NMS-P937, a 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivative as potent and selective Polo-like kinase 1 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2011.03.054

As part of our drug discovery effort, we identified and developed 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as PLK1 inhibitors. We now report the optimization of this class that led to the identification of NMS-P937, a potent, selective and orally available PLK1 inhibitor. Also, in order to understand the source of PLK1 selectivity, we determined the crystal structure of PLK1 with NMS-P937. The compound was active in vivo in HCT116 xenograft model after oral administration and is presently in Phase I clinical trials evaluation.

Full text of DOI:10.1016/j.bmcl.2011.03.054

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2969–2974
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
NMS-P937, a 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivative
as potent and selective Polo-like kinase 1 inhibitor
Italo Beria a, , Roberto T. Bossi a, Maria Gabriella Brasca a, Michele Caruso a, Walter Ceccarelli a,
⇑
Gabriele Fachin a, Marina Fasolini a, Barbara Forte a, Francesco Fiorentini b, Enrico Pesenti a,
Daniele Pezzetta b, Helena Posteri a, Alessandra Scolaro a, Stefania Re Depaolini a, Barbara Valsasina a
a Nerviano Medical Sciences srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, MI, Italy
b Accelera srl, Viale Pasteur 10, 20014 Nerviano, MI, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of our drug discovery effort, we identified and developed 4,5-dihydro-1H-pyrazolo[4,3-h]quinazo-
line derivatives as PLK1 inhibitors. We now report the optimization of this class that led to the identification
of NMS-P937, a potent, selective and orally available PLK1 inhibitor. Also, in order to understand the source
of PLK1 selectivity, we determined the crystal structure of PLK1 with NMS-P937. The compound was active
in vivo in HCT116 xenograft model after oral administration and is presently in Phase I clinical trials
evaluation.
Received 21 February 2011
Revised 14 March 2011
Accepted 14 March 2011
Available online 21 March 2011
Keywords:
PLK1
Ó 2011 Elsevier Ltd. All rights reserved.
Polo-like kinase
Kinase inhibitor
In vivo activity
Phase I clinical trials
Antimitotics form the basis of the therapy for patients with both
solid tumors and hematological malignancies. However, current
antimitotic drugs affect both dividing and non-dividing cells. One
of the emerging next generation antimitotic targets is Polo-like
kinase 1 (PLK1).1 Among the four members of PLK family, PLK1 is
the best characterized and it is recognized to be a key component
of the cell cycle control machinery with important roles in the mi-
totic entry, centrosome duplication, bipolar mitotic spindle forma-
tion, transition from metaphase to anaphase, cytokinesis and
maintenance of genomic stability.2–5 PLK1 is often over-expressed
in many different tumor types and over-expression often correlates
with poor prognosis.6,7 As an antimitotic target, PLK1 is only ex-
pressed in dividing cells while it is not expressed in differentiated
postmitotic cells like neurons, where instead expression of PLK2
and PLK3 was reported. This indicates a potentially better safety
profile for a PLK1 specific inhibitor.1,8 Thus, PLK1 is thought to be
a promising target for anti-cancer therapy and indeed some PLK1
inhibitors are currently under evaluation in clinical trials.9
Efforts to improve the solubility and pharmacokinetic profile of
lead compound 2 by a focused expansion at the 50- and 1-positions
of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline scaffold led to
the identification of a new potent, selective and orally bioavailable
inhibitor.
Compounds modified at the 50-position, were prepared by react-
ing the iodo amide 3 with the suitable anilines 4a–f and Pd(OAc)2
under Buchwald–Hartwig conditions, to obtain final compounds
5a–b and intermediates 5c–f (Scheme 1).12 Intermediates 5e–f were
then converted to final compounds 5g–h, by removal of the benzyl
protecting group with BCl3. Reaction of bromo intermediate 5c with
the suitable primary or secondary amines under Buchwald–Hartwig
conditions, catalyzed by Pd2(dba)3 and 2-dicyclohexylphosphino-
20-(N,N-dimethylamino)-biphenyl, gave compounds 5i–l and the
protected intermediate 5m that was converted to the final
compound 5n by treatment with 4 M HCl (Scheme 2). Synthesis of
In the recent past we have identified the 4,5-dihydro-1H-pyraz-
olo[4,3-h]quinazoline template as a good scaffold to obtain potent
and selective PLK1 inhibitors 1 and 2 (Fig. 1).10,11 Subsequently
work continued on the class in order to discover a specific and
orally available PLK1 inhibitor that is suitable for clinical trials.
