Design and optimization of (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones as potent PLK4 inhibitors with oral antitumor efficacy

Article abstract of DOI:10.1016/j.bmcl.2016.08.063

Previous efforts from our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we examined variations on this theme wherein ‘directly-linked’ aromatics, pendant from the indazole core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of 22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.

Full text of DOI:10.1016/j.bmcl.2016.08.063

Accepted Manuscript
Design and optimization of (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3’-
indolin]-2’-ones as potent PLK4 inhibitors with oral antitumor efficacy
Sze-Wan Li, Yong Liu, Peter B. Sampson, Narendra Kumar Patel, Bryan T.
Forrest, Louise Edwards, Radoslaw Laufer, Miklos Feher, Fuqiang Ban, Donald
E. Awrey, Richard Hodgson, Irina Beletskaya, Guodong Mao, Jacqueline M.
Mason, Xin Wei, Xunyi Luo, Reza Kiarash, Erin Green, Tak W. Mak, Guohua
Pan, Henry W. Pauls
PII:
S0960-894X(16)30896-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.063
BMCL 24189
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 July 2016
17 August 2016
19 August 2016
Please cite this article as: Li, S-W., Liu, Y., Sampson, P.B., Patel, N.K., Forrest, B.T., Edwards, L., Laufer, R.,
Feher, M., Ban, F., Awrey, D.E., Hodgson, R., Beletskaya, I., Mao, G., Mason, J.M., Wei, X., Luo, X., Kiarash, R.,
Green, E., Mak, T.W., Pan, G., Pauls, H.W., Design and optimization of (3-aryl-1H-indazol-6-
yl)spiro[cyclopropane-1,3’-indolin]-2’-ones as potent PLK4 inhibitors with oral antitumor efficacy, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.08.063
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design and optimization of (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-
ones as potent PLK4 inhibitors with oral antitumor efficacy
Sze-Wan Li*, Yong Liu, Peter B. Sampson, Narendra Kumar Patel, Bryan T. Forrest, Louise
Edwards, Radoslaw Laufer, Miklos Feher, Fuqiang Ban, Donald E. Awrey, Richard Hodgson,
Irina Beletskaya, Guodong Mao, Jacqueline M. Mason, Xin Wei, Xunyi Luo, Reza Kiarash, Erin
Green, Tak W. Mak, Guohua Pan, Henry W. Pauls*
Campbell Family Institute for Breast Cancer Research, University Health Network, Princess Margaret
Cancer Research Tower, MaRS Centre, 101 College Street, Toronto, Ontario, MG5 1L7, CANADA
* Corresponding authors Tel.: +14165817626, email: swli@uhnresearch.ca (Sze-Wan Li),
henry.pauls@cogeco.ca (Henry W. Pauls)
Keywords: Polo-Like kinase 4, PLK4 inhibitors, spiro[cyclopropane-1,3'-indolin]-2'-ones, (1H-
indazol-6-yl)-methylene)indolin-2-ones, antitumor agent
Graphical abstract

Abstract
Previous efforts by our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-
6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon
IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we
examined variations on this theme wherein directly-linked aromatics, pendant from the indazole
core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series
which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-
ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in
rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of
22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.
The family of Polo-like kinases (PLKs) comprises of five highly conserved
serine/threonine protein kinases. They play a vital role in cell division and checkpoint regulation
of mitosis.1 Overexpression of PLKs found in human tumors coupled with their essential roles in
cell division makes them potential anticancer targets.2 In particular, PLK1 has been studied
extensively with several inhibitors currently in clinical trials.3 In contrast to PLKs 1–3, PLK4 is
structurally divergent, containing only one polo-box domain instead of two.4 PLK4 is essential in
the centriole duplication pathway.5,6,7 It has been shown that PLK4 is up-regulated in breast
cancer, and overexpression of PLK4 is associated with a poor prognosis.8,9,10 Hence, our
laboratory is interested in the discovery of PLK4 inhibitors as novel therapeutic agents.11
Previously, we described PLK4 inhibitors based on an (E)-3-((1H-indazol-6-
yl)methylene)-indolin-2-one core 1 (Figure 1); further elaboration culminated in highly potent

