Cytotoxic cardiac glycosides and coumarins from Antiaris toxicaria

Abstract

Eight new cardiac glycosides/aglycones (antiaritoxiosides A–G, 1–7, and antiarotoxinin B, 8), two new coumarins (anticarins A–B, 41–42), and two new flavanones (antiarones L–K, 43–44) were isolated from trunk bark of Antiaris toxicaria together with 53 known compounds. The new structures were established by extensive analysis of spectroscopic data. Compound 1 (10-carboxy and 3α-hydroxy) and compounds 3–6 (10-hydroxy) contain unique substituents that are rarely found in cardiac glycosides. The cytotoxic effects of isolated compounds against ten human cancer cell lines, KB, KB-VIN, A549, MCF-7, U-87-MG, PC-3, 1A9, CAKI-1, HCT-9 and S-KMEL-2, were tested using the sulforhodamine B assay. Five compounds (12, 16, 20, 22, and 31) showed significant cytotoxicity against all ten cancer cell lines, with notable potency at the ng/mL level against some cell lines, which merits further development as clinical trial candidates.

1. Introduction

Cardiac glycosides are naturally occurring compounds with a steroidal framework and are used for the treatment of congestive heart failure and as anti-arrhythmic agents. They also have been used as arrow poisons, abortifacients, emetics, diuretics, and heart tonics.¹ Mechanistically, their anti-arrhythmic activity results from inhibition of Na⁺/K⁺-ATPase, and leads to an increased intracellular calcium concentration. Recent research indicated that binding of cardiac glycosides to Na⁺/K⁺-ATPase can activate multiple downstream signal transduction pathways that regulate many important physiological and pathological states.² Less well known are the emerging role of this compounds category in the prevention and/or treatment of proliferative diseases, such as cancer. Because cardenolide-like compounds are often quite toxic, however, the potential use of such compounds for the treatment of cancer was abandoned 40 years ago. Even so, studies have revealed that some cardiac glycosides can selectively inhibit human tumor cell growth, but not normal cellular proliferation through complex cell signal transduction mechanisms.¹ Specifically, cardiac glycosides can inhibit growth of cancer cells, induce apoptosis via activation of caspase-3, induce cytochrome C release from mitochondria, and generate reactive oxygen species. In addition, clinical trials of cardiac glycosides and extracts containing them have been initiated to assess the anticancer potential.² Therefore, the use of certain cardiac glycosides may represent a worthwhile approach for control of cancer, despite their narrow therapeutic index.

In a previous investigation of bioactive compounds with potential therapeutic value, we reported cardiotonic principles from Antiaris toxicaria (Pers.) Lesch.³ As a continuation of our anticancer research program based on natural products, an ethanolic extract of A. toxicaria (Moraceae) was found to show significant cytotoxicity against KB cells. A. toxicaria, also called ‘upas tree’, is widely distributed in tropical rain forests of Southeast Asia. Phytochemical studies have shown that this plant contains cardiac glycosides,^3,4 prenylaurones,⁵ chalcones,⁶ flavanones,⁶ and dihydrochalcones.⁷ Prior investigations also indicated that this species exhibited a broad spectrum of biological activities, such as anti-tumor,^8–12 anti-arrhythmic,³ and 5-lipoxygenase inhibition effects.¹² Therefore, an intensive investigation of this cytotoxic extract was carried out and led to the isolation of 12 new compounds, together with 53 known compounds. Herein, we describe the isolation, structural elucidation, and antitumor activity of these isolates against ten human cancer cell lines. Preliminary structure–activity relationship (SAR) correlations are also described. In addition, based on the structures of 10-hydroxy-19-nor-cardenolides and 19-nor-cardenolides identified from this plant, a biogenetic pathway of cardiac glycoside has been proposed.

2. Results and discussion

Twelve new compounds, including eight cardiac glycosides/ aglycones (1–8), two coumarins (41–42) and two flavanones (43–44) (Fig. 1), together with 53 known compounds, were isolated from an EtOH extract of fresh trunk bark of A toxicaria through column chromatography on silica gel, Diaion HP-20, Sephadex H-20 and semipreparative HPLC. The subfractions were examined by H₂SO₄ solution spray on TLC for cardiac glycosides, which show as green spots, and subjected to a series of column chromatographic steps in order to obtain cardenolides/aglycones. The structures of new compounds were elucidated on the basis of spectroscopic methods, including 2D NMR techniques.

Figure 1.

Chemical structures for new compounds.

The HRFAB mass spectrum of 1 showed a molecular ion-related [M+H]⁺ peak at m/z 567.2808, corresponding to the molecular formula C₂₉H₄₂O₁₁. The NMR signals were due to one methyl, eleven methylene, eight methine and six quaternary carbons as determined by DEPT 135 spectroscopy (Table 1). Compound 1 showed a UV absorption maximum at 216 nm and IR absorption at 1734 cm ⁻¹ (γ-lactone carbonyl), which were indicative of a butenolactone system.¹³ The ¹H NMR spectrum of 1 (Table 2) showed characteristic signals for a butenolactone ring at δ_H 4.98 and 5.27 (each 1H, d, J = 18.1 Hz, H-21a, and b) and 6.08 (s, H-22), as well as a methyl singlet at δ_H 0.98 (s, H-18), suggesting 1 to be a cardenolide with a C-18 methyl group. A strong carbonyl absorption in the IR spectrum at 1734 cm⁻¹ and a carbon signal at δ_C 176.7 in the ¹³C NMR spectrum suggested that a carboxylic acid was present at C-19. Comparison of the ¹H and ¹³C NMR spectra of 1 with those of antiaroside R (9),⁴ which was isolated from the n-BuOH soluble fraction, showed that the two structures were very similar, except for the differences in glycosidic signals and orientation at C-3. An anomeric proton signal at δ_H 6.08 (s) in the ¹H NMR spectrum and signals at δ_C 99.8, 72.7, 72.8, 74.3, 69.9, and 18.6 in the ¹³C NMR spectrum indicated the presence of α-rhamnose. The α-orientation of C-3 was deduced from the coupling constant values of H-3 (dddd, J = 11.0, 11.0, 5.4, 5.4 Hz). This assignment was supported by downfield shifts of H-2a, H-3, and H-4a from δ_H 1.43 (m), 4.37 (br s, W_1/2 = 15.7 Hz), and 1.67 (d, J =12.7 Hz) in 9 to δ_H 2.19 (m), 4.81 (dddd, J = 11.0, 11.0, 5.4, 5.4 Hz), and 2.19 (m) in 1. HMBC correlation of H-1′ with C-3 [δ_C 72.9/δ_H 6.08 (s)] inferred that the rhamnose unit was linked to C-3. Hence, the structure shown was established for 1 (antiaritoxioside A).

Table 1. ¹³C NMR data for antiaritoxioside A-G (1–7) and antiarotoxinin B (8).

Carbon	1‡	2‡	3‡	4‡	5	6	7‡	8
1	36.3	26.9	25.3	26.9	26.7	22.0	23.6	25.8
2	22.4	28.7	27.4	28.6	27.9	26.4	26.8	28.6
3	72.9	73.9	73.8	73.8	73.2	72.3	74.1	67.8
4	32.1	35.6	34.8	35.6	35.1	30.0	36.1	36.1
5	72.9	74.7	73.1	74.2	74.3	30.4	73.4	74.5
6	43.3	35.8	35.1	35.9	36.5	33.2	40.8	37.8
7	28.4	24.3	24.5	24.8	24.7	26.0	21.3	24.4
8	42.3	40.7	41.3	40.4	40.4	48.2	41.2	39.0
9	41.0	40.2	39.4	37.5	37.7	42.0	37.3	40.0
10	55.7	74.4	74.4	74.7	74.4	34.0	39.2	41.3
11	23.5	21.6	22.8	30.8	30.7	22.1	26.1	22.0
12	39.9	40.0	40.4	74.7	74.7	39.7	31.4	40.3
13	50.2	50.1	50.3	56.7	56.9	50.1	51.9	47.8
14	84.8	84.6	85.0	85.0	85.1	83.8	86.7	83.8
15	33.4	33.0	33.0	33.4	33.4	32.2	33.7	33.1
16	27.2	27.5	27.2	27.9	29.2	22.1	34.3	19.0
17	51.5	51.3	51.3	46.4	46.2	51.4	87.8	51.5
18	16.3	16.3	16.3	10.3	10.3	16.4	13.1	15.5
19	176.7						17.0	17.4
20	176.1	176.3	176.4	176.7	176.8	176.0	174.0	71.1
21	73.8	73.8	73.8	74.0	74.1	73.6	73.8	65.8
22	117.6	117.7	117.6	117.5	117.4	117.6	116.6
23	174.6	174.8	174.6	174.7	174.8	174.5	173.2
1′	99.8	100.8	99.3	100.7	99.4	99.8	99.8
2′	72.7	72.6	72.2	72.6	69.8	72.9	69.6
3′	72.8	73.0	73.1	72.9	73.6	72.9	73.8
4′	74.3	73.9	73.8	73.7	73.7	74.1	73.3
5′	69.9	70.7	70.8	70.7	70.0	70.0	70.0
6′	18.6	18.6	18.8	18.6	17.0	18.6	17.3

δ Value in pyridine-d₅ (100 MHz).

