Phytochemistry 103 (2014) 8–12

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Cytotoxic dimeric quinolone–terpene alkaloids from the root bark of Zanthoxylum rhetsa Monira Ahsan a, Mohammad Rashedul Haque a, Md. Belayet Hossain a, Sheikh Nazrul Islam b, Alexander I. Gray c, Choudhury Mahmood Hasan a,⇑ a b c

Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh Institute of Nutrition and Food Science, University of Dhaka, Dhaka 1000, Bangladesh Department of Pharmaceutical Sciences, University of Strathclyde, SIBS Building, 27 Taylor Street, Glasgow G4 ONR, UK

a r t i c l e

i n f o

Article history: Received 24 August 2013 Received in revised form 6 February 2014 Available online 22 April 2014 Keywords: Chelerybulgarine Dihydrochelerythrine 20 -Episimulanoquinoline 2,11-Didemethoxyvepridimerine Rhetsidimerine

a b s t r a c t Four quinolone–terpene alkaloids, chelerybulgarine (1), 20 -episimulanoquinoline (3), 2,11-didemethoxyvepridimerine B (4), and rhetsidimerine (5) were isolated from the root bark of Zanthoxylum rhetsa DC. Chelerybulgarine (1) is a C–C linked terpene alkaloid where the C-6 of dihydrochelerythrine is linked to C-11 of the sesquiterpenoid 10b-methoxybulgarene. 20 -Episimulanoquinoline is a dimeric alkaloid containing dihydrochelerythrine and 8-methoxy-N-methylflindersine moieties, whereas 2,11-didemethoxyvepridimerine B and rhetsidimerine are dimeric prenylated quinolone alkaloids. Seven of the isolated compounds exhibited weak cytotoxicity when tested against a panel of six human stomach-cancer cell lines. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Zanthoxylum rhetsa DC (Syn. Zanthoxylum budrunga; Bengali name, Bazna; Family, Rutaceae) is a medium-sized tree with pale, corky bark that grows in Bangladesh, India, Myanmar, Thailand, Malaysia and other tropical countries. The fruits and stem bark are used in the treatment of asthma, bronchitis, heart complaints and rheumatism. The essential oil is used to treat cholera and as an antiseptic and disinfectant (Yusuf et al., 1994). A number of alkaloids, including canthine-6-one (Ahsan et al., 2001), 8-methoxy-N-methylfindersine (Ahsan et al., 2000), dihydrochelerythrine (Ahsan et al., 2001), chelerythrine, rhetine, rhetsine, rhetsinine (Chatterjee et al., 1959), dihydroavicine (Joshi et al., 1991), dictamnine, arborine (Ruangrungsi et al., 1981) and others have been isolated from the stem bark of the plant. In addition, reports have identified the coumarins suberosin (Ewing et al., 1950) and xanthyletin as well as the lignin esamin in the plant (Ahsan et al., 2000). The essential oil of the fruit and seed contains mainly monoterpenoids (Jirovetz et al., 1998; Shankaracharya et al., 1994). We report the isolation and structural elucidation of five dimeric alkaloids, including four new quinolone–terpene alkaloids,

from Z. rhetsa. In addition, we isolated two known alkaloids, three lignans, and lupeol. The methanol extraction of Z. rhetsa root bark produced compounds 1–5, 8-methoxy-N-methylflindersine (Ahsan et al., 2000), rhetsine (evodiamine) (Chatterjee et al., 1959), piperitol-c, c-dimethylallyl ether, xanthoxylol-c,c-dimethylallyl ether (Fumiko et al., 1973), sesamin (Nissanka et al., 2001), and lupeol. 12

H 3CO

8

6a

7

10

11' 6

H

6'

N 5

8'

H CH3

H

13'

14'

10a 10b

4

O

1

12a

Hd Hc 4b

18a

6 6a

N CH3

N

6

CH3

6

7

Hb

Ha O

19

15

14

He H Hf g O

O

4'a

10'a 10'b

8'

5' 6'

6a

O

8

CH3 OCH3 N9 10 9a

13a

Hc

13

7a

O Hd

7

H3C

6

N5

6a

Hg Hb 19

18 17

4a

4b

O

4

16

1 2

He

Hf 20 21

5

Ha

14

O

16a

H 3CO

13b

15

7'

N CH3 OCH3 3

O

10' 8'

5' 6'

7'

N 6a CH3 OCH3

11 13a

O

CH3 1'

