WO2006002233A2 - Alvaradoins e-n, new antitumor and cytotoxic anthracenone g-glycosides - Google Patents

Abstract

This invention relates to anthracenone G-glycoside compounds from the leaves of Alvardoa Haijiensis which have anticancer activity. This invention is also related to the treatment of cancer in animals with the isolated anthracenone G-glycoside compounds. The G-glycoside compounds of the present invention are summarized by the following structure (I):

Description

TITLE OF THE INVENTION

ALVARADOINS E-N₅ NEW ANTITUMOR AND CYTOTOXIC ANTHRACENONE G-GLYCOSIDES

This application claims priority to U.S. provisional application No. 60/581,379 filed on June 22, 2004, the entire contents of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to anthracenone G-glycoside compounds from the leaves of Alvardoa Haijiensis which have anticancer activity. This invention is also related to the treatment of cancer in animals with the isolated anthracenone G-glycoside compounds.

DISCUSSION OF THE BACKGROUND The genus Alvaradoa Liebm. was recently established in the family Picramniaceae and constitutes the subfamily Alvaradoideae, and was formerly classified in the family Simaroubaceae. This genus can be found as a shrub or a small-to-medium-sized tree up to 15 meters high and is composed of seven species distributed in tropical America from southern Florida and Mexico through Central America and the West Indies to South America. (Jacobs, H. Comparative phytochemistry of Picramnia and Alvaradoa, genera of the newly established family Picramniaceae. Biochem. Syst. Ecol. 2003, 31, 773-783; Fernando, E. S.; Quinn, C. J. Picramniaceae, a new family, and a recircumscription of Simaroubaceae. Taxonomy 1995, 44, 177-181; Fernando, E. S.; Gadek, P. A.; Quinn, C. J. Simaroubaceae, an artificial construct: Evidence from mc^ sequence variation. Am. J. Bot. 1995, 82, 92-103; Cronquist, A. Studies in the Simaroubaceae-IV. Resume of the American genera. Brittonia 1944, 5, 128-147). These species grow in different climates and varying altitudes from sea level to above 5000 feet. The medicinal properties and phytochemical constituents of only two of these species have been studied to date. An extract from the leaves of A. amorphides, used to treat skin rashes and head fever, yielded the compounds chaparrin (quassinoid), chrysophanol and chrysophanein (anthraquinones). (Heywood, V. H.; Moore, D. M.; Steam, W. T.; Richardson, I. B. K. Flowering Plants of the World; Oxford University Press: New York, 1993; pp 199-200; Soto de Villatoro, B.; Gonzalez, F. G.; Polonsky, J.; Baskevitch- Varon, Z. Chrysophanic acid, chrysphanein and chaparrin from Alvaradoa amorphides. Phytochemistry 1974, 13, 2018-2019). From the aerial parts ofA.jamaicensis, the anthracenone C-glycosides, alvaradoins A-D, were isolated and tested for activity against Mycobacterium tuberculosis but found inactive. (Harding, W. W.; Henry, G. E.; Lewis, P. A.; Jacobs, H.; McLean, S.; Reynolds, W. F. Alvaradoins A-D. Anthracenone C arabinosides from Alvaradoa jamaicensis. J. Nat. Prod. 1999, 62, 98-101). While similar anthracenone C- glycosides have been isolated from a related genus, Picramnia, to the best of our knowledge, this class of compounds has not been explored previously for their potential anticancer activity. (Hernandez-Medel, M. D. R.; Garcia-Salomones, L; Santillan, R.; Trigos, A. An anthrone from Picramnia antidesma. Phytochemistry 1996, 49, 2599-2601; Hernandez-Medel, M. D. R.; Lopez-Marquez, O.; Santillan, R.; Trigos, A. Mayoside, an oxanthrone from Picramnia hirsuta. Phytochemistry 1996, 43, 279-281; Hernandez-Medel, M. D. R.; Ramirez- Corzas, C. O.; Rivera-Dominguez, M. N.; Ramirez-Mendez, J.; Santillan, R.; Rojas-Lima, S. Diastereomeric C-glycosyloxanthrones from Picramnia antidesma. Phytochemistry 1999, 50, 1379-1383; Solis, P. N.; Ravelo, A. G.; Gonzalez, A. G.; Gupta, M. P.; Phillipson, J. D. Bioactive anthraquinone glycosides from Picramnia antidesma ssp. fessonia. Phytochemistry 1995, 38, 477-480; Rodriguez-Gamboa, T.; Fernandes, J. B.; Fo, E. R.; Da Silva, M. F. D. G. F.; Vieira, P. C; Castro C, O. Two anthrones and one oxanthrone from Picramnia teapensis. Phytochemistry 1999, 51, 583-586). The invention of the present application involves the investigation of the plant kingdom for novel anticancer agents. Initial studies showed that the chloroform-soluble extract obtained from the leaves ofAlvardoa Haijiensis Urb. exhibited promising cytotoxicity against KB (human oral epidermoid carcinoma) cells (97% inhibition at 2μm/mL). Using this cell line to monitor subsequent bioactivity-directed fractionation, ten new anthracenone G-glycoside compounds (Alvaradoin E-N) were obtained, all of which displayed strong anticancer and/or cytotoxic activities.

SUMMARY OF THE INVENTION One object of the invention is to provide novel compounds for a method of treatment of cancer by isolating and purifying potential anticancer compounds from plants. Another object of the invention is use of bioactivity-guided fractionation of the chloroform extract from the leaves ofAlvardoa Haijiensis using the KB human cancer cell line to identify new anticancer compounds. Another objective of the invention is the isolation and identification often new anticancer anthracenone G-glycoside compounds (Alvaradoin E-N). The object compounds of the present invention are summarized by the following structure (I):

where R₁ is -H- or -OH R₂ is -OH or -OCOCHC(CH₃)₂ R₃ is -OH or OCOCHC(CH₃)₂, and R₄ Is -OH Or -OAc.

