Benzylated Dihydroflavones and Isoquinoline-Derived Alkaloids from the Bark of Diclinanona calycina (Annonaceae) and Their Cytotoxicities

Diclinanona calycina R. E. Fries popularly known as “envira”, is a species of the Annonaceae family endemic to Brazil. In our ongoing search for bioactive compounds from Annonaceae Amazon plants, the bark of D. calycina was investigated by classical chromatography techniques that yielded thirteen compounds (alkaloids and flavonoids) described for the first time in D. calycina as well as in the genus Diclinanona. The structure of these isolated compounds were established by extensive analysis using 1D/2D-NMR spectroscopy in combination with MS. The isolated alkaloids were identified as belonging to the subclasses: simple isoquinoline, thalifoline (1); aporphine, anonaine (2); oxoaporphine, liriodenine (3); benzyltetrahydroisoquinolines, (S)-(+)-reticuline (4); dehydro-oxonorreticuline (3,4-dihydro-7-hydroxy-6-methoxy-1-isoquinolinyl)(3-hydroxy-4-methoxyphenyl)-methanone) (5); (+)-1S,2R-reticuline Nβ-oxide (6); and (+)-1S,2S-reticuline Nα-oxide (7); tetrahydroprotoberberine, coreximine (8); and pavine, bisnorargemonine (9). While the flavonoids belong to the benzylated dihydroflavones, isochamanetin (10), dichamanetin (11), and a mixture of uvarinol (12) and isouvarinol (13). Compound 5 is described for the first time in the literature as a natural product. The cytotoxic activity of the main isolated compounds was evaluated against cancer and non-cancerous cell lines. Among the tested compounds, the most promising results were found for the benzylated dihydroflavones dichamanetin (10), and the mixture of uvarinol (12) and isouvarinol (13), which presented moderate cytotoxic activity against the tested cancer cell lines (<20.0 µg·mL−1) and low cytotoxicity against the non-cancerous cell line MRC-5 (>25.0 µg·mL−1). Dichamanetin (11) showed cytotoxic activity against HL-60 and HCT116 with IC50 values of 15.78 µg·mL−1 (33.70 µmol·L−1) and 18.99 µg·mL−1 (40.56 µmol·L−1), respectively while the mixture of uvarinol (12) and isouvarinol (13) demonstrated cytotoxic activity against HL-60, with an IC50 value of 9.74 µg·mL−1, and HCT116, with an IC50 value of 17.31 µg·mL−1. These cytotoxic activities can be attributed to the presence of one or more hydroxybenzyl groups present in these molecules as well as the position in which these groups are linked. The cytotoxic activities of reticuline, anonaine and liriodenine have been previously established, with liriodenine being the most potent compound.

Although the Annonaceae family is considered a primitive and well-studied family, few phytochemical and/or pharmacological studies have been carried out with its species [3]. Phytochemical and/or pharmacological studies have focused mainly on species of the genera Annona, Asimina and Cananga, due to their great economic importance, and on some species of the genera Duguetia, Guatteria and Xylopia [5]. Despite the great growth in the last 20 years in relation to phytochemical and pharmacological studies, the number of species investigated is still very small in relation to the large number of recognized species. Currently, according to the Web of Science, Scopus, and SciFinder scientific databases, only about 15% of the Annonaceae species described have any corresponding phytochemical and/or pharmacological study.
Among the little studied species are those belonging to the genus Diclinanona Diels. This genus belongs to the tribe Annoneae, of the subfamily Annonoideae, and occurs only in tropical South America (mainly in the Amazon region). It is a genus consisting of only three species, Diclinanona calycina (Diels) R. E. Fries Diclinanona matogrossensis Maas and Diclinanona tessmannii Diels, which occur as trees [22][23][24]. D. calycina (synonymy Xylopia calycina Diels) is an 8 to 30 m tall tree, popularly known as "envireira" and "envira", distributed throughout the Amazon basin in Brazil, Peru and Venezuela [23]. D. calycina is superficially similar to Xylopia for its flowers with elongate and narrow petals, but it is different for its woody, indehiscent, globose and thick-walled monocarps [23,24].
Previous studies with D. calycina report only pharmacological studies. The first study describes the investigation of the antimicrobial activity of methanolic, chloroform and aqueous extracts against the microorganisms Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method [25]. The second one reports the investigation of the antimicrobial activity of organic (dichloromethane:methanol 1:1) and aqueous extracts against the microorganism Enterococcus faecalis using the microdilution broth assay (MDBA) and disk diffusion assay (DDA) [26]. Thus, in our continuous search for new bioactive natural products from Annonaceae from Amazon rainforest, this study aimed to investigate the phytochemical and pharmacological properties of the bark of D. calycina. In this report, thirteen compounds (nine alkaloids and four benzylated dihydroflavones) were isolated and identified for the first time in D. calycina, as well as in the genus Diclinanona. In addition, the cytotoxicity of the main compounds was investigated against B16-F10, HepG2, K562, and HL-60 tumor cell lines using the Alamar blue assay.

