Chemical Constituents of the Leaves of Thujopsis dolabrata and Their Cytotoxicity

Abstract

Five podophyllotoxin derivatives (1–5), two diterpenoids (6 and 7), three diterpenoid xylosides (8–10), a flavanonol glycoside (11), flavonol (12), and biflavonoid (13) were isolated from the leaves of Thujopsis dolabrata (Cupressaceae). Compounds 1, 6, and 8 were named (−)-β-isopeltatin, epi-nootkastatin 2, and acetoxyanticopalol 15-O-β-D-xylopyranoside, respectively. The structures of the isolated compounds were determined based on a detailed analysis of NMR spectroscopic data and through chromatographic and spectroscopic analyses following specific chemical transformations. The isolated compounds (1–5 and 7–11) were evaluated for their cytotoxicity toward HL-60 human promyelocytic leukemia cells and Caki-1 human kidney carcinoma cells. The podophyllotoxin derivatives (1–5) exhibited cytotoxicity against both HL-60 and Caki-1 cells with IC₅₀ values ranging from 0.00069 to 5.4 µM, and the diterpenoid derivatives (7–10) demonstrated cytotoxicity against HL-60 cells with IC₅₀ values ranging from 4.5 to 11 µM. HL-60 cells treated with 8 exhibited apoptosis characteristics, such as accumulation of sub-G₁ cells and nuclear chromatin condensation.

Introduction

Thujopsis dolabrata (Thunb. ex L.f.) Siebold & Zucc. (Cupressaceae) is an evergreen tree native to Japan, and its leaves, called “Asunaro-yo,” have been used as a folk medicine for the treatment of jaundice.^1,2) T. dolabrata is a rich source of essential oils such as (−)-thujopcene, cedrol, and hinokitiol. Furthermore, lignans, flavanol glycosides, terpenoid glycosides, and phenylpropanoid glycosides have also been isolated from the plant.^3–5) As part of our systematic studies on biologically active components originating from natural sources,^6–8) we conducted a phytochemical investigation on the leaves of T. dolabrata. This resulted in the isolation of five podophyllotoxin derivatives (1–5), two diterpenoids (6, 7), three diterpenoid xylosides (8–10), a flavanonol glycoside (11), flavonol (12), and biflavonoid (13), of which 1, 6, and 8 were named (−)-β-isopeltatin, epi-nootkastatin 2, and acetoxyanticopalol 15-O-β-D-xylopyranoside, respectively. The structures of the isolated compounds were determined based on a detailed analysis of NMR spectroscopic data and through chromatographic and spectroscopic analyses following specific chemical transformations. Among the isolated compounds, 1–5 and 7–11 were evaluated for their cytotoxicity against HL-60 human promyelocytic leukemia cells and Caki-1 human kidney carcinoma cells. The apoptosis-inducing activities of 8 in HL-60 cells were also investigated.

Results and Discussion

Dried leaves of T. dolabrata (5.0 kg) were extracted with MeOH. The concentrated MeOH extract, which demonstrated cytotoxicity against HL-60 cells with an IC₅₀ value of 0.13 µg/mL, was passed through a porous-polymer polystyrene resin (Diaion HP-20) column and successively eluted with 30% MeOH, 50% MeOH, 100% MeOH, EtOH, and EtOAc. Because the fractions eluted with MeOH and EtOH exhibited potent cytotoxicity against HL-60 cells with IC₅₀ values of 0.051 and 0.055 µg/mL, respectively, each fraction was subjected to multiple chromatographic steps over silica (Si) gel and octadecylsilanized (ODS) Si gel to result in 1–14, among which 1, 6, 8, and 11 are previously undescribed. The known compounds were identified as (−)-deoxypodophyllotoxin (2),⁹⁾ (−)-β-peltatin (3),¹⁰⁾ (−)-podorhizol aetate (4),¹¹⁾ (−)-podorhizol (5),¹²⁾epi-nootkastatin 1 (7),¹³⁾ anticopalol 15-O-β-D-xylopyranoside (9),⁴⁾ isoagatholal 15-O-β-D-xylopyranoside (10),⁴⁾ quercetin (12),¹⁴⁾ and amentoflavone (13)¹⁵⁾ by comparing the physical and spectroscopic data with those reported in literature (Fig. 1).

