Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma

European Journal of Medicinal Chemistry 223 (2021) 113653

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Si113-prodrugs selectively activated by plasmin against hepatocellular

and ovarian carcinoma

Enrico Rango ^a^,¹^,², Lucia D'Antona ^b¹, Giulia Iovenitti , Annalaura Brai ,

,

a

c

a

Arianna Mancini , Claudio Zamperini , Claudia Immacolata Trivisani ,

a

d

Stefano Marianelli , Anna Lucia Fallacara , Alessio Molinari , Annarita Cianciusi ,

d

b

,

**

, Elena Dreassi ^a, Maurizio Botta ^a

,

*

, c, e

Silvia Schenone , Nicola Perrotti

a

Dipartimento di Biotecnologie, Chimica e Farmacia, Universit aꢀ degli Studi di Siena, Via Aldo Moro 2, 53100, Siena, Italy

Dipartimento di Scienze della Salute, Universit aꢀ ”Magna Graecia” di Catanzaro, Viale Europa, 88100, Catanzaro, Italy

Lead Discovery Siena S.r.l., Via Vittorio Alﬁeri 31, 53019, Castelnuovo Berardenga, Siena, Italy

Dipartimento di Farmacia, Universit aꢀ degli Studi di Genova, Viale Benedetto XV 3, Genoa, 16132, Italy

b

c

d

e

Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology Temple University, BioLife

Science Building, Suite 333, 1900 North 12th Street, Philadelphia, PA, 19122, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 18 March 2021

Received in revised form

Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its

potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian

carcinoma models. But the drawback is the low water solubility which prevents its further development.

In this context, we successfully overcame this limitation by synthesizing two novel prodrugs intro-

ducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high afﬁnity with

plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian

carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties,

plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was

evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the

presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue

distribution conﬁrmed a better proﬁle highlighting the importance of the prodrug approach. Finally, the

prodrug antitumor efﬁcacy was evaluated in an HCC xenografted murine model, where a signiﬁcant

reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with

paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an

impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of

Si113 prodrugs and pave the way for their further development against resistant cancer.

1

June 2021

Accepted 13 June 2021

Available online 17 June 2021

Keywords:

Sgk1

Kinase inhibitors

HCC

Ovarian carcinoma

Prodrugs

Plasmin-activated prodrugs

Targeted therapy

ADME

Pharmacokinetic

©

1

. Introduction

threonine kinase family. In the last years, this protein gained more

and more attention as a molecular target in oncology, being

involved at different levels in the development and progression of

different types of cancer [3], including breast [4,5], prostate [6],

ovarian [7], non-small-cell lung cancer (NSCLC) [8], hepatocellular

carcinoma (HCC) [9,10] and malignant glioma [11]. Sgk1 is activated

by cAMP, insulin [12], growth factors [13,14], and steroids [15] and

participates in a wide variety of pathways and mechanisms pro-

moting tumor cell proliferation and conferring resistance to

apoptosis. Sgk1 has been demonstrated to be an Akt-independent

Compound Si113 (Fig. 1a), a pyrazolo[3,4-d]pyrimidine deriva-

tive, has been identiﬁed as a potent inhibitor of Sgk1 [1,2] (serum

and glucocorticoid-regulated kinase 1) which belongs to the serine/

*

Corresponding author.

*

* Corresponding author.

E-mail addresses: perrotti@unicz.it (N. Perrotti), elena.dreassi@unisi.it

(

E. Dreassi).

2þ

1

critical PI3K downstream effector [16,17], to regulate Ca -depen-

dent tumor growth [18], through the control of potassium channels,

to contribute cell survival, proliferation, and differentiation by

Both authors contributed equally to this work.

Present addresses: Dipartimento di Farmacia, Universit ꢀa degli Studi di Genova,

2

Viale Benedetto XV 3, Genoa 16132, Italy.

https://doi.org/10.1016/j.ejmech.2021.113653

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Abbreviations

PAMPA

MR

DMSO

MD

Parallel Artiﬁcial Membrane Permeability Assay

Membrane retention

Dimethyl sulfoxide

Molecular dynamic

Pharmacokinetic

Paclitaxel

Acetonitrile

Human liver microsomes

Dulbecco's Modiﬁed Eagle's Medium

Fetal Bovine Serum

%

Sgk1

HCC

TP

SCLC

MDM2

LK

Serum and Glucocorticoid-regulated Kinase 1

Hepatocellular Carcinoma

D-Ala-Leu-Lys tripeptide

Non-small-cell lung cancer

Mouse Double Minutes 2

Linker

PK

TAX

ACN

HLM

DMEM

FBS

EDC

DMAP

TFA

1-Ethyl-3(3dimethylaminopropyl)carbodiimide

4-Dimethylaminopyridine

Triﬂuoroacetic acid

I.P.

Intraperitoneal

LKTP

ADME

Linker-Tripeptide

Absorption distribution, metabolism, and excretion

%ME

%RE

Matrix effect percentage

Recovery percentage

Fig. 1. Chemical structures of (a) Si113, (b) ProSi113-TP and (c) ProSi113-LKTP.

Black: parent drug Si113; Red: tripeptide sequence, TP; Blue: linker, LK; TFA: Triﬂuoroacetic acid.

(

phosphorylation of Mouse Double Minutes 2 (MDM2) [19]. In

addition, Sgk1 is implicated in conferring resistance to chemo- and

radiotherapy, affecting cellular sensitivity to taxol, by regulating the

expression of RANBP1, which is also involved in the alteration of

mitotic stability [20].

As a potent inhibitor of Sgk1 (IC50 value of 600 nmol/L) [1], Si113

was found to induce apoptosis and to reduce proliferation in

different cancer cell lines, in which this protein is overexpressed/

overactivated: RKO colorectal adenocarcinoma cells [2], MCF-7

breast cancer cells [2], A-172, (LI)PARI, ADF, and T98G glioblas-

toma multiforme cells [11], HepG2 and HuH-7 HCC cell lines [21],

A2780 ovarian cancer cells [22], Ishikawa, HEC1B, and AN3CA

endometrial cancer cell lines [23]. Furthermore, Si113 showed a

good safety proﬁle, being non-toxic on human ﬁbroblast cells up to

In particular, the inhibition of Sgk1 by Si113 has been proved to

strongly reduce HCC cellular viability, with a dramatic increase in

apoptosis and altered cell cycle. In addition, Si113 was intraperi-

toneally administered at a dosage of 8 mg/kg in human HCC cell-

xenografted mice, arresting cancer growth, inducing high levels

of necrosis in tumor tissues, and synergizing with radiotherapy

[21]. Moreover, in in vitro and in vivo preclinical models of ovarian

cancer, Si113 inhibits tumor cell proliferation, synergizes with

paclitaxel, and rescues paclitaxel sensitivity in resistant cells [22].

The aim of this work is the improvement of the efﬁcacy and the

beneﬁts of the Si113 therapy in HCC and ovarian cancer, thanks to

the application of a prodrug strategy, which allows for the activa-

tion of the parent drug locally at the tumor site. Indeed, most

conventional chemotherapeutics are characterized by poor phar-

macokinetic proﬁles and non-speciﬁc distribution in body tissues.

a concentration of 50 mM [11].

2

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Accordingly, the development of rationally modiﬁed prodrugs can

lead to the realization of targeted cancer therapy, avoiding off-

target effects and enhancing the efﬁcacy of conventional chemo-

therapy [24e26]. In this context, the aminoacidic sequence D-Ala-

Leu-Lys (TP) has been selected, synthesized, and introduced as pro-

moiety in the structure of Si113. Indeed, this tripeptide sequence

has been proved to have a high afﬁnity for a speciﬁc reactive site of

plasmin [27], a protease recognized to be overexpressed in many

tumors, including HCC [28] and ovarian cancer [29,30]. The plasmin

system has a primary role in extracellular matrix degradation, tis-

sue inﬁltration, angiogenesis, invasion, metastasis, and drug resis-

tance [31]. Plasminogen is plasmin's inactive precursor, and it is

locally converted by two types of high-afﬁnity activators, urinary-

type (uPA) and tissue-type (tPA). The conversion takes place

when plasmin is bound to plasminogen-receptors, in its cell

surface-associated form. This allows greater proteolytic activity and

protection from inactivation for cell surface-associated plasmin

DMAP, then Si113 (2) was added, and the reaction mixture was

stirred at room temperature for 72 h to obtain the intermediate 3.

