,
which possessed a terminal alkyne, and the biotinyl azide 20.
Coupling of these compounds successfully yielded the desired
2. Results and discussion
2.1. Chemistry
To prepare the photoaffinity probe 4, we first synthesized 5-
(2,2-dimethylpent-4-yn)oxazole-4-carboaldehyde 10 from methyl
isobutyrate 6 in six steps, as described in Scheme 1. After the start-
ing material 6 was treated with lithium diisopropylamide (LDA) at
ꢀ78 °C, 3-bromopropyne was introduced to an
a-carbon of com-
pound 6 to give the ester 7.16 The ester 7 was hydrolyzed under
O
O
c, d
e, f
O
a
b
O
O
EtO
H
O
O
O
OH
N
N
O
6
7
8
9
10
Scheme 1. Synthesis of 5-(2-methylpent-4-yn-2-yl)oxazole-4-carboaldehyde 10. Reagents and conditions: (a) 3-bromopropyne, LDA, THF, ꢀ78 °C to rt, 59%; (b) 1 M NaOH,
CH3OH, 65 °C, 92%; (c) DCC, CH2Cl2, rt; (d) ethyl isocyanoacetate, DBU, THF, rt, 69% over two steps; (e) LiAlH4, THF, ꢀ78 °C; (f) MnO2, acetone, rt, 50% over two steps.
O
O
O
OH
O
O
O
COOH
O
a
b
c
N
H
H
11
12
13
14
Scheme 2. Synthesis of 3-benzoylbenzaldehyde 14. Reagents and conditions: (a) MeONHMeꢁHCl, EDCꢁHCl, Et3N, DMF, rt, 86%; (b) LiAlH4, THF, ꢀ78 °C, 98%; (c) Dess–Martin
periodinane, THF, rt, 87%.

Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
597
O
O
O
Ac
N
Ac
a
b
N
HN
N
OHC
+
O
O
O
Ac
NH
N
NH
N
O
O
N
O
O
O
15
10
16
17
d
4
O
HN
HN
NH
H
NH
H
H
O
O
c
H
N
H
N
H
N
H
N3
HO
N
H
N
S
S
3
3
3
3
H
O
O
O
O
18
20
Scheme 3. Synthesis of photoaffinity probe 4. Reagents and conditions: (a) Cs2CO3, DMF, rt, 57%; (b) 14, Cs2CO3, DMF, 60 °C, 61%; (c) 3-azido-propylamine 19, EDCꢁHCl,
HOBtꢁH2O, DMF/DMSO, rt, then HPLC purification, 25%; (d) 5 mol % Cu(OAc)2, 15 mol % sodium ascorbate, H2O/t-butanol/DMF, MW, 80 °C, 15 min, then HPLC purification,
63%.
basic conditions to afford the corresponding carboxylic acid 8.
After the carboxylic acid 8 was converted to the corresponding
anhydride with N,N0-dicyclohexylcarbodiimide (DCC), the resultant
anhydride and ethyl isocyanoacetate were condensed in the pres-
ence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford oxazol-
ecarboxylate 9.17 This ester 9 was subsequently reduced with
LiAlH4 to yield the corresponding oxazole carbinol, followed by
oxidation with MnO2 to give the desired aldehyde 10.
Scheme 2 shows that compound 17 was synthesized from
3-benzoylbenzaldehyde 14 as follows: the purchased benzoylben-
zoic acid 11 was converted to the corresponding Weinreb amide
12, followed by reduction with LiAlH4 to obtain benzhydryl alcohol
13. This alcohol 13 was then oxidized with Dess–Martin period-
inane18 to yield the desired aldehyde 14.
To obtain the biotin-tagged derivative 4 as a photoaffinity
probe, we separately synthesized two units, namely, DKP part 17
and biotinyl azide 20, which were then conjugated by Huisgen’s
cycloaddition, as described in Scheme 3. During synthesis of the
DKP part 17 core structure of the anti-microtubule activity, oxazol-
ecarboxaldehyde 10 was condensed to N,N-diacetyl piperazine-
2,5-dione 15 in the presence of Cs2CO3 in N,N-dimethylformamide
(DMF) under an argon atmosphere19 to give mono-dehydroDKP 16.
