Cytotoxic C-Methylated Chalcones from Syzygium samarangense.

Abstract

The flavonoids 2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), 2′,4′-dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4) and 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethyldihydrochalcone (5), isolated from Syzygium samarangense. (Blume) Merr. & L.M. Perry (Myrtaceae), were subjected to cytotoxicity testing using the dimethylthiazoldiphenyl tetrazolium (MTT) assay. The cell lines used were the Chinese hamster ovarian (CHO-AA8) and the human mammary adenocarcinoma, (MCF-7 and SKBR-3). Among the test compounds, 2 exhibited significant differential cytotoxicity against the MCF-7 cell line with an IC₅₀ of 0.0015 ± 0.0001 nM. It was also cytotoxic against the SKBR-3 cell line with an IC₅₀ of 0.0128 ± 0.0006 nM. Doxorubicin, the positive control, had an IC₅₀ of 2.60 ± 0.28 × 10⁻⁴ nM against the MCF-7 cell line and an IC₅₀ of 2.76 ± 0.52 × 10⁻⁵ nM against the SKBR-3 cell line. When tested in a mechanism-based yeast bioassay for detecting DNA-damaging agents using genetically engineered Saccharomyces cerevisiae. RS322Y (RAD52) mutant strain and (LF15/11) (RAD+) wild-type strain, 2 showed significant selective cytotoxicity against the RAD52 yeast mutant strain. It had an IC₁₂ of 0.1482 nM, as compared with the positive control, streptonigrin, which had an IC₁₂ of 0.0134 nM. Hence, 2 is a cytotoxic natural product with potential anticancer application.

Keywords:

• CHO-AA8
• cytotoxicity
• 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone
• DNA damaging agent
• MCF-7
• SKBR-3
• Syzygium samarangense

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Erratum

Introduction

Second to lung cancer, breast cancer is reported to be the leading cause of cancer deaths and is the most common cancer in women today (McPherson et al., 2000). In the Philippines, the breast is reported to be the second leading site of cancer for both sexes occurring more frequently in females and rarely in males. The incidence of breast cancer in the Philippines is observed to be the highest when compared with other Asian countries (Parkin et al., 2002).

Syzygium samarangense. (Blume) Merr. & L.M. Perry (Myrtaceae) has been reported to have immunostimulant (Srivastava et al., 1995) and antibacterial (Santos, 1981) activities. A number of compounds have been isolated from this plant (Nonaka et al., 1992; Srivastava et al., 1995; Amor et al., 20042005) that have been reported to have antidiabetic (Nonaka et al., 1992; Magno, et al., 2005), prolyl endopeptidase inhibitory (Amor et al., 2004), and spasmolytic (Amor et al., 2005) activities.

This study focused on the cytotoxic activity of the isolated flavonoids using the dimethylthiazoldiphenyl tetrazolium (MTT) assay against the CHO-AA8, MCF-7, and SKBR-3 cell lines. In addition, 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2) (Fig. 1) was tested in a mechanism-based yeast bioassay, which is based on the differential response of DNA repair-deficient (RAD52) and repair-proficient (RAD +) yeast strains to the test sample (Gunatilaka et al., 1992, 1994).

Figure 1 Chemical structures of 2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), 2′,4′-dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4) and 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethyldihydrochalcone (5).

Materials and Methods

Plant material

Leaves of S. samarangense. were sampled from Parañaque, Metro Manila. This was authenticated, and a voucher specimen with accession no. 14258 was submitted to the Dr. Jose Vera Santos Herbarium, Institute of Biology, University of the Philippines, in Diliman.

Extraction and isolation

The ground leaves of S. samarangense. (2.9 kg) were extracted at room temperature with methanol and subsequently partitioned between H₂O:hexane (1:6 v/v), from which the hexane extract was obtained and concentrated (140 g oily residue) in vacuo.. This was subjected to fractionation by normal phase column chromatography (NPCC) on silica gel (1:10 m/m) employing gradient elution (10% increments) with hexane, dichloromethane-hexane, dichloromethane, methanol-dichloromethane, and finally with methanol, giving fractions 1–4.

