Kinesin spindle protein (KSP) inhibitors. Part 8: Design and synthesis of 1,4-diaryl-4,5-dihydropyrazoles as potent inhibitors of the mitotic kinesin KSP

Article abstract of DOI:10.1016/j.bmcl.2007.07.074

Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlig

Full text of DOI:10.1016/j.bmcl.2007.07.074

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5677–5682
Kinesin spindle protein (KSP) inhibitors. Part 8: Design
and synthesis of 1,4-diaryl-4,5-dihydropyrazoles as potent
inhibitors of the mitotic kinesin KSP
Anthony J. Roecker,a,* Paul J. Coleman,a Swati P. Mercer,a John D. Schreier,a
Carolyn A. Buser,b Eileen S. Walsh,b Kelly Hamilton,b Robert B. Lobell,b Weikang Tao,b
Ronald E. Diehl,b Vicki J. South,b Joseph P. Davide,b Nancy E. Kohl,b Youwei Yan,c
Lawrence C. Kuo,c Chunze Li,d Carmen Fernandez-Metzler,d Elizabeth A. Mahan,d
Thomayant Prueksaritanontd and George D. Hartmana
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, USA
cDepartment of Structural Biology, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, USA
dDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, USA
Received 30 May 2007; revised 16 July 2007; accepted 17 July 2007
Available online 21 August 2007
Abstract—Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-
dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also
highlighted.
ꢀ 2007 Elsevier Ltd. All rights reserved.
O
In the preceding communications, several classes of KSP
N
Me
inhibitors, including dihydropyrazoles, dihydropyrroles,
and pyrazolobenzoxazines, have been described.1 Sev-
eral members of each of these classes of compounds
have demonstrated excellent biochemical and cellular
potencies against KSP, aqueous solubility, limited
susceptibility to P-glycoprotein-mediated efflux, and
potency in a xenograft mouse model of cancer. Inspired
from the recent forays into the dihydropyrazolobenzox-
azine class of compounds (1, Fig. 1), herein we describe
the design and synthesis of a novel class of KSP inhibi-
tors, namely the 1,4-diaryl-4,5-dihydropyrazoles.2 This
novel class of KSP inhibitors maintained the aforemen-
tioned properties of the previous classes of KSP inhibi-
tors as well as comparable potency in vivo.
Crystallographic evidence of the similarities in binding
mode of the dihydropyrazolobenzoxazine and 1,4-dia-
N
O
N
F
N
Me
(1)
O
KSP IC50 = 1.6 nM
excise
constraint
O
N
Me
N
F
F
N
N
copper-mediated
coupling
Me
(2)
Keywords: KSP inhibitors; Antimitotics; Pgp; Kinesins; 1,4-Diaryl-4,5-
dihydropyrazoles; Copper-mediated N-arylation; Kinesin spindle pro-
tein; Cancer.
O
Figure 1. Potent dihydropyrazolobenzoxazine KSP inhibitor (1)
inspired efforts to synthesize and study its 1,4-diaryl-4,5-dihydropy-
razole analog (2).
*
Corresponding author. Tel.: +1 215 652 8257; fax: +1 215 652
7310; e-mail: anthony_roecker@merck.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.074

5678
A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5677–5682
ryl-4,5-dihydropyrazole class of inhibitors to an alloste-
ric site of the KSP enzyme will also be demonstrated.
HN
N
(a)
CO2Me
CO2Me
(b)
3
4
Chemistry. Inspiration for the synthesis of 1,4-diaryl-4,5-
dihydropyrazoles as KSP inhibitors was derived from
compound 1 as shown in Figure 1. Compound 1 is a
highly potent member of the dihydropyrazolobenzox-
azine class of KSP inhibitors (IC50 = 1.6 nM), the details
of which are covered in the preceding publication.1g
Excision of the central methyleneoxy constraint affords
a target 1,4-diaryl-4,5-dihydropyrazole (2) containing
only a single stereocenter. This seemingly simplified
structure could not be easily synthesized in a manner
analogous to compound 1. Compound 1 was synthesized
through an intramolecular [3 + 2] cycloaddition utilizing
the methyleneoxy subunit as a linker.1g The application
of an intermolecular version of this cycloaddition pro-
cess to the synthesis of 2 was daunting,3 thus an alterna-
tive synthetic approach was sought.
F
F
(c)
X
N
N
X
N
N
6
5
CO2Me
CO2Me
X = F, Cl, Br, CF3, H
(d)
F
OH
F
(e)
X
N
N
X
N
N
Me
N
7
8
Me
O
N
OMe
O
OMe
One novel approach relied on a copper-mediated N-ary-
lation using pyrazolines and boronic acids for the for-
mation of the key pyrazoline-aryl bond. First disclosed
by Lam, Chan, and Evans, this methodology allows a
variety of heterocyclic nucleophiles including anilines,
anilides, imidazoles, pyrazoles, triazoles, and indazoles
to be arylated by boronic acids in good to excellent
yields.4 However, the use of pyrazolines as nucleophiles
in these copper-mediated reactions has been scarce and
only recently has progress been made utilizing di- and
triarylbismuth reagents.5 With this concept in mind we
began an effort to synthesize the 1,4-diaryl-4,5-dihydro-
pyrazole targets such as 2.
(f)
R1
N
OH
F
R2
F
(g)
X
N
N
X
N
N
Me
+ 10
9
Me
O
O
Scheme 1. General synthesis of 1,4-diaryl-4,5-dihydropyrazole KSP
inhibitors. Reagents and reaction conditions: (a) CH2N2, Et2O,
0–25 ꢁC, 12 h, 98%; (b) ArB(OH)2, Cu(OAc)2, Et3N, CH2Cl2, 24 h,
5–27%; (c) LHMDS, allyl bromide, À78 to À5 ꢁC, THF, 2 h, 50–72%;
(d) 1.0 M NaOH, 25 ꢁC, THF, 24 h, aqueous workup, then
MeN(OMe)HCl, EDC, HOAt, Et3N, THF:DMF (1:1), 16 h,
80–94%; (e) 9-BBN, THF, 55 ꢁC, then H2O2, NaOH, 25 ꢁC, 1 h, 58–
72%; (f) MeLi, THF, 0 ꢁC, 1 h, 38–67%; (g) Dess–Martin periodinane,
NaHCO3, CH2Cl2, 1 h, then R1R2NH, NaBH(OAc)3, Et3N, 1,2-
dichloroethane, 2 h, 54–80%.
The implementation of our strategy is shown in Scheme
1. The synthesis in racemic form begins with the addi-
tion of diazomethane to methyl cinnamate (3) to afford
pyrazoline 4 in near quantitative yield and as a single
regioisomer as precedented in the literature.6 The pyrazo-
line (4) was then exposed to the requisite boronic acid,
copper (II) acetate, and triethylamine in dichlorometh-
ane in air overnight to yield arylated products 5.
Although the yields were low, the only byproduct was
retro-cycloaddition resulting in re-isolation of methyl-
cinnamate which was readily separated from N-arylpy-
razoline products (5). Compound 5 was then allylated
(LHMDS, allyl bromide)7 in good yield to afford 6
and subsequently transformed into the Weinreb amide
(7) in a two-step procedure. Compound 7 was treated
with 9-BBN followed by oxidation to afford regioselec-
tive hydroboration product 8. Compounds of type 8
were then advanced to the final targets (10) through
transformation of the Weinreb amide to a methyl ketone
(MeLi), oxidation of the primary alcohol (Dess–Martin
periodinane), and reductive amination with various
amines.
2 bearing the same amine as dihydropyrazolobenzox-
azine 1 maintains excellent intrinsic potency (3.8 nM)
while suffering a 16-fold loss in cellular potency
(78 nM). Modulation of the amine to a tertiary hetero-
cyclic amine (11) or thiomorpholine dioxide (12) affor-
ded compounds with diminished cellular potency as
compared to 2. Potency in cells was regained when the
acetylpiperazine was replaced with various tertiary
amines such as morpholine 12 (53 nM), 3-fluoroazeti-
dine 14 (40 nM), pyrrolidine 15 (15 nM), and bridged
morpholine 16 (11 nM). The best substituent in the sub-
set tested was dimethylamine (17) having biochemical
potency of 2.1 nM and cellular potency of 9.4 nM.
From previous work in the 3,5-diaryl-4,5-dihydropyraz-
ole series,1a it was shown that the 5-position of the aro-
matic ring could have a significant effect on KSP potency.
The results of a similar investigation in the 1,4-diaryl-
4,5-dihydropyrazole series are shown in Table 2.
Compounds containing a halogen at the 5-position (2,
18, and 19) showed good intrinsic potency and up to a
twofold improvement in cellular activity as shown for
In vitro SAR and X-ray crystallographic analysis. Our
investigations into the effect of the amine on the intrin-
sic8 and cellular potency9 of 1,4-diaryl-4,5-dihydropy-
razoles are shown in Table 1. In general, the intrinsic
potency of the compounds did not vary greatly with
modulation of the amine. It is notable that compound

A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5677–5682
Table 2. SAR of the 5-position of the western aryl ring
5679
Table 1. Sidechain amine SAR
R
N
O
R
N
F
N
F
F
N
N
X
N
(+)
N
O
(+)
O
a
KSP IC50 (nM) Cell EC50 (nM)
a
Compound Amine
a
KSP IC50 (nM)
a
Cell EC50 (nM)
Compound
X
2
3.8
4.0
16.5
4.2
5.2
3.8
1.2
2.1
78
567
138
53
N
NAc
O
2
18
19
20
21
22
F
Cl
3.8
3.9
78
46
N
Br
H
4.7
38
11
N
N
21.8
10.1
2.0
368
208
27
CF3
CH3
12
SO2
O
a See Refs. 8,9 for the details of these assays. All compounds listed were
assayed as racemates.
13
N
N
N
N
N
The determination of the stereochemistry of 25 was
aided by the high resolution crystal structure of com-
pound 22 (synthesized in an analogous fashion to 25,
Scheme 2) in an allosteric binding site of KSP.11 Com-
pound 22 in racemic form was co-crystallized with
KSP as shown in Figure 2, and only the R-antipode
docks in the allosteric binding site. As expected, com-
pound 1 and compound 22 show many similar interac-
14
40
F
15
15
16b
11
O
OH
F
17
9.4
a See Refs. 8,9 for the details of these assays. All compounds listed were
assayed as racemates.
b Approximately 1:1 mixture of diastereomers.
Br
N
N
Me
N
OMe
23
O
compound 19 (38 nM). Deletion of the 5-position substi-
tuent afforded a sixfold decrease in intrinsic potency
concomitant with a fivefold decrease in cell potency.
Incorporation of a trifluoromethyl (21) group was also
deleterious with respect to potency. Finally, changing
the 5-position fluorine to a methyl group afforded com-
pound 22 which gave a twofold and threefold improve-
ment in intrinsic (2.0 nM) and cellular (27 nM) potency
as compared to 2.
(a), (b)
OH
F
Me
N
N
24
Me
O
With these results in hand, a more advanced target
was sought containing the optimal substituents in both
the western aryl ring and the amine substituent. The
synthesis of such a compound is shown in Scheme 2.
Advanced intermediate 23 (see compound 8, Scheme
1) was treated under Stille coupling conditions [tetram-
ethyltin, LiCl, and Pd(PPh3)4], and the Weinreb amide
was subsequently transformed into the methyl ketone
via the action of methyllithium affording 24 in accept-
able yield. Oxidation of the primary alcohol in 24 with
Dess–Martin reagent and reductive amination with
dimethylamine gave 25. Compound 25 was then
separated into its enantiomers using a ChiralPak AD
column to afford the active enantiomer shown as
R-(+)-25.10
(c), (d)
NMe2
F
Me
N
N
25
Me
O
Scheme 2. Synthesis of the 5-methyl derivative on the western aryl ring
(25). Reagents and reaction conditions: (a) Me4Sn, LiCl, Pd(PPh3)4,
DMF, 105 ꢁC, 1 h, 93%; (b) MeLi, THF, 0 ꢁC, 1 h, 64%; (c) Dess–
Martin periodinane, NaHCO3, CH2Cl2, 1 h, then dimethylamine,
NaBH(OAc)3, Et3N, 1,2-dichloroethane, 2 h, 53%; (d) ChiralPak AD
separation.

5680
A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5677–5682
Table 3. Comparison of key data for compounds 1 and 25
tions with the allosteric binding site of the enzyme: (1)
the key hydrophobic interactions of both aryl rings;
(2) the projection of the methyl ketone moiety into a sol-
vent exposed area of the enzyme; and (3) the use of the
unoccupied space above the plane of the core of the
compounds by a tethered amine. By analogy with com-
pound 22 as well as many other Merck KSP inhibitors,
compound 25 was assigned the stereochemistry of R.
With enantiomerically pure 25 in hand we sought to
study its in vitro and in vivo properties.
O
Me
N
N
Me
N
Me
F
O
N
Me
N
N
F
N
Me
25
Me
O
1
O
Property
1
25
In vitro and in vivo comparison of 1 and 25. The key data
for the comparison of compounds 1 and 25 are shown in
Table 3. Compared to compound 1, compound 25 dem-
onstrated excellent activity giving an eightfold increase
in intrinsic potency. Moreover, the KSP IC50 of
0.2 nM represents significant additivity of the amine
and western aryl ring SAR. Compound 25 was particu-
larly potent in cells (3.2 nM) showing approximately
equivalent potency to dihydropyrrolobenoxazine 1
(5.0 nM). The cellular activity also shows additivity with
respect to compounds 17 and 22. Compound 25 main-
tained adequate aqueous solubility at moderate pH.
Next, we studied the hERG inhibition properties, Pgp
susceptibility, and in vivo efficacy of compound 25 as
compared with compound 1.
a
KSP IC50 (nM)
a
Cell EC50 (nM)
1.6
5.0
0.2
3.2
>4
Solubility (pH 4, mg/mL)
MDR ratiob
>4
1.0
1.6
3036
67
c
hERG IC50 (nM)
10,000
100
M2B (ꢀEC99; nM)d
a See Refs. 8,9 for the details of these assays.
b See Ref. 12 for a more detailed discussion of this assay.
c See Ref. 13 for an assay description and relevant references therein.
d See Ref. 14 for a discussion of this assay.
pound 25 was measured in a nude mouse model contain-
ing a human xenograft from an A2780 cell line (in vivo
mitotic arrest assay).14 Compound 25, dosed as a solu-
tion in pH 4 buffer, provides maximum induction of
phospho-histone H3 with a dose of 3.5 mpk and
67 nM plasma concentrations. This level of in vivo po-
tency may translate well in humans as compound 25
has similar protein binding in mouse (93%) and human
(96%) plasma. This result also represents comparable
potency with respect to compound 1 which demon-
strated a similar in vivo effect with a plasma concentra-
tion of 100 nM.
As discussed in the two preceding letters, we have cho-
sen to target compounds that are similarly potent in
Pgp-overexpressing cell lines and normal KB cell lines
with the hope of avoiding resistance due to Pgp-medi-
ated efflux. A detailed analysis of Pgp and KSP inhibi-
tors has been published from our group.1e Compound
25 showed an MDR ratio of 1.6 which compares very
favorably to compound 1 (1.0).12 A second important
design criterion of KSP inhibitors has been to minimize
affinity for hERG with the hope of diminishing the pos-
sibility of cardiotoxicity. Although compound 25 has
approximately a threefold increase in binding affinity
for hERG as compared to compound 1, the eightfold
increase in KSP IC50 actually increases the off-target
window (hERG IC50/KSP IC50) from approximately
6000 to 15,000.13 Finally, the in vivo potency of com-
In conclusion, we have described a novel series of 1,4-
diaryl-4,5-dihydropyrazole KSP inhibitors designed
from the dihydropyrazolobenzoxazine class of KSP
inhibitors. These compounds were synthesized using a
novel approach through a copper-mediated arylation
reaction between pyrazolines and aryl boronic acids.
The SAR between the western aryl ring and sidechain
amine was additive and prompted the synthesis of com-
pound 25 in enantiopure form. Compound 25 was
exceptionally potent with good cellular activity, demon-
strated aqueous solubility at moderate pH, showed good
potency in Pgp-overexpressing cells, and demonstrated
full mitotic arrest in a mouse xenograft model of cancer
with low plasma exposure. Taken together, the last three
letters have described three distinct classes of KSP inhib-
itors that possess good to excellent intrinsic potency,
aqueous solubility, low MDR ratios, limited hERG
affinity, and excellent in vivo potency. Additional series
of KSP inhibitors will be the topic of future publications
and will be described in due course.
Acknowledgments
The authors thank Dr. Chuck Ross, Dr. Harri Ramjit,
and Joan Murphy for high resolution mass spectral
Figure 2. Overlay of X-ray structures of R-22-ADP (cyan) and 1-ADP
(green) in the allosteric KSP binding site.

A. J. Roecker et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5677–5682
5681
7. Jacobson, R. M.; Eur. Pat. Appl., 1985, EP 153127 A2.
analyses. The authors also thank Maricel Torrent for
assistance with the figures in this manuscript.
8. KSP inhibitory activity was measured using a standard
ATPase assay. IC50 values are reported as averages of at
least two determinations; standard deviations are 25–
50%.
