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

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

by Manu Sharmaa), Pritam Dev Sharma*a), Mohinder Pal Bansalb), and Jaswant Singhc)

a) University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India(phone: þ91-172-2534117; fax: þ91-172-2541141; e-mail: [email protected])

b) Department of Biophysics, Panjab University, Chandigarh-160014, Indiac) Division of Pharmacology, Regional Research Laboratory, Jammu-180004, India

Five new derivatives of the pentacyclic triterpenoid lantadene A (¼22b-angeloyloxy-3-oxoolean-12-en-28-oic acid; 1) from the leaves ofLantana camaraL. were synthesized, characterized, and screened fortheir cytotoxicities against four human cancer cell lines. The three most-potent compounds, i.e., 1, 4, and6, with IC50 values in the range of ca. 20–29 mm, were further studied for their in vivo tumor-inhibitorypotential upon oral administration in two-stage squamous cell carcinogenesis, using female Swiss albinomice, papilloma being induced by 7,12-dimethylbenz[a]anthracene (DMBA), and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). The results are discussed in terms of structure–activityrelationship.

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 hugeamount of biomass, and, currently, there is much interest to exploit its natural productsin drug research [2]. Pentacyclic triterpenoids are known to have antibacterial, anti-inflammatory, antitumor, and anti-AIDS activities, and their unique structuralvariability is responsible for their interesting biological properties [3] [4]. Thetriterpenoids of L. camara L. have attracted considerable interest, mainly because oftheir cytotoxicity. Most of the triterpenoids isolated from this species are pentacyclic,belong to the oleanane series, and are named as lantadenes.

Lantadene A (¼22b-angeloyloxy-3-oxoolean-12-en-28-oic acid; 1)1) is the mostabundant pentacyclic triterpenoid (0.7% content on a dry-weight basis) in Lantanacamara var. aculeate (red variety) [5]. Compound 1 has been reported to inhibit theactivation of the Epstein–Barr virus in Raje cells induced by 12-O-tetradecanoylphor-bol-13 acetate (TPA), as well as to act as a tumor inhibitor in a two-stagecarcinogenesis mouse model [6] [7]. There is constant search for new biologicallyactive agents, particularly from plants, that could be used as chemotherapeuticanticancer drugs [8].

Lantadene A (1) is a pentacyclic triterpenoid derived biosynthetically by cyclizationof squalene. It is a potential antitumor compound, and its different functional groupsare responsible for its interesting biological and pharmacological activities. Quitesurprisingly, no systematic studies on the structure–activity relationship (SAR) of 1have been reported by chemical variation. The chemopreventive effect of the leaf

CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)932

F 2007 Verlag Helvetica Chimica Acta AG, ZIrich

1) Angeloyl (Ang)¼ (2Z)-2-methylbut-2-enoyl.

extract of L. camara L. towards skin carcinogenesis in Swiss albino mice has beenreported in one of our earlier studies [9]. In continuation of this research, we hereinreport five new derivatives of lantadene A, compounds 2–6, with minor functionalchanges introduced at positions 17 and 22, to evaluate the cytotoxicities and tumor-inhibitory profiles of these congeners.

Results and Discussion. – 1. Chemistry. For the synthesis of 2–6, the parentcompound 1 was isolated from the leaves of L. camara var. aculeate, and thenchemically 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 thecorresponding free acid 2 in 45% yield. Then, the COOH group of 2 in position 17 wasesterified 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 byreacting 1 with oxalyl chloride. The latter was then quenched with ammonia to providethe corresponding amide 5 in 65% yield. Finally, dehydration of 5 by exposure tothionyl chloride gave the desired nitrile 6 in 42% yield. The structures of all thesecompounds were unambiguously confirmed spectroscopically and by elementalanalysis (see Exper. Part).

