Two 8 C-methylated Flavonols from the Leaves of Vellozia candida Mikan ( Velloziaceae )

Do extrato etéreo das folhas de Vellozia candida Mikan (Velloziaceae) foram isolados os flavonóis 3',4',5,7-tetraidroxi-3,6-dimetoxi-8-metilflavona e 3’,4’,5-triidroxi-3,6,7-trimetoxi-8-metilflavona. Estes compostos foram identificados através de seus dados espectrais, incluindo RMN 2D e EMEM. O derivado 5-hidroxi-3,3’,4’,6,7-pentametoxi-8-metilflavona foi obtido por metilação usando diazometano.


Introduction
The flavonol aglycones constitute a very diverse group of plant secondary metabolites.A number of these natural products are lipophilic and may show close R f -values in TLC, with overlapping spots, making it impossible to identify them in mixtures containing several related components.A preliminary MS spectrum of a plant fraction and/or crude extract, and also data from GC, HPLC, TMS (Tandem Mass Spectrometry) can supply important structural information on natural compounds and thereby allow an optimization of the investigation and avoid unnecessary isolation, especially of known compounds.The Velloziaceae family contains C-methyl and Cprenylated flavonoids which are relatively rare in nature. 1,2ecently, we applied high temperature high resolution gas chromatography (HT-HRGC) coupled to mass spectrometry (HT-HRGC-MS) in the prospection and identification of the monoisoprenylated flavonol 3,5,4'-trimethoxy-3'-hydroxy-6,7-(2"-isopropenyldihydrofuran)-flavone from Vellozia graminifolia, and the importance of this technique to determine the presence of lipophilic flavonols was demonstrated. 3 previous phytochemical analysis of V. candida, collected on the Corcovado mountain, in the city of Rio de Janeiro, Brazil, resulted in the identification of several rosane diterpenoids, 4,5 including a velloziolide with a new skeleton 6 and a bioactive seco-rosane. 7In this paper we describe the characterization of the 8-C-methylated flavonols 3',4',5,7-tetrahydroxy-3,6-dimethoxy-8methylflavone (1) and its known 7-O-methyl derivative, 3',4',5-trihydroxy-3,6,7-trimethoxy-8-methylflavone (2), isolated from a specimen of V. candida collected in Barra da Tijuca, a coastal area of the city of Rio de Janeiro.The structural determination of these compounds as components of a mixture was based on spectral data, including 2D NMR techniques, such as heteronuclear correlation 1 H-13 C-COSY-n J CH (n=1, HMQC = 1 H-detected Heteronuclear Multiple Quantum Coherence; n=2 and 3, HMBC = 1 H-detected Heteronuclear Multiple Bond Connectivity) and tandem mass spectrometry, together with chemical transformation and comparative analysis of chemical shifts of previously reported analogous flavonols isolated from the Velloziaceae family. 8

Results and Discussion
Chromatography of the Et 2 O extract of V. candida on a silica gel column yielded initially a fraction containing aliphatic carboxylic acids and sterols, which were characterized by GC/MS analysis (see Experimental).The other fractions obtained from the same column were combined on the basis of TLC comparison and subsequently chromatographed on Sephadex LH-20 to give a mixture of two lipophilic flavonoids.This mixture was subjected to methylation with diazomethane and yielded 5-hydroxy-3,6,7,3',4'-pentamethoxy-8-methylflavone (3) as the only product of known structure.
The mixture of flavonols 1 and 2 showed IR absorption bands for a conjugated carbonyl group (ν max 1651 cm -1 ) and for aromatic ring (ν max 1602 and 1559 cm -1 ).The comparative analysis of the HBBD-and DEPT-13 C NMR spectra was used to recognize the number of signals corresponding to quaternary, methine and methyl carbon atoms (Table 1).The 1 H NMR spectrum showed signals for aromatic hydrogens H-2', H-5' and H-6' in the B-ring of a 3',4'disubstituted flavonoid, along with signals for methoxyl, methyl and chelated hydroxyl groups (Table 1).
In accordance with the 1 H and 13 C NMR spectral data of 1, the HMBC spectrum confirmed a completely substituted A-ring through the heteronuclear long range couplings via two ( 2 J CH ) and three ( 3 J CH ) bonds between carbon atoms C-6 (δ C 130.24) and hydrogens of both HO-5 [δ H 12.76, s; 3 13 C NMR (75 MHz) data for flavonols 1 and 2 (CDCl 3 , δ in ppm, J in parentheses in Hz) Including results of heteronuclear 2D experiments 1 H-13 C-COSY-n J CH (HMQC and HMBC) * 1 2 Hx 13 C-COSY-n J CH NMR spectra, as shown in Table 1.
The electrospray ionization (ESI) mass spectrum at collision energies of 10 eV and 20 eV of the mixture isolated from V. candida shows peaks due to protonated molecular ions ([M+H] + ) for 1 (m/z 361, 1a) and for the related minor 8-C-methylated flavonol 2 (m/z 375, 2a) (Figure 1).The difference of 14 daltons between the protonated molecular ions [M+H] + of 1 (m/z 361 [360+H] + ) and 2 (375 [374+H] + ) was attributed to a biomethylation of one hydroxyl group of 1 (dimethoxylated) to produce 2 (trimethoxylated).This is in accordance with the selective methylation of the mixture which afforded only one product containing a chelated hydroxyl group at C-5 [δ H 12.58 (s)], a relatively strong intramolecular hydrogen bond involving a sixmembered ring.
The CID spectra of 1a and 2a show peaks at m/z 345 (1) and 359 (2), which were attributed to ionic fragments 1b and 2b formed from quasimolecular ions by elimination of one molecule of methane ([M+H] + -16 daltons), consistent with literature data for 3-methoxy flavonoids. 9hus, the CID spectra at collision energies of 10 and 20 eV allowed the observation of peaks corresponding to [M+H] + .At 30 and 40 eV these ionic species are absent and the peaks observed can be used to determine the fragmentation mechanisms for 1a and 2a in the mass spectrum (Scheme 1).
Tandem mass spectrometry, a technique based in tandem mass analyzers (ions formed are separated by mass analysis and secondary mass spectrum of each ion may be obtained, MS/MS) used for the separation and identification of ions (such as those representing components in complex mixture), proved to be a rapid and powerful technique for the characterization of flavonoids. 10tructural information of the components of a sample may be obtained by fragmentation of selected [M+H] + in CID.

