Phytochemistry 117 (2015) 317–324

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Biologically active vallesamine, strychnan, and rhazinilam alkaloids from Alstonia: Pneumatophorine, a nor-secovallesamine with unusual incorporation of a 3-ethylpyridine moiety Jun-Lee Lim a, Kae-Shin Sim b, Kien-Thai Yong b, Bi-Juin Loong c, Kang-Nee Ting c, Siew-Huah Lim a, Yun-Yee Low a, Toh-Seok Kam a,⇑ a

Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Biomedical Sciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia b

a r t i c l e

i n f o

Article history: Received 22 January 2015 Received in revised form 22 April 2015 Accepted 22 June 2015

Keywords: Alstonia pneumatophora Alstonia rostrata Apocynaceae Indole alkaloids NMR Cytotoxicity Vasorelaxation

a b s t r a c t Four alkaloids comprising two vallesamine, one strychnan, and one pyranopyridine alkaloid, in addition to 32 other known alkaloids were isolated from two Malayan Alstonia species, Alstonia pneumatophora and Alstonia rostrata. The structures of these alkaloids were determined using NMR and MS analyses, and in one instance, confirmed by X-ray diffraction analysis. The nor-6,7-secovallesamine alkaloid, pneumatophorine, is notable for an unusual incorporation of a 3-ethylpyridine moiety in a monoterpenoid indole. The rhazinilam-type alkaloids (rhazinicine, nor-rhazinicine, rhazinal, and rhazinilam) showed strong cytotoxicity toward human KB, HCT-116, MDA-MB-231, and MRC-5 cells, while pneumatophorine, the uleine alkaloid undulifoline, and the strychnan alkaloids, N4-demethylalstogustine and echitamidine, induced concentration dependent relaxation in phenylephrine-precontracted rat aortic rings. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction

2. Results and discussion

Plants of the genus Alstonia are distributed over the tropical regions of Central America, Africa, and Asia (Markgraf, 1974; Sidiyasa, 1998). These plants are typically shrubs or trees and are usually rich in alkaloids (Kam, 1999; Kam and Choo, 2006). About seven species (local name Pulai) occur in Peninsular Malaysia (Middleton, 2011), some of which are used in traditional medicine (Burkill, 1966; Perry and Metzger, 1980). A number of the Alstonia bisindole alkaloids have been shown to possess antiproliferative and antimalarial properties (Kam et al., 2008; Keawpradub et al., 1999; Wright et al., 1993). As part of our systematic study of the Malaysian members (Ku et al., 2011; Lim et al., 2011, 2012, 2013; Tan et al., 2010a, 2014), we investigated the alkaloid content of two species, viz., Alstonia pneumatophora Backer ex Den Berger and Alstonia rostrata C.E.C. Fisch, and herein report the results. Plants belonging to the former species are usually encountered in swampy areas (Middleton, 2011).

The basic fraction from the EtOH extract of A. pneumatophora yielded a total of 22 alkaloids, of which three (1, 4, 5) are new, while a total of 21 alkaloids were isolated from A. rostrata, of which one, the monoterpene alkaloid 6 is new (Fig. 1). Compound 1 (pneumatophorine) was isolated as a light yellowish oil with [a]25 D 41 (CHCl3, c 0.57). The IR spectrum showed bands due to NH/OH (3380 cm1) and ester carbonyl functions (1725 cm1), while the UV spectrum showed characteristic indole absorptions at 231, 282, and 290 nm. The ESIMS showed an [M+H]+ peak at m/z 434, the odd mass (M+ m/z 433) indicating the presence of a third nitrogen. This was confirmed by 13C NMR and HRESIMS data, which established the molecular formula as C26H31N3O3. The 1H (Table 1) and 13C NMR (Table 2) spectra of 1 appeared complex and indicated the presence of two sets of signals with very similar or coincident chemical shifts, corresponding to the presence of two unresolvable components of nearly identical structure (1a:1b = 0.55:0.45 in CDCl3). This was especially true for the 13 C NMR spectrum where the majority of the signals appear to occur in pairs with very similar chemical shifts. Thus of a total of

⇑ Corresponding author. E-mail address: [email protected] (T.-S. Kam). http://dx.doi.org/10.1016/j.phytochem.2015.06.024 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of compounds 1–6.

