2422 J. Agric. Food Chem., Vol. 57, No. 6, 2009
Saiz-Urra et al.
by column chromatography on silica gel, with increasing gradients of
ethyl acetate on petroleum ether, to give compound 13 (125 mg) (62%):
oil; [R]25D +4 (c 2.1 CHCl3); IR (neat, KBr) νmax 3398 (OH) cm-1; 1H
NMR (CDCl3, 400 MHz) δ 0.81 (s, 3H, H-13R), 0.92 (s, 3H, H-15),
0.99 (s, 3H, H-14ꢀ), 0.95 (m, 1H, H-12a), 1.10 (m, 1H, H-11a),
1.17-1.31 (5H, H-6a, H-7a, H-6b, H-7b, H-5ꢀ), 1.40-1.55 (3H, H-3a,
H-10R, H-12b), 1.6-1.7 (2H, H-3b, H-11b), 1.92 (m, 1H, H-10ꢀ),
2.65-2.59 (2H, H-2′a, H-2′b), 3.27 (bs, 1H, H-9ꢀ), 3.39 (dd, 1H, J )
5.6, 4.2 Hz, H-2R), 3.62-3.50 (2H, H-1′a, H-1′b); 13C NMR (CDCl3,
50 MHz) δ 20.6 (C-6), 24.9 (C-2′), 25.4 (C-13), 26.0 (C-10), 26.8 (C-
11), 28.3 (C-15), 31.3 (C-14), 33.1 (C-7), 34.6 (C-8*), 36.3 (C-12),
37.1 (C-4*), 44.3 (C-1*), 44.4 (C-3), 50.4 (C-5), 71.9 (C-1′), 75.0 (C-
9), 88.6 (C-2) (*, interchangeable signals); EIMS m/z (%) 298 (4) [M]+,
280 (20), 220 (49), 204 (74), 203 (100); HRFABMS (thioglycerol
matrix) (m/z) [M + Na]+ calcd for C17H30O2SNa, 321.1864; found,
321.1862.
(148 mg) (94%): oil; [R]25 -4 (c 6.4 CHCl3); IR (neat, KBr) νmax
D
3456 (OH), 1601, 1598, 1431 (CdC aromatic, NO2) 1369 (NO2) cm-1
;
1H NMR (CDCl3, 400 MHz) δ 0.82 (s, 3H, H-13R), 0.91 (s, 3H, H-15),
0.98 (s, 3H, H-14ꢀ), 0.96 (d, 1H, J ) 12.6 Hz, H-12a), 1.08 (m, 1H,
H-11ꢀ), 1.30-1.22 (3H, H-6a, H-7a, H-11a), 1.38-1.31 (3H, H-6b,
H-7b, H-5ꢀ), 1.54-1.50 (3H, H-3a, H-10R, H-3b), 1.56 (d, 1H, J )
12.6 Hz, H-12b), 1.66 (m, 1H, H-11b), 1.93 (m, 1H, H-10ꢀ), 3.28 (bs,
1H, H-9ꢀ), 3.51 (dd, 1H, J ) 5.6, 4.4 Hz, H-2R), 3.84-3.78 (2H, H-2′a,
H-2′b), 4.18-4.14 (2H, H-1′a, H-1′b), 6.95 (d, 2H, J ) 9.4 Hz, H-4′,
H-8′), 8.15 (d, 2H, J ) 9.4 Hz, H-5′, H-7′); 13C NMR (CDCl3, 50
MHz) δ 20.5 (C-6), 25.3 (C-13), 25.9 (C-10), 26.6 (C-11), 28.3 (C-
15), 31.2 (C-14), 32.9 (C-7), 34.6 (C-8*), 36.4 (C-12), 37.0 (C-4*),
44.3 (C-1*), 44.4 (C-3), 50.4 (C-5), 68.4 (C-1′), 68.5 (C-2′) 75.0 (C-
9), 89.2 (C-2′), 114.6 (2C, C-4′, C-8′), 125.7 (2C, C-5′, C-7′) 140.2
†
(C-3′†), 164.0 (C-6′†) (*, , interchangeable signals); EIMS m/z (%)
404 (16) [M + 1]+, 358[M + 1 - 46]+ (50), 266 (11), 262 (15), 220
(10), 190 (60), 176 (100); HREIMS (m/z) [M + 1]+ calcd for
C23H34NO5, 404.2438; found, 404.2441.
