Design, synthesis and preliminary biological evaluation of brain targeting l-ascorbic acid prodrugs of ibuprofen

Article abstract of DOI:10.1016/j.cclet.2013.01.022

l-Ascorbic acid (AA, vitamin C) exhibits a high concentration in the brain. The transportation of AA in brain is mainly mediated by the glucose transporter 1 (GLUT₁) and the Na⁺-dependent vitamin C transporter SVCT₂. While l-ascorbic acid C6O conjugation has been investigated as a tool to enhance brain drug delivery, C5O conjugation and C5O & C6O conjugation as brain targeting tools have not been reported. In this letter, ibuprofen was linked directly to C5O, C6O and C5O & C6O positions of l-ascorbic acid with eater bonds, providing prodrug 1, 2 and 3, respectively, to improve their targeting abilities in the brain. Prodrug 1, 2 and 3 were synthesized in facile ways with good yields. And the preliminary evaluation in vivo illustrated that prodrug 2 had a better targeting ability than prodrug 1. Moreover, prodrug 3, whose C5O & C6O positions were both modified, had good targeting ability for brain which will provide an important evidence for our further study on C5O- & C6O- di-derivatives of l-ascorbic acid.

Full text of DOI:10.1016/j.cclet.2013.01.022

Chinese Chemical Letters 24 (2013) 117–119
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Design, synthesis and preliminary biological evaluation of brain targeting
-ascorbic acid prodrugs of ibuprofen
L
Xue-Ying Wu a, Xiao-Cen Li b, Jie Mi b, Jing You b, Li Hai b,
*
a College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China
b Key Laboratory of Drug Targeting and Drug Delivery System of the Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University,
Chengdu 610041, China
A R T I C L E I N F O
A B S T R A C T
Article history:
L
-Ascorbic acid (AA, vitamin C) exhibits a high concentration in the brain. The transportation of AA in
brain is mainly mediated by the glucose transporter 1 (GLUT1) and the Na+-dependent vitamin C
transporter SVCT2. While -ascorbic acid C6–O conjugation has been investigated as a tool to enhance
brain drug delivery, C5–O conjugation and C5–O & C6–O conjugation as brain targeting tools have not
been reported. In this letter, ibuprofen was linked directly to C5–O, C6–O and C5–O & C6–O positions of
Received 4 November 2012
Received in revised form 17 December 2012
Accepted 31 December 2012
Available online 4 February 2013
L
L
-
ascorbic acid with eater bonds, providing prodrug 1, 2 and 3, respectively, to improve their targeting
abilities in the brain. Prodrug 1, 2 and 3 were synthesized in facile ways with good yields. And the
preliminary evaluation in vivo illustrated that prodrug 2 had a better targeting ability than prodrug 1.
Moreover, prodrug 3, whose C5–O & C6–O positions were both modified, had good targeting ability for
brain which will provide an important evidence for our further study on C5–O- & C6–O- di-derivatives of
Keywords:
Vitamin C
Brain target
Prodrug
GLUT1 transporter
SVCT2 transporter
Ibuprofen
L-ascorbic acid.
ß 2013 Li Hai. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
1. Introduction
mainly based on two distinct ways: the glucose transporter GLUT1
can transport the oxidized form of AA, dehydro-ascorbic acid
Over the past few decades, one of the crucial problems in drug
delivery into the central nervous system (CNS) is the low
permeability of drugs due to the blood–brain barrier (BBB).
Difficulties in crossing the BBB often hinder the bioavailability of
drugs in the treatment of CNS diseases, such as Alzheimer’s disease
(AD). Numerous studies have suggested that chronic intake of
nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen,
could reduce the risk or prevent the onset of AD [1,2]. Because of
the poor permeability of NSAIDs, the higher doses are required to
achieve satisfactory therapeutic effects against AD, which may
result in serious gastrointestinal and renal toxicity [3]. Therefore, a
new promising strategy should be developed to improve brain
delivery of ibuprofen for the treatment of AD.
