Intermolecular interactions between doxorubicin and β-cyclodextrin 4-methoxyphenol conjugates

Article abstract of DOI:10.1021/jp2091363

Newly synthesized derivatives of β-cyclodextrin, mono(6-deoxy-6-(1-1, 2,3-triazo-4-yl)-1-propane-3-O-(4-methoxyphenyl))β-cyclodextrin (1) and mono(6-deoxy-6thio(1-propane-3-O-(4-methoxyphenyl))) β-cyclodextrin (2) were designed to be receptors of the anticancer drug doxorubicin, which could potentially decrease the adverse effects of the drug during treatment. In both aqueous and aqueous dimethyl sulfoxide (DMSO) solutions, doxorubicin forms an inclusion complex with the new cyclodextrin derivatives with formation constants of K_s = 2.3 × 10⁴ and K_s = 3.2 × 10⁵ M^-1 for cyclodextrins 1 and 2, respectively. The stabilities of the complexes are 2-3 orders of magnitude greater than those with native β-cyclodextrin, and the flexibility of the linker of the side group of the cyclodextrins contributes to this stability. In a hydrogen-bond- accepting solvent, such as pure DMSO, an association that includes hydrogen bonding and chloride ions is favored over the binding of doxorubicin in the cavity of the cyclodextrin derivative. This contrasts with an aqueous medium in which a strong inclusion complex is formed. Cyclic voltammetry, UV-vis, ¹H NMR, and molecular modeling studies of solutions in DMSO and of solutions in water/DMSO demonstrated that the two different modes of intermolecular interaction between doxorubicin and the cyclodextrin derivative depended on the solvent system being utilized.

Full text of DOI:10.1021/jp2091363

Article
pubs.acs.org/JPCB
Intermolecular Interactions between Doxorubicin and β-Cyclodextrin
4-Methoxyphenol Conjugates
Olga Swiech, Anna Mieczkowska, Kazimierz Chmurski, and Renata Bilewicz*
Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
ABSTRACT: Newly synthesized derivatives of β-cyclodextrin,
mono(6-deoxy-6-(1−1,2,3-triazo-4-yl)-1-propane-3-O-(4-
methoxyphenyl))β-cyclodextrin (1) and mono(6-deoxy-6thio-
(1-propane-3-O-(4-methoxyphenyl))) β-cyclodextrin (2) were
designed to be receptors of the anticancer drug doxorubicin,
which could potentially decrease the adverse effects of the drug
during treatment. In both aqueous and aqueous dimethyl
sulfoxide (DMSO) solutions, doxorubicin forms an inclusion
complex with the new cyclodextrin derivatives with formation constants of Ks = 2.3 × 104 and Ks = 3.2 × 105 M−1 for
cyclodextrins 1 and 2, respectively. The stabilities of the complexes are 2−3 orders of magnitude greater than those with native β-
cyclodextrin, and the flexibility of the linker of the side group of the cyclodextrins contributes to this stability. In a hydrogen-
bond-accepting solvent, such as pure DMSO, an association that includes hydrogen bonding and chloride ions is favored over the
binding of doxorubicin in the cavity of the cyclodextrin derivative. This contrasts with an aqueous medium in which a strong
inclusion complex is formed. Cyclic voltammetry, UV−vis, 1H NMR, and molecular modeling studies of solutions in DMSO and
of solutions in water/DMSO demonstrated that the two different modes of intermolecular interaction between doxorubicin and
the cyclodextrin derivative depended on the solvent system being utilized.
the dermal barrier is rapidly accomplished without irreversible
tissue damage. DMSO enhances the return of tissue-entrapped
drug to the circulation and is able to scavenge anthracycline-
generated oxygen radicals to some extent.
