Novel ATP-competitive kinesin spindle protein inhibitors

Article abstract of DOI:10.1021/jm070435y

Kinesin spindle protein (KSP), an ATPase responsible for spindle pole separation during mitosis that is present only in proliferating cells, has become a novel and attractive anticancer target with potential for reduced side effects compared to currently available therapies. We report herein the discovery of the first known ATP-competitive inhibitors of KSP, which display a unique activity profile as compared to the known loop 5 (L5) allosteric KSP inhibitors that are currently under clinical evaluation. Optimization of this series led to the identification of biphenyl sulfamide 20, a potent KSP inhibitor with in vitro antiproliferative activity against human cells with either wild-type KSP (HCT116) or mutant KSP (HCT116 D 130V). In a murine xenograft model with HCT116 D130V tumors, 20 showed significant antitumor activity following intraperitoneal dosing, providing in vivo proof-of-principle of the efficacy of an ATP-competitive KSP inhibitor versus tumors that are resistant to the other known KSP inhibitors.

Full text of DOI:10.1021/jm070435y

J. Med. Chem. 2007, 50, 4939-4952
4939
Novel ATP-Competitive Kinesin Spindle Protein Inhibitors
Cynthia A. Parrish,*,† Nicholas D. Adams,† Kurt R. Auger,‡ Joelle L. Burgess,† Jeffrey D. Carson,§ Amita M. Chaudhari,†
Robert A. Copeland,§ Melody A. Diamond,‡ Carla A. Donatelli,† Kevin J. Duffy,† Leo F. Faucette,‡ Jeffrey T. Finer,|
William F. Huffman,† Erin D. Hugger, Jeffrey R. Jackson,‡ Steven D. Knight,† Lusong Luo,§ Michael L. Moore,†
Ken A. Newlander,† Lance H. Ridgers,† Roman Sakowicz,| Antony N. Shaw,† Chiu-Mei M. Sung,‡ David Sutton,‡
Kenneth W. Wood,| Shu-Yun Zhang,‡ Michael N. Zimmerman,† and Dashyant Dhanak†
From the Departments of Medicinal Chemistry, Biology, Enzymology and Mechanistic Pharmacology, and Drug Metabolism and
Pharmacokinetics, Oncology Center of Excellence for Drug DiscoVery, GlaxoSmithKline, 1250 South CollegeVille Road,
CollegeVille, PennsylVania 19426, and Cytokinetics, Inc., 280 East Grand AVenue, South San Francisco, California 94080
ReceiVed April 12, 2007
Kinesin spindle protein (KSP), an ATPase responsible for spindle pole separation during mitosis that is
present only in proliferating cells, has become a novel and attractive anticancer target with potential for
reduced side effects compared to currently available therapies. We report herein the discovery of the first
known ATP-competitive inhibitors of KSP, which display a unique activity profile as compared to the known
loop 5 (L5) allosteric KSP inhibitors that are currently under clinical evaluation. Optimization of this series
led to the identification of biphenyl sulfamide 20, a potent KSP inhibitor with in vitro antiproliferative
activity against human cells with either wild-type KSP (HCT116) or mutant KSP (HCT116 D130V). In a
murine xenograft model with HCT116 D130V tumors, 20 showed significant antitumor activity following
intraperitoneal dosing, providing in vivo proof-of-principle of the efficacy of an ATP-competitive KSP
inhibitor versus tumors that are resistant to the other known KSP inhibitors.
Introduction
Kinesin spindle protein (KSP or HsEg5) has recently been the
focus of intense interest as a novel biological target for
anticancer therapy. The role of this kinesin in mitosis has been
elucidated using both biological (antibody microinjection,
siRNA)11,12 and small molecule inhibitor approaches.13 KSP
activity has been shown to be required early in mitosis for the
establishment of a functional, bipolar mitotic spindle. In a
number of human cancer cell lines, failure of spindle assembly
has been associated with mitotic arrest, failure of the mitotic
checkpoint, and subsequent apoptosis.
The kinesins are a family of motor proteins involved in a
number of key cellular functions including subcellular organelle
positioning, vesicular trafficking, and mitotic spindle assembly
and function.1-4 In general, these proteins couple the energy of
ATP hydrolysis to a directed mechanical force along microtu-
bules (MTsa). A sub-group of kinesins, the mitotic kinesins,
are unique in that their expression is limited to the mitotic phase
of cell division during which they act, often in complex with
other proteins, to help establish the mitotic spindle, segregate
and align chromosomes, and aid in cytokinesis.5
The mitotic spindle is a well-known and pharmaceutically
validated target, and agents such as the taxanes (paclitaxel,
docetaxel) and vinca alkaloids (vincristine, vinblastine, vinorel-
bine), which interfere with MT dynamics, are important
chemotherapeutics currently in clinical use as anticancer agents
(Chart 1).6,7 However, because MTs are also present in other
nondividing cells, including post-mitotic neurons, undesirable
side effects, such as peripheral neuropathy, are often observed
in patients undergoing treatment with these drugs.8 In contrast,
mitotic kinesins are absent from post-mitotic neurons, and small
molecule inhibitors of these enzymes may lead to novel
anticancer agents lacking the neuropathic side effects of the
antitubulins.9
Following the discovery of monastrol (1, Figure 1),13 several
other small molecule KSP inhibitors have been reported from
a variety of structural classes.9,14-24 Of these, ispinesib (2) was
the first compound to enter human clinical trials and is currently
The mitotic kinesin family is comprised of at least 11
members with either overlapping or unique roles in mitosis.10
* Corresponding author. Telephone: 610-917-6007. Fax: 610-917-4171.
E-mail: cynthia.a.parrish@gsk.com.
† Department of Medicinal Chemistry.
‡ Department of Biology.
§ Department of Enzymology and Mechanistic Pharmacology.
| Cytokinetics, Inc.
Department of Drug Metabolism and Pharmacokinetics.
a Abbreviations: KSP, kinesin spindle protein; L5, loop 5; siRNA, small
interfering RNA; MT, microtubule; SAR, structure-activity relationship;
CTG, CellTiter-Glo; LHS, left-hand side; RHS, right-hand side; MTD,
maximum tolerated dose; CR, complete regression; PR, partial regression.
Figure 1. Some reported KSP inhibitors.
10.1021/jm070435y CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/29/2007

4940 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Parrish et al.
Chart 1
undergoing Phase II evaluation in various solid tumor types.7,25
Ispinesib is a potent (subnanomolar Ki) KSP inhibitor with
impressive in vivo efficacy in a variety of preclinical animal
tumor xenograft models.26,27 Biochemical and crystallographic
characterization of 2 has established that the mechanism of
action of this class of inhibitor is ATP-uncompetitive and MT-
noncompetitive.28 The compound binds to KSP at an induced
fit binding site similar to that reported for 129 and other inhibitors
(3, 4).16,17,19,20
) 6 µM), analysis of inhibitor-treated SKOV3 cells by immu-
nofluorescence confirmed a monopolar spindle phenotype,
consistent with intracellular KSP inhibition. Importantly, the
compound displayed similar potency against both wild-type KSP
and mutant (D130V) KSP, suggesting that biphenyl 5 was
interacting with KSP at a site distinct from the L5 inhibitor
binding site. This article describes the synthesis and in vitro
and in vivo characterization of a series of analogs of 5 displaying
a unique mode of inhibition against KSP.
Acquired resistance to chemotherapeutic agents as a result
of either amino acid mutations of the target or at a cellular level
by, for example, up-regulation of efflux mechanisms is a
common occurrence. Indeed, the generation of a colorectal tumor
cell line resistant to quinazolinone KSP inhibitors such as 2
has been reported (designated HCT116 D130V).28 In these cells,
DNA sequencing of KSP revealed a D130V mutation in the
loop 5 (L5) region of the motor domain.30,31 This region forms
one side of a binding pocket shared by all of the currently
reported KSP inhibitors (e.g., Figure 1). We reasoned that
identification of KSP inhibitors that rely on accessing a distinct
binding site and engaging different amino acids could represent
a unique and complementary approach to the L5-targeted
inhibitors. In addition, if this binding mode also resulted in a
different mode of enzyme inhibition (e.g., ATP-competitive),
it could allow for novel pharmacology because the association
of KSP to MTs is strongly influenced by the occupancy of the
ATP-binding pocket.32
To discover inhibitors with these properties, cell-active KSP
inhibitors identified from a large library of compounds were
screened versus the mutant (D130V) KSP. Chemically attractive
hits were subsequently triaged using both the biochemical mode
of inhibition studies as well as the cellular assays to rule out
compounds with nonspecific or off-target effects.
The biphenyl methylsulfonamide 5 satisfied many of our
criteria and was selected for further investigation (Figure 2).
The biochemical potency of 5 (KSP Ki ) 120 nM) was
remarkable in view of its relatively simple structure and, under
steady-state conditions, the compound was determined to be
an ATP-competitive, MT-uncompetitive inhibitor (Figure 3).
Although biphenyl 5 showed only modest activity in inhibiting
the proliferation of human ovarian cancer cells (SKOV3 IC50
Results and Discussion
Chemistry. The relative simplicity of 5 as a lead was
attractive because it offered an opportunity to modulate simul-
taneously both potency and developability properties. In addi-
tion, the ease of synthesis allowed for the rapid preparation of
analogs to survey structure-activity relationship (SAR) trends.
A general synthetic procedure for the preparation of various
biphenyl analogs reported in this paper is outlined in Scheme
1. Functionalized biphenylamines were prepared via a Suzuki
cross-coupling reaction between a bromoaniline and a phenyl
boronic acid. Acylation of the aniline product with a sulfonyl
chloride, isocyanate, sulfamoyl chloride, or carbamyl chloride
provided several of the analogs described. Alternatively, acy-
lation of a bromoaniline under similar conditions was carried
out prior to Suzuki cross-coupling to yield the target biphenyl
analogs directly. When performed in a parallel synthesis manner,
these two complementary routes were sufficient to quickly
access a large number of biphenyl analogs with varied substitu-
tion around the two halves (Tables 1 and 2).33-35
Biological Evaluation of Biphenyl Inhibitors. The ability
of the biphenyl derivatives to inhibit KSP enzymatic activity
was determined using the previously reported ATPase assay.33,36
Biochemical mechanism of action studies, shown for 5 (Figure
3), were also performed on compounds 16, 20, 29, and 30 and
confirmed ATP-competitive inhibition (data not shown). Com-
pounds were assayed for their antiproliferative activity against
the human ovarian cancer cell line, SKOV3, using a lumines-
cence-based readout (CTG). Finally, to assess the cellular
phenotype resulting from inhibitor treatment, an automated cell
imaging protocol was employed.
Initial studies established that major modifications to the
biphenyl core, such as the introduction of one to three spacer
atoms (e.g., O, S, C, N) between the two phenyl rings, were
not tolerated (data not shown). This suggested that when bound
to KSP, the molecule preferred to maintain a relatively linear
conformation. We hypothesized that the methylsulfonamide
moiety was important in making key hydrogen bond interactions,
and this idea was supported by the large decrease in inhibitory
activity observed when this group was moved to the meta-
position (6, Table 1) or removed altogether (7). Similarly,
methylation of the sulfonamide (8) or removal of the sulfonyl
group to give methylamine 9 afforded analogs with decreased
Figure 2. Novel biphenyl KSP inhibitor.

ATP-CompetitiVe Kinesin Spindle Protein Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4941
Figure 3. Mode of inhibition studies of biphenyl 5 to KSP by varying the ATP concentration in the presence of saturating MT (left) and by
varying the MT concentration in the presence of saturating ATP (right). The Lineweaver-Burk plots indicate 5 is competitive with respect to ATP
but uncompetitive with respect to MT.
Scheme 1a
a Reagents and conditions: (i) Pd(PPh3)4 (cat.), aq K2CO3 soln, DMF, 100 °C; (ii) Y-Cl (Y ) SO2R1, SO2NR1R2, or CONR1R2); (iii) R1sNdCdO,
CH2Cl2; (iv) PdCl2(dppf) (cat.), aq Na2CO3 soln, CH3CN, µwave, 100 °C.
biochemical potency, highlighting a need for a hydrogen bond
donor and potentially a hydrogen bond acceptor. Extending the
methyl group of the sulfonamide to an ethyl (10) or elaborating
it further (data not shown) also led to large decreases in KSP
inhibitory activity, consistent with a high degree of steric
constraint in this region.
to have a similar pKa for the anilino nitrogen (e.g., 20 and 23)38
also showed a 100-fold difference in KSP inhibition, perhaps
reflecting the greater importance of the terminal amino group
as a weakly acidic hydrogen bond donor. Consistent with this
hypothesis, methylation of the anilino nitrogen (24) or replace-
ment with a methylene (25) or oxygen (27) led to analogs with
reasonable to very good KSP inhibitory activity. Interestingly,
perhaps indicative of subtle differences between the urea and
the sulfamide subseries, sulfamate 27 showed only a 3-fold
decrease in potency as compared to the parent sulfamide (20),
in contrast to the 18-fold decrease observed when introducing
the same substitution in the ureas (i.e., carbamate 28 compared
to the parent urea 16).
With the identification of the sulfamide as an effective
methylsulfonamide replacement, we proceeded to evaluate the
effects of additional substituents on the left phenyl ring (LHS),
with an aim to increase potency further. Sterically undemanding
substituents ortho to the urea or sulfamide were tolerated, with
the presence of a fluorine atom providing a more potent KSP
inhibitor (29, 30). As noted above, this effect appeared to be
unrelated to the enhanced acidity of the sulfamide group, as
indicated by the decreased potency of analogs with more
electron-withdrawing groups (31, 37, 38, 39).38 Replacement
of the fluorine atom (e.g., 32, 34, 35, 37) provided biphenyl
analogs with good to excellent KSP inhibitory activity, with
the primary amino group (35) providing a compound with the
best activity of the fluorine replacements. Based on a 60-fold
reduction in potency of the methylamine 36 compared to the
corresponding primary amine 35, it seemed likely that either
both anilino hydrogens were involved in making critical
interactions with KSP or the binding pocket could not favorably
accommodate the additional methyl group. However, the
unexpectedly good activity of methyl ether 33, an isosteric
Replacement of the methylsulfonamide moiety led to biphenyl
analogs encompassing a wider range of inhibitory activity. While
some polar groups with hydrogen bond donating capabilities
(11, 12) had modest KSP inhibitory activity, others were at best
equipotent to 5 (e.g., 13). Better results were obtained when
the methylsulfonamide in 5 was replaced with a urea moiety
(16), giving a compound that had approximately 4-fold higher
potency in inhibiting KSP. This increased biochemical potency
also translated to improved cellular activity (Table 2). To probe
further the SAR of the urea group, the isosteric thiourea (17)
and guanidine (18) analogs were prepared. Although the basic
guanidine 18 lacked inhibitory activity, the thiourea 17 led to
another 4-fold increase in biochemical potency. We therefore
prepared the corresponding thiourea bioisostere, cyanoguanidine
19.37 This analog had measurable KSP inhibitory activity but
was approximately 20-fold less potent than the urea (16) and
80-fold less potent than 17. Substitution of the urea thiocarbonyl
moiety with a sulfonyl group proved to be more feasible and
provided the sulfamide 20, which was approximately equipotent
to 17.
Methylation or substitution of the various hydrogen bond
donors in the urea and sulfamide analogs was subsequently
investigated (21-28). Mono- or bismethylation on the terminal
nitrogen led to analogs with significantly decreased KSP
inhibitory activity, potentially the result of substantial steric
constraint, as observed in earlier attempts to extend the
methylsulfonamide (e.g., 5 to 10). However, analogs predicted

