Acetylcholinesterase Inhibitory Alkaloids from the Whole Plants of

Article pubs.acs.org/jnp

Acetylcholinesterase Inhibitory Alkaloids from the Whole Plants of Zephyranthes carinata Guanqun Zhan, Junfei Zhou, Junjun Liu, Jinfeng Huang, Hanqi Zhang, Rong Liu, and Guangmin Yao* Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: Eleven new alkaloids (1−11), classified as the 12-acetylplicamine (1), N-deformyl-seco-plicamine (2), plicamine (3−6), 4a-epi-plicamine (7), seco-plicamine (8), and lycorine (9−11) framework types, along with 15 known alkaloids (12−26) were isolated from the whole plants of Zephyranthes carinata. The structures of the new alkaloids 1− 11 were established by extensive spectroscopic data interpretation. The absolute configurations of 9 and 10 were defined by single-crystal X-ray diffraction analysis. Zephycarinatines A (1), B (2), and G (7) represent the first examples of 12-acetylplicamine, N-deformyl-seco-plicamine, and 4a-epi-plicamine alkaloids, respectively. Alkaloids 6, 11, 17, and 20−23 exhibited AChE inhibitory activities with IC50 values ranging from 1.21 to 184.05 μM, and a preliminary structure−activity relationship is discussed. nature,11a,d and this is the first report of these types of alkaloids in the whole plants of Z. carinata. Herein, the isolation, structure determination, AChE inhibitory activities, and a preliminary structure−activity relationship (SAR) of alkaloids 1−26 are reported.

A

maryllidaceae, perennial herbs with bulbs, comprises more than 100 genera and 1200 species that are distributed widely in the tropical and subtropical regions of the world.1 Alkaloids are the major chemical constituents of Amaryllidaceae plants. To date, more than 600 Amaryllidaceae alkaloids representing 22 skeletal types have been reported,2 and some of them exhibited a wide variety of biological activities including acetylcholinesterase (AChE) inhibitory, analgesic, antibacterial, antifungal, antimalarial, antitumor, antiviral, and cytotoxic activities.2 Galanthamine, widely occurring in the Amaryllidaceae plants, has been approved by the FDA as an AChE inhibitor to treat Alzheimer’s disease.3 Thus, the Amaryllidaceae alkaloids are an important resource for new drug discovery. Zephyranthes carinata Herbert (Amaryllidaceae), previously known as Z. grandif lora, is native to Mexico and naturalized in South China as an ornamental.1 The whole plants of Z. carinata, called Sai-Fan-Hong-Hua in Chinese, are used as a traditional Chinese medicine to treat swelling, snake bites, and stomach bleeding in China.4 A literature survey revealed that previous phytochemical studies on Z. carinata have focused on the bulbs,5−10 leading to the isolation of alkaloids. However, there are no reports on the chemical constituents of the whole plants of Z. carinata. In the course of a search for new AChE inhibitors from Chinese Amaryllidaceae plants,2a,b,10 the whole plants of Z. carinata, collected at Laifeng, Hubei Province of China, were investigated, leading to the isolation of 11 new (1−11) and 15 known alkaloids (12−26). Zephycarinatines A (1), B (2), and G (7) represent the first examples of 12-acetylplicamine, Ndeformyl-seco-plicamine, and 4a-epi-plicamine alkaloids, respectively. Plicamine and seco-plicamine type alkaloids are rare in © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Zephycarinatine A (1) was obtained as a colorless, viscous liquid. The 13C NMR data and the sodium adduct ion detected at m/z 463.2205 [M + Na]+ in the HRESIMS established the molecular formula of 1 as C25H32N2O5 (calcd for C25H32N2O5Na, 463.2209). The IR spectrum revealed the presence of carbonyl (1693 cm−1) and methylenedioxy (934 cm−1) functionalities. The 1H NMR data of 1 (Table 1) revealed the presence of two aromatic protons (δH 6.59, s, H-7; 6.80, s, H-10), two olefinic protons (δH 5.71, d, J = 10.2 Hz, H-1; 6.22, dd, J = 10.2, 4.3 Hz, H-2), a methylenedioxy group (δH 5.91, d, J = 1.0 Hz; 5.89, d, J = 1.0 Hz), an oxymethine (δH 3.85, ddd, J = 4.5, 4.3, 4.2 Hz, H-3), an OCH3 group (δH 3.46, s), an NCH3 group (δH 2.83, s), an acetyl group (δH 2.25, s, CH3-19), and two methyl groups (δH 0.93, d, J = 6.4 Hz, CH3-16; 0.90, d, J = 6.4 Hz, CH3-17). The 13C NMR data of 1 (Table 2) exhibited 25 carbon resonances assignable via DEPT and HSQC data to five methyls (δC 57.0, OCH3; 29.7, C-19; 28.3, NCH3; 23.5, C-16; 23.0, C-17), five methylenes including a methylenedioxy (δC 102.6, OCH2O), seven methines including an oxymethine (δC 72.4, C-3) and four sp2 methines (δC 133.0, C-1; 129.5, C-2; 106.6, C-7; 109.6, CReceived: April 7, 2017 Published: September 12, 2017 2462

DOI: 10.1021/acs.jnatprod.7b00301 J. Nat. Prod. 2017, 80, 2462−2471

Journal of Natural Products

Article

Chart 1

10), two oxygenated tertiary sp2 carbons (δC 148.35, C-8; 148.36, C-9), two tertiary sp2 carbons (δC 129.4, C-6a; 130.5, C-10a), a nitrogenated tertiary carbon (δC 82.1, C-12), a quaternary carbon (δC 49.0, C-10b), and two carbonyl carbons (δC 173.0, C-11; 210.4, C-18). The NMR data of 1 closely resemble those of compound 12 (N-isopentyl-5,6-dihydroplicane), which was previously isolated from the whole plants of Z. candida and whose structure was defined by single-crystal X-ray diffraction analysis.11a The major differences included the presence of an acetyl group (δH 2.25, s, 3H, H-19; δC 210.4, C-18; 29.7, C-19) and a nitrogenated tertiary carbon (δC 82.1, C-12) in 1, instead of a methine (δC 67.2, C-12) in 12. Thus, zephycarinatine A (1) is the 12-acetyl derivative of 12. HMBC correlations from H3-19 (δH 2.25, s) to the carbonyl (δC 210.4, C-18) and the nitrogenated tertiary carbon (δC 82.1, C-12) confirmed the location of the acetyl group at C-12. The 2D NMR experiments including 1H−1H COSY, HSQC, and HMBC confirmed the 2D structure of 1 (Figure 1). The relative configuration of 1 was determined by NOESY data and coupling constant analyses, as well as comparison of these data with those of 12. Similar to compound 12, H-4a in 1 was randomly assigned as β-orientated. The small coupling constants of H-3 with H2-4 (J = 4.5 and 4.2 Hz, respectively) and the large coupling constant of H-4a (J = 9.6 Hz) with H-4α established the α-orientation of H-3.4a The cross-

