The Triterpenoids from Munronia pinnata and Their Anti-Proliferative Effects

Six new tirucallane-type triterpenoids, named munropenes A–F (1–6), were extracted from the whole plants of Munronia pinnata using a water extraction method. Their chemical structures were determined based on detailed spectroscopic data. The relative configurations of the acyclic structures at C-17 of munropenes A–F (1–6) were established using carbon–proton spin-coupling constants (2,3JC,H) and inter-proton spin-coupling constants (3JH,H). Furthermore, the absolute configurations of munropenes A–F (1–6) were determined through high-performance liquid chromatography (HPLC), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) analyses. The antiproliferative effects of munropenes A–F were evaluated in five tumor cell lines: HCT116, A549, HepG2, MCF7, and MDAMB. Munropenes A, B, D, and F (1, 2, 4, and 6) inhibited proliferation in the HCT116 cell line with IC50 values of 40.90, 19.13, 17.66, and 32.62 µM, respectively.

Numerous structurally diverse compounds have been extracted from this plant, including limonoids, triterpenoids, flavonoids, lignans, sterols, sesquiterpenoids, and diterpenoids [3,5,6].These compounds exhibit a wide range of bioactivities, such as anti-inflammatory, antiproliferative, anti-tobacco mosaic virus, and insect antifeedant activities [3,[7][8][9], and have various roles in preserving food, flavoring, and treating various illnesses.In the early stages of our research, phytochemical study on the aerial parts of Munronia pinnata was isolated six novel limonoids [10].In this study, six triterpenoids, named munropenes A-F (1-6) (Figure 1) were obtained from the whole plants of Munronia pinnata using a water extraction method, and their antiproliferative activities against several tumor cell lines, including HCT116, A549, HepG2, MCF7, and MDAMB acquired from the cell bank of Chinese Academy of Sciences (Shanghai, China) were also carried out.

