Enantiomeric Pairs of Meroterpenoids with Diverse Heterocyclic

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2018, 81, 1810−1818

Enantiomeric Pairs of Meroterpenoids with Diverse Heterocyclic Systems from Rhododendron nyingchiense Guang-Hui Huang,† Zhu Hu,† Chun Lei,† Pei-Pei Wang,‡ Jing Yang,§ Jing-Ya Li,‡ Jia Li,‡ and Ai-Jun Hou*,†

J. Nat. Prod. 2018.81:1810-1818. Downloaded from pubs.acs.org by QUEEN MARY UNIV OF LONDON on 08/24/18. For personal use only.

†

Department of Pharmacognosy, School of Pharmacy, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 201203, People’s Republic of China ‡ National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China S Supporting Information *

ABSTRACT: Eight enantiomeric pairs of new meromonoterpenoids (1a/1b−8a/8b) and four known compounds (9− 12) were isolated from Rhododendron nyingchiense. Their structures were established by spectroscopic methods, quantum chemical calculations, and X-ray crystallography. The enantiomeric pairs were acquired from scalemic mixtures by chiral-phase HPLC and showed diverse heterocyclic frameworks. Compounds 1a/1b possess a rare 6/7/5/5 heterocyclic system, and 2a/2b incorporate a new 6/6/3/5 heterocyclic system featuring a quinone motif. Compounds 3a/3b represent the first meroterpenoids with a 6/6/5 ring system from the Rhododendron genus. Putative biosynthetic pathways of these compounds are proposed. Compounds 1b, 2a−4a, 8a, 8b, and 11 exhibited weak inhibitory effects on PTP1B, with IC50 values ranging from 5.7 ± 0.5 to 61.0 ± 4.8 μM.

T

he Rhododendron genus in the Ericaceae family has rich resources in China, including 409 endemic species.1 Grayanane diterpenoids are known as the characteristic metabolites of this genus,2 but meroterpenoids have been investigated by natural products chemists in recent years. More than 40 structurally diverse meroterpenoids have been reported, and some of them exhibited anti-HIV and antiHSV-1 activities and the ability to inhibit protein tyrosine phosphatase 1B (PTP1B) and histamine release.3,4 Our previous research on the plant Rhododendron capitatum resulted in the discovery of seven enantiomeric pairs of meroterpenoids, (+)-/(−)-rhodonoids A−G, which were acquired from naturally occurring scalemic mixtures.3 These compounds have soon afterward been synthesized due to their unique structural motifs and interesting bioactivities.5 As a continuing investigation into the Rhododendron species, we focused on R. nyingchiense R. C. Fang et S. H. Huang, which is endemic in Nyingchi County from the Tibet Autonomous Region. No research has been previously reported on this plant. Fractionation of the ethanol extract of R. nyingchiense provided eight enantiomeric pairs of new meromonoterpenoids present as scalemic mixtures (1−8), including (−)-/(+)-nyingchinoids A (1a/1b), C (3a/3b), D (4a/4b), and H (8a/8b) and (+)-/(−)-nyingchinoids B (2a/2b), E (5a/5b), F (6a/6b), and G (7a/7b). Four known compounds, © 2018 American Chemical Society and American Society of Pharmacognosy

confluentin (9), (E)-4-(3,7-dimethylocta-2,6-dienyl)-5-methylbenzene-1,3-diol (10), grifolin (11), and grifolinone A (12), were also isolated. The structures were assigned by spectroscopic methods, X-ray diffraction, and quantum chemical NMR and electronic circular dichroism (ECD) calculations. These terpene−shikimate hybrids displayed diverse heterocyclic frameworks. Compounds 1a/1b incorporate a rare 6/7/5/5 heterocyclic system. Compounds 2a/2b possess a new 6/6/3/5 heterocyclic system and a quinone moiety that is rare in meroterpenoids. Compounds 3a/3b represent the first meroterpenoids with a 6/6/5 ring system from the Rhododendron plants. Putative biosynthetic pathways toward 1a/1b−3a/3b via precursor 10 are proposed. Compounds 1b, 2a−4a, 8a, 8b, and 11 exhibited in vitro inhibition of PTP1B. Herein, the structural characterization, putative biosynthesis, and bioactivity profiling of the isolates are discussed.

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RESULTS AND DISCUSSION Nyingchinoid A (1), an optically active compound ([α]25D −26.5), was assigned a molecular formula of C17H22O4 with Received: April 4, 2018 Published: August 1, 2018 1810

DOI: 10.1021/acs.jnatprod.8b00273 J. Nat. Prod. 2018, 81, 1810−1818

Journal of Natural Products

Article

Chart 1

Table 1. 1H and

13

C NMR Spectroscopic Data for 1−4 in CDCl3 1a

δΗ (J in Hz)

no. 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16

2.13, dd (14.4, 3.0) 4.91, d (3.0)

6.40, d (3.0) 6.32, d (3.0) a 2.57, dt (15.0, 9.0) b 2.37, ddd (15.0, 10.0, 1.2) a 1.77, m b 1.34, m 3.42, ddd (14.4, 12.6, 6.0)

2b δC 81.3 64.3 96.3 138.7 134.0 113.0 151.9 109.7 151.5 47.4 21.4

1.52, s

52.1 80.2 29.8

1.17, s 1.28, s 2.23, s

22.9 24.9 16.6

3a

δΗ (J in Hz)

δC

2.20, dd (8.0, 4.0) 4.03, d (4.0)