INTRODUCTION
■
Anthracycline drugs have been used for nearly fifty years for the
treatment of many malignancies, and hundreds of analogs of
the first anthracycline antibiotics, doxorubicin and daunor-
ubicin, have been synthesized and evaluated. The clinical effects
are associated with modification of the DNA structure primarily
through intercalating complexes and covalent bonding.1,2
Multiple molecular mechanisms have been proposed to explain
the cytostatic and cytotoxic effects induced by these drugs and
the disturbances caused in processes such as replication,
transcription, or immobilization of DNA, repair mechanisms,
and apoptosis. The specific toxicity induced by anthracyclines is
caused by the formation of reactive oxygen species from the
redox reactions of these drugs. A semiquinone is formed as a
result of an electron transfer to the quinone group of the drug.
When the semiquinone is transformed back to a quinone,
oxygen is reduced to a reactive oxygen species with a different
level of toxicity: superoxide (O2•−), hydrogen peroxide (H2O2),
and the especially toxic hydroxyl radical (HO•) (Scheme 1).
An equally dangerous reaction is the formation of hydroxyl
radicals by free iron cations in the Fenton reaction3 (Scheme
1). In addition, the semiquinone can break the glycosidic bond
in the drug molecule, leading to the formation of an aglycone
molecule, which penetrates the membrane and releases an even
larger amount of reactive oxygen species.4,5
To prevent the adverse action of active oxygen species, the
quinone group responsible for their production can be blocked
until the delivery of drug molecules to the pathologically
changed cells occurs. Such blockage can be achieved by
complexing the anthracycline molecule with appropriate
nanoscale carriers. Common drug nanoparticulate carriers
include pegylated liposomes and anionic polymers. The
cationic drug forms complexes with polyelectrolytes, e.g.,
polyglutamates, polyacrylates, polyaspartates, or dextran sulfate.
Electrostatic interactions, aromatic stacking, and hydrogen
bonding play a role in such complexes.8
Blockage can also be achieved by complexing the
anthracycline molecule using cyclic oligosaccharides, such as
cyclodextrins (CDs), as receptors.9 Previous studies of
anthracycline−CD complexes have shown that the doxorubicin
molecule fits into the cavity of the CD from the quinone side.10
The limitation with using CD as the carrier for anthracycline
drugs is the low stability of the complex. The stability constants
are many orders less than those of the drug−DNA complexes,
e.g., 2.1 × 102 M−1 and 5.4 × 105 M−1 for β-cyclodextrin
(βCD)−Dox and Dox−DNA, respectively.11,12 Modification of
CD with an appropriate functional group can increase the
stability of the CD−drug complex, and stability constants that
A serious complication associated with the treatment of
cancer patients is the extravasation of anthracycline agents
during administration.6,7 Recently, the topical application of
dimethyl sulfoxide (DMSO) has been proposed to decrease the
effects of doxorubicin on subcutaneous tissue. DMSO
penetrates easily into tissues, and the transfer of DMSO across
Received: September 21, 2011
Revised: January 26, 2012
Published: January 27, 2012
© 2012 American Chemical Society
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Scheme 1. Toxic Reaction of Anthracyclines
Scheme 2. Syntheses of βCD(1) and βCD(2)
are an order of magnitude greater than that of the drug−DNA
complex have been reported by Thiele et al. These authors
reported increased stabilities for the inclusion compounds of
three chemotherapeutic agents, camptothecin (CPT), docetaxel
(DOC), and idarubicin (IDA), and a model compound
1,4-dihydroxyanthraquinone (DHA) with heptakis-6-substi-
tuted βCD derivatives.13 Yamanoi et al. prepared a βCD-
conjugated with two arbutin moieties and reported, based on
surface plasmon resonance measurements, an extremely high
association constant of 1.4 × 108 M−1 with doxorubicin, which
was caused by the stacking effect between the substituent and
doxorubicin.14
linkers (Scheme 2) and to evaluate their potential as ligands for
drug complexation. Using cyclic voltammetry, UV−vis,
1H NMR, and molecular modeling methods, we investigated
the modes of interaction of the CD with the drug and the
factors that affect the stability of the complex. Because DMSO
is often employed in the drug treatment and is useful for the
solubilization of CD derivatives, we selected mixed water−
DMSO solutions and pure DMSO as the solvents.