The second aldehyde 14 was introduced, by a similar aldol reac-
tion, to mono-dehydroDKP 16 to afford alkynyl di-dehydroDKP
17. In the synthesis of the linker part, biotin-Acp-Acp-OH 18
(Acp: aminocaproyl) was prepared by Fmoc-based solid phase
chemistry using the procedure described previously.12 3-Azido
propylamine 19 was prepared from 3-bromo-1-propylamine
hydrobromide by azidation.20 Azidation to yield compound 19
Compound 5 (Fig. 1), a negative control compound for the
photoaffinity labeling study, was also prepared. Synthesis of this
compound has been described previously.11
2.2. Biological activity of probe 4
The biological activity of the synthetic photoaffinity probe 4 was
evaluated first by performing a binding assay against purified por-
cine tubulin (0.5
ation constant (Kd) of the probe 4 was calculated to be 17.9
(Fig. 2). The binding affinity of probe 4 was about 17 times and
5 times less than the binding affinities of plinabulin 1 (1.06
M)12
and colchicine (3.70 M, Fig. S1 in the Supplementary data), respec-
l
M) in MES buffer (pH 6.8) at 37 °C.22 The dissoci-
l
M
l
l
tively. Compound 5, the negative control, did not bind to tubulin at
all (Fig. S2 in the Supplementary data). The binding affinity of probe
4 was found to be significant. Next, an in vitro cytotoxicity assay
was performed based on the XTT/PMS method.23 In this method,
probe 4 showed low but significant cytotoxicity against the HT-29
human colon cancer cell lines with an IC50 value of 9.8 lM (Table 1),
725 times less potent than plinabulin 1 (13.5 nM, Table 1). The
higher molecular weight and greater hydrophilicity of probe 4
probably resulted in poor cellular uptake, which reduced the cyto-
toxicity. These findings together indicated that probe 4 may be
valuable in a subsequent photoaffinity labeling study.
2.3. Tubulin photoaffinity labeling
The binding site of probe 4 was investigated by photoaffinity
labeling to tubulin. Purified porcine tubulin was photo-irradiated
at 365 nm in the presence or absence of probe 4 on ice after incuba-
tion at 37 °C in MES buffer (containing 1 mM GTP, pH 6.8) for the
appropriate duration. The sample was then separated by SDS–PAGE
using a 10% polyacrylamide gel. The protein was transferred to a
PVDF membrane, and the photolabeled protein, to which was
attached probe 4, was visualized using a streptavidin–HRP/ECL sys-
tem. Non-specific binding was very low in the absence of photo-
irradiation (lanes 1 and 9 in Fig. 3), suggesting that chemical
modification of tubulin through covalent bond formation (e.g.,
Michael addition reaction) with probe 4 did not occur. In the
photo-irradiated samples, UV irradiation time- or dose-dependent
labeling was observed (lanes 2–4 and 10–12 in Fig. 3, respectively).
Non-specific photolabeling by the benzophenone moiety on probe 4
was assessed by photoaffinity labeling with compound 5, which did
not show any binding activity to tubulin in the binding assay
was confirmed by observation of
a strong absorption at
2098 cmꢀ1 in the FT-IR spectrum. Coupling between the biotinyl
peptide 18 and azido propylamine 19 was performed using the
EDC–HOBt method, and the crude products were purified by pre-
parative HPLC to afford the desired biotinyl azide 20.
Finally, Huisgen’s cycloaddition was performed between the
alkynyl didehydroDKP 17 and the biotinyl azide 20 in the presence
of 5 mol % Cu(OAc)2 and 15 mol % sodium ascorbate in water/
t-butanol/DMF under microwave irradiation to shorten the reac-
tion time and increase the chemical yield.21 After microwave irra-
diation, a new single peak was detected in the reverse phase HPLC
analysis. Thus, the reaction mixture was purified by preparative
HPLC to afford the desired probe 4 in 63% yield. The observed rel-
atively low yield in the CuI-catalyzed cycloaddition was due to the
low solubility of the biotinyl azide 20.

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Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
as a negative control (5–100 equiv, lanes 17–20 in Fig. 3C). This re-
sult was consistent with a previous report that PLH, an original lead
compound of plinabulin 1, competed with [3H]colchicine for tubu-
lin.6 When vinblastine, an anti-microtubule agent that recognizes a
binding site (the vinblastine site) that is distinct from the colchicine
binding site, was used in high concentration as a competitor, mod-
erate inhibition was observed (lanes 26–28 in Fig. 3D). This could be
attributed to an allosteric effect of vinblastine binding to its own
binding site, which induces small conformational changes at the
colchicine binding site,24 or a vinblastine-induced paracrystal for-
mation of tubulin,25 leading to the inhibition of probe 4 binding.
Therefore, it has been comprehensively demonstrated that plinabu-
lin and colchicine recognized the probe 4 binding site, suggesting
that the new probe 4 may be used effectively for investigating the
properties of the plinabulin binding site. In our previous study,
we developed other plinabulin chemical probes KPU-244-B2 (3)
and -B3, which featured a biotin tag at the benzophenone moiety,
in contrast with probe 4, which features a biotin tag at the opposite
oxazole side.11,12 In both cases, it was concluded that these chemi-
cal probes recognized tubulin in a similar manner.
