Structure-Based Molecular Networking for the Target Discovery of

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Structure-Based Molecular Networking for the Target Discovery of Oxahomoaporphine and 8‑Oxohomoaporphine Alkaloids from Duguetia surinamensis

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Weider H. P. Paz,†,‡ Rodolfo N. de Oliveira,‡ Gabriel Heerdt,§ Célio F. F. Angolini,⊥ Lívia S. de Medeiros,∥ Valdenizia R. Silva,□ Luciano S. Santos,□ Milena B. P. Soares,□ Daniel P. Bezerra,□ Nelson H. Morgon,# Jackson R. G. S. Almeida,¶ Felipe M. A. da Silva,‡ Emmanoel V. Costa,*,‡ and Hector H. F. Koolen*,† †

Metabolomics and Mass Spectrometry Research Group, Amazonas State University, Manaus 690065-130, Brazil Department of Chemistry, Federal University of Amazonas, Manaus 69077-000, Brazil § Department of Chemistry, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil ⊥ Center of Human and Natural Sciences, Federal University of ABC, 09210-580 Santo André, Brazil ∥ Department of Chemistry, Federal University of São Paulo, 09920-540 Diadema, Brazil □ Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador 40296-710, Brazil # Institute of Chemistry, University of Campinas, Campinas 13083-970, Brazil ¶ Center for Study and Research of Medicinal Plants, Federal University of Vale do São Francisco, Petrolina 56304-205, Brazil ‡

S Supporting Information *

ABSTRACT: In addition to seven known alkaloids (2, 6−11) and 1,2,4trimethoxybenzene (1), three isoquinoline-derived alkaloids (3−5), namely, duguetinine (3), a compound based on an unprecedented oxahomoaporphine scaffold, and two new 8-oxohomoaporphine alkaloids, duguesuramine (4) and 11-methoxyduguesuramine (5), and a new asarone-derived phenylpropanoid (10) were isolated from the bark of Duguetia surinamensis. The isolation workflow was guided by HPLC-HRESIMS/MS and molecular networking-based analyses. Twenty-four known alkaloids were dereplicated from the D. surinamensis alkaloid-rich fraction network and were assigned by manual MS/MS interpretation. Their cytotoxic potential was evaluated.

he Amazon region is a remarkably iconic center of flora diversity and endemism. An example is the family Annonaceae, for which the taxa are frequent components of tropical rain forests worldwide and comprise more than 34 genera and 2450 species in the neotropics.1 Many of them, such as Annona cherimola (cherimoya), A. squamosa (sugarapple), A. montana (mountain soursop), A. mucosa (wild sugarapple), and A. muricata (soursop), have economic importance.2 In addition, several other genera, including Bocageopsis,3 Duguetia,4 Onychopetalum,5 Unonopsis,6 Uvaria,7 and Xylopia,8 are of interest due to the presence of biologically active compounds. Among these genera, Duguetia comprises 94 species, with 66 of them occurring in Brazilian Amazonia and Cerrado ecosystems.9 Several metabolites from this genus have been described, mainly alkaloids such as benzylisoquinolines, aporphines, oxoaporphines, tetrahydroprotoberberines, protoberberines, and azahomoaporphines.10 Duguetia surinamensis R. E. Fries (synonym D. caudata), known popularly as “araticum” and “biribarana”, is distributed

T

© XXXX American Chemical Society and American Society of Pharmacognosy

throughout the whole of Amazonia and the Guyanas.11 To date, this species has not been studied chemically and biologically. In a continuing effort to study the phytochemistry of the Amazonian Annonaceae,12−14 three new alkaloids with unprecedented oxahomoaporphine (3) and 8-oxohomoaporphine (4 and 5) skeletons and a phenylpropanoid (10) were obtained together with seven (1, 2, 6−9, and 11) known compounds, all isolated from the bark of D. surinamensis (Figure 1).

