New iboga-type alkaloids from Ervatamia hainanensis

Abstract

Seven new iboga-type alkaloids (1–7) and six known ones (8–13) were obtained from Ervatamia hainanensis. Their structures with absolute configurations were determined by spectroscopic data, Mosher's method, single crystal X-ray diffraction and electric circular dichroism (ECD) analyses. The relationship between the absolute configuration of C-7 in iboga-type 7-hydroxyindolenine alkaloids and the Cotton effects in the ECD spectrum was established for the first time. The neural activities of these compounds were evaluated, and compound 11 exhibited protective effects against MPP⁺ (1-methyl-4-phenylpyridinium)-induced damage in primary cortical neurons.

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Introduction

The plant Ervatamia hainanensis Tsiang, mainly distributed in the Guangdong and Hainan provinces of China, is used as a folk medicine for the treatment of snakebites, stomach ache, rheumatoid arthritis, and hepatitis.¹ The genus Ervatamia, belonging to the Apocynaceae family, is a rich source of structurally complex and biologically active monoterpenoid indole alkaloids (MIAs).^2–4 Some of these alkaloids had been reported to show promising AChE inhibitory,⁵ Aβ aggregation inhibitory,⁶ anti-addiction and antitumor activities.^7,8 The development of natural products as drug candidates to prevent neuronal death is considered a feasible strategy to address the multiple pathological aspects of neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD).^9–12 Hence, it is worth exploring whether some MIAs possess remarkable neuroprotective or neuritogenic activities. Our group had reported the isolation of a series of new MIAs from several herbal medicines in recent years.^13–16 Our previous study on E. hainanensis had reported the first example of a cyano-substituted oxindole alkaloid.¹⁵ In a continuing investigation, seven new iboga-type alkaloids (1–7) and six known ones (8–13) were isolated from E. hainanensis. The absolute configurations of the new compounds were determined by comprehensively using Mosher's method, single crystal X-ray diffraction and electronic circular dichroism (ECD) analyses. The relationship between the stereochemistry of C-7 in iboga-type 7-hydroxyindolenine alkaloids and the Cotton effects in the associated ECD spectrum is established. The isolated alkaloids were screened for neuritogenic activities^17–19 and neuroprotective effects in neuronal cell lines and cultured cortical neurons (Fig. 1).

Fig. 1 Chemical structures of 1–13.

Results and discussion

3-Oxo-7R-coronaridine hydroxyindolenine (1) was obtained as light yellow blocks (CHCl₃/MeOH), possessed a molecular formula of C₂₁H₂₄N₂O₄ as established by its HRESIMS (m/z 369.1808 [M + H]⁺, calcd for C₂₁H₂₅N₂O₄: 369.1809). The IR spectrum revealed the characteristic absorption bands for hydroxyl (3221 cm⁻¹), carbonyl (1741 cm⁻¹) and the ortho-disubstituted benzene ring (1566, 1471, 1435, 755 cm⁻¹). The ¹H NMR spectrum of 1 displayed the characteristic signals for a benzene ring [δ_H 7.37 (1H, overlapped), 7.35 (1H, overlapped), 7.30 (1H, td, J = 7.5, 1.1 Hz), 7.23 (1H, td, J = 7.5, 1.1 Hz)], a hydroxyl [δ_H 5.91 (1H, br s)], a bridgehead proton adjacent to a nitrogen [δ_H 4.01 (1H, br s)], a methoxyl [δ_H 3.74 (3H, s)] and an ethyl side chain [δ_H 1.34 (1H, m), 1.18 (1H, m), 0.86 (3H, t, J = 7.4 Hz)]. The ¹³C NMR and DEPT-135 spectra of 1 exhibited twenty-one carbon signals including two carbonyls, seven olefinic carbons, two methyl, five methylene, three methine and two quaternary carbons. The ¹³C NMR data of 1 resembled those of 3-oxo-7S-coronaridine hydroxyindolenine (9),²⁰ except for upfield shift for two methylene (C-5, C-6) and two methine (C-17, C-21), suggesting that 1 possess a hydroxyindolenine iboga-type structure. With the aid of ¹H–¹H COSY, HSQC and HMBC experiments, the ¹H and ¹³C NMR signals of 1 were assigned as shown in Table 1. Furthermore, the presence of three spin systems (Fig. 2) and HMBC correlations between H-21 and C-2/C-3/C-5/C-22, between H-6β and C-2, as well as between H-9 and C-7, confirming the planar structure of 1 which was identical to that of 9. In the NOESY spectrum, correlations between H-15α and H-17α, between H-21 and H-5α/H-6α indicated these protons were the same α-orientation. In particular, the β-orientation of 7-OH was deduced by the NOESY correlations between 7-OH and H-5β/H-6 (Fig. 3). Finally, the absolute configuration of 1 was established by a single crystal X-ray diffraction experiment using Cu Kα radiation. The small Flack parameter of 0.04(13), allowed an unambiguous assignment of the absolute configuration of 1 as 7R,14R,16S,20S,21S (Fig. 4). The absolute configurations of iboga-type 7-hydroxyindolenine alkaloids were reported for the first time.

