Chemical Composition of the Bark of Tetrapterys mucronata and

Article pubs.acs.org/jnp

Chemical Composition of the Bark of Tetrapterys mucronata and Identification of Acetylcholinesterase Inhibitory Constituents Marcos Marçal Ferreira Queiroz,† Emerson Ferreira Queiroz,*,‡ Maria Luiza Zeraik,† Samad Nejad Ebrahimi,§ Laurence Marcourt,‡ Muriel Cuendet,‡ Ian Castro-Gamboa,† Matthias Hamburger,§ Vanderlan da Silva Bolzani,*,† and Jean-Luc Wolfender‡ †

Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, NuBBE, Instituto de Química, Universidade Estadual Paulista (UNESP), Araraquara, São Paulo, Brazil ‡ School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30, Quai Ernest-Ansermet, CH-1211, Geneva 4, Switzerland § Division of Pharmaceutical Biology, University of Basel, Klingelbergrstrasse 50, CH-4056, Basel, Switzerland S Supporting Information *

ABSTRACT: The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called “Ayahuasca”, was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16−18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 μM.

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were isolated and fully characterized, providing important information for the chemotaxonomy of the previously poorly studied genus Tetrapterys.

he Malpighiaceae is an angiosperm family of trees, shrubs, and vines occurring in the tropical and subtropical forests and savannas of the New and Old Worlds and comprises approximately 1300 species distributed in 77 genera.1 The genus Tetrapterys has 90 species distributed in tropical Central America and South America.2 Certain Tetrapterys species, such as T. multiglandulosa and T. acutifolia, are known to be toxic.3 The vine Tetrapterys mucronata Cav. is of particular interest because it is used occasionally in the preparation of “Ayahuasca”, a psychotropic plant decoction that has a long cultural history in Brazil.4,5 Since this plant is known to act on the central nervous system, the activity of its constituents was investigated on the inhibition of acetylcholinesterase, a target enzyme for the treatment of Alzheimer’s disease.6,7 Natural products, such as galantamine or alkaloid-related synthetic compounds (e.g., rivastigmine), are used for the treatment of patients with mild to moderate Alzheimer’s disease.6 There is an urgent need for the discovery of more effective compounds with fewer undesirable side effects for this type of therapy.7 In the present study, the compounds responsible for the acetylcholinesterase activity inhibition of the crude ethanolic extract of T. mucronata were identified. In addition, 22 polar compounds (1−22) from the stem bark ethanolic extract, including six new natural products (9, 10, 16−18, and 20), © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

To eliminate tannins prior to biological evaluation and isolation, the ethanolic extract of the bark of T. mucronata was filtered over polyamide using ethanol and methanol as eluents.8,9 In a preliminary screening procedure, the tannin-free dried ethanolic extract of T. mucronata stem bark at 10 μg/spot showed significant in vitro acetylcholinesterase (AChE) inhibition using a bioautography assay.10 To localize the active compounds, the extract was microfractionated in a 96-well plate using reversed-phase HPLC.11,12 The fractions collected in the 96-well plates were dried and transposed to TLC for the AChE inhibition evaluation. This procedure enabled the chromatographic zone of the HPLC chromatograms to be defined in which the activity was concentrated (Figure 1). Special Issue: Special Issue in Honor of Otto Sticher Received: December 3, 2013 Published: February 12, 2014 650

dx.doi.org/10.1021/np401003p | J. Nat. Prod. 2014, 77, 650−656

Journal of Natural Products

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Chart 1

1,2,3,4-tetrahydro-β-carboline (7).16 Similarly, the main peak (3), which presented a characteristic PDA-UV spectrum of a flavanol ([M + H]+ at m/z 291.0864; molecular formula C15H15O6), was dereplicated as catechin, which is distributed widely in plants and described already as a constituent of other plants in the Malpighiaceae family.17 For the other compounds, the PDA-UV and MS data were insufficient for their effective dereplication. To ensure the effective isolation of most of the secondary metabolites detected in the HPLC-UV profiles, including those that displayed AChE inhibition activity, the isolation of the compounds was performed by directly transferring the analytical HPLC conditions to medium-pressure liquid

To obtain preliminary information on its chemical composition, the metabolite profiling of the ethanolic extract was established by UHPLC-TOFMS and HPLC-PDA-ESIMS. The positive ion high-resolution (HR)-ESIMS of the four peaks detected in the LC-UV trace (254 nm) (1, 5−7) displayed [M + H]+ ions at m/z 205.1347 [M + H]+ (C12H17N2O), m/z 205.1341 [M + H]+ (C12H17N2O), m/z 219.1498 [M + H]+ (C13H19N2O), and m/z 217.1349 [M + H]+ (C13H17N2O) and revealed the presence of alkaloids bearing two nitrogen atoms. These compounds were identified as the following four known alkaloids: 5-hydroxy-N,N-dimethyltryptamine, also called bufotenine (1),13 5-methoxy-N-methyltryptamine (5),14 5-methoxyN,N-dimethyltryptamine (6),15 and 2-methyl-6-methoxy651



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Figure 1. HPLC-PDA metabolite profiling (UV 254 nm) of the ethanolic extract after polyamide filtration of the extracts of T. mucronata. The chromatographic zones that were positive in the at-line AChE profiling are highlighted in gray.

group in C-3 and to the second indole group in C-2′. The presence of an N,N-dimethylethyl group was supported by the HMBC correlations between the two methyl groups at δH 2.21 and the methylene at δC 59.7 (C-11′) and by the weak COSY correlation between the latter and the H-10′ methylene. The N,N-dimethylethyl group was positioned at C-3′ based on the weak HMBC correlations between H-10′ and the carbons C-2′ and C-3′. The absolute configuration of 9 was established by comparing experimental and calculated ECD spectra. The ECD spectrum of 9 showed a positive Cotton effect (CE) around 231 nm and a negative CE around 210 nm. Conformational analysis gave five conformers (a 73.3%, b 15.5%, c 4.4%, d 4.0%, e 2.8%) within a 2 kcal/mol energy window from the global minimum in the gas phase (Figure S1, Supporting Information). The comparison of calculated ECD spectra for two possible stereoisomers is shown in Figure 2.

chromatography (MPLC-UV). This MPLC-UV separation combined with further semipreparative HPLC-UV resulted in the isolation of 22 compounds (1−22). The structural elucidation of the new compounds (9, 10, 16−18, and 20) was based on the interpretation of their 1D- and 2D-NMR spectra and by HRMS analysis. Several known compounds were identified as the following: 5-hydroxy-N,N-dimethyltryptamine (bufotenine) (1),13 gentisic acid (2),18 (+)-catechin (3),17 gentisic acid 5-O-β-xyloside (4),19 5-methoxy-N-methyltryptamine (5),14 5-methoxy-N,Ndimethyltryptamine (6),15 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline (7),16 vanillic acid (8),20 (Z)-3-methoxy-4,5(methylenedioxy)cinnamic acid (11),21 (E)-3-methoxy-4,5(methylenedioxy)cinnamic acid (12),22 salicylic acid (13),23 lyoniside (14),24 trans-N-feruloyltyramine (15),25 grossamide (19),26 cannabisin F (21),27 and smilaside L (22).28 In addition, two new dimeric indolic alkaloids (9 and 10), a new glycosylated lignan (16), and three new phenanthrene derivatives (17, 18, and 20) were purified, and their structural elucidation is described below. Compound 9 was isolated as an amorphous solid. The HRESIMS showed a molecular ion at m/z 394.2122 [M + H]+ (calcd for C23H27N3O3, 394.2131). Analysis of the 1H and HSQC spectra indicated the presence of two methyl groups (linked to a nitrogen atom), three methylene groups (two of which were linked to a heteroatom), two methine groups (one of which was oxygenated), eight olefinic groups, and two exchangeable protons. COSY and HMBC NMR correlations allowed the identification of two indole rings. The first ring was proposed as a 3-monosubstituted indole characterized by its four aromatic signals at δH 7.53 (1H, d, J = 8.0 Hz, H-4), 6.93 (1H, dd, J = 8.0, 7.5 Hz, H-5), 7.02 (1H, dd, J = 8.0, 7.5 Hz, H6), and 7.31 (1H, d, J = 8.0 Hz, H-7) and by the two doublets at δH 7.37 and 10.86 (J = 2.4 Hz) assigned to the H-2 and NH protons, respectively. The second indole ring was established as a 2,3,5-trisubstituted indole, as shown by the HMBC NMR correlations from H-4′ to C-5′ (δC 149.6) and C-8′ (δC 129.9), from H-6′ to C-4′ (δC 101.3) and C-8′ (δC 129.9), from H-7′ to C-5′ (δC 149.6) and C-9′ (δC 127.9), and from NH-1′ to C2′ (δC 136.9), C-3′ (δC 107.6), and C-9′ (127.9). The chemical shift of C-5′ (δC 149.6) indicated a substitution by a hydroxy group at this position. The COSY correlation between the methine H-11 (δH 4.19) and both the methylene H-12 (δH 3.28) and the methine H-10 (δH 4.55) suggested the presence of a 1,2-dioxygenated propyl chain. The HMBC correlations from H-10 to C-2′, C-3′, C-2, C-3, and C-9 indicated that the methine H-10 of this propyl chain is linked to the first indole

Figure 2. Comparison of experimental and calculated ECD spectra of 9 and two possible stereoisomers. The calculation was achieved with TDDFT at the B3LYP/6-31** level in the MeOH.

