Targeted Isolation of Indolopyridoquinazoline Alkaloids from

Article pubs.acs.org/jnp

Targeted Isolation of Indolopyridoquinazoline Alkaloids from Conchocarpus fontanesianus Based on Molecular Networks Rodrigo Sant’Ana Cabral,†,‡ Pierre-Marie Allard,‡ Laurence Marcourt,‡ Maria Cláudia Marx Young,† Emerson Ferreira Queiroz,*,‡ and Jean-Luc Wolfender‡ †

Nucleus of Research in Physiology and Biochemistry, Botany Institute of São Paulo, Avenida Miguel Estefano, 3687, 04301-012, São Paulo, Brazil ‡ School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland S Supporting Information *

ABSTRACT: A dichloromethane-soluble fraction of the stem bark of Conchocarpus fontanesianus showed antifungal activity against Candida albicans in a bioautography assay. Off-line high-pressure liquid chromatography activity-based profiling of this extract enabled a precise localization of the compounds responsible for the antifungal activity that were isolated and identified as the known compounds flindersine (17) and 8methoxyflindersine (18). As well as the identification of the bioactive principles, the ultra-high-pressure liquid chromatography−high-resolution mass spectrometry metabolite profiling of the dichloromethane stem bark fraction allowed the detection of more than 1000 components. Some of these could be assigned putatively to secondary metabolites previously isolated from the family Rutaceae. Generation of a molecular network based on MS2 spectra indicated the presence of indolopyridoquinazoline alkaloids and related scaffolds. Efficient targeted isolation of these compounds was performed by geometric transfer of the analytical high-pressure liquid chromatography profiling conditions to preparative medium-pressure liquid chromatography. This yielded six new indolopyridoquinazoline alkaloids (5, 16, 19−22) that were assigned structurally. The medium-pressure liquid chromatography separations afforded additionally 16 other compounds. This work has demonstrated the usefulness of molecular networks to target the isolation of new natural products and the value of this approach for dereplication. A detailed analysis of the constituents of the stem bark of C. fontanesianus was conducted.

T

were not established.8 Furoquinoline and quinolone alkaloids and coumarins exhibiting acetyl cholinesterase-inhibitory properties have also been described from this plant.9 As part of a continuing search for antifungal agents of natural origin,10,11 the CH2Cl2-soluble fractions of the stem bark and leaves of C. fontanesianus showed antifungal activity against Candida albicans. This microorganism colonizes the skin and mucosal surfaces and can cause a widespread spectrum of diseases, ranging from benign mucosal infections such as oral thrush to fatal disseminated candidiasis.12 The compounds responsible for the antifungal activity of the CH2Cl2 stem bark and leaf fractions of C. fontanesianus were identified, and metabolite profiling by UHPLC-HRESIMS2 and molecular networks highlighted several unknown indolopyridoquinazoline analogues that were isolated. Twenty-two compounds were isolated using this approach, with six (5, 16, 19−22) of them being new indolopyridoquinazoline analogues. The isolated compounds were then evaluated individually against C. albicans.

he plant genus Conchocarpus, belonging to the family Rutaceae, consists of shrubs or small trees up to seven meters tall, with 45 species that are distributed from Nicaragua to northern Bolivia and Brazil.1 In Brazil, species of this genus can be found in almost all biomes, mainly in the Amazonian, Atlantic Rainforest, and Cerrado regions.2 Only a few species in this genus have been studied up to now from a phytochemical viewpoint. Two acridone alkaloids were isolated from Conchocarpus paniculatus,3 with flavonoids, alkaloids, and steroids reported in C. heterophyllus4 and alkaloids and amides from C. gaudichaudianus. Phytochemical investigation of leaves and stems of C. marginatus and C. inopinatus have led to the identification of coumarins and acridone and quinoline alkaloids.5 Conchocarpus fontanesianus (A. St.-Hil.) Kallunki & Pirani, popularly known as “pitaguará”, is a shrub or small tree, reaching 1−3 m in height. The species is native, endemic, and distributed in the Brazilian Atlantic Rainforest region in the states of Rio de Janeiro and São Paulo.1,6,7 A previous study on the leaf extract of C. fontanesianus reported their antifungal, cytotoxic, and antimicrobial properties, but the active principles © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 27, 2016

A

DOI: 10.1021/acs.jnatprod.6b00379 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

■

RESULTS AND DISCUSSION During a preliminary antifungal screening procedure, an ethanolic extract from the stem bark of C. fontanesianus exhibited weak activity at 600 μg/spot against a hypersensitive mutant strain of C. albicans (DSY2621) in a bioautography assay.10 Bioautography with the hypersusceptible strain was used to detect the presence of minor antifungal compounds with high sensitivity. To identify the active compounds, a crude ethanol extract of the stem bark was partitioned with CH2Cl2 and a MeOH−water (7:3) mixture. The CH2Cl2 and hydromethanolic fractions were submitted to biological assay. Antifungal activity was identified in the CH2Cl2 fraction (100 μg/spot), while the hydromethanolic fraction was inactive. A follow-up investigation of the constituents in the CH2Cl2 fraction was undertaken to reveal the constituents active against C. albicans. To obtain the active compounds, the CH2Cl2 fraction (50 mg) was microfractionated using reversed-phase HPLC.13,14 Aliquots of the 60 microfractions produced were assayed by bioautography to localize the activity (Figure 1), and two HPLC peaks at 39.5 min (17) and 41.8 min (18) were found to be the antifungal principles. Metabolite profiling by UHPLC-

HRESIMS revealed that the molecular formula of peak 17 was C14H13NO2 (HRESIMS [M + H]+ at m/z 228.1019, calcd for C14H14NO2, 228.1019; Δppm = 0.02) and that of 18 was C15H15NO3 (HRESIMS [M + H]+ at m/z 258.1126, calcd for C15H16NO3, 257.1125; Δppm = 0.54). A cross search of these molecular formulas in the Dictionary of Natural Products15 within the genus Conchocarpus did not yield any proposed compound identification, while many isomers for both molecular formulas were found to have been reported for other genera in the Rutaceae family. The identification of these active principles thus required their targeted isolation, and they were purified in one step by semipreparative HPLC from the leaf CH2Cl2 fraction, which contained them in high yields (Figure 1). The antifungal agents were identified as flindersine (17)16 and 8-methoxyflindersine (18),17 furoquinoline alkaloids previously described in other members of the Rutaceae family but never reported in the genus Conchocarpus.5 Both compounds exhibited a similar minimal inhibitory quantity (MIQ) of 15 μg against the C. albicans-sensitive mutant strain DSY2621, but were inactive against a wild strain of this yeast. The antifungal activity of flindersine (17) against C. albicans has B



Article

Figure 1. (A) HPLC-PDA metabolite profiling (UV 254 nm) of the CH2Cl2 extract of the stem bark and (B) leaves of C. fontanesianus. Dashed areas correspond to the chromatographic zone with off-line antifungal activity against C. albicans.