MATERIALS AND METHODS
■
Chemicals and Reagents. βCD hydrate, 4-butyn-1-ol, 1,3-
dibromopropane, and 4-methoxyphenol were purchased from
Aldrich and used as supplied. p-Toluenesulfonic anhydride was
synthesized according to a literature procedure.15 Tris[(1-
Our goal was to prepare novel βCD derivatives that
contained electron-rich aromatic substituents with two different
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benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (TBTA) was pre-
pared using the method of Sharpless.16 Doxorubicin hydro-
chloride salt was purchased from LC Laboratories (Woburn,
USA). The remainder of the compounds used in this work for
the syntheses were purchased from Aldrich and Fluka. The
buffers were prepared using water from a Milli-Q ultrapure
water system. Britton-Robinson buffer (BR, pH = 7) was
prepared in the usual way by the addition of appropriate
amounts of 0.2 M sodium hydroxide to orthophosphoric acid,
acetic acid and boric acid (0.04 M solutions). The ionic
strength was increased to 0.2 M by adding an appropriate
amount of potassium perchlorate. The pH was measured using
a PHM240 MeterLab pH meter (Radiometer Copenhagen).
Preparation of βCD 4-Methoxyphenol Conjugates (1)
and (2). Mono(6-O-tosyl) βCD was prepared from βCD
hydrate according to the procedure of Bitmann et al.15 using p-
toluenesulfonic acid anhydride and an aqueous solution of
sodium hydroxide. The βCD monotosylate was used to prepare
the mono(6-azido-6-deoxy-) and mono(6-deoxy-6-iodo)βCD
derivatives by the nucleophilic displacement of the tosylate with
an azide or iodide anion, respectively, at elevated temperature
in dimethylformamide (Scheme 2).
4-Butyn-1-ol was coupled with p-methoxyphenol under
Mitsunobu conditions to afford alkyne derivative A.17 The
latter was used for azide−alkyne coupling with the monoazido
βCD derivative in the presence of Cu(I) and TBTA, which was
used as a Cu(I) stabilizer, in a DMSO:water mixture under an
atmosphere of argon.
Mono(6-deoxy-6-(1−1,2,3-triazo-4-yl)-1-propane-3-O-(4-
methoxyphenyl)) βCD (1) was isolated in 96% yield. TOF MS
ES+ m/z 1350.51 [M+Na]+; 1H NMR (200 MHz,
CD3SOCD3) δ 7.82 (s 1H (1,2,3 triazoyl H)), 6.85 (s 4H
aryl), 5.76 (bs 20H OH), 4.86−4.83 (7H 2 br d H-1), 3.95 (t
2H O−CH2), 3.68 (s 3H O−CH3), 3.63−3.31 (2 m 36 H
remaining sugar H), 2.74(t 2H CH2-4(1,2,3 triazole)), 2.00 (t
2H CH2-CH2-4(1,2,3 triazole)); 13C NMR (50.28 MHz,
CD3SOCD3) δ 153.01, 152.48, 115.19, 114.43 (phe) 101.81
(C1), 81.31 (C4) 72.58−70.18 (C2,C3,C5), 55.18 (O-CH3)
67.12(O-CH2), 59.66 (C-6), 28.47 (CH2-CH2−CH2), 21.47
(CH2-4(1,2,3 triazole)),
3-O-(4-Methoxyphenyl)-propane-1-thiol (B) was prepared
from 4-methoxyphenol and 1,3-dibromopropane followed by
the exchange of bromine with thiol utilizing thiourea. Thiol B
was treated with sodium methoxide under an argon
atmosphere, and subsequently, S-alkylated was treated with
the mono(6-deoxy-6-iodo)-βCD derivative in DMF to produce
mono(6-deoxy-6thio(1-propane-3-O-(4-methoxyphenyl)))-
βCD (2) in 83,6% yield. TOF MS ES+ m/z 1338.4 [M+Na]+;
1H NMR (500 MHz CD3SOCD3) δ6.84 (s, 4H phe), 5.76 (bs
20H OH), 4.86−4.83 (7H 2d H-1), 3.95 (t 2H O−CH2), 3.69
(s 3H O−CH3), 3.65−3.2 2 (m 36 H sugar H), 2.67 (t 2H
CH2-S), 2.50 (t 2H C-6H2-S), 1.89 (m 2H CH2−CH2-CH2);
13C NMR (125.8 MHz, CD3SOCD3) δ 154.14, 152.29, 115.26,
114.42 (phe) 102.12−101.46 (C1) 81.30−81.22 (C4) 78.88−
