Five new quinoline alkaloids from Sauropus hirsutus Beille and their cytotoxicity

Abstract Chemical investigation of the whole plant of Sauropus hirsutus Beille led to the isolation of eight quinolines and two known flavonoids. Furthermore, five quinolines were new, two were reported in plant for the first time and one was known. Cytotoxicity evaluation against cholangiocarcinoma, KKU-M156, showed that the most active compound was 4-hydroxy-6-methoxy-7,8-methylenedioxyquinaldine (IC50 20.54 ± 6.82 µM) which was a little more active than the cisplatin standard (IC50 24.39 ± 1.14 µM). Graphical Abstract


Introduction
The Sauropus genus comprise about 40 species and about 3-5 species have been studied for their chemical components such as S. androgynus, S. bacciformis, S. rostratus and S. brevipes (Liang et al. 2016;Chen et al. 2017;Mahardiani et al. 2020;Huong et al. 2021). S. androgynus is one of the plants in this genus that has been investigated (Bose et al. 2018). It is a vegetable grown in Thailand, Malaysia, Indonesia, Vietnam and Southwest China. This plant contains a large amount of various nutrients, and it has been reported that the leaves contain alkaloid papaverine which may cause dizziness, drowsiness and constipation (Lai et al. 2000;Soka et al. 2010).
S. hirsutus Beille belongs to the family Euphorbiaceae. It is a prostrate woody herb to small shrub which can grow up to 30 cm-2 m high (Welzen 2007). This plant is distributed in Southeast Asia such as Thailand, Laos and Cambodia and is called Phak wan nok in Thai. This plant usually grows in deciduous, dry dipterocarpus and bamboo forests. It is used as an ointment to heal wounds and as a remedy for edema, and by oral administration for syphilis and intestinal hemorrhage caused by overwork (Ayano et al. 2018). It is the first time to study phytochemical contents and cytotoxicity of isolated compounds from this plant. In our chemical screening on S. hirsutus, alkaloids were detected from this plant. Thus, the chemical investigation of S. hirsutus was determined and found eight quinoline alkaloids and two flavonoids. Cytotoxic evaluations against cholangiocarcinoma, KKU-M156, and human liver cancer cell line, HepG2, of all isolated compounds were determined by using the MTS-based assay.