26 carbons as required by the molecular formula, 24 of the resonances appeared as pairs with very similar chemical shifts (average Dm for all paired 13C signals is 0.11 ppm, Table 2) and were therefore indistinguishable, while three resonances were overlapped or coincident. In the 1H NMR spectrum, 12 resonances were overlapped or coincident, while 12 resonances appeared as pairs with very similar chemical shifts (average Dm for all paired 1H signals is 0.05 ppm, Table 1). Assignment of the 1H resonances into the two respective sets required spectra obtained at 600 MHz, combined with the application of DEPT and 2-D HSQC experiments. The possibility that these components correspond to a pair of equilibrating conformers was ruled out based on the following observations. First, variable temperature NMR experiments showed that the spectrum remained unchanged when the temperature was raised (no evidence of any signal broadening up to 80 °C in toluene-d8). In addition, the ratio of the two components as determined by 1H NMR spectroscopy remained constant as the temperature was increased or lowered. Furthermore, the ratio of the two components was also essentially invariant when the spectra were obtained in different solvents (1a:1b = 0.55:0.45 in CDCl3, CD2Cl2, toluene-d8, benzene-d6) (Garrido et al., 2003). Treatment of the mixture with Ac2O/pyridine yielded a mixture of the corresponding O-acetyl derivatives (2a, 2b), which also proved resistant to chromatographic resolution and which also occurred in essentially the same proportions (2a:2b  6:4) as determined by 1H NMR. These observations constituted strong evidence that the two components shown in the NMR spectrum do not correspond to a pair of equilibrating conformers, but represents a mixture of two diastereomers which could not be further resolved. Attempts to separate the two components by conventional column chromatography, TLC, radial chromatography, Sephadex LH20, and HPLC, including chiral-phase HPLC, were in every instance singularly unsuccessful. Similar behavior has also been previously documented with other alkaloids (Lim and Kam, 2009; Lim et al., 2009; Tan et al., 2012). The 1H NMR data of pneumatophorine (Table 1, 1a) showed the presence of eight aromatic resonances (d 7.08–8.39), an indolic NH (d 9.88), a methyl ester group (d 3.74), a hydroxymethyl group (d 4.20, 4.40; d, J = 12 Hz), a CHMe (d 1.26, 3.28), and an ethylidene side-chain (d 1.64, 5.54). The 13C NMR data (Table 2, 1a) showed

a total of 26 carbon resonances, comprising three methyl, four methylene, twelve methine, four quaternary, one ester carbonyl, and two tertiary carbons linked to the indolic nitrogen (corresponding to C-2 and C-13). The presence of a hydroxymethyl group was supported by the ready conversion of 1 to its O-acetyl derivative 2. The NMR data of 2 also showed the same doubling of signals corresponding to the presence of two diastereomers (See Section 4). Of the nine aromatic hydrogen resonances, four are contiguous as shown by the COSY spectrum and can be readily assigned to the aromatic hydrogens (H-9, H-10, H-11, H-12) of the indole moiety. The NOE observed between the aromatic doublet at d 7.36 and the indolic NH at d 9.88, facilitated the assignment of this doublet to H12, and the doublet at d 7.52 to H-9. Furthermore an NOE was observed between H-9 and the aromatic-H singlet at d 6.48 (dC 101.94, Fig. 2), indicating that these hydrogens are proximate and suggesting that this relatively shielded aromatic H is due to H-7 of a 6,7-seco monoterpenoid indole (Tan et al., 2010b; Yamauchi et al., 1990; Zeches et al., 1987). This was further supported by the COSY spectrum, which showed long-range coupling between this hydrogen to the indolic NH. Of the remaining four aromatic resonances, three correspond to a CH–CH–CH fragment as shown by the COSY data (Fig. 3), while one corresponds to an isolated aromatic hydrogen. Since five additional aromatic carbon resonances were observed and the MS data required the presence of a third nitrogen, a 3-substituted pyridine moiety is suggested. The relatively deshielded aromatic resonance at d 8.34 (dC 149.2) is characteristic of the a-hydrogen (a-carbon) of a pyridine moiety, likewise, the aromatic resonance at d 8.39 (dC 148.6). These assignments were supported by the HMBC data (Fig. 3). The alkyl substituent at C-30 of the pyridine moiety is readily deduced to be a CHCH3 from the 1H NMR spectrum. This group is attached to N-4 of the monoterpenoid indole as shown by the HMBC data (Fig. 3). The identity of the monoterpenoid indole half of the alkaloid was deduced by linking the various substructures shown by the COSY spectrum with the help of the HMBC data (Fig. 3). The presence of an aromatic hydrogen at C-7 alluded to earlier suggested a nor-6,7-seco-vallesamine type alkaloid, of which a number occur in Alstonia (Tan et al., 2010b; Yamauchi et al., 1990; Zeches et al., 1987). The quaternary carbon at ca. dC 58 is characteristic of C-

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J.-L. Lim et al. / Phytochemistry 117 (2015) 317–324 Table 1 H NMR spectroscopic data for compounds 1, 3–6.

Table 2 C NMR spectroscopic data for compounds 1, 3–6.

1

H

1aa

1ba

3b

4b

5b

6b

C

1aa

1ba

3b

4b

5b

6b

1 3

– 2.01 m 2.39 m

– 2.01 m 2.39 m

– 2.58 m 2.70 m

– 3.93 m

8.25 s 8.51 d (6)

4







– 1.99 td (12, 3) (b) 2.81 m (a) –



5









6









7 9 10

6.48 br s 7.52 d (8) 7.08 m

6.52 br s 7.54 d (8) 7.08 m

11

7.16 m

7.16 m

12

7.36 d (8) 1.69 m 1.86 m

7.35 d (8) 1.69 m 1.86 m

6.42 s 7.51 d (8) 7.07 t (8) 7.15 t (8) 7.38 d (8) 1.48 m 1.85 m

6.26 br s 7.53 br d (7.5) 7.07 td (7.5, 1) 7.13 td (7.5, 1) 7.31 br d (7.5) 1.53 m (a) 1.62 m (b)

2.86 m (b) 3.09 m (a) 1.85 dd (13, 7) (a) 2.94 m (b) – 7.16 d (7.5)

8.04 d (6) –

3.49 br d (7) – 4.20 d (12) 4.40 d (12) 1.64 d (7)

3.57 br d (7) – 4.27 d (12) 4.43 d (12) 1.64 d (7)

3.42 br d (8) – 4.21 d (11) 4.39 d (11) 1.61 d (7)