Figure 2. Molecular network of tandem MS/MS data acquired by UHPLC-HRESIMS/MS data dependent metabolite profiling of the crude CH2Cl2 extract of the bark of Conchocarpus fontanesianus. (A) Full molecular network of the extract. (B) Within this network, a cluster could be related mainly to parent ions corresponding to molecular formula containing three nitrogen atoms, represented as blue spots. (C) Evaluation of this cluster allowed highlighting dereplicated indolopyridoquinazolines such as paraensine and focusing on ions of interest potentially corresponding to new alkaloids of this class.

been described already,18 while such activity for 8-methoxyflindersine (18) is described here for the first time. Since the genus Conchocarpus has been poorly studied phytochemically, a detailed metabolite profiling procedure was performed on an ethanol-soluble stem bark extract of C. fontanesianus by UHPLC-HRESIMS, in order to obtain preliminary information on its secondary metabolite composition. An automated peak picking procedure of the UHPLCHRESIMS data led to the establishment of a peak list constituted by 1098 features (characterized by a given m/z at

a specific retention time). The 200 most intense features (based on peak area) of this peak list were retained for dereplication. Among them, 16 were redundant as adducts, dimers, or complexes (Table S1, Supporting Information). This peak list was then dereplicated with a ±3 ppm mass accuracy tolerance against a subset of the Dictionary of Natural Products database constituted by all compounds isolated from species of the Rutaceae family (4212 compounds), allowing the putative annotation of 77 compounds. In doing this, chemotaxonomy was considered as an orthogonal filter and a level 3 C



Article

identification was assigned according to Schymanski et al.19 In addition, for all features of the peak list, a molecular formula calculation was produced independently using element count heuristic filters,20 ring double bond equivalent (RDBE) restrictions, and an isotopic pattern filter within a range of 5 ppm. The molecular formula calculation matched the 77 previously annotated features. For the 107 remaining features, a unique molecular formula could be attributed in most cases (Table S1, Supporting Information). Thus, from the 200 initial features, after filtering, 172 molecular formulas were calculated. Of these, 125 were determined as unique, indicating 27% of isomerism within the whole set. From these unique molecular formulas, 57 of them were assigned to compounds previously reported in the Rutaceae family, with 68 being new to the family. Within the 125 unique molecular formulas obtained, 38 of these (>30%) were found to contain three nitrogen atoms. Twenty of these molecular formulas could be assigned putatively to compounds previously described within the Rutaceae, of which three were described in the genus C o n c h o c a r p u s prev io us ly (3-(2-(7,7-dimethyl-3,7dihydropyrano[3,2-e]indol-1-yl)ethyl)quinazoline-2,4(1H,3H)dione, 3-(2-(7,7-dimethyl-3,7-dihydropyrano[3,2-e]indol-1-yl)ethyl)-1-hydroxyquinazoline-2,4(1H,3H)-dione, and 3-(2-(7,7dimethyl-3,7-dihydropyrano[3,2-e]indol-1-yl)ethyl-1-methylquinazoline-2,4(1H,3H)-dione).21 Within this set, all dereplicated compounds with three nitrogen atoms were found to be indolopyridoquinazoline alkaloids. The 18 remaining compounds of this type could not be identified (Table S2, Supporting Information). Since these compounds may have potentially interesting biological properties and may be new natural products, they were studied in more depth. To investigate further the structural similarities occurring among the compounds containing three nitrogen atoms, the CH2Cl2 fraction was further analyzed by data-dependent UHPLC-HRESIMS2. All HRESIMS2 fragmentation spectra were processed globally and organized as molecular networks (MN). This approach has been employed successfully for clustering compounds sharing similar tandem MS2 spectra22 and has been recently implemented as an efficient tool for the dereplication of natural products.23,24 In the present case, MN revealed that most of the three nitrogen atom containing features belonged mainly to the same cluster among the full MN (Figure S1, Supporting Information). Within this cluster it was possible to link these particular molecular formulas to dereplicated features representing an indolopyridoquinazoline scaffold (Figure 2). Furthermore, when annotating the generated MN by spectral matching against an in silico database of Rutaceae metabolites using a recently developed dereplication strategy,25 it was indeed possible to confirm the link of this cluster to indolopyridoquinazoline alkaloids (Figure S2, Supporting Information), thus motivating the phytochemical investigation of the bark of C. fontanesianus to verify the occurrence of new members of this particular alkaloid class. The feature information from this cluster (m/z vs tR) provided an efficient way to localize these alkaloids in the metabolite profile of the crude stem bark extract by displaying their extracted ion chromatograms (XIC) (Figure S3, Supporting Information), allowing their targeted isolation. A large-scale isolation of the CH2Cl2-soluble extract of the stem bark of C. fontanesianus was performed in order to isolate these potentially novel compounds, establish their structures, and thus validate the approach taken. Isolation was conducted