N
O
NH2
HN
N
N
1 R = -OMe
2 R = -OCF3
R
N
N
1
5'
N
⇑
Corresponding author. Tel.: +39 331 581516; fax: +39 331 581347.
E-mail address: italo.beria@nervianoms.com (I. Beria).
Figure 1. Structure of PLK1 inhibitors 1 and 2.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.054

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I. Beria et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2969–2974
R1 =
NH2
N
N
(a) 1-Methyl-1,2,3,6-tetrahydro-pyridin-4-yl
(b) 1-Methyl-piperidin-4-yl
(c) Bromine
O
O
a
CF3O
HN
N
+
I
N
NH2
NH2
CF3O
N
N
R1
N
N
(d) Nitro
4a-f
R1
b
3
(e) (R)-2-benzyloxymethyl-4-methyl-piperazin-1-yl
5a-d
(f) (S)-2-benzyloxymethyl-4-methyl-piperazin-1-yl
(g) (R)-2-oxymethyl-4-methyl-piperazin-1-yl
(h) (S)-2-oxymethyl-4-methyl-piperazin-1-yl
5e-f
5g-h
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, ( )-BINAP, K2CO3, anilines 4a–f, DMF, 80 °C, 20–62%; (b) under nitrogen atmosphere, BCl3, DCM, ꢀ78 °C then rt, 12 h,
quantitative.
N
R1 =
N
O
O
HN
N
(i) 4-(3-Dimethylamino-propyl)-piperazin-1-yl
(j) (2-Diethylamino-ethyl)-methyl-amino
(k) 3-(4-Methyl-piperazin-1-yl)-propylamino
(l) 2-(1-Methyl-pyrrolidin-2-yl)-ethylamino
(m) N-Boc-pyrrolidin-2-ylamino
a
HN
N
NH2
CF3O
N
N
NH2
CF3O
N
N
Br
R1
(n) Pyrrolidin-2-ylamino
5c
5i-l
5m
5n
b
Scheme 2. Reagents and conditions: (a) primary or secondary amines, Pd2(dba)3, 2-dicyclohexylphosphino-20-(N,N-dimethylamino)-biphenyl, LiN(TMS)2, THF, reflux, 10–
70%; (b) 4 M HCl in dioxane, dioxane, rt, quantitative.
N
N
O
O
HN
N
a
HN
N
NH2
CF3O
N
NH2
N
CF3O
N
N
R1
R1
5d R1 = NO2
R1 = NH2
5o
b
R1 = (S)-Pyrrolidine-2-carbonyl-amino
R1 = 1-Methyl-piperidine-4-carbonyl-amino
5p
5q
Scheme 3. Reagents and conditions: (a) Fe, NH4Cl, MeOH/H2O, reflux, quant.; (b) O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU), HOBt, DIPEA,
acids, 4 h, rt, 60%, then to yield 5p DCM, TFA, rt, 30 min, 30% (2 steps).
derivatives 5p–q were carried out by reducing the nitro intermedi-
ate 5d to the amino derivative 5o with Fe and NH4Cl then, coupling
5o with suitable acids in the presence of O-(benzotriazol-1-yl)-
then selective alkylation at the 1-position of the pyrazolo ring with
alkyl halogens under basic conditions, yielded, either directly or
through removal of the protecting group, the final compounds 7a
and 7e–g (Scheme 4).12
N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU) as
a
condensing agent to directly yield the final derivative 5q or, in two
steps, after removal of the BOC protecting group, the compound 5p
(Scheme 3). Modifications at the 1-position of the 4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline nucleus, to install the piperazino
residue were performed reacting the advanced intermediate 612
with 4-methyl-piperazine under Buchwald–Hartwig conditions,
Table 1 summarizes the structure–activity relationship (SAR) of
derivatives modified at the 50-position of the hit compound 2. All
the compounds, with the exception of 5o and 5p–q, showed an
increased solubility with respect to the hit 2. The compounds
wherein the 1-methyl piperazin-4-yl appendix has been replaced
with either 1-methyl-1,2,3,6-tetrahydro-pyridine-4-yl (5a) or with
R2 =
N
N
O
O
NH2
(a) = -(CH2)3-N-(CH3)2
HN
N
N
HN
N
(b) = -(CH2)2-NH-Boc
(c) = -(CH2)3-NH-Boc
(d) = -(CH2)2-O-THP
(e) = -(CH2)2-NH2
(f) = -(CH2)3-NH2
NH2
CF3O
N
N
CF3O
N
N
a, b
H
R2
Br
N
6
7a
(g) = -(CH2)2-OH
c
7b-c
7d
7e-f
7g
d
Scheme 4. Reagents and conditions: (a) 4-methyl-piperazine, Pd2(dba)3, 2-dicyclohexylphosphino-20-(N,N-dimethylamino)-biphenyl, LiN(TMS)2, THF, reflux, 70%; (b)
Cs2CO3, R2-Cl or R2-Br, DMF, rt, 20–60%; (c) 4 N HCl in dioxane, rt, 4 h, quant.; (d) TsOH, EtOH, rt, 60%.

I. Beria et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2969–2974
2971
Table 1
SAR: variation at the 50-position of the aniline residue
N
O
NH2
HN
N
N
O
N
F3C
R1
R1
PLK1 IC50
(lM)
PLK2 IC50
(l
M)
PLK3 IC50
(
lM)
A2780 IC50
(l
M)
Solubility pH 7 (lM)
a
a
a
a
Compound
2
N
N
N
N
0.003 0.001
0.003 0.001
0.006 0.003
3.519 0.063
2.254 0.77
>10
1.439 0.078
0.323 0.062
>10
0.021 0.005
0.034 0.006
0.090 0.002
72
148
203
5a
5b
5i
N
N
0.042 0.010
0.044 0.011
3.855 0.467
1.453 0.106
1.315 0.182
0.681 0.219
0.526 0.041
0.348 0.008
174
79
N
H
N
H
N
5p
O
N
N
5j
0.060 0.028
>10
>10
1.136 0.077
162
H
H
N
N
5n
5o
0.083 0.021
0.087 0.033
0.762 0.034
1.280 0.464
0.239 0.008
0.154 0.024
2.209 0.296
1.074 0.123
171
31
NH2
N
N
N
H
5k
5l
0.106 0.044
0.149 0.051
1.364 0.341
0.429 0.017
0.713 0.064
0.485 0.046
1.635 0.416
2.549 0.411
186
157
N
H
N
N
N
N
N
5g
5h
5q
0.154 0.032
0.201 0.012
0.224 0.043
>10
>10
0.598 0.186
1.615 0.475
1.353 0.180
121
166
33
HO
HO
1.494 0.211
1.499 0.337
3.519 0.708
0.705 0.073
H
N
N
O
a
Values are means of three experiments. Compounds 5c and 5d showed PLK1 IC50 >1 lM and were not further profiled.
1-methyl piperidin-4-yl (5b) residues maintain the PLK1 activity,
the selectivity profile versus PLK2–3 and the antiproliferative ef-
fect on A2780 cells line of the hit 2. The other compounds,
although more soluble than 2, showed from 14-fold (5i) to 74-fold
(5q) decreased PLK1 activity and a more drastic reduction of the
cytotoxic activity on cells, ranging from 15-fold (5p) to more than
120-fold (5l).
