DSC studies on the interaction of lipophilic cytarabine prodrugs with DMPC multilamellar vesicles

Article abstract of DOI:10.1007/s10973-019-08780-x

Cytarabine (1-β-d-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is used for the treatment of both acute and chronic myeloblastic leukemias and non-Hodgkin lymphoma. It has a very short plasma half-life and a very low oral bioavailability. To overcome these disadvantages, much effort has been focused on the design of cytarabine prodrugs. In this study, we have synthesized four different cytarabine prodrugs in order to increase the drug lipophilicity and the affinity of the prodrugs toward the biological membranes, as well as the lipophilic carriers. Differential scanning calorimetry was used to study the interaction of cytarabine and its prodrugs with multilamellar vesicles (MLVs) made of dimyristoylphosphatidylcholine (DMPC) and used as a model of biomembranes as well as a lipophilic carrier. The results showed that the 4-N-acetyl-2′,3′-5′-acetyl derivative and the prodrug with short chain fatty acids do not have a significant affinity with MLVs, whereas the prodrugs with long chain fatty acids have a stronger affinity with the MLVs with respect to cytarabine. The entity of the affinity depends on the fatty acids length. The increased affinity could be due to the fatty acid moieties which allow the molecule to insert among the phospholipid molecules. These results provide information on the interaction of these prodrugs with biomembranes and could be useful to design liposomes as carriers for the prodrugs.

Full text of DOI:10.1007/s10973-019-08780-x

Journal of Thermal Analysis and Calorimetry
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DSC studies on the interaction of lipophilic cytarabine prodrugs
with DMPC multilamellar vesicles
Jhon Fernando Berrio Escobar Diana Margarita Marquez Fernandez Cristiano Giordani1,2 •
1 •
1 •
3
3
Francesco Castelli
•
Maria Grazia Sarpietro
Received: 19 October 2018 / Accepted: 1 September 2019
Ó Akad e´ miai Kiad o´ , Budapest, Hungary 2019
Abstract
Cytarabine (1-b-D-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is used for the treatment of both
acute and chronic myeloblastic leukemias and non-Hodgkin lymphoma. It has a very short plasma half-life and a very low
oral bioavailability. To overcome these disadvantages, much effort has been focused on the design of cytarabine prodrugs.
In this study, we have synthesized four different cytarabine prodrugs in order to increase the drug lipophilicity and the
affinity of the prodrugs toward the biological membranes, as well as the lipophilic carriers. Differential scanning
calorimetry was used to study the interaction of cytarabine and its prodrugs with multilamellar vesicles (MLVs) made of
dimyristoylphosphatidylcholine (DMPC) and used as a model of biomembranes as well as a lipophilic carrier. The results
0
0
0
showed that the 4-N-acetyl-2 ,3 -5 -acetyl derivative and the prodrug with short chain fatty acids do not have a significant
affinity with MLVs, whereas the prodrugs with long chain fatty acids have a stronger affinity with the MLVs with respect
to cytarabine. The entity of the affinity depends on the fatty acids length. The increased affinity could be due to the fatty
acid moieties which allow the molecule to insert among the phospholipid molecules. These results provide information on
the interaction of these prodrugs with biomembranes and could be useful to design liposomes as carriers for the prodrugs.
Keywords Cytarabine Á Prodrugs Á DSC Á Biological membranes Á DMPC
Introduction
membrane consists of one or more lipids representing the
main membrane components. Phospholipids have often
been used for such studies, and among them, phos-
phatidylcholine is a predominant phospholipid in most of
the mammalian plasma membranes [6].
Drugs are known to interact with biomembranes producing
changes in their physical characteristics and functions.
Therefore, it is very important to understand the molecular
mechanism of interaction of these molecules with
biomembranes [1]. However, as biomembranes are very
intricate structures with complex behaviors, studies are
often carried out with model membranes with selected and
controlled composition [2–5]. The simplest model
By increasing the temperature, fully hydrated saturated
0
phosphatidylcholines form different phases: gel (Lb ,
0
ordered) phase, ripple (Pb ) phase and liquid crystalline (L,
disordered) phase. The pre-transition is associated with
lamellar to periodic gel transition leading to a ripple
structure. Then, in the main transition, the lipid acyl chains
in an all-trans configuration (ordered) undergo a chain
melting transition to a liquid crystalline (disordered) phase
with the chains having both trans as well as gauche con-
figurations [7, 8]. The temperature at which this transition
&
Maria Grazia Sarpietro
mg.sarpietro@unict.it
1
Grupo Productos Naturales Marinos, Facultad de Ciencias
Farmac e´ uticas y Alimentarias, Universidad de Antioquia,
Calle 70 No 52-21, Medell ´ı n, Colombia
occurs is called transition temperature (T ). In physiolog-
m
ical conditions, in most of the biomembranes, the lipids are
in the liquid crystalline state.
