Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2011.07.054

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) th

Full text of DOI:10.1016/j.bmc.2011.07.054

Bioorganic & Medicinal Chemistry 19 (2011) 5446–5453
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of tritylthioethanamine derivatives with potent
KSP inhibitory activity
Delany Rodriguez a, Chinnasamy Ramesh b, Lauren H. Henson a, Lori Wilmeth a, Bj K. Bryant b,
Samuel Kadavakollu b, Rebecca Hirsch a, Johnelle Montoya a, Porsha R. Howell a, Jon M. George b,
David Alexander b, Dennis L. Johnson b, Jeffrey B. Arterburn b, Charles B. Shuster a,
⇑
a Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA
b Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of
this motor results in mitotic arrest and apoptosis. S-Trityl- -cysteine is an allosteric inhibitor of verte-
Received 4 June 2011
Revised 21 July 2011
Accepted 25 July 2011
Available online 30 July 2011
L
brate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer prop-
erties, however, poor pharmacological properties have limited the use of this compound. We have
modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield
new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model
systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a prom-
ising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as
research tools, with activities that provide impetus for further development as anti-tumor agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Kinesin Spindle Protein (KSP)
Mitosis
Kinesin
Anti-mitotic
Cell division
1. Introduction
attachments to the spindle, the cell arrests in mitosis, and
eventually dies by apoptosis.16 Although there are reports that
Natural and synthetic small molecules that target the microtu-
bule cytoskeleton have proven invaluable for investigating the
mechanisms of cell division. Modulators of microtubule polymeri-
zation (such as colcimid and paciltaxel) interfere with cell division
by altering microtubule dynamics, resulting in extended mitotic
arrest and cell death.5,6 Microtubule disrupters have proven to be
effective chemotherapeutics, but because microtubules are essen-
tial for many cellular functions, their toxicity imposes limitations
to their use as anti-cancer agents. More recently, attention has
turned toward kinesins as potential targets for anti-proliferative
drugs.7–11 There are roughly 14 classes of kinesin motors,12 whose
functions range from vesicular transport to cell division, and RNA
interference analysis of 25 Drosophila kinesins revealed that four
members were involved in mitotic spindle assembly.13 Most prom-
inent of these is the class five kinesin, Kinesin Spindle Protein (KSP;
also known as Kif11 or Eg5), a plus end-directed motor that sepa-
rates the spindle poles early in mitosis.14 KSP is antagonized by a
minus end-directed kinesin known as KifC1,15 and inhibition of
KSP results in spindle collapse and formation of a monopolar spin-
dle. Because chromosomes are unable to establish bioriented
KSP plays a role in neuronal development,17 there are no other
known functions outside of mitosis in adults, making KSP an
attractive target for chemotherapeutic intervention.
Anti-cancer drug screening and chemical biological approaches
have identified a number of KSP inhibitors, including monastrol,18
S-trityl-
K85824 (Fig. 1) that exhibit no activity against the other kinesin
family members. The allosteric site located between helix 3
L
-cysteine (STLC, 1),19,20 3QC,21 MK-0731,22 ispinesib,23 and
a
and loop 5 of the KSP motor domain in vertebrate organisms has
been identified as the common site of action for monastrol, 1 and
ispinesib.1,4,25–29 The specificity of these compounds for KSP is
associated with the extended loop 5 found only in class 5 kine-
sins.29 Kinetic analyses indicate that these allosteric inhibitors
act through a mechanism whereby ADP release and motor rebind-
ing to the microtubule are blocked.16,23,30 Loop 5 undergoes dy-
namic conformational shifts during the ATP binding and
hydrolysis cycle, and it is thought that interactions with small mol-
ecules such as monastrol ‘‘lock’’ this loop into an ADP bound-like
conformation.1,4,31,32 While monastrol has not demonstrated effi-
cacy at pharmacologically relevant concentrations, several com-
pounds that exhibit anti-cancer activity have advanced to clinical
trials.22,33–38 However, recent characterization of drug-resistant
variants of KSP raises concern that tumor cells may develop resis-
tance over time.1,3,4
⇑
Corresponding author.
E-mail address: cshuster@nmsu.edu (C.B. Shuster).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.054

D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
5447
O
O
H3CO
S
OH H3CO
S
OH
NH2
NH2
2
3
Figure 2. Enantiomers of S-4-methoxytritylcysteine evaluated as KSP inhibitors.
in situ reduction of cysteine to cysteinol with BH3-THF, followed
by quenching the excess borane reagent with DMF and selective
S-alkylation with the appropriate trityl chloride. This efficient,
two-step procedure avoids the formation of disulfide byproducts,
and provided convenient access to the enantiomeric compounds
5 and 6 derived from L- and D-cysteine respectively. The achiral
ethanamine derivative 7 was prepared using the published proce-
dure for S-alkylation of cysteamine with 4-methoxytrityl chloride
in trifluoroacetic acid.43
Figure 1. Representative structures of known KSP inhibitors, including STLC,
monastrol, K858, 3QC, ispinesib, and MK-0731.
3. Results
The cysteine derivative 1 represents a relatively simple scaffold
with potent and reversible KSP inhibitory activity. Recent struc-
ture–activity analyses of STLC derivatives have been described
and revealed the critical role of the trityl group for inhibitory activ-
ity.39,40 The amphiphilic character of 1 results in poor water solubil-
ity and reduced permeability that affect bioavailability. We were
interested in identifying related structures with increased inhibi-
tory activity and improved physicochemical properties that would
confer better water solubility and other drug-like characteristics.
Herein, we describe the development of a new class of small, con-
formationally flexible KSP inhibitors based on the 2-(tritylthio)eth-
anamine scaffold. These compounds were effective inhibitors of
human KSP in both biochemical and cell-based assays, as well as
in nonmammalian model organisms. Two of the lead thioethan-
amine compounds 5 and 7 exhibited elevated anti-proliferative
activity against the NCI60 panel of tumor cell lines and demonstrate
the potential of this scaffold for the development of new anti-
cancer therapeutics.
Compounds 1–7 were tested for their ability to block human
KSP activity in a biochemical ATPase assay44 and a phenotypic as-
say in cultured HeLa cells (Table 1). The two assays provided a
complementary assessment of direct inhibition of the motor, as
well as indirectly reflecting membrane permeability and demon-
strating efficacy in whole cells. The parent molecule 1 blocked
the ATPase activity of recombinant human KSP with an IC50 of
0.62 lM, and in mitotic cells generated the stereotypical arrange-
ment of microtubules radiating from the collapsed spindle poles
with chromosomes arranged in a rosette pattern (Fig. 3, panel G).