Finally, the N-deprotection was performed with TFA affording the

desired product ProSi113-TP (4) with a 99% yield.

For the synthesis of ProSi113-LKTP, the following steps have

been performed. First, the ethyl 5-aminopentanoate hydrochloride

(LK, 6) was obtained starting from the 5-aminovaleric acid (5) and

2

SOCl in anhydrous EtOH, with a 99% yield (Scheme 2).

Then, LK (6) was coupled with TP (1) in presence of EDC,HCl/

DMAP and subsequently O-deprotected using a 5% NaOH aqueous

solution obtaining the derivative 8 (LKTP), (Scheme 3).

Finally, to synthesize ProSi113-LKTP the same synthetic

approach used for Si113-TP was applied: LKTP (8) was coupled with

Si113 (2) using EDC,HCl/DMAP as activating system to give the

intermediate 9 protected. The latter was then reacted with TFA

affording the desired product ProSi113-LKTP (10) with 99% yield

(Scheme 4).

[

32]. Moreover, the selected amino acid sequence (TP) has been

extensively studied for the design of doxorubicin prodrugs [33]. A

clear improvement in the in vivo toxicity proﬁle was achieved with

the prodrugs approach, in contrast to the toxicity proﬁle of the

parent drug doxorubicin.

2.2. In vitro ADME assays

In vitro ADME properties were assessed, and the results are re-

ported in the following Table 1. The ADME properties of Si113

(called 6c in the manuscript) were previously determined and

published in Radi et al. (2011) [36]. First, we have evaluated the

thermodynamic water solubility of Si113 prodrugs. As expected, the

water solubility has increased signiﬁcantly for both prodrugs,

which were 2500-times more soluble than the parent drug. After

that, parallel artiﬁcial membrane permeability assay (PAMPA) was

performed to evaluate the ability of these compounds to reach the

cytoplasmic district in which our target enzyme is expressed. The

results suggest for both prodrugs, a lower membrane permeability

10-fold less than the parental compound Si113 essentially due to

their high degree of hydrophilicity. Moreover, both prodrugs pro-

vided good values of metabolic stability in human liver microsomes

(ꢀ95%).

Being a tumor-related protease, plasmin has been already

exploited in prodrug approaches, as a parent drug-releasing

mediator [27,33,34]. We already explored the ability of the above-

mentioned TP to target plasmin. TP was integrated as a targeting

portion in a Si113-encapsulating liposomal system, which was

successfully tested in in vitro models of HCC [35]. We now report

the synthesis and preclinical characterization of plasmin-activated

Si113 prodrugs, designed to be cleaved by plasmin, allowing for the

release of the parent drug Si113 speciﬁcally at the tumor site. The

alcohol on C-6 sidechain was chosen to readily synthesize the

corresponding esters, avoiding protection and deprotection steps

and ensuring rapid hydrolysis and thus a fast release of the parent

drug. The amine in C-4 position was also evaluated, but the insta-

bility of carbamate was not compatible with TP deprotection steps,

while the synthesis of the corresponding amide was not successful

in acceptable yields (data not shown). All the prodrugs have been

docked into plasmin pocket, to determine their binding modes (for

details see supporting information). The ﬁrst prodrug, named

ProSi113-TP (Fig. 1b) is composed of the parent drug (black portion)

and TP (red portion), connected by an ester link (where the cleav-

age takes place), while the second, ProSi113-LKTP (Fig. 1c), presents

four carbon atoms introduced as linker (LK, blue portion) between

drug Si113 and TP.

Si113-prodrugs have been characterized in terms of stability in

polar media, human plasma, and human plasmin solution. In

addition, Si113 and its prodrugs have been characterized for their

water solubility, passive permeability, and metabolic stability. The

in vitro antitumor activity was evaluated in HCC HepG2 and Huh-7,

and human ovarian carcinoma A2780 cell lines. In vivo pharmaco-

kinetic proﬁle and preliminary 24 h biodistribution of Si113 and

prodrugs were evaluated. Finally, their antitumor efﬁcacy was

evaluated in HCC and ovarian carcinoma mice models, also in

combination with paclitaxel.

Finally, in order to evaluate the hydrolysis rate of prodrugs,

stability tests were performed in polar solvents (DMSO, MeOH, and

Tris buffer), human plasma, and human plasmin solution (Table 2).

Prodrugs showed to be stable in polar solvents for more than 48 h.

To verify the ability of ProSi113-TP and ProSi113-LKTP to release the

corresponding parental drug Si113, both prodrugs were incubated

ꢁ

at 37 C in human plasma or plasmin solution for 24 h as described

in the materials and methods section. ProSi113-TP, presenting only

an ester as a hydrolyzable group, showed after 24 h a higher hy-

drolysis rate (65%) with half-life (t1/2) value equal to 0.3 h in plasma

and a lower hydrolysis rate in plasmin solution (40%) while main-

taining a good half-life time (t1/2 ¼ 1.5 h). On the other hand,

ProSi113-LKTP, presenting two hydrolyzable sites, showed a plasma

hydrolysis rate about of 35% and a higher hydrolysis rate (64%) in

plasmin solution with comparable half-life values in human plasma

and plasmin solution, equal to 4.6 and 3.1 h, respectively.

2.3. In vitro biological assay

2.3.1. In vitro evaluation of Sgk1 kinase inhibition

The interaction of Si113, ProSi113-TP and ProSi113-LKTP with

Sgk1 was then tested in kinase assays. Sgk1 kinase activity was

measured as radioactivity incorporated into speciﬁc target peptide

(GRPRTSSFAEGKK), in the presence of increasing drugs and pro-

2

. Results

2.1. Synthetic strategy

drugs concentrations (0.6, 3, and 12.5

obtained, Si113 is able to inhibit Sgk1 kinase activity in a dose-

dependent manner showing inhibition of 75% as early as 0.6

(increasing to more than 90% at 12.5 M). ProSi113-TP is able to

signiﬁcantly reduce Sgk1 activity by 80% only at a concentration of

12.5 M, while ProSi113-LKTP reaches to reduce activity by 73% at a

mM) for 30 min. From the data

Si113-prodrugs have been synthesized starting from com-

pounds Si113 and TP, which were previously synthesized and

published by our group [35,36].

ProSi113-TP (4) was synthesized following the procedure re-

ported in Scheme 1: TP (1) was activated for 30 min using EDC,HCl/

mM

m

3

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Scheme 1. Preparation of ProSi113-TP.

ꢂ6

for 12 h) and/or insulin (10 M) for the following 30 min. Cells

were then lysed and Sgk1 protein was immunoprecipitated using

an anti-Sgk1 antibody (#07e315, EMD Millipore). Sgk1 activity was

then assayed in the immune complexes. Insulin increased signiﬁ-

cantly Sgk1 activity, as expected. Incubation of HepG2 cells in the

presence of Si113 led approximately to a 35% inhibition of insulin-

stimulated Sgk1 activity, thus suggesting that the molecule was

able to permeate the cells and inhibit intracellular Sgk1 activity.

Similarly, HepG2 cells incubation in the presence of ProSi113-TP

and ProSi113-LKTP led approximately to a 40% inhibition of

insulin-stimulated Sgk1 activity, suggesting that also the prodrugs,

if hydrolyzed in an intact cellular environment, can permeate the

membrane and inhibit the activity of Sgk1 (Fig. 3).

Scheme 2. Preparation of linker (LK).

concentration of 12.5

ference between the two prodrugs is observed at lower concen-

trations, where already at 0.6 M ProSi113-LKTP showed a

mM, both compared to the control. The dif-

m

reduction in enzyme activity of approximately 53% in contrast to

ProSi113-TP which is only 18% (Fig. 2).

2

.4. In silico studies

.4.1. Molecular docking

2

.3.2. Inhibition of Sgk1 kinase activity in an intact cell

environment

We investigated the effects of Si113, ProSi113-TP and ProSi113-

LKTP on the enzymatic activity of endogenous Sgk1. HepG2 cells

were incubated in the presence or absence of molecules (12,5

To further conﬁrm our results, docking studies have been per-

formed on Si113, ProSi113-TP, and ProSi113-LKTP, using the previ-

ously published docking protocol (see supporting information for

more details) [1]. The binding mode analysis of Si113 (Fig. 4a)

shows that the alcoholic group of the compound interacts with the

mM

4

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Scheme 3. Preparation of LKTP.

backbone of Asp177 and Ile179 of Sgk1. The pyrazolo[3,4-d]py-

rimidine core is inserted into a lipophilic area constituted by

molecules, and the -NH group of phenethylamine is also involved in

H-bond interactions with the backbone of Ile179. During the

simulation, TP interacts with Glu183, Glu226, Glu262, and Ile104.

