Kinesin spindle protein (KSP) inhibitors with 2,3-fused indole scaffolds

Article abstract of DOI:10.1021/jm100476d

Mitotic kinesin spindle protein (KSP) is involved in the assembly of the bipolar spindle during cell division. On the basis of a common 2,3-fused indole substructure within the complex frameworks of terpendole E and other KSP inhibitors, the carbazoles with a bulky alkyl group were identified as a novel KSP inhibitory scaffold. Additionally, among several naturally occurring cell growth inhibitors with 2,3-fused indole structures,β-carboline alkaloids, harman and harmine, showed moderate inhibition of KSP.

Full text of DOI:10.1021/jm100476d

5054 J. Med. Chem. 2010, 53, 5054–5058
DOI: 10.1021/jm100476d
Kinesin Spindle Protein (KSP) Inhibitors with 2,3-Fused Indole Scaffolds
Shinya Oishi,*,† Toshiaki Watanabe,† Jun-ichi Sawada,‡ Akira Asai,‡ Hiroaki Ohno,† and Nobutaka Fujii*,†
†Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, and ‡Graduate School of Pharmaceutical
Sciences, University of Shizuoka, Suruga-ku, Shizuoka 422-8526, Japan
Received April 19, 2010
Mitotic kinesin spindle protein (KSP) is involved in the assembly of the bipolar spindle during cell
division. On the basis of a common 2,3-fused indole substructure within the complex frameworks of
terpendole E and other KSP inhibitors, the carbazoles with a bulky alkyl group were identified as a
novel KSP inhibitory scaffold. Additionally, among several naturally occurring cell growth inhibitors
with 2,3-fused indole structures, β-carboline alkaloids, harman and harmine, showed moderate
inhibition of KSP.
Introduction
Mitosis is a highly regulated process to separate the repli-
cated sister chromatids into the daughter cells during cell
division. Among a number of kinesin motor proteins with a
catalytic motor domain driven by ATP hydrolysis, kinesin
spindle protein (KSP,a also known as Eg5), a member of the
kinesin-5 family, is associated with mitotic spindle assembly in
the early stages of mitosis.1 A bipolar homotetramer of KSP
cross-links and moves antiparallel microtubules apart to
prompt centrosome separation.2 Impairment of KSP induces
mitotic arrest in prometaphase without affecting microtu-
bules, resulting in apoptotic cell death.3 Accordingly, signifi-
cant efforts have been made in developing KSP inhibitors as
potential antitumor agents4 since the identification of the first
KSP inhibitor, monastrol.5
Figure 1. Structures of the reported KSP inhibitors 1-3 and the
potential KSP inhibitory 2,3-fused indole scaffold 4.
Terpendole E 1 is a fungal-derived KSP inhibitor,6 which
was originally identified as a minor isolate of the acyl-CoA:
cholesterol acyltransferase(ACAT) inhibitor.7 Thecorestruc-
ture of 1 consists of indole and diterpene moieties. HR22C162
is also a cell membrane-permeable KSP inhibitor with a
tetrahydro-β-carboline structure that is observed in a number
of natural products.8 Furthermore, moderate in vitro KSP
inhibitory activity of N-substituted carbazole 3 was recently
reported.9,10 Although these three compounds apparently
include an exquisite complex framework with cyclic and/or
acyclic accessory substituents, it was envisaged that a 2,3-fused
indole 4 would be the common minimal scaffold for KSP
inhibitory activity (Figure 1).11,12
Recently, we have accomplished the facile preparation of
carbazole derivatives, which represent 2,3-fused indole deri-
vatives. Of note, carbazoles were obtained via one-pot con-
secutive reactions including N-arylation of anilines and the
oxidative biaryl coupling in the presence of a palladium cata-
lyst (Scheme 1).13 A combination of aryl triflates 5 and
Scheme 1. One-Pot Synthesis of Carbazoles by N-Arylation
and Oxidative Biaryl Coupling
substituted anilines 6 can improve the structural diversity of
carbazole derivatives 7. By exploiting this carbazole library,
the current study was undertaken to identify the minimal
chemical space for KSP inhibitory activity from the precedent
inhibitors. Furthermore, the structure-activity relationship
study on carbazole-based KSP inhibitors and identification of
KSP inhibitory alkaloids were also conducted.
