The discovery of tetrahydro-β-carbolines as inhibitors of the kinesin Eg5

Article abstract of DOI:10.1016/j.bmcl.2009.11.012

A series of tetrahydro-β-carbolines were identified by HTS as inhibitors of the kinesin Eg5. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of removing potential metabolic liabilities and improving cellular potency.

Full text of DOI:10.1016/j.bmcl.2009.11.012

Bioorganic & Medicinal Chemistry Letters 20 (2010) 157–160
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of tetrahydro-b-carbolines as inhibitors of the kinesin Eg5
*
Paul A. Barsanti , Weibo Wang, Zhi-Jie Ni, David Duhl, Nathan Brammeier, Eric Martin,
Dirksen Bussiere, Annette O. Walter
Global Discovery Chemistry/Oncology and Exploratory Chemistry, Novartis Institutes for Biomedical Research, 5400 Hollis Street, Emeryville, CA 94608, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetrahydro-b-carbolines were identified by HTS as inhibitors of the kinesin Eg5. Molecular
modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a
focus of removing potential metabolic liabilities and improving cellular potency.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 15 September 2009
Revised 30 October 2009
Accepted 5 November 2009
Available online 12 November 2009
Keywords:
Eg5
KSP
Kinesin
Eg5 is a member of the kinesin family of proteins, which hydro-
lyze ATP as they migrate along microtubules. Kinesins play pivotal
roles in chromosome motility, spindle assembly/function, and in
the transport of vesicles and organelles.1 Eg5 (also known as Kine-
sin-5, BimC, KIF11, KSP) is essential in mitosis, and is expressed
primarily in dividing cells. Inhibition of Eg5 blocks centrosome
separation and thereby inhibits cell division. Current anti-mitotic
drugs stop cell division through direct effects on microtubule sta-
bility. Eg5 inhibition, on the other hand, blocks mitosis by prevent-
ing spindle formation without directly affecting microtubule
dynamics. Eg5 inhibitors are specific to dividing cells, and thus lack
the neuronal toxicity of taxanes and vinca alkaloids.2 Eg5 and its
inhibitors have been the subject of several reviews.3 We describe
herein the discovery of potent tetrahydro-b-carboline inhibitors
of Eg5 that were identified from optimization of high-throughput
screening (HTS) hits.
O
1
OEt
O
N
EtO
N
3
R
R
O
NH2
O
N
H
N
H
NH2NH2
3-hydroxybenzaldehyde
R
NH2
AcOH, 100 o
C
EtOH, RT
N
H
n
1.
HO
R
NHBoc
R
n
NH2
NH
N
N
H
O
N
H
O
HOAT , EDC.HCL
Et3N, DMA
Synthesis of the tetrahydro-b-carbolines began with Fisher In-
dole synthesis utilizing the bis-ethoxy acetal 1 which followed
by phthalimide deprotection afforded the tryptamine derivatives
(Scheme 1). Subjection of the tryptamines with aldehydes under
standard Pictet–Spengler conditions afforded the corresponding
tetrahydro-b-carbolines in good yields. Acylation followed by
Boc-deprotection afforded the final compounds in excellent
yields.4
OH
OH
2. 10% TFA / CH2Cl2
Scheme 1. Synthesis of N-acyl-tetrahydro-b-carbolines.
chemotypes, one of which was the tetrahydro-b-carboline (TH-b-
carboline) 2, which had an Eg5 IC50 of 2.5 M (Fig. 1). Shortly there-
l
Concurrent to our internal fast-follower5 Eg5 program, we also
ran an Eg5 HTS assay in order to pursue potentially more diverse
chemotypes. Our primary Eg5 screening assay employed a stan-
dard ATPase assay.6a Our HTS efforts yielded several intriguing
after, it was found that addition of a single methyl group in the aryl
ring to give compound 3 led to a 10-fold increase in Eg5 binding
affinity. In conjunction with our internal Eg5 co-crystallography ef-
forts in our pyrimidinone series, we were successful in obtaining a
co-crystal structure7 of 3, which induces a new pocket as it did for
the pyrimidinone series8 (Fig. 2a). This is consistent with all
reported co-crystal structures in the PDB which show similar
* Corresponding author.
E-mail address: paul.barsanti@novartis.com (P.A. Barsanti).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.012

