Acetogenins from the Stem of Uvaria rufa and Their Cytotoxic Activity

Abstract

Four new adjacent bis-tetrahydrofuran acetogenins, bullacin C (7), uvarirufin (9), uvariasolins III (12) and IV (13), along with eleven known acetogenins, were isolated from the stem of Uvaria rufa. Their structures were elucidated based on spectroscopic data analysis, including 1D and 2D NMR, HRESIMS and MALDI-MS/MS of the lithium adducts. Absolute configurations were assigned using Mosher ester analysis and ECD measurements. Uvarirufin (9) possesses a unique C-39 skeleton among acetogenins. Most tested acetogenins exhibited cytotoxicity against human cancer cell lines (HCT 116, 22Rv1, MDA-MB-435, OVCAR3). Squamocin (8) and uvarirufin (9) were found to be the most potent, with IC₅₀ value of 1.2 μM for both in HCT 116 colon cancer cells. Additionally, a new application of Dragendorff’s reagent is proposed herein for the TLC detection of acetogenins.

Annonaceous acetogenins are a series of C-35/C-37 polyketide compounds found in plants of the Annonaceae family.¹ Structurally, they consist of a long aliphatic chain bearing a terminal methylated α,β-unsaturated γ-lactone ring (sometimes rearranged to a ketolactone), with tetrahydrofuran (THF) and less often tetrahydropyran (THP) rings, as well as oxygenated functional groups, such as hydroxyl, acetoxy, keto and epoxy groups, along with double bonds located along the hydrocarbon chain.² They can be classified into mono-THF, adjacent bis-THF, nonadjacent bis-THF, linear and other types of acetogenins.^2,3 The biogenetic pathway of acetogenins is proposed to originate from long chain (C-32/C34) fatty acids combined with a 2-propanol unit at C-2, forming the lactone moiety.^1,4 Among the biological activities reported for the Annonaceous acetogenins, such as pesticidal, antiparasitic, immunosuppressive and neurotoxic effects, their cytotoxic and antitumor activities have received the most attention.¹ Acetogenins are potent inhibitors of complex I (NADH : ubiquinone oxidoreductase) in the mitochondrial electron transport system and they decrease the ATP production, leading to apoptosis.² Both in vitro cytotoxic activity against cancer cells and in vivo antitumor effects have been reported for the members of the acetogenin class, and they have been proposed as potential anticancer drug candidates.^1,3,5

Since the discovery of the first Anonaceous acetogenin, uvaricin, from Uvaria acuminata in 1982, over 500 acetogenins have been isolated and identified from different parts of the Annonaceae plants of the genera Annona, Asimina, Goniothalamus, Rollinia, Uvaria and others.^1,6,7 While there were no compounds of the acetogenin class reported previously from Uvaria rufa, our continued efforts to identify bioactive secondary metabolites of this plant ⁸ led to the isolation of fifteen acetogenins, including a new acetogenin (9) with a unique C-39 skeleton. MALDI-MS/MS analysis of the lithiated species of acetogenins was employed herein, as it was previously proved to be a valuable tool for structural identification of these compounds.⁹ This ionization method provided the MS/MS spectra containing informative fragments, especially the B and Y ions generated by a fragmentation across a tetrahydrofuran ring,¹⁰ allowing for unambiguous placement of the bis-THF ring system along the alkyl chain. Cytotoxic activity of the isolated compounds against several human cancer cell lines was evaluated using the MTT assay. Furthermore, a detection method of acetogenins using Dragendorff’s reagent is proposed.

RESULTS AND DISCUSSION

The hexane and ethyl acetate fractions of a methanol stem extract of Uvaria rufa were separated by column chromatography to yield 15 acetogenins, including four new compounds (7, 9, 12, 13). The isolated acetogenins, namely squamocin-K (1),¹¹ neoannonin (synonym: squamocin-J) (2),^11,12 isodesacetyluvaricin (3),¹³ desacetyluvaricin (4),¹⁴ narumicin I (synonym: uvarigrandin A) (5),^13,15 narumicin II (6),¹³ bullacin C (7), squamocin (8),¹⁶ uvarirufin (9), uvariasolin I (10),¹⁷ uvariasolin II (11),¹⁷ uvariasolin III (12), uvariasolin IV (13), panalicin (14),¹⁸ and microcarpacin(-A) (15),¹⁹ were characterized by spectroscopic methods, including NMR, HRESIMS, MALDI-MS/MS and ECD data, and by chemical transformations. The known compounds were identified by comparison of their spectroscopic data with those reported in the literature. All isolated acetogenins belong to the adjacent bis-THF group, and many of them are hydroxylated at C-5 and/ or C-6, which seems to be a characteristic feature of acetogenins from Uvaria species.^{1,13,17,18,20} Among the new structures, uvarirufin (9) was found to possess a unique C-39 skeleton, as opposed to the known acetogenins having C-37 or C-35 skeleton.