2
3
Instituto de F ´ı sica, Universidad de Antioquia, Calle 70 No
2-21, Medell ´ı n, Colombia
5
The thermotropic properties of the model membranes
are affected by any changes in the lipid packing within the
Dipartimento di Scienze del Farmaco, Universit a` degli Studi
di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
123

J. F. B. Escobar et al.
bilayer. Exogenous molecules in the bilayer can perturbate
and disrupt the lipid packing. Then, the study of the ther-
motropic properties of the membrane model can give
indication on the kind of interaction of the lipid with the
exogenous molecules in the bilayer.
compounds obtained were characterized by nuclear mag-
netic resonance spectroscopy on a Bruker NMR-300
spectrometer for per-acetylated cytarabine and an NMR-
600 Ascend spectrometer for derivatives with fatty and
butyric acids, where dimethylsulfoxide-d6 and deuterated
chloroform, was used as solvent by the aliphatic and fatty
acids, respectively, and the tetramethylsilane (TMS) was
used as internal standard.
Cytarabine has been widely used in cancer chemother-
apy since the 1960s against soft (leukemia and lymphoma
non-Hodgkin) and solid (breast cancer, among others)
tumors. This drug exhibits several action mechanisms such
as disruption of nucleic acids synthesis, incorporation and
demethylation of DNA fragments and inhibition of neces-
sary nucleoside synthesis. However, the cytarabine shows
many side effects as gastrointestinal disorder, neurotoxic-
ity, nephrotoxicity and hepatotoxicity, among others
Mass spectra analysis was performed on an Agilent
6300-LC/MS equipment (electrospray ionization (ESI) and
Ion-Trap analyzer) and Thermo Scientific Exactive Plus
EMR (ESI and Orbitrap analyzer), both in positive mode
and direct injection method.
[
9, 10].
Most side effects are due to pharmacodynamic and
Per-acetylated reaction
pharmacokinetic problems, and many of these disadvan-
tages are related to the physicochemical properties such as
the higher polarity and chemical lability to acid medium.
Among the pharmacodynamic disadvantages, there is low
cellular incorporation across transporter channels, intra-
cellular inactivation and degradation by several enzymes,
higher elimination through transporter channels and vesi-
cles. Some of the principal pharmacokinetic problems are
low selectivity by cancer cell, higher chemical lability and
biological degradation and inactivation in blood plasma
The cytarabine (300 mg) was dissolved in an acetic
anhydride/DMF/TEA (1:1:1) mixture, and after the DMAP
(5 mol%) is added. The reaction mixture was stirred at
room temperature for 8 h (see reaction scheme) [11].
0
0
0
4-N-acetyl-3 ,4 ,6 -O-triacetyl-cytarabine
The product was isolated as yellow solid in quantitative
1
yield. H NMR (300 MHz; CDCl ), d ppm (multiplicity;
3
0
0
integration; J (Hz); position) 1.89 (s; 3H; H-2 acetyl
0
[
11, 12].
In this paper, interaction of cytarabine and cytarabine
linked to O-3 arabinofuranosyl ring), 2.09–1.98 (m; 6H;
0
0
0
0
H-2 acetyl linked to O-4 and O-6 arabinofuranosyl ring),
0
0
prodrugs with model membranes was investigated by DSC.
As model membranes, we used multilamellar vesicles
2.21 (s; 3H; H-2 acetyl linked to 4-N arabinofuranosyl
0
ring), 4.16 (t; 1H; J = 10.5; H-5 arabinofuranosyl ring),
0
(
MLVs) of dimyristoylphosphatidylcholine (DMPC). The
4.33 (t; 2H; J = 12.3; H-6 arabinofuranosyl ring), 5.04 (d;
0
effects of cytarabine and cytarabine derivatives on the
thermotropic behavior of MLVs were monitored.
1H; J = 12.2; H-4 arabinofuranosyl ring), 5.49 (d; 1H;
0
J = 2.5; H-3 arabinofuranosyl ring), 6.29 (d; 1H; J = 3.4;
0
H-2 arabinofuranosyl ring), 7.43 (d; 1H; J = 7.6 Hz; H-5
cytosine ring), 7.88 (d; 1H; J = 7.6 Hz; H-6 cytosine ring).
Materials and methods
ESI-HRMS (positive mode) m/z: Calculated for
?
C H N O : 411.3634; found, 434.1173 [M ? Na] .
1
7 21 3 9
Chemicals
NMR carbon characterization and 2D correlation spec-
troscopy were reported previously [11].
