A Lanostane Triterpenoid and Three Cholestane Sterols from Tilia kiusiana

Abstract

Kiusianins A–D (1–4) were isolated from the leaves of a Japanese endemic plant, Tilia kiusiana, together with 14 known compounds. The structures of a new lanostane-type triterpenoid 1 and three new cholestane-type sterols 2–4 were elucidated by spectroscopic methods, including two dimensional (2D) NMR. All the compounds isolated were evaluated for their cytotoxicity against two human cancer cell lines, HeLa and HL-60.

Tilia kiusiana is an endemic Japanese plant species found on the islands of Honshu, Shikoku, and Kyushu in Japan. For our screening program to discover new biologically active compounds from Japanese trees, we have constructed an extracts library having more than one hundred species. All the samples in the library were tested for activity against murine leukemia P388 cells. We investigated the constituents of the leaves of the Japanese tree, Tilia kiusiana MAKINO et. SHIRAS (Tiliaceae), because its methanol extract showed cytotoxic activity against P388 cells at 30 µg/mL. This pharmacological property of T. kiusuana led us to investigate the components of Tilia kiusiana, resulting in the isolation of four new compounds 1–4 named kiusianins A–D.

Herein, we describe the isolation and structure elucidation of kiusianins A–D (1–4) and the cytotoxic evaluation of all the isolated compounds against two human cancer cell lines, cervical cancer cells HeLa and promyelocytic leukemia cells HL-60.

The MeOH extract of the leaves of T. kiusiana was partitioned between n-hexane and 90% MeOH. The MeOH-soluble material was purified by octadecyl silica (ODS) column chromatography followed by silica gel column chromatography and reverse-phase HPLC to afford new triterpene 1 and three new sterols 2–4, together with 3β-hydroxy-cycloart-24-en-26-al (5),¹⁾ oleanolic acid (6),²⁾ arjunolic acid (7),³⁾ 2α,3β-dihydroxy-24-nor-4 (23),12-oleanadien-28-oic acid (8),⁴⁾ 20-hydroxy-cholest-4-en-3-one (9), guggulsterol III (10),^5,6) guggulsterol І (11),^5,7) (8R)-3β,6β-hydroxy-cholest-4-en-3-one (12),⁸⁾ 3β,5α-dihydroxy-cholest-7-ene-6-one (13),⁹⁾ (8R)-3β-8α-dihydroxy-polypoda-13E,17E,21-triene (14),¹⁰⁾ phytol (15),¹¹⁾ aphanamol II (16),¹²⁾ linarin (17),¹³⁾ and (3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one (18).¹⁴⁾

Compound 1 ([α]_D²⁰+22.5 (c 0.12; CHCl₃)) was formulated as C₃₀H₄₈O₂ based on high resolution-electron impact-mass spectra (HR-EI-MS) (m/z 440.3660, calcd: 440.3655). The IR spectrum showed the presence of a hydroxyl group, an aldehyde, and an olefin at 3415, 1690, and 1633 cm⁻¹, respectively. The structure of 1 was deduced from the detailed analysis of the ¹H- and ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT), and further aided by two dimensional (2D) NMR experiments (¹H–¹H correlation spectroscopy (COSY), ¹H-detected heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC)). The ¹H- and ¹³C-NMR and HMQC spectra (Tables 1, 2) of 1 indicated the presence of one aldehyde group (δ_H 9.40, s, H-26; δ_C 195.4, C-26), whose proton signal had an HMBC correlation with C-25 of one trisubstituted double bond attached to a methyl group [(δ_H 6.50, br t, J=7.6 Hz, H-24, δ_C 155.5, C-24; δ_C 139.2, C-25; δ_H 1.75, s, H₃-27, δ_C 9.2, C-27)], indicating the presence of a 6-methyl-2-en-1-al side chain. NMR analysis further identified the presence of one sp³ oxymethine (δ_C 79.3, C-3), one trisubsituted double bond [δ_H 5.27, dd, J=6.8 and 2.9 Hz, H-7, δ_C 118.1, C-7; δ_C 145.7, C-8], two sp³ methines (δ_C 50.7, C-5; δ_C 36.1, C-20), four sp³ quaternary carbons [δ_C 39.0 (C-4), 35.0 (C-10), 51.2 (C-13), 43.6 (C-14)], nine methylenes, and five methyl groups [δ_H 0.82, δ_C 21.9 (C-18); δ_H 0.75, δ_C 13.1 (C-19); δ_H 0.98, δ_C 27.3 (C-28), δ_H 0.97, δ_C 27.6 (C-29), δ_H 0.86, δ_C 14.7 (C-30)]. From the data provided, three of the seven degrees of unsaturation were accounted for; therefore, compound 1 must contain four rings.

