Synthesis and inclusion ability of a bis-β-cyclodextrin pseudo-cryptand towards Busulfan anticancer agent

Article abstract of DOI:10.1016/j.tet.2006.10.070

The synthesis of a C₂-symmetric receptor including two β-cyclodextrins connected by urea linkers to a chiral diaza-crown ether organising platform is reported. This molecular system, long thought to be a potent selective carrier for chiral/achi

Full text of DOI:10.1016/j.tet.2006.10.070

Tetrahedron 63 (2007) 1706–1714
Synthesis and inclusion ability of a bis-b-cyclodextrin
pseudo-cryptand towards Busulfan anticancer agent
a
Stephane Menuel, Jean-Pierre Joly, Blandine Courcot, Josias Elysee,
b,y
c,z
c
ꢀ
ꢀ
Nour-Eddine Ghermanid,x and Alain Marsuraa,
*
a
´
´
´
ꢁ
´
Groupe GEVSM, Unite Mixte de Recherches CNRS, Structure et Reactivite des Systemes Moleculaires Complexes,
´
´
Universite Henri Poincare-Nancy 1, 5 rue A. Lebrun, B.P. 403, F-54001 Nancy Cedex, France
b
´
´
´
Groupe SUCRES, Unite Mixte de Recherches CNRS, Structure et Reactivite des Systeꢁmes Moleculaires Complexes,
Universite Henri Poincare-Nancy 1, Campus Victor Grignard, B.P. 239, F-54506 Vandoeuvre-les-Nancy, France
´
´
´
ꢁ
cLaboratoire SPMS, UMR CNRS 8580 B236, Ecole Centrale Paris, 1, Grande Voie des Vignes, F-92295 Cha^tenay-Malabry, France
d
´
´
Laboratoire de Physique Pharmaceutique UMR 8612, Universite Paris XI, Faculte de Pharmacie, 5 rue J. B. Clement,
´
F-92296 Cha^tenay-Malabry Cedex, France
Received 11 September 2006; accepted 26 October 2006
Available online 16 November 2006
Abstract—The synthesis of a C2-symmetric receptor including two b-cyclodextrins connected by urea linkers to a chiral diaza-crown ether
organising platform is reported. This molecular system, long thought to be a potent selective carrier for chiral/achiral organic/inorganic guests
at the supramolecular level, was found to be an efficient complexing tool towards the Busulfan anticancer agent.
Ó 2006 Published by Elsevier Ltd.
1. Introduction
greatly enhanced binding abilities as a result of cooperative
binding of one guest molecule by the two hydrophobic cav-
ities located in close vicinity.4 Much effort has been devoted
to the design and synthesis of bis-CDs with a large panel of
structures and the investigation of their inclusion behaviour
with model guests.5 Metal ions have also been introduced as
additional recognition sites to enhance the binding selectiv-
ity of chromogenic bis-CDs and CD derivatives.6 Particu-
larly, coupling of a diaza-crown ether with a CD was early
investigated by Pikramenou et al.7 to develop light emitter
chemosensors upon recognition of a guest.8 Some rare ex-
amples of enantiopure C-substituted-aza-crown ethers have
been reported over three decades,9 but there is so far no con-
tribution in which a CD is covalently associated with a chiral
diaza-crown ether in a heterotopic co-receptor. The underly-
ing idea was to use the known high propensity of CDs to
form stable inclusion complexes with hydrophobic guests,
notably in the case of CD dimers. This feature was expected
to give a pseudo-cryptand framework by closing together the
CD side arms through the formation of a 1:1 inclusion com-
plex with a suitable organic guest. As illustrated in Figure 1,
the novelty of our approach is supported by the symmetry of
the chiral aza-crown organising platform and functionalisa-
tion of the latter with ureido-b-CD cavities. More precisely,
such a podand architecture associates in a close spatial prox-
imity: (i) one metallic complexing site (aza-crown cavity),
(ii) two polar pockets (threonyl and tertiary nitrogen moie-
ties) and (iii) two hydrophobic cavities (b-CDs).
Cyclodextrins (CDs), a class of natural cyclic oligosaccha-
rides with six, seven or eight D-glucose units linked by
a-1,4-glucose bonds, are known to accommodate various
guest molecules into their hydrophobic cavity in aqueous
solution.1 If natural CDs are themselves of great interest as
molecular hosts, much of their utility in supramolecular
chemistry derives from their structural modification.1b On
the other hand, unmodified CDs may be considered as
molecular scaffolds on which functional groups and/or other
substituents of increasing sophistication can be assembled
with controlled geometry.2 Enhancing the binding abilities
of CDs first introduced by Breslow’s work3 has been a per-
manent challenge for three decades and different strategies
have been proposed to reach a high level of reaction rates.
Increased binding and catalytic power was achieved with
capped, dimeric or tetrameric CDs coupled by different
linkers. For example, bridged CDs are known to exhibit
Keywords: b-Cyclodextrin; Diaza-crown ether; Pseudo-cryptand; Busulfan;
Inclusion; Molecular modelling.
* Corresponding author. Fax: +33 3 83 68 23 45; e-mail addresses: jean-
pierre.joly@sucres.uhp-nancy.fr; courcot@spms.ecp.fr; noureddine.
ghermani@cep.u-psud.fr; alain.marsura@pharma.uhp-nancy.fr
y
z
x
Fax: +33 3 83 68 47 80.
Fax: +33 1 41 13 14 37.
Fax: +33 1 46 83 58 82.
0040–4020/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.tet.2006.10.070

S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
1707
acid followed by a reductive deprotection (H2, Pearlman’s
catalysts) and one-pot phosphine imide polymer-supported
coupling reaction11 with 6A-azido-6A-deoxy-per-O-acetyl-
ated-b-CD 7 (method A) afforded the desired bis-ureido-CD
chiral co-receptor 8 in a 24% moderate yield.
( )
OH
HO
( )
6
6
OH
HO
14 14
( ) ( )
HYDROPHOBIC GUEST
A higher yield (50%) in 8 was achieved using the direct
coupling of the diaza-crown ether 6 with the 6A-isocyana-
to-6A-deoxy-peracetylated-b-CD 7bis (method B). The target
compound 9 was isolated from the peracetylated precursor 8
H
H
H
OH
N
N
H3C
N
O
O
OH
H
O
N
ꢀ
after a deacetylation step using Zemplen conditions (Scheme
O
O
O
H3C
1). All the new compounds were fully characterised by FTIR,
1H and 13C NMR, 2D NMR, elemental analysis and/or MS.