Table 1 ¹H and ¹³C NMR data of compounds 1–3, 6 and 7 (δ in ppm, J in Hz)^a

Position	1^b			2^b			3^b			6^c			7^c
		δH	δC		δH	δC		δH	δC		δH	δC		δH	δC
a Overlapped resonances are reported without designating multiplicity.b Measured in DMSO-d6.c Measured in CD3OD.
2			184.8			189.4			190.5			142.8			143.3
3			173.6		3.99 br s	50.3		4.28 m	80.3	a	3.58 dt (12.0, 3.0)	49.5	a	3.58 dt (11.5, 2.8)	49.6
										b	3.08 d (12.0)		b	3.09 d (11.5)
5	α	2.94 dd (12.4, 6.7)	39.1	α	3.43 t (13.5)	47.4	α	3.22 t (13.0)	45.4			177.8			176.1
	β	4.59 td (12.4, 6.7)		β	2.97 d (13.5)		β	3.16 m
6	α	1.89 d (13.0)	33.1	α	1.93 d (14.5)	33.2	α	1.90	34.6		5.54 s	71.0		5.14 s	80.3
	β	2.27		β	1.78 t (14.5)		β	1.56 d (13.5)
7			85.8			86.6			86.8			107.6			105.1
8			142.1			142.6			142.8			128.8			129.4
9		7.37	122.2		7.37 d (7.4)	121.9		7.34 d (7.4)	121.8		7.58 d (7.8)	118.5		7.59 d (7.6)	118.5
10		7.23 td (7.5, 1.1)	126.7		7.32 t (7.4)	128.9		7.21 t (7.4)	126.1		7.07 dt (7.8, 1.0)	120.9		7.08 t (7.6)	121.2
11		7.30 td (7.5, 1.1)	129.1		7.24 t (7.4)	126.4		7.29 t (7.4)	128.7		7.10 dt (7.8, 1.0)	123.0		7.11 t (7.6)	123.2
12		7.35	120.2		7.40 d (7.4)	120.2		7.36 d (7.4)	120.0		7.28 d (7.8)	112.2		7.29 d (7.6)	112.3
13			150.8			151.4			151.4			137.1			137.1
14		2.44 m	38.1		2.11 m	30.3		1.66 m	33.6		2.17 br s	29.9		2.19 m	29.9
15	α	1.81 t (10.8)	29.3	α	1.71 t (11.0)	27.9	α	1.38	23.8	α	1.92 dt (12.8, 2.0)	30.7	α	1.93 m	30.6
	β	1.23 m		β	1.24		β	1.35 m		β	1.33 dt (12.8, 2.0)		β	1.34 m
16			58.0			56.6			56.8			52.0			52.0
17	α	2.55 d (12.7)	29.9	α	2.82 d (12.5)	35.5	α	2.81 dd (14.5, 2.0)	35.8	α	3.03 ddd (14.0, 3.8, 2.0)	34.9	α	1.55 m	34.8
	β	2.25		β	2.09 m		β	1.92 dd (14.5, 2.0)		β	1.53 m		β	3.03 dt (14.0, 2.0)
18		0.86 t (7.4)	11.2		0.85 t (7.3)	11.5		0.81 t (7.3)	11.7		1.03 t (7.4)	12.5		1.01 t (7.4)	12.3
19	a	1.34 m	27.0	a	1.47 m	26.7	a	1.50 m	26.3	a	1.59 m	29.8	a	1.58 m	29.7
	b	1.18 m		b	1.40 m		b	1.40 m		b	1.49 m		b	1.49 m
20		1.57 m	35.8		1.26	36.8		1.14 m	36.7		1.75 m	36.7		1.72 m	36.6
21		4.01 br s	55.7		4.02 br s	54.1		4.04 br s	55.9		5.83 d (2.8)	51.7		5.60 d (2.7)	51.9
22			172.6			171.8			172.3			174.7			174.4
23		3.74 s	52.4		3.57 s	52.7		3.54 s	52.4		3.69 s	53.2		3.68 s	53.3
24						120.6								3.51 s	57.5
7-OH		5.91 s			6.02 s			5.87 s

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Fig. 2 Key ¹H–¹H COSY and HMBC correlations of 1 and 2.

Fig. 3 Key NOESY correlations of 1 and 2.

Fig. 4 X-ray crystal structures of 1 and 9.

