Ajmaline, Oxindole, and Cytotoxic MacrolineâAkuammiline Bisindole

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Ajmaline, Oxindole, and Cytotoxic Macroline−Akuammiline Bisindole Alkaloids from Alstonia penangiana Joanne Soon-Yee Yeap,† Suerialoasan Navanesan,‡ Kae-Shin Sim,‡ Kien-Thai Yong,‡ Subramaniam Gurusamy,§ Siew-Huah Lim,† Yun-Yee Low,† and Toh-Seok Kam*,† †

Department of Chemistry and ‡Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia § School of Chemical and Life Sciences, Nanyang Polytechnic, 180 Ang Mo Kio Avenue 8, Singapore 569830, Singapore S Supporting Information *

ABSTRACT: Examination of the EtOH extract of the Malayan Alstonia penangiana resulted in the isolation of 10 new alkaloids, comprising two ajmaline (1, 2), four macroline oxindole (3−6), and four macroline− akuammiline bisindole alkaloids (7−10). The structures of these alkaloids were determined based on analysis of the spectroscopic data and, in the case of the oxindole 6 and the bisindole alkaloid 7, also confirmed by X-ray diffraction analysis. The bisindole alkaloids 7 and 8 showed pronounced in vitro growth inhibitory activity against an array of human cancer cell lines, including KB, vincristine-resistant KB, PC-3, LNCaP, MCF7, MDA-MB231, HT-29, HCT 116, and A549 cells with IC50 values in the 0.3−8.3 μM range.

T

he genus Alstonia (Apocynaceae) comprises about 43 species that are distributed over the tropical and subtropical regions of the world, including Africa, Asia, the Pacific Islands, and Australia.1,2 About eight species occur in Peninsular Malaysia (or Malaya),2−4 and several of these species (local name Pulai) are used in Traditional Medicine, for example in the treatment of malaria and dysentery.5,6 Plants of this genus, which are shrubs and trees, have been shown to be rich sources of alkaloids, especially indole alkaloids, and additionally, a particular feature of the alkaloid composition is the preponderance of the macroline unit.7,8 In continuation of the phytochemical studies of this genus,9−12 we investigated the alkaloid composition of A. penangiana. This species was first described by Sidayasa.2 It has a limited distribution, occurring only in Penang (island),2,4 and in addition, the alkaloid content has not been previously examined. We report herein the first phytochemical study of this species, including the structure elucidation and biological activity of the new alkaloids.

to an unsubstituted indole moiety (δ 6.25−6.92, 4H), two additional singlets, each accounting for two aromatic hydrogens (δ 7.07, s, 2H; 7.09, s, 2H), six aromatic methoxy groups (δ 3.89−3.94), a methyl ester (δ 3.49), and an ethylidene side chain (δ 1.56, 5.32). The presence of the additional nonindole aromatic resonances is suggestive of additional aromatic units and corresponds to the presence of two trimethoxy-substituted aromatic moieties. The two aromatic units are identical and are deduced to be 3,4,5-trimethoxy-substituted from the presence of six aromatic methoxy groups and the observation of two sets of aromatic two-proton singlets. The 13C NMR data (Table 2) gave a total of 41 carbon resonances comprising eight methyls, three methylenes, 14 methines, an ester carbonyl (δ 172.0), a conjugated ester carbonyl (δ 163.7), a conjugated amide carbonyl (δ 169.3), a tertiary carbon linked to an indolic nitrogen (C-13), six oxygenated tertiary carbons, and six quaternary carbon atoms. The oxymethine resonance at δH 6.18 (δC 75.7) is reminiscent of H-17/C-17 in a vincamajine ester derivative. This was supported by the 2D NMR data (Figure 1), which confirmed a vincamajine-type core skeleton with the ester function constituted from linking of one of the trimethoxybenzoyl (or eudesmoyl) groups to the C-17 oxygen (three-bond correlation from H-17 to the ester carbonyl, Figure 1). The second trimethoxybenzoyl moiety must be attached to the indolic N-1, in view of the absence of an indolic hydrogen or an N-1−Me in the 1H NMR spectrum and the presence of an amide carbonyl in the 13C NMR spectrum of 1. The relative

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RESULTS AND DISCUSSION Compound 1 (vincamaginine A) was isolated as a light yellowish oil, with [α]25D −172 (c 0.2, CHCl3). The UV spectrum (215, 275, and 298 nm) showed the presence of Nacyl dihydroindole and trimethoxybenzoyl chromophores, while the IR spectrum showed bands due to ester carbonyl (1733 cm−1) and amide carbonyl (1652 cm−1) groups. The HRESIMS data ([M + H]+ m/z 741.3027) established the molecular formula as C41H44N2O11, corresponding to 21 indices of hydrogen deficiency. The 1H NMR data of 1 (Table 1) showed, apart from the four aromatic resonances due © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 23, 2018

A

DOI: 10.1021/acs.jnatprod.8b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR Spectroscopic Data of 1−6a 1b

2b

4.33, d (5.0) 4.56, dd (10.0, 5.0) 3.73, d (5.0) 1.95, d (12.0)

4.34, d (5.0) 4.57, dd (10, 5.0) 3.74, d (5.0) 1.96, d (12.0)

2.72, dd (12.0, 5.0) 6.92, dd (7.5, 1.0) 6.71, t (7.5)

15

6.89, td (7.5, 1.0) 6.25, d (7.5) 1.72, dd (14.5, 10.0) 2.40, dd (14.5, 5.0) 3.58, d (5.0)

2.73, dd (12.0, 5.0) 6.92, dd (7.5, 1.0) 6.70, td (7.5, 1.0) 6.88, td (7.5, 1.0) 6.25, d (7.5) 1.72, dd (14.5, 10.0) 2.40, dd (14.5, 5.0) 3.58, d (5.0)

16 17

6.18, s

6.17, s

position 2 3 5 6

9 10 11 12 14

3c

4c

5b

3.22, m

3.15, m

3.83, m

4.46, m

3.42, br d (7.0) 2.30, dd (14.5, 7.0) 2.47, d (14.5)

3.50, br d (7.0) 2.39, m

5.05, br d (8.0) 2.21, d (14.0)

4.65, br d (8.0) 2.21, d (14.0)

8.07, m

2.52, dd (14.0, 1.0) 8.14, m

3.62, br d (7.5) 2.19, dd (14.0, 7.5) 2.92, dd (14.0, 1.0) 7.14, dd (8.0, 1.0)

2.62, dd (14.0, 8.0) 7.56, d (8.0)

2.66, dd (14.0, 8.0) 7.56, d (8.0)

7.14, m

7.25, m

7.02, td (8.0, 1.0)

7.12, t (8.0)

7.09, t (8.0)

7.15, m

7.25, m

7.28, td (8.0, 1.0)

7.35, t (8.0)

7.33, t (8.0)

6.85, m 1.32, ddd (15.0, 12.0, 4.0) 2.35, ddd (15.0, 7.0, 2.0) 3.00, m

6.89, m 1.39, ddd (15.0, 12.0, 3.0) 2.39, m 3.09, ddd (12.0, 7.0, 2.0)

6.82, d (8.0) 1.33, ddd (14.0, 11.0, 3.0) 2.44, ddd (14.0, 7.0, 3.0) 3.75, ddd (11.0, 7.0, 2.0)