2ꢀ-(2-(Imidazol-1-yl)ethoxy)cloVan-9R-ol (16). Sodium imidazole
(195 mg) was added to a solution of compound 11 (330 mg) in dry
dimethylformamide (10 mL), and the reaction mixture was left stirring
under nitrogen atmosphere for 48 h. The resulting reaction mixture
was diluted with ethyl acetate (50 mL), washed with brine (2 × 50
mL), and dried over anhydrous Na2SO4. Evaporation of the solvent
afforded a crude reaction product, which was purified by column
chromatography on silica gel, with increasing gradients of methanol
2ꢀ-(2-(Phenylthio)ethoxy)cloVan-9R-ol (19). Thiophenol (200 mg)
and NaH (47 mg) were added to a solution of compound 11 (129 mg)
in dry DMF (5 mL) and left stirring under N2 atmosphere for 48 h.
The resulting reaction mixture was diluted with ethyl acetate (50 mL),
washed with brine (2 × 50 mL), and dried over anhydrous Na2SO4.
Evaporation of the solvent afforded a crude reaction product, which
was purified by column chromatography on silica gel, with increasing
gradients of ethyl acetate on petroleum ether, to give compound 19
on ethyl acetate, to give compound 16 (220 mg) (70%); oil; [R]25
D
+13 (c 5.4 CHCl3); IR (neat, KBr) νmax 3239 (OH) cm-1; H NMR
1
(CDCl3, 400 MHz) δ 0.75 (s, 3H, H-13R), 0.82 (m, 1H, H-12a), 0.89
(s, 3H, H-15), 0.92 (s, 3H, H-14ꢀ), 1.1-0.95 (2H, H-7a, H-11a),
1.1-1.25 (5H, H-3a, H-5ꢀ, H-6a, H-6b, H-7a), 1.25-1.6 (4H, H-3b,
H-10a, H-11b, H-12b), 1.88 (m, 1H, H-10b), 3.25 (bs, 1H, H-9ꢀ), 3.29
(dd, 1H, J ) 5.6, 9.6 Hz; H-2R), 3.63-3.58 (2H, H-2′a, H-2′b),
4.02-3.97 (2H, H-1′a, H-1′b), 6.92 (bs, 1H, H-3′), 6.95 (bs, 1H, H-4′),
7.47 (bs, 1H, H-5′); 13C NMR (CDCl3, 50 MHz) δ 20.5 (C-6), 25.2
(C-13), 26.0 (C-10), 26.7 (C-11), 28.3 (C-15), 31.1 (C-14), 32.9 (C-
7), 34.5 (C-8*), 36.2 (C-12), 37.1 (C-4*), 44.0 (C-3), 44.4 (C-1*), 47.4
(C-2′), 50.3 (C-5), 69.4 (C-1′), 74.6 (C-9), 89.1 (C-2), 119.4 (C-4′),
128.71 (C-5′), 137.48 (C-3′) (*, interchangeable signals); EIMS m/z
(%) 333 (97) [M + H]+, 275 (96), 203 (10), 164 (5), 149 (5), 112
(60), 96 (95), 82 (100); HREIMS (m/z) [M + 1]+ calcd for C20H33O2N2,
333.2542; found, 333.2533.