inhibitors such as 2.12 Subsequent optimization yielded a (1R,2S)-2-(1H-indazol-6-
yl)spiro[cyclopropane-1,3'-indolin]-2'-one core4 as that found in compound 3.13,14 Compounds 2
and 3 share a (E)-2-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)vinyl moiety,15,16 pendant from the
indazole, that serves to fill a hydrophobic channel which terminates at the protein/solvent
interface. Based on our PLK4 homology model (Figure 2),17,18 we reasoned that “directly-linked”
aromatics, such as the pyridin-3-yl moiety found in compound 4a, would provide a viable
alternative to populate the hydrophobic channel. Advantages to pursuing the directly-linked
modification include reduced molecular weight and log P, and potentially enhanced selectivity
profile and physicochemical properties. Specifically, we were interested in attenuating the off-
target kinase activity which was observed in the early indazole based compounds12 and
improving the pharmacokinetic profile. Herein, we describe our efforts to develop potent and
selective PLK4 inhibitors with optimized drug-like properties, starting from the (E)-3-((1H-
indazol-6-yl)methylene)indolin-2-one core 1.
Figure 1. Structure of PLK4 Inhibitors.
Directly-linked analogs such as compound 4a (E-isomer) were docked and scored in our
previously described PLK4 homology model.12 As was observed in previous docking
experiments with the vinyl linked system, the Z-isomers scored poorly. For compound 4a (E-
isomer), hydrogen bonding interactions were observed between the indazole and the backbone
carbonyl and NH of Glu90 and Cys92 respectively. Within the PLK4 active site, the indolinone
heteroatoms are involved in hydrogen-bonding interactions to Lys41 and Asp154 (Figure 2A).

Leu89, the gatekeeper residue, is situated between the hinge region and the active site of PLK4.
The pyridyl ring makes close contacts with the wall of the hydrophobic channel (Figure 2B).
Figure 2. Binding mode of compound 4a in the 3COK-based PLK4 homology model. Hydrogen
bonds are shown as dashed lines. (A) H-bonds of the indazole moiety to Glu90 and Cys92 of the
hinge region and H-bonds of the oxindole to Lys41 and Asp154. Leu89 is the gatekeeper residue
of PLK4. (B) An orthogonal view of PLK4 active site surface with compound 4a. The pyridyl
moiety extends toward the enzyme/solvent interface, making hydrophobic interactions with the
side chain of Leu18.
These observations prompted the synthesis of a number of directly-linked 3-((3-aryl-1H-
indazol-6-yl)methylene)indolin-2-ones (Scheme 1). The compounds were prepared using
precursors previously described in the preparation of arylvinyl compounds.12 The heteroaryl
moiety was installed via Suzuki-Miyaura cross coupling using either unprotected 5a or SEM-
protected 3-iodo-1H-indazole-6-carbaldehyde 5b.19 Removal of the SEM group, as necessary,
with TBAF provided the aryl indazoles 6a, which were subjected to Knoevenagel condensation
with oxindoles to afford the desired PLK4 inhibitors 4a–g.20 Note that Knoevenagel
condensation yielded a mixture of E- and Z-geometric isomers wherein the E-isomer was
predominant.

