Anti-HIV terpenoids from Daphne aurantiaca Diels. stems

Abstract

Thirteen new sesquiterpenoids, including six guaiane type auranticanols A–F (1, 2, and 4–7) and seven carotene type auranticanols G–M (18–24) were isolated from the stems of Daphne aurantiaca Diels., along with fourteen known sesquiterpenoids (3, 8–17, 25–27) and two known tigliane diterpenoids (28, 29), and their structures were elucidated by extensively analyzing their MS and NMR spectroscopic data. A bioassay of anti-HIV activity indicated that compounds 11, 14, 19, and 28 showed definite activities with EC₅₀ values of 2.138, 0.286, 1.773 and 0.000282 μg mL⁻¹ and SI > 93.545, 93.787, 10.243, and 65 177.305, respectively.

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Introduction

Named after a Greek myth, the genus Daphne (Thymelaeceae) was laureated for their sweet-scented flower, silky fiber bark, and officinal usage.^1,2 Nowadays, scientists have been focusing on their diverse constituents such as diterpenoids, biflavans, lignans, and sesquiterpenoids, which possess beneficial bioactivities to human beings.^3,4 Our previous studies on this genus indicated Daphne may be a potential AIDS remedy as it is rich in chemical components with strong anti-HIV activity.^5,6 One member of the genus Daphne is the alpine meadows plant “Daphne aurantiaca Diels.”, which decorates the Southwest China Hengduan Mountain highland with dazzling yellow flowers in blooming summer. The local people in the Shangri-La Tibetan minority community use this plant for making religious paper, as a pest repellent, and as a remedy for traumatic injuries.^7,8 A previous chemical investigation on the bark of D. aurantiaca showed the presence of sesquiterpenoids, diterpenoids, and phenols, with anti-inflammatory activity.^9–12 Bioactive compounds from this plant in Shangri-La were reported, known as the three novel sesquiterpenoids daphnauranols A–C with antifeedant activities.¹³ However, there is no systematic chemical analysis of this plant. To elucidate the full composition of this D. aurantiaca Diels., our further chemical study on their stems led to the isolation of thirteen new sesquiterpenoids: six guaiane type auranticanols A–F (1, 2, and 4–7) and seven carotane type auranticanols G–M (18–24), together with sixteen known sesquiterpenoids including eleven guaiane ones: chamaejasmone D (3),¹⁴ torelolone (8),¹⁵ virginolide (9),¹⁶ 14α, 15β, 1(H)α, 5(H)α, 7(H)α-guai-11(13)-ene-8β,12-diol (10),¹⁷ 4α,5α,8α,11(H)α-2-oxo-guai-1(10)-en-12,8-olide-7α-ol (11),⁹ 4α,5α,8α,15β,11(H)α-2-oxo-guai-1(10)-en-12,8-olide-7β-ol (12),⁹ 4α,5β-guai-9(10),7(11)-diene-12,8-olide-1α,7α-diol (13),⁹ 3-oxo-guai-4-ene-11β,12-diol (14),⁹ 1α,4α,5α,8α,11(H)β-2-oxo-guai-12,8-olide-7β-ol (15),⁹ 1α,10β-3-oxo-guai-4,11-diene-7β-ol (16),¹⁸ and 5α,7(H)α-6-oxo-guai-1(10)-ene-4β-ol (17),¹⁹ and three carotane ones: dauca-3,11-dien-2β,15-diol (25),⁹ [1R-(1α,3aα,6α,8aα)]-felikiol (26),²⁰ and styxone B (27),²¹ and two tigliane diterpenoids: 12-O-benzoylphorbol-13-octanoate (28)⁹ and phorbol 13-monoacetate (29)²² (Fig. 1). Herein, the isolation process, structural elucidation, proposed biogenetic pathways, and anti-HIV activity assay of these compounds are described.

Fig. 1 The structures of compounds 1–29.

Results and discussion

Structural elucidation of 1–2, 4–7, and 18–24

Auranticanol A (1) was obtained as a colorless oil and defined with the molecular formula C₁₅H₂₀O₃ from HRESIMS (m/z 271.1305 [M + Na]⁺, calcd for C₁₅H₂₀O₃Na, 271.1310), with six degrees of unsaturation. Its IR spectrum revealed the absorptions of hydroxyl (3433 cm⁻¹), and carbonyls and double bond (overlapped 1750, 1721, and 1619 cm⁻¹). The ¹H NMR spectrum (Table 1) of 1 exhibited signals of three methyls [δ_H 0.91 (3H, s, H-13), 1.23 (3H, s, H-14), and 0.90 (3H, d, J = 7.2 Hz, H-15)] and an olefinic proton [δ_H 5.39 (1H, t, J = 2.1 Hz, H-2)]. The ¹³C NMR (DEPT) spectroscopic data (Table 1) showed three methyls, three methylenes, four methines (one olefinic and one formyl), and five quaternary carbons (one olefinic, one oxygenated, and one carbonyl). The ¹H and ¹³C NMR data of 1 were similar to those of chamaejasmone D (3),¹⁴ a rare distorted guaiane skeleton, except for the markedly different shifts at δ_C 60.5 (s, C-11), and 207.5 (d, C-12) instead of δ_C 49.0 (s, C-11) and 65.8 (t, C-12) in chamaejasmone D, indicating that C-12 was dehydrogenated to form an aldehyde group in 1. The HMBC (Fig. 2) correlations of 1 from H-12 [δ_H 9.63 (1H, s)], H-13 [δ_H 0.91 (3H, s)], H-14 [δ_H 1.23 (3H, s)], and H-6 [δ_H 1.91 (1H, dd, J = 4.2, 12.2 Hz) and 1.53 (1H, dd, J = 10.2, 12.2 Hz)] to C-11 confirmed the hypothesis. The other correlations in the HMBC and ¹H ¹H COSY spectrum (Fig. 2) further verified the planar structure of 1. The relative configuration of 1 was elucidated on the basis of a ROESY experiment and the hypothesis that the same type of natural products might have the same stereochemistry for one plant origin. Compound 1 supposedly had the α-orientations of H-4, H-5, 7-OH, and Me-14 as those of chamaejasmone D by its ROESY experiment (Fig. 3) and comparison of their similar ¹³C NMR data. Thus, the structure of 1 was assigned as shown and named auranticanol A.