Results and Discussion
In this investigation, compounds The IR spectrum showed the presence of carbonyl functionalities at 1748 cm −1 and 1689 cm −1 for 1 and 2. The 1 H NMR spectrum revealed the existence of one trisubstituted olefin, eight sp 3 methines, seven sp 3 methylenes, and six singlet methyls (including one acetyl methyl).The 13 C NMR spectrum displayed 32 signals, including 3 ester carbonyls, 2 olefinic, 2 oxygenated tertiary, and 3 quaternary carbon signals (Table 1).These data indicate a tetracyclic triterpene structure for compounds 1 and 2, with the primary difference being the configuration at C-7.The tetracyclic ring moiety includes an α,β-unsaturated-ε-caprolactone ring (C-1-C-5, C-10) with a formyl group and a methoxy group at C-4, as well as three methyl groups at C-8, C-10, and C-13.The 1 H- and H-15 and C-14 (Figure 2).From the IR spectrum and the degree of unsaturation of compound 1, it was determined that an α,β-unsaturated-ε-caprolactone ring is present.Furthermore, the presence of an acetoxy group at C-11 was elucidated by the heteronuclear multiple bond correlation (HMBC) between H-1 and the acetoxy carbonyl carbon.The presence of a 1,4,5,6-tetrahydroxy-6-methyl-heptanol moiety at C-17 was suggested based on the 1 H-1 H COSY cross-peaks of H-17/H-20 and H 2 -21/H-20/H 2 -22/H-23/H-24, as well as the HMBC correlations between H 3 -26 and C-24, C-25, and C-26, and H-21 with C-20 and C-22.In short, the planar structure of 1 was established as described.
The molecular formula of munropene D (compound 4) was determined to be C38H62O14 through HRESIMS analysis, showing a peak at m/z 741.4010 ([M − H] − , Δ−5.7 mmu).The 1D NMR spectra of compound 4 (Table 1) exhibited signals originating from a glucose group, indicating its structural similarity to that of compound 3, except for the modifications at the C-5 and C-7 positions.The attachment of a methylethylene moiety at C-5 was confirmed by the HMBC correlation of H3-29 with C-4, C-5, and C-28.
The sugar moiety was obtained through acid hydrolysis, followed by treatment with L-cysteine methyl ester and o-tolylisothiocyanate, resulting in a reaction mixture that produced a peak during HPLC analysis identical to that of the derivative of authentic D-glu- cose prepared using the same procedure [17].Hence, the glucose moiety of compound 4 was determined to be D-glucose.The β-glycosidic linkage of the D-glucosyl moiety at C-7 was observed, revealing the molecular formula of compound 3 to be C 32 H 52 O 13 , suggesting the presence of seven degrees of unsaturation.The 1D NMR spectra of compound 3 (Table 1) were similar to those of compound 1, except for signals related to ring A. By comparing the degrees of unsaturation and molecular formula of 3 with those of compound 1, it was concluded that compound 3 was a ring A-seco munropene A (1).This conclusion was further supported by the 1H NMR chemical shifts of H-1 and H-2 in compound 3 (Figure 2).Therefore, a possible biosynthetic pathway for munropene C (compound 3) was proposed, suggesting that it might be generated through the hydrolysis of munropene A (compound 1) in ring A (Figure S2).
The molecular formula of munropene D (compound 4) was determined to be C 38 H 62 O 14 through HRESIMS analysis, showing a peak at m/z 741.4010 ([M − H] − , ∆−5.7 mmu).The 1D NMR spectra of compound 4 (Table 1) exhibited signals originating from a glucose group, indicating its structural similarity to that of compound 3, except for the modifications at the C-5 and C-7 positions.The attachment of a methylethylene moiety at C-5 was confirmed by the HMBC correlation of H 3 -29 with C-4, C-5, and C-28.
The sugar moiety was obtained through acid hydrolysis, followed by treatment with L-cysteine methyl ester and o-tolylisothiocyanate, resulting in a reaction mixture that produced a peak during HPLC analysis identical to that of the derivative of authentic Dglucose prepared using the same procedure [17].Hence, the glucose moiety of compound 4 was determined to be D-glucose.The β-glycosidic linkage of the D-glucosyl moiety at C-7 was concluded based on the coupling constant value of the anomeric proton (H-1 , J = 7.8 Hz), as well as the HMBC correlation of H-1 with C-7 (Figure 2).
Munropenes E (compound 5) and F (compound 6) were isolated as optically active colorless amorphous solids.Their optical rotations were determined as  2) displayed resonances corresponding to a trisubstituted olefin, a 1,2-disubstituted olefin, seven sp 3 methines, seven sp 3 methylenes, six singlet methyls, and a glucosyl moiety.The 13 C NMR spectrum exhibited 36 signals, including 1 ketone carbonyl, 4 olefinic, 1 oxygenated tertiary, and 4 quaternary carbon signals (Table 2).These data indicate that compounds 5 and 6 are isomers of each other and closely related to compounds 1 and 2, except for changes occurring in the A ring and at C-7.The glucose moiety of compounds 5 and 6 was determined to be D-glucose through similar HPLC analyses as performed for compound 4. The comparison of the 1D NMR spectroscopic data of compounds 5 and 6 with those of compound 4 indicated that the β-glycosidic linkage of the D-glucosyl moiety was attached at C-7, which was confirmed by the HMBC correlation of H-1 with C-7, as well as similar HPLC analyses as conducted for compound 4. The A rings of these two compounds were assigned as α, β-unsaturated hexane ketones with one methyl and one methanol group at C-4, elucidated by 1 H-1 H COSY cross-peaks of H-1/H-2 and the HMBC correlations of H3-29 with C-3, C-4, C-5, and C-28, as well as H-1 with C-3, C-5, and C-10.Additionally, the HMBC correlations of H 3 -19 with C-1, C-10, C-5, and C-9 supported this assignment and allowed for the connectivity between ring A and ring B.
The relative configurations of compounds 5 and 6 in the aglycone moieties were deduced to be similar to those of compounds 1 and 2, respectively, based on the resemblance of their 1D NMR data (Tables 1 and 2) and ROESY correlations (Figure S2).The ECD spectra of compounds 5 and 6 indicated a similar Cotton effect at 237 nm and 203 nm.According to the octant rule [18], the positive Cotton effect observed in compounds 5 and 6, attributed to the exciton coupling of α, β-unsaturated hexane ketone, suggested the absolute configuration of 4S*, 5R*, and 10R* in compounds 5 and 6.The absolute configuration of compound 6 was confirmed by comparing the experimental ECD spectrum with the TDDFT calculated spectrum.The experimental ECD spectrum of compound 5 correlated well with the calculated spectrum of a possible enantiomer with the 4S*, 5R*, 7R*, 8R*, 9R*, 10R*, 13S*, 17R*, 20R*, 23S*, and 24S* configurations (Figure 7), confirming the assignment of the absolute configuration of compound 5 as mentioned above.Thus, the structures of compounds 5 and 6 were elucidated as shown in Figure 1.

