Identification of novel, potent and selective inhibitors of Polo-like kinase 1

Article abstract of DOI:10.1016/j.bmcl.2011.11.052

A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good selectivity against other kinases, and excell

Full text of DOI:10.1016/j.bmcl.2011.11.052

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1247–1250
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel, potent and selective inhibitors of Polo-like kinase 1
Shaoqing Chen a, , David Bartkovitz a, Jianping Cai a, Yi Chen a, Zhi Chen a, Xin-Jie Chu a, Kang Le a,
⇑
Nam T. Le a, Kin-Chun Luk a, Steve Mischke a, Goli Naderi-Oboodi a, John F. Boylan b,
,
Tom Nevins b, Weiguo Qing b,à, Yingsi Chen c, Peter M. Wovkulich a,
⇑
a Discovery Chemistry, Hoffmann-La Roche, Inc., Pharma Research & Early Development, Small Molecule Research, 340 Kingsland Street, Nutley, NJ 07110, United States
b Discovery Oncology, Hoffmann-La Roche, Inc., Pharma Research & Early Development, Small Molecule Research, 340 Kingsland Street, Nutley, NJ 07110, United States
c Discovery Technologies, Hoffmann-La Roche, Inc., Pharma Research & Early Development, Small Molecule Research, 340 Kingsland Street, Nutley, NJ 07110, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis
and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good
selectivity against other kinases, and excellent in vitro cellular potency. It showed strong antitumor activ-
ity in xenograft mouse models.
Received 1 October 2011
Revised 9 November 2011
Accepted 14 November 2011
Available online 1 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Polo-like kinase 1 (PLK1)
Pyrimidodiazepines
Kinase inhibitor
Antitumor agent
The Polo-like kinases (PLKs), members of the serine/threonine
family of kinases, play a variety of roles in G2/M phase transition
and have been identified as key regulators of cell mitosis.1,2 PLKs
are highly conserved from yeast to human and five Polo-like
kinases, PLK1, PLK2 (Snk), PLK3 (Fnk), PLK4 and PLK5 have been
identified.1a,3 Among them, PLK1 is the best characterized. PLK1
is overexpressed in many solid tumors of various origin (breast,
lung, colon, stomach, ovary, smooth muscle, and esophagus) and
in non-Hodgkin lymphomas.1a,4 The overexpression studies of
PLK1 in NIH3T3 cell line showed these cells can form tumors in
nude mice due to PLK1 overexpression.5 Loss of PLK1 expression
can induce pro-apoptotic pathways and inhibit growth.6 An shRNA
screen in mutant KRAS cells shows that PLK1 has a synthetic lethal
interaction with this oncogene.7 Inhibition of PLK1 kinase activity
by a small molecule represents a novel approach to target mitosis
and may be clearly differentiated from other mitosis-targeting
agents on the market. The crystal structure of the kinase domain
for PLK1 has been reported.8 PLK1 is a validated target for cancer
and several PLK1 inhibitors have shown promising results in phase
I or II clinical trials.9 Our own efforts to find inhibitors of PLK1
resulted in the identification of pyrimidodiazepines as a novel
and potent class of ATP-competitive inhibitors of PLK1. This paper
outlines the synthesis, structure–activity relationships, and in vitro
and in vivo activity of this series of compounds.
Our interest in the diazepine ring system was to explore
whether it offered improved physical and biological properties
over the previously reported pteridines,9a as well as providing
additional vectors for substitution.
The general synthetic approach for the preparation of this series
is shown in Scheme 1.10 Starting from b-amino ester 21, regioselec-
tive substitution of the 4-chloro group of 2,4-dichloro-5
-nitro-pyrimidine gave 22. Reduction of the nitro group with
in situ cyclization gave pyrimidodiazepinone 23. Alkylation of the
lactam nitrogen and substitution of the 2-chloro group gave carbox-
ylic acid 25, which was coupled with a series of amines to give the
final products.
A key step in the synthesis of difluoro-analog 7 (RO3280) was
the preparation of the intermediate
27 (Scheme 2).10 This was prepared by the reaction of cyclopentyl-
amine with formaldehyde and benzotriazole11 to give N-(
-amino-
a,a-difluoro-b-amino ester
a
alkyl)-benzotriazole 26. Reformatsky reaction12 of ethyl
bromodifluoroacetate with intermediate 26 provided the desired
intermediate difluoro b-amino ester 27. It was subsequently found
that the b-amine ester could also be prepared by reductive amina-
tion of the aldehyde equivalent (ethyl hemiacetal form)13 and so-
dium cyanoborohydride in the presence of chlorotrimethylsilane.
We explored the structure–activity relationships for substitu-
tions around the ring system, as well as the portions of the
⇑
Corresponding authors. Tel.: +1 973 235 4636; fax: +1 973 235 6084.
E-mail address: shaoqing.chen@roche.com (S. Chen).
Present address: Celgene Research, 4550 Towne Centre Court, San Diego, CA
92121, United States.
à
Present address: Hutchison Medi Pharma, 720 Cai Lun Road, Zhangjiang Hi-Tech
Park, Shanghai 201203, China.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.052

