Seco-Dendrobine-Type Alkaloids and Bioactive ... - ACS Publications

Jan 17, 2018 - State Key Laboratory of Phytochemistry and Plant Resources in West China and Yunnan Key Laboratory of Natural Medicinal. Chemistry ...
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Article Cite This: J. Nat. Prod. 2018, 81, 227−235

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Seco-Dendrobine-Type Alkaloids and Bioactive Phenolics from Dendrobium findlayanum Dan Yang,†,‡,§ Zhong-Quan Cheng,‡ Liu Yang,† Bo Hou,† Jing Yang,† Xiao-Nian Li,† Cheng-Ting Zi,†,§ Fa-Wu Dong,† Zheng-Hua Liu,† Jun Zhou,† Zhong-Tao Ding,§ and Jiang-Miao Hu*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Guilin Normal College, Guilin 541001, People’s Republic of China § School of Chemical Sciences and Technology of Yunnan University, Kunming 650091, People’s Republic of China S Supporting Information *

ABSTRACT: Investigation of the 95% EtOH extract of stems of Dendrobium f indlayanum afforded four new seco-dendrobines, findlayines A−D (1−4); two known dendrobines, dendrobine (5) and 2-hydroxydendrobine (6); and four new phenolic compounds, dendrofindlaphenols A−C (7, 9, and 10) and 6″-de-O-methyldendrofindlaphenol A (8). Compounds 1 and 2 are the first seco-dendrobines possessing a sevenmembered lactam moiety, with 3 and 4 derived from the oxidative cleavage of the C-2−C-3 bond of dendrobine. The structures were established using spectroscopic methods and by comparison with literature data. The absolute configurations of 1−4 were confirmed via single-crystal X-ray diffraction data. Cytotoxic activity assays against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cell lines revealed IC50 values ranging from 2.3 to 5.3 μM for compound 7, from 19.4 to 34.4 μM for 8, and from 49.4 to 96.8 μg/mL for the EtOAc extract. An assay of the inhibition of NO production with RAW 264.7 cells indicated that 8 had an IC50 value of 21.4 μM, and the EtOAc extract, 10.5 μg/mL. The EtOAc extract possessed DPPH radical scavenging activity of 69.93% at 100 μg/mL. Dendrobium is a large and diverse genus of Orchidaceae with approximately 1500 species distributed throughout the world. In China, the stems of several Dendrobium species have been used for thousands of years in the folk medicine “Shihu” for the treatment of cancer, chronic atrophic gastritis, skin aging, fever, and cardiovascular disease and more recently as a high-quality health food.1−6 The main chemical components of Dendrobium are sesquiterpenoids, alkaloids, phenolic compounds, and polysaccharides with antitumor, anti-inflammatory, and antioxidant effects.5−16 To date, 19 dendrobine-type alkaloids have been reported from Dendrobium species.17,18 Some of the phenolic compounds, such as moscatilin,7 erianin,9,10 and chrysotoxine,9,10 have been reported to possess antitumor effects. D. f indlayanum Par. et Rchb. f., a perennial herb belonging to the genus Dendrobium, is distributed mainly in southern China. Previous phytochemical research indicated the presence of several dendrobine-type alkaloids and sesquiterpenoids from this species.19,20 To enrich the alkaloid composition of the genus Dendrobium and find more potent bioactive compounds, D. f indlayanum plant materials were collected from Wenshan County, Yunnan Province, People’s Republic of China, and chemically analyzed. As a result, four seco-dendrobine-type alkaloids, findlayines A−D (1−4); two known related dendrobine-type alkaloids, dendrobine (5)18 and 2-hydroxydendrobine (6);19 and four new phenolic compounds, dendrofindlaphenols © 2018 American Chemical Society and American Society of Pharmacognosy

A−C (7, 9, and 10) and 6″-de-O-methyldendrofindlaphenol A (8), were isolated. Compounds 1−10 and the EtOAc and n-BuOH fractions were evaluated for their cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480), their inhibition of nitric oxide (NO) production, their effect on the reactive oxygen species (ROS)

