Design, synthesis and biological evaluation of multivalent glucosides with high affinity as ligands for brain targeting liposomes

Article abstract of DOI:10.1016/j.ejmech.2013.10.007

The new bifunctional cluster glucosides were designed and synthesized as liposome ligands for preparing novel liposome to achieve the effective delivery of drug formulations to brain by GLUT1. Docetaxel-loaded five liposomes were prepared successfully and tested in the animals. Results from the in vivo distribution study after i.v. administration of these five liposomes and blank-docetaxel indicated that the coupled liposomes Lip-1, Lip-2, Lip-3, Lip-5 exhibited excellent transport ability across the BBB. In particular, they significantly increased the level of docetaxel in brain compared to blank-docetaxel and Lip. Among them, Lip-5 showed higher brain concentration. Both pharmacokinetics and distribution study in mice confirmed that this novel brain targeting drug delivery system was a promising carrier to enhance brain delivery capacity for CNS drugs.

Full text of DOI:10.1016/j.ejmech.2013.10.007

European Journal of Medicinal Chemistry 72 (2014) 110e118
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of multivalent glucosides
with high affinity as ligands for brain targeting liposomes
,
Boyi Qu a, Xiaocen Li a, Mei Guan b, Xun Li a, Li Hai a, Yong Wu a
*
a Key Laboratory of Drug Targeting of Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
b West China Hospital, Sichuan University, No. 37, GuoXue Road, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The new bifunctional cluster glucosides were designed and synthesized as liposome ligands for pre-
paring novel liposome to achieve the effective delivery of drug formulations to brain by GLUT1.
Docetaxel-loaded five liposomes were prepared successfully and tested in the animals. Results from the
in vivo distribution study after i.v. administration of these five liposomes and blank-docetaxel indicated
that the coupled liposomes Lip-1, Lip-2, Lip-3, Lip-5 exhibited excellent transport ability across the BBB.
In particular, they significantly increased the level of docetaxel in brain compared to blank-docetaxel and
Lip. Among them, Lip-5 showed higher brain concentration. Both pharmacokinetics and distribution
study in mice confirmed that this novel brain targeting drug delivery system was a promising carrier to
enhance brain delivery capacity for CNS drugs.
Received 21 July 2013
Received in revised form
30 September 2013
Accepted 4 October 2013
Available online 20 November 2013
Keywords:
Cholesterylated glucosides
Brain targeting
GLUT1 transporter
Synthesis
Ó 2013 Elsevier Masson SAS. All rights reserved.
Liposome
1. Introduction
In order to facilitate the delivery of drugs to the brain and
achieve active targeting, many CNS delivery techniques have been
In modern society, central nervous systems diseases, such as
Parkinson’s disease, brain tumor and other neurodegenerative
disorders, have become one of the most dangerous threats to hu-
man health, due to the dramatic increase of their incidence rates,
especially in younger people, and their low recovery rates. So, the
treatment for CNS diseases has become a pressing problem that
needs to be solved.
The greatest obstacle of drugs delivery into brain is the presence
of bloodebrain barrier (BBB) between blood circulation system and
central nervous system [1]. The BBB functions as not only a physical
barrier but also a biochemical barrier which plays an important role
in its further protecting action towards the brain microenviron-
ment [2]. Moreover, the BBB is often the rate-limiting factor in
determining the permeation of therapeutic drugs into the brain [3],
because the BBB is formed by brain vessel endothelial cells linked
together by tight junctions [4]. Therefore, the rate of drugs crossing
BBB is relevant to its physico-chemical characteristics such as
molecular size, lipid solubility, charge, hydrogen bonding, and
ionization profile [5].
adopted. Because of the high transport affinity and capacity be-
tween the substance being transported and components of the
membrane, carrier-mediated transporter (CMT) system seems to be
one of the most promising methods. The CMT system usually ex-
presses on both the luminal and abluminal membranes of the brain
capillary endothelium [6,7]. Among all the physiological transport
systems for nutrients and endogenous compounds in CMT, glucose
transporter 1 (GLUT1) expressed on the surface of brain capillary
endothelial cells is considered as one of the most efficient transport
systems [8,9]. GLUT1 can transport glucose, an active small mole-
cule and an essential energy resource for brain function sustenance
across the BBB into brain effectively. This unique character also
makes itself a potential transporter of brain-targeting drugs [10e
12]. To date, several different glycoconjugates serving as candi-
dates for the treatment of CNS diseases have been reported, which
could increase the permeability of BBB by carrier mediated trans-
port GLUT1, such as dopamine [13,14] and 7-chlorokynurenic acid
[15,16]. Recently, structureeactivity relationship studies show that
analogs with substituents at the C-6 position of glucose reflect a
stronger affinity to GLUT1 than those substituted at other positions
[17]. Consequently, based on the above studies, glucose could be an
effective means to improve drugs permeability through the BBB.
However, preparation of the macromolecular drugs and ionic drugs
* Corresponding author. Tel./fax: þ86 2885503666.
E-mail addresses: wyong@scu.edu.cn, pro_wuy_2002@sohu.com (Y. Wu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.007

B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
111
into prodrugs is not ideal, so we should pay more efforts to solve
these drawbacks.
Herein these four designed ligands were described in detail. The
objective of our study was to propose a novel brain targeting de-
livery system mediated by GLUT1. Docetaxel, as a unique anticancer
active drug, was used to model drug, which was stable and PH-
independent. Furthermore, the brain biodistribution and pharma-
cokinetics of these liposomes in mice after i.v administration were
discussed.
As the drug carrier, liposome has many advantages such as non-
toxicity, biocompatibility and biodegradability [18]. The modified
liposomes can carry hydrophilic, lipophilic and also amphoteric
drug molecules to the diseased tissues or target organs [3,19,20].