All solvents were employed of reactive grade. The
cytarabine 99% (Alfa Aesar) was employed as substrate;
acetic anhydride and butyric, myristic and stearic acids
Percylation reaction with butyric acid
9
9% (Merck) were employed as agent by per-acylation
0
The DIC (1.5 equivalents with respect to theoretical
amount of aliphatic acid) was dissolved in a butyric acid/
DMF/TEA (1:1:1) mixture; this was stirred at room tem-
perature for 6 h for a pre-activation of the coupling agent,
after the cytarabine (300 mg) and DMAP (5 mol%) were
added. The reaction mixture was stirred at room tempera-
ture for 8 h (see reaction scheme in Fig. 1) [11].
reaction; N,N -dicyclohexylcarbodiimide (DCC) 99%
0
(
Alfa Aesar) and N,N -diisopropylcarbodiimide (DIC) were
used as coupling agent, and 4-N,N-dimethylamino-pyridine
DMAP) 99% (Alfa Aesar) was employed as nucleophilic
catalyst.
The per-acylated derivatives of cytarabine were purified
(
by column and preparative thin-layer chromatography in
normal phase, with ethyl acetate/acetone and hexane/ethyl
acetate mixtures as eluent systems for derivatives with
aliphatic and fatty acids as substituents, respectively. All
1
23

DSC studies on the interaction of lipophilic cytarabine prodrugs with DMPC multilamellar…
Fig. 1 Scheme reaction for
O
1"
synthesis of cytarabine per-
acylated with long and short
aliphatic acids
NH2
HN
4
R
2"
5
4
N
R
O
3
5
N
3
2
HO
6
RCOOH, DIC, DMAP
6
'
N
O
O
6
1
6'
2
O
N
1
O
Chloroform/DMF/TEA, rt
1
'
O
5
'
2'
HO
1
'
5
'
2'
HO
4
'
3
'
4
'
3'
OH
R
O
DIC, DMAP
Butyric
acid/DMF/TEA,rt
Acetic
anhydride/DMF/TEA,
O
DMAP, rt
O
O
3
"
1
"
1
"
HN
2"
HN
4"
2
"
5
4
5
4
N
3
N
O
O
3
2
6
6'
6
6'
2
O
N
1
O
N
O
O
O
1
O
O
1
'
1'
O
2'
5'
2'
5'
O
O
4'
4
'
3
'
3
'
O
O
O
O
0
0
0
4
-N-butyryl-3 ,4 ,6 -tributyryl-cytarabine
room temperature for 6 h for a pre-activation, after DMAP
5 mol%) and cytarabine (300 mg) was added [13]. The
(
1
The product was isolated as yellow solid (60% yield). H
reaction mixture was stirred at room temperature for 7 days
(see reaction scheme).
NMR (600 MHz, DMSO-d6) d ppm (multiplicity; inte-
gration; J (Hz); position):1.3–1.0 (m; 20H; H-3 y H-4
00
00
0
0
0 0
4-N-myristoyl-4 ,6 -O-dimyristoyl-cytarabine
butanoyl or butyryl chain), 1.8–1.6 (m; 8H; H-2 (a pro-
0
tons) butanoyl chain), 3,96 (m; 1H; H-6 arabinofuranosyl
0
1
The product was isolated as white solid (30% yield). H
ring), 4,05 (m; 1H; H-6 arabinofuranosyl ring), 5.62 (m;
0
0
1
H; H-5 arabinofuranosyl ring), 5.67 (d; 1H; H-3 arabi-
0
NMR (600 MHz; CDCl ), d ppm (multiplicity; integration;
3
00
nofuranosyl ring), 5,72 (d; 1H; H-4 arabinofuranosyl ring),
0
J (Hz); position): 0.92 (t; 9H; J = 6.6; H-14 myristoyl
00 00
6
,05 (d; 1H; J = 4.5 Hz; H-2 arabinofuranosyl ring), 7,61
chain), 1.32 (m; 48H; H-4 to H-13 myristoyl chain), 1.70
00
(m; 6H; H-3 (b protons) myristoyl chain), 2.40 (m; 6H;
(
d; 1H; J = 8.1 Hz; H-5 cytosine ring), 7,78 (d; 1H;
0
0
0
J = 8,2; H-6 cytosine ring). ESI-HRMS (positive mode)
H-2 (a protons) myristoyl chain), 4.14 (m; 2H; H-6 ara-
0
m/z: Calculated for C H N O : 523.5759; found,
2
binofuranosyl ring), 4.44 (m; 1H; H-5 arabinofuranosyl
0
5 37 3 9
?
46.4113 [M ? Na] .
5
ring), 4.76 (d; 1H; J = 2.8; H-3 arabinofuranosyl ring),
0
5.15 (m; 1H; H-4 arabinofuranosyl ring), 6.08 (d; 1H;
0
Per-acylation of amine and two hydroxyls groups
J = 3.2; H-2 arabinofuranosyl ring), 7.45 (d; 1H; J = 7.8;
H-5 cytosine ring), 7.90 (d; 1H; J = 7.8; 6 cytosine ring).
ESI-HRMS (positive mode) m/z: Calculated for
The esterification reaction for amine group (H N-4) cyto-
2
0
0
?
sine ring and hydroxyl groups (OH-4 and OH-6 ) of ara-
binofuranosyl ring of cytarabine with fatty was performed
by Steglich esterification. DCC (1.5 equivalents with
respect to fatty acid) was aggregated to fatty acid (4.5
equivalents with respect to nucleoside) solution in Chlo-
roform/DMF/TEA (1:2:1) mixture; this mix was stirred at
C H N O : 874.2835; found, 897.7647 [M ? Na] .
