Phytochemistry Letters 13 (2015) 200–205 Aryltetralols from Holostylis reniformis and syntheses of lignan analogous Marcos D.P. Pereiraa, Matheus R. Ferreiraa, Gisele B. Messianoa,1, Isabela Penna Cerávolob , Lucia M.X. Lopesa,*, Antoniana U. Krettlib,** a Institute of Chemistry, São Paulo State University, UNESP, C.P. 355, 14801-970, Araraquara, SP, Brazil bCentro de Pesquisas René Rachou/FIOCRUZ, Av. Augusto de Lima 1715, 30190-002, Belo Horizonte, MG, Brazil A R T I C L E I N F O Article history: Received 18 March 2015 Received in revised form 26 May 2015 Accepted 4 June 2015 Available online 25 June 2015 Keywords: Holostylis reniformis Aristolochiaceae Lignans Aryltetralol Arytetralene Antiplasmodial activity A B S T R A C T Two new lignans, an aryltetralol and its methyl ether analogous, were isolated from Holostylis reniformis (Aristolochiaceae) together with futokadsurin C and (�)-80-epi-aristoligone. The latter was also obtained as an enantiomeric mixture by synthesis and was transformed into aryltetralols and aryltetralenes that were subjected to chiral-HPLC separations. The compound structures were determined by spectroscopic methods. Several of these lignans had their antiplasmodial activity (against Plasmodium falciparum, W2 clone, anti-HRPII) and toxicity to mammalian kidney cells (MDL50) evaluated. (�)-Cyclogalgravin and (�)-aristoligol exhibited activity (IC50� 10.8 and 8.4 mM, respectively), the latter exhibited lower toxicity. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Phytochemistry Letters journal homepa ge: www.elsev ier .com/locate /phytol 1. Introduction Holostylis reniformis Duch. (Aristolochiaceae) synthesizes a variety of seco compounds, including lignans and sesquiterpenes (da Silva and Lopes, 2004, 2006; Lopes et al., 2012; Martins et al., 2014; Pereira et al., 2012). More than 25 aryltetralone and furan lignans without oxygenation at C-9,90 have been isolated from extracts of this species (da Silva and Lopes, 2004, 2006; Lopes et al., 2012), and isoeugenol is shown to be their biosynthetic intermediate (da Silva and Lopes, 2004, 2006; Messiano et al., 2008, 2009). The anti-Plasmodium falciparum activities and toxicities of lignans and extracts obtained from H. reniformis have been demonstrated (da Silva et al., 2004; da Silva and Lopes, 2004; de Andrade-Neto et al., 2007). As part of our continuing studies on the chemical constituents of H. reniformis, in this paper we report the results of the in vitro antiplasmodial and toxicity evaluation of two new natural lignans (1 and 2) and of aryltetralone, aryltetralol, * Corresponding author. Fax: +55 16 3301 9692. ** Corresponding author. Fax: +55 31 3295 3115. E-mail addresses: mpelicon@bol.com.br (M.D.P. Pereira), matheusrf_sud@hotmail.com (M.R. Ferreira), gbaraldi@ifsp.edu.br (G.B. Messiano) , ceravolo@cpqrr.fiocruz.br (I.P. Cerávolo), lopesxl@iq.unesp.br (L.M.X. Lopes), akrettli@cpqrr.fiocruz.br (A.U. Krettli). 1 Present address: Instituto Federal de São Paulo, R. Stéfano D’Avassi 625, 15991- 502 Matão, SP, Brazil. http://dx.doi.org/10.1016/j.phytol.2015.06.001 1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All righ and aryltetralene lignans obtained by regioselective syntheses. These lignans belong to 2,70-cyclolignan subclass of lignans or arylnaphthalene type of lignans. Lignans of this subclass, without oxygenation at C-9,90, have already been obtained by syntheses using commercially available compounds (Kuo and Lin, 1993; Takeya et al., 1983; Yvon et al., 2001), via transformations of 7,70-epoxylignans (furan lignans) (Blears and Haworth, 1958; Crossley and Djerassi, 1962; Purushothaman et al., 1984), and by biotransformations from fungi (Messiano et al., 2010). However, stereoselective syntheses of 2,70-cyclolignans normally involve more than 10 steps (Peng et al., 2013; Rye and Barker, 2011), and those regioselective carried out in fewer steps showed poor yields (Barba et al., 1990; Iguchi et al., 1978). Aiming to obtain lignan analogous to advances in pharmacological studies of these lignans, this report describes a sequence for the syntheses of aryltetralone, aryltetralol, and aryltetralene lignans in four, five, and six steps, successively by improving a route for obtaining aryltetralone lignans proposed by Müller and Vajda (1952). 