Table 1. ¹H-NMR Data for Kiusianins A–D (1–4) in CDCl₃

Position	1	2	3	4
	δH mult. (J, Hz)	δH mult. (J, Hz)	δH mult. (J, Hz)	δH mult. (J, Hz)
1	1.70 m	2.03 m	1.85 m	1.86 m
	1.16 dt (13.0, 4.3)	1.70 m	1.36 m	1.36 m
2	1.66 m, 2H	2.41 m	1.85 m	1.84 m
		2.34 m	1.38 m	1.37 m
3	3.24 dd (11.4, 4.1)
4		5.73 s	2.22 m	2.22 m
			1.45 m	1.44 m
5	1.32 dd (12.0, 5.6)		2.25 m	2.26 m
6	2.15 m	2.39 m	3.63 m	3.62 m, 2H
	1.98 m	2.28 m
7	5.27 dd (6.8, 2.9)	1.86 m	5.73 t (2.0)	5.74 br t (2.2)
		1.05 m
8		1.52 m
9	2.20 m	0.96 m	2.16 m, 2H	2.19 m
11	1.53 m, 2H	1.57 m	1.79 m	1.82 m
		1.42 m	1.64 m	1.63 m
12	1.80 m	1.77 m	2.14 m	2.04 m
		1.22 m	1.39 m	1.92 m
14		1.13 m	2.05 m	2.15 m
15	1.64 m	1.72 m	1.62 m	1.69 m, 2H
	1.49 m	1.23 m	1.50 m
16	1.95 m	1.80 m	1.94 m	1.83 m, 2H
	1.27 m	1.72 m	1.33 m
17	1.50 m	2.27 m	1.31 m	2.26 m
18	0.82 s	0.58 s	0.61 s	0.50 s
19	0.75 s	1.18 s	0.87 s	0.87 s
20	1.50 m		1.37 m
21	0.93 d (6.0)	4.18 d (11.5)	0.94 d (6.4)	4.90 br s
		4.04 br d (11.5)		4.82 br s
22	1.25 m, 2H	5.42 t (7.3)	1.39 m	2.02 m
			1.02 m	1.98 m
23	2.40 m	2.16 q (7.5), 2H	1.34 m	1.46 m
	2.28 m		1.15 m	1.42 m
24	6.50 br t (7.6)	1.26 m, 2H	1.14 m, 2H	1.18 m, 2H
25		1.57 m	1.52 m	1.53 m
26	9.40 s	0.90 s	0.87 d (6.6)	0.89 d (6.6)
27	1.75 s	0.90 s	0.87 d (6.6)	0.87 d (6.6)
28	0.98 s
29	0.97 s
30	0.86 s

Table 2. ¹³C-NMR Data for Kiusianins A–D (1–4) in CDCl₃

Position	1	2	3	4
	δC	δC	δC	δC
1	37.2	35.7	36.9	36.8
2	27.7	34.0	30.5	30.4
3	79.3	199.5	199.7	199.7
4	39.0	123.8	30.3	30.3
5	50.7	171.4	53.3	53.3
6	24.0	32.9	70.8	70.7
7	118.1	31.9	123.1	123.0
8	145.7	36.0	164.0	163.6
9	49.0	53.9	50.1	50.1
10	35.0	38.7	38.2	38.2
11	18.1	20.9	21.9	21.9
12	33.8	38.0	38.9	37.3
13	51.2	43.4	44.5	45.1
14	43.6	55.4	55.7	55.3
15	34.0	24.1	22.6	22.5
16	28.3	25.5	27.7	25.2
17	52.9	55.0	56.3	55.9
18	21.9	12.8	12.4	13.2
19	13.1	17.4	13.3	13.2
20	36.1	137.5	36.0	148.1
21	18.2	61.2	18.8	110.1
22	34.7	131.4	36.0	37.8
23	26.1	25.8	23.8	26.2
24	155.5	39.6	39.4	38.8
25	139.2	27.7	28.0	27.9
26	195.4	22.5	22.6	22.7
27	9.2	22.5	22.8	22.7
28	27.3
29	27.6
30	14.7