70.96 (C2, C3, C5), 55.18 (O-CH3) 66.13(O-CH2), 33.06
(CH2-CH2−CH2), 20.61 (CH2−S), 28.78−28.69 (C-6)
Solubility of the Compounds. The solubility of
doxorubicin was determined by LC Laboratory (Woburn,
USA) at 10 mg/mL and 100 mg/mL in water and DMSO,
respectively. The solubility of native βCD in water is 18.5 mg/
mL, and its solubility increases in an irregular manner after the
addition of DMSO. At less than 30% DMSO, the solubility
remains constant (20 mg/mL). Between 30 and 40% DMSO,
the solubility rapidly increases to 770 mg/mL. From 40 to 86%,
the solubility is constant but then decreases to 500 mg/mL in
100% DMSO.18 The solubilities of βCD(1) and βCD(2) were
determined using UV−vis spectroscopy. The values for the
solubility in pure water are less than that of native βCD and
equal to 2.18 mg/mL and 0.28 mg/mL for βCD(1) and
βCD(2), respectively. In a mixture of water/DMSO (2:1), the
solubility increases to 107.9 mg/mL for βCD(1) and 6.10 mg/
mL for βCD(2).
Electrochemical Measurements. Electrochemical meas-
urements were performed using a PGSTAT Autolab (Eco
Chemie BV, Utrecht, Netherlands). All electrochemical experi-
ments were performed in a three-electrode arrangement with a
silver/silver chloride (Ag/AgCl) electrode in a saturated
solution of KCl as the reference, platinum foil as the counter
and an Au electrode (BAS, 2 mm diameter) or GC electrode
(BAS, 3 mm in diameter) as the working electrodes. The
working electrodes were polished mechanically with 1.0, 0.3,
and 0.05 μm of alumina powder on a Buehler polishing cloth.
Prior to measurements, the buffer solutions were purged with
purified argon for 30 min, and all experiments were performed
at room temperature. Milli-Q ultrapure water (resistivity 18.2
MΩ/cm) was used. Experiments in DMSO were performed
using a 0.5 M solution of tetrabutylammonium hexafluor-
ophosphate.
Spectroscopic Measurements. UV−vis spectroscopic
measurements were performed using a UV−vis EVOLU-
1
TION60 spectrophotometer with a 1-cm acryl cell. H NMR
spectra were obtained with a Bruker Avance 500 MHz (1H
frequency) spectrometer. All spectroscopic analyses were
conducted at room temperature. Experiments in DMSO were
performed in the absence of daylight.
Molecular Modeling. All theoretical calculations were
performed with YASARA19 using force field AMBER0320 with
periodic boundary conditions. The system consisted of ca. 2700
atoms including CD, the hydrochloride salt of doxorubicin
(Dox), and solvent (water or DMSO). Several system
configurations were considered including Dox placed inside
CD and Dox at the entrance of CD. During the preparation
stage, the models of molecules were parametrized and partial
charges were obtained using semiempirical methods.21 Each
simulation lasted 100 ns and was preceded by energy
minimization.
Calculation of Formation Constants from Spectros-
copy and Voltammetric Data. The formation constants of
the donor−acceptor associate were calculated using the
Benesi−Hildebrand method from the UV−vis data assuming
one-to-one associates22
A
ε
ε
D
1
0
D
− ε
=
+
A − A
ε
ε
− ε [CD]·K
(1)
0
D−A
D
D−A D s
where A is the observed absorption and A0 the absorption of
free doxorubicin. Ks is the formation constant. According to eq
1, the ratio of the slope and the intersection from the A0/(A−
A0) vs 1/[CD] plot provides the value of the formation
constant.