Received: February 20, 2017 Published: January 17, 2018 227

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carbon signals, including four methyl (one N-CH3), three methylene, four methine, two quaternary carbons, two carbonyl carbons, and an oxygenated olefinic tertiary carbon. Compared with the known dendrobine-type alkaloid dendrobine (5),18 the HMBC correlations from H3-16 (δH 2.96) to C-11 (δC 52.1, t) and C-15 (δC 171.6, s) and from H2-11 (δH 3.42, 2.93) to C-15 indicated that C-11 of compound 1 was connected with C-15 via a nitrogen atom, creating a seven-membered lactam moiety (Figure 1). One carbonyl carbon (δC 197.9), an oxygenated

levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and their 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The isolation and structural elucidation of the new compounds together with the biological evaluation of the isolates and fractions will be described herein.

■

RESULTS AND DISCUSSION Compound 1 was obtained as cubic crystals. The molecular formula was assigned as C16H23NO3, with six indices of hydrogen deficiency, from the [M + H]+ (m/z 278.1753, calcd 278.1751) ion in HRESIMS. The 13C NMR data (Table 2) exhibited 16 Table 1. 1H NMR Spectroscopic Data for Compounds (1−4) (δ in ppm, J in Hz) position

2a,c

3a,c

4a,b

Figure 1. Selected HMBC (blue →), and key ROESY (red ↔) correlations of 1.

1 2 3 4 5 6 7

8 9 10 11

12 13 14 15 16 17 NH a

1a,b

3.65, d (7.5) 2.39, t (6.8) 2.00, m

3.52, d (7.2) 2.45, t (6.6) 2.06, m

1.69, m 1.98, m 1.59, m 2.33, t (6.4) 1.17, s 3.42, d (15.2) 2.93, d (7.2)

1.77, m 2.02, m 1.82, m 2.39, t (6.0) 1.22, s 3.13, d (15.0) 2.92, d (7.2)

5.31, d (8.4) 2.29, m 2.51, dd (7.2, 3.6) 2.03, m 2.67, dd (12.6, 6.6) 1.76, m 1.65, dd (6.0, 6.0) 1.72, m 2.45, m 1.35, s 3.39, t (9.6) 3.05, dd (7.8, 7.2)

2.84, m 1.15, d (7.2) 1.09, d (7.2)

2.89, m 1.20, d (6.6) 1.15, d (6.6)

1.75, m 1.01, d (6.6) 0.98, d (6.6)

2.98, s

2.76, s

olefinic carbon (δC 143.7), and a quaternary olefinic carbon (δC 133.2) in the 13C NMR spectrum of 1 established an α,β-unsaturated carbonyl moiety (C-2−C-3−C-4), which was assigned by the HMBC correlations from H3-10 (δH 1.17) to C-2 (δC 197.9, s) and from H-12 (δH 2.84) to C-3 (δC 143.7, s) and C-4 (δC 133.2, s). Thus, the 2D structure of 1 was defined. From the ROESY correlations of H3-10/H-6 and H3-10/H-9, it could be inferred that H-9, H-6, and H3-10 of 1 were cofacial (Figure 1). Single-crystal diffraction analysis of compound 1 was performed. The refined values of the Flack [0.01(18)] and Hooft [0.03(6)] parameters for 767 Bijvoet pairs confirmed that compound 1, findlayine A, had a (1R,5R,6S,9S) configuration (Figure 2).21,22

2.82, m 2.28, m 1.96, m 2.16, m 1.26, m 2.38, dd (8.0, 4.0) 1.41, s 3.59, t (10.0) 3.01, dd (10.0, 6.0) 2.29, m 1.19, d (6.8) 1.05, d (6.8) 2.87, s 3.73, s

6.21, s

Recorded in CDCl3. bRecorded at 400 MHz. cRecorded at 600 MHz.

