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Globoidnan A: a lignan from Eucalyptus globoidea inhibits HIV integrase
PHYTOCHEMISTRY Phytochemistry 65 (2004) 3255–3259 www.elsevier.com/locate/phytochem
Globoidnan A: a lignan from Eucalyptus globoidea inhibits HIV integrase Simon P.B. Ovenden a, Jin Yu a, Soo San Wan a, Gian Sberna a, R. Murray Tait a, David Rhodes b,1, Susan Cox b,1, Jonathan Coates b,1, Neville G. Walsh c, Barbara M. Meurer-Grimes a,* a Cerylid Biosciences Ltd., 576 Swan Street, Richmond, Vic. 3121, Australia Amrad Corporation Limited, 576 Swan Street, Richmond, Vic. 3121, Australia Royal Botanic Gardens Melbourne, Private Bag 2000, South Yarra, Vic. 3141, Australia b
c
Received 30 July 2004; received in revised form 12 October 2004
Abstract An HTS campaign aimed at the identification of inhibitors of HIV integrase showed that the methanol extract from the buds of a Eucalyptus globoidea was active. Bioassay guided fractionation of this extract resulted in the purification and structural elucidation of the lignan, globoidnan A (1) as the only compound in the extract responsible for the inhibition of HIV integrase. The compound was found to inhibit the combined 3 0 processing and strand transfer activity of HIV integrase with an IC50 = 0.64 lM. 2004 Published by Elsevier Ltd. Keywords: Phenylpropanoid; Cyclolignan; Lignan; Globoidnan A; Eucalyptus globoidea; HIV-integrase; 3-(3,4-dihydroxyphenyl) lactic acid; 4-(3,4Dihydroxyphenyl)-6,7-dihydroxy-2-naphthalene carboxylic acid
1. Introduction HIV/AIDS has become an epidemic of global proportions, with an estimated 40 million people living with the disease at the end of 2003, of which 26.6 million are living in Sub-Saharan Africa (WHO web site, 2004). This growing health epidemic has focussed the energies of many research organisations on identifying new HIV molecular targets and their inhibitors. The HIV genome encodes three enzymes essential to its replication: reverse transcriptase, protease and integ* Corresponding author. Tel.: +61 3 9208 4074; fax: +61 3 9208 4126. E-mail address: [email protected] (B.M. MeurerGrimes), [email protected] (B.M. Meurer-Grimes). 1 Present address: Avexa Limited, 576 Swan Street, Richmond, Vic. 3121, Australia.
0031-9422/$ - see front matter 2004 Published by Elsevier Ltd. doi:10.1016/j.phytochem.2004.10.006
rase (Au et al., 2001). Over the past 17 years, antiviral compounds have been developed to inhibit the activities of reverse transcriptase and protease. Whilst these have proven effective in controlling virus replication in the majority of treated individuals, viruses resistant to many of these inhibitors are becoming more common place (Pomerantz and Horn, 2003). To circumvent the development of resistant viruses, new targets are being examined. One such target is integrase, the enzyme responsible for inserting the HIV proviral DNA into host DNA. This enzyme functions in a two-step manner, it initially removes a dinucleotide unit from the 3 0 ends of the proviral DNA (termed ‘‘3 0 -processing’’), then the 3 0 -processed strands are transferred from the cytoplasm to the host nucleus, where they are introduced into the host DNA (termed ‘‘strand-transfer’’ or ‘‘integration’’) (Lin et al., 1999). Therefore, effecting the mechanism of action of this enzyme selectively would
3256
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be a significant contribution towards discovering novel therapeutics for treatment of HIV/AIDS. During an HTS campaign conducted to find novel inhibitors of HIV integrase, in particular the 3 0 -processing reaction, a library of natural product extracts were screened, and the MeOH extract from the buds from of Eucalyptus globoidea were found to be active. Bioassay guided fractionation of this crude MeOH extract allowed for the purification of the new lignan, globoidnan A (1) as the sole compound responsible for the activity. Discussed below is the structure elucidation and biological activity of 1.
2. Results and discussion Globoidnan A (1) was isolated as a brown oil which gave a [M H] ion peak in the mass spectrum at m/z 491.0967, suggestive of the molecular formula C26H20O10 [D1.1 mmu] and equating to 17 double bond equivalents. Analysis of the NMR data (Table 1) for 1 immediately suggested a highly aromatised molecule, as the 13C NMR chemical shifts suggested that 22 of the 26 carbons were aromatic. The 1H–1H-COSY and gHMBC correlations (Table 1) identified resonances consistent with a 1,3-disubstituted naphthalene-6,7-diol moiety (13C: 149.4, 147.4, 138.1, 129.5, 128.3, 127.5, 123.5, 122.5, 111.5, 108.0 ppm; 1H: d 8.18, 7.47, 7.32,
Table 1 NMR data (500 MHz, d6-DMSO) for globoidnan A (1) No.