^‡δ Value in pyridine-d₅ (75 MHz).

Table 2. ¹H NMR data for antiaritoxioside A-G (1–7) and antiarotoxinin B (8).

Proton	1	2‡	3	4	5	6	7	8
1a/b	1.76 (br d, 13.6)/3.29 (ddd, 13.6, 13.6, 5.7)	1.90 (m)/2.19 (m)	2.04 (m)/2.44 (m)	1.95 (m)/2.27 (br dd, 8.4, 2.3)	1.92 (m)/2.46 (br s dd, 14.5, 2.9)	1.23 (m)/2.07 (m)	1.32 (ddd, 13.5, 13.5, 3.8)/2.21 (m)	1.45 (t, 11.7)/2.21 (td, 12.5, 4.4)
2a/b	2.19 (m)/2.27 (m)	1.67 (m)/2.04 (m)	1.81 (m)/2.14 (m)	1.69 (br d, 14.8)/2.00 (m)	1.81 (br d, 13.1)/2.17 (m)	1.52 (m)	1.92 (m)/2.07 (m)	1.81 (m)
3	4.81 (dddd, 11.0, 11.0, 5.4, 5.4)	4.29 (br s, 6.6)†	4.40 (br s, 15.7)†	4.27 (br s, 7.9)†	4.48 (br s, 7.7)†	4.18 (br s, 9.9)†	4.52 (br s, 9.8)†	4.44 (br s, 11.3)
4a/b	2.19 (m)/2.45 (br d, 12.8)	1.90 (m)/2.11 (m)	1.81 (m)/2.04 (m)	1.97 (m)/2.15 (m)	1.92 (m)/2.09 (br d, 9.7)	1.23 (m)/1.48 (br t, 4.0)	1.56 (m)/1.97 (m)	1.78 (m)/2.32 (dd, 14.9, 2.5)
5						2.07 (m)
6a/b	2.19 (m)/2.27 (m)	1.55 (m)/2.25 (m)	1.65 (m)/2.14 (m)	1.56 (dd, 11.2, 6.3)/1.97 (m)	1.63 (br d, 13.0)/2.28 (m)	1.78 (m)/2.02 (m)	1.49 (m)/2.07 (m)	1.62 (br d, 13.0)/2.03 (m)
7a/b	2.27 (m)/2.68 (br d, 11.8)	1.23 (m)/2.33 (br. d, 9.8)	1.65 (m)/2.15 (m)	1.32 (br d, 13.2)/ 2.29 (br s dd, 9.9, 3.3)	2.12 (m)/2.32 (m)	1.55 (m)/1.78 (m)	1.30 (m)/1.64 (br t, 13.5)	1.40 (m)/2.36 (td, 13.3, 2.0)
8	2.27 (m)	2.36 (d, 12.3)	2.15 (m)	2.48 (br t, 11.8)	2.48 (br dd, 12.8, 2.9)	1.29 (m)	1.99 (m)	1.76 (dt, 12.2, 11.8)
9	2.27 (m)	1.63 (m)	1.65 (m)	1.78 (ddd, 8.7, 8.7, 3.0)	1.81 (br d, 13.1)	1.24 (m)	2.39 (m)	2.03 (m)
10						1.43 (m)
11a/b	1.88 (m)	1.55 (m)/1.90 (m)	1.38 (m)/2.26 (m)	2.15 (m)/2.24 (m)	2.12 (m)/2.25 (m)	1.66 (br d, 2.3)/1.84 (m)	1.84 (br t, 13.9)/2.25 (m)	1.40 (m)
12a/b	1.46 (m)	1.40 (m)	1.38 (m)	−/3.74 (br t, 7.8)	−/3.76 (br t, 9.7)	1.31 (m)	2.17 (m)/ 2.39 (m)	1.42 (t, 11.9)
15a/b	1.93 (m)/2.19 (m)	1.90 (m)/2.04 (m)	1.81 (m)/2.04 (m)	1.73 (m)/1.97 (m)	1.92 (m)	1.35 (m)	1.19 (m)/1.49 (m)	1.89 (m)/2.09 (dt, 12.6, 9.8)
16a/b	1.93 (m)/2.03 (dd, 20.0, 10.8)	2.04 (m)	1.81 (m)/2.04 (m)	2.15 (m)	2.12 (m)	1.94 (m)/2.07 (m)	1.97 (m)/2.21 (m)	1.92 (m)/2.26 (m)
17	2.76 (dd, 9.1, 3.2)	2.78 (br d, 7.7)	2.79 (br t,2.5)	3.76 (br t, 7.4)	3.76 (br t, 9.7)	2.76 (br d, 8.8)		2.21 (m)
18	0.98 (s)	1.05 (s)	1.15 (s)	1.28 (s)	1.29 (s)	0.99 (s)	1.26 (s)	1.31 (s)
19							1.17 (s)	1.19 (s)
20								4.36 (t, 6.5)
21a/b	4.98 (d, 18.1)/5.27 (d, 18.1)	5.13 (d, 18.2)/5.30 (d, 18.2)	5.01 (d, 18.0)/5.29 (d, 18.0)	5.09 (d, 17.9)/5.23 (d, 17.9)	5.03 (d, 18.1)/5.33 (d, 18.1)	5.02 (d, 17.8)/5.31 (br d, 17.8)	5.07 (dd, 18.2, 1.4)/5.21 (dd, 18.2, 1.4)	3.89 (dd, 10.5, 6.1)/4.08 (dd, 10.5, 7.2)
22	6.08 (s)	6.12 (s)	6.12 (s)	6.22 (s)	6.23 (s)	6.12 (s)	6.23 (d, 1.4)
1′	5.44 (br s)	5.49 (br s)	5.40 (d, 5.8)	5.33 (br s)	5.43 (d, 8.1)	5.41 (br s)	5.39 d, 8.1)
2′	4.27 (br s)	4.54 (br s)	3.96 (d, 4.7)	4.48 (br s)	4.50 (dd, 8.1, 3.1)	4.54 (m)	4.46 (br d, 8.1)
3′	4.44 (dd, 8.9, 3.4)	4.44 (dd, 9.0, 3.2)	4.67 (m)	4.38 (dd, 8.6, 2.8)	4.77 (t, 3.1)	4.54 (m)	4.70 (m)
4′	4.19 (dd, 8.9, 8.9)	4.31 (m)	3.66 (d, 6.2)	4.21 (br t, 8.6)	4.13 (d, 3.1)	4.29 (m)	4.11 (br s)
5′	4.32 (dq, 8.9, 6.1)	4.22 (m)	4.34 (dq, 6.2, 4.6)	4.18 (dq, 8.6, 5.9)	4.61 (q, 6.6)	4.29 (m)	4.59 (q, 6.4)
6′a/b	1.59 (d, 6.1)	1.63 (d, 6.0)	1.59 (d, 4.6)	1.61 (d, 5.9)	1.53 (d, 6.6)	1.67 (d, 4.7)	1.56 (d, 6.4)

δ Value in pyridine-d₅ (400 MHz), coupling constants in Hz are given in parentheses.

^†W_1/2 (Hz): width of 1/2 peak high.

^‡δ Value in pyridine-d₅ (300 MHz), coupling constants in Hz are given in parentheses.

A molecular formula of C₂₈H₄₂O₁₀ was deduced for compound 2, 12 mass units less than that of periplorhamnoside (11).¹⁴ The ¹H and ¹³C NMR data of the two compounds were similar except for the C-19 and C-10 signals. The carbon signal at δ_C 17.2 (C-19) in 11 was absent in 2. Furthermore, the carbon signal at δ_C 41.2 (C-10) in 11 was present at δ_C 74.4 (C-10) in 2. Thus, both the NMR and MS data were indicative of a hydroxy rather than methyl group at C-10 in 2. The β-orientation of the C-3 monosaccharide unit was deduced from the W_1/2 constant of H-3 (br s, W_1/2 = 6.6 Hz). Therefore, the structure of 2 was determined as demethyl-10-hydroxy-periplorhamnoside and 2 was named antiaritoxioside B.

Compound 3 showed a pseudo molecular ion peak at m/z 539.2855 in its HRFABMS and had the same molecular formula, C₂₈H₄₂O₁₀, as toxicarioside M (10),¹⁵ which was also isolated from the n-BuOH extract of this plant. The UV, IR and NMR data of 3 strongly resembled those of 10, consistent with a general structure containing a central cardenolide moiety dioxygenated at C-3, and C-5 and a hydroxy group in the 10-position. The sole significant differences were in certain glycosidic signals (Tables 1 and 2). An anomeric proton signal at δ_H 5.40 (d, J = 5.8 Hz) in the ¹H NMR spectrum and signals at δ_C 99.3, 72.2, 73.1, 73.8, 70.8, and 18.8 in the ¹³C NMR spectrum indicated the presence of β-allomethylose. An NOE effect from H-1′ to H-3 suggested that the allomethylose unit was attached to C-3. These data agreed with the replacement of the antiarosyl unit in 10 by an allomethylosyl unit in 3. Thus, the structure of 3 was established for antiaritoxioside C.

A molecular formula of C₂₈H₄₃O₁₁ was deduced for compound 4, 12 mass units less than that of β-antiarin (34),¹⁶ isolated from the CHCl₃ soluble fraction. The ¹H and ¹³C NMR data of the two compounds were similar except for the absence of a C-19 aldehyde group and a downfield shift of C-10 from δ_C 55.0 in 34 to δ_C 74.7 in 4. These data suggested that the aldehyde group in 34 was replaced by a hydroxy group in 4. This conclusion was supported by ³J_CH HMBC correlations from H-1b and H-11b to C-10. The β-orientations of the C-3 and -12 OH groups were deduced from the coupling constants of H-3 (δ_H 4.27, br. s, W_1/2 = 7.9 Hz) and H-12 (δ_H 3.74, br t, J = 7.8 Hz). Thus, 4 was determined as 10-hydroxy-19-nor-β-antiarin and named antiaritoxioside D.

Compound 5 had the same molecular formula, C₂₈H₄₃O₁₁, as 4. The UV, IR and NMR data also strongly resembled those of 4, consistent with a structure containing a 19-nor-cardenolide moiety tetraoxygenated at C-3, C-5, C-10 and C-12. The sole significant differences occurred in glycosidic signals of the molecules (Tables 1 and 2). The sugar proton signals of 5 indicated the presence of a β-antiarosyl moiety. These data were consistent with the replacement of the rhamnosyl unit in 5 by an antiarosyl unit in 5. The β-orientations of C-3 and C-12 were deduced by the W_1/2 of H-3 (br s, 7.7 Hz) and H-12 coupling constant (br t, J = 9.7 Hz). Thus, the structure of 5 was assigned as shown, and 5 was named antiaritoxioside E.

The HRFAB mass spectrum of 6 showed a molecular ion-related [M+Na]⁺ peak at m/z 529.2774, corresponding to the molecular formula C₂₈H₄₂O₈. The ¹H and ¹³C NMR data (Tables 1 and 2) indicated that 6 was a 19-nor-cardenolide monosaccharide with a hexose sugar unit. Analyses of COSY, HMQC and HMBC spectra indicated that the sugar was α-rhamnose. Comparison of the ¹H and ¹³C NMR spectra of 6 with those of malayoside (29),^3,16 showed that the two structures were very similar, except for that 6 did not contain the C-19 aldehyde group found in 29 based on the presence of a methine (−CH) proton at δ_H 1.43 (m), upfield shift of C-10 from δ_C 51.2 to δ_C 34.0, and absence of CHO signals. Compound 6 was thus assigned to be 19-nor-malayoside and named antiaritoxioside F.

Compound 7 (C₂₉H₄₅O₁₀) was 16 mass units larger than 11.¹⁴ The ¹H/¹³C NMR spectra of 7 and 11 were very similar, except for the sugar moiety signals and one oxygenated carbon in 7. The spectra of 11 showed a proton signal at δ_H 2.81 (dd, J = 8.9, 5.1 Hz) and a carbon signal at δ_C 51.4 (C-17), while in 7, the former signal was absent and the latter signal was replaced by an oxygenated carbon signal at δ_C 87.8 (C-17). Thus, an OH group was attached at C-17 in 7, and this postulate was supported by a downfield shift of C-16 from δ_C 27.35 in 11 to δ_C 34.3 in 7, together with HMBC correlations of H-18 to C-17. An anomeric proton signal at δ_H 5.39 (d, J = 8.1 Hz) and carbon signals at δ_C 99.8, 69.6, 73.8, 73.7, 70.0, and 17.3 indicated the presence of an antiarosyl moiety. An NOE effect between H-3 and H-1′ placed the antiarosyl unit on C-3, and a W_1/2 coupling constant of 9.8 Hz for H-3 indicated α-orientation. Therefore, the structure 7 was established for antiaritoxioside G.

Compound 8 (C₂₁H₃₆O₅) had 21 signals in the ¹³C NMR spectrum corresponding to two methyl, ten methylene, five methine, and four quaternary carbon atoms. The ¹H and ¹³C NMR spectra of 8 displayed signals characteristic of the steroid skeleton of a cardenolide. However, the absence of typical signals for the olefinic group and carbonyl carbon of the butenolactone ring, suggested that the five-membered lactone in 8 was absent. This hypothesis was confirmed by the presence of a methylene group at δ_C 65.8/ δ_H 3.89 (1H, dd, J = 10.5, 6.1 Hz) and 4.08 (1H, dd, J = 10.5, 7.2 Hz, H-21) and one methine at δ_C 71.1/δ_H 4.36 (1H, dd, J = 7.2, 6.1 Hz, H-20), as well as the absence of any significant UV absorption. In addition to three OH groups on the steroid skeleton (C-3, C-5 and C-14), two OH groups were assigned at C-20 and C-21, based on carbon signals at δ_C 71.1 and 65.8, respectively. Orientation of the C-3 OH was also determined by the W_1/2 of H-3 (br s, W_1/2 = 11.3 Hz). The above analysis established the structure of 8 as shown. This compound was reported as an intermediate in the synthesis of periplogenin by Peoghenghi et al.¹⁷ It has been isolated from a natural source for the first time and named antiarotoxinin B.

Compound 41 was obtained as colorless syrup, and its molecular formula was determined to be C₁₅H₁₈O₆ on the basis of HREIMS ([M]⁺, m/z 294.1100). The IR spectrum showed absorption bands at 3400 and 1711 cm⁻¹ for a hydroxyl group and α, β-unsaturated lactone, respectively. UV absorptions at 316 and 258 nm,¹⁸ and two AB type system protons at δ_H 7.94 and 6.32 (each 1H, d, J = 9.6 Hz, H-4 & -3) suggested that 41 was a coumarin derivative.¹⁹ The ¹H NMR spectrum of 41 also showed an aromatic proton at δ_H 7.06 (1H, s), three hydroxy protons at δ_H 9.60, 4.39 and 4.23 (each 1H, D₂O exchangeable), one methoxy group at δ_H 3.79 (3H, s) and one 2,3-dihydroxyisopentyl group at δ_H 1.12 and 1.10 (each 3H, s), 3.36 (1H, m), 2.95 (1H, d, J = 13.2 Hz) and 2.39 (1H, dd, J = 13.2, 10.4 Hz). The ¹³C NMR spectrum of 41 revealed the presence of 15 carbons resonances including two methyl, one methoxy, one methylene, four methine and seven quaternary carbons. Comparison of the ¹H and ¹³C NMR spectra of 41 with those of skimminan, which was isolated from Skimmia anquetelia by Sharma et al.,²⁰ showed that the two structures were very similar, except for the absence of signals for a sugar and one additional methoxy group (δ_H 3.79, δ_C 60.5) in the former. The HMBC spectrum showed correlations of the methoxy group at δ_H 3.79 with C-7 (δ_C 149.9) and of H-1′ (δ_H 2.95, and 2.39) with C-5 (δ_C 119.7), C-6 (δ_C 131.0) and C-7 (δ_C 149.9). Therefore, the methoxy group was attached at C-7 and the 2, 3-dihydroxyisopentyl moiety was located at C-6. This assignment was further confirmed by NOE effects between H-4 (δ_H 7.94) and H-5 (δ_H 7.06), as well as H-5 and H-1′ for 41. From the above data, new compound 41 was elucidated as 8-hydroxy-7-methoxy-6-[2′, 3′-dihydroxyisopentyl]coumarin, and has been designated as anticarin A.

Compound 42, ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +38.2° (c 0.25, MeOH), was isolated as colorless needles. The HRFABMS exhibited a pseudo-molecular ion at m/z 425.1367 for [M+H]⁺, consistent with the molecular formula C₂₀H₂₄O₁₀. UV absorptions at 326, 302 (sh), 259, 247 and 220 (sh) nm,¹⁸ and IR band at 1709 (OC=O) cm⁻¹, as well as two AB type protons at δ_H 7.96 (1H, d, J = 9.2 Hz, H-4) and 6.26 (1H, d, J = 9.2 Hz, H-3) suggested that 42 was a coumarin derivative.¹⁹ In the ¹H NMR spectrum, the presence of two additional AB type protons at δ_H 7.54 and 6.76 (each 1H, d, J = 8.8 Hz), two vicinal methine protons at δ_H 4.84 (1H, d, J = 2.4 Hz) and 3.94 (1H, dd, J = 4.4, 2.4 Hz), and two methyl groups at δ_H 1.27 (6H, s), indicated that 42 was a khellactone glycoside.²¹ The signal at δ_H 4.84 was assigned as H-4′ based on its correlations to C-7 (δ_C 156.6), C-9 (δ_C 155.0), C-8 (δ_C 108.6), and C-1″ (δ_C 103.8) in the HMBC spectrum, whereas the signal at δ_H 3.94 was assigned as H-3′ based on its correlation to C-3′ in the HMQC spectrum, as well as long range correlations of two methyl groups (δ_H 1.27) to C-3′ (δ_C 70.6). An anomeric proton signal at δ_H 4.71 (1H, d, J = 8.0 Hz, H-1″) in the ¹H NMR spectrum and signals at δ_C 103.8, 74.0, 76.8, 70.3, 77.3 and 61.5 in the ¹³C NMR spectrum suggested the presence of β-glucose. The coupling constant of the anomeric doublet (δ_H 4.71, 8.0 Hz) suggested a β-glucosidic linkage for the khellactone. The FAB-MS spectrum showed an ion fragment at m/z 263[M⁺-162], also indicating the presence of a hexose moiety. The β-glucose unit was placed at C-4′ on the basis of HMBC correlation between H-1″ of the glucose unit and C-4′ of the aglycone moiety, together with NOE correlation between H-1″ and H-4′. Accordingly, the structure of 42 was established as 4′-β-glucosyl-khellactone, and 42 has been named anticarin B.

Compound 43 was obtained as colorless obtained as colorless needles. The molecular formula formula was established as C₂₁H₂₂O₆ from m/z 370.1412 [M]⁺ in HREIMS. The IR spectrum of 43 showed absorption bands at 3456 and 3175 cm⁻¹ for one or more hydroxy group(s) and 1638 cm⁻¹ for a conjugated carbonyl functionality. The presence of a flavanone skeleton was evident from UV [226 (sh), 288, and 328 (sh) nm],¹⁸ and ¹H [δ_H 2.66 (1H, dd, J =17.2, 2.8 Hz, H-3α), 3.16 (1H, dd, J = 17.2, 13.2 Hz, H-3β) and 5.62 (1H, dd, J = 13.2, 2.8 Hz, H-2)] and ¹³C [δ_C 79.9 (C-2), 43.1 (C-3) and 197.4 (C-4)] NMR spectra. The ¹H NMR spectrum further showed three hydroxy groups [δ_H 12. 17, 9.69 and 7.55 (each 1H, s)], two singlet aromatic protons [δ_H 5.96 (2H, s)], AB system aromatic protons [δ_H 6.90 (1H, d, J =8.4 Hz) and 7.06 (1H, d, J = 8.4 Hz)], a methoxy moiety [δ_H 3.86 (3H, s)], and an isoprenyl group [δ_H 1.67 (3H, s), 1.63 (3H, s), 3.52 (2H, d, J = 6.4 Hz) and 5.13 (1H, t, J = 6.4 Hz)]. The locations of these substituents were determined by HMQC and HMBC experiments. Based on ³J-correlations between the proton at δ_H 5.96 with C-5 (δ_C 165.1), C-7(δ_C 167.2), and C-10 (δ_C 103.0) in the HMBC experiment and with C-6 in a HMQC spectrum, the singlet aromatic protons were assigned to H-6 and H-8, respectively, of ring-A HMBC correlations of the proton at δ_H 6.90 to C-1′ (δ_C 130.5) and C-3′ (δ_C 144.8) and at δ_H 7.06 to C-2 (δ_C 79.9) and C-2′ (δ_C 126.9) suggested that the AB system aromatic protons belonged H-5′ and H-6′, respectively. Combined with HMBC correlations of H-1″ (δ_H 3.52) to C-1′ (δ_C 130.5), C-2′ (δ_C 126.9), C-3′ (δ_C 144.8), and C-2″ (δ_C 124.0), and methoxy protons (δ_H 3.86) to C-4′ (δ_C 148.0) implied that the isoprenyl group was attached to C-2′, while the methoxy group was located at C-4′. These assignments were further confirmed by an NOE effect between the methoxy group and H-5′ (δ_H 6.90). Finally, a hydroxy group was attached to C-3′. The S-configuration of C-2 was determined by a positive Cotton effect observed at 312 nm and a negative one at 286 nm in the circular dichroism (CD) spectrum of 43.²² Based on these observations, 43 was established as (2S)-3′,5,8-trihydroxy-4′-methoxy-6,7-methylenedioxy-2′-prenylflavanone, and named antiarone L.

The molecular formula of 44 was determined as C₂₅H₂₆O₆ from a molecular ion peak at m/z 423.1733 [M+H]⁺ in its HRESIMS. The UV (324, 286, and 231 nm),^{18 1}H, and ¹³C NMR spectra indicated that 44 was also a flavanone. The ¹H and ¹³C NMR spectra of 44 closely resembled those of 43 except that signals for a 2,2-dimethylpyran ring unit were present at δ_H 6.40 (1H, d, J = 9.8 Hz, H-4″′), 5.73 (1H, d, J = 9.8 Hz, H-3″′), 1.20 and 1.18 (6H, s, 2″′-CH₃) and δ_C 122.7 (C-4″′), 131.3 (C-3″′), 77.5 (C-2″′), 29.1 & 32.4 (2″′-CH₃) in 44, and the methoxy group found 43 was absent. Correlations of H-1″ [δ_H 3.48 (2H, d, J = 6.8 Hz)] to C-1′ (δ_C 130.1), C-2′ (δ_C 124.0) and C-3′ (δ_C 143.8) in the HMBC spectrum indicated an isoprenyl group was attached to C-2′, whereas long range correlations of H-4″′ (δ_H 6.40) to C-4′ (δ_C 140.5), C-5′ (δ_C 133.1) and C-6′ (δ_C 120.0) and of H-3″′ (δ_H 5.73) to C-5′ suggested that the 2,2-dimethylpyran ring was fused to C-5′ and 4′. The absolute stereochemistry of 44 was deduced to be 2S because of a positive Cotton effect at 314 nm and a negative one at 290 nm in the circular dichroism (CD) spectrum of 44.²² Thus, the structure of compound 44 was assigned as (2S)-2″′,2″′-dimethylpyrano-3′,5,8-trihydroxy-2′-prenylflavanone, and named antiarone K.

