The discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5

Article abstract of DOI:10.1016/j.bmcl.2010.01.110

Here we describe the discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5 originally found during a high-throughput screening (HTS) campaign sampling our in-house compound collection. The compounds optimized subsequently and characterized herein were potently inhibiting the ATPase activity of Kinesin-5 and also exhibited consistent cellular activity, in that cells arrested in mitosis and apoptosis induction could be observed. X-ray crystallographic data demonstrated that these inhibitors bind in an allosteric pocket of Kinesin-5 distant from the nucleotide and microtubule binding sites. The selected clinical candidate EMD 534085 caused strong growth inhibition in human tumor xenograft models using Colo 205 colon carcinoma cells at doses below 30 mg/kg administered twice weekly without showing severe toxicity as determined by loss of body weight.

Full text of DOI:10.1016/j.bmcl.2010.01.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1491–1495
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines
(HHPQs) as potent and selective inhibitors of the mitotic kinesin-5
b
c
d
e
b
Kai Schiemann a, , Dirk Finsinger , Frank Zenke , Christiane Amendt , Thorsten Knöchel , David Bruge ,
*
Hans-Peter Buchstaller b, Ulrich Emde b, Wolfgang Stähle b, Soheila Anzali f
a MS-RTC-MedChem DA35, Merck Serono, Merck KGaA, Frankfurter Strasse 250, D-64293 Darmstadt, Germany
b MS-RTC-MedChem, Merck Serono, Merck KGaA, Frankfurter Strasse 250, D-64293 Darmstadt, Germany
c MS-ROB-Cellular Pharmacology, Merck Serono, Merck KGaA, Frankfurter Strasse 250, D-64293 Darmstadt, Germany
d MS-ROV-DA-in vivo Pharmacology, Merck Serono, Merck KGaA, Frankfurter Strasse 250, D-64293 Darmstadt, Germany
e MS-RES, Merck Serono, Merck KGaA, Frankfurter Strasse 250, D-64293 Darmstadt, Germany
f PC-RT Computer Modeling, Merck KGaA, Frankfurter Strasse 250, D-64293 Darmstadt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we describe the discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as
potent and selective inhibitors of the mitotic kinesin-5 originally found during a high-throughput screen-
ing (HTS) campaign sampling our in-house compound collection. The compounds optimized subse-
quently and characterized herein were potently inhibiting the ATPase activity of Kinesin-5 and also
exhibited consistent cellular activity, in that cells arrested in mitosis and apoptosis induction could be
observed. X-ray crystallographic data demonstrated that these inhibitors bind in an allosteric pocket of
Kinesin-5 distant from the nucleotide and microtubule binding sites. The selected clinical candidate
EMD 534085 caused strong growth inhibition in human tumor xenograft models using Colo 205 colon
carcinoma cells at doses below 30 mg/kg administered twice weekly without showing severe toxicity
as determined by loss of body weight.
Received 17 December 2009
Revised 19 January 2010
Accepted 20 January 2010
Available online 25 January 2010
Keywords:
Kinesin-5
Eg5 inhibitors
Kinesin spindle protein
KSP
Ó 2010 Elsevier Ltd. All rights reserved.
Anti mitotic agents
SAR
EMD 534085
The process of mitosis is a highly attractive point of therapeutic
intervention in cancer therapy and a variety of antimitotic drugs
are successfully being used in the clinic.1 Especially microtubule
targeting inhibitors are highly efficacious; however, these drugs
are associated with a variety of side effects,2 including peripheral
neuropathies.3 Moreover, the clinical efficacy of antimitotic drugs
has been hampered due to the development of drug resistance.
