Flemingin-Type Prenylated Chalcones from the Sarawak Rainforest

Aug 18, 2015 - (10) Investigations of Desmodium species have identified prenylated flavonoids ..... surrounding villages near Bintulu, Sarawak, Malays...
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Flemingin-Type Prenylated Chalcones from the Sarawak Rainforest Plant Desmodium congestum Karlee A. Rees,† Cindy Bermudez,† David J. Edwards,† Alysha G. Elliott,† Jovita E. Ripen,‡ Cynthia Seta,‡ Johnny X. Huang,† Matthew A. Cooper,† James A. Fraser,§ Tiong Chia Yeo,‡ and Mark S. Butler*,† †

Institute for Molecular Bioscience and §School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia 4072, Australia ‡ Sarawak Biodiversity Centre, KM 20 Jalan Borneo Heights, Semengoh, Locked Bag No. 3032, 93990 Kuching, Sarawak, Malaysia S Supporting Information *

ABSTRACT: In an ongoing program to identify new antiinfective leads, an extract derived from whole plant material of Desmodium congestum collected in the Sarawak rainforest was found to have anti-MRSA activity. Bioassay-guided isolation led to the isolation of two new prenylated chalcones, 5′-Omethyl-3-hydroxyflemingin A (1) and 5′-O-methylflemingin C (2), which were closely related to the flemingins previously isolated from various Flemingia species. Chalcones 1 and 2, which were determined to be 4:6 enantiomeric mixtures by chiral HPLC, exhibited moderate activity against a panel of Gram-positive bacteria and were also cytotoxic to the HEK293 human embryonic kidney cell line. he fight against antibiotic resistance is ongoing, with innovative drug candidates desperately needed to boost the antibiotic pipeline.1−4 Although synthetic-derived compounds have been developed as antibiotics, natural products remain an important source of new lead compounds for antibiotic drug development and other therapeutic areas.1,5,6 In particular, underexplored natural sources could provide greater opportunities to identify novel lead compounds, and one such habitat is the tropical rainforests of Sarawak in Malaysia.7 The Sarawak Biodiversity Centre (SBC) has undertaken a program to assemble a large collection of often rare and endemic plant and microbial species, while preserving the traditional knowledge associated with the plant samples.7 As part of our search for new anti-infective lead compounds, extracts derived from plants, fungi, and actinomycetes collected by the SBC were tested for antibacterial and antifungal activity. A 1:1 CHCl3−MeOH extract from Desmodium congestum (Wight) Benth. (Fabaceae) displayed activity against methicillin-resistant Staphylococcus aureus (MRSA) but was inactive against the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae and the fungi Cryptococcus neoformans and Candida albicans. Desmodium is a genus of herbs and small shrubs of over 350 species that are widely distributed across subtropical and tropical climates.8,9 Desmodium has been used in traditional medicine for many illnesses including infection, as well as a feedstock in agriculture.10 Investigations of Desmodium species have identified prenylated flavonoids and isoflavonoids with antimicrobial activity11−13 and prenylated chalcones with cytotoxic activity.14,15 No chemical investigations of D. congestum have been reported previously.

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© XXXX American Chemical Society and American Society of Pharmacognosy

The extract was separated using C18 reversed-phase MPLC, and the active fractions were found to be complex mixtures of flavonoids. HPLC separation of these fractions led to the isolation of two brightly orange colored compounds, 5′-Omethyl-3-hydroxyflemingin A (1) and 5′-O-methylflemingin C (2) (Figure 1). The flemingins were first described by Perkin in

Figure 1. Structures of 5′-O-methyl-3-hydroxyflemingin A (1) and 5′O-methylflemingin C (2) isolated in this study and the previously reported flemingins A (3) and C (4).18

1898 as a dye constituent from a purplish, resinous powder that covered the seedpods of the Indian plant Flemingia congesta (Fabaceae).16 The flemingins are also found in seedpod scrapings from East African F. rhodocarpa,17 and the structures of flemingins A (3), B, and C (4) were elucidated as prenylated Received: May 8, 2015