The spectroscopic data are in perfect agreement with the as-
signed structures. For instance, the FTIR spectrum of 8 shows
a medium absorption band at 1675 cmꢀ1 characteristic of an
urea carbonyl bond, also corroborated by the 13C NMR cor-
responding signal at 161 ppm (C]O urea) indicating that the
CDs have been coupled with the diaza-crown. The MS of
8 displays the partially deacetylated monocharged ion
fragment at m/z¼3998.19 [Mꢀ9CH3CO]+ and the partially
deacetylated discharged ion at m/z¼2067.26 [Mꢀ5CH3CO+
5H+]2+. The 13C NMR signal of the urea carbonyl bond in
the final deacetylated compound 9 was upfield shifted to
133 ppm likely through the formation of hydrogen bonds
with the numerous close-space free hydroxyls of the CD.
Altogether, these results prove undoubtedly the proposed
molecular structure.
H
POLAR POCKET
H
POLAR/APOLAR COMPLEXING SITE
Figure 1. Schematic representation of the hetero tritopic co-receptor con-
cept.
We expected this structure to form in fine supramolecular
complexes with convenient chiral or achiral organic/inor-
ganic ligands.
2. Results and discussion
2.1. Synthesis
An unprecedented scale-up chiral diaza-crown ether 5 five-
step preparation (w4.6 g)10 starting from L-threonine amino
Cl
O
CO2H
CO2Bn
CH2OH
H2N
H
Bn2N
Bn2N
O
i
ii
iii
OH
OH
OH
H
Bn2N
OH
H
H
H
H
H
H
H3C
H3C
L-threonine
H3C
1
H3C
2
3
H
H
H3C
OH
R
)
H3C
(
AcO
OH
6
N
Cl
O
N
H
O
O
O
vi
v
O
O
Bn O
iv
+
O
b
O
H
N
Bn O
N
H2N
H
OH
H
HO
CH3
HO
CH3
)
H3C
(OAc 14
H
H
6
7 : R = N
5
4
3
7bis: R = NCO
HO
H
O
O
O
(OAc)6
NH
N
H C
H
3
N
(AcO)6
vii
vii a
viii
CH3
H
OH
N
O
N
O
O
b
b
8
(OAc)14
(OAc)14
HO
O
O
O
H
H3C
NH
(OH)6
H
N
CH3
N
(HO)6
H
OH
O
O
O
b
b
9
(OH)14
(OH)14
Scheme 1. Synthesis of water-soluble host 9. Reagents and conditions: (i) BnBr, Na2CO3, EtOH/H2O, 80 ꢁC, 5 h, 78%; (ii) LiAlH4, THF/Et2O, rt, 5 h, 86%; (iii)
2,20-bis-dichlorodiethylether, NaOH, NBu4HSO4, 4 ꢁC, 14 h, 61%; (iv) H2, HCO2H, Pd(OH)2, MeOH, rt, 14 h, 99%; (v) NaI, Na2CO3, ref. MeCN, 4 d then
BnBr, 18 h, 43%; (vi) H2, Pd(OH)2, MeOH/CH2Cl2, rt, 14 h, 98%; (vii) CO2, PPh3 on resin, rt, 24 h, method A, THF, 24%; method B, DMF, 50.4%; (viii)
MeOH/Na catalytic (95%).

1708
S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
Figure 2. Side and face views of the backbone (without hydrogen atoms) and spacefill (with hydrogen atoms) structures with lowest energy after dynamic
simulations: S1 conformation in vacuum; S2 conformation in water.
2.2. Conformation of the free bis-CD-aza-crown 9 in
water
In both S1 and S2 conformations, the two CD wide rims face
each other, strongly connected each other through multiple
hydrogen bonds involving secondary hydroxyl groups. The
closest distances between O-donor and O-acceptor atoms
It is well known that elucidation of the crystal structure is
one of the most convincing methods of unequivocally illus-
trating the geometrical CD derivatives. Unfortunately, isola-
tion of suitable single crystals for X-ray crystallography of
this kind of strongly modified CDs is also often a big diffi-
culty too and many attempts to prepare such single crystals
from 8 or 9 failed. To elucidate the possible conformations,
we performed molecular modelling calculations on the di-
mer 9. The converged structures of the bis-CD ligand in
both vacuum (S1) and water medium (S2) obtained by using
dynamic molecular method at the MM3 force field level are
depicted in Figure 2.
˚
were found equal to 3.05 and 2.88 A for S1 and S2 conforma-
tions, respectively. This shows that in water medium, the at-
traction between the two CDs is more pronounced as could
be expected from the known hydrophobicity of the cavities
of these fragments.
Figure 3 enlightens how the crown ether can be distorted in
the present system.
A higher distortion is found in S2 conformation, which dis-
plays a close atomic distance for both urea and crown
Figure 3. Close view of the crown ether moiety with geometrical features for S1 and S2 conformations.

S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
1709
oxomethylene oxygen atoms. These calculations are in good
agreement with previous results obtained with the ureido-
cyclam tri- and tetra-substituted-b-CD ligand family12 by
molecular-dynamics computations, which have shown a spa-
tial ‘bouquet’ conformation adopted by these kind of mole-
cules. This essentially arises from strong intramolecular
hydrogen bonds between urea functions and also from the
spatial distribution of the CDs that face each other with their
wide rims strongly connected on the same side of the crown.
2.3. Interaction of 9 with Busulfan as guest
The formulation of molecules with a trend to crystallise is a
major problem in pharmacy. Typically, one of the main side
effects observed clinically with high dose rate of Busulfan
(1,4-butanediol-dimethylsulfonate, a powerful antitumoural
agent in leukaemias13) is the hepatic veno-occlusive compli-
cation due to microcrystallisation in the microvenous system
of the liver.14 In the course of developing a more appropriate
formulation based on supramolecular strategy, the possibil-
ity of encapsulating the Busulfan molecule into the bis-CD
host 9 was investigated. Signs of interaction were first de-
tected by chemical-induced shifts (CIS) of some protons
of the guest signals compared to those of the free compound.
