322
R. Castelli et al. / European Journal of Medicinal Chemistry 103 (2015) 312e324
H
2
SO
organic layer is washed with half-saturated brine and then brine.
The organic phase is dried over Na SO and evaporated under
4
(50 mL). The aqueous layer is discarded, and the
buffer (11.3 g/L citric acid, 9.7 g/L sodium phosphate, pH 5.0) and
0.02% H (30% m/m in water), added immediately before use.
The reaction was stopped with 3N HCl 100 L/well and the
2 2
O
2
4
m
reduced pressure. The crude material thus obtained is purified by
silica gel column chromatography eluting at first with a 5% solu-
tion of methanol in dichloromethane, then with a mixture of
DCM:MeOH:AcOH ¼ 95:5:0.5 v/v/v to furnish 10 (215.5 mg, 79%)
absorbance was measured using an ELISA plate reader (Sunrise,
TECAN, Switzerland) at 450 nm. IC50 values were determined us-
ing one-site competition non-linear regression analysis with
Prism software (GraphPad Software Inc.). Ki were calculated using
Schild analysis. To assess the selectivity of compound 10, all EphA
(R&D Systems SMPK1) and EphB (R&D Systems SMPK2) receptors
were incubated overnight similarly to EphA2 as previously
described; biotinylated ephrin-A1-Fc or biotinylated ephrin-B1-Fc
as a white amorphous solid. R
t
HPLC (C18 150 ꢂ 4.6 mm, 5
mm,
1
H
mL/min,
O þ 0.1% HCOOH v/v): 11.95 min; m.p.: 204e207 C (dec.); H
/CD : 7.53 (d, 1H, J ¼ 7.9 Hz), 7.33
OD ¼ 1:1, 400 MHz)
l
¼ 254 nm, A:B ¼ 7:3 (A: MeOH þ 0.1% v/v HCOOH; B:
ꢁ
1
2
NMR (CDCl
3
3
d
(
d, 1H, J ¼ 8.1 Hz), 7.09 (dt, 1H, J ¼ 0.9, 8.1 Hz), 7.04 (s, 1H), 7.01 (dt,
D
(R&D Systems BT473) at their K values were used towards EphAs
or EphBs, respectively.
1
3
3
H, J ¼ 0.9, 7.9 Hz), 5.31 (d, 1H, J ¼ 4.8 Hz), 3.45e3.38 (m, 1H),
.37e3.33 (m, 1H), 3.21 (dd, 1H, J ¼ 6.8, 14.8 Hz), 2.26e2.13 (m,
H), 2.06e1.90 (m, 3H), 1.85e1.60 (m, 4H), 1.57e1.27 (m, 8H),
5.3.2. Phosphorylation of EphA2 in PC3 cells
1
.25e1.01 (m, 6H), 0.98 (s, 3H), 0.86 (d, 3H, J ¼ 6.5 Hz) 0.63 (s, 3H);
EphA2 phosphorylation was measured in cell lysates using
DuoSet IC Sandwich ELISA (R&D Systems DYC4065) following
manufacturer's protocol. Briefly, 96-well ELISA high binding plates
(costar 2592) were incubated overnight with 100 mL/well of the
specific capture antibody diluted in sterile PBS at the proper
working concentrations. Next day the wells were washed and
blocked for 1 h and 100 mL/well of lysates were added for 2 h. Then,
13
®
C NMR (CDCl
37.4, 128.5, 124.1, 122.2, 119.7, 119.0, 112.1, 110.3, 72.0, 57.6, 56.6,
4.1, 51.1, 43.1, 42.6, 40.6, 38.1, 37.3, 36.3, 33.8, 32.8, 32.7, 32.5, 31.9,
3
/CD
3
OD ¼ 1:1, 100 MHz) d: 175.8, 175.0, 141.7,
1
5
2
5
8.8, 28.1, 25.0, 21.8, 19.9, 18.8, 12.3; MS-ESI, calc. for C35
H
48
N
2
O
4
:
þ
60.36, found: 561.6 [M þ H ].
5
.3. Pharmacology
wells were incubated with the specific detection antibody and the
phosphorylation level was revealed utilizing a standard HRP format
and tetra-methylbenzidine through a colorimetric reaction read at
450 nm. Each step was performed at room temperature and fol-
lowed by washing each well.