4' 10'

CH3 OCH3 N 9a 10

7a

N

2'

10'a 10'b

O

4

11'

O

16

4

5

6a

H OCH3

11'

4'a

O

1 2

4a

10a

7

H3CO

13b

16b

18 17

5

6a

10

4b

2

8

O5

9

O

O

4a

10b

O

10a

H OCH3

12a

11

4

1'

1

4

7

12

2

2'

2

11 12

10

O

1

4a

4b

4'

4b 4a

1

http://dx.doi.org/10.1016/j.phytochem.2014.03.008 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

1' 5'

OCH3

9

H3 CO

10'

H3 CO

E-mail address: [email protected] (C.M. Hasan).

OCH 3

H 3'

2

⇑ Corresponding author. Mobile: +880 1819253698; fax: +880 2 8615583.

12a

11 10b

11 13

9

M. Ahsan et al. / Phytochemistry 103 (2014) 8–12

and pyridine-d5 provided the correlations necessary to assign all the protons and carbons. These results led to a structure for compound 1, 6-(10b-methoxybulgarene-11-yl) dihydrochelerythrine, which was given the trivial name chelerybulgarine. The molecular formulas of simulanoquinoline (2) and 20 -episimulanoquinoline (3) were determined as C37H34N2O7 by HRFABMS. The base peaks for both compounds were found at m/ z 348 (C21H18NO4), indicating the presence of a dihydrochelerythrine moiety. The 1H NMR data from compounds 2 and 3 (Table 1) further supported the presence of the alkaloid. The remaining proton signals indicated the presence of a methoxy group, an Nmethyl group, three adjacent aromatic protons, two doublets at d 5.98 and 7.31 (J = 10 Hz) and a methyl group. Together, these signals suggested the presence of an 8-methoxy-N-methylflindersine moiety. Three mutually coupled aliphatic protons, at d 5.20, 2.15 and 2.02 for compound 2 and at d 5.21, 2.17 and 1.96 for compound 3, indicated that C-6 of the dihydrochelerythrine unit was linked to Me-20 of the 8-methoxy-N-methylflindersine moiety. The 1H and 13 C data of compounds 2 and 3 were almost identical, but significant differences (0.15–25 ppm) were observed in the 1H NMR of the 2-quinolone nucleus, particularly for the proton resonances of Me-20 , H-30 , H-40 , 70 -methoxy and H-100 . There were two chiral centres, C-2’ and C-6, but no 1H NMR differences for the dihydrochelerythrine moiety, which suggested that 2 and 3 were epimers of each other at C-20 . The NOESY spectra of both compounds showed similar correlations between the N-Me and H-5, H-5 and H-110 and C-20 Me and both H-30 and H-100 . The only noticeable difference between compounds 2 and 3 was the correlation between the C-20 Me and H-6 in compound 3. The dimeric alkaloid simulanoquinoline was first identified in Zanthoxylum simulans (Shwu-Jen and Ih-Sheng, 1993) but its absolute configuration was not determined. A direct comparison of the 1H NMR data

Results and discussion HRFABMS analysis of compound 1 showed a [M+H]+ peak at m/z 584 (C37H46NO5). The 1H NMR spectrum (Pyridine-d5, Table 1) indicated a dihydrochelerythrine and a sesquiterpenoid moiety. The presence of the dihydrochelerythrine moiety was confirmed by several characteristic 1H NMR signals: i.e. (a) methylenedioxy protons at d 6.20 and 6.17; (b) two methoxy groups at d 4.05 and 3.83; (c) an N-methyl group at d 2.68; (d) two pairs of orthocoupled aromatic protons at d 7.10, 7.80, 8.00 and 7.70; (e) two aromatic proton singlets at d 7.39 and 7.86; and (f) a benzylic proton as a doublet of doublet, at d 4.95. The remaining proton signals suggested a cadinene-type sesquiterpenoid moiety. The 1H NMR spectrum also showed three methyl groups, two of which appeared as doublets at d 0.75 and 0.52, the third appeared as a singlet at d 1.10, an olefinic proton at d 5.10, eleven proton multiplets between d 0.94–2.56 and a methoxy group at d 3.30. A NOESY experiment showed correlations between H-60 and both H-70 and the C-100 methyl group, indicating that they were all on the same side of the molecule. From these data, it was inferred that the sesquiterpene moiety is 10b-methoxybulgarene. This identification was further substantiated by 1H NMR (CDCl3) data from the sesquiterpene moiety of 1 which differed from any of the frequently occurring, natural cadinene sesquiterpenoids previously reported (Borg-Karlson et al., 1981), e.g., ()-torreyol, ()-a-cadinol, ()muurolol or (+)-T-cadinol. Unfortunately, no published NMR data on bulgarene were available for comparison. Three mutually coupled aliphatic protons at d 4.95, 2.39, and 2.26 were attributed to H-6 of the dihydrochelerythrine unit and to 2-H-11 of the sesquiterpenoid moiety. These data suggested a C-C linkage between C-6 of the alkaloid and C-11 of the terpenoid that was confirmed by 1H–1H COSY experiments. 2D NMR studies in CDCl3

