Discovery of TAK-960: An orally available small molecule inhibitor of polo-like kinase 1 (PLK1)

Article abstract of DOI:10.1016/j.bmcl.2013.02.083

Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with

Full text of DOI:10.1016/j.bmcl.2013.02.083

Accepted Manuscript
Discovery of TAK-960: an orally available small molecule inhibitor of polo-
like kinase 1 (PLK1)
Zhe Nie, Victoria Feher, Srinivasa Natala, Christopher McBride, Andre
Kiryanov, Benjamin Jones, Betty Lam, Yan Liu, Stephen Kaldor, Jeffrey
Stafford, Kouki Hikami, Noriko Uchiyama, Tomohiro Kawamoto, Yuichi
Hikichi, Shin-ichi Matsumoto, Nobuyuki Amano, Lilly Zhang, David Hosfield,
Robert Skene, Hua Zou, Xiaodong Cao, Takashi Ichikawa
PII:
S0960-894X(13)00268-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.02.083
BMCL 20201
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
22 October 2012
14 February 2013
19 February 2013
Please cite this article as: Nie, Z., Feher, V., Natala, S., McBride, C., Kiryanov, A., Jones, B., Lam, B., Liu, Y.,
Kaldor, S., Stafford, J., Hikami, K., Uchiyama, N., Kawamoto, T., Hikichi, Y., Matsumoto, S-i., Amano, N., Zhang,
L., Hosfield, D., Skene, R., Zou, H., Cao, X., Ichikawa, T., DiscoveryofTAK-960:anorallyavailablesmallmolecule
inhibitor of polo-like kinase 1 (PLK1), Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/
10.1016/j.bmcl.2013.02.083
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Discovery of TAK-960: an orally available
small molecule inhibitor of polo-like kinase 1
(PLK1)
Leave this area blank for abstract info.
Zhe Nie, Victoria Feher, Srinivasa Natala, Christopher McBride*, Andre Kiryanov, Benjamin Jones, Betty Lam,
Yan Liu, Stephen Kaldor, Jeffrey Stafford, Kouki Hikami, Noriko Uchiyama, Tomohiro Kawamoto, Yuichi
Hikichi, Shin-ichi Matsumoto, Nobuyuki Amano, Lilly Zhang, David Hosfield, Robert Skene, Hua Zou,
Xiaodong Cao and Takashi Ichikawa

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of TAK-960: an orally available small molecule inhibitor of polo-like
kinase 1 (PLK1)
Zhe Niea,c, Victoria Fehera,d, Srinivasa Natalaa, Christopher McBridea, *, Andre Kiryanova, Benjamin Jonesa,
Betty Lama, Yan Liua, Stephen Kaldora, c, Jeffrey Stafforda, c, Kouki Hikamib, Noriko Uchiyamab,
Tomohiro Kawamotob, Yuichi Hikichib, Shin-ichi Matsumotob, Nobuyuki Amanob, Lilly Zhanga, David
Hosfielda,e, Robert Skenea, Hua Zoua, Xiaodong Caoa,f and Takashi Ichikawab
aTakeda California, 10410 Science Center Drive, San Diego 92121, USA
bTakeda Pharmaceutical Company, Ltd, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, 251-855, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Using structure-based drug design, we identified and optimized a novel series of
pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate
TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced
solid malignancies.
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
TAK-960
PLK1 inhibitor
Structure-based drug design
Antitumor activity
Multidrug resistance
Polo-like kinase 1 (PLK1) is a serine/threonine kinase that
controls entry into and progression through mitosis at multiple
stages.1 Over-expression of PLK1 is associated with poor
prognosis and survival rates in a number of human cancers.1d,2
Several reports have demonstrated that PLK1 depletion (by α-
PLK1 antibody injection or siRNA interference) causes mitotic
arrest and apoptosis in cancer cell lines, but not in normal diploid
cells or non-dividing cells.3 PLK1 was therefore considered an
attractive target for treatment of proliferative diseases.4
Currently, small molecule PLK1 inhibitors are being evaluated in
human clinical trials.5 In one such trial, clinical responses were
reported when patients were treated with the investigational IV
drug, volasertib.6
proteins such as the mitotic kinases.3d Herein, we report the
discovery of TAK-960, an orally available, potent and selective
PLK1 inhibitor currently in a phase I clinical trial in patients with
advanced non-hematologic malignancies.
Following a screen of in-house assets, we identified a
compound with an N-arylpyrimidin-2-amine hinge binding motif
as an inhibitor of PLK1 and utilized this as our starting point.
From this hit we designed a novel series of ATP-competitive 2-
aryl pyrimidodiazepinone derivatives (Figure 1) as potential
inhibitors of PLK1.
Anti-mitotic agents that target tubulin, such as taxanes, have
been clinically validated. However, these drugs have several
drawbacks including development of peripheral neuropathy,7 the
need for pretreatment to reduce anaphylaxis,8 and the potential
for tumors to become refractory/resistant.2b Consequently, there
Figure 1. Structural representation of N-arylpyrimidin-2-amine and 2-aryl
pyrimidodiazepinone scaffold
is a need for novel anti-mitotic drugs that target non-microtubule
———
c Present address: Quanticel Pharmaceuticals Inc.
d Present address: UC San Diego, Department of Chemistry and Biochemistry
* Corresponding author. Tel.: +1-858-731-3665; fax: +1-858-550-0526; e-mail: chris.mcbride@takeda.com
e Present address: Ben May Cancer Institute, University of Chicago
f Present address: Viva Biotech, Ltd.