Fig. 1. Chemical Structures of 1–13

Compound 1 was obtained as an amorphous solid with a molecular formula of C₂₂H₂₂O₈ as determined from its high resolution (HR)-electrospray ionization (ESI)-time of flight (TOF)-MS data (m/z: 437.1208 [M + Na]⁺, Calcd for C₂₂H₂₂NaO₈: 437.1212). The IR spectrum of 1 demonstrated absorption bands for a hydroxy group (3431 cm⁻¹) and an ester carbonyl group (1770 cm⁻¹). The UV spectrum demonstrated an absorption maxima indicative of a conjugated system (292 and 239 nm). The ¹H-NMR spectrum of 1 comprised signals for two tetrasubstituted aromatic rings [δ_H 6.91 (1H, s, H-5) and 6.64 (1H, s, H-2); 6.50 (2H, s, H-2′ and H-6′)], one methylenedioxy group [δ_H 5.99 and 5.96 (each 1H, d, J = 1.3 Hz)], three methoxy groups [δ_H 3.83 (3H, s, MeO-4′) and 3.76 (3H × 2, s, MeO-3′ and 5′)], one methylene group [δ_H 3.06 (1H, dd, J = 16.0, 5.4 Hz, H-7α) and 2.85 (1H, dd, J = 16.0, 11.6 Hz, H-7β)], two methine groups [δ_H 2.89 (1H, d, J = 14.0 Hz, H-8′) and 2.67 (1H, m, H-8)], and one oxymethylene group [δ_H 4.42 (1H, dd, J = 8.5, 7.4 Hz, H-9β) and 3.95 (1H, dd, J = 10.6, 8.5 Hz, H-9α). In addition, a γ-lactone carbonyl carbon was observed at δ_C 174.4 in the ¹³C-NMR spectrum of 1 (Table 1). These NMR spectral features of 1 were closely related to those of (−)-deoxypodophyllotoxin (2).⁹⁾ However, the molecular formula of 1 was greater than that of 2 by one oxygen atom. When the ¹H- and ¹³C-NMR spectra of 1 were compared with those of 2, the signal assigned to the H-7′ methine proton/carbon [δ_H 4.55 (1H, d, J = 4.9 Hz, H-7′)/δ_C 45.1 (C-7′)] in 2 was not presented in 1. Instead, a quaternary carbon with an oxygen atom was observed (δ_C 77.0). Analysis of the ¹H-detected heteronuclear multiple-bond connectivity (HMBC) correlations between the quaternary carbon (δ_C 77.0) and H-5/H-8/H-2′/H-6′/H-8′ allowed us to determine the planar structure of 1, as shown in Fig. 2. The large ³J value between H-8 and H-8′ (J = 14.0 Hz) indicates that the H-8–C-8–C-8′–H-8′ bond is trans-oriented. In the phase-sensitive (PH) nuclear Overhauser effect spectroscopy (NOESY) spectrum, NOE correlations between H-8′ and H-7β/H-9β and those between H-8 and H-2′/H-7α/H-9α indicated that H-7β, H-9β, and H-8′ were located on the same face, whereas H-7α, H-8, H-9α, and H-2′ were oriented on the opposite side, as shown in Fig. 2. The circular dichroism (CD) spectral feature of 1, which demonstrated a positive Cotton effect at 290 nm, was essentially analogous to that of burseranin [Δε 291.9 nm (+1.19)], confirming the 7′S configuration.¹⁶⁾ Thus, the absolute configurations at C-7′, C-8′, and C-8 were deduced as 7′S, 8′S, and 8S, respectively. Accordingly, the structure of 1 was determined as shown in Fig. 1 and named (−)-β-isopeltatin. Although 1 has been reported as an intermediate in the synthesis of dehydroanhydropicropodophyllin,^17,18) this is its first isolation from the plant as a natural product.