In consideration for the fact that the probes possessing biotin
tags at different positions on a common structure may recognize
tubulin in a similar manner, it was speculated that the interfacial
structure formed by the a- and b-tubulin subunits included a space
for accepting not only the DKP core structure, but also the biotin
linkers located on different parts of the tubulin. The binding modes
of KPU-244-B2 (3) and -B3 were previously analyzed in a docking
study.12 Hence, in the present study, the binding mode of probe 4
at the colchicine binding site was assessed by performing a similar
docking study using the molecular modeling package MOE 2009. 10
(Chemical Computing Group, Inc., Montreal, Canada). Tubulin pro-
teins were modeled according to the crystallographic data of the
complex with colchicine (PDB ID, 1SA0). Because probe 4 success-
fully formed a photolabel and exhibited functions similar to probe
3, probe 4 was assumed to dock proximal to the colchicine binding
site of the tubulin dimer, and initial conformation of 4 was built
using previous result of 2.12 Next, an energy minimization routine
and MD simulation were successively performed. In the obtained
model, probe 4 was found to be positioned at the boundary region
between the a- and b-tubulin subunits, a region that partially over-
Figure 2. Tubulin binding assay based on fluorescence quenching. (A) Fluorescence
quenching on tubulin by probe 4. (B) Increase of tubulin-probe 4 binding complex.
lapped with the colchicine binding pocket (Fig. 4). The 2,2-dimethyl
group on probe 4 covered the colchicine binding pocket, indicated
in green on the molecular surface shown in Fig. 4, and the benzo-
phenone moiety was situated slightly closer to the b-tubulin than
in the case of probe 3 docking. In addition, the model suggested that
the biotin linker on probe 4 protruded from the interfacial region
away from the tubulin molecule through the interfacial space
Table 1
Tubulin binding activity and cytotoxicity of compounds
a
b
Compounds
Kd
(lM)
IC50 (nM)
Plinabulin 1
Probe 4
Colchicine
1.06 0.18
17.9 3.4
3.70 0.18
13.5 2.2
9788 1680
16.6 0.9
formed between the
a- and b-subunits, although the orientation
of the biotin linker on probe 4 differed from that of probe 3. There-
fore, probe 4 appears to specifically recognize tubulin and functions
as an effective chemical probe in a manner similar to that of probe
3. Our anti-microtubule diketopiperazine derivatives may bind at
a
Porcine tubulin concentration: 0.5
IC50 against HT-29 cells. Experiments were triplicated.
l
M.
b
described above. In this case, only weakly stained bands were de-
tected, in contrast with the case of probe 4 labeling, and the staining
was not UV irradiation-time-dependent. It would appear that the ob-
served weak bands could be attributed to non-specific photolabeling.
A competitive photoaffinity labeling study using probe 4 was
carried out in the presence or absence of plinabulin 1, colchicine,
the boundary region formed between the a- and b-tubulin subunits
around the colchicine binding site, but not inside the site, to effec-
tively disrupt the interaction between the two tubulin subunits,
thereby producing microtubule depolymerization. Further analysis
using the photoaffinity probes is now under way to identify the
modified amino acid residues that react with the photoaffinity
probe. These findings would help us understand the mechanism
of binding between plinabulin derivatives and tubulin.
vinblastine, or D-biotin to elucidate the binding mode of probe 4
on tubulin (Fig. 3 and densitometry analysis is shown in Fig. S6, Sup-
plementary data). Dose-dependent inhibition of photoaffinity label-
ing was observed in the presence of plinabulin 1 (1–50 equiv, lanes
21–24 in Fig. 3D), indicating that probe 4 recognized the same bind-
ing site as plinabulin 1. Photoaffinity labeling with probe 4 was also
inhibited dose-dependently (5–100 equiv) in the presence of col-
3. Conclusion
We designed and synthesized a new bioactive photoaffinity
probe KPU-252-B1 (4) possessing a biotin tag on the oxazole ring
chicine (lanes 13–16 in Fig. 3C) but not in the presence of D-biotin

Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
599
Figure 3. Photoaffinity labeling of tubulin. (A) Photoaffinity labeling with probe 4 (2
with probe 4 or compound 5 at 37 °C in MES buffer, followed by photo-irradiation for the appropriate time (0–30 s). (B) Photoaffinity labeling in the presence of different
concentrations of probe 4 (1–4 M). (C) Photoaffinity labeling with probe 4 (2 M) in the absence (lanes 13 and 17) or presence (10–200 M, lanes 14–16) of colchicine or
-biotin (10–200 M, lanes 18–20). Samples were photo-irradiated for 30 s. (D) Photoaffinity labeling with probe 4 (2 M) in the absence (lanes 21 and 25) or presence of
pliabulin 1 (2–100 M, lanes 22–24) or vinblastine (10–200 M, lanes 26–28). Samples were photo-irradiated for 30 s.
