Design, synthesis and biological evaluation of enzymatically cleavable NSAIDs prodrugs derived from self-immolative dendritic scaffolds for the treatment of inflammatory diseases

Article abstract of DOI:10.1016/j.bmc.2013.05.006

It has been reported that delivery systems based on dendritic prodrugs of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) improved the properties of drug molecules and reduced the side effects and irritation on the gastric mucosa. To find a more effective way in NSAIDs dendritic prodrugs, in this paper, three different dendritic scaffolds of enzymatically cleavable naproxen conjugates have been synthesized in a convergent approach and well characterized by NMR and MS techniques. These self-immolative dendritic NISADs prodrugs programmed to release multiple molecules of the potent naproxen after a single enzymatic activation step, and in 50% human plasma, the drug released from the compound T3 reaching 47.3% after 24 h in vitro assay. Moreover, all prodrugs were also found to maintain more significant anti-inflammatory activity, no significant cytotoxicity against HEK293 cells and less degree of ulcerogenic potential in vivo than their monomeric counterpart naproxen. These results provided an effective entry to the development of new dendritic NSAIDs prodrugs.

Full text of DOI:10.1016/j.bmc.2013.05.006

Bioorganic & Medicinal Chemistry 21 (2013) 4192–4200
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of enzymatically
cleavable NSAIDs prodrugs derived from self-immolative
dendritic scaffolds for the treatment of inflammatory diseases
Jinbao Wei a, , Jianyou Shi c, , Jing Zhang d, Gu He b, Junzhu Pan a, Jun He b, Rui Zhou a, Li Guo a,
,
⇑
Liang Ouyang b,
⇑
a Key Laboratory of Drug Targeting and Drug Delivery System of Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy,
Sichuan University, Chengdu 610041, PR China
b State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, PR China
c Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu 610072, PR China
d Test Centre for Feed Quality Supervision and Inspection of Agriculture Ministry, Chengdu 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
It has been reported that delivery systems based on dendritic prodrugs of Nonsteroidal Anti-Inflamma-
tory Drugs (NSAIDs) improved the properties of drug molecules and reduced the side effects and irritation
on the gastric mucosa. To find a more effective way in NSAIDs dendritic prodrugs, in this paper, three dif-
ferent dendritic scaffolds of enzymatically cleavable naproxen conjugates have been synthesized in a
convergent approach and well characterized by NMR and MS techniques. These self-immolative dendritic
NISADs prodrugs programmed to release multiple molecules of the potent naproxen after a single enzy-
matic activation step, and in 50% human plasma, the drug released from the compound T3 reaching 47.3%
after 24 h in vitro assay. Moreover, all prodrugs were also found to maintain more significant anti-inflam-
matory activity, no significant cytotoxicity against HEK293 cells and less degree of ulcerogenic potential
in vivo than their monomeric counterpart naproxen. These results provided an effective entry to the
development of new dendritic NSAIDs prodrugs.
Received 1 April 2013
Revised 26 April 2013
Accepted 3 May 2013
Available online 15 May 2013
Keywords:
Naproxen
Dendritic prodrug
Self-immolative
Drug release
Anti-inflammatory
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction
tiple end-groups. With the features of monodispersity, spherical
macromolecules with well-defined three dimensional structures
Nowadays, nonsteroidal anti-inflammatory drugs (NSAIDs) are
widely used in clinical to treat arthritis, fever and relieve pain.1–3
However, the NSAIDs have been reported to cause a series of side
effects, gastrointestinal toxicity in particular, which has an inter-
wovenness connection with their pharmacological activities.4–7
Many drug delivery systems (DDSs) were designed to circumvent
this drawback meanwhile preserving their satisfactory pharmaco-
logical activities. Among all the DDSs that improved the anti-
inflammatory effect of small molecule NSAIDs, nontoxic prodrugs
based on drug carriers were focused widely.8–12 With the develop-
ment of materials-based approaches, the dendritic prodrugs have
been considered as a promising entry to find prodrugs on the basis
of drug delivery.13–18 Dendrimers, a kind of synthetic artificial
macromolecules, are highly branched tree-like polymers with mul-
and functionalized end-groups, the dendrimers have many unique
properties that significantly differ from those corresponding linear
polymers.19–23
In traditional approaches of dendrimer-based drug delivery, the
bioactive substances were covalently attached to termini and
thereby independent cleavages were required for their release.
The activities of drug molecules were typically attenuated or elim-
inated when they incorporated into the delivery system.24,25 One
example is PAMAM based NSAIDs dendritic prodrugs reported by
D’Emanuele and co-workers.26,27 Conjugation of naproxen with
G0 PAMAM dendrimer increased the permeability and biocompat-
ibility appreciably, enhanced the potency and diminished the gas-
trointestinal side-effects of naproxen at the same time.28–31
Although the preliminary results were so exciting, the release
properties of dendritic prodrug in vitro and potency in vivo were
a challenge for us. As for the current research, NSAIDs dendritic
prodrugs were mainly focused on the PAMAM, not extending to
more scaffolds. Therefore, a distinct release profile might be found
when different dendritic chains or scaffolds were conjugated to a
model drug molecule.
⇑
Corresponding authors. Tel./fax: +86 028 85503817.
E-mail addresses: rosaguoli@yahoo.com.cn (L. Guo), ouyangliang@scu.edu.cn
(L. Ouyang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.006

J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
4193
Another update is that, recently an emerging class of dendri-
mers has been built which allows for the simultaneous release of
all end-groups upon a single triggering event.32,33 Shabat and co-
workers provided an efficient entry to obtain such special mole-
cules with excellent results: a self-immolative dendritic linker
acted as a molecular amplifier and conjugate was designed for acti-
vation by a triggering unit driven by proteins, enzyme or other bio-
active molecules in biological system.34–36 This strategy not only
increased the loading capacity of drug molecules per [a] polymer
molecule by two times or more than that of the classic conjugate,
but also enhanced the ability to receive and translate a biological
signal into an amplified response if the targeted or secreted en-
zyme that initiates drug release was present at a relatively low le-
vel in the malignant tissue and lead to the specific drug
delivery.37,38 Thus, the above-mentioned challenge could be over-
come by using self-immolative dendritic prodrugs which allowed
the release of several drug units after a single enzymatic activation
step.39,40 In this case, we tried to design and synthesize three dif-
ferent dendritic naproxen scaffolds with a single triggering and
self-immolative linkers, and compare drug release profiles
in vitro with different dendritic structures. The cytotoxicity
in vitro, anti-inflammatory and ulcerogenicity in vivo of the new
dendritic prodrugs were evaluated by the MTT method, xylene-in-
duced mice auricle tumefaction model and acute ulcerogenesis as-
say respectively. Through a series of experiments, we hope to find
effective dendritic prodrugs used in NSAIDs DDSs.