6.88, d (8.0) 1.53, td (14.0, 4.0) 1.87, ddd (14.0, 6.0,1.0) 2.98, m

6.85, d (8.0) 1.57, td (14.0, 4.0) 1.87, ddd (14.0, 6.0, 1.0) 2.98, m

3.98, dd (12.0, 2.0) 4.67, d (12.0)

4.01, dd (12.0, 2.0) 4.67, d (12.0)

3.94, dd (11.0, 2.0) 4.52, d (11.0)

2.26, m 3.50, t (12.0)

2.26, m 3.61, t (10.0)

3.67, m 1.28, d (6.0) 4.00, m 1.30, m 1.42, ddd (14.0, 10.0, 3.0)

3.79, dd (10.0, 5.0) 1.28, d (6.0) 4.00, m 1.30, m 1.42, ddd (14.0, 10.0, 3.0)

3.19, s 8.14, s

3.16, s 8.22, s

1.56, d (7.0) 5.32, q (7.0)

1.55, d (7.0) 5.31, q (7.0)

2.24, s

2.27, s

2.26, s

21

3.51, m 3.51, m

3.51, m 3.51, m

9.81, s

7.67, s

7.59, s

a

3.25, s 3.49, 7.07, 7.09, 3.89,

s s s s

3.92, s 3.94, s

3.47, 7.10, 7.09, 3.93,

6bb

3.15, m

18 19 20

N(1)−Me N(4)−CHO CO2Me 2′, 6′ 2″, 6″ 3′-OMe, 5′-OMe, 3″OMe, 5″-OMe 4′-OMe 4″-OMe

6ab

s s s s

3.93, s

Assignments based on COSY, HSQC, and NOESY. bCDCl3, 600 MHz. cCDCl3 (with a drop of methanol-d4), 400 MHz.

confirmed by the observed base-induced bathochromic shift in the UV spectrum (Experimental Section). Another notable difference is in the resonances due to the aromatic methoxy groups. In compound 1, there are three discernible aromatic methoxy resonances (a 12-proton singlet at δ 3.89 due to 4 × OMe groups; two three-proton singlets at δ 3.92 and 3.94 due to two OMe groups), while in 2, there is only one overlapped aromatic methoxy resonance (a 15-proton singlet at δ 3.93 due to 5 × OMe groups) (Table 1). The 13C NMR spectra of the two compounds were also highly similar except for changes in the carbon shifts of some of the aromatic carbons (1′, 3′, 4′, 5′) of the “upper” or syringoyl unit in 2 when compared to 1 (Table 2). The identification of the acid residue in the C-17 ester function in 2 as syringoyl (4′-hydroxy-3′,5′-dimethoxybenzoyl) instead of eudesmoyl (3′,4′,5′-trimethoxybenzoyl) is based on the three-bond correlations from H-2′/H-6′ to the ester carbonyl in the HMBC spectrum (Figure 3). These observations (MS, UV, 1H and 13C NMR, HMBC) lead to the identification of vincamaginine B (2) as 17-O-4′-hydroxy-3′,5′-

configuration of 1 is deduced to be similar to those in vincamajine and its derivatives from the NOESY data. The orientation of H-17 is α from the observed H-17/H-14β, H-15 NOEs, while the geometry of the 19,20-double bond is E from the observed H-18/H-15 and H-19/H-21 NOEs (Figure 2). Compound 2 (vincamaginine B) was isolated as a light yellowish oil, with [α]25D −379 (c 0.2, CHCl3). The UV spectrum (214, 280, and 297 nm) was similar to that of 1 and is consistent with the presence of N-acyldihydroindole and trimethoxybenzoyl chromophores. The IR spectrum was also similar to absorption bands at 1733 and 1651 cm−1 due to ester carbonyl and amide carbonyl functionalities, respectively. The HRESIMS data ([M + H]+ m/z 727.2873) gave the molecular formula C40H42N2O11 (14 mass units less than 1, or replacement of CH3 with H compared with 1). The 1H NMR data (Table 1) of 2 are similar to those of 1 with highly similar chemical shifts. A notable difference is the absence of an aromatic methoxy group in 2 (5 × OMe) when compared to 1 (6 × OMe). These observations suggest that in 2 an aromatic OMe substituent has been replaced by OH, which was B



Article

Table 2. 13C NMR Spectroscopic Data of 1−6a

a

position

1b

2b

3c

4c

5b

6ab

6bb

2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 N(1)−Me CO2Me CO2Me N(4)−CHO 19-OMe 1′ 1″ 2′, 6′, 2″, 6″ 3′, 5′, 3″, 5″ 4′ 4″ 3′-OMe, 5′-OMe, 3″-OMe, 5″-OMe 4′-OMe 4″-OMe O-CO N-CO

70.6 52.9 61.6 35.5 57.0 130.4 123.7 123.0 127.8 116.0 144.8 23.1 30.0 59.0 75.7 12.7 117.3 136.2 55.4

70.5 52.9 61.6 35.5 57.0 130.5 123.7 123.0 127.8 116.0 144.8 23.1 30.0 59.0 75.6 12.7 117.3 136.2 55.4

185.0 63.4 61.3 37.4 57.8 129.5 125.8 123.2 128.2 109.9 141.6 32.9 32.2 67.6 71.6 16.6 171.4 116.0 190.5

184.4 63.5 61.2 37.5 57.7 129.4 125.8 123.3 128.1 109.6 141.2 33.3 32.9 67.9 71.4 25.0 197.1 119.6 156.9

177.5 66.7 61.7 38.3 58.0 137.2 120.8 122.4 128.0 107.9 142.4 34.6 33.3 68.8 71.0 25.8 197.0 119.6 155.2 26.6

179.6 61.7 51.0 37.3 53.9 127.3 124.7 123.0 129.0 108.4 144.5 33.8 27.6 42.1 59.7 25.0 64.8 40.8

179.8 56.8 55.3 37.1 54.1 127.6 124.6 122.6 128.8 108.3 144.7 32.0 27.2 43.0 59.7 24.7 64.8 41.1

26.3

26.4

51.8 172.0

51.8 172.1 159.4

159.6

124.3 130.3 106.6 153.0 142.6 141.3 56.0 61.0 60.9 163.7 169.3

120.2 130.3 106.4 146.8 139.7 141.3 56.5 61.0 163.8 169.4

Assignments based on DEPT, HSQC, and HMBC. bCDCl3, 150 MHz. cCDCl3 (with a drop of methanol-d4), 100 MHz.

Figure 1. COSY and selected HMBCs of 1. Figure 2. Selected NOEs (ROEs) of 1 and 2.

dimethoxybenzoyl-N(1)-3″,4″,5″-trimethoxybenzoylvincamajine. Four new macroline-type oxindoles were isolated. Two of these, alstonisinines A (3) and B (4), correspond to the socalled type A and type B isomers, respectively.12,13 Compound 3 (alstonisinine A) was isolated as a white, amorphous solid,

with [α]25D +183 (c 0.2, MeOH). The UV spectrum (EtOH) showed characteristic oxindole absorption maxima at 225 and 261 nm, while the IR spectrum showed the presence of NH/ OH (3210 cm−1), conjugated formyl carbonyl (1682 cm−1, δC 190.5), and lactam carbonyl (oxindole) functions (1605 cm−1, C



Article

Chart 1

d, J = 12 Hz) due to the geminal protons of an oxymethylene corresponding to C-17, and a methyl linked to a trisubstituted double bond (δ 2.24). The 13C NMR data (Table 2) showed 19 carbon resonances, comprising a methyl, three methylenes, eight methines, a tertiary carbon linked to an indolic nitrogen, two tertiary carbons linked to oxygen, one lactam (oxindole) carbonyl, and three quaternary carbon atoms. The COSY spectrum (Figure 4) showed the presence of CHCH2 and CHCH2CH partial structures, which are characteristic of C-5− C-6 and C-3−C-14−C-15 of a macroline oxindole skeleton. The presence of the C-17 oxymethylene, the Δ19,20 olefinic moiety, to which the methyl and formyl groups are attached, led to completion of the fifth or E ring, which is fused to ring D Figure 3. COSY and selected HMBCs of 2.