3S-Cyano-7S-coronaridine hydroxyindolenine (2) was isolated as yellow power. Its molecular formula was assigned as C₂₂H₂₅N₃O₃ by its HRESIMS (m/z 380.1973 [M + H]⁺, calcd for C₂₂H₂₆N₂O₃: 380.1969). The IR spectrum of 2 showed absorption bands corresponding to hydroxyl (3445 cm⁻¹), cyano (2262 cm⁻¹), carbonyl (1736 cm⁻¹) and aromatic ring (1557, 1458 cm⁻¹). The NMR spectra of 2 exhibited similarity to that of 7S-coronaridine hydroxyindolenine (8).²¹ The main difference was the appearance of resonances for a methine [δ_H 3.99 (1H, br s), δ_C 50.3] and a cyano carbon (δ_C 120.6) as well as the absence of a methylene, indicating that 2 was a cyano substituted derivative of 8. In addition, the HMBC correlations between H-3/H-14 and C-24 further confirmed the cyano group was attached to C-3 position (Fig. 2). The 3S* and 7S* configurations of 2 were revealed by NOE correlations between H-3 and H-6β/H-17β, and between 7-OH and H-6α/H-21 (Fig. 3). Owing to the absolute stereochemistry of 9 proved by X-ray diffraction experiment (Fig. 4), as well as the ECD curves of 2 showing the same Cotton effects as those of 9 (Fig. 5), the absolute configuration of 2 could be identified as 3S,7S,14R,16S,20S,21S (Fig. 6).

Fig. 5 CD spectra of 2–3 and 8–9.

Fig. 6 Key ¹H–¹H COSY, HMBC correlations and NOESY correlations of 4.

3R-Hydroxy-7S-coronaridine hydroxyindolenine (3) was assigned the molecular formula C₂₁H₂₆N₂O₄ by its HRESIMS (m/z 371.1973 [M + H]⁺, calcd for C₂₁H₂₇N₂O₄: 371.1965), and it was suggested to be a hydroxylated derivative of 8 according to analysis of the NMR data and comparison with those of 8. The HMBC cross-peaks between H-21 and C-2/C-3/C-5/C-17 revealed that the hydroxyl group was located at C-3. In addition, the NOE correlations between H-3 and H-6β/H-17β, and between 7-OH and H-6α/H-21 indicated S* configuration at C-3. The absolute configuration of 3 was also defined based on a good agreement of ECD spectra by comparing with those of 8 (Fig. 5).

3S-(24S-Hydroxyethyl)-coronaridine (4), colorless crystals, displayed C₂₃H₃₀N₂O₃ as the molecular formula based on HRESIMS (m/z 383.2328 [M + H]⁺, calcd for C₂₃H₃₁N₂O₃: 383.2329). Comparison of the ¹³C NMR data of 4 with those of coronaridine (11)²² showed considerable similarity, except for the additional resonances for two methines (δ_C 71.8, 67.9) and a methyl (δ_C 21.8), as well as the absent resonance for a methylene in 4, suggesting that 4 was a derivative of 11 with a hydroxyethyl group. The ¹H–¹H COSY correlations between H-24 and H-25/H-3 (Fig. 7) as well as the HMBC correlations between H-21/H-25 and C-3 demonstrated that the hydroxyethyl group was located at C-3 (Fig. 7). In the NOESY spectrum, correlations between H-21 and H-6α, and between H-3 and H-6β/H-17β suggested the presence of 3S* configuration. Then, the Mosher's method²³ was used to further support the absolute configuration of 4. It was treated with (R)- and (S)-MTPA-Cl, and interpretation of the ¹H NMR chemical shift differences (Δδ = δ_S − δ_R) between (S)- and (R)-MTPA ester (4a and 4b) established S configuration at C-24 (Fig. 8). Fortunately, suitable crystals for single-crystal X-ray diffraction experiment on the Cu Kα radiation were obtained. The final refinement data resulted in a small Flack parameter of 0.0(4), allowing the unambiguous assignment of the absolute configuration of 4 as 3S,14R,16S,20S,21S,24S.

Fig. 7 X-ray crystal structures of 4 and 6.

Fig. 8 Δδ values (Δδ = δ_S − δ_R) for the MTPA esters of 4 and 5.

3S-(24R-Hydroxyethyl)-coronaridine (5) showed the same molecular formula as 4, C₂₃H₃₀N₂O₃, established by its HRESIMS (m/z 383.2328 [M + H]⁺, calcd for C₂₃H₃₁N₂O₃: 383.2329). The ¹H and ¹³C NMR spectra of 5 were very similar to those of 4, except for upfield shift at C-14, C-21 and C-24, and lowfield shift at C-3. Moreover, the ¹H–¹H COSY, HSQC, HMBC and NOESY analyses further defined that 5 and 4 were a pair of epimers with a different configuration at C-24. The ECD spectrum of 5 exhibited similar Cotton effects to those of 4 and 11 (Fig. 9), indicating the presence of 3S,14R,16S,20S,21S configuration in 4.^24,25 The 24R absolute configuration was also determined by the Mosher's method²³ (Fig. 8). It is worth mentioning that the planar structure of 4 and 5 was previously reported, however, its relative configuration was not defined.²⁶

Fig. 9 CD spectra of 4, 5 and 11.