2. Results and discussion The acetone extract of H. reniformis roots was subjected to column chromatography followed by HPLC to yield two minor new lignans (1 and 2, Fig. 1) together with the known compounds (�)-futokadsurin C (3) and (�)-80-epi-aristoligone (4a). They were analyzed by spectroscopic methods, and the known compounds ts reserved. http://crossmark.crossref.org/dialog/?doi=10.1016/j.phytol.2015.06.001&domain=pdf mailto:mpelicon@bol.com.br mailto:matheusrf_sud@hotmail.com mailto:matheusrf_sud@hotmail.com mailto:gbaraldi@ifsp.edu.br mailto:ceravolo@cpqrr.fiocruz.br mailto:lopesxl@iq.unesp.br mailto:akrettli@cpqrr.fiocruz.br mailto:akrettli@cpqrr.fiocruz.br http://dx.doi.org/10.1016/j.phytol.2015.06.001 http://dx.doi.org/10.1016/j.phytol.2015.06.001 http://www.sciencedirect.com/science/journal/18743900 www.elsevier.com/locate/phytol H3CO R 1 O OR OCH3 OR 2 R R1 R2 1 H H CH3 2 CH3 H CH3 5 H CH3 CH3 6 CH3 CH3 CH3 7 CH3 CH3 H O OCH3 OCH3 O O H3CO H3CO O OCH3 OCH3 3 4a 1 7 9 3 1' 3' 7' 9' Fig. 1. Chemical structures of compounds 1-3, 4a, and 5–7. M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205 201 were identified by comparison with data reported in the literature (da Silva and Lopes, 2004, 2006; Konishi et al., 2005; Kuo et al., 1989) (Fig. 1). The 1H and 13C NMR, IR, and UV spectroscopic data for compounds 1 and 2 were very similar to those reported for the aryltetralol derivatives, previously isolated from H. reniformis and obtained by microbial transformation of the aryltetralin lignans 5-7 (da Silva and Lopes, 2006; Messiano et al., 2008, 2010). Compounds 1 and 2 were also suggested to be aryltetralol lignans based on the HRMS spectra. They displayed quasi-molecular ions at m/z 381.1663 [M + Na]+ and at m/z 371.1445 [M � H]�, respectively, which were consistent with the molecular formula C21H26O5 for 1 and C22H28O5 for 2 (14 m higher than 1). The IR spectra of both compounds showed characteristic absorptions of hydroxyl groups at �3440 cm�1. The 1H and 13C NMR spectra of both compounds (Tables 1 and 2, Figs. S1–S4) suggested the presence of a veratryl group (C ring), and one methoxyl group linked to 1,2,4,5- tetrasubstituted aromatic ring (A). These spectra also showed signals for two methyl groups and four methine carbons, of which one is oxygenated. In addition, signals for hydroxyl groups at dH� 5.4 were observed. Compound 2 showed additional resonances reminiscent of an aliphatic methoxyl group (dC 56.0; dH 3.40). The differences between the chemical shifts of carbons and hydrogens on the B ring of 1 and 2, particularly of CH-7 Table 1 1H NMR spectroscopic data for compounds 1 and 2 (CDCl3, 11.7 T). Position 1 H dH (J in Hz)a gNOESY 3 6.28, d (1.0) 70 6 6.78, s 7, OCH3-5 7 4.38, d (4.0) 6, 8, 9 8 1.99, ddq (4.0, 3.5, 7.5) 7, 9, 80 , 90 9 0.83, d (7.5) 7, 8, 70, 90 20 6.55, d (2.0) 70, 80 , OCH3-30 50 6.70, d (8.0) OCH3-40 60 6.56, dd (8.0, 2.0) 50 , 70, 80 70 3.36, br d (9.5) 3, 9, 20 , 60 , 90 80 2.32, ddq (9.5, 3.5, 7.0) 8, 20 , 60 , 90 90 0.81, d (7.0) 8, 9, 80 OCH3-30 3.73, s 20 OCH3-40 3.78, s 50 OCH3-5 3.81, s 6 OCH3-7 OH-4 5.49, br s a Multiplicities were determined with the assistance of 1H–1H COSY spectra and sim (1: dC 74.1; dH 4.38; 2: dC 83.4; dH 3.92) and CH-8 (1: dC 39.4; dH 1.99; 2: dC 34.6; dH 2.19), suggested that these compounds differ by the presence of a methoxyl group at C-7 in 2 instead of a hydroxyl group in 1. Furthermore, gHMBC experiments supported correlations between C-7 and OCH3-7, H-6, and 3H-9. The magnitude of the coupling constants of the methine hydrogens in the B ring evidenced trans diaxial, cis axial–equatorial, and trans diequatorial positions for H-70,80 (J = 10.0 � 0.5 Hz), H-80,8 (J = 3.5 Hz), and H-7,8 (J = 3.7 � 0.3 Hz), respectively. Moreover, gNOESY experiments showed spatial interactions between H-7 and 3H-9, as well as between H-70 and H-3, H-20, H-60, 3H-9, and 3H-90 for both compounds. Furthermore, spatial interactions of H-6 (d 6.68) with H-7, OCH3-7 (d 3.40), and OCH3-5 (d 3.84) were observed in gNOESY experiments of 2. Both compounds showed very similar CD curves with a positive Cotton effect at l � 290 nm, consistent with a 70R configuration (da Silva et al., 2004). Thus, the absolute configuration of 1 and 2 was determined as (7R,70R,8S,80S), which was identical to that previously determined for 5–7 (da Silva and Lopes, 2006; Messiano et al., 2010). The synthesis of aryltetralone (4 and 11), aryltetralol (12), and aryltetralene (13) lignans was achieved from four to six steps, successively, starting from stable and easily available materials. Improving the scheme proposed by