51 91 3 8
0
0
4-N-stearoyl-4 ,6 -O-distearoyl-cytarabine
1
The product was isolated as white solid (25% yield). H
NMR (600 MHz; CDCl ), d ppm (multiplicity; integration;
3
123

J. F. B. Escobar et al.
0
0
J (Hz); position): 0.87 (t; 9H; J = 6.5; H-18 stearoyl
00
and as solvent used for reaction mixtures, thus increasing
significantly the effective collisions among the substrate
and reagent.
00
chain), 1.25 (m; 48H; H-4 to H-17 stearoyl chain), 1.56
00
(
m; 6H; H-3 (b protons) stearoyl chain), 2.32 (m; 6H;
0
0
0
0
0
H-2 (a protons) stearoyl chain), 4.09 (m; 2H; H-6 ara-
The esterification on OH-4 and OH-6 positions is very
easy by the fact they showed isomerism E (these are anti-
0
binofuranosyl ring) 4.35 (m; 1H; H-5 arabinofuranosyl
0
0
ring), 4.72 (m; 1H; J = 2.9; H-3 arabinofuranosyl ring),
0
oriented among themselves) and the hydroxyl in 6 is far
5
.12 (m; 1H; H-4 arabinofuranosyl ring), 6.05 (d; 1H;
away from cytosine ring. However, the per-acylation of all
the hydroxyl groups of this nucleoside with fatty acids was
not possible to obtain due to two reasons: first, the per-
acylation on all of them is very complex, because the OH-
0
J = 3.4; 2 arabinofuranosyl ring), 7.49 (d; 1H; J = 7.6;
H-5 cytosine ring), 7.93 (d; 1H; J = 7.6; H-6 cytosine
ring). ESI-HRMS (positive mode) m/z: Calculated for
?
0
C H N O : 1042.6025; found, 1065.9217 [M ? Na]
6
3 is syn-oriented in relation to the cytosine ring (these
3 115 3 8
?
and 1043.9202 [M ? H] .
groups showed isomerism Z); therefore, the steric inter-
actions among the long acyl chain and nitrogenous base are
strong. Moreover, the Steglich esterification requires the
MLVs preparation
0
formation of O-acyl-isourea intermediate, starting of N,N -
MLVs containing different molar fractions of compounds
were prepared as follows. Solutions of DMPC and each of
the compounds were separately prepared in chloroform/
methanol (1:1). Aliquots of the DMPC (containing
dicyclohexylcarbodiimide and the carboxylic acid. This
intermediate presents a reactivity similar to the corre-
sponding carboxylic anhydride but the fatty O-acyl-isourea
intermediate is a bulky compound; the steric effects cause a
decrease in effective shocks between the intermediate and
nucleophilic catalyst (DMAP), therefore in the amount of
amide quaternary type (intermediate 2) formed. Further-
0
.01032 mmol) were delivered in glass tubes. Aliquots of
each compound were added to have the following molar
fraction of compound: 0.015, 0.03, 0.045, 0.06, 0.09, 0.12,
0
0
.15 and 0.18. Then, phospholipid/compound films were
prepared, evaporating the solvents under nitrogen flow, at
7 °C. The films were kept in a freeze dryer for at least
more, when the OH-3 hydroxyl group attacks as nucle-
ophile on carbonyl of intermediate 2, they will show steric
effects among this group and the cytosine ring.
3
four hours to completely eliminate the solvents. 168 lL of
a TRIS solution (5 mM, pH 7.4) was added to the film. The
sample was kept in a water bath at 37 °C for 1 min and
vortexed for 1 min, for three times; finally, it was left 1 h at
Cytarabine is a highly polar substance; consequently, it
shows high solubility on polar solvents as water, methanol
and ethanol, among others; due to this, the amount dis-
solved in the solvent of the mix reaction is low. Never-
theless, the per-acylated products are highly soluble in the
mixture reaction; this phenomenon causes the displacement
to the right of the equilibrium between solid and dissolved
substrates, therefore reaction yield improves. The per-
acylated derivatives of cytarabine show lower polarity than
the nucleoside; the polarity decreases with the chain length
increasing. As a result, the derivatives with fatty acids are
more soluble on nonpolar solvents (hexane/ethyl acetate
mix, chloroform, among others) than the per-acylated
compounds with short chain that show higher solubility on
medium-polar solvents such as ethyl acetate, ethyl acetate/
acetone mix.
37 °C. MLV without compound were also prepared as a
Ref. [14–16].
