Alkaloids with Different Carbon Units from Myrioneuron faberi

Article pubs.acs.org/jnp

Alkaloids with Different Carbon Units from Myrioneuron faberi Ming-Ming Cao,†,‡ Yu Zhang,† Sheng-Dian Huang,† Ying-Tong Di,† Zong-Gen Peng,§ Jian-Dong Jiang,§ Chun-Mao Yuan,† Duo-Zhi Chen,† Shun-Lin Li,† Hong-Ping He,† and Xiao-Jiang Hao*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China ‡ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, People’s Republic of China § Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 10050, People’s Republic of China S Supporting Information *

ABSTRACT: Three new Myrioneuron alkaloids, myrifamines A−C (1−3), with unique skeletons were isolated from Myrioneuron faberi. The absolute configuration of 1 was confirmed by single-crystal X-ray diffraction analysis, and the stereochemistry of the other two alkaloids was determined using a combination of ROESY experiments and calculated and experimental electronic circular dichroism spectra. Myrifamine C (3) is the first example of a symmetric dimer among the Myrioneuron alkaloids. Known alkaloids myrionamide (4) and schoberine (5) were also isolated, and experimental NMR and Xray diffraction data suggest their structural revision. Compound 2 showed significant inhibitory activity toward the hepatitis C virus in vitro, with a therapeutic index (CC50/EC50) greater than 108.7.

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Myrioneuron alkaloids constitute a family of nitrogen-containing polycyclic skeletons produced by plants of the genus Myrioneuron R. Br. (Rubiaceae).1−4 The lysine-based origin of these compounds has been suggested by Bodo and coworkers.5,6 One feature of the Myrioneuron alkaloids is their different carbon frameworks, mainly in multiples of fivemembered carbon atom chains (C10, C15, C20, and C35).2−6 During our ongoing investigation of these structurally unique and biologically interesting alkaloids, myrifamines A−C (1−3), with different carbon units (C 16 , C17, and C31 ), and myrionamide (4) and schoberine (5), with normal C15 frameworks, were obtained. On the basis of X-ray diffraction analyses, the structures of myrionamide and “schoberine”6 are suggested to be 4 and 5, respectively. Herein, the isolation, structural elucidation, anti-hepatitis C virus (HCV) activity, and proposed biosynthetic pathways of 1−5, as well as the structural revision of myrionamide (4) and deoxomyrionamide (4a), are discussed. © XXXX American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION

Myrifamine A (1) was isolated as colorless orthorhombic crystals (acetone) with [α]21 D −353 (c 0.2, MeOH). Its molecular formula, C16H24N2O, was established by 13C NMR and HREIMS (m/z 260.1884 [M]+, calculated for C16H24N2O, 260.1889) data, corresponding to six indices of hydrogen deficiency. The 13C NMR and DEPT data (Table 1) revealed 16 carbon signals comprising three sp2 carbon atoms (2 × CH and 1 × qC) and 13 sp3 carbon atoms (4 × CH and 9 × CH2). The sp2 carbon signal (δC 188.4) combined with the 1H NMR signal (1H, δH 9.42, s) indicated the existence of a formyl group, and the remaining two sp2 carbon atoms (CH, δC 149.6 and qC, δC 113.5) constituted one double bond. This information, coupled with IR absorption maxima at 1642 and 1598 cm−1 and a UVmax absorption at 297 nm, suggests the presence of a conjugated formyl group in 1, accounting for two of six indices of hydrogen deficiency, with the remaining four Received: June 18, 2015

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DOI: 10.1021/acs.jnatprod.5b00543 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H and 13C NMR Spectroscopic Data for Myrifamines A−C (1−3) Recorded at 600 and 125 MHz in Pyridine-d5 1 position 2 3

δC, type 149.6, CH 113.5, qC

4

20.5, CH2

5 6

30.0, CH 30.2,a CH2

7

20.0, CH2

8

30.1,a CH2

9 10 11

34.8, CH 64.3, CH 62.9, CH2

13 14

80.5, CH 28.6, CH2

15

24.5, CH2

16

25.7, CH2

17

55.8, CH2

20 21

188.4, CH

2 δH, (J in Hz)

