Synthesis and cytotoxic activity of benzopyranoxanthone analogues of benzo[b]acronycine and psorospermine

Article abstract of DOI:10.1248/cpb.49.675

Condensation of 3-hydroxy-2-naphthalenecarboxylic acid with phloroglucinol afforded 1,3-dihydroxy-12H-benzo[b]xanthen-12-one. Construction of an additional dimethylpyran ring onto this skeleton, by alkylation with 3-chloro-3-methyl-1-butyne followed by Claisen rearrangement, gave access to a series of benzo[b]pyrano[2,3-i]xanthen-6-ones and benzo[b]pyrano[3,2-h]xanthen-7-ones related to psorospermine and benzo[b]acronycine. In contrast with what is observed in the pyridoacridone and benzopyridoacridone series, the linear benzo[b]-pyrano[2,3-i]xanthen-6-one derivatives were more potent than their angular benzo[b]pyrano[3,2-h]xanthen-7-one isomers. cis-3,4-Diacetoxy-5-methoxy-2,2-dimethyl-3,4-dihydro-2H,6H-benzo[b]pyrano [2,3,i]xanthen-6-one, the most active among the new compounds, was more potent than acronycine in inhibiting the proliferation of L1210 murine leukemia cells.

Full text of DOI:10.1248/cpb.49.675

June 2001
Chem. Pharm. Bull. 49(6) 675—679 (2001)
675
Synthesis and Cytotoxic Activity of Benzopyranoxanthone Analogues of
Benzo[b]acronycine and Psorospermine
a
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Chavalit SITTISOMBUT, Nadine COSTES, Sylvie MICHEL, Michel KOCH, François TILLEQUIN,
Bruno PFEIFFER,b Pierre RENARD,b Alain PIERRÉ,c and Ghanem ATASSI
c
Laboratoire de Pharmacognosie de l’Université René Descartes,a U.M.R./C.N.R.S. n°8638, Faculté des Sciences
Pharmaceutiques et Biologiques, 4, Avenue de l’Observatoire, F-75006 Paris, France, A.D.I.R. et Compagnie,b 1 rue Carle
Hébert, F-92415 courbevoie Cedex, France, and Institut de Recherche Servier,c 11 rue des Moulineaux, F-92150 Suresnes,
France. Received September 22, 2000; accepted February 13, 2001
Condensation of 3-hydroxy-2-naphthalenecarboxylic acid with phloroglucinol afforded 1,3-dihydroxy-12H-
benzo[b]xanthen-12-one. Construction of an additional dimethylpyran ring onto this skeleton, by alkylation with
3-chloro-3-methyl-1-butyne followed by Claisen rearrangement, gave access to a series of benzo[b]pyrano[2,3-
i]xanthen-6-ones and benzo[b]pyrano[3,2-h]xanthen-7-ones related to psorospermine and benzo[b]acronycine. In
contrast with what is observed in the pyridoacridone and benzopyridoacridone series, the linear benzo[b]-
pyrano[2,3-i]xanthen-6-one derivatives were more potent than their angular benzo[b]pyrano[3,2-h]xanthen-7-
one isomers. cis-3,4-Diacetoxy-5-methoxy-2,2-dimethyl-3,4-dihydro-2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one,
the most active among the new compounds, was more potent than acronycine in inhibiting the proliferation of
L1210 murine leukemia cells.
Key words benzopyranoxanthone; acronycine; synthesis; cytotoxicity
Several simple and fused xanthone derivatives have been
reported as promising compounds in the search for new anti-
cancer candidates. Indeed, xanthone-4-acetic acid derivatives
induced tumor-growth delays and regressions when evaluated
against the sc colon 38 model in mice.1,2) Some natural xan-
thones have shown an intensive inhibitory effect against
topoisomerases I and II in the course of in vitro experi-
ments.3) The furanoxanthone psorospermine (1), isolated
from Psorospermum febrifugum,4—6) exhibited significant cy-
totoxic and antineoplastic properties due to its interaction
with DNA.7,8) The tetracyclic basic core of psorospermine is
closely related to that of the pyranoacridone alkaloid
acronycine (2). This latter is an efficient anticancer agent,
with a broad spectrum of activity including numerous solid
tumors.9—14) The structural relationships between the psoros-
permine and acronycine series stimulated the synthesis of
pyrano[2,3-c]xanthen-7-one and pyrano[3,2-b]xanthen-6-one
derivatives.15,16) Some of those compounds exhibited cyto-
toxic activities within the same range of magnitude as
pyrano[3,2-h]xanthen-7-ones and benzo[b]pyrano[2,3-i]-
xanthen-6-ones related to psorospermine and benzo[b]-
acronycine.