enolysis, compound 3 was coupled with compound 1 in the
presence of EDCI and HOBT. At last, the polyester dendron T1
was obtained through repeating the convergent synthesis method,
as described above. In the condensation reaction with naproxen,
condensing agent such as DCC or EDCI could promote the comple-
tion of the reaction accompanied by the catalysis of DMAP, so we
used EDCI/DMAP as the coupling reagents in most of the ester bond
condensation reactions considering the water-solubility of EDCI,
the convenience of post-processing and better yield (>80%). Due
to the congestion degree of the structures and the steric hindrance
effect, the reaction activity of the third generation dendritic com-
pound T1 was relatively low and the Rf value of different genera-
tions of naproxen dendrons in TLC layer tended to decrease, so
we used different polar eluents (petroleum ether/ethyl acetate,
petroleum ether/acetone) to purify different generations of na-
proxen dendrons by silica gel column chromatography to obtain
compound T1 with complete structures.
The synthesis route of target molecule T2 was outlined in
Scheme 2. First, naproxen was directly conjugated to Lys-OBzl with
IBCF/NMM to obtain the intermediate 6. Then, after a series of
repeating deprotection and condensation steps, we finally obtained
compound T2 by convergence method. To prepare first generation
intermediate 6, we found that a variety of peptide synthesis meth-
ods could be applied, among which IBCF/NMM showed a better
yield. Mixed anhydride method (IBCF/NMM as the representative
coupling reagent) had many advantages in the reaction, such as
less side reactions, easier to operate and the shorter reaction time.
However, using this method could not obtain the second genera-
tion compound 8, suggesting that mixed anhydride method was
very sensitive to the steric hindrance and the congestion degree
of the structures of these compounds. Therefore, we used EDCI/
HOBT as the coupling reagents, and finally succeeded in obtaining
the second generation compound 8 and the third generation target
molecule T2.
2. Results and discussion
2.1. The design of dendritic prodrugs
In our previous work, NSAIDs prodrugs with aspartate or succi-
nate based dendrons were studied: a sustained release enhance-
ment profile was found when the different chains were
conjugated to a model drug molecule.29 Thus, we decided to study
the different dendritic prodrugs T1–T3 designed for the selective
targeting of more naproxens. The design of our dendritic prodrugs
was based on three classic types of dendrons: polyamide–ester,
polylysine and Newkcome type dendron. All of the dendritic com-
pounds were connected to a triggering group (that can be activated
either chemically or enzymatically) and enzymatic cleavable bonds
served as the ‘handle’ for attachment of an end-unit (naproxen).
The release of the end-unit could be initiated by the removal of
the triggers and then a spontaneous enzymatic cleavage reaction
took place, leading to the release of the end-unit. In this case,
one benzyl ester or amide which presented an enzyme substrate
of arylesterase41 in human plasma was designed as a triggering
group. In order to verify our assumption, a peptidase, cathepsin B
which was always used in the enzymatic release test,42 was taken
as a control compared with the arylesterase in human plasma. In
addition, different dendritic scaffolds such as polyester, polyamide
and polyamide–ester were conjugated to the model drug naprox-
en. We preliminarily planed to investigate the release capacities
of the different bond-type dendritic drugs in vitro and screen out-
standing dendritic carriers, which will lay a foundation for further
molecular design.
The synthesis route of the target molecule T3 was outlined in
Scheme 3. First, naproxen was directly conjugated to Cbz-pro-
tected tris through an ester bond condensation to obtain the inter-
mediate 10. After the removal of the protecting groups by catalytic
hydrogenolysis, intermediate 10 experienced an amide bond con-
densation reaction with succinic acid monobenzyl ester to produce
compound 12. Then, after the removal of protecting groups, com-
pound 12 reacted with Cbz-protected tris through
a multi-
branched condensation reaction to obtain the target molecule T3.
The purposes of introducing succinate chains were not only to
add more cleavable chemical bonds as a self-immolative moiety,
but also to extend the length of carbon chains and reduce the dif-
ficulties of the reaction. In the synthetic process of these dendritic
prodrugs, the steric hindrance was still the greatest difficulty we
encountered, and we ultimately obtained the target product by
EDCI/HOBT with acceptable yield after a variety of condensing
agents had been tried.
The high degree of symmetry in these dendrons enabled facile
confirmation of both structure and purity by NMR techniques. For
example, in the 1H NMR spectrum of dendron T3, the naproxen pro-
tons observed the resonance signals at 1.52 (d), 3.72 (q), and 3.88
(s) ppm were clearly distinguishable from the resonances arising
from the cores (tris protons) at 2.10 (m) and 4.00 (m) ppm. Integra-
tion of the respective areas of the core protons and methoxy protons
of naproxen confirmed the complete coupling of the central core
and the model drugs. Furthermore, the structures of these dendrons
were further verified by ESI-MS (electrospray ionization mass spec-
trometry), all of the ESI-MS spectra displayed a very prominent peak
corresponding to the dendrons combined with protons or sodium
cations. Moreover, the result of elemental analysis was also in good
agreement with those of the signed structures.
2.2. Chemistry
The synthetic procedure of the target molecules T1 could be
outlined by Scheme 1. First, benzyl acrylate was conjugated to
diethanolamine through a simple Michael addition and obtained
the intermediate 1 with free hydroxyls. Second, the compound 2
was obtained by a condensation reaction of compound 1 with na-
proxen. After the removal of the benzyl group by catalytic hydrog-

4194
J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
OH
OH
CH3OH
O
O
HN
OH
Naproxen
O
N
O
EDCI/DMAP
1
OH
O
O
O
O
O
O
O
O
O
O
H2/Pd-C
O
O
O
O
HO
N
O
N
O
N
O
O
O
O
O
O
O
N
O
3
2
O
O
O
O
O
O
O
N
O
O
O
O
O
O
N
O
O
O
O
O
O
O
1
i, , EDCI/DMAP
O
1
i, , EDCI/DMAP
O
N
O
ii, H2/Pd-C
HO
N
O
O
N
O
O
O
O
O
O
O
O
T1
4
O
O
O
O
O
N
N
O
O
O
O
O
N
O
O
O
Scheme 1. The synthesis of target dendritic molecule T1.
O
O
H
N
H
N
O
HO
O
O
NH2
O
H2/Pd-C
Naproxen
O
O
NH2
IBCF/NMM
O
HN
HN
O
O
O
O
5
6
7
O
O
NH
O
O
HN
O
O
O
O
H
N
O
HN
N
H
O
O
O
O
HN
H
N
O
i, H2/Pd-C
ii, 5
5, EDCI/HOBT
O
N
O
H
, EDCI/HOBT
O
O
O
O
H
NH
H
N
HN
N
O
N
O
H
HN
H
O
O
N
O
O
O
O
NH
H
HN
N
O
HN
O
O
O
O
O
8
T2
HN
O
O
Scheme 2. The synthesis of target dendritic molecule T2.
2.3. Biological evaluation
a 0.02 M phosphate buffer at pH 2, 7, and 8 (37 °C). The evolution of
the mixture was followed by HPLC over a period of 10 days. Table 1
shows the percentage of conjugate remaining after hydrolysis of the
amide bond in compound T2 and the ester or amide–ester bonds in
compounds T1 and T3. The stabilities of all conjugates were high
with approximately >95% of the conjugates remaining at all pH
One of the essential prerequisites for the use of dendritic pro-
drugs is that they should be stable in the physiological pH, such that
we can get more accurate data of their release in different microen-
vironment.43 The chemical stability of prodrugs was investigated in

J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
4195
O
O
O
O
O
O
O
O
O
OH
O
O
OH
OH
OH
IBCF/NMM, BzlO
H2/Pd-C
Naproxen
H
N
O
O
HN
Cbz
Cbz
H2N
O
O
EDCI/ DMAP
O
O
O
O
O
11
10
9
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
i, H2/Pd-C
NH
H
N
HN
O
O
O
O
O
ii, 9, EDCI/ HOBT
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
O
O
HN
O
O
O
O
O
O
O
O
12
T3
O
O
Scheme 3. The synthesis of target dendritic molecule T3.
Table 1
Chemical stability of dendritic conjugates at pH 2, 7 and 8 (37 °C)
Compound
% Conjugate remaininga
pH 2
48 h
pH 7
48 h
pH 8
48 h
240 h
240 h
240 h
T1 (ester)
T2 (amide)
T3 (amide–ester)
95.7 1.2
97.1 0.7
97.2 1.3
72.3 4.8
93.1 0.4
89.9 2.1
96.2 0.4
97.6 0.9
99.1 0.2
95.9 0.7
96.1 1.6
98.4 2.5
97.3 1.9
91.2 0.8
94.45 3.1
79.4 7.4
89.4 2.6
79.4 4.1
a
Data are represented as mean SD.
values after 48 h. The direct linkage of naproxen to the T2 dendron
resulted in a very stable amide prodrug under all pH conditions and
even at pH 8 more than 89% of the conjugate remained intact after
10 days. The two ester or amide–ester conjugates T1 and T3 after 10
days of incubation at all pH values were less stable than T2 possibly
because the less chemical stable of the ester bonds. However, most
importantly, both ester conjugates showed good stability under
physiological conditions (pH 7.4 and 37 °C) after 10 days of incuba-
tion. So, they may still be considered to be a sufficiently stable ester
in enzymatic drug release assay.
The drug release profile was one of the most important factors
to be considered for the conjugates. Since the conjugates contained
different covalent bonds between the drugs and the dendritic scaf-
folds, they should be enzymatically and/or hydrolytically degrad-
able to release the drug molecules to inflammatory tissue44 and
the different release capabilities in vitro of the three types of na-
proxen prodrugs can be compared. The precipitate (T1–T3) was
incubated in the presence of cathepsin B at pH 7 or 50% (v/v in
PBS) human plasma (with a small amount of Twain as hydrotropy
agent) at 37 °C. All compounds are dissolved in solvent with clean-
ing state and the hydrolytic release of naproxen in the solution was
monitored by RP-HPLC.
The results from these in vitro assays (Fig. 1) suggested several
trends. First, the three different types of prodrugs were able to re-
lease the parent drugs in vitro. Specifically, the drug released rap-
idly from the compound T3 in 50% human plasma, reaching 57.1%
after 48 h and almost 80% within five days, whereas the drug re-
lease in cathepsin B was much slower, implying 4.6% and 16.1%
in the same period, respectively. Second, in 50% human plasma,
the drug released from the compound T3 reaching 47.3% after
24 h. The release property of compound T3 was much better than
compounds T1 and T2. For compound T1, it might be due to the
C–N bond’s relative stability under various environments causing
the release performance decline. Indeed, T1 showed only ca. 18%
of activation after 120 h of incubation in human plasma.
The trends observed with in vitro drug release data would sug-
gest that the three linkages were relatively stable in cathepsin B
but labile under human plasma conditions to allow the transport
of the prodrug and effective release of the free active drug from
the carrier. Prodrug T3 with benzyl ester or amide had a more rapid
activation pathway in human plasma, possibly because a benzyl
ester or amide linkages were better substrates of aryl ester hydro-
lase in human plasma and as more suitable triggers, the eliminate
process was accelerated by a self-immolative disassembly pathway

4196
J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
30
25
20
15
10
5
(a)
(b)
80
T1
T2
T3
T1
70
60
50
40
30
20
10
0
T2
T3
0
0
12
24
36
48
60
72
84
96
108
120
0
12
24
36
48
60
72
84
96
108
120
Time (h)
Time (h)
Figure 1. Release of Naproxen from compounds T1–T3 in different conditions (a) in cathepsin B at pH 7; (b) in human plasma at pH 7. The percentage of drug release was
deduced from the difference between the initial amounts of compounds and that of regenerated drug Naproxen. Error bars represented the mean and standard deviation of
three independent experiments.
or the ester bond was more instable than the amide bond. A plau-
sible mechanism for the drug release could be explained as fol-
lows: when incubated with the enzyme, prodrug T3 was rapidly
cleaved leading to the release of intermediate with benzyl re-
moved, while cathepsin B might not be very good to initiate this
reaction.45 Prodrugs disappeared concomitantly with the appear-
ance of naproxen in 120 h. Furthermore, a LC–MS analysis of den-
dritic prodrug T3 after 120 h at 37 °C in human plasma at pH 7.0
was taken. The solution was evaluated by an Agilent 6430 LC–MS
system. The mobile phase was a mixture of methanol and 0.2% for-
mic acid aqueous solution (linear gradient beginning with 20:80 v/
v, reaching 60:40 v/v within 5 min.). From the results monitored by
HPLC–MS, 120 h after the addition of human plasma, tris (M.wt.
121), succinic acid (M.wt. 118) and naproxen were detected in
the mixture (see Supporting Information). The results shown in
LC–MS study provided a proof that tris and succinic acid linkages
were the final eliminated products after enzymatic cleavage of
dendritic T3 (Fig. 2).
120
100
80
60
40
20
0
NAP
T1
T2
T3
2
4
8
16
32
Concentrations (ug/mL)
Figure 3. Cytotoxicity of dendrons against HEK293 cells over a 24 h incubation
period determined by MTT assay. Cell viability was expressed as a percentage of the
control cell culture. Values are represented as mean SD (n = 3).
After in vitro drug release, several pharmacodynamics experi-
ments were taken. First, cytotoxicity is one of the most important
factors to be considered in selecting dendritic prodrugs for bio-
medical applications. The toxicity of compounds T1–T3 against
HEK293 cells was evaluated by MTT assays and naproxen was ta-
ken as a positive control. The results are shown in Figure 3. The as-
says demonstrated that neither all dendritic conjugates nor
naproxen was significantly toxic against HEK293 cells in the con-
by a slight degree. However, we did not observe a significant differ-
ence in the toxicity of dendrons with positive control naproxen. So,
not any obvious toxicity was found in cell culture experiment, as
we expected.
Finally, Table 2 revealed the in vivo acute anti-inflammatory
activity of the target conjugates at a dose of 10 mg/kg q.d. in xy-
lene-induced mice auricle tumefaction model.46 Compared with
saline group, entire dendritic prodrugs exhibited good anti-inflam-
matory activity with the inhibition percentage of auricle tumefac-
tion ranging from 39.62% to 49.64%. It was worth pointing out that
centration range of 2–32 lg/ml. With the augment of tested con-
centration, the cytotoxicity of these compounds increased only
Human plasma
Cathepsin B
(arylester hydrolase)
Trigger cleavage
Enzymatic cleavage
NAP
H2N
NAP
Dendron T3
OH
OH
OH
O
OH
Enzymatic cleavable
dendritic bond
+
CO2
OH
O
H2N
+
HO
Figure 2. Human plasma-catalyzed drug release mechanism.

J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
4197
Table 2
In vivo inhibitory activity of dendritic prodrug in xylene-induced mice auricle tumefaction assay and ulcer index
Compound
na
Dosage (
l
M/kg)b
Degree of tumefaction
Inhibition percentagec
Ulcer indexd
––
8.26
6.93
6.21
Blank
Naproxen
T1
T2
T3
10
10
10
10
10
––
15.67 2.59
10.87 1.73
9.46 3.74*
8.12 3.07*
7.89 2.66*
––
200, q.d.
25, q.d.
25, q.d.
30.63
39.62
48.18
49.64
22.2, q.d.
5.94
a
Values are the means of the indicated number of experiments (n).
Mice were treated with 0.9% NaCl solution, naproxen and test compounds one time a day for five days, respectively. The administration of the dendritic prodrug is same
b
molar amount of native naproxen.
c
Percentage of inhibition = (1 À tumefaction degree of blank group/tumefaction degree of test group) Â 100%.
d
Ulcerogenic activity was evaluated after oral administration of test compounds at the dosage of 200 mg/kg.
*
P < 0.05 comparing with blank group.
significant anti-inflammatory activity was achieved for T1
(39.62%), T2 (48.18%) and T3 (49.64%) with potent inhibition per-
centage higher than the contrast drug naproxen (30.63%). On the
other side, the most common side effects associated with the
long-term administration of NSAIDs are gastrointestinal erosions,
ulcer formation, and sometimes severe bleeding. It was therefore
essential to evaluate the potential in vivo ulcerogenicity of pro-
drugs T1–T3 in comparison to the corresponding contrast drug na-
proxen. The severity of gastric damage, assessed using an
ulcerogenicity assay, was expressed as an ulcer index (UI) and
the UI for each test compound is calculated by adding the total
length (L, in mm) of individual gastric lesions in each stomach
and averaging over the number of animals in each group
(n = 10): UI = (L1 + L2 + L3 + L4 + L5 + L6 + L7 + L8 + L9 + L10)/10. As
shown in Table 2, the results indicated that these compounds
T1–T3 (ulcer index ranges from 5.94 to 6.93) cause less gastric
ulceration and disruption of gastric epithelial cells as compared
to naproxen (ulcer index 8.26). The results from the preliminary
in vivo assay indicated that the dendritic prodrugs, especially com-
pound T3, exhibited improved anti-inflammatory activities with
reducing gastric ulcerogenicity which were consistent with our
in vitro release experimental results. We speculated that the den-
dritic prodrugs might make the drug half-life longer and would
form a continuous and stronger effect compared with native na-
proxen so exhibited better anti-inflammatory activity.
were visualized by irradiation with UV light and/or by treatment
with a solution of phosphomolybdic acid (20 wt% in ethanol) fol-
lowed by heating. Column chromatography was performed by
using silica gel with eluent given in parentheses. 1H NMR analysis
was performed using CDCl3 or DMSO-d6 as a solvent at room tem-
perature. The chemical shifts were expressed in relative to TMS
( = 0 ppm) and the coupling constants J in Hz. The purity of com-
pounds screened in biological assays was determined to be
P97% by HPLC (Agilent 1100 HPLC system) analysis with a photo-
diode array detector, An Atlantis C18 (150 Â 4.6 mm, i.d. 5
lm)
(Waters, Milford, Mass, USA) was used with a gradient elution of
methanol and HPLC-grade water as mobile phase at a flow rate
of 1 ml/min.
4.1.1. Synthesis of compound 1
Benzyl acrylate (0.9 g, 5.5 mmol) and diethanolamine (0.85 g,
8.1 mmol) dissolved in methanol (20 ml), were stirred at room
temperature overnight. The solution was concentrated under vac-
uum, then the residue was extracted with ethyl acetate (30 ml)
and washed with brine (10 ml). The organic layer was dried over
anhydrous Na2SO4. After concentration, the compound 1 was ob-
tained as colorless oil 1.14 g (yield 77.7%). 1H NMR (400 MHz,
CDCl3): 2.54 (t, 2H, J = 6.4 Hz, COCH2), 2.63 (t, 4H, J = 5.2 Hz, N–
CH2Â2), 2.86 (t, 2H, J = 6.4 Hz, N–CH2), 3.59 (t, 4H, J = 5.2 Hz,
CH2–OÂ2), 5.14 (s, 2H, ph-CH2), 7.34–7.37 (m, 5H, ph-H).
4.1.2. Synthesis of compound 2
3. Conclusions
To the solution of compound 1 (150 mg, 0.56 mmol) and na-
proxen (260 mg, 1.13 mmol) dissolved in anhydrous CH2Cl2
(15 ml), EDCI (233 mg, 1.13 mmol) and DMAP (14 mg,
0.013 mmol) were added at room temperature. After stirring for
24 h at rt, the solution was concentrated under vacuum. The resi-
due was then extracted with ethyl acetate (30 ml) and washed suc-
cessively with 1M HCl (10 ml), 1M NaHCO3 (10 ml), and brine
(10 ml). The organic layer was dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The residue was purified
by silica-gel column chromatography using PE-EA (5:1–3:1, v/v)
as an eluent to obtain compound 2 as colorless oil 440 mg (yield
85.3%). 1H NMR (400 MHz, CDCl3): 1.54 (d, 6H, J = 7.2 Hz, Nap-
CH3Â2), 2.30 (t, 2H, J = 6.8 Hz, COCH2), 2.60 (t, 4H, J = 6 Hz, N–
CH2Â2), 2.73 (t, 2H, J = 6.8 Hz, N–CH2), 3.78 (q, 2H, J = 7.2 Hz,
NapCOCHÂ2), 3.91 (s, 6H, NapOCH3Â2), 3.99 (t, 4H, J = 6 Hz,
CH2–OÂ2), 5.05 (s, 2H, ph-CH2), 7.08–7.68 (m, 17H, Nap-
phH + ph-H). ESI-MS (m/z): calcd for 691.8 obsd 692.2 ([M+H]+).
In conclusion, we developed a new self-immolative dendritic
NISADs prodrug programmed to release multiple molecules of
the potent naproxen after a single enzymatic activation step. Upon
human plasma activation, the dendritic prodrug is much easier to
release than its positive counterpart as well as cathepsin B at the
same concentration. The preliminary in vivo biological activities
of these compounds evidenced our rational design of improved
anti-inflammatory prodrugs with reduced gastric toxicity (ulcerog-
enicity). This targeting system could represent an important step
toward the quest for the magic bullet in the anti-inflammatory
therapy. Further developments in this direction including preli-
minary study of biodistribution and pharmacokinetic analysis of
these conjugates in vivo are now underway in our laboratory.
4. Experimental
4.1. Chemistry
4.1.3. Synthesis of compound 3
Compound 2 (400 mg) was dissolved in methanol (10 ml), and
10% Pd/C (100 mg) were added into the solution. The reaction mix-
ture was stirred at room temperature under a H2 atmosphere. After
24 h, the mixture was passed through a membrane filter to remove
General: all reactions requiring anhydrous conditions were per-
formed under an Ar or N2 atmosphere. Chemicals and solvents
were either A.R. grade or purified by standard techniques. Thin
layer chromatography (TLC): silica gel plates GF254; compounds

4198
J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
the catalyst and then evaporated under vacuum to afford com-
pound 3 as colorless oil 250 mg (yield 91.7%). 1H NMR (400 MHz,
CDCl3): 1.48 (d, 6H, J = 7.2 Hz, NapCH3Â2), 2.34 (t, 2H, J = 6.8 Hz,
COCH2), 2.62 (t, 4H, J = 6 Hz, N–CH2Â2), 2.71 (t, 2H, J = 6.8 Hz, N–
CH2), 3.72 (q, 2H, J = 7.2 Hz, NapCOCHÂ2), 3.89 (s, 6H, Nap-
OCH3Â2), 4.01 (t, 4H, J = 6 Hz, CH2–OÂ2), 7.09–7.48 (m, 12H,
Nap-phH). ESI-MS (m/z): calcd for 601.8 obsd 600.2 ([MÀH]À).
(m, 18H, lysCH2Â9), 3.35 (br s, 6H, lysN–CH2Â3), 3.87 (m, 4H,
NapCOCHÂ4), 3.98 (s, 12H, NapOCH3Â4), 4.42 (br s, 3H, lysCHÂ3),
5.21 (m, 2H, ph-CH2), 7.10–7.80 (m, 29H, Nap-phH + ph-H). ESI-MS
(m/z): calcd for 1341.63 obsd 1365.2 ([M+Na]+).
4.1.9. Synthesis of compound T2
According to the same procedure of preparation of 49 and 51.
After increased generation and debenzylation, compound 42 was
afforded as white solid (37% yield in two steps). 1H NMR
(400 MHz, CDCl3): 1.52 (d, 24H, J = 7.2 Hz, NapCH3Â8), 1.24–1.91
(m, 42H, lysCH2Â21), 3.29 (br s, 14H, lysN–CH2Â7), 3.87 (m, 8H,
NapCOCHÂ8), 3.92 (s, 24H, NapOCH3Â8), 4.46 (br s, 7H, lysCHÂ7),
5.11 (m, 2H, ph-CH2), 7.10–7.80 (m, 53H, Nap-phH + ph-H). ESI-MS
(m/z): calcd for 2708.4275 obsd 2709.4671 ([M+H]+).
4.1.4. Synthesis of compound 4
The same procedure as described above for preparation of 3
from compound 1. Compound 4: pale yellow oil (78% total yield
in two steps). 1H NMR (400 MHz, CDCl3): 1.52 (d, 12H, J = 7.2 Hz,
NapCH3Â4), 2.2–2.4 (m, 6H, COCH2Â3), 2.70–2.90 (m, 12H, N–
CH2Â6), 3.79 (q, 4H, J = 7.2 Hz, NapCOCHÂ4), 3.96 (s, 12H, Nap-
OCH3Â2), 3.98–4.13 (br s, 12H, CH2–OÂ6), 5.10 (s, 2H, ph-CH2),
7.08–7.68(m, 29H, Nap-phH + ph-H). ESI MS (m/z): calcd for
1433.66 obsd 1433.26 ([M]+).
4.1.10. Synthesis of compound 10
Z-protected tris (0.1 g, 0.39 mmol) was dissolved in CH2Cl2
(15 ml), then naproxen (0.3 g, 1.3 mmol), EDCI (0.27 g, 1.3 mmol)
and DMAP (0.02 g, 0.13 mmol) were successively added into the
solution. After stirred overnight at room temperature, the solution
was concentrated under vacuum. The concentrated solution was
dissolved in ethyl acetate (30 ml) and washed with 1M HCl
(10 ml), 1M NaHCO3 (10 ml), brine (10 ml). The organic layer was
dried over anhydrous Na2SO4. After concentration, the crude prod-
uct was purified by a silica gel chromatography column using PE-
EA (5:1–3:1, v/v) to obtained compound 53 as white solid 0.34 g
(yield 94.4%) mp:120–122 °C. ESI-MS (m/z): calcd for 891.4 obsd
914.2 ([M+Na]+).
4.1.5. Synthesis of compound T1
The same procedure as described above for preparation of com-
pound 4. Compound T1: pale yellow oil (yield: 64.4%). 1H NMR
(400 MHz, CDCl3): 1.54 (d, 24H, J = 7.2 Hz, NapCH3Â8), 2.22–2.39
(m, 14H, COCH2Â7), 2.71–2.92 (m, 28H, N–CH2Â14), 3.73 (m,
14H, COCH2Â7), 3.79 (q, 8H, J = 7.2 Hz, NapCOCHÂ8), 3.96 (s,
24H, NapOCH3Â8), 3.98–4.03 (br s, 28H, CH2–OÂ14), 5.10 (s, 2H,
ph-CH2), 7.08–7.68 (m, 53H, Nap-phH + ph-H). ESI-TOF MS (m/z):
calcd for 2922.3736 obsd 2923.3805 ([M+H]+).
4.1.6. Synthesis of compound 6
Naproxen (0.24 g, 1 mmol) dissolved in anhydrous THF (10 ml)
was cooled at À15 °C, then added NMM (0.1 g, 1 mmol) and IBCF
(0.13 g, 1 mmol), stirred for 5 min. Lysine benzyl ester p-toluene-
sulfonate salt (0.3 g, 0.52 mmol) dissolved in the mixed solution
NMM and THF was added to the reaction. After stirred overnight,
the solution was concentrated under vacuum. The concentrated
solution was taken up in ethyl acetate (30 ml) and washed with
1M HCl (10 ml), 1M NaHCO3 (10 ml), brine (10 ml). The organic
layer was dried over anhydrous Na2SO4. After concentration, The
crude product was purified by recrystallization using PE-EA to ob-
tained compound 49 as white solid 0.55 g (yield 84%). 1H NMR
(400 MHz, CDCl3): 1.48 (d, 6H, J = 7.2 Hz, NapCH3Â2), 1.37–1.82
(m, 6H, lysCH2Â3), 3.25 (br s, 2H, lysN–CH2), 3.88 (m, 2H, NapC-
OCHÂ2), 3.99 (s, 6H, NapOCH3Â2), 4.32 (br s, 1H, lysCH), 5.22
(m, 2H, ph-CH2), 7.04–7.83 (m, 17H, Nap-phH + ph-H). ESI-MS
(m/z): calcd for 660.3199 obsd 661.3297 ([M+H]+).
4.1.11. Synthesis of compound 12
Succinic acid monobenzyl ester (74 mg, 0.36 mmol) dissolved in
anhydrous THF (10 ml) was cooled at À15 °C, then added NMM
and IBCF (0.07 ml, 0.48 mmol), stirred for 5 min, compound 11
(0.28 g, 0.36 mmol) dissolved in THF was added to the reaction.
After stirred at room temperature for 3 h, the solution was concen-
trated under vacuum. The residue was then taken up in ethyl ace-
tate (30 ml) and washed with 1M HCl (10 ml), 1M NaHCO3 (10 ml),
and brine (10 ml). The organic layer was dried over anhydrous
Na2SO4. After concentration, the residue was purified by silica-gel
column chromatography using PE-EA (3:1–2:1, v/v) as an eluent
to obtain compound 12 as white solid 150 mg (yield 40.5%).
mp:115–117 °C 1H NMR (400 MHz, CDCl3): 1.47 (d, 9H,
J = 7.6 Hz, NapCH3Â3), 1.70–1.72 (m, 2H, COCH2), 2.19–2.31 (m,
2H, COCH2), 3.72 (q, 3H, J = 7.6 Hz, NapCOCHÂ3), 3.87 (s, 9H, Nap-
OCH3Â3), 4.13 (s, 6H, tris-CH2Â3), 5.04 (s, 2H, ph-CH2), 7.04–7.64
(m, 23H, Nap-phH + phH). ESI-MS (m/z): calcd for 948.1 obsd 970.2
([M+Na]+).
4.1.7. Synthesis of compound 7
Compound 6 was dissolved in methanol (10 ml), and 10% Pd/C
(100 mg) were added into the solution. The reaction mixture was
stirred at room temperature under a H2 atmosphere. After 24 h,
the mixture was filtrated to remove the catalyst and then evapo-
rated under vacuum to afford compound 7 as white waxy solid
250 mg (yield 89.1%).
4.1.12. Synthesis of compound T3
According to the same procedure of preparation of 53. Com-
pound 45: yield 49%. 1H NMR (400 MHz, CDCl3): 1.52 (d, 27H,
J = 7.6 Hz, NapCH3Â9), 2.10–2.72 (m, 6H, SA-COCH2Â3), 3.72 (q,
9H, J = 7.6 Hz, NapCOCHÂ9), 3.88 (s, 27H, NapOCH3Â9), 4.00–
4.11 (m, 24H, tris-CH2Â12), 7.04–7.64 (m, 59H, Nap-phH + phH).
ESI-MS (m/z): calcd for 2783.1539 obsd 2784.1666 ([M+H]+).