Table 1 H NMR spectroscopic data of compounds 1, 2, and 3.a

1

Position

1 4 6 9 10 11 12 OCH3-7 OCH3-8 NCH3-5 OCH2O 10 20 30 40 50 60 70 80 90 100 110 120 CH2-20 CH3-20 CH3-130 CH3-140 CH3-150 OCH3-70 OCH3-100 NCH3-60 a

1

2

3

CDCl3

Pyridine-d5

Pyridine-d5

Pyridine-d5

7.08 7.61 4.65 6.94 7.55 7.72 7.46 3.95 3.93 2.59 6.01 1.54 1.22 2.46 – 4.87 1.65 0.80 1.55 1.56 – 1.93 1.40 – – 0.65 0.52 1.07 – 3.30 –

7.39 7.86 4.95 7.10 7.80 8.00 7.70 4.05 3.83 2.68 6.20 1.77 1.28 2.56 – 5.10 1.72 0.98 1.57 1.66 – 2.26 1.46 – – 0.75 0.52 1.10 – 3.30 –

7.36 7.83 5.20 7.11 7.78 8.02 7.70 3.99 3.84 2.70 6.14 – – 5.98 7.31 – – – 7.10 7.20 7.84 – – 2.15 1.47 – – – 3.75 – 4.04

7.32 7.74 5.21 7.10 7.76 8.00 7.68 3.83 3.78 2.75 6.07 – – 5.74 7.12 – – – 7.11 7.19 7.66 – – 2.17 1.70 – – – 3.73 – 3.99

s s dd (11.3, 4.3) d (8.6) d (8.6) d (8.6) d (8.6) s s s d, 6.04 d (1.2) m m, 2.09 m m, 2.07 m br.s m m m, 0.95 m m, 1.84 m m, 2.10 m m

d (6.9) d (6.9) s s

s s dd (11.6, 4.2) d (8.3) d (8.3) d (8.4) d (8.4) s s s br.s, 6.17 br.s br.d (11.3) m, 2.32 dd (12.9, 5.2) m, 2.18 m br.s m m m, 0.94 m m, 1.88 br.d (11.5) dd (13.9, 11.6), 2.39 br.d (13.9) m

d (6.8) d (6.8) s s

Spectra recorded at 400 MHz in CDCl3. J values (Hz) in parentheses.

s s dd (10.4, 2.4) d (8.8) d (8.8) d (8.4) d (8.4) s s s d (1.0), 6.12 d (1.0)

d (10.0) d (10.0)

dd (8.0, 1.2) t (8.0 Hz) dd (8.0, 1.2)

dd (14.5, 10.2), 2.02 dd (14.5, 2.0) s

s s

s s dd (10.4, 2.2) d (8.8) d (8.8) d (8.4) d (8.8) s s s d (1.0), 6.03 d (1.0)

d (10.0) d (10.0)

dd (8.0, 1.2) t (8.0) dd (8.0, 1.6)