2.51 m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 CO2Me CO2Me N4Me 20 30 40 50 60 70 80

– 135.97c 46.24d – – – 101.94 127.66e 120.30f 119.78g 121.85h 111.19 136.27 26.32i 39.35 57.78j 66.00 13.92k 126.20l 133.16m 57.96n 52.64o 174.35p – 149.24 133.16q 134.96r 123.36s 148.64t 61.40u 19.27v

– 135.99c 46.39d – – – 101.98 127.71e 120.33f 119.80g 121.87h 111.19 136.55 26.26i 39.19 57.87j 66.00 13.85k 126.45l 133.23m 58.16n 52.61o 174.21p – 149.24 133.23q 134.78r 123.44s 148.71t 61.80u 19.20v

– 136.3 52.1 – – – 102.1 127.6 120.3 119.9 121.9 111.4 136.1 28.5 39.3 57.4 65.9 13.9 126.2 134.2 52.1 52.7 174.5 – – – – – – – –

– 139.6 55.6 – – – 99.8 128.5 120.1 119.9 121.5 110.8 135.7 32.8 44.2 46.6 73.4 67.9 124.5 137.2 65.6 – – 45.7 – – – – – – –

– 171.9 60.7 – 53.8 42.9 56.5 135.3 119.9 121.3 127.8 109.8 144.2 32.0 30.1 98.2 – 22.2 69.7 45.1 47.3 51.3 168.6 – – – – – – – –

144.5 – 150.1 117.4 134.3 106.3 160.0 – 73.9 125.5 20.6 – – – – – – – – – – 51.5 165.4 – – – – – – – –

19

5.54 m

5.54 m

20 21

– 2.71 d (12) 2.89 d (12) 3.74 s 9.88 br s

– 2.87 d (12) 2.92 d (12) 3.73 s 9.84 br s

– 8.34 br s 7.48 br t (7) 7.11 m 8.39 m 3.28 m 1.26 br d (8)

– 8.34 br s 7.48 br t (7) 7.11 m 8.41 m 3.28 m 1.27 br d (8)

5.53 (7) – 3.00 s 3.00 s 3.77 9.92 s – – –

14

15 16 17

18

CO2Me NH N4Me 20 40 50 60 70 80 a b

13

– – – –

q

br br s br

6.88 t (7.5) 7.11 td (7.5, 1) 6.79 d (7.5) 1.37 m (R) 2.02 dt (13, 3) (S) 3.07 m



7.75 s – 5.42 q (7) – 1.66 d (7) –



321 m 3.89 dd (12, 9) 3.99 dd (12, 3.5) (b) 4.21 m (a) 4.34 br d (17) (b) 5.56 br s

– –

– –

1.35 d (6)



3.59 m



– 2.63 d (13) (a) 3.21 d (13) (b) – 8.40 br s

1.80 m 3.22 dd (12, 4.5) (a) 2.29 t (12) (b) 3.75 s 9.01 br s

– –

2.26 s – –

– – –

– – –

– – – –

– – – –

– – – –

a b c-v

CDCl3, 150 MHz. CDCl3, 100 MHz. Interchangeable.

3.84 s –

CDCl3, 600 MHz. CDCl3, 400 MHz.

16, attached to the indole moiety at C-2 and geminally-substituted by methyl ester and hydroxymethyl groups (Perera et al., 1984; Zeches et al., 1987). It remained to link the ethylpyridine substituted piperidine moiety with an ethylidene side-chain at C-20, to the quaternary C-16 via C-15. The structure is in complete agreement with the HMBC (Fig. 3) and NOE (Fig. 2) data. It transpires that pneumatophorine (1) is the 3-ethylpyridine substituted derivative of yunnanensine (3), recently reported from the Chinese Ervatamia yunnanensis (Luo et al., 2007). Since yunnanensine is the nor-6,7-seco derivative of vallesamine, the relative stereochemistry at the stereogenic centers in 1, 3, and vallesamine can be assumed to be similar (i.e., 15S, 16S) which was also consistent with the NOE data of 1 and 3. The NOE data (Fig. 2) also provided confirmation of the E-geometry of the C-19–C-20 double bond, from the observed H-21/H-19 and H-18/H-15 NOEs. Also

Fig. 2. Selected NOEs of 1.

included is the better-resolved 600 MHz NMR data for 3 (which was also isolated in this study) for the purpose of comparison with those of 1. It can be seen from a comparison of the NMR data of 1 and 3 that the main chemical shift differences observed for the monoterpenoid indole portion of the molecules are those of H3/C-3 and H-21/C-21 (both positions a to N-4) which are consistent with N-4 being the site of substitution by the 3-ethylpyridine moiety. Similarly, comparison of the NMR data of the two diastereomers 1a and 1b also established a close correspondence of the chemical shifts with the exception of the chemical shift of C-70 (Dm = 0.4 ppm), indicating that 1a and 1b correspond in all

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Fig. 3. Selected COSY and HMBC data of 1.