Table 1 ¹H and ¹³C NMR data of compounds 1, 2, and 4

No.	1^b		2^a		4^b
	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC
a ^1H NMR data measured at 500 MHz.b ^1H NMR data at 400 MHz in CDCl3. All ^13C NMR data measured at 100 MHz in CDCl3.
1		145.2, s		149.9, s		106.7, s
2	5.39 (1H, t, 2.1)	122.3, d	5.12 (1H, t, 1.7)	117.1, d	3.61 (1H, ddd, 1.9, 7.3, 15.4), 3.57 (1H, ddd, 1.7, 6.9, 15.4)	60.1, t
3	2.60 (1H, ddd, 1.8, 7.1, 15.2), 1.99 (1H, ddd, 1.8, 1.9, 15.2)	41.0, t	2.59 (1H, ddd, 1.7, 7.0, 15.2), 1.92 (1H, ddd, 1.7, 1.9, 15.2)	40.9, t	1.70 (1H, m), 1.24 (1H, m)	39.7, t
4	2.46 (1H, m)	33.2, d	2.44 (1H, m)	33.3, d	1.45 (1H, m)	32.3, d
5	2.70 (1H, m)	42.2, d	3.06 (1H, m)	41.6, d	2.09 (1H, m)	54.5, d
6	1.91 (1H, dd, 4.3, 12.2), 1.53 (1H, dd, 10.2, 12.2)	32.0, t	1.72 (2H, m)	29.4, t	2.63 (1H, dd, 10.0, 13.3), 1.69 (1H, dd, 6.9, 13.3)	37.3, t
7		83.0, s		86.3, s		86.7, s
8		217.0, s	4.57 (1H, dd, 4.0, 9.8)	76.9, d		199.1, s
9	2.34 (1H, d, 16.9), 2.24 (1H, d, 16.9)	46.3, t	2.15 (1H, dd, 9.8, 13.2), 1.48 (1H, dd, 4.0, 13.2)	42.4, t	5.91 (1H, d, 1.4)	126.1, d
10		42.7, s		43.9, s		166.8, s
11		60.5, s		48.2, s		147.8, s
12	9.63 (1H, s)	207.5, d	4.11 (1H, d, 15.4), 3.50 (1H, d, 15.4)	68.4, t	5.39 (1H, d, 1.2), 5.26 (1H, d, 1.2)	112.2, t
13	0.91 (3H, s)	9.7, q	0.98 (3H, s)	13.7, q	4.25 (1H, d, 14.8), 4.01 (1H, d, 14.8)	62.8, t
14	1.23 (3H, s)	17.3, q	0.95 (3H, s)	16.3, q	2.12 (1H, d, 1.4)	20.1, q
15	0.90 (3H, d, 7.2)	17.4, q	0.91 (3H, d, 7.2)	17.3, q	1.15 (3H, d, 6.4)	18.6, q

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Fig. 2 Key ¹H ¹H COSY ([thick line, graph caption]) and HMBC (H → C) correlations of 1, 2, 4–7 and 18–24.

Fig. 3 Key ROESY correlations of 1, 2, 4–7 and 18–24.

Auranticanol B (2), obtained as a colorless oil, had the molecular formula C₁₅H₂₄O₃ from HRESIMS (m/z 275.1641 [M + Na]⁺, calcd for C₁₅H₂₄O₃Na, 275.1631). The ¹³C NMR (DEPT) spectroscopic data (Table 1) showed three methyls, four methylenes (one oxygenated), four methines (one olefinic and one oxygenated), and four quaternary carbons (one olefinic and one oxygenated). The ¹H and ¹³C NMR data of 2 were similar to those of 3, except for the remarkably different shift at δ_C 76.9 (d, C-8) in 2, replacing δ_C 220.5 (s, C-8) in 3, indicating that the carbonyl group in C-8 was hydrogenated to be an oxygenated methylene in 2. The HMBC (Fig. 2) correlation of 1 from H-8 [δ_H 4.57 (1H, dd, J = 4.0, 9.8 Hz)] to C-11 (δ_C 48.2) and the ¹H ¹H COSY correlations of H-8 with H-9 [δ_H 2.15 (1H, m) and 1.93 (1H, m)] further confirmed this assignment. The relative configuration of 2, also elucidated by the ROESY experiment (Fig. 3) and above biogenesis hypothesis, was determined to be the same as those of 1 and 3 with α-orientations of H-4, H-5, 7-OH, and Me-14. The α-orientation of 8-OH was proposed by the ROESY correlations of H-8/H-12 [δ_H 4.11 (1H, d, J = 15.4 Hz) and 3.50 (1H, d, J = 15.4 Hz)]. Therefore, the structure of compound 2 was elucidated as shown and named auranticanol B.

Auranticanols C (4) and D (5) were assigned the molecular formula C₁₅H₂₂O₅ and C₁₅H₂₂O₄ according to the analysis of HRESIMS (m/z 305.1370 [M + Na]⁺, calcd for C₁₅H₂₂O₅Na, 305.1364) and (m/z 289.1418 [M + Na]⁺, calcd for C₁₅H₂₂O₄Na, 289.1415), respectively. The ¹³C NMR and DEPT data of 4 and 5 (Tables 1 and 2) showed the carbon resonances were similar to those of 10. When compared with the ¹³C NMR spectroscopic data of 10 (δ_C 39.0 d, 46.2 d, 81.2 d, 34.7 t, and 31.5 d), the C-1, C-7, C-8, C-9, and C-10 carbon signals of 4 and 5 were shifted downfield to (δ_C 106.7 s, 86.7 s, 199.1 s, 126.1 d, and 166.8 s, respectively) in 4 and (δ_C 87.1 s, 84.3 s, 206.0 s, 124.3 d, and 154.3 s, respectively) in 5. This suggested that 4 and 5 were both derived from 10 via oxidations of C-1 and C-7 to oxygenated quaternary carbons and C-8 to a carbonyl group, and the formation of a double bond between C-9 and C-10. This hypothesis was confirmed by the HMBC correlations of 4 (Fig. 2) from H-14 [δ_H 2.12 (3H, d, J = 1.4 Hz)], H-9 [δ_H 5.91 (1H, d, J = 1.4 Hz)], and H-6 [δ_H 2.63 (1H, dd, J = 10.0, 13.3 Hz) and 1.69 (1H, dd, J = 6.9, 13.3 Hz)] to C-1, from H-6 to C-8, and from H-9, H-12 [δ_H 5.39 (1H, d, J = 1.2 Hz), 5.26 (1H, d, J = 1.2 Hz)], and H-13 [δ_H 4.25 (1H, d, J = 14.8 Hz) and 4.01 (1H, d, J = 14.8 Hz)] to C-7. The assignment of 5 was also confirmed by similar HMBC and ¹H ¹H COSY correlations (Fig. 2). The only difference between 4 and 5 was the chemical group at C-1: a hydroxyl group in 5 and a hydroperoxyl in 4. This was confirmed by their assigned molecular formulas and chemical shifts at C-1. The similar ROESY correlations (Fig. 3) of 4 and 5 to 10 indicated that 4 and 5 possessed the same relative configuration which were determined to have α-orientations of OH (OOH)-1, H-5 and OH-7 like 10, as evidenced by their similar ¹³C NMR data and them having the same biogenesis origin. The α-orientation of Me-15 in 4 and 5 was proved by the NOE of H-5 [δ_H 2.09 (1H, m)]/H-15 [δ_H 1.15 (3H, d, J = 6.4 Hz)] in 4 and H-5 [δ_H 1.97 (1H, m)]/H-3α [δ_H 1.91 (1H, m)] and H-3α/H-15 [δ_H 1.04 (3H, d, J = 6.9 Hz)] in 5. Thus, the structures of 4 and 5 were assigned and named auranticanols C and D, respectively.