dd (15.0, 10.5), 1.96 dd (15.0, 2.0) s

s s

10

M. Ahsan et al. / Phytochemistry 103 (2014) 8–12

between this alkaloid and compounds 2 or 3 could not be made, because the initial 1H NMR of simulanoquinoline was taken in CDCl3, while Pyridine-d5 was used in this study. This discrepancy prevented us from determining whether 2 or 3 was the new alkaloid. However, we did conclude that 2 or 3 was a new epimer of the known dimeric alkaloid and could be named 20 -episimulanoquinoline. This is the first detailed report on 2D NMR of 13C NMR for these dimeric alkaloids. Compounds 2 and 3 showed single spots on TLC plates, but 1H NMR spectra revealed that they were mixtures of the same components in ratios of approximately 2:1 and 3:1, respectively. 1H NMR analysis of 3, performed in pyridine-d5 24 h following the initial experiment, revealed that 3 was a mixture with 2 in a ratio of 1:1. These data suggested that, to some extent, the two epimers are interchangeable. The molecular formula of compound 4 was identified as C32H34N2O6 by HRFABMS and showed a peak at [M+1]+ m/z 543. Data from 1H, 13C, HMBC and COSY experiments showed the presence of two 8-methoxy-N-methylquinolone units joined by a C10 moiety, consisting of three tertiary methyls (dC 21.2, 29.4, and 29.8; dH 1.35, 2.13, and 1.71), two methylenes (dC 40.3, 31.2), three methines (dC 52.9, 27.1, 26.4) and two oxygen bearing saturated carbons (dC 82.0, 79.0) (Tables 2 and 3). It was evident from spectroscopic properties that compound 4 was similar to vepridimerine B (Ngadjui et al., 1982) although it lacked methoxyl groups at C-2 and C-11. The large coupling constant between Hd and He (12.5 Hz) suggested a trans relationship. The relative configuration was confirmed by a NOESY experiment. Thus, the new compound 4 was characterised as 2,11-didemethoxyvepridimerine B.

The molecular formula of compound 5, as identified by HRFABMS, was C32H34N2O6. The 1H and 13C data (Tables 2 and 3) from this compound were similar to those of compound 4 and indicated the presence of two 2-quinolone units joined by a C10 moiety. There were several differences between 4 and 5: (a) the C10 moiety of compound 5 contained only one oxygen bearing saturated carbon at d 72.1 instead of two; (b) the oxygen atom attached to C-13b directly linked the two quinolone units; and (c) the C5 units of the two 2-quinolone units were joined differently and left a 2-methyl-2-propene chain, which was confirmed by HMBC. The two methyls at d 1.44 and 1.63 were correlated and showed 2J correlation to C-4 at dc 135.6 and 3J correlation to the C-20 methine carbon at d 125.6. A molecular model of compound 5 revealed that the appearance olefinic proton He (d 3.70) at such a high field resulted from its position above the benzene ring. The COSY experiment showed all the expected 1H–1H couplings. The HMQC and HMBC (Table 3) spectra showed all the expected 2J and 3J correlations, which facilitated assignment of all protons and carbons. The relative configuration was determined by NOESY. Hb, Hc, Hd and Hg all showed strong mutual interactions, which indicated that they were all co-facial. Hf showed a strong correlation to the aromatic proton H-1 at d 7.76. All the expected correlations were observed for the N-Me, the OMe and the aromatic protons. The aromatic proton at d 7.76 showed a strong interaction with Hf while the methoxy, N-methyl groups, and the protons of the quinolone rings showed all the expected interactions. Based on the above findings, the structure of compound 5 was identified and assigned the trivial name rhetsidimerine.

Table 2 C NMR spectroscopic data (pyridine-d5) of compounds 1, 2, 3, 4, and 5.a

13

Position

1 2 3 4 4a 4b 6 6a 7 7a 8 9 9a 10 10a 10b 11 12 12a 13 13a 13b 15 16 16a 16b 17 18 18a 19 20 21 10 a

dC in ppm (Hz)

Position

CDCl3

Pyridine-d5

1

1

2

3

4

5

104.3 147.6 147.8 102.0 127.8 140.4 55.6 130.9 145.9 – 152.3 111.2 – 118.9 125.2 123.9 119.9 123.5 131.1 – – – – – – – – – – – – – 47.1

105.1 148.6 148.8 102.5 128.5 141.0 56.4 131.2 146.8 – 153.3 112.4 – 119.8 125.7 124.8 120.9 124.5 132.8 – – – – – – – – – – – – – 47.9

105.5 149.2 148.6 101.9 128.1 140. 7 54.7 130.5 146.3 – 153.5 112.8 – 119.7 125.6 124.9 121.0 124.7 132.3 – – – – – – – – – – – – – –

105.3 149.0 148.5 102.0 128.1 140.8 54.8 130.3 146.3 – 153.3 112.7 – 119.8 125.6 124.8 120.9 124.7 132.2 – – – – – – – – – – – – – –

