Melokhanines AâJ, Bioactive Monoterpenoid Indole Alkaloids with

Article pubs.acs.org/jnp

Melokhanines A−J, Bioactive Monoterpenoid Indole Alkaloids with Diverse Skeletons from Melodinus khasianus Gui-Guang Cheng,†,‡,§,# Dan Li,†,§,# Bo Hou,†,§,# Xiao-Nian Li,†,§ Lu Liu,†,§ Ying-Ying Chen,†,§ Paul-Keilah Lunga,†,§ Afsar Khan,†,§,⊥ Ya-Ping Liu,†,§ Zhi-Li Zuo,*,†,§ and Xiao-Dong Luo*,†,§ †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, People’s Republic of China ‡ Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, 650500, People’s Republic of China § Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming, 650201, People’s Republic of China ⊥ Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan S Supporting Information *

ABSTRACT: The new melokhanines A−J (1−10) and 22 known (11−32) alkaloids were isolated from the twigs and leaves of Melodinus khasianus. The new compounds and their absolute configurations were elucidated by extensive analysis of spectroscopic, X-ray diffraction, and computational data. Melokhanine A (1), composed of a hydroxyindolinone linked to an octahydrofuro[2,3-b]pyridine moiety, is an unprecedented monoterpenoid indole alkaloid. Melokhanines B−H (2−8) possess a new 6/5/5/6/6 pentacyclic indole alkaloid skeleton. Alkaloids 1−16, 25−27, 31, and 32 showed the best antibacterial activity against Pseudomonas aeruginosa (MIC range 2−22 μM). Among the seven dermatophytes tested, compound 1 showed significant inhibitory activity against Microsporum canis, M. ferrugineum, and Trichophyton ajelloi (MIC range 38−150 μM), i.e., half the efficacy of the positive control, griseofulvin.

T

(11),9 rhazinal (12),10 leuconodine C (13),11 leuconodine E (14),11 (+)-eburnamonine (15),12 eburnamenine (16),13 Omethyl-16-epivincanol (17),14 O-methylvincanol (18),15 14,15dehydrovincanol (19),16 16-epi-14,15-dehydrovincanol (20),17 14,15-dehydrovincamine (21),18 14,15-dehydroepivincamine (22),18 melodinine F (23),19 (+)-eburnamonine N(4)-oxide (24),12 melodinines A−C (25−27),5c leuconicine B (28),19 leuconicine E (29),19 decarbomethoxydihydrogambirtannine (30),20 ajmalicine (31),21 and isositsirikine (32) 22 by comparison with reported data. The alkaloids were tested for antimicrobial activity, while the new compounds were also tested for their in vitro cytotoxicity against SMMC-7721, MCF7, HL-60, SW480, and A-549 human cancer cell lines.

he genus Melodinus (Apocynaceae), with 53 species, is widely distributed in the tropical and subtropical regions, of which 12 plants are found in the south of China. Plants of Melodinus have been used for the treatment of rheumatic heart diseases and meningitis in children in Traditional Chinese Medicine.1 Alkaloids from Melodinus plants were reported to possess antitumor,2 antifertility,3 and antibacterial activities.4 Previous chemical investigations of the genus Melodinus resulted in a series of structurally diverse monoterpenoid indole alkaloids and their dimeric forms;5 among these, total syntheses of melotenine A,6 melohenine B,7 and melodinine E8 have been carried out to solve the structures of their complex polycyclic skeletons. In an ongoing search for novel indole alkaloids with pharmacological activities, 10 new compounds, melokhanines A−J (1−10), and 22 known compounds (11−32) were isolated from M. khasianus. Melokhanine A (1) is an unprecedented alkaloid bearing a hydroxyindolinone moiety connected to a fused piperidine-tetrahydrofuran heterocycle via an ethylene linker. Alkaloids 2−8 are rare 6/7-seco rearranged spiro-indolinone-type alkaloids including a 6/5/5/6/6 ring architecture. Compound 9 is a unique 6/5/7/6/6 pentacyclic andranginine-type alkaloid, and compound 10 is a 2,7-seco aspidosperma alkaloid with a rare 6/9/6/5 tetracyclic ring system. The known alkaloids were identified as leuconolam © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Melokhanine A (1) was obtained as an amorphous powder. The molecular formula of 1 was established as C19H26N2O3 by HREIMS (m/z 330.1946 [M]+) in association with 13C and DEPT NMR spectra. The formula indicated eight indices of hydrogen deficiency. The IR spectrum suggested the presence of a carbonyl group (1719 cm−1) and aromatic ring (1622 and 1471 cm−1). The 1H NMR signals at δH 7.39 (d, J = 7.5 Hz, HReceived: January 5, 2016

A

DOI: 10.1021/acs.jnatprod.6b00011 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR Spectroscopic Data of Compounds 1−5a (δ in ppm and J in Hz) position NH 3a 3b 5a 5b 6a 6b 9 10 11 12 14a 14b 15a 15b 16a 16b 17a 17b 18 19a 19b 21 OMe 22

1 9.32, 2.73, 2.53, 3.06, 2.68, 2.32, 1.80, 7.39, 7.02, 7.24, 6.90, 1.59, 1.49, 1.52, 1.35, 3.86, 3.74, 1.73, 1.62, 0.89, 1.66, 1.27, 4.06,

s m td (11.1, 2.9) m ddd (13.4, 5.4, 4.0) ddd (14.4, 10.8, 5.4) ddd (14.4, 4.9, 4.0) d (7.5) td (7.5, 1.1) td (7.5, 1.1) d (7.5) ovb ov ov m dd (16.6, 8.3) ddd (9.4, 8.3, 3.2) dt (12.2, 9.4) ov t (7.5) dq (14.7, 7.5) dq (14.7, 7.5) s

2

3

5

3.06, 1.92, 3.16, 2.18, 2.12, 2.02, 7.50, 6.84, 7.48, 7.37, 1.70, 1.58, 1.41, 1.08, 5.51,

m m t (8.1) m m m d (7.4) t (7.4) t (7.4) d (7.4) m ov br d (13.4) td (13.4, 4.4) dd (10.2, 5.4)

3.02, 2.04, 2.98, 2.29, 1.91, 1.87, 7.47, 6.84, 7.55, 7.25, 1.61, 1.42, 1.47, 1.03, 5.05,

m td (12.0, 2.6) m ddd (11.5, 8.2, 6.0) m m d (7.5) t (7.5) t (7.5) d (7.5) m m m td (13.4, 4.8) m

2.92, 1.77, 3.02, 2.04, 1.96, 1.86, 7.34, 6.69, 7.36, 7.29, 1.56, 1.43, 1.27, 0.93, 5.47,

br d (12.6) td (12.6, 3.0) t (8.1) td (10.3, 8.0) m ddd (12.8, 10.8, 8.0) d (7.6) t (7.6) t (7.6) d (7.6) ovb m br d (3.4) td (13.4, 4.3) dd (10.2, 5.5)

3.15, 2.29, 3.08, 2.43, 2.16, 1.97, 7.74, 7.34, 7.76, 8.28, 1.76, 1.62, 1.74, 1.30,

m td (11.0, 2.3) m ddd (11.5, 8.7, 5.4) dd (12.5, 5.4) m d (7.5) t (7.5) t (7.5) d (7.5) ov m ov td (14.0, 4.0)

1.75, 1.61, 0.50, 0.71, 0.71, 1.86, 3.57,

m ov t (7.4) dq (14.4, 7.4) dq (14.4, 7.4) s s

2.36, 1.80, 0.56, 0.58, 0.54, 1.96, 3.29,

dd (13.6, 6.5) dd (13.6, 8.8) ov ov ov s s

1.60, 1.51, 0.36, 0.56, 0.56, 1.70,

ov ov t (7.3) dq (14.2, 7.5) dq (14.2, 7.5) s

3.45, 2.05, 0.71, 0.91, 0.73, 2.48,

d (15.2) m ov m ov s

3.81, dq (14.6, 7.4) 3.49, dq (14.6, 7.4) 1.17, t (7.4)

23 a

4

Compounds 1, 2, 4, and 5 were measured in acetone-d6, 3 was measured in DMSO-d6. bov: overlap.

