Synthesis, characterization and potential application of monoacyl-cyclodextrins

Article abstract of DOI:10.1016/j.carres.2009.11.009

Although the preparation of cyclodextrin (CD) monoesters with a variety of carboxylic acids has been already described in the literature, the direct regioselective CD acylation has proved to be critical, often requiring to be replaced with a more elaborat

Full text of DOI:10.1016/j.carres.2009.11.009

Carbohydrate Research 345 (2010) 191–198
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis, characterization and potential application of monoacyl-cyclodextrins
Katia Martina, Devendra Sharad Puntambekar, Alessandro Barge, Marina Gallarate, Daniela Chirio,
*
Giancarlo Cravotto
Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Via Giuria 9, 10125 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 August 2009
Received in revised form 3 November 2009
Accepted 5 November 2009
Available online 11 November 2009
Although the preparation of cyclodextrin (CD) monoesters with a variety of carboxylic acids has been
already described in the literature, the direct regioselective CD acylation has proved to be critical, often
requiring to be replaced with a more elaborate synthetic process. In this paper we describe the one-step
preparation of several monoacylated CDs from acyclic or aromatic carboxylic acid derivatives. The ability
of b-CD to enclose cupric ions in a sandwich-type manner was exploited to lead to high regioselectivity in
the acylation of b-CD with benzoyl chloride, cinnamoyl chloride and phenyl acetyl chloride in water. Long
Keywords:
chain aliphatic monoesters of
a-, b- and c-CD were best prepared in DMF. The results of our study
Cyclodextrin
Regioselectivity
Monoacylation
NMR study
Silibinin
showed that solvent and general conditions determined an overwhelming regioselectivity of acylation.
1H, 13C and 2D NMR experiments could easily discriminate the position of the ester. Monoacylated CDs
were evaluated as a carrier of silibinin, the inclusion complexes were prepared and characterized by ther-
mal analysis.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
often involves elaborate multistep protection/deprotection
strategy.
Cyclodextrins (CD) are seductive molecules transcending tradi-
tional barriers that separate many subdisciplines of chemical sci-
ence. They are considered the most important host compounds
because of the well-documented ability of the parent CDs to form
inclusion complexes with a wide variety of guest species in an
aqueous solution.1 For this reason, CD derivatives are employed
in many fields:2 pharmaceuticals, agrochemicals, analytical chem-
istry, cosmetics and toiletry, as well as in food industries.3
Native b-CD is known to be parenterally nephrotoxic because it
removes some cholesterol from the cell walls and transport it to
the kidneys where the changes in the ‘solution composition’ results
in the precipitation of the cholesterol/CD complex (pin-like crys-
tals). This cholesterol removal is a common property of cyclodex-
trin derivatives, in various extents, and it is true particularly for
some b-CD derivatives such as methylated b-CD.4,5 With the aim
to mitigate these drawbacks, new CD derivatives are in the offing.
The introduction of suitable functional groups on CD hydroxyls
may prevent crystallization because it gives rise to amorphous
products, and increases the solubility by breaking the 2-OH/3-OH
hydrogen bond. The CD regioselective mono- or poly-functionali-
zation in position 2, 3 or 6 still remains a challenging mission.
The hydroxyl groups in the three different positions compete for
the reagents and make selective derivatization a daunting task that
The regioselective acylation of CDs represents a versatile but
difficult means of obtaining series of new derivatives. The derivati-
zation of the primary –OH group (the most basic and nucleophilic),
can be driven with a stoichiometric amount of acyl chloride in
pyridine, or to avoid the use of such an unpleasant solvent, by dis-
placement of the tosyl group from the 6I-deoxy-6I-(p-tolylsulfo-
nyl)-b-CD with a carboxylate.6,7
Only very few examples are known of monoacylation at the sec-
ondary face of CDs. By the addition of the acyl chloride in acetoni-
trile to the CD aqueous alkaline solution under precise pH control,
the monoacylation occurred mainly on the secondary face and both
2- and 3-monoacyl derivatives were isolated.8 Recently a mono-
benzoylation of b-CD by using N-benzoylimidazole and carbonate
buffer in DMF was described, the authors observed an acyl migra-
tion between the C-2 and C-3 hydroxyl groups.9 Lin and coworkers
reported the regioselective C-2 acylation by enzyme-catalyzed
transesterification of the vinyl esters.10,11 In all those cases, the
monoacylated derivatives were obtained after time-consuming
purifications.
On the basis of the literature data and our previous experience
we experimented with several reaction conditions and solvents for
the synthesis of regioselective monoacylated CD derivatives. This
paper reports the most promising results and the bidimensional
NMR spectra to assign the position of the ester unambiguously.
We recently showed that triglyceride-based solid lipid nanopar-
ticles (SLNs) can be prepared in the presence of monoacyl
c
a- and
* Corresponding author. Tel.: +39 011 6707684; fax: +39 011 6707687.
E-mail address: giancarlo.cravotto@unito.it (G. Cravotto).
-CD derivatives.12 Along this research line, in this paper, a new
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.11.009

192
K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
OX
OH
O
O
RCOCl
O
O
O
(or anhydride)
OY
O
O
HO
OH
OH
O
HO
HO
OH
HO
6
Reagents and conditions
as described in Table 1
OH
OH
6
O
O
O
5 X = R, Y = H
6 X = H, Y = H
1 X = R, Y = H
2 X = H, Y = R
3 X = R, Y = H
4 X = H, Y = R
Scheme 1. Synthesis of monoacylated b-CD 1–6.
series of monoacylated CD derivatives were preliminarily tested
for their ability to form inclusion complexes with silibinin (S), cho-
sen as model drug, in the perspective of a future SLN development.