7.27, s

2.51, 2.30, 2.02, 1.52, 1.30, 1.30, 1.23, 1.29, 0.75, 1.57, 2.67, 2.64, 1.66, 3.02, 1.70, 1.55, 1.68, 1.17, 1.38, 1.38, 2.63, 1.84, 9.42,

dd dd m m m m m m dq m dd m m dd m m m m m m m m s

22

δC, type 150.1, CH 114.9, qC

(16.2, 6.0) (16.2, 12.6)

7.49, s

20.7, CH2 29.9, CH 30.0,b CH2 19.9, CH2 30.2,b CH2

(12.0, 3.6) (10.8, 4.8)

(10.2, 2.4)

3 δH, (J in Hz)

34.2, CH 64.8, CH 63.2, CH2 82.0, CH 37.0, CH 28.5, CH2 25.0, CH2 55.7, CH2 189.1, CH 73.6, CH2

2.53, 2.36, 2.09, 1.49, 1.28, 1.33, 1.26, 1.33, 0.77, 1.61, 2.69, 2.69, 1.71, 3.18, 2.29,

dd (16.8, 6.0) dd (16.8, 6.0) m m m m m m dq (12.6, 3.6) m m m d (9.6) d (10.2) m

2.00, 1.35, 1.49, 1.49, 2.67, 1.90, 9.49, 3.42, 3.35,

m m m m m qd (12.0, 3.6) s dd (9.6, 3.0) dd (9.6, 6.0)

δC, type 168.8, qC 33.8, CH2 22.8, CH2 33.5, CH 31.6, CH2 20.9, CH2 29.9, CH2 38.3, CH 57.3, CH 53.2, CH2 71.3, CH 27.6, CH 32.7, CH2 20.4, CH2 54.3, CH2

34.7, CH2

δH, (J in Hz) 2.55, 2.41, 1.84, 1.24, 1.96, 1.60, 1.60, 1.36, 1.24, 1.43, 0.94, 1.78, 3.22, 2.83, 2.50, 5.60,

ddd (17.4, 4.8, 1.2) ddd (17.4, 12.0, 6.6) qd, (13.2, 5.4) m m m m ddd (13.2, 6.6, 3.0) m m qd (12.6, 3.6) ddt (12.6, 10.8, 3.6) dd (10.8, 5.4) m dd (14.4, 3.6) d (10.8)

2.13, 1.96, 1.08, 1.67, 1.06, 2.92, 2.84,

m m m qt (13.8, 3.6) m td (13.8, 2.4) t (10.8)

1.25, m 1.25, m

59.5, CH3

Interchangeable δC values.

a,b

Figure 2. 1H−1H COSY (bold) and key HMBC correlations of 1 (H to C) (A) and key ROESY correlations of 1 (B).

Along with the featured nitrogenated carbon atoms CH-13 (δH 3.02 dd, 10.2, 2.4 Hz), CH-10 (δH 2.67 dd, 10.8, 4.8 Hz), CH211 (δH 2.64 m; 1.66 m), and CH2-17 (δH 2.63 m; 1.84 m), this finding suggested that 1 possesses the same basic tetracyclic skeleton as dehydroschoberine.7 In the HMBC spectrum, crosspeaks of H-2 (1H, δH 7.27, s) to C-10 (CH, δC 64.3) and C-13 (CH, δC 80.5), H-10 to C-2 (CH, δC 149.6) and C-13, and H13 to C-2 and C-10 indicated that C-2, C-10, and C-13 are linked through N-1, as shown in Figure 2A. Furthermore, the presence of the C-3 formyl group was deduced via HMBC correlations from H-20 (1H, δH 9.42, s) to C-3 (qC, δC 113.5), C-2, and C-4 (CH2, δC 20.5). Thus, the 2D structure of 1 was constructed as shown in Figure 2A.

Figure 1. Structures of compounds 1−5.

assumed to be due to the presence of a tetracyclic system (Figure 1). Detailed 2D NMR (HSQC, 1H−1H COSY, and HMBC experiments) data revealed that 1 possesses two spin-coupling systems: a, H2-4/H-5/H2-6(H-10)/H2-7/H2-8/H-9/H-10(H211), and b, H-13/H2-14/H2-15/H2-16/H2-17 (Figure 2A). B