Compound 3 (75 mg) was crystallized out of fraction 1, eluted with hexane and 10% dichloromethane-hexane. Compound 3 was subsequently purified by recrystallization in 5% dichloromethane-hexane.

Sequential NPCC of fraction 2, eluted with 20% to 50% dichloromethane-hexane on silica gel employing gradient elution (10% increments) with hexane and dichloromethane-hexane yielded 2 (3 g).

Sequential NPCC of fraction 3, eluted with 60% dichloromethane-hexane to 30% methanol-dichloromethane on silica gel employing gradient elution (10% increments) with hexane, dichloromethane-hexane, dichloromethane, and methanol-dichloromethane yielded 1 (100 mg) and 4 (3 mg).

Hydrogenation of compound 2

Hydrogenation (Laswell & Hufford, 1977) was carried out on a Parr hydrogenation apparatus model 3911 (shaker type hydrogenator; Parr Instrument Company, Moline, Illinois, USA) with 66CA 50 mL reaction bottle. Analytical grade diethyl ether was used as solvent. Sample weighing 30 mg was placed in the reaction vessel together with 30 mL of solvent and 20 mg of catalyst, 10% palladium in carbon. Pressure was maintained at 30 psi and reaction time was 2 h. The mixture in the reaction bottle was filtered and the filtrate concentrated in vacuo.. The crude product was chromatographed on silica gel and purified through a dropper column using isocratic elution with dichloromethane. Hydrogenation of 2 yielded 5 (9 mg).

2′-Hydroxy-4′,6′-dimethoxy-5′-methylchalcone (1)

M.p. 145°C. UVλ_max (MeOH) = 344.0 nm; λ_max (AlCl₃, HCl) = 374.6 nm; λ_max (NaOMe) = 339.6 nm; λ_max (NaOAc, H₃BO₃) = 349.8 nm. FT-IR (KBr): ν_max = 3130, 2941, 2860, 1625, 1563, 1428, 1332, 1223, 1142, 980, 872, 791, 749, 706 cm⁻¹. EIMS (70 eV, rel. int.%): C₁₈H₁₈O₄ HR-MS 298.11989 (calcd. 298.120500, 75.31), 281.0 (9.39), 270.0 (13.49), 221.1 (100), 195.1 (42.69), 179.1 (15.47), 165.0 (15.87), 136.0 (35.29), 103.1 (47.11), 91.0 (18.46), 77.0 (60.09), 51.0 (48.51).

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2)

M.p. 125–126°C. UV λ_max (MeOH) = 335.8 nm; λ_max (AlCl₃, HCl) = 368.8 nm; λ_max (NaOMe) = 409.8 nm; λ_max (NaOAc, H₃BO₃) = 421.4 nm. FT-IR (KBr): ν_max = 3335, 2945, 2860, 1629, 1548, 1424, 1359, 1312, 1231, 1169, 1115, 985, 911, 818, 760, 691 cm⁻¹. EIMS (70 eV, rel. int.%): C₁₈H₁₈O₄ 298.1 (100), 221.0 (93.12), 194.0 (80.67), 166.1 (24.11), 136.0 (20.47), 103.1 (33.04), 83.0 (49.84), 77.0 (21.92), 69.1 (14.18).

2′,4′-Dihydroxy-6′-methoxy-3′-methylchalcone (3)

M.p. 198–203°C. UV λ_max (MeOH) = 346.0 nm; λ_max (AlCl₃, HCl) = 372.2 nm; λ_max (NaOMe) = 389.8 nm; λ_max (NaOAc, H₃BO₃) = 370.2 nm. FT-IR (KBr): ν_max = 3142, 2933, 2725, 1625, 1536, 1447, 1339, 1231, 1150, 1119, 976, 864, 795, 760, 699 cm⁻¹. EIMS (70 eV, rel. int.%): C₁₇H₁₆O₄ 284.1 (100), 267.1 (41.15), 256.1 (37.33), 207.1 (99.1), 181.1 (62.20), 165.0 (42.87), 151.1 (37.39), 122.0 (55.53), 103.1 (51.14), 77.0 (47.71).

