Design, synthesis, and biological evaluation of pyrazolopyrimidine- sulfonamides as potent multiple-mitotic kinase (MMK) inhibitors (part I)

Article abstract of DOI:10.1016/j.bmcl.2011.06.129

A novel class of pyrazolopyrimidine-sulfonamides was discovered as selective dual inhibitors of aurora kinase A (AKA) and cyclin-dependent kinase 1 (CDK1). These inhibitors were originally designed based on an early lead (compound I). SAR development has led to the discovery of potent inhibitors with single digit nM IC₅₀s towards both AKA and CDK1. An exemplary compound 1a has demonstrated good efficacy in an HCT116 colon cancer xenograft model.

Full text of DOI:10.1016/j.bmcl.2011.06.129

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5633–5637
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of pyrazolopyrimidine-
sulfonamides as potent multiple-mitotic kinase (MMK)
inhibitors (part I)
Lin Zhang a, , Junhua Fan a, Jer-Hong Chong a, Angela Cesena a, Betty Y. Y. Tam a, Charles Gilson a,
⇑
Christina Boykin a, Deping Wang b, Dikran Aivazian a, Doug Marcotte b, Guangqing Xiao b,
Jean-Yves Le Brazidec a, Jinhua Piao a, Karen Lundgren a, Kevin Hong a, Khang Vu a, Khanh Nguyen a,
Liang-Shang Gan b, Laura Silvian b, Leona Ling b, Min Teng a, Mitchell Reff a, Nicole Takeda a, Noel Timple a,
Qin Wang b, Ron Morena a, Samina Khan a, Shuo Zhao a, Tony Li a, Wen-Cherng Lee b, Arthur G. Taveras b,
Jianhua Chao a
a Biogen Idec, 5200 Research Place, San Diego, CA 92122, United States
b Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of pyrazolopyrimidine-sulfonamides was discovered as selective dual inhibitors of aurora
kinase A (AKA) and cyclin-dependent kinase 1 (CDK1). These inhibitors were originally designed based
on an early lead (compound I). SAR development has led to the discovery of potent inhibitors with single
digit nM IC50s towards both AKA and CDK1. An exemplary compound 1a has demonstrated good efficacy
in an HCT116 colon cancer xenograft model.
Received 4 April 2011
Revised 7 June 2011
Accepted 10 June 2011
Available online 13 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keyword:
Kinase inhibitors
H
H
N
N
Mitotic kinases play important roles in the development and
progression of cancer by acting as key regulators of cell prolifera-
tion. There are three families of serine/threonine mitotic kinases:
aurora kinases (AKA, AKB, and AKC),1 polo-like kinases (PLK1),2
and cyclin-dependent kinases (CDK1).3 These mitotic kinases are
involved in cell cycle arrest and apoptosis of tumor cells, and fre-
quently overexpressed in many tumors. Inhibition of any single
mitotic kinase could lead to inhibition of tumor growth; inhibiting
multiple mitotic kinases (MMK) simultaneously may present a par-
ticularly unique opportunity in cancer therapy. These MMK inhib-
itors can be used as a single agent or in combination with other G2/
M active agents. In our search of small molecule inhibitors target-
ing AKA/AKB and CDK1, we have identified a novel class of pyraz-
olopyrimidine-sulfonamides as potent inhibitors. Herein we report
our results and findings.
N
N
Me
N
H
N
N
N
N
O
H
N
O
O
OH
O
S
N
N
F
Cl
MLN8237
VX680
O
NH
O
N
N
F
OH
OH
O
H2N
F
P
HN
N
NH
O
O
N
N
NH
N
N
O
H
O
S
F
N
R547
AZD1152
O
A number of aurora kinase inhibitors have been reported in lit-
eratures, which have demonstrated anti-tumor activities in preclin-
ical models. A few of them have entered clinical evaluations,
including VX-680, AZD1152, and MLN8237 (Fig. 1).4–6 CDK inhibi-
tors such as R547 have also demonstrated anti-tumor activities
and have been evaluated in clinical trials.7 In our earlier disclosure,8
N
N
N
N
N
N
N
N
HN
N
HN
N
N
HN
N
N
O
O
O
N
O
N
S
O
HN
S
S
S
O
O
O O
I
1a
2
⇑
Corresponding author.