9), 7.24 (td, J = 7.5, 1.1 Hz, H-11), 7.02 (td, J = 7.5, 1.1 Hz, H10), and 6.90 (d, J = 7.5 Hz, H-12) revealed the presence of an ortho-disubstituted phenyl ring (Table 1). In the HMBC spectrum, correlations of H-9 (δH 7.39) with C-11 (δC 129.6, d), C-13 (δC 142.4, s), and C-7 (δC 76.9, s) suggested the latter carrying an −OH group, judging from its chemical shift. A deshielded carbon resonance at δC 180.1 was assigned to the C2 amide carbonyl, which revealed the presence of an oxindole moiety. The correlations of H2-5 (δH 3.06, 2.68) with H2-6 (δH 2.32, 1.80) in the 1H−1H COSY spectrum revealed a −CH2CH2− fragment corresponding to the C-5/C-6 linkage.

The HMBC correlations of H2-5 with C-7 and of H2-6 with C2, C-7, and C-8 (δC 133.1) established the partial structure 1a (Figure 1). The 13C and DEPT NMR spectra displayed nine carbon signals corresponding to a methyl (δC 9.1), six methylenes (δC 63.7, 44.7, 36.3, 27.4, 27.4, and 21.7), a methine (δC 98.5), and a quaternary carbon (δC 42.5), which required two rings based on the indices of hydrogen deficiency (Table 3). Further analysis of the 1H−1H COSY and HSQC spectra indicated that 1 possessed a deshielded partial spin system (H2-3/H2-14/H215). A methylene carbon at δC 44.7 (C-3), with corresponding B



Article

21 (δH 4.06) with Me-18 (δH 0.89) and H2-19 (δH 1.66, 1.27) placed them on the same side, suggesting a cis configuration of the C/D ring junction (Figure 2), which established the relative configuration of 1. The absolute configuration of 1 was assigned by timedependent density functional theory (TDDFT) calculations of the electronic circular dichroism (ECD),23 NMR chemical shifts,24 vibrational circular dichroism (VCD),25 and specific rotation26 data. A conformational search was carried out employing the CHARMM force field, which showed 231 conformers for 1 within a 5 kcal·mol−1 energy window. Conformational analysis suggested that two of the minimum energy conformers (Figure S1, Supporting Information) accounted for >90% of the conformational itinerary. The ECD spectra of the four main conformers were computed at the TDDFT/B3LYP/6-311++G(2d,2p) level in the gas phase, COSMO, and PCM models. In addition to solvent effects, calculated percentages of transitions, excitation energies, oscillator strengths, and rotatory strengths were also simulated (Table S1, Supporting Information). The π→π* (1Lb) electronic transitions from HOMO to LUMO (Figure 3) of the hydroxyindoline moiety in 1 afforded a rotatory strength at 267.8 nm, which was in accordance with the weak positive Cotton effect (CE) at 266 nm in its experimental ECD spectrum (Figure 4). The negative rotatory strength at 242.7 nm was attributed to the other π→π* (1La) electronic transitions of the same group (∼53% contributions), which was in agreement with the experimental negative CE at 241 nm in the ECD spectrum. The second positive Cotton effect was complicated, as another mixed transition orbital of the condensed skeleton shifted to a higher unoccupied molecular

Figure 1. 1H−1H COSY (bold) and HMBC (arrows) correlations of compounds 1, 2, and 9.

protons at δH 2.73 and 2.53 in the 1H NMR spectrum, was assigned to a carbon bearing a nitrogen atom. The HMBC correlations of H-3a (δH 2.73) with C-21 (δC 98.5) and of H-21 (δH 4.06) with C-3 (δC 44.7) indicated the presence of a −CH2−N−CH− fragment (C-3/N-4/C-21). Accordingly, the presence of a six-membered C-ring was established by the correlations of H-15a (δH 1.52, m) with C-20 (δC 42.5, s) and C-21 (δC 98.5, d) in the HMBC spectrum. The deshielded signal at δC 98.5 was assigned to a carbon (C-21) bearing a nitrogen and an oxygen atom. Furthermore, the 1H−1H COSY cross-peaks of H2-16 (δH 3.86, 3.74) with H2-17 (δH 1.73, 1.62) indicated the presence of a −CH2CH2− fragment. The HMBC correlations of H2-16 and H2-17, respectively, with C-20 (δC 42.5) and C-21 (δC 98.5) indicated the presence of a tetrahydrofuran D-ring. In addition, the linkage of C-18/19/ 20 was also indicated by the correlations of Me-18 (δH 0.89) with C-19 (δC 27.4) and C-20 (δC 42.5) in the HMBC spectrum. Therefore, the partial structure of an octahydrofuro[2,3-b]pyridine moiety (1b) was established. Finally, the correlations of H2-5 with C-3 and C-21 indicated the linkage of 1a and 1b through C-5/N-4. The ROESY correlations of H-

Table 2. 1H NMR Spectroscopic Data of Compounds 6−10a (δ in ppm and J in Hz) position NH 3a 3b 5a 5b 6a 6b 7 9 10 11 12 14a 14b 15a 15b 16a 16b 17a 17b 18a 18b 19a 19b 21 OMe a

6 ovb ov ov m ov m

7

8

9 10.78, 3.79, 3.54, 3.85, 3.53, 3.28, 3.28,

s ddd (12.4, 12.4, 3.3) ov m ov m m

3.10, 2.27, 2.99, 2.45, 2.10, 2.06,

m m m m ov ov

3.11, 2.28, 3.00, 2.42, 2.09, 2.06,

dd (10.1, 4.3) m m ov ov m

7.57, d (7.7) 6.83, t (7.7) 7.43 t (7.7) 6.90 d (7.7) 1.64, m 1.59, m 1.80, br d (13.4) 1.02, td (13.4, 4.3) 6.61, d (7.0)

7.56, 6.87, 7.48, 6.95, 2.19, 1.63, 2.08, 1.26, 4.39,

d (7.8) t (7.8) t (7.8) d (7.8) m m ov m d (4.6)

7.54, 6.85, 7.48, 7.09, 2.12, 1.65, 2.03, 1.26, 4.72,

d (7.8) t (7.8) t (7.8) d (7.8) ov m m m d (4.5)

7.54, 7.11, 7.18, 7.47, 2.21, 2.21, 3.90,

d (8.0) t (8.0) t (8.0) d (8.0) m m t (2.7)

5.36, d (7.0)

4.22, d (4.6)

3.96, d (4.5)

0.55, t (7.3)

0.57, t (6.6)

0.60, t (6.8)

0.89, dq (14.2, 7.3) 0.62, dq (14.2, 7.3) 2.52, s

0.61, 0.52, 2.40, 3.49,

0.66, q (6.8) 0.54, q (6.8) 2.43, ov

3.12, 1.94, 2.45, 2.35, 6.31,

br d (12.0) td (12.0, 4.6) m dt (10.6, 4.6) d (5.5)