A

DOI: 10.1021/acs.jnatprod.5b00410 J. Nat. Prod. XXXX, XXX, XXX−XXX

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chalcones in 1968.18 Analogs have been subsequently isolated from other Flemingia spp.19−21 The molecular formula of both 1 and 2 was determined to be C26H28O6 on the basis of HRESIMS and supported by the presence of 26 distinct carbon resonances in their 13C NMR spectra (Table 1). Analysis of the 1H, 13C, and 2D NMR data of Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for 5′O-Methyl-3-hydroxyflemingin A (1) and 5′-OMethylflemingin C (2) 1 position

δC, type

1 2 3 4

122.3, 144.7, 143.3, 116.9,

5

120.2, CH

6

122.2, CH

1′ 2′ OH-2′ 3′ 4′ 5′ 6′ OCH3-5′ 1″ 2″ 3″ 4″

112.2, C 156.6, C

5″ 6″

C C C CH

2 δH, mult. (J in Hz)

6.89, dd (1.1, 8.0) 6.80, dd (8.0, 8.0) 7.15, dd (1.1, 8.0)

δC, type 122.9, 149.5, 117.5, 119.0,

C C CH CH

C C C CH CH3 CH CH C CH2

27.2, CH3 22.7, CH2

7″

123.7, CH

8″ 9″ 10″ CO α β

132.0, 25.7, 17.6, 192.2, 122.0, 139.5,

C CH3 CH3 C CH CH

(δH 3.87) correlated to H-5′ (δC 141.0), while the phenolic proton (δH 13.56) had correlations with C-1′ (δC 112.2), C-2′ (δC 156.6), and C-3′ (δC 110.2). These, in addition to correlations from H-1″ (δH 6.79) to C-2′ (δC 156.6) and C-4′ (δC 151.1), H-2″ (δH 5.57) to C-3″ (δC 80.8), and H-6′ (δH 7.25) to the carbonyl (δC 192.2) supported the structure proposed. ROESY correlations from H-6′ (δH 7.25) to the methyl ether (δH 3.87) and H-α (δH 7.71) and from H-α to H6 (δH 7.15) corroborated the assignments (Figure 2). Chalcone 1 is as a new member of the flemingins with a C-5′ methyl ether and a catechol for the pendant aromatic ring and was assigned the name 5′-O-methyl-3-hydroxyflemingin A (1). Chalcone 2, which was isomeric with 1, differed from the latter compound only in the pendant aromatic ring substitution pattern. The NMR data supported the presence of a 1,2,4trisubstituted aromatic ring: H-3 (δH 6.73, d, J = 8.5 Hz), H-4 (δH 6.78, dd, J = 2.5, 8.5 Hz), and H-6 (δH 7.06, d, J = 2.5 Hz). An HMBC correlation from H-β (δH 8.05) to C-6 (δC 115.1) allowed the structure of 2 to be assigned as the 5′-O-methyl ether derivative of flemingin C (Figure 2). 5′-O-Methylflemingin C (2) was unstable and degraded to multiple degradation products over a few days. Chiral HPLC analysis of 1 and 2 showed that they were a mixture of enantiomers in approximate ratios of 4:6. Chalcones 1 and 2 both had small positive optical rotations in MeOH, but accurate [α]D measurements could not be obtained due to the intense orange coloration, which allowed only 0.01 mg/mL solutions to be analyzed. These enantiomeric mixtures could be formed by cyclization and/or oxidation of homoflemingin,18 by either nonspecific enzymatic or in situ nonenzymatic cyclization processes. Chalcones 1 and 2 were moderately active against Grampositive bacteria (MICs in the 16−32 μg/mL range) but were inactive (MIC > 32 μg/mL) against the Gram-negative bacterium E. coli (Table 2). As both compounds displayed cytotoxicity against the HEK293 human embryonic kidney cell line with IC50 values of 12 ± 1.2 μg/mL (37 ± 3.7 μM) for 1 and 7.3 ± 1.2 μg/mL (21 ± 3.5 μM) for 2, there was no interest in pursuing these compounds further as antibacterial leads. The catechol and 1,4-hydroquinone moieties in 1 and 2, respectively, which can oxidize to form reactive ortho- and paraquinones, could be contributing to the cytotoxicity activity. Although no antibacterial activity has been reported for the flemingins, moderate cytotoxicity has been reported for flemingin C (3).21 Chalcones have been reported to have a variety of biological activities including antibacterial, antifungal, antiprotozoal, anti-inflammatory, antiulcer, antioxidant, and cytotoxic effects.22−24

6.73, d (8.5) 6.78, dd (2.5, 8.5)

149.6, C 115.1, CH

7.06, d (2.5)

112.1, C 156.6, C 13.56, s

110.2, 151.1, 141.0, 113.4, 57.5, 116.6, 127.2, 80.8, 41.4,

Figure 2. Key HMBC and ROESY correlations for 5′-O-methyl-3hydroxyflemingin A (1) and 5′-O-methylflemingin C (2).

