Small-molecular, non-peptide, non-ATP-competitive polo-like kinase 1 (Plk1) inhibitors with a terphenyl skeleton

Article abstract of DOI:10.1016/j.bmc.2012.11.054

Polo-like kinase (Plk) 1 is a serine-threonine protein kinase that plays a role in cell division, and its overexpression is highly correlated with aggressiveness and prognosis of many cancers. We have designed, synthesized and evaluated a series of terphe

Full text of DOI:10.1016/j.bmc.2012.11.054

Accepted Manuscript
Small-molecular, non-peptide, non-ATP-competitive polo-like kinase 1 (Plk1)
inhibitors with a terphenyl skeleton
Yusuke Mita, Tomomi Noguchi-Yachide, Minoru Ishikawa, Yuichi Hashimoto
PII:
S0968-0896(12)00949-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.11.054
BMC 10463
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 November 2012
29 November 2012
30 November 2012
Please cite this article as: Mita, Y., Noguchi-Yachide, T., Ishikawa, M., Hashimoto, Y., Small-molecular, non-
peptide, non-ATP-competitive polo-like kinase 1 (Plk1) inhibitors with a terphenyl skeleton, Bioorganic &
Medicinal Chemistry (2012), doi: http://dx.doi.org/10.1016/j.bmc.2012.11.054
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Small-molecular, non-peptide, non-ATP-competitive polo-like kinase 1 (Plk1) inhibitors
with a terphenyl skeleton
Yusuke Mita, Tomomi Noguchi-Yachide, Minoru Ishikawa and Yuichi Hashimoto
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan
Abstract
Polo-like kinase (Plk) 1 is a serine-threonine protein kinase that plays a role in cell
division, and its overexpression is highly correlated with aggressiveness and prognosis
of many cancers. We have designed, synthesized and evaluated a series of terphenyl
compounds as inhibitors of the kinase activity of Plk 1. Some of them act as
non-ATP-competitive Plk 1 inhibitors.
Keyword
Polo-like kinase; Inhibitor; Small molecule; Terphenyl skeleton; Non-peptide;
Non-ATP-competitive
1. Introduction
Polo-like kinases (Plk) function in mitosis and in maintaining DNA integrity. There
are four family members, i.e., Plk1, Plk2 (also called Snk), Plk3 (also called Prk or Fnk)
and one more distantly related member, Plk4 (also called Sak).1 Plk1 regulates diverse
cellular and biochemical events at multiple stages of M phase, including centrosome
maturation, bipolar spindle formation, DNA damage adaptation, mitotic entry, activation
of anaphase-promoting complex, and cytokinesis.2-5 Plk2 appears to contribute to S
phase entry6 and plays a role in maintaining cell viability after spindle poisoning.7 Plk3
regulates mitotic entry and cytokinesis.8-9 In contrast to the role of Plk1-3 during the cell
cycle, Plk4 appears to be a key regulator of centriole duplication.10-13 Among the Plk
family members, Plk1 has been especially well studied, because it is thought to be an
attractive anticancer drug target.14
Plk1 is a serine-threonine protein kinase that is expressed only in dividing cells, with
peak expression during G2/M.15 Plk1 is essential for almost every step of mitosis

through its different protein-binding and phosphorylation activities, and its cellular
localization changes during mitotic progression.14 Overexpression of Plk1 is strongly
correlated with aggressiveness and prognosis of many cancers.16 Thus, Plk1 has been
studied as a potential drug target for anticancer therapy, and many small-molecular
ATP-competitive Plk1 inhibitors have been reported (Fig. 1).17-23
On the other hand, it is known that Plk1 has a unique non-catalytic domain called the
polo-box domain (PBD) in the C-terminal region, in addition to the catalytic kinase
domain in the N-terminal region. The PBD recognizes phosphorylated peptide motifs of
interacting proteins and contributes to PBD-dependent subcellular localization of Plk1,1
which is important for correct mitosis. Recently, several compounds, such as
poloxipan,24 purpurogoline25 and phosphorylated pentapeptide PLHSpT analogs (pT
indicates phosphorylated threonine)(Fig. 2),26-28 have been shown to inhibit interactions
between PBD of Plk1 and the corresponding phosphorylated peptide motif, causing
delocalization of Plk1, which results in dysfunction of mitosis. The binding modes of
PLHSpT peptide analogs of PBD were analyzed by X-ray crystallography.26 In this
report, based on the X-ray crystallographical data, we designed, synthesized and
evaluated a series of terphenyl compounds as mimetics of the PLHSpT peptide analogs,
and generated non-peptide and ATP non-competitive Plk1 inhibitors, which
unexpectedly works in the different mechanism of PLHSpT peptide analogs.
2. Results and Discussion
The X-ray structure of the complex of Plk1 PBD and PLHSpT pentapeptide indicated
that the Plk1 PBD binding pocket is stabilized by four types of binding,26 involving (i)
the core SpT motif, (ii) the N-terminal hydrophobic residue, (iii) glycerol (a buffer
component) and (iv) a network of contacting water molecules. Based on this
information, we designed non-peptide templates corresponding to the Plk1-bound
conformation of the phosphorylated pentapeptide PLHSpT (Fig. 3). We choose a
terphenyl skeleton as the scaffold because the terphenyl skeleton would be able to fix
the pharmacophore rigidly, and synthesize substituted analogs easily. We thought that
terphenyl compounds bearing two hydrophilic groups and a hydroxyalkyl group might
mimic PLHSpT peptide. In other words, we thought that the hydrophilic groups would
correspond to phosphoric acid and the carbonyl oxygen atom of the Pro residue

interacting with the guanidinium moiety of Arg516 of Plk1 PBD, and the hydroxyalkyl
group would bind to Plk1 PBD like ethylene glycol.
We synthesized terphenyl compound 5, which contains two carboxyl groups as
hydrophilic groups, in 4 steps from para-crezole 1 (Scheme 1). Introduction of iodine
atoms with ICl-pyridine, followed by nucleophilic substitution of 2-iodoethanol,
afforded 3. Suzuki coupling reaction of
3
and commercially available
4-cyanophenylboronic acid pinacol ester gave terphenyl compound 4, which contains
two nitrile groups. Hydrolysis of the nitrile groups afforded terphenyl compound 5 with
two carboxylic groups. Terphenyl analogs 6 and 7 bearing tetrazole moieties (bioisoster
of carboxylic acid) as the hydrophilic groups were synthesized by cyclization of sodium
azide and dinitrile 4.
To investigate the inhibitory activity on the interaction between Plk1 PBD and the
phosphorylated peptide motif, we constructed a modified ELISA-based inhibition assay
based on a previously reported assay system.26 In this assay, biotinylated
phosphopeptide (biotin-GGGGG-PLHSpT) was fixed on an avidin-coated plate via
avidin-biotin interaction. Then, test samples were incubated with HeLa cell lysate,
which contains Plk1 protein, on the phosphopeptide-fixed plate. Non-bound Plk1, other
cell components and samples were washed out, then the 1st antibody for Plk1, 2nd
antibody conjugated with HRP, 3,3’,5,5’-tetramethylbenzidine (TMB) solution and stop
solution were added to the plate. The amount of oxidized TMB was determined by
measuring the absorbance at 450 nm, as a measure of the intensity of the interaction
between Plk1 PBD and phosphorylated peptide. PLHSpT pentapeptide, used as a
positive control, inhibited the interaction between Plk1 PBD and phosphorylated
peptide fixed on the plate with an IC50 value of 5.27 μM, as expected. Dicarboxylic acid
5 showed no effect on the interaction between Plk1 PBD and phosphorylated peptides
(Fig. 4). However, terphenyl compounds bearing nitrile group(s) or tetrazole group(s),
especially monotetrazole 6, unexpectedly resulted in enhanced interaction between Plk1
PBD and phosphorylated peptide (Fig. 4). We could not investigate the inhibitory
activity of compound 4 in higher concentration range because the maximum solubility
of 4 was about 100 μM under the buffer conditions for our ELISA-based assay system.
It is unknown why terphenyl compounds increase the interaction between Plk1 PBD
and phosphorylated peptide substrate in our assay system. One possible interpretation
would be that they bind Plk1 and change the conformation of Plk1 in a way that favors

recognition of the phosphorylated peptide motif. This idea led us evaluate whether
terphenyl compounds inhibit Plk1 kinase activity.
To investigate inhibition of Plk1, we used a CycLex Polo-like kinase 1
Assay/Inhibitor Screening Kit (CycLex Co. Ltd.), which employs recombinant Plk1.
The phospho-threonine specific polyclonal antibody used in this assay kit has been
demonstrated to recognize the Plk1-related phospho-threonine residue in the plate-fixed
protein. Interestingly, the assay showed that 6 dose-dependently inhibited the kinase
activity of Plk1, and the IC50 of 6 was calculated to be 151 μM. On the other hand,
PLHSpT peptide did not inhibit the kinase activity of Plk1 (Fig. 5). These results
suggested that 6, which has a monotetrazole moiety, might interact with a domain of
Plk1 other than the PLHSpT-binding site to inhibit the kinase activity of Plk1. In order
to examine the structure-activity relationship (SAR) of the terphenyl compounds, we
further designed and synthesized a range of terphenyl analogs. These were synthesized
by Suzuki coupling reaction of halide 3 and appropriate boronic acid pinacol esters. The
boronic acid pinacol esters were synthesized as described below.
To optimize the alkyl chain length of the carboxylic acid, compounds 12b-c (and 12a
= 5) were synthesized as shown in Scheme 2. Protection of the carboxyl group of 8a-c
by benzyl ester, followed by substitution of the bromine atom with boronic acid pinacol
ester gave 10a-c. Terphenyl compound 16 which had carboxylic acids at the
meta-positions was similarly synthesized (Scheme 3). Condensation reaction of
2-(4-bromophenyl)ethanoic acid (8b) (Scheme 4) and methylamine, followed by
substitution of the bromine atom with boronic acid pinacol ester gave 18, which is a
precursor of 19. Bromination of 2,6-difluorophenol (20) (Scheme 5) followed by
substitution of the bromine atom with boronic acid pinacol ester and Suzuki coupling
reaction with 3 afforded 22, which has two difluorophenyl groups (bioisoster of
carboxylic acid).29 Terphenyl compound 24 (Scheme 6) without alkyl alcohol was
synthesized in 2 steps, i.e., Suzuki coupling reaction and deprotection of the carboxylic
acids.
Table 1 shows the results of evaluation of the inhibition ratio at 300 μM terphenyl
analogs. Compounds 6, 7, 12b and 22 showed moderately strong inhibition of Plk1
kinase activity, while compounds 4, 5, 12c, and 24 showed weak inhibition. Compounds
3, 16, and 19 showed no inhibition of Plk1 kinase activity at the investigated
concentrations. These results suggest that hydrophilic groups are necessary for

Plk1-inhibitory activity, and lipophilic acidic groups, such as tetrazole and
difluorophenol, might be preferable to inhibit Plk1 activity.
In order to obtain insights into the mechanism of PLK1 inhibition, we determined the
IC50 values of 6 and staurosporine, the latter of which is an ATP-competitive kinase
inhibitor, for PLK1 in the presence of various concentrations of ATP. A typical data set
is shown in Fig. 6 and Table 2. The measured IC50 values of 6 were independent of the
ATP concentration, indicating a non-ATP-competitive mechanism of action. On the
other hand, a high concentration of ATP resulted in a dramatic decrease of the inhibitory
efficacy of staurosporine, that is, the IC50 value of staurosporine was over five times
higher (129 μM) in the presence of 3.12 mM ATP as compared to that observed in the
presence of 62.5 μM ATP (22.4 μM). These results suggested that terphenyl compounds
might interact with Plk1 protein at a domain other than the PLHSpT peptide-binding
site, and inhibit Plk1 kinase activity in a non-ATP-competitive manner.
3. Conclusion
Plk 1 is a serine-threonine protein kinase that plays a critical role in cell division.
Overexpression of Plk1 is strongly correlated with aggressiveness and prognosis of
many cancers, so Plk1 is a potential target for anticancer therapy. Here, we designed a
series of terphenyl compounds based on the X-ray structure of the complex of Plk1
PBD and PLHSpT pepntapeptide, anticipating that they would inhibit the interaction
between Plk1 PBD and phosphorylated protein substrate, and worked as
non-ATP-competitive inhibitors. Contrary to our expectation, these compounds did not
inhibit but rather increased the interaction between Plk1 PBD and the phosphorylated
peptide motif. Investigation of the mechanism of Plk1 kinase inhibition showed that the
terphenyl compounds are non-ATP-competitive inhibitors of Plk1 kinase. The
phosphorylated peptide did not inhibit Plk1 kinase activity under the same assay
conditions. These results suggested that terphenyl compounds interact with Plk1 protein
at a domain other than the PLHSpT peptide-binding site, consequently inhibiting Plk1
kinase activity in a non-ATP-competitive manner.
4. Experimental section

4.1. Synthesis
4.1.1 General
Melting points were determined by using a Yanagimoto hot-stage melting point
apparatus and are uncorrected. 1H-NMR spectra were recorded on a JEOL JNM-GX500
(500 MHz) spectrometer. Chemical shifts are expressed in parts per million relative to
tetramethylsilane. Mass spectra were recorded on a JEOL JMS-DX303 spectrometer.
Elemental analyses were carried out in the Microanalytical Laboratory, Faculty of
Pharmaceutical Sciences, University of Tokyo, and were within ±0.4% of theoretical
values.
4.1.2. General Procedure A (GP-A)
Under an Ar atmosphere, DMF was added to a mixture of 3 (or 2), the corresponding
boronic acid pinacol ester, K3PO4, and Pd(dppf)Cl2. The reaction mixture was stirred for
o
an appropriate time at 80 C, then filtered through Celite. The filtrate was diluted with
H2O and extracted with AcOEt. The organic layer was washed with H2O, dried over
MgSO4, and concentrated. The resulting residue was purified by silica gel
chromatography to afford the target material.
4.1.3 General Procedure B (GP-B)
Benzyl bromide and K2CO3 were added to a solution of carboxylic acid 8 in DMF at
0 oC, then the atmosphere was replaced with Ar. The reaction mixture was stirred for an
appropriate time at 100 oC, then the reaction was quenched with H2O and the whole was
extracted with AcOEt. The organic layer was washed with H2O and brine, dried over
MgSO4, and concentrated. The resulting residue was purified by silica gel
chromatography to afford the target material.
4.1.4. General Procedure C (GP-C)
Under an Ar atmosphere, 1,4-dioxane was added to a mixture of aryl bromide,
bis(pinacolate)diboron, KOAc, and (PPh3)2PdCl2. The reaction mixture was stirred for
o
an appropriate time at 100 C, then filtered through filter paper or Celite. The filtrate
was diluted with H2O and brine, and extracted with AcOEt. The organic layer was
washed with H2O and brine, dried over MgSO4, and concentrated. The resulting residue
was purified by silica gel chromatography to afford the target material.

4.1.5. 2,6-Diiodo-4-methylphenol (2)
ICl-pyridine complex (5.46g, 22.6 mmol) was added to a stirred solution of p-cresol
(1.11g, 10. 3 mmol) in CH2Cl2 (66 mL) and H2O (33 mL) at 0 oC. The reaction mixture
was stirred for 7 h at rt, then the reaction was quenched with 0.3 M Na2S2O5 aq at rt,
and the mixture was stirred until its color changed to yellow. At that point, it was
extracted with CH2Cl2. The organic layer was dried over MgSO4 and concentrated. The
resulting residue was purified by silica gel chromatography (hexane / AcOEt = 20 / 1) to
afford 2 (2.54 g, 7.06 mmol, 69%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ: 7.49 (s, 2H), 5.57 (s, 1H), 2.22 (s, 3H). FAB-MS m/z:
360 (M+).
4.1.6. 2-(2,6-Diiodo-4-methylphenoxy)ethanol (3)
2-Iodoethanol (467 μL, 6.00 mmol) was added to a solution of 2 (1.08 g, 3. 00 mmol)
o
and Cs2CO3 (3.04 g, 9.33 mmol) in DMF (10 mL) at 0 C. The reaction mixture was
stirred for 9.5 h at 80 oC, then the reaction was quenched with H2O, and the mixture was
extracted with AcOEt. The organic layer was washed with H2O, dried over MgSO4, and
concentrated. The resulting residue was purified by silica gel chromatography (hexane /
AcOEt = 3 / 1) to give 3 (1.02 g, 2.51 mmol, 84 %) as a white solid.
1H-NMR (500 MHz, CDCl3) δ: 7.60 (s, 2H), 4.15-4.12 (m, 2H), 4.06-4.02 (m, 2H),
2.33-2.29 (m, 1H), 2.25 (s, 3H). FAB-MS m/z: 404 (M+).
4.1.7. 2’-(2-Hydroxyethoxy)-5’-methyl-(1,1’:3’,1-terphenyl)-4,4’’-dinitrile (4)
This compound was prepared by means of GP-A, with 3 (198 mg, 490 μmol),
4-cyanophenylboronic acid pinacol ester (228 mg, 995 μmol), K3PO4 (447 mg, 2.11
mmol), Pd(dppf)Cl2 (39.6 mg, 54.1 μmol) and DMF (10 mL). Purification by silica gel
chromatography (hexane / AcOEt = 4 / 1 to 2 / 1) afforded 4 (113 mg, 319 μmol, 65 %)
as a pale yellow solid.
o
1
mp 210-212 C. H-NMR (500 MHz, CDCl3) δ: 7.76-7.70 (m, 8H), 7.20 (s, 2H),
3.32-3.25 (m, 4H), 2.42 (s, 3H), 1.07-1.03 (m, 1H). FAB-MS m/z: 354 (M+), 355
(M+H+). FAB-HRMS m/z: 354.1373 (Calcd for C23H18N2O2: 354.1368).
4.1.8. 2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dicarboxylic acid

(5 = 12a)
KOH (438 mg, 7.81 mmol) was added to a solution of 4 (47.0 mg, 133 μmol) in
EtOH (2.5 mL). The reaction mixture was refluxed for 6.5 h, then acidified with 2 N
HCl aq. to pH 1. The mixture was extracted with AcOEt and the organic layer was
washed with H2O, dried over MgSO4, and concentrated. The resulting residue was
purified by PTLC (CHCl3 / MeOH = 5 /1) to afford 5 (= 12a) (6.90 mg, 17.6 μmol,
13 %) as a white solid.
mp 276-279 oC. 1H-NMR (500 MHz, CD3OD) δ: 8.09 (d, J = 8.6 Hz, 4H), 7.74 (d, J =
8.6 Hz, 4H), 7.26 (s, 2H), 3.24-3.22 (m, 4H), 2.42 (s, 3H), 1.20-1.15 (m, 1H). FAB-MS
m/z: 392 (M+), 393 (M+H+). FAB-HRMS m/z: 392.1255 (Calcd for C23H20O6:
392.1260).
4.1.9. 2'-(2-Hydroxyethoxy)-5'-methyl-(1,1':3':1''-terphenyl)-4''-(1H-tetrazol-5-yl)
-4-nitrile (6) and 2-[5'-methyl-4,4''-ditetrazol-5-yl-(1,1':3',1''-terphenyl)-2'-
oxy]ethanol (7)
Under an Ar atmosphere, DMF (1.0 mL) was added to a mixture of 4 (33.0 mg, 93.1
μmol), NH4Cl (18.9 mg, 353 μmol), LiCl (2.50 mg, 59.0 μmol), NaN3 (20.7 mg, 318
o
μmol). The reaction mixture was stirred for 18 h at 100 C, then the reaction was
quenched with H2O and the mixture was extracted with AcOEt. The organic layer was
dried over MgSO4 and concentrated. The resulting residue was purified by PTLC
(CHCl3 / MeOH = 3 / 1) to afford 6 (15.9 mg, 40.0 μmol, 43 %, white solid) and 7 (4.50
mg, 10.2 μmol, 11 %, colorless oil).
6: mp 218-221 oC. 1H-NMR (500 MHz, CD3OD) δ: 8.11 (d, J = 8.0 Hz, 2H), 7.84 (d,
J = 8.6 Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.30 (s, 1H), 7.24 (s,
1H), 3.26-3.23 (m, 4H), 2.43 (s, 3H). FAB-MS m/z: 397 (M+), 398 (M+H+).
FAB-HRMS m/z: 397.1542 (Calcd for C23H19N5O2: 397.1539). Anal. C, 66.72; H, 5.00;
N, 16.70 (Calcd for C23H19N5O2•H2O: C, 66.49; H, 5.09; N, 16.86).
7: 1H-NMR (500 MHz, CD3OD) δ: 8.12 (d, J = 7.9 Hz, 4H), 7.80 (d, J = 7.9 Hz, 4H),
7.27 (s, 2H), 3.28-3.25 (m, 4H), 2.44 (s, 3H). FAB-MS m/z: 440 (M+), 441 (M+H+).
FAB-HRMS m/z: 440.1706 (Calcd for C23H20N8O2: 440.1709).
4.1.10. Benzyl 4-bromobenzoate (9a)
This compound was prepared by means of GP-B, with 4-bromobenzoic acid (4.33 g,

21.5 mmol), benzyl bromide (2.58 mL, 21.7 mmol), K2CO3 (6.11 g, 44.2 mmol), and
DMF (25 mL). Purification by silica gel chromatography (hexane / AcOEt = 10 / 1 to 5 /
1) afforded 9a (5.13 g, 17.6 mmol, 82 %) as a white solid.
1H-NMR (500 MHz, CDCl3) δ: 7.93 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H),
7.45-7.33 (m, 5H), 5.35 (s, 2H). FAB-MS m/z: 183, 185 (M-OBn+).
4.1.11. Benzyl 2-(4-bromophenyl)acetate (9b)
This compound was prepared by means of GP-B, with 4-bromophenylacetic acid
(1.07 g, 4.99 mmol), benzyl bromide (564 μL, 5.00 mmol), K2CO3 (1.39 g, 10.0 mmol),
and DMF (10 mL). Purification by silica gel chromatography (hexane / AcOEt = 10 / 1)
afforded 9b (1.11 g, 3.62 mmol, 73 %) as a pale yellow oil.
1H-NMR (500 MHz, CDCl3) δ: 7.44 (d, J = 8.6 Hz, 2H), 7.37-7.29 (m, 5H), 7.16 (d, J
= 8.6 Hz, 2H), 5.13 (s, 2H), 3.62 (s, 2H). FAB-MS m/z: 304, 306 (M+), 305, 307
(M+H+).
4.1.12. Benzyl 3-(4-bromophenyl)propionate (9c)
This compound was prepared by means of GP-B, with 3-(4-bromophenyl)propionic
acid (1.15 g, 5.02 mmol), benzyl bromide (594 μL, 5.00 mmol), K2CO3 (1.43 g, 10.4
mmol), and DMF (10 mL). Purification by silica gel chromatography (hexane / AcOEt
= 10 / 1) afforded 9c (1.34 g, 4.18 mmol, 83 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 7.38 (d, J = 8.6 Hz, 2H), 7.36-7.32 (m, 3H), 7.30-7.27
(m, 3H), 7.05 (d, J = 8.6 Hz, 2H), 5.10 (s, 2H), 2.92 (t, J = 7.9 Hz, 2H), 2.67 (t, J = 7.9
Hz, 2H). FAB-MS m/z: 319, 321 (M+).
4.1.13. Benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (10a)
This compound was prepared by means of GP-C, with 9a (510 mg, 1.75 mmol),
bis(pinacolate)diboron (488 mg, 1.92 mmol), KOAc (700 mg, 7.13 mmol),
(PPh3)2PdCl2 (63.5 mg, 90.5 μmol) and 1,4-dioxane (18 mL). Purification by silica gel
chromatography (hexane / AcOEt = 1 / 0 to 1 / 1) afforded 10a (381 mg, 1.13 mmol,
64 %) as a yellow solid.
1H-NMR (500 MHz, CDCl3) δ: 8.05 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 7.6 Hz, 2H), 7.45
(d, J = 7.3 Hz, 2H), 7.40 (dd, J = 7.3, 7.3 Hz, 2H), 7.35 (d, J = 7.3 Hz, 1H), 5.37 (s, 2H),
1.35 (s, 12H). FAB-MS m/z: 339 (M+H+).

4.1.14. Benzyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetate
(10b)
This compound was prepared by means of GP-C, with 9b (630 mg, 2.06 mmol),
bis(pinacolate)diboron (536 mg, 2.11 mmol), KOAc (788 mg, 8.03 mmol),
(PPh3)2PdCl2 (39.5 mg, 56.3 μmol) and 1,4-dioxane (20 mL). Purification by silica gel
chromatography (hexane / AcOEt = 1 / 0 to 5 / 1) afforded 10b (410 mg, 1.16 mmol,
56 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 7.77 (d, J = 7.3 Hz, 2H), 7.35-7.28 (m, 7H), 5.12 (s,
2H), 3.68 (s, 2H), 1.34 (s, 12H). FAB-MS m/z: 352 (M+), 353 (M+H+).
4.1.15. Benzyl 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propionate
(10c)
This compound was prepared by means of GP-C, with 9c (637 mg, 2.00 mmol),
bis(pinacolate)diboron (534 mg, 2.10 mmol), KOAc (791 mg, 8.06 mmol),
(PPh3)2PdCl2 (47.8 mg, 68.1 μmol) and 1,4-dioxane (20 mL). Purification by silica gel
chromatography (hexane / AcOEt = 1 / 0 to 5 / 1) afforded 10c (392 mg, 1.07 mmol,
54 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 7.73 (d, J = 7.9 Hz, 2H), 7.37-7.28 (m, 5H), 7.20 (d, J
= 7.9 Hz, 2H), 5.10 (s, 2H), 2.98 (t, J = 7.3 Hz, 2H), 2.68 (t, J = 7.3 Hz, 2H), 1.34 (s,
12H). FAB-MS m/z: 366 (M+), 367 (M+H+).
4.1.16.
2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dicarboxylic
acid, dibenzyl ester (11a)
This compound was prepared by means of GP-A, with 3 (189 mg, 469 μmol), 10a
(348 mg, 1.03 mmol), K3PO4 (404 mg, 1.90 mmol), Pd(dppf)Cl2 (34.6 mg, 47.2 μmol)
and DMF (5.0 mL). Purification by silica gel chromatography (toluene / AcOEt = 10 /
1) afforded 11a (186 mg, 325 μmol, 69 %) as a pale yellow oil.
1H-NMR (500 MHz, CDCl3) δ: 8.15 (d, J = 8.3 Hz, 4H), 7.69 (d, J = 8.3 Hz, 4H),
7.47 (d, J = 7.3 Hz, 4H), 7.41 (dd, J = 7.3, 7.3 Hz, 4H), 7.36 (d, J = 7.3 Hz, 2H), 7.19 (s,
2H), 5.39 (s, 4H), 3.29-3.25 (m, 2H), 3.25-3.21 (m, 2H), 2.41 (s, 3H), 1.08-1.04 (m, 1H).
FAB-MS m/z: 572 (M+), 573 (M+H+). FAB-HRMS m/z: 572.2200 (Calcd for C37H32O6:
392.2199).

4.1.17. 2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-diacetic acid,
dibenzyl ester (11b)
This compound was prepared by means of GP-A, with 3 (102 mg, 252 μmol), 10b
(190 mg, 539 μmol), K3PO4 (237 mg, 1.12 mmol), Pd(dppf)Cl2 (20.3 mg, 27.7 μmol)
and DMF (2.5 mL). Purification by silica gel chromatography (toluene / AcOEt = 8 / 1)
afforded 11b (94.7 mg, 158 μmol, 63 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 7.57 (d, J = 8.0 Hz, 4H), 7.38-7.31 (m, 14H), 7.15 (s,
2H), 5.16 (s, 4H), 3.73 (s, 4H), 3.30-3.28 (m, 2H), 3.22-3.17 (m, 2H), 2.40 (s, 3H),
1.09-1.05 (m, 1H). FAB-MS m/z: 600 (M+), 601 (M+H+).
4.1.18. 2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dipropionic acid,
dibenzyl ester (11c)
This compound was prepared by means of GP-A, with 3 (102 mg, 252 μmol), 10c
(223 mg, 609 μmol), K3PO4 (243 mg, 1.14 mmol), Pd(dppf)Cl2 (23.3 mg, 31.8 μmol)
and DMF (2.5 mL). Purification by silica gel chromatography (toluene / AcOEt = 8 / 1)
afforded 11c (94.7 mg, 158 μmol, 63 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 7.52 (d, J = 8.0 Hz, 4H), 7.38-7.32 (m, 10H),
7.27-7.24 (m, 4H), 7.13 (s, 2H), 5.13 (s, 4H), 3.29-3.26 (m, 2H), 3.21-3.17 (m, 2H),
3.02 (t, J = 7.9Hz, 4H), 2.73 (t, J = 7.9 Hz, 4H), 2.39 (s, 3H), 1.08-1.04 (m, 1H).
FAB-MS m/z: 628 (M+), 629 (M+H+).
4.1.19.
2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dicarboxylic
acid (5 = 12a)
Under an Ar atmosphere, 10 % Pd/C (21.5 mg) was added to a solution of 11a (75.8
mg, 132 μmol) in 1,4-dioxane (3.0 mL). The Ar atmosphere was replaced with H2. The
reaction mixture was stirred for 18 h at rt, then filtered through Celite. The filtrate was
concentrated to afford 5 (= 12a) (41.8 mg, 107 μmol, 81 %) as a white solid. See data
for 5.
4.1.20. 2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-diacetic acid
(12b)
2 N NaOH aq (60.0 μL, 120 μmol) was added to a stirred solution of 11b (18.0 mg,

30.0 μmol) in MeOH (1.0 mL) at rt. The reaction mixture was stirred for 1 d at rt, then
concentrated. The resulting residue was dissolved in AcOEt / MeOH (10 /1), and 2 N
HCl aq. was added to the solution. The organic layer was partitioned and the water layer
was extracted with AcOEt / MeOH (10 / 1). The combined organic layer was washed
with H2O, dried over MgSO4, and concentrated. The resulting residue was purified by
PTLC (CHCl3 / EtOH / H2O / NH4OH = 4 / 4 / 1 / 1) to afford 12b (Rf 0.00; 9.80 mg,
23.3 μmol, 78 %) as a yellow oil.
1H-NMR (500 MHz, CD3OD) δ: 7.53 (d, J = 7.6 Hz, 4H), 7.36 (d, J = 7.6 Hz, 4H),
7.12 (s, 2H), 3.55 (s, 4H), 3.25-3.20 (m, 4H), 2.37 (s, 3H). FAB-MS m/z: 420 (M+), 421
(M+H+). FAB-HRMS m/z: 420.1568 (Calcd for C25H24O6: 420.1568).
4.1.21. 2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dipropionic acid
(12c)
2 N NaOH aq. (92.5 μL, 185 μmol) was added to a stirred solution of 11c (29.1 mg,
46.3 μmol) in MeOH (1.0 mL) at rt. The reaction mixture was stirred for 1 d at rt, then
concentrated. The resulting residue was dissolved in Et2O, and 2 N HCl aq. was added
to the solution. The organic layer was partitioned and the water layer was extracted with
Et2O. The combined Et2O layer was washed with H2O, dried over MgSO4, and
concentrated. The resulting residue was recrystallized (EtOH / hexane) to afford 12c
(7.80 mg, 17.4 μmol, 38 %) as white needles.
mp 176-179 oC. 1H-NMR (500 MHz, CD3OD) δ7.52 (d, J = 8.0 Hz, 4H), 7.29 (d, J =
8.0 Hz, 4H), 7.11 (s, 2H), 3.22-3.18 (m, 4H), 2.96 (t, J = 7.3Hz, 4H), 2.64 (t, J = 7.9 Hz,
4H), 2.37 (s, 3H). FAB-MS m/z: 448 (M+), 449 (M+H+). FAB-HRMS m/z: 448.1885
(Calcd for C27H28O6: 448.1886).
4.1.22. Benzyl 3-bromobenzoic acid (14)
This compound was prepared by means of GP-B, with 3-bromobenzoic acid (5.03 g,
25.0 mmol), benzyl bromide (3.00 mL, 25.3 mmol), K2CO3 (6.96 g, 50.4 mmol), and
DMF (25 mL). Purification by silica gel chromatography (hexane / AcOEt = 10 / 1)
afforded 14 (6.87 g, 23.9 mmol, 96 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 8.20 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 6.6
Hz, 1H), 7.46-7.29 (m, 6H), 5.63 (s, 2H). FAB-MS m/z: 291, 293 (M+H+).