13
C (d, m)a
1 2 3 4 4a 5 6 7 8 8a 9 10 20 30 40 50 60 100 200 300 400 500 600 700
138.1 (s) 122.5 (d) 123.5 (s) 127.5 (d) 128.3 (s) 111.5 (d) 147.4 (s) 149.4 (s) 108.0 (d) 129.5 (s) 165.8 (s) 131.3 (s) 116.9 (d) 145.0 (s) 145.1 (s) 116.6 (d) 120.4 (d) 129.5 (s) 115.8 (d) 144.9 (s) 143.8 (s) 115.3 (d) 120.1 (d) 36.5 (t)
800 900
74.3 (d) 171.0 (s)
1
1
H–1H COSY
gHMBC 1H to
7.47 (br s)
H-4
C-4, C-8a, C-9, C-1 0
8.18 (br s)
H-2
C-2, C-5, C-8a, C-9
H (d, m, J (Hz))
13
C
7.32 (s)
C-4, C-6, C-7, C-8a
7.22 (s)
C-1, C-4a, C-6, C-7
6.81 (d, 1.8)
H-6 0
C-1, C-4 0 , C-6 0
6.87 (d, 8.5) 6.65 (dd, 8.5, 1.8)
H-6 0 H-5 0 , H-6 0
C-1 0 , C-3 0 C-1, C-2 0 , C-4 0
6.71 (br s)
H-600
H-300 , H-400 , H-600 , H-700
6.60 6.57 3.08 2.91 5.11
H-600 H-200 , H-500 Hb-700 , H-800 Ha-700 , H-800 H2-700
H-100 , H-300 H-200 , H-400 , H-700 C-100 , C-200 , C-600 C-100 , C-200 , C-600
(d, 8.5) (br d, 8.5) (dd, 14.5, 3.3) (dd, 14.5, 9.5) (dd, 9.5, 3.3)
7.22) as well as an alkylated 3,4-bis-catechol (13C: 144.9, 143.8, 129.5, 120.1, 115.3, 115.8, 74.3, 36.5 ppm; 1H: d 6.71, 6.60, 6.57, 5.11, 3.08, 2.91) and a mono-substituted 3,4-bis-catechol (13C: 145.1, 145.0, 131.3, 120.4, 116.9, 116.6 ppm; 1H: d 6.87, 6.81, 6.65). These data, along with the observation of two carbonyl 13C resonances (13C: 171.0, 165.8 ppm) consistent with the presence of ester and carboxylic acid moieties, accounted for all of the available double bond equivalents. Further analysis of the gHMBC data for 1 revealed several diagnostic correlations. The observation of gHMBC correlations from d 6.81 (H-2 0 ) and d 6.65 (H-6 0 ) into 138.1 ppm (C-1), as well as from d 7.47 (H-2) into 131.3 ppm (C-1 0 ) established that the monosubstituted 3,4-bis-catechol was attached at the C-1 position of the 1,3-disubstituted naphthalene-6,7-diol. Additional gHMBC correlations from d 7.47 (H-2) and d 8.18 (H-4) into 165.8 ppm (C-9) placed an ester carbonyl at C-3 of the 1,3-disubstituted naphthalene6,7-diol, and hence established the substitution pattern of the 1,3-disubstituted naphthalene-6,7-diol moiety as shown. The point of attachment for the 3-(3,4-dihydroxyphenyl) lactic acid moiety to the 1,3-disubstituted naphthalene-6,7-diol at C-3 was elucidated through the observed NMR chemical shift data for C-800 (13C: 74.3 ppm; 1H: d 5.11), which was consistent with an ester methine. Thus the carboxylic acid functionality had to be attached at the only remaining position, C-800 , leading to the proposed gross structure of 1 as shown below. Therefore 1 is a novel ester composed of (3,4-dihydroxyphenyl) lactic acid and 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy2-naphthalene carboxylic acid. An attempt was made to determine the absolute stereochemistry of 1 through hydrolysis to form the known compound (3,4-dihydroxyphenyl) lactic acid. However, only decomposition product was produced and as a consequence the absolute stereochemistry remains unassigned at this time. The structure of 1-(3,4-dihydroxyphenyl)-naphthalene-6,7-diol, and associated carboxylic acid analogues from other natural sources has been reported previously (Cullmann et al., 1999; Rischmann et al., 1989; Tazaki et al., 1995). However, this is the first report of an alkylated aryl side chain attached through an ester functionality as shown in 1. Interestingly, this is the first report of this type of cyclolignan from a higher plant, with all previous reports of this structure class referring to their occurrence in liverworts. A great number of HIV integrase inhibitors have previously been isolated from natural sources, including examples from plants (Kim et al., 1998), lichens (Neamati et al., 1997) and fungi (Singh et al., 1998, 2002a,b; Herath et al., 2004) resulting in several granted patents (Neamati, 2002). IC50 values of these compounds against HIV integrase range from 1 to 20 lM.
