Phytochemistry Letters 2 (2009) 88–90

A benzopyrroloisoquinoline alkaloid from Ficus fistulosa

G. Subramaniam, Kenny K.H. Ang, Siewbee Ng, Antony D. Buss, Mark S. Butler *

MerLion Pharmaceuticals, 1 Science Park Road, The Capricorn #05-01, Singapore Science Park II, Singapore 117528, Singapore

A R T I C L E I N F O

Article history:

Received 6 November 2008

Received in revised form 23 December 2008

Accepted 30 December 2008

Available online 15 January 2009

Keywords:

Ficus fistulosa

Moraceae

Alkaloid

Antifungal activity

A B S T R A C T

A new benzopyrroloisoquinoline alkaloid, fistulosine (1), was isolated from the stem-bark of Ficus

fistulosa (Moraceae) collected in Singapore, along with three known phenanthroindolizidine alkaloids,

(�)-13aa-antofine (2), (�)-14b-hydroxyantofine (3) and (�)-13aa-secoantofine (4). (�)-13aa-Antofine

(2) accounted for the antifungal activity against Aspergillus fumigatus and Candida albicans originally

observed in the crude alkaloid extract.

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1. Introduction

There are approximately 800 plant species of the genus Ficus

(Moraceae), which predominantly grow in lowland tropicalrainforests (Harrison, 2005; Lansky et al., 2008). An alkaloidenriched extract from the stem-bark of Ficus fistulosa Reinw ex.Blume (Moraceae), a small dioecious tree that is abundant in forestmargins and old hedges around Singapore (Corlett, 1987),displayed excellent antifungal activity against Aspergillus fumigatus

and Candida albicans. Previous phytochemical investigations of F.

fistulosa collected from Vietnam identified 16 non-alkaloidalcompounds (Tuyen et al., 1998; Zhang et al., 2002). In this paper,we report the structure elucidation of a new benzopyrroloisoqui-noline alkaloid, fistulosine (1), and the isolation of three knownphenanthroindolizidine alkaloids, (�)-13aa-antofine (2), (�)-14b-hydroxyantofine (3) and (�)-13aa-secoantofine (4). (�)-13aa-Antofine (2) accounted for the antifungal activity observed in thealkaloid extract.

2. Results and discussion

Separation of the alkaloid extract using centrifugal TLC oversilica afforded (�)-13aa-antofine (2) (Capo and Saa, 1989;Baumgartner et al., 1990; Wu et al., 2002; Stærk et al., 2002,2005) as the antifungal component. Also isolated were two knownalkaloids, (�)-14b-hydroxyanofine (hydroxyantofine) (3) (Wie-grebe et al., 1970; Toribio et al., 2006) and (�)-13aa-secoantofine(4) (Stærk et al., 2002), and a new alkaloid, fistulosine (1).

* Corresponding author. Tel.: +65 6829 5611; fax: +65 6829 5601.

E-mail address: mark@merlionpharma.com (M.S. Butler).

1874-3900/$ – see front matter � 2009 Phytochemical Society of Europe. Published by