5-Oxo-6S-hydroxycoronaridine (6) was crystallized as colorless blocks from aqueous methanol, and possessed a molecular formula of C₂₁H₂₄N₂O₄ as deduced by its HRESIMS (m/z 369.1811 [M + H]⁺, calcd for C₂₁H₂₅N₂O₄: 369.1809). The ¹H NMR spectrum of 6 showed signals for an ortho-disubstituted benzene ring [δ_H 7.58 (1H, d, J = 7.8 Hz), 7.28 (1H, d, J = 7.8 Hz), 7.10 (1H, td, J = 7.8, 1.0 Hz), 7.07 (1H, td, J = 7.8, 1.0 Hz)], a bridgehead proton adjacent a nitrogen [δ_H 5.83 (1H, d, J = 2.8 Hz)], a methoxyl [δ_H 3.69 (3H, s)], as well as an ethyl [δ_H 1.59 (1H, m), 1.49 (1H, m), 1.03 (3H, t, J = 7.4 Hz)]. The ¹³C NMR spectrum of 6 closely matched those of 5-oxocoronaridine (10),^20,27 except that an oxygenated methine signal (δ_C 71.0) replaced the methylene signal (δ_C 32.5) in 10, indicating 6 was a hydroxyl substitute of 10. The assignment of the hydroxyl group at C-6 was deduced by the HMBC correlations between H-6 and C-2/C-8. The NOE correlations between H-3a and H-6/H-17β suggested that H-6 was a β-orientation. Furthermore, X-ray diffraction analysis using Cu Kα radiation confirmed the presence of 6S,14R,16S,20S,21S configurations in 6 with a small Flack parameter of 0.01(6) (Fig. 7).

5-Oxo-6S-methoxycoronaridine (7) was established to be a methylated derivative of 5-oxo-6S-hydroxycoronaridine (6) based on the HRESIMS (m/z 383.1966 [M + H]⁺, calcd for C₂₂H₂₇N₂O₄: 383.1965). The HMBC correlations between H-6 and C-2/C-8, and between H-24 and C-6 indicated that the methoxyl group (C-24) was linked to C-6 position. Comparing the CD data of 7 with that of 6, the same Cotton effects occurred at 280, 250, 230 nm indicated that they possessed the same absolute configuration (Fig. 10).

Fig. 10 CD spectra of compounds 6 and 7.

Six known alkaloids were respectively identified as 7S-coronaridine hydroxyindolenine (8),²¹ 3-oxo-7S-coronaridine hydroxyl indolenine (9),²⁰ 5-oxocoronaridine (10),^20,27 coronaridine (11),²² 3-oxocoronaridine (12)²⁷ and pseudoindoxyl coronaridine (13)^20,28 based on their spectroscopic data and physiochemical characteristics upon comparison with values reported in the literatures.

Iboga-type alkaloids were reported to possess AChE inhibitory and anti-addiction activities which were crucial to the nervous system.^5,7 To screen the potential bioactivity of iboga-type alkaloids on the nervous system, we tested the neural activities of these alkaloids except 2 and 10 using three in vitro approaches, neuritogenic activity screening using Neuro-2a cells, neuroprotective activity screening against corticosterone-induced damage of PC12 cells or MPP⁺ (1-methyl-4-phenylpyridinium)-induced damage of cultured cortical neurons. No positive hits were identified in the first two screens. However, it was noteworthy that compound 11 was found to have significant protective effect in MPP⁺-injured primary cortical neurons (Fig. 11). MPP⁺ is a toxic agent in Parkinson's disease (PD) by interfering with mitochondria function and etc.,²⁹ thus causing neuronal death. Further studies will be needed to determine the mechanisms underlying the protective effect of this iboga-type alkaloid and whether it could reverse the symptoms in mouse models of PD.

Fig. 11 Evaluation of neuroprotective effects of iboga-type alkaloids in MPP⁺-injured primary cortical neurons. BDNF (200 ng mL⁻¹) was used as a positive control. The results are expressed as mean ± SEM (n = 3) of three independent experiments. ***P < 0.001 as compared with control (DMSO); #P < 0.05 as compared with the MPP⁺ alone. One-way ANOVA followed by Bonferroni's multiple comparison test.

In summary, thirteen iboga-type alkaloids including 7 new ones were isolated from E. hainanensis. In these new compounds, 1 represents a rare 7R-hydroxyindolenine alkaloid in genus Ervatamia. Comparison of the ECD curves (Fig. 12) of 1 with that of its 7S-epimer (9) indicated that the Cotton effects between 280 and 230 nm were dominated by the C-7 absolute configuration. This deduction can be useful for later absolute configuration identification of iboga-type 7-hydroxyindolenine alkaloids. 4 and 5 were a pair of new 3-hydroxyethyl substituted epimers. Moreover, the investigations on the neural activities of these iboga-type alkaloids revealed that 11 has neuroprotective actions in MPP⁺-injured primary cortical neurons. This finding may provide a potential candidate for treating PD, and much more in vivo research is needed to validate it as a useful drug.