The calculated ECD for the 10R,11S stereoisomer showed an excellent fit with the experimental data, with a positive CE around 228 nm and a negative CE around 210 nm. The other stereoisomer, 10S,11S, displayed a mirror image. The positive CE around 228 nm in the experimental spectrum was likely due to a π → π* transition in the extended π-system of the indole moiety. On the basis of these data, the absolute configuration of C-10 was established unambiguously as 10R. For the second 652



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which is a known phenanthrene derivative,31 but now has been isolated for the first time from a natural source. The NMR data of 18 exhibited many similarities to those of 17. The two ortho aromatic protons, H-8 and H-9, were replaced by two methylene protons at δH 2.64 (δC 28.1) and δH 2.66 (δC 29.6), respectively. The HMBC correlations between the aromatic proton H-1 at δH 6.64 (d, J = 2.6 Hz) and the methylene C-10 at δC 29.7, and between H-8 at δH 6.99 (d, J = 8.0 Hz) and the other methylene C-9 at δC 28.1 indicated that 18 corresponds to the 9,10-dihydro derivative of 17. The molecular formula of C14H10O2, which was determined based on the HRESIMS at m/z 211.0755 [M − H]− (calcd C14H12O2, 211.0759), supported this inference. On the basis of the results obtained, compound 18 was identified as 2,6-dihydroxy-9,10dihydrophenanthrene, a new phenanthrene derivative. The HRESIMS of 20 displayed an [M − H]− ion at m/z 223.0745, in agreement with the molecular formula, C15H10O2 (calcd 223.0759). Compared with that of 17, the HRESIMS indicated that 20 contains an extra methylene group. The 1H NMR spectrum showed that an additional methyl group was observed at δH 2.30, and two noncoupled protons at δH 7.58 and 7.85 replaced the AMX system of 17. The position of this methyl at C-7 was assigned by long-range HMBC correlations of the protons with C-6 (δC 155.2), C-7 (δC 124.6), and C-8 (δC 129.8). This assignment was confirmed by the dipolar correlation between the methyl group and the H-8 proton. On the basis of these results, 20 was identified as 7-methyl-2,6phenanthrenediol, a new phenanthrene derivative. From the AChE HPLC profiling of the crude plant extract (Figure 1), the compounds present in these active zones were isolated and their biological activities evaluated. Compounds 1, 5, 6, 9, and 10 inhibited AChE activity with IC50 values below 15 μM, whereas the other isolated compounds showed IC50 values above 20 μM (Table 1). These results indicate that the

stereogenic center, only the relative configuration could be determined, due to free rotation around C-10/C-11 and high conformational flexibility. The 3JH−H coupling constant of 6.4 Hz between H-10 and H-11 is also in a good agreement with the free rotation around C-10 and C-11. On the basis of these results, 9 (mucronatin A) was identified as a new dimeric indole alkaloid. Compound 10 was isolated as an amorphous solid. The 1H NMR spectrum of 10 demonstrated a close resemblance to that of 9. The only difference observed was the presence of a NH2 group instead of the N,N-dimethyl group, which indicated a 5hydroxytryptamine moiety. The HRESIMS showed a molecular ion at m/z 364.1667 [M − H]− (calcd for C21H23N3O3, 364.1661), which is in agreement with this hypothesis. Due to the restricted amount of compound 10 available, its CD spectrum could not be measured to determine its relative configuration. On the basis of the results obtained, 10 was identified as a new dimeric indole alkaloid named mucronatin B, with the structure shown. The HRESIMS of 16 displayed a [M − H]− ion at m/z 687.2276, in agreement with a molecular formula of C34H40O15 (calcd for 687.2289). The 1H NMR spectrum of 16 showed close similarities to analogous data for lyoniside, a lignan glycoside (14) isolated from the same plant extract.24 The primary differences were the presence in 16 of three additional aromatic protons at δH 7.34 (1H, d, J = 2.1 Hz, H-2‴), 6.78 (1H, d, J = 8.2 Hz, H-5‴), and 7.21 (1H, dd, J = 8.2, 2.1 Hz, H6‴) and the downfield shift of the H-9′ methylene proton. The HMBC correlations from H-2‴ to C-6‴ (δC 121.5), C-4‴ (δC 150.5), and C-7‴ (δC 165.5), from H-6‴ to C-2‴ (δC 116.0), C-4‴, and C-7‴, from H-5‴ to C-1‴ (δC 120.3) and C-3‴ (δC 144.9), and from H-9′ to C-7‴ indicated that a 2,3dihydroxybenzoic acid group is linked to C-9′. All of the other data (coupling constants and NOE correlations) observed for compound 16 were similar to those of lyoniside (14), except for the optical rotation, which was negative [α]25D −4.1 (c 0.1, MeOH) for 16 and positive [α]25D +3.1 (c 0.1, MeOH) for 14. These data were in good agreement with those reported for lyoniside ([α]25D +27.3) and nudiposide ([α]25D −63.9) by G. Dada et al.,29 suggesting that 16 should have the same stereochemistry as nudiposide.29 On the basis of these results, 16 was identified as a new analogue of the lignan nudiposide, named nudiposide-9′-dihydroxybenzoic acid. The 1H NMR data of 17 exhibited eight aromatic protons distributed in three different spin systems attributed to three aromatic rings. The first system consisted of an ABX system at δH 7.15 (1H, d, J = 2.5 Hz, H-1), 7.13 (1H, dd, J = 8.8 and 2.5 Hz, H-3), and 8.38 (1H, d, J = 8.8 Hz, H-4). The second system was an AMX system at δH 7.85 (1H, d, J = 2.3 Hz, H-5), 7.04 (1H, dd, J = 8.6 and 2.3 Hz, H-7), and 7.71 (1H, d, J = 8.6 Hz, H-8). The third system was an isolated ortho coupling system at δH 7.59 (1H, d, J = 8.8 Hz, H-9) and 7.40 (1H, d, J = 8.8 Hz, H-10). The HMBC spectrum showed correlations from H-9 to C-8 (δC 130.3), C-13 (δC 132.5), and C-11 (δC 134.3) and from H-10 to C-1 (δC 111.7), C-12 (δC 122.6), and C-14 (δC 124.7). The data demonstrated that these aromatic rings are arranged as a phenanthrene unit.30 The chemical shifts of carbons C-2 and C-6 (δC 156.8 and 157.2, respectively) indicated the phenanthrene to be hydroxylated at these two positions. These observations were in agreement with the molecular formula, C14H12O2, as suggested by the HRESIMS [M − H]− ion at m/z 209.0596. On the basis of these observations, 17 was identified as a 2,6-phenanthrenediol,