90.9 43.7 63.7

6.28, br s 6.00, d (1.6) 2.08, 1.88, 1.91, 1.43, 2.55,

m mc mc m q (8.0)

4b

δΗ (J in Hz)

54.7 149.0 131.9 187.7 115.3 167.2 40.5 25.5

1.24, s

53.8 72.4 26.0

1.35, s 1.30, s 1.86, br s

30.4 27.3 16.0

2.08, m a 2.33, dd (13.8, 4.8) b 2.06, mc

6.26, d (2.4) 6.21, d (2.4) 2.04, 1.77, 1.95, 1.68, 2.74,

mc m m m m

a 4.91, s b 4.73, s 1.76, s 1.39, s 2.16, s

δC 86.0 45.3 20.3 116.2 137.2 109.7 154.2 102.4 155.1 38.0 25.0

δΗ (J in Hz)

δC

2.56, dd (9.6, 8.0) 3.08, d (9.6)

83.4 39.3 38.0

6.28, d (2.4) 6.24, d (2.4) 1.98, 1.60, 1.72, 1.63, 2.41,

m mc m mc td (8.0, 3.0)

116.2 138.6 111.0 154.4 102.9 154.7 39.5 25.5

48.9 144.7 111.2

1.38, s

46.6 39.7 34.1

23.4 28.4 19.2

0.66, s 1.32, s 2.11, s

18.7 26.8 20.2

a

600 MHz (1H) and 150 MHz (13C). b400 MHz (1H) and 150 MHz (13C). cThe signals are overlapped.

additional rings should be present in the structure of 1. It was therefore speculated to possess a tetracyclic system, one of the rings being a benzene moiety. The 2D structure of 1 was assembled via the 2D NMR data. The 1H−1H COSY and HSQC spectra provided the C(4)H− C(3)H−C(11)H−C(10)H2−C(9)H2 structural unit shown in bold lines (Figure 1). The H-4/C-12; H3-13, 14/C-11, C-12; H-3/C-2, C-12; H2-9/C-2, C-3; and H3-15/C-2, C-3, C-9 HMBC correlations (Figure 1) established a cyclopentane and a furan ring. The H3-16/C-4a, C-5, C-6; H-6/C-4a, C-7, C-8; and H-8/C-4a, C-7, C-8a HMBC correlations constructed a trioxygenated methylphenyl moiety. These data delineated three rings, and the remaining one was identified as a 1,4-

seven indices of hydrogen deficiency, as suggested by the (+)-HRESIMS (m/z 291.1593 [M + H]+; calcd for C17H23O4, 291.1591). The IR spectrum exhibited absorptions for hydroxy (3463 cm−1) and aromatic (1611 and 1463 cm−1) functionalities. The NMR data (Table 1) displayed signals for a 1,2,3,5tetrasubstituted benzene ring (δH 6.40, H-6; 6.32, H-8; δC 138.7, C-4a; 134.0, C-5; 113.0, C-6; 151.9, C-7; 109.7, C-8; 151.5, C-8a), an acetal moiety (δH 4.91, H-4; δC 96.3, C-4), four methyl singlets (δH 1.52, H3-13; 1.17, H3-14; 1.28, H3-15; 2.23, H3-16; δC 29.8, C-13; 22.9, C-14; 24.9, C-15; 16.6, C16), and two oxygenated tertiary carbons (δC 81.3, C-2; 80.2, C-12), as well as resonances for two methylenes and two methines. Based on the indices of hydrogen deficiency, three 1811



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named (−)-nyingchinoid A and (+)-nyingchinoid A, respectively. Nyingchinoid B (2), an optically active compound ([α]25D +12.6), had the same molecular formula (C17H22O4) as 1 based on the (+)-HRESIMS and 13C NMR data. The IR spectrum displayed absorptions for hydroxy (3460 cm−1) and conjugated carbonyl (1659 cm−1) groups. The NMR data indicated the presence of four methyl singlets, two methylenes, three methines (one oxygenated), two trisubstituted double bonds, a conjugated carbonyl carbon, and three oxygenated tertiary carbons (Table 1). Additionally, a tetracyclic system was required to satisfy the indices of hydrogen deficiency. The H3-16/C-4a, C-5, C-6; H-6/C-4a, C-8; and H-8/C-4a, C-8a HMBC correlations (Figure 1) and the shielded ketocarbonyl (δC 187.7, C-7) due to the conjugated double bonds revealed the presence of a quinone methide moiety. The chemical shifts of C-4 (δC 63.7) and C-4a (δC 54.7) supported the presence of a 4,4a-oxirane unit. A H-4/H-3/H-11/H2-10/H2-9 spin system was evident in the 1H−1H COSY spectrum (Figure 1). The H4/C-2, C-3 and H3-15/C-2, C-3 HMBC correlations together with the two “unassigned” oxygenated tertiary carbons at C-8a (δC 167.2) and C-2 (δC 90.9) indicated the presence of a tetrahydropyran moiety. The presence of a cyclopentane moiety carrying a 2-hydroxypropyl unit at C-11 was indicated by the H-3/C-9, C-12; H-9/C-2; H3-15/C-9; and H3-13, 14/ C-11, C-12 HMBC correlations. Compound 2 was thus defined as a meromonoterpenoid with a new 6/6/3/5 heterocyclic system, comprising a quinone moiety and a 2,5dioxatricyclo[5.3.0.04,6]decane motif. The ROESY correlation of H-3/H3-15 implied their synperiplanar relationship, assigning their β-orientation (Figure 2). The H-3/H-4 and H-3/H3-13, H3-14 ROESY correlations suggested their cis-relationship and an αorientation for the 4,4a-oxirane moiety and H-11, but further evidence was required to prove this. In order to unambiguously assign the relative configurations at C-4, C-4a, and C-11, calculations of the 13C NMR chemical shifts for structures 2a′ (2S, 3S, 4R, 4aR, 11S), 2b′ (2S, 3S, 4S, 4aS, 11S), 2c′ (2S, 3S, 4R, 4aR, 11R), and 2d′ (2S, 3S, 4S, 4aS, 11R) were performed at the mPW1PW91/6-31G(d,p) level with the GIAO method