acronycine, when tested against L1210 murine leukemia
cells.16)
Based on a hypothesis of bioactivation of acronycine into
the corresponding epoxide17,18) and of intercalation within
DNA,19) we recently prepared the pentacyclic 6-methoxy-
3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]pyrano[3,2-h]-
acridin-7-one (“benzo[b]acronycine”) (3) and a series of cor-
responding cis-1,2-dihydrodiol diesters, exemplified by diac-
etate 4 and carbonate 5.20) These latter compounds were 20 to
1000 fold more potent than acronycine in inhibiting L1210
cell proliferation. In vivo, against P388 leukemia, acronycine
was only marginally active, while compounds 4 and 5 were
significantly active at doses 16 fold lower. Against the sc
colon 38 adenocarcinoma model in mice, compounds 4 and 5
were also highly efficient, inhibiting by more than 80% the
tumor growth. These promising results prompted us to pre-
pare pentacyclic xanthone isosters and analogues, in which
the acridone nitrogen is replaced by an oxygen atom. We re-
port here the synthesis and biological properties of benzo[b]-
Chemistry Construction of the tetracyclic 1,3-dihy-
droxy-12H-benzo[b]xanthen-12-one (6) basic core was ob-
tained by condensation of 3-hydroxy-2-naphthalenecar-
boxylic acid (7) with phloroglucinol (8) in the presence of
zinc chloride and phosphoryl chloride.21,22) Chelation of the
1-hydroxy group of 6 by the carbonyl at the 12-position per-
mitted the selective O-alkylation of the 3-hydroxy group with
3-chloro-3-methylbut-1-yne (9),23,24) to give the correspond-
ing propargylic ether 10. Methylation of the 1-hydroxy group
of 10, carried out with dimethylsulfate in the presence of
sodium hydride gave 11 in almost quantitative yield. When
thermal cyclization was performed on the hydroxy ether 10,
for 2 h at 130 °C in dimethylformamide (DMF), the linear 5-
hydroxy-2,2-dimethyl-2H,6H-benzo[b]pyrano[2,3-i]xanthen-
6-one (12) was the major reaction product, which could be
subsequently methylated to afford the desired 5-methoxy-
To whom correspondence should be addressed. e-mail: tillequi@pharmacie.univ.paris5.fr
© 2001 Pharmaceutical Society of Japan

676
Vol. 49, No. 6
2,2-dimethyl-2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one
(13). In contrast, Claisen rearrangement of the methoxy ether
11, under similar conditions, exclusively gave the angular 6-
methoxy-3,3-dimethyl-3H,7H-benzo[b]pyrano[3,2-h]xan-
then-7-one (14), most probably due to the increased steric
hindrance of the methoxy group at 5-position of 11, when
compared with the chelated hydroxy group of 10. The linear
structures of benzopyranoxanthones 12 and 13, and the angu-
lar structure of benzopyranoxanthone 14 were unambigu-
ously ascribed from phase-sensitive nuclear Overhauser and
exchange spectroscopy (NOESY) experiments performed on
these three compounds.20,25) The (Ϯ)-cis-diol 15 was conve-
niently obtained by catalytic osmium tetroxide oxidation of
14, using N-methylmorpholine N-oxide to regenerate the oxi-
dizing agent.26) Isosters of the highly active compounds 4 and
5 were obtained by treatment of diol 15 with either acetic an-
hydride or N,NЈ-carbonyldiimidazole, affording the diacetate
16 and the cyclic carbonate 17, respectively. Under similar
conditions, oxidation of the linear 5-methoxy-2,2-dimethyl-
2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one (13) gave the
corresponding cis-diol 18, which was subsequently converted
into the diacetate 19 and the cyclic carbonate 20.
Pharmacology The study of the biological properties of
the new benzopyranoxanthone derivatives was carried out in
vitro on L1210 murine leukemia cell line. The results (IC50)
are reported in Table 1. Most angular compounds were de-
void of significant antiproliferative activity. In contrast, linear
derivatives significantly inhibited the proliferation of L1210
cells. The most cytotoxic compound, cis-3,4-diacetoxy-5-
methoxy-2,2-dimethyl-3,4-dihydro-2H,6H-benzo[b]pyrano-
[2,3-i]xanthen-6-one (19), was more potent than acronycine
in inhibiting the proliferation of L1210 murine leukemia
cells.
Chart 1. Synthesis of Benzo[b]pyrano[3,2-h]xanthen-7-ones and Benzo-
[b]pyrano[2,3-i]xanthen-6-ones
Chart 2. Synthesis of 6-Methoxy-3,3-dimethyl-3,7-dihydrobenzo[b]pyrano[3,2-h]xanthen-6-one and of 5-Methoxy-2,2-dimethyl-2,6-dihydrobenzo[b]-
pyrano[2,3-i]xanthen-6-one Analogues

June 2001
677
Table 1. Inhibition of L1210 Cell Proliferation by Benzoxanthone 6 and
Benzopyranoxanthones 11—20 in Comparison with Acronycine (2)
7.66 (1H, t, Jϭ8 Hz, C8-H), 7.97 (1H, s, C6-H), 7.99 (1H, d, Jϭ8 Hz, C7-
H), 8.17 (1H, d, Jϭ8 Hz, C10-H), 8.76 (1H, s, C11-H), 11.16 (1H, s, D2O
exch., C3-OH), 12.87 (1H, s, D2O exch., C1-OH). 13C-NMR (75 MHz,
DMSO-d6) d: 181.4 (s, C-12), 167.6 (s, C-3), 164.5 (s, C-1), 158.9 (s, C-4a),
152.3 (s, C-5a), 137.4 (s, C-6a), 130.7 (d, C-10), 130.5 (d, C-8), 130.2 (s, C-
10a), 128.2 (2d, C-7, C-11), 127.0 (d, C-9), 120.7 (s, C-11a), 114.2 (d, C-6),
102.8 (s, C-12a), 98.9 (d, C-2), 95.4 (d, C-4). DCI-MS m/z: 279 [MH]ϩ.
