Chemical Constituents from the Aerial Parts of Cyrtopodium paniculatum

Abstract

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.

1. 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 [1,2,3], 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 CH₂Cl₂ 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.

2. Results and Discussion

Structure Elucidation

The pseudobulbs and the leaves from C. paniculatum were studied separately. The freshly cut pseudobulbs (7.5 kg) were ground and macerated in water to get rid of their sugar content, and then in ethanol to afford a crude extract (60 g). The alcoholic crude extract was suspended in water and sequentially extracted with cyclohexane, CH₂Cl₂ and n-BuOH. The CH₂Cl₂ extract was concentrated to dryness and subjected to silica gel column chromatography, Sephadex LH-20 and preparative thin layer chromatography (PTLC) to afford eight new compounds 1–8 (Figure 1) together with 28 previously described compounds 9–35 which were identified as para-ethoxybenzyl alcohol (9) [18,19], 3,4,6-trimethoxyl-9,10-dihydrophenanthrene-2,7-diol (10) [20], confusarin (11) [21], erianthridin (12) [22], para-hydroxybenzaldehyde (13) [18], nudol (14) [23], cephathrene-B (15) [24], gigantol (16) [25], batatasin III (17) [26], ephemeranthoquinone (18) [27], coelonin (19) [28], 2,4-dimethoxy-phenanthrene-3,7-diol (20) [29], lusianthridin (21) [30], densiflorol B (22) [31], denthyrsinin (23) [32], bleformins A (24) and B (25) [33], chrysoeriol (26) [34,35], vanillic alcool (27) [36], gastrodigenin (28) [37,38], 1-(4-hydroxybenzyl)-4-methoxyl-9,10-dihydrophenanthrene-2,7-diol (29) [39], shancidin (30) [40], blestriarenes A (32) and B (31) [41], velutin (33) [42], (+)-syringaresinol (34) [43] and (+)-balanophonin (35) [44], respectively, by comparison of their UV, MS, and NMR data with literature data.

In parallel, the leaves (70 g) were ground, lyophilized and successively extracted by maceration with cyclohexane, CH₂Cl₂ and n-BuOH. The CH₂Cl₂ extract (1.23 g) was fractionated using vacuum liquid chromatography (VLC) and semi-preparative RP-HPLC to afford gastrodigenin (28), coelonin (19), ephemeranthoquinone (18), lusianthridin (21), 1-(4-hydroxybenzyl)-4-methoxyl-9,10-dihydrophenanthrene-2,7-diol (29) and trans-feruloyltyramine (36) [45].

Compound 1 was obtained as a brown amorphous powder and showed a [M + H]⁺ peak at m/z 331.1188 (calcd. for C₁₈H₁₉O₆ 331.1176) in the HRESIMS, indicating a molecular formula of C₁₈H₁₈O₆ and suggesting the existence of ten degrees of unsaturation. The UV maximal absorptions at 216, 261, 281, 312 and 344 nm indicated the presence of a phenanthrene derivative [46,47,48]. The IR spectrum exhibited a broad peak at 3370 cm⁻¹, characteristic of hydroxyl groups, and 1616, 1576, 953 and 860 cm⁻¹, characteristic of aromatic rings. Comparison of the HRMS and the ¹H-NMR data of 1 to those of cephathrene-B (15) [16] indicated substantial similarities between the two compounds, thus suggesting that compound 1 is structurally related to cephathrene-B. Extensive analysis of the 1D NMR of 1 (

Table 1. ¹H (500 MHz) and ¹³C (125 MHz), HMBC and NOESY spectroscopic data of compounds 1–3 (δ in ppm, J in Hz) in acetone-d₆.

No.	1				2				3
	δH	δC	HMBC	NOESY	δH	δC	HMBC	NOESY	δH	δC	HMBC	NOESY
1		142.5			6.59 (s)	112.7	2, 3, 4, 4a, 10	10, 10′		119.7
2		138.5				149.9				147.9
3		149.5				141.1				142.3
4	8.90 (s)	103.9	2, 3, 4b, 10a	3/5-OCH3		152.1				150.6
4a		124.2				120.2				127.8
4b		118.8				124.3				125.5
5		152.3			8.09 (d, 8.6)	129.2	4a, 7, 8, 8a, 9	4-OCH3, 6	9.21 (d, 9.4)	124.4	4a, 4b, 7	4-OCH3, 6
6		142.7			6.76 (dd, 8.6, 2.7)	114.6	4b, 7, 8	5	7.24 (d, 9.4)	118.2	4b, 7, 8	5
7		150.2				157.1				147.2
8	7.17 (s)	109.7	4b, 5, 6, 7, 9	7-OH, 9	7.11 (d, 2.7)	112.8	4b, 6, 7, 9	9		142.1
8a		131.0				143.3				128.8
9	7.50 (d, 8.9)	125.3	4b, 8, 10a	8, 10	4.63 (dd, 10.6, 4.6)	68.9	4b, 8, 8a, 10a, 10	8, 10, 10′	7.87 (d, 9.5)	120.3	8, 4b, 10a	8-OCH3
10	7.88 (d, 8.9)	120.1		9, 1-OCH3	2.70 (dd, 14.3, 10.6)	40.1	1, 4a, 8a, 9, 10a	1, 9, 10	7.90 (d, 9.5)	124.3	1, 4a, 8a	2′/6′, 7′
10					2.83 (dd, 14.3, 4.6)	40.1	1, 4a, 8a, 9, 10a	1, 9, 10′
10a		122.6	1, 4a, 8a			132.1				129.2
1-OCH3	3.99 (s)	61.1	1	2-OH, 10
3-OCH3	4.06 (s)	56.3	3	2-OH, 4	3.85 (s)	61.1	3		4.06 (s)	61.6	3
4-OCH3					3.72 (s)	60.3	4		3.95 (s)	60.1	4
5-OCH3	4.03 (s)	60.5	5	4
6-OCH3	4.02 (s)	61.4	6	7-OH
8-OCH3									3.90 (s)	61.5	8
2-OH	7.91 (s)		1, 2, 3	1/3-OCH3
7-OH	8.39 (s)		6, 7, 8	6-OCH3, 8
9-OH					4.32 (brs)
1′										133.0
2′/6′									7.08 (d, 8.6)	130.1	3′/5′, 4′	3′/5′, 7′, 10
3′/5′									6.68 (d, 8.6)	115.9	2′/6′, 1′	2′/6′
4′/										156.3
7′									4.41 (s)	30.9	1, 10a, 1′, 2′/6′	10