Müller and Vajda (1952), via Reformatsky, by using veratrylacetone and a-bromopropianate, 2 dH (J in Hz)a gNOESY 6.29, d (0.5) 70 6.68, s 7, OCH3-5, OCH3-7 3.92, d (3.5) 6, 8, 9, OCH3-7 2.19, ddq (3.5, 3.5, 7.0) 7, 9, 80 , OCH3-7 0.79, d (7.0) 7, 8, 70, 90 6.55, d (2.0) 70, 80 , OCH3-30 6.71, d (8.0) OCH3-40 6.63, dd (8.0, 2.0) 70 3.30, br d (10.5) 3, 9, 20 , 60 , 90 2.35, ddq, (10.5, 3.5, 7.0) 8, 20 , 90 0.81, d (7.0) 80 3.73, s 3.80, s 3.84, s 3.40, s 7, 8 5.37, br s ulation using ACD/C + 1H NMR predictors (ACD, 2010). Table 2 13C NMR spectroscopic data for compounds 1 and 2 (CDCl3, 11.7 T). Position 1 2 H dC, typea gHMBCb dC, typea gHMBCb 1 128.6, C H-3, 7, 70 126.0, C H-3 2 133.0, C H-6, 7, 70 134.0, C H-6 3 115.6, CH H-70 115.5, CH 4 145.3, C H-3 and/or H-6 145.3, C H-6 5 145.4, C H-3 and/or H-6, OCH3-5 144.9, C H-3, OCH3-5 6 111.4, CH H-7 112.2, CH H-7 7 74.1, CH H-9 83.4, CH H-6, 9, OCH3-7 8 39.4, CH H-9 34.6, CH H-70, 90 9 11.7, CH3 H-7 11.1, CH3 10 138.1, C H-50 , 70 138.4, C H-50 20 112.4, CH H-60 , 70 112.2, CH H-60 , H-70 0 30 148.9, C H-50 , OCH3-30 148.9, C H-50 , OCH3-30 40 147.5, C H-20 , 60 , OCH3-40 147.4, C H-20 , 60 , OCH3-40 50 111.0, CH 110.8, CH 60 121.7, CH H-20 , 50 , 70 121.8, CH H-20 70 48.7, CH H-3, 20 , 60 , 90 48.8, CH H-3, 20 , 90 80 35.1, CH H-7, 9, 70 35.3, CH 90 16.8, CH3 H-70 17.2, CH3 H-70 OCH3-30 55.9, CH3 56.0, CH3 OCH3-40 55.9, CH3 55.9, CH3 OCH3-5 55.9, CH3 55.9, CH3 OCH3-7 56.0, CH3 a Chemical shifts and multiplicities were determined with the assistance of DEPT and gHMQC experiments. b gHMBC correlations, optimized for 8 Hz, are from carbon stated to the indicated hydrogen(s). 202 M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205 two pairs of aryltetralone enantiomers (4 + 11) were obtained, after HPLC separation, in four steps with good yield (87.5%). The major bottleneck in 4 + 11 synthesis was the lactonization after Reformatsky reaction. By extracting the intermediate lactone (9) with CHCl3 instead of distilling it (Müller and Vajda, 1952), the yield of this step increased from 28% to 99.8%. After separation by HPLC, each pair of enantiomers (4 and 11) was evaluated in vitro for their antiplasmodial activity. The immunoenzymatic test with monoclonal antibodies to the parasite protein histidine- and alanine-rich (HRPII), as shown in SI, was used to assess the inhibition of growth of P. falciparum in the presence of these compounds (Krettli et al., 2009; Noedl et al., 2002). Enantiomers 11 and enantiomers 4 showed to be inactive (IC50� 82 mM, Table S1), suggesting that the stereochemistry is very important for the antiplasmodial activity of this type of lignans. Aiming to obtain pure enantiomers to advance in the biological tests and chemical transformations, to evaluate the importance of C-7 carbonyl group for the activity, samples of 4 were analyzed by chiral-HPLC by using several conditions in analytical scales (columns, solvents and flows). Unfortunately, no suitable condition to separate the enantiomers 4 in a semi- preparative scale was achieved. Thus, samples of 4 + 11 and the natural aryltetralone 4a [(�)-80-epi-aristoligone] were individually subjected to reduction with NaBH4 in methanol to give 12 and 12a, respectively. The structure of 12a was confirmed by comparison of its 1D and 2D NMR spectra and optical activity ([a]D = �27 (CHCl3, c 0.10)) with those of an authentic sample of (�)-aristoligol (12a) [a]D = �20 (CHCl3, c 0.10) (da Silva and Lopes, 2006). Interestingly, the stereochemistry of C-7 of 12a is different from that of the natural aryltetralol (1). Finally, subjecting 12 and 12a to dehydra- tion with p-toluenesulfonic acid, the products 13 and 13a were obtained in 83.3% and 85% yield, respectively (Fig. 2). A better chromatographic condition, with lower tR, was achieved for the separation of the enantiomers 13 than for the aryltetralones 4 using chiral columns (Fig. S5). Thus, 13 was subjected to semi-preparative chiral-HPLC to give (�)-cyclo- galgravin 13a (tR = 14.3 min, 50.9%) and (+)-cyclogalgravin 13b (tR = 13.4 min, 49.1%). The similarity of the 1H and 13C NMR data and optical activities of 13a ([a]D = �96 (CHCl3; c 0.21), [u]290� 10330) with those from the natural product ([a]D = �106 (CHCl3; c 1.07), [u]290� 1254) (da Silva and Lopes, 2006) confirmed the assigned relative and absolute configurations for 13a. The compound 12a and 13a exhibited the highest activity (IC50 8.4 and 10.8 mM, respectively), whereas 13b showed moderate activity (IC50 30 mM) (Table S1). The natural aryltetralols 1 and 5 also showed moderate activity (IC50 33.6 and 33.1 mM, respectively), while the C-7 OCH3 analogous 2 and 6 were inactive (Table S1). These results suggest that steric effects also have influence on the activity. Among the tested lignans, 12a and 13a exhibited good selectivity index (SI > 28). 