# A New Pyrrolidone Alkaloid and Other Constituents fromRourea oligophlebia Stems

Authors: Thuc Ngoc Dinh; Mai Vu Thi Ha; Luong Ngo Xuan; Huan Trinh Thi; Huong Nguyen Thi; Mai Nguyen Thi Ngoc; Giap Tran Huu; Oanh Vu Thi Kim; Thanh Le Nguyen
Journal: Journal of Chemistry
 (2021)

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## Abstract

Phytochemical study ofRourea oligophlebia stems led to the isolation of a new 2-pyrrolidone alkaloid (R,S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1), together with 14 known compounds including friedelin (2), friedanol (3), taraxerol (4), vanillin (5), coniferyl aldehyde (6), apigenin (7), 7α-hydroxy-3β-sitosterol (8), coniferyl alcohol (9), scopoletin (10), emodin (11), protocatechuic acid (12), catechin (13), procyanidin A1 (14), and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15). Several isolated compounds were evaluated for cytotoxicity and antimicrobial activity. Compound 11 exhibited good antimicrobial activity on Gram (+) strains and moderate cytotoxicity against KB, Hep-G2, and LU cancer cell lines. Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 over KB and LU cancer cell lines, while compound 7 exhibited similar effects on KB, HepG-2, and MCF-7 cell lines with IC50 values of 36.46 ± 0.81, 32.00 ± 0.58, and 32.03 ± 0.61 µg/mL, respectively.


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## Body

## 1. Introduction

The genus ofRourea is a group of climbing shrubs belonging to the family of Connaraceae which are widely distributed in the Amazon, Pacific region, Africa, and Asia [1]. Chemical investigations have revealed that Rourea species contain a substantial number of biologically active constituents, including flavonoids, phytosteroids, triterpenes, lipids, phenolic acids, and coumarins [1–6]. The Rourea plant extracts and their active components exhibited interesting biological activities such as hypoglycemic activity [1, 5, 7], antibacterial activity [1, 8], antinociceptive activity [9], antiplasmodial activity [3, 10], and antioxidant activity [1, 4, 8]. Rourea oligophlebia Merr. is a species found on mountains of central provinces of Vietnam. According to folk medicine, R. oligophlebia stems have been used for the treatment of bleeding and bone fractures [11]. A preliminary study of R. oligophlebia revealed the presence of triterpene, sterol, and phenolic constituents [12]. In this study, we described the isolation and identification of a new pyrrolidone alkaloid (R,S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1) (Figure 1) and fourteen known compounds including friedelin (2), friedanol (3), taraxerol (4), vanillin (5), coniferyl aldehyde (6), apigenin (7), 7α-hydroxy-3β-sitosterol (8), coniferyl alcohol (9), scopoletin (10), emodin (11), protocatechuic acid (12), catechin (13), procyanidin A1 (14), and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15) from the stems of R. oligophlebia (Figure S1). The isolated compounds were evaluated for antimicrobial activity and cytotoxicity.Figure 1
Structure of new compound isolated fromR. oligophlebia.


## 2. Materials and Methods

### 2.1. General Experimental Procedures

The NMR data including1H-NMR, 13C-NMR, 15N-NMR, HSQC, and HMBC spectra were recorded by a Bruker AM500 FT-NMR spectrometer using TMS as an internal standard. The HR-ESI-MS was obtained using an Agilent 6530 Accurate Mass Q-TOF LC/MS system. Column chromatography (CC) was performed on silica gel (Merck, 230–400 mesh) or Sephadex LH-20 (Sigma Aldrich). Thin layer chromatography used precoated silica gel plates (Merck 60 F254). Compounds were visualized by spraying with 10% aqueous H2SO4.


### 2.2. Plant Materials

The plant stems were collected from the Ben En National Park, Thanh Hoa province, Vietnam, in 2018. The plant was identified by Dr. Do Ngoc Dai, Nghe An University of Economics, asRourea oligophlebia Merr. (Connaraceae). A voucher specimen (MT-202) was deposited at Hong Duc University, Faculty of Natural Sciences.