Table 1. AChE Inhibition of the Active Compounds from T. mucronata Bark IC50 (μM)a

compound 1 5 6 9 10 galanthamineb tacrineb

11.4 12.5 14.0 11.7 12.7 2.4 0.09

± ± ± ± ± ± ±

0.2 0.3 0.2 0.4 0.3 0.2 0.02

a The values shown are the means ± standard deviations obtained from three independent experiments. bPositive control.

presence of a tryptamine alkaloid nucleus is important for the activity shown.32 The AChE inhibition of bufotenine (1) was previously described,33 whereas such activity of 5, 6, 9, and 10 is reported herein for the first time.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured in methanolic solutions on a JASCO polarimeter in a 1 cm tube. The ECD spectra were recorded in MeOH with a Chirascan CD spectrometer. The UV spectra were measured on a HACH UV−vis DR/4000 instrument. The NMR spectroscopic data were recorded on a 500 MHz Varian Inova spectrometer. Chemical shifts are reported in parts per million (δ) using the residual CD3OD signal (δH 3.31; δC 49.0) or the DMSO-d6 signal (δH 2.50; δC 39.5) as internal standards for 1H and 13C NMR, respectively, and coupling constants (J) are 653



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reported in Hz. Complete assignments were performed based on 2DNMR experiments (COSY, TOCSY, NOESY, HSQC, and HMBC). HRESIMS were performed on a Waters Acquity UPLC system coupled to a Waters Micromass LCT Premier time-of-flight mass spectrometer (Milford, MA, USA), equipped with an electrospray interface (ESI). HPLC-PDA-ESIMS analyses were conducted on an HP 1100 system equipped with a photodiode array detector (Agilent Technologies, Santa Clara, CA, USA) connected to a Finnigan MAT LCQ ion-trap mass spectrometer (Finnigan, San Jose, CA, USA), equipped with a Finnigan electrospray interface. The HPLC conditions were as follows: X-Bridge C18 column (5 μm, 250 × 4.6 mm i.d.; Waters); solvent system CH3CN−H2O, both containing 0.1% formic acid; gradient mode 5% to 100% CH3CN in 60 min, 100% CH3CN for 5 min; flow rate 1 mL/min; injection volume 20 μL; sample concentration 10 mg/mL in CH3CN. The UV absorbance was measured at 254 nm, and the UV-PDA spectra were recorded between 190 and 600 nm (step 2 nm). The ESIMS conditions were as follows: capillary voltage 30 V; capillary temperature 200 °C; source voltage 4.5 kV; source current 80 μA; nitrogen was used as the sheath gas; positive and negative ion modes. The spectra (180−1200 Da) were recorded every 3 s. UHPLC-TOF-HRMS metabolite profiling of the extracts was performed on a Micromass-LCT Premier time-of-flight (TOF) mass spectrometer (Waters) equipped with an electrospray interface and coupled to an Acquity UPLC system (Waters). The ESI conditions were as follows: capillary voltage 2800 V, cone voltage 40 V, MCP detector voltage 2400 V, source temperature 120 °C, desolvation temperature 300 °C, cone gas flow 20 L/h, desolvation gas flow 600 L/h. Detection was performed in the positive ion mode with a m/z range of 100−1000 Da and a scan time of 0.5 s in the W-mode. The MS was calibrated using sodium formate. Leucine enkephalin (SigmaAldrich, Steinheim, Germany) was used as an internal reference at 2 μg/mL and infused through a Lock Spray probe at a flow rate of 10 μL/min aided by a second LC pump. The separation was performed on an Acquity BEH C18 UPLC column (50 mm × 1 mm i.d.; 1.7 μm, Waters), using a linear gradient (solvent system: A = 0.1% formic acid−water, B = 0.1% formic acid−acetonitrile; gradient 5−95% B in 4 min; flow rate 0.3 mL/min). The temperature was set to 30 °C. The injection volume was constant (1 μL). At-line HPLC AChE profiling was carried out by microfractionation performed with a semipreparative X-Bridge C18 column (5 μm, 250 × 10 mm i.d.; Waters). The solvent system used a mixture of MeOH (A) and H2O (B), and the following gradient was applied: 5% to 100% A in 84 min followed by 100% A for 10 min. The flow rate was 2 mL/ min, and the injection volume was 200 μL (10 mg of extract was injected into the column). The UV absorbance was measured at 254 nm. The different peaks from the semipreparative HPLC-UV microfractionation were collected with a Gilson collector (FC204). The fractions were collected every minute in a 96-well plate (2 mL per well). After collection, the plate was evaporated to dryness using N2. The contents of the plate were suspended in 40 μL of MeOH and used for the evaluation of the acetylcholinesterase inhibition by bioautography.10 A 10 μL sample from each well was deposited on the TLC plate, and the assay was performed without migration of the compounds. Plant Material. The stem bark from Tetrapterys mucronata was collected in May 2012 in São Paulo, São Paulo State, Brazil. The botanical material was identified by Rafael Felipe Almeida. Voucher specimen number SP146620, representing this collection, was deposited in the “Herbarium Maria Eneyda P. K. Fidalgo”, Instituto Botânico de São Paulo, São Paulo, Brazil. Extraction and Isolation. The dried stem bark powder of T. mucronata (500 g) was extracted exhaustively with EtOH (3 × 1 L for one week) by mechanical agitation. The extent of the extraction was monitored by TLC using the Godin and Dragendorff reagents. The extract was filtered and concentrated to dryness by rotatory evaporation to produce 48 g of a dried EtOH extract (yield 9.6% w/w). UHPLC-TOFMS and HPLC-PDA-ESIMS were used for the metabolite profiling of the EtOH extract.