The 40-methoxytrityl-derivative 2 exhibited increased potency as
expected from recent reports.39,40 The enantiomeric compound 3
derived from D-cysteine gave similar results in both assays, con-
firming the lack of stereochemical effects on inhibitory activity
previously demonstrated for 1.16
Evaluation of the inhibitory activity of the reduced cysteinol
derivative 4 revealed an increase in potency associated with trans-
formation of the carboxylic acid functional group to a primary alco-
hol that was comparable to incorporation of the 4’-methoxy-trityl
group in 2. An additive effect was observed for 4-methoxy-substi-
tuted cysteinol compound 5 that incorporates both of these modi-
fications to achieve increased KSP inhibitory activity in both the
biochemical and cellular assays. The enantiomer 6 derived from
2. Chemistry
A series of STLC analogs have recently been evaluated in attempts
to characterize structure–activity effects on KSP inhibition.39–41
While structural alterations of the trityl group generally result in re-
duced inhibitory activity, the substitution of methyl, chloro, and
methoxy groups in the para-position of the triphenylmethyl
appendage was shown to increase the potency of STLC analogs. Car-
boxylic amides were found to function with similar inhibitory con-
centration values to the acids, but have not exhibited increased
potency in the cellular assays.42 Additionally, the individual R- and
D-cysteine displayed equivalent activity in both biochemical and
cell-based assays. The simple ethanamine compound 7 derived
from cysteamine displayed the highest potency with an
EC50 = 115 nM.
The parent compound 1 affects bipolar spindle formation with-
out altering interphase microtubule dynamics or organization
(Fig. 3B).16 As illustrated in Figure 3, interphase microtubule mor-
phology was normal for compounds 4, 5 and 7 tested in the pres-
S-enantiomers of trityl-cysteine derived from L- and D-cysteine have
previously been shown to inhibit KSP with equivalent potency.16
Therefore, we focused our investigation on a series of novel thioe-
thanamine compounds 4–7 related to 1, but lacking the carboxylic
group, seeking to achieve increased KSP inhibitory properties, more
favorable physicochemical characteristics and increased cell perme-
ability by eliminating the zwitterionic structure of the parent com-
pound. The individual enantiomers of 40-methoxytrityl derivatives 2
and 3 were prepared to allow comparison of their activities (Fig. 2)
with 4–7.
ence of 1 lM compound (Fig. 3C–E), whereas spindle morphology
mirrored that of the parent compound (Fig. 3H–J). Additionally, it
has been established that 1 displays no activity against other kine-
sin family members.16,19 Compounds 4 and 5 were similarly tested
for inhibitory activity against a selected panel of mitotic kinesins
(mammalian centromere-associated kinesin (MCAK), centromeric
protein E (CENP-E) and chromokinesin), with no measurable inhi-
bition detected (not shown), suggesting that selectivity for class 5
kinesins was retained by the cysteinol derivatives.
KSP is a bipolar, homotetrameric motor whose nucleotide
hydrolysis cycle is disrupted when treated with allosteric inhibi-
The thioethanamine derivatives were synthesized by the proce-
dures shown in Scheme 1. Compounds 4–6 were prepared by

5448
D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
O
R
i
ii
HS
OH
S
OH
HS
OH
NH2
NH2
NH2
OCH3
OCH3
S
OH
S
OH
S
OH
NH2
NH2
NH2
4
5
6
OCH3
iii
NH2
NH3Cl
S
HS
7
Scheme 1. Reagents and conditions: (i) BH3-THF 0 °C – RT, DMF quench (ii) 4, 88% yield from
L
-cysteine and trityl chloride; 5, 91% yield from
L-cysteine and 4-methoxytrityl
chloride; 6, 92% yield from
D
-cysteine and 4-methoxytrityl chloride; (iii) 7, 96% yield from 4-methoxytrityl chloride, CF3CO2H.43
cells,46 KSP was observed clustering at the center of the monopole
upon treatment with either compound (Fig. 4A, D, G, J, M, and P).
Both compounds displayed a dose-dependent depletion of KSP
from the spindle, with 5 exhibiting a 1.63-fold increase in clear-
ance of KSP over 1 (Fig. 4F J, M, and P), consistent with the bio-
chemical- and cell-based assays (Table 1). Thus, the behavior of
KSP in the presence of this cysteinol derivative was consistent with
a mechanism of action by which these allosteric modulators pro-
mote disengagement of the motor from its microtubule track.
The hydrophobic domain created by an extended loop 5 and al-
pha helix 3 in the KSP motor domain accommodates a structurally
diverse group of allosteric KSP inhibitors (Fig. 1) and is unique to
class 5 kinesins.1 Moreover, it has been proposed that the nonpolar
nature of this pocket is unique to vertebrates.1 Sequence align-
ments comparing the binding pockets of human, Xenopus laevis,
and sea urchin Strongylocentrotus purpuratus revealed a high
degree of sequence conservation between urchin and vertebrate
KSP proteins, particularly at residues identified by mutational anal-
ysis to be critical for inhibition by monastrol and STLC analogs
(Fig. 5A).1–4 In contrast, the Drosophila homolog Klp61F was more
Table 1
IC50 values for inhibition of microtubule-stimulated KSP ATPase activity and EC50
values for mitotic arrest in cell-based assays for compounds 1–7
Compound
ATPase activity [
lM]
Cell-based assay [lM]
1
2
3
4
5
6
7
0.62
0.27
0.23
0.31
0.127
0.130
0.136
1.72
0.23
0.60
0.294
0.190
0.160
0.115
tors such as 1, monastrol and ispinesib.16,23,30 Kinetic analysis of
motor inhibition predicts that KSP remains locked in the ADP
bound state,16,23,45 blocking motor rebinding and leading to a pre-
dicted loss of motor from spindle microtubules. To determine
whether the cysteinol derivatives had a similar effect on KSP
dynamics, cells were treated with 1 or 5 and probed for KSP local-
ization with anti-KSP antibodies, using EB1 antibodies to label
microtubules (Fig. 4). Similar to reports of monastrol-treated
Figure 3. STLC analogs block bipolar spindle formation without affecting interphase microtubule organization. HeLa cells were incubated in the presence of 0.1% DMSO (A
and F) or 1 M STLC 1 (B and G), 4 (C and H), 5 (D and I), 7 (E and J) and then fixed and stained for microtubules (green) and DNA (blue).
l

D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
5449
Figure 4. Dose-dependent depletion of KSP from monopolar spindles in 1 and 5-treated cells. HeLa cells were incubated in either 1 (A–I) or 5 (J–R) for 4 h and the fixed and
processed for KSP (green), the microtubule-binding protein EB1 (red) and DNA (blue) localization, and images were acquired using equivalent exposure times for the green
channel.
