The synthesis of amphipathic prodrugs of 1,2-diol drugs with saccharide conjugates by high regioselective enzymatic protocol

Article abstract of DOI:10.1016/j.bmc.2006.11.039

A facile, high regioselective enzymatic synthesis approach for the preparation of amphipathic prodrugs with saccharides of mephenesin and chlorphenesin was developed. Firstly, transesterification of two drugs with divinyl dicarboxylates with different car

Full text of DOI:10.1016/j.bmc.2006.11.039

Bioorganic & Medicinal Chemistry 15 (2007) 1741–1748
The synthesis of amphipathic prodrugs of 1,2-diol drugs with
saccharide conjugates by high regioselective enzymatic protocol
Jing Quan, Zhichun Chen, Chengyou Han and Xianfu Lin*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
Received 29 October 2006; revised 22 November 2006; accepted 27 November 2006
Available online 30 November 2006
Abstract—A facile, high regioselective enzymatic synthesis approach for the preparation of amphipathic prodrugs with saccharides
of mephenesin and chlorphenesin was developed. Firstly, transesterification of two drugs with divinyl dicarboxylates with different
carbon chain length was performed under the catalysis of Candida antarctica lipase acrylic resin and Lipozymeꢀ in anhydrous ace-
tone at 50 ꢁC, respectively. A series of lipophilic derivatives with vinyl groups of mephenesin and chlorphenesin were prepared. The
influences of different organic solvents, enzyme sources, reaction time, and the acylation reagents on the synthesis of vinyl esters
were investigated. And then, protease-catalyzed high regioselective acylation of D-glucose and D-mannose with vinyl esters of
mephenesin and chlorphenesin gave drug–saccharide derivatives in good yields. The studies of lipophilicity and hydrolysis in vitro
of prodrugs verified that drug–saccharide derivatives had amphipathic properties, and both lipophilic and amphipathic drug deriv-
atives had obvious controlled release characteristics.
ꢂ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Glycoconjugates of drugs contain a large number of
functional groups that are nearly indistinguishable
chemically. However, it is well known that the deriva-
tions of drugs at specific group have clear therapeutic
advantages, including high activity and reduced toxici-
ty.11 Therefore, it is crucial to have good selectivity in
synthesis procedure. Enzyme-catalyzed reactions are
widely recognized as superior to conventional chemical
methods in selective modification of polyfunctional sub-
strates owing to mild reaction conditions, high catalytic
efficiency, inherent selectivity, and simple downstream
processing. Therefore, many research groups have paid
much effort in the area of enzymatic synthesis of drug
derivatives.12–16
Amphipathic prodrugs are one of the most effective sur-
face-active drugs and attracting much interest in recent
years. They can obviously affect the absorption of drugs
through cell membranes and biological barriers. In par-
ticular, it would like to self-assemble into ordered aggre-
gates such as micelles and vesicles to deliver themselves
in vivo.1–4 For example, many stable non-covalent drug-
surfactant conjugates are amphipathic, and generally
used to enhance the permeability of cell membranes.5–7
Some amphipathic macromolecule drugs are designed
for improving pharmaceuticals delivery properties and
their therapeutic effect.8,9 Saccharides are highly hydro-
philic and play an important role in biological recogni-
tion processes. A great number of drugs in use today
have relied on carbohydrates for their therapeutic
action. The incorporation of carbohydrates offers many
opportunities for preparing amphipathic prodrugs with
bioactive moieties and spans a wide range of drug
types.10
In pursuit to synthesize amphipathic small molecule pro-
drugs with sugar moieties, we have selected mephenesin
and chlorphenesin as substrates. As one type of 1,2-diol
drugs, they have centrally active muscle relaxant effect
and sedative properties. In addition, mephenesin is
effectively antitoxic agent for deltamethrin.17–20 Chlor-
phenesin is also used to treat injuries and other painful
muscular conditions along with rest and physical
therapy.21–23 The two drugs exhibit a short half-life in
the blood stream and a high overall clearance rate.
Amphipathic glycoconjugates of mephenesin and chlor-
phenesin maybe overcome these disadvantages and
possess better therapeutic effect.
Keywords: Amphipathic prodrug; Enzymatic synthesis; Mephenesin;
Chlorphenesin.
*
Corresponding author. Tel.: +86 571 87953001; fax: +86 571
87952618; e-mail: llc123@zju.edu.cn
0968-0896/$ - see front matter ꢂ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.039

1742
J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
Herein, we have developed a facile and high regioselec-
tive enzymatic method to prepare amphipathic drug–
saccharide derivatives. The lipophilicity and in vitro
hydrolysis of prodrugs were investigated. Results
showed that the derivatives of mephenesin and chlor-
phenesin containing sugar moiety had amphipathic
characteristics, which would like to permeate biomem-
branes and well solubilize in water. And then both lipo-
philic and amphipathic derivatives had obvious
sustained release characteristics compared with their
parent drugs. These characteristics will have potential
value for clinic symptom. After optimizing the reaction
conditions, fast reaction rate and good yields were
achieved.
2.2. Effect of enzyme
In order to identify suitable enzymes with high transe-
sterification activity in the synthesis of drug vinyl esters,
ten commercially available enzymes were tested for the
transesterification of mephenesin and chlorphenesin
with divinyl adipate in acetone at 50 ꢁC. The screening
results are presented and compared in Table 1. The cor-
responding control experiment in the absence of enzyme
formed drug vinyl esters in yields less than 0.5%.
Enzymes derived from various sources such as bacteria,
yeast, and molds show different properties, including
stability in organic solvent, activity, and specificity. As
shown in Table 1, the yield of 1b catalyzed by the ten en-
zymes ranged from 0.8% to 78.5%. Most of lipases were
appropriate for the transesterification of mephenesin
and divinyl adipate except lipase from C. cylindracea
with a yield of 16.5%. The yield of the 2b catalyzed by
the ten enzymes ranged from 1.9% to 72.5%. Two
proteases in this research showed low activity for two
drugs.