Therefore, it is of great interest to identify novel antimitotic thera-
peutics with alternate mechanisms of actions to either eliminate or
reduce neurotoxicities as well circumvent drug resistance. Several
potential new targets (e.g., mitotic kinases, kinesin ATPases) have
been identified and are currently in preclinical and clinical devel-
opment by many pharmaceutical companies.4
The mitotic kinesin-5 (KSP, KIF11, Eg5) belongs to this group of
novel antimitotic targets. Kinesins are ATP-dependent motor pro-
teins which transport cargoes along the microtubule or participate
in chromosome or spindle movements.5 Mitotic kinesins function
during mitosis only. The mitotic kinesin-5 is essential for proper
mitotic spindle formation.6 Kinesin-5 interacts with the spindle
and is thought to stabilize the bipolar architecture of the spindle
apparatus.7 The first identified inhibitor against Kinesin-5 was
Monastrol, discovered by the groups of Stuart Schreiber and Tim
Mitchison.8 Analysis of Monastrol’s mode of action has shown that
the activity to hydrolyze ATP is absolutely required for its proper
function.9 The typical phenotype caused by kinesin-5 inhibition
such as M phase arrest with characteristic monoastral spindles in
cancer cells was nicely shown applying EMD 534085 and other
kinesin-5 inhibitors.9c
Here, we describe the discovery and optimization of hexahydro-
2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhib-
itors of the mitotic kinesin-5 originally identified in the high-
throughput screening (HTS). We were able to further optimize
our initially discovered inhibitors to high potency and selectivity
in line with consistent cellular mechanism of action. Protein X-
ray structures of kinesin-5 in complex with ADP and several
HHPQs demonstrate that these inhibitors bind in an allosteric
pocket of Kinesin-5 distant from the nucleotide and microtubule
binding sites.
Small molecule inhibitors of Kinesin-5 were found in a HTS of
our in-house compound collection. An in vitro ATPase assay in
the presence of microtubules measuring the compound’s ability
* Corresponding author. Tel.: +49 6151728139; fax: +49 6151723129.
E-mail address: kai.schiemann@merck.de (K. Schiemann).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.110

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K. Schiemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1491–1495
2
5
protein. The potency dropped when the steric bulkiness was re-
1O
3
4
X=Y=H
1
2
IC50 > 10 µM
IC50 = 300 nM
duced from tert-butyl to isopropyl (14) to ethyl (12) to methyl
(11) and chlorine (18) or—in opposite—was increased (e.g., n-Pr,
13). The CF3 derivative appeared to be equally potent (16). In
agreement with structural information obtained by protein X-ray
crystallography, all attempts to introduce more polar substituents,
like nitriles (19, 20), amines (23), alcohols (24) or even acid func-
tions (22) let to less active or inactive compounds. All efforts to
vary the position of the hydrophobic group in the western part
or to introduce additional substituents also reduced the potency
dramatically. Only fluorine in 7-position was accepted and in-
10
7
10b
9
X
X=tert.-Bu, Y=H
4a
3
4
X=tert.-Bu, Y=F IC50 = 870 nM
X=tert.-Bu, Y=Cl
Y
8
N
IC50 > 10 µM
6
Figure 1. HHPQs as kinesin-5 inhibitors.
to prevent the hydrolysis of ATP to ADP was established, providing
a simple and robust biochemical assay to determine the potency of
enzyme inhibition.10
In an initial HTS screen around 1000 HHPQs out of 232,000
compounds were tested, of which 40 confirmed hits were identi-
fied. A R-group analysis showed clearly that the 9-position of the
HHPQs plays an important role for the activity as indicated for
compound 2 with a tert-butyl group (Fig. 1, IC50 = 300 nM) com-
Table 1
Western part SAR
2
1O
3
R1
10
10b
4
9
4a
pared to the non-substituted compound 1 (IC50 >10 lM). As well,
8
steric restrictions in the phenyl substituent of the molecule were
identified; the potency dropped dramatically by exchanging a flu-
oro by a chloro substituent (compounds 3 and 4).
N
5
7
6
10-30
The HHPQs were readily assembled by modification of the Pova-
rov reaction11 as shown in Scheme 1. The inverse electron-demand
aza Diels–Alder reaction worked well with a wide range of anilines
and aromatic aldehydes with dihydropyran in a one-pot reaction
using 1 equiv of TFA at room temperature. The product mixture
contained 2 diastereomers mainly in favor of the trans-product.
