880
Vol. 60, No. 7
hydropyrazin-2-yl)oxy)methoxy)-4-oxobutyl)-1H-1,2,3-tri- OBDTM 19×150mm (5mm, 12nm), gradient: milli-Q water
azol-1-yl)-3-hydroxybutanoic Acid (11) To a solution of (TFA 0.1%):CH3CN (TFA 0.1%)=30:70 to milli-Q water
alkyne 10 (10mg) in DMF/tBuOH (1:1) (0.60mL) were added (TFA 0.1%):CH3CN (TFA 0.1%)=40:60 over 40min, Flow
a solution of (R)-4-azido-3-hydroxybutanoic acid (4) (2.8mg) rate: 9.00mL/min, UV: 365, 230nm) to give the triazole 13
in H2O (0.30mL), CuSO4·5H2O (5mol%) and sodium ascor- (11mg, 79%). IR (KBr) νmax: 3131, 1749, 1659, 1623, 1586,
1
bate (15mol%) at room temperature. The mixture was stirred 1353, 1204, 1135cm−1; H-NMR (300MHz, CD3OD) δ: 8.27 (s,
°
for 10min at 50 C under microwave irradiation. Then the 1H), 8.09 (s, 1H), 8.07 (s, 1H), 7.88 (s, 1H), 7.40–7.37 (m, 3H),
mixture was cooled to rt and solvent was removed in vacuo. 7.27 (s, 1H), 6.58 (s, 1H), 6.16 (s, 2H), 5.48 (d, J=9.2Hz, 1H),
The residue was diluted with AcOEt and water and extracted 4.10 (t, J=9.4Hz, 1H), 3.97 (d, J=3.2Hz, 1H), 3.81–3.65 (m,
with AcOEt. The extracts were washed with brine, dried over 4H), 2.73 (t, J=7.6Hz, 2H), 2.50 (t, J=7.3Hz, 2H), 2.01–1.96
Na2SO4, filtered and concentrated. The residual mixture was (m, 2H), 1.41 (s, 9H); 13C-NMR (100MHz, CD3OD) δ: 173.62,
purified through reverse-phase HPLC (SunFireTMPrepC18 161.65, 153.26, 148.18, 141.94, 136.04, 134.91, 132.95, 132.48,
OBDTM 19×150mm (5mm, 12nm), gradient: milli-Q water 130.74, 130.56, 129.56, 122.15, 101.07, 90.17, 83.36, 79.92,
(TFA 0.1%):CH3CN (TFA 0.1%)=35:65 to milli-Q water 75.33, 71.41, 70.39, 62.47, 34.04, 33.09, 30.50, 25.46; HR-MS
(TFA 0.1%):CH3CN (TFA 0.1%)=75:25 over 40min, Flow (ESI) m/z Calcd for C32H40N7O9 [M+H]+ 666.2888, Found
rate: 9.00mL/min, UV: 365, 230nm) to give the triazole 11 666.2886.
(10mg, 79%). IR (KBr) νmax: 3180, 3050, 2972, 2925, 1663,
(R)-4-(4-(Carboxymethyl)-1H-1,2,3-triazol-1-yl)-3-hy-
1205, 1134cm−1; H-NMR (500MHz, CD3OD) δ: 8.08 (s, 1H), droxybutanoic Acid (14) 14 was synthesized from 5-hexy-
8.07 (s, 1H), 7.80 (s, 1H), 7.61 (s, 1H), 7.39–7.30 (m, 3H), 7.23 noic acid in 3 steps.12) 1H-NMR (300MHz, CD3OD) δ: 7.81 (s,
(s, 1H), 6.71 (s, 1H), 6.15 (s, 2H), 4.45–4.30 (m, 3H), 2.71 (t, 1H), 4.57–4.49 (m, 1H), 4.44–4.36 (m, 2H), 2.76 (t, J=7.5Hz,
J=7.6Hz, 2H), 2.51–2.47 (m, 3H), 2.39 (dd, J=16, 7.3Hz, 1H), 2H), 2.57–2.43 (m, 2H), 2.35 (t, J=7.3Hz, 2H), 1.96 (quint,
1.99 (quint, J=7.4Hz, 2H), 1.42 (s, 9H); 13C-NMR (125MHz, J=7.5Hz, 2H); HR-MS (ESI) m/z Calcd for C10H16N3O5
CD3OD) δ: 174.26, 173.69, 161.47, 154.03, 147.82, 142.28, [M+H]+ 258.1090, Found 258.1096.
1
136.36, 135.05, 132.98, 132.77, 131.22, 130.15, 129.46, 129.19,
Water Solubility and Stability of Prodrug Derivatives.