peak of H3-19 (δH 2.25, s) and H-1 (δH 5.71, d, J = 10.2 Hz) in the NOESY spectrum suggested that the acetyl group in 1 was αorientated. The absolute configuration of 1 was assigned to be the same as that of 12 by their similar ECD spectra (Figure 2). Thus, the structure of 1, zephycarinatine A, was defined as 12αacetyl-N-isopentyl-5,6-dihydroplicane. Zephycarinatine A (1) is the first example of a 12-acetylplicamine alkaloid. Zephycarinatine B (2) possessed the elemental composition C22H30N2O4 as established by the protonated molecular ion at m/z 387.2290 [M + H]+ (calcd for C22H31N2O4, 387.2284) in the HRESIMS spectrum and the 13C NMR data. The NMR data (Tables 1 and 2) of 2 were similar to those of N-isopentyl-11,12seco-5,6-dihydroplicane, a known alkaloid isolated from the whole plants of Z. candida,11a but for the absence of a formyl group in 2. Therefore, 2 is an N-2-deformyl analogue of Nisopentyl-11,12-seco-5,6-dihydroplicane. The 2D structure of 2 was confirmed by 1H−1H COSY, HSQC, and HMBC data (Figure S1, Supporting Information), and the relative configuration was determined to be the same as that of N-isopentyl11,12-seco-5,6-dihydroplicane by 1D and 2D NMR data analysis. The absolute configurations of 2 and N-isopentyl-11,12-seco-5,6dihydroplicane were identical based on their similar ECD data (Figure 2). Thus, the structure of 2, zephycarinatine B, was defined as N-deformyl-N-5-isopentyl-11,12-seco-5,6-dihydropli2463



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Table 1. 1H NMR [δ, mult, (J in Hz)] Data for Compounds 1−5 in Methanol-d4 (400 MHz) no.

1

2

3

4

5

1 2 3 4α 4β 4a 6α 6β 7 10 12 13a 13b 14a 14b 15 16 17 18 19 20 OCH2O

5.71, d (10.2) 6.22, dd (10.2, 4.3) 3.85, ddd (4.5, 4.3, 4.2) 1.85, ddd (13.9, 9.6, 4.2) 2.11, ddd (13.9, 4.5, 4.3) 3.72, dd (9.6, 4.3) 3.93, d (15.4) 3.67, d (15.4) 6.59, s 6.80, s

5.61, d (10.0) 6.18, dd (10.0, 3.9) 3.98, ddd (4.8, 4.6, 3.9) 1.87, ddd (13.8, 4.8, 3.2) 2.19, ddd (13.8, 9.6, 4.6) 2.93, dd (9.6, 3.2) 4.72, d (16.2) 4.33, d (16.2) 6.73, s 6.73, s

5.77, d (9.8) 6.18, dd (9.8, 5.4) 4.02, ddd (5.4, 3.4, 3.1) 1.78, ddd (13.5, 12.5, 3.1) 2.58, ddd (13.5, 4.7, 3.4) 4.14, dd (12.5, 4.7)

5.83, d (9.8) 6.16, dd (9.8, 5.4) 4.00, ddd (5.4, 3.0, 3.0) 1.73, ddd (13.4, 12.4, 3.0) 2.59, ddd (13.4, 4.7, 3.0) 4.14, dd (12.4, 4.7)

3.14, ddd (13.6, 10.6, 5.3) 2.52, ddd (13.6, 10.6, 4.8) 1.58, m 1.36, m 1.55, m 0.93, d (6.4) 0.90, d (6.4)

3.60, ddd (13.4, 13.2, 6.8) 3.45, overlap 1.49, m

7.40, s 6.83, s 4.40, s 4.29, ddd (13.6, 9.0, 6.9) 3.20, ddd (13.6, 8.7, 5.6) 1.51, m

7.42, s 6.84, s 4.40, s 3.30, s

5.82, ddd (10.5, 1.8, 1.8) 5.72, br d (10.5) 3.79, m 2.05, ddd (12.4, 10.2, 1.8) 2.85, overlap 3.96, overlap 4.33, d (15.7) 3.71, d (15.7) 6.65, s 6.76, s 3.29, s 2.94, overlap 2.84, overlap 2.91, overlap

1.60, m 0.96, d (6.5) 0.95, d (6.5)

1.62, m 0.96, d (6.5) 0.95, d (6.5)

5.94, d (1.1) 5.95, d (1.1) 3.44, s 2.22, s

6.04, d (1.0) 6.03, d (1.0) 3.51, s 2.79, s

OCH3-3 NCH3

7.26, overlap 7.23, overlap 7.15, m 7.23, overlap 7.26, overlap 5.91, d (1.0) 5.90, d (1.0) 3.45, s 2.87, s

2.25, s 5.89, d (1.0) 5.91, d (1.0) 3.46, s 2.83, s

6.04, d (1.0) 6.03, d (1.0) 3.50, s 2.80, s

Table 2. 13C NMR Data for Compounds 1−10 in Methanol-d4 (100 MHz) no.

