Synthesis and evaluation of biphenyl compounds as kinesin spindle protein inhibitors

Article abstract of DOI:10.1002/cbdv.201200400

Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)) assays, combined with fluorescence-assisted cell sorting (FACS) and Western blot studies analyzing cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds. Copyright

Full text of DOI:10.1002/cbdv.201200400

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein
Inhibitors
by Jason P. Holland*a)b), Albert Kangb), Susan Cohrsc), Svetlana V. Selivanovab), Selena Milicevic
Sephtonb), Thomas Betzelb), Daniel Freyd), Mara Wieserd), Rolf Jaussid), Richard A. Kammererd),
Roger Schiblib)c), and Eliane Fischer*c)
a) Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, and
Department of Radiology, Harvard Medical School, 55 Fruit St., White 427, Boston, 02114, MA, USA
(phone: þ1-(617)-726-6107; fax: þ1-(617)-726-6165; e-mail: holland.jason@mgh.harvard.edu)
b) Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences,
ETH Zꢀrich, CH-8093 Zꢀrich
c) Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, CH-5232 Villigen PSI
(phone: þ41-56-310-2857; fax: þ41-56-310-2849; e-mail: eliane.fischer@psi.ch)
d) Laboratory of Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen PSI
Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar
spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a
novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new
biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting
KSP expression. Antiproliferation (MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide)) assays, combined with fluorescence-assisted cell sorting (FACS) and Western blot studies
analyzing cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range
of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds
with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group
with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using
confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far,
compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster
formation. These studies confirm that chemical scope remains for improving the potency and treatment
efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.
Introduction. – Hyperproliferation – the increased rate of cellular division – is a
primary hallmark of cancer [1]. Many anticancer agents used in the clinic inhibit cell-
cycle progression during either the synthesis (S) or mitotic (M) phase. Subsequent
activation of checkpoints typically induces cell-cycle arrest or programmed cell death
(apoptosis). Antiproliferative drugs can be subdivided into three main categories [2]: i)
drugs that induce DNA damage and/or inhibit DNA repair mechanisms (e.g.,
topoisomerase-IIa inhibitors); ii) drugs that interfere with DNA synthesis in the S
phase (e.g., DNA intercalators (doxorubicin) and cross-linkers (cisplatin)); and iii)
drugs that disrupt mitosis by inhibiting various components or functions of the mitotic
spindle. This third class includes the taxanes (paclitaxel), epothilones, and Vinca
alkaloids, each of which bind to microtubules (MTs) and disrupt the normal dynamic
instability of the bipolar spindle through either stabilizing or destabilizing interactions.
The efficacy of MT-targeted drugs resides in their ability to activate the M-phase
ꢁ 2013 Verlag Helvetica Chimica Acta AG, Zꢀrich

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
539
spindle checkpoint. Spindle checkpoint activation prevents the onset of anaphase and
typically leads to cell death by mitotic catastrophe or mitotic slippage, followed by
apoptosis during the subsequent growth phase (G1 phase).
Although MT-targeted drugs have proven successful in the clinic [3], treatment
often induces a range of unwanted side-effects including severe peripheral neuropathy
and hematopoietic toxicity. These side-effects derive from the fact that MTs also play
important roles in resting and differentiated cells like, for example, facilitating
intracellular transport of proteins or vesicles. In efforts to circumvent the observed
side-effects, new antimitotic agents directed toward crucial M-phase-specific kinase
and kinesin motor proteins associated with bipolar spindle function have been the
subject of intense investigations (Fig. 1) [2].
Fig. 1. Structures of selected allosteric KSP inhibitors
Recently, kinesin spindle protein (KSP, Kif11, hsEg5, or kinesin-5) has emerged as a
potential antimitotic target for drug discovery [4][5]. KSP is an ATP-dependent motor
protein that binds MTs and is required at early stages in mitosis (during prophase and
prometaphase) for both bipolar spindle formation and proper segregation of sister

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chromatids [6]. Inhibition of KSP results in an unusual cell phenotype in which the two
centrosomes fail to migrate to opposite poles of the cell, thereby leading to the
formation of a characteristic monopolar (monoastral) spindle [4][7][8]. Systematic
analysis of KSP mRNA expression levels revealed that, relative to neurons and
fibroblasts, KSP is highly expressed in proliferating human tissue. Importantly, KSP
protein levels were also shown to be overexpressed in various tumor tissues [8][9].
Preclinical studies have shown that kinesin inhibitors have fewer side-effects than MT-
targeted agents, including reduced neuronal toxicity, and are also effective antiprolif-
eration agents against paclitaxel-resistant tumors [10]. Further, several allosteric, ATP-
noncompetitive KSP inhibitors such as ispinesib [11] and ARRY-520 [12] have been
investigated in Phase-I/II clinical trials (Fig. 1).
In 2007, Luo et al. reported the first ATP-competitive, allosteric KSP inhibitors
based on biphenyl derivatives [13]. Chemical optimization identified two lead
compounds, GSK-1 and GSK-2, which were both characterized as ATP-competitive,
but MT-noncompetitive KSP inhibitors with Ki values of 1.8ꢀ0.2 and 8.8ꢀ0.7 nm,
respectively. Interestingly, the binding mode of these biphenyl compounds was found to
be distinct from previous ATP-noncompetitive inhibitors such as the dihydropyrimi-
dine (S)-monastrol and quinazolinone-derivative (R)-ispinesib. Further, GSK-1 and
GSK-2 were shown to be potent antimitotic agents with increased activity in ispinesib-
resistant strains harboring the D130V mutant KSP; GSK-1 inhibits the proliferation of
parental HCT-116 colorectal cancer cells (IC50 36 nm) but showed increased potency
(IC50 0.5 nm) toward ispinesib-resistant HCT116-D130V cells [13].
Following the discovery of biphenyl derivatives as novel KSP inhibitors, further
studies probed the chemical scope of interaction with KSP by conducting structureꢁ
activity relationships (SARs) [14][15]. Parrish et al. synthesised 58 compounds with
varying substituents on both phenyl rings [14]. They found that the para-trifluoro-
methyl (CF3) substituent was a conserved feature of the pharmacophore required for
KSP inhibition. This effect was also noted to be due to steric contributions related to
the size rather than electronic properties of the CF3 group. In addition, they found that
the most potent analogs had a urea or sulfamide group in the para-position of the
second phenyl ring.
Here, we present data evaluating several biphenyl compounds as potential KSP
inhibitors. Specifically, we report the synthesis of a new, more active thiourea derivative
and also the effect of functionalizing the urea group with aliphatic or aromatic moieties.
The new compounds were studied by using a range of techniques including chemical
characterization and protein inhibition assays in vitro. The effect of our compounds on
various human cancer cells (prostate cancer cells: PC-3 and DU-145; ovarian cancer
cells: SKOV-3; and breast cancer cells: MCF-7) was evaluated by using cellular growth
inhibition assays (MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-disphenyl-2H-tetrazolium
bromide)), cell-cycle analysis with fluorescence-assisted cell sorting (FACS), and
Western blot studies, as well as confocal fluorescence microscopy studies. From the
outset, our hypothesis was that chemical modification of the terminal substituent on the
biphenyl system could be made so as to facilitate the introduction of an [18F]- or [11C]-
radiolabel for developing a novel positron-emitting radiotracer for noninvasive
positron-emission tomography (PET) imaging of the drug pharmacokinetics and
KSP expression in vivo. The aim of this work was to identify a lead biphenyl compound

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
541
that retained high biological activity and specificity toward KSP inhibition which could
potentially be developed as radiolabelled analog.
Results and Discussion. – Synthesis of Biphenyl Derivatives. The synthesis of
biphenyl derivatives 6–13 was accomplished in two-steps by using procedures modified
from previous reports (Scheme) [14–16]. Where appropriate, the chemical identity and
purity of all compounds has been fully characterized by using a range of techniques
1
including thin-layer chromatography (TLC), melting points, H-, 13C-, and 19F-NMR
spectroscopy, high-resolution mass spectrometry, IR spectroscopy, and high-perform-
ance liquid chromatography (HPLC; cf. the Exper. Part). In the first step, the CꢁC
bond linking the two aromatic groups in compounds 4 and 5 was formed by reaction of
either compound 1 or 2 with compound 3 in a classical Pd-catalyzed ([Pd(PPh3)4])
Suzuki cross-coupling [17]. Compound 4 was isolated as a deep-red amorphous powder
in 87% yield, whereas compound 5 was obtained as a white amorphous powder (77%).
For synthesis of compounds 6–13, the second step involved functionalization of the
anilinic NH2 group by reaction with chlorosulfonyl isocyanate (ClSO2NCO), ammo-
Scheme. Chemical Synthesis of Biphenyl Derivatives 6-13.
a) Standard Suzuki coupling reactions with [Pd(PPh3)4,] K2CO3 (aq.), DMF, 1008, 24 h. b) For
compounds 6 and 8: ClSO2NCO, r.t., 3 h; for 7 and 10: ammonium thiocyanate (NH4SCN), 1408, 15 h;
for 9 and 11: ClSO2NCO, 708, 5 h; for 12: PhCOCl, 508, 18 h; for 13: 3-methylbutanoyl chloride, r.t., 24 h.
Lit. ¼Yields reported in [14].

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
nium thiocyanate (NH4SCN), benzoyl chloride (PhCOCl), or 3-methylbutanoyl
chloride. In general, for compounds where R1 ¼F, reaction yields were moderate to
high (53–99%). However, due to solubility issues and difficulties associated with
purification, yields for the NO2 derivatives 9 and 10 were lower (21–43%), and it was
not possible to isolate compound 11 using our standard chromatography methods.
Nucleophilic aromatic substitution (SNAr) of the NO2 group as R1 by 18F represents a
potential (if in this case challenging) route to producing an [18F]-radiolabelled
compound. Unfortunately, lack of access to the radiolabelling precursor hindered our
efforts, and this route was not pursued further.
Density-Functional Theory (DFT) Calculations. Interestingly, unlike the ATP-
noncompetitive KSP inhibitors (Fig. 1) for which extensive characterization of the
binding mode and mechanism for KSP inhibition have been provided by the use of
biochemical assays and crystallography [9], no crystal structure exists of a biphenyl
compound bound to KSP. This is likely because biphenyl compounds are only active
against MT-bound KSP. The heterogeneous chemical nature of MT samples makes co-
crystallization difficult. In their SAR studies, Parrish et al. noted that linker atoms
inserted between the two phenyl groups were not tolerated, and they proposed that, for
an active conformation, the biphenyl group must remain close to planar [14]. To
investigate this possibility further, we conducted DFT calculations to probe the
electronic structure and preferred geometry of the biphenyl compounds (Fig. 2).
Fig. 2. DFT-Optimized geometry of compound 7 showing the twisted orientation between the two planes
formed by the phenyl groups. Dihedral angle f(C(1)ꢁC(2)ꢁC(3)ꢁC4))ꢁ39.08.
The geometry of compound 7 was optimized by using Gaussian09 with the
restricted B3LYP exchange-correlation functionals and the double-z 6-31þ þG(d,p)
all electron basis set [18]. Contrary to the supposition by Parrish et al., we found that, in
the absence of a protein interaction, the biphenyl derivatives prefer a twisted
orientation with a dihedral angle f of ꢁ39.08 formed between the intersecting planes
of the two aromatic rings. All other bond lengths and angles were found to be within
normal parameters for small organic molecules [19]. In the absence of crystallographic
data, the precise binding mode to KSP remains uncertain. However, we note that,
although our DFT calculations predict a twisted geometry, a planar orientation cannot

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
543
be precluded, since stabilizing interactions between the biphenyl ligand and KSP may
overcome the associated rotational energy barrier.
Kinesin Motor Protein Inhibition Assays. After successful isolation of the biphenyl
compounds, we first investigated their ability to inhibit the enzymatic ATP-hydrolysis
activity of KSP, as well as their selectivity for KSP vs. a panel of selected kinesin motor
proteins (Fig. 3 and Table 1). Kinesins investigated included centromeric protein-E
(CENP-E), mitotic centromere-associated kinesin (MCAK), kinesin heavy chain
(KHC), and kinesin protein KIFC3. In addition, (S)-monastrol and (R)-ispinesib were
used as positive controls, and the inactive stereoisomer (S)-ispinesib was used as a
negative control for KSP inhibition.
Fig. 3. Bar chart showing the specificity of compound 7 toward inhibition of KSP ATPhydrolysis in vitro.
(S)-Monastrol and (R)-ispinesib were used as positive controls, while the inactive enantiomer, (S)-
ispinesib, was used as a negative control.
Table 1. Kinesin Motor Protein ATP-Hydrolysis Activity [%] Relative to Untreated Controls
Compound (conc. [mm])
KSP
CENP-E
MCAK
KHC
KIFC3
(S)-Monastrol (10)
(R)-Ispinesib (1)
(S)-Ispinesib (1)
6 (1)
7 (1)
8 (1)
9 (1)
10 (1)
12 (100)
13 (100)
10.8ꢀ1.4
8.8ꢀ1.4
99.4ꢀ1.1
4.4ꢀ1.4
3.7ꢀ1.7
5.5ꢀ2.0
95.1ꢀ3.2
94ꢀ4.0
97.7ꢀ2.2
98 ꢀ3.7
97ꢀ2.1
98.5ꢀ1.0
101.5ꢀ4.3
96.8ꢀ2.3
99.1ꢀ1.1
99.9ꢀ1.1
98.4ꢀ2.2
99.1ꢀ1.7
98.7ꢀ2.0
99.2ꢀ1.4
99.1ꢀ1.5
98.3ꢀ2.6
97.3ꢀ2.8
97.9ꢀ2.5
96.4ꢀ2.8
100.7ꢀ3.7
101.3ꢀ3.0
98.9ꢀ1.8
98.4ꢀ1.9
98.6ꢀ1.3
100.4ꢀ1.3
96.7ꢀ3.2
99.2ꢀ2.3
98.3ꢀ2.6
99.1ꢀ1.2
98.4ꢀ2.2
96.0ꢀ4.1
99.6ꢀ2.2
98.1ꢀ1.8
99.4ꢀ3.0
99.1ꢀ1.6
98.3ꢀ2.0
97.3ꢀ1.7
98.8ꢀ2.7
95.1ꢀ4.0
98.2ꢀ2.1
98.7ꢀ2.2
98.9ꢀ1.4
97.7ꢀ1.0
98.4ꢀ3.1
These assays indicated that only compounds 6–8 displayed activity and high
selectivity towards KSP inhibition in vitro. These data demonstrate that NO2
derivatives, i.e., compounds 9 and 10, as well as the terminally substituted compounds
12 and 13 failed to inhibit ATP-hydrolase activity of KSP or other kinesins studied.
Unfortunately, these data also suggest that addition of a bulky aliphatic or aromatic (as
typically required when using radiolabelled prosthetic groups for derivatizing small

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molecules or peptides) is a not feasible route toward developing [18F]- or [11C]-
radiolabelled compounds for this class of molecules.
Cellular-Growth-Inhibition Assays. We next investigated the potency of compounds
6–8 toward cellular-growth inhibition in a range of human cancer cell lines. Previous
work on KSP inhibition has been focused on the use of drugs like ispinesib to inhibit the
growth of colorectal carcinomas (using HCT116 cells) and ovarian cancer (SKOV-3
cells) [8]. In this work, we were interested in evaluating the potential of biphenyl
compounds to inhibit KSP in different cell lines, namely prostate cancer lines PC-3 and
DU-145, and the breast cancer cell line MCF-7. SKOV-3 Cells and treatment with (S)-
monastrol were used as positive controls. Inhibition of cellular proliferation was
determined by using the standard MTT assay, and data on the drug concentration at
which proliferation was inhibited by 50% (growth-inhibition (GI50) values) are
compiled in Table 2. An example of the growth-inhibition curves generated for
compounds 6–8 in DU-145 cells is shown in Fig. 4.
Fig. 4. Growth-inhibition curves derived from MTT assays of compounds 6–8 in DU-145 cells
Table 2. The GI50 Values [mm] for Various Biphenyl Derivatives Tested in Four Human Cancer Cell Lines
Compound
DU-145
PC-3
MCF-7
SKOV-3
(S)-Monastrol
6
7
8
9
159ꢀ57
57.0ꢀ17.8
1.76ꢀ0.54
0.62ꢀ0.24
39.3ꢀ23.8
458ꢀ474
42.7ꢀ21.3
0.46ꢀ0.15
0.37ꢀ0.16
0.38ꢀ0.17
NM
11.4ꢀ2.6
1.59ꢀ0.54
0.52ꢀ0.16
0.45ꢀ0.16
NM
1.19ꢀ0.52
0.15ꢀ0.05
0.97ꢀ0.29
NMa)
10
NM
418ꢀ410
NM
NM
a) NM¼Not measured.
The MTT data indicate that after 44 h incubation, biphenyl compounds 6–8 were
found to be active toward all four cell lines. In particular, the novel thiourea compound
7 was found to inhibit DU-145 cellular proliferation with a GI50 value of 0.15ꢀ0.05 mm.
In comparison, the published urea analog (6; GSK-2) was found to be eight fold less
potent (GI50 1.19ꢀ0.52 mm) in the same cells. Interestingly, this result was not

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545
reproduced across all cell lines. In general, PC-3 and SKOV-3 cells were found to be less
sensitive toward treatment with biphenyl drugs than DU-145 and MCF-7 cells. In
addition, we also tested NO2 derivatives 9 and 10 in PC-3 cells; both compounds were
found to be inactive at mm concentrations, thus confirming our results from the protein
inhibition assays (vide supra). First, these antiproliferative data indicate that minor
structural or electronic changes in the biphenyl compound, such as replacing O with S
(urea to thiourea) can induce comparatively large effects in their ability to inhibit
cellular growth. Second, our data also point toward a pronounced cell-line dependence
in the efficacy of KSP inhibition using biphenyl-based drugs. Reasons for this cell-line
dependence remain unclear.
Cell-Cycle Analysis. To confirm that the antiproliferative effects observed in the
MTT assays were the result of inhibition of cellular proliferation in the M phase, we
investigated changes in cell-cycle population numbers upon treatment with the
biphenyl drugs. Cells were treated with 1.0 mm concentrations of compounds 6–8 for a
period of 24 h before staining with propidium iodide, and using fluorescence-assisted
cell sorting (FACS) to analyse the number of cells in various stages of the cell cycle (G1,
S, and G2/M phases). FACS Data for treatment of cells with the most potent compound
7 are presented in Table 3.
Table 3. Percentage of Cells in the G1, S, and G2/M Phases of the Cell Cycle upon Treatment with Either
Vehicle (< 1% DMSO in medium) or Compound 7 (1.0 mm) at 37 8C for 24 h
Cell phase
Cell line
DU-145
Control
PC-3
MCF-7
Control
SKOV-3
Control
Treated
Control
Treated
Treated
Treated
G1
S
G2/M
57.8
6.5
31.6
35.9
11.6
47.9
39.6
12.1
39.8
13.9
14.9
62.6
54.9
20.0
21.7
23.4
11.9
60.8
56.9
19.7
20.0
5.8
14.8
69.5
In each cell line, treatment with compound 7 led to a dramatic shift in the number of
cells found within the G2/M phase of the cell cycle compared to vehicle-treated (<1%
DMSO in medium) controls. For example, in SKOV-3 cells, treatment with 7 induced
an increase in the G2/M-phase population from 20 to ca. 70% of cells. Somewhat
surprising was the small shift observed for the more sensitive DU-145 cells (G2/M-
phase control, 32%; treated, 48%). For DU-145 cells, we noted that a greater number
of the cells were found to be apoptotic, and hence, excluded from the cell-cycle analysis
by FACS gating. This observation is consistent with the MTTassays which revealed that
DU-145 cells are particularly sensitive toward KSP inhibition. After only 24 h
incubation, FACS data indicated that these drugs induce M-phase arrest in all cell lines,
but, at this point, the cellular response diverges: DU-145 cells undergo rapid apoptosis
(likely in the M phase due to mitotic catastrophe), whereas less sensitive lines such as
PC-3 and SKOV-3 cells appear to remain in G2/M-phase arrest or undergo mitotic
slippage, followed by apoptosis in the G1 phase. These data are consistent with the
known mechanism of action of KSP inhibition with other inhibitors, including
monastrol, ispinesib, and progenitor biphenyl compounds [4][8][13].

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Western-Blot Protein Analysis. As confirmation of the induced M-phase arrest, we
measured relative changes in the concentration of three protein markers displaying
characteristic expression levels in different stages of the cell cycle by using Western
blotting (Fig. 5). Here, we interrogated the change in expression levels of cyclin B1,
cyclin E, and phospho-histone H3 (PHH3) as markers of the G2/M and G1/S phases and
of mitosis, respectively. In all experiments, glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was used as a protein loading control. The Western blot data provide further
demonstration of the G2/M-phase cell-cycle arrest induced up on treatment with
compound 7. With the exception of PHH3 levels in DU-145, markedly increased levels
of both cyclin B1 and PH3 were observed across all cell lines after treatment.
Furthermore, although control DU-145 and PC-3 cells showed only low levels of cyclin
E, treatment did induce a loss of this protein marker, consistent with cell-cycle arrest in
the G2/M phase and the hypothesis that cell lines which are particularly sensitive to
Fig. 5. Western-blot data showing the relative change in cell-cycle protein marker expression between cells
treated with compound 7 and control cells. a) Relative expression levels of cyclin B1 and cyclin E as
markers of the G2/M phase and G1/S phase, respectively. Numbers indicate Lanes 1–8: DU-145 (Lanes 1
and 2); PC-3 (Lanes 3 and 4); MCF-7 (Lanes 5 and 6); SKOV-3 (Lanes 7 and 8). b) Relative expression
of phosphor-histone 3 (PH3) as a mitosis marker lanes. DU-145 (Lanes 1 and 2); PC-3 (Lanes 3 and 4);
SKOV-3 (Lanes 5 and 6). In both gels, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used
as a protein loading control.

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KSP inhibition (like DU-145) undergo apoptosis resulting from mitotic catastrophe, as
opposed to mitotic slippage and G1-phase apoptosis.
Confocal Fluorescence Microscopy Studies. After having established the relative
sensitivity of various human cancer cell lines toward treatment with biphenyl drugs and
elucidated the likely mechanism of action, we next investigated the induced phenotypic
effects using confocal fluorescence microscopy. The primary roles of KSP are
associated with bipolar spindle formation and force generation on interpole MTs.
Inhibition of KSP is known to induce M-phase arrest during prophase/prometaphase,
which prevents separation of the two centrosome to opposite poles of the cell. Thus,
KSP inhibition leads to a failure to establish a functional bipolar spindle, and gives rise
to a characteristic monoaster phenotype [4].
Human cancer cells were grown for 22 h on 8-well-chambered microscope slides,
before treating with either compound 7 (1.0 mm) or vehicle (<1% DMSO in medium)
at 378C for 24 h. Cells were then fixed and permeabilized before staining with an anti-
a-tubulin antibody (and a secondary antibody labelled with Alexa 568), to probe the
structure of the MT network, and Hoechst 33342 to stain for DNA. Selected confocal
fluorescence microscopy images are presented in Fig. 6.
In the control (vehicle-treated) SKOV-3 cells, we observed cells undergoing normal
mitotic division in which a well-defined bipolar spindle, with the chromosomes aligned
on the metaphasic plate, was clearly visible (Fig. 6,d). In contrast, upon examination of
cells treated with compound 7, we observed that the majority of cells in the samples
treated with compound 7 exhibited a monoaster phenotype (Fig. 6,a–c). Results were
confirmed by using (R)-ispinesib and (S)-ispinesib as positive and negative controls,
respectively (data not shown).
Interestingly, we also observed that not all of the cells in the treated samples
displayed a monoaster spindle (Fig. 6,b). Due to the restricted expression of KSP in
the early stages of mitosis, treatment with KSP inhibitors such as compound 7 induces
M-phase arrest in mitotic cells but fails to affect cells in interphase. Thus, cells which did
not enter M phase during the treatment window were not affected by the drugs. These
data are congruent with the results obtained from FACS and Western blot analysis
where we observed an increase in the accumulation of cells in the G2/M phase. Taken
together, our experimental data provide compelling evidence that the antiproliferative
effect of biphenyl dervatives 6–8 in a range of human cancer cell lines resides in their
inhibition of KSP.
Conclusions. – Here, we reported the synthesis and in vitro characterization of a
range of biphenyl compounds as inhibitors of KSP and potential antimitotic agents.
Replacement of the urea functionality with a thiourea led to an increased potency of
the compounds toward growth inhibition in a range of human cancer cell lines.
Structural modification centered on functionalizing the terminal amine group with a
bulky aliphatic or aromatic group was found to be detrimental to KSP inhibition
activity. The nature of the antiproliferative effects observed for compounds 6–8 was
evaluated further by using a range of assays in vitro, including kinesin motor protein
inhibition assays, FACS, Western blot analysis, and confocal fluorescence microscopy.
These data confirmed that compound 7, to the best of our knowledge, the most potent
antiproliferative biphenyl derivative identified thus far, exerts its pharmacologic effect

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Fig. 6. Representative confocal fluorescence-microscopy images showing the effects of treatment of
SKOV-3 cells with compound 7 at 1.0 mm after incubation at 378 for 22 h. a) A collection of four cells
displaying the characteristic monoaster phenotype indicative of KSP inhibition and M-phase arrest. b)
Two SKOV-3 cells in the same sample. The cell above displays a monoaster spindle whereas the control
cell (below) remains in the growth phase. c) Magnified view of the monoaster formation showing MTs
radiating from the center of the cell and a diffuse pattern of DNA staining. d) A representative image of
a vehicle-treated SKOV-3 cell undergoing normal mitosis (anaphase). For full microscopy details,
including magnification powers, see the Exper. Part.

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549
via inhibition of KSP and induced M-phase arrest. Unfortunately, our structureꢁactiv-
ity-relationship data indicate that appending the biphenyl compounds with bulky
aliphatic or aromatic substituents is not a viable route for radiolabelling this class of
molecules. Due to the complications of synthesizing the NO2 derivatives and the likely
low yields from attempts at direct [18F]-radiolabelling by an SNAr mechanism, we
conclude that these two approaches are not suitable for radiolabelling these biphenyl
compounds. Alternative approaches involving [18F]-radiolabelling of the CF3 group are
under development.
Funded in part by the ETH Fellowship Program (FEL-01-10-1; JPH). We thank Dr. Aristeidis
Chiotellis and Cindy Fischer for helpful discussions and advice regarding chemical synthesis and
chromatography. We also thank Drs. Tobias Schwarz and Joaquim Hehl at the Light Microscopy Centre,
ETH Zurich, for technical support regarding fluorescence microscopy studies.
Experimental Part
General. Standard laboratory techniques were employed when handling H2O-sensitive reagents. All
solvents and reagents were used as received from the supplier unless otherwise noted. Flash column
chromatography (FC) was performed by using silica gel (BDH) according to the method of Still et al.
[20]. TLC was performed on aluminium plates pre-coated with silica gel (0.2 mm, Merck 60 F254),
visualized by using UV fluorescence (254 and 366 nm) and KMnO4 staining. RP-HPLC was performed
on a Merck Hitachi HPLC (monitoring at a wavelength of 254 nm) equipped with a LUNA 5m C18
column (5 mm, 250ꢂ4.60 mm), eluting with an isocratic mobile phase of 60% MeCN and H2O with 0.1%
CF3COOH (TFA) at a flow rate of 1.0 ml/min unless otherwise specified. M.p.: Bꢀchi 535 melting-point
apparatus. IR Spectroscopy: Jasco FT/IR-6200, OMNILAB spectrometer. 1H-, 13C-, and 19F-NMR
spectra: at 400, 100, and 376 MHz, resp., on a Bruker Ultra-Shield AV400 spectrometer; chemical shifts
(d(H), d(C), and d(F)) are expressed in ppm, referenced to the residual solvent resonance of
1
(D6)DMSO or CDCl3 for H- and 13C-NMR, and to the internal standard resonance of CFCl3 for 19F-
NMR, coupling constants (J) to the nearest 0.1 Hz. ESI-MS (m/z (rel. %)) and HR-ESI-MS: Bruker
Daltonics maxis (UHR-TOF) Compass 1.3 spectrometer.
Syntheses. 3-Fluoro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-amine (4). To 4-bromo-2-fluoroaniline (2;
1.20 g, 6.32 mmol, 1.2 equiv.) and [4-(trifluoromethyl)phenyl]boronic acid (1; 1.00 g, 5.27 mmol,
1.0 equiv.) were added 2.0m aq. K2CO3 (8.78 ml, 55.3 g K2CO3 in 200 ml H2O) and DMF (8.78 ml) at
r.t., and the mixture was stirred for 15 min at this temp. to give a pale yellow soln. [Pd(PPh3)4] (304 mg,
263 mmol, 0.05 equiv.) was then added, and the mixture was heated at 1258 for 24 h under N2. The
mixture was subsequently cooled to r.t., poured onto 50% aq. NaHCO3, then extracted with Et2O (3ꢂ
70 ml), dried (Na2SO4), filtered on a pad of silica gel with Celite, and concentrated under reduced
pressure. Purification by FC (0–15% AcOEt/hexane) gave 4 (1.11 g, 87%). White amorphous powder. Rf
(30% AcOEt/hexane) 0.57. M.p. 93–948. 1H-NMR ((D6)DMSO): 7.80 (d, J ¼ 8.2, F3CC(CH)2); 7.71 (d,
J ¼ 8.3, F3C(CH)2(CH)2CC); 7.45 (dd, J ¼ 2.1, 13.1, FCCH); 7.33 (dd, J ¼ 2.0, 8.3, H2NCCHCHCC); 6.86
(q, J ¼ 8.4, 9.4, H2NCCH); 5.45 (br. s, NH2). 13C-NMR ((D6)DMSO): 151.9 (CNH2); 149.6 (CF); 143.4
(CCCHCF); 137.1 (CCF3); 137.0 (FCCHCC); 126.0 (F3CC(CH)4C); 125.6 (CF3); 123.1 (2 C,
CCCHCH); 116.35 (d, J ¼ 10.0, H2NCCH); 113.3 (d, J ¼ 20, FCCH). 19F-NMR ((D6)DMSO): ꢁ60.7
(s, CF3); ꢁ134.7 (dd, J ¼ 9.5, 13.3, arom. F). ESI-MS: 255 (100, [MþH]þ ). HR-ESI-MS: 256.2227
([MþH]þ , C13H10F4Nþ ; calc. 256.0744). Data are in accordance with those in [14].
3-Nitro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-amine (5). To 4-bromo-2-nitroaniline (3; 500 mg,
2.30 mmol, 1.0 equiv.) and 1 (525 mg, 2.76 mmol, 1.2 equiv.) were added 2.0m aq. K2CO3 (3.78 ml) and
DMF (3.78 ml) at r.t., and the mixture was stirred for 15 min. [Pd(PPh3)4] (128 mg, 0.111 mmol,
0.05 equiv.) was then added, and the mixture was heated at reflux under N2 and stirred at 1008 for 21 h.
The mixture was subsequently cooled, poured onto 50% aq. NaHCO3, extracted with Et2O (3ꢂ70 ml),
dried (Na2SO4), filtered on a pad of silica gel with Celite, and concentrated under reduced pressure.
Purification by FC (10–50% AcOEt/hexane) gave compound 5 (501 mg, 77%). Deep-red amorphous

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
powder. Rf (70% AcOEt/hexane) 0.30. M.p. 154–1558. 1H-NMR ((D6)DMSO): 7.68 (s,
F3CC(CH)2)(CH)2); 6.93 (d, J ¼ 2.1, O2NCCH); 6.82 (dd, J ¼ 2.1, 8.0, H2NCCHCHCC); 6.60 (d, J ¼
8.0, H2NCCH); 4.69 (s, NH2). 13C-NMR ((D6)DMSO): 145.2 (CNH2); 136.1 (O2NCCHC); 135.2
(O2NCCHCC); 128.7 (CCF3); 127.0 (CNO2); 125.7 (F3CC(CH)4C); 125.5 (CF3); 116.2, (CCCHCH);
114.5 (H2NCCH); 112.5 (O2NCCH). 19F-NMR ((D6)DMSO): ꢁ60.6 (s, CF3). ESI-MS: 283 (100, [Mþ
H]þ ). HR-ESI-MS: 283.1177 ([MþH]þ , C13H10F3N2Oþ2 ; calc. 283.0689).
N-[3-Fluoro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]urea (6). To a soln. of 4 (100 mg, 0.392 mmol,
1.0 equiv.) in CH2Cl2 (4.67 ml) was added ClSO2NCO (52.8 ml, 0.608 mmol, 1.55 equiv.). The mixture was
stirred at r.t. for 3 h, during which a white precipitate was formed. The reaction was then quenched with
H2O (4.67 ml), and the mixture was stirred at r.t. for 18 h. The mixture was filtered, washed with H2O
(20 ml), and dried in vacuo. Purification by FC (20–70% AcOEt/hexane) gave 6 (71.2 mg, 61%). White
amorphous powder. Rf (50% AcOEt/hexane) 0.21. Anal. HPLC: 99.3% purity, tR 7.43 min. M.p. 286–
2878. 1H-NMR ((D6)DMSO): 8.50 (d, J ¼ 2.2, NH); 8.29 (t, J ¼ 8.6, 8.6, HNCCH); 7.86 (d, J ¼ 8.2,
F3CC(CH)2); 7.77 (d, J ¼ 8.3, F3C(CH)2(CH)2CC); 7.64 (dd, J ¼ 2.1, 12.9, FCCH); 7.51 (dd, J ¼ 1.8, 8.6,
HNCCHCHCC); 6.26 (s, NH2). 13C-NMR ((D6)DMSO): 155.5 (C¼O); 153.0 (CNH); 150.6 (CF); 142.7
(CCCHCF); 131.6 (CCF3); 131.5 (FCCHCC); 126.9 (F3CC(CH)4C); 125.7 (CF3); 122.9 (CCCHCH);
120.3 (HNCCH); 113.1 (FCCH). 19F-NMR ((D6)DMSO): ꢁ60.9 (s, CF3); ꢁ130.0 (t, J ¼ 10.9, 11.4,
arom. F). ESI-MS: 299 (100, [MþH]þ ). HR-ESI-MS: 299.0799 ([MþH]þ , C14H11F4N2Oþ ; calc.
299.0802).
N-[3-Fluoro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]thiourea (7). To a soln. of 4 (1.10 g, 4.33 mmol,
1.0 equiv.) in H2O (8.66 ml) and 1m HCl (8.66 ml) was added NH4SCN (822 mg, 10.8 mmol, 2.5 equiv.),
and the mixture was heated at 1408 for 5 h to give a white solid in a colorless soln. The mixture was then
cooled to r.t. and poured onto ice (2.62 g), washed with cold H2O (20 ml), and placed in an ice/water bath
overnight. The white precipitate was collected by filtration and dried in vacuo. The residue was purified
by FC (20–60% AcOEt/hexane) to give 7 (1.03 g, 76%). White amorphous powder. Rf (50% AcOEt/
hexane) 0.32. Anal. HPLC: 99.8% purity, tR 7.64 min. M.p. 186–1878. 1H-NMR ((D6)DMSO): 9.50
(s, NH); 7.94 (mc, F3CC(CH)2(CH)2CCCHCHCNH); 7.81 (d, J ¼ 8.4, F3C(CH)2(CH)2CC); 7.69 (dd,
J ¼ 2.0, 12.0, FCCH); 7.57 (dd, J ¼ 1.8, 8.4, HNCCHCHCC). 13C-NMR ((D6)DMSO): 182.0 (C¼S);
156.9 (CNH); 154.5 (CF); 142.4 (CCCHCF); 136.8 (CCF3); 136.7 (FCCHCC); 127.7 (HNCCH);
127.3 (F3CC(CH)4C); 125.8 (CF3); 122.5 (CCCHCH); 114.2 (FCCH). 19F-NMR ((D6)DMSO):
ꢁ61.0 (s, CF3); ꢁ122.8 (s, arom. F). ESI-MS: 315 (100, [MþH]þ ). HR-ESI-MS: 315.0556 ([MþH]þ
, C14H11F4N2Sþ ; calc. 315.0574).
N-[3-Fluoro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]sulfamide (8). To a soln. of ClSO2NCO (37.2 ml,
0.427 mmol, 1.09 equiv.) in MeCN (0.91 ml) at 08 was added H2O (7.69 ml, 0.427 mmol, 1.09 equiv.). The
mixture was stirred at 08 for 1 min, then allowed to warm to r.t., and stirred for a further 3 h at the same
temp. Then, the mixture was cooled to 08, and a soln. of 4 (100 mg, 0.392 mmol, 1.0 equiv.) in pyridine
(63.4 ml, 0.784 mmol, 2.0 equiv.) and MeCN (0.90 ml) was added dropwise over 2 min. The mixture was
allowed to warm to r.t. and stirred for an additional 15 h. The pale-yellow soln. was subsequently diluted
with H2O (50 ml), washed with sat. aq. NaCl (10 ml), extracted with AcOEt (3ꢂ50 ml), dried (Na2SO4),
and concentrated under reduced pressure. Purification by FC (10–30% AcOEt/hexane) gave 8 (69.4 mg,
53%). White amorphous powder. Rf (30% AcOEt/hexane) 0.33. Anal. HPLC: 98.9% purity, tR 7.74 min.
M.p. 151–1528. 1H-NMR ((D6)DMSO): 9.34 (br. s, NH); 7.92 (d, J ¼ 8.2, F3CC(CH)2); 7.80 (d, J ¼ 8.4,
F3C(CH)2(CH)2CC); 7.66 (dd, J ¼ 1.9, 12.1, FCCH); 7.58 (mc, HNC(CH)2CC); 7.23 (s, NH2). 13C-NMR
((D6)DMSO): 155.2 (CNH); 152.8 (CF); 142.4 (CCCHCF); 134.8 (CCF3); 134.7 (FCCHCC); 126.9
(F3CC(CH)4C); 125.7 (CF3); 123.4 (CCCHCH); 122.9 (HNCCH); 114.15 (FCCH). 19F-NMR
((D6)DMSO): ꢁ60.9 (s, CF3); ꢁ124.4 (dd, J¼7.2, 12.2, arom. F). ESI-MS: 357 (100, [MþNa]þ ).
HR-ESI-MS: 357.0289 ([MþNa]þ , C13H10F4N2NaO2Sþ ; calc. 357.0291). Data in accordance those in
[14].
N-[3-Nitro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]urea (9). To a soln. of 5 (100 mg, 0.355 mmol,
1.0 equiv.) in CH2Cl2 (4.20 ml), was added ClSO2NCO (61.6 ml, 0.710 mmol, 2.0 equiv.) at 708, and the
mixture was heated at reflux the same temp. for 5 h during which a dark-red soln. was obtained. The
reaction was then quenched with H2O (4.20 ml) to give a white precipitate, and the mixture was stirred at
r.t. for a further 48 h. The mixture was then filtered, washed with H2O (20 ml) and sat. aq. NaHCO3, and

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
551
dried in vacuo. Purification by FC (1–15% MeOH/CH2Cl2) gave 9 (49.4 mg, 43%). White amorphous
powder. Rf (70% AcOEt/hexane) 0.23. Anal. HPLC: 91.5% purity, tR 8.86 min, eluted with isocratic 40%
MeCN/H2O with 0.1% TFA. M.p. 315–3188. 1H-NMR ((D6)DMSO): 8.24 (d, J ¼ 2.2, NH); 7.99 (s,
O2NCCH); 7.83 (s, F3C(CH)2(CH)2CC); 7.80 (s, HNCCH); 6.18 (s, H2N). 13C-NMR ((D6)DMSO): 156.6
(C¼O); 144.1 (CNH); 141.7 (CNO2); 132.6 (CCCHCNO2); 131.9 (CCF3); 131.1 (NO2CCHCC); 126.8
(F3CC(CH)4C); 125.7 (CF3); 122.8 (HNCCH); 121.4 (HNCCHCH); 121.4 (O2NCCH). 19F-NMR
((D6)DMSO): ꢁ60.9 (s, CF3). ESI-MS: 326 (100, [MþH]þ ). HR-ESI-MS: 326.2238 ([MþH]þ
,
C14H11F3N3Oþ3 ; calc. 326.2086).
N-[3-Nitro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]thiourea (10). To
a soln. of 5 (100 mg,
0.355 mmol, 1.0 equiv.) in H2O (423 ml) and 1.0m HCl (355 ml) was added NH4SCN (54.0 mg, 0.710 mmol,
2.0 equiv.), and the mixture was heated at 1508 for 18 h. The mixture was subsequently cooled to r.t.,
poured onto ice (2.32 g), washed with cold H2O (20 ml) and sat. aq. NaHCO3, and placed in an ice-bath
for 1 h. The brown precipitate formed was filtered on a pad of silica gel and dried in vacuo. The residue
was purified by FC (60–90% AcOEt/hexane) to give 10 (25.0 mg, 21%). Brown amorphous powder. Rf
(90% AcOEt/hexane) 0.83. Anal. HPLC: 98.2% purity, tR 12.4 min, eluted with isocratic 50% MeCN/
H2O with 0.1% TFA. M.p. 306–3088. 1H-NMR ((D6)DMSO): 12.7 (br. s, NH2); 7.91 (d, 7.1, F3CC(CH)2);
7.83 (d, J ¼ 7.3, F3C(CH)2(CH)2); 7.54 (d, J ¼ 7.8, HNCCH); 7.45 (s, O2NCCH); 7.29 (d, 8.0,
HNCCHCH). 13C-NMR ((D6)DMSO): 169.1 (C¼S); 144.2 (CNO2); 142.4 (CNH); 133.1 (O2NCCHCC);
132.6 (CCF3); 132.4 (O2NCCHCC); 127.4 (F3CC(CH)4C); 125.8 (CF3); 121.8 (HNCCH); 109.9
(HNCCHCH); 107.7 (O2NCCH). 19F-NMR ((D6)DMSO): ꢁ61.2 (s, CF3). ESI-MS: 342 (100, [Mþ
H]þ ). HR-ESI-MS: 342.9828 ([MþH]þ , C14H11F3N3O2Sþ ; calc. 342.9711).
N-[3-Fluoro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]benzamide (12). To a soln. of 4 (70.0 mg,
0.274 mmol, 1.0 equiv.) in Et3N (37.9 ml) and CH2Cl2 (1.26 ml) was added PhCOCl (47.9 ml, 0.411 mmol,
1.5 equiv.), and the mixture was heated at 508 for 18 h. The mixture was subsequently cooled to r.t.,
washed with CH2Cl2 (50 ml) and sat. aq. NaHCO3, and extracted with CH2Cl2 (3ꢂ30 ml). The combined
org. layers were washed with brine and concentrated in vacuo. Purification by FC (0–40% AcOEt/
hexane) gave 12 (107 mg, 99%). White amorphous powder. Rf (20% AcOEt/hexane) 0.37. Anal. HPLC:
98.6% purity, tR 10.3 min, eluted with isocratic 70% MeCN/H2O with 0.1% TFA. M.p. 210–2118.
1H-NMR ((D6)DMSO): 10.23 (br. s., NH); 8.01 (d, J ¼ 7.2, C(O)C(CH)2); 7.97 (d, J ¼ 8.2, F3CC(CH)2);
7.83 (d, J ¼ 8.3, F3CC(CH)2(CH)2); 7.80 (d, J ¼ 8.4, HNCCH); 7.75 (mc, FCCH); 7.65 (s, HNCCHCH);
7.62 (d, J ¼ 7.4, C(O)C(CH)2(CH)2CH); 7.55 (t, J ¼ 7.6, 7.4, C(O)C(CH)2(CH)2). 13C-NMR
((D6)DMSO): 165.5 (C¼O); 157.0 (CF); 154.5 (HNC); 142.4 (FCCHCC); 137.0 (FCCHCC); 136.9
(F3CC); 133.8 (C(O)C); 131.9 (C(O)C(CH)2(CH)2CH); 128.4 (C(O)(CH)2(CH)2CH); 127.8
(C(O)(CH)2(CH)2CH); 127.4 (F3CC(CH)2(CH)2); 127.1 (HNCCH); 125.8 (CF3); 122.8 (HNCCHCH);
114.3 (FCC). 19F-NMR ((D6)DMSO): ꢁ61.4 (s, CF3). ESI-MS: 360 (100, [MþH]þ ). HR-ESI-MS:
360.1010 ([MþH]þ , C20H14F4NOþ ; calc. 360.1006).
N-[3-Fluoro-4’-(trifluoromethyl)[1,1’-biphenyl]-4-yl]-3-methylbutanamide (13). To a soln. of 4
(70.0 mg, 0.274 mmol, 1.0 equiv.) in Et3N (37.9 ml) and CH2Cl2 (1.26 ml) was added 3-methylbutanoyl
chloride (50.6 ml, 0.411 mmol, 1.5 equiv.), and the mixture was stirred at r.t. for 24 h. The mixture was
subsequently cooled to r.t. and washed with CH2Cl2 (50 ml) and sat. aq. NaHCO3, and extracted with
CH2Cl2 (3ꢂ30 ml). The combined org. layers were washed with brine (30 ml) and concentrated in vacuo.
Purification by FC (0–40% AcOEt/hexane) gave 13 (90.1 mg, 88%). White amorphous powder. Rf (20%
AcOEt/hexane) 0.38. Anal. HPLC: 98.5% purity, tR 10.4, eluted with isocratic 70% MeCN/H2O with
0.1% TFA. M.p. 159–1608. 1H-NMR ((D6)DMSO): 9.76 (br. s, NH); 8.03 (t, J ¼ 8.3, 8.4, HNCCH); 7.92
(d, J ¼ 8.2, F3CC(CH)2); 7.80 (d, J ¼ 8.3, F3CC(CH)2(CH)2); 7.69 (dd, J ¼ 12.2, 1.7, FCCH); 7.57 (dd, J ¼
8.4, 1.3, HNCCHCH); 2.29 (d, J ¼ 7.2, C(O)(CH)2); 2.09 (dt, J ¼ 13.6, 6.8, C(O)(CH)2CH); 0.95 (d, J ¼
6.6, Me2CH). 13C-NMR ((D6)DMSO): 171.2 (C¼O); 155.0 (CF); 152.6 (HNC); 142.4 (FCCHCC); 135.2
(FCCHCC); 127.2 (F3CC(CH)2(CH)2); 126.5 (F3CC); 125.8 (CF3); 124.4 (HNCCH); 122.7
(HNCCHCH); 44.9 (C(O)C); 25.6 (C(O)CH2C); 22.2 (Me2CH). 19F-NMR ((D6)DMSO): ꢁ60.8 (s,
CF3). ESI-MS: 340 (100, [MþH]þ ). HR-ESI-MS: 340.1323 ([MþH]þ , C18H18F4NO; calc. 340.1319).
Density-Functional Theory (DFT) Calculations. Calculations were conducted using DFT [21][22] as
implemented in the Gaussian09, suite of ab initio quantum chemistry programs [18]. Geometry
optimizations and vibrational frequency calculations were performed with a restricted scheme using the

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
hybrid-DFT B3LYP exchange-correlation functionals and the all electron double-z 6-31þ þG(d,p) basis
set by Pople and co-workers for all atoms. Normal SCF and geometry convergence criteria were used
throughout, and no symmetry constraints were imposed. Harmonic frequency analysis based on
analytical second derivates was used to characterize the optimized geometries as local minima.
Cloning, Expression, and Purification of the Human KSP (Kif11/Eg5) Motor Domain. A synthetic
cDNA fragment encoding residues 1–368 of human Kif11 and codon-optimized for bacterial expression
was cloned into pSTCm1, a modified pET47b vector. The plasmid was transformed to chemically
competent BL21[DE3] bacterial cells, plated onto LB-Kan plates, and incubated overnight at 378. A
single colony was picked and used to inoculate a 100-ml LB-Kan overnight culture which was grown at
378 in an incubator shaker (200 rpm). 4L of LB-Kan were inoculated with 20 ml (1:50) of the overnight
culture and grown in an incubator shaker at 378 and 200 rpm to an OD of 0.6. The temp. was lowered to
208 before induction with 1 mm IPTG, and incubation was continued overnight at 208 and 200 rpm.
Cells were harvested by centrifugation, and the pellet was taken up in 50 ml of lysis buffer, 50 mm
Tris (pH7.5), 500 mm NaCl, 10 mm 1H-imidazole, 10 mm B-mercapthoethanol, 1 protease inhibitor
cocktail tablet (Complete EDTA free; Roche Diagnostics). Cells were lysed on ice by using a Vibra Cell
sonication device (Sonics) equipped with a large prep. sonication tip by applying three 1-min sonication
bursts at 100% amplitude. The lysate was cleared by 25000g centrifugation step (1 h) and passed through
a 0.45-mm filtration step.
The protein was subsequently purified using IMAC, followed by a size-exclusion chromatography on
an ꢁKTA Xpress chromatography machine. The filtered supernatant was applied on a 5-ml HisTrap
crude FF column and washed with 20CV (column volumes) of wash buffer 1 (50 mm Tris (pH7.5),
500 mm NaCl, 10 mm 1H-imidazole, 10 mm b-mercaptoethanol) and 20CV wash buffer 2 (50 mm Tris
(pH7.5), 500 mm NaCl, 60 mm 1H-imidazole, 10 mm b-mercaptoethanol). The protein was eluted using
5CV of elution buffer (50 mm Tris (pH7.5), 500 mm NaCl, 500 mm 1H-imidazole, 10 mm b-mercapto-
ethanol). The eluted protein was loaded onto a 16/60 HiLoad Superdex200 size-exclusion column
equilibrated with 20 mm Tris·HCl (pH7.5) and 150 mm NaCl. The total yield was ca. 15 mg of pure
protein.
Kinesin Motor Protein ATP-Hydrolase Inhibition Assay. Inhibition of the ATP-hydrolase activity of
various kinesin motor proteins was measured by using the Kinesin ATPase End-Point Biochem Kit,
BK053 (Cytoskeleton, Denver, USA). Generation of inorg. phosphate (Pi) during MT-activated ATP-
hydrolase activity of kinesin motor proteins was measured at 650 nm. The concentration of the motor
proteins (2.5 mg of total protein) used in the assay was kept as low as possible to minimize possible
crowding effects on MTs. Compounds were diluted to give final concentrations of 1.0 mm and were
incubated at r.t. for 10–20 min. Assays were conducted in accordance with the manufacturerꢂs protocol.
Antiproliferative MTT Assays (in vitro). DU-145, PC-3, MCF-7, and SKOV3 cell lines, cell culture
media, and additives were obtained from BioConcept (CH-Allschwil). Cells were grown in plastic tissue
culture flasks. For PC-3 cells, Dulbeccoꢂs Modified Eagleꢂs Medium (DMEM) with 10% Fetal Calf
Serum (FCS; v/v) was supplemented with glutamine (2 mm) and antibiotics (penicillin (100 units/ml),
streptomycin (100 mg/ml), and fungizone (0.25 mg/ml)). For DU-145 cells, Minimum Essential Medium
Eagle (MEM) with 10% FCS (v/v), glutamine (2 mm), and antibiotics was supplemented with sodium
pyruvate (1 mm), sodium bicarbonate (1.5 g/l), and 1% non-essential amino acids (NEAA). For MCF-7
cells, DMEM with Nutrient Mixture F-12 Ham (Hamꢂs F-12) and 10% FCS (v/v) was supplemented with
glutamine (2 mm) and antibiotics (penicillin (100 units/ml), streptomycin (100 mg/ml), and fungizone
(0.25 mg/ml)). For SKOV-3 cells, DMEM with 10% FCS and glutamine (2 mm) was supplemented with
antibiotics (penicillin (100 units/ml), streptomycin (100 mg/ml), and fungizone (0.25 mg/ml)). All the cells
were maintained at 378 with 5% CO2.
Test compounds, in DMSO stock solns., were prepared in concentrations ranging from 1.0 nm to
1.0 mm. The test compounds were serially diluted (1:4) with growth medium and then diluted into the
cell assay plate with a final DMSO concentration of ꢃ0.2%. Cells were plated in duplicate 96-well plates
with a density of 2000 cells/well with the exception of MCF-7, where cell density was 1000 cells/well, and
allowed to adhere for 24 h. Cells were then exposed to the test compounds which were added directly to
the media. Appropriate controls for culture media and vehicle were used throughout to measure non-
specific background, and to serve as a reference to each cell line. Plates were incubated at 378 for 44 h,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
553
then treated with MTT soln., and cultured for 2–4 h. After removing the media, DMSO (100 ml/well) was
added to solubilize the purple formazan product. Electronic absorbance was then measured at 560 nm
with Perkin-Elmer VictorTM X3 Multilabel Plate Reader. The extent of growth inhibition was determined
by nonlinear regression analysis of a plot of absorbance (560 nm) vs. drug concentration (mm), and
analyzed with GraphPad Prism 5. Data are reported as the average of four quadruplet runs, and the
statistical limits for the data are reported to be within the 95% confidence interval.
Flow Cytometry. DU-145, PC-3, MCF-7, and SKOV3 cell lines were grown in DMEM supplemented
with 10% fetal bovine serum and 1 unit/ml penicillin-streptomycin. Cells were plated onto 100 mm2