2. Results and discussion
2.1. Enzymatic synthesis of drug vinyl esters
Transesterifications of mephenesin and chlorphenesin
with divinyl dicarboxylates catalyzed by Candida antarc-
tica lipase acrylic resin and Lipozymeꢀ, respectively, are
shown in Scheme 1. The products were purified by flash
chromatography and analyzed by 13C NMR and 1H
NMR to identify the esterified position. Based on the
general strategy described by Yoshimoto et al.,24 acyla-
tion of a hydroxyl group of substrate will lead to the
O-acylated carbon (*CH2OCOR) downfield shift, while
the neighboring carbon (*CCH2OCOR) upfield shift in
13C NMR. Characterization of the products 1a and 2a
revealed that mephenesin and chlorphenesin were
acylated at C-1 positions, respectively. The signal for
C-1 of mephenesin shifted downfield from 64.1 to
65.7 ppm and that of C-2 shifted upfield from 70.7 to
68.6 ppm. The signal for C-1 of chlorphenesin shifted
downfield from 63.8 to 65.5 ppm and that of C-2 shifted
upfield from 70.5 to 68.4 ppm. Likewise, analysis of 13C
NMR spectra of other products (1b–1d and 2b–2d)
showed that acylations occurred at the primary hydrox-
All investigated enzymes for the transesterification were
found to catalyze exclusively the formation of the
mono-substituted 1-O-vinyladipoyl-substrate in the
initial reaction stage. After 24 h, the disubstituted
products of two drugs were found in the yields in
0.3–4.3% with all investigated enzymes (the data not
shown), and had no obvious increase with extending the
reaction time. The best result was obtained from Lipo-
zymeꢀ for chlorphenesin and Candida antarctica lipase
acrylic resin for mephenesin. Therefore, we selected
C. antarctica lipase acrylic resin and Lipozymeꢀ for fur-
ther investigation.
2.3. Effect of organic solvents
Reaction media play a crucial role on maintaining en-
zyme catalytic activity and stability.25 To optimize reac-
tion conditions for enzymatic transesterification of
mephenesin and chlorphenesin, sixteen different solvents
1
yl of mephenesin and chlorphenesin. The analysis of H
NMR spectra also confirmed the products’ structure.
O
C
O
C
OH
1
COOCH=CH2
(CH2)n
O
C
O
C
O
(CH2)n
R'
HO
O
O
(CH2)n
CH=CH2
*
2
HO
O
HO
O
COOCH=CH2
3
*
*
Alkaline protease
Pyridine, 50 oC
3a-3d
5
6
Enzyme
50 oC
acetone,
1a-1d
2a-2d
R
R
R
1, 2
R =
O
ortho- CH3
para- Cl
mephenesin
1
2
O
HO O
O
chlorphenesin
HO
HO
HO
R' =
HO
OH
OH
OH
D-Glucose
1a, 2a, 3a, 1aG, 2aG, 2aM n = 2
D-Mannose
1b, 2b, 3b, 1bG, 2bG
1c, 2c, 3c
n = 4
n = 7
n = 8
1d, 2d, 3d
2aG-2bG
2aM
1aG-1bG ,
Scheme 1. High regioselective enzymatic synthesis of drug vinyl esters and amphipathic drug–saccharide derivatives.

J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
1743
Table 1. Effect of enzyme on synthesis of vinyl esters of mephenesin
and chlorphenesin
The solvents with logP value ranging from ꢀ1.3 to 1.2
were appropriate to the transesterification. The yields
of 1b and 2b were 49.8–78.5% and 40.2–72.5%, respec-
tively (entries 1–10). The solvents with logP greater than
1.2 are unfavorable for the reaction except tetrachloro-
methane (logP 2.9, entry 13), which was due to its good
solubility for drugs. The best results were obtained in
anhydrous acetone which was a green solvent with lower
toxicity and easier processing. Consequently, anhydrous
acetone was chosen as reaction medium in the following
investigations (Table 2).
Enzyme
Yield (%)a
1b 2b
<0.5 <0.8
0.8 1.9
28.7 33.9
Control, no enzyme
Alkaline protease from Bacillus subtilis
Amano protease PS from Aspergillus melleus
Lipozymeꢀ immobilized lipase from Mucor miehei 56.0 72.5
Lipase from porcine pancreas
Lipase from hog pancreas
56.4 70.7
57.7 67.6
16.5 24.8
65.5 21.8
61.1 34.5
34.3 41.3
78.5 65.4
Lipase from Candida cylindracea
Lipase Type VII from Candida rugosa
Lipase AY30
2.4. Influence of chain length of acylation agent on initial
reaction rates and yields
Amano Lipase M from Mucor javanicusr
Candida antarctica lipase acrylic resin
Conditions: Enzyme (15 mg mLꢀ1), acetone (2 mL), mephenesin or
chlorphenesin (1 mmol), divinyl adipate (6 mmol), 50 ꢁC, 250 rpm, 2 h.
a Determined by HPLC.
The influence of the chain length of acylation agent on
yields was evaluated. The enzymatic reactivity was
found to decrease as the length of the chain of the divi-
nyl dicarboxylates increased. As shown in Figure 1, the
equilibrium time of reaction prolonged with the chain
length increasing. The yield of 1a had already been
greater than 82% after 30 min, while the yields of 1c–
1d were only 65.0% and 61.3% even after prolonged
4 h, respectively, and had no obvious increase with
extending the reaction time. The reaction result of 2a–
2d was similar to that of mephenesin. Comparing with
acylation agents 3b–3d, the reactivity of 3a was the high-
est. The initial reaction rate in the transesterification of
Table 2. Effect of solvent on synthesis of vinyl esters of mephenesin
and chlorphenesin
Entry
Solvent
logP
Yield (%)a
1b
2b
1
2
DMSO
DMF
ꢀ1.3
ꢀ1.0
ꢀ0.5
ꢀ0.39
ꢀ0.23
0.46
0.6
63.9
61.2
64.5
65.2
78.5
58.4
59.0
66.7
58.2
49.8
12.5
24.1
45.3
5.7
41.1
50.6
52.3
48.4
72.5
40.2
44.8
27.8
27.1
46.2
14.9
10.8
22.6
3.2
3
Dioxane
Acetonitrile
Acetone
4
5
3a with 1 and 2 was high up to 326.4 and 283.8 mM hꢀ1
,
6
Tetrahydrofuran
Dichloromethane
Pyridine
7
respectively (Table 3).