1

2

3

4

5

6

7

8

9

10

1 2 3 4 4a 6 6a 7 8 9 10 10a 10b 11 12 13 14 15 16 17 18 19 20 OCH2O OCH3 NCH3-3 OCH3-2 OCH3-7 OCH3-8

133.0 129.5 72.4 28.5 64.4 51.7 129.4 106.6 148.35 148.36 109.6 130.5 49.0 173.0 82.1 53.4 39.1 27.7 23.5 23.0 210.4 29.7

133.4 129.8 74.6 29.8 63.8 51.3 126.7 106.4 148.6 148.9 108.5 132.9 54.6 171.4 47.4 37.0 27.4 23.1 23.0

133.6 127.9 72.7 30.0 61.6 163.8 124.2 108.4 149.6 153.5 107.3 137.7 45.8 172.3 64.2 47.2 37.6 27.3 23.2 22.9

133.9 127.6 72.7 30.2 61.8 164.2 123.9 108.3 149.6 153.5 107.5 137.8 45.8 172.4 66.3 36.5

132.0 127.8 72.9 29.3 58.8 59.3 129.4 108.2 147.9 148.4 105.5 133.5 46.9 173.7 70.1 56.6 61.5

135.7 127.7 73.4 27.0 72.9 49.7 127.6 108.6 147.8 148.3 109.2 133.1 47.2 56.9 67.6 53.7 37.5 27.8 23.2 23.1

139.5 126.8 74.9 30.4 62.4 56.6 131.1 107.4 148.4 148.4 108.3 131.9 47.3 165.3 57.17 58.5 36.8 28.1 23.5 23.2

68.1 81.9 120.8 139.0 72.9 68.7 125.0 112.4 149.8 150.1 109.9 127.1 36.7 27.5 69.2

66.3 81.4 119.6 140.7 80.8 69.9 124.4 114.0 147.9 148.5 112.9 130.4 40.0 30.7 72.2

102.6 57.0 28.3

102.8 56.8 35.1

103.8 57.3 28.7

103.8 57.2 28.6

132.2 127.6 73.0 29.3 58.8 58.2 129.43 108.3 148.0 148.4 105.5 133.5 46.9 173.7 69.7 56.0 34.1 142.1 130.1 129.39 127.0 129.39 130.1 102.6 56.6 27.8

102.3 56.6 27.8

102.4 56.4 40.5

102.5 57.19 31.1 58.1 56.7 56.8

58.5

cane. Zephycarinatine B (2) is the first example of an Ndeformyl-seco-plicamine alkaloid.

56.8

Zephycarinatines C (3) and D (4) were isolated as colorless, viscous liquids. Their molecular formulas were determined to be 2464



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oxide in 5 and 6 was determined by the NOESY correlations between H-12 and H2-13. Based on their similar ECD spectra (Figure 3), the absolute configurations of 5 and 6 are the same as that of 16. Compounds 5 and 6 represent only the third and fourth examples of N-oxide-type plicamine alkaloids.11a Zephycarinatine G (7) was isolated as a colorless, viscous liquid. The 13C NMR data and the protonated molecular ion at m/z 385.2492 in the HRESIMS established its molecular formula as C23H32N2O3 (calcd for C23H33N2O3, 385.2491). The NMR data (Tables 2 and 3) of 7 resembled those of 12, except that the C-11 carbonyl group (δC 174.3) in 12 was replaced by a methylene group (δH 2.42, dd, J = 9.6, 9.3 Hz, H-11α; 3.16, dd, J = 9.6, 7.5 Hz, H-11β; δC 56.9, C-11) in 7. These differences suggested that compound 7 is an 11-deoxy derivative of 12. The 1 H−1H COSY correlation from H2-11 to H-12 and the HMBC correlations from NCH3 to C-4a and C-11 confirmed the presence of the methylene at C-11. The NOESY correlation between H-1 and H-12 in 7 suggested that H-12 is α-oriented. NOESY correlations from H-4a to H-12 and H-3 established the α-orientation of H-4a and H-3. The similar ECD spectra of 7 and 12 (Figure 3) suggested the same absolute configuration of C10b. Hence, the structure of 7, zephycarinatine G, was defined as 4a-epi-11-deoxo-11,11-dihydro-N-isopentyl-5,6-dihydroplicane. Plicamine-type alkaloids are rare in nature, with only 12 analogues being reported, nine of which were isolated from the whole plants of Z. candida.11a,d In the reported plicamine-type alkaloids, the orientation of H-4a is β, while H-4a is α-orientated in compound 7. Zephycarinatine G (7) represents the first example of a 4a-epi-plicamine-type alkaloid. Zephycarinatine H (8) was obtained as a colorless, viscous liquid. A molecular formula of C23H32N2O4 was assigned on the basis of the 13C NMR and HRESIMS data. The NMR data (Tables 2 and 3) of 8 were similar to those of N-isopentyl-11,12seco-5,6-dihydroplicane, a known alkaloid from the whole plants of Z candida,11a except for the presence of a methylene group (δH 2.99, d, J = 12.7 Hz, H-12α; 2.24, d, J = 12.7 Hz, H-12β; δC 57.17, C-12) in 8 replacing the carbonyl group (δC 168.8, C-12) in Nisopentyl-11,12-seco-5,6-dihydroplicane. Thus, zephycarinatine H (8) is a 12-deoxy derivative of N-isopentyl-11,12-seco-5,6dihydroplicane. The correlations from H2-12 to C-6 and C-10b in the HMBC spectrum verified the deduction. The relative and absolute configurations of 8 were established to be the same as those of N-isopentyl-11,12-seco-5,6-dihydroplicane based on their similar coupling constants, NOESY, and ECD data (Figure 3). Zephycarinatine H (8) is the first example of a 12-deoxy-5,6dihydro-seco-plicamine-type alkaloid. Galanthine N-β-oxide (9) was obtained as colorless needles. Its molecular formula, C18H23NO5, was assigned from the HRESIMS and 13C NMR data. The NMR data (Tables 2 and 3) of 9 were similar to those of compound 17 (galanthine),12 except that the C-6 (δC 68.7) and C-12 (δC 69.2) resonances in 9 were deshielded by 10 ppm relative to those (δC 57.6, C-6; 54.8, C-12) in 17. These characteristics demonstrated that compound 9 was the N-oxide11a,13 of 17. It was further proved by the molecular formula of 9, which contains one more oxygen atom than 17. The larger coupling constant of J4a,10b = 11.3 Hz indicated that H-4a and H-10b were in an anti-relationship, while the smaller coupling constant J1,10b suggested the syn-relationship of H-1 and H-10b. Thus, the relative configuration of 9 was determined except for the orientations of the N-oxide and H-2. The βorientation of the N-oxide and α-orientation of H-2 in 9 were defined via single-crystal X-ray diffraction analysis (Figure 4), and the absolute configuration of 9 was assigned as

Figure 1. 1H−1H COSY, key HMBC, and NOESY correlations of 1.