Table 1. ¹H- and ¹³C-NMR Chemical Shift Assignments for 1, 6, 8, and 11

1a)			6a)			8a)			11a)
Position	δH	δC	Position	δH	δC	Position	δH	δC	Position	δH	δC
1	—	128.3	1α	1.33 m	44.1	1α	1.02 m	38.8	2	5.23 d (10.2)	83.6
2	6.64 s	107.6	β	2.12 m		β	1.79 m		3	4.77 d (10.2)	77.5
3	—	147.7	2α	1.50 m	19.6	2α	1.44 m	18.9	4	—	195.8
4	—	147.3	β	1.62 m		β	1.51 m		5	—	165.5
5	6.91 s	107.3	3α	1.25 ddd (13.3, 13.3, 3.2)	42.1	3α	1.01 m	36.2	6	5.90 d (2.0)	97.4
6		135.3	β	1.41 br d (13.3)		β	1.72 m		7	—	169.0
7α	3.06 dd (16.0, 5.4)	33.0	4	—	33.9	4	—	37.3	8	5.88 d (2.0)	96.4
β	2.85 dd (16.0, 11.6)		5	2.05 dd (11.7, 4.7)	45.1	5	1.24 dd (12.6, 1.6)	56.2	9	—	164.2
8	2.67 m	35.5	6α	2.18 ddd (13.3, 11.7, 4.6)	33.8	6α	1.82 m	24.4	10	—	102.5
9α	3.95 dd (10.6, 8.5)	71.6	β	2.12 ddd (13.3, 8.8, 4.7)		β	1.34 m		1′	—	128.4
β	4.42 dd (8.5, 7.4)		7	4.76 dd (8.8, 4.6)	107.5	7α	1.92 ddd (12.6, 12.6, 4.4)	38.4	2′	7.33 d (8.6)	130.3
OCH2O	5.99 d (1.3)	101.4	8	—	152.7	β	2.38 m		3′	6.81 d (8.6)	116.3
	5.96 d (1.3)		9	—	135.7	8	—	147.7	4′	—	159.4
1′	—	138.8	10	—	41.5	9	1.57 m	56.2	5′	6.81 d (8.6)	116.3
2′	6.50 s	106.8	11	7.03 d (8.9)	126.5	10	—	39.5	6′	7.33 d (8.6)	130.3
3′	—	152.4	12	6.40 d (8.9)	111.3	11a	1.60 m	21.8	Xyl1″	3.81 d (6.0)	103.1
4′	—	137.8	13	—	152.6	b	1.46 m		2″	3.22 dd (7.8, 6.0)	73.4
5′	—	152.4	14	—	124.5	12a	2.15 m	38.5	3″	3.19 dd (7.8, 7.4)	75.8
6′	6.50 s	106.6	15	3.87 sept (7.2)	24.1	b	1.80 m		4″	3.47 m	70.8
7′	—	77.0	16	1.36 d (7.2)	20.9	13	—	142.5	5″a	3.91 dd (11.8, 4.7)	65.9
8′	2.89 d (14.0)	53.3	17	1.38 d (7.2)	20.6	14	5.30 dd (7.5, 6.5)	119.2	b	3.03 dd (11.8, 8.4)
9′	—	174.4	18	0.98 s	33.5	15a	4.30 dd (11.5, 6.5)	65.5
3′-OMe	3.76 s	56.3	19	1.01 s	23.3	b	4.13 dd (11.5, 7.5)
4′-OMe	3.83 s	60.8	20	1.39 s	24.5	16	1.66 s	16.5
5′-OMe	3.76 s	56.3	7-OMe	3.54 s	57.1	17a	4.82 br s	106.8
						b	4.51 br s
						18	0.95 s	27.5
						19a	4.21 d (11.0)	66.8
						b	3.84 d (11.0)
						20	0.68 s	15.3
						Ac	2.02 s	21.0
								171.4
						Xyl1′	4.34 d (6.5)	101.4
						2′	3.40 dd (7.5, 6.5)	72.5
						3′	3.53 dd (7.8, 7.5)	75.0
						4′	3.69 m	69.6
						5′a	3.99 dd (11.5, 4.5)	64.5
						b	3.29 dd (11.5, 9.0)

a) The spectra of 1, 6, 8 were measured in CDCl₃, and those of 11 in CD₃OD.