by direct transfer of the analytical HPLC conditions to MPLC with the same reversed-phase material.26 This resulted in the purification of six new natural products of the indolopyridoquinazoline class (5, 16, 19−22). The purification protocol also yielded 16 additional known secondary metabolites (coumarins, lignans, amides, furoquinoline alkaloids) (1−4, 6−10, 11−15, 17, 18), with some of these being reported here for the first time in the genus Conchocarpus. The 16 known compounds were identified by comparison of their physical and spectroscopic data with published values as dimorphamide C (1),27 N-cis-feruloyltyramine (2),28 paprazine (3),29 confusameline (4),30 moupinamide (6),29 (−)-syringaresinol (7),31 aegelinol (8),32 haplopine (9),33 1-methyl-2phenyl-4-quinolone (10),34 skimmianine (11),17 γ-fagarine (12),35 grossamide (13),28 10-hydroxyrutaecarpine (14),36 cannabisin F (15),37 flindersine (17),16 and 8-methoxyflindersine (18).17 The structure elucidation of the six new indolopyridoquinazoline alkaloids (5, 16, 19−22) (Figure 2) is described below. The UHPLC-HRESIMS2 dereplication procedure indicated that 5 is an indolopyridoquinazoline alkaloid with a [M + H]+ at m/z 320.1036 (calcd for C18H14N3O3, 320.1035; Δppm = 0.3). The NMR data showed close similarities to values reported for 10-hydroxyrutaecarpine (14).36 For compound 5, two methylene signals were observed at δH 4.52 (2H, t, J = 6.8 Hz, H-5) and 3.14 (2H, t, J = 6.8 Hz, H-6). The 1H and COSY spectra indicated the presence of two 1,2,4-trisubstituted aromatic rings at δH 6.95 (1H, d, J = 2.3 Hz, H-9), 6.85 (1H, dd, J = 8.8, 2.3 Hz, H-11), and 7.31 (1H, d, J = 8.8 Hz, H-12) for the first ring and δH 7.59 (1H, d, J = 8.7 Hz, H-16), 7.28 (1H, dd, J = 8.7, 2.8 Hz, H-17), and 7.54 (1H, d, J = 2.8 Hz, H19) for the second one. Long-range HMBC correlations from H-12 to the quaternary carbons at δC 127.1 (C-8) and the oxygenated carbon at 152.2 (C-10) and from H-6 to C-8 allowed a hydroxy group to be positioned at C-10. The NOESY correlation between H-6 and H-9 confirmed the position of these protons in the structure. A second hydroxy group was placed at C-18 from the correlations between H-16 and the signals at δC 157.8 (C-18) and 122.7 (C-20) and between H-17 and C-15 at δC 142.4 (linked to a nitrogen). On the basis of these data, 5 was proposed as 10,18-dihydroxyrutaecarpine, a new indolopyridoquinazoline alkaloid. The indolopyridoquinazoline alkaloid 16 was isolated as an amorphous solid (m/z 414.1821 [M]+ (calcd for C25H24N3O3, 414.1818; Δppm = 0.7)) (Table S1, Supporting Information). Analysis of the 1H and HSQC NMR spectra indicated resonances for two methylene groups (δH/δC 4.51/43.0 and 3.51/21.6), a methoxy group (δH/δC 3.99/55.8), a N-CH3 group (δH/δC 4.32/41.5), and a spin system with three aromatic signals at δH 6.64 (1H, d, J = 7.8 Hz, H-10), 7.42 (1H, t, J = 8.5, 7.8 Hz, H-11), and 7.18 (1H, d, J = 8.5 Hz, H-12) characteristic of a 1,2,3-trisubstituted aromatic ring. Also observed were a spin system corresponding to two ortho aromatic signals at δH 7.78 (1H, d, J = 9.2 Hz, H-16) and 7.44 (1H, d, J = 7.79 Hz, H-17) and a 2,2-dimethylchromene moiety, as indicated by the proton signals at δH 1.49 (6H, s, 2 × CH3), 7.94 (1H, d, J = 10.3 Hz, H-22), and 6.05 (1H, d, J = 10.3 Hz, H-23). The methoxy group was located at C-9 according to the HMBC and NOESY spectrum correlations from H-11 and OCH3-9 to C-9 (δC 157.4) and from OCH3-9 to H-10. The HMBC correlations from H-22 to C-18 (δC 154.6) and C-24 (δC 77.6), from H-23 to C-19 (δC 122.4), from H-17 to C-15 (δC 136.3) and C-19, and from H-16 to CD



Article

Figure 3. Key HMBC correlations of compounds 16, 19, 20, and 21.

The HRESIMS of 21 (m/z 370.1555 [M + H]+ (calcd for C23H20N3O2, 370.1556; Δppm = 0.1)) indicated one oxygen to be missing when compared to 19. The substitution of the aromatic group linked to the pyrimidone group was different, as shown by the four aromatic protons at δH 7.67 (1H, dd, J = 8.3, 1.2 Hz, H-16), 7.81 (1H, ddd, J = 8.3, 7.1, 1.5 Hz, H-17), 7.47 (1H, ddd, J = 8.0, 7.1, 1.2 Hz, H-18), and 8.16 (1H, dd, J = 8.0, 1.5 Hz, H-19). This indicated that 21 has no hydroxy group on the aromatic ring. On the basis of these results, 21 (fontanesine B) was identified as a new indolopyridoquinazoline alkaloid, as shown. Compound 22 was also found to be an indolopyridoquinazoline alkaloid derivative. Its NMR data were close to those of 19, except for the position of the free hydroxy group at C-16. This assignment was obtained by HMBC correlations from H-19 (δH 7.55, 1H, dd, J = 7.9, 1.3 Hz) and H-5 (δH 4.41, 2H, t, J = 6.9 Hz) to C-21 (δC 160.0) and from H-18 (δH 7.29, 1H, t, J = 7.9 Hz) to C-16 (δC 151.8) and C-20 (δC 120.8) (Figure 3). The HRESIMS of 22 confirmed this supposition from the presence of a quasimolecular ion at m/z 386.1507 [M + H]+ (calcd for C23H20N3O3, 386.1505; Δppm = 0.5). On the basis of these results, compound 22 (fontanesine C) was identified as a new indolopyridoquinazoline alkaloid, as shown.

18 and C-20 (δC 115.1) indicated that the chromene moiety is linked to C-18 and C-20. The HMBC correlations from H-12 to C-8 and C-10 (δC 116.7 and 101.4, respectively), from the methylene H-6 to C-2 (δC 119.8), C-7 (δC 132.2), and C-8, and from the methylene H-5 to C-3 (δC 149.3) and C-7 were used to locate the 1,2,3-trisubstituted aromatic ring position (part of an indole group) and the two methylene groups (Figure 3). Finally, the HMBC and NOESY correlations from the Nmethyl group to C-3, C-15, and H-16 permitted the various substructures to be linked. Compound 16 was established as a new indolopyridoquinazoline alkaloid and was given the name conchacarpine A. The 1H NMR spectrum of 19 (m/z 386.1515 [M + H]+ (calcd for C23H20N3O3, 386.1505; Δppm = 2.7)) exhibited two methylene signals at δH 4.41 (2H, t, J = 6.8 Hz, H-5) and 3.29 (2H, t, J = 6.8 Hz, H-6), three aromatic signals characteristic of a 1,2,4-trisubstituted aromatic ring at δH 7.56 (1H, d, J = 8.8 Hz, H-16), 7.28 (1H, dd, J = 8.8, 2.9 Hz, H-17), and 7.48 (1H, d, J = 2.9 Hz, H-19), two aromatic signals characteristic of a 1,2,3,4-tetrasubstituted aromatic ring at δH 6.74 (1H, d, J = 8.7 Hz, H-11) and 7.23 (1H, d, J = 8.6 Hz, H-12), and protons typical of a 2,2-dimethylchromene moiety at δH 6.86 (1H, d, J = 9.7 Hz, H-22), 5.74 (1H, d, J = 9.7 Hz, H-23), and 1.38 (6H, s, 2 × CH3). Compared to 16, the chromene group of 19 was linked to the indole part as shown by the HMBC interactions from H-22 to C-8 (δC 121.0) and C-10 (δC 146.1), from H-12 to C-8 and C-10, from H-23 to C-9 (δC 112.5), and from H-11 to C-9 and C-13 (δC 134.2) (Figure 3). The two methylenes were also linked to the indole portion (H-6 correlated with C-2 (δC 128.2), C-7 (δC 115.3), and C-8). The long-range correlations from H-5 and H-19 to C-21 (δC 160.2) were used to position the phenol group and the pyrimidone moiety. Compound 19 was thus identified as a new indolopyridoquinazoline alkaloid named fontanesine A. The NMR data of compound 20 were related closely to those observed for 16. The differences between these compounds were related to the lack of the N-Me group in 20 and the substitution of the indole moiety. The presence of the aromatic protons at δH 6.91 (1H, d, J = 2.3 Hz, H-9), 6.83 (1H, dd, J = 8.8, 2.3 Hz, H-11), and 7.27 (1H, d, J = 8.8 Hz, H12) was characteristic of a 1,2,4-trisubstituted aromatic ring. The HMBC correlations between H-12 and an oxygenated carbon at δC 152.3 (C-10) suggested the presence of a free hydroxy group at C-10 (Figure 3). This interference was confirmed by the molecular formula deduced from its [M + H]+ peak at 386.1501 (calcd for C23H20N3O3, 386.1505; Δppm = 0.6). On the basis of these results, 20 was identified as a new indolopyridoquinazoline alkaloid named conchacarpine B.