E-mail address: lin.zhang.ge@sbcglobal.net (L. Zhang).
Figure 1. Structures of VX680, MLN8237, AZD1152, R547, I, 1a and 2.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.129

5634
L. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5633–5637
N
OH
N
N
N
a
N
b
S
N
N
H
+
S
N
3
4
5
N
H2N
N
N
N
d
N
S
N
N
c
O
N
N
O
H
7
6
O
S
N
O
H
O
N
S
N
R
N
N
O
N
e
N
N
N
H
N
N
H
N
1a to 1g
8
Scheme 1. Synthesis of pyrazolo-pyrimidines 1. Reagents and conditions: (a) DIAD, PPh3, THF, À70 °C to rt, 65%; (b) oxone, 1:1 MeOH/H2O, rt, 85%; (c) benzene-1,4-diamine,
DMSO, 120 °C, 78%; (d) CH3SO2Cl, DIEA, CHCl3, rt, 87%; (e) RCl, K2CO3, DMF, 70 °C, 30–85%.
fonamides as represented by 2 and 10, which have demonstrated
improved drug properties with comparable inhibitory activities
to the mono-sulfonamide-phenyl class.
N
BocHN
N
N
N
N
N
b, c
S
N
N
O
O
N
H
N
O
a
The synthesis of monosulfonamidophenyl pyrazolo-pyrimidine
series of compounds 1 (a–g) was outlined in Scheme 1. Starting
with thiomethyl pyrazolopyrimidine 3 and cycloheptanol 4, key
building block 5 was prepared using the procedures described by
Adams.9 Oxidation of 5 using OxoneÒ afforded methyl sulfone 6,
which was subsequently converted to phenylaniline intermediate
7 by reacting with 1,4-diaminobenzene under a standard condition
(DMSO, 120 °C). Treatment of 7 with methanesulfonyl chloride un-
der a mild basic condition (DIEA) resulted in monomethylsulfona-
mide 8. Compound 8 has served as a template for SAR exploration.
A variety of N-substituted mono-sulfonamides 1 were prepared by
reacting 8 with appropriate halides in the presence of K2CO3 at an
elevated temperature.
9
6
H
O
S
N
O
N
N
S
N
N
O
N
d
R
N
N
N
N
H
N
N
N
H
N
2
10a to 10f
Scheme 2. Synthesis of 10. Reagents and conditions: (a) 2-Bocamino-5-amino
pyridine, DMSO, 120 °C, 70%; (b) TFA, CH2Cl2, rt, 95%; (c) CH3SO2Cl, DIEA, CHCl3, rt,
86%; (d) RCl, K2CO3, DMF, 70 °C, 30–87%.
The synthetic approach used for the preparation of mono-sul-
fonamide-pyridyl pyrazolo-pyrimidine analogs 10 is summarized
in Scheme 2. Methylsulfone intermediate 6 was reacted with 2-
Boc-amino-5-amino pyridine under elevated temperature to afford
key pyridyl intermediate 9. By removing the Boc protection group
followed by sulfonylation, 9 was converted to mono-methylsulf-
onamide 2. N-Alkylation of 2 with various halides afforded the de-
sired pyridyl analogs 10 (a–f).
To investigate the biochemical and cellular activities of this
compound class, the pyridyl nitrogen was positioned at different
locations within the ring. The 2-pyridyl bissulfonamide 13 can be
compared directly to both the earlier phenyl lead I (Fig. 1) and to
the 3-pyridyl analogs 10 (a–f). The 2-pyridyl 5-bissulfonamide
compound 13 was prepared following the route depicted in
Scheme 3. Displacement of the sulphone moiety in methyl sul-
phone 6 with 2-amino-5-boc-amino pyridine yielded 11. Upon
deprotection and reaction with methanesulfonyl chloride, 3-pyri-
dyl bissulfonamide 13 was obtained.
we mentioned bissulfonamide compounds such as I (Fig. 1) which
were capable of inhibiting both aurora and CDK1 kinases and acting
as an MMK inhibitor.