Many publications described this reaction by variation of the
Brønsted12 or Lewis13 acid catalyst and solvent.14 Others induce
the reaction photochemically15 or describe solid-supported16 or
stereoselective approaches17 to the compound class. In our case
the two diastereomers could be easily separated by chromatogra-
phy or crystallization. Since we could show that the trans-isomer
is more stable and potent than the cis-isomer (easily forms com-
pound 9, also isolated in various yields as a side product during
the reaction) the SAR is described for the racemic trans-isomer.
For example, trans-isomer 31 (Table 2) expressing a kinesin-5
activity of IC50 = 40 nM is five times more potent than its corre-
sponding cis-isomer, as well as trans-isomer 44, which is 3 times
more potent than its cis-isomer.
Looking at the SAR shown in Table 1 one can easily see that a
spherical hydrophobic substituent at the C-9 position gives the
most potent compounds in accordance to the protein X-ray struc-
ture of kinesin-5 in complex with EMD 534085 (Fig. 2),18 which
can also be compared to co-crystal structures of several other
inhibitors described in the literature.19 The aromatic portion in
the western part in addition with a non-lipophilic tert-butyl substi-
tuent at C-9 (compound 15, Table 1) fills-up perfectly the hydro-
phobic pocket of the allosteric binding site of the kinesin-5
Entry
R1
Kinesin-5
IC50 (nM)
Proliferation
in HCT116
IC50 (nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
9-Me
9-Et
9-n-Pr
9-i-Pr
9-tert-Bu
9-CF3
9-SF5
9-Cl
9-CN
9-CH2CN
9-OCF3
9-COOH
9-NMe2
9-(CH2)2OH
8-tert-Bu
7-F,9-CF3
7-F,8-Cl
8,9-Di-Me
7,9-Di-Me
8,10-Di-Me
>10,000
420
130
360
130
120
110
910
720
n.d.a
840
370
1800
170
250
280
3400
1400
n.d.a
580
>10,000
n.d.a
8600
>10,000
n.d.a
130
>10,000
500
3700
>10,000
5300
10,000
>10,000
90
540
1600
1500
n.d.a
n.d.a
1250
>10,000
>10,000
a
Not determined.
R1
H
a.
O
+
+
R2
O
NH2
5
6
7
O
O
HO
R1
R1
R1
+
+
N
H
R2
N
H
R2
N
R2
trans-8
cis-8
9
Scheme 1. Synthesis of basic HHPQs. Reagents and conditions: (a) trifluoroacetic
acid (1 equiv), acetonitrile, 0–5 °C?rt, 4 h.
Figure 2. X-ray structure of EMD 534085 in the allosteric binding pocket of
kinesin-5.

K. Schiemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1491–1495
1493
Table 2
Eastern part SAR
ment of the protein, which could be nicely shown by analyzing
the X-ray structures of kinesin-5 in complex several HHPQ inhibi-
tors (Fig. 2), a position was identified where modifications of these
quite small molecules can be done to overcome the mentioned lia-
bilities without loosing the desired activity. The hydroxyl function
of a aza Diels–Alder product using commercially available 3,4-
dihydro-2H-pyran-2-methanol as dienophile—often used in solid-
supported chemistry—was supposed to point directly into the
aqueous environment. However, these compounds comprised an
additional stereogenic center.
O
N
R2
15, 31-50
Entry
R2
Kinesin-5
IC50 (nM)
Proliferation
in HCT116
IC50 (nM)
Surprisingly, in the course of the aza Diels–Alder reaction only
two of the likely four diastereomers (in theory 8 isomers possible)
were formed in the reaction, in most cases in a 1:1 mixture as
shown in Scheme 2. So far, theoretical consideration did not offer
a convincing explanation for this fact. However, as the solubility
of cis-53 is much better than of its diastereomer trans-53, these
two compounds could be easily separated by crystallization.
trans-53 was separated by chiral HPLC into the enantiomeres20
or the two antipodes could be separated on a later stage as diaste-
reomeric salts through crystallization.21 The alcohol function of the
enantiomerically pure compound 54 was then activated by selec-
tive mesylation and subsequently substituted with amines. The
amines for R4 = H were further derivatized to the amides, sulfona-
mides, ureas, and carbamates, as described.