PREFERRED EMBODIMENTS OF THE INVENTION A preferred embodiment of the invention comprises anthracenone G-glycoside compounds having the structure (I):

where R₁ is -H- or -OH R₂ is -OH or -OCOCHC(CH₃)₂ R₃ is -OH or -OCOCHC(CH₃)₂, and R₄ is -OH or -OAc. Other preferred embodiments of the invention comprises compounds having structure (I) where (i) R₁ is -H, R₂ and R₃ are -OH and R₄ is -OAc (Alvaradoin E and F) (ii) R₁ is -H, R₂ is -OCOCHC(CH₃)_2s R₃ is -OH and R₄ is OAc (Alvaradoin G and H) (iii) R₁ and R₃ are -OH, R₂ is -OCOCHC(CH₃)₂, R₄ is -OAC (Alvaradoin I and J) (iv) R₁ is -H, R₂ and R₄ are -OH and R₃ is -OCOCHC(CH3)₂ (Alvaradoin K and L) (v) R₁ is -H, R₂ and R₄ are -OH and R₂ is -OCOCHC(CH₃)₂ (Alvaradoin M) and (vi) R₁, R₂ and R₃ are -OH and R₄ is -OAc (Alvaradoin N) Another preferred embodiment of the invention comprises R and S isomers of compounds with structures (I)(i) where Alvaradoin E has the S configuration at C-IO and Alvaradoin F has the R configuration at C-IO. The embodiment includes the individually isolated R and S isomers, a racemic mixture of the two or a mixture of each in any proportion. Another preferred embodiment of the invention comprises the R and S isomers of the compounds with the structure (I)(U) where Alvaradoin G has the R configuration at C-IO and Alvaradoin H has the S configuration at C-IO. The embodiment includes the individually isolated R and S isomers, a racemic mixture of the two or a mixture of each in any proportion. Another preferred embodiment of the invention comprises the R and S isomers of the compound with the structure (I)(Ui) where Alvaradoin I has the R configuration at C-IO and Alvaradoin J has the S configuration at C-IO. The embodiment includes the individually isolated R and S isomers, a racemic mixture of the two or a mixture of each in any proportion. Another preferred embodiment of the invention comprises the R and S isomers of the compound with the structure (I)(iv) where Alvaradoin K has the R configuration at C-IO and Alvaradoin L has the S configuration at C-IO. The embodiment includes the individual isolated R and S isomers, a racemic mixture of the two or a mixture of each in any proportion. Another preferred embodiment of the invention comprises the S isomer of the compounds with the structure (I)(v) where Alvaradoin M has the S configuration at C-10. Another preferred embodiment of the invention comprises the S isomer of the compounds with the structure (I)(vi) where Alvaradoin N has the S configuration at C-10. A further embodiment of the invention includes mixtures of Alvaradoin E through N in any proportion. This includes mixtures of Alvaradoin E through N as obtained by extraction from the leaves of Alvaradoa Haitiensis. The Alvaradoin compounds E through N were characterized by several techniques including melting point, optical rotation, circular dichroism, UV spectroscopy, IR spectroscopy, NMR spectroscopy, and mass spectroscopy. The compounds were separated and purified by chromatography. Melting points were measured either on a Kofler hot-stage apparatus or a Mel-Temp II digital thermometer melting point apparatus and are uncorrected. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter at 25 °C and the [α]o values are given in 10^"1 deg cm² g^"1. Circular dichroism spectra were obtained from an Aviv CD Stopped Flow Model 202. UV spectra were recorded on a Varian Cary 3 G UV- Visible spectrophotometer and IR (NaCl or KBr pellet) spectra were recorded on a Nicolet Avatar 360 FT-IR spectrometer. NMR experiments were performed on a Bruker AMX 500 spectrometer with TMS as an internal standard. EIMS and ESIMS were recorded on HP5989A and Finnigan LCQ instruments, respectively. High-resolution MS measurements were obtained via direct insertion probe EI or fast-atom bombardment on a VG70S magnetic sector instrument (Micromass, Beverly, MA) or by an Applied Biosystems (Framingham MA) TOF/TOF mass spectrometer, equipped with a Nd: YAG laser operating at 355 nm and 200 Hz. This instrument was operated in the reflectron mode, and the matrix employed was 2,5-dihydroxybenzoic acid prepared at a concentration of 9 mg/mL in 70:30 (v.v) acetonitrile-0.1% trifluoroacetic acid. Column chromatography was carried out on Si gel 60 (230-400 mesh, Merck, Darmstadt, Germany). Fractions were monitored by TLC (silica gel 60 F254 plates, 0.25 mm thickness) with visualization under UV light (254 and 365 nm) and with 1% sulfuric acid in EtOH. Preparative HPLC was carried out on a Varian Prostar 210 pump system attached to a YMC (Wilmington, NC) Diol NP (250 x 25 mm, i.d., 5 μm) column or a YMC ODS-A (250 x 25 mm, i.d., 5 μm) column. The peaks were detected at 365 and 420 nm using a Prostar 330 PDA detector and data were recorded by Star Chromatography Workstation ver. 5.51 software system. The flow-rate was 7 and 10