Antiaroside R (9),⁴ toxicarioside M (10),¹⁵ periplorhamnoside (11),¹⁴ antiaroside A (12),³ peripalloside (13),¹⁴ cheiranthoside (14),²³ strophanthidol (15),²⁴ convallatoxal (16),¹⁶ cannogenol (17),²⁵ antiaroside B (18),³ strophanthidin (19),¹⁴ convallatoxin (20),¹⁶ strophathojavoside (21),²⁶ desglucocheirotoxin (22),²⁴ strophalloside (23),¹⁴ convalloside (24),²⁷ glucostrophalloside (25),²⁸ cheirotoxin (26),²⁹ antiaroside C (27),³ antiaroside D (28),³ malayoside (29),^3,16 antiarigenin (30),¹⁴ α-antiarin (31),¹⁶ antialloside (32),²⁶ toxicarioside B (33),³⁰ β-antiarin (34),¹⁶ antiaroside E (35),³ antiaroside F (36),³ antiaroside G (37),³ antiaroside H (38),³ antiaroside I (39),³ antiarotoxinin A (40),³ (+)-marmesin (45),³¹, decursinol (46),²¹ (R)-peucedanol (47),³² oxypeucedanin hydrate (48),³³ (2S)-pinocembrin (49),³⁴ antiarones F (50),⁶ I (51),⁶ vanillic acid (52),³⁵ p-hydroxybenzoic acid (53),³⁶, vanillin (54),³⁵ (3S,4R)-hydroxymellein (55),³⁷ 2, 4,6-trihydroxy-5-methoxyactophenone (56),³⁸ 6-acetoxy-2,4-dihydroxyacetophenone (57),³⁹ syringic aicd (58),⁴⁰ koaburaside (59),⁴¹ vanilloyl β-d-glucose (60),⁴² uridine (61),⁴³ xanthine (62),⁴⁴ adenosine (63),³⁶ 3-hydroxy-5-methoxybenzalacetophenone (64),⁴⁵ and β-sitosterol (65)³⁵ were also isolated and identified by comparison of their physical and spectroscopic properties with those reported in the literature.

Cardenolides 11–40 and coumarins 41, 42, and 45 were assayed for anti-proliferative effects against 10 human cancer cell lines, KB, KB-VIN, A549, MCF-7, U-87-MG, PC-3, 1A9, CAKI-1, HCT-9, and S-KMEL-2.⁴⁶ The cytotoxic data are shown in Table 3. Among the tested isolates, compounds 11, 12, 16, 17, 19–25, 29, 31–35, and 42 exhibited potent effects against all cancer cell lines. Among them, compounds 12, 16, 20, 22, and 31 showed ng/mL potency against one or more cancer cell lines. Compound 12 exhibited an ED₅₀ of 0.0097 μg/mL against KB cancer cell lines. Compound 16 demonstrated ED₅₀ values of 0.0040, 0.0076, and 0.0061 μg/mL against A549, 1A9 and CAKI-1 cells, respectively. Compound 20 showed ED₅₀ values of 0.0027, 0.0065, 0.0031, 0.0014 and 0.0013 μg/mL against A549, MCF-7, CAKI-1, HCT-9 and s-KMEL-2 cells, respectively. Compound 22 exhibited an ED₅₀ of 0.0092 μg/mL against A549 cells. Finally, compound 31 showed an ED₅₀ of 0.0044 μg/mL against PC-3 cells. Common structural features in all five compounds included a 5β-OH, 13β-Me, 14β-OH, and 17β-α, β-unsaturated-γ-butyrolactone. Compounds 20, 22, and 31 contained a C-18 β-CHO, while 12 and 16 contained β-CH₃ and −CH₂OH, respectively. Compound 31 contained a C-12 β-OH, while compounds 12, 16, 20, and 22 were unsubstituted. All five compounds contained a 3β-sugar moiety, 3β-O-β-d-antiarose in 12, 22, and 31, 3β-O-α-l-rhamnose in 16 and 20.

Table 3. Cytotoxicity (ED₅₀ μg/mL) of isolated compounds from A. toxicaria^a.

Compound	KB*	KB-VIN	A549	MCF-7	U-87-MG	PC-3	1A9	CAKI-1	HCT-9	S-KMEL-2
11	0.054	0.020	0.012	0.018	0.082	0.021	0.012	0.014	0.044	0.017
12	0.0097	0.068	0.044	0.063	0.15	0.078	0.020	0.018	0.090	0.073
14	1.5	1.7	0.12	0.58	1.9	0.60	0.53	0.51	0.94	0.16
15	2.7	3.0	0.14	0.69	2.9	0.81	0.66	0.67	6.4	0.57
16	0.025	0.014	0.0040	0.012	0.036	0.013	0.0076	0.0061	0.020	0.014
17	0.45	0.50	0.050	0.21	0.50	0.15	012	0.11	0.46	0.70
19	0.61	0.69	0.11	0.38	0.72	0.17	0.15	0.14	0.51	0.13
20	0.014	0.013	0.0027	0.0065	0.014	0.094	0.030	0.0031	0.0014	0.0013
21	0.066	0.051	0.016	0.042	0.078	0.052	0.013	0.014	0.067	0.064
22	0.044	0.018	0.0092	0.015	0.054	0.018	0.012	0.014	0.044	0.015
23	0.081	0.050	0.015	0.024	0.043	0.063	0.013	0.014	0.085	0.063
24	0.2	0.18	0.070	0.15	0.40	0.095	0.072	0.071	0.20	0.42
25	0.12	0.083	0.038	0.096	0.15	0.084	0.057	0.065	0.13	0.12
27	1.5	1.6	0.48	1.4	1.7	0.42	0.39	0.32	1.5	0.26
28	1.7	1.9	0.38	1.0	4.2	0.53	0.74	0.93	1.9	0.31
29	0.30	0.32	0.080	0.19	0.37	0.11	0.076	0.073	0.18	0.62
30	5.3	5.1	0.59	1.3	4.6	1.2	0.12	1.3	4.5	0.41
31	0.14	0.15	0.059	0.10	0.017	0.0044	0.058	0.072	0.15	0.13
32	0.065	0.075	0.046	0.12	0.15	0.085	0.062	0.068	0.11	0.12
33	0.13	0.14	0.058	0.092	0.13	0.091	0.063	0.070	0.11	0.12
34	0.067	0.061	0.013	0.018	0.071	0.050	0.014	0.015	0.066	0.065
35	0.49	0.57	0.070	0.42	0.73	0.16	0.13	0.13	0.35	0.12
36	1.8	2.2	0.54	1.3	4.5	0.89	0.97	1.2	3.4	0.32
37			5.3	17.5
38	5.9	7.1	0.76	3.1	7.3	1.8	1.4	1.5	2.5	1.3
39	2.0	2.3	0.36	1.3	3.6	0.90	0.83	1.1	2.9	0.30
40			4.3	15.0
41			10.8	>20		1.5	1.5	1.5	6.7	1.3
42	5.8	4.1	1.2	3.1	7.4
45			>20	>20

^aCompounds 1–9 were not tested due to insufficient quantities remaining.

^*Cell line, ED₅₀ in μg/mL.