Chemistry
Chemical investigation of the whole plant of S. hirsutus using chromatographic methods led to the isolation of five new quinolines (1-5), two first time reported from a natural source, quinolines (6-7) (Nandhakumar et al. 2007;Chandrashekarappa et al. 2013), a known quinoline (8) (Graves et al. 1938) and two known flavonoids, aromadend (9) (Pitchuanchom et al. 2021) and 3,5,7,3 0 ,5 0 -pentahydroxyflavan (10) (Jiang et al. 2014;Zeng et al. 2021) (Figure 1). Compound 1 was obtained as a white solid. The molecular formula C 12 H 11 NO 3 was deduced from HRESIMS (m/z found as 218.0871 [M þ H] þ , calcd. 218.0817). The 13 C NMR showed two methyl, three aromatic methine, a methylene and six quaternary aromatic carbons. The oxygenated carbon signals at d C 162.9, d C 147.6 and d C 140.3 were assigned as C-4, C-7 and C-8, respectively. The nitrogenated carbons C-2 and C-8a showed signals at d C 161.4 and d C 135.3, respectively. Three methine carbons were observed at d C 99.3, d C 115.8 and d C 108.9 of C-3, C-5 and C-6. The 1 H NMR showed two doublet signals at d H 7.70 (1H, J ¼ 8. 4 Hz, H-5) and 7.09 (1H, J ¼ 8. 4 Hz, H-6), which were located at carbons at d C 115.8 and d C 108.9, respectively, in the HMQC experiment. In this spectrum, the singlet signal at d H 6.49 (H-3) correlated to carbon at d C 99.3. The methoxy group at d H /d C 4.00/55.7 displayed long-range correlation to C-4 (d C 162.9). The singlet signal at d H 2.69 of the methyl group correlated with carbons at C-2 (d C 161.4) and C-3 (d C 99.3) in the HMBC spectrum ( Figure S1). Long-range correlations between H-3 and C-2, C-4, C-4a (d C 116.5) and CH 3 (d C 26.0) were observed in this spectrum. Cross-peaks were observed between H-5 and C-4, C-7 and C-8a, while those between H-6 and C-4a, C-7 and C-8 were evident in the HMBC data. A methylenedioxy group at d H /d C 6.20/102.4, showed long-range correlations with C-7 and C-8. From all the data, compound 1 was concluded as 4-methoxy-7,8methylenedioxyquinaldine.
Compound 2 was obtained as a white solid and assigned the molecular formula C 13 H 13 NO 4 from its HRESIMS (m/z found 248.0989 [M þ H] þ , calcd. 248.0923). The 1 H and 13 C NMR spectra of compound 2 were similar to compound 1, except for the addition of a methoxy group and lack of an aromatic proton. The 13 C NMR spectrum of compound 2 showed the presence of an additional oxygenated carbon at the C-6 position (d C 144.0). In the 1 H NMR spectrum, compound 2 showed two singlet signals at d H 6.50 (H-3) and d H 7.11 (H-5). Comparing the chemical shift at C-5 position of compounds 1 and 2, those of compound 2 (d H /d C 7.11/95.8) showed higher field shift than compound 1 (d H /d C 7.70/115.8). These evident indicated that H-5 in compound 2 was located on the ortho position to methoxy group. The HMBC spectrum displayed the correlation between H-5 and C-4 (d C 161.8), C-6 (d C 144.0) and C-7 (d C 137.8) which confirmed a methine aromatic proton at the C-5 position. Thus compound 2 was deduced as 4,6-dimethoxy-7,8-methylenedioxyquinaldine.
Compound 3 was given the molecular formula C 13 H 15 NO 3 on the basis of its HRESIMS data (m/z found 234.1125 [M þ H] þ , calcd. 234.1130). Its 1 H and 13 C NMR spectra were similar to compound 1, except for the absence of a methylene group and the addition of two methoxy groups. These NMR data revealed the presence of three methoxy groups at d H /d C 4.02/55.9 (OCH 3 -4), d H /d C 4.00/56.9 (OCH 3 -7) and d H /d C 4.09/61.9 (OCH 3 -8). The HMBC correlations between the protons of the methoxy group at d H 4.02 (OCH 3 -4) and the oxygenated aromatic carbon at d C 163. 2 (C-4), between protons at d H 4.00 (OCH 3 -7) and the carbon at d C 152.7 (C-7) and between protons at d H 4.09 (OCH 3 -8) and the carbon at d C 142.3 (C-8) were observed. Cross-peaks in the HMBC experiment were observed between H-5 (d H 7.85, d, J ¼ 9.2 Hz) and C-4 (d C 163.2), C-7 (d C 152.7) and C-8a (d C 143.3) and between H-6 (d H 7.21, d, J ¼ 9.2 Hz) and C-4a (d C 115.7), C-8 (d C 142.3) and C-7 (d C 152.7). Thus, the structure of compound 3 was assigned as 4,7,8-trimethoxyquinaldine.  -5), which was located at the carbon d C 95.6, correlated with C-4 (d C 162.0), C-7 (d C 144.7) and C-8a (d C 139.2) in the HMBC experiment. From all data, the structure of compound 4 was identified as 4,6,7,8-tetramethoxyquinaldine.
Compound 5 possessed the molecular formula of C 12 H 11 NO 4 resolved by the HRESIMS experiment (m/z found 234.0746 [M þ H] þ , calcd. 234.0766). The 13 C and 1 H NMR spectra of compound 5 were similar to compound 2, except for the presence of a hydroxyl group at the C-4 position in compound 5 while compound 2 contained a methoxy group. Due to the containing of hydroxyl group at C-4 position, compound 5 was more polar than the others. The nitrogenated carbons C-2 and C-8a resonated at d C 151.8 and 122.7, respectively. In the HMBC experiment, correlations between H-5 (d H 7.31, s) and C-4 (d C 179.1), C-6 (d C 144.1), C-7 (d C 140.9) and C-8a (d C 122.7) were observed. The structure of compound 5 was concluded as 4-hydroxy-6-methoxy-7,8methylenedioxyquinaldine.

Biological activity
The cytotoxic activity of all isolated compounds was evaluated against cholangiocarcinoma (KKU-M156) and human liver cancer cell line (HepG2) using MTS assay (Ganguly et al. 2006). The results indicated that compound 5 was the most active against KKU-M156 cells (IC 50 20.54 ± 6.82 mM), and a little more active than the cisplatin standard (IC 50 24.39 ± 1.14 mM) (Table S3). On the other hand, compound 5 was inactive against HepG2 (IC 50 >100 mM). It seems that this compound selected KKU-M156 cells. All isolated compounds exhibited inactive against HepG2 cells (IC 50 ! 100 mM). Comparing between compounds 2 and 5, it seems the 4-hydroxyl group may play an important role for cytotoxicity against the KKU-M156 cell line. Compounds 5 and 8 contained hydroxyl group at C-4 position but compound 8 showed no activity against KKU-M156 cells. It means methylenedioxy at C-7/C-8 and methoxy group at C-6 may necessary for the activity. Comparing cytotoxicity against KKU-M156 cells between compounds 1 and 5, it was concluded that the hydroxyl at C-4 position and methoxy group at C-6 position were essential for cytotoxicity. Compound 2 (IC 50 ¼ 91.12 ± 36.04 mM) which displayed a bit more active than compound 1 (IC 50 > 100 mM) indicated that the methoxy group at C-6 position enhanced the activity.