Thus, the novel liposomal technology for brain targeting is
attracting more and more attention of researchers. The modified
liposome, in which the multiple surfaces and lipid membrane
bilayer are prone to the connection of the monoclonal antibodies or
functional ligands, showed efficient drug delivery ability [21].
Therefore, this strategy could be utilized in the design of glucosyl
liposome ligands, which are able to cross BBB via GLUT1 as drug
carriers in targeting delivery system.
2. Chemistry
The synthetic route of di-antennary glucosides ligand 24 and the
tri-antennary glucosides ligand 25 has already been reported in the
previous article [24]. The synthetic route of 24 and 25 outlined in
Schemes 1 and 2.
Ligand contains glucose molecule could mediate liposomes
through the BBB. Compared to the immune liposomes, which have
good targeting properties but their use is restricted because of their
own immunogenicity, expensive preparation process and complex
operation extremely. Glucosyl modified liposomes showed poten-
tial application with brain targeting, high transfer efficiency, good
in vivo cycling stability and easy preparation. Based on the previ-
ously mentioned ideas, we designed a series of ligands 23, 24, 25
and 26 with ester linkage between glucose and cholesterol de-
rivatives (Fig. 1). Using post-insertion technique [22], the lipophilic
steroidal portion was embedded into the liposome membrane,
while exposing hydrophilic glucose residue outside the membrane
for recognizing GLUT1 and connected with the ethylene glycols,
which reduced the non-specific binding of proteins and other
bioactive molecules [23]. Simultaneously, we assumed that the
more the quantity of glucose residues exposed to the liposome
surface, the stronger the affinity for GLUT1 showed.
Based on the assumption above, we focused on the quin-
antennary glucosides ligand 26 with five glucose residues and
one steroid unite, which linked glucose residue via ethylene glycols
at C-6 position of glucose [17].This ligand, briefly, still maintained
its two functional properties: the lipophilic steroid inset into lipid
bilayer, while the five glucose residues were exposed on the surface
of liposome for recognizing by GLUT1 and then transported across
the BBB to fulfill the blood-to-brain delivery. In order to observe the
effect of glucose residues exposed to the liposome surface, we also
synthesized the single-antennary glucoside ligand 23, the reported
di-antennary glucosides ligand 24 and tri-antennary glucosides 25
ligand [24] as the reference.
In order to successfully complete our synthetic work, glucosy-
lated derivatives have been first synthesized as previously reported
[24]. The synthetic route of glucosylated derivative 3 was demon-
strated in Scheme 3. The hydroxyl of -glucose 1 was full protected,
D
then selectively deprotected at C-6 position to get reported com-
pound 2 [25].Finally compound 2 coupled with succinic anhydride
to give acid 3 by using DMAP as catalyst.
Next, the synthetic pathway of single-antennary glucoside
ligand 23 has been display in Scheme 4. The reaction of available 6
and glucosylated derivative 3 to achieve ester 16 used DCC as
dehydrating agent for ester condensation. Then, debenzylation of
ester 16 reacted under a mild hydrogen reduction condition with
10%Pd/C in a mixture of MeOH and CH2Cl2 to afford the desired
product 23.
The synthetic route used to prepared the quin-antennary glu-
cosides ligand 26 has been illustrated in Scheme 5. The inositol 17
was chosen as the six-branch skeleton, under the conditions of 40%
KOH, coupled with redistilled acrylonitrile to produce hexacyano
compound 18 at a suitable temperature. The temperature should be
controlled below 20 ꢀC to reduce side reactions and increase yield.
Hexacyano 18 was converted with H2SO4 and EtOH to get hexaester
compound 19 in reflux, which was reduced by treating with LiAlH4
using normal operation. The resulting hexol 20 in THF was coupled
with cholesteryl carboxylate derivatives 12 at the same mole by
dealing with condensing agent to give pentaol 21, conventional acid
as a catalyst used in this reaction is not ideal. For the purpose of the
key intermediate 22, large excess moles of glucosylated derivatives
3 were added in the reaction to ensure a good yield in the presence
of DCC as dehydrating agent. Finally, debenzylation of 22 with 10%
Fig. 1. The structure of multivalent glucosides ligand 23, 24, 25, 26.

112
B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
Scheme 1. Reagents and conditions: (a) TsCl, pyridine, 50 ꢀC, 8 h; (b) HOCH2 (CH2OCH2)2CH2OH, dioxane, reflux, 8 h; (c) TsCl, triethylamine, THF, rt, 36 h; (d) 2-phenyl-1,3-dioxan-
5-ol 8, NaH, THF, reflux, 8 h; (e) TsOH,CH3OH, rt, overnight; (f) glucosylated derivatives 3 DCC, DMAP, rt, CH2Cl2, 20 h; (g) H2, 10%Pd/C, CH2Cl2, CH3OH, 12 h.
Pd/C in THF and MeOH gave the last desired compound 26. All the
title compounds and important intermedium were characterized
by their respective IR, 1H NMR, 13C NMR and MS.
3. Results and discussion
3.1. Preparation of liposomes
Scheme 3. Reagents and conditions: (a) i. NaH, BnBr, DMF, rt, 24 h; ii. HOAceAc2O
(5:1), ZnCl2, 90 min; iii. NaOMeeMeOH, 5 h (b) succinic anhydride, DMAP, CH2Cl2, rt,
overnight.
