Synthesis and SAR of thiophene containing kinesin spindle protein (KSP) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.04.076

We have identified and synthesized a series of thiophene containing inhibitors of kinesin spindle protein. SAR studies led to the synthesis of 33, which was co-crystallized with KSP and determined to bind to an allosteric pocket previously described for o

Full text of DOI:10.1016/j.bmcl.2007.04.076

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3562–3569
Synthesis and SAR of thiophene containing
kinesin spindle protein (KSP) inhibitors
Anthony B. Pinkerton,a,* Tom T. Lee,b Timothy Z. Hoffman,a Yan Wang,b
Mehmet Kahraman,a Travis G. Cook,a Daniel Severance,a Timothy C. Gahman,a
Stewart A. Noble,a Andrew K. Shiaub and Robert L. Davisb
aDepartment of Medicinal Chemistry, Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
bDepartment of Biology, Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
Received 21 March 2007; revised 18 April 2007; accepted 18 April 2007
Available online 29 April 2007
Abstract—We have identified and synthesized a series of thiophene containing inhibitors of kinesin spindle protein. SAR studies led
to the synthesis of 33, which was co-crystallized with KSP and determined to bind to an allosteric pocket previously described for
other known KSP inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Anti-mitotic chemotherapeutics, such as the taxanes and
vinca alkaloids, represent one of the main classes of
effective cancer therapies, and are broadly used against
a wide range of cancer types.1 However, mechanism-
related toxicities2 and acquired resistance have stimu-
lated considerable interest in developing anti-mitotics
that target mechanisms other than direct microtubule
inhibition. Kinesin spindle protein (KSP, also known
as Eg5) is a member of a superfamily of force-generating
motor proteins associated with microtubules.3a Like
other kinesins, KSP contains a catalytic ATPase activity
required for directed movement along microtubules. In
dividing cells, the effects of KSP inhibition are limited
to mitosis, while the only reported expression of KSP
in non-dividing cells is in subsets of post-mitotic neu-
rons, and its function there is unclear.3b Inhibition of
KSP during mitosis leads to lack of formation or disrup-
tion of the bipolar mitotic spindle, sustained mitotic ar-
rest, and subsequent induction of apoptosis both in vitro
and in vivo.4 Numerous small molecule inhibitors of the
KSP ATPase have been recently described,5 for example
quinazolinone 1,5l dihydropyrazole 2a,5a and dihydro-
pyrrole 2b,5b and clinical trials have been initiated to
validate KSP as a target for cancer therapeutics.6
Ultra high-throughput screening of the Kalypsys com-
pound collection identified a series of thiophene contain-
ing KSP inhibitors of the general structure 3, with
ATPase IC50s in the low micromolar range and effects
on cultured cells consistent with selective targeting of
KSP. Thus, a medicinal chemistry effort was initiated
to optimize these screening hits and determine their
mode of binding to KSP.
The compounds described herein were synthesized as
outlined in Scheme 1.7 Cyclic ketones 4 were reacted
with ethyl cyanoacetate (5) and sulfur with a catalytic
amount of morpholine in ethanol to give amino thioph-
enes 6 in moderate to good yields.8 Ester hydrolysis fol-
lowed by activation of the resulting acid with
triphosgene and reaction with an amine afforded amino-
thiophene amides 9. Reductive amination of the pendant
amine provided KSP inhibitors such as 10. Additionally,
6 could be reacted with acid chlorides (7) to give amides
8 in moderate to excellent yields. Hydrolysis of the free
acid and coupling to an amine gave KSP inhibitors of
the general structure 11.
To investigate the SAR and improve the potency three
main areas were examined: (1) investigation of the pen-
dant amide connected to the amino thiophene (N-
linked), (2) substitution of the thiophene core, and (3)
replacements of the diethylamide. Three in vitro assays
were used to drive the SAR: (a) a biochemical assay that
Keywords: Eg5; KSP; Kinesin spindle protein; Anti-mitotics;
Thiophenes.
*
Corresponding author. Tel.: +1 858 754 3457; fax: +1 858 754
3301; e-mail: apinkerton@kalypsys.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.076

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3562–3569
3563
F
F
O
R1
R2
F
F
NH2
S
O
N
N
H
R3
N
N
O
N
N
N
N
O
Cl
O
N
O
1
2a
2b
3
measures microtubule-stimulated KSP ATPase activity,
(b) a cell-based assay that measures mitotic arrest main-
tained by KSP inhibition (MPM-2 cytoblot), and (c) a
cell-based assay that measures cytotoxicity due to pro-
longed mitotic arrest, itself a result of continuous KSP
inhibition.9–11 In general, trends were consistent among
the three assays.
(18), methyl thiazole (19), furan (20), and phenyl (21),
all showing a log or more loss of activity. Removing
the amide carbonyl of 15 to give amine derivative 22 also
led to a log loss of activity. To further drive medicinal
chemistry efforts, we determined the co-crystal structure
of KSP with cyclohexyl analog 15, Mg2+, and ADP to a
12
˚
resolution of 1.85 A (Fig. 1).
We initially examined substituent effects on the thio-
phene core as well as the effect of the pendant amide
(Table 1). From the initial screen, a 2-thiophene amide
at R3 was found to be the most active analog (for exam-
ple compound 14), and thus was used as a starting point.
As shown, only very weak activity was observed with an
unsubstituted thiophene core (12). Some improvement
was observed with dimethyl substitution (13), but more
dramatic effects were observed with fused aliphatic rings
appended onto the thiophene (14–16). In particular,
cyclohexyl analog 15, with activity in the ATPase assay
of 1.7 lM and the MPM-2 cytoblot assay of 0.48 lM,
became a scaffold for further optimization. Attempts
to optimize the R3 amide from the starting 2-thiophene
were less successful, with the 3-thiophene (17), thiazole
As shown in Figure 1A, compound 15 binds in a similar
fashion to known KSP inhibitor 2b at an allosteric si-
te.5b The cyclohexylthiophene core of 15 occupies the
same hydrophobic pocket as the difluorophenyl group
of 2b. One ethyl of the diethylamide group projects into
solvent and the other in the pocket formed by Tyr211,
Leu214, and Glu215. The pendant 2-thiophene amide
of 15 fills the same pocket as the phenyl group of 2b,
leaving no extra space to accommodate any substituents
on the thiophene ring. Using this information, we turned
our focus to optimization of the diethylamide group.
Initial exploration around the diethyl amide (Table 2)
indicated that the hydrophobic interactions provided
by amide substitution were required for activity. This
conclusion is supported by the following observations:
O
S
X
S
O
X
R4
R4
N
H
R3
R4
a
NH2
OEt
b
X
n
n
O
EtO
n
CN
4
OEt
O
O
8
Cl
R3
O
5
6
7
c
d,e
O
S
X
S
X
R4
R4
R3
NH2
R5
N
H
n
R5
n
N
N
11
O
O
9
R6
R6
f
S
X
R4
N
H
R7
R5
n
N
O
10
R6
Scheme 1. Reagents and conditions: (a) S, morpholine, EtOH, 50 ꢁC, 40–95%; (b) Et3N, CH2Cl2, rt, 50–80%; (c) i—NaOH, EtOH, 70 ꢁC, 72%;
ii—triphosgene, THF, rt, quant.; iii—R5R6NH, DMF, 60 ꢁC, 50–90%; (d) LiOH, THF/H2O, 1:1, 60 ꢁC, 30–70%; (e) R5R6NH, HATU, DMF, rt,
20–60%; (f) R7CHO, NaBH(OAc)3, CH2Cl2, rt, 20–50%.

3564
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3562–3569
Table 1. Optimization of core and pendant amide
R1
R2
S
R3
N
H
N
O
R1
R2
R3
ATPase IC50 (lM)
MPM-2 Cytoblot EC50 (lM)
Cytotoxicity 60 h EC50 (lM)
a
a
a
Compound
O
12
–H
–H
>100
NAb
NAb
>30
S
S
S
S
S
O
O
O
O
13
14
15
16
17
18
19
20
21
22
–CH3
–CH3
23(5)
29(11)
2.2(0.2)
0.48(0.13)
5.8(6.8)
4.3(0.8)
>30
–(CH2)3–
9.9(2.1)
1.7(0.4)
4.1(0.9)
13(2)
7.1(1.0)
2.0(0.2)
13(5)
29.4(0.1)
NAb
–(CH2)4–
–(CH2)5–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
O
O
S
S
S
76(19)
17(4)
N
O
>30
>30
N
O
O
O
23(5)
20(7)
>30
33(9)
34(18)
>30
>30
S
20(8)
>30
a Value represents the mean of three experiments with standard deviations shown in parentheses.
b NA, not active <30 lM.
Figure 1. X-ray structures of compounds in the KSP allosteric binding site. (A) 2b (yellow, PDB code 2FL6) and 15 (green, PDB code 2PG2); (B) 33
(blue, PDB code 2UYI) and 37 (orange, PDB code 2UYM). Protein surface is shown for KSP/ADP/15 (A) and KSP/ADP/33 (B). Compounds are
shown in thick sticks, and protein residues are shown in thin sticks.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3562–3569
3565
Table 2. Effect of amide substitution
O
S
S
N
H
R4
O
R4
OEt
ATPase IC50 (lM)
MPM-2 Cytoblot EC50 (lM)
NAb
Cytotoxicity 60 h EC50 (lM)
NAb
a
a
a
Compound
23
>100
24
25
15
26
27
28
29
30
31
>100
NAb
NAb
OH
>100
NAb
NAb
NH2
1.7(0.4)
6.0(1.6)
3.9(0.8)
2.8(0.7)
4.3(0.9)
27(6)
0.48(0.13)
3.6(1.2)
1.1(0.1)
0.54(0.30)
1.8(0.4)
37(29)
2.0(0.2)
12(2)
NEt2
NMe2
3.1(1.6)
5.1(2.2)
7.2(2.7)
>30
N
N
N
O
N
N
NH
N
8.6(1.8)
7.6(1.4)
>30
a Value represents the mean of three experiments with standard deviations shown in parentheses.
b NA, not active <30 lM.
first, an unsubstituted amide abolished activity (25). Sec-
ond, unbranched amide substitution, which has fewer
conformations allowing contact (30), was less potent
than a fully branched substitution (31). The fact that
the ester (23) and acid (24) are inactive is consistent with
this notion. Third, cyclization of the branched amide
(27–29) gave comparable activity. Finally, the dimeth-
ylamide 26, reduced in size, showed diminished potency.
vs 1.7 lM) and the MPM-2 cytoblot assay (0.10 lM vs
0.48 lM). Co-crystallization of 33 showed that the
methyl group occupied an additional part of the main
hydrophobic pocket (Fig. 1B). In addition, 33 was re-
solved into its enantiomers and one isomer was found
to be 10 fold more active than the other.15 Addition of
another methyl group at position 2 (37) of the cyclo-
hexylthiophene was tolerated but led to a loss of activity
compared to 15. An X-ray structure shows that the axial
˚
Lastly we turned our attention to further optimization
of the thiophene core (Table 3). From the crystal struc-
ture of 15, we surmised that there was additional space
in the hydrophobic pocket occupied by the cyclohexyl
ring, in particular space occupied by the halogens in 1
and 2a/2b. To test this hypothesis we added alkyl groups
to various positions on the cyclohexyl ring to potentially
enhance favorable hydrophobic interactions. These and
other substituted cycloalkylthiophene compounds were
synthesized and tested as racemic mixtures unless other-
wise noted. Satisfyingly, we found that a methyl group
in the 2 position (33) gave a three- to fourfold boost
in potency over 15 in both the ATPase assay (0.48 lM
methyl group in 37 makes close contact (3.2 A) to the
polar main chain oxygen of Leu214 as shown in Figure
1B. Furthermore, the Ca atoms of 213–218 move an
˚
average of 0.34 A away from 37, resulting in slight open-
ing of the pocket as compared to 33. Attempts to in-