8
0.65
0.79
1.2
9
2-Methyl-2-propanol
1,2-Dichloroethane
Isopropyl ether
Toluene
2.5. Enzymatic regioselective synthesis of amphipathic
drug–saccharide derivatives
10
11
12
13
14
15
16
1.9
2.2
Saccharides are hydrophilic compounds and play an
important role in living systems. They are particularly
effective for the improvement of drugs water-solubility,
and dissolution behavior. Our approach is based on a
strategy of two-step enzymatic acylation. In the first
step, mephenesin and chlorphenesin were reacted with
divinyl dicarboxylate to give active acyl derivatives,
which were then used as an acyl donor in the second
step. Then, we synthesized successfully products 1aG–
1bG, 2aG–2bG, and 2aM using alkaline protease from
Bacillus subtilis as the catalyst, which was confirmed to
be an efficient catalyst in the acylation of sugars in our
Tetrachloromethane
Cyclohexane
Hexane
2.9
3.4
3.9
4.9
3.8
4.4
4.1
5.2
Octane
Conditions: Candida antarctica lipase acrylic resin or Lipozymeꢀ
(15 mg mLꢀ1), solvent (2 mL), mephenesin or chlorphenesin (1 mmol),
divinyl adipate (6 mmol), 50 ꢁC, 250 rpm, 2 h.
a Determined by HPLC.
with logP value ranging from ꢀ1.3 to 4.9 were screened.
There was a certain correlation between the yields and
logP of solvents.
90
85
80
75
70
65
60
90
80
70
60
50
divinylsuccinate
divinyladipate
divinylsebacate
divinylazelate
divinylsuccinate
divinyladipate
divinylazelate
divinylsebacate
55
50
45
40
30
0
50
100
150
200
250
0
50
100
150
200
250
Time (min) mephenesin
Time (min) chlorphenesin
Figure 1. Progress curve of enzymatic acylation of mephenesin and chlorphenesin with different divinyl dicarboxylates.

1744
J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
Table 3. Influence of chain length of acylation agent on the initial reaction rates and yields of the transesterification
Acylation agents
Time (min)
Yieldsa(%)
Initial rate (mM hꢀ1
)
Mephenesin
Chlorphenesin
Mephenesin
Chlorphenesin
2a
2b
2c
2d
60
120
120
120
86.2
78.5
64.1
60.4
84.7
72.5
56.8
51.9
326.4
260.4
87.0
283.8
111.0
85.8
84.6
52.8
Conditions: Candida antarctica lipase acrylic resin or Lipozymeꢀ (15 mg mLꢀ1), mephenesin or chlorphenesin (1 mmol), divinyl dicarboxylates
(6 mmol), acetone (2 mL), 50 ꢁC, 250 rpm.
a Determined by HPLC.
previous researches.26 The position of acylation in enzy-
matically prepared drug–saccharide derivatives was ver-
ified by 13C NMR and 1H NMR. Glucose and mannose
were both substituted at C-6 position. The alkaline pro-
tease from B. subtilis showed an effective regioselectivity
in the transesterification of saccharides with vinyl esters
of mephenesin and chlorphenesin.
ous solubility, which was attributed to modifications of
prodrug with carbon chain and glucose functional
groups. For chlorphenesin–mannose derivative 2aM, it
was also amphipathic properties. The value of D7.4 (dis-
tribution coefficient in an n-octanol/PBS) was 1.18,
which was similar to the value of D7.4 2aG 1.04. With
the increased carbon chain of drug–saccharide deriva-
tives, 1bG and 2bG had greater lipophilicity with values
of D7.4 3.86 and 4.31, respectively, than 1aG and 2aG.
The amphipathy of drug–saccharide derivatives was
obvious when 1a and 2a were used as acylation agents.
It was the four carbon chain length of drug vinyl esters
that were compatible with the structure of amphipathic
drugs.
Anomeric mixtures were obtained for D-glucose
(a:b = 40:60) in good yields 70.2–78.4% and D-mannose
(a:b = 60:40) in yield 64.8%. We monitored the forma-
tion of products by TLC. Results indicated that D-glu-
cose reacted faster than D-mannose. No product was
detected in the absence of enzyme.
2.7. Hydrolysis studies in vitro
2.6. Lipophilicity of the prodrugs
The kinetics of chemical hydrolysis of 1a and 2a as well
as its glucose derivatives 1aG and 2aG was investigated
in simulated gastric fluid (hydrochloric acid buffer, pH
1.2) and simulated phosphate buffer of pH 7.4 at 37 ꢁC.
The variation of ester concentration was monitored by
HPLC, and the results indicated that the reaction dis-
played pseudo-first-order kinetics. The rate constants
(kobsd) for the individual hydrolysis reaction were calcu-
lated from the linear regression equations. The corre-
sponding half-life for the respective prodrugs was then
calculated. The results are summarized in Table 5.
Lipophilicity is well known as a prime physicochemical
descriptor of drugs with relevance to their biological
properties. The apparent partition coefficients (logP)
of the prodrugs and the parent drugs were investigated
in an n-octanol/phosphate buffer solution (PBS) of pH
7.4. The concentration of the compounds in n-octanol
and PBS layer was determined by HPLC. The results
are listed in Table 4.
The prodrugs 1a and 2a with logP 2.16 and 2.44 were
more lipophilic than their parent drugs 1 and 2, which
would be characterized by enhanced intestinal absorp-
tion relative to the parent drug. With the longer carbon
chain of vinyl esters, the prodrugs 1b–1d and 2b–2d
should have more lipophilicity than 1a and 2a.
The hydrolysis rates of prodrugs 1aG and 2aG were
slower than those of 1a and 2a in two buffer solutions.