Finally, the analysis of the trajectory of ProSi113-LKTP shows that,

after the assessment of the compound into the binding site, it stably

interacts with amino acids that constitute the pocket (RMSD shows

the increase of the RMSD values at the beginning of the simulation,

Fig. 5c). The ester link forms long-lasting contacts with the back-

bone of Ile179, while the TP is involved in hydrogen bond in-

teractions with Glu183, Glu226, and Ile179.

Val112, Ala125, Ile179, Leu229, and Thr239, while cation-

p in-

teractions are established between the phenyl group and Lys127.

ProSi113-TP (Fig. 4b) shows a different orientation with respect to

Si113 due to the steric hindrance of TP. The parent drug hydro-

phobically interacts with residues that constitute the binding site,

without establishing other proﬁtable interactions. Lys and Ala of TP

establish hydrogen bond interactions with Glu226 and Leu229,

respectively. The presence of a four-carbon atom linker in ProSi113-

LKTP (Fig. 4c) allows the parent drug moiety to maintain the spatial

orientation of Si113. The carbonyl group is involved in hydrogen

bond interactions with the backbone of Ile179. Lys and Ala of TP are

involved in hydrogen bond interactions with Glu183.

2

.5. HCC

.5.1. In vitro cytotoxicity assays

HepG2 and Huh-7 HCC cell lines were plated (see material and

2

.4.2. Molecular dynamics

To further explore the stability and the dynamic behaviour of

methods section). After 24 h, when cells were approximately 60%

conﬂuent, increasing concentration (0.1 M, 1 M, 10 M, and

100 M) of Si113, ProSi113-TP and ProSi113-LKTP were added, and

cell viability was estimated after 24, 48, and 72 h (Table 3). In hu-

man HCC cell lines, Si113 yielded a signiﬁcant dose-dependent

reduction in the number of viable cells with IC50 values (after

72 h) of 3.15 and 0.58 mM on HepG2 and Huh-7 cell lines, respec-

tively. Regarding prodrugs, on HepG2 cell line, ProSi113-TP and

ProSi113-LKTP at 72 h showed IC50 values equal to 9.49 and

m

the compounds bound to Sgk1, 50 ns of molecular dynamics (MD)

simulation were conducted using AMBER 16 software (see sup-

porting information for more details). The analysis of the trajec-

tories showed that Si113 is stably bound to the binding site of Sgk1

and is not subjected to great conformational changes. The Si113

RMSD plot shows a RMSD value which is stabilized around 3.5 Å

m

(

Fig. 5a). During the simulation, the -OH group is involved in

hydrogen bond interactions with the backbone of Asp177 and with

the side chain of Thr159. Other hydrogen bond interactions are

established between the -NH group of the phenethylamine and the

side chain of Thr239, and between the pyrazolo moiety and the side

chain of Glu183. The MD simulation of ProSi113-TP shows that the

compound is partially solvent-exposed and subjected to confor-

mational changes that increase the RMSD values, oscillating around

3.92

reduction in cell viability with an IC50 value of 13.85

On the contrary, ProSi113-LKTP showed a signiﬁcant reduction

already after 24 h to reach an IC50 value equal to 0.74 M after 72 h.

In addition, we performed cytotoxicity tests in the presence of

plasmin (0.2 U/well, ﬁnal concentration) to assess whether the

activity of the prodrugs increases due to the hydrolytic action of

plasmin. In this regard, ProSi113-TP showed increased cytotoxicity

after 24 h with up to 2 times higher reduction in cell viability for

m

M, respectively. On Huh-7, ProSi113-TP showed a slight

mM after 72 h.

m

4

.5 Å and reaching a value of 6.5 Å (Fig. 5b). The pyrimidine moiety

and the two amino groups form hydrogen bonds with water

5

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Scheme 4. Preparation of ProSi113-LKTP.

Table 1

In vitro ADME assays of Si113 and its prodrugs.

Assays

Measurement Units

g/mL

Si113

0.4

ProSi113-TP

ProSi113-LKTP

Water Solubility

m

>1000

ꢂ6

PAMPA

10 cm/s

(%)

2.6

(86.2)

0.2

(0)

0.2

(0)

a

(

%MR )

Metabolic Stability^b

%

84

ꢀ95

a

Membrane Retention (%MR) expressed as percentage of compound unable to reach the acceptor compartment.

b

Expressed as percentage of unmodiﬁed compound.

Table 2

Stability assays of prodrugs in polar media, human plasma and human plasmin solution.

Stability in:

Unit

ProSi113-TP

ProSi113-LKTP

DMSO, MeOH and Tris Buffer

Human Plasma

Human Plasmin solution

h

>48

65 (0.3)

40 (1.5)

>48

35 (4.6)

64 (3.1)

Hydrolysis (t1/2a_,_h₎

%

a

Half-life expressed as the amount of time it takes before half of the prodrugs are hydrolyzed.

6

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

concentration of the compounds was determined by HPLC-MS/MS

analysis. Table S1 (supporting information) shows the recovery and

matrix effect data, which suggests that the developed analysis

method could be successfully applied to the pharmacokinetic and

biodistribution studies. The plasma concentration-time curves

(expressed as nmol of compound/mL of plasma) of Si113 and its

prodrugs are reported in Fig. 6, while the corresponding PK pa-

rameters are shown in Table 5.

Following intraperitoneal administration, Si113 was rapidly

detected after 0.5 h (Tmax) with a Cmax of 46.27 nmol/mL while the

observed Cmax for ProSi113-TP and ProSi113-LKTP after 0.25 h were

8.09 and 2.66 nmol/mL, respectively. It is interesting to note how

Si113 derived from the hydrolysis of ProSi113-LKTP reaches a Cmax

value of 21.31 nmol/mL after 1.50 h. In contrast, the Si113 derived

from ProSi113-TP showed a 5-fold lower Cmax value compared with

the Si113 derived from ProSi113-LKTP (Table 5). However, ProSi113-

TP showed an AUC0/24h value almost 4-fold higher than ProSi113-

LKTP. Regarding the AUC0/24h values of Si113 resulting from the

hydrolysis of each prodrug, Si113 released from ProSi113-LKP

showed an AUC0/24h 6-fold higher than Si113 released from

ProSi113-TP and the same trend is true for the parent drug Si113.

The plasma half-life (t1/2) and MRT values remain approximately in

the same range after treatment with ProSi113-TP (t1/2: 15.30 h,

MRT: 18.41 h) and ProSi113-LKTP (t1/2: 21.82 h, MRT: 31.55 h). On

the contrary, Si113 showed a t1/2 and MRT values of 2.73 h and

1.13 h respectively, while Si113 deriving from the hydrolysis of

prodrugs showed for Si113 released by ProSi113-TP a two-fold

higher half-life value (t1/2: 4.13 h) compared to Si113 released by

ProSi113-LKTP (t1/2: 2.08 h). The same results were observed for the

MRT values.

Fig. 2. Sgk1 kinase inhibition activity of Si113, ProSi113-TP and ProSi113-LKTP in

enzymatic assay. A Bonferroni's one-way multiple comparisons test (ANOVA) was

performed to test the signiﬁcance of the observed differences. *indicates statistically

signiﬁcant differences between each treatment vs CTRL (*p < 0.05, **p < 0.01 and

***p < 0.001).

Si113 and its prodrugs were detected in all tissues collected from

mice treated and sacriﬁced after 24 h (Fig. 7). ProSi113-LKTP

showed higher tissue concentrations than Si113 and ProSi113-TP

in all organs analyzed. In particular, in the hepatic tissue, for

ProSi113-LKTP, it has been detected a concentration (11.05 nmol/g),

almost 14-fold higher than that recorded for ProSi113-TP

(0.81 nmol/g) and the same trend has also been observed for the

drug released from prodrugs. The tissue concentration of Si113

resulting from ProSi113-LKTP (1.72 nmol/g) was almost 3-fold

higher than that obtained from the hydrolysis of ProSi113-TP

(0.65 nmol/g). On the other hand, the tissue concentrations of

parent drug Si113 were comparable to those of Si113 released by

ProSi113-LKTP hydrolysis. The same trend was also observed in

renal tissue, where ProSi113-LKTP showed a higher tissue con-

centration (3.01 nmol/g) than ProSi113-TP (1.77 nmol/g), while

similar Si113 levels for both parent drug and the drug derived by

prodrugs hydrolysis were observed.

