Design, synthesis and biological evaluation of brain targeting l-ascorbic acid prodrugs of ibuprofen with "lock-in" function

Article abstract of DOI:10.1016/j.ejmech.2014.05.072

A novel brain targeting l-ascorbic acid derivatives with "lock-in" function were designed and synthesized as prodrugs to achieve the effective delivery of ibuprofen to brain by glucose transporter 1 (GLUT₁) and the Na⁺-dependent vitamin C transporter SVCT₂. Ibuprofen-loaded four prodrugs were tested in the animals. Results from the in vivo distribution study after i.v. administration of these four prodrugs and naked ibuprofen indicated that four prodrugs exhibited excellent transport ability across the BBB and significantly increased the level of ibuprofen in brain. Among them, prodrugs 4 showed higher brain concentration. Both biodistribution data and pharmacokinetic parameters suggested that l-ascorbic acid thiamine disulfide delivery system was a promising carrier to enhance CNS drug's delivery ability into brain.

Full text of DOI:10.1016/j.ejmech.2014.05.072

European Journal of Medicinal Chemistry 82 (2014) 314e323
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 brain targeting
-ascorbic acid prodrugs of ibuprofen with “lock-in” function
L
,
,
Yi Zhao a 1, Boyi Qu a 1, Xueying Wu b, Xiaocen Li a, Qingqing Liu a, Xiuxiu Jin a, Li Guo 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, PR China
b School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel brain targeting L-ascorbic acid derivatives with “lock-in” function were designed and synthesized
Received 16 April 2014
Received in revised form
30 May 2014
Accepted 31 May 2014
Available online 2 June 2014
as prodrugs to achieve the effective delivery of ibuprofen to brain by glucose transporter 1 (GLUT1) and
the Naþ-dependent vitamin C transporter SVCT2. Ibuprofen-loaded four prodrugs were tested in the
animals. Results from the in vivo distribution study after i.v. administration of these four prodrugs and
naked ibuprofen indicated that four prodrugs exhibited excellent transport ability across the BBB and
significantly increased the level of ibuprofen in brain. Among them, prodrugs 4 showed higher brain
concentration. Both biodistribution data and pharmacokinetic parameters suggested that L-ascorbic acid
thiamine disulfide delivery system was a promising carrier to enhance CNS drug's delivery ability into
brain.
Keywords:
L-Ascorbic acid
Brain targeting
Ibuprofen
© 2014 Elsevier Masson SAS. All rights reserved.
Thiamine disulfide
Synthesis
Prodrugs
1. Introduction
therapeutic effect, ibuprofen in the CNS needs to reach a higher
concentration. However, because of the poor permeability of
Over the past few decades, with the rapid development of
medical science, many diseases are gradually being overcome. But
the treatment of central nervous system (CNS) diseases, such as
Alzheimer's disease (AD) and Parkinson's disease, is still a major
challenge for the medical field. One of the crucial problems in
drug delivery into the CNS is the low permeability of drugs due to
the bloodebrain barrier (BBB). The BBB plays an important role in
its further protecting action towards the brain microenvironment,
the function of BBB is not only a physical barrier but also a
biochemical barrier [1]. Difficulties in crossing the BBB often
hinder the bioavailability of drugs in the treatment of CNS dis-
eases, such as those diseases mentioned above. Numerous studies
[2,3] have suggested that chronic intake of nonsteroidal anti-
inflammatory drugs (NSAIDs), such as ibuprofen, could reduce
the risk or even delay the onset of CNS diseases [4,5]. Therefore,
ibuprofen may be seen as a very promising drug for the treatment
of CNS diseases [6e8]. In order to achieve the satisfactory
NSAIDs, the larger doses are required to achieve the desired
therapeutic effect. This will cause a lot of gastrointestinal adverse
effects and some toxicity, and may cause some damage to the body
[9]. Therefore, the application of ibuprofen has been greatly
restricted, and a new promising strategy should be developed to
overcome the problems of low BBB permeability of ibuprofen and
also to improve brain delivery of ibuprofen for the treatment of
CNS diseases.
L
-Ascorbic acid (AA), which is usually mentioned as vitamin C, is
essential for many enzymatic reactions, and it serves to scavenge
free radicals in order to protect tissues from oxidative damage [10].
In recent studies, the highest concentration of AA can be found in
brain tissue, where it acts as an antioxidant and plays an important
physiological function [11,12]. According to the recent researches
on AA, the transport and storage mechanisms of AA in the brain
have been clearly explained. It is generally believed that the
transportation of AA in the brain is mainly based on two distinct
ways: the facilitative sugar transporters of the GLUT type can
transport the oxidized form of AA, dehydro-ascorbic acid (DHAA),
which is then reduced into AA, and will retain in the brain and
enhance to its CNS level [13,14]; while Naþ-dependent vitamin C
transporter SVCT2 transports AA directly into brain [15]. Based on
* Corresponding authors.
E-mail address: wyong@scu.edu.cn (Y. Wu).
These authors contributed equally to this study and share first authorship.
1
http://dx.doi.org/10.1016/j.ejmech.2014.05.072
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
315
the above two transport ways, let us have a deeper understanding
of the AA.
Recently, much studies reported that AA could be used as a
carrier to improve its BBB permeation properties and then to pro-
mote brain drug delivery [16,17]. It was reported that the hydroxyl
groups of enediol lactone in C2 and C3 are the important reaction
sites and are required for the reducing properties of AA in the redox
process while the C6-hydroxyl group of AA is not critical for the
transport [18,19]. Because of these reports, most researchers
focused their attention on the preparation of C6eO-modified AA,
while we are also more curious about the preparation of C5eO-
modified AA, and also C5&C6eO-di-derivatized AA (Fig. 1). Our
previous work gave the answer for these ideas: it was showed that
C5eO-ibuprofen-AA (Prodrug 2), had the similar or even better
targeting ability than C6eO-ibuprofen-AA (Prodrug 1), which
indicated that C5eOH of AA is a preferred position for modification.
Fig. 2. The structure of prodrug 4.
prodrug 1, 2, 3 [20] as the reference. In this paper these four
designed prodrugs were described in detail. Furthermore, the brain
biodistribution and pharmacokinetics of these prodrugs in mice
after i.v. administration were discussed.
The RE, relative uptake efficiency ((AUC0et)pro/(AUC0et)ibu) were
enhanced to 2.03, 2.25 times than that of ibuprofen. Furthermore,
C5eO-&C6eO-di-ibuprofen-AA (Prodrug 3), whose C5eO&C6eO
positions were both modified, also had good targeting ability for
brain. And the RE was enhanced to 3.51 times than that of ibuprofen
[20]. Therefore, the above results indicated that C5&C6-hydroxyl
groups of AA may be both not critical for the transport.
2. Chemistry
The C6eO-modified AA prodrug 1, C5eO-modified AA prodrug 2
and C5&C6eO-di-derivatized AA prodrug 3 were synthesized ac-
cording to the reported procedures in the reference 20. The syn-
thetic routes of prodrug 1, 2, 3 were outlined in Schemes 1 and 2.