Fig. 2. Key HMBC and NOE Correlations of 1

Compound 6 (C₂₁H₃₂O₃) was obtained as an amorphous solid. The ¹H-NMR spectrum of 6 contained signals for a 1,2,3,4-tetrasubstituted aromatic ring [δ_H 7.03 (1H, d, J = 8.9 Hz, H-11) and 6.40 (1H, d, J = 8.9 Hz, H-12)], one methoxy group [δ_H 3.54 (3H, s, MeO-7)], three tertiary methyl groups [δ_H 1.39 (3H, s, Me-20), 1.01 (3H, s, Me-19), and 0.98 (3H, s, Me-18)], one isopropyl group [δ_H 3.87 (1H, sept, J = 7.2 Hz, H-15), 1.38 (3H, d, J = 7.2 Hz, Me-17), and 1.36 (3H, d, J = 7.2 Hz, Me-16)], and one acetal group [δ_H 4.76 (1H, dd, J = 8.8, 4.6 Hz, H-7)]. These spectral features of 6 were similar to those of the known abietane diterpenoid 7. However, the molecular formula of 6 contained one more oxygen atom than that of 7. A comparison of the ¹H- and ¹³C-NMR spectra of 6 with those of 7 suggest that the signals for the oxymethine proton/carbon at H-7/C-7 [δ_H 4.41 (1H, br s)/δ_C 74.8] in 7 were displaced by those for an acetal proton/carbon [δ_H 4.76/δ_C 107.5] in 6, and the aromatic carbon signal at C-8 (δ_C 133.2) in 7 was shifted downfield by 19.5 ppm (δ_C 152.7) in 6. In the HMBC spectrum of 6, the methoxy protons correlated with the C-7 acetal carbon, and in turn, the H-7 acetal proton exhibited a ³J_C,H correlation with the C-8 aromatic carbon, which implied that C-7 and C-8 were bonded through an oxygen atom. In the ¹H–¹H correlation spectroscopy (COSY) spectrum, spin-coupling correlations were observed from H-7 to H-5 [δ_H 2.05 (1H, dd, J = 11.7, 4.7 Hz)] via H₂-6 [δ_H 2.18 (1H, ddd, J = 13.3, 11.7, 4.6 Hz, H-6α) and 2.12 (1H, ddd, J = 13.3, 8.8, 4.7 Hz, H-6β)]. Therefore, the presence of the rearranged seven-membered B-ring moiety with an acetal group in 6 was evident. In the PHNOESY spectrum of 6, NOE correlations between Me-20 and H-7/Me-19, Me-18 and H-6α, and those between Me-19 and H-6β were indicative of the 5α-H (A/B trans ring fusion) and 7α-methoxy configurations (Fig. 3). Therefore, the structure of 6 was determined as shown in Fig. 1 and named epi-nootkastatin 2. Compound 6 is a rare type of abietane diterpene with a seven-membered B-ring. One related diterpenoid named nootkastain 2 has been isolated from Chamaecyparis nootkatensis (Cupressaceae).¹³⁾ In a synthetic study of diterpenoids, the B-ring of a diterpene with a carbonyl group at C-7 was converted to ε-caprolactone by Baeyer–Villiger oxidation. Then, reduction of the ε-caprolactone moiety with diisobutylaluminium hydride (DIBAL) led to the corresponding C-7 hydroxy derivative.¹⁹⁾ Compound 6 was presumed to be produced through the similar biosynthetic reactions.

Fig. 3. Key HMBC and NOE Correlations of 6

Compound 8 (C₂₇H₄₄O₇) was obtained as an amorphous solid. The IR spectrum of 8 revealed absorption bands for hydroxy groups (3386 cm⁻¹) and a carbonyl group (1738 cm⁻¹). The ¹H- and ¹³C-NMR spectra of 8 exhibited signals for three methyl groups [δ_H 1.66 (3H, s, Me-16)/δ_C 16.5 (C-16), δ_H 0.95 (3H, s, Me-18)/δ_C 27.5 (C-18), and δ_H 0.68 (3H, s, Me-20)/δ_C 15.3 (C-20)]; two oxymethylene groups [δ_H 4.30 (1H, dd, J = 11.5, 6.5 Hz, H-15a) and 4.13 (1H, dd, J = 11.5, 7.5 Hz, H-15b)/δ_C 65.5 (C-15), and δ_H 4.21 (1H, d, J = 11.0 Hz, H-19a) and 3.84 (1H, d, J = 11.0 Hz, H-19b)/δ_C 66.8 (C-19)]; an olefinic group [δ_H 5.30 (1H, dd, J = 7.5, 6.5 Hz, H-14)/δ_C 119.2 (C-14) and 142.5 (C-13)]; an exomethylene group [δ_H 4.82 (1H, br s, H-17a) and 4.51 (1H, br s, H-17b)/δ_C 106.8 (C-17)]; and an acetyl group [δ_H 2.02 (3H, s)/δ_C 21.0 and 171.4 (Ac-19)], and a β-D-xylopyranosyl group [δ_H 4.34 (1H, d, J = 6.5 Hz, H-1′); δ_C 101.4, 72.5, 75.0, 69.6, and 64.5 (C-1′–5′)]. These spectral features of 8 were similar to those of the known diterpenoid β-D-xylopyranoside 10. However, the ¹H- and ¹³C-NMR spectral data suggest that the aldehyde group [δ_H 9.74 (1H, s)/δ_C 208.1] in 10 was replaced by an acetoxymethylene group in 8. Enzymatic hydrolysis of 8 with naringinase yielded 19-acetylagathadiol (8a)²⁰⁾ and D-xylose. The identification of D-xylose was carried out by direct HPLC analysis of the hydrolysate using an optical rotation detector. The anomeric configuration of the xylosyl moiety was ascertained as β by the relatively large ³J_H-1,H-2 value (6.5 Hz). In the HMBC spectrum of 8, long-range correlations were observed between the H₂-19 oxymethylene protons and the acetyl carbonyl carbon, and between the anomeric proton of the xylosyl moiety and C-15 of the aglycone (δ_C 65.5) (Fig. 4). Accordingly, the structure of 8 was determined to be 19-O-acetoxy-15-hydroxylabda-8(17),13E-dien-15-yl β-D-xylopyranoside as shown in Fig. 1 and named acetoxyanticopalol 15-O-β-D-xylopyranoside.

Fig. 4. Key HMBC Correlations of 8 and 11

Compound 11 (C₂₀H₂₀O₁₀) was obtained as an amorphous solid, with [α]_D²⁵ +71.2 (c 0.10, MeOH). The UV maximum at 295 nm and absorption bands for the hydroxy groups (3370 cm⁻¹) and a carbonyl group (1642 cm⁻¹) in the IR spectrum suggest that 11 is a flavanonol analogue. The ¹H-NMR spectrum of 11 displayed signals for two oxymethine protons [δ_H 5.23 (1H, d, J = 10.2 Hz, H-2) and 4.77 (1H, d, J = 10.2 Hz, H-3)] assignable to H-2 and H-3 of the flavanonol skeleton, meta-coupling aromatic protons [δ_H 5.90 (1H, d, J = 2.0 Hz, H-6) and 5.88 (1H, d, J = 2.0 Hz, H-8)], a 1,4-disubstituted aromatic ring [δ_H 7.33 (2H, d, J = 8.6 Hz, H-2′and H-6′) and 6.81 (2H, d, J = 8.6 Hz, H-3′and H-5′)], and an anomeric proton [δ_H 3.81 (1H, d, J = 6.0 Hz, H-1″). These data suggest that 11 is a flavanonol monoglycoside. Enzymatic hydrolysis of 11 with naringinase yielded (2R,3R)-dihydrokaempferol (11a) ([α]_D +21.2; ³J_H-2,H-3 = 10.2 Hz)²¹⁾ and D-xylose. In the HMBC spectrum of 11, long-range correlations were observed between H-1′ of Xyl (δ_H 3.81) and C-3 of the aglycone (δ_C 77.5). Accordingly, the structure of 11 was determined as (2R,3R)-dihydrokaempferol 3-O-β-D-xylopyranoside (Fig. 1).

Among the isolated compounds, 1–5 and 7–11 were evaluated for their cytotoxicity against HL-60 and Caki-1 cells (Table 2). Cisplatin and etoposide were used as the positive controls. The cytotoxic activities of (−)-deoxypodophyllotoxin (2) and (−)-β-peltatin (3) toward HL-60 cells have been reported.²²⁾ As expected, the podophyllotoxin derivatives 1–5 exhibited potent cytotoxicity against both HL-60 and Caki-1 cells with IC₅₀ values ranging from 0.00069 to 5.4 µM. Diterpenoids 7–10 exhibited cytotoxicity against HL-60 cells with IC₅₀ values ranging from 4.5 to 11 µM. Compound 5 was the C₆–C_7′ bond cleavage derivative of 1, and its cytotoxicity against HL-60 and Caki-1 cells (IC₅₀ 5.4 µM for HL-60 cells; IC₅₀ 1.6 µM for Caki-1 cells) was less than that of 1 (IC₅₀ 0.015 µM for HL-60 cells; IC₅₀ 0.41 µM for Caki-1 cells). This suggests that the C₆–C_7′ bond cleavage in the podophyllotoxin derivatives attenuated the cytotoxicity.