### 2.3. Extraction and Isolation

The stem powder (5.3 kg) ofRourea oligophlebia was extracted successively with n-hexane, ethyl acetate, and methanol (12 L × 3 times, 24 hours/time) at room temperature. The combined extracts were evaporated in vacuo to obtain n-hexane residue (17.6 g), ethyl acetate residue (10 g), and MeOH residue (300 g), respectively.The ethyl acetate residue (10 g) was separated on the silica gel CC, eluted withn-hexane/ethyl acetate gradient (0–100% ethyl acetate) to afford 15 fractions E1–E15. Compounds 2 (15 mg), 3 (5.5 mg), and 4 (4.5 mg) were obtained from E1 fraction (0.2 g), E3 fraction (0.12 g), and E4 fraction (0.15 g), respectively, by crystallization in n-hexane. Fraction E10 (0.5 g) was fractionated by Sephadex CC, eluted with CH2Cl2/MeOH (1/9, v/v) to afford 4 subfractions E10.1–E10.4. Subfraction E10.2 (17 mg) was purified by preparative TLC using CH2Cl2/MeOH (95/5, v/v) as eluent to yield 5 (2 mg) and 6 (2.5 mg). Fraction E10.3 (25 mg) was purified by silica gel CC, eluted with CH2Cl2/MeOH (95/5, v/v) to obtain compound 7 (15 mg). Fraction E12 (0.9 g) was fractionated by Sephadex CC, eluted with CH2Cl2/MeOH (1/9, v/v) to give 3 subfractions E12.1–12.3. Compound 8 (6.1 mg) was obtained from E12.1 fraction (85 mg) by crystallization in n-hexane. Fraction E12.2 (30 mg) was chromatographed by silica gel CC, eluted with CH2Cl2/MeOH (95/5, v/v) to yield 9 (15 mg). Fraction E13 (0.7 g) was purified by Sephadex CC, eluted with CH2Cl2/MeOH (1/9), to give two subfractions E13.1–E13.2. Subfraction E13.2 (20 mg) was purified by preparative TLC using CH2Cl2/MeOH (95/5, v/v) as an eluant to afford compound 10 (3 mg). The MeOH extract (300 g) was chromatographed on a silica gel CC and eluted with a gradient solvent system of n-hexane-ethyl acetate (100 : 1–0 : 1, v/v) to afford 14 fractions M1–M14, respectively. Fraction M6 (0.8 g) was separated by Sephadex LH-20 CC eluting with CH2Cl2/MeOH (1/9, v/v) to yield compound 11 (7 mg). Fraction M12 (0.6 g) was purified by Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1/9, v/v) to afford compound 12 (6 mg). Compound 13 (12 mg) was obtained from M13 fraction (1.1 g) by purification on Sephadex LH-20 CC, eluted with CH2Cl2/MeOH (1/9, v/v). Fraction M14 was purified by silica gel CC and eluted with CH2Cl2/acetone (9 : 1, v/v) to give 3 fractions M14.1–M14.3. Fraction M14.1 (0.5 g) was subjected to a Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1/9, v/v) to yield 14 (5.7 mg). Fraction M14.2 (2 g) was separated by Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1/9, v/v) to give 3 fractions M14.2.1–M14.2.3. Fraction M14.2.1 (0.45 g) was purified by Sephadex LH-20 CC, followed by separation with silica gel CC, eluted with CH2Cl2/MeOH (9/1, v/v) to yield compound 1 (20 mg). Fraction M14.2 (0.28 g) was subjected to silica gel CC eluted with CH2Cl2/MeOH (9/1, v/v) to yield 2 subfractions M14.2.2.1-M14.2.2.2. Fraction M14.2.2.2 (84 mg) was purified by Sephadex LH-20 CC, eluting with CH2Cl2/MeOH (1/9, v/v) to yield compound 15 (7 mg).(R,S)-N-(5-Hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1): colorless oil; αD25∼0 (c 0.4, MeOH); IR νmax (KBr) 3239, 3022, 1706, 1664, 1211 cm−1; HR-ESI-MS (positive-ion mode) m/z 141.0673 [(M-H2O) + H]+ (calcd. for C6H9N2O2+, 141.0659); 1H and 13C-NMR data (Table 1).Table 1
1H and 13C NMR data of 1.
C1 (acetone-d6)1 (MeOH-d4)δCaδHb (m, J, Hz)δCaδHb (m, J, Hz)2172.4—175.0—327.32.46–2.39 (m, H-3a)27.82.59–2.50 (m, H-3a)2.38–2.32 (m, H-3b)2.38–2.32 (m, H-3b)427.12.27–2.21 (m, H-4a)27.12.27–2.21 (m, H4a)1.88–1.81 (m, H-4b)1.88–1.81 (m, H4b)583.45.22 (br s)84.25.22 (dd,J = 3.5 Hz, 6.5 Hz)7169.6—172.1—820.61.96 (s)20.52.06 (s)OH—5.47 (br d,J = 5.5)——NH—9.16 (br s)——