δH, mult. (J in Hz)

7.25, 3.87, 6.79, 5.57,

s s d (10.2) d (10.2)

1.69, m; 1.87, ma 1.49, s 2.12, br dt (8.2, 8.2)a 5.09, tqq (7.2, 1.3, 1.3)a 1.65, br s 1.56, br s 7.71, d (15.5) 8.05, d (15.5)

13.54, s 110.2, 151.2, 141.0, 113.3 57.6, 116.6, 127.2, 80.9, 41.5,

C C C CH3 CH CH C CH2

27.1, CH3 22.7, CH2 123.7, CH 132.0, 25.7, 17.6, 192.0, 121.8, 139.1,

C CH3 CH3 C CH CH

7.24, 3.87, 6.79, 5.58,

s s d (10.1) d (10.1)

1.69, m; 1.88, ma 1.49, s 2.13, br dt (7.9, 7.9)a 5.09, tqq (7.1, 1.3, 1.3)a 1.65, br s 1.56, br s 7.60, d (15.6) 8.05, d (15.6)

a Although the chemical shift difference between the H-4″ methylene protons was around 0.18 ppm, their 1JHH displayed small second-order effects: one side of the multiplet was a dd (7.2, 10.2 Hz), and the other side a dd (8.7, 8.7 Hz).

1 identified four different 1H/1H spin systems: isoprene C-4″ to C-10″, one Z double bond (H-1″, δH 6.79, J = 10.2 Hz and H-2″, δH 5.57, J = 10.2 Hz), one E double bond (H-α, δH 7.71, J = 15.5 Hz, and H-β, δH 8.05, J = 15.5 Hz), and a 1,2,3trisubstituted aromatic group [H-4 (δH 6.89, dd, J = 1.1, 8.0 Hz), H-5 (δH 6.80, dd, J = 8.0, 8.0 Hz), and H-6 (δH 7.15, dd, J = 1.1, 8.0 Hz)]. Also present in the 1H NMR spectrum of 1 were singlet resonances associated with one hydrogen-bonded phenolic proton (OH-2′, δH 13.56), one aromatic methine (H6′, δH 7.25, δC 113.4), one methyl ether (OCH3-5′, δH 3.87, δC 57.5), and one methyl group (H-5″, δH 1.49, δC 27.2). HMBC correlations were used to join the different fragments and to clarify the central ring structure (Figure 2). The methyl ether B

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Table 2. MIC (μg/mL) Data for 5′-O-Methyl-3-hydroxyflemingin A (1) and 5′-O-Methylflemingin C (2) against Various Bacteria MIC (μg/mL) (n = 2)

a

organism

strain type

resistance phenotype

1

2

VANa

DAPb

S. aureus S. aureus S. aureus Streptococcus pneumoniae Enterococcus faecalis E. faecium E. coli

ATCC 43300 NRSA-NRS 17 NRSA-NRS 1 ATCC 700677 clinical isolate ATCC 51559 ATCC 25922

MRSA GISA GISA, MRSA MDR VanA MDR-VanA FDA control

32 >32 32 32 16 32 >32

32 >32 32 32 16 32 >32

1 8 8 2 >32 >32

1.2 8 16 2 8 16

COLc

0.25

Vancomycin (VAN). bDaptomycin (DAP). cColistin (COL).

In conclusion, two new flemingin-type 18 prenylated chalcones, 5′-O-methyl-3-hydroxyflemingin A (1) and 5′-Omethylflemingin C (2), were isolated from the Sarawak plant D. congestum using anti-MRSA bioassay-guided isolation and were found to be enantiomeric mixtures using chiral HPLC. Although 1 and 2 displayed Gram-positive antibacterial activity, they also had moderate cytotoxicity, which precluded further development as antibacterial leads. The identification of flemingin-type prenylated chalcones in D. congestum is also taxonomically interesting, as previously they had been reported only from Flemingia spp.