For the calculation of the formation constant of CD:Dox
complexes from cyclic voltammetry experiments, the Osa
equation was used23
2
2
(I
− I
)
2
Dox
obs
2
I
=
+ I
obs
Dox:CD
K ·[CD]
(2)
s
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where Iobs is the observed reduction peak current and IDox and
I
Dox:CD are the reduction peak currents for the free doxorubicin
and the inclusion complex, respectively. Ks is the complex
formation constant, and [CD] is the concentration of CD. The
value of Ks was calculated from the slope of the linear plot of
I2 vs (ID2 ox − Io2bs)/[CD].
obs
RESULTS AND DISCUSSION
■
The Interaction of βCD Derivatives with Doxorubicin
in DMSO Solutions. Because DMSO is often used to decrease
the adverse effects of doxorubicin treatment, we initially studied
the intermolecular interactions of Dox and CDs using pure
DMSO as the solvent. The UV−vis spectra of doxorubicin in
the presence of increasing concentrations of βCD(1) in DMSO
solution are shown in Figure 1. The absorption peaks at
Figure 2. Photograph of the DMSO solutions of Dox, βCD(2), the
mixture of native βCD with Dox, and the mixture of βCD(2) with
Dox.
To better understand the interactions of CDs with
1
doxorubicin in DMSO, H NMR experiments were performed
for βCD(2) in DMSO-d6 (Figure 3C).
The peak in the proton spectrum of the hydroxyl groups at
carbons C2 and C3 of CD change from a broad singlet at 5.55
ppm to a group of at least three multiplets as the concentration
of doxorubicin is increased. The same changes were observed
for the protons of the hydroxyl groups at the C6 carbon of CD
and reflect hydrogen-bonding interaction between the hydroxyl
groups of CD and Dox. The aromatic protons of doxorubicin at
the C1 and C2 positions of ring A do not show a change in
chemical shifts (Figure 3B).
The 1H NMR spectrum of pure Dox in DMSO-d6 shows two
peaks downfield with chemical shifts of 13.28 ppm and 14.06
ppm. These downfield chemical shifts indicate the formation of
intramolecular hydrogen bonds between the hydroxyl groups
on the C ring of doxorubicin and its quinone groups in ring B
(Figure 3A). Even the small addition of a CD derivative
removes the intramolecular hydrogen bonding in the Dox
molecule because of the intermolecular binding with the C2,
C3, and C6 hydroxyl groups of βCD (Figure 3B).
The absence of changes in the chemical shift of the protons
of the methoxy group on the phenyl in the side chain of CD
(6.85 ppm) when the molar ratio of Dox to βCD is altered
indicates that the phenyl substituent resides in the cavity of the
CD both in the absence and presence of doxorubicin (Figure
4). Taking the resonance structures of 4-methoxyphenyl units
into consideration, the oxygen atoms of the substituent would
be expected to donate electron density into the phenyl ring and
gain a partial positive charge. Their interactions with the
neighboring hydroxyl groups at the C2, C3, and C6 carbons of
CD (Figure 3B) increase the acidity of these hydroxyl groups.
Such changes in the CD favor the breaking of the intra-
molecular hydrogen bond at the B and C rings (Figure 3A) of
doxorubicin. The free phenolic OH groups of ring C may lose
their protons when reacting with proton acceptors that are
present in the solution, with naked chloride ions or with the
solvent itself. This change in the chromophore structure results
in the transition to a blue color.
Figure 1. (A) UV−vis spectra of 5 × 10−5 M doxorubicin solution in
DMSO with increasing concentrations of βCD(1): 5.0 × 10−5, 1.0 ×
10−4, 2.5 × 10−4, 5 × 10−4, 6 × 10−4, 7.5 × 10−4, 8.5 × 10−4, 1.0 ×
10−3, 1.25 × 10−3, 1.5 × 10−3, 1.8 × 10−3, 1.9 × 10−3, 2.1 × 10−3, and
2.8 × 10−3 M.
wavelengths 479 and 496 nm decrease after the addition of
βCD(1). However, there are two new peaks at wavelengths 594
and 642 nm, which appear and increase upon the addition of
βCD(1).