divergent, particularly within loop 5. To determine whether 1 or
the cysteamine- or cysteinol derivatives were effective in sea urch-
in eggs or Drosophila S2 cells, cells were treated over a three log or-
der range of concentrations, and scored for bipolar spindle
formation by immunofluorescence. Compounds (1–7) were
screened in S2 cells, however, no activity was detected at doses
weakly active with EC50 P 100 lM. The response to all compounds
was phenotypically identical, causing spindle collapse following
nuclear envelope breakdown resulting in a large monopolar spin-
dle (Fig. 5B, panels B–D). Thus, the observed activity of these com-
pounds in the highly ionic environment of seawater provides an
additional assessment of the compounds biological efficacy using
an invertebrate model organism, and the differences in activity be-
tween the compounds 1 and 5 in sea urchin eggs parallel the activ-
ities measured in human cultured cells.
up to 100 lM and treatments extending up to 7 h (not shown).
In contrast, monopolar spindles were detected in S. purpuratus
eggs treated during the first cell cycle following fertilization
(Fig. 5B). Compound 5 blocked bipolar spindle formation with an
Compounds 5 (MSTCO, NSC 747880) and 7 (MSTNH2, NSC
753791) were evaluated for anti-proliferative activity against the
NCI60 tumor panel. Growth inhibitory concentrations (GI50) of 5
EC50 of 28 lM, whereas the parent molecule 1 and monastrol were
and 7 ranged from 10 nM to 3 lM across the panel, with average
GI50’s of 360 and 350 nM for 5 and 7, respectively (Table 2). This
represented roughly a 143- and 35-fold average increase in
potency over Monastrol (NSC 716782) and 1 (NSC 83265), respec-
tively. There were, however, individual tumor lines such as mela-
noma M14 where 5 and 7 displayed a 1200-fold increase in
activity over monastrol and the ovarian SK-OV-3 line where 5
and 7 displayed a 1250-fold increase in potency over the parent
compound 1. Thus, the tritylthioethanamine derivatives 5 and 7
had activities over a broad range of tumor types and represented
a significant increase in anti-proliferative potency.
The possible binding modes of derivatives 5–7 were evaluated
by molecular docking experiments using the crystal structure of
human KSP (PBD: 2FME), and Genetic Optimization for Ligand
Docking (Gold) scores were derived from 30 poses for each com-
pound, with the highest scores illustrated in Figure 6. All three
molecules docked into the binding pocket in the same manner,
with the ammonium group forming hydrogen-bonding interac-
tions with GLU 116 and GLY 117, consistent with previous docking
studies for 1.39 Indeed, a structural of overlay of 1 with 7 revealed
an identical alignment within the binding pocket (Fig. 6D). Simi-
larly, the aromatic rings of the trityl groups for all three molecules
fit into the previously described hydrophobic pockets (Hy1, com-
prised of TYR 211 and ALA 218 of
of 2 at the base of loop 5; and Hy3, comprised of ILE 136 from
2, LEU 160, LEU 214 from 3). The highest scoring poses of 5, 6
a3; Hy2, comprised of ALA 133
a
a
a
and 7 gave Gold scores of 85.74, 83.61, and 85.79, respectively.
No specific interactions of the primary alcohol groups of 5 and 6
with the backbone of KSP were identified in these binding models.
Previously described studies as well as data reported here (Ta-
ble 1) demonstrate an increase of potency for methoxytrityl deriv-
atives of 1.39,40 While no specific interactions were identified for
Figure 5. Efficacy of STLC derivatives in non-vertebrate organisms. (A) Alignment
of the loop 5 (top) and helix 3 (bottom) of kinesin 5 homologs from Homo sapiens
(NM_004523), Xenopus laevis (NM_001085956), Strongylocentrotus purpuratus
(AF292395), and Drosophila melanogaster (NM_057470). Asterisks denote the
residues reported to be required for drug inhibition.1–4 (B) Micrographs of S.
purpuratus eggs incubated in 100
lM Monastrol, 1, or 5 and 0.1% DMSO as control.
Eggs were fixed and processed for microtubule (green) and DNA (blue) localization.

5450
D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
ents are oriented towards the Hy3 hydrophobic pocket.39 The trit-
Table 2
GI50 values [l
M] from NCI60 panel screens* of Monastrol, 1, 5, and 7
ylthioethanamine derivatives described here are able to establish
additional interactions of the methoxy substituent with hydropho-
bic pockets 1 and 3, and preserve hydrogen bonding with the polar
backbone contacts GLU116 and GLY117 that were identified as
part of the pharmacophore.
Cell line
CCRF-CEM
Monastrol
1
5
7
Leukemia
Leukemia
Leukemia
Leukemia
Leukemia
Leukemia
NSC Lung
NSC Lung
NSC Lung
NSC Lung
NSC Lung
NSC Lung
NSC Lung
Colon
Colon
Colon
Colon
Colon
Colon
Colon
CNS
CNS
31.6
25.1
31.6
31.6
31.6
31.6
50.1
63.1
63.1
50.1
63.1
39.8
31.6
31.6
39.8
31.6
39.8
79.4
31.6
39.8
63.1
31.6
50.1
79.4
39.8
31.6
79.4
63.1
25.1
31.6
79.4
39.8
63.1
39.8
50.1
50.1
63.1
79.4
63.1
63.1
63.1
79.4
63.1
39.8
100
1.58
2.51
1.58
7.94
3.16
6.31
5.01
3.16
19.9
100
2.51
1.26
0.5
3.16
2.51
0.5
2.51
3.98
5.01
1.58
3.98
0.5
0.79
2.51
1.99
1
6.31
1.26
0.398
1.5
12.6
0.63
1.26
1.99
3.98
2.51
12.6
100
1.99
100
1.26
19.9
15.8
3.16
6.31
100
12.6
0.1
0.16
0.25
0.04
0.50
0.31
0.13
0.10
0.39
0.66
1.74
0.51
0.06
0.04
0.21
0.24
0.04
0.05
0.35
0.31
0.07
0.15
0.18
0.25
0.34
0.60
0.05
0.50
0.22
0.03
0.06
0.64
0.05
0.70
0.40
0.15
0.15
1.40
0.57
0.12
0.08
0.40
0.69
0.19
0.13
1.56
0.50
0.35
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.19
0.06
0.28
0.03
0.01
0.33
0.51
0.51
2.75
0.26
0.11
0.22
0.08
0.05
0.03
0.21
0.34
0.22
0.07
0.11
0.28
0.26
0.25
0.26
0.19
0.49
0.36
0.02
0.24
0.62
0.06
0.54
0.25
0.26
0.2
The steric volume and hydrophobicity of the triphenylmethyl
group are major determinants of the physicochemical and pharma-
cological properties of these compounds. It is intriguing to note the
presence of the trityl moiety in a variety of compounds that have
been identified as potential anti-cancer agents and function
through different mechanisms of action.48 50-O-Tritylinosine func-
tions an allosteric inhibitor of the angiogenic enzyme thymidine
phosphorylase.49 The anti-fungal imidazole agent clotrimazole
and synthetic analogs exhibit anti-cancer activity in cell culture
and animal models, with treatment resulting in G1-phase arrest
in the cell cycle, affecting intracellular Ca2++ levels and inducing
detachment of mitochondrial-bound hexokinase that inhibits gly-
colysis.50–53 Triphenylmethylamide derivatives have been shown
to exhibit activity against multiple melanoma cell lines, causing
G1-phase arrest and inducing apoptosis.54,55 The related trityl-
phosphonate compound TPMP-III-2 exhibits anti-cancer activity
and induces arrest in the M-phase of the cell cycle with character-
istic formation of fragmented mitotic spindles, through inhibition
of tubulin polymerization.48 In contrast, inhibition of KSP by STLC
derivatives results in M phase cell cycle arrest and is characterized
by the formation of the monopolar spindle. Synthetic diphenyl-
heterocyclic carbinols derivatives were recently shown to exhibit
anti-proliferative effects against carcinoma cell lines.56 The cyste-
amine and cysteinol derivatives described herein are shown to be
effective inhibitors of KSP and exhibit the archetypal monopolar
phenotype, demonstrating the importance of both the trityl- and
ethanamine elements of the pharmacophore.
