Three New Compounds from Piper montealegreanum Yuncker (Piperaceae)

Três novos compostos: dois flavonóides [(S)-8-formil-3’,5-diidroxi-7-metoxi-6-metilflavanona (1) e 3’-formil-3,4’,6’-triidroxi-2’-metoxi-5’-metilchalcona (2)] e um fenilpropanóide [3,4-metilenodioxi-5-metoxi-7,8-diidrocinamato de etila (3)] foram isolados dos ramos secos de Piper montealegreanum. As estruturas desses compostos foram estabelecidas através das técnicas espectroscópicas UV, IV, EM e RMN (H e C, 1D e 2D), além da interpretação dos dados de RMN de H e C dos derivados metilados dos compostos 1 e 2.


Results and Discussion
Compound 1 was obtained as orange-yellow crystals.The MS spectrum presented a molecular ion peak at m/z 327.0887 (M-1) − , in LC-MS-IT-TOF apparatus (ion traptime of flight liquid chromatography mass spectrometry).The 1 H NMR spectrum showed the presence of four singlets at d H 12.63 (1H), 10.15 (1H), 3.99 (3H) and 2.05 (3H) consistent with the presence of chelated hydroxyls, aldehyde, methoxyl and methyl groups, respectively.The presence of three signals at d H , 5.43 (dd,1H, J 10.8 and 4.6 Hz), 2.97 (dd, 1H, J 17.0 and 10.8 Hz), 2.85 (dd,1H, J 4.6 and 17.0 Hz), and also signals for four coupled aromatic protons at d H , 7.26 (t, 1H, J 8.0 Hz), 6.85 (brd, 1H, J 8.0 Hz) and 6.93 (m, 2H), suggested a flavanone nucleus 14 with a 3'-monosubstituted B ring, deduced by analysis of the multiplicity and coupling constants of the aromatic protons. 15The above data and UV spectrum, with l max at 266 nm, reinforced a flavanone nature for compound 1. 13 Since no further aromatic protons were evident, ring A should be fully substituted.The presence of the signal to methine carbon at d C 193.8 in the 13 C NMR APT (attached proton test) spectrum was taken as a proof for the presence of an aldehyde group, and the low-field hydroxyl hydrogen [d H Vol. 22, No. 8, 2011   12.63 (s, 1H)] evidenced the chelated hydroxyl at C-5 with the C=O of the a,b-unsaturated carbonyl group. 13n the HMBC spectrum, the presence of cross peaks at d H 12.63 with d C 166.  13,16 The above data and UV absorption bands (l max ) at 317 and 282 nm suggested a chalcone structure for compound 2. 13 Signals for four coupled aromatic protons: a triplet at d H 7.33 (1H, J 7.8 Hz), a multiplet at d H 7.28-7.23(m, 2H) and a double double doublet at d H 6.97 (1H, J 7.8, 2.0 and 1.6 Hz) suggested a 3'-substituted B ring. 15 Additionally, the 1 H NMR spectrum showed tree singlets at d H , 2.01 (3H), 4.01 (3H) and 10.17 (1H), consistent with the presence of methyl, methoxyl and aldehyde groups, respectively, besides two hydroxyl group singlets at d H 12.83 and 14.21.The two low-field hydroxyl protons evidenced a formyl group at C-3'.This conclusion is achieved since the down-field shift of OH-4' (d H 12.83) can be explained by hydrogen-bonding with the oxygen atom of the formyl substituent at a neighboring carbon atom and at the same time that the down-field shift of OH-6' (d H 14.21) is caused by chelation between the 6'-hydroxyl proton and the carbonyl oxygen of the a,b-unsaturated carbonyl group function. 13This intramolecular hydrogen bonding corroborated the assignments of the signals at 1 Three New Compounds from Piper montealegreanum Yuncker (Piperaceae) J. Braz.Chem.Soc.1612 coupling constant (16.0 Hz) observed in 2a indicated the E-isomer for the double bond. 16The placement of the other groups in the A-ring was made on the basis of the HMBC correlations (Figure 3  Formyl flavonoids have been reported from a few species in the plant kingdom.Early reports included a description of 2',4-dihydroxy-4'-methoxy-5'formylchalcone from Psoralea corylifolia (Fabaceae). 17ts isomeric compound, with methoxy group at the 2'-position, has been reported from the same species. 18',4',6'-trihydroxy-3'-formylchalcone has been reported from Psidium acutangulum (Myrtaceae) 19 and its retrochalcone derivative was obtained from Anredera scandens (Basellaceae). 203'-formyl-4',6'-dihydroxy-2'-methoxy-5'-methylchalcone and (2S)-8-formyl-5hydroxy-7-methoxy-6-methylflavanone were isolated from Cleistocalyx operculatus (Myrtaceae). 13he compound 3 was obtained as yellow powder.The 1 H spectrum showed two doublets in d H 6.37 (J 1.4 Hz, 1H) and d H 6.34 (J 1.4 Hz, 1H) typical of meta aromatics hydrogens in addition to two singlets in d H 5.91 (2H) and 3.86 (3H) typical of methylenedioxy and methoxyl groups, respectively.These data suggested the presence of tetrasubstituted aromatic ring.The signals in d H 4.11 (q, 2H, J 7.0 Hz) and 1.23 (t, 3H, J 7.0 Hz) supported the presence of ethyl group attached to heteroatom and also two signals at d H 2.84 (t, 2H, J 7.5 Hz) and 2.55 (t, 2H, J 7.5 Hz) typical of methylene hydrogens.
The 13