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RESULTS AND DISCUSSION To prioritize the isolation of the previously undescribed alkaloids, the alkaloid-rich extract of D. surinamensis trunk bark was analyzed by a high-performance liquid chromatography instrument coupled to high-resolution electrospray ionization Received: March 28, 2019

A

DOI: 10.1021/acs.jnatprod.9b00287 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Chemical structures of compounds 1−11.

Figure 2. Annotation of the molecular networking of the alkaloid-rich fraction derived from D. surinamensis bark methanol extract, which shows aporphines (blue dots), benzylisoquinolines (orange dots), tetrahydroisoquinolines (dark green dots), and oxoaporphines (red dots). Silver dots represent nonalkaloid and/or chimeric ions, and light green dots are from tetrahydroprotoberberine-like compounds. New compounds duguetinine and 11-methoxyduguesuramine were targeted upstream during the initial interpretation of product ion spectra from HPLC-HRESIMS/MS analysis and were further isolated.

Analysis of the molecular network revealed that aporphine alkaloids (blue nodes) are the main group in the alkaloid extract of D. surinamensis (Figure 2). Several product ion spectra within this group of the network could not be assigned to known structures due to different patterns of neutral losses. In particular, the precursor ions at m/z 356.1 and m/z 366.1 were potentially of interest for MS-guided isolation due to the absence of similar spectra in our database, the alkaloid gasphase fragmentation literature, and the libraries available at GNPS. Further analysis enabled the annotation of 24 nodes according to their HRMS spectra and typical MS/MS

tandem mass spectrometry (HPLC-HRESIMS/MS). Then, MS/MS data were visualized by molecular networking and subsequently dereplicated by comparison with data from previously published papers15−18 and spectra from our inhouse Annonaceae alkaloid database. In the generated molecular network (MN), several clusters of connected nodes were observed, from which the largest (95 nodes) was of interest (Figure 2) due to the presence of matches with known alkaloids. In the MN, the different patterns of product ion spectra for the alkaloids of D. surinamensis (Supporting Information) enabled the arrangement into groups representing different classes of isoquinoline-derived alkaloids. B



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Table 1. NMR Spectroscopic Data (1H 500 MHz, 13C 125 MHz) for Compounds 3−5 duguetinine (3)a δC, type

position 1 1a 2 3 3a 3b 4

141.7, C 120.6, C 147.3, C 107.9, CH 129.2, C 125.5, C 29.4, CH2

5

43.8, CH2

6a 7 8

84.3, CH

8a 9 10 11 12 12a (OCH2O)1-2

128.4, 112.3, 148.8, 148.6, 111.6, 127.3, 100.9,

OCH3-10 OCH3-11 N-CH3

56.0, CH3 56.1, CH3 41.1, CH3

66.7, CH2 C CH C C CH C CH2

duguesuramine (4)a

δH mult. (J in Hz)

6.67, s

Hα: 2.71, Hβ: 3.01, Hα: 3.22, Hβ: 2.67, 4.72, s

dd (3.7, 16.7) ddd (6.8, 12.5, 16.7) dt (3.7, 12.5) dd (6.8, 12.5)

Hα: 4.22, d (11.5) Hβ: 4.44, d (11.5) 6.96, s

7.20, s Hα: 5.93, d (1.3) Hβ: 6.07, d (1.3) 3.94, s 3.93, s 2.55, s

δC, type

δH mult. (J in Hz)

11-methoxyduguesuramine (5)a δC, type

δH mult. (J in Hz)

6.63, s

3.11, m

142.9, C 111.6, C 143,7, C 108.9, CH 129.5, C 126.6, C 36.0, CH2

55.7, CH2

3.68, m

55.6, CH2

3.66, m

106.3, C 152.4, CH 183.5, C

8.24, s

105.8, C 152.0, CH 182.9, C

8.23, s

143.0, C 111.6, C 143.9, C 108.6, CH 129.4, C 125.9, C 36.0, CH2

6.64, s

133.0, 108.1, 158.7, 120.6, 128.8, 127.4, 100.6,

C CH C CH CH C CH2

7.93, d (2.9) 7.21, dd (2.9, 8.9) 8.63, d (8.9) 6.09, s

55.4, CH3

3.94 s

47.4 CH3

3.31, s

125.8, 107.8, 148.6, 152.2, 108.9, 128.5, 100.6,

C CH C C CH C CH2

55.9, CH3 56.0, CH3 47.3, CH3

3.09, m

7.92, s

8.21, s 6.09, s 4.03, s 4.02, s 3.29, s

a

Recorded in CDCl3.