3.06, 2.10, 3.04, 2.30, 2.11, 1.88,

q (6.6) q (6.6) s s

10 2.99, 2.51, 2.92, 2.70, 2.59, 1.49, 3.76, 7.13, 6.99, 7.16, 8.11, 1.75, 1.59, 1.80, 1.20, 3.59, 2.34, 1.73, 1.73, 0.73,

m td (12.4, 3.6) td (12.6, 6.0) dd (12.6, 6.8) m dd (12.4, 6.0) d (9.7) d (7.4) t (7.4) t (7.4) d (7.4) ov m m m dd (14.0, 8.6) ddd (14.0, 5.0, 2.8) ov ov t (7.4)

1.30, dq (14.0, 7.4) 1.08, dq (14.0, 7.4)

4.38, s 3.27, s

Compounds 6−8 and 10 were measured in CDCl3; 9 was measured in DMSO-d6 and methanol-d4. bov: overlap. C



Article

Table 3. 13C NMR Spectroscopic Data of Compounds 1−10a (δ in ppm) position 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17a 18 19 20 21 22 23 OMe

1 180.1, 44.7, 51.1, 34.4, 76.9, 133.1, 124.8, 122.5, 129.6, 110.4, 142.4, 21.7, 27.4, 63.7, 36.3, 9.1, 27.4, 42.5, 98.5,

2 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH2 CH2 CH3 CH2 C CH

75.5, 51.4, 53.6, 35.2, 203.5, 122.3, 123.9, 118.5, 136.9, 113.8, 160.7, 21.9, 34.4, 86.3, 34.8, 6.8, 33.9, 35.7, 74.7,

3 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH CH2 CH3 CH2 C CH

55.6, CH3

72.6, 50.8, 52.3, 37.2, 204.5, 118.0, 124.0, 118.8, 137.6, 109.9, 161.4, 21.5, 32.7, 84.6, 31.5, 6.8, 28.6, 34.7, 71.2,

4 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH CH2 CH3 CH2 C CH

75.5, 51.4, 53.6, 35.2, 203.6, 122.2, 123.9, 118.5, 136.9, 113.6, 160.8, 21.9, 34.4, 84.5, 35.0, 6.8, 33.9, 35.7, 74.8, 63.9, 14.5,

5 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH CH2 CH3 CH2 C CH CH2 CH3

74.9, 50.5, 51.2, 40.3, 199.9, 122.4, 123.9, 124.6, 136.5, 119.2, 152.1, 21.9, 31.8, 169.7, 37.8, 6.2, 29.6, 38.0, 68.6,

6 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 C CH2 CH3 CH2 C CH

53.3, CH3

74.1, 51.6, 52.5, 40.1, 204.4, 120.7, 124.8, 119.8, 136.9, 111.7, 159.2, 23.2, 32.9, 126.3, 124.9, 7.6, 34.3, 37.8, 75.3,

7 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH CH CH3 CH2 C CH

71.4, 49.5, 50.6, 37.1, 204.0, 117.9, 123.6, 119.1, 136.6, 110.3, 161.1, 23.0, 32.0, 88.1, 71.6, 6.1, 27.8, 36.1, 69.9,


52.7, CH3

71.3, 49.5, 50.6, 37.8, 203.8, 117.8, 123.7, 118.9, 136.7, 110.5, 161.0, 22.7, 31.6, 79.6, 73.4, 7.6, 27.9, 35.8, 69.6,


139.9, 51.4, 60.8, 20.1, 111.1, 128.2, 118.5, 120.0, 122.3, 112.3, 136.3, 30.4, 78.5, 50.3, 33.1, 23.4, 136.0, 129.6, 65.6, 176.4,

10 C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH C CH2 CH2 CH C CH C

172.6, 50.8, 52.2, 33.3, 47.0, 133.8, 123.5, 123.4, 127.4, 114.4, 143.0, 19.9, 32.1, 32.8, 29.1, 7.4, 31.5, 40.4, 97.5,

C CH2 CH2 CH2 CH C CH CH CH CH C CH2 CH2 CH2 CH2 CH3 CH2 C C

55.6, CH3

a

Compounds 1, 2, 4, and 5 were measured in acetone-d6, 3 was measured in DMSO-d6, 6−8 and 10 were measured in CDCl3, and 9 was measured in DMSO-d6 and methanol-d4.

Figure 2. ROESY correlations of 1, 2, and 9.

Figure 4. Key structure of compound 1 in the PCM model used for ECD calculations at the TDDFT/B3LYP/6-311++G(2d,2p) level. All the calculated spectra were wavelength-corrected to match the experimental ECD spectrum (Calcd 1 in the gas phase; Calcd 2 with the PCM model in MeOH; Calcd 3 with the COSMO model in MeOH; Exp experimental trace in MeOH).

experimental data (Figures S2 and S3, Supporting Information).27 The predicted VCD data for (7R,20R,21S)-1 at the B3LYP/6-311++G(2d,2p) level showed a close correlation to the experimental data (Figure 5). The VCD bands originating from the stretching modes in the 1000−1600 cm−1 region have the same signs and magnitudes for (7R,20R,21S)-1.28 The results showed that the frequencies at 1000−1100 cm−1 and 1300−1400 cm−1 in the VCD mainly resulted from different stretching vibrations of bonds at C-20 and C-21 (Figure 5). Furthermore, the specific rotation was calculated at the B3LYP/ 6-311++G(2d,2p) level of theory using the PCM model in CH3OH. The computed specific rotation for the (7R,20S,21R) configuration was −0.30 and for the (7R,20R,21S) diastereomer +78.12. The latter was closer to the experimental value of +72. Data analysis showed that all the conformers contributed to the total magnitude of specific rotation, while the four major conformers have positive signs (Table S2, Supporting Information). The deviation in specific rotation of the conformers a1 and a2 occurred due to free rotation of the C-

Figure 3. Molecular orbitals (MOs) of the most stable conformers involved in each transition calculated at the TDDFT/B3LYP/6-311+ +G(2d,2p) level with the PCM model in CH3OH.

orbital at approximately 219 nm (∼39% contributions). Although the qualitative analysis of ECD results confirmed the R configuration at C-7 in 1, the absolute configurations at C-20 and C-21 were still unclear. For this purpose, the 13C NMR chemical shifts were calculated at the mPW1PW91/6311++G(2d,2p) level. The calculated 13C NMR values of C-7, C-20, and C-21 (78.7, 44.4, and 95.8 ppm) in (7R,20R,21S)-1 were close to the experimental values of 76.9, 42.5, and 98.5 ppm, respectively. For the (7R,20S,21R) diastereomer, the computed values were 79.8 (C-7), 50.1 (C-20), and 102.1 (C21) ppm. The corrected mean absolute errors of chemical shifts were less than 5.0 ppm and in good agreement with the D



Article

Figure 6. ORTEP drawing of 2. Figure 5. Comparison of the experimental VCD spectrum for (+)-1 with the predicted population-weighted spectrum for the [7R,20R(S),21S(R)] configuration at the B3LYP/6-311++G(2d,2p) level.

