Targeted Isolation of Monoterpene Indole Alkaloids from Palicourea

Article Cite This: J. Nat. Prod. 2017, 80, 3032-3037

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Targeted Isolation of Monoterpene Indole Alkaloids from Palicourea sessilis Luiz C. Klein-Júnior,*,†,‡,¶ Sylvian Cretton,§,¶ Pierre-Marie Allard,§ Grégory Genta-Jouve,⊥ Carolina S. Passos,§ Juliana Salton,† Pablo Bertelli,† Marion Pupier,∥ Damien Jeannerat,∥ Yvan Vander Heyden,‡ André L. Gasper,# Jean-Luc Wolfender,§ Philippe Christen,§ and Amélia T. Henriques† †

Laboratory of Pharmacognosy and Quality Control of Phytomedicines, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul-UFRGS, 90610-000, Porto Alegre/RS, Brazil ‡ Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Center for Pharmaceutical Research (CePhaR), Vrije Universiteit Brussel, B-1090 Brussels, Belgium § School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland ⊥ Faculté des Sciences Pharmaceutiques et Biologiques, C-TAC, UMR 8638 CNRS, Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France ∥ Department of Organic Chemistry, University of Geneva, 1211 Geneva 4, Switzerland # Herbarium Dr. Roberto Miguel Klein, Department of Natural Sciences, Universidade Regional de Blumenau, 89012-900, Blumenau/SC, Brazil S Supporting Information *

ABSTRACT: Phytochemical investigation of the alkaloid extract of Palicourea sessilis by LC-HRMS/MS using molecular networking and an in silico MS/MS fragmentation approach suggested the presence of several new monoterpene indole alkaloids. These compounds were isolated by semipreparative HPLC, and their structures confirmed by means of HRMS, NMR, and ECD measurements as 4-N-methyllyaloside (3), 4-N-methyl-3,4-dehydrostrictosidine (4), 4β-hydroxyisodolichantoside (6), and 4α-hydroxyisodolichantoside (7), as well as the known alkaloids alline (1), N-methyltryptamine (2), isodolichantoside (5), and 5-oxodolichantoside (8). In addition, the acetylcholinesterase inhibitory activity of the compounds was evaluated up to 50 μM.

P

psychollatine,8−10 lyaloside,11,12 and vallesiachotamine13,14 have already been described for both genera. Our research group has been studying the chemical aspects of these genera for several years, and the pharmacological potential of these monoterpene indole alkaloids has also been explored.13,15−20 Recently, Psychotria vellosiana Benth. was renamed as Palicourea sessilis (Vell.) C.M.Taylor, taking into account morphological and ecological aspects.3 A previous chemical study has led to a report of squalene, stigmasterol, sitosterol, lupeol, and scopoletin in an ethanol extract of the aerial parts of the plant.21 Taking this into account, in the present study, the leaves of P. sessilis were submitted to acid−base extraction. The alkaloid fraction was analyzed by LC-HRMS/MS. Fragmentation data of the alkaloid-enriched fraction were organized as molecular networks in order to classify metabolites in clusters of similar scaffolds.22 The generated data were then annotated

alicoureeae (Psychotrieae s. l.) is a complex tribe of the Rubioideae (Rubiaceae) subfamily and was defined in 2006 by Robbrecht and Manen.1 This tribe includes the genera Carapichea Aubl., Chassalia Comm. ex Poir., Geophila Bergeret, Hymenocoleus Robbr., Margaritopsis C.Wright, Notopleura (Benth.) Bremek., Rudgea Salisb., Palicourea Aubl., and Psychotria L. subg. Heteropsychotria Steyerm. Among them, the two latter are the most difficult ones to distinguish. Although they have been considered as different genera for several years, it has recently been demonstrated that, in fact, several Psychotria subg. Heteropsychotria species belong to the genus Palicourea.2,3 In addition to common morphological and molecular characteristics of the genera Psychotria subg. Heteropsychotria and Palicourea, some chemical aspects are also very similar. Psychotria s. str. is characterized by the occurrence of pyrrolidinoindoline alkaloids, such as hodgkinsine and calycanthine.4 On the other hand, Psychotria subg. Heteropsychotria and Palicourea are characterized by monoterpene indole alkaloids.5 Compounds such as brachycerine,6,7 © 2017 American Chemical Society and American Society of Pharmacognosy

