Design, synthesis, and neuroprotective effects of dual-brain targeting naproxen prodrug

Article abstract of DOI:10.1002/ardp.201700382

A new dual-targeting naproxen prodrug conjugated with glucose and ascorbic acid for central nervous system (CNS) drug delivery was designed and synthesized in order to effectively deliver naproxen to the brain. Naproxen could be released from the prepared prodrugs when incubated with various buffers, mouse plasma, and brain homogenate. Also, the prodrug showed superior neuroprotective effect in vivo over naproxen. Our results suggest that chemical modification of therapeutics with warheads of glucose and ascorbic acid represents a promising and efficient strategy for the development of brain targeting prodrugs by utilizing the endogenous transportation mechanism of the warheads.

Full text of DOI:10.1002/ardp.201700382

Received: 6 December 2017
Revised: 28 February 2018
Accepted: 6 March 2018
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DOI: 10.1002/ardp.201700382
FULL PAPER
Design, synthesis, and neuroprotective effects of dual-brain
targeting naproxen prodrug
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Linhui Wang
Li Zhang
Yi Zhao
Qiuyi Fu
Wenjiao Xiao
Runxin Lu
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Li Hai
Li Guo
Yong Wu
Key Laboratory of Drug Targeting and Drug
Delivery System of Education Ministry,
Department of Medicinal Chemistry, West
China School of Pharmacy, Sichuan University,
Chengdu, P. R. China
Abstract
A new dual-targeting naproxen prodrug conjugated with glucose and ascorbic acid for
central nervous system (CNS) drug delivery was designed and synthesized in order to
effectively deliver naproxen to the brain. Naproxen could be released from the
prepared prodrugs when incubated with various buffers, mouse plasma, and brain
homogenate. Also, the prodrug showed superior neuroprotective effect in vivo over
naproxen. Our results suggest that chemical modification of therapeutics with
warheads of glucose and ascorbic acid represents a promising and efficient strategy for
the development of brain targeting prodrugs by utilizing the endogenous transporta-
tion mechanism of the warheads.
Correspondence
Prof. Li Guo and Dr. Yong Wu, 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, P. R. China.
Email: guoli@scu.edu.cn (L.G.);
wyong@scu.edu.cn (Y.W.)
Funding information
National Natural Science Foundation of China,
Grant numbers: 21472130, 81573286,
K E Y W O R D S
drug delivery, dual-brain targeting, neuroprotective, synthesis
81773577
[5–9]
1
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INTRODUCTION
exogenous substance into CNS.
Generally, the capability of
molecules to cross the barriers by passive diffusion is related to their
molecular weight, lipid solubility, charge, hydrogen bonding, ioniza-
With the continuous improvement of the social medical level and the
human life quality, the society has begun entering into aging and the
population ratio of old people is increasing quickly in most of
countries. Meanwhile, the incidence of degenerative disease,
including Alzheimer and Parkinson diseases (AD and PD), has also
significantly increased during the past few decades. Numerous
studies suggest that NSAIDs (nonsteroidal anti-inflammatory drugs),
such as naproxen, indometacin, and ibuprofen, have been used as
neuroprotective agents to treat central nervous system (CNS)
[
10–12]
tion profile, and physicochemical characteristics.
Thus, all the
macromolecular drugs and over 98% of micromolecular drugs could
not cross the barriers to achieve the effective concentration in
[
13–15]
brain.
Therefore, there is a huge amount of demand of, not
only for ibuprofen but in general, maneuver that could increase the
delivery of drugs into brain for the treatment of CNS diseases. In this
paper, we will explore the possibility to develop a brain-targeting
naproxen prodrug which uses glucose and ascorbic acid as the
mediator to improve the brain permeability.
[
1–4]
diseases.
For instance, naproxen would be a potential agent to
treat CNS disorders owing to reducing the odds of CNS diseases.
Nevertheless, the property of naproxen determined its poor ability
to cross the two physiological barriers, blood-brain barrier (BBB)
and blood-cerebrospinal fluid barrier (BCSFB), which could prevent
It seems that carrier-mediated transporter (CMT) system is an
excellent strategy to facilitate the delivery of drugs into brain, due to its
high transport affinity between the transporter and substance. There
are plenty of physiological transport systems for nutrients and
endogenous compounds, such as monocarboxylic acid transporter
(
1
MCT ), sodium-dependent vitamin C transporter 2 (SVCT
2
), neutral
Linhui Wang, Li Zhang, and Yi Zhao contributed equally to this work.
amino acid transporter (LAT
1
), and glucose transporter (GLUT
1
).