Scheme 1. Synthesis of 3-((3-aryl-1H-indazol-6-yl)methylene)indolin-2-one. Reagents and
Conditions: (i) arylboronic acid pinacol ester, 10 mol% PdCl2(PPh3)2, 7:1 DME/EtOH, 2 M aq.
Na2CO3, 120 °C; (ii) TBAF, THF, 60 °C; (iii) oxindole, piperidine, MeOH, 60 °C.
As shown in Table 1, the introduction of pyridin-3-yl group at the C3-position of
indazole (4a) resulted in a 10-fold improvement in potency as compared to the unsubstituted lead
compound 1. The position of the nitrogen was important in that the 4-pyridyl analog 4b was an
order of magnitude less potent than the 3-pyridyl analog. Pyrrole substitution (4c) also enhanced
potency vis a vis the core 1, but indole 4d and thiazole 4e had reduced PLK4 potency, suggesting
that size and electrostatic factors were important for optimal activity. Our binding model
indicated that the increase in PLK4 activity was due to the hydrophobic interaction between the
side chain of Leu18 and pyridyl ring (Figure 2).12 The model, and results with the vinyl linked
analogs, indicated that further extensions towards the solvent interface, e.g. polar and/or basic
moieties, would be tolerated.17,18 Accordingly, compound 4f, with a (4-methylpiperazin-1-
yl)pyridin-3-yl substituent, was prepared as a potent PLK4 inhibitor. In keeping with established
SAR,12 further increases in potency and selectivity were realized by incorporation of a methoxy
group at the 5' position to give compound 4g.
Table 1. In vitro activity and calculated physicochemical properties of 3-((3-aryl-1H-indazol-6-
yl)methylene)indolin-2-one PLK4 inhibitors
IC50 (µM)
GI50 (μM)
Entrya
R1
H
R2
MDA-MB-
468
PLK4 FLT3
H
H
H
H
0.29
0.049
4.6
0.022
5.0
1
2
0.0014 0.013
0.029 0.027
4a
4b
0.22
0.12
9.9
H
0.064 0.050
7.7
4c

H
0.54
0.49
ND
ND
4d
4eb
4f
H
H
0.072
ND
1.3
1.3
0.013 0.017
OMe 0.0024 0.028
4g
aE/Z mixtures wherein E predominates; bE isomer.
Analogs 4f and 4g exhibited good PLK4 potency while maintaining similar FLT3
selectivity as compound 2; however, the enzyme inhibitory activity did not translate into cancer
cell growth inhibitory activity. Both compounds showed modest inhibition of cell growth in
human breast cancer cell lines. In addition, the inherent E/Z-isomerization of this class of
inhibitors was a significant liability for future drug development.12 Our approach to alleviate this
problem was to replace the double bond linker with a bioisosteric cyclopropane ring.13,14
Compounds were prepared in a convergent manner wherein the installation of the aryl moiety
pendant from the indazole was the final step (Scheme 2). ((1H-Indazol-6-yl)-methylene)indolin-
2-ones 7 were readily obtained via Knoevenagel condensation between oxindoles and 1H-
indazole-6-carbaldehyde.12,19,20 Subsequent cyclopropanation of 7 under Corey-Chaykovsky
conditions21 gave the desired major diastereomer 8, which was readily separable from the minor
diastereomer that did not possess inhibitory activity against PLK4.13 Elaboration of the C3
position of the indazole was achieved by Suzuki-Miyaura cross coupling reaction to give the
racemic 9 in moderate to good yields.