Conclusions
The flavonoid substitution patterns are useful in the determination of the generic limits within the family Velloziaceae. 11In the genus Vellozia it is common to find flavonols with C-prenylation, 6-hydroxylation and Cmethylation which are rarely found in the other genera of this family.In the case of V. candida Mikan, after the characterization of the new 3',4',5,7-tetrahydroxy-3,6dimethoxy-8-methylflavone (1) by NMR spectroscopy (Table 1), it was possible to recognize its known O-methyl ether derivative (2) in the mixture with the aid of tandem mass spectrometry.

General
Column chromatography was performed with silica gel 60 (Merck 70-230 mesh).Merck silica gel F254 was used for TLC plates and spots visualized by UV (254 and 360 nm).NMR spectra in CDCl 3 solution were recorded at 300 MHz for 1 H and 75 MHz for 13 C on a Bruker AC 300 spectrometer, TMS as internal standard or referenced to the solvent signals CHCl 3 , at δ H 7.25 and CHCl 3 , at δ C 77.00.Samples were initially diluted in aqueous/ acetonitrile solution and subsequently with acetic acid to provide solutions of 10 mM HOAc (50/50).The ESI/MS/ Scheme 1. CID mass fragmentation of 1 and 2. Elimination of CH 2 O and CH 4 showed in this scheme may involve heterolytic (as indicated with full arrow corresponding to a electron par) or homolytic (fish-hook corresponding to a single electron) bond cleavages MS spectra were acquired using a Quattro II (MicroMass, UK) triple quadrupole mass spectrometer equipped with an electrospray ionization source.The spectra were recorded and processed by MassLynx v. 3.2 software (MicroMass, UK).The acquisitions were accomplished in positive ion mode (ES+) from m/z 50 to 550 at 2 s/scan.The cone voltage was 40V and source temperature maintained at 100°C.Each sample was delivered to the electrospray source through an infusion pump (Pump 22, Harvard Apparatus, So.Natick, MA) at a flow rate of 10 µL min -1 .The protonated molecule, [M+H] + , was selected at Q1 as precursor ion, and product ion scans were made at Q3, after collisionally induced dissociation at q2. Argon was used as collision gas (Gas Cell pressure was 1.7x10 -3 mBar) and different collision energies, from 10 to 40 eV, were applied.

Plant material
Vellozia candida Mikan was collected in Barra da Tijuca, Rio de Janeiro, Brazil and identified by Prof. Nanuza L. de Menezes of the Universidade de São Paulo (USP), São Paulo-SP, Brazil.A voucher specimen has been deposited at the herbarium of the Instituto de Botânica of USP.

Extraction and isolation
Dried and powdered leaves were extracted with ethyl ether at room temperature.After concentration of the solvent, under vacuum, an oily residue (6.9 g) was obtained.This extract was re-dissolved in methanol, filtered to remove waxes and evaporated at reduced pressure to yield an oily material (3.6 g), which was chromatographed on a silica gel column (45 g) with a gradient of EtOAc in hexane.A total of 12 fractions of ca 100 mL each were collected and combined on the basis of TLC comparison.Fraction 2, eluted with hexane/EtOAc (1:1) contained palmitic acid (40 %), stearic acid (20 %), b-sitosterol (3 %) and stigmasterol (12 %) which have been characterized through fragmentation patterns obtained by HRGC-MS. 12Fractions 4, 5 and 6, eluted with EtOAc/MeOH (7:3 to 5:5), were combined and chromatographed on a Sephadex LH-20 column using methanol as eluent.Fractions 2-9 (15 mL each) were again combined and rechromatographed on a Sephadex LH-20 column using methanol as eluent.Fractions 2-6 (15 mL each) furnished a mixture of 1 and 2 as a yellow oil (19.6 mg).
* Multiplicity of signals of carbon atoms deduced by comparative analysis of HBBD-and DEPT-13 C. 2D 1 H-1 H-COSY NMR also used for these assignments peak correlating the signals at δ