Chemical Constituents from the Aerial Parts of Cyrtopodium paniculatum

We report the first phytochemical study of the neotropical orchid Cyrtopodium paniculatum. Eight new compounds, including one phenanthrene 1, one 9,10-dihydro-phenanthrene 2, one hydroxybenzylphenanthrene 3, two biphenanthrenes 4–5, and three 9,10 dihydrophenanthrofurans 6–8, together with 28 known phenolic compounds, mostly stilbenoids, were isolated from the CH2Cl2 extract of its leaves and pseudobulbs. The structures of the new compounds were established on the basis of extensive spectroscopic methods.


Introduction
The family Orchidaceae comprises 820 genera with almost 35,000 described species and can be regarded as the largest family of flowering plants. The phytochemical and biological investigations of this family has been mainly conducted in relation with their traditional uses and focused on a large number of Asian orchids from the genus Arundina, Bletilla, Dendrobium, Gastrodia and Pleione at the expense of the New World orchids In fact, the first report on a phytochemical study of South American orchids was in the late 1990s and was mainly focused on species in the genus Scaphyglottis [1-3], Maxillaria [4][5][6][7] and Cyrtopodium [8][9][10].
This genus Cyrtopodium includes 47 endemic species, distributed from Southern Florida to Central America, and they are terrestrial or epiphytic orchids, well recognized by their ovoid to fusiform pseudobulbs as well as their showy flowers [11][12][13][14]. To the best of our knowledge, only three Cyrtopodium species have been explored: C. cardiochilum Lindl. and C. andersonnii R.Br. for their polysaccharidic content, which displays anti-inflammatory and gastroprotective activities [8,10], and C. macrobulbon (La Llave & Lex.) G.A. Romero & Carnevali, a species traditionally used for treating urinary infections and whose stilbenoids are considered as its bioactive constituents [9]. C. paniculatum (Ruiz & Pav.) Garay has not yet received any attention since no report on its medicinal uses or chemical composition has been found in the literature. This provides a substantial basis for a detailed phytochemical investigation of this unexplored species. Therefore, in our continuing search for bioactive secondary metabolites from orchids [15][16][17], we carried out an investigation on the CH 2 Cl 2 extracts from the leaves and pseudobulbs of C. paniculatum. This investigation led to the isolation of eight new compounds 1-8, together with 28 known compounds. In this paper, we describe the structure elucidation of these new compounds, which was performed by means of NMR, HRESIMS data and CD spectrum determination.
The positioning of the substituents on the 9,10-dihydrophenanthrene unit was deduced based on a HMBC cross peak from H-4 to C-2 , as well as H-8 and 7 -OCH 3 to C-7 that ascertained the position of the hydroxyl on C-2 and the methoxyl on C-7 . The remaining carbon (C-7), because of its proton-free environment, didn't allow any HMBC correlations and was thus directly assigned based on the 13 C chemical shift of C-7 (δ C 153.5) which favors a hydroxyl substitution. The two monomers were connected through a C-8-C-3 linkage as indicated by HMBC correlations from H-4 to C-8 and nOe correlations between H-4 and H-9 ( Figure 3).