4.1.8. Synthesis of compound 8
Compound
7 (0.57 g, 1 mmol) and compound 5 (0.3 g,
0.5 mmol) were dissolved in DCM (15 mL), then added EDCI
(0.23 g, 1.13 mmol) and HOBt (0.15 g, 1 mmol), stirred in room
temperature for 16 h. After stirred overnight, the solution was con-
centrated under vacuum. The concentrated solution was taken up
in ethyl acetate (30 ml) and washed with 1M HCl (10 ml), 1M NaH-
CO3 (10 ml), brine (10 ml).The organic layer was dried over anhy-
drous Na2SO4. The crude product was purified by silica-gel
column chromatography using PE-EA (1:1–1:2, v/v) as an eluent
to obtain compound 8 0.33 g as colorless oil. (yield 50%). 1H NMR
(400 MHz, CDCl3): 1.52 (d, 12H, J = 7.2 Hz, NapCH3Â4), 1.25–1.92
4.2. HPLC analysis, chemical stability and drug release studies
The HPLC system consisted of a photodiode array detector and a
set of Model LC-10AT liquid chromatography including a mano-
metric module as well as a dynamic mixer from Agilent 1100 HPLC
system. The mobile phase is a mixture of methanol and 0.05 mol/l
potassium dihydrogen phosphate (adjusted to pH 3.0 with phos-
phoric acid) (60:40) which was filtered through a 0.45 mm mem-
brane filter before use. A Waters XTerra RP18 column (250 mm,
4.6 mm, 5 lm) was eluted with the mobile phase at flow rate of

J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
4199
1.0 ml/min. The eluate was monitored by measuring the absorp-
tion at 256 nm with a sensitivity of AUFS 0.01 at 25 °C. The reten-
tion time (RT) of Naproxen is 10.265 min. The retention time (RT)
of compound T3 is 7.43 min.
calculated by adding the total length (L, in mm) of individual gas-
tric lesions in each stomach and averaging over the number of ani-
mals in each group (n = 10): UI = (L1 + L2 + L3 + L4 + L5 + L6
+ L7 + L8 + L9 + L10)/10.
The conjugates (0.1 lM) was incubated at 37 °C in 20 mM phos-
phate buffer at different pH 2.0, 7.0, 8.0, respectively, (1 ml). Stabil-
ity in phosphate buffer was monitored by analytical HPLC
conditions mentioned above.
Acknowledgments
Financial support from National Natural Science Foundation of
China (No. 81102325) and China Postdoctoral Science Foundation
(No. 2012T50781) is gratefully acknowledged.
The conjugates (0.1 lM) were incubated in 50% (v/v in PBS) hu-
man plasma (with a small amount of Twain as hydrotropy agent) at
37 °C and the hydrolytic release of Naproxen in the solution was
monitored by RP-HPLC. The concentration of Naproxen was ana-
lyzed using the HPLC conditions mentioned above.
Supplementary data
Supplementary data (full synthetic procedures and character-
ization data, as well as methods for drug release experiments)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.05.006.
4.3. In vitro cytotoxicity assay
The cytotoxic effects of compounds T1–T3 and naproxen were
determined using the standard MTT assay. Briefly, HEK293 cells
(2500/well) were seeded in 96-well plates and cultured for 24 h,
followed by treatment with the target compounds for another
48 h. Twenty microliter of 5 mg/ml MTT was added per well and
incubated for another 2.5 h at 37 °C. Then the supernatant fluid
was removed and MTT formazan precipitate was dissolved in
150 ll of DMSO, shaken mechanically for 15–20 min. The optical
density of each well was measured at 570 nm, using a SpectraMAX
M5 microplate spectrophotometer.
References and notes
1. Harder, S.; Thurmann, P. Clin. Pharmacokinet. 1996, 30, 416.
2. Heyneman, C. A.; Lawless-Liday, C.; Wall, G. C. Drugs 2000, 60, 555.
3. Whitehouse, M. W. Curr. Med. Chem. 2005, 12, 2931.
4. Silverstein, F. E.; Faich, G.; Goldstein, J. L.; Simon, L. S.; Pincus, T.; Whelton, A.;
Makuch, R.; Eisen, G.; Agarwal, N. M.; Stenson, W. F.; Burr, A. M.; Zhao, W. W.;
Kent, J. D.; Lefkowith, J. B.; Verburg, K. M.; Geis, G. S. J. Am. Med. Assoc. (JAMA)
2000, 284, 1247.
5. Arakawa, T.; Watanabe, T.; Tanigawa, T.; Tominaga, K.; Otani, K.; Nadatani, Y.;
Fujiwara, Y. Curr. Med. Chem. 2012, 19, 77.
6. Abdel-Tawab, M.; Zettl, H.; Schubert-Zsilavecz, M. Curr. Med. Chem. 2009, 16,
2042.
7. Al-Turki, D. A.; Abou-Zeid, L. A.; Shehata, I. A.; Al-Omar, M. A. Int. J. Pharmacol.
2010, 6, 813.
4.4. In vivo studies
4.4.1. Anti-inflammatory assay
According to the requirements of the National Act on the usage
of experimental animals (PR China), the Sichuan University Animal
Ethical Experimentation Committee, approved all procedures of
our in vivo studies. Male Kunming mice (18–22 g) which were
housed in room temperature of 25 2 °C and humidity of 60 5%
were used through the studies. Naproxen was administered
(10 mgl/kg, t.i.d.) as a positive comparator. The test compounds
were administered at the same dose (10 mgl/kg, t.i.d.), respec-
tively. Fifty mice were randomly divided into 5 groups and were
treated with 0.9% NaCl solution, naproxen and test compounds
for five days, respectively. The buninoid filter with diameter of
7 mm is infiltrated by xylene. After 1 h of the last administration,
the filter was clung to the right ear of the mice for 30 s. After
30 min the mice were executed by decollation and the ears were
slotted wafers by 7 mm hole puncher. The inhibition percent of
auricle tumefaction was calculated using the following formula.
Percent of inhibition (%) = (1 À a/b) Â 100%, where a means tume-
faction degree of control group and b means tumefaction degree
of test group.
8. Quan, L. D.; Thiele, G. M.; Tian, J.; Wang, D. Exp. Opin. Ther. Patents 2008, 18,
723.
9. Cai, J.; Duan, Y. B.; Yu, J.; Chen, J. Q.; Chao, M.; Ji, M. Eur. J. Med. Chem. 2012, 55,
409.
10. Ren, K.; Purdue, P. E.; Burton, L.; Quan, L. D.; Fehringer, E. V.; Thiele, G. M.;
Goldring, S. R.; Wang, D. Mol. Pharm. 2011, 8, 1043.
11. Fan, W.; Wu, Y.; Li, X. K.; Yao, N.; Li, X.; Yu, Y. G.; Hai, L. Eur. J. Med. Chem. 2011,
46, 3651.
12. Huang, Z. J.; Velazquez, C. A.; Abdellatif, K. R. A.; Chowdhury, M. A.; Reisz, J. A.;
DuMond, J. F.; King, S. B.; Knaus, E. E. J. Med. Chem. 2011, 54, 1356.
13. Pan, J. Z.; Wen, M.; Yin, D. Q.; Jiang, B.; He, D. S.; Guo, L. Tetrahedron 2012, 68,
2943.