δC 185.0). The HRMS data ([M + H]+ m/z 341.1511) gave the molecular formula as C19H20N2O4. The 1H NMR data (Table 1) of 3 showed the presence of four aromatic resonances (δ 6.85−8.07) due to an unsubstituted indole moiety, a formyl H (δ 9.81), a pair of deshielded signals (δ 3.98, H-17b, dd, J = 12, 2 Hz; 4.67, H-17a,

Figure 4. COSY and selected HMBCs of 3. D



Article

at C-15 and C-16. Examination of the NMR data, however, indicated that the resonance due to H-16 in the macroline oxindoles (usually at ca. δ 1.98)14 was absent and furthermore C-16 in 3 is an oxygenated tertiary carbon (δC 67.6). This indicates that a hydroxy group is attached to C-16, as in the case of 16-hydroxyalstonal.15 The relative configurations at C-3, C-5, C-15, and C-16 are assumed to follow that of the macroline alkaloids from the similarity of the NMR data, including the NOESY data. The configuration of the spirocyclic C-7 was assigned as S from the diagnostic C-2 (δ 185.0) and C8 (δ 129.5) chemical shifts as well as the H-9/H-15 NOEs,12,16 while the α-orientation of the C-16 hydroxy group was indicated by the H-17β/H-14β NOEs (Figure 5).

Figure 6. Selected NOEs of 4.

an indolic nitrogen, a lactam (oxindole) carbonyl, a conjugated ketone, an oxygenated tertiary carbon, and three quaternary carbon atoms. The 1H and 13C NMR data of 5 showed many features in common with 16-hydroxyalstonisine,15 except for the C-2 (δ 177.5) and C-8 (δ 137.2) resonances, compared to δ 182.2 and 128.9, respectively, seen in 16-hydroxyalstonisine. These values are diagnostic of the configuration of spirocyclic C-7 in unsubstituted oxindoles (vide supra) and indicated that 5 is the (7R)-diastereomer of (7S)-16-hydroxyalstonisine.12,14 This conclusion was also supported by the observed H-9/H-6β NOE (Figure 7) as well as the observed shielding of H-9 in 5 Figure 5. Selected NOEs of 3.

Compound 4 (alstonisinine B) was isolated as a white, amorphous solid, with [α]25D +113 (c 0.1, MeOH). The UV spectrum (MeOH) showed oxindole absorption maxima at 210 and 253 nm, while the IR spectrum showed bands at 3295, 1702, and 1609 cm−1 due to NH/OH, conjugated ketocarbonyl (δC 197.1), and lactam carbonyl (oxindole, δC 184.4) functions, respectively. HRMS data ([M + H]+ m/z 341.1500) gave the molecular formula C19H20N2O4, which indicated that 4 was isomeric with 3. The 1H (Table 1) and 13C NMR (Table 2) data of 4 were similar to those of 3 except for a few differences, such as the absence of the methyl and formyl resonances (δ 2.24 and 9.81, respectively, in 3), which in 4 were replaced by acetyl and olefinic proton resonances (δ 2.27 and 7.67, respectively, in 4). Corresponding changes were seen for the resonances of C-18 (δC 25.0), C-19 (197.1), and C-21 (156.9) in the 13C NMR data (Table 2). In common with 3, C-16 in 4 is also hydroxy substituted (δC 67.9), and the configuration of the spirocyclic C-7 is S from the observed C-2 and C-8 shifts at δ 184.4 and 129.4, respectively, and from the NOEs between H-9 and H-15 (Figure 6). Alstonisinine B (4) is therefore the type B isomer of alstonisinine A (3, type A) and corresponds to the N(1)-demethyl derivative of 16-hydroxyalstonisine.15 Compound 5 (alstonisinine C) was obtained as a light yellowish oil with [α]25D +180 (c 0.4, CHCl3). The IR spectrum showed bands due to OH/NH (3403 cm−1), lactam carbonyl (1708 cm−1), and conjugated ketocarbonyl (1616 cm−1) groups, while the UV spectrum showed oxindole absorption maxima at 225 and 256 nm. The HRMS data ([M + H]+ m/z 355.1666) established the molecular formula as C20H22N2O4. The 1H NMR data (Table 1) of 5 showed the presence of four aromatic resonances of an unsubstituted indole moiety (δ 6.82−7.28), an olefinic proton (δ 7.59), an N-methyl (δ 3.25), and an acetyl group (δ 2.26). The 13C NMR data (Table 2) accounted for all 20 carbon atoms comprising two methyls, three methylenes, eight methines, a tertiary carbon bonded to


by ca. 1 ppm to δ 7.14, compared to H-9 in the 7S diastereomeric oxindoles (δ H-9 ca. 8.1, e.g., compounds 3 and 4). The lower field resonance of H-9 in the related 7S pentacyclic oxindoles (e.g., 3 and 4) is caused by the anisotropic effect of the proximate Δ20,21 bond. Such an effect is absent in the pentacyclic 7R oxindoles such as 5, due to the change in configuration at the spirocenter, which results in the same double bond being too remote from the aromatic ring to exert anisotropy on H-9.14 Compound 6 (alstonoxine F) was obtained as a light yellowish, amorphous solid with [α]25D −20 (c 0.3, CHCl3). The UV spectrum is typical of an oxindole chromophore (214, 255, and 289 nm), while the IR spectrum showed bands at 3392, 1705, and 1646 cm−1 due to OH, lactam carbonyl, and formamide functionalities. HRESIMS measurements ([M + H]+ m/z 359.1971) gave the molecular formula C20H26N2O4. The 1H NMR data (Table 1, 6a) showed the presence of four aromatic resonances of an unsubstituted indole moiety (δ 6.88−7.56), an N-1−Me (δ 3.19), a formamide-H (δ 8.14), a pair of downfield signals (δ 3.50, t, J = 12 Hz; 3.67, m) due to the geminal protons of an oxymethylene corresponding to C17, an oxymethine (δH 4.0, δC 64.8), and a methyl group (δ 1.28, d, J = 6 Hz), the latter two constituting part of a 2hydroxypropyl side chain. The 13C NMR spectrum (Table 2, 6a) showed a total of 20 carbon resonances, comprising two E



