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Current Medicinal Chemistry, 2014, 21, 2691-2701

2691

Developments of Kinesin Spindle Protein Inhibitors as Antitumor Agents

Based on the Five-membered Heterocycle Scaffolds
Guo-Dong Zhao1, Ren-Zhong Wan*,2 and Zhao-Peng Liu*,1
1

Department of Organic Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China; 2College of Animal Science & Veterinary Medicine,
Shandong Agricultural University, Taian 271018, P. R. China
Abstract: Kinesin spindle protein (KSP) plays an essential role in centrosome separation and formation of the bipolar
mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for
developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Small molecule
KSP inhibitors have demonstrated their potential as novel antimitotic agents. Several KSP inhibitors have progressed into
clinical trials and many others are in preclinical developments. Recently, KSP inhibitors of wide structural diversity have
appeared in literatures. This review will summarize the developments of KSP inhibitors based on the five-membered heterocycle scaffolds in recent 10 years. These small molecule KSP inhibitors were classified as dihydropyrazoles, dihydropyrroles, thiophenes, dihydrothiadiazoles, thiazoles and fused pyrroles, their structure-activity relationships were discussed.

Keywords: Antitumor agents, ATP competitive inhibitors, ATP uncompetitive inhibitors, cancer, Eg5, heterocycles, kinesin,
kinesin spindle protein, KSP, KSP inhibitors, structure-activity relationships.
INTRODUCTION
Mitotic kinesins are enzymes essential for assembly and
function of the mitotic spindle. These microtubule-based
motor proteins travel along microtubules reaching into every
corner of the cell. They are regarded as biological machines
that transduce chemical energy into mechanical forces and
motion [1, 2]. Kinesins utilize the energy from ATP hydrolysis to power their movement unidirectionally along microtubules and to transport molecular cargo to specific destinations. Mitotic kinesins play essential roles during all phases
of mitosis. During mitosis, kinesins organize microtubules
into the bipolar structure that is the mitotic spindle. They
mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle
associated with specific phases of mitosis [3, 4]. To date,
there are at least 12 kinesins involved in mitosis and meiosis
which are responsible for spindle and chromosomal movement. Among them, Kinesin spindle protein (KSP, HsEg5) is
a slow, plus end-directed motor of the kinesin-5 subfamily
[5-7]. KSP is mainly consisted of three domains: the motor
domain, the coiled-coil domain and the C-terminal domain.
The motor domain starts with the residue Val21 and ends
with the residue Lys357 and before the motor domain there
is a 20 residue long stretch. A short linker (Asn358Pro363)
connects the motor to a predicted interrupted coiled-coil
*Address correspondence to these authors at the Department of Organic
Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P.
R. China; Tel: +86-531-88382006; Fax: +86-531-88382548;
E-mail: liuzhaop@sdu.edu.cn; and College of Animal Science & Veterinary
Medicine, Shandong Agricultural University, Taian 271018, P. R. China;
Tel: +86-538-8233881; Fax: +86-538-8241419; E-mail: wrzh@sdau.edu.cn
1875-533X/14 $58.00+.00

domain (Gly364Val523, Cys684Lys716 and Gln782

Arg826). The C-terminal domain of KSP ranges from
Tyr829 to Leu1057, containing a potential p34cdc2 phosphorylation site Thr927. The phosphorylation of Thr927 in
vivo is cell cycle-dependent and is increased strongly in M
phase [8]. The motor domain has six major -sheets surrounded by six
-helices with an ATP binding pocket and a
microtubule binding interface [9, 10]. KSP forms a homotetrameric structure capable of binding antiparallel microtubules and sliding them apart. It acts during the early stages of
mitosis and is responsible for centrosome separation and
bipolar spindle assembly, which are essential for proper segregation of chromosomes [11-13]. Failure of KSP function
leads to cell cycle arrest in mitosis with monoastral microtubule arrays. The important role of KSP in mitotic progression makes it an ideal candidate for drug discovery [7, 14].
Furthermore, it is most abundant in proliferating human tissues and is highly expressed in tumors of the breast, colon,
lung, ovary, and uterus [15]. Therefore, targeting KSP provides a novel route for the manipulation of the cell cycle,
alternative to antimitotic agents that target tubulin. Microtubule-directed drugs like paclitaxel (Taxol) belong to the most
successful anticancer agents that play a central role in the
therapy of breast, lung, ovarian, bladder, head and neck cancers, and also contribute dramatically to a better quality of
life of cancer patients, and represent milestones in modern
chemotherapy. However, microtubule agents have adverse
effects like peripheral neuropathy, which may be caused by
the abnormal aggregation of microtubules in the neuronal
cells [16]. Agents which target KSP selectively are expected
to act only on cells undergoing cell division, thus making
KSP inhibitors mitosis-specific drugs, likely to have fewer
side effects than drugs inhibiting other essential microtubule 2014 Bentham Science Publishers

2692 Current Medicinal Chemistry, 2014, Vol. 21, No. 23

Zhao et al.
H2N

O
S
N

F
S

N
N

NH2

OH
F

ARRY-520
Phase II for plasma cell leukemia

O
N

MK-0731
Phase I for advanced solid tumor

H
N
H
N
O

N
N

F3C

N
S

N
O
NHEt

HN

NH2

N
H

Ispinesib
Phase II for non-small-cell lung cancer

O
Cl

H
N

N
AZD4877
Phase II for bladder cancer

N
N

Cl

S
O

EMD534085
Phase I for lymphoma

LY2523355
Phase II for metastatic breast cancer
O

F
Cl

Cl

N
O

O
NH2

ARQ 621
Phase I for metastatic solid tumors

NH2

SB-743921
Phase II for Hodgkin's disease

Fig. (1). Representative KSP inhibitors in clinical trials.