crease the size beyond methyl, as in 39, were
detrimental. Substitutions at other positions of the
cyclohexylthiophene were less favorable than position
2 (compare 33 to 32, or 34; 37 to 38; or 39 to 40). An
energy penalty likely occurs by rearranging adjacent res-
idues in order to avoid close contacts incurred by these
substitutions. A similar boost in potency was observed
when a methyl group was incorporated on the cyclopen-

3566
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3562–3569
Table 3. Core optimization
O
S
N
H
CORE
O
N
a
a
a
Compound
CORE
ATPase IC50 (lM)
MPM-2 Cytoblot EC50 (lM)
Cytotoxicity 60 h EC50 (lM)
32(1)
33(2)
34(3)
7.5(1.6)
4.7(1.4)
6.3(1.7)
1.0(0.7)
6.1(0.9)
S
H3C 3
0.48(0.11)
5.9(1.1)
0.10(0.03)
2.6(1.0)
2
1
S
35(1)
36(2)
8.1(1.0)
3.5(0.6)
5.4(1.9)
1.5(0.4)
36(19)
7.7(4.9)
2
H3C
1
S
37
38
3.1(0.9)
12(1)
1.7(0.8)
14(7)
6.0(2.6)
24(4)
S
S
3
2
39(2)
40(3)
7.4(1.0)
69(1)
1.9(0.4)
>30
12(2)
7.3(1.2)
1
S
O
41
42
43
44
24(4)
17(5)
9.6(9.5)
NAb
>30
>30
S
S
3.3(0.9)
>100
S
HN
NAb
NAb
S
>100
NAb
a Value represents the mean of three experiments with standard deviations shown in parentheses.
b NA, not active <30 lM.
tyl analog, for example activity was improved to 3.5 lM
(for 36) from 9.9 lM (for 14) in the ATPase assay. At-
tempts to incorporate heteroatoms into the ring led to
a range of results, from a minor effect for sulfur (42),
to a detrimental effect with oxygen (41) and complete
loss of activity with nitrogen (43). This suggests that po-
lar substituents are not tolerated in the hydrophobic
pocket.5a Somewhat surprisingly, benzothiophene ana-
log 44 showed no activity, although subsequent model-
ing into the active site indicated that the aromatic ring
was not as well tolerated. Naphthalene and benzofuran
analogs also showed no activity (data not shown).
tion. No additional effect on cell morphology prior to
induction of apoptosis was observed.
Flow cytometry of non-synchronous A-549 cells treated
for 18 h with 15 lM compound 15 demonstrated a
majority with a 4 N (G2/M) DNA content, consistent
with mitotic arrest. For comparison, compounds 1,
and 2a (as a racemic mixture), generated a similar profile
at 1 lM (Table 4A). Cells previously treated for 18 h
were washed and cultured for an additional 24 h in the
absence of compounds (Table 4B). In this case, the effect
of previous compound 15 treatment was mostly revers-
ible, as was the case for compound 2a. By contrast,
the effect of previous compound 1 treatment was not
reversible, with most cells continuing to have a 4 N
DNA content, and some increase in the sub-2 N fraction
associated with apoptotic and dead cells.
To confirm that the cytotoxic activity was a consequence
of KSP inhibition, we further characterized the cellular
activity of compounds 15 and 33 (Fig. 2). Non-synchro-
nously dividing A-549 lung cancer cells were treated for
18 h with vehicle alone (A), compound 33 (B), or com-
pound 15 (not shown). Incubation with compound 33
or 15 resulted in the accumulation of cells with the typ-
ical monopolar spindle morphology due to KSP inhibi-
A-549 cells have been reported to aberrantly exit mitosis
after prolonged mitotic arrest due to KSP inhibition by
treatment with 200 lM monastrol. However, this exit

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3562–3569
3567
Figure 2. Imaging of cells following treatment with DMSO vehicle (A) or 10 lM compound 33 (B).16 Separate images of microtubules (green) and
DNA (blue) are overlaid. (A) shows a single normal mitosis in the upper left, while (B) shows numerous cells with the monopolar spindle phenotype.