Table 2. 13C NMR Spectroscopic Data for Compounds (1−4) (δ in ppm)

a

position

1a,b

2a,c

3a,c

4a,b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

52.4, C 197.9, C 143.7, C 133.2, C 51.2, CH 43.4, CH 26.8, CH2 23.7, CH2 49.2, CH 21.8, CH3 52.1, CH2 30.3, CH 19.0, CH3 19.5, CH3 171.6, C 39.8, CH3

53.7, C 198.3, C 143.8, C 133.2, C 50.4, CH 43.8, CH 27.1, CH2 24.4, CH2 49.7, CH 23.0, CH3 44.3, CH2 30.8, CH 19.4, CH3 19.8, CH3 175.1, C

52.2, C 177.1, C 101.5, CH 54.3, CH 44.3, CH 52.4, CH 30.4, CH2 28.8, CH2 46.7, CH 28.1, CH3 53.1, CH2 31.2, CH 19.7, CH3 20.0, CH3 176.3, C 29.7, CH3

59.0, C 178.2, C 169.2, C 143.7, C 129.4, C 51.6, CH 30.2, CH2 34.4, CH2 44.2, CH 26.8, CH3 54.9, CH2 31.8, CH 21.4, CH3 20.6, CH3 169.6, C 30.2, CH3 51.9, CH3

Figure 2. ORTEP drawing of compound 1.

Compound 2 was obtained as colorless needles with a molecular formula of C15H21NO3, with six indices of hydrogen deficiency, based on the ion peak at m/z 264.1594 ([M + H]+, calcd 264.1594) in the HRESIMS. An amide proton (δH 6.21) was inferred from the 1H NMR data (Table 1). By comparing the 13 C NMR spectroscopic data with those of 1 and further referring to the molecular formula of C15H21NO3, it showed that the N-methyl group (δH 2.98) in 1 was replaced by an N-H group in 2. Thus, the 2D structure of 2 was defined. Comparing the ROESY spectra of 1 and 2 revealed that the relative configuration of 2 was identical to that of 1. The absolute configuration of findlayine B (2) was defined as (1R,5R,6S,9S) by X-ray diffraction (Figure 3) analysis.

Recorded in CDCl3. bRecorded at 100 MHz. cRecorded at 150 MHz. 228

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Compound 4 was obtained as cubic crystals and had a molecular formula of C17H25NO5, with six indices of hydrogen deficiency, according to HRESIMS (m/z 324.1805 [M + H]+, calcd 324.1805). Detailed comparison of the NMR data (Tables 1 and 2) of compounds 4 and 3 suggested the presence of a carbonyl carbon signal at δC 169.2 (C-3) and a methoxy group (δC 51.9, C-17) in 4, together with a substituted olefinic moiety with chemical shifts at δC 143.7 (C-4) and 129.4 (C-5). The HMBC correlations from H-12 (δH 2.29) to C-3 (δC 169.2, s), C-4 (δC 143.7, s), and C-5 (δC 129.4, s) and from H3-13 (δH 1.19) and H3-14 (δH 1.05) to C-4 (δC 143.7) confirmed that C-4 and C-5 were olefinic quaternary carbons and C-3 was oxygenated to a hydroxy carbonyl group in 4. The correlation from H3-17 (δH 3.73) to C-15 (δC 169.6, s) implied the location of a methoxy group at C-15 in 4 (Figure 6). In the ROESY

Figure 3. ORTEP drawing of compound 2.

Compound 3 was isolated as colorless needles and determined to possess a molecular formula of C16H25NO4 based on HRESIMS ([M + Na]+ m/z 318.1678, calcd 318.1676), with five indices of hydrogen deficiency. The NMR spectra (Tables 1 and 2) showed four methyl (one N-CH3), three methylene, six methine (one oxygenated), one quaternary, and two carbonyl carbons, suggesting a dendrobine-type alkaloid similar to 5.18 Compared to 5, the C-2−C-3 bond was cleaved and oxygenated in compound 3, which was indicated by the HMBC correlations from H-6 (δH 2.03), H-9 (δH 2.45), H3-10 (δH 1.35), H2-11 (δH 3.29; 3.05), and H3-16 (δH 2.76) to C-2 (δC 177.1, s) and from H-12 (δH 1.75) and H-5 (δH 2.51) to C-3 (δC 101.5, d) (Figure 4).

Figure 6. Selected HMBC (blue →) and key ROESY (red ↔) correlations of 4.

spectrum, correlations of H3-10/H-6 and H3-10/H-9 were observed (Figure 6). Single-crystal X-ray diffraction analysis confirmed the absolute configuration of findlayine D (4) as (1R,6R,9S) (Figure 7).