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Arch Pharm Chem Life Sci. 2018;e1700382.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
https://doi.org/10.1002/ardp.201700382

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WANG ET AL.
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GLUT
1
and SVCT
2
expressed on the surface of brain capillary
cell cytotoxicity, targeting efficiency, and neuroprotective effect
in vivo of the novel prodrug has been studied and compared with
prodrugs G-Nap and V-Nap.
endothelial cells and choroid plexus epithelium cells, respectively,
[3,8]
are considered as the most efficient transportation systems.
As we all know, glucose is an essential substance for human and
provides almost the large and uninterrupted energy demand for the
brain. According to recent researches on glucose, it is generally
believed that the transportation of glucose across the BBB is mediated
2
2
2
|
RESULTS AND DISCUSSION
Chemistry
.1
|
[16]
mainly by GLUT
1
.
Prodrugs of agents conjugated with glucose have
been proved as a novel method to facilitate the distribution of drugs
.1.1 Synthesis and characterization of the prodrugs
|
[8]
in brain. Moreover, it has been widely reported that C-6 position
glycosylation is an effective way to heighten the accumulation of drugs
The synthetic route of conjugate prodrug G-V-Nap is illustrated in
Scheme 1. Firstly, we synthesized the glycosylated derivative 5 and
the synthetic route is demonstrated in Scheme 1. Briefly, D-glucose 1
was totally etherified with chlorotrimethylsilane (TMSCl) and
hexamethyl disilazane in pyridine to give penta-O-trimethylsilyl-d-
glucopyranose 2, which was treated by the mixture of acetone,
acetic acid, and methanol to deprotect the C-6 TMS group
selectively to obtain compound 3. Then, the linker 4-(benzyloxy)-
4-oxobutanoic acid was connected to intermediate 3 to generate
compound 4, which deprotected the benzyl group to form glucose-
TMS derivative 5. The synthesis of intermediate 9 started from the
available material Vc. Briefly, in the presence of acetyl chloride, the
hydroxyl groups of the 5,6-positions of Vc reacted with acetone to
generate the 5,6-position isopropylidene protected Vc ketol 7. The
[
17]
in brain.
drugs could be released from glucose-conjugated drugs, which were
transported by the specific transporter, GLUT , to largely increase the
These evidences all indicated that
Our previous studies have also suggested that the parent
1
[8,13]
concentration in the brain.
glucose could be applied to design novel brain-targeted drugs.
On the other hand, vitamin C (Vc), also known as i-ascorbic acid,
[12]
has an important role in many enzymatic reactions.
In recent
studies, it was found that the concentration of Vc in brain is the
[3]
highest. The transportation mechanisms of Vc across the barriers
have been expressly illustrated. One is through the sugar transporter
GLUT
form of Vc and can be reduced to Vc in brain). The other way is through
the transporter SVCT which transports Vc directly into brain. It was
also reported that the C2-OH and C3-OH are critical for the
1
, which can transport dehydro-vitamin C (DHVC, the oxidized
2
2 3
ketol 7, under the conditions of K CO in acetone, was coupled with
[
3]
transportation of Vc, while the C5-OH and C6-OH are not essential.
benzyl bromide to yield 2,3-O-di-benzyl protected derivative 8 in
reflux, which deprotected the isopropylidene group under the action
of HCl to give intermediate 9, which then coupled with naproxen to
get compound 10 by using DCC as a condensing agent. The other
5-hydroxy group of intermediate 10 coupled with glucose-TMS
In the previous work of our lab, we have designed and synthesized
ibuprofen prodrugs coupled with Vc. The results suggested that these
[3,12,14]
prodrugs had good brain targeting ability.
Therefore, conjuga-
tion of potential CNS-active drugs with Vc is also an effective strategy
for brain targeting delivery of drugs.
derivative
5 by using DCC as dehydrating agent for ester
Our group has been devoted to designing the brain-targeted drugs
over a long period of time, and we have coupled drugs with glucose or
condensation to get compound 11. Finally, compound 11 readily
underwent a deprotection reaction to get compound 12 when
subjected to the condition of trifluoroacetic acid in dichloromethane,
then the 2,3-O-di-benzyl groups of 6 was removed under hydrogen
catalyzed by Pd/C to provide the target prodrug G-V-Nap. All the
title compounds and important intermediates were characterized
[3,8,12–14]
vitamin C to demonstrate their brain targeting ability.
