Kinesin spindle protein (KSP) inhibitors. Part 6: Design and synthesis of 3,5-diaryl-4,5-dihydropyrazole amides as potent inhibitors of the mitotic kinesin KSP

Article abstract of DOI:10.1016/j.bmcl.2007.07.046

3,5-Diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjust

Full text of DOI:10.1016/j.bmcl.2007.07.046

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5390–5395
Kinesin spindle protein (KSP) inhibitors. Part 6: Design
and synthesis of 3,5-diaryl-4,5-dihydropyrazole amides
as potent inhibitors of the mitotic kinesin KSP
Paul J. Coleman,a,* John D. Schreier,a Christopher D. Cox,a Mark E. Fraley,a
Robert M. Garbaccio,a Carolyn A. Buser,b Eileen S. Walsh,b Kelly Hamilton,b
Robert B. Lobell,b Keith Rickert,b Weikang Tao,b Ronald E. Diehl,b Vicki J. South,b
Joseph P. Davide,b Nancy E. Kohl,b Youwei Yan,c Lawrence Kuo,c
Thomayant Prueksaritanont,d Chunze Li,d Elizabeth A. Mahan,d
Carmen Fernandez-Metzler,d Joseph J. Salatae 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
eDepartment of Cellular Safety, Merck Research Laboratories, PO Box 4, Sumneytown Pike, West Point, PA 19486, USA
Received 1 June 2007; revised 27 July 2007; accepted 30 July 2007
Available online 6 August 2007
Abstract—3,5-Diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These
enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Care-
ful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining
good aqueous solubility.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Small molecule inhibitors of kinesin spindle protein
are anti-mitotic agents that have clinical potential
for the treatment of cancer.1 Unlike anti-mitotic treat-
ments that directly target tubulin (e.g., taxanes, vinca
alkaloids), KSP inhibitors act upon a motor protein
(KSP or Hs Eg5) that drives the separation of centro-
somes during mitosis.2 Inhibition of KSP halts mitosis
affording a characteristic monoastral phenotype which
eventually leads to cell death in transformed cells.3
Compared with current tubulin-targeting agents, KSP
inhibitors may offer distinct advantages in having a
novel resistance profile and a reduced potential for
neurotoxicity. Several KSP inhibitors are currently in
clinical trials to evaluate this mechanism as a potential
cancer therapy.4
Previous publications from this laboratory have de-
scribed efforts to design and synthesize small molecule
KSP inhibitors based on diaryl dihydropyrazole and
dihydropyrrole scaffolds.5 Preferred aryl substitutions
in these two series have been established with consider-
able overlap in structure–activity relationships between
the two. We have recently reported an important ad-
vance in the design of these enzyme inhibitors where
incorporation of a weakly basic amine tethered to the
2-position of the dihydropyrrole core conferred aqueous
solubility, enhanced enzyme inhibitory potency, and
maintained cellular activity in a P-glycoprotein (Pgp)
over-expressing cell line.
Keywords: Cancer; Kinesin spindle protein; KSP inhibitors; Antimit-
otics; Dihydropyrazoles; P-Glycoprotein.
*
An important design consideration for our leading
KSP inhibitors is to maintain cellular potency in
Corresponding author. Tel.: +1 215 652 4618; fax: +1 215 652
7310; e-mail: paul_coleman@merck.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.046