The targeted fractionation of the alkaloid-rich fraction of D. surinamensis allowed the purification of three unprecedented alkaloids (3−5) and one new phenylpropanoid (10), which were confirmed by extensive structure elucidation, in addition to seven (1, 2, 6−9, and 11) known compounds. Compound 3 was obtained as a brown, amorphous powder, [α]25 D +40.1 (c 0.1, MeOH). The protonated molecular ion at m/z 356.1502 [M + H]+ (calcd for C20H22NO5, 356.1492) in the HRESIMS, together with the 13C NMR spectroscopic data, supported a molecular formula of C20H21NO5 (11 degrees of unsaturation). The 1H NMR spectrum of 3 showed aromatic, methylenedioxy bridge, methoxy group, methylene, and methyl hydrogen resonances, which are indicative of an aporphine derivative12 (Table 1). The 1H NMR spectra showed proton signals at δΗ 6.07 (d, J = 1.3 Hz) and 5.93 (d, J = 1.3 Hz) connected to the carbon at δC 100.9 as confirmed by HSQC to indicate a methylenedioxy bridge, common in aporphinoids.29 The COSY correlations among δΗ 2.71 and 3.01 (H-4) with δΗ 2.67 and 3.22 (H-5), in addition to the key HMBC correlations of δΗ 6.67 (s, H-3) with δC 141.7 (C-1), 125.5 (C-3b), and 29.4 (C-4) and correlations of δΗ 4.72 (s, H-6a) with δC 120.6 (C-1a) and 129.2 (C-3a) confirmed a tetrahydroisoquinoline moiety (rings A and B) (Figure 3A). A dihydrobenzo[c]oxepine core (ring C) was found to be linked to the isoquinoline system. This observation was possible according to the HMBC correlation between δΗ 4.22 and 4.44 (d, 11.5 Hz) with δC 128.4 (C-8a) and 127.3 (C-12a), from δΗ 6.96 (s, H-9) with δC 66.7 (C-8), and from the N-methyl group at δΗ 2.55 (s) with δC 84.3 (C-6a) (Figure 3A). This set of data led us to propose an unprecedented subtype of aporphine alkaloid named oxahomoaporphine related to previously described azahomoaporphines from

fragmentation already described for the main isoquinolines found in Annonaceae, such as benzylisoquinolines, aporphines, and oxoaporphines.15−18 Moreover, the first neutral losses of these compounds were analyzed to annotate them according to the substitution pattern of the nitrogen atom of the isoquinoline moiety. Therefore, nonsubstituted (−17 Da), Nmethyl (−31 Da), N-formyl (−45 Da), and N-methyl N-oxide (−47 Da) analogues (Figure 2) were dereplicated among the different formed clusters. Since the fragmentation of N-methyl N-oxide-derivatives was not discussed so far, a mechanistic proposal is provided herein (Figure 2). The dereplicated alkaloids were the benzylisoquinolines coclaurine (12a, m/z 286.139),18 N-methylcoclaurine (12b, m/z 300.155),18 O-methylarmepavine (12c, m/z 328.185),19 reticuline (12d, m/z 330.165),18 N,O-dimethylcoclaurine (12e, m/z 314.170), 18 N-formylreticuline (12f, m/z 344.149), 20 N-methylcoclaurine N-oxide (12g, m/z 316.149),21 and reticuline N-oxide (12h, m/z 346.160),21 the oxoaporphines subsessiline (13a, m/z 338.101)17 and 9methoxy-O-methylmoschatoline (13b, m/z 338.112),17 the aporphines asimilobine (14a, m/z 268.129),15,22 laurotetanine (14b, m/z 328.149),15 norglaucine (14c, m/z 342.164),15 Nmethylasimilobine (14d, m/z 282.144),15 N-methylaurotetanine (14e, m/z 342.164),15 isoboldine (14f, m/z 328.149),15 glaucine (14g, m/z 356.181),15 isolaureline (14h, m/z 310.139),15 arcabucoine (14i, m/z 354.164),23 N-formylglaucine (14j, m/z 370.195),24 N-methylasimilobine N-oxide (14k, m/z 298.139),25 isolaureline N-oxide (14l, m/z 326.134),26 and stepharine (14m, m/z 298.139),27 and the tetrahydroisoquinoline heliamine (15, m/z 194.113) 28 (Supporting Information). C