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Consequently, the orientation of H-9 (α) and H-5 (β), the chair conformations of rings B, C, and D, and the half-chair conformation of ring A were determined. Notably, compared to myriberine A,2 H-13 of 1 possesses the opposite orientation. Myrifamine B (2) was obtained as a white powder with [α]24 D −439 (c 0.1, MeOH). The molecular formula C18H28N2O2 was established via its 13C NMR and HREIMS (m/z 304.2146 [M]+, calculated for C18H28N2O2, 304.2151) data, corresponding to six indices of hydrogen deficiency. The IR absorptions at 1639 and 1602 cm−1 and the UVmax absorption at 297 nm were deduced as signals of an α,β-unsaturated formyl moiety, as in the case of 1. 13C NMR and DEPT data (Table 1) revealed 18 carbon signals comprising three sp2 carbon atoms (one formyl group and one trisubstituted double bond) and 15 sp3 carbon atoms (5 × CH, 9 × CH2 and 1 × CH3). In addition to the two indices of hydrogen deficiency accounting for the α,βunsaturated formyl moiety, the remaining four indices of hydrogen deficiency were assumed to be due to the presence of a tetracyclic system in 2 (Figure 1). The 1H NMR and 13C NMR data (Table 1) of compound 2 resembled those of 1, indicating that the two compounds are analogues. Compared to 1, the 13C NMR and DEPT spectra of 2 showed two additional carbon signals (CH3-22, δC 59.5 and CH2-21, δC 73.6), which were elucidated as a fragment “CH3OCH2−” and resolved by HMBC correlations from H322 (3H, δH 3.27 s) to C-21 (CH2, δC 73.6) to reveal the location of the “CH3OCH2−” moiety. As a result, the linkage between C-21 and C-14 was confirmed by 1H−1H COSY correlations of H-14 (1H, δH 2.29 m) and H2-21 (1H, δH 3.42 dd, 9.6, 3.0 Hz; 1H, 3.35 dd, 9.6, 6.0 Hz) and HMBC correlations from H2-21 to C-13 (CH, δC 82.0), C-14 (CH, δC 37.0), and C-15 (CH2, δC 28.5) (Figure 4A). Therefore, the 2D structure of 2 was established as shown in Figure 4A. The relative configuration of 2 was elucidated via its ROESY spectrum (Figure 4B) and 1H−1H vicinal coupling constants. In the ROESY spectrum (Figure S2.12), correlations of H-13 (1H, δH 3.46 d, 10.2 Hz)/H-10 (1H, δH 3.08 dd, 10.8, 4.2 Hz) indicated that these protons are β-oriented. Then, β-oriented H-5 and α-oriented H-9 were resolved by comparing the coupling constants of H-10 in 2 and 1 (dd, 10.8, 4.2 Hz for H10 in 2; dd, 10.8, 4.8 Hz for H-10 in 1) (Table 1 and Figures S2.12 and S1.6). Because the coupling constant of 10.2 Hz between H-13 (1H, δH 3.46 d, 10.2 Hz) and H-14 (1H, δH 2.29, m) requires a trans relationship between these two protons, the orientation of H-14 was deduced. Thus, the orientations of the hydrogens at the stereocenter of 2 were elucidated as 5β, 9α, 10β, 13β, and 14α. The absolute configuration of 2 was defined by comparing its similar electronic circular dichroism (ECD) spectra with those of 1 due to their asymmetrically perturbed α,β-unsaturated formyl chromophore. Thus, the absolute configuration of 2 was deduced as 5S, 9S, 10R, 13S, 14R (Figure 1). Myrifamine C (3) was obtained as an amorphous powder. The molecular formula C31H48N4O2 was established by its 13C NMR and HREIMS (m/z 508.3777 [M]+, calculated for C31H48N4O2, 508.3777) data, which indicated 10 indices of hydrogen deficiency. However, the 13C NMR spectra of 3 exhibited only 16 carbon signals rather than 31 (Figure S3.2). Further analysis of the integration of the 1H NMR spectrum suggested that 3 is a symmetric dimer, in which CH2-21 bridged two C15 moieties (Figure 1). This conclusion was also supported by the observation of a fragment ion peak at m/z 263 in its mass spectrum (Figure 6). The IR absorption at 1623

Figure 3. X-ray ORTEP drawing of compound 1.


Figure 5. ECD spectra of compounds 1 and 2 (in MeOH).