based processes. Currently, several KSP inhibitors enter

clinical evaluations for the treatments of various cancers
(Fig. 1) [17-24].
Most of the KSP inhibitors are the allosteric and ATP
uncompetitive. They bind to KSP and cause both local and
distal conformational changes that allow ATP binding, but
prevent ADP releasing. One representative allosteric KSP
inhibitor is the MK-0731 that forms three main interactions
with KSP (Fig. 2). The difluorophenyl group extends into a
hydrophobic pocket formed by lipophilic residue Leu214,
Ala218, Phe239 and Ile136; the unsubstituted phenyl ring
sits orthogonally to a small region sandwiched between
Pro137 (back) and Arg119 (front); the urea moiety extends
toward the solvent-exposed region located above the
Trp127/Tyr211
-stacking interaction that seals the protein
cavity. In addition, the hydroxymethyl group of MK-0731
forms a hydrogen bond with the carbonyl group of Gly117 at
the roof of the cavity (dOH-O = 2.5 ) [25]. Other KSP inhibitors are competitive with ATP, but uncompetitive with microtubules [26].
In recent years, structurally diverse small molecule KSP
inhibitors have been investigated. In this review, we made a
summary of the developments of KSP inhibitors as antitumor
agents based on the five-membered heterocycle scaffolds.
These five-membered heterocycle KSP inhibitiors include
dihydropyrazoles, dihydropyrroles, thiophenes, dihydrothiadiazoles, thiazoles and fused pyrroles. The structureactivity relationships of these KSP inhibitiors along with

their selectivity and binding modes with the KSP were discussed.
1. 4,5-DIHYDROPYRAZOLES
3,5-Diaryl-4,5-dihydropyrazoles were identified as potent
and cell-active KSP inhibitors through the optimization of
high-throughput screening (HTS) hits [27]. In a series of 3-(2chlorophenyl)-5-(3-hydroxyphenyl)-4,5-dihydropyrazoles, the
N1-acetyl analogue 1 (Fig. 3) exhibited the best KSP binding
ability with an IC50 of 450 nM. Increasing the size of the acyl
group from acetyl to propionyl or isobutyryl reduced potency
(~2.5-fold), but the pivaloyl analogue restored the potency
(460 nM). The replacement of the acetyl group with a 4aminobutanoyl or a benzoyl group had a detrimental effect on
the potency. N1-Methylation or the addition of an oxygen
spacer to the acetyl group by formation of a methyl carbamate
resulted in a loss of measurable activity. Moving the hydroxyl
group from 3-position to 2- or 4-position on the C5 phenyl
ring decreased the potency. Replacing the 3-hydroxyl group
with methoxy, cyano, amino, or acetamido groups also reduced the potency, possibly because there was a hydrogenbonding interaction between the phenolic hydroxyl group and
carbonyl oxygen of Glu118. Additionally, the alkyl replacements for the 5-aryl group led to loss of activity, since the aryl
ring might form a hydrophobic interaction between Pro137
and Arg119, which was beneficial to the potency [27].
Among the N1-acetyl-3-aryl-5-phenyl-4,5-dihydropyrazoles, the 3-(2-chlorophenyl)-substituted analogue inhibited

Developments of Kinesin Spindle Protein Inhibitors

Current Medicinal Chemistry, 2014, Vol. 21, No. 23 2693

Fig. (2). Docking of MK-0731 into the allosteric binding site of KSP using SYBYL-X 1.3 (PDB: 3CJO).

Cl
3
2 N

F
4
5

H2N

OH

OH
N

N 1
COCH3

( )n
N

COR

COCH3

2: R = Me; 3: R = MeNH; 4: R =Me2N

NHEt
O

6: n = 3; 7: n = 4

R
H2N

11: R =

H2N

NAc

F
F

F
F

12: R =

13: R =

F
N
N

CH3

NMe2

O
(S)-8

N
O

14: R =

9: R = NH2; 10: R = Me2N

S
CH3

15

R
COCH3
N

CH3

N
N
16

KSP IC50 (nM)

17: X = F, R =
O

18: X = F, R = Me2N
19: X = Me, R = Me2N

NAc

9
10
11
12
13
14
15
16
17
18
19
(R)-19

0.9
1.4
2.8
1.8
1.0
1.8
0.82
1.6
3.8
2.1
2.0
0.2

MDR ratio
88
4.4
2.5
1.7
3.4
1.1
5.2
1.0

1.6

Fig. (3). 4,5-dihydropyrazoles.

the KSP with an IC50 of 3600 nM. Whereas exchange of

chlorine for fluorine was tolerated (3600 nM), other modest
modifications such as bromo, methyl, methoxy, and
trifluoromethyl resulted in substantial losses in potency. The
3-chlorophenyl derivative was somewhat less potent (9800
nM), whereas the 4-chloro analog was inactive. In addition,

the 2,3-, 2,4-, 2,6-, and 3,5-dihalogenated analogues were

uniformly less active than the best monohalogenated compounds, while the 2,5-difluoro derivative 2, with an IC50 of
94 nM, displayed an impressive 40-fold boost in potency
relative to the 2-chlorophenyl analogue. The replacement of
the N1 acetyl in 2 with a thioacetyl group led to a modest

