Two-step enzymatic selective synthesis of water-soluble ketoprofen-saccharide conjugates in organic media

Article abstract of DOI:10.1016/j.bmc.2009.01.040

Ketoprofen-saccharide conjugates were synthesized by selectively enzymatic hydrolysis and acylation. Firstly, the (S)-ketoprofen vinyl ester was prepared by enzymatic hydrolysis of (R,S)-ketoprofen vinyl ester. Then enzymatic transesterification of (S)-ke

Full text of DOI:10.1016/j.bmc.2009.01.040

Bioorganic & Medicinal Chemistry 17 (2009) 1905–1910
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Two-step enzymatic selective synthesis of water-soluble ketoprofen–saccharide
conjugates in organic media
*
*
Hai-Yang Wang, Chao Li, Na Wang , Kun Li, Xing-Wen Feng, Ting He, Xiao-Qi Yu
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ketoprofen–saccharide conjugates were synthesized by selectively enzymatic hydrolysis and acylation.
Firstly, the (S)-ketoprofen vinyl ester was prepared by enzymatic hydrolysis of (R,S)-ketoprofen vinyl
ester. Then enzymatic transesterification of (S)-ketoprofen vinyl ester with a series of saccharides were
performed by the catalysis of a commercial protease from Bacillus licheniformis (BLP) in organic medium
mixture of pyridine and tert-butanol. The ketoprofen was selectively conjugated onto the primary hydro-
xyl group of saccharides and with high yield after 72 h. Partition coefficient determination showed that
all the products have better water solubility than parent ketoprofen. Chemical hydrolysis experiment
indicated that 50% ketoprofen could be release from ketoprofen glucoside and maltoside in aqueous buf-
fer (pH 7.4) within 48 h.
Received 5 December 2008
Revised 20 January 2009
Accepted 21 January 2009
Available online 23 January 2009
Keywords:
Biocatalysis
Regioselectivity
Ketoprofen
Saccharide
Protease
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Carbohydrate plays an important role in biological molecular
recognition and exhibits several desirable biological properties
Ketoprofen is one of the commercially available 2-arylpropionic
acids non-steroidal anti-inflammatory drugs (NSAIDs), which is
widely used for alleviation of pain and inflammation associated
with tissue injury.1 However, previous pharmacological study of
ketoprofen has indicated that the gastrointestinal (GI) ulceration
and hemorrhage due to the locally direct contact effect induced
by the acidic moiety.2 An alternative method of avoiding GI side ef-
fects is to administer the drug via the transdermal route. However,
the therapeutic efficacy of ketoprofen, especially its application
onto the skin, is mainly limited by its poor aqueous solubility
and low permeability.3
such as non-toxicity, good biocompatibility and biodegradabil-
ity.12–14 Since a good hydrophilic–lipophilic balance of the prodrug
is essential to give an acceptable bioavailability,15,16 the selection
of hydroxyl compound (alcohol) is important in the study of pro-
drug. Sugars are hydrophilic compounds and it is particularly effec-
tive for the improvement of drugs dissolution behavior to form
ester prodrugs. Multivalent sugar-based therapeutic agents have
been reported in recent years.17–19 Thus we considered it worthy
of interest to evaluate sugar as a potential carrier for improving
drug dissolution behavior.
However, preparation of optically active ketoprofen prodrug
containing sugar has still received limited attention. The prepara-
tion of optically active ketoprofen has become an important sub-
ject due to the bioactivity of ketoprofen is mainly S-
enantiomer.20 The application of enzymes in the resolution of keto-
profen has great advantages such as the simplicity of process and
the high enantioselectivity of biocatalysis. An irreversible enzy-
matic resolution method has been developed to prepare optically
active ketoprofen vinyl ester in our previous study.21,22
Herein, we wish to report the two-step selectively enzymatic
synthesis optically active ketoprofen–saccharide conjugates with
high water solubility. (S)-Ketoprofen vinyl ester (2) was prepared
from resolution of (R,S)-ketoprofen vinyl ester (1) by using
LipozymeÒ immobilized from Mucor miehei (MML). The protease
from Bacillus licheniformis (BLP) was used to catalyze the transeste-
rification of (S)-ketoprofen vinyl ester with a series of saccharides
in organic media. (S)-ketoprofen was regioselectively conjugated
onto the primary hydroxyl group of saccharides through the
Therefore, various methods such as salt formation, solid disper-
sion with a polymer and the prodrug approach have been used to
increase its solubility and bioavailability.4–6 Among these methods,
preparation of ester prodrug, especially for its functionalization
with appropriate hydrophilic groups is one of the most effective
and promising way to enhance the solubility of drug and then to
exploit their properties as well as possible. Furthermore, prepara-
tion of ester prodrugs can temporarily mask the acidic moiety
and decrease the side effects.7 Appropriately designed prodrugs,
such as oligoethylene ester of ketoprofen,8 sugar-surfactant/keto-
profen catanionic assembly,9 ketoprofen acyloxyalkyl ester10 and
amide derivative of ketoprofen with L
-cysteine ethyl ester11, have
been reported to show good water stability, rapid enzymatic
hydrolysis and increased flux through excised human skin.