This observation was also supported by the ROESY correlations of H-16 (δH 5.05) with H2-19 (δH 0.58 and 0.54). Thus, the structure of melokhanine C (3) was elucidated as shown. The HREIMS data of alkaloid 4 suggested a molecular formula of C21H28N2O2, which indicated one more methylene group in 4 than 2. Analysis of the NMR spectroscopic data of 4 (Tables 1 and 3) indicated that it was homologue of 2, with the difference being the replacement of the C-16 methoxy group in 2 by an ethoxy group in 4. The deduction was supported by the HMBC correlations from H2-22 (δH 3.81 and 3.49) to C-16 (δC 84.5) and Me-23 (δC 14.5) and from H-16 (δH 5.47) to C-22 (δC 63.9). Detailed analysis of the NMR spectroscopic data showed that the remaining parts of 4 were the same as those of 2. Thus, the structure of 4, melokhanine D, was elucidated as shown. The molecular formula of melokhanine E (5), C19H22N2O2, was established by the HREIMS ion at m/z 310.1689 [M]+ (calcd 310.1681). The IR band observed at 1676 cm−1 and the 13 C NMR signal at δC 169.7 suggested the presence of a lactam carbonyl group. Comparison of the NMR spectroscopic data of 5 with those of 2 (Tables 1 and 3) showed that they were structural analogues, except for the absence of the methoxy group and the C-16 oxymethine group in 5. These were replaced by a lactam carbonyl group (δC 169.7) in the piperidine E-ring of 5, as supported by the MS data and the key HMBC correlations of H2-17 (δH 3.45 and 2.05) with C-16 (δC 169.7) and C-21 (δC 68.6). Alkaloid 6, obtained as a yellow oil, showed a protonated molecule at m/z 295 [M + H]+ in the ESIMS spectrum, while the HREIMS data indicated a molecular formula of C19H22N2O. The similar IR and UV spectra of 6 compared to 2−5 indicated the presence of similar functional groups. Its NMR spectra were similar to those of 2−5 except for the presence of two olefinic methines (δC 126.3 and 124.9) in 6. The two olefinic carbons were assigned to C-16 and C-17 via the correlations of H-16 (δH 6.61) with C-2 (δC 74.1), C-20 (δC 37.8), and C-17 (δC 124.9), in combination with correlations of H-17 (δH 5.36) with C-15 (δC 32.9) and C-21 (δC 75.3) in the HMBC spectrum. Hence, the structure 6 was established, and the compound named melokhanine F. Alkaloid 7 displayed a molecular ion at m/z 342.1946 in the HREIMS, indicating a molecular formula of C20H26N2O3, differing from 2 by the addition of 16 mass units. Its IR spectrum showed absorption of a hydroxy group (3440 cm−1). The NMR spectroscopic data (Tables 2 and 3) showed a close correlation with those of 2, except for the presence of an additional hydroxymethine (δC 71.6) in 7 instead of a

20 ethyl group. Evidence from 1H−1H COSY, HSQC, HMBC, and ROESY spectra and computations established the absolute configuration of compound 1, melokhanine A, as (7R,20R,21S). Melokhanine B (2) was isolated as pale yellow crystals. Its molecular formula was assigned as C20H26N2O2 on the basis of HREIMS (m/z 326.1985 [M]+), requiring nine indices of hydrogen deficiency. The UV absorptions at 238, 259, and 403 nm suggested a pseudoindoxyl chromophore,29 which was supported by a typical chemical shift at δC 203.5 for C-7 in the 13 C NMR spectrum. The four aromatic proton signals [δH 7.50 (d), 7.48 (t), 7.37 (t), and 6.84 (d)] as well as the HMBC correlation of H-9 with C-7 suggested an indolone fragment. The nitrogenated tertiary carbon resonance at δC 75.5 was readily assigned to C-2, on the basis of HMBC correlations of H2-6 (δH 2.12, 2.02) with C-7 (δC 203.5) and C-2 (δC 75.5) and of H2-5 (δH 3.16, 2.18) with C-2 (δC 203.5) and C-6 (δC 35.2). Additionally, the 13C and DEPT NMR spectra of 2 revealed resonances for one methoxy group (δC 55.6), a methyl group (δC 6.8), seven methylenes (δC 21.9, 33.9, 34.4, 34.8, 35.2, 51.4, and 53.6), two methines (δC 74.7 and 86.3), and one quaternary carbon (δC 35.7). The spectroscopic data of 2 were closely related to those of meloyunine B, a 6/7-seco rearranged spiro-indolinone alkaloid previously isolated from M. yunnanensis.5i Analysis of the 1H−1H COSY and HSQC data revealed that the −NCH2CHCH− moiety in meloyunine B was replaced by an −NCH2CH2CH2− fragment in 2 (Figure 1). This observation was further confirmed by the correlations of H2-15 (δH 1.41 and 1.08) with C-14 (δC 21.9), C-3 (δC 51.4), and C-21 (δC 74.7) in the HMBC spectrum and the MS analysis. The ROESY correlations of H-21 (δH 1.86) with Me-18 (δH 0.50) and H2-19 (δH 0.71) showed that these groups were cofacial, which suggested a cis D/E ring junction. Furthermore, the ROESY correlation of H-16 (δH 5.51) with H-6a (δH 2.12) suggested they were located on the opposite side. Crystals of 2 were obtained from CH3OH−H2O (9:1), which were analyzed by X-ray diffraction with Cu Kα radiation (Figure 6). Thus, the alkaloid 2 has an absolute configuration of (2R,16R,20R,21R). The molecular formula of melokhanine C (3), C20H26N2O2, was similar to that of compound 2, as indicated by HREIMS ([M]+ at m/z 326.1994). The similar UV and IR spectra of compounds 3 and 2 displayed characteristic features of an indole chromophore. Comparison of the NMR spectroscopic data of 3 and 2 indicated that they shared the same 2D structure, with the exception of the β-orientation of H-16 in 3. E



Article

The isolated compounds were tested for their antimicrobial activity against five bacterial strains (Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 10541, Escherichia coli ATCC 11775, Staphylococcus aureus ATCC 25922, and Klepsiella pneumoniae ATCC 13883) using the broth microdilution method.31 The alkaloids 1−16, 25−27, 31, and 32 showed the best antibacterial activity against P. aeruginosa (MIC range 2−22 μM), while compounds 2 (MIC = 5 μM), 5 (MIC = 5 μM), 11 (MIC = 2 μM), and 15 (MIC = 2 μM) showed the best activity against E. faecalis (Table 4). In