Activity data concerning compounds modified at position 1 of
the pyrazole ring of the hit compound 2 are summarized in Table
2. Although replacement of methyl residue with either primary
(7e and 7f) or tertiary (7a) amines increases the solubility of the
compounds it also led to a reduction of potency in both the bio-
chemical and cellular assays. In particular, the best compound 7e
showed a 10-fold loss of activity on PLK1 and a more than 50-fold
decreased cytotoxicity in A2780 cell lines. This heavy loss in cell
activity, shared also by the other amine derivatives 7a and 7f,
could be explained with a decreased ability of the molecules to en-
ter into the cells, due to their higher hydrophilicity, as confirmed
by in vitro permeability data. For example, the hit compound 2 re-
sulted indeed to be highly permeable (Papp = 50 ꢁ 10ꢀ6 cm/s),
while compounds 7e–f resulted to be low/medium permeable
(Papp = 5 ꢁ 10ꢀ6 cm/s and 4 ꢁ 10ꢀ6 cm/s, respectively).13 Replace-
ment of the methyl residue with the 2-hydroxyethyl residue (7g)
resulted instead very profitable. Actually, compound 7g showed
the same activity of hit 2, both in the biochemical assay and in
cells. Moreover, it exhibited an increased solubility and an im-
proved selectivity for PLK1 with respect to the other two isoforms
PLK2-3, and also on a larger kinases panel, where at least 300-fold
selectivity was found.14
The most promising compounds 5a, 5b and 7g were further
evaluated in vitro for ADME properties and data were compared
with those of hit 2 (Table 3). Among the three players, compound

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I. Beria et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2969–2974
Table 2
SAR: variation at the 1-position of the pyrazole ring
N
O
HN
N
N
NH2
N
O
N
F3C
R2
N
a
a
a
a
Compound
R2
PLK1 IC50
(lM)
PLK2 IC50
>10
(
lM)
PLK3 IC50
(lM)
A2780 IC50
(lM)
Solubility pH 7 (lM)
7g
2
7e
7d
7f
7a
–(CH2)2–OH
–CH3
–(CH2)2–NH2
–(CH2)2–O–THP
–(CH2)3–NH2
–(CH2)3–N-(CH3)2
0.002 0.001
0.003 0.001
0.033 0.012
0.043 0.003
0.052 0.021
0.279b
>10
0.042 0.007
0.021 0.005
1.307 0.388
0.126 0.027
0.846 0.186
2.501b
201
72
202
35
186
200
3.519 0.063
1.439 0.078
>10
1.002 0.264
>10
>10
>10
>10
>10
>10
a
Values are means of three experiments.
Single data.
b
Table 3
In vitro ADMEa properties of selected compounds
Compound
Solubility 10% Tween 80
(mg/mL)
Permeability
PAMPAc
Clint (mL/min/kg) rat
Clint (mL/min/kg)
HLMd (1
M)
Caco-2b (Papp
)
(Papp 10ꢀ6 cm/s)
hepatocytes (1
l
M)
l
2
>3.2
3.7
3.8
High
Moderate
High
50.00
46.41
36.78
49.59
600 15
638 21
603 12
25.70 0.20
24.50 0.31
15.95 0.06
16.90 0.15
5a
5b
7g
>3.4
Moderate
165
7
a
b
c
Absorption, distribution, metabolism and excretion.
Permeability class has been ascribed using Ranitidine and N-acetyl-DL-phenylalaninamide as low or high permeable reference compounds, respectively.
Parallel artificial membrane permeability assay.
Human liver microsomes.
d
Table 4
In vivo pharmacokinetic parametersa standard deviation of selected compounds in CD1 nu/nu miceb
Compound
PK data (iv), dosec: 10 mg/kg
PK data (po), dosec: 10 mg/kg
AUC1
(l
M h)
CL (L/h/kg)
Vss (L/kg)
t1/2 (h)
Cmax
(l
M)
AUC1
(
lM h)
t1/2 (h)
Fd (%)
2
7g
4.97 0.71
8.57 1.18
4.21 0.45
2.36 0..33
3.39 0.03
1.71 0.11
0.92 0.12
0.89 0.01
0.29 0.04
0.64 0.24
1.04 0.10
2.04 0.50
1.44 0.29
1.60 0.57
21
24
a
Data of compounds 2 and 7 were compared statistically using Student’s t-test; P <1% for CL and Vss, P = 1.1% for AUC1 and P >5% for t1/2 after iv administration were found;
P = 2.5% for AUC1, and P >5% for Cmax and t1/2 after oral administration were calculated.
b
n = 3 animals per study.