doi:10.1016/j.phytol.2008.12.006

The molecular formula of fistulosine (1), C19H23NO3, which wasdetermined on the basis of a (+)-HRESIMS quasi molecular ion peakat m/z 314.1765 [M+H]+, indicated the presence of nine degrees ofunsaturation. In addition, 1 displayed UV absorption maxima at238, 282, 314 and 326 nm (log e 3.70, 3.38, 2.70 and 2.59), whichwas characteristic of a naphthalene chromophore (An et al., 2001).The 13C NMR spectrum (Table 1) displayed 19 carbon resonances(two methyl, six methylenes, four methines and seven quaternarycarbons), which was in agreement with the proposed molecularformula. The 1H NMR spectrum (Table 1) showed the presence ofthree aromatic singlet resonances at dH 7.11, 7.15 and 7.56 and twomethoxy resonances at dH 4.00 (s, 3H) and 4.02 (s, 3H). Themethoxy resonances showed HMBC correlations to dC 150.5 and150.0, respectively, which suggested that they were attached to anaromatic ring. Analysis of the COSY and HSQC experiments showedthe presence of an isolated methylene, a CH2OH unit and aCH2CHCH2CH2CH2 fragment. The observed HMBC correlationsfrom H-12 to C-6a, C-11a and C-12a, and H-7 to C-6a, C-12a and C-11a allowed the six membered ring C to be assembled. The otherHMBC correlations from H-9 to C-10 and C-7, and H-11 to C-9, C-10, C-11a and C-12 complete the pyrolidine ring D. The remainingCH2OH unit was attached to C-6 based on HMBC correlations fromH-13 to C-5, C-6 and C-6a. These NMR data indicated that thestructure of 1 is similar to vincetene (5) (Budzikiewicz et al., 1979;Jirovsky et al., 2001) and alkaloid 6 (An et al., 2001) except fordifferent substitutions at C-6.

Given that the known alkaloids 2, 3 and 4 isolated in this studyhad negative optical rotation values and their absolute configura-tions have been assigned, an R stereochemistry was tentatively

assigned to H-11a in (�)-fistulosine (1) by analogy. Although theabsolute configuration of the secondary alcohol present invincetene (5) has been determined by degradation studies

Elsevier B.V. All rights reserved.

Table 11H (500 MHz) and 13C NMR (125 MHz) assignments for fistulosine (1) (CDCl3)a.

Position dC dH, mult (J in Hz) HMBC (H to C)

1 103.2 7.15, s C-2, 4a, 12b

2 150.5

3 150.0

4 108.0 7.11, s C-3, 4a, 5, 12b

4a 128.6

5 124.7 7.56, s C-4, 6a, 13, 12b

6 134.9

6a 129.9

7 53.8 3.43, d (15) C-6, 6a, 9, 11a, 12a

4.28, d (15)

9 55.7 2.35, q (9) C-7, 10

3.37, m

10 22.5 1.90, m C-9, 11

2.01, m

11 32.2 1.72, m C-9, 10, 11a, 12

2.23, m

11a 61.2 2.43, m

12 34.0 2.87, dd (15, 11) C-6a, 11, 11a, 12a

3.37, m

12a 129.9

12b 128.2

13 64.1 4.70, s C-5, 6, 6a

2-OMe 56.8 4.02, s C-2

3-OMe 56.8 4.00, s C-3

a Assignments based on COSY, HSQC and HMBC.

G. Subramaniam et al. / Phytochemistry Letters 2 (2009) 88–90 89

(Jirovsky et al., 2001), the H-11a stereochemistry of the knownalkaloids 5 and 6 remains unassigned. Alkaloid 6 also has anegative optical rotation, while no optical rotation has beenreported for vincetene (5).

(�)-13aa-Antofine (2) exhibited antifungal activity against A.

fumigatus ATCC 46645 and C. albicans ATCC 900280 with an IC50

of 4 mM in both assays, while fistulosine (1), (�)-14b-hydro-xyanofine (3) and (�)-13aa-secoantofine (4) were inactive. (�)-13aa-Antofine (2) has been reported to have antifungal,antibacterial (Capo and Saa, 1989; Baumgartner et al., 1990;Lansky et al., 2008) and cytotoxic activities against variouscancer cell lines (Wu et al., 2002; Stærk et al., 2002; Toribioet al., 2006; Lansky et al., 2008). The cytotoxic activity had beenproposed to be caused through inhibition of protein and nucleicacid synthesis (Xi et al., 2005). (�)-13aa-Antofine (2) was notfurther pursued as a potential antifungal agent due tocytotoxicity concerns.