Article

nine of the hydrogens for which the resonances are overlapped or coincident, the resonances for the remaining 11 hydrogens, though close, are clearly distinguishable. In the 13C NMR data (Table 2), only two carbon resonances were coincident. In the 1 H NMR spectrum, the resonances of H-3 and H-5 are notably separated or distinct. The same was true for C-3 and C-5 in the 13 C NMR spectrum. It was previously suggested that the major rotamer 6a was one in which the carbonyl CO and H-5 are approximately syn-coplanar, resulting in the deshielding of H-5, whereas the minor rotamer 6b was one in which the carbonyl CO and H-3 are approximately syn-coplanar, resulting in the deshielding of H-3 (Table 1).14 This proposal was consistent with the NOEs observed for NCHO/H-3 in the major rotamer and NCHO/H-5 in the minor rotamer (Figure 9). The DFT calculations are in agreement with the proposed major and minor rotamers (Figure 9) with the major rotamer estimated to be more stable than the minor rotamer by ca. 4.4 kcal mol−1 (18.3 kJ mol−1). Since repeated attempts at crystallization only gave amorphous solids, the absolute configuration was determined by ECD measurements in conjunction with TDDFT calculations (Figure 10). Eventually, however, a crystal was found among the amorphous solids obtained during crystallization from MeCN−MeOH, which was suitable for Xray diffraction analysis. The X-ray crystal structure (Figure 11) vindicated all the above, in addition to facilitating assignment of the (19S) configuration and establishment of the absolute configuration. Compound 7 (angustilongine A) was initially obtained in amorphous form and subsequently as light yellowish block crystals from CH2Cl2−hexanes, with mp >199 °C (dec) and [α]25D +3 (c 0.4, CHCl3). The IR spectrum showed bands due to OH (3360 cm−1) and ester (1741 cm−1) functions, while the UV spectrum indicated a composite of indole and dihydroindole chromophores (212, 232, 255, and 295 nm). The HRESIMS data ([M + H]+ m/z 707.4180) established the molecular formula as C43H54N4O5. Analysis of the 1H NMR and 13C NMR data indicated a bisindole alkaloid constituted from the union of macroline and akuammiline moieties. The 1H NMR data of 7 (Table 3) showed the presence of four aromatic resonances of an unsubstituted indole moiety (δ 7.04−7.37, macroline), two isolated aromatic singlets of another indole moiety substituted at C-10′ and C-11′ (δ 6.40, 5.29; akuammiline), an aromatic methoxy group (δ 3.62, akuammiline), a methyl ester group (δ 3.74, akuammiline), an ethylidene side chain (δ 5.38, 1.48, akuammiline), three methyl singlets corresponding to two N-1−Me (δ 3.37, macroline; 2.29,

methyls, four methylenes, 10 methines, a tertiary carbon linked to an indolic nitrogen, a lactam (oxindole) carbonyl, and two quaternary carbon atoms. The presence of the formamide function is supported by the resonance at δ 159.4 in the 13C NMR spectrum. The COSY spectrum showed the presence of HOCH2CHCHCH2 and CH3CH(OH)CH2CHCH2CH partial structures (Figure 8). These fragments and the HMBC data

Figure 8. COSY and selected HMBCs of 6.

(Figure 8) indicated the presence of an E-ring-seco macroline oxindole belonging to the alstonoxine series as shown in 6.12,14,16 The configuration at the spirocyclic C-7 was assigned as S from the NOE observed between H-9 and H-15 (Figure 9).12,16 The formamide group, not unexpectedly, gives rise to

Figure 9. Optimized geometries of major (6a) and minor (6b) conformers of 6 at the B3LYP/6-31G(d) level and their key NOEs.

the presence of a pair of rotamers, similar to alstofoline.14 As was noted in the previous instance, although two separate bands are seen during preparative radial chromatography, equilibration is rapid in solution at room temperature, resulting in an equilibrium mixture of two rotamers, with one form predominating. This was clearly seen in the 1H and 13C NMR spectra of 6. The 1H NMR spectrum showed two sets of signals corresponding to the two conformers with a 10-fold predominance of the major rotamer (Table 1, 6a). Except for

Figure 10. Experimental ECD spectrum of 6 and calculated ECD spectra of (3S,5S,7S,15S,16R,19S)-6 and (3R,5R,7R,15R,16S,19R)-6. F



Article

Table 3. 1H NMR Spectroscopic Data of 7−10a position

7

8 3.91, m 2.91, d (7.0)

3.73, m 3.05, m

3.93, m 2.96, m

2.41, d (16.0)

2.46, d (16.0)

2.16, m

9 10

3.75, m 2.84, br d (7.0) 2.19, br d (16.0) 3.13, dd (16.0, 7.0) 7.37, d (7.5) 7.04, t (7.5) 7.09, m

12 14

7.08, m 1.13, m

15 16 17

2.84, m 1.27, m 2.08, m 3.45, dd (11.5, 5.0) 4.45, t (11.5)

2.35, m 1.90, m 2.32, m 3.97, dd (12.0, 5.0) 4.18, t (12.0)

3.25, dd (16.0, 7.0) 7.48, d (7.5) 7.10, td (7.5, 1.0) 7.19, td (7.5, 1.0) 7.28, d (7.5) 1.81, dd (15.0, 2.0) 2.78, m 1.89, m 1.92, m 3.94, dd (11.0, 3.4) 4.24, t (11.0)

3.14, dd (16.0, 7.0) 7.35, d (7.7) 7.06, m

11

3.28, dd (16.0, 7.0) 7.50, d (8.0) 7.11, td (8.0, 1.0) 7.20, td (8.0, 1.0) 7.30, d (8.0) 1.53, m

1.09, 3.17, 1.33, 5.36, 3.37,

1.24, 2.61, 1.65, 5.44, 3.62,

3 5 6

Figure 11. X-ray crystal structure of 6.

18 19 20 21 N(1)− Me N(4)− Me 2′ 3′

akuammiline) and an N-4−Me (δ 2.27, macroline), an oxymethylene due to C-17 (δ 3.45, 4.45; macroline), and a methyl doublet that is part of a CH3CHCH fragment (δ 1.09, macroline). A downfield singlet at δ 5.36 (δC 93.7) was reminiscent of a methine of a hemiacetal moiety.17 The observed NOE (Figure 13) between the aromatic singlet at δ 5.29 and N-1′−Me (δ 2.29) allowed the assignment of this aromatic resonance to H-12′ of the akuammiline unit and the other aromatic singlet at δ 6.40 to H-9′. The 13C NMR data (Table 4) accounted for a total of 43 carbon atoms comprising seven methyls, seven methylenes, 18 methines, one oxygenated tertiary carbon, three tertiary carbons bonded to an indolic nitrogen, an ester carbonyl, and six quaternary carbon atoms. Of these, the deshielded resonance at δ 156.2 was due to a methoxy-substituted aromatic carbon (C-11′, 3J from H-9′ to C-11′ in the HMBC spectrum, Figure 12), while the oxymethylene carbon resonance at δ 59.6 (C-17) and another downfield signal at δ 93.7 (C-21) were consistent with the presence of a hemiacetal function in ring E.17 The substitution of the aromatic methoxy group at C-11′ suggested that the bisindole is branched from C-10′ of the akuammiline unit, while the olefinic carbon resonances at δ 140.0 (C-20′) and 118.6 (C-19′) are consistent with the presence of the ethylidene side chain in the akuammiline half. Assignment of the NMR resonances with the 2D NMR data (COSY, HMBC) led to identification of the macroline (NCHCH2, NCHCH2CHCHCH2O, CH3CHCH, OCHOH) and akuammiline (NCH2CH2, CHCH2CHCH, CH3CHC, NCH2) moieties linked by the methine C-19 as shown in Figure 12. The three-bond correlation from CH3-18 to C-20 and C-10′ is consistent with the link from C-19 of the macroline unit to C-10′ of the akuammiline unit. The resonance of the methyl ester attached to C-16′ was seen at δ 3.74, the lack of unusual shielding indicating that the carbomethoxy group is directed away from the aromatic ring. The observed NOE between H-14β and H-20α requires both to be equatorially oriented (Figure 13). The substitution at C20 is therefore β. In addition, the resonance of H-21 was a