(128 mg) (92%): oil; [R]25 -3 (c 3.5 CHCl3); IR (neat, KBr) νmax
D
3399 (OH), 1523, 1442 (CdC aromatic); 1H NMR (CDCl3, 400 MHz)
δ 0.82 (s, 3H, H-13R), 0.93 (s, 3H, H-15), 1.00 (s, 3H, H-14ꢀ), 0.95
(m, 1H, H-12a), 1.2-1.4 (6H, H-5ꢀ, H-6a, H-7a, H-6b, H-7b, H-11a),
1.40-1.55 (3H, H-3a, H-10R, H-12b), 1.55-1.7 (2H, H-3b, H-11b),
1.94 (m, 1H, H-10ꢀ), 3.10-3.06 (2H, H-2′a, H-2′b), 3.27 (bs, 1H,
H-9ꢀ), 3.39 (dd, 1H, J ) 5.6, 4.5 Hz; H-2R), 3.65-3.61 (2H, H-1′a,
H-1′b), 7.15 (t, 1H, J ) 7.0 Hz; H-6′), 7.26 (t, 2H, J ) 7.0 Hz; H-5′,
H-7′), 7.35 (d, 2H, J ) 7.0 Hz; H-4′, H-8′); 13C NMR (CDCl3, 50
MHz) δ 20.8 (C-6), 25.4 (C-13), 26.3 (C-10), 27.0 (C-11), 28.6 (C-
15), 31.5 (C-14), 33.3 (C-7), 33.7 (C-2′), 34.9 (C-8*), 36.6 (C-12),
37.3 (C-4*), 44.6 (C-1*), 44.7 (C-3), 50.7 (C-5), 69.4 (C-1′), 75.3 (C-
9), 89.0 (C-2), 126.1 (C-6′), 129.1 (2C, C-5′, C-7′), 129.3 (2C, C-4′,
C-5′), 136.7 (C-3′) (*, interchangeable signals); EIMS m/z (70 eV) 374
[M]+ (20), 203 (33), 156 (40), 138 (58), 110 (100); HREIMS (m/z)
[M]+ calcd for C23H34SO2, 374.2281; found, 374.2269.
2ꢀ-(2-(1,2,4-Triazol-1-yl)ethoxy)cloVan-9R-ol (20). Sodium triazole
(60 mg) was added to a solution of compound 11 (200 mg) in dry
dimethylformamide (10 mL), and the reaction mixture was left stirring
under nitrogen atmosphere for 30 h. The resulting reaction mixture
was diluted with ethyl acetate (50 mL), washed with brine (2 × 50
mL), and dried over anhydrous Na2SO4. Evaporation of the solvent
afforded a crude reaction product, which was purified by column
chromatography on silica gel, with increasing gradients of methanol
2ꢀ-(2-Mercaptoethylthio)cloVan-9R-ol (22). BF3 ·Et2O (518 mg, 0.45
mL) was added dropwise to a solution of caryophyllene oxide (201
mg) in dry Et2O (4 mL). The resulting solution was kept stirred, under
nitrogen atmosphere, at -63 °C for 2 h. Then, 1,2-ethanedithiol (782
mg) was added, and the reaction was kept stirring for a further 1 h and
then allowed to reach room temperature. The resulting reaction mixture
was diluted with diethyl ether (50 mL), washed with brine (2 × 50
mL), and dried over anhydrous Na2SO4. Evaporation of the solvent
afforded a crude reaction product, which was purified by column
chromatography on silica gel, with increasing gradients of ethyl acetate
on ethyl acetate, to give compound 20 (160 mg) (75%): oil; [R]25
D
+12 (c 4 CHCl3); IR (neat, KBr) νmax 3422 (OH) cm-1
;
1H NMR
(CDCl3, 400 MHz) δ 0.73 (s, 3H, H-13R), 0.80 (s, 3H, H-15), 0.82
(m, 1H, H-12a), 0.86 (s, 3H, H-14ꢀ), 0.95-1.65 (11H, H-3a, H-3b,
H-5ꢀ, H-6a, H-6b, H-7a, H-7b, H-10a, H-11a, H-11b, H-12b), 1.85
(m, 1H, H-10b), 3.10 (bs, 1H, H-9ꢀ), 3.20 (dd, 1H, J ) 5.6, 9.6 Hz;
H-2R), 3.71-3.66 (2H, H-2′a, H-2′b), 4.30-4.24 (2H, H-1′a, H-1′b),
7.86 (s, 1H, H-4′), 8.34 (s, 1H, H-3′); 13C NMR (CDCl3, 50 MHz) δ
21.8 (C-6), 25.8 (C-13), 27.1 (C-10), 28.1 (C-11), 29.1 (C-15), 31.8
(C-14), 34.4 (C-7), 35.7 (C-8*), 37.3 (C-12), 38.3 (C-4*), 45.2 (C-3),
46.0 (C-1*), 51.4 (C-2′), 51.9 (C-5), 68.7 (C-1′), 75.7 (C-9), 90.4 (C-
2), 145.7 (C-4′), 151.9 (C-3′) (*, interchangeable signals); EIMS m/z
(%) 334 (18) [M + 1]+, 316 (5), 276 (24), 237 (5), 203 (7), 180 (4),
163 (7), 114 (25), 97 (100); HREIMS (m/z) [M + 1]+ calcd for
C19H32O2N3, 334.2495; found, 334.2496.