In this
paper, we will explore the possibility to develop a dual-brain targeting
naproxen prodrug (G-V-Nap) which uses glucose and ascorbic acid as
the mediator to improve the brain permeability (Figure 1). The stability,
1
by their respective IR, H NMR and MS.
The synthesis of prodrugs G-Nap and V-Nap are illustrated in
Scheme 2. Briefly, the prepared saccharide 3 condensed with naproxen
by DCC and DMAP to give intermediate 13, which was then
deprotected by trifluoroacetic to afford prodrug G-Nap. What is
more, the prodrug V-Nap was obtained by removing the
2
,3-O-di-benzyl groups of compound 10 under hydrogen catalyzed
by 10% Pd/C.
2
.2
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Pharmacology
Cytotoxicity
2
.2.1
|
Cytotoxicity is one of the most critical factors to be considered in
selecting conjugates for biomedical applications. The toxicity of the
prodrugs against PC12 cells was evaluated by MTT assays and the
FIGURE 1 Structure of the naproxen prodrug G-V-Nap

WANG ET AL.
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SCHEME 1 Synthesis of prodrug G-V-Nap. Reagents and conditions: (a) HMDS, TMSCl, pyridine, 0°C to r.t.; (b) AcOH, CH
3
OH, acetone,
CO
, 0°C
0
°C; (c) 4-(benzyloxy)-4-oxobutanoic acid, DCC, DMAP, CH
acetone, reflux; (g) HCl, CH CN, 30°C; (h) naproxen, DCC, DMAP, CH
to r.t., (k) Pd/C, H , CH OH, r.t.
2
Cl
2
; (d) Pd/C, H
2
, methanol, r.t.; (e) acetone, acetyl chloride, r.t.; (f) BnBr, K
Cl , r.t.; (j) TFA, CH Cl /CH NO
2
3
,
3
2
Cl , r.t.; (i) 5, DCC, DMAP, CH
2
2
2
2
2
3
2
2
3
results are shown in Figure 2. At the dose level of 0–500 µM,
Table 1. It could be seen that all the prodrugs appeared to be highly
stable in pH 2.5 buffer solution, moderately stable in pH 5.0 and 7.4,
and instable in pH 8.0 buffer solution. According to this result, the slow
hydrolysis of the prodrugs ensured that the parent drug naproxen
could be slowly released and sustained in the physiological environ-
ment at pH 7.4.
the prodrugs had limited toxicity on PC12 cells.
2
.2.2 In vitro stability in various buffers
|
In order to investigate the chemical stability of prodrugs G-V-Nap,
G-Nap, and V-Nap, they were incubated in phosphate buffers of pH
2
3
.5, 5.0, 7.4, and 8.0, respectively. These solutions were maintained at
7°C, and the aliquots were withdrawn in predetermined time
2
.2.3 Metabolic stability in plasma and brain
|
homogenate
intervals, then the concentrations of the conjugates were determined
by HPLC method. The pseudo first order rate constants (Kdisapp) and
half-lives (t1/2) of these prodrugs in aqueous solutions were calculated
by linear regression of the peak area against time, and are illustrated in
The metabolic stability of the prodrugs was studied in the mice plasma
and brain homogenate at 37°C. The Kdisapp and t1/2 of the prodrugs in
plasma and brain homogenate were calculated by linear regression of
SCHEME 2 Synthesis of prodrugs G-Nap and V-Nap. Reagents and conditions: (a) naproxen, DCC, DMAP, CH
CH Cl , 0°C; (c) Pd/C, H , CH OH, r.t.
2 2 3 2
Cl , r.t.; (b) TFA, CH NO ,
2
2
2
3

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WANG ET AL.
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TABLE 2 Metabolic stability of prodrugs in mice plasma extracts and
brain homogenate at 37°C
Kinetic constants
−
1
Prodrug
Biological matrix
Plasma
Brain
K
disapp (min
)
t1/2 (min)
−3
−2
−3
−2
−2
−2
G-V-Nap
9.86 × 10
3.76 × 10
8.60 × 10
2.27 × 10
1.17 × 10
3.52 × 10
70.28
18.43
80.64
30.56
59.16
19.72
G-Nap
V-Nap
Plasma
Brain
Plasma
Brain
FIGURE 2 Cell viability upon addition of prodrugs in PC12 cells.
Cell viability was expressed as a percentage of the control cell
culture. Values are represented as mean ± SD (n = 3)
demonstrated that the prodrugs possessed favorable physicochemical
properties, we proceeded with the neuroprotective effect studies
in vivo.