Figure 4. Selected HMBC (blue →), and key ROESY (red ↔) correlations of 3.

In the ROESY spectrum, cross-peaks of H3-10/H-6, H3-10/ H-9, H-5/H-12, and H-12/H-3 were observed (Figure 4). A seco-dendrobine-type alkaloid with a cleaved C-2−C-3 bond is quite unusual; thus, single-crystal diffraction analysis of compound 3 was performed to verify its absolute configuration. The final refinement of Cu Kα data resulted in a Flack parameter of 0.09(18) and a Hooft parameter of 0.02(6) for 1041 Bijvoet pairs, allowing an unambiguous assignment of the absolute configuration of findlayine C (3) as (1R,3R,4R,5R,6S,9S) (Figure 5). Figure 7. ORTEP drawing of compound 4.

Compound 7, a white, amorphous powder, had a molecular formula of C27H30O8 based on HRESIMS (m/z 505.1838 [M + Na]+, calcd 505.1833), with 13 indices of hydrogen deficiency. The IR absorptions at 3455, 1611, and 1465 cm−1 indicated the presence of hydroxy and aromatic functional groups. The presence of four methoxy, three methylene (one oxygenated), two oxygenated methine, and 18 aromatic carbons were inferred from the 13C NMR and DEPT spectra (Table 3), reflecting a structure similar to that of dendronophenol B.23 The difference between these compounds was that the methoxy group was connected to C-6″ (δC 157.8, s) in 7 instead of C-5″ in dendronophenol B,23 which was supported by the two pairs of equivalent aromatic protons H-4″,8″ (2H, δH 7.09, d, J = 8.4 Hz) and H-5″,7″ (2H, δH 6.82, d, J = 8.4 Hz) and the HMBC cross-peaks

Figure 5. ORTEP drawing of compound 3. 229

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Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 7 and 8 (δ in ppm, J in Hz) 7a δH

position 1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 3‴ 4‴ 5‴

MeO-6 MeO-2′ MeO-6′ MeO-6″

6.52, br s 6.32, br s

6.68, br s 6.68, br s 2.81, m 2.81, m 7.09, d (8.4) 6.82, d (8.4) 6.82, d (8.4) 7.09, d (8.4) 4.95, d (8.0) 4.00−3.97, m 3.90, dd (3.2, 12.4) 3.54, d (12.4) 3.86, s 3.92, s 3.92, s 3.79, s

replacing the methoxy group at C-6″ in 7; the molecular formula of C26H28O8 further confirmed this difference. The relative configuration of 8 was identical to that of 7 based on the ROESY correlations between H-3‴ and H-5‴ and from H-4‴ to H-3′ and H-5′ (Figure 8). Futhermore, ECD calculations permitted assignment of the (3‴S,4‴S) absolute configuration of 8 (Figure 85, Supporting Information). Therefore, the structure of compound 8, 6″-de-O-methyldendrofindlaphenol A, was defined as 4′-[(3S,4S)-4-(hydroxymethyl)-1,2-dioxetan-3-yl]-4(4-hydroxyphenethyl)-2′,6,6′-trimethoxy-[1,1′-biphenyl]-2-ol. Compound 9 was obtained as an amorphous powder and displayed a molecular ion peak at m/z 475.1734 ([M + Na]+, calcd 475.1727) in its HRESIMS spectrum, corresponding to a molecular formula of C26H28O7. On the basis of its NMR data (Table 4), the structure of 9 was similar to that of dendrocandin

8b δC 130.9, C 144.1, C 109.5, CH 134.5, C 104.7, CH 148.4, C 135.2, C 147.2, C 104.0, CH 127.3, C 104.0, CH 147.2, C 38.0, CH2 37.0, CH2 133.7, C 129.3, CH 113.7, CH 157.8, C 113.7, CH 129.3, CH 76.4, CH 78.2, CH 61.5, CH2

56.0, CH3 56.4, CH3 56.4, CH3 55.2, CH3

δH

6.39, d (1.6) 6.33, d (1.6)

