New Neolignans from Krameria tomentosa A . St .-Hil

A investigação fitoquímica das raízes de Krameria tomentosa A. St.-Hil. levou ao isolamento de cinco neolignanas, duas delas com estruturas inéditas [1,1’-(E)-propenil4-metóxi-3,4’-oxineolignana (ottomentosa) e ácido 2-(2’-hidróxi-4’,6’-dimetoxifenil) benzofurano-5-carboxílico (sobralina)], além de três compostos conhecidos [eupomatenoide 6, di-hidrocarinatidina e 2-(2’,4’-di-hidroxifenil)-5-(E)-propenilbenzofurano]. A caracterização estrutural dos compostos isolados foi estabelecida com base na espectroscopia no infravermelho, espectrometria de massas, ressonância magnética nuclear uni e bidimensional, além de comparação com dados espectrais descritos na literatura.


Results and Discussion
Compound 1 was isolated in the form of a colorless oil.The high resolution mass spectrum (HS-ESI-MS) utilizing the ESI+ ionization mode showed the peak of the cationized molecule at m/z 303.1372 [M + Na] + , compatible with the molecular formula C 19 H 20 O 2 Na (calc.303.1356).The IR spectrum showed absorptions in the region of 1603-1441 cm -1 , characteristic of C=C stretching of the aromatic ring, as well as absorptions of 1269-982 cm -1 , characteristic of C-O stretching.The 1 H nuclear magnetic resonance (NMR) spectrum showed in the region of hydrogens in aromatic systems two connected doublets, one at d H 7.23 (J 9.0 Hz) and the other at d H 6.85 (J 9.0 Hz), compatible with the hydrogens of an AA'XX' system.In the same region, there was a doublet of doublets at d H 7.04 (J 2.5 and 8.5 Hz) coupled with the doublets at d H 6.95 (J 2.5 Hz) and at d H 6.89 (J 8.5 Hz), compatible with the hydrogens of an AMX system.The set of signals at d H 6.33, 6.10 and 1.84, as well as the set at d H 6.25, 6.00 and 1.80, characterized the hydrogens of two propenyl units.The 13 C APT NMR (attached-proton-test NMR) spectrum displayed 17 signals, corresponding to 19 carbon atoms.From these, 5 were assigned to non-hydrogenated carbons, from which 3 were oxygenated, 11 to methine carbons, 2 to methyl carbons and 1 to a methoxyl carbon.According to the data described, it was possible to suggest that the phenylpropanoid units of 1 were connected forming the skeleton of an oxyneolignan, 17,18 showing an oxygenated substitute, this being a methoxyl.The correlation of the hydrogens at d H 3.80 (OMe) with the hydrogen at d H 6.85 (H-3'/5'), observed in the NOESY (nuclear Overhauser effect spectroscopy) spectrum, made it possible to infer that the methoxy group was connected to C-4.In the heteronuclear multiple-quantum correlation (HMQC) spectrum, direct correlations can be seen between the hydrogens at d H 7.23 (d) and 6.85 (d) with the carbons at d C 126.9 and 117.3, assigning them to C-2'/C-6' and C-3'/C-5', respectively.Chemical shifts at d H 6.95 (d), 6.89(d) and 7.04 (dd) showed correlations with the carbons at d C 117.9, 112.8 and 122.2, respectively, in which they were thereby assigned to carbons C-2, C-5 and C-6.In the heteronuclear multiplebond correlation (HMBC) spectrum, correlations were observed between the hydrogens at d H 6.95 (H-2) and 1.80 (H-9) with the carbon at d C 129.9 (C-7), as well as the correlation between the hydrogen at d H 6.33 (H-7') with the carbon at d C 126.9 (C-2'/6'), confirming the insertion of the propenyl group more shielded in the ring of the AMX system and less shielded in the AA'XX' system.The shifts of the non-hydrogenated, non-oxygenated carbons were confirmed by the correlations between the signal at d H 7.04 (H-6) with the carbon at d C 150.3, assigned to C-4, and between the hydrogens at d H 7.23 (H-2'/H-6') and 6.85 (H-3'/H-5') with the carbon at d C 156.8, assigned to C-4'.Homonuclear correlation spectroscopy (COSY) showed correlations of the signal at d H 7.04 (H-6) with 6.95 (H-2) and 6.89 (H-5) and of the signal at d H 6.85 (H-3'/5') with 7.23 (H-2'/6').Table 1 gives a compilation of the chemical shifts and correlations observed in the spectra of one-and two-dimensional 1 H and 13 C NMR for compound 1.After the analysis of all the spectral data, this compound was determined to be 1,1'-(E)-propenyl-4-methoxy-3,4'-oxyneolignan, reported here for the first time and given the trivial name ottomentosa.
Compound 2 was isolated in the form of a white powder, with a melting point of 262-264 °C.The high resolution mass spectrum utilizing the ESI-ionization mode showed the peak of the deprotonated molecule at m/z 313.0708 [M -H] -, compatible with the molecular formula C 17 H 13 O 6 (calc.313.0706).The IR spectrum revealed the presence of a wide band between 3472 and 2650 cm -1 and a band at 1690 cm -1 , characteristic of O-H and C=O stretching of a carboxyl group, respectively.Absorption was also observed at 1624-1458 cm -1 , characteristic of C=C From these, 9 were assigned to non-hydrogenated carbons, 6 to methine carbons and 2 to methoxyl carbons.Based on comparison with 13 C NMR spectral data of the neolignans 2-(2'-hydroxy-4'-6'-dimethoxyphenyl)-5-(E)-propenylbenzofuran and krametosan, also isolated from K. tomentosa, 14  ) and 6.24 (d, J 2.5), corresponding to the hydrogens H-3' and H-5', respectively.It was also observed two singlets at d H 3.81 and 3.83, the first referring to the methoxyl at C-4' and the second to the methoxyl at C-6'.These assignments were confirmed by the direct correlations observed in the HMQC spectrum.The HMBC spectrum showed correlations between the hydrogens at d H 8.31 (H-4) and 7.97 (H-6) with the signal at d C 168.1, assigned to the carbon of the carbonyl, confirming its insertion at C-5. Correlations were observed between the hydrogen at d H 8.31 (H-4) and the signal at d C 107.5, assigned to C-3, and between the hydrogens at d H 7.03 (H-3), 8.31 (H-4) and 7.97 (H-6) and the signal at d C 157.4, assigned to C-7a.Table 2 gives a compilation of the chemical shifts and the correlations found in the spectra of one-and two-dimensional 1 H and 13 C NMR for this compound, which is reported here for the first time.This compound was given the trivial name sobraline.
C o m p o u n d 3 ( 2 -( 2 ' , 4 ' -d i h y d r o x y p h e ny l ) -5-(E)-propenylbenzofuran) was isolated in the form of colorless crystals, showing a melting point of 181-184 °C.This substance has already been isolated from other species of the genus Krameria, [4][5][6][7][8] but this is the first report

