Optimization of EphA2 antagonists based on a lithocholic acid core led to the identification of UniPR505, a new 3α-carbamoyloxy derivative with antiangiogenetic properties

Article abstract of DOI:10.1016/j.ejmech.2020.112083

The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5β-cholan-24-oic acids and 5β-cholan-24-oyl L-β-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5β-cholan-24-oyl]-L-β-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.

Full text of DOI:10.1016/j.ejmech.2020.112083

European Journal of Medicinal Chemistry 189 (2020) 112083
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Optimization of EphA2 antagonists based on a lithocholic acid core led
to the identification of UniPR505, a new 3a-carbamoyloxy derivative
with antiangiogenetic properties
,
,
Matteo Incerti a 1, Simonetta Russo a 1, Miriam Corrado a, Carmine Giorgio a,
Vigilio Ballabeni a, Paola Chiodelli b, Marco Rusnati b, Laura Scalvini a,
Donatella Callegari a, Riccardo Castelli a, Federica Vacondio a, Francesca Ferlenghi a,
Massimiliano Tognolini a **, Alessio Lodola a
,
, *
a Department of Food and Drug, University of Parma, 43124, Parma, Italy
b Department of Molecular and Translational Medicine, University of Brescia, 25123, Brescia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The EphA2 receptor has been validated in animal models as new target for treating tumors depending on
angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on
structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor,
Received 29 July 2019
Received in revised form
16 January 2020
Accepted 19 January 2020
Available online 22 January 2020
namely 5
develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a
result of our exploration, we identified UniPR505, N-[3 -(Ethylcarbamoyl)oxy-5 -cholan-24-oyl]-
b-cholan-24-oic acids and 5b-cholan-24-oyl L-b-homotryptophan conjugates, with the aim to
a
b
L-b-
homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block
EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.
© 2020 Elsevier Masson SAS. All rights reserved.
Keywords:
EphA2
Lithocholic acid
Protein-protein interaction
SAR
Molecular modelling
Angiogenesis
1. Introduction
a migratory phenotype, and the formation of new blood vessels by
angiogenesis [7] and vasculogenic mimicry [8,9]. In this scenario,
The EphA2 receptor is a member of the Eph receptor super-
family of receptor tyrosine kinases [1] whose biological activity is
mainly controlled by the ephrin-A1 membrane protein [2]. The
EphA2 receptor is overexpressed in a large number of cancers,
including brain, prostate, breast, colon, pancreatic and lung cancers
[3,4]. Compelling evidences demonstrate that its uncontrolled
activation concurs to cancer progression [5] by sustaining an effi-
cient self-renewal of tumor-propagating cells [6], the acquisition of
the EphA2 receptor has emerged as a promising target for the
development of new, alternative antiangiogenic therapies [10,11].
Three key strategies can be used to inhibit the tumorigenic activity
of the EphA2 receptor. The first is based on ATP-mimicking agents
targeting the intracellular kinase domain of Eph receptor [12,13].
The second targets the SAM domain of EphA2 [14,15], an intracel-
lular region of the receptor endowed with a regulatory role on its
kinase activity [16]. The third relies on molecular agents targeting
the extracellular ligand-binding domain (LBD) of EphA2 [17,18],
thus preventing its direct interaction with ephrin-A1 [19,20].
Regarding the first approach, kinase inhibitors targeting Eph
receptors were initially synthetized starting from the benzamide
scaffold of nilotinb (Chart 1). This approach led to the dual EphB4/
VEGFR inhibitor NVP-BHG712 [10] and the two multi-kinase in-
hibitors (ALW-II class) developed by the Gray group at the Harvard
Medical School in Boston [21]. While not specifically selective for
the Eph kinases, these compounds were used to corroborate the
Abbreviations: PPI, protein-protein interaction; EphA2, ephrin receptor A2; LCA,
lithocholic acid; ELISA, enzyme-linked immunosorbent assay; CAM, chorioallantoic
membrane.
* Corresponding author.
