932

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

Synthesis, Cytotoxicity, and Antitumor Activity of Lantadene-A Congeners

by Manu Sharma a ), Pritam Dev Sharma* a ), Mohinder Pal Bansal b ), and Jaswant Singh c )
a

) University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India (phone: 91-172-2534117; fax: 91-172-2541141; e-mail: pritamdevsharma@homail.com) b ) Department of Biophysics, Panjab University, Chandigarh-160014, India c ) Division of Pharmacology, Regional Research Laboratory, Jammu-180004, India

Five new derivatives of the pentacyclic triterpenoid lantadene A ( 22b-angeloyloxy-3-oxoolean-12en-28-oic acid; 1) from the leaves of Lantana camara L. were synthesized, characterized, and screened for their cytotoxicities against four human cancer cell lines. The three most-potent compounds, i.e., 1, 4, and 6, with IC50 values in the range of ca. 20 29 mm, were further studied for their in vivo tumor-inhibitory potential upon oral administration in two-stage squamous cell carcinogenesis, using female Swiss albino mice, papilloma being induced by 7,12-dimethylbenz[a ]anthracene (DMBA), and promoted by 12-Otetradecanoylphorbol-13-acetate (TPA). The results are discussed in terms of structure activity relationship.

Introduction. Lantana camara L. is one of the most-noxious weeds of the world, growing wild in tropical and subtropical regions [1]. Its wild growth provides a huge amount of biomass, and, currently, there is much interest to exploit its natural products in drug research [2]. Pentacyclic triterpenoids are known to have antibacterial, antiinflammatory, antitumor, and anti-AIDS activities, and their unique structural variability is responsible for their interesting biological properties [3] [4]. The triterpenoids of L. camara L. have attracted considerable interest, mainly because of their cytotoxicity. Most of the triterpenoids isolated from this species are pentacyclic, belong to the oleanane series, and are named as lantadenes. Lantadene A ( 22b-angeloyloxy-3-oxoolean-12-en-28-oic acid; 1) 1) is the most abundant pentacyclic triterpenoid (0.7% content on a dry-weight basis) in Lantana camara var. aculeate (red variety) [5]. Compound 1 has been reported to inhibit the activation of the Epstein Barr virus in Raje cells induced by 12-O-tetradecanoylphorbol-13 acetate (TPA), as well as to act as a tumor inhibitor in a two-stage carcinogenesis mouse model [6] [7]. There is constant search for new biologically active agents, particularly from plants, that could be used as chemotherapeutic anticancer drugs [8]. Lantadene A (1) is a pentacyclic triterpenoid derived biosynthetically by cyclization of squalene. It is a potential antitumor compound, and its different functional groups are responsible for its interesting biological and pharmacological activities. Quite surprisingly, no systematic studies on the structure activity relationship (SAR) of 1 have been reported by chemical variation. The chemopreventive effect of the leaf
1)

Angeloyl (Ang) (2Z)-2-methylbut-2-enoyl.

 2007 Verlag Helvetica Chimica Acta AG, Zrich

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

933

extract of L. camara L. towards skin carcinogenesis in Swiss albino mice has been reported in one of our earlier studies [9]. In continuation of this research, we herein report five new derivatives of lantadene A, compounds 2 6, with minor functional changes introduced at positions 17 and 22, to evaluate the cytotoxicities and tumorinhibitory profiles of these congeners.

Results and Discussion. 1. Chemistry. For the synthesis of 2 6, the parent compound 1 was isolated from the leaves of L. camara var. aculeate, and then chemically transformed by a series of standard procedures, as outlined in the Scheme. First, the angeloyl (Ang) group of 1 was cleaved off by basic hydrolysis to afford the corresponding free acid 2 in 45% yield. Then, the COOH group of 2 in position 17 was esterified with ethereal diazomethane to furnish the corresponding Me ester 3 in 80% yield. Similarly, 1 was treated with ethereal diazomethane to afford the ester 4 in 76% yield. To access compounds 5 and 6, the acid chloride 7 was prepared quantitatively by reacting 1 with oxalyl chloride. The latter was then quenched with ammonia to provide the corresponding amide 5 in 65% yield. Finally, dehydration of 5 by exposure to thionyl chloride gave the desired nitrile 6 in 42% yield. The structures of all these compounds were unambiguously confirmed spectroscopically and by elemental analysis (see Exper. Part ). 2. Pharmacology. Compound 1 and its derivatives 2 6 were studied for their cytotoxicities against several human cancer cell lines (HL-60, HeLa, Colon 502713,
Scheme

