New Class of Betulinic Acid-Based Nanoassemblies of Cabazitaxel, Podophyllotoxin, and Thiocolchicine

Article abstract of DOI:10.1021/acsmedchemlett.9b00668

Betulinic acid is validated as a new self-assembly inducer for the formation of nanoparticles (NPs) in combination with different drugs. The target compounds are characterized by the presence of anticancer drugs acting on tubulin dynamics and of a linker that could be a carbon chain or a triazole-based one. Nanoparticles formed are characterized and their biological activity is evaluated.

Full text of DOI:10.1021/acsmedchemlett.9b00668

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Letter
New class of betulinic acid-based nanoassemblies
of cabazitaxel, podophyllotoxin and thiocolchicine
Eleonora Colombo, Laura Polito, Michele Biocotino, Paola Marzullo,
Mariafrancesca Hyeraci, LISA DALLA VIA, and Daniele Passarella
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.9b00668 • Publication Date (Web): 28 Feb 2020
Downloaded from pubs.acs.org on February 28, 2020
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New class of betulinic acid-based nanoassemblies of cabazitaxel,
podophyllotoxin and thiocolchicine
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Eleonora Colombo,a Laura Polito,b Michele Biocotino,a Paola Marzullo,a Mariafrancesca Hyeraci,c Lisa Dalla Via, c
Daniele Passarella*,a
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a Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy. Email: daniele.passarella@unimi.it
b CNR-ISTM, Via G. Fantoli 16/15, 20138, Milano, Italy
c Dipartimento di Scienze del Farmaco - Università degli Studi di Padova - Via F. Marzolo 5, 35131 Padova, Italy
Betulinic acid is validated as a new self-assembly inducer for ability to form self-assembled nanoparticles and their ability to affect
the formation of nanoparticles (NPs) in combination to ovarian carcinoma cell viability are here reported.
different drugs. The target compounds are characterized by
BETULINIC ACID
the presence of anticancer drugs acting on tubulin dynamics
and of a linker that could be a carbon chain or a triazole-based
Self-assembly
one. Nanoparticles formed are characterized and their
O
Drug
Linker
biological activity is evaluated.
O
O
1
Keywords: Self-assembled nanoparticles, cancer, betulinic acid,
cabazitaxel, podophyllotoxin, thiocolchicine.
O
O
OH
OH
O
SMe
Drug
O
O
=
O
O
O
For several years we have been interested in the use of
nanotechnology to improve the properties of both anticancer and
O
NH
O
MeO
HO
O
O
MeO
MeO
O
O
O
NH2
O
neuroprotective drugs1-8
.
We designed conjugates able to
O
N
O
O
spontaneously assemble in water forming nanoparticles that can
release the drug in cellular media2, fluorescent hetero-NPs3,4 and
hetero-NPs bearing two different drugs4-6. These compounds present
the general structure of a drug conjugated through a linker to a self-
assembly inducer, that was either squalene2-5, 4-(1,2-diphenylbut-1-
en-1-yl)aniline7 or 20-hydroxyecdisone6. The choice of the self-
assembly inducer is important for the formation of nanoparticles, and
here is where we set our interest. In fact, the possibility to have a
moiety that not only is able to induce the aggregation, but also
possesses some biological activity towards the same target could be
useful to further improve the pharmacological properties of the drug.
O
2
3
4
O
O
N
N
Linker
=
O
O
5
6
Figure 1: Structure of the designed conjugate compounds.
To assess the capability of betulinic acid to act as a self-assembly
inducer, we conjugated it to three different drugs, all interacting with
microtubules as either stabilizers or destabilizers: N-desacetyl
thiocolchicine (2), cabazitaxel (3) and podophyllotoxin (4).
All these moieties were attached to betulinic acid through sebacic acid
as a linker. Moreover, an additional triazole-based linker was used
with N-desacetyl thiocolchicine to verify its influence in the formation
and in the biological properties of nanoparticles12. (Figure 2)
Our goal was to identify a pharmacologically active compound that
will be able to act as a self-assembly inducer in the formation of
nanoparticles. Our previous results regarding the use of 20-
hydroxyecdysone as a self-assembly inducer moved us to consider
betulinic acid, a natural product derived from plane tree bark, that has
shown beneficial properties for tumor therapy. In fact, the pentacyclic
triterpenoid betulinic acid exhibits a wide range of biological and
medicinal properties such as antivenom, anti-HIV, antibacterial,
antimalarial, anti-inflammatory, anthelmintic, antinociceptive, anti-
HSV-1 and anticancer activities9. It has been reported to induce
different forms of cell death, such as apoptosis, necrosis and
autophagy, in different types of cancers. In addition, nontumor cells,
such as fibroblasts and lymphocytes, resulted less affected by
betulinic acid than tumor cells10. The use of betulinic acid in
nanoformulations is really interesting11, also because other
pentacyclic triterpenoids were previously used as self-assembly
inducers, like oleanolic acid and ursolic acid. We decided to consider
betulinic acid for its possible dual activity (selective cytotoxic
compound and self-assembly inducer) and to incorporate it in
conjugated compounds using as second building block well-known
tubulin binders (Figure 1). Synthesis of the planned compounds, their
For this synthesis, betulinic acid needed to be modified, as its acidic
moiety could give side reaction in the conjugation steps. It was reacted
with trimethylsilyl diazomethane to form its methyl ester 11, that was
subsequently used for the synthesis of the two series of conjugates.