The following specific structure-activity correlations were noted in these studies. Changing the orientation of the glycoside from 3β in potent cardenolide 20 to 3α in 27 significantly decreased the cytotoxicity, for example, from 0.0027 to 0.48 μg/mL against A549, from 0.0065 to 1.4 against MCF-7, from 0.0031 to 0.31 μg/mL against CAKI-1, from 0.0014 to 1.5 μg/mL against HCT-9, and from 0.0013 to 0.26 μg/mL against s-KMEL-2. Compound 28 with a 3α-diglycoside was also one of the least potent cardenolides. Activity decreased dramatically when the 3β-O-α-l-rhamnose of 16 (ED₅₀ values 0.0.0040–0.036 μg/mL) was changed to 3β-OH in 15 (ED₅₀ values 0.14–6.5 μg/mL). Replacement of the C-10 CH₃ of 12 with COOH in 38 together with addition of a 12β-OH decreased cytotoxicity from 0.0097 to 5.9 μg/mL against KB cells. Similarly, changing the C-10 CH₂OH of 16 to H in 39 decreased cytotoxicity from 0.0040 to 0.36 μg/mL against A549 cells. Compounds showing the lowest potency against the cancer cell lines included 14, 15, 27, and 28 with ED₅₀ values ranging from 0.12 to 4.2 μg/mL, as well as 30 and 36–40 with ED₅₀ values of 0.36–17.5 μg/mL. Notably, a C-10 COOH, COO-glucose, or H was found in 36–39 rather than CHO, CH₃, or CH₂OH as found in the more potent compounds. While the cytotoxic effects of new compounds 1–6 were not examined in this report,⁴⁶ the cytotoxic effects of structurally similar cardiac glycosides have been reported.^4,10 Our observations were generally consistent with the prior findings (for example, compound 9 with COO-glucose at C-10 and compounds with α-oriented substituents at C-3 exhibited lower cytotoxicity).⁴ In summary, from our investigation, the β-orientation of C-3, C-3 monosaccharide, and C-10 substituents (CHO, CH₃, or CH₂OH) are crucial factors in the cytotoxicity effects of cardiac glycosides. Prior reports also indicated that compounds with C-10 OH may exhibit significant cytotoxicity.¹⁰

Among the three tested coumarins, compound 41 and the structurally related linear dihydrofuranocoumarin 45 showed no cytotoxic activity, whereas the angular dihydropyranocoumarin 42 exhibited broad and significant cytotoxicity against KB-VIN, A549, MCF-7, PC-3, 1A9, CAKI-1 and S-KMEL-2 cell lines with ED₅₀ values ranging from 1.2 to 4.1 μg/mL.

It is a definite possibility that the structurally similar cardiac glycoside metabolites were derived biogenetically from a general catabolic process. A possible biogenetic transformation pathway is presented in Scheme 1 for the first time on the basis of evidence from natural sources. The proposed biosynthesis could explain the catabolic progression of structurally interesting cardiac glycosides.

Scheme 1.

Biosynthetic pathway of cardiac glycosides.

3. Conclusions

In summary, this paper described the isolation and structure elucidation of 12 new compounds, including eight cardiac glycosides/aglycones, antiaritoxiosides A-G (1–7) and antiarotoxinin B (8), two coumarins, anticarin A–B, and two flavanones, antiarones L–K. The cytotoxic activity of cardiac glycosides and coumarins against ten cancer cell lines also reported. Compounds 12, 16, 20, 22, and 31 showed significant cytotoxicity against all 10 cancer cell lines, with notable potency at the ng/mL level against some cell lines. SAR correlations were also identified. While many cardiac glycosides are reported in the literature, compounds with 10-carboxy (e.g., 1, 9) and 3α-hydroxy (e.g., 1) substituents are rare. Furthermore, the C-10 substituents, including COOH, CHO, CH₂OH, CH₃, OH and H, of cardiac glycosides can help to explain the catabolic progression of structurally interesting cardiac glycosides.

4. Experimental

4.1. General procedures

Optical rotations were measured with a JASCO DIP-370 polarimeter. IR spectra were determined as KBr discs on a Shimazu FTIR-8501 spectrophotometer. UV spectra were acquired on a Hitachi UV-3210 spectrophotometer. The ¹H and ¹³C NMR spectra were recorded on Bruker Avance 300 (¹H at 300 MHz) and AMX 400 (¹H at 400 MHz) spectrometers. The ¹H and ¹³C chemical shifts (ppm) were referenced to the solvent peaks for pyridine-d₅ at δ_H 8.71 and δ_C 149.9, DMSO-d₆ at δ_H 2.49 and δ_C 39.5, acetone-d₆ at δ_H 2.05 and δ_C 29.9, 206.7, and methanol-d₄ at δ_H 3.31 and δ_C 49.0. ESIMS, FABMS and HRMS were taken with a VG 70–250 S spectrometer by direct inlet system. Merck silica gel 60 (Merck 70–230, 230–400 mesh) was used for column chromatography. Glass sheets of silica gel 60 F₂₅₄ (Merck 0.2 mm thick) were used for TLC Melting points were measured on a Yanagimoto MP-S3 micromelting point apparatus and are uncorrected.

4.2. Plant material

Trunk bark of A toxicaria was collected from Yunnan, China, and authenticated by C.S. Kuoh (Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan). A voucher specimen (NCKUWu 92012) has been deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan, R.O.C.

4.3. Extraction and isolation

Trunk bark of A toxicaria (6.0 kg) was cut into small pieces and extracted with 95% EtOH (20 L × 3). Evaporation of the solvent under reduced pressure provided 239.0 g of crude extract, which was partitioned between CHCl₃−H₂O, and n-BuOH−H₂O, successively, to yield CHCl₃ (AT-C, 65.1 g), n-BuOH (AT-B, 100.2 g), and H₂O (AT-W, 73.7 g) fractions. The CHCl₃ fraction was subjected to silica gel CC using increasing polarity mixtures of n-hexanes-acetone as eluant to give 14 fractions (AT-C-1 ∼ 14). AT-C-2 was recrystallized to obtain 65 (42.5 mg). AT-C-3 was chromatographed on silica gel using n-hexane–diisopropyl ether (3:1) to obtain 56 (1.2 mg), 57 (1.7 mg), and 64 (11.5 mg). AT-C-4 was chromatographed on silica gel using n-hexane–diisopropyl ether (3:1) to obtain 43 (12.4 mg), 49 (3.1 mg), 50 (10.0 mg), 51 (175.4 mg), 55 (1.2 mg), and 64 (2.4 mg). AT-C-5 was chromatographed on silica gel eluted with n-hexane–diisopropyl ether (1:1) to give 43 (18.0 mg), 44 (1.0 mg), 49 (3.7 mg), 50 (17.1 mg), and 55 (2.4 mg). AT-C-6 was chromatographed on silica gel using diisopropyl ether−MeOH (40:1) to obtain 7 (8.7 mg), 19 (103.2 mg), 46 (3.1 mg), 52 (8.7 mg), 54 (12.3 mg), and 65 (12.5 mg). AT-C-7 was chromatographed on silica gel and eluted with n-hexane–diisopropyl ether (1:1) to give 15 (6.6 mg) and 21 (7.0 mg). AT-C-8 was chromatographed on silica gel using EtOAc−MeOH (20:1) to obtain 6 (2.1 mg), 8 (19.6 mg), 17 (3.2 mg), 29 (8.8 mg), 35 (37.6 mg), and 39 (7.5 mg). AT-C-9 and 10 were chromatographed on silica gel using EtOAc−MeOH (20:1) to obtain 29 (21.4 mg), and 20 (117.2 mg), respectively.

The n-BuOH fraction was subjected to Diaion HP-20 CC eluting with a H₂O−MeOH gradient system to give 12 fractions (AT-B-1 ∼ 12). AT-B-4 and 5 were chromatographed on silica gel using CHCl₃−MeOH (9:1) to obtain 4 (1.3 mg), 5 (1.5 mg), 30 (58.9 mg), 31 (11.0 mg), 32 (4.3 mg), 34 (7.3 mg), 37 (34.7 mg), 38 (147.4 mg), 52 (10.0 mg), 53 (10.2 mg), and 54 (5.5 mg), successively. AT-B-6 was chromatographed on silica gel using CHCl₃−MeOH (9:1) to obtain 31 (118.4 mg). AT-B-7 and 8 were chromatographed on silica gel using CHCl₃−MeOH−H₂O (9:1:005) to obtain 1 (3.0 mg), 2 (3.8 mg), 10 (0.5 mg), 14 (9.1 mg), 16 (6.1 mg), 18 (12.3 mg), 20 (431.2 mg), 22 (4.3 mg), 23 (5.9 mg), 24 (91.2 mg), 25 (18.3 mg), 26 (0.5 mg), 27 (112.3 mg), 28 (12.2 mg), 36 (5.9 mg), 41 (25.3 mg), 42 (5.8 mg), 47 (6.7 mg), and 58 (2.7 mg), successively. AT-B-9 and 10 were chromatographed on silica gel using CHCl₃−MeOH−H₂O (9:1:0.05) to obtain 3 (0.3 mg), 11 (10.9 mg), 12 (9.8 mg), 13 (1.9 mg), 20 (12.5 mg), 22 (29.9 mg), 23 (17.8 mg), 33 (13.5 mg), 40 (0.7 mg), 45 (1.2 mg), 48 (0.9 mg), and 53 (2.8 mg), successfully.