Table 2 ¹H and ¹³C NMR data of compounds 5–7^a

No.	5^b		6^a		7^a
	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC
a ^1H, ^13C NMR data measured at 400 and 100 MHz, respectively. ^a in CDCl3. ^b in CD3OD.
1		87.1, s		136.0, s		144.1, s
2	2.06 (1H, m), 1.89 (1H, m)	35.7, t		206.8, s	2.37 (1H, m), 2.18 (1H, m)	30.6, t
3	1.91 (1H, m), 1.26 (1H, m)	30.3, t	2.38 (1H, dd, 7.5, 17.2), 2.03 (1H, dd, 3.2, 17.2)	47.6, t	1.68 (1H, m), 1.34 (1H, m)	33.7, t
4	2.74 (1H, m)	36.1, d	2.33 (1H, m)	32.2, d	2.11 (1H, m)	39.6, d
5	1.97 (1H, m)	50.4, d	2.82 (1H, m)	40.9, d	2.83 (1H, m)	41.2, d
6	1.85 (1H, dd, 5.3, 14.2), 1.78 (1H, dd, 11.4, 14.2)	32.0, t	1.74 (1H, m), 1.60 (1H, m)	24.9, t	1.72 (1H, m), 1.37 (1H, dd, 9.6, 12.0)	36.9, t
7		84.3, s	2.84 (1H, m)	44.4, d		80.4, s
8		206.0, s	4.66 (1H, ddd, 3.3, 7.6, 7.8)	78.5, d		103.9, s
9	5.80 (1H, s)	124.3, d	2.77 (1H, m), 2.58 (1H, dd, 1.8, 17.2)	39.9, t	2.73 (1H, d, 15.6), 2.28 (1H, d, 15.6)	42.0 t
10		154.3, s		146.3, s		122.5, s
11		152.2, s	2.75 (1H, m)	38.5, d		155.7, s
12	5.29 (1H, d, 1.3), 5.22 (1H, d, 1.3)	112.2, t		179.7, s	4.46 (1H, d, 13.2), 4.32 (1H, d, 13.2)	67.9, t
13	4.14 (1H, d, 15.2), 4.11 (1H, d, 15.2)	62.2, t	1.27 (3H, d, 7.9)	12.6, q	5.15 (1H, d, 1.2), 4.95 (1H, d, 1.2)	104.4, t
14	1.98 (1H, s)	21.2, q	2.24 (3H, s)	21.9, q	1.67 (3H, s)	22.4, q
15	1.04 (3H, d, 6.9)	16.1, q	0.89 (3H, d, 7.2)	15.3, q	0.93 (3H, d, 7.1)	15.4, q

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Auranticanol E (6) was defined with the molecular formula C₁₅H₂₀O₃ from HRESIMS (m/z 271.1314 [M + Na]⁺, calcd for C₁₅H₂₀O₃Na, 271.1310). The ¹³C NMR data (Table 2) of 6 were similar to those of 9, except for the markedly different shifts at δ_C 136.0 (s, C-1), 146.3 (s, C-10), 47.6 (d, C-3), 32.2 (d, C-4), 38.5 (d, C-11), and 12.6 (q, C-13) instead of the corresponding carbons at δ_C 62.6 (d, C-1), 27.6 (d, C-10), 130.4 (d, C-3), 180.9 (s, C-4), 140.7 (s, C-11), and 118.6 (t, C-13) in compound 9, indicating that the olefinic carbons C-3, C-4, C-11 and C-13 were saturated and C-1–C-10 were dehydrogenated to form a double bond in 6. The HMBC (Fig. 2) correlations of 6 from H-3 [δ_H 2.38 (1H, dd, J = 7.5, 17.2 Hz) and 2.03 (1H, dd, J = 3.2, 17.2 Hz)] and H-14 [δ_H 2.24 (3H, s)] to C-1, from H-5 [δ_H 2.78 (1H, m)] and H-9 [δ_H 2.77 (1H, m) and 2.57 (1H, dd, J = 1.8, 17.2 Hz)] to C-10, and from H-13 [δ_H 1.27 (3H, d, J = 7.9 Hz)] to C-12 (δ_C 179.7 s), together with the key ¹H ¹H COSY correlations H-4 [δ_H 2.35 (1H, m)]/H-5, H-11 [δ_H 2.75 (1H, m)]/H-13 [δ_H 1.27 (3H, d, J = 7.9 Hz)] further verified the hypothesis. The other correlations in the HMBC and ¹H ¹H COSY spectrum further confirmed the atom connectivity in 6 (Fig. 2). The configuration of the skeleton in 6 was elucidated by the ROESY experiment (Fig. 3) and determined to possess the α-orientations of H-5 and H-8 like 9. The β-orientations of H-7, Me-13, and Me-15 were elucidated by the NOE of H-5/H-3α [δ_H 2.38 (1H, dd, J = 7.5, 17.2 Hz)], H-3β [δ_H 2.03 (1H, dd, J = 3.2, 17.2 Hz)]/H-15 [δ_H 0.89 (3H, d, J = 6.9 Hz)], H-4/H-6α [δ_H 1.74 (1H, m)], H-6β [δ_H 1.60 (1H, m)]/H-13, H-7 [δ_H 2.84 (1H, m)]/H-13, and H-8 [δ_H 4.66 (1H, ddd, J = 3.3, 7.6, 7.8 Hz)]/H-11. Thus, the structure of 6 was assigned as shown and named auranticanol E.

Auranticanol F (7) was defined with the molecular formula C₁₅H₂₂O₃ from HRESIMS (m/z 273.1459 [M + Na]⁺, calcd for C₁₅H₂₂O₃Na, 273.1466). The comparison of its 1D NMR data (Table 2) with those of 5 suggested that 7 had a similar skeleton to 5. The differences were the remarkably different shifts at δ_C 144.1 (s, C-1), 103.9 (s, C-8), 42.0 (t, C-9), and 122.5 (s, C-10) in 7 instead of δ_C 87.1 (s, C-1), 206.0 (s, C-8), 124.3 (d, C-9), and 154.3 (s, C-10) in 5, revealing that the double bond at C-9–C-10 in 5 was moved to C-1–C-10, and the carbonyl C-8 in 5 was linked to C-12 via an oxygen atom forming a hemiketal group in 7. The key HMBC (Fig. 2) correlations of 7 from H-3 [δ_H 1.34 (1H, m), 1.68 (1H, m)], H-9 [δ_H 2.73 (1H, d, J = 15.6 Hz) and 2.28 (1H, d, J = 15.6 Hz)] and H-14 [δ_H 1.67 (3H, s)] to C-1 and from H-12 [δ_H 4.46 (1H, d, J = 13.2 Hz) and 4.32 (1H, d, J = 13.2 Hz)] to C-8 further supported this hypothesis. The other correlations in the HMBC and ¹H ¹H COSY spectrum (Fig. 2) further confirmed the atom connectivity in 7. The α-orientations of H-5 and OH-7 in 7 were elucidated by the ROESY experiment (Fig. 3) and determined to be the same as those of 5 for its biosynthesis origin. And the β-orientations of OH-8 and Me-15 were elucidated by the key NOE of H-15 [δ_H 0.93 (3H, d, J = 6.9 Hz)]/H-6β [δ_H 1.72 (1H, m)], H-6α [δ_H 1.37 (1H, dd, J = 9.6, 12.0 Hz)]/H-12 [δ_H 4.32 (1H, d, J = 13.2 Hz)], and H-6β/H-12. Thus, the structure of 7 was assigned as shown and named auranticanol F.

