An Azafluorenone Alkaloid and a Megastigmane from Unonopsis lindmanii ( Annonaceae )

O alcalóide azafluorenona 5,8-dimetóxi-7-hidróxi-1-metil-4-azafluoren-9-ona e o megastigmano (–)-(5R*, 6S*)-megastigman-3-ona-10,7-olidio foram isolados das partes aéreas de Unonopsis lindmanii (Annonaceae), juntamente com os compostos conhecidos (3S*, 5S*, 8R*)-3,5-dihidróxi-megastigma-6,7-dien-9-ona (grasshopper ketone), N-trans-feruloiltiramina, (–)-anonaína, (–)-asimilobina, liriodenina e (–)-siringaresinol. Este é o primeiro relato da presença de megastigmanos em Annonaceae. As estruturas dos compostos foram elucidadas com base em dados espectroscópicos.


Results and Discussion
The ethanolic extract of the heartwood of U. lindmanii was chromatographed on a silica gel column and then on a Sephadex LH-20 column to afford compound 1.The positive HRESIMS of 1 revealed a pseudo-molecular ion at m/z 272.0957 [M + H] + , consistent with the molecular formula C 15 H 13 NO 4 .The presence of a carbonyl group was demonstrated by an IR band at 1649 cm -1 .The 1 H nuclear magnetic resonance (NMR) spectrum of 1 (Table 1) showed three signals of aromatic hydrogens at d H 6.91 (s, H-6), 6.86 (d, J 5.2 Hz, H-2) and 8.23 (d, J 5.2 Hz, H-3), two methoxyl groups at d H 3.92 (s, 5-OCH 3 ) and 4.01 (s, 8-OCH 3 ), and a signal of a methyl bonded to an aromatic ring at d H 2.59 (s, 1-CH 3 ).The 13 C NMR spectrum of 1 (Table 1) contained fifteen signals attributed to a conjugated carbonyl (d C 190.7), three aromatic protonated sp 2 carbons bound to hydrogens, eight sp 2 aromatic carbons without hydrogens attached, two methoxyl groups and one methyl.Atomic connectivity was established using COSY, one-bond (HSQC), long-range (HMBC) 1 H- 13 C NMR correlation experiments and nuclear Overhauser effect spectroscopy (NOESY) (Figure 2).The 13 C NMR spectrum of 1 showed a signal for only one methoxyl group attached to ortho-disubstituted carbon at d C 61.1, which was correlated in HSQC with the 1 H NMR signal at d H 4.01 and in HMBC with the signal at d C 142.5.These correlations imply that the aromatic hydrogen at d H 6.91 must not be vicinal to this methoxyl group.The second methoxyl signal at d H 3.92 showed one-bond correlation with the 13 C NMR signal at d C 56.5 and long-range correlation with the signal at d C 156.4.In the HMBC experiment, the aromatic hydrogen at d H 6.91 showed strong correlations with the carbons at d C 119.2, 142.5, 147.2 and 156.4,suggesting two possible structures for this compound: 5,8-dimethoxy-7-hydroxy or 5,8-dimethoxy-6-hydroxy.A comparison between the 1 H NMR chemical shifts with the 5,8-dimethoxy-6-hydroxy-1-methyl-azafluorenone (kinabaline) 32 indicated that H-7 displays a higher field resonance (d H 6.34) when compared to that (d H 6.91) in compound 1.This evidence allowed the assignment of the resonance at d The compound 1a was obtained via reduction of 1 with NaBH 4 .In the 1 H NMR spectrum of 1a (Table 1), the presence of a signal at d H 5.70 (s, H-9) was consistent  ).This observation allowed us to determine the correct position of hydrogen H-6 (d H 6.97).Consequently, the structure of 1a corresponds to that displayed in Figure 1.
Compound 2 was isolated as a brownish-yellow amorphous solid from the CHCl 3 leaf extract, after chromatographic steps using silica gel column.From HRESIMS spectrum of 2, a pseudo-molecular ion peak at m/z 225.1551 [M + H] + was obtained, corresponding to the molecular formula C 13 H 20 O 3 .In the 1 H NMR spectrum of 2 (Table 2), two methyl singlets were observed at d H 0.76 and 1.03 (H-11 and H-12, respectively), and a doublet at d H 1.04 (d, J 6.4 Hz) was attributed to the methyl H-13 hydrogen.The signal at d H 2.05 (dd, J 13.4 and 2.0 Hz) was assigned to H-2 equatorial hydrogen, with geminal coupling constant of 13.4 Hz and a 4 J Wequatorial-equatorial coupling of 2.0 Hz with the H-4 equatorial , appearing as a double doublet, while the H-2 axial appeared as a doublet at d H 2.25 (d, J 13.4 Hz) with geminal coupling constant of 13.4 Hz.The signals at d H 2.00 (d, J 11.2 Hz) and 2.29 (dd, J 11.2 and 2.0 Hz) were assigned to the methylenic hydrogens H-4 axial/equatorial , respectively, supported by HSQC spectrum.The analysis of the 13 C NMR spectrum revealed the presence of 13 carbons, suggesting a megastigmane skeleton.The signals at d C 211.0 and 176.3 were attributed to the carbonyl at C-3 and the carboxyl at C-10, the last signal indicating a possible lactone ring.A signal at d C 71.9 was assigned to C-7, that one at d C 52.1 to C-6, and the signals at d C 20.6, 29.9 and 20.9 assigned to the three methyl carbons C-11, C-12 and C-13, respectively.Correlations between C-3 and H-2 and H-4 were visualized in the HMBC spectrum, as well as 3 J C-H correlations between C-10 and H-7.The C-13 signal at d C 20.9, which had its position confirmed through HMBC and HSQC experiments, was consistent with the equatorial position of this group, in comparison with spectral data from previously described analogues. 33n the NOESY experiment, some important correlations were observed between H-11 (d H 0.76), which is in axial position, and H-5 (d H 1.79), confirming the configuration at position 5; between H-11 and H-8 (d H 1.62) and between H-5 and H-7 (d H 4.14), indicating the position of the lactone ring (Figure 3).These assignments were checked by COSY, HSQC and HMBC analyses, and the relative configuration was based on correlations in the NOESY experiment, confirming the structure of 2 as (-)-(5R*, 6S*)-megastigman-3-one-10,7-olide.

Conclusions
Although alkaloids are a common theme in Annonaceae, the occurrence of azafluorenones is rare and restricted to this family.Some hypotheses for the biosynthesis of these alkaloids are raised based on different sources, some suggesting their origin from oxaporphinic alkaloids, supported by the co-occurrence of these compounds. 34Other authors indicated a possible route from a polyketide pathway 34 or from a shikimic acid intermediate bound to a glutamic acid unit as a base to the skeleton of these molecules. 35espite several proposals, the biosynthetic pathway to the formation of azafluorenones remains unknown.This study  contributed to the expansion of the chemical characterization of the Unonopsis genus since the compounds grasshopper ketone, (-)-syringaresinol and N-trans-feruloyltyramine are being described for the first time in Unonopsis.To the best of our knowledge, this is the first time that the presence of megastigmanes in Annonaceae is described, indicating the importance of continuing the investigation of this family as a source of novel molecules.

General procedures
Silica gel (70-230 mesh, Merck) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC), whereas silica gel 60 GF 254 was employed for analytical (0.50 mm) and preparative (1.0 mm) thin layer chromatography (TLC).The 1 H and 13 C NMR spectra were obtained at 300 and 75 MHz, respectively, on a Bruker DPX-300 spectrometer using CDCl 3 (Aldrich) and acetone-d 6 (Aldrich) as solvents and tetramethylsilane (TMS) as an internal standard.The IR spectra were obtained on a Perkin-Elmer 783 spectrometer and the specific optical rotations on a Perkin-Elmer 341 MS polarimeter.Mass spectra were obtained in Agilent Ultra Q-TOF mass spectrometer with electrospray ionization.

Plant material
Heartwood and leaves of Unonopsis lindmanii were collected in March 2005 in Pantanal (Mato Grosso do Sul, Brazil) and identified by Dr. Renato Mello Silva (University of São Paulo, Brazil).A voucher specimen (No. 4730) was deposited in the Herbarium GC/MS (Universidade Federal do Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil).