Synthesis and pharmacological evaluation of a new targeted drug carrier system: β-Cyclodextrin coupled to oxytocin

Article abstract of DOI:10.1016/j.bmcl.2008.02.017

β-Cyclodextrin (β-CD) was monofunctionalized into its carboxylic derivative and then conjugated to the N-side of oxytocin (OT), a nonapeptide involved in human behavior and myometrium contraction. On isolated rat myometrium, this conjugate (β-CD-OT) partly preserves the contracting activity of OT (EC₅₀ = 0.40 μM vs 1.7 nM). Moreover, the contraction induced frequency is also lowered by β-CD-OT. This novel hydrophilic targeted carrier could form a host-guest complex with prostaglandins and their derivatives used as labor inducers or with anticancer drugs used in cervix and endometrial cancer. This strategy can improve the solubility, the stability, and/or the biological activity of these drugs as well as reducing their side-effects.

Full text of DOI:10.1016/j.bmcl.2008.02.017

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1855–1858
Synthesis and pharmacological evaluation of a new targeted
drug carrier system: b-Cyclodextrin coupled to oxytocin
a
a
b
´
Carine Bertolla, Stephanie Rolin, Brigitte Evrard,
Lionel Pocheta and Bernard Masereela,*
aDrug Design and Discovery Center, University of Namur, FUNDP, 61 rue de Bruxelles, 5000 Namur, Belgium
` `
Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Liege, Sart-Tilman, 4000 Liege, Belgium
b
Received 4 January 2008; revised 6 February 2008; accepted 7 February 2008
Available online 10 February 2008
Abstract—b-Cyclodextrin (b-CD) was monofunctionalized into its carboxylic derivative and then conjugated to the N-side of oxy-
tocin (OT), a nonapeptide involved in human behavior and myometrium contraction. On isolated rat myometrium, this conjugate
(b-CD-OT) partly preserves the contracting activity of OT (EC50 = 0.40 lM vs 1.7 nM). Moreover, the contraction induced fre-
quency is also lowered by b-CD-OT. This novel hydrophilic targeted carrier could form a host–guest complex with prostaglandins
and their derivatives used as labor inducers or with anticancer drugs used in cervix and endometrial cancer. This strategy can
improve the solubility, the stability, and/or the biological activity of these drugs as well as reducing their side-effects.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Cyclodextrins (CDs) are water soluble cyclic oligosac-
charides composed, for the most common species, of
six (a-), seven (b-), or eight (c-) a (1 ! 4)-linked
D-glucopyranose units. These units are arranged in a
torus-shaped structure with hydrophilic outside and a
hydrophobic cavity. Due to these structural features,
CDs are able to form host–guest inclusion complexes
with hydrophobic molecules possessing the appropriate
size and shape. Initially, this property was used in the
food and cosmetic industry where CDs were used as
stabilizer for flavoring agents and labile fragrances, and
to reduce unpleasant odor and taste.1 In the same time
CDs were used in pharmaceutical formulations generally
to improve drug properties such as solubility, stability,
and absorption.2 As CDs are chiral entities, they are also
used for asymmetric synthesis3 and chiral separation in
chemistry.4 More recently, peptides were grafted on func-
tionalized CDs to target a receptor or an enzyme involved
in pathologies. . .5 These conjugates can host a guest-drug
which is then specifically driven to the target.
social behavior, including maternal care and aggression,
pair bonding, sexual behavior, social memory and sup-
port, and human trust, and downregulates stress re-
sponses, including anxiety.6 The nonapeptide OT is
also involved in various reproductive functions as uter-
otonic activity and milk-ejection during labor and lacta-
tion, respectively. All these activities are mediated
through a seven transmembrane domains receptor
belonging to the G protein-coupled receptors family.7
Actually, OT is only approved as labor or abortive
inducer.
Selective and mono-functionalization of b-CD is a major
challenge due to the presence of seven identical 6-primary
and 14 2,3-secondary hydroxyl functions of the glucose
units. The synthetic pathway is depicted in Scheme 1. Un-
der nitrogen, the 1-tosyl-1H-imidazole was prepared at
room temperature by adding 1-tosyl chloride to a suspen-
sion of imidazole in CH2Cl2 (yield 88%).8 Then, sodium
hydroxide (1.1 N) and 1-tosylimidazole were added to
an aqueous solution of b-CD 1. After stirring for 1 h at
room temperature, hydrochloric acid, 0.1 N, was added.