The ¹H–¹H COSY spectrum revealed the presence of four segments; C-1–C-3, C-5–C-7, C-9–C-12, and C-15–C-24 (bold lines in Fig. 2). The HMBC correlations (Fig. 1) from the remaining five methyl signals (H₃-18, H₃-19, H₃-28, H₃-29, and H₃-30) to the corresponding quaternary carbons and carbons of each segments (Fig. 2) means that 1 is lanostane-type triterpenoid having the same C-24–C-27 side chain as that in previously known compound 5.¹⁾ Furthermore, the nuclear Overhauser effect spectroscopy (NOESY) correlation of H-3/ H-5 and the coupling constant of H-3 (δ_H 3.24, dd, J=11.4, 4.1 Hz) indicated that 3-OH was in an equatorial β-orientation. For the other NOESY correlations (Fig. 3), the cross-peaks of H₃-19/H₃-30, H-3/H-29, and H-3/H-5/H-9 indicated that H₃-19 was in a β-axial configuration and H-5, H-9, and H₃-29 were in an α-axial configuration. Moreover, the consecutive cross-peaks of H₃-19/H-18/H₃-21 and H-17/H₃-28 indicated that H₃-28 and H-17 were in an axial α-configuration, and the side chain was β-oriented. Meanwhile, the NOESY correlation between H-24 and H-26 indicated an E-geometry for the Δ^24,25 double bond. Thus, the relative configuration of compound 1 was assigned as 3β-hydroxy-lanostane-7,24E-dien-26-al and named kiusianin A.

Fig. 1. Chemical Structure of Compounds 1–4

Fig. 2. Key ¹H–¹H COSY (Bold) and HMBC (Arrow) Correlations for Kiusianins A–C (1–3)

Fig. 3. Key NOESY Correlations for Kiusianins A–C (1–3)

The molecular formula of compound 2 was established as C₂₇H₄₂O₂ by HR-EI-MS, indicating seven degrees of unsaturation. The IR spectrum revealed the presence of a hydroxyl group, an α,β-unsaturated ketone and an olefin group at 3410, 1678, and 1631 cm⁻¹, respectively. The ¹H- and ¹³C-NMR, and HMQC spectra of 2 indicated the presence of one ketone carbonyl, two sp² quaternary carbons, two sp³ quaternary carbons, two sp² methines, five sp³ methines, ten methylenes, one oxymethylene, and four methyls. Because three of the seven degrees of unsaturation have been accounted for, it was conducted that compound 2 contained four rings. The spectroscopic data disclosed that compound 2 was closely related to chloestane-4,20(22)-dien-3-one.¹⁵⁾ The only difference was that the olefinic methyl group at C-20 was replaced by a hydroxyl methyl group. This was supported by the HMBC correlation of H₂-21 (δ_H 4.04 br d, 4.18 br d, ABq, J=11.5 Hz; δ_C 61.2) to C-17 and C-22 (δ_C 131.4) as well as of H-22 (δ_H 5.42, t, J=7.3 Hz) to C-17 and C-21. The aforementioned data and detailed analysis of ¹H–¹H COSY and HMBC analyses (Fig. 2) suggested that 2 was 21-hydroxy-4,20(22)-dien-3-one. The NOESY correlations indicated that the relative configuration of 2 was identical to that of cholesterol. An E-geometry for the Δ^{20, 22} double bond was evident from the observation of NOE between H-21 and H-23. Thus, the relative configuration of compound 2 was assigned as 21-hydroxy-cholest-4,20-dien-3-one and named kiusianin B.

Compound 3 was formulated as C₂₇H₄₄O₂ by HR-EI-MS, and its IR spectrum showed the presence of a hydroxyl group, an olefin, and a ketone at 3392, 1732, and 1667 cm⁻¹, respectively. The NMR data (Tables 1, 2) disclosed that 3 was cholest-7-en-3-one with one hydroxyl group attached. The position of the hydroxyl group was unambiguously assigned to C-6 on the basis of HMBC correlation of H-5 (δ_H 2.25) to C-6 (δ_C 70.8) and ¹H–¹H COSY cross-peak of H-6/H-7. The NOESY spectrum of 3 showed cross-peaks of H-5/H-9 and H-6, indicating that the hydroxyl group was in a β-configuration. The other NOESY correlations (Fig. 3) showed that the relative configuration of 3 was identical to that of cholesterol. Thus, the relative configuration of compound 3 was elucidated as 6β-hydroxy-cholest-7-en-3-one and named kiusianin C.