Glucosides-modified liposomes (Lip-1, Lip-2, Lip-3, Lip-5) and
unmodified liposome Lip loaded with docetaxel were prepared by
the lipid film hydration-ultrasound method with various modified
PBS rehydration solution (PH ¼ 6.5, 0.02 M) for 90 min at 37 ꢀC with
constant mixing. Followed, it was further intermittently sonicated
by a probe sonicator at 200 W for 100 s. The final concentration of
docetaxel was 0.2 mg/mL.
ligands (23, 24, 25, 26) or without ligand. Briefly, lipid materials
(phospholipid and cholesterol in a molar ratio of 2:1) and ligand,
docetaxel were dissolved in chloroform, and then chloroform was
removed by vacuum evaporation and stored in vacuo overnight to
remove the solvent completely. The lipid film was then incubated in
Conventional liposomes were evaluated as the control in this
study. Liposome suspensions were diluted with PBS rehydration
Scheme 2. Reagents and conditions: (a) TsCl, pyridine, 50 ꢀC, 8 h; (b) i. HOCH2 (CH2OCH2)2CH2OH, dioxane, reflux, 8 h; ii. t-butyl bromoacetate, NaH, THF, reflux, 8 h; iii. TsOH,
toluene, reflux, 2 h; (c) pentaerythritol derivatives 13, DCC, DMAP, rt, CH2Cl2, overnight; (d) glucosylated derivatives 3, DCC, DMAP, rt, CH2Cl2, 20 h; (e) H2, 10%Pd/C, THF, CH3OH,
14 h.

B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
113
Scheme 4. Reagents and conditions: (a) i. DCC, DMAP, rt, CH2Cl2, overnight; (b) H2, 10%Pd/C, THF, CH3OH, 10 h.
Scheme 5. Reagents and conditions: (a) acrylonitrile, 40%KOH, 10 ꢀC, overnight; (b) H2SO4, EtOH, reflux, 30 h; (c) LiAlH4, anhydrous THF, 6 h; (d) cholesteryl carboxylate derivatives
12, DCC, DMAP, rt, CH2Cl2, 20 h; (e) glucosylated derivatives 3, DCC, DMAP, rt, CH2Cl2, 20 h; (f) H2, 10%Pd/C, THF, CH3OH, 14 h.
solution, and the average diameters, polydispersity indices (PDI)
and zeta-potentials of Lip, Lip-1, Lip-2, Lip-3, Lip-5 were
determined.
approximately). A good baseline separation of docetaxel, diazepam,
and other major components from mouse plasma and brain tissue
samples was achieved under this chromatographic condition.
3.2. Characterization of modified liposomes of different ligands
3.3.2. Pharmacokinetics in plasma and brain of each liposome
For in vivo study, Lip, Lip-1, Lip-2, Lip-3, Lip-5 and docetaxel
original drug were injected through caudal vein of the mice with a
single dose equivalent to 5 mg/kg body weight of docetaxel. At
30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h after injection, blood sample
was collected from the eye socket of mice, and placed in heparin
tubes. Then blood and brain were collected to analyze the con-
centration of docetaxel at different intervals by HPLC method.
In order to demonstrate the in vivo behavior of the different
liposomes, the plasma pharmacokinetics of docetaxel and various
liposomes were assessed (Fig. 2). Pharmacokinetics parameters of
docetaxel in mice were reported in Table 2 after i.v. administration
of each liposome (Lip, Lip-1, Lip-2, Lip-3, Lip-5) and docetaxel.
The result showed that the area under the concentrationetime
profile (AUC0et) of docetaxel in the five types of liposomes was
much larger than that of naked docetaxel within 24 h. Free doce-
taxel of liposomes Lip, Lip-1, Lip-2, Lip-3, Lip-5 and docetaxel
presented with an area ratio of 1.14, 1.85, 2.24, 2.72 and 2.53, and
the maximum concentration for free docetaxel was 1.12, 2.52, 3.92,
4.71 and 5.42 times that of the five types of docetaxel liposomes.
These data indicated that the liposomes showed certain stability
which increased the chance to be transported across BBB. The mean
residence times (MRT) of docetaxel in plasma after i.v. adminis-
tration of Lip, Lip-1, Lip-2, Lip-3, Lip-5 were 1.12, 1.04,1.07,1.04 and
1.01 times that of docetaxel, respectively, the MRT was the same as
the blank-docetaxel or a slight increase. The half lives (t1/2) and
MRT of docetaxel by these liposomes indicated that the liposomes
can prolong the circulation time of docetaxel, by delaying its
For the five types of liposomes, encapsulation efficiencies of
docetaxel were all greater than or nearly 80%, respectively. Briefly,
the lipid materials (Soybean phospholipids and cholesterol) and
ligand were in a molar ratio of 8:4:1. The average particle sizes of all
liposomes were close to or less than 120 nm, with the value of PDI
less than 0.3, fully complied with the conditions. The composition
and characterization of all liposomes were shown in Table 1.
3.3. In vivo studies
3.3.1. Methodology evaluation
The chromatograms of blank mouse plasma and brain tissues
showed no peaks at the retention time of docetaxel (12 min
approximately) and the internal standard diazepam (8 min
Table 1
The composition and characterization of different docetaxel-loaded liposomes
(n ¼ 3).
Molar ratio Size (nm)
of lipids
PDI
Encapsulating
efficiency (EE, %)
Zeta (mv)
and ligand
Lip
2:1:0
121.6 ꢁ 5.6 0.219 ꢁ 0.024 93.54 ꢁ 2.41
118.3 ꢁ 8.4 0.264 ꢁ 0.019 84.79 ꢁ 3.46
108.4 ꢁ 6.1 0.245 ꢁ 0.022 82.83 ꢁ 5.37
105.5 ꢁ 7.4 0.232 ꢁ 0.027 80.21 ꢁ 7.36
115.1 ꢁ 8.8 0.277 ꢁ 0.034 78.89 ꢁ 9.45
ꢂ64.1 ꢁ 9.2
ꢂ44.8 ꢁ 1.6
ꢂ32.6 ꢁ 3.5
ꢂ29.5 ꢁ 2.2
ꢂ27.4 ꢁ 3.6
Lip-1 8:4:1
Lip-2 8:4:1
Lip-3 8:4:1
Lip-5 8:4:1

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B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
2 > Lip-1 > Lip. The results indicated that Lip also delivered
docetaxel across the BBB, but to a significantly lower extent than
other liposomes, which may be the reason that Lip was not the
ligand-modified liposome, but rather rely on the nature of the
liposome itself. The higher REs and CEs of modified liposomes
demonstrated that exposing glucose residues outside the mem-
brane of coupled liposomes could recognize GLUT1. Moreover, the
more the quantity of glucose residues exposed to the liposome
surface, the stronger the affinity for GLUT1, which means the
stronger ability to transport docetaxel into CNS. Therefore, the Lip-
5 showed the best affinity. Details above indicated that more
docetaxel were distributed into brain.