Fig. 3. Inhibition of Sgk1 kinase isolated from cell lysates. A Bonferroni's one-way

multiple comparisons test (ANOVA) was performed to test the signiﬁcance of the

observed differences. *indicates statistically signiﬁcant differences between each

treatment vs its respective CTRL (**p < 0.01 and ***p < 0.001).

2

.5.3. Human hepatocellular carcinoma xenograft mice model

HepG2 cells (2.5 ꢃ 10 ) were implanted in the ﬂanks of nude

6

both HCC cell lines whereas, ProSi113-LKTP showed increased

cytotoxicity against Hep-G2 cell line with up to 4 times higher

reduction in cell viability.

female mice for in vivo treatment with vehicle alone, Si113,

ProSi113-TP and ProSi113-LKTP at a dosage of 9.5 mg/kg/day for

Si113 and 21 mg/kg/day for both prodrugs. Twenty mice were used,

randomly subdivided into four batches. Treatment started when

3

2

.5.2. In vivo administration of Si113 and prodrugs in male healthy

mice

Preliminary pharmacokinetic and biodistribution tests were

tumor volume reached 100 mm , as assessed by manual caliper

measurement. The drugs (or vehicle) were administered daily, and

tumor growth was monitored every 7 days. Mice were sacriﬁced on

day 21. Tumors were then excised and weighed. An impressive and

signiﬁcant reduction in tumor growth was recorded in all treated

batches compared to controls (Fig. 8a). Measurements of the tumor

weights further conﬁrmed the effectiveness of the treatment

(Fig. 8b) showing a signiﬁcant reduction in mean tumor mass

weight of approximately 93 and 87% for ProSi113-TP and ProSi113-

LKTP treatments, respectively.

performed. For this purpose, plasma and tissue distribution proﬁle

of Si113 and both prodrugs were quantiﬁed after intraperitoneal

administration. Si113, previously dissolved in DMSO [37], was

administered as a single dose of 9.5 mg/kg in a ﬁnal volume of

5

0

m

L. Both prodrugs were solubilized in saline solution at a single

dose of 21 mg/kg in a ﬁnal volume of 100 L. After treatments, mice

were sacriﬁced, and blood and organs were collected. The

m

7

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Fig. 4. Binding mode of (a) Si113 (green), (b) ProSi113-TP (blue) and (c) ProSi113-LKTP (orange) to the ATP binding pocket of Sgk1 (PDB ID: 3HDN).

2

.6. Ovarian carcinoma

xenografted mice and Fig. 9b: non-xenografted mice) at 24 h af-

ter treatment with Si113 (9.5 mg/kg) and ProSi113-TP (21 mg/kg).

Comparable plasma levels were detected in xenografted mice

group with a higher concentration of Si113 resulting from the hy-

drolysis of ProSi113-TP (0.51 nmol/mL) than the parent compound

(0.20 nmol/mL). The same trend was observed in the non-

xenografted group with an almost two-fold lower plasma concen-

tration almost two-fold lower for both the parental Si113 and the

ProSi113-TP derived Si113 compared with the plasma levels

observed in the A2780-xenografted mice group. Moreover, Pro-

Si113-TP shows higher tissue concentrations than the parental drug

in all organs examined. Interestingly the concentrations of

ProSi113-TP detected in the tumor mass (5.69 nmol/g) were higher

than the concentrations of Si113 released from the hydrolysis of

ProSi113-TP (1.26 nmol/g). In contrast, the parental drug Si113

showed a tissue concentration (2.20 nmol/g) lower than its prodrug

(Fig. 9a). The same is observed in ovarian tissue where Pro-Si113-TP

showed a tissue concentration 2-fold higher than Si113 (4.11 and

1.89 nmol/g, respectively) and the same trend is detected in the

ovaries of non-xenografted mice (Fig. 9b). Whereas comparable

tissue concentrations of Si113, which was obtained after hydrolysis

of ProSi113-TP, were observed in the ovaries of both animal groups

(1.25e1.26 nmol/mL).

.6.1. In vitro cytotoxicity assays

Preliminary cytotoxicity tests have been performed against

A2780 ovarian cancer cell line. In recent work, D'Antona et al. (2019)

22] evaluated the activity of the Si113 inhibitor on ovarian cancer,

[

indicating that it has the ability to inhibit cell proliferation, enhance

the effects of paclitaxel-based chemotherapy and counteract the

development of paclitaxel resistance and restore paclitaxel sensi-

tivity in resistant A2780 ovarian cancer cells. In addition, in vivo

preclinical studies on a xenograft model of nude mice implanted

with paclitaxel-resistant human ovarian cancer cells have shown

that the Si113 inhibitor has a synergistic action with paclitaxel in the

treatment of xenografted ovarian cancer cells. For this reason, given

the results obtained in this type of cancer, the cytotoxicity proﬁle of

prodrugs towards this type of cancer has been evaluated. Table 4

shows that Si113 yielded a signiﬁcant dose-dependent reduction in

the number of viable cells with IC50 values, after 72 h of 3.47

Similarly, prodrugs showed comparable cytotoxicity proﬁles with

IC50 values (after 72 h) equal to 5.14 and 7.10 M for ProSi113-TP and

ProSi113-LKTP, respectively. On the contrary, only ProSi113-TP

showed an increased cytotoxicity proﬁle up to 2 times higher in

mM.

m

presence of plasmin with an IC50 value of 26.05 mM.

2

.6.2. Preliminary tissue distribution in nude female mice

Preliminary plasma and tissues concentrations proﬁle was per-

formed in two groups of nude female mice (Fig. 9a: A2780-

2.6.3. Ovarian carcinoma xenograft mice model

Based on the in vitro studies, we generated xenografts for in vivo

6

experiments by implanting 2.5 ꢃ 10 paclitaxel-resistant A2780TC

8

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Fig. 5. RMSD plot of Si113, ProSi113-TP and ProSi113-LKTP. The line indicates the ligand ﬂuctuation. (a) The Si113 RMSD plot shows a RMSD value which is stabilized around 3.5 Å.

b) The ProSi113-TP RMSD plot is characterised by values that oscillate around 4.5 Å and that reaches the value of 6.5 Å. The variations in the RMSD values are caused by

(

conformational variation of the molecule, that is partially exposed to the solvent. (c) The ProSi113-LKTP RMSD shows the increase of the RMSD values at the beginning of the

simulation. After the assessment of the compound into the binding site, it stably interacts with the amino acids that constitute the binding site.

section, and the mice were sacriﬁced on day 21. Tumors were

Table 3

In vitro cytotoxicity assays of Si113 and both prodrugs against HepG2 and Huh-7

HCC cell lines.

excised, weighed and immediately frozen in liquid nitrogen. As

expected, based on the known chemoresistance of A2780TC cells,

Paclitaxel treatment did not affect tumor growth. As previously

published in D'Antona L. et al. (2019) [22] and here reported for

comparison with ProSi113-TP, the mice group treated with Si113

showed a signiﬁcant reduction in tumor volume on day 21

compared with the control group, and a more substantial effect was

observed in the mice treated with Si113 in combination with

Paclitaxel (Fig. 10a [22]). The mice group treated with ProSi113-TP

alone does not show a signiﬁcant reduction compared with pacli-

taxel treatment alone but showed a substantial reduction (>95%) of

tumor volume when used in combination with paclitaxel (Fig. 10c).

Measurements of the tumor weights further conﬁrmed the effec-

tiveness of the treatment (Fig. 10b [22] and 10c).

Cell lines

HepG2

Si113

ProSi113-TP

IC50^a

M ± S.D.)

ProSi113-LKTP

(m

24 h

48 h

72 h

15.87 ± 1.27

8.29 ± 0.54

3.15 ± 0.08

12.10 ± 1.32

9.49 ± 0.87

4.40 ± 0.39

14.69 ± 1.37

5.63 ± 0.40

3.92 ± 0.12

2

4 h þ plasmin

6.75 ± 0.32

3.60 ± 0.07

Huh-7

IC50^a

(mM ± S.D.)