* Corresponding authors. Tel.: +86 28 85460576; fax: +86 28 85415886 (X.-Q.Y.).
E-mail address: xqyu@tfol.com (X.-Q. Yu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.040

1906
H.-Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 1905–1910
Table 1
ester-linkage and afforded ester type ketoprofen–saccharide conju-
gates. The obtained ketoprofen–saccharide conjugates have better
water solubility than parent ketoprofen and thus suitable for
potentially pharmaceutical application.
The acylation position and yields of ketoprofen–saccharide conjugates
Entry
Compound
Acylation position
Yielda (%)
1
2
3
4
5
6
7
3a
3b
3c
3d
3e
3f
1
6
6
73.5
73.8
87.4
37.1
22.8
33.2
50.0
2. Results and discussion
60
60
6
2.1. Selective enzymatic synthesis of ketoprofen–saccharide
conjugates
3g
10
Reaction condition: 3.2 mmol (S)-ketoprofen vinyl ester, 0.8 mmol saccharide,
400 L BLP, 40 mL pyridine/t-butanol (1:1, v/v), 50 °C, 96 h.
Determined by HPLC.
Two-step enzymatic selective synthesis of ketoprofen–saccha-
ride conjugates was shown in Scheme 1. At the first step, optically
active (S)-ketoprofen vinyl ester was prepared by enzymatic irre-
versible resolution. LipozymeÒ immobilized from M. miehei was
used to catalyze the hydrolysis of (R,S)-ketoprofen vinyl ester in
the solvent composed of 2.5% water in dioxane. We obtained bio-
active S-enantiomer with good optical purity under the optimized
conditions based on the previous studies.21,22 The (S)-ketoprofen
vinyl ester obtained was used in the subsequent transesterification
with a series of saccharides catalyzed by BLP under mild condi-
tions. The products were purified by silica gel chromatography
and characterized by MS and NMR spectrometries. Vinyl ester
had been chosen as active acyl substrate in the enzymatic hydroly-
sis and transesterification reaction because the vinyl alcohols could
instantly tautomerize to volatile acetaldehyde, which is thermody-
namically favourable to product synthesis and make the process
irreversible.
After the reaction conditions such as enzyme sources, solvent
and reaction time were optimized, we obtained a series of ketopro-
fen–saccharide conjugates with good yields. The acylation position
and yields of products are shown in Table 1. The acylation position
in compounds 3a–g was characterized by NMR. According to the
general strategy described by Yoshimoto et al.23, acylation of a hy-
droxyl group of sugar would lead to the O-acylated carbon down-
field while the adjacent carbon upfield in the 13C NMR. Analysis of
13C NMR spectra of all the products revealed that the reactions oc-
curred on the primary hydroxyl position of the saccharides. For
l
a
example, in the 13C NMR spectrum of 3b, the peak of C6
sults in downfield shift from 60.72 (60.94) ppm to 64.9
(65.0) ppm, while the peak of C5 (C5b) results in an upfield shift
a (C6b) re-
a
a
from 73.2 (76.9) ppm to 69.7 (74.0) ppm, which shows that the
acylation position of 3b is the C-6 hydroxyl of glucose. In the 13C
NMR spectrum of 3e, the peak of C60 results in a downfield shift
from 62.4 ppm to 64.5 ppm, while the peak of C50 results in a up-
field shift from 77.2 ppm to 72.9 ppm, which shows that the acyl-
ation position of 3e is the C60 hydroxyl of lactose.
2.2. Selection of biocatalysts
One of the most important parameters for enzyme-catalyzed
reaction is the selection of biocatalyst. Eight commercial enzymes
had been tested for their ability to catalyze the transesterification
of (S)-ketoprofen vinyl ester with glucose in pyridine, which is
widely used in the synthesis of sugar esters due to its good solubil-
ity for sugar. The results were listed in Table 2. It clearly shows that
no product can be detected without enzyme participation. The best
result was obtained with protease from Bacillus licheniformis (BLP).