The formed precipitate was collected and washed with
water and acetone to afford the mono-6-deoxy-6-(4-to-
syl)-b-cyclodextrin 2 (yield 33%).8,9
In the present report, we describe the functionalization
of b-CD and its coupling to oxytocin (OT). The CNS re-
lease of OT, a very abundant neuropeptide, modulates
The monotosylated b-CD 2 was then converted to the
mono-6-deoxy-6-formyl-b-cyclodextrin 3 through a
Swern oxidation.10 This formylation was performed un-
Keywords: Cyclodextrin; Oxytocin; Drug targeting; Uterus.
*
Corresponding author. Tel.: +32 81 724338; fax: +32 81 724299;
e-mail: bernard.masereel@fundp.ac.be
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.017

1856
C. Bertolla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1855–1858
O
O
O
S
CH3
OH
HO
H
HO
HO
6
6
6
a,b
c
O
3
3
2
3
2
2
(OH)7 (OH)7
(OH)7 (OH)7
1
(OH)7 (OH)7
3
2
O
O
NH-Gly-Leu-Pro-Cys-Asn-Gln-Ile-Tyr-Cys-COOH
OH
HO
d
HO
6
6
e,f
3
3
2
2
(OH)7 (OH)7
4
(OH)7 (OH)7
5
Scheme 1. Reagents and conditions: (a) 1-tosylimidazole, NaOH 1.1 N, 1 h, rt; (b) HCL 0.1 N; (c) DMSO, collidine, microwaves, 20 min, 200 ꢁC; (d)
phosphate buffer, pH 6.0, Br2, 5 days; (e) DCC, HOBt, DMF, 0 ꢁC, 1 h; (f) oxytocin, DMF, 48 h.
der microwaves by heating (200 ꢁC, 20 min) the tosylate
derivative in DMSO with collidine as a non-nucleophilic
base (yield 60%).11 The mono-6-deoxy-6-formyl-b-cyclo-
dextrin 3 obtained was then oxidized to the corresponding
carboxylic acid 4 by bromine at pH 6.0, which represent
the optimized conditions.12 Following this long reaction
(5 days), preparative HPLC gave pure mono-6-deoxy-6-
carboxy-b-cyclodextrin 4 (yield 20%).13
Finally, the b-cyclodextrinyl carboxylic acid 4 was acti-
vated by reaction with 1-hydroxybenzotriazole (HOBt)
and dicyclohexylcarbodiimide (DCC) in DMF (0ꢁ,
1 h). This activated ester was then condensed with oxy-
tocin in DMF (25 ꢁC, 48 h). The mono-6-deoxy-6-oxyt-
Figure 1. Isolated rat uterine contractile response to OT or b-CD-OT.
ocinyl-b-cyclodextrin 5 (b-CD-OT) was then isolated by
preparative HPLC (yield 30%).14 The LC/MS analysis of
the collected conjugate revealed the absence of uncou-
pled oxytocin as well as the absence of CD derivatives.
Each synthesized molecule was purified by preparative
LC/MS and characterized by analytical LC/MS, elemen-
tal analyses, and NMR spectroscopy, their structures
being thus confirmed.
As OT is a highly potent contracting agent of uterine
muscle, the contractile potency of the mono-6-deoxy-6-
oxytocinyl-b-cyclodextrin was examined on myome-
trium isolated from rats pretreated with b-estradiol ben-
zoate (500 lg kgꢀ1, ip) and compared with OT.15 As
shown in Figure 1, the OT concentration
(EC50 = 1.7 nM) required to induce the half-maximal
tension is 230-times lower than the conjugate b-CD-
OT (EC50 = 0.40 lM).
Figure 2. Frequency of uterine contractions induced by OT or b-CD-
OT.
The frequency of contractions induced by the conjugate
b-CD-OT is also lower than that induced by OT alone.
Indeed, the OT concentration which induces at least one
contraction per minute is 1 nM while the required con-
centration of b-CD-OT is 0.6 lM (Fig. 2).
is less potent than OT, but remains active at a submi-
cromolar concentration. This conjugate has a double
interest. First, it has been previously demonstrated that
CDs could form a host–guest complex with several pros-
taglandins and derivatives, and then increase their sta-
bility, their solubilization and their half-life.16 So the
inclusion of approved uterotonic drugs such as dino-
The b-CD-OT is a novel drug carrier system targeting
specifically the OT receptor. This b-CD-OT conjugate

C. Bertolla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1855–1858
1857
7. Gimpl, G.; Fahrenholz, F. Physiol. Rev. 2001, 81, 629.
8. Byun, H. S.; Zhong, N.; Bittman, R. Org. Synth. 2000, 77,
225.