The HPLC metabolite profiling procedure revealed the presence of tannins, which can alter the baseline of the HPLC trace (Figure 1) and interfere with biological assays. To remove the tannins from the T. mucronata ethanolic extract, solid-phase extraction using polyamide was conducted.9 The extraction was optimized at the analytical scale using various solvent systems (EtOH, MeOH, and EtOAc) with a ratio of 0.5 g of polyamide per 65 mg of dry extract. The optimal solvent system consisted of EtOH (6 mL) followed by MeOH (2 mL). The efficiency of the tannin removal was controlled using the HPLC-PDA analysis of the ethanolic extract before and after polyamide treatment. The HPLC analysis of the ethanolic tannin-free extract demonstrated a normal baseline profile, suggesting satisfactory tannin removal without significant loss of the remaining constituents (Figure 1). This procedure was scaled up using 153 g of polyamide per 20 g of extract with EtOH (1.5 L) and then MeOH (0.4 L) for elution. The combined eluents were evaporated to dryness, yielding 8.5 g of extract that was tannin free (42.5% yield). The tannin-free ethanolic extract (8 g) was fractionated by mediumpressure liquid chromatography using Zeoprep C18 as the stationary phase (15−25 μm, 460 × 49 mm i.d., Zeochem) and a linear acidic (0.1% formic acid) MeOH−H2O gradient (5% to 100% MeOH over 48 h). These conditions were optimized on an HPLC column packed with the same stationary phase. The extract, prepared by mixing 8 g of the extract with 30 g of the Zeoprep C18 stationary phase, was introduced into the MPLC column by dry injection. The mixture was conditioned in a dry-load cell (11.5 × 2.7 cm i.d.). The dry-load cell was connected subsequently between the pumps and the MPLC column. The flow rate was set to 4 mL/min, and UV detection was performed at 254 nm. The MPLC separation yielded 270 fractions, which were analyzed by TLC and HPLC-UV. Nine compounds were isolated in one step with the following yields: fraction 8 yielded 1 (80.0 mg), fraction 12 yielded 4 (9.0 mg), fraction 18 yielded 5 (25.8 mg), fraction 23 yielded 6 (66.5 mg), fraction 43 yielded 7 (3 mg), fraction 131 yielded 14 (23.7 mg), fraction 204 yielded 15 (28.0 mg), fraction 252 yielded 19 (21.2 mg), and fraction 264 yielded 20 (8.0 mg). The fractions having similar profiles as determined by HPLC-DAD analysis were combined to give 22 fractions (A−V). Fractions H, K, Q, and R were selected for further purification. The final fractionation steps were performed by semipreparative HPLC-UV using a μBondapak, C18 prepacked column (100 × 25 mm i.d.) (Waters) with MeOH−H2O− 0.1% formic acid as the solvent system for the isocratic elution. The flow rate was 10 mL/min, and the UV absorbance was detected at 254 nm. Fraction E (180 mg) was purified with 11% of MeOH to yield 2 and 3 (identified as a mixture, 35.7 mg); fraction H (116.2 mg) was purified with 14% MeOH to yield 8 (12.0 mg), 9 (2.0 mg), and 10 (3.0 mg); fraction K (142.0 mg) was purified with 18% MeOH to yield 11 (11.1 mg), 12 (20.5 mg), and 13 (13.8 mg); fraction Q (55.0 mg) was purified with 23% MeOH to yield 16 (2.6 mg) and 17 (10.2 mg); fraction R (66.3 mg) was purified with 25% MeOH to yield 18 (6.5 mg); and fraction U (57.3 mg) was purified with 33% MeOH to yield 21 (15.0 mg) and 22 (12.7 mg). Mucronatin A (9): amorphous powder; [α]25D −14.5 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 203 (3.79); 220 (3.74); 281 (3.23) nm; ECD (MeOH, c 0.9 mM, 0.1 cm) [θ]210 −1171, [θ]231 +8091; 1H NMR (DMSO-d6, 500 MHz) δ 2.21 (6H, s, 2 × CH3), 2.26 (1H, m, H-11′b), 2.38 (1H, m, H-11′a), 2.78 (2H, m, H-10′), 3.28 (2H, m, H12), 4.19 (1H, dt, J = 6.4, 5.7 Hz, H-11), 4.55 (1H, d, J = 6.4 Hz, H10), 6.46 (1H, dd, J = 8.5, 2.2 Hz, H-6′), 6.68 (1H, d, J = 2.2 Hz, H4′), 6.93 (1H, dd, J = 8.0, 7.5 Hz, H-5), 7.02 (1H, dd, J = 8.0, 7.5 Hz, H-6), 7.08 (1H, d, J = 8.5 Hz, H-7′), 7.31 (1H, d, J = 8.0 Hz, H-7), 7.37 (1H, d, J = 2.4 Hz, H-2), 7.53 (1H, d, J = 8.0 Hz, H-4), 10.04 (1H, s, NH-1′), 10.86 (1H, d, J = 2.4 Hz, H-1); 13C NMR (DMSO-d6, 126 MHz) δ 21.7 (C-10′), 35.0 (C-10), 44.8 (2 × CH3), 59.7 (C-11′), 64.1 (C-12), 73.5 (C-11), 101.3 (C-4′), 107.6 (C-3′), 109.4 (C-6′), 110.7 (C-7′), 110.9 (C-7), 114.8 (C-3), 117.8 (C-5), 117.9 (C-4), 120.4 (C-6), 122.8 (C-2), 126.4 (C-9), 127.9 (C-9′), 129.9 (C-8′), 136.9 (C-2′), 135.4 (C-8), 149.6 (C-5′); HRESIMS m/z 394.2122 [M + H]+ (calcd for C23H27N3O3, 394.2131). Mucronatin B (10): amorphous powder; [α]25D −13.4 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 201 (3.76), 218 (3.72), 278 (3.20) 654