When sebacic acid was used as a linker, compound 11 was conjugated
to a monoprotected sebacic acid to give product 13 that, upon
deprotection by TBAF, gave us the desired betulinic acid-linker 14.
This moiety was then conjugated, under the same conditions, to the
three above mentioned drugs, leading to final compounds 7, 8 and 9.
(Scheme 1)
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MeO
MeO
MeO
OMe
O
O
NH2
O
O
O
HO
O
O
O
O
O
O
MeS
O
11
2
O
4
4
O
4
MeO
O
N3
17
15
O
HO
Cl
O
OMe
5
O
a
b)
)
O
O
MeO
MeO
MeO
O
BocHN
O
HO
O
O
AcO
9
O
BzO
OMe
N3
5
10
11
12
13
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N
O
O
8
7
H
O
O
4
O
O
O
MeS
16
18
OMe
O
c
)
O
O
O
O
MeO
MeO
MeO
O
OMe
O
MeO
MeO
MeO
4
4
O
O
N
N
O
N
O
4
N
HN
H
O
N
N
5
N
5
O
H
N
O
H
OMe
OMe
O
MeS
10
MeS
MeO
O
MeS
9
10
a) DIPEA, PPy, 0°C to rt, 80%; b) DCC, DMAP, CH2Cl2, 20h, quant; c) CuSO4-5H2O, sodium
ascorbate, DABCO, AcOH, H2O/t-BuOH 1,2:1, 3h, 51%.
Figure 2: Structure of the conjugates.
Scheme 2: Synthesis of thiocolchicine-triazole conjugate.
O
O
HO
Si
4
O
Synthetized all the desired conjugates, we evaluated the successful
formation of nanoparticles by dynamic light scattering (DLS)
measurements (Table 1). All the compounds gave a stable and
monodisperse suspension of NPs, characterized by hydrodynamic
diameters (HD) in the range of 320-560 nm and a negative Z-Potential
(< -25.0 mV).
12
OH
a
O
b
O
O
HO
HO
1
11
Si
O
O
c
Table 1: Hydrodynamic diameter and Z-potential of nanoformulated
compounds 7, 8, 9, 10.
O
O
O
O
HO
O
O
O
4
4
Compound Polidispersity
index (PI)
Hydrodinamic
Diameters (nm)
322±11
Z-Potential
(mV)
-34.02±0.48
-35.53±0.51
-41.03±0.65
-25.66±3.84
O
O
13
14
7
8
9
0.088±0.022
0.126±0.010
0.074±0.012
0.173±0.015
503±104
558±49
329±59
O
d
10
O
O
O
Drug
O
4
O
The ability of the building blocks (desacetylthiocolchicine,
podophyllotoxin and cabazitaxel), of conjugates and of the NPs to
affect cell viability was assayed by trypan blue exclusion test on
ovarian carcinoma cell line, A2780. The obtained results, expressed
as GI50 values (M), are shown in Table 2.
7:
8:
9:
drug= podophyllotoxin;
drug= cabazitaxel;
drug= N-desacetyl thiocolchicine.
a) (CH3)3SiCHN2, MeOH/PhCH3, rt, 30h, 95%; b) DCC, DMAP, CH2Cl2, rt, overnight,
92%; c) TBAF, THF, rt, 20h, 93%; d) drug, DCC, DMAP, CH2Cl2, rt, 26h, 66-95%
Scheme 1: Synthesis of conjugates with sebacic liker.
For what regards the conjugate bearing the triazole moiety, instead,
compound 11 was reacted with acyl chloride 15 (prepared following
a procedure already reported in literature12) to give the corresponding
ester. Meanwhile, the chosen drug, i.e. N-desacetyl thiocolchicine,
was conjugated to 17, thus obtaining azide 18. Having in our hands
the azide and the alkyne moieties, we performed a 1,3-dipolar
cycloaddition to obtain triazole 10 as the final compound. (Scheme 2)
Table 2: Cell growth inhibition of A2780 cells in the presence of tested
compounds and NPs.