) indicated the presence of two mutually ortho-coupled aromatic protons at δ_H 7.88 (1H, d, J = 8.9 Hz, H-10) and 7.50 (1H, d, J = 8.9 Hz, H-9), thus suggesting the presence of a tetra-substituted aromatic ring. Additional aromatic proton signals at δ_H 8.90 (1H, s, H-4) and 7.17 (1H, s, H-8) afforded two penta-substituted aromatic rings. Two hydroxyl signals at δ_H 7.91 (1H, s, 2-OH) and 8.39 (1H, s, 7-OH) were observed. Four methoxyl groups were presumed based on the ¹H-NMR resonances at δ_H 3.99 (3H, s, 1-OCH₃), 4.06 (3H, s, 3-OCH₃), 4.03 (3H, s, 5-OCH₃) and 4.02 (3H, s, 6-OCH₃) and was supported by the corresponding ¹³C-NMR signals at δ_C 61.1, 56.3, 60.5 and 61.4, respectively.

HMBC correlations from 1-OCH₃ to C-1, 3-OCH₃ to C-3, 5-OCH₃ to C-5 and 6-OCH₃ to C-6 confirmed the position of the methoxyls assigned to C-1, C-3, C-5 and C-6 respectively. In the same way, the locations of the hydroxyl signals at δ_H 7.91 (1H, s, 2-OH) and 8.39 (1H, s, 7-OH) were positioned on C-2 and C-7 respectively, due to HMBC correlations from 2-OH to C-1, C-2 and C-3 and 7-OH with C-6, C-7, and C-8. Furthermore, NOESY correlations between 2-OH and 1/3-OCH₃; 7-OH with 6-OCH₃ and H-8 confirmed the positions of the hydroxyl and methoxyl substituents. Additional HMBC correlations from H-4 to C-2, C-3, C-4b, and C-10a; H-8 with C-4b, C-5, C-6, C-7 and C-9; from H-9 to C-4b, C-8 and C-10a and H-10 to C-1, C-4a and C-8a established the link between the three aromatic moieties. Therefore, 1 was assigned as 1,3,5,6-tetramethoxylphenanthrene-2,7-diol and named cyrtopodin.

Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined as C₁₆H₁₆O₅ as deduced from HRESIMS at m/z 311.0884 [M + Na]⁺; (calcd. for C₁₆H₁₆NaO₅ 311.8900), suggesting the presence of nine degrees of unsaturation. The UV spectrum showed absorption maxima at 220, 262 and 282 nm suggesting a 9,10 dihydrophenanthrene moiety [49,50]. The IR spectrum showed a broad absorption at 3227 cm⁻¹, indicating the presence of hydroxyl groups and at 1611, 1584, 999, 946, 870 and 828 cm⁻¹, characteristic of aromatic rings. The ¹H-NMR spectra (Table 1) showed a close resemblance to those of erianthridin (12) [22], except for the presence of an additional hydroxyl at position 9. This was supported by signals of the ¹H and ¹³C resonance belonging to the methylene protons at δ_H 2.70 (1H, dd, J = 14.3, 10.6 Hz, H-10 ax) and 2.83 (1H, dd, J = 14.3, 4.6 Hz, H-10 eq), a deshielded methine resonance at δ_H 4.63 (1H, dd, J = 10.6, 4.6 Hz, H-9 ax), and deshielded carbon resonance at δ_C 68.9 (C-9). The absolute configuration of compound 2 was obtained with the help of circular dichroism (CD). The CD spectrum of 2 showed a negative Cotton effect at 236 nm and a positive Cotton effect at 281 nm, suggesting a 9S configuration for compound 2 which agrees with previous reports [51,52]. Therefore, 2 was identified as (+)-(S)-3,4-dimethoxyl-9,10-dihydrophenanthrene-2,7,9-triol, and named (S)-9-hydroxyerianthridin.