Scale bar = 20lm.
Table 4. Flow cytometry of compound-treated A-549 cells
DNA content
Cell cycle phase
<2 N
Apoptotic
2 N
G1
>2 N, <4 N
S
4 N
G2/M
Aa
DMSO
1, 1 lM
2a, 1 lM
15, 15 lM
7
3
2
3
64
17
14
18
13
12
11
14
12
61
64
56
Bb
DMSO
1, 1 lM
2a, 1 lM
15, 15 lM
0.5
10
5
58
8
17
7
16
64
24
25
46
47
14
12
4
a Cells were treated for 18 h with DMSO vehicle or the listed compounds and processed for flow cytometry.17 Table values are expressed as
percentages. The remaining percentage of the sample is in multi-cell aggregates and not listed.
b Cells were treated for 18 h with DMSO vehicle or the listed compounds then washed and cultured in the absence of compounds for an additional
24 h before processing for flow cytometry.17 Table values are expressed as percentages. The remaining percentage of the sample is in multi-cell
aggregates and not listed.
2. Quasthoff, S.; Hartung, H. P. J. Neurol. 2002, 249, 9.
3. (a) Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Curr.
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A.; Lewis, E.; Fritsch, A.; Lee, Y.; Mak, J.; Moody, R.;
Turincio, R.; Chabala, J. C.; Gonzales, P.; Roth, S.;
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Heald, R. Cell 2000, 102, 399; (d) Sharp, D. J.; Rogers, G.
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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. Bioorg. Med. Chem. Lett. 2005, 15, 2041; (b) 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. Bioorg.
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does not occur until at least 36 h of continuous arrest,
and is followed by a G1-like arrest with a 4 N DNA con-
tent.18 Our results suggest shorter periods of mitotic ar-
rest are insufficient for A-549 cells to commit to mitotic
slippage, at least for compounds 15 and 2a. Finally, the
cell viability assay was used to demonstrate that signifi-
cant apoptosis does not begin until at least 24 h after
incubation of compounds 15 or 33, consistent with other
KSP inhibitors.19
In conclusion, a new class of thiophene containing com-
pounds that are inhibitors of KSP have been developed
from a uHTS screening lead. These compounds have
been shown to have sub-micromolar activity in second-
ary cellular assays and a cell phenotype consistent with
KSP inhibition. Structural information gained from
co-crystal structures with KSP should help in the further
development of these and other structural classes of
KSP inhibitors.
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10. Mitotic arrest maintained by KSP inhibition was assayed
using A-549 human non-small cell lung carcinoma cells
(CCL-185, ATCC). If A-549 cells synchronized in mitosis
by nocodazole treatment are replated in the absence of
nocodazole, ꢀ100% of the cells will exit mitosis, and
daughter cells will acquire a flat morphology within 4 h.
However, if KSP is inhibited immediately after nocodazole
washout, cells will remain in mitotic arrest, which can be
demonstrated by measuring levels of the MPM-2 phos-
pho-epitope13. Compounds in DMSO and arrayed in 11
point 1/2 log dilution dose response (final top concentra-
tion of 96 lM, 1% DMSO in all assay wells) were added
by passive pin transfer to culture medium (with 1% fetal
bovine serum) previously dispensed to black 384-well
assay plates. A-549 cells treated for 10 h with 1 lM
nocodazole were harvested, washed, and dispensed to the
compound-pinned assay plates at 3 · 105 cells/ml. Four
hours later, cells were washed and fixed with 2% parafor-
maldehyde in phosphate-buffered saline for 10 min at
room temperature. Fixed cells were then processed for a
typical ‘cytoblot’ assay14, using MPM-2 primary antibody
(Upstate Cell Signaling), donkey anti-mouse IgG ðFab02Þ
horseradish peroxidase conjugate (Jackson ImmunoRe-
search), and a chemiluminescent substrate (POD, Roche
Applied Science). Signal detection, data normalization,
and data analysis were similar to that in Ref. 9.
