Structurally novel C₁₇-sesquiterpene lactones from Ainsliaea pertyoides

Abstract

Pertyolides A (1) and B (2), two unprecedented C₁₇-eudesmanolides with extended side chains, and one rare C₁₇-guaianolide, pertyolide C (3), along with 15 known sesquiterpene lactones were isolated from the whole plant of Ainsliaea pertyoides. Their structures were elucidated by extensive spectroscopic analysis. The absolute configuration of compound 1 was assigned by X-ray crystallography. All isolates were evaluated for their cytotoxic activities against four human tumor cell lines A549, HCT116, MGC803, CCRF-CEM, and only alantolactone (5) showed significant inhibitions against the tumor cell lines tested.

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The genus Ainsliaea (family Compositae), consisting of nearly 70 species, widely spread throughout the South East Asian region, are receiving much attention due to their remarkable biological activities and chemical diversities.^1–5 As an important part of our research for screening bioactive constituents from natural sources, great efforts of our group have been devoted to the phytochemical investigations on plants of the genus Ainsliaea, which resulted in a series of biologically active sesquiterpenoid lactone dimmers and triterpenoid derivatives with new carbon frameworks, as well as other types of constituents such as triterpenes, steroids, flavonoids, etc.^6–8

With the aim of discovering bioactive natural products we carried out an investigation on the chemical constituents of Ainsliaea pertyoides, one of the 48 Chinese endemic species, which is a medicinal herb growing mainly in southeast of China and only a few natural products have been discovered.⁹ It is an ethnomedicinal plant used in traditional Chinese medicine for the treatment of various diseases, including bone fracture and ankylosing spondylitis. From this study, we have isolated two unprecedented C₁₇-eudesmanolides with extended side chains pertyolides A (1) and B (2), one new C₁₇-guaianolide pertyolide C (3) (Fig. 1) and 15 known sesquiterpene lactones (Fig. S1, ESI†). It is worthwhile to mention that compounds 1 and 2 are two unusual eudesmane-type sesquiterpenoid with a C₁₇ skeleton. Herein we describe their isolation, structural elucidation, possible biosynthetic pathway, and cytotoxicities.

Fig. 1 Chemical structures of 1–3.

The EtOAc-soluble fraction of the 95% EtOH extract of A. pertyoides was subjected to repeating column chromatography (CC) on macroporous resin, silica gel, ODS, Sephadex LH-20, and semi-preparative HPLC to yield three new (1–3) and 15 known sesquiterpenoids (4–18). By comparison of the NMR and MS data with previously reported data in the literatures, 15 known compounds were characterized as macrophyllilactone F (4),¹⁰ alantolactone (5),^11,12 isoalantolactone (6),¹¹ 11,13-dihydroisoalantolactone (7),¹³ 13-hydroxy-8(αH)-eudesma-5,7(11)-dien-8,12-olide (8),¹⁴ 11α,13-dihytdrosantamarin (9),⁶ 3α-hydroxy-11αH-guaia-4(15),10(14)-dien-12,6α-olide (10),¹⁵ zaluzamin C (11),¹⁶ diaspanolide A (12),¹⁷ dihydroestafiatll (13),⁶ 4β,14-dihydrozaluzanin C (14),¹⁸ 11α,13-dihydrozaluzanin C (15),¹⁹ zauzanin C (16),²⁰ estafiatone (17),²¹ and dihydroestafiatone (18).²¹

Pertyolide A (1) was obtained as a colorless crystal. The molecular formula was assigned as C₁₇H₂₄O₄ by the HRESIMS ion peak at m/z 315.1579 [M + Na]⁺ (calcd 315.1567), implying six degrees of unsaturation.‡ The IR spectrum suggested the presence of hydroxy (3353 cm⁻¹), γ-lactone (1766 cm⁻¹), ketone (1705 cm⁻¹) and olefinic (1643 cm⁻¹) functionalities. The ¹H NMR spectrum of 1 displayed diagnostic signals for two tertiary methyls [δ_H 0.78 (3H, s) and 2.34 (3H, s)] and two olefinic protons [δ_H 4.43 (1H, s) and 4.79 (1H, s)] (Table 1). The ¹³C NMR and DEPT spectroscopic data revealed 17 carbon resonances that were distinguished via DEPT and HSQC data to be two methyls, five methylenes (including one sp² methylene at δ_C 106.5), three methines (one oxygenated at δ_C 77.7), and five quaternary carbons (including one oxygenated at δ_C 79.7, one sp² methine at δ_C 149.4, one keto group at δ_C 210.5, and one ester carbonyl at δ_C 175.5) (Table 2). Its ¹H and ¹³C NMR spectra displayed characteristic resonances of a eudesmane-type sesquiterpenoid lactone and exhibited a great similarity with those of compound 6, except for the reduction of exocyclic olefinic bond [δ_C 79.7; δ_H/δ_C 2.64 (1H, d, J = 17.5 Hz), 3.02 (1H, d, J = 17.5 Hz)/42.2] and the presence of an additional methyl (δ_H/δ_C 2.34 (3H, s)/32.0) and an additional ketone (δ_C 210.5) in 1. The HMBC correlations from H₂-13 to C-11 (δ_C 79.7), C-12 (δ_C 175.5) and C-16 (210.5) and from H₃-17 (δ_H 2.34) to C-16 (δ_C 210.5) linked two surplus carbons at C-13 as acetyl group. The ¹H–¹H COSY spectrum indicated the H-1/H-2/H-3 and H-5/H-6/H-7/H-8/H-9 proton sequences (Fig. 2). The HMBC cross-peaks between H-6/C-11, H-9/C-8, and H-9/C-7 indicated that a γ-lactone function was formed between C-8 and C-11 (Fig. 2). In the HMBC spectrum, the olefinic protons at δ_H 4.43 (s) and 4.79 (s) were correlated with C-4 (δ_C 149.4), C-3 (δ_C 36.9) and C-5 (δ_C 46.7), indicating the location of the exomethylene double bond at C-4 and proton signal assignment to H₂-15. Moreover, the HMBC correlations from H₃-14 to C-1, C-5, C-9, and C-10 confirmed that CH₃-14 was connected to C-10. These evidences support the C₁₇-eudesmanolide skeleton of 1 with an extended acetyl.

Table 1 ¹H NMR spectroscopic data for compounds 1–3^a

No.	1, δH (J in Hz)	2, δH (J in Hz)	3, δH (J in Hz)
a Data were recorded in CDCl3 at 500 MHz for ^1H NMR.
1a	1.23, m	1.15, m	2.88, q (8.4)
1b	1.53, m	1.62, m
2a	1.26, m	1.40, m	2.44, m
2b	1.60, m	1.80, m	1.76, m
3a	2.00, m	1.56, m	5.56, m
3b	2.34, m
4		2.46, m
5	1.81, d (12.4)		2.75, t (9.2)
6a	1.06, q (12.4)	4.94, d (3.5)	4.37, t (9.2)
6b	1.42, m
7	2.38, m	3.01, dd (5.4, 3.5)	1.94, m
8a	5.04, m	5.13, dt (5.4, 3.0)	1.64, m
8b			1.81, m
9a	1.46, dd (15.6, 4.5)	2.14, dd (14.9, 3.0)	2.03, m
9b	2.20, dd (15.6, 2.6)	1.51, dd (14.9, 3.0)	2.50, m
13a	2.64, d (17.5)	2.65, d (17.5)	2.62, d (16.7)
13b	3.02, d (17.5)	2.95, d (17.5)	2.80, d (16.7)
14a	0.78, s	1.22, s	4.93, s
14b			4.91, s
15a	4.79, s	1.13, d (7.7)	5.43, br s
15b	4.43, s		5.30, br s
17	2.34, s	2.33, s	2.32, s
2′			2.23, dd (7.1, 1.7)
3′			2.12, m
4′, 5′			0.97, d (6.5)

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Table 2 ¹³C NMR spectroscopic data for compounds 1–3