Compound 3 was obtained as a yellow amorphous powder. It showed a [M + H]⁺ signal at m/z 407.1508 (calcd. for C₂₄H₂₃O₆ 407.1890) in the HRESIMS, indicating a molecular formula of C₂₄H₂₂O₆ and fourteen degrees of unsaturation. Its UV spectrum showed maximal absorptions at 212, 268, 289, 297, 301 and 314 nm suggesting a phenanthrene moiety. The 1D-NMR data indicated that the structure of 3 comprised a confusarin (11) [21] and gastrodigenin (28) [38] subunits. This hypothesis was supported by the 1D and 2D-NMR spectral data (Table 1). In the ¹H-NMR spectrum, six aromatic protons signals at δ_H 9.21 (1H, d, J = 9.4 Hz, H-5), 7.24 (1H, d, J = 9.4 Hz, H-6), 7.87 (1H, d, J = 9.5 Hz, H-9), 7.90 (1H, d, J = 9.5 Hz, H-10) and three methoxyl proton signals at δ_H 4.06 (3H, s, 3-OCH₃), 3.95 (3H, s, 4-OCH₃) and 3.90 (3H, s, 8-OCH₃) were observed, which corresponds to the resonances attributable to the confusarin moiety.

Additional aromatic signals of two equivalent set of mutually coupling protons at δ_H 7.08 (2H, d, J = 8.6 Hz, H-2′/6′) and 6.68 (2H, d, J = 8.6 Hz, H-3′/5′) indicated the presence of a di-substituted aromatic ring which was attributed to the resonance belonging to the gastrodigenin moiety. The linkage between the confusarin and gastrodigenin subunits took place via a methylene bridge at δ_H 4.41 (s, H-7′) and was confirmed by the HMBC correlations from H-7′ to C-2, C-10a, C-2′/6′ (Figure 2). Thus, compound 3 was identified as 1-(4′-hydroxybenzyl)-3,4,8-trimethoxylphenanthrene-2,7-diol and named gastrodiconfusarin.

Compound 4 was obtained as a pale yellow amorphous powder. The HRESIMS showed a [M + H]⁺ molecular ion peak at m/z 511.1742 indicating a molecular formula of C₃₁H₂₆O₇ (calcd. C₃₁H₂₇O₇ for 511.1751) which is consistent with nineteen degrees of unsaturation. Its UV spectrum showed maximal absorptions at 213, 263, 296, 310, 349 and 367 nm. The IR spectrum showed typical absorption bands of a hydroxyl group at 3245 cm⁻¹ and of aromatic rings at 1608, 1585, 948, 865 and 830 cm⁻¹. In 1D-NMR spectrum (

Table 2. ¹H (500MHz) and ¹³C (125 MHz), HMBC and NOESY spectroscopic data of compounds 4–5 (δ in ppm, J in Hz) in acetone-d₆.

No.	4				5
	δH	δC	HMBC	NOESY	δH	δC	HMBC	NOESY
1	7.11 (s)	109.5	2, 3, 4a, 10	10		118.4
2		150.0				148.1
3		142.9				142.6
4		152.4				151.4
4a		119.4				119.4
4b		124.8				124.5
5	9.42 (d, 9.2)	128.1	4a, 7, 8a	4-OCH3, 6	9.42 (d, 9.2)	129.3	4a, 7, 8b	4-OCH3, 6
6	7.31 (d, 9.2)	117.7	4b, 8	5	7.20 (dd, 9.2, 2.7)	117.6	4b, 7, 8	5
7		153.5				156.1
8		121.0			7.21 (d, 2.7)	112.2	4b, 6, 9	9
8a		133.5				134.5
9	7.43 (s)	125.6	4b, 8, 10a	10, 4′	7.41 (d, 9.0)	126.6	4b, 8, 10a	8, 10
10	7.43 (s)	127.2	1, 4a, 8a	1, 9	7.44 (d, 9.0)	126.0	1, 4a, 8a	9, 4′
10a		130.1				129.8
3-OCH3	4.01 (s)	61.4	3		4.06 (s)	61.3	3
4-OCH3	4.00 (s)	60.2	4	5	4.00 (s)	60.1	4	5
1′	6.92 (s)	116.1	2′, 3′, 4a′, 10′	10′	6.89 (s)	115.7	2′, 3′, 4a′, 10′	10′
2′		154.6				154.3
3′		120.6				121.1
4′	8.28 (s)	133.2	8, 2′, 4b′, 10a′	9	8.27 (s)	133.0	1, 2′, 4b′, 10a′	10
4a′		126.2				126.1
4b′		115.8				115.8
5′		156.0				155.9
6′	6.39 (d, 2.6)	101.6	4b′, 7′, 8′	7′-OCH3	6.38 (d, 2.6)	101.6	4b′, 7′, 8′	7′-OCH3
7′		159.5				159.4
8′	6.42 (d, 2.6)	106.1	4b′, 6′, 7′, 9′	7′-OCH3, 9′	6.42 (d, 2.6)	106.1	4b′, 6′, 7′, 9′	7′-OCH3, 9′
8a′		141.5				141.5
9′	2.80 (brs)	30.7	4b′, 8′, 10a′	8′	2.80 (brs)	30.7	4b′, 8′, 10a′	8′
10′	2.80 (brs)	31.6	1′, 4a′, 8a′′	1′	2.80 (brs)	31.7	1′, 4a′, 8a′′	1′
10a′		139.6				139.5
7′-OCH3	3.73 (s)	55.4	7′	6′, 8′	3.74 (s)	55.4	7′	6′, 8′