methylene in 2. The hydroxy group in 7 was placed at C-17, based on HMBC correlations from H-17 (δH 4.22) to C-15 (δC 32.0), C-20 (δC 36.1), and C-21 (δC 69.9). Biosynthetically, H219 and H-21 are β-oriented in eburnan alkaloids.20 The ROESY correlations of Me-18 (δH 0.57) with H-16 (δH 4.39), H-17 (δH 4.22), and H-21 (δH 2.40) placed H-16 and H-17 at the same side (β-orientation), which was further supported by the absence of cross-peaks between OMe-16 and H-17. Thus, the structure of melokhanine G was assigned as 7. The HREIMS data of melokhanine H (8) revealed a molecular formula of C19H24N2O3, indicating the loss of a methylene group compared to compound 7. Analysis of the spectroscopic data (Tables 2 and 3) of 8 suggested that it had a structure closely related to 7, except for replacement of MeO16 by HO-16 in 8. The HMBC correlations of H-17 (δH 3.96) to C-15 (δC 31.6), C-21 (δC 69.6), and C-16 (δC 79.6) also supported the observation. The ROESY correlations and the similar coupling constants suggested that alkaloid 8 had the same relative configuration as 7. Considering the co-occurrence of vincamine-type alkaloids 2−8, the same absolute configuration (20R,21R) was assigned to 3−8 as that of 2. The molecular formula of alkaloid 9 was established as C21H24N2O3 by HREIMS data (m/z 352.1785 [M]+, calcd 352.1787). The 13C NMR spectrum indicated an unsubstituted indole A-ring [δC 139.9 (s, C-2), 136.3 (s, C-13), 128.2 (s, C8), 122.3 (d, C-11), 120.0 (d, C-10), 118.5 (d, C-9), 112.3 (d, C-12), and 111.1 (s, C-7)] and signals for one methoxy (δC 55.6), six sp3 methylenes (δC 60.8, 51.4, 33.1, 30.4, 23.4, and 20.1), one sp2 methine (δC 136.0), two sp3 methines (δC 78.5 and 65.6), one carbonyl (δC 176.4), and two quaternary carbons (δC 129.6 and 50.3). These data were similar to those of andransinine,30 the absolute configuration of which was established by X-ray analysis. The difference between the two compounds involves replacement of the methyl ester and ethoxy groups in andransinine with a carboxylic and a methoxy group in 9. H-15 was assigned a β-equatorial orientation based on its coupling constant (J = 2.7 Hz) as well as the ROESY correlations of the hydrogens of the methoxy group with H-21 and H-17b. The observed ROESY correlations of H-21/H-3a, H-6a, and H-17b and of H-15/H-19 (Figure 2) and the negative sign of the specific rotation indicated that 9 possessed the same absolute configuration as andransinine.30 Thus, the structure of 9, melokhanine I, was defined as shown. Melokhanine J (10) possessed a molecular formula of C19H26N2O2, as determined by the HREIMS ion at m/z 314.2001 [M]+ (calcd 314.1994). The absorption maxima at 282 and 252 nm in the UV spectrum suggested an indole chromophore, while the IR spectrum showed bands for −OH (3426 cm−1) and lactam carbonyl (1684 cm−1) functionalities. The 13C NMR spectrum of alkaloid 10 displayed signals for 19 carbons, including one methyl, five methines, eight methylenes, one carbonyl, three quaternary carbons, and a secondary carbon carrying O- and N-functionalities. The resonances at δC 172.6 and 97.5 could be assigned to the C-2 lactam carbonyl and a hydroxy-substituted C-21. The structure of alkaloid 10 was closely related to leuconolam (11),9 except for the replacement of an α,β-unsaturated carbonyl moiety by three sp3 carbons in 10. This was supported by the HMBC correlations between H7 (δH 3.76) and C-6 (δC 33.3), C-5 (δC 52.2), and C-9 (δC 123.5). Thus, the structure of 10, melokhanine J, was defined as shown. On the basis of HRESIMS data (Table S6 and Figure S82, Supporting Information), melokhanines A−J (1−10) are natural products rather than artifacts of isolation.

Table 4. Antibacterial Activity (MIC, μM) of the Isolated Alkaloidsa

a

compound

P. aeruginosa ATCC 27853

E. faecalis ATCC 10541

E. coli ATCC 11775

S. aureus ATCC 25922

K. pneumoniae ATCC 13883

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 25 26 27 31 32 cefotaxime

19 5 19 4 2 2 18 19 4 5 5 2 19 2 5 22 2 2 4 4 2 0.4

75 5 150 147 5 42 73 75 71 39 2 19 19 >150 2 22 >150 66 >150 71 18 0.4

19 150 75 147 >150 >150 73 150 71 >150 >150 >150 76 >150 85 >150 >150 66 >150 142 >150 1.7

75 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150 19 >150 >150 >150 >150 >150 >150 >150 >150 0.4

150 >150 >150 >150 >150 >150 >150 150 >150 >150 >150 77 >150 >150 >150 >150 69 133 69 >150 >150 1.7

MIC > 150 μM is not active.

addition, alkaloid 1 was evaluated for its antifungal activity against Microsporum canis CBS113480, M. ferrugineum CBS457.80, M. gypseum CBS118893, Trichophyton mentagrophytes ATCC4439, T. ajelloi E1501, T. terrestre, and Epidermophyton f loccosum CBS566.94 (Table 5). Compound 1 exhibited inhibitory activities against most of the tested fungal strains, and the best activities were observed against M. canis, M. ferrugineum, and T. ajelloi with MIC values of 38, 75, and 150 μM, while the MIC values of griseofulvin (positive control) were 18, 35, and 71 μM, respectively. Table 5. Antifungal Activities of Compound 1 (MIC in μM)

F

fungus

compound 1

griseofulvin

M. canis CBS113480 M. ferrugineum CBS457.80 M. gypseum CBS118893 T. mentagrophytes ATCC4439 T. ajelloi E1501 T. terrestre E. f loccosum CBS566.94

38 75 150 150 150 >150 150

18 35 9 18 71 35 9



Article

16-epivincanol (17, 67 mg), and isositsirikine (32, 90 mg) were isolated from the subfraction III-5 via silica gel column chromatography (CHCl3−acetone, 15:1−5:1) and further separated on a preparative C18 HPLC column with CH3CN−H2O (2:3−3:2). Fr. IV (6.5 g) was chromatographed by MPLC with CH3OH−H2O (2:3− 4:1) to afford four subfractions. Subfraction IV-1 (230 mg) was purified on preparative HPLC (CH3OH−H2O, 5.5:4.5−7:3) to yield melokhanine G (7, 4 mg), melokhanine H (8, 3 mg), and rhazinal (12, 26 mg). 16-epi-14,15-Dehydrovincanol (20, 37 mg) and leuconicine B (28, 91 mg) were obtained from subfraction IV-2 (1.2 g) by preparative HPLC eluted with a gradient of CH3OH−H2O (3:2) and Sephadex LH-20. Fraction V (17.4 g) was subjected to silica gel CC, eluting with CHCl3−CH3OH (15:1−10:1), to give (+)-eburnamonine N(4)-oxide (24, 92 mg), melodinine F (23, 42 mg), and a mixture (9.2 g). Five subfractions (V-1−5) were obtained from the mixture by MPLC. Subfraction V-1 (3.7 g) was further separated by preparative HPLC, eluted with a gradient of CH3OH−H2O (3:7−4:1), purified by silica gel CC (CHCl3−CH3OH, 10:1), to afford melodinine A (25, 50 mg), melodinine B (26, 162 mg), melodinine C (27, 66 mg), and leuconicine E (29, 18 mg). Subfraction V-3 (4.2 g) was separated by MPLC (CH3OH−H2O, 1:4−7:3) to yield subfractions V-3-1−3. Melokhanine I (9, 16 mg) was crystallized (CH3OH−H2O, 4:1) from subfraction V-3-1. Melokhanine A (1): amorphous powder; [α]D21 +72 (c 0.2, CH3OH); ECD (c 0.3 mM, MeOH) λmax (Δε) 210 (+3.0), 241 (−1.8), 266 (+0.18); UV (CH3OH) λmax (log ε) 288 (4.41), 252 (3.77), 208 (3.17); IR (KBr) νmax 3431, 2935, 1719, 1622, 1471, 1381, 1331, 1187, 1118, 1023, 754 cm−1; VCD data, see Table S4, Supporting Information; 1H (600 MHz) and 13C NMR (150 MHz) data (acetone-d6), see Tables 1 and 3; HREIMS m/z 330.1946 [M]+ (calcd for C19H26N2O3, 330.1943). Melokhanine B (2): pale yellow crystals (CH3OH−H2O, 9:1); mp 154−156 °C; [α]24 D −10 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 403 (3.49), 259 (3.80), 238 (4.41), 194 (4.06); IR (KBr) νmax 2934, 2801, 2782, 1696, 1607, 1479, 1460, 1305, 1120, 1007, 760 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data (acetone-d6), see Tables 1 and 3; HREIMS m/z 326.1985 [M]+ (calcd for C20H26N2O2, 326.1994). Melokhanine C (3): amorphous powder; [α]24 D −207 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 397 (3.41), 256 (3.74), 236 (4.33), 195 (4.01); IR (KBr) νmax 2933, 2855, 2795, 1703, 1613, 1479, 1466, 1323, 1165, 1073, 969, 752 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (DMSO-d6), see Tables 1 and 3; HREIMS m/z 326.1994 [M]+ (calcd for C20H26N2O2, 326.1994). Melokhanine D (4): amorphous powder; [α]D24 −49 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 403 (3.67), 259 (4.01), 238 (4.59), 194 (4.25); IR (KBr) νmax 3433, 2936, 2853, 2782, 1700, 1609, 1479, 1460, 1302, 1263, 1116, 757 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data (acetone-d6), see Tables 1 and 3; HREIMS m/ z 340.2146 [M]+ (calcd for C21H28N2O2, 340.2151). Melokhanine E (5): amorphous powder; [α]24 D −317 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 363 (3.28), 349 (3.38), 318 (3.39), 256 (4.05), 235 (4.52), 201 (4.17); IR (KBr) νmax 3427, 2929, 2786, 1721, 1676, 1605, 1462, 1333, 1298, 1097, 765 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data (acetone-d6), see Tables 1 and 3; HREIMS m/z 310.1689 [M]+ (calcd for C19H22N2O2, 310.1681). Melokhanine F (6): colorless oil; [α]24 D −562 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 410 (3.47), 277 (3.98), 245 (4.39), 195 (4.19); IR (KBr) νmax 3440, 2939, 2924, 1706, 1631, 1607, 1465, 1321, 1260, 1184, 1152,1128, 763, 729 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Tables 2 and 3; HREIMS m/z 294.1728 [M]+ (calcd for C19H22N2O, 294.1732). Melokhanine G (7): colorless oil; [α]18 D −81 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 391 (3.11), 232 (4.16), 204 (4.17); IR (KBr) νmax 3450, 2928, 2858, 1708, 1638, 1550, 1464, 1384, 1276, 1122, 1043, 753 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Tables 2 and 3; HREIMS m/z 342.1946 [M]+ (calcd for C20H26N2O3, 342.1943). Melokhanine H (8): colorless oil; [α]23 D −9 (c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 378 (2.45), 362 (3.5), 232 (4.39), 203 (3.56);