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replaced by an olefin signal at δΗ 8.24 (s, H-7) and a typical signal of an α,β-unsaturated ketone at δC 183.5 (C-8), indicative of a cyclohept-2-en-1-one moiety (ring C). The substitution pattern of ring D was deduced from the typical ortho constant coupling of 8.9 Hz between δΗ 7.21 (dd, H-11) and 8.63 (d, H-12), in addition to key HMBC correlations (Figure 4A). Therefore, compound 4 was designated as a previously undescribed 8-oxohomoaporphine alkaloid and was named duguesuramine.

Figure 4. Key 2D NMR correlations of compounds (A) 4 and (B) 5.

Compound 5 was obtained as an orange, amorphous powder. Its molecular formula was determined to be C21H19NO5 (13 degrees of unsaturation) from the HRESIMS data (m/z 366.1345 [M + H]+, calcd 366.1336). The 1H and 13 C NMR spectra (Table 1) were similar to those of 4. The main differences in the NMR data were the absence of the C11 hydrogen at δΗ 7.21 (dd) present in 4, which was replaced by a methoxy group signal at δΗ 4.02 (s). The substitution pattern of ring D was confirmed by the key HMBC correlations of OCH3-11, δΗ 7.92 (s, H-9), and δΗ 8.21 (s, H-12) resonances with δC 152.2 (C-11) (Figure 4B). Therefore, compound 5 was designated as a previously undescribed 8-oxohomoaporphine alkaloid and was named 11-methoxyduguesuramine. Compound 10 was obtained as a white, amorphous powder, 1 [α]25 D +71.6 (c 0.3, CHCl3). The H NMR spectrum of 10 showed aromatic and methoxy group hydrogen resonances in addition to methylene and methine (both oxygenated signals), indicative of a tetrasubstituted benzenoid derivative.32 The planar structure of this substance was found to be 1-(2,4,5trimethoxyphenyl)ethane-1,2-diol based on 13C NMR resonances33,34 and key HMBC correlations of the methoxy group hydrogens with the arene carbons (Figure 5A). The proposed structure was supported by the deduced chemical formula of C11H16O5Na (4 degrees of unstauration) from the HRESIMS data (m/z 251.0893 [M + Na]+, calcd 251.0889) of 10 (m/z 251 [M + Na]+). The specific rotation of 10 was dextrorotatory, indicating the α-orientation of the C-7-hydroxy group. Optical rotation calculations carried out at the same level of theory (TD-DFT) using the CAM-B3LYP/6-311+ +G(d,p) basis set showed that the theoretical R isomer agreed with the experimental data ([α]25 D +70.8, Δ = 0.83) (Figure 5B). Moreover, the coupling values (J7,8 = 8.0 Hz)34 (Figure 5A) indicated that the absolute configuration of 10 was 7R. Therefore, compound 10 was designated as a new asaronederived phenylpropanoid and was named (+)-surinasarone. By NMR data analysis and comparison with the reported spectroscopic data, the seven known compounds were identified as 1,2,4-trimethoxybenzene (1),33 dicentrine (2),35

Figure 3. (A) Key 2D NMR correlations of compound 3. (B) Experimental ECD and calculated ECD spectra of 3.