proton-free environment, didn't allow any HMBC correlations and was thus directly assigned based on the 13 C chemical shift of C-7 (δC 153.5) which favors a hydroxyl substitution. The two monomers were connected through a C-8-C-3′ linkage as indicated by HMBC correlations from H-4′ to C-8 and nOe correlations between H-4′ and H-9 ( Figure 3).  This was also supported by the downfield shift in the 13 C-NMR signal of C-8 and C-3′ as compared to their respective monomeric units (113.0 to 121.0 ppm for nudol (14) and 113.3 to 120.6 ppm for lusianthridin (21)). The optical rotation of compound 4 was zero, and no Cotton effects was  This was also supported by the downfield shift in the 13 C-NMR signal of C-8 and C-3 as compared to their respective monomeric units (113.0 to 121.0 ppm for nudol (14) and 113.3 to 120.6 ppm for lusianthridin (21)). The optical rotation of compound 4 was zero, and no Cotton effects was observed in the CD spectrum, inferring that 4 is a racemic mixture. On the basis of the above data, 4 was identified as 3,4,7 -trimethoxy-9 ,10 -dihydro-[1,3 -biphenanthrene]-2,2,7,7 -tetraol and named lusidol A.  511.1751) which indicated that it has the same molecular formula as compound 4. The UV and IR spectra were identical to those of 4. Comparison of the NMR spectral data of these two compounds revealed their structural similarities, suggesting that the structure of 5 comprises the same phenanthrene (nudol) and 9,10-dihydrophenanthrene (lusianthridin) moieties ( Table 3). The only difference was observed in the linkage that connects the two units: in 6 the connection was via a C-1-C-3 linkage as opposed to the C-8-C-3 linkage in 4 ( Figure 3). This was confirmed by the presence of 1 H signal observed at δ H 7.21 (d, J = 2.7 Hz, H-8) in 5. The NOESY correlations from H-8 to H-9, H-10 to H-4 , as well as subsequent HMBC correlations from H-10 and H-4 to C-1; H-8 to C-4b, C-6 and C-9 confirmed the position of this linkage. The optical rotation experiment result was zero, thus suggesting that 5 is a racemic compound. Therefore, compound 5 was identified as a 3,4,7 -trimethoxy-9 ,10 -dihydro-[1,3 -biphenanthrene]-2,2 ,5 ,7-tetraol and named lusidol B.
Compound 6 was obtained as a white amorphous powder. Its molecular formula was determined to be C 33 H 32 NO 7 based on its HRESIMS [M + H] + molecular ion peak at m/z 554.2169 (calcd for C 33 H 32 NO 7 554.2173). Its UV spectrum showed maximal absorption bands at 208, 281, 305 and 318 nm, indicative of a dihydrophenanthrene derivative. Its IR spectrum exhibited absorption bands at 3241 cm −1 (hydroxyl), 1704 and 1519 cm −1 (amide), 1199, 1118, 1015, 950, 814 and 773 cm −1 (aromatic rings). Analysis of the 13 C-NMR and DEPT-135 spectra of 6 disclosed the presence of signals belonging to 24 aromatic carbons, comprising of eleven methine, seven quaternary, and six oxygenated tertiary carbons. The 1 H-NMR spectrum (Table 3) showed resonances for three aromatic protons as an ABX system owing to a 9,10-dihydrophenanthrene moiety at δ and a amide carbonyl at δ C 172.3, thus achieving a hydroxybenzylethylamide moiety. The connection between the three partial structures of 9,10-dihydrophenanthrene, 4 -hydroxy-3 -methoxyphenyl and hydroxybenzylethylamide moiety was achieved through a central furan ring as observed by the presence of two coupled methines at δ H 4.11 (1H, d, J = 6.6 Hz, H-2 ) and the second being oxygenated at δ H 5.67 (1H, d, J = 6.6 Hz, H-3 ). This was confirmed by extensive analysis of HMBC and NOESY spectra (Figure 4). The oxymethine proton at H-3 (δ H 5.67) showed HMBC correlations to C-2, C-4 , C-5 , C-9 and the amide carbonyl at C-1 ; H-2 to C-1, C-2, C-1 , C-3 , and C-4 . Additional HMBC correlations were observed from the two aromatic protons at H-5 to C-3 and H-9 to C-3 .
This was further supported by NOESY correlations between H-2 to H-10, H-5 , H-9 and 1 -NH; H-3 to H-5 , H-9 , and 1 -NH. The assignments and locations of the two methoxyl groups at δ H 3.88 (3H, s, 4-OCH 3 ) and 3.82 (3H, s, 8 -OCH 3 ) as well as the three hydroxyl groups on position C-7, C7 and C-7 were confirmed by HMBC and NOESY analysis and supported by the characteristic chemical shift of the carbon bearing the groups. The hydroxyl bearing carbon position was settled in C-7 based on the HMBC correlation between H-5 and C-7 and by the characteristic chemical shift at δ C 156.2. The positioning of the methoxyl on C-4 was affirmed from the NOESY cross peaks between H-3, H-5 and 4-OCH 3 .    The relative configuration of the methine protons H-2 and H-3 on the furan ring was defined as transbased on NOESY correlations between H-2 and H-5 /9 , suggesting these protons are on the same side of the furan ring. This was also supported by the coupling constant of 6.6 Hz, typical of trans-dihydrophenanthrenofuran derivatives [55,56], which allows the possibility of two enantiomeric stereoconfigurations, either (2 R, 3 R) or (2 S, 3 S). The absolute configurations of compound 6 could not be ascertained as its optical rotation was zero, suggesting a racemization of the trans form. The 3D structure of 6 ( Figure 4) obtained from a minimized energy MM2 algorithm suggested that a S,S-trans configuration was more appropriate for this compound. Thus, the structure of 6 should be 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-N-(4-hydroxyphenethyl)-10-methoxy-2,3,4,5-tetrahydrophenanthro[2,1-b]furan-3-carboxamide, and it was named moupilonin.