Results and Discussion
Chemistry. A series of carbazole derivatives 8-10, 11e,
and 12-15 were prepared by palladium-catalyzed one-pot
N-arylation-oxidative biaryl coupling (Scheme 1),13 except
for the compounds indicated below. Carbazoles 12c, 14b,
and 14f were synthesized by palladium-catalyzed intramole-
cular C-H arylation of N-phenyl-2-haloaniline derivatives,
which were obtained by N-arylation using o-chloroaniline
*To whom correspondence should be addressed. Phone: þ81-75-753-
4551. Fax: þ81-75-753-4570. E-mail: soishi@pharm.kyoto-u.ac.jp (S.O.);
nfujii@pharm.kyoto-u.ac.jp (N.F.).
a Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; CENP-E,
centromere-associated protein E; KSP, kinesin spindle protein; MKLP-1,
mitotic kinesin-like protein 1.
r
pubs.acs.org/jmc
Published on Web 06/03/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5055
16a/substituted bromobenzene 17a or substituted anilines
16b,c/o,o-dihalobenzenes 17b,c (Scheme 2). Alternatively,
arylation of an enamine intermediate prepared from 2-bro-
moaniline 16d and cyclohexane-1,3-dione 18 conferred cyclo-
hexenone-fused indole derivative 15c. Carbazoles 12f, 12j,
and 15a were obtained by simple transformations such as
tert-butyl modification of 12d, saponification of 10f, and
N-methylation of 8f, respectively. Several carbazoles or the
related derivatives (11a,f, 12g, 13b, 14d,e,g, 15b,e,f) were
prepared according to previous literature.14
Identification of 2- or 3-Substituted Carbazoles and
β-Carboline Alkaloids as KSP Inhibitors. A small library
of substituted carbazoles 8-10, which were prepared by
one-pot carbazole synthesis, was screened using a micro-
tubule-activated KSP ATPase assay. Among the 28 com-
pounds examined, several carbazoles exhibited potent or
moderate inhibition at 20 μM (Table 1). The most potent
carbazoles 8e (IC50 = 0.30 μM), 8f (IC50 = 0.26 μM), and
9e (IC50 = 0.95 μM) possess a bulky t-Bu or CF3 groups at
the carbazole 2- or 3-position. In contrast, these carbazoles
did not inhibit the ATPase activity of the other motor
proteins, centromere-associated protein E (CENP-E), kid,
KIF-4, and mitotic kinesin-like protein 1 (MKLP-1) even at
20 μM, indicating the effects of the compounds are KSP-
selective without direct binding to microtubules.
Scheme 2. Synthesis of Carbazole Derivatives
Inhibition of KSP activity by reported cell proliferation
inhibitors15 with a 2,3-fused indole substructure or related
scaffolds was also investigated. Two topoisomerase II in-
hibitors, azatoxin 11a14d and ellipticine 11b,15a showed no
inhibitory activity. Tryprostatin A 11c,15b fumitremorgin C
11d,15b murrayafoline A 11e,15c and a substituted carbazole
11f,14g which were reported to arrest the cell cycle in the G2/
M-phase, did not inhibit the KSP motor activity (Figure 2).
Interestingly, moderate inhibitory activity of KSP using
β-carboline alkaloids, harman 11g (IC50 = 32 μM) and
harmine 11h (IC50 = 38 μM), was observed. These com-
pounds were previously reported to exhibit cytotoxicity,
mutagenic, and neurotoxic effects by binding to DNA or
through topoisomerase I/II inhibition.16 This novel KSP
inhibitory effect may be a secondary mode-of-action for
the potent cytotoxicity of aromatic β-carboline alkaloids.
The bioactivities of the KSP inhibitory compounds 8e,f,
9e, and 11g,h on topoisomerases I and II were examined
(Figure 3) because the chemical space around the 2,3-fused
indole derivatives appears to be shared among the known
KSP and topoisomerase inhibitors. Harmine 11h inhi-
bited the DNA relaxation mediated by topoisomerases,
whereas no inhibitory activities on either topoisomerases
were observed when carbazoles 8e,f and 9e were present,17
Table 1. Structures and KSP Inhibitory Activities of the Substituted Carbazoles Screened
a 20 μM of each carbazole was employed for the KSP ATPase assay.

5056 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Oishi et al.
supporting the notion that these carbazoles are specific
KSP inhibitors.