P. J. Coleman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5390–5395
5391
Table 1. Dihydropyrazole Inhibitors: Amides vs. Ureas
chemoresistant cell lines including cell lines that
overexpress Pgp. Pgp is an ATP-dependent efflux
pump that recognizes a broad set of substrates and
reduces cellular concentrations of chemotherapeutic
agents. This efflux transporter is upregulated by the
human MDR1 gene. Overexpression confers a mul-
ti-drug resistance phenotype to cells and may play a
role in clinical resistance to known anti-mitotic
agents.6
Compound KSP
MDR
IC50 (nM) ratioa
a
NH2
F
9
10
11
12
2.2
2.0
0.9
1.4
1204
491
88
F
F
F
N
O
NMe2
NH2
In a recent publication, we disclosed that Pgp trans-
port correlated with amine basicity for dihydropyrrole
inhibitors.5e Indeed, incorporation of b,b-difluorosub-
stitution to provide compound 1 moderates basicity,
preserves aqueous solubility, and maintains potency
in a multidrug resistant cell line (MDR+). However
difluorination of 1 is associated with a general increase
in lipophilicity and modest in vivo activity. In this pa-
per, we describe how dihydropyrazole amides were
modified by appending weakly basic amines in anal-
ogy to the efforts we have reported in the dihydropyr-
role urea series. These pyrazole amides have distinct
advantages with an overall reduced propensity for
Pgp efflux, enhanced physical properties, and an im-
proved in vivo profile.
F
N
N
O
NMe2
NH2
F
N
N
O
Me
Our efforts reported in this manuscript are focused on
dihydropyrazole amides (e.g., 12) as KSP inhibitors
(Table 1). A straightforward synthesis of dihydropy-
razole amides is shown in Scheme 1.7 Commercially
available 3-pentyn-1-ol 2 was protected as a tetrahy-
dropyranyl ether. Lithiation of the alkynyl ether with
n-butyllithium followed by acylation with Weinreb
amide 3 provided adduct 4 in excellent yield. Diphe-
nyl cuprate addition to 4 provided adduct 5 as a mix-
ture of olefin isomers in good yield. Treatment of 5
with hydrazine hydrate in pyridine with microwave
heating followed by cooling to 0 ꢁC and treatment
with acetyl chloride provided the acetylated dihydro-
pyrazole 6. Acidic deprotection of 6 with TsOH
afforded primary alcohol 7 in good overall yield.
Me
N
Me
F
4.4
F
N
N
O
Me
All values are reported for n = 3 or greater with standard deviations
within 25–50% of the reported value.
a See Refs. 9 and 10 for assay details.
Racemic alcohol
7
could be resolved by chiral
MDR ratio is attained when the primary amine in
11 is replaced with a tertiary amine as in 12.11
HPLC to provide the desired 5S-enantiomer.8 Oxida-
tion of the primary alcohol with Dess–Martin peri-
odinane gave an aldehyde that was transformed by
reductive alkylation to provide the KSP inhibitors
11–21.
Based on these data, we refocused our efforts on com-
pounds related to 12 where various tertiary amines
were introduced onto the C-5 side chain. Potency
for these analogs is shown in Table 2. Enzyme po-
tency was maintained across a diverse set of substi-
tuted tertiary amines.
As shown in Table 1, pyrazole urea 10 is equipotent9
to pyrrole urea 9 with an improved MDR ratio. The
MDR ratio is calculated by comparing the cellular po-
tency (G2/M arrest) of a KSP inhibitor in a wild-type
KB-3-1 human epidermoid carcinoma cell line versus
the cellular potency in a Pgp-over-expressing KB cell
line.10 The ratio of these numbers (Pgp+EC50/wt
EC50) is reported as the MDR ratio. In this assay,
Taxol has an MDR ratio of ca. 25 · 103. In our view,
a compound with an MDR ratio <10 is desirable, and
a value of 1 is ideal. Replacing the dimethyl urea with
an acetyl group in 11 maintains potency and further
reduces the MDR ratio. Presumably, as a hydrogen-
bond acceptor, the urea in 10 is a contributor to
Pgp susceptibility. A further improvement in the
The crystal structures of the binary KSP-ADP and
ternary KSP-ADP-inhibitor complexes have been
solved for multiple compounds in the dihydropyrazole
series, including compound 14. These structures reveal
a unique loop, denoted L5, that is flexible and sol-
vent-exposed in the binary structure. The L5-loop
folds down to trap the inhibitor in a surface pocket
in the ternary structure. This novel binding site is dis-
tinct from the ATP and microtubule binding sites and
gives rise to allosteric enzyme inhibition.12 Tethered
tertiary amines such as the morpholine in 14 are
exposed to solvent in the solid state. Consistent with