Anal. Calcd for C17H10O4: C, 73.37; H, 3.62. Found: C, 73.15; H, 3.54.
1-Hydroxy-3-(1,1-dimethylpropargyloxy)-12H-benzo[b]xanthen-12-
one (10) A solution of 1,3-dihydroxy-12H-benzo[b]xanthen-12-one (6)
(2 g, 7.19 mmol) in dry DMF (160 ml) was stirred and heated at 70 °C for 3 d
under nitrogen, in the presence of potassium carbonate (2 g), potassium io-
dide (2.4 g) and 3-chloro-3-methylbut-1-yne (9) (3.7 g, 21 mmol). Water
(100 ml) was added and the precipitate was filtered and dried. Flash column
chromatography (solvent: cyclohexane/dichloromethane 50 : 50 to 5 : 95,
then dichloromethane) gave 10 (1.31 g, 53%) as a yellow amorphous solid.
IR (NaCl film) cmϪ1: 3055, 2979, 2924, 1684, 1639, 1600, 1472, 1446,
1193, 1176, 813. UV lmax (MeOH) nm (log e): 263 (3.43), 284 (3.25), 343
(3.00). DCI-MS m/z: 345 [MH]ϩ. 1H-NMR (300 MHz, C6D6) d: 1.60 (6H, s,
C(CH3)2), 3.10 (1H, s, C3Ј-H), 7.02 (1H, d, Jϭ2 Hz, C2-H), 7.10 (1H, d,
Jϭ2 Hz, C4-H), 7.14 (1H, td, Jϭ8,1 Hz, C9-H), 7.27 (1H, td, Jϭ8, 1 Hz, C8-
H), 7.50 (1H, dd, Jϭ8, 1 Hz, C7-H), 7.52 (1H, s, C6-H), 7.64 (1H, dd, Jϭ8,
1 Hz, C10-H), 8.96 (1H, s, C11-H), 13.64 (1H, s, D2O exch., C1-OH). Anal.
Compound
IC50 (mM)
2
6
27
100
109
Insoluble
42
11
12
13
14
15
16
17
18
19
20
96
55
13
52
28
7
16
Results and Discussion
Considering the structure–activity relationships in the
acronycine series, it appears that angular pyranoxanthone de-
rivatives are less potent than their acridone counterparts in Calcd for C22H16O4: C, 76.73; H, 4.68. Found: C, 76.75; H, 4.59.
1-Methoxy-3-(1,1-dimethylpropargyloxy)-12H-benzo[b]xanthen-12-
one (11) Sodium hydride (0.3 g of 50% oil dispersion, 6 mmol) was slowly
added to an ice-cooled suspension of 1-hydroxy-3-(1,1-dimethylpropargy-
loxy)-12H-benzo[b]xanthen-12-one (10) (1 g, 2.9 mmol) in DMF (50 ml).
inhibiting the proliferation of L1210 cells. Indeed, the only
significantly active compound, cis-1,2-diacetoxy-6-methoxy-
3,3-dimethyl-1,2-dihydro-3H,7H-benzo[b]pyrano[3,2-h]xan-
then-7-one (16), was less potent than the corresponding 1,2-
diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahy-
drobenzo[b]pyrano[3,2-h]acridin-7-one (4), belonging to the
benzo[b]acronycine series.20)
Dimethylsulfate (1.1 ml, 12 mmol) was added and the reaction mixture was
stirred under nitrogen at room temperature for 3 h. After addition of water
(100 ml) and neutralization with 10% sulfuric acid, the mixture was ex-
tracted with dichloromethane (4ϫ50 ml). The organic layers were evapo-
rated under reduced pressure. Flash chromatography (solvent: cyclohexane,
then cyclohexane/acetone 99 : 1 to 90 : 10) gave 11 (0.85 g, 82%) as a pale
yellow amorphous solid. IR (KBr) cmϪ1: 3200, 3003, 2944, 2106, 1739,
1658, 1628, 1600, 1576, 1466, 1350, 1261, 1141, 1113, 873, 820, 744. UV
lmax (MeOH) nm (log e): 259 (4.91), 288 (sh.), 328 (4.33). 1H-NMR
(300 MHz, DMSO-d6) d: 1.80 (6H, s, C(CH3)2), 2.79 (1H, s, C3Ј-H), 4.02
(3H, s, OCH3), 6.55 (1H, d, Jϭ2 Hz, C2-H), 7.03 (1H, d, Jϭ2 Hz, C4-H),
7.45 (1H, td, Jϭ8, 1 Hz, C9-H), 7.56 (1H, td, Jϭ8, 1 Hz, C8-H), 7.76 (1H, s,
C6-H), 7.86 (1H, dd, Jϭ8, 1 Hz, C7-H), 8.04 (1H, dd, Jϭ8, 1 Hz, C10-H),
8.85 (1H, s, C11-H). 13C-NMR (75 MHz, DMSO-d6) d: 175.8 (s, C-12),
161.8 (s, C-1), 161.7 (s, C-3), 159.4 (s, C-4a), 151.5 (s, C-5a), 136.0 (s, C-
6a), 129.6 (d, C-10), 128.5 (2d, C-8, C-11), 128.0 (d, C-7), 126.8 (s, C-10a),
125.1 (d, C-9), 122.5 (s, C-11a), 112.4(d, C-6), 106.9 (s, C-12a), 98.4 (d, C-
4), 97.8 (d, C-2), 84.7 (s, C-2Ј), 75.3 (s, d, C-1Ј, C-3Ј), 56.3 (q, OCH3), 29.5
(q , 2C, C-2Ј (CH3)2). DCI-MS m/z: 359 [MH]ϩ. Anal. Calcd for C23H18O4:
In strong contrast with what is observed in the pyri-
doacridone (acronycine, benzo[b]acronycine) series, all the
linear pyranoxanthone derivatives were more potent than
their angular isomers. For instance, both diacetate 19 and
cyclic carbonate 20 were more potent than their angular
counterparts 16 and 17, and also than acronycine (2) in in-
hibiting the proliferation of L1210 murine leukemia cells.
