New Natural Products from Siphoneugena densiflora Berg ( Myrtaceae ) and their Chemotaxonomic Significance

Siphoneugena Berg (Myrtaceae) é um pequeno gênero eugenióide segregado de Eugenia por Berg. A investigação fitoquímica de Siphoneugena densiflora foi realizada com o objetivo de se descobrir metabólitos secundários que possam ser considerados marcadores quimiotaxonômicos e auxiliar na distinção entre os dois gêneros. Cinco taninos hidrolisáveis inéditos foram isolados do extrato metanólico da casca da raiz e caracterizados como 4-O-α-L2”-Oe seu isômero 4-O-α-L-3”-O-acetilramnopiranosídeo do ácido elágico, siphoneugenina, constituído por uma nova aglicona de estrutura dibenzo-1,4-dioxínica, 4-O-β-D-6”e 4-O-β-D3”,6”-di-O-acetilglucopiranosídeo do ácido 3,4’-di-O-metilelágico, juntamente com o 4-O-αL-4”-O-acetilramnopiranosídeo do ácido elágico, eschweilenol C, sitosterol, daucosterol, ramnose, casuarinina, castalagina e ácidos elágico, gálico e siríngico. Do extrato metanólico das folhas, além dos compostos conhecidos como a quercetina, quercitrina, guiajaverina, reinoutrina, chebulosídeo II, ácidos terminólico, madecássico e asiático, lupeol, αe β-amirina, foi isolado um novo triterpeno pentacíclico nomeado como 2α,3β,6β-triidroxiolean-12-en-28ato de β-D-glucopiranosila. Todas as substâncias foram identificadas através da análise dos dados espectrais e químicos obtidos, além da comparação com dados relatados na literatura.