DSC analysis
1
20 lL of the MLVs dispersion containing 5 mg of DMPC
was put into an aluminum calorimetric pan (160 lL). The
pan was sealed and submitted to the following analysis: a
-
1
heating scan from 5 to 37 °C, at the rate of 2 °C min ; a
-
1
cooling scan from 37 to 5 °C, at the rate of 4 °C min ; the
heating and cooling scan were repeated for at least three
times.
The per-acylated compounds present polar and nonpolar
domains, represented by nucleoside and aliphatic sub-
stituents, respectively. Therefore, they have an amphiphilic
behavior; this property can give an advantage when they
interact with the amphiphilic phospholipids; this effect
becomes stronger with the increase in the chain. (See the
results obtained in the calorimetric analysis.)
Results and discussion
Chemicals
Synthesis
The per-acetylated products of cytarabine with acetic acid
and butyric acid were obtained in quantitative and 60%
yields, respectively; this was possible because acetic
anhydride and butyric acid are employed in higher amounts
NMR data
The per-acylated derivatives were identified by comparison
13
1
of H-NMR,
1
C-NMR, H- H COSY and HSQC
1
1
23

DSC studies on the interaction of lipophilic cytarabine prodrugs with DMPC multilamellar…
1
cytarabine data (300 MHz and 600 MHz; DMSO-d6). H-
with those of DMPC-MLVs containing the compounds
1
3
NMR and C-NMR signals for the monosaccharide rings
and nitrogenous bases of the nucleoside derivatives pre-
sented a significant deviation on their chemical shift from
the respective substrates (cytarabine). The NMR data and
masses founded with the MS spectrometry fit with the
structures proposed.
(Fig. 2). The calorimetric curve of DMPC-MLVs is char-
acterized by a pre-transition peak that originates from the
heat absorption during the passage from the ordered (gel)
state to a ripple phase and by a peak at 24.5 °C, due to the
heat absorption during the so-called lipid melting, due to
the passage from the ripple phase to the disordered (liquid
crystalline) phase [17].
The COSY H–H spectrums of the per-acylated deriva-
tives of cytarabine in CDCl show at high field that the
3
The calorimetric curves of DMPC-MLVs prepared with
different amounts of cytarabine do not show significant
differences with respect to that of DMPC-MLVs: the pre-
transition peak is always present, and the main transition
peak remains almost unchanged (Fig. 2a). This is an evi-
dence that cytarabine does not interact with the phospho-
lipidic bilayers of DMPC-MLVs.
0
0
signals for the H-5 and H-6 protons of arabinofuranosyl
0
0
ring are not overlapped, neither are the H-3 and H-4
protons. Therefore, it is possible to observe two couplings,
0
0
first coupling among H-5 and H-6 protons, a second weak
0
0
coupling between H-3 and H-4 signals. Moreover, the
0
H-3 proton is coupled with a high deshielding signal,
0
corresponding to the anomeric proton (H-2 ) of the arabi-
As the calorimetric curves of DMPC-MLVs prepared
0
0
0
nofuranosyl ring. The chemical shift of proton of arabi-
with 4-N-acetyl-3 ,4 -6 -acetyl derivative are regarded,
what it is observed is the disappearance of the pre-transi-
tion peak at 0.045 molar fraction, whereas the main tran-
sition peak is almost unaffected (Fig. 2b). This behavior
suggests that the compound interacts mainly with the polar
part of the DMPC-MLVs phospholipids.
0
0
0
nofuranosyl ring in the 4-N-acetyl-3 ,4 ,6 -O-triacetyl-
cytarabine is higher than in the per-acylated derivatives
with fatty acids. At downfield, it is observed an intense
coupling between two highly deshielding signals, H-5 and
H-6 protons of cytosine ring.
The HSQC spectrums of the per-acylated derivatives of
0
In the calorimetric curves of DMPC-MLVs prepared
0
0
0
cytarabine in CDCl show at high field a coupling by H-5
3
with the 4-N-butyryl-3 ,4 -6 -butyryl derivative, the pre-
transition peak is gradually abolished; the main transition
peak moves toward lower temperatures (Fig. 2c). This
indicates that the derivative interacts with the polar head as
well as with the apolar tails of the phospholipids. The
proton of the arabinofuranosyl ring with a deshielding
0
carbon, corresponding to C-5 of the monosaccharide. In
addition, it is possible to notice a coupling between the
0
H-6 proton of arabinofuranosyl ring and a shielding car-
0
0
0
bon, corresponding to C-6 of the monosaccharide, as well
0
effect of 4-N-myristoyl-4 ,6 -O-dimyristoyl-cytarabine on
DMPC-MLVs is very strong: the pre-transition peak is no
more present since the smallest molar fraction of com-
pound; the main transition peak shifts toward lower tem-
peratures and broadens (Fig. 2d). This is a clear evidence
of a strong interaction of the compound with the phos-
pholipids and in particular with the polar head and with the
apolar chains.
the expected couplings between the proton signals H-4 ,
0
0
H-3 and H-2 of arabinofuranosyl ring and the carbons
corresponding to the same position of the monosaccharide.