Scheme 2. Synthesis of racemic and optically active 2-(3-aryl-1H-indazol-6-
yl)spiro[cyclopropane-1,3'-indolin]-2'-ones. Reagents and Conditions: (i) Me3SOI, NaH, DMF,
rt; (ii) arylboronic acid pinacol ester, 5 mol% Pd(PPh3)4, 2:1 PhCH3/EtOH, 2 M aq. Na2CO3, 120
°C; (iii) chiral preparative SFC; (iv) NaH, THF, rt; (v) DMSO, t-BuOK, THF, O2 (1 atm), rt; (vi)
I2, K2CO3, DMF, rt.
Application of the bioisostere strategy to one of the best compounds from Table 1, 4f
yielded compound 9a. We were pleased to see that 9a was a potent inhibitor of PLK4 with
improved cancer cell growth inhibitory activity (Table 2). In addition, the replacement of the
alkene with cyclopropane improved selectivity over FLT3. Compound 9a constituted an
excellent starting point for further optimization.
Table 2. In vitro activity and calculated physicochemical properties of racemic 2-(3-aryl-1H-
indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one PLK4 inhibitors
IC50 (μM)
GI50 (μM)
Entry
R1
R2
MDA-MB-
468
PLK4
0.054
FLT3
0.20
H
F
0.77
0.6
9a
9b
9c
0.0059 0.098
OMe 0.0010
OMe 0.0021
0.26
0.32
0.12
0.38
9d
OMe 0.0046
OMe 0.0017
1.20
0.24
0.67
0.89
9e
9f
Previous studies showed that substitution of the oxindole ring in the 5ˈ-position improved
potency;12 we explored the effect of this modification to the current series in more detail. As
shown in Table 2, the replacement of 5ˈ-hydrogen with fluorine and methoxy (9b and 9c
respectively) resulted in a marked improvement in potency against PLK4. Compound 9c, the
most effective, is a nanomolar inhibitor of PLK4 with over two orders of magnitude selectivity

against FLT3 and potently attenuates the growth of MDA-MB-468 cells. Additional variations
on this theme were explored; compounds 9d–f were of similar potency and selectivity but none
were superior to 9c in the cell viability assay.
Our previous publication with styryl-1H-indazol-6-yl-spiro[cyclopropane-1,3'-indolin]-2'-
ones has shown that the 1R,2S enantiomer is substantially more active against PLK4 than the
1S,2R enantiomer.13,14 Hence, we decided to prepare enantiomerically pure adducts using the
chemistry reported.13,14 Enantiomerically pure spiro[cyclopropane-1,3'[3H]indol]-2'(1'H)-ones
12a and 12b were first isolated by preparative chiral SFC separations of racemic 8, wherein R=
OMe (Scheme 2). A stereoselective synthesis was subsequently developed.14 The key reaction
utilized a double SN2 displacement of chiral dimesylate 10 with benzyl-protected oxindole 11 to
give the protected spirocyclopropane with high enantiomeric excess (>98%). Subsequent
oxidative debenzylation22 followed by iodination gave the desired enantiomer 12a in good
yields. The final products (1R,2S stereochemistry) were obtained by Suzuki-Miyaura cross
coupling reaction using conditions employed in the racemic synthesis. The 1S,2R enantiomer 14
was prepared similarly from 12b.
Comparison of 13a and 14 (Table 3) demonstrates a 40 fold increase in the PLK4 activity
of the 1R,2S stereoisomer as compared to the much weaker 1S,2R stereoisomer. The former
recapitulates the cell activity of the racemate 9c whereas the latter showed much weaker
inhibition of cancer cell proliferation. Subsequent analoging efforts were focused on the 1R,2S
enantiomer. Although 13a inhibited PLK4 potently it was not as effective as typical arylvinyl
based analogs in the cell viability assay.14 We postulated that the lower potency could be due to
the moderate permeability of the compounds. To address this concern, we replaced the mildly
basic and polar pyridine of 13a with a phenyl moiety to yield 13b. Both the lipophilicity (log D)