probability to the 70 R/70 S epimers of pneumatophorine (1). While incorporation of pyridine moieties have been encountered in indole alkaloids (Brown and Chapple, 1976; Murray et al., 1972; Sainsbury and Webb, 1975), 1 represents a rare instance of incorporation of a 3-ethylpyridine moiety in a monoterpenoid indole alkaloid (Arambewela and Madawela, 2001; Erdelmeier et al., 1992; Majumdar et al., 1972). Compound 4 was isolated as a light yellowish oil with [a]25 D 62 (CHCl3, c 0.19). The IR spectrum showed an absorption band at 3339 cm1 due to an NH function, while the UV spectrum showed characteristic indole absorptions at 219, 281, and 289 nm. The ESIMS showed an [M+H]+ peak at m/z 283, and HRESIMS established the molecular formula as C18H22N2O. The 1H NMR spectrum (Table 1) showed the presence of four aromatic resonances at d 7.07–7.53 due to the four adjacent aromatic hydrogens of an indole moiety, an indolic NH at d 8.40, an N-Me at d 2.26, a broad singlet at d 5.56 due to an olefinic hydrogen, and a relatively shielded aromatic singlet at d 6.26. The latter signal was also observed in compound 1 and, as in the case of 1, long range coupling was also observed between this aromatic-H and the indolic NH at d 8.40, providing strong support for the assignment of this resonance to H-7 of a 6,7-seco monoterpenoid indole alkaloid. The 13C NMR data (Table 2) showed a total of 18 carbon resonances, comprising one methyl, five methylene, eight methine, two quaternary, and two tertiary carbons linked to the indolic nitrogen (corresponding to C-2 and C-13). Two olefinic carbon resonances were seen at d 124.5 (methine) and 137.2 (quaternary), the former was associated with the olefinic-H signal at d 5.56. In addition, two oxymethylenes were observed at d 67.9 (dH 4.21,

Fig. 4. Selected COSY and HMBC data of 4.

Fig. 5. Selected NOEs of 4.

Fig. 6. Selected COSY and HMBC data of 5.

4.34) and 73.4 (dH 3.89, 3.99), as well as an isolated aminomethylene at d 65.6 (dH 2.63, 3.21; d, J = 13 Hz). The COSY spectrum showed the presence of NCH2, NCH2CH2CHCHCH2O and @CHCH2O partial structures which are readily linked by the HMBC data to reveal the 6,7-secoangustilobine B type ring system as shown in structure 4 (Fig. 4). The main difference compared to 6,7-secoangustilobine B (Zeches et al., 1987) and losbanine (Yamauchi et al., 1990) is the lack of the C-16 carbomethoxy group, which in 4 is replaced by a hydrogen,

Fig. 7. Selected NOEs of compound 5.

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seen at d 3.21. This has also resulted in a corresponding change, in the adjacent H-17 resonances (d 3.89, J = 12, 9 Hz; d 3.99, J = 12, 3.5 Hz), in which the additional coupling due to the presence of H-16 was evident in the 1H NMR spectrum of 4 compared to 6,7secoangustilobine B and losbanine, where the H-17 signals were doublets with J = 11 or 12 Hz (Yamauchi et al., 1990; Zeches et al., 1987). The relative configurations at the two stereogenic centers in 4 are similar to those of the related alkaloids, 6,7-secoangustilobine B and losbanine, as shown by the NOESY data (Fig. 5). Compound 4 is therefore 16-decarbomethoxy-6,7-secoangustilobine B. Compound 5 was obtained as a light yellowish oil, and subsequently as colorless block crystals from CHCl3-hexanes (mp 195– 199 °C) with [a]25 D +491 (CHCl3, c 0.29). The IR spectrum showed absorption bands due to NH/OH (3359 cm1) and a,b-unsaturated carbonyl (1705 cm1) functions, while the UV spectrum showed absorption maxima at 232, 292, and 326 nm characteristic of b-anilinoacrylate chromophores. The ESIMS showed an [M+H]+ peak at m/z 341, and HRESIMS established the molecular formula as C20H24N2O3. The 1H NMR spectrum (Table 1) showed the presence of four aromatic resonances of an indole moiety (d 6.79–7.16), an indolic NH (d 9.01), a CH3CHOH side chain (d 1.35, 3.59), and a methyl ester group (d 3.75). The 13C NMR data (Table 2) showed a total of 20 carbon resonances, comprising two methyl, four methylene, eight methine, three quaternary, and two tertiary carbons linked to the indolic nitrogen (corresponding to C-2 and C-13). The four aromatic methine carbon resonances can be readily assigned to aromatic carbons of the indole moiety (C-9, C-10, C-11, C-12), while two olefinic carbon resonances seen at d 171.9 (tertiary carbon linked to N-1) and 98.2 (quaternary) are assigned to C-2 and C-16, respectively, as they formed part of the b-anilinoacrylate chromophore. The ester carbonyl resonance was observed at d 168.6 while the oxymethine of the CH3CHOH side chain was observed at d 69.7 (dH 3.59). The COSY spectrum (Fig. 6) showed the presence of NCH2CH(CHOHCH3)CHCH2CH and NCH2CH2 partial structures. The latter corresponds to C-5–C-6 while the former corresponds to C-21–C-20–(C-19–C-18)–C-15–C-14–C-3 of a strychnan ring system (akuammicine-type) as indicated from the HMBC data (Fig. 6). The relative configurations at C-3, C-7, and C-15 are similar to the akuammicine-type alkaloids as shown by the NOE data (Fig. 7). The configuration of C-20 can be inferred from the 13C resonances of C-2, C-14, and C-16, which have been previously shown to be of diagnostic significance in the assignment of C-20

Fig. 8. X-ray crystal structure of compound 5.