2694 Current Medicinal Chemistry, 2014, Vol. 21, No. 23

loss in potency (250 nM), whereas with a methanesulfonyl

(1800 nM) or N-methylformyl (3: 3100 nM) group resulted
in a more drastic loss. However, the N,N-dimethylurea 4
restored the potency (84 nM). Incorporating the 3-hydroxyl
functionality on the 5-phenyl ring of 2 showed an additive
effect, the resulting compound 5 inhibited KSP with an IC50
of 51 nM. Moreover, the (S)-enantiomer of 5 was >2000-fold
selective for KSP over a panel of eight structurally and functionally related mitotic and transport kinesins. The inhibition
of KSP by (S)-5 was not competitive with either ATP or microtubules, suggesting an allosteric mode of action wherein
the binding site of the inhibitor was remote from both the
nucleotide and microtubule binding sites. In addition, (S)-5
did not affect tubulin polymerization in vitro at 20
M, distinguishing it mechanistically from the taxanes. (S)-5 also
induced apoptosis and generated aberrant mitotic spindles in
human ovarian carcinoma cells at low nanomolar concentrations (IC50 = 15 nM) [27].
The introduction of another substituent at the C5-position
of 1,3,5-trisubstituted-4,5-dihydropyrazoles was helpful to
improve the KSP binding potency, aqueous solubility and
effective resistance to Pgp. Installing a methyl group at the
C5 position of 3 led to a ten-fold improvement in the KSP
inhibition potency (284 nM). The introduction of hydroxyalkyl group also improved the potency, but was less effective
than the hydrophobic methyl group. In addition, the aminoalkyl group was more beneficial than the hydroxyalkyl
group and the linker of three or four carbons between the
nitrogen and C5 carbon atom was preferred. In the Nethylurea series, compound 6 with an aminopropyl substituent at the C5 was the most potent (44 nM), while the aminobutyl analogue 7 was only slightly less potent than 6 (67
nM). Addition of one or two alkyl groups to form a secondary or tertiary amine (including the large bicyclic amino
group) in 7 was well tolerated. This indicated that the steric
effects of the aminoalkyl groups were not crucial for the
KSP binding potency. However, reducing the basicity of the
amine by tying up the lone pair of electrons in the form of
either an N-oxide or an amide (benzamide, moderately basic
heterocyclic amides) eliminated the potency enhancement
gained from the amino substituent. Therefore, a portion of
the potency enhancement gained from the C5 alkylamine
was resulted from the basicity of the amine, and its ability to
form a critical hydrogen bonding interaction. The X-ray
crystal structure of compound 6 in the allosteric site of KSP
indicated a hydrogen-bonding interaction between the primary amino and the backbone carbonyl of Gly117, which
may partly account for the observed boost in potency [28].
Incorporating an aminopropyl group into the dimethylurea 4 at the C5 position provided a ten-fold boost in potency (racemate 8: 8 nM). Further cyclization at the ureido
site to form a 4-, 5-, or 6-membered ring resulted in modest
losses of potency. Compared with (S)-5, the enantiomer (S)-8
exhibited a significant increase in potency, both against the
isolated enzyme (1.9 nM) and in cells (5.2 nM vs 25 nM),
and reduced logP from 3.1 to 1.2. The aqueous solubility was
dramatically enhanced from 0.029 mg/mL in (S)-5 at pH 6.5
to greater than 12 mg/mL at pH 4 for (S)-8. (S)-8 also had
improved dog pharmacokinetics relative to (S)-5, as evidenced by a reduction in clearance and a significant increase
in half-life (14.7 h vs 1.0 h). In addition, unlike in a related

Zhao et al.

series of inhibitors where the introduction of a basic amine

resulted in greater binding affinity for the potassium channel
hERG, the hERG binding of (S)-8 was not significantly increased with respect to (S)-5 (19.3
M vs 26.4
M) [28].
Unfortunately, (S)-8 was a good substrate for Pgp efflux
(MDR ratio = 491) [29]. Replacing the dimethyl urea with an
acetyl group in (S)-8 maintained potency (0.9 nM) and reduced the MDR ratio (88). A further improvement in the
MDR ratio was attained when the primary amine in 9 was
replaced with a tertiary amine as in 10. Therefore, various
tertiary amines were introduced onto the C5 side chain and
the resulting compounds maintained the high enzyme potency (1.44.0 nM) and cellular activities (2.355 nM). The
MDR ratios correlated closely with amine basicity for these
compounds. Lowering the pKa (< 8) of the tertiary amine
caused a significant attenuation in the MDR ratio. Among
them, compounds 1114 had the favorable MDR ratios. KSP
inhibitor 11 possessed excellent intrinsic (2.8 nM) and cellular (6.0 nM) potencies and a low Pgp susceptibility (MDR
ratio = 2.5). In vivo, compound 11 could induce maximal
mitotic arrest with a plasma concentration of 56 nM. However, a potential limiting feature of 11 and several structurally related tertiary amines from this series was a propensity
to induce QTc prolongation in a canine cardiovascular model
at low micromolar (< 10
M) exposures. The ECG changes
associated with 11 were not predicted by hERG binding
(IC50 > 30,000 nM), but the blockade of the IKr current by
compound 11 could be measured in a classical patch clamp
assay. Fortunately, the primary amine 9 was not a potent IKr
inhibitor and a single -fluorine substitution at the side chain
reduced its MDR ratio. The (5S,2S) diastereomer 15 was a
potent KSP inhibitor (0.82 nM) and exhibited high cellular
potency (3.4 nM) with a good MDR ratio (5.2). In vivo, inhibitor 15 provided a full mitotic arrest with circulating
plasma levels of 38 nM and had highly favorable pharmacokinetics with a plasma half-life of 9 h in dogs. Unlike appended tertiary amines in this series, compound 15 did not
cause QTc changes in the dog with exposures as great as 40