The Alvaradoin compounds E through N were extracted from the leaves of A. haitiensis. The leaves were collected under a Consultant Agreement in February 1996 by F. J. and R. G. at Cordillera Central, San Cristobal Province, Dominican Republic and dried. Voucher specimens (#2047 and 7357) have been deposited at the Field Museum of Natural History, Chicago, IL. The dried and ground leaves (5408 g) of A. haitiensis were extracted with MeOH (6 L x 2) for 24 h at room temperature. The extracts were combined and concentrated in vacuo. The concentrated MeOH solution was diluted with H₂O to give a MeOH-H₂O (9:1) solution (2 L), defatted with hexane (2 Lx 2), and then concentrated in vacuo. The aqueous MeOH fraction was dissolved in CHCl₃-MeOH (4:1, 1 L) and partitioned further with H₂O (I L x 2). The organic fraction was washed using 1% saline solution until there was no evidence of tannins and concentrated in vacuo to afford 169 g of crude extract that inhibited 99% of KB cell growth when a concentration of 20 μg/mL was tested. This extract was purified by low- pressure column chromatography with Si gel (1300 g) using gradient mixtures of 50-M00% CHCl₃ in hexane then 0->10% MeOH in CHCl₃, resulting in 17 pooled fractions (F01-F17). Of these, F07-F14 showed significant inhibition of KB cancer cells (93-96% inhibition at 2 μg/mL). A precipitate from F07 and F08 [eluted with CHCl₃-MeOH (99:1); 93-94% inhibition at 2 μg/mL] was recrystallized from CHCl₃-MeOH (4:1) to yield 1.6 g of solid, which contained an approximate 3:2 mixture of Alvaradoin G and H, according to the ¹H NMR spectrum. The compounds were isolated by gradient normal-phase HPLC with a YMC- Diol NP column under the following conditions: A:B (A = 19:1, CHCl₃-isopropanol; B = hexane) (33:67->40:60 for 28 min) then isocratic (40:60 for 17 min) to yield pure compounds Alvaradoin G and H. F09 [eluted with CHCl₃-MeOH (98.5:1.5); KB, 95% inhibition at 2 /ig/mL] was subjected to Si gel chromatography using hexane-acetone (100:0->0:100, gradient mixtures), affording 14 fractions (F15-F28). F26 [eluted with hexane-acetone (3:2); 97% inhibition at 0.2 μg/mL] was subjected to purification by multiple reversed-phase preparative HPLC YMC ODS-A C₁₈ column chromatography carried out under the following conditions: MeCN-H₂O (45:55->60:40 for 45 min) then (60:40^100:0 for 5 min) resulting in four impure fractions (F29-F32). Further purification of F29 (YMC ODS-A, 50% MeCN in H₂O) was achieved using normal-phase preparative HPLC YMC-Diol NP with solvent system A:B (A = 19:1, CHCl₃-isopropanol; B = hexane) (65:35— >90:10 for 50 min) to yield pure compounds Alvaradoin K and L. In turn, F30 was subjected to normal-phase preparative HPLC YMC- Diol NP with solvent system A:B (A = 9:1, CHCl₃-isopropanol; B = hexane) (30:70→40:60 for 60 min) to yield pure compounds Alvaradoin I, J and M. F13 [eluted with CHCl₃-MeOH (19:1); KB, 96% inhibition at 2 μg/mL] was further subjected to Si gel chromatography using hexane-acetone (90:10-^0:100, gradient mixtures), resulting in 11 fractions (F33-F43). A precipitate was formed from F38 [eluted with hexane- acetone (55:45); 96% inhibition at 2 μg/mL] to give a yellow solid (1.2 g). The solid was subjected to purification by preparative HPLC YMC-Diol NP under the following conditions: MeOH-CHCl₃ (2:98->6:94 for 40 min), affording pure compounds Alvaradoin E, F and N. Alvaradoin E [(105)-C-(5 '-O-acetyl)-_/?-D-xylopyranosyl-l ,8-dihydroxy-3-methyl- anthracen-9(10H)-one, I]: yellow solid (219.5 mg, yield 0.0017% w/w); t_τ 14.12 min in 50:50^100:0 MeOH-H₂O over 25 min and t_r 13.32 min in 2:98^4:96 MeOH-CHCl₃ over 20 min, with the YMC-Diol NP column; mp 194-196 ⁰C; [α]_D -16.8° (c 0.07, methanol); UV (MeOH) λ_max (log ε) 358 (4.97), 297 (4.85), 268 (4.79), 202 (5.43) nm; CD (MeOH) [θ\ (nm) -5.87 x 10-⁶ (323), +8.66 x 10^"6 (297), -5.40 x 10^"6 (268); IR (KBr) v_max 3428, 2921, 1740, 1636, 1618, 1457, 1294, 1234, 1139, 1021, 959, 806, 761 cm ¹; ¹H and ¹³C NMR data, see Table 1; HMBC data, see Table 1; ESIMS m/z 453 [M + Na]⁺; HRFABMS m/z 469.0916 [M + K]⁺ (calcd for C₂₂H₂₂O₉K, 469.0901). Alvaradoin F [(10i?)-C-(5'-O-acetyl)-^-D-xylopyranosyl-l,8-dihydroxy-3-methyl- anthracen-9(10H)-one, 2]: yellow solid (93.5 mg, yield 0.00070% w/w); t_τ 13.95 min in 50:50^100:0 MeOH-H₂O over 25 min and t_t 14.39 min in 2:98->4:96 MeOH-CHCl₃ over 20 min, with the YMC-Diol NP column; mp 210-213 °C; [α]_D -107.7° (c 0.05, methanol); UV (MeOH) λ_max (log ε) 357 (5.03), 296 (4.94), 268 (4.89), 203 (5.46) nm; CD (MeOH) [θ\ (nm) +4.73 x 10^'6 (353), -2.44 x 1(T⁷ (299), +2.63 x 10^"6 (257); IR (KBr) v_max 3423, 2924, 1750, 1635, 1617, 1457, 1293, 1230, 1139, 1021, 961, 800, 770 cm^'1; ¹H and ¹³C NMR data, see Table 1; HMBC data, see Table 1; ESIMS m/z 453 [M + Na]⁺; HRFABMS m/z 469.0916 [M + K]⁺ (calcd for C₂₂H₂₂O₉K, 469.0901). Alvaradoin G [(I Oi?)-C-(5 '-O-acetyl-3 '-O-senecioyl)-^-D-xylopyranosyl- 1 ,8- dihydroxy-3-methyl-anthracen-9(10H)-one, 3]: brown solid (6.9 mg, yield 0.000052% w/w); t_τ 16.64 min in 50:50^ 100:0 MeOH-H₂O over 25 min and t_r 17.11 min in 38:62 A:B (A = 19:1 CHCl₃-isopropanol, B = hexane) over 30 min, with the YMC-Diol NP column; mp 197- 200 ⁰C; [α]_D -85.0° (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 360 (3.99), 295 (3.92), 268 (3.94), 205 (4.67) nm; CD (MeOH) [θ\ (nm) +3.11 x 10^"6 (351), -2.03 x 10^"7 (296), +1.62 x 10^'6 (258); IR (NaCl) v_max 3446, 2990, 1759, 1693, 1616, 1596, 1286, 1220, 1127, 1018, 756; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, C-3, CH₃-Il, C-4; H-4/C-2, C-10, CH₃-Il; H-5/C-6, C-IO, C-7; H-6/C-5a, C-8; H- 7/C-5; H-10/C-4a, C-5a, C-4, C-5, C-Ia, C-8a, C-I¹; CH₃-I l/C-2, C-3, C-4; H-1VC-2', C-4a, C-5a; H-2VC-3'; H-37C-2¹, C-I"; H-4VC-3¹; H-5VC-1', C-3', C-4¹, C-I"'; H-2'VC-l", C-4", C- 5"; CH₃-4'7C-2", C-3", C-5"; CH₃-5"/C-2", C-3", C-4"; CH₃-2^M'/C-1"'; OH-l/C-1, C-Ia; OH-8/C-8, C-8a; OH-2'/C-r, C-2'; OH-4VC-3', C-4', C-5'; EIMS m/z 512 [M]⁺ (2), 452 (1), 352 (5) 280 (5), 265 (6), 240 (100), 165 (9); HRFABMS m/z 551.1336 [M + K]⁺ (calcd for C₂₇H₂₈O₁₀K, 551. 1319). Alvaradoin H [(105)-C-(5 '-O-acetyl-3 '-O-senecioyl)-^-D-xylopyranosyl- 1,8- dihydroxy-3-methyl-anthracen-9(10H)-one, 4]: yellow solid (12 mg, yield 0.000090% w/w); t_t 16.72 min in 50:50-> 100:0 MeOH-H₂O over 25 min and t_t 15.45 min in 38:62 A:B (A = 19:1 CHCl₃-isopropanol, B = hexane) over 30 min, with the YMC-Diol NP column; mp 240- 243 °C; [α]_D -26.0° (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 358 (4.06), 297 (3.98), 268 (3.98), 207 (4.77) nm; CD (MeOH) [0] (nm) -5.18 x 10^"6 (322), +3.46 x 10^"6 (296), -6.00 x 10-⁶ (268); IR (NaCl) v_max 3460, 3026, 2977, 1747, 1693, 1617, 1598, 1294, 1228, 1156, 1022, 752; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, C-Ia, CH₃-Il, C-4; H-4/C-2, C-10, CH₃-Il; H-5/C-10; H-6/C-5a, C-8; H-7/C-5, C-8; H-10/C-4a, C-5a, C-4, C-5, C-Ia, C-8a, C-I'; CH₃-I l/C-2, C-3, C-4; H-17C-2¹, C-4a; H-27C-3'; H-3VC-2'; H-47C-3'; H-5VC-1', C-3', C-4', C-I"'; H-27C-1", C-4", C-5"; CH₃-4"/C-2", C-3", C-5"; CH₃-5"/C-2", C-3", C-4"; CH₃^¹¹VC-I¹"; OH-l/C-1, C-Ia, C-2; OH-8/C-8, C-8a; OH-27C-1¹, C-2¹; OH-4VC-4¹, C-5¹; EIMS m/z 512 [M]⁺ (1), 452 (2), 352 (5), 280 (34), 265 (45), 240 (100), 165 (18); HRFABMS m/z 551.1324 [M + K]⁺ (calcd for C₂₇H₂₈O₁₀K, 551.1319). Alvaradoin I [(10i?)-C-(5'-O-acetyl-3'-O-senecioyl)-^-D-xylopyranosyl-l,8,10- trihydroxy-3-methyl-anthracen-9-one, 5]: yellow solid (6.0 mg, yield 0.000045%w/w); t_τ 13.24 min in 50:50^100:0 MeOH-H₂O over 25 min and t_τ 9.92 min in 20:80^60:40 A:B (A = 9:1 CHCla-isopropanol, B = hexane) over 10 min, with the YMC-Diol NP column; mp 148- 149 °C; [α]_D -11.7° (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 369 (3.89), 300 (3.71), 213 (4.50) nm; CD (MeOH) [θ\ (mn) -1.73 x 10⁶ (332), +7.61 x 10⁶ (301), -8.98 x 10⁶ (268); IR (NaCl) v_max 3394, 3016, 2980, 2917, 1719, 1642, 1220, 1142, 757; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, C-Ia, CH₃-Il,