Introduction
The genus Siphoneugena comprises only eight species and belongs to Myrtaceae family.It is one of the specialized genera segregated from Eugenia by Berg. 1 Siphoneugena densiflora Berg, commonly known as Uvatinga, occurs in montane grasslands ("campos rupestres"), deciduous forests ("cerradões") and gallery forests in Southern Goiás State, Distrito Federal, Minas Gerais and São Paulo States of Brazil. 1 That species is the first one of the genus which has been chemically studied.Generic delimitation is a severe problem in Eugeniinae subtribe, 1 and chemosystematics must be used to refine, reinforce or revise taxonomic systems based on morphological and anatomical characters.This paper deals with the results of a phytochemical survey of extracts from S. densiflora, reporting the isolation and structural elucidation of six new secondary metabolites (1-6), together with twenty one known compounds (7-27).In addition, some isolated compounds are being brought into focus as useful chemotaxonomic features to differentiate the investigated genus from Eugenia.
The small amount and the difficulty of separation, led us to characterize compounds 1, 2 and 7 as a mixture.The integration of the proton signals at δ 7.81 (1), δ 7.79 (2) and δ 7.78 (7) in the 1 H NMR spectrum beside cross peaks of these protons with carbon at δ 149.2 (C-4, 1, 2 and 7), a 2 J (C,H) correlation observed in the HMBC spectrum (Table 2), and with carbons at δ 110.7 or 110.3 (C-5, 1, 2 and 7), a J (C,H) correlation observed in the HSQC spectrum, suggested that the ratio of these compounds in the mixture was about 1:1:1.Their 1 H NMR spectrum (Table 1) was very similar to that of 8, except Scheme 1.
for the signals correspondent to acetyl groups.The position of acetyl groups was determined carefully by cross peak of the signals at δ 5.42 (H-2", 1), 5.23 (H-3", 2) and 5.03 (H-4", 7) with carbons at δ 74.1, 75.7 and 75.6 (C-2", C-3" and C-4" of 1, 2 and 7, respectively, J (C,H)) and a 3 J (C,H) correlation with the ester carbon at δ 173.2 (1, 2 and 7).The chemical shift of ester carbon was confirmed by cross peak ( 2 J (C,H) correlation) with methyl protons at δ 2.11 (1), 2.16 (2) and 2.18 (7) in the HMBC spectrum. 1H 13 C long-range correlation from the anomeric protons at δ 5.56 (1), 5.55 (2) and 5.54 (7) to C-4 (δ 149.2) of the ellagic acid moiety ascertained that the rhamnose residue was attached to C-4 of the aglycone.Their molecular formula was determined to be C 22 H 18 O 13 from the pseudo molecular ion peak at m/z 489 [M-H] -, the fragment ion peaks at m/z 447 [M-H-H 2 CCO] -and at m/z 301 [M-H-H 2 CCO-rha] -, observed in the ESI-MS negative ion mode.Thus, the structure of 1 was assigned as ellagic acid 4-Oα-L-2"-O-acetylrhaminopyranoside; 2 as ellagic acid 4-O-α-L-3"-O-acetylrhaminopyranoside and 7 as ellagic acid 4-O-α-L-4"-O-acetylrhaminopyranoside. 2 Siphoneugenin (3) was isolated as a white powder.IR spectrum displayed characteristic absorptions for hydroxyl (3386 cm -1 ), methoxyl (2925 cm -1 ), acid carbonyl (1705 cm -1 ) and aromatic ring (1613, 1507, 1425 cm -1 ) groups. 1 H NMR spectrum (Table 3) disclosed two singlets in the aromatic region at δ 7.12 and 6.39; a doublet at δ 4.99 (J 7.6 Hz) and superimposable signals between δ 4.39-3.40indicated the presence of a sugar moiety at β configuration; two singlets at δ 3.72 and 3.63 shown the presence of methoxyl groups. 13C NMR spectrum (Table 3) revealed 20 signals.Carbon signal at δ 167.5 confirmed the presence of acid carbonyl observed in IR spectrum.Carbon signals at δ 101.9, 76.7-64.7 helped to determine the stereochemistry and nature of sugar moiety as β-D-glucopyranose.Signals at δ 56.3 and 60.8 confirmed two methoxyl groups attached to one and di-ortho substituted carbon, respectively.The compound was found to have the composition C 21 H 22 O 14 by analysis of its 13 C NMR spectrum and from the pseudo molecular ion peak at m/z 497 [M-H] -, the fragment ion peaks at m/z 453 [M-H-CO 2 ] -and at m/z 335 [M-H-glu] -, observed in the ESI-MS negative ion mode.HMBC spectrum (Table 3) showed a cross peak between anomeric proton signal at δ 4.99 and aromatic carbon at δ 154.9 confirming the linkage site of the monosaccharide at C-4' of the aglycone.The results obtained from NOE difference spectra of 3 performed by irradiation at δ 3.72 revealing enhancement at δ 6.39 (H-6') and absence of NOE by irradiation at δ 3.63 were used to locate the methoxyl groups at 5' and 3' positions, respectively. 1H 13 C HSQC correlation of aromatic proton signal at δ 6.39 (H-6') with a shielded carbon signal at δ 95.5 and cross peaks of this proton signal with two aromatic carbons in the HMBC spectrum (C-4' and C-2', Table 3) suggested the presence of a dibenzo-1',4dioxin structure. 3,16All of the above mentioned data and symmetry of the molecule suggested several possible isomers.We proposed a biosynthetic route for formation of 3 through phenolic oxidative coupling between gallic and syringic acid units, followed by an oxidation of aromatic ring, 17 but this could not be assumed to be the correct structure.A strategy was considered to solve the problem of the structural assignment, between 3 and 3a.UV spectral shift reagents were used to find the positions of hydroxyl groups.When the spectrum was run in methanol containing aluminum chloride, there was a bathochromic shift of 14 nm, indicating the presence of a quelate hydroxyl group, 18 but this result was not sufficient to distinguish between the two proposed structures.Nevertheless, addition of boric acid and sodium acetate caused no shift change in UV absorptions, confirming that no ortho-dihydroxyphenol system (cathecol) was present at the molecule.Accordingly the  spectroscopic and chemical evidence obtained, the structure of siphoneugenin was deduced to being that shown in formula 3.