The SAR of these modifications is summarized in Table 3. Due to
poor PK properties both the phenolic systems (65) and the thio-
phene derivatives (69) were abandoned, although these com-
pounds showed a single-digit activity for kinesin-5 and also a
strong activity in the HCT116 proliferation assay.22 Looking at
compounds with R1 = CF3 and R2 = Ph all modifications let to highly
potent derivatives (57–62) with potencies below 20 nM in the ATP-
ase assay. Equally potent are the cyclopropyl (70) and isopropyl
(71) derivatives. Interestingly, a putative analog within this series
to Ispinesib23 (Cytokinetics) was much less active than the other
derivatives (compound 63). The highest potency obtained in the
Kinesin-5 assay and the proliferation assay was found for the 7-F,
9-Br or Cl substituted derivatives (72–76), outreached by the sul-
fonamide 76 with IC50 values in the range of 1 nM.
15
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Ph
3-OH-Ph
4-OH-Ph
120
40
34
95
250
40
32
320
3-OH,4-OH-Ph
3-F, 4-OH-Ph
3-F,4-OMe-Ph
4-(NH-COMe)-Ph
3-NH2-Ph
4-COOH-Ph
2-F-Ph
4-F-Ph
17
19
>10,000
>10,000
2300
>10,000
170
270
350
6800
25
120
300
>10,000
530
1100
>10,000
>10,000
n.d.a
n.d.a
7000
n.d.a
260
970
3-F-Ph
3-Cl-Ph
1800
>10,000
20
130
880
>10,000
1600
2000
n.d.a
n.d.a
2-Thiophenyl
3-Thiophenyl
5-Cl-2-Thiophenyl
2-Imidazolyl
2-Thioazolyl
3-Pyridyl
c-Hexyl
(CH2)4OH
a
Not determined.
creased the potency of the HHPQ kinase-5 inhibitors slightly as
exemplified by combination with the trifluoro methyl group of
compound 26.
In the eastern part of the molecule an aromatic moiety was
shown to be crucial for the activity, for example, c-hexyl (49, Ta-
ble 2) or aliphatic alcohols (50) were inactive. In the protein X-
ray structure (Fig. 2) the phenyl ring positions deeply into the
pocket. In analogy to Monastrol, in which the phenol moiety forms
a hydrogen bond to the carbonyl oxygen of the backbone amide
bond of Glu118, the corresponding derivatives in the HHPQ series
were synthesized. And indeed, the 3-phenolic derivative 31 was
three times more potent than the corresponding unsubstituted
phenylic compound. Interestingly the 4-phenol 32 was equally po-
tent and by increasing the acidity of the phenol by introducing
fluorine in ortho position (34) the activity was increased again by
a factor of 2. Since larger substituents let to inactive compounds
the steric freedom of inhibitors in this part of the allosteric pocket
is quite limited, for example, the methoxy derivative of the highly
potent phenol 34 expressed decreased activity at least by three or-
ders of magnitude. The anilinic function like in 37 was still toler-
ated with some loss of activity but amides or acids were
completely inactive. Even fluorine-substituted phenyl let to
slightly reduced potency (39–41).
The expansion into the aqueous environment did not only im-
prove the potency of the series members, it also improved the sol-
ubility, increased the metabolic stability and offered the chance to
improve the pharmacokinetic properties.
In a low dose PK of EMD 534085 in mice the clearance was
found to be 1.8 L/h/kg on average, the volume of distribution was
7.4 L/kg and the half life around 2.5 h. The bioavailability in high
dose experiments (>10 mg/kg) was always above 50% in mice.
The antitumor activity of EMD 534085 was determined in vivo
using the subcutaneously growing human xenograft Colo205 colon
carcinoma model. Two doses of EMD 534085 (15 mg/kg and
30 mg/kg) were applied twice weekly by ip injection when tumors
reached a mean size of approximately 70 mm3. EMD 534085
caused a dose-dependent antitumor effect, which was resulting
in a complete growth inhibition for the 30 mg/kg dose group (T/
C = 19, p <0.001 Anova test) and in a reduced growth rate for the
lower dose group (T/C = 62). Both doses of EMD 534085 were well
tolerated as no body weight loss was observed (Fig. 3).