Auranticanol G (18) was defined with the molecular formula C₁₅H₂₄O₂ from HRESIMS (m/z 259.1673 [M + Na]⁺, calcd for C₁₅H₂₄O₂Na, 259.1673). The ¹H and ¹³C NMR data (Table 3) of 18 were similar to those of 25, except for the signals at δ_C 143.7 (s, C-1), 68.8 (d, C-2), and 128.0 (d, C-7) instead of δ_C 143.8 (s, C-1), 127.4 (d, C-2), and 70.0 (d, C-7) in 25, indicating that the hydroxyl group at C-7 moved to C-2 and the double bond moved to C-1–C-7 in 18. The HMBC (Fig. 2) correlations of 18 from H-2 [δ_H 4.23 (1H, d, J = 1.9, 4.1 Hz)], H-3 [1.83 (1H, ddd, J = 1.9, 4.4, 14.8 Hz) and 1.54 (1H, ddd, J = 4.1, 11.3, 14.8 Hz)], H-6 [2.13 (2H, m)], and H-14 [δ_H 4.03 (1H, d, J = 16.0 Hz) and 3.93 (1H, d, J = 16.0 Hz)] to C-1 and from H-2 to C-4 (δ_C 46.2 d) and the ¹H ¹H COSY correlations of H-6/H-7 [δ_H 5.67 (1H, m)] and H-2/H-3 confirmed this structural change. The other correlations in the HMBC and ¹H ¹H COSY spectrum (Fig. 2) further determined the atom connectivity in 18. The relative configurations of C-4 and C-5 in 18 were determined to be the same as those of 25 based on their similar NMR data and the hypothesis that the skeleton of carotene had the same stereochemistry for the probable common biogenesis. The α-orientations of H-2 and H-10 were elucidated by the NOE of H-2/H-4 [δ_H 2.53 (1H, ddd, J = 1.6, 11.3, 13.2 Hz)] and Me-13 [δ_H 1.70 (3H, s)]/Me-15 [δ_H 0.86 (3H, s)] (Fig. 3). Thus, the structure of 18 was assigned as shown and named auranticanol G.

Table 3 ¹H and ¹³C NMR data of compounds 18–20

No.	18^a		19^b		20^a
	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC
a ^1H NMR data measured at 500 MHz.b ^1H NMR data at 400 MHz in CDCl3. All ^13C NMR data measured at 100 MHz in CDCl3.
1		143.7, s		136.5, s		142.2, s
2	4.23 (1H, dd, 1.9, 4.1)	68.8, d	6.41 (1H, d, 0.9)	143.8, d	5.73 (1H, dd, 7.7, 8.0)	128.9, d
3	1.83 (1H, ddd, 1.9, 4.4, 14.8), 1.54 (1H, ddd, 4.1, 11.3, 14.8)	32.1, t	4.37 (1H, dd, 0.9, 11.4)	70.0, d	2.07 (1H, m), 1.90 (1H, m)	26.7, t
4	2.53 (1H, ddd, 4.4, 11.3, 13.2)	46.2, d	2.38 (1H, dd, 11.2, 11.4)	57.1, d	1.86 (1H, m)	50.0, d
5		43.8, s		41.3, s		41.8, s
6	2.13 (2H, m)	42.9, t	2.63 (1H, d, 15.5), 2.49 (1H, d, 15.5)	58.5, t	2.09 (1H, dd, 2.2. 15.5), 1.47 (1H, dd, 8.6, 15.5)	51.2, t
7	5.67 (1H, m)	128.0, d		202.3, s	4.55 (1H, dd, 2.2, 8.6)	70.0, d
8	1.53 (1H, ddd, 4.2, 5.0, 16.4), 1.51 (1H, m)	43.6, t	1.54 (2H, m)	42.6, t	1.57 (1H, m), 1.41 (1H, m)	42.0, t
9	1.75 (2H, m)	29.5, t	1.85 (1H, m), 1.76 (1H, m)	29.5, t	1.69 (2H, m)	28.0, t
10	2.98 (1H, m)	51.7, d	3.12 (1H, m)	48.5, d	2.87 (1H, m)	49.6, d
11		148.9, s		148.1, s		147.0, s
12	4.78 (1H, d, 1.3), 4.71 (1H, d, 1.3)	113.7, t	4.97 (1H, d, 1.4), 4.95 (1H, d, 1.4)	114.4, t	4.79 (1H, d, 1.4), 4.68 (1H, d, 1.4)	113.1, t
13	1.70 (3H, s)	23.3, q	1.88 (3H, s)	23.4, q	1.67 (3H, s)	23.3, q
14	4.03 (1H, d, 16.0), 3.93 (1H, d, 16.0)	69.1, t	4.18 (1H, d, 16.0), 4.13 (1H, d, 16.0)	64.2, t	4.16 (2H, brs)	69.8, t
15	0.86 (3H, s)	19.4, q	0.98 (3H, s)	19.4, q	0.89 (3H, s)	18.5, q

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Auranticanol H (19) was formulated as C₁₅H₂₂O₃ from HRESIMS (m/z 273.1460 [M + Na]⁺, calcd for C₁₅H₂₂O₃Na, 273.1466). 19 had similar ¹³C NMR data (Table 3) to those of 25, and the differences were δ_C 70.0 (d, C-3) and 202.3 (s, C-7), replacing δ_C 28.2 (t, C-3) and 70.0 (d, C-7) in 25, indicating that in 19 C-3 and C-7 were oxidized to an oxygenated methine and a carbonyl group, respectively. The HMBC correlations and ¹H ¹H COSY correlations (Fig. 2) of 19 confirmed the hypothesis and the atom connectivity in 19. The relative configuration of C-4 and C-5 in 19 was also presumptively determined to be the same as that of 25 for the biogenesis hypothesis. The α-orientations of OH-3 and H-10 were elucidated by the ROESY experiment. Thus, the structure of 19 was assigned as shown and named auranticanol H.

Auranticanol I (20) was established to have the molecular formula C₁₅H₂₄O₂ from HRESIMS (m/z 259.1673 [M + Na]⁺, calcd for C₁₅H₂₄O₂Na, 259.1673). The ¹H and ¹³C NMR data (Table 3) of 20 were closely similar to those of 25, except for a little difference in the shift at C-10 in 20, indicating that 20 and 25 were epimers at C-10. Its HMBC and ¹H ¹H COSY correlations (Fig. 2) accorded with the atom connectivity in 20. The relative configuration of C-4 and C-5 in 20 was determined to be the same as that of 19. And the α-orientations of OH-7 and H-10 were determined by the key ROESY (Fig. 3) correlations of H-15 [δ_H 0.89 (3H, s)] with H-13 [δ_H 1.67 (3H, s)] and H-7 [δ_H 4.55 (1H, d, J = 2.2, 8.6 Hz)]. Thus, the structure of 20 was assigned as shown and named auranticanol I.

Auranticanol J (21) was defined with the molecular formula C₁₅H₂₂O₂ from HRESIMS (m/z 257.1516 [M + Na]⁺, calcd for C₁₅H₂₂O₂Na, 257.1517). The ¹³C NMR data (Table 4) of 21 were similar to those of 20, except for the remarkably different shift at δ_C 203.6 (s, C-7) instead of δ_C 70.2 (d, C-7) in 20, indicating that the C-7 was oxidated to be a carbonyl group in 21. The HMBC and ¹H ¹H COSY (Fig. 2) correlations further confirmed the above hypothesis and the atom connectivity in 21. The relative configuration of 21 was elucidated by the ROESY experiment (Fig. 3) and biogenetically determined to be the same as 19. Thus, the structure of 21 was assigned as shown and named auranticanol J.