934

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

Lung A-549), as determined by colorimetric assay (see Exper. Part ). As can be seen from the Table, the Me ester 4 exhibited a slightly higher cytotoxicity than the parent compound 1 ( P < 0.05). In contrast, the amide 5 and the nitrile 6 showed moderate cytotoxicities, lower than that of 1. Removal of the Ang moiety by ester hydrolysis, affording the free acid 2, significantly decreased the cytotoxic activity ( IC50 > 100 mm ). When 2 was esterified to 3, a better cytotoxicity profile was obtained ( P < 0.01). Finally, the cytotoxicity of the nitrile 6 was more similar to that of the amide 5 than to that of the parent acid 1.
Table. Cytotoxicities of Compounds 1 6 against Different Human Cancer Cell Lines. The averaged values ( n 3) are expressed in terms of IC50 (in mg/ml). For details, see Exper. Part. Drug 1 2 3 4 6 7 Paclitaxel c )
a

HL-60 19.8 0.10 a ) > 100 70.6 0.05 a ) 19.3 0.14 a ) 37.6 0.12 b ) 27.3 0.14 a ) <2

HeLa 23.3 0.08 b ) > 100 > 100 21.5 0.05 a ) 39.5 0.05 b ) 26.4 0.10 a ) <2

Colon 502713 21.3 0.05 b ) > 100 72.4 0.05 21.1 0.08 b ) 42.0 0.08 a ) 28.1 0.10 a ) <2

Lung A-549 21.8 0.10 a ) > 100 84.2 0.05 19.2 0.05 a ) 44.2 0.10 b ) 28.6 0.08 b ) <2

) P < 0.05 rel. to control. b ) P < 0.01 rel. to control. c ) Positive control.

Based on the above in vitro cytotoxicity results, the ester 4 and the nitrile 6 were selected for the determination of their tumor-inhibitory potential against two-stage carcinogenesis in Swiss albino mice, as induced by topical application of 7,12dimethylbenz[a ]anthracene (DMBA), and promoted by 12-O-tetradecanoylphorbol13-acetate (TPA). The onset of papilloma formation (36.3%) was observed in the 5th week in the DMBA/TPA-treated mice. There was gradual rise in the incidence of cancer, reaching 100% during the 8th week. When the methyl ester 4 (50 mg/kg body weight) was administered orally, the occurrence of DMBA/TPA-induced papillomas was delayed by four weeks, in contrast to only three weeks for the parent compound 1 or the nitrile 6. As evident from Fig. 1, compound 4 showed a significant decrease in the incidence of cancer (13.6%; P < 0.001) after 20 weeks, compared with the DMBA/TPA-treated control group (100%). The overall papilloma incidence in 1 and 6 were 18.1% ( P < 0.001) and 24.9% ( P < 0.01), respectively. The survival rate of the mice ( Fig. 2 ) was significantly lower (37.5%) in the DMBA/TPA-treated control group lacking drug administration, as compared to the vehicle-treated group. The absolute survival rates were 87.5% for animals treated with 1 or 4, and 75% for those treated with 6. The average body weight of the DMBA/TPA-treated mice did not differ from that of the acetone-treated mice (negative control) throughout the study. However, there was a slight increase, at the end of the experiment, in the average body weight of the mice to which 1, 4, or 6 had been orally administered ( Fig. 3 ). The maximal tolerated dose of these drugs was 50 mg/kg body weight, administered twice a week, starting one week before initiation, followed by treatment over 20 weeks thereafter.

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

935

Fig. 1. Effect of compounds 1, 4, and 6 on skin-papilloma formation in mice. Single and double asterisks (*) refer to P < 0.001 and P < 0.01, resp., relative to control (DMBA/TPA treatment alone).

Fig. 2. Effect of compounds 1, 4, and 6 on the survival rate of the tested mice. Single and double asterisks (*) refer to P < 0.01 and P < 0.05, resp., relative to control (DMBA/TPA treatment alone).