No.	1	2	3
a Attached protons were deduced by DEPT experiment.
1	42.2^a	42.3	44.3
2	22.8	16.9	36.4
3	36.9	32.9	75.5
4	149.4	38.7	148.4
5	46.7	152.8	50.3
6	21.2	113.7	83.0
7	47	47.1	52.0
8	77.7	77.2	25.2
9	41.4	42.7	34.6
10	34.7	33.1	148.2
11	79.7	79.2	76.5
12	175.5	175.5	176.0
13	42.2	43.6	44.4
14	18.0	28.8	114.0
15	106.5	23.2	114.5
16	210.5	210.4	210.4
17	32.0	32.0	32.3
1′			173.1
2′			43.8
3′			25.9
4′			22.5
5′			22.6

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Fig. 2 Key ¹H–¹H COSY (), HMBC (), and NOESY () correlations of 1.

The relative configuration of 1 was deduced from the NOESY spectrum (Fig. 2) and biogenetic consideration. On the basis of biogenetic considerations, the H-7 was predicted to be α-oriented. The NOESY correlations of H-7α/H-5 and H-7α/H-8 confirmed H-5, H-7 and H-8 to be α-oriented. The NOESY correlations of H-14/H-6β and H-6β/H₂-13 indicated that Me-14 and the side chain at C-11 were β-oriented, while 11-OH was α-oriented. The X-ray crystallographic data corroborated the planar structure and relative configuration of 1 as elucidated via NMR data and further allowed the assignment of its absolute configuration as 5S, 7S, 8R, 10R, 11R [Fig. 3, Flack parameter: 0.02 (7)], being a unprecedented C₁₇ skeleton eudesmanolide.

Fig. 3 X-ray crystallographic structure of 1.

Pertyolide B (2) was obtained as a colorless oil and exhibited the same molecular formula C₁₇H₂₄O₄ as that of 1, as established by HRESIMS at m/z 315.1577 [M + Na]⁺ (calcd 315.1567), requiring six degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3440 cm⁻¹), γ-lactone (1774 cm⁻¹) and ketone (1718 cm⁻¹) groups. The ¹H and ¹³C NMR data of 2 showed a close structural resemblance to 1 (Tables 1 and 2), differing in the locations of the double bond [δ_H 4.94 (d, J = 3.5 Hz, H-6), δ_C 152.8 (C-5), 113.7 (C-6)]. The HMBC correlations from H-6 to C-4, C-7, C-8 and C-10 supported the occurrence of the double bond at C-5 and C-6 in 2 (Fig. 4), instead of the exocyclic double bond between C-4 and C-15 in 1. The H-1/H-2/H-3/H-4/H-15 and H-6/H-7/H-8/H-9 proton spin systems were deduced from the ¹H–¹H COSY correlations (Fig. 4). The HMBC spectrum showed the following correlations: H-6 with C-7 and C-8; H-13 with C-7, C-11, C-12 and C-16; H-17 with C-13 and C-16; H-14 to C-1, C-5, C-9 and C-10; and H-15 to C-3, C-4 and C-5. These data further supported the structure of 2.

Fig. 4 Key ¹H–¹H COSY (), HMBC (), and NOESY () correlations of 2.

The relative configurations of 2 was established as shown via excellent NMR comparisons with 1 at all stereo centers, and were further validated by NOESY experiment (Fig. 4). Similar to 1, H₃-14 and H₂-13 were determined to be β-oriented via the NOESY correlations of H₃-14/H₂-13, while H-7 and H-8 were determined to be α-oriented via the NOESY correlation of H-7/H-8 and biogenetic considerations. In addition, the new chiral center of C-4 was established on the basis of the NOESY correlations of H₃-15/H₃-14 and H-4/H-6, and the H₃-15 was assigned as β-oriented. Thus, the structure of 2 was thereby deduced and named pertyolide B.