Conclusions
Considering the wealth of neolignans in the species of the family Krameriaceae, this study comes to confirm the predominance of this class of secondary metabolites in the family and contributes to the expansion of their chemical knowledge with the isolation of two new neolignans.

General experimental procedures
Melting points were obtained by the digital apparatus, model MQAPF-302 from Microchemical and were not corrected.IR spectra were recorded on a BOMEM-MB 100 spectrophotometer.One-dimensional ( 1 H and 13 C) and two-dimensional (gHMQC, gHMBC, gCOSY and gNOESY) NMR analyses were performed on a VARIAN-System spectrometer operating at 500 MHz ( 1 H) and 125 MHz ( 13 C).CDCl 3 or CD 3 COCD 3 was used as the solvent with TMS as an internal standard.HR-ESI-MS was obtained using the microTOF-II system from Bruker.Conventional chromatographic methods were used for column chromatography (CC) (silica gel 60, Merck, 0.063-0.20 and 0.04-0.063mm).Medium pressure liquid chromatography (MPLC) was performed using the Buchi system of binary gradient flash separation, in which the chromatograph was equipped with two pump modules (C-601 and C-605), controller module (C-615), Knauer UV detector and columns packed with silica gel (Merck, 0.063-0.20 and 0.04-0.063mm).