Analysis of a homology model of PLK1 identified that within
the ATP binding pocket of the target there is a “roof pocket”
created by the surrounding residues C67, L130, K82, and A80.
This pocket is an uncommon feature among kinases. Based on
this observation, we proposed a hypothesis that an appropriate
substituent at C-7 of the puckered diazepinone would interact
favorably with the hydrophobic “roof pocket” resulting in
improved enzyme potency and selectivity (Figure 2). Structure-
activity relationships (SAR) for this core, relating to the N-5 and
N-9 and certain substitutions around the ring of the C-2 aniline
have been previously disclosed.9a,b,10
((cyclopentylamino)methyl)-2-fluorobutanoate 5 which was used
as shown in Scheme 1 to transform to the pyrimidodiazapinone.
The individual enantiomers were separated by chiral
chromatography to give 12 and 13.
Scheme 2. Reagents and conditions: (a) HCHO, HNMe2, water, reflux; (b)
MSA, MeOH, DCM, reflux; (c) mCPBA, 4,4’-thiobis(2-t-butyl-6-
methylphenol), DCE, reflux; (d) cyclopentylamine, EtOH, reflux; (e) BnBr,
K2CO3, MeCN, reflux; (f) DAST, DCM; (g) Pd(OH)2, H2, TFA, EtOH
For the allyl substitution at C-7, deprotonation of compound 6
by LDA followed by alkylation with allyl bromide provided
intermediate 7. This intermediate was then modified at the 2
position in the same manner which has been previously described
and is shown in Scheme 3. Subsequent separation of enantiomers
by chiral chromotography gave compounds 15-16.
Figure 2. Homology model of C-7 gem dimethyl pyrimidodiazapinone
docked in PLK1 active site bound to the hinge residue C133
The results of our “roof pocket”-targeted designs are
summarized in Table 111 and described below. The synthesis of
compounds 10-11 has been disclosed by our group9 as well as
another lab which used a similar procedure10 so will not be
reviewed here in detail. Scheme 1 shows a general steps for
synthesis and has been included to aid the following discussion.
Scheme 3. Reagents and conditions: (a) LDA/THF, allyl bromide, -78oC-rt.;
(b) (1) 4-amino-3-methoxybenzoic acid, iPrOH, conc. HCl (cat.), 100oC; (2)
1-methylpiperidin-4-amine, DIEA, HATU, DMF.
7-Vinyl substituted analogs were synthesized as shown in
Scheme 4. Compound 6 was deprotonated by LDA and quenched
with acetaldehyde to generate the alcohol intermediate, which
was then converted to the mesylate. Upon treatment with a base,
ethylidene intermediate 8 was obtained. Deprotonation of 8 using
LDA/HMPA,
followed
by
quenching
with
N-
fluorobenzenesulfonimide (NFSi) resulted in fluorination at
exclusively at the desired site (9)13. This intermediate was
substituted via an SNAr reaction and modified by amide coupling
reactions. The enantiomerically pure products were separated
through chiral chromotography to give compounds 16-17 and
their configurations were determined later by obtaining co-crystal
structures.
Scheme 1. Synthetic strategy for pyrimidodiazapinones
For the synthesis of compounds 10-11, the necessary -amino
acid esters were commercially available or previously described
in the literature12. For the synthesis of compounds 12-13 we
followed the synthetic route outlined in Scheme 2 to obtain the
requisite -amino acid ester. This route starts with ethyl malonic
acid 1 which was treated with dimethylamine in formic acid and
water. The product was then esterified by treatment with
methanesulfonic acid (MSA) in MeOH and DCM, followed by
epoxidation using mCPBA and 4,4’-thiobis(2-t-butyl-6-
methylphenol) to give 3. The epoxide was opened with
cyclopentylamine and the resulting amine was protected by
reaction with benzyl bromide to give 4. The alcohol was then
treated with DAST to effect conversion to the fluoro ester
Scheme 4. Reagents and conditions: (a) (1) LDA/THF, CH3CHO, -78oC-rt.;
(2) MsCl, Et3N, DCM; (3) 60% NaH in THF, 0oC-rt; (b) LDA:HMPA(1:1) in
THF, NFSi, -78oC-rt; (c) (1) 4-amino-3-methoxybenzoic acid, iPrOH, conc.
HCl (cat.), 100oC; (2) 1-methylpiperidin-4-amine, DIEA, HATU, DMF.
which,
following
deprotection
gave
ethyl
2-