Lipase from Candida antarctica (CAL-B), protease from Bacillus sp.,
and lipase from Candida rugosa also exhibited moderate activities
(Table 2, entries 3–5), while other proteases and lipases showed
O
CH3
OH
O
(R)-ketoprofen
O
CH3
O
MML
O
+
BLP / ROH
O
CH3
O
dioxane / water
O
CH3
pyridine / t-butanol
O
R
O
(R, S)-ketoprofen vinyl ester (1)
O
3a-g
(S)-ketoprofen vinyl ester (2)
CH3CHO
3c: R=
3a: R=
3b: R=
3d: R=
OH
CH2
O
OH
O
HO
HO
H
H
HO
HO
O
HO
HO
HO
H
OH
O
HO
OH
OH
OH
O
HO
OH
OH
H
OH
OH
CH2OH
mannitol
glucose
mannose
maltose
OH
OH
3f: R=
HO
3g: R=
3e: R=
HO
O
O
O
O
HO
HO
O
HO
HO
OH
O
HO
O
HO
O
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
OH
lactose
sucrose
trehalose
Scheme 1. Enzymatic synthesis of ketoprofen–saccharide conjugates.

H.-Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 1905–1910
1907
Table 2
2.4. Time course of acylation of saccharides
Effect of enzyme sources on the transesterification reaction
The time course of the transesterification of ketoprofen vinyl es-
ter with different saccharides under the optimal condition was
shown in Figure 1. After 72 h the yield reached the plateau and
ketoprofen mannoside could be obtained with the highest yield
(87.4%). Obviously, mono-saccharides esters (3a, 3b and 3c) were
obtained with higher yields than di-saccharides esters (3d, 3e, 3f
and 3g). Thus, the optimal reaction time is 72 h.
Entry
Enzyme
Yielda (%)
1
2
3
4
5
6
7
8
9
Protease from Bacillus licheniformis
Protease from Bacillus subtilis
Protease from Bacillus sp.
17.2
Trace
12.1
12.0
13.0
10.0
8.0
Lipase from Candida rugosa
Lipase from acrylic resin from Candida antarctica
Lipase from porcine pancreas
LipozymeÒ immobilized from Mucor miehei
Amano lipase M from Mucor javanicus
No enzyme
9.0
0
2.5. Partition coefficient
Reaction conditions: 0.16 mmol (S)-ketoprofen vinyl ester, 0.04 mmol glucose,
20 mg ( L) enzyme, 2 mL pyridine, 50 °C, 72 h.
Octanol–water partition coefficient (logP) is one of the essential
physicochemical parameters for the estimation bioavailability of a
prodrug molecule. Thus, logP of compound 3a–g were determined
and compared to that of the parent drug (Table 4). The results indi-
cated that these ketoprofen–saccharide conjugates exhibited a
lower partition coefficient than ketoprofen, which implied their
water solubility was increased. And ketoprofen–disaccharide con-
jugates have better water solubility than ketoprofen–monosaccha-
ride conjugates. The improvement of aqueous solubility may
facilitate biomembrane transport and make ketoprofen prodrugs
process higher bioavailability.
l
a
Determined by HPLC.
quite low catalytic activities for the reaction. Thus, we chose BLP
for the further investigation of the transesterification of ketoprofen
vinyl ester with other mono- and di-saccharides.
2.3. Effect of reaction media
Enzymatic synthesis of sugar ester is limited by the fact that
the activities of biocatalysts may obviously decrease in polar sol-
vents while the sugar is soluble. Owing to the solubility of sac-
charides, polar solvents such as DMF, DMSO or pyridine usually
are used in the enzyme-catalyzed acylation of sugars.24,25 Ter-
tiary alcohol or mixtures solvents as reaction media have also
been reported.26–28 Since BLP showed the best activity for the
transesterification of ketoprofen vinyl ester with glucose, it was
selected as the biocatalyst for further optimization study to im-
prove the yield.
Both the CLOGP and molinspiration programs are unable to dis-
tinguish between stereoisomers, such as ketoprofen glucoside and
ketoprofen maltoside. The difference of experimental partition
coefficients between stereoisomers of ketoprofen–saccharide con-
jugates clearly indicated that spatial structure of compound plays
an important role in the physicochemical properties of these
compounds.