In hydrochloric acid buffer, the hydrolysis rate of pro-
drug 1aG was 5.04 · 10ꢀ2 hꢀ1, while that of 1a was
7.92 · 10ꢀ2 hꢀ1. The half-life time of 1aG was also long-
er than 1a in pH 1.2. Compared with 1a and the parent
drug, 1aG was of sustained release, especially more obvi-
ous in PBS. The half-life time of 1aG in PBS was 6-fold
Compared with the parent drugs, the prodrugs 1aG and
2aG had not only the lipid solubility but also the aque-
Table 4. The lipophilic parameters of the prodrugs and the parent
drugs
Drug
kdetection (nm)
Lipophilicitya
logP
1
228
230
228
230
230
232
230
232
230
12.2
27.5
143.4
1.10
3.86
277.1
1.04
4.31
1.18
1.09
1.44
2.16
0.04
0.59
2.44
0.02
0.63
0.07
Table 5. Kinetic data for chemical hydrolysis of the prodrugs
2
1a
Compound
pH 1.2a
pH 7.4b
1aG
1bG
2a
kobsd (hꢀ1) · 102 t1/2 (h) kobsd (hꢀ1) · 102 t1/2 (h)
1a
7.92
5.04
8.07
5.04
8.75
13.75
8.59
3.49
19.86
92.90
18.83
84.31
2aG
2bG
2aM
1aG
2a
0.746
3.68
0.822
2aG
13.75
a hydrochloric acid buffer (0.2 M).
b phosphate buffer (0.2 M).
Eluent: methanol/water (80:20, v/v).
a D7.4, distribution coefficient in n-octanol/phosphate buffer (pH 7.4).

J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
1745
more than that in pH 1.2. The hydrolysis of chlorphen-
4.2. Analytical methods
esin prodrugs was similar to that of mephenesin pro-
drugs. Presumably, it was the amphipathy of 1aG and
2aG that acted as self-stabilizing property due to
protection of the hydrolytically labile prodrug linkage
especially in PBS, and extension of hydrolysis half-lives
in vitro.
The process of reactions was monitored by TLC on sil-
ica with petroleum ether/EtOAc (2:1, v/v) as solvent for
the monoester compounds (1a–1d, 2a–2d) and EtOAc/
methanol/water (17:2:1, v/v) for the drug–saccharide
1
derivatives (1aG–1bG, 2aG–2bG, 2aM). The H NMR
and 13C NMR spectra were recorded with TMS as inter-
nal standard using a Bruker AMX-500 MHz spectrom-
1
3. Conclusion
eter. H NMR and 13C NMR spectra were recorded at
500 and 125 MHz, respectively. Chemical shifts were ex-
pressed in ppm and coupling constants (J) in Hz. IR
spectra were measured with a Nicolet Nexus FTIR 670
spectrophotometer. Analytical HPLC was performed
using an Agilent 1100 series with a reversed-phase
Shim-Pack VP-ODS column (150 · 4.6 mm) and a UV
detector (275 nm). Methanol/water (80:20, v/v) was used
as a mobile phase, while the flow rate was adjusted to
In conclusion, in order to achieve amphipathic and
small molecule prodrugs with saccharide bioactive
moieties, we selected mephenesin and chlorphenesin as
substrates. A highly regioselective enzymatic synthesis
approach was described by the transesterification of
mephenesin and chlorphenesin with divinyl dicarboxy-
lates containing different carbon chain length. The influ-
ences of enzyme source, organic solvent, reaction time,
and chain length of acylation agent on the initial reac-
tion rates and yields were systematically studied. After
obtaining eight drug analogues with vinyl groups, we
achieved a series of amphipathic drug–saccharide deriv-
atives by sequential regioselective acylation with D-glu-
cose and D-mannose. Through investigation of the
lipophilicity and hydrolysis in vitro, drug–saccharide
derivatives showed amphipathy and obvious sustained
release characteristics compared with parent drugs.
The strategy was efficient and facile for synthesis of
amphipathic small molecule prodrugs with saccharide.
The further research of other relevant amphipathic pro-
drug derivatives is in progress.
1 mL minꢀ1
.
4.3. General procedure for synthesis of drug vinyl esters
The reaction was initiated by adding 15 mg/mL
Lipozymeꢀ to 20 mL acetone containing 5 mmol drugs
and 30 mmol divinyl dicarboxylates (3a–3d), respective-
ly. The suspension was kept at 50 ꢁC and shaken at
250 rpm. The reaction was monitored by TLC (petro-
leum ether/ethyl acetate 3:2, v/v). The reactions were
terminated by filtering off the enzyme and then the
filtrate was evaporated under reduced pressure. The
products were isolated by silica gel column chromatog-
raphy with an eluent consisting of petroleum ether/ethyl
acetate (2:1, v/v). Enzymatic synthesis of vinyl esters of
mephenesin and chlorphenesin is shown in Scheme 1.
4. Experimental
4.1. Materials
4.4. Synthesis of 1-O-vinylsuccinyl-mephenesin (1a)
The reaction time was 1 h and the product yield was
85.2%. 1H NMR (CDCl3, d, ppm): 7.25 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.15 (t, 2H, Ar–H),
6.88 (d, 1H, Ar–H), 6.81 (d, 1H, Ar–H), 4.90 (dd, 1H,
J = 14.1 Hz, J = 6.3 Hz, @CH2), 4.58 (dd, 1H,
J = 6.3 Hz, J = 1.5 Hz, @CH2), 4.38–4.26 (m, 3H,
–CH(OH)CH2–), 4.02 (d, 2H, J = 5.0 Hz, –OCH2CH–),
2.72 (m, 4H, –CH2CH2–), 2.58 (br, 1H, –CH(OH)–),
2.22 (s, 3H, –CH3). 13C NMR (CDCl3, d, ppm): 173.7,
171.0 (C@O), 156.5, 131.0, 127.1, 126.9, 121.2, 111.1
(Ar, mephenesin), 141.4 (–O–CH@), 98.1 (@CH2), 68.7
(C-3, mephenesin), 68.6 (C-2, mephenesin), 65.7 (C-1,
mephenesin), 29.2, 29.1 (–CH2–), 16.3 (C-4, mephenesin).
IR (KBr, cmꢀ1): 3495 (OH), 1750, 1742 (O–C@O), 3091,
1647 (C@C), 1603, 1591, 1496, 753, 714 (Ar). ESI-MS
(m/z): 331 [M+Na]+.