5′

6′

9′ 12′ 14′

15′ 16′ 18′ 19′ 21′

N(1′)− Me CO2Me′ 11′-OMe CH2Cl

d (7.0) m m s s

d (7.0) m m d (2.9) s

9

10

7.18, m

6.19, s 3.52, s

7.18, m 1.27, br d (13.0) 3.10, m 1.37, m 2.09, m 3.48, dd (11.0, 4.0) 4.59, br t (11.0) 1.08, d (7.0) 3.24, m 1.33, m 5.40, s 3.37, s

1.03, d (7.0) 3.39, q (7.0)

2.27, s

2.32, s

2.26, s

2.30, br s

2.24, br s 3.96, br d (5.0) 2.56, dd (13.0, 6.5) 3.63, m

2.48, s 4.27, m

2.82, s 4.61, br d (5.0) 3.27, dd (12.0, 6.0) 4.39, m

2.54, br s 4.57, br d (5.0)

0.86, br d (13.0) 2.91, m 6.40, s 5.29, s 1.54, br d (13.0) 2.32, m

1.26, m

3.51, m 2.78, d (3.6) 1.48, dd (7.0, 2.0) 5.38, br q (7.0) 2.90, m

3.58, m 2.85, d (4.0) 1.48, dd (7.0, 2.0) 5.47, br q (7.0) 3.03, m

3.89, br d (16.0) 2.29, s

4.00, m

3.74, s 3.62, s

3.67, s

2.71, m 3.91, m

3.03, 6.45, 6.13, 1.64,

m s s m

2.35, m

2.62, s

1.37, dd (16.0, 6.0) 3.03, m 6.80, s 6.14, s 1.41, m 1.72, td (13.0, 4.0) 3.60, m 2.93, d (3.6) 1.57, dd (7.0, 2.0) 5.63, br q (7.0) 3.80, m 4.44, br d (14.0) 2.79, s 3.79, s 3.61, s

3.27, m 4.13, br t (12.0) 0.85, m 3.13, m 6.38, s 5.67, s 1.91, br d (14.0) 3.35, br d (14.0) 3.65, m 2.83, d (3.0) 1.56, dd (7.0, 2.0) 5.79, br q (7.0) 4.21, br d (14.0) 5.06, br d (14.0) 2.41, br s 3.79, 3.72, 5.89, 6.36,

s br s d (9.0) d (9.0)

a

Assignments based on COSY, HSQC, and NOESY. CDCl3, 600 MHz.

G



Article

Table 4. 13C NMR Spectroscopic Data of 7−10a



singlet (J20−21 = 0 Hz), requiring H-21 to be also equatorial or β-oriented (and the 21-OH to be axial or α-oriented, Figure 13). Determination of the configuration at C-19 by NMR however was not possible on account of free rotation about the C-19−C-20 bond. Angustilongine A (7) is related to the known angusticraline by loss of the 10-methoxy substituent.13 The configuration at C-19 in angusticraline, however, was not determined in the previous report. In the present instance, the configuration at C-19 as well as the absolute configuration of 7 was established by X-ray diffraction analysis (Figure 14). Angustilongine B (8) was isolated as a light yellowish oil with [α]25D −33 (c 0.1, CHCl3). The IR spectrum showed an absorption band at 1736 cm−1 due to the presence of an ester carbonyl function but no band due to OH, which was seen in 7, while the UV spectrum was similar to that of 7, showing a composite of absorption bands for indole and dihydroindole chromophores (229, 250, and 294 nm). The HRMS data showed an [M + H]+ ion at m/z 675.3932, which analyzed for C42H50N4O4 + H. The 1H and 13C NMR data of 8 showed a general similarity to those of compound 7, indicating a bisindole with similar monomeric halves (macroline and akuammiline) to compound 7. As in the case of 7, the 1H NMR data (Table 3) showed the presence of an unsubstituted indole moiety (δ 7.11−7.50, macroline), another indole ring substituted at C-10′ and C-11′ (δ 6.45, 6.13; akuammiline), a methyl ester group (δ 3.67, akuammiline), an ethylidene side chain (δ 5.47, 1.48; akuammiline), three methyl singlets, corresponding to two N-1−Me (δ 3.62, macroline; 2.62, akuammiline), an N-4−Me (δ 2.32, macroline), a methyl doublet (δ 1.24, C-18, macroline) that is part of a CH3CHCHCH(O)−O fragment, an oxymethylene due to C17 (δ 3.97, 4.18; macroline), and a deshielded H (δ 5.44) due to an acetal function (macroline). The 13C NMR data (Table

a b

position

7

8

9

10

2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 N(1)−Me N(4)−Me 2′ 3′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 18′ 19′ 20′ 21′ N(1′)−Me CO2Me′ CO2Me′ 11′-OMe CH2Cl

132.9 54.2 55.3 22.2 106.5 126.3 117.6 118.2 120.4 108.6 136.8 32.5 25.3 38.2 59.6 19.5 NDb 48.2 93.7 28.6 41.8 78.9 47.4 51.0 31.7 42.4 132.3 118.6 124.3 156.2 92.9 151.6 33.9 34.4 52.9 13.0 118.6 140.0 54.9 33.2 51.4 173.0 55.2

133.0 53.5 54.4 23.0 106.5 126.5 118.0 118.2 120.9 108.8 137.1 32.9 27.7 39.4 67.5 20.7 32.9 39.1 97.5 29.0 41.7 68.0 47.6 50.1 30.0 42.3 133.0 120.4 118.2 151.0 98.6 152.5 32.9 33.8 52.7 13.1 120.9 137.1 54.4 34.3 51.5 172.5

133.5 53.6 55.2 22.9 106.2 126.6 118.0 118.7 120.6 108.8 137.0 33.5 28.6 40.6 66.4 20.4 34.1 119.4 140.1 29.0 41.7 79.1 70.1 66.8 29.9 40.8 130.0 120.3 126.0 156.5 94.7 151.0 31.7 32.2 51.9 13.5 124.0 130.0 72.5 34.7 52.0 172.0 55.6

132.3 54.4 55.5 22.4 106.2 126.1 117.8 118.5 129.8 108.6 136.9 31.7 25.1 38.2 59.2 20.2 NDb 46.9 93.3 28.9 41.6 76.7 58.7 57.2 28.0 40.3 129.9 119.1 126.1 156.9 94.8 150.2 31.7 32.2 51.4 13.5 126.1 128.2 63.5 35.3 52.0 171.6 55.6 72.6

Assignments based on DEPT, HSQC, and HMBC. CDCl3, 150 MHz. Not detected.