M [29].
Rearrangement of the pyrazole nitrogens in 11 and cyclization led to the dihydropyrazolobenzoxazine 16 that displayed favorable balance of properties (KSP IC50: 1.6 nM;
cell EC50: 5.0 nM; MDR ratio: 1.0; hERG > 30
M; aqueous
solubility: > 4 mg/mL at pH = 4.0; and dog pharmacokinetics: t1/2: 5.6 h, CL: 14 mL/min/kg) [30]. Furthermore, compound 16 induced maximal mitotic block with a plasma exposure of 100 nM, but it showed no effect on QTc interval
when dosed up to 35
M plasma levels. KSP inhibitor 16
represented a highly optimized lead with ideal properties for
a cancer drug.
Replacement of the fluoro atom in 16 with a chlorine or
methyl group did not have much influence on the potency
and MDR ratio. In addition, potency was not greatly altered
by changing the 4-acetylpiperazinyl group of 16 with dimethylamino, hydroxyl, amino or a variety of cyclic amines.
However, these changes had significant impact on hERG
binding potency and the MDR ratio, depending on the basicity of the resulting compounds. With respect to hERG
binding, amines with reduced basicity generally showed
weaker binding, whereas strongly basic amines engendered a
stronger interaction with this ion channel. Similar trends

Developments of Kinesin Spindle Protein Inhibitors

were seen for the MDR ratio, wherein amines with reduced
basicity, and tertiary amines tended to have improved potency against the Pgp-overexpressing cell line with lower
MDR ratios [30]. The X-ray structure analysis showed that
both aromatic rings in 16 formed hydrophobic interactions
with the nonpolar pocket of KSP, the tethered amine at C4
occupied the unoccupied region above the plane of the core
dihydropyrazole scaffold, and the C3 acetyl group projected
into the solvent exposed area of the enzyme [31].
Further modifications by removing the ring strain in 16
gave a racemate 17 that maintained excellent intrinsic potency (3.8 nM) while suffering a 16-fold loss in cellular potency (78 nM). Modulation of the amine to a tertiary heterocyclic amine or thiomorpholine dioxide afforded compounds
with diminished cellular potency as compared to 17. Potency
in cells was regained when the acetylpiperazine was replaced
with various tertiary amines such as morpholine (53 nM), 3fluoroazetidine (40 nM), pyrrolidine (15 nM), and bridged
morpholine (11 nM). The dimethylamine analogue 18 exhibited excellent biochemical potency of 2.1 nM and cellular
potency of 9.4 nM. Replacement of the 5-F in 17 with 5-Cl
or 5-Br improved the cellular activity (up to twofold). Deletion of the 5-F substituent in 17 afforded a sixfold decrease
in intrinsic potency concomitant with a fivefold decrease in
cell potency. Incorporation of a trifluoromethyl group was
also deleterious with respect to potency. However, changing
the 5-position fluorine to a methyl group afforded compound
19 which gave a twofold and threefold improvement in intrinsic (2.0 nM) and cellular (27 nM) potency as compared to
17. Especially, the enantiomer (R)-19 exhibited excellent
biological activities. (R)-19 was eightfold more potent than
dihydropyrazolobenzoxazine 16 in KSP inhibition (0.2 nM).
It was particularly potent in cells (3.2 nM) showing approximately equivalent potency to 16 (5.0 nM). (R)-19 also
demonstrated good aqueous solubility (> 4 mg/mL) at moderate pH of 4, showed good potency in Pgp-overexpressing
cells (MDR ratio of 1.6), and exhibited full mitotic arrest in a
mouse xenograft model of cancer with low plasma exposure
(EC99 = 67 nM) [31].
2. DIHYDROPYRROLES
To mimic the 3,5-diaryl-4,5-dihydropyrazole pharmacophore, a series of 2,4-diaryl-2,5-dihydropyrroles were developed as potent KSP inhibitors. Introduction of basic amide and urea moieties at N1 position of the 2,4-diaryl-2,5dihydropyrroles could improve the KSP binding potency and
aqueous solubility, while combination of C2 phenol group
with neutral N1 side chain could minimize the hERG binding
affinity to improve their selectivity [32]. The 4-piperidinyl
urea 20 (Fig. 4) was the most potent among the urea series
and had a good aqueous solubility (> 10 mg/mL pH = 5).
The crystal structure of compound 20 bound to the allosteric
site of KSP revealed that the N1 piperidinyl group projected
into the polar solvent exposed region (above Trp127 and
Tyr211) away from the hydrophobic pocket inside and reduced the energy for desolvation. The N-methyl group directed into a hydrophobic region below the dihydropyrrole
scaffold that was benefit to the potency. However, compound 20 had a low IKr potassium channel hERG binding
IC50 value. High hERG binding affinity could cause alteration of cardiac ventricular repolarization, prolongation of QT

Current Medicinal Chemistry, 2014, Vol. 21, No. 23 2695

interval, cardiac arrhythmia, or even sudden death [33]. In

addition, the highly basic compounds were found to bind
more tightly with hERG than the less basic ones. The nonbasic acylated derivative 21 exhibited much lower affinity to
hERG than 20. The incorporation of branched
- or -amino
amides provided low molecular weight KSP inhibitors with
excellent potency and solubility properties. In the case of the