Bullacin C (7) was obtained as a white solid. The molecular formula was determined to be C₃₇H₆₆O₇ based on the positive ion HRESIMS that revealed a protonated molecular ion at m/z 623.4883 [M + H]⁺ and a sodium adduct ion at m/z 645.4707 [M + Na]⁺. An IR absorption at 1755 cm⁻¹ due to the conjugated carbonyl, UV maximum at 208 nm, and signals in the ¹H NMR spectrum at δ_H 7.02 (H-35), 5.00 (H-36), 1.41 (H-37), and in the ¹³C NMR spectrum at δ_C 174.0 (C-1), 134.2 (C-2), 149.3 (C-35), 77.6 (C-36), and 19.4 (C-37) indicated the presence of a methylated α,β-unsaturated γ-lactone ring (Tables 1 and 2).^21–23 The signal of one proton at δ_H 3.40 (H-15) and signals between δ_H 3.80–3.96 integrating for five protons (H-16, 19, 20, 23, 24), and those at δ_C 83.4 (C-16), 83.0 (C-23), 82.6 (C-20), 82.5 (C-19), 74.2 (C-15) and 71.5 (C-24) in the ¹H and ¹³C NMR spectra are characteristic of the presence of an adjacent bis-THF ring system flanked by two OH groups, with a threo/trans/threo/trans/erythro relative configuration (Tables 1 and 2).^23,24 A third OH appeared in the ¹H NMR spectrum at δ_H 3.62 (H-6) and in the ¹³C NMR at δ_C 71.7 (C-6). Its position at C-6 was predicted based on the chemical shifts at H-3 (δ_H 2.30) and H-35 (δ_H 7.02) which are more shielded than with C-5 hydroxylated (δ_H-3 ca. 2.40, δ_H-35 ca. 7.05) and C-4 hydroxylated (δ_H-3 ca. 2.51, 2.38, δ_H-35 ca. 7.17) acetogenins but more deshielded than in acetogenins without an OH group near the lactone ring (δ_H-3 ca. 2.26, δ_H-35 ca. 6.99).^24,25 The placement of the hydroxy group at C-6 was further confirmed by a TOCSY correlation between H-3 (δ_H 2.30) and H-6 (δ_H 3.62), indicating the proximity of the OH group to the lactone part. Further corroboration came from the peak in the MALDI-MS/MS at m/z 175 corresponding to a lithiated A₁ ion, whereas in C-5 hydroxylated narumicins this ion is showing at m/z 161 (Figure 1). The location of the bis-THF unit with flanking OH groups was determined to be between C-15 and C-24 based on the diagnostic B and Y ions in the MALDI-MS/MS spectrum of the lithium-cationized species at m/z 415 (B₂), 345 (B₁), 275 (Y₂), 205 (Y₁) (Figure 1). The absolute configurations of the carbinol centers of compound 7 were determined by analysis of the ¹H NMR and COSY data of the (S)-(MTPA) (7s) and (R)-MTPA (7r) ester derivatives (Table 3). The absolute configurations at C-15 and C-24 in 7 were determined to be R and S, respectively, by comparison of Δδ_S-R values from H-14 to H-25 with those of acetogenins with known configurations.^26–29 The stereochemistry of C-6 was established as S based on the positive Δδ_S-R value for H-3, H-35 and H-36 matching those of other C-6 hydroxylated acetogenins with 6S configuration, such as 6-OH desacetyluvaricin ³⁰ and bullacin,³¹ but opposite to bullacin B with 6R configuration.³² The absolute configuration at C-36 in the lactone ring of 7 was determined to be S based on ECD spectrum which showed a negative n−π* Cotton effect at 238 nm (Δε = −1.50) and a positive π−π* Cotton effect at 208 nm (Δε = 13.23).^33–35

Table 1.

¹³C NMR Data (100 MHz, CDCl₃) of Compounds 7, 9, 12 and 13 in CDCl₃ ^a

	7		9		12		13
position	δ C		δ C		δ C		δ C
1	174.0		174.0		174.4		174.4
2	134.2		134.5		134.0		134.0
3	25.3		25.3		21.8		21.8
4	23.8		27.6		29.4–29.9		29.4–29.8
5	36.9		29.3		73.5		73.5
6	71.7		29.5–29.9		74.8		74.8
7	37.8		29.5–29.9		31.8		31.8
8	25.8		29.5–29.9		26.0		26.1
9	29.5–29.8		29.5–29.9		29.4–29.9		29.4–29.8
10	29.5–29.8		29.5–29.9		29.4–29.9		29.4–29.8
11	29.5–29.8		29.5–29.9		29.4–29.9		29.4–29.8
12	29.5–29.8		29.5–29.9		29.4–29.9		29.4–29.8
13	25.8		29.5–29.9		25.7		25.7
14	33.6		29.5–29.9		33.6		33.4
15	74.2		25.8		74.3		74.2
16	83.4		33.5		83.4		83.4
17	28.5b		74.3		28.5b		28.5b
18	29.0b		83.4		29.1b		29.1b
19	82.5c		28.5b		81.9c		82.4c
20	82.6c		29.1b		81.9c		82.7c
21	29.1b		82.4c		29.1b		29.1b
22	24.7b		82.7c		28.5b		24.6b
23	83.0		29.1b		83.3		83.0
24	71.5		25.0b		74.2		71.5
25	32.6		82.9		33.6		32.6
26	26.2		71.6		25.8		26.2
27	29.5–29.8		32.7		29.4–29.9		29.4–29.8
28	29.5–29.8		22.2		29.4–29.9		29.4–29.8
29	29.5–29.8		37.4		29.4–29.9		29.4–29.8
30	29.5–29.8		71.9		29.4–29.9		29.4–29.8
31	29.5–29.8		37.7		29.4–29.9		29.4–29.8
32	32.1		25.8		32.1		32.0
33	22.8		29.5–29.9		22.8		22.8
34	14.3		32.0		14.3		14.3
35	149.3		22.8		150.0		150.0
36	77.6		14.2		77.8		77.8
37	19.4		149.0		19.3		19.3
38			77.5
39			19.4

^aChemical shifts were calibrated using solvent residual signal (δ_C 77.16).

^{b, c}Assignments may be interchanged within the column.

Table 2.