Table 4 ¹H and ¹³C NMR data of compounds 21–24^a

No.	21^b		22^c		23^a		24^c
	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC
a ^a,c 1H NMR data measured at 400 MHz in CDCl3 and ^b 1H NMR data at 500 MHz. ^a 13C NMR data measured at 100 MHz in CDCl3 and ^b,c 13C NMR data 125 MHz in CDCl3.
1		139.6, s		136.5, s		137.2, s		150.8, s
2	6.50 (1H, dd, 2.1, 6.7)	143.4, d	6.36 (1H, dd, 7.8, 8.3)	140.7, d	5.40 (1H, dd, 7.7, 8.5)	124.6, d	1.37 (1H, m), 1.21 (1H, m)	41.0, t
3	2.39 (1H, ddd, 3.0, 6.7, 14.8), 2.14 (1H, ddd, 2.1, 13.3, 14.8)	30.0, t	3.14 (1H, m), 2.50 (1H, m)	30.1, t	2.48 (1H, m), 2.01 (1H, m)	41.0, t	1.53 (1H, m), 1.15 (1H, m)	24.2, t
4	2.25 (1H, ddd, 3.0, 13.2, 13.3)	48.4, d	2.37 (1H, m)	48.9, d	2.37 (1H, m)	50.0, d	1.71 (1H, m)	49.9, d
5		40.4, s	—	40.7, s		44.4, s		35.9, s
6	2.70 (1H, d, 16.3), 2.44 (1H, d, 16.3)	58.8, t	2.70 (1H, d, 15.5), 2.41 (1H, d, 15.5)	58.8, t	2.15 (1H, m), 1.84 (1H, m)	30.1, t	1.52 (1H, m), 1.31 (1H, m)	21.6, t
7		203.6, s		203.1, s	1.58 (1H, m), 1.16 (1H, m)	41.0, t	1.39 (1H, m), 1.21 (1H, m)	41.7 t
8	1.56 (1H, ddd, 4.4, 5.1, 19.0), 1.41 (1H, ddd, 4.2, 11.5, 19.0)	41.8, t	1.54 (1H, m), 1.36 (1H, m)	41.6, t	1.38 (1H, m), 1.25 (1H, m)	27.4, t	1.55 (2H, m)	23.4, t
9	1.74 (2H, m)	28.6, t	1.66 (1H, m), 1.38 (1H, m)	26.9, t	1.59 (1H, m), 1.50 (1H, m)	26.0, t	2.25 (1H, m), 1.93 (1H, m)	36.8, t
10	2.91 (1H, ddd, 2.2, 13.2, 13.6)	49.2, d	2.51 (1H, m)	47.4, d	2.51 (1H, m)	47.4, d	1.58 (1H, m)	43.0, d
11		145.7, s		76.9, s		75.9, s	—	74.7, s
12	4.87 (1H, d, 1.3), 4.72 (1H, d, 1.3)	113.4, t	3.39 (1H, d, 10.9), 3.26 (1H, d, 10.9)	70.3, t	3.55 (1H, d, 10.8), 3.42 (1H, d, 10.8)	68.3, t	3.68 (1H, d, 10.9), 3.61 (1H, d, 10.9)	65.7, t
13	1.69 (3H, s)	23.8, q	1.19 (3H, s)	21.2, q	1.18 (3H, s)	24.4, q	3.68 (1H, d, 10.9), 3.61 (1H, d, 10.9)	65.7, t
14	4.15 (1H, d, 12.5), 4.09 (1H, d, 12.5)	66.6, t	1.81 (3H, s)	21.9, q	1.64 (3H, s)	28.3, q	4.67 (1H, d, 1.9), 4.38 (1H, d, 1.9)	105.3, t
15	0.89 (3H, s)	19.1, q	0.91 (3H, s)	19.1, q	0.78 (3H, s)	17.3, q	0.66 (3H, s)	16.2, q

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Auranticanol K (22) was established to have the molecular formula C₁₅H₂₄O₃ from HRESIMS (m/z 275.1620 [M + Na]⁺, calcd for C₁₅H₂₄O₃Na, 275.1623). The ¹³C NMR data (Table 4) of 22 were also similar to those of 21, the main differences were the signals δ_C 76.9 (s, C-11), 70.3 (t, C-12), and 21.9 (q, C-14) were substituted for δ_C 145.7 (s, C-11), 113.4 (t, C-12), and 66.6 (t, C-14) in 21, indicating that C-11 and C-12 were oxygenated and linked hydroxyl groups and C-14 was deoxygenized in 22. This was supported by the key HMBC (Fig. 2) correlations from H-13 [δ_H 1.19 (3H, s)] to C-10 (δ_C 47.4, d), C-11 and C-12, and from H-14 [δ_H 1.19 (3H, s)] to C-7 (δ_C 203.1, s), C-1 (δ_C 136.5, s), and C-2 (δ_C 140.7, d). The configurations of C-4, C-5, and C-10 in 22 were elucidated by the ROESY experiment (Fig. 3) and determined to be the same as those of 21 due to their similar ¹³C NMR data. Therefore, the structure of 22 was assigned as shown and named auranticanol K.

Auranticanol L (23) was formulated as C₁₅H₂₆O₂ from HRESIMS (m/z 261.1829 [M + Na]⁺, calcd for C₁₅H₂₆O₂Na, 261.1829). The ¹³C NMR data (Table 4) of 23 were similar to those of 22, except for the carbon shift at δ_C 41.9 (t, C-7) replacing δ_C 203.1 (s, C-7) in 22, indicating that the carbonyl group in C-7 was deoxygenized to form the methylene in 23. The HMBC and ¹H ¹H COSY correlations (Fig. 2) further confirmed the hypothesis and the atom connectivity in 23. The configuration of 23 was also biogenetically determined to be the same as 22 by the ROESY experiment (Fig. 3). Thus, the structure of 23 was assigned as shown and named auranticanol L.

Auranticanol M (24) was defined with the molecular formula C₁₅H₂₄O₃ from HRESIMS (m/z 253.1808 [M − H]⁻, calcd for C₁₅H₂₅O₂, 253.1803). The comparison of the similar ¹³C NMR data (Table 4) of 24 with those of 23 showed the carbon signals at δ_C 65.7 (t, C-13), 150.8 (s, C-1), 41.0 (t, C-2), and 105.3 (t, C-14) in 24 replaced those of δ_C 24.4 (q, C-13), 137.2 (s, C-1), 124.6 (t, C-2), and 28.3 (q, C-14) in 23, indicating that C-13 in 23 was oxygenated to a methylol in 24, and the double bond of C-1–C-2 in 23 moved to C-1–C-14 in 24. This deduction was proved by the HMBC and ¹H ¹H COSY (Fig. 2) correlations. The relative configuration of 24 was biogenetically elucidated to be the same as 23 by the ROESY experiment (Fig. 3) and the similar NMR data of those chiral carbons. Thus, the structure of 24 was assigned as shown and named auranticanol M.

The isolates from the stems of D. aurantiaca mainly were divided into two types of sesquiterpenoid, guaiane and carotene. The new guaiane sesquiterpenoids were plausibly derived from the guaiane skeletons of 11 and 12 via chemical reactions or transformations like hydrolysis, hydrogenation, oxidation, electrophilic addition,²³ H [1,3] σ migration,²⁴ and electron migration.^25,26 The new carotane sesquiterpenoids may be generated from the known natural product 25 via changes like isomerization, rearrangement, oxidation, hydrogenation, H [1,3] σ migration,²⁴ and electron migration.^25,26

Bioactivity evaluation

Twenty compounds (1–7, 11–14, 18–24, 28, and 29) were tested for anti-HIV bioactivity (Table 5). They were evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC₅₀) and the cytotoxicity assay against the C8166 cell line by MTT methods. Two guaiane type sesquiterpenoids 11 and 14 showed moderate activities with EC₅₀ values of 2.138 μg mL⁻¹ and 0.286 μg mL⁻¹ and SI values of SI > 93.545 and SI = 93.787, respectively. The carotene type sesquiterpenoid 19 also showed moderate activities with an EC₅₀ value of 1.773 μg mL⁻¹ and SI = 10.243. The tigliane diterpenoid 28 showed better anti-HIV bioactivities than the positive control with an EC₅₀ value of 0.000282 μg mL⁻¹ and SI = 65177.305 (positive control 3′-azido-3′-deoxythymidine with an EC₅₀ value of 0.001656 μg mL⁻¹ and SI = 593 164.855). The other 16 compounds showed weak anti-HIV bioactivities with EC₅₀ values that ranged from 12.530 to 136.937 μg mL⁻¹.