149.1 114.7 122.7 116.2 119.6 156.0 82.0 52.9 26.4 115.4 163.1 – 131.4 149.0 – – 114.7 122.7 – 116.4 119.6 154.8 79.0 40.3 27.1 108.8 163.9 – 131.6 31.2 – – –

117.0 125.9 115.0 150.9 130.9 – 171.0 45.5 29.8 114.4 163.4 – 131.3 149.2 – – 114.8 122.8 – 115.9 118.2 151.2 72.1 – 98.9 – 41.3 37.4 – 40.6 125.6 135.6 –

20 30 40 40 a 50 60 60 a 70 80 90 100 100 a 100 b 110 120 130 140 150 NCH3-5 NCH3-9 NCH3-18 NCH3-60 OCH3-1 OCH3-4 OCH3-7 OCH3-8 OCH3-10 OCH3-70 CH3-20 CH3-6 CH3-15 CH3-21 O-CH2-O

Spectra recorded at 100 MHz in CDCl3. Assignments were made based on HMQC and HMBC.

dC in ppm (Hz) CDCl3

Pyridine-d5

1

1

2

3

4

5

22.8 28.8 136.0 – 124.5 39.8 – 45.9 21.7 35.7 76.5 – – 43.3 25.7 21.3 15.1 17.4 43.0 – – – – – 61.3 56.0 – – – – – – 101.1

23.5 29.5 137.0 – 125.4 40.3 – 46.5 22.3 36.4 76.5 – – 44.3 26.3 21.3 15.5 17.8 43.3 – – – – – 61.4 56.3 – – – – – – 102.2

81.6 128.1 117.1 106.5 162.5 – 132.2 149.3 115.4 123.1 116.3 118.8 155.4 46.0 – – – – 43.1 – – 35.5 – – 61.2 56.3 – 57.1 28.9 – – – 102.3

81.7 127.2 118.6 106.5 162.4 – 132.0 149.1 115.2 122.9 116.5 119.0 155.7 44.6 – – – – 43.0 – – 35.5 – – 61.0 56.2 – 57.1 27.7 – – – 102.2

– – – – – – – – – – – – – – – – – – – 35.1 35.6 – 57.1 – – – 57.1 – – 29.4, 21.2 29.8 – –

– – – – – – – – – – – – – – – – – – 35.2 35.3 – – – 56.4 – – 57.0 – – – 25.8 26.3, 18.9 –

M. Ahsan et al. / Phytochemistry 103 (2014) 8–12

11

Table 3 H NMR spectroscopic data (pyridine-d5), 1J, 2J, and 3J heteronuclear interactions through HMQC and HMBC experiments on compound 5.a

1

a

Proton

1

1 2 3 a b c d e f g 11 12 13 NCH3-5 NCH3-9 OCH3-4 OCH3-10 CH3-15 CH3-21 CH3-21

7.76 7.39 7.17 1.90 2.54 4.51 3.34 3.70 1.32 1.81 6.96 7.00 7.54 3.36 3.90 3.75 3.64 1.33 1.44 1.63

H NMR dd (8.0, 1.2) t (8.0) dd (8.0, 1.2) br d (12.7) dd (13.5, 10.5) dd (10.5, 1.5) dd (12.0, 9.5) dd (9.5, 1.5) m dd (13.2, 9.8) dd (8.0, 1.8) d (8.0) dd (8.0, 1.8) s s s s s s s

1

2/3

117.0 125.9 115.0 40.6 40.6 29.8 37.4 125.6 41.3 41.3 114.8 122.8 115.9 35.2 35.3 56.4 57.0 25.8 26.3 18.9

98.9 (C-16a), 115.0 (C-3), 130.9 (C-4a) 115.0 (C-3), 131.7 (C-4b), 150.9 (C-4) 117.0 (C-1), 130.9 (C-4a), 150.9 (C-4) 72.1 (C-15) 114.4 (C-7a), 29.8 (C-7) 45.5 (C-6a), 72.1 (C-15), 98.9 (C-16a), 114.4 (C-7a), 151.2 (C-13b), 163.4 (C-8) 29.8 (C-7), 41.3 (C-17), 45.5 (C-6a), 125.6 (C-20), 135.6 (C-21), 98.9 (C-16a) – – 37.4 (C-18), 45.5 (C-6a) 115.9 (C-13), 149.2 (C-10), 131.3 (C-9a) 115.9 (C-13), 118.2 (C-13a), 149.2 (C-10) 114.8 (C-11), 131.3 (C-9a), 151.2 (C-13b) 130.9 (C-4a), 171.0 (C-6) 131.3 (C-9a), 163.4 (C-8) 150.9 (C-4) 149.2 (C-10) 41.3 (C-17), 72.1 (C-15) 18.9 (CH3-21), 45.5 (C-6a), 125.6 (C-20), 135.6 (C-21) 26.3 (CH3-21), 125.6 (C-20), 135.6 (C-21)

J Interactions

J Interactions

Spectra recorded at 400 MHz in CDCl3. J values (Hz) in parentheses.