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P. J. Coleman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5390–5395
F
KSP IC50 = 5.2 nM
CHF2
NH2
Mitotic Arrest EC50 = 22.5 nM
MDR Ratio:
F
(Pgp+Cell EC50/Wt Cell EC50) = 5
2
N
O
NMe2
In Vivo Mitotic Arrest = 99% of
control at 1425 nM (70 mpk)
1
F
F
O
F
Me
O
N
3
OMe
OH
F
(a), (b)
4
2
THPO
(c)
F
O
F
F
Ph
F
OTHP
(d)
N
N
O
Ph
Me
(e)
6
OTHP
5
F
F
(f)
Ph
F
Ph
O
F
NR1R2
OH
N
N
N
N
O
7
Me
Me
8
Scheme 1. General synthesis of 1,4-diaryl-4,5-dihydropyrazole KSP inhibitors. Reagents and conditions: (a) DHP, TsOH; (b) n-BuLi, then 3, THF
ꢁ78 ꢁC, 92%; (c) PhLi, CuBr–DMS, THF, 95%; (d) H2 NNH2–H2O, pyridine, 90 ꢁC, 30 min then cool to 0 ꢁC, AcCl; (e) TsOH, MeOH, then chiral
HPLC 79% for three steps; (f) Dess–Martin periodinane, DCM, 80% R1R2NH; NaBH(OAc)3.
this mode of binding is the fact that increasing the ste-
ric bulk of the tertiary amine had no significant im-
pact on enzyme potency. Cellular potency was
maintained in most instances. Interestingly, single fluo-
rine substitution was tolerated in 19; however, installa-
tion of a second fluorine atom in 20 decreased cell
potency, presumably due to reduced cell permeability
as a result of greater lipophilicity.
graft implants via Alzet osmotic pumps.13 The mice
were then sacrificed after 22 h and the tumors were re-
moved and sectioned. The sections were stained for
phospho-histone H3, an inducible marker of mitotic ar-
rest. Minimal plasma concentrations associated with
maximal mitotic arrest in the tumor were determined.
In this model KSP inhibitor 13 induced maximal mitotic
arrest with a plasma exposure of 56 nM. The concentra-
tion of compound 13 was not measured in the tumor.
Compound 13 has similar protein binding in both mice
(92%) and human (91%) plasma suggesting that com-
pound 13 could provide a similar level of pharmacody-
namic potency in human. This level of in vivo potency
represents a >20-fold improvement over pyrrole urea
1.14
As previously noted in a related series of pyrrole-based
KSP inhibitors,5e the MDR ratio correlated closely with
amine basicity (Table 3) for compounds in this class.
Lowering the pKa (below ꢀ8) of the tertiary amine
causes a significant attenuation in the MDR ratio. Inter-
estingly, these pyrazole amides had a greater tolerance
for basicity than the corresponding pyrrole ureas. KSP
inhibitor 13 possessed excellent intrinsic and cellular
potencies and a low Pgp susceptibility (MDR ra-
tio = 2.5). This compound was chosen for in vivo evalu-
ation in a pharmacodynamic model of G2/M arrest.
A potential limiting feature of 13 and several structur-
ally related tertiary amines from this series was a pro-
pensity to induce QTc prolongation in a canine
cardiovascular model at low micromolar (<10 lM)
exposures.15 The ECG changes associated with 13 were
not predicted by hERG binding (IC50 > 30,000 nM);
however blockade of the IKr current by 13 could be
Aqueous solutions of compound 13 of increasing doses
were administered to nude mice bearing A2780 xeno-