4. Conclusion
For developing a brain-specific drug liposomal carrier, in the
present work, we designed and successfully synthesized a serial of
multivalent glucosides with high affinity as ligands for brain tar-
geting liposomes. According to the in vivo study, after i.v. admin-
istration of the coupled liposomes Lip-1, Lip-2, Lip-3, Lip-5, the
results showed that the concentrations of docetaxel in brain at
different time points were significantly higher than that of
uncoupled Lip and naked-docetaxel with the same dose. The
coupled liposomes exhibited excellent transport ability across the
BBB, among which the biodistribution data had indicated higher
brain concentration and AUC0et of Lip-5 than those of other three
ones. Lip-5 seemed to be easier to be transported into CNS, which
was attributed to the larger quantity of glucose residues. So, Lip-5
Fig. 2. The concentration curve of docetaxel in plasma versus time after i.v. injection of
Lip, Lip-1, Lip-2, Lip-3, Lip-5 and Docetaxel in mice. Data represent the mean ꢁ SD
(n ¼ 3).
metabolic rate in vivo. So it is obvious that all the liposomes can
keep the certain concentration of docetaxel in plasma, which might
increase the chance for the drug to be delivered across BBB.
3.3.3. Distribution in brain of different liposomes in mice
For further evaluation of the possibility of the glucose-mediated
liposome’s being transported across BBB, the distributions in brain
of Lip, Lip-1, Lip-2, Lip-3, Lip-5 and docetaxel were determined.
The distribution of docetaxel was measured at 30 min, 1 h, 2 h, 4 h,
8 h, 16 h, and 24 h after i.v. injection. The concentrations of doce-
taxel in brain versus time curves were displayed in Fig. 3. After i.v.
administration of Lip, Lip-1, Lip-2, Lip-3, Lip-5 and docetaxel, the
pharmacokinetic parameters, RE and CE, of docetaxel in brain were
reported in Table 3.
was
a potent brain targeting liposome ligand which could
enhance liposome’s ability of delivering drug into brain.
Generally, this work presented a way that the synthesis of
liposome ligand may be used as potential means to improve the
therapeutic efficiency in brain diseases.
In brain, it is obvious that all of the liposomes could be delivered
to the brain following i.v. administration. At different time interval,
the concentration of docetaxel released from coupled liposomes
was much higher than that from uncoupled liposomes and blank-
docetaxel during 24 h. The AUC0et and Cmax of docetaxel in brain
after i.v. administration of liposomes were fairly higher than that
after the injection of blank-docetaxel, and it is worth noting that it
showed an increasing trend with the increase of the number of
glucose residues. The relative uptake efficiencies (REs) were
enhanced to 1.80, 2.63, 4.53, 5.70 and 6.09 times that of naked
docetaxel for liposomes Lip, Lip-1, Lip-2, Lip-3, Lip-5 respectively.
The concentration efficiencies (CEs) were also enhanced to 1.94,
4.11, 5.32, 8.19 and 9.38 times that of docetaxel. So these data
further proved our conjecture that all the ligand-modified lipo-
somes can deliver docetaxel across the BBB, and the pharmacoki-
netic parameters suggested that the brain targeted abilities of these
designed liposomes had a descending trend as Lip-5 > Lip-3 > Lip-
5. Experimental protocols
5.1. Chemistry
All liquid reagents were distilled before use. All unspecified
reagents were from commercial resources. TLC was performed
using precoated silica gel GF254 (0.2 mm), while column chroma-
tography was performed using silica gel (100e200 mesh). The
melting point was measured on a YRT-3 melting point apparatus
(Shantou Keyi instrument & Equipment Co. Ltd, Shantou, China). IR
spectra were obtained on a Perkin Elmer983 (Perkin Elmer, Nor-
walk, CT, USA). Elemental analyses were performed by Atlantic
Microlab (Atlanta, GA, USA). 1H NMR spectra were taken on a Varian
INOVA400 (Varian, Palo Alto, CA, USA) using CDCl3, d-DMSO and
D2O as solvent. Chemical shifts are expressed in
tetramethylsilane (TMS) functioning as the internal reference, and
coupling constants (J) were expressed in Hz. Mass spectra were
d (ppm), with
Table 2
Pharmacokinetic parameters of docetaxel in blood after administration of docetaxel and liposomes (n ¼ 3).
Compounds
AUC(0et) (ng/ml h)
MRT(h)
T1/2 (h)
Tmax (h)
Cmax (ng/ml)
Docetaxel
Lip
Lip-1
Lip-2
Lip-3
Lip-5
3800.578 ꢁ 173.40
4320.478 ꢁ 93.19
9.664 ꢁ 0.17
10.862 ꢁ 0.44*
10.043 ꢁ 0.70
10.310 ꢁ 0.11
10.076 ꢁ 0.21
9.751 ꢁ 0.28
16.24
4
208.633 ꢁ 13.33
20.134
19.891
30.166
26.347
35.987
3.333
233.133 ꢁ 22.18***
526.725 ꢁ 57.54***
817.575 ꢁ 78.33***
983.175 ꢁ 56.57***
1129.95 ꢁ 93.40***
7044.894 ꢁ 68.78**
8509.774 ꢁ 502.60**
10322.43 ꢁ 555.59**
9625.14 ꢁ 625.06**
1
1
1
1
*P < 0.05 versus Docetaxel.