Figure 2. Comparison of ECD spectra of 1−4, 12, and N-isopentyl11,12-seco-5,6-dihydroplicane in MeOH.

C23H28N2O5 and C19H21N2O5, respectively, by the HRESIMS and 13C NMR data. Comparison of their NMR data (Tables 1 and 2) with those of 12 and 15,11a respectively, revealed the presence of the carbonyls in 3 and 4 replacing the C-6 methylenes in 12 and 15, respectively. Therefore, zephycarinatines C (3) and D (4) should be the 6-oxo derivatives of 12 and 15, respectively, which were confirmed by HMBC correlations from H2-13 to C-6 and C-12. The relative configurations of 3 and 4 were the same as those of 12 and 15 based on their coupling constants and NOESY data. The ECD spectra of 3 and 4 (Figure 2) exhibited positive Cotton effects at 208 and 225 nm, which were similar to those of 12, suggesting the 10bS absolute configurations in 3 and 4. Thus, the structures of 3 and 4 were defined as shown. The molecular formulas of zephycarinatines E (5) and F (6) were assigned as C23H28N2O5 and C20H24N2O6, respectively, on the basis of their HRESIMS and 13C NMR data. The NMR data of 5 and 6 (Tables 1, 2, and 3) exhibited resonances for a phenethyl group (δH 2.94, H-13a; 2.84, H-13b; 2.91, H-14; 7.26, H-16, H-20; 7.23, H-17, H-19; 7.15, H-18; δC 56.0, C-13; 34.1, C-14; 142.1, C-15; 130.1, C-16, C-20; 129.39, C-17, C-19; 127.0, C-18) and a 2-hydroxyethyl group (δH 3.98, H-13a; 2.56, H-13b; 3.75, H-14; δC 56.6, C-13; 61.5, C-14), respectively. The NMR spectra of 5 and 6 were similar to those of compound 16 (bliquine N-oxide),11a except for the presence of N-phenethyl and N-2-hydroxyethyl groups in 5 and 6, respectively, replacing the N-p-hydroxylphenethyl group in 16. HMBC correlations from H2-13 to C-6 and C-12 established the location of these groups at N-5 in 5 and 6. Similar to 16, the β-orientation of the N2465



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Table 3. 1H NMR [δ, mult, (J in Hz)] Data for Compounds 6−10 in Methanol-d4 (400 MHz) no.

6

7

8

1 2 3 4α 4β 4a 6α 6β 7 10 10b 11α 11β 12α 12β 13a 13b 14 15 16 17 OCH2O

5.93, ddd (10.5, 1.4, 1.4) 5.78, br d (10.5) 3.78, overlap 2.05, ddd (14.4, 10.2, 2.1) 2.83, overlap 3.95, overlap 4.34, d (15.8) 3.68, d (15.8) 6.62, s 6.76, s

5.57, ddd (10.1, 1.7, 1.7) 5.94, ddd (10.1, 1.7, 1.7) 3.91, dddd (10.6, 5.5, 1.7, 1.7) 1.62, ddd (13.9, 10.6, 3.3) 2.23, dddd (13.9, 5.5, 3.7, 1.7) 2.02, dd (3.7, 3.3) 3.86, d (15.4) 3.57, d (15.4) 6.69, s 6.84, s

5.52, d (9.9) 6.00, dd (9.9, 4.9) 3.99, ddd (4.9, 4.4, 2.7) 2.10, ddd (13.7, 2.7, 2.7) 2.38, ddd (13.7, 12.7, 4.4) 4.05, dd (12.7, 2.7) 3.74, d (14.5) 3.05, d (14.5) 6.54, s 6.71, s 7.55, s

3.27, s

2.42, dd (9.6, 9.3) 3.16, dd (9.6, 7.5) 3.36, dd (9.3, 7.5)

OCH3-3 NCH3 OCH3-2 OCH3-7 OCH3-8

3.98, overlap 2.56, ddd (13.0, 5.6, 5.6) 3.75, overlap

5.89, d (1.0) 5.90, d (1.0) 3.43, s 2.87, s

2.44, m 1.40, m 1.52, m 0.88, d (6.5) 0.87, d (6.5) 5.89, d (0.9) 5.92, d (0.9) 3.45, s 2.28, s

2.99, d (12.7) 2.24, d (12.7) 2.51, ddd (13.3, 9.2, 6.8) 2.26, overlap 1.45, m 1.59, m 0.94, d (6.4) 0.93, d (6.4) 5.91, s

9

10

4.78, overlap 3.81, overlap 5.83, br s

4.64, br s 3.76, br s 5.72, br s

4.04, br d (11.3) 4.77, d (14.7) 4.56, d (14.7) 6.78, s 7.05, s 3.32, br d (11.3) 2.82, dddd (17.0, 8.4,1.8, 1.8) 3.03, dddd (17.0, 10.2, 8.4, 1.8) 3.87, overlap 3.78, overlap

3.91, br d (12.4) 4.50, d (13.2) 4.36, d (13.2) 7.01 s 7.00, s 2.60, br d (12.4) 3.23, m 2.67, dd (14.0, 5.7) 3.84, overlap 3.66, ddd (13.5, 10.8, 5.7)

3.51, s 3.82, s 3.88, s

3.51, s

3.45, s 2.64, s

3.88, s

Figure 4. ORTEP drawing of galanthine N-β-oxide (9).