** Corresponding author.
E-mail addresses: massimiliano.tognolini@unipr.it (M. Tognolini), alessio.
lodola@unipr.it (A. Lodola).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.112083
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

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M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
Chart 1. Relevant kinase inhibitors targeting the intracellular kinase domain of Eph receptors.
role played by the Eph-ephrin system in angiogenesis and in insulin
homeostasis. A second generation of Eph kinase inhibitors was
developed by Caflisch and Nevado at the University of Zurich. This
includes three distinct classes of compounds exemplified by i. the
pyrimidoisoquinolinone PYR-B1 [22], ii. the xanthine XAN-B9 [23],
and iii. the pyrrolo[3,2-b]quinoxaline QUI-B4 [24]. These inhibitors
displayed nanomolar activity on EphB4 and were also featured by a
fair degree of selectivity, according to a kinome scan assay.
around 3
(3 -carbamoyloxy-5
jugate (B-series) by modification of the substituent at the nitrogen
atom of 3 -carbamoyloxy group in the search for new EphA2
antagonists.
a
-carbamoyloxy-5
b
-cholan-24-oic acid (A-series) and N-
a
b-cholan-24-oyl)-
L-b-homo-tryptophan con-
a
2. Chemistry
While the development of agent targeting the SAM domain is
still in its infancy with only few active peptides being available [25],
the disruption of Eph-ephrin interaction is reaching a maturity
stage with several peptidomimetics [26] and small molecules re-
ported in the literature [27]. Within the pool of small molecules
targeting EphA2 LBD [28], bile acid conjugates and related de-
rivatives have emerged as promising EphA2 antagonists useful for
treating highly vascularized tumors (Chart 2) [29]. Besides lith-
Lithocholic acid (LCA, 1) was purchased from Sigma (Milano,
Italy) while compounds 3 and 12 were synthesized according to the
procedure described in Refs. [31,35]. The 3
cholan-24-oic acid 4 and its corresponding
conjugate 13 were synthesized following the steps reported in
Scheme 1. The inversion of configuration of the 3 -hydroxyl group
of LCA resulted from the Mitsunobu reaction between LCA methyl
b-carbamoyloxy-5
b-
L-b-homo-tryptophan
a
ester 4e and acetic acid. The obtained 3
b
-acetyl derivative was then
-carbamoyloxy-5 -cholan-
ocholic acid (LCA, 5
inhibitor of the EphA2-ephrin-A1 association [30], its
tryptophan conjugate, known as UniPR129 (N-(3 -hydroxy-5
cholan-24-oyl)- -homotryptophan, 2) was identified as a potent
b
-cholan-24-oic acid 1), a weak competitive
hydrolyzed to give compound 4c. The 3
b
b
L-b-homo-
24-oic acid 4 was synthesized reacting the methyl ester 4d with
potassium isocyanate, and the following hydrolysis of the ester
a
b-
L-b
group with NaOH. The N-(3
b-carbamoyloxy-5b-cholan-24-oyl)-L-
inhibitor of this protein-protein interaction [31,32]. The use of
compounds 1 and 2 as pharmacological tools has been limited by
their suboptimal pharmacodynamic and pharmacokinetic proper-
ties [33]. Our recent investigations demonstrated that the
b
-homo-tryptophan 13 was obtained by conjugation of compound
4 with the L-b-homo-tryptophan methyl ester hydrochloride, using
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(EDCI) as coupling agent. The resulting amide 13a was then hy-
drolyzed with NaOH to give the title compound 13.
replacement of the 3a-hydroxyl group with the 3a-carbamoyloxy
one on the nucleus of compound 2 led to inhibitors featuring better
pharmacodynamics or pharmacokinetics properties. However, the
success of this exploration was only partial as compound 12
(UniPR502), displayed cytotoxic effects on HUVEC cells at high
doses [34]. Here, we tried to exploit the aforementioned carba-
moyloxy functionality to introduce a point for chemical diversifi-
Compounds 5e11 and 14e20 were synthesized according to the
procedure reported in Scheme 2. The N-substituted carbamates at
position 3 of LCA 5e11 were obtained reacting the LCA methyl ester
4e with the corresponding N-alkyl/aryl-isocyanate. The obtained
methyl ester derivatives 5a-11a were hydrolyzed to yield com-
pounds 5e11. These compounds were then coupled with the L-b-
homo-tryptophan methyl ester hydrochloride following the same
procedure described in the Scheme 1, to provide the corresponding
cation into the 5b-cholan-24-oic acid scaffold, to design better Eph-
ephrin antagonist. We report the result of our SAR investigation

M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
3
Chart 2. Structures of EphA2 receptor antagonists featured by a the 5b-cholan-24-oic acid scaffold.