Lipozymeꢀ (EC 3.1.1.1, an immobilized preparation of
lipase from Mucor miehei, 42 U/g), lipase from porcine
pancreas (EC 3.1.1.3, Type II, powder, 30–90 U/mg),
lipase from C. cylindracea (EC 3.1.1.3, powder, 2.8
U/mg), and lipase from hog pancreas (EC 3.1.1.3,
powder, 2.4 U/mg) were purchased from Fluka. C. ant-
arctica lipase acrylic resin (EC 3.1.1.3, an immobilized
preparation of lipase from C. antarctica on macropo-
rous acrylic resin, 10,000 U/g,) and lipase Type VII from
Candida rugosa (EC 3.1.1.3, powder, 706 U/mg) were
purchased from Sigma. Amano Lipase M from Mucor
javanicus (EC 3.1.1.3, powder, 10 U/mg) and Amano
Protease PS from Aspergillus melleus (EC 3.4.21.63,
powder, 100 U/mg) were purchased from Aldrich.
Lipase AY30 (EC 3.1.1.3, powder) was purchased from
Acro¨s. Alkaline protease from B. subtilis (EC 3.4.21.14,
a crude preparation of the alkaline serine protease,
100 U/mg) was purchased from Wuxi Enzyme Co. Ltd
(Wuxi, PR China). Mephenesin and chlorphenesin
were purchased from Alfa Aesar, a Johnson Matthey
Company. All the enzymes were used directly in
commercial preparations without further purification.
All solvents were of analytical grade and were dried by
4.5. Synthesis of 1-O-vinyladipoyl-mephenesin (1b)
The reaction time was 2 h and the product yield was
78.5%. 1H NMR (CDCl3, d, ppm): 7.25 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.15 (t, 2H, Ar–H),
6.88 (d, 1H, Ar–H), 6.81 (d, 1H, Ar–H), 4.87 (dd, 1H,
J = 14.1 Hz, J = 6.3 Hz, @CH2), 4.56 (dd, 1H,
J = 6.3 Hz, J = 1.5 Hz, @CH2), 4.35–4.26 (m, 3H,
–CH(OH)CH2–), 4.02 (d, 2H, J = 5.0 Hz, –OCH2CH–),
˚
storing over activated 3 A molecular sieves before use.
All other reagents were used as received.

1746
J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
3.24 (br, 1H, –CH(OH)–), 2.39 (m, 4H, 2-CH2), 2.22 (s,
3H, –CH3), 1.68 (m, 4H, 2-CH2). 13C NMR (CDCl3, d,
ppm): 173.5, 170.6 (C@O), 156.5, 130.9, 127.0, 126.9,
121.1, 111.2, (Ar, mephenesin), 141.4 (–O–CH@), 98.0
(@CH2), 68.7 (C-3, mephenesin), 68.6 (C-2, mephenesin),
65.7 (C-1, mephenesin), 33.8, 33.5, 24.3, 24.0
(–CH2–), 16.3 (C-4, mephenesin). IR (KBr, cmꢀ1):
3498 (OH), 1749, 1738 (O–C@O), 3092, 1647 (C@C),
1597, 1591, 1493, 750, 719 (Ar). ESI-MS (m/z): 359
[M+Na]+.
phenesin), 141.4 (–O–CH@), 98.1 (@CH2), 69.2 (C-3,
chlorphenesin), 68.5 (C-2, chlorphenesin), 65.5 (C-1,
chlorphenesin), 29.2, 29.1 (–CH2–). IR (KBr, cmꢀ1):
3528 (OH), 1762, 1740 (O–C@O), 1647 (C@C), 1597,
1499, 836, 806 (Ar). (ESI-MS (m/z): 351 [M+Na]+.
4.9. Synthesis of 1-O-vinyladipoyl-chlorphenesin (2b)
The reaction time was 2 h and the product yield was
72.5%. 1H NMR (CDCl3, d, ppm): 7.27 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.22 (d, 2H, Ar–H),
6.84 (d, 2H, Ar–H), 4.88 (dd, 1H, J = 14.1 Hz,
J = 6.3 Hz, @CH2), 4.57 (dd, 1H, J = 6.3 Hz,
J = 1.5 Hz, @CH2), 4.32–4.21 (m, 3H, –CH(OH)CH2–),
3.98 (m, 2H, –OCH2CH–), 3.05 (br, 1H,
–CH(OH)–), 2.39 (m, 4H, 2-CH2–), 1.68 (m, 4H,
2-CH2–). 13C NMR (CDCl3, d, ppm): 173.5, 170.6
(C@O), 157.1, 129.5, 129.5, 126.3, 116.0, 116.0 (Ar,
chlorphenesin), 141.2 (–O–CH@), 98.0 (@CH2), 69.2
(C-3, chlorphenesin), 68.5 (C-2, chlorphenesin), 65.4
(C-1, chlorphenesin), 33.8, 33.5, 24.3, 24.0 (–CH2–). IR
(KBr, cmꢀ1): 3515 (OH), 1760, 1736 (O–C@O), 1647
(C@C), 1592, 1497, 836, 806 (Ar). ESI-MS (m/z): 379
[M+Na]+.
4.6. Synthesis of 1-O-vinylazeloyl-mephenesin (1c)
The reaction time was 2 h and the product yield was
64.1%. 1H NMR (CDCl3, d, ppm): 7.28 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.15 (t, 2H, Ar–H),
6.89 (d, 1H, Ar–H), 6.81 (d, 1H, Ar–H), 4.87 (dd, 1H,
J = 14.1 Hz, J = 6.3 Hz, @CH2), 4.56 (dd, 1H,
J = 6.3 Hz, J = 1.5 Hz, @CH2), 4.35–4.26 (m, 3H,
–CH(OH)CH2–), 4.02 (d, 2H, J = 5.0 Hz, –OCH2CH–),
2.68 (br, 1H, –CH(OH)–), 2.36 (m, 4H, 2-CH2), 2.23 (s,
3H, –CH3), 1.63 (m, 4H, 2-CH2), 1.31 (m, 6H, 3-CH2).