Fig. 12 CD spectra of compounds 1, 8 and 9.

Experimental

General experimental procedures

Melting points were determined using an X-5 micro melting point apparatus (uncorrected). Optical rotations were measured on a JASCO P-1020 digital polarimeter at 25 °C. UV spectra were obtained using a JASCO V-550 UV/VIS spectrophotometer. IR data were determined on a JASCO FT/IR-480 plus infrared spectrometer in KBr discs. ECD spectra were measured with a Chirascan spectrometer (Applied Photophysics Ltd). HRESIMS spectra were acquired using an Agilent 6210 ESI-TOF mass spectrometer. NMR spectra were recorded on Bruker AV-400 or AV-600 spectrometers. Single-crystal data were performed using an Agilent Gemini S Ultra diffractometer and Cu Kα radiation. Silica gel (200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, P. R. China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), and ODS (YMC, Kyoto, Japan) were utilized for column chromatography (CC). Preparative HPLC (pHPLC) was carried out on an Agilent 1260 system (G1310B Iso pump and G1365D MWD VL detector) with a Waters Xbridge™ C₁₈ OBD reversed-phase column (19 × 250 mm, 5 μm, USA). All solvents used in CC and HPLC were of analytical grade (Shanghai Chemical Plant, Shanghai, P. R. China) and chromatographic grade (Fisher Scientific, New Jersey, USA), respectively. (R)-(−)- and (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) were obtained from Sigma-Aldrich.

Plant material

The dried twigs and leaves of E. hainanensis were collected in Jinfeng Mountain, Hainan Province, P. R. China, in September 2010 and identified by Dr Shi-Man Huang, Hainan University, Haikou, P. R. China. A voucher specimen (no. 2010091601) was deposited at the herbarium of the College of Pharmacy, Jinan University, Guangzhou, P. R. China.

Extraction and isolation

The air-dry leaves and twigs (21.0 kg) of E. hainanensis were powdered and extracted with 95% EtOH (50 L × 3) at room temperature. The extract was suspended in water (6 L) and acidified with 0.5% HCl to pH 3. The acidic suspension was partitioned with CHCl₃ and then basified with ammonia to pH 9–10 and partitioned with CHCl₃ to obtain the crude alkaloids (150.0 g). The crude alkaloid extract was subjected to silica gel CC (200–300 mesh) and eluted with CHCl₃–MeOH (1 : 0–0 : 1) to give 12 fractions (Fr. A–L). Fr. B (11.5 g) was separated by a silica gel CC (300–400 mesh) and eluted with CHCl₃–Me₂CO (1 : 0–0 : 1) to afford 17 fractions (Fr. B1–B17). Subfraction B7 (0.6 g) and B10 (0.5 g) were separated by repeated Sephadex LH-20 CC eluted with CHCl₃–MeOH (3 : 7) to afford 7 (5.0 mg) and 3 (7.1 mg), respectively. Fr. B9 (2.0 g) was successively chromatographed on Sephadex LH-20 CC to obtain 11 (72.8 mg) and subfraction B9b (580.0 mg). Fr. B9b was then purified on pHPLC with MeOH–H₂O–Et₂NH (6 : 4 : 0.0001) to afford 13 (5.5 mg, t_R = 39.2 min), 8 (40.0 mg, t_R = 65.8 min), 10 (4.2 mg, t_R = 80.0 min) and 6 (7.3 mg, t_R = 84.5 min). Fr. B11 (1.3 g) was further separated by ODS CC with MeOH–H₂O (1 : 9–1 : 0), then subjected to pHPLC with MeOH–H₂O–Et₂NH (7 : 3 : 0.0001) to yield 4 (5.2 mg, t_R = 114.0 min), 5 (9.0 mg, t_R = 125.4 min), 2 (11.6 mg, t_R = 24.5 min), 9 (13.0 mg, t_R = 16.8 min) and 12 (36.8 mg, t_R = 32.5 min). Fr. B12 (0.8 g) was purified using Sephadex LH-20 CC (MeOH) and pHPLC with MeOH–H₂O–Et₂NH (6 : 4 : 0.0001) to yield 1 (12.3 mg, t_R = 22.5 min).

X-ray crystallographic analysis of 1, 4, 6 and 9

The structures were solved by direct methods and refined by full-matrix least-squares on F² using SHELXL-97 package software. CCDC 1444994–1444997 ESI† for 1, 4, 6 and 9, respectively.

Crystal data of 1: C₂₁H₂₄N₂O₄·C₂H₃N (fw = 409.48); monoclinic, space group P2₁; a = 10.07340(18) Å, b = 9.87893(13) Å, c = 11.00555(18) Å, a = 90°, β = 107.6571(19)°, γ = 90°; V = 1043.61(3) ų, T = 173(2) K, Z = 2, D_c = 1.303 g cm⁻³, F(000) = 436. A total of 16 047 reflections were collected in the range 5.22 ≤ θ ≤ 62.73, of which 3274 unique reflections with I > 2σ(I) were collected for the analysis. Final R = 0.0243 and R_w = 0.0634, and the goodness of fit on F² was equal to 1.048; Flack parameter = 0.04(13).