Scheme 1. Reagents and conditions: i) H2SO4, CH3OH, r.t., 4 h; ii) PPh3, DIAD, CH3COOH, THF, 0 ꢀC e r.t., overnight; iii) KOH(aq) 15%, EtOH, reflux, 3 h; iv) H2SO4, CH3OH, r.t., 4 h; v)
NCOK, CF3COOH, toluene, reflux, overnight; vi) NaOH(aq) 15%, EtOH, r.t. 1 h; vii) NMM, EDCI, CH2Cl2, r.t., overnight; viii) NaOH(aq) 15%, EtOH, r.t. 1 h.
amide conjugates 14a-20a. The final products 14e20 were obtained
by hydrolysis of the methyl ester.
[36] were used as template structures to develop two focused series
of N-primary carbamates that were then evaluated for their ability
to prevent ephrin-A1 binding to EphA2 by using a previously
validated ELISA binding assay [32]. The IC50 values for the reference
and newly synthetized compounds are reported in Tables 1 and 2
(A- and B-series, respectively). Previous investigations have
shown that compound 3 [35] and 12 [36] likely engage the EphA2
receptor with a different binding mode, i.e. with the free-carboxyl
group forming a salt bridge with Arg159 in case of 3 or with
Arg103 as for compound 12 (Fig. 1). In line with these findings, a
3. Results and discussion
3.1. SAR analysis of 3-carbamoyloxy derivatives of 5
b-cholan-24-
oic acid and 5 -cholan-24-oyl-L- -homo-tryptophan conjugates
b
b
3
a-carbamoyloxy-5
b
-cholan-24-oic acid 3 [35] and N-(3
a-car-
bamoyloxy-5 -cholan-24-oyl)-L-b
b
-homo-tryptophan conjugate 12

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M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
Scheme 2. Reagents and conditions: i) RNCO, toluene, reflux, overnight; ii) NaOH(aq) 15%, EtOH, r.t. 1 h; iii) NMM, EDCI, CH2Cl2, r.t., overnight; iv) NaOH(aq) 15%, EtOH, r.t. 1 h.
different profile for the two newly explored series is expected in
term of SAR.
As a first step in our exploration, we investigated the role of the
absolute configuration of the carbamoyloxy group at position 3 of
inserted on the nitrogen of the 3
acid. The obtained compound 5 showed an IC50 of 30
a
-carbamoyloxy-5
b
-cholan-24-oic
M, slightly
M).
m
higher than that of the reference compound 3 (IC50 ¼ 17
m
Further, a remarkable improvement of potency was obtained with
the elongation of two carbon units of the ethyl substituent. The N-
butyl(carbamoyloxy)-derivative 6 resulted indeed more potent
the steroid scaffold. We thus synthesized the 3b-carbamoyloxy
derivatives of both free acid derivative and -homo-tryptophan
L-b
conjugate, compound 4 (Table 1) and 13 (Table 2). The inversion of
configuration of the stereocenter in position 3 was tolerated in the
case of the A-series, while it led to a loss of inhibitory potency in the
case of the B-series. Then, we focused our attention on the syn-
than compound 3, with an IC50 of 3.7
tion of a N-hexyl substituent provided a compound with a good
potency (7, IC50 ¼ 3.3 M). A further elongation of the linear alkyl
chain was still tolerated, as revealed by the IC50 value of the octyl-
derivative 8 (IC50 ¼ 4.5 M). Introducing a small branched alkyl
substituent (isopropyl) resulted in compound 9, which turned out
to be endowed with an IC50 equal to 12 M, thus less potent than
mM. Similarly, the introduc-
m
thesis of compounds with the
a
configuration at the position 3 of
m
the steroidal nucleus of both A- and B- series.
We next interrogated the effect on the inhibitory potency of
substituents with varying stereoelectronic properties at the nitro-
m
the linear alkyl chain-derivatives 6e8. The inclusion of the iso-
propyl group into a cyclohexyl substituent led to compound 10,
gen atom of 3a-carbamoyloxy group. At first, an ethyl group was
Fig. 1. Binding mode of 3 (left panel) [35] and 12 (right panel) [36] within the LBD domain of the EphA2 [37] deduced by free-energy simulations. EphA2 surface is coloured
according to the electrostatic potential (red, negative; blue positive; green neutral), while ligand carbon atoms are represented with cyan tube. (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of this article.)