S is the major active component of silymarin, a mixture of flavo-
lignans extracted from the milk thistle Silybum marianum, which
presents potent antioxidant activity. Because of the pervasive role
that oxidative/nitrosative stress is now known to play in patho-
physiology, the flavolignans in silymarin have received increasing
attention in treatment and prevention of a variety of liver disor-
ders13 and in various forms of neoplasies, such as breast, prostate
and skin cancer.14,15 In the literature, the preparation of inclusion
complexes of S16 and other flavonoids such as dioclein17 and baic-
alin18 with b-CD was described, to enhance their low bioavailabil-
ity after oral administration, related to their low water solubility.
monoacyl derivatives, namely the benzoyl (Bz), cinnamoyl (Cin)
and phenylacetyl (PhAc) (Scheme 1). The formation of inclusion
complexes with solvents or reagents may strongly perturb the reg-
ioselectivity, competing with nucleophilicity and basicity of the
three types of hydroxyl groups. As shown in Table 1, the major role
of the solvent in the conversion rate and selectivity emerged. The
benzoylation in pyridine with a stoichiometric amount of BzCl
gave selectively the mono(6-O-benzoyl)-b-CD, while the main
product in DMF was the mono(2-O-benzoyl)-b-CD. The reactivity
in DMF was confirmed with the mixed carbonic acid anhydride
prepared from ethyl chloroformate. In fact, both the reactions in
DMF gave mainly the derivative acylated on the secondary face
at the C-2 besides only 2% of mono(6-O-benzoyl)-b-CD and traces
of diacylated-b-CD. The same reaction repeated in water with Bz
mixed anhydride, BzCl or Bz imidazole failed in all cases. When
BzCl was added to an aqueous NaOH solution of b-CD and CuSO4
the reaction gave the mono(6-O-benzoyl)-b-CD in a 30% yield.
The product was isolated by precipitation without the need for
purification by chromatography or by soxhlet extraction. In general
a slow dropwise addition of the corresponding acyl chloride or
anhydride to the CD solution favours the monosubstitution.
As reported in the literature, in an alkaline aqueous solution, b-
CD forms a stable copper-CD assembly,19,20 in which the two CD
tori are joined by copper(II) ions in a sandwich-type complex.21
In this metal-bridged complex the CD secondary face is masked
so that new regioselective synthetic protocols can be envisaged,
2. Results and discussion
Hydrophobic monoacyl-CDs can be employed as a novel slow-
release drug carrier because of their ability to form nanoparticles,
vesicles or more elaborate SLN to be proposed for cosmetic or
pharmaceutical formulations. Encouraging results on the incorpo-
ration of curcumin in SLN by CD inclusion,12 prompted us to fur-
ther investigate the improved synthetic protocols for the
preparation of CD monoesters. A comprehensive scenario of sol-
vents, catalysts and experimental conditions was explored with
the aim to optimize the synthetic protocols of the three common
Table 1
Assessment of esterification on b-CD
Entry
Reagent
Reaction condition
Product (yield %)
1
2
3
4
5
BzCl
5 h, rt, Pyridine
DMAP, 2 h, rt, DMF
DMAP, 2 h, rt, DMF
NaOH, 3 h, rt, water
NaOH, 8 h, rt, water
NaOH, 8 h, rt, water
1 (8%)a
Bz anhydrideb
BzCl
1 (3%), 2 (14.5%)
1 (7%), 2 (13%)
Bz anhydrideb
Bz imidazole
BzCl
—
c
c
—
c
6
—
7
8
9
10
11
12
13
14
BzCl
CuSO4, NaOH, 4 h, rt, water, CH3CN
CuSO4, NaOH, 5 h, rt, water, CH3CN
DMAP, 2 h, rt, DMF
CuSO4, NaOH, 4 h, rt, water, CH3CN
DMAP, 2 h, rt, DMF
DMAP, 2 h, rt, DMF
DMAP, 2 h, rt, DMF
CuSO4, NaOH, 4 h, rt, water, CH3CN
1 (30%)
—
c
Bz anhydrideb
CinCl
4 (15%), 3 (3%)
3 (31%)
4 (20%), 3 (1%)
5 (15%), 6 (5%)
5 (17%), 6 (2%)
6 (29%)
CinCl
Cin anhydrideb
PhAcCl
PhAc anhydrideb
PhAcCl
a
b
c
Yield as referred by Tong et al.6
Mixed carbonic acid anhydride prepared from the corresponding acid with ethyl chloroformate.
Starting material.

K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
193
Table 2
Assessment of esterification on b-CD
Entry
CD
Reagent
Reaction condition
Product (yield %)
a
a
a
1
2
3
4
5
6
7
b
b
b
b
b
a
c
Propanoyl Cl
CuSO4, water, CH3CN, 5 h, rt
CuSO4, water, CH3CN, 5 h, rt
CuSO4, water, CH3CN, 5 h, rt
DMF, 2 h, rt
DMAP, Et3N, DMF, 2 h, rt
DMAP, Et3N, DMF, 2 h, rt
DMAP, Et3N, DMF, 2 h, rt
—
—
—
Ethylmalonyl Cl
Isostearoyl Cl
Isostearic anhydrideb
Isostearoyl Cl
7 (16), 8 (5)
7 (25), 8 (10)
9 (40), 10 (13)
11 (16), 12 (7)
Isostearoyl Cl
Isostearoyl Cl
a
Starting material.
b
Mixed carbonic acid anhydride prepared from the corresponding acid with ethyl chloroformate.
as referred to the synthesis of the mono(6-p-toluensulfonyl)-b-
CD.22
anhydride prepared with ethyl chloroformate gave mainly the
mono(6-O-isostearoyl derivative)-b-CD (7) in addition to the regio-
isomer 8 and traces of disubstituted derivatives (Table 2, entry 4).
Higher conversion was observed when the reaction was repeated
in DMF in the presence of isostearoyl chloride (Table 2, entry 5).
Scheme 2 reports all the monoisostearoyl derivatives obtained un-
der these conditions.