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gradient elution from 90% H2O−CH3CN to 98% CH3CN over 9.5 min, then an 11.5 min hold at 98% CH3CN) to yield 2 (2.4 mg) and 1 (1.7 mg) from each respective fraction. 5′-O-Methyl-3-hydroxyflemingin A (1): unstable orange oil (2.4 mg, 0.006% dry weight); UV (MeOH) λmax (log ε) 282 (1.69), 368 (1.69), 671 (−1.19) nm; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3), see Table 1; (+)-ESIMS m/z 437.3 [M + H]+; (−)-ESIMS m/z 435.1 [M − H]−; (+)-HRESIMS m/z 437.1970 (calcd for C26H29O6, 437.1959). 5′-O-Methylflemingin C (2): unstable orange oil (1.7 mg, 0.004% dry weight); UV (MeOH) λmax (log ε) 277 (1.77), 402 (1.40), 670 (−1.19) nm; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3), see Table 1; (+)-ESIMS m/z 437.2 [M + H]+; (−)-ESIMS m/z 435.2 [M − H]−; (+)-HRESIMS m/z 437.1959 (calcd for C26H29O6, 437.1959). Antimicrobial Assays. The tested bacteria strains (detailed in Table 2) were cultured in Muller-Hinton broth (MHB) (Bacto Laboratories, cat. no. 211443) at 37 °C overnight with shaking. A sample of each culture was then diluted 40-fold in fresh MHB broth and incubated with shaking at 37 °C for 2−3 h. The compounds were serially diluted 2-fold across the wells, with concentrations ranging from 0.03 to 64 μg/mL, plated in duplicate. The resultant mid-log phase cultures were diluted to the final concentration of 5 × 105 CFU/ mL; then 50 μL was added to each well of the compound-containing 96-well plates (Corning; cat. no. 3370, polystyrene plates), giving a final compound concentration range of 0.015−32 μg/mL. Plates were covered and incubated at 37 °C for 24 h. MICs were determined visually, being defined as the lowest concentration showing no visible growth. Vancomycin·HCl (Sigma 861987), daptomycin (Molekula 64342447), and colistin sulfate (Sigma C4661) were used as controls. All of this work was conducted in a level 2 biosafety cabinet. Cytotoxicity Assay. HEK293 human embryonic kidney cells (ATCC CRL-1573) were seeded as 3000 cells/well in clear-bottom 384-well plates (Corning 3712) in a volume of 20 μL in DMEM medium (GIBCO-Invitrogen, cat. no. 11995-073), in which 10% FBS was added. Cells were incubated for 24 h at 37 °C in 5% CO2 to allow cells to attach to the plates. Compounds were dissolved in DMSO at 1.28 mg/mL, a dilution series of 1:3-fold steps in cell culture medium was created, and 20 μL of each dilution was added to the cell culture wells, resulting in 40 μL as the final volume. The final DMSO concentration was 1%. The cells were incubated with the compounds for 24 h at 37 °C, 5% CO2. After the incubation, 10 μM resazurin (dissolved in PBS) was added to each well, and the plates were incubated for a further 2 h at 37 °C, 5% CO2. The fluorescence intensity was read using a Polarstar Omega (BMG Technologies) plate reader with excitation/emission at 560/590 nm. The data from four replicates were analyzed using GraphPad Prism software (version 6), and the results were calculated using the following equation: percent viability = (FITEST − FINEGATIVE/FI1%DMSO − FINEGATIVE) × 100%. Tamoxifen (Sigma T5648) was the positive control and had an IC50 of 51 μM.