Pertyolide C (3) gave the molecular formula C₂₂H₃₀O₆ as determined by the HRESIMS ion at m/z 408.2387 [M + NH₄]⁺ (calcd 408.2386) and from the ¹³C NMR data. The IR spectrum implied the presence of hydroxyl (3394 cm⁻¹), ester (1729 cm⁻¹), γ-lactone (1772 cm⁻¹) groups. The ¹H and ¹³C NMR spectra of 3 revealed structural fragments similar to those found in compound 11 (Tables 1 and 2). Some characteristic signals between 3 and 11 are very similar in their chemical shifts, such as the two exocyclic double bonds [δ_H 4.93 (1H, s, H-14a), 4.91 (1H, s, H-14b), 5.43 (1H, s, H-15a), 5.30 (1H, s, H-15b) and δ_C 148.2 (C-10), 114.0 (C-14), 148.4 (C-4), 114.5 (C-15)], an isovalerate moiety [δ_H 2.23 (2H, d, J = 7.1 Hz, H-2′), 2.12 (1H, m, H-3′), 0.97 (6H, d, J = 6.5 Hz, H-4′, 5′) and δ_C 173.1 (C-1′), 43.8 (C-2′), 25.9 (C-3′), 22.5 (C-4′), 22.6 (C-5′)], and one lactone ring [δ_H 4.37 (1H, t, J = 9.7 Hz, H-6) and δ_C 176.0 (C-12), 83.0 (C-6)]. However, the major changes of the chemical shifts were about C-11 and C-13. The proton and carbon signals for a methylene (δ_H 2.62, 2.80; δ_C 44.4), an oxygenated quaternary carbon (δ_C 76.5), and an acetyl group (δ_H 2.32; δ_C 32.3, 210.4) were observed in 3, instead of those of the Δ (ref. 11) double bond in compound 11. In addition, HMBC correlations (Fig. 5) observed between H-13 and C-11/C-16 and Me-17 and C-16/C-13, indicated the existence of a C4 side chain in 3. Moreover, an HMBC correlation between H-13 (δ_H 5.37) and C-12 showed that the C4 side chain was connected to C-11. In the ¹H–¹H COSY experiment (Fig. 5), a long fragment was deduced by the correlations of H-3/H-2/H-1 and H-5/H-6/H-7/H-8/H-9. Additionally, the HMBC correlations of H-14 to C-1 and C-9, and the correlations of H-15 to C-3 and C-5 further confirmed the planar structure of 3.

Fig. 5 Key ¹H–¹H COSY (), HMBC (), and NOESY () correlations of 3.

Finally, the relative stereochemistry of 3 was established by a NOESY experiment (Fig. 5). The cis ring junction at C-1 and C-5 was confirmed from the NOESY correlation of H-1/H-5. On the other hand, H-1, H-3, H-5 and H-7 were determined to be α-oriented via the NOESY correlation of H-1/H-3, and H-5/H-7. In addition, the C4 side chain was established to be α-oriented on the basis of the key NOESY correlations between H₂-13(δ_H 2.62, 2.80) and H-7 (δ_H 1.94). Thus, the structure of compound 3 was established and named pertyolide C.

Pertyolides A and B are two unprecedented C₁₇-eudesmanolides. To date, no any similar eudesmane sesquiterpenoid has been reported with C₁₇ skeleton. Therefore, the possible biogenetic pathway for pertyolides A and B will be an interesting topic. Our group have reported four unprecedented C₁₇-pseudoguaianolides with an extended side chains from Inula hookeri.²² Five natural 11,16-oxetane lactones, structurally appertained to C₁₇-guaianolides, also have been reported from Centaurea genus previously.²³ The photoaddition of acetaldehyde to guaianolides could provide C₁₇-guaianolides. Consequently, a possible biogenetic pathway for pertyolides A and B was proposed as shown in Scheme 1. In this biogenetic pathway, two precursor compounds 5 and 6 could be converted into C₁₇ eudesmanolides 1 and 2 by an addition reaction with acetyl-CoA. To date, there is no report about that an acetyl moiety was attached to the eudesmane skeleton at C-13 by a carbon–carbon linkage. Therefore, the formation of 1–2 would provide a new insight into the structural transformation of eudesmanes.

Scheme 1 Plausible biogenetic pathway to pertyolides A (1) and B (2).