A549/ATCC
EKVX
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
786-0
CNS
CNS
CNS
CNS
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Melanoma
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Renal
1.62
0.39
0.42
0.08
0.44
0.49
0.45
0.32
0.46
1.1
Methoxytritylethanamine derivatives demonstrated improved
efficacy in blocking KSP ATPase activity and bipolar spindle assem-
bly in HeLa cells (Table 1). The zwitterionic character and neutral
overall charge of carboxylic derivatives 1–3 represents major phys-
icochemical differences, with respect to compounds 4–7, that pos-
sess positive charges due to their protonated amine groups at
physiological pH. In order to evaluate our lead compounds against
an expanded panel of tumor cell types, 5 and 7 were submitted to
the NCI Developmental Therapeutics Program to be screened
against the NCI60 tumor panel.
Renal
Renal
Renal
Renal
Renal
Average
ACHN
CAKI-1
SN12C
TK-10
UO-31
100
51.5
0.36
*
Cell lines shown are those common to all four screens.
Examination of the growth inhibitory activities in cell lines
common to screens of monastrol, 1, 5, and 7 revealed that the eth-
anamine derivatives showed greater efficiency in blocking tumor
cell growth in comparison to 1 and monastrol (Table 2). Leukemia
and colon tumor cell lines were generally the most susceptible,
with average GI50 values 6230 nM for both compounds. Indeed,
the differential sensitivity of hematological neoplasms to KSP
inhibitors has been noted for other compounds, and several of
these compounds have advanced through preclinical evalua-
tion.47,57–61 Thus, the cysteinol and cysteamine scaffolds hold
promise for further development as therapeutically useful anti-
proliferative agents.
the methoxy group and any side chains within the binding pocket,
5 and 7 demonstrated a slight preference for positioning the
methoxyphenyl group in pocket Hy3 (Fig. 6A and C), while 6 pre-
ferred to occupy Hy1 (Fig. 6B), as illustrated by structural overlays
of 5 and 6 (Fig. 6E). However, no poses showed the methoxyphenyl
ring occupying pocket Hy2, the area nearest loop 5.
4. Discussion
The potential of kinesin inhibitors for use in the clinical arena is
an area of active investigation, with several candidate drugs in clin-
ical trials.34,37,38,47 This study has identified S-trityl-derivatives of
cysteinol and cysteamine as promising classes of selective KSP
inhibitors. These compounds incorporate a small, flexible thioe-
thanamine scaffold and exhibited increased activity as inhibitors
of both purified recombinant motor activity as well as bipolar spin-
dle assembly (Table 1). The increased potency of para-substitution
of the trityl group in derivatives of 1 was maintained in the trit-
ylthioethanamine compounds developed in this study. Previous
modeling studies of 1 with KSP have indicated that these substitu-
As an additional means of studying the interaction between
these compounds and the KSP motor domain, analogs were tested
against diverse species that contain varying degrees of conserva-
tion within the inhibitor-binding pocket (Fig. 5). A single report
has described monastrol- and 1 efficacy in a brown algae,62 but
there have been no other examples of KSP inhibitors functioning
in invertebrate cells. Examination of the loop 5 and helix 3 reveal
that while sea urchin KSP shares a high degree of identity with hu-
man KSP throughout the binding pocket, including the residues
identified as being essential for inhibition by 1, as well as monas-
trol and ispinesib (Fig. 5A, asterisks). In contrast, the Drosophila

D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
5451
Figure 6. Docking studies of compounds 5–7. Highest scoring poses of 5 (A), 6 (B), and 7 (C) in the allosteric binding site of KSP (PDB: 2FME). All inhibitors bind to the same
pocket formed by loop 5, 2 and 3. Helix 3 is shown to the left and loop 5 defines the right side and upper lid of the site. (D) Overlay of optimal poses for 1 and 7; (E) overlay
of the R- and S-enantiomers 5 and 6.
a
a
a
homolog Klp61F shares only partial identity with echinoderm and
vertebrate loop 5, with notable variation at positions 116, 117,
and 130, residues identified as crucial for monastrol- and STLC
inhibition in human KSP.1–4 Indeed, whereas we found that deriv-
atives of STLC were effective in sea urchin eggs (Fig. 5B), Drosoph-
ila S2 cells were resistant to all compounds tested (not shown).