The synthetic route of prodrug 1 and 3 started from the available
material AA. Briefly, in the presence of acetyl chloride, the hydroxyl
groups of the 3, 4 positions of AA reacted with acetone to generate
the 3,4-position isopropylidene protected AA ketol 5. The ketol 5,
under the conditions of K2CO3 in acetone, was coupled with benzyl
bromide to yield 2,3-O-di-benzyl protected derivative 6 in reflux,
which deprotected the isopropylidene group under the action of
HCl to give intermediate 7. Then, coupled with the different number
of equivalents of ibuprofen, intermediate 7 to get compound 8 or
compound 9 by using DCC as a condensing agent, respectively.
Finally, the debenzylation of the 2,3-O-di-benzyl groups of 8 and 9
with 10% Pd/C reached the prodrug 1 and 3, respectively.
The synthesis of prodrug 2 was different from prodrug 1 and 3,
where it focused on the protection of C6-hydroxyl group of AA
with suitable protecting group, thus exposing C6-hydroxyl group
to couple with ibuprofen. Initially, we tried to introduce a phenyl
group at the C3, C4 and C6 hydroxyl group at the same time, which
can be removed together at the last step. Unfortunately, due to the
difficult to control, we did not get a satisfactory yield of the
product. Consequently, trityl chloride was chosen to selectively
protect the C6-hydroxyl group of intermediate 7 to obtain com-
pound 10. Similarly, compound 10 was coupled with ibuprofen
used DCC as dehydrating agent for ester condensation to get
compound 11. After the detritylation of compound 11, 2,3-O-di-
benzyl derivative 12 was obtained. Finally, the 2,3-O-di-benzyl
groups of 12 was removed under hydrogen catalyzed by 10% Pd/C
to provide prodrug 2.
Although our previous work has shown that our design is
feasible, our group is still exploring how to further improve the
carrier's BBB permeation. GLUT1 and SVCT2 are bidirectional
transporters [21,22] which mediate the blood-to-brain and brain-
to-blood transport of AA in either direction across the BBB. This
indicated that the AA mediated ibuprofen would be pumped out to
the outside of the brain after their entry, which prevented the
concentration of prodrugs in the brain. In order to avoid the bidi-
rectional delivery, a lipophilic thiamine disulfide system (TDS) [23]
with the ability of “lock-in” attracted our attention. TDS is more
stable and convenient for conservation in the air, which can be
reduced by disulfide reductase and then ring-closed to be a thia-
zolium once entered the CNS, and then “locked” in the brain and
can not be transported outside across the BBB. Then, followed by
simple hydrolysis, the prodrug system can release the active drug to
play a therapeutic role [24]. This TDS modified prodrug system
seems to be a good way for the delivery, which not only can in-
crease the lipophilic prodrug itself, making it easy to cross the BBB,
but also prevent the bidirectional delivery of AA, improving the
brain drug concentration. Hence, in the present study TDS was
introduced into AA delivery system to form a novel brain targeting
L-ascorbic acid prodrug (Prodrug 4, Fig. 2) with ester linkage be-
tween ibuprofen and AA moieties with “lock-in” function. After
changing of the behavior of prodrugs form bidirectional to unidi-
rectional, this will enhance the concentration of the active drugs in
brain (Fig. 3).
Based on the assumption above, we focused on the AA prodrug
of ibuprofen with “lock-in” function, which linked TDS at C-6 po-
sition of AA and C5eO-&C6eO-di-ibuprofen-modified AA. In order
to observe the effect of prodrug 4, we also synthesized the reported
For the synthesis of the prodrug 4, the key intermediate acid 18
is essential to achieve the “lock-in” functions. The synthetic route
used to prepare key intermediate acid 18 has been illustrated in
Fig. 1. The structure of prodrugs 1, 2, 3.

316
Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
Fig. 3. Distribution, sequential metabolism and brain “lock-in” pathways of prodrug 4.
Scheme 1. The synthetic route of prodrug 1 and 3. Reagents and conditions: (a) Acetone, acetyl chloride, r.t.; (b) BnBr, K2CO3, acetone, reflux; (c) HCl, CH3CN, 30 ꢀC; (d) DCC, DMAP,
overnight; (e) 10% Pd/C, H2, 0.4 MPa.
Scheme 3. It was started from the commercially available materials
thiazole alcohol derivative 13, which was reacted with tert-butyl
bromoacetate in acetonitrile to yield tert-butyl ester 14. Then, by
the reaction of benzyl bromide with sodium thiosulfate to afford
sodium S-benzyl sulfothioate 15, which was coupled with tert-
butyl ester 14 under alkaline conditions to obtain the ring-
opened compound 16. TBDPS-Cl was coupled with the hydroxy
group of compound 16 to produce the TBDPS-protected compound
17. Finally, after the hydrolysis of the compound 17, the key inter-
mediate acid 18 was successfully obtained.
Scheme 2. The synthetic route of prodrug 2. Reagents and conditions: (a) TrtCl, Et3N, DCM, r.t.; (b) DCC, DMAP, overnight; (c) HCl, CH3CN, 50 ꢀC; (d) 10% Pd/C, H2, 0.4 MPa.

Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
317
Scheme 3. The synthetic route of key intermediate acid 18. Reagents and conditions: (a) tert-butyl bromoacetate, CH3CN, 85 ꢀC, 20 h, reflux; (b) S-benzyl sulfothioate 15, NaOH, EA,
25 ꢀC, 18 h; (c) imidazole, TBSCl, DCM, r.t., 3 h; (d) TFA, DCM, r.t., 5 h.
The synthetic pathway of prodrug 4 has been displayed in
Scheme 4. AA as the starting material first reacted with 2-
methoxyethoxymethyl chloride (MEMCl) to give the 2,3-hydroxy
group protected AA 14. The small steric hindrance 6-hydroxy
group of AA coupled with the key intermediate 18 by using DCC
as dehydrating agent for ester condensation to get ester 20. To take
off the TBDPS protecting group selectively, a number of conditions
including different kinds and concentrations of the acids were
tried. Finally the condition of 50% AcOH was chosen to take off
TBDPS group and one MEME group at 2-hydroxy to produce the
intermediate 21 with about 60% yield. Then, the exposed 2-
hydroxyl group of intermediate 21 reacted with MEMCl again to
yield the diol 22. Subsequently, the diol 22 coupled with ibuprofen
by condensation reaction also as the previous methods to obtain
the C5eO-&C6eO-di-ibuprofen ester 23. Finally, under the condi-
tions of 1% HCleMeOH, we removed the 2,3-hydroxy protecting
group to reached anticipated prodrug 4. 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. Stability in different buffer solutions
In order to investigate the chemical stability of several pro-
drugs, prodrugs 1, 2, 3, 4 were incubated in pH 2.21, 5.86, 7.33 and
7.88 phosphate buffers, respectively. These solutions were main-
tained in 37 ꢀC, and the aliquots were withdrawn in pre-
determined time intervals, then the concentrations of the
prodrugs were determined by HPLC method. The half-lives (t1/2
)
and the pseudo first order rate constants (Kdisapp) of these pro-
drugs in aqueous solutions which calculated by linear regression
of Ln of peak area against time in hours are illustrated in Table 1.
All the prodrugs appeared to be highly stable in PH 2.21 buffer
solution, and moderately stable in pH 5.86 and 7.43, and instable
in pH 7.88 buffer solution. According to this result, the slow hy-
drolysis of the prodrugs at pH 7.4 ensured that ibuprofen parent
drug could be sustained and slowly released in the physiological
environment.