2.6. Chemical hydrolysis of prodrug
Firstly, four pure solvents (DMF, DMSO, pyridine and t-butanol),
which were commonly used in the sugar esterification, were inves-
tigated for the transesterifications of glucose with (S)-ketoprofen
vinyl ester (Table 3, entries 1–4). The reaction carried out in pyri-
dine or t-butanol attained considerable yields, while almost no
product was detected in DMF or DMSO. Therefore we considered
using binary mixtures of solvent as reaction media in order to im-
prove the yield of product. The best result was obtained in the mix-
ture of equal volume pyridine and t-butanol (Table 3, entry 7) and
the yield of 6-O-ketoprofen glucoside reached 70% after 72 h. In
addition, different ratios of pyridine to t-butanol (from 1/3 to 3/
1) were examined by the catalysis of BLP at 50 °C. Obviously, differ-
ent ratios of pyridine to t-butanol (Table 3, entries 7–11) obtained
the better yields than single solvent, and the highest yield was
reached at a ratio of 1/1 up to 70%.
An essential prerequisite for successful prodrug is that prodrug
reconversion into the parent drug occurs in vivo. The designed es-
ters should undergo enzymatic hydrolysis to release the parent
drug after entering circulation. Different models have been devel-
oped to assess the susceptibility of ester prodrugs in undergoing
bioconversion in vivo. In this experiment, the ketoprofen maltoside
(3d) and glucoside (3b) were hydrolysis in the phosphate buffer at
pH 7.4. As shown in Figure 2, the release amount of (S)-ketoprofen
is 47.5% from maltose ester and 37.5% from glucose ester within
24 h, almost 90% of (S)-ketoprofen can be released within 7 days.
90
3a
3b
80
3c
3d
70
Table 3
3e
3f
60
Effect of reaction media on the yield of 6-O-ketoprofen glucoside
3g
50
40
30
20
10
0
Entry
Solvents
Yielda (%)
1
2
3
4
5
6
7
8
Pyridine
t-Butanol
DMF
DMSO
DMF/t-butanol (1:1, v/v)
Pyridine/DMF (1:1, v/v)
Pyridine/t-butanol (1:1, v/v)
Pyridine/t-butanol (3:1, v/v)
Pyridine/t-butanol (2:1, v/v)
Pyridine/t-butanol (1:2, v/v)
Pyridine/t-butanol (1:3, v/v)
17.2
10.1
4.4
0
16.9
Trace
70.0
46.2
47.4
50.0
38.3
9
10
11
0
20
40
60
80
100
120
140
160
time (h)
Reaction conditions: 0.16 mmol (S)-ketoprofen vinyl ester, 0.04 mmol glucose,
20 L BLP, 2 mL solvent, 50 °C, 72 h.
Figure 1. Time course of the saccharides with (S)-ketoprofen vinyl ester catalyzed
by BLP. Reaction conditions: 3.2 mmol (S)-ketoprofen vinyl ester, 0.8 mmol
saccharide, 400 lL BLP, 40 mL pyridine/t-butanol (1:1, v/v), 50 °C.
l
a
Determined by HPLC.

1908
H.-Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 1905–1910
Table 4
was purchased from Fluka. Amano lipase M from M. javanicus
was purchased from Amano. Racemic ketoprofen was obtained
from Wuhan Gang Zheng Biology Technology Co. Ltd. (China). All
organic solvents were analytical grade and were dried over 3 Å
molecular sieves for 24 h prior to use.
Partition coefficients (logP) of ketoprofen and ketoprofen–saccharide conjugates
Compound
Predicted
CLOGPa
Experimentalc
Molinspirationb
Ketoprofen
2.76
1.16
1.00
1.00
ꢁ1.18
ꢁ1.18
ꢁ0.05
ꢁ0.64
3.59
0.79
1.25
1.25
ꢁ0.68
ꢁ0.68
ꢁ0.46
ꢁ0.83
3.12d
0.78
1.26
3a
3b
3c
3d
3e
3f
4.2. Analytical methods
1.00
ꢁ0.13
ꢁ0.43
ꢁ0.25
ꢁ0.24
All reactions were monitored by TLC on silica gel plates eluted
with ethyl acetate/methanol/water (17/2/1, v/v). 1H NMR and 13C
NMR spectra were obtained on a Bruker DMX 400. Spectra were
run in DMSO-d6 and referenced to an internal TMS standard. High
resolution mass spectrometry (HRMS) data were recorded on a
Bruker Daltonics Bio TOF mass spectrometer. The quantitative
analysis of samples was made by HPLC on a reverse phase column
3g
a
Calculated using CLOGP software.
b
Calculated using molinspiration (http://www.molinspiration.com/cgi-bin/
properties).
c
At pH 7.0 phosphate buffer.