Dosed as HCl in situ salt/glucosate.
Bioavailability.
c
d
Table 5
In vivo intravenous activity of compounds 2 and 7g on HCT116 tumor in nude micea
Compound
Treatment
Dose (mg/kg)
%TGImax (day)
%BWLmax (day)
2
7g
1–2 bid ꢁ 3 weekly cycles
1–3 bid ꢁ 2 weekly cycles
30
45
81 (32)
83 (24)
14 (30)
16 (15)
a
n = 7 animals per study.
7g came out as the best, showing similar permeability, in both
Caco-2 and PAMPA assay, with respect to 5a and 5b but higher
metabolic stability in rat hepatocytes (Clint = 165 vs 638 and
603 mL/min/Kg, respectively). In view of its better overall profile,
compound 7g was selected for further evaluation. In vivo pharma-
cokinetic properties of 7g were evaluated in Harlan nu/nu mice,
following intravenous (iv) and oral (po) dosing and data were com-
pared with those of compound 2 (Table 4).11,15 Compound 7g
showed a pharmacokinetic profile slightly better than 2 with high-
er AUC and Cmax, lower clearance, and acceptable oral bioavailabil-
ity (F = 24%).
The mechanism of action of compound 7g was evaluated in
different cell lines by fluorescent activated cell sorting (FACS),
western blot and immunocytochemical analysis and in analogy
to 5-dihydro-1H-pyrazolo[4,3-h]quinazoline analogs,10,11 resulted
in agreement with PLK1 inhibition (Valsasina et al., manuscript
in preparation).
Efficacy of 7g was evaluated in vivo in CD1 nu/nu mice xeno-
grafted with human HCT116 colon adenocarcinoma cells. The com-
pound 7g when administered intravenously (iv) at 45 mg/kg, bid at
days 1, 2, 3, in a weekly scheduling ꢁ 2 cycles, showed a good tu-
mor growth inhibition (TGImax = 83%, day 24) with acceptable and

I. Beria et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2969–2974
2973
Table 6
in vivo activity on additional tumor models and for toxicological
studies that led to its selection as clinical candidate.
In vivo oral activity of compounds 2 and 7g on HCT116 tumor in nude micea,b
The 2.2 Å crystal structure of the PLK1 kinase domain (residues
36–345) in complex with NMS-P937 was solved to fully character-
ize the interactions between the enzyme and inhibitor and to bet-
ter understand the source of the PLK1 selectivity.16 As expected,
NMS-P937 binds in the ATP-pocket and most of the interactions
are similar to those found in the past with a close analog.10 Specif-
ically, NMS-P937 makes a series of donor–acceptor–donor hydro-
gen bonds with the PLK1 hinge residues (Glu131 & Cys133) and
the amide moiety hydrogen bonds with Lys82 and Asp194
(Fig. 3). The pyrazolo-quinazoline core of NMS-P937 is sandwiched
between Cys67 and Phe183 and the attached ethyl hydroxyl ex-
tends into the ribose pocket. The 20-trifluoromethoxy group binds
in a pocket formed by Arg57 and the hinge segment Leu132-
Cys133-Arg134 and multipolar interactions are present between
fluorine atoms and the guanidinium group of Arg57 and the back-
bone carbonyl of Arg134. Presumably, the ‘fit’ of the 20-trifluoro-
methoxy group in this pocket plays a crucial role in obtaining
PLK selectivity versus kinases bearing a residue bulkier in the posi-
tion corresponding to Leu132, such as those present in Aurora-A
and CDK2. In addition, the 50-methylpiperazine moiety contributes
to the PLK1 selectivity with respect to PLK 2–3 since it establishes a
polar interaction with the side chain of Glu140 and the same type
of interaction is hampered in both PLK2 and PLK3 where Glu140 is
replaced by histidine.10,17
Compound
Dose (mg/kg)
%TGImax (day)
%BWLmax (day)
Death
2
7g
90
90
120
59 (18)
71 (36)
84 (36)
14 (15)
10 (36)
18 (29)
3
0
0
a
n = 7 animals per study.