With the aim to obtain new hydrophobic CD derivatives and to
compare their ability to form an inclusion complex with silibinin
(2R,3R)-2-[(2R,3R)-2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-
2-(hydroxymethyl)-1,4-benzodioxin-6-yl]-2,3-dihydro-3,5,7-trihy-
droxy-4H-1-benzopyran-4-one) (Fig. 1), mono (6-O-isostearoyl
The acylation protocol was optimized by the addition of 8 equiv
of BzCl dissolved in CH3CN to a basic aqueous solution of CuSO4
and b-CD. The monoacyl derivative was isolated by precipitation
after addition of 1 N HCl sol. to neutralize the reaction mixture
and NMR experiments confirmed the ester moiety on the primary
face. The same reaction conditions were used with cinnamoyl chlo-
ride and phenylacetyl chloride (DMF vs water). In these two cases,
DMF disfavours the attack on the primary face while the mono-
(6-acyl)-b-CD derivative was obtained when the reaction was
performed in water in the presence of CuSO4. Surprisingly, with
phenylacetyl chloride the regiochemistry was inverted: mono-
(6-O-phenylacetyl)-b-CD (5) was rather selectively obtained in
DMF while mono(2-O-phenylacetyl)-b-CD (6) was obtained in
water, only traces of the C-6 esterified derivative were detected.
Few studies in the literature describe the binding constants of b-
CD with benzoic acid and phenylacetic acid, so the existence of
intracavity inclusion of these derivatives has been proved.23,24
Braga et al. published the characterization of the crystal structure
of the inclusion complex isolated in water from b-CD with S-(+)-
ibuprofen (a phenylacetic derivative).25 By the combination of
powder X-ray diffraction (XRD) and single crystal X-ray diffraction,
they confirmed the formation of a head-to-head dimer of b-CD
molecules. The formation of a phenylacetyl-CD assembly that
masks the primary face, clearly unables the attack on the C-6 posi-
tion. These data explain the inverse 2?6 regioselectivity observed
in water, respectively with phenylacetyl chloride and benzoyl
chloride.
derivative)-
a
and
c-CD were also synthesized. As described for
the b-CD, also the
a
- and -CD can be esterified in DMF affording
c
the ester on the primary face as the main product. All the products
were characterized by mass spectrometry and NMR spectroscopy.
NMR spectra enable the unambiguous determination of the regio-
chemistry as shown in Table 2 and in the experimental part.
As reported in the well-known NMR study of Yoshimoto,26 the
*
shift of O-acylated carbon ( CH2OCOR) is somewhat downfielded
*
while the adjacent carbon Cb ( CCH2OCOR) would move markedly
upfield in 13C NMR. Furthermore, throughout all positional iso-
mers, the anomeric proton signal in 1H NMR of the acylated glu-
cose unit resonates downfield with
coupling pattern.
a characteristic doublet
CH2OH
O
To broaden the scope, we extended our study to the esterifica-
tion of CD with some aliphatic carboxylic acids; a few examples are
listed in Table 2, which includes comparative data for different al-
kyl acylating agents. Esterification with propanoyl, ethylmalonyl
and isostearyl chloride in water with CuSO4, failed. When the reac-
tion was performed in DMF, the isostearoyl-mixed carbonic acid
HO
O
O
OCH3
OH
O
OH
OH
Figure 1. Chemical structure of silibinin.
OX
OH
O
O
1
1
O
O
O
O
5
OY
C17H35COCl
Et3N, DMAP, DMF
5
OH
O
6
HO 6
2
HO
OH
3OH
OH
2
HO
n
HO
n
3 OH
O
4
4
OX
YO
7
8
9
n = 6, m = 13, X = COC17H35, Y = H
α, β
n = 6, m = 13, X = H,
Y = COC17H35
- or -
or γ
n = 5, m = 11, X = COC17H35, Y = H
HO
10 n = 5, m = 11, X = H,
Y = COC17H35
n
OH
11 n = 7, m = 15, X = COC17H35, Y = H
12 n = 7, m = 15 , X = H, Y = COC17H35
m
Scheme 2. Synthesis of monoisostearoyl-a-, b-, c-CD 7–12.

194
K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
the upshift of the C-50 adjacent carbon. On the contrary, the
mono(2-O-phenylacetyl)-b-CD (6) showed the downfield shift of
the C-20 signal at 73.55 ppm while the two upfield peaks referred
to the C-10 at 98.87 and to the C-30 at 70.26 ppm. As described pre-
viously, even the 1H NMR spectra provided substantial informa-
tion. The mono(6-O-acyl)-b-CD (1) and 3 showed a downfield
signal coming from the proton linked to the C-60 atom, detected
by homo- and hetero-nuclear 2D NMR experiments and overlaid
by the OH(6) signal. The 1H NMR spectrum of product 6 showed
a C-10 proton downfield doublet (d = 5.25 ppm) separate from the
other six C-1 protons (multiplet d = 5.06 ppm), even the C-3 pro-
tons show a downfield triplet at 4.14 ppm.
A preliminary evaluation on the ability of acyl-b-CD to include S
was performed by characterizing the S-CD inclusion complexes of a
set of different acyl-CDs with differential scanning calorimetry
(DSC). The S transition peak was monitored: DSC analysis was used
to ascertain the formation of inclusion complexes and to compare
them with the plain physical mixtures. The thermograms reported
in Figures 2A and B showed the formation of S complexes with all
CD-derivatives tested.
Table 3
Chemical shift of 13C NMR of monosubstituted derivatives 1, 3, 6
Carbon atom
1
3
6
DMSO-d6
DMSO-d6
DMSO-d6
C-1
102.65
102.02
101.61
103.02
102.97
102.41
102.21
102.14
98.87
72.71
72.32
72.25
73.55
73.66
73.47
70.26
81.39
81.27
81.10
80.70
72.54
72.44
C-10
C-2
72.44
72.25
73.26
73.46
C-20
C-3
73.1
73.89
73.09
C-30
C-4
82.58
82.44
81.29
83.14
82.72
82.19
82.00
72.91
72.10
72.02
69.75
60.77
C-5
72.28
72.13
72.01
69.07
59.97
59.68
C-50
C-6
60.28
In the isostearoyl CD series (Fig. 2A), the S transition peak
(158.37 °C) disappeared completely in 2:1 CD:S molar ratio inclu-
sion complexes prepared with each CD, probably indicating a com-
plete inclusion of the drug in the CD cavity. On the other hand,
C-60
C@O
64.06
64.47
165.69
166.97
174.18
40.76 (CH2–Ph)
134.32 (Cipso
129.82 (Cortho
128.15(Cmeta
126.76(Cpara
133.47 (Cipso
)
145.42 (Ph–CH@
with
ready observed at 1:1 CD:S molar ratio. Probably the bigger size of
the -CD should favour the inclusion of S molecule. In the S-CD
c-CD a complete disappearance of the transition peak was al-
129.85 (Cpara
128.82 Cortho
129.55 (Cmeta
)
)
)
134.93 (Cipso
129.22 (Cpara
129.87 (Cortho
129.25 (Cmeta
118.86 (CH–CO)
)
)
)
)
c
)
)
)
derivatives physical mixture, the S transition peak was still pres-
ent, and no significant modification was noted, indicating only
slight interactions.
)
As the complete ethanol removal at room temperature by N2
stream can be questioned, and considering that the presence of
ethanol has been described to influence the complexation of drugs
As an example, analysis of the 13C NMR spectra of compounds 1,
3 and 5 revealed that an esterification occurred at the C-6 primary
hydroxyl position, while compounds 2, 4 and 6 referred to the acyl-
ated in C-2. In Table 3, the chemical shifts of 13C NMR of b-CD
derivatives 1, 3 and 6 obtained by performing the reaction in water
are listed. In the 13C NMR spectrum of products 1 and 3 a peak at
64.06 or 64.24 ppm referred to the downshift of the C-60 substi-
tuted carbon, at 69.07 and 69.51 ppm the NMR spectra evidenced
with CD,27 the complex prepared by dissolving S and
a-isostearoyl-
CD (1:2 molar ratio) in acetone was analyzed by DSC. The obtained
curve is quite superimposable to that obtained in ethanol.
In the b-CD series (Fig. 2B), a complete disappearance of the S
transition peak was observed in all S/CD derivatives 1:2 molar ratio
complexes, confirming the results obtained with isostearoyl b-CD.
Figure 2A. DSC thermograms of S-isostearoyl (a, b, c)-CD 7, 9 and 11.

K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
195
Figure 2B. DSC thermograms of S-acyloyl b-CD 1, 3 and 6.
To confirm the S/isostearoyl
c
-CD 1:1 inclusion complex forma-
by performing DSC on the S-complex with mono(6-Bz)-b-CD,
mono(6-Cin)-b-CD and mono(2-PheAc)-b-CD, and mono(6-isostea-
royl)a-, b- and c-CD.
tion, the stoichiometry of the complex was evaluated by the con-
tinuous variation method (Job’s plot).28 The r ratio was plotted
against the difference of absorbance (
and in the absence of isostearoyl -CD. The results, reported in
A at r value 0.5, indicating a
D
A) of drug in the presence
Such promising results will require further investigations for a
future development of SLN-containing S/CD complexes in drug
delivery.
c
D
Figure 3 evidenced a maximum
1:1 stoichiometry.
As also reported in the literature,29 the DSC curves of the raw
material compared with those obtained by simply mixing acyl-
CDs and S, and with those of inclusion complexes, confirm not only
an interaction between flavonoid and acyl-CDs, but also a real
inclusion.
4. Experimental
Commercially available reagents and solvents were used with-
out further purification. Native CDs were kindly provided by Wac-
ker Chemie. In accordance with the well-established procedure,
the mixed carbonic acid anhydrides were prepared from the corre-
sponding acid with ethyl chloroformate in dichlorometane. Reac-
tions were monitored by TLC on Merck 60 F254 (0.25 mm)
plates, which were visualized by UV inspection and/or by heating
after a spray with 5% H2SO4 in ethanol. IR spectra were recorded
with a Shimadzu FT-IR 8001 spectrophotometer. NMR spectra
were recorded with a Bruker 300 Avance (300 MHz and 75 MHz
for 1H and 13C, respectively) at 25 °C; chemical shifts are calibrated
to the residual proton and carbon resonances of the solvent:
DMSO-d6 (dH = 2.54, dC = 39.5). Chemical shifts (d) are given in
ppm, coupling constants (J) in hertz. ESI-mass spectra were per-
formed on a Waters Micromass ZQ spectrometer equipped with
ESI source. A DSC7 Perkin Elmer was used for differential scanning
calorimetry measurements.
3. Conclusions
In summary, the direct synthesis of a series of monoacylated
CDs in water and DMF has been described. From this study, it
emerged that a general procedure for the direct regioselective CD
monoacylation is not available. On the basis of CD type and acylat-
ing agent, ad hoc tailored methods have to be applied to achieve
regioselective monoacylation. Good regioselectivity was observed
when the reactions were performed DMF by using mixed anhy-
drides or in water/CuSO4 with aromatic acyl chlorides. The intrigu-
ing influence of the solvent often showed complementary
regioselectivity. The ability to include S was successfully evaluated
4.1. Mono(6-O-benzoyl)-b-CD (1)
b-CD (1 g, 0.88 mmol) was dissolved in distilled water (25 ml)
at 60 °C. A copper sulfate solution (0.66 g, 2.65 mmol) in water
(15 ml) was added. The reaction mixture was stirred at room tem-
perature for 2 h then a NaOH solution (0.85 g, 21.25 mmol) was
added. A benzoyl chloride (0.8 ml, 6.95 mmol) solution in CH3CN
(7.5 ml) was added dropwise and stirred for 4 h at room tempera-
ture. During the addition of benzoyl chloride the solution became
opalescent. After 4 h, 1 N HCl was added to neutralize the reaction
mixture. Precipitate was observed after the addition of HCl and it
was filtered. The precipitate was dissolved in methanol and the de-
sired monoacyl derivative was precipitated using acetone (3 vol-
umes). Further recrystallization using hot methanol yielded
Figure 3. Continuous variation plot of S-monoisostearoyl
aqueous solution.
c-CD in 1.0% DMSO

196
K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
367 mg (33% yield) of pure product. Analytical data were in accor-
dance with published data.6
4.5. Mono(6-O-phenylacetyl)-b-CD (5)
b-CD (0.2 g, 0.176 mmol) and ethylphenylacetylcarbonate
(55 mg, 0.26 mmol) were dissolved in 4 ml of DMF. A catalytic
amount of DMAP was added. The solution was left under stirring
for 2 h at room temperature. The solution was partially dried under
vacuum therefore acetone was added and the precipitate was fil-
tered. The desired product was isolated by reverse-phase chroma-
tography with a gradient from water/methanol 97:3 to methanol
100% to yield 36 mg (17% yield) of the desired mono-(6-O-phenyl-
acetyl)-b-CD.