Table 1. SAR of representative C-7 substitutions
Compd.
Structure (R1, R2)
X
PLK1
t1/2off
(min)
1
HT-29
IC50 (nM)
EC50 (nM)
10
11
12
13
14
15
16
17
18
CH
CH
CH
CH
CH
CH
CH
CH
N
12
5
32
5
170
39
2
5
100
1
ND
66
180
8
70
5
ND
170
ND
131
43
6
170
3
270
16
As seen in Table 1, as the size of the 7-substituent increased
(such as allyl), the activity increased over the unsubstituted
compound 10 and only S enantiomers were tolerated. This result
is consistent with our hypothesis that occupying the “roof
pocket” would improve binding affinity. We then determined the
cellular activity and dissociation rates (t1/2off) from PLK1 for
selected inhibitors as shown in Table 1.14,15 Compounds such as
11 and 16 showed slow off-rates, which we hypothesized may
lead to a longer residence time at the target and a prolonged
pharmacologic effect in cells or tissues.16
optimization efforts to improve compound 11 and evaluated the
influence of substituents on the aniline moiety. This resulted in
the identification of compound 24 (TAK-960) as our
development candidate.
Analysis of the co-crystal structures17 of compounds in this
series with PLK1 demonstrated that appropriate C-7 substituents
interact favorably with a “roof pocket” and adjacent water
molecules and these features are believed to contribute to
improved enzyme potency and slow dissociation kinetics. For
example, co-crystal structure of a 7-fluoro-7-vinyl analog
(compound 18) illustrates the roles of the C-7 substituents
(Figure 3). The 7-vinyl group indeed occupies the “roof pocket”
and makes hydrophobic interactions with adjacent protein
residues. Additionally, we observed water-mediated interactions
with the salt bridge residues K82 and D194.
Figure 3. Co-crystal structure of compound 18 in PLK1 active site PDB 4J52
For the purpose of cellular assay of our compounds, HT-29
cells were selected as they have been shown to be sensitive to
PLK1 inhibitors.18 Compound 11 (a 7,7-difluoro-8,9-dihydro-
The synthesis of TAK-960 is shown in Scheme 5. Treatment
of methyl 2,5-difluoro-4-nitrobenzoate (19) with KOH in MeOH
gave 2-fluoro-5-methoxy-4-nitrobenzoic acid (20) which was
then reduced under an atmosphere of hydrogen using Pd/C as a
catalyst to yield the requisite aniline 21. The SNAr conditions
used previously to introduce the aniline to the
pyrimidodiazepinone core led to significant decarboxylation
products, so in this case we utilized a Pd cross coupling reaction
to affect the transformation from 22 to 23. A subsequent amide
coupling reaction produced TAK-960 (24). Compounds 25-30
were prepared in a similar fashion, using the corresponding
aniline in the SNAr or cross coupling reaction.
5H-pyrimido[4,5-b][1,4]diazepin-6(7H)-one
analog)
demonstrated 5 nM inhibition of proliferation of a HT-29 cell
line with slow dissociation kinetics from PLK1. This compound
served as a valuable tool to understand the consequences of
PLK1 inhibition in vitro and in vivo and demonstrated efficacy in
a number of animal models targeting multiple tumor types.10b,11
However, only 11% of compound 11 was absorbed when dosed
orally in rat which was consistent with the finding that it is a
substrate for P-glycoprotein (MDR1). Thus, we shifted our lead