The relative configuration of 1 was elucidated via its ROESY spectrum. The correlations of H-13/H-10, H-10/H-8β (1H, δH 0.75, dq, 12.0, 3.6 Hz), and H-8β/H-11β (1H, δH 0.75, m) indicated that these protons are cofacial; they were arbitrarily assigned as β-oriented (Figure 2B). However, as the orientations of H-9 and H-5 and the ring conformations could not be established via the ROESY data, 1 was crystallized from acetone to produce orthorhombic crystals with the space group P212121, which was then analyzed by X-ray crystallography. The final refinement of the Cu Kα data resulted in a Flack8 parameter of 0.0(2) and a Hooft9 parameter of −0.04(5), which allowed the unambiguous assignment of the absolute configuration of 1 as (5S, 9S, 10R, 13S) (Figure 3). C



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Figure 6. Mass spectrum of 3.


2D NMR (HSQC, 1H−1H COSY, and HMBC experiments) data revealed that 3 possesses two spin coupling systems: a, H23/H2-4/H-5/H2-6(H-10)/H2-7/H2-8/H-9/H-10(H2-11), and b, H-13/H-14/H2-15(H2-21)/H2-16/H2-17 (Figure 7A). In the HMBC spectrum, cross-peaks of H-13 (1H, δH 5.60, d, 10.8 Hz) to C-2 (δC 168.8) and C-10 (CH, δC 57.3) and H-10 (1H, δH 3.22, dd, 10.8, 5.4 Hz) to C-2 indicated that C-2, C-10, and C-13 (CH, δC 71.3) are linked through N-1, as shown in Figure 7A. In addition, HMBC correlations of H-17 (1H, δH 2.92, td, 13.8, 2.4 Hz and 1H, δH 2.84, t, 10.8 Hz) to C-11 (CH2, δC 53.2) and C-13 revealed that C-11, C-13, and C-17 (CH2, δC 54.3) are connected through N-12. According to this analysis, the two spin coupling fragments are connected via N-1 and N12. For 3, the same A, B, and C ring systems as in myrionamide6 were recognized by the similarity of their NMR data, and the unusual 1H−1H COSY correlations of H14 (1H, δH 2.13, m)/H2-21 (2H, δH 1.25, m) revealed that one C15 structural moiety was attached to another through a C-21 (δC 34.7) methylene bridge. The relative configuration of 3 was established via its ROESY spectrum and 1H−1 H vicinal coupling constants. The orientation of H-13 was arbitrarily assigned as α, and the coupling constant of 10.8 Hz between H-13 and H-14 indicated a trans relationship of these two protons. In addition, the presence of NOE correlations of H-14/H-10/H-11β (δH 2.83, m) suggested a β-oriented H-10. The NOE correlation of H10/H-11β indicated a 1,3-cis-diaxial relationship between H-10 and H-11β, requiring that H-9 be α-axial (Figure 7B). The

Figure 8. Calculated and experimental ECD spectra of 3 (solid line, experimental in MeOH; dashed line, at the B3LYP/6-31G(d,p) level with the IEFPCM model in MeOH).

cm−1 suggested the existence of a lactam carbonyl. Carbon signals in the 13C NMR and DEPT spectra of 3 can be classified as one lactam carbonyl, five methines (two nitrogenated), and nine methylenes (two nitrogenated) in each monomeric unit and one shared methylene bridge. Because of the C-2 (2′) carbonyl group, in each monomeric unit, the remaining eight indices of hydrogen deficiency are assumed to be due to the presence of the two tetracyclic systems, as shown in Figure 1. D



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Table 2. 1H and 13C NMR Spectroscopic Data for 4, 4a, and 5 Recorded in CDCl3 at 400 and 100 MHz 4 no.

δC, type

2

168.4, qC

3

33.0, CH2

4

22.0, CH2

5

32.8, CH

6

30.6, CH2

7

20.6, CH2

8

30.1, CH2

9 10 11

37.7, CH 55.2, CH2 53.0, CH2

13 14

68.4, CH 23.3, CH2

15

24.4, CH2

16

20.1, CH2

17

53.9, CH2

4a δH (J in Hz)

δC, type 39.8, CH2

2.39, 2.27, 1.88, 1.38, 2.00, 1.87, 1.66, 1.59, 1.44, 1.30, 1.51, 1.04, 1.74, 3.29, 2.67, 2.49, 5.09, 1.77, 1.40, 1.77, 1.51, 1.51, 1.18, 2.79, 2.72,

m m m m m m m m m m m qd m dd m dd dd m m m m m m m m

δH (J in Hz)