2. Pharmacology. Compound 1 and its derivatives 2–6 were studied for theircytotoxicities against several human cancer cell lines (HL-60, HeLa, Colon 502713,

CHEMISTRY & BIODIVERSITY – Vol. 4 (2007) 933

Scheme

Lung A-549), as determined by colorimetric assay (see Exper. Part). As can be seenfrom the Table, the Me ester 4 exhibited a slightly higher cytotoxicity than the parentcompound 1 (P<0.05). In contrast, the amide 5 and the nitrile 6 showed moderatecytotoxicities, 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 2was esterified to 3, a better cytotoxicity profile was obtained (P<0.01). Finally,the cytotoxicity of the nitrile 6was more similar to that of the amide 5 than to that of theparent acid 1.

Based on the above in vitro cytotoxicity results, the ester 4 and the nitrile 6 wereselected for the determination of their tumor-inhibitory potential against two-stagecarcinogenesis in Swiss albino mice, as induced by topical application of 7,12-dimethylbenz[a]anthracene (DMBA), and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). The onset of papilloma formation (36.3%) was observed in the 5thweek in the DMBA/TPA-treated mice. There was gradual rise in the incidence ofcancer, reaching 100% during the 8th week. When the methyl ester 4 (50 mg/kg bodyweight) was administered orally, the occurrence of DMBA/TPA-induced papillomaswas delayed by four weeks, in contrast to only three weeks for the parent compound 1or the nitrile 6.

As evident from Fig. 1, compound 4 showed a significant decrease in the incidenceof cancer (13.6%; P<0.001) after 20 weeks, compared with the DMBA/TPA-treatedcontrol 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) wassignificantly lower (37.5%) in the DMBA/TPA-treated control group lacking drugadministration, as compared to the vehicle-treated group. The absolute survival rateswere 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 thatof the acetone-treated mice (negative control) throughout the study. However, therewas a slight increase, at the end of the experiment, in the average body weight of themice to which 1, 4, or 6 had been orally administered (Fig. 3). The maximal tolerateddose of these drugs was 50 mg/kg body weight, administered twice a week, starting oneweek before initiation, followed by treatment over 20 weeks thereafter.

Table. Cytotoxicities of Compounds 1–6 against Different Human Cancer Cell Lines. The averagedvalues (n¼3) are expressed in terms of IC50 (in mg/ml). For details, see Exper. Part.

Drug HL-60 HeLa Colon 502713 Lung A-549

1 19.8�0.10a) 23.3�0.08b) 21.3�0.05b) 21.8�0.10a)2 >100 >100 >100 >1003 70.6�0.05a) >100 72.4�0.05 84.2�0.054 19.3�0.14a) 21.5�0.05a) 21.1�0.08b) 19.2�0.05a)6 37.6�0.12b) 39.5�0.05b) 42.0�0.08a) 44.2�0.10b)7 27.3�0.14a) 26.4�0.10a) 28.1�0.10a) 28.6�0.08b)Paclitaxelc) <2 <2 <2 <2

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


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


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).

of papilloma formation is most likely due to drug pretreatment. This allows the mice tointerfere with the initiation of carcinogenesis, which is a relatively rapid process, as wellas to better respond upon continuous treatment to TPA promotion, which is a slowprocess. The increased survival rate would then be associated with the low papillomaburden as a result of carcinogenesis inhibition. Finally, the observed slight gain in bodyweight upon drug treatment could be a result of faster recovery from the effect ofDMBA/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 inactivity. The a,b-unsaturated C¼O group of the Ang moiety is strongly electrophilic,and seems to play an important role in binding to the receptor site. This wouldrationalize the strongly reduced activity of the corresponding acid 2. Methylation of the17-COOH group, in turn, resulted in higher activities, as observed for 3 and 4 comparedto 2 and 1, respectively. This effect may be attributed to the increased lipophilicity andbetter bioavailability of the esters. Further, the nitrile 6 and amide 5 were less cytotoxicthan 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 Meester group at C(17), giving rise to a better papilloma-inhibition profile than the parentcompound 1. In summary, compound 4, with two ester functions, calls for furthermodifications to be developed into an antitumor lead compound.

We thank the Indian Council of Medical Research, New Delhi, for financial assistance and for aSenior Research Fellowship (to M. S.).


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

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 I2vapor. Melting points (m.p.): B/chi apparatus; uncorrected. IR Spectra: Perkin-Elmer-1710 spectrom-eter; in cm�1. 1H- and 13C-NMR Spectra: Bruker AC-300 apparatus, at 300/75 MHz, resp.; d in ppm, J inHz. MS:Micromass 70-VSE apparatus; inm/z (rel. %). Elemental analysis agreed within �0.04% of thecalc. 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 theHerbarium 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 byfiltration through muslin cloth, and decolorized with activated charcoal (20 g), which afforded a golden-yellow 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 solventwas removed under reduced pressure, and the residual solid was recrystallized from MeOH to afford apre-purified lantadene fraction (1.06 g, 1.06%) in colorless, crystalline form. Part of this fraction (1 g) waspurified by CC (30 g SiO2, 60–120 mesh; CHCl3, then CHCl3/MeOH 99.5 :0.5). The fractions enriched in1 were pooled, the solvent was removed in vacuo, and the resulting solid residue was recrystallized twicefrom MeOH to obtain pure 1 (0.45 g). Colorless crystals. M.p. 285–2888. The spectroscopic data of 1were 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) ethanolicKOH soln. (170 ml) was heated at reflux for 6 h. The solvent was removed in vacuo, and the residue wasdiluted with H2O (15 ml). The mixture was acidified to pH 1 to 2 with conc. aq. HCl, and extracted withEt2O (3�15 ml). The combined org. layers were washed with 4% (w/v) aq. Na2CO3 soln. (3�15 ml) andH2O (15 ml), dried (Na2SO4), and concentrated under reduced pressure. The resulting residue wasrecrystallized from MeOH/H2O to afford 2 (0.45 g, 45%). Colorless crystals. M.p. 2348. IR (KBr): 3590and 3480 (COOH), 1720 (C¼O). 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 ¼ 3.7, H�C(18)); 3.50 (s, OH); 3.91 (t, J ¼ 3, H�C(22)); 4.80(br., 22-OH); 5.35 (t, J ¼ 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 (Mþ ), 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) wasadded in excess an etheral soln. of diazomethane3), and the mixture was kept overnight. The excessdiazomethane 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 toafford 3 (0.12 g, 80%). Colorless crystals. M.p. 179–1808. IR (KBr): 3595 (COOH), 1718 (C¼O).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 ¼ 3.7, H�C(18)); 3.59 (s, MeOOC); 3.7 (t, J¼3, H�C(22)); 4.80 (br., 22-OH); 5.28 (t, J¼3.5,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 (Mþ ), 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) Diagnostic signals only.3) Warning: hazardous and explosive reagent!

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 C¼O), 1715 (keto C¼O), 1677 (ester C¼O). 1H-NMR(CDCl3)2): 5.98 (dd, J¼7.1, 1.2, H�C(3’)); 5.38 (t, J¼3.5, H�C(12)); 4.97 (t, J¼3, H�C(22)); 3.54 (s,MeOOC); 3.07 (dd, J¼14.2, 4.0, H�C(18)); 1.96 (dd, J¼7.1, 1.6, Me(4’)); 1.76 (d, J¼1.6, 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 (Mþ ), 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, H9.60; 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.tovernight. The solvent was removed under reduced pressure, and the residue was co-evaporated withCHCl3 (3�15 ml) to afford 7 (0.12 g, 90%). Crystalline solid. M.p. 1068. IR (KBr): 2951 (CH), 1807(COCl), 1738 (ester C¼O), 1714 (keto C¼O), 778 (C�Cl). 1H-NMR (CDCl3)2): 5.55 (dd, J¼7.1, 1.2,H�C(3’)); 5.38 (t, J¼3.5, H�C(12)); 5.07 (t, J¼3, H�C(22)); 3.04 (dd, J¼14.2, 4.0, H�C(18)); 1.96(dd, J¼7.1, 1.6, Me(4’)); 1.76 (d, J¼1.6, 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 wasevaporated under reduced pressure, and the resulting solid was repeatedly recrystallized from aq. MeOHto afford 5 (0.13 g, 65%). Amorphous powder. M.p. 133–1358. IR (KBr): 3495 (NH), 2951 (CH), 1745(ester C¼O), 1714 (keto C¼O), 1699 (CONH2). 1H-NMR (CDCl3)2): 6.27 (br. s, NH2); 5.55 (dd, J¼7.1,1.2, H�C(3’)); 5.37 (t, J¼3.5, H�C(12)); 5.04 (t, J¼3, H�C(22)); 3.06 (dd, J¼14.2, 4.0, H�C(18)); 1.96(dd, J¼7.1, 1.6, Me(4’)); 1.76 (d, J¼1.6, 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 (Mþ ), 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 heatedat reflux for 2.5 h. The solvent was distilled off, and the residue was co-evaporated twice with CH2Cl2 toremove traces of SOCl2. The resulting semi-solid residue was purified by CC (SiO2; CHCl3, then CHCl3/MeOH 99.7 :0.3), followed by recrystallization fromMeOH, to afford 6 (0.07 g, 42%). M.p. 186–1888. IR(KBr): 2949 (CH), 2261 (CN), 1737 (ester C¼O), 1714 (keto C¼O). 1H-NMR (CDCl3)2): 5.55 (dd, J¼7.1, 1.2, H�C(3’)); 5.37 (t, J¼3.5, H�C(12)); 5.03 (t, J¼3, H�C(22)); 3.06 (dd, J¼14.2, 4.0, H�C(18));1.96 (dd, J¼7.1, 1.6, Me(4’)); 1.76 (d, J¼1.6, 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)); 15.62 (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));


166.26 (C(1’)); 217.63 (C(3)). MS: 533.3 (Mþ ), 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 RPMImedium supplemented with 10% fetalbovine serum, 100 mg/ml streptomycin, and 100 IU/ml penicillin. The cells (2�104 cells/0.1 ml) wereincubated at 378 for 48 h in a humidified atmosphere containing 5% CO2 in the presence or absence ofserially diluted drug, using 96-well culture plates (Costar, USA). During the last 4 h of incubation, MTT(¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) soln. was added. The resultingformazan 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 (n¼3) in at least oneexperiment.

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 performedaccording to the guidelines set by theWorld Health Organization (WHO), Geneva, Switzerland, and theIndian National Science Academy (INSA), NewDelhi, India. Depilatory creamwas used to remove hairsfrom the back of the mice. The animals were left for 2 d, and then divided into eight groups. Animals ingroup I (n¼10) were treated with 100 ml vehicle (acetone). The acetone was topically applied on thedepilated back of each mouse, twice weekly over 20 weeks. The animals of group II (n¼15) weretopically treated with 7,12-dimethylbenz[a]anthracene (DMBA; 100 nmol/100 ml acetone) on thedepilated back of each mouse for two weeks, and cancer was promoted twice weekly by application of12-O-tetradecanoylphorbol-13 acetate (TPA; 1.7 nmol/100 ml acetone) for the next 18 weeks. Theanimals of group III (n¼10) were orally treated with 1 suspended in H2O/carboxymethyl cellulose at adose of 50 mg/kg body weight. Similarly, compounds 4 and 6 were tested in groups IVand V, resp. (n¼10each), and the animals were orally treated at the same dose (50 mg/kg body weight). Treatment startedone week before DMBA application, and continued over 20 weeks thereafter. Body weight, incidence ofskin papillomas, and total number of surviving animals after the 20-week treatment were recorded atweekly intervals. Only those papillomas that persisted for two weeks or more were taken intoconsideration for final evaluation of the data. The data were analyzed by means of StudentPs t-test and thec2 test. A statistical value of P<0.05 was considered significant.

REFERENCES

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Received January 1, 2007


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