C-4; H-4/C-la, C-2, C-IO, CH₃-I l; H-5/C-7, C-8a, C-10; H-6/C-5a, C-8; H-7/C-5, C-8a; CH₃-I l/C-2, C-3, C-4; H-l'/C-4a, C-5a, C-IO, C-2¹, C-3', C-5'; H-27C-3¹; H-3VC-2¹, C-4¹, C- 1"; H-5VC-1¹, C-3¹, C-I"¹; H-2^M/C-4^M, C-5"; CH₃-4"/C-2", C-3", C-5"; CH₃-5"/C-2", C-3", C-4"; CH₃-2"7C-1"'; OH-l/C-1, C-Ia; OH-8/C-7, C-8, C-8a; OH-4VC-4¹, C-5¹; EIMS m/z 528 [M]⁺ (3), 468 (1), 315 (3), 298 (8), 273 (14), 256 (100), 240 (2), 213 (24), 83 (53); HRMALDI m/z 551.1518 [M + Na]⁺ (calcd for C₂₇H₂₈O₁₁Na, 551.1529). Alvaradoin J [(105)-C-(5 '-O-acetyl-3 '-O-senecioyl)-^-D-xylopyranosyl-l ,8, 10- trihydroxy-3-methyl-anthracen-9-one, 6]: yellow solid (10.4 mg, yield 0.000078% w/w); t_r 13.27 min in 50:50^100:0 MeOH-H₂O over 25 min and t_t 10.39 min in 20:80->60:40 A:B (A = 9:1 CHCl₃-isopropanol, B = hexane) over 10 min, with the YMC-Diol NP column; mp 138-139 °C; [α]_D -56.7° (c 0.05, MeOH); UV (MeOH) λ_max log ε) 369 (3.88), 300 (3.68), 213 (4.48) nm; CD (MeOH) [θ\ (nm) +2.10 x 10⁶ (353), -1.40 x 10⁷ (299), +4.29 x 10⁶ (260); IR (NaCl) v_max 3417, 3021, 2980, 2923, 1723, 1604, 1287, 1217, 1143, 754; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, C-Ia, CH₃-I l, C-4; H-4/C-la, C-2, C-3, C-10, CH₃-I l; H-5/C-6, C-7, C-8a, C-10; H-6/C-5a, C-8; H-7/C-5, C-8, C-8a; CH₃-I l/C-2, C-3, C-4; H-17C-4a, C-5a, C-10, C-2', C-3¹, C-5¹; H-2VC-3¹; H-37C-2¹, C-I"; H-5VC-1¹, C-3¹. C-4¹, C-I¹"; H-27C-4", C-5"; CH₃-47C-2", C-3", C-5"; CH₃-5"/C-2", C-3", C-4"; CH₃-2"7C-1'"; OH-l/C-1, C-Ia, C-2; OH-8/C-7, C-8; OH-10/C-4a, C-10, C-I¹; OH-47C-4', C-5¹; EIMS m/z 528 [M]⁺ (2), 468 (1), 315 (2), 298 (2), 285 (3), 273 (15), 256 (100), 240 (3), 213 (23), 83 (48); HRMALDI m/z 551.1525 [M + Na]⁺ (calcd for C₂₇H₂₈O₁₁Na, 551.1529). Alvaradoin K [(10i?)-C-(4'-O-senecioyl)-^-D-xylopyranosyl-l,8-dihydroxy-3- methyl-anthracen-9(10)-one, 7]: yellow solid (14 mg, yield 0.00011% w/w); t_τ 17.56 min in 50:50^100:0 MeOH-H₂O over 25 min and t_τ 10.95 min in 80:20-^95:5 A:B (A = 19:1 CHCl₃-isopropanol, B = hexane) over 15 min, with the YMC-Diol NP column; mp 143-144 ⁰C; [α]_D -65.0° (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 359 (3.87), 297 (3.77), 210 (4.42) nm; CD (MeOH) [θ\ (run) +1.47 x 10⁶ (351), -8.51 x 10⁶ (298); IR (NaCl) v_max 3451, 3020, 2975, 2936, 1690, 1603, 1291, 1229, 1074, 754; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, CH₃-Il, C-4; H-4/C-10, CH₃-Il; H-5/C-6, C-IO; H-6/C-5a, C-8; H-7/C-5; H-10/C-4a, C-5a, C-Ia, C-8a, C-I¹, C-2¹; CH₃-I l/C-2, C-3, C-4; H- r/C-10, C-2', C-3¹, C-5'; H-2VC-10, C-I¹, C-3¹; H-4VC-2¹, C-3¹, C-I¹¹; H-5VC-1¹, C-3¹, C-4¹; H- 27C-4", C-5"; CH₃-4"/C-2", C-3", C-5"; CH₃-5"/C-2", C-3", C-4"; OH-l/C-1, C-Ia, C-2; OH-8/C-8, C-8a; OH-5VC-4¹, C-5'; ESIMS m/z 493 [M + Na]⁺; HRMALDI m/z 493.1475 [M + Na]⁺ (calcd for C₂₅H₂₆O₉Na, 493.1474). Alvaradoin L [(105)-C-(4'-O-senecioyl)-^-D-xylopyranosyl-l,8-dihydroxy-3-methyl- anthracen-9(10)-one, 8]: yellow solid (14 mg, yield 0.00011% w/w); t_x 15.91 min in 50:50^100:0 MeOH-H₂O over 25 min and t_r 11.63 min in 80:20^95:5 A:B (A = 19:1 CHCl₃-isopropanol, B = hexane) over 10 min, with the YMC-Diol NP column; mp 141-142 ⁰C; [α]_D -26.7° (C 0.09, MeOH); UV (MeOH) λ_max (log ε) 360 (3.92), 298 (3.82), 210 (4.47) nm; CD (MeOH) [θ\ (nm) -3.19 x 10⁶ (327), +3.64 x 10⁶ (298), -4.90 x 10⁶ (268); IR (NaCl) v_max 3436, 3020, 2975, 2924, 1691, 1603, 1291, 1231, 1079, 755; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, C-Ia, CH₃-I l, C-4; H-4/C-la, C-IO, CH₃-I l; H-5/C-7, C- 8a; H-6/C-5a, C-8; H-7/C-5, C-8; H-10/C-4a, C-5a, C-4, C-Ia, C-8a, C-I¹, C-2¹; CH₃-I l/C-2, C-3, C-4; H-l'/C-4a, C-5a, C-10, C-2¹; H-4VC-2¹, C-I"; H-5'/C-r, C-3¹, C-4¹; H-2"/C-l", C-4", C-5"; CH₃-4"/C-2", C-3", C-5"; CH₃-5"/C-2", C-3", C-4"; OH-l/C-1, C-Ia; OH-8/C-7, C-8, C-8a; OH-27C-1¹; OH-3YC-2¹, C-3¹; OH-5VC-4¹, C-5¹; ESIMS m/z 493 [M + Na]⁺; HRMALDI m/z 493.1452 [M + Na]⁺ (calcd for C₂₅H₂₆O₉Na, 493.1474). Alvaradoin M [(105)-C-(3 '-O~senecioyl)-_/?-D-xylopyranosyl-l,8-dihydroxy-3- methyl-anthracen-9(10)-one, 9]: yellow solid (6.8 mg, yield 0.000051% w/w); t_x 15.91 min in 50:50^100:0 MeOH-H₂O over 25 min and t_x 11.91 min in 20:80-^60:40 A:B (A = 9:1 CHCl₃-isopropanol, B = hexane) over 15 min, with the YMC-Diol NP column; mp 153-154 ⁰C; [α]_D -32.0° (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 360 (3.86), 297 (3.74), 212 (4.42) nm; CD (MeOH) [θ\ (nm) -1.58 x 10⁶ (322), +4.97 x 10⁵ (301), -2.65 x 10⁶ (268); IR (NaCl) v_max 3452, 3019, 2974, 2926, 1699, 1603, 1292, 1230, 1079, 756; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H-2/C-1, CH₃-11, C-4; H-4/C-la, C-IO₅ CH₃-I l; H-5/C-7, C-8a, C-IO; H-6/C-5a, C-8; H-7/C-5; H-10/C-4a, C-5a, C-4, C-5, C-Ia, C-I¹; CH₃-I l/C-2, C-3, C-4; H-l'/C-4a; H-3VC-2'; H-2"/C-4", C-5"; CH₃-4"/C-2", C-3", C-5"; CH₃-57C-2", C-3", C-4"; OH-1/C-la; OH-8/C-7, C-8a; OH-27C-1¹; OH-5VC-4¹, C-5¹; ESIMS m/z 493 [M + Na]⁺; HRMALDI m/z 493.1461 [M + Na]⁺ (calcd for C₂₅H₂₆O₉Na, 493.1474). Alvaradoin N [(10S)-C-(5 '-O-acetyl)-^-D-xylopyranosyl- 1,8,10-trihydroxy-3-methyl- anthracen-9-one, 10]: yellow solid; (6.1 mg, yield 0.000046% w/w); t_r 9.64 min in 50:50^100:0 MeOH-H₂O over 25 min and t_r 17.58 min in 2:98^4:96 MeOH-CHCl₃ over 20 min, with the YMC-Diol NP column; mp 139-140 ⁰C; [α]_D -56.0° (c 0.05, MeOH); UV (MeOH) λ_max log ε) 368 (3.70), 301 (3.54), 211 (4.13) nm; CD (MeOH) [θ\ (nm) +4.61 x 10⁶ (351), -4.72 x 10⁸ (299), +1.51 x 10⁸ (259); IR (NaCl) v_raax 3400, 2925, 2854, 1743, 1628, 1607, 1285, 1219, 1071, 756 cm^"1; ¹H and ¹³C NMR data, see Tables 2 and 3; HMBC H- 2/CH₃-I l, C-4; H-4/C-10, CH₃-I l; H-5/C-10; H-6/C-5a, C-8; H-7/C-5; CH₃-I l/C-2, C-3, C- 4; H-17C-5a, C-IO, C-2¹, C-3¹; H-2VC-1¹, C-3'; H-37C-2'; H-4VC-3'; H-57C-1¹, C-3¹, C-I"; H- 2"/C-I"; OH-l/C-1, C-2; OH-8/C-7, C-8; OH-10/C-4a, C-IO, C-I'; OH-3VC-3', C-4¹; OH-4VC-3¹, C-4¹, C-5¹; ESIMS m/z 445 [M - 1]⁺; HRMALDI m/z 469.1097 [M + Na]⁺ (calcd for C₂₂H₂₂O₁₀Na, 469.1111). Table 1. ¹HNMR data for Alvaradoin E and F (acetone-efe) Alvaradoin E Alvaradoin F position δ_H δc HMBC ROESY δc HMBC ROESY