9. Mitotic arrest was measured by assessing the mitosis
specific phosphorylation of nucleolin using an antibody-
coated, bead-based assay. In this assay, total nucleolin is
captured on a streptavidin-coated paramagnetic bead
coupled with biotinylated nucleolin monoclonal IgG1
antibody 4E2 (Research Diagnostics, Inc.). Nucleolin
phosphorylation is detected by an antibody complex
consisting of a phospho-specific nucleolin IgM monoclo-
nal antibody, TG3 (Applied NeuroSolutions, Inc.) and a
goat anti-mouse IgM labeled with a ruthenium Tris–
bipyridyl complex (BV-TAG Technology, BioVeris
Corp.). The complex is quantitated via electrochemilumi-
nescence based on the excitation/emission properties of the
Tris–bipyridyl complex using a BioVeris M-series ana-
lyzer. EC50 for KSP-induced nucleolin phosphorylation
was determined after treatment with a compound in a 7-
point, half-log dilution series for 16 h. The values of EC50
are reported as average of at least two independent
determinations; standard deviations are within 25–50%
of EC50 values.
References and notes
1. (a) 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. 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.; 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. Med. Chem. Lett.
2006, 16, 1775; (c) Garbaccio, R. M.; Fraley, M. E.;
Tasber, E. S.; Olson, C. M.; Hoffman, W. F.; Torrent, M.;
Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.;
Lobell, R. B.; Tao, W.; South, V. J.; Yan, Y.; Kuo, L. C.;
Prueksaritanont, T.; Slaughter, D. E.; Shu, C.; Heim-
brook, D. C.; Kohl, N. E.; Huber, H. E.; Hartman, G. D.
Bioorg. Med. Chem. Lett 2006, 16, 1780; (d) 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. Bioorg. Med. Chem. Lett. 2006, 16, 3175; (e) Cox, C.
D.; Breslin, M. J.; Whitman, D. B.; Coleman, P. J.;
Garbaccio, R. M.; Fraley, M. E.; Buser, C. A.; Walsh, E.
S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao, W.;
Abrams, M. T.; South, V. J.; Davide, J. P.; Kohl, N. E.;
Hartman, G. D. Bioorg. Med. Chem. Lett. 2007, 17, 2697;
(f) Coleman, P. J.; Schreier, J. D.; Cox, C. D.; Fraley, M.
E.; Garbaccio, R. M.; Buser, C. A.; Walsh, E. S.;
Hamilton, K.; Lobell, R. B.; Rickert, K.; Tao, W.; Diehl,
R. E.; South, V. J.; Davide, J. P.; Kohl, N. E.; Yan, Y.;
Kuo, L.; Salata, J. J.; Hartman, G. D. Bioorg. Med. Chem.
Lett. 2007, previous letter #1; (g) Garbaccio, R. M.;
Tasber, E. S.; Neilson, L.; Coleman, P. J.; Fraley, M. E.;
Olson, C.; Bergman, J.; Torrent, M.; Buser, C. A.; Walsh,
E. S.; Hamilton, K.; Lobell, R. B.; Tao, W.; South, V. J.;
Diehl, R. E.; Davide, J. P.; Yan, Y.; Kuo, L. C.; Li, C.;
Prueksaritanont, T.; Slaughter, D. E.; Salata, J. J.; Kohl,
N. E.; Huber, H. E.; Hartman, G. D. Bioorg. Med. Chem.
Lett. 2007, previous letter #2.
10. Separation of 25 was performed on a ChiralPak AD
(4.6 mm · 250 mm for analytical; then 250 · 5 cm for
preparatory scale, 99:1 hexanes:EtOH, 0.1% diethylamine
as a modifier) column to afford analytically pure material
(98.5% e.e.). The first isomer to elute was the (À) antipode
(Rt = 6.77 min),
and
it
was
‘inactive’
(KSP
IC50 = 221 nM). The second isomer to elute was the (+)
antipode (Rt = 9.03 min), and it was active (KSP
IC50 = 0.2 nM). Data for 25: NMR (500 MHz, CDCl3):
d = 7.39 (dd, J = 8.0, 1.5 Hz, 1H), 7.32–7.28 (m, 4H),
7.24–7.19 (m, 1H), 6.95 (dd, 12.5, 8.5 Hz, 1H), 6.84–6.78
(m, 1H), 4.38 (dd, J = 11.5, 3.5 Hz, 1H), 4.27 (dd, J = 11.5,
3.5 Hz, 1H), 2.47 (s, 3H), 2.46–2.35 (m, 3H), 2.35 (s, 3H),
2.26–2.19 (m, 1H), 2.22 (s, 6H), 1.58–1.49 (m, 1H), 1.34–
1.25 (m, 1H) ppm. HRMS (ES) calcd M+H for
C23H28FN3O 382.2294. Found: 382.2289.