P. J. Coleman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5390–5395
Table 3. Mitotic arrest MDR ratio as a function pKa
5393
Table 2. Side-chain amine SAR
a
Compound
pKa
Mitotic arrest MDR ratio
R
F
16
18
15
14
17
13
10.1
9.4
8.0
7.1
7.1
6.7
31
10
3.4
F
1.7
1.1
2.5
N
N
O
Me
a Values were determined with a Sirius GLpKatitrator, average of n = 3
determinations.
Compound
R
KSP
IC50 (nM)
Cell potencya
(nM)
11
0.9
1.4
2.8
1.8
1.0
1.0
1.8
1.3
1.0
4.0
2.0
2.6
2.3
6.0
11
NH2
functional inhibitor of IKr and efforts were refocused
on compounds related to 11 that lacked a tertiary
amine.
Me
N
Me
12
13
14
15
16b
17
18
19
20
21
In order to attenuate the Pgp efflux issues associated
with primary amine 11, single b-fluorine substitution
was probed proximal to the appended amine. Both
the (5S,20S) and (5S,20R) b-fluoroamine diastereomers
were studied in detail. Preparation of the (5S,20S) dia-
stereomer is shown in Scheme 2. Primary alcohol 7 was
efficiently oxidized to the carboxylic acid with chro-
mium trioxide and transformed to acyl oxazolidinone
22. Diastereoselective fluorination of the sodium eno-
late of 23 with (PhO2S)2NF gave compound 23 with
greater than 20:1 selectivity for the 20S-fluoro substitu-
tion.17 Notably, racemic substituted pyrazole 7 could
be carried through this route and the diastereomers
associated with compound 23 could be separated on
silica gel. Transformation of 23 to amine 24 was
straightforward.
O
N
N
N
O
O
Me
H
2.8
2.4
7.2
3.1
1.8
55
N
H
N
Although both diastereomers were potent KSP inhibi-
tors,18 the 5S,20S diastereomer 24 had superior phar-
macokinetic properties (dog T1/2 = 9 h vs 4.3 h) and
was the subject of additional study. Compound 24
had reduced potential for IKr blockade based on its
N
N
F
potency in
a
functional patch clamp assay
(EC50 > 30 lM). Indeed when evaluated in the same
cardiovascular dog model as compound 13, KSP
inhibitor 24 showed no propensity for ECG interval
changes with exposures as great as 40 lM. When eval-
uated in the mouse xenograft at 3 mpk, compound 24
provided a full mitotic arrest with circulating plasma
levels of 38 nM. As predicted from our previous
observations, potency for compound 24 was main-
tained in a Pgp over-expressing KB cell line (MDR
ratio = 5). Summary data for compound 24 relative
to compound 1 are shown in Table 4.
F
N
N
F
F
We have demonstrated that pyrazole amides such as 24
are potent KSP inhibitors that have a lower intrinsic
susceptibility for Pgp efflux relative to pyrrole ureas such
as 1 with a greater tolerance of basicity on the appended
side chain. Because of this, single b-fluorine substitution
preserves cellular potency in Pgp over-expressing cells
without compromising physical properties and in vivo
activity. Additionally, KSP inhibitor 24 has highly
favorable pharmacokinetics with a plasma half-life of
9 h in dogs. Unlike appended tertiary amines in this ser-
ies, compound 24 does not cause QTc changes in the
Me
N
7.0
OMe
a EC50 for G2/M block in A2780 cell line.
b Approximately 1:1 mixture of diastereomers.
measured in a classical patch clamp assay.16 This activ-
ity reduced the potential safety margins associated with
13. Fortunately, the primary amine 11 was not a potent

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P. J. Coleman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5390–5395
F
F
Ph
O
Me
Ph
O
F
Ph
O
F
(a), (b)
N
OH
N
N
N
N
O
O
Me
Me
7
22
(c)
F
F
F
Ph
O
Me
O
Ph
O
Ph
F
F
F
(d), (e),
(f), (g)
N
NH2
N
N
N
N
O
O
Me
Me
24
23
Scheme 2. Reagents: (a) H5lO6/CrO3, H2O/MeCN, 99%; (b) SOCl2 then (4R,5S)-4-methyl-5-phenyl-2-oxazolidinone/n-BuLi, 79%; (c) NaHMDS,
(PhO2S)2NF, 39% (50% theoretical); (d) LiBH4; (e) MsCl, Et3N; (f) NaN3, DMF, 47% (three steps); (g) Pd/C, H2, 90%.
Table 4. Comparative data for compounds 1 and 24
F
F
CHF2
NH2
F
Ph
Ph
NH2
F
F
N
N
O
N
O
NMe2
Me
1
24
KSP IC50
Mitotic arrest (EC50
5.2 nM
22.5 nM
1425 nM
5.2
0.82 nM
3.4 nM
)
In vivo mitotic arrest (EC50
)
38 nM
5.2
T1/2 = 9.0 h Vdss = 6.3 L/kg Cl = 11.1 mL/min/kg
MDR ratio
Dog PK (iv, 0.25 mpk)
T1/2 = 3.2 h Vdss = 2.8 L/kg Cl = 16.5 mL/min/kg
E.; Arrington, Kenneth L.; Lobell, Robert B. Mol. Cell.
Biol. 2007, 27, 689.
4. (a) Jackson, J. R.; Patrick, D. R.; Dar, M. M.; Huang, P.
dog. Additional studies on related dihydropyrazole-
based KSP inhibitors will be described in the following
two papers.
´
S. Nat. Rev. Cancer 2007, 7, 107; (b) Sorbera, L. A.; Bolos,
N.; Serradell, N.; Bayes, M. Drugs Future 2006, 31, 778;
´
(c) Duhl, David M.; Renhowe, Paul A. Curr. Opin. Drug
Discov. Devel. 2005, 8, 431.
Acknowledgments
5. (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.
The authors thank Dr. Chuck Ross, Dr. Harri Ramjit,
and Joan Murphy for high resolution mass spectral
analyses. We thank Matt Zrada for determining pKa
values. We also acknowledge Lazlo Kiss for high-
throughput electrophysiology studies.
References and notes
1. Miglarese, Mark R.; Carlson, Robert O. Expert Opin.
Investig. Drugs 2006, 15, 1411.
2. Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R.
W.; Schreiber, S. L.; Mitchison, T. J. Science 1999, 286,
971.
3. Tao, Weikang; South, Victoria J.; Diehl, Ronald E.;
Davide, Joseph P.; Sepp-Lorenzino, Laura; Fraley, Mark