and polar surface area (PSA) of 13b trended in the desired direction (Table 4); we were gratified
to see that this replacement resulted not only in a marked improvement in cell potency, but also
in a substantial enhancement in mouse oral exposure (Table 5). As anticipated, the replacement
of pyridine with phenyl group had no adverse effect on CYP450 inhibition. To evaluate the
kinase selectivity of these compounds as compared to their vinyl counterparts, PLK4 inhibitors
3, 13a and 13b were profiled against a panel of 14 kinases at a screening concentration of 0.1
M. We were pleased to see that replacement of vinyl aryl moiety with a “directly-linked” aryl
group improved the kinase selectivity as illustrated by Figure 3. Compound 13b was further
evaluated against a large panel of kinases. Of the 274 kinases tested, only 15 were inhibited
above 50% at a screening concentration of 0.1 M.
Table 3. In vitro activity and calculated physicochemical properties of (1S,2R)- and (1R,2S)-5ˈ-
methoxy2-(3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one PLK4 inhibitors
IC50 (nM)
AURKA
GI50 (μM)
Entry
R1
PLK4
13
FLT3
17
KDR
36
AURKB
25
MDA-MB-468
MCF-7
5.4
HCC1954
5.9
510
3.7
4f
0.69
0.94
0.45
97
45
44
14,000
9,400
5600
>1,000
10
6.9
6.9
0.12
0.10
0.001
<0.01
3.3
1.4
3.6
13a
>1,000
>1,000
0.015
<0.01
13b
13c
1.2
230
>5000
>1,000
13
0.0058
0.03
8.8
13d
1.5
1.6
200
62
ND
ND
ND
ND
8.5
0.22
0.15
1.42
0.24
13e
13f
>1,000
0.031
0.0063
1.2
30
77
ND
>1,000
>1,000
7
0.068
3.7
1.7
5.4
3.0
5.9
13g
14
6,500
>50,000
2,100

Table 4. Calculated physicochemical data for selected PLK4 inhibitors24
2
3
4g 13a 13b 13c 13d 13e 13f 13g
PSA
77
86
86
86
3.3
5.6
73
4.0
4.7
82
2.6
5.7
82
3.4
4.4
70
4.0
5.2
73
4.8
3.9
94
4.0
4.8
Log D at
pH 7.4
4.5 4.2 3.5
LLE*
4.0 3.9 4.8
*LLE = pIC50 – clog P
Table 5. eADME and murine in vivo data for selected PLK4 inhibitors
Rat PK
(IV 1 mg/kg)
Rat PK
(PO 5 mg/kg)
CYP450 % inh
Microsomal
t1/2 (min)
Mouse PK
(PO 25 mg/kg)
at 1 M
Entr
y
Cmax
(g/mL)
< LOD
ND
ND
0.071
0.71
< LOD
0.11
1.1
AUC
CL
t1/2
(h)
AUC
(g.h/mL)
0.17
0.33
0.094
0.27
0.27
ND
ND
0.31
AUC
mouse/
human
4/24
9/16
4/13
8/20
9/19
>60/59
43/>60
15/>60
28/>60
2C9/2C19/3A4
F%
(L/h.kg)
(g.h/mL)
ND
(g.h/mL)
< LOD
0.25
67/83/55
73/93/76
60/64/19
34//30/6
35/33/3
ND
44/54/-3
37/60/6
41/28/11
6.4
3.1
10.7
3.8
5.1
2.4
1.3
1.6
1.3
ND
ND
1.6
1.4
<1
15
2
10
22
ND
ND
11
11
2
3
4g
13a
13b
13c
13d
13f
13g
ND
ND
0.024
0.14
0.26
ND
ND
0.14
1.83
ND
0.17
3.0
4.1
ND
ND
3.3
0.31
0.13
0.25
0.27
4.6
0.22
<LOD: below limit of detection; ND: not determined.
Figure 3. Heat map showing % inhibition of kinases at a screening concentration of 0.1 M by
PLK4 inhibitors 3, 13a, and 13b.
Modifications of the solubilizing piperazine ring were undertaken with the view to
scanning physiochemical properties in hopes of identifying a compound with improved ADME
properties. We expected potency and selectivity would be maintained since modifications were
limited to the solvent exposed region of the molecules. To assess selectivity these new PLK4
inhibitors were subjected to profiling against four other kinases, namely FLT3, AURKA,