158
P. A. Barsanti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 157–160
Table 1
R
H3C
SAR of the aryl ring for compounds 5–14
N
N
NH2
N
N
H
H
R
5
O
O
N
NH2
N
H
OH
OH
O
(+)
-
2
µ
µ
4
R=H;
IC50 = 2.5
M
OH
R=CH3; 3 IC50 = 0.2
M
IC50 = 58 nM
Figure 1. Lead series evolution.
Compounda
R
Eg5 IC50
(l
M)
Cell GI50 (lM)
5
6
7
8
9
10
11
12
13
14
H
F
0.63
0.76
0.020
0.011
>25
n.d.
n.d.
0.13
0.10
n.d.
Me
Et
iPr
Ph
Cl
1.7
n.d.
0.039
0.027
4.5
0.525
0.496
n.d.
Br
OH
CF3O
0.2
>2
n.d., not determined.
a
All compounds tested as racemic mixtures.
basic primary amine. Although these interactions were never
confirmed by X-ray, this did lead to a substantial increase in bind-
ing affinity to Eg5.
The combination of ease of synthesis, rapid increase in potency
and the ability to embark on a structure based drug design pro-
gram encouraged us to explore the structure–activity relationship
(SAR) of compounds in this series. It was apparent from early SAR
that hydrophobic groups in the fifth position of the b-carboline nu-
cleus were optimal and followed the trend of Et > Me ꢀ Br > Cl in
binding affinity, however, the Cl and Br had poor cell activity (Table
1). In addition, di-substitution was not well tolerated by the active
site.
With these data in hand, we turned our attention to the effect of
the amine sidechain, the results of which are summarized in Table
2. In varying the length of the sidechain, we found that a two car-
bon tether is optimal in the acyclic series, consistent with model-
ing. In general, a 4–5-fold difference in potency was noted
between the biochemical and cellular assays. Notably, compound
20 demonstrated similar potency in both biochemical and cell as-
says. Deletion of the amine as in compound 15 resulted in a greater
than 10-fold loss in binding affinity. Replacement of the amide car-
bonyl to a sulfonamide linkage such as in compound 19 resulted in
approximately a 10-fold loss in Eg5 binding affinity. The loss in po-
tency observed in capping the basic amine such as in the sulfon-
amide 23 is consistent with the observations from modeling of
the co-crystal structures in that the amine is involved in a key
hydrogen bond.
In order to try to pick up further hydrophobic interactions, var-
ious alkyl substituents were introduced proximal to the amine.
Interestingly, the enzyme distinguishes between the two different
diastereoisomers 24 and 25.
As part of the hit-to-lead process, we sought to identify any is-
sues that might arise from the pharmacokinetic profile, especially
since the series contained a phenol, a well known potential meta-
bolic liability. Indeed, the rat PK of 4 confirmed our suspicions
since it exhibited high clearance and no oral bioavailability (Table
3), whilst the direct phenyl analogue 26 had much improved PK
properties.
Figure 2a. Co-crystal structure of 3 bound to Eg5; Phenol of carboline displaces
sidechain of Tyr211 to a new position. Loop 5 translocates approximately 10 A to
form the top of the pocket.
structural movements in the protein upon binding compounds in
the same allosteric site.9
There is a strong hydrogen bond between the phenolic –OH and
the backbone carbonyl of Glu118, and worth about 10Â in potency
relative to –H or 100Â relative to –Me. Additional possible hydro-
gen bonds from the carboline –NH and amide carbonyl have sub-
optimal geometries and are inconsistent from analog to analog,
suggesting that most of the binding is hydrophobic (Fig. 2b).
Interestingly, although compound 3 was soaked into the crystal
as the racemic mixture, it is the R enantiomer that the protein se-
lects. Following modeling overlays with our potent in-house pyri-
midinone series of Eg5 inhibitors, an amino functionality was
introduced in an attempt to pick up additional hydrogen bonds
to Glu215 or Tyr211, leading to the TH-b-carboline 4 whereby
the acetyl moiety has been chain extended to terminate with a
mainchain
carbonyl,
Glu-116
Leu-140
O
Possible H-bond
Phe239
H-bond
donor
Donor (geometry
suboptimal)
mainchain
carbonyl,
Glu-118
H
N
O
OH
(R)
O
H-N
N
H-bond
mainchain amide
Arg-119
acceptor
(suboptimal geometry)
Leu214
To this end, we investigated phenol replacements (Table 4).
Modeling suggested that cyclohexyl would be a suitable replace-
ment, and indeed whilst better than most, it still exhibited a
Figure 2b. Schematic depicting key interactions in co-crystal structure of 3 bound
in an allosteric site of Eg5.