2
.2.4 Brain targeting efficiency in vivo
|
the peak area against time (Table 2). As expected, the three prodrugs
It is obvious that the prodrugs could be delivered to the brain after i.v.
administration (Figure 3). The concentration of naproxen was
much higher when prodrugs were administrated than when naked
naproxen was used at any interval during 8 h. The AUC0−t and Cmax of
naproxen for these prodrugs were fairly higher than that of naked
naproxen (Table 3). The relative uptake efficiencies (REs) were
enhanced for naproxen from prodrugs G-V-Nap, G-Nap, and V-Nap,
for which the values were 2.10, 2.38, and 2.63 times higher,
respectively, than that of the naked naproxen. The concentration
efficiencies (CEs) were also increased to 4.13, 4.20, and 5.24 times
higher than that of naproxen. The higher REs and CEs of G-V-Nap
demonstrated that the G-V moiety was a suitable brain targeting
modifying group, which improved the concentration of naproxen in the
brain.
showed considerable stability in plasma, where t1/2 were found to be
7
0.28, 80.64, and 59.16 min for prodrug G-V-Nap, G-Nap, and V-Nap,
respectively. It is relatively reasonable to give enough time for the
compounds to be distributed and reach the brain before complete
decomposition.
As shown in Table 2, all the prodrugs had a faster metabolic rate in
brain homogenate than in plasma. The t1/2 of the G-V-Nap, G-Nap, and
V-Nap in brain homogenate were 18.43, 30.56, and 19.72 min,
respectively, which indicated that these prodrugs were hydrolyzed
rapidly in the brain. It may be attributed to the abundant esterase in
brain that could degrade the prodrugs to release the parent drug
naproxen, avoiding the aggregation effects of prodrugs and improving
the concentration of naproxen in the brain. As the above studies in vitro
TABLE 1 Chemical stability of prodrugs at 37°C
Kinetic constants
−1
Prodrug
pH value
Kdisapp (h
)
t1/2 (h)
−
2
G-V-Nap
2.5
6.45 × 10
2.65 × 10
3.77 × 10
4.15 × 10
3.45 × 10
2.76 × 10
4.84 × 10
1.11 × 10
6.45 × 10
2.65 × 10
3.77 × 10
4.15 × 10
10.75
2.62
−1
−1
−1
−2
−2
−2
−1
−2
−1
−1
−1
5
7
8
.0
.4
.0
1.84
1.67
G-Nap
V-Nap
2.5
20.07
25.08
14.33
6.27
5
7
8
.0
.4
.0
2.5
10.75
2.62
5
7
8
.0
.4
.0
FIGURE 3 Concentration curves in brain homogenate versus
time after administration of naproxen (Nap), G-V-Nap, G-Nap, and
V-Nap (n = 3)
1.84
1.67

WANG ET AL.
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TABLE 3 Pharmacokinetic parameters of naproxen in brain homogenate (n = 3)
Parameters
Naproxen
Nap from G-V-Nap
6313.13 ± 730.88
165.01 ± 28.90
45
Nap from G-Nap
5170.64 ± 170.33
181.88 ± 10.28
40
Nap from V-Nap
AUC(0−t) (μg/g · min)
MRT (min)
2624.08 ± 767.24
5507.41 ± 574.57
172.14 ± 13.96
40
167.91 ± 27.28
T
max (min)
max (μg/g)
15
C
13.66 ± 2.70
38.71 ± 2.56
2.40
25.60 ± 2.99
1.97
29.56 ± 1.23
2.09
RE
CE
–
–
2.83
1.87
2.16
2
.2.5
|
Neuroprotective effect
sham group). Though not significant different with naproxen, the SOD
activity of prodrug G-Nap group was significantly higher than saline
group. Moreover, the SOD activity of V-Nap and G-V-Nap groups had
significant raise compared with saline group or naproxen group, which
indicated their delighted neuroprotective function. The MDA level of
ischemia rats (saline 250.43%) was more than twice that in the sham
group, whilenaproxen ortheprodrugscouldsuppresstheincreaseinthe
MDA content caused by the injury with cerebral ischemia shown in
Figure 5B (naproxen 193.41%, G-Nap 164.60%, V-Nap 142.14%, G-V-
Nap 135.71%). On the contrary, the levels of GSH in administration
groups were significantly increased than that in the saline group
In the in vivo study, the brain ischemic injury model was established by
ligating bilateral carotid arteries. TTC (2,3,5-triphenyltetrazolium
chloride) staining is commonly used to quantify experimental tissue
infarctions. TTC, a marker of mitochondrial oxidative enzyme function,
can accept a proton system and reduce itself to a red insoluble
formazan product in normal brain sections. As shown in Figure 4A, five
brain sections of saline group, almost pale, indicated that the ischemia
model was successfully established with serious brain damage. The
increased neuroprotective function by administration of naproxen or
these prodrugs can be seen from Figure 4A with the red-staining rate
compared with the saline group. What is more, the prodrug G-V-Nap
had the optimal therapeutic effect with the highest red-staining rate
(
Figure 5C). The TTC staining, SOD activity, MDA, and GSH level all
indicated that these prodrugs, especially G-V-Nap, had superior
neuroprotective function. Moreover, the results also suggested that
the glucose-vitamin C system (G-V) could act as a vector to enhance
the delivery of CNS drugs into brain. Given its efficiency and ease of
synthesis, this approach may be applied in the design of other brain
targeting drugs.