Guatteria sagotiana and Meiogyne virgata.30 The substitution pattern of ring D was deduced by the absence of multiplicity for H-9 and H-12, the carbon resonances of C-10 and C-11 (δC 148.8 and 148.6, respectively), and HMBC correlations of H-9 with C-8 and C-11 and of H-12 with C-1a (Figure 3A). The configuration of 3 was assigned via electronic circular dichroism (ECD) with the aid of polarimetry measurements. The ECD spectrum of 3 displayed a negative Cotton effect at λ 229 nm (Δε −204) (Figure 3B). Since ECD data for oxahomoaporphines are not available, a screening of two enantiomeric structural possibilities was performed via a timedependent density functional theory (TD-DFT) approach using the triple-ζ Pople basis set [6-311++G(d,p)].31 The agreement between the experimental and calculated spectra (Figure 3B) and the dextrorotatory behavior in MeOH led to the assignment of the absolute configuration of C-6a as S. Therefore, compound 3 was designated as a previously undescribed oxahomoaporphine alkaloid and was named duguetinine. Compound 4 was obtained as an orange, amorphous powder. Its molecular formula was determined to be C20H17NO4 (13 degrees of unsaturation) from the HRESIMS data (m/z 336.1233 [M + H]+, calcd 336.1230). The 1H and 13 C NMR spectra (Table 1) were similar to those of 3. The main differences in the NMR data were related to the absence of the C-6a hydrogen at δΗ 4.72 present in 3, which was D



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compound 7 in the HCT-8, SF-295, and MDA/MB435 tumor cell lines, respectively, and IC50 values of 1.9, 1.6, and 7.3 μM for compound 11 in the same cell lines, respectively. The cytotoxic potential of compound 6 has not been previously investigated (Table 3).

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific optical rotations were measured with a JASCO P-2000 polarimeter. ECD spectra were obtained on a JASCO J-720 spectrometer, and measurements were performed by combining 15 scans at a scanning speed of 50 nm min−1 from 100 to 450 nm with a bandwidth of 1 nm.12 1D and 2D NMR experiments were acquired in CDCl3 at 293 K on a Bruker AVANCE III HD NMR spectrometer operating at 11.75 T (1H and 13C at 500 and 125 MHz, respectively). All 1H and 13C NMR chemical shifts (δ) are presented in ppm relative to the tetramethylsilane signal at 0.00 ppm as an internal reference, and the coupling constants (J) are given in Hz. LC-MS analysis was performed with an Agilent iFunnel Q-ToF 6550 LC-MS system. HPLC separations were performed on an analytical C18 column (Poroshell 120 SB-Aq 2.7 μm, 2.1 × 100 mm, Agilent). Elution was conducted with a mobile phase consisting of 0.1% formic acid solution (A) and MeOH + 0.1% formic acid (B) following the gradient 5% B held for 3 min, 5% to 100% B for 17 min, then maintaining 100% B for 3 min (not acquired) at a flow rate of 350 μL/min. Column re-equilibration was performed in 5% B for 3 min. Column chromatography was performed on silica gel (Merck, 70−230 mesh). TLC analysis (analytical and preparative) was performed using precoated silica gel 60 F254 plates (0.25 mm, Merck), and spots were visualized by exposure under UV254/365 light, by spraying with p-anisaldehyde reagent followed by heating on a plate, or by spraying with Dragendorff’s reagent. Plant Material. The trunk bark from a flowering plant of Duguetia surinamensis R. E. Fries was collected in June 2015 on the Adolpho Ducke Reserve (02°55′37.4″ S, 059°58′36.0″ W), Manaus, Amazonas State, Brazil, and identified at the Herbarium of the National Institute of Research of Amazonia (INPA), where a voucher (number 9282) was deposited. Molecular Networking. Product ion spectra arising from the HPLC-HRESIMS/MS analysis of the alkaloid-rich fraction from D. surinamensis were analyzed and organized in molecular networks by using the GNPS platform (http://gnps.ucsd.edu).47 The MS/MS data were converted to the .mzXML format with MS-Convert40 and then uploaded on the GNPS Web platform. Parameters for molecular network generation were set as follows: the precursor ion mass tolerance of 0.05 Da, product ion tolerance of 0.5 Da, and fragment ions below 10 counts were removed from the MS/MS spectra. Molecular networks were generated using four minimum matched peaks and a cosine score of 0.65. Data were visualized using Cytoscape 3.7.0 software. Dereplication of known alkaloids was performed by manual interpretation of MS/MS spectra in comparison with our in-house Annonaceae alkaloid database. The MS/MS molecular network is accessible at the GNPS Web site with the following link: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task= b1820c8d49ba4ff8bfbf55825d195088. Extraction and Isolation. The dried and powdered bark (2.67 kg) of D. surinamensis was extracted with n-hexane followed by MeOH to yield a lipid-free MeOH extract (340 g). An aliquot of the MeOH extract (300 g) was subjected to an acid−base extraction procedure,14 yielding an alkaloid-containing fraction (16.2 g, 4.76% w/w). A portion (10 g) of the alkaloid fraction was subjected to 10% NaHCO3-treated silica gel column chromatography12 and eluted with gradient systems of n-hexane−CH2Cl2 (100:0 to 10:90), CH2Cl2− EtOAc (100:0 to 10:90), and EtOAc−MeOH (100:0 to 50:50). The eluted fractions were evaluated and pooled according to TLC analysis, resulting in seven major fractions. Fraction 1 (45.2 mg, m/z 356.1), eluted with n-hexane−CH2Cl2 (85:15), was purified by preparative TLC and eluted with CH2Cl2−MeOH (95:5, 3×), to yield 1 (8.3 mg), 2 (21.4 mg), and 3 (1.5 mg). Fraction 3 (4.50 g), eluted with n-