1 - 162.9 - - - 162.9 - - Ia - 118.3 - - - 116.1 - - 2 6.73 (IH, s) 117.1 1, 4, CH₃-Il CH₃-Il 6.70 (IH, s) 116.6 1, 1a, 4, CH₃-Il CH₃-Il 3 - 148.0 - - - 148.7 - - 4 7.04 (IH, s overlap) 122.4 Ia, 2, 10, CH₃-Il H-2¹, OH-2¹, H-IO, 6.90 (IH, s) 120.2 Ia, 10, CH₃-Il H-I¹, H-10, CH₃-Il CH₃-Il 4a - 142.3 - - - 146.7 - - S 7.04 (IH, d overlap, 7.5) 119.1 7, 8a, 10 H-6, H-IO, H-I' 7.18 (IH, d, 7.5) 121.2 8a, 10 H-2¹, OH-2 5a - 146.7 - - - 142.4 - - 6 7.56 136.7 5a, 8 H-5, H-7 7.53 136.1 5a, 8 H-5, H-7 (IH, dd overlap, 8.0, 7.9) (IH, dd overlap, 8.0, 7.9) 7 6.85 OH, d, 8.4) 116.4 5, 8 H-6 6.88 (IH, d, 7.9) 116.8 5 H-6 8 - 163.2 - - - 163.1 - - 8a - 116.1 - - - 118.3 - - 9 - 195.0 - - - 195.0 - - 10 4.66 (IH, d, 2.3) 44.0 4, 5, 1', 4a, 5a, Ia, 8a H-4, H-5, H-I¹, H-2' 4.66 (IH, d, 2.3) 43.9 4, 5, 1', 4a, 5a, Ia, 8a H-I¹ CH₃-I l 2.38 (3H, s) 22.2 2, 3, 4 H-2, H-4 2.40 (3H, s) 22.0 2, 3, 4 H-2, H-4 1' 3.84 (IH, dd, 9.8, 2.4) 81.3 4a, 5a H-2', H-3¹, H-5, H-IO 3.86 (IH, dd, 9.9, 2.5) 81.4 5a H-4 2' 3.59-3.62 (IH, m) 68.2 3' H-4, OH-2', H-I', H-IO 3.59-3.61 (IH, m) 68.3 3¹ OH-2¹ 3' 3.65-3.69 (2H, m) 73.1 l', 2' H-I¹, OH-2¹, OH-4', 3.66-3.70 (2H, m) 73.1 2' - 4' 3.65-3.69 (2H, m) 70.6 2' OH-4', H-5' 3.66-3.70 (2H, m) 70.5 2' H-5' 5' 5.63 (IH, s) 94.4 l', 3', 1" H-4', OH-4¹ 5.62, s 94.4 I¹, 3', 1" H-4¹, OH-4 1" - 168.3 - - - 168.3 - - CH₃-2¹¹ 1.76 (IH, s) 20.4 1" - 1.78 (IH, s) 20.4 1" - OH-I 12.01 (IH, s) - 1, 1a, 2 - 11.92 (IH, s) - 1, 1a, 2, - OH-8 11.92 (IH, s) - 7, 8, 8a - 12.04 (IH, s) - 7, 8, 8a - OH-2¹ 4.50 (IH, d, 4.8) - I¹, 2' H-4, H-2', H-3¹ 4.43 (IH, d, 4.7) - l', 2' H-2¹ OH-3' 3.97 (IH, d, 6.8) - y - 3.96 (IH, d, 6.9) - 3', 4' - OH-4' 4.09 (IH, d, 3.7) - 4', 5' H-4¹, H-5¹ 4.08 (IH, d, 3.6) - 4', 5' H-5¹ Table 2. ¹H NMR data of Alvaradoin G-N (acetone-^) Position Alvaradoin G Alvaradoin H Alvaradoin I Alvaradoin J Alvaradoin K Alvaradoin L Alvaradoin M Alvaradoin N¹ H-2 6.72 (s) 6.73 (s) 6.80 (s) 6.75 (S) 6.70 (s) 6.76 (s) 6.71 (S) 6.73, s n o H-4 6.96 (s) 7.01 (S) 7.28 (s) 7.36 (s) 6.91 (s overlap) 7.03 (s) 6.99 (s) 7.38, s H-5 7.16 (d, 7.S) 7.07 (d, 7.5) 7.50 (d, 7.6) 7.41 (dd, 7.6, 1.0) 7.15 (d, 7.4) 7.04 (d, 7.6) 7.06 (d, 7.4) 7.41, (dd, 7.8, 0.9) H-6 7.58 (d, 7.7) 7.56 (dd, 8.1, 7.2) 7.62 (bit, 8.1, 7.8) 7.63 (t, 8.0) 7.57 (t, 7.9) 7.55 (t, 8.1, 7.8) 7.53 (t, 7.9) 7.62, (t, 7.8, 8.1) H-7 6.89 (d, 8.3) 6.85 (d, 8.1) 6.90 (d, 8.3) 6.95 (dd, 8.3, 0.8) 6.91 (d overlap, 7.9) 6.84 (d, 8.4) 6.83 (d, 7.6) 6.93, (dd, 8.4, 0.9) H-IO 4.71 (brs) 4.69 (s) - - 4.68 (brs) 4.68 (brs) 4.70 (d, 2.1) - CH₃-Il 2.41 (S) 2.38 (s) 2.43 (S) 2.43 (S) 2.39 (s) 2.43 (s) 2.38 (s) 2.41, s H-Γ 4.01 (dd,) 3.99 (dd, 9.9, 2.1) 3.72 (d, 9.7) 3.74 (d, 9.6) 4.05 (dd, 9.9, 2.3) 4.05 (dd, 9.8, 2.1) 4.15 (dd, 9.9, 2.3) 3.57 (overlap) H-2' 3.87 (m) 3.84-3.92 (m) 3.95 (brt, 9.3, 9.2) 3.93 (m) 3.65 (brt, 9.7, 9.5) 3.70 (brt, 9.6) 3.89 (m) 3.63 (overlap) H-3¹ 4.98 (dd, 8.8, 3.6) 4.96 (dd, 9.6, 3.3) 4.96 (dd, 9.1, 3.2) 4.97 (dd, 9.1, 3.3) 3.96 (m) 3.97 (m) 5.07 (dd, 9.7, 3.1) 3.69 (overlap) H-4' 3.87 (m) 3.84-3.92 (m) 3.84 (m) 3.84 (m) 4.88 (m) 4.89 (m) 3.81 (m) 3.63 (overlap) H-₅' 5.62 (dd, 5.5, 1.6) 5.63(d, 4.8) 5.57 (brs) 5.56 (d, 1.8) 4.81 (brd, 6.8) 4.81 (brd, 4.2) 4.81 (m) 5.55 (d, 1.5) H-2" 5.67 (dd, 2.8, 1.4) 5.66 (dd, 2.4, 1.3) 5.62 (s) 5.62 (m) 5.32 (brs) 5.33 (brs) 5.66 (m) - CH₃-4" 2.14 (S) 2.12 (S) 2.12 (S) 2.11 (d, 1.1) 1.90 (s) 1.89 (s) 2.13 (d, 1.2) - CH₃-5" 1.89 (s) 1.88 (s) 1.89 (s) 1.88 (d 1.2) 1.84 (s) 1.85 (S) 1.88 (d, 1.2) - CH₃-2¹" 1.83 (S) 1.80 (s) 1.79 (s) 1.81 (s) - - - 1.75, s OH-I 11.95 (s) 12.00 (s) 11.93 (s overlap) 11.85 (s) 11.85 (s) 11.86 (s) 11.95 (s) 11.84, s OH-8 12.06 (s) 11.91 (s) 11.92 (s overlap) 12.04 (s) 11.95 (s) 11.95 (s) 11.85 (s) 12.03, s OH-10 - - 6.41 (s) 6.40 (brs) - - - 6.54, s OH-2' 4.83 (d, 6.0) 4.81 (d, 5.7) 5.73 (brs) 5.63 (brs) 4.40 (brs) 4.44 (brs) 4.54 (d, 6.1) 5.65 (d, 2.1) OH-3' - - - - 4.14 (brs) 4.12 (brs) - 4.05 (d, 6.6) OH-4' 4.47 (d, 4.0) 4.42 (d, 4.5) 4.46 (brs) 4.40 (d, 4.5) - - 3.85 (d, 4.4) 4.08 (d, 3.6) OH-5' - - - - 5.74 (d, 4.4) 5.70 (d, 4.5) 5.44 (d, 4.3) - Table 3. "C NMR data of Alvaradoin G-N (acetone-^ carbon Alvaradoin G Alvaradoin H Alvaradoin I Alvaradoin J Alvaradoin K Alvaradoin L Alvaradoin M Al^ar C-I 163.4 162.8 163.2 162.9 162.7 163.2 162.5 1 C-Ia 116.3 116.4 114.9 114.9 116.2 116.4 116.5 C-2 117.1 117.2 118.1 117.7 116.5 116.9 116.9 1 C-3 148.2 148.0 148.1 148.8⁶ 148.7 147.8 147.8 1 C-4 120.5 122.2 120.0 118.5 120.7 122.5 122.4 1 C-4a 146.5 142.2 145.8 148.9' 146.9 143.2 143.2 1 C-5 121.2 119.2 117.7 118.8 121.2 119.6 119.6 1 C-5a 142.4 146.3 149.0 145.8 143.3 147.1 147.1 1 C-6 136.3 136.7 136.9 136.3 135.9 136.9 136.9 1 C-7 116.7 116.5 117.2 118.0 116.6 116.3 116.2 1 C-8 163.3 163.2 162.6 162.9 162.9 162.6 163.1 1 C-8a 118.4 117.1 117.1 116.9 118.5 118.3 118.3 1 C-9 193.1 194.9 194.0 193.9 195.1 195.2 195.2 1 C-IO 44.2 44.0 76.3 76.2 44.6 44.6 44.4 CH₃-Il 22.3 22.1 22.4 22.2 22.1 22.2 22.2 c-r 81.9 81.6 79.8 79.9 79.2 79.2 79.4 C-2¹ 65.9 65.6 67.3 67.3 69.1 69.1 66.2 C-3¹ 74.9 74.8 74.5 74.4 70.5 70.6 75.0 C-4¹ 68.7 68.5 68.2 68.1 73.3 73.4 70.5 C-5¹ 94.6 94.4 94.1 94.0 92.9 93.0 95.7 C-I" 166.5 166.0 166.2 166.2 166.0 166.1 166.5 C-2" 116.9 116.7 116.6 116.6 116.8 116.9 117.1 C-3" 157.7 157.8 158.0 157.9 157.0 156.9 157.2 CH₃-4" 20.2 20.1 20.1 2Cl 20.3 20.3 20.0 CH₃-5" 27.3 27.2 27.2 27.2 27.3 27.4 27.2 C-I¹" 168.4 168.0 168.2 168.1 - - - 1 CH₃-2¹" 20.6 20.4 20.4 20.3 - - - - "Data are based on DEPT, HSQC, and HMBC experiments. 'Signals may be interchangeable. Another embodiment of the invention comprises a method of treating tumors or cancer with compounds having structure I. The Alvaradoin compounds E through N were tested in a human oral epidermoid carcinoma (KB) cell line using established protocols. (See, E. K.; Kim, N. C; Ward, M. C; Wall, M. E.; Navarro, H. A.; Burgess, J. P.; Kawanishi, K.; Kardono, L. B. S.; Riswan, S.; Rose, W. C; Fairchild, C. R.; Farnsworth, N. R.; Kinghorn, A. D. Cytotoxic prenylated xanthones and the unusual compounds anthraquinoben). The results are summarized in Table 4 below.