6.70, br s 6.70, br s 2.74, m 2.74, m 6.94, d (8.4) 6.65, d (8.4) 6.65, d (8.4) 6.94, d (8.4) 4.82, d (8.4) 4.00−3.97, m 3.70, dd (3.6, 12.4) 3.45, dd (3.6, 12.4) 3.77, s 3.84, s 3.84, s

δC 132.5, C 145.5, C 110.6, CH 133.9, C 106.3, CH 149.7, C 137.1, C 149.4, C 105.8, CH 128.7, C 105.8, CH 149.4, C 39.2, CH2 38.3, CH2 135.6, C 130.5, CH 116.0, CH 156.4, C 116.0, CH 130.5, CH 77.8, CH 79.8, CH 62.1, CH2

Table 4. 1H and 13C NMR Spectroscopic Data for Compound 9 (δ in ppm, J in Hz)a position 1 2 3 4 5 6 2′ 3′ 5′ 6′ 7′ 8′ 9′ 10′ 11′

56.6, CH3 56.8, CH3 56.8, CH3

a

Recorded at 400 and 100 MHz for 1H and 13C NMR in CDCl3, respectively. bRecorded at 400 and 100 MHz for 1H and 13C NMR in methanol-d4, respectively.

1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ MeO-2 MeO-5′ MeO-6″

from MeO-6″ (δH 3.79) to C-6″ in 7 (Figure 8). The relative configuration of 7 was the same as that of dendronophenol B23 on the basis of the ROESY correlations between H-3‴ and H-5‴ and from H-4‴ to H-3′ and H-5′ (Figure 8). In addition, electronic circular dichroism (ECD) calculations were used to assign the (3‴S,4‴S) absolute configuration (Figure 85, Supporting Information). Consequently, the structure of compound 7, dendrofindlaphenol A, was defined as 4′-[(3S,4S)-4-(hydroxymethyl)-1,2-dioxetan-3-yl]-2′,6,6′-trimethoxy-4-(6″-methoxyphenethyl)-[1,1′-biphenyl]-2-ol. Compound 8 was isolated as an amorphous powder. The molecular formula, C26H28O8, was deduced from the [M + Na]+ ion peak in HRESIMS (m/z 491.1685, calcd 491.1676), with 13 indices of hydrogen deficiency. The 13C NMR data (Table 3) were similar to those of 7, with the difference due to a hydroxy group in 8

δH

δC 146.2, C 146.8, C 109.6, CH 128.2, C 120.7, CH 114.6, CH 76.2, CH 78.3, CH 148.3, C 104.7, CH 134.4, C 109.5, CH 144.2, C 130.9, C 61.6, CH2

6.96, br s 6.96, d (8.0) 6.93, d (8.0) 4.94, d (8.0) 3.99, m 6.31, d (1.6) 6.50, d (1.6)

3.50, dd (2.4, 12.4) 3.86, m 2.80, m 2.83, m

38.0, CH2 37.0, CH2 133.7, C 129.3, CH 113.7, CH 157.8, C 113.7, CH 129.3, CH 56.0, CH3 55.2, CH3 56.0, CH3

7.09, d (8.4) 6.82, d (8.4) 6.82, d (8.4) 7.09, d (8.4) 3.91, s 3.79, s 3.85, s

a Recorded at 400 and 100 MHz for 1H and respectively.

13

C NMR in CDCl3,

B,24 except for the absence of MeO-2 in dendrocandin B.24 This was supported by the protons of a 1,3,4-trisubstituted aromatic moiety [H-3 (δH 6.96, br s), H-5 (δH 6.96, d, J = 8.0 Hz),

Figure 8. Selected HMBC (blue →) and key ROESY (red ↔) correlations of 7. 230

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Figure 9. Selected HMBC (blue →) and key ROESY (red ↔) correlations of 9.