This suggests that the mechanism of action of pyranoxan-
thone derivatives should be different from that of the corre-
sponding pyranoacridones.
Experimental
Chemistry The melting points were determined on a Leica VM appara- C, 77.08; H, 5.06. Found: C, 77.02; H, 4.98.
tus and are not corrected. IR spectra (nmax in cmϪ1) were obtained on a
5-Hydroxy-2,2-dimethyl-2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one
Perkin-Elmer 257 instrument. UV spectra (lmax in nm) were determined in (12) A solution of 1-hydroxy-3-(1,1-dimethylpropargyloxy)-12H-benzo-
spectroscopic grade MeOH on a Beckman Model 34 spectrophotometer. 1H- [b]xanthen-12-one (10) (0.5 g, 1.45 mmol) in DMF (40 ml) was heated under
NMR [d (ppm), J (Hz)] and 13C-NMR spectra were recorded at 300 and nitrogen at 130 °C for 2 h. Water (50 ml) was added and the mixture was ex-
75 MHz respectively, using a Bruker AC-300 spectrometer. When necessary, tracted with dichloromethane (3ϫ40 ml). The organic layers were evapo-
the signals were unambiguously assigned by two-dimensional (2D) NMR rated under reduced pressure. Flash chromatography (solvent: cyclohexane,
techniques: 1H–1H correlation spectroscopy (COSY), 1H–1H NOESY, then cyclohexane/acetone 99 : 1 to 95 : 5) gave 12 (0.42 g, 85%) as a yellow
13C–1H heteronuclear chemical shift correlation (HETCOR), and 13C–1H amorphous solid. IR (KBr) cmϪ1: 3455, 3066, 2974, 2925, 2857, 1724,
correlation spectroscopy via long range couplings (COLOC). These experi- 1639, 1612, 1472, 1343, 1243, 1176, 1087, 834, 777. UV lmax (MeOH) nm
ments were performed using standard Bruker microprograms. Mass spectra (log e): 235 (sh.), 265 (4.58), 285 (4.48), 343 (4.15). 1H-NMR (300 MHz,
were recorded with a Nermag R-10-10C spectrometer using electron impact CDCl3) d: 1.45 (3H, s, C2-CH3), 1.52 (3H, s, C2-CH3), 5.61 (1H, d,
(MS) and/or desorption chemical ionization (DCI-MS; reagent gas: NH3) Jϭ10 Hz, C3-H), 6.34 (1H, s, C14-H), 6.74 (1H, d, Jϭ10 Hz, C4-H), 7.47
techniques. Flash column chromatographies were conducted using Silica gel (1H, td, Jϭ8, 1 Hz, C9-H), 7.59 (1H, td, Jϭ8, 1 Hz, C10-H), 7.77 (1H, s,
60 Merck (35—70 mm) with an overpressure of 300 mbars.
C12-H), 7.86 (1H, dd, Jϭ8, 1 Hz, C11-H), 8.01 (1H, dd, Jϭ8, 1 Hz, C8-H),
1,3-Dihydroxy-12H-benzo[b]xanthen-12-one (6) To a mixture of 3-hy- 8.78 (1H, s, C7-H), 13.22 (1H, s, D2O exch., C5-OH). 13C-NMR (75 MHz,
droxy-2-naphthalenecarboxylic acid (7) (5 g, 26 mmol), phloroglucinol (8) CDCl3) d: 181.2 (s, C-6), 161.4 (s, C-5), 158.1 (s, C-14a), 157.5 (s, C-13a),
(3.55 g, 26 mmol) and freshly fused zinc chloride (11 g), phosphoryl chlo- 151.8 (s, C-12a), 136.6 (s, C-11a), 129.6 (d, C-8), 129.5 (s, C-7a), 129.1 (d,
ride (50 ml) was added. The reaction mixture was stirred at 70 °C for 4 h, C-10), 127.4 (d, C-7), 127.2 (d, C-3), 127.0 (d, C-11), 125.6 (d, C-9), 120.1
poured onto ice (150 g) and left overnight. After filtration, the precipitate (s, C-6a), 115.4 (d, C-4), 113.2 (d, C-12), 104.2 (2s, C-4a, C-5a), 95.2 (d, C-
was washed with saturated aqueous sodium bicarbonate and water, and 14), 78.4 (s, C-2), 28.4 (q ,C2-CH3), 26.9 (q, C2-CH3). DCI-MS m/z: 345
dried. Purification by flash column chromatography (solvent: cyclohexane, [MH]ϩ. Anal. Calcd for C22H16O4: C, 76.73; H, 4.68. Found: C, 76.65; H,
then cyclohexane/acetone 99 : 1 to 80 : 20) gave 6 (6.52 g, 90%) as reddish 4.68.