Out of the nine compounds tested for cytotoxicity, seven were cytotoxic in multiple cancer cell lines. Among the active compounds, 1 showed the highest cytotoxicity (ED50 from 37.47 to 45.46 lM) followed by a mixture of sesamin and xanthoxylolc,c-dimethylallyl ether (ED50 from 47.44 to 91.56 lM), 8-methoxy-N-methylflindersine (ED50 from 60.96 to 79.37 lM) and 4 (ED50 from 83.26 to 94.28 lM). Lupeol, evodiamine, and 5 showed poor cytotoxicity in the cell lines and cell mortality was nil for compounds 2 and 3. However, compared to vinblastine, all the compounds tested were weakly cytotoxic. Even then, the cytotoxicity of compound 1 and the mixture of sesamin and xanthoxylolc,c-dimethylallyl ether were comparable to those for diterpenes from Scoparia dulcis (Ahsan et al., 2003). Comparison of ED50 values between the pure compounds and positive controls were performed by an independent sample t-test. Experimental

EtOAc in petroleum ether, were collected and concentrated. Lupeol (8 mg) was isolated as crystals and the supernatant was subjected to PTLC (silica gel, EtOAc/toluene: 10/90) to obtain compound 1 (7 mg, Rf 0.52 in EtOAc/toluene, 5:95). Fractions 42–50, eluted with 8% EtOAc in petroleum ether, were collected and concentrated to produce rhetsine (6 mg, Rf 0.31 in EtOAc/toluene, 30:70) as yellow needles. The supernatant was chromatographed by preparative TLC to obtain sesamin and xanthoxylol-c,c-dimethylallyl ether as a mixture (5 mg, Rf 0.50 in EtOAc/toluene, 30:70) including piperitol-c,c-dimethylallyl ether (4 mg, Rf 0.48 in EtOAc/toluene, 30:70) and 8-methoxy-N-methylflindersine (4 mg, Rf 0.44 in EtOAc/toluene, 15:85). Fractions 75–79 were further fractionated on Sephadex LH-20 and preparative TLC over silica gel (EtOAc/ toluene, 15:85, multiple development) and produced compounds 2 (5 mg, Rf 0.42 in EtOAc/toluene, 25:75), 3 (6 mg, Rf 0.38 in EtOAc/toluene, 25:75), 4 (6 mg, Rf 0.37 in EtOAc/toluene, 20:80) and 5 (8 mg, Rf = 0.22 in EtOAc/toluene, 20:80).

General

Chelerybulgarine (1)

Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 1H NMR (400 MHz) and 13C NMR (100 MHz), 1H–1H COSY, NOESY, HMQC and HMBC spectra were measured on Bruker AMX-400 and DPX-400 instruments. HRFABMS data were recorded on a JEOL AX505 mass spectrometer. FABMS were measured using a matrix of glycerol/nitrobenzoyl alcohol on the same mass spectrometer with xenon as the atom source.

Colourless gum; [a]25 D 76.4 (CHCl3); UV: kmax (MeOH) 229, 284, 314; IR: mmax (NaCl film) 3391, 2931, 1599, 1426, 1367, 1283, 1239, 1077, 1039, 756 cm1. 1H and 13C NMR data, see Tables 1 and 2; FABMS: m/z 584 [M+H]+ (100), 571 (97), 570 (54); HRFABMS m/z 584.34121 (calculated for C37H46NO5, 584.34430).

Plant material Root bark of Z. rhetsa was collected from Tongi, Gazipur in August of 1999. A voucher specimen (DACB 37864) was deposited in the National Herbarium, Dhaka, Bangladesh.