Figure 1. Key HMBC and 1H−1H COSY correlations for 1−6.

dioxepine moiety with two O atoms between C-4a and C-4 and between C-8a and C-2, as supported by their diagnostic chemical shifts and the H-4/H3-16 and H-8/H3-15 NOESY correlations (Figure 2). Therefore, the structure of 1 was defined as a meromonoterpenoid incorporating the same benzo[c]-2,5,7-trioxatricyclo[7.2.1.06,12]dodecane ring system as rasumatranin D.6 Compound 1 is the second compound possessing such a 6/7/5/5 heterocyclic system. The coupling constants of H-3/H-4 (J = 3.0 Hz) and H-3/ H-11 (J = 14.4 Hz) suggested 3,4-cis and 3,11-trans configurations. Furthermore, the H-3/H3-14, H3-15; H-4/H315; and H-11/H3-13 NOESY correlations (Figure 2) assigned a β-orientation for H-3, H-4, and H3-15 and an α-orientation for H-11. Chiral-phase HPLC analysis revealed that 1 was a scalemic mixture with two enantiomers in the ratio of 6.5:1 (Figure 3). The two enantiomers 1a ([α]25D −36.2) and 1b ([α]25D +37) were obtained by chiral-phase resolution. Their NMR and MS spectra are shown in Figures S22−S27, Supporting Information, and the ECD curves in Figure 3. A single-crystal X-ray diffraction experiment with Cu Kα radiation for 1a defined its (2S, 3R, 4S, 11S) absolute configuration [absolute configuration parameter: 0.15(7)] (Figure 4). Thus, the absolute configuration of 1b was assigned as (2R, 3S, 4R, 11R). Compounds 1a and 1b were

Figure 2. Key NOESY correlations for 1 and 3−6 and ROESY correlations for 2. 1812



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Figure 3. Chiral separation chromatograms (columns A and C) and ECD spectra (columns B and D) for enantiomers 1a/1b−8a/8b.

Figure 4. X-ray ORTEP plots for 1a and 3a−6a.

the experimental and calculated chemical shifts. The relative configuration of 2 was thus elucidated as 2S*, 3S*, 4R*, 4aR*, 11S*. By chiral-phase HPLC analysis of 2 (Figure 3), two

(Supporting Information). Model compound 2a′ showed a lower LAD (largest absolute deviation) and MAD (mean absolute deviation) than 2b′, 2c′, and 2d′ by comparison of 1813


Journal of Natural Products Table 2. 1H and

13

Article

C NMR Spectroscopic Data for 5−8 in CDCl3a 5

no. 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16 OMe-12

δΗ (J in Hz) 3.87, d (3.6) 3.20, t (3.6)

6.14, d (2.4) 6.13, d (2.4) a 1.97, td (13.2, 4.8) b 1.77, br d (13.2) a 1.56, qd (13.2, 4.2) b 1.31, br d (13.2) 2.75, dt (12.6, 3.6) a 4.72, br s b 4.42, br s 1.75, s 1.37, s 2.08, s

6 δC 76.3 72.5 40.2 113.6 139.6 108.8 154.8 99.5 156.9 34.2 22.6 41.2 147.4 111.7 23.2 26.1 19.8

δΗ (J in Hz) a 1.81, dd (13.2, 3.0) b 1.70, dt (13.2, 3.0) 3.46, br s

6.17, d (2.4) 6.14, d (2.4) 1.99, 1.53, 1.46, 1.37, 1.83,

br d (13.2) td (13.2, 4.2) br d (13.2) qd (13.2, 4.2) dt (12.6, 3.0)

7 δC 74.5 39.5 29.2 115.9 139.0 108.9 154.7 100.2 157.9 40.7 21.6

1.12, s

52.1 77.5 25.5

0.74, 1.32, 2.34, 3.27,

19.3 28.7 19.9 48.5

s s s s

δΗ (J in Hz) 1.82, dd (13.2, 3.0) 1.72, dt (13.2, 3.0) 3.50, br s

6.18, d (2.4) 6.16, d (2.4) 2.00, 1.52, 1.54, 1.35, 1.69,

br d (13.2) td (13.2, 4.8)b br d (13.2)b qd (13.2, 4.2) dt (13.2, 3.0)

8 δC

δΗ (J in Hz)

δC

74.3 39.5

3.74, dd (9.0, 3.0)

77.1 72.2

29.7 115.6 138.6 109.1 154.8 100.5 158.0 40.7 21.4

1.23, s

54.1 73.6 31.5

0.94, s 1.32, s 2.38, s

25.1 28.6 20.7

3.36, t (3.0)