Table 5. 1H and 13C NMR Spectroscopic Data for Compound 10 (δ in ppm)a

and H-6 (δH 6.93, d, J = 8.0 Hz)] in 9 in place of the two-proton singlet at δH (6.68, 2H, s) in the spectrum of dendrocandin B,24 as well as the HMBC cross-peak from MeO-2 (δH 3.91) to C-2 (δC 146.8, s) in 9 (Figure 9). The ROESY correlation between H-3′ and MeO-5′ indicated the positions of C-2′ and C-3′ in the 1,4-dioxane ring. The coupling constant (J2′,3′ = 8.0 Hz) between H-2′ and H-3′ and the ROESY correlations of H-2′ with H-11′ and of H-3′ with H-3 and H-5 indicated a threo configuration of the 1,4-dioxane ring, which was identical to those in dendrocandin B (Figure 9).24−27 The absolute configuration was defined via the sign of the specific rotation in comparison with dendrocandin B,24 (−)-aiphanol, and (+)-aiphanol.26 The negative specific rotation of 9 permitted assignment of the (2′S,3′S) absolute configuration. Therefore, the structure of compound 9, dendrofindlaphenol B, was defined as 4-{(2S,3S)-3-(hydroxymethyl)-5-methoxy-7-(4-methoxyphenethyl)-2,3-dihydrobenzo[b][1,4]dioxin-2-yl}-2-methoxyphenol. Compound 10 was isolated as an amorphous powder. Its molecular formula, C27H32O8, with 12 indices of hydrogen deficiency, was deduced from its HRESIMS data ([M + Na]+ m/z 507.2000, calcd 507.1989). Based on its NMR spectroscopic data (Table 5), the structure of compound 10 was similar to dendrocandin M,28 except that a methoxy group was attached to C-6″ (δC 157.9, s) in 10 instead of the hydroxy group in dendrocandin M,28 which was confirmed by the HMBC correlation from MeO-6″ (δH 3.79) to C-6″ (Figure 10). The relative configurations of C-1 and C-2 in 10 were concluded to be erythro29,30 and confirmed by the small coupling constant (J < 1 Hz) between H-1 and H-2 in CDCl3. The absolute configuration was established as (1S,2R) on the basis of the negative specific rotation.31 Thus, the structure of 10 was defined as (1S,2R)-2-[2,6-dimethoxy-4-(4-methoxyphenethyl)phenoxy]1-(4-hydroxy-3-methoxyphenyl)propane-1,3-diol and named dendrofindlaphenol C. The structures of the known compounds 5 and 6 were elucidated by comparison of their spectroscopic data with literature data.18,19 Considering the previous results of cytotoxicity assays of the extracts and compounds from Dendrobium species,7−11 compounds 1−10 and the EtOAc and n-BuOH fractions were evaluated for their in vitro cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480),32 with cisplatin and paclitaxel as the positive controls. Compound 7 showed moderate cytotoxic activity against all five tumor cell lines, with IC50 values of 2.3, 5.1, 4.4, 4.7, and 5.3 μM, respectively; compound 8 exhibited weak cytotoxic activity against the HL-60, SMMC-7721, A-549, and MCF-7 cell lines, with IC50 values of 23.5, 34.4, 20.8, and 19.4 μM, respectively; and the EtOAc fraction exhibited cytotoxic activity against the HL-60, SMMC-7721, A-549, and MCF-7 cell lines, with respective IC50 values of 49.4, 61.3, 96.8, and 90.1 μg/mL (Table 6). Notably, compound 7 showed stronger activities against the SMMC-7721,

position 1 2 3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ MeO-2′ MeO-6′ MeO-6″ MeO-3‴ a

δH

δC

5.00, br s 4.12, m 3.85, m 3.46, m

72.4, CH 87.0, CH 60.6, CH2 132.5, C 152.9, C 105.4, CH 138.5, C 105.4, CH 152.9, C 38.6, CH2 36.9, CH2 133.4, C 129.4, CH 113.7, CH 157.9, C 113.7, CH 129.4, CH 131.3, C 108.2, CH 146.5, C 144.7, C 114.1, CH 118.7, CH 56.0, CH3 56.0, CH3 55.3, CH3 55.9, CH3

6.40, br s 6.40, br s 2.87, m 2.87, m 7.07, d (8.4) 6.82, d (8.4) 6.82, d (8.4) 7.07, d (8.4) 6.96, d (1.6)

6.85, d (8.0) 6.74, d (8.0) 3.83, s 3.83, s 3.79, s 3.89, s

Recorded at 400 and 100 MHz for 1H and respectively.