3. Experimental

3.1. General

Optical rotations were recorded on a PerkinElmer model 341polarimeter. UV spectra were scanned on a Pharmacia BiotechUltrospec 2000 and IR spectra on a PerkinElmer Spectrum One FT-IR spectrophotometer. NMR spectra were collected on a BrukerAvance DRX-500 NMR spectrometer, using 5-mm BBI (1H, G-COSY,multiplicity-edited G-HSQC and G-HMBC spectra) or BBO (13Cspectra) probeheads equipped with z-gradients. Spectra werecalibrated to residual protonated solvent signals. HRESIMS valueswere collected on an Applied Biosystems Mariner TOF massspectrometer, using sodium trifluoroacetate as an internalstandard for both positive- and negative-ionization modes.Chromatographic separation was performed on a Chromatotron,model no. 7924T (Harrison Research, Palo Alto, CA, USA). TLC was

G. Subramaniam et al. / Phytochemistry Letters 2 (2009) 88–9090

carried out on silica gel 60F254 precoated plates sprayed withDragendoff’s reagent for detection.

3.2. Plant material

The stem-bark of F. fistulosa Reinw ex. Blume (Moraceae) wascollected in the secondary forest along a minor road off Old UpperThomson Road leading to the Upper Pierce Reservoir Park,Singapore, in August 1994. The plant was identified by Haji Sidekand a voucher specimen (W20501) has been deposited at theSingapore Botanic Gardens herbarium.

3.3. Extraction and isolation

Dried and milled stem-bark (500 g) were extracted with MeOH(3� 5 L) and dried by rotary evaporation. The crude extract (43 g)was acidified with 5% HCl and filtered through kieselguhr to removenon-alkaloidal substances. The filtrate was basified with 25%ammonia solution to about pH 10 with cooling and the liberatedalkaloids were extracted with CHCl3. The crude alkaloidal mixture(260 mg) was subjected to centrifugal TLC (Chromatotron) usingCHCl3 with increasing proportions of MeOH to give (�)-fistulosine(1) (6.3 mg), (�)-antofine (2) (4.5 mg), (�)-14b-hydroxyanofine(hydroxyantofine) (3) (6.0 mg) and (�)-secoantofine (4) (3.5 mg).

3.3.1. Fistulosine (1)

Light yellowish amorphous powder; [a]D25 �118 (c 0.08,

MeOH); UV lMeOHmax nm (log e): 238 (370), 282 (3.38), 314

(2.70), 326 (2.59); IR nfilmmax cm�1: 3360; NMR data, see Table 1;

(+)-HRESIMS m/z 314.1765 (calcd. for C19H24NO3, 314.1756).

3.4. Biological assay

Frozen stocks of A. fumigatus ATCC 46645 spores, which hadbeen stored at �80 8C, were diluted with assay medium (RPMI1640 with L-glutamine buffered to pH 7.0 with 0.165 Mmorpholinepropanesulfonic acid buffer) to 2.5 � 104 spores/mL.45 mL of the spore suspension was added to 384-well microtiterplates that contained 5 mL of either the test sample or 12.5% DMSO(for 0% inhibition control). Control wells for 100% inhibitionconsisted of 45 mL of assay medium and 5 mL of 12.5% DMSO.Plates were then incubated at 35 8C for 24 h. After the incubation,10 mL of 0.025% resazurin solution (BDH) with 1 mM menadionewas added and the plates further incubation in the dark for 2 h atroom temperature. Fluorescence was measured using SpectraFluorPlus (Tecan) with an excitation wavelength of 530 nm and anemission wavelength of 590 nm. For susceptibility testing of C.

albicans ATCC 900280, the assay procedures were similar to those

described for A. fumigatus, except that an exponential phaseculture at 2.5 � 104 cfu/mL in assay medium was used and theplates were incubated for 20 h prior to resazurin addition.Amphotericin B was used as a standard in both the C. albicans

and A. fumigatus assays and had IC50 3.0 � 2.1 and 5.4 � 2.4 mM,respectively.

Acknowledgments

The authors would also like to acknowledge the contribution ofEugene Tang and Haji Sidek, former employees of MerLionPharmaceuticals, for plant collection and identification.

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