M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
5
Table 1
Table 2
IC50 values for 3-substituted derivatives of 5
b
-cholan-24-oic acid 3e11 obtained
IC50 values for 5
b
-cholan-24-oyl-
L
-b
-homo-tryptophan derivatives 12e20 measured
from EphA2-ephrin-A1 displacement experiments.
in EphA2-ephrin-A1 displacement experiments.
Cpd
1
R-
IC50
(
m
M)a
Cpd
2
R-
IC50 (m
M)a
79b (67e93)
0.91 (0.80e1.1)b
3
17 (8.7e35)
12
1.2 (0.85e1.6)
4
5
9.1 (6.1e14)
30 (20e44)
13
14
5.2 (3.0e9.1)
0.95 (0.60e1.5)
6
3.7 (2.9e4.6)
3.3 (2.4e4.5)
4.5 (3.2e6.3)
12 (8.9e14)
2.7 (2.1e3.4)
15
16
17
18
19
1.1 (0.70e1.8)
1.5 (1.2e2.0)
15 (13e17)
7
8
9
7.1 (3.9e13)
1.2 (0.94e1.5)
10
11
38 (21e68)
20
5.5
a
a
Values are the mean from at least three independent experiments. Numbers in
Values are the mean from at least three independent experiments. Numbers in
brackets denote the 95% confidence interval for IC50
.
brackets denote the 95% confidence interval for IC50
.
b
b
Value from Ref. [36].
Value from reference [36].
featuring a higher potency (IC50 ¼ 2.7
mM) and supporting the
tryptophan conjugate 12, prototypical member of the B-series. In
this case, the substitution with ethyl- and butyl-groups led to
importance of undertaking lipophilic interaction at the EphA2 re-
ceptor. However, the replacement of a cyclohexyl substituent with a
phenyl one was barely tolerated, as indicated by the low inhibitory
highly potent compounds, 14 and 15, with an IC50 value of 0.95
and 1.1 M, respectively, thus in range with the reference com-
pound 12 (IC50 ¼ 1.2 M). The elongation of the linear alkyl chain
provided by hexyl substituent led to compound 16, showing a
M) with respect to the shorten N-
mM
m
potency of 11 (IC50 ¼ 30
mM). It could be devised that, in addition to
lipophilicity, a mutual stereoelectronic complementarity between
the inhibitor and EphA2 receptor is indeed critical.
m
comparable potency (IC50 ¼ 1.5
m
Docking of 5e11 within the ligand binding domain of EphA2 (see
Experimental section for details) reveals that these compounds can
assume a pose comparable to that of 3, i.e. with the carboxylic
group interacting with Arg159 and the chain emerging from the
carbamic nitrogen atom pointing toward a lipophilic pocket
delimited by two cysteines (Cys70 and Cys188) involved in a di-
sulfide bridge (Fig. 2). The most active inhibitors of this series (7, 8
and 10) undertakes several productive contacts within the EphA2
receptors. This is not the case for compound 11 that due to the
accommodation assumed by its phenyl ring undertakes several
clashes with the protein surface.
alkyl substituted-carbamates. The further lengthening of the alkyl
chain obtained with the octyl substituent resulted detrimental for
binding EphA2 receptor. As demonstrated by compound 17, that
displayed a marked decrease of potency (IC50 ¼ 15
mM), the hy-
drophobic pocket of EphA2 receptor accounts limited space to host
the alkyl-substituent of the carbamate-moiety. The introduction of
an iso-propyl group led to
a less potent derivative (18,
IC50 ¼ 7.1
m
M) with respect to the reference compound 12. Analo-
gously, a cycloalkyl-substituent was tolerated better than a free
branched alkyl group, as demonstrated by the cyclohexyl-
derivative 19 that showed an inhibitory potency (IC50 ¼ 1.0
mM)
The same linear and branched substituents were inserted on the
nitrogen of the N-(3a-carbamoyloxy-5b-cholan-24-oyl)-L-b-homo-
similar to that of the best compounds of the series. Finally, the

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M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
The docking poses for compounds 14e20 reinforce the concept
that the -homo-tryptophan moiety is important for the activity,
L-b
as its aromatic eNH group is involved in a hydrogen bond with
Asp53. In agreement with this model, a previously reported SAR
study around UniPR129 showed that the replacement of the indole
with a thiophen or a naphthyl ring led to a moderate reduction in
the inhibitory potency [36]. On the same line, a significant lost in
the potency was observed inverting the chirality of the
bearing the indol-3-ylmethyl chain.