-amino amides, only the S configuration of the
stereocenter imparted a high level of potency. These basic
amides (e.g., 22 and 23) demonstrated moderate levels of
hERG binding. Compound 23 exhibited good pharmacokinetic profiles and aqueous solubility, had moderate plasma
clearance in rats, dogs and monkeys with half-lives ranging
from 1 to 4 h, respectively [32]. Further incorporating a
meta-hydroxyl group on the phenyl ring at C2 afforded
highly potent (IC50 < 3 nM) KSP inhibitors (24 and 25) with
excellent cell-based activity, however, the hERG binding
was only minimally improved by the presence of the phenol
alone [34]. A combination of the phenol group at C2 and
neutral N1 side chains decreased the binding affinity to
hERG, but retained KSP inhibitory activity with IC50 < 10
nM. KSP inhibitors 2629, that possessed neutral
-hydroxy
amides or ureas, displayed excellent potency and reduced
hERG binding. A key limitation to these inhibitors was that,
with the absence of the basic amine, aqueous solubility was
negligible. A phosphate prodrug strategy circumvented this
problem [35]. When the phenolic phosphate of 28 was dosed
to rats and dogs, it provided approximately 100% of the
AUC seen with direct dosing of the parent (iv), indicative of
complete conversion and bioequivalence of prodrug to parent
28 [34].
Further incorporating a hydroxymethyl group at the C2
position of the dihydropyrrole 20 reduced the hERG binding
affinity, but the resulting analogue 30 (Fig. 5) was easily
pumped out of the cells by P-glycoprotein (Pgp) (with a high
MDR ratio) [25]. The MDR ratio is expressed as the ratio of
cell potency between paired cell lines that have high and low
expression of Pgp, respectively, and is used as a surrogate
measure of Pgp susceptibility. It is regarded that a compound
with an MDR ratio <10 is desirable, and a value of 1 is ideal.
The introduction of different alkyl groups at the piperidinyl
N atom in 30 could affect the pKa values of the resulting
derivatives. It was found that the compounds with a pKa less
than 6.5 had low KSP inhibition potency, while those with
pKa values more than 8 (31) had high MDR ratios. On the
other hand, compounds 32 and 33 with 6.5 < pKa < 8 exhibited low MDR ratio and high KSP inhibition potency [25].
Unfortunately, the cyclopropylamine 33 was a timedependent inhibitor of CYP3A4 (a member of the cytochrome P450 mixed-function oxidase system), and compound 32, though with good formulation properties and high
potency in vivo (EC90 = 100 nM), had acute toxicity with the
depletion of white blood cells. The toxicity of 32 might be
caused by its hydrolysis to generate a toxin, the fluoroacetate. Placement of the fluorine in a metabolically benign location on the piperidine ring to simultaneously avoid toxic
metabolite formation and tune the pKa of the nitrogen generated a potent analogue MK-0731 with an optimized in vitro
and in vivo profile [25]. MK-0731 exhibited superior potency
in both the enzymatic KSP assay and a cell-based assay, and
displayed little affinity for binding to the hERG channel.

2696 Current Medicinal Chemistry, 2014, Vol. 21, No. 23

4
R

Zhao et al.

R2

OH

2
N

N1

R1

CH3

NH2

OH

22: R1 = i-Pr, R2 = H; 23: R1 = c-Pr, R2 = H;

24: R1 = i-Pr, R2 = OH; 25: R1 = c-Pr, R2 = OH

20: R = H; 21: R = Ac

H
N
R

KSP IC50
(nM)

Cell EC50
(nM)

hERG IC50
(M)

2.6
50
3.6
2.0
1.2
0.5
2.9
1.3
7.0
4.0

6.8
69
12
8.6
3.3
3.0
3.1
2.6
22
9.1

1.3
>10
2.4
3.5
8.5
5.6
18
7.8
33
15

20
21
22
23
24
25
26
27
28
29

OH

CH3
28: R = Me; 29: R = CH2CH2OH

26: R = c-Pr; 27: R = t-Bu

Fig. (4). 1,2,4-Trisubstituted dihydropyrroles.

H2N

H2N

HO
F
R

N
N
CH3

N
H3C

CH3

30-33
R

H3C

CH3

34

KSP IC50
MDR ratio
(nM)

pKa

O
3
5

hERG IC50
(M)

30

7.4

345.1

9.8

19.2

31

6.2

21.2

8.8

14.6

32

Me
CH2CH2F

5.0

4.5

7.6

13.8

33

cyclopropyl

5.9

1.2

7.5

15.2
20.5

MK-0731

2.2

4.5

7.6

34

2.2

1200

10.3

7.1

35

5.2

5.0

7.0

15.9

Fig. (5). 1,2,2,4-Tetrasubstituted dihydropyrroles.

Consistent with its favorable pKa, MK-0731 also had significant activity in Pgp-overexpressing cells, with potencies
of 4.2 and 18.9 nM in the parental and Pgp-overexpressing
cell lines, respectively, resulting in an MDR ratio of 4.5. It
had the ability to induce a mitotic block with an IC50 of 19
nM in cells that were refractory to paclitaxel due to Pgp
overexpression. In addition, it was >20000-fold selective for
KSP over a panel of eight structurally and functionally related kinesins. The inhibition of KSP by MK-0731 was not
competitive with either ATP or microtubules. Unlike taxanes
and the epothilones, MK-0731 had no effect on microtubule
polymerization in vitro at 20 M. It induced apoptosis in
A2780 cells with an EC50 of 2.7 nM. In mouse xenograft
assays, MK-0731 induced dose-dependent mitotic arrest in
tumors and inhibited tumor growth comparable to paclitaxel.