6.19, d (2.4) 6.17, d (2.4) 2.03, 1.57, 1.45, 1.30, 2.32,

ddd (13.2, 4.2, 2.4) td (13.2, 4.2) qd (13.2, 4.2) br d (13.2) dt (12.6, 3.0)

4.71, 4.39, 1.75, 1.41, 2.10,

br s br s s s s

41.3 109.3 141.7 109.7 155.1 99.9 157.1 39.0 22.6 49.7 146.4 111.9 23.2 23.9 19.8

a

600 MHz (1H) and 150 MHz (13C). bThe signals are overlapped.

and H-11/H3-15 correlations indicated that they were cofacial and β-oriented. Compound 3 was a scalemic mixture with a ratio of approximately 3.5:1 (Figure 3) and a specific rotation of [α]25D −9. Compounds 3a and 3b, a pair of enantiomers with identical NMR and MS spectra (Figures S56−S61, Supporting Information) and opposite specific rotations (3a: [α]25D −14.2; 3b: [α]25D +14.6) and ECD data (Figure 3), were obtained by chiral-phase HPLC separation. An X-ray crystallography study assigned the absolute configuration of 3a as (2S, 3R, 11R) [absolute configuration parameter: 0.02(10)] (Figure 4). The absolute configuration of 3b was accordingly defined as (2R, 3S, 11S). Compounds 3a and 3b were named (−)-nyingchinoid C and (+)-nyingchinoid C, respectively. Nyingchinoid D (4), [α]25D −9.6, was assigned the same molecular formula as 3 by means of the (+)-HRESIMS and 13 C NMR data. Analysis of its NMR data (Table 1) and HMBC spectrum (Figure 1) revealed a 6/6/5/4 (benzo[c]-2oxatricyclo[5.2.1.05,10]decane) tetracyclic system, consistent with that of rhodonoid B.3a Being a meromonoterpenoid, a C13 methyl group was present in 4 rather than the 4methylpent-3-en-2-one moiety in rhodonoid B. Moreover, it had the same benzene motif as 3, which is different from rhodonoid B.3a The differences were verified by the HMBC correlations shown in Figure 1. The coupling constants of H3/H-4 (J = 9.6 Hz) and H-3/H-11 (J = 8.0 Hz) and the NOESY correlations of H-3/H3-15 and H-11/H3-15 (Figure 2) indicated that 3 had the same relative configurations at C-2, C-3, C-4, and C-11 as its analogues, rhodonoids B, E, and F3 and rhododaurichromanic acid A,4a assigning a β-orientation for H-3, H-4, H-11, and H3-15. Chiral-phase HPLC separation afforded two enantiomers (4a and 4b) with a peak area ratio of approximately 3:1 (Figure 3). The enantiomeric relationship was confirmed by their specific rotations (4a: [α]25D −15.6;

enantiomers in an approximate ratio of 5:1 (2a and 2b) were obtained. Their specific rotations (2a: [α]25D +17; 2b: [α]25D −16.8) and ECD data (Figure 3) were opposite, and their NMR and MS data (Figures S40−S45, Supporting Information) were identical. The absolute configurations of 2a and 2b were established by comparison of their experimental ECD data with those calculated for 2a′ (2S, 3S, 4R, 4aR, 11S) and ent-2a′ (2R, 3R, 4S, 4aS, 11R), respectively (Supporting Information). The ECD spectrum of 2a matched well with that of 2a′, and the ECD curve for 2b showed consistency with that of ent-2a′ (Figure 3). Hence, the absolute configurations of 2a and 2b were elucidated as (2S, 3S, 4R, 4aR, 11S) and (2R, 3R, 4S, 4aS, 11R), and the compounds were named (+)-nyingchinoid B and (−)-nyingchinoid B, respectively. Nyingchinoid C (3) gave an [M + H]+ ion at m/z 259.1689 (calcd 259.1693) in the (+)-HRESIMS, which established the molecular formula of C17H22O2. The 1H and 13C NMR spectra supported by the DEPT and HSQC data indicated the presence of a 1,2,3,5-tetrasubstituted benzene moiety, a double bond, three methyl singlets, three methylenes, two methines, and an oxygenated tertiary carbon (Table 1). These data satisfied five indices of hydrogen deficiency, and two extra rings were required to fulfill the remaining indices of hydrogen deficiency. The HMBC data facilitated construction of the 2D structure of 3. The H2-4/C-2, C-3, C-4a, C-5, C-8a, C-11; H315/C-2, C-3; H-6/C-4a, C-7, C-8; H-8/C-4a, C-8a; and H316/C-4a, C-5, C-6 HMBC correlations proved the presence of a chromane moiety (Figure 1). The H-3/C-9, C-10; H2-9/C-2, C-10, C-11; H3-15/C-9; H2-13/C-11, C-12, C-14; and H3-14/ C-11 HMBC correlations defined the cyclopentane moiety with an isopropenyl group at C-11. Thus, the structure of 3 was elucidated as a meromonoterpenoid with a benzo[c]-2oxadicyclo[4.3.0]nonane scaffold. Its relative configuration was assigned by the NOESY spectrum (Figure 2). The H-3/H3-15 1814