Compounds from the bissulfonamide class have generally
exhibited poor solubility. To improve physiochemical properties
and inhibitory activities, we modified the bissulfonamide scaffold
of I and discovered that mono-sulfonamide-phenyl pyrazolopyrim-
idine analogs such as 1a improve solubility while maintaining inhi-
bition of both aurora and CDK kinases in biochemical and cellular
assays. Furthermore, our SAR studies led us to mono-pyridyl sul-
N
BocHN
N
N
N
N
b
N
S
N
N
O
N
H
N
O
a
11
6
The biochemical and cellular activities of the N-substituted
mono-sulfonamido-phenyl analogs 1 are summarized in Table 1.
Compounds 1a, 1b, 1c, 1e, 1f, and 1h demonstrate potent enzy-
matic inhibition, with single digit or low double digit nM potency
against AKA and CDK1 in biochemical FRET assays.10 In a G2/M cell
cycle arrest assay, where flow cytometry is used to measure the
degree of G2/M arrest in K562 cells,11 these compounds exhibited
inhibitory IC50s below 100 nM. Compounds 1d and 1g showed
slightly weaker activity in the G2/M arrest assay (IC50 of 300 nM)
or in the MTS phenotypic growth assay compared to the others
O
O
S
N
H2N
O
N
N
N
S
N
N
O
N
N
N
N
N
c
H
N
H
N
12
13
Scheme 3. Reagents and conditions: (a) tert-butyl 6-aminopyridin-3-ylcarbamate,
DMSO, 120 °C, 65%; (b) TFA, CH2Cl2, rt, 90%; (c) CH3SO2Cl (2 equiv), DIEA, CHCl3, rt,
72%.

L. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5633–5637
5635
M)
Table 1
AKA, CDK1, G2M arrest and MTS assay results for compounds 1a–1h
No.
R
AKAa IC50
(
lM)
CDKb IC50
0.023
(l
M)
G2Mc IC50
(
lM)
MTSd IC50
0.160
(l
1a
0.014
0.10
N
O
1b
1c
0.005
0.006
0.005
0.006
0.10
0.10
0.170
0.078
N
O
OH
1d
1e
0.005
0.013
0.09
0.30
0.03
0.240
0.077
0.005
OH
1f
0.020
0.030
0.005
0.023
0.10
0.30
0.060
0.250
NH2
1g
N
1h
0.009
0.006
0.10
0.092
N
a
b
c
AKA inhibition assay, measures inhibition of phosphorylation of a Lats2 substrate protein by aurora kinase A protein.
CDK1 inhibition assay, a FRET-based assay that measures inhibition of phosphorylation of a peptide substrate by CDK1/cyclin B.
G2/M cell cycle arrest assay, a flow cytometric assay for measuring the percentage of cells in G2/M arrest after treatment with mitotic kinase inhibitors.
d
MTS assay for cellular cytotoxicity in HCT116 cells, a phenotypic cellular assay, which measures growth inhibition in cells treated with inhibitors.12
Table 2
AKA, CDK1, G2M arrest and MTS assay results for compounds 10a to 10f, and 13
b
No.
R
AKAa IC50
0.017
(
lM)
CDK IC50
(l
M)
G2Mc IC50
0.3
(
lM)
MTSd IC50
0.210
(l
M)
2
H
0.007
10a
0.150
0.031
2.0
0.500
N
O
10b
10c
0.350
0.260
0.010
0.010
1.0
0.3
0.125
0.220
N
N
10d
10e
0.110
0.180
0.004
0.003
0.3
0.3
0.145
0.119
OH
NH2
10f
13
0.095
>10
0.013
>10
0.3
>3
0.320
N/A
N
a–d
See footnote under Table 1.

5636
L. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5633–5637
Figure 2. X-ray structure of inhibitors 2 and 10e in aurora A (2.8 Å). Graphics were prepared using PyMOL.14
Table 3
ADMET properties of compound 1a and 2
No.