From in-built database of CLOGP.
d
(Welchrom-C18, 5
l
m, 4.6 ꢀ 150 mm, Welchrom) using shimadzu
LC-2010AHT equipped with a UV detector at 254 nm. For the anal-
ysis of sugar esters of ketoprofen, methanol/water 50:50 (v/v) was
used as eluent (flow rate, 0.8 mL/min).
Based on the data of the chemical hydrolyses, it was concluded that
the derivatives could effectively release the ketoprofen.
4.3. Synthesis of (R,S)-ketoprofen vinyl ester (1)
3. Conclusions
Ketoprofen vinyl ester was synthesized and purified as de-
scribed by Yang et al.29 (R,S)-ketoprofen (0.02 mol) and Hg(OAc)2
(0.22 g) were dissolved in 80 mL vinyl acetate. After stirring the
mixture for 30 min at room temperature, 0.5 mL of concentrated
H2SO4 was added dropwise and the solution was refluxed for 6 h.
Then the mixture was cooled to room temperature and NaOAc
(5 g) was added to quench the catalyst. The resulting mixture
was filtered and the filtrate was concentrated under reduced pres-
sure. The crude product was purified by silica gel column chroma-
tography (petroleum ether/ethyl ether 20/1, v/v).
In conclusion, a selectively enzymatic method for ketoprofen
modification with different saccharides was described. Seven sac-
charide conjugates of ketoprofen were synthesized using the pro-
tease from B. licheniformis in a mixture of pyridine/t-butanol (1:1,
v/v) at 50 °C. Ketoprofen was selectively conjugated onto the pri-
mary hydroxyl group of saccharides and afforded ester type keto-
profen–saccharide conjugates. Their lower partition coefficients
show an increased hydrophilicity, and the ability to release keto-
profen in the buffer suggests that the derivatives are anticipated
to be new candidates for potential high bioavailability prodrugs.
4.4. Enzymatic preparation of (S)-ketoprofen vinyl ester (2)
4. Experimental
4.1. Materials
1 g of (R,S)-ketoprofen vinyl ester was added to 20 mL mixture
dioxane/water (97.5/2.5, v/v). The reaction was initiated by adding
200 mg LipozymeÒ immobilized from M. miehei and the suspen-
sion was shaken at 25 °C for 4 d. The solution was filtered and con-
centrated. The product was purified by silica gel column
chromatography (petroleum ether/ethyl ether 20/1, v/v). The enan-
tiomer of ketoprofen and ketoprofen vinyl ester were analyzed
using a chiral column (chiralcel OD-H, 0.46 cm ꢀ 25 cm, DAICEL)
and were detected at 254 nm. The mobile phase was n-hexane/iso-
propanol (90/10, v/v) with a flow rate of 0.8 mL/min. (S)-ketopro-
fen vinyl ester: light yellow liquid; (yield: 90%; ee: 90%). 1H NMR
(CDCl3): 7.81–7.46 (m, 9H, ArH), 7.26 (dd, J = 6.3 Hz, J = 14.0 Hz,
1H, –CH@CH2), 4.58 (dd, J = 1.6 Hz, J = 6.2 Hz, 1H, –CH@CH2), 3.87
(m, 1H, –C6H4CH), 1.58 (d, J = 7.2 Hz, 3H, –CH3).
Protease from B. licheniformis, protease from Bacillus sp., prote-
ase from B. subtilis, lipase from acrylic resin from C. antarctica, li-
pase from C. rugosa and lipase from porcine pancreas were
purchased from Sigma. LipozymeÒ immobilized from M. miehei
100
90
80
70
60
50
40
30
4.5. General method for the enzymatic synthesis of ketoprofen
sugar conjugates
The typical reaction mixture consisted of 4 mmol (S)-ketoprofen
vinyl ester, 1 mmol of saccharide, 200 lL of protease from Bacillus
licheniformis and 25 mg/mL of 3 Å molecular sieves in 20 mL of
reaction medium mixture of 1:1 pyridine and t-butanol. The mix-
tures were kept at 50 °C and shaken at 200 rpm. The reaction mix-
ture was then filtered and removed the organic solvent in vacuum.
All reactions were detected by TLC plates using ethyl acetate/meth-
anol/water (17:2:1, v/v) as eluent.
20
10
0
maltoseester
glucoseester
0
100
200
300
400
500
4.5.1. 1-O-Ketoprofen mannitol ester (3a)
time (h)
Yellow solid. 1H NMR (DMSO-d6): 7.76–7.52 (m, 9H, Ar–H), 4.77
(s, 1H, 2-OH), 4.41 (d, J = 4.0 Hz, 1H, 3-OH), 4.39 (m, 2H, 4-OH, 5-
OH), 4.28 (d, J = 8.0 Hz, 1H, 6-OH), 4.14 (d, J = 4.0 Hz, 1H, 1-H),
Figure 2. Hydrolysis of ketoprofen glucoside and maltoside at pH 7.4 phosphate
buffer.