At downfield, a coupling is detected for the proton signal of
the H-5 cytosine ring with deshielding carbon, corre-
sponding to C-5 of the same nitrogenous base and a second
coupling among the proton signal with highest deshielding
of the cytosine ring (H-6) and a higher deshielding carbon,
position C-6 of cytosine ring.
0
0
4-N-stearoyl-4 ,6 -O-distearoyl-cytarabine causes deep
variations in the DMPC-MLVs calorimetric curve. In fact,
the pre-transition peak is no more present since the smallest
molar fraction of compound. The main transition peak
shifts toward lower temperature and broadens. At 0.015
molar fraction of compound, the main transition peak is
well-defined; as the compound molar fraction increases, the
main transition peak is proceeded by a shoulder (Fig. 2e).
Calorimetric analysis
In this research, we investigated the effect of cytarabine
and four cytarabine prodrugs on the thermotropic behavior
of model biomembranes represented by DMPC-MLVs to
get insight into the possible interaction mechanism
between the investigated compounds and DMPC-MLVs.
To this purpose, DMPC-MLVs were prepared with
different molar fraction of the compounds. Empty DMPC-
MLVs were also prepared, for comparison with those
containing cytarabine and the four cytarabine prodrugs, and
used as reference.
0
0
This behavior indicates that also 4-N-stearoyl-4 ,6 -O-dis-
tearoyl-cytarabine interacts with the polar as well as with
the apolar part of the phospholipids.
The transition temperature (transition temperature, Tm)
values associated with the main transition peak of DMPC-
MLVs prepared with different amount of compounds are
reported in Fig. 3 as a function of the molar fraction of
compound. The compounds affect the transition tempera-
ture differently. The transition temperature of DMPC-
The effect of the compounds on the DMPC-MLVs was
studied comparing the calorimetric curve of DMPC-MLVs
123

J. F. B. Escobar et al.
0
0
0
0
0
0
0
0
0
.18
.15
.12
.09
.06
.045
03
Endo
.015
.00
5
mW
a
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Temperature/°C
0
0
0
0
0
0
0
0
.18
.15
.12
.09
.06
.045
03
0.18
0.15
0.12
0.09
0.06
0.045
003
Endo
Endo
.015
0.015
0.00
5
mW
5
mW
0.00
b
c
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Temperature/°C
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Temperature/°C
0
0
0
0
0
0
0
0
0
.18
.15
.12
.09
.06
.045
03
0.18
0.15
0.12
0.09
0.06
0.045
003
Endo
Endo
.015
.00
0.015
0.00
5
mW
5
mW
d
e
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Temperature/°C
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Temperature/°C
1
23

DSC studies on the interaction of lipophilic cytarabine prodrugs with DMPC multilamellar…
bFig. 2 Calorimetric curves, in heating mode, of DMP-MLV prepared
with increasing molar fractions of a cytarabine, b 4-N-acetyl-3 ,4 ,6 -
cytarabine
0
0
0
4-N-acetyl-3',4',6'-triacetyl-cytarabine
4-N-butyroyl-3',4',6'-tributyroyl-cytarabine
4-N-myristoyl-4',6'-O-dimyristoyl-cytarabine
4-N-stearoyl-4',6'-O-distearoyl-cytarabine
0
0
0
O-triacetyl-cytarabine, c 4-N-butyroyl-3 ,4 ,6 -O-tributyroyl-cytara-
0
0
bine, d 4-N-myristoyl-4 ,6 -O-dimyristoyl-cytarabine and e 4-N-
0
0
stearoyl-4 ,6 -O-distearoyl-cytarabine. The labels at the right side of
each curve represent the molar fraction of compound. The curve 0.00
belongs to DMPC-MLV prepared without compound
6
5
4
3
2
1
0
cytarabine
4-N-acetyl-3',4',6'-triacetyl-cytarabine
4-N-butyroyl-3',4',6'-tributyroyl-cytarabine
4-N-myristoyl-4',6'-O-dimyristoyl-cytarabine
4-N-stearoyl-4',6'-O-distearoyl-cytarabine
2
2
2
2
2
2
2
6
5
4
3
2
1
0
0
0.05
0.1
0.15
0.2
Molar fraction
Fig. 4 DT1/2 (peak width at half height) of DMPC-MLV prepared
with increasing molar fractions of compound, as a function of the
molar fraction of compound
increases up to 0.045 molar fraction; with regard to 4-N-
0
0
myristoyl-4 ,6 -O-dimyristoyl-cytarabine, it increases up
0.12 molar fraction.