4.1.23. Benzyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (15)
This compound was prepared by means of GP-C, with 14 (1.03 g, 3.54 mmol),
bis(pinacolate)diboron (902 mg, 3.55 mmol), KOAc (1.06 g, 10.8 mmol), (PPh3)2PdCl2
(47.8 mg, 68.1 μmol) and 1,4-dioxane (35 mL). Purification by silica gel
chromatography (hexane / AcOEt = 15 / 1 to 10 / 1) afforded 15 (998 mg, 2.95 mmol,
83 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 8.50 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.3
Hz, 1H), 7.48-4.32 (m, 6H), 5.38 (s, 2H), 1.35 (s, 12H). FAB-MS m/z: 338 (M+), 339
(M+H+).
4.1.24.
2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-3,3''-dicarboxylic
acid (16)
Under an Ar atmosphere, DMF (4.5 mL) was added to a mixture of 3 (169 mg, 418
μmol), 15 (315 mg, 931 μmol), K3PO4 (362 mg, 1.71 mmol), and Pd(dppf)Cl2 (30.7 mg,
o
41.9 μmol). The reaction mixture was stirred for 6 h at 80 C, then the reaction was
quenched with H2O and brine, and the mixture was extracted with AcOEt. The organic
layer was dried over MgSO4, and concentrated. The resulting residue was purified by
silica gel chromatography (toluene / AcOEt = 10 / 1) to afford a mixture of dicarboxylic
acid and dibenzyl ester (203 mg) as a pale yellow oil. In the next reaction, 2 N NaOH aq.
(600 μL, 1.20 mmol) was added to a solution of the mixture (203 mg) in MeOH (3.0
mL) at rt. The reaction mixture was stirred for 23 h at rt, then the reaction was quenched
with H2O, and the whole was acidified with 2 N HCl aq. at 0 oC. The resulting solution
was extracted with AcOEt, and the water layer was extracted with AcOEt / MeOH (10 /
1). The organic layers were combined and washed with H2O. The organic layer was
dried over MgSO4 and concentrated. The resulting residue was purified by
reprecipitation (EtOH / hexane / CH2Cl2) to afford 16 (70.8 mg, 180 μmol, 43 % (2
steps)) as a white solid.
o
1
mp 240-242 C. H-NMR (500 MHz, CD3OD) δ: 8.28 (s, 2H), 8.02 (d, J = 7.3 Hz,
2H), 7.89 (d, J = 7.3 Hz, 2H), 7.55 (dd, J = 7.3, 7.3 Hz, 2H), 7.24 (s, 2H), 3.24-3.15 (m,
4H), 2.43 (s, 3H). FAB-MS m/z: 393 (M+H+). FAB-HRMS m/z: 392.1256 (Calcd for
C23H20O6: 392.1260).
4.1.25. 2-(4-Bromophenyl)-N-methylethanamide (17)

Methylamine (40 % in MeOH) (600 μL, 5.88 mmol) was added to a solution of
4-bromophenylacetic acid (1.01 g, 4.69 mmol), HOBt•H2O (836 mg, 5.46 mmol),
DIPEA (2.50 mL, 14.3 mmol), and EDCI (1.09 g, 5.76 mmol) in CH2Cl2 (25 mL) at rt.
The reaction mixture was stirred for 23 h, then the reaction was quenched with sat.
NaHCO3 aq. and the mixture was partitioned. The organic layer was washed with sat.
NaHCO3 aq. and H2O, dried over MgSO4, and concentrated. The resulting residue was
purified by silica gel chromatography (CHCl3 / MeOH = 10 / 1 to 5 / 1) to afford 17
(344 mg, 1.51 mmol) as a white crystals.
1H-NMR (500 MHz, CDCl3) δ: 7.48 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.6 Hz, 2H),
5.45-5.20 (m, 1H), 3.52 (s, 2H), 2.78 (d, J = 4.9 Hz, 3H). FAB-MS m/z: 228, 230
(M+H+).
4.1.26. 2-[4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-N-
methylethanamide (18)
This compound was prepared by means of GP-C, with 17 (229 mg, 1.00 mmol),
bis(pinacolate)diboron (264 mg, 1.04 mmol), KOAc (392 mg, 3.99 mmol),
(PPh3)2PdCl2 (33.3 mg, 47.4 μmol) and 1,4-dioxane (10 mL). Purification by silica gel
chromatography (hexane / AcOEt = 1 / 1 to 1 / 2) afforded 18 (127 mg, 462 μmol,
64 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 7.80 (d, J = 7.3 Hz, 2H), 7.26-7.25 (m, 2H), 5.35-5.16
(m, 1H), 3.60 (s, 2H), 2.73 (d, J = 4.9 Hz, 3H), 1.33 (s, 12H). FAB-MS m/z: 275 (M+),
276 (M+H+).
4.1.27. 2'-(2-Hydroxyethoxy)-5'-methyl-N4,N4’’-dimethyl-[1,1':3',1''-terphenyl]
-4,4''-diacetamide (19)
This compound was prepared by means of GP-A, with 3 (88.6 mg, 219 μmol), 18
(127 mg, 462 μmol), K3PO4 (186 mg, 876 μmol), Pd(dppf)Cl2 (15.3 mg, 20.9 μmol) and
DMF (2.5 mL). Purification by silica gel chromatography (CHCl3 / MeOH = 10 / 1),
PTLC (CHCl3 / MeOH = 10 / 1) and reprecipitation (AcOEt) afforded 19 (13.4 mg, 30.0
μmol, 14 %) as a white solid.
mp 234-236 oC. 1H-NMR (500 MHz, CDCl3) δ: 7.56 (d, J = 7.9 Hz, 4H), 7.34 (d, J =
7.9 Hz, 4H), 7.13 (s, 2H), 3.53 (s, 4H), 3.22-3.18 (m, 4H), 2.73 (s, 6H), 2.67 (s, 3H);
MS (FAB) 446 (M+), 447 (M+H+). FAB-HRMS m/z: 446.2202 (Calcd for C27H30N2O4:

446.2206).
4.1.28. 4-Bromo-2,6-difluorophenol (21)
N-Bromosuccinimide (3.60 g, 20.2 mmol) was added to a stirred solution of
o
2,5-difluorophenol (2.61 g, 20.1 mmol) in DMF (10 mL) at 0 C. The reaction mixture
was stirred for 24 h at rt. with shielding from light, then the reaction was quenched with
H2O, and the mixture was extracted with AcOEt. The organic layer was washed with
H2O, dried over MgSO4, and concentrated. The resulting residue was purified by silica
gel chromatography (hexane / AcOEt = 4 / 1) to afford 21 (3.72 g, 17.8 mmol, 88 %) as
pale yellow needles.
1H-NMR (500 MHz, CDCl3) δ: 7.09 (d, J = 6.7 Hz, 2H), 5.23 (s, 1H). FAB-MS m/z:
256 (M+), 257 (M+H+).
4.1.29. 2'-(2-Hydroxyethoxy)-5'-methyl-[1,1':3',1''-terphenyl]-3,3’’,5,5’’-tetrafluoro
-4,4''-diol (22)
Under an Ar atmosphere, 1,4-dioxane (20 mL) was added to a mixture of 21 (1.09 g,
5.23 mmol), bis(pinacolate)diboron (1.33 g, 5.24 mmol), KOAc (1.96 g, 20.0 mmol),
and (PPh3)2PdCl2 (82.1 mg, 117 μmol). The reaction mixture was stirred for 10.5 h at
100 oC, and filtered through Celite. The filtrate was diluted with H2O and extracted with
AcOEt. The organic layer was washed with H2O, dried over MgSO4, and concentrated.
The resulting residue was purified by silica gel chromatography (hexane / AcOEt = 3 /
1) to afford a mixture of pinacol ester boronic acid (373 mg) as a white solid. This
mixture was used in the next step without further purification. In the next step, DMF (7
mL) was added to pinacol ester boronic acid (373 mg, mixture), 3 (263 mg, 651 μmol),
K3PO4 (578 mg, 2.72 mmol), and Pd(dppf)Cl2 (47.3 mg, 64.6 μmol) under an Ar
o
atmosphere. The reaction mixture was stirred for 9 h at 80 C, and filtered through
Celite. The filtrate was diluted with H2O and extracted with AcOEt. The water layer was
acidified with 2 N HCl aq to pH 1, and extracted with AcOEt. The combined organic
layer was washed with H2O, dried over MgSO4, and concentrated. The resulting residue
was purified by silica gel chromatography (hexane / AcOEt = 1 / 0 to 1/ 2) and
recrystallization (CH2Cl2 / hexane) to afford 22 (53.9 mg, 132 μmol, 3 % (2 steps)) as
colorless crystals.
o
1
mp 123 C. H-NMR (500 MHz, CDCl3) δ: 7.20 (d, J = 8.6 Hz, 4H), 7.11 (s, 2H),

5.17-5.14 (m, 2H), 3.42-3.34 (m, 4H), 2.39 (s, 3H), 1.28-1.25 (m, 1H). FAB-MS m/z:
408 (M+). FAB-HRMS m/z: 408.0981 (Calcd for C21H16F4O4: 408.0985).
4.1.30.
2'-Hydroxy-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dicarboxylic
acid,
dibenzyl ester (23)
This compound was prepared by means of GP-A, with 2 (269 mg, 822 μmol), 9a
(556 mg, 1.64 mmol), K3PO4 (719 mg, 3.38 mmol), Pd(dppf)Cl2 (62.2 mg, 85.0 μmol)
and DMF (8.5 mL). Purification by silica gel chromatography (hexane / AcOEt = 1 / 0
to 5 / 1) afforded 23 (126 mg, 238 μmol, 28 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ: 8.17 (d, J = 8.3 Hz, 4H), 7.26 (d, J = 8.3 Hz, 4H),
7.48-7.45 (m, 4H), 7.42-7.38 (m, 4H), 7.37-7.33 (m, 2H), 7.12 (s, 2H), 5.39 (s, 4H),
5.13 (s, 1H), 2.37 (s, 3H). FAB-MS m/z: 528 (M+), 529 (M+H+).
4.1.31. 2'-Hydroxy-5'-methyl-[1,1':3',1''-terphenyl]-4,4''-dicarboxylic acid (24)
Under an Ar atmosphere, 10 % Pd/C (21.5 mg) was added to a solution of 23 (70.2
mg, 133 μmol) in 1,4-dioxane (2.0 mL). Then atmosphere was replaced with H2. The
reaction mixture was stirred for 22 h at rt, then filtered through Celite. The filtrate was
reprecipitated (EtOH / hexane) to afford 24 (5.10 mg, 14.6 μmol., 11 %) as a white
solid.
o
mp decomposed (248 C). 1H-NMR (500 MHz, CD3OD) δ: 8.06 (d, J = 8.3 Hz, 4H),
7.64 (d, J = 8.3 Hz, 4H), 7.11 (s, 2H), 3.35 (s, 1H), 2.35 (s, 3H). FAB-MS m/z: 348 (M+),
349 (M+H+). FAB-HRMS m/z: 348.0995 (Calcd for C21H16O5: 348.0998).
4.2. Biology
4.2.1. ELISA-based PBD-binding inhibition assay
Biotin-GGGGG-PLHSpT peptide (PH Japan) was first diluted with coating solution
(KPL Inc., 50-84-01) to the final concentration of 0.1 μM, and then 100 μl of the
resulting solution was immobilized onto a 96-well streptavidin-coated plate (Thermo
Fisher Scientific Inc., 236004) for 90 min. To block unoccupied sites, wells were
washed 3 times with PBST (PBS (pH 7.4) + 0.05% Tween 20) and incubated with 100
μl of blocking buffer (PBS + 1% BSA (Sigma, A2153)) for 90 min. HeLa lysate was
prepared by homogenization with a sonicator (SHIMADZU, USP-600A) in
homogenization buffer (20 mM Tris-maleate (pH 7.0), 150 mM NaCl, 5 mM EGTA,