39.8, CH2

24.2, CH2

35.1, CH

1.55, m 1.30, m 1.93, m

34.7, CH

2.87, 2.76, 1.73, 1.52, 1.52, 1.28, 1.92,

29.8, CH2

1.66, m

31.2, CH2

1.52, m

20.1, CH2

1.39, m

19.9, CH2

1.37, m

30.2, CH2

1.51, 0.82, 2.17, 2.53, 2.81, 1.72, 2.91, 1.54,

m m m dd (11.0, 4.6) m m m m

29.9, CH2

1.52, 0.80, 2.17, 2.60, 2.87, 1.18, 3.07, 1.68,

1.74, 1.52, 1.78, 1.30, 2.78, 1.90,

m m m m m m

24.6, CH2

(12.8, 3.6)

(11.2, 5.6) (11.2, 2.4)

δC, type

2.84, m 2.74, m 1.53, m

25.3, CH2

(10.8, 5.2)

5 δH (J in Hz)

27.0, CH 65.0, CH 63.5, CH2 82.6, CH 31.5, CH2 26.1, CH2 24.6, CH2 56.2, CH2

25.6, CH2

26.8, CH 64.8, CH 62.6, CH2 82.7, CH 29.1, CH2 24.1, CH2

m m m m m m m

m qd (12.0, 5.2) m dd (11.2, 4.6) m m m m

1.78, m 1.28, m 1.52, m

24.8, CH2 55.9, CH2

2.85, m 1.96, m

Figure 9. Structural revision of myrionamide and X-ray ORTEP drawing of compound 4.

proton at the C-5 stereogenic center (δH 1.96, m) could be assigned as β-oriented based on JH‑10/H‑5 (5.4 Hz, cis). Finally, H-9 and H-13 were assigned as α-oriented, and H-5, H-10, and H-14 were assigned as β-oriented (Figure 1). To determine the absolute configuration of 3, the ECD spectrum was calculated using the TD-DFT-B3LYP/6-311G +(2d,p) theory of B3LYP/6-31G(d,p)-optimized geometries with the IEFPCM model (in MeOH) in the Gaussian 09 program package.10 The calculated ECD curve of 3 compared well to the experimentally recorded curve (Figure 8). Thus, the absolute configuration of 3 was determined as 5S, 9S, 10R, 13R, and 14R. Myrionamide (4) was recrystallized from acetone and exhibited a triclinic pattern with a P1̅ space group. The 13C NMR data (Table 2) and melting point (61−62 °C) of 4 were similar to those reported for myrionamide,6 which indicated that they share the same structure. However, an X-ray

Figure 10. X-ray ORTEP drawing of compound 5.

Scheme 1. Reduction of Myrionamide

diffraction experiment revealed that 4 possesses an α-oriented H-13 (Figure 9), which was supported by the ROESY correlation cross-peak: H-13 (δH 5.09, dd, 11.2, 2.4 Hz)/H-9 E



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between 1b and formaldehyde12,13 may result from microbial degradation reactions14 and is followed by oxidation to yield intermediate 1c, then affording 1. Subsequent dehydrogenation and oxidation produce the key intermediate 2a. An intermolecular “aldol-condensation-like” reaction between 2a and formaldehyde followed by methylation yields intermediate 2b, which could be selectively reduced to 2. In the second pathway, myrionidine is oxidized to intermediate 3a. Subsequently 3a condenses with formaldehyde and is dehydrated to afford intermediate 3b. The dimeric intermediate 3c is produced from 3a and 3b via a Michael-type addition. Finally, the symmetric dimer 3 is generated by selective reduction. Known alkaloids 4 and 5 could be the reduction products of 3a and 1a, respectively.

Table 3. Anti-HCV Activities of Compounds 1−5 CC50 (μM)

EC50 (μM)