(
77.57%) compared with the other treatment groups (naproxen
4
3.52%, G-Nap 50.29%, V-Nap 59.70%) shown in Figure 4B.
Superoxide dismutase (SOD) is one of the most important anti-
oxidases. Malonaldehyde (MDA), a degradation product of lipid
peroxidation, has been widely used as an index of cell membrane
damage. Glutathione (GSH) is a substrate of glutathione peroxidase,
which could scavenge reactive oxygen species (ROS) in vivo. In this
study, we used SOD, MDA, and GSH as antioxidant markers. As shown
in Figure 5A, the naproxen administration did not obviously reverse the
decrease of SOD activity in the ischemia injury animals (76.05% of the
3
|
CONCLUSION
In order to develop an efficient brain targeting drug delivery system
to greatly improve the brain permeability, we designed, synthesized,
FIGURE 4 The neuroprotective effect on brain ischemia model. (A) TTC staining of the brain sections, (B) percentage of TTC staining rate
compared with the sham group. ***p < 0.001, “a” indicates significance at p < 0.05 versus saline group, “b” indicates significance at p < 0.05
versus naproxen group, “c” indicates significance at p < 0.05 between G-Nap or V-Nap group and G-V-Nap group. Data are presented as
mean ± SD (n = 5)

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WANG ET AL.
|
FIGURE 5 The neuroprotective effect of naproxen and the prodrugs. (A) SOD activity, (B) MDA level, (C) GSH level. **p < 0.01,
**p < 0.001, “a” indicates significantly at p < 0.05 versus saline group, “b” indicates significantly at p < 0.05 versus naproxen group, “c”
indicates significantly at p < 0.05 between G-Nap or V-Nap group and G-V-Nap group. Data are presented as mean ± SD (n = 5)
*
and evaluated a dual-targeting prodrug. Using naproxen as the
model parent drug, we added both glucose and vitamin C onto it. The
G-V moiety significantly increased the naproxen uptake in brain and
the neuroprotective effect compared with the naked naproxen or
the singly modified prodrugs in vivo. This suggested that both
4.1.2 Synthesis of compounds 2–9
|
The synthesis of compounds 2–9 was reported in our previous
[
3,8]
work.
1 2
glucose and vitamin C transporters (GLUT and SVCT ) might be
4
.1.3 Synthesis of compound 10
|
involved to help the co-modified prodrug to penetrate the
BBB. Therefore, the G-V modification represents a promising
strategy for the development of future brain-targeting drug delivery
systems.
2 2
To a solution of naproxen (0.59 g, 2.55 mmol) in CH Cl (20 mL) was
added DCC (1.16 g, 5.61 mmol) and DMAP (0.17 g, 1.40 mmol), and
the reaction was stirred at −5°C for 30 min. Then compound 9
(
2 2
1.00 g, 2.81 mmol) in CH Cl (10 mL) was added dropwise. After
stirring for another 10 h at room temperature, the mixture was
filtered and the filtrate was concentrated. Then the residue was
4
4
4
|
EXPERIMENTAL
Chemistry
purified by chromatography to give 10 (0.80 g, 55.2%) as a white
.1
|
1
solid. H NMR (400 MHz, CDCl
3
) δ: 1.58 (d, 3H, J = 6.8 Hz), 3.44–
3
4
.49 (m, 1H), 3.90 (s, 3H), 3.96–4.02 (m, 1H), 4.15–4.21 (m, 1H),
.1.1 General
|
.29–4.35 (m, 1H), 4.49–4.50 (m, 1H), 5.02–5.16 (m, 4H), 7.08–7.70
+
(
34 32 8
m, 16H). HRMS: (ESI+) calculated for C H O Na [M+Na]
Naproxen and ibuprofen were obtained from National Institute for
Food and Drug Control. All liquid reagents were distilled before use.