Table 3 Cytotoxic effects of compounds against KB cells. Compound

Pneumatophorine (1) 16-Decarbomethoxy-6, 7-secoangustilobine B (4) 19-Epiechitamidine (5) Rostracine (6) Rhazinicine Akuammicine Alstolobine A Alstolucine A Alstolucine B Alstolucine D Angustilobine B Echitamidine 19-Epi-N4-Demethylalstogustine 15-Hydroxyangustilobine A N4-Demethylalstogustine N4-Demethyl-12-methoxyalstogustine Neozeylanicine Nor-6,7-secoangustilobine A Scholaricine 4,6-Secoangustilobinal A Vinervine Verapamil Vincristine

IC50, lg/mL KB/Sa

KB/VJ300a

KB/VJ300(+)b

>30 >30

>30 >30

>30 >30

>30 16.2 1.0 >30 >30 >30 >30 >30 >30 >30 >30 28.5 >30 >30 >30 >30 >30 >30 >30 >30 5.0

>30 20.4 2.50 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 5.1

>30 22.0 1.85 >30 >30 >30 >30 >30 >30 >30 >30 26.2 >30 >30 >30 >30 >30 >30 >30 2.9 6.6

a KB/S and KB/VJ300 are vincristine-sensitive and vincristine-resistant human oral epidermoid carcinoma cell lines, respectively. b With added vincristine, 0.1 lg/mL, which did not affect the growth of the KB/ VJ300 cells.

configuration in the akuammicine-type alkaloids (Tan et al., 2010b). The 13C resonances of C-2, C-14, and C-16 of 5, which were observed at d 171.9, 32.0, and 98.2, allowed the assignment of C-20 configuration as S. This was also in agreement with the observed H-21b/H-5b, H-6b and H-20/H-14S NOEs (axial H-20 in chair ring D, Fig. 7). The remaining configuration to be determined is that of C-19. Examination of the NMR data showed that while the 13C NMR data of 5 are essentially similar to those of echitamidine (Zeches et al., 1984), the 1H NMR data showed differences for H15, H-18, H-19, and H-21, suggesting that 5 may be the 19R epimer of echitamidine. This was confirmed by X-ray analysis which showed that compound 5 is 19-epiechitamidine (Fig. 8). Compound 6 (rostracine) was isolated as an optically inactive light yellowish oil, [a]25 D 0 (c 0.15, CHCl3). The IR spectrum showed an absorption band at 1713 cm1 due to a conjugated ester carbonyl function. The ESIMS showed an [M+H]+ peak at m/z 206, and HRESIMS established the molecular formula as C11H11NO3. The 1H NMR spectrum showed a pair of AB doublets at d 8.04 and 8.51 (d, J = 6 Hz) due to two adjacent aromatic hydrogens, another singlet at d 8.25 due to an isolated aromatic-H, an olefinic singlet at d 7.75, a 3H singlet at d 3.84 due to a methyl ester group, a one-H quartet at d 5.42 coupled to a methyl doublet at 1.66 (J = 7 Hz) due to a CH3CH–O group. The deshielded olefinic singlet at d 7.75 (dC 160) indicated oxygenation at this carbon, suggesting that it is part of an enol ether functionality, while the deshielded aromatic singlet at d 8.25 (dC 144.5) is reminiscent of the a-hydrogen of a pyridine moiety. These features sufficed to allow the assembly of the molecule, which is entirely consistent with the HMBC (H-7/CO2Me, C-9; Me-11/C-10; H-1, H-3/C-5; H-4/C-6) and NOE data (H-1/Me-11). Rostracine (6) is therefore a new monoterpene alkaloid of the pyranopyridine-type as exemplified by gentianine (Cordell, 1998). The new alkaloids as well as a number of previously tested known Alstonia alkaloids (Tan et al., 2010b) were screened for cytotoxicity against KB cells. Most did not show any cytotoxicity toward KB cells, except for rhazinicine (Table 3). As such, the rhazinilam-type alkaloids (rhazinicine, nor-rhazinicine, rhazinal, and

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Table 4 Cytotoxicity of nor-rhazinicine, rhazinicine, rhazinal, and rhazinilam against seven human cell lines. Compound

Nor-rhazinicine Rhazinicine Rhazinal Rhazinilam Cisplatin Vincristine

IC50 (lg/ml) KB/S

MRC-5

HCT 116

MDA-MB-231

PC-3

A549

MCF7

5.10 1.00 0.08 0.17 – 1 (ng/mL)

19.00 4.20 0.20 0.55 1.10 –

6.30 1.80 0.15 0.38 2.80 –

12.20 4.10 0.16 0.58 2.20 –

>30 >30 >30 >30 3.10 –

>30 >30 >30 >30 4.70 –

>30 >30 >30 >30 5.00 –

KB: human oral epidermoid carcinoma; MRC-5: normal human lung fibroblast; HCT-116: human colorectal carcinoma; MDA-MB-231: estrogen insensitive human breast adenocarcinoma; PC-3: human prostate carcinoma; A549: human lung carcinoma; MCF-7: estrogen sensitive human breast adenocarcinoma.