11. See Ref. 1a for a discussion of the unique allosteric
binding site of KSP. More than 20 structurally related
inhibitors have been crystallized in the allosteric binding
pocket of KSP and all have had the orientation shown at
the relevant stereocenter of 25. See Ref. 1a for the details
of the co-crystallization procedure. The coordinates of the
co-crystal structures have been deposited with RCSB
Protein Data Bank under the access codes 2Q2Y and
2Q2Z.
2. See also, Coleman, P. J.; Mercer, S. P.; Roecker, A. J. WO
2006 068933 A2.
12. Relative to the parental KB-3-1 cells, KB-V-1 cells,
originally derived by culturing KB-3-1 cells in the presence
of the Pgp substrate vinblastine (J. Biol. Chem. 1986, 261,
7762), express >500-fold higher levels of Pgp mRNA and
protein. The compound potency (IC50) for induction of
mitotic arrest was determined by evaluating the levels of
the mitotic marker phospho-nucleolin after 16 h incuba-
tion with the test compound in an 11-point half-log
dilution series. The ratio of IC50 obtained in KB-V-1 cells
vs. that in KB-3-1 cells is defined as the MDR ratio. As a
general guideline, we considered compounds with MDR
ratios <10 to be of interest for their ability to enter and kill
cells that overexpress Pgp. Verapamil, a competitive
inhibitor of Pgp, restores the activity of Taxol and our
KSP inhibitors in the KB-V-1 cell line to nearly that
observed in the parental KB-3-1 line, confirming that Pgp
efflux is responsible for the observed resistance to drug-
mediated mitotic arrest. All compounds presented in this
manuscript have MDR ratios of less than 4.4.
3. Intermolecular cycloadditions of this type are well prec-
edented in the literature, however, the products either give
the undesired regiochemistry for compound 2 or products
that are difficult to convert to compounds similar to 2. See
(a) Shimizu, T.; Hayashi, Y.; Nishio, T.; Teramura, K.
Bull. Chem. Soc. Jpn. 1984, 57, 787; (b) Barluenga, J.;
Fernandez-Mari, F.; Gonzalez, R.; Aguilar, E.; Revelli, G.
A.; Viado, A. L.; Fananas, F. J.; Olano, B. Eur. J. Org.
Chem. 2000, 9, 1773.
4. (a) Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J.;
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13. The hERG IC50 values are reported as averages of at least
two independent determinations and were acquired by
radioligand binding competition experiments using mem-
brane preparations from human embryonic kidney cells
that stably express hERG. For assay details, see: Bilodeau,
M. T. et al. J. Med. Chem. 2004, 47, 6363 and references
therein. It should be noted that the ratios of hERG IC50/
KSP EC50 or hERG IC50/KSP EC99 could also be used to
determine off-target window. Compound 1 has approxi-
mately a twofold better ratio than compound 25 via this
analysis.
14. Athymic nude mice (nu/nu) were xenografted subcutane-
ously with the human ovarian carcinoma cell line A2780 and
the resulting tumors were allowed to reach 200–300 mm3
before mice were surgically implanted with Alzet mini-
pumps (Durect Corporation) filled with the appropriate
KSP inhibitors according to manufacturer’s recommenda-
tions. Prior to the implant, pumps containing KSP inhib-
itors were primed by incubation in 37 ꢁC water bath for 3 h,
so that the pumps would discharge KSP inhibitors at a
constant rate of 8 lL/h after subcutaneous implantation.
Mice were euthanized 22 h post pump implant and blood
and tumors were harvested. Blood was collected in EDTA
Vacutainers and processed for plasma to determine phar-
macokinetics. Tumors were fixed in 10% neutral buffered
formalin and then processed and embedded in paraffin.
Paraffin embedded tumors were sectioned 5 lm thick and
used in a phospho-histone H3 immunohistochemistry assay
designed to determine the percentage of cells blocked in
mitosis compared to control treated tumors. After paraffin
removal, re-hydration, and antigen retrieval, sections were
incubated with anti-phospho-histone H3 (ser10; Upstate).
Following incubation with a biotinylated secondary anti-
body, staining of antigen positive nuclei was accomplished
using avidin:biotin complexed horseradish peroxidase and
diaminobenzidine reagent. Sections were imaged using an
Olympus BX51 microscope with a motorized stage and
Image-Pro Analysis software. Quantization of the sections
was accomplished by measuring the percentage of positively
stained nuclei (black) per unit area.