¹H NMR Data (400 MHz, CDCl₃) of Compounds 7, 9, 12 and 13 ^a

	7		9		12		13
position	δH (J in Hz)		δH (J in Hz)		δH (J in Hz)		δH (J in Hz)
3	2.30, m		2.26, m		2.39, m; 2.53, m		2.39, m; 2.53, m
4	1.63, m; 1.70, m		1.53, m		1.70, m		1.70, m
5	1.34–1.55, m		1.25, m		3.56, m		3.56, m
6	3.62, m		1.25, m		3.62, m		3.62, m
7	1.34–1.55, m		1.25, m		1.34–1.47, m		1.34–1.47, m
8	1.27, m		1.25, m		1.26, m		1.25, m
9	1.27, m		1.25, m		1.26, m		1.25, m
10	1.27, m		1.25, m		1.26, m		1.25, m
11	1.27, m		1.25, m		1.26, m		1.25, m
12	1.27, m		1.25, m		1.26, m		1.25, m
13	1.27, m		1.25, m		1.26, m		1.25, m
14	1.34–1.47, m		1.25, m		1.34–1.47, m		1.34–1.47, m
15	3.40, m		1.25, m		3.39, m		3.39, m
16	3.80–3.96, m		1.34–1.47, m		3.80–3.90, m		3.80–3.96, m
17	1.55–2.03, m		3.39, m		1.55–2.03, m		1.55–2.03, m
18	1.55–2.03, m		3.80–3.96, m		1.55–2.03, m		1.55–2.03, m
19	3.80–3.96, m		1.55–2.03, m		3.80–3.90, m		3.80–3.96, m
20	3.80–3.96, m		1.55–2.03, m		3.80–3.90, m		3.80–3.96, m
21	1.55–2.03, m		3.80–3.96, m		1.55–2.03, m		1.55–2.03, m
22	1.55–2.03, m		3.80–3.96, m		1.55–2.03, m		1.55–2.03, m
23	3.80–3.96, m		1.55–2.03, m		3.80–3.90, m		3.80–3.96, m
24	3.80–3.96, m		1.55–2.03, m		3.39, m		3.80–3.96, m
25	1.34–1.47, m		3.80–3.96, m		1.34–1.47, m		1.34–1.47, m
26	1.27, m		3.80–3.96, m		1.26, m		1.25, m
27	1.27, m		1.34–1.47, m		1.26, m		1.25, m
28	1.27, m		1.28, 1.65, m		1.26, m		1.25, m
29	1.27, m		1.34–1.55, m		1.26, m		1.25, m
30	1.27, m		3.60, m		1.26, m		1.25, m
31	1.27, m		1.34–1.55, m		1.26, m		1.25, m
32	1.27, m		1.25, m		1.26, m		1.25, m
33	1.27, m		1.25, m		1.26, m		1.25, m
34	0.88, t (6.8)		1.25, m		0.88, t (6.8)		0.87, m
35	7.02, m		1.25, m		7.07, m		7.07, m
36	5.00, m		0.88, m		5.02, m		5.01, m
37	1.41, d (6.8)		6.98, m		1.41, d (6.8)		1.41, d (6.8)
38			4.99, m
39			1.40, d (6.8)

^aChemical shifts were calibrated using solvent residual signal (δ_H 7.26).

Figure 1.

MALDI-MS/MS diagnostic fragment ions of the lithiated molecules [M + Li]⁺ of the isolated acetogenins (1–15).

Table 3.

¹H NMR (400 MHz, CDCl₃) chemical shifts of the (S)- and (R)-MTPA esters of 7, 9, 12 and 13.

7 (15R, 24S, 6S)					9 (17R, 26S, 30S)					12 (15R, 24R, 5R, 6S)					13 (15R, 24S, 5R, 6S)
	7s	7r	Δδ S-R			9s	9r	Δδ S-R			12s	12r	Δδ S-R			13s	13r	Δδ S-R
14	1.61	1.46	+0.15		16	1.60	1.46	+0.14		14	1.57	1.47	+0.10		14	1.60	1.46	+0.14
15	5.05	5.01	+0.04		17	5.04	5.01	+0.03		15	5.00	5.01	−0.01		15	5.04	5.01	+0.03
16	4.02	3.98	+0.04		18	4.01	3.98	+0.03		16	3.93	3.98	−0.05		16	4.02	3.98	+0.04
17	1.93	2.00	−0.07		19	1.92	2.00	−0.08		17	1.79	1.95	−0.16		17	1.93	1.98	−0.05
	1.51	1.53	−0.02			1.51	1.56	−0.05			1.44	1.54	−0.10			1.52	1.54	−0.02
18	1.79	1.87	−0.08		20	1.79	1.87	−0.08		18	1.72	1.90	−0.18		18	1.80	1.86	−0.06
	1.67	1.71	−0.04			1.63	1.75	−0.12			1.65	1.83	−0.18			1.68	1.71	−0.03
19	3.79	3.81	−0.02		21	3.77	3.79	−0.02		19	3.77	3.92	−0.15		19	3.79	3.81	−0.02
20	3.79	3.63	+0.16		22	3.77	3.62	+0.15		20	3.77	3.92	−0.15		20	3.79	3.63	+0.16
21	1.79	1.75	+0.04		23	1.79	1.75	+0.04		21	1.72	1.90	−0.18		21	1.80	1.75	+0.05
	1.67	1.64	+0.03			1.63	1.63	0.00			1.65	1.83	−0.18			1.68	1.63	+0.05
22	1.81	1.77	+0.04		24	1.82	1.70	+0.12		22	1.79	1.95	−0.16		22	1.81	1.77	+0.04
	1.70	1.63	+0.07			1.67	1.60	+0.07			1.44	1.54	−0.10			1.70	1.63	+0.07
23	3.98	3.91	+0.07		25	3.94	3.86	+0.08		23	3.93	3.98	−0.05		23	3.97	3.91	+0.06
24	5.23	5.21	+0.02		26	5.19	5.12	+0.07		24	5.00	5.01	−0.01		24	5.23	5.21	+0.02
25	1.51	1.57	−0.06		27	1.54	1.53	+0.01		25	1.57	1.47	+0.10		25	1.48	1.57	−0.09
34	0.86	0.86	0.00		36	0.84	0.86	−0.02		34	0.86	0.87	−0.01		34	0.86	0.86	0.00
35	6.94	6.84	+0.10		37	6.96	6.96	0.00		35	7.00	6.91	+0.09		35	6.99	6.91	+0.08
36	4.97	4.94	+0.03		38	4.97	4.97	0.00		36	4.97	4.96	+0.01		36	4.98	4.96	+0.02
37	1.37	1.37	0.00		39	1.38	1.38	0.00		37	1.38	1.37	+0.01		37	1.38	1.37	+0.01
3	2.26	2.18	+0.08		3	2.24	2.24	0.00		3	2.32	2.16	+0.16		3	2.32	2.16	+0.16

