1356
X. Zhao et al. / European Journal of Medicinal Chemistry 41 (2006) 1352–1358
To this reaction mixture was added 20.0 ml of acetonitrile. The
mixtures were incubated in a thermoconstanter orbital shaker at
yield of 54.0 mg (26.3%), white solid, R : 0.71 (chloroform/
methanol/water, 65:15:2); H NMR data are as follows
f
1
2
0
00 rpm and 50 °C. Reactions were initiated by addition of
.2 g Novozym 435 lipase and terminated after 144 h. At the
(500 MHz, CDCl ): δ 7.19 (d, 2H, J = 8.01 Hz, H-2′, H-6′),
7.08 (d, 2H, J = 7.94 Hz, H-3′, H-5′), 4.77 (d, 1H,
J = 3.72 Hz, H-1, β-H), 4.27 (3H, H-6a, H-6b, H-5), 3.70 (q,
3
end of the reaction, the mixture was extracted with dichloro-
methane by stirring at ambient temperature for 20 min. Hereby,
the immobilized lipase was separated by flotation from the
reaction mixture allowing an easy recovering of the biocatalyst.
After removal of organic solvent in vacuum, the crude products
were purified by silica gel chromatography (ethyl acetate/
methanol, 10:1, V/V).
After purification of the synthesized products by silica gel
liquid chromatography, identity of products was confirmed by
MS using Perkin–Elmer SCIEX API 100 for ESI measure-
ments and by H NMR in CDCl using Brücker AM 500 spec-
trometer.
Optical rotations were measured at 589 nm (sodium line)
and 25 °C in a WZZ-1S automatic polarimeter (Shanghai Pre-
cision Scientific Instrument Co. Ltd., China).
1H, ROOC-CH(CH )-R′), 3.70 (3H, OH-2,3,4), 3.39 (5H, H-2,
3
H-3, H-4, RO-CH CH CH ), 2.40 (d, 2H, J = 7.08 Hz, CH
2
2
3
CH(CH ) ), 1.83 (m, 1H, J = 6.75 Hz, CH CH(CH ) ), 1.48
2
3 2
2
3 2
(
3H, J = 6.96 Hz, ROOC-CH(CH )-R′), 0.88 (9H, CH CH(CH
3
2
3)2
, RO-CH CH CH ). MS: m/z = 843.4 (2M + Na).
2
2
3
After acylation of propyl glucopyranoside with ibuprofen,
optical rotation of residual ibuprofen was analyzed. The speci-
25
fic rotations ([α]D (c = 1.0 in ethanol)) of residual ibuprofen
and (S)-ibuprofen were +19.4° and +56.0°, respectively. Then,
the optical purity of product was 34.6%, and the ratio of R-
enantiomer and S-enantiomer in the product was 2.06.
1
3
6.3. HPLC procedure for the analysis of ester prodrugs
of ibuprofen
6
.2.1. Methyl 6-O-(2′-(4′-isobutylphenyl) propionyl) D-α-
glucopyranoside (II)
Quantitative analysis of glucopyranoside ester prodrugs and
ibuprofen was determined by HPLC on a reversed-phase col-
umn (Zorbax SB-C18, 5 um, 4.6 × 250 mm, Agilent, USA)
using Agilent 1100 series (USA) equipped with a DAD detec-
tor at 254 nm. Elution was conducted with mixture of 20 mM
sodium acetate solution (pH 2.5) and acetonitrile at a flow rate
of 1.0 ml min and ambient temperature. The compositions of
the eluent were adjusted for each compound in order to provide
an appropriate retention time and separation of ester prodrugs
and ibuprofen. The volume of the injected sample was 10 μl.
Quantification of the compounds was carried out by measuring
the peak areas in relation to those of standards chromato-
graphed under the same conditions. The concentration of ibu-
profen was determined from the peak area based on calibration
curve prepared using standard ibuprofen, as well as ibuprofen
glucopyranoside esters solution in organic medium.
Synthesis was performed as described above starting from
1
mmol of racemic ibuprofen and 0.5 mmol of methyl α-D-glu-
copyranoside with yield of 65.1 mg (34.1%), white solid, the
spectroscopic data were reported earlier in [23].
−
1
6
.2.2. Ethyl 6-O-(2′-(4′-isobutylphenyl) propionyl) D-
glucopyranoside (III)
Synthesis was performed as described above starting from
1
mmol of racemic ibuprofen and 0.5 mmol of ethyl glucopyr-
anoside (mixtures of α- and β-ethyl glucopyranoside) with
yield of 83.6 mg (42.2%), white solid, R : 0.69 (chloroform/
methanol/water, 65:15:2); H NMR data are as follows
f
1
(
500 MHz, CDCl ): δ 7.19 (d, 2H, J = 8.05 Hz, H-2′, H-6′),
3
7
.07 (d, 2H, J = 7.94 Hz, H-3′, H-5′), 4.74 (d, 0.67H,
J = 3.76 Hz, H-1, β-H), 4.38 (d, 0.33H, H-1, α-H), 4.14–4.31
(
-
3H, H-6a, H-6b, H-5), 3.73 (q, 1H, J = 7.13, ROOC-CH(CH3)
R′), 3.58 (3H, OH-2,3,4), 3.40 (2H, H-2, H-4), 3.22 (2H, RO-
6.4. Hydrolysis study in aqueous buffer
CH CH ), 3.07 (t, 1H, J = 9.45 Hz, H-3), 2.38 (d, 2H,
2
3
J = 7.15 Hz, CH CH(CH ) ), 1.78 (m, 1H, J = 6.74 Hz,
2
3 2
CH CH(CH ) ), 1.43 (d, 3H, J = 7.10 Hz, ROOC-CH(CH )-
The chemical hydrolysis of the ester prodrugs was studied at
2
3 2
3
R′), 1.22 (m, 3H, RO-CH CH ), 0.83 (d, 6H, J = 6.60 Hz,
pH 3.0–10.0 using HCl, acetate, phosphate, and NaOH buffer.
The total buffer concentration was 20 mM and constant ionic
strength of 0.5 M for each sample was maintained by adding
KCl. Samples for hydrolysis were prepared by adding the com-
pound to 20 mM buffer solution. The solutions were sealed in
screw-capped glass vials and then placed into a thermostati-
cally controlled water bath at 37 °C. At appropriate intervals,
samples were taken and assayed for the presence of ester and
hydrolysis product by HPLC according to the previously
described method. Pseudo first-order rate constants (Kobs) for
the individual reactions were determined from the slope of lin-
ear plots of the logarithm of residual ester concentration
against time. The corresponding half-life (t1/2) was then
2
3
CH CH(CH ) ). MS: m/z = 397.1 (M + H).
2
3 2
After acylation of ethyl glucopyranoside with ibuprofen,
optical rotation of residual ibuprofen was analyzed. The speci-
2
5
fic rotations ([α]D (c = 1.0 in ethanol)) of residual ibuprofen
and (S)-ibuprofen were +20.6° and +56.0°, respectively. Then,
the optical purity of product was 36.8%, and the ratio of R-
enantiomer and S-enantiomer in the product was 2.16.
6
.2.3. Propyl 6-O-(2′-(4′-isobutylphenyl) propionyl) D-
glucopyranoside (IV)
Synthesis was performed as described above starting from
1
mmol of racemic ibuprofen and 0.5 mmol of propyl gluco-
pyranoside (mixtures of α- and β-propyl glucopyranoside) with
obtained from the equation: t1/2 = 0.693/kobs
.