■

EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured in methanolic solutions on a JASCO polarimeter in a 1 cm tube. UV spectra were measured on a HACH UV−vis DR/4000 instrument (Loveland, CO, USA). NMR spectroscopic data were recorded on a 500 MHz Varian (Palo Alto, CA, USA) INOVA NMR 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 reported in Hz. Complete assignments were obtained based on 2D NMR experiments (COSY, NOESY, HSQC, and HMBC). For compounds 5 and 16, the 13C NMR information was obtained indirectly from 2D NMR analysis. 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). HRESIMS2 analysis was performed on a Thermo Dionex Ultimate 3000 UHPLC system interfaced to a Q-Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. HPLC-PDA-ESIMS analysis was conducted on an HP 1200 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 ESI. Plant extraction was performed by accelerated solvent extraction on an ASE300 apparatus (Dionex, Sunnyvale, CA, USA). MPLC was performed using a Büchi system equipped with a C-660 module pump, C-640 UV detector, and C-684 fraction collector from Büchi E



Article

Consensus spectra that contained less than one spectrum 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 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 GNPS spectral libraries. 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 clustered spectra of the network was searched against the ISDB (In Silico DataBase) restricted to metabolites of the Rutaceae family. Generation of the ISDB has been described by Allard et al.25 Spectral matching parameters were the following: TOLERANCE = 0.005, SCORE_TRESHOLD = 0.2, TOP_K_RESULTS = 3. Molecular networks were visualized using Cytoscape 2.8 and ChemViz 1.3 for structure display. Plant Material. The stem bark and leaves from Conchocarpus fontanesianus were collected in March 2012 at the Ecological Station Jureia-Itatins (license SMA, process no. 260.108-007.532 2009), ́ São Paulo State, Brazil (24°22′58.5″ S, Nucleus Arpoador/Peruibe, 47°01′07.5″ W, 38 m above sea level) and identified by Dra. Inês Cordeiro. A voucher specimen (RSCabral 09) has been deposited at the Herbário do Estado “Maria Eneyda P. Kaufmann Fidalgo (SP)”, Instituto Botânico de São Paulo, São Paulo, Brazil. Extraction. The dried stem bark (1500 g) and leaves (800 g) of C. fontanesianus were extracted individually with ethanol (60 °C, 1500 psi) by accelerated solvent extraction on an ASE300 apparatus (Dionex). The extracts were concentrated to dryness by rotatory evaporation, yielding 43 and 54 g of the stem bark and leaf crude extracts, respectively. These extracts were submitted to liquid/liquid extraction with methanol (MeOH)−water (H2O) (7:3, v/v) and subsequent partition using organic solvents of increasing polarity: nhexane, CH2Cl2, and EtOAc. This yielded the following extracts: nhexane (stem bark 1.9 g; leaves 3.4 g), CH2Cl2 (stem bark 6 g; leaves 12 g), and EtOAc (stem bark 2.5 g; leaves 2.3 g). HPLC-Microfractionation of the Dichloromethane Fraction of the Stem Bark and Leaves. The microfractionation of the CH2Cl2 fraction of the stem bark was performed by semipreparative HPLC-UV with an X-Bridge RP C18 column (5 μm, 150 × 19 mm, i.d.; Waters). The flow rate was set at 17 mL/min, and the injection volume was 200 μL (20 mg of the extract). The solvent system used was H2O (A) and MeOH (B), with both of them containing 0.01% formic acid. The mobile phase gradient used was 40% to 64% B in 27 min, 64% to 78% B in 45 min, 78% to 100% B in 54 min, keeping 100% B until 60 min. In order to localize the chromatogram zone containing bioactive constituents, fractions were collected every minute. Each separation yielded 60 fractions. Fractions obtained were evaluated in an antifungal bioautography assay according to the methodology described by Favre-Godal et al.10 Using this procedure, it was possible to correlate the antifungal activity to two chromatographic peaks at the retention time 39.5 min (17) and 41.8 min (18). Purification of Compounds 17 and 18 from the Dichloromethane Fraction of the Leaves Using Semipreparative HPLC. The purification of the CH2Cl2 fraction of the leaves was performed by semipreparative HPLC-UV with an X-Bridge RP C18 column (5 μm, 150 × 19 mm, i.d.; Waters). The flow rate was set to 17 mL/min, and the injection volume was 200 μL (five injections, 20 mg of the extract). The solvent system used was H2O (A) and MeOH (B), with both of them containing 0.01% formic acid. The gradient used for the separation of the leaf CH2Cl2 fraction was 40% to 70% B in 48 min, 70% to 100% B in 54 min, keeping 100% B until 60 min. Using this approach, two compounds were isolated in one step with the following yields: 17 (5.2 mg) and 18 (6.4 mg). Purification of the Dichloromethane Fraction of the Stem Bark Using MPLC and Semipreparative HPLC. The stem bark CH2Cl2 fraction (6 g) was fractionated by MPLC using Zeoprep C18 as stationary phase (15−25 μm, 920 × 49 mm i.d., Zeochem) and an acidic (0.1% formic acid) H2O (A) and MeOH (B) gradient: 45% B in 3.5 h, 45% to 80% B in 14.5 h, 80% to 100% B in 8 h, and 100% B for 1 h. These conditions were optimized on an HPLC column (15−25