Structural Elucidation of the Compounds
Having discovered the presence of nitrogen-containing compounds in the methanolic extract using Dragendorff's reagent, it was subjected to acid-base treatment according to the methodology of Costa et al. [12] resulting in alkaloidal and neutral fractions. A high concentration of nitrogen-containing compounds was observed in the alkaloidal fraction that was subjected to chromatographic analysis. The successive chromatographic separations, as described in the Extraction and Isolation section, led to the isolation and identification of thirteen chemical constituents (1−13, Figure 1), nine isoquinoline-derived alkaloids 1−9 and four benzylated dihydroflavones 10−13. The structures of these isolated compounds ( Figure 1) were established by extensive analysis using 1D and 2D NMR spectroscopy in combination with MS (Supplementary Data), as well as comparison with data from the literature. Compound 5 was obtained as a brown amorphous powder and tested positive for Dragendorff's reagent. It showed a protonated molecule at m/z 328 [M + H] + in the LR-ESI (+)MS compatible with the molecular formula C 18 H 17 NO 5 . The 1 H and 13 C-NMR spectra of 5 (Table 1) were consistent with those of reticuline (4) [27], except for the absence of the nitrogen-bonded methyl group (CH 3 −N) and the signal of the methine group in position 1, which was replaced by a signal at δ C 165.1 in the typical 13 C-NMR spectrum of the imine group conjugated to a carbonyl group at δ C 192.8. In the 1 H-NMR spectrum, three downfield hydrogens at δ H 6.88 (1H, d, J = 8.4 Hz, H-5 ), δ H 7.57 (1H, d, J = 2.0 Hz, H-2 ) and δ H 7.60 (1H, dd, J = 8.4 and 2.0 Hz, H-6 ), revealed the presence of an ABX coupling system. Two singlet signals at δ H 6.90 (1H, s, H-8) and 6.71 (1H, s, H-5) indicated the presence of 1,2,4,5-tetrasubstituted phenyl ring. In addition, the 1 H-NMR spectrum of 5 also showed signals for two methoxy groups resonating at δ H 3.93 (3H, s) and δ H 3.95 (3H, s), and signals for two methylene groups resonating at δ H 2.79 (2H, t, J = 7.8 Hz) and δ H 3.89 (2H, t, J = 7.8 Hz), attributed to H-4 and H-3, respectively (Table 1). These data indicated that alkaloid 5 has a benzyltetrahydroisoquinoline skeleton [28][29][30].
These groups were established based on the two and three-bond 1 H-13 C correlation map from HMBC-NMR experiment ( Figure 2 and Table 1). This analysis revealed that the hydrogen at δ H 3.89 (H-3) showed three-bond 1 H-13 C correlation with the carbons at δ C 130.1 (4a) and δ C 165.0 (C-1) and two-bond 1 H-13 C correlation with the carbon at δ C 25.4 (C-4), confirming the presence of imine group in the molecule. On the other hand, the signals at δ H 7.57 (H-2 ) and δ H 7.60 (H-6 ) showed three-bond 1 H-13 C correlation with the carbons at δ C 124.3 (C-6 ), δ C 151,4 (C-4 ) and δ C 192.8 (C-7 ), and δ C 116.0 (C-2 ), δ C 151.4 (C-4 ) and δ C 192.8 (C-7 ), respectively, establishing the carbonyl group in the molecule ( Figure 2 and Table 1). Therefore, based on these NMR data, compound 5 was established as the benzyltetrahydroisoquinoline alkaloid 3,4-dihydro-7-hydroxy-6methoxy-1-isoquinolinyl)(3-hydroxy-4-methoxyphenyl)-methanone, which was named as dehydro-oxonorreticuline. This alkaloid is described for the first time in the literature as a natural product. Its first and only record was described by Dörnyei et al. in 1982 [31] as a product of synthetic origin. Only the 1 H-NMR data are described with some undetermined multiplicities. Thus, the complete assignments for all 1 H-and 13 C-NMR chemical shifts were established by one-bond (HSQC) and two and three-bond (HMBC) 1 H-13 C-NMR correlation experiments, and were described in the Table 1.  Compounds 6 and 7 were identified as the benzyltetrahydroisoquinoline alkaloids (+)-1S,2R-reticuline-N β -oxide and (+)-1S,2S-reticuline-N α -oxide, respectively. The 1 H-NMR data of these alkaloids were compared with the data described by Lee et al. [28] using the same deuterated solvent (CD 3 OD) and some inconsistencies were observed (Table 1), mainly in relation to H-1, H-8, and H 3 C-NO. There are no 13 C-NMR data described for these alkaloids in the literature. Based on the limited 1 H-and 13 C-NMR data, as well as the ambiguities observed for these molecules, 1 H and 13 C 1D and 2D NMR experiments were performed to determine their correct assignments and multiplicities.
The correct position of the hydrogen H-8 and H-1 of both isomers 6 and 7 was established based on the three-bond 1 H-13 C correlation map from HMBC-NMR experiment ( Figure 2). For the isomer 6 the analysis revealed that the hydrogen at δ H 5.78 (H-8) showed three-bond 1 H-13 C correlation with the carbons at δ C 79.8 (C-1), δ C 120.6 (4a), and δ C 149.4 (C-6), while the hydrogen at δ H 4,13 (H-1) showed three-bond 1 H-13 C correlation with the carbons at δ C 60.7 (C-3), δ C 115.7 (C-8), δ C 120.6 (C-4a), and 131.2 (C-1 ), thus establishing the correct positions of the hydrogens H-8 and H-1 for isomer 6. This attribution can be further confirmed by the analysis of the hydrogen at δ H 6.76 (H-5) that showed three-bond 1 H-13 C correlation with the carbons at δ C 27.0 (C-4), δ C 127.1 (C-8a), and δ C 145.7 (C-7) ( Figure 2). For the isomer 7 the analysis revealed that the hydrogen at δ H 6.30 (H-8) showed three-bond 1 H-13 C correlation with the carbons at δ C 79.4 (C-1), δ C 122.8 (4a), and δ C 148.9 (C-6), while the hydrogen at δ H 4.46 (H-1) showed three-bond 1 H-13 C correlations with the carbons at δ C 62.9 (C-3), δ C 115.5 (C-8), δ C 122.8 (C-4a), and 131.4 (C-1 ), thus establishing the correct positions of the hydrogens H-8 and H-1 for isomer 7. This attribution was also confirmed by the analysis of the hydrogen at δ H 6.71 (H-5) that showed three-bond 1 H-13 C correlations with the carbons at δ C 26.3 (C-4), δ C 126.9 (C-8a), and δ C 146.1 (C-7) ( Figure 2). The NOESY experiment was also carried out to establish the correct stereochemistry of isomers 6 and 7. In this experiment the strong NOE correlation of H-1 (δ H 4.13) and H 3 C-N (δ H 3.15) indicated the β-orientation of the oxygen in the isomer 6. On the other hand, no obvious NOE correlation between H-1 (δ H 4.46) and H 3 C-N (δ H 3.20) could be found in the 2D-NOESY experiment for 7, indicated the α-orientation of the oxygen in the isomer 7 ( Figure 2). These small differences in the chemical shifts of these isomers are clearly observed due to the stereochemistry of the nitrogen affected by the α and β position of oxygen. Comparisons of the 1 H-and 13 C-NMR data obtained for compounds 6 and 7 with data of molecules with close structures such as hexapetaline A and hexapetaline B [30] support the data described in Table 1 without ambiguity.
From a chemophenetic (a new term for plant chemosystematics/plant chemotaxonomy) point of view, it is important to note that the presence of C-benzylated flavanones and C-benzylated dihydrochalcones are a special type of flavonoids derived from the well-known flavanone pinocembin and have been described particularly in species of the genus Uvaria belonging to the Annonaceae family [3,[38][39][40][41][42]. The presence of these compounds in D. calycina suggest close chemophenetic relationships with Uvaria. On the other hand, further investigations should be carried out with other parts of the plant, as well as other species of Diclinanona to confirm this chemophenetic relationship. Flavanones and chalcones are widespread in the higher plants, but the addition of benzyl groups is quite rare and seems to be limited in the Annonaceae to the genus Uvaria [3,[39][40][41][42] and now to the genus Diclinanona. Benzyl groups presumably arise from a C 6 −C 1 pathway, but o-hydroxy functionality is unusual. The absence of substituents in B-ring in all these flavonoids of Uvaria and Diclinanona can be linked with the previous observation concerning the flavonoids of Popowia cauliflora [38].
The isoquinoline-derived alkaloids isolated and described in this work have already been registered in several species of Annonaceae in different genera. Some of these, such as liriodenine and anonaine, are considered chemophenetic markers, and the presence of these alkaloids in D. calycina further reinforces the relationship of these chemophenetic markers in the Annonaceae family [5][6][7]15,20,27,[33][34][35]43].
It is worth mentioning that, according to Zidorn [44], the chemophenetic studies are defined as studies aimed to describe the array of specialized secondary metabolites in a given taxon, as already observed in several published works [5][6][7]15,20,27,[33][34][35][43][44][45][46]. Thus, chemophenetic studies contribute to the phenetic description of taxa, similar to anatomical, morphological and karyological approaches, which have already been recognized as of great importance for the establishment "natural" systems, and which continue to be of extreme importance for the description of classified organisms with the help of modern molecular methods [44].