The signals of both the sulfomethyl and methylene of the
butyl chain are slightly shifted downfield (ꢀ0.005 ppm)
and upfield (+0.014 ppm), respectively as shown in Figure 4.
0
0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9
R = [Busulfan] / [Busulfan] + [9]
1
Figure 5. Job plot corresponding to the chemical shift displacement of the
sulfomethyl and methylene protons of Busulfan for [Busulfan/9] in D2O at
300 K.
performed in parallel and that failed to obtain inclusion com-
plexes of 9 in water at 298 K with some hydrophobic guest
molecules, for example, bis-nitrophenyl phosphate, which
are well known to form currently [1:1] inclusion complexes
with bis-CD systems.16
Elsewhere, two-dimensional NMR spectroscopy has re-
cently become a very valuable method for the study of the
structures of CD dimers and their complexes in solution17
since one can conclude on the spatial proximity of two pro-
tons if an NOE/ROE cross peak is detected between the
relevant proton signals in the 2D NOESY or 2D ROESY
spectrum. So it was possible to estimate the local spatial in-
teractions and the orientation of the Busulfan guest molecule
inside the bis-CD-aza-crown host using the assigned ROE
correlations (Fig. 6).
Stoichiometry of the complex [Busulfan/9] could be con-
firmed by the continuous variation method known as Job plot
illustrated in Figure 5.
A value of R¼0.5 was reached at the maximum, which
strengthens the 1:1 stoichiometry for the complex with an
apparent complexation constant (Ka) of ca. 1600 molꢀ1 at
300 K. On the other hand, H3 and H5 proton signals located
inside the CD cavity of the host remained unchanged. This
suggests that the Busulfan is not embedded in CDs hydro-
phobic cavities but is likely in interaction with the ureas
and crown ether part of 9. This feature is in fair agreement
with recent results on the interaction of Busulfan by
b-CD15 and the above results of molecular modelling show
that the CD cavities are strongly connected in water by their
wide rims so that they probably prohibit a free access to
Busulfan. This feature is consistent with some attempts we
The 2D ROESY spectrum of the [Busulfan/bis-CD-aza-
crown] 1:1 complex in D2O displays on one hand two cross
peaks between the methylene protons of the crown and the
methylene protons of the Busulfan-butyl chain and on the
other hand, between the methyl protons of the Busulfan and
the methylene protons of the CD (C-6 connected to the urea
N–H atom). These results corroborate the above-observed
chemical-induced shifts and indicate that the Busulfan is
(a)
(b)
0
1.95
1.90
3.30
3.25
3.20
[ppm]
[ppm]
Figure 4. 1H NMR spectrum of [Busulfan/aza-crown-bis-b-CD] [1:1] complex at 400 MHz; (a) view of b-methylene protons of the Busulfan-butyl chain;
(b) view of sulfomethyl protons of Busulfan.

1710
S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
(a)
(b)
CH2 Busulphan
CH3 Sulfomethyl
3.58
3.60
3.62
C-6 CyD
3.52
CH2
Crown
3.56
3.60
3.64
3.64
3.66
3.68
3.70
3.18 3.16 3.14 3.12 3.10
(ppm)
2.00 1.96 1.92 1.88 1.84
(ppm)
Figure 6. Sectional 1H NMR 2D ROESY spectrum of the [Busulfan/bis-CyD-aza-crown] 1:1 complex in D2O (8.57ꢂ10ꢀ3 M) at 298 K, mixing time 400 ms.
(a) Cross peak between CH2 b of Busulfan and CH2 of the crown; (b) cross peak between the CH3 ester of Busulfan and CH2 of the CyD C-6 linker.
connected to the ureas N–H atoms at each extremity of the
crown, probably by H-bonds with its two ester oxygens,
that consequently forced the sulfomethyl protons to be lo-
cated in close proximity to the CD C-6 methylene atom con-
nected to the urea N–H nitrogens. Thus, concerning the
Busulfan central lipophilic butyl chain, it should lie across
the crown ether macrocycle in close proximity with the oxo-
ethylene bridges. Considering the dimension of the bis-
ureido crown ether existing cavity (which was estimated
above the crown ether moiety to form a pseudo-cryptand
molecular system. It was established experimentally that
the new host interact efficiently with the Busulfan antitu-
moural agent thus strongly enhancing its water solubility.
The 1D and 2D NMR results clearly established that the
lipophilic guest is not embedded in the hydrophobic CD cav-
ities, but connected across the aza-crown macrocycle to the
urea functions at each extremity of the crown ether, likely by
hydrogen bonds with the sulfomethyl oxygen atoms. In light
of these first investigations, a deeper study on the stability of
the inclusion complex of Busulfan in biological media along
its anti-neoplasic activity is now under way. The new molec-
ular devices introduced here should contribute to the future
development of CD-based nano-biomaterials.
˚
between 5.1 to 5.4 A), the distance between the two O3 ester
˚
oxygens (w5.2 A), the conformation and the electron density
map of the guest,15 there is a high probability of N–H/O3
strong H-bond formation between N–H of ureas and ester
oxygen atoms in the [Busulfan/9] inclusion complex as illus-
trated in Figure 7.
4. Experimental
4.1. General comments
All new compounds gave satisfactory spectroscopic data. 1H
and 13C NMR spectra were recorded on Bruker DRX-400
and AC 250 spectrometers, FTIR spectra on a Perkin–Elmer
1600 and a Bruker Vector22 spectrometers. Mps were deter-
€
mined on a Buchi apparatus in capillary tubes and are uncor-
rected. Optical rotations were measured on a Perkin–Elmer
141 automatic polarimeter in a 1 dm cell at rt. In all experi-
ments, DMF was dried over CaSO4, filtered off, distilled over
CaH2 and flushed with argon to eliminate dimethylamine.
Electrospray mass spectra in the positive ion mode were
obtained on a Q-TOF Ultima Global hybrid quadrupole/
time-of-flight instrument (Waters-Micromass, Manchester,
UK) equipped with a pneumatically assisted electrospray
(Z-spray) ion source and an additional sprayer (Lock Spray)
for the reference compound. The source and desolvation
temperatures were kept at 80 and 150 ꢁC, respectively.