(Flawil, Switzerland). The system was controlled by the software Sepacore Control. Semipreparative HPLC was performed on a Spot Prep Armen instrument (Saint-Avé, France). UHPLC-TOF-HRESIMS Analysis. UHPLC-TOF-HRESIMS metabolite profiling was performed on a Micromass-LCT Premier timeof-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 2450 V, cone voltage 40 V, MCP detector voltage 2400 V, source temperature 120 °C, desolvation temperature 300 °C, cone gas flow 20 L/h, and desolvation gas flow 800 L/h. Detection was performed in the positive-ion mode (PI) with an m/z range of 100−1300 Da and a scan time of 0.3 s in the W-mode. The MS was calibrated using sodium formate. Leucine enkephalin (Sigma-Aldrich, 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 (1.7 μm, 150 mm × 2.1 mm i.d.; Waters), using a gradient (solvent system: A = 0.1% formic acid−water, B = 0.1% formic acid−acetonitrile; gradient: 5−95% B (0−30 min), 95% B (30−40 min), 95−5% B (40−40 min), and 5% B (40−50 min); flow rate 0.46 mL/min). The temperatures in the autosampler and in the column oven were fixed at 10 and 40 °C, respectively. The injection volume was constant (2 μL). Data-Dependent UHPLC-HRESIMS2 Analysis. Chromatographic separation was performed on a Thermo Dionex Ultimate 3000 UHPLC system interfaced to a Q-Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. The LC conditions were as follows: BEH C18 column (1.7 μm, 100 × 2.1 mm i.d.; Waters); mobile phase: (A) water with 0.1% formic acid; (B) acetonitrile with 0.1% formic acid; flow rate 600 μL/min; injection volume 1 μL; linear gradient of 2−98% A over 8 min. Full-scan MS analysis was performed in the positive and negative modes with a mass range of 150−1300 amu at a resolution of 70 000 full width at half-maximum (fwhm) (at m/z 200). The ion injection time used was 200 ms. In the positive mode, the diisooctyl phthalate C24H38O4 [M + H]+ ion (m/z 391.28429) was used as internal lock mass. The optimized HESI-II parameters were the following: source voltage 4.0 kV (PI), sheath gas flow rate (N2) 50 units; auxiliary gas flow rate: 12 units; spare gas flow rate 2.5; capillary temperature 266.25 °C (PI); S-Lens RF level 50. The mass analyzer was calibrated according to the manufacturer’s directions using a mixture of caffeine, methionine-arginine-phenylalanine-alanine-acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an acetonitrile−MeOH−water solution containing 1% acetic acid by direct injection. The datadependent MS2 events were performed on the five most intense ions detected by full-scan MS (Top5 experiment). The isolation window width was 1 m/z for the MS2, and the normalized collision energy was set to 60, 80, and 100 units. In the data-dependent MS2 experiment, full scans were acquired at a resolution of 35000 fwhm and maximum injection time of 50 ms, and MS2 scans at 17 500 fwhm and maximum injection time of 50 ms. After being acquired in the MS2 scan, parent ions were placed in a dynamic exclusion list for 5.0 s. MS Data Processing. ThermoRAW data were converted to mzXML using ProteoWizzard.38 Data treatment and the dereplication process were performed using mzMine 2.14.2.39 The Dictionary of Natural Products was used as the database for the dereplication (http://dnp.chemnetbase.com/).15 The conversion of the proprietary files to open MS format (.mzXML) was performed using ProteoWizzard.38 The data treatment steps (cropping, peak-picking, chromatogram deconvolution, and isotopic filtering) were performed using MzMine 2.14.240 and allowed to generate a list of 1098 features (characterized by a given m/z at a specific retention time). Generation of the Molecular Network and ISDB Spectral Library Search. A molecular network was created using the online workflow at Global Natural Products Social molecular networking (GNPS) (http://gnps.ucsd.edu). The data were then clustered with MS-Cluster with a parent mass tolerance of 1.0 Da and an MS2 fragment ion tolerance of 0.5 Da to create consensus spectra. F



Article

μm, 250 × 4.6 mm i.d., Zeochem, Uetikon am See, Switzerland) packed with the same stationary phase. The extract, prepared by mixing 6 g of the CH2Cl2 fraction from the stem bark with 10 g of 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 10 mL/ min, and UV detection was performed at 254 nm. The MPLC separation yielded 95 fractions, which were analyzed by UHPLC-TOFHRESIMS in order to localize the potential new indolopyridoquinazoline alkaloids. Using this strategy, fractions 78 and 85 were selected for purification by normal-phase semipreparative HPLC-UV using an SIHP column (7 μm, 10 × 250 mm i.d.; Interchim, Montluçon, France) with n-hexane−EtOAc as the solvent system for a linear gradient elution. The flow rate was 5 mL/min, and UV absorbance was detected at 254 nm. Fraction 78 (124.8 mg) was purified with a linear gradient (35% to 55% EtOAc in 47 min) and led to the isolation of alkaloids 19 (25.9 mg) and 20 (4.3 mg). Fraction 85 (45.8 mg) was purified with a linear gradient (20% to 32% EtOAc in 36 min) and afforded alkaloids 21 (4.6 mg) and 22 (3.1 mg). Using the same approach, fraction 57 was selected and purified by semipreparative HPLC-UV on an X-Bridge C18 column (5 μm, 19 × 150 mm i.d.; Waters) with acidified MeOH−H2O (0.1% formic acid) as mobile phase system. The separation was performed by isocratic elution with 46% MeOH in 60 min. The flow rate was 10 mL/min, and the UV absorbance was detected at 254 nm. Purification of fraction 57 yielded 4 (5.3 mg) and 5 (3.5 mg). Fraction 13 (146.5 mg) was purified using the same column by isocratic elution with 40% MeOH in 60 min and yielded 12 (19.2 mg), 14 (4.1 mg), and 15 (2.5 mg). Fractions 42 to 44 were combined and purified by semipreparative HPLC-UV on an X-Bridge C18 column (5 μm, 19 × 150 mm i.d.; Waters) with acidified MeOH−H2O (0.1% formic acid) as mobile phase system. The separation was performed by isocratic elution with 46% MeOH in 60 min. The flow rate was 10 mL/min, and the UV absorbance was detected at 254 nm. Purification of fraction 42/44 yielded 16 (9.2 mg). The MPLC fractions 8, 20, and 68 were found to contain pure compounds and yielded, respectively, 1 (17.3 mg), 6 (87.7 mg), and 18 (83.8 mg). MPLC fractions 4, 5, 6, 7, 10, and 13, containing other polar metabolites, were selected for further purification using the reversed-phase semipreparative HPLC-UV conditions described above. Fraction 4 (129.1 mg) was purified in the isocratic mode with 36% MeOH and yielded 2 (2.7 mg); fraction 5 (135.4 mg) was purified with 30% MeOH and yielded 3 (17.2 mg) and 8 (1.2 mg); fraction 6 (41.7 mg) was purified with 29% MeOH to give 9 (5.2 mg); fraction 7 (75.8 mg) was purified with 38% MeOH and yielded 10 (4.2 mg); fraction 10 (81.5 mg) was purified with 42% MeOH and yielded 11 (12.4 mg) and 13 (4.1 mg); fraction 13 (146.5 mg) was purified with 40% MeOH to give 12 (19.2 mg), 14 (4.1 mg), and 15 (2.5 mg); fraction 18 (28.8 mg) was purified with 36% MeOH and yielded 7 (3.1 mg); and finally fraction 63 (64.5 mg) was purified with 50% MeOH and yielded 17 (4.1 mg). 10,18-Dihydroxyrutaecarpine (5): amorphous powder; UV (MeOH) λmax (log ε) 338 (8.9) nm; 1H NMR (CD3OD, 500 MHz) δ 3.14 (2H, t, J = 6.8 Hz, H-6), 4.52 (2H, t, J = 6.8 Hz, H-5), 6.85 (1H, dd, J = 8.8, 2.3 Hz, H-11), 6.95 (1H, d, J = 2.3 Hz, H-9), 7.28 (1H, dd, J = 8.7, 2.8 Hz, H-17), 7.31 (1H, d, J = 8.8 Hz, H-12), 7.54 (1H, d, J = 2.8 Hz, H-19), 7.59 (1H, d, J = 8.7 Hz, H-16); 13C NMR (CD3OD, 126 MHz) δ 20.2 (C-6), 42.4 (C-5), 103.8 (C-9), 110.6 (C-19), 113.5 (C-12), 116.5 (C-11), 117.4 (C-7), 122.7 (C-20), 125.0 (C-17), 127.1 (C-8), 128.6 (C-2), 129.2 (C-16), 135.0 (C-13), 142.4 (C-15), 144.1 (C-3), 152.2 (C-10), 157.1 (C-18); HRESIMS m/z 320.1036 [M + H]+ (calcd for C18H14N3O3, 320.1035) (Δppm = 0.3). Conchacarpine A (16): amorphous powder; UV (MeOH) λmax (log ε) 255 (3.9), 339 (3.9), 366 (3.8) nm; 1H NMR (CD3OD, 500 MHz) δ 1.49 (6H, s, CH3-25, 26), 3.51 (2H, t, J = 6.9 Hz, H-6), 3.99 (3H, s, OCH3-9), 4.32 (3H, s, NCH3-14), 4.51 (2H, t, J = 6.9 Hz, H-5), 6.05 (1H, d, J = 10.3 Hz, H-23), 6.64 (1H, d, J = 7.8 Hz, H-10), 7.18 (1H, d, J = 8.5 Hz, H-12), 7.42 (1H, t, J = 8.5, 7.8 Hz, H-11), 7.44 (1H, d, J = 9.2 Hz, H-17), 7.78 (1H, d, J = 9.2 Hz, H-16), 7.94 (1H, d, J = 10.3 Hz, H-22); 13C NMR (DMSO-d6, 126 MHz) δ 21.6 (C-6), 27.7

