Design and synthesis of 2-amino-pyrazolopyridines as Polo-like kinase 1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.08.095

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.

Full text of DOI:10.1016/j.bmcl.2008.08.095

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5648–5652
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 2-amino-pyrazolopyridines as Polo-like kinase
1 inhibitors
Raymond V. Fucini a, , , Emily J. Hanan b, , Michael J. Romanowski c, Robert A. Elling c, Willard Lew b,
*
Kenneth J. Barr b, Jiang Zhu b, Joshua C. Yoburn b, Yang Liu b, Bruce T. Fahr b, Junfa Fan b, Yafan Lu b,
Phuongly Pham b, Ingrid C. Choong b, Erica C. VanderPorten a, Minna Bui b, Hans E. Purkey b,
Marc J. Evanchik a, Wenjin Yang b
a Department of Biology, Sunesis Pharmaceuticals, Inc., 395 Oyster Point Boulevard Suite 400, South San Francisco, CA 94080, USA
b Department of Chemistry, Sunesis Pharmaceuticals, Inc., 395 Oyster Point Boulevard Suite 400, South San Francisco, CA 94080, USA
c Department of Structural Biology, Sunesis Pharmaceuticals, Inc., 395 Oyster Point Boulevard Suite 400, South San Francisco, CA 94080, 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 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors
based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar
inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal
structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 12 June 2008
Revised 25 August 2008
Accepted 26 August 2008
Available online 29 August 2008
Keywords:
2-Amino-pyrazolopyridine
Polo-like kinase (Plk)
SAR
Kinase inhibitor
Polo-like kinases (Plks) belong to a highly conserved family of
serine/threonine kinases that play critical roles during multiple
stages of mitosis.1–3 Four Plks have been identified in mammals
(Plk1–4), each containing an N-terminal catalytic domain and a
C-terminal domain containing one or two highly conserved
sequences called polo box domains (PBDs).1 The best-characterized
human Plk is Plk1. Plk1 is expressed maximally during late G2 and
M phases of the cell cycle and contributes to the regulation of
centrosome maturation, bipolar spindle formation, and
cytokinesis.1–4 Inhibition of Plk1 activity with small molecules
leads to the formation of aberrant mitotic spindle poles, which in
turn triggers the spindle assembly checkpoint (SAC), resulting in
mitotic arrest and strong induction of apoptosis.1,5–7 Plk1 is
overexpressed in a broad range of human tumors and this
overexpression is positively correlated with aggressiveness and
poor prognosis in many cancers.1,4,5 Plk1 is thus considered a good
target for chemotherapeutic intervention.1,4,5
in complex with the kinase domain of zebrafish Plk1 (zPlk1), a
homolog that possesses a single conserved amino acid substitution
(zPlk1 Ile118 = hPlk1 Leu132) for a residue lining the active site
compared to human Plk1.16
The identification of 1 as a Plk1 inhibitor prompted further
investigation into this class of compounds. A SAR study was ini-
tiated, with a particular focus on the phenyl group linked to the
pyrazole nitrogen. Compounds 1–22 were synthesized as shown
in Scheme 1. Commercially available 2,4-dichloro-nicotinic acid
methyl ester was first hydrolyzed to the carboxylic acid and
then converted to acyl chloride 2 by reaction with oxalyl chlo-
ride. Treatment of 2 with copper iodide and methyl lithium at
low temperature yielded methyl ketone 3.8 Cyclization of 3 to
pyrazole
4 was effected through treatment with hydrazine.
Reaction of 4 with aryl halides in the presence of copper iodide,
potassium carbonate, and N,N-dimethylglycine resulted in N-
arylation of the pyrazole ring.9 Standard Buchwald–Hartwig
amination conditions provided the desired compounds 1 and
6–22.10
As part of our drug discovery efforts, we identified 2-amino-
pyrazolopyridine
1 as a moderate inhibitor of Plk1. In this
communication, we describe the synthesis and structure–activity
relationship (SAR) development of this novel chemical series, and
present an X-ray crystal structure of a small-molecule inhibitor
The SAR of these compounds is detailed in Table 1. Moving a
methyl or fluoro group around the phenyl ring indicated that
substitution at the 3-position is preferred. However, the
electron-donating methoxy group is not well tolerated except in
the 4-position. Further exploration of the 3-position confirmed
the preference for relatively small electron withdrawing groups,
with the 3-chloro (compound 15) being the most active as
* Corresponding author. Tel.: +1 650 266 3500; fax: +1 650 266 3505.
E-mail addresses: info@sunesis.com, rfucini@sunesis.com (R.V. Fucini).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.095

