Wan-Bai Su,
Xiao-Meng Hou,
Ling Zhang,
Li Yan,
Zhi-Yang Tang,
Yang Yu,
Jin-Song Liu,
Yun-Peng Sun
* and
Guo-Kai Wang
*
a, School of Pharmacy, Anhui University of Chinese Medicine, Anhui Province Key Laboratory of Bioactive Natural Products, Hefei 230012, P.R. China. E-mail: sunyp@ahtcm.edu.cn; wanggk@ahtcm.edu.cn
First published on 18th July 2025
Three undescribed pimarane-type diterpenoids, muhenrins A–C (1–3), along with a known analogue were isolated from the petroleum ether extract of Munronia henryi (Meliaceae). Their structures, including absolute configurations, were elucidated by means of various spectroscopic methods (IR, UV, HR-ESI-MS, NMR), single-crystal X-ray diffraction, ECD and NMR calculations. Muhenrin A (1) features a unique 6,7-seco pimarane skeleton, and its putative biosynthetic pathways have been proposed. Compounds 1–4 showed weak inhibitory activity against NO production in LPS-induced RAW 264.7 cells, with inhibition rates ranging from 13.73 – 32.35% at 50 μM concentrations.
Pimarane diterpenoids are characterized by a 4, 4, 10, 13-tetramethylperhydrophenanthrene core skeleton and are classified into pimarane, isopimrane, ent-pimarane and ent-isopimrane based on their stereochemical configurations.9 These compounds primarily occur in plants from the Lamiaceae, Zingiberaceae, and Cupressaceae families, as well as in fungi and marine organisms. To date, over 360 pimarane-type molecules have been reported, demonstrating notable cytotoxicity, anti-inflammatory, and antimicrobial activities.10 However, such diterpenoids are rarely documented in Meliaceae plants, with only minor quantities isolated from Dysoxylum, Guarea, and Chukrasia species.11 This study reports, for the first time, the presence of pimarane-type diterpenoids in Munronia plants, including compound 1, which features a unique 6,7-seco pimarane skeleton (Fig. 1). Additionally, their cytotoxic and anti-inflammatory activities are evaluated.
No | 1a | 2a | 3b | |||
---|---|---|---|---|---|---|
δC | δH (J in Hz) | δC | δH (J in Hz) | δC | δH (J in Hz) | |
a 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD.b 1H NMR (600 MHz) and 13C NMR (150 MHz) data in CDCl3.c overlapped. | ||||||
1α | 36.5 | 1.49, mc | 35.0 | 0.96, dd (13.2, 3.7) | 39.1 | 1.00, m |
1β | 1.37, m | 2.69, br d (13.2) | 1.66, m | |||
2α | 19.8 | 1.50, mc | 18.5 | 1.52, m | 19.0 | |
2β | 1.62, mc | 1.70, qt (13.7, 3.5) | 1.48, mc | |||
3α | 42.0 | 1.19, mc | 40.9 | 1.20, dd (13.6, 3.9) | 41.9 | 1.17, td (13.3,4.5) |
3β | 1.53, mc | 1.46, br d (13.4) | 1.45, mc | |||
4 | 34.5 | 33.1 | 33.3 | |||
5 | 58.6 | 2.55, s | 50.1 | 1.59, dd (15.0, 3.2) | 52.0 | 3.87, t (8.0) |
6α | 177.1 | 35.7 | 2.56, dd (17.8, 3.2) | 32.6 | 2.00, m | |