Figure 3. Comparison of ECD spectra of 5−8, 12, 16, and N-isopentyl11,12-seco-5,6-dihydroplicane in MeOH.

data, the relative configuration of 10, except for the N-oxide, was the same as 18. Compounds 10 and 9 had similar structures; however, the resonances of H-6α (δH 4.50, d, J = 13.2 Hz) and H6β (δH 4.36, d, J = 13.2 Hz) in 10 were deshielded relative to those of 9 (δH 4.77, d, J = 14.7 Hz, H-6α; 4.56, d, J = 14.7 Hz, H6β). In addition, the C-4a (δC 80.8), C-6 (δC 69.9), C-10b (δC 40.0), C-11 (δC 30.7), and C-12 (δC 72.2) resonances of 10 were deshielded relative to those of 9 (δC 72.9, C-4a; 68.7, C-6; 36.7, C-10b; 27.5, C-11; 69.2, C-12). These differences may be caused by the orientation of the N-oxide. Thus, the N-oxide moiety in 10 was deduced to be in the α-orientation, instead of the βorientation in 9. Finally, the structure of 9 was unambiguously

1S,2S,4aS,5S,10bS using the Flack parameter14 of 0.1(1) for 1618 Friedel pairs and the Hooft parameter15 of 0.04(4) for 1617 Bijvoet pairs. Carinatine N-α-oxide (10) was obtained as colorless needles (MeOH), mp 212 °C. The HRESIMS and 13C NMR data established the molecular formula of 10 as C17H21NO5, which is one more oxygen atom than compound 18 (carinatine).6 Comparison of the 13C NMR data (Table 2) of 10 with those of 18 revealed that C-6 and C-12 in 10 were deshielded by ca. 10 ppm compared to those of 18. Therefore, compound 10 is the Noxide of 18. By comparing the coupling constants and NOESY 2466



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defined by single-crystal X-ray diffraction analysis (Figure 5), and the absolute configuration was assigned as 1S,2S,4aS,5R,10bS by the Flack parameter14 of 0.1(3) for 2068 Friedel pairs and Hooft parameter15 of 0.1(1) for 2068 Bijvoet pairs.

Figure 5. ORTEP drawing of carinatine N-α-oxide (10).

Zephycarinatine I (11), a yellow powder, possessed an elemental composition of C17H15NO3 as established by the HRESIMS and 13C NMR data. The NMR data of 11 (Table 4) Figure 6. Structures and differences in ppm between calculated and experimental 13C NMR shifts for 11a and 11b.

Table 4. 1H (400 MHz) and 13C NMR (100 MHz) Data for Compound 11 (Methanol-d4) and DFT Calculation of 13C NMR for 11a and 11b 11 no. 1 2 3 4 4a 6 6a 7 8 9 10 10a 10b 11 12 OCH3-2 OCH3-8

δH (mult, J) 7.00, s 7.00, s

8.17, s 6.66, s

6.81, s

3.43, t (6.5) 4.65, t (6.5) 3.91, s 3.73, s

11a

4) were calculated by the method of Crews and co-workers,16 and the differences between the corrected and experimental 13C NMR chemical shifts for 11a and 11b analyzed.17 As shown in Figure 6, the MAE (mean absolute error) and MD (maximum deviation)16 for 11b were 1.6 and 3.7 ppm, respectively, which are acceptable in terms of MAE < 2.2 and MD < 5.16 However, the MAE and MD for 11a were 2.6 and 11.8 ppm, which were considerably higher than the standard MAE < 2.2 and MD < 5. Thus, the structure of 11 could be defined as 11b. Compounds 12−26 were identified as N-isopentyl-5,6dihydroplicane (12), 11a N-phenethyl-5,6-dihydroplicane (13), 11a obliquine (14), 18 N-methyl-5,6-dihydroplicane (15),11d bliquine N-oxide (16),11a galanthine (17),12 carinatine (18),5 lycorine (19),19 pseudolycorine (20),20 galanthamine (21),21 lycoramine (22),22 3-epi-lycoramine (23),23 11βhydroxygalanthamine (24),24 lycoramine N-oxide (25),25 and 11β-hydroxygalanthamine N-oxide (26),11a respectively, on the basis of spectroscopic data analysis and comparison with reported data. Known alkaloids 12−16, 20, and 23−26 were isolated from Z. carinata for the first time. The known Amaryllidaceae alkaloids may be classified according to 22 framework types.2 However, dinitrogenous Amaryllidaceae alkaloids such as plicamine and seco-plicamine types are rare, with only 12 plicamine and three seco-plicaminetype alkaloids thus far reported.11a,d This is the first report of the presence of dinitrogenous Amaryllidaceae alkaloids in Z. carinata. Similar to the biosynthetic pathway of plicamine-type alkaloids,2f the pathways toward 1 and 2 (Scheme 1) in Z. carinata may begin with L-tyrosine and L-phenylalanine to generate a benzylphenethylamine alkaloid via a Schiff base formation reaction.2f A galanthamine-type alkaloid intermediate (27) may form via phenol-oxidative p−p coupling. Hydrolysis

11b

δC

δC (calcd)

δC (cor)

δC (calcd)

δC (cor)

101.3 162.2 116.3 138.1 132.9 138.4 117.0 108.4 155.9 171.5 108.1 132.4 122.7 28.3 54.8 56.8 55.9

89.8 144.6 101.6 123.2 118.3 121.6 101.6 108.0 142.8 160.0 100.5 119.3 109.1 21.7 44.9 46.3 47.5

101.1 158.7 113.5 136.1 131.0 134.5 113.5 120.2 156.8 174.9 112.3 132.0 121.3 29.5 53.8 55.3 56.5

90.2 144.3 100.8 123.0 118.3 120.4 101.8 96.8 142.6 157.7 98.5 118.7 108.7 21.9 44.9 46.3 45.9

102.7 160.1 113.9 137.5 132.5 134.7 115.0 109.6 158.2 174.3 111.5 132.9 122.2 30.0 54.4 55.9 55.5

were similar to those of zephgrabetaine10a but for the location of the methoxy group at C-8 in 11 instead of at C-9 in zephgrabetaine. The HMBC correlation from the O-methyl group to C-8 together with the NOESY correlation between OCH3 and H-7 confirmed the location of OCH3 at C-8. Thus, the structure of 11 may be considered as 11a (Figure 6). However, 11a is a zwitterionic compound or inner salt and is an isomer of 11b (Figure 6). To determine the structure of 11, the 13C NMR chemical shifts of isomers 11a and 11b (Table 4) were calculated16 by using Gaussian09 at the B3LYP/6-31G* level.2b The corrected 13C NMR chemical shifts for 11a and 11b (Table 2467