(DHAA), which is then reduced into AA and enhanced to its CNS
level; while Na+-dependent Vitamin C transporter SVCT2 trans-
ports AA directly into brain [5].
It was reported that the C6-hydroxyl group of AA is not critical
for the transport while the hydroxyl groups of enediol lactone in C2
& C3 are the reaction sites in the redox process of AA and are
required for the reducing properties of AA [6]. Recent researches
revealed that AA can be used as a tool to improve brain drug
delivery [7]. While most researchers focused their attention on the
preparation of C6–O-modified AA, we became interested in the
preparation of C5–O-derivative of AA as well as C5&C6–O-di-
derivatives of AA.
In account for all of the above and to overcome the low
permeability of ibuprofen in brain, we have designed and
synthesized C6–O–ibuprofen-AA (1), C5–O–ibuprofen-AA (2) as
well as C5–O- & C6–O- di-ibuprofen forms of AA (3). In this paper,
we describe the synthesis of these three brain targeting ibuprofen
prodrugs and evaluate their preliminary biological activities.
L-Ascorbic acid (AA), which is familiar to us as vitamin C, is an
essential nutrient for all mammals. The highest concentration of
AA can be found in brain tissue, where it acts as an antioxidant and
is essential for many enzymatic reactions [4]. In recent years,
considerable effort has been made to investigate the transport and
accumulation mechanism of AA, especially in brain tissue. It is
generally believed that the transportation of AA in the brain is
2. Experimental
The synthetic route of prodrug 1 and 3 is outlined in Scheme 1.
The starting material is readily available AA. After stirring in
acetone for 20 h, isopropylidene protecting group was introduced
* Corresponding author.
E-mail address: smile@scu.edu.cn (L. Hai).
1001-8417/$ – see front matter ß 2013 Li Hai. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.01.022

118
X.-Y. Wu et al. / Chinese Chemical Letters 24 (2013) 117–119
HO
HO
HO
O
O
HO
3217, 2955, 2868, 1762, 1710, 1665, 1511, 1364, 1171, 1035, 819,
667; MS (m/z): 387.4 ([M+Na]+); mp: 126–128 8C. 2: 1H NMR
(400 MHz, CDCl3): 0.83–0.88 (q, 6H, J = 6.8, 13.2 Hz, –(CH3)2), 1.39
(d, 3H, J = 6.8 Hz, –CH3), 1.77–1.80 (m, 1H, –CH(CH3)2), 2.38 (d, 2H,
J = 7.2 Hz, –CH2–Ph–), 3.67–3.71 (m, 1H, –CH–CH3), 3.72 (s, 2H,
6CH2–), 4.98 (s, 1H, 5C–H), 5.18 (s, 1H, 4C–H), 7.03 (d, 2H, J = 8 Hz,
O
O
O
O
a
b
O
O
O
O
O
O
c
HO
BnO
OH
HO
OH
BnO
OBn
OBn
d
6
5
4
L-Ascorbic acid
O
O
Ar–H), 7.11 (d, 2H, J = 7.6 Hz, Ar–H); IR (KBr, cmꢀ1):
v
3419, 3056,
3025, 2956, 2929, 2870, 1902, 1799, 1738, 1513, 1381, 1167, 1071,
848, 631; MS (m/z): 387.4 ([M+Na]+). 3: 1H NMR (400 MHz, CDCl3):
O
O
O
d
O
O
HO
O
O
7
d
BnO
e
OBn
O
BnO
e
OBn
8
0.87–0.88 (d, 12H, J = 6.4 Hz, 2ꢁ –(CH3)2), 1.37–1.45 (m, 6H, 2ꢁ –
CH3), 1.77–1.85 (m, 2H, 2ꢁ –CH(CH3)2), 2.43 (d, 4H, J = 6.4 Hz, 2ꢁ –
CH2–Ph–), 3.59–3.66 (m, 2H, 2ꢁ –CH–CH3), 4.22–4.37 (m, 2H, 6CH2–),
4.61–4.67 (m, 1H, 5C–H), 5.35–5.39 (m, 1H, 4C–H), 7.04–7.16 (m, 8H,
O
O
O
O
HO
O
O
Ar–H); IR (KBr, cmꢀ1):
v 3439, 3294, 3089, 3049, 2955, 2930, 2865,
HO
OH
O
2725, 1905, 1743, 1654, 1537, 1513, 1381, 1202, 1072, 1022, 849, 630,
596; MS (m/z): 575.3 ([M+Na]+), 591.5 ([M+K]+).
