Journal of Medicinal Chemistry
Article
as determined by high-performance liquid chromatography and
elemental analyses.
4-Methylumbellifer-7-yl-β-D-glucopyranoside (9): 55% over-
all yield; Rf = 0.39 (8/2 CH2Cl2/MeOH); mp 210−212 °C; 1H NMR
(400.13 MHz, DMSO-d6) δ 2.41 (s, 3H), 3.17 (dd, 1H, J = 14.2, 8.8
Hz), 3.29 (dd, 2H, J = 11.9, 7.4 Hz), 3.40−3.53 (m, 2H), 5.08 (d, 1H,
J = 5.3 Hz), 6.25 (s, 1H), 7.03 (d, 1H, J = 2.4 Hz), 7.05 (dd, 1H, J =
9.2, 2.4 Hz), 7.71 (d, 1H, J = 9.2 Hz); 13C NMR (100 MHz, DMSO-
d6) δ 18.35, 60.86, 69.85, 73.35, 76.70, 77.36, 100.21, 103.42, 111.92,
113.60, 114.29, 126.63, 153.56, 154.61, 160.33, 160.37; MS (ESI+) m/
z 361.38 [M + Na]+. Anal. Calcd for C16H18O8: C, 56.80; H, 5.36.
Found: C, 56.85; H, 5.41.
4-Methylumbellifer-7-yl-β-D-galactopyranoside (10): 64%
overall yield; Rf = 0.35 (8/2 CH2Cl2/MeOH); mp 248 °C; 1H
NMR (400.13 MHz, DMSO-d6) δ 2.41 (s, 3H), 3.44 (ddd, 1H, J = 9.2,
5.5, 3.3 Hz), 3.48−3.65 (m, 3H), 3.68 (t, 1H, J = 6.3 Hz), 3.72 (t, 1H,
J = 3.8 Hz), 4.99 (d, 1H, J = 7.7 Hz), 6.25 (s, 1H), 7.02 (d, 1H, J = 2.4
Hz), 7.05 (dd, 1H, J = 9.1, 2.4 Hz), 7.70 (d, 1H, J = 9.1 Hz); 13C NMR
(100 MHz, DMSO-d6) δ 18.15, 60.39, 68.13, 69.87, 73.22, 75.71,
100.60, 103.15, 112.24, 112.85, 114.79, 126.17, 153.89, 154.75, 160.19,
160.19; MS (ESI+) m/z 361.35 [M + Na]+. Anal. Calcd for C16H18O8:
C, 56.80; H, 5.36. Found: C, 56.75; H, 5.31.
Synthesis of 2,3,4,6-Tetra-O-acetyl-D-mannopyranose (2). D-
Mannose pentaacetate (1) (10.25 × 10−3 mol) was dissolved in dry
CH2Cl2 (40 mL). Morpholine (41 × 10−3 mol) was then added, and
the mixture was stirred under an N2 atmosphere at room temperature
overnight. The mixture was then washed twice with 40 mL of 1 N HCl
and 3 × 20 mL of water, dried (MgSO4), and concentrated under
vacuum to give the 2,3,4,6-tetra-O-acetyl-D-mannopyranose (2).
Synthesis of 2,3,4,6-Tetra-O-acetyl-D-mannopyranosyl Trichlor-
oacetimidate (3). The 2,3,4,6-tetra-O-acetyl-D-mannopyranose (2)
(4.31 × 10−3 mol) was dissolved in dry CH2Cl2 (38 mL).
Trichloroacetonitrile (43.1 × 10−3 mol) was added, and the mixture
was stirred under an N2 atmosphere at 0 °C for 1 h. Then
diazabicyclo[5.4.0]undec-7-ene (DBU) (0.86 × 10−3 mol) was added,
and the mixture was stirred under an N2 atmosphere at 0 °C for 30
min and concentrated under vacuum. The crude 2,3,4,6-tetra-O-acetyl-
D-mannopyranosyl trichloroacetimidate (3) was used without further
purification in the next step.