24 h

48 h

72 h

2.05 ± 0.12

1.88 ± 0.07

0.58 ± 0.03

>100

35.66 ± 2.62

13.85 ± 1.19

6.83 ± 0.37

1.58 ± 0.05

0.74 ± 0.03

2

4 h þ plasmin

46.27 ± 2.17

6.72 ± 0.72

a

IC50 was evaluated as dose-response inhibition of MTT assay. Data are

means ± S.D. of three independent experiments each done in triplicate.

3

. Materials and methods

cells into the ﬂanks of nude mice. Thirty xenografted mice were

randomly divided into four experimental groups: I) treatment with

the drug vehicle, II) treatment with Paclitaxel (TAX) at a dosage of

3.1. Synthesis and characterization of compounds

5

mg/kg, III) treatment with ProSi113-TP (21 mg/kg) and IV) treat-

Compounds Si113 and TP were previously synthesized and

published by our group [35,38].

ment with ProSi113-TP in combination with Paclitaxel at the same

dosages reported above respectively. The treatments were started

All commercially available chemicals were used as purchased

from Sigma Aldrich. CH Cl , was dried over sodium hydride, while

72 h after cell implantation, and the drugs (or vehicles) were

2

administered ﬁve days per week. The tumor volumes were

measured every 7 days for 21 days, as indicated in the Methods

MeOH and EtOH were dried over magnesium and iodine as indi-

cator. Anhydrous reactions were run under a positive pressure of

9

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Fig. 6. Plasma concentration-time curves (mean ± S.E.M., n ¼ 3) after intraperitoneal administration of (a) Si113 9.5 mg/kg, (b) ProSi113-TP 21 mg/kg and (c) ProSi113-LKTP 21 mg/

kg. The plasma concentrations in the y-axis are expressed as log10 scale. *Si113 marked with asterisk represents the amount of drug detected deriving from the hydrolysis of

prodrugs.

Table 4

3.2. In vitro ADME assays

In vitro cytotoxicity assays of Si113 and ProSi113-TP against A2780 ovarian carci-

noma cell line.

3.2.1. Chemicals

Cell line

A2780

Si113

ProSi113-TP

ProSi113-LKTP

All solvents and reagents were from Sigma-Aldrich Srl (Milan,

Italy). Dodecane was purchased from Fluka (Milan, Italy). Pooled

male donors 20 mg/mL HLM were from BD Gentest-Biosciences

_I_C₅₀a

(mM ± S.D.)

24 h

48 h

72 h

25.64 ± 1.72

4.56 ± 0.27

3.47 ± 0.19

41.58 ± 3.36

11.30 ± 2.49

5.14 ± 0.34

21.43 ± 0.97

22.06 ± 1.07

7.10 ± 0.41

(

San Jose, California). Milli-Q quality water (Millipore, Milford,

MA, USA) was used. Hydrophobic ﬁlter plates (MultiScreen-IP, clear

plates, 0.45 m diameter pore size), 96-well microplates, and 96-

well UV-transparent microplates were obtained from Millipore

Bedford, MA, USA).

m

2

4 h þ plasmin

26.05 ± 1.55

21.60 ± 1.56

a

IC50 was evaluated as dose-response inhibition of MTT assay. Data are

means ± S.D. of three independent experiments each done in triplicate.

(

3

.2.2. UV/LC-MS methods

LC analyses for water solubility, blood samples and organs

2

dry N . TLC was carried out using Merck TLC silica gel 60 F254.

Chromatographic puriﬁcations were performed on columns packed

collected were performed by LC-MS/MS system consisted of a

Varian apparatus (Varian Inc) including a vacuum solvent degass-

ing unit, two pumps (212-LC), a Triple Quadrupole MSD (Mod. 320-

LC) mass spectrometer with ES interface and Varian MS Worksta-

tion System Control Vers. 6.9 software. Chromatographic separa-

tion was obtained using a Pursuit C18 column (50 ꢃ 2.0 mm)

1

with Merck silica gel 60, 23e400 mesh, for ﬂash technique. H NMR

13

and C NMR spectra were recorded at 400 MHz on a NMR Bruker

Advance DPX400. Chemical shifts are reported relative to tetra-

methylsilane at 0.00 ppm.

Mass spectra (MS) data were obtained using an Agilent 1100 LC/

MSD VL system (G1946C) with a 0.4 mL/min ﬂow rate using a bi-

(Varian) with 3 mm particle size and gradient elution with a binary

nary solvent system of 95/5 ¼ MeOH/H

2

O. UV detection was

solution; (eluent A: ACN, eluent B: Water, both eluents were acid-

iﬁed with formic acid 0.1% v/v). The analysis started with 5% of A

monitored at 254 nm. MS were acquired in positive and negative

mode scanning over the mass range 100e1500 m/z. The following

ion source parameters were used: drying gas ﬂow, 9 mL/min;

(

from t ¼ 0 to t ¼ 3 min), then A was increased to 95% (from t ¼ 3 to

t ¼ 12 min), then kept at 95% (from t ¼ 12 to t ¼ 20 min) and ﬁnally

ꢁ

nebulizer pressure, 40 psi; drying gas temperature, 350 C.

return to 5% of eluent A in 1.0 min. The ﬂow rate was 0.2 mL/min

All target compounds possessed a purity of ꢀ95% veriﬁed by UV/

and injection volumes were 5

positive mode and parameters were: detector 1850 V, drying gas

mL. The instrument operated in

LC-MS method, as reported in UV/LC-MS methods section.

10

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Table 5

a

Plasma pharmacokinetic parameters of Si113 and its prodrugs evaluated using a non-compartment model.

Plasma Pharmacokinetic Parameters^a

Parameters (Unit)

Dosage

Si113ⁱ

9

.5 mg/kg

21 mg/kg

Si113

ProSi113-TP

ProSi113-LKTP

Si113ⁱ

b

t

1/2 (h)

2.73

0.5

15.30

0.25

8.09

64.58

92.26

18.41

5.02

4.13

1.00

4.16

27.65

27.95

2.55

4.48

0.75

21.82

0.25

2.66

18.41

35.71

31.55

18.51

0.59

2.08

1.50

21.31

167.46

167.49

1.88

c

T

max (h)

d

C

max (nmol/mL)

AUC0/24h (nmol/mL,h)

AUC0/∞ (nmol/mL,h)

MRT0/∞ (h)

/F ((mg/kg)/(nmol/mL))

CL/F ((mg/kg)/(nmol/ml)/h))

46.27

35.48

35.52

1.13

1.05

0.28

e

f

g

V

z

0.38

0.13

h

0.23

a

Calculated with PKSolver.

b

t

1/2: half-life.

c

d

e

f

T

C

max: time of maximum concentration observed.

max: maximum concentration observed.

AUC: area under the curve.

MRT: mean residence time.

V: volume of distribution.

CL: clearance.

g

h

i

Si113 marked with an asterisk represents the drug deriving from the hydrolysis of each prodrug.

ꢁ

pressure 25.0 psi, desolvation temperature 300.0 C, nebulizing gas

4

tested compound. Quantiﬁcation of the single compound was made

by comparison with appropriate calibration curves realized with

standard solutions in methanol.

LC analyses of PAMPA, Metabolic Stability and Stability Tests (in

DMSO, MeOH, Tris-buffer, human plasma and human plasmin so-

lution) were performed by UV/LC-MS with an Agilent 1100 LC/MSD

5.0 psi, needle 5000 V and shield 600 V. Nitrogen was used as

nebulizer gas and drying gas. Collision induced dissociation was

performed using Argon as the collision gas at a pressure of 1.8

mTorr in the collision cell. The transitions as well as the capillary

voltage and the collision energy used are appropriated for each

Fig. 7. Tissue distribution concentration proﬁle (mean ± S.E.M., n ¼ 3) in male healthy mice at 24 h after treatment with (a) Si113 9.5 mg/kg, (b) ProSi113-TP 21 mg/kg and (c)

ProSi113-LKTP 21 mg/kg *Si113 marked with an asterisk represents the amount of drug detected deriving from the hydrolysis of prodrugs.

11

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Fig. 8. HCC in vivo efﬁcacy on HepG2-xenografted mice model. (a) tumor volume and (b) tumor weight (mean ± S.D., n ¼ 5). A Bonferroni's one-way multiple comparisons test

ANOVA) was performed to test the signiﬁcance of the observed differences. *indicates statistically signiﬁcant differences between each treatment vs CTRL (*p < 0.05, **p < 0.01).