lM) or compound 5 (2 lM) at different irradiation times. Porcine tubulin was incubated
l
l
l
D
l
l
l
l
of a potent plinabulin derivative KPU-244 (2) via the CuI-catalyzed
Huisgen’s cycloaddition reaction. Probe 4 exhibited significant
binding affinity toward tubulin and cytotoxicity against an HT-29
cells. Furthermore, probe 4 photolabeled tubulin in a dose- or UV
irradiation-time-dependent manner, and labeling was inhibited
by addition of colchicine or plinabulin. These results revealed that
probe 4 serves as an effective chemical probe of our anti-microtu-
bule diketopiperazines, and its recognition site is located around
the intradimer space between the
a- and b-tubulin units, near
the colchicine binding site. These results are consistent with our
previous findings based on a photoaffinity labeling study using
other plinabulin chemical probes. Therefore, our synthetic probes
may contribute to an understanding of the tubulin depolymeriza-
tion activity of diketopiperazine-based anti-microtubule agent,
plinabulin.
4. Experimental
4.1. General
Reagents and solvents were purchased from Wako Pure Chem-
ical Ind., Ltd (Osaka, Japan), Nakalai Tesque (Kyoto, Japan), and Al-
drich Chemical Co. Inc. (Milwaukee, WI, USA) and were used
without further purification. Porcine tubulin was purchased from
Cytoskeleton, Inc. (Denver, Colorado, USA). All other chemicals
were of the highest commercially available purity. Analytical
thin-layer chromatography (TLC) was performed on Merck silica
gel 60F254 pre-coated plates. Preparative HPLC was performed
using a C18 reverse-phase column (19 ꢂ 100 mm; SunFire™ Prep
C18 OBD™ 5 lm) with a binary solvent system: a linear gradient
of CH3CN in 0.1% aqueous TFA at a flow rate of 10 mL/min, detected
at UV 230 nm and 365 nm. Solvents used for HPLC were HPLC-
grade. 1H and 13C NMR spectra were recorded on either a JEOL
JNM-AL300 or a Varian Mercury 300 spectrometers at 300 and
75 MHz, or a BRUKER AV600 spectrometer at 600 MHz. Chemical
shifts were recorded as d values in parts per million (ppm) down-
field from tetramethylsilane (TMS). High-resolution mass spectra
(EI, CI) were recorded on a JEOL JMS-GCmate, and high-resolution
mass spectra (ESI) were recorded on a micromass Q-Tof Ultima API.
Photoaffinity labeling was performed using UV Spot Light Source
L9588-01 (Hamamatsu Photonics).
Figure 4. Superimposition of molecular dynamics simulated probe 4 (purple sticks)
and probe 3 (KPU-244-B2, white sticks) in a tubulin heterodimer constructed from
KPU-244 (2, yellow sticks). (A) Full view of probe 4.
a- and b-Tubulin are
represented by the magenta and green ribbons, respectively. The colchicine binding
site on b-tubulin is represented by the green surface. (B) Close-up view of the
binding area.

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Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
4.2. Synthesis of compounds 4, 7–10, 12–14, 16, 17, 19, and 20
4.2.1. Methyl 2,2-dimethyl pent-4-ynoate (7) [CAS No. 86101-49-
34 mmol), and the mixture was stirred at room temperature over-
night. After the MnO2 was removed by filtering, the solvent was re-
moved in vacuo and the residual oil was purified by silica gel
column chromatography using hexane/EtOAc (4:1–3:1) as an elu-
ent to give a yellow oil of compound 10 (530 mg, 50%). 1H NMR
(300 MHz, CDCl3) d 1.54 (s, 6H), 1.94 (t, 1H, J = 2.6 Hz), 2.72 (d,
2H, J = 2.6 Hz), 7.80 (s, 1H), 10.07 (s, 1H); HRMS (ESI): m/z
178.0856 (M+H+) (calcd for C10H12NO2: 178.0868).
16
7]
To a solution of methyl isobutyrate (8 g, 78 mmol) in anhydrous
THF (50 mL) was added dropwise a 1.8 M solution of LDA solution
in THF (47 mL, 85.8 mmol) at ꢀ78 °C under an argon atmosphere,
and the mixture was stirred for 1 h at the same temperature. To
this solution was then slowly added a solution of 3-bromopropyne
(9.7 g, 82 mmol) in anhydrous THF (8 mL) at ꢀ78 °C under an ar-
gon atmosphere. The cooling bath was removed, the mixture was
stirred overnight at room temperature, and the reaction mixture
was quenched with water (50 mL). The organic layer was sepa-
rated, and the aqueous layer was extracted once with Et2O. The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. Distillation of the residual oil
at 55–60 °C (20 mmHg) gave a colorless oil (6.5 g, 59%) of com-
pound 7. 1H NMR (300 MHz, CDCl3) d 1.28 (s, 6H), 2.01 (t, 1H,
J = 2.6 Hz), 2.45 (d, 2H, J = 2.6 Hz), 3.70 (s, 3H).