The other chemicals and reagents were obtained from commercial
sources.
5
91.1995, found: 591.1992. Elemental analysis: C, 71.82; H, 5.67,
found: C, 71.88; H, 5.61.
The melting point was measured on a YRT-3 melting point
4
.1.4 Synthesis of compound 11
|
apparatus (Shantou Keyi instrument
& Equipment Co. Ltd.,
Shantou, China). Infrared (IR) spectra were obtained on a Perkin
2 2
To a solution of compound 5 (0.48 g, 0.85 mmol) in CH Cl (10 mL) was
1
Elmer983 (Perkin Elmer, Norwalk, CT). H NMR spectra were taken
added DCC (0.23 g, 1.23 mmol) and DMAP (14 mg, 0.11 mmol), and the
on a Varian INOVA 400 (Varian, Palo Alto, CA) using CDCl
3
,
reaction was stirred at −5°C for 30 min. Then compound 10 (0.32 g,
dimethylsulfoxide-d (DMSO-d and as solvent. High
6
6
)
D
2
O
2 2
0.56 mmol) in CH Cl (5 mL) was added dropwise. After stirring
resolution mass spectroscopy data of the product were collected
on a Waters Micromass GCT or a Bruker Apex IV FTMS instrument.
Reversed-phase chromatography performed on C18 chro-
matographic analysis (ODS-C18 column, 4.6 mm × 200 mm, 5 mm,
SinoChrom, Dalian, China) was carried out using the high
performance liquid chromatography (HPLC) system (Shimadzu,
overnight at room temperature, the mixture was filtered and the
filtrate was concentrated. Then the residue was purified by
1
chromatography to give 11 (0.25 g, 39.7%) as a white solid. H NMR
3 3 3
(400 MHz, CDCl ) δ: 0.07–0.18 (s, 36H, −(SiCH ) × 4), 1.55 (dd, 3H,
J = 7.2 4.0 Hz), 2.32–2.55 (m, 4H), 3.34–3.42 (m, 2H), 3.75–3.98 (m,
4H), 3.90 (s, 3H), 4.26–4.35 (m, 3H), 4.58–4.62 (m, 1H), 5.00 (d, 1H,
Kyoto, Japan) consisted of
Shimadzu) and Allchorom plus data operator.
NMR spectra of selected compounds and the InChI codes of the
investigated compounds are provided as Supporting Information.
a
RF-530 fluorescence detector
J = 2.8 Hz), 5.05–5.19 (m, 4H), 5.34–5.39 (m, 1H), 7.08–7.70 (m, 16H).
+
(
HRMS: (ESI+) calculated for C56
H
78
O
16Si
4
Na [M+Na] 1141.4265,
found: 1141.4260. Elemental analysis: C, 60.08; H, 7.02; Si, 10.03,
found: C, 60.17; H, 6.89; Si, 10.15.

WANG ET AL.
| 7 of 9
4
.1.5
|
Synthesis of compound 12
in a 5% CO
USA).
2
humidified environment incubator (Thermo Scientific,
To a solution of compound 11 (0.24 mg, 0.21 mmol) in CH
CH NO (5 mL/5 mL) was added trifluoroacetic acid (0.32 mL,
.25 mmol) at 0°C and then the reaction was stirred for 3 h at room
2 2
Cl /
The cytotoxicity of prodrugs and naproxen was assayed in PC12
cells using the (3,4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium
3
2
4
(
MTT) assay. Briefly, PC12 cells were seeded in 96-well plates at a
temperature. The mixture was concentrated and the residue was
4
density of 6 × 10 cells/well and cultured for 24 h at 37°C. The cells
were then treated with prodrugs and naproxen (10, 20, 50, 100, 200,
and 500 µM), respectively, and incubated for 24 h. After incubation,
purified by chromatography to give compound 12 (0.12 mg, 68.5%) as
1
a clear oil. H NMR (400 MHz, CDCl
3
) δ: 1.53 (d, 3H, J = 5.6 Hz),
2
4
7
.25–2.37 (m, 4H), 3.26–3.56 (m, 8H), 3.80–3.99 (m, 5H), 3.87 (s, 3H),
2
0 µL MTT solution (5.0 mg/mL) was added to the medium and
.24–4.34 (m, 4H), 4.53–4.57 (m, 1H), 4.99–5.12 (m, 4H), 5.27 (br, 1H),
incubated for another 4 h at 37°C. After removal of the culture
medium, the reduced MTT dye was solubilized by DMSO (100 µL) and
the absorbance was read at 490 nm wavelength on an automatic
microplate spectrophotometer. The cell viability = A490 nm for the
treated cells/A490 nm for the control cells × 100%.