(

Fig. 9. Plasma and tissue concentrations (mean ± S.E.M., n ¼ 2, plasma: nmol/mL; tissues: nmol/g) proﬁle at 24 h after treatment with Si113 in red, ProSi113-TP in grey (Si113*

represent the detected drug-derived from hydrolysis of Pro-Si113-TP - in blue) in (a) A2780-xenografted and (b) non-xenografted female mice model.

VL system (G1946C) (Agilent Technologies, Palo Alto, CA) using a

‘acceptor solution’ (50% v/v DMSO and phosphate buffer). The

sandwich plate was assembled and incubated for 5 h at room

temperature under gentle shaking. After the incubation time, the

sandwich was disassembled and the amount of compound in both

the donor and acceptor wells was measured by UV/LC-MS. For each

compound, the determination was performed in three independent

experiments.

Permeability (Papp) was calculated according to the following

equation obtained from Wohnsland and Faller [40] and Sugano

et al. [41] equation with some modiﬁcation in order to obtain

permeability values in cm/s:

Phenomenex Kinetex C18-100 Å column (150 ꢃ 4.6 mm, 5

mm

particle size) at room temperature. Analyses were carried out with

the same chromatographic conditions reported above with a ﬂow

rate of 0.6 mL/min and an injection volume of 20

mL.

3.2.3. Water solubility

Each solid compound (1 mg) was added to 1 mL of distilled

ꢁ

water. Each sample was mixed at 20 C, in a shaker water bath for

2

4 h [39]. The resulting suspension was ﬁltered through a 0.45 mm

nylon ﬁlter (Acrodisc). The concentration of the compound in so-

lution was determined by LC-MS/MS by comparison with the

appropriate calibration curve that was obtained from samples of

the compound dissolved in methanol at different concentrations.

For each compound, the determination was performed in three

independent experiments.

V_D,V_A

P

app

¼

ꢂ lnð1 ꢂ rÞ

ðV þ V ÞAt

D

A

3

A D

where V is the volume in the acceptor well (cm ), V is the volume

3

.2.4. Parallel artiﬁcial membrane permeability assay (PAMPA)

Each ‘donor solution’ was prepared from a solution of the

in the donor well (cm ), A is the “effective area” of the membrane

2

(cm ), t is the incubation time (s) and r the ratio between drug

appropriate compound (DMSO, 1 mM) diluted with phosphate

buffer (pH 7.4, 25.0 mM) up to a ﬁnal concentration of 500 M. The

donor wells were ﬁlled with 150 L of ‘donor solution’. The ﬁlters

were coated with 5 L of a solution of phosphatidylcholine in

dodecane 1% (w/v) and the lower wells ﬁlled with 300 L of

concentration in the acceptor and equilibrium concentration of the

m

drug in the total volume (V

D

þV

A

). Drug concentration was esti-

m

mated by using the peak area integration.

Membrane retentions (%MR) were calculated according to the

following equation:

m

12

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

Fig. 10. Ovarian carcinoma in vivo efﬁcacy on A2780TC-xenografted mice mode (mean ± S.D., n ¼ 5). Tumor volume after treatment with (a) Paclitaxel (TAX), Si113 and Si113 in

combination with TAX and (c) TAX, ProSi113-TP and ProSi113-TP in combination with TAX. Tumor weight after treatment with (b) TAX, Si113 and Si113 in combination with TAX and

(

d) TAX, ProSi113-TP and ProSi113-TP in combination with TAX. Tumor volume and tumor weight represented in ﬁgure (a) and (b) are previously published in D'Antona L. et al.

2019). A Bonferroni's one-way multiple comparisons test (ANOVA) was performed to test the signiﬁcance of the observed differences. *indicates statistically signiﬁcant differences

between each treatment vs CTRL (*p < 0.05, **p < 0.01, ***p < 0.001).

compound, the determination was performed in three independent

experiments.

½

r ꢂ ðD þ AÞꢄ

%

MR ¼

,100

Eq

3

.2.6. Stability tests

where r is the ratio between drug concentration in the acceptor and

equilibrium concentration, D, A, and Eq represented drug concen-

tration in the donor, acceptor, and equilibrium solution,

respectively.

.2.6.1. In polar solvents. Each prodrug was dissolved at r.t. in

DMSO, MeOH or Tris-buffer (100 mM, pH 7.4) up to a ﬁnal concen-

tration of 500 M. Aliquot samples (20 L) were taken at ﬁxed time

points (0.0, 0.25, 0.50, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 24.0 and

8 h) and were analyzed by UV/LC-MS. For each compound, the

determination was performed in three independent experiments.

m

4

3.2.5. Metabolic stability in HLM (Human liver microsomes)

The incubation mixture (total volume of 500 L) was constituted

m

by the following components: human liver microsomes (0.2 mg/

þ

mL, 5

mM,

phosphate dehydrogenase and MgCl

m

L), a NADPH regenerating system (NADPH 0.2 mM, NADPH

-glucose-6-phosphate mM,

48 mM), 50

3.2.6.2. In human plasma. Pooled human plasma (1.5 mL, 55.7

protein/mL) [42], phosphate buffer (1.4 mL, pH 7.4, 25 mM) and a

mg

1

D

4

unit/mL glucose-6-

M of each

compound in DMSO and phosphate buffer (pH 7.4, 25 mM, up to a

2

m

prodrug dissolved in DMSO (100

tube that was incubated at 37 C. At set time points (0.0, 0.25, 0.50,

m

L, 3.0 mM) were mixed in a test

ꢁ

ﬁnal volume of 500

m

L). The mixture was incubated at 37 C for 1 h.

1.0, 3.0, and 24.0 h), samples of 150

mL were taken, mixed with

The reaction was cooled down and quenched with acetonitrile

1.0 mL). After centrifugation (4000 rpm for 10 min), the super-

natant was taken, dried under nitrogen ﬂow, suspended in 100

of methanol and analyzed by UV/LC-MS to determine the per-

centage of the compound that was not metabolized. For each

600 L of cold acetonitrile and centrifuged at 5000 rpm for 15 min

m

(

[43]. The supernatant was removed and analyzed by UV/LC-MS to

monitor the hydrolysis process of the prodrug. For each compound,

the determination was performed in three independent

experiments.

mL

13

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

3

.2.6.3. In human plasmin solution. Human plasmin (10

m

L, 150

L, pH 7.4, 100 mM) and a solution of the

L, 7.5 mM) were mixed in a test tube and

m

g/

3.4. Animals

mL), Tris-buffer (970

prodrug in DMSO (20

incubated at 37 C. At set time points (0.0, 0.25, 0.50,1.0, 3.0, 6.0 and

m

Naive BALB/C mice (aged 4e6 weeks, Charles Rivers - Milan,

Italy), and female nude mice (aged 4 weeks, ENVIGO - Udine, Italy)

were maintained under pathogen-free conditions and given food

and water at libitum. The adaption period to the environment was

not less than seven days. All the procedures used on animals in this

study were approved by Institutional Animal Use and Care Com-

mittee at Universit aꢀ degli Studi “Magna Graecia” di Catanzaro and

Universit aꢀ degli Studi di Siena and authorized by the Italian Min-

ꢁ

2

4.0 h), samples of 50 mL were taken, mixed with 200 mL of cold

acetonitrile and centrifuged at 5000 rpm for 15 min [34,44]. The

supernatant was removed and analyzed by UV/LC-MS to monitor

the hydrolysis process of the prodrug. For each compound, the

determination was performed in three independent experiments.

istry of Health, according to Legislative Decree 116/92, which

implemented the European Directive 86/609/EEC on laboratory

animal protection in Italy. Methods for all the conducted experi-

ments were performed in accordance with regulations, standards

and guidelines of the Animal Use and Care Committee of Universitaꢀ

degli Studi “Magna Graecia” di Catanzaro and Universit aꢀ degli Studi

3

.3. In vitro biological assays

.3.1. Cell cultures

Human HCC cell lines HepG2 and Huh-7 and Ovarian carcinoma

cell line A2780, kindly provided by Prof. Ali O. Gure (Bilkent Uni-

versity, Ankara, Turkey), were cultured in DMEM (Life Technologies,

Inc., Grand Island, NY) supplemented with 10% fetal bovine serum

di Siena.