Table 5 Vasorelaxation effects of compounds on phenylephrine-induced contraction in isolated rat aortic rings. Compound name

Emax

EC50

Log EC50

Undulifoline Pneumatophorine (1) N4-Demethylalsotogustine Echitamidine 20S-Tubotaiwine Rostracine (6) 15-Hydroxyangustilobine A 19-Epiechitamidine (5) 19,20-E-Vallesamine 6,7-Secoangustilobine B Isoprenaline (control)

125.5 ± 16.44 125.0 ± 5.4 138.8 ± 9.65 128.4 ± 14.06 132.4 ± 4.8 110.4 ± 14.61 185.1 ± 78.5 63.01 ± 9.3 77.44 ± 12.1 18.02 ± 1.3 79.74 ± 4.2

9.158  107 9.38  106 4.488  106 1.333  105 3.254  106 6.976  106 7.649  105 3.15  105 1.497  105 1.194  107 7.57  108

6.038 ± 0.37 5.030 ± 0.08 5.348 ± 0.15 4.875 ± 0.19 5.488 ± 0.07 5.156 ± 0.26 4.116 ± 0.39 4.500 ± 0.23 4.825 ± 0.24 6.923 ± 0.22 7.121 ± 0.13

rhazinilam, from the present as well as previous studies) were further tested against a panel of human cancer cell lines and were found to show strong cytotoxicity toward KB, HCT-116, MDAMB-231, and MRC-5 cells (Table 4). On the other hand, a number of alkaloids including, pneumatophorine (1), the uleine alkaloid, undulifoline, the strychnan alkaloids, echitamidine and N4demethylalstogustine, as well as rostracine (6), 20S-tubotaiwine, and 15-hydroxyangustilobine A, induced concentration dependent relaxation in rat aortic rings pre-contracted with phenylephrine (Table 5). 3. Concluding remarks The present investigation of A. rostrata resulted in the isolation of 21 alkaloids including one new monoterpene alkaloid. The known alkaloids include corynanthean (1 isolation), aspidospermatan (1), uleine (1), akuammiline (1), vallesamine (1), rhazinilam-leuconoxine (6), strychnan (4), vallesiachotaman (2) and monoterpene alkaloids (3). This result differs from that of a previous study of the same plant (A. undulifolia) collected in the same general area but at a different time where only eight alkaloids were reported, including undulifoline which was new (Massiot et al., 1992). In the case of A. pneumatophora, in addition to the three new alkaloids, the known alkaloids include corynanthean (1), aspidospermatan (1), uleine (1), akuammiline (2), vincamine (1), vallesamine (7), strychnan (3), and monoterpene alkaloids (3). This result differs markedly from a study of the same plant collected in Malaysia by Japanese investigators, but in a different location (Forest Research Institute of Malaysia, Kuala Lumpur). First, there is a predominance of vallesamine-type alkaloids compared to strychnan-type alkaloids in the present sample, whereas the reverse was the case in the earlier sample investigated. Second, in addition to a number of new strychnan-type alkaloids, other new indole alkaloids were reported for the previous sample (Koyama et al., 2010a,b; Koyama et al., 2012). These results show that seasonal as well geographical factors do affect alkaloidal composition in the same species. In the present report, the unusual

incorporation of a 3-ethylpyridine moiety in a monoterpenoid indole isolated from of A. pneumatophora is particularly noteworthy, in addition to the biological effects (antiproliferation, vasorelaxation) shown by the alkaloids isolated. 4. Experimental 4.1. General Melting points were determined on a Mel-Temp melting point apparatus and are uncorrected. Optical rotations were determined on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Perkin–Elmer RX1 FT-IR spectrophotometer. UV spectra were obtained on a Shimadzu UV-3101PC spectrophotometer. 1H and 13 C NMR spectra were recorded in CDCl3 using TMS as internal standard on JEOL 400, or Bruker 400 and 600 MHz spectrometers. ESIMS and HRESIMS were obtained on an Agilent 6530 Q-TOF and JEOL DART-TOF mass spectrometers. 4.2. Plant material Plant material was collected near Sabak Bernam in Selangor (A. pneumatophora) and Kedah (A. rostrata), Malaysia, and identification was confirmed by Dr. K.T. Yong (Institute of Biological Sciences, University of Malaya, Malaysia) and Dr. Richard C.K. Chung (Forest Research Institute, Malaysia). Herbarium voucher specimens of A. pneumatophora (K717, herbarium No. KLU 48184) and A. rostrata (K676, herbarium No. FRI 10361, FRI 32548, KLU 47649) were deposited at the Herbarium of the University of Malaya (KLU) and Herbarium of Forest Research Institute, Malaysia (KEP). 4.3. Extraction and isolation Extraction of the ground stem-bark materials (A. pneumatophora, 30 kg; A. rostrata, 26 kg), were carried out in the standard manner by partitioning the concentrated EtOH extract with dilute acid (5% tartaric acid). The alkaloid mixture was initially fractionated by column chromatography (CC) on silica gel using CHCl3 with increasing proportions of MeOH followed by re-chromatography of the appropriate partially resolved fractions using radial chromatography (Chromatotron, Harrison Research). Solvent systems used for radial chromatography were: Et2O–hexanes, Et2O, EtOAc–hexanes, EtOAc, CHCl3–hexanes, CHCl3, CHCl3– MeOH. The yields (mg kg1) of the alkaloids from the bark extract of A. pneumatophora were as follows: 1 (0.7), 4 (0.01), 5 (0.9), yunnanensine (3) (0.01), 19,20-E-vallesamine (8.6), 15-hydroxyangustilobine A (0.02), 4,6-secoangustilobine A (0.01), nor-6,7secoangustilobine A (0.02), angustilobine B (0.1), 6,7-secoangustilobine B (37.5), akuammicine (0.02), N(4)-demethylalstogustine (0.3), N4-demethylalstogustine N-oxide (0.3), 20S-tubotaiwine (0.4), 16R,19E-isositsirikine (0.4), rhazimol (0.6), strictamine