H.-Y. Wang et al. / Bioorg. Med. Chem. 17 (2009) 1905–1910
1909
3.95 (m, 2H, -CH, 6-H), 3.69 (m, 1H, 3-H), 3.66–3.53 (m, 3H, other H
of mannitol), 3.48 (m, 1H, 5-H), 3.43 (m, 1H, 6-H), 1.45 (d,
J = 6.8 Hz, 3H, -CH3). 13C NMR (DMSO-d6): 195.6 (CO), 173.8
(CHCO), 141.2, 137.1, 137.0, 132.7, 131.9, 129.6, 128.7 (CH), 71.3
(C-5), 71.2 (C-3), 69.6 (C-4), 68.4 (C-2), 67.8 (C-1), 63.8 (C-6),
44.4 (CH), 18.7 (CH3). HRMS (m/z): Calcd for C22H26O8: 418.1628;
Found: 418.1636.
80.9 (C-4b), 75.3 (C-5b), 75.1 (C-3b), 75.0 (C-2b), 73.2 (C-30), 72.9
(C-50), 71.7 (C-5 ), 70.6 (C-20), 70.2 (C-2
), 70.7 (C-3 ), 68.7 (C-
40), 64.5 (C-60), 60.9 (C-6
& C-6b), 44.7 (CH), 18.9 (CH3). HRMS:
a
a
a
a
Calcd for C28H34O13: 578.1999; Found: 578.1998.
4.5.6. 6-O-Ketoprofen trehaloside (3f)
White powder. 1H NMR (DMSO-d6): 7.76–7.51 (m, 9H, Ar–H),
5.05 (s, 1H, 10-H), 4.90 (s, 1H, 1-H), 4.83 (m, 2H), 4.77 (m, 2H),
4.69 (m, 2H), 4.35 (m, 1H), 4.33–4.04 (m, 2H), 3.96 (m, 2H, –CH,
6-H), 3.63 (s, 1H, 2-H), 3.52–3.47 (m, 4H, 4-H), 3.23 (d, J = 4.0 Hz,
2H), 3.14 (m, 2H, 40-H), 1.43 (d, J = 6.8 Hz, 3H, –CH3). 13C NMR
(DMSO-d6): 195.6 (CO), 173.3 (CHCO), 141.1, 137.1, 137.0, 132.7,
131.9, 129.6, 128.7, 128.5, 128.4 (CH), 93.3 (C-10), 93.2 (C-1), 72.8
(C-30), 72.7 (C-3), 72.5 (C-50), 71.5 (C-20), 71.4 (C-2), 70.1 (C-40),
70.1 (C-4), 69.7 (C-5), 63.5 (C-6), 60.7 (C-60), 44.4 (CH), 18.5
(CH3). HRMS: Calcd for C28H34O13: 578.1999; Found: 578.1982.
4.5.2. 6-O-Ketoprofen glucoside (3b)
Yellow solid. 1H NMR (DMSO-d6): 7.75–7.52 (m, 9H, Ar–H), 6.66
(s, 0.4H, 1b-OH), 6.33 (s, 0.6H, 1
(m, 1.2H, other b-OH), 4.78 (s, 0.5H, 3
0.6H, 2
-OH), 4.42–4.35 (m, 0.7H, 6b-H and 60b-H), 4.28 (d,
J = 8.0 Hz, 0.6H, 6
-H), 4.03 (m, 0.6H, 60a-H), 3.94 (m, 1H, –CH),
3.78 (m, 0.6H, 5
a-OH), 5.04 (m, 0.6H, 1
a-H), 4.95
a-OH), 4.51 (d, J = 6.1 Hz,
a
a
a
-H), 3.55 (m, 0.7H, 3b-H and 4b-H), 3.30–2.90
(m, 4.4H, other H of glucose), 1.45 (d, J = 6.8 Hz, 3H, –CH3). 13C
NMR (DMSO-d6): 196 (CO), 174 (CHCO), 141.6, 137.7, 133.3,
132.4, 130.2, 129.4, 128.6 (CH), 97.4 (C-1b), 92.8 (C-1
3b), 75.2 (C-2b), 74.0 (C-5b), 73.3 (C-3 ), 72.6 (C-2
), 70.6 (C-4b), 69.7 (C-5 ), 65.0 (C-6b), 64.9 (C-6 ), 44.8 (CH),
a), 76.9 (C-
4.5.7. 10-O-Ketoprofen sucroside (3g)
a
a), 71.0 (C-
White powder. 1H NMR (DMSO-d6): 7.76–7.51 (m, 9H, Ar–H),