Crystal data of 4: C₂₃H₃₀N₂O₃ (fw = 382.49); monoclinic, space group P2₁; a = 9.7015(8) Å, b = 10.7426(8) Å, c = 10.0282(10) Å, a = 90°, β = 107.663(10)°, γ = 90°; V = 995.86(16) ų, T = 173(2) K, Z = 2, D_c = 1.276 g cm⁻³, F(000) = 418. A total of 4010 reflections were collected in the range 4.63 ≤ θ ≤ 62.72, of which 2101 unique reflections with I > 2σ(I) were collected for the analysis. Final R = 0.0468 and R_w = 0.1104, and the goodness of fit on F² was equal to 1.071; Flack parameter = 0.0(4).

Crystal data of 6: C₂₁H₂₄N₂O₄ (fw = 368.42); orthorhombic, space group P2₁2₁2₁; a = 7.44984(14) Å, b = 11.4893(2) Å, c = 21.3736(4) Å, a = β = γ = 90°; V = 1829.44(6) ų, T = 173(2) K, Z = 4, D_c = 1.338 g cm⁻³, F(000) = 784. A total of 14 469 reflections were collected in the range 4.37 ≤ θ ≤ 62.70, of which 2879 unique reflections with I > 2σ(I) were collected for the analysis. Final R = 0.0262 and R_w = 0.0676, and the goodness of fit on F² was equal to 1.084; Flack parameter = 0.01(6).

Crystal data of 9: C₂₁H₂₄N₂O₄ (fw = 368.42); orthorhombic, space group P2₁2₁2₁; a = 8.78022(19) Å, b = 13.8217(4) Å, c = 15.0748(4) Å, a = β = γ = 90°; V = 1829.43(8) ų, T = 173(10) K, Z = 4, D_c = 1.338 g cm⁻³, F(000) = 784. A total of 14 636 reflections were collected in the range 4.33 ≤ θ ≤ 62.94, of which 2827 unique reflections with I > 2σ(I) were collected for the analysis. Final R = 0.0289 and R_w = 0.0703, and the goodness of fit on F² was equal to 1.066; Flack parameter = 0.13(9).

Preparation of (R) and (S)-MTPA esters

The (S)- and (R)-MTPA ester derivatives of 4 and 5 were prepared in a manner described previously.^30,31 Two portions of 4 or 5 (each 1 mg) were added into two NMR tubes and dried completely. Then, pyridine-d₅ was added to both tubes (each 0.5 mL). Finally, (S)-MTPA-Cl or (R)-MTPA-Cl (each 10 μL) was injected into the NMR tubes separately under N₂ gas protection and sufficiently mixed with the dissolved sample at room temperature. The ¹H NMR data of the (S)- and (R)-MTPA esters (4a, 4b, 5a and 5b) were recorded after 3 h.

The ¹H NMR data of the 4a (400 MHz, pyridine-d₅): δ 3.609 (1H, s, H-21), 3.174 (1H, m, H-17α), 2.998 (1H, J = 8.3 Hz, H-3), 2.995 (1H, m, H-5β), 2.878 (1H, m, H-6β), 2.874 (1H, m, H-6α), 1.943 (1H, d, m, H-17β), 1.781 (1H, brs, H-14), 1.709 (1H, m, H-19a), 1.589 (1H, m, H-15α), 1.240 (3H, d, J = 6.2 Hz, H-25).

The ¹H NMR data of the 4b (400 MHz, pyridine-d₅): δ 3.632 (1H, s, H-21), 3.114 (1H, m, H-17α), 3.11 (1H, m, H-5β), 2.911 (1H, J = 8.8 Hz, H-3), 2.989 (1H, m, H-6α), 2.951 (1H, m, H-6β), 1.917 (1H, d, m, H-17β), 1.732 (1H, brs, H-14), 1.725 (1H, m, H-19a), 1.545 (1H, m, H-15α), 1.263 (3H, d, J = 6.2 Hz, H-25).

The ¹H NMR data of the 5a (400 MHz, pyridine-d₅): δ 3.949 (1H, s, H-21), 3.433 (1H, m, H-6α), 3.099 (1H, m, H-17α), 3.081 (1H, m, H-5α), 2.827 (1H, d, J = 7.4 Hz, H-3), 1.887 (1H, m, H-17β), 1.717 (1H, brs, H-14), 1.600 (1H, m, H-15α), 1.474 (3H, d, J = 6.1 Hz, H-25), 0.863 (3H, t, J = 7.4 Hz, H-18).