### 2.4. Cytotoxicity Assay

The cytotoxicity assays were carried out in triplicate against KB, HepG-2, LU, and MCF-7 cell lines (American Type and Culture Collection, ATCC). Cells were maintained in Dulbecco’s D-MEM medium, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100μg/mL), and gentamicin (10 μg/mL). Stock solutions of compounds were prepared in DMSO/H2O (1/9), and the cytotoxicity assays were carried out against cancer cell lines (3 × 103 cells/mL) using a modification of the published method [13]. After 72 h of incubation at 37°C in air/CO2 (95 : 5) with or without test compounds, cell growth was estimated by colorimetric measurement at 540 nm with a Titertek Multiskan photometer. Ellipticine was used as a positive compound.


### 2.5. Antimicrobial Assay

The antimicrobial activity was evaluated by the microdilution method previously described by Hadacek [14] and expressed as IC50 (50% inhibitory concentration) values. Six bacterial strains and a fungus were used for the test: 3 strains of Gram (+) bacteria: Staphylococcus aureus ATCC 13709, Bacillus subtilis ATCC 6633, and Lactobacillus fermentum N4; 3 strains of Gram (−) bacteria: Salmonella enterica ATCC12228, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 15442; fungus: Candida albicans ATCC 10231. Bacterial solutions with a concentration of 5 × 105 CFU/ml and a fungal solution with a concentration of 1 × 103 CFU/ml were prepared. The isolated compounds were diluted in DMSO at the following dilution concentrations: 128, 64, 32, 16, 8, and 4 μg/mL. The positive controls were ampicillin for Gram (+) bacterial strains, cefotaxime for Gram (−) bacterial strains, and nystatin for fungus. IC50 values were determined based on the measured turbidity by optical Biotech spectra and raw data software.


## 2.1. General Experimental Procedures

The NMR data including1H-NMR, 13C-NMR, 15N-NMR, HSQC, and HMBC spectra were recorded by a Bruker AM500 FT-NMR spectrometer using TMS as an internal standard. The HR-ESI-MS was obtained using an Agilent 6530 Accurate Mass Q-TOF LC/MS system. Column chromatography (CC) was performed on silica gel (Merck, 230–400 mesh) or Sephadex LH-20 (Sigma Aldrich). Thin layer chromatography used precoated silica gel plates (Merck 60 F254). Compounds were visualized by spraying with 10% aqueous H2SO4.


## 2.2. Plant Materials

The plant stems were collected from the Ben En National Park, Thanh Hoa province, Vietnam, in 2018. The plant was identified by Dr. Do Ngoc Dai, Nghe An University of Economics, asRourea oligophlebia Merr. (Connaraceae). A voucher specimen (MT-202) was deposited at Hong Duc University, Faculty of Natural Sciences.