P. J. Coleman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5390–5395
5395
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.; Coleman, P. J.; Garbaccio, R. M.;
Fraley, M. E.; Zrada, M. M.; Buser, C. A.; Walsh, E. S.;
Hamilton, K.; Lobell, R. B.; Tao, W.; Abrams, M. T.;
South, V. J.; Huber, H. H.; Kohl, N. E.; Hartman, G. D.
Bioorg. Med. Chem. Lett. 2007, 17, 2697.
300 mm3 before mice were surgically implanted with Alzet
mini-pumps (Durect Corporation) filled with the appro-
priate KSP inhibitors according to manufacturer’s recom-
mendations. Prior to the implant, pumps containing KSP
inhibitors 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 implanta-
tion. 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
pharmacokinetics. 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 immunohisto-
chemistry 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 antibody, 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 Anal-
ysis software. Quantization of the sections was accom-
plished by measuring the percentage of positively stained
nuclei (black) per unit area. All procedures performed on
these animals were in accordance to established guidelines
and were reviewed and approved by the Institutional
Animal Care and Use Committee (IACUC).
6. Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-
Genthe, C.; Gottesman, M. M. Nat. Rev. Drug Disc. 2006,
5, 219.
7. Cox, C. D.; Breslin, M. J.; Mariano, B. J. Tetrahedron
Lett. 2004, 45, 1489.
8. The enantiomers were resolved by preparative HPLC with
a 10 cm Chiralpak AD column using an eluant of 92%
hexanes (with 0.1% diethylamine), 4% MeOH, and 4%
EtOH at 150 mL/min. The first peak to elute was the
inactive (R)-isomer and the second to elute was the (S)-
isomer. The absolute configuration was assigned by X-ray
crystallography of
7 + ADP + KSP.
a
ternary complex of (S)-
9. KSP Inhibitory potency was measured with an ATPase
assay. For details, see Breslin, M. J.; Coleman, P. J.; Cox,
C. D.; Culberson, C. J.; Hartman, G. D.; Mariano, B. J.;
Torrent, M. PCT WO 079973 A2, 2003.
10. 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
versus 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.
11. Passive permeability differences may also play a role in the
observed differences in the MDR ratios. Tertiary amines
such as 12 have high Papp values (31.8 · 10ꢁ6 cm/s) versus
primary amines such as 11 (12 · 10ꢁ6 cm/s). For a full
discussion, see Ref. 5e.
12. Yan, Y.; Sardana, V.; Xu, B.; Homnick, C.; Halczenko,
W.; Buser, C. A.; Schaber, M.; Hartman, G. D.; Huber, H.
E.; Kuo, L. C. J. Mol. Biol. 2004, 335, 547.
14. A partial explanation for the improved in vivo activity of
compound 13 versus 1 may be related to nonspecific
binding in plasma. Performing the ATPase assay in the
presence of human serum (+30%) increases the IC50 for
compound 1 from 5 to 84 nM. When the same serum shift
experiment is done with compound 13, the IC50 increases
from 2 to 20 nM.
15. Blockade of the hERG channel is associated with drug-
induced prolongation of the QTc interval of the ECG, an
observation that is linked to potentially fatal ventricular
arrhythmias (Torsades-de-Pointes). The hERG IC50 values
were determined by radioligand competition experiments
using membrane preparations from HEK cells that stably
express hERG. For assay details, see: Bilodeau, M. T.
et al. J. Med. Chem. 2004, 47, 6363, and references
therein.
16. A Herg patch clamp IC50 for compound 13 was deter-
mined at 7 lM. For a description of the assay, see: Wang,
J.; Della Penna, K.; Wang, H.; Karczewski, J.; Connolly,
T. M.; Koblan, K. S.; Bennett, P. B.; Salata, J. J. Am. J.
Physiol. 2003, 284, H256.
17. Davis, F. A.; Kasu, P. V. N. Tetrahedron Lett. 1998, 39,
6135.
18. The 5S,20R diastereomer has a KSP IC50 = 2.6 nM and
had an EC50 (A2780) = 10.5 nM. Plasma half-life in the
dog was 4.3 h.
13. 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–