The ¹H NMR data of the 5b (400 MHz, pyridine-d₅): δ 4.008 (1H, s, H-21), 3.410 (1H, m, H-6α), 3.141 (1H, m, H-17α), 3.035 (1H, m, H-5α), 2.918 (1H, d, J = 7.2 Hz, H-3), 2.034 (1H, m, H-17β), 2.014 (1H, brs, H-14), 1.546 (1H, m, H-15α), 1.395 (3H, d, J = 6.4 Hz, H-25), 0.887 (3H, t, J = 7.2 Hz, H-18).

Structure characterization

3-Oxo-7R-coronaridine hydroxyindolenine (1). Light yellow blocks (CHCl₃/MeOH); mp 182–183 °C; [α]²⁵_D −71.2 (c 0.80, CHCl₃); UV (CHCl₃) λ_max (log ε) 240 (3.44), 275 (3.58) nm; CD (CH₃CN) λ_max (Δε) 208 (0), 221 (+26.3), 237 (0), 260 (−10.9), 294 (0) nm; IR (KBr) ν_max 3221, 2956, 2933, 2872, 1741, 1662, 1566, 1528, 1471, 1435, 1266, 1227, 1148, 982, 755 cm⁻¹; HRESIMS m/z: 369.1808 [M + H]⁺ (calcd for C₂₁H₂₅N₂O₄, 369.1809); ¹H and ¹³C NMR data, see Table 1.

3S-Cyano-7S-coronaridine hydroxyindolenine (2). Pale yellow power; [α]²⁵_D −96.1 (c 0.80, CHCl₃); UV (CHCl₃) λ_max (log ε) 242 (3.45), 261 (3.43), 289 (3.41) nm; CD (CH₃CN) λ_max (Δε) 212 (−10.5), 234 (0), 260 (+15.4), 281 (0), 300 (−6.7), 334 (0) nm; IR (KBr) ν_max 3445, 2955, 2931, 2871, 2262, 1736, 1657, 1458, 1436, 1361, 1251, 1167, 1086, 980, 882, 755 cm⁻¹; HRESIMS m/z: 380.1973 [M + H]⁺ (calcd for C₂₂H₂₆N₃O₃, 380.1969); ¹H and ¹³C NMR data, see Table 1.

3R-Hydroxy-7S-coronaridine hydroxyindolenine (3). Pale yellow power; [α]²⁵_D −32.7 (c 0.83, CHCl₃); UV (CHCl₃) λ_max (log ε) 242 (3.62), 295 (3.47), 336 (3.37) nm; CD (CH₃CN) λ_max (Δε) 227 (0), 250 (+17.3), 282 (0), 337 (−11.2) nm; IR (KBr) ν_max 3396, 2965, 2932, 2873, 1727, 1661, 1590, 1464, 1437, 1235, 1187, 1094, 753 cm⁻¹; HRESIMS m/z: 371.1973 [M + H]⁺ (calcd for C₂₁H₂₇N₂O₄, 371.1965); ¹H and ¹³C NMR data, see Table 1.

3S-(24S-Hydroxyethyl)-coronaridine (4). Colorless blocks (MeOH); mp 210–211 °C; [α]²⁵_D −112.1 (c 0.81, CHCl₃); UV (CHCl₃) λ_max (log ε) 241 (4.16), 283 (4.18) nm; CD (CH₃CN) λ_max (Δε) 207 (+4.9), 224 (0), 245 (+4.9), 259 (0), 280 (−7.5), 320 (0) nm; IR (KBr) ν_max 3383, 2965, 2943, 2903, 1732, 1547, 1480, 1440, 1253, 1232, 1147, 1077, 910, 750 cm⁻¹; HRESIMS m/z: 383.2328 [M + H]⁺ (calcd for C₂₃H₃₁N₂O₃, 383.2329); ¹H and ¹³C NMR data, see Table 2.

Table 2 ¹H and ¹³C NMR data of compounds 4 and 5 (CD₃OD, δ in ppm, J in Hz)^a

Position	4			5
		δH^b	δC^c		δH^b	δC^c
a Overlapped resonances are reported without designating multiplicity.b Measured at 400 MHz.c Measured at 100 MHz.
2			138.8			138.8
3		2.64 d (6.5)	67.9		2.50 d (6.7)	68.7
5		3.43 m	56.9	α	3.44 m	56.0
				β	3.12 m
6	α	2.96 m	22.4		3.03 m	22.5
	β	3.12 m
7			111.1			110.9
8			129.6			129.4
9		7.40 d (8.0)	118.7		7.38 d (8.0)	118.7
10		6.96 t (8.0)	119.6		6.95 t (8.0)	119.6
11		7.02 t (8.0)	122.2		7.01 t (8.0)	122.2
12		7.23 d (8.0)	111.5		7.22 d (8.0)	111.6
13			137.6			137.7
14		1.80 br s	31.1		2.07 m	29.0
15	α	1.56 m	28.2	α	1.53 m	28.4
	β	1.25 m		β	1.47 m
16			56.0			55.9
17	α	2.88 dd (13.6, 2.0)	39.0	α	2.88 dd (13.6, 2.4)	39.1
	β	1.85 td (13.6, 2.0)		β	1.85 m
18		0.92 t (7.6)	12.2		0.90 t (7.3)	12.2
19	a	1.69 m	27.9	a	1.62 m	27.9
	b	1.47 m		b	1.42 m
20		1.35 m	39.4		1.34 m	39.3
21		3.71	59.4		3.76 m	58.3
22			176.3			176.3
23		3.70 s	52.9		3.66 s	52.9
24		3.74 dd (13.0, 6.8)	71.8		3.78 m	68.8
25		1.10 d (6.8)	21.8		1.18 d (6.0)	20.9