2′,4′-Dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4)

M.p. 171–173°C. UV λ_max (MeOH) = 292.4 nm; λ_max (AlCl₃, HCl) = 313.8 nm; λ_max (NaOMe) = 330.0 nm; λ_max (NaOAc, H₃BO₃) = 295.4 nm. FT-IR (KBr): ν_max = 3331, 2956, 2875, 1648, 1613, 1571, 1428, 1305, 1204, 1119, 838, 787, 695 cm⁻¹. EIMS (70 eV, rel. int.%): C₁₇H₂₀O₄ 286.1 (71.38), 269.1 (6.84), 181.1 (100), 154.1 (54.11), 138.0 (6.97), 104.1 (5.42), 91.0 (22.51), 77.0 (6.75).

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyldihydrochalcone (5)

M.p. 65–68°C. UV λ_max (MeOH) = 281.2 nm; λ_max (AlCl₃, HCl) = 305.0 nm; λ_max (NaOMe) = 337.4 nm; λ_max (NaOAc, H₃BO₃) = 340.0 nm. FT-IR (KBr): ν_max = 3323, 2945, 2864, 1606, 1567, 1420, 1355, 1308, 1223, 1146, 1108, 992, 826, 753, 702 cm⁻¹. EIMS (70 eV, rel. int.%): C₁₈H₂₀O₄ 300.1 (64.16), 283.2 (8.68), 269.2 (32.68), 195.1 (100), 168.1 (59.01), 152.1 (33.38), 91.1 (55.87), 83.0 (25.30), 77.0 (21.08).

Table 1 summarizes the ¹H NMR data of compounds 1–5 and Table 2 summarizes the ¹³C NMR data of compounds 1–4.

Table 1.. ¹H NMR data given in ppm of compounds 1–5.

	Compounds
C	1^a.	2^a.	3^b.	4^c.	5^a.
1	—	—	—	—	—
2	7.60 (2H, m)	7.63 (2H, m)	7.71 (2H, m)		7.16–7.31 (5H, m)
6
3	7.40 (3H, m)	7.41 (3H, m)	7.44 (3H, m)	∼7.26 (5H, m)
4
5
β.	7.76 (1H, d, J. = 15.6 Hz)	7.84 (1H, d, J. = 15.6 Hz)	7.74 (1H, d, J. = 15.6 Hz)	3.00 (2H, t, J. = 7.8 Hz)	3.02 (2H, t, J. = 7.75 Hz)
α.	7.89 (1H, d, J. = 15.6 Hz)	8.00 (1H, d, J. = 15.6 Hz)	8.02 (1H, d, J. = 15.6 Hz)	3.32 (2H, t, J. = 7.8 Hz)	3.40 (2H, t, J. = 7.75 Hz)
C=O	—	—	—	—	—
1′	—	—	—	—	—
2′	—	—	—	—	—
3′	—	—	—	—	—
4′	—	—	—	—	—
5′	5.99 (1H, s)	—	6.15 (1H, s)	5.92 (1H, s)	—
6′	—	—	—	—	—
3′-CH3	2.04 (3H, s)	2.15 (3H, s)	2.00 (3H, s)	2.05 (3H, s)	2.09 (3H, s)
5′-CH3	—	2.17 (3H, s)	—	—	2.11 (3H, s)
6′-OCH3	3.90 (3H,s)	3.66 (3H, s)	3.92 (3H, s)	3.82 (3H, s)	3.66 (3H, s)
2′-OH	No signal	No signal	14.51(1H, s)	No signal	13.45
4′-OH	—	No signal	No signal	No signal	No signal
4′-OCH3	3.95 (3H, s)	—	—	—	—

^a.In CDCl₃ at 300 MHz.

^b.In C₃D₆O at 500 MHz.

^c.In 30% CD₃OD-CDCl₃ at 500 MHz.

Table 2.. ¹³C NMR given in ppm and DEPT data of compounds 1–4.