3. Experimental 3.1. General experimental procedures One-dimensional (1H, 13C, DEPT, HOMODEC, TOCSY, and gNOESY) and two-dimensional (1H–1H gCOSY, gNOESY, gHMQC, gHSQC, and gHMBC) NMR experiments were performed on a Varian INOVA 500 spectrometer (11.7 T) at 500 MHz (1H), and 126 MHz (13C), using deuterated solvents (CDCl3 and DMSO-d6) (P 99.9% D) as an internal standards for 13C NMR chemical shifts and residual solvent as an internal standard for 1H NMR. d values are reported relative to TMS. Mass spectra (ESI-MS) were obtained on a LCQ Fleet—Thermo Scientific, and flow injection into the electro- spray source was used for LC-ESI-MS. High-resolution mass spectra (HRMS) were obtained on a Bruker Daltonics ultrOTOFq (ESI- TOFMS). IR spectra were obtained on a PerkinElmer 1600 FT-IR spectrometer using KBr discs. Optical rotations were measured on a PerkinElmer 341-LC polarimeter. Ultraviolet (UV) absorptions were measured on a PerkinElmer UV–vis Lambda 14P diode array spectrophotometer. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectrometer, using 0.2 mm cell. HPLC analyses were performed using a Shimadzu liquid chromatograph (SPD-10 Avp), equipped with UV–vis and 341-LC polarimeter detectors, and using a Jasco LC-NetII/ADC, equipped with photodiode array (MD- 2018 Plus) and CD (2095 Plus) detectors, and chromatograms were acquired at 270 nm and 254 nm. The columns RP-18 (C18, Varian), Chiralpak1 IC (DAICEL), Lux 5 m Cellulose-1 (Phenomenex1), and b-CD BR Chiralpak1 (YMC) having a particle size of 5 mm, were used for analytical analysis (250 � 4.6 mm) and for semi- preparative analysis (250 � 10 mm). All reactions were monitored by TLC using 0.25 mm E. Merk silica gel plates (60 PF254) with UV, I2 vapor, or 10% H2SO4—heat as developing agent. All reactions were carried out under N2 atmosphere with freshly distilled solvents under anhydrous conditions unless otherwise noted. Reagents were purchased from Sigma–Aldrich Co., and used without purification, except where noted. Solvents employed were HPLC grade from Mallinckrodt. All yields refer to chromatographically and spectroscopically (1H and 13C NMR, aD) homogenous material unless otherwise stated. Ultrapure water was obtained from Milli-Q Gradient A10 from Millipore. 3.2. Plant materials The plant materials were collected in Ituiutaba, MG, Brazil, in February 2010, and identified as Holostylis reniformis Duch. by Dr. Vinícius C. Souza and Dr. Lindolpho Cappellari Jr. A voucher specimen (ESA 110,744/2010) was deposited at the herbarium of the Escola Superior de Agricultura, Luiz de Queiroz (ESALQ), Piracicaba, SP, Brazil. Authorization CGEN/MMA number 10,586/ 2012-1. The material was separated according to the plant parts and dried (�45 �C). Fig. 2. Total syntheses of aryltetralenes 13a and 13b. M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205 203 204 M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205 3.3. Extraction and isolation of the chemical constituents The roots (3.7 kg) were ground and exhaustively extracted successively at room temperature with hexanes, acetone, and ethanol [4 � (�200 mL, 2 days and shaken manually every 12 h for 2 min) each solvent] (da Silva and Lopes, 2006; Messiano et al., 2009). The residues were extracted with ethanol in a Soxhlet apparatus and the extracts were individually concentrated. The crude acetone extract (6.6 g) was subjected to CC (40.0 � 5.0 cm, silica gel 60H, 203.5 g, n-hexanes/EtOAc gradient, 19:1 to 100% EtOAc) to give 31 fractions (ca. 120 mL each), as previously described (da Silva et al., 2004). After semi-preparative HPLC (C18, MeOH/H2O, 7:3, flow rate: 8 mL/min) fractions 16 and 19 gave 3 (8.2 mg) and 4a (1140.0 mg), respectively. Fraction 23 (280.0 mg) gave 1 (43.5 mg) and 2 (8.9 mg) after semi-preparative HPLC (C18, MeOH/H2O, 3:2, flow rate: 8 mL/min). 