4.2. Mono(2-O-benzoyl)-b-CD (2)
b-CD (0.2 g, 0.176 mmol) and ethylbenzoylcarbonate (51 mg,
0.264 mmol) were dissolved in 4 ml of DMF. A catalytic amount
of 4-dimethylaminopyridine (DMAP) was added. The solution
was left under stirring for 2 h at room temperature. The solution
was partially dried under vacuum therefore acetone was added
and the precipitate was filtered. The desired product was isolated
by reverse-phase chromatography with a gradient from water/
methanol 97:3 to methanol 100%.
Compound 6 is a white powder; Rf = 0.42 (iPrOH/H2O/AcOEt/
NH4OH 5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2924, 1732, 1660, 1475,
1372, 1089, 1028 and 754; 1H NMR (300 MHz; DMSO-d6): 7.30
(m, 5H, aromatic protons), 5.84–5.73 (m, 14H, O(2)H, O(3)H),
4.86 (br s, 7H, H-1), 4.76–4.68 (m, 6H, O(6)H), 4.46 (m,2H, CH2Ph),
31 mg (14.5% yield) of mono-(2-O-benzoyl) b-CD were recov-
ered and characterized. Analytical data were in accordance with
reported values.8
0
0
4.38 (m, 1H, H-6A ), 4.12 (m, 1H, H-6B ) 3.9–3.2 (m, overlaps with
H2O, H-6, H-3, H-2, H-4, H-5); 13C NMR (75 MHz; DMSO-d6):
172.15 (CO), 135.2 (Cipso), 130.30 (Cortho), 129.10 (Cmeta), 127.58
(Cpara), 102.86 (C1), 82.65 (C4), 82.65 (C4), 73.88(C3), 73.27 (C2),
72.90 (C5), 68.78 (C50), 63.77 (C60), 61.12 (C6); m/z (ESI) calcd for
C50H76O36 1275.41 [M+Na]+, found 1275.61 [M+Na]+.
4.3. Mono(6-O-cinnamoyl)-b-CD (3)
b-CD (1 g, 0.88 mmol) was dissolved in distilled water (45 ml)
at 60 °C. A copper sulfate solution (0.66 g, 2.65 mmol) in water
(65 ml) was added. The reaction mixture was stirred at room tem-
perature for 2 h then a NaOH solution (0.88 g, 21.25 mmol) in
water (25 ml) was added. A solution of t-cinnamoyl chloride
(0.8 ml, 7 mmol) in CH3CN (7.5 ml) was added dropwise and stir-
red for 4 h at room temperature. After 4 h, 1 N HCl was added to
neutralize the reaction mixture. Precipitate was observed after
the addition of HCl, so that the reaction mixture was filtered to re-
move copper salt. To the filtrate acetone was added to precipitate
b-CD. The resultant filtrate was evaporated and the desired product
was precipitated in water to yield 337 mg (31% yield) of the de-
sired cinnamoyl derivative.
4.6. Mono(2-O-phenylacetyl)-b-CD (6)
b-CD (1 g, 0.88 mmol) was dissolved in distilled water (45 ml)
at 60 °C. A copper sulfate solution (0.66 g, 7 mmol) in water
(65 ml) was added. The reaction mixture was stirred at room tem-
perature for 2 h then a NaOH solution (0.88 g, 21.25 mmol) in
water (25 ml) was added. Then, phenylacetyl chloride (0.9 ml,
6.95 mmol) solution in CH3CN (7.5 ml) was added dropwise and
stirred for 4 h at room temperature. 1 N HCl was added to neutral-
ize the reaction mixture and precipitate was observed. To the
filtrate solution, acetone was added to precipitate b-CD. The resul-
tant filtrate was evaporated and the desired product was isolated
by reverse-phase chromatography with a gradient from water/
methanol 97:3 to methanol 100% to yield 305 mg (29% yield) of
the desired mono-(2-O-phenylacetyl)-b-CD.
Compound 6 is a light yellow powder; Rf = 0.52 (iPrOH/H2O/
AcOEt/NH4OH 5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2925, 1730, 1659,
1475, 1372, 1089, 1028 and 754; 1H NMR (300 MHz; D2O): 7.32
(m, 5H, aromatic protons), 5.25 (d, 1H, J = 3.6 Hz, H-10), 5.06–5.01
(m, 6H, H-1), 4.8 (m, overlaps with H2O,H-20), 4.16 (t, 1H,
J = 7.8 Hz, H-30), 4.0–3.48 (m, 35H, H-6, H-3, H-2, H-4, H-5, CH2CO);
13C NMR (75 MHz; DMSO-d6): as shown in Table 3, m/z (ESI) calcd
for C50H76O36 1275.41 [M+Na]+, found 1275.26 [M+Na]+.
Compound 3 is a white powder; Rf = 0.4 (iPrOH/H2O/AcOEt/
NH4OH 5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2925, 1736, 1653, 1471,
1369, 1086, 1028 and 754; 1H NMR (300 MHz; DMSO-d6): 7.69
(m, 3H, ortho, –CH–CO), 7.46 (m, 3H, meta, para), 6.72 (d, 1H,
J = 15.7 Hz, Ph–CH@), 5.76 (m, 14H, O(2)H, O(3)H), 4.86 (br s, 7H,
0
0
H-1), 4.54–4.43 (m, 7H, O(6)H, H-6A ), 4.22 (m, 1H, H-6B ), 3.96
(m, 1H, H-50), 3.7–3.2 (m, overlaps with H2O, H-6, H-3, H-2, H-4,
H-5); 13C NMR (75 MHz; DMSO-d6): as shown in Table 3, m/z
(ESI) calcd for C51H76O36 1287.40 [M+Na]+, found 1287.75 [M+Na]+.