	Compounds
C	1^a.		2^a.		3^b.		4^c.
	^13C	DEPT	^13C	DEPT	^13C	DEPT	^13C	DEPT
1	135.6	C	135.3	C	136.4	C	141.8	C
2	128.3	CH	128.9	CH	129.0	CH	128.3	CH
3	128.8	CH	128.4	CH	129.7	CH	128.3	CH
4	128.0	CH	130.2	CH	130.7	CH	125.8	CH
5	128.8	CH	128.4	CH	129.7	CH	128.3	CH
6	128.3	CH	128.9	CH	129.0	CH	128.3	CH
β.	141.9	CH	142.9	CH	142.3	CH	31.0	CH2
α.	129.9	CH	126.7	CH	128.6	CH	45.7	CH2
C=O	193.0	C	193.4	C	193.1	C	204.5	C
1′	106.3	C	106.6	C	104.4	C	104.9	C
2′	164.2	C	162.0	C	166.4	C	164.9	C
3′	106.1	C	109.0	C	106.0	C	104.4	C
4′	163.6	C	159.3	C	161.8	C	161.9	C
5′	86.3	CH	109.0	C	91.5	CH	90.2	CH
6′	161.1	C	158.8	C	163.3	C	160.9	C
3′-CH3	7.3	CH3	8.2	CH3	7.36	CH3	7.00	CH3
5′-CH3	—	—	7.6	CH3	—	—	—	—
6′-OCH3	55.8	CH3	62.3	CH3	56.1	CH3	55.2	CH3
4′-OCH3	55.6	CH3	—	—	—	—	—	—

^a.In CDCl₃ at 400 MHz.

^b.In C₃D₆O at 500 MHz.

^c.In 30% CD₃OD-CDCl₃ at 500 MHz.

DEPT = Distortionless Enhancement by Polarization Transfer.

Culture of mammalian cell lines

CHO-AA8 (Chinese hamster ovarian cells) and human breast cancer cells (MCF-7 and SKBR-3), were obtained from the American Type Culture Collection (Rockville, MD, USA). The CHO-AA8 cells were grown as a monolayer in alpha-minimum essential medium (α-MEM), MCF-7 cells in minimum essential medium (MEM), and SKBR-3 in McCoy's 5a medium containing 10% fetal bovine serum (FBS) and 1X of antibiotic-antimycotic penicillin-streptomycin and fungizone (PSF). Cell cultures were maintained at 37°C in a humidified 5% CO₂ atmosphere.

In vitro. tumor cytotoxicity assay

Cytotoxicity toward CHO-AA8, MCF-7, and SKBR-3 cells was assessed in a MTT-microtiter plate tetrazolium cytotoxicity assay. This assay was originally described by Mossmann (1983) and has since been modified by others (Denizot & Lang, 1986; Buttke et al., 1993). Media were initially removed from culture flasks of CHO-AA8, MCF-7, and SKBR-3 cells. The CHO-AA8, MCF-7 and SKBR-3 cells adhering to the culture flasks were washed twice by phosphate-buffered saline (PBS), pH 7.4. Five milliliters of 0.5X trypsin-EDTA was added to the culture flask to facilitate the harvesting of the adherent cells. The flask was incubated at 37°C in a humidified 5% CO₂ atmosphere. After 10 min incubation with trypsin, the harvested cells were centrifuged at 3000 rpm for 3 min. The number of cells per milliliter was determined using a hemocytometer. Cells were seeded (20,000 cells/well) in 200 µL of growth medium in 96-well microtiter plates (Costar) and allowed to attach for 24 h. Cells were treated with the samples at 50, 5, and 0.5 µg/mL. The samples were tested in quadruplicate and were solubilized in 100% DMSO with a final DMSO concentration of 1.0% or less in each well. The treated cells were incubated for 72 h. The media was removed after 72 h and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H.-tetrazolium bromide (MTT) solution (15 µL, 5 mg/mL in PBS, pH 7.4) was added followed by a 3-h incubation. Viable cells reduce MTT to a purple formazan product. After removal of media, the formazan product was solubilized by addition of DMSO (100 µL) to each well. The absorbance for each well at 570 nm was measured using an SLT Model Tecan Spectra III ELISA plate reader. The cell growth in the DMSO control wells was used to determine the zero inhibition growth level for each experiment. Doxorubicin was used as the positive control.