Figure 5. (A) Key 2D NMR correlations of compound 10 and the corresponding Newman projection 2J coupling constant. (B) Optimized structures of possible 7R and 7S enantiomers and calculated specific rotations.

7-hydroxynordicentrine (6),36 (−)-duguetine (7),37 pallidine (8),38 dicentrinone (9),39,40 and duguetine N-oxide (11).37 Unequivocal assignment and full NMR data for compounds 2, 6, 7, 9, and 11 at 500 MHz are made available in Tables S1 and S2 (Supporting Information) since previously published data are outdated and some signals are missing. Compounds 4 and 5 are analogues of the benzyltetrahydroisoquinoline alkaloids and are similar to homoaporphine alkaloids, which contain an extra carbon between the tetrahydroisoquinoline and the pendant aromatic rings. The cycloheptane motif has been proposed to originate from the condensation of dopamine (from tyrosine) and 4-hydroxydihydrocinnamaldehyde (from phenylalanine).41 Further hydroxylation, oxidation, reduction, and methylation steps then result in the substitution patterns for 4 and 5, herein referred to as 8-oxohomoaporphine derivatives. Interestingly, so far, homoaporphines have only been isolated from plants in the family Liliaceae.42,43 On the other hand, compound 3 displayed an unusual oxahomoaporphine skeleton bearing an oxepane moiety. It is plausible that the biosynthetic formation of 3 may occur via hydroxylation of the α′-position of (S)benzylisoquinoline derivatives (e.g., N,O-dimethylcoclaurine) followed by isomerization as a key step to access the oxepane motif (Scheme 1). The inhibitory effects of compounds 1, 2, 4−9, and 11 on the growth of five tumor cell lines and one normal cell line (MRC-5) was measured by the Alamar blue method after 72 h of incubation. The alkaloids 2, 6, 7, and 11 were active, with IC50 values below 10 μM, against all assayed cell lines. 7Hydroxynordicentrine (6) exhibited the most pronounced activities, with IC50 values of 2.0 and 4.0 μM against HL-60 human acute promyelocytic leukemia and HCT116 human colon carcinoma cell lines, respectively. Dicentrine (2) and (−)-duguetine (7) were active with IC50 values of 3.7−5.0 μM against different cell lines. Moreover, duguetine N-oxide (11) showed a considerably lower cytotoxicity (Table 3). In previous studies, compound 2 displayed IC50 values ranging from 16.0 to 55.0 μM in different tumor cell lines.44,45 Silva et al.46 reported the cytotoxic potential of compounds 7 and 11, finding IC50 values of 1.4, 1.4, and 12.4 μM for E