4.4. Mono(2-O-cinnamoyl)-b-CD (4)
b-CD (0.5 g, 0.44 mmol) and t-ethylcinnamoylcarbonate (145 mg,
0.65 mmol) were dissolved in 10 ml of DMF. A catalytic amount of
DMAP was added. The solution was left under stirring for 2 h at
room temperature. The solution was partially dried under vacuum
therefore acetone was added and the precipitate was filtered. The
desired product was isolated by reverse-phase chromatography
with a gradient from water/methanol 97:3 to methanol 100%.
Hundred and eighteen milligrams (20% yield) of mono-(2-O-
cinnamoyl)-b-CD were recovered and characterized.
Compound 4 is a light yellow powder; Rf = 0.5 (iPrOH/H2O/
AcOEt/NH4OH 5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2925, 1736, 1653,
1471, 1369, 1086, 1028 and 754; 1H NMR (300 MHz; DMSO-d6):
7.69 (m, 3H, ortho), 7.58 (d, 1H, J = 15.9 Hz, CH–CO), 7.43 (m, 3H,
meta, para), 6.57 (d, 1H, J = 15.9 Hz, Ph–CH@), 5.74 (m, 13H,
O(2)H, O(3)H), 5.08 (m, 1H, H-10), 4.75 (br s, 6H, H-1), 4.58–4.24
(m, 7H, O(6)H, H-20), 3.83 (m, 1H, H-30), 3.6–3.2 (m, overlaps with
H2O, H-6, H-3, H-2, H-4, H-5); 13C NMR (75 MHz; DMSO-d6):
167.15 (CO), 143, 7 (Ph–CH@), 135.25 (Cipso), 130.87 (Cmeta),
129.74 (Cpara), 129.01 (Cortho), 120.64 (@CH–CO), 102.87 (C1),
100.01 (C10), 82.44 (C4), 73.93 (C20), 72.86 (C3), 72.63 (C2), 72.06
(C5), 70.35 (C30), 60.78 (C6); m/z (ESI) calcd for C51H76O36
1287.40 [M+Na]+, found 1287.36 [M+Na]+.
4.7. General procedure for the synthesis of monoisostearoyl-
a-,
b-, -CD derivatives
c
CD (0.51 mmol), and triethylamine (56 ll, 0.76 mmol) were dis-
solved in dry DMF (3 ml). A catalytic amount of DMAP was added.
The solution was cooled down to ꢀ15 °C and the isostearoyl chlo-
ride (0.22 g, 0.76 mmol) previously diluted with DMF (2 ml), was
added dropwise. The reaction mixture was stirred for 2 h at room
temperature, and finally filtered on a sintered glass funnel to re-
move triethylammonium chloride. The solution was dried under
vacuum and the crude product was purified by reverse-phase chro-
matography (from water/methanol 97:3 to methanol 100%) to give
pure compounds 7–12.
4.7.1. Mono(6-O-isostearoyl)-b-CD (7)
White powder (25%); Rf = 0.57 (iPrOH/H2O/AcOEt/NH4OH
5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2926, 1736, 1653, 1369, 1157,
1028; 1H NMR (300 MHz; DMSO-d6): 5.78–5.69 (m, 14H, O(2)H,
O(3)H), 4.83 (br s, 7H, H-1), 4.47–4.38 (m, 6H, O(6)H), 3.35 (m,

K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
197
A0
B0
1H, H-6 ), 4.073.35 (m, 1H, H-6 ), 3.82 3.35 (m, 1H, H-50), 3.63 (m,
29H, H-3, H-5, H-6), 3.5–3.2 (m, overlaps with H2O, H-2,H-4), 2.30
(m, 2H, CH2CO), 1.49 (m, 2H, CH2), 1.28 (m, 23H, CH2, CH), 0.89 (m,
8H, CH3, CH2); 13C NMR (75 MHz; DMSO-d6): 172.12 (CO), 101.94
(C1), 81.53 (C4), 73.08 (C3), 72.45 (C2), 72.054 (C5), 69.05 (C50),
63.52 (C60), 59.93 (C6), 31.31 (CH2CO), 29.04 (CH2), 28.76 (CH2),
24.52 (CH2), 22.12 (CH2), 15.20 (CH2), 14.03 (CH3); m/z (ESI) calcd
for C60H104O36 1400.63, found 1423.55 [M+Na]+.
4.7.6. Mono(2-O-isostearoyl)-c-CD (12)
Light yellow powder (7%); Rf = 0.65 (iPrOH/H2O/AcOEt/NH4OH
5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2926, 1735, 1655, 1369, 1157,
1028; 1H NMR (300 MHz; DMSO-d6): 5.80–5.75 (m, 15H, O(2)H,
O(3)H), 5.13 (d, 1H, J = 3.2 Hz, H-10), 4.90 (br s, 7H, H-1), 4.73 (d,
J = 3.2 Hz, H-20), 4.62–4.32 (m, 8H, O(6)H), 4.05–4.01 (m, 1H, H-
30), 4.0–3.15 (m, overlaps with H2O, H-2,H-4, H-3, H-5, H-6), 2.15
(m, 2H, CH2CO), 1.67 (m, 2H, CH2), 1.2–1.3 (m, 22H, CH2), 0.92
(m, 8H, CH3, CH2); 13C NMR (75 MHz; DMSO-d6): 172.12 (CO),
103.65 (C1), 96.75 (C10), 91.95 (C4), 73.13 (C3), 72.77 (C20), 72.15
(C2), 71.60 (C5), 69.83 (C30), 59.30 (C6), 32.15 (CH2CO), 29.13
(CH2), 27.82 (CH2), 24.15 (CH2), 22.55 (CH2), 16.23 (CH3), 14.33
(CH3) m/z (ESI) calcd for C66H114O411562.68, found 1585.73
[M+Na]+.