MK-0731 also inhibited tumor growth in cell lines that were

resistant to paclitaxel because of either tubulin mutations or
Pgp-overexpression. In a Phase I open-label clinical trial to
test safety and pharmacokinetic parameters in patients with
taxane-refractory tumors, MK-0731 was well-tolerated and
exhibited dose-proportional exposure following a 24 h continuous infusion. It was a low clearance compound (100
250
mL/min on average) with a half-life of 4
10 h in humans. At
the MTD (maximum tolerated dose) of 17 mg/m2/day every
21 days, MK-0731 was well-tolerated with the anticipated
dose-limiting toxicity of neutropenia [24, 25]. On the basis
of its favorable efficacy, pharmacokinetic, and safety profile,
MK-0731 entered a Phase I clinical trial in patients with taxane refractory solid tumors.

Developments of Kinesin Spindle Protein Inhibitors

Current Medicinal Chemistry, 2014, Vol. 21, No. 23 2697

Installation of an aminopropyl side chain to the C2 position of the dihydropyrrole scaffold resulted in a potent, water
soluble KSP inhibitor (34), but the aminopropyl group induced susceptibility to cellular efflux by P-glycoprotein
(Pgp). Substituting the primary amine in 34 with an ethyl, a
fluoroethyl, or a difluoroethyl group decreased the KSP
binding potency (5-fold). In addition, the ethylated analogue
remained a good substrate for Pgp efflux (MDR ratio > 135),
the fluoroethyl compound had a significant improvement in
the MDR ratio (= 32), while the difluoroethyl analogue had a
good MDR ratio of almost unity (= 3). Trifluoroethylation of
the primary amino group continued the trend of reduced
MDR ratio (= 1), but also suffered a substantial reduction in
KSP potency (55-fold). However, by carefully modulating
the basicity of the amino group through introducing a
difluoromethyl substituent at the -position of the primary
amine group led to compound 35 that maintained potency
against KSP and had greatly improved efficacy in a Pgpoverexpressing cell line [36]. The discovery that cellular
efflux by Pgp could be overcome by carefully modulating
the basicity of an amine might be of general use to medicinal
chemists attempting to transform leading compounds into
cancer cell- or CNS-penetrant drugs.

same pocket as the C2 phenyl group in the dihydropyrrole

series, leaving no extra space to accommodate any substituents on the thiophene ring.
In the optimization of the diethylamide group in 36, it
was found that the ethyl ester, the acid and an unsubstituted
amide abolished activity, the dimethylamide (reduced in
size) showed diminished potency, unbranched amide substitution was less potent than a fully branched substitution. In
addition, cyclization of the branched amide gave comparable
activity. These results indicated that the hydrophobic interactions provided by amide substitution were required for activity [37].
To explore the additional space in the hydrophobic
pocket occupied by the cyclohexyl ring, alkyl groups were
added to various positions on the cyclohexyl ring to potentially enhance favorable hydrophobic interactions. A methyl
group in the 5 position (37) gave a three- to fourfold boost in
potency over 36 in both the ATPase assay (0.48
M vs 1.7

M) and the MPM-2 cytoblot assay (0.10
M vs 0.48
M).
Addition of another methyl group at position 5 of the cyclohexylthiophene was tolerated but led to a reduced activity
(IC50: 3.1
M, EC50: 1.7
M) compared to 36. Increasing the
size beyond methyl was detrimental. Substitutions at other
positions of the cyclohexylthiophene were less favorable
than position 5. A similar boost in potency was observed
when a methyl group was incorporated on the cyclopentyl
analogue (IC50: 3.5
M vs 9.9
M). Incorporating heteroatoms into the cyclohexyl ring led to a range of results, from a
minor effect for sulfur, to a detrimental effect with oxygen
and complete loss of activity with nitrogen. This suggested
that polar substituents were not tolerated in the hydrophobic
pocket. In addition, benzothiophene, naphthalene and benzofuran analogues showed no activity [37].

3. THIOPHENES
Ultra high-throughput screening of the Kalypsys compound collection identified the thiophene scaffold for the
development of novel KSP inhibitors [37]. The analogue that
had no substituents at the thiophene C4 and C5 positions was
inactive (IC50 > 100
M) in microtubule-stimulated KSP
ATPase activity assay. Some improvement was observed
with dimethyl substitution at the thiophene C4 and C5 positions, but more dramatic effects were observed with fused
aliphatic 5- to 7-membered rings appended onto the thiophene. In particular, the cyclohexyl analogue 36 (Fig. 6),
with activity in the ATPase assay of 1.7
M and the MPM-2
cytoblot assay of 0.48
M, became a scaffold for further
optimization. Replacements of the 2-thiophene in the amide
portion of 36 with the 3-thiophene, thiazole, methyl thiazole,
furan, and phenyl led to a log or more loss of activity. Removing the amide carbonyl of 36 also led to a log loss of
activity. The co-crystal structure of KSP with 36, Mg2+, and
ADP revealed that the cyclohexylthiophene core of 36 occupied the same hydrophobic pocket as the difluorophenyl
group in the 1,2,4-trisubstituted dihydropyrroles (e.g. 28).
One ethyl of the diethylamide group projected into solvent
and the other in the pocket formed by Tyr211, Leu214, and
Glu215. The pendant 2-thiophene amide of 36 filled the