The cytotoxicity of melokhanines A−J (1−10) was evaluated against five human cancer cell lines, MCF-7 (breast cancer), SMMC-7721 (hepatocellular carcinoma), HL-60 (myeloid leukemia), A-549 (lung cancer), and SW480 (colon cancer), using the reported MTT assay,32 with cisplatin as positive control. However, none of the alkaloids showed potential cytotoxic activity (IC50 > 40 μM).

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-4 micro melting points apparatus. Optical rotations were obtained on a Horiba SEPA-300 polarimeter. UV spectra were recorded with a Shimadzu UV-2401A spectrophotometer. ECD spectra were measured in MeOH on a JASCO 810 instrument. VCD data were acquired on a BioTools DualPEM ChiralIR-2X FTVCD spectrometer. A Bruker FT-IR Tensor 27 was used to record IR spectra with KBr pellets. NMR spectra were recorded on an AVANCE III-600 MHz spectrometer, a Bruker DRX-500 MHz spectrometer, or an AV-400 MHz spectrometer with tetramethylsilane as an internal standard. HREIMS data were obtained on an API QSTAR time-offlight spectrometer. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph (Agilent Technologies Co. Ltd., Palo Alto, CA, USA) coupled with Zorbax SB-C18 columns (9.4 × 150 and 21.2 × 250 mm). MPLC was carried out on a Büchi pump system coupled with C18 silica gel-packed glass columns (15 × 230 and 26 × 460 mm). Silica gel (200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Chromatores C18 (20−45 μm, Fuji Silysia Chemical Ltd., Tokyo, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden) were used for column chromatography. Plant Material. The leaves and twigs of Melodinus khasianus were collected in July 2010 in Pu’er city, Yunnan Province of China, and were identified by Mr. J. Y. Cui, Xishuangbanna Tropical Plant Garden. A voucher specimen (no. Cui20100720) has been deposited at Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. Extraction and Isolation. The powdered sample (9.5 kg) was extracted with 90% aqueous CH3OH (50 L) three times (2 days each time) at room temperature. The solvent was evaporated in vacuo, and the residue was partitioned between EtOAc and HCl (pH 2−3). The acidic aqueous solution was adjusted to basic pH with 5% ammonia, to give a total alkaloidal extract (70 g) after partitioning with EtOAc. The extract was passed over a silica gel column, eluting with CHCl3− CH3OH (1:0 to 0:1), to give seven fractions. Fraction I (3.4 g) was separated on a silica gel column (petroleum ether−acetone, 10:1−5:1) to yield three subfractions (I-1−3). Subfraction I-1 (390 mg) was separated by preparative HPLC with a gradient of CH3OH−H2O (3:2−4:1), then separated on semipreparative HPLC (CH3OH−H2O, 3:1) to give melokhanine B (2, 160 mg), melokhanine D (4, 16 mg), and melokhanine E (5, 20 mg). Fraction II (10.5 g) was chromatographed over a silica gel column with a gradient of petroleum ether−acetone (from 8:1 to 2:1), then by MPLC, eluted with CH3OH−H2O (2:3−1:0), to afford melokhanine J (10, 5 mg), (+)-eburnamonine (15, 230 mg), eburnamenine (16, 66 mg), 14,15dehydrovincamine (21, 305 mg), decarbomethoxydihydrogambirtannine (30, 96 mg), and a mixture (4.1 g). The mixture was separated on a silica gel column (petroleum ether−EtOAc, 6:1−2:1) and preparative HPLC with a gradient of CH3OH−H2O (3:2−4:1) to yield melokhanine C (3, 37 mg), melokhanine F (6, 180 mg), leuconolam (11, 14 mg), leuconodine C (13, 20 mg), and 14,15dehydroepivincamine (22, 32 mg). Fraction III (17 g) was decolorized on an MCI gel column (CH3OH−H2O, 9:1) and separated on MPLC with CH3OH−H2O (2:3−4:1) to obtain seven subfractions. The third subfraction (278 mg) was separated on a silica gel column chromatography (petroleum ether−acetone, 5:1−2:1) to yield melokhanine A (1, 16 mg) and ajmalicine (31, 36 mg). Subfraction III-4 (1.4 g) was separated by MPLC with CH3OH−H2O (from 2:3 to 1:0), purified by silica gel column chromatography (CHCl3−Me2CO, 15:1−5:1), to afford O-methylvincanol (18, 42 mg) and 14,15dehydrovincanol (19, 26 mg). Leuconodine E (14, 11 mg), O-methylG