4.2.5. 3-Benzoyl-N-methoxy-N-methylbenzamide (12)
To the solution of 3-benzoylbenzoic acid 11 (850 mg,
3.76 mmol) in DMF (40 mL) were added N,O-dimethyl hydroxyl-
amine hydrochloride (385 mg, 3.95 mmol), Et3N (0.55 mL,
3.95 mmol), and EDCꢁHCl (757 mg, 3.95 mmol). After the mixture
was stirred for 5 h at room temperature, the solvent was removed
in vacuo and the residue was dissolved in EtOAc, washed with 10%
citric acid, 10% NaHCO3, and brine, dried over Na2SO4. The solvent
was removed in vacuo to give a colorless oil of compound 12
(0.95 g, 86%). 1H NMR (300 MHz, CDCl3) d 3.37 (s, 3H), 3.56 (s,
3H), 7.46–7.60 (m, 4H), 7.78–7.81 (m, 2H), 7.91 (dd, 2H, J = 1.6,
7.5 Hz), 8.09 (t, 1H, J = 1.5 Hz); 13C NMR (75.5 MHz, CDCl3) d
33.5, 61.2, 128.2, 128.4, 129.7, 130.0, 131.9, 132.0, 132.7, 134.2,
137.1, 137.4, 168.9, 196.0; HRMS (EI): m/z 269.1045 (M+) (calcd
for C16H15NO3: 269.1052).
4.2.2. 2,2-Dimethyl pent-4-ynoic acid (8)
A suspension of compound 7 (1.0 g, 7.13 mmol) in methanol
(10 mL) and 1 M NaOH (11 mL) was stirred for 4 h at 60 °C. After
cooling to room temperature, the reaction mixture was acidified
by addition of excess 6 M HCl and extracted five times with Et2O.
The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo to give a colorless oil of com-
pound 16 (0.83 g, 92%). 1H NMR (300 MHz, CDCl3) d 1.32 (s, 6H),
2.04 (t, 1H, J = 2.6 Hz), 2.47 (d, 2H, J = 2.6 Hz).
4.2.6. 3-(Hydroxy(phenyl)methyl)benzaldehyde (13)
To a solution of Weinreb amide 12 (505 mg, 1.87 mmol) in
anhydrous THF (15 mL) was added portionwise LiAlH4 (85.4 mg,
2.25 mmol) at ꢀ78 °C under an argon atmosphere, and the mixture
was stirred for 2.5 h at the same temperature. After the mixture
was quenched with H2O at ꢀ78 °C, EtOAc was added and the
resulting precipitate was removed by Celite filtration. The filtrate
was washed with H2O and brine, dried over Na2SO4, and concen-
trated in vacuo to give an oil of compound 13 (390 mg, 98%). 1H
NMR (300 MHz, CDCl3) d 2.39 (d, 1H), 5.92 (d, 1H), 7.27–7.37 (m,
5H), 7.49 (t, 1H, J = 7.7 Hz), 7.66 (d, 1H, J = 7.7 Hz), 7.78 (d, 1H,
J = 7.7 Hz), 7.92 (s, 1H), 9.98 (s, 1H); 13C NMR (75.5 MHz, CDCl3)
d 75.7, 126.6, 127.6, 128.0, 128.8, 129.2, 132.5, 136.5, 143.2,
144.9, 192.3; HRMS (CI): m/z 213.0924 (M+H)+ (calcd for
4.2.3. Ethyl 5-(2-methylpent-4-yn-2-yl)oxazole-4-carboxylate (9)
To a solution of compound 8 (0.8 g, 6.34 mmol) in anhydrous
dichloromethane (4 mL) was added DCC (654 mg, 3.17 mol), and
the mixture was stirred for 4 h at room temperature. After dicyclo-
hexylurea was filtered off in a glass filter and rinsed with a small
amount of dichloromethane, the organic solvent was concentrated
in vacuo and the resulting DCU was removed on a glass filter. The
filtrate was concentrated in vacuo to give a yellow oil of the corre-
sponding anhydride (0.61 g, 82%). This oil was used in the next
reaction without further purification. According to the report by
Suzuki et al.,17 to the solution of ethyl isocyanoacetate (271 mg,
2.4 mmol) in anhydrous THF (9 mL) were added DBU (365 mg,
2.4 mmol) and the anhydride (0.56 g, 2.4 mmol), and the mixture
was stirred at room temperature overnight. After the solvent was
removed in vacuo, the residue was extracted with EtOAc, washed
with 10% Na2CO3, 10% citric acid, and brine, dried over Na2SO4,
and concentrated in vacuo. The residual oil was purified by silica
gel column chromatography using hexane/EtOAc (6:1–5:1) as the
eluent to give a colorless oil of compound 9 (367 mg, 69%). 1H
NMR (300 MHz, CDCl3) d 1.42 (t, 3H, J = 7.1 Hz), 1.54 (s, 6H), 1.90
(t, 1H, J = 2.6 Hz), 2.83 (d, 2H, J = 2.6 Hz), 4.39 (q, 2H, J = 7.1 Hz),
7.75 (s, 1H); HRMS (ESI): m/z 222.1124 (M+H+) (calcd for
C14H13O2: 213.0915).