compound

mean

SD

mean

SD

1 2 3 4 5 VX-950

>100 >100 >100 >100 95.89 47.83

0.00 0.00 0.00 0.00 6.72 1.25

3.27 0.92 10.53 30.61 7.35 0.129

2.21 0.26 3.14 2.44 0.56 0.011

SI >30.6 >108.7 >9.5 >3.3 13.0 370.8

(δH 1.74, m). As a result, the structure of myrionamide should be revised as 4. Schoberine (5) was also obtained as crystals (mp 69−70 °C), exhibiting a monoclinic pattern with a P21 space group. Its single-crystal structure indicated the same structure as reported for “schoberine” (Figure 10); however, the chemical shifts of C13 of these two compounds showed a maximum ΔδH = 0.23 value, calling into question the orientation of H-13 of “schoberine” reported in 2008.6 In addition, the reduction product of 4 with LiAlH4, deoxomyrionamide (4a), showed 13C NMR data (Table 2) identical to those of “schoberine” (Scheme 1).6 This reveals that “schoberine” reported in 2008 was indeed a new alkaloid with the same structure as 4a instead of the schoberine reported in 1989.11 For comparison, the NMR data of 4, 4a, and 5 were recorded in the same solvent (CDCl3), and their 1D NMR data assignments were performed based on their 2D NMR spectra (HSQC, 1H−1H COSY, and HMBC experiments). Moreover, the 13C NMR chemical shift of C-9 of myrionamide6 should be corrected from δC 34.7 to 37.4. The P1̅ space group of 4 proved it to be a racemic mixture, and the subsequent separation of (±)-4 using a chiral column by HPLC also generated two enantiomer peaks. The inhibitory effects of compounds 1−5 on the life cycle of hepatitis C virus in vitro were evaluated.2,3,12 Compound 2 exhibited significant inhibitory activity, with a selective index (SI) higher than 108.7 (as compared to the 307.8 of VX-950 as a positive control), as shown in Table 3. A tentative biosynthetic pathway for alkaloids 1−3 is proposed in Scheme 2. These compounds might be derived from myrionidine.6 In one pathway, myrionidine is transformed into intermediates 1a and 1b. Intermolecular condensation

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured using a Yuhua X-4 digital microdisplaying melting point apparatus. Optical rotations were measured with a Jasco P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. ECD spectra were recorded with an Applied Photophysics Chirascan spectrometer. A Tenor 27 spectrophotometer was used for the recording of IR spectra as KBr pellets. 1D and 2D NMR spectra were recorded using Bruker AVANCEIII-600 spectrometers with TMS as an internal standard. HREIMS was performed with an API QSTAR time-of-flight spectrometer. X-ray data were collected using a Bruker APEX DUO instrument. Semipreparative HPLC was performed using an Agilent 1100 liquid chromatograph with a Waters X-Bridge C18 (4.6 × 250 mm) column. Column chromatography (CC) was performed using silica gel (200− 300 mesh and 300−400 mesh, Qingdao Marine Chemical, Inc., Qingdao, P. R. China). Plant Material. The aerial parts of M. faberi were collected in October 2011 from Sichuan Province, People’s Republic of China, after its flowering phase. The plant samples were identified by Prof. Xun Gong of the State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS). A voucher specimen was deposited at KIB, CAS, under the accession number KIB H20111001. Extraction and Isolation. The air-dried, powdered leaves and stems (30 kg) of M. faberi were extracted three times with 50 L of 95% EtOH. After removing saccharides using a macroporous resin (D101), the crude alkaloids (223 g) were subjected to normal-phase silica gel chromatography (200−300 mesh; CHCl3/MeOH, 20:1 → 0:1), yielding four fractions (Fr 1−4). Fr 1 (12.9 g) was further subjected to normal-phase Si gel (200−300 mesh; PE/EtOAc = 5:1) to give three