Simulanoquinoline (2) 1 13 Gum; [a]25 C NMR data, see Tables 1 and D 0.5 (CHCl3); H and 2; FABMS m/z 619 [M+H]+ (94), 589 (69), 525 (100), 375 (35), 364 (30), 349 (71), 348 (100), 256 (28), 239 (36); HRFABMS m/z 619.24334 (calculated for C37H35N2O7, 619.24443).

Extraction and isolation

20 -Episimulanoquinoline (3)

The sun dried root bark (620 g) was powdered and extracted with MeOH (1.5 L). The solvent was evaporated under reduced pressure in a rotary evaporator to obtain a gummy residue (10 g) that was chromatographed on a silica gel (Kiesel gel 60H, mesh 70–230) and column eluted with petroleum ether containing increasing amounts of EtOAc. Fractions 29–31, eluted with 5%

Gum; [a]25 D 4.0 (CHCl3); IR (film) mmax 3435, 2998, 2964, 2938, 2838, 1653, 1624, 1492, 1466, 1443, 1364, 1274, 1241, 1190, 1076, 1038, 757 cm1; 1H and 13C NMR data, see Tables 1 and 2; FABMS m/z 619 [M+H]+ (100), 618 (79), 529 (50), 395 (16), 364 (29), 349 (35), 348 (100), 272 (28), 256 (14); HRFABMS m/z 619.24524 (calculated for C37H35N2O7, 619.24443).

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M. Ahsan et al. / Phytochemistry 103 (2014) 8–12

2,11-Didemethoxyvepridimerine B (4)

Appendix A. Supplementary data

Gum; [a]25 D 1.3 (CHCl3); UV: 219, 237, 255, 282, 291 nm; IR (film) mmax 3460, 2969, 2935, 1630, 1593, 1577, 1483, 1464, 1385, 1255, 1072, 755 cm1; 1H and 13C NMR data, see Tables 1 and 2; FABMS m/z 543 [M+H]+ (100), 521 (15), 474 (51), 369 (26), 277 (60); HRFABMS m/z 543.24808 (calculated for C32H35N2O6, 543.24951).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 03.008.

Rhetsidimerine (5) Gum; [a]25 D 2.9 (CHCl3); UV: 218, 248, 255, 282, 291 nm; IR (film) mmax 3392, 2965, 2930, 1673, 1657, 1626, 1596, 1489, 1464, 1391, 1353, 1309, 1254, 1167, 1082, 1065, 749 cm1; 1H and 13C NMR data, see Tables 1 and 2; FABMS m/z 543 [M+H]+ (100), 481 (12), 411 (10), 377 (6), 272 (6); HRFABMS m/z 543.24828 (calculated for C32H35N2O6, 543.24951). Cytotoxicity assay A panel of six human stomach-cancer cell lines, SCL, SCL-6, SCL370 6, SCL-9, Kato-3 and NUGC-4 (Islam, 1994; Sekiguchi et al., 1978; Akiyama et al., 1988) were used to test the cytotoxicity of the nine pure compounds isolated from Z. rhetsa. The MTT assay, as described by Mosmann (1983), was employed to estimate cell mortality. A series of serial dilutions (250, 125, 62.5, 31.25 and 15.63 lg/mL) of the pure compounds and vinblastine were tested against each of the cell lines. For every concentration, three replicate analyses were performed. The percentage of cell mortality for each concentration was estimated, and ED50 values were determined in units of lM. Vinblastine sulphate (Sigma Chemicals Co, USA) was used as positive control. RPMIC (RPMI-1640 complete medium) was used to culture the cancer cells to confluence, and RPMIC-DMSO (RPMIC containing 0.25% DMSO) was used to prepare the test materials and to culture the cells in presence of the test materials. Both RPMIC and RPMIC-DMSO were negative controls. Cells grown in RPMIC or RPMIC-DMSO showed no differences and were considered to exhibit 100% cell survival (that is, cell mortality was nil) and used to estimate cell mortality and consequently, to determine the ED50 for the compounds tested. The SPSS (12.5 version) software package was used to analyse the data. Descriptive statistics were performed, and values were expressed as the mean ± standard deviation.

Acknowledgement M.A. thanks the Association of Commonwealth Universities for the award of a Fellowship.

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Cytotoxic dimeric quinolone-terpene alkaloids from the root bark of Zanthoxylum rhetsa.

Four quinolone-terpene alkaloids, chelerybulgarine (1), 2'-episimulanoquinoline (3), 2,11-didemethoxyvepridimerine B (4), and rhetsidimerine (5) were ...
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