1.5 mM EDTA, 0.5 mM Na3VO4 and 20 mM disodium 4-nitrophenyl phosphate),
centrifuged at 105000 g for 60 min at 4 oC (Beckman, Optima L-70 Ultracentrifuge) and
the supernatant diluted with homogenization buffer to the assay concentration (5.0 x 106
cells / mL for HeLa cells). Then, 225 μL / well of the resulting lysate was applied a
96-well plate (IWAKI, 3860-096) and 25 μL / well of DMSO solution of 10 x test
compounds was added to the plate . Pre-incubation was carried out for 30 min at rt.
Blocking buffer was removed from the streptavidin-coated plate and the plate was
washed 3 times with PBST. Then, 100 μL / well mixture of HeLa lysate and test
compounds were added to the plate in duplicate and incubation was continued for 60
min at rt. To terminate the reaction, the ELISA plate was washed 3 times with PBST.
For detection of bound biotin-GGGGG-PLHSpT peptide and Plk1, plates were
incubated for 1 h with 100 μl / well of monoclonal anti-Plk1 antibody (Novus
Biologicals, NB100-74502 or Thermo Fisher Scientific Inc., MA1-848) at a
concentration of 1 μg / mL in the blocking buffer. The plates were washed 3 times, then
100 μl / well of an HRP-conjugated secondary antibody (Cell Signaling Technology,
Inc., #7076) diluted to 1:500 in the blocking buffer was added to each well and
incubation was continued for 1 h at rt. Afterwards, the plates were washed 5 times with
PBST and incubated with 100 μL / well of 3,3′,5,5′-tetramethylbenzidine solution
(TMB) (Cell Signaling Technology, Inc., #7004S) for 15 min at rt. The reactions were
terminated by the addition of 100 μL / well stop solution (Cell Signaling Technology,
Inc., #7002S) and the optical density for each sample was measured at 450 nm by using
a plate reader (PerkinElmer Co., Ltd., ARVO-SX).
4.2.2. Assay for inhibitory activity of Plk1
In this assay, a CycLex Polo-like kinase 1 Assay/Inhibitor Screening Kit (CycLex Co.
Ltd., CY-1163) was used and the assay was performed according to the kit instructions.
We used recombinant Plk1 enzyme (CycLex Co. Ltd., CY1163E), which is the same as
the constitutive active form of human full-length Plk1 with the mutation T210D in the
active-site loop of kinase domain, together with an N-terminal GST tag. To investigate
the mechanism of inhibition of Plk1 by terphenyl compounds, assay was performed at a
normal ATP concentration (62.5 μM final concentration) and at a high ATP
concentration (3.12 mM final concentration).

Acknowledgement
The work described in this paper was partially supported by Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and Technology of
Japan, and the Japan Society for the Promotion of Science.

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Figure 1. Plk1 kinase inhibitors under clinical trial
Figure 2. Compounds known to inhibit the interaction between Plk1 PBD and
phosphorylated peptide.
Figure 3. Binding mode of phosphorylated pentapeptide PLHSpT and Plk1 PBD, and
illustration of the designed terphenyl compounds.
Figure 4. ELISA-based assay for evaluating the binding of Plk1 PBD and
phosphorylated peptide. Plk1 kinase activity was calculated based on the value of the
DMSO-treated sample, taken as 100 %. The maximum solubility of compound 4 was
about 100 μM under the buffer conditions for our assay system.
Figure 5. Terphenyl compound 6 dose-dependently inhibited Plk1 kinase activity. Plk1
kinase activity was calculated based on the value of the DMSO-treated sample, taken as
100 %.
Figure 6. Terphenyl compound
6
inhibited Plk1 kinase activity in
a
non-ATP-competitive manner. Plk1 kinase activity was calculated based on the value of
the DMSO-treated sample, taken as 100 %.
o
Scheme 1. Reagents and conditions: (a) ICl-pyridine, CH2Cl2 / H2O = 2 / 1, 0 C to rt;
(b) 2-iodoethanol, Cs2CO3, DMF, 0 oC to 80 oC; (c) 4-cyanophenylboronic acid pinacol
ester, PdCl2(dppf), K3PO4, DMF, 80 oC; (d) KOH, EtOH, reflux; (e) NH4Cl, LiCl, NaN3,
DMF, 100 oC.
o
o
Scheme 2. Reagents and conditions: (a) benzyl bromide, K2CO3, DMF, 0 C to 100 C;
o
(b) (Ph3)2PdCl2, Bis(pinacolate)diboron, KOAc, 1,4-dioxane, 100 C; (c) 10a-c,
o
PdCl2(dppf), K3PO4, DMF, 80 C; (d) 2N NaOH aq., MeOH, rt, or H2, Pd/C,
1,4-dioxane, rt.

o
Scheme 3. Reagents and conditions: (a) benzyl bromide, K2CO3, DMF, 100 C; (b)
o
(Ph3)2PdCl2, bis(pinacolate)diboron, KOAc, 1,4-dioxane, 100 C; (c) 15, PdCl2(dppf),
K3PO4, DMF, 80 oC; (d) 2N NaOH aq., MeOH, rt.
Scheme 4. Reagents and conditions: (a) MeNH2 in MeOH, HOBt•H2O, DIPEA, EDCI,
CH2Cl2, rt; (b) (Ph3)2PdCl2, bis(pinacolate)diboron, KOAc, 1,4-dioxane, 100 oC; (c) 18,
PdCl2(dppf), K3PO4, DMF, 80 oC.
o
Scheme 5. Reagents and conditions: (a) NBS, DMF, 0 C to rt; (b) (Ph3)2PdCl2,
o
bis(pinacolate)diboron, KOAc, 1,4-dioxane, 100 C; (c) 3, PdCl2(dppf), K3PO4, DMF,
80 oC.
o
Scheme 6. Reagents and conditions: (a) 9a, PdCl2(dppf), K3PO4, DMF, 80 C, (b) H2,
Pd/C, 1,4-dioxane, rt.
Table 1. Inhibition ratio of Plk1 kinase activity
a inhibition ratio at 300 μM
b inhibition ratio at 250 μM (300 μM could not be tested because of poor solubility)
Table 2. IC50 values of 6 and staurosporine at normal and high ATP concentrations

(table 1)

(table 2)

(Figure 4)
900
800
700
600
500
400
300
200
100
0
3 μM
10 μM 30 μM 100 μM 0.1 mM 0.3 mM 0.8 mM 1 mM 0.1 mM 0.3 mM 0.8 mM 1 mM 0.1 mM 0.3 mM 0.8 mM 1 mM
4
5
6
7

(Figure 5)
120
100
80
60
40
20
0
PLHSpT 100 μM
30 μM
100 μM
300 μM
1000 μM
6