Nitrogen was used as the drying and nebulising gas at flow
rates of 350 and 50 L/h, respectively. The capillary voltage
was 3 kV, the cone voltage 100 V and the RF lens1 energy
was optimised for each sample (30–150 V). Lock mass cor-
rection, using appropriate cluster ions of an orthophosphoric
acid solution (0.1% in 50:50 acetonitrile/water) or of a
sodium iodide solution (2 mg/mL in 50/50 propan-2-ol/
water+0.05 mg/mL caesium iodide), was applied for accurate
mass measurements. The mass range was typically 50–
4050 Da and spectra were recorded at 4 s/scan in the profile
mode at a resolution of 104 (FWMH). Data acquisition and
Figure 7. Postulated interactions of Busulfan with the bis-ureido-aza-crown
moiety of 9.
Lastly, the Busulfan inclusion mode was also unambigu-
ously supported by the IR spectrum of the complex in which
the characteristic C]O urea frequency is up-shifted from
1685 to 1642 cmꢀ1. Interestingly, the solubility of the com-
plex in water was estimated to be at least 10 g Lꢀ1. This is
a fairly good solubility to an almost water insoluble drug,
which is at the origin of its major side effects.14
3. Conclusions
A water-soluble C2-symmetric receptor including two b-
CDs connected by urea linkers to a chiral diaza-crown ether
organising platform has been synthesised in eight steps and
characterised. Its inclusion properties towards Busulphan
have been experimentally evaluated. Molecular modelling
simulations, either in vacuum or in water as solvent, gave a
convergent set of unexpected conformations in which the CD
cavities are tightly connected together by their wide rims

S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
1711
processing were performed with MassLynx 4.0 software.
Busulphan was purchased from Sigma–Aldrich (Schnell-
NCHHPh); 3.85 (1H, m, H-b); 3.80 (2H, d, J¼13.2 and
5.8 Hz, CH2OD); 3.72 (2H, d, NCHHPh); 2.62 (1H, d,
J¼8.8 Hz, H-a); 1.15 (3H, d, J¼5.8 Hz, CH3). 13C NMR
(100 MHz, CDCl3, 25 ꢁC): d (ppm) 139.5 (C arom.);
129.4, 128.7, 127.5 (CH arom.); 65.5 (C-b); 64.8 (C-a);
59.1 (PhCH2); 54.7 (CH2–OH); 20.4 (CH3). Elemental anal-
ysis calcd (%) for C18H23NO2 (285.39): C 75.76, H 8.12,
N 4.91; found: C 75.83, H 8.12, N 4.99.
dorf, Germany) and compounds
7
and 7bis were
synthesised according to the literature.18
4.2. Synthesis of host 9
4.2.1. (2S,3R)-2-(N,N-Dibenzyl)amino-3-hydroxy-
benzyl-butanoate 1. Benzyl bromide (66 mL, 0.55 mol,
3.3 equiv) was dropped over 2 h on a mechanically stirred
dispersion of L-threonine (98%, 20.2 g, 166 mmol) and
38.75 g of dry Na2CO3 (195 mmol, w2.2 equiv) in 75% aq
EtOH (200 mL) below 25 ꢁC. The resulting mixture was
then refluxed for 5 h (the formation of 1 being monitored
by SiO2 TLC: Rf¼0.43; AcOEt/cyclohexane, 1:4), cooled
to rt, concentrated under reduced pressure and partitioned
between CH2Cl2 and H2O (2ꢂ200 mL). The aqueous phase
was extracted with CH2Cl2 (2ꢂ100 mL). The combined
organic phases were washed with satd aq NaHCO3
(50 mL), H2O (2ꢂ50 mL), dried over MgSO4 and finally
concentrated under vacuum to afford the crude ester 1 as
a pale yellow syrup used for the next step without further
purification. Yield w67 g (172 mmol, 78%). An analytical
sample was isolated by LC (SiO2, AcOEt/hexane, 1:9) as
a colourless gum: [a]D20 ꢀ149 (c 2.5, CHCl3). MS (70 eV,
EI+) calcd for C25H27NO3 (389): found m/z (%) 344 (32)
4.2.3. (2R,3R)-1-[(2-Chloroethoxy)-2-ethyl]-2-N,N-di-
benzylamino-butan-3-ol 3. To a mechanically stirred
dispersion of diol 2 (57.1 g, w200 mmol) and 98%
N(butyl)4HSO4 (69.3 g, 1 equiv), 2,20-bis-dichloro-diethyl-
ether (650 mL) in 2-L Morton flask was slowly added chilled
50% M/v NaOH (0.7 L) over 15 min. The two-phase system
was vigorously stirred below 6 ꢁC for 14 h, the reaction be-
ing monitored by SiO2 TLC: ethyl acetate/n-hexane/toluene;
1:1:1, Rfw0.52. The resulting emulsion was partitioned be-
tween H2O (0.7 L) and CH2Cl2 (0.6 L). The isolated aque-
ous phase was extracted with CH2Cl2 (3ꢂ100 mL) and the
combined organic phases washed with H2O (3ꢂ50 mL),
dried over MgSO4, concentrated under reduced pressure,
the excess of reagent being removed by distillation under va-
cuo around 50 ꢁC (w85% recovery) under an efficient fume
board. The residue was purified by HPLC (SiO2, ethyl ace-
tate/n-hexane; 1:4) to yield the pure ether 3 (47.8 g, 61%)
as a colourless gum: [a]2D0 ꢀ73.0 (c 3, CHCl3); MS (70 eV,
EI+) calcd for C22H30ClNO3 (391.19): found m/z 392.0
[M+H]+, 346.0 [MꢀC2H5O]ꢃ+; HRMS (70 eV, EI+) calcd
for C22H30ClNO3 (391.1914): found m/z 392.1976 [M+H]+.
