Three new cytotoxic annonaceous acetogenins from the seeds of Annona squamosa

Abstract Three new annonaceous acetogenins, annotemoyin L (1), annotemoyin Y (2) and annotemoyin X, (3) were isolated from the seeds of Annona squamosa Linn. Their structures were ascertained by chemical methods and spectral data. The cytotoxic activities of compounds against three multidrug-resistant cancer cell lines were evaluated, and compound 3 exerted strong cytotoxicity against SMMC 7721/ADR (IC50 0.163 μM), A549/T (IC50 0.064