General experimental procedures
Melting points were determined on a SANYO Gallenkamp (Leicester, UK) melting point apparatus and are uncorrected. UV spectra were measured on an Agilent 8453 UV-VIS spectrophotometer (Waldbronn, Germany). IR spectra were recorded by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) (Germany) Bruker TENSOR 27. The NMR spectra were measured in CDCl 3 or CD 3 OD on a Varian Mercury Plus spectrometer (400 MHz) (California, USA) and Bruker AVANCE NEO (400 MHz) (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR) spectrometer. HRESIMS data was measured on a Micromass Q-TOF 2 hybrid quadrupole time of flight (Q-TOF) mass spectrometer with a Z-spray ES source (Micromass, Manchester, UK). Column chromatography was done with silica gel 60 (0.063-0.200 mm, 0.040-0.063 mm and less than 0.063 mm), Sephadex LH-20 and RP-18 column chromatography. Thin layer chromatography (TLC) was carried out on MERCK silica gel 60 F 254 TLC aluminium sheets. Preparative thin layer chromatography (PLC) was carried out on glass supported silica gel plates using silica gel 60 PF 254 for preparative layer chromatography. All commercial grade solvents were routinely distilled prior to use.

Extraction and isolation
The air-dried Sauropus hirsutus (1 kg) was powdered (mesh size ffi 1-2 mm) and extracted by maceration with hexane (1.5 L) three times at room temperature, for up to 3 days each, and then evaporated in vacuo. Then sequential extractions in the same manner as the previous method with EtOAc (3 Â 1.5 L, 3 days each) and MeOH (3 Â 1.5 L, 3 days each) were done. After evaporation in vacuo, three dry crude extracts, hexane (26 g, 2.6%), EtOAc (40 g, 4.0%) and MeOH (250 g, 25%) were obtained. The crude hexane extract (26 g) was subjected to column chromatography (CC), subsequently eluted with a gradient system of hexane, hexane-EtOAc and EtOAc-MeOH to obtain three fractions (H1-H3). Fraction H3 was subjected to silica gel CC and eluted with an isocratic system of 35% EtOAc:hexane to give 1 (12.8 mg) and 2 (5.5 mg).

Cytotoxic activity assay
3.5.1. Cytotoxicity assays Cell toxicity effects were studied by using the MTS-based assay kit (CellTiter 96V R AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) and the assay was performed according to the manufacturer's instructions. Different concentrations of tested compounds and cisplatin were prepared with serum-free media and the final concentration of DMSO content in each sample was kept below 0.2% (v/v). Briefly, KKU-M156 and HepG2 cells were placed onto each well of a 96-well microplate and preincubated overnight before the cells were further treated with the tested compounds and cisplatin. After the 24 h-incubation period, MTS reagent was then added to each well and the microplate was incubated for 4 h before the absorbance was measured at 490 nm through a microplate reader (Sunrise TM microplate absorbance reader). Cell cytotoxicity effects of all compounds were calculated as percentage absorbance of controls. The IC 50 value was calculated by a non-linear curve-fitting program from triplicate assay of three separate experiments (Ganguly et al. 2006).

Conclusion
Chemical investigation of the whole plant of S. hirsutus led to the isolation of ten compounds. They are five new quinoline derivatives (compounds 1-5), three known quinolines (two of which, compounds 6 and 7, were found in plants for the first time) together with two flavonoid derivatives (compounds 9 and 10). The cytotoxicity testing against two cancer cell lines, KKU-M156 and HepG2 cells was examined. The results exhibited that compound 5 was the most active against KKU-M156 cells with an IC 50 value of 20.54 ± 6.82 mM while the cisplatin standard showed an IC 50 value of 24.39 ± 1.14 mM. The structure activity relationship indicated that the hydroxyl group at C-4 position, methoxy group at C-6 and methylenedioxy group at C-7/C-8 may play an important role for cytotoxicity. Khon Kaen University for T. Sribuhom is acknowledged (Grant no. 591471). This work was partially supported by the Thailand Research Fund and Khon Kaen University [RSA6280050].