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Scheme 1. Proposed Biosynthetic Pathway of Compounds 3−5

356.1492, Δm/z theoretical = −2.47 ppm); the MS/MS spectrum is deposited in the GNPS spectral library,https://gnps.ucsd.edu/ ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00005436042#%7B%7D. Duguesuramine (4): orange amorphous powder; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 336.1233 [M + H]+ (calcd for C20H18NO4, 336.1230, Δm/z theoretical = −1.24 ppm); the MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.edu/ ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00005436044#%7B%7D. 11-Methoxyduguesuramine (5): orange amorphous powder; for 1 H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 366.1345 [M + H]+ (calcd for C21H20NO5, 366.1336, Δm/z theoretical = −2.21 ppm); the MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.

hexane−CH2Cl2 (60:40), was selected and purified by silica gel CC (treated with 10% NaHCO3 solution) using the same methodology as described above for the initial column chromatography of the alkaloid fraction to give 14 subfractions. Subfraction 3 (140 mg, m/z 366.1), eluted with n-hexane−CH2Cl2 (65:35), was purified by preparative TLC and eluted with CH2Cl2−MeOH (95:5, 3×) to give 4 (0.8 mg) and 5 (2.4 mg). Subfraction 11 (94.5 mg), eluted with CH2Cl2− EtOAc (80:20) after purification by preparative TLC (eluted with CH2Cl2−MeOH, 95:5, 3×), gave 6 (3.4 mg) and 7 (4.8 mg). Subfraction 12 (94.5 mg), eluted with CH2Cl2−EtOAc (65,35), was purified by preparative TLC and eluted with CH2Cl2−MeOH (90,10, 3×) to give 8 (5.0 mg), 9 (17.9 mg), 10 (1.4 mg), and 11 (9.4 mg). Duguetinine (3): brown amorphous powder; [α]25 D +40.1 (c 0.58, MeOH); ECD (c 0.58, MeOH) λmax (Δε) −204 (229); for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 356.1502 [M + H]+ (calcd for C20H22NO3, F



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Table 2. NMR Spectroscopic Data (1H 500 MHz, 13C 125 MHz) for Compound 10

Collection (ATCC). The cells were cultured as recommended by ATCC guidelines, and a mycoplasma stain kit (Sigma-Aldrich) was used to confirm the cells were free from contamination. Cell viability was quantified by the Alamar blue method as previously described50 with minor modifications.51 Doxorubicin (purity ≥95%, doxorubicin hydrochloride, Laboratory IMA S.A.I. C) was used as the positive control. The half-maximal inhibitory concentration (IC50) values with their 95% confidence intervals were obtained by nonlinear regression using GraphPad Prism (Intuitive Software for Science).

(+)-surinasarone (10)a δC, type

position 1 2 3 4 5 6 7 8

119.8, C 150.7, C 97.4, CH 149.1, C 143.2, C 111.2, CH 70.8, CH 66.7, CH2

OCH3-2 OCH3-4 OCH3-5

56.1, CH3 56.2, CH3 56.6, CH3

δH mult. (J in Hz)

6.52, s

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6.95, s 5.00, dd (3.5, 8.0) Hα: 3.77, dd (3.5, 11.1) Hβ: 3.65, dd (8.0, 11.1) 3.82, s 3.88, s 3.85, s

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00287. LC-MS/MS chromatograms, MS/MS spectra of dereplicated compounds, ECD, 1D NMR, 2D NMR, and HRESIMS of compounds 3−5 and 10 (PDF)

a

Recorded in CDCl3.