Table 2. Cytotoxic Activities of 1–5 and 7–11 against HL-60 and Caki-1 Cells

Compounds	IC50 (µM)a)
	HL-60	Caki-1
1	0.015 ± 0.0078	0.41 ± 0.013
2	0.0045 ± 0.000060	0.012 ± 0.00030
3	0.00069 ± 0.000015	0.0027 ± 0.000072
4	0.82 ± 0.010	4.3 ± 0.34
5	5.4 ± 0.43	1.6 ± 0.28
7	11.0 ± 0.51	>20
8	4.5 ± 0.014	>20
9	8.9 ± 0.78	>20
10	6.8 ± 0.54	>20
11	>20	>20
Cisplatin	1.1 ± 0.064	3.2 ± 0.082
Etoposide	0.63 ± 0.0062	—

a) The data are present as the mean ± S. E. M. of three experiments performed in triplicate.

The new diterpenoid 8 was found to be cytotoxic to HL-60 cells with an IC₅₀ value of 4.5 µM. Next, the apoptosis-inducing activities of 8 in HL-60 cells were investigated (Fig. 5). An increase in the sub-G₁ cell population in HL-60 cells treated with 8 was revealed via cell cycle analysis using a flow cytometer (Fig. 5A). The nuclear chromatin condensation and formation of apoptotic bodies, which are the morphological hallmarks of apoptotic cells, were observed in HL-60 cells treated with 8 after staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Fig. 5B). These observations suggest that 8 induced apoptosis in HL-60 cells.

Fig. 5. Compounds 8 or Etoposide Induced Apoptosis in HL-60 Cells

A, Cell cycle progression of HL-60 cells treated with 8 or etoposide. HL-60 cells were treated with 20 µM of 8 or 15 µM of etoposide for 24 h. The cell cycle distribution was analyzed by a flow cytometer. B, Morphology of HL-60 cells treated with 8 or etoposide. HL-60 cells were stained with DAPI after treatment with 20 µM of 8 or 15 µM of etoposide for 24 h. The cells were stained with DAPI and observed by a fluorescence microscopy.

Conclusion

The leaves of T. dolabrata yielded five podophyllotoxin derivatives (1–5), two diterpenoids (6 and 7), three diterpenoid xylosides (8–10), a flavanonol glycoside (11), flavonol (12), and biflavonoid (13), of which 1, 6, and 8 were named (−)-β-isopeltatin, epi-nootkastatin 2, and acetoxyanticopalol 15-O-β-D-xylopyranoside, respectively. The podophyllotoxin derivatives 1–5 demonstrated potent cytotoxicity against both HL-60 and Caki-1 cells, and diterpene derivatives 7–10 were found to be cytotoxic to HL-60 cells. Compound 8 induced apoptosis in HL-60 cells.

Experimental

Optical rotations (OR) were measured using a P-1030 (JASCO, Tokyo, Japan) automatic digital polarimeter. IR spectra were recorded on a FT-IR 620 spectrophotometer (JASCO). UV spectra were measured using a V-630 UV-Vis spectrophotometer (JASCO). CD spectra were measured using a J-720 spectrophotometer (JASCO). NMR spectra were recorded on a DRX-500 spectrometer (500 MHz for ¹H-NMR, Bruker, Billerica, MA, U.S.A.) using standard Bruker pulse programs at 300 K. Herein, chemical shifts are presented as δ values with reference to tetramethylsilane as an internal standard. HR-ESI-TOF-MS data were recorded on an LCT mass spectrometer (Waters-Micromass, Manchester, U.K.). Diaion HP-20 (50 mesh, Mitsubishi-Chemical, Tokyo, Japan), Si gel Chromatorex BW-300 (300 mesh, Fuji-Silysia Chemical, Aichi, Japan), and COSMOSIL 75C₁₈-OPN (75 µM particle size, Nacalai Tesque, Kyoto, Japan) were used for columun chromatography (CC). TLC was conducted on precoated Si gel 60 F₂₅₄ or RP₁₈ F₂₅₄S plates (0.25 mm thick, Merck, Darmstadt, Germany), and spots were detected by spraying the plates with a 10% H₂SO₄ aqueous solution, followed by heating. HPLC was performed using a system consisting of a DP-8020 pump (Tosoh, Tokyo, Japan), a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and Rheodyne™ injection port (Thermo Fisher Scientific, Waltham, MA, U.S.A.). HL-60 cells (JCRB0085) and Caki-1 cells (JCRB0801) were obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank (Osaka, Japan). The following materials and reagents were used for the cell culture assays: SH-1300 microplate reader (Corona Electric, Ibaraki, Japan), 96-well flat-bottom plates (Iwaki Glass, Chiba, Japan), fetal bovine serum (FBS), 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution, RPMI 1640 medium, Minimum Essential Medium (MEM), etoposide, cisplatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), naringinase (EC 3.2.1.40) (Sigma-Aldrich, St. Louis, MO, U.S.A.), penicillin G sodium salt, streptomycin sulfate, and phosphate buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). All other chemicals used were of biochemical reagent grade.