11. A-549 cells were dispensed to white 384-well assay plates
at 1 · 105 cells/ml in medium with 1% fetal bovine serum.
Compounds in DMSO and arrayed in 11 point 1/2 log
dilution dose response (final top concentration of 96 lM,
1% DMSO in all assay wells) were added by passive pin
transfer. After 60 h incubation, cell viability was measured
by dispensing an equal volume of ATPLite reagent
(Perkin-Elmer). Signal detection, data normalization,
and data analysis were similar to that in Ref. 9.
6. (a) Duhl, D. M.; Renhowe, P. A. Curr. Opin. Drug Discov.
Dev. 2005, 8, 431; (b) Coleman, P. J.; Fraley, M. E. Expert
Opin. Ther. Patents 2004, 14, 1659; (c) Jackson, J. R.;
Patrick, D. R.; Dar, M. M.; Huang, P. S. Nat. Rev. Cancer
2007, 7, 107.
12. An N-terminal His6-tagged catalytically active fragment of
KSP (1–368) was expressed in Escherichia coli and purified
over a Ni–NTA column. The His6 tag was removed, and the
protein was further purified over a HiTrap Q column.
Crystals were prepared at 4 ꢁC using the hanging drop
method. Protein [10 mg/mL in 50 mM Pipes (pH 6.8), 1 mM
ATP, 2 mM MgCl2, 1 mM EGTA, and 1 mM Tris
(2-carboxyethyl)phosphine hydrochloride] was mixed with
2 mM compound and incubated on ice for 1 h. The initial
drop contained equal volumes of complex and well buffer
[100 mM Bis–Tris (pH 6.0), 200 mM ammonium sulfate,
and 18–20% PEG3350], and 10 mM SrCl2 was also added to
the drop. Crystallization was improved by microseeding,
and the seeded drops were left at 4 ꢁC for 2–3 days. Crystals
were soaked in cryoprotecting solution (well buffer with
PEG concentration increased to 35%) before freezing in
liquid nitrogen. Data were collected at the Advanced Light
Source beam line 5.0.2 (LBL). Structures were determined
by molecular replacement using the KSP/ADP/monastol
complex (PDB ID:1Q0B) as the search model. Crystallo-
graphic data are as follows. KSP/ADP/15: resolu-
7. All final compounds displayed spectral data (NMR, MS)
that were consistent with the assigned structure.
8. Andersen, H. S.; Olsen, O. H.; Iversen, L. F.; Sorensen, A.
L. P.; Mortensen, S. B.; Christensen, M. S.; Branner, S.;
Hansen, T. K.; Lau, J. F.; Jeppesen, L.; Moran, E. J.; Su,
J.; Bakir, F.; Judge, L.; Shahbaz, M.; Collins, T.; Vo, T.;
Newman, M. J.; Ripka, W. C.; Moller, N. P. H. J. Med.
Chem. 2002, 45, 4443.