1H NMR (250 MHz, CDCl3, 25 ꢁC): d (ppm) 7.37–7.20
(10H, m, arom.); 4.10 (1H, br s, OH); 3.96 (2H, d,
J¼13.1 Hz, 2ꢂCHH–N); 3.85–3.58 (13H, m, H-b, ClCH2,
4ꢂOCH2, 2ꢂCHH–N); 2.62 (1H, m, H-a); 1.11 (3H, d,
ꢃ
[MꢀC2H5O] +. IR (film): 1730 cmꢀ1. H NMR (250 MHz,
CDCl3, 25 ꢁC): d (ppm) 7.15–7.55 (15H, m, arom.); 5.28
(1H, d, J¼12.4 Hz, CO2CHHPh); 5.22 (1H, d, CO2CHHPh);
4.07 (1H, m, H-b); 4.00 (2H, d, J¼13.2 Hz, NCHHPh); 3.50
(1H, s, OH); 3.39 (2H, d, NCHHPh); 3.11 (1H, d, J¼9.5 Hz,
H-a); 1.09 (3H, d, J¼5.8 Hz, CH3). 13C NMR (100 MHz,
CDCl3, 25 ꢁC): d (ppm) 139.5 (C arom.); 135.7 (C arom.);
129.1, 128.7, 128.6, 128.5, 127.4 (CH arom.); 67.2 (C-b);
66.3 (PhCH2O); 63.2 (C-a); 54.8 (PhCH2N); 19.2 (CH3).
Elemental analysis calcd (%) for C25H27NO3 (389.49): C
77.09, H 6.99, N 3.60; found: C 76.91, H 6.81, N 3.67.
1
13
J¼5.8 Hz, CH3). C NMR (100 MHz, CDCl3, 25 ꢁC):
d (ppm) 139.3 (C arom.); 129.2, 128.5, 127.2 (CH arom.);
71.4, 70.8, 70.7 (3ꢂOCH2), 68.1 (N–CH–CH2–O); 64.0
(C-b); 63.7 (C-a); 54.5 (PhCH2); 42.9 (CH2Cl); 19.7 (CH3).
Elemental analysis calcd (%) for C22H30ClNO3 (391.94):
C 67.42, H 7.72, N 3.55; found: C 67.29, H 7.68, N 3.65.
4.2.2. (2R,3R)-2-N,N-Dibenzylamino-1,3-butane-diol 2.
To a mechanically stirred suspension of 95% LiAlH4
(7.8 g,w195 mmol, 1.15 equiv) in THF (450 mL) under Ar
and cooled below 4 ꢁC was carefully dropped a solution of
crude ester 1 (66.2 g, 170 mmol) in abs ether (150 mL) over
2 h. The mixture was then stirred for 5 h at rt and refluxed for
one more hour. After completion of the reaction (checked by
SiO2 TLC: ethyl acetate/n-hexane; 1:1, Rfw0.29), the mix-
ture was cooled to 0 ꢁC, quenched by slow addition of ethyl
acetate (50 mL), satd aq Na2SO4 (35 mL) and stirred over-
night at rt in open air. The suspension was filtered through
a sintered glass and the remaining salts thoroughly washed
with a CH2Cl2/ethanol mixture (1:1; 250 mL). The filtrates
were concentrated under reduced pressure and the resi-
due dissolved in CH2Cl2 (400 mL), washed with H2O
(3ꢂ50 mL), dried over MgSO4, concentrated under reduced
pressure and finally stored at 4 ꢁC in the dark. The resulting
solids were isolated by filtration and crystallised twice from
ethyl acetate/hexanes to yield pure alcohol 2 (ca. 32 g, 66%
over two steps) as white crystals (an extra 20% crop could be
obtained from the mother liquors by preparative chromato-
graphy on SiO2 with ethyl acetate/hexanes; 1:2): mp¼90–
91 ꢁC; [a]2D0 ꢀ55 (c 1, CHCl3). MS (70 eV, EI+): m/z 286.1
[M+H]+. 1H NMR (250 MHz, CDCl3/3D2O): d (ppm)
7.20–7.42 (m, 10H, arom.); 3.99 (2H, d, J¼13.2 Hz,
4.2.4. (2R,3R)-1-[(2-Chloroethoxy)-2-ethyl]-2-amino-
butan-3-ol 4. Ar was bubbled through a solution of tertiary
amine 3 (16.5 g,w42 mmol) and HCO2H (1.6 mL, 1 equiv)
in abs MeOH (100 mL) for 10 min at rt to remove O2. Moist-
ened Pearlman’s catalyst (20% Pd(OH)2/C, 435 mg, ca.
0.02 equiv) was added to the suspension, which was imme-
diately saturated with H2 (w1 atm) and magnetically stirred
for 14 h. Na2CO3 (4.45 g, 1 equiv) was added to the suspen-
sion, which was stirred for one more hour. The resulting
mixture was filtered through a Celite pad, the reactor and
pad exhaustively rinsed with a mixture of CH2Cl2/EtOH
(1:1; 200 mL), the filtrates concentrated under reduced pres-
sure to yield the primary amine 4 (8.7 g, 98%) as a pale
yellow syrup: Rf ¼0.31 (SiO2 TLC, CH2Cl2/MeOH, 95:5);
[a]2D0ꢀ0.2 (c 4, CHCl3); HRMS (70 eV, EI+) calcd for
1
C8H18ClNO3 (211.0975): found m/z 212.1038 [M+H]+. H
NMR (250 MHz, CDCl3, 25 ꢁC): d (ppm) 3.74–3.46 (10H,
m, 4ꢂOCH2, CH2Cl); 3.38 (1H, m, H-b); 2.68 (1H, m,
H-a); 2.3 (3H, br s, NH2, OH); 1.14 (3H, d, J¼6.6 Hz,
13
CH3). C NMR (100 MHz, CDCl3, 25 ꢁC): d (ppm) 74.2,
71.3, 70.5, 70.45 (4ꢂOCH2); 67.8 (C-b); 56.3 (C-a); 42.8
(CH2Cl); 20.1 (CH3).

