4.1 General experimental procedures

A JASCO P-1020 digital polarimeter measured at 590 nm (Na lamp D line) was used to measure the specific optical rotation values. A JASCO J-815 spectropolarimeter was utilized to provide the ECD spectra. A Perkin–Elmer Spectrum One spectrometer [attenuated total reflectance (ATR)] was used to obtain FT-IR spectra. A Bruker AVANCE 600 spectrometers was used to record 1D and 2D NMR spectra. A Bruker MicroTOFLC spectrometer was used to record the ESITOFMS (positive mode) data. The stationary phases for column chromatography (CC) are silica gel 60 (Merck, 70–200 mesh ASTM), Sephadex LH-20 gel (Ambersham Biosciences), and silica gel 60 RP-18 (Merck, 40–63 µm). Preparative TLC was performed with silica gel 60 PF254. TLC plate detection was carried out on silica gel precoated aluminum-backed plate (silica gel 60 F254, Merck). A VertiSep™ UPS RP-C18 column (21.2 × 250 mm) on Waters 600 Delta HPLC instrument connected to a Water photodiode array detector was used for purification with the eluent flow rate at 12 mL/min.

4.2 Plant materials

The twigs and stems of Casearia grewiifolia Vent. (Salicaceae) were collected from the Sai Yok National Park, Kanchanaburi Province, Thailand (14.2495, 99.0270) in December 2020. One of the authors (T.T.) authenticated the plant, and a voucher specimen (SY16) has been deposited at the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Thailand.

4.3 Extraction and isolation

Air dried twigs and stems powder of C. grewiifolia (8.9 kg) were macerated two times with CH2Cl2 at ambient temperature, and the resulting solvent was concentrated under vacuum to afford 50.0 g of the CH2Cl2 extract. A 48 g of CH2Cl2 extract was developed to column chromatography (CC) with silica gel with the mobile phase of hexane–acetone (a gradient from 100:0 to 0:100) and acetone-MeOH (a gradient from 100:0 to 0:100) provide sixteen fractions (A1–A16). The Sephadex LH-20 CC was then applied for fraction A6 (13.6 g) using CH2Cl2–MeOH in a ratio of 1:1 as eluent to give five fractions (B1–B5). Fraction B3 (2.7 g), was fractionated by silica gel CC with the mobile phase of hexane–EtOAc (a gradient from 100:0 to 0:100) providing seven fractions (C1–C7). A gradient elution of hexane and acetone was applied to silica gel CC for further separated fraction C5 (1.5 g) to provide eleven fractions (D1–D11). Using HPLC approach for purification of fraction D3 (69.0 mg) and eluting with a CH3CN–H2O (70:30 to 100:0 for 180 min) gave compounds 10 (2.0 mg) and 11 (2.6 mg). Compound 4 (2.0 mg) was afforded from the purification of fraction D5 (1.5 g) by HPLC, using an isocratic system of CH3CN in H2O (73:27) as eluent. Compounds 6 (1.3 mg), 9 (25.6 mg), and 5 (28.5 mg) were obtained from fraction D6 (782.5 mg) by the RP 18 HPLC column using an isocratic system of CH3CN–H2O (72:27) as mobile phase. Fraction B4 (3.4 g) was chromatographed by CC with gel filtration, Sephadex LH-20, eluting with the mobile phase of CH2Cl2–MeOH in ratio of 1:1., then followed by RP 18 HPLC column, eluting with an isocratic system of CH3CN–H2O (65:35) to give additional yields of compounds 11 (5.7 mg), 9 (21.5 mg), and 5 (42.6 mg). A 1.6 g of fraction A8 was subjected by CC with a gel filtration, Sephadex LH-20, eluting with CH2Cl2–MeOH in a ratio of 1:1 and then purified by silica gel CC with a gradient of MeOH in CH2Cl2 as eluent to afford sixteen fractions (E1–E16). After purifying fraction E10 (15.9 mg) with RP 18 HPLC column, using a mobile phase of MeOH–H2O (a gradient from 60:40 to 80:20 in 140 min), compounds 3 (19.5 mg) and 15 (8.5 mg) were obtained. The eluent of CH2Cl2–MeOH (1:1) was applied on Sephadex LH-20 CC for the fractionations of fraction E12 (231.9 mg) to give three fractions (F1–F3). Subsequently, fraction F2 (139.9 mg) was applied on preparative TLC plates, eluting with the mobile phase of hexane:acetone:EtOAc in a ratio of 8:1:1 yielding the three bands (G1–G3). Compound 12 (17.5 mg) was also obtained from band G1, while band G2 (47.4 mg) was subjected to RP 18 HPLC column using a mixture of CH3CN in H2O (a gradient from 70:30 to 80:20 for 60 min) as eluent to give compounds 2 (6.4 mg) and 1 (1.8 mg). Compounds 8 (11.0 mg), 7 (29.5 mg), and 16 (6.5 mg) was obtained from fraction E13 (181.2 mg), purifying by RP 18 HPLC column with a gradient elution of CH3CN in H2O (60:40 to 80:20 for 90 min) at flow rate of 10 mL/min. The Sephadex LH-20 CC with eluent of CH2Cl2–MeOH (1:1) and silica gel CC with a gradient elution of MeOH in CH2Cl2 were used for fractionation of fraction A12 (5.9 g), yielding twelve fractions (H1–H12). Fraction H10 (424.4 mg) was applied on preparative TLC plates, eluting with a mixture of hexane:acetone:MeOH in the ratio of 70:30:1 to provide three bands (I1–I3). Band I2 (152.4 mg) was further purified with HPLC with a gradient elution of CH3CN in H2O (65:35 to 70:30 for 140 min) to afforded compounds 18 (3.0 mg) and 14 (2.5 mg). Compound 17 (8.2 mg) was received from band I3 (198.5 mg) by HPLC, eluting with an isocratic system of CH3CN–H2O (65:35). Purification of fraction H11 (709.3 mg) with RP 18 HPLC column which was eluted with an isocratic system of CH3CN in H2O (80:20) afford compounds 14 (20.0 mg) and 13 (6.0 mg). The eluent, a mixture of CH2Cl2 and MeOH (1:1), was applied to Sephadex LH-20 CC for the purification of fraction A13 (3.06 g) provided four fractions (J1–J4). Further purification of fraction J3 (1.1 g) by silica gel CC with a gradient elution of MeOH in CH2Cl2 provided seven fractions (K1–K7) were obtained. Compounds 19 (23.5 mg) and 20 (21.0 mg) were purified from fractions K4 (52.0 mg) and K6 (35.0 mg), respectively, were applied on preparative TLC plates, eluting with MeOH:CH2Cl2 in the ratio of 1:99 and 2:98 respectively.