13C NMR (CDCl3, d, ppm): 174.2, 170.8 (C@O), 156.5,
131.0, 127.0, 127.0, 121.1, 111.3, (Ar, mephenesin),
141.4 (–O–CH@), 98.0 (@CH2), 68.7 (C-3, mephenesin),
68.6 (C-2, mephenesin), 65.7 (C-1, mephenesin), 34.3,
34.0, 29.0, 29.0, 29.0, 25.0, 24.7 (–CH2–), 16.3
(C-4, mephenesin). IR (KBr, cmꢀ1): 3495 (OH),
4.10. Synthesis of 1-O-vinylazeloyl-chlorphenesin (2c)
The reaction time was 2 h and the product yield was
56.8%. 1H NMR (CDCl3, d, ppm): 7.27 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.22 (d, 2H, Ar–H),
6.84 (d, 2H, Ar–H), 4.88 (dd, 1H, J = 14.1 Hz,
J = 6.3 Hz, @CH2), 4.57 (dd, 1H, J = 6.3 Hz,
J = 1.5 Hz, @CH2), 4.32–4.21 (m, 3H, –CH(OH)CH2–),
3.99 (m, 2H, –OCH2CH–), 3.60 (br, 1H, –CH(OH)–),
2.36 (m, 4H, 2-CH2–), 1.64 (m, 4H, 2-CH2–), 1.32 (m,
6H, 3-CH2–). 13C NMR (CDCl3, d, ppm): 174.2, 170.8
(C@O), 157.1, 129.7, 129.6, 126.4, 116.0, 116.0 (Ar,
chlorphenesin), 141.2 (–O–CH@), 98.0 (@CH2), 69.3
(C-3, chlorphenesin), 68.4 (C-2, chlorphenesin), 65.3
(C-1, chlorphenesin), 34.3, 34.0, 29.0, 29.0, 29.0, 25.0,
24.7 (–CH2–). IR (KBr, cmꢀ1): 3520 (OH), 1757, 1734
(O–C@O), 1645 (C@C), 1592, 1497, 836, 806 (Ar). ESI-
MS (m/z): 421 [M+Na]+.
1748,
1740
(O–C@O),
3092,
1647
(C@C),
1603, 1591, 1496, 753, 714 (Ar). ESI-MS (m/z): 401
[M+Na]+.
4.7. Synthesis of 1-O-vinylsebacoyl-mephenesin (1d)
The reaction time was 2 h and the product yield was
60.0%. 1H NMR (CDCl3, d, ppm): 7.28 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.15 (t, 2H, Ar–H),
6.89 (d, 1H, Ar–H), 6.80 (d, 1H, Ar–H), 4.87 (dd, 1H,
J = 14.1 Hz, J = 6.3 Hz, @CH2), 4.56 (dd, 1H,
J = 6.3 Hz, J = 1.5 Hz, @CH2), 4.35–4.26 (m, 3H,
–CH(OH)CH2–), 4.02 (d, 2H, J = 5.0 Hz, –OCH2CH–),
2.68 (br, 1H, –CH(OH)–), 2.36 (m, 4H, 2-CH2), 2.23 (s,
3H, –CH3), 1.63 (m, 4H, 2-CH2), 1.28 (m, 8H, 4-CH2).
13C NMR (CDCl3, d, ppm): 174.2, 171.1 (C@O), 156.5,
131.0, 127.0, 127.0, 121.2, 111.2, (Ar, mephenesin),
141.3 (–O–CH@), 98.1 (@CH2), 68.7 (C-3, mephenesin),
68.6 (C-2, mephenesin), 65.7 (C-1, mephenesin), 34.3,
34.1, 29.2, 29.2, 29.1, 29.1, 25.0, 24.7 (–CH2–), 16.4
(C-4, mephenesin). IR (KBr, cmꢀ1): 3495 (OH), 1750,
1742 (O–C@O), 3093, 1647 (C@C), 1603, 1591, 1496,
753, 714 (Ar). ESI-MS (m/z): 415 [M+Na]+.
4.11. Synthesis of 1-O-vinylsebacoyl-chlorphenesin (2d)
The reaction time was 2 h and the product yield was
51.9%. 1H NMR (CDCl3, d, ppm): 7.27 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.22 (d, 2H, Ar–H),
6.84 (d, 2H, Ar–H), 4.88 (dd, 1H, J = 14.1 Hz,
J = 6.3 Hz, @CH2), 4.57 (dd, 1H, J = 6.3 Hz,
J = 1.5 Hz, @CH2), 4.30–4.22 (m, 3H, –CH(OH)CH2–),
3.99 (m, 2H, –OCH2CH–), 2.80 (br, 1H, –CH(OH)–),
2.36 (m, 4H, 2-CH2–), 1.63 (m, 4H, 2-CH2–), 1.27 (m,
8H, 4-CH2–). 13C NMR (CDCl3, d, ppm): 174.2,
171.18 (C@O), 157.2, 129.5, 129.5, 126.4, 116.0, 116.0
(Ar, chlorphenesin), 141.3 (–O–CH@), 98.1 (@CH2),
69.3 (C-3, chlorphenesin), 68.5 (C-2, chlorphenesin),
65.4 (C-1, chlorphenesin), 34.3, 34.1, 29.2, 29.2, 29.1,
29.1, 25.0, 24.7 (–CH2–). IR (KBr, cmꢀ1): 3519 (OH),
1757, 1734 (O–C@O), 1642 (C@C), 1592, 1497, 832,
803 (Ar). ESI-MS (m/z): 435 [M+Na]+.
4.8. Synthesis of 1-O-vinylsuccinyl-chlorphenesin (2a)
The reaction time was 1 h and the product yield was
84.7%. 1H NMR (CDCl3, d, ppm): 7.26 (dd, 1H,
J = 6.3 Hz, J = 14.1 Hz, –CH@), 7.23 (d, 2H, Ar–H),
6.85 (d, 2H, Ar–H), 4.91 (dd, 1H, J = 14.1 Hz,
J = 6.3 Hz, @CH2), 4.58 (dd, 1H, J = 6.3 Hz,
J = 1.5 Hz, @CH2), 4.35-4.24 (m, 3H, –CH(OH)CH2–),
4.00 (m, 2H, –OCH2CH–), 2.74 (m, 4H, –CH2CH2–).