4) showed a total of 42 carbon resonances, comprising six methyls, seven methylenes, 18 methines, an oxygenated tertiary carbon, three tertiary carbons linked to nitrogen, an ester carbonyl (δ 172.5), and six quarternary carbon atoms. A distinctly deshielded resonance at δ 97.5 (δH 5.44) is attributed to the carbon of an acetal group. In common with bisindole 7, bisindole 8 also displayed an oxymethylene carbon resonance at δ 67.5 corresponding to C-17 of the macroline unit and olefinic carbon resonances (δ 137.1, 120.9) consistent with the presence of an ethylidene side chain in the lower (akuammiline) half. The main difference in the NMR data is the absence of resonances due to the C-11′ OMe group in 8, although C11′ still retains oxygen substitution (δ 151.0, 3J from H-9′ to CH



Article


an [M + H]+ ion at m/z 705.4041, which analyzed for C43H52N4O5 + H. The UV spectrum (230, 252, and 295 nm) was similar to those of 7 and 8, suggesting the presence of similar chromophores, while the IR spectrum indicated the presence of an ester carbonyl (1741 cm−1). Examination of the 1 H, 13C, and 2D NMR (Figure 17) data indicated a macroline−

Figure 14. X-ray crystal structure of 7.

11′ in the HMBC spectrum) (Figure 15). In addition, the molecular formula of 8 differs from that of 7 by 32 mass units


akuammiline bisindole that bears a close similarity to 7. Thus, the 1H NMR data (Table 3) also showed the presence of four aromatic hydrogens (δ 7.10−7.48, macroline), two isolated aromatic singlets (δ 6.14, 6.80, 10′,11′-disubstituted akuammiline), an aromatic methoxy group (δ 3.61, akuammiline), a methyl ester group (δ 3.79, akuammiline), three methyl singlets corresponding to two N-1−Me (δ 3.52, 2.79) and an N-4−Me (δ 2.26), a methyl doublet that is part of a CH3CH fragment (δ 1.03, C-18, macroline), an oxymethylene due to C-17 (δ 3.94, 4.24; macroline), and an ethylidene side chain (δ 5.63, 1.57, akuammiline). The 13C NMR data (Table 4) showed a total of 43 carbon resonances comprising seven methyls, seven methylenes, 17 methines, an oxygenated tertiary carbon, three tertiary carbons linked to nitrogen, an ester carbonyl (δ 172.0), and seven quarternary carbon atoms. There are, however, several notable differences in the NMR data of 9 when compared to 7. First, the resonance due to H-20 was not seen in the spectrum of 9, while H-21 is an olefinic-H at δ 6.19. In the 13C NMR spectrum, C-20 of 9 is a quaternary olefinic carbon at δ 119.4, while C-21 is an oxygen-substituted olefinic methine at δ 140.1. These changes suggest the presence, in 9, of a trisubstituted double bond involving C-20 and C-21 constituting part of an enol ether containing ring E. Another notable observation is that the resonances due to H-3′, H-5′, and H-21′ of 9 are significantly deshielded when compared to


(or loss of CH3 and OH compared to 7) as well as requiring the presence of an additional ring. Bond formation between the oxygen at C-11′ and the hemiacetal C-21 in 7 (demethylation followed by elimination of water) forges an additional ring F, which incorporates an acetal function as a result of fusion of two tetrahydropyran moieties as shown in 8. The structure is entirely consistent with the full HMBC (Figure 15) and NOESY (Figure 16) data. Since additional ring formation has resulted in the incorporation of the methyl-bearing C-19 as part of a new tetrahydropyran ring, determination of the relative configuration at C-19 is now possible based on NMR data. The E/F ring junction is cis based on the J20−21 coupling of 2.9 Hz. Additionally, the observed NOEs for H-18/H-16 and for H-9′/ H-19, H-18 (Figure 16) allow the orientation of H-19 to be assigned as α or equatorial (19S). The assignment of the configuration of C-19 in 8 as S by NMR data is also consistent with its purported origin from 7 (for which the absolute configuration has been established by X-ray analysis, Figure 14, 19S) since the configurational integrity of C-19 is not affected by the transformation from 7 to 8. Compound 9 (angustilongine C) was obtained as a yellowish oil with [α]25D +16 (c 0.1, CHCl3). The HRESIMS of 9 showed I



Article

Scheme 1. Putative Biosynthetic Pathway to 7, 8, and 9

Table 5. Cytotoxic Effects of Bisindole Alkaloids 7 and 8a IC50 ± SD (μM)b compound angustilongine A (7) angustilongine B (8) vincristine cisplatin verapamil

KB/S

KB/VJ300

KB/VJ300c

PC-3

LNCaP

MCF7

MDA-MB231

HT-29

HCT 116

A549

2.0 ± 0.5

>10

>10

>10

6.9 ± 0.1

3.8 ± 0.5

5.2 ± 0.9

0.7 ± 0.1

6.6 ± 1.9

8.3 ± 0.1

0.7 ± 0.3

6.7 ± 0.3

0.9 ± 0.2

7.2 ± 0.3

3.9 ± 0.4

3.8 ± 0.2

3.3 ± 0.4

0.3 ± 0.0

5.2 ± 0.5

6.5 ± 0.1

0.3 ± 0.1 (nM)

3.9 ± 0.9 >10

7.1 ± 0.9

6.2 ± 0.6

3.8 ± 0.4

>10

4.5 ± 0.6

>10

0.1 ± 0.1

a

KB: human oral epidermoid carcinoma; KB/S: vincristine-sensitive KB carcinoma; KB/VJ300: vincristine-resistant KB carcinoma; PC-3 and LNCaP: human prostate carcinoma; MCF7 and MDA-MB-231: human breast adenocarcinoma; HT-29 and HCT 116: human colorectal carcinoma; A549: human lung carcinoma. bData expressed as mean ± SD of three independent experiments. cWith added vincristine, 0.1 μM, which did not affect the growth of the KB/VJ300 cells.

those of 7. The same is true of the C-3′, C-5′, and C-21′ resonances of 9 versus 7. These observations and the molecular formula of 9 suggest that 9 is the N-4′-oxide of the parent bisindole, 9a, which is as yet unknown and which was not isolated in this study. Compound 10 (angustilongine D) was obtained as a light yellowish, amorphous solid with [α]25D +12 (c 0.3, CHCl3). The HRESIMS showed an M+ peak at m/z 755.3949, which was consistent with the molecular formula C44H56N4O5Cl ([M + 2]+ isotope peak seen at m/z 757.3948), while the UV and IR data were similar to those of 7. The 1H NMR (Table 3) and 13 C NMR (Table 4) data were also similar to those of 7 except for the presence of two additional signals due to a pair of geminal hydrogens (δ 5.89 and 6.36, J = 9 Hz; δC 72.6) consistent with the presence of a CH2Cl group linked to N-4′, which were absent in the 1H NMR spectrum of 7. In addition, the downfield shift of the resonances for C-3′, C-5′, and C-21′ (δ 58.7, 57.2, and 63.5, respectively) of the akuammiline half in 10 indicated that they are linked to a quaternary nitrogen (N4′). Compound 10 is therefore the chloromethylene adduct of 7, an artifact probably formed during crystallization of 7 in solvent mixtures containing dichloromethane.11