(CH3-25, 26), 41.5 (NCH3-14), 43.0 (C-5), 55.8 (OCH3-9), 77.6 (C24), 101.4 (C-10), 106.8 (C-12), 115.1 (C-20), 116.7 (C-8), 119.4 (C16), 119.8 (C-2), 120.4 (C-22), 122.4 (C-19), 126.0 (C-17), 131.6 (C11), 132.2 (C-7), 135.7 (C-23), 136.3 (C-15), 144.8 (C-13), 149.3 (C3), 154.4 (C-18), 157.4 (C-9); HRESIMS m/z 414.1821 [M]+ (calcd for C25H24N3O3, 414.1818) (Δppm = 0.7). Fontanesine A (19): amorphous powder; UV (MeOH) λmax (log ε) 216 (8.6), 348 (8.0), 362.5 (8.4), 382.5 (8.2) nm; 1H NMR (DMSOd6, 500 MHz) δ 1.38 (6H, s, CH3-25, 26), 3.29 (2H, t, J = 6.8 Hz, H6), 4.41 (2H, t, J = 6.8 Hz, H-5), 5.74 (1H, d, J = 9.7 Hz, H-23), 6.74 (1H, d, J = 8.7 Hz, H-11), 6.86 (1H, d, J = 9.7 Hz, H-22), 7.23 (1H, d, J = 8.7 Hz, H-12), 7.28 (1H, dd, J = 8.8, 2.9 Hz, H-17), 7.48 (1H, d, J = 2.9 Hz, H-19), 7.56 (1H, d, J = 8.8 Hz, H-16), 11.62 (1H, s, H-1); 13 C NMR (DMSO-d6, 126 MHz) δ 20.8 (C-6), 27.0 (CH3-25, 26), 40.6 (C-5), 75.0 (C-24), 109.6 (C-19), 112.5 (C-9), 112.6 (C-12), 115.0 (C-11), 115.3 (C-7), 119.2 (C-22), 121.0 (C-8), 121.6 (C-20), 123.8 (C-17), 128.1 (C-16), 128.2 (C-2), 130.0 (C-23), 134.2 (C-13), 140.5 (C-15), 142.5 (C-3), 146.1 (C-10), 155.7 (C-18), 160.2 (C-21); HRESIMS m/z 386.1515 [M + H]+ (calcd for C23H20N3O3, 386.1505) (Δppm = 2.7). Conchacarpine B (20): amorphous powder; UV (MeOH); λmax (log ε) 241 (4.19), 310 (4.11), 358 (4.3) nm; 1H NMR (CD3OD, 500 MHz) δ 1.44 (6H, s, CH3-25, 26), 3.10 (2H, t, J = 6.9 Hz, H-6), 4.43 (2H, t, J = 6.9 Hz, H-5), 5.80 (1H, d, J = 10.2 Hz, H-23), 6.83 (1H, dd, J = 8.8, 2.3 Hz, H-11), 6.91 (1H, d, J = 2.3 Hz, H-9), 7.17 (1H, d, J = 8.7 Hz, H-17), 7.27 (1H, d, J = 8.8 Hz, H-12), 7.45 (1H, d, J = 8.7 Hz, H-16), 7.96 (1H, d, J = 10.2 Hz, H-22); 13C NMR (CD3OD, 126 MHz) δ 20.5 (C-6), 27.7 (CH3-25, 26), 42.3 (C-5), 76.7 (C-24), 104.0 (C-9), 113.7 (C-12), 116.6 (C-20), 116.7 (C-11), 117.8 (C-7), 120.8 (C-19), 122.1 (C-22), 125.3 (C-17), 127.3 (C-8), 128.4 (C-2), 128.7 (C-16), 132.6 (C-23), 135.3 (C-13), 144.7 (C-15), 144.8 (C-3), 152.3 (C-10), 152.8 (C-18), 163.4 (C-21); HRESIMS m/z 386.1503 [M + H]+ (calcd for C23H20N3O3, 386.1505) (Δppm = −0.4). Fontanesine B (21): amorphous powder; UV (MeOH) λmax (log ε) 355.5 (8.4) nm; 1H NMR (DMSO-d6, 500 MHz) δ 1.40 (6H, s, CH325, 26), 3.33 (2H, t, J = 6.9 Hz, H-6), 4.43 (2H, t, J = 6.9 Hz, H-5), 5.77 (1H, d, J = 9.8 Hz, H-23), 6.77 (1H, d, J = 8.7 Hz, H-11), 6.88 (1H, d, J = 9.8 Hz, H-22), 7.25 (1H, d, J = 8.7 Hz, H-12), 7.47 (1H, ddd, J = 8.0, 7.1, 1.2 Hz, H-18), 7.67 (1H, dd, J = 8.3, 1.2 Hz, H-16), 7.81 (1H, ddd, J = 8.3, 7.1, 1.5 Hz, H-17), 8.16 (1H, dd, J = 8.0, 1.5 Hz, H-19), 11.72 (1H, s, H-1); 13C NMR (DMSO-d6, 126 MHz) δ 20.7 (C-6), 27.0 (CH3−25, 26), 40.6 (C-5), 75.0 (C-24), 112.6 (C-9), 112.7 (C-12), 115.6 (C-11), 116.5 (C-7), 119.2 (C-22), 120.6 (C-20), 120.9 (C-8), 125.9 (C-18), 126.4 (C-16), 126.5 (C-19), 127.9 (C-2), 130.1 (C-23), 134.3 (C-17), 134.4 (C-13), 145.2 (C-3), 146.2 (C-10), 147.4 (C-15), 160.5 (C-21); HRESIMS m/z 370.1555 [M + H]+ (calcd for C23H20N3O2, 370.1556) (Δppm = 0.1). Fontanesine C (22): amorphous powder; UV (MeOH) λmax (log ε) 251 (4.38), 335 (4.42), 354 (4.5), 386 (4.48) nm; 1H NMR (DMSOd6, 500 MHz) δ 1.40 (6H, s, CH3-25, 26), 3.35 (2H, t, J = 6.9 Hz, H6), 4.41 (2H, t, J = 6.9 Hz, H-5), 5.78 (1H, d, J = 9.8 Hz, H-23), 6.79 (1H, d, J = 8.7 Hz, H-11), 6.88 (1H, d, J = 9.8 Hz, H-22), 7.20 (1H, dd, J = 7.9, 1.3 Hz, H-17), 7.29 (1H, t, J = 7.9 Hz, H-18), 7.29 (1H, d, J = 8.7 Hz, H-12), 7.55 (1H, dd, J = 7.9, 1.3 Hz, H-19), 9.23 (1H, s, OH-16), 11.40 (1H, s, NH-1); 13C NMR (DMSO-d6, 126 MHz) δ 20.5 (C-6), 26.6 (CH3-25,26), 40.2 (C-5), 74.8 (C-24), 112.1 (C-18), 112.5 (C-9), 115.2 (C-11), 115.3 (C-7), 115.9 (C-19), 116.5 (C-17), 118.8 (C-22), 120.8 (C-20), 121.0 (C-8), 126.2 (C-12), 128.1 (C-2), 129.9 (C-23), 133.8 (C-13), 135.8 (C-15), 143.1 (C-3), 145.9 (C-10), 151.8 (C-16), 160.0 (C-21); HRESIMS m/z 386.1507 [M + H]+ (calcd for C23H20N3O3, 386.1505) (Δppm = 0.5). Yeast Strains. Candida albicans DSY2621 and parent wild-type CAF2-1 (ura3Δ::imm434/URA3) were obtained from Prof. Dominique Sanglard (Institute of Microbiology, University of Lausanne and University Hospital Center). The C. albicans hypersusceptible strain DSY2621 was constructed by targeted deletions of genes encoding membrane efflux transporters (cdr1Δ::hisG/cdr1Δ::hisG, cdr2Δ::hisG/ cdr2Δ::hisG, f lu1Δ::hisG/f lu1Δ::hisG, mdr1Δ::hisG/mdr1Δ::hisG) and calcineurin subunit A (cmp1Δ::hisG/cmp1Δ::hisG-URA3-hisG).41 The yeast strains were maintained on Sabouraud agar (peptone from G