Article

Scheme 1. Putative Biosynthetic Pathways toward 1 and 2

oxide,30 were reported. In this study, two new lycorine N-oxide alkaloids (9 and 10) were isolated from Z. carinata, and the absolute configurations of the N-oxide moieties in 9 and 10 were determined to be R and S, respectively, by single-crystal X-ray diffraction analysis. This is the first determination of the absolute configuration of the N-oxide moiety in lycorine-type alkaloids by single-crystal X-ray diffraction analysis, although the absolute configuration of the N-oxide moiety in pancratinine D was determined by molecular mechanics calculations.30 Galanthine N-β-oxide (9) possessing an R-configuration of the N-oxide moiety showed positive and negative Cotton effects at 227 and 268 nm, respectively, in the ECD spectrum, while carinatine N-αoxide (10), with an S-configuration of the N-oxide moiety, exhibited negative and positive Cotton effects at 237 and 286 nm,

and intramolecular cyclization of the amino group to C-4a would transform the galanthamine-type intermediate (27) into intermediate 28. Upon subsequent Schiff base formation, reduction, oxidation, intramolecular Schiff base formation, and oxidation, plicamine-type (12) and seco-plicamine-type alkaloids (N-isopentyl-11,12-seco-5,6-dihydroplicane) may be formed. Acetyl coenzyme A (Ac-CoA) would transform 12 into zephycarinatine A (1). In addition, zephycarinatine B (2) may be formed from N-isopentyl-11,12-seco-5,6-dihydroplicane via oxidization and subsequent decarboxylation. Lycorine-type alkaloids are common in nature; however, lycorine N-oxides are rare. Heretofore, only five lycorine Noxides, 3-O-acetylnarcissidine N-oxide,26 ungiminorine Noxide,27 lycorine N-oxide,28 pancratinine D,29 and incartine N2468



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chromatography (CC). Thin-layer chromatography (TLC) was carried out with silica gel GF254 (Yantai Chemical Industry Research Institute, China) and RP-C18 F254 plates (Merck, Germany). Plant Material. The whole plants of Z. carinata were collected in October 2013 at Laifeng County, Hubei Province, China, and authentificated by Professor Jianping Wang at this Institute. A voucher specimen (No. 20131003) has been deposited at this institute. Extraction and Isolation. The dried whole plants of Z. carinata (10 kg) were extracted with 95% aqueous EtOH at 40 °C (4 × 25 L, each 2 days) to afford a crude residue (537 g) after evaporation of the solvent under vacuum. The residue was suspended in H2O (4 L), adjusted to pH 2 with 0.3 M HCl, and extracted with CHCl3 (5 × 4 L) to remove the nonalkaloid components. The aqueous phase was adjusted to pH 10 using NH3 solution in H2O (25%) and extracted by CHCl3 (4 × 3 L) to afford 22 g of CHCl3 extract. The CHCl3 extract (22 g) was fractionated via silica gel CC eluting with CH2Cl2/MeOH (1:0 to 5:1) to yield five fractions (Fr.A−E). Fr.A was subjected to silica gel CC using a gradient system of petroleum ether/acetone (15:1, 5:1) to provide two subfractions (Fr.A1 and Fr.A2). Compounds 1 (2.4 mg, tR 44.2 min) and 7 (2.7 mg, tR 46.6 min) were isolated by RP HPLC (MeOH/H2O/ Et2NH, 74.8:25:0.2) from Fr.A1. Compounds 5 (2.9 mg, tR 33.2 min), 12 (15.6 mg, tR 36.6 min), and 13 (13.2 mg, tR 40.9 min) were purified by RP HPLC (MeOH/H2O/Et2NH, 72.8:27:0.2) from Fr.A2. Fr.B was chromatographed on Sephadex LH-20 (MeOH) and purified by silica gel CC (petroleum ether/acetone, 12:1, 5:1) to give two subfractions (Fr.B1 and Fr.B2). Purification of Fr.B1 by RP HPLC (MeOH/H2O/ Et2NH, 34.8:65:0.2) afforded compounds 4 (2.3 mg, tR 19.5 min), 15 (13.3 mg, tR 22.5 min), 6 (2.5 mg, tR 28.8 min), 14 (15.3 mg, tR 51.6 min), and 16 (13.0 mg, tR 77.5 min). Fr.B2 was separated using RP HPLC (MeCN/H2O/Et2NH, 48.8:50:0.2) to afford compounds 2 (4.3 mg, tR 42.7 min), 8 (13.2 mg, tR 45.3 min), and 3 (2.6 mg, tR 47.3 min). Fr.C was chromatographed on Sephadex LH-20 (MeOH) and silica gel CC (CH2Cl2/MeOH, 80:1 to 5:1) to yield compounds 23 (3.7 mg) and 10 (10.9 mg), Fr.C1, and Fr.C2. Fr.C1 was subjected to RP HPLC (MeCN/H2O/Et2NH, 22.8:77:0.2) to yield compounds 17 (28.9 mg, tR 16.7 min) and 11 (4.3 mg, tR 21.2 min). Fr.C2 was separated by RP HPLC (MeOH/H2O/Et2NH, 37.8:62:0.2) to give 26 (17.3 mg, tR 18.6 min), 24 (25.6 mg, tR 40.5 min), 21 (78.9 mg, tR 44.8 min), and 22 (64.3 mg, tR 48.4 min). The MeOH-insoluble solid was filtered from Fr.D to yield 19 (1.2 g). The filtrate of Fr.D was purified in the same way as Fr.C to obtain 18 (15.8 mg) and 20 (17.9 mg). In a similar manner, compounds 9 (16.3 mg, tR 14.2 min) and 25 (14.7 mg, tR 23.1 min) were isolated from Fr.E by RP HPLC (MeOH/H2O/Et2NH, 19.8:80:0.2). Zephycarinatine A (1): colorless, viscous liquid; [α]25 D +206 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 210 (4.37), 220 (4.17), 234 (3.91), 290 (3.66) nm; ECD (MeOH) 208 (Δε, +27.71), 243 (Δε, +3.43), 287 (Δε, +1.73) nm; IR (KBr) νmax 2952, 2929, 1693, 1485, 1393, 1240, 1087, 1038, 934 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 463.2205 [M + Na]+ (calcd for C25H32N2O5Na, 463.2209). Zephycarinatine B (2): colorless, viscous liquid; [α]25 D +38 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 208 (4.28), 225 (3.76), 292 (3.58) nm; ECD (MeOH) 204 (Δε, +19.31), 236 (Δε, +4.19), 288 (Δε, +1.72) nm; IR (KBr) νmax 2954, 2930, 1639, 1481, 1425, 1235, 1089, 1038, 935 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 387.2290 [M + H]+ (calcd for C22H31N2O4, 387.2284). Zephycarinatine C (3): colorless, viscous liquid; [α]25 D +108 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 207 (4.17), 226 (4.37), 234 (4.20), 303 (3.69) nm; ECD (MeOH) 206 (Δε, +12.89), 225 (Δε, +29.35), 254 (Δε, +1.93), 261 (Δε, +2.77) nm; IR (KBr) νmax 2952, 2930, 1707, 1650, 1471, 1267, 1083, 1036, 932 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 435.1899 [M + Na]+ (calcd for C23H28N2O5Na, 435.1896). Zephycarinatine D (4): colorless, viscous liquid; [α]25 D +176 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.40), 306 (3.62); ECD (MeOH) 208 (Δε, +18.60), 224 (Δε, +31.15), 244 (Δε, +1.80), 261 (Δε, +5.51) nm; IR (KBr) νmax 3422, 2930, 2824, 1705, 1650, 1612, 1477, 1389, 1268, 1079, 1036, 931 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 357.1436 [M + H]+ (calcd for C19H21N2O5, 357.1451).