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Table 3. In Vitro Cytotoxicity of Compounds 1, 2, 4−9, and 11 against Five Human Tumor and Normal Cell Lines compoundb

MCF-7

HCT116

HepG2

HL-60

MRC-5

2 6 7 11 doxorubicinc

>10 10.0 >10 >10 1.6

8.8 4.0 >10 9.2 0.3

>10 9.1 5.0 >10 0.04

3.7 2.0 4.8 9.6 0.04

>10 >10 >10 >10 2.4

AUTHOR INFORMATION

Corresponding Authors

*Tel (E. V. Costa): +5592981388085. E-mail: emmanoelvc@ gmail.com. *Tel (H. H. F. Koolen): +5592981667646. E-mail: hkoolen@ uea.edu.br. ORCID

Gabriel Heerdt: 0000-0003-4573-0616 Lívia S. de Medeiros: 0000-0001-8743-3340 Daniel P. Bezerra: 0000-0002-6774-2063 Jackson R. G. S. Almeida: 0000-0002-0867-1357 Emmanoel V. Costa: 0000-0002-0153-822X Hector H. F. Koolen: 0000-0002-0181-348X

Data are presented as IC50 values in μM and their respective 95% confidence interval obtained by nonlinear regression from at least three independent experiments performed in duplicate as measured by the Alamar blue assay after 72 h of incubation. MCF-7: human breast adenocarcinoma cells; HCT116: human colon carcinoma cells; HepG2: human hepatocellular carcinoma cells; HL-60: human acute promyelocytic leukemia cells; MRC-5: human lung fibroblasts. b Compounds 1, 4, 5, 8, and 9 were inactive (IC50 > 10 μM). c Positive control. a

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to dedicate this work to Prof. Dra. Maria Lúcia Belém-Pinheiro from UFAM, Brazil, for her contributions for more than 30 years to the study of the chemistry of natural product from the Amazon region. We are also grateful to Prof. Dr. A. C. Webber of UFAM for botanical identification, M. Sc. Renan Galaverna from UNICAMP, ́ Brazil, for the ECD measurements, and Central Analitica (UFAM) for the NMR and MS analysis. We acknowledge the funding foundations FAPEAM, CAPES, CNPq, FINEP, and FAPESB.

edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00005436043#%7B%7D. (+)-Surinasarone (10): white, amorphous powder; [α]25 D +71.6 (c 0.3, CHCl3); for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 2; HRESIMS m/z 251.0893 (calcd for C11H16O5Na, 251.0889, Δm/z theoretical = +0.80 ppm). TD-DFT Calculations. Theoretical calculations began with ground-state structure optimization using DFT. The R and S configurations were fully optimized without constraints with the exchange−correlation functional CAM-B3LYP.48 The TD-DFT calculations were carried out from the minimal ground-state structures by the same functions. The triple-ζ Pople basis set 6-311++G(d,p) was used to represent the carbon, oxygen, and hydrogen atoms.31 Solvent effects for methanol were introduced in all the calculations through the SMD continuum solvation method.31 The final energy values for the ground-state structures were corrected by adding the free energy correction at 300 K and 1 atm. All the calculations were performed using the Gaussian 09 suite of programs (revision D1).49 Theoretical ECD spectra were generated by calculating the excitation energies and rotatory strengths (intensities) of each excited state. The calculated rotatory strengths from the first 10 singlet → singlet electronic transitions were simulated into an ECD curve using Gaussian band shapes with half-width at 0.67 eV.49 Cytotoxicity Assay. The MCF-7 (human breast adenocarcinoma), HCT116 (human colon carcinoma), HepG2 (human hepatocellular carcinoma), HL-60 (human acute promyelocytic leukemia), B16-F10 (mouse melanoma), and MRC-5 (human lung fibroblast) cell lines were obtained from the American Type Culture

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