1
O
O
O
HO
OH
3
Scheme 1. Synthesis of prodrug 1 and 3. Reagents and conditions: a. acetone, acetyl
chloride, r.t., 73.3%; b. BnBr, K2CO3, acetone, reflux, 44.1%; c. HCl, CH3CN, 30 8C,
100%; d. DCC, DMAP, overnight, 43.6% and 25%; e. 10% Pd/C, H2, 0.4 MPa, 74.7% and
75.8%.
3. Results and discussion
To evaluate the brain targeted ability of these prodrugs, an in
vivo study was taken. Kunming mice were randomly divided into
groups, and the mice were injected with the samples of ibuprofen
and prodrug 1, 2 and 3 through the tail vein at an equivalent dose of
48 mmol/g body weight, respectively. Then, mice from each group
were sequentially sacrificed at 5, 10, 30, 45, 60, 90, 120 and
240 min after administration. Their brains and blood were
to afford ketol 4. Subsequently, ketol 4 and benzyl bromide were
refluxed in acetone for about 4 h to give 2,3-O-di benzyl protected
derivative 5, which afforded 6 after cleavage of the isopropylidene
protecting group. Then, intermediate
6 was coupled with
ibuprofen to give 7 as well as 8 by using DCC as a condensing
agent. Finally, the 2,3-O-di benzyl groups of 7 and 8 were removed
under hydrogen catalyzed by 10% Pd/C to provide the title
compounds 1 and 3, respectively.
Although Dai and Tang [8] has reported the synthesis of prodrug
1 catalyzed by Novozyme 435 lipase, we chose this method which
could provide prodrug 1 and 3 in one step.
collected and analyzed by HPLC (UV detector,
l = 219 nm). The
concentrations of ibuprofen in plasma are shown in Fig. 1 and the
concentrations of ibuprofen in brain are demonstrated in Fig. 2.
Clearly, the ibuprofen concentrations of prodrug 1, 2 and 3 in
plasma were higher than that of ibuprofen. It can be calculated that
the AUC0–t of ibuprofen in plasma after i.v. administration prodrug
1, 2 and 3 were 2.71, 5.05, 3.75 times that of ibuprofen. And the
Cmax of ibuprofen in plasma after i.v. administration prodrug 1, 2
The synthetic route of prodrug 2 was demonstrated in Scheme 2.
We predicted that selecting a suitable protecting group was
essential to the synthesis of C5–O–ibuprofen-AA. At the very
beginning, we chose benzyl group in order to remove all the
protecting groups when ibuprofen was coupled with the AA.
However, we failed to introduce a phenyl group even in the presence
of NaH. Consequently, trityl chloride was used to selectively block
the C6-hydroxyl group of intermediate 6 and produce compound 9
in good yield [9]. Then, compound 9 was coupled with ibuprofen to
give 10 by using DCC as a condensing agent. After detritylation of
compound 10, 2,3-O-di benzyl derivative 11 was obtained. Finally,
the debenzylation of 11 was reduced to the title compound 2
catalyzed by 10% Pd/C under hydrogen.