respectively. Thus, ECD data could be used to establish the absolute configuration of the N-oxide moiety in lycorine-type alkaloids. Since the EtOH extract and the alkaloid extract of the bulbs of Z. carinata showed anti-AChE activities,9 alkaloids 1−26 were evaluated for their AChE inhibitory activities using galanthamine (21) as a positive control. The results (Table 5) revealed that 6 Table 5. AChE Inhibitory Activities of Alkaloids 1−26a compound

IC50 (μM)b

compound

IC50 (μM)

6 11 17 20

126.16 ± 2.72 35.61 ± 1.90 6.10 ± 0.26 187.63 ± 22.72

21c 22 23

1.27 ± 0.09 20.07 ± 1.91 57.90 ± 2.28

For alkaloids 1−5, 7−10, 12−16, 18, 19, and 24−26, IC50 > 200 μM. IC50 values are expressed as the mean ± SD (n = 3). cGalanthamine (21) was used as a positive control.

a b

showed weak AChE inhibitory activity (IC50 = 126.16 ± 2.72 μM), while alkaloids 1−5, 7, 8, and 12−16 did not exhibit significant AChE inhibitory activities (IC50 > 200 μM), suggesting that the presence of the N-2-hydroxyethyl group in plicamine-type alkaloids may be essential for activity. The known lycorine-type alkaloid 17 exhibited a significant AChE inhibitory effect (IC50 = 6.10 ± 0.26 μM) compared to 18−20, indicating that the 9-OCH3 group may be essential for the AChE inhibitory activity in lycorine-type alkaloids. Galanthine N-β-oxide (9) did not show significant AChE inhibitory activity (IC50 > 200 μM) compared to galanthine (17), revealing that the N-oxide moiety may be a deactivating group toward AChE inhibitory activity. The new lycorine-type alkaloid 11, featuring an aromatic C-ring, showed moderate AChE inhibitory activity (IC50 = 35.61 ± 1.90 μM) compared to 18 (IC50 > 200 μM), suggesting that an aromatic C-ring in lycorine-type alkaloids may increase the AChE inhibitory activity.31 Galanthamine-type alkaloids 21−23 showed AChE inhibitory activities with IC50 values of 1.27 ± 0.09, 20.07 ± 1.91, and 57.90 ± 2.28 μM, respectively, while 24− 26 did not show AChE inhibitory activities (IC50 values >200 μM), which were in accordance with reported data.11a

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained with a Beijing Tech X-5 micromelting point apparatus without correction. Optical rotations of compounds 4, 6, and 11 were measured in MeOH with a 3 mL cell on a PerkinElmer 341 polarimeter, and those of the other compounds were measured in MeOH with a 0.7 mL cell on an Autopol IV automatic polarimeter. UV and ECD spectra were recorded using Varian Cary 50 and JASCO J-810 instruments, respectively. FT-IR spectra were recorded using a Bruker Vertex 70 instrument. NMR spectra were recorded on a Bruker AM-400 spectrometer operating at 400 (1H) and 100 (13C) MHz, and the residual peaks of methanol-d4 (δH 3.31 and δC 49.15) were used as references. The HRESIMS spectra of compounds 4, 6, 9, and 11 were obtained in a positive ion mode on a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer, and the HRESIMS spectra of the others were obtained on a Bruker micrOTOF II spectrometer. Single-crystal X-ray diffraction data were collected using a Bruker SMART APEX-II CCD diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.541 78 Å). HPLC was accomplished on an Agilent 2100 quaternary system with a UV detector using a reversed-phased (RP) C18 column (5 μm, 10 × 250 mm, Weltch Ultimate XB-C18). Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), silica gel (100−200 mesh, 200−300 mesh, Qingdao Haiyang Chemical Co. Ltd., China), and ODS (45 μm, YMC Co. Ltd., Japan) were used for column 2469