Scheme 4. The synthetic route of prodrug 4. Reagents and conditions: (a) MEMCl, DIEA, DCM, r.t., 1 h; (b) intermediate acid 18, DCC, DMAP, r.t., overnight; (c) 50% AcOH, 50ꢀC, 3.5h;
(d) MEMCl, DIEA, DCM, r.t., 1 h; (e) ibuprofen, DCC, DMAP, DCM, r.t., overnight; (f) 1% HCleEtOH, 70 ꢀC, 1 h.

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Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
Table 1
3.3. In vivo studies
Chemical stability of prodrugs at 37 ꢀC.
3.3.1. Methodology evaluation
Compound
Prodrug 1
PH value
Kdisapp (hꢁ1
)
t1/2 (h)
The chromatograms of blank mouse plasma and brain tissues
showed no peaks at the retention time of ibuprofen and the in-
ternal standard naproxen. A good baseline separation of ibuprofen,
naproxen, and other major components from mouse plasma and
brain tissue samples was achieved under this chromatographic
condition.
2.21
5.86
7.33
7.88
2.21
5.86
7.33
7.88
2.21
5.86
7.33
7.88
2.21
5.86
7.33
7.88
1.73 ꢂ 10ꢁ2
3.76 ꢂ 10ꢁ2
4.67 ꢂ 10ꢁ2
8.42 ꢂ 10ꢁ2
1.61 ꢂ 10ꢁ2
3.36 ꢂ 10ꢁ2
4.30 ꢂ 10ꢁ2
7.15 ꢂ 10ꢁ2
1.76 ꢂ 10ꢁ2
3.66 ꢂ 10ꢁ2
5.58 ꢂ 10ꢁ2
8.65 ꢂ 10ꢁ2
1.29 ꢂ 10ꢁ2
2.89 ꢂ 10ꢁ2
4.27 ꢂ 10ꢁ2
6.87 ꢂ 10ꢁ2
40.1
18.4
14.8
8.2
43.0
20.6
16.1
9.7
39.5
18.9
12.4
8.1
53.9
23.9
16.2
10.1
Prodrug 2
Prodrug 3
Prodrug 4
3.3.2. Pharmacokinetics in plasma and brain of each prodrug
For in vivo study, prodrugs 1, 2, 3, 4 and ibuprofen original drug
were injected through caudal vein of the mice with a single dose
equivalent to 48 mmol/g body weight of ibuprofen respectively.
Mice from each group were sequentially sacrificed at 5 min, 10 min,
30 min, 45 min, 60 min, 90 min, 120 min and 240 min after injec-
tion, blood sample was collected from the eye socket of mice, and
placed in heparin tubes. Then blood and brain were collected to
analyze the concentration of ibuprofen at different intervals by
HPLC method.
For better expression of the in vivo behavior of each prodrug, the
plasma pharmacokinetics of ibuprofen and various prodrugs in
mice were assessed (Fig. 4). Pharmacokinetics parameters of
ibuprofen in mice were reported in Table 3 after i.v. administration
of ibuprofen and each prodrug (Prodrugs 1, 2, 3, 4).
The curves showed that the ibuprofen concentrations of pro-
drugs 1, 2, 3 and 4 were higher than that of ibuprofen and also
showed slowly declined concentrations compared to the fast
metabolism of ibuprofen drug. Because the prodrugs had certain
stability in plasma, the degradation rate slowed down, increasing
the chances of prodrugs to be rapidly transported across the BBB
and the effective concentration of ibuprofen drug. On the other
hand, the calculated result showed that the AUC0et of ibuprofen in
four types of prodrugs were much higher than that of naked
ibuprofen within 240 min after i.v. administration. Free ibuprofen
of prodrugs 1, 2, 3 and 4 presented with an area ratio of 2.71, 5.05,
3.75, and 8.42, and the Cmax for free ibuprofen of prodrugs were
2.72, 4.26, 7.40, and 8.67 times that of ibuprofen original drugs.
These data further indicated that the prodrugs could be maintained
stable in plasma. The mean residence times (MRT) of ibuprofen in
plasma after i.v. administration of prodrugs 1, 2, 3, 4 were 1.06, 1.17,
0.95 and 1.12 times that of ibuprofen, respectively. The half lives (t1/
2) and MRT of ibuprofen in these prodrugs indicated that all the
prodrugs could keep relatively high concentration in plasma during
the text time, so they all could delay the plasma clearance of
ibuprofen which is a well-known quick metabolized drug. Among
them prodrug 4 showed the best stability, probably due to its
3.2. Metabolism studies of the prodrugs
3.2.1. Metabolic stability
To investigate the metabolic stability of the prodrugs 1, 2, 3, 4 in
mice plasma extract and brain homogenate at 37 ꢀC, the pharma-
cokinetics of these prodrugs were detected. The half-lives (t1/2) and
the pseudo first order rate constants (Kdisapp) of these prodrugs in
aqueous solutions which calculated by linear regression of Ln of
peak area against time in minutes, the metabolic stability data for
these compounds were summarized in Table 2. As expected, these
four kinds of prodrugs 1, 2, 3, 4 showed considerable stability in
plasma, where t1/2 were found to be 51.4e44.2 min. Therefore,
these prodrugs have sufficient time to reach the brain before
decomposition and also gain more opportunities to get through
BBB to release the carried parent drug while in the process of de-
livery and circulation.
In brain homogenate prodrugs 1, 2, 3 showed a moderate sta-
bility, illustrating that these three compounds were metabolized
with a low rate and rarely hydrolyzed, probably due to the weak-
ness or loss of esterase in brain. For this reason, it may take some
time to release the active drug after the prodrugs were transported
into the brain. Furthermore, it may cause that a portion of the
prodrugs were excreted again to the outside of the brain, which
prevented the concentrating of parent drug in brain effectively. In
contrast, prodrug 4 converted swiftly after entering the brain, and
almost completely converted in 30 min, resulting from the disulfide
reductase which catalyzed the reduction and cyclization reaction of
prodrug 4 to become the thiazolium. Upon completion of this
transformation, the drug would be “locked” in the brain and would
not be transported outside the brain again, thereby improved the
concentration of active drug.
300
Ibuprofen concentration in Plasma
250
200
150
100
50
Table 2
Metabolic stability of prodrugs in mice plasma extracts and brain homogenate at
37 ꢀC.
Compound
Prodrug 1
Biological matrix
Kdisapp (minꢁ1
)
t1/2 (min)
Blood
Brain
Blood
Brain
Blood
Brain
Blood
Brain
1.49 ꢂ 10ꢁ2
3.46 ꢂ 10ꢁ2
1.35 ꢂ 10ꢁ2
4.16 ꢂ 10ꢁ2
1.57 ꢂ 10ꢁ2
2.48 ꢂ 10ꢁ2
1.84 ꢂ 10ꢁ2
5.46 ꢂ 10ꢁ2
46.6
20.1
51.4
16.7
44.2
27.9
27.8
12.7
Prodrug 2
Prodrug 3
Prodrug 4
0
Time(min)
Fig. 4. The concentration curves of ibuprofen in plasma versus time after i.v. injection
of prodrugs and ibuprofen in mice.

Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
319
Table 3
The pharmacokinetic parameters of ibuprofen in blood after administration of ibuprofen and prodrugs. *P < 0.05 versus ibuprofen; **P < 0.01 versus ibuprofen; ***P < 0.001
versus ibuprofen.