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(0.3), N4-demethylechitamine (10.6), 16-epivincamine (3.7), alstilobanine C (0.3), neozeylanicine (0.02), venoterpine (1.9), and cantleyine (26.0). The yields (mg kg1) of the alkaloids from the bark extract of A. rostrata were as follows: 6 (1.7), (-)-alstolucine B (0.06), echitamidine (26.1), akuammicine (3.2), 20S-tubotaiwine (7.7), 19,20E-vallesamine (11.4), N4-chloromethylechitamidine chloride (26.1), leuconolam (0.8), 6,7-dehydroleuconoxine (0.4), O-methylleuconolam (0.5), leuconoxine (1.3), mersicarpine (0.5), rhazinicine (0.01), tetrahydroalstonine (0.03), N4-demethylechitamine (36.6), E- & Z-vallesiachotamine (0.5), neozeylanicine (1.6), venoterpine (2.3), cantleyine (12.7) and undulifoline (2.8). 4.4. Characterization data 4.4.1. Pneumatophorine (1) Light yellowish oil; [a]25 D 41 (c 0.57, CHCl3); UV (EtOH) kmax (log e) 231 (4.13), 282 (3.91), 290 (3.84) nm; IR (dry film) mmax 3380, 1725 cm1; for 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 2, respectively; HRESIMS m/z 434.2449 [M+H]+ (calcd for C26H32N3O3, 434.2439). 4.4.2. 16-Decarbomethoxy-6,7-secoangustilobine B (4) Light yellowish oil; [a]25 D 62 (c 0.19, CHCl3); UV (EtOH) kmax (log e) 219 (4.24), 281 (3.22), 289 (3.20) nm; IR (dry film) mmax 3339 cm1;for 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 2, respectively; HRESIMS m/z 283.1810 [M+H]+ (calcd for C18H23N2O, 283.1805). 4.4.3. 19-Epichitamidine (5) Light yellowish oil and subsequently colorless block crystals from CHCl3-hexanes; mp 195–199 °C; [a]25 D +491 (c 0.29, CHCl3); UV (EtOH) kmax (log e) 232 (3.99), 292 (3.75), 326 (3.84) nm; IR (dry film) mmax 3359, 1705 cm1; for 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 2, respectively; HRESIMS m/z 341.1862 [M+H]+ (calcd for C20H25N2O3, 341.1860). 4.4.4. Rostracine (6) Light yellowish oil; [a]25 D 0 (c 0.15, CHCl3); UV (EtOH) kmax (log e) 289 (3.03); IR (dry film) mmax 1713 cm1; for 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 2, respectively; HRESIMS m/z 206.0807 [M+H]+ (calcd for C11H12NO3, 206.0812). 4.4.5. Acetylation of pneumatophorine (1) To a solution of 1 (6.5 mg, 0.015 mmol), pyridine (2.4 lL, 0.030 mmol), and CH2Cl2 (2 mL), was added Ac2O (4.7 lL, 0.015 mmol), and the mixture was stirred at rt for 30 min. Na2CO3 solution (5%, 5 mL) was then added and the mixture extracted with CH2Cl2 (3  5 mL). The combined organic extracts was washed with H2O (3  10 mL), dried (Na2SO4), concentrated in vacuo, and the residue was purified by radial chromatography (SiO2, 5% MeOH/CHCl3, NH3–saturated) to give the O-acetyl derivative (5) (2.7 mg, 42%) as a light yellowish oil; [a]25 D 87 (c 0.1, CHCl3); UV (EtOH) kmax (log e) 233 (4.14), 281 (3.82), 290 (3.74) IR (dry film) mmax 3342, 1732, 1703 cm1; 1H NMR (CDCl3, 600 MHz) 2a: d 9.70 (1H, br s, NH), 8.38 (1H, d, J = 9 Hz, H-60 ), 8.30 (1H, br s, H-20 ), 7.50 (1H, d, J = 8 Hz, H-9), 7.42 (1H, m, H-40 ), 7.37 (1H, d, J = 8 Hz, H-12), 7.17 (1H, t, J = 8 Hz, H-11), 7.08 (1H, m, H-10), 7.06 (1H, m, H-50 ), 6.28 (1H, br s, H-7), 5.62 (1H, q, J = 7 Hz, H-19), 4.80 (1H, d, J = 11 Hz, H-17a), 4.69 (1H, d, J = 11 Hz, H-17b), 3.80 (3H, s, CO2Me), 3.30 (1H, m, H-15), 3.27 (1H, m, H-70 ), 2.82 (1H, d, J = 12 Hz, H-21a), 2.47 (1H, m, H-3a) 2.41 (1H, d, J = 12 Hz, H-21b), 1.88 (3H, s, OCOMe), 1.69 (3H, m, H-3b, H-14a, H-14b), 1.66 (3H, d, J = 7 Hz, H-18), and 1.21 (3H, d, J = 7 Hz, H-80 ); 2b: d 9.70 (1H, br s, NH), 8.39 (1H, d, J = 9 Hz, H-60 ), 8.33 (1H, br s, H-20 ), 7.54 (1H, d, J = 8 Hz, H-9), 7.42 (1H, m, H-40 ), 7.36 (1H, d, J = 8 Hz, H-12), 7.17 (1H, t, J = 8 Hz, H-11), 7.08 (1H, m, H-10), 7.06 (1H, m, H-50 ), 6.30