Scheme 2. Putative Biosynthetic Pathway of 1−3

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fractions (Fr 1A−1C). Fr 1A was subjected to normal-phase Si gel (200−300 mesh; PE/EtOAc = 20:1) to afford 4 (350 mg) and 5 (110 mg). Fr 1C was separated using a Waters X-Bridge C18 column (4.6 × 250 mm) (MeCN/H2O = 60:40) to give compounds 1 (30 mg) and 2 (1 mg). Fr 2 (32.4 g) was subjected to normal-phase Si gel (200−300 mesh; PE/EtOAc = 5:1) to yield four fractions (Fr 2A−2D). Fr 2B (1.2 g) was repeatedly purified on normal-phase Si gel, resulting in 3 (2 mg) after Waters X-Bridge C18 (4.6 × 250 mm) chromatography (MeCN/H2O = 40:60). Myrifamine A (1): colorless crystals (acetone); mp 142.0−143.0 °C; [α]21 D −353 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 297 (3.75), 205 (2.97); IR (KBr) νmax 3431, 2926, 2853, 1642, 1598, 1441, 1330, 1293, 1276, 1242, 1189, 1119, and 1043 cm−1; ECD (0.000 25 M, MeOH) λmax (Δε) 275 (+2.22), 304 (−6.99); 1H and 13C NMR data, Table 1; positive ESIMS m/z 261 [M + H]+; positive HREIMS m/z 260.1884 [M]+, calculated for C16H24N2O, 260.1889. Myrifamine B (2): white solid; [α]24 D −439 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 297 (3.83), 206 (3.13); IR (KBr) νmax 3424, 2926, 2854, 1639, 1602, 1441, 1384, 1296, 1275, 1187, 1116, and 1066 cm−1; ECD (0.000 24 M, MeOH) λmax (Δε) 275 (+2.72), 304 (−9.53); 1H and 13C NMR data, Table 1; positive ESIMS m/z 305 [M + H]+; positive HREIMS m/z 304.2146 [M]+, calculated for C18H28N2O2, 304.2151. Myrifamine C (3): amorphous powder; [α]23 D −17 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (3.10); IR (KBr) νmax 3431, 2928, 2852, 1623, 1445, 1383, 1318, 1231, 1172, 1100, and 1077 cm−1; ECD (0.000 79 M, MeOH) λmax (Δε) 210 (−2.92); 1H and 13C NMR data, Table 1; positive ESIMS m/z 509 [M + H]+, 531 [M + Na]+; positive HREIMS m/z 508.3777 [M]+, calculated for C31H48N4O2, 508.3777. Bioassay. Cells. Huh7.5 human liver cells (kindly provided by Vertex Pharmaceuticals, Boston, MA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, CA, USA) supplemented with 10% inactivated fetal bovine serum (Invitrogen) and 1% penicillin−streptomycin (Invitrogen). The cells were cultured at 37 °C in 5% CO2. HCV Infection and Treatment. The Huh7.5 cells were seeded into 96-well plates (Costar) at a density of 3 × 104 cells/cm2; after 24 h, the cells were infected with HCV viral stock (chimeric HCV FL-J6/JFH/ JC1, approximately 45 IU per cell) and simultaneously treated with compounds 1−5 or solvent as the control. The culture medium was removed at 72 h after inoculation, and intracellular RNA was extracted with an RNeasy mini kit (Qiagen). The intracellular HCV RNA and internal control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were quantified using AgPath-ID One-Step RT-PCR kit (Applied Biosystems). The results were calculated with 2ΔΔCT. The half maximal effective concentration (EC50) was calculated using the Reed and Muench method.15,16 X-ray Crystal Structure Analysis. Colorless crystals of 1 were obtained from acetone. Intensity data were collected using a Bruker APEX DUO diffractometer equipped with an APEX II CCD and Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT software. The structure was solved by direct methods using SHELXL-97.17 Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystal data: 3(C16H24N2O)· H2O, M = 799.13, orthorhombic, a = 9.7135(3) Å, b = 13.9213(4) Å, c = 31.7069(8) Å, α = β = γ = 90.00°, V = 4287.6(2) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.617 mm−1, 19 888 reflections measured, 7428 independent reflections (Rint = 0.0353). The final R1 values were 0.0450 (I > 2σ(I)). The final wR(F2) values were 0.1280 (I > 2σ(I)). The final R1 values were 0.0452 (all data). The final wR(F2) values were 0.1283 (all data). The goodness of fit for F2 was 1.083. The Flack parameter was 0.0(2), and the Hooft parameter was −0.04(5) for 3200 Bijvoet pairs. Crystallographic data (excluding structure factor tables) for 1 were deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 926408. Crystallographic data for 4 and 5 were also deposited with the Cambridge Crystallographic Data Center with

CCDC numbers of CCDC 902353 and CCDC 1059225, respectively. Copies of these data can be obtained free of charge by application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. + 44 (0) (1223) 336 033; e-mail: [email protected]].

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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.5b00543. 1D and 2D NMR, ESIMS, HRESIMS, IR, and ECD spectra of 1−3; 1D NMR and ESIMS spectra of 4 and 5; computational methods for ECD of 3 (PDF) X-ray crystallographic data of 1 (CIF) X-ray crystallographic data of 4 (CIF) X-ray crystallographic data of 5 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (X.-J. Hao): [email protected]. Fax: +86-87165223070. Tel: +86-871-65223263. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported financially by the National Natural Science Foundation of China (No. 21372228) and Applied Basic Research Projects of Yunnan (No. 2014FB166).

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REFERENCES

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