Compounds
AUC(0et)
(
m
g/ml$min)
MRT (h)
Cmax (ng/ml)
Tmax (h)
Ibuprofen
Prodrug 1
Prodrug 2
Prodrug 3
Prodrug 4
3405.999 517.582**
9228.515 171.038
17210.306 452.971**
12786.652 1280.341***
30116.755 767.929**
78.89 4.113
83.414 4.432
91.942 4.01
75.088 4.419
88.606 4.84
28.429 2.297
6.667
23.333
23.333
5
77.376 5.786**
121.147 9.224**
210.488 21.952***
246.528 26.776***
30
unique chemical structure extending the metabolic processes of
ibuprofen.
compared to the prodrug 2, but also indicated that C5&C6-hydroxyl
groups of AA are both not critical for the transport. Among all the
prodrugs, prodrug 4 showed the best targeting ability for brain,
which may be attributed to the characteristics of the compound.
The higher REs and CEs of modified prodrug 4 demonstrated that
TDS modified prodrug not only increases the lipophilic drug itself,
but also prevent the bidirectional delivery of the drug, improving
the brain ibuprofen concentration. Details above also indicated that
more ibuprofen was distributed into brain.
3.3.3. Distribution in brain of different prodrugs in mice
For further evaluation of the possibility of the AA-TDS-ibuprofen
prodrugs being transported across BBB, the distributions in brain of
prodrugs 1, 2, 3, 4 and ibuprofen original drug were determined.
The distribution of ibuprofen was measured at 5 min, 10 min,
30 min, 45 min, 60 min, 90 min, 120 min and 240 min after i.v.
injection. The concentrations of ibuprofen in brain versus time
curves were displayed in Fig. 5. After i.v. administration of prodrugs
1, 2, 3, 4 and ibuprofen, the pharmacokinetic parameters, relative
uptake efficiencies (RE) and concentration efficiencies (CE) of
ibuprofen in brain were reported in Table 4.
4. Conclusion
In order to develop a more optimal brain targeting drug delivery
system, we designed and successfully synthesized a serial of brain
targeting -ascorbic acid-prodrugs 1, 2, 3 and 4 of ibuprofen. After
L
In brain, it is obvious that all the four prodrugs could be deliv-
ered to the brain after i.v. administration. The concentration of
released ibuprofen from the four prodrugs at any interval was much
higher than that from naked ibuprofen during 240 min. The AUC0et
and Cmax of ibuprofen after i.v. administration of prodrugs were
fairly higher than that after the injection of naked ibuprofen, and it
was worth noting that the curves showed an increasing trend from
prodrug 1e4. The relative uptake efficiencies (REs) were enhanced
to 2.03, 2.25, 3.52, and 4.07 times that of naked ibuprofen for
prodrugs 1, 2, 3, 4, respectively. The concentration efficiencies (CEs)
were also enhanced to 2.62, 4.19, 4.96, and 7.46 times that of
ibuprofen. Therefore, these data further indicated our design is
feasible, that all four kinds of prodrugs could maintain sufficient
levels and deliver ibuprofen into the brain. The pharmacokinetic
parameters suggested that the brain targeted abilities of these
designed prodrugs had a descending trend as prodrug 4 > prodrug
3 > prodrug 2 > prodrug 1.
i.v. administration of the prodrugs 1, 2, 3 and 4, the results showed
that the concentrations of ibuprofen in brain were significantly
higher than that of naked ibuprofen with the same dose. The pro-
drugs exhibited excellent transport ability across the BBB, among
which the biodistribution data and pharmacokinetic parameters
indicated that prodrugs 4 had higher brain concentration and
AUC0et than those of other four ones. Prodrugs 4 seemed to be
easier to be transported into CNS. Briefly, the L-ascorbic acid moiety
of prodrugs 4 could be recognized by the GLUT and SVCT2, which
efficiently transported this prodrug into the brain, then TDS
modified prodrug was locked in the brain through reduction and
cyclization processes, contributing to the brain bioavailability of
ibuprofen. These expected results strongly suggest that prodrug 4
was a potent brain targeting prodrug which could enhance drug's
delivery ability into brain.
Generally, this work presented a way that the synthesis of L-
ascorbic acid-TDS-prodrugs may be used as potential means to
improve the therapeutic efficiency in brain diseases.
The result highlighted that prodrug 2, C5eO-ibuprofen-AA, had
a better targeting ability than prodrug 1, which indicated C5eOH of
AA a preferred position for modification than C6eOH. Additionally,
the targeting ability for brain of prodrug 3, whose C5eO&C6eO
positions were both modified, only showed a marginally improved
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 an 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
Ibuprofen concentration in Brain
90
80
70
60
50
40
30
20
10
0
Ibuprofen
Prodrug1
Prodrug2
Prodrug3
Prodrug4
D2O as solvent. Chemical shifts are expressed in
d (ppm), with
tetramethylsilane (TMS) functioning as the internal reference, and
coupling constants (J) were expressed in Hz. Mass spectra were
recorded on an Agilent 1946B ESI-MS instrument (Agilent, Palo
Alto, CA, USA). Ibuprofen and naproxen were obtained from Na-
tional Institute for Food and Drug Control. Soybean phospholipids
Time (min)
Fig. 5. The concentration curves of ibuprofen in brain versus time after i.v. injection of
prodrugs and ibuprofen in mice.

320
Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
Table 4
The pharmacokinetic parameters of ibuprofen in brain after administration of ibuprofen and prodrugs. *P < 0.05 versus ibuprofen; **P < 0.01 versus ibuprofen; ***P < 0.001
versus ibuprofen.
Compounds
AUC(0et)
(
m
g/g$min)
MRT (min)
Cmax
(m
g/g)
Tmax (min)
Re
CE
Ibuprofen
Prodrug 1
Prodrug 2
Prodrug 3
Prodrug 4
1856.215 160.2364
3767.9842 167.8195**
4183.3895 319.0054**
6526.7422 468.2708***
7557.692 655.9545***
116.826 5.111
106.204 5.435
101.489 2.907
109.097 2.174
90.279 6.508
10.6812 1.4904
27.9772 2.047
44.7925 2.645
53.0288 7.3462*
79.6932 9.0269**
25
45
55
5
e
e
2.029928753
2.25372034
3.516156372
4.071560676
2.619293712
4.193583118
4.964685616
7.46107179
55
(SPC) were purchased from Kelong Chemical. Cholesterol (CHOLE)
was purchased from Bio Life Science& Technology Co., Ltd
(Shanghai, China).
4CeH), 6.98e7.16 (m, 10H, AreH); 7.32e7.37 (m, 8H, AreH). MS (m/
z): 755.3 ([MþNa]þ).