Article

meat, 5.0 g/L; peptone from casein, 5.0 g/L; D-(+)-glucose, 40.0 g/L; agar−agar, 15.0 g/L; Merck) Petri dishes. Bioautography. Inhibitory activity of compounds 17 and 18 against Candida albicans was evaluated using the method described by Fabre-Godal et al.10 The C. albicans strains were cultivated in Sabouraud broth medium overnight at 36 °C. An inoculum of 105 cells/mL (an optical density (OD) equal to 1 at 630 nm, corresponding to approximately l07 cells/mL) was prepared in malt agar (malt extract, 30.0 g/L; peptone from soymeal, 3.0 g/L; agar− agar, 15.0 g/L; Merck). The molten medium was maintained in a water bath at 45 °C. The OD at 630 nm of the C. albicans culture was measured with a UV/vis spectrophotometer (Synergy H1, Biotek, equipped with Gen 5.2 software). Approximately 20 mL of the inoculum (either DSY2621 or CAF2-1) was quickly distributed over the TLC plate surface with a sterile pipet. The solidified TLC plates were incubated overnight at 36 °C in polyethylene boxes lined with moist chromatography paper. The TLC plates were sprayed with an aqueous solution (2.5 mg/mL) of thiazolyl blue ([methylthiazolyl tetrazolium chloride; MTT; Fluka]) and incubated for 6 h at 36 °C. Inhibition zones were observed against a purple background. For the MIQ determination, 10 μL aliquots of different concentrations (from 0.01 to 10 mg/mL in MeOH) of the pure compounds were spotted manually on the TLC plate as well as 10 μL of only MeOH. Then, the TLC plate without elution was submitted to the same procedure described above. The MIQ was defined as the test compound quantity at which inhibition was observed. Miconazole was used as a positive control (MIQ at 0.0006 μg).