crystals, mp 278—279 °C. IR (KBr) cmϪ1: 3355, 3055, 1664, 1603, 1480,
1344, 1252, 1158, 1079, 899, 813, 738. UV lmax (MeOH) nm (log e): 259
(4.89), 330 (sh.), 344 (4.28). 1H-NMR (300 MHz, DMSO-d6) d: 6.17 (1H, d,
5-Methoxy-2,2-dimethyl-2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one
(13) Sodium hydride (0.19 g of 50% oil dispersion, 4 mmol) was slowly
added to an ice-cooled solution of 5-hydroxy-2,2-dimethyl-2H,6H-benzo[b]-
Jϭ2 Hz, C2-H), 6.36 (1H, d, Jϭ2 Hz, C4-H), 7.52 (1H, t, Jϭ8 Hz, C9-H), pyrano[2,3-i]xanthen-6-one (12) (0.69 g, 2 mmol) in DMF (50 ml). Di-

678
Vol. 49, No. 6
methylsulfate (0.56 ml, 6 mmol) was added and the reaction mixture was 1.48 (3H, s, C3-CH3), 1.56 (3H, s, C3-CH3), 2.15 (6H, s, C1-OCOCH3, C2-
stirred under nitrogen at room temperature for 2 h. After addition of water OCOCH3), 4.01 (3H, s, OCH3), 5.31 (1H, d, Jϭ5 Hz, C2-H), 6.32 (1H, s,
(200 ml), the mixture was extracted with ethyl acetate (3ϫ30 ml). The or- C5-H), 6.71 (1H, d, Jϭ5 Hz, C1-H), 7.48 (1H, ddd, Jϭ9, 8, 1 Hz, C10-H),
ganic layers were washed with 10% aqueous NaOH and evaporated under 7.59 (1H, ddd, Jϭ9, 8, 1 Hz, C11-H), 7.68 (1H, s C13-H), 7.87 (1H, dd,
reduced pressure. Flash chromatography (solvent: cyclohexane, then cyclo- Jϭ8, 1 Hz, C12-H), 8.05 (1H, dd, Jϭ8, 1 Hz, C9-H), 8.85 (1H, s, C8-H).
hexane/acetone 99 : 1 to 96 : 4) gave 13 (0.63 g, 88%) as a yellow amorphous 13C-NMR (75 MHz, CDCl3) d: 179.6 (s, C-7), 170.0 (s, C2-OCOCH3),
solid. IR (KBr) cmϪ1: 3062, 2980, 2930, 2857, 1735, 1662, 1627, 1601, 169.6 (s, C1-OCOCH3), 162.8 (s, C-6), 159.0 (2s, C-4a, C-14a), 151.0 (s, C-
1470, 1364, 1257, 1078, 888, 832, 777. UV lmax (MeOH) nm (log e): 238
13a), 136.1 (s, C-12a), 129.7 (d, s, C-9, C-8a), 128.7 (d, C-11), 128.1 (d, C-
8), 127.0 (d, C-12), 125.4 (d, C-10), 122.3 (s, C-7a), 112.6 (d, C-13), 107.3
(sh.), 279 (4.69), 323 (4.14). 1H-NMR (300 MHz, CDCl3) d: 1.50 (6H, s,
C2-(CH3)2), 4.00 (3H, s, OCH3), 5.71 (1H, d, Jϭ10 Hz, C3-H), 6.64 (1H, s, (s, C-6a), 103.4 (s, C-14b), 95.3 (d, C-5), 76.6 (s, C-3), 71.2 (d, C-2), 60.9
C14-H), 6.76 (1H, d, Jϭ10 Hz, C4-H), 7.46 (1H, td, Jϭ8, 1 Hz, C9-H), 7.57
(1H, td, Jϭ8, 1 Hz, C10-H), 7.77 (1H, s, C12-H), 7.86 (1H, dd, Jϭ8, 1 Hz,
(d, C-1), 54.6 (q, OCH3), 26.0 (q, C3-CH3), 21.7 (q, C3-CH3), 20.9 (q, C2-
OCOCH3), 20.7 (q, C1-OCOCH3). DCI-MS m/z: 477 [MH]ϩ. Anal. Calcd
C11-H), 8.20 (1H, dd, Jϭ8, 1 Hz, C8-H), 8.85 (1H, s, C7-H). 13C-NMR for C27H24O8: C, 68.06; H, 5.08. Found: C, 68.12; H, 5.21.