1–2 daily ꢁ 4 weekly cycles treatment.
b
Control
2.0
Compound 7g
Median tumor
weight (g)
1.0
0.0
10
15
20
25
30
35
Days
Figure 2. In vivo oral antitumor activity of 7g at 60 mg/kg, once a day, 1–10
consecutive days (blue line) on HCT116 xenograft model.
In summary, we report the identification of NMS-P937, a new
PLK1 specific inhibitor that was found to be highly potent on the
target and highly selective versus PLK2 and PLK3 isoforms, as well
as in a wide kinase panel. The compound showed good solubility
and PK properties suitable for either iv or oral administration.
When tested in vivo by oral administration, NMS-P937 showed
good activity and good tolerability also after prolonged treatment.
For its favorable characteristics, NMS-P937 was selected for clinical
development. NMS-P937 is the first selective PLK1 inhibitor given
orally to enter Phase I clinical studies.
Acknowledgments
We thank the group of Assay Development and Biochemical
Screening for the biochemical assay on kinase panel, Dario Ballinari
and the group of Cell Screening for cell proliferation assay, Paolo
Cappella for FACS analysis, Jay Bertrand for crystal structure and
discussion, Daniele Donati and Eduard R. Felder for their useful
comments on the manuscript.
Figure 3. Compound 7g bound to PLK1. Hydrogen bonds are shown as red dashed
lines.
Supplementary data
reversible body weight loss (BWLmax = 16%, day 15) (Table 5).
Higher activity and better tolerability was shown by compound
7g, with respect to 2, after oral administration (po) (Table 6). When
given orally at 90 mg/kg, once a day at days 1, 2, in a weekly sched-
uling ꢁ 4 cycles, compound 2 showed low activity (TGImax = 59%)
with 3/7 death. At the same dose and scheduling, compound 7g
exhibited good activity (TGImax = 71%, day 36) without any death.
In addition, the dose could be increased to 120 mg/kg, without
death and with an improvement of activity (TGImax = 84%, day
36). The better safety profile of 7g in comparison with 2 was
reaffirmed in the 1–10 daily oral scheduling treatment, where
partial regression on 6/7 animals was obtained at 60 mg/kg with
TGImax = 89% at day 23 (Fig. 2). At the same daily dose of 7g
(60 mg/kg), compound 2 showed toxicity with 5 out of 7 death at
five days after the end of the treatment. In view of its better selec-
tivity and safety profile, together with its suitability for a more
flexible scheduling, compound 7g (NMS-P937) was selected for
Supplementary data (mechanism of action by FACS analysis and
kinase profile of compound 7g) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.03.054.
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MAP-KAPK2, MELK, MET, MNK2, MPS1, MST4, NEK6, NIM, P38 , PAK4, PDGFR,
PDK1, PERK, PIM1, PIM2, PKA , PKCb, RET, ROS1, SULU1, SYK, TLK2, TRKA,
a
a
10. Beria, I.; Ballinari, D.; Bertrand, J. A.; Borghi, D.; Bossi, R. T.; Brasca, M. G.;
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TYK2, VEGFR2, VEGFR3 and ZAP70.
15. The pharmacokinetic profiles of the compounds were investigated in overnight
fasted male nu/nu mice following a single dose given intravenously (iv) or
orally (po). Blood samples of each mouse were taken from saphenous vein at
different time and after centrifugation plasma samples were analyzed by LC/
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16. Coordinates of the PLK1 complex with compound 7g have been deposited in
the Protein Data Base under accession code 2YAC, together with the
corresponding structure factor file.
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CDK2/A, CDK2/E, CDK4/D1, CDK5/P25, CHK1, CK2, EEF2K, EGFR1, EphA2, ERK2,
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