4.4 Compound characterization4.4.1 Grewiifopene A (1)

Optically active colorless oil; [α] 25D + 29.6 (c 0.30, CHCl3); ECD (c 0.06 μM, acetonitrile) λmax (Δε): 204 (+ 30.94), 226 (− 0.98), 236 (+ 0.45); IR (ATR) νmax 2970, 2933, 1729, 1456, 1373, 1226, 1182, 1138, 1098, 1074, 1019, 951, 897, 733 cm−1; HRESIMS (positive mode) m/z 613.2990 (calcd for C32H46NaO10 [M+Na]+, 613.2983).

4.4.2 Grewiifopene B (2)

Optically active colorless oil; [α] 25D + 52.0 (c 0.67, CHCl3); ECD (c 0.06 μM, acetonitrile) λmax (Δε): 203 (+ 20.34), 223 (+ 0.24), 228 (− 0.29), 243 (+ 0.32); IR (ATR) νmax 2959, 2931, 1733, 1457, 1372, 1226, 1184, 1096, 1074, 1019, 998, 979, 950, 897, 734 cm−1; HRESIMS (positive mode) m/z 613.2981 (calcd for C32H46NaO10 [M+Na]+, 613.2983).

4.4.3 Grewiifopene C (3)

Optically active colorless oil; [α] 25D + 58.7 (c 2.30, CHCl3); ECD (c 0.06 μM, acetonitrile) λmax (Δε): 191 (+ 24.76), 227 (+ 0.60), 237 (+ 1.42); IR (ATR) νmax 2960, 2930, 1744, 1371, 1217, 1167, 1100, 1071, 1023, 999, 946, 917, 900, 734 cm−1; HRESIMS (positive mode) m/z 641.2931 (calcd for C33H46NaO11 [M+Na]+, 641.2932).

4.4.4 Grewiifopene D (4)

Optically active colorless oil; [α] 25D − 108.3 (c 0.07, CHCl3); ECD (c 0.28 μM, acetonitrile) λmax (Δε): 200 (− 2.33), 220 (− 0.34); IR (ATR) νmax 2965, 2930, 1755, 1731, 1454, 1374, 1225, 1179, 1151, 1100, 1076, 1024, 995, 936, 898 cm−1; HRESIMS (positive mode) m/z 555.2932 (calcd for C30H44NaO8 [M+Na]+, 555.2928).