The bisindoles 7−9 possess a similar mode of connection of the monomeric units to that in lumutinines C and D9 and perhentisine A.11 A plausible pathway to these alkaloids is shown in Scheme 1, involving conjugate addition of cabucraline (12) via its nucleophilic C-10′ onto the hypothetical macroline 11 (a ring E-seco-talcarpine), to give the hydroxy-aldehyde 13. Ring closure to the hemiacetal 7 followed by ketalization gives 8. Alternatively dehydration of 7 yields 9a. Angustilongines A (7) and B (8) showed pronounced in vitro growth inhibitory activity against an array of human cancer cell lines, including KB, vincristine-resistant KB, PC-3, LNCaP, MCF7, MDA-MB-231, HT-29, HCT 116, and A549 cells (Table 5). Particularly strong effects were shown by both compounds against HT-29 cells, while compound 8 was shown to reverse multidrug resistance in vincristine-resistant KB cells.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Mel-Temp melting point apparatus or an Electrothermal IA9100 digital melting point apparatus and are uncorrected. Optical rotations were determined on a JASCO P-1020 automatic digital polarimeter. UV spectra were obtained on a Shimadzu UV-2600 spectrophotometer. ECD spectra were measured with a JASCO J-815 CD spectrometer. IR spectra were recorded on a J



Article

Angustilongine A (7): light yellowish block crystals (CH2Cl2− hexanes); mp >199 °C (dec); [α]25D +3 (c 0.4, CHCl3); UV (EtOH) λmax (log ε) 212 (4.64), 232 (4.54), 255 (3.90), and 295 (3.99) nm; IR (dry film) νmax 3360, 1741 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 707.4180 [M + H]+ (calcd for C43H54N4O5 + H, 707.4172). Angustilongine B (8): light yellowish oil; [α]25D −33 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 229 (4.48), 250 (3.87), and 294 (3.92) nm; IR (dry film) νmax 1736 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRDARTMS m/z 675.3932 [M + H]+ (calcd for C42H50N4O4 + H, 675.3910). Angustilongine C (9): light yellowish oil; [α]25D +16 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 230 (4.58), 252 (4.03), and 295 (4.03) nm; IR (dry film) νmax 1741 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 705.4041 [M + H]+ (calcd for C43H52N4O5 + H, 705.4016). Angustilongine D (10): light yellowish, amorphous solid; [α]25D +12 (c 0.3, CHCl3); UV (EtOH) λmax (log ε) 212 (4.70), 230 (4.55), 250 (3.96), and 295 (4.04) nm; IR (dry film) νmax 3392, 1739 cm−1; 1 H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 755.3949 M+ (calcd for C44H56N4O5Cl, 755.3939). Computational Method. The conformations of compound 6 (3S, 5S, 7S, 15S, 16R, 19S) were obtained by Spartan’14 software18 using the MMFF94 force field. Conformers occurring within a 10 kcal mol−1 energy window from the global minimum were imported into the Gaussian 09 software19 for DFT-level geometry optimization and frequency calculation using the B3LYP functional with a basis set of 631G(d). TDDFT electronic circular dichroism (ECD) calculations were performed at the B3LYP/6-311++G(d,p) level with the optimized conformers using a PCM solvation model for MeOH. The ECD curve for each optimized conformer was weighted by Boltzmann distribution after UV correction, and the overall ECD curves were produced by SpecDis, version 1.64, software.20 X-ray Crystallographic Analysis of 6 and 7. X-ray diffraction analysis was carried out on a Rigaku Oxford (formerly Agilent Technologies) SuperNova Dual diffractometer with Mo Kα (λ = 0.710 73 Å) or Cu Kα (λ = 1.541 84 Å) radiation at 100 K or rt. The structures were solved by intrinsic phasing methods (SHELXT-2014) and refined with full-matrix least-squares on F2 (SHELXL-2018). All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Crystallographic data for 6 and 7 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 (0)1223-336033, or e-mail: [email protected]. uk). Crystallographic data of 6: colorless needles, C20H26N2O4·H2O, Mr = 376.44, monoclinic, space group P21, a = 8.2777(8) Å, b = 11.3247(9) Å, c = 10.2020(7) Å, β = 94.708(8)°, V = 953.13(14) Å3, Z = 2, Dcalcd = 1.312 g cm−3, crystal size 0.2 × 0.03 × 0.01 mm3, F(000) = 404, Cu Kα radiation (λ = 1.541 84 Å), T = 293(2) K. The final R1 value is 0.0714 (wR2 = 0.1866) for 3766 reflections [I > 2σ(I)]. The Flack,21 Hooft,22 and Parsons23 parameters were x = 0.1(4), y = 0.1(3), and z = 0.1(2), respectively. For the inverted structure, the Flack, Hooft, and Parsons parameters were x = 0.9(4), y = 0.9(3), and z = 0.9(2), respectively, from which it follows that the correct enantiomer is the one depicted in Figure 11. CCDC number: 1822775. Crystallographic data of 7: light yellowish block crystals, C43H54N4O5·2CHCl3, Mr = 945.63, monoclinic, space group P21, a = 9.4917(6) Å, b = 12.8453(9) Å, c = 18.5275(10) Å, β = 99.113(6)°, V = 2230.4(2) Å3, Z = 2, Dcalcd = 1.408 gcm−3, crystal size 0.50 × 0.25 × 0.16 mm3, F(000) = 992, Mo Kα radiation (λ = 0.710 73 Å), T = 100(2) K. The final R1 value is 0.0445 (wR2 = 0.1216) for 7713 reflections [I > 2σ(I)]. The absolute configuration of compound 7 was determined on the basis of the Flack21 parameter [x = 0.03(7)], refined using 2364 Friedel pairs. CCDC number: 1822774. Cytotoxicity Assays. Cytotoxicity assays were carried out following the published procedures.16