However, because Klp61F contains a serine residue position 129
that is a proline in vertebrate KSP homologs, it is possible that
this variation alters the structure of loop 5 and thus Hy2. Thus,
understanding the structural differences between vertebrate and
invertebrate KSP homologs may lead to opportunities for the ra-
tional design of inhibitors that selectively target nonmammalian
pathogens.
acetate/hexanes or methanol/dichloromethane as eluent. Reverse
phase chromatography employed C-18 columns and water–aceto-
nitrile or water–methanol mobile phase. NMR spectra were ac-
quired at ambient temperatures (18 2 °C) unless otherwise
noted. Reactions were monitored by thin-layer chromatography
on silica gel (60 Å pore size, 5–17 lm) polyester backed sheets that
were visualized under a UV lamp, iodine vapor, phosphomolybdic
acid, or anisaldehyde. The 1H NMR spectra in CDCl3 were refer-
enced to TMS unless otherwise noted. The 13C {1H} NMR spectra
were recorded at 75 or 100 MHz and referenced relative to the
13C {1H} peaks of the solvent. NMR spectra are reported as ppm
(d), (multiplicity, coupling constants (Hz), and number of protons).
Infrared spectra were recorded as KBr pellets or neat films and are
reported in cmꢀ1. Melting points are uncorrected. The purity of all
compounds used in biological studies was determined to be >95%
by analytical HPLC equipped with Photodiode Array (PDA) and
5. Conclusions
ESI-MS detection. The compounds (1 mg/mL CH3CN, 20
l
L) were
Derivatization of both the cysteine and triphenylmethyl groups
of known KSP inhibitor 1 has provided a new class of compounds
that display increased potency in both biochemical and cell-based
assays. The structure-based mechanism of action exhibited by this
class of triphenylmethyl derivatives with respect to 1 is main-
tained by these compounds, eliciting cell cycle arrest and produc-
ing the characteristic monoaster spindle phenotype that results
from KSP inhibition. The cysteinol and cysteamine derivatives 5
and 7 displayed an average thirty five-fold increase anti-prolifera-
tive activity against the NCI60 panel of cancer cell lines in compar-
ison with 1, as well as broadened species specificity. The potent
activity and conformational flexibility of these small molecule allo-
steric inhibitors of KSP offers real promise for applications as re-
search tools and potential for further development as clinically
useful anti-tumor agents.
injected into a Waters SymmetryÒ C18
5
lm 3.0 ꢁ 150 mm column
and eluted as specified. Compound STLC (1) was obtained from
Novabiochem, Compounds 2,40 3,40 and 743 were prepared follow-
ing reported procedures. The other compounds were synthesized
as follows.
6.1.1. General procedure for the preparation of S-triarylmethyl-
cysteinol (4–6)
Borane-THF (4 mL, 4 mmol) was added dropwise to cysteine
(0.121 g, 1 mmol) in dryTHF (5 mL) at 0 °C under an argon atmo-
sphere, and stirred at room temperature for 7 h. The reaction mix-
ture was quenched with dry DMF (1 mL) and stirred for 1 h.
Triarylmethylchloride (0.5 mmol) was added to the reaction mix-
ture and stirred at room temperature for 3 h. The volatiles were re-
moved in vacuo. The residue was dissolved in CH2Cl2 (20 mL),
washed with H2O (20 mL), saturated NaCl solution (10 mL), and
dried over Na2SO4. The solvents were evaporated under reduced
pressure and the residue was purified by silica gel chromatography
using CH3OH/CH2Cl2 (3:97) as eluent to isolate the product.
6. Experimental
6.1. General methods
All reactions were performed in an efficient fume hood. Com-
mercially available solvents and reagents were used without fur-
ther purification. Preparative chromatographic separations were
performed using medium pressure flash chromatography and ethyl
6.1.2. (R)-2-amino-3-(tritylthio)propan-1-ol (4)
From
L-cysteine and trityl chloride; yield (88%) as colorless vis-
cous oil. IR (KBr, cmꢀ1): 3425, 3054, 2917, 1593, 742; 1H NMR

5452
D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
(300 MHz, CDCl3) d 7.44–7.39 (m, 6H), 7.31–7.17 (m, 9H), 3.38 (dd,
J = 10.86, 4.26 Hz, 1H), 3.15 (dd, J = 10.71, 6.89 Hz, 1H), 2.61–2.53
(m, 1H), 2.31 (dd, J = 12.47, 5.14 Hz, 1H), 2.20 (dd, J = 12.47,
7.63 Hz, 1H), 1.52 (s, 3H); 13C NMR (75 MHz, CDCl3) d 144.60,
129.50, 127.84, 126.67, 66.77, 65.53, 51.99, 36.62. HPLC–MS: Elu-
tion with CH3CN/H2O (20:80) exhibited a single peak at 3.92 min.
ESI-MS m/z [ES+] calcd for C22H24NOS [M+H]+ 350.44; found
350.16.
bonate, and 1.0 mM sodium pyruvate (Sigma Chemical Co., St. Louis,
MO) in 5% CO2 at 37 °C in a humidified incubator. STLC and its deriv-
atives were screened for KSP inhibition by incubating cells for four
hours with either carrier alone (0.1% DMSO) or DMSO-solublized
compound in doses ranging from 50 nM to 100 lM. Cells were then
processed for tubulin and DNA localization and 100 mitotic cells
were scored for bipolar spindle formation per condition. EC50 values
were calculated using Graphpad Prism software using a four param-
eter nonlinear regression as described above. Growth inhibitory
activity of tumor cell lines (NCI60 panel) was performed by the
Developmental Therapeutics Program, Division of Cancer Treatment
and Diagnosis, National Cancer Institute, U.S.A.
6.1.3. (R)-2-amino-3-((4-methoxyphenyl)diphenylmethylthio)
propan-1-ol (5)
From L-cysteine and 4-methoxytrityl chloride; yield (91%) as
colorless viscous oil. ½a D20
ꢂ
+6.91° (c 0.57, CHCl3); IR (Neat, cmꢀ1):
3447, 2925, 1508, 1250, 1033; 1H NMR (300 MHz, CDCl3) d 7.42–
7.17 (m, 12H), 6.81 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.44–3.39 (dd,
J = 10.71, 4.11 Hz, 1H), 3.21–3.15 (m, 1H), 2.65–2.55 (m, 1H),
2.36–2.19 (m, 2H), 1.55 (s, 3H); 13C NMR (75 MHz, CDCl3) d
158.10, 144.95, 136.76, 130.72, 129.46, 127.87, 126.64, 113.14,
66.36, 65.72, 55.11, 52.06, 36.85. HPLC–MS: Elution with CH3CN/
H2O (20:80), exhibited a single peak at 2.18 min. ESI-MS m/z
[ES+] calcd for C29H26NO2S [M+H]+ 380.09; found 380.16. HRMS
calcd 380.1679; found 380.1678.
6.4. Sea urchin embryo culture
S. purpuratus sea urchins were obtained from Marinus (Long
Beach, CA) and maintained in chilled aquarium at 10 °C. Gametes
were obtained by intracoloemic injection of 0.5 M KCl and eggs
were fertilized in artificial seawater in the presence of 2 mM 3-
amino triazole to prevent hardening of the fertilization envelope.