In summary, administration of lantadene A (1) or of its congeners 4 or 6 showed a significant delay in the onset of papilloma formation and an overall reduction of papillomas in mice, a slight increase in the average body weight upon treatment, and a significantly higher survival rate in comparison to DMBA/TPA treatment alone, indicating the strong chemopreventive activity of these compounds. The retarded onset

936

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

Fig. 3. Effect of compounds 1, 4, and 6 on the average body weight of the tested mice. Single and double asterisks (*) refer to P < 0.01 and P < 0.05, resp., relative to control (DMBA/TPA treatment alone).

of papilloma formation is most likely due to drug pretreatment. This allows the mice to interfere with the initiation of carcinogenesis, which is a relatively rapid process, as well as to better respond upon continuous treatment to TPA promotion, which is a slow process. The increased survival rate would then be associated with the low papilloma burden as a result of carcinogenesis inhibition. Finally, the observed slight gain in body weight upon drug treatment could be a result of faster recovery from the effect of DMBA/TPA or, alternatively, better papilloma control. 3. Structure Activity Relationship. From the cytotoxicity profiles of compounds 1 6, it is evident that removal of the Ang moiety of 1 gives rise to a significant decrease in activity. The a,b-unsaturated C O group of the Ang moiety is strongly electrophilic, and seems to play an important role in binding to the receptor site. This would rationalize the strongly reduced activity of the corresponding acid 2. Methylation of the 17-COOH group, in turn, resulted in higher activities, as observed for 3 and 4 compared to 2 and 1, respectively. This effect may be attributed to the increased lipophilicity and better bioavailability of the esters. Further, the nitrile 6 and amide 5 were less cytotoxic than the parent acid 1, the nitrile being more active, though, than the amide. The two-stage in vivo carcinogenesis studies further reflect the importance of a Me ester group at C(17), giving rise to a better papilloma-inhibition profile than the parent compound 1. In summary, compound 4, with two ester functions, calls for further modifications to be developed into an antitumor lead compound.
We thank the Indian Council of Medical Research, New Delhi, for financial assistance and for a Senior Research Fellowship (to M. S. ).