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4b: [α]25D +15.2), ECD spectra (Figure 3), and NMR and MS data (Figures S72−S77, Supporting Information). The absolute configuration of 4a was determined as (2S, 3S, 4S, 11R) [absolute configuration parameter: 0.16(10)] by X-ray diffraction data analysis (Figure 4). The absolute configuration of 4b was thus assigned as (2R, 3R, 4R, 11S). Compounds 4a and 4b were assigned the trivial names (−)-nyingchinoid D and (+)-nyingchinoid D, respectively. Nyingchinoid E (5) was obtained as an optically active compound ([α]25D +14). Its molecular formula was established as C17H22O3 based on the (+)-HRESIMS and 13C NMR data. The NMR (Table 2) and HMBC data (Figure 1) indicated a meromonoterpenoid structure with the same 6/6/6 (benzo[c]2-oxadicyclo[3.3.1]nonane) tricyclic system as rhodonoid G.3b The benzene moiety in 5 was consistent with those in 3 and 4. The deshielded chemical shifts of H-3 (δH 3.87) and C-3 (δC 72.5) and the H-3/C-2, C-9, C-11 and H3-15/C-3 HMBC correlations indicated the presence of a hydroxy group at C-3. Additionally, an isopropenyl group was evident from the NMR data (δH 4.72 and 4.42, H2-13; 1.75, H3-14; δC 147.4, C-12; 111.7, C-13; 23.2, C-14), which was located at C-11 based on the H3-14/C-11 and H2-13/C-11 HMBC correlations. The cyclohexane ring in 5 possesses the same chair conformation with equatorial H-4 and H3-15 as those in rhodonoid G3b and rasumatranins A−C.6 The H-11/H-9a NOESY correlation indicated their 1,3-diaxial relationship, assigning a βorientation for H-11 and H-9a (Figure 2). The coupling constants of H-4/H-11 (J = 3.6 Hz) and H-11/H-10a (J = 12.6 Hz) further validated that H-4 was equatorial and H-11 was axial. By comparing with rasumatranin B,6 C-9 and C-11 in 5 were shielded by ΔδC 5.6 and 9.0, respectively. These variations were ascribed to the γ-gauche effect of HO-3, indicating that HO-3 was axially oriented.6 The NOESY crosspeaks of H3-15 with axial H-9a and equatorial H-9b confirmed an equatorial orientation for H3-15. The structure of 5 and its relative configuration were hence elucidated. Two enantiomers (5a and 5b) with a peak area ratio of approximately 8:1 were acquired by chiral-phase separation (Figure 3) and corroborated by their specific rotations (5a: [α]25D +19.8; 5b: [α]25D −19.5), ECD curves (Figure 3), and NMR and MS spectra (Figures S88−S93, Supporting Information). The absolute configuration of 5a was determined as (2S, 3S, 4S, 11R) [absolute configuration parameter: 0.11(6)] by X-ray crystallographic data analysis (Figure 4), and the compound was named (+)-nyingchinoid E. The absolute configuration of 5b was established as (2R, 3R, 4R, 11S), and the compound was named (−)-nyingchinoid E. Nyingchinoid F (6), a compound with optical activity ([α]25D +13.4), had the molecular formula C18H26O3, as deduced from its (+)-HRESIMS and 13C NMR data. It is also a meromonoterpenoid possessing the same 6/6/6 tricyclic scaffold as 5, as supported by the 1D and 2D NMR spectra. The differences were the absence of a hydroxy group at C-3 and the presence of 2-methoxypropyl group at C-11 in 6 instead of the isopropenyl group. These changes were corroborated by the methylene resonances for H2-3 and C-3 (Table 2) and the HMBC correlations of H3-13, 14/C-11, C12 and OCH3/C-12 (Figure 1). The relative configurations at C-2, C-4, and C-11 were consistent with those of 5, as evidenced by the similar coupling constants and NOESY data (Figure 2). By chiral-phase resolution (Figure 3), the two enantiomers 6a and 6b (7:1) were obtained. The specific rotations (6a: [α]25D +18.1; 6b: [α]25D −17.2), ECD curves