The H₂O fraction was subjected to Diaion HP-20 CC eluting with a H₂O−MeOH gradient system to give 10 fractions (AT-W-1 ∼ 10). AT-W-2 and 3 were chromatographed on silica gel using EtOAC−MeOH−H₂O (4:1:0.5) to obtain 61 (2.3 mg), and 62 (10.7 mg). AT-W-4 was chromatographed on silica gel using EtOAC−MeOH−H₂O (4:1:0.5) to obtain 59 (22.4 mg), 60 (29.0 mg), and 63 (1.2 mg). AT-W-5 was chromatographed on silica gel using CHCl₃−MeOH−H₂O (4:1:0.005) to obtain 59 (12.6 mg). AT-W-6 and 7 was chromatographed on silica gel using EtOAC−MeOH−H₂O (4:1:0.5) to obtain 58 (17.3 mg). AT-W-8 and 9 were chromatographed on Sephadex LH-20 using a H₂O−MeOH gradient system to obtain 9 (0.7 mg), 20 (43.2 mg), 24 (26.8 mg), 28 (26.4 mg), 31 (20.2 mg) and 32 (1.3 mg), successfully.

4.4. Antiaritoxioside A (1)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +189.13° (c 0.01 in MeOH); UV λ_max (MeOH)/nm (log ε) 216 (4.21); IR ν_max/cm⁻¹ (KBr) 3400, 2930, 1734, 1651, 1506, 1456, 1394, 1229, 1026; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 567 (13%) [M+H]⁺; HRFABMS m/z 567.2808 (C₂₉H₄₃O₁₁ [M+H]⁺ requires 567.2805).

4.5. Antiaritoxioside B (2)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −9.52° (c 0.04 in MeOH); UV λ_max(MeOH)/nm (log ε) 215 (4.31); IR ν_max/cm⁻¹ (KBr) 3440, 2937, 1744, 1647, 1634, 1454, 1032; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 539 (8%) [M+H]⁺; HRFABMS m/z 539.2855 (C₂₈H₄₃O₁₀ [M+H]⁺ requires 539.285).

4.6. Antiaritoxioside C (3)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −242.2° (c 0.003 in MeOH); UV λ_max (MeOH)/nm (log ε) 213 (4.14); IR ν_max/cm⁻¹ (KBr) 3410, 2928, 1734, 1647, 1636, 1541, 1456, 1076, 1036; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 539 (6%) [M+H]⁺; HRFABMS m/z 539.2855 (C₂₈H₄₃O₁₀ [M+H]⁺ requires 539.2856).

4.7. Antiaritoxioside D (4)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −45.88° (c 0.01 in MeOH); UV λ_max (MeOH)/nm (log ε) 214 (4.17); IR ν_max/cm⁻¹ (KBr) 3410, 2932, 1742, 1649, 1454, 1377, 1029; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 555 (3%) [M+H]⁺; HRFABMS m/z 555.2805 (C₂₈H₄₃O₁₁ [M+H]⁺ requires 555.2805).

4.8. Antiaritoxioside E (5)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −69.72° (c 0.02 in MeOH); UV λ_max (MeOH)/nm (log ε) 271 (3.63), 218 (4.36); IR ν_max/cm⁻¹ (KBr) 3410, 2945, 1732, 1640, 1630, 1452, 1385, 1171, 1109, 1078,1028, 997; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 555 (8%) [M+H]⁺; HRFABMS m/z 555.2802 (C₂₈H₄₃O₁₁ [M+H]⁺ requires 555.2805).

4.9. Antiaritoxioside F (6)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −15.28° (c 0.02 in MeOH); UV λ_max (MeOH)/nm (log ε) 216 (4.32); IR ν_max/cm⁻¹ (KBr) 3410, 2926, 1742, 1645, 14474, 1377, 1049; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 529 (7%) [M+Na]⁺, 507 (5%) [M+H]⁺; HRFABMS m/z 529.2774 (C₂₈H₄₂-NaO₈ [M+Na]⁺ requires 529.2777).

4.10. Antiaritoxioside G (7)

Colorless powder (CHCl₃−MeOH); mp 227–228 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −57.69° (c 0.01 in MeOH); UV λ_max (MeOH)/nm (log ε) 215 (4.62); IR ν_max/cm⁻¹ (KBr) 3350, 2949, 1753, 1383, 1286, 1175, 1101, 1022, 991; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; FABMS m/z 553 (2%) [M+H]⁺; HRFABMS m/z 553.3014 (C₂₉H₄₅O₁₀ [M+H]⁺ requires 553.3012).

4.11. Antiarotoxinin B (8)

Colorless powder (CHCl₃−MeOH); mp >280 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −31.34° (c 0.02 in MeOH); IR ν_max/cm⁻¹ (KBr) 3261, 2936, 2882, 1470, 1448, 1383, 1088, 1047; ¹H NMR data (pyridine-d₅) see Table 2; ¹³C NMR data (pyridine-d₅) see Table 1; EIMS m/z 368 (8%) [M]⁺, 350 (28%), 319 (39%), 301 (41%), 296 (100%), 290 (15%), 283 (20%), 272 (14%), 147 (12%), 121 (15%), 107 (28%), 97 (39%); HREIMS m/z 368.2560 (C₂₁H₃₆O₅ [M]⁺ requires 368.2563).

4.12. Anticarin A (41)

Colorless syrup; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +16.3° (c 0.058, MeOH); UV λ_max (MeOH)/nm (log ε) 316 (3.60), 258 (3.48); IR ν_max/cm⁻¹ (KBr) 3400, 2926, 1711, 1607, 1572, 1450, 1279; ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.60 (1H, br s, D₂O exchangeable, OH), 7.94 (1H, d, J = 9.6 Hz, H-4), 7.06 (1H, s, H-5), 6.32 (1H, d, J = 9.6 Hz, H-3), 4.39 (1H, d, J = 6.0 Hz, D₂O exchangeable, 2′-OH), 4.23 (1H, br s, D₂O exchangeable, 3′-OH), 3.79 (3H, s, OCH₃), 3.36 (1H, m, H-2′), 2.95 (1H, d, J = 13.2 Hz, H-1′), 2.39 (1H, dd, J = 13.2, 10.4 Hz, H-1′), 1.12 (3H, s, H-4′), 1.10 (3H, s, H-5′); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 160.3 (C-2), 149.9 (C-7), 145.0 (C-4), 142.3 (C-9), 137.2 (C-8), 131.0 (C-6), 119.7 (C-5), 115.0 (C-10), 114.0 (C-3), 77.8 (C-2′), 71.9 (C-3′), 60.5 (OCH3), 31.4 (C-1′), 26.6 (C-5′), 24.8 (C-4′); EIMS m/z 294 (28%) [M]⁺, 261 (7%), 236 (79%), 235 (37%), 221 (23%), 205 (100%), 193 (56%), 177 (20%), 163 (25%), 147 (22%), 59 (84%); HREIMS m/z 294.1100 (C₁₅H₁₈O₆ [M]⁺ requires 294.1103).