Table 5 Summary of anti-HIV-1 activity of compounds 1–7, 11–14, 18–24, 28, and 29

No.	CC50 (μg mL^−1)	EC50 (μg mL^−1)	SI	No.	CC50 (μg mL^−1)	EC50 (μg mL^−1)	SI
1	155.641	61.511	2.530	18	95.424	23.352	4.086
2	103.979	49.779	2.089	19	18.16	1.773	10.243
3	>200	136.937	>1.461	20	26.361	15.446	1.707
4	>200	80.952	>2.471	21	15.419	12.530	1.231
5	>200	77.034	>2.596	22	163.974	73.895	2.219
6	175.041	77.000	2.273	23	61.010	60.604	1.007
7	147.926	69.606	2.125	24	151.059	45.484	3.321
11	>200	2.138	>93.545	28	18.38	0.000282	65 177.305
12	>200	24.246	>8.249	29	>200	17.808	>11.231
13	62.348	37.932	1.644	3′-Azido-3′-deoxythymidine	982.281	0.001656	593 164.855
14	26.823	0.286	93.787

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Conclusion

A series of terpenoids including a number of diterpenoids and a few sesquiterpenoids have been isolated from the Daphne species. The plant D. aurantiaca was also in accordance with this common law as both types of terpenoids were isolated.³ From our results, the chemical constitutes of this plant had more sesquiterpenoids and less diterpenoids. The genus Daphne exhibited obvious anti-HIV-1 activities according to previous studies, however no compound from this plant was identified to be responsible for this activity. Among the isolates tested here, three sesquiterpenoids including two guaianes and one carotene showed moderate activities with a low SI value. Nevertheless, one tigliane diterpenoid showed a better anti-HIV bioactivity with an EC₅₀ value of 0.000282 μg mL⁻¹ when compared to the positive control 3′-azido-3′-deoxythymidine even though the SI was lower. The other tigliane diterpenoid without a long-chain fatty acid and benzoate revealed limited activity, which suggested the esterification with an organic acid, especially a long-chain lipophilic acid, at C-12 and C-13 in the tigliane diterpenoid is required for the anti-HIV bioactivity.

Experimental section

General

The optical rotations were obtained on a Horiba SEAP-300 polarimeter (Kyoto, Japan). Mass spectra were measured on a Bruker HCT/Esquire (Billerica, USA) and a VG Auto Spec-3000 mass spectrometer (Manchester, UK). And the UV spectra were obtained on a Hitachi UV 210A spectrophotometer (Tokyo, Japan). IR spectra were acquired on a Bio-Rad FTS-135 spectrometer (Berkeley, USA) with KBr pellets. 1D and 2D NMR spectra were measured using a Bruker AV-400 or a DRX-500 (Billerica, USA) instrument with TMS as an internal standard. Column chromatography (CC) was performed on Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), reverse phase-18 (RP-18) (40–70 μm, Fuji Silysia Chemical Ltd, Nagoya, Japan) and hydroxypropyl Sephadex (Sephadex LH-20) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Fractions were monitored by TLC and spots were visualized by heating after spraying with 5% H₂SO₄ in EtOH (bp 77–79 °C).

Plant material

Daphne aurantiaca Diels. stems were obtained from the Shangri-La Yunnan Province, People’s Republic of China. The voucher specimen (HUANG0005) was identified by Prof. Dr Y. Niu (Kunming Institute of Botany, Chinese Academy of Sciences) and deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, People’s Republic of China.

Extraction and isolation

The stems of D. aurantiaca (4.5 kg) were crushed and extracted with 95% EtOH refluxing at 80 °C (3 hours, 3 × 20 L). The filtrated EtOH solution was concentrated to give the concrete (1.6 kg). The concrete was suspended in 3 L water and then extracted with EtOAc (3 × 3 L). After concentration, the EtOAc extract (303 g) was firstly subjected to a silica gel (200–300 mesh) column (15 × 120 cm) eluted with CHCl₃/MeOH (50 : 1–1 : 1) to afford fractions A–D. Fraction A (79 g) was defatted with a Sephadex LH-20 column (MeOH/CHCl₃ 1 : 1) and then separated repeatedly with a RP-18 column eluting with MeOH/H₂O (1 : 5–1 : 0) to afford fractions A1–A7. Fractions A1–A7 were purified repeatedly by silica gel column (petroleum ether/acetone, 4 : 1) and Sephadex LH-20 (MeOH) column chromatography to yield 6 (56.3 mg), 9 (6.3 mg), 2 (21.3 mg), 11 (156.3 mg), 12 (143.2 mg), 14 (15.6 mg), and 15 (4.9 mg), respectively. Fraction B (110 g) was then subjected to a silica gel column eluted with petroleum ether/acetone (10 : 1–1 : 1) to give four fractions B1–B4. Fraction B1 was separated repeatedly with a RP-18 column with MeOH/H₂O (1 : 5–5 : 1) to afford fractions B1a–B1e. Fractions B1a–B1e were separated repeatedly with a silica gel column (petroleum ether/acetone, 3 : 1) and Sephadex LH-20 column (MeOH) to yield 16 (2.0 mg), 17 (2.4 mg), 21 (11.3 mg), 25 (2.3 mg), and 27 (1.8 mg), respectively. Fraction B2 was separated repeatedly with a RP-18 column (MeOH/H₂O 1 : 5–5 : 1) to afford fractions B2a–B2f. Fractions B2a–B2f were purified repeatedly by silica gel column (petroleum ether/acetone 2 : 1) and Sephadex LH-20 (MeOH) column chromatography to yield 3 (34.5 mg), 7 (460 mg), 8 (3.3 mg), 10 (2.1 mg), and 13 (4.7 mg), respectively. Fraction B3 was separated repeatedly with a RP-18 column (MeOH/H₂O 1 : 5–5 : 1) to afford fractions B3a–B3g. Fractions B3a–B3g were separated repeatedly with a silica gel column (petroleum ether/acetone 2 : 1) and Sephadex LH-20 column (MeOH) to yield 18 (15.7 mg), 19 (4.2 mg), 20 (782.4 mg), 22 (5.6 mg), 23 (10.3 mg), and 26 (2.7 mg), respectively. Fraction B4 combined with fraction C (10 g) was separated repeatedly with a RP-18 column (MeOH/H₂O 1 : 5–5 : 1) to afford fractions C1–C4. Fractions C1–C4 were separated repeatedly with a silica gel column (petroleum ether/acetone 2 : 1) and Sephadex LH-20 column (MeOH) to yield 1 (23.4 mg), 4 (5.3 mg), 5 (13.1 mg), and 24 (9.6 mg), respectively. Lastly the fraction D (79 g) was separated repeatedly with a Sephadex LH-20 column (MeOH), and separated repeatedly with a RP-18 column (MeOH/H₂O 1 : 5–9 : 1) to obtain fractions D1–D2. Fractions D1–D2 were separated repeatedly with silica gel column (petroleum ether/acetone 2 : 1) and Sephadex LH-20 (MeOH) column chromatography to yield 28 (22.3 mg) and 29 (35.9 mg), respectively.