**P < 0.01 versus Docetaxel.
***P < 0.001 versus Docetaxel.

B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
115
The reaction mixture was stirred overnight at room temperature.
The solution was then concentrated under vacuum, the resulting
syrup was purified by the column chromatography with petroleum
ethereacetone as eluent to get a colorless semi-solid. (65%) 1H NMR
(400 MHz, CDCl3,
d
ppm):7.27e7.39 (m, 20H þ CHCl3, AreH), 4.86
(d, 2H, CH2Ar, J ¼ 10.8 Hz), 4.79 (d, 2H, CH2Ar, J ¼ 10.8 Hz), 4.72 (d,
2H, CH2Ar, J ¼ 10.8 Hz), 4.57 (d, 2H, CH2Ar, J ¼ 10.8 Hz), 4.52 (d, 1H,
H-1, J ¼ 7.6 Hz), 4.37e4.40 (m, 1H, H-6), 4.29e4.34 (m, 1H, H-2),
4.22e4.26 (m, 2H, H-60, H-5), 3.54e3.57 (m, 2H, H-4, H-3), 2.63e
2.65 (m, 4H, COCH2CH2CO) MS (m/z): 663.3 ([M þ Na]þ); [
(c ¼ 1.0, CHCl3).
a
]
20 þ35ꢀ
D
5.1.4. Synthesis of glucose-cholesterol derivatives (16)
A
solution of compound 3 (1.57 g, 2.46 mmol) in THF,
Fig. 3. The concentration curve of docetaxel in brain versus time after i.v. injection of
Lip, Lip-1, Lip-2, Lip-3, Lip-5 and Docetaxel in mice. Data represent the mean ꢁ SD
(n ¼ 3).
DCC(610.5 mg, 2.95 mmol), and catalytic amount of DMAP were
added in reaction bottle. The reaction was activated for half an hour,
followed by the addition of alcohol 6 (1.27 g, 2.46 mmol) in THF. The
solution was stirred at room temperature overnight, then the white
precipitate was filtered off and THF was evaporated under reduced
pressure, the crude oil was purified on a silica-gel chromatography
recorded on an Agilent 1946B ESI-MS instrument (Agilent, Palo
Alto, CA, USA). Docetaxel and diazepam were obtained from Na-
tional Institute for Food and Drug Control. Soybean phospholipids
(SPC) were purchased from Kelong Chemical. Cholesterol (CHOLE)
was purchased from Bio Life Science& Technology Co., Ltd
(Shanghai, People’s Republic of China).
column to yield colorless oil 16. (70%) IR (KBr):
n
3025, 2974, 2887,
ppm):
1748, 1592, 1242, 1112 cmꢂ1 1H NMR (400 MHz, CDCl3,
d
7.27e7.39 (m, 20H þ CHCl3, AreH), 5.34 (m, 1H, chol H-6), 4.86 (d,
2H, CH2Ar, J ¼ 10.8 Hz), 4.79 (d, 2H, CH2Ar, J ¼ 10.8 Hz), 4.72 (d, 2H,
CH2Ar, J ¼ 10.8 Hz), 4.57 (d, 2H, CH2Ar, J ¼ 10.8 Hz), 4.52 (d, 1H, H-1,
J ¼ 7.6 Hz), 4.37e4.40 (m, 1H, H-6), 4.29e4.34 (m, 1H, H-2), 4.22e
4.26 (m, 2H, H-60, H-5), 3.62e3.68 (m, 12H, OCH2CH2O), 3.54e3.57
(m, 2H, H-4, H-3), 3.18 (m, 1H, chol H-3), 2.63e2.65 (m, 4H,
COCH2CH2CO), 2.37e0.66 (remaining chol protons) with 0.67 (s,
3H, CH3-18), 0.86 (d, 6H, J ¼ 6.8 Hz, CH3-26, CH3-27), 0.91 (d, 3H,
J ¼ 6.8 Hz, CH3-21), 0.99 (s, 3H, CH3-19); MS (m/z): 1163.6
([M þ Na]þ). Anal. Calcd for C71H96O12 C, 74.70; H, 8.48. Found: C,
74.66; H, 8.52.
5.1.1. Synthesis of di-antennary glucosides compound 24 [24]
The known cholesteryl tosylate 5 [26] and tosyl chloride was
refluxed for 8 h with triethylene glycol in dry dioxane to get alcohol
6 [27]. Alcohol 6 was converted with tosyl chloride to give tosylate
7 in triethylamine and THF. Tosylate 7 reacted with 2-phenyl-1,3-
dioxan-5-ol 8, which was prepared by the reaction of glycerol
with benzaldehyde according to the literature procedure [28], in
the presence of NaH in refluxing THF to afford the 1,3-dioxane
compound 9. Then, under the conditions of TsOH, compound 9
was deprotected to achieve 1,3-diol compound 10. Condensation of
intermediate 10 and glucosylated derivative 3 in the presence of
DCC as dehydrating agent gave diester 11. Finally, debenzylation of
diester 11 was achieved by catalyzed with 10%Pd/C under hydrogen
in MeOH and CH2Cl2 to afford the desired product 24.