General procedures
Melting points (mp) were determined on a MQAPF-302 melting point digital apparatus.UV spectra were recorded on a Vankel-50 UV-Vis spectrophotometer.IR spectra were obtained on an FT-IR-1750 spectrophotometer, Perkin-Elmer apparatus.The spectra mass were obtained on a SHIMADZU LCMS-IT-TOF (225-07100-34) equipped with a Z-spray ESI (electrospray) source and operated in negative mode. 1 H and 13 C NMR (1D and 2D) spectra were recorded on a Varian Mercury 200 spectrometer in CDCl 3 and (CD 3 ) 2 CO, with TMS as internal standard.Sephadex LH-20 and silica gel 60 (PF 254 art.7749 and art.7731) were purchased from Merck.
The methylated flavonoids were obtained by treatment of the sample, dissolved in dry propanone, with 1.1 equiv. of dimethyl sulphate (Me 2 SO 4 ) and 1.1 equiv. of potassium carbonate (K 2 CO 3 ) to each free hydroxyl.The reactions were carried at room temperature during 12 h.After removal of the solvent in vacuum, the residue was suspended in H 2 O (50 mL), treated with 5 mL of ammonia and extracted with CHCl 3 (3 × 15 mL).The CHCl 3 solution was dried with Na 2 SO 4 , filtrated and concentrated to dryness. 13

Plant material
Branches of Piper montealegreanum Yuncker was collected in Belém (Pará State, Brazil), in December 2002 and identified in the Botanical Garden, Rio de Janeiro (Rio de Janeiro State, Brazil).A voucher specimen (MSP-010) was deposited at Emilio Goeldi Museum, Belém.
Compound 1a and 2a. 1 H and 13 C NMR spectral data, see Table 2.
d 7.78 (d, 1H) and d 7.84 (d, 1H) to a and b positions, respectively. 16Spectral analysis of 2a showed H a and H b signals with a marked difference in chemical shifts [d 7.29 (d, 1H, J 16.0 Hz, H-7) and d 6.98 (d, 1H, J 16.0 Hz, H-8)] related with the absence of the intramolecular hydrogen bonding with the C=O of the a,b-unsaturated carbonyl group.The
(s, 1H) / d C 166.6 and d C 109.5; d H 4.01 / d C 168.5 that evidenced the hydroxyl, formyl, methyl and methoxyl groups at C-6'-4', C-3', C-5' and C-2', respectively.The hydroxyl group in the C-3 was confirmed for the methylated derivative of 2 (Figure 1) which showed correlation between d H 3.80 / d C 55.3 in the HMQC spectrum and d H 3.80 / d C 159.9 (C-3) in the HMBC spectrum.Other correlations observed in the HMQC spectrum of 2a were d H 3.86 / d C 62.7 and d H 3.75 / d C 61.9.In the HMBC spectrum, the signals at d H 3.86 and 3.75 showed correlations with d C 163.0 and 161.8, respectively, providing support for the presence of the methoxyl groups at the C-6' and C-4' position.