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3S-(24R-Hydroxyethyl)-coronaridine (5). Pale yellow power; [α]²⁵_D −42.6 (c 0.82, CHCl₃); UV (CHCl₃) λ_max (log ε) 242 (3.82), 284 (3.77) nm; CD (CH₃CN) λ_max (Δε) 225 (0), 244 (+4.7), 260 (0), 279 (−4.8), 307 (0) nm; IR (KBr) ν_max 3393, 2956, 2938, 2872, 1752, 1652, 1616, 1585, 1541, 1508, 1456, 1258, 1147, 1078, 746 cm⁻¹; HRESIMS m/z: 383.2328 [M + H]⁺ (calcd for C₂₃H₃₁N₂O₃, 383.2329); ¹H and ¹³C NMR data, see Table 2.

5-Oxo-6S-hydroxycoronaridine (6). Colorless blocks (MeOH); mp 171–173 °C; [α]²⁵_D +6.7 (c 1.0, CHCl₃); UV (CHCl₃) λ_max (log ε) 219 (4.68), 285 (3.88) nm; CD (CH₃CN) λ_max (Δε) 204 (−8.6), 210 (0), 221 (+16.9), 242 (+3.8), 249 (+5.0), 262 (0), 275 (−2.1), 298 (0) nm; IR (KBr) ν_max 3459, 3252, 2953, 2930, 2874, 1739, 1622, 1540, 1457, 1442, 1245, 1200, 1148, 1087, 741 cm⁻¹; HRESIMS m/z: 369.1811 [M + H]⁺ (calcd for C₂₁H₂₅N₂O₄, 369.1809); ¹H and ¹³C NMR data, see Table 1.

5-Oxo-6S-methoxy-coronaridine (7). Pale yellow power; [α]²⁵_D −7.7 (c 0.80, CHCl₃); UV (CHCl₃) λ_max (log ε) 242 (3.62), 281 (3.60) nm; CD (CH₃CN) λ_max (Δε) 204 (−5.2), 210 (0), 222 (+9.0), 265 (0), 279 (−1.9), 310 (0) nm; IR (KBr) ν_max 3223, 2976, 2939, 2883, 1738, 1628, 1580, 1457, 1436, 1351, 1241, 1198, 1088, 735 cm⁻¹; HRESIMS m/z: 383.1966 [M + H]⁺ (calcd for C₂₂H₂₇N₂O₄, 383.1965); ¹H and ¹³C NMR data, see Table 1.

Neuronal culture and measurement of cell viability

Primary cortical neurons were isolated from E18 Sprague-Dawley (SD) rat embryos as previously described.³² Dissociated neurons were fed with Neurobasal medium (Life Technologies) supplemented with 2% B27 (Life Technologies). Neuronal cells were seeded on 96 well dishes (1 × 10⁵ per well), which was pre coated with poly-L-lysine (0.1 mg mL⁻¹, Sigma) for 24 h at 37 °C. MPP⁺ (Sigma) was dissolved in PBS (pH 7.4) and was prepared as a 100 mmol L⁻¹ stock immediately before use. All the compounds were first dissolved in DMSO and were then diluted in culture medium to the indicated final concentrations. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, sigma) assay.¹⁹ At the end of the treatment, MTT solution (final concentration, 5 mg mL⁻¹) was added to each well for 4 h at 37 °C. Subsequently, the dark blue formazan crystals formed in intact cells were solubilized with 100 μL DMSO. After shaking at room temperature for 10 min, absorbance at 595 nm was measured with a DTX880 multimode detector (Beckman Coulter, Brea, CA, USA). Cell viability was expressed as a percentage of the non-treated control. The GraphPad Prism 5.0 software was used to perform the statistics. Data were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. P < 0.05 was considered to be statistically significant.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81373935, 81172947), the Ministry of Science and Technology of China (No. 2013BAI11B05, 2013DFM30080 and 2012ZX09103201-056), the Program for New Century Excellent Talents in University (NCET-12-0676) and the Science and Technology Planning Project of Guangdong Province (No. 2013A022100028).

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Footnotes

† Electronic supplementary information (ESI) available: UV, IR, NMR, HRESIMS and ECD spectra of compounds 1–7. CCDC 1444994–1444997. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra00185h

‡ Z. W. L. and X. J. H. contributed equally to this work.

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