b-carbon
3.2. Activity on EphA2 phosphorylation in human prostate
adenocarcinoma (PC3) cells
Among all the synthetized compounds, we focused our atten-
tion on those belonging to the B-series which displayed higher
inhibitory potency in the displacement assay, and we selected an-
tagonists with an IC50 value lower than 5 mM. These compounds
were then tested for their ability to interfere with EphA2 activation
in human prostate adenocarcinoma (PC3) cells, which naturally
overexpress the EphA2 receptor [38]. PC3 cells were incubated for
20 min with increasing concentration of compounds 12, 14e16 and
Fig. 2. Docking of compound 5e11 within the LBD domain of the EphA2. EphA2 sur-
face is coloured in white, while ligand carbon atoms are represented with different
colours. The most actives are in cyan (compound 8) and in magenta (compound 10).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
19, further stimulated for 20 min with ephrin-A1 (0.25 mg/ml) and
then evaluated for EphA2 phosphorylation using an ELISA assay. All
the compounds dose-dependently inhibited EphA2 phosphoryla-
tion, with 14 emerging as the most potent compound of the series,
replacement of the N-cyclohexyl group with a N-phenyl one led to
compound 20 which had a slightly higher IC50 value, indicating that
aromatic substituents are tolerated by EphA2 receptor at this po-
sition. Overall, this exploration points out that while it is possible to
with an IC50 equal to 1.5 mM, that is very close to the inhibitory
activity displayed in the cell-free displacement assay (Fig. 4). All the
compounds did not alter EphA2 phosphorylation on their own
(data not shown) and did not exert any aspecific toxic effect on PC3
cells, as assessed by LDH assay after 2h of compound incubation
(Fig. S1, supplementary data). Taken together, these results indicate
the specificity of the observed reduction in the phospho-EphA2
level, that can be genuinely linked to a direct action of the com-
pounds at the EphA2 receptor.
reach single-digit micromolar concentration with 3-substituted 5
cholan-24-oic acid derivatives (as for compounds 3, 6 or 10), sub-
micromolar potency can be reached only with 5 -cholan-24-oyl-
-homo-tryptophan conjugates (as for compound 14).
Docking of 14e20 within the LBD of EphA2 shows that these
compounds are able to assume the same binding pose of the parent
compound 12, i.e. with the carboxylic group of the -homo-
b-
b
L-
b
L-b
tryptophan moiety forming a salt bridge with Arg103 and the
lipophilic substituent at the carbamic nitrogen atom pointing to-
ward the exit of the EphA2 binding pocket, flanking Ile58 (Fig. 3).
Such accommodation accounts for the tolerance of EphA2 with
respect to the size and shape of the substituent in position 3 of
3.3. Molecular mechanism of action of compound 14 at the EphA2
receptor
Among all the compounds tested in the EphA2 phosphorylation
assay, we focused our attention on compound 14 whose mecha-
nism of action was investigated at a molecular level. To this aim,
binding curves describing ephrin-A1 association to EphA2 up to
saturation were built in the presence of increasing concentrations
of compound 14 (Fig. 5). The parallel rightward shift of saturation
nearly all the tested L-b-homo-tryptophan conjugates (i.e. com-
pounds 14e16, 18e20) and for the loss of activity displayed by 17, in
light of its long octyl chain which extends beyond the EphA2 pocket
reaching the bulk solvent.
Fig. 3. Docking of compounds 14e20 within the LBD domain of the EphA2. EphA2
surface is coloured in white, while ligand carbon atoms are represented with different
colours. The most actives are in magenta (compound 14) and in orange (compound 15).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
Fig. 4. Concentration-dependent inhibition of ephrin-A1-induced activation of EphA2.
Cells were pretreated for 20 min with 0.3% DMSO or with the indicated concentrations
of compounds and then stimulated for 20 min with ephrin-A1-Fc 0.25
mg mL-1.
Phospho-EphA2 levels are relative to ephrin-A1-Fc
means st.dev of at least 3 independent experiments.