S.P.B. Ovenden et al. / Phytochemistry 65 (2004) 3255–3259
The activity of 1 compares favourably with previously reported natural product derived inhibitors of HIV integrase, with an IC50 = 0.64 lM in our combined 3 0 processing/strand transfer HIV integrase assay. In assays designed to measure the inhibitory effect of compounds on separate 3 0 processing (Chow, 1997) or strand transfer activities (Hwang et al., 2000), globoidnan A (1) was determined to inhibit HIV integrase with IC50Õs of 7 and 1.5 lM, respectively. Globoidnan A (1) was also assessed for its ability to inhibit the replication of HIV-1NL4-3 in the HuT78 T-cell line. No antiviral or toxic effects were observed after 6 days at concentrations up to 50 lM, the highest dose tested. Although no in vitro activity was observed for compound 1 in the whole cell anti-HIV assay, the enzyme HIV-integrase is considered a valid target for HTS efforts to identify novel small molecule inhibitors of viral replication. Enzyme based assays are usually more robust, time and cost efficient, and less prone to erratic screening results. In addition, they have the potential to yield inhibitors with known mechanisms of action, thus reducing downstream development cost for novel drug candidate molecules. Globoidnan A (1) shares two structural motifs that were previously noted for inhibitors of HIV integrase. Compounds containing a diketoacid motif, such as L()-chicoric acid, played a major role in validating HIV integrase as a target for therapeutic intervention (Lin et al., 1999; Long et al., 2004) and have been used as scaffolds for the preparation of HIV integrase inhibitors (Pais et al., 2002). Compounds with the diketoacid motif have been demonstrated to possess activity in whole cell assay systems (Lin et al., 1999), which was not observed for 1. In addition, the bis-catechol structural motif has been frequently associated with inhibition of HIV integrase (Dupont et al., 2001). The structure of 1 contains three biscatechol moieties. In most cases, inhibitors with this structural feature are active in cell-free assay systems, but lack selective activity in whole cell assay systems or display unacceptably high levels of toxicity (Lin et al., 1999). Interestingly, despite the presence of the three bis-catechol moieties in 1, no cytotoxicity was observed.
O HO
5
O
4 3 9 O
OH 9'' 7'' 1''
HO
2''
1 1'
8 6'
2''
OH OH 1
OH OH
3257
3. Experimental 3.1. General SPE was performed using Varian Megabond Elute C18 SPE cartridges (10 g, 50 lm). HPLC was performed on either a Waters Delta Prep 4000 chromatography system equipped with a Waters 2487 dual wavelength UV detector, a Waters prep LC system controller and a Waters Fraction Collector, or system equipped with a Waters 600 Controller, a Waters 996 Photodiode Array detector, Waters 717 plus autosampler and a Waters Fraction Collector II. All data generated from these chromatographic systems were collected using Waters Millenium32 data collection package. All NMR spectra were collected on a Varian Unity Inova 500 MHz spectrometer in the solvents indicated, with spectra referenced to residual 1H in the deuterated NMR solvents. Optical rotations were performed on a Jasco Dip1000 digital polarimeter, while infrared spectra were acquired on a Bio-Rad FTS-165 Fourier-transform infrared spectrometer. Low resolution mass spectral data were collected on a ThermoFinnigan LCQ ion trap mass spectrometer, with an ESI probe. High resolution mass measurements were collected on a Bruker BioApex FT mass spectrometer. 3.2. Plant material The buds of a specimen of Eucalyptus globoidea (Myrtaceae) were collected from the south side of Cape Conran, 13 km from Marlo, Victoria, Australia in April 1995 (3747 0 5600 S, 14840 0 4600 E). A voucher specimen (MEL2025404) has been lodged with the National Herbarium of Victoria. 3.3. Extraction and isolation The ground and dried buds of a specimen of Eucalyptus globoidea (7 g) were extracted twice with MeOH (500 ml), concentrated in vacuo and subjected to a C18 SPE column (20% stepwise gradient elution from 0% MeOH to 100% MeOH in H2O). Biological activity was located in the 40% and 60% MeOH/H2O fractions. These were combined and further purified on a C18 preparative HPLC [16 ml/min, gradient elution from 20% MeCN:H2O (+0.1% formic acid) to 100% MeCN (+0.1% formic acid) over 25 min through a Varian C18 250 · 50 mm 5 lm preparative HPLC axial compression column] and C8 semi-preparative HPLC [6 ml/min, gradient elution from 20% (MeCN:H2O + 0.1% formic acid) to MeCN (+0.1% formic acid) over 20 min through a Waters C8 Symmetry 250 · 25 mm 7 lm HPLC column] to yield the new lignan globoidnan A (1) (1.9 mg, 0.03% yield) as the compound responsible for the activity.
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3.3.1. Globoidnan A (1) Brown oil. [a]D + 14.4 (c 0.118, MeOH); IR (film) mmax 3200, 2956, 2924, 2852, 1720, 1701 cm1; UV (PDA, MeCN/H2O) kmax 224, 260, 320 nm; 1H (500 MHz, d6-DMSO) and 13C (125 MHz, d6-DMSO) NMR data see Table 1; HRESIMS [M H] m/z 491.0967 (calculated for C26H19O10 491.0978). 3.3.2. HIV-integrase assays Natural product extracts were reconstituted in 2 ll of DMSO and incubated in 18 ll of 30 nM substrate DNA and 300 nM HIV integrase in assay buffer (containing 20 mM Tris–HCl (pH 7.5), 25 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 5 mM DTT, 50 lg/ml nuclease-free BSA and 0.05% Tween 20) for 90 min at 37 C. Both positive and negative control wells have HIV integrase and DMSO as vehicle, whilst negative control wells do not contain substrate DNA. The substrate DNA for the strand transfer assay is as previously reported (Hwang et al., 2000) and for the combined 3 0 processing/strand transfer assay, a modified 5 0 -DIG labelled oligonucleotide is used where an additional 2 nucleotides, G and T, are added to the 3 0 end to complement the BIO labelled oligonucleotide sequence. For the combined 3 0 processing/strand transfer reaction, the reaction time was extended to 90 min to provide time for the 3 0 processing (removal) of the two additional nucleotides from the substrate DNA to occur prior to it under going strand transfer. After incubation, a further 30 ll of 33 mM Tris–HCl (pH 7.5, containing 664 mM NaCl, 16.6 mM EDTA and 0.166 mg/ml sonicated salmon sperm DNA) was added to each well and the 50 ll reaction volume transferred to 384-well plates coated with streptavidin. Following a 60 min incubation at room temperature, the streptavidin-coated plates were washed three times with 30 mM NaOH, 200 mM NaCl and 1 mM EDTA. Plates were washed twice with 10 mM Tris–HCl (pH 8.0, containing 1 mM EDTA and 0.1 mg/ml nucleasefree BSA) prior to the addition of 50 ll of 0.075 U/ml of anti-digoxigenin alkaline phosphatase, Fab fragments in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA and 0.1 mg/ ml nuclease-free BSA buffer and incubated for 60 min at 37 C. Plates were then washed four times with phosphate-buffered saline containing 0.1% Tween-20 and once with phosphate-buffered saline, and wells were then developed in 50 ll of p-nitrophenyl phosphate solution (1 mg/ml in 0.1 M Tris–HCl) and measured for absorbance at 405 nm. The 3 0 processing assay was conducted as described previously (Chow, 1997) using a 33P-end labelled HU5V1 substrate. Processed products were measured following their separation from substrate in a denaturing acrylamide gel (Chow, 1997). The whole cell antiviral assay was conducted by infecting 50,000 HuT78 T-cells in RPMI + 10% heat
inactivated fetal calf serum, in each well of a 96 well plate, with 65 TCID50 units of HIV-1NL4-3 in the presence of different concentrations of globoidnan A (1). After an overnight infection in a total volume of 100 ll, and equal volume of fresh media containing 1 was added. After an additional 48 h, 100 ll of media was removed and replaced with 100 ll fresh media containing 1. A sample of culture supernatant was diluted 1 in 2000 and p24 measured using a p24 ELISA assay (Vironostika HIV-1 Antigen kit, Organon Teknika). For cytotoxicity assays, cells were passaged in the presence of compound as described for the infection assay: however, no virus was added. The viability of cells was assessed using the Cytolux assay (Perkin–Elmer Life Sciences) following the manufacturerÕs instructions.
Acknowledgements We acknowledge P.C. Jobson for the collection of the plant material, Wendy Love for the preparation of the dried plant material for chemical isolation, Simon Harris, Effie Tsotsis and Dimuthu Goonasekara for their help in conducting biological screening. We acknowledge Dr. Marisa Spiniello and Associate Professor Mark Rizzacasa at the School of Chemistry, The University of Melbourne, for their help in the acquisition of optical rotation and IR spectra, and Sally Duck at the Department of Chemistry, Monash University, for the high resolution mass measurement.
References Au, T.K., Lam, T.L., Ng, T.B., Fong, W.P., Wan, D.C.C., 2001. A comparison of HIV-1 integrase inhibition by aqueous and methanol extracts of Chinese medicinal herbs. Life Sci. 68, 1687–1694. Chow, S.A., 1997. In vitro assays for activities of retroviral integrase. Methods 12, 306–317. Cullmann, F., Schmidt, A., Schuld, F., Trennheuser, M.L., Becker, H., 1999. Lignans from the liverworts Lepidozia incurvata, Chiloscyphus polyanthos and Jungermannia exsertifolia ssp. cordifolia. Phytochemistry 52, 1647–1650. Dupont, R., Jeanson, L., Mouscadet, J.-F., Cotelle, P., 2001. Synthesis and HIV-1 integrase inhibitory activities of catechol and bis-catechol derivatives. Bioorg. Med. Chem. Lett. 11, 3175–3178. Herath, K.B., Jayasuriya, H., Bills, G.F., Polishook, J.D., Dombrowski, A.W., Guan, Z., Felock, P.J., Hazuda, D.J., Singh, S.B., 2004. Isolation, structure, absolute stereochemistry, and HIV-1 integrase inhibitory activity of integrasone, a novel fungal polyketide. J. Nat. Prod. 67, 872–874. Hwang, Y., Rhodes, D., Bushman, F., 2000. Rapid microtitre assays for poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1 integrase: application to inhibitor isolation. Nucl. Acid Res. 28, 4884–4892. Kim, H.J., Woo, E.R., Shin, C.G., Park, H., 1998. A new flavonol glycoside gallate ester from Acer okamotoanum and its inhibitory activity against human immunodeficiency virus-1 (HIV-1) integrase. J. Nat. Prod. 61, 145–148.