Received: August 8, 2017 Published: November 9, 2017 3032

DOI: 10.1021/acs.jnatprod.7b00681 J. Nat. Prod. 2017, 80, 3032−3037

Journal of Natural Products

Article

side (GPX57-T), mitragynaline (LDR57-M), 9-methoxymitralactonine (KZO51-E), apodantheroside (JGV09-S), and genipin (JSW21-B). In order to further document the alkaloid profile of P. sessilis, three molecular ions at m/z 541.21, 543.23, and 561.24 were selected. The occurrence of these features within cluster A (mostly constituted by strictosidine-type monoterpene indole alkaloids) and the absence of annotation when dereplicated against all generated databases indicated possible novel analogues. Furthermore, the variable dereplication results against the in silico spectral database constituted by metabolites of the Rubiaceae+Loganiaceae suggested that these novel features should be members of the monoterpene indole alkaloid family (see Table S1, Supporting Information). The alkaloid fraction was submitted to semipreparative HPLC-DAD separation. Eight compounds were isolated, and their structural identification is described below. The MS fragmentation spectra of all isolated compounds were uploaded on the GNPS spectral library. They are accessible using the following links: 1 [http:// gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum. jsp?SpectrumID=CCMSLIB00003740006], 2 [http://gnps. ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00003740007], 3 [http://gnps.ucsd.edu/ P r o t e o S A F e /g n p s l i b r a r y s p e c t r u m .j s p ? S p e c t r u m ID = CCMSLIB00003740000], 4 [http://gnps.ucsd.edu/ P r o t e o S A F e /g n p s l i b r a r y s p e c t r u m .j s p ? S p e c t r u m ID = CCMSLIB00003740001], 5 [http://gnps.ucsd.edu/ P r o t e o S A F e /g n p s l i b r a r y s p e c t r u m .j s p ? S p e c t r u m ID = CCMSLIB00003740002], 6 [http://gnps.ucsd.edu/ P r o t e o S A F e /g n p s l i b r a r y s p e c t r u m .j s p ? S p e c t r u m ID = CCMSLIB00003740003], 7 [http://gnps.ucsd.edu/ P r o t e o S A F e /g n p s l i b r a r y s p e c t r u m .j s p ? S p e c t r u m ID = CCMSLIB00003740004], and 8 [http://gnps.ucsd.edu/ P r o t e o S A F e /g n p s l i b r a r y s p e c t r u m .j s p ? S p e c t r u m ID = CCMSLIB00003740005]. Compounds 1, 2, 5, and 8 were identified as alline,26 N-methyltryptamine,27 isodolichantoside,28,29 and 5-oxodolichantoside,30 respectively, by comparison of their experimental data with the literature. HRESIMS of compound 3 showed a molecular ion peak at m/z 541.2188 [M]+ (calcd for 541.2181), indicating a molecular formula of C28H33N2O9+. The application of 1H and 2D NMR experiments (HSQC, HMBC, and COSY) revealed the presence of a secologanin moiety28,31 and a βcarboline nucleus with six aromatic methines at δC 136.0, 133.1, 124.0, 123.1, 116.8, and 113.6 ppm (C-5, C-11, C-9, C-10, C-6, and C-12, respectively), five aromatic quaternary carbons at δC 145.5, 143.1, 137.3, 133.3, and 121.1 ppm (C-13, C-3, C-2, C-7, and C-8), and an N-methyl unit at δC 44.9 ppm. HMBC correlations from the N-methyl signal at δH 4.50 ppm to C-3 and C-5 and from H-14 to C-3 and C-15 (δC 35.5 ppm, CH) and the COSY correlations between H-10 (δH 7.48 ppm), H-9 (δH 8.38 ppm) and H-11 (δH 7.82 ppm), H-12 (δH 7.75 ppm) and H-11 led to the conclusion that 3 is a new alkaloid, named 4-N-methyllyaloside (Figure 1). Lyaloside is a monoterpene indole alkaloid dereplicated in P. sessilis (see Figure S1, Supporting Information) and previously isolated from Palicourea adusta and Pauridiantha lyalli.32,33 HRESIMS of compound 4 showed a molecular ion peak at m/z 543.2349 [M]+ (calcd for 543.2337), indicating a molecular formula of C28H35N2O9+. The application of 1H and 2D NMR spectra showed similarities with those of 3 except for the two methylene carbons C-5 and C-6 at δC 54.5 and 20.3 ppm, respectively. HMBC correlations from the N-methyl unit