fold drop in potency. The ketone of the pyrimidodiazapinone ring
makes a water mediated interaction with D194.
Scheme 5. Reagents and conditions: (a) KOH, MeOH, 80oC; (b) H2, Pd/C,
AcOH, MeOH; (c) compound 21, Pd(OAc)2, XANTPHOS, Cs2CO3,
DMA/dioxane, microwave, 160oC; (d) 1-methylpiperidin-4-amine, HATU,
DIEA, DMF.
In order to determine the MDR1 profiles of these compounds,
the efflux ratio was calculated using a pig kidney cell line and
compared to that of an MDR1-transfected cell line as shown in
Table 2. The MDR1 susceptibility was lowered for both the 2 and
6 fluorobenzamides (TAK-960 and 26) by greater than 15-fold
compared to the 3-fluorobenzamide (compound 25) and by 5-fold
when compared to the des-fluoro analog (compound 11). The
ortho-fluoro groups are hypothesized to form pseudo intra-
molecular H-bond interactions with the adjacent amide NH. This
lowers the number of H-bond donors present in the drug and
restricts the rotation around the aryl-amide C-C bond, limiting the
available conformers. The reduced MDR1 susceptibility in the
ortho-fluoro amides is consistent with one or both of these factors
interfering with efflux. In support of this hypothesis, analogs
Figure 4. Co-crystal structure of TAK-960 in PLK1 active site PDB code
4J53
The pharmacokinetics of compound 11 and TAK-960 were
evaluated in rats (Table 3). Both compounds exhibited an
acceptable clearance and high volume of distribution. Oral
administration of TAK-960 gave higher plasma exposure and 5-
fold higher oral bioavailability than those of compound 11. This
finding is likely attributed to the fact that this compound is not a
substrate for MDR1.
Table 3. Selected rat PK parameters for compd 11 and 24 (TAK-960)19
Compd
CL
Vdss
(mL/kg)
19600
13700
t1/2
(h)
3.3
5.4
F (%)
(mL/min/kg)
11
24
51
42
11
54
with various ortho substitutions were prepared.
Those
compounds carrying ortho substitutions capable of forming H-
bond with amide NH such as F, OMe and Cl have relatively low
MDR1 efflux, whereas, compounds with a Me group or H at this
position are more susceptible to MDR1 efflux (Table 2).
TAK-960 (24) is a potent and selective PLK1 inhibitor with
slow dissociation kinetics (t1/2 off = 184 min.). It inhibits
proliferation of various cancer cell lines including MDR1-
expressing tumors, such as K562ADR, HCT-15 and
COLO320DM. Oral administration of TAK-960 demonstrated
potent antitumor activity in various xenograft models, including a
disseminated model of AML and MDR1 expressing
hematological tumors.18 Preclinical data demonstrates the
therapeutic potential of TAK-960 in the treatment of diverse
human malignancies.
Table 2. SAR of substitutions on C-2 aniline
In summary, we have identified
a
novel series of
pyrimidodiazepinone analogs as potent and selective PLK1
inhibitors. Structure-driven modifications to the core at the C-7
position impart slow dissociation kinetics and favorable cellular
potency due to an interaction with an uncommon “roof pocket” in
the ATP binding pocket of the target. The C-7 difluoro analog,
compound 11, was selected for further optimization and
fluorination ortho to the amide moiety improved the MDR1
profile and oral bioavailability, giving TAK-960. TAK-960 is
currently undergoing Phase I evaluation in adult patients with
advanced non-hematologic malignancies.
Structure
(R, R’)
HT29
EC50 (nM)
LLC-MDR ER
/ LLC ER
PLK1
IC50 (nM)
Cmpd
11
24
25
26
27
28
29
30
OMe, H
4
2
81
26
1
1
15
190
5
3
264
40
16
7
10
OMe, 2-F
OMe, 3-F
OMe, 6-F
H, H
OMe, 2-Cl
H, 2-OMe
H, 2- Me
0.9
49
2.9
45
2.4
4.8
25
95
70
LLC: Pig kidney cell line; LLC-MDR: MDR1 transfectant; ER: Efflux ratio
Acknowledgments
The co-crystal structure of TAK-960 with PLK1 (Figure 4)
demonstrated that the amide NH twists to make an interaction
with the backbone ketone of Leu59, stabilizing the P-loop. The
lowest energy conformation of TAK-960 maintains the amide NH
proximal to the Leu59 compared to compound 11 which is rotated
180 degrees, leaving it with a much higher energy barrier to
access to this interaction with the P-loop which is reflected the 13
The authors would like to acknowledge the assistance of Dr.
Jason Yano for the help in preparation of co-crystal figures for
this manuscript. We also wish to thank Dr. Steve Gwaltney, Dr.
Gavin Hirst and Dr. Matt Marx for their close examination of
drafts and help in preparation of this paper.