dishes at a cell density of 1.0ꢂ106 cells/dish, and 24 h later, they were treated with 1.0 mm of the biphenyl
compound. Cells were harvested 24 h after treatment. The culture medium was collected, and the floating
cells were collected by short centrifugation (5 min at 1000 rpm). Attached cells were washed once with
ice-cold PBS (5 ml), detached by treatment with trypsin/EDTA soln. (3 ml) at 378 for ca. 5 min, and,
together with the detached floating cells, were transferred into 15 ml Falcon tubes. The cell culture dish
was washed with DMEM (10 ml) to collect all the remaining cells. After centrifugation for 5 min at
1000 rpm at r.t., the supernatant was discarded, and the cell pellet was washed twice with 10 ml PBS. The
supernatant was discarded, and the cells were resuspended in 4 ml of ice-cold fixation soln. (70 vol%
EtOH, 30 vol% H2O) and stored at ꢁ208 for 24 h. Thereafter, PBS (10 ml) stored at 48 was added to the
cells before centrifugation for 5 min at 1000 rpm. The supernatant was discarded, and the cell pellet was
washed once with PBS (10 ml; 48). The cell pellet was resuspended in 1.0 ml of staining soln. containing
0.1 mg/ml RNAse A (Sigma-Aldrich), 50 mg/ml propidium iodide (PI) from 2.5 mg/ml stock soln., and
0.05% Triton X-100 (Fluka) in PBS. After incubation at 378 for 40 min in the dark, PBS (3 ml) was
added, and the cell pellet was collected by centrifugation for 5 min at 1500 rpm, followed by the removal
of the supernatant. The pellet was suspended in PBS (500 ml) and analyzed using the Millipore easyCyte
HT flow cytometry system (excitation, 488 nm, emission, 583 nm). Data were analyzed by cell-cycle
analysis software (Flowjo, TreeStar Inc.).
Western Blot. DU-145, PC-3, MCF-7, and SKOV-3 cells were grown in 10-cm dishes to ca. 70%
confluence in DMEM containing 10% FCS and incubated at 378 for 24 h. Cells were then treated with
1.0 mm of biphenyl compounds and incubated at 378 for 24 h. Cells were washed twice with PBS and lysed
on ice in a lysis buffer consisting of 0.1m NaCl, 0.05m Tris ·His (pH 7.5), 1% Triton X-100, 0.001m
ethylenediaminetetraacetic acid (EDTA), 0.01m b-glycerophosphate, 0.02m sodium vanadate, and
protease inhibitor cocktail CompleteTM (Roche Diagnostics, Switzerland).
Lysates were cleared by centrifugation, and 20 mg of protein, as determined by the ꢃAdvanced
Protein Assayꢂ (Sigma-Aldrich, CH-Buchs), were separated by SDS-PAGE (7.5%) under reducing
conditions and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore AG, CH-Zug)
with a semi-dry blotting device (BioRad Laboratories AG, CH-Reinach). Incubations with primary
antibodies (Cyclin B1, Cyclin E, and Phospho-Histone H3) were performed according to the
manufacturerꢂs protocol at 48 overnight in TBST (0.14m NaCl, 0.02m Tris·1.1 HCl (pH 7.5), 0.1%
Tween 20) containing 2% bovine serum album (BSA). Detection was accomplished with secondary
HRP-linked antibodies (3 ml in 15 ml for 30 min) from Cell Signalling Technologyꢄ (Bioconcept) and the
ECL chemiluminescence kit from Pierce (Perbio Science Switzerland SA, CH-Lausanne).
Confocal Fluorescence Microscopy. Permeabilization Buffer (prepared fresh). A 10ꢂpermeabiliza-
tion buffer was prepared as a stock soln. (1.54m NaCl, 15.44 mm KH2PO4, 28.58 mm Na2HPO4 ·7 H2O,
and 5% Triton X-100). To 10ꢂ permeabilization buffer (5 ml) were added 40 ml deionized H2O. The pH
was adjusted to pH7.2, and then the soln. was diluted to 50 ml to make a 1.0ꢂ permeabilization buffer
stock soln. Stock solns. can be kept for several weeks in a refrigerator. To 1.0ꢂ permeabilization buffer
(2.4 ml) were added 9.6 ml PBS to give 0.2ꢂ permeabilization buffer used as working soln.
Blocking Buffer. A soln. of PBS containing 1% BSA and 0.3% Tween-20 was prepared.
Confocal Fluorescence Microscopy. Cells lines, cell culture media, and additives were obtained from
BioConcept. Cells were maintained at 378 with 5% CO2 and were placed in 8-well Lab-Tek II Chamber
Slides (NUNC, VWR, LabShop, Batavia, IL) with a density of 5000 cells/well, and allowed to adhere for
24 h and to grow to 80% confluence. Cells were then exposed to 1.0 mm concentrations of biphenyl
compounds and incubated for 22 h at 378. All plates contained wells with culture media only to serve as a
reference control to the SKOV-3 cell line.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Cells were washed with PBS (3ꢂ400 ml), incubated with freshly prepared, pre-warmed 4%
paraformaldehyde (400 ml) for 15 min at 378, washed with PBS (3ꢂ400 ml), incubated with a freshly
made 0.2ꢂ permeabilization buffer (300 ml) for 15 min at 378, washed with PBS (3ꢂ400 ml), incubated
with 1 drop of ImageitFX (Invitrogen ꢃSignal Enhancerꢂ) for 30 min, washed with PBS (3ꢂ400 ml) and
incubated with primary antibody (250 ml, Mouse a-tubulin (1:1000 dilution in blocking buffer; Sigma-
Aldrich)) at 378 for 2 h. Then, cells were washed with PBS (3ꢂ400 ml), incubated with secondary
antibody (250 ml, Goat a-Mouse Alexa 568 a-tubulin staining (Invitrogen), 1:2000 dilution in blocking
buffer) at 378 for 1.5 h then washed with PBS (3ꢂ400 ml), incubated with Hoechst 33342 1:100000 in
deionized H2O for 10 min, and finally washed with deionized H2O (3ꢂ400 ml). Slides were drained, and
all areas surrounding the tissue were dried, mounted onto microscope slides with one drop of Prolong
Gold (Invitrogen), sealed with nail polish (Gemey, Express Finish), and kept in the dark at 48.
Images were captured using a Zeiss LSM 510 laser-scanning confocal microscope, 63ꢂ oil DIC Plan-
Apochromat objective, 1.4NA. The microscope was incubated in an EMBL incubator box GP 168 at
36.58. Solid-state (561–10 nm) and dDiode (405–430 nm) laser lines were used to image Alexa 568 a-
tubulin staining and Hoechst 33342, resp. Images were processed using AIM LSM4.0 software (Carl
Zeiss) and ImageJ (National Institutes of Health, USA).
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Received November 25, 2012