Caco-2 Papp (Â10À6 cm/s) AP to BL
26.5
Efflux ratio
1.1
LM stability %Qh M/R/H
96/94/95
CYP IC50
(
lM)
hERG% inh (10
40
lM)
1a
3A4 = 0.04
2C19 = 0.03
2C9 = 0.001
2D6 and 1A2 >10
3A4, 2D6, 1A2, 2C9, 2C19 >10
2
20.1
1.5
33/30/39
6.5
in this series. In general, analogs with smaller N-substituents (R)
are more potent in both biochemical assays against AKA and
CDK1 and in cellular assays.
9
8
7
Control 10ml/kg IP QDx5
Compound 1a 15mg/kg
Compound 1a 30mg/kg
6
The in vitro activity data of N-substituted mono-sulfonamido-
pyridyl analogs 10 was reported in Table 2. 2-Pyridyl sulfonamides
generally maintained biochemical activities, though weaker than
the corresponding phenyl analogs (1). However, the placement of
nitrogen was important. Moving the nitrogen from the ortho posi-
tion of the sulfonamide (10) to the meta position (13) resulted in a
loss of all activity. This is likely due to the unfavorable electrostatic
repulsion between the hinge and the nitrogen at this position.
In the pyridyl series, the requirement of the N-substituent
group was slightly different from the phenyl series, and the unsub-
stituted (R = H) mode seemed to be preferred. This was particularly
evident in monitoring the AKA inhibitory activity, as compound 2
exhibited the most potent AKA inhibition observed to date. Co-
crystal structures of compounds 2 and 10e with AKA were ob-
tained (Fig. 2, a and b) to provide insight into the SAR observed.13
The N of pyridine in compound 2 can form water-bridged hydrogen
bonds with both R137 and the NH of the sulfonamide. In contrast,
no sulfonamide proton is available for the water-bridged hydrogen
bond network in compound 10e, and the water is also not observed
in electron density maps. This difference in water structure could
explain the 10-fold more potent activity observed for compound 2.
5
4
3
2
1
0
-1
-2
-3
-4
Dose QDx5
12 16 20 24 28 32 36 40
Days Post Tumor Inoculation
Figure 4. Body weight change after administration of 1a in HCT116 nu/nu mouse
models.
We speculated that the diaminophenyl arrangement in com-
pounds like 1 may lead to the generation of reactive metabolites
while in the pyridyl analogs, this problem could be alleviated.
The pyridyl compound 2 also exhibited improved pharmacokinetic
properties (Table 3). Both phenyl compound 1a and pyridyl analog
2 were compared in Caco-2 cell permeability assays,15 liver micro-
some stability assays,16 CYP17 and hERG18 inhibition assays. Com-
pound 1a showed high clearance in mouse, rat and human liver
microsomes. In contrast, compound 2 was quite stable, with a
%Qh in the range of 30–40% hepatic blood flow for mouse, rat
and human liver microsomes. The phenyl compound 1a has meta-
bolic liabilities, as it inhibits CYP450s (3A4, 2C19 and 2C9) and
2000
Control 10ml/kg IP QDx5
Compound 1a 15mg/kg
1500
hERG channels at <10 lM concentrations. The pyridyl compound
Compound 1a 30mg/kg
2, on the other hand, was less encumbered by these issues.
To establish a correlation between in vitro and in vivo activity,
an early lead compound phenyl sulfonamide 1a was evaluated in
an HCT116 mouse colon xenograft mouse model. This compound
demonstrated a 50% tumor growth inhibition without causing
body weight loss after a 21-day treatment regime (IP, daily, Figs.
3 and 4). This result was encouraging as we continue our efforts
in modifying the compounds to achieve better in vitro activity
and an improved metabolic profile, ultimately to realize better
in vivo efficacy.
1000
500
daily dose
10%DMA, 20%cyclodextran
0
12 16 20 24 28 32 36 40
Days
Figure 3. Efficacy results of 1a in HCT116 nu/nu mouse tumor xenograft models.

L. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5633–5637
secondary development reaction,
5637
a
site-specific protease (Development
In conclusion, a novel class of pyrazolopyrimidine-sulfonamides
(phenyl 1 or pyridyl 10) was identified, which shows dual inhibi-
tion of aurora kinase A (AKA) and cyclin-dependent kinase 1
(CDK1). The analogs generally have good enzymatic and cellular
inhibitory activities. The N-substituted mono-sulfonamidophenyl
analogs are more potent in cellular phenotypic and G2/M arrest as-
says than the N-substituted mono-sulfonamidopyridyl analogs but
the pyridyl analogs have better metabolic properties. The pyridyl
nitrogen ortho to the sulfonyl group is required to maintain activ-
ity. A prototypic compound (1a) demonstrated encouraging effi-
cacy in an HCT116 colon cancer xenograft model, which warrants
further SAR development of this class of MMK inhibitors.
Reagent) is added and the development buffer quenches the kinase reaction.
Cleavage disrupts FRET between donor and acceptor fluorophores on the non-
phosphorylated substrate, while uncleaved, phosphorylated substrate
maintains FRET. The fluorescence is measured at 445–520 nm and the
%
phosphorylated is determined from the ratio of coumarin emission to
fluorescein emission. EC50s were determined by GraphPad analysis.
11. G2/M arrest assay: 2 Â 104 K562 cells were treated with varying
concentrations of compound for 24 h. The cells were then stained by
propidium iodide staining buffer (1% sodium citrate, 0.1% Triton X-100,
50 lg/ml propidium iodide in PBS) and analyzed by flow cytometry (5000
events were collected). The Waston model was applied to obtain cell cycle data
(FlowJo) and the concentration at which the ratio of G2 peak to M peak was
greater than 1 was determined to be the minimum concentration at which G2/
M arrest was achieved.
12. Cell proliferation assay: Approximately 2 Â 103 Hct116 or K562 cells were
seeded in a 96 well plate. After the cells were allowed to adhere overnight, cells
were treated with a final concentration of 10–0.01 lM of compound for 5 days.
Acknowledgments
Cell viability was assessed using the tetrazolium-based MTS reagent (Promega)
with absorbance measured at 490 nm. EC50s were determined by GraphPad
analysis.
Special thanks to Dr. Adeela Kamal, Dr. Srinivas R. Kasibhatla,
Dr. Francis J. Burrows, and Dr. Marcus F. Boehm for their contribu-
tions to this program.
13. The coordinates for the X-ray co-crystal structures of compounds 2 and 10e
with AKA at 2.8 Å resolution have been deposited in the PDB. The PDB ID codes
are 3R22 and 3R21, respectively.
14. The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.
15. Bidirectional Transport Assay using Caco-2: Caco-2 cells were purchased from
ATCC. Caco-2 cells were grown with media containing high glucose DMEM
supplemented with 10% FBS and 1% Non-essential amino acids. Caco-2 cells
from passage 25–50 were seeded on a 24-well transwell plate (BD Biosciences,
San Diego, CA). Cells were washed and pre-incubated for 30 min at 37 °C with
transport buffer containing Hank’s Balanced Salt Solution (HBSS), 25 mM
HEPES, and 1% DMSO. Compounds were then added either to the apical
chamber to measure transport to the basolateral chamber (A–B transport) or to
the basolateral chamber to measure transport to the apical chamber (B–A
transport) and incubated for 2 h at 37 °C with gentle shaking.
References and notes
1. Andrews, P. D.; Knatko, E.; Moore, W. J.; Swedlow, J. R. Curr. Opin. Cell Biol. 2003,
15, 672.
2. Steegmaier, M.; Hoffmann, M.; Baum, A.; Lénárt, P.; Petronczki, M.; Krssák, M.;
Gürtler, U.; Garin-Chesa, P.; Lieb, S.; Quant, J.; Grauert, M.; Adolf, G.; Kraut, N.;
Peters, J.; Rettig, W. Curr. Biol. 2007, 17, 316.
3. Benson, C.; Kaye, S.; Workman, P.; Garrett, M.; Walton, M.; de Bono, J. Br. J.
Cancer 2005, 92, 7.