(Figure 3), and NMR and MS spectra (Figures S104−S109, Supporting Information) confirmed their enantiomeric relationship. An X-ray diffraction measurement established the absolute configuration of 6a as (2S, 4S, 11R) [absolute configuration parameter: −0.03(8)] (Figure 4). The absolute configuration of 6b was then assigned as (2R, 4R, 11S). Therefore, the structures of compounds 6a and 6b were assigned and named (+)-nyingchinoid F and (−)-nyingchinoid F, respectively. Nyingchinoid G (7) was acquired as an optically active compound ([α]25D +6.7). Inspection of its NMR data (Table 2) implied that its structure resembled that of 6 with the difference being the appendage at C-11, where the 2hydroxypropyl group in 7 replaced the 2-methoxypropyl moiety in 6. This was confirmed by its molecular formula C17H24O3 assigned from the (+)-HRESIMS data and the shielded C-12 (ΔδC −3.9) and deshielded C-11, C-13, and C14 (ΔδC +2.0, +6.0, and +5.8, respectively). The 2D structure of 7 was confirmed by the HMBC data (Figure S1, Supporting Information). The relative configuration of 7 was identical to that of 6, as evidenced by the NOESY data (Figure S1, Supporting Information). Chiral-phase analysis of 7 showed a scalemic mixture with a 2:1 approximate ratio (Figure 3). The two resulting enantiomers 7a and 7b showed the anticipated opposite specific rotations (7a: [α]25D +16.2; 7b: [α]25D −15.8) and ECD curves (Figure 3) and identical NMR and MS data (Figures S120−S125, Supporting Information). The absolute configurations of 7a and 7b were established as (2S, 4S, 11R) for 7a and (2R, 4R, 11S) for 7b by comparison of their ECD data with those of 6a and 6b, respectively. Compounds 7a and 7b were therefore structurally characterized and named (+)-nyingchinoid G and (−)-nyingchinoid G, respectively. Nyingchinoid H (8) shared the same molecular formula (C17H22O3) as 5, as deduced from its (+)-HRESIMS and 13C NMR data. The 1H−1H COSY and HMBC spectra (Figure S2, Supporting Information) revealed that the two compounds possessed the same 2D structure, inferring them to be stereoisomers. Different from 5, the H-3/H-9b and H-3/H11 NOESY cross-peaks indicated that they were axial and βoriented (Figure S2, Supporting Information). Additionally, the chemical shifts of C-9 and C-11 were deshielded without the γ-gauche effect of HO-3, which were similar to those of rasumatranin B.6 These data showed that 8 was a 3-epimer of 5. Compound 8 was also a scalemic mixture with a specific rotation of −8.6, showing two chiral-phase HPLC peaks in a 10:1 approximate ratio (Figure 3). The enantiomeric pair 8a and 8b were acquired and verified by their specific rotations (8a: [α]25D −12; 8b: [α]25D +11.3), ECD data (Figure 3), and NMR and MS spectra (Figures S137−S140, Supporting Information). Their absolute configurations were established as (2S, 3R, 4S, 11R) for 8a and (2R, 3S, 4R, 11S) for 8b, as depicted by the similarity of their ECD curves to 5a and 5b, respectively. Compounds 8a and 8b were therefore structurally characterized and named (−)-nyingchinoid H and (+)-nyingchinoid H, respectively. Four known compounds were isolated and identified as confluentin (9),7 (E)-4-(3,7-dimethylocta-2,6-dienyl)-5-methylbenzene-1,3-diol (10),8 grifolin (11),9 and grifolinone A (12)10 (see the Supporting Information for details). Plausible biosynthetic pathways toward 1a/1b−3a/3b were proposed. Their biosynthetic precursor may be 10, a coisolated terpene−shikimate adduct. Compound 10 would 1815



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Scheme 1. Putative Biosynthetic Pathways toward the Formation of 1a/1b−3a/3b

Plant Material. The aerial parts of Rhododendron nyingchiense R. C. Fang et S. H. Huang were collected in Nyingchi County from the Tibet Autonomous Region, People’s Republic of China, in August 2015. The plant material was authenticated by Prof. Jian Luo, College of Agriculture, Tibet University. A voucher specimen (TCM 15-08-25 Hou) has been deposited at the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Fudan University. Extraction and Isolation. The plant sample (9 kg) was ground and percolated with 95% EtOH at room temperature to give a crude extract (2.3 kg), which was partitioned between H2O and EtOAc. The EtOAc extract (650 g) was subjected to Diaion HP-20 column chromatography (CC) (EtOH−H2O, from 1:9 to 9:1) to provide fractions A−D. Fraction C (200 g) was separated over a silica gel column (CH2Cl2−MeOH, from 100:1 to 15:1) to give fractions C1− C9. Fraction C2 (15.0 g) was separated by silica gel CC eluted with petroleum ether−EtOAc (from 50:1 to 1:1) to give fractions C2a− C2e. The main fraction C2b (2.4 g) was further chromatographed on ODS gel (MeOH−H2O, from 8:2 to 9:1), then by HPLC (MeOH− H2O, 8:2) to yield 4 (70.0 mg). Using procedures similar to fraction C2, fraction C3 (10.0 g) yielded 1 (37.0 mg), and fraction C4 (8.0 g) afforded 2 (7.0 mg) and 3 (12.0 mg). Fraction C5 (16.0 g) was separated into fractions C5a−C5d by silica gel CC (petroleum ether− Me2CO, from 20:1 to 1:1). The main fraction C5c (2.8 g) was purified by ODS CC (MeOH−H2O, from 7:3 to 8:2), followed by HPLC (MeOH−H2O, 7:3) to afford 6 (40.0 mg), 7 (12.0 mg), and 10 (62.0 mg). By a process similar to fraction C5, fraction C7 (12.0 g) provided 5 (10.0 mg), 8 (8.0 mg), and 12 (35.0 mg). Fraction C8 (15.0 g) gave the four fractions C8a−C8d using the same CC conditions as fraction C5. The major component C8b (4.0 g) afforded 9 (20.0 mg) by ODS CC (MeOH−H2O, from 6:4 to 7:3), then by HPLC (MeOH−H2O, 6:4). Another major component, C8c (2.0 g), gave 11 (30.0 mg) using the same means as fraction C8b. A 3 mL/ min flow rate was applied to the semipreparative HPLC. Chiral-Phase Separation. Compounds 1−8 were subjected to chiral-phase HPLC separation using n-hexane−2-propanol (7.5:2.5 for 1; 6.5:3.5 for 2; 8:2 for 3, 4, 6, and 7; 9:1 for 5; and 7:3 for 8) as the mobile phase at a flow rate of 4.0 mL/min, a Daicel chiralpak IC column at room temperature/20 °C, and a UV detector (210 nm for