Article

IR (KBr) νmax 3440, 2939, 2924, 1706, 1631, 1607, 1465, 1321, 1260, 1184, 1152, 1128, 763, 729 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Tables 2 and 3; HREIMS m/z 328.1786 [M]+ (calcd for C19H24N2O3, 328.1787). Melokhanine I (9): colorless crystals, (CH3OH); mp 162−166 °C; [α]26 D −80 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 283 (3.93), 222 (4.47), 203 (4.34); IR (KBr) νmax 3429, 3255, 2929, 1611, 1463, 1356, 1324, 1261, 1088, 1071, 937, 743 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4 and DMSO-d6), see Tables 2 and 3; HREIMS m/z 352.1785 [M]+ (calcd for C21H24N2O3, 352.1787). Melokhanine J (10): amorphous powder; [α]18 D −47 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 282 (3.52), 252 (4.01), 204 (4.40); IR (KBr) νmax 3426, 2945, 1684, 1631, 1482, 1462, 1398, 1347, 1122, 1099, 750 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CDCl3), see Tables 2 and 3; HREIMS m/z 314.2001 [M]+ (calcd for C19H26N2O2, 314.1994). X-ray Crystallographic Analysis of 2. The intensity data for melokhanine B (2) were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. Structure was resolved by direct methods, expanded by Fourier techniques, and refined by the program and full-matrix leastsquares calculations. All non-hydrogen atoms have been refined anisotropically, and all hydrogen atoms were fixed at calculated positions. Crystallographic data for melokhanine B (2) have been placed in the Cambridge Crystallographic Data Centre as supplementary publications (number: CCDC 1436267). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, U.K.; fax: (+44) 1223-336-033; or [email protected]). Crystallographic data for melokhanine B (2): C20H26N2O2, M = 326.43, orthorhombic, a = 8.9399(2) Å, b = 12.3247(2) Å, c = 15.5926(3) Å, V = 1718.02(6) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.645 mm−1, 16 431 reflections measured, 3124 independent reflections (Rint = 0.0340). The final R1 values were 0.0274 (I > 2σ(I)). The final wR(F2) values were 0.0697 (I > 2σ(I)). The final R1 values were 0.0274 (all data). The final wR(F2) values were 0.0698 (all data). The goodness of fit on F2 was 1.076. Flack parameter = 0.10(17).33 The Hooft parameter is 0.10(4) for 1298 Bijvoet pairs.34 Computational Details. The ECD spectrum of 1 recorded in CH3OH shows two positive Cotton effects at 210 nm (Δε = +3.0) and 266 nm (Δε = +0.18) and a negative CE at 241 nm (Δε = −1.8) (ECD data, Table S3, Supporting Information). Discovery Studio 3.5 was used to study the features of the conformational distributions. The molecular geometries were fully optimized without imposing any symmetry constraints. The energies and geometries of the stationary points on the potential energy surface were calculated using the DFT (B3LYP) method together with 6-31+G(d) and 6-311++G(2d,2p) basis sets. The averages were obtained by Boltzmann distribution, using the relative standard free energies as weighting factors at 298.15 K. The ECD calculations were performed using TDDFT at the B3LYP/6-311++G(2d,2p) level in CH3OH using the PCM model, the COSMO model, and the gas phase. The ECD Cotton effect was approximated by a Gaussian distribution, and the fitting curve is formulated as35 ⎡

Δε(σ ) = {σ0/(2.296 × 10−39 π Δσ )} × R exp⎣⎢

⎤ { σ−σ Δσ }⎦⎥

−

0

basis set (acetone, ε = 20.7), and NMR values were averaged on the basis of Boltzmann populations. The molecular orbital information (NBO plot files) was calculated using the Gaussian 09 software package.36 The optimized conformer was selected for MO analysis. NBO plot files were used to generate the corresponding cube file by Multiwfn 3.1.37 Lastly, the isosurfaces of the MOs were visualized using VMD software.38 Antimicrobial Assay. The isolated compounds were screened for their antimicrobial activity against five bacterial strains (Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 10541, Escherichia coli ATCC 11775, Staphylococcus aureus ATCC 25922, and Klepsiella pneumoniae ATCC 13883). In addition, compound 1 was evaluated for antifungal activity against the following fungal strains: Microsporum canis CBS113480, M. ferrugineum CBS457.80, M. gypseum CBS118893, Trichophyton mentagrophytes ATCC4439, T. ajelloi E1501, T. terrestre, and Epidermophyton f loccosum CBS566.94. The strains were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), Centraalbureau voor Schimmelcultures (CBS, Holland), Ecole Nationale Vétérinaire d’Alfort (France), and Centre Pasteur of Yaounde (Cameroon). The MIC values were determined by the broth microdilution method as described.31 The tests were conducted for three independent replicates. Cefotaxime and griseofulvin were used as positive controls in the antibacterial and antifungal assays,39 respectively. Cytotoxicity Assay. The cytotoxicity of the new alkaloids against the MCF-7, SMMC-7721, HL-60, A-549, and SW480 cell lines was assessed using the MTT method in 96-well microplates.32 Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h, with cisplatin (Sigma, USA) as a positive control. IC50 values of the compounds were calculated following Reed and Muench’s method.40

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00011. 1D and 2D NMR, IR, UV, HREIMS, and UHPLCESIMS analysis of 1−10; computational conformation analysis and DFT-calculated spectroscopic data (13C NMR, ECD, VCD, and specific rotations) of 1 (PDF) X-ray crystallographic data for 2 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-871-65223177. Fax: +86-871-65223188. E-mail: [email protected] (X.-D. Luo). *E-mail: [email protected] (Z.-L. Zuo). Author Contributions #

G.-G. Cheng, D. Li, and B. Hou have contributed equally to this work. Notes

The authors declare no competing financial interest.

■

2

ACKNOWLEDGMENTS This project was financially supported by the National Natural Science Foundation of China (81225024). We also thank the National Supercomputing Center for providing computational resources.

VCD calculation of each optimized geometry was carried out at the B3LYP/6-311++G(2d,2p)//B3LYP/6-31+G(d) level considering Lorentzian with half-width 8 cm−1. The population-weighted VCD spectra were generated from the four optimized conformers in Gibbs free energies. The 6-311G++(2d,2p) basis set was adopted for accurate calculations of the electric dipole−electric dipole polarizability. NMR shielding constants were calculated by the GIAO method with the SCRF-mPW1PW91/6-311++G(2d,2p)//B3LYP/6-311++G(2d,2p)

■

REFERENCES

(1) (a) Li, P. T.; Leeuwenberg, A. J. M.; Middleton, D. J. Flora of China; Science Press: Beijing, 1977; Vol. 16, pp 147−150. (b) He, Y. L.; Chen, W. M.; Feng, X. Z. J. Nat. Prod. 1994, 57, 411−414.