AURKB and KDR. Subsequently, compounds of interest were moved into rodent PK studies.
Removing the N-methyl substituent attached to piperazine resulted in a potent PLK4 inhibitor
13c with much improved microsomal stability (Table 5). The disubstituted piperazine 13d with
higher lipophilicity was prepared to address the potential loss in permeability of the desmethyl
analogs (Table 4);23 potency and selectivity against FLT3 and AURKB were maintained. It
should be noted that the spiro[cyclopropane-1,3'[3H]indol]-2'(1'H)-ones (13a–d, 14) all showed
excellent selectivity against AURKA and KDR. In fact, the KDR selectivity appeared to be
driven by the bioisosteric replacement of the alkene with the cyclopropane linker; consequently
KDR was dropped from our routine selectivity panel. Replacing the piperazine with a 4-fluoro
piperidine gave a potent and selective inhibitor 13e with a mildly basic solubilizing element (pKa
= 7.7).24 However this compound had modest cancer cell growth inhibition. Replacing the
terminal methyl with ethyl (13f) or hydroxyethyl (13g) resulted in compounds with similar PLK4
potency and somewhat attenuated kinase selectivity against FLT3 and AURKB. Nevertheless,
both compounds showed improved human microsomal stability (Table 5) and were taken into
mouse PK studies.
Our ultimate goal was to develop an orally efficacious PLK4 inhibitor. The beneficial
effects of the alkene to cyclopropane transformation are made clear by comparison of
compounds 4g and 13a. Aside from imparting configurational stability, 13a exhibits improved
ligand-lipophilicity efficiency, cell activity, KDR selectivity and reduced CYP inhibition (Tables
4 & 5). Moreover, a 5-fold increase in rat bioavailability was observed. Further improvements in
exposure were seen upon conversion of the pyridyl to phenyl ring. Compound 13b demonstrated
an order of magnitude increase in mouse oral exposure vis a vis 13a, although the increase in rat
bioavailability was modest. The desmethyl analog 13c had much improved microsomal stability

in mouse but poor oral exposure. The addition of flanking methyl groups only marginally
improved the oral exposure (13d). The methyl and ethyl analogs (13b and 13f) possessed the
best combination of properties and hence were selected for in vivo efficacy evaluation.
The in vivo antitumor activity of compounds 13b and 13f was examined with mouse
xenograft studies using the MDA-MB-468 human breast cancer cell line.25,26 Two dosage
regimens (6.25 mg/kg BID and 37.5 mg/kg QD) were chosen for the first xenograft study with
compound 13b. Compound 13b were administered orally to mice for 21 days, and the observed
tumor growth inhibition is summarized in Figure 4. A dose response was seen and compound
13b showed the best response at 37.5 mg/kg QD with a 96% inhibition of tumor growth at day
21. Based on this encouraging result, a high dose of compound 13f was used in a subsequent
xenograft study. Compound 13f was also orally efficacious, albeit to a lesser extent, with a 48%
tumor growth inhibition. Both compounds were tolerated during the course of the experiment
with less than 20% decrease in body weight during the course of the study. The promising results
from in vivo studies provide a strong basis for further development of PLK4 inhibitors as
antimitotic agents.
Figure 4. Tumor volume vs days of treatment with compounds 13b and 13f in an MDA-MB-468
mouse xenograft model (PO dosing)*
350
Control
300
13f - 37.5 mg/kg, QD, P.O.
13b - 6.25 mg/kg, BID, P.O.
250
13b - 37.5 mg/kg, QD, P.O.
200
150
100
50
0
0
4
8
12
16
20
24
Day
*Results are expressed as mean ± standard error (n = 8 per group); p < 0.06 compared to treatment with vehicle.