3.3.1. (�)-(7R,70R,8S,80S)-4,7-Dihydroxy-30,40,5-trimethoxy-2,70- cyclolignan [(�)-4-O-demethyl-7-hydroxyisogalbulin, 1] Yellow oil (CHCl3); ½a�25D �38 (c 0.7, CHCl3); UV lMeOH max see Fig. S6; IR (KBr) nmax 3434, 1590, 1513, 1463 cm�1; 1H and 13C NMR (CDCl3) see Tables 1 and 2; ESIMS 20 eV, positive mode, m/z (rel. int.): 359 [M + H]+ (100); HRESIMS (probe) 4.5 eV, positive mode, m/z (rel. int.): 381.1663 [M + Na]+ (100) (calcd for C21H26O5Na, 381.1672); CD (CHCl3, c 0.50): [u]307 �5626, [u]296 +22895, [u]279 �44311, [u]260 �14739, [u]241 �141569. 3.3.2. (7R,70R,8S,80S)-4-Hydroxy-30,40,5,7-tetramethoxy-2,70- cyclolignan [(�)-4-O-demethyl-7-methoxyisogalbulin, 2] Yellow oil (CHCl3); ½a�25D �26 (c 0.6, CHCl3); UV lMeOH max see Fig. S6; IR (KBr) nmax 3446, 1590, 1520, 1430 cm�1; 1H and 13C NMR (CDCl3) see Tables 1 and 2; HRESIMS (probe) 4.5 eV, negative mode, m/z (rel. int.): 371.1445 [M � H]� (100) (calcd for C22H27O5, 371.1853); CD (CHCl3, c 0.57): [u]297 +5494, [u]278 �18353, [u]245 �113950. 3.3.3. (7R,70R,8S,80S)-7-Hydroxy-30,40,4,5-tetramethoxy-2,70- cyclolignan [(–)-holostylol, 12a] Yellow oil (CHCl3); ½a�25D �28 (c 0.10, CHCl3) lit. (da Silva et al., 2006) ½a�25D �20 (c 1.6, CHCl3). 1H NMR (CDCl3, 500 MHz): d 6.16 (1H, s, H-3), 7.08 (1H, s, H-6), 4.93 (1H, d, J = 4.5 Hz, H-7), 2.16 (1H, ddq, J = 3.0, 4.5, 7.0 Hz, H-8), 0.86 (3H, d, J = 7.0 Hz, H-9), 6.48 (1H, d, J = 2.0 Hz, H-20), 6.72 (1H, d, J = 8.0 Hz, H-50), 6.58 (1H, dd, J = 8.0, 2.0 Hz, H-60), 3.48 (1H, d, J = 10.0 Hz, H-70), 2.01 (1H, ddq, J = 3.0, 10.0, 7.0 Hz, H-80), 0.84 (3H, d, J = 7.0 Hz, H-90), 3.54 (s, OCH3-4), 3.84 (s, OCH3-5), 3.74 (s, OCH3-30), 3.81 (s, OCH3-40). 3.3.4. (70R,80S)-30,40,4,5-Tetramethoxy-2,70-cyclolignan-7-eno [(�)-cyclogalgravin, 13a] Yellow oil (CHCl3); ½a�25D � 103 (c 0.10, CHCl3), lit. (da Silva et al., 2006) ½a�25D �106 (c 1.07, CHCl3). 1H NMR (CDCl3, 300 MHz): d 6.49 (1H, s, H-3), 6.56 (1H, s, H-6), 6.08 (1H, br s, H-7), 1.73 (3H, d, J = 1,2 Hz, H-9), 6.60 (1H, d, J = 2.1 Hz, H-20), 6.65 (1H, d, J = 8.4 Hz, H- 50), 6.49 (1H, dd, J = 8.4, 2.1 Hz, H-60), 3.62 (1H, d, J = 3.3 Hz, H-70), 2.33 (1H, dq, J = 7.2, 3.3 Hz, H-80),1.02 (3H, d, J = 7.2 Hz, H-90), 3.76 (s, OCH3-4), 3.82 (s, OCH3-5); 3.72 (s, OCH3-30), 3.72 (s, OCH3-40). 3.4. Syntheses and chemical transformations 3.4.1. Benzenebutanoic acid, b-hydroxy-3,4-dimethoxy- a,b-dimethyl-, ethyl ester (8): a-Bromopropionate (2.89 g, 17.4 mmol) was dropwise added to a stirred solution of veratrylacetone (3.08 g, 17.2 mmol) in 9 mL of dry benzene (under N2) and zinc (previously washed with acetone and activated at 100 �C for 8 h (Chavan, 2004)). The stirring solution was heated until the reflux began, this being maintained for 4 h. Then, the mixture was cooled and the ester (4.24 g, 16.89 mmol, 98.2%) extracted with CHCl3 (3 � 50 mL). 3.4.2. a,b-Dimethyl-g-(3,4-dimethoxyphenyl) butyrolactone (9) The ester (3.92 g, 15.6 mmol) was dissolved in CH3CO2H (13 mL) and then ice-cooling. To this cooling and stirring solution sulfuric acid (3 mL) was added by drops over 1 h. After 4 h on steam-bath, the organic portion was successively extracted with CHCl3 (3 � 50 mL), neutralized with NaHCO3, washed with water and dried (CaCl2), and concentrated at reduced pressure to give the lactone 9 (3.43 g, 15.6 mmol, 99.8%). 3.4.3. 4,4-Bis(3,4-dimethoxyphenyl)-2,3-dimethyl-butyric acid (10) A solution of AlCl3 (2.23 g, 16.8 mmol) in veratrol (1 mL, 7.97 mmol) was slowly dropwise to veratrol (1 mL, 7.97 mmol, 15 min) and the lactone 9 (2.2 g, 8.78 mmol). The resulting mixture was then stirred at 80 �C for 3 h, and then the complex was decomposed by dropwise addition of EtOH (5 mL) at room temperature, followed by addition of 10% HCl (10 mL). After disappearance of solid particles, the mixture was extracted with CHCl3 (3 � 50 mL), washed with 8% NaHCO3 (3 � 50 mL) and then with 10% HCl until a white solution was obtained. The removal of solvents under reduced pressure yielded the intermediary acid 10 (3.20 g, 8.24 mmol, 93.8 %). 3.4.4. 80-epi-Aristoligone and aristoligone (4 + 11) The acid 10 (3.02 g, 8.1 mmol) was dissolved in dry benzene (6 mL), then PCl5 (1.98 g, 9.5 mmol) was added to this solution, which was kept stirring in ice-bath for 30 min. Then, the stirring mixture was warmed up to 40 �C and occasionally shacked until the reactants dissolved. After that, the mixture was chilled and a solution of SnCl4 (2.84 g, 10.9 mmol) in dry benzene (4 mL) was added to it. In a few minutes, a red precipitate was observed, which was dissolved with concentrate HCl. A mixture comprising the enantiomer pairs 4 + 11 (2.85 g, 7.7 mmol, 95.1%) was obtained by extraction with CHCl3, followed by removal of the solvents. 