All heterocyclic derivatives containing basic function were infe-
rior compared to compound 15. Thiazolyl or furanyl derivatives
showed already sub-
lM activity, whereas thiophene derivatives
especially linked at 2-position (43) was one of the most active
compounds. Further attempts to increase potency of these hetero-
cycles by substitution failed (e.g., 45).
Conversely to the increased potency, the necessity to fill the
hydrophobic protein pocket of the kinesin-5 protein led to poorly
soluble compounds, poor PK properties and metabolic instability.
As the tetrahydropyran ring points towards the aqueous environ-
To evaluate selectivity of our identified inhibitors inhibition of
other available kinesin family members were tested using the
kinesin ATPase endpoint assay format. The superfamily of kine-
sin ATPases consists of more than hundred representatives
which can be ordered into 14 different families. In the human
genome 44 kinesins have been identified. EMD 534085 did not
inhibit any other tested kinesins (BimC, CEN-PE, Chromokinesin,
KHC, KIF3C, KIFC3, MKLP-1, and MCAK) at 1 lM or 10 lM con-
centration. Thus, EMD 534085 appears to selectively inhibit
kinesin-5. However, the list of available and tested kinesins is

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K. Schiemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1491–1495
OH
OH
O
O
OH
R1
R1
R1
H
b.
+
+
+
O
R2
O
R2
N
H
R2
NH2
N
H
5
6
52
trans-53
cis-53
c.
R3
R3
N
N
OR
OH
R5
R4
chiral
chiral
R1
chiral
chiral
R1
O
O
O
O
R1
R1
f.
R4 = H
e.
+
HNR3R4
N
H
R2
N
H
R2
N
H
R2
N
H
R2
56
58-61, 63, 71, 74, 76
EMD 534085
57, 62, 64-70, 72, 73, 75
54 (R=H)
55 (R=SO2CH3)
ent-54
d.
Scheme 2. Synthesis of highly potent enantiomerically pure HHPQs. Reagents and conditions: (b) trifluoroacetic acid (1 equiv), acetonitrile, 0–5 °C?rt, 8 h; (c) HPLC:
ChiralpakÒ AD (ethanol); (d) methane sulfonyl chloride (1 equiv), triethyl amine (2 equiv), methylene chloride, rt, 16 h; (e) HNR3R4 (1 equiv), 1-methyl-2-pyrrolidinone,
100 °C, 15 h; (f) various conditions to form amides, sulfonamides, ureas, and carbamates as illustrated in Table 3.
700
limited and inhibition of other currently inaccessible kinesins
control
cannot be ruled out.
T/C
62
19
600
EMD 53408855**HHCCll,,1155mmgg//kkgg,,qq33--44dd,,ipip
EMD 53408855**HHCCll,,3300mmgg//kkgg,,qq33--44dd,,ipip
In conclusion, we have described the synthesis and properties of
potent and selective kinesin-5 inhibitors in the HHPQ series with
high cellular activity. Starting from a high-throughput screen, we
were able to rapidly identify Kinesin-5 inhibitors with potencies
in the low nanomolar range that possess favorable physical and
biological properties. The challenging synthesis due to the high
number of stereogenic centers was optimized, although the overall
yield is low because of two steps involving diastereomeric and
enantioselective separations. The documented SAR could be nicely
explained with the X-ray structure of kinesin-5 in complex with
EMD 534085, which binds to the allosteric pocket of kinesin-5.
Intraperitonal administration of EMD 534085 enabled significant
systemic exposure in mice leading to a significant tumor growth
reduction without toxic side effects. In combination with a con-
vincing advanced profile and low side effects EMD 534085 was se-
lected for clinical trials.
500
400
300
200
100
0
Mean + SEM
8
10
12
14
16
18
20
22
24
26
days after tumor cell injection
Figure 3. In vivo xenograft model of EMD 534085 in Colo 205 cells.