Structure-Activity Relationship Study of Carbazole-Based
Inhibitors. On the basis of the advantageous potency
and selectivity for KSP inhibition by 8e,f and 9e, a further
structure-activity relationship study was performed for
carbazole derivatives (Table 2). KSP ATPase inhibition
and antiproliferative effects were evaluated. Carbazoles
12a,b and CF2H-substituted 12c, which include a smaller
number of Me- and F-substituting groups on the benzyl
position, exhibited less potent KSP inhibitory activity
compared with carbazoles 8e and 8f, respectively. Insertion
of an ether linkage between the carbazole core and the
bulky alkyl group (12e,f) and modification with carbonyl
groups (12g-i) were ineffective in improving the bioacti-
vity. Moderate to potent KSP inhibitory activity of 10f and
12k with the secondary accessory 6-CO2Me and 7-CF3
groups, respectively, was observed, while no cell growth
inhibition was exhibited at 60 μM. For the carbazole
3-position, modification with the t-Bu group provided the
best inhibitor 13a testified by in vitro KSP and cell pro-
liferation assays. 1-Substituted carbazoles 14a-f and
1-phenyl-β-carboline 14g, which resemble the podophyllo-
toxin-like scaffold, showed no or less potent KSP inhibitory
activity.
Investigation of additional modifications onto 2- or 3-sub-
stituted carbazole derivatives was also conducted (Table 3).
N-Methylation of carbazole 8f led to a significant decrease in
the inhibitory activity [IC50 (15a) = 5.6 μM]. Cyclohexene- or
cyclohexenone-fused derivatives 15b,c failed to inhibit the
KSP ATPase even at 20 μM. Carbazoles with an additional
saturated or aromatic fused ring structure at 2,3- or 1,2-
positions exhibited moderate KSP inhibitory activity. These
results indicate that the planar tricyclic structure of carba-
zoles with a single bulky substituting group is the minimal
scaffold for KSP inhibition and that t-Bu- and CF3-groups at
the 2- or 3-position are the most favorable accessory groups.
These carbazoles 8e,f, 9e, and 13a exhibited up to 30-fold
more potency in KSP ATPase inhibition compared with the
known KSP inhibitor 2, while similarly or less potent inhibi-
tion against HeLa cell growth was observed. The low cell
membrane permeability or unspecific binding to the secondary
biomolecules could be attributable to the incompatible results.
Figure 2. Structures of the natural products or the related com-
pounds with a 2,3-fused indole substructure evaluated for KSP
inhibitory activity.
Figure 3. Effects of carbazoles 8e,f, 9e, and β-carbolines 11g,h (100
μM) on DNA topoisomerases I [topo I, (a)] and II [topo II, (b)].
Lane 1: DNA marker; lane 2, supercoiled DNA; lane 3, DNA/
enzyme; lane 4, DNA/enzymeþ9e; lane 5, DNA/enzymeþ8e; lane 6,
DNA/enzymeþ8f; lane 7, DNA/enzymeþ11g; lane 8, DNA/en-
zymeþ11h; lane 9, DNA/enzymeþcamptothecin (for topo I) or
þAMSA (for topo II).
Table 2. Structure-Activity Relationships of Carbazoles That Inhibit KSP
a IC50 values were derived from the dose-response curves generated from triplicate data points. b Inhibition of microtubule-activated KSP ATPase
activity. c Cytotoxic activity against HeLa cells after 48-h exposure to the compound. d Less than 50% inhibition at 20 μM. e Less than 50% inhibition
at 60 μM. f Not tested.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5057
Table 3. Structure-Activity Relationships of Carbazoles and the
Related Compounds for Inhibition of KSP
the complex structures in KSP specific inhibitors and by using
a library of carbazole derivatives prepared by the one-pot
carbazole synthesis. In addition, among several naturally
occurring cell growth inhibitors with 2,3-fused indole struc-
tures, moderate KSP inhibition of two β-carboline alkaloids,
harman and harmine, was demonstrated. An additional struc-
ture-activity relationship study accentuated that carbazoles
with a bulky alkyl group at the 2- or 3-position are the key
substructures relevant to the potent and selective KSP inhibi-
tory scaffold. Many natural secondary metabolites often have
complicated structures and bind to multiple target molecules
with moderate potency. This approach can also be used to
identify the lead scaffold within natural products for further
optimization processes, as well as to reveal the undefined
mode of action of natural products.