4942 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Table 1. KSP Inhibitory Activity of LHS Analogs
Parrish et al.
Table 2. Biochemical and Cellular Data for RHS Analogs
KSP IC50
(nM)
cmpd
R1
R2
R3
R4
5
6
7
8
9
H
NHSO2CH3
H
H
N(CH3)SO2CH3
NHCH3
NHSO2CH2CH3
NH2
OH
SO2NH2
C(O)NH2
CO2H
NHC(O)NH2
NHC(S)NH2
NHC(NH)NH2
NHC(NCN)NH2
NHSO2NH2
NHC(O)NHCH3
NHC(O)N(CH3)2
NHSO2N(CH3)2
N(CH3)SO2NH2
CH2SO2NH2
CH2SO2NHCH3
OSO2NH2
OC(O)NH2
NHC(O)NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
NHSO2NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
161
NHSO2CH3
6000
5320
>20 000
3726
2518
1649
700
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
345
>20 000
>20 000
39
11
>20 000
840
18
>20 000
>20 000
1795
1422
118
>20 000
64
716
15
5
196
141
66
349
F
CN
CH3
OCH3
OH
NH2
NHCH3
Cl
24
1448
67
685
502
H
F
H
analog of 36 that lacks hydrogen bond donating capabilities,
suggested that the binding site may be flexible enough to
compensate for alternative key interactions. Substitution close
to the biphenyl juncture at R4 with even a fluorine atom (38)
led to a significant decrease in KSP inhibitory potency,
indicating the binding site may be sensitive to the planarity of
the molecule.
Having established that urea or sulfamide were close to being
optimal groups on the left phenyl for KSP inhibition, we next
investigated expanding the SAR around the right phenyl ring
(RHS) of the biphenyl core. It quickly became apparent that
there was a very high preference for a trifluoromethyl group in
the para-position (Table 2). Removal (44), transposition to the
meta-position (45), or replacement of the trifluoromethyl group
(16) with the smaller methyl group (46) afforded analogs either
devoid of or with significantly less KSP affinity. Nonetheless,
replacements such as an isopropyl (47) or a t-butyl (48) were
better tolerated, particularly in the biochemical KSP inhibition
assay. This suggested that the molecular volume associated with
the trifluoromethyl group rather than the electronic nature of
the group was the more critical element in imparting activity.
Reasons for the approximately 7-fold reduction in cellular
activity are unclear, as the analogs, especially 47, which displays
a similar clogP,39 encompass similar permeability and solubility
properties as the parent (16). The use of a halogen (40, 49) in
a NT ) not tested.
lieu of the trifluoromethyl group led to more modest inhibitors
in the biochemical KSP assay.
Since electronegative fluorine atoms have been reported as
weak hydrogen bond acceptors,40 we investigated this region
for such effects. Homologation of the trifluoromethyl group by
a methylene (41) provided an analog with negligible KSP
inhibitory activity. However, homologation by either an oxygen
(42) or sulfur (50) atom was tolerated, although each led to
some decrease in biochemical activity as compared to 5.
Furthermore, conversion of 50 to the corresponding sulfone (51)
led to a more potent inhibitor, exhibiting similar biochemical

ATP-CompetitiVe Kinesin Spindle Protein Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4943
Table 3. Biological Evaluation of Optimized Biphenyls vs Wild-Type
Table 4. Pharmacokinetic Profile of 20 in the Conscious Nude Mouse
and Mutant KSP
a
Cmax
Clast
DNAUC(0-t)
route
ip
(µg/mL)
(µg/mL)
(µg h/mL/mg/kg)
10.49
8.73
2.35
a Taken at 24 h.
not discriminate between wild-type and mutant KSP. In contrast,
quinazolinone 63,14 an analog of 2, was more than 350-fold
more potent versus wild-type compared to the mutant enzyme.
As expected, 63 was far less effective in inhibiting KSP
containing the D130V mutation in the L5 region where this
compound binds. This profile of activity was also reproduced
in cell culture where sulfamides 20 and 30 both showed
antiproliferative activity versus wild-type (HCT116) and mutant
(HCT116 D130V) KSP cell lines, whereas 63 was poorly active
in cells containing the L5-mutated KSP. Surprisingly, sulfamides
20 and 30 were of much higher potency in inhibiting the
proliferation of the mutant cell line relative to the wild-type
cell line. We rationalized that this could be due to differences
in the catalytic properties between the mutant and the wild-
type KSP enzymes. Indeed, biochemical characterization of the
KSP D130V enzyme showed that the Km for ATP was
approximately 20-fold higher than that for wild-type KSP.43
Because both 20 and 30, like 5, are ATP-competitive inhibitors,
the reduced affinity of the mutant enzyme toward ATP would
have the effect of increasing its sensitivity toward the biphenyl
inhibitors.44,45
WT KSP
Ki (nM)
D130V KSP
Ki (nM)
HCT116 IC50
(nM)
HCT116 D130V
IC50 (nM)
cmpd
20
30
63
6.2 ( 0.4
3.5 ( 0.2
<1
7 ( 1
6 ( 1
350 ( 10
403 ( 225
294 ( 151
2.0 ( 1.0
5.4 ( 1.5a
3.1 ( 0.8a
1372 ( 631
a Data from one experiment (run in duplicate).
and cellular potency to the parent trifluoromethyl analog (16).
The increased potency of sulfone 51 over thioether 50 was not
merely due to the presence of the oxygen atoms, as evidenced
by the lack of activity of ethylsulfone 52, an analog that would
have been expected to show moderate activity based on the
ability to replace the trifluoromethyl group with alkyl groups.
To mimic the electron-withdrawing effect of the sulfone, the
corresponding trifluoromethylketone 53 was prepared but,
surprisingly, lacked KSP inhibitory activity. Such electron-
deficient ketones are known to readily form hydrates in aqueous
media,41,42 and the resulting ketal is presumably not compatible
with KSP inhibition. The incorporation of additional groups
capable of participating in hydrogen bonds, such as a carboxylic
acid, carboxamide, methyl ether, methyl ketone, or phosphonate,
afforded compounds without significant KSP activity (data not
shown).
In Vivo Evaluation of Biphenyl Inhibitors. In view of its
exquisite cellular potency against the HCT116 D130V colorectal
tumor line, sulfamide 20 made an excellent candidate for in
vivo evaluation using this cell line in a xenograft model. Prior
to in vivo experiments, the pharmacokinetic profile of 20 was
determined (Table 4). Following ip administration at a nontoxic
dose (82 mg/kg), sulfamide 20 led to a maximum concentration
in plasma of 10.49 µg/mL that was sustained for 24 h (Clast
)
8.73 µg/mL). Although the elimination half-life could not be
estimated, the data indicate sulfamide 20 exhibits a long half-
life and good exposure at biologically active doses in the mouse
(vide infra).
As with the left-hand side, additional substitution on the right-
hand side ring was interrogated, with substitution near the
biphenyl junction again being detrimental to KSP inhibitory
activity. However, substitution ortho to the trifluoromethyl group
was tolerated, although the substituent range investigated was
small. With a fluorine atom (54), both biochemical and cellular
potencies were similar to the parent (20), whereas the nitro
analog (55) showed similar biochemical but decreased cellular
activity. Reduction of the nitro group to an amine (56) led to a
reduction in both biochemical and cellular potency. Interestingly,
cyclization of the 3,4-position to afford benzodioxoles or
benzodioxins led to analogs showing good KSP inhibitory
activity only when fluorinated (58, 61, 62), although the cellular
activity of these analogs was diminished as compared to the
monocyclic parent (20). Generally, the presence of fluorine
atoms in this region potentiated biochemical activity, indicating
that an electronic contribution from the alkylfluorides could not
be excluded (e.g., 57 vs 58, 60 vs 61). However, despite our
efforts at modification, the balance of electronic and steric
properties of the trifluoromethyl group uniquely seemed to
provide analogs with the best biochemical and cellular activity.
Although in general the lead inhibitor 5 was resistant to a
variety of structural changes, we were able to increase signifi-
cantly the KSP inhibitory activity as well as introduce cellular
activity to the compound by making smaller, targeted modifica-
tions. Two of the most potent compounds identified from this
investigation (20, 30) were evaluated for their activity versus
the mutant form of KSP (KSP D130V). As shown in Table 3,
both compounds were highly potent enzyme inhibitors and did
Sulfamide 20 was evaluated for antitumor activity against
advanced HCT116 D130V solid tumors implanted in nude mice
in doses ranging from 16 mg/kg up to 125 mg/kg delivered ip
every 4 days for a total of three doses (Figure 4). Relative to
placebo-treated animals, tumors in mice treated with 20 showed
either regressions or significant tumor growth delay. Indeed, at
the highest dose tested (MTD > 125 mg/kg), complete tumor
regressions (CR) were observed in three of five animals dosed,
whereas at lower doses, tumor growth delays of at least 19 days
(16 mg/kg dose) were observed. In contrast and as expected
due to its decreased activity in cells carrying the mutant KSP,
the quinazolinone 63 showed only a modest delay in tumor
growth (10 days) under similar conditions at its MTD (10 mg/
kg). These results demonstrate for the first time that an ATP-
competitive KSP inhibitor can induce significant antitumor
effects and can overcome resistance due to binding site
mutations generated in vitro by allosteric quinazolinone KSP
inhibitors.
The in vivo efficacy of 20 versus tumor cell lines carrying
wild-type KSP was also investigated using the Colo205 col-
orectal tumor cell line, historically one of the more sensitive
cell lines to KSP inhibitors. In marked contrast to its activity
versus tumor lines bearing a mutant KSP, sulfamide 20 did not
show significant antitumor activity at doses up to its MTD (20
mg/kg) versus Colo205 tumors that contain wild-type KSP

4944 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Parrish et al.
Figure 4. In vivo efficacy of 20 against advanced HCT116 D130V xenografts in nude mice (dosed ip q4dx3, indicated by arrows).
Figure 5. In vivo efficacy of 20 against advanced Colo205 xenografts in nude mice (dosed ip q4dx3, indicated by arrows). Dosage of 20 at 40
mg/kg was toxic.
(Figure 5). In line with its superior activity relative to 20 in
wild-type cell culture, the quinazolinone 2 was very effective,
leading to three complete and two partial (PR) tumor regressions
of five animals dosed. The lack of efficacy for 20 in a wild-
type model versus a mutant-KSP model may be rationalized
by its more modest potency in Colo205 cells (IC50 ) 181 nM)
as compared to HCT116 D130V cells (IC50 ) 5.4 nM). These
results suggest that in vivo efficacy with ATP-competitive
inhibitors versus a tumor cell line carrying wild-type KSP may
require substantially more potent inhibitors to overcome the high
cellular ATP concentration, and further studies directed toward
this will be reported in due course.
fied here by the quinazolinones, are significantly less effective
at inhibiting KSP containing select point mutations in the L5
region, the ATP-competitive biphenyls are potent inhibitors of
KSP D130V and represent a way to overcome resistance
generated by the L5 binders. Indeed, the biphenyl analogs have
demonstrated antitumor activity in an HCT116 D130V mouse
xenograft model. Although the reported inhibitors could be
useful to treat emergent L5-mutated tumors, it would be more
desirable to develop ATP-competitive KSP inhibitors with
efficacy versus both wild-type and mutant KSP tumors. The
development and characterization of more potent ATP-competi-
tive KSP inhibitors will be the subject of future publications.
Experimental Section
Conclusion
Chemistry. General Methods. Unless otherwise noted, starting
materials and reagents were purchased from commercial sources
and used without further purification. Air- or moisture-sensitive
reactions were carried out under a nitrogen atmosphere. Anhydrous
solvents were obtained from Sigma-Aldrich. Microwave irradiation
was carried out in a Personal Chemistry Emrys Optimizer micro-
wave. Flash chromatography was performed using silica gel (EM
Science, 230-400 mesh) under standard techniques or using silica
gel cartridges (RediSep normal phase disposable flash columns)
on an Isco CombiFlash. Reverse phase HPLC purification was
We have identified a novel biphenyl series of KSP inhibitors
that display good biochemical and cellular activity and exhibit
a unique mode of action. These ATP-competitive inhibitors
contrast with the previously reported ATP-uncompetitive class
of KSP inhibitors both in mechanism and in activity versus KSP
D130V, the L5 mutant KSP generated in cell culture with
resistance to a quinazolinone inhibitor. All data are consistent
with a binding site distinct from the previously reported L5
allosteric KSP inhibitors. Whereas previous inhibitors, exempli-