## 2.3. Extraction and Isolation

The stem powder (5.3 kg) ofRourea oligophlebia was extracted successively with n-hexane, ethyl acetate, and methanol (12 L × 3 times, 24 hours/time) at room temperature. The combined extracts were evaporated in vacuo to obtain n-hexane residue (17.6 g), ethyl acetate residue (10 g), and MeOH residue (300 g), respectively.The ethyl acetate residue (10 g) was separated on the silica gel CC, eluted withn-hexane/ethyl acetate gradient (0–100% ethyl acetate) to afford 15 fractions E1–E15. Compounds 2 (15 mg), 3 (5.5 mg), and 4 (4.5 mg) were obtained from E1 fraction (0.2 g), E3 fraction (0.12 g), and E4 fraction (0.15 g), respectively, by crystallization in n-hexane. Fraction E10 (0.5 g) was fractionated by Sephadex CC, eluted with CH2Cl2/MeOH (1/9, v/v) to afford 4 subfractions E10.1–E10.4. Subfraction E10.2 (17 mg) was purified by preparative TLC using CH2Cl2/MeOH (95/5, v/v) as eluent to yield 5 (2 mg) and 6 (2.5 mg). Fraction E10.3 (25 mg) was purified by silica gel CC, eluted with CH2Cl2/MeOH (95/5, v/v) to obtain compound 7 (15 mg). Fraction E12 (0.9 g) was fractionated by Sephadex CC, eluted with CH2Cl2/MeOH (1/9, v/v) to give 3 subfractions E12.1–12.3. Compound 8 (6.1 mg) was obtained from E12.1 fraction (85 mg) by crystallization in n-hexane. Fraction E12.2 (30 mg) was chromatographed by silica gel CC, eluted with CH2Cl2/MeOH (95/5, v/v) to yield 9 (15 mg). Fraction E13 (0.7 g) was purified by Sephadex CC, eluted with CH2Cl2/MeOH (1/9), to give two subfractions E13.1–E13.2. Subfraction E13.2 (20 mg) was purified by preparative TLC using CH2Cl2/MeOH (95/5, v/v) as an eluant to afford compound 10 (3 mg). The MeOH extract (300 g) was chromatographed on a silica gel CC and eluted with a gradient solvent system of n-hexane-ethyl acetate (100 : 1–0 : 1, v/v) to afford 14 fractions M1–M14, respectively. Fraction M6 (0.8 g) was separated by Sephadex LH-20 CC eluting with CH2Cl2/MeOH (1/9, v/v) to yield compound 11 (7 mg). Fraction M12 (0.6 g) was purified by Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1/9, v/v) to afford compound 12 (6 mg). Compound 13 (12 mg) was obtained from M13 fraction (1.1 g) by purification on Sephadex LH-20 CC, eluted with CH2Cl2/MeOH (1/9, v/v). Fraction M14 was purified by silica gel CC and eluted with CH2Cl2/acetone (9 : 1, v/v) to give 3 fractions M14.1–M14.3. Fraction M14.1 (0.5 g) was subjected to a Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1/9, v/v) to yield 14 (5.7 mg). Fraction M14.2 (2 g) was separated by Sephadex LH-20 CC and eluted with CH2Cl2/MeOH (1/9, v/v) to give 3 fractions M14.2.1–M14.2.3. Fraction M14.2.1 (0.45 g) was purified by Sephadex LH-20 CC, followed by separation with silica gel CC, eluted with CH2Cl2/MeOH (9/1, v/v) to yield compound 1 (20 mg). Fraction M14.2 (0.28 g) was subjected to silica gel CC eluted with CH2Cl2/MeOH (9/1, v/v) to yield 2 subfractions M14.2.2.1-M14.2.2.2. Fraction M14.2.2.2 (84 mg) was purified by Sephadex LH-20 CC, eluting with CH2Cl2/MeOH (1/9, v/v) to yield compound 15 (7 mg).(R,S)-N-(5-Hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1): colorless oil; αD25∼0 (c 0.4, MeOH); IR νmax (KBr) 3239, 3022, 1706, 1664, 1211 cm−1; HR-ESI-MS (positive-ion mode) m/z 141.0673 [(M-H2O) + H]+ (calcd. for C6H9N2O2+, 141.0659); 1H and 13C-NMR data (Table 1).Table 1
1H and 13C NMR data of 1.
C1 (acetone-d6)1 (MeOH-d4)δCaδHb (m, J, Hz)δCaδHb (m, J, Hz)2172.4—175.0—327.32.46–2.39 (m, H-3a)27.82.59–2.50 (m, H-3a)2.38–2.32 (m, H-3b)2.38–2.32 (m, H-3b)427.12.27–2.21 (m, H-4a)27.12.27–2.21 (m, H4a)1.88–1.81 (m, H-4b)1.88–1.81 (m, H4b)583.45.22 (br s)84.25.22 (dd,J = 3.5 Hz, 6.5 Hz)7169.6—172.1—820.61.96 (s)20.52.06 (s)OH—5.47 (br d,J = 5.5)——NH—9.16 (br s)——