The molecular formula of compound 4 was established as C₂₇H₄₂O₂ by HR-EI-MS. The IR spectrum showed the presence of a hydroxyl group, a ketone carbonyl group, and an olefin at 3398, 1730, 1665, and 1632 cm⁻¹, respectively. The ¹H- and ¹³C-NMR (Tables 1, 2) and HMQC data of compound 4 resembled those of compound 3, except for the presence of an exo-methylene in compound 4. Detailed 2D NMR analysis (HMQC, HMBC) revealed that compound 4 contained a terminal double bond at the C-20 position, in place of the C-20 methyl in compound 3. This was substantiated by the HMBC correlations of the exo-methylene protons (δ_H 4.82 and 4.90) to C-17 (δ_C 55.9) and C-22 (δ_C 37.8). The NOESY data supported that the relative configuration of 4 was the same as that of 3. Thus, the relative configuration of compound 4 was elucidated as 6β-hydroxy-cholest-7,20-diene-3-one and named kiusianin D.

All the compounds isolated in the present phytochemical study were evaluated for cytotoxicity (Table 3). As results, guggulsterol III (10) and linarin (17) exhibited significant cytotoxicity in vitro against human promyelocytic leukemia cells (IC₅₀: 10=5.4 µM; 17=5.0 µM). New compounds 2–4 showed moderate cytotoxicity against HL-60 cells (IC₅₀: 2=11.9 µM; 3=11.1 µM; 4=14.1 µM), and new compound 1 and known compounds 8, 9, and 11–15 showed weak activity against HL-60 cells with IC₅₀ values in the range of 20.5–30.1 µM. New compounds 1 and 2 and known compounds 10, 14–15, and 17 showed weak activity against HeLa cells with IC₅₀ values in the range of 22.7–30.6 µM. On the other hand, compounds 5, 16 and 18 did not show cytotoxicity against HeLa and HL-60 cells (IC₅₀ >60 µM).

Table 3. Cytotoxicity of 1–18 against HeLa and HL-60 Cells (IC₅₀; µM)

	HL-60	HeLa
1	20.5	30.2
2	11.9	29.6
3	11.1	>60
4	14.1	>60
5	>60	>60
6	41.1	>60
7	38.5	>60
8	28.0	>60
9	30.1	>60
10	5.4	25.2
11	24.9	>60
12	24.4	>60
13	25.3	>60
14	21.7	22.7
15	26.1	25.3
16	>60	>60
17	5.0	30.6
18	>60	>60
VCR	0.013
Taxol		0.002

Experimental

General

¹H- and ¹³C-NMR spectra were obtained on Bruker AVANCE 500 with cryo-platoform and AMX500 spectrometer using tetramethylsilane as the internal standard. HR-EI-MS spectrum was obtained on JEOL HX-100 and JEOL JMS-GCMATE spectrometer. UV and IR spectra were obtained on Shimadzu UV-260 and JASCO FT/IR-5300 spectrometers, respectively. Column chromatography was performed using ODS (YMC ODS-AM) and silica gel (Wako-gel C-300, Wako Pure Chemical Industries, Ltd. (Osaka, Japan)). MPLC was performed using silica-gel {Lobar® Fertigsäule Gröβe B, LiChroprep® Si 60 (40–63 µm), Merck (310×25 mm)}. HPLC was performed using YMC-Pack ODS-AM (250×10 mm) and YMC-Pack SIL (250×10 mm). Fractions were monitored by TLC, and spots were visualized using the anisaldehyde reagent.

Plant Material

Plants of T. kiusiana were collected from Tama Forest Science Garden, Forestry and Forest Products Research Institute, Tokyo, Japan, in September 2008. The plant was identified by K. Iwamoto (Tama Forest Science Garden, Forestry and Forest Products Research Institute). A voucher specimen (JSS06022-1) has been deposited at the Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University.