S.P.B. Ovenden et al. / Phytochemistry 65 (2004) 3255–3259 Lin, Z., Neamati, N., Zhao, H., Kiryu, Y., Turpin, J.A., Aberham, C., Strebel, K., Kohn, K., Witvrouw, M., Pannecouque, C., Debyser, Z., De Clercq, E., Rice, W.G., Pommier, Y., Burke Jr., T.R., 1999. Chicoric acid analogues as HIV-1 integrase inhibitors. J. Med. Chem. 42, 1401–1414. Long, Y.Q., Jiang, X.H., Dayam, R., Sanchez, T., Shoemaker, R., Sei, S., Neamati, N., 2004. Rotational design and synthesis of novel dimeric diketoacid-containing inhibitors of HIV-1 integrase: implications for binding to two metal ions on the active site of integrase. J. Med. Chem. 47, 2561–2573. Neamati, N., Hong, H., Mazumder, A., Wang, S., Sunder, S., Nicklaus, M.C., Milne, G.W.A., Proksa, B., Pommier, Y., 1997. Despides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching. J. Med. Chem. 40, 942–951. Neamati, N., 2002. Patented small molecules inhibitors of HIV-1 integrase: a 10-year saga. Expert Opin. Ther. Patents 12, 709– 724. Pais, G.C.G., Zhang, X., Marchand, C., Neamati, N., Cowansage, K., Svarovskaia, E.S., Pathak, V.K., Tang, Y., Nicklaus, M., Pommier, Y., Burke Jr., T.R., 2002. Structure activity of 3-aryl-1,3diketo-containing compounds as HIV-1 integrase inhibitors. J. Med. Chem. 45, 3184–3194. Pomerantz, R.J., Horn, D.L., 2003. Twenty years of therapy for HIV-1 infection. Nat. Med. 9, 867–873.
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Rischmann, M., Mues, R., Geiger, H., Laas, H.J., Eicher, T., 1989. Isolation and synthesis of 6,7-dihydroxy-4-(3,4-dihydroxyphenyl)naphthalene-2-carboxylic acid from Pellia epiphylla. Phytochemistry 28, 867–869. Singh, S.B., Zink, D.L., Goetz, M.A., Dombrowski, A.W., Polishook, J.D., Hazuda, D.J., 1998. Equisetin and a novel opposite stereochemical homolog phomasetin, two fungal metabolites as inhibitors of HIV-1 integrase. Tetrahedron Lett. 39, 2243–2246. Singh, S.B., Zink, D.L., Bills, G.F., Pelaez, F., Teran, A., Collado, J., Silverman, K.C., Lingham, R.B., Felock, P., Hazuda, D.J., 2002a. Discovery, structure and HIV-1 integrase inhibitory activities of integracins, novel dimeric alkyl aromatics from Cytonaema sp. Tetrahedron Lett. 43, 1617–1620. Singh, S.B., Zink, D.L., Quamina, D.S., Pelaez, F., Teran, A., Felock, P., Hazuda, D.J., 2002b. Integrastatins: structure and HIV-1 integrase inhibitory activities of two novel racemic tetracyclic aromatic heterocycles produced by two fungal species. Tetrahedron Lett. 43, 2351–2354. Tazaki, H., Adam, K.P., Becker, H., 1995. Five lignan derivatives from in vitro cultures of the liverwort Jamesoniella autumnalis. Phytochemistry 40, 1671–1675. World Health Organisation, 2004. ‘‘AIDS and Sexually Transmitted Diseases: World AIDS Campaign 2003 Fact Sheets’’. Accessed 30/ 06/2004. Available from:.
Globoidnan A: a lignan from Eucalyptus globoidea inhibits HIV integrase Simon P.B. Ovenden a, Jin Yu a, Soo San Wan a, Gian Sberna a, R. Murray Tait a, David Rhodes b,1, Susan Cox b,1, Jonathan Coates b,1, Neville G. Walsh c, Barbara M. Meurer-Grimes a,* a Cerylid Biosciences Ltd., 576 Swan Street, Richmond, Vic. 3121, Australia Amrad Corporation Limited, 576 Swan Street, Richmond, Vic. 3121, Australia Royal Botanic Gardens Melbourne, Private Bag 2000, South Yarra, Vic. 3141, Australia b
c
Received 30 July 2004; received in revised form 12 October 2004
Abstract An HTS campaign aimed at the identification of inhibitors of HIV integrase showed that the methanol extract from the buds of a Eucalyptus globoidea was active. Bioassay guided fractionation of this extract resulted in the purification and structural elucidation of the lignan, globoidnan A (1) as the only compound in the extract responsible for the inhibition of HIV integrase. The compound was found to inhibit the combined 3 0 processing and strand transfer activity of HIV integrase with an IC50 = 0.64 lM. 2004 Published by Elsevier Ltd. Keywords: Phenylpropanoid; Cyclolignan; Lignan; Globoidnan A; Eucalyptus globoidea; HIV-integrase; 3-(3,4-dihydroxyphenyl) lactic acid; 4-(3,4Dihydroxyphenyl)-6,7-dihydroxy-2-naphthalene carboxylic acid
1. Introduction HIV/AIDS has become an epidemic of global proportions, with an estimated 40 million people living with the disease at the end of 2003, of which 26.6 million are living in Sub-Saharan Africa (WHO web site, 2004). This growing health epidemic has focussed the energies of many research organisations on identifying new HIV molecular targets and their inhibitors. The HIV genome encodes three enzymes essential to its replication: reverse transcriptase, protease and integ* Corresponding author. Tel.: +61 3 9208 4074; fax: +61 3 9208 4126. E-mail address: [email protected] (B.M. MeurerGrimes), [email protected] (B.M. Meurer-Grimes). 1 Present address: Avexa Limited, 576 Swan Street, Richmond, Vic. 3121, Australia.