þ
0.3% DMSO. Data are the

M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
7
Table 3
doseeresponse curves with no significant alteration of the maximal
IC50 values of compound 14 measured in displacement experi-
ments for EphA1-EphA8 and EphB1-EphB4, EphB6, in the pres-
ence of biotinylated ephrin-A1-Fc or ephrin-B1-Fc.
response suggests a competitive mechanism of action for com-
pound 14. Relevant to note, with an inhibitory constant (Ki) equal to
0.3 mM, compound 14 is among the most potent antagonists of the
EphA2 receptor reported in the literature, slightly superior to its
close analogue 12, and comparable to the reference antagonist
UniPR129 [32], which were reported to have Ki values of 0.8 and
Receptor subtype
IC50 (m
M)a
EphA1
EphA2
EphA3
EphA4
EphA5
EphA6
EphA7
EphA8
EphB1
EphB2
EphB3
EphB4
EphB6
>30
0.95 (0.60e1.5)
4.5 (2.8e7.1)
6.4 (3.1e13)
4.4 (2.5e7.9)
18 (9.2e36)
>30
7.7 (4.0e15)
>30
14 (6.5e29)
11 (4.9e25)
>30
0.4 mM, respectively.
3.4. Selectivity profile of compound 14 on a panel of Eph receptors
Beside EphA2-EphinA1, other Eph-ephrin signaling axes are
involved in physiological and pathological processes, prompting us
to evaluate the selectivity of compound 14. To this aim, its ability to
interfere with ephrin binding to EphA1-EphA8 and EphB1-EphB4
receptor subtypes was evaluated in a displacement assay where
biotinylated ephrin-A1-Fc and ephrin-B1-Fc were employed as
EphA or EphB receptor ligands, respectively. Diverging from pre-
>30
bNumbers in brackets denote the 95% confidence interval for IC50
.
a
Values are the mean from at least three independent
experiments.
viously reported 3a-hydroxy-5b-cholan-24-oyl amino acid conju-
gates (including 12, able to engage all the Eph receptors with
comparable inhibitory potency [34]), compound 14 demonstrated a
moderate preference for the EphA2 (Table 3). Further, the lack of
activity on some receptors (i.e. EphA1, EphA7, EphB1, EphB4 and
EphB6) suggests that the position 3 on the 5b-cholan-24-oyl core
could be exploited for improving receptor selectivity.
3.5. Antiangiogenic properties of compound 14 on chicken egg CAM
assay
The EphA2-ephrin-A1 signaling axis has been reported to be
critical for angiogenesis [39]. Compounds able to disrupt EphA2-
ephrin-A1 interaction are expected to dramatically hamper the
formation of blood vessels and are potentially useful for cancer
treatment. Fig. 6 shows that compound 14 dose-dependently in-
hibits the ability of HUVE cells to organize themselves in relatively
large vessels in the presence of a polymeric scaffold known as
Matrigel [40]. The IC50 for this process was close to 3 mM, consistent
with the activity observed in both the displacement assay and the
phosphorylation test. Again, at the tested concentrations, com-
pound 14 did not display cytotoxic effects on HUVECs when tested
with LDH assay after 2 and 16 h of incubation and with MTT after
Fig. 6. Angiogenesis assay for compound 14. The number of polygons measured is
relative to PBS solution with 1% DMSO, used as the control. The histogram shows the
means of three independent experiments SEM. One-way ANOVA followed by Tukey’s
post-test was performed to compare the control to all other conditions. *P < 0.05,
**P < 0.01.
16 h up to 30 mM (Fig. S2, supplementary data).
In light of these encouraging results, the antiangiogenic prop-
erties of compound 14 were investigated in the chick chorioallan-
growth factors. It is also possible to evaluate the inhibitory effect of
small molecules [41]. As the VEGF/VEGFR and Eph-ephrin systems
cooperate in promoting the formation of blood vessels [42,43] we
evaluated the effect of compound 14 on VEGF-dependent neo-
vascularization. VEGF alone determined a substantial increase in
the number of vessels that was significantly blocked by compound
14 (Fig. 7). On the opposite, compound 14 has no effect on the basal,
unstimulated angiogenesis of the CAM, further adding to the
specificity of its inhibitory effect (Fig. 7).