General Experimental Protocols
The Jasco P-1020 polarimeter was used to measure optical rotation.Infrared (IR) spectra were obtained using a Tensor 27 spectrometer and a Nicolet Fourier transform infrared spectrometer (Thermo Fisher, Waltham, MA, USA) with KBr pellets.Circular dichroism (CD) spectra were recorded using a J-810 CD spectrometer.MS spectra were measured using an LC/MS-IT-TOF mass spectrometer.Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE III-HD 500 spectrometer with MeOH (δ H 3.30 and δ C 49.0) serving as the internal standard.The countercurrent chromatography (CCC) experiment was conducted using a TBE-300C machine (manufactured by Tauto Biotechnique, located in Shanghai, China).HPLC analysis was performed using an Agilent 1260 InfinityIILC system (Agilent Technologies, Santa Clara, CA, USA).The columns utilized were Agilent Poroshell 120 SB-C18 (4 mm, 4.6 mm × 150 mm, Agilent, Santa Clara, CA, USA), ChromCore 120-C18 (5 mm, 10 mm × 250 mm, NanoChrom, Suzhou, China), and Agilent ZORBAX SB-C18 (5 mm, 9.4 mm × 250 mm, Agilent, Santa Clara, CA, USA).Silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and MCI gel (Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography.Thin-layer chromatography (TLC) analyses were performed using preloaded silica gel 60 F254 plates from Merck Millipore in Germany.The spots were visualized by heating the silica gel plate, which was sprayed with a mixture of 10% H 2 SO 4 and ethanol.

Plant Material
Botanical samples of Munronia pinnata (Wall.)W. Theob.were collected in July 2021 from Jingxi City, located in the Guangxi Zhuang Autonomous Region.The plant material was identified by one of the authors, X.-Q.Li.Voucher specimens have been preserved at the herbarium of the Center for Natural Products Chemistry Studies, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region, and Chinese Academy of Sciences (21-GX-001).

Extraction and Isolation
Munronia pinnata (25 kg), which had been air-dried and powdered, was extracted three times with 95% ethanol (250 L) under reflux conditions.The resulting mixture was then filtered to remove any insoluble components.The filtrate was concentrated under reduced pressure to obtain the extract.The extract was further extracted using petroleum ether and EtOAc, yielding a remaining water layer.To obtain fraction Fr 3 (34 g), the water layer was subjected to macroporous resin column chromatography with elution using 20%, 40%, and 80% ethanol.Additionally, the 80% fraction was subjected to gel column chromatography with methanol elution, yielding fraction Fr2 (15 g).Fr2 was separated by C 18 column chromatography with a methanol gradient elution (MeOH-H 2 O, 30:70-50:50 gradient system) to obtain seven fractions (Fr2.1-Fr2.7).Fr2.