5.1.5. Synthesis of prodrug 1
5.1.1. Synthesis of compound 5 [25]
To a solution of compound 8 (200 mg) in MeOH (15 ml) in an
autoclave vessel was added 10% Pd/C (30 mg), and hydrogen
pressure of 4 bar was maintained for 4 h at room temperature. Pd/C
was filtered and the filtrate was concentrated to afford prodrug 1 as
To a stirred suspension of L-ascorbic acid (20 g, 114 mmol) in
distilled acetone (150 ml) was added acetyl chloride (0.4 ml,
5.7 mmol). After stirred at room temperature for 20 h, the mixture
was filtrated. The filter cake was washed with cooled acetone and
dried to afford 5 as a white solid, which was used without purifi-
cation. Yield (73.3%), m.p: 202e204 ꢀC.
a white solid (74.7%) m.p: 126e128 ꢀC. IR (KBr, cmꢁ1): 3393, 3217,
n
2955, 2868, 1762, 1710, 1665, 1511, 1364, 1171, 1035, 819, 667; 1H
NMR (400 MHz, DMSO-d6):
0.83e0.86 (t, 6H, J ¼ 6 Hz, (CH3)2),
d
1.38e1.40 (d, 3H, J ¼ 6.8 Hz, CH3), 1.79 (m, 1H, CH(CH3)2), 2.40 (d,
2H, J ¼ 7.2 Hz, CH2Ph), 3.76e3.81 (q, 1H, J ¼ 6.8, 13.6 Hz, CHCH3),
3.91 (s, 1H, 5CeOH), 3.97e3.81 (m, 2H, 6-CH2), 4.47 (q, 1H, J ¼ 1.2,
8 Hz, 5CeH), 5.33 (s, 1H, 4CeH), 7.10 (d, 2H, J ¼ 8 Hz, AreH), 7.19 (d,
2H, J ¼ 7.6 Hz, AreH), 8.41 (bs, 1H, 2CeOH), 11.09 (bs, 1H, 3CeOH);
13C NMR (CDCl3, ppm): 18.11 (1C), 22.32 (1C), 22.36 (1C), 30.11 (1C),
44.83 (1C), 44.99 (1C), 66.84 (1C), 68.75 (1C), 74.34 (1C), 118.36
(1C), 127.15 (1C), 127.24 (1C), 128.64 (1C), 129.37 (1C), 137.20 (1C),
140.66 (1C), 153.66(1C), 174.98 (1C), 179.93 (1C) .MS (m/z): 387.4
([MþNa]þ).
5.1.2. Synthesis of compound 6 [25]
Compound 5 (6 g, 27.75 mmol) and K2CO3 powder (11.3 g,
81.5 mmol) were suspended in acetone (distilled, 100 ml). After
reflux, benzyl bromide (8.74 ml, 73.6 mmol) was added and the
mixture was allowed to reflux for another 4 h. The solvent was
removed under reduced pressure. The residue was treated with
proper H2O and Et2O. After filtrated, the title compound 6 was
afford as a white solid, which was used without purification. Yield
(44.1%).
5.1.3. Synthesis of compound 7 [25]
5.1.6. Synthesis of prodrug 3
The tetra protected -ascorbic acid compound 6 was dissolved in
L
To a solution of compound 9 (280 mg) in MeOH (15 ml) in an
autoclave vessel was added 10% Pd/C (40 mg), and hydrogen
pressure of 4 bar was maintained for 5 h at room temperature. Pd/C
was filtered and the filtrate was concentrated to afford prodrug 2 as
CH3CN (200 ml), then the aqueous solution of HCl (20 ml, 2 mol/L)
was added and the reaction mixture was stirred for 3 h at 30 ꢀC. The
solvent was removed under reduced pressure and the residue was
diluted in EtOAc. The organic layer was washed successively with
H2O and brine, then was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to afford compound 7 as a
yellow oil which slowly solidified. Yield (98%), m.p: 67e69 ꢀC.
a colorless semi-solid (75.8%) IR (KBr,
n
cmꢁ1): 3439, 3294, 3089,
3049, 2955, 2930, 2865, 2725, 1905, 1743, 1654, 1537, 1513, 1381,
1202, 1072, 1022, 849, 630, 596; 1H NMR (400 MHz, CDCl3, ppm):
d
0.87e0.88 (d, 12H, J ¼ 6.4 Hz, 2 ꢂ (CH3)2), 1.37e1.45 (m, 6H,
2 ꢂ CH3), 1.77e1.85 (m, 2H, 2 ꢂ CH(CH3)2), 2.43 (d, 4H, J ¼ 6.4 Hz,
2 ꢂ CH2Ph), 3.59e3.66 (m, 2H, 2 ꢂ CHCH3), 4.22e4.37 (m, 2H,
6CH2), 4.61e4.67 (m, 1H, 5CeH), 5.35e5.39 (m, 1H, 4CeH),
7.04e7.16 (m, 8H, AreH); 13C NMR (CDCl3, ppm): 18.11 (1C), 20.81
(1C), 22.22 (1C), 22.26 (1C), 22.33 (1C), 22.36 (1C), 30.12 (1C), 30.16
(1C), 44.78 (1C), 44.94 (1C), 45.49 (1C), 49.78 (1C), 61.97 (1C), 69.26
(1C), 71.08 (1C), 118.76 (1C), 126.84 (1C), 127.12 (1C), 127.19 (1C),
127.29 (1C), 129.25 (1C), 129.29 (1C), 129.35 (1C), 129.59 (1C),
136.98 (1C), 138.64 (1C), 140.48 (1C), 140.78(1C), 154.05(1C), 173.94
(1C), 175.73 (1C), 179.97 (1C). MS (m/z): 575.3 ([MþNa]þ), 591.5
([MþK]þ).
5.1.4. Synthesis of compound 8 and 9
To a stirred solution of ibuprofen (0.94 g, 4.57 mmol) in DCM
(30 ml) was added DCC (1.14 g, 5.48 mmol) and DAMP (55.8 mg,
0.457 mmol) and the mixture was stirred for 30 min. Compound 7
(1.5 g, 4.21 mmol) was added dropwise in DCM (10 ml) and the
reaction mixture was stirred overnight at room temperature and
then filtrated. The filtrate was concentrated under reduced pres-
sure. The residue was purified by column chromatography with
petroleumeethylacetate to give compound 8 as a pale-yellow oil
(43.6%), as well as compound 9 as a colorless oil (25%). Compound
8: 1H NMR (400 MHz, CDCl3, ppm):
d
0.82e0.85 (m, J ¼ 6 Hz (CH3)2),
1.39e1.41 (d, 3H, J ¼ 6.8 Hz, CH3), 1.76e1.82 (m, 1H, CH(CH3)2), 2.40
(d, 2H, J ¼ 7.2 Hz, CH2Ar), 3.77e3.81 (m, 1H, CHCH3), 3.97e3.81 (m,
2H, 6-CH2), 4.46e4.48 (m, 1H, Hz, 5CeH), 4.86e4.92 (d, 2 ꢂ 2H,
J ¼ 11.2 Hz, OCH2Ar), 5.23 (s, 1H, 4CeH), 7.07 (m, 4H, AreH),7.10 (d,
2H, J ¼ 8 Hz, AreH), 7.19 (d, 2H, J ¼ 7.6 Hz, AreH),7.37 (m, 6H,
AreH); MS (m/z): 567.3 ([MþNa]þ); compound 9: 1H NMR
5.1.7. Synthesis of compound 10 [25]
To a stirred solution of (6) (670 mg, 1.88 mmol) and Et3N
(0.57 ml, 4.08 mmol) in DCM (15 ml) was added TrtCl (704 mg,
2.45 mmol) in DCM (10 ml) at 0 ꢀC. Then the reaction mixture was
stirred overnight at room temperature. The reaction mixture was
washed successively with H2O and brine, then was dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The residue was purified by column chromatography with
petroleumeethylacetate to afford compound 10 as a white solid
(68.4%) m.p: 68e70 ꢀC.