5.1.5. Synthesis of single-antennary glucosides compound 23
The prepared compound 16 (1.8 g, 1.58 mmol) was dissolve in
the mixture of methanol and THF, then 10%Pd over activated carbon
(180 mg) was added. The reaction was agitated at room tempera-
ture under H2 for 10 h, the reaction was filtered and the filtrate was
concentrated under vacuum. The residue was purified on a silica-
gel chromatography column, using dichloromethaneemethanol
5.1.2. Synthesis of tri-antennary glucosides compound 25 [24]
The cholesteryl carboxylate derivative 12 was prepared from
cholesterol 4 via 3 steps as described in the literature [27]. Tetrol 13
[29] was coupled with cholesteryl carboxylate derivative 12 by
dealing with DCC/DMAP in CH2Cl2 to get triol 14. Similarly, as the
same method to achieve 24 from 10, we readily got the second
desired product 25 from triol 14.
as eluent, to get desired ligand 23 as semi-solid. (76%) IR (KBr):
n
3476, 2960, 2892, 1746, 1262, 1086 cmꢂ1 1H NMR (400 MHz, DMSO,
d
ppm): 4.54 (d, 1H, H-1, J ¼ 6.4 Hz), 4.32e4.30 (m, 1H, H-6), 4.29e
4.34 (m, 1H, H-2), 4.27e4.28 (m, 2H, H-60, H-5), 3.72e3.78 (m, 12H,
OCH2CH2O), 3.64e3.67 (m, 2H, H-4, H-3), 2.93 (m, 1H, chol H-3),
2.55e2.56 (m, 4H, COCH2CH2CO), 2.37e0.66 (remaining chol pro-
tons) with 0.62 (s, 3H, CH3-18), 0.83 (d, 6H, J ¼ 6.8 Hz, CH3-26, CH3-
27), 0.89 (d, 3H, J ¼ 6.8 Hz, CH3-21), 0.97 (s, 3H, CH3-19); 13C NMR
(CD3OD-d6, ppm): 173.2 (1C, COCH2), 171.6 (1C, COCH2), 93.4 (1C, C-
1), 82.3 (1C, chol C-3), 75.2 (1C), 74.2 (1C), 72.3 (1C), 71.4 (1C), 70.8
(1C), 70.13 (1C), 69.5 (1C), 68.4 (1C), 67.2 (1C), 64.5 (1C), 63.4 (1C, C-
5.1.3. Synthesis of glucosylated derivatives (3)
The prepared known compound 2 [25] (2.56 g, 4.75 mmol) was
dissolved in CH2Cl2 (50 ml), followed by adding catalytic amount of
DMAP and Succinic Anhydride (0.523 g, 5.23 mmol) to the system.
Table 3
Pharmacokinetic parameters of docetaxel in brain after administration of docetaxel and liposomes (n ¼ 3).
Compounds
AUC(0et) (ng/g h)
MRT (h)
T1/2 (h)
Tmax (h)
Cmax (ng/g)
171.9 ꢁ 10.27
Re
CE
Docetaxel
Lip
Lip-1
Lip-2
Lip-3
Lip-5
3365.934 ꢁ 125.15
6045.388 ꢁ 358.51*
8839.033 ꢁ 322.14**
15260.34 ꢁ 868.95**
19180.276 ꢁ 1288.42**
20526.546 ꢁ 1020.61***
11.437 ꢁ 0.15
11.026 ꢁ 0.39
10.321 ꢁ 0.47
10.939 ꢁ 0.68
9.914 ꢁ 0.37
10.226 ꢁ 0.50
98.108
35.675
29.219
23.123
18.407
24.439
4
e
e
1.667
334.369 ꢁ 29.48*
707.696 ꢁ 162.14
914.424 ꢁ 129.23*
1408.03 ꢁ 153.05**
1613.023 ꢁ 163.79*
1.79605
2.62603
4.53376
5.69835
6.09832
1.94514
4.11691
5.31951
8.19098
9.3835
1
1
1
1
*P < 0.05 versus Docetaxel.
**P < 0.01 versus Docetaxel.
***P < 0.001 versus Docetaxel.

116
B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
6), 29.8 (1C, CH2CO), 29.5 (1C, CH2CO), 12.7e56.4 (remaining chol
protons) 56.4 (1C), 55.8 (1C), 55.2 (1C), 42.5 (1C), 41.7 (1C), 40.3
(1C), 38.8 (1C), 37.9 (1C), 37.3 (1C), 37.1 (1C), 35.3 (1C), 33.2 (1C),
33.1 (1C), 30.2 (1C), 28.4 (1C), 27.9 (1C), 25.3 (1C), 25.1 (1C), 23.1
(1C), 22.6 (1C), 22.3 (1C), 21.2 (1C),19.8 (1C),13.3 (1C), 12.8 (1C); MS
(m/z): 802.5 ([M þ Na]þ). Anal. Calcd for C43H74O12 C, 65.96; H, 9.52.
Found: C, 65.92; H, 9.57.
0.89 (d, 3H, J ¼ 6.8 Hz, CH3-21), 0.97 (s, 3H, CH3-19). MS (m/z):
1109.74 ([M þ Na]þ).
5.1.10. Synthesis of key intermediate compound 22
To a solution of compound 3 (4.7 g, 7.36 mmol) in CH2Cl2, DCC
(1.83 g, 8.83 mmol) and catalytic amount of DMAP were added. The
reaction mixture was stirring for half an hour, followed by addition
of the solution Pentaol 21 (1.0 g, 0.92 mmol) in CH2Cl2 dropwise.