prostone (=prostaglandin E2) or carboprost (=15-methyl
PGF2a) could improve the stability and reduce the sys-
temic side-effects of these drugs since they will be mainly
driven to the uterus level, an OT-receptor rich tissue in
case of pregnancy and delivery. The other interest of this
conjugate is to form a host–guest complex with antican-
cer drugs used in the therapy of cervix cancer. This strat-
egy could drive the anticancer to the uterus and so limit
the side-effects of these anticancer drugs. Indeed, it has
been reported that when paclitaxel, docetaxel, and doxo-
rubicine, all used in endometrium and cervix cancer, form
a complex with CD, their antitumoral activity is im-
proved.17 So, these drugs can probably be included as a
guest in the OT-bCD carrier. By using this strategy, their
solubility and their tumor concentration will be increased
and as well as their toxicity will be lowered. As OT
contains lipophilic amino acid residues, we examined its
possibility to be inserted into the cavity of b-CD. It seems
that this ‘cup and ball’ (well known as ‘bilboquet’) effect is
unlikely. Indeed, the larger internal diameter of b-CD is
9. Mono-6-deoxy-6-(p-tosylsulfonyl)-b-cyclodextrin (2). In
an E-flask equipped with a magnetic stirrer, sodium
hydroxide (8.8 g; 220 mmol) was added to a suspension
of b-CD (1, 25 g; 22 mmol) in distilled water. Then, 1-(p-
toluenesulfonyl)imidazole) (5 g; 22.5 mmol) was added.
After 1 h, hydrochloric acid, 0.1 N, was added dropwise to
reach pH 5–6. The white formed precipitate was collected,
washed with hot water (200 mL), with cold water
(200 mL), with acetone (100 mL), and dried to give
9.23 g (yield = 33%) of the title compound 2. 1H NMR
(400 MHz, DMSO-d6)d 2.44 (s, 3H), 3.20–3.69 (overlap
with D2O, 40 H), 4.17–4.22 (m, 1H), 4.33–4.40 (m, 2H),
4.45–4.49 (m, 2H), 4.53 (br s, 3H), 4.78 (br s, 2H), 4.85 (br
s, 5H), 5.65–5.86 (m, 14H), 7.44 (d, 2H), 7.76 (d, 2H); m/z
1311 [M+Na]+. Anal. Calcd for C49H76O37SÆ3.5H2O: C,
43.23; H, 6.17. Found: C, 43.05; H, 5.94.
10. Tidwell, T. T. Synthesis 1990, 10, 857.
11. Mono-6-deoxy-6-formyl-b-cyclodextrin (3). A microwave
vial containing DMSO (10 mL) was filled with b-cyclo-
dextrin monotosylate (2, 1.02 g; 0.78 mmol) and collidine
(1 mL; 7.76 mmol). The vial was sealed with a teflon
septum and irradiated at 2.450 GHz and 200 ꢁC for 20 min
(Biotage AB microwave oven, Uppsala). After heating, the
vial was cooled, and its content added to acetone
(200 mL). The formed precipitate was collected and
dissolved in water (10 mL). This solution was added
dropwise to ethanol (100 mL). The formed precipitate was
collected, washed with ethanol, purified by preparative
LC/MS (Econosphere-Alltech column, 250 cm · 22 mm;
particle size 10 lm; mobile phase acetic acid 0.1%: CH3CN
only 6.5 A18 whereas the diameter of the cyclic sequence
˚
19
˚
Cys-Asn-Gln-Ile-Tyr-Cys of OT is 11 A. Moreover,
the distance between the C atom of the carbonyl linking
˚
oxytocin and the C-terminal atom of proline is 6 A. As
compared to the height of the torus-shaped b-CD
˚
(7.9 A), this spacer is too short and the geometrical
constraints are too strong to host OT. The great interest
of this carrier has to be confirmed by the preparation of
a drug/b-CD-OT complex.
(80:20 v/v); flow rate: 5 mL minꢀ1
) to give 2.63 g
(yield = 60%) of the titled compound 3. 1H NMR
(400 MHz, DMSO-d6) (note: this contains both the
aldehyde and covalent hydrate form of the compound) d
3.26–3.81 (overlap with D2O, 40H), 4.16 (d, 1H), 4.38–
4.45 (m, 6H), 4.79 (br s, 6H), 4.85–4.90 (d,1), 5.11 (t,
0.6H), 5.48 (d, 0.6H), 5.49 (d, 0.6H), 5.68–5.85 (m, 12H),
9.67 (s, 0.3); m/z 1133.1 [M+H]+, 1155.3 [M+Na]+. Anal.
Calcd for C42H68O35Æ4H2O: C, 41.86; H, 6.31. Found: C,
42.06; H, 6.02.
Acknowledgment
This research was financed by a grant of the Region
Wallonne (WALEO/NANOPHAR No. 215211). The
authors thank C. Cauvin for her help in distance
measurements.
References and notes
12. Yoon, J.; Hong, S.; Martin, K. A.; Czarnik, A. W. J. Org.
Chem. 1995, 60, 2792.