Uvarirufin (9) was isolated as a waxy solid. The HRESIMS exhibited a protonated molecular ion at m/z 651.5200 [M + H]⁺ and a sodium adduct peak at m/z 673.5019 [M + Na]⁺, corresponding to a molecular formula of C₃₉H₇₀O₇. IR absorption bands at 3423 and 1755 cm⁻¹ indicated the presence of hydroxy and carbonyl groups. The NMR data (Tables 1 and 2) was consistent with an α,β-unsaturated γ-lactone and adjacent bis-THF rings with two flanking hydroxyls in a threo/trans/threo/trans/erythro relative configuration. The chemical shifts at H-3 (δ_H 2.26) and H-37 (δ_H 6.98) indicated the absence of an OH group near the lactone ring. The third hydroxy group appeared in the ¹H NMR spectrum at δ_H 3.60 (H-30) and in the ¹³C NMR at δ_C 71.9 (C-30). The ¹H and ¹³C NMR spectra of 9 were similar to those of squamocin (8),¹⁶ showing a signal at δ_C 22.2, indicative of the presence of a 1,5-diol functionality.¹¹ Similar to squamocin (8), analysis of the MALDI-MS/MS spectrum of 9 showed Y ions at m/z 291 (Y₂) and 221 (Y₁), indicating the same substructure of the methyl terminal part (Figure 1). However, the B ions in the spectrum of 9 at m/z 427 (B₂) and 357 (B₁) suggested that the fatty acid chain on the lactone ring side in 9 is two methylene units longer than in squamocin (8). Thus, the location of the bis-THF unit with flanking OH groups was determined to be between C-17 and C-26. A TOCSY correlation between H-30 (δ_H 3.60) and H-26 (δ_H 3.87) confirmed the presence of the OH group in the vicinity of the bis-THF ring unit. With MALDI-MS/MS data indicating the position of the third OH group on the methyl terminal part, this TOCSY correlation between the geminal proton on hydroxyl-bearing carbon (H-30) and H-26 (δ_H 3.87, indicative of erythro configuration), confirmed the erythro relative configuration on the methyl terminal side of the bis-THF unit (between C-25 and C-26, rather than C-17 and C-18). The absolute configurations at C-17, C-26 and C-30 in 9 were determined to be R, S and S, respectively, by Mosher ester analysis and comparison of Δδ_S-R values of 9 (Table 3) with literature values for squamocin.³⁶ ECD data indicated 38S absolute configuration in the lactone ring of 9. Almost all known acetogenins reported so far possess a C-37 or C-35 skeleton.¹ The few known acetogenins with 39 carbon atoms bear an acetoxy group in place of one of the hydroxy groups, for example uvaricin with an acetoxy group at C-24.^1,7 On the contrary, compound 9 does not possess any acetoxy groups, as evidenced by ¹H and ¹³C NMR data and HRESIMS. Instead, the C-39 skeleton of 9 featuring a longer alkyl chain is unique among Annonaceous acetogenins, as no other known acetogenins possess such skeleton. The biogenetic pathway of C-35/C-37 acetogenins is proposed to originate from long chain fatty acids in the polyketide pathway. The C-32 dotriacontanoic (lacceroic) acid or C-34 tetratriacontanoic (ghedoic) acid is combined with a three-carbon precursor at C-2 to form a methyl-substituted α,β-unsaturated γ-lactone of C-35 and C-37 acetogenins, respectively.^1,2,6,23,37 It is therefore proposed that C-39 skeleton of 9 is derived from a C-36 hexatriacontanoic acid (Figure S2). Further biosynthetic steps are expected to be analogous to those of other bis-THF acetogenins.^2,6,23,37 Tetrahydrofuran rings could possibly be formed through a 5-exo-trig or 5-exo-tet mechanism.³⁸