6β | 2.44, dd (17.8, 15.0) | 1.29, mc | ||||
7 | 174.9 | 204.1 | 72.5 | 3.99, br s | ||
8 | 135.3 | 141.1 | 139.4 | |||
9 | 41.6 | 2.78, br s | 156.0 | 50.3 | 1.93, m | |
10 | 42.7 | 39.4 | 38.4 | |||
11α | 21.5 | 1.60, mc | 200.3 | 26.3 | 1.80, m | |
11β | 2.06, br d (14.7) | 1.53, m | ||||
12α | 33.8 | 1.54, mc | 45.7 | 2.53, d (17.0) | 73.2 | 3.57, dd (12.3, 3.9) |
12β | 1.74, td (13.0, 3.0) | 2.65, d (17.0) | ||||
13 | 38.9 | 41.7 | 42.7 | |||
14 | 144.7 | 6.33, s | 68.9 | 4.55, d (2.6) | 124.9 | |
15 | 147.1 | 5.73, dd (17.4, 10.6) | 143.9 | 5.75, dd (17.6, 10.9) | 146.2 | 5.80, dd (17.4, 10.8) |
16a | 112.7 | 4.82, d (17.6) | 113.9 | 4.93, d (17.6) | 114.1 | 5.14, d (17.4) |
16b | 4.96, d (10.6) | 5.04, d (10.9) | 5.15, d (10.8) | |||
17 | 27.2 | 1.14, s | 22.4 | 1.16, s | 17.5 | 1.08, s |
18 | 34.7 | 1.03, s | 33.0 | 0.89, s | 33.8 | 0.91, s |
19 | 24.5 | 1.16, s | 21.4 | 0.93, s | 22.2 | 0.87, s |
20 | 22.5 | 1.21, s | 17.3 | 1.32, s | 14.8 | 0.82, s |
The 2D NMR spectra revealed key structural features of compound 1 (Fig. 2). In the HMBC spectrum, H3-18 (δH 1.03, 3H, s) showed correlations to C-19 (δC 24.5), C-3 (δC 42.0), C-4 (δC 34.5), and C-5 (δC 58.6). These correlations, together with the 1H–1H COSY cross-peaks between H-1/H-2/H-3, confirmed the intact nature of ring A. Furthermore, HMBC correlations of H3-17 (δH 1.14, s) to C-12 (δC 33.8) and C-14 (δC 144.7), along with those of H-14 (δH 6.33, s) to C-9 (δC 41.6) and C-12, combined with the 1H–1H COSY cross-peaks of H-9/H-11/H-12, established the complete ring C structure. Furthermore, the HMBC correlations between H-14 and δC 174.9, and between H-5 and δC 177.1 suggested that the two carboxyl groups belong to C-7 and C-6, respectively. This assignment was further supported by the molecular formula derived from HR-MS analysis. Consequently, compound 1 was identified as a 6,7-seco pimarane-type diterpenoid, as illustrated in the Fig. 1. Notably, the 13C NMR spectrum of 1 exhibits signal broadening, likely attributable to steric hindrance from the carboxyl group restricting free rotation about the C9–C10 single bond.13
The relative configuration of compound 1 was partially established through ROESY correlations (Fig. 2). The cross-peaks between H3-18/H-5/H-3α indicated α-orientation for both H3-18 and H-5, while the correlation between H3-19 and H-3β supported β-orientation for H3-19. However, the stereoconfiguration of compound 1 could not be determined by ROESY due to the free rotation about the C9–C10 single bond. The absolute configuration was ultimately determined by X-ray crystallographic analysis of single crystals (Fig. 3) obtained from methanol solution, which established the 5S, 9R, 10R, 13R configuration [Flack parameter = −0.06(13)].