Table 4. Cytotoxic Activity of Isolates from A. haitiensis against the KB Cell Line

Compound KB^σ Alvaradoin E 0.050 ± 0.019

Alvaradoin F 0.065 ± 0.026 Alvaradoin G 0.652 ± 0.152

Alvaradoin H 1.074 ± 0.430 Alvaradoin I 12.500 ± 3.030 Alvaradoin J 15.909 ± 3.409

Alvaradoin K 0.272 ± 0.072

Alvaradoin L 0.591 ± 0.104

Alvaradoin M 0.383 ± 0.043 Alvaradoin N 2.935 ± 1.298

CPT^δ 0.0036 ± 0.0029

^αHuman oral epidermoid carcinoma. Results are expressed as EC₅₀ values as μM. Mean ± SEM determined from three separate experiments. ^έCPT denotes camptothecin control (μM ) as typical average value.

Alvaradoin E and F were evaluated in an in vivo test system using the murine P-388 lymphocytic leukemia model, administered i.p./i.p., as described previously. (Rose, W. C; Schurig, J. E.; Meeker, J. B. Correlation of in vitro cytotoxicity with preclinical in vivo antitumor activity. Anticancer Res. 1988, 8, 355-367). For this, a positive response is noted when the treated animals live 25% longer than the untreated controls (T/C = 125%). Indeed, compound 1 displayed a T/C of 125% when both the tumor cells and the compound were injected at the intraperitoneal (i.p.) site. However, compound 2 displayed a T/C of 100%, indicating that it is not active in vivo. In vitro studies conducted on Alvaradoin E and Alvaradoin F are summarized in Table 5. Both compounds demonstrated significant inhibition of KB cell growth (Table 1). These compounds also exhibited significant inhibitory activity against a panel of in vitro cancer cell lines, human lung carcinoma (LuI), human colon carcinoma (Col2), hormone-dependent human prostate carcinoma (LNCaP) and telomerase-immortalized (hTERT-RPEl). Against all four cell lines, 1 and 2 were equipotent, and this suggests that the confirmation at position 10 is not essential for anticancer activity in vitro (however, 1 is more potent than 2 in vivo; discussed later). Against the KB cells, both compounds were approximately an order of magnitude less active than the positive control, camptothecin.

Table 5. Summary of in vitro data¹

IC_5O values (concentration required to inhibit cell growth by 50%) in μM. ²EC₅o values (μM) for assay performed in the Natural Products Laboratory at Research Triangle Institute. ³IiTERT-RPE 1 is a human retinal pigment epithelial (RPE) cell line that expresses human telomerase reverse transcriptase (hTERT). ⁴CPT denotes camptothecin control as typical average value. Data represents two independent experiments, with each concentration tested in triplicate.