(400 MHz, CDCl3, ppm):
d
0.86e0.89 (d, 12H, J ¼ 6.4 Hz, 2 ꢂ (CH3)2),
1.35e1.43 (m, 6H, 2 ꢂ CH3), 1.74e1.82 (m, 2H, 2 ꢂ CH(CH3)2),
2.36e2.43 (d, 4H, J ¼ 6.4 Hz, 2 ꢂ CH2Ph), 3.58e3.64 (m, 2H,
2 ꢂ CHCH3), 4.27e4.35 (m, 2H, 6-CH2), 4.56e4.61 (m, 1H, 5CeH),
4.84e5.07 (d, 2 ꢂ 2H, J ¼ 11.2 Hz, OCH2Ar), 5.30e5.35 (m, 1H,

Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
321
5.1.8. Synthesis of compound 11
5.1.13. Synthesis of compound 18
To a stirred solution of ibuprofen (0.65 g, 3.15 mmol) in DCM
(22 ml) was added DCC (0.65 g, 3.15 mmol) and DAMP (32 mg,
0.26 mmol). After the mixture was stirred for 30 min, compound 10
(1.57 g, 2.62 mmol) in DCM (10 ml) was added dropwise. The re-
action mixture was refluxed overnight and then filtrated. The
filtrate was concentrated under reduced pressure. The residue was
purified by column chromatography with petroleumeethylacetate
to give compound 11 as a pale-yellow oil. (77.5%) MS (m/z): 809.4
([MþNa]þ).
To a solution of compound 17 (6.4 g) in DCM (300 ml) was added
TFA (12 ml), the reaction mixture was stirred for 5 h at room
temperature. The reaction was monitored by TLC, controlling the
amount of the impurities. The reaction solution was washed with
H2O and brine, then was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography to yield compound 18 as a yellow syrup.
(38.6%) 1H NMR (400 MHz, CDCl3, ppm):
d 1.03 (s, 9H, SiC(CH3)3),
1.88 (s, 3H, NCCH3), 2.70 (t, 2H, SCCH2), 3.72 (t, 2H, CH2CH2O), 3.79
(s, 2H, SCH2Ar), 4.02 (s, 2H, COCH2N), 7.18e7.20 (m, 2H, AreH),
7.23e7.27 (m, 3H, AreH), 7.35e7.42 (m, 6H, AreH), 7.63e7.65 (m,
4H, AreH), 7.93 (s, 1H, CHO). MS (m/z): 602.1 ([MþNa]þ).
5.1.9. Synthesis of compound 12
To a stirred solution of compound 11 (1.6 g, 2.03 mmol) in
CNCH3 (30 ml) was added HCl (7 ml, 1.5 mol/L). After the reaction
mixture was stirred for 4 h at 50 ꢀC, the solvent was removed under
reduced pressure and the residue was diluted in EtOAc. The organic
layer was washed successively with H2O and brine, then was dried
over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by column chromatography with
petroleumeethylacetate to give compound 12 as a yellow semi-
5.1.14. Synthesis of compound 19 [26]
In 30 ml of DCM was added ascorbic acid (1.5 g, 8.52 mmol) and
DIPEA (3.693 ml, 20.5 mmol), the reaction mixture was stirred for
15 min at room temperature. Then, MEMCl (2.1 ml, 18.74 mmol)
was added to the reaction solution, the reaction mixture was stirred
for 30 min again at room temperature. The reaction solution was
washed with H2O, the organic layer was separated, dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The residue was purified by column chromatography to obtain
a pale-yellow oil (70.3%).
solid. (78.6%) 1H NMR (400 MHz, CDCl3, ppm):
d 0.79e0.81 (m,
6H, (CH3)2), 1.46e1.48 (d, 3H, J ¼ 7.2 Hz, CH3), 1.66e1.71 (m, 1H,
CH(CH3)2), 2.27 (d, 2H, J ¼ 7.2 Hz, CH2Ph), 3.68e3.73 (m, 1H,
CHCH3), 3.86 (s, 2H, 6-CH2), 4.58e4.61 (s, 1H, 5CeH), 4.85e4.94 (d,
2 ꢂ 2H, J ¼ 10.8 Hz, OCH2Ar), 5.16 (s, 1H, 4CeH), 6.99 (d, 2H,
J ¼ 8 Hz, AreH), 7.06e7.08 (m, 2H, AreH), 7.17 (d, 2H, J ¼ 8 Hz,
AreH), 7.35e7.41 (m, 8H, AreH); MS (m/z): 567.4 ([MþNa]þ).
5.1.15. Synthesis of compound 20
To a stirred solution of compound 18 (2 g, 2.113 mmol) in DCM
(distilled, 20 ml) was added DCC (524.8 mg, 2.536 mmol) and
DAMP (25.8 mg, 0.2113 mmol), then the mixture was stirred for
30 min. Compound 19 (745 mg, 2.113 mmol) was added dropwise in
DCM (distilled, 20 ml) and the reaction mixture was refluxed
overnight and then filtrated. The filtrate was concentrated under
reduced pressure. The residue was purified by column chroma-
tography to reach the compound 20 as colorless oil. (52.1%) 1H NMR
5.1.10. Synthesis of prodrug 2
To a solution of compound 12 (100 mg) in MeOH (10 ml) in an
autoclave vessel was added Pd/C (10%, 15 mg), and hydrogen
pressure of 4 bar was maintained for 3.5 h at room temperature. Pd/
C was filtered and the filtrate was concentrated to afford prodrug 2
as a white solid. (80.7%) IR (KBr,
2929, 2870, 1902, 1799, 1738, 1513, 1381, 1167, 1071, 848, 631; 1H
NMR (400 MHz, CDCl3, ppm):
0.83e0.88 (q, 6H, J ¼ 6.8, 13.2 Hz,
n
cmꢁ1): 3419, 3056, 3025, 2956,
(400 MHz, CDCl3, ppm): d 1.04 (s, 9H, SiC(CH3)3), 1.91 (s, 3H,
NCCH3), 2.72 (t, 2H, SCCH2), 3.35 (s, 3H, OCH3), 3.37 (s, 3H, OCH3),
3.55e3.58 (m, 4H, OCH2CH2O), 3.73 (t, 2H, CH2CH2O), 3.81 (s, 2H,
SCH2Ar), 3.84e3.90 (m, 4H, OCH2CH2O), 4.02 (s, 2H, COCH2N),
4.10e4.17 (m, 2H, 6-CH2), 4.38e4.42 (m, 1H, 5CeH), 4.73 (s, 1H,
4CeH), 5.26 (s, 2H, OCH2O), 5.36 (d, 1H, J ¼ 5.6 Hz, OCH2O), 5.76 (d,
1H, J ¼ 6 Hz, OCH2O), 7.19e7.21 (m, 2H, AreH), 7.24e7.27 (m, 3H,
AreH), 7.36e7.45 (m, 6H, AreH), 7.64e7.65 (m, 4H, AreH), 7.92 (s,
1H, CHO). MS (m/z): 936.3 ([MþNa]þ).
d
(CH3)2), 1.39 (d, 3H, J ¼ 6.8 Hz, CH3), 1.77e1.80 (m, 1H, CH(CH3)2),
2.38 (d, 2H, J ¼ 7.2 Hz, CH2Ph), 3.67e3.71 (m, 1H, CHCH3), 3.72 (s,
2H, 6-CH2), 4.98 (s, 1H, 5CeH), 5.18 (s, 1H, 4CeH), 7.03 (d, 2H,
J ¼ 8 Hz, AreH), 7.11 (d, 2H, J ¼ 7.6 Hz, AreH); 13C NMR (CDCl3,
ppm): 18.32 (1C), 22.72 (1C), 22.76 (1C), 30.81 (1C), 44.83 (1C),
46.78 (1C), 62.64 (1C), 69.65 (1C), 71.34 (1C), 118.56 (1C), 127.19
(1C), 127.48 (1C), 129.34 (1C), 129.67 (1C), 138.64 (1C), 140.78 (1C),
154.36(1C),174.92 (1C),178.64 (1C) .MS (m/z): 387.4 ([MþNa]þ); MS
(m/z): 387.4 ([MþNa]þ).