Then the reaction mixture was kept stirring at room temperature
for 20 h. The generated white precipitate was filtered off and CH2Cl2
was evaporated under reduced pressure to get the crude oil, which
was further purified on a silica-gel chromatography column to give
colorless oil. (65%) IR (KBr, cmꢂ1): nmax 3061, 2970, 2885, 1741, 1594,
5.1.6. Synthesis of hexacyano compound 18
The inositol 17 (1.8 g, 0.01 mol) was dissolved in 1.2 ml 40%KOH
solution, then redistilled acrylonitrile was added dropwise in an ice
bath. After the dropwise addition, the reaction was controlled the
temperature at 10 ꢀC, overnight. TLC monitoring of the reaction is
completed, adding dilute HCl adjust the reaction to neutral. Then,
the resultant was extracted with DCM (30 ml) and the organic layer
was dried over anhydrous Na2SO4, and concentrated under reduced
pressure. Purification of the crude product by recrystallization form
MeOH to reach white solid. (83%) Mp: 124e126 ꢀC. 1H NMR
1262, 1115; 1H NMR (400 MHz, CDCl3, ppm):
d 7.27e7.42 (m,
80H þ CHCl3, AreH), 5.30 (m, 1H, chol H-6), 4.97 (d, 5H, CH2Ar,
J ¼ 10.8 Hz), 4.94 (d, 5H, CH2Ar, J ¼ 10.8 Hz), 4.87 (d, 5H, CH2Ar,
J ¼ 10.8 Hz), 4.78 (d, 5H, CH2Ar, J ¼ 10.8 Hz), 4.72 (d, 5H, CH2Ar,
J ¼ 10.8 Hz), 4.67 (d, 5H, CH2Ar, J ¼ 10.6 Hz), 4.64 (d, 5H, CH2Ar,
J ¼ 10.8 Hz), 4.58 (d, 5H, CH2Ar, J ¼ 10.6 Hz), 4.52 (d, 5H, H-1,
J ¼ 7.6 Hz), 4.26e4.31 (m, 5H, H-6), 4.14e4.22 (m, 10H, H-2, H-5),
4.13 (m, 2H, CH2OCO), 4.11e4.12 (m, 2H, OCOCH2O), 3.67e3.75 (m,
12H, OCH2CH2O), 3.62e3.65 (m, 18H, CHOCH2), 3.58e3.62 (m, 10H,
CH2OCO), 3.45e3.57 (m, 15H, H-3, H-4, H-60), 3.17 (m, 1H, chol H-3),
2.59e2.62 (m, 20H, COCH2CH2CO), 1.84e1.86 (m, 12H, CH2), 2.37e
0.66 (remaining chol protons) with 0.68 (s, 3H, CH3-18), 0.88 (d, 6H,
J ¼ 6.8 Hz, CH3-26, CH3-27), 0.91 (d, 3H, J ¼ 6.8 Hz, CH3-21), 1.01 (s,
3H, CH3-19); MS (m/z): 4221.1 [M þ Na]þ; Anal. Calcd for
(400 MHz, CDCl3,
d ppm): 2.64 (m, 12H, CH2CN), 3.2e3.7 (m, 6H,
CHO), 4.02 (m, 12H, CH2O). MS (m/z): 490.22 ([M þ 1]þ).
5.1.7. Synthesis of hexaester compound 19
In an ice bath, H2SO4 (5 ml) was adding dropwise into ethanol
(15 ml), the mixture was stirred enough. Then in the solution of
H2SO4eEtOH was added the hexacyano compound 18, the reaction
was heated to 90 ꢀC and maintained for 30 h. The ethanol was
removed under vacuum, then the residue was partitioned between
ethyl acetate and water (1:1, v/v). The aqueous layer was the
extracted with ethyl acetate, the combined organic layer was
washed by 5%Na2CO3, then dried with anhydrous Na2SO4, and
concentrated. The residue was purified on a silica gel chromatog-
raphy column, using petroleum ethereacetone as eluent, to get
compound 19 as colorless oil. (65%) 1H NMR (400 MHz, CDCl3,
C249H296O57: C, 71.19, H, 7.12; Found: C, 71.24, H, 7.15.
5.1.11. Synthesis of the quin-antennary glucosides compound 26
The prepared compound 22 (2.7 g, 0.968 mmol) was dissolve in
the mixture of methanol and THF Then 10%Pd over activated carbon
(270 mg) was added. The reaction was agitated under H2 for 14 h,
the reaction was filtered and the filtrate was evaporated under
vacuum. The residue was purified on a silica gel chromatography
column, using dichloromethaneemethanol as eluent, to get desired
d
ppm): 1.24e1.26 (m, 18H, COOCH2CH3), 2.54e2.56 (m, 12H,
CH2CO), 3.42e3.92 (m, C 6H þ 12H, HOCH2), 4.12 (m,12H, COOCH2).
MS (m/z): 781.42 ([M þ 1]þ).