9. Microtubule-stimulated KSP ATPase activity was deter-
mined by measuring production of ADP (ADPQuest
Assay, DiscoverX). Affinity purified, catalytically active
human KSP protein fragment (residues 1–386, C-terminal
His6-tagged) in 20 ll of assay buffer was dispensed to
black 384-well assay plates. Compounds in DMSO and
arrayed in 11 point 1/2 log dilution dose response (final
top concentration of 96 lM, 1% DMSO in all assay wells)
were added by passive pin transfer, followed by addition
of 5 ll assay buffer containing taxol-stabilized bovine
brain microtubules (Cytoskeleton) and ATP. The final
concentrations of KSP, microtubules, and ATP were
15 nM, 500 nM, and 30 lM, respectively. After a 2-h
room-temperature incubation, ADPQuest assay reagents
were added according to the manufacturer’s instructions,
and the resulting fluorescence signal was read on a
Molecular Devices Acquest plate reader. Raw fluorescence
data were normalized to negative (DMSO) and positive
˚
˚
˚
tion = 1.85 A, space group = C2, a = 161.1 A, b = 80.4 A,
˚
c = 69.3 A, b = 96.8ꢁ, R/Rfree = 0.210/0.248; KSP/ADP/33:
resolution = 2.1 A, space group = P212121, a = 69.5 A,
˚
˚
˚
b = 80.1 A, c = 159.0 A, R/Rfree = 0.240/0.294; KSP/ADP/
˚
˚
37: resolution = 2.11 A, space group = C2, a = 160.8 A,
˚
˚
˚
b = 80.5 A, c = 68.8 A, b = 96.2ꢁ, R/Rfree = 0.208/0.272.
13. Davis, F. M.; Tsao, T. Y.; Fowler, S. K.; Rao, P. N. Proc.
Natl. Acad. Sci. U.S.A. 1983, 80, 2926.
14. Stockwell, B. R.; Haggarty, S. J.; Schreiber, S. L. Chem.
Biol. 1999, 6, 71.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3562–3569
3569
15. Compound 33 was resolved using a Chiralpak IA column
(4.6 mm · 250 mm) eluting 10% i-PrOH/90% heptane/
0.1% TFA, 1 mL/min. The first eluting isomer showed
ATPase activity of 0.48 lM versus 3.9 lM for the second
eluting isomer. The absolute stereochemistry was not
determined; the resolution of the crystal structure was not
sufficient for definitive assignment.
17. A-549 cells in medium with 1% fetal bovine serum were
treated with DMSO vehicle (<=0.15 % final) alone or
containing compounds as specified for 18 h. To test
reversibility of mitotic arrest, previously treated cells were
washed three times with medium containing 1% fetal
bovine serum and then replated in the same for an
additional 24 h. Cells were harvested, washed twice with
phosphate-buffered saline containing 2% fetal bovine
serum, and then resuspended in 1 ml of the same wash
buffer. For fixation, 3 ml of ꢁ20 ꢁC absolute ethanol was
added slowly, followed by a 1-h incubation on ice. Fixed
cells were then washed twice with phosphate-buffered
saline and resuspended in staining buffer (phosphate-
buffered saline containing 3.8 mM sodium citrate, 50 lg/ml
propidium iodide, and 100 lg/ml RNase A) for a mini-
mum of 1 h at 4 ꢁC. Cells were analyzed on a BD LSR
flow cytometer.
16. A-549 cells were plated at 1 · 105 cells/ml into glass
coverslip chambers in medium with 1% fetal bovine
serum. DMSO vehicle (1% final) alone or containing
compounds (10 lM final) was added and cells incubated
for 18 h. Cells were then washed and fixed with 100% ice-
cold methanol for 10 min at room temperature. Fixed
cells were processed for immunofluorescence microscopy
of microtubules using anti-alpha tubulin mAb (DM1A,
Sigma) and donkey anti-mouse IgG ðFab02Þ Alexa 488
(Invitrogen). Chromosomal DNA was stained with 40-6-
diamidino-2-phenylindole (DAPI, Sigma). Cells were
imaged on a Zeiss Axiovert 2 with an ORCA-ER CCD
camera (Hamamatsu) and OpenLab software (Improvi-
sion). Images were imported into Adobe Photoshop for
labeling and minor alterations in contrast.
18. Vijapurkar, U.; Wang, W.; Herbst, R. Cancer Res. 2007,
67, 238.
19. Tao, W.; South, V. J.; Zhang, Y.; Davide, J. P.; Farrell, L.;
Kohl, N. E.; Sepp-Lorenzino, L.; Lobell, R. B. Cancer
Cell 2005, 8, 49.