46
.06–7.68 (m, 16H). HRMS: (ESI+) calculated for C44H O16Na
+
[
M+Na] 853.2684, found: 853.2680. Elemental analysis: C, 63.61;
H, 5.58, found: C, 63.69; H, 5.52.
4
.1.6 Synthesis of prodrug G-V-Nap
|
To a solution of compound 12 (0.12 g, 0.14 mmol) in methanol (10 mL),
Pd/C (10 mg, 10%) was added. Then, the mixture was stirred in
hydrogen atmosphere at room temperature for 2 h. Pd/C was filtered,
4.2.2 In vitro stability in various buffers
|
The in vitro chemical stability of prodrugs was investigated by
incubating the prodrugs in pH 2.5, 5.0, 7.4, and 8.0 phosphate buffers,
respectively. Briefly, 1 mL of the prodrugs methanol solution (250 µg/
mL) was added into 4 mL different buffer separately. The entire system
was kept at 37 ± 0.5°C with continuous shaking at 100 rpm/min. A
total of 200 µL medium samples were taken at the following time
points: 0, 0.5, 1, 2, 3, 6, 12, and 24 h, and then replaced by equivalent
fresh medium. The samples were analyzed by high performance liquid
chromatography (Shimadzu, Japan). The analysis was accomplished on
a SinoChrom ODS-C18 column (200 mm × 4.6 mm, 5 mm), thermo-
stated at 35°C. The mobile phase was composed of methanol/water
and the filtrate was concentrated to give prodrug G-V-Nap (80 mg,
1
8
8.9%) as a white solid. H NMR (400 MHz, CD
3
OD) δ: 1.54 (t, 3H,
J = 7.2 Hz), 2.29–2.42 (m, 4H), 3.11–5.40 (m, 15H), 3.88 (s, 3H), 7.11 (d,
1
7
6
H, J = 8.8 Hz), 7.19 (s, 1H), 7.37 (d, 1H, J = 8.8 Hz), 7.66 (s, 1H),
+
34
.70–7.73 (m, 2H). HRMS: (ESI+) calculated for C30H O16Na [M+Na]
73.1745, found: 673.1748. Elemental analysis: C, 55.39; H, 5.27,
found: C, 55.30; H, 5.36.
4
.1.7 Synthesis of prodrug G-Nap
|
(
73:27), and the pH was adjusted to 2.86 with 10% phosphoric
acid. The UV wavenumber was 238 nm and the flow rate was set at
.0 mL/min with a 20 µL injection volume.
[13]
The synthesis of prodrug G-Nap was reported in our previous work.
1
4
.1.8 Synthesis of prodrug V-Nap
|
4
.2.3 Metabolic stability in plasma and brain
|
To a solution of compound 10 (0.15 g, 0.27 mmol) in methanol (10 mL),
Pd/C (20 mg, 10%) was added. Then, the mixture was stirred in
hydrogen atmosphere at room temperature for 2 h. Pd/C was filtered,
homogenate
The preliminary metabolic stability of prodrugs was studied by
incubating in plasma and brain homogenate, respectively. A total of
and the filtrate was concentrated to give prodrug V-Nap (85 mg,
1
8
0.8%) as a white solid. H NMR (400 MHz, CD
3
OD): δ: 1.55 (d, 3H,
1
mL of the prodrugs methanol solution (250 µM) was added into 4 mL
plasma or brain homogenate. Then the entire system was kept at
7 ± 0.5°C with continuous shaking at 100 rpm/min. A total of 200 µL
J = 7.2 Hz), 3.88 (s, 3H), 3.94 (dd, 1H, J = 7.2,14.0 Hz), 4.01–4.04 (m,
1
9
3
H), 4.14–4.29 (m, 2H), 4.50–4.51 (m, 1H), 7.09 (dd, 1H, J = 2.4 Hz,
3
.2 Hz), 7.19 (d, 1H, J = 2.0 Hz), 7.39 (d, 1H, J = 8.4 Hz), 7.67–7.73 (m,
samples were taken at the following time points: 0, 0.5, 1, 2, 3, 6, 12,
and 24 h, and then replaced by plasma or brain homogenate. The
internal standard (ibuprofen, 10 µL, 30 µg/mL) was added to each
sample followed by 200 µL acetonitrile. After centrifuging for 15 min,
the supernatants of the samples were analyzed by HPLC method
described before.