Plant Material

The leaves of Thujopsis dolabrata (Lot No. AAH3005) were purchased from UCHIDA WAKANYAKU Co., Ltd,. (Tokyo, Japan) in Dec., 2011. A voucher specimen was deposited in our laboratory (voucher no. KS-2011-003, Department of Medicinal Pharmacognosy).

Extraction and Isolation

Dried leaves of T. dolabrata (5.0 kg) were extracted with MeOH (18 L). After removal of the solvent under reduced pressure, the MeOH extract (1.1 kg) was passed through a Diaion HP-20 column and successively eluted with H₂O–MeOH (7 : 3), H₂O–MeOH (1 : 1), MeOH, EtOH, and EtOAc. The fractions eluted with MeOH and EtOH exhibited potent cytotoxicity against HL-60 cells with IC₅₀ values of 0.051 and 0.055 µg/mL, respectively. The MeOH eluate fraction (240 g) was subjected to Si CC (80 mm i.d. × 280 mm) eluted with stepwise gradient mixtures of n-hexane–EtOAc (19 : 1; 9 : 1; 6 : 1; 4 : 1; 1 : 1), which produced 14 fractions (A–N). Fraction I was separated by Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH (49 : 1; 9 : 1) to resulted in 13 subfractions I-A–I-M. Fraction I-C was purified by ODS Si CC (20 mm i.d. × 250 mm), eluted with MeCN–H₂O (1 : 1) and Si CC, eluted with n-hexane–EtOAc (1 : 1) to yield 2 (64.5 mg) and 4 (2.1 mg). Fraction I-E was purified by Si CC, eluted with n-hexane–acetone (2 : 1) and n-hexane–EtOAc (4 : 1), and subjected to HPLC (10 mm i.d. × 250 mm) using MeOH-H₂O (7 : 3) as the mobile phase to yield 1 (6.0 mg), 3 (13.6 mg), 5 (63.7 mg), and 6 (1.5 mg). Fraction I-M was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH-H₂O (1 : 1; 6 : 4) to yield 12 (48.7 mg). Fraction K was purified by Si CC (20 mm i.d. × 250 mm) eluted with n-hexane–EtOAc (49 : 1; 19 : 1) and ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH-H₂O (8 : 2; 9 : 1) and MeCN–H₂O (7 : 3; 1 : 1) to yield 8 (44.7 mg), 9 (64.7 mg), 10 (10.7 mg), 11 (28.3 mg), and 13 (43.7 mg). The EtOH eluate fraction (35 g) was subjected to Si CC (80 mm i.d. × 280 mm) and eluted with n-hexane–EtOAc (19 : 1; 6 : 1) to result in 10 fractions (A–J). Fraction E was purified by Si CC (20 mm i.d. × 250 mm), eluted with n-hexane–acetone (20 : 1) and ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH-H₂O (6 : 1) and MeCN–H₂O (6 : 1; 3 : 1) to yield 7 (2.8 mg). Fraction J was purified by Si CC (20 mm i.d. × 250 mm) and eluted with n-hexane–EtOAc (19 : 1; 6 : 1) and n-hexane–acetone (5 : 1; 2 : 1; 1 : 1) to yield 2 (32.0 mg).

Compound 1: Amorphous solid; [α]_D²⁵ −51.7 (c = 0.10, MeOH); UV (MeOH) λ_max nm (log ε): 292 (3.70), 239 (4.40); IR (film) ν_max: 3431 (OH), 1770 (C=O) cm⁻¹; ¹H- (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃) spectra, see Table 1; HR-ESI-TOF-MS m/z 437.1208 [M + Na]⁺ (Calcd for C₂₂H₂₂NaO₈: 437.1212).