3.4.5. Aryltetralol 12 To a stirring solution of 4 + 11 (2.00 g, 5.40 mmol) in MeOH (20 mL) a methanol solution of NaBH4 (204.2 mg, 5.40 mmol, 20 mL) was added. The mixture was stirred in an ice-bath for 4 h. Following reduction, the excess of NaBH4 was quenched by dropwise addition of MeOH and water. The organic phase resulting from EtOAc (3 � 30 mL) extraction was washed with water, dried (MgSO4), and concentrated to yield 12 (2.01 g, 5.40 mmol, 100.0%). 3.4.6. (�)-(70R,80S)- and (+)-(70S,80R)-30,40,4,5-Tetramethoxy-2,70- cyclolignan-7-ene [(�)-cyclogalgravin, 13a and (+)-cyclogalgravin, 13b] To a stirring solution of the alcohol 12 (1.93 g, 5.20 mmol) in dry benzene (15 mL) TsOH (0.72 g, 0.40 mmol) was slowly added. After stirring under reflux, this solution was cooled, extracted with CHCl3 and the solvent removed under reduced pressure to give 13 (1.53 g, 4.33 mmol, 83.3%). This product was subjected to chiral-HPLC analysis [cellulose tris-(3,5-dichlorophenylcarbamate column) to give (�)-13a (0.78 g, 2.20 mmol, ee > 99.9%), and (+)-13b (0.75 g, 2.13 mmol, ee > 99.9%). (�)-13a: yellow oil (CHCl3); ½a�25D �96 (c 0.21, MeOH); (+)-13b: Yellow oil (CHCl3); ½a�25D +106 (c 0.20, MeOH), lit. (Messiano et al., 2010)] ½a�25D �106 (c 1.07, CHCl3); CD (CHCl3, c 0.3): [u]296 +24783, [u]290 +10406, [u]274 +71184, [u]249 �15366, [u]240 +543, [u]226 +71706, [u]220–52760, [u]210 �153856, [u]201 +113946. M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205 205 3.4.7. HPLC separation of 4 + 11 Aliquots (30 mg) of 4 + 11 were subjected to semi-preparative HPLC (20 times) using RP-C18 ODS Chrompack column, UV (l 254 nm) detector, flow rate of 8 mL/min, and MeOH/H2O (7:3, v/v) as mobile phases. 3.4.8. Chiral-HPLC analysis of 4 (4a + 4b) and 13 (13a + 13b) Aryltetralone (4, 4 mg) and aryltetralene (13, 4 mg) lignans were subjected to HPLC (44 times each sample) using Chiralpak1 IC column, UV (l 270 nm) and polarimeter (l 365 nm) detectors, flow rate of 0.5 mL/min and 0.7 mL/min for 4 and 13, respectively, and n-hexanes/EtOAc (17:3, v/v for 4 and 87:13, v/v for 13) as mobile phases (Fig. S5). 4. Conclusions Two new lignans were isolated from H. reniformis. Aryltetr- alone, aryltetralol, and aryltetralene lignans were synthesized by a regioselective route in four, five, and six steps, successively, with good overall yields (81.0%, 80.9%, and 67.6%). (�)-Cyclogalgravin (13a) and (�)-aristoligol (12a) exhibited activity (IC50� 10.8 and 8.4 mM, respectively), the latter exhibited lower toxicity. Acknowledgments The authors thank Vinicius C. Souza and Lindolpho Capellari Jr. for plant identification, and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCT/MS/PRO- NEX, Brazil) for financial support and fellowships. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2015.06.001. References Advanced Chemistry Development, 2010. ACD/C + H NMR predictors and DB: ChemSketch Window. Version 12.01. ACD, Toronto 1CD-ROM. Barba, I., Chinchilla, R., Gomez, C., 1990. Electrochemical methoxylation of allyl and propenyl derivatives of phenol and phenol ethers. J. Org. Chem. 55, 3270–3272. Blears, J.G., Haworth, R.D., 1958. The constituents of natural phenolic resins. 23. A synthesis of galgravin. J. Chem. Soc. 1985–1987. Chavan, S.P., 2004. Reformatsky reaction of a-chloroesters with carbonyl compounds with commercially available zinc. J. Chem. Res. Synop. 6, 406–407. Crossley, N.S., Djerassi, C., 1962. Naturally occurring oxygen heterocyclics. 11. Veraguensin. J. Chem. Soc. 1459–1462. da Silva, T., Krettli, A.U., de Andrade-Neto, V.F., Lopes, L.M.X., 2004. Set of lignans and lignan extracts comprises constituents of malaria prevention compositions. BR200404986-A. Patent PI0404986-1, 2004. Lignanas, lignanas ariltetralônicas, extratos, processos de obtenção de lignanas, processo de obtenção de extratos, uso de lignanas, uso de extratos e composição farmacêutica para prevenir e tratar malária. Rev. Prop. Ind. 1774. da Silva, T., Lopes, L.M.X., 2004. Aryltetralone lignans and 7,8-seco-lignans from Holostylis reniformis. Phytochemistry 65, 751–759. da Silva, T., Lopes, L.M.X., 2006. Aryltetralol and aryltetralone lignans from Holostylis reniformis. Phytochemistry 67, 929–937. de Andrade-Neto, V.F., da Silva, T., Xavier Lopes, L.M., do Rosario, V.E., de Pilla Varotti, F., Krettli, A.U., 2007. Antiplasmodial activity of aryltetralone lignans fro Holostylis reniformis. Antimicrob. Agents Chemother. 