ATP-CompetitiVe Kinesin Spindle Protein Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4945
conducted on a Gilson HPLC (monitoring at a wavelength of 214
) 8.5 Hz), 7.79 (d, 2H, J ) 8.6 Hz), 7.55 (d, 2H, J ) 8.6 Hz),
3.29 (s, 3H), 3.00 (s, 3H). MS m/z 330.0 [M + H]+. Anal.
(C15H14F3NO2S) C, H, N.
or 254 nm) with a YMC ODS-A C18 column (5 µm, 75 × 30
1
mm), eluting with 5-90% CH3CN in H2O with 0.1% TFA. H
NMR spectra were recorded on a Bruker Avance 400 MHz
spectrometer. Chemical shifts (δ) are reported in ppm relative to
an internal solvent reference. Apparent peak multiplicities are
described as s (singlet), br s (broad singlet), d (doublet), dd (doublet
of doublets), t (triplet), q (quartet), or m (multiplet). Coupling
constants (J) are reported in hertz (Hz) after the integration. Mass
spectra were recorded on an Applied Biosystems MSD Sciex API
150EX single quadrupole mass spectrometer with an electrospray
ionization (ESI) source. Analytical HPLC was conducted on an
Agilent 1100 Series HPLC with a Zorbax Eclipse XDB-C18 column
(5 µm, 4.6 × 150 mm) using a 10 min gradient (10-90% CH3-
CN/H2O with 0.1% TFA). The retention time (tR) is expressed in
min at a UV detection of 254 nm. Elemental analyses were
performed by Quantitative Technologies, Inc., Whitehouse, NJ.
General Method for Suzuki Cross-Coupling. Method A: 4′-
(Trifluoromethyl)-4-biphenylamine (11). A solution of 4-bro-
moaniline (29 mmol), 4-trifluoromethylphenyl boronic acid (35
mmol), and tetrakis(triphenylphosphine)palladium(0) (1.4 mmol)
in 2 M aq potassium carbonate (50 mL) and DMF (50 mL) was
heated at 100 °C for 17 h. The reaction mixture was cooled, poured
into half-saturated aq sodium bicarbonate (400 mL), and extracted
with (3 × 400 mL) diethyl ether. The combined organic layers
were dried over sodium sulfate and concentrated in vacuo.
Purification of the residue by silica gel chromatography (10-30%
ethyl acetate/hexanes) provided the title product as a white powder
(70%). 1H NMR (DMSO-d6): δ 7.76 (d, 2H, J ) 8.1 Hz), 7.69 (d,
2H, J ) 8.3 Hz), 7.45 (d, 2H, J ) 8.6 Hz), 6.67 (d, 2H, J ) 8.6
Hz), 5.41 (s, 2H). MS m/z 238.2 [M + H]+. Analytical HPLC 98.5%
purity, tR ) 5.39.
Methyl[4′-(trifluoromethyl)-4-biphenylyl]amine (9). To a solu-
tion of 4′-(trifluoromethyl)-4-biphenylamine (4.21 mmol) and
pyridine (6.31 mmol) in dichloromethane (30 mL) was added
4-nitrophenyl formate (4.21 mmol). The reaction mixture was stirred
at room temperature for 18 h. The reaction mixture was dissolved
in ethyl acetate (150 mL) and washed with water (3 × 100 mL)
and brine (1 × 100 mL). The organic layer was dried over sodium
sulfate and was concentrated in vacuo. Purification of the residue
by reverse phase HPLC afforded the intermediate [4′-(trifluoro-
methyl)-4-biphenylyl]formamide as a white solid (85%). MS m/z
266.2 [M + H]+. A solution of this formamide (3.75 mmol) in 1
M lithium aluminum hydride in tetrahydrofuran (10 mL) was stirred
for 18 h at room temperature. The reaction was quenched with
saturated sodium sulfate solution (10 mL), extracted into ethyl
acetate (3 × 10 mL), and concentrated in vacuo. The residue was
purified on reverse phase HPLC to afford the title product as a
brown solid (15%). 1H NMR (DMSO-d6): δ 7.78 (d, 2H, J ) 8.0
Hz), 7.70 (d, 2H, J ) 8.0 Hz), 7.55 (d, 2H, J ) 8.0 Hz), 6.70 (d,
2H, J ) 8.0 Hz), 2.74 (s, 3H). MS m/z 252.2 [M + H]+. Analytical
HPLC 94.5% purity, tR ) 6.16.
N-[4′-(Trifluoromethyl)-4-biphenylyl]ethanesulfonamide (10).
The title compound was prepared following Method B using
1
ethanesulfonylchoride (88%). H NMR (DMSO-d6): δ 10.02 (s,
1H), 7.86 (d, 2H, J ) 8.3 Hz), 7.79 (d, 2H, J ) 8.3 Hz), 7.72 (d,
2H, J ) 8.6 Hz), 7.34 (d, 2H, J ) 8.6 Hz), 3.15 (q, 2H, J ) 7.3
Hz), 1.21 (t, 3H, J ) 7.3 Hz). MS m/z 330.0 [M + H]+. Anal.
(C15H14F3NO2S) C, H, N.
4′-(Trifluoromethyl)-4-biphenylol (12). The title compound was
prepared as a white solid following Method A using 4-bromophenol
with purification by reverse phase HPLC (46%). 1H NMR (DMSO-
d6): δ 9.75 (s, 1H), 7.81 (d, 2H, J ) 8.4 Hz), 7.75 (d, 2H, J ) 8.5
Hz), 7.58 (dd, 2H, J ) 6.7, 1.9 Hz), 6.89 (dd, 2H, J ) 6.7, 1.9
Hz). MS m/z 239.2 [M + H]+. Analytical HPLC 99.6% purity, tR
) 7.77.
4′-(Trifluoromethyl)-4-biphenylsulfonamide (13). The title
compound was prepared as a yellow solid following Method A
using 4-bromobenzenesulfonamide with purification by reverse
phase HPLC (50%). 1H NMR (DMSO-d6): δ 7.76 (d, 2H, J ) 8.4
Hz), 7.64 (d, 2H, J ) 8.6 Hz), 7.52 (d, 2H, J ) 8.8 Hz), 7.48 (d,
2H, J ) 8.6 Hz), 7.23 (s, 2H). MS m/z 302.2 [M + H]+. Anal.
(C13H10F3NO2S) C, H, N.
4′-(Trifluoromethyl)-4-biphenylcarboxamide (14). The title
compound was prepared as a white solid following Method A using
4-bromobenzamide with purification by reverse phase HPLC (35%).
1H NMR (DMSO-d6): δ 8.08 (s, 1H), 8.01 (d, 2H, J ) 8.6 Hz),
7.97 (d, 2H, J ) 8.1 Hz), 7.86-7.83 (m, 4H), 7.46 (s, 1H). MS m/z
266.2 [M + H]+. Anal. (C14H10F3NO) C, H, N.
4′-(Trifluoromethyl)-4-biphenylcarboxylic Acid (15). The title
compound was prepared following Method A using 4-bromobenzoic
acid with purification by reverse phase HPLC (6%). 1H NMR
(DMSO-d6): δ 12.10 (s, 1H), 8.07 (d, 2H, J ) 8.4 Hz), 7.98 (d,
2H, J ) 8.2 Hz), 7.88 (m, 4H). MS m/z 267.2 [M + H]+. Anal.
(C14H9F3O2) C, H.
General Method for Sulfonylation. Method B: N-[4′-(Tri-
fluoromethyl)-4-biphenylyl]methanesulfonamide (5). To a solu-
tion of 4′-(trifluoromethyl)-4-biphenylamine (0.42 mmol) in dichlo-
romethane (2.0 mL) was sequentially added pyridine (0.84 mmol)
and methanesulfonylchloride (0.63 mmol). The reaction mixture
was stirred at room temperature for 18 h and then concentrated in
vacuo. Purification by reverse phase HPLC afforded the title product
as a white solid (47%). 1H NMR (DMSO-d6): δ 9.99 (s, 1H), 7.88
(d, 2H, J ) 8.2 Hz), 7.81 (d, 2H, J ) 8.4 Hz), 7.75 (d, 2H, J ) 8.6
Hz), 7.34 (d, 2H, J ) 8.6 Hz), 3.06 (s, 3H). MS m/z 316.2 [M +
H]+. Anal. (C14H12F3NO2S) C, H, N.
N-[4′-(Trifluoromethyl)-3-biphenylyl]methanesulfonamide (6).
The intermediate 4′-(trifluoromethyl)-3-biphenylamine was prepared
following Method A using 3-bromoaniline (83%). 1H NMR
(CDCl3): δ 7.59 (s, 3H), 7.19 (m, 2H), 6.91 (dd, 1H, J ) 7.7, 1.6
Hz), 6.83 (t, 1H, J ) 1.9 Hz), 6.66 (dd, 1H, J ) 7.9, 2.3 Hz), 3.72
(s, 2H). MS m/z 238.2 [M + H]+. The title compound was prepared
1
from this aniline following Method B (45%). H NMR (DMSO-
d6): δ 9.92 (s, 1H), 7.85 (s, 4H), 7.52-7.47 (m, 3H), 7.29 (d, 1H,
J ) 6.8 Hz), 3.06 (s, 3H). MS m/z 316.2 [M + H]+. Anal.
(C14H12F3NO2S) C, H, N.
4-(Trifluoromethyl)biphenyl (7). The title compound was
prepared as a white solid following Method A using 4-bromoben-
1
zene (55%). H NMR (DMSO-d6): δ 7.91 (d, 2H, J ) 8.2 Hz),
7.83 (d, 2H, J ) 8.3 Hz), 7.75 (d, 2H, J ) 7.1 Hz), 7.53 (m, 2H),
7.45 (t, 1H, J ) 7.3 Hz). MS m/z 223.2 [M + H]+. Analytical
HPLC 97.3% purity, tR ) 9.78.
General Method for Urea Formation. Method C: N-[4′-
(Trifluoromethyl)-4-biphenylyl]urea (16). To a solution of 4′-
(trifluoromethyl)-4-biphenylamine (0.84 mmol) in dichloromethane
(10.0 mL) was added chlorosulfonylisocyanate (1.3 mmol). The
reaction mixture was stirred at room temperature for 3 h, during
which time a white precipitate formed. The reaction mixture was
quenched with water (10.0 mL) and stirred 18 h. The mixture was
filtered, washed with water, and the solid was dried in vacuo.
Recrystallization of the solid from hot isopropanol afforded the title
compound as a white powder (54%). Alternatively, the product
could be purified by reverse phase HPLC or by flash chromatog-
N-Methyl-N-[4′-(trifluoromethyl)-4-biphenylyl]methanesulfon-
amide (8). Sodium hydride (0.15 mmol of a 60% dispersion in
mineral oil) was carefully added to a solution of N-[4′-(trifluorom-
ethyl)-4-biphenylyl]methanesulfonamide (0.13 mmol) in DMF (2
mL). After 20 min, methyl iodide (0.19 mmol) was added, and the
reaction mixture was stirred at room temperature for 16 h. The
reaction mixture was poured into water (75 mL) and extracted with
(3 × 70 mL) ethyl acetate. The combined organic layers were dried
over sodium sulfate and concentrated in vacuo. Purification of the
residue by silica gel chromatography (10-35% ethyl acetate/
hexanes) provided the title product as a white solid (quantitative).
1H NMR (DMSO-d6): δ 7.92 (d, 2H, J ) 8.1 Hz), 7.83 (d, 2H, J
1
raphy (50-90% ethyl acetate/hexanes). H NMR (DMSO-d6): δ
8.73 (s, 1H), 7.85 (d, 2H, J ) 8.3 Hz), 7.77 (d, 2H, J ) 8.4 Hz),