1248
S. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1247–1250
NO2
R2
R1
COOEt
R2
R1
NO2
Cl
N
a
N
COOEt
R4
N
Cl
N
N
R4
Cl
N
H
21
22
O
R3
N
H
N
O
N
R2
R1
R2
R1
b
c
N
Cl
N
N
Cl
N
N
R4
R4
24
23
R3
R3
N
O
R6
O
O
R6
O
R5
N
e
R2
R1
d
HO
N
R2
R1
N
H
N
N
N
N
R4
N
N
N
R4
H
H
25
1-20
Scheme 1. Reagents and conditions: (a) NaHCO3, EtOAc, 0 °C to rt, 18 h; (b) Fe, HOAc, 80 °C, 2 h; (c) R3I, Cs2CO3, DMF, rt, 3 h; (d) 4-amino-3-methoxy-benzoic acid (for
compounds 1–19) or 4-amino-6-fluoro-3-methoxy-benzoic acid (for compound 20), EtOH/H2O/HCl, reflux, 18 h; (e) R5-NH2, HBPyU, DIEA, DMF, rt, 12 h.
F
tion may be the more potent one, a series of matched pairs of di-
fluoro and dimethyl analogs with substitutions elsewhere on the
ring system were prepared and evaluated. In general, the difluoro
compounds were 2- to 15-fold more potent (typically 3-fold) in
the biochemical assay and 2- to 6-fold more potent (typically 3-
fold) in the cell-based assay (data not shown). We found that di-
fluoro-analog 7 (RO3280) gave the best results in both enzymatic
(PLK1, IC50 = 3 nM) and cellular (lung cancer cell line H82,
EC50 = 6 nM) assays. The increased potency for difluoro and
COOEt
a
b
F
N
N
N
H
NH2
H
N
N
26
27
N
O
O
c
N
N
N
N
F
H
F
N
N
H
OMe
Table 2
Structure-activity relationship of pyrimidodiazepinone at R3 and R4 positions
7 (RO3280)
R3
Scheme 2. Reagents and conditions: (a) Benzotriazole, HCHO, ether, rt, 12 h; (b)
BrCF2COOEt, Zn, TMSCl, THF, À10 °C, 20 min, then compound 26, À10 °C to rt, 3 h;
(c) conditions (a) to (e) in Scheme 1.
N
O
O
N
N
H
N
F
F
N
H
N
N
R4
template expected to extend out of the hinge-binding region.
Small, di-substitutions, such as dimethyl, cyclopropyl, difluoro
and dichloro for R1 and R2 gave analogs with good potency (Table
1). Because this initial dataset suggested that the difluoro substitu-
OMe
Compd
R3
R4
PLK1
IC50 (nM)
H82 cell
EC50 (nM)
7
8
Me
H
3
6
Table 1
Structure–activity relationship of pyrimidodiazepinone at R1 and R2 positionsa
Me
234
138
355
NT
NT
NT
N
O
O
N
N
H
N
R1
R2
9
Et
N
H
N
N
OMe
10
Propyl
H-⁄
123
6
NT
15
Compd
R1, R2
PLK1
IC50 (nM)
H82 cell
EC50 (nM)
11
12
Me
Me
*
1
2
3
4
5
6
7
H,H
Me,H
Et,H
di-Me
c-propyl
di-Cl
30
34
103
15
10
4
48
53
127
23
13
10
6
13
14
15
Me
Me
Me
3
7
10
8
*
*
di-F
3
*
20
236
a
See Ref. 10 for assay details.

S. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1247–1250
1249
Table 3
Structure–activity relationship of pyrimidodiazepinone at R5 and R6 positions
O
R6
O
N
R5
N
F
F
N
H
N
N
Compd
R6
R5
PLK1
H82 cell
IC50 (nM)
EC50 (nM)
16
17
18
2-OMe
2-OMe
2-OMe
–OH
–NH2
–NHMe
18
4
6
390
27
51
NH*
19
20
2-OMe
39
6
NT
12
2-OMe,5-F
N
NH*
Figure 1. Tumor growth inhibition by intravenous (IV) administration of RO3280 in
nude mouse implanted with HT-29 human colorectal tumors.
Table 4
Selectivity of RO3280 against a panel of kinasesa
Ambit gene symbol
Kd (nM)
biochemical and cell-based assays were seen when the R5 group
contained a basic amine. Several substitutions on phenyl ring at
R6 position were well tolerated (e.g., compound 20).
The most promising analogs were examined in both an in-house
kinase selectivity panel and KINOMEscan™ from Ambit Biosci-
ences.14 Compound RO3280 was found to be a potent and selective
PLK1 inhibitor. It shows no inhibitory activity against a panel of 48
in-house kinases and inhibited 13 out of 293 kinases in the Ambit
ALK
230
1100
87
100
70
CAMKK1
CAMKK2
DAPK1
DAPK3
FER
GAK
MYLK
PTK2
53
87
170
84
130
560
51
PTK2B
RPS6KA6 (KinDom.2)
TTK
panel more than 85% at 1 lM. Of the kinases read as a hit in the
Ambit panel, Kd values were determined for the more active ki-
nases, data summarized in the Table 4. From the Kd measurements,
binding to PLK1 was more than 500-fold tighter than to any of the
other kinases in the Ambit panel as shown in Table 4.
PLK1
0.09
a
Ambit selectivity screen is a panel of 318 kinases
(wild type + mutants). Kd values were determined for
the kinases where the inhibition was >85% at 1 M.
l
As shown in Table 1, RO3280 exhibits an EC50 value of 6 nM in a
cell-based assay using the H82 lung cancer cell line. Table 5 shows
the strong anti-proliferative activity of this compound in a panel of
tumor cell lines including lung, colon cancer, breast cancer, pros-
tate and skin cancer. An initial assessment of EC50 showed no cor-
relation with MDR1 or MRP1 status, but does appear to correlate
with p21 expression (Western blot, data not shown).15 A more
comprehensive description of the biological profiling of RO3280,
as well as the development and characterization of resistant muta-
tions (HT-29) will be the topic of a subsequent publication.
RO3280 was evaluated for its in vivo antitumor activity in a ser-
ies of mouse xenograft models, of which the HT-29 colorectal can-
cer cell line is representative. As showed in Figure 1, RO3280
displayed robust antitumor activity ranging from 72% tumor
growth inhibition when dosed once weekly at 40 mg/Kg, to com-
plete tumor regression when dosed more frequently.
dichloro substitution at this position may be a consequence of
additional interaction with nearby water molecules in the binding
pocket according to computer modeling, that analysis will be de-
ferred pending a crystallographic determination.
Further exploration of the structure activity relationships in our
difluoro series (R1 = R2 = F) is summarized in Table 2 (R3 and R4
variations) and Table 3 (R5 and R6 variations). The optimal substi-
tution for R3 is methyl, with activity dropping off significantly
either for no substitution (R3 = H) or for larger groups. The substi-
tution at R4, which is expected to extend into a hydrophobic pock-
et, tolerates
a much wider range of modification, including
branched and cycloalkyl groups. Larger alkyl group at this position
led to a loss in potency.
Table 3 shows the SAR in the R5 and R6 positions. Simple amide
derivatives give good potency in the biochemical assay, but lose
some potency in the cell-based assay. The best activities in the
In summary, we report the discovery of a novel class of pyrim-
idodiazepinones as potent and selective ATP-competitive PLK1
inhibitors. RO3280 exhibits potent antiproliferative activity in a
Table 5
Inhibitory activity on various tumor cells for RO3280
Tumor cell line (EC50, nM)
Colon
Colo205
16
Lung
H69
7
Breast
MDA-MB-468
19
Prostate
PC-3
12
Skin
A375
70
H82
6
A549
82
HCT-116
60
RKO
18
SW480
45
HT-29
10
SW620
10
MTLn3
20

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ities in an in vivo HT-29 colorectal xenograft mouse model.
Acknowledgments
The authors would like to thank Dr. Paul Gillespie for reading
the manuscript and his support, Dr. Aruna Railkar and Jiakui Li
for formulation work.
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