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

937

Experimental Part General. Column chromatography (CC) was performed on silica gel (60 120 or 100 200 mesh). Thin-layer chromatography (TLC) was performed on silica-gel-G plates; visualization by exposure to I2 vapor. Melting points (m.p.): Bchi apparatus; uncorrected. IR Spectra: Perkin-Elmer-1710 spectrometer; in cm 1. 1H- and 13C-NMR Spectra: Bruker AC-300 apparatus, at 300/75 MHz, resp.; d in ppm, J in Hz. MS: Micromass 70-VSE apparatus; in m/z (rel. %). Elemental analysis agreed within 0.04% of the calc. values. Plant Material. The leaves of Lantana camara L. were collected in September 2004 from Palampur, Himachal Pradesh, India. A shade-dried voucher specimen (No. LC-097/UIPS) was deposited at the Herbarium of the Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India. Extraction and Isolation of Lantadene A (1). The shade-dried, powdered leaves of L. camara (100 g) were extracted with MeOH (500 ml) for 24 h, with intermittent shaking. The extract was separated by filtration through muslin cloth, and decolorized with activated charcoal (20 g), which afforded a goldenyellow soln. After solvent removal under reduced pressure, the residue (23 g) was suspended in MeOH/ H2O 1 : 7, and further extracted with CHCl3 (2 15 ml). The org. layer was dried over Na2SO4, the solvent was removed under reduced pressure, and the residual solid was recrystallized from MeOH to afford a pre-purified lantadene fraction (1.06 g, 1.06%) in colorless, crystalline form. Part of this fraction (1 g) was purified by CC (30 g SiO2 , 60 120 mesh; CHCl3 , then CHCl3/MeOH 99.5 : 0.5). The fractions enriched in 1 were pooled, the solvent was removed in vacuo, and the resulting solid residue was recrystallized twice from MeOH to obtain pure 1 (0.45 g). Colorless crystals. M.p. 285 2888. The spectroscopic data of 1 were found to be identical with those reported previously [10]. (22b )-22-Hydroxy-3-oxoolean-12-en-28-oic Acid (2 ). A soln. of 1 (1.00 g) in 10% (w/v ) ethanolic KOH soln. (170 ml) was heated at reflux for 6 h. The solvent was removed in vacuo, and the residue was diluted with H2O (15 ml). The mixture was acidified to pH 1 to 2 with conc. aq. HCl, and extracted with Et2O (3 15 ml). The combined org. layers were washed with 4% (w/v ) aq. Na2CO3 soln. (3 15 ml) and H2O (15 ml), dried (Na2SO4 ), and concentrated under reduced pressure. The resulting residue was recrystallized from MeOH/H2O to afford 2 (0.45 g, 45%). Colorless crystals. M.p. 2348. IR (KBr): 3590 and 3480 (COOH), 1720 (C O). 1H-NMR (CDCl3 ) 2 ): 0.85 (s, Me); 0.89 (s, Me); 1.04 (s, 2 Me); 1.09 (s, Me); 1.11 (s, Me); 1.15 (s, Me); 3.05 (dd, J 3.7, H C(18)); 3.50 (s, OH); 3.91 (t, J 3, H C(22)); 4.80 (br., 22-OH); 5.35 (t, J 3.4,H C(12)). 13C-NMR (CDCl3 ): 15.1 (C(25)); 16.7 (C(26)); 19.5 (C(6)); 21.5 (C(24)); 23.6 (C(16)); 24.4 (C(11)); 25.8 (C(27)); 26.5 (C(23)); 26.8 (C(30)); 27.7 (C(15)); 30.1 (C(20)); 32.3 (C(7)); 33.8 (C(29)); 34.2 (C(2)); 36.8 (C(10)); 38.1 (C(21)); 39.1 (C(18)); 39.2 (C(8)); 39.3 (C(1)); 42.0 (C(14)); 45.8 (C(19)); 46.9 (C(9)); 47.5 (C(4)); 52.2 (C(17)); 55.4 (C(5)); 76.6 (C(22)); 121.2 (C(12)); 143.3 (C(13)); 180.5 (C(28)); 217.6 (C(3)). MS: 470.3 ( M ), 452.3 (100), 409.7, 407.7, 248.1, 246.1, 203.1, 190.1, 189.1. Anal. calc. for C30H46O4 (470.68): C 76.55, H 9.85; found: C 76.55, H 9.84. Methyl (22b )-22-Hydroxy-3-oxoolean-12-en-28-oate ( 3 ). To compound 2 (0.15 g, 0.35 mmol) was added in excess an etheral soln. of diazomethane 3 ), and the mixture was kept overnight. The excess diazomethane was destroyed by adding a few drops of AcOH, until the yellow color had disappeared. The solvent was then removed under reduced pressure, and the residue was crystallized from MeOH to afford 3 (0.12 g, 80%). Colorless crystals. M.p. 179 1808. IR (KBr): 3595 (COOH), 1718 (C O). 1 H-NMR (CDCl3 )2 ): 0.85 (s, Me); 0.89 (s, Me); 1.04 (s, 2 Me); 1.09 (s, Me); 1.11 (s, Me); 1.15 (s, Me); 3.05 (dd, J 3.7, H C(18)); 3.59 (s, MeOOC); 3.7 (t, J 3, H C(22)); 4.80 (br., 22-OH); 5.28 (t, J 3.5, H C(12)). 13C NMR (CDCl3 ): 15.1 (C(25)); 16.7 (C(26)); 19.5 (C(6)); 21.5 (C(24)); 23.6 (C(16)); 24.3 (C(11)); 25.8 (C(27)); 26.5 (C(23)); 26.8 (C(30)); 27.7 (C(15)); 30.1 (C(20)); 32.1 (C(7)); 33.8 (C(29)); 34.2 (C(2)); 36.6 (C(10)); 38.1 (C(21)); 39.1 (C(18)); 39.2 (C(8)); 39.4 (C(1)); 42.0 (C(14)); 45.8 (C(19)); 46.9 (C(9)); 47.5 (C(4)); 51.1 (COOMe )); 52.1 (C(17)); 55.4 (C(5)); 76.6 (C(22)); 121.2 (C(12)); 143.3 (C(13)); 180.5 (C(28)); 217.4 (C(3)). MS: 484.3 ( M ), 466.3 (100), 409.7, 407.7, 248.1, 246.1, 203.1, 201.1, 190.1, 189.1, 133.0. Anal. calc. for C31H48O4 (484.71): C 76.82, H 9.98; found: C 76.82, H 9.99.
2) 3)

Diagnostic signals only. Warning: hazardous and explosive reagent!