4946 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Parrish et al.
7.64 (d, 2H, J ) 8.7 Hz), 7.54 (d, 2H, J ) 8.7 Hz), 5.94 (s, 2H).
J ) 4.4 Hz), 2.66 (d, 3H, J ) 4.3 Hz). MS m/z 295.2 [M + H]+.
Anal. (C15H13F3N2O) C, H, N.
MS m/z 281.2 [M + H]+. Analytical HPLC 97.7% purity, tR
)
6.53.
N,N-Dimethyl-N′-[4′-(trifluoromethyl)-4-biphenylyl]urea (22).
Sodium hydride (7.00 mmol of a 60% suspension in mineral oil)
was added to an ice-cooled, stirred solution of 4-bromophenyl
isocyanate (3.53 mmol) and guanidine hydrochloride (31.4 mmol)
in DMF (30 mL) under nitrogen. After stirring at room temperature
for 18 h, the mixture was poured into water (300 mL) and acidified
with 1 M aq hydrochloric acid. The solid was filtered, washed with
water and ether, then dried to give N-[amino(imino)methyl]-N′-(4-
bromophenyl)urea as a white solid (47%) contaminated with a small
N-[4′-(Trifluoromethyl)-4-biphenylyl]thiourea (17). A solution
of 4′-(trifluoromethyl)-4-biphenylamine (0.84 mmol) in water (1
mL) and 1 M hydrochloric acid solution (0.84 mL) was treated
with ammonium thiocyanate (0.84 mmol). The reaction mixture
was heated at 110 °C for 2 h. The homogeneous reaction solution
was cooled and poured onto ice (4 g). A white precipitate formed
and was collected by filtration. Purification of the residue by reverse
1
phase HPLC yielded the title product (21%). H NMR (CD3CN):
1
amount (13%) of bisacylated guanidine. H NMR (DMSO-d6) δ
δ 8.56 (s, 1H), 7.93 (d, 2H, J ) 8.6 Hz), 7.87 (d, 2H, J ) 8.6 Hz),
10.41 (br s, 1H), 9.09 (br s, 1H), 7.54 (d, 2H, J ) 9.1 Hz), 7.50 (d,
2H, J ) 9.1 Hz). MS m/z 257 [M + H]+. A stirred mixture of the
urea (1.00 mmol), 4-(trifluoromethyl)phenylboronic acid (2.00
mmol), and tetrakis(triphenylphosphine)palladium(0) (0.05 mmol)
in DMF (3 mL) and 2 M aq potassium carbonate (3 mL) was heated
at 100 °C under argon for 5 h, then cooled and diluted with 1 M
aq hydrochloric acid (45 mL). The precipitate was filtered, washed
with water and dried. The crude product was purified by silica gel
chromatography (50-70% ethyl acetate/hexanes) to give the title
7.81 (d, 2H, J ) 8.6 Hz), 7.60 (d, 2H, J ) 8.6 Hz), 6.57 (br s, 2H).
MS m/z 297.5 [M + H]+. Analytical HPLC 87.8% purity, tR
6.86.
)
N-[4′-(Trifluoromethyl)-4-biphenylyl]guanidine (18). A solu-
tion of 4′-(trifluoromethyl)-4-biphenylamine (0.30 mmol) in pyri-
dine (2.0 mL) was treated with 1H-pyrazole-1-carboxamidine
hydrochloride (0.32 mmol). The reaction mixture was stirred at
room temperature for 19 h, heated at 50 °C for 22 h, and then
heated at 100 °C for 7 h. The mixture was cooled, poured into
water (50 mL) and brine (20 mL), and extracted with (3 × 60 mL)
ethyl acetate. The combined organic layers were dried over sodium
sulfate and concentrated in vacuo. Purification of the residue by
reverse phase HPLC provided the trifluoroacetate salt of the title
1
compound as a solid (55%). H NMR (DMSO-d6) δ 8.46 (s, 1H),
7.87 (d, 2H, J ) 8.2 Hz), 7.77 (d, 2H, J ) 8.3 Hz), 7.64 (m, 4H),
2.96 (s, 6H). MS m/z 309 [M + H]+. Analytical HPLC 99.7%
purity, tR ) 7.51.
N,N-Dimethyl-N′-[4′-(trifluoromethyl)-4-biphenylyl]sul-
famide (23). To a solution of 4′-(trifluoromethyl)-4-biphenylamine
(0.42 mmol) in dichloromethane (2.0 mL) was sequentially added
triethylamine (1.26 mmol) and dimethylaminesulfamoyl chloride
(0.63 mmol). The reaction mixture was stirred at room temperature
for 18 h and then concentrated in vacuo. Purification by reverse
1
product as a white solid (26%). H NMR (DMSO-d6): δ 9.91 (s,
1H), 7.92 (d, 2H, J ) 8.2 Hz), 7.84 (m, 4H), 7.58 (br s, 3H), 7.38
(d, 2H, J ) 8.6 Hz). MS m/z 280.2 [M + H]+. Anal. (C14H12F3N3‚C2-
HF3O2) C, H, N.
N-Cyano-N′-[4′-(trifluoromethyl)-4-biphenylyl]guanidine (19).
A solution of 4′-(trifluoromethyl)-4-biphenylamine (0.32 mmol) and
sodium dicyanamide (0.35 mmol) in water (2.0 mL) with 1 N aq
hydrochloric acid (0.35 mmol) was heated at 60 °C for 5.5 h. The
reaction mixture was poured into water (55 mL) and brine (15 mL)
and extracted with (3 × 50 mL) ethyl acetate. The combined organic
layers were dried over sodium sulfate and concentrated in vacuo.
Purification of the residue by silica gel chromatography (60-100%
ethyl acetate/hexanes) afforded the title product as an ivory solid
(26%). 1H NMR (DMSO-d6): δ 9.25 (s, 1H), 7.87 (d, 2H, J ) 8.1
Hz), 7.80 (d, 2H, J ) 8.3 Hz), 7.71 (d, 2H, J ) 8.9 Hz), 7.51 (d,
2H, J ) 8.9 Hz), 7.12 (s, 2H). MS m/z 305.2 [M + H]+. Analytical
HPLC 97.5% purity, tR ) 6.76.
General Method for Sulfamide Formation. Method D: N-[4′-
(Trifluoromethyl)-4-biphenylyl]sulfamide (20). To an ice-cooled
solution of chlorosulfonylisocyanate (0.47 mmol) in acetonitrile (1.0
mL) was added water (0.47 mmol). The reaction mixture was stirred
at 0 °C for 1 min and then allowed to warm to room temperature
slowly. After 3 h, the reaction mixture was cooled to 0 °C, at which
point a solution of 4′-(trifluoromethyl)-4-biphenylamine (0.43
mmol), pyridine (0.86 mmol), and acetonitrile (1.0 mL) was added
dropwise. The reaction mixture was stirred at 0 °C for 1 min and
then allowed to warm to room temperature slowly. After 15 h, the
reaction mixture was diluted with water (60 mL) and brine (10
mL) and extracted with (3 × 50 mL) ethyl acetate. The combined
organic layers were dried over sodium sulfate and then concentrated
in vacuo. Purification by silica gel chromatography (20-50% ethyl
acetate/hexanes) afforded the title compound as a white solid (57%).
1H NMR (DMSO-d6): δ 9.75 (s, 1H), 7.85 (d, 2H, J ) 8.1 Hz),
7.77 (d, 2H, J ) 8.3 Hz), 7.68 (d, 2H, J ) 8.6 Hz), 7.28 (d, 2H,
J ) 8.6 Hz), 7.22 (s, 2H). MS m/z 317.2 [M + H]+. Anal.
(C13H11F3N2O2S) C, H, N.
1
phase HPLC afforded the title product as a white solid (35%). H
NMR (DMSO-d6) δ 10.15 (s, 1H), 7.88 (d, 2H, J ) 8.2 Hz), 7.79
(d, 2H, J ) 8.4 Hz), 7.71 (d, 2H, J ) 8.7 Hz), 7.51 (d, 2H, J ) 8.7
Hz), 2.73 (s, 6H). MS m/z 345.2 [M + H]+. Anal. (C15H15F3N2O2S)
C, H, N.
N-Methyl-N-[4′-(trifluoromethyl)-4-biphenylyl]sulfamide (24).
To a solution of N-[4′-(trifluoromethyl)-4-biphenylyl]sulfamide
(0.36 mmol) in DMF (2.0 mL) was added sodium hydride (0.38
mmol). The reaction mixture was stirred at room temperature for
10 min and was treated with methyl iodide (0.47 mmol). After 18
h, the reaction mixture was diluted with water (10 mL) and brine
(10 mL) and extracted with (3 × 20 mL) ethyl acetate. The
combined organic layers were dried over sodium sulfate and then
concentrated in vacuo. Purification of the residue by reverse phase
HPLC afforded the title compound as a white solid (57%). 1H NMR
(DMSO-d6): δ 7.90 (d, 2H, J ) 7.6 Hz), 7.82 (d, 2H, J ) 7.6 Hz),
7.76 (d, 2H, J ) 7.1 Hz), 7.48 (d, 2H, J ) 7.2 Hz), 7.12 (s, 2H),
3.16 (s, 3H). MS m/z 331.2 [M + H]+. Anal. (C14H13F3N2O2S) C,
H, N.
1-[4′-(Trifluoromethyl)-4-biphenylyl]methanesulfonamide (25).
To a solution of 1-bromo-4-(bromomethyl)benzene (4.00 mmol)
in acetonitrile (12.0 mL) was added a solution of sodium sulfite
(4.80 mmol) in water (8.0 mL).46 The biphasic mixture was heated
at reflux (87 °C oil bath) for 16 h and then concentrated in vacuo
to remove the acetonitrile. The resultant solid was filtered and rinsed
with water (2 × 5 mL) and dichloromethane (3 × 5 mL). The
sodium salt of (4-bromophenyl)methanesulfonic acid was dried to
constant weight to afford a white powder (54%). 1H NMR (DMSO-
d6): δ 7.44 (d, 2H, J ) 8.4 Hz), 7.25 (d, 2H, J ) 8.4 Hz), 3.66 (s,
2H). MS m/z 248.8 [M - H]-. This intermediate (0.73 mmol) was
slurried in DMF (5.0 mL), cooled to 0 °C, and treated dropwise
with thionyl chloride (2.71 mmol). After 15 min, the cooling bath
was removed and the reaction mixture was warmed to room
temperature. After 2 h, the reaction mixture was carefully poured
into ice cold water (75 mL), and the organics were extracted with
(3 × 50 mL) ethyl acetate. The combined organic layers were dried
over sodium sulfate and concentrated in vacuo. The crude residue
was carefully treated with aq ammonium hydroxide solution (4.0
mL). After 24 h at room temperature, a white precipitate was filtered
and rinsed with water. Concentration in vacuo provided the crude
N-Methyl-N′-[4′-(trifluoromethyl)-4-biphenylyl]urea (21). To
a solution of 4′-(trifluoromethyl)-4-biphenylamine (0.421 mmol)
in dichloromethane (2 mL) was added methyl isocyanate (0.505
mmol). After stirring 18 h at room temperature, the reaction mixture
was quenched with water (2 mL), stirred for 1 h, and concentrated
in vacuo. The residue was purified by reverse phase HPLC to afford
1
the title product as a white solid (22%). H NMR (DMSO-d6): δ
8.70 (s, 1H), 7.85 (d, 2H, J ) 8.3 Hz), 7.77 (d, 2H, J ) 8.4 Hz),
7.64 (d, 2H, J ) 8.7 Hz), 7.54 (d, 2H, J ) 8.7 Hz), 6.08 (q, 1H,