Compounds 4 and 5 have also been characterized as a mixture.Their 1 H NMR spectrum (Table 1) was closely related to that of 1, in particular the signals arising from aromatic rings and acetyl groups.However, significant differences were revealed as the presence of two sugar anomeric doublets at δ 4.94 and 4.60 (J 7.6 Hz), the signals at δ 3.83 and δ 3.48 indicating two methoxyl groups, the signals relative to a CH 2 OAc group, and the lack of the signal relative to the methyl group linked to C-5" of compound 1.Furthermore, the negative ESI-MS spectrum displayed two [M-H] -peaks at m/z 533 and 575, respectively, whose are 44 and 86 u larger than that of compound 1.These data suggested that compound 4 and 5 had a glucose moiety at β configuration, being compound 5 with an additional acetyl group.The location of acetyl groups was determined at the same way for compound 1.The results acquired from NOE difference spectra of 4 and 5 carried out by irradiation at δ 3.83 showing signal enhancement at δ 6.74 (H-5') and absence of NOE by irradiation of signal at δ 3.48 were used to locate the methoxyl groups at C-4' and C-3, respectively.In addition, 1 H 13 C HSQC correlations of signals at δ 3.83 and 3.48 with carbons at δ 56.7 and 61.3, respectively, along with HMBC correlations (Table 2) between proton signal at δ 3.83 and carbon at δ 148.2 (C-4'), proton signal at δ 6.74 (H-5') and carbon at δ 137.4 (C-3'); and proton signal at δ 7.59 (H-5) and carbons at δ 141.0 (C-3) and 162.2 (C-7) confirmed the linkages of methoxyl groups at carbons C-3 and C-4' of the aglycone.Long-range correlation between anomeric proton at δ 4.94 with C-4 at δ 154.1 revealed the bonding site of the sugar.Thus, compound 4 was characterized as 3,4'-di-O-methylellagic acid 4-O-β-D-6"-O-acetylglucopyranoside and compound 5 as 3,4'-di-O-methylellagic acid 4-O-β-D-3", 6"-di-O-acetylglucopyranoside.
Compound 6 was obtained as a white amorphous powder.The 1 H NMR, 1 H 13 C HSQC and 13 C (DEPT) NMR spectra (Table 4) indicated the presence of seven tertiary methyl groups, an olefinic proton H-12 coupled with a carbon at δ 123.5, a quaternary carbon at δ 143.9 (C-13), and a β-18 proton at δ 3.19 (dd, 4.1 and 13.2 Hz), that are typical signals of an oleanene skeleton.Moreover, an anomeric proton signal at δ 6.28, in addition with several signals between δ 4.01-4.45,confirmed the presence of a hexose unit.The relative stereochemistry and nature of the sugar moiety were determined as β-D-glucopyranose on the basis of the characteristic J 1,2 coupling constant of its anomeric proton (J 8.0 Hz) and typical 1 H and 13 C NMR shifts. 19The ester linkage was located at C-28 with the δ shifted downfield from 180.1 (its aglycone) 20 to 176.9, and C-1' anomeric carbon at δ 96.2.In addition, HMBC spectrum (Table 4) displayed a cross peak between C-28 and H-1', confirming the linkage site of the monosaccharide at C-28. 1 H-1 H COSY (Table 4) couplings between proton signal at δ 2.30 (H-1a) with 1.39 (H-1b) and 4.29 (H-2), respectively, and 1 H- 13 C HSQC correlation of the carbon signal at δ 50.4 with the proton signal at δ 2.30 (H-1a) became possible the doubtless assignments for H-1a and H-1b, which are changed with H19a and H19b in the literature. 20Furthermore, the negative ESI-MS spectrum displayed a pseudo molecular ion at m/z 649 [M-H] -and a fragment ion peak at m/z 487 [M-Hglu] -, and the molecular formula was determined as C 36 H 58 O 10 .Thus, compound 6 was assigned as 28-β-Dglucopyranosyl-6β-hydroxymaslinate.
The occurrence of triterpenes in leaves of Myrtaceae is high and frequent, 21 but terminolic (25) and madecassic (26) acids have only been recently reported in Syzygium, 14 and compound 25 in Myrtus. 22hebuloside II (21), a pentacyclic triterpene glucoside, has been previously isolated from Terminalia chebula (Combretaceae), 12 but this is the first report of this compound from the family Myrtaceae.The glycosidic pattern of the found flavonols (17-20) is a simple one and this is in keeping with the primitive morphological features of the family Myrtaceae. 23Tannin 7, initially found in Combretaceae, 2 and tannin 8, in Lecythidaceae, 3 are being reported for the first time in Myrtaceae together with compounds 1 and 2, although some methoxylated derivatives were reported for Eucalyptus globulus. 24,25Similar compounds to tannins 4 and 5 were found in Eucalyptus polyanthemos. 26ccording to Hillis and Yazaki, 27 those ellagitannins have rarely been reported possibly to the difficulties in their isolation, purification and identification.In addition, some of them present poor solubility.All the tannins found in this phytochemical survey, except for 15 and 16, have not yet been reported to occur in Eugenia and might be considered as chemical features for taxonomic purposes, justifying the segregation of Siphoneugena from that genus by Berg. 1 Moreover, tannin 3 presents a new aglycone type for hydrolysable tannins and might also be taken into account as a valuable taxonomic character.