5.27 (d, J = 4.0 Hz, 1H, 1-H), 5.15 (s, 1H, 3-OH), 4.82–4.76 (m,
4H), 4.39 (s, 2H, 30-OH), 4.28–4.20 (m, 1H), 4.04–3.89 (m, 2H, –
CH, 30-H), 3.80–3.71 (m, 1.5H, 6-H), 3.67–3.63 (m, 2H, 10-H),
3.55–3.51 (m, 3H, 40b-H, 5-H), 3.44 (m, 2.5H, 4-H, 3-H), 3.16–
3.10 (m, 2H), 1.46 (d, J = 6.8 Hz, 3H, –CH3). 13C NMR (DMSO-d6):
196.2 (CO), 173.4 (CHCO), 141.4, 137.6, 137.4, 133.2, 132.4,
130.1, 129.3, 129.0, 128.9 (CH), 102.5 (C-20), 92.5 (C-1), 83.1 (C-
50), 77.0 (C-30), 73.5 (C-40), 73.4 (C-3), 73.2 (C-5), 71.8 (C-2), 70.2
(C-4), 63.1 (C-10), 62.3 (C-60), 60.9 (C-6), 44.8 (CH), 18.8 (CH3).
HRMS: Calcd for C28H34O13: 578.1999; Found: 578.1983.
4a
a
a
19.4 (CH3). HRMS: Calcd for C22H24O8: 416.1471; Found: 416.1463.
4.5.3. 6-O-Ketoprofen mannoside (3c)
Yellow solid. 1H NMR (DMSO-d6): 7.75–7.52 (m, 9H, Ar–H), 6.35
(d, J = 4.4 Hz, 0.64H, 1
4.89–4.85 (m, 1.7H, H-1 and 4-OH), 4.65 (d, J = 4.0 Hz, 0.3H, 3b-
OH), 4.62 (d, J = 4.1 Hz, 0.7H, 3 -OH), 4.56 (d, J = 6.8 Hz, 1H, 2
OH), 4.52 (d, J = 4.0 Hz, 0.3H, 2b-OH), 4.45 (d, J = 11.5 Hz, 0.7H,
-H), 4.30–4.11 (m, 0.6H, 6b-H and 60b-H), 4.03 (m, 0.3H, 5b-H),
a-OH), 6.22 (d, J = 8.4 Hz, 0.24H, 1b-OH),
a
a-
6
a
3.95 (m, 1.7H, 60a-H, -CH), 3.55 (m, 0.7H, 5
a-H), 3.45–3.3 (m, 2H,
2-H, 3-H), 3.29 (m, 1H, 4-H), 1.43 (d, J = 6.8 Hz, 3H, –CH3). 13C
NMR (DMSO-d6): 196.1 (CO), 174.1 (CHCO), 141.6, 137.6, 137.4,
133.2, 132.4, 130.1, 129.3, 129.1, 128.9 (CH), 94.6 (C-1), 74.5 (C-
4.6. n-Octanol/water partition coefficient (logP)
Partition coefficient was determined in n-octanol–water system
using the shake flask method. Equal volumes of n-octanol and
phosphate buffer (pH 7.0) were saturated for a period of 24 h. Solu-
tions of the prodrugs were prepared in the pre-saturated n-octanol
phase, and it was shaken with the phosphate buffer at 30 °C for 1 h
in a 10 mL tube. Two layers were separated by centrifugation. After
the two phases were separated, the concentrations of prodrugs in
octanol phase (C0) and the concentrations of prodrugs in water
phase (C1) are detected by HPLC. The partition coefficients (logP)
can be calculated with following equation:
5b), 74.0 (C-3b), 72.0 (C-2b), 71.8 (C-2a), 70.9 (C-5a), 70.8 (C-
3a
), 67.6 (C-4 ), 67.3 (C-4b), 65.4 (C-6b), 65.3 (C-6a
a
), 44.8 (CH),
19.4 (CH3). HRMS: Calcd for C22H24O8: 416.1471; Found: 416.1480.