Auranticanol A (1). Colorless oil; C₁₅H₂₀O₃, [α]¹⁸_D −51.26 (c 0.241, MeOH); ESIMS positive m/z [M + Na]⁺ 271 (100); HRESIMS m/z [M + Na]⁺ 271.1305 (calcd for C₁₅H₂₀O₃Na, 271.1310); UV (MeOH) λ_max (log ε) 203 (3.69); IR (KBr) ν_max 3433, 2963, 2931, 2874, 1751, 1721, 1619, 1453, 1404, 1382, 1065, 963, 934 cm⁻¹; ¹H and ¹³C NMR data see Table 1.

Auranticanol B (2). Colorless oil; [α]²⁰_D −29.09 (c 0.115, MeOH); ESIMS positive m/z [M + Na]⁺ 275 (75); HRESIMS m/z [M + Na]⁺ 275.1631 (calcd for C₁₅H₂₄O₃Na, 275.1623); UV (MeOH) λ_max (log ε) 203 (2.73), 237 (2.36); IR (KBr) ν_max 3405, 2959, 2919, 2876, 2839, 1628, 1450, 1374, 1161, 1106, 1046, 1021, 997, 982, 685 cm⁻¹; ¹H and ¹³C NMR data see Table 1.

Auranticanol C (4). Colorless oil; [α]²⁰_D +72.95 (c 0.176, MeOH); ESIMS positive m/z [M + Na]⁺ 305 (100); HRESIMS m/z [M + Na]⁺ 305.1370 (calcd for C₁₅H₂₂O₅Na, 305.1364); UV (MeOH) λ_max (log ε) 204 (3.57), 224 (3.76); IR (KBr) ν_max 3428, 2959, 2932, 2879, 1682, 1626, 1434, 1385, 1166, 1031, 1022, 984, 916 cm⁻¹; ¹H and ¹³C NMR data see Table 1.

Auranticanol D (5). Colorless oil; [α]¹⁸_D + 38.47 (c 0.261, MeOH); ESIMS positive m/z [M + Na]⁺ 289 (100); HRESIMS m/z [M + Na]⁺ 289.1418 (calcd for C₁₅H₂₂O₄Na, 289.1415); UV (MeOH) λ_max (log ε) 202 (3.59), 235 (3.59), 307 (2.33); IR (KBr) ν_max 3427, 2958, 2935, 2873, 1675, 1636, 1440, 1379, 1246, 1182, 1115, 1065, 1009, 912 cm⁻¹; ¹H and ¹³C NMR data see Table 2.

Auranticanol E (6). Colorless oil; [α]¹⁸_D −127.70 (c 0.496, MeOH); ESIMS positive m/z [M + Na]⁺ 271 (100); HRESIMS m/z [M + Na]⁺ 271.1314 (calcd for C₁₅H₂₀O₃Na, 271.1310); UV (MeOH) λ_max (log ε) 221 (3.95), 251 (3.85); IR (KBr) ν_max 2957, 2929, 1767, 1708, 1629, 1452, 1409, 1381, 1340, 1256, 1166, 1098, 1054, 1019, 1003, 942, 889 cm⁻¹; ¹H and ¹³C NMR data see Table 2.

Auranticanol F (7). Colorless needle crystal, mp 123–124 °C; [α]¹⁸_D −9.12 (c 0.23, MeOH); ESIMS positive m/z [M + Na]⁺ 273 (100); HRESIMS m/z [M + Na]⁺ 273.1459 (calcd for C₁₅H₂₂O₃Na, 273.1466); UV (MeOH) λ_max (log ε) 203 (3.93), 293 (1.93); IR (KBr) ν_max 3539, 3387, 2961, 2946, 2873, 1430, 1330, 1236, 1217, 1157, 1115, 1087, 1034, 985, 895, 825, 651, 618, 572 cm⁻¹; ¹H and ¹³C NMR data see Table 2.

Auranticanol G (18). Colorless oil; [α]¹⁷_D +0.36 (c 0.206, MeOH); ESIMS positive m/z [M + Na]⁺ 259 (85); HRESIMS m/z [M + Na]⁺ 259.1673 (calcd for C₁₅H₂₄O₂Na, 259.1673); UV (MeOH) λ_max (log ε) 202 (3.66); IR (KBr) ν_max 3427, 2951, 2926, 2855, 1634, 1452, 1379, 1120, 1085, 890 cm⁻¹; ¹H and ¹³C NMR data see Table 3.

Auranticanol H (19). Colorless oil; [α]¹⁷_D −0.54 (c 0.343, MeOH); ESIMS positive m/z [M + Na]⁺ 273 (85); HRESIMS m/z [M + Na]⁺ 273.1460 (calcd for C₁₅H₂₂O₃Na, 273.1466); UV (MeOH) λ_max (log ε) 202 (3.73), 236 (3.72), 312 (2.58), 492 (1.26); IR (KBr) ν_max 3418, 2955, 2879, 1647, 1452, 1382, 1232, 1122, 1085, 1023, 994, 883 cm⁻¹; ¹H and ¹³C NMR data see Table 3.

Auranticanol I (20). Colorless needle crystal mp 74–75 °C; [α]¹⁶_D +19.30 (c 0.228, MeOH); ESIMS positive m/z [M + Na]⁺ 259 (30), [2M + Na]⁺ 495 (100); HRESIMS m/z [M + Na]⁺ 259.1673 (calcd for C₁₅H₂₄O₂Na, 259.1673); UV (MeOH) λ_max (log ε) 202 (3.88), 221 (3.99); IR (KBr) ν_max 3428, 2956, 2930, 2878, 1694, 1635, 1449, 1417, 1384, 1204, 1138, 1053 cm⁻¹; ¹H and ¹³C NMR data see Table 3.

Auranticanol J (21). Colorless oil; [α]¹⁸_D +110.86 (c 0.288, MeOH); ESIMS positive m/z [M + Na]⁺ 257 (100); HRESIMS m/z [M + Na]⁺ 257.1516 (calcd for C₁₅H₂₂O₂Na, 257.1517); UV (MeOH) λ_max (log ε) 202 (3.94), 222 (4.05); IR (KBr) ν_max 3425, 2956, 2885, 1642, 1448, 1421, 1381, 1287, 1238, 1220, 1087, 1005, 892 cm⁻¹; ¹H and ¹³C NMR data see Table 4.

Auranticanol K (22). Colorless oil; [α]¹⁶_D +34.52 (c 0.270, MeOH); ESIMS positive m/z [M + Na]⁺ 275 (85); HRESIMS m/z [M + Na]⁺ 275.1620 (calcd for C₁₅H₂₄O₃Na, 275.1623); UV (MeOH) λ_max (log ε) 201 (3.91), 222 (4.17), 240 (3.82); IR (KBr) ν_max 3427, 2950, 2928, 2878, 1645, 1636, 1455, 1385, 1283, 1245, 1202, 1126, 1053 cm⁻¹; ¹H and ¹³C NMR data see Table 4.