R. V. Fucini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5648–5652
5649
Scheme 1. Reagents and conditions: (a) LiOH, THF/water, 25 °C, 1 h, 100%; (b) oxalyl chloride, DMF/DCM, 25 °C, 1 h; (c) CuI, MeLi, THF, À78 °C, 95%; (d) hydrazine, Et3N, DMA,
80 °C, 2 h, 75%; (e) R–I or R–Br, CuI, K2CO3, N,N-dimethylglycine, DMSO, 100 °C, 12–24 h; (f) R2NH2, Pd2dba3, rac-BINAP, NaOtBu, toluene, 100 °C, 2–15 h.
Table 1
tion while extending larger groups at the 5-position in an attempt
to generate hydrogen-bonding interactions with the protein’s cat-
SAR for phenyl substitution of 1a
alytic machinery. A selection of this expanded series of compounds
is shown in Table 2, and their syntheses are depicted in Schemes
2–4.
The bis-substituted phenyl compounds 28–30 were synthesized
from intermediate 4 (Scheme 2). Copper-mediated arylation of 4
provided benzyl alcohol 23. Dess–Martin oxidation to 24 followed
by condensation with the appropriate phosphonate gave the
a,b-
unsaturated ester 25.11 Rhodium-catalyzed hydrogenation pro-
vided intermediate 26. Amination conditions as described above
generated compound 27, and TFA hydrolysis of the tert-butyl ester
yielded compound 28. HATU-mediated amide bond formation
yielded compounds 29 and 30.
Compound
R
Plk1 IC50 (lM)
1
6
7
8
H
1.301
7.685
0.474
4.528
14.451
5.928
0.703
4.516
0.464
3.241
0.121
1.476
0.412
0.977
0.225
0.149
0.641
0.274
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-F
3-F
4-F
3-Cl
3-iPr
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Tritylation of intermediate 4 followed by amination of the pyr-
idine ring yielded 32 (Scheme 3). Subsequent detritylation to 33
and copper-mediated arylation with 1,3-dibromo-5-chlorobenzene
provided compound 22 (an alternate synthesis to that shown in
Scheme 1).9 Palladium-catalyzed Heck coupling with N,N-bis-
Boc-allylamine generated compound 34.12 Rhodium-catalyzed
hydrogenation of the alkene followed by amino-deprotection
yielded desired compound 36.
Compounds 37 and 38 were synthesized from compound 22,
which was derivatized through Suzuki couplings with aryl boronic
acids (Scheme 4).13 Des-methyl compound 39 was synthesized in
the same fashion, starting with 4,6-dichloropyridine-3-
carbaldehyde.
3-CN
3-N(CH3)2
3-SO2CH3
3,5-CH3
3,5-Cl
3-Cl,5-Br
a
See Supplementary data for assay details.
As shown in Table 2, propionic acid 28 was highly active against
compared with isopropyl, cyano, N,N-dimethylamino, or methyl
sulfone groups. Compounds with 3,5-bis-substitution (20–22) also
showed good activity, with bis-methyl compound 22 having 10-
fold better activity than compound 1.
The observation that 3-chloro substitution on the right-hand
phenyl ring provides an improvement in activity, and that 3,5-
bis-substitution is tolerated, led us to maintain 3-chloro substitu-
the Plk1 enzyme (0.021
lM) as was propionic amide 29
(0.032 M). The secondary amide 30 retains most of this activity,
l
as does the propyl-amine 36. Rigidifying the alkyl chain with a
phenyl ring also maintains activity, as long as the hydrogen-bond
donor is in the proper position. For example, benzamide 38 pro-
vides 5-fold better enzymatic activity as compared to acetamide
37, which has a one atom shift in hydrogen-bond donor position.
Table 2
SAR for bis-substitution of phenyl ringa
R1
R2
Plk1 IC50
(lM)
HCS EC50
(l
M)
b
Compound
15
28
29
30
36
37
38
39
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
0.121
0.021
0.032
0.048
0.059
0.207
0.042
0.050
>20
>20
3-Propionic acid
3-Propionamide
3-(N-2-Hydroxyethyl)-propionamide
3-Propyl-amine
Phenyl-2-N-acetamide
2-Benzamide
9.35
9.20
5.80
>20
3.64
2.43
2-Benzamide
a
See Supplementary data for assay details.
Mitotic arrest as measured by doubling of DNA content in High Content Screening (HCS) of HCT116 cells.
b