Compound 2 was obtained as a white powder. Its molecular formula was established as C20H28O3 by HR-ESI-MS (m/z 315.1960 [M − H]−), corresponding to seven degrees of unsaturation. The 1H NMR spectrum of 2 (Table 1) displayed signals characteristic of a terminal double bond (δH 5.75, dd, J = 17.6, 10.9 Hz; 5.04, d, J = 10.9 Hz; 4.93, d, J = 17.6 Hz) and four tertiary methyl groups (δH 1.32, 1.16, 0.93 and 0.89, each 3H, s). The 13C spectrum of 2, combined with the HSQC spectrum, revealed the presence of seven quaternary carbons (including two keto carbonyls at δC 204.1 and 200.3, and two olefinic carbons at δC 156.0 and 141.4), three methine carbons (one olefinic at δC 143.9, and one oxygenated at δC 69.0), six methylene carbons (one olefinic at δC 113.9) and four methyl carbons. These signals account for four degrees of unsaturation, while the remaining three unsaturations suggest that 2 is a tricyclic diterpenoid. The spin systems identified in the 1H–1H COSY spectrum (Fig. 4), specifically H-1/H-2/H-3 and H-5/H-6, along with key HMBC correlations (H3-18 to C-3/C-4/C-5; H-20 to C-1/C-5/C-9/C-10; and H-6 to C-7/C-8/C-10), were crucial for establishing the structures of rings A and B. Combined with the terminal double bond feature and HMBC correlations from H-17 to C-12, C-14, C-15, compound 2 was identified as a pimarane-type diterpenoid, exhibiting a planar structure similar to known compound 12β-Hydroxy-7,11-dioxopimal-8,15-dien.14 The sole structural difference involves the position of hydroxyl group substitution in 2, the hydroxyl group is located at C-14, whereas in 12β-hydroxy-7,11-dioxopimar-8,15-dien, it is positioned at C-12. This distinction was confirmed by the HMBC correlations observed between H-14 and C-7/C-8.
The ROESY spectrum revealed key spatial relationships: correlations between H3-19 (δH 0.93), H-6β (δH 2.44), H3-20 (δH 2.69) indicated their cofacial arrangement and β-orientation. Similarly, the H3-18 (δH 0.89)/H-6α (δH 2.56) correlation suggested their cofacial α-orientation (Fig. 4). The α-orientation of H-5 was further supported by its small coupling constant (J5,6α = 3.2 Hz). While the ROESY correlation between H3-17(δH 1.16) and H-14 (δH 4.55) confirmed their cofacial arrangement, two possible relative configurations (13S*, 14R* and 13R*, 14S*) remained. To resolve this ambiguity, we calculated the 13C chemical shifts for both epimers (Fig. 5A). DP4+ analysis of both 1H and 13C NMR data unequivocally identified (13S*, 14R*)-2 as the correct structure (100% probability, Fig. S3†).15,16 The absolute configuration (5S, 10S, 13S, 14R) was ultimately confirmed by excellent agreement between experimental and calculated ECD spectra (Fig. 5B).
![]() | ||
Fig. 5 Correlations between experimental and calculated 13C NMR chemical shifts of (13S*, 14R*)-2 (A) and ECD calculations for (13S, 14R)-2 (B). |
Compound 3 was isolated as a white powder. Its molecular formula (C20H32O2) was established by 13C NMR and HR-ESI-MS (observed [M − H]− at m/z 327.2295), implying five degrees of unsaturation. The 1H NMR spectrum (Table 1) displayed characteristic methyl group signals (δH 1.08, 0.91, 0.87, 0.82, each, 3H, s) and olefinic proton signals (δH 5.80, dd, J = 17.4, 10.8 Hz; 5.54, d, J = 2.0 Hz; 5.15, d, J = 10.8 Hz; 5.14, d, J = 17.4 Hz), suggesting a pimarane-type diterpenoid skeleton analogous to compound 2. Comparative analysis of 1D and 2D NMR data revealed close structural similarity to (5S, 9R, 10S, 12R, 13R)-12-hydroxyisopimara-8(14),15-dien-7-one,17 with only differences of the absence of a carbonyl carbon and the presence of an additional oxygenated carbon. The observed mass difference of 2 suggested reduction of the C-7 carbonyl group to a hydroxyl group in compound 3. This structural modification was confirmed by HMBC correlations from H-7 (δH 3.99) to C-5 (δC 52.0), C-8 (δC 139.4), C-9 (δC 50.3), along with 1H–1H COSY cross-peaks between H-5/H-6/H-7 (Fig. 6).