ATP-CompetitiVe Kinesin Spindle Protein Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4947
intermediate 1-(4-bromophenyl)methanesulfonamide as a white
powder (56%). 1H NMR (DMSO-d6): δ 7.59 (d, 2H, J ) 8.3 Hz),
7.32 (d, 2H, J ) 8.3 Hz), 6.87 (s, 2H), 4.26 (s, 2H). MS m/z 169.2
[M - SO2NH2]+. The title compound was prepared as a white
glassy solid from this intermediate following Method A, with
purification by silica gel chromatography (25-50% ethyl acetate/
hexanes) and then recrystallization from hot isopropanol (43%).
1H NMR (DMSO-d6): δ 7.92 (d, 2H, J ) 8.4 Hz), 7.84 (d, 2H, J
) 8.3 Hz), 7.77 (d, 2H, J ) 8.1 Hz), 7.51 (d, 2H, J ) 8.3 Hz),
6.91 (s, 2H), 4.34 (s, 2H). MS m/z 235.2 [M - SO2NH2]+; 428.2
[M + CF3CO2-]-. Anal. (C14H12F3NO2S) C, H, N.
(s, 2H). MS m/z 335.2 [M + H]+. Anal. (C13H10F4N2O2S) H, N,
C: calcd, 46.71; found, 45.91. Analytical HPLC 99.5% purity, tR
) 7.00.
N-[3-Cyano-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (31).
The intermediate 4-amino-4′-(trifluoromethyl)-3-biphenylcarboni-
trile was prepared following Method A using 2-amino-5-bromoben-
zonitrile (87%). 1H NMR (DMSO-d6): δ 7.86-7.83 (m, 3H), 7.77-
7.73 (m, 3H), 6.90 (d, 1H, J ) 8.8 Hz), 6.38 (s, 2H). MS m/z 263.2
[M + H]+. The title compound was prepared from this aniline
following Method D (45%). 1H NMR (DMSO-d6): δ 9.77 (s, 1H),
8.21 (d, 1H, J ) 1.5 Hz), 8.08 (d, 1H, J ) 8.3 Hz), 7.97 (d, 2H,
J ) 8.3 Hz), 7.83 (d, 2H, J ) 8.3 Hz), 7.71 (d, 1H, J ) 8.3 Hz),
7.38 (s, 2H). MS m/z 342.0 [M + H]+, 364.2 [M + Na]+. Anal.
(C14H10F3N3O2S) C, H, N.
N-[3-Methyl-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (32).
Crude intermediate 3-methyl-4′-(trifluoromethyl)-4-biphenylamine
was prepared following Method A using 4-bromo-2-methylaniline.
MS m/z 252.5 [M + H]+. The title compound was prepared as a
white solid from this aniline following Method D (25%). 1H NMR
(DMSO-d6): δ 8.68 (s, 1H), 7.88 (d, 2H, J ) 8.4 Hz), 7.80 (d, 2H,
J ) 8.4 Hz), 7.59 (s, 1H), 7.56 (dd, 1H, J ) 8.3, 2.2 Hz), 7.50 (d,
1H, J ) 8.3 Hz), 7.02 (s, 2H), 2.37 (s, 3H). MS m/z 331.2 [M +
H]+. Anal. (C14H13F3N2O2S) C, H, N.
N-Methyl-1-[4′-(trifluoromethyl)-4-biphenylyl]methanesulfon-
amide (26). The sodium salt of (4-bromophenyl)methanesulfonic
acid (0.37 mmol) was slurried in DMF (2.0 mL), cooled to 0 °C,
and treated dropwise with thionyl chloride (1.35 mmol). After 10
min, the cooling bath was removed and the reaction mixture was
warmed to room temperature. After 2 h, the reaction mixture was
carefully poured into ice cold water (65 mL), and the organics were
extracted with (3 × 50 mL) ethyl acetate. The combined organic
layers were dried over sodium sulfate and concentrated in vacuo.
The crude residue was carefully treated with a 40% solution of
methylamine in water (2.0 mL). After 3.5 h at room temperature,
the reaction mixture was heated at 40 °C for 16 h. A white
precipitate was filtered and rinsed with water. Concentration in
vacuo provided the crude intermediate 1-(4-bromophenyl)-N-
N-[3-(Methoxy)-4′-(trifluoromethyl)-4-biphenylyl]sulfamide
(33). 3-Methoxy-4-nitro-4′-(trifluoromethyl)biphenyl was prepared
following Method A using 5-chloro-2-nitroanisole (42%). 1H NMR
(DMSO-d6): δ 8.03 (dd, 3H, J ) 8.2, 4.9 Hz), 7.89 (d, 2H, J )
8.3 Hz), 7.64 (d, 1H, J ) 1.8 Hz), 7.48 (dd, 1H, J ) 8.3, 1.8 Hz),
4.05 (s, 3H). MS m/z 298.2 [M + H]+. To a solution of this biphenyl
(1.96 mmol) in acetic acid (10 mL) was added zinc dust (13.7
mmol). The reaction mixure was stirred at room temperature for 2
h, filtered through celite, and the filter pad was washed with acetic
acid (5 mL) and ethanol (5 mL). The filtrate was concentrated in
vacuo, and the residue was taken up in ethyl acetate (15 mL) and
washed with saturated aqueous sodium carbonate solution (10 mL).
The organic layer was dried over magnesium sulfate and concen-
trated in vacuo to give the intermediate [3-(methoxy)-4′-(trifluo-
romethyl)-4-biphenylyl]amine as an off-white solid (quantitative).
1H NMR (DMSO-d6): δ 7.82 (d, 2H, J ) 8.1 Hz), 7.71 (d, 2H, J
) 8.3 Hz), 7.17 (d, 1H, J ) 2.0 Hz), 7.12 (dd, 1H, J ) 8.1, 1.8
Hz), 6.73 (d, 1H, J ) 8.1 Hz), 5.06 (s, 2H), 3.87 (s, 3H). MS m/z
268.0 [M + H]+. The title compound was prepared as a white solid
1
methylmethanesulfonamide as a white powder (33%). H NMR
(DMSO-d6): δ 7.59 (d, 2H, J ) 8.3 Hz), 7.33 (d, 2H, J ) 8.3 Hz),
6.95 (q, 1H, J ) 4.6 Hz), 4.34 (s, 2H), 2.57 (d, 3H, J ) 4.6 Hz).
MS m/z 169.2 [M - SO2NHCH3]+. The title compound was
prepared as a white powder from this intermediate following
1
Method A (31%). H NMR (DMSO-d6): δ 7.92 (d, 2H, J ) 8.3
Hz), 7.83 (d, 2H, J ) 8.3 Hz), 7.77 (d, 2H, J ) 8.4 Hz), 7.51 (d,
2H, J ) 8.4 Hz), 6.99 (br s, 1H), 4.41 (s, 2H), 2.61 (br s, 3H). MS
m/z 235.2 [M - SO2NHCH3]+; 442.2 [M + CF3CO2-]-. Analytical
HPLC 99.6% purity, tR ) 7.56.
4′-(Trifluoromethyl)-4-biphenylyl Sulfamate (27). The title
compound was prepared as a white powder from 4′-(trifluoro-
methyl)-4-biphenylol following Method D (25%). 1H NMR (DMSO-
d6): δ 8.05 (br s, 2H), 7.90 (d, 2H, J ) 8.3 Hz), 7.84 (d, 4H, J )
8.6 Hz), 7.41 (d, 2H, J ) 8.6 Hz). MS m/z 318.2 [M + H]+.
Analytical HPLC 94.26% purity, tR ) 7.55.
1
from this aniline following Method D (19%). H NMR (DMSO-
4′-(Trifluoromethyl)-4-biphenylyl Carbamate (28). The in-
termediate 4′-(trifluoromethyl)-4-biphenylol was prepared as a white
d6): δ 8.20 (s, 1H), 7.92 (d, 2H, J ) 8.2 Hz), 7.80 (d, 2H, J ) 8.2
Hz), 7.50 (d, 1H, J ) 8.3 Hz), 7.35 (s, 1H), 7.31 (d, 1H, J ) 8.3
Hz), 7.18 (s, 2H), 3.92 (s, 3H). MS m/z 267.2 [M - SO2NH2]+.
Anal. (C14H13F3N2O3S) C, H, N.
1
solid following Method A using 4-bromophenol (71%). H NMR
(CDCl3): δ 7.63 (m, 4H), 7.48 (d, 2H, J ) 8.7 Hz), 6.92 (d, 2H,
J ) 8.7 Hz), 4.82 (s, 1H). The title compound was prepared as a
white powder (88%) from this phenol following Method C with
purification by silica gel chromatography (20-45% ethyl acetate/
hexanes). 1H NMR (DMSO-d6): δ 7.89 (d, 2H, J ) 8.3 Hz), 7.82
(d, 2H, J ) 8.4 Hz), 7.74 (d, 2H, J ) 8.5 Hz), 7.25 (m, 3H), 6.99
(br s, 1H). MS m/z 282.2 [M + H]+. Anal. (C14H10F3NO2) C, H,
N.
N-[3-Fluoro-4′-(trifluoromethyl)-4-biphenylyl]urea (29). The
intermediate [3-fluoro-4′-(trifluoromethyl)-4-biphenylyl]amine was
prepared following Method A using 4-bromo-2-fluoroaniline (93%).
1H NMR (DMSO-d6): δ 7.80 (d, 2H, J ) 8.3 Hz), 7.71 (d, 2H, J
) 8.4 Hz), 7.46 (dd, 1H, J ) 12.9, 2.0 Hz), 7.34 (dd, 1H, J ) 8.3,
2.0 Hz), 6.85 (m, 1H), 5.48 (s, 2H). MS m/z 256.0 [M + H]+. The
title compound was prepared as a white solid from this aniline
following Method C (56%). 1H NMR (DMSO-d6): δ 8.52 (d, 1H,
J ) 2.0 Hz), 8.30 (app t, 1H, J ) 8.6 Hz), 7.90 (d, 2H, J ) 8.2
Hz), 7.79 (d, 2H, J ) 8.3 Hz), 7.66 (dd, 1H, J ) 12.9, 2.0 Hz),
7.53 (dd, 1H, J ) 8.6, 1.8 Hz), 6.29 (s, 2H). MS m/z 299.2 [M +
H]+. Analytical HPLC 95.4% purity, tR ) 6.84.
N-[3-Hydroxy-4′-(trifluoromethyl)-4-biphenylyl]sulfamide
(34). To a solution of [3-(methoxy)-4′-(trifluoromethyl)-4-biphen-
ylyl]amine (0.68 mmol) in 1-methyl-2-pyrrolidinone (0.34 mL) was
added potassium carbonate (0.034 mmol) and thiophenol
(0.68 mmol). The reaction mixture was irradiated in the microwave
at 220 °C for 15 min. The reaction mixture was diluted with ethyl
acetate (10 mL) and concentrated in vacuo. Purification of
the residue by silica gel chromatography (20-65% ethyl acetate/
hexanes) afforded the intermediate 4-amino-4′-(trifluoro-
1
methyl)-3-biphenylol as a brown solid (82%). H NMR (DMSO-
d6): δ 9.25 (s, 1H), 7.70 (s, 4H), 7.03-6.98 (m, 2H), 6.68 (d, 1H,
J ) 8.0 Hz), 4.86 (s, 2H). MS m/z 254.2 [M + H]+. To a
solution of the phenol (0.395 mmol) in pyridine (2.0 mL) was
added sulfamide (0.435 mmol). The reaction mixture was heated
at reflux for 2 h. The reaction mixture was then cooled to
room temperature and concentrated in vacuo. Purification of the
residue by reverse phase HPLC afforded the title compound as a
1
light brown solid (22%). H NMR (DMSO-d6): δ 9.97 (s, 1H),
7.99 (s, 1H), 7.79 (s, 4H), 7.43 (d, 1H, J ) 8.1 Hz), 7.19-7.10 (m,
4H). MS m/z 333.2 [M + H]+. Analytical HPLC 96.9% purity, tR
) 6.30.
N-[3-Fluoro-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (30).
The title compound was prepared from [3-fluoro-4′-(trifluoro-
methyl)-4-biphenylyl]amine following Method D (30%). 1H NMR
(DMSO-d6): δ 9.42 (s, 1H), 7.92 (d, 2H, J ) 8.3 Hz), 7.80 (d, 2H,
J ) 8.3 Hz), 7.67 (d, 1H, J ) 11.1 Hz), 7.62-7.56 (m, 2H), 7.25
N-[3-Amino-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (35).
3-Nitro-4′-(trifluoromethyl)-4-biphenylamine was prepared follow-
ing Method A with 4-bromo-2-nitroaniline (79%). 1H NMR