5650
R. V. Fucini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5648–5652
Scheme 2. Reagents and conditions: (a) (3,5-dichlorophenyl)-methanol, CuI, K2CO3, N,N-dimethylglycine, DMSO, 100 °C, 16 h, 30%; (b) Dess–Martin, DCM, 12 h, 40%; (c)
(diethoxy-phosphoryl)acetic acid tert-butyl ester, LiCl, DBU, THF, 6 h, 78%; (d) Rh/Al2O3, H2, EtOAc/iPrOH, 4 h, 71%; (e) (S)-
a-methylbenzylamine, Pd2dba3, rac-BINAP, NaOtBu,
toluene, 100 °C, 18 h, 13%; (f) TFA/DCM, 1 h, 98%; (g) H2NR, HATU, DIEA, DMF, 2 h, 80–90%.
Scheme 3. Reagents and conditions: (a) trityl chloride, NEt3, DCM, 0–20 °C, 3 h, 89%; (b) (S)-a-methylbenzylamine, Pd2dba3, rac-BINAP, NaOtBu, toluene, 100 °C, 24 h; (c)
HBr, HOAc, 20 °C, 1 h, 39% (2 steps); (d) 1,3-dibromo-5-chlorobenzene, CuI, N,N-dimethylglycine, K2CO3, DMSO, 100 °C, 18 h, 50%; (e) N,N-bis-Boc-allylamine, Pd(OAc)2, P(o-
tol)3, NEt3, DMA, 100 °C, 18 h; (f) Rh/Al2O3, H2, EtOAc/iPrOH, 5 h, 75%; (g) TFA/DCM, 1 h, 90%.
Limited SAR studies were also performed on the left-hand ami-
no group of compound 1 (data not shown). It was determined that
neither the des-methyl-benzylamine nor the (R)-enantiomer of
compound 1 retained any activity. Minimal substitution around
the benzyl ring was tolerated, with most substitution resulting in
loss of activity. While the (S)-a-methyl-benzyl amine could be ex-
changed for a variety of substituted anilines and result in compa-
rable activity, no significant improvements were observed.
Plk1 inhibition leads to a mitotic arrest and cell death, thus we
tested the effects of inhibitors on cell cycle progression and viabil-
ity in the HCT 116 colorectal cancer cell line. Mitotic arrest was
determined as a doubling of DNA content (4 N) and increased lev-
els of phospho-histone H3, a marker of productive entry into mito-
sis. Monastrol, an inhibitor of the mitotic kinesin Eg5 that causes
cells to arrest in mitosis with monopolar spindles, was used as a
reference. As shown in Figure 1, administration of monastrol or
compound 39 resulted in comparable EC50 values for both mitotic
endpoints (Fig. 1a). Fluorescence microscopy imaging of cells trea-
ted with either monastrol or 39 revealed that DNA was organized
in aster-like morphologies characteristic of monopolar spindle and
Scheme 4. Reagent and condition: (a) aryl boronic acid, Pd(PPh3)4, Na2CO3,
toluene/ethanol, 110 °C, 16 h, 28–65%.
Finally, removal of the methyl group on the pyrazole ring is toler-
ated with no loss of activity (compound 39).
Several heterocyclic variations were explored, and it was found
that a variety of substituted thiophene rings may be attached to
the pyrazole 1-nitrogen with activity comparable to that of analo-
gous phenyl compounds (data not shown). All other synthesized
heterocyclic replacements for the phenyl ring were found to result
in decreased activity.