51, 2346–2350. Iguchi, M., Nishiyama, A., Hara, M., Terada, Y., Yamamura, S., 1978. Anodic-oxidation of E-isoeugenol and Z-isoeugenol. Chem. Lett. 1015–1018. Krettli, A.U., Adebayo, J.O., Krettli, L.G., 2009. Testing of natural products and synthetic molecules aiming at new antimalarials. Curr. Drug Targets 10, 261–270. Konishi, T., Konoshima, T., Daikonya, A., Kitanaka, S., 2005. Neolignans from Piper futokadsura and their inhibition of nitric oxide production. Chem. Pharm. Bull. 53, 121–124. Kuo, Y.H., Lin, S.T., 1993. Studies on chromium trioxide-based oxidative coupling reagents and synthesis of lignan-cagayanone. Chem. Pharm. Bull. 41,1507–1512. Kuo, Y.H., Lin, S.T., Wu, R.E., 1989. 3 New lignans from the nutmeg of Myristica cagayanesis. Chem. Pharm. Bull. 37, 2310–2312. Lopes, L.M.X., Pereira, M.D.P., da Silva, T., Krettli, A.U., 2012. Sesquiterpenes from Holostylis reniformis. Pharm. Microbiol. 50, 643–644. Martins, G.F., Pereira, M.D.P., Lopes, L.M.X., da Silva, T., Vieira e Rosa, P. de T., Barbosa, F.P., Messiano, G.B., Krettli, A.U., 2014. Intraspecific variability of Holostylis reniformis: concentration of lignans, as determined by maceration and supercritical fluid extraction (SFE-CO2), as a function of plant provenance and plant parts. Quim. Nova 37, 281–287. Messiano, G.B., da Silva, T., Nascimento, I.R., Lopes, L.M.X., 2008. Biosynthesis of antimalarial aryltetralone lignans fro Holostylis reniformis. Planta Med. 74, 924–924. Messiano, G.B., da Silva, T., Nascimento, I.R., Lopes, L.M.X., 2009. Biosynthesis of antimalarial lignans from Holostylis reniformis. Phytochemistry 70, 590–596. Messiano, G.B., Wijeratne, E.M.K., Lopes, L.M.X., Gunatilaka, A.A.L., 2010. Microbial transformations of aryltetralone and aryltetralin lignans by Cunninghamella echinulata and Beauveria bassiana. J. Nat. Prod. 73, 1933–1937. Müller, A., Vajda, M., 1952. Dimeric propenyl phenol ethers. 15. The synthesis of 1-veratryl-2,3-dimethyl-6,7-dimethoxytetralin. J. Org. Chem. 17, 800–806. Noedl, H., Wernsdorfer, W.H., Miller, R.S., Wongsrichanalai, C., 2002. Histidine rich protein II, a novel approach to antimalarial drug susceptibility testing. Antimicrob. Agents Chemother. 46, 1658–1664. Peng, Y., Luo, Z.B., Zhang, J.J., Luo, L., Wang, Y.W., 2013. Collective synthesis of several 2,70-cyclolignans and their correlation by chemical transformations. Org. Biomol. Chem. 11, 7574–7586. Pereira, M.D.P., da Silva, T., Lopes, L.M.X., Krettli, A.U., Madureira, L.S., Zukerman- Schpector, J., 2012. 4,5-Seco-guaiane and a nine-membered sesquiterpene lactone from Holostylis reniformis. Molecules 17 (14), 046–14057. Purushothaman, K.K., Sarada, A., Connolly, J.D., 1984. Structure of malabaricanol—a lignan from the aril of Myristica malabarica Lam. Indian J. Chem. Sect. B 23, 46–48. Rye, C.E., Barker, D., 2011. Asymmetric synthesis of (+)-galbelgin, (�)-kadangustin J, (�)-cyclogalgravin and (�)-pycnanthulignenes A and B, three structurally distinct lignan classes, using a common chiral precursor. J. Org. Chem. 76, 6636–6648. Takeya, T., Matsumoto, H., Kotani, E., Tobinaga, S., 1983. New reagent systems containing CrO3 provide precursors for syntheses of neo-lignans. Chem. Pharm. Bull. 31, 4364–4367. Yvon, B.L., Datta, P.K., Le, T.N., Charlton, J.L., 2001. Synthesis of magnoshinin and cyclogalgravin: modified Stobbe condensation reaction. Synthesis 1556–1560. http://dx.doi.org/10.1016/j.phytol.2015.06.001 http://dx.doi.org/10.1016/j.phytol.2015.06.001 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0005 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0005 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0010 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0010 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0015 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0015 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0020 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0020 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0025 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0025 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0030 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0030 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0030 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0030 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0030 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0030 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0035 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0035 