Uvariasolin III (12) was obtained as a waxy solid. The molecular formula was determined to be C₃₇H₆₆O₈ based on the HRESIMS (m/z 639.4840 [M + H]⁺, m/z 661.4666 [M + Na]⁺). The existence of hydroxy and carbonyl groups in 12 was evidenced by IR absorption bands at 3408 and 1736 cm⁻¹, respectively. Signals exhibited in the ¹H and ¹³C NMR (Tables 1 and 2) revealed typical resonances for the methylated α,β-unsaturated γ-lactone. The signals at δ_H 3.39 integrating for two protons (H-15, 24) and between δ_H 3.80–3.90 integrating for four protons (H-16, 19, 20, 23), and those at δ_C 83.4, 83.3 (C-16, 23), 81.9 (C-19), 81.9 (C-20), 74.3 and 74.2 (C-15, 24) in the ¹H and ¹³C NMR spectra indicated the presence of an adjacent bis-THF ring system flanked by two OH groups, with a threo/trans/threo/trans/threo relative configuration (Tables 1 and 2).^23,24 This relative configuration can result in pseudo-symmetry across the THF pattern and only two signals between δ_C 81 and δ_C 84 ppm and one signal around δ_C 74 ppm can sometimes be observed,²³ as in the case of narumicin I (5) (Table S2). However, the presence of two additional OH groups on one side of the OH-flanked bis-THF ring unit disturbs the symmetry. As a result, the ¹³C NMR spectrum of 12 displayed six distinct carbon signals in this region. The additional OH groups appeared in the ¹H NMR spectrum at δ_H 3.56 (H-5) and δ_H 3.62 (H-6), and in the ¹³C NMR at δ_C 73.5 (C-5) and 74.8 (C-6). Their chemical shifts were in agreement with the presence of two vicinal OH groups with an erythro configuration (ca. δ_H 3.60). In case of a vicinal diol with a threo configuration, as in uvariasolin I (10) and II (11), the geminal protons on hydroxyl-bearing carbons appear at ca. δ_H 3.40, similarly to the methine protons geminal to hydroxyls flanking the THF rings in threo configuration (Table S1).^17,39–41 The position of the vicinal diol was predicted at C-5 and C-6 based on the chemical shifts at H-35 (δ_H 7.07) and H-3 (δ_H 2.39. 2.53) which are more deshielded than in acetogenins with OH only at C-5 or only at C-6, but more shielded than in C-4 hydroxylated acetogenins.²⁵ The position of the hydroxy groups at C-5 and C-6 was further confirmed by the X ions in the MALDI-MS/MS analysis at m/z 519 (X₄) and 489 (X₃) (Figure 1). Similar to compound 7 and narumicins (5, 6), the bis-THF unit with flanking OH groups in 12 was located between C-15 and C-24 based on the same Y ions [m/z 275 (Y₂), 205 (Y₁)]. B ions at m/z 431 (B₂) and 361 (B₁) showed masses 16 units higher than narumicins and confirmed that both additional OH groups are on the lactone ring side (Figure 1). The absolute configuration at C-36 in the lactone ring of 12 was determined to be S based on ECD data. Analysis of the ¹H NMR and COSY data of the (S)-MTPA (12s) and (R)-MTPA (12r) esters (Table 3) indicated the absolute configurations of the carbinol centers of 12 at both C-15 and C-24 to be R by comparison of Δδ_S-R values from H-14 to H-25 with those of acetogenins with known configurations.^26–29 Since the relative configuration of the vicinal diol in 12 was erythro, the effects of the C-6-MTPA and C-5-MTPA should have combined to give a large negative or large positive Δδ_S-R value at H-3.⁴¹ Large positive Δδ_S-R value at H-3 (Table 3) indicated the 6S/5R absolute configuration based on positive value at H-3 for both (6S)-bullacin C (7) (Table 3) and (5S)-uvarigrandin A.^15,42 It is worth noting that, due to a switch in the relative priority of the groups,⁴³ 5S configuration becomes 5R when placed next to 6-OH. The positive Δδ_S-R value for lactone ring protons (H-35, 36, 37) matched those of (5S)-uvarigrandin A,¹⁵ as C-5-MTPA is closer to the lactone ring and should have greater influence than C-6-MTPA.

Uvariasolin IV (13) was obtained as a waxy solid and in the HRESIMS peaks at m/z 639.4839 [M + H]⁺ and m/z 661.4659 [M + Na]⁺ were observed, leading to a proposed molecular formula of C₃₇H₆₆O₈. The UV and IR data were similar to 12 and they both showed the same fragmentation pattern in the MALDI-MS/MS spectra, suggesting the same planar structure. The only difference between 12 and 13 was the stereochemistry of the bis-THF ring unit. The ¹H NMR and ¹³C NMR chemical shifts from C-15 to C-24 (Tables 1 and 2) were similar to those of 7, indicating the threo/trans/threo/trans/erythro relative configuration. The absolute configuration was determined as 5R, 6S, 15R, 24S by Mosher ester analysis (Table 3), and 36S by ECD analysis.

One of the commonly used TLC reagents for detection of acetogenins is Kedde’s reagent.⁴⁴ Kedde’s reagent is reportedly diagnostic for the α,β-unsaturated γ-lactone subunit but it was originally developed for the detection of cardiac glycosides and 17-ketosteroids. Positive reaction is indicated by faint red-pink chromophores which fade quickly.^2,6,23,44,45 Indeed, in this study a lactone-containing andrographolide standard showed a faint pink positive reaction (Figure 2B). A strong pink coloration on the TLC plate was also observed after staining with Kedde’s reagent for fraction E9.3, similarly to vanillin–sulfuric acid staining (Figure 2A and B). However, ¹H NMR of this fraction indicated the absence of acetogenin signature peaks, whereas acetogenin signals were observed in fraction E9.1 (Figure 2E–F). The latter in turn showed positive color reaction with Dragendorff’s reagent (orange spot, Figure 2C), which is often used for the detection of alkaloids and other nitrogen-containing compounds.⁴⁶ False positive reactions of nonalkaloid compounds with Dragendorff’s reagent containing conjugated carbonyl (ketone or aldehyde) or lactone functions have been reported.^47–49 Positive reaction with this reagent in the present study was consistently associated with the presence of acetogenins in the fractions (Figure 2D) and as pure compounds even at low concentrations. Therefore, we propose the use of Dragendorff’s reagent for the detection of acetogenins, as it seems more reliable than Kedde’s reagent (no reaction with acetogenins but strong reaction with other compounds in this study), vanillin–sulfuric acid reagent (greyish color for low concentrations and strong yellow for high concentrations of acetogenins) or UV-based TLC detection (weak UV absorption⁶). Acetogenin-rich fractions can be readily distinguished from alkaloid-rich fractions by their characteristic fatty acid chain signals in ¹H NMR between 0–1.5 ppm (Figure 2E).