938

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

Methyl 22-{[(2Z )-2-Methylbut-2-enoyl]oxy}-3-oxoolean-12-en-28-oate (4 ). Prepared from 1 (0.17 g, 0.35 mmol) in analogy to 3. Yield after crystallization from MeOH: 0.12 g, 76%. Colorless crystals. M.p. 140 1428. IR (KBr): 2960 (CH), 1738 (ester C O), 1715 (keto C O), 1677 (ester C O). 1H-NMR (CDCl3 )2 ): 5.98 (dd, J 7.1, 1.2, H C(3)); 5.38 (t, J 3.5, H C(12)); 4.97 (t, J 3, H C(22)); 3.54 (s, MeOOC); 3.07 (dd, J 14.2, 4.0, H C(18)); 1.96 (dd, J 7.1, 1.6, Me(4)); 1.76 (d, J 1.6, 2-Me); 1.18, 1.05, 1.04, 1.02, 1.00, 0.90, 0.86 (7 Me). 13C-NMR (CDCl3 ): 15.09 (C(25)); 15.64 (2-Me); 16.83 (C(26)); 19.59 (C(6)); 20.56 C(4); 21.45 (C(24)); 23.50 (C(11)); 24.18 (C(16)); 25.79 (C(27)); 26.13 (C(30)); 26.44 (C(23)); 27.57 (C(15)); 30.03 (C(20)); 32.17 (C(7)); 33.67 (C(29)); 34.11 (C(2)); 36.76 (C(10)); 37.71 (C(21)); 38.41 (C(1)); 38.41 (C(18)); 39.21 (C(8)); 41.97 (C(14)); 46.87 (C(19)); 47.42 (C(4)); 47.73 (C(9)); 50.60 (C(17)); 51.3 ( MeOOC)); 55.29 (C(5)); 75.88 (C(22)); 122.46 (C(12)); 127.61 (C(2)); 138.88 (C(3)); 143.10 (C(13)); 166.26 (C(1)); 180.10 (C(28)); 217.66 (C(3)). MS: 566.4 ( M ), 466.4 (100), 407.4, 262.1, 247.1, 233.1, 203.1, 198.1, 133.0, 83.0, 55.0. Anal. calc. for C36H54O5 (566.81): C 76.28, H 9.60; found: C 76.28, H 9.63. 28-Chloro-3,28-dioxoolean-12-en-22-yl (2Z )-2-Methylbut-2-enoate ( 7 ). Compound 1 (0.14 g, 0.27 mmol) was treated with redistilled oxalyl chloride (4 ml) in anh. CH2Cl2 (25 ml), and stirred at r.t overnight. The solvent was removed under reduced pressure, and the residue was co-evaporated with CHCl3 (3 15 ml) to afford 7 (0.12 g, 90%). Crystalline solid. M.p. 1068. IR (KBr): 2951 (CH), 1807 (COCl), 1738 (ester C O), 1714 (keto C O), 778 (C Cl). 1H-NMR (CDCl3 )2 ): 5.55 (dd, J 7.1, 1.2, H C(3)); 5.38 (t, J 3.5, H C(12)); 5.07 (t, J 3, H C(22)); 3.04 (dd, J 14.2, 4.0, H C(18)); 1.96 (dd, J 7.1, 1.6, Me(4)); 1.76 (d, J 1.6, 2-Me); 1.18, 1.05, 1.04, 1.02, 1.00, 0.90, 0.86 (7 Me). 13C NMR (CDCl3 ): 15.09 (C(25)); 15.64 (2-Me); 16.83 (C(26)); 19.59 (C(6)); 20.56 (C(4)); 21.45 (C(24)); 23.50 (C(11)); 24.18 (C(16)); 25.79 (C(27)); 26.13 (C(30)); 26.44 (C(23)); 27.57 (C(15)); 30.03 (C(20)); 32.17 (C(7)); 33.67 (C(29)); 34.11 (C(2)); 36.76 (C(10)); 37.71 (C(21)); 38.41 (C(1)); 38.41 (C(18)); 39.21 (C(8)); 41.97 (C(14)); 46.87 (C(19)); 47.42 (C(4)); 47.73 (C(9)); 50.60 (C(17)); 55.29 (C(5)); 75.88 (C(22)); 122.46 (C(12)); 127.61 (C(2)); 138.88 (C(3)); 143.10 (C(13)); 166.26 (C(1)); 179.30 (C(28)); 217.66 (C(3)). 