The assignment of the substitution pattern was achieved based on HMBC correlations from 4-OCH 3 to C4 and 3 /5 -OCH 3 to C-3 /5 suggested that the methoxy groups were attached to C-4 and C-3 /5 , respectively. NOESY correlations between 4-OCH 3 and H-5, and between H-2 /H-6 to 3 /5 -OCH 3 also validated this assignment. The hydroxyl groups were assigned to positions C-7 and C-4 based on HMBC long range correlations between 7-OH to C-7 (and additional correlation to C6 and C8), and 4 -OH to C-4 (and additional correlations to C3 /5 ). The relative stereoconfiguration of 7 was established on the basis of NOESY experiments, which displayed cross peaks between H-12 on the furan ring and H-2 /6 indicated that both protons reside on the same side. In addition, the coupling constant of 5.4 Hz between H-11 and H-12 was in agreement with the reported literature values for the relative trans-configuration [57]. The absolute configurations of C-11 and C-12 were confirmed by CD determination: a negative Cotton effect at 284 nm suggested a 11S,12R form, which is consistent with previous reports [58,59]. The 3D structure was generated with an optimized energy minimization procedure using MM2, and suggested also a trans form as a 11S,12R. Therefore compound 7 was identified as (+)-(9S,10R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-10-(hydroxymethyl)-11-methoxy-5,6,9,10-tetrahydrophenanthro[2,3-b]furan-3-ol and named cyrtonesin A .
Compound 8 was obtained as a white amorphous powder and its molecular formula was established as C 26 (Table 4) indicated the striking similarity between compounds 7 and 8. The difference between the two compounds is the positioning of the furan ring on the dihydrophenanthrene chore. In compound 7, the furan ring was positioned as -C2-C3-C12-C-11-O-, while in compound 8 it is positioned as a -C-1-C2-O-C-11-C-12-. This was corroborated by the presence of a singlet proton resonance at δ H 6.56 (1H, s, H-3) as well as HMBC correlations (Figure 5a) from H-3 to C-2 and C-4a as well as NOESY correlations (Figure 5b) between 4-OCH 3 , H-3 and H-5 also validated the proton in position C-3. The methylene at position C-13 also had NOESY correlation with the proton H-10 on the dihydrophenanthrene subunit. The relative configuration between H-11 and H-12 was assumed to be trans because of the NOESY correlation between H-12 to H-2 /H-6 as well as the coupling constant of 3.3 Hz supporting a trans-configuration as previously reported in dihydrophenanthrenofuran derivatives [54]. The CD spectrum of 8 showed a negative Cotton effect at 273 nm, allowing the assignment of a 11S, 12R. Based on the above evidence, the structure of compound 8 was determined to be (+)-(2S,3R)-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(hydroxymethyl)-10-methoxy-2,3,4,5-tetrahydrophenanthro[2,1-b]furan-7-ol.