H



Article

(2) (a) He, X.; Zhou, Y.; Huang, Z. Acta Chim. Sin. 1992, 50, 96− 101. (b) Yan, K. X.; Feng, X. Z. Chin. Tradit. Herb. Drugs 1998, 29, 793−795. (3) Guo, L. W.; Zhou, Y. L. Phytochemistry 1993, 34, 563−566. (4) (a) Au, K. S.; Gray, D. E. Biochem. Pharmacol. 1969, 18, 2673. (b) Lu, Y.; Knoo, T. J.; Wiart, C. Pharmacol. Pharm. 2014, 5, 773−780. (5) (a) Feng, T.; Cai, X. H.; Li, Y.; Wang, Y. Y.; Liu, Y. P.; Xie, M. J.; Luo, X. D. Org. Lett. 2009, 11, 4834−4837. (b) Feng, T.; Li, Y.; Liu, Y. P.; Cai, X. H.; Wang, Y. Y.; Luo, X. D. Org. Lett. 2010, 12, 968−971. (c) Feng, T.; Cai, X. H.; Liu, Y. P.; Li, Y.; Wang, Y. Y.; Luo, X. D. J. Nat. Prod. 2010, 73, 22−26. (d) Feng, T.; Li, Y.; Wang, Y. Y.; Cai, X. H.; Liu, Y. P.; Luo, X. D. J. Nat. Prod. 2010, 73, 1075−1079. (e) Liu, Y. P.; Li, Y.; Cai, X. H.; Li, X. Y.; Kong, L. M.; Cheng, G. G.; Luo, X. D. J. Nat. Prod. 2012, 75, 220−224. (f) Liu, Y. P.; Zhao, Y. L.; Feng, T.; Cheng, G. G.; Zhang, B. H.; Li, Y.; Cai, X. H.; Luo, X. D. J. Nat. Prod. 2013, 76, 2322−2329. (g) Cai, X. H.; Li, Y.; Liu, Y. P.; Li, X. N.; Bao, M. F.; Luo, X. D. Phytochemistry 2012, 83, 116−124. (h) Li, J. L.; Lunga, P. K.; Zhao, Y. L.; Qin, X. J.; Yang, X. W.; Liu, Y. P.; Luo, X. D. Chin. J. Nat. Med. 2015, 13, 307−310. (i) Cai, X. H.; Jiang, H.; Li, Y.; Cheng, G. G.; Liu, Y. P.; Feng, T.; Luo, X. D. Chin. J. Nat. Med. 2011, 9, 259−263. (j) Cai, X. H.; Li, Y.; Su, J.; Liu, Y. P.; Li, X. N.; Luo, X. D. Nat. Prod. Bioprospect. 2011, 1, 25−28. (k) Wang, D. W.; Luo, X. D.; Jiang, B. China Tradit. Herb Drugs 2012, 43, 653−657. (6) Zhao, S.; Sirasani, G.; Vaddypally, S.; Zdilla, M. J.; Andrade, R. B. Angew. Chem., Int. Ed. 2013, 52, 8309−8311. (7) Lancefield, C. S.; Zhou, L.; Lébl, T.; Slawin, A. M. Z.; Westwood, N. J. Org. Lett. 2012, 14, 6166−6169. (8) Umehara, A.; Ueda, H.; Tokuyama, H. Org. Lett. 2014, 16, 2526− 2529. (9) Goh, S. H.; Ali, A. R. M.; Wong, W. H. Tetrahedron 1989, 45, 7899−7920. (10) Kam, T. S.; Tee, Y. M.; Subramaniam, G. Nat. Prod. Lett. 1998, 12, 307−310. (11) Gan, C. Y.; Low, Y. Y.; Thomas, N. F.; Kam, T. S. J. Nat. Prod. 2013, 76, 957−964. (12) Kam, T. S.; Tan, P. S.; Chuah, C. H. Phytochemistry 1992, 31, 2936−2938. (13) Bartlett, M. F.; Taylor, W. I. J. Am. Chem. Soc. 1960, 82, 5941− 5946. (14) Pfäffli, P.; Hauth, H. Helv. Chim. Acta 1978, 61, 1682−1695. (15) Baassou, S.; Mehri, H.; Plat, M. Ann. Pharm. Fr. 1987, 45, 49− 56. (16) Pegnyemb, D. E.; Ghogomu, R. T.; Sondengam, B. L. Fitoterapia 1999, 70, 446−448. (17) Batchily, F.; Baassou, S.; Mehri, H.; Plat, M.; Sevenet, T.; Pusset, J. Ann. Pharm. Fr. 1985, 43, 359−364. (18) Zhang, Y. W.; Yang, R.; Cheng, Q.; Ofuji, K. Helv. Chim. Acta 2003, 86, 415−419. (19) Gan, C. Y.; Low, Y. Y.; Etoh, T.; Hayashi, M.; Komiyama, K.; Kam, T. S. J. Nat. Prod. 2009, 72, 2098−2103. (20) Peube-Locou, N.; Plat, M.; Koch, M. Phytochemistry 1973, 12, 199−200. (21) Melchio, J.; Bouquet, A.; Pais, M.; Goutarel, R. Tetrahedron Lett. 1977, 18, 315−316. (22) Kutney, J. P.; Brown, R. T. Tetrahedron 1966, 22, 321−336. (23) Pescitelli, G.; Bari, L. D.; Berova, N. Chem. Soc. Rev. 2011, 40, 4603−4625. (24) (a) Lodewyk, M. W.; Tantillo, D. J. J. Nat. Prod. 2011, 74, 1339−1343. (b) Barone, G.; Duca, D.; Silvestri, A.; Gomez-Paloma, L.; Riccio, R.; Bifulco, G. Chem. - Eur. J. 2002, 8, 3240−3245. (c) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J. Chem. Rev. 2011, 112, 1839−1862. (d) Qiu, S.; De Gussem, E.; Abbaspour Tehrani, K.; Sergeyev, S.; Bultinck, P.; Herrebout, W. J. Med. Chem. 2013, 56, 8903−8914. (25) (a) Devlin, F. J.; Stephens, P. J.; Cheeseman, J. R.; Frisch, M. J. J. Am. Chem. Soc. 1996, 118, 6327−6328. (b) Freedman, T. B.; Cao, X.; Dukor, R. K.; Nafie, L. A. Chirality 2003, 15, 743−758. (26) (a) Rosenfeld, L. Z. Eur. Phys. J. A 1928, 52, 161−174. (b) Condon, E. U. Rev. Mod. Phys. 1937, 9, 432−457. (c) Stephens, P.

J.; Devlin, F. J.; Cheeseman, J. R.; Frisch, M. J.; Mennucci, B.; Tomasi, J. Tetrahedron: Asymmetry 2000, 11, 2443−2448. (27) (a) Barone, G.; Gomez-Paloma, L.; Duca, D.; Silvestri, A.; Riccio, R.; Bifulco, G. Chem. - Eur. J. 2002, 8, 3233−3239. (b) Li, X. N.; Zhang, Y.; Cai, X. H.; Feng, T.; Liu, Y. P.; Li, Y.; Ren, J.; Zhu, H. J.; Luo, X. D. Org. Lett. 2011, 13, 5896−5899. (28) (a) Krautmann, M.; de Riscala, E. C.; Burgueño-Tapia, E.; Mora-Pérez, Y.; Catalán, C. A. N.; Joseph-Nathan, P. J. Nat. Prod. 2007, 70, 1173−1179. (b) Mazzeo, G.; Santoro, E.; Andolfi, A.; Cimmino, A.; Troselj, P.; Petrovic, A. G.; Superchi, S.; Evidente, A.; Berova, N. J. Nat. Prod. 2013, 76, 588−599. (29) (a) Lim, K. H.; Raja, V. J.; Bradshaw, T. D.; Lim, S. H.; Low, Y. Y.; Kam, T. S. J. Nat. Prod. 2015, 78, 1129−1138. (b) Gan, C. Y.; Yoganathan, K.; Sim, K. S.; Low, Y. Y.; Lim, S. H.; Kam, T. S. Phytochemistry 2014, 108, 234−242. (30) Low, Y. Y.; Gan, C. Y.; Kam, T. S. J. Nat. Prod. 2014, 77, 1532− 1535. (31) (a) Zhang, H. X.; Lunga, P. K.; Li, Z. J.; Dai, Q.; Du, Z. Z. Fitoterapia 2014, 95, 147−153. (b) Pan, Y. M.; Zhang, Y.; Wang, X. N.; Chen, H. P.; Li, S. L.; Di, Y. T.; Chen, D. Z.; Guo, L. L.; Hao, X. J.; He, H. P. Nat. Prod. Bioprospect. 2015, 1, 55−59. (32) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (33) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, A39, 876−881. (34) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr. 2008, 41, 96−103. (35) (a) Stephens, P. J.; Harada, N. Chirality 2010, 22, 229−233. (b) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis version 1.53; University of Würzburg: Würzburg, Germany, 2012. (36) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision C.01; Gaussian, Inc.: Wallingford, CT, 2010. (37) Lu, T.; Chen, F. J. Comput. Chem. 2012, 33, 580−592. (38) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33−38. (39) Tereschuk, M. L.; Riera, M. V. Q.; Castro, G. R.; Abdala, L. R. J. Ethnopharmacol. 1997, 56, 227−232. (40) Reed, L.; M, H. Am. J. Hygiene 1938, 27, 493−497.

I


Melokhanines AâJ, Bioactive Monoterpenoid Indole Alkaloids with

Recommend Documents

Melokhanines AâJ, Bioactive Monoterpenoid Indole Alkaloids with