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References and notes
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Figure 1. Graphical representation of
compounds 26a–d as a function on the time (A) and as a function of concentration
(B).
% DPPH radical scavenging activity of
16. Kabbe, H. J. Synthesis 1978, 886.
17. Data for 40-hydroxyspiro[cyclopentane-1,80-80Y-pyrano[2,3-h]-2Y-benzo[b]-
pyran]-20-one (8a). Mp > 250 °C (EtOH). 1H NMR (DMSO-d6, 400 MHz)
d
We have further investigated the ability of the new derivatives
to scavenge superoxide anions, generated by an enzymic (xan-
thine–xanthine oxidase) system, by measurement of the reduction
product of nitro blue tetrazolium (NBT), as previously described.15
(ppm): 1.5–2.1 (m, 8H, cyclopentane-H), 5.42 (s, 1H, H-30), 5.95 (d, 1H,
J = 10.0 Hz, H-90), 6.72 (d, 1H, J = 10.0 Hz, H-100), 6.74 (d, 1H, J = 8.7 Hz, H-60),
7.55 (d, 1H, J = 8.7 Hz, H-50). 13C NMR (DMSO-d6, 50 MHz) d (ppm): 23.59 and
38.70 (cyclopentane-C), 88.44 (C-80), 88.81 (C-30), 109.46 and 109.64 (C-40a
and C-100a), 113.11 (C-60), 115.69 (C-100), 123.74 (C-50), 130.62 (C-90), 149.87
(C-100b), 156.26 (C-60a), 162.29 (C-20), 166.56 (C-40). Anal. Calcd for C16H14O4:
C, 71.10; H, 5.22. Found: C, 70.86; H, 5.14.
The incubation system contained 200 lV xanthine and 600 lV
NBT in 0.1 phosphate buffer (pH 7.4). The reaction started with
the addition of 0.07 U mlꢀ1 of xanthine oxidase, which is consid-
ered to be an important biological source of superoxide radicals.28
The tested compounds were dissolved in 0.1% dimethylformamide
(DMF) in buffer, and added to the reaction mixture (final concen-
tration 0.5 mM). DMF was tested and found not to interfere with
the assay at the concentration used. We found again that the cis-
diols 26a–d and the adamantyl-substituted chromene 12 were ac-
tive and among them compounds 26a and 26b are more potent
superoxide anion radical scavengers than the reference compounds
4-hydroxycoumarin, 7-hydroxycoumarin and allopurinol at the
same concentration (Table 1).
In conclusion a synthetic methodology for the preparation of 4-
hydroxy spiropyranocoumarins as well as their dihydropyrano cis-
diols counterparts was developed. The evaluation of the free radi-
cal scavenging activity of the new compounds by means of two dif-
ferent tests, the interaction with DPPH free radical and the
quenching of superoxide anions generated by the enzymic xan-
thine–xanthine oxidase system, revealed that the spirocyclopentyl
substituted cis-diol 26a is the most potent radical scavenger pre-
senting high activity in both assays.
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20Y,80Y-pyrano[2,3-h]benzo[b]pyran]-20-one (26a). Mp > 250 °C (EtOH). 1H
NMR (DMSO-d6, 400 MHz) d (ppm): 1.4–2.1 (m, 8H, cyclopentane-H), 3.78 (d,
1H, J = 3.9 Hz, H-90), 4.86 (d, 1H, J = 3.9 Hz, H-100), 5.43 (s, 1H, H-30), 6.71 (d, 1H,
J = 8.8 Hz, H-60), 7.59 (d, 1H, J = 8.8 Hz, H-50). 12.17 (s, 1H, OH). 13C NMR (DMSO-
d6, 50 MHz) d (ppm): 23.60, 24.98, 32.09, 36.14 (cyclopentane-C), 61.02 (C-100),
68.92 (C-90), 88.67 (C-30), 89.95 (C-80), 108.81 (C-40a), 112.56 (C-100a), 113.85 (C-
60), 124.02 (C-50), 154.43 (C-100b), 156.90 (C-60a), 162.73 (C-20), 168.68 (C-40).
Anal. Calcd for C16H16O6: C, 63.15; H, 5.30. Found: C, 62.94; Y, 5.22.
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