■

(5) Bellete, B. S.; de Sa, I. C. G.; Mafezoli, J.; Cerqueira, C. D.; da Silva, M. F. G. F.; Fernandes, J. B.; Vieira, P. C.; Zukerman-Schpector, J.; Pirani, J. R. Quim. Nova 2012, 35, 2132−2138. (6) Groppo, M.; Pirani, J. R.; Salatino, M. L. F.; Blanco, S. R.; Kallunki, J. A. Am. J. Bot. 2008, 95, 985−1005. (7) Pirani, J. R. In Flora Fanerogâmica do Estado de São Paulo; Wanderley, M. G. L.; Shepherd, G. J.; Giulietti, A. M.; Melhem, T. S.; Bittrich, V.; Kameyama, C., Eds.; Hucitec: São Paulo, 2002. (8) Agripino, D. G.; Lima, M. E. L.; Silva, M. R.; Meda, C. I.; Bolzani, V. S.; Cordeiro, I.; Young, M. C. M.; Moreno, P. R. H. Biota Neotrop. 2004, 4, 1−15. (9) Cabral, R. S.; Sartori, M. C.; Cordeiro, I.; Queiroga, C. L.; Eberlin, M. N.; Lago, J. H. G.; Moreno, P. R. H.; Young, M. C. M. Rev. Bras. Farmacogn. 2012, 22, 374−380. (10) Favre-Godal, Q.; Dorsaz, S.; Queiroz, E. F.; Conan, C.; Marcourt, L.; Wardojo, B. P. E.; Voinesco, F.; Buchwalder, A.; Gindro, K.; Sanglard, D.; Wolfender, J. L. Phytochemistry 2014, 105, 68−78. (11) Queiroz, M. M. F.; Queiroz, E. F.; Zeraik, M. L.; Marti, G.; Favre-Godal, Q.; Simoes-Pires, C.; Marcourt, L.; Carrupt, P. A.; Cuendet, M.; Paulo, M. Q.; Bolzani, V. S.; Wolfender, J. L. Phytochem. Lett. 2014, 10, lxxxviii−xciii. (12) Lim, C. S. Y.; Rosli, R.; Seow, H. F.; Chong, P. P. Eur. J. Clin. Microbiol. Infect. Dis. 2012, 31, 21−31. (13) Potterat, O.; Hamburger, M. Nat. Prod. Rep. 2013, 30, 546−564. (14) Wolfender, J. L.; Queiroz, E. F. Chimia 2012, 66, 324−329. (15) Buckingham, J. E. Dictionary of Natural Products on DVD [Ressource electronique]; CRC Press: Boca Raton, FL, 2013; pp 1 DVD-ROM + . (16) Ahmad, S. J. Nat. Prod. 1984, 47, 391−392. (17) Jackson, G. E.; Campbell, W. E.; Davidowitz, B. Spectrosc. Lett. 1990, 23, 971−982. (18) Duraipandiyan, V.; Ignacimuthu, S. J. Ethnopharmacol. 2009, 123, 494−898. (19) Schymanski, E. L.; Jeon, J.; Gulde, R.; Fenner, K.; Ruff, M.; Singer, H. P.; Hollender, J. Environ. Sci. Technol. 2014, 48, 2097−2098. (20) Kind, T.; Fiehn, O. BMC Bioinf. 2007, 8, 105. (21) Cortez, L. E. R.; Cortez, D. A. G.; Fernandes, J. B.; Vieira, P. C.; Ferreira, A. G.; da Silva, M. F. G. F. Heterocycles 2009, 78, 2053−2059. (22) Yang, J. Y.; Sanchez, L. M.; Rath, C. M.; Liu, X. T.; Boudreau, P. D.; Bruns, N.; Glukhov, E.; Wodtke, A.; de Felicio, R.; Fenner, A.; Wong, W. R.; Linington, R. G.; Zhang, L. X.; Debonsi, H. M.; Gerwick, W. H.; Dorrestein, P. C. J. Nat. Prod. 2013, 76, 1686−1699. (23) Kleigrewe, K.; Almaliti, J.; Tian, I. Y.; Kinnel, R. B.; Korobeynikov, A.; Monroe, E. A.; Duggan, B. M.; Di Marzo, V.; Sherman, D. H.; Dorrestein, P. C.; Gerwick, L.; Gerwick, W. H. J. Nat. Prod. 2015, 78, 1671−1682. (24) Klitgaard, A.; Nielsen, J. B.; Frandsen, R. J. N.; Andersen, M. R.; Nielsen, K. F. Anal. Chem. 2015, 87, 6520−6526. (25) Allard, P. M.; Peresse, T.; Bisson, J.; Gindro, K.; Marcourt, L.; Pham, V. C.; Roussi, F.; Litaudon, M.; Wolfender, J. L. Anal. Chem. 2016, 88, 3317−3323. (26) Challal, S.; Queiroz, E. F.; Debrus, B.; Kloeti, W.; Guillarme, D.; Gupta, M. P.; Wolfender, J. L. Planta Med. 2015, 81, 1636−1643. (27) Karim, A.; Fatima, I.; Hussain, S.; Malik, A. Helv. Chim. Acta 2011, 94, 528−533. (28) Yoshihara, T.; Yamaguchi, K.; Takamatsu, S.; Sakamura, S. Agric. Biol. Chem. 1981, 45, 2593−2598. (29) Atta-ur-Rahman; Bhatti, M. K.; Akhtar, F.; Choudhary, M. I. Phytochemistry 1992, 31, 2869−2872. (30) Wattanapiromsakul, C.; Forster, P. I.; Waterman, P. G. Phytochemistry 2003, 64, 609−615. (31) Stocklin, W.; Desilva, L. B.; Geissman, T. A. Phytochemistry 1969, 8, 1565−1569. (32) Elasaad, K.; Alkhatib, R.; Hennebelle, T.; Norberg, B.; Wouters, J. Crystals 2012, 2, 1441−1454. (33) Moulis, C.; Gleye, J.; Fouraste, I.; Stanislas, E. Planta Med. 1981, 42, 400−402.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00379. Additional information (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel (E. F. Queiroz): +41 223793641. Fax: +41 223793399. Email: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors extend their gratitude to the São Paulo State Research Foundation (FAPESP) for fellowship support (BEPE process no. 2013/05480-0) to R.S.C. The authors are thankful to Dra. I. Cordeiro from the Botanical Institute of Estado de São Paulo for the botanical identification, Dr. Q. Favre-Godal for his advice on the antifungal assays, and Dr. M. M. F. Queiroz for the UV measurements. The authors are thankful to the Swiss National Science Foundation for providing financial support for this project, which aims to identify new antifungal compounds of natural origin (Grant CR2313_143733 to J.L.W. and E.F.Q.).

■

REFERENCES

(1) Kallunki, J.; Pirani, J. R. Kew Bull. 1998, 53, 257−334. (2) Pirani, J. R. In Lista de Espécies da Flora do Brasil, Jardin Botânico do Rio de Janeiro, Rio de Janeiro, Brazil; available at http:// floradobrasil.jbrj.gov.br, 2010. Accessed on April 27, 2016. (3) Vieira, P. C.; Kubo, I.; Kujime, H.; Yamagiwa, Y.; Kamikawa, T. J. Nat. Prod. 1992, 55, 1112−1117. (4) Ambrozin, A. R. P.; Vieira, P. C.; Fernandes, J. B.; Fernandes, M. F. G. Quim. Nova 2008, 31, 740−743. H



Article

(34) Biavatti, M. W.; Vieira, P. C.; da Silva, M. F. D. F.; Fernandes, J. B.; Victor, S. R.; Pagnocca, F. C.; Albuquerque, S.; Caracelli, I.; Zukerman-Schpector, J. J. Braz. Chem. Soc. 2002, 13, 66−70. (35) Ito, A.; Shamon, L. A.; Yu, B. Y.; Mata-Greenwood, E.; Lee, S. K.; Van Breemen, R. B.; Mehta, R. G.; Farnsworth, N. R.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. J. Agric. Food Chem. 1998, 46, 3509−3516. (36) Hu, C. Q.; Li, K. K.; Yang, X. W. J. Asian Nat. Prod. Res. 2012, 14, 634−639. (37) Sakakibara, I.; Ikeya, Y.; Hayashi, K.; Okada, M.; Maruno, M. Phytochemistry 1995, 38, 1003−1007. (38) Kessner, D.; Chambers, M.; Burke, R.; Agus, D.; Mallick, P. Bioinformatics 2008, 24, 2534−2536. (39) Pluskal, T.; Castillo, S.; Villar-Briones, A.; Oresic, M. BMC Bioinf. 2010, 11, 395. (40) Katajamaa, M.; Miettinen, J.; Oresic, M. Bioinformatics 2006, 22, 634−636. (41) Pascual, A.; Nieth, V.; Calandra, T.; Bille, J.; Bolay, S.; Decosterd, L. A.; Buclin, T.; Majcherczyk, P. A.; Sanglard, D.; Marchetti, O. Antimicrob. Agents Chemother. 2007, 51, 137−143.

I


Targeted Isolation of Indolopyridoquinazoline Alkaloids from

Recommend Documents