Extraction and Isolation

The leaves of T. kiusiana (dry weight, 187 g) were crushed and then extracted with MeOH at room temperature for two weeks and then evaporated in vacuo. The MeOH extracts (13 g) were chromatographed using an ODS column (CH₃CN/H₂O, 20 : 80→100 : 0, MeOH and CHCl₃) to obtain seven fractions (F1-1–7). The F1-3 fraction (CH₃CN–H₂O, 60 : 40, 240 mg) was purified using by silica gel column chromatography (c.c.) (CHCl₃–MeOH, 90 : 10→0 : 100) and HPLC (MeOH–H₂O, 70 : 30) to afford compound 17 (3.4 mg). The F1-4 fraction (CH₃CN–H₂O, 80 : 20, 150 mg) was separated by silica gel c.c. (CHCl₃–MeOH, 100 : 0→0 : 100) to obtain nine fractions (F2-1–9). The F2-2 fraction (18 mg) was further purified by HPLC (MeOH–H₂O, 70 : 30) to isolate 7 (3.5 mg). The F2-6 fraction (5.0 mg) was further purified on HPLC (MeOH–H₂O, 85 : 15) to isolate 8 (1.5 mg). The F1-6 fraction (MeOH, 1.8 g) was separated using fractionated with a silica gel c.c. (CHCl₃–MeOH, 100 : 0→0 : 100) to obtain six fractions (F3-1–6). The F3-2 fraction (410 mg) was separated using silica gel c.c. (CHCl₃–MeOH, 98 : 2→0 : 100) to obtain four fractions (F4-1–4). The F4-3 fraction (15 mg) was further purified using HPLC (MeOH–H₂O, 95 : 5) to isolate 15 (3.3 mg). The F4-3 fraction was further purified using HPLC (MeOH–H₂O, 95 : 5) to afford kiusianin A (1, 4.3 mg) and 5 (2.6 mg).

The second portion of MeOH extracts (8.4 g) was partitioned between n-hexane and 90% aqueous MeOH. The 90% aqueous MeOH-soluble materials (5.0 g) was subjected to silica gel column chromatography (n-hexane–EtOAc, 80 : 20→0 : 100, and then EtOAc–MeOH, 95 : 5→0 : 100) to obtain fractions (F5-1–13). The F5-2 fraction (456 mg) was separated using silica gel c.c. (n-hexane–EtOAc, 90 : 10→0 : 100, and then MeOH) to obtain eight fractions (F6-1–8). The F6-1 fraction (120 mg) was separated using silica gel c.c. (CHCl₃-MeOH, 98 : 2→0 : 100) to obtain five fractions (F7-1–5). The F7-2 fraction (15 mg) was further purified by HPLC (MeOH–H₂O, 93 : 7) to afford 9 (2.4 mg) and 11 (0.60 mg). The F7-4 fraction was purified using HPLC (MeOH–H₂O, 90 : 10) to afford 14 (7.5 mg). The F6-3 fraction (95 mg) was subjected to a silica gel c.c. (n-hexane–EtOAc, 70 : 30→0 : 100) to obtain two fractions (F8-1–2). The F8-1 fraction (63 mg) was separated using HPLC (n-hexane–EtOAc, 70 : 30) to obtain four fractions (F9-1–4). The F9-2 fraction (7.5 mg) was purified using HPLC (MeOH–H₂O, 95 : 5) to afford 6 (4.5 mg). The F9-4 fraction (28 mg) was separated on ODS column (MeOH–H₂O, 30 : 70→0/100, MeOH and CHCl₃) to obtain five fractions (F10-1–5). The F10-4 fraction (4 mg) was purified using HPLC (MeOH–H₂O, 85 : 15) to afford kiusianin B (2, 0.50 mg). The F10-5 fraction (6 mg) was further purified using HPLC (CH₃CN) to afford compound 12 (1.2 mg). The F6-4 fraction (61 mg) was subjected to silica gel c.c. (CHCl₃–MeOH, 98 : 2→0 : 100) to obtain five fractions (F11-1–5). The F11-4 fraction (15 mg) was separated using silica gel c.c. (CHCl₃–MeOH, 100 : 0→0 : 100) to obtain three fractions (F12-1–3). The F11-2 fraction (3.1 mg) was further purified using HPLC (MeOH–H₂O, 95 : 5) to afford compound 18 (1.0 mg). The F6-5 fraction (28 mg) was separated using with silica gel c.c. (CHCl₃–MeOH, 100 : 0→0 : 100) to obtain two fractions (F12-1–2). The F12-1 fraction (16 mg) was separated using HPLC (MeOH–H₂O, 95 : 5) to afford 10 (2.0 mg). The F5-3 fraction (110 mg) was subjected to MPLC (n-hexane–EtOAc, 80 : 20→0 : 100, EtOAc–MeOH, 90 : 10→100 : 0, and then CHCl₃–MeOH, 90 : 10) to obtain 12 fractions (F13-1–12). The F13-7 fraction (9.1 mg) was further purified using HPLC (MeOH/H₂O, 95/5) to afford kiusianin C (3, 0.50 mg) and kiusianin D (4, 0.50 mg). The F5-5 fraction (150 mg) was separated using MPLC (n-hexane–EtOAc, 50 : 50→0 : 100) to obtain four fractions (F14-1–4). The F14-2 fraction (15 mg) was further purified using HPLC (MeOH–H₂O, 95 : 5) to afford compound 13 (0.88 mg). The final portion of MeOH extract of T.kiusiana (14 g) was partitioned between n-hexane and 90% aqueous MeOH. The 90% aqueous MeOH-soluble material was obtained on drying and partitioned between EtOAc and water. The EtOAc-soluble material was subjected to silica gel column chromatography (n-hexane–EtOAc, 80 : 20→0 : 100, and then EtOAc–MeOH, 95 : 5→0 : 100) to obtain 13 fractions (F15-1–13). The F15-1 fraction (24 mg) was further purified by HPLC (CH₃CN) to afford 16 (0.78 mg).