Similar changes in the spectra were observed for βCD(2).
The shift for βCD(1) was observed only when the
concentration ratio of Dox:βCD(1) was 1:10 or higher. A
change in the color of the solution from orange to dark blue
occurred (Figure 2) but only in hydrogen-bond-accepting
solvents, such as DMSO. Even a small addition of water leads
to the disappearance of the blue color. With native CD, the
blue-colored species is not formed (Figure 2). Similar spectral
changes have been observed when anthracyclines were reacted
with KO2 crown ether complex in DMSO24 and have been
ascribed to the formation of the anthracycline phenolate anion.
Anthracyclines are known to produce blue species in basic
solutions that is caused by ionization of the phenolic proton. It
has been postulated that similar reactions of superoxide with
anthracyclines in vivo may play a role in the antitumor activity
of these drugs.25
The spectrum of free doxorubicin showed two proton peaks
from the amino group of doxorubicin, a doublet at 7.81 ppm,
which corresponds to the NH3 group, and a broad singlet at
The proton acceptor nature of DMSO, which promotes the
dissociation of the compounds and the generation of naked
anionic forms, is well described in the literature.26,27 Proton
donor properties of the two CDs 1 and 2 were clearly different,
with βCD(2) as the better proton donor.
+
4.57 ppm corresponding to the NH2 group. The presence of
+
these two signals indicates an equilibrium between NH3 and
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Figure 3. (A) The structure of doxorubicin showing the intramolecular hydrogen bonds. (B) The structure of βCD and (C) NMR spectra for Dox,
βCD(2), and the βCD(2) + Dox mixture.
Molecular modeling confirmed that the substituent, and not
doxorubicin, is self-included in the cavity of the CD in DMSO
(Figure 4). The trajectories for this system in DMSO also
suggest that the naked chloride ion can interact with the
hydroxyl groups of the sugars in the CD ring and with the
amine group of doxorubicin to form hydrogen-bonded
complexes. These complexes in DMSO were favored for
βCD(2), while those for βCD(1) with the less flexible side
chains dissociated after the complexes were formed. For
comparison, modeling was also performed for the native CDs
and doxorubicin and showed only sporadic and short-lived
complexes in DMSO. This result confirms the inclusion of the
side arm substituents of the CD derivatives in DMSO. In the
case with the more flexible side arm (βCD(2)), the aromatic
moiety enters the cavity more easily than with βCD(1). Thus,
the hydrogen atoms of the βCD(2) hydroxyl groups become
more acidic, and their tendency to form hydrogen-bonded
complexes increases.
Figure 4. Molecular modeling of the βCD(2) interaction with
doxorubicin in DMSO solution in the absence of water.
NH2. The ratio of these signals changes from 2:1 to 1:2 for free
Dox and Dox:βCD(2) (1:1), respectively. These changes reflect
the interaction of the doxorubicin amino group with βCD(1)
and βCD (2) in aprotic solvents. Simultaneously, the chemical
shifts of the protons on the sugar of doxorubicin were observed.
These 1H NMR observations confirm that with pure DMSO-
d6 as the solvent doxorubicin does not reside in the cavity of
CD but interacts with CD solely by means of hydrogen bonds
because the hydroxyl groups at the C2, C3, and C6 positions of
CD are good hydrogen bond donors in DMSO.
Interaction of βCD Derivatives with Doxorubicin in a
Mixed Solution of Water:DMSO. Because of the limited
solubility of CD complexes in water, experiments were
conducted in a mixture of Britton−Robinson buffer and
DMSO (2:1 ratio). After the addition of βCD(1) or βCD(2)
to the solution of doxorubicin in the mixed solvent, a decrease
of the voltammetric peak current was observed that indicates
formation of the complex. The dependence of the reduction
peak current of Dox on the concentration of βCD(1) is shown
in Figure 5A.