4948 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Parrish et al.
(DMSO-d6): δ 8.31 (d, 1H, J ) 2.3 Hz), 7.89-7.84 (m, 3H), 7.80-
7.75 (m, 2H), 7.65 (s, 2H), 7.15 (d, 1H, J ) 8.8 Hz). MS m/z 283.2
[M + H]+. The intermediate N-[3-nitro-4′-(trifluoromethyl)-4-
biphenylyl]sulfamide was prepared from the aniline following
Hz), 7.62 (d, 2H, J ) 8.8 Hz), 7.07 (s, 2H). MS m/z 353.2 [M +
H]+. Analytical HPLC 95.4% purity, tR ) 8.80.
N-(4′-Chloro-4-biphenylyl)methanesulfonamide (40). The title
compound was prepared as a yellow solid (82%) from N-(4-
bromophenyl)methanesulfonamide47 and (4-chlorophenyl)boronic
1
Method D (69%). H NMR (DMSO-d6): δ 9.62 (s, 1H), 8.44 (d,
1
1H, J ) 2.3 Hz), 8.19 (dd, 1H, J ) 8.8, 2.3 Hz), 8.00 (d, 2H, J )
8.1 Hz), 7.86 (dd, 3H, J ) 8.5, 2.6 Hz), 7.76 (s, 2H). MS m/z
362.0 [M + H]+. A solution of the sulfamide (0.73 mmol) in ethanol
(10 mL) was reduced in a Parr shaker at 50 psi H2 with platinum-
(IV) oxide (0.01 mmol). After 15 min, the reaction mixture was
filtered through celite and concentrated in vacuo. Purification by
reverse phase HPLC afforded the title product as a white solid
acid following Method A. H NMR (DMSO-d6): δ 9.91 (s, 1H),
7.67 (m, 4H), 7.51 (d, 2H, J ) 8.5 Hz), 7.30 (d, 2H, J ) 8.6 Hz),
3.03 (s, 3H). MS m/z 282.0 [M + H]+. Anal. (C13H12ClNO2S) H,
N. Analytical HPLC 95.4% purity, tR ) 7.36.
N-[4′-(2,2,2-Trifluoroethyl)-4-biphenylyl]methanesulfon-
amide (41). To a solution of 4-bromobenzyl bromide (20 mmol)
in 1-methyl-2-pyrrolidinone (25 mL) was added copper(I) iodide
(5.2 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (39
mmol). The reaction mixture was purged with nitrogen and heated
at 80 °C for 24 h. After cooling, the reaction mixture was diluted
with water, and the organics were extracted with hexanes, filtered
through a pad of celite, and concentrated in vacuo. Purification of
the residue by silica gel chromatography (hexanes) gave the
intermediate 4-(2,2,2-trifluoroethyl)bromobenzene as a white solid
(61%, contained ∼15% 4-bromobenzyl bromide). 1H NMR
(CDCl3): δ 7.51 (d, 2H, J ) 8.3 Hz), 7.19 (d, 2H, J ) 8.3 Hz),
3.35 (q, 2H, J ) 10.7 Hz). Intermediate 4-[4′-(2,2,2-trifluoroethyl)-
4-biphenylyl]amine was prepared as a yellow solid from this
bromide and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
following Method A (100%). 1H NMR (CDCl3): δ 7.54 (d, 2H, J
) 8.4 Hz), 7.37 (d, 2H, J ) 8.5 Hz), 7.34 (d, 2H, J ) 8.5 Hz),
6.64 (d, 2H, J ) 8.4 Hz), 5.26 (s, 2H), 3.63 (q, 2H, J ) 11.6 Hz).
MS m/z 252.0 [M + H]+. The title compound was prepared from
4-[4′-(2,2,2-trifluoroethyl)-4-biphenylyl]amine following Method B.
Purification of the residue by silica gel chromatography (35% ethyl
acetate/hexanes), trituration with hexanes, filtration, and concentra-
tion in vacuo afforded the title product as a white solid (94%). 1H
NMR (CDCl3): δ 9.88 (s, 1H), 7.67 (d, 2H, J ) 8.6 Hz), 7.65 (d,
2H, J ) 8.1 Hz), 7.44 (d, 2H, J ) 8.1 Hz), 7.30 (d, 2H, J ) 8.6
Hz), 3.68 (m, 2H, J ) 11.6 Hz), 3.03 (s, 3H). MS m/z 330.2 [M +
H]+. Anal. (C15H14F3NO2S) C, H, N.
1
(81%). H NMR (DMSO-d6): δ 8.40 (s, 1H), 7.80-7.74 (m, 4H),
7.24 (d, 1H, J ) 8.1 Hz), 7.03 (d, 1H, J ) 2.0 Hz), 6.91 (s, 2H),
6.88 (dd, 1H, J ) 8.2, 2.2 Hz), 5.21 (s, 2H). MS m/z 332.2 [M +
H]+. Anal. (C13H12F3N3O2S) C, H, N.
N-[3-(Methylamino)-4′-(trifluoromethyl)-4-biphenylyl]sul-
famide (36). N-Methyl-4-nitro-4′-(trifluoromethyl)-3-biphenylamine
was prepared following Method A using 5-chloro-N-methyl-2-
1
nitroaniline (68%). H NMR (DMSO-d6): δ 8.31 (d, 1H, J ) 4.8
Hz), 8.19 (d, 1H, J ) 9.1 Hz), 8.01 (d, 2H, J ) 8.1 Hz), 7.88 (d,
2H, J ) 8.3 Hz), 7.22 (d, 1H, J ) 1.5 Hz), 7.03 (dd, 1H, J ) 8.8,
1.8 Hz), 3.07 (d, 3H, J ) 5.0 Hz). MS m/z 297.2 [M + H]+. To a
solution of this biphenyl (1.09 mmol) in acetic acid (7 mL) was
added zinc dust (7.63 mmol). The reaction mixure was stirred at
room temperature for 2 h, filtered through celite, and the filter pad
was washed with acetic acid (10 mL) and ethanol (10 mL). The
filtrate was concentrated in vacuo, and the residue was taken up in
ethyl acetate (10 mL) and washed with saturated aqueous sodium
bicarbonate solution (10 mL). The organic layer was dried over
magnesium sulfate and concentrated in vacuo to give the intermedi-
ate 4-amino-4′-(trifluoromethyl)-3-biphenylyl]methylamine as an
1
off-white solid (77%). H NMR (DMSO-d6): δ 7.77 (d, 2H, J )
8.3 Hz), 7.69 (d, 2H, J ) 8.4 Hz), 6.85 (dd, 1H, J ) 7.8, 2.0 Hz),
6.71 (d, 1H, J ) 1.8 Hz), 6.63 (d, 1H, J ) 8.1 Hz), 4.82 (s, 2H),
4.79 (d, 1H, J ) 4.8 Hz), 2.80 (d, 3H, J ) 5.0 Hz). MS m/z 267.0
[M + H]+. A solution of the aniline (0.38 mmol) and sulfamide
(1.9 mmol) in 1,4-dioxane (1.0 mL) was irradiated in the microwave
at 150 °C for 10 min. The reaction mixture was concentrated in
vacuo. Purification of the residue by reverse phase HPLC afforded
the title compound as a pink solid (5%). 1H NMR (DMSO-d6): δ
8.40 (s, 1H), 7.86 (d, 2H, J ) 8.3 Hz), 7.78 (d, 2H, J ) 8.6 Hz),
7.26 (d, 1H, J ) 8.1 Hz), 6.93-6.85 (m, 3H), 6.82 (d, 1H, J ) 1.5
Hz), 5.41 (br s, 1H), 2.80 (s, 3H). MS m/z 346.2 [M + H]+.
Analytical HPLC 97.3% purity, tR ) 6.18.
N-[4′-(Trifluoromethoxy)-4-biphenylyl]methanesulfon-
amide (42). The title compound was prepared from N-(4-bro-
mophenyl)methanesulfonamide47 and 4-(trifluoromethoxy)phenyl
boronic acid following Method A. Purification by reverse phase
HPLC provided the title product as a white powder (25%). 1H NMR
(DMSO-d6): δ 10.10 (s, 1H), 8.16 (d, 2H, J ) 8.6 Hz), 8.13 (d,
2H, J ) 8.6 Hz), 7.85 (d, 2H, J ) 8.4 Hz), 7.36 (d, 2H, J ) 8.3
Hz), 3.08 (s, 3H). MS m/z 332.4 [M + H]+. Anal. (C14H12F3NO3S)
C, H, N.
N-[3-Chloro-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (37).
Crude intermediate 3-chloro-4′-(trifluoromethyl)-4-biphenylamine
was prepared following Method A using 4-bromo-2-chloroaniline.
MS m/z 272.0 [M + H]+. The title compound was prepared as a
white solid from this aniline following Method D with purification
N-{4′-[(Trifluoromethyl)sulfonyl]-4-biphenylyl}methanesul-
fonamide (43). N-[4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl]methanesulfonamide was prepared in 30% yield with
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline following
Method B with purification by flash chromatography (50-100%
ethyl acetate/hexanes). 1H NMR (DMSO-d6): δ 10.05 (s, 1H), 7.62
(d, 2H, J ) 8.5 Hz), 7.20 (d, 2H, J ) 8.5 Hz), 3.03 (s, 3H), 1.28
(s, 12H). MS m/z 298.2 [M + H]+. The title compound was
prepared from this boronate and 1-bromo-4-[(trifluoromethyl)-
sulfonyl]benzene following Method A with purification by reverse
1
by reverse phase HPLC (35%). H NMR (DMSO-d6): δ 9.96 (s,
1H), 7.82 (d, 2H, J ) 8.1 Hz), 7.65 (d, 2H, J ) 8.1 Hz), 7.40-7.36
(m, 4H), 7.20 (dd, 1H, J ) 8.4, 2.2 Hz). MS m/z 351.2 [M + H]+.
Anal. (C13H10ClF3N2O2S) C, H, N.
N-[2-Fluoro-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (38).
Crude intermediate 2-fluoro-4′-(trifluoromethyl)-4-biphenylamine
was prepared following Method A using 4-bromo-3-fluoroaniline.
MS m/z 256.2 [M + H]+. The title compound was prepared as a
white solid from this aniline following Method D with purification
by reverse phase HPLC (35%). 1H NMR (DMSO-d6): δ 10.04 (s,
1H), 7.82 (d, 2H, J ) 8.3 Hz), 7.75 (d, 2H, J ) 8.1 Hz), 7.53 (t,
1H, J ) 8.7 Hz), 7.38 (s, 2H), 7.12 (dd, 1H, J ) 13.2, 2.2 Hz),
7.06 (dd, 1H, J ) 8.5, 2.2 Hz). MS m/z 335.2 [M + H]+. Anal.
(C13H10F4N2O2S) C, H, N.
N-[3,5-Difluoro-4′-(trifluoromethyl)-4-biphenylyl]sulfamide
(39). Crude intermediate 3,5-difluoro-4′-(trifluoromethyl)-4-biphen-
ylamine was prepared following Method A using 4-bromo-2,6-
difluoroaniline. MS m/z 274.0 [M + H]+. The title compound was
prepared as a white solid from this aniline following Method D
with purification by reverse phase HPLC (16%). 1H NMR (DMSO-
d6): δ 8.95 (s, 1H), 7.99 (d, 2H, J ) 8.3 Hz), 7.85 (d, 2H, J ) 8.3
1
phase HPLC (61%). H NMR (DMSO-d6): δ 10.13 (s, 1H), 8.18
(d, 2H, J ) 8.6 Hz), 8.12 (d, 2H, J ) 8.7 Hz), 7.85 (d, 2H, J ) 8.7
Hz), 7.36 (d, 2H, J ) 8.7 Hz), 3.09 (s, 3H). MS m/z 380.2 [M +
H]+. Anal. (C14H12F3NO4S2) C, H, N.
N-4-Biphenylylurea (44). The title compound was prepared as
a white powder from 4-biphenylamine following Method C (27%).
1H NMR (DMSO-d6): δ 8.64 (s, 1H), 7.62 (d, 2H, J ) 8.4 Hz),
7.55 (d, 2H, J ) 8.8 Hz), 7.49 (d, 2H, J ) 8.8 Hz), 7.43 (t, 2H, J
) 7.7 Hz), 7.30 (m, 1H), 5.90 (s, 2H). MS m/z 213.2 [M + H]+.
Analytical HPLC 95.0% purity, tR ) 5.23.
N-[3′-(Trifluoromethyl)-4-biphenylyl]urea (45). The intermedi-
ate 3′-(trifluoromethyl)-4-biphenylamine was prepared as a brown
solid following Method A using 3-trifluoromethylphenyl boronic
acid (48%). 1H NMR (CDCl3): δ 7.76 (s, 1H), 7.69 (m, 1H), 7.50
(m, 2H), 7.41 (d, 2H, J ) 8.6 Hz), 6.76 (d, 2H, J ) 8.5 Hz), 3.60
(br s, 2H). MS m/z 238.0 [M + H]+. The title compound was