R. V. Fucini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5648–5652
5651
Figure 1. Phenotypic and mechanistic assessment of cellular Plk inhibition. (a) Dose–response curves from cell cycle assays for compound 39 and monastrol.8 EC50 values are
shown in parentheses ( M); (b) Western analysis of B23-nucleophosmin phosphorylation (pB23) as compared to total B23 in HeLa cells. Cells were synchronized with
300 nM nocodazole and treated with DMSO (D), 2 M monastrol (M) or 5 M 39 for 16 h; (c) fluorescence microscopy of Hoechst 3342-stained nuclei shows prometaphase
arrested cells with monopolar spindles in monastrol-treated (M) and 39-treated cells compared to control cells (D).
l
l
l
A co-crystal structure of 38 with zPlk1 further elucidates the
SAR of this series (Fig. 2).16 The pyrazolopyridine ring is sand-
wiched between Phe169 (hPlk1 Phe183) at the bottom of the pur-
ine-binding pocket and Cys53 (hPlk1 Cys67) on the b4 strand in the
back of the G-loop. The pyridine nitrogen forms a hydrogen-bond
with the amide nitrogen of Cys119 (hPlk1 Cys133) (dis-
tance = 3.0 Å) located in the hinge region. Additionally, the proton
of the pyrazolopyridine 2-amino group forms a direct hydrogen-
bonding interaction with the carbonyl of the same amino acid
residue (distance = 2.7 Å). The interaction of 38 with the protein
appears to be stabilized by the tight packing of the 3-chloro group
against a hydrophobic pocket formed by the b-strands on each side
of the G-loop and the side chain of the catalytic lysine 68 (hPlk1
Lys82) on the b5 strand. This interaction explains the SAR for this
position as seen in Table 1. The observed water-mediated hydro-
gen-bonding interaction between the 2-benzamide nitrogen and
Asp180 (hPlk1 Asp194) of the DFG-loop explains the SAR described
in Table 2, where precise placement of a hydrogen-bond donor sig-
nificantly improves inhibitory activity. Additional lipophilic inter-
actions are observed between the terminal benzamide ring of 38
and the bottom of the purine pocket. Co-crystal structures of three
additional analogs with zPlk1 were obtained, all demonstrating
similar interactions (see Supplementary data, Fig. S1).
In conclusion, we have disclosed a novel series of 2-amino-pyr-
azolopyridines that act as inhibitors of Plk1. Key SAR and binding
elements have been identified, and initial hit 1 has been improved
to achieve several compounds with Plk1 enzymatic activity
<50 nM. Moreover, the primary mechanism of this series in cells
has been shown to be that of Plk1 inhibition. Crystal structures
of inhibitors in this series with zPlk1 have confirmed their binding
mode and provided insight to inform future generations of Plk
inhibitors.
Figure 2. Structure of pyrazolopyridine 38 in the zPlk1 active site.
mitotic arrest14 (Fig. 1c). Biochemical activity for 39 is greater than
50-fold selective for Plk1 over isoforms Plk2 and Plk3, and over
150-fold selective over the highly homologous kinase Aurora A
(see Supplementary data, Table S1).
To further support the conclusion that these responses are a re-
sult of Plk inhibition and not another mitotic regulator, we moni-
tored phosphorylation of B23-nucleophosmin, a known substrate
for Plk1.15 Western analysis of nocodazole-synchronized HeLa cells
shows a marked decrease in B23 phosphorylation in 39-treated
cells compared to monastrol-treated or control cells (Fig. 1b).
Together, these data support a mechanism of action for these
2-amino-pyrazolopyridines as functioning through inhibition of
cellular Plk1. As shown in Table 2, several of the more active
Supplementary data
analogs induce mitotic arrest with an EC50 less than 10 lM. These
compounds also exhibit a cytotoxic effect, consistent with Plk1
inhibition, as demonstrated by comparable activities observed in
an MTT assay (data not shown).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.095.

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