4.4.5 Grewiifopene E (12)

Optically active colorless oil; [α] 25D + 66.2 (c 1.72, CHCl3); ECD (c 0.05 μM, acetonitrile) λmax (Δε): 200 (+ 28.49), 229 (+ 0.70), 236 (+ 1.17), 238 (+ 1.05), 250 (+ 0.62); IR (ATR) νmax 3503, 2959, 2927, 1733, 1465, 1457, 1371, 1291, 1226, 1188, 1167, 1093, 1023, 996, 956, 902, 735 cm−1; HRESIMS (positive mode) m/z 641.3303 (calcd for C34H50NaO10 [M+Na]+, 641.3296).

4.4.6 Grewiifopene F (13)

Optically active colorless oil; [α] 25D + 26.8 (c 0.41, CHCl3); ECD (c 0.06 μM, acetonitrile) λmax (Δε): 200 (+ 6.29), 229 (− 0.22), 279 (+ 0.61), 312 (+ 0.90); IR (ATR) νmax 3388, 2957, 2925, 2854, 1724, 1632, 1604, 1515, 1446, 1372, 1264, 1168, 1097, 1044, 1006, 991, 948, 900, 833, 731 cm−1; HRESIMS (positive mode) m/z 675.3138 (calcd for C37H48NaO10 [M+Na]+, 675.3140).

4.4.7 Grewiifopene G (14)

Optically active colorless oil; [α] 25D + 54.1 (c 0.39, CHCl3); ECD (c 0.07 μM, acetonitrile) λmax (Δε): 190 (+ 15.69), 201 (+ 7.82), 206 (+ 7.93), 231 (+ 0.31), 299 (+ 0.72), 340 (+ 0.02); IR (ATR) νmax 3406, 2960, 2928, 1711, 1632, 1604, 1515, 1443, 1373, 1227, 1168, 1094, 1024, 996, 903, 833, 735 cm−1; HRESIMS (positive mode) m/z 703.3098 (calcd for C38H48NaO11 [M+Na]+, 703.3089).

4.4.8 Grewiifopene H (15)

Optically active colorless oil; [α] 25D + 58.7 (c 0.98, CHCl3); ECD (c 0.06 μM, acetonitrile) λmax (Δε): 196 (+ 23.82), 239 (− 1.61), 259 (+ 0.47); IR (ATR) νmax 2963, 2930, 1748, 1434, 1370, 1216, 1105, 1071, 1060, 999, 982, 962, 931, 917 cm−1; HRESIMS (positive mode) m/z 641.2943 (calcd for C33H46NaO11 [M+Na]+, 641.2932).

4.4.9 Grewiifopene I (16)

Optically active colorless oil; [α] 25D + 40.7 (c 0.62, CHCl3); ECD (c 0.07 μM, acetonitrile) λmax (Δε): 204 (+ 19.80), 246 (− 0.30), 256 (+ 0.26); IR (ATR) νmax 3456, 2965, 2930, 2878, 1749, 1729, 1461, 1373, 1227, 1177, 1150, 1099, 1074, 1023, 1002, 959, 946, 925, 890, 735 cm−1; HRESIMS (positive mode) m/z 541.2782 (calcd for C29H42NaO8 [M+Na]+, 541.2772).

4.4.10 Grewiifopene J (17)

Optically active colorless oil; [α] 25D + 116.7 (c 0.46, CHCl3); ECD (c 0.06 μM, acetonitrile) λmax (Δε): 209 (+ 9.20), 238 (− 5.48), 290 (+ 2.36); IR (ATR) νmax 3407, 2962, 2929, 1727, 1632, 1604, 1515, 1441, 1371, 1329, 1226, 1168, 1093, 1024, 998, 981, 960, 918, 891, 833, 734 cm−1; HRESIMS (positive mode) m/z 703.3090 (calcd for C38H48NaO11 [M+Na]+, 703.3089).

4.4.11 Grewiifopene K (18)

Optically active colorless oil; [α] 25D + 74.0 (c 0.29, CHCl3); ECD (c 0.09 μM, acetonitrile) λmax (Δε): 198 (+ 8.60), 223 (+ 0.10), 236 (− 1.91); IR (ATR) νmax 3374, 2958, 2926, 1728, 1632, 1604, 1515, 1448, 1372, 1227, 1161, 1095, 1025, 998, 894, 834, 736 cm−1; HRESIMS (positive mode) m/z 703.3090 (calcd for C38H48NaO11 [M+Na]+, 703.3089).