ATP-CompetitiVe Kinesin Spindle Protein Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4949
prepared as a tan solid from this aniline following Method C (55%).
1H NMR (DMSO-d6): δ 8.70 (s, 1H), 7.95 (m, 1H), 7.91 (s, 1H),
7.68-7.63 (m, 4H), 7.54 (d, 2H, J ) 8.6 Hz), 5.92 (s, 2H). MS m/z
281.0 [M + H]+. Anal. (C14H11F3N2O) C, H, N.
borolan-2-yl)aniline (85%). 1H NMR (DMSO-d6): δ 8.06-7.97 (m,
4H), 7.59 (d, 2H, J ) 8.8 Hz), 6.69 (d, 2H, J ) 8.6 Hz), 5.71 (s,
2H). MS m/z 302.2 [M + H]+. The title compound was prepared
1
as a white solid from this aniline following Method C (35%). H
NMR (DMSO-d6): δ 8.83 (s, 1H), 8.19-8.02 (m, 4H), 7.75 (d, 2H,
J ) 8.8 Hz), 7.59 (d, 2H, J ) 8.6 Hz), 6.00 (s, 2H). MS m/z 345.2
[M + H]+. Anal. (C14H11F3N2O3S) C, H, N.
N-(4′-Methyl-4-biphenylyl)urea (46). The intermediate 4′-
methyl-4-biphenylamine was prepared following Method A using
4-methylphenyl boronic acid (37%). 1H NMR (CDCl3):
δ
7.42-7.35 (m, 4H), 7.18 (d, 2H, J ) 8.3 Hz), 6.73 (d, 2H, J ) 8.6
Hz), 3.67 (br s, 2H), 2.35 (s, 3H). MS m/z 184.0 [M + H]+. The
title compound was prepared as a tan solid from this aniline
following Method C (59%). 1H NMR (DMSO-d6): δ 8.60 (s, 1H),
7.52-7.46 (m, 6H), 7.23 (d, 2H, J ) 8.1 Hz), 5.86 (s, 2H), 2.33
(s, 3H). MS m/z 227.2 [M + H]+. Analytical HPLC 99.3%, tR )
5.89.
N-(4′-Isopropyl-4-biphenylyl)urea (47). The intermediate 4′-
isopropyl-4-biphenylamine was prepared following Method A using
4-isopropylphenyl boronic acid (38%). 1H NMR (CDCl3): δ 7.45
(d, 2H, J ) 8.3 Hz), 7.38 (d, 2H, J ) 8.6 Hz), 7.24 (d, 2H, J ) 8.1
Hz), 6.73 (d, 2H, J ) 8.6 Hz), 3.68 (br s, 2H), 2.92 (septet, 1H, J
) 6.9 Hz), 1.26 (d, 6H, J ) 6.9 Hz). MS m/z 212.2 [M + H]+.
The title compound was prepared as a tan solid from this aniline
following Method C (44%). 1H NMR (DMSO-d6): δ 8.59 (s, 1H),
7.53-7.45 (m, 6H), 7.29 (d, 2H, J ) 8.3 Hz), 5.86 (s, 2H), 2.91
(septet, 1H, J ) 6.9 Hz), 1.23 (d, 6H, J ) 6.8 Hz). MS m/z 255.2
[M + H]+. Anal. (C16H18N2O) H, N, C: calcd, 75.56; found, 74.82.
Analytical HPLC 94.6% purity, tR ) 7.09.
N-[4′-(Ethylsulfonyl)-3-fluoro-4-biphenylyl]urea (52). The title
compound was prepared as a white solid following Method E with
1
4-(ethanesulfonyl)benzeneboronic acid (36%). H NMR (DMSO-
d6): δ 8.55 (d, 1H, J ) 2.5 Hz), 8.31 (app t, 1H, J ) 8.8 Hz), 7.96
(d, 2H, J ) 8.9 Hz), 7.91 (d, 2H, J ) 8.6 Hz), 7.69 (dd, 1H, J )
12.9, 2.0 Hz), 7.56 (dd, 1H, J ) 8.5, 1.8 Hz), 6.30 (s, 2H), 3.33 (q,
2H, J ) 7.3 Hz), 1.12 (t, 3H, J ) 7.3 Hz). MS m/z 323.0 [M +
H]+. Anal. (C15H15FN2O3S) C, H, N.
N-[4′-(Trifluoroacetyl)-4-biphenylyl]urea (53). The intermedi-
ate 1-(4′-amino-4-biphenylyl)-2,2,2-trifluoroethanone was prepared
as a bright yellow solid following Method A with 2,2,2-trifluoro-
1-(4-iodophenyl)ethanone49 and 4-(4,4,5,5-tetramethyl-1,3,2-di-
oxaborolan-2-yl)aniline at 80 °C for 1.5 h (50%). 1H NMR (DMSO-
d6): δ 8.03 (d, 2H, J ) 7.6 Hz), 7.87 (d, 2H, J ) 8.8 Hz), 7.57 (d,
2H, J ) 8.8 Hz), 6.66 (d, 2H, J ) 8.4 Hz), 5.65 (s, 2H). MS m/z
266.0 [M + H]+. The title compound was prepared as a yellow
solid from this aniline following Method C with purification by
silica gel chromatography (ethyl acetate) followed by recrystalli-
zation from acetonitrile (13%). TLC Rf ) 0.33 (EtOAc). 1H NMR
(DMSO-d6): δ 8.80 (s, 1H), 8.10 (d, 2H, J ) 8.0 Hz), 7.96 (d, 2H,
J ) 8.8 Hz), 7.73 (d, 2H, J ) 8.8 Hz), 7.57 (d, 2H, J ) 8.8 Hz),
5.98 (s, 2H). MS m/z 341.2 [M + H + H2O]+. Anal. (C15H11F3N2O2)
C, H, N.
N-(4′-t-Butyl-4-biphenylyl)urea (48). The intermediate 4′-t-
butyl-4-biphenylamine was prepared following Method A using 4-t-
butylphenyl boronic acid (51%). 1H NMR (CDCl3): δ 7.46 (d, 2H,
J ) 8.5 Hz), 7.40 (m, 4H), 6.73 (d, 2H, J ) 8.5 Hz), 3.69 (br s,
2H), 1.33 (s, 9H). MS m/z 226.2 [M + H]+. The title compound
was prepared as a tan solid from this aniline following Method C
N-[3′-Fluoro-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (54).
The intermediate 3′-fluoro-4′-(trifluoromethyl)-4-biphenylamine was
prepared as a white powder following Method A using 4-bromo-
2-fluoro-1-(trifluoromethyl)benzene and 4-(4,4,5,5-tetramethyl-
1
(55%). H NMR (DMSO-d6): δ 8.60 (s, 1H), 7.55-7.43 (m, 8H),
5.86 (s, 2H), 1.31 (s, 9H). MS m/z 269.2 [M + H]+. Analytical
HPLC 99.1% purity, tR ) 7.52.
1
1,3,2-dioxaborolan-2-yl)aniline (86%). H NMR (DMSO-d6): δ
General Method for Suzuki Cross-Coupling. Method E:
N-(4′-Bromo-3,3′-difluoro-4-biphenylyl)urea (49). The intermedi-
ate N-(4-bromo-2-fluorophenyl)urea was prepared in 70% yield
following Method C using 4-bromo-2-fluoroaniline and was
purified by recrystallization from hot ethyl acetate/methanol (5:1).
1H NMR (DMSO-d6): δ 8.45 (d, 1H, J ) 2.0 Hz), 8.13 (app t,
1H, J ) 9.0 Hz), 7.50 (dd, 1H, J ) 11.1, 2.3 Hz), 7.28 (m, 1H),
6.25 (s, 2H). MS m/z 233.0 [M + H]+. A solution of the urea (0.54
mmol), 4-bromo-3-fluorobenzeneboronic acid (0.64 mmol), and
dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)-
dichloromethane adduct (0.02 mmol) in DMF (2 mL) and 2 M aq
sodium carbonate (1.61 mmol) was irradiated in the microwave at
110 °C for 7 min. The resulting dark solution was treated with
sodium sulfate (∼500 mg) and decolorizing charcoal (∼50 mg)
and filtered through a syringe fitted with a nylon filter disc. The
filtrate was purified by reverse phase HPLC and the subsequent
precipitated product was collected, filtered, washed with water, and
dried in vacuo to afford the title product as a tan solid (10%).
7.69 (m, 2H), 7.58 (d, 1H, J ) 8.3 Hz), 7.52 (d, 2H, J ) 8.6 Hz),
6.68 (d, 2H, J ) 8.3 Hz), 5.75 (s, 2H). MS m/z 256.2 [M + H]+.
The title compound was prepared as a white solid from this aniline
following Method D with purification by reverse phase HPLC
(24%). 1H NMR (DMSO-d6): δ 9.83 (s, 1H), 7.82 (m, 2H), 7.76-
7.69 (m, 3H), 7.27 (m, 4H). MS m/z 335.2 [M + H]+. Anal.
(C13H10F4N2O2S) H, N. Analytical HPLC 95.2% purity, tR ) 7.01.
N-[3′-Nitro-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (55).
The intermediate 3′-nitro-4′-(trifluoromethyl)-4-biphenylamine was
prepared following Method A using 4-bromo-2-nitro-1-(trifluo-
romethyl)benzene and 4-aminophenyl boronic acid (78%). 1H NMR
(DMSO-d6): δ 8.27 (d, 1H, J ) 1.6 Hz), 8.06 (dd, 1H, J ) 8.6,
1.2 Hz), 7.94 (d, 1H, J ) 8.4 Hz), 7.59 (d, 2H, J ) 8.6 Hz), 6.68
(d, 2H, J ) 8.6 Hz), 5.66 (br s, 2H). MS m/z 283.2 [M + H]+. The
title compound was prepared from this aniline following Method
1
D (74%). H NMR (DMSO-d6): δ 9.90 (s, 1H), 8.41 (d, 1H, J )
1.5 Hz), 8.19 (d, 1H, J ) 8.3 Hz), 8.06 (d, 1H, J ) 8.6 Hz), 7.81
(d, 2H, J ) 8.6 Hz), 7.31 (s, 2H), 7.30 (d, 2H, J ) 8.6 Hz). MS
m/z 362.2 [M + H]+. Analytical HPLC 96.4% purity, tR ) 6.75.
MS m/z 328.9 [M + H]+. Analytical HPLC 100.0% purity, tR
)
6.82.48
N-{4′-[(Trifluoromethyl)thio]-4-biphenyl}urea (50). The in-
termediate 4′-[(trifluoromethyl)thio]-4-biphenylamine was prepared
as a tan solid following Method A using 4-bromophenyl trifluo-
romethyl sulfide and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)aniline with purification by reverse phase HPLC (45%). 1H NMR
(DMSO-d6): δ 8.07-7.99 (m, 4H), 7.61 (d, 2H, J ) 8.6 Hz), 6.71
(d, 2H, J ) 8.3 Hz), 5.74 (br s, 2H). MS m/z 270.2 [M + H]+. The
title compound was prepared as a white solid from this aniline
following Method C (55%). 1H NMR (DMSO-d6): δ 8.62 (s, 1H),
7.71-7.65 (m, 4H), 7.53 (d, 2H, J ) 8.8 Hz), 7.44 (d, 2H, J ) 8.6
Hz), 5.85 (s, 2H). MS m/z 313.0 [M + H]+. Analytical HPLC 94.3%
purity, tR ) 7.26.
N-[3′-Amino-4′-(trifluoromethyl)-4-biphenylyl]sulfamide (56).
To a stirred solution of N-[3′-nitro-4′-(trifluoromethyl)-4-biphenylyl]-
sulfamide (0.78 mmol) in acetic acid (10 mL) was added zinc
powder (5.4 mmol). The suspension was stirred at room temperature
for 18 h and then filtered through a pad of celite. The filter pad
was rinsed with acetic acid, and the filtrate was concentrated in
vacuo. The residue was taken up in ethyl acetate, washed
sequentially with 1 N aqueous sodium carbonate solution and brine,
dried over sodium sulfate, and concentrated in vacuo. Purification
by flash chromatography on silica gel (40-60% ethyl acetate/
dichloromethane), trituration with hexanes, and drying in vacuo
provided the title compound as a white solid (85%). 1H NMR
(DMSO-d6): δ 9.70 (s, 1H), 7.52 (d, 2H, J ) 8.6 Hz), 7.37 (d, 1H,
J ) 8.3 Hz), 7.24 (d, 2H, J ) 8.6 Hz), 7.19 (br s, 2H), 7.07 (s,
1H), 6.87 (d, 1H, J ) 8.3 Hz), 5.63 (s, 2H). MS m/z 332.2 [M +
H]+. Anal. (C13H12F3N3O2S) C, H, N.
N-{4′-[(Trifluoromethyl)sulfonyl]-4-biphenyl}urea (51). The
intermediate 4′-[(trifluoromethyl)sulfonyl]-4-biphenylamine was
prepared as a yellow solid following Method A using 4-chlorophen-
yl trifluoromethyl sulfone and 4-(4,4,5,5-tetramethyl-1,3,2-dioxa-