Figure 2.

Detection of acetogenins by TLC and ¹H NMR in U. rufa fractions. TLC staining with vanillin – sulfuric acid reagent (CH₂Cl₂ : MeOH, 9:1) (A), Kedde’s reagent (CH₂Cl₂ : MeOH, 9:1) (B), Dragendorff’s reagent (CH₂Cl₂ : MeOH, 9:1, C; hexanes : EtOAc, 1:1, D). ¹H NMR of subfraction E9.1 (400 MHz, CDCl₃) (E) and E9.3 (400 MHz, CD₃OD) (F). E – ethyl acetate fractions, H – hexanes fractions, AGL – andrographolide.

Bioactivity data obtained for compounds 2–9 and 12–15 are summarized in Table 4. Most tested acetogenins displayed cytotoxic activity against the melanoma (MDA-MB-435), ovarian cancer (OVCAR3), colorectal carcinoma (HCT 116) and prostate carcinoma (22Rv1) cell lines, with IC₅₀ values in the low micromolar range. According to previously described structure–activity relationships of acetogenins, the presence of the adjacent bis-THF ring system and the α,β-unsaturated γ-lactone ring at the end of the chain are important for the activity.² Among the isolated acetogenins, squamocin (8) and uvarirufin (9) exhibited the most potent activity, both with the lowest IC₅₀ value of 1.2 μM in HCT 116 cells, comparable to etoposide control. This is in consistence with earlier observations, indicating that three hydroxyl groups (two flanking the THF rings) are optimal for the most potent activity.² Previous observations suggest that the C-35 or C-37 skeleton present in almost all acetogenins has evolved because of its optimal chain length with respect to mitochondrial membrane dimensions (target enzyme site), providing the highest activity and protection of plant against herbivores.² Indeed, shorter acetogenins were found less active.² However, the unique C-39 skeleton of uvarirufin (9), with alkyl chain elongated by two carbon atoms compared with squamocin (8), does not seem to compromise activity, as the two compounds show similar IC₅₀ values across different cell lines. Dihydroxylated acetogenins (2–4) seem to have weaker cytotoxic activity. The tetrahydroxylated panalicin (14) showed cytotoxicity comparable to that of 8 and 9, suggesting the importance of 1,5-diol functionality for bioactivity. Additionally, narumicin II (6) was tested for inhibitory activity against Mycobacterium tuberculosis (H₃₇Rv), as previously described,⁵⁰ but showed no inhibition up to 100 μg/mL.

Table 4.

Cytotoxic activity of the isolated acetogenins (IC₅₀ in μM).

Compound	OVCAR3a	MDA-MB-435 a	HCT 116b	22Rv1b
2	ND	ND	ND	18.3
3	>10.0	4.1	2.8	7.8
4	>10.0	5.3	29.1	16.5
5	4.6	4.1	5.5	7.0
6	3.5	4.6	9.2	17.5
7	ND	ND	10.0	12.5
8	4.5	4.6	1.2	6.4
9	4.4	4.5	1.2	5.6
12	3.7	4.4	2.7	5.5
13	>10.0	4.6	15.0	8.3
14	4.5	4.4	1.8	8.2
15	>10.0	4.0	5.4	38.3
Paclitaxel	5.6 nM	2.6 nM	ND	ND
Etoposide	ND	ND	1.8 μM	2.3 μM
Vinblastine sulfate	ND	ND	1.0 nM	1.0 nM

^acell viability determined using MTS assay

^bcell viability determined using MTT assay

EXPERIMENTAL SECTION

General Experimental Procedures.

Optical rotations were measured using a Rudolph Autopol IV automatic polarimeter at the sodium D-line at room temperature. Concentrations (c) are given in g/100 mL. UV spectra were collected on a Shimadzu UFLC system equipped with a PDA detector. The ECD measurements were made using a JASCO J-810 spectrometer. IR spectra were recorded on a Nicolet 380 FTIR spectrometer and analyzed with OMNIC software. NMR spectra were acquired using Bruker Avance 400 MHz instruments and processed with MestReNova software v14.2. Chemical shifts were reported on the δ scale in ppm and the solvent residual signal (chloroform: δ_H 7.26, δ_C 77.16) was used as chemical shift reference. Coupling constants (J) are reported in Hz. HRESIMS data were obtained from a Bruker Impact II Q-TOF mass spectrometer using positive electrospray ionization and sodium formate as a calibrant. HPLC purification was performed on a Shimadzu UFLC system using semi-preparative HPLC columns: Agilent Eclipse XDB-C8 (250 × 9.4 mm, 5 μm) and Agilent SB-C18 column (250 × 9.4 mm, 5 μm).

Plant Material.