General Experimental Procedures
Optical rotations in methanol (MeOH) were recorded with a P-2000 polarimeter (Jasco, Tokyo, Japan) at 589 nm. 1D and 2D NMR experiments were acquired in CDCl 3 (chloroform-d) or CDCl 3 plus drop of CD 3 OD (methanol-d 4 , and CD 3 COCD 3 (acetone-d 6 ) at 298 K on an AVANCE III HD NMR spectrometer (Bruker, Billerica, MA, USA) operating at 11.75 T ( 1 H and 13 C at 500 and 125 MHz, respectively) and on a Bruker AVANCE III 600 NMR spectrometer operating at 14.1 T ( 1 H and 13 C at 600 and 150 MHz respectively). All 1 H-and 13 C-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. The NMR spectrometer was equipped with a 5-mm multinuclear inverse detection probe (1D and 2D NMR experiments) with z-gradient. One-bond (HSQC) and two and three-bond (HMBC) 1 H-13 C-NMR correlation experiments were optimized for average coupling constant 1 J (C,H) and LR J (C,H) of 140 and 8 Hz, respectively. For low resolution mass spectrometry (LR-MS) analysis the samples of the isolated compounds were resuspended in methanol (HPLC grade), creating the stock solutions (1 mg·mL −1 ). Aliquots (5 µL) of the stock solutions were further diluted to 5 µg·mL −1 and analyzed by direct infusion into a triple quadrupole mass spectrometer, model TSQ Quantum Access (Thermo Scientific, San Jose, CA, USA), equipped with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) sources. ESI-MS conditions: spray voltage, 5 kV; sheath gas, 10 arbitrary unit (arb); auxiliary gas, 5 arb; sweep gas, 0 arb; capillary temp, 250 • C; capillary voltage, 40 V; tube lens, 70 V; mass range, m/z 100 to 1000. APCI-MS conditions: discharge current, 5 µA; vaporizer temperature, 350 • C; sheath gas pressure, 25 arbitrary unit (arb); ion sweep gas pressure, 0.0 arb; aux gas pressure, 10 arb; capillary temperature, 250 • C; tube lens offset, 70 V; skimmer offset, 0 V; mass range, m/z 100 to 1000. Argon was used as collision gas, and the MS/MS spectra were obtained using collision energies ranging from 25 to 30 eV. Silica gel 60 (Sigma-Aldrich, San Luis, MO, USA, 70−230 mesh) was used for the column chromatography (CC), while silica gel 60 F 254 (Macherey-Nagel, Düren, Germany, 0.25 mm, aluminum) was used for analytical and preparative with thin layer chromatography (PTLC) (Macherey-Nagel, 1.00 mm, glass). Compounds were visualized by exposure under UV 254/365 light, by spraying with p-anisaldehyde reagent followed by heating on a hot plate, and by spraying Dragendorff's reagent.

Plant Material
In the present investigation, the botanical material (bark) of D. calycina was collected in 27

Cells
HL-60 (human promyelocytic leukemia), MCF-7 (human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma), HCT116 (human colon carcinoma) and MRC-5 (human lung fibroblast) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured as recommended by ATCC animal cell culture guide. All cell lines were tested for mycoplasma using a mycoplasma stain kit (Sigma-Aldrich) to validate the use of cells free from contamination.

Cytotoxicity Assay
For cytotoxicity assay, cell viability was quantified by Alamar blue method, as previously described [47][48][49]. For all experiments, cells were plated in 96-well plates. Chemical constituents were dissolved in dimethyl sulfoxide (DMSO, Vetec Química Fina Ltda., Duque de Caxias, RJ, Brazil) and added to each well and incubated for 72 h. Doxorubicin (doxorubicin hydrochloride, purity ≥ 95%, Laboratory IMA S.A.I.C., Buenos Aires, Argentina) was used as a positive control. At the end of treatment, 20 µL of a stock solution (0.312 mg/mL) of resazurin (Sigma-Aldrich Co.) was added to each well. Absorbances at 570 nm and 600 nm were measured using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Half-inhibitory concentration (IC 50 ) was obtained by nonlinear regression with 95% confidence intervals (CI 95%) using the software GraphPad Prism (Intuitive Software for Science; San Diego, CA, USA).
The results obtained in this work indicate that the species of the family Annonaceae are a promising source of biologically active compounds with cytotoxic properties, and suggest the continuation of their investigation in other models of biological assays.