13C NMR (CDCl3, d, ppm): 173.7, 171.0 (C@O),
157.2, 129.6, 129.6, 126.3, 116.0, 116.0 (Ar, chlor-

J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
1747
4.12. General procedure for synthesis of amphipathic
drug–saccharide derivatives
4.15. Synthesis of 6-O-(1-O-chlorphenesin-vinylsuccinyl)-
D-glucose (2aG)
A mixture of 1a–1b or 2a–2b (2 mmol) and D-glucose
(4 mmol) or D-mannose, alkaline protease from B. sub-
tilis (0.25 g, 25 mg/mL), and 10 mL pyridine was shaken
at 250 rpm for 36 h at 50 ꢁC. The reactions were termi-
nated by filtering off the enzyme. The pyridine was evap-
orated. Formations of the drug–saccharide derivatives
were confirmed by TLC. The product was isolated by
silica gel chromatography with an eluent consisting of
ethyl acetate/methanol/water (17:2:1, by vol) to give
products (1aG–1bG, 2aG–2bG, 2aM). Regioselective
enzymatic synthesis of drug–saccharide derivatives is
shown in Scheme 1.
The isolated yield of product was 78. 4%. 1H NMR
(D2O, d, ppm): 6.88 (d, 2H, Ar–H), 6.56 (d, 2H, Ar–
H), 5.12 (d, 0.40H, J = 3.60, H-1 of a-D-glucose), 4.55
(d, 0.60H, J = 7.91, H-1 of b-D-glucose), 4.31–4.12 (m,
2H, H-6, 60 of b-D-glucose, H-6, 60 of a-D-glucose),
4.03-3.18 (m, 9H, –OCH2CHCH2–,a H or b H of D-glu-
cose), 2.52 (m, 4H, 2-CH2–). 13C NMR (D2O, d, ppm):
174.2, 174.1 (C@O), 157.1, 129.5, 129.5, 126.0, 116.2,
116.2 (C–Ar, chlorphenesin), 96.4 (C1 of b-D-glucose),
92. 5 (C1 of a-D-glucose), 76.1 (C3 of b-D-glucose),
74.5 (C2 of b-D-glucose), 73.7 (C5 of b-D-glucose),
73.0 (C3 of a-D-glucose), 71.8 (C2 of a-D-glucose),
70.1 (C4 of a-D-glucose), 70.0 (C4 of b-D-glucose),
69.9 (C5 of a-D-glucose), 69.5 (C-3, chlorphenesin),
67.8 (C-2, chlorphenesin), 65.8 (C-1, chlorphenesin),
63.7 (C6 a, b of D-glucose), 29.2, 29.0 (–CH2–). IR
(cmꢀ1): 3364 (O–H), 1739 (C@O). ESI-MS (m/z): 487
[M+Na]+.
4.13. Synthesis of 6-O-(1-O-mephenesin-vinylsuccinyl)-D-
glucose (1aG)
The isolated yield of product was 77.7%. 1H NMR
(D2O, d, ppm): 6.88 (d, 1H, Ar–H), 6.78 (d, 1H, Ar–
H), 6.79 (t, 2H, Ar–H), 5.12 (d, 0.40H, J = 3.60, H-1
of a-D-glucose), 4.58 (d, 0.60H, J = 7.91, H-1 of b-D-glu-
cose), 4.35–4.12 (m, 2H, H-6, 60 of b-D-glucose, H-6, 60
of a-D-glucose), 4.03–3.18 (m, 9H,–OCH2CHCH2–, a H
or b H of D-glucose), 2.53 (m, 4H, 2-CH2–), 1.96 (s, 3H,
-CH3). 13C NMR (D2O, d, ppm): 174.2, 174.1 (C@O),
156.5, 130.8, 127.1, 126.8, 121.0, 111.7 (C–Ar, mephen-
esin), 96.4 (C1 of b-D-glucose), 92.4 (C1 of a-D-glucose),
75.9 (C3 of b-D-glucose), 74.4 (C2 of b-D-glucose), 73.7
(C5 of b-D-glucose), 73.0 (C3 of a-D-glucose), 71.8 (C2
of a-D-glucose), 70.1 (C4 of a-D-glucose), 70.0 (C4 of
b-D-glucose), 69.4 (C5 of a-D-glucose), 68.6 (C-3,
mephenesin), 67.9 (C-2, mephenesin), 66.0 (C-1,
mephenesin), 63.7 (C6 a, b of D-glucose), 29.2, 29.1
(–CH2–), 16.3 (C-4, mephenesin). IR (cmꢀ1): 3340
(OH), 2960, 2850 (CH), 1735 (C@O), 1468 (CH2),
1149 (C–O), 1058 (C–O–H). ESI-MS (m/z): 467
[M+Na]+.
4.16. Synthesis of 6-O-(1-O-chlorphenesin-vinyladipoyl)-
D-glucose (2bG)
The isolated yield of product was 70. 2%. 1H NMR
(D2O, d, ppm): 6.88 (d, 2H, Ar–H), 6.56 (d, 2H, Ar–
H), 5.12 (d, 0.40H, J = 3.60, H-1 of a-D-glucose), 4.55
(d, 0.60H, J = 7.91, H-1 of b-D-glucose), 4.31–4.12 (m,
2H, H-6, 60 of b-D-glucose, H-6, 60 of a-D-glucose),
4.03-3.18 (m, 9H, –OCH2CHCH2–,a H or b H of D-glu-
cose), 2.52 (m, 4H, 2-CH2–). 13C NMR (D2O, d, ppm):
174.2, 174.1 (C@O), 157.1, 129.5, 129.5, 126.0, 116.2,
116.2 (C–Ar, chlorphenesin), 96.4 (C1 of b-D-glucose),
92. 5 (C1 of a-D-glucose), 76.1 (C3 of b-D-glucose),
74.5 (C2 of b-D-glucose), 73.7 (C5 of b-D-glucose),
73.0 (C3 of a-D-glucose), 71.8 (C2 of a-D-glucose),
70.1 (C4 of a-D-glucose), 70.0 (C4 of b-D-glucose),
69.9 (C5 of a-D-glucose), 69.5 (C-3, chlorphenesin),
67.8 (C-2, chlorphenesin), 65.8 (C-1, chlorphenesin),
63.7 (C6 a, b of D-glucose), 29.2, 29.0 (–CH2–). IR
(cmꢀ1): 3364 (O–H), 1739 (C@O). ESI-MS (m/z): 515
[M+Na]+.
4.14. Synthesis of 6-O-(1-O-mephenesin-vinyladipoyl)-D-
glucose (1bG)
The isolated yield of product was 73.6%. 1H NMR
(D2O, d, ppm): 6.88 (d, 1H, Ar–H), 6.78 (d, 1H, Ar–
H), 6.79 (t, 2H, Ar–H), 5.12 (d, 0.40H, J = 3.60, H-1
of a-D-glucose), 4.58 (d, 0.60H, J = 7.91, H-1 of b-D-glu-
cose), 4.35–4.12 (m, 2H, H-6, 60 of b-D-glucose, H-6, 60
of a-D-glucose), 4.03–3.18 (m, 9H,–OCH2CHCH2–, a H
or b H of D-glucose), 2.37 (m, 4H, 2-CH2), 1.98 (s, 3H,
–CH3), 1.66 (m, 4H, 2-CH2). 13C NMR (D2O, d, ppm):
174.2, 174.1 (C@O), 156.5, 130.8, 127.1, 126.8, 121.0,
111.7 (C–Ar, mephenesin), 96.4 (C1 of b-D-glucose),
92.4 (C1 of a-D-glucose), 75.9 (C3 of b-D-glucose),
74.4 (C2 of b-D-glucose), 73.7 (C5 of b-D-glucose),
73.0 (C3 of a-D-glucose), 71.8 (C2 of a-D-glucose),
70.1 (C4 of a-D-glucose), 70.0 (C4 of b-D-glucose),
69.4 (C5 of a-D-glucose), 68.6 (C-3, mephenesin), 67.9
(C-2, mephenesin), 66.0 (C-1, mephenesin), 63.7 (C6 a,
b of D-glucose), 33.8, 33.5, 24.3, 24.0 (–CH2–), 16.3
(C-4, mephenesin). IR (cmꢀ1): 3338 (O–H), 2963, 2850
(C–H), 1734 (C@O), 1468 (CH2), 1149 (C–O), 1058
(C–O–H). ESI-MS (m/z): 495 [M+Na]+.
4.17. Synthesis of 6-O-(1-O-chlorphenesin-vinylsuccinyl)-
D-mannose (2aM)
6-O-(1-O-Chlorphenesin-vinylsuccinyl)-D-mannose was
synthesized by the same method as for 6-O-(1-O-chlor-
phenesin-vinylsuccinyl)-D-glucose. The isolated yield of
1
product was 64. 8%. H NMR (D2O, d, ppm): 6.98 (d,
2H, Ar–H), 6.62 (d, 2H, Ar–H), 5.07 (s, 0.6H, H-1 of
a-D-mannose), 4.76 (s, 0.4H, H-1 of b-D-mannose),
4.31–4.18 (m, 2H, H-6, 60 of b-D-mannose, H-6, 60 of
a-D-mannose), 4.10-4.04 (m, 3H, –CH(OH)CH2–), 3.88
(m, 2H, –OCH2CH–), 3.77–3.51 (m, 4H, a H or b H
of D-mannose), 2.56 (m, 4H, 2-CH2–). 13C NMR
(D2O, d, ppm): 174.4, 174.3 (C@O), 157.0, 129.5,
129.5, 125.8, 116.2, 116.1 (C–Ar, chlorphenesin), 94.4
(C1 of a-D-mannose), 94.0 (C1 of b-D-mannose), 73.9
(C5 of b-D-mannose), 73.1 (C3 of b-D-mannose), 71.3
(C2 of b-D-mannose), 70.8 (C2 of a-D-mannose), 70.4

1748
J. Quan et al. / Bioorg. Med. Chem. 15 (2007) 1741–1748
(C3 of a-D-mannose), 70.3 (C5 of a-D-mannose), 68.9
(C-3, chlorphenesin), 67.6 (C-2, chlorphenesin), 67.1
(C-1, chlorphenesin), 66.9 (C4 of a-D-mannose), 66.8
(C4 of b-D-mannose), 64.3 (C6 a, b of D-mannose),
28.9, 28.9 (-CH2-). IR (cmꢀ1): 3360 (O–H), 1736
(C@O). ESI-MS (m/z): 487 [M+Na]+.
References and notes
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3. Lambert, D. M. Eur. J. Pharm. Sci. 2000, 11, s15.
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4.18. Determination of distribution coefficients
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6. Martins, L. M. S.; Mamizuka, E. M.; Carmona-Ribeiro,
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The distribution coefficient (D) of the prodrugs and the
parent drugs was determined by dissolving 5 mg of the
respective compound in 2.5 mL of n-octanol and adding
to an equal volume of phosphate buffer (pH 7.4) in
screw-capped test tube. The solutions were then mixed
for 15 min and centrifuged at 1 · 104 rpm for 5 min.
The layers were separated and aliquots of 50 lL were
diluted to 500 lL. Samples of 20 lL were taken and ana-
lyzed by HPLC. All experiments were conducted in trip-
licate and the mean values were taken. The values of
D7.4 for the respective compounds were then calculated
and the results are listed in Table 4.
4.19. Hydrolysis in aqueous solutions in vitro
A 0.2 M hydrochloric acid buffer of pH 1.2 as non-enzy-
matic simulated gastric fluid (SGF) and a 0.2 M phos-
phate buffer of pH 7.4 were used in this study. Stock
solution of the respective ester prodrug (25 mM) was
prepared in dioxane. An aliquot (1 mL) was added to
9 mL of the appropriate thermostated (37 ꢁC) aqueous
buffer solution (pH 7.4) to initiate the hydrolysis reac-
tion. Samples of 20 lL were taken at fixed intervals
and diluted to 200 lL with methanol, then analyzed by
HPLC. Pseudo-first-order rate constants (kobsd) for the
hydrolysis of the ester prodrugs were then calculated
from the slopes of the lineate plots of log (% residual
prodrug) versus time.
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Acknowledgments
The financial support from the National Natural Science
Foundation of China (Nos. 20474056, 20572099) and
the Zhejing Provincial Science and Technology Council
(Project No. 2004C24009) is gratefully acknowledged.
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