Auranticanol L (23). Colorless oil; [α]¹⁶_D −21.86 (c 0.304, MeOH); ESIMS positive m/z [M + Na]⁺ 261 (100); HRESIMS m/z [M + Na]⁺ 261.1829 (calcd for C₁₅H₂₆O₂Na, 261.1829); UV (MeOH) λ_max (log ε) 202 (3.48), 237 (2.46), 299 (1.85), 362 (1.85); IR (KBr) ν_max 3421, 2962, 2921, 2853, 1705, 1631, 1450, 1379, 1130, 1045, 872, 809 cm⁻¹; ¹H and ¹³C NMR data see Table 4.

Auranticanol M (24). Colorless oil; [α]¹⁶_D +19.40 (c 0.311, MeOH); ESIMS negetive m/z [M + Cl]⁻ 289 (95); HRESIMS m/z [M − H]⁻ 253.1808 (calcd for C₁₅H₂₅O₂, 253.1803); UV (MeOH) λ_max (log ε) 202(3.94), 221(4.10); IR (KBr) ν_max 3432, 2927, 2854, 1676, 1641, 1458, 1380, 1203, 1186, 1140, 1044, 886 cm⁻¹; ¹H and ¹³C NMR data see Table 4.

Anti-HIV assay

Anti-HIV activity of the compounds was evaluated by the cytopathic effects of HIV-1 (EC₅₀) and the cytotoxicity assay against the C8166 cell line (IC₅₀) with MTT methods as described in the literature and earlier research.^5,6,27 AZT (3′-azido-3′-deoxythymidine) was used as a positive control. The concentration of the antiviral sample reducing HIV-1 replication by 50% (EC₅₀) was calculated and determined with the dose–response standard curve. The selectivity index (SI) was calculated with the ratio of IC₅₀/EC₅₀.

Acknowledgements

This work was financed by NSFC (National Natural Science Foundation of China 31300294), Special Fund for Agro-Scientific Research in the Public Interest (201303117), National Support Science and Technology Subject (2013BAI11B04), Fundamental Scientific Research Funds for CATAS (ITBB2015ZD02), and Natural Science Foundation of Hainan Province (214039). The authors thank Dr Y.L. Huang (Department of Epigenetics and Molecular Carcinogenesis, UT MD Anderson Cancer Center, USA), Liwen Tian (Southern Medical University, China), Zhikai Guo (The Scripps Research Institute, USA), and Dr F. Jacques (Xishuangbanna Tropical Garden CAS, China) for the proofreading of this paper.

References and notes

1. Y. Wang, M. G. Gilbert, B. Mathew, C. D. Brickell and L. Nevling, Flora of China, Science Press, Beijing, 2007, vol. 13, pp. 230–245.
2. The Families and Genera of Angiosperms in China, ed. C. Y. Wu, A. M. Lu, T. YanCheng, C. Z. Rui and D. Z. Li, Science Press, Beijing, 2003, pp. 588–592.
3. W. C. Xu, J. G. Shen and J. Q. Jiang, Chem. Biodiversity, 2011, 8, 1215–1233.
4. R. P. Borris, G. Blaskó and G. A. Cordell, J. Ethnopharmacol., 1988, 24, 41–91.
5. S. Z. Huang, X. J. Zhang, X. Y. Li, H. Z. Jiang, Q. Y. Ma, P. C. Wang, Y. Q. Liu, J. M. Hu, Y. T. Zheng, J. Zhou and Y. X. Zhao, Planta Med., 2012, 78, 182–185.
6. S. Z. Huang, X. J. Zhang, X. Y. Li, L. M. Kong, H. Z. Jiang, Q. Y. Ma, Y. Q. Liu, J. M. Hu, Y. T. Zheng, Y. Li, J. Zhou and Y. X. Zhao, Phytochemistry, 2012, 75, 99–107.
7. Flora of Yunnan, ed. C. A. S. Kunming Institute of Botany, Science Press, Kunming, 1997, vol. 8, pp. 219–231.
8. Yunnan institute of materia medica, The annals of national medicine in Yunnan, The nationalities publishing house of Yunnan, Kunming, 2009, pp. 5–6.
9. S. Liang, Y. H. Shen, Y. Feng, J. M. Tian, X. H. Liu, Z. Xiong and W. D. Zhang, J. Nat. Prod., 2010, 73, 532–535.
10. S. Liang, S. X. Liu, H. Z. Jin, L. Shan, S. C. Yu, Y. H. Shen, H. L. Li, Q. Y. Wu, Q. Y. Sun and W. D. Zhang, Chem. Commun., 2013, 49, 6968–6970.
11. S. Liang, J. M. Tian, Y. Feng, X. H. Liu, Z. Xiong and W. D. Zhang, Chem. Pharm. Bull., 2011, 59, 653–656.
12. Y. X. Zhao, S. Z. Huang, Q. Y. Ma, W. L. Mei and H. F. Dai, Molecules, 2012, 17, 10046–10051.
13. S. Z. Huang, X. N. Li, Q. Y. Ma, H.-F. Dai, L. C. Li, X. H. Cai, Y. Q. Liu, J. Zhou and Y. X. Zhao, Tetrahedron Lett., 2014, 55, 3693–3696.
14. L. P. Liu, K. Han, W. Chen, Y. Y. Zhang, L. J. Tong, T. Peng, H. Xie, J. Ding and H. B. Wang, Bioorg. Med. Chem., 2014, 22, 4198–4203.
15. D. C. Kim, J. A. Kim, B. S. Min, T. S. Jang, M. K. Na and S. H. Lee, Helv. Chim. Acta, 2010, 93, 692–697.
16. W. Herz and P. Santhanam, J. Org. Chem., 1967, 32, 507–510.
17. Z. Cekan, V. Herout and F. Sorm, Chem. Listy, 1957, 51, 756–763.
18. G. Blay, B. Garcia, E. Molina and J. R. Pedro, Tetrahedron, 2007, 63, 9621–9626.
19. A. Garcia-Granados, A. Molina and E. Cabrera, Tetrahedron, 1986, 42, 81–87.
20. J. G. Diaz, B. M. Fraga, A. G. Gonzalez, M. G. Hernandez and A. Perales, Phytochemistry, 1986, 25, 1161–1165.
21. J. Peng, S. G. Franzblau, F. Zhang and M. T. Hamann, Tetrahedron Lett., 2002, 43, 9699–9702.
22. M. Neeman and O. D. Simmons, Can. J. Chem., 1979, 57, 2071–2072.
23. L. Fitjer, A. Malich, C. Paschke, S. Kluge, R. Gerke, B. Rissom, J. Weiser and M. Noltemeyer, J. Am. Chem. Soc., 1995, 117, 9180–9189.
24. I. G. Collado, J. R. Hanson and A. J. Macias-Sanchez, Nat. Prod. Rep., 1998, 15, 187–204.
25. P. M. Dewick, in Medicinal Natural Products, John Wiley & Sons, Ltd, 2001, pp. 7–34.
26. Natural product chemistry :a mechanistic, biosynthetic, and ecological approach, ed. K. B. G. Torssell, Apotekarsocieteten, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 1–480.
27. S. Z. Huang, X. Zhang, Q. Y. Ma, H. Peng, Y. T. Zheng, J. M. Hu, H. F. Dai, J. Zhou and Y. X. Zhao, Fitoterapia, 2014, 95, 34–41.

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Footnote

† Electronic supplementary information (ESI) available: 1D and 2D NMR spectra and mass spectra of the new compounds. See DOI: 10.1039/c5ra17099k

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