(

FBS), 2 mM L-Glutamine (Invitrogen-Gibco) and 10000 units/mL

3

.4.1. In vivo administration of Si113 and prodrugs for PK and BD

evaluation

For both in vivo studies in healthy male mice and nude female

Penicillin-Streptomycin (Aurogene), respectively. Cells were

cultured at 37 C in a humidiﬁed atmosphere of 5% CO

ꢁ

2

and 95% air.

4

Brieﬂy, for each cell line, 2.5 ꢃ 10 cells were seeded in 12-well

mice for PK and preliminary BD studies, Si113 was dissolved in

DMSO [37] while its prodrugs were dissolved in saline solution. The

compounds were administered intraperitoneally as a single dose of

plates. The day after seeding, compounds dissolved in DMSO were

added at increasing concentrations (0.1, 1.0, 10.0 and 100.0

incubated for 24, 48 and 72 h at 37 C in 5% v/v CO

prodrugs could be activated by plasmin, in vitro cytotoxicity assays

have been also performed in presence of the enzyme. First, 24 h

after seeding, our compounds were added by using serum-free

DMEM to which plasmin has been added at a ﬁnal concentration

m

M) and

ꢁ

2

. To verify if

9

.5 and 21 mg/kg in a ﬁnal volume of 100

prodrugs, respectively. Healthy male mice, at several time points

0.25, 0.5, 1, 1.5, 2 and 24 h), after drug administration, were treated

i.p. with heparin (5000 U/kg) and sacriﬁced under CO . Blood, liver,

mL for Si113 and its

(

2

spleen, lungs, and kidneys were collected. Three animals were used

for each time point. Similarly, two groups of nude female mice (two

animals for each experimental group), xenografted mice group

ꢁ

of 0.2 U/well [43]. Then, cells were incubated for 24 h at 37 C in 5%

v/v CO

Counter (Beckman Coulter). IC50 was calculated by GraphPad Prism

.0 software using the best ﬁtting sigmoid curve. For each com-

pound, the determination was performed in triplicate.

2

. Cell number and viability were evaluated using Z2 Coulter

6

(

2.5 ꢃ 10 A2780TC cells subcutaneously) and non-xenografted

6

group, were treated intraperitoneally as a single dose of 9.5 and

1 mg/kg in a ﬁnal volume of 100 L for Si113 and ProSi113-TP. After

4 h, mice were treated i.p. with heparin (5000 U/kg) and sacriﬁced

2

m

2

under CO , blood, and organs were collected. The dosage of the

prodrugs was chosen to obtain the same molar ratio as Si113

administered at 9.5 mg/kg.

All blood and organ samples were processed, and LC-MS/MS

quantitative analyses were performed. Matrix effect %ME (ioniza-

tion suppression or ionization enhancement) and recovery (%RE)

were evaluated (see Supporting Information for more details). The

pharmacokineticparameters were calculated by non-compartmental

analysis using PKSolver software [45].

3

.3.2. In vitro evaluation of Sgk1 kinase inhibition

The evaluation of Sgk1 inhibition was carried out following the

same analytical procedures: active kinase (Upstate) was incubated

in kinase buffer containing ATP (10 M), protein kinase A inhibitor

1 mM; Sigma), dithiothreitol (2 mM), [g-32P]ATP (0.02 mCi/sam-

m

(

ple), tris HCl pH 7.5 (100 mM) in the absence and in presence of

Sgktide (KKRNRRLSVA) peptide substrate (1 mM) and our com-

pounds at concentration of 0.6 mM, 3 mM, 12.5 mM. The reaction was

allowed to occur for 30 min at room temperature, with gentle

agitation, prior to stopping with 10 mL of stopping solution (ATP,

3.4.1.1. Sample preparation. Blood samples were centrifuged at

1

mM; bovine serum albumin, 2%; HCl, 0.6% w/v). The reaction mix

was centrifuged in an Eppendorf microcentrifuge for 10 min at

4,000 rpm. Supernatants (10 L) were applied to a 2.1-cm diam-

5000 rpm for 20 min to separate the plasma fraction, which was

subsequently collected in a test tube. 1.0 mL of acetonitrile solution

containing Si34 [46] (a pyrazolo[3,4-d]pyrimidine compound

already used as internal standard, IS) at a concentration of 5 mg/mL

was added to each sample to denature protein component. Samples

were centrifuged at 5000 rpm for 20 min, and the supernatant was

recovered and analyzed by LC-MS/MS. Liver and the other organs

were homogenized using a T10 basic ULTRA-TURRAX® homoge-

nizer (Bioclass, Pistoia, Italy); in order to extract the compound

from the tissue, acetonitrile IS solution was added (1.5 mL at the

1

m

eter p81 Whatman paper. After drying at room temperature, the

ﬁlter was washed four times with phosphoric acid (25 mM), once

with acetone, and then counted in a scintillation counter to mea-

sure the radioactivity incorporated in the substrate peptide.

3

.3.3. Inhibition of Sgk1 kinase activity in an intact cell

environment

Cell cultures were serum-starved and treated with 12.5

Si113 and prodrugs for 18 h before stimulation. 1 M insulin was

added 30 min before lysis [Lysis buffer: 50 mM Tris-HCl, pH 7.4,

.15 M NaCl, 0.5% IGEPAL, 25 mM NaF,1 mM DTT,1 mM Na VO plus

m

M of

concentration of 5 mg/mL) to 200 mg of tissue, homogenate and

m

then centrifuged at 5000 rpm for 20 min. The supernatant was

recovered, ﬁltered and analyzed by LC-MS/MS. The quantiﬁcation

of each compound was performed by reference to the appropriate

calibration curve.

0

3

4

protease inhibitor cocktail 10 X (Sigma-Aldrich, St. Louis, MO)]. In

these experiments, endogenous Sgk1 was immunoprecipitated by

means of a Sgk1-speciﬁc rabbit polyclonal antibody (#07e315,

EMD Millipore). Immunoprecipitates were employed in the kinase

assay reaction as described above.

3.4.2. In vivo efﬁcacy on HCC xenografted mice model

Si113 was dissolved in DMSO [37] while its prodrugs were dis-

solved in saline solution. The compounds were administered

14

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

intraperitoneally into xenografted nude female mice at the dose of

plasmin solution where ProSi113-TP, in human plasma, showed a

higher percentage of hydrolysis of the drug but a shorter t1/2 value

than ProSi113-LKTP; the opposite trend is observed in human

plasmin solution. Indeed, the presence of a four-carbon linker in

ProSi113-LKTP makes the cleavage site more accessible to plasmin

(overexpressed in the tumor district), allowing greater hydrolysis

and consequent release of the Si113 parent drug. The in vitro inhi-

bition activity of Skg1 was evaluated for Si113 and both prodrugs.

As expected, the prodrugs showed pronounced inhibitory activity

only at the highest concentration tested, unlike Si113 which

showed inhibition of Sgk1 in a dose-dependent manner (inhibition

9

.5 and 21 mg/kg for Si113 and its prodrugs respectively to obtain

the same molar ratio as Si113 administered. At 6 weeks of age, the

6

mice were subcutaneously injected with 2.5 ꢃ 10 HepG2 cells

suspended in 200 mL of a 1:1 solution containing RPMI without

serum and Matrigel™ solution (BD Collaborative Research) in the

dorsal posterior-lateral right region. The mice were randomly

assigned to four groups of ﬁve animals and then administered

vehicle alone (DMSO or saline solution), Si113, ProSi113-TP or

ProSi113-LKTP for ﬁve days/week. The tumor volumes were

measured every 7 days using caliper. Speciﬁcally, two perpendic-

ular diameters (a ¼ smaller diameter; b ¼ larger diameter) were

measured, and the tumor volume was calculated in accordance

of Sgk1 activity by 75% as early as 0.6

90% at 12.5 M). It was interesting to note a signiﬁcant inhibition of

Sgk1 by ProSi113-LKTP already at 0.6 M by 53% and then

increasing to 73% at 12.5 M. In contrast, ProSi113-TP did not show

a marked inhibition at 0.6 M (only 18%) but only at the highest

tested concentration of 12.5 M showed inhibition of Sgk1 activity

mM increasing to more than

m

with formula V ¼

p

/6 ꢃ a2 ꢃ b. The mice were placed under general

m

anesthesia and sacriﬁced by vertebral dislocation.