4.5.4. 60-O-Ketoprofen maltoside (3d)
White powder. 1H NMR (DMSO-d6): 7.76–7.55 (m, 9H, Ar–H),
6.78 (m, 0.3H, 1b-OH), 6.35 (m, 0.3H, 1
0.3H, 2 -OH), 5.52–5.47 (m, 1H, 2b-OH), 5.44–5.34 (m, 1H, 3-
OH), 5.18 (m, 0.5H, 1
-H), 5.03 (m, 1H, 40-OH), 4.97 (m, 1H, 10-
H), 4.92 (m, 1H, 30-OH), 4.61 (m, 0.3H, 1b-H), 4.52–4.48 (m, 1H,
6b-OH), 4.45–4.20 (m, 1H, 6 -OH), 4.14–4.08 (m, 0.5H), 4.04–
3.98 (m, 1.5H, -CH), 3.76–3.59 (m, 3H, 50a-H, 50b-H, 5
-H, 6 -H),
3.59–3.36 (m, 4H, 30a-H, 30b-H, 3b-H), 3.32–3.19 (m, 3H, 4b-H,
a-OH), 5.62 (d, J = 4.0 Hz,
a
a
P ¼ C0V0=C1V1
a
V0 is the volume of octanol phase and V1 is the volume of water
phase.
CLOGP and molinspiration software were used to predict the
partition coefficients for these prodrugs.
a
a
4a a
-H, 20a-H, 20b-H, 2b-H, 2 -H), 3.10–2.93 (m, 1.5H, 40a-H, 40b-
H), 1.45 (d, J = 6.8 Hz, 3H, -CH3). 13C NMR (DMSO-d6): 196.1 (CO),
174.0 (CHCO), 141.5, 137.6, 137.4, 133.2, 132.4, 130.1, 129.4,
129.1, 128.9 (CH), 101.2 (C-10), 97.3 (C-1b), 92.6 (C-1
a
), 81.4 (C-
), 81.1 (C-4b), 77.0(C-3b), 76.7 (C-5b), 73.9 (C-30), 73.6 (C-3
),
72.8 (C-20), 72.6 (C-2 ), 71.1 (C-50), 70.8 (C-5 ), 70.4 (C-40), 64.9
(C-60), 60.2 (C-6
,C-6b), 44.7 (CH), 19.2 (CH3). HRMS: Calcd for
4.7. Chemical hydrolysis study in aqueous buffers
4a
a
The chemical hydrolysis rates of ester prodrugs 3b and 3d were
determined by monitoring the production of ketoprofen with HPLC
methods described above. The chemical hydrolysis of the ester pro-
drugs was studied at pH 7.4 using phosphate buffer. 15 mg of sam-
ple for hydrolysis was dissolved in 70 mL of buffer solution, which
were shaken at 37 °C. At appropriate time intervals, samples were
withdrawn and diluted with mobile phase for direct analysis by
HPLC.
a
a
a
C28H34O13: 578.1999; Found: 578.2011.
4.5.5. 60-O-Ketoprofen lactoside (3e)
White powder. 1H NMR (DMSO-d6): 7.75–7.54 (m, 9H, Ar–H),
6.70 (d, J = 4.0 Hz, 0.5H, 1b-OH), 6.37 (s, 0.5H, 1
1H, 20-OH), 4.95–4.90 (m, 2H, 1 -H, 2b-OH), 4.80 (m, 1H, 30a-OH,
30b-OH), 4.60 (m, 1H, 2
-OH, 6b-OH), 4.47 (m, 1H, 4 -OH, 3b-
OH), 4.35–3.97 (m, 5H, -CH, other H of lactose), 3.72 (m, 2H, 5
H), 3.64–3.58 (m, 3H, 3 -H, 6
-H, 50a-H), 3.34 (m, 2.5H, 50b-H,
-H, 4
a-OH), 5.18 (s,
a
a
a
Acknowledgments
a
-
a
a
20-H), 3.27 (m, 2H, 2
a
a-H), 3.00 (m, 0.5H, 2b-H), 1.45 (d,
Authors gratefully acknowledge the National Natural Science
Foundation of China (Nos. 20725206, 20732004 and 20702034)
for financial support and the Sichuan University Analytical & Test-
ing Center for NMR analysis.
J = 6.8 Hz, 3H, –CH3). 13C NMR (DMSO-d6): 196.1 (CO), 174.0
(CHCO), 141.5, 137.6, 137.4, 133.2, 132.7, 132.5, 130.1, 129.3,
128.9 (CH), 104.0 (C-10), 97.2 (C-1b), 92.5 (C-1
a), 81.3 (C-4a),

1910
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15. Chang, C. S.; Su, C. C.; Zhuang, J. R.; Tsai, S. W. J. Mol. Catal. B: Enzym. 2004, 30,
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