## 2.4. Cytotoxicity Assay

The cytotoxicity assays were carried out in triplicate against KB, HepG-2, LU, and MCF-7 cell lines (American Type and Culture Collection, ATCC). Cells were maintained in Dulbecco’s D-MEM medium, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100μg/mL), and gentamicin (10 μg/mL). Stock solutions of compounds were prepared in DMSO/H2O (1/9), and the cytotoxicity assays were carried out against cancer cell lines (3 × 103 cells/mL) using a modification of the published method [13]. After 72 h of incubation at 37°C in air/CO2 (95 : 5) with or without test compounds, cell growth was estimated by colorimetric measurement at 540 nm with a Titertek Multiskan photometer. Ellipticine was used as a positive compound.


## 2.5. Antimicrobial Assay

The antimicrobial activity was evaluated by the microdilution method previously described by Hadacek [14] and expressed as IC50 (50% inhibitory concentration) values. Six bacterial strains and a fungus were used for the test: 3 strains of Gram (+) bacteria: Staphylococcus aureus ATCC 13709, Bacillus subtilis ATCC 6633, and Lactobacillus fermentum N4; 3 strains of Gram (−) bacteria: Salmonella enterica ATCC12228, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 15442; fungus: Candida albicans ATCC 10231. Bacterial solutions with a concentration of 5 × 105 CFU/ml and a fungal solution with a concentration of 1 × 103 CFU/ml were prepared. The isolated compounds were diluted in DMSO at the following dilution concentrations: 128, 64, 32, 16, 8, and 4 μg/mL. The positive controls were ampicillin for Gram (+) bacterial strains, cefotaxime for Gram (−) bacterial strains, and nystatin for fungus. IC50 values were determined based on the measured turbidity by optical Biotech spectra and raw data software.


