Journal of Natural Products
Article
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(TLC) 0.36 (n-hexane−-acetone, 70:30); H NMR (acetone-d6, 500
MHz) δ 7.45 (2H, bs, OH), 7.10 (2H, d, J = 2.0 Hz, H-4), 6.83 (2H, d,
J = 2.0 Hz, H-6), 4.29 (4H, t, J = 7.0 Hz, H2-8), 3.93 (6H, s, 3-OCH3),
2.93 (2H, t, J = 7.0 Hz, H2-7), 2.05 (6H, s, OCOCH3); 13C NMR
(acetone-d6, 125 MHz) δ 170.0 (C, C-9), 147.8 (C, C-3), 142.3 (C, C-
2), 128.8 (C, C-5), 125.4 (C, C-1), 123.7 (CH, C-6), 111.2 (CH, C-
4), 64.8 (CH2, C-8), 55.5 (CH3, OCH3-3), 34.6 (CH2, C-7),20.0
(CH3, OCOCH3); HRESIMS m/z 417.2201 [M − H]− (calcd for
C22H25O8, 417.4306).
(3H, s, CH3-10′); 13C NMR (acetone-d6, 125 MHz) δ 171.0 (C, C-9/
9′), 148.5 (C, C-3), 147.4 (C, C-2′), 142.1 (C, C-2), 141.4 (C, C-3′),
131.2 (C, C-5′), 131.1 (C, C-5), 127.5 (C, C-1′), 126.3 (C, C-1),
124.5 (CH, C-6), 122.9 (CH, C-6′), 115.6 (CH, C-4′), 112.2 (CH, C-
4), 65.8 (CH2, C-8/8′), 56.6 (CH3, OCH3-3), 35.56 (CH2, C-7), 35.3
(CH2, C-7′), 20.9 (CH3, OCOCH3); ESIMS m/z 403.0 [M − H]−
(calcd for C21H23O8, 403.1); anal. C 62.40, H 5.96%, calcd for
C21H24O8, C 62.37, H 5.98%.
Flash chromatography afforded also a more polar product with a
13.7% yield, which showed the same spectroscopic data as 16.
Enzymatic Butanolysis of 16 and 20. Candida antarctica lipase
(10.0 mg) and n-butyl alcohol (0.05 mL) were added to a solution of a
substrate (16 and 20, 10.0 mg) in MTBE (1.20 mL). The resulting
mixture was stirred (400 rpm) at 40 °C, and the progress of each
reaction was monitored by TLC (CH2Cl2−MeOH, 90:10). After the
completion of each reaction, the enzyme was filtered off and the filtrate
was evaporated in vacuo. The crude mixtures were purified by flash
chromatography on Diol silica gel (CH2Cl2−MeOH, 98:2 → 90:10).
5,5′-Bis(2-hydroxyethyl)-[1,1′-biphenyl]-2,2′,3,3′-tetraol (17): yel-
low oil (6.5 mg, 82.7%); Rf (TLC) 0.25 (CH2Cl2−MeOH, 90:10); 1H
NMR (acetone-d6, 500 MHz) δ 6.76 (2H, s, H-6), 6.70 (2H, s, H-4),
3.73 (4H, t, J = 7.5 Hz, H2-8), 2.71 (4H, t, J = 7.5 Hz, H2-7); 13C
NMR (acetone-d6, 125 MHz) δ 146.7 (C, C-3), 140.8 (C, C-2), 132.7
(C, C-5), 127.4 (C, C-1), 123.1 (CH, C-4), 115.7 (CH, C-6), 64.2
(CH2, C-8), 39.9 (CH2, C-7); ESIMS m/z 305.2 [M − H]− (calcd for
C16H17O6, 305.1); anal. C 62.76, H 5.95%, calcd for C16H18O6, C
62.74, H 5.92%.
5,5′-Bis(2-hydroxyethyl)-[1,1′-biphenyl]-2,2′-diol (22): yellow oil
(20.1 mg, 19.1%); Rf (TLC) 0.28 (CH2Cl2−MeOH, 94:6); H NMR
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(acetone-d6, 500 MHz) δ 7.13 (2H, d, J = 1.5 Hz, H-6), 7.06 (2H, dd, J
= 1.5, 7.5 Hz, H-4), 6.86 (2H, d, J = 8.0 Hz, H-3), 3.70 (4H, t, J = 7.0
Hz, H2-8), 2.74 (4H, t, J = 7.0 Hz, H2-7); 13C NMR (acetone-d6, 125
MHz) δ 153.0 (C, C-2),132.9 (CH, C-6), 132.3 (C, C-5), 130.0 (CH,
C-4), 127.0 (C, C-1), 117.3 (CH, C-3), 64.14 (CH2, C-8), 39.54
(CH2, C-7); HRESIMS m/z 273.1705 [M − H]− (calcd for C16H17O4,
273.2968).