toic membrane (CAM), which is
a
model of in vivo
neovascularization. In the CAM model, it is possible to follow the
formation of primitive vessels and their differentiation to a func-
tional arteriovenous system in the presence of soluble angiogenic
Differently to what observed for the EphA2 antagonist 14
(UniPR505), the parent compound 12 (UniPR502) promoted the
growth of new vessels when tested alone in the CAM model (Fig. S3,
supplementary data). We ascribed this phenotypic response to the
low selectivity displayed by UniPR502 in light of its ability to
target all the members of the Eph receptor family with comparable
potency. In this respect, the higher selectivity displayed by com-
pound 14 (Table 3), with a moderate preference for EphA2, leads to
an improved pharmacological profile when this compound is tested
on endothelial cells.
Fig. 5. Saturation curves of biotinylated ephrin-A1-Fc on EphA2 built in presence of
increasing concentration of compound 14. The inhibition constant (Ki) was calculated
using Prism non-linear regression analysis. Data are the means st.dev. of three in-
dependent experiments.

8
M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
Fig. 7. (a) Number of vessels per egg in presence of VEGF (positive control) or PBS solution (negative control) and/or after adding 10
mM of compound 14. Data are the mean SEM
of 3 independent experiments ***: p < 0.001, one-way ANOVA followed by Dunnett’s post-hoc test. (b) Representative photographs of CAMs incubated with VEGF and 14.
4. Conclusions
characterized as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), bs (broad signal). Mass spectra were recorded on an
Applied Biosystem API-150 EX system spectrometer using an ESI
interface. Compounds 3e23 were prepared according to the syn-
thetic procedures described in the Supplementary Data. The purity
of each compound was assessed by HPLC/MS and elemental anal-
ysis (ThermoQuest FlashEA 1112 elemental analyzer for C, H, and
N). All tested compounds were >9 5% pure by HPLC analysis and
had acceptable C, H, N analysis results (within 0.4 of theoretical
values).
The EphA2 receptor is a key factor in several cancers where its
activity correlates with tumor stage and progression particularly in
the case of glioblastoma [29]. Although some promising agents that
target the EphA2 receptor kinase domain or that bind and mask its
ectodomain have been already described, no drugs acting specif-
ically on this receptor have been approved so far. In the present
work, we describe the synthesis and pharmacological character-
ization of compound 14, a new EphA2 antagonist with sub-
micromolar affinity for the EphA2 LBD domain and able to block
ephrin-dependent signals in cancer cells at low micromolar con-
centration. Consistently with its activity on the Eph-ephrin system,
compound 14 markedly blocks the formation of new blood vessels
in a CAM assay, in the presence of growth factors. Compared to
previously reported EphA2 antagonists featured by a 5
oyl core [36] or by a
14 displays higher selectivity for the EphA2 receptor and can be
thus considered as one of the most interesting antagonist reported
so far, which deserves further investigations in animal models of
cancer.
5.2. Molecular modelling
Docking studies were performed with Glide [46] using the
atomic coordinates of the EphA2 receptor (pdb code: 3HEI [37])
taken from equilibrated complexes (by plain molecular dynamics
simulations) of its ligand binding domain with 1 (LCA, see Ref. [35])
or 2 (UniPR129, see Ref. [36]). Compounds 5e11 were docked in the
EphA2 structure equilibrated with 1, while 14e20 were docked in
the EphA2 structure equilibrated with 2. In both cases, docking
grids were centered in the channel of the EphA2 receptor delimited
by Arg103, Phe108, Phe156 and Arg159. Dimensions of enclosing
and bounding boxes were set to 20 and 10 A on each side,
respectively, and van der Waals radii of protein atoms were not
scaled during grid generation. The structures of the docked com-
pounds were built in Maestro [47] and then energy-minimized
with MacroModel [48] applying the OPLS3e force field [49] to an
energy gradient of 0.01 kcal/(mol A). Docking simulations were
performed with Glide in Standard Precision mode [50]. In case of
compounds 5e11, only poses superimposable to that 1 were
retained for visual inspection and analysis, while for 14e20 only
poses similar to that of compound 2 were considered.