EXPERIMENTAL SECTION

General Experimental Procedures. Chiroptical measurements ([α]D) were obtained on a JASCO P-1010 polarimeter in a 100 × 2 mm cell, while UV spectra were recorded on a Varian Cary 50 Bio UV−vis spectrophotometer. 1H (600 MHz) and 13C (150 MHz) NMR spectra were obtained in CDCl3 using a Bruker Avance-600 spectrometer equipped with a TXI cryoprobe using standard pulse sequences with the resonances referenced to TMS (δH 0.00 and δC 0.0 ppm). High-resolution mass spectrometry (HRMS) was performed on a Bruker Micro TOF mass spectrometer using (+)-ESI calibrated to sodium formate. Compound purity was analyzed using a Shimadzu LCMS 2020 LC/MS with a SPD-M20A UV−vis detector (λ = 200− 400 nm), using a Zorbax Eclipse XDB phenyl column (3.0 × 100 mm, 3.5 μm, flow rate 1 mL/min, 40 °C). Chiral LC-MS analyses were conducted using an Agilent Technologies 1200 Series Instrument with a G1316A UV−vis detector (λ = 210 and 254 nm), 1200 Series ELSD, and 6110 quadrupole ESIMS, using a Phenomenex Lux Cellulose-2 column (4.6 × 250 mm, 3 μm, flow rate 1 mL/min, room temperature). The eluents were 0.05% formic acid in water and 0.05% formic acid in CH3CN. The MPLC and HPLC purifications were achieved using a Gilson PLC 2020 personal purification system. Plant Material. Desmodium congestum was collected from the tropical rainforest surrounding villages near Bintulu, Sarawak, Malaysia, in September 2014. The plant was selected based on its traditional use in healing various diseases, including sores and antiinflammatory activity. The plant was identified by one of the authors (J.E.R.), and a voucher specimen (SABC4003) was deposited at the herbarium of the Sarawak Biodiversity Centre, Malaysia. Extraction and Isolation. Whole plant material of D. congestum was dried (290 g, dry weight), ground to a powder, and extracted with CH2Cl2−MeOH (1:1). The solvent was removed by rotary evaporation to give approximately 7 g of crude extract. A 1 g aliquot was dissolved in CH3CN, and the soluble material (230 mg) was separated by MPLC using a Biotage 80 g C18 SNAP cartridge (30 mL/ min, isocratic 0.1% formic acid, isocratic elution of 90% H2O−CH3CN for 3 min, then a gradient elution from 90% H2O−CH3CN to 98% CH3CN over 15 min, then an 8 min hold at 98% CH3CN) to yield six fractions. Fraction 4 (test tubes 28−32, 23.7 mg) and fraction 5 (test tubes 33−39, 54.7 mg) were subjected to HPLC (Agilent Zorbax C18 5 μm, 250 × 9.4 mm semipreparative column, 5 mL/min, isocratic 0.1% formic acid, isocratic elution of 90% H2O−CH3CN for 2 min, then a C

DOI: 10.1021/acs.jnatprod.5b00410 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Note

(18) Cardillo, G.; Merlini, L.; Mondelli, R. Tetrahedron 1968, 24, 497−510. (19) Cardillo, B.; Gennaro, A.; Merlini, L.; Nasini, G.; Servi, S. Phytochemistry 1973, 12, 2027−2031. (20) Saxena, V. K. Asian J. Chem. 1995, 7, 307−310. (21) Gumula, I.; Alao, J. P.; Ndiege, I. O.; Sunnerhagen, P.; Yenesew, A.; Erdélyi, M. J. Nat. Prod. 2014, 77, 2060−2067. (22) Katsori, A.-M.; Hadjipavlou-Litina, D. Expert Opin. Ther. Pat. 2011, 21, 1575−1596. (23) Sahu, N. K.; Balbhadra, S. S.; Choudhary, J.; Kohli, D. V. Curr. Med. Chem. 2012, 19, 209−225. (24) Rozmer, Z.; Perjési, P. Phytochem. Rev. 2014, DOI: 10.1007/ s11101-014-9387-8.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00410. HPLC-ESIMS-PDA data, chiral HPLC analysis, and NMR spectra (1H, 13C, COSY, ROESY, edHSQC, and HMBC) of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-7-3346-2992. Fax: +61-7-3346-2090. E-mail: m. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank the community who participated in the SBC Traditional Knowledge Documentation Program, D. L. Paterson (UQCCR, University of Queensland) for his kind donation of clinically derived E. faecalis vanA strain, and R. Pelingon (IMB, University of Queensland) for running the HRESIMS. The MIC screening was done in collaboration with CO-ADD (Community for Open Antimicrobial Drug Discovery). M.A.C. is an Australian NHMRC Principal Research Fellow. This work was supported by NHMRC Project Grant APP1049716.

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