124.46, 122.93, 104.18, 83.25, 68.06, 56.29, 40.11, 34.07, Water Solubility Prodrug derivatives were saturated in
33.14, 31.00, 25.57, 25.40; high resolution (HR)-MS (electro- distilled water and shaken vigorously. The saturated solu-
spray ionization (ESI)) m/z: Calcd for C30H36N7O7 [M+H]+ tions were sonicated for 15min at 25 C and filtered through
°
606.2676, Found 606.2704.
the centrifugal filter (0.45nm filter unit, Ultrafree®-MC, Mil-
Sodium (R)-4-(4-(4-((((3Z,6Z)-6-Benzylidene-3-((5-(tert- lipore). The filtrate was injected into HPLC to analyze the
butyl)-1H-imidazol-4-yl)methylene)-5-oxo-3,4,5,6-tetra- water solubility.
hydropyrazin-2-yl)oxy)methoxy)-4-oxobutyl)-1H-1,2,3-tri-
Hydrolysis of 12 in the Presence of Esterase 12 (500µg)
azol-1-yl)-3-hydroxybutanoate (12) The triazole 11 was was dissolved in a 10mM PBS (500µL, pH 7.4) solution and
eluated with a solvent of H2O/CH3CN (1:1) through an ion the solution was separated to three eppendorf tubes by each
exchange resin at room temperature. The eluent was filtrated 50, 50 and 300µL. The two 50µL solutions were used as the
with H2O/CH3CN (1:1) and concentrated. The residue was negative control without the enzyme, one was incubated at rt
°
lyophilized to give the title compound 12 as a yellow solid. and the other was incubated at 37 C. To a solution of 10mM
IR (KBr) νmax: 3141, 3075, 2954, 1647, 1579, 1440, 1396, 1219, PBS (300µL, pH 7.4) was added a suspension of the porcine
1
1126cm−1; H-NMR (500MHz, CD3OD) δ: 8.07 (s, 1H), 8.06 liver esterase (6.0µL) (PLE; carboxylic–ester hydrolase; EC
(s, 1H), 7.65 (s, 1H), 7.63 (s, 1H), 7.39–7.30 (m, 3H), 7.21 (s, 3.1.1.1; E-2884), which is a model esterase having a good cor-
1H), 6.75 (s, 1H), 6.14 (s, 2H), 4.44 (dd, J=15, 2.8Hz, 1H), relation with plasma esterase in the stability assay and was
4.31–4.22 (m, 2H), 2.71 (t, J=7.5Hz, 2H), 2.48 (t, J=7.3Hz, obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.)
2H), 2.36 (dd, J=15, 5.0Hz, 1H), 2.27 (dd, J=15, 7.2Hz, 1H), as a suspension in a 3.2M (NH4)2SO4 solution (pH 8.0) con-
1.99 (quint, J=4.5Hz, 2H), 1.41 (s, 9H); 13C-NMR (125MHz, taining 40 units of enzyme per mL. Then the solution was
CD3OD) δ: 178.83, 173.72, 161.41, 154.24, 147.77, 142.38, separated to six eppendorf tubes by 50µL, followed by incu-
°
136.45, 135.09, 133.12, 132.70, 130.03, 129.42, 128.75, 124.28, bation at 37 C. DMSO (150µL) was added to each tubes and
°
121.83, 105.07, 83.24, 69.22, 56.56, 42.47, 34.10, 33.15, 31.15, the mixtures were sonicated for 15min at 25 C and filtered
25.63, 25.46; HR-MS (ESI) m/z Calcd for C30H35N7O7Na through the centrifugal filter (0.45nm filter unit, Ultrafree®-
[M+H]+ 628.2496, Found 628.2479.
MC, Millipore). The filtrate was injected into HPLC to ana-
(((3Z,6Z)-6-Benzylidene-3-((5-(tert-butyl)-1H-imida- lyze the conversion to the parent drug.
zol-4-yl)methylene)-5-oxo-3,4,5,6-tetrahydropyrazin-2-yl)-
oxy)methyl
4-(1-((2R,3R,4S,5R,6R)-3,4,5-Trihydroxy-6-
Acknowledgments This work was supported by Grants
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3- from MEXT (Ministry of Education, Culture, Sports, Sci-
triazol-4-yl)butanoate (13) To a solution of alkyne 10 ence and Technology) of Japan, including a Grant-in-Aid for
(10mg) in DMF/tBuOH/H2O (1:1:1) (0.40mL) were add- Scientific Research (B) (No. 20390036), a Grant-in-Aid for
ed 1-azide-1-deoxy-β-D-galactopyranoside (6) (8.9mg), Research Activity Start-up (No. 21890262), and a Grant-in-Aid
CuSO4·5H2O (5mol%) and sodium ascorbate (15mol%) at for Young Scientists (B) (No. 21790118).
°
room temperature. The mixture was stirred for 10min at 50 C
under microwave irradiation. Then the mixture was cooled to
rt and solvent was removed in vacuo. The residue was filtered
through silica gel pad and concentrated. The residual mixture
was purified through reverse-phase HPLC (SunFireTMPrepC18