1712
S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
4.2.5. (2R,11R)-1,10-Diaza-1,10-N,N0-benzyl-2,11-bis-
[(20R)-20-hydroxyethyl]-4,7,13,16-tetraoxa-cyclooctade-
cane 5. Ar was bubbled through a solution of primary amine
4 (8.5 g,w40 mmol) in abs MeCN (200 mL) for 10 min at rt
to remove O2. Anhydrous Na2CO3 (5.51 g, 1.3 equiv) and
NaI (6.0 g, 1 equiv) were slowly added to the solution and
the resulting suspension magnetically stirred and heated to
gentle reflux for 20 h under Ar. The mixture was allowed
to cool to rt and benzyl bromide (1.23 mL, 1.5 equiv) was
dropped over 5 min on the mixture, which was refluxed
again for 10 h. After complete cooling, the reaction mixture
was concentrated under reduced pressure and the residue
partitioned between H2O and CH2Cl2 (2ꢂ250 mL). The iso-
lated aqueous phase was extracted with CH2Cl2 (3ꢂ75 mL)
and the combined organic phases washed with satd NH4Cl,
H2O twice (50 mL), dried over MgSO4, concentrated under
reduced pressure and the residue purified by LC (SiO2, ethyl
acetate/n-hexane/HN(iso-Pr)2; 800:199:1) to yield pure
crown ether 5 (4.58 g, 43%) as a pale yellow gum: Rf ¼
0.29 (SiO2 TLC, ethyl acetate); [a]2D0 ꢀ71.6 (c 1.2, CHCl3).
HRMS (70 eV, EI+) calcd for C30H46N2O6 (530.3356): found
m/z 530.3353 [M]ꢃ+, 485.2 (100%) [MꢀC2H5O]ꢃ+. 1H NMR
(400 MHz, CDCl3, 25 ꢁC): d (ppm) 7.35–7.20 (10H, m,
Harom); 4.20 (2H, sl, OH); 3.93 (2H, d, J¼13.3 Hz,
2ꢂPhCHH); 3.70–3.45 (18H, m, 2ꢂPhCHH, 7ꢂOCH2,
2ꢂH-b); 3.40 (2H, m, J¼6.2 and 8.0 Hz); 3.03 (4H, m,
J¼14.5 Hz, 2ꢂOCH2N); 2.62 (2H, 2ꢂH-a); 1.14 (6H, d,
filtration, the polymer was washed by DMF (3ꢂ30 mL)
and the solution was concentrated to dryness. The crude
product was filtered and washed with ether, then purified
on a ChromatotronÒ (silica gel, CH2Cl2/MeOH; 98:2). A
white amorphous powder (0.038 mmol, 0.101 g, 24%) was
isolated.
Method B: 6A-isocyanato-6A-deoxy-peracetyl-b-cyclodex-
trin 7bis (0.779 g, 0.389 mmol, 2.1 equiv) into anhyd DMF
(40 mL) was added to a DMF (10 mL) solution of diaza-
crown ether 6 (0.065 g, 0.186 mmol). The mixture was
stirred at rt for 24 h under argon. The mixturewas then evapo-
rated to dryness and the residue treated by MeOH (2 mL).
The final product was precipitated from the methanolic solu-
tion by ether, filtered on a sintered glass and dried in a dessi-
cator over anhydrous KOH. A white amorphous powder
(0.408 g, 0.153 mmol, 50.4%) was obtained: [a]1D8 +93.5 (c
0.1, MeOH). FTIR (KBr): n¼1751 cmꢀ1 (C]O acetate),
1
n¼1675 cmꢀ1 (C]O urea). H NMR (400 MHz, CDCl3,
25 ꢁC): d (ppm) 5.42–5.23 (m, 14H, H-3 CDa,b); 5.20–5.05
(m, 14H, H-1 CDa,b); 4.92–4.73 (m, 14H, H-2 CDa,b);
4.68–4.50 (m, 14H, H-5 CDa,b); 4.40–3.80 (m, 32H, H-6a,
crown); 3.88–3.44 (m, 34H, H-6b, crown); 2.20–1.98 (m,
120H, CH3 acetates); 1.25–1.17 ppm (m, 6H, CH3 crown).
13C NMR (100 MHz, CDCl3, 25 ꢁC): d (ppm) 171.0–170.0
(multiple s, C]O acetates); 161.0 (C]O, urea); 97.0
(C-1); 76.0 (C-4); 72.0, 71.0, 70.0 (C-2, C-3, C-5); 63.0
(C-6); 41.0 (CH, CH2, crown); 23.0 (CH3, crown); 21.0
(CH3, acetates). MS-MALDI (a-cyano matrix) m/z:
3998.19 [Mꢀ9CH3CO]+, 2067.26 [Mꢀ5CH3CO+5H+]2+.
13
J¼6.1 Hz, 2ꢂCH3). C NMR (100 MHz, 25 ꢁC, CDCl3):
d (ppm) 139.6 (C arom.); 129.0, 128.4, 127.2 (CH arom.);
70.8, 70.5, 68.4 (OCH2); 67.3 (C-b); 63.6 (C-a); 56.1
(PhCH2); 50.4 (N–CH2 aliph.); 19.7 (CH3).
4.2.8. (2R,11R,19R,21R)-2,11-Bis-(2-hydroxyethyl)-1,10-
N,N0-bis-[cyclomaltoheptaosyl-6A-deoxy-6A-ureido]-
4,7,13,16-tetraoxa-1,10-diaza-cyclooctadecane 9. The
4.2.6. (2R,11R)-2,11-Bis-[(20R)-20-hydroxyethyl]-4,7,13,
16-tetraoxa-1,10-diaza-cyclooctadecane 6. After Ar was
bubbled for 5 min through a solution of N,N0-dibenzyl-
1,10-diaza-crown ether 5 (1.064 g, 2 mmol) and 80 mL of
formic acid (1 equiv) in abs MeOH at rt to remove O2,
20% Pd(OH)2 on charcoal (200 mg) was added and the sus-
pension immediately stirred under 1 atm of H2 for 14 h. The
catalyst was removed by filtration through basic alumina
using MeOH/CH2Cl2 (1:1) for washings, and volatiles
were evaporated under reduced pressure to yield the crude
aza-crown 6 (680 mg, 98% yield), which was used without
further purification for the next step. Colourless wax:
mp<50 ꢁC; [a]D20 ꢀ42.0 (c 0.65, CHCl3). HRMS (70 eV,
EI+) calcd for C16H34N2O6 (350.2417): found m/z
peracetylated bis-b-CD-crown ether
7
(0.049 mmol,
0.214 g) was dissolved in anhyd MeOH (50 mL). The solu-
tion was chilled in an ice bath at 0 ꢁC and a 1 M MeONa
solution (3.5 mL) was added drop by drop. The mixture
was stirred 1 h under argon at 0 ꢁC and then 1 h at rt. Small
amounts of IRN77Ò ion exchange resin were added until
neutralisation at pH¼7.0. The resulting suspension was fil-
tered off, the filtrate was evaporated to dryness, the solid
product was dissolved into distilled water and finally lyophi-
lised to yield 8 as an orange amorphous powder (0.125 g,
0.053 mmol, 95%): [a]D18 +88 (c 0.05, H2O). FTIR (KBr):
n¼1685 cmꢀ1 (C]O urea). 1H NMR (400 MHz, D2O,
25 ꢁC): d (ppm) 5.09 (m, 14H, H-1 CDab); 4.08–3.93 (m,
14H, H-3 CDab); 3.94–3.78 (complex m, 36H, H-6 CDa;
H-5 CDab); 3.77–3.72 (m, 14H, CH2 crown); 3.71–3.51
(complex m, 42H, H-2 CDab, H-4 CDab, H-6 CDb, CH2
crown); 3.42–3.25 (m, 2H, CHb crown); 1.20 (dd, 6H,
1
351.2479 [M+H]+. H NMR (250 MHz, CDCl3, 25 ꢁC):
d (ppm) 3.50–3.70 (20H, m); 3.42 (2H, dd, J¼4.4 and
9.5 Hz); 3.05 (2H, m, J¼5.8 Hz); 2.58 (2H, m, J¼11.7 Hz);
2.34 (2H, m); 1.18 (6H, d, J¼5.8 Hz, 2ꢂCH3). 13C NMR
(100 MHz, CDCl3, 25 ꢁC): d (ppm) 71.1, 70.7, 70.0, 68.6
(4ꢂOCH2); 65.8 (C-b); 64.2 (C-a); 47.0 (CH2N); 19.8(CH3).