undergo cyclization to give intermediate i, which after oxidation would produce intermediate ii. Through Baeyer− Villiger oxidation and epoxidation, intermediate iii would be formed (Scheme 1). This intermediate would be transformed into 1a/1b by cyclization, reduction, and dehydration reactions. Compound 10 would also generate 2a/2b and 3a/ 3b through intermediate i as proposed in Scheme 1. Compounds 1a−8a, 1b, 4b, 6b−8b, and 11 were evaluated for their ability to inhibit PTP1B in vitro, using oleanolic acid11 as positive control (IC50 = 2.5 ± 0.2 μM). Compounds 1b, 2a−4a, 8a, 8b, and 11 exhibited inhibitory effects with IC50 values of 43.6 ± 2.7 (1b), 38.1 ± 2.9 (2a), 61.0 ± 4.8 (3a), 58.2 ± 4.9 (4a), 48.1 ± 0.3 (8a), 29.0 ± 9.2 (8b), and 5.7 ± 0.5 (11) μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were recorded on an SGM X-4 apparatus. Optical rotations were measured on a Rudolph Autopol IV polarimeter. UV and ECD spectra were obtained on a JASCO J-810 instrument. IR spectra were measured on a Nicolet iS5 spectrometer. NMR spectra were obtained on Varian Mercucy Plus 400 MHz and Bruker Avance 600 MHz spectrometers with CDCl3 as solvent. ESIMS and HRESIMS data were acquired on an Agilent 1100 LC/MSD and an AB 5600+ Q TOF mass spectrometer, respectively. Single-crystal X-ray diffraction experiments were performed on a Bruker APEX-II CCD detector using graphitemonochromated Cu Kα radiation (λ = 1.54178 Å). Silica gel (200− 300 mesh, Qingdao Haiyang Chemical Co., Ltd., China), ODS gel (50 μm, YMC Co., Japan), and Diaion HP-20 (Mitsubishi Chemical Co., Japan) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., China) were used for TLC. Semipreparative HPLC and chiral-phase separation were performed on a Shimadzu Essentia LC-16 with a UV detector (210 and 254 nm), using a YMC C18 column (150 × 10 mm, 5 μm, YMC Co., Japan) and a Daicel Chiralpak IC column (250 × 20 mm, 5 μm, Daicel Chiral Technologies (China) Co., Ltd.), respectively. 1816



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Nyingchinoid F (6): white, amorphous powder; [α]25D +13.4 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 207 (4.52), 231 (4.16), 282 (4.03) nm; IR (KBr) νmax 3456, 2937, 1611, 1438, 1196, 1181, 1132, 1076, 973 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 291.2 [M + H]+, 313.2 [M + Na]+; positive HRESIMS m/z 291.1947 [M + H]+ (calcd for C18H27O3, 291.1955). (+)-Nyingchinoid F (6a): colorless crystals (MeOH); mp 177−179 °C; [α]25D +18.1 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 205 (−3.35), 237 (+1.25), 253 (+0.12), 282 (+1.80) nm. (−)-Nyingchinoid F (6b): white, amorphous powder; [α]25D −17.2 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 204 (+5.49), 237 (−1.20), 254 (+0.21), 284 (−1.77) nm. Nyingchinoid G (7): white, amorphous powder; [α]25D +6.7 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 208 (4.49), 233 (4.14), 283 (4.00) nm; IR (KBr) νmax 3424, 2928, 1614, 1460, 1198, 1135, 1076 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 277.2 [M + H]+, 299.2 [M + Na]+; positive HRESIMS m/z 277.1795 [M + H]+ (calcd for C17H25O3, 277.1798). (+)-Nyingchinoid G (7a): white, amorphous powder; [α]25D +16.2 (c 0.2, MeOH); ECD (MeOH) λmax (Δε) 207 (−4.25), 238 (+1.17), 258 (+0.08), 282 (+1.20) nm. (−)-Nyingchinoid G (7b): white, amorphous powder; [α]25D −15.8 (c 0.2, MeOH); ECD (MeOH) λmax (Δε) 206 (+6.70), 237 (−1.14), 256 (+0.06), 281 (−1.18) nm. Nyingchinoid H (8): white, amorphous powder; [α]25D −8.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.28), 232 (4.04), 284 (3.69) nm; IR (KBr) νmax 3441, 2923, 1606, 1458, 1196, 1181, 1132, 1076 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 275.2 [M + H]+; positive HRESIMS m/z 275.1644 [M + H]+ (calcd for C17H23O3, 275.1642). (−)-Nyingchinoid H (8a): white, amorphous powder; [α]25D −12 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 206 (−10.50), 238 (+2.20), 251 (−0.05), 284 (+1.63) nm. (+)-Nyingchinoid H (8b): white, amorphous powder; [α]25D +11.3 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 208 (+12.60), 237 (−2.16), 252 (−0.04), 284 (−1.65) nm. X-ray Crystallographic Analysis. The crystals of 1a were obtained from MeOH−H2O (10:1), and those of 3a, 4a, 5a, and 6a from MeOH. The method used for structure analysis and the detailed crystallographic data are described in the Supporting Information. PTP1B Activity Assay. A colorimetric assay used to measure PTP1B inhibition was the same as that described previously.12,13 Oleanolic acid and DMSO were used as the positive and negative controls, respectively.