Twenty minutes after fertilization, eggs were stripped of their fer-
tilization envelops by passage through 80 lm Nitex, and cultured
either in the presence of 0.1% DMSO or KSP inhibitors. At the time
of first mitosis, eggs were fixed and processed for microtubule and
DNA localization as previously described.63 To generate EC50 values
for KSP inhibitor activity in sea urchin eggs, embryos were treated
over a three log order range of concentrations from 100 nM to
6.1.4. (S)-2-amino-3-((4-methoxyphenyl)diphenylmethylthio)
propan-1-ol (6)
From D-cysteine and 4-methoxytrityl chloride; yield (92%) as
colorless viscous oil. ½a D20
ꢂ
ꢀ6.04° (c 0.57, CHCl3); IR (Neat, cmꢀ1):
3447, 2925, 1508, 1250, 1033; 1H NMR (300 MHz, CDCl3) d 7.42–
7.17 (m, 12H), 6.81 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.44–3.39 (dd,
J = 10.71, 4.11 Hz, 1H), 3.21–3.15 (m, 1H), 2.65–2.55 (m, 1H),
2.36–2.19 (m, 2H), 1.54 (s, 3H); 13C NMR (75 MHz, CDCl3) d
158.10, 144.89, 136.70, 130.72, 129.46, 127.87, 126.67, 113.14,
66.36, 65.72, 55.20, 52.06, 36.81. HPLC–MS: Elution with 20–80%
CH3CN in H2O (gradient 1.5% minꢀ1), exhibited a single peak at
2.18 min. ESI-MS m/z [ES+] calcd for C29H26NO2S [M+H]+ 380.09;
found 380.16.
100 lM, processed for microtubule localization, and then scored
for bipolar spindle formation (100 cells scored per condition).
6.5. Immunofluorescence
Hela cells were incubated in the presence of compounds dis-
solved in DMSO at the specified concentration for 4 h and then
fixed by immersion in 100% MeOH for a minimum of thirty min-
utes at ꢀ20 °C before rehydration in phosphate-buffered saline
(PBS) for 10 min. Hela cells were blocked by incubation in PBS con-
taining 5% Bovine Serum Albumin (PBS-BSA) for 1 hour at room
temperature. Cells were then probed with either mouse anti-tubu-
lin (Sigma, Co, St. Louis, MO), or mouse anti-KSP (Abcam, Cam-
bridge, MA) in blocking buffer overnight at 4 °C. To counterstain
the microtubule cytoskeleton in KSP localization experiments, cells
were also probed for the presence of the microtubule end-binding
protein EB1 using a custom rabbit polyclonal antibody against
whole recombinant human EB1 (Pro-Sci, Poway, CA). Primary anti-
bodies were detected using AlexaFluor-conjugated secondary anti-
bodies (Molecular Probes, Eugene OR). After washing, cells were
mounted in 90% glycerol/1ꢁ PBS and imaged using a Zeiss 200 M
inverted microscope equipped with epifluorescence optics and an
Apotome structured illumination module, a 100 W mercury arc
fluorescent light source and DAPI, FITC, and TRITC filter sets.
Images were acquired using a 63ꢁ 1.4 NA Plan-aprochromat objec-
tive, an Axiocam MrM 12 bit CCD camera driven by Axiovision 4.5
software. Images were exported as 8 bit tif images and figures were
prepared using Adobe PhotoshopÒ software.
6.2. Analysis of analogs using an in vitro kinesin end-point
ATPase assay
Inhibitory activity of STLC analogs were measured using a
microtubule-activated ATPase end-point assay from Cytoskeleton
(Denver, CO). The reagents and kinesin motor domains were
added to wells of a 96-well microtitre dish (Greiner Bio-One,
Monroe, NC) in a total reaction volume of 30 lL. Reactions were
started by the addition of 2 mM ATP (Sigma Co., St Louis, MO),
incubated at room temperature for 5 min, and terminated by
the addition of 70 lL of CytoPhos. The reactions were incubated
for an additional 10 min at room temperature and absorbance
was measured at 650 nm using a microplate reader Elx 800™
(BIO-TEKÒ Winooski, VT) at 650 nm. All kinesin domain motors
were used at a final concentration of 0.08
derivatives were assayed over a range of concentrations between
0.25 M to 10 M. Controls of DMSO were used at a final concen-
lg/lL. STLC and its
l
l
tration of 0.1%. All conditions were performed in triplicate, and
raw data was entered into Graphpad Prism software, and IC50
values were calculated using
regression.
a
four parameter nonlinear
6.6. In silico docking studies
All docking calculations were performed on a PC equipped with
a 1.6 GHz Core 2 duo processor and 4 GB of RAM, running Win-
dows 7. Ligand structures were generated using Spartan’ 10.0
(Wavefunction Inc., Irvine, CA), including expected protonation
states at physiological pH, and geometry optimized. Binding site
preparation of the KSP complexes and docking studies were per-
formed with Gold Suite 5.0.1 (The Cambridge Crystallographic Data
6.3. Mammalian cell culture
HeLa cells (American Type Culture Collection, Manassas, VA)
were cultured in minimum essential medium with Earle’s BSS
(Lonza, Walkersville, MD), supplemented with 10% fetal bovine
serum, 1% penicillin/streptomycin, fungicide, 1.5 g/L sodium bicar-

D. Rodriguez et al. / Bioorg. Med. Chem. 19 (2011) 5446–5453
5453
23. Lad, L.; Luo, L.; Carson, J. D.; Wood, K. W.; Hartman, J. J.; Copeland, R. A.;
Sakowicz, R. Biochemistry 2008, 47, 3576.
24. Nakai, R.; Iida, S.; Takahashi, T.; Tsujita, T.; Okamoto, S.; Takada, C.; Akasaka, K.;
Ichikawa, S.; Ishida, H.; Kusaka, H.; Akinaga, S.; Murakata, C.; Honda, S.; Nitta,
M.; Saya, H.; Yamashita, Y. Cancer Res. 2009, 69, 3901.
25. Brier, S.; Lemaire, D.; Debonis, S.; Forest, E.; Kozielski, F. Biochemistry 2004, 43,
13072.
26. Brier, S.; Lemaire, D.; DeBonis, S.; Kozielski, F.; Forest, E. Rapid Commun. Mass
Spectrom. 2006, 20, 456.