13
CH3 crown). C NMR (100 MHz, DMSO-d6, 25 ꢁC):
d (ppm) 133.0 (C]O urea); 129.0 (C-1); 74.0 (C-4); 72.0
(C-2); 70.0 (C-3); 67.0 (C-5); 63.0 (C-6); 61.0 (C–H crown),
52.0–46.0 (CH2 crown); 24.0 (CH3 crown); Elemental anal-
ysis calcd (%) for C102H172N4O76$7H2O (2796.58): C 43.81,
H 6.70, N 2.00; found: C 43.75, H 6.81, N 1.96.
4.2.7. (2R,11R,19R,21R)-2,11-Bis-(2-hydroxyethyl)-1,10-
N,N0-bis-{[hexakis-(2,3,6-tri-O-acetyl)]-2,3-di-O-acetyl-
cyclomaltoheptaosyl-6A-deoxy-6A-ureido}-4,7,13,16-tet-
raoxa-1,10-diaza-cyclooctadecane 8. Method A: Polysty-
rene bound triphenylphosphine resin (2.7 g), 6A-azido-6A-
deoxy-peracetyl-b-CD 7 (0.562 g, 0.156 mmol) and 6
(0.0547 g, 0.156 mmol) in freshly distilled DMF (40 mL;
previously flushed 20 min by argon) were placed into a
solid-phase peptide synthesis vessel at rt. The mixture was
then stirred for 24 h under continuous CO2 bubbling. After
4.3. Preparation of [Busulfan/9] inclusion complex
A solution of Busulfan (0.0048 g, 0.019 mmol) in DMSO
(0.5 mL) was added under argon to a solution of 9 (0.047 g,

S. Menuel et al. / Tetrahedron 63 (2007) 1706–1714
1713
4. De Jong, M. R.; Knegtel, M. A. R.; Grootenhuis, P. D. J.;
Huskens, J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2002,
41, 1004–1008.
0.018 mmol) in H2O (25 mL) at rt. The mixture was stirred
18 h more and then lyophilised to yield a yellow amorphous
powder: [a]2D0 +83 (c 0.1, H2O); FTIR (KBr): n¼1642 cmꢀ1
5.09 (m, 14H, H-1 CDa,b); 4.42 (m, 4H, CH2 Busulfan);
4.08–3.93 (m, 14H, H-3 CDa,b); 3.94–3.78 (m, 36H,
H-6 CDa, H-5 CDa,b); 3.77–3.72 (m, 14H, CH2 crown);
3.71–3.52 (m, 40H, H-2 CDa,b, H-4 CDa,b, H-6 CDb, CH2
crown); 3.49–3.39 (m, 2H, CHb crown); 3.23 (s, 6H, CH3
Busulfan); 1.95 (m, 4H, CH2b Busulfan); 1.29–1.10 (m, 6H,
CH3 crown).
1
(C]O urea). H NMR (400 MHz, D2O, 25 ꢁC): d (ppm)
5. (a) Breslow, R.; Halfon, S.; Zhang, B. Tetrahedron 1995, 51,
377–388; (b) Breslow, R.; Zhang, B. J. Am. Chem. Soc. 1996,
118, 8495–8496; (c) De Jong, M. R.; Engbersen, J. F. J.;
Huskens, J.; Reinhoudt, D. N. Chem.—Eur. J. 2000, 6, 4034–
4040; (d) Venema, F.; Nelissen, H. F. M.; Berthault, P.;
Birlirakis, N.; Rowan, A. E.; Feiters, M. C.; Nolte, R. J. M.
Chem.—Eur. J. 1998, 4, 2237–2250; (e) Shiu, S. H.; Myles,
D. C.; Garell, R. L.; Stoddart, J. F. J. Org. Chem. 2000, 65,
2792–2796.
6. (a) Zhang, B.; Breslow, R. J. Am. Chem. Soc. 1997, 119,
1676–1681; (b) Sallas, F.; Marsura, A.; Petot, V.; Pinter, I.;
Kovacs, J.; Jicsinszky, L. Helv. Chim. Acta 1998, 81,
632–645; (c) Peirera Silva, M. J. J.; Haider, J. M.; Chavarot,
M.; Ashton, P. R.; Williams, R. M.; De Cola, L.; Heck, R.;
Marsura, A.; Pikramenou, Z. Supramol. Chem. 2003, 15,
563–571.
7. (a) Pikramenou, Z.; Nocera, D. G. Inorg. Chem. 1992, 31, 532–
536; (b) Pikramenou, Z.; Johnson, K. M.; Nocera, D. G.
Tetrahedron Lett. 1993, 34, 3531–3534.
8. To our knowledge, only two reports in the literature deal with
achiral azacrowns linked to CDs: (a) Suzuki, I.; Ito, M.; Osa,
T.; Anzai, J.-i. Chem. Pharm. Bull. 1999, 47, 151–155; (b)
Lock, J. S.; May, B. L.; Clements, P.; Lincoln, S. F.; Easton,
C. J. Org. Biomol. Chem. 2004, 2, 1381–1386.
9. (a) Wudl, F.; Gaeta, F. J. Chem. Soc., Chem. Commun. 1972,
107; (b) de Vries, E. F. J.; Steenwinkel, P.; Brusses, J.; Kruse,
C. G.; Van der Gen, A. J. Org. Chem. 1993, 58, 4315–4325;
(c) Zhu, X.-C.; Yan, H.; Chen, Y.-Y.; Wu, C.-Y.; Lu, X.-R.
J. Chromatogr., A 1996, 753, 269–277; (d) Joly, J.-P.;
4.4. Molecular modelling calculations
A coarse skeleton of the ether crown was initially prepared
using ChemDraw and Chem3D software packages19 and
the structure of the native b-CD (native b-CD) was taken
from the Cambridge Structural Database by means of
ConQuest 1.8.20 In order to build up the final ligand, two
CDs were attached through the urea groups to the ether
crown after removing one of their primary hydroxyl groups.
The molecular modelling calculations were carried out on
a Pentium-4 personal computer using the TINKER pro-
gramme and its implemented MM3 force field.21 The energy
minimisation MINIMIZE option of the TINKER pro-
gramme was first used to find a starting geometrical state
of the system. The respective conformational minima
were sought without constraints for the ligand in vacuum
(structure S1) and in water solvent (structure S2). Structure
S1 was minimised to a final RMS gradient equal to
0.0948 kJ Aꢀ1 molꢀ1 by a modified version of the algorithm
˚
of Jorge Nocedal based on a quasi-Newton method (596
cycles).22 No cut-off option was used in this case. The
structure S2 was obtained with the same method (final
€
Schroder, G. Tetrahedron Lett. 1997, 38, 8197–8198; (e) Joly,
J.-P.; Walker, O.; Mutzenhardt, P. Magn. Reson. Chem. 2001,
39, 212–214; (f) He, Y.; Xiao, Y.; Meng, L.; Zeng, Z.; Wu,
X.; Wu, C.-T. Tetrahedron Lett. 2002, 43, 6249–6253; (g)
Lobach, A. V.; Leus, O. N.; Titova, N. Y.; Luk’yanenko,
N. G. Russian J. Org. Chem. 2003, 39, 1037–1041; (h)
Correa, W. H.; Scott, J. L. Molecules 2004, 9, 513–519.
10. (a) Template Synthesis of Macrocyclic Compounds; Gerbeleu,
N. V., Arion, V. B., Burgess, J., Eds.; Wiley-VCH: Germany,
1999; (b) Menuel, S.; Joly, J.-P.; Marsura, A. Proceedings
of the 12th International Cyclodextrin Symposium; APGI:
Toulouse, France, 2004; pp 153–158; (c) Menuel, S.; Joly,
J.-P.; Marsura, A. E.S.F. Conference on Supramolecular
Chemistry, Obernay (Strasbourg), France, 2005.
RMS gradient equal to 0.0950 kJ Aꢀ1 molꢀ1 (1475
˚
3
˚
cycles)) considering a cubic box of 25ꢂ25ꢂ25 A contain-
ing 383 water molecules. In this case, the periodic boundary
˚
conditions were imposed with a cut-off radius of 9 A. A sto-
chastic annealing procedure (chosen temperature 1000 K,
time step 0.02 ps, 95 snapshots) applied to conformation
S2 gives very similar results showing the robustness of
the MINIMIZE method implemented in the TINKER
programme.
Acknowledgements
11. Porwanski, S.; Kryczka, B.; Marsura, A. Tetrahedron Lett.
2002, 43, 8441–8443.
Financial supports from the CNRS and the French ‘Minis-
ꢁ
tere de l’Education et de la Recherche’ are gratefully
acknowledged.
ꢀ
12. Charbonnier, F. Ph.D. thesis, University Henri Poincare, Nancy
1, France, 1999.
13. Galton, D. A. G. Lancet 1953, 1, 208–213.
14. Vassal, G.; Koscielny, S.; Challine, D.; Valteau-Couanet, D.;
Boland, I.; Deroussent, A.; Lemerle, J.; Gouyette, A.;
Hartmann, O. Cancer Chemother. Pharmacol. 1996, 37, 247–
253.
References and notes
ꢀ
15. Ghermani, N. E.; Spasojevic-de-Bire, A.; Bouhmaida, N.;
1. For a general overview, see: (a) Szjetli, J. Chem. Rev. 1998,
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Cyclodextrins, Scaffolds and Templates for Supramolecular
Chemistry; ICP: London, 1999; pp 1–226 and references cited
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11, 2649–2653.
3. Emert, J.; Breslow, R. J. Am. Chem. Soc. 1975, 97, 670–672.
Ouharzoune, S.; Bouligand, J.; Layre, A.; Gref, R.; Couvreur,
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5883; (b) See Ref. 6a; (c) Breslow, R.; Zhang, B. J. Am. Chem.
Soc. 1994, 116, 7893–7894.
17. Liu, Y.; Sung, Y.; Chen, Y.; Li, X.-Q.; Ding, F.; Zhong, R.-Q.
Chem.—Eur. J. 2004, 10, 3685–3696.

1714
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18. (a) Charbonnier, F.; Humbert, T.; Marsura, A. Tetrahedron Lett.
1998, 39, 3481–3484; (b) Charbonnier, F.; Marsura, A.;
Roussel, K.; Kovacs, J.; Pinter, I. Helv. Chim. Acta 2001, 84,
535–551.
20. Damodharan, L.; Pattabhi, V.; Nagarajan, K. Mol. Cryst. Liq.
Cryst. 2004, 423, 17–35.
21. Ponder, J. W. TINKER, Version 4.2, Software Tools for
Molecular design, June 2004.
22. Nocedal, J. Math. Program. 1989, 45, 503–528.
19. ChemDraw Ultra 5.0, CambridgeSoft.