4950 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Parrish et al.
General Method for Suzuki Cross-Coupling. Method F:
4-(1,3-Benzodioxol-5-yl)aniline. A solution of 3,4-(methylene-
dioxy)phenylboronic acid (5.14mmol), 4-iodoaniline (3.42 mmol),
dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)-dichlo-
romethane adduct (0.10 mmol), and cesium carbonate (17.1 mmol)
in DMF (10 mL) and water (3 mL) was heated at 100 °C for 20 h.
The reaction mixture was cooled, poured into brine (50 mL), and
extracted with (3 × 50 mL) ethyl acetate. The combined organic
layers were dried over magnesium sulfate and decolorizing charcoal,
filtered through celite, and concentrated in vacuo. Purification of
the residue by reverse phase HPLC and neutralization of the
collected fractions afforded the title product as an off-white solid
(60%). MS m/z 214.1 [M + H]+.
N-[4-(2,2,3,3-Tetrafluoro-2,3-dihydro-1,4-benzodioxin-6-yl)-
phenyl]sulfamide (61). A solution of 5-bromo-2,2,3,3-tetrafluoro-
1,4-benzodioxane (1.20 mmol), [4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]amine (1.32 mmol), cesium carbonate
(2.08 mmol), and dichloro[1,1′-bis(diphenylphosphino)ferrocene]-
palladium(II)-dichloromethane adduct (0.02 mmol) in DMF (6 mL)
was irradiated in the microwave at 110 °C for 10 min. The reaction
mixture was diluted with diethyl ether and washed with saturated
aqueous sodium bicarbonate solution. The organic layer was
concentrated to give 0.275 g of crude [4-(2,2,3,3-tetrafluoro-2,3-
dihydro-1,4-benzodioxin-6-yl)phenyl]amine. MS m/z 300.0 [M +
H]+. The title compound was prepared from this aniline following
1
Method D with purification by reverse phase HPLC (23%). H
NMR (DMSO-d6): δ 9.73 (s, 1H), 7.75 (s, 1H), 7.69-7.58 (m, 3H),
7.53 (m, 1H), 7.31-7.15 (m, 4H). MS m/z 379.2 [M + H]+. Anal.
(C14H10F4N2O4S) C, H, N.
N-[4-(1,3-Benzodioxol-5-yl)phenyl]sulfamide (57). The title
compound was prepared from 4-(1,3-benzodioxol-5-yl)aniline fol-
1
lowing Method D (39%). H NMR (DMSO-d6): δ 9.57 (s, 1H),
N-[4-(2,2,4,4-Tetrafluoro-4H-1,3-benzodioxin-6-yl)phenyl]sul-
famide (62). The intermediate [4-(2,2,4,4-tetrafluoro-4H-1,3-ben-
zodioxin-6-yl)phenyl]amine was prepared following Method A
using 6-bromo-2,2,4,4-tetrafluoro-4H-1,3-benzodioxin and 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (75%). 1H NMR (DMSO-
d6): δ 8.07 (s, 1H), 8.02 (d, 1H, J ) 8.8 Hz), 7.61 (d, 2H, J ) 8.8
Hz), 7.52 (d, 1H, J ) 8.8 Hz), 6.93 (d, 2H, J ) 8.8 Hz). MS m/z
300.2 [M + H]+. The title compound was prepared from this aniline
following Method D with purification by reverse phase HPLC
7.52 (d, 2H, J ) 8.1 Hz), 7.23-7.17 (m, 3H), 7.14 (s, 2H), 7.09
(dd, 1H, J ) 8.1, 1.8 Hz), 6.97 (d, 1H, J ) 8.1 Hz), 6.05 (s, 2H).
MS m/z 293.2 [M + H]+. Anal. (C13H12N2O4S) H, N, C: calcd
53.42; found 52.79. Analytical HPLC 98.0% purity, tR ) 5.32.
N-[4-(2,2-Difluoro-1,3-benzodioxol-5-yl)phenyl]sulfamide (58).
The intermediate 4-(2,2-difluoro-1,3-benzodioxol-5-yl)aniline was
prepared following Method F using 5-bromo-2,2-difluoro-1,3-
benzodioxole, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
aniline, and tetrakis(triphenylphosphine)palladium(0) (50%). 1H
NMR (DMSO-d6): δ 7.57 (d, 1H, J ) 1.5 Hz), 7.41-7.28 (m, 4H),
6.65-6.58 (m, 2H), 5.30 (s, 2H). MS m/z 250.2 [M + H]+. The
title compound was prepared from this aniline following Method
D (31%). 1H NMR (DMSO-d6): δ 9.67 (s, 1H), 7.69 (s, 1H), 7.58
(d, 2H, J ) 8.6 Hz), 7.45 (s, 2H), 7.23 (d, 2H, J ) 8.8 Hz), 7.18
(s, 2H). MS m/z 329.3 [M + H]+. Anal. (C13H10F2N2O4S) C, H,
N.
N-[4-(2,2-Dimethyl-1,3-benzodioxol-5-yl)phenyl]sulfamide (59).
A solution of 3,4-O-isopropylidinebromobenzene50 (2.62 mmol),
[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]amine (3.14
mmol), and dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium-
(II)-dichloromethane adduct (0.26 mmol) in acetonitrile (2.6 mL)
and 2 M aqueous sodium carbonate solution (2.62 mL) was
irradiated in the microwave at 150 °C for 8 min. The reaction
mixture was filtered through celite, concentrated in vacuo, and
purified by reverse phase HPLC to afford the intermediate 4-(2,2-
dimethyl-1,3-benzodioxol-5-yl)aniline (32%). MS m/z 242.0 [M +
H]+.
1
(12%). H NMR (DMSO-d6): δ 9.74 (s, 1H), 8.13 (s, 1H), 8.07
(d, 1H, J ) 8.4 Hz), 7.70 (d, 2H, J ) 8.8 Hz), 7.56 (d, 1H, J ) 8.8
Hz), 7.26 (d, 2H, J ) 9.2 Hz), 7.23 (s, 2H). MS m/z 379.2 [M +
H]+. Anal. (C14H10F4N2O4S) C, H, N.
Biology. Biochemical Characterization. The KSP ATPase assay
was performed utilizing 15 µM ATP and 25 nM KSP motor domain
as previously described.33,36 Unless indicated, data are reported as
the average of two or more experiments (each run in duplicate).
Statistical limits for the data are reported as the standard deviation
of two or more experiments or the standard error within one
experiment. Mode of inhibition studies and inhibition constant
calculations were conducted on all key analogs as described
previously (selected results presented).36
Inhibition of Cellular Proliferation. SKOV3, Colo205,
and HCT116 cell lines were obtained from the ATCC. The HCT116
D130V derivative was created by continuous exposure to a
quinazolinone KSP inhibitor.28 Cells were grown in plastic
tissue culture flasks containing RPMI 1640 medium supplemented
with 10% fetal calf serum (v/v) and were maintained at 37 °C
in a humidified atmosphere containing 5% carbon dioxide. Test
compounds in DMSO stock solutions were serially diluted in
DMSO, diluted with growth medium, and then diluted into the
cell assay plate with a final DMSO concentration of e0.2%. Cells
were plated in duplicate 96-well plates at 1000 cells/well and
were allowed to adhere for 24 h. Test compounds were added to
the wells. All plates contained a DMSO control and wells with no
cells to subtract nonspecific background. Cell assay plates were
incubated at 37 °C for 72 h and then harvested and read using
the CellTiter-Glo (CTG) Luminescent Cell Viability Assay (Pro-
mega #G7571) on a Wallac EnVision 2100 Multichannel Reader
to determine the number of viable cells. The extent of growth
inhibition was determined by comparison of the number of
cells remaining after 72 h exposure to the growth of cells in
control wells that had been treated with vehicle alone (0.2%
DMSO). Dose-response curves were analyzed with XLFit or
Grafit and the value from the cells treated with DMSO alone was
considered 100%. Unless indicated, data are reported as the average
of two or more experiments (each run in duplicate). Statistical
limits for the data are reported as the standard deviation of
two or more experiments or the standard error within one
experiment. Cellular phenotype resulting from inhibitor treatment
Approximately half of the 4-(2,2-dimethyl-1,3-benzodioxol-5-
yl)aniline was dissolved in dioxane (1 mL) and was treated with
sulfamide (3.3 mmol). The reaction mixture was irradiated in the
microwave at 150 °C for 10 min. The reaction mixture was
concentrated in vacuo and the residue was purified by reverse phase
HPLC. Precipitation of the product from water and filtration with
1
a hexanes wash provided the title compound (36 mg). H NMR
(DMSO-d6): δ 9.55 (s, 1H), 7.49 (d, 2H, J ) 8.6 Hz), 7.19 (d, 2H,
J ) 8.6 Hz), 7.13-7.09 (m, 3H), 7.04 (m, 1H), 6.87 (d, 1H, J ) 8.1
Hz), 1.66 (s, 6H). MS m/z 321.2 [M + H]+. Anal. (C15H16N2O4S)
C, H, N.
N-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)phenyl]sulfamide (60).
A solution of 6-bromo-2,3-dihydro-1,4-benzodioxin (1.20 mmol),
[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]amine (1.32
mmol), cesium carbonate (2.08 mmol), and dichloro[1,1′-bis-
(diphenylphosphino)ferrocene]palladium(II)-dichloromethane ad-
duct (0.02 mmol) in DMF (6 mL) was irradiated in the microwave
at 110 °C for 10 min. The reaction was cooled, diluted with diethyl
ether, and extracted with aqueous sodium bicarbonate solution. The
organic layer was concentrated to give 0.223 g of crude [4-(2,3-
dihydro-1,4-benzodioxin-6-yl)phenyl]amine (83%). MS m/z 228.0
[M + H]+. The title compound was prepared from this aniline
following Method D with purification by reverse phase HPLC
was assessed using
protocol.
a Cytometrix automated cell imaging
1
(25%). H NMR (DMSO-d6): δ 9.55 (s, 1H), 7.54-7.43 (m, 2H),
7.22-7.16 (m, 2H), 7.15-7.04 (m, 4H), 6.90 (d, 1H, J ) 8.3 Hz),
4.27 (s, 4H). MS m/z 307.2 [M + H]+. Anal. (C14H14N2O4S) C,
H, N.
In Vivo Mouse Studies. All in vivo studies were performed in
accordance with protocols approved by the GSK Institutional
Animal Care and Use Committee and met or exceeded the standards

ATP-CompetitiVe Kinesin Spindle Protein Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4951
(14) Finer, J. T.; Bergnes, G.; Feng, B.; Smith, W. W.; Chabala, J. C.
Methods and compositions utilizing quinazolinones as KSP kinesin
modulators. WO2001030768A1, May 3, 2001.
of the American Association for the Accreditation of Laboratory
Animal Care (AAALAC), the United States Department of Health
and Human Services, and all local and federal animal welfare laws.
For xenograft models, Colo205 and HCT116 D130V tumors were
grown subcutaneously in female athymic nude mice (CD-1 Nu/Nu
mice). Compounds were formulated in 2% Cremophor EL, 2% N,N-
dimethylacetamide, and 96% acidified water (pH 5.0) and admin-
istered intraperitoneally on a q4dx3 schedule (three doses each
separated by 4 days). Tumors were measured twice weekly and
the tumor volume was calculated using the following formula
[(length × width2) × 0.5]. Data are presented as the median of
five animals. The median tumor volume at the start of the efficacy
studies was 100-200 mm3. Delays in tumor growth were deter-
mined by comparison of the times when compound-treated mice
and placebo-treated mice each reached a median tumor volume of
1000 mm3. Objective responses were defined as follows: complete
regression (CR), three consecutive measurements of 13.5 mm3 or
less; partial regression (PR), three consecutive measurements of
e50% of initial volume.
(15) Finer, J. T.; Bergnes, G.; Feng, B.; Smith, W. W.; Chabala, J. C.;
Morgans, D. J., Jr. Preparation of N-acylquinazolinonealkylamines
as KSP kinesin inhibitors. WO2001098278A1, Dec 27, 2001.
(16) Cox, C. D.; Breslin, M. J.; Mariano, B. J.; Coleman, P. J.; Buser, C.
A.; Walsh, E. S.; Hamilton, K.; Huber, H. E.; Kohl, N. E.; Torrent,
M.; Yan, Y.; Kuo, L. C.; Hartman, G. D. Kinesin spindle protein
(KSP) inhibitors. Part 1: The discovery of 3,5-diaryl-4,5-dihydro-
pyrazoles as potent and selective inhibitors of the mitotic kinesin
KSP. Bioorg. Med. Chem. Lett. 2005, 15, 2041-2045.
(17) Fraley, M. E.; Garbaccio, R. M.; Arrington, K. L.; Hoffman, W. F.;
Tasber, E. S.; Coleman, P. J.; Buser, C. A.; Walsh, E. S.; Hamilton,
K.; Fernandes, C.; Schaber, M. D.; Lobell, R. B.; Tao, W.; South,
V. J.; Yan, Y.; Kuo, L. C.; Prueksaritanont, T.; Shu, C.; Torrent,
M.; Heimbrook, D. C.; Kohl, N. E.; Huber, H. E.; Hartman, G. D.
Kinesin spindle protein (KSP) inhibitors. Part 2: The design,
synthesis, and characterization of 2,4-diaryl-2,5-dihydropyrrole in-
hibitors of the mitotic kinesin KSP. Bioorg. Med. Chem. Lett. 2006,
16, 1775-1779.
(18) Garbaccio, R. M.; Fraley, M. E.; Tasber, E. S.; Olson, C. M.;
Hoffman, W. F.; Arrington, K. L.; Torrent, M.; Buser, C. A.; Walsh,
E. S.; Hamilton, K.; Schaber, M. D.; Fernandes, C.; Lobell, R. B.;
Tao, W.; South, V. J.; Yan, Y.; Kuo, L. C.; Prueksaritanont, T.;
Slaughter, D. E.; Shu, C.; Heimbrook, D. C.; Kohl, N. E.; Huber, H.
E.; Hartman, G. D. Kinesin spindle protein (KSP) inhibitors. Part 3:
Synthesis and evaluation of phenolic 2,4-diaryl-2,5-dihydropyrroles
with reduced hERG binding and employment of a phosphate prodrug
strategy for aqueous solubility. Bioorg. Med. Chem. Lett. 2006, 16,
1780-1783.
(19) Cox, C. D.; Torrent, M.; Breslin, M. J.; Mariano, B. J.; Whitman,
D. B.; Coleman, P. J.; Buser, C. A.; Walsh, E. S.; Hamilton, K.;
Schaber, M. D.; Lobell, R. B.; Tao, W.; South, V. J.; Kohl, N. E.;
Yan, Y.; Kuo, L. C.; Prueksaritanont, T.; Slaughter, D. E.;
Li, C.; Mahan, E.; Lu, B.; Hartman, G. D. Kinesin spindle protein
(KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-
3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of
the mitotic kinesin KSP. Bioorg. Med. Chem. Lett. 2006, 16, 3175-
3179.
(20) Kim, K. S.; Lu, S.; Cornelius, L. A.; Lombardo, L. J.; Borzilleri, R.
M.; Schroeder, G. M.; Sheng, C.; Rovnyak, G.; Crews, D.; Schmidt,
R. J.; Williams, D. K.; Bhide, R. S.; Traeger, S. C.; McDonnell, P.
A.; Mueller, L.; Sheriff, S.; Newitt, J. A.; Pudzianowski, A. T.; Yang,
Z.; Wild, R.; Lee, F. Y.; Batorsky, R.; Ryder, J. S.; Ortega-Nanos,
M.; Shen, H.; Gottardis, M.; Roussell, D. L. Synthesis and SAR of
pyrrolotriazine-4-one based Eg5 inhibitors. Bioorg. Med. Chem. Lett.
2006, 16, 3937-3942.
(21) Hotha, S.; Yarrow, J. C.; Yang, J. G.; Garrett, S.; Renduchintala, K.
V.; Mayer, T. U.; Kapoor, T. M. HR22C16: A potent small-molecule
probe for the dynamics of cell division. Angew. Chem., Int. Ed. 2003,
42, 2379-2382.
(22) Sunder-Plassmann, N.; Sarli, V.; Gartner, M.; Utz, M.; Seiler, J.;
Huemmer, S.; Mayer, T. U.; Surrey, T.; Giannis, A. Synthesis and
biological evaluation of new tetrahydro-b-carbolines as inhibitors
of the mitotic kinesin Eg5. Bioorg. Med. Chem. 2005, 13, 6094-
6111.
(23) Sarli, V.; Huemmer, S.; Sunder-Plassmann, N.; Mayer, T. U.; Giannis,
A. Synthesis and biological evaluation of novel Eg5 inhibitors.
ChemBioChem 2005, 6, 2005-2013.
For pharmacokinetic studies, compound was administered as a
single intraperitoneal dose to female athymic nude mice as described
above. At each timepoint (5, 10, 60, 120, 240, 360, and 1440 min),
three mice were bled by cardiac puncture under terminal anesthesia.
Plasma samples were analyzed by HPLC/MS/MS after protein
precipitation with acetonitrile. Data was analyzed using WinNonlin
version 3.2 with noncompartmental analysis.
Acknowledgment. We gratefully acknowledge Stanley J.
Schmidt, Michael G. Darcy, Jian Jin, Deana F. Wang, Yonghui
Wang, and Dongchuan Shi for early SAR contributions. We
also thank Jim Bulgarelli, Eric Manning, and Ganesh Moorthy
for analytical mouse PK support, Yan Lee (Cytokinetics) for
initial biochemical characterization of compound 5, Hong Lu
for technical assistance, and Chao Han for helpful discussions.
Supporting Information Available: Full elemental analyses
on noted compounds and statistical limits for biological data in
Tables 1 and 2. This material is available free of charge via the
Internet at http://pubs.acs.org.
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JM070435Y