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nm; 1H NMR (DMSO-d6, 500 MHz) δ 2.88 (2H, brt, J = 7.0 Hz, H11′), 2.96 (2H, brt, J = 7.0 Hz, H-10′), 3.32 (2H, m, H-12), 4.27 (1H, dt, J = 7.0, 6.1 Hz, H-11), 4.56 (1H, d, J = 7.0 Hz, H-10), 6.51 (1H, dd, J = 8.5, 2.1 Hz, H-6′), 6.74 (1H, d, J = 2.1 Hz, H-4′), 6.95 (1H, dd, J = 8.0, 7.5 Hz, H-5), 7.03 (1H, dd, J = 8.0, 7.5 Hz, H-6), 7.11 (1H, d, J = 8.5 Hz, H-7′), 7.31 (1H, d, J = 8.0 Hz, H-7), 7.42 (1H, d, J = 2.3 Hz, H-2), 7.60 (1H, d, J = 8.0 Hz, H-4), 10.27 (1H, s, NH-1′), 10.91 (1H, d, J = 2.3 Hz, NH-1); 13C NMR (DMSO-d6, 126 MHz) δ 22.6 (C10′), 35.0 (C-10), 64.0 (C-12), 73.4 (C-11), 101.3 (C-4′), 105.4 (C3′), 109.9 (C-6′), 110.9 (C-7′), 111.0 (C-7), 114.4 (C-3), 117.9 (C-4 and C-5), 120.5 (C-6), 122.6 (C-2), 126.5 (C-9), 127.8 (C-9′), 129.9 (C-8′), 135.4 (C-8), 138.1 (C-2′), 150.1 (C-5′); HRESIMS m/z 364.1667 [M − H]− (calcd for C21H23N3O3, 364.1661). Nudiposide-9′-dihydroxybenzoic acid (16): amorphous powder; [α]25D −4.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.43), 264 (2.57), 300 (sh) nm; 1H NMR (DMSO-d6, 500 MHz) δ 1.93 (1H, m, H-8′), 2.05 (1H, dq, J = 10.2, 6.2 Hz, H-8), 2.64 (2H, dd, J = 15.3, 11.3 Hz, H-7′b), 2.71 (2H, dd, J = 15.3, 4.8 Hz, H-7′a), 3.01 (1H, dd, J = 11.8, 10.7 Hz, H-5″b), 3.02 (1H, dd, J = 8.8, 7.3 Hz, H-2″), 3.11 (1H, t, J = 8.8 Hz, H-3″), 3.24 (3H, s, OCH3-5′), 3.28 (1H, ddd, J = 10.7, 8.8, 5.7 Hz, H-4″), 3.32 (1H, dd, J = 10.4, 6.1 Hz, H-9b), 3.64 (6H, s, OCH3-3, OCH3-5), 3.67 (1H, dd, J = 11.8, 5.7 Hz, H-5″a), 3.70 (1H, dd, J = 10.4, 6.1 Hz, H-9a), 3.77 (3H, s, OCH3-3′), 4.13 (1H, dd, J = 11.1, 6.1 Hz, H-9′b), 4.13 (1H, d, J = 7.3 Hz, H-1″), 4.25 (1H, dd, J = 11.1, 4.1 Hz, H-9′a), 4.29 (1H, d, J = 6.3 Hz, H-7), 6.33 (2H, s, H-2 and H-6), 6.58 (1H, s, H-2′), 6.78 (1H, d, J = 8.3 Hz, H5‴), 7.21 (1H, dd, J = 8.3 and 2.1 Hz, H-6‴), 7.34 (1H, d, J = 2.1 Hz, H-2‴); 13C NMR (DMSO-d6, 126 MHz) δ 32.0 (C-7′), 35.3 (C-8′), 40.7 (C-7), 44.7 (C-8), 55.4 (OCH3-3′), 55.8 (OCH3-3 and OCH3-5), 58.3 (OCH3-5′), 65.5 (C-5″), 66.2 (C-9′), 68.5 (C-9), 69.2 (C-4″), 73.0 (C-2″), 76.5 (C-3″), 103.7 (C-1″), 106.5 (C-2′), 105.7, (C-2 and C-6), 115.0 (C-5‴), 116.0 (C-2‴), 120.3 (C-1‴), 121.5 (C-6‴), 124.2 (C-6′), 127.0 (C-1′), 133.3 (C-4), 136.8 (C-1), 137.2 (C-4′), 144.9 (C-3‴), 146.2 (C-5′), 146.9 (C-3′), 147.5 C-3 and C-5), 150.5 (C4‴), 165.5 (C-7‴); HRESIMS m/z 687.2276 [M − H]− (calcd for C34H40O15, 687.2289). 