Table 3
SAR of the optimized enantiomerically pure HHPQs
Entry
R1
R2
R3
R4 or R5
Kinesin-5
IC50 (nM)
Proliferation
in HCT116
IC50 (nM)
57
58
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
tert-Bu
tert-Bu
tert-Bu
c-Pr
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4-OH-Ph
4-F-Ph
Ph
H
H
H
H
H
Me
Ph
H
20
11
8
14
4
4
11
30
110
12
28
25
23
24
29
190
5
28
5
8
4
3
C@ONH(CH2)3OH
C@ON(CH)2NMe2
C@OO(CH)2NMe2
SO2(CH2)2NHMe
SO2(CH2)3NH2
(CH2)2NH2
EMD 534085
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
7
19
65
6
4
58
4
19
5
11
2
3
6
C@O-4-Me-Ph
(CH2)2NH2
–N@CH-N@CH-
H
H
H
Et
H
H
H
H
H
H
H
H
Me
Me
Me
(CH2)2OH
Me
Ph
2-Thiophenyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
i-Pr
SO2(CH2)3NMe2
Me
(CH2)2NHC@OMe
C@O(CH2)3NMe2
(CH2)2OH
SO2(CH2)2NMe2
7-F, 9-Br
7-F, 9-Br
7-F, 9-Br
7-F, 9-Cl
7-F, 9-Cl
7
5
7
2
16
19
1

K. Schiemann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1491–1495
1495
16. Kiselyov, A. S.; Smith, L., II; Virgilio, A.; Armstrong, R. W. Tetrahedron 1998,
7987.
Acknowledgment
17. (a) Akiyama, T.; Morita, H.; Fuchibe, K. J. Am. Chem. Soc. 2006, 128, 13070; (b)
Magesh, C. J.; Makesh, S. V.; Perumal, P. T. Bioorg. Med. Chem. Lett. 2004, 14,
2035; (c) Sundarajan, G.; Prabagaran, N.; Varghese, B. Org. Lett. 2001, 3, 1973.
18. Crystals of Kinesin-5 (amino acids 1–368) in complex with Monastrol were first
obtained by vapor diffusion by mixing equal volumes of reservoir solution
(17–18% w/v PEG3350, 0.23–0.26 M ammonium citrate pH 7.0) and protein
solution (10 mg/ml Kinesin 5, 50 mM PIPES, 2 mM MgCl2, 1 mM EGTA, 1 mM
ATP, 1 mM TCEP, 8 mM Monastrol, pH 6.8). Crystals of Kinesin-5 (amino acids
1–368) in complex with EMD 534085 were subsequently obtained by soaking
crystals of Kinesin-5 in complex with Monastrol for 3 days in reservoir solution
containing 20 mM EMD 534085. X-ray diffraction data were collected at 100 K
at beamline X06SA of the Swiss Light Source (Paul-Scherrer-Institut, 5232
Villigen, Switzerland) to 2.0 Å resolution (R-sym = 0.061, completeness = 88,3%)
in space group C2 with cell dimensions a = 161.0 Å, b = 79.7 Å, c = 65.5 Å,
We thank Dr. Mireille Krier for the illustrations of the inhibitor–
protein-interactions.
References and notes
1. (a) Jordan, M. A.; Wilson, L. Cancer 2004, 4, 253; (b) Pellegrini, F.; Budman, D. R.
Cancer Invest. 2005, 23, 264.
2. Zhou, J.; Giannakakou, P. Curr. Med. Chem. Anticancer Agents 2005, 5, 65.
3. Bergnes, G.; Brejc, K.; Belmont, L. Curr. Top. Med. Chem. 2005, 5, 127.
4. (a) Jackson, J. R.; Patrick, D. R.; Dar, M. M.; Huang, P. S. Nat. Rev. Cancer 2007, 7,
107; (b) Warner, S. L.; Gray, P. J.; Von Hoff, D. D. Semin. Oncol. 2006, 33, 436; (c)
Duhl, D. M.; Renhowe, P. A. Curr. Opin. Drug Discov. Devel. 2005, 8, 431.