1, 3−8, and 254 nm for 2). Eight pairs of enantiomers were obtained, including 1a (18.0 mg), 1b (3.2 mg), 2a (3.7 mg), 2b (0.8 mg), 3a (5.0 mg), 3b (1.8 mg), 4a (15.0 mg), 4b (3.2 mg), 5a (4.5 mg), 5b (1.2 mg), 6a (18.0 mg), 6b (3.2 mg), 7a (5.2 mg), 7b (1.5 mg), 8a (3.6 mg), and 8b (1.0 mg). The peak area ratios were 6.5:1 (1a:1b), 5:1 (2a:2b), 3.5:1 (3a:3b), 3:1 (4a:4b), 8:1 (5a:5b), 7:1 (6a:6b), 2:1 (7a:7b), and 10:1 (8a:8b). Nyingchinoid A (1): white, amorphous powder; [α]25D −26.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.54), 284 (3.26) nm; IR (KBr) νmax 3463, 2967, 2930, 1611, 1463, 1364, 1347, 1308, 1284, 1195, 1139, 1071, 1025, 960, 847 cm−1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 291.2 [M + H]+, 313.2 [M + Na]+, 603.4 [2 M + Na]+; negative ESIMS m/z 289.2 [M − H]−; positive HRESIMS m/z 291.1593 [M + H]+ (calcd for C17H23O4, 291.1591). (−)-Nyingchinoid A (1a): colorless crystals (MeOH−H2O, 10:1); mp 173−175 °C; [α]25D −36.2 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 230 (+2.95), 280 (−0.80) nm. (+)-Nyingchinoid A (1b): white, amorphous powder; [α]25D +37 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 230 (−2.91), 280 (+0.80) nm. Nyingchinoid B (2): colorless gum; [α]25D +12.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223 (3.60), 256 (4.02), 308 (3.36) nm; IR (KBr) νmax 3460, 2963, 2925, 1659, 1607, 1464, 1422, 1383, 1196, 1180, 1132, 1077 cm−1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 313.2 [M + Na]+, 603.2 [2 M + Na]+; negative ESIMS m/z 289.0 [M − H]−, 325.0 [M + Cl]−; positive HRESIMS m/z 313.1408 [M + Na]+ (calcd for C17H22O4Na, 313.1410). (+)-Nyingchinoid B (2a): colorless gum; [α]25D +17 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 223 (−1.76), 256 (+6.90), 332 (−1.26) nm. (−)-Nyingchinoid B (2b): colorless gum; [α]25D −16.8 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 223 (+1.75), 256 (−6.66), 332 (+1.27) nm. Nyingchinoid C (3): white, amorphous powder; [α]25D −9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.45), 232 (3.90) nm; IR (KBr) νmax 3416, 2928, 1612, 1450, 1332, 1196, 1180, 1133, 1093, 1076, 838 cm−1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 259.2 [M + H]+; positive HRESIMS m/z 259.1689 [M + H]+ (calcd for C17H23O2, 259.1693). (−)-Nyingchinoid C (3a): colorless crystals (MeOH); mp 166−168 °C; [α]25D −14.2 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 203 (−3.74), 233 (+0.42) nm. (+)-Nyingchinoid C (3b): white, amorphous powder; [α]25D +14.6 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 203 (+3.71), 233 (−0.05) nm. Nyingchinoid D (4): white, amorphous powder; [α]25D −9.6 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 207 (4.36), 232 (4.06), 284 (3.93) nm; IR (KBr) νmax 3406, 2925, 1612, 1458, 1196, 1181, 1133, 1076 cm−1; 1H NMR and 13C NMR data, see Table 1; positive ESIMS m/z 259.2 [M + H]+; positive HRESIMS m/z 259.1690 [M + H]+ (calcd for C17H23O2, 259.1693). (−)-Nyingchinoid D (4a): colorless crystals (MeOH); mp 128− 130 °C; [α]25D −15.6 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 214 (+1.72), 234 (+2.30), 252 (+0.20), 281 (+1.10) nm. (+)-Nyingchinoid D (4b): white, amorphous powder; [α]25D +15.2 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 210 (−3.06), 234 (−2.32), 252 (+0.05), 281 (−1.06) nm. Nyingchinoid E (5): white, amorphous powder; [α]25D +14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.38), 234 (4.12), 284 (4.01) nm; IR (KBr) νmax 3387, 2925, 2852, 1596, 1451, 1196, 1133, 1076 cm−1; 1H NMR and 13C NMR data, see Table 2; positive ESIMS m/z 275.2 [M + H]+; positive HRESIMS m/z 275.1631 [M + H]+ (calcd for C17H23O3, 275.1642). (+)-Nyingchinoid E (5a): colorless crystals (MeOH); mp 198−201 °C; [α]25D +19.8 (c 0.2, MeOH); ECD (MeOH) λmax (Δε) 208 (−5.60), 237 (+1.85), 251 (+0.31), 283 (+1.24) nm. (−)-Nyingchinoid E (5b): white, amorphous powder; [α]25D −19.5 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 208 (+8.60), 235 (−1.86), 252 (+0.26), 284 (−1.22) nm.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00273. Key 2D NMR correlations of 7 and 8; physical and spectroscopic data of known compounds; NMR and ECD calculations; and raw MS, NMR, and IR spectra (PDF) X-ray crystallographic data for 1a (CIF) X-ray crystallographic data for 3a (CIF) X-ray crystallographic data for 4a (CIF) X-ray crystallographic data for 5a (CIF) X-ray crystallographic data for 6a (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 21 51980138. E-mail: [email protected]. ORCID

Ai-Jun Hou: 0000-0002-4514-0846 1817



Article

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the Shanghai Commission of Science and Technology (14431902800) and the National Key Research and Development Program of China (2016YFC1305500).

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REFERENCES

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Enantiomeric Pairs of Meroterpenoids with Diverse Heterocyclic

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