2,6-Phenanthrenediol (17): amorphous powder; UV (MeOH) λmax (log ε) 204 (2.95), 253 (2.54), 272 (2.30) nm; 1H NMR (DMSO-d6, 500 MHz) δ 7.04 (1H, dd, J = 8.6, 2.3 Hz, H-7), 7.13 (1H, dd, J = 8.8, 2.5 Hz, H-3), 7.15 (1H, d, J = 2.5 Hz, H-1), 7.40 (1H, d, J = 8.8 Hz, H10), 7.59 (1H, d, J = 8.8 Hz, H-9), 7.71 (1H, d, J = 8.6 Hz, H-8), 7.85 (1H, d, J = 2.3 Hz, H-5), 8.38 (1H, d, J = 8.8 Hz, H-4); 13C NMR (DMSO-d6, 126 MHz) δ 106.1 (C-5), 111.7 (C-1), 116.5 (C-7), 117.3 (C-3), 122.6 (C-12), 123.1 (C-10), 124.7 (C-14), 124.8 (C-4), 127.3 (C-9), 130.3 (C-8), 132.5 (C-13), 134.3 (C-11), 156.8 (C-2), 157.2 (C-6); HRESIMS m/z 209.0596 [M − H]− (calcd for C14H10O2, 209.0603). 2,6-Dihydroxy-9,10-dihydrophenanthrene (18): amorphous powder; UV (MeOH) λmax (log ε) 203 (2.10), 273 (0.92), 317 (sh) nm; 1 H NMR (DMSO-d6, 500 MHz) δ 2.64 (2H, m, H-9), 2.66 (2H, m, H-10), 6.55 (1H, d, J = 8.0, 2.4 Hz, H-8), 6.64 (1H, d, J = 2.5 Hz, H1), 6.69 (1H, d, J = 8.0, 2.4 Hz, H-4), 6.99 (1H, dd, J = 8.7 Hz, H-7), 7.04 (1H, d, J = 2.4 Hz, H-5), 7.47 (1H, d, J = 8.4 Hz, H-3); 13C NMR (DMSO-d6, 126 MHz) δ 28.0 (C-9), 29.5 (C-10), 109.7 (C-5), 129.1 (C-7), 113.5 (C-8), 126.6 (C-14), 135.5 (C-13), 156.7 (C-6), 139.0 (C-11), 125.6 (C-12), 115.2 (C-1), 157.3 (C-2), 125.1 (C-3), 114.3 (C-4); HRESIMS m/z 211.0755 [M − H]− (calcd for C14H12O2, 211.0759). 7-Methyl-2,6-phenanthrenediol (20): amorphous powder; UV (MeOH) λmax (log ε) 209 (2.68), 228 (2.73), 253 (2.97), 273 (2.63), 287 (sh) nm; 1H NMR (DMSO-d6, 500 MHz) δ 2.30 (s, 3H, CH3), 7.13 (1H, dd, J = 8.7, 2.5 Hz, H-3), 7.14 (1H, d, J = 2.5 Hz, H1), 7.38 (1H, d, J = 8.8 Hz, H-10), 7.54 (1H, d, J = 8.8 Hz, H-9), 7.58 (1H, s, H-8), 7.85 (1H, s, H-5), 8.26 (1H, d, J = 8.7 Hz, H-4); 13C NMR (DMSO-d6, 126 MHz) δ 16.2 (CH3), 104.8 (C-5), 111.0 (C-1), 116.8 (C-3), 122.2 (C-12), 122.5 (C-10), 123.7 (C-4), 124.0 (C-14), 124.6 (C-7), 126.5 (C-9), 129.8 (C-8), 129.9 (C-13), 133.2 (C-11), 155.2 (C-6), 155.7 (C-2); HRESIMS m/z 223.0745 [M − H]− (calcd for C15H10O2, 223.0759).