5. Kull, F. J. Essays Biochem. 2000, 35, 61.
a
= 90°, b = 96.9°, c = 90°. The structure of Kinesin-5 in complex with EMD
534085 was determined by difference Fourier analysis and refined to an
R-factor of 0.234 and R-free (5% of data) of 0.258. The refined model consists of
two copies of Kinesin-5 (visible residues 16–271 and 288–362), ADP (as a result
of ATP hydrolysis under the experimental conditions) and EMD 534085, related
by a twofold non-crystallographic symmetry axis plus water molecules. The
coordinates have been deposited with RCSB Protein Data Bank under the
accession code 3L9H.
6. (a) Weil, D.; Garcon, L.; Harper, M.; Dumenil, D.; Dautry, F.; Kress, M.
Biotechniques 2002, 33, 1244; (b) Blangy, A.; Lane, H. A.; d’Herin, P.; Harper,
M.; Kress, M.; Nigg, E. A. Cell 1995, 83, 1159.
7. Sawin, K. E.; LeGuellec, K.; Philippe, M.; Mitchison, T. J. Nature 1992, 359, 540.
8. Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.;
Mitchison, T. J. Science 1999, 286, 971.
9. (a) Kapoor, T. M.; Mayer, T. U.; Coughlin, M. L.; Mitchison, T. J. J. Cell. Biol. 2000,
150, 975; (b) Maliga, Z.; Kapoor, T. M.; Mitchison, T. J. Chem. Biol. 2002, 9, 989;
(c) Orth, J. D.; Tang, Y.; Shi, J.; Loy, C. T.; Amendt, C.; Wilm, C.; Zenke, F. T.;
Mitchison, T. J. Mol. Cancer Ther. 2008, 7, 3480.
10. Hydrolysis of ATP was coupled to an ATP regeneration system using an
enzyme-coupling to pyruvate kinase and lactate dehydrogenase. As readout
the consumption of NADH was measured by a decrease in NADH absorption
over time. Tubulin (Cytoskeleton Inc., Denver, Colorado) was polymerized in
PEM buffer (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA pH 6.8) by stepwise
addition of Paclitaxel and spun through a PEM/40% glycerol cushion to separate
the polymerized from non-polymerized tubulin. The Paclitaxel-stabilized
microtubules were washed and resuspended in Kinesin-5 reaction buffer
(20 mM PIPES, 3 mM MgCl2, 1 mM EGTA, 10 mM NaCl, 20 mM KCl, 0.1% BSA pH
19. (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) 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. Bioorg. Med. Chem. Lett. 2006, 16, 3937; (c) 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.
20. For example, putative separation of 1.3 g of trans-53 with R1 = Et and R2 = Ph was
carried out on two successive connected 250 ꢀ 5 cm IM Chiralpak AD columns
with MeOH as mobile phase (flow: 80 mL/min, k = 254 nm). The first isomer to
6.8) supplemented with 10
carried out at room temperature in 140
1 mM DTT, 3 mM ATP, 0.15 mM NADH, 0.48 units pyruvate kinase, 0.47 Units
lactate dehydrogenase, 10.5 g Paclitaxel-stabilized microtubules and 260 ng
l
M Paclitaxel. The Kinesin-5 ATPase reaction was
elute (tR = 8.0 min, 634 mg), the (ꢁ) antipode (½a D20
ꢁ27.1, (c 0.94, MeOH)), was
ꢂ
l
L Kinesin-5 reaction buffer containing
active (IC50 = 100 nM) in the ATPase assay. The second isomer to elute
(tR = 13.8 min, 615 mg), the (+) antipode ((½a D20
ꢂ
+27.4, (c 0.94, MeOH)), was
inactive (IC50 >10 lM). Each isomer was >99% ee by analytical HPLC under the
l
recombinant Kinesin-5. The total concentration of the Kinesin-5 protein in the
reaction was approximately 50 nM. Serial dilutions of compounds were added
to the reaction to determine the inhibitory concentration at which 50% of the
enzymatic activity was inhibited (IC50). DMSO was used as a solvent for
compounds and kept constant in the assay at a concentration of 0.5%. A
reaction without the Kinesin-5 ATPase served as a blank control. The ATPase
reaction was monitored by measuring absorption at 340 nm over time
(Mithras, LB 940 photometer). The reaction velocity was calculated by linear
regression from the time kinetic record.