+
H). HRMS: (ESI+) calculated for C20
H
20
O
8
Na [M+Na] 411.1056,
found: 411.1059. Elemental analysis: C, 61.85; H, 5.19, found: C,
1.96; H, 5.10.
6
4
.2
|
Pharmacology
Evaluation of cytotoxicity
4
.2.1
|
4.2.4 Brain targeting efficiency in vivo
|
The PC12 cells were purchased from the American Type Culture
Collection (Rockville, MD, USA). Cells were cultured in
DMEM (Hyclone, USA) supplemented with 10% FBS (Gibco, USA),
Kunming mice weighing 20–22 g were purchased from DOSSY
Experimental Animal Center of Chengdu (Sichuan, China). All animal
procedures for this study were approved by the Experiment Animal
Administrative Committee of Sichuan University (P. R. China). The
1
00 U/mL penicillin and streptomycin. Cells were cultured at 37°C

8
of 9
WANG ET AL.
|
aqueous solutions of naproxen and prodrugs (G-V-Nap, G-Nap, and
V-Nap) were prepared with 0.1% Tween-80 to give a final concentra-
tion of 1 mg/mL. The mice were injected with the naproxen or prodrugs
solutions (10 mg/kg, calculated as naproxen) through the tail vein.
At appropriate time interval (5, 10, 15, 30, 45, 60, 90, 120, 240,
through a middle incision of the neck and careful separation from the
vagal nerves. Then, the arteries were enduringly ligated with bulldog
clamps for 1.5 h (the arteries in sham-operated group were only
exposed without ligation). The brains were isolated, washed with PBS,
frozen at −20°C for 30 min, and cut into 2 mm slices. The brain slices
were stained in 1% TTC at 37°C for 30 min in dark. Stained sections
were photographed with digital camera and the staining rate was
calculated by ImageJ (National Institutes of Health, USA).
4
80 min), the animals were killed by cervical dislocation. The brain
tissue was removed, and then washed with saline for three times to
remove the remained blood and rolled over on the filter paper to
remove the main vessel. All the brain tissues were homogenized with
twice the amount of saline. An aliquot of 10 µL of internal standard
Furthermore, the remained brain was adequately homogenized
with ice-cold saline to determine the levels of SOD, MDA, and GSH.
The activity of SOD, the content of MDA, and the level of GSH were
measured by using commercial assay kits (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). The tests were performed
according to the manufacturer's instructions.
(
ibuprofen, 30 µg/mL) was added into 200 µL brain homogenate,
the mixture was vortexed with 200 µL methanol for 5 min. Then an
aliquot of 50 µL of 6 M NaOH aqueous solution was added to the
spiking mixture. After 10 min of hydrolysis at room temperature,
naproxen was released from the prodrugs, and 50 µL of 6 M HCl was
added to neutralize NaOH. Followed by vortexing for 5 min, 0.6 mL
acetonitrile was added and the mixture was centrifuged at
4
.2.6 Statistical analysis
|
1
5000 rpm for 10 min. The separated supernatant was evaporated
All the data were presented as mean ± standard deviation (SD).
Statistical comparisons were performed by analysis of variance
(ANOVA) for multiple groups followed by Student's t-test. The
threshold for significance was p < 0.05.
to dryness at 40°C under air flow. The residues were redissolved in
5
2
0 µL methanol and centrifuged at 15000 rpm for 10 min, and then
0 µL of the supernatant was injected into the HPLC system for
analysis.
The area under the curve (AUC0−t) and the maximal concentra-
ACKNOWLEDGMENT
tion (Cmax) were calculated by Data and Statistics (DAS, Shanghai,
China). The statistical analysis of the samples was performed by
using a Student's t-test with p-values <0.05 as the minimal level of
significance. The relative uptake efficiency (RE) and concentration
efficiency (CE) were calculated to evaluate the brain targeting
property of prodrugs. The values of REbrain and CEbrain are defined as
follows:
This work was supported by the National Natural Science Foundation
of China (Nos. 81573286, 81773577, and 21472130).
CONFLICT OF INTEREST
The authors declare no competing financial interest.
RE ¼ ðAUC0ꢀtÞPro=ðAUC0ꢀtÞNap
CE ¼ ðCmaxÞPro=ðCmaxÞNap
,
ORCID
:
Yong Wu
http://orcid.org/0000-0003-0719-4963
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