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0040 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0040 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0045 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0045 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0045 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0050 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0050 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0055 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0055 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0055 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0060 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0060 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0060 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0065 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0065 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0070 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0070 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0075 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0075 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0080 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0080 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0080 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0080 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0080 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0085 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0085 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0085 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0090 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0090 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0095 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0095 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0095 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0100 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0100 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0105 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0105 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0105 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0110 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0110 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0110 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0115 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0115 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0115 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0120 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0120 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0120 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0125 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0125 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0125 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0125 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0130 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0130 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0130 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0135 http://refhub.elsevier.com/S1874-3900(15)30007-0/sbref0135 Aryltetralols from Holostylis reniformis and syntheses of lignan analogous 1 Introduction 2 Results and discussion 3 Experimental 3.1 General experimental procedures 3.2 Plant materials 3.3 Extraction and isolation of the chemical constituents 3.3.1 (−)-(7R,7′R,8S,8′S)-4,7-Dihydroxy-3′,4′,5-trimethoxy-2,7′-cyclolignan [(−)-4-O-demethyl-7-hydroxyisogalbulin, 1] 3.3.2 (7R,7′R,8S,8′S)-4-Hydroxy-3′,4′,5,7-tetramethoxy-2,7′-cyclolignan [(−)-4-O-demethyl-7-methoxyisogalbulin, 2] 3.3.3 (7R,7′R,8S,8′S)-7-Hydroxy-3′,4′,4,5-tetramethoxy-2,7′-cyclolignan [(–)-holostylol, 12a] 3.3.4 (7′R,8′S)-3′,4′,4,5-Tetramethoxy-2,7′-cyclolignan-7-eno [(−)-cyclogalgravin, 13a] 3.4 Syntheses and chemical transformations 3.4.1 Benzenebutanoic acid, β-hydroxy-3,4-dimethoxy-α,β-dimethyl-, ethyl ester (8): 3.4.2 α,β-Dimethyl-γ-(3,4-dimethoxyphenyl) butyrolactone (9) 3.4.3 4,4-Bis(3,4-dimethoxyphenyl)-2,3-dimethyl-butyric acid (10) 3.4.4 8′-epi-Aristoligone and aristoligone (4+11) 3.4.5 Aryltetralol 12 3.4.6 (−)-(7′R,8′S)- and (+)-(7′S,8′R)-3′,4′,4,5-Tetramethoxy-2,7′-cyclolignan-7-ene [(−)-cyclogalgravin, 13a and (+)-cycl... 3.4.7 HPLC separation of 4+11 3.4.8 Chiral-HPLC analysis of 4 (4a+4b) and 13 (13a+13b) 4 Conclusions Acknowledgments Appendix A Supplementary data Appendix A Supplementary data