The use of “directly-linked” aromatic moieties was investigated in the quest of improving
the drug-like properties of PLK4 inhibitors with the (E)-3-((1H-indazol-6-yl)methylene)indolin-
2-one scaffold. Computational modelling suggested such modification should be well tolerated,
and this work led to nanomolar PLK4 inhibitors. Cell growth inhibitory activity and kinase
selectivity were further enhanced by replacing the alkene with a bioisosteric cyclopropane ring.
Marked improvement in oral mouse exposure was achieved by substituting a pyridyl with a
phenyl moiety, pendant from the indazole. Further optimization led to the discovery of
compounds 13b and 13f. Both compounds are orally efficacious in inhibiting tumor growth in
xenograft studies. Compound 13b, for instance, resulted in a 96% inhibition of tumor growth in
an MDA-MB-468 triple-negative breast cancer xenograft model. The results reported herein
illustrate the potential of this class of compounds for cancer therapy.
Acknowledgements
The Princess Margaret Cancer Foundation, the Canadian Institute of Health Research,
and the Canadian Foundation for Innovation are acknowledged for financial support.
References and notes
1 Barr, F. A.; Silljé, H. H. W.; Nigg, E. A. Nat. Rev. Mod. Cell. 2004, 5, 429.
2 Schoffski, P. Oncologist 2009, 14, 559.
3
Frost, A.; Kross, K.; Steinbild, S.; Hedbom, S.; Unger, C.; Kaiser, R.; Trommeshauser, D.;
Munzert, G. Curr. Oncol 2012, 19, 28.
4 Sillibourne, J. E.; Bornens, M. Cell Div. 2010, 5, 25.
5 Bettencourt-Dias, M.; Rodrigues-Martins, A.; Carpenter, L.; Riparbelli, M.; Lehmenn, L.; Gatt,
M .K.; Carmo, N.; Balloux, F.; Callani, G.; Glover, D. M. Curr. Biol. 2005, 15, 2199.

6 Habedanck, R.; Stierhof, Y.-D.; Wilkinson, C. J.; Nigg, E. A. Nat. Cell Biol. 2005, 7, 1140.
7
Kleylein-Sohn, J.; Westendorf, J.; Le Clech, M.; Habedanck, R.; Stierhof, Y.-D.; Nigg, E. A.
Dev. Cell. 2007, 13, 190.
8
Hu, Z.; Fan, C.; Oh, D. S.; Marron, J. S.; He, X.; Qaqish, B. F.; Livasy, C.; Carey, L. A.;
Reynolds, E.; Dressler, L.; Nobel, A.; Parker, J.; Ewend, M. G.; Sawyer, L. R.; Wu, J.; Liu, Y.;
Nanda, R.; Tretiakova, M.; Orrico, A. R.; Dreher, D.; Palazzo, J. P.; Perreard, L.; Nelson, E.;
Mone, M.; Hansen, H.; Mullins, M.; Quackenbush, J. F.; Ellis, M. J.; Olopade, O. I.; Bernard, P.
S.; Perou, C. M. BMC Genomics 2006, 7, 96.
9
van di Vijver, M. J.; He, Y. D.; van 't Veer, L.; Dai, H.; Hart, A. A. M.; Voskuil, D. W.;
Schreiber, G. J.; Peterse, J. L.; Roberts, C.; Marton, M. J.; Parrish, M.; Atsma, D.; Witteveen, A.;
Glas, A.; Delahaye, L.; van der Velde, T.; Bartelink, H.; Rodenhuis, S.; Rutgers, E. T.; Friend, S.
H.; Bernards, R. N. Engl. J. Med. 2002, 347, 1999.
10 Miller, L. D.; Smeds, J.; George, J.; Vega, V. B.; Vergara, L.; Ploner, A.; Pawitan, Y.; Hall, P.;
Klaar, S.; Liu, E. T.; Bergh, J. Proc. Natl. Acad. Sci. 2005, 102, 13550.
11 Mason, J. M.; Lin, D.C.; Wei, X.; Che, Y.; Yao, Y.; Kiarash, R.; Cescon, D. W.; Fletcher, G.
C.; Awrey, D. E.; Bray, M. R.; Pan, G.; Mak, T. W. Cancer Cell. 2014, 26, 163.
12 Laufer, R.; Forrest, B.; Li, S.-W.; Liu, Y.; Sampson, P.; Edwards, L.; Lang, Y.; Awrey, D. E.;
Mao, G.; Plotnikova, O.; Leung, G.; Hodgson, R.; Beletskaya, I.; Mason, J. M.; Luo, X.; Wei,
X.; Yao, Y.; Feher, M.; Ban, F.; Kiarash, R.; Green, E.; Mak, T. W.; Pan, G.; Pauls, H. W. J.
Med. Chem. 2013, 56, 6069.
13
Sampson, P. B.; Liu, Y.; Patel, N. K.; Feher, M.; Forrest, B.; Li, S.-W.; Edwards, L.; Laufer,
R.; Lang, Y.; Ban, F.; Awrey, D. E.; Mao, G.; Plotnikova, O.; Leung, G.; Hodgson, R.; Mason,
J.; Wei, X.; Kiarash, R.; Green, E.; Qiu, W.; Chirgadze, N. Y.; Mak, T. W.; Pan, G.; Pauls, H.
W. J. Med. Chem. 2015, 58, 130.
14
Sampson, P. B.; Liu, Y.; Forrest, B.; Cumming, G.; Li, S.-W.; Patel, N. K.; Edwards, L.;
Laufer, R.; Feher, M.; Ban, F.; Awrey, D. E.; Mao, G.; Plotnikova, O.; Hodgson, R.; Beletskaya,
I.; Mason, J. M.; Luo, X.; Nadeem, V.; Wei, X.; Kiarash, R.; Madeira, B.; Huang, P.; Mak, T.W.;
Pan, G.; Pauls, H. W. J. Med. Chem. 2015, 58, 147.
15 Csandy, G. A.; Laib, R. J.; Filser, J. G. Toxicol. Lett. 1995, 75, 217.
16 Kumar, N.; Windisch, W.; Ammon, H. L. Pharm. Res. 1995, 12, 1708.
17
Liu, J.; Yang, C.; Simpson, C.; DeRyckere, D.; Van Deusen, A.; Miley, M. J.; Kireev, D.;
Norris-Drouin, J.; Sather, S.; Hunter, D.; Korboukh, V. K.; Patel, H. S.; Janzen, W. P.; Machius,
M.; Johnson, G. L.; Earp, H. S.; Graham, D. K.; Frye, S. V.; Wang, X. ACS Med. Chem. Lett.
2012, 3, 129.