Acid Hydrolysis and Sugar Analysis of Munropenes D-F (Compounds 4-6)
Compounds 4-6 (1.5 mg each) were subjected to hydrolysis using 2.0 M HCl (2.0 mL) for a duration of 2 h at a temperature of 90 • C. To establish neutral conditions, anion exchange resin (IRA 400) was added and subsequently removed through filtration.The resulting filtrate was then subjected to vacuum concentration and dried under vacuum conditions.The resultant residue was dissolved in pyridine (1.0 mL) supplemented with L-cysteine methyl ester hydrochloride (1.0 mg) and heated at 60 • C for 1 h.Subsequently, o-torylisothiocyanate (1.0 mg) was added to the mixture, which was then stirred at 60 • C for an additional hour.Reversed-phase HPLC was used to directly analyze the reaction mixture, and the retention times of reference compounds and carbohydrate derivatives were compared, which was performed under the following conditions: detection wavelength of 250 nm, mobile phase consisting of 25% acetonitrile-water with 0.1% formic acid, and utilizing an Agilent Poroshell 120 SB-C18 column (4 mm, 4.6 mm × 150 mm, Agilent, Santa Clara, CA, USA).The absolute conformation of the sugar moiety was ascertained through comparison with D-glucose (t R = 9.55 min).

Cytotoxicity Assay
The cytotoxicity of munropenes A-F (1-6) in A549, HepG2, HCT116, MCF7, and MDAMB was tested using the Cell Counting Kit-8 (CCK-8).A 100 µL cell suspension (2 × 10 5 cells/mL) was seeded into 96-well plates.Following incubation for 24 h, the cells were treated with various concentrations (5,10,20,40,80, or 160 µM) of each specific compound, while the control cells received an equal volume of DMSO.Subsequently, after an additional 24 h of culture, 10 µL CCK-8 was added and incubated for an additional 2 h.The absorbance value at 450 nm was detected using a microplate reader, enabling the calculation of the cell survival rate.

Conclusions
The phytochemical study on the whole plants of a Chinese traditional medicine plant Munronia pinnata (Meliaceae) led to the isolation of six new tirucallane-type triterpenoids, munropenes A-F (compounds 1-6).Tirucallane-type triterpenoids are known as major components of plants belonging to Meliaceae, but they had not been systematically studied in M. pinnata.In the present paper, munropenes A and B (1 and 2) were identified as tirucallane-type triterpenoids with an α,β-unsaturated-ε-caprolactone moiety in ring A, while munropenes C and D (compounds 3 and 4) were categorized as ring A seco-tirucallanetype triterpenoids.Additionally, munropenes D, E, and F (compounds 4, 5, and 6) were determined to be glycosides of tirucallane-type triterpenoids based on 1D and 2D-NMR, HR-ESI-MS, IR, single-crystal X-ray diffraction, ECD, and J-based configuration analyses.Munropenes A, B, D, and F (compounds 1, 2, 4, and 6) was moderately cytotoxic to the HCT116 cell line, but did not show any cytotoxicity in the A549, HepG2, MCF7, and MDAMB cell lines.Furthermore, munropenes C (compound 3) and E (compound 5) exerted no cytotoxicity against all tested cell lines, including HCT116, A549, HepG2, MCF7, and MDAMB cells.

Figure 5 .
Figure 5. X-ray crystal structure of munropene A (1). Munropene C (compound 3) was obtained as an optically active, colorless amorphous solid.The specific rotation [α]D 20.0 = −50.51(c 0.10, MeOH) indicated its optical activity.From the HRESIMS, a sodiated molecular ion at m/z 643.3221 ([M − H] − , Δ−11.4 mmu) was observed, revealing the molecular formula of compound 3 to be C32H52O13, suggesting the presence of seven degrees of unsaturation.The 1D NMR spectra of compound

Figure 7 .
Figure 7. Experimental and calculated ECD spectra of 5.Figure 7. Experimental and calculated ECD spectra of 5.

Figure 7 .
Figure 7. Experimental and calculated ECD spectra of 5.Figure 7. Experimental and calculated ECD spectra of 5.
in CD 3 OD.