0031-9422/$ - see front matter 2004 Published by Elsevier Ltd. doi:10.1016/j.phytochem.2004.10.006
rase (Au et al., 2001). Over the past 17 years, antiviral compounds have been developed to inhibit the activities of reverse transcriptase and protease. Whilst these have proven effective in controlling virus replication in the majority of treated individuals, viruses resistant to many of these inhibitors are becoming more common place (Pomerantz and Horn, 2003). To circumvent the development of resistant viruses, new targets are being examined. One such target is integrase, the enzyme responsible for inserting the HIV proviral DNA into host DNA. This enzyme functions in a two-step manner, it initially removes a dinucleotide unit from the 3 0 ends of the proviral DNA (termed ‘‘3 0 -processing’’), then the 3 0 -processed strands are transferred from the cytoplasm to the host nucleus, where they are introduced into the host DNA (termed ‘‘strand-transfer’’ or ‘‘integration’’) (Lin et al., 1999). Therefore, effecting the mechanism of action of this enzyme selectively would
3256
S.P.B. Ovenden et al. / Phytochemistry 65 (2004) 3255–3259
be a significant contribution towards discovering novel therapeutics for treatment of HIV/AIDS. During an HTS campaign conducted to find novel inhibitors of HIV integrase, in particular the 3 0 -processing reaction, a library of natural product extracts were screened, and the MeOH extract from the buds from of Eucalyptus globoidea were found to be active. Bioassay guided fractionation of this crude MeOH extract allowed for the purification of the new lignan, globoidnan A (1) as the sole compound responsible for the activity. Discussed below is the structure elucidation and biological activity of 1.
2. Results and discussion Globoidnan A (1) was isolated as a brown oil which gave a [M H] ion peak in the mass spectrum at m/z 491.0967, suggestive of the molecular formula C26H20O10 [D1.1 mmu] and equating to 17 double bond equivalents. Analysis of the NMR data (Table 1) for 1 immediately suggested a highly aromatised molecule, as the 13C NMR chemical shifts suggested that 22 of the 26 carbons were aromatic. The 1H–1H-COSY and gHMBC correlations (Table 1) identified resonances consistent with a 1,3-disubstituted naphthalene-6,7-diol moiety (13C: 149.4, 147.4, 138.1, 129.5, 128.3, 127.5, 123.5, 122.5, 111.5, 108.0 ppm; 1H: d 8.18, 7.47, 7.32,
Table 1 NMR data (500 MHz, d6-DMSO) for globoidnan A (1) No.
13
C (d, m)a
1 2 3 4 4a 5 6 7 8 8a 9 10 20 30 40 50 60 100 200 300 400 500 600 700
138.1 (s) 122.5 (d) 123.5 (s) 127.5 (d) 128.3 (s) 111.5 (d) 147.4 (s) 149.4 (s) 108.0 (d) 129.5 (s) 165.8 (s) 131.3 (s) 116.9 (d) 145.0 (s) 145.1 (s) 116.6 (d) 120.4 (d) 129.5 (s) 115.8 (d) 144.9 (s) 143.8 (s) 115.3 (d) 120.1 (d) 36.5 (t)
800 900
74.3 (d) 171.0 (s)
1
1
H–1H COSY
gHMBC 1H to
7.47 (br s)
H-4
C-4, C-8a, C-9, C-1 0
8.18 (br s)
H-2
C-2, C-5, C-8a, C-9
H (d, m, J (Hz))
13
C
7.32 (s)
C-4, C-6, C-7, C-8a
7.22 (s)
C-1, C-4a, C-6, C-7
6.81 (d, 1.8)
H-6 0
C-1, C-4 0 , C-6 0
6.87 (d, 8.5) 6.65 (dd, 8.5, 1.8)
H-6 0 H-5 0 , H-6 0
C-1 0 , C-3 0 C-1, C-2 0 , C-4 0
6.71 (br s)
H-600
H-300 , H-400 , H-600 , H-700
6.60 6.57 3.08 2.91 5.11
H-600 H-200 , H-500 Hb-700 , H-800 Ha-700 , H-800 H2-700
H-100 , H-300 H-200 , H-400 , H-700 C-100 , C-200 , C-600 C-100 , C-200 , C-600
(d, 8.5) (br d, 8.5) (dd, 14.5, 3.3) (dd, 14.5, 9.5) (dd, 9.5, 3.3)
7.22) as well as an alkylated 3,4-bis-catechol (13C: 144.9, 143.8, 129.5, 120.1, 115.3, 115.8, 74.3, 36.5 ppm; 1H: d 6.71, 6.60, 6.57, 5.11, 3.08, 2.91) and a mono-substituted 3,4-bis-catechol (13C: 145.1, 145.0, 131.3, 120.4, 116.9, 116.6 ppm; 1H: d 6.87, 6.81, 6.65). These data, along with the observation of two carbonyl 13C resonances (13C: 171.0, 165.8 ppm) consistent with the presence of ester and carboxylic acid moieties, accounted for all of the available double bond equivalents. Further analysis of the gHMBC data for 1 revealed several diagnostic correlations. The observation of gHMBC correlations from d 6.81 (H-2 0 ) and d 6.65 (H-6 0 ) into 138.1 ppm (C-1), as well as from d 7.47 (H-2) into 131.3 ppm (C-1 0 ) established that the monosubstituted 3,4-bis-catechol was attached at the C-1 position of the 1,3-disubstituted naphthalene-6,7-diol. Additional gHMBC correlations from d 7.47 (H-2) and d 8.18 (H-4) into 165.8 ppm (C-9) placed an ester carbonyl at C-3 of the 1,3-disubstituted naphthalene6,7-diol, and hence established the substitution pattern of the 1,3-disubstituted naphthalene-6,7-diol moiety as shown. The point of attachment for the 3-(3,4-dihydroxyphenyl) lactic acid moiety to the 1,3-disubstituted naphthalene-6,7-diol at C-3 was elucidated through the observed NMR chemical shift data for C-800 (13C: 74.3 ppm; 1H: d 5.11), which was consistent with an ester methine. Thus the carboxylic acid functionality had to be attached at the only remaining position, C-800 , leading to the proposed gross structure of 1 as shown below. Therefore 1 is a novel ester composed of (3,4-dihydroxyphenyl) lactic acid and 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy2-naphthalene carboxylic acid. An attempt was made to determine the absolute stereochemistry of 1 through hydrolysis to form the known compound (3,4-dihydroxyphenyl) lactic acid. However, only decomposition product was produced and as a consequence the absolute stereochemistry remains unassigned at this time. The structure of 1-(3,4-dihydroxyphenyl)-naphthalene-6,7-diol, and associated carboxylic acid analogues from other natural sources has been reported previously (Cullmann et al., 1999; Rischmann et al., 1989; Tazaki et al., 1995). However, this is the first report of an alkylated aryl side chain attached through an ester functionality as shown in 1. Interestingly, this is the first report of this type of cyclolignan from a higher plant, with all previous reports of this structure class referring to their occurrence in liverworts. A great number of HIV integrase inhibitors have previously been isolated from natural sources, including examples from plants (Kim et al., 1998), lichens (Neamati et al., 1997) and fungi (Singh et al., 1998, 2002a,b; Herath et al., 2004) resulting in several granted patents (Neamati, 2002). IC50 values of these compounds against HIV integrase range from 1 to 20 lM.