17. The PLK1 kinase domain (residues 13-345) was cloned into the
pFastBacHTb vector, and recombinant baculovirus was generated and
expressed in fusion with an N-terminal 6x poly-histidine tag and TEV
cleavage site. Large scale production of recombinant protein was
carried out in Spodoptera frugiperda Sf9 cells. The cell pellet was
suspended into lysis buffer consisting of 25 mM HEPES (pH 8.0), 1 M
NaCl, 20 mM imidazole, 3 Roche Complete tablets. The lysate was
centrifuged, and clarified supernatant was batch bound with 10 ml of
Probond Ni resin (Invitrogen). The protein was then eluted with buffer
containing an additional 250 mM imidazole. After tag cleavage, the
protein was further purified by size-exclusion chromatography utilizing
a S3000 column equilibrated in 25 mM HEPES (pH 7.2), 50 mM NaCl,
0.25 mM TCEP, 1% Glycerol. Fractions containing the protein of
interest were pooled, concentrated to 12 mg/ml and flash-frozen in
liquid nitrogen for storage at -80oC. For crystallization, the PLK1
samples were incubated with 1 mM compound. Crystals were obtained
from solutions containing 0.95 mM Succinic Acid, 1.7% PEG
2000mme, 0.4 mM Zn(OAc)2, and 100 mM HEPES (pH 7.0).
Diffraction data were collected at the Advanced Light Source (ALS)
beamline 5.0.3 and Stanford Synchrotron Radiation Lightsource (SSRL)
beamline 9-2. PLK1 co-crystals of compounds 18 and 24 diffracted to
resolutions of 2.3 to 2.5Å, and subsequent data reduction was conducted
using the HLK2000 software package. The structures were determined
by molecular replacement using the program MOLREP, employing the
coordinates of PLK1 (2OWB) as a search model. Subsequent structure
refinement and model re-building were conducted utilizing REFMAC
and XtalView software packages.
References and notes
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14. Cell proliferation assay: HT-29 human colon adenocarcinoma cells
were seeded into 96-well plates at 3,000 cells/well in DMEM
(Dulbecco's Modified Eagle's Medium) plus 10% fetal calf serum
(FCS). After 24 hours, cells were treated with serial dilutions of PLK1
inhibitors, and 72 hours later, the number of viable cells was assessed
using the CellTiter-Glo Assay (Promega).
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designed to determine the dissociation rate of a compound from an
enzyme. Inhibitors were mixed with PLK1 kinase domain (residues 21-
351) in a reaction buffer, which consisted of 25 mM HEPES (pH7.5),
10 mM MgAcetate, 0.05% Brij35, 0.01% BSA, 1 mM DTT and
incubated at room temperature for 60 min. to allow for sufficient
binding. The PLK1/inhibitor complex was then diluted into reaction
buffer containing 1 mM ATP and 1-μM FAM-mTOR peptide, and this
initiation of the enzymatic reaction was considered time zero (t0). The
dissociation rate was then measured on a Caliper LabChip 3000 (LC-
3000) from Caliper Life Sciences Inc. with off-chip assays using a
TC372 chip.
16. Copeland, R.A.; Pompliano, D.L.; Meek, T.D. Nat. Rev. Drug Discov.
2006, 5, 730.

Figure for abstract

Figure 1

Figure 2

Scheme 1

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Scheme 4

Table 1 Picture

Figure 3

Scheme 5

Table 2 picture

Figure 4