4. Harrington, E. A.; Bebbington, D.; Moore, J.; Rasmussen, R.; Ajose-Adeogun, A.;
Nakayama, T.; Graham, J.; Demur, C.; Hercend, T.; Diu-Hercend, A.; Su, M.;
Golec, J. M. C.; Miller, K. M. Nat. Med. 2004, 10, 262.
5. Wilkinson, R. W.; Odedra, R.; Heaton, S. P.; Wedge, S. R.; Keen, N. J.; Crafter, C.;
Foster, J.; Brady, M. C.; Bigley, A.; Brown, E.; Byth, K.; Barrass, N. C.; Mundt, K.
E.; Foote, K.; Heron, N.; Jung, F. H.; Mortlock, A. A.; Boyle, F. T.; Green, S. Clin.
Cancer Res. 2007, 13, 3682.
6. Zhang, M.; Huck, J.; Hyer, M.; Ecsedy, J.; Manfredi, M. J. Clin. Oncol. 2009, 27,
8553.
7. DePinto, W.; Chu, X.-J.; Yin, X.; Smith, M.; Packman, K.; Goelzer, P.; Lovey, A.;
Chen, Y.; Qian, H.; Hamid, R.; Xiang, Q.; Tovar, C.; Blain, R.; Nevins, T.; Higgins,
B.; Luistro, L.; Kolinsky, K.; Felix, B.; Hussain, S.; Heimbrook, D. Mol. Cancer Ther.
2006, 5, 2644.
8. Kasibhatla, S. R.; Hong, K.; Zhang, L.; Boehm, M. F.; Fan, J.; Le Brazidec, J. Patent
WO2008094602, 2008; Patent WO2008094575, 2008.
16. Test compounds were incubated in Duplicate Matrix MultiScreen mintubes
(Matrix Technologies, Hudson, NH) with liver microsomes (Xenotech, Lenexa,
KS). Each assay is performed in 50 mM potassium phosphate buffer, pH 7.4,
and 2.5 mM NADPH. Compounds were tested at a final assay concentration of
1.0
Compounds were preincubated for 5 min at 37 °C and the metabolic
reactions were initiated by the addition of NADPH. Aliquots of 80 L were
removed from the incubation mix at 0, 5 and 30 min after the start of the
reaction for screening data. Each aliquot was added to 160 L acetonitrile for
lM. The protein concentration in the reaction mix was 1 mg/mL.
l
l
extraction by protein precipitation. These samples were mixed for 1 min by
vortexing, and a volume of the mixture was filtered through wells in 0.25 mm
glass fiber filter plates by centrifugation at 3000 rpm for 5 min. Sample extracts
were analyzed by LC–MS–MS to determine parent compound levels. Percent
loss of parent compound was calculated from the peak area at each time point
to determine the half-life for test compounds (T1/2, min) and clearance (T1/2
expressed as percent hepatic blood flow, %Qh).
9. Adams, J.; Kasparec, J.; Silva, D.; Yuan, C. WO03/029209A2, 2003.
10. FRET based AKA and CDK1 Z-lyte Kinase Assays: AKA and CDK1 kinase assays
were performed using the Z-lyte platform according to manufacturer’s
instructions (Invitrogen). Briefly, in the primary kinase reaction, synthetic Z-
17. Cytochrome P450 inhibition was determined with the use of fluorescent
probes (2C9: 7-methoxy-4-trifluoromethylcoumarin; 2D6: 3-[2-(N, N-diethyl-
N-methylamino)ethyl]-7-methoxy-4-methylcoumarin;
3A4:
7-
lyte Peptide Substrate (final concentration 2
fluorophore (coumarin) and an acceptor fluorophore (fluorescein). The specific
kinase (final concentration 3 nM) then transfers the -phosphate of ATP to a
single tyrosine, serine, or threonine residue on the substrate. Phosphorylation
is suppressed in the presence of a kinase inhibitor (30–0.017 M). In the
lM) is labeled with a donor
benzyloxyquinoline and 7-benzyloxy-4-(trifluoromethyl)-coumarin).
18. hERG assessment used the automated PatchXpress 7000A patch clamp robot
(Molecular Devices).
c
l