S.P.B. Ovenden et al. / Phytochemistry 65 (2004) 3255–3259
The activity of 1 compares favourably with previously reported natural product derived inhibitors of HIV integrase, with an IC50 = 0.64 lM in our combined 3 0 processing/strand transfer HIV integrase assay. In assays designed to measure the inhibitory effect of compounds on separate 3 0 processing (Chow, 1997) or strand transfer activities (Hwang et al., 2000), globoidnan A (1) was determined to inhibit HIV integrase with IC50Õs of 7 and 1.5 lM, respectively. Globoidnan A (1) was also assessed for its ability to inhibit the replication of HIV-1NL4-3 in the HuT78 T-cell line. No antiviral or toxic effects were observed after 6 days at concentrations up to 50 lM, the highest dose tested. Although no in vitro activity was observed for compound 1 in the whole cell anti-HIV assay, the enzyme HIV-integrase is considered a valid target for HTS efforts to identify novel small molecule inhibitors of viral replication. Enzyme based assays are usually more robust, time and cost efficient, and less prone to erratic screening results. In addition, they have the potential to yield inhibitors with known mechanisms of action, thus reducing downstream development cost for novel drug candidate molecules. Globoidnan A (1) shares two structural motifs that were previously noted for inhibitors of HIV integrase. Compounds containing a diketoacid motif, such as L()-chicoric acid, played a major role in validating HIV integrase as a target for therapeutic intervention (Lin et al., 1999; Long et al., 2004) and have been used as scaffolds for the preparation of HIV integrase inhibitors (Pais et al., 2002). Compounds with the diketoacid motif have been demonstrated to possess activity in whole cell assay systems (Lin et al., 1999), which was not observed for 1. In addition, the bis-catechol structural motif has been frequently associated with inhibition of HIV integrase (Dupont et al., 2001). The structure of 1 contains three biscatechol moieties. In most cases, inhibitors with this structural feature are active in cell-free assay systems, but lack selective activity in whole cell assay systems or display unacceptably high levels of toxicity (Lin et al., 1999). Interestingly, despite the presence of the three bis-catechol moieties in 1, no cytotoxicity was observed.
O HO
5
O
4 3 9 O
OH 9'' 7'' 1''
HO
2''
1 1'
8 6'
2''
OH OH 1
OH OH
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3. Experimental 3.1. General SPE was performed using Varian Megabond Elute C18 SPE cartridges (10 g, 50 lm). HPLC was performed on either a Waters Delta Prep 4000 chromatography system equipped with a Waters 2487 dual wavelength UV detector, a Waters prep LC system controller and a Waters Fraction Collector, or system equipped with a Waters 600 Controller, a Waters 996 Photodiode Array detector, Waters 717 plus autosampler and a Waters Fraction Collector II. All data generated from these chromatographic systems were collected using Waters Millenium32 data collection package. All NMR spectra were collected on a Varian Unity Inova 500 MHz spectrometer in the solvents indicated, with spectra referenced to residual 1H in the deuterated NMR solvents. Optical rotations were performed on a Jasco Dip1000 digital polarimeter, while infrared spectra were acquired on a Bio-Rad FTS-165 Fourier-transform infrared spectrometer. Low resolution mass spectral data were collected on a ThermoFinnigan LCQ ion trap mass spectrometer, with an ESI probe. High resolution mass measurements were collected on a Bruker BioApex FT mass spectrometer. 3.2. Plant material The buds of a specimen of Eucalyptus globoidea (Myrtaceae) were collected from the south side of Cape Conran, 13 km from Marlo, Victoria, Australia in April 1995 (3747 0 5600 S, 14840 0 4600 E). A voucher specimen (MEL2025404) has been lodged with the National Herbarium of Victoria. 3.3. Extraction and isolation The ground and dried buds of a specimen of Eucalyptus globoidea (7 g) were extracted twice with MeOH (500 ml), concentrated in vacuo and subjected to a C18 SPE column (20% stepwise gradient elution from 0% MeOH to 100% MeOH in H2O). Biological activity was located in the 40% and 60% MeOH/H2O fractions. These were combined and further purified on a C18 preparative HPLC [16 ml/min, gradient elution from 20% MeCN:H2O (+0.1% formic acid) to 100% MeCN (+0.1% formic acid) over 25 min through a Varian C18 250 · 50 mm 5 lm preparative HPLC axial compression column] and C8 semi-preparative HPLC [6 ml/min, gradient elution from 20% (MeCN:H2O + 0.1% formic acid) to MeCN (+0.1% formic acid) over 20 min through a Waters C8 Symmetry 250 · 25 mm 7 lm HPLC column] to yield the new lignan globoidnan A (1) (1.9 mg, 0.03% yield) as the compound responsible for the activity.
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3.3.1. Globoidnan A (1) Brown oil. [a]D + 14.4 (c 0.118, MeOH); IR (film) mmax 3200, 2956, 2924, 2852, 1720, 1701 cm1; UV (PDA, MeCN/H2O) kmax 224, 260, 320 nm; 1H (500 MHz, d6-DMSO) and 13C (125 MHz, d6-DMSO) NMR data see Table 1; HRESIMS [M H] m/z 491.0967 (calculated for C26H19O10 491.0978). 3.3.2. HIV-integrase assays Natural product extracts were reconstituted in 2 ll of DMSO and incubated in 18 ll of 30 nM substrate DNA and 300 nM HIV integrase in assay buffer (containing 20 mM Tris–HCl (pH 7.5), 25 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 5 mM DTT, 50 lg/ml nuclease-free BSA and 0.05% Tween 20) for 90 min at 37 C. Both positive and negative control wells have HIV integrase and DMSO as vehicle, whilst negative control wells do not contain substrate DNA. The substrate DNA for the strand transfer assay is as previously reported (Hwang et al., 2000) and for the combined 3 0 processing/strand transfer assay, a modified 5 0 -DIG labelled oligonucleotide is used where an additional 2 nucleotides, G and T, are added to the 3 0 end to complement the BIO labelled oligonucleotide sequence. For the combined 3 0 processing/strand transfer reaction, the reaction time was extended to 90 min to provide time for the 3 0 processing (removal) of the two additional nucleotides from the substrate DNA to occur prior to it under going strand transfer. After incubation, a further 30 ll of 33 mM Tris–HCl (pH 7.5, containing 664 mM NaCl, 16.6 mM EDTA and 0.166 mg/ml sonicated salmon sperm DNA) was added to each well and the 50 ll reaction volume transferred to 384-well plates coated with streptavidin. Following a 60 min incubation at room temperature, the streptavidin-coated plates were washed three times with 30 mM NaOH, 200 mM NaCl and 1 mM EDTA. Plates were washed twice with 10 mM Tris–HCl (pH 8.0, containing 1 mM EDTA and 0.1 mg/ml nucleasefree BSA) prior to the addition of 50 ll of 0.075 U/ml of anti-digoxigenin alkaline phosphatase, Fab fragments in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA and 0.1 mg/ ml nuclease-free BSA buffer and incubated for 60 min at 37 C. Plates were then washed four times with phosphate-buffered saline containing 0.1% Tween-20 and once with phosphate-buffered saline, and wells were then developed in 50 ll of p-nitrophenyl phosphate solution (1 mg/ml in 0.1 M Tris–HCl) and measured for absorbance at 405 nm. The 3 0 processing assay was conducted as described previously (Chow, 1997) using a 33P-end labelled HU5V1 substrate. Processed products were measured following their separation from substrate in a denaturing acrylamide gel (Chow, 1997). The whole cell antiviral assay was conducted by infecting 50,000 HuT78 T-cells in RPMI + 10% heat
inactivated fetal calf serum, in each well of a 96 well plate, with 65 TCID50 units of HIV-1NL4-3 in the presence of different concentrations of globoidnan A (1). After an overnight infection in a total volume of 100 ll, and equal volume of fresh media containing 1 was added. After an additional 48 h, 100 ll of media was removed and replaced with 100 ll fresh media containing 1. A sample of culture supernatant was diluted 1 in 2000 and p24 measured using a p24 ELISA assay (Vironostika HIV-1 Antigen kit, Organon Teknika). For cytotoxicity assays, cells were passaged in the presence of compound as described for the infection assay: however, no virus was added. The viability of cells was assessed using the Cytolux assay (Perkin–Elmer Life Sciences) following the manufacturerÕs instructions.
Acknowledgements We acknowledge P.C. Jobson for the collection of the plant material, Wendy Love for the preparation of the dried plant material for chemical isolation, Simon Harris, Effie Tsotsis and Dimuthu Goonasekara for their help in conducting biological screening. We acknowledge Dr. Marisa Spiniello and Associate Professor Mark Rizzacasa at the School of Chemistry, The University of Melbourne, for their help in the acquisition of optical rotation and IR spectra, and Sally Duck at the Department of Chemistry, Monash University, for the high resolution mass measurement.
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