4. DIHYDROTHIADIAZOLES
LY2523355 (Fig. 1), also known as KF89617 or Litronesib, was a dihydrothiadiazole analogue developed by Kyowa
Hakko Kirin [17, 38]. Litronesib is an ATP-noncompetitive,
allosteric, reversible KSP inhibitor (IC50 < 0.1 M) that has
no observed effect on microtubule formation. It was effective
against MV-4-11, HCT-116, A549 and SK-OV-3 cell lines
(GI50 < 10 M) [39, 40]. Litronesib has been investigated in
clinical trials for the treatment of multiple cancers by Eli
Lilly and Company [23]. The main toxicities of it were neutropenia and leucopenia. Grade 4 neutropenia was observed,
but all recovered to above 500/mm2 within 7 days. Japanese
patients at 4 or 5 mg/m2/day required G-CSF support, and
F

S
1
S

7
6

O
2

5
4
O

1
S

NH

NH

3N

H3C
N(Et)2

36

Fig. (6). Thiophenes and dihydrothiadiazoles.

O
37

N(Et)2

38

5
N
6

H2N

2698 Current Medicinal Chemistry, 2014, Vol. 21, No. 23

there was no DLT (dose limiting toxicity) up to 5 mg/m2/day

in phase I trial [23].
ARRY-520 (Fig. 1), also known as Filanesib, was discovered and optimized by structure-based design by Array
BioPharma [41]. It is a potent inhibitor of human KSP (IC50
= 6 nM) with in vitro antiproliferative EC50s ranging from
0.4 nM to 14.4 nM for a variety of human and rodent tumor
cell lines. ARRY-520 caused accumulation of cells in the
G2/M phase of the cell cycle, as measured by FACS (fluorescence-activated cell sorting) analysis. In vivo study, the
antitumor efficacy of ARRY-520 was greater than that of
paclitaxel in several subcutaneous HT-29 (colon), HCT-116
(colon) and A2780 (ovarian) mouse xenograft models. It was
also more potent than that vincristine in K562 model.
ARRY-520 had modest efficacy against subcutaneous HCT15 xenografts (resistant to paclitaxel due to overexpression
of p-glycoprotein), but the efficacy was also greater than that
of paclitaxel [19, 41, 42]. The MTD of ARRY-520 was 4.5
mg/m2. Plasma pharmacokinetic analyses revealed low
clearance of ARRY-520 (~3 L/hour), a volume of distribution of ~450 L, and a median terminal half-life of >90 hours
in phase I trial [43]. ARRY-520 demonstrated single-agent
activity in refractory myeloma, and was generally well tolerated with hematologic toxicity. Neutropenia was reversible
and not cumulative in phase II trial, and ARRY-520 will
enter phase III clinical trial soon [20, 44].
The spiro-fused dihydrothiadiazole 38 (Fig. 6) with the
(5S,6R) configurations potently inhibited KSP ATPase activity with an IC50 value
5 nM. On the other hand, its antipode with the (5R,6S) configurations did not show any inhibitory activity (> 3000 nM). The cis-isomers that had the
(5S,6S) and (5R,6R) configurations displayed IC50 values 675
nM and 23 nM, respectively. Furthermore, 38 potently induced pharmacodynamic marker, Histone H3 (p-HH3 EC50 =
1 nM), in A2780 human ovarian carcinoma cells. The oral
bioavailability of 38 was 21% and the oral dosing of 50
mg/kg yielded an AUC value of 3.9 M h [45].
5. THIAZOLES
Unlike the ATP uncompetitive inhibitors (dihydropyrazoles, dihydropyrroles, thiophenes and dihydrothiadiazoles)
that shared a common induced-fit allosteric binding site between helix 3 and the L5 insertion loop, the thiazole KSP
inhibitors were ATP competitive [46]. Thiazole 39 (Fig. 7)
was identified as a small molecule inhibitor by highthroughput screening of a subset of the Merck sample collection (~2.5  105 compounds) against the ATPase activity of
KSP (IC50 = 11 M). Substitution on the linkage to the
phenyl group proved to be a key potency-enhancing feature.
Replacing the methyl substituent on the benzylic methylene
in 39 with a cyclopropyl group led to a large increase in potency (40, IC50 = 790 nM). Further modification of 40 by
introducing a 4-fluoro atom in the benzyl ring provided analogue 41 with a modest enhancement to potency (IC50 = 540
nM). Substitution of the cyclopropane ring with a hydroxymethyl group was not tolerated. Small, lipophilic substituents (methyl and halogen) were tolerated at multiple
positions on the benzyl ring (2-Me: 1500 nM; 3-Me: 2400
nM; 3-Cl: 970 nM). Halogen substitution in the ortho and
para positions had various effects on potency (2-Br: 1000

Zhao et al.

nM; 2-F: 300 nM; 4-Br: 2400 nM; 4-Cl: 290 nM), although
multiple substitutions did not lead to further improvement
(2,6-F: 280 nM; 2,4-F: 330 nM; 2,4,6-F: 230 nM). Substitutions on the pyridyl ring in 40 reduced the potency (2-OH:
1400 nM; 2-MeO: 1800 nM; 3-Cl: 7000 nM). Replacement
of the 4-pyridyl in 40 with 3-pyridyl or substituted phenyl
ring also led to reduced potency (IC50: 8304400 nM).
CH3

1
S

N
1
2
3
N

N
39

40

N
41

Fig. (7). Thiazoles.