(75 MHz, CDCl3) d: 175.5 (s, C-6), 159.6 (s, C-5), 159.0 (s, C-14a), 156.6
(؎)-cis-1,2-Di-O-carbonyloxy-6-methoxy-3,3-dimethyl-1,2-dihydro-
(s, C-13a), 151.4 (s, C-12a), 136.1 (s, C-11a), 129.8 (d, C-3), 129.6 (d, s, C- 3H,7H-benzo[b]pyrano[3,2-h]xanthen-7-one (17) To a solution of (Ϯ)-
8, C-7a), 128.5 (d, C-10), 127.9 (d, C-7), 126.8 (d, C-11), 125.2 (d, C-9), cis-1,2-dihydroxy-6-methoxy-3,3-dimethyl-1,2-dihydro-3H,7H-ben-
122.1 (s, C-6a), 115.9 (d, C-4), 112.6 (d, C-12), 111.7 (s, C-4a), 109.5 (s, C- zo[b]pyrano[3,2-h]xanthen-7-one (15) (0.1 g, 0.25 mmol) in 2-butanone
5a), 100.6 (d, C-14), 77.9 (s, C-2), 62.6 (q, OCH3), 28.4 (q, 2C, C-2(CH3)2). (10 ml), N,NЈ-carbonyldiimidazole (0.33 g, 1.7 mmol) was added. The reac-
DCI-MS m/z: 359 [MH]ϩ. Anal. Calcd for C23H18O4: C, 77.08; H, 5.06. tion mixture was refluxed for 24 h and after cooling, 5% aqueous Na2CO3
Found: C, 76.92; H, 5.15.
(10 ml) was added. The mixture was extracted with EtOAc (3ϫ10 ml). The
6-Methoxy-3,3-dimethyl-3H,7H-benzo[b]pyrano[3,2-h]xanthen-7-one combined organic layers were dried over anhydrous Na2SO4 and evaporated
(14) A solution of 1-methoxy-3-(1,1-dimethylpropargyloxy)-12H-benzo- under reduced pressure. The crude product was purified by flash chromatog-
[b]xanthen-12-one (11) (0.6 g, 1.67 mmol) in DMF (40 ml) was heated under
raphy (solvent: dichloromethane, then dichloromethane/methanol 99 : 1 to
nitrogen at 130 °C for 1.5 h. The cooled mixture was diluted with water 90 : 10) to give 17 (0.018 g, 17%) as a pale yellow amorphous solid. IR
(50 ml) and extracted with ethyl acetate (3ϫ50 ml). The organic layers were (NaCl film) cmϪ1: 3010, 2982, 1787, 1668, 1630, 1582, 1484, 1416, 1382,
evaporated under reduced pressure. Flash chromatography (solvent: cyclo- 1260, 1194, 1110, 888, 838, 780. UV lmax (MeOH) nm (log e): 260 (2.87),
1
hexane, then cyclohexane/acetone 99 : 1 to 70 : 30) gave 14 (0.56 g, 93%) as 326 (2.46). H-NMR (300 MHz, DMSO-d6) d: 1.37 (3H, s, C3-CH3), 1.60
a yellowish amorphous solid. IR (KBr) cmϪ1: 3064, 2984, 2941, 2857, 1738,
(3H, s, C3-CH3), 3.96 (3H, s, OCH3), 5.22 (1H, d, Jϭ8 Hz, C2-H), 6.40 (1H,
1665, 1632, 1576, 1459, 1393, 1258, 1141, 873, 828, 754. UV lmax (MeOH) d, Jϭ8 Hz, C1-H), 6.52 (1H, s, C5-H), 7.56 (1H, ddd, Jϭ9, 8, 1 Hz, C10-H),
nm (log e): 261 (4.60), 285 (sh.), 328 (3.96). 1H-NMR (300 MHz, CDCl3) d: 7.67 (1H, ddd, Jϭ9, 8, 1 Hz, C11-H), 8.04 (1H, s, C13-H), 8.07 (1H, dd,
1.51 (6H, s, C3-(CH3)2), 4.02 (3H, s, OCH3), 5.63 (1H, d, Jϭ10 Hz, C2-H), Jϭ8, 1 Hz, C12-H), 8.18 (1H, dd, Jϭ8, 1 Hz, C9-H), 8.76 (1H, s, C8-H).
6.29 (1H, s, C5-H), 6.93 (1H, d, Jϭ10 Hz, C1-H), 7.45 (1H, td, Jϭ8, 1 Hz, DCI-MS m/z: 419 [MH]ϩ. Anal. Calcd for C24H18O7: C, 68.90; H, 4.34.
C10-H), 7.56 (1H, td, Jϭ8, 1 Hz, C11-H), 7.77 (1H, s, C13-H), 7.85 (1H, dd,
Jϭ8, 1 Hz,, C12-H), 8.03 (1H, dd, Jϭ8, 1 Hz, C9-H), 8.84 (1H, s, C8-H).
Found: C, 68.82; H, 4.27.
(؎)-cis-3,4-Dihydroxy-5-methoxy-2,2-dimethyl-3,4-dihydro-2H,6H-
13C-NMR (75 MHz, CDCl3) d: 175.9 (s, C-7), 162.1 (s, C-6), 159.4 (s, C- benzo[b]pyrano[2,3-i]xanthen-6-one (18) Oxidation of 13 (0.14 g, 0.39
4a), 154.1 (s, C-14a), 151.3 (s, C-13a), 136.0 (s, C-12a), 129.7 (d, C-9), mmol) under conditions similar to those described for the preparation of 15
128.5 (d, s, C-11, C-8a), 128.1 (d, C-8), 127.0 (d, C-2), 126.8 (d, C-12), from 14, afforded 18 (0.05 g, 33%) as a pale yellow amorphous solid. IR
125.2 (d, C-10), 122.5 (s, C-7a), 115.5 (d, C-1), 112.4 (d, C-13), 106.2 (s, C-
(KBr) cmϪ1: 3450, 2980, 2934, 1664, 1445, 1353, 1248, 1187, 1136, 1107,
6a), 102.3 (s, C-14b), 95.3 (d, C-5), 78.3 (s, C-3), 56.4 (q, OCH3), 28.3 (q, 834, 754, 689. UV lmax (MeOH) nm (log e): 259 (4.66), 328 (4.18). 1H-
2C, C3-(CH3)2). DCI-MS m/z: 359 [MH]ϩ. Anal. Calcd for C23H18O4: C,
NMR (300 MHz, DMSO-d6) d: 1.45 (6H, s, C3-(CH3)2), 3.60 (1H, dd, Jϭ7,
5 Hz, C3-H), 3.94 (3H, s, OCH3), 4.83 (1H, t, Jϭ5 Hz, C4-H), 5.14 (2H, m,
77.08; H, 5.06. Found: C, 77.02; H, 5.14.