13

C NMR in CDCl3,

A-549, MCF-7, and SW-480 cell lines than cisplatin. In addition, all compounds (IC50 > 40 μM for all the tested cell lines) and fractions (IC50 > 100 μg/mL for all the tested cell lines) were noncytotoxic in the tested systems. Considering the folk use of D. f indlayanum as both a medicinal herb and a high-quality health food and that some phenolic compounds from Dendrobium species exert significant inhibitory effects on NO production,12,13 compounds 1−10 and the EtOAc and n-BuOH fractions were tested for their inhibition of NO production in LPS-stimulated RAW 264.7 cells in an MTT assay.33 The EtOAc fraction showed activity with an IC50 value of 10.5 μg/mL, compound 8 showed weak activity with an IC50 value of 21.4 μM, and the other compounds did not exhibit inhibitory activity against NO production at 25 μM (Table 7). Furthermore, the level of NO is an essential parameter of oxidative stress. Thus, the level of NO using the NO-sensitive fluorescent probe DAF-FMDA detected via flow cytometry was measured. The results indicated that the level of NO was 231

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Figure 10. Selected HMBC (blue →) correlations of 10.

Table 6. IC50 ± SD Values (μM) of the Isolates and IC50 Values (μg/mL) of the EtOAc Partition from D. f indlayanum for Human Tumor Cell Lines compound/partition

HL-60

SMMC-7721

A-549

MCF-7

SW480

7a 8a EtOAc partitionb DDPc paclitaxelc

2.3 ± 0.2 23.5 ± 2.0 49.4 ± 3.3 1.0 2σ(I)); the final wR(F2) values were 0.0940 (I > 2σ(I)); the final R1 values were 0.0376 (all data); the final wR(F2) values were 0.0940 (all data); the goodness of fit on F2 was 1.090; Flack parameter = 0.09(18); the Hooft parameter is 0.02(6) for 1041 Bijvoet pairs. Crystallographic data for findlayine D (4): C17H25NO5, M = 323.38, orthorhombic, space group P212121, Z = 4, a = 9.8684(8) Å, b = 9.9030(8) Å, c = 17.2493(14) Å, α = β = γ = 90.00°, V = 1685.7(2) Å3, T = 100(2) K, μ(Cu Kα) = 0.768 mm−1; 8889 reflections measured, 2928 independent reflections (Rint = 0.0436); the final R1 values were 0.0503 (I > 2σ(I)); the final wR(F2) values were 0.1684 (I > 2σ(I)); the final R1 values were 0.0503 (all data); the final wR(F2) values were 0.1685 (all data); the goodness of fit on F2 was 1.083; Flack parameter = 0.1(3); the Hooft parameter is 0.04(6) for 1189 Bijvoet pairs.