225
200
175
150
125
100
75
50
25
Ibuprofen
1
2
3
Prodrug
Prodrug
Prodrug
0
0
25
50
75
100 125 150 175 200 225 250
Time (min)
Selected data for compound 1: 1H NMR (400 MHz, DMSO-d6):
d
0.83–0.86 (t, 6H, J = 6 Hz, –(CH3)2), 1.38–1.40 (d, 3H, J = 6.8 Hz, –
CH3), 1.79 (m, 1H, –CH(CH3)2), 2.40 (d, 2H, J = 7.2 Hz, –CH2–Ph–),
3.76–3.81 (q, 1H, J = 6.8, 13.6 Hz, –CH–CH3), 3.91 (s, 1H, 5C–OH),
3.97–3.81 (m, 2H, 6CH2–), 4.47 (q, 1H, J = 1.2, 8 Hz, 5C–H), 5.33 (s,
1H, 4C–H), 7.10 (d, 2H, J =8 Hz, Ar–H), 7.19 (d, 2H, J = 7.6 Hz, Ar–H),
Fig. 1. The concentration of ibuprofen in plasma versus time after i.v. injection in
mice. Error bars show the value of SD (n = 3).
8.41 (bs, 1H, 2C–OH), 11.09 (bs, 1H, 3C–OH); IR (KBr, cmꢀ1):
v
3393,
60
Ibuprofen
Prodrug 1
Prodrug2
50
40
30
20
10
0
HO
HO
BnO
TrtO
HO
BnO
TrtO
O
O
O
a
b
O
O
O
O
3
Prodrug
OBn
OBn
O
BnO
10
HO
OBn
9
6
HO
O
c
O
d
O
O
O
O
O
O
HO
OH
BnO
OBn
0
25
50
75
100 125 150 175 200 225 250
Time (min)
2
11
Scheme 2. Synthesis of prodrug 2. Reagents and conditions: a. TrtCl, Et3N, DCM, r.t.,
68.4%; b. DCC, DMAP, overnight, 77.5%; c. HCl, CH3CN, 50 8C, 78.6%; d. 10% Pd/C, H2,
0.4 MPa, 80.7%.
Fig. 2. The concentrations of ibuprofen in brain versus time after i.v. injection in
mice. Error bars show the value of SD (n = 3).

X.-Y. Wu et al. / Chinese Chemical Letters 24 (2013) 117–119
119
and 3 were 2.72, 4.26, 7.40 times that of ibuprofen. The curves
Acknowledgments
displayed that ibuprofen could be quickly metabolized while
prodrug 1, 2 and 3 showed slowly declined concentrations. It is
worth noting that the three prodrugs can keep relatively high
concentrations of ibuprofen in plasma, which increased their
chances to be transported across BBB. And the decomposition of
these prodrugs may extend the metabolism course of ibuprofen.
Data also showed that the AUC0–t and Cmax of ibuprofen in brain
after i.v. administration of prodrug 1, 2 and 3 were significantly
higher than that after the injection of naked ibuprofen. The RE,
This work was supported by the Ph.D. Programs Foundation of
Ministry of Education of China (No. 20110181130011) and the
National Natural Science Foundation of China (No. 81072532).
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/(AUC0–t)ibu), and CE, concen-
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testing time. The results also highlighted prodrug 2, C5–O–
ibuprofen-AA, had a better targeting ability than prodrug 1, which
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modified, had good targeting ability for brain, which indicated that
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In conclusion, we have successfully synthesized three brain
targeting L-ascorbic acid-prodrugs of ibuprofen 1, 2 and 3, and
provided a facile method for the preparation of these three
prodrugs. The preliminary biological evaluation in vivo of prodrug
1, 2 and 3 was taken, which highlighted their potential as
promising brain targeting candidates. The results also illustrated
that prodrug 2 had a better targeting ability than prodrug 1.
Furthermore, prodrug 3 had good targeting ability for brain which
will provide an important evidence for our further study on C5–O-
& C6–O- di-derivatives of L-ascorbic acid.