against an in silico fragmented spectral database according to a previously described dereplication workflow.23 Various taxonomically restricted spectral databases were used in order to refine the annotation process. The annotation procedure allowed defining a cluster related to the monoterpene indole alkaloid family. Using the annotated molecular network as a guide it was possible to focus isolation efforts on previously undescribed compounds. Additionally, compounds were isolated and identified by MS and NMR studies and evaluated in vitro for their modulatory effect on acetylcholinesterase (AChE).

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RESULTS AND DISCUSSION The leaves of P. sessilis were extracted using an acid−base extraction strategy, leading to an enriched alkaloid fraction. The latter was analyzed by LC-HRMS/MS, and the data were used to generate a molecular network.24 The annotation of the molecular network using the Global Natural Product Social Molecular Networking (GNPS) experimental spectral libraries afforded eight hits, with two of them being alkaloids. The generated molecular network is available at the following address (http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task= 4bfd51cf7eba4facbc3fcb8a8b75c06f). Recently, an experimental spectral database focused on the monoterpene indole alkaloid scaffold has been constituted. It covers more than 50% of the 42 known monoterpene indole alkaloid skeletons and has proven to be a useful tool to target members of this alkaloid group.25 Unfortunately, this experimental database is not publicly available, thus limiting its use. Therefore, to further annotate the molecular network, the experimental fragmentation patterns acquired on the studied P. sessilis extract were compared to in silico spectra of natural products present in the Dictionary of Natural Products (http://dnp.chemnetbase.com), using a computationally generated spectral database.23 Structures depicted over the specific cluster A of the molecular network (Figure S1, Supporting Information) were found to be mostly related to the monoterpene indole alkaloid family. In order to further refine the annotation process, various taxonomically restricted in silico spectral libraries were built. The first one was restricted to molecules found only in the Rubiaceae family, and the second to molecules found in the Rubiaceae and Loganiaceae families. The dereplication table (Table S1, Supporting Information) shows the annotation of cluster A against the theoretical spectral database restricted to (i) molecules found in the Rubiaceae family; (ii) molecules found in the Rubiaceae and Loganiaceae families; (iii) all metabolites reported in the Dictionary of Natural Products; and finally (iv) molecules found in the Rubiaceae and Loganiaceae families but using a variable dereplication mode. For the latter, a modified tolerant spectral match was used, allowing compounds having different parent ion mass but sharing MS/ MS spectral similarities to be highlighted. In the four cases, 20.7%, 28.0%, 37.8%, and 86.6% of the nodes of the considered cluster were annotated, respectively. In cluster A it was possible to highlight various alkaloids mostly belonging to the strictosidine scaffold. The iridoid monoterpene and the glycosylated iridoid moiety of these alkaloids could also be detected. The dereplicated compounds were 18,19-dehydrocorynoxinic acid A (PPC18-C), tetrahydroalstonine (BBS49-W), 5-carboxytetrahydroalstonine (CFL71-B), strictosidine (CCF30-P), isodolichantoside (CCF35-U), 5-carboxystrictosidine (CFL68-F), 3,4-dehydrostrictosidine (OHZ46-F), lyaloside (CDJ73-F), 5-oxodolichantoside (RHR22-O), 14-oxolyalo3033