In vitro/in vivo studies conducted on Alvaradoin E and Alvaradoin F are summarized in Table 6. The hollow fiber studies are a preliminary in vitro/in vivo assessment of cancer chemotherapeutic efficacy. For this, discrete cell lines are distributed in hollow fibers that are inserted into an animal. The animal is treated with test compound, and after several days of treatment, the fibers are removed and examined for cell survival. The results are shown in Table 6 against three different cell lines, KB, LNCaP and Col2. To summarize, both compounds display growth inhibition of tumor cells in this in vitro/in vivo assay. Table 6. Percent growth inhibition of in vivo hollow fiber model

KB Col2 LNCaP

Alvaradoin E (1) 40.0-58.1 31.2-73.8 60.3-79.3

Alvaradoin F (2) 31.9-57.5 21.0-71.6 59.3-72.4

PBS² 0 0 0

Results are expressed as a range of percent inhibition at the following concentrations 0.195 mg/kg, 0.39 mg/kg, 0.78 mg/kg, and 1.56 mg/kg. ²PBS denotes phosphate buffered-saline control.

Several additional experiments have been completed including DAPI

(diamidinophenolindole) assay, mitochondrial membrane potential, annexin V-FITC and

TUNEL assays. These indicate a unique mechanism of action that results in arrest of the cell

cycle in the G2 phase. Also, induction of apoptosis has been observed in the LNCaP cell line

(Table 7).

Table 7. Average percentage of apoptotic LNCaP cells¹ after drug treatment

DAPI assay performed at University of Illinois at Chicago. ²Results are expressed as % values. Data represents the mean of the two independent experiments with each concentration tested in triplicate. The invention also embodies pharmaceutical composition containing compounds having structure I. The Alvaradoin E-N compounds and mixtures thereof are administered in a dose which is effective to inhibit the growth of tumors. The compounds of the present invention may be administered as a pharmaceutical composition containing the Alvaradoin E-N compounds and a pharmaceutically acceptable carrier or diluent. The active materials can also be mixed with other active materials which do no impair the desired action and/or supplement the desired action. The active material according to the present invention can be administered by any route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form. For the purposes of parenteral therapeutic administration, the active ingredient may be incorporated into a solution or suspension. The solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Another mode of administration of the compounds of this invention is oral. Oral compositions will generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the aforesaid compounds may be incorporated with excipients and used in the form of tablets, gelatine capsules, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Compositions may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents. Tablets containing the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil. The tablets, pills, capsules, troches and the like may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added. When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically or veterinarially pure and non-toxic in the amounts used. Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylethyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono oleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame, saccharin, or sucralose. Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water may be formulated from the active ingredients in admixture with a dispersing, suspending and/or wetting agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. The pharmaceutical composition of the invention may also be in the form of oil in water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono oleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent. The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables. Sterilization may be performed by conventional methods known to those of ordinary skill in the art such as by aseptic filtration, irradiation or terminal sterilization (e.g. autoclaving). Aqueous formulations (i.e., oil in water emulsions, syrups, elixers and injectable preparations) may be formulated to achieve the pH of optimum stability. The determination of the optimum pH may be performed by conventional methods known to those of ordinary skill in the art. Suitable buffers may also be used to maintain the pH of the formulation. The compounds of this invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable nonirritating excipient which is solid at ordinary temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to release the drug. Non limiting examples of such materials are cocoa butter and polyethylene glycols. They may also be administered by intranasal, intraocular, intravaginal, and intrarectal routes including suppositories, insufflation, powders and aerosol formulations. The compounds of the present invention may also be administered in the form of liposome or microvesicle preparations. Liposomes are microvesicles which encapsulate a liquid within lipid or polymeric membranes. Liposomes and methods of preparing liposomes are known and are described, for example, in U.S. 4,452,747, U.S. 4,448,765, U.S. 4,837,028, U.S. 4,721,612, U.S. 4,594,241, U.S. 4,302,459 and U.S. 4,186,183. The disclosures of these U.S. patents are incorporated herein by reference. Suitable liposome preparations for use in the present invention are also described in WO-9318749-A1, J-02056431-A and EP-276783- A.

Claims

WE CLAIM:

Claim 1. An anthracenone G-glycoside compound having the structure (I):

wherein R₁ is -H- or -OH R₂ is -OH or -OCOCHC(CH₃)₂ R₃ is -OH or -OCOCHC(CH₃)₂, and R₄ is -OH or -OAc.

Claim 2. The compound as claimed in Claim 1, wherein R₁ is -H, R₂ and R₃ are -OH and R₄ is -OAc.

Claim 3. The compound as claimed in Claim 1, wherein R₁ is -H, R₂ is -OCOCHC(CH₃)₂, R₃ is -OH and R₄ is OAc.

Claim 4. The compound as claimed in Claim 1, wherein R₁ and R₃ are -OH, R₂ is - OCOCHC(CH₃)₂ and R₄ is -OAc.

Claim 5. The compound as claimed in Claim I₃ wherein R₁ is -H, R₂ and R₄ are - OH and R₃ is -OCOCHC(CH3)₂.

Claim 6. The compound as claimed in Claim 1, wherein R₁ is — H, R₂ and R₄ are — OH and R₂ is -OCOCHC(CH₃)₂.

Claim 7. The compound as claimed in Claim 1, wherein R₁, R₂ and R₃ are -OH and R₄ is -OAc.

Claim 8. The compound as claimed in Claim 2, wherein the compound has the S configuration at C-IO, the R configuration at C-IO, a racemic mixture of the two configurations or a mixture of each configuration in any proportion.

Claim 9. The compound as claimed in Claim 3, wherein the compound has the S configuration at C-IO, the R configuration at C-IO, a racemic mixture of the two configurations or a mixture of each configuration in any proportion.

Claim 10. The compound as claimed in Claim 4, wherein the compound has the S configuration at C-IO, the R configuration at C-IO, a racemic mixture of the two configurations or a mixture of each configuration in any proportion.

Claim 11. The compound as claimed in Claim 5, wherein the compound has the S configuration at C-IO, the R configuration at C-IO, a racemic mixture of the two configurations or a mixture of each configuration in any proportion.

Claim 12. The compound as claimed in Claim 6, wherein the compound has the S configuration at C-10.

Claim 13. The compound as claimed in Claim 7, wherein the compound has the S configuration at C-10.

Claim 14. A composition comprising the compound as claimed in Claim 1, wherein the composition is obtained from the leaves oϊAlvardoa Haiβensis.

Claim 15. A pharmaceutical composition comprising, the compound as claimed in Claim 1 and a pharmaceutically acceptable carrier and/or diluents.

Claim 16. A method of treating cancer comprising, administering the pharmaceutical composition as claimed in Claim 15 to an animal.

Claim 17. A method for inhibiting the growth of tumors comprising, administering the compound as claimed in Claim 1 to a tumor.

Claim 18. A method for inhibiting the growth of tumors comprising, administering the pharmaceutical composition as claimed in Claim 15 to a tumor.

Claim 19. The method as claimed in Claim 16 wherein the animal is human.