On the basis of the spectroscopic results, the formation
constants of doxorubicin phenolate in the presence of βCD(1)
and βCD(2) were calculated using eq 1. The values obtained
for Ks were 121.1 6.2 M−1 and 6768 41 M−1 for βCD(1)
and βCD(2), respectively. The formation constant in the case
of βCD(2) that has a more flexible linker is more than an order
of magnitude greater than that of the CD with a triazole in the
linker.
The formation constants of 1:1 CD complexes calculated
using eq 2 were found to be 2.3 × 104 0.2 and 3.2 0.2 ×
105 M−1for βCD(1) and βCD(2), respectively (Figure 5).
Molecular modeling confirmed the formation of the
inclusion complex between doxorubicin and βCD(1) or
βCD(2) (Figure 6). The systems with water have lower
energy, and the average distance between the aromatic groups
(phenyl or 1,2,3-triazole) of the side chain of the modified CD
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Figure 5. (A) Dependence of reduction peak current for 5 × 10−5 M Dox on the concentration of βCD(1) recorded in Britton−Robinson
buffer:DMSO (2:1). v = 50 mV/s. (B) The plot of the Osa eq 2 for Dox in the presence of βCD(1).
for βCD(1) and βCD(2), respectively. The stability constants
of the doxorubicin complexes with the CD derivatives that have
a single pendant 4-methoxyphenyl-terminated arm are 2−3
orders of magnitude greater than those of the complexes with
native βCD. The formation of the inclusion complex requires
the presence of water. In dry DMSO, formation of the complex
does not occur because doxorubicin cannot compete with the
side chain of CD for space in the cavity. The blue species
detected in DMSO are the phenolate anions that are formed as
a result of proton abstraction from one of the weakly acidic
phenolic groups at C6 or C11 of anthracycline. Interestingly, in
DMSO some interactions of doxorubicin with modified CD
also exist, and they involve the more acidic hydrogen atoms of
the CD hydroxyl groups and chloride anions. Modeling
performed for the native CDs and doxorubicin showed only
sporadic and short-lived complexes in DMSO, which confirms
the importance of the inclusion of the side arm substituent of
the CD derivative in the mechanism in DMSO. On the basis of
Figure 6. Molecular modeling of the structure of the βCD(2)
doxorubicin complex in water.
1
the H NMR, UV−vis spectroscopy, and molecular modeling
results, the formation of a hydrogen-bonded associate with the
self-included aromatic group of the CD side-chain is proposed
to explain the behavior of the doxorubicin−CD derivative
system in dry DMSO.
and the aromatic ring A of doxorubicin (Figure 4) is smaller
than that in pure DMSO.
In the case of βCD(1), interactions between both the
aromatic triazole and phenyl groups with ring A of doxorubicin
may occur. The electron deficient ring A of doxorubicin can
interact with the aromatic groups of the side chain in βCD(1).
The 1,2,3-triazole moieties, which reside close to the inclusion
cavity, should be more prone to interactions. In contrast, the
larger stability constant for βCD(2) indicates that the flexibility
of the linker plays a dominant role in these interactions.
Molecular modeling of βCD(2) revealed the possibility of
strong π−π interactions between the aromatic phenyl ring of
the CD side group and ring A of doxorubicin as shown in
Figure 6.
AUTHOR INFORMATION
■
Corresponding Author
*Phone/Fax: +48 22 8220211/+48 22 8225996. E-mail:
bilewicz@chem.uw.edu.pl.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Polish Ministry of Sciences
and Higher Education and the National Center for Research
and Development (NCBiR), Grant No. NR05-0017-10/2010
(PBR-11), and by the Faculty of Chemistry, University of
Warsaw. We would like to thank Alexander Debinski for help
with the molecular modeling and discussion of the results.
CONCLUSIONS
■
In this study, we have shown that newly designed and
synthesized derivatives of βCDs form strong inclusion
complexes with doxorubicin in mixed water:DMSO solutions
with high formation constants, 2.3 × 104 M−1and 3.2 × 105 M−1
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