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C-12, respectively). A deshielding of the carbons C-3 (δC 74.1 ppm, CH) and C-5 (68.2 ppm, CH2) and the N-methyl signal (δC 47.6 ppm) was noticed in comparison to the chemical shifts of isodolichantoside (5). This observation suggested an electronegative atom bonded to the nitrogen atom. In accordance with this result, the IR spectrum showed a broad absorption band at 3325 cm−1 characteristic for an alcohol or N-oxide function,34 and the HRESIMS revealed the presence of an additional oxygen atom compared to 5. These data led to the conclusion that 6 is a hydroxylamine derivative of 5. NMR chemical shifts of compound 7 are close to those of 6 except in C-5 (61.8 ppm, CH2), C-14 (33.1 ppm, CH2), and the N-methyl signal (δC 55.6 ppm). HRESIMS data of 6 and 7 are identical. Thus, the difference between these two compounds lies in the orientation of the hydroxylamine group. In order to fully elucidate the stereochemistry of 6 and 7, the absolute configuration at C-3 was first established by comparison between experimental and calculated ECD spectra. As drawn in Figure 2, three Cotton effects (CEs) with alternative signs are observed on the experimental spectrum. Only two theoretical spectra exhibited three CEs with alternative signs, compounds a and d, with a 30 nm shift for the latter. In order to discriminate the like−unlike relationship between C-3 and N-4, a CP3 calculation35 was undertaken. The NMR prediction indicated a β-orientation (like) for 6 and αorientation (unlike) for 7 with a very high probability (87.3%). These compounds were named 4β-hydroxyisodolichantoside and 4α-hydroxyisodolichantoside, respectively. Different Psychotria and Palicourea monoterpene indole alkaloids already demonstrated the ability to inhibit the activity of some enzymes related to neurodegeneration, such as acetylcholinesterase (AChE), butyrylcholinesterase (BChE), MAO-A, catechol-O-methyltransferase (COMT), and sirtuins.13,15,16,20 However, it is not expected that monoterpene

Figure 1. Structures of the new compounds.

at δH 3.74 ppm to C-3 (δC 168.7 ppm, C) and C-5, as well as the COSY correlations between H-5 (δH 4.15 ppm) and H-6 (δH 3.30 ppm) led to the conclusion that 4 is the dihydro derivative of 3 and was named 4-N-methyl-3,4-dehydrostrictosidine. HRESIMS of compound 6 showed a molecular ion peak at m/z 561.2451 [M]+ (calcd for 561.2443) indicating a molecular formula of C28H37N2O10+. As previously described for 3 and 4, 1 H and 2D NMR experiments allowed identifying a secologanin moiety and a β-carboline nucleus with four aromatic methines at δC 121.9, 119.3, 117.7, and 111.2 ppm (C-11, C-10, C-9, and

Table 1. 1H NMR Data of New Compounds (500 MHz, in CD3OD) δH (J in Hz) position 3 5 6 9 10 11 12 14 15 17 18 19 20 21 1′ 2′ 3′ 4′ 5′ 6′ CO2CH3 NCH3 a

3 8.50, 8.50, 8.38, 7.48, 7.82, 7.75, 3.59, 3.53, 7.57, 5.42, 6.08, 2.86, 6.11, 4.90, 3.28, 3.46, 3.31, 3.47, 3.73, 2.90, 4.50,

s s d (8.1) d (7.6) ddd (1.02, 7.0, 8.2) d (8.4) m; 3.81, dd (3.6, 13.8) m s d (10.8); 5.49, m m m d (9.3) d (7.9) m m m m m; 4.05, dd (2.1, 11.8) s s

4 4.15, 3.30, 7.74, 7.24, 7.50, 7.50,

m m dt (1.0, 8.2) ddd (1.9, 6.0, 8.1) m m

a

3.35, 7.53, 5.42, 6.00, 2.83, 5.94, 4.85, 3.26, 3.43, 3.43, 3.29, 3.71, 3.14, 3.74,

m s d (10.7); 5.47, d (17.4) m m d (9.3) m m m m m m; 4.02, dd (2.2, 11.9) s s

6 4.91, 3.80, 3.12, 7.47, 7.06, 7.16, 7.41, 2.14, 3.26, 7.64, 5.32, 6.22, 2.92, 5.74, 4.72, 3.20, 3.38, 3.33, 3.28, 3.66, 3.80, 2.97,

m m; 3.86, m m d (7.9) ddd (7.4) ddd (1.2, 7.0, 8.3) d (8.0) m; 2.81, m m s dd (2.1, 10.3); 5.39, dd (1.2, 16.8) m m d (6.3) d (8.0) m m m m m; 3.90, dd (2.1, 12.2) s s