b-cholan-24-
D
5-cholen-24-oyl structure [44,45], compound
5. Experimental section
5.1. Chemistry
Unless otherwise noted, reagents and solvents were purchased
from commercial suppliers and were used without purification. The
progress of the reaction was monitored by thin-layer chromatog-
raphy with F254 silica-gel precoated sheets (Merck, Darmstadt,
Germany). UV light and a solution of ammonium molybdate and
ceric sulfate in aqueous sulfuric acid (5% v/v) were used for
detection. Flash chromatography was performed using Merck
silica-gel 60 (Si 60, 40e63
mm, 230e400 mesh ASTM). Dichloro-
5.3. Pharmacology
methane was dried by distillation over calcium hydride. All re-
actions were carried out using flame-dried glassware under
atmosphere of nitrogen. Melting points were determined on a
Gallenkamp melting point apparatus and were not corrected. The
1H NMR and 13C NMR spectra were recorded on a Bruker Avance
5.3.1. ELISA assays and IC50 determination on EphA2-ephrin-A1
binding
ELISA assays were performed as previously described [30].
Briefly, 96-well ELISA high binding plates (Costar #2592) were
400 spectrometer (400 MHz); chemical shifts (
d
scale) are reported
coated with 100 mL/well of 1 m
g mLꢁ1 (EphA2-Fc (R&D, Minneap-
in parts per million (ppm). 1H NMR spectra are reported in the
following order: multiplicity, approximate coupling constants (J
value) in Hertz (Hz) and number of protons; signals were
olis, MN, USA 639-A2) and incubated overnight at 4 ꢀC. The wells
were washed 3 times with washing buffer solution and blocked for
1 h at 37 ꢀC with a solution containing PBS þ0.5% BSA. Afterwards,
̊

M. Incerti et al. / European Journal of Medicinal Chemistry 189 (2020) 112083
9
the wells were washed again, and compounds were added at
and the untreated cells.
proper concentrations in 1% DMSO and incubated at 37 ꢀC for 1 h.
Then, biotinylated ephrin-A1-Fc (R&D Systems, BT602) was added
at its KD value for displacement studies or in a range from 1 to
2000 ng mLꢁ1 for saturation studies. Binding of biotinylated eph-
rin-A1-Fc to EphA2 was detected after 4 h through a standard
Streptavidin-HRP-tetramethylbenzidine reaction and read in an
ELISA plate reader (Sunrise, TECAN, Switzerland) at 450 nm. IC50
and Ki values were determined using one-site competition non-
linear regression analysis with Prism software (GraphPad Soft-
ware Inc.). The selectivity of compound 14 was measured by coating
the plate with all the EphA (R&D Systems, #SMPK1) and EphB (R&D
Systems, #SMPK2) receptors and biotinylated ephrin-A1-Fc or
biotinylated ephrin-B1-Fc (R&D Systems, #BT473) were used to
study the binding on EphAs or EphBs, respectively, as described
above.
5.3.5. Chick-embryo chorioallantoic membrane (CAM) assay
Alginate plugs containing VEGF (4.5 pmol/embryo) (R&D Sys-
tems, 293-VE) with or without Compound 14 were placed on the
CAM of fertilized white Leghorn chicken eggs at day 11. After 3 days,
microvessels converging toward the implant were counted.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
ꢀ
This work was supported by Ministero dell’Universita e della
5.3.2. Cell cultures
Ricerca, “Futuro in Ricerca” program(project code: RBFR10FXCP) to
AL and Associazione Italiana Ricerca sul Cancro, Italy (AIRC IG
15211) to MT. This research benefits from the HPC (High Perfor-
mance Computing) facility of the University of Parma, Italy - http://
www.hpc.unipr.it.
HUVE cells (Life Technologies, Waltham, MA, USA, CC035C)
were grown in MEM 200 (Gibco, Thermo Fisher Scientific, Wal-
tham, MA, USA, 097144) supplemented with LSGS kit (Gibco,
Thermo Fisher Scientific, S003K), 1% penicillinestreptomycin so-
lution (Euroclone, Milan, Italy, ECM0010D) and 10% fetal bovine
serum (FBS, Euroclone, Milan, Italy, ECS0170L). PC3 human prostate
adenocarcinoma cells (ECACC, Port Down, UK) were cultivated in
Ham-F12 (Carlo Erba, Italy FA30WL0136500), supplemented with
7% FBS and 1% penicillinestreptomycin solution. Both cells were
maintained in a humidified atmosphere of 95% air, 5% CO2 at 37 ꢀC.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112083.
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