2ꢀ-(2-(4-Nitrophenoxy)ethoxy)cloVan-9R-ol (17). p-Nitrophenol (278
mg) and NaH (49 mg) were added to a solution of compound 11 (135
mg) in dry DMF (5 mL) and left stirring under N2 atmosphere for 24 h.
The resulting reaction mixture was diluted with ethyl acetate (50 mL),
washed with brine (2 × 50 mL), and dried over anhydrous Na2SO4.
Evaporation of the solvent afforded a crude reaction product, which
was purified by column chromatography on silica gel, with increasing
gradients of ethyl acetate on petroleum ether, to give compound 17
on petroleum ether, to give compound 22 (40 mg) (8%): oil; [R]25
D
+4 (c 2.0 CHCl3); IR (neat, KBr) νmax 3390 (OH) cm-1; H NMR
1
(CDCl3, 400 MHz) δ 0.83 (s, 3H, H-13R), 0.88 (d, 1H, J ) 12.8 Hz,
H-12a), 0.96 (s, 3H, H-15), 0.99 (m, 1H, H-11a), 1.03 (s, 3H, H-14ꢀ),
1.08 (m, 1H, H-7a), 1.22-1.44 (3H, H-6a, H-6b, H-7b), 1.52-1.60
(3H, H-3a, H-5ꢀ, H-10R), 1.64 (d, 1H, J ) 12.8 Hz,; H-12b), 1.68-1.78
(2H, H-3b, H-11b), 1.93 (m, 1H, H-10ꢀ), 2.66-2.78 (5H, H-2′a, H-2′b,
H-1′a, H-1′b, H-2R), 3.28 (bs, 1H, H-9ꢀ); 13C NMR (CDCl3, 50 MHz)
δ 20.6 (C-6), 24.5 (C-13), 25.1 (C-2′), 25.9 (C-10), 28.3 (C-15), 28.7
(C-11), 30.8 (C-14), 33.0 (C-7), 34.8 (C-8*), 35.8 (C-12), 36.6 (C-1′),
39.1 (C-4*), 44.7 (C-1*), 48.3 (C-3), 51.2 (C-5), 55.8 (C-2), 74.8 (C-
9) (*, interchangeable signals); EIMS m/z (%) 314 (18) [M]+, 296 (36)
[M - 18]+, 281 (10) [M + 1 - 34]+, 253 (22), 235 (16), 203 (100);
HRFABMS (thioglycerol matrix) (m/z) [M - H]+ calcd for C17H29OS2,
313.1660; found, 313.1657.
Microorganism and Antifungal Assays. The culture of B. cinerea
employed in this work, B. cinerea (UCA 992), was obtained from grapes
from the Domecq vineyard, Jerez de la Frontera, Ca´diz, Spain. This
culture of B. cinerea has been deposited at the Mycological Herbarium