4.2.7. 3-Benzoylbenzaldehyde (14)
To a solution of aldehyde 13 (102 mg, 0.48 mmol) in THF (5 mL)
was added Dess–Martin periodinane (305 mg, 0.72 mmol), and the
mixture was stirred for 2 h at room temperature. After the mixture
was quenched by the addition of satd NaHCO3 (1.5 mL) and satd
Na2S2O3 (1.5 mL), EtOAc was added, and the organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residual oil was purified by silica gel column chromatography
using hexane/EtOAc (4:1) as an eluent to give a colorless oil of
compound 14 (88 mg, 87%). 1H NMR (300 MHz, CDCl3) d 7.51
(dd, 2H, J = 7.7, 7.1 Hz), 7.61–7.71 (m, 2H), 7.79–7.83 (m, 2H),
8.07–8.13 (m, 2H), 8.28 (t, 1H, J = 1.6 Hz), 10.09 (s, 1H); 13C NMR
(75.5 MHz, CDCl3) d 128.6, 129.2, 130.0, 131.3, 132.6, 133.0,
135.4, 136.3, 136.8, 138.5, 191.4, 195.4; HRMS (EI): m/z 210.0684
(M+) (calcd for C14H10O2: 210.0681).
C12H16NO3: 222.1130).
4.2.4. 5-(2-Methylpent-4-yn-2-yl)oxazole-4-carboaldehyde (10)
To a solution of ester 9 (1.31 g, 6.0 mol) in anhydrous THF
(50 mL) was added portionwise LiAlH4 (225 mg, 6.0 mmol) at
ꢀ78 °C under an argon atmosphere, and the bath temperature
was gradually increased to ꢀ35 °C with stirring for 2.5 h. After
the mixture was quenched with satd NH4Cl at ꢀ78 °C, EtOAc was
added and the resulting precipitate was removed by Celite filtra-
tion. The filtrate was washed with H2O and brine, dried over
Na2SO4, and concentrated in vacuo to give an oil of the correspond-
ing oxazole alcohol (1.22 g). This oil was used in the next oxidation
without further purification. To a solution of the oxazole alcohol
(1.22 g, 6.8 mmol) in acetone (54 mL) was added MnO2 (3.0 g,
4.2.8. (Z)-1-Acetyl-3-((5-(2-methylpent-4-yn-2yl)oxazol-4-
yl)methylene)piperazine-2,5-dione (16)
To
a solution of aldehyde 10 (46.5 mg, 0.262 mmol) in
anhydrous DMF (5 mL) was added N,N0-diacetyl-2,5-diketopiperaz-
inedione (15, 78 mg, 0.394 mmol). The solution was repeatedly
evacuated for short periods of time to remove oxygen, then flushed
with argon. To this solution was added Cs2CO3, and the evacua-
tion–flushing process was repeated again. An argon atmosphere
was maintained throughout the reaction. The resultant mixture

Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
601
was stirred at room temperature overnight. After the solvent was
removed in vacuo, the residue was extracted with EtOAc, washed
with H2O and brine, dried over Na2SO4, and concentrated in vacuo.
The residual oil was purified by silica gel column chromatography
using CHCl3/MeOH (100:1) as an eluent to give a pale yellow pow-
der of compound 16 (47 mg, 57%). 1H NMR (300 MHz, CDCl3) d 1.55
(s, 6H), 2.00 (t, 1H, J = 2.6 Hz), 2.59 (d, 2H, J = 2.6 Hz), 2.65 (s, 3H),
4.48 (s, 2H), 7.08 (s, 1H), 7.87 (s, 1H), 11.23 (br s, 1H); HRMS (ESI):
m/z 316.1300 (M+H+) (calcd for C16H18N3O4: 316.1297).
and purified by preparative HPLC (with a linear gradient of 35–
80% CH3CN in 0.1% aq TFA over 40 min) to give a pale yellow
powder of the desired probe 4 (3.0 mg, 63%). 1H NMR (600 MHz,
DMSO-d6) d 1.15–1.37 (m, 10H), 1.42 (s, 6H), 1.42–1.52 (m, 7H),
1.57–1.63 (m, 1H), 1.79–1.84 (m, 2H), 1.99–2.04 (m, 6H), 2.57 (d,
1H, J = 12 Hz), 2.81 (dd, 1H, J = 5.2, 12 Hz), 2.92 (q, 2H, J = 6.0 Hz),
2.96–3.00 (m, 6H), 3.06–3.10 (m, 1H), 4.12 (dd, 1H, J = 4.4,
7.7 Hz), 4.23 (t, 2H, J = 6.9 Hz), 4.30 (dd, 1H, J = 5.0, 7.5 Hz), 6.36
(br s, 1H), 6.42 (br s, 1H), 6.48 (s, 1H), 6.85 (s, 1H), 7.57–7.60 (m,
3H), 7.65 (s, 1H), 7.66 (s, 1H), 7.69–7.76 (m, 4H), 7.79–7.83 (m,
4H), 8.60 (s, 1H), 10.55 (s, 1H), 11.10 (s, 1H); HRMS (ESI): m/z
1018.4969 (M+H+) (calcd for C53H68N11O8S: 1018.4973).
4.2.9. (3Z,6Z)-3-(3-Benzoylbenzylidene)-6-((5-(2-methyl-pent-
4-yn-2-yl)oxazol-4-yl)methylene)piperazine-2,5-dione (17)
To a solution of monodehydroDKP 16 (4 mg, 0.0127 mmol) in
anhydrous DMF (2 mL) were added Cs2CO3 (8.3 mg, 0.0254 mmol)
and aldehyde 14 (5.34 mg, 0.0254 mmol) under an argon atmo-
sphere, and the mixture was stirred for 3 h at 60 °C. After the sol-
vent was removed in vacuo, the residue was extracted with EtOAc,
washed with 10% citric acid, 5% NaHCO3, and brine, dried over
Na2SO4, and concentrated in vacuo. The residual oil was purified
by preparative HPLC (with a linear gradient of 50–90% CH3CN in
0.1% aq TFA over 40 min) to give a pale yellow powder of dide-
hydroDKP 17 (3.6 mg, 61%). 1H NMR (300 MHz, DMSO-d6) d 1.47
(s, 6H), 2.60 (d, 2H, J = 2.3 Hz), 2.89 (t, 1H, J = 2.3 Hz), 6.72 (s,
1H), 6.87 (s, 1H), 7.56–7.84 (m, 9H), 8.66 (s, 1H), 10.62 (s, 1H),
11.14 (s, 1H); HRMS (ESI): m/z 466.1760 (M+H+) (calcd for
4.3. Tubulin binding assay
Fluorescence spectra were measured at 37 °C as described
previously.11 Porcine tubulin (0.5
lM) in MES buffer (0.1 M
MES, 0.5 mM MgCl2, 1 mM EGTA, 1 mM GTP, pH 6.8) was incu-
bated with different concentrations of the test compounds
(0–12 lM, 1% DMSO) at 37 °C for 1 h. After incubation, the fluores-
cence of each solution was measured (excitation at 295 nm, emis-
sion at 300–450 nm) using an FP-750 Spectrofluorometer (JASCO,
JAPAN).
4.4. In vitro cytotoxicity assay
C28H24N3O4: 466.1767).
The human cell line HT-29 (colorectal adenocarcinoma) was ob-
tained from ATCC. HT-29 cells were maintained in McCoy’s 5A
medium (modified, Invitrogen) containing 10% fetal bovine serum
20
4.2.10. 3-Azido-propylamine (19) [CAS No. 109-76-2]
To a solution of 3-bromo-1-propylamine hydrobromide (1.6 g,
7.3 mmol) in water (5 mL) was slowly added a solution of sodium
azide (1.6 g, 24.6 mmol) in water (7.5 mL), and the mixture was re-
fluxed overnight. After cooling to room temperature, about one-
half of the water was removed by evaporation in vacuo, and the
remaining residue was diluted with Et2O. This biphasic mixture
was cooled to 0 °C and KOH pellets (2.0 g) were slowly added.
The phases were separated and the aqueous layer was extracted
twice with Et2O. The combined organic layers were dried over
MgSO4 and concentrated in vacuo to give a yellow oil of 19
(412 mg, 56%). 1H NMR (300 MHz, CDCl3) d 1.29 (br s, 2H), 1.69–
1.78 (m, 2H), 2.81 (t, 2H, J = 6.9 Hz), 3.38 (t, 2H, J = 6.7 Hz).
supplemented with 1% penicillin/streptomycin at 37 °C in
a
humidified 5% CO2 atmosphere. For the growth inhibition assays,
cells were plated in 96-well plates at 5000 cells/well the day before
compound addition. Stock solutions of compounds were prepared
in DMSO. Serially diluted compounds were added to the cells,
resulting in a final concentration range of 20
two hours later, 0.1 mg/mL XTT (Sigma) solution in PBS buffer con-
taining 25 M phenazine methosulfate (Wako) was added to each
lM–2 pM. Seventy-
l
well and the cells were incubated for an additional 1 h. The absor-
bance of the formazan product was measured at 492 nm on a plate
reader (TECAN SAFIRE). To compensate for the non-specific absorp-
tion, the absorbance at 690 nm was measured.
4.2.11. N-
D-Biotinyl-Acp-Acp-amino-propylazide (20)
To a solution of
D-biotin-Acp-Acp-OH (54 mg, 0.114 mmol) in
4.5. Tubulin photoaffinity labeling
DMF/DMSO (5 mL) were added HOBtꢁH2O (16 mg, 0.114 mmol),
EDCꢁHCl (22 mg, 0.114 mmol), and 3-azido propylamine (23 mg,
0.228 mol), and the mixture was stirred at room temperature over-
night. The resultant precipitates were collected and washed with
EtOAc. The precipitate was then purified by preparative HPLC (with
a linear gradient of 10–60% CH3CN in 0.1% aq TFA over 40 min) to
give a white powder of compound 20 (16 mg, 25%). 1H NMR
(600 MHz, DMSO-d6) d 1.18–1.38 (m, 10H), 1.42–1.54 (m, 7H),
1.58–1.65 (m, 3H), 2.00–2.04 (m, 6H), 2.57 (d, 1H, J = 12 Hz), 2.82
(dd, 1H, J = 5.0, 12 Hz), 2.99 (q, 4H, J = 6.0 Hz), 3.07–3.11 (m, 3H),
3.32–3.37 (m, 2H, overlapping with H2O), 4.11–4.13 (m, 1H), 4.30
(dd, 1H, J = 5.0, 7.7 Hz), 6.36 (s, 1H), 6.42 (s, 1H), 7.71 (t, 1H,
J = 5.0 Hz), 7.74 (t, 1H, J = 5.0 Hz), 7.83 (t, 1H, J = 5.0 Hz); HRMS
(ESI): m/z 553.3287 (M+H+) (calcd for C25H45N8O4S: 553.3284).
Tubulin photoaffinity labeling by probe 4 and compound 5 were
performed using the same procedure as previously described.11,12
Briefly, a solution of each compound was added to a tubulin solu-
tion (2 lM) in MES buffer (pH 6.8) containing 1 mM GTP and 2%
DMSO, and the solution was incubated at 37 °C for 30 min, fol-
lowed by UV irradiation at 365 nm at a distance of 10 cm on ice
for an appropriate time using a UV irradiator.
4.6. SDS–PAGE, Western blotting
SDS–PAGE and Western blotting of photolabeled tubulin were
performed using the same procedure as described previously.11,12
Photolabeled tubulin was separated by SDS–PAGE in 10% polyacryl-
amide gels and transferred to PVDF membrane. The membrane was
blocked with 5% (w/v) skim milk in PBS buffer containing 0.1% (v/v)
Tween 20 (pH 7.4) and incubated with streptavidin–horseradish
peroxidase conjugate (GE healthcare) for 1 h at room temperature
to detect the photolabeled protein. After washing, the membrane
was incubated with ECL Western blotting detection reagents (GE
healthcare), and the chemiluminescence was visualized by using
an imaging system (LAS-1000plus, FUJIFILM).
4.2.12. KPU-252-B1 (4)
To a solution of compounds 20 (2.6 mg, 0.0047 mmol) and com-
pound 17 (2.2 mg, 0.0047 mmol) in H2O/t-butanol/DMF (1:1:1,
0.3 mL) was added Cu(OAc)2 (5 mol %, 0.042 mg) and sodium
ascorbate (15 mol %, 0.14 mg). The reaction mixture was stirred
for 15 min at 80 °C under microwave irradiation. After cooling to
room temperature, the reaction mixture was diluted with DMSO

602
Y. Yamazaki et al. / Bioorg. Med. Chem. 19 (2011) 595–602
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D
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Competitive photoaffinity labeling was performed using the
same procedure as described previously.11 Tubulin (2
M) was
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for 15 min at 37 °C, followed by addition of probe 4 (2 M, final
concentration 2% DMSO). After incubation at 37 °C for 30 min,
samples were UV irradiated at 365 nm at a distance of 10 cm on
ice for 30 s, followed by separation by SDS–PAGE. Photolabeled
tubulin was visualized in a manner similar to that described above.
l
D
l
12. Yamazaki, Y.; Sumikura, M.; Hidaka, K.; Yasui, H.; Kiso, Y.; Yakushiji, F.;
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This research was supported by grants from MEXT (Ministry of
Education, Culture, Sports, Science and Technology), Japan, includ-
ing the Grant-in Aid for Young Scientists (B) 21790118 and the
Grant-in Aid for Scientific Research (B) 20390036. We are grateful
to Professor H. Toyoda, Mr. H. Kikuchi, and Mr. Y. Yoshino at the
Tokyo University of Pharmacy and Life Sciences, and Dr. S. Neute-
boom at Nereus Pharmaceuticals Inc., who provided HT-29 cells
and gave helpful suggestions for the cytotoxicity assay. We also
thank Professor Y. Sida and Ms. C. Sakuma of the Tokyo University
of Pharmacy and Life Sciences for mass spectra and NMR
measurements.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.10.055.
References and notes
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