Compound
N-Desacetylthiocolchicine (2)
A2780 cells (GI50 µM)a
0.0121 ± 0.0003
2.6 ± 0.1
10
NP-10
9
3.0 ± 0.1
14.5 ± 0.6
9.5 ± 1.2
NP-9
Podophyllotoxin (4)
0.0085 ± 0.0005
16.9 ± 0.3
7
NP-7
Cabazitaxel (3)
17.0 ± 3.2
0.000327 ± 0.000023
0.35 ± 0.01
8
NP-8
12.0 ± 2.3
17.6 ± 1.7
Betulinic methyl ester (11)
a Values are the mean ± SD of at least three independent experiments.
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Table 3: Data relative to Figure 3a.
All starting drugs are very effective in inducing cytotoxicity, as
2
3
4
5
6
7
8
9
expected, with GI50 values in the nanomolar range. Otherwise, 11, the
methyl ester of betulinic acid, appears significantly less active.
Interestingly, the antiproliferative capacity is similarly maintained
both by the conjugates as monomers and by the corresponding NPs,
even though to a lesser extent than native drugs. Indeed, for all NPs
were obtained GI50 values in the micromolar range. In particular, 10
and 9 exert the highest cytotoxicity, while 8 formulated as
nanoparticle demonstrates the most pronounced decrease in biological
effect with respect to the starting cabazitaxel, by about five orders of
magnitude. This result may be attributed to a slower disaggregation of
the nanoparticles or cleavage of the ester moiety. Moreover, it is
noteworthy the difference in antiproliferative effect between 10 and
9. In detail, 10 shows a GI50 value about three times lower than that
of 9, which highlights the crucial role played by the linker and, in
particular, by the insertion of the triazole moiety.
Control
N-Desac. (2)
[40nM]
15.5%
10
9
[5 µM]
[30 µM]
PreG0
G0/G1
S
0.6%
69.1%
12.8%
17.2%
6.5%
6.2%
11.4%
16.7%
55.8%
15.8%
10%
17.2%
8.7%
G2/M
67.1%
67.4%
Table 4: Data relative to Figure 3b.
10
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Podop. (4)
[15 nM]
7
Cabaz. (3)
[7.5 nM]
17.4%
30.8%
15.8%
8
[40 µM]
4.2%
56.9%
11.5%
27.3%
[30 µM]
PreG0
G0/G1
S
9.8%
39.4%
16.7%
33.2%
12.4%
30.5%
26.4%
30.5%
G2/M
35.5%
N-Desac. (2) [40 nM]
Control
The maintenance of the cytotoxic effect by the NPs prompts us to
investigate if the mechanism of action is also retained. For this
purpose, cytofluorimetric analyses were performed on A2780 stained
with propidium iodide and incubated with test agents, and the effect
on cell cycle was examined. The cytograms of DNA content and the
percentages of cells in the different phases of cell cycle are reported
in Error! Reference source not found.a-b and in Error! Reference
source not found. and Table 4. The obtained results indicate that the
NPs are able to induce an increase in G2/M, thus acting, in accordance
with the starting drugs, as microtubule-targeting agents.
9 [30 μM]
10 [5 μM]
Conclusions
A class of betulinic acid-based conjugate compounds of cabazitaxel,
podophyllotoxin and thiocolchicine were prepared. The obtained
compounds self-assemble and form nanoassemblies that we
characterized in this study. Both the conjugates as monomers and the
relative nanoparticles were tested for their antiproliferative activity,
obtaining good results even though all the products result less active
then the native drugs. In particular, while the thiocolchicine-based
conjugates maintain an interesting activity, especially the compound
that presents the triazole ring-based linker, the one bearing cabazitaxel
was the most affected by the conjugation. A reasonable explanation
of that can be attributed to a sluggish disaggregation of said
nanoparticles, to a limited interaction of the drug conjugate or to a
partial hydrolysis of the ester bond that connects the native drug to the
linker. We consider relevant that the studies regarding the biological
mechanism inducing the detected cytotoxicity show an increase in
G2/M, that confirms the maintenance of the activity of the native
microtubules-tubulin binders cabazitaxel, podophyllotoxin and
thiocolchicine. The introduction of a proper self-immolative linker
could secure the release of the native drugs and the improvement of
3a
Podop. (4) [15 nM]
7 [30 μM]
Cabaz. (3) [7.5 nM]
8 [30 μM]
the biological activity. The described results are
a further
demonstration of the easy obtainment of self-assembled nanoparticles
by simple chemical functionalization of known anticancer drugs and
of the possible modulation of their activity by varying the nature of
the self-assembly inducer and linker.
Supporting information
3b
This material is available free of charge via the internet at
http://pubs.acs.org.: experimental details regarding synthesis of
Figure 3a-b: Cell cycle distribution of A2780 cells incubated in the
presence of the test agents for 24h at the indicated concentrations.
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conjugates, nanoparticles preparation and characterization, and
biological evaluation.
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