Experimental
General experimental procedures IR spectra were measured on a Bomem MB-102 spectrophotometer in KBr pellets.UV spectra were obtained on a Varian Cary 500 Scan/UV-Vis-Nir

Extraction
The air-dried powdered root bark (0.4 kg) and leaves (0.9 kg) of S. densiflora were extracted with hexane and MeOH through percolation processing at room temperature.Crude extracts were obtained after filtration and removal of the solvents under vacuum at 40 o C. The methanolic extracts of root bark (RME, 73.2 g) and leaves (LME, 70.0 g) were dissolved in H 2 O/MeOH 1:1 (v/v) and fractionated through liquid-liquid partition using solvents of increasing polarity (CH 2 Cl 2 , EtOAc).The solvents were removed to yield the residues RMED (0.9 g), RMEAc (10.5 g), LMED (9.5 g) and LMEAc (14.6 g).

Table 1 .
1 H NMR spectral data of compounds 1, 2, 4, 5 and 7 Recorded in CD 3 OD, chemical shifts values are reported as δ from TMS at 400 MHz, signal multiplicity and coupling constants (Hz) are shown in parentheses; b Values are interchangeable within the same line; c Recorded in acetone-d 6 at 400 MHz; * Signals overlapped.
a Recorded in CD 3 OD at 400 MHz (J 8.0 Hz); b Recorded in acetone-d 6 at 400 MHz (J 8.0 Hz).a

Table 3 .
13C NMR,1H NMR and HMBC spectral data for 3 a 13Spectra recorded in acetone-d 6 ; 400 MHz for 1 H NMR and HMBC (J 8.0 Hz); 100 MHz for13C NMR; b Signal multiplicity and coupling constants (Hz) are shown in parentheses; nd = signal not detected.

Table 4 .
13C (DEPT) NMR,1H NMR, HMBC and COSY spectral data for 6 a Spectra recorded in C 5 D 5 N; 400 MHz for 1 H NMR, HMBC (J 8.0 Hz) and COSY; 50 MHz for13C NMR; b Signal multiplicity and coupling constants (Hz) are shown in parentheses.spectrophotometer.Optical rotation was obtained on a Perkin Elmer 241 polarimeter.NMR spectra were recorded in acetone-d 6 , D 2 O, CD 3 OD or pyridine-d 5 , using TMS as internal reference, employing a Bruker DRX-400 ( 1 H, 400 MHz;13C, 50 or 100 MHz).Pharmacie Fine Chemical Co.Ltd.).Spots were visualized under ultraviolet light of wavelength 254 nm and by spraying vanillin sulfuric acid solution and heating, or FeCl 3 acid solution. a