7 4.94, 3.57, 3.04, 7.46, 7.05, 7.14, 7.39, 2.02, 3.40, 7.55, 5.29, 5.80, 2.97, 5.58, 4.66, 3.22, 3.37, 3.28, 3.26, 3.65, 3.69, 3.29,

t (5.2) m; 3.78, m m; 3.14, m dt (1.0, 7.9) ddd (1.1, 7.0, 8.1) ddd (1.2, 7.2, 8.3) dt (1.0, 8.1) dt (5.34, 15.1); 2.54, dt (5.0, 14.8) m s dd (2.0, 10.4); 5.43, dd (1.3, 17.2) m m d (3.8) d (7.9) dd (7.9, 9.2) m m m m; 3.90, dd (2.1, 12.0) s s

Signal too weak to be measured. 3034



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Figure 2. TDDFT-predicted ECD spectra for the like−unlike diastereoisomers of 6 at C-3 and N-4. temperature to 40 °C, and the injection volume to 1 μL. Semipreparative chromatography was performed on an Armen Spot System (Saint-Avé, France) with an Xselect CSH C18 column (150 × 10 mm i.d.; 5 μm, Waters). MS Data Treatment, Molecular Network Generation, and Annotation. Thermo .RAW files were converted to .mzXML format using MSConvert GUI of Proteowizzard.36 A molecular network was created using the online workflow at GNPS. The data were then clustered with MS-Cluster with a parent mass tolerance of 0.05 Da and an MS/MS fragment ion tolerance of 0.05 Da to create consensus spectra. Consensus spectra that contained less than two spectra were discarded. A network was then created where edges were filtered to have a cosine score above 0.7 and more than six matched peaks. Further edges between two nodes were kept in the network if and only if each of the nodes appeared in each other’s respective top 10 most similar nodes. The spectra in the network were then searched against the spectral libraries GNPS. All matches kept between network spectra and library spectra were required to have a score above 0.6 and at least six matched peaks. The generated molecular network was then annotated against in silico spectral libraries constituted of compounds of the Dictionary of Natural Products and restricted at various taxonomical levels. The generation of these libraries has been previously described.23 The spectral library search was achieved using the open-source tool Tremolo.37 When using parent mass as filter, the parent mass tolerance was set to 0.0005 Da. In variable dereplication mode it was set to 200 Da. The spectral similarity score threshold was set at 0.2. ECD and NMR Computational Details. All calculations have been performed with the Gaussian 09 program.38 Geometry optimization has been achieved using density functional theory (DFT) with the B3LYP functional and the 6-31G(d) basis set in the gas phase. Vibrational analysis was completed at the same level to confirm a minimum. NMR prediction was performed using the mPW1PW91/6-31+G(d,p) level, while rotational strengths were evaluated using the B3LYP/6-311+G(d,p) method for six excited states. ECD curves were constructed on the basis of rotatory strength dipole velocity (Rvel) with a half-band of 0.2 eV using SpecDis v1.61.39 Plant Material. Leaves of P. sessilis were collected in Blumenau/ SC, Brazil (lat −26.928333°, long −49.048889°; February 2015). The plant material was identified by the botanist A. L. Gasper (FURB/ Brazil), and a voucher specimen was deposited at the Dr. Roberto

indole alkaloids with a glucose moiety (3−8) would inhibit AChE activity, as previously demonstrated,13 probably due to the bulkiness of the structure, hampering the interaction. On the other hand, it is known that quaternary β-carboline alkaloids (3, 4, 6, 7) demonstrate higher potency, such as prunifoleine and 14-oxoprunifoleine (IC50 10 and 3.4 μM, respectively).13 Taking this into account, alkaloids 1−8 were assayed for their modulatory effect on AChE at 10, 25, and 50 μM, using 0.1 μM tacrine as positive control. Only compounds 1 (53.7 ± 2.8%, at 50 μM) and 2 (49.8 ± 2.4%, at 50 μM) exhibited a moderate inhibitory effect, indicating that they can better interact with the active site of the enzyme. Tacrine inhibited AChE on the order of 57.8 ± 1.8% at 0.1 μM.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1030 polarimeter (Easton, MD, USA; EtOH, c in g/100 mL). The ECD spectra were recorded in MeOH on a JASCO J-815 CD spectrometer, and the UV spectra, also in MeOH, on a PerkinElmer Lambda-25 UV−vis spectrophotometer (Wellesley, MA, USA). IR spectra were measured on a PerkinElmer Spectrum 100 spectrometer. 1H and 13C NMR spectra were recorded on a Bruker Avance III 500 MHz NMR spectrometer (Rheinstetten, Germany) equipped with a 5 mm cryogenic DCH (1H/13C) probe. Chemical shifts are reported in parts per million (δ) using the residual CD3OD signals (δH 3.31; δC 49.0) as internal standards for 1H and 13C NMR, respectively, and coupling constants (J) are reported in Hz. HRMS were obtained on a Q Exactive Plus Hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) using electrospray ionization in the positive-ion mode. The spray voltage was set at 3.5 kV; the sheath gas flow rate (N2) at 50 units; the capillary temperature at 320 °C; the S lens RF level at 50; and the probe heater temperature at 425 °C. UHPLC was performed on an Ultimate 3000 UHPLC System (Thermo Scientific). The separation was performed on an Acquity BEH C18 UPLC column (100 × 2.1 mm i.d.; 1.7 μm, Waters), using a gradient (MeCN and H2O both containing 0.1% formic acid) from 5% to 98% MeCN in 6 min, followed by an isocratic washing step at 98% MeCN for 2 min. After the washing step, the column was re-equilibrated with 5% MeCN for 3 min prior to the next injection. The flow rate was set to 0.4 mL/min, the column 3035



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Miguel Klein Herbarium under the number 47383 (available at http:// furb.jbrj.gov.br/v2/consulta.php). Extraction and Isolation. The leaves were air-dried and milled separately. The plant material (10.1 g) was submitted to static maceration using ethanol for 48 h (1:30, m/v), three times. The crude dried extract was suspended in 1 M HCl and washed six times with CH2Cl2 in order to remove nonpolar compounds. Then, the extract was alkalinized with NH4OH to pH 9. Finally, the alkaloid fraction (50 mg) was obtained by partitioning the aqueous layers with dichloromethane.40 The partitioning procedure was repeated until a negative reaction with Mayer’s reagent. On removal of the solvent, the final fractionation step was performed with 30 mg of the fraction by semipreparative HPLC using a Xselect CSH C18 column (150 × 10 mm i.d.; 5 μm, Waters) and MeCN/H2O/0.1% formic acid as solvents using an optimized gradient of 10% to 30% MeCN in 30 min. The flow rate was set to 5.5 mL/min and UV absorbance measured was at 220 nm. Fraction 2 yielded 1 (0.5 mg),26 fraction 5 yielded 2 (0.8 mg),27 fraction 13 yielded 3 (0.5 mg), fraction 15 yielded 4 (0.4 mg), fractions 18−20 yielded 5 (9.4 mg),28,29 fractions 23 and 24 yielded 6 (1.0 mg), fractions 27 and 28 yielded 7 (1.0 mg), and fraction 33 yielded 8 (0.9 mg).30 4-N-Methyllyaloside (3): pale yellow oil; UV (MeOH) λmax (log ε) 222 (5.4), 310 (2.4), 372 (1.0) nm; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 541.2188 [M]+ (calcd for C28H33N2O9+, 541.2181); MS/MS spectra [http://gnps.ucsd.edu/ProteoSAFe/ gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB00003740000]. 4-N-Methyl-3,4-dehydrostrictosidine (4): pale yellow oil; UV (MeOH) λmax (log ε) 221 (5.4), 357 (2.6) nm; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 543.2349 [M]+ (calcd for C28H35N2O9+, 543.2337); MS/MS spectra [http://gnps.ucsd.edu/

ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00003740001]. 4β-Hydroxyisodolichantoside (6): pale yellow oil; UV (MeOH) λmax (log ε) 224 (5.4) nm; IR (CHCl3) νmax 3325, 2936, 2836 1648, 1451, 1413, 1107, 1018 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 561.2451 [M]+ (calcd for C28H37N2O10+, 561.2443); MS/MS spectra [http://gnps.ucsd.edu/ProteoSAFe/ gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB00003740003]. 4α-Hydroxyisodolichantoside (7): pale yellow oil; UV (MeOH) λmax (log ε) 224 (5.4) nm; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 561.2451 [M]+ (calcd for C28H37N2O10+, 561.2443); MS/MS spectra [http://gnps.ucsd.edu/ProteoSAFe/ gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB00003740004]. Acetylcholinesterase Assay. Ellman’s method,41 modified by Di Giovanni et al.,42 was used in order to estimate alkaloid effects on AChE activity on 96-well plates. Tacrine was used as positive control at 0.1 μM. Each well was filled with 158 μL of Ellman’s reagent (5,5′dithiobis(2-nitrobenzoic acid) in 0.1 M phosphate buffer, pH 7.4), acetylthiocholine iodide 0.33 mM (ATCI) solution (20 μL), and the test alkaloid (10, 25, or 50 μM) or tacrine solutions in DMSO (2 μL). The corresponding volume of DMSO was used as negative control. Electric eel AChE solution (20 μL, 1 UI/mL in 0.1 M phosphate buffer pH 7.4, containing human serum albumin at 1 mg/mL) was used to start the reactions. Assays were performed in triplicate at room temperature with a microplate spectrophotometer following the rate of increase in absorbance at 412 nm for 10 min (intervals of 40 s between readings).

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00681. Molecular network (cluster A) and the associated dereplication table (Figure S1 and Table S1) as well as 1 H and 13C NMR spectra of compounds 3, 4, 6, and 7 (Figures S1−S17) (PDF) (XLSX)

Table 2. 13C NMR Data of New Compounds (125 MHz, in CD3OD) δC position 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1′ 2′ 3′ 4′ 5′ 6′ CO2CH3 NCH3 a

3 137.3, C 143.1, CH 136.0, CH 116.8, CH 133.3, C 121.1, C 124.0, CH 123.1, CH 133.1, CH 113.6, CH 145.5, C 32.3, CH2 35.5, CH 108.6, C 155.5, C 120.1, CH2 135.2, CH 44.9, CH 96.7, CH 168.2, C 100.5, CH 74.6, CH 78.3, CH 71.6, CH 78.5, CH 62.9, CH2 50.1 43.7

4 129.2, C 168.7, C 54.5, CH2 20.3, CH2 125.3, C 125.3, C 122.5, CH 123.0, CH 130.1, CH 113.8, CH 142.9, C 32.3, CH2 35.3, CH 108.2, C 155.2, C 120.5, CH2 135.0, CH 44.9, CH 96.7, CH 168.3, C 100.3, CH 74.5, CH 78.4, CH 78.4, CH 71.6, CH 62.7, CH2 51.4 42.4

6

7

74.1, CH 68.2, CH2 19.4, CH2 104.7, C 125.8, C 117.7, CH 119.3, CH 121.9, CH 111.2, CH 137.2, C 28.6, CH2 31.9, CH 109.3, C 153.6, C 119.0, CH2 134.8, CH 45.0, CH 96.2, CH 169.1, C 99.1, CH 73.0, CH 76.5, CH 77.1, CH 70.2, CH 61.4, CH2 51.0 47.6

131.4, C 73.3, CH 61.8, CH2 18.9, CH2 103.5, C 125.8, C 117.7, CH 119.1, CH 121.9, CH 111.0, CH 137.2, C 33.1, CH2 30.2, CH 109.9, C 152.7, C 119.4, CH2 133.9, CH 44.5, CH 96.1, CH 168.5, C 98.4, CH 73.1, CH 76.4, CH 77.0, CH 70.3, CH 61.4, CH2 51.5 55.6

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ASSOCIATED CONTENT

S Supporting Information *

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AUTHOR INFORMATION

Corresponding Author

*E-mail (L.C. Klein-Júnior): [email protected]. Phone/fax: +55 51 3308 5258. ORCID

Luiz C. Klein-Júnior: 0000-0003-2243-1701 Jean-Luc Wolfender: 0000-0002-0125-952X Author Contributions ¶

L. C. Klein-Júnior and S. Cretton contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by The National Council for Scientific and Technological Development (CNPq), Fundaçaõ de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Brazil, and by The Fund for Scientific Research (FWO), Vlaanderen, Belgium. L.C.K.J., J.S., and A.T.H. thank CNPq for the fellowships. The authors are grateful to Prof. C. M. Taylor for her contribution to the Psychotria and Palicourea taxonomy.

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