## 3. Results and Discussion

### 3.1. Structure Elucidation

Compound1 was isolated as colorless oil. Its molecular formula of 1 was deduced as C6H10N2O3 from the [(M-H2O)+H]+ peak at m/z 141.0673 (C6H9N2O2, calcd. for 141.0659) in the positive-ion HR-ESI-MS spectrum (Figure S2). The molecular formula of C6H10N2O3 was further confirmed by combined 1H, 13C, and 15N-NMR spectra as ten protons, six carbons, and two signals of nitrogen (δN 199.99 and 165.86) were observed (Figure S3). The IR spectrum suggested that 1 contained carbonyl amide groups (1706, 1664 cm−1) and hydroxyl and amine groups (3239 cm−1) (Figure S4), which appeared in the 1H-NMR spectrum (in acetone-d6) at δH 5.47 (br d, J = 5.5 Hz) and 9.16 (br s), respectively (Figure S5). The 1H-NMR spectrum (in MeOH-d4) showed signals of an oxymethine group at δH 5.22 (1H, dd, J = 3.5, 6.5 Hz, H-5), two methylene groups (H-3, H-4), and a methyl singlet of an acetyl group at δH 2.06 (3H, s, H-8) (Figure S6). The 13C-NMR spectrum of 1 displayed six carbon signals including two signals of carbonyl group at δC 175.0 (C-2) and 172.1 (C-7), an oxymethine group at δC 84.2 (C-5), two methylene groups at δC 27.1 (C-3) and 27.8 (C-4), and a methyl signal at δC 20.5 (C-8) (Figures S7−S10). The HMBC cross-peaks (in acetone-d6) of the hydroxyl group (δH 5.47) to C-5 (δC 84.2) confirmed the free hydroxyl group was placed at C-5 position. In the HMBC spectrum (in MeOH-d4), the correlations of H-3 (δH 2.59–2.50 and 2.38–2.32), H-4 (δH 2.27–2.21 and 1.88–1.81), and H-5 (δH 5.47) to C-2 (δC 175.0) suggested a 5-hydroxy-pyrrolidin-2-one moiety in the structure of 1(Figures S11 and S12). In addition, methyl protons H-8 (δH 2.06) have only correlated with the carbonyl carbon C-7 (δC 172.1) (Figure 2). Based on the optical rotation value of nearly zero and the CD spectrum (Figure S13), compound 1 may be regarded as a racemate. Therefore, the structure of 1 was assigned as (R, S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide. It is noted that alkaloids containing 5-hydroxy-2-pyrrolidone fragment were rarely found in nature. To our knowledge, only few examples are brachystemidine D isolated from Brachystemma calycinum [15], longistrobin and isolongistrobin from Macrorungia longistrabus [16], and lepiota and (R)-5-hydroxypyrrolidin-2-one from the mushroom Macrolepiota neomastoidea [17].Figure 2
Key HMBC correlations of the new compound1.The known compounds were elucidated as friedelin (2), friedanol (3) [12], taraxerol (4) [18], vanillin (5), coniferyl aldehyde (6) [19], apigenin (7) [20], 7α-hydroxy-3β-sitosterol (8) [21], coniferyl alcohol (9) [22], scopoletin (10) [23], emodin (11) [24], protocatechuic acid (12), catechin (13) [25], procyanidin A1 (14) [26], and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15) [27]. Compounds 2-3, 6–9, 11-12, and 15 were reported for the first time from Rourea genus.


### 3.2. Biological Activities

Several isolated compounds were evaluated for antimicrobial activity and cytotoxicity. Only emodin (11) showed good antimicrobial activity against Gram (+) strains S. aureus, B. subtilis, and L. fermentum with IC50 values of 4.51, 15.83, and 28.94 µg/mL, respectively, but exhibited no activity against Gram (−) strains and fungus at the concentration of 128 µg/mL. Other compounds were also inactive (IC50 > 128 µg/mL) in antimicrobial activity test. The cytotoxicity of isolated compounds was evaluated against KB, HepG-2, LU, and MCF-7 cancer cell lines (Table S1). Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 with IC50 ranging from 18.73 ± 0.40 µg/mL to 43.63 ± 0.90 µg/mL over KB and LU cancer cell lines. Compound 7 exhibited similar effect on KB, HepG-2 and MCF-7 cell lines. Among the tested compounds, emodin (11) had the best cytotoxicity on LU cancer cell line with IC50 values of 21.04±0.52 µg/mL, respectively.