Considering that some sesquiterpenoids from Ainsliaea species, such as ainsliatrimer A, have been reported to have significant anti-tumor activity,^4,5 all the isolates were evaluated for cytotoxicity against four human tumor cell lines A549, HCT116, MGC803 and CCRF-CEM (Table 3). Only alantolactone (5) exhibited significant inhibition against A549, HCT116, MGC803 and CCRF-CEM cell lines with IC₅₀ values of 3.56, 2.23, 2.89 and 14.67 μM, respectively. The compound 6 exhibited moderate activity toward HCT116 and MGC803 cells, with IC₅₀ values of 14.4 and 15.1 μM, respectively. From these results, only compound 5 exhibited significant inhibitions against the tumor cell lines.

Table 3 IC₅₀ values (μM) of compounds 1–18 against four human tumor cell lines (mean ± SD, n = 3)

Compounds	IC50
	A549	HCT116	MGC803	CCRF-CEM
a Other compounds, including 1–4, 7–18. b Positive control.
5	3.56 ± 0.22	2.23 ± 0.18	2.89 ± 0.24	14.67 ± 0.51
6	>20	14.4 ± 0.98	15.51 ± 1.15	>20
Others^a	>20	>20	>20	>20
Doxorubicin^b	0.02 ± 0.002	0.12 ± 0.01	0.13 ± 0.01	<0.001

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In summary, we have discovered three irregular sesquiterpene lactones named pertyolides A–C (1–3) that possess unusual C₁₇-sesquiterpenoid frameworks and 15 known compounds from A. Pertyoides. To the best of our knowledge, 1–2 represent the first two C₁₇-eudesmanolides with extended side chains from nature. It is noteworthy that compound 5 exhibited much stronger inhibitory activity against four tumor cell lines than any sesquiterpene lactones from A. pertyoides.

Acknowledgements

The work was supported by program NCET Foundation, NSFC (81373301, 81230090), Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03) and the National Key Technology R&D Program of China (2012BAI29B06).

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Footnotes

† Electronic supplementary information (ESI) available: The extraction scheme, spectroscopic data, bioassay methods, computational method, and crystallographic data of 1. CCDC 1405733. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra16551b

‡ Pertyolide A (1): colorless crystal from petroleum ether/acetone (1 : 1). m.p. 136–138 °C; [α]²⁵_D +42.0 (c 0.100, CH₃OH); UV (CH₃OH) λ_max 202 nm; IR (KBr) ν_max 3354, 2924, 2852, 1766, 1705, 1643, 1442, 1263, 1173, 1090, 887, 802, 636 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; ESIMS m/z 315.2 ([M + Na]⁺), 291.0 ([M − H]⁻); positive HRESIMS m/z 315.1579 ([M + Na]⁺, calcd 315.1567). Crystal data for pertyolide A (1): C₁₇ H₂₄ O₄: M 292.36, orthorhombic, space group P 21 21 21, a = 6.38940(10), b = 11.8924(2), c = 19.9718(4) Å, α = 90°, β = 90°, γ = 90°, V = 1517.56(5) ų, Z = 4, μ = 0.727 mm⁻¹, 9899 reflections measured, 2731 unique [R(int) = 0.0285]. The final wR(F²) was 0.0896 (all data). CCDC 1405733. Pertyolide B (2): colorless oil; [α]²⁵_D +1.0 (c 0.100, CH₃OH); UV (CH₃OH) λ_max 201 nm; IR (KBr) ν_max 3440, 2925, 2852, 1774, 1718, 1664, 1458, 1363, 1192, 1063, 970, 891, 667 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; ESIMS m/z 293.2 ([M + H]⁺), 315.2 ([M + Na]⁺); positive HRESIMS m/z 315.1577 ([M + Na]⁺, calcd 315.1567). Pertyolide C (3): colorless oil; [α]²⁵_D +17.0 (c 0.100, CH₃OH); UV (CH₃OH) λ_max 202 nm; IR (KBr) ν_max 3394, 2924, 2852, 1772, 1729, 1647, 1466, 1367, 1292, 1259, 1169, 1074, 903 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; ESIMS m/z 413.3 ([M + Na]⁺); positive HRESIMS m/z 408.2387 ([M + NH₄]⁺, calcd 408.2386).

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