The stem of Uvaria rufa (Dunal) Blume (Annonaceae) was collected on April 4 2000, through ethnobotanical studies from Ban Hinhlath, Sanamxai District, Attapeu Province in Laos (106°28.98’N 14°45.03’E) by Keokongtone Parakosonh, D.T. Kien and M.V. Sinh under the auspices of the Vietnam-Laos International Cooperative Biodiversity Group (ICBG) Memorandum of Agreement (1998–2003). The sample was identified by Dr. Djaja Djendoel Soejarto, College of Pharmacy, University of Illinois Chicago, USA. The dried stem pieces were stored in cool and dark conditions to prevent degradation. A voucher herbarium specimen has been deposited at the John G. Searle Herbarium (F) of the Field Museum of Natural History, Chicago, IL, USA, under the accession number FM-2246536.

Extraction and Isolation.

The dried stem of U. rufa (2.6 kg) was exhaustively extracted with MeOH by maceration at room temperature to yield 267 g of crude extract. The crude extract was partitioned into hexanes-soluble (8 g), EtOAc-soluble (20 g), n-BuOH-soluble (35 g) and H₂O-soluble (128 g) fractions by solvent partitioning (Figure S1). The hexanes-soluble fraction was separated on a silica gel column, eluted with hexanes–EtOAc gradient (100:0 to 0:100), followed by MeOH wash, to afford 10 fractions (H1–10). Fraction H8 was subjected to a C₁₈ open column chromatography (80–100% MeOH–H₂O) to yield 12 subfractions. Subfraction H8.6 was purified by semi-preparative HPLC on a C₈ column (MeCN–H₂O, 80:20; 4 mL/min) to afford neoannonin (2, 2.17 mg, t_R = 20.7 min) and squamocin-K (1, 1.44 mg, t_R = 22.2 min). Semi-preparative HPLC of subfraction H8.8 (C₈ column, MeCN–H₂O, 85:15; 4 mL/min) yielded desacetyluvaricin (4, 5.25 mg, t_R = 21.2 min) and isodesacetyluvaricin (3, 3.46 mg, t_R = 22.3 min). The EtOAc-soluble fraction was subjected to silica gel column chromatography and eluted with hexanes–EtOAc gradient (100:0 to 0:100), then MeOH, to afford 11 fractions (E1–11). Fraction E9 was separated into 4 subfractions on a silica gel column (CH₂Cl₂–MeOH, 90:10–100% MeOH). This solvent system was found suitable for separating acetogenins (eluting early) from more polar components. Subfraction E9.1 was chromatographed over a Sephadex LH-20 gel open column eluted by MeOH to give 6 subfractions. Subfraction E9.1.3 was applied to an MCI gel reversed phase column eluted with a gradient of 50–100% MeOH–H₂O, to yield 13 subfractions. After passing subfraction E9.1.3.11 through another silica gel open column (petroleum ether–EtOAc), the acetogenin-rich fractions were purified by semi-preparative HPLC on a C₈ column (MeCN–H₂O, 80:20; 4 mL/min) to afford bullacin C (7, 0.89 mg, t_R = 10.3 min), narumicin II (6, 4.93 mg, t_R = 11.3 min) and narumicin I (5, 8.79 mg, t_R = 12.4 min). Additional amount of 7 was isolated from other fractions. Fraction E10 was separated on a silica gel column eluted with CH₂Cl₂–MeOH (90:10–100% MeOH), to give 11 subfractions. An MCI open column was employed for further separation of subfraction E10.3, eluted with 70–100% MeOH–H₂O, to give 8 subfractions. Squamocin (8, 22.61 mg, t_R = 9.8 min) was isolated from subfraction E10.3.6 by semi-preparative HPLC (C₈ column, MeCN–H₂O, 80:20; 4 mL/min), along with additional amounts of narumicins. Subfraction E10.3.8 was purified on a semi-preparative C₈ column (MeCN–H₂O, 80:20; 4 mL/min) to yield uvarirufin (9, 4.63 mg, t_R = 15.6 min). Fraction E11 was applied to a silica gel column eluted with CH₂Cl₂–MeOH (90:10–100% MeOH), to give 9 subfractions. Subfraction E11.4 was chromatographed over a C₁₈ open column, eluted with 60–100% MeOH–H₂O to yield 11 subfractions. Subfraction E11.4.6 yielded microcarpacin (15, 9.56 mg, 10.0 min) and panalicin (14, 4.36 mg, 11.8 min) after purification on a semi-preparative C₈ column (MeCN–H₂O, 60:40; 4 mL/min). Further purification of 15 was performed on a semi-preparative C₁₈ column (MeCN–H₂O, 70:30). Subfractions E11.4.8 and E11.4.9 were subjected to semi-preparative HPLC on a C₈ column (MeCN–H₂O, 65:35; 4 mL/min) and afforded uvariasolin IV (13, 8.55 mg, 16.1 min), uvariasolin II (11, 3.31 mg, 17.0 min), uvariasolin III (12, 3.67 mg, 18.2 min) and uvariasolin I (10, 8.29 mg, 19.6 min). Further purification of 11 was achieved using a semi-preparative C₁₈ column (MeCN–H₂O, 75:25).

Bullacin C (7).

White solid; [α] +8.0 (c 0.10, MeOH); ECD (c 0.05, MeOH) λ_max (Δε) 238 (−1.50), 208 (+13.23) nm; UV (MeCN/H₂O) λ_max 208 nm; IR (neat) ν_max 3438, 2924, 2853, 1755, 1459, 1373, 1319, 1199, 1067, 1028, 955, 875 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 623.4883 [M + H]⁺ (calcd for C₃₇H₆₇O₇, 623.4887); m/z 645.4707 [M + Na]⁺ (calcd for C₃₇H₆₆O₇Na, 645.4706).