(؎)-cis-1,2-Dihydroxy-6-methoxy-3,3-dimethyl-1,2-dihydro-3H,7H- D2O exch., C3-OH, C4-OH), 6.68 (1H, s, H-14), 7.52 (1H, t, Jϭ8 Hz, C9-
benzo[b]pyrano[3,2-h]xanthen-7-one (15) Compound 14 (0.2 g, 0.556 H), 7.65 (1H, t, Jϭ8 Hz, C10-H), 7.98 (1H, s, H-12), 8.00 (1H, d, Jϭ8 Hz,
mmol) was added to a solution of osmium tetroxide (2.5% in 2-methyl-2- C11-H), 8.18 (1H, d, Jϭ8 Hz, C8-H), 8.78 (1H, s, H-7). DCI-MS m/z: 393
propanol, 0.4 ml) and 4-methylmorpholine N-oxide monohydrate (0.076 g, [MH]ϩ. Anal. Calcd for C23H20O6: C, 70.40; H, 5.14. Found: C, 70.29; H,
2.7 mmol) in tert-BuOH–tetrahydrofuran (THF)–H2O (10 : 3 : 1, 15 ml). The 5.06.
reaction mixture was stirred at room temperature for 24 h. After addition of
(؎)-cis-3,4-Diacetoxy-5-methoxy-2,2-dimethyl-3,4-dihydro-2H,6H-
saturated aqueous solution of sodium bisulfite (20 ml), the mixture was benzo[b]pyrano[2,3-i]xanthen-6-one (19) Acetylation of 18 (0.03 g,
stirred for 1 h and then extracted with dichloromethane (6ϫ15 ml). The 0.076 mmol) under conditions similar to those described for the preparation
combined organic layers were dried with sodium sulfate, filtered and evapo- of 16, gave 19 (0.026 g, 72%) as a pale yellow amorphous solid. IR (KBr)
rated under reduced pressure. The crude product was purified by flash chro- cmϪ1: 3010, 2934, 1747, 1663, 1606, 1464, 1375, 1355, 1241, 1191, 1142,
matography (solvent: dichloromethane, then dichloromethane/methanol 1103, 1055, 838, 756, 671. UV lmax (MeOH) nm (log e): 260 (4.74), 324
1
99 6: 1 to 90 : 10) to give 15 (0.13 g, 59%) as a pale yellow amorphous solid.
(4.24). H-NMR (300 MHz, CDCl3) d: 1.48 (3H, s, C2-CH3), 1.50 (3H, s,
IR (NaCl film) cmϪ1: 3329, 2924, 1658, 1599, 1581, 1477, 1455, 1400,
C2-CH3), 2.12 (6H, s, C3-OCOCH3, C4-OCOCH3), 4.02 (3H, s, OCH3),
1300, 1268, 1111, 870, 814. UV lmax (MeOH) nm (log e): 260 (3.56), 330 5.26 (1H, d, Jϭ5 Hz, C3-H), 6.46 (1H, d, Jϭ5 Hz, C4-H), 6.73 (1H, s, C14-
1
(3.00). H-NMR (300 MHz, DMSO-d6) d: 1.40 (3H, s, C3-CH3), 1.43 (3H, H), 7.49 (1H, td, Jϭ8, 1 Hz, C9-H), 7.61 (1H, td, Jϭ8, 1 Hz, C10-H), 7.80
s, C3-CH3), 3.67 (1H, dd, Jϭ8, 4 Hz, C2-H), 3.85 (3H, s, OCH3), 5.02 (1H,
(1H, s C12-H), 7.89 (1H, dd, Jϭ8, 1 Hz, C11-H), 8.05 (1H, dd, Jϭ8, 1 Hz,
t, Jϭ4 Hz, C1-H), 5.08 (1H, d, Jϭ8 Hz, D2O exch., C2-OH), 5.43 (1H, d, C8-H), 8.85 (1H, s, C7-H). 13C-NMR (75 MHz, CDCl3) d: 181.2 (s, C-6),
Jϭ4 Hz, D2O exch., C1-OH), 6.33 (1H, s, C5-H), 7.53 (1H, ddd, Jϭ9, 8,
1 Hz, C10-H), 7.64 (1H, ddd, Jϭ9, 8, 1 Hz, C11-H), 8.02 (1H, dd, Jϭ9,
169.9 (2s, C1-OCOCH3, C2-OCOCH3), 162.2 (s, C-5), 159.7 (s, C-14a),
159.3 (s, C-13a), 151.5 (s, C-12a), 136.5 (s, C-11a), 129.8 (d, C-8), 128.8 (s,
1 Hz, C12-H), 8.07, (1H, s, C13-H), 8.20 (1H, dd, Jϭ9, 1 Hz, C9-H), 8.71 C-7a), 128.3 (2d, C-10, C-7), 127.0 (d, C-11), 125.4 (d, C-9), 122.1 (s, C-
(1H, s, C8-H). DCI-MS m/z: 393 [MH]ϩ. Anal. Calcd for C23H20O6: C, 6a), 112.7 (d, C-12), 110.0 (s, C-4a), 109.9 (s, C-5a), 101.0 (d, C-14), 77.0
70.40; H, 5.14. Found: C, 70.22; H, 5.21.