General Procedures
Optical rotations were measured with a P-2000 polarimeter (Jasco, Lisses, France) and circular dichroïsm spectra were recorded on a Jasco J-510 spectropolarimeter apparatus (Jasco). UV spectra were recorded on a UV-2401 PC spectrometer (Shimadzu, Kyoto, Japan). IR spectra were obtained on a 380 FT-IR spectrophotometer (Thermo Electron Corporation, Saint-Herblain, France).The 1D and 2D NMR spectra were recorded on a Bruker 500 MHz Avance III spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a DCH 13 C/ 1 H Cryoprobe (Bruker Biospin, Fallanden, Switzerland). Acetone-d 6 and methanol-d 4 (Euriso-Top, Saint-Aubin, France) were used as deuterated solvents and their protonated residual signals were used as internal standard at 2.05 ppm and 3.31 ppm respectively, relative to TMS. The HRESIMS analysis were performed on a HPLC-DAD/UV-MS Agilent 1200 series coupled to a 6520 Q-ToF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The acquisition of mass spectra was conducted in ESI positive ion mode. Column chromatography and vacuum liquid chromatography were carried out using 40-60 µm silica gel (Sigma Aldrich, St-Louis, MO, USA). The obtained fractions were monitored by TLC and the spots were visualized under UV light (254 nm) and by 2% sulfuric vanillin reagent. Sephadex LH-20 (Sigma Aldrich, St-Louis, MO, USA), was used for gel chromatography. RP-HPLC experiments were conducted on a Gilson LC system (Gilson Inc., Limburg an der Lahn, Germany) equipped with a semi preparative Kinetex Axia C-18 column (100 mm × 21.2 mm i.d, 5 µm; Phenomenex, Torrance, CA, USA). Experiments were conducted at a wavelength of 280 nm. Preparative TLC was performed on a glass supported silica gel 60F254 (0.25 mm thickness; Merck, Darmstadt, Germany). Analytical grade solvents of HPLC quality were purchased from Sigma Aldrich.

Plant Material
Fifteen fresh specimens of C. paniculatum (Ruiz & Pav.) Garay were purchased from the orchid farm Orquidea del Valle, Ginebra, Colombia in October 2013 and imported to France, according to the Convention of Natural Trades in Endangered Species (CITES). The voucher specimens (No 58054 and 58056) were deposited at the Herbarium of CUVC, Universidad del Valle, Cali, Colombia.
Fresh leaves were lyophilized and grinded to afford 70 g of dried powder and were successively extracted with cyclohexane, CH 2 Cl 2 and CH 3

Conclusions
Due to extreme environmental and territorial conditions as well as predation, plant species have evolved to produce complex secondary metabolites as a means of survival. In orchids, stilbenoids and phenanthrene derivatives are produced as major phytoalexins produced in response to various biotic and abiotic stressors, mostly fungal, bacterial and worm attacks [60][61][62][63][64][65][66]. The phytochemical study of CH 2 Cl 2 extract from the aerial parts of C. paniculatum led us to the isolation of an long list of polyphenolic derivatives, mainly stilbenoids. Monomeric stilbenoid derivatives, comprising bibenzyls, 9,10-dihydrophenanthrenes, phenanthrenes and phenanthrenequinones, are well represented in this orchid. The second set of isolated compounds consisted of phenolic adducts (4-hydroxybenzyl) coupled to a 9,10-dihydrophenanthrene or phenanthrene derivative. This type of 4-hydroxybenzyl adduct (gastrodigenin) is very common in orchids and mostly found in the genus Arundina [67,68], Bletilla [69][70][71][72] and Pleione [40,73,74]. The third set of isolated compounds was a combination of a 9,10-dihydrophenanthrene and a phenylpropane derivative (trans-feruloyltyramine for 6, synapyl alcohol for 7 and 8). The connection between the two units allows a new cyclization leading to a furan ring. It is noteworthy that this kind of dihydrophenanthrene derivative is considered as a chemotaxonomic marker for the species in the genus Pleione [59,73,[75][76][77], but has been also found in the genus Bulbophyllum [55] and Cremastra [54]. The last set of compounds were the dimers, occurring as either homo-(two units of 9,10-dihydrophenanthrene or phenanthrene) or as heterodimers (combinations of a 9,10-dihydrophenanthrene and a phenanthrene unit) having a C-C bond linkage. These biphenanthrene derivatives occur mostly in species of the genus Bletilla [33,41,71,[78][79][80], Bulbophyllum [55,[81][82][83] and Cremastra [54,[84][85][86][87][88]. The existence of dimers together their respective monomers in the same species provides support for a proposed biogenetic pathway for biaryl-derived compounds occurring from their corresponding monomers as a result of free radical [56] or an enzymatic oxidative phenolic coupling reaction [82,[89][90][91]. This study reveals for the first time the chemical diversity of phenolic constituents produced by an endemic South-American orchid, Cyrtopodium paniculatum.