0
0.05
0.1
0.15
0.2
Molar fraction
The enthalpy variation (DH) values are reported in
Fig. 5 as a function of compound molar fraction. All the
compounds cause a decrease in this parameter. However,
the extent of the decrease is different. It is very small for
Fig. 3 Transition temperature of DMPC-MLV prepared with increas-
ing molar fractions of compound, as a function of the molar fraction
of compound
0
0
0
cytarabine and the 4-N-acetyl-3 ,4 -6 -acetyl derivative and
0
0
0
bigger for the other compounds. The enthalpy variation
MLVs prepared with cytarabine, 4-N-acetyl-3 ,4 -6 -tri-
0
0
0
acetyl derivative and 4-N-butyryl-3 ,4 -6 -tributyryl
derivative do not variate in a significant way, even at high
molar fraction of compounds. From a general point of
cytarabine
4
-N-acetyl-3',4',6'-triacetyl-cytarabine
4
-N-butyroyl-3',4',6'-tributyroyl-cytarabine
0
0
view, both 4-N-myristoyl-4 ,6 -O-dimyristoyl-cytarabine
4-N-myristoyl-4',6'-O-dimyristoyl-cytarabine
4-N-stearoyl-4',6'-O-distearoyl-cytarabine
0
0
and 4-N-stearoyl-4 ,6 -O-distearoyl-cytarabine, cause a
variation of the transition temperature. For both com-
pounds, the transition temperature decreases by 12% at the
highest molar faction. The transition temperature decreases
almost gradually up to 0.06 molar fraction; however, sig-
nificant differences exist between the two compounds for
higher molar fractions: the entity of transition temperature
5
0
0
4
30
2
0
0
0
0
0
decrease is smaller for 4-N-myristoyl-4 ,6 -O-dimyristoyl-
1
cytarabine, whereas the transition temperature remains
0
0
almost constant for 4-N-stearoyl-4 ,6 -O-distearoyl-cytara-
bine. Plotting the DT1/2 (peak width at half height) as a
function of compound molar fraction (Fig. 4), it results that
– 10
0
0
0
0
0.05
0.1
0.15
0.2
cytarabine, the 4-N-acetyl-3 ,4 -6 -acetyl derivative and the
0 0 0
Molar fraction
4
-N-butyryl-3 ,4 -6 -butyryl derivative, has a very small
effect on this parameter, whereas the two derivatives with
long chains strongly affect it. The DT1/2 increases up to a
certain molar fraction, and then it remains almost constant;
0
0
Fig. 5 Enthalpy variation, as DDH/DH (DDH = DH - DH , where
DH is the enthalpy variation of DMPC-MLV prepared with the
0
compound and DH is the enthalpy variation of pure DMPC-MLV), of
DMPC-MLV prepared with increasing molar fractions of compound,
as a function of the molar fraction of compound
0
0
with regard to 4-N-stearoyl-4 ,6 -O-distearoyl-cytarabine, it
123

J. F. B. Escobar et al.
values decrease occurs at small molar fraction of com-
pound; afterward, the values remain almost constant.
By taking together all the results, it permits to make a
hypothesis on the interaction occurring between the tested
compounds and the DMPC-MLVs. The first data taken in
consideration is the presence or not of the pre-transition
peak. For all the compounds, we assist to the disappearance
of the peak; as this behavior indicates that one compound is
able to interact with the polar head of the phospholipid, we
can argue that all the compounds show this kind of inter-
action. Other important data regard the main transition
peak and the parameters related to it (transition tempera-
ture, enthalpy variation, sharpness, DT1/2). The trend of the
transition temperature is an indication of the localization of
the compound with respect to the phospholipid. Usually,
compounds that are able to penetrate the phospholipid
bilayer lower the gel-to-fluid phase transition [18]. The
results obtained could suggest that cytarabine and the two
prodrugs with short chains, as cause an insignificant vari-
ation on the transition temperature, are localized in prox-
imity of the polar heads of the phospholipids not
penetrating the DMPC bilayers. On the other hand, the two
prodrugs with the long chains, which provoke the decrease
in the transition temperature, penetrate into the DMPC
bilayers.
voids remain among the DMPC chains with a reduction in
the van der Waals interactions and, hence, of the enthalpy
variation.
Comparing the structure of the two derivatives with
myristic and stearic acid moieties, it should be expected a
0
0
stronger effect of 4-N-stearoyl-4 ,6 -O-distearoyl-cytara-
bine, with longer acyl chains, with respect to 4-N-myris-
0
0
toyl-4 ,6 -O-dimyristoyl-cytarabine, with shorter acyl
chains. But, actually, the effect caused by 4-N-myristoyl-
0
0
4 ,6 -O-dimyristoyl-cytarabine is stronger than that of 4-N-
0
0
stearoyl-4 ,6 -O-distearoyl-cytarabine. We can imagine that
to accommodate among the apolar part of DMPC mole-
cules, some acyl chain of prodrugs bends, so only a part of
0
0
it fits. In the case of 4-N-myristoyl-4 ,6 -O-dimyristoyl-
cytarabine (whose chains are of the same length of those of
DMPC, 14 carbon atoms), the part of the chain which fits is
shorter than 14 carbon atoms, leaving some void among the
0
0
DMPC apolar tails. In the case of 4-N-stearoyl-4 ,6 -O-
distearoyl-cytarabine (whose chains are 4 carbon atoms
longer than that of DMPC), even if only a part of the chain
fits, this part is of a length that leaves less empty spaces
0
0
than 4-N-myristoyl-4 ,6 -O-dimyristoyl-cytarabine does.
Conclusions
If both Tm and DH strongly decrease, the compound is
inserted into the hydrophobic core of the bilayer [19]. In
addition, the decrease in the enthalpy indicates the reduc-
tion in the intra- and inter-molecular van der Waals inter-
actions [20]. During the phase transition, the phospholipids
show a cooperative behavior that has been explained as the
coexistence of clusters of lipids with properties that are
characteristic of the specific phases. In other words, the
lipids do not melt independently of each other but undergo
the transition as a cooperative domain. The width at half
height (DT1/2) of the main phase transition peak is an
important calorimetric parameter. The value of the DT1/2 is
inversely proportional to the size of the cooperative unit.
The broadening of the main transition peak caused by the
presence of a compound indicates a destabilization of the
phospholipid assemblies, suggesting that the compound
intercalates within the lipid bilayer. This could be in rela-
tion to a reduction in the average number of phospholipid
lipid molecules undergoing the transition as a cooperative
unit [21].
The methods here employed, i.e., acetic anhydride and the
agent coupling DIC, allowed to obtain the per-acylation of
all hydroxyl and amine groups of substrates Ara-C with the
aliphatic acid of short chain.
Steglich esterification method allowed to obtain per-
acylated derivatives of cytarabine with fatty acids. The two
hydroxyl groups esterification was possible due to the OH-
0
0
6
and OH-4 positions that are anti-oriented among
themselves; therefore, the aliphatic chain could have been
arranged like scissor for minimize the steric effects.
0
However, the esterification by agent coupling on OH-3
with fatty acid is too unfavorable, because the hydroxyl
group of this position is syn-oriented with respect to
cytosine ring and the intermediate formed by fatty acid and
DMAP is very bulky.
In this work, we have used cytarabine and designed four
synthesized cytarabine prodrugs such as 4-N-acetyl-
0
0
0
0
0
0
3 ,4 ,6 -triacetyl-cytarabine, 4-N-butyroyl-3 ,4 ,6 -tributy-
0
0
royl-cytarabine, 4-N-myristoyl-4 ,6 -O-dimyristoyl-cytara-
0
0
0
0
0
As already noted, the 4-N-butyryl-3 ,4 -6 -butyryl
derivative has a stronger effect on the enthalpy variation
rather than on the transition temperature. This behavior can
be explained hypothesizing that the molecule localizes
among the polar heads of DMPC with butyryl chains
toward the acyl chains of the DMPC; the derivative spans
the DMPC molecules and because of the different length
between the chains of the derivatives and the DMPC, some
bine and 4-N-stearoyl-4 ,6 -O-distearoyl-cytarabine in
order to increase the drugs lipophilicity and investigate
their interaction with a model of lipid bilayer by means of
DSC.
The most significant changes in the T , DT and
m
1/2
0
0
DH have been found in 4-N-myristoyl-4 ,6 -O-dimyristoyl-
0
0
cytarabine and 4-N-stearoyl-4 ,6 -O-distearoyl-cytarabine
prodrugs.
1
23

DSC studies on the interaction of lipophilic cytarabine prodrugs with DMPC multilamellar…
These results suggest that the chain lengths of the pro-
drug can play an important role in the mixing behavior of
the prodrugs with DMPC. In particular, the prodrugs with
short acyl chains are able to incorporate relatively poorly
among the DMPC molecules; on the other hand, prodrugs
with long acyl chains can better incorporate into the DMPC
molecules.
10. Chik F, Machnes Z, Szyf M. Synergistic anti-breast cancer effect
of a combined treatment with the methyl donors-adenosyl
0
methionine and the DNA methylation inhibitor 5-aza-2 -deoxy-
cytidine. Carcinogenesis. 2014;35:138–44.
11. Berr ´ı o Escobar JF, Pastrana Restrepo MH, Galeano Jaramillo E,
´
Ma
rquez Fern a´ ndez DM, M a´ rquez Fern a´ ndez ME, Mart ´ı nez
Mart ´ı nez A. Synthesis and cytotoxic activity of per-acetylated
and halogenated derivatives of nucleosides in breast cancer cells.
Ars Pharm. 2017;58:145–54.
1
2. Bzowska A, Kulikowska E, Shugar D. Purine nucleoside
phophorylases: properties, functions and clinical aspects. Phar-
macol Ther. 2000;88:349–425.
Funding Authors thank financing to the University of Antioquia
(
2
Project CODI CIQF-155 2012-2014, Strategy of Sustainability
014-2015).
13. Berr ´ı o Escobar JF, Arango Carmona VH, Galeano Jaramillo E,
M a´ rquez Fern a´ ndez DM, M a´ rquez Fern a´ ndez ME, Camargo
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