14. Cheng, Y. Y.; Xu, T. W. Eur. J. Med. Chem. 2005, 40, 1188.
15. Stach, M.; Maillard, N.; Kadam, R. U.; Kalbermatter, D.; Meury, M.; Page, M. G.
P.; Fotiadis, D.; Darbre, T.; Reymond, J. L. MedChemComm 2012, 3, 86.
16. Calderon, M.; Welker, P.; Licha, K.; Fichtner, I.; Graeser, R.; Haag, R.; Kratz, F. J.
Controlled Release 2011, 151, 295.
17. El Kazzouli, S.; Mignani, S.; Bousmina, M.; Majoral, J. P. New J. Chem. 2012, 36,
227.
18. Cheng, Y. Y.; Zhao, L. B.; Li, Y. W.; Xu, T. W. Chem. Soc. Rev. 2011, 40, 2673.
19. Johansson, E. M. V.; Kadam, R. U.; Rispoli, G.; Crusz, S. A.; Bartels, K. M.; Diggle,
S. P.; Camara, M.; Williams, P.; Jaeger, K. E.; Darbre, T.; Reymond, J. L.
MedChemComm 2011, 2, 418.
20. Newkome, G. R.; Shreiner, C. Chem. Rev. 2010, 110, 6338.
21. Svenson, S. Eur. J. Pharm. Biopharm. 2009, 71, 445.
22. Svenson, S.; Chauhan, A. S. Nanomedicine 2008, 3, 679.
23. Sella, E.; Shabat, D. J. Am. Chem. Soc. 2009, 131, 9934.
24. Zhu, S. J.; Hong, M. H.; Tang, G. T.; Qian, L. L.; Lin, J. Y.; Jiang, Y. Y.; Pei, Y. Y.
Biomaterials 2010, 31, 1360.
25. Zhang, Y. Q.; Sun, Y. H.; Xu, X. P.; Zhang, X. Z.; Zhu, H.; Huang, L. L.; Qi, Y. J.;
Shen, Y. M. J. Med. Chem. 2010, 53, 3262.
26. Najlah, M.; Freeman, S.; Attwood, D.; D’Emanuele, A. Int. J. Pharm. 2006, 308,
175.
4.4.2. Acute ulcerogenesis assay
Kunming mice of either sex were divided into control and dif-
ferent test groups of ten animals each group (18–22 g). Ulcerogenic
activity was evaluated after oral administration of test compounds
at the dose of 200 mg/kg. Naproxen and the test compounds were
suspended and administered in a 1% methylcellulose solution.
Food and water were removed 24 h before administration of test
compounds. All animals were sacrificed after 4 h of drug adminis-
tration. Their stomachs were removed, immerged in 10% formalde-
hyde solution and 30 min later cut out along the greater curvature
of the stomach, gently rinsed with water, and placed on ice. The
number and the length of ulcers observed in each stomach were
determined by using magnifier lenses. In this assay, the severity
of each gastric lesion is measured along its greatest length
(1 mm, rating of 1; 1–2 mm, rating of 2; and >2 mm, rating accord-
ing to their length in millimeter). The UI for each test compound is
27. Najlah, M.; Freeman, S.; Attwood, D.; D’Emanuele, A. Int. J. Pharm. 2007, 336,
183.
28. Ouyang, L.; Ma, L. F.; Li, Y. H.; Pan, J. Z.; Guo, L. Arkivoc 2010, 256.
29. Ouyang, L.; Zhang, J.; Pan, J. Z.; Yan, L. N.; Guo, L. Drug Delivery 2009, 16, 348.
30. Ouyang, L.; Huang, W. C.; He, G.; Guo, L. Lett. Org. Chem. 2009, 6, 272.
31. Ouyang, L.; Ma, L. F.; Jiang, B.; Li, Y. H.; He, D. S.; Guo, L. Eur. J. Med. Chem. 2010,
45, 2705.
32. Wang, R. E.; Costanza, F.; Niu, Y. H.; Wu, H. F.; Hu, Y. G.; Hang, W.; Sun, Y. Q.;
Cai, J. F. J. Controlled Release 2012, 159, 154.
33. Wong, A. D.; DeWit, M. A.; Gillies, E. R. Adv. Drug Delivery Rev. 2012, 64, 1031.
34. Avital-Shmilovici, M.; Shabat, D. Bioorg. Med. Chem. 2010, 18, 3643.
35. Sella, E.; Weinstain, R.; Erez, R.; Burns, N. Z.; Baran, P. S.; Shabat, D. Chem.
Commun. 2010, 46, 6575.

4200
J. Wei et al. / Bioorg. Med. Chem. 21 (2013) 4192–4200
36. Erez, R.; Segal, E.; Miller, K.; Satchi-Fainaro, R.; Shabat, D. Bioorg. Med. Chem.
2009, 17, 4327.
41. Celli, N.; Dragani, L. K.; Murzilli, S.; Pagliani, T.; Poggi, A. J. Agric. Food Chem.
2007, 55, 3398.
37. Grinda, M.; Clarhaut, J.; Tranoy-Opalinski, I.; Renoux, B.; Monvoisin, A.;
Cronier, L.; Papot, S. ChemMedChem 2011, 6, 2137.
42. Calderon, M.; Graeser, R.; Kratz, F.; Haag, R. Bioorg. Med. Chem. Lett. 2009, 19,
3725.
38. Grinda, M.; Clarhaut, J.; Renoux, B.; Tranoy-Opalinski, I.; Papot, S.
MedChemComm 2012, 3, 68–70.
39. Legigan, T.; Clarhaut, J.; Renoux, B.; Tranoy-Opalinski, I.; Monvoisin, A.;
Berjeaud, J. M.; Guilhot, F.; Papot, S. J. Med. Chem. 2012, 55, 4516.
40. Sella, E.; Shabat, D. Chem. Commun. 2008, 5701.
43. Calderon, M.; Quadir, M. A.; Sharma, S. K.; Haag, R. Adv. Mater. 2010, 22, 190.
44. Shrivastava, P. K.; Singh, R.; Shrivastava, S. K. Chem. Pap. 2010, 64, 592.
45. Avital-Shmilovici, M.; Shabat, D. Soft Matter 2010, 6, 1073.
46. Huang, B. Z.; Du, D.; Zhang, R.; Wu, X. H.; Xing, Z. H.; He, Y.; Huang, W. Bioorg.
Med. Chem. Lett. 2012, 22, 7330.