4.5 Preparation of 2-methylbutyric acid benzyl esters

A solution of benzyl alcohol (31.8 mg, 0.3 mmol) in CH2Cl2 1 mL was added to R/S-2-methylbutanoic acid (20 mg, 0.2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (57 mg, 0.3 mmol), and dimethylaminopyridine (DMAP) (1 mg, 0.008 mmol). The reaction mixture was stirred at room temperature for 3 h, then evaporated and purified by PTLC with 5% EtOAc-hexnae to afford R/S-2-methylbutyric acid benzyl esters (32 mg, 85%): colorless oil; 1H NMR (400 MHz, CDCl3) 7.37–7.29 (m, H-2–H-6), 5.12 (s, H2-7), 2.45 (sext, J = 7.0 Hz, H-2′), 1.70 (m, J = 7.4 Hz H-3′a), 1.49 (m, H-3′b), 1.16 (d, J = 7.0 Hz, Me-5′), 0.89 (t, J = 7.5 Hz, Me-4′); 13C NMR (100 MHz, CDCl3): 176.6 (C-1′), 136.3 (C-1), 128.5 (C-2, C-6), 128.07 (C-3, C-5), 128.1 (C-4), 66.0 (C-7), 41.1 (C-2′), 26.8 (C-3′), 16.6 (C-5′), 11.6 (C-4′) (Figs. S119 and S120, Supporting Information). The corresponding S-2-methylbutyric acid benzyl ester (30 mg, 85%) was obtained by the same procedure using (S)-2-methylbutanoic acid.

4.6 Transesterification of compound 5

Corymbulosin N (5) (28.1 mg, 0.053 mmol), benzyl alcohol (0.5 mL, 4.8 mmol), and 4-dimethylaminopyridine (0.65 mg, 0.053 mmol) was heated to 120 °C in seal tube and stirred for 48 h. The reaction was allowed to cool to room temperature and then was purified with column chromatography with 5% EtOAc in hexane as eluent to remove a large amount of benzyl alcohol. The resulting was analyzed by chiral HPLC.

4.7 ECD computational methods

Spartan’ 20 with MMFF94 molecular mechanics model (within 5 kcal/mol energy window) was used for a conformational search and Guassian 16 Rev. C.01 program was used for all DFT calculations [24, 25]. Further optimization of the obtained low-energy conformers was calculated using wB97XD/cc-PVDZ level of theory with IEFPCM of methanol solvent model. All optimized conformers were confirmed to be a true minimum of electronic potential energy by means of the vibrational frequency calculation at the same level without any detection of imaginary frequencies. Each conformer with the over 2% population was subjected to the ECD calculations, according to Boltzmann distribution law base on Gibbs free energies. Using TD-DFT calculations at the CAM-B3LYP/def2-SVP level of theory and the application of the IEFPCM of methanol solvent model [26] were computed the simulated ECD spectra of 1, 4, 8, and 15. Thirty excited states of each conformer were calculated and the resulting ECD curves were developed using SpecDis with Boltzmann average all conformers and overlapping Gaussian function with an exponential half-width (σ = 0.35) [27, 28]. For the related enantiomers of 1, 4, 8, and 15, their theoretical ECD spectra derived from the direct inversion of their simulated ECD spectra.

4.8 Cytotoxicity assays

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [29] was employed for the cytotoxic evaluation against five human cancer cell lines, including HepG2, A549, MDA-MB-231, HeLa, and HuCCA-1, while the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide) method [30] was used for MOLT-3 cancer cell. Doxorubicin and etoposide were used as positive controls (Table 4).

4.9 Antibacterial susceptibility testing4.9.1 Tested microorganisms

The tested strains, Staphylococcus aureus (S. aureus) ATCC6538, Staphylococcus epidermidis (S. epidermidis) ATCC12228, and Bacillus cereus (B. cereus) ATCC11778 were obtained from the American Type Culture Collection (ATCC).

4.9.2 Inoculum preparation

All tested species were grown on Mueller Hinton Agar (MHA) and incubated at 37 °C for 18–24 h. The bacterial colonies suspension in sterile normal saline was diluted until they reached a 1 × 106 CFC/mL concentration, which was then compared to the McFarland scale. This step provided a standard bacterial suspension (1 × 106 CFU/mL), which was used for the following assays.

4.9.3 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The broth microdilution technique was used for the MIC and MBC determination, which was expressed according to Clinical and Laboratory Standards Institute (CLSI) standard M07 guideline [31]. The compounds were prepared as a stock solution by dissolving in dimethyl sulfoxide (DMSO) with the concentration ranging from 0.195 to 200 µg/mL. DMSO and penicillin G or gentamicin were used as normal and standard drug control. The minimum inhibitory concentration (MIC) refers to the lowest antibacterial compound concentration at which no visual growth was observed, while the complete death of bacteria compared to the initial bacterial inoculum refers to the MBC (minimum bactericidal concentration). Three replicates were performed for each experiment.

4.10 Aromatase (CYP19) inhibition assay

The aromatase assay used a method previously designed by Stresser and co-workers [32]. Letrozole, a positive control, exhibited an IC50 value of 1.4 ± 0.3 nM for CYP19 inhibition.