PerkinElmer RX1 FT-IR or Spectrum 400 FT-IR/FT-FIR spectrophotometer. 1H and 13C NMR spectra were recorded in CDCl3 using tetramethylsilane as internal standard on JEOL JNM (ECA or ECX 400 MHz) and BrukerAvance III spectrometers (400 or 600 MHz). ESIMS and HRESIMS were obtained on an Agilent 6530 Q-TOF spectrometer, and DARTMS were recorded on a JEOL Accu TOFDART mass spectrometer. Plant Material. Plant material (A. penangiana) was collected in Penang (Bukit Bendera), Malaysia, and was identified by one of the authors (K. T. Yong). Herbarium voucher specimens (KLU 49466, KLU 49468) are deposited at the Herbarium, University of Malaya. Extraction and Isolation. Extraction of alkaloids from the dried and ground leaf material (3 kg) was carried out in the usual manner by partitioning the concentrated ethanolic extract with 3% tartaric acid. The basic fraction obtained was first chromatographed over SiO2 (MeOH−CHCl3) to furnish five main fractions (I−V). Fraction II was rechromatographed over SiO2 (MeOH−CH2Cl2) to furnish eight subfractions (IIa−IIh), after which rechromatography of fraction IIb using LH-20 (MeOH) gave three subfractions, IIbi−IIbiii. A further purification of fraction IIbi by repeated preparative radial chromatography (SiO2; EtOAc−hexanes, MeOH−CHCl3) gave 1 and 2. Column chromatography of fraction III (SiO2; MeOH−CHCl3) gave eight subfractions (IIIa−IIIh). Repeated purification of fraction IIIc by preparative radial chromatography (SiO2; EtOAc−MeOH, MeOH− CHCl3) gave 6 and 7−10. Processing of a second batch of leaf material (2.4 kg, MeOH) and initial chromatographic fractionation of the basic fraction as before gave eight main fractions (I−VIII). Repeated purification of fraction IV by preparative radial chromatography (SiO2; Et2O−hexanes, Et2O, EtOAc, CHCl3−MeOH) gave 6. Compounds 3−5 were obtained following repeated fractionation of fraction V by preparative radial chromatography (SiO2; EtOAc−hexanes, CHCl3− hexanes, Et2O−hexanes, CHCl3, Et2O−MeOH, CHCl3−MeOH). All solvent systems used for preparative radial chromatography were NH3saturated. The yields (mg kg−1) of the alkaloids from the leaf were as follows: 1 (1.53), 2 (1.03), 3 (1.29), 4 (0.67), 5 (2.88), 6 (3.70), 7 (20.13), 8 (0.8), 9 (0.47), and 10 (1.7). Vincamaginine A (1): light yellowish oil; [α]25D −172 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 215 (4.74), 275 (4.37), and 298 sh (4.10) nm; IR (dry film) νmax 1733, 1652 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 741.3027 [M + H]+ (calcd for C41H44N2O11 + H, 741.3023). Vincamaginine B (2): light yellowish oil; [α]25D −379 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 214 (4.72), 280 (4.42), and 297 sh (4.31) nm, addition of 0.1 M NaOH resulted in a shift from 280 to 333 nm; IR (dry film) νmax 1733, 1651 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 727.2873 [M + H]+ (calcd for C40H42N2O11 + H, 727.2867). Alstonisinine A (3): white, amorphous solid; [α]25D +183 (c 0.2, MeOH); UV (EtOH) λmax (log ε) 225 (4.12) and 261 (4.18) nm; IR (ATR) νmax 3210, 1682, 1605 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRDARTMS m/z 341.1511 [M + H]+ (calcd for C19H20N2O4 + H, 341.1501). Alstonisinine B (4): white, amorphous solid; [α]25D +113 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (4.19) and 253 (3.99) nm; IR (ATR) νmax 3295, 1702, 1609 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRDARTMS m/z 341.1500 [M + H]+ (calcd for C19H20N2O4 + H, 341.1501). Alstonisinine C (5): light yellowish oil; [α]25D +180 (c 0.4, CHCl3); UV (EtOH) λmax (log ε) 225 (3.99) and 256 (4.09) nm; IR (dry film) νmax 3403, 1708, 1616 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRDARTMS m/z 355.1666 [M + H]+ (calcd for C20H22N2O4 + H, 355.1658). Alstonoxine F (6): light yellowish, amorphous solid and subsequently colorless needles (MeCN−MeOH); mp >169 °C (dec); [α]25D −20 (c 0.3, CHCl3); UV (EtOH) λmax (log ε) 214 (4.34), 255 (3.69), and 289 (3.30) nm; ECD (MeOH), λmax (Δε) 210 (−18.94), 232 (16.82), 257 (−5.95), 278 (−0.57), and 290 (−0.90); IR (dry film) νmax 3392, 1705, 1646 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 359.1971 [M + H]+ (calcd for C20H26N2O4 + H, 359.1971). K



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(19) 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, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision C.01; Gaussian Inc.: Wallingford, CT, 2010. (20) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y. SpecDis, Version 1.64; University of Wuerzburg: Germany, 2015. (21) (a) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, 39, 876−881. (b) Flack, H. D.; Bernardinelli, G. J. Appl. Crystallogr. 2000, 33, 1143−1148. (c) Flack, H. D.; Bernardinelli, G. Chirality 2008, 20, 681−690. (22) (a) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr. 2008, 41, 96−103. (b) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr. 2010, 43, 665−668. (23) (a) Parsons, S.; Flack, H. D.; Wagner, T. Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 2013, 69, 249−259. (b) Parsons, S.; Wagner, T.; Presly, O.; Wood, P. A.; Cooper, R. I. J. Appl. Crystallogr. 2012, 45, 417−429.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00170. 1 H and 13C NMR spectra for compounds 1−10 (PDF) X-ray crystallographic data in CIF format for compound 6 (CIF) X-ray crystallographic data in CIF format for compound 7 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 603-79674266. Fax: 603-79674193. E-mail: tskam@um. edu.my. ORCID

Toh-Seok Kam: 0000-0002-4910-6434 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the University of Malaya (PG126-2015A) and MOHE Malaysia (FP043-2015A) for financial support and K.W. Chong for TDDFT calculations.

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REFERENCES

(1) Markgraf, F. Blumea 1974, 22, 20−29. (2) Sidiyasa, K. Taxonomy, Phylogeny, and Wood Anatomy of Alstonia (Apocynaceae); Rijksherbarium/Hortus Botanicus: The Netherlands, 1998; Blumea Supplement 11, pp 1−230. (3) Whitmore, T. C. In Tree Flora of Malaysia; Longman: London, 1972; Vol. 2, pp 7−12. (4) Middleton, D. J. In Flora of Peninsular Malaysia, Series II: Seed Plants; Kiew, R., Chung, R. C. K., Saw, L. G., Soepadmo, E., Boyce, P. C., Eds.; Forest Research Institute Malaysia: Kepong, Malaysia, 2011; Vol. 2. (5) Burkill, I. H. In A Dictionary of Economic Products of the Malay Peninsula; Ministry of Agriculture and Co-operatives: Kuala Lumpur, 1966. (6) Perry, L. M.; Metzger, J. In Medicinal Plants of East and Southeast Asia; MIT Press: Cambridge, 1980. (7) Kam, T. S. In Alkaloids: Chemical and Biological Perspectives; Pelletier, S. W., Ed.; Pergamon: Amsterdam, 1999; Vol. 14, pp 285− 435. (8) Kam, T. S.; Choo, Y. M. In The Alkaloids; Cordell, G. A., Ed.; Academic Press: Amsterdam, 2006; Vol. 63, pp 181−337. (9) Lim, S. H.; Tan, S. J.; Low, Y. Y.; Kam, T. S. J. Nat. Prod. 2011, 74, 2556−2562. (10) Lim, S. H.; Low, Y. Y.; Tan, S. J.; Lim, K. H.; Thomas, N. F.; Kam, T. S. J. Nat. Prod. 2012, 75, 942−950. (11) Tan, S. J.; Lim, K. H.; Subramaniam, G.; Kam, T. S. Phytochemistry 2013, 85, 194−202. (12) Tan, S. J.; Lim, J. L.; Low, Y. Y.; Sim, K. S.; Lim, S. H.; Kam, T. S. J. Nat. Prod. 2014, 77, 2068−2080. (13) Ghedira, K.; Zeches-Hanrot, M.; Richard, B.; Massiot, G.; Le Men-Olivier, L.; Sevenet, T.; Goh, S. H. Phytochemistry 1988, 27, 3955−3962. (14) Kam, T. S.; Choo, Y. M. Tetrahedron 2000, 56, 6143−6150. (15) Kam, T. S.; Choo, Y. M. J. Nat. Prod. 2004, 67, 547−552. (16) Lim, S. H.; Low, Y. Y.; Sinniah, S. K.; Yong, K. T.; Sim, K. S.; Kam, T. S. Phytochemistry 2014, 98, 204−215. (17) Lim, S. H.; Low, Y. Y.; Subramaniam, G.; Abdullah, Z.; Thomas, N. F.; Kam, T. S. Phytochemistry 2013, 87, 148−156. (18) Spartan’14; Wavefunction, Inc.: Irvine, CA, 2014. L


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