ROESY correlations observed among H3-18/H-5/H-9/H-12, H-5/H-7, and H3-18/H-6α (δH 2.00) established their cofacial arrangement and α-orientation. Complementary ROESY cross-peaks between H3-17/H-11β (δH 1.53)/H3-20/H-6β (δH 1.29) confirmed their β-orientation. The absolute configuration of 3 was determined to be 5S, 7S, 9R, 10S, 12R, 13R through comparison of experimental and calculated ECD spectra (Fig. S2†).
In addition to the aforementioned compounds, a known pimarane-type diterpenoid was isolated from M. henryi. Based on comparison of its spectroscopic data with literature values,17 this compound was identified as (5S, 9R, 10S, 12R, 13R)-12-hydroxyisopimara-8(14),15-dien-7-one (4).
According to previous literature, pimarane-type diterpenoids have demonstrated significant cytotoxic and anti-inflammatory potential.18–20 Initially, all compounds were tested for anti-inflammatory activity using L-NMMA (50 μM) as a positive control (52.75 ± 1.28% inhibition). At 50 μM concentration, compounds 1–4 exhibited no significant inhibitory effects on NO production in LPS-induced RAW 264.7 macrophages, showing inhibition rates ranging from 13.73% to 32.35%.
Subsequently, the cytotoxicity of these compounds was evaluated against human colon cancer cells (HCT-166) and human liver cancer cells (Hep3B). None of the compounds exhibited significant cytotoxicity against either cancer cell line.
The initial biological activity assays did not yield significant results. Future studies should consider alternative cell models or explore additional biological activities, such as antibacterial or antiviral effects.24,25
The PE fraction (363 g) was fractionated by silica gel column chromatography using a stepwise gradient of PE-EtOAc (100:
0 → 0
:
100, v/v) to yield eight fractions (A–H). Fraction F (27.1 g) was further purified by ODS column chromatography with a MeOH–H2O gradient (40
:
60 → 100
:
0, v/v), yielding six subfractions (Fa–Ff). Subfraction Fc was chromatographed on ODS with isocratic elution (MeCN–H2O, 45
:
55, v/v) to afford nine fractions (Fca–Fci). Final purification of Fcc by semi-preparative HPLC (MeCN–H2O, 65
:
35, 8.0 mL min−1) yielded compound 4 (4.81 mg, tR = 36 min).
Similarly, subfraction Fb was separated by ODS chromatography (isocratic MeCN–H2O, 45:
55) into thirteen fractions (Fba–Fbn). Fraction Fbk was purified by semi-preparative HPLC (MeCN– H2O 57
:
43, 8.0 mL min−1) to give Fbka (4.71 mg, tR = 43 min), which was further purified (MeOH–H2O, 75
:
25, 8.0 mL min−1) to afford compound 3 (2.58 mg, tR = 28 min). Fraction Fbn was processed similarly (MeCN–H2O, 67
:
33 → MeOH–H2O, 55
:
45) to yield compound 2 (2.55 mg, tR = 32 min) via intermediate fraction Fbna (5.31 mg, tR = 45 min).
Fraction H (24.2 g) was fractionated by MCI gel column chromatography using a MeOH–H2O gradient (20:
80 → 100
:
00, v/v), yielding six subfractions (Ha–Hf). Subfraction Hf was further purified by Sephadex LH-20 column chromatography (MeOH) to afford four fractions (Hfa–Hfd). Final purification of Hfb by semi-preparative HPLC (MeCN–H2O gradient: 0–30 min, 55
:
45; 30–35 min, 55
:
45 → 85
:
15; 35–50 min, 85
:
15; flow rate 8.0 mL min−1) yielded compound 1 (6.75 mg, tR = 43 min).
Footnote |
† Electronic supplementary information (ESI) available. ESI figures, biological assays, experimental and computational details, and original spectra (NMR, MS, UV, and ECD) of 1–3. CCDC 2452064. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5ra04525h |
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