Uvarirufin (9).

Waxy solid; [α] +8.0 (c 0.10, MeOH); ECD (c 0.05, MeOH) λ_max (Δε) 238 (−1.60), 208 (+12.65) nm; UV (MeCN/H₂O) λ_max 207 nm; IR (neat) ν_max 3423, 2924, 2853, 1755, 1460, 1319, 1199, 1066, 1028, 954, 873 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 651.5200 [M + H]+ (calcd for C₃₉H₇₁O₇, 651.5200); m/z 673.5019 [M + Na]⁺ (calcd for C₃₉H₇₀O₇Na, 673.5019).

Uvariasolin III (12).

Waxy solid; [α] +13.0 (c 0.10, MeOH); ECD (c 0.05, MeOH) λ_max (Δε) 239 (−1.39), 210 (+13.10) nm; UV (MeCN/H₂O) λ_max 208 nm; IR (neat) ν_max 3408, 2922, 2852, 1736, 1465, 1322, 1202, 1065, 1032, 951, 884, 720 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 639.4840 [M + H]⁺, (calcd for C₃₇H₆₇O₈, 639.4836); m/z 661.4666 [M + Na]⁺ (calcd for C₃₇H₆₆O₈Na, 661.4655).

Uvariasolin IV (13).

Waxy solid; [α] +9.0 (c 0.10, MeOH); ECD (c 0.05, MeOH) λ_max (Δε) 239 (−1.71), 209 (+14.97) nm; UV (MeCN/H₂O) λ_max 208 nm; IR (neat) ν_max 3444, 2924, 2853, 1754, 1465, 1319, 1200, 1065, 953, 877 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 639.4839 [M + H]⁺, (calcd for C₃₇H₆₇O₈, 639.4836); m/z 661.4659 [M + Na]⁺ (calcd for C₃₇H₆₆O₈Na, 661.4655).

MALDI-MS/MS sample preparation and data acquisition.

The samples were prepared according to a previously described procedure,⁹ with modifications. Lithium iodide solution was prepared at 10 mg/mL in MeOH. Stock solutions of acetogenins were prepared in MeOH at a concentration of 2 mg/mL and diluted 4× with lithium iodide solution. The samples were further diluted 5× with 70% CH₃CN in H₂O, then mixed with matrix (2,5-dihydroxybenzoic acid (DHB) at 5 mg/mL in 70% CH₃CN + 0.1% TFA) in 1:1 ratio and applied to the MALDI plate. The MALDI-MS/MS experiments were carried out on an AB SCIEX 4800 MALDI TOF/TOF instrument in a CID on mode.

Preparation of Mosher Esters.

The Mosher ester derivatives were prepared based on the previously described procedure.⁴³ To a dried acetogenin (0.5 mg) in a 4 mL vial were sequentially added anhydrous pyridine (5 μL), anhydrous CDCl₃ (100 μL) and (R)-(−)-α-methoxy-α-trifluoromethylphenylacetyl chloride ((R)-(−)-MTPA-Cl, 5 μL). The mixture was stirred on a shaker at room temperature for 5 hours to give the (S)-MTPA ester, then further diluted with CDCl₃ and directly transferred to an NMR tube for acquisition of ¹H NMR and ¹H-¹H COSY spectra. The (R)-Mosher ester of the acetogenin was prepared from (S)-(+)-MTPA-Cl using the same method.

TLC Reagents.

General staining was performed using modified vanillin–sulfuric acid reagent. Dragendorff’s reagent was prepared according to Munier.⁴⁶ Kedde’s reagent was prepared as previously described.^44,45 The reagent recipes can be found in the Supporting Information.

Cell Culture and Cell Viability.

The cytotoxic activity against MDA-MB-435 and OVCAR3 cancer cell lines was evaluated after 72 hours of incubation with test substance, according to the previously described procedures.⁵¹ A commercial absorbance assay (CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay, Promega Corp, Madison, WI, USA) was used to assess cell viability relative to the solvent (DMSO) control. Paclitaxel was used as a positive control.

The 22Rv1 and HCT 116 cancer cell lines were cultured at 37°C in 5% CO₂ in RPMI 1640 medium and McCoy’s medium, respectively, supplemented with fetal bovine serum (10% v/v), penicillin (100 units/mL), and streptomycin (100 μg/mL). For cytotoxicity assay the cells were harvested by trypsinization and 5000 cells per well were seeded in 96-well plates. After 24 hours of incubation (37°C in 5% CO₂), the test compounds dissolved in DMSO were diluted with a fresh medium and added to the appropriate wells at the final concentrations of 100, 20, 4, 0.8, 0.16, and 0.032 μM (total volume: 200 μL) and 0.5% DMSO was used as a vehicle control. After 72 hours of incubation (37°C in 5% CO₂) in the presence of test substance, the test media was replaced with media containing MTT reagent (thiazolyl blue tetrazolium bromide, Alfa Aesar),⁵² at a final concentration of 0.5 mg/mL. After 3 hours of incubation, the media was discarded, DMSO was added to dissolve the formazan crystals and the plate was put on a plate shaker for 10 min, protected from light. The absorbance reading was taken at a wavelength of 570 nm and the viability was calculated with respect to DMSO control. Etoposide and vinblastine were used as a positive control. All compounds were tested in duplicate, and the experiment was performed independently three times. IC₅₀ values were calculated using GraphPad Prism 9.5.1 software.