Experimental Section
General Procedure for the Synthesis of Carbazoles. Toluene
(0.4 mL) was added to a flask containing aryl triflate 5 (0.20 mmol),
aniline 6 (0.22 mmol), Pd(OAc)2 (10 mol %), XPhos (15 mol %),
and Cs2CO3 (0.24 mmol) under an argon atmosphere. The mixture
was stirred at 100 °Cfor1-2 h and then stirred at room temperature
for 5 min. AcOH (1.6 mL) was added to the mixture, and an oxygen
balloon was connected to the reaction vessel. The reaction mixture
was stirred at 100 or 120 °C for 7-20 h. After cooling, the reaction
mixture was diluted with ethyl acetate, washed with saturated
NaHCO3, dried over MgSO4, and concentrated in vacuo. Crude
material was purified by flash chromatography to afford the desired
carbazole. The purity of the compounds was determined as >95%
by HPLC analysis or combustion analysis. The characterization
data of newly synthesized carbazoles are shown in the Supporting
Information.
a IC50 values were derived from the dose-response curves generated
from triplicate data points. b Inhibition of microtubule-activated KSP
ATPase activity. c Cytotoxic activity against HeLa cells after 48 h
exposure to the compound. d Less than 50% inhibition at 20 μM. e Less
than 50% inhibition at 60 μM. f Not tested.
KSP ATPase Assay. The microtubules-stimulated KSP
ATPase reaction was performed in a reaction buffer [20 mM
PIPES-KOH (pH 6.8), 25 mM KCl, 2 mM MgCl2, 1 mM
EGTA-KOH (pH 8.0)] containing 38 nM of the KSP motor
domain and 350 nM microtubules in 96-well half area plates
(Corning). Each chemical in DMSO at different concentrations
was diluted 12.5-fold with the chemical dilution buffer [10 mM
Tris-OAc (pH7.4), 0.04% (v/v) NP-40]. After preincubation of
9.7 μL of the enzyme solution with 3.8 μL of each chemical
solution at 25 °C for 30 min, the ATPase reaction was initiated
by the addition of 1.5 μL of 0.3 mM ATP and followed by
incubation at 25 °C for a further 15 min. The reaction was
terminated by the addition of 15 μL of the Kinase-Glo Plus
reagent (Promega). The ATP consumption in each reaction was
measured as the luciferase-derived luminescence by ARVO
Light (PerkinElmer). At least three experiments were performed
per condition, and the averages and standard deviations of
inhibition rates in each condition were evaluated.
Figure 4. Monopolar spindle formation by treatment of HeLa cells
with carbazole derivatives (8e,f, 9e: 80 μM; 13a: 16 μM). Chromo-
somes are colored in blue, and R-tubulin in red.
Growth Inhibition Assay. HeLa cells were cultured in DMEM
medium (Wako) supplemented with 10% (v/v) FCS and anti-
biotics at 37 °C in a 5% CO2 incubator. Growth inhibition
assays using HeLa cells were performed in 96-well plates
(Greiner). HeLa cells were seeded at 5000 cells/well in 50 μL
of DMEM and placed for 6 h. Chemicals in DMSO were diluted
100-fold with the culture medium in advance. Following the
addition of 40 μL of the fresh culture medium, 30 μL of the
chemical diluents were also added to the cell cultures. The final
volume of DMSO in the medium was equal to 0.25% (v/v). The
cells under chemical treatment were incubated for a further 72 h.
The wells in the plates were washed twice with DMEM medium
(phenol-red minus) supplemented with 5% (v/v) FCS and
antibiotics. After 1 h incubation with 100 μL of the medium,
the cell culture in each well was supplemented with 20 μL of the
MTS reagent (Promega), followed by incubation for an addi-
tional 40 min. Absorbance at 590 nm of each well was measured
using a VersaMax plate reader (Molecular Devices).
However, high rates of HeLa cells were arrested in the
M-phase after 20 h exposure to 32 μM of the compounds
[relative mitotic index (% induction of 0.1 μM taxol): 8e,
37%; 8f, 70%; 9e, 57%; 13a, 76%]. The arrested cells in the
presence of the carbazoles 8e,f, 9e, and 13a had monopolar
spindles, the typical phenotype of KSP suppression, as a
major phenotype (Figure 4). It is of note that another
phenotype of cell cycle arrest at the G2/M-phase with
incomplete chromosome condensation was also observed
as a quite minor population following treatment with
carbazole 13a (Figure 4: 13a-2).18
Conclusions
We have identified a novel inhibitory carbazole scaffold
against mitotic kinesin KSP by comparative classification of

5058 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Oishi et al.
Acknowledgment. This research was supported in part by
the Targeted Protein Research Program and Grants-in-Aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. We are
indebted to Tomoko Kurose for excellent technical assistance.
T.W. is grateful for the JSPS Research Fellowships for Young
Scientists.
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