Plant material
The botanical material utilized was collected in the municipality of Santa Rita, Paraíba State, Brazil, in June 2010.Its botanical identification was carried out by Prof. Dr. Maria de Fátima Agra and a dried specimen is deposited in the Herbário Professor Lauro Pires Xavier of UFPB under No. 3271.

Extraction and isolation
The roots of K. tomentosa (3.5 kg), dried and pulverized, were extracted with 95% EtOH at ambient temperature.The extract obtained was concentrated in a rotary evaporator under reduced pressure at 40 °C, yielding 685.0 g ethanolic extract.A portion (100.0 g) was suspended in MeOH:H 2 O (7:3) and partitioned with hexane, CH 2 Cl 2 and EtOAc to obtain the hexane (2.5 g), dichloromethane (5.4 g) and ethyl acetate (6.5 g) extracts.The hexane extract (2.5 g) was separated by CC, utilizing silica gel 60 (0.063-0.200 mm) and the eluents hexane and EtOAc and MeOH, pure or in binary mixtures, in increasing order of polarity, resulting in 67 fractions of 100 mL each, which were analyzed by analytical TLC.Fraction 14 yielded compound 4 (16.6 mg).Fractions 1-2 (168.3 mg) were submitted to another CC utilizing similar conditions as before, providing 25 subfractions of 10 mL each.Subfractions 10-15 gave the neolignan 1 (35.4 mg).Fractions 27-35 (99.3 mg) were rechromatographed as before, from which 55 subfractions of 10 mL each were collected.Subfractions 33-37 yielded compound 5 (26.2 mg).
The dichloromethane extract (5.0 g) was submitted to MPLC, with the column packed with silica gel 60 (0.063-0.200 mm), utilizing a flow rate of 30 mL min -1 and mobile phase of the solvents hexane and EtOAc and MeOH, pure or in binary mixtures, in increasing order of polarity.A total of 81 fractions of 100 mL each was collected, which were concentrated in rotary evaporator and combined, after analysis by analytical TLC, to form 24 groups.Fractions 42-45 provided compound 3 (22.7 mg).Fractions 67-81 (675.3 mg) were submitted to another MPLC, utilizing a column packed with silica gel 60 (0.04-0.063 mm) and flow rate of 30 mL min -1 .From this column, 53 subfractions of 100 mL each were collected, which were analyzed by analytical TLC and combined into 10 groups.Subfractions 5-6 yielded compound 2 (8.5 mg).

Figure 1 .
Figure 1.Neolignans isolated from the roots of K. tomentosa.
it was possible to make the following considerations: (i) the signals at d C 153.5, 107.5, 130.1 and 157.5 were assigned to carbons C-2, C-3, C-3a and C-7a, respectively, of the benzofuran ring; (ii) the signals at d C 100.6, 158.6, 95.1, 163.4,91.6 and 160.8 were assigned to carbons C-1', C-2', C-3', C-4', C-5' and C-6', respectively; (iii) the absence of signals at approximately d C 131.0, 124.6 and 18.5 suggestive of a propenyl unit plus the presence of the signal at 168.0 (referring to a carbonyl) and the information obtained from the IR spectrum indicate that compound 2 is possibly the trinor-neolignan 2-(2'-hydroxy-4',6'-dimethoxyphenyl)benzofuran-5-carboxylic acid.The 1 H NMR spectrum of this compound indicated the presence of a signal at d H 7.03 (d, J 0.5 Hz) characteristic of H-3, as well as signals at d H 8.31 (d, J 1.7 Hz), 7.97 (dd, J 1.7 and 8.5 Hz) and 7.57 (d, J 8.5 Hz), assigned to the hydrogens H-4, H-6 and H-7, respectively, and signals at d H 6.23 (d, J 2.5

H 6 .
50 (H-5') and the carbons at d C 104.0 and d C 103.9 assigned to C-3 and C-3', respectively.C o m p o u n d s 4 ( e u p o m a t e n o i d 6 ) a n d 5 (dihydrocarinatidin) were identified based on direct comparison with NMR data described in the literature.4,19