ligand 26 as semi-solid. (70%) 1H NMR (400 MHz, DMSO,
nmax 3454, 2973, 2896, 1752, 1251, 1098; 1H NMR (400 MHz, CDCl3,
ppm):
4.56 (d, 5H, H-1, J ¼ 7.2 Hz), 4.26e4.31 (m, 5H, H-6), 4.30e
d ppm):
5.1.8. Synthesis of hexol compound 20
To a suspensions of LiAlH4 (0.414 g, 10.89 mmol) in dry THF at
0 ꢀC, the prepared Hexaester in THF (1.0 g, 1.281 mmol) was added
dropwise into the reaction. The reaction mixture was stirred at
25 ꢀC for 20 h. Then, in ice bath the LiAlH4 was destroyed by
sequentially added H2O, 15%NaOH, H2O(1:1:3) and stirred for
30 min, the reaction was filtered and the filtrate was evaporated
under vacuum. The residue was purified on a silica gel chroma-
tography column, using dichloromethaneemethanol as eluent, to
get compound 20 as colorless oil. (73%) 1H NMR (400 MHz, DMSO,
d
4.32 (m, 5H, H-6), 4.26e4.28 (m, 5H, H-2), 4.24 (m, 2H, CH2OCO),
4.14e4.16 (m, 2H, OCOCH2O), 4.07e4.09 (m, 5H, H-5), 3.67e3.79
(m, 12H, OCH2CH2O), 3.63e3.67 (m, 18H, CHOCH2), 3.53e3.58 (m,
10H, CH2OCO), 3.42e3.57 (m, 15H, H-3, H-4, H-60), 3.07 (m, 1H, chol
H-3), 2.52e2.54 (m, 20H, COCH2CH2CO), 1.78e1.81 (m, 12H, CH2),
2.37e0.62 (remaining chol protons) with 0.62 (s, 3H, CH3-18), 0.84
(d, 6H, J ¼ 6.8 Hz, CH3-26, CH3-27), 0.88 (d, 3H, J ¼ 6.8 Hz, CH3-21),
0.94 (s, 3H, CH3-19); 13C NMR (CD3OD-d6, ppm): 175.2 (5C, COCH2),
174.7 (5C, COCH2), 172.7 (1C, OCOCH2), 95.1 (5C, C-1), 82.8 (1C, chol
C-3), 79.2 (5C, CHO), 78.5 (1C), 77.6 (5C), 76.5 (5C), 74.9 (5C), 74.2
(1C), 73.7 (5C), 72.4 (1C), 71.3 (1C), 70.8 (5C), 70.2 (1C), 69.5 (1C),
69.3 (5C), 68.7 (1C), 68.4 (1C), 64.8 (1C), 63.8 (1C), 63.5 (5C, C-6),
29.8 (5C, CH2CO), 29.6 (5C, CH2CO), 29.2 (5C, CH2CHCH2), 29.0 (1C),
12.6e58.2 (remaining chol protons) 58.2 (1C), 57.9 (1C), 57.6 (1C),
46.6 (1C), 43.5 (1C), 41.1 (1C), 40.1 (1C), 38.5 (1C), 37.9 (1C), 37.4
(1C), 37.2 (1C), 36.8 (1C), 33.2 (1C), 33.1 (1C), 30.3 (1C), 28.7 (1C),
28.1 (1C), 25.3 (1C), 24.9 (1C), 23.3 (1C), 23.0 (1C), 22.2 (1C), 20.1
(1C), 19.5 (1C), 12.4 (1C), 12.6 (1C); MS (m/z): 2422.1 [M þ Na]þ;
Anal. Calcd for C109H178O57: C, 54.54, H, 7.47; Found: C, 54.48, H,
7.52.
d
ppm): 1.86 (m, 12H, CH2), 3.31e3.89 (m, 18H, CHOCH2; 12H,
CH2O). MS (m/z): 529.31 ([M þ 1]þ).
5.1.9. Synthesis of pentaol compound 21
A solution of cholesteryl carboxylate derivatives 12 (4.5 g,
7.8 mmol) in THF, DCC (2.1 g, 10.14 mmol), and catalytic amount of
DMAP were added in reaction bottle. The reaction was activated for
half an hour, followed by the addition of the prepared hexol com-
pound 20 (4.12 g, 7.8 mmol) in THF. The solution was stirred
overnight at room temperature, then the white precipitate was
filtered off and THF was evaporated under reduced pressure, the
crude oil was purified on a silica-gel chromatography column to
yield product colorless oil 21. (54%) 1H NMR (400 MHz, CDCl3,
d
ppm): 5.31 (m, 1H, chol H-6), 4.25 (m, 2H, CH2OCO), 4.15 (s, 2H,
5.2. Biodistribution studies in vivo
OCOCH2O), 3.82e3.97 (m, 6H, CHO), 3.71e3.78 (m, 12H, OCH2
-
CH2O), 3.44e3.71 (m, 12H, CHOCH2; 10H, CH2OH), 3.16 (m, 1H, chol
H-3), 1.82e1.86 (m, 12H, CH2), 2.37e0.66 (remaining chol protons)
with 0.65 (s, 3H, CH3-18), 0.84 (d, 6H, J ¼ 6.8 Hz, CH3-26, CH3-27),
5.2.1. Test animals
Adult Kunming mice weighing 20e22 g were obtained from the
animal center of Sichuan University. The animals were left for two

B. Qu et al. / European Journal of Medicinal Chemistry 72 (2014) 110e118
117
days to acclimatize to animal room conditions and were main-
RE ¼ ðAUC0ꢂtÞs=ðAUC0ꢂtÞc
CE ¼ ðCmaxÞs=ðCmaxÞc;
tained on standard pellet diet and water ad libitum. Food was
withdrawn on the day before the experiment, but free access to
water was allowed. Since the experiment could be completed
within 24 h, there was no significant change in the mices’ body
weight during the experiment. All animals received human care,
and the study protocols complied with the guidelines of Sichuan
University animal ethical experimentation committee. Throughout
the experiments, the animals were handled according to the re-
quirements of the National Act on the use of experimental animals
(People’s Republic of China).
where S and C represented sample (the liposomes) and control
(docetaxel), respectively.
Acknowledgments
This work was supported by the National Natural
Science Foundation of China (81072532), the Ph.D. Programs
Foundation of Ministry of Education of China (20110181130011)
and Science & Technology Pillar Program of Sichuan Province of
China (2011SZ0014).
5.2.2. HPLC analysis of liposomes
The waters liquid chromatographic system employed was an
LC-10A liquid chromatographic system (Shimadzu Japan). The
Appendix A. Supplementary data
analysis was carried out on
a SinoChrom ODS-C18 column
(200 mm ꢃ 4.6 mm, 5 mm), thermostated at 25 ꢀC. The solution of
acetonitrile-0.1%phosphoric acid solution (55:45) was used as the
mobile phase at a flow rate of 1.0 mL/min and the UV detector was
set to monitor the signal at 230 nm corresponding to the maximum
absorbance for the docetaxel.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.007.
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