All reagents were from Gibco Laboratories (Grand Island, NY, USA) except for PBS, which was from Sigma Chemical Co. (St. Louis, MO, USA).

Analysis of MTT result

Average absorbance for each set of quadruplicate drug-treated wells was compared with the average absorbance of the control wells (containing DMSO, which is the solvent of the sample) to determine the fraction of growth inhibition (fractional survival) at a particular drug dose. Fractional survival (fs) was determined using the following formula (Norberg-King, 1993):

Mechanism-based yeast bioassay

Two types of genetically engineered yeast, Saccharomyces cerevisiae. (provided by Mr. L. Faucette of Smithkline Beecham Pharmaceuticals), RS322Y (RAD52) mutant strain and LF15 (RAD+) wild-type strain, were palted on YPD agar (7 mm layer) in 9 × 9 cm plates (wells are 6 mm in diameter, 9 wells in a plate) (Gunatilaka et al., 19921994). Various concentrations of 100 µL samples in DMSO-MeOH (1:1) were placed in each well. Bioassays were done in triplicate. Plates were read after incubation of 48 h at 30°C. Any agent that is cytotoxic (>12-mm inhibition) to the mutant strain and not cytotoxic (<12-mm inhibition) to wild type is considered a potential DNA damaging agent. Streptonigrin was the positive control used.

Results and Discussion

The flavonoids isolated from the hexane extract of Syzygium. samarangense. belong to the chalcone and dihydrochalcone subclass and were identified from spectral data (Tables 1 and 2) as 2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), and 2′,4′-dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4) (Fig. 1). Compound 5 (Fig. 1) was synthesized by reduction of the C_α.–C_β. double bond of 2.

Compound 1 was previously isolated from Didymocarpus aurentiaca. (Adityachaudhury et al., 1976) and its ¹³C NMR data obtained in CDCl₃ at 300 MHz summarized in carbon-13 NMR of flavonoids (Agrawal, ed., 1989). Srivastava et al. (1995) reported the isolation of compound 2 from the same plant and obtained its ¹³C NMR data in CDCl₃ at 100 MHz. Compound 3 was previously isolated from Sterculia urens. (Anjaneyulu & Raju, 1984). They obtained the ¹H NMR data in 30% CD₃OD-CDCl₃ but ¹³C NMR data were not available. Compound 4 was previously isolated from Myrica gale. (Mathiesen et al., 1997). Comparison of spectral data available for these compounds shows a difference and the most likely reason for this are the differences in experimental conditions.

Compound 2 was the major compound isolated from the hexane extract of S. samarangense.. Previously, the same compound was tested against six other cell lines, namely, SMMC-7721, 8898, HeLa, SPC-A-1, 95-D, and GBC-SD, and was found to be active against SMMC-7721 (Ye et al., 2004). However, this is the first report of the cytotoxic activity of compound 2 against MCF-7 and SKBR-3 cell lines as well as the cytotoxic activity of the other structurally related chalcones and dihydrochalcones. Compound 2 is also reported to have leishmanicidal and trypanocidal properties (Salem & Werbovetz, 2005).

Fractional survival and IC₅₀ values of the tested compounds are given in Table 3. Test compounds with fractional survival values of less than 0.4 are considered cytotoxic, between 0.4 to 0.6 are slightly cytotoxic, and greater than 0.6 are non-cytotoxic. Fractional survival values are used to determine the differential cytotoxicity and activity index of a sample. Differential cytotoxicity is expressed as the ratio of the IC₅₀ of the normal cell line (CHO-AA8) over the cancer cell lines (MCF-7 and SKBR-3). If the ratio is greater than 1, then the compound is more toxic to cancer cells compared with normal cells, and vice versa if the ratio is less than 1. The activity index, which is the ratio of the IC₅₀ of the standard drug, doxorubicin, over the IC₅₀ of the test compound, is determined to test for the efficacy of the test sample. If the activity index is greater than 1, then it indicates that the compound is more toxic than doxorubicin for a specific cell line.

Table 3.. Fractional survival and IC₅₀ values of compounds 1–5 against CHO-AA8, MCF-7, and SKBR-3 cell lines.