5.1.16. Synthesis of compound 21
In 100 ml of 50% AcOH was added compound 20 (900 mg,
0.984 mmol), the reaction mixture was stirred for 3.5 h at 50 ꢀC. The
reaction was monitored by TLC, and was adjusted to neutral with
saturated sodium carbonate solution. The reaction solution was
extracted with ethylacetate, and then the organic layer was washed
successively with H2O and brine, filtered and concentrated under
reduced pressure. The residue was purified by column chroma-
tography to get the compound 21 as a pale-yellow oil. (58.4%) 1H
5.1.11. Synthesis of compound 14e16 [17]
The synthesis of TDS compound 14e16 were reported in our
previous work [17].
5.1.12. Synthesis of compound 17
To a solution of compound 16 (6 g, 0.015 mol) and imidazole
(4.2 g, 0.062 mol) in DCM (70 ml) was added TBDPS-Cl (6 ml), then
the reaction mixture was stirred for 3 h at 20 ꢀC. After the reaction
was completed, the reaction solution was washed with H2O and
brine, then was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to give the pure semi-solid
NMR (400 MHz, CDCl3, ppm): d 2.02 (s, 3H, NCCH3), 2.72 (t, 2H,
SCCH2), 3.45 (s, 3H, OCH3), 3.69e3.73 (m, 4H, OCH2CH2O), 3.89 (s,
2H, SCH2Ar), 3.97 (m, 2H, CH2CH2O), 4.05 (s, 2H, COCH2N),
4.16e4.23 (m, 2H, 6-CH2), 4.38e4.42 (m, 1H, 5CeH), 4.74 (s, 1H,
4CeH), 4.95 (s, 2H, OCH2O), 7.27e7.35 (m, 5H, AreH), 8.02 (s, 1H,
CHO). MS (m/z): 610.1 ([MþNa]þ).
product. (82.6%) 1H NMR (400 MHz, CDCl3, ppm):
d 0.97 (s, 9H,
SiC(CH3)3), 1.38 (s, 9H,OC(CH3)3), 1.86 (s, 3H, NCCH3), 2.65 (t, 2H,
SCCH2), 3.66 (t, 2H, CH2CH2O), 3.73 (s, 2H, SCH2Ar), 3.85 (s, 2H,
COCH2N), 7.12e7.14 (m, 2H, AreH), 7.17e7.21 (m, 3H, AreH),
7.29e7.35 (m, 6H, AreH), 7.57e7.59 (m, 4H, AreH), 7.92 (s, 1H,
CHO). MS (m/z): 658.2 ([MþNa]þ).
5.1.17. Synthesis of compound 22
In 5.6 ml of DCM was added compound 21 (70 mg, 0.1192 mmol)
and DIPEA (25 ul, 0.12 mmol), the reaction mixture was stirred for
15 min at room temperature. Then, MEMCl (15 ul, 0.13 mmol) was
added to the reaction solution, the reaction mixture was stirred for

322
Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
30 min again at room temperature. The reaction solution was
washed with H2O, and the organic layer was separated, dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The residue was purified by column chromatography to obtain
(200 mm ꢂ 4.6 mm, 5 mm), thermostated at 25 ꢀC. The mobile
phase was composed of methanol/water (70:30), and the pH was
adjusted to 2.86 with 10% phosphoric acid at a flow rate of 1.0 ml/
min and the UV detector was set to monitor the signal at 219 nm
corresponding to the maximum absorbance for the ibuprofen de-
rivatives. The apparent pseudo first order rate constants of disap-
pearance of prodrugs (Kdisapp, hꢁ1 or minꢁ1) was determined by
calculation from linear regression of the natural logarithm of
absorbance against time in hours or minutes.
a pale-yellow oil. (85.3%) 1H NMR (400 MHz, CDCl3, ppm):
d 1.96 (s,
3H, NCCH3), 2.72 (t, 2H, SCCH2), 3.37 (s, 3H, OCH3), 3.38 (s, 3H,
OCH3), 3.55e3.57 (m, 4H, OCH2CH2O), 3.72 (t, 2H, CH2CH2O),
3.82e3.86 (m, 4H, OCH2CH2O), 3.89 (s, 2H, SCH2Ar), 4.12 (s, 2H,
COCH2N), 4.16e4.18 (m, 2H, 6-CH2), 4.41e4.45 (m, 1H, 5CeH), 4.73
(s, 1H, 4CeH), 5.24 (s, 2H, OCH2O), 5.38 (d, 1H, J ¼ 6 Hz, OCH2O),
5.74 (d, 1H, J ¼ 6 Hz, OCH2O), 7.26e7.34 (m, 5H, AreH), 8.03 (s, 1H,
CHO). MS (m/z): 698.2 ([MþNa]þ).
5.2.2. Stability in different buffer solutions
The stability in phosphate buffer solution was investigated at
four pH values: 2.21, 5.86, 7.33 and 7.88. Precisely, 1 ml methanol
5.1.18. Synthesis of compound 23
solution (250
4 ml different buffer at 37 ꢀC. After mixing, it was kept in a 37 1 ꢀC
constant water bath, and the 200 l sample was withdrawn at the
following time points: 0, 1, 3, 6, 12, and 24 h. The disappearance of
ibuprofen esters was monitored by HPLC method as described.