Findlayine C (3): cubic crystals (MeOH); mp 128−129 °C; [α]20 D −25 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.18) nm; IR (KBr) νmax 3428, 2963, 1769, 1658, 1503, 1463, 1406, 1365, 1305, 1268, 1168, 1115, 1017 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESIMS m/z 318 [M + Na]+; positive-ion HRESIMS [M + H]+ m/z 318.1678 (calcd for 318.1676). Findlayine D (4): cubic crystals (MeOH); mp 145−146 °C; [α]20 D +18 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (2.83) nm; IR (KBr) νmax 3427, 2959, 1729, 1625, 1457, 1403, 1330, 1266, 1233, 1209 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESIMS m/z 346 [M + Na]+; positive-ion HRESIMS [M + H]+ m/z 324.1805 (calcd for 324.1805). Dendrofindlaphenol A (7): white, amorphous power; [α]21 D −8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209.2 (5.25) nm; ECD (c 0.08, MeOH) λ (Δε) 203 (+5.02), 208 (−5.35), 216 (−5.18); IR (KBr) νmax 3455, 2931, 1616, 1511, 1465, 1340, 1245, 1222, 1117, 1037, 830 cm−1; 1 H and 13C NMR data, see Tables 3 and 4; positive-ion ESIMS m/z 505 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 505.1838 (calcd for 505.1833). 6″-De-O-methyldendrofindlaphenol A (8): white, amorphous power; [α]21 D −9 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 209.4 (5.12) nm; ECD (c 0.08, MeOH) λ (Δε) 200 (+1.25), 204 (−3.33), 212 (−4.91); IR (KBr) νmax 3442, 2936, 1601, 1513, 1463, 1341, 1220, 1116, 1049, 831 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive-ion ESIMS m/z 491 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 491.1685 (calcd for 491.1676). Dendrofindlaphenol B (9): white, amorphous power; [α]21 D −8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209.4 (3.26) nm; IR (KBr) νmax 3441, 2930, 1602, 1512, 1454, 1360, 1243, 1118, 1034, 826 cm−1; 1 H and 13C NMR data, see Tables 3 and 4; positive-ion ESIMS m/z 475 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 475.1734 (calcd for 475.1727). Dendrofindlaphenol C (10): white, amorphous power; [α]21 D −11 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203.6 (3.33) nm; IR (KBr) νmax 3442, 2935, 1611, 1513, 1461, 1425, 1243, 1123, 1033, 827 cm−1; H and 13C NMR data, see Tables 3 and 4; positive-ion ESIMS m/z 507 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 507.2000 (calcd for 507.1989). X-ray Crystal Structure Analysis. Colorless crystals of 1, 2, 3, and 4 were obtained in MeOH, respectively. Intensity data were collected at 100 K on an Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97.34 Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 1054043 for 1, CCDC 1407880 for 2, CCDC 1407879 for 3, and CCDC 1054044 for 4. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. + 44(0) (1223) 336 033); e-mail: [email protected]]. Crystallographic data for findlayine A (1): C16H23NO3, M = 277.35, monoclinic, space group P21, Z = 2, a = 8.1168(3) Å, b = 9.4717(3) Å, c = 9.3676(3) Å, α = 90.00°, β = 98.3700(10)°, γ = 90.00°, V = 712.51(4) Å3, T = 100(2) K, μ(Cu Kα) = 0.713 mm−1, 7212 reflections measured, 2138 independent reflections (Rint = 0.0318); the final R1 values were 0.0367 (I > 2σ(I)); the final wR(F2) values were 0.0922 (I > 2σ(I)); the final R1 values were 0.0367 (all data); the final wR(F2) values were 0.0922 (all data); the goodness of fit on F2 was 1.123; Flack parameter = 0.01(18); Hooft parameter is 0.03(6) for 767 Bijvoet pairs. Crystallographic data for findlayine B (2): C15H21NO3, M = 263.33, orthorhombic, space group P212121, Z = 4, a = 7.9580(5) Å, b = 9.8360(6) Å, c = 17.1200(11) Å, α = β = γ = 90.00°, V = 1340.07(15) Å3, T = 100(2) K, μ(Cu Kα) = 0.730 mm−1; 5332 reflections measured, 1966 independent reflections (Rint = 0.0886); the final R1 values were 0.1019 (I > 2σ(I)); the final wR(F2) values were 0.2698 (I > 2σ(I)); the

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00150. 1D and 2D NMR, HRESIMS, IR, UV, and [α]D spectra of the new compounds (1−4 and 7−10) and possible pathways for the biogenesis of 1−4 and 7−10 (PDF) Crystallographic data (CIF) (CIF) (CIF) (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 87165223264. Fax: +86 87165223261. E-mail: [email protected]. ORCID

Jiang-Miao Hu: 0000-0002-9013-8489 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the National Key Research and Development Program of China (2017YFD0201402), the Yunnan Provincial Science and Technology Department (Nos. 2015HB093 and 2015FB168 and the Academician Free Exploration Project), the Guangxi Natural Science Foundation (2017GXNSFBA198033), and the Innovative Team & Outstanding Talent Program of Colleges and Universites in Guangxi. The authors are grateful to the staff of the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for the measurements of all spectra.

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on January 17, 2018, with errors in the abstract graphic, structure graphic, and Figure 4. The corrected version was reposted on January 22, 2018. 235

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