Compound 6: Amorphous solid; [α]_D²⁵ 60.3 (c = 0.07, MeOH); UV (MeOH) λ_max nm (log ε): 206.5 (4.51); IR (film) ν_max: 3413 (OH) cm⁻¹; ¹H- (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃) spectra, see Table 1; HR-ESI-TOF-MS m/z 355.2239 [M + Na]⁺ (Calcd for C₂₁H₃₂NaO₃: 355.2249)

Compound 8: Amorphous solid; [α]_D²⁵ −14.3 (c = 0.21, CHCl₃); IR ν_max (film) cm⁻¹: 3386 (OH), 1738 (C=O); ¹H- (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃) spectra, see Table 1; HR-ESI-TOF-MS m/z 503.2980 [M + Na]⁺ (Calcd for C₂₇H₄₄NaO₇: 503.2985).

Enzymatic Hydrolysis of 8

Compound 8 (10.1 mg) was treated with naringinase (EC 232-962-4, Sigma-Aldrich, 11.7 mg) in a HOAc/KOAc buffer (pH 4.3, 5 mL) at room temperature for 18 h. The reaction mixture was separated via silica gel chromatography, eluted with n-hexane–EtOAc (1 : 3) and MeOH to yield 19-acetylagathadiol (8a, 1.6 mg) and the sugar fraction (1.1 mg) The sugar fraction was analyzed by HPLC under the following conditions. Capcell Pak NH₂ UG80 column (4.6 mm i.d. × 250 mm, 5 µm, Shiseido, Tokyo, Japan); solvent: MeCN–H₂O (17 : 3); detected by OR; flow rate: 1.0 mL/min. HPLC analysis of the sugar fraction revealed the presence of D-xylose (t_R = 8.8 min, positive optical rotation).

Compound 11: Amorphous solid; [α]_D²⁵ 71.2 (c = 0.10, MeOH); UV (MeOH) λ_max nm (log ε): 294.5 (3.69), 208.0 (3.95); IR ν_max (film) cm⁻¹: 3370 (OH), 1642 (C=O); ¹H- (500 MHz, CD₃OD) and ¹³C-NMR (125 MHz, CD₃OD) spectra, see Table 1; HR-ESI-TOF-MS m/z 443.0966 [M + Na]⁺ (Calcd for C₂₀H₂₀NaO₁₀: 443.0954).

Enzymatic Hydrolysis of 11

Compound 11 (15.1 mg) was subjected to the enzymatic hydrolysis described for 8 to yield (2R,3R)-dihydrokaempferol (11a, 6.2 mg) and the sugar fraction (2.5 mg). HPLC analysis of the sugar fraction under the same conditions used for 8 revealed the presence of D-xylose.

Cell Culture and Assay for Cytotoxic Activity against HL-60 and Caki-1 Cells

HL-60 cells and Caki-1 cells were cultured in an RPMI 1640 medium and MEM, respectively, containing heat-inactivated 10% (v/v) FBS supplemented with L-glutamine, 100 unit/mL penicillin G sodium salt, and 100 µg/mL streptomycin sulfate in a humidified incubator at 37 °C with 5% CO₂. The cells (HL-60 cells: 4 × 10⁴ cells/mL and Caki-1 cells: 2 × 10⁴ cells/mL) were washed and resuspended in the medium, and the cell suspension (HL-60 cells: 196 µL/well, Caki-1 cells: 100 µL/well) was seeded into 96-well flat bottom plates (Iwaki Glass, Chiba, Japan). The cells were continuously treated with each compound for 72 h, and cell viability was measured with an MTT reduction assay.²³⁾ Dose-response curves were plotted for 1–5 and 7–10 and the concentrations resulting in IC₅₀ were calculated.

Cell Cycle Analysis

HL-60 cells (1 × 10⁶ cells) were treated with 20 µM of 8 or 15 µM of etoposide for 24 h. The cells were collected and fixed in 70% EtOH at −20 °C overnight. Next, the cells were washed with PBS in 1 mL of FxCycle™ (PI)/RNase Staining Solution (Thermo Fisher Scientific) for 15 min. The DNA content was measured using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, U.S.A.).

DAPI Staining

HL-60 cells (1 × 10⁵ cells/well) were plated on coverslip bottom 96-well plates. The cells were treated with 20 µM of 8 or 15 µM of etoposide for 20 h. The cells were fixed with 1% glutaraldehyde for 30 min at room temperature before staining with DAPI (0.5 µg/mL in PBS). They were then observed immediately using a CKX41 fluorescence microscope.

Acknowledgments

This work was financially supported in part by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI (Grant Numbers JP26860069 and JP18K06735).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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