4.13. Anticarin B (42)

Colorless needles (MeOH); mp. 265–267 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +38.2° (c 0.2523, MeOH); UV λ_max (MeOH)/nm (log ε) 326 (4.18), 302 (3.92, sh), 259 (3.46), 247 (3.50), 220(4.13, sh), 204 (4.67); IR ν_max/cm⁻¹ (KBr) 3300, 1709, 1605, 1592, 1541, 1306, 1126, 1076, 1036; ¹H NMR (400 MHz, DMSO-d₆) δ_H 7.96 (1H, d, J = 9.2 Hz, H-4), 7.54 (1H, d, J = 8.8 Hz, H-5), 6.76 (1H, d, J = 8.8 Hz, H-6), 6.26 (1H, d, J = 9.2 Hz, H-3), 5.29 (1H, d, J = 4.4 Hz, D₂O exchangeable, 3′-OH), 4.93 (2H, m, D₂O exchangeable, 3″, 4″-OH), 4.84 (1H, br. d, J = 2.4 Hz, H-4′), 4.71 (1H, d, J = 8.0 Hz, H-1″), 4.52 (1H, br s, D₂O exchangeable, 4′-OH), 4.42 (1H, t, J = 6.0 Hz, D₂O exchangeable, 6″-OH), 3.94 (1H, dd, J = 4.4, 2.4 Hz, H-3′), 3.76 (1H, br. dd, J = 12.0, 6.0 Hz, H-6″), 3.50 (1H, br s dd, J = 12.0, 6.0 Hz, H-6″), 3.25 (1H, m, H-5″), 3.22 (1H, t, J = 8.4 Hz, H-3″), 3.05 (1H, m, H-4″), 2.90 (1H, t, J = 8.4 Hz, H-2″), 1.27 (6H, s, 20-CH₃ × 2); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 160.1 (C-2), 156.6 (C-7), 155.0 (C-9), 144.8 (C-4), 129.1 (C-5), 114.1 (C-6), 112.0 (C-3, 10), 108.6 (C-8), 103.8 (C-1″), 78.5 (C-2′), 77.3 (C-5″), 76.8 (C-3″), 74.0 (C-2″), 71.4 (C-4′), 70.6 (C-3′), 70.3 (C-4″), 61.5 (C-6″), 24.9 & 24.3 (2′-CH₃ × 2); FABMS m/z 425 (23%) [M+H]⁺, 371 (8%), 307 (10%), 263 (75%), 245 (31%), 219 (74%), 154 (100%), 136 (86%); HRFABMS m/z 425.1367 (C₂₀H₂₅O₁₀ [M+H]⁺ requires 425.1369).

4.14. Antiarone L (43)

Colorless needles (acetone); mp. 131–133°C; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −3.0° (c 0.53, MeOH); UV λ_max (MeOH)/nm (log ε) 328 (3.63, sh), 288 (4.31), 226 (4.44, sh), 206 (4.86); IR ν_max/cm⁻¹ (KBr) 3456, 3175, 2964, 2924, 1638, 1605, 1497, 1460, 1375, 1277, 1163, 1082; CD (c 5.4 × 10⁻⁵, MeOH) [θ]₂₀₈ −9580, [θ]₂₂₄ +38450, [θ]₂₈₆ −22450, [θ]₃₁₂ +52330; ¹H NMR (400 MHz, acetone-d₆) δ_H 12.17 (1H, br s, D₂O exchangeable, 5-OH), 9.69 (1H, br s, D₂O exchangeable, 8-OH), 7.55 (1H, br s, D₂O exchangeable, 3′-OH), 7.06 (1H, d, J =8.4 Hz, H-60), 6.90 (1H, d, J = 8.4 Hz, H-5′), 5.96 (2H, s, H-6&8), 5.62 (1H, dd, J = 13.2, 2.8 Hz, H-2), 5.13 (1H, t, J = 6.4 Hz, H-2″), 3.86 (3H, s, OCH₃), 3.52 (2H, d, J = 6.4Hz, H-1″), 3.16 (1H, dd, J = 17.2, 13.2 Hz, H-3α), 2.66 (1H, dd, J = 17.2, 2.8 Hz, H-3β), 1.67&1.63 (each 3H, s, H-4″&5″); ¹³C NMR (acetone-d₆, 100 MHz) δ_C 197.4 (C-4), 167.2 (C-7), 165.1 (C-5), 164.4 (C-9), 148.0 (C-4′), 144.8 (C-3′), 131.4 (C-3″), 130.5 (C-1′), 126.9 (C-2′), 124.0 (C-2″), 118.2 (C-6′), 109.5 (C-5′), 103.0 (C-10), 96.7 (C-8), 95.7 (C-6), 79.9 (C-2), 56.2 (OCH₃), 43.1 (C-3), 25.7 & 17.9 (C-4″ & 5″), 25.0 (C-1″); EIMS m/z 370 (100%) [M]⁺, 352 (10%), 219 (12%), 153 (26%), 125 (18%); HREIMS m/z 370.1412 (C₂₁H₂₂O₆ [M]⁺ requires 370.1416).

4.15. Antiarone K (44)

Colorless powder (acetone); mp 171–173 °C; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −44.7° (c 0.001, MeOH); UV λ_max (MeOH)/nm (log ε) 324 (3.41, sh), 286 (4.06), 231 (4.35, sh), 213 (4.25); IR ν_max/cm⁻¹ (KBr) 3600–3000, 2963, 2926, 2855, 1639, 1522, 1466, 1456, 1340, 1271, 1161, 1085; CD (c 5.0 × 10⁻⁵, MeOH) [θ]₂₁₀ −10580, [θ]₂₂₉ +39460, [θ]₂₉₀ −25380, [θ]₃₁₄ +52190; ¹H NMR (acetone-d₆, 400 MHz) δ_H 12.18 (1H, br s, D₂O exchangeable, 5-OH), 7.70 (1H, br s, D₂O exchangeable, 3′-OH), 6.56 (1H, s, H-6′), 6.40 (1H, d, J = 9.8 Hz, H-1″′), 5.95 (2H, s, H-6 & 8), 5.73 (1H, d, J = 9.8 Hz, H-2″′), 5.60 (1H, dd, J = 13.4, 2.6 Hz, H-2), 5.13 (1H, t, J = 6.8 Hz, H-2″), 3.48 (2H, d, J = 6.8 Hz, H-1″), 3.17 (1H, dd, J = 17.0, 13.4Hz, H-3α), 2.64 (1H, dd, J = 17.0, 2.6 Hz, H-3β), 1.63&1.67 (each 3H, s, H-4″&5″), 1.18 & 1.20 (each 3H, s, 2″′-CH₃ × 2); ¹³C NMR (acetone-d₆, 100 MHz) δ_C 197.4 (C-4), 167.2 (C-7), 165.2 (C-5), 164.5 (C-9), 143.8 (C-3′), 140.5 (C-4′), 133.1 (C-5′), 131.3 (C-3″ & 3″′), 120.1 (C-1′), 127.1 (C-2′), 124.0 (C-2″), 122.7 (C-4″′), 120.0 (C-6′), 103.0 (C-10), 96.7 (C-8), 95.7 (C-6), 77.5 (C-2″′), 77.0 (C-2), 43.2 (C-3), 32.4 & 29.1 (C-2″′-CH₃ × 2), 25.7&17.9 (C-4″& 5″), 25.2 (C-1″); EIMS m/z 436 (100%) [M]⁺; 418 (10%), 286 (25%), 255 (18%); 153 (26%); HREIMS m/z 436.1889 (C₂₆H₂₈O₆ [M]⁺ requires 436.1886).

4.16. In vitro cytotoxicity assay

The sulforhodamine B assay was used according to the procedures developed and validated at NCI.⁴⁷ Doxorubicin was used as the positive control antitumor drug. The in vitro anticancer activities are expressed as ED₅₀ values, which is the test compound concentration (μg/mL) that reduced the cell number by 50% after 72 h of continuous treatment. The values were interpolated from dose-response data. Each test was performed in triplicate with variation less than 5%. The ED₅₀ values determined in each of independent tests varied less than 10%. Compound stock solutions were prepared in DMSO with the final solvent concentration ≤ 1% DMSO (v/v), a concentration without effect on cell replication. The cells were cultured at 37 °C in RPMI-1640 supplemented with 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2% (w/v) sodium bicarbonate, 10% (v/v) fetal bovine serum, and 100 μg/mL kanamycin in a humidified atmosphere containing 5% CO₂.