Synthesis of 4-Methylumbellifer-7-yl-2,3,4,6-tetra-O-acetyl-α-D-
mannopyranose (5). The crude 2,3,4,6-tetra-O-acetyl-D-mannopyr-
anosyl trichloroacetimidate (3) (4.31 × 10−3 mol) was dissolved in dry
CH2Cl2 (38 mL). 7-Hydroxy-4-methyl coumarin (4) (4.31 × 10−3
mol) and boron trifluoride metherate (BF3·Me2O) (0.86 × 10−3 mol)
were then added, and the mixture was stirred under an N2 atmosphere
at room temperature overnight. Twenty milliliters of CH2Cl2 was
further added, and the solution was washed with water, dried over
anhydrous MgSO4, and concentrated under vacuum. The crude
product (5) was then purified by crystallization from MeOH or by
silica gel column chromatography [5/5 (v/v) EP/AcOEt] to give the
expected compound in 60% yield.
4-Methylumbellifer-7-yl-β-D-xylopyranoside (11): 45% over-
1
all yield; Rf = 0.58 (8/2 CH2Cl2/MeOH); mp 223 °C; H NMR
(400.13 MHz, DMSO-d6) δ 2.40 (s, 3H), 3.27 (d, 2H, J = 2.3 Hz),
3.40 (m, 2H), 3.76 (m, 1H), 5.12 (d, 1H, J = 3.9 Hz), 6.25 (s, 1H),
7.01 (d, 1H, J = 2.4 Hz), 7.03 (dd, 1H, J = 9.2, 2.4 Hz), 7.70 (d, J = 9.2
Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 18.13, 62.73, 69.27,
72.95, 76.32, 100.32, 102.74, 112.74, 113.36, 114.13, 126.47, 153.32,
155.3, 159.32, 160.05; MS (ESI+) m/z 331.32 [M + Na]+. Anal. Calcd
for C15H16O7: C, 58.44; H, 5.23. Found: C, 58.49; H, 5.20.
4-Methylumbellifer-7-yl-β-D-melibiopyranoside (12): 47%
1
overall yield; Rf = 0.1 (8/2 AcOEt/MeOH); mp 103−105 °C; H
Synthesis of 4-Methylumbellifer-7-yl-α-D-mannopyranoside (6).
The 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl coumarin (5) (0.59 ×
10−3 mol) was added to a solution of MeONa (0.88 × 10−3 mol) in
dry MeOH (5 mL). The mixture was stirred at room temperature for
30 min. The product (6) was then purified by crystallization or by
silica gel column chromatography [5/5 (v/v) EP/AcOEt]. The
reaction is quantitative.
NMR (400.13 MHz, DMSO-d6) δ 2.41 (s, 3H), 3.18 (dd, 1H, J = 25.6,
13.2 Hz), 3.32 (m, 3H), 3.40 (dd, 2H, J = 10.7, 6.3 Hz), 3.55 (m, 6H),
4.65 (d, 1H, J = 3.4 Hz), 5.00 (d, 1H, J = 7.3 Hz), 6.26 (s, 1H), 7.04
(d, 1H, J = 2.4 Hz), 7.10 (dd, 1H, J = 8.8, 2.4 Hz), 7.71 (d, 1H, J = 8.8
Hz); 13C NMR (100 MHz, DMSO-d6) δ 20.66, 59.99, 60.08, 68.25,
68.35, 69.88, 70.09, 71.14, 74.32, 75.05, 77.26, 98.89, 100.02, 104.67,
111.38, 112.53, 114.23, 126.55, 154.17, 160.28, 166.57, 173.79; MS
(ESI+) m/z 523.16 [M + Na]+. Anal. Calcd for C22H28O13: C, 52.80;
H, 5.64. Found: C, 52.75; H, 5.61.
4-Methylumbellifer-7-yl-α-D-mannopyranoside (6): 51%
1
overall yield; Rf = 0.24 (9/1 CH2Cl2/MeOH); mp 132−134 °C; H
NMR (400.13 MHz, DMSO-d6) δ 2.4 (d, 3H, J = 0.8 Hz), 3.33 (m,
1H), 3.47 (m, 1H), 3.51 (t, 1H, J = 9.4 Hz), 3.57 (m, 1H), 3.69 (dd,
1H, J = 9.2 Hz), 3.86 (d, 1H, J = 1.2 Hz), 5.53 (d, 1H, J = 1.6 Hz),
6.24 (d, 1H, J = 1.2 Hz), 7.09 (d, 1H, J = 2.4 Hz), 7.11 (dd, 1H, J =
8.8, 2.4 Hz), 7.70 (d, 1H, J = 8.8 Hz); 13C NMR (100 MHz, DMSO-
d6) δ 18.82, 61, 66.95, 70.43, 71, 76.06, 99.48, 104.31, 112.38, 114.38,
114.79, 127.14, 160.80, 159.83, 155.02, 154.05; MS (ESI+) m/z 339.24
[M + H]+, 361.29 [M + Na]+, 699.37 [2M + Na]+. Anal. Calcd for
C16H18O8: C, 56.80; H, 5.36. Found: C, 56.84; H, 5.33.