4.7.2. Mono(2-O-isostearoyl)-b-CD (8)
Light yellow powder (10%); Rf = 0.63 (iPrOH/H2O/AcOEt/NH4OH
5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2926, 1736, 1653, 1369, 1157,
1028; 1H NMR (300 MHz; DMSO-d6): 5.68–5.42 (m, 14H, O(2)H,
O(3)H), 5.02 (d, 1H, J = 3.2 Hz, H-10), 4.84 (br s, 6H, H-1), 4.54(m,
7H, O(6)H), 4.40 (d, J = 3.2 Hz, H-20), 4.05 (m, 1H, H-30), 3.8–3.6
(m, 28H, H-3, H-5, H-6), 3.45–3.33 (m, overlaps with H2O, H-2,H-
4), 2.32 (m, 2H, CH2CO), 1.48 (m, 2H, CH2), 1.2–1.4 (m, 23H, CH2,
CH), 0.72 (m, 8H, CH3, CH2); 13C NMR (75 MHz; DMSO-d6):
174.12 (CO), 103.14 (C1), 100.1 (C10), 82.5 (C4), 73.87 (C3), 73.74
(C2), 73.154 (C5), 69.86 (C30), 60.1 (C6), 31.27 (CH2CO), 29.12
(CH2), 28.56 (CH2), 23.73 (CH2), 22.42 (CH2), 15.47 (CH2), 14.63
(CH3) m/z (ESI) calcd for C60H104O36 1400.63, found 1423.43
[M+Na]+.
4.8. Silibinin/CD derivatives inclusion complexes
S/CD-derivatives complexes at 1:1 and 1:2 molar ratios were
prepared. Appropriate amounts of CD derivatives and S were dis-
persed/solubilized in 3 ml of ethanol. The organic solvent was then
removed by keeping the suspension under a nitrogen stream for
30 min.
4.9. DSC analysis
4.7.3. Mono(6-O-isostearoyl)-a-CD (9)
White powder (40%); Rf = 0.56 (iPrOH/H2O/AcOEt/NH4OH
5:3:1:1); mmax (KBr)/cmꢀ1: 2926, 2856, 1736, 1659, 1153, 1080,
949, 704; 1H NMR (300 MHz; DMSO-d6): 5.67–5.42 (m, 12H,
O(2)H, O(3)H), 4.85 (br s, 6H, H-1), 4.62–4.42 (m, 7H, O(6)H,
H-60), 4.0–3.2 (m, overlaps with H2O, H-2,H-4, H-3, H-5, H-6),
2.49 (m, 2H, CH2CO), 1.55 (m, 2H, CH2), 1.0–1.42 (m, 23H, CH2,
CH), 0.88 (m, 8H, CH3, CH2); 13C NMR (75 MHz; DMSO-d6):
173.22 (CO), 101.98 (C1), 82.08 (C4), 73.29 (C3), 72.23 (C2), 72.1
(C5), 68.48 (C50), 62.53 (C60), 60.01 (C6), 32.21 (CH2CO), 29.14
(CH2), 28.57 (CH2), 24.47 (CH2), 21.72 (CH2), 15.20 (CH2), 13.73
(CH3) m/z (ESI) calcd for C54H94O31 1238.58, found 1262.32
[M+Na]+.
An appropriate amount of each sample, corresponding to 0.7 mg
S was placed in conventional aluminium pans and a scan speed of
10 °C minꢀ1 was used operating in 120–180 °C temperature range.
(DSC7 Perkin Elmer).
4.10. Continuous variation method (Job’s plot)
Equimolar (0.05 mM) solutions of S and isostearoyl
V/V DMSO aqueous solution were added in varying quantities so as
to get different r values: r = [S]/([S] + [isostearoyl -CD]) at a 2 ml
c-CD in 1.0%
c
final volume. Samples were analyzed using UV spectrophotometer
at 286 nm.
4.7.4. Mono(2-O-isostearoyl)-a-CD (10)
Acknowledgements
Light yellow powder (13%); Rf = 0.63 (iPrOH/H2O/AcOEt/NH4OH
5:3:1:1); mmax (KBr)/cmꢀ1 3420, 2926, 1736, 1653, 1369, 1157,
1028; 1H NMR (300 MHz; DMSO-d6): 5.67–5.43 (m, 11H, O(2)H,
O(3)H), 5.03 (d, 1H, J = 3.2 Hz, H-10), 4.86 (br s, 5H, H-1), 4.75 (d,
1H, J = 3.2 Hz, H-20), 4.6–4.35 (m, 6H, O(6)H), 4.11–4.05 (m, 1H,
H-30), 4.05–3.23 (m, overlaps with H2O, H-2,H-4, H-3, H-5, H-6),
2.27 (m, 2H, CH2CO), 1.57 (m, 2H, CH2), 1.2–1.3 (m, 23H, CH2,
CH), 0.92 (m, 8H, CH3, CH2); 13C NMR (75 MHz; DMSO-d6):
173.12 (CO), 103.74 (C1), 98.75 (C10), 82.22 (C4), 73.18 (C3),
73.22 (C20), 72.15 (C2), 71.75 (C5), 69.91 (C30), 60.0 (C6), 31.32
(CH2CO), 29.42 (CH2), 28.76 (CH2), 23.43 (CH2), 22.52 (CH2),
15.67 (CH2), 14.33 (CH3) m/z (ESI) calcd for C54H94O31 1238.58,
found 1262.12 [M+Na]+.
Regione Piemonte and the University of Turin (Project Nano-
IGT—Converging technologies) are warmly acknowledged for the
financial support. D. S. P. is indebted to the University of Turin
for the young researcher fellowship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.11.009.
References
1. Szejtli, S. Cyclodextrin and their Inclusion Complexes, Akademiai Kiado,
Budapest,1982.
2. Szejtli, J. Pure Appl. Chem. 2004, 76, 1825–1845.
3. Cravotto, G.; Binello, A.; Baranelli, E.; Carraro, P.; Trotta, F. Curr. Nutr. Food Sci.
2006, 2, 343–350.