P. A. Barsanti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 157–160
159
Table 2
Table 4
Sidechain SAR
Phenol replacements
R1
R1
N
N
NH2
R2
N
H
N
H
R2
O
(+)
-
(+)
-
OH
Compounda
R1
R2
Eg5 IC50
0.175
(lM)
Compounda R1
R2
Eg5 IC50
HCT-116 EC50
(l
M)b
(
l
M)b
26
CH3
15
16
17
CH3
0.46
1.4
O
NH2
NH2
27
28
CH3
CH3
11.0
>25
CH3
CH3
0.049
0.020
0.25
0.083
O
O
OCH3
NH2
NH2
NHSO2CH3
18
19
CH3
Cl
0.086
0.186
0.43
O
S
0.256c
O
O
29
30
CH3
>25
>25
NH
O
O
N
20
21
CH3
CH3
0.10
0.9
0.07
2.0d
O
N
Cl
NH
O
N
O
O
31
32
33
Cl
6.6
22
23
Cl
Cl
0.86
>2.0d
0.87e
NH
NHSO2CH3
NH2
0.155
O
O
O
Cl
0.23
0.10
NH2
NH2
24f
25f
Cl
Cl
0.76
8.1
n.d
n.d
CH3
F
a
All compounds tested as racemic mixtures.
n.d., not determined.
a
All compounds tested as racemic mixtures.
See Refs. 6a,b for assay details.
COLO-205 cell line.
KB3.1 cell line.
MDA-MB-435 cell line.
b
c
d
e
f
In order to establish that the observed cellular activity was
indeed through Eg5 inhibition, we initiated several mechanistic
studies. U2OS cells were left untreated or treated with compound
3 at 1 lM for 8 h before being stained with DAPI (DNA) and an
anti-b-tubulin antibody (b-tubulin).
Approximately 1:1 mixture of diastereoisomers.
Representative pictures of normal (bipolar spindle) and abnor-
mal (mono-polar spindle) mitotic figures are shown (Fig. 3). Com-
pound 3 has no effect on microtubules in the interphase, and
demonstrates the mono-polar spindle formation in mitosis which
is the phenotypic signature of Eg5 inhibition.
In summary, we have identified a novel class of small molecule
Eg5 inhibitors. These inhibitors were potent in the biochemical as-
says, and demonstrated activity in cells consistent with the ex-
pected mechanism of action. The phenolic metabolic liability was
replaced with a phenyl group which greatly improved the PK prop-
erties of the series. Further optimization of this series will be re-
ported in due course.
Table 3
Rat PK
Compound
T
(h)
Cl (mL/min/kg)
Vdss (L/kg)
%F
½
4
26
10.7
9.5
171
26
94
18
1
32
10-fold loss in Eg5 binding affinity. The 3-fluorophenyl 33 turned
out to be a reasonable replacement with only a modest decrease
in binding affinity.

160
P. A. Barsanti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 157–160
References and notes
1. Endow, S. A. BioEssays 2003, 25, 1212.
2. Chu, Q. S .; Holen, K. D.; Rowinsky, E. K.; Wilding, G.; Volkman, J. L.; Orr, J. B.;
Williams, D. D.; Hodge, J. P.; Sabry, J. 40th ASCO Annual Meeting, New Orleans,
LA, USA, 2004, 22(14S), Abs 2078.
3. (a) Zhang, Y.; Xu, W. Anti-Cancer Agents Med. Chem. 2008, 8, 698; (b) Jiang, C.;
You, Q.; Li, Z.; Guo, Q. Exp. Opin. Ther. Patents 2006, 16, 1517.
4. For detailed experimental procedures see Wang, W.; Ni, Z.; Barsanti, P.; Pecchi,
S.; Xia, Y.; Brammeier, N.; Treutle, M.; Jazan, E.; Wayman, K.; Dibble, D.; Cheng, J.
WO05070930, 2005.
5. (a) Wang, W.; Lagniton, L. M.; Constantine, R. N.; Burger, M. T. WO04111058,
2004.; (b) Wang, W.; Constantine, R. N.; Lagniton, L. M.; Pecchi, S.; Burger, M. T.;
Desai, M. C. WO04113335, 2004.
6. (a) Purified Eg5 enzyme is put into solution at 3 nM along with 50
lg/ml
purified bovine brain tubulin, 250 M ATP, and inhibitors of Eg5 in a final
l
concentration of 2.5% DMSO. The assay is incubated for 2 h.; (b) The compounds
anti-proliferative activity on cells was assessed by determining the
concentration of inhibitor that results in 50% growth inhibition (GI50, using
CellTiter-GloÒ to measure ATP) after 72 h incubation.
7. The coordinates have been deposited with RCSB Protein Data Bank under
accession code 3K3B.
8. Wang, W.; Constantine, R.; Lagniton, L. US05228002, 2005.
9. (a) 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; (b) 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; (c) Cox, C. D.; Torrent, M.; Breslin,
M. J.; Mariano, B. J.; Whitman, D. B.; Coleman, P. J.; Buser, C. A.; Walsh, E. S.;
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Figure 3. U2OS cells were treated with compound 3 at 1 lM for 8 h. Chromosomes
are visualized using DAPI staining (blue) and microtubules are visualized using b-
tubulin antibodies (red).
Acknowledgments
We thank Vincent Le and Cornelia Bellamacina for their help in
collecting and interpreting the crystallographic data.