), the presence of two deshielded aromatic proton signals at δ_H 9.42 (1H, d, J = 9.2 Hz, H-5) and δ_H 8.28 (1H, s, H-4′) suggested that 4 was a dimeric molecule formed from the fusion of a phenanthrene unit [23,46,53] and a 9,10-dihydrophenanthrene subunit [28,54]. The ¹H spectrum showed resonances for a pair of ortho-coupled aromatic protons at δ_H 9.42 (1H, d, J = 9.2 Hz, H-5) and 7.31 (1H, d, J = 9.2 Hz, H-6), a broad singlet integrated for two aromatic protons at δ_H 7.43 (2H, s, H-9, H-10), two meta-coupled aromatic protons at δ_H 6.39 (1H, d, J = 2.6 Hz, H-6′) and 6.42 (d, J = 2.6 Hz, H-8′) and three isolated aromatic protons at δ_H 7.11 (1H, s, H-1), 6.92 (1H, s, H-1′) and 8.28 (1H, s, H-4′). Additional signals belonging to three methoxyl groups at δ_H 4.01 (3H, s, 3-OCH₃), 4.00 (3H, s, 4-OCH₃), 3.73 (3H, s, 7′-OCH₃) and two methylene groups emerging as a broad singlet at δ_H 2.80 (4H, s, H-9′ and H-10′), were also observed. The position of the hydroxyl and methoxyl functions and the linkage between the two subunits were ascertained based on the comprehensive analysis of ¹³C, HMBC and NOESY spectra (Figure 3). HMBC correlations from H-1 and 3-OCH₃ to C-3, as well as HMBC cross peak from 4-OCH₃ to C-4 and NOESY cross peak between 4-OCH₃ and H-5 confirmed the location of the two methoxyl group on position C-3 and C-4 of the phenanthrene unit.

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′-OCH₃ 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 ¹³C 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).

This was also supported by the downfield shift in the ¹³C-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.

Compound 5 was obtained as a pale yellow amorphous powder. Its molecular formula was determined as C₃₁H₂₆O₇ by the HRESIMS [M + H]⁺ peak at m/z 511.1740 (calcd. for C₃₁H₂₇O₇ 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. ¹H (500 MHz), ¹³C (125 MHz), HMBC and NOESY spectroscopic data of compound 6 (δ in ppm, J in Hz) in acetone-d₆.

No.	6
	δH (J, Hz)	δC	HMBC	NOESY
1		116.7
2		160.4
3	6.54 (s)	93.6	1, 2, 4, 4a	4-OCH3
4		159.2
4a		117.6
4b		125.7
5	8.04 (d, 9.4)	130.0	4a, 8a, 7	4-OCH3, 6
6	6.68 (dd, 9.4, 2.5)	113.6	4b, 8	5
7		156.2
8	6.68 (d, 2.5)	115.0	4b, 6, 9	9
8a		139.8
9	2.55, 2.60 (m)	30.7	8	8, 10
10	2.44, 2.55 (m)	27.5	8a	9, 2′
10a		137.2
4-OCH3	3.88 (s)	56.1	4	3, 5
1′		172.3
2′	4.11 (d, 6.6)	58.6	1, 2, 1′, 3′, 4′	10, 3′, 5′, 9′, 1′′-NH
3′	5.67 (d, 6.6)	89.8	2, 1′, 2′, 4′, 5′, 9′	2′, 5′, 9′, 1′′-NH
4′		133.8
5′	6.82 (dd, 8.3, 1.5)	119.5	3′, 7′, 9′	2′, 3′
6′	6.82 (d, 8.3)	115.9	4′, 8′
7′		147.6
8′		148.5
9′	6.99 (d, 1.5)	110.3	3′, 5′, 7′	2′, 3′, 8′-OCH3
8′-OCH3	3.82 (s)	56.4	8′	9′
2′′	3.46 (td, 7.1, 5.5)	41.9	1′, 3′′, 4′′	1′′-NH, 3′′
3′′	2.72 (brt, 7.1)	35.5	2′′, 5′′/9′′	2′′
4′′		130.9
5′′/9′′	7.01 (d, 8.4)	130.6	3′′, 9′′/5′′, 6′′	6′′/8′′
6′′/8′′	6.73 (d, 8.4)	116.1	4′′, 8′′/6′′	5′′/9′′
7′′		156.8
1′′-NH	7.28 (t, 5.5)			2′, 3′, 2′′