Centre: Cambridge U.K., 2010) and Hermes 1.4.1. Docking was per-
formed in the binding site of PDB code: 2FME. All the water mole-
cules present in the binding site were allowed to be replaced by the
ligand or to change orientation. The standard function Gold score
was used for ranking the poses, including 30 solutions for each li-
gand generated. Ligand Scout v 3.0 (Inte:Ligand GmbH: Vienna,
Austria, 2010) was used for visual inspection of the docked poses
and for producing three-dimensional visual representations.
27. Maliga, Z.; Kapoor, T. M.; Mitchison, T. J. Chem. Biol. 2002, 9, 989.
28. Maliga, Z.; Xing, J.; Cheung, H.; Juszczak, L. J.; Friedman, J. M.; Rosenfeld, S. S. J.
Biol. Chem. 2006, 281, 7977.
Acknowledgments
29. Turner, J.; Anderson, R.; Guo, J.; Beraud, C.; Fletterick, R.; Sakowicz, R. J. Biol.
Chem. 2001, 276, 25496.
30. Cochran, J. C.; Gilbert, S. P. Biochemistry 2005, 44, 16633.
31. Behnke-Parks, W. M.; Vendome, J.; Honig, B.; Maliga, Z.; Moores, C.; Rosenfeld,
S. S. J. Biol. Chem. 2011, 286, 5242.
32. Kim, E. D.; Buckley, R.; Learman, S.; Richard, J.; Parke, C.; Worthylake, D. K.;
Wojcik, E. J.; Walker, R. A.; Kim, S. J. Biol. Chem. 2010, 285, 18650.
33. Beer, T. M.; Goldman, B.; Synold, T. W.; Ryan, C. W.; Vasist, L. S.; Van
Veldhuizen, P. J., Jr.; Dakhil, S. R.; Lara, P. N., Jr.; Drelichman, A.; Hussain, M. H.;
Crawford, E. D. Clin. Genitourin. Cancer 2008, 6, 103.
34. Lee, C. W.; Belanger, K.; Rao, S. C.; Petrella, T. M.; Tozer, R. G.; Wood, L.; Savage,
K. J.; Eisenhauer, E. A.; Synold, T. W.; Wainman, N.; Seymour, L. Invest. New
Drugs 2008, 26, 249.
35. Blagden, S. P.; Molife, L. R.; Seebaran, A.; Payne, M.; Reid, A. H.; Protheroe, A. S.;
Vasist, L. S.; Williams, D. D.; Bowen, C.; Kathman, S. J.; Hodge, J. P.; Dar, M. M.;
de Bono, J. S.; Middleton, M. R. Br. J. Cancer 2008, 98, 894.
36. Kathman, S. J.; Williams, D. H.; Hodge, J. P.; Dar, M. Cancer Chemother.
Pharmacol. 2009, 63, 469.
The authors would like to thank Dr. Kathy Hanley for her assis-
tance in statistical analyses. This work is supported by National
Science Foundation MCB0818729 (CBS), National Center for
Research Resources P20 RR16480 (JBA and CBS), the Howard
Hughes Medical Institute’s Undergraduate Education Improvement
Program at NMSU, the Cowboys for Cancer Research Foundation,
and an Interdisciplinary Research Grant from the NMSU Vice Pres-
ident for Research (JBA and CBS). We acknowledge the NCI Devel-
opmental Therapeutics Program for providing biological data
including NCI60 screening services, see http://dtp.cancer.gov/.
Supplementary data
37. Knox, J. J.; Gill, S.; Synold, T. W.; Biagi, J. J.; Major, P.; Feld, R.; Cripps, C.;
Wainman, N.; Eisenhauer, E.; Seymour, L. Invest. New Drugs 2008, 26, 265.
38. Tang, P. A.; Siu, L. L.; Chen, E. X.; Hotte, S. J.; Chia, S.; Schwarz, J. K.; Pond, G. R.;
Johnson, C.; Colevas, A. D.; Synold, T. W.; Vasist, L. S.; Winquist, E. Invest. New
Drugs 2008, 26, 257.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.054.
39. Debonis, S.; Skoufias, D. A.; Indorato, R. L.; Liger, F.; Marquet, B.; Laggner, C.;
Joseph, B.; Kozielski, F. J. Med. Chem. 2008, 51, 1115.
References and notes
40. Ogo, N.; Oishi, S.; Matsuno, K.; Sawada, J.; Fujii, N.; Asai, A. Bioorg. Med. Chem.
Lett. 2007, 17, 3921.
1. Maliga, Z.; Mitchison, T. J. BMC Chem. Biol. 2006, 6, 2.
2. Marshall, C. G.; Torrent, M.; Williams, O.; Hamilton, K. A.; Buser, C. A. Arch.
Biochem. Biophys. 2009, 484, 1.
3. Tcherniuk, S.; van Lis, R.; Kozielski, F.; Skoufias, D. A. Biochem. Pharmacol 2009,
79, 864.
41. Kaan, H. Y.; Weiss, J.; Menger, D.; Ulaganathan, V.; Tkocz, K.; Laggner, C.;
Popowycz, F.; Joseph, B.; Kozielski, F. J. Med. Chem. 2011, 54, 1576.
42. Hapuarachchige, S.; Montano, G.; Ramesh, C.; Rodriguez, D.; Henson, L. H.;
Williams, C. C.; Kadavakkollu, S.; Johnson, D. L.; Shuster, C. B.; Arterburn, J. B. J.
Am. Chem. Soc. 2011, 133, 6780.
43. Riddoch, R. W.; Schaffer, P.; Valliant, J. F. Bioconjugate Chem. 2006, 17, 226.
44. Funk, C. J.; Davis, A. S.; Hopkins, J. A.; Middleton, K. M. Anal. Biochem. 2004, 329,
68.
45. Cochran, J. C.; Gatial, J. E., 3rd; Kapoor, T. M.; Gilbert, S. P. J. Biol. Chem. 2005,
280, 12658.
46. Kapoor, T. M.; Mayer, T. U.; Coughlin, M. L.; Mitchison, T. J. J. Cell Biol. 2000, 150,
975.
4. Brier, S.; Lemaire, D.; DeBonis, S.; Forest, E.; Kozielski, F. J. Mol. Biol. 2006, 360,
360.
5. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
6. Zhou, J.; Giannakakou, P. Curr. Med. Chem. Anticancer Agents 2005, 5, 65.
7. Bergnes, G.; Brejc, K.; Belmont, L. Curr. Top. Med. Chem. 2005, 5, 127.
8. Duhl, D. M.; Renhowe, P. A. Curr. Opin. Drug Discov. Devel. 2005, 8, 431.
9. Huszar, D.; Theoclitou, M. E.; Skolnik, J.; Herbst, R. Cancer Metastasis Rev. 2009,
28, 197.
47. Carol, H.; Lock, R.; Houghton, P. J.; Morton, C. L.; Kolb, E. A.; Gorlick, R.;
Reynolds, C. P.; Maris, J. M.; Keir, S. T.; Billups, C. A.; Smith, M. A. Pediatr. Blood
Cancer 2009, 53, 1255.
48. Palchaudhuri, R.; Nesterenko, V.; Hergenrother, P. J. J. Am. Chem. Soc. 2008, 130,
10274.
49. Casanova, E.; Hernandez, A. I.; Priego, E. M.; Liekens, S.; Camarasa, M. J.;
Balzarini, J.; Perez-Perez, M. J. J. Med. Chem. 2006, 49, 5562.
50. Al-Qawasmeh, R. A.; Lee, Y.; Cao, M. Y.; Gu, X.; Vassilakos, A.; Wright, J. A.;
Young, A. Bioorg. Med. Chem. Lett. 2004, 14, 347.
51. Benzaquen, L. R.; Brugnara, C.; Byers, H. R.; Gatton-Celli, S.; Halperin, J. A. Nat.
Med. 1995, 1, 534.
52. Khalid, M. H.; Tokunaga, Y.; Caputy, A. J.; Walters, E. J. Neurosurg. 2005, 103, 79.
53. Snajdrova, L.; Xu, A.; Narayanan, N. J. Biol. Chem. 1998, 273, 28032.
54. Dothager, R. S.; Putt, K. S.; Allen, B. J.; Leslie, B. J.; Nesterenko, V.; Hergenrother,
P. J. J. Am. Chem. Soc. 2005, 127, 8686.
55. Palchaudhuri, R.; Hergenrother, P. J. Bioorg. Med. Chem. Lett. 2008, 18, 5888.
56. Benaka Prasad, S.; Vinaya, K.; Ananda Kumar, C.; Swarup, S.; Rangappa, K. Med.
Chem. Res. 2010, 19, 220.
10. Knight, S. D.; Parrish, C. A. Curr. Top. Med. Chem. 2008, 8, 888.
11. Zhang, Y.; Xu, W. Anticancer Agents Med. Chem. 2008, 8, 698.
12. Lawrence, C. J.; Dawe, R. K.; Christie, K. R.; Cleveland, D. W.; Dawson, S. C.;
Endow, S. A.; Goldstein, L. S.; Goodson, H. V.; Hirokawa, N.; Howard, J.;
Malmberg, R. L.; McIntosh, J. R.; Miki, H.; Mitchison, T. J.; Okada, Y.; Reddy, A.
S.; Saxton, W. M.; Schliwa, M.; Scholey, J. M.; Vale, R. D.; Walczak, C. E.;
Wordeman, L. J. Cell Biol. 2004, 167, 19.
13. Goshima, G.; Vale, R. D. J. Cell Biol. 2003, 162, 1003.
14. Blangy, A.; Lane, H. A.; d’Herin, P.; Harper, M.; Kress, M.; Nigg, E. A. Cell 1995,
83, 1159.
15. Mountain, V.; Simerly, C.; Howard, L.; Ando, A.; Schatten, G.; Compton, D. A. J.
Cell Biol. 1999, 147, 351.
16. Skoufias, D. A.; DeBonis, S.; Saoudi, Y.; Lebeau, L.; Crevel, I.; Cross, R.; Wade, R.
H.; Hackney, D.; Kozielski, F. J. Biol Chem. 2006, 281, 17559.
17. Haque, S. A.; Hasaka, T. P.; Brooks, A. D.; Lobanov, P. V.; Baas, P. W. Cell Motil.
Cytoskeleton 2004, 58, 10.
18. Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.;
Mitchison, T. J. Science 1999, 286, 971.
57. Shi, J.; Orth, J. D.; Mitchison, T. Cancer Res. 2008, 68, 3269.
58. Kantarjian, H. M.; Padmanabhan, S.; Stock, W.; Tallman, M. S.; Curt, G. A.; Li, J.;
Osmukhina, A.; Wu, K.; Huszar, D.; Borthukar, G.; Faderl, S.; Garcia-Manero, G.;
Kadia, T.; Sankhala, K.; Odenike, O.; Altman, J. K.; Minden, M. Invest New Drugs
2011. doi:10/1007/s10637-011-9660-2.
59. Shimizu, M.; Ishii, H.; Ogo, N.; Unno, Y.; Matsuno, K.; Sawada, J.; Akiyama, Y.;
Asai, A. Cancer Lett. 2010, 298, 99.
60. Tunquist, B. J.; Woessner, R. D.; Walker, D. H. Mol. Cancer Ther. 2010, 9, 2046.
61. Carter, B. Z.; Mak, D. H.; Woessner, R.; Gross, S.; Schober, W. D.; Estrov, Z.;
Kantarjian, H.; Andreeff, M. Leukemia 2009, 23, 1755.
19. DeBonis, S.; Skoufias, D. A.; Lebeau, L.; Lopez, R.; Robin, G.; Margolis, R. L.;
Wade, R. H.; Kozielski, F. Mol. Cancer Ther. 2004, 3, 1079.
20. Zee-Cheng, K. Y.; Cheng, C. C. J. Med. Chem. 1970, 13, 414.
21. Tarby, C. M.; Kaltenbach, R. F., 3rd; Huynh, T.; Pudzianowski, A.; Shen, H.;
Ortega-Nanos, M.; Sheriff, S.; Newitt, J. A.; McDonnell, P. A.; Burford, N.;
Fairchild, C. R.; Vaccaro, W.; Chen, Z.; Borzilleri, R. M.; Naglich, J.; Lombardo, L.
J.; Gottardis, M.; Trainor, G. L.; Roussell, D. L. Bioorg. Med. Chem. Lett. 2006, 16,
2095.
22. Cox, C. D.; Coleman, P. J.; Breslin, M. J.; Whitman, D. B.; Garbaccio, R. M.; Fraley,
M. E.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao,
W.; Davide, J. P.; Diehl, R. E.; Abrams, M. T.; South, V. J.; Huber, H. E.; Torrent,
M.; Prueksaritanont, T.; Li, C.; Slaughter, D. E.; Mahan, E.; Fernandez-Metzler,
C.; Yan, Y.; Kuo, L. C.; Kohl, N. E.; Hartman, G. D. J. Med. Chem. 2008, 51, 4239.
62. Peters, N. T.; Kropf, D. L. BMC Plant Biol. 2006, 6, 19.
63. George, O.; Bryant, B. K.; Chinnasamy, R.; Corona, C.; Arterburn, J. B.; Shuster, C.
B. ACS Chem. Biol. 2008, 3, 167.