## 3.1. Structure Elucidation

Compound1 was isolated as colorless oil. Its molecular formula of 1 was deduced as C6H10N2O3 from the [(M-H2O)+H]+ peak at m/z 141.0673 (C6H9N2O2, calcd. for 141.0659) in the positive-ion HR-ESI-MS spectrum (Figure S2). The molecular formula of C6H10N2O3 was further confirmed by combined 1H, 13C, and 15N-NMR spectra as ten protons, six carbons, and two signals of nitrogen (δN 199.99 and 165.86) were observed (Figure S3). The IR spectrum suggested that 1 contained carbonyl amide groups (1706, 1664 cm−1) and hydroxyl and amine groups (3239 cm−1) (Figure S4), which appeared in the 1H-NMR spectrum (in acetone-d6) at δH 5.47 (br d, J = 5.5 Hz) and 9.16 (br s), respectively (Figure S5). The 1H-NMR spectrum (in MeOH-d4) showed signals of an oxymethine group at δH 5.22 (1H, dd, J = 3.5, 6.5 Hz, H-5), two methylene groups (H-3, H-4), and a methyl singlet of an acetyl group at δH 2.06 (3H, s, H-8) (Figure S6). The 13C-NMR spectrum of 1 displayed six carbon signals including two signals of carbonyl group at δC 175.0 (C-2) and 172.1 (C-7), an oxymethine group at δC 84.2 (C-5), two methylene groups at δC 27.1 (C-3) and 27.8 (C-4), and a methyl signal at δC 20.5 (C-8) (Figures S7−S10). The HMBC cross-peaks (in acetone-d6) of the hydroxyl group (δH 5.47) to C-5 (δC 84.2) confirmed the free hydroxyl group was placed at C-5 position. In the HMBC spectrum (in MeOH-d4), the correlations of H-3 (δH 2.59–2.50 and 2.38–2.32), H-4 (δH 2.27–2.21 and 1.88–1.81), and H-5 (δH 5.47) to C-2 (δC 175.0) suggested a 5-hydroxy-pyrrolidin-2-one moiety in the structure of 1(Figures S11 and S12). In addition, methyl protons H-8 (δH 2.06) have only correlated with the carbonyl carbon C-7 (δC 172.1) (Figure 2). Based on the optical rotation value of nearly zero and the CD spectrum (Figure S13), compound 1 may be regarded as a racemate. Therefore, the structure of 1 was assigned as (R, S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide. It is noted that alkaloids containing 5-hydroxy-2-pyrrolidone fragment were rarely found in nature. To our knowledge, only few examples are brachystemidine D isolated from Brachystemma calycinum [15], longistrobin and isolongistrobin from Macrorungia longistrabus [16], and lepiota and (R)-5-hydroxypyrrolidin-2-one from the mushroom Macrolepiota neomastoidea [17].Figure 2
Key HMBC correlations of the new compound1.The known compounds were elucidated as friedelin (2), friedanol (3) [12], taraxerol (4) [18], vanillin (5), coniferyl aldehyde (6) [19], apigenin (7) [20], 7α-hydroxy-3β-sitosterol (8) [21], coniferyl alcohol (9) [22], scopoletin (10) [23], emodin (11) [24], protocatechuic acid (12), catechin (13) [25], procyanidin A1 (14) [26], and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15) [27]. Compounds 2-3, 6–9, 11-12, and 15 were reported for the first time from Rourea genus.


## 3.2. Biological Activities

Several isolated compounds were evaluated for antimicrobial activity and cytotoxicity. Only emodin (11) showed good antimicrobial activity against Gram (+) strains S. aureus, B. subtilis, and L. fermentum with IC50 values of 4.51, 15.83, and 28.94 µg/mL, respectively, but exhibited no activity against Gram (−) strains and fungus at the concentration of 128 µg/mL. Other compounds were also inactive (IC50 > 128 µg/mL) in antimicrobial activity test. The cytotoxicity of isolated compounds was evaluated against KB, HepG-2, LU, and MCF-7 cancer cell lines (Table S1). Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 with IC50 ranging from 18.73 ± 0.40 µg/mL to 43.63 ± 0.90 µg/mL over KB and LU cancer cell lines. Compound 7 exhibited similar effect on KB, HepG-2 and MCF-7 cell lines. Among the tested compounds, emodin (11) had the best cytotoxicity on LU cancer cell line with IC50 values of 21.04±0.52 µg/mL, respectively.


## 4. Conclusions

In conclusion, fifteen compounds were isolated from the stems ofRourea oligophlebia, among which a new alkaloid (R,S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1) was identified along with fourteen known compounds 2–15. Compound 11 showed good antimicrobial activity on Gram (+) strains and moderate cytotoxicity against KB, Hep-G2, and LU-1 cell lines. Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 over KB and LU cancer cell lines, while compound 7 was moderately active on KB, HepG-2, and MCF-7 cell lines.



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*Source: 6659106-2021-08-18.xml*