Article

Zephycarinatine E (5): colorless, viscous liquid; [α]25 D +74 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.31), 227 (4.06), 292 (3.58) nm; ECD (MeOH) 209 (Δε, +19.26), 228 (Δε, +10.85) nm; IR (KBr) νmax 2926, 2855, 1694, 1655, 1609, 1482, 1457, 1391, 1088, 1035, 932 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 449.2049 [M + H]+ (calcd for C23H29N2O5, 449.2077). Zephycarinatine F (6): colorless, viscous liquid; [α]25 D +278 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.77), 230 (4.13), 250 (3.88), 293 (3.92) nm; ECD (MeOH) 208 (Δε, +34.05), 227 (Δε, +11.61), 266 (Δε, +0.58), 288 (Δε, +1.48) nm; IR (KBr) νmax 3383, 2930, 2892, 2824, 1691, 1484, 1242, 1089, 1036, 931 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 389.1698 [M + H]+ (calcd for C20H25N2O6, 389.1713). Zephycarinatine G (7): colorless, viscous liquid; [α]25 D +72 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 207 (4.23), 224 (3.95), 243 (3.69), 292 (3.54) nm; ECD (MeOH) 206 (Δε, +17.66), 225 (Δε, +9.69) nm; IR (KBr) νmax 2950, 2868, 1652, 1612, 1480, 1245, 1096, 1038, 933 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 385.2492 [M + H]+ (calcd for C23H33N2O3, 385.2491). Zephycarinatine H (8): colorless, viscous liquid; [α]25 D +33 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 208 (4.36), 224 (4.04), 242 (3.75), 293 (3.60) nm; ECD (MeOH) 206 (Δε, +19.31), 233 (Δε, +5.53), 286 (Δε, +2.43) nm; IR (KBr) νmax 2952, 2933, 2871, 2820, 2764, 1667, 1485, 1240, 1082, 1036, 934 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 401.2438 [M + H]+ (calcd for C23H33N2O4, 401.2440). Galanthine N-β-oxide (9): colorless needles (MeOH); mp 223 °C; [α]25 D −51 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 206 (4.47), 251 (3.71), 305 (3.43) nm; ECD (MeOH) 216 (Δε, +0.16), 227 (Δε, +0.65), 268 (Δε, −1.06) nm; IR (KBr) νmax 3265, 2933, 2830, 1518, 1460, 1343, 1258, 1230, 1085, 958, 878 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 334.1641 [M + H]+ (calcd for C18H24NO5, 334.1655). Carinatine N-α-oxide (10): colorless needles (MeOH); mp 212 °C; [α]25 D +137 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 207 (4.55), 214 (4.01), 234 (3.68), 282 (3.49) nm; ECD (MeOH) 209 (Δε, +23.91), 237 (Δε, −2.68), 286 (Δε, +0.41) nm; IR (KBr) νmax 3178, 2938, 1610, 1458, 1330, 1092, 1074, 957, 929, 818 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 320.1493 [M + H]+ (calcd for C17H22NO5, 320.1498). Zephycarinatine I (11): yellow powder; UV (MeOH) λmax (log ε) 207 (4.14), 264 (4.28), 280 (4.15), 300 (3.96), 408 (4.14) nm; IR (KBr) νmax 3425, 2971, 1619, 1509, 1465, 1434, 1259, 1154, 878, 759, 619 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 282.1129 [M + H]+ (calcd for C17H16NO3, 282.1130). Single-Crystal X-ray Diffraction Analysis. The intensity data collection, structure solution, reduction, and hydrogen atom treatment for galanthine N-β-oxide (9) and carinatine N-α-oxide (10) were performed as described in a previous paper.11a The ORTEP drawing crystal structures for 9 and 10 are shown in Figures 4 and 5, respectively. The crystallographic data for the structures of 9 (CCDC 1542685) and 10 (CCDC 1542687) have been deposited in the Cambridge Crystallographic Data Centre. Crystallographic data of 9: C18H23NO5·3H2O, M = 387.42, monoclinic, T = 298(2) K, P2(1), a = 9.5350(3) Å, b = 7.4498(2) Å, c = 14.7161(4) Å, α = γ = 90°, β = 103.4880(10)°, V = 1016.51(5) Å3, Z = 2, Dcalcd = 1.266 Mg/m3, absorption coefficient 0.835 mm−1, F(000) = 416, crystal size 0.12 × 0.10 × 0.10 mm3, reflections collected 21 116, independent reflections 3521 [R(int) = 0.0272], final R indices [I > 2σ(I)] R1 = 0.0315, wR2 = 0.0816, R indices (all data) R1 = 0.0317, wR2 = 0.0817. Flack parameter = 0.1(1), 1618 Friedel pairs. Hooft parameter = 0.04(4), 1617 Bijvoet pairs. Crystallographic data of 10: 2C17H21NO5·3H2O, M = 692.74, monoclinic, T = 298(2) K, P2(1), a = 25.998(2) Å, b = 12.1491(11) Å, c = 11.3547(11) Å, α = γ = 90°, β = 103.780(5)°, V = 3483.2(5) Å3, Z = 4, Dcalcd = 1.321 Mg/m3, absorption coefficient 0.848 mm−1, F(000) = 1480, crystal size 0.12 × 0.10 × 0.10 mm3, reflections collected 17 506, independent reflections 4530 [R(int) = 0.0710], final R indices [I > 2σ(I)] R1 = 0.0625, wR2 = 0.1614, R indices (all data) R1 = 0.0679, wR2 =

0.1682. Flack parameter = 0.1(3), 2068 Friedel pairs. Hooft parameter = 0.1(1), 2068 Bijvoet pairs. NMR Data Calculations. The NMR data calculations of 11a and 11b were performed by using Gaussian09 at the B3LYP/6-31G* level as previously described.2b The relative errors between the computed and measured 13C NMR were calculated by the method of Crews and coworkers.16 Acetylcholinesterase Inhibitory Activity Testing. The AChE inhibitory activities of 1−26 were evaluated as previously described.4a Galanthamine was used as a positive control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00301. 1 H−1H COSY, key HMBC, and NOESY correlations of 2−11; calculated NMR of 11; HRESIMS, IR, UV, ECD, and NMR spectra for the new compounds 1−11; data fitting curves for IC50 calculation of compounds 6, 11, 17, and 20−23; scheme of the relationship in biosynthesis for isolated skeletons (PDF) X-ray crystallographic data for compound 9 (CIF) X-ray crystallographic data for compound 10 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Junjun Liu: 0000-0001-9953-8633 Guangmin Yao: 0000-0002-8893-8743 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (31170323), the Foundation of the Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences (2008DP173091-2016-01), and the Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148). We acknowledge the Analysis and Measurement Centre at Huazhong University of Science and Technology for IR and ECD testing.

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REFERENCES

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