(s, C-2), 71.3 (d, C-3), 62.6 (q, OCH3), 60.9 (d, C-4), 26.2 (q, C2-CH3), 21.9
(؎)-cis-1,2-Diacetoxy-6-methoxy-3,3-dimethyl-1,2-dihydro-3H,7H- (q, C2-CH3), 20.9 (q, C3-OCOCH3), 20.6 (q, C4-OCOCH3). DCI-MS m/z:
benzo[b]pyrano[3,2-h]xanthen-7-one (16) An ice-cooled mixture of 477 [MH]ϩ. Anal. Calcd for C27H24O8: C, 68.06; H, 5.08. Found: C, 68.15;
acetic anhydride (0.5 ml, 5 mmol) and pyridine (2 ml) was added to (Ϯ)- H, 4.99.
cis-1,2-dihydroxy-6-methoxy-3,3-dimethyl-1,2-dihydro-3H,7H-
(؎)-cis-3,4-Di-O-carbonyloxy-5-methoxy-2,2-dimethyl-3,4-dihydro-
benzo[b]pyrano[3,2-h]xanthen-7-one (15) (0.035 g, 0.09 mmol). After stir- 2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one (20) Treatment of 18 (0.1 g,
ring at room temperature for 3 d, the mixture was poured onto cold water 0.255 mmol) with N,NЈ-carbonyldiimidazole (0.207 g, 1.275 mmol) under
(10 ml). The precipitate was filtered, washed with water (2ϫ5 ml) and dried conditions similar to those described for the preparation of 17, gave 20
in vacuo over P2O5 to give 16 (0.016 g, 38%) as an amorphous pale yellow (0.05 g, 47%) as a white yellowish amorphous solid. IR (KBr) cmϪ1: 3057,
solid. IR (NaCl film) cmϪ1: 3058, 2948, 2363, 2342, 1760, 1667, 1600,
2944, 1812, 1663, 1613, 1468, 1375, 1357, 1252, 1190, 1175, 1078, 1031,
1588, 1515, 1481, 1420, 1342, 1215, 1110, 906, 878, 821, 747. UV lmax 918, 878, 847, 758. UV lmax (MeOH) nm (log e): 262 (4.58), 324 (4.03). 1H-
1
(MeOH) nm (log e): 260 (3.54), 327 (3.00). H-NMR (300 MHz, CDCl3) d: NMR (300 MHz, CDCl3) d: 1.32 (3H, s, C2-CH3), 1.65 (3H, s, C2-CH3),

June 2001
679
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4.16 (3H, s, OCH3), 4.79 (1H, d, Jϭ8 Hz, C3-H), 6.08 (1H, d, Jϭ8 Hz, C4-
H), 6.77 (1H, s, C14-H), 7.50 (1H, td, Jϭ8, 1 Hz, C9-H), 7.61 (1H, td, Jϭ8,
1 Hz, C10-H), 7.81 (1H, s C12-H), 7.90 (1H, dd, Jϭ8, 1 Hz, C11-H), 8.05
(1H, dd, Jϭ8, 1 Hz, C8-H), 8.88 (1H, s, C7-H). 13C-NMR (75 MHz, CDCl3)
d: 175.5 (s, C-6), 163.0 (s, C-5), 160.4 (s, C-14a), 158.7 (s, C-13a), 153.8 (s,
O–CO–O), 151.4 (s, C-12a), 136.5 (s, C-11a), 129.7 (2d, C-7, C-8), 129.1
(d, C-10), 128.4 (d, C-7), 127.0 (d, C-11), 125.7 (d, C-9), 121.9 (s, C-6a),
113.0 (d, C-12), 110.8 (s, C-5a), 108.4 (s, C-4a), 102.2 (d, C-14), 78.0 (d, C-
3), 76.4 (s, C-2), 68.4 (d, C-4), 64.2 (q, OCH3), 24.2 (q, C2-CH3), 22.4 (q,
C2-CH3). DCI-MS m/z: 419 [MH]ϩ. Anal. Calcd for C24H18O7: C, 68.90; H,
4.34. Found: C, 68.92; H, 4.40.
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Biological Pharmacology Cytotoxicity: Murine leukemia L1210 cells
from the American Type Culture Collection (Rockville Pike, MD) were
grown in RPMI medium 1640 supplemented with 10% fetal calf serum,
2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10 mM
HEPES buffer (pH 7.4). The cytotoxicity was measured by microculture
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nine graded concentrations in triplicate of the test drug. Results are ex-
pressed as IC50 (mean, nϭ3), which is defined as the drug concentration in-
hibiting the absorbance by 50% with respect to that of untreated cells.
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