13. Mono-6-deoxy-6-carboxy-b-cyclodextrin (4). Bromine
(4 mL; 0.90 mmol) was added to a solution of 6-deoxy-6-
formyl-b-cyclodextrin (3, 1.02 g; 0.90 mmol) in 0.1 M
phosphate buffer (10 mL; pH 6.0). The solution was
stirred for 5 days at room temperature in the dark. Then,
excess of bromine was extracted by diethyl ether
(4 · 20 mL). The aqueous phase was added to acetone
(600 mL), and the formed precipitate was collected and
purified by preparative LC/MS according to the above
described procedure to give 0.21 g (yield = 20%) of the title
compound 4. 1H NMR (400 MHz, DMSO-d6) d 3.37–3.74
(overlap with D2O, 40 H), 3.83 (d, 1H), 4.38–4.45 (m, 6H),
4.73–4.97 (m, 7H); 5.63–5.75 (m, 14H); m/z 1150.1
[M+H]+, 171.2 [M+Na]+. Anal. Calcd for C42H68O36Æ
4H2O: C, 41.31; H, 6.23. Found: C, 41.55; H, 6.11.
14. Mono-6-deoxy-6-oxytocinyl-b-cyclodextrin (5). 6-Deoxy-
6-carboxy-b-cyclodextrin (4, 0.52 g, 0.45 mmol) was dis-
solved in DMF (3 mL) and the solution cooled to 0 ꢁC.
Then, HOBt (88 mg, 0.54 mmol) in DMF (2 mL) and
DCC (111 mg, 0.54 mmol) in DMF (2 mL) were added.
The solution was stirred and the temperature maintained
at 0–4 ꢁC. After 1 h, oxytocin (0.54 mg, 0.54 mmol)
dissolved in DMF (5 mL) was added to the reactive
mixture, which was stirred for 48 h at room temperature.
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C. Bertolla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1855–1858
At the end of the reaction, 200 mL of acetone was added,
OT). When a stable tension was obtained, cumulative
increasing concentrations of OT or b-CD-OT (ranging
from 10ꢀ13 to 10ꢀ5 M) were added to the bath until
tension and frequency are stable. Contractile response of
myometrial tissue segments was recorded isometrically
with a force–displacement transducer IT1 (EMKA Tech-
nologies, Paris, France). The EC50 value of each drug was
assessed for at least three concentration–response curves
and corresponded to the concentration which elicited 50%
the maximal tension. The EC50 values were calculated by
nonlinear regression analysis (GraphPad Prism software).
The results are expressed as means SEM, n P 3.
the formed precipitate was collected, washed with acetone,
dried, and purified by preparative LC/MS according to the
above described procedure to give 0.21 g (yield = 30%) of
1
the title compound 5. H NMR (DMSO-d6) d 0.77–0.84
(m, 12H, OT), 2.29–2.50 (overlap with DMSO, 14H, OT),
3.08–3.93 (overlap with D2O, 41H (CD) + 11H (OT)),
4.38–4.45 (m, 6H), 4.53–4.70 (br m, 11H, OT), 4.73–4.97
(m, 7H, CD); 5.63–5.75 (m, 14H, CD), 5.91 (br s, 4H, OT),
7.10 (d, 2H, OT) 7.70 (d, 2H, OT), 8.46 (s, 8H, CONH); m/
z 2138.1 [M+H]+, 1070.1 [M+2H]2+
.
15. Female rats (Wistar, 200–250 g, Charles River Laborato-
ries, Bruxelles, Belgium) are pretreated with b-estradiol
benzoate (500 lg kgꢀ1, ip, Sigma, Brussels, Belgium) 24 h
before the experiment. Rats were killed with pentobarbital
(80 mg kgꢀ1, ip, Ceva Sante Animale, Brussels, Belgium)
and myometrial tissue was removed and carefully trimmed
of surrounding connective tissue. Uterine segments were
placed longitudinally in a 20 mL tissue bath (EMKA
Technologies, Paris, France) containing Krebs solution
(mM: NaCl 118, KCl 5.4, CaCl2 2.5, MgCl2Æ6 H2O 1.5,
NaHCO3 25, NaH2PO4 1.2, glucose 10; pH 7.4) contin-
uously bubbled with a mixture of O2:CO2 (95:5%) at
37 ꢁC. Each uterine segment was placed under optimum
resting tension of 1 g and allowed to equilibrate for 1 h
before the experiment. During this equilibrium period, the
buffer was renewed every 15 min before exposing the
uterine segments to the contractile agent (OT or b-CD-
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19. Pymol software (v 1.0) was used to measure the distances
from the crystal structure of oxytocin deposited with the
RCSB Protein Data Bank; 1npo. doi:10.2210/pdb1npo/
pdb.