4. Frijlink, H. W.; Eissens, A. C.; Hefting, N. R.; Poelstra, K.; Lerk, C. F.; Meijer, D. K.
F. Pharm. Res. 1991, 8, 9–16.
5. Bekers, O.; Uijtendaal, E. V.; Beijnen, J. H.; Bult, A.; Underberg, W. J. M. Drug Dev.
Ind. Pharm. 1991, 17, 1503–1549.
6. Tong, L. H.; Hou, Z. J.; Inoue, Y.; Tai, A. J. Chem. Soc., Perkin Trans. 2 1992, 1253–
1257.
7. Hoshino, T.; Miyauchi, M.; Kawaguchui, Y.; Yamaguchi, H.; Harada, A. J. Am.
Chem. Soc. 2000, 122, 9876–9877.
8. Hao, A. Y.; Yong, L. H.; Zhang, F. S.; Gao, X. M. Carbohydr. Res. 1995, 277, 333–
337.
9. Wang, Z.; Lu, R. J. Inclusion Phenom. 2009, 63, 373–378.
10. Xiao, Y. M.; Wu, Q.; Wang, N.; Lin, X. F. Carbohydr. Res. 2004, 339, 1279–1283.
4.7.5. Mono(6-O-isostearoyl)-c-CD (11)
White powder (16%); Rf = 0.58 (iPrOH/H2O/AcOEt/NH4OH
5:3:1:1); mmax (KBr)/cmꢀ1: 2925, 2859, 1738, 1661, 1153, 1080,
949, 704; 1H NMR (300 MHz; DMSO-d6): 5.81–5.79 (m, 16H,
O(2)H, O(3)H), 4.90 (br s, 8H, H-1), 4.62–4.42 (m, 9H, O(6)H, H-
60), 4.0–3.2 (m, overlaps with H2O, H-2,H-4, H-3, H-5, H-6), 2.49
(m, 2H, CH2CO), 1.55 (m, 2H, CH2), 1.0 -1.42 (m, 22H, CH2), 0.88
(m, 8H, CH3, CH2); 13C NMR (75 MHz; DMSO-d6): 175.22 (CO),
101.55 (C1), 83.08 (C4), 75.29 (C3), 72.56 (C2), 71.65 (C5), 69.64
(C50), 63.54 (C60), 60.68 (C6), 33.25 (CH2CO), 28.25 (CH2), 27.63
(CH2), 24.20 (CH2), 20.65 (CH2), 15.23 (CH2), 14.76 (CH3) m/z
(ESI) calcd for C66H114O41 1562.68, found 1575.32. [M+Na]+.

198
K. Martina et al. / Carbohydrate Research 345 (2010) 191–198
11. Wang, N.; Wu, Q.; Xiao, Y. M.; Chen, C. X.; Lin, X. F. Bioorg. Med. Chem. 2005, 13,
3667–3671.
21. Fuchs, R.; Habermann, N.; Klüfers, P. Angew. Chem., Int. Ed. Engl. 1993, 32, 852–
854.
12. Chirio, D.; Gallarate, M.; Trotta, M.; Carlotti, M. E.; Calcio Gaudino, E. C.;
Cravotto, G. J. Inclusion Phenom. 2009. doi:10.1007/s10847-009-9597-7.
13. Wellington, K.; Jarvis, B. BioDrugs 2001, 15, 465–489.
14. Kren, V.; Walterova, D. Biomed. Papers Med. Faculty Univ. Palacky, Olomoic,
Czechoslovakia 2005, 149, 29–41.
15. Ramasamy, K.; Agarwal, R. Cancer Lett. 2008, 269, 352–362.
16. Arcari, M.; Brambilla, A.; Brandt, A.; Caponi, R.; Corsi, G.; Di Rella, M.; Solinas, F.
Boll. Chim. Farm. 1992, 131, 205–209.
22. Defaye, J.; Crouzy, S.; Evrard, N.; Law, H. PCT Int. Appl. WO9961483, 1999;
Chem Abstr. 2000, 132, 24077.
23. Gadre, A.; Ru-Diger, V.; Schneider, H.-J.; Connors, K. A. J. Pharm. Sci. 1997, 2,
236–243.
24. Rekharsky, M. V.; Mayhew, M. P.; Goldberg, R. N. J. Phys. Chem. B 1997, 101, 87–
100.
25. Braga, S. S.; Gonçalves, I. S.; Herdtweck, E.; Teixeira-Dias, J. J.-C. New J. Chem.
2003, 27, 597–601.
17. Rezende, B. A.; Cortes, S. F.; De Sousa, F. B.; Lula, I. S.; Schmitt, M.; Sinisterra, R.
D.; Lemos, V. L. Int. J. Pharm. 2009, 367, 133–139.
26. Yoshimoto, K.; Itatani, Y.; Shibata, K.; Tsuda, Y. Chem. Pharm. Bull. 1980, 28,
208–219.
18. Li, J.; Zhang, M.; Chao, J.; Shuang, S. Spectrochim. Acta, Part A 2009, 73, 752–
756.
27. Li, R.; Quan, P; Liu, D.-F.; Wei, F.-D.; Zhang, Q.; Xu, Q.-W. AAPS Pharm. Sci. Tech.
2009, DOI: 10.1208/s12249-009-9317-z.
19. Matsui, Y.; Kurita, T.; Yagi, M.; Okayama, T.; Mochida, K.; Date, Y. Bull. Chem.
Soc. Jpn. 1975, 48, 2187–2191.
20. Klufers, P.; Piotrowski, H.; Uhlendorf, J. Chem. Eur. J. 1997, 3, 601–608.
28. Jadhav, G. S.; Vavia, P. R. Int. J. Pharm. 2008, 352, 5–16.
29. Ficarra, R.; Tommasini, S.; Raneri, D.; Calabrò, M. L.; Di Bella, M. R.; Rustichelli,
C.; Gamberin, M. C.; Ficarra, P. J. Pharm. Biomed. Anal. 2002, 29 1005-1014.