same conditions. 1H NMR (500 MHz, DMSO) d = 7.47–7.34 (m, 4H), 7.34–7.27 (m,
1H), 6.88 (s, 1H), 6.83 (dd, J = 8.2, 1.9, 1H), 6.51 (d, J = 8.2, 1H), 5.86 (s, 1H), 4.56 (t,
J = 5.8, 1H), 4.48 (d, J = 11.4, 1H), 4.31 (d, J = 2.2, 1H), 3.62–3.53 (m, 1H), 3.47–3.41
(m, 1H), 3.39–3.33 (m, 1H), 2.44 (q, J = 7.6, 2H), 1.88–1.78 (m, 1H), 1.64–1.55 (m,
1H), 1.53–1.37 (m, 1H), 1.34–1.23 (m, 2H), 1.12 (t, J = 7.6, 3H).
21. Schiemann, K.; Emde, U.; Schlueter, T.; Saal, C.; Maiwald, M. PCT
WO2007147480A2, 2007.
22. HCT116 cells (ATCC CCL-247) were cultured in MEM alpha medium with 1 mM
sodium pyruvate, 2 mM glutamine, and 10% fetal bovine serum. 10,000 cells
11. Povarov, L. S.; Grigos, V. I.; Karakhanov, R. A.; Mikhailov, B. M. Br. Acad. Sci. USSR
Ch+ 1964, 163 (Izv. An. SSSR Khi+ 1964, 179).
12. (a) Baudelle, R.; Melnyk, P.; Déprez, B.; Tartar, A. Tetrahedron 1998, 54, 4125;
(b) Yadav, J. S.; Subba Reddy, B. V.; Srinivas, R.; Madhuri, C.; Ramalingam, T.
Synlett 2001, 2, 240; (c) Nagarajan, R.; Magesh, C. J.; Perumal, P. T. Synthesis
2004, 1, 69.
13. (a) Mahesh, M.; Ventakeshwar Reddy, C.; Srinivasa Reddy, K.; Raju, P. V. K.;
Narayana Reddy, V. V. Synth. Commun. 2004, 34, 4089; (b) Ma, Y.; Qian, C.-T.;
Xie, M.-H.; Sun, J. J. Org. Chem. 1999, 64, 6462; (c) More, S. V.; Sastry, M. N. V.;
Yao, C.-F. Synlett 2006, 9, 1399.
were plated in a volume of 100
l
L in 96-well plates and incubated o/n at 37 °C
at 10% CO2. On the next day, the medium was removed and 100
lL of fresh
medium including serial dilutions of compounds were added to the culture
plates and incubation was continued further for 48 h. The concentration of the
compound solvent DMSO was kept constant at 0.5%. At the end of the
compound incubation period the medium was removed and wells were
washed briefly with 200
lL 1ꢀ PBS. 100 lL crystal violet staining solution
(0.5% crystal violet in methanol) was added per well and incubated for 15 min
at room temperature. The staining solution was removed and wells were
washed thoroughly with deionized water. Plates were dried at room
temperature o/n or at 37 °C for 1 h. The remaining dye in the wells was
14. (a) Vidiš, A.; Ohlin, C. A.; Laurenczy, G.; Küsters, E.; Sedelmeier, G.; Dyson, P. J.
ˇ
Adv. Synth. Catal. 2005, 347, 266; (b) Jurcik, V.; Wilhelm, R. Org. Biomol. Chem.
extracted with 200
550 nm.
lL methanol and absorption was determined at 540 or
2005, 3, 1239.
15. Zhang, W.; Guo, Y.; Liu, Z.; Jin, X.; Yang, L.; Liu, Z.-L. Tetrahedron 2005, 61,
23. Sorbera, L. A.; Bolos, J.; Serradell, N.; Bayes, M. Drugs Future 2006, 31, 778.
1325.