mm/ml) of the synthesized compound was added into
To a stirred solution of ibuprofen (55.7 mg, 0.27 mmol) in DCM
(distilled, 5 ml) was added DCC (55.9 mg, 0.27 mmol) and DAMP
(1.98 mg, 0.0162 mmol). After the mixture was stirred for 30 min,
compound 22 (73 mg, 0.108 mmol) was added dropwise in DCM
(distilled, 10 ml) and the reaction mixture was refluxed overnight,
and then filtrated. The filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography with
dichloromethane-methanol to yield compound 23 as a white semi-
m
5.2.3. Stability in plasma extract and brain homogenate
Blood was drawn from mice though orbital sinus and was
collected in a heparinized tube paved with heparin sodium. Sam-
ples were centrifuged at 15,000 rpm for 15 min to separate plasma
which was diluted with double volumes of water. The brain was
removed and homogenized in cold phosphate buffer of pH 7.4 with
proportion of 1:5 (w/v). Samples were then placed on ice and used
solid. (87.5%) 1H NMR (400 MHz, CDCl3, ppm):
d 0.88e0.89 (d, 12H,
J ¼ 6.4 Hz, 4 ꢂ (CH3)2), 1.45e1.46 (d, 6H, J ¼ 6.8 Hz 2 ꢂ CH3),
1.58e1.62 (m, 2H, 2 ꢂ CH(CH3)2), 1.85 (s, 3H, NCCH3), 2.43 (d, 4H,
J ¼ 6.4 Hz, 2 ꢂ CH2Ph), 2.70 (t, 2H, SCCH2), 3.35 (s, 3H, OCH3), 3.37
(s, 3H, OCH3), 3.45e3.47 (m, 4H, OCH2CH2O), 3.55 (t, 2H, CH2CH2O),
3.64e3.68 (m, 4H, OCH2CH2O), 3.69e3.72 (m, 2H, 2 ꢂ CHCH3), 3.83
(s, 2H, SCH2Ar), 4.13 (s, 2H, COCH2N), 4.34e4.39 (m, 2H, 6-CH2),
4.95e4.94 (m, 1H, 5CeH), 5.03 (s, 1H, 4CeH), 5.28 (s, 2H, OCH2O),
5.23 (d, 1H, J ¼ 6.4 Hz, OCH2O), 5.43 (d, 1H, J ¼ 6 Hz, OCH2O),
7.06e7.17 (m, 8H, AreH), 7.22e7.31 (m, 5H, AreH), 7.78 (s, 1H,
CHO); MS (m/z): 1074.44 ([MþNa]þ).
immediately.1 ml methanol solution (250
was added to 4 ml of plasma, or brain homogenate and gently
vortexed. Samples were incubated at 37 ꢀC and 200
l aliquots were
removed after 0, 5, 15, 30, 60, and 90 min, respectively. Then 200
mm/ml) of the compound
m
ml
of acetonitrile was added to each aliquot and vortexed. Samples
were centrifuged for 15 min to remove proteins and the superna-
tants were analyzed by HPLC method as described.
5.1.19. Synthesis of prodrug 4
5.3. Biodistribution studies in vivo
In 1.44 ml of 1% HCleEtOH was added compound 23 (60 mg,
58.6 mmol), and the reaction mixture was stirred for 1 h at room
temperature at 70 ꢀC. The reaction was monitored by TLC and was
concentrated under reduced pressure. The residue was purified by
column chromatography with dichloromethane-methanol to get
prodrug 4 as a white semi-solid. (61.3%) 1H NMR (400 MHz, CDCl3,
5.3.1. Test animals
Adult Kunming mice weighing 20e22 g were obtained from the
animal center of Sichuan University. The animals were left for two
days to acclimatize to animal room conditions and were main-
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).
ppm):
d
0.87e0.88 (d, 12H, J ¼ 6.4 Hz, 4 ꢂ (CH3)2), 1.44e1.46 (d, 6H,
J ¼ 6.8 Hz 2 ꢂ CH3), 1.58e1.61 (m, 2H, 2 ꢂ CH(CH3)2), 1.81 (s, 3H,
NCCH3), 2.42 (d, 4H, J ¼ 6.4 Hz, 2 ꢂ CH2Ph), 2.68 (t, 2H, SCCH2), 3.39
(t, 2H, CH2CH2O), 3.63e3.69 (m, 2H, 2 ꢂ CHCH3), 3.84 (s, 2H,
SCH2Ar), 4.12 (s, 2H, COCH2N), 4.37e4.39 (m, 2H, 6-CH2), 4.51e4.52
(m, 1H, 5CeH), 5.45 (s, 1H, 4CeH), 7.04e7.09 (m, 4H, AreH),
7.13e7.16 (m, 4H, AreH), 7.21e7.29 (m, 5H, AreH), 7.78 (s,1H, CHO);
13C NMR (CDCl3, ppm): 18.12 (1C), 18.59 (1C), 20.81 (1C), 22.23 (1C),
22.28(1C), 22.34 (1C), 22.46 (1C), 29.65 (1C), 30.11 (1C), 30.16 (1C),
44.24 (1C), 44.91 (1C), 45.03 (1C), 45.49 (1C), 45.76 (1C), 46.24 (1C),
62.45 (1C), 63.32 (1C), 70.23 (1C), 71.87 (1C), 102.25 (1C), 118.72
(1C), 126.84 (1C), 127.09 (1C), 127.12 (1C), 127.19 (1C), 127.29 (1C),
127.75 (1C), 128.42 (1C), 128.60 (1C), 128.81 (1C), 129.25 (1C),
129.39 (1C), 129.45 (1C), 129.69 (1C), 136.07 (1C), 137.47 (1C),
138.84 (1C), 139.86 (1C), 140.48 (1C), 140.66 (1C), 153.98 (1C),
162.95 (1C), 167.62 (1C), 173.64 (1C), 174.71 (1C), 179.27 (1C). MS
(m/z): 898.4 ([MþNa]þ).
5.3.2. HPLC analysis of prodrugs and ibuprofen
The waters liquid chromatographic system was carried out ac-
cording to the Section 5.2.1. The mobile phase was composed of
methanol/water (70:30), and the pH was adjusted to 2.86 with 10%
phosphoric acid at a flow rate of 1.0 ml/min and the UV detector
was set to monitor the signal at 219 nm corresponding to the
maximum absorbance for the ibuprofen derivatives.
5.2. In vitro studies
5.3.3. Sample preparation
Blood was collected from the eye socket of mouse into a tube
containing heparin, and centrifuged at 5000 rpm for 5 min. The
supernatant was collected as plasma sample. The animals were
killed by cervical dislocation, and the organs were removed and
flushed with water for three times to remove the blood remained
5.2.1. Instrumentation and method
The waters liquid chromatographic system employed was an LC-
10A liquid chromatographic system (Shimadzu Japan). The analysis
was carried out on
a
SinoChrom ODS-C18 column

Y. Zhao et al. / European Journal of Medicinal Chemistry 82 (2014) 314e323
323
[27] and then the brains were roll over on the filter paper carefully to
remove the main vessel. All the tissues were homogenized with
triple amount of water. An aliquot of 50 l of internal standard
(naproxen) was added into 500 l plasma or 500 l organ homog-
enate, the mixture containing ibuprofen was vortexed with 1.5 ml
methanol for 5 min. Hydrolysis was performed, before analyzing
prodrugs. Then an aliquot of 35 ml of 6 M NaOH aqueous solution
was added to the spiking mixture. After 10 min of hydrolysis at room
temperature, ibuprofen was released from the prodrugs, and 35 ml
of 6 M HCl was added to neutralize NaOH. Subsequently, the mixture
was vortexed with 1.5 ml methanol for another 5 min. After that, the
mixture was centrifuged at 15,000 rpm for 10 min. The separated
supernatant was evaporated to dryness at 40 ꢀC under air flow. The
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RE ¼ ðAUC0ꢁtÞs=ðAUC0ꢁtÞc
CE ¼ ðCmaxÞs=ðCmaxÞc;
where s and c represented sample (the prodrugs) and control
(ibuprofen), respectively.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (81072532 &, No. 81102324), the Ph.D. Pro-
grams Foundation of Ministry of Education of China
(20110181130011) and Science & Technology Pillar Program of
Sichuan Province of China (2011SZ0014).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.072.