A new alkaloid and a new benzaldehyde derivative from the twig of Zanthoxylum rhetsa (Roxb.) DC.

Abstract A new alkaloid, 2-acetyl-4-methoxyfuro[2,3-b]quinoline (1), and a new benzaldehyde derivative, (2′S)-4-(2′-hydroxy-3′-methyl-3′-butenoxy)benzaldehyde (2), were isolated from the twig of Zanthoxylum rhetsa (Roxb.) DC. along with twenty-six known compounds (3−28). Their structures were determined by spectroscopic analysis (1D and 2D NMR spectroscopy and HRMS analysis) and comparison with data reported in the literature. Thirteen of the known compounds were evaluated for their cytotoxic activities against human cancer cell lines that included MDA-MB-231, SW1353, A549, and HCT116. (±)-8-Acetonyldihydronitidine (15) showed moderate cytotoxicity toward the SW1353 cancer cell line with an IC50 value of 18.90±0.39 µg/mL, and exhibited weak cytotoxic activity against MDA-MB-231, A549 and HCT116 cell lines with IC50 values of 49.86−71.32 µg/mL.


Introduction
The Zanthoxylum genus comprises more than 250 species worldwide, five of which have been identified in the northern part of Thailand (Suksathan et al. 2009).
Compound 1 was isolated as a brown powder.The positive HRESIMS analysis of 1 showed an [M þ Na] þ ion at m/z 264.0632, suggesting the molecular formula of 1 was C 14 H 11 NO 3 .The IR spectrum showed absorption bands for keto carbonyl at 1681 cm À1 , aromatic groups at 1581 cm À1 and ether bond at 1213 cm À1 .Analysis of 1D NMR and HMQC spectra of 1 revealed the presence of one methoxy, one methyl, five aromatic methines, and seven quaternary carbons (one keto carbonyl carbon and six aromatic quaternary carbons).According to the ten degrees of unsaturation, this structure contained three additional rings except for one ketone group (Supplementary material, Table S1).Except for the presence of an acetyl group in 1, the aforementioned data is highly similar to the corresponding data of dictamine ( 7), a furoquinoline alkaloid, which was first isolated from the leaves of Skimmia repens (Tanaka et al. 1985).The HMBC correlations from the aromatic proton at d H 7.80 (H-3) to C-1a (d C 162.7), C-2 (d C 150.7), C-3a (d C 103.8) and the keto carbonyl carbon (C-9, d C 190.0), and from the methyl proton at d H 2.69 to C-2 (d C 150.7) and C-9 (d C 190.0) suggested that the acetyl group was located at C-2 of the furoquinoline moiety (Supplementary material, Figure S2).Therefore, compound 1 was identified as 2-acetyl-4-methoxyfuro[2,3-b]quinoline.

Plant material
The twigs of Z. rhetsa (Roxb.)DC. were collected in April 2020 from Amphoe Klongtom, Krabi Province, Thailand.Identification was made by Dr. Jareansak SAE WEI, Division of Biological Science, Faculty of Science, Prince of Songkla University.The specimen (Titiya 01) has been deposited at the Herbarium of Division of Biological Science (Biology), Faculty of Science, Prince of Songkla University, Thailand.

Extraction and isolation
Air-dried twigs of Z. rhetsa (7 kg) were extracted with acetone for three days at room temperature.The solvent was evaporated under reduced pressure to give a crude acetone extract (80.87 g).The crude extract was separated by quick column chromatography over silica gel 60H, and eluted with a gradient of hexane, hexane-acetone, acetone and finally pure methanol to afford 20 fractions [A2T].Fraction N (0.8566 g) was purified by CC over silica gel and eluted with 60% CH 2 Cl 2 /hexane to yield 28 subfractions [N1ÀN28].Subfraction N18 (0.0238 g) was further purified by CC over silica gel and eluted with 20% EtOAc/hexane to give 2 (0.0039 g).Subfraction N21 (0.0219 g) was separated by CC over silica gel and eluted with 20% EtOAc/hexane to provide 16 subfractions [N21AÀN21P].Subfraction N21N (0.0010 g) was further purified by preparative TLC and eluted with 100% CH 2 Cl 2 to provide 1 (0.0009 g).The known compounds 3228 were isolated from fractions J, K, L, M and O by CC and preparative TLC techniques (for the details see Supplemental material).

Cytotoxic activity
Cell proliferation or cell viability of the cell lines was measured by the MTT colorimetric assay.Breast cancer cell (MDA-MB-231), chondrosarcoma cell (SW1353), lung cancer cell (A549) and colon cancer cell (HCT116) were obtained from the American Type Culture Collection.This study was carried out in accordance with a previously reported procedure (Daus et al. 2022).