18
Kilkiewicz, K. L.; Weinberg, L. R.; Albom, M. S.; Angeles, T. S.; Cheng, M.; Ghose, A. K.;
Roemmele, R. C.; Theroff, J. P.; Underiner, T. L.; Zificsak, G. A.; Dorsey, B. D. Bioorg. Med.
Chem. 2010, 18, 4351.
19 Crestey, F.; Collot, V.; Stiebing, S.; Rault, S. Synthesis 2006, 20, 3506.
20
Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M.; Garaliene,
V.; Farruggia, G.; Masotti, L. Bioorg. Med. Chem .2004, 12, 1121.
21 Corey, E. J.; Chaykovsky, M. J. Am. Chem. Soc. 1965, 87, 1353.
22 Haddach, A. A.; Kelleman, A.; Deaton-Rewolinski, M. V. Tet. Lett. 2002, 43, 399.
23 Chambers, M. S.; Hobbs, S. C.; Graham, M. I.; Watt, A. P.; Fletcher, S. R.; Bakers, R.;
Freedman, S. B.; Patel, S.; Smith, A. J.; Matassa, V. G. Bioorg. Med. Chem. Lett. 1995, 5, 2303.
24
ACD/Structure Designer, version 9, Advanced Chemistry Development, Inc., Toronto, ON,
Canada, 2015.
25 Martin, J. L.; deSilva, H. C.; Lin, M. Z.; Scott, C. D.; Baxter, R. C. Mol. Cancer Therapeutics
2014, 13, 316.
26 Liu, C.-Y.; Hung, M.-H.; Wang, D.-S; Chu, P.-Y.; Su, J.-C.; Teng, T.-H.; Huang, C.-T.; Chao,
T.-T.; Wang, C.-Y.; Shiau, C.-W.; Tseng, L.-M.; Chen, K.-F. Breast Cancer Res. 2014, 16, 431.