m

3

.4.3. In vivo efﬁcacy on ovarian carcinoma xenografted mice

model

Paclitaxel was used at a concentration of 5 mg/kg/day, while

ProSi113-TP was administered in saline solution at a dosage of

equal to 80%. Moreover, the inhibition of Sgk1 kinase was evaluated

in an intact cell environment with and without insulin stimulation

of Sgk1. Si113 and its prodrugs showed similar inhibition of insulin-

stimulated Sgk1 activity, thus suggesting that the prodrugs if hy-

drolyzed in an intact cellular environment, reach the inhibitory

activity of Si113. To obtain further clariﬁcation on the inhibitory

activity of the two prodrugs on Sgk1, a molecular docking study was

performed. The results suggest that the presence of a four-carbon

atom linker in ProSi113-LKTP allows the moiety of the parent

drug to maintain the spatial orientation of Si113. In contrast to

ProSi113-TP which, not possessing an elongated spacer, is unable to

maintain the spatial orientation of the Si113 parent drug. This may

2

1 mg/kg/day. At 6 weeks of age, the mice were subcutaneously

6

injected with 2.5 ꢃ 10 A2780TC cells suspended in 200

mL of a 1:1

solution containing RPMI without serum and Matrigel™ solution

(

BD Collaborative Research) in the dorsal posterior-lateral right

region. The mice were randomly assigned to four groups of ﬁve

animals and then treated intraperitoneally with vehicle alone (sa-

line solution), ProSi113-TP and ProSi113-TP in association with

Paclitaxel for ﬁve days/week. The tumor volumes were measured

every 7 days using caliper as described above. The mice were placed

under general anesthesia and sacriﬁced by vertebral dislocation.

explain the activity of ProSi113-LKTP shown at 0.6 mM on Sgk1 as

opposed to ProSi113-TP. Besides, molecular dynamic simulations

conﬁrmed the binding mode stability of Si113 in contrast to the two

prodrugs, which did not form a stable complex.

3

.4.4. Statistical analysis

t-test and Bonferroni's Multiple Comparison Test (One-way

ANOVA) was performed to evaluate the differences between groups

using GraphPad Prism 8 software (San Diego, CA), and differences

were considered signiﬁcant at *p < 0.05, **p < 0.01, and

The in vitro cytotoxicity was evaluated on HCC HepG2 and Huh-

7 cell lines. The same trend was observed for Si113 and ProSi113-

LKTP after 72 h, where they showed comparable IC50 values on

both cell lines unlike ProSi113-TP which showed a cytotoxic effect

*

**p < 0.001.

3- and almost 14-fold lower than the parent drug and Si113-LKTP

4

. Discussion and conclusions

on HepG2 and Huh-7 cell lines, respectively. Cytotoxic activity

was also evaluated after 24 h in the presence of human plasmin.

HepG2 and Huh-7 cells treated in presence of plasmin with

ProSi113-TP showed IC50 values 2- and 3-fold lower than cells

treated without plasmin. In contrast, ProSi113-LKTP showed an

increased cytotoxic effect 4-fold higher on HepG2 cell line in

presence of plasmin, but similar IC50 values on Huh-7 were ob-

tained. The same evaluations were conducted on the ovarian car-

cinoma A2780 cell line obtaining comparable IC50 values after 72 h

for Si113 and both prodrugs. On the other hand, in presence of

human plasmin, ProSi113-TP showed increased anticancer activity

compared to cells treated without plasmin. To complete the pre-

clinical proﬁle of Si113 and its prodrugs, in vivo pharmacokinetic

properties were evaluated in healthy male mice treated intraperi-

toneally at a dosage of 9.5 and 21 mg/kg for Si113 and prodrugs,

respectively. Following administration, Si113 was rapidly detected

showing Cmax of 46.27 nmol/mL while the observed Cmax for

ProSi113-TP and ProSi113-LKTP was 8.09 and 2.66 nmol/mL,

respectively. Moreover, the Si113 obtained from hydrolysis of

ProSi113-LKTP showed a Cmax value 4-fold higher than Si113

released from the ProSi113-TP prodrug.

Si113, a Sgk1 inhibitor [1] with potent antiproliferative and pro-

apoptotic effect on different cancer cell lines [2,11,21e23], has been

selected for prodrugs development to achieve not only an

enhancement of its ADME properties, but also an active target

system that allows the release of the parent drug locally into the

tumor site. In this context, we have synthesized in good yield two

plasmin-activated Si113 prodrugs, including a small amino acid

sequence D-Ala-Leu-Lys (TP): ProSi113-TP and ProSi113-LKTP. The

rationale of this strategy is to exploit the high afﬁnity of TP for a

speciﬁc reactive site of plasmin [27], a protease recognized as

overexpressed in many tumors, including HCC [28] and ovarian

cancer [29,30]. ProSi113-TP is composed by the parent drug and TP

portion, connected by ester group, while ProSi113-LKTP presents an

ester linker elongated with a chain of four carbon atoms between

Si113 and TP. Si113-prodrugs have been fully characterized in terms

of their in vitro ADME properties. The analysis of thermodynamic

solubility demonstrated an improved water solubility for both

prodrugs in comparison to the respective drug Si113, validating this

approach as an excellent strategy to overcome the poor water

solubility of the parent drug. Both prodrugs showed a decrease in

passive permeability compared to the parent drug; this was pre-

dictable given its high hydrophobic nature. In addition, both pro-

drugs also showed good metabolic stability, as well as Si113. The

ability to release the active compound from each prodrug has been

conﬁrmed by hydrolysis assays in human plasma and human

The plasma half-life remains in the same range after treatment

with ProSi113-TP (15.30 h) and ProSi113-LKTP (21.82 h). On the

other hand, Si113 showed a t1/2 value more than 5-fold lower than

its prodrugs. Si113 and its prodrugs were detected in all tissues

analyzed at 24 h after administration. ProSi113-LKTP showed a

higher tissue concentration than Si113 and ProSi113-TP in hepatic

15

E. Rango, L. D'Antona, G. Iovenitti et al.

European Journal of Medicinal Chemistry 223 (2021) 113653

tissue and the same trend is observed in kidneys, lungs, and splenic

tissue. Starting from the in vitro activity proﬁle, ProSi113-TP was

selected for a preliminary 24 h biodistribution study in female nude

mice. The test was conducted to assess drug uptake in the ovaries

and tumor mass. In two groups of animals, A2780-xenografted and

non-xenografted mice, in ovaries, ProSi113-TP was found 2-fold

higher than Si113 (4.11 and 1.89 nmol/g, respectively) and the

same trend is observed in the ovaries of non-xenografted mice.

Moreover, higher concentrations of ProSi113-TP were detected in

the tumor mass (5.69 nmol/g). In contrast, the parental drug Si113

showed almost 3-fold lower tissue concentration (2.20 nmol/g)

than its prodrug. Finally, their antitumor efﬁcacy was evaluated in

HCC and ovarian carcinoma mice models. In HCC murine model, an

impressive reduction in tumor growth was recorded in all treated

compared to control with a reduction in tumor weight of around

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 manuscript was realized in memory of Maurizio Botta,

deceased on August 2, 2019.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113653.

9

0%. Concerning the antitumor effectiveness on the paclitaxel-

resistant ovarian carcinoma mouse model, as expected, treatment

with paclitaxel had no effect on tumor growth. The same was

achieved following treatment with ProSi113-TP alone, which

showed no signiﬁcant reduction compared to paclitaxel treatment,

but a substantial reduction in tumor volume was obtained when it

is used in combination with paclitaxel resulting in a reduction in

tumor mass weight of approximately 85% compared to control.

In conclusion, the results conﬁrmed not only the promising

antitumor action of Si113 already widely studied, but also a valid

active target strategy through a prodrug approach, able to improve

the uptake in cancer cells and not less, improve the pharmacoki-

netic properties of the parent drug suffering to low water solubility.

This has made it possible to obtain prodrugs that can be formulated

in aqueous solutions, more manageable, and well-tolerated in vivo

by systemic route. Therefore, we believe that the developed pro-

drugs, especially ProSi113-TP, given its remarkable in vivo anti-

tumor activity on HCC and especially in association with pacli-

taxel on paclitaxel-resistant ovarian carcinoma, a promising alter-

native to the common chemotherapeutic agents. For this reason,

through further studies and investigations, these compounds could

be the subject of future clinical trials.

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17

Article Doi

Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma

DOI: 10.1016/j.ejmech.2021.113653

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