5,5′-Bis(2-hydroxyethyl)-3,3′-dimethoxy-[1,1′-biphenyl]-2,2′-diol
(23): yellow oil (34.2 mg, 43.5%); Rf (TLC) 0.40 (CH2Cl2−MeOH,
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94:6); H NMR (acetone-d6, 500 MHz) δ 8.15 (2H, bs, OH), 6.86
(2H, d, J = 2.0 Hz, H-4), 6.74 (2H, d, J = 2.0 Hz, H-6), 3.89 (6H, s,
OCH3-3), 3.77 (4H, t, J = 7.0 Hz, H2-8), 2.78 (4H, t, J = 7.0 Hz, H2-
7); 13C NMR (acetone-d6, 125 MHz) δ 150.4 (C, C-3), 144.5 (C, C-
2), 132.7 (C, C-5), 128.2 (C, C-1), 126.2 (CH, C-6), 113.9 (CH, C-
4), 65.8 (CH2, C-8), 58.0 (CH3, OCH3-3), 41.5 (CH2, C-7);
HRESIMS m/z 333.2061 [M − H]− (calcd for C18H21O6, 333.3570).
Synthesis of [5,5′,6,6′-Tetrahydroxy-(1,1′-biphenyl)-3,3′-
diyl]bis(ethane-2,1-diyl) Diacetate (16). A 0.2 M solution of 15
(175 mg, 0.48 mmol) in MeOH (2.4 mL) was stirred with IBX
(161.23 mg, 1.2 equiv) at 0 °C for 30 min. Finally, a solution of
Na2S2O4 (83.5 mg, 0.47 mmol in 2.4 mL of H2O) was added, and the
resulting mixture was stirred at rt for 10 min. After the evaporation of
the solvent under vacuum, the residue was solubilized with ethyl
acetate (20 mL) and partitioned with a saturated NaHCO3 solution (3
× 20 mL). The combined aqueous phase was extracted with ethyl
acetate (1 × 50 mL). The organic phases were washed with saturated
NaCl and dried over Na2SO4. After filtration, the solvent was
evaporated in vacuo. The flash chromatography on Diol silica gel,
eluted with n-hexane−CHCl3 (30:70 → 0:100) and CHCl3−MeOH
(99:1 → 97:3) gave the expected product 16 (22.6 mg, 11.6%) as a
brown oil: Rf (TLC) 0.51 (CH2Cl2−MeOH, 93:7); 1H NMR
(acetone-d6, 500 MHz) δ 6.81 (2H, d, J = 2.0 Hz, H-6), 6.73 (2H,
d, J = 2.0 Hz, H-4), 4.22 (4H, t, J = 7.0 Hz, H2-8), 2.83 (4H, t, J = 7.0
Hz, H2-7), 1.98 (6H, s, OCOCH3); 13C NMR (acetone-d6, 125 MHz)
δ 171.0 (C, C-9), 146.8 (C, C-3), 141.1 (C, C-2), 131.5 (C, C-5),
127.4 (C, C-1), 122.5 (CH, C-4), 115.2 (CH, C-6), 65.7 (CH2, C-8),
35.3 (CH2, C-7), 20.9 (CH3, OCOCH3); ESIMS m/z 389.0 [M − H]−
(calcd for C20H21O8, 389.1); anal. C 61.49, H 5.65%, calcd for
C20H22O8, C 61.53, H 5.68%.
Synthesis of [5,6,6′-Trihydroxy-5′-methoxy-(1,1′-biphenyl)-
3,3′-diyl]bis(ethane-2,1-diyl) Diacetate (20). A 0.06 M solution of
19 (171.6 mg, 0.41 mmol) in THF (6.8 mL) was stirred with IBX
(172.1 mg, 1.5 equiv) at rt for 3 h. Then, a solution of Na2S2O4 (71.38
mg, 0.61 mmol in 6.8 mL of H2O) was added, and the mixture was
stirred at rt for 10 min. After the evaporation of the solvent under
vacuum, the residue was solubilized with ethyl acetate (20 mL) and
partitioned with a saturated NaHCO3 solution (3 × 20 mL). The total
aqueous phase was partitioned with ethyl acetate (1 × 50 mL), and
finally the combined organic layer was washed with a saturated NaCl
solution. The organic phase was dried over Na2SO4, filtered, and
evaporated under vacuum. The crude mixture was purified by flash
chromatography on Diol silica gel, eluting with n-hexane−CHCl3
(15:85 → 0:100) and CHCl3−MeOH (99:1 → 93:7), affording the
product 20 (33.1 mg, 18.3%) as a brown oil: Rf (TLC) 0.46 (CH2Cl2−
MeOH, 95:5); 1H NMR (acetone-d6, 500 MHz) δ 6.94 (1H, d, J = 1.5
Hz, H-4), 6.83 (1H, d, J = 1.5 Hz, H-6), 6.78 (1H, d, J = 1.5 Hz, H-4′),
6.70 (1H, d, J = 1.5 Hz, H-6′), 4.25 (2H, t, J = 7.0 Hz, H2-8), 4.22
(2H, t, J = 7.0 Hz, H2-8′), 3.91 (3H, s, OCH3-3′), 2.91 (2H, t, J = 7.0
Hz, H2-7), 2.84 (2H, t, J = 7.0 Hz, H2-7′), 1.99 (3H, s, CH3-10), 1.98
5,5′-Bis(2-hydroxyethyl)-3′-methoxy-[1,1′-biphenyl]-2,2′,3-triol
(21): yellow oil (6.6 mg, 82.4%); Rf (TLC) 0.30 (CH2Cl2−MeOH,
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90:10); H NMR (acetone-d6, 500 MHz) δ 6.90 (1H, s, H-4), 6.79
(1H, s, H-6), 6.74 (1H, d, J = 1.5, H-4′), 6.66 (1H, d, J = 1.5 Hz, H-
6′), 3.89 (3H, s, OCH3-3), 3.75 (2H, t, J = 7.1 Hz, H2-8), 3.71 (2H, t, J
= 7.1 Hz, H2-8′), 2.75 (2H, t, J = 7.1 Hz, H2-7), 2.71 (2H, t, J = 7.1
Hz, H2-7′); 13C NMR (acetone-d6, 125 MHz) δ 147.4 (C, C-3), 147.3
(C, C-2′), 141.5 (C, C-2), 140.9 (C, C-3′), 132.7 (C, C-5′), 132.5 (C,
C-5), 127.4 (C, C-1′), 126.3 (C, C-1), 124.5 (CH, C-6), 122.2 (CH,
C-6′), 115.7 (CH, C-4′), 112.3 (CH, C-4), 64.2 (CH2, C-8), 64.1
(CH2, C-8′), 56.5 (CH3, OCH3-3), 40.1 (CH2, C-7), 39.9 (CH2, C-
7′); ESIMS m/z 319.1 [M − H]− (calcd for C17H19O6, 319.1); anal. C
63.71, H 6.32%, calcd for C17H20O6, C 63.74, H 6.29%.
α-Glucosidase Inhibition Activity Assay. The inhibitory activity
on yeast α-glucosidase was assessed through a slight modification of a
previously reported method employing pNP-α-G as substrate.50 Stock
solutions at different concentrations of the compounds (1, 2, 6, 7, 12,
13, 15−17, 19−23) were prepared in MeOH (in the range 0.5−100
μM). For each assay, different amounts (10, 20, 30, 40, 60 μL) of each
sample, the α-glucosidase from Saccharomyces cerevisiae solution (10
U/mL; 50 μL), and finally a 6.0 × 10−3 M pNP-α-G solution (0.05 M
buffer Na2HPO4−KH2PO4, pH 7.2; 30 μL) were added in a 5 mL
volumetric flask. The final concentration of MeOH did not exceed
1.5%. The solutions were incubated at 37 °C for 30 min and reactions
stopped by adding 1 M Na2CO3 solution (200 μL). Enzymatic activity
was quantified by measuring absorbance at 405 nm. The assay was
performed in triplicate with five different concentrations; quercetin
and acarbose were used as positive controls. The inhibition percentage
was calculated by the equation
Inhibition% = [(Acontrol − Asample)/Acontrol]× 100
The IC50 value is the concentration that inhibits 50% of α-
glucosidase activity.
Kinetics of α-Glucosidase Inhibition. The inhibitory mode of
compound 15 on α-glucosidase (0.05 U/mL) was measured with
increasing concentrations of pNP-α-G (0, 0.15, 0.33, 0.50, 0.80, 1.00,
1.25, 1.50, and 2.00 mM solutions in 0.05 M buffer Na2HPO4−
KH2PO4, pH 7.2) as a substrate in the absence and presence of 15.51
Optimal amounts of the test compound were determined on the basis
of the α-glucosidase inhibition assay (1.1, 3.3, and 5.5 μM). The
reactions were incubated at 37 °C for 20 min, and the optical density
was read at regular time intervals of 1 min. The experiment was
performed in duplicate. The mode of inhibition was resolved by
H
J. Nat. Prod. XXXX, XXX, XXX−XXX