28-Amino-3,28-dioxoolean-12-en-22-yl (2Z )-2-Methylbut-2-enoate ( 5 ). The acid chloride 7 (0.20 g, 0.41 mmol) was dissolved in NH3-sat. MeOH (20 ml) and kept at r.t. overnight. The mixture was evaporated under reduced pressure, and the resulting solid was repeatedly recrystallized from aq. MeOH to afford 5 (0.13 g, 65%). Amorphous powder. M.p. 133 1358. IR (KBr): 3495 (NH), 2951 (CH), 1745 (ester C O), 1714 (keto C O), 1699 (CONH2 ). 1H-NMR (CDCl3 )2 ): 6.27 (br. s, NH2 ); 5.55 (dd, J 7.1, 1.2, H C(3)); 5.37 (t, J 3.5, H C(12)); 5.04 (t, J 3, H C(22)); 3.06 (dd, J 14.2, 4.0, H C(18)); 1.96 (dd, J 7.1, 1.6, Me(4)); 1.76 (d, J 1.6, 2-Me); 1.18, 1.05, 1.04, 1.02, 1.00, 0.90, 0.86 (7 Me). 13C-NMR (CDCl3 ): 15.05 (C(25)); 15.62 (2-Me); 16.83 (C(26)); 19.59 (C(6)); 20.54 (C(4)); 21.45 (C(24)); 23.50 (C(11)); 24.18 (C(16)); 25.74 (C(27)); 26.13 (C(30)); 26.44 (C(23)); 27.57 (C(15)); 30.03 (C(20)); 32.17 (C(7)); 33.67 (C(29)); 34.09 (C(2)); 36.76 (C(10)); 37.71 (C(21)); 38.37 (C(1)); 38.39s (C(18)); 39.21 (C(8)); 41.97 (C(14)); 46.87 (C(19)); 47.46 (C(4)); 47.73 (C(9)); 50.60 (C(17)); 55.29 (C(5)); 75.88 (C(22)); 122.46 (C(12)); 127.61 (C(2)); 138.88 (C(3)); 143.10 (C(13)); 166.26 (C(1)); 184.40 (C(28)); 217.63 (C(3)). MS: 551.4 ( M ), 451.4 (100), 407.4, 247.1, 245.1, 233.1, 218.1, 203.1, 183.1, 133.0, 83.0, 55.0. Anal. calc. for C35H53NO4 : C 76.18, H 9.68, N 2.54; found: C 76.18, H 9.70, N 2.54. 28-Nitrilo-3,28-dioxoolean-12-en-22-yl (2Z )-2-Methylbut-2-enoate (6 ). A soln. of the amide 5 (0.17 g, 0.35 mmol) in CH2Cl2 (2.75 ml) was treated with redist. SOCl2 (0.38 mmol), and the mixture was heated at reflux for 2.5 h. The solvent was distilled off, and the residue was co-evaporated twice with CH2Cl2 to remove traces of SOCl2 . The resulting semi-solid residue was purified by CC (SiO2 ; CHCl3 , then CHCl3/ MeOH 99.7 : 0.3), followed by recrystallization from MeOH, to afford 6 (0.07 g, 42%). M.p. 186 1888. IR (KBr): 2949 (CH), 2261 (CN), 1737 (ester C O), 1714 (keto C O). 1H-NMR (CDCl3 )2 ): 5.55 (dd, J 7.1, 1.2, H C(3)); 5.37 (t, J 3.5, H C(12)); 5.03 (t, J 3, H C(22)); 3.06 (dd, J 14.2, 4.0, H C(18)); 1.96 (dd, J 7.1, 1.6, Me(4)); 1.76 (d, J 1.6, 2-Me); 1.18, 1.05, 1.04, 1.02, 1.00, 0.90, 0.86 (7 Me). 13 C-NMR (CDCl3 ): 15.09 (C(25)); 15.62 (2-Me); 16.83 (C(26)); 19.59 (C(6)); 20.54 (C(4)); 21.45 (C(24)); 23.50 (C(11)); 24.18 (C(16)); 25.79 (C(27)); 26.13 (C(30)); 26.44 (C(23)); 27.57 (C(15)); 30.03 (C(20)); 32.17 (C(7)); 33.67 (C(29)); 34.09 (C(2)); 36.76 (C(10)); 37.71 (C(21)); 38.39 (C(1)); 38.41 (C(18)); 39.21 (C(8)); 41.97 (C(14)); 46.87 (C(19)); 47.42 (C(4)); 47.73 (C(9)); 50.60 (C(17)); 55.29 (C(5)); 75.88 (C(22)); 122.46 (C(12)); 122.72 (C(28)); 127.61 (C(2)); 138.88 (C(3)); 143.10 (C(13));