All culture media and supplements were purchased from
Euroclone (Milan, Italy). Recombinant proteins and antibodies were
from R&D systems. Cells were purchased from ECACC (Porton
Down, UK). Leupeptin, aprotinin, NP40, MTT, tween20, BSA and
salts for solutions were from Applichem (Darmstadt, Germany);
analytical grade extraction solvents, bile acids, EDTA and sodium
orthovanadate were from Sigma (St. Louis, MO, USA). Mouse
plasma was obtained from male mice (Charles River Laboratories,
Milan, Italy). Animals were housed, handled, and cared accordingly
to the European Community Council Directive 2010/63/UE, in Ital-
ian regulations (DL 26/2014) and with local ethical committee
guidelines for animal research. Pooled plasma was obtained by
cardiac puncture, collected into heparinized tubes, centrifuged
5.3.3. Phosphorylation of the EphA2 kinase domain
The kinase activity of the EphA2 kinase domain (Millipore, 14-
560) was evaluated with the EphA2 kinase assay/inhibitor assay kit
from Abnova (KA0061). This screening kit uses a horseradish
peroxidase coupled anti-phosphotyrosine monoclonal antibody as
a reporter molecule in a 96-wells ELISA format. The manufacturer
ꢁ
ꢁ
(
1900 g, 4 C, 10 min) and stored at ꢀ70 C until use. Mouse liver S
9
5.3.4. In vitro angiogenesis
fractions were obtained from the same mice, transcardially
perfused with 20 mL ice-cold KCl 0.15 M. Livers were removed,
weighted, sliced into small pieces, and homogenized on ice with an
ice-cold phosphate-buffered saline (PBS) solution (0.01 M, pH 7.4,
Twenty-four well tissue culture plates were coated with BD
ꢁ
Matrigel (80
m
L/well) and incubated for 30 min at 37 C in order to
form a thin layer of gel on the bottom of the wells. HUVEC were
treated with compound 10 (or DMSO as control) and 3.2ꢂ105 cells/
well were seeded on Matrigel. After 15 h of incubation the cells
were fixed with 3.7% formaldehyde for 15 min at room tempera-
ture. Photographs were taken through a digital camera mounted on
a microscope and the number of polygons formed was counted.
Data were normalized to control (100%).
2
4
0% w/v). The S
9
fraction was obtained by centrifugation (9000 g,
ꢁ
ꢁ
C, 30 min) and stored at ꢀ70 C until use. Protein content was
quantified by the colorimetric Bicinchoninic Acid assay (Pierce,
Rockford, IL, USA), employing bovine serum albumin (BSA) as
standard.
5.3.1. ELISA assays and Ki/IC50 determination on EphA2
5.4. Pharmacokinetic studies
ELISA assays were performed as previously described [23]. 96-
well ELISA high binding plates (Costar #2592) were incubated
5.4.1. Single dose pharmacokinetic study in mice
ꢁ
ꢀ1
overnight at 4 C with 100
m
L/well of 1
m
g mL EphA2-Fc (R&D
Compounds 10 and 3 were formulated in 0.5% methylcellulose
(10/90 v/v) for the oral administration. Test compounds were orally
dosed as a single esophageal gavage at 30 mg/kg to male mice. Each
group consisted of three mice. Blood samples were collected via tail
puncture at 6e8 time points in the following range: 5 minꢀ6 h (oral
6
39-A2) diluted in sterile phosphate buffered saline (PBS, 0.2 g/
ꢀ1
2 4 2 4
L KCl, 8.0 g/L NaCl, 0.2 g L KH PO , 1.15 g/L Na HPO , pH 7.4).
Next day the wells were washed with washing buffer (PBS þ 0.05%
tween20, pH 7.5) and blocked with blocking solution (PBS þ 0.5%
ꢁ
BSA) for 1 h at 37 C. Compounds were added to the wells at
route). Whole blood samples were centrifuged at 5000 rpm for
ꢁ
ꢁ
proper concentration in 1% DMSO and incubated at 37 C for 1 h.
10 min, and the resulting plasma samples were stored at ꢀ20
C
Biotinylated ephrin-A1-Fc (R&D Systems BT602) was added at
pending analysis. Compounds 10 and 3 were dosed in mouse
plasma by HPLC-ESI-MS/MS employing a Thermo Accela UHPLC
gradient system coupled to a Thermo TSQ Quantum Access Max
triple quadrupole mass spectrometer (Thermo Italia, Milan, Italy)
equipped with a heated electrospray ionization (H-ESI) ion source.
(see Supplementary data for details on the HPLC-ESI-MS/MS
method) Xcalibur 2.1 software (Thermo Italia, Milan, Italy) was
ꢁ
3
7 C for 4 h at its K
D
value in displacement assays or in a range
from 1 to 2000 ng/mL in saturation studies. Then wells were
washed and incubated with 100 L/well Streptavidin-HRP (Sigma
m
S5512) for 20 min at room temperature, washed again and
finally incubated at room temperature with 0.1 mg/mL tetrame-
thylbenzidine (Sigma T2885) reconstituted in stable peroxide