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(1H, br s, H-7), 5.52 (1H, q, J = 7 Hz, H-19), 4.81 (1H, d, J = 11 Hz, H-17a), 4.70 (1H, d, J = 11 Hz, H-17b), 3.78 (3H, s, CO2Me), 3.30 (1H, m, H-15), 3.27 (1H, m, H-70 ), 2.96 (1H, d, J = 12 Hz, H-21a), 2.63 (1H, m, H-3a), 2.50 (1H, d, J = 12 Hz, H-21b), 1.91 (3H, s, OCOMe), 1.69 (3H, m, H-3b, H-14a, H-14b), 1.63 (3H, d, J = 7 Hz, H18), and 1.20 (3H, d, J = 7 Hz, H-80 ); ESIMS m/z 476 [M+H]+; HRESIMS m/z 476.2525 [M+H]+ (calcd for C28H34N3O4, 476.2549). 4.4.6. X-ray crystallographic analysis of compound 5 X-ray diffraction analysis was carried out on a Bruker SMART APEX II CCD area detector system equipped with a graphite monochromator and a Mo Ka fine-focus sealed tube (k = 0.71073 Å), at 150 K. The structures were solved by direct methods (SHELXS-97) and refined with full-matrix least-squares on F2 (SHELXL-2014). 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 compound 5 have been deposited with the Cambridge Crystallographic Data Centre (Deposition No.: CCDC 1042682). These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail: [email protected]). Crystallographic data of 5: colorless block crystals, C21H24N2O3, Mr = 341.19, orthorhombic, space group P212121, a = 8.52750(10)Å, b = 13.9829(2)Å, c = 14.5187(2)Å, Z = 4, Dcalc = 1.292 g cm3, crystal size 0.1  0.2  0.2 mm3, F(0 0 0) = 720, T = 150 K. The final R1 value is 0.0327 (wR2 = 0.0868) for 4565 reflections [I > 2r(I)]. CCDC number: 1042682. 4.4.7. Cytotoxicity assays Cytotoxicity assays were carried out following the procedure that has been described previously (Lim et al., 2014). 4.4.8. Vasorelaxation activity Evaluation of vasorelaxation activity was carried out following the procedure that has been described previously (Yap et al., 2015). Acknowledgments We thank the University of Malaya and MOHE Malaysia (HIRF005) for financial support. References Arambewela, L.S.R., Madawela, G., 2001. Alkaloids from Rauvolfia canescens. Pharm. Biol. 39, 239–240. Brown, R.T., Chapple, C.L., 1976. Anthocephalus alkaloids: cadamine and isocadamine. Tetrahedron Lett. 19, 1629–1630. Burkill, I.H., 1966. A Dictionary of Economic Products of the Malay Peninsula. Ministry of Agriculture and Co-operatives, Kuala Lumpur. Cordell, G.A., 1998. The monoterpene alkaloids. In: Cordell, G.A. (Ed.), The Alkaloids: Chemistry and Biology, vol. 52. Academic Press, San Diego, pp. 260–327. Erdelmeier, C.A., Regenass, U., Rali, T., Sticher, O., 1992. Indole alkaloids with in vitro antiproliferative activity from the ammoniacal extract of Nauclea orientalis. Planta Med. 58, 43–48. Garrido, L., Zubia, E., Maria, J.O., Salva, M.J., 2003. Haouamines A and B: A new class of alkaloids from the ascidian Aplidium haouarianum. J. Org. Chem. 68, 293–299. Kam, T.S., 1999. Alkaloids from Malaysian flora. In: Pelletier, S.W. (Ed.), Alkaloids: Chemical and Biological Perspectives, vol. 14. Pergamon, Amsterdam, pp. 285– 435. Kam, T.S., Choo, Y.M., 2006. Bisindole alkaloids. In: Cordell, G.A. (Ed.), The Alkaloids: Chemistry and Biology, vol. 63. Academic Press, Amsterdam, pp. 181–337. Kam, T.S., Tan, S.J., Ng, S.W., Komiyama, K., 2008. Bipleiophylline, an unprecedented cytotoxic bisindole alkaloid constituted from the bridging of two indole moieties by an aromatic spacer unit. Org. Lett. 10, 3749–3752. Keawpradub, N., Eno-Amooquaye, E., Burke, P.J., Houghton, P.J., 1999. Cytotoxic activity of indole alkaloids from Alstonia macrophylla. Planta Med. 65, 311–315. Koyama, K., Hirasawa, Y., Nugroho, A., Toshio, K., Teh, C.H., Chan, K.L., Morita, H., 2010a. Alsmaphorazines A and B, novel indole alkaloids from Alstonia pneumatophora. Org. Lett. 12, 4188–4191.

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Biologically active vallesamine, strychnan, and rhazinilam alkaloids from Alstonia: Pneumatophorine, a nor-secovallesamine with unusual incorporation of a 3-ethylpyridine moiety.

Four alkaloids comprising two vallesamine, one strychnan, and one pyranopyridine alkaloid, in addition to 32 other known alkaloids were isolated from ...
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