CHEMISTRY & BIODIVERSITY Vol. 4 (2007)

939

166.26 (C(1)); 217.63 (C(3)). MS: 533.3 ( M ), 433.3 (100), 407.3, 229.1, 214.1, 203.1, 199.1, 133.0, 83.0, 55.0. Anal. calc. for C35H51NO3 (533.78): C 78.75, H 9.63, N 2.62; found: C 78.75, H 9.67, N, 2.62. Colorimetric Cytotoxicity Assay. The four cell lines (HL-60, HeLa, Colon 502713, and Lung A-549) were obtained from NCCS, Pune, India, and maintained in RPMI medium supplemented with 10% fetal bovine serum, 100 mg/ml streptomycin, and 100 IU/ml penicillin. The cells (2 104 cells/0.1 ml) were incubated at 378 for 48 h in a humidified atmosphere containing 5% CO2 in the presence or absence of serially diluted drug, using 96-well culture plates (Costar, USA). During the last 4 h of incubation, MTT ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) soln. was added. The resulting formazan product was extracted with DMSO, and the UV/VIS absorbance was measured at 540 nm [11]. The data represent mean values (including standard deviations) of triplicate assays ( n 3) in at least one experiment. In vivo Antitumor Activity. Squamous cell carcinogenesis was induced in female Swiss albino mice (LACCA) according to a well-established method [12]. Animal care and handling was strictly performed according to the guidelines set by the World Health Organization ( WHO ), Geneva, Switzerland, and the Indian National Science Academy ( INSA ), New Delhi, India. Depilatory cream was used to remove hairs from the back of the mice. The animals were left for 2 d, and then divided into eight groups. Animals in group I ( n 10) were treated with 100 ml vehicle (acetone). The acetone was topically applied on the depilated back of each mouse, twice weekly over 20 weeks. The animals of group II ( n 15) were topically treated with 7,12-dimethylbenz[a ]anthracene (DMBA; 100 nmol/100 ml acetone) on the depilated back of each mouse for two weeks, and cancer was promoted twice weekly by application of 12-O-tetradecanoylphorbol-13 acetate (TPA; 1.7 nmol/100 ml acetone) for the next 18 weeks. The animals of group III ( n 10) were orally treated with 1 suspended in H2O/carboxymethyl cellulose at a dose of 50 mg/kg body weight. Similarly, compounds 4 and 6 were tested in groups IV and V, resp. ( n 10 each), and the animals were orally treated at the same dose (50 mg/kg body weight). Treatment started one week before DMBA application, and continued over 20 weeks thereafter. Body weight, incidence of skin papillomas, and total number of surviving animals after the 20-week treatment were recorded at weekly intervals. Only those papillomas that persisted for two weeks or more were taken into consideration for final evaluation of the data. The data were analyzed by means of Students t-test and the c2 test. A statistical value of P < 0.05 was considered significant.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] O. P. Sharma, J. Vaid, P. D. Sharma, J. Chem. Ecol. 1989, 17, 2283. E. L. Ghisalberti, Fitoterapia 2000, 71, 467. J. Liu, J. Ethnopharmacol. 1995, 49, 57. K. Sakurawi, F. Yasuda, T. Tozyo, M. Nakamura, T. Sato, J. Kikuchi, Y. Terui, T. Iwata, K. Takahashi, T. Konoike, S. Mihara, M. Fujimoto, Chem. Pharm. Bull. 1996, 44, 343. M. Sharma, P. D. Sharma, Chem. Nat. Comp. 2006, 42, 442. A. Inada, T. Nakanishi, H. Tokuda, H. Nishino, A. Iwashina, O. P. Sharma, Planta Med. 1995, 61, 558. A. Inada, T. Nakanishi, H. Tokuda, H. Nishino, A. Iwashina, O. P. Sharma, Planta Med. 1997, 63, 272. S. F. Guyer, F. Afaq, H. Mukhtar, Photodermatol. Photoimmunol. Photomed. 2003, 19, 56. M. Sharma, P. D. Sharma, M. P. Bansal, Pharm. Biol. 2006, in press. P. J. Beeby, Aust. J. Chem. 1978, 31, 1313. W. K. Liu, J. C. K. Ho, G. W. Qin, C. T. Che, Biochem. Pharmacol. 2002, 63, 955. M. A. Azuine, S. V. Bhide, Nutr. Cancer 1992, 17, 77. Received January 1, 2007