	CHO-AA8		MCF-7		SKBR-3
Test compounds	Fractional survival^a.	IC50 (nM)	Fractional survival^a.	IC50 (nM)	Fractional survival^a.	IC50 (nM)
1^b.	0.575 ± 0.002	0.0208 ± 0.0001	0.227 ± 0.027	0.0192 ± 0.0023	0.394 ± 0.045	0.0121 ± 0.0003
2^b.	0.101 ± 0.002	0.0801 ± 0.0082	0.052 ± 0.001	0.0015 ± 0.0001	0.310 ± 0.003	0.0128 ± 0.0006
3^b.	0.456 ± 0.011	Not done	0.488 ± 0.013	Not done	0.335 ± 0.080	Not done
4^c.	Not active		0.195 ± 0.023	Not done	Not active
5^c.	0.033 ± 0.001	0.0130 ± 0.0017	0.054 ± 0.002	0.0093 ± 0.0008	0.075 ± 0.008	2.213 ± 0.209
Doxorubicin	—	2.88 × 10^−4 ± 0.24 × 10^−4	—	2.60 ± 0.28 × 10^−4	—	2.76 ± 0.52 × 10^−5

^a.n = 4.

^b.Active dose of 50 µg/mL.

^c.Active dose of 5 µg/mL.

Based on fractional survival values (Table 3), 2 and 5 are cytotoxic against normal and breast cancer cells at concentrations of 0.1678 nM and 0.0167 nM, respectively. Compounds 1 and 3 showed slight toxicity against normal and breast cancer cells at a concentration of 0.1678 nM, while 4 is cytotoxic at 0.0175 nM. However, 1, 3, 4, and 5 did not exhibit a significant differential toxicity against the breast cancer cell lines. Compound 2 appears to be the most promising because it exhibited significant differential cytotoxicity toward the MCF-7 breast cancer cells. Based on its activity index, its cytotoxicity is almost one third that of the positive control, doxorubicin.

Compound 2 was subjected to a mechanism-based yeast bioassay utilizing Saccharmoyces cerevisiae. yeast mutant strain (RAD52) deficient in DNA damage repair and recombination pathways and a wild-type strain (RAD +) that is not deficient in DNA repair pathways. Compound 2 exhibited selective cytotoxicity toward the RAD52 mutant strain with an IC₁₂ of 0.0134 nM compared with streptonigrin, the positive control, which has an IC₁₂ of 0.1482 nM. This suggests that the mechanism of cytotoxic activity of 2 lies in its ability to damage DNA that is incapable of repair through recombination pathways.

The chalcones and dihydrochalcone isolated are rare C-methylated compounds, which are less than 5% of the total number of reported naturally occurring chalcones and dihydrochalcones (The Dictionary of Natural Products., 2004). Among the chalcones (1, 2 and 3), the presence of C-5′-CH₃ in 2 appears to be important for cytotoxic activity. Hydrogenation of 2 to yield 5 (Fig. 1), a dihydrochalcone, resulted in a decrease in activity. This suggests that the C_α.–C_β. double bond is essential for activity. It is noteworthy that the most active dihydrochalcone, 5, also possesses a C-5′-CH₃. The other dihydrochalcone, 4, is less cytotoxic than its chalcone counterpart, 1, which is cytotoxic against the two cancer cell lines (MCF-7 and SKBR-3). Thus, dihydrochalcones were consistently less active in terms of differential cytotoxicity and activity index than the corresponding chalcones. The presence of a C-5′-CH₃in 5 may be the reason why it is more cytotoxic than 4.

This study establishes the potential of compound 2 as an antitumor agent, specifically against human mammary adenocarcinoma MCF-7. Also, it is a potential anticancer agent whose mechanism of action may be through inhibition of topoisomerases, which are involved in repairing damaged DNA in recombination pathways.

Acknowledgments

E.C.A. thanks Ms. Hanshella Magno for the hexane extract, UNESCO for the travel grant to Pakistan, and the Department of Science & Technology-Education, Science & Engineering Program (DOST-ESEP) and the Commission on Higher Education (CHED) for the scholarship and research funding.