Isolation of Monoterpene Dihydrochalcones from Piper montealegreanum Yuncker (Piperaceae)

Four new compounds were isolated from the branches of Piper montealegreanum Yuncker, a shrub found in the Amazon rainforest, including two new dihydrochalcones named claricine (1) and maisine (2), a cinnamic acid derivative 3 and a phenylalkanoid 4, along with a porphyrin identified as the known compound phaeophytin a (5). The structures were established using spectroscopic experiments, including 1D and 2D NMR and HRESIMS experiments, performed on the two monoterpene dihydrochalcones and their monoacetyl derivatives. The structural diversity of these substances is very important for the Piper genus chemotaxonomy.

Piper montealegreanum is a branching shrub upper with elongate internodes and oblong or lance elliptic leaves with an acuminate apex and subequilaterally rounded. It presents spikes of 3 mm thick and up to 9 cm long; drupes are oblong or obovoid, laterally compressed. It was first recorded in 1908 in the city of Monte Alegre, Pará-Brazil [11].

Results and Discussion
Chromatographic procedures led to the isolation of five compounds from P. montealegreanum ( Figure 1). Compounds 1 and 2 were identified as monoterpene dihydrochalcones. One of the most characteristic features of the structural chemistry of chalcones and dihydrochalcones is their ability to act as dienophiles in enzyme-catalyzed Diels-Alder reactions [13]. In the biosynthetic route proposed by Hano et al. [14] it was possible predicted how an initial adduct reacted with the 2′,4′,2,4tetrahydroxychalcone to form the monoterpene dihydrochalcone (Scheme 1). Few plant families such as the Anonnaceae [15], Moraceae [16] and Piperaceae [17] have a group of adducts. The crinatusins, obtained from the stems of Cyathocalyx crinatus, showed lethal toxicity in a brine shrimp bioassay [15]. Palodesangrens A-E, obtained from the bark of Brosimum rubescens, effectively inhibited the formation of a 5a-dihydrotestosterone androgen receptor complex implicated in androgen-dependent diseases [18]. Cytotoxicity toward KB cells was recorded for both fissistin and isofissistin [19]. The general metabolism of phenylpropanoid 3 can generate an enormous array of secondary metabolites (flavonoids, coumarins, lignins, hydrolyzable tannins) based on the few intermediates of the shikimate pathway as the core unit [20]. Phenylalkanoids like 4 act as chemotaxonomy markers and exhibit antifungal activity [21]. The porphyrinic compounds (such as phaeophytin a) are derived from chlorophyll a and are widely present in the plant kingdom [22].
The various compounds isolated from different Piper species have been classified under twelve categories, viz. alkaloids/amides, propenylphenols, lignans, neolignans, terpenes, steroids, kawapyrones, piperolides, chalcones and dihydrochalcones, flavones, flavanones and miscellaneous compounds [4]. Most of the chalcones and dihydrochalcones isolated from the Piper species are oxygenated at the C-2′, C-4′ and C-6′ positions and the B-ring is unsubstituted in all the chalcones and dihydrochalcones, except asebogenin [23], flavokawains A and C [24] in which C-4 position is also oxygenated. Five new unusual monoterpene-substituted dihydrochalcones, adunctins A-E [25], were isolated from the leaves of P. aduncum. Various propenylphenols have been isolated so far from different Piper species, such as ω-hydroxyisodillapiole [26] and safrole [27]. Besides the above mentioned groups of compounds, several long chain alkanes, alcohols, acids, esters and oxygenated cyclohexanes have been isolated from Piper species (e.g., villiramulin B) [28]. The general metabolism of phenylpropanoid 3 can generate an enormous array of secondary metabolites (flavonoids, coumarins, lignins, hydrolyzable tannins) based on the few intermediates of the shikimate pathway as the core unit [20]. Phenylalkanoids like 4 act as chemotaxonomy markers and exhibit antifungal activity [21]. The porphyrinic compounds (such as phaeophytin a) are derived from chlorophyll a and are widely present in the plant kingdom [22].
The various compounds isolated from different Piper species have been classified under twelve categories, viz. alkaloids/amides, propenylphenols, lignans, neolignans, terpenes, steroids, kawapyrones, piperolides, chalcones and dihydrochalcones, flavones, flavanones and miscellaneous compounds [4]. Most of the chalcones and dihydrochalcones isolated from the Piper species are oxygenated at the C-2 , C-4 and C-6 positions and the B-ring is unsubstituted in all the chalcones and dihydrochalcones, except asebogenin [23], flavokawains A and C [24] in which C-4 position is also oxygenated. Five new unusual monoterpene-substituted dihydrochalcones, adunctins A-E [25], were isolated from the leaves of P. aduncum. Various propenylphenols have been isolated so far from different Piper species, such as ω-hydroxyisodillapiole [26] and safrole [27]. Besides the above mentioned groups of compounds, several long chain alkanes, alcohols, acids, esters and oxygenated cyclohexanes have been isolated from Piper species (e.g., villiramulin B) [28]. The UV spectrum (in MeOH) showed two absorption bands at λmax 274 and 338 (sh) nm. The use of AlCl3 produced bathochromic shift to λmax 299, 313 and 371 (sh) nm, respectively, suggesting the presence of a flavonoid-sustaining chelated hydroxyl group and/or ortho-dihydroxy system [13,29].
The 13 C-NMR spectrum revealed carbon signals (Table 1)    The UV spectrum (in MeOH) showed two absorption bands at λ max 274 and 338 (sh) nm. The use of AlCl 3 produced bathochromic shift to λ max 299, 313 and 371 (sh) nm, respectively, suggesting the presence of a flavonoid-sustaining chelated hydroxyl group and/or ortho-dihydroxy system [13,29].
This data, involving a comparison with 1 H-and 13 C-NMR data of hostmanin D [30], suggested 1 to be a prenylated dihydrochalcone with a totally substituted ring A. This deduction was supported by differences justified by the presence of formyl and hydroxyl groups at rings A and B of 1, in agreement with HRESITOFMS (negative mode) compatible with the proposed fragmentation summarized in Scheme 2.
Compound 4 was isolated as a yellow oil. The IR spectrum showed bands at νmax 2924 and 2852 Spectral analyses of 2a showed a magnetic deshielding effect at CH-2, CH-4 and CH-6 for δ C 120.30, 119.35 and 124.97, respectively. The absorptions at δ C 170.30 (C-10) and 21.08 (CH 3 -11) confirmed the presence of the acetate group. A marked difference was also observed in the chemical shift at CH-6" for δ C 71.17, suggesting the presence of the acetate group at C-6 and a shield of C-7" and CH 2 -5" by γ-effect of the carbonyl carbon.

General Procedures
UV spectra were recorded on a Vankel-50 UV-Vis spectrophotometer (Varian, Palo Alto, CA, USA) and AlCl 3 was used as shift reagent. IR spectra were obtained on an FT-IR-1750 spectrophotometer (Perkin-Elmer, Waltham, MA, USA) using KBr disks. The spectra mass were obtained on a LCMS-IT-TOF (225-07100-34) (Shimadzu, Tokay, Japan) 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 Mercury 500 and 200 MHz spectrometer (Varian, Palo Alto, CA, USA) in CDCl 3 using TMS as internal standard. Sephadex LH-20 and silica gel 60 (PF 254 art. 7749 and art. 7731) were purchased from Merck (Kenilworth, NJ, USA). Hexane, chloroform, ethyl acetate and methanol were of analytical grade and were purchased from Merck. The acetylated flavonoids were obtained by dissolving the samples in pyridine and acetic anhydride (1:1-v/v), and the mixture was maintained under stirring for 48 h. The reaction progress was monitored by TLC. After this period, the mixture was treated with ice-cold distilled water until the formation of a precipitate. The precipitate was filtered and washed several times with distilled water, dissolved in chloroform, and dried with anhydrous sodium sulfate. The remaining solvent was evaporated in a rotating evaporator [34].

Extraction and Isolation
A powered material of P. montealegreanum (1.3 kg) was treated with EtOH (4 × 2.0 L) at room temperature (30 • C) and for three consecutive days, then the solvent was removed under reduced pressure to furnish the crude ethanolic extract (CEE, 115 g), which was partitioned in a series of solvents to yield hexane (44 g), chloroform (37 g), ethyl acetate (8 g) and hydroalcoholic (4.5 g) fractions. A portion (9.5 g) of the hexane fraction was chromatographed over a silica gel column using a gradient of solvents with increasing polarity (hexane, chloroform and methanol) yielding 109 fractions. The fractions 38-42 (1.169 mg) were rechromatographed using Sephadex LH-20 and eluted with methanol. Of the 19 fractions obtained, fractions 12-13 and 14 were purified over silica gel column and preparative TLC to yield 1 (9 mg) and 2 (6 mg), respectively. Fraction 10-11 was chromatographed on Sephadex LH-20 and purified by preparative TLC yielding 4 (12 mg). The supernatant of 12-13 was chromatographed by preparative TLC to obtain a mixture of 1 and 3 (14 mg). 11.0 g of chloroform phase, eluted over Sephadex LH-20 column with a binary mixture of methanol−chloroform (1:1-v/v), provided compound 5 (25 mg).

Conclusions
The present study, based on the phytochemical investigations of P. montealegreanum, using chromatographic and spectroscopic techniques, led to the identification of five substances, including two flavonoids with rare skeletons, called monoterpene dihydrochalcones. The acetylated derivatives of the dihydrochalcones claricine (1) and maisine (2) were prepared to identify their structures. The cinnamic acid derivative, ethyl-3,4,5-trimethoxy-7,8-dihydrocinnamate (3) and the phenylalkanoid, (5-(3E,5E)-undeca-3,5-dienyl)benzo[d] [1,3]dioxole (4) were isolated for the first time as natural products, along with the known compound phaeophytin a (5). Monoterpene dihydrochalcones are unusual compounds restricted to a few plant genus, such as Piper. Besides that, phenylpropanoids are precursors of some classes of secondary metabolites, such as flavonoids. Thus, these structures are important for the chemotaxonomy of the Piper genus. These data corroborate with previous studies on the species evaluating the biological properties of the isolated products.