CA Inhibition. An Applied Photophysics stopped-flow instrument
has been used for assaying the CA-catalyzed CO2 hydration activity.
Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes
(pH 7.5) as a buffer and 20 mM Na2SO4 (for maintaining a constant
ionic strength), following the initial rates of the CA-catalyzed CO2
hydration reaction for a period of 10−100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor, at least six
traces of the initial 5−10% of the reaction have been used for
determining the initial velocity. The uncatalyzed rates were
determined in the same manner and subtracted from the total
observed rates. Stock inhibitor solutions (0.1 mM) were prepared in
distilled−deionized water, and dilutions of up to 0.01 nM were made
thereafter with distilled−deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min to 72 h at room
temperature (15 min) or 4 °C (all other incubation times) prior to the
assay, to allow the formation of the E−I complex or the eventual active
site-mediated hydrolysis of the inhibitor. Data reported in Table 1
show the inhibition after incubation for 6 h, which led to the
completion of the in situ hydrolysis of the coumarin and formation of
the 2-hydroxycinnamic acids.1,2 The inhibition constants were
obtained by nonlinear least-squares methods using PRISM 3, as
reported previously,1,2 and represent the mean from at least three
different determinations. CA isofoms were recombinant ones obtained
in house as reported previously.1,2
4-Methylumbellifer-7-yl-α-L-rhamnopyranoside (7): 58%
1
overall yield; Rf = 0.4 (9/1 CH2Cl2/MeOH); mp 207−209 °C; H
NMR (400.13 MHz, CDCl3) δ 1.14 (d, 3H, J = 6.4 Hz), 2.35 (d, 1H, J
= 1.2 Hz), 3.86 (q, 1H, J = 5.3 Hz), 5.10 (t, 1H, J = 10 Hz), 5.42 (d,
1H, J = 3.6 Hz), 5.44 (t, 1H, J = 2.3 Hz, H2), 5.45 (t, 1H, J = 2.2 Hz),
6.13 (d, 1H, J = 0.8 Hz), 7.02 (d, 1H, J = 2.4 Hz), 7.06 (dd, 1H, J =
8.8, 2.4 Hz), 7.47 (d, 1H, J = 8.8 Hz); 13C NMR (100 MHz, CDCl3) δ
21.05, 21.11, 69, 69.27, 69.51, 70.1, 95, 104.26, 113.23, 113.61, 125,
152.52, 155.10, 158.61, 170.15, 170.31; MS (ESI+) m/z 345.31 [M +
Na]+, 667.39 [2M + Na]+. Anal. Calcd for C16H18O7: C, 59.62; H,
5.63. Found: C, 59.58; H, 5.65.
4-Methylumbellifer-7-yl-β-D-ribopyranoside (8): 60% overall
yield; Rf = 0.45 (8/2 AcOEt/MeOH); 1H NMR (400.13 MHz,
DMSO-d6) δ 2.38 (d, 3H, J = 1.2 Hz), 3.91 (m, 1H), 4.03 (m, 1H),
4.70 (t, 1H, J = 5.4 Hz), 5.07 (d, 1H, J = 6 Hz), 5.61 (d, 1H, J = 2 Hz),
6.23 (s, 1H), 6.77 (d, 1H, J = 2 Hz), 6.96 (dd, 1H, J = 8.4, 2 Hz), 7.68
(d, 1H, J = 8.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ 18.09, 62.518,
70.40, 74.46, 84.81, 103.27, 105.05, 111.55, 113.36, 113.84, 126.46,
153.32, 155.3, 159.32, 160.05; MS (ESI+) m/z 331.26 [M + Na]+,
639.25 [2M + Na]+. Anal. Calcd for C15H16O7: C, 58.44; H, 5.23.
Found: C, 58.40; H, 5.25.
Pharmacological Inhibitors. For in vivo studies, the glycosyl
coumarin 6 was dissolved in a 37.5% PEG400/12.5% ethanol/50%
saline mixture prior to injection. Solutions were heated gently (∼40
°C) to completely dissolve the compound. Solutions of the inhibitor
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dx.doi.org/10.1021/jm200983e | J. Med. Chem. 2011, 54, 8271−8277