). 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 ¹H 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 C₃₃H₃₂NO₇ based on its HRESIMS [M + H]⁺ molecular ion peak at m/z 554.2169 (calcd for C₃₃H₃₂NO₇ 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 ¹³C-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 ¹H-NMR spectrum (Table 3) showed resonances for three aromatic protons as an ABX system owing to a 9,10-dihydrophenanthrene moiety at δ_H 8.04 (1H, d, J = 9.4 Hz, H-5), 6.68 (1H, dd, J = 9.4, 2.5 Hz, H-6), 6.68 (1H, d, J = 2.5 Hz, H-8) and one aromatic proton singlet at δ_H 6.54 (1H, s, H-3). Two methylene proton signals at δ_H 2.45–2.55 (2H, m, H-9) and 2.55–2.60 (2H, m, H-10) and the corresponding carbon signals at δ_C 30.7 and 27.5, respectively, were characteristic of the methylene groups of a 9,10-dihydrophenanthrene skeleton. A 4′-hydroxy-3′-methoxyphenyl moiety comprising of three aromatic protons at δ_H 6.82 (1H, dd, J = 8.3, 1.5 Hz, H-5′), 6.82 (1H, d, J = 8.3 Hz, H-6′) and 6.99 (1H, d, J =1.5 Hz, H-9′) that formed the second ABX system. The presence of an A₂B₂ system characteristic of a symmetrical hydroxybenzyl ring was inferred by the resonance at δ_H 7.01 (2H, d, J = 8.4 Hz, H-5′′/9′′), 6.73 (2H, d, J = 8.4 Hz, H-6′′/8′′), as well as signals attributed to an ethylamide moiety at δ_H 3.46 (1H, td, J = 7.1, 5.5 Hz, H-2′′), 2.72 (1H, brt, J = 7.1 Hz, H-3′′), 7.28 (1H, t, J = 7.28, 1′-NH) 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-OCH₃) and 3.82 (3H, s, 8′-OCH₃) 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-OCH₃.

The relative configuration of the methine protons H-2′ and H-3′ on the furan ring was defined as trans- based 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.

Compound 7 was obtained as a white amorphous powder. Its molecular formula was determined as C₂₆H₂₆O₇ as deduced from its HRESIMS [M + H]⁺ ion peak at m/z 451.1753 (calcd. for C₂₆H₂₇O₇ 451.1751), suggesting the presence of fourteen degrees of unsaturation. The UV spectrum showed absorption maxima at 210, 274 and 284 nm, suggesting that compound 7 has a dihydrophenanthrene skeleton. The IR absorption bands at ν_max at 3303, 1601, 1452 and 773 cm⁻¹ were characteristic to a hydroxyl and aromatic moieties.

The ¹H-NMR spectrum (

Table 4. ¹H (500MHz), ¹³C (125 MHz), HMBC and NOESY spectroscopic data of compounds 7–8 (δ in ppm, J in Hz) in acetone-d₆.

No.	7				8
	δH (J, Hz)	δC	HMBC	NOESY	δH (J, Hz)	δC	HMBC	NOESY
1	6.56 (s)	105.9	2, 3, 4a, 10			117.4
2		160.5				160.3
3		118.7			6.56 (s)	93.5	2, 4a	4-OCH3
4		156.0				158.9
4a		120.9				116.8
4b		125.4				125.8
5	8.03 (d, 9.2)	128.9	7, 8a	4-OCH3, 6	8.04 (d, 9.3)	130.0	7, 8a	4-OCH3, 6
6	6.73 (dd, 9.2, 2.8)	114.2		5, 7-OH	6.68 (dd, 9.3, 2.7)	113.6	4b, 8	5
7		156.7				156.1
8	6.73 (d, 2.8)	115.2		9, 7-OH	6.69 overlapped	115.0	4b, 6, 9	9
8a		140.2				139.8
9	2.69 (m)	30.8	4b, 10, 10a	8	2.59-2.64 (m)	30,4		8
10	2.67 (m)	31.5	8a, 9, 1	1	2.59-2.71 (m)	27.5		12
10a		142.1				137.1
4-OCH3	3.60	60.0	4	5, 12	3.87 (s)	56.0	4	3, 5
7-OH	8.29 (brs)		6, 7, 8	6, 8	8.22 (brs)			11, 12
11	5.66 (d, 5.4)	88.4	2, 13, 2′/ 6′	12, 13, 2′/6′	5.71 (d, 3.4)	88.2	2, 13, 2′	10, 11, 13, 2′/6′
12	3.73 (m)	54.3		11, 13, 2′/6′	3.54 (dt, 8.7, 3,4)	54.5		12
13	4.08 (ddd, 10.4, 4.7, 4.2)	63.5		12	3.63 (m)	64.6		12
	3.83 (m)			11, 12	3.90 (m)			-
13-OH	4,17 (dd, 6.1, 4.7)			13	4.22 (brt 5.3)			-
1′		133.9				134.4
2′/6′	6.75 (s)	104.3	11, 1′, 4′, 6′/2′, 3′/5′	11, 12, 3′/5′-OCH3	6.69 (s)	103.9	11, 1′, 4′, 6′/2′, 3′/5′	11, 12, 3′/5′-OCH3
3′/5′		148.8				148.8
4′		136.6				136.4
3′/5′-OCH3	3.80 (s)	56.7	3′/5′	2′/6′	3.78 (s)	56.7	3′/5′	2′/6′
4′-OH	7.24 (brs)		3′/5′, 4′		7.23 (brs)		3′/5′