Computational Methods. Conformational analysis of 9 was performed with Schrödinger MacroModel 9.1 (Schrödinger, LLC, New York) employing the OPLS2005 (optimized potential for liquid simulations) force field in H2O. Conformers within a 2 kcal/mol energy window from the global minimum were selected for geometrical optimization and energy calculation applying DFT with the B3LYP/6-31G** level of theory in the gas phase with the Gaussian 09 program package.34 Vibrational evaluation was done at the same level to confirm minima. Excitation energy (denoted by wavelength in nm), rotator strength dipole velocity (Rvel), and dipole length (Rlen) were calculated in MeOH by TD-DFT/B3LYP/6-31G**, using the SCRF method, with the CPCM model. ECD curves were obtained on the basis of rotator strengths with a half-band of 0.25 eV using SpecDis v1.61.35 Acetylcholinesterase Activity Assay. The AChE inhibitory activity of compounds 1, 5, 6, 9, and 10 was evaluated using the method described by Ellman and associates 36 with certain modifications.37,38 This method is based on the amount of thiocholine released when AChE hydrolyzes the substrate acetylthiocholine iodide. The product thiocholine reacts with Ellman’s reagent [5,5′-dithiobis(2nitrobenzoic acid), DTNB] to produce a yellow compound [5-thio-2(nitrobenzoate)], which can be detected at 412 nm.32 The test compounds dissolved in DMSO were added to the wells of a 96-well plate to reach concentrations ranging from 40.0 to 0.04 μM. Subsequently, 0.44 mM acetylthiocholine and 0.15 M Ellman’s reagent (DTNB dissolved in 0.1 M phosphate buffer pH 7.4) were added. The enzymatic reaction was initiated by 0.01 U AChE from Electrophorus electricus (Sigma-Aldrich, St. Louis, MO, USA), and the plate was incubated at room temperature for 6 min. The absorbance was measured at 412 nm using a microplate reader (PowerWavex, BioTek Instrument, Winooski, VT, USA). The final DMSO concentration was 1% in each well. Each concentration was tested in triplicate. The percent inhibition was calculated using the following formula: (control absorbance − sample absorbance)/control absorbance × 100. The 50% inhibitory concentration (IC50) was determined from at least six different concentrations using GraphPad Prism 5.0 software.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: ++ 41 22 379 3641. Fax: ++41 22 379 33 99. E-mail: [email protected]. *Tel: ++55 16 3301 9660. Fax: ++55 16 3322 2308. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors extend their gratitude to the São Paulo State Research Foundation (FAPESP) for fellowship support (2010/ 09780-0 to M.M.F.Q.) and for funding the program FP7PEOPLE-2010-IRSES, “ChemBioFigh”. The authors are thankful to Ms. R. Felipe de Almeida from the Botanical Institute of Estado de São Paulo for the botanical identification. The authors are also thankful to Dr. W. Milliken at the Royal Botanic Gardens, Kew, Richmond, UK, for the photograph of T. mucronata presented in the graphical abstract.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry. 655



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P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A02; Gaussian, Inc.: Wallingford, CT, 2009. (35) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis version 1.61; University of Wuerzburg: Germany, 2013. (36) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88−95. (37) Di Giovanni, S.; Borloz, A.; Urbain, A.; Marston, A.; Hostettmann, K.; Carrupt, P. A.; Reist, M. Eur. J. Pharm. Sci. 2008, 33, 109−119. (38) Atta-ur-Rahman; Choudhary, M. I.; Thomsen, W. J. Bioassay Techniques for Drug Development; Taylor & Francis e-Library: Amsterdam, 2005.

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on February 12, 2014, with errors in the names and/or structures of compounds 11, 12, 15, 19, and 22 and ref 22. These were corrected in the version posted on March 7, 2014.

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Chemical Composition of the Bark of Tetrapterys mucronata and

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