Kiusianin A (1)

Colorless, amorphous solid; [α]_D²⁰+22.5 (c 0.12, CHCl₃); UV (MeOH) λ_max (log ε) 231 (3.21) nm; IR (KBr) λ_max 3415, 1739, 1687, 1633 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-EI-MS m/z 440.3660 [M]⁺ (Calcd for C₃₀H₄₈O₂, 440.3655).

Kiusianin B (2)

Colorless, amorphous solid; [α]_D²⁰+111.8 (c 0.017, CHCl₃); UV (MeOH) λ_max (log ε) at 240 (3.84) nm; IR (KBr) λ_max 3410, 1737, 1678, 1631 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-EI-MS m/z 398.3175 [M]⁺ (Calcd for C₂₇H₄₂O₂, 398.3185).

Kiusianin C (3)

Colorless, amorphous solid; [α]_D²⁰+114.5 (c 0.017, CHCl₃); IR (KBr) λ_max 3392, 1734, 1667 cm⁻¹; ¹H and ¹³C-NMR data, see Tables 1 and 2; HR-EI-MS m/z 400.3342 [M]⁺ (Calcd for C₂₇H₄₄O_2, 400.3342).

Kiusianin D (4)

Colorless, amorphous solid; [α]_D²⁰+72.3 (c 0.017, CHCl₃); IR (KBr) λ_max 3398, 1730, 1665, 1632 cm⁻¹; ¹H and ¹³C-NMR data, see Tables 1 and 2; HR-EI-MS m/z 398.3175 [M]⁺ (Calcd for C₂₇H₄₂O₂, 398.3185).

Cell Cultures and Cytotoxic Activity

The cell lines used in this study were HeLa (human cervical cancer) cells and HL-60 (human promyelocytic leukemia) cells. Both cell lines were purchased from the RIKEN BioResource Center (Tokyo, Japan). HeLa cells were grown as a monolayer in MEM, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in a humidified atmosphere of 5% CO₂. HL-60 cells were maintained in RPMI-1640, supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C in a humidified atmosphere of 5% CO₂. HeLa and HL-60 cells were seeded in 96-well plates at a density 5×10³ cells/well (90 µL/well) and 8×10³ cells/well (90 µL/well), respectively. The precultures were incubated for 24 h (HeLa cells) and 2 h (HL-60 cells). The medium containing test compounds (10 µL; final DMSO concentration was 0.2%) was then added, and the cells were incubated for 48 h. The cultures were added to CCK-8 (Cell Counting Kit-8, DOJINDO, Kumamoto, Japan) solution (10 µL) and incubated for 2 h (HeLa cells) and 4 h (HL-60 cells) at 37°C. After incubation, their optical densities at 450 nm were measured using a microplate reader (Model 680, BIO RAD, Japan).

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