These thiazole molecules were found to be competitive

with ATP and uncompetitive with microtubules in steadystate ATPase assays. Furthermore, these molecules also
showed selectivity for KSP over other kinesins and other
ATP-utilizing enzymes. They were active in cells, and led to
the formation of monoastral spindles, the phenotype observed with several other classes of KSP inhibitors. Treatment of human A2780 cells with compound 41 led to inhibition of proliferation and induction of caspase 3 activity with
EC50s of 1.7 M and 1.1 M, respectively [46]. One of the
novel features of ATP-competitive inhibitors as compared to
L5 binding inhibitors was the uncompetitive binding of inhibitor to KSP with respect to microtubules. Thus, inhibition
of KSP with the thiazole compounds should lead to a thiazoleKSPmicrotubule complex, whereas inhibition of KSP
with the allosteric L5 binding inhibitors was observed to lead
to an inhibitornucleotideKSP complex. Since no L5 binding inhibitor was observed to stall the motor on the microtubule as was predicted for the thiazole inhibitors, the thiazole
KSP inhibitors may represent distinct tools for understanding
the function of KSP, and developing novel treatments for
proliferative diseases such as cancer. However, there was on
hard evidence that the thiazoles bind within the orthosteric
site, and some benzimidazoles ATP competitive KSP inhibitors were reported to bind to a distinct binding pocket in the
KSP motor domain [46, 47].
6. FUSED PYRROLES
Some fused indole and carbazole compounds are also potent KSP inhibitors. As shown in (Fig. 8), the R1 of ethyl
group in compound 42 was optimal following the trend of Et
> Me ~ Br > Cl in binding affinity, and it was more beneficial for cell activity than Br or Cl. The two carbon tether
between nitrogen and carbonyl carbon of R2 (42) was preferred in the acyclic series [48]. The amino group at the terminal region of the hydrophobic chain of the compound 43
was preferred, and the two carbon atom chain attached to the
amino group is the best choice. Substitutions on the C3 position of compound 43 led to poor cellular activity [49]. Replacing the carbon atom with nitrogen at the  (44) or  position was favored, and introducing an accessory anilide group
at the  position of the carbazole could also improve the KSP
inhibitory activity. Likely, the urea group at the  position of

Developments of Kinesin Spindle Protein Inhibitors

Current Medicinal Chemistry, 2014, Vol. 21, No. 23 2699

3
R1

R2

CF3

1
N
H
42: R1 = Et, R2 = COCH2CH2NH2
43: R1 = Cl, R2 = CH2CH2NH2HCl

OH

X
R 

N
H

44: X = N
45: R = NHCONH2, X = C

42
43
44
45

KSP IC50 Cell IC50

(nM)
(M)
11
0.1
7
8.7
0.81
52
85
0.38

Fig. (8). Fused pyrroles.

the carbazole (45) was more potent than that on the  position. Carbazole derivatives fused with a five- to sevenmembered lactam ring at the ,-positions exhibited similar
activity, suggesting the great flexibility of the lactam carbonyl placement [50]. Insertion of an ether linkage between
the carbazole core and the CF3 group, and replacing the CF3
group with carbonyl groups could not improve the potency.

-Substituted carbazoles showed no or less KSP inhibitory
activity, and the t-Bu- or CF3- group at the - or - position
of carbazole core was the most favorable accessory group
[51].

tinct tools for understanding the function of KSP, and serve

as novel leads in the development of anticancer drugs.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts of interest.
ACKNOWLEDGEMENTS
This work was partially supported by the National Natural Science Foundation of China (NSFC, Grant No.
81072517).

CONCLUSION
Anti-mitotic chemotherapeutics, such as the taxanes and
vinca alkaloids, represent one of the main classes of effective
cancer therapies, and are broadly used against a wide range
of cancer types. However, mechanism related toxicities and
acquired resistance have stimulated considerable interest in
developing anti-mitotics that target mechanisms other than
direct microtubule inhibition. Kinesin spindle protein (KSP)
plays an essential role in centrosome separation and
formation of the bipolar mitotic spindle. Its exclusive
involvement in the mitotic spindle of proliferating cells
presents an opportunity for developing new anticancer agents
with reduced side effects relative to antimitotics that target
tubulin. A large number of KSP inhibitors based on many
distinct chemical scaffolds are currently in development.
This review summarized the recent developments of KSP
inhibitors based on the five-membered heterocycle scaffolds.
Dihydropyrroles and dihydropyrazoles represent the most
common chemical scaffolds that target an allosteric pocket in
the motor domain of KSP. These inhibitors do not inhibit
any other kinesins and their high specificity is based on a
particularly long loop L5 region in the motor domain, the
length of which is unique for KSP. They inhibit or slow
down ADP release, but do not interfere with ATP binding
during the ATP hydrolysis cycle. As discussed above, introducing suitable substituents and carefully modulating the
basicity of the dihydropyrroles and dihydropyrazoles could
minimize the hERG binding affinity to improve their selectivity, reduce their susceptibility to cellular efflux by Pglycoprotein (Pgp), and lead to potent KSP inhibitors with
good aqueous solubility and highly favorable pharmacokinetics. These modification strategies might be of general use
to medicinal chemists attempting to transform leading compounds into anticancer drugs. Compared with the allosteric
KSP inhibitors, the ATP competitive KSP inhibitors with
five-membered heterocycle scaffolds are less studied. The
thiazole ATP competitive KSP inhibitors may represent dis-

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Received: May 03, 2013

Revised: December 29, 2013

Accepted: February 26, 2014

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