) showed resonances for four aromatic protons at δ_H 8.03 (1H, d, J = 9.2 Hz, H-5), 6.73 (1H, dd, J = 9.2, 2.8 Hz, H-6), 6.73 (1H, d, J = 2.8 Hz, H-8), and 6.56 (1H, s, H-1), as well as two methylenes at δ_H 2.69 (2H, m, H-9) and 2.67 (2H, m, H-10), which corresponds to resonances attributed to a 9,10-dihydrophenanthrene ring. A singlet aromatic proton integrating for two protons at δ_H 6.75 (2H, s, H-2′/H-6′) suggested the presence of a symmetrical 1′,3′,4′,5-tetrasubstituted aromatic ring. Three methoxy groups at δ_H 3.80 (6H, s, 3′/5′-OCH₃), 3.60 (3H, s, 4-OCH₃) and two hydroxyl groups at δ_H 8.29 (1H, s, 7-OH) and 7.24 (1H, s, 4′-OH), were also noticed. The linkage between the 9,10-dihydrophenanthrene unit and 1′,3′,4′,5-tetrasubstituted aromatic moiety of compound 7 was achieved through a furan ring as supported by signals assigned to an oxymethine proton at δ_H 5.66 (1H, d, J = 5.4 Hz, H-11), a methine at δ_H 3.73 (1H, m, H-12) and an oxygenated methylene at δ_H 3.83 (1H, m, H-13) and δ_H 4.08 (1H, ddd, J = 10.4, 4.7, 4.2 Hz, H-13) which coupled to an hydroxyl group at δ_H 4.17 (1H, dd, J = 6.1, 4.7 Hz, 13-OH). Analysis of the COSY spectrum (Figure 5) validated the OH-CH₂(13)-CH(12)-CH(11) proton spin systems. Furthermore, HMBC correlations from H-11 to C-2, C-2′/6′; H-2′/6′ to C-11 as well as NOESY cross peaks between H-11 and H-2′/6′; H-2′/6′ and H-12 supported the structure as shown, connecting the phenyl to the furan ring at C-11.

The assignment of the substitution pattern was achieved based on HMBC correlations from 4-OCH₃ to C4 and 3′/5′-OCH₃ to C-3′/5′ suggested that the methoxy groups were attached to C-4 and C-3′/5′, respectively. NOESY correlations between 4-OCH₃ and H-5, and between H-2′/H-6′ to 3′/5′-OCH₃ 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 C₂₆H₂₆O₇ by the HREIMS peak at m/z 451.1748 [M + H]⁺ (calcd. for C₂₆H₂₇O₇ 451.1751). The mass, UV, IR and NMR spectra (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-OCH₃, 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-tetra-hydrophenanthro[2,1-b]furan-7-ol. Compound 8 was named cyrtonesin B.

3. Experimental Section

3.1. 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 ¹³C/¹H Cryoprobe (Bruker Biospin, Fallanden, Switzerland). Acetone-d₆ and methanol-d₄ (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.

3.2. 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.

3.3. Extraction and Isolation

The pseudobulbs (7.5 kg) were cut into small pieces, ground and soaked in demineralized water for 30 min to get rid of the mucilage content. The remaining part was then macerated with ethanol and the filtrates were evaporated under reduced pressure to obtain a crude ethanolic extract (60 g). The ethanolic extract was suspended in water and successively partitioned with cyclohexane, CH₂Cl₂ and n-BuOH to yield after solvent removal a cyclohexane extract (0.60 g), a CH₂Cl₂ extract (1.67 g) and an n-BuOH extract (7.88 g). The CH₂Cl₂ extract was subjected to silica column chromatography (cyclohexane/EtOAc; 100:0 to 0:100, EtOAc/CH₃OH 100:0 to 0:100) to afford 24 fractions (A–X). Fraction D (24.4 mg) was subjected to preparative thin layer chromatography (PTLC; CHCl₃/CH₃OH 94:6) to afford compound 9 (6.3 mg). Fraction F (51.6 mg) was fractionated on Sephadex LH-20 (CH₃OH) to obtain sub-fractions F1-F8. Sub-fractions F4 (7.4 mg) and F5 (25.7 mg) were further purified using PTLC (CHCl₃/CH₃OH 94:6) to afford 10 (4 mg), 11 (5 mg), 12 (5 mg) and 13 (0.9 mg). Fraction G (165.4 mg) was purified using Sephadex LH-20 (CH₃OH) to obtain eight sub-fractions (G1–G8). Sub-fraction G8 was obtained as a pure compound 14 (2.8 mg). Fraction H (71.6 mg) was also subjected to Sephadex LH-20 (CH₃OH) to obtain four sub-fractions (H1–H4). H1 (12.2 mg) led to the isolation of 15 (1.4 mg) and 16 (1.3 mg) on PTLC eluting with CHCl₃/CH₃OH (94:6). Sub-fraction H2 (4.7 mg) led to the isolation of 17 (1 mg) and 18 (0.9 mg) using PTLC (CHCl₃/CH₃OH). Fraction I (87 mg) was purified on Sephadex LH-20 (CH₃OH) to obtain seven sub-fractions (I1-I7). Sub-fraction I1 (25.6 mg) led to the isolation of 19 (13.1 mg), while sub-fraction I2 (13.3 mg) afforded compounds 20 (5.3 mg) and 21 (4.1 mg) using PTLC eluting with CHCl₃/CH₃OH (94:6). Sub-fraction I7 (4.2 mg) was purified on a PTLC (CHCl₃/CH₃OH; 94:6) to obtain compound 1 (1.3 mg). Fraction J (32.2 mg) was fractioned on Sephadex LH-20 (CH₃OH) and PTLC (CHCl₃/CH₃OH; 94:6) to obtain 22 (0.9 mg) and 23 (1 mg). Fraction K (29.5 mg) was purified on Sephadex LH-20 (CH₃OH) affording 7 sub-fractions (K1–K7). Sub-fraction K6 (4.7 mg) was subjected to a PTLC eluting with CHCl₃/CH₃OH (94:6) affording compounds 3 (0.6 mg), 24 (2 mg) and 25 (0.6 mg). Fraction L (26.6 mg) was also subjected to Sephadex LH-20 (CH₃OH) to yield 8 sub-fractions (L1–L8). Sub-fraction L5 (2.2 mg) was purified using PTLC with CHCl₃/CH₃OH (94:6) as the eluting solvent to yield 26 (0.6 mg). Fraction M (90.0 mg) was subjected to Sephadex LH-20 and eluted with CH₃OH to afford 10 sub-fractions (M1–M10). Compounds 27 (2.4 mg) and 28 (6.7 mg) were obtained from sub-fraction M2 (19.9 mg) by PTLC using CHCl₃/CH₃OH as the mobile phase. Sub- fraction M7 (13.2) was further purified by PTLC using CHCl₃/CH₃OH (94:6) to obtain 29 (2.1 g) and 30 (2.6 mg). Sub-fraction M8 (2.9 mg) was passage over PTLC that was eluted with CHCl₃/CH₃OH (94:6) to yield compound 31 (1.1 mg). Sub-fraction M9 (11.6 mg) was purified by PTLC with CHCl₃/CH₃OH (94:6) to yield compounds 4 (0.7 mg), 5 (0.6 mg) and 32 (0.9 mg). Fraction O (71.7 mg) was subjected to Sephadex LH-20 eluting with CH₃OH, to obtain 5 sub-fractions (O1–O5). Sub-fraction O3 (9.8 mg) was purified by PTLC with CHCl₃/CH₃OH (94:6) to obtain compound 2 (2.8 mg). Sub-fraction O5 (2.4 mg) was purified by PTLC with CHCl₃/CH₃OH (94:6) to give compound 33 (0.7 mg). Fraction P (63 mg) was fractionated on Sephadex LH-20 (CH₃OH) to obtain six sub-fractions (P1–P6). Sub-fraction P4 (3.7 mg) was further subjected to PTLC eluting with CHCl₃/CH₃OH (94:6) to obtain compounds 6 (0.6 mg), 7 (0.9 mg) and 8 (0.6 mg). Fraction Q (19.5 mg) was purified on Sephadex LH-20 (CH₃OH) affording six sub-fractions. Compounds 34 (3 mg) and 35 (0.8 mg) were obtained from sub-fraction Q4 (7 mg) through a PTLC with CHCl₃/CH₃OH (94:6) as the eluting solvent.

Fresh leaves were lyophilized and grinded to afford 70 g of dried powder and were successively extracted with cyclohexane, CH₂Cl₂ and CH₃OH (1 g/15 mL × 3) to afford a cyclohexane extract (6.99 g), a CH₂Cl₂ extract (1.23 g) and a CH₃OH extract (4.98 g). The CH₂Cl₂ extract was subjected to vacuum liquid chromatography (VLC) using cyclohexane, EtOAc and CH₃OH to afford six main fractions (A–F). Fraction E (98 mg) was subjected to semi-preparative HPLC (40%–45% B in 5 min, 45% isocratic mode for 30 min, B = CH₃OH/0.05% formic acid, A = H₂O/0.05% formic acid) to afford compounds 13 (3.5 mg), 18 (0.7 mg), 19 (1 mg), and 21 (1.7 mg). Fractions F (158.4 mg) was also subjected to semi- preparative RP-HPLC (35% B for 35 min) to afford compound 36 (9.6 mg).

3.4. Compound Characterization

Cyrtopodin (1). Brown amorphous powder (1.3 mg); UV (CH₃OH) λ_max (log ε): 216 (3.96), 261 (4.38), 281 (4.02), 312 (3.48), 344 (2.52); IR (FT-IR) ν_max: 3370, 2935, 2831, 1616, 1573, 1465, 1268, 1118, 1061, 1021, 953, 860, 770 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 1; HRESIMS: m/z 331.1188 [M + H]⁺ (Calcd. for C₁₈H₁₉O₆, 331.1176).

(+)-9S-Hydroxyerianthridin (2). White amorphous powder (2.8 mg); ${\left[\mathsf{\alpha }\right]}_D^{25}$ + 6.4 (c 0.09, CH₃OH); CD (CH₃OH): nm (Δε): 236.2 (−20.69), 280.9 (+5.39); (UV (CH₃OH) λ_max (log ε): 220 (4.01), 262 (3.91), 282 (4.00), 348 (2.28), 364 (2.26); IR (FT-IR) ν_max: 3227, 2936, 2834, 1611, 1584, 1446, 1412, 1348, 1213, 1112, 1066, 999, 946, 870, 828 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 1; HRESIMS: m/z 311.0884 [M + Na]⁺; (Calcd. for C₁₆H₁₆O₅Na, 311.8900).

Gastrodiconfusarin (3). Yellow amorphous powder (0.6 mg); UV (CH₃OH) λ_max (log ε): 212 (4.11), 268 (4.09), 289 (3.63), 297 (4.00), 301 (3.59), 314 (3.45); IR (FT-IR) ν_max: 3336, 2931, 2936, 1606, 1512, 1454, 1352, 1221, 1106, 1017, 822, 773 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 1; HRESIMS m/z 407.1508 [M + H]⁺ (Calcd. for C₂₄H₂₃O₆, 407.189).

Lusidol A (4). Pale yellow amorphous powder (0.7 mg); ${\left[\mathsf{\alpha }\right]}_D^{25}$ 0 (c 0.04, CH₃OH); UV (CH₃OH) λ_max (log ε): 213 (4.80), 263 (4.82), 296 (4.47), 310 (4.40), 349 (3.58), 367 (3.55); IR (FT-IR) ν_max: 3245, 2931, 2835, 1608, 1585, 1451, 1393, 1351, 1210, 1162, 1118, 1076, 999, 948, 865, 830 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 2; HRESIMS: m/z 511.1742 [M + H]⁺ (Calcd. for C₃₁H₂₇O₇, 511.1751).

Lusidol B (5). Pale yellow amorphous powder (0.6 mg); ${\left[\mathsf{\alpha }\right]}_D^{25}$ 0 (c 0.04, CH₃OH); UV (CH₃OH) λ_max (log ε): 212 (4.72), 264 (4.66), 297 (4.37), 309 (4.29), 350 (3.28), 367 (3.27). IR (FT-IR) ν_max: 3271, 2931, 2835, 1608, 1585, 1451, 1393, 1351, 1210, 1162, 1118, 1076, 999, 948, 865, 830; 527 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 2; HRESIMS: m/z 511.1740 [M + H]⁺ (Calcd. for C₃₁H₂₇O₇, 511.1751).

(±)-Moupilonin (6). White amorphous powder (0.9 mg); ${\left[\mathsf{\alpha }\right]}_D^{25}$ 0 (c 0.07, CH₃OH); UV (CH₃OH) λ_max (log ε): 208 (4.37), 281 (3.97), 305 (3.73), 318 (3.51); IR (FT-IR) ν_max : 3241, 2935, 2829, 1704, 1519, 1447, 1349, 1237, 1199, 1118, 1015, 950, 814, 773 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 3; HRESIMS: m/z 554.2169 [M + H]⁺ (Calcd. for C₃₃H₃₂NO₇, 554.2173).

(+)-Cyrtonesin A (7). White amorphous powder (0.6 mg); ${\left[\mathsf{\alpha }\right]}_D^{25}$ 27 (c 0.04, CH₃OH); CD (CH₃OH): nm (Δε): 233.1 (+0.84), 284.2 (−0.95); UV (CH₃OH) λmax (log ε): 210 (4.30), 274 (3.73), 284 (3.76); IR (FT-IR) ν_max: 3303, 2935, 2831, 1601, 1452, 1359, 1239, 1217, 1114, 1068, 1033, 1008, 977, 951, 824, 773 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 4; HRESIMS: m/z 451.1753 [M + H]⁺ (Calcd. for C₂₆H₂₇O₇ 451.1751).

(+)-Cyrtonesin B (8). White amorphous powder (0.6 mg); ${\left[\mathsf{\alpha }\right]}_D^{25}$ 26 (c 0.02, CH₃OH); CD (CH₃OH): nm (Δε): 235.0 (+1.11), 272.8 (−7.26); UV (CH₃OH) λ_max (log ε): 207 (4.29), 292 (3.73), 301 (3.77); IR (FT-IR) ν_max: 3320, 2936, 2825, 1599, 1460, 1354, 1239, 1221, 1060, 1035, 1008, 977, 955, 825, 773 cm⁻¹; ¹H-NMR and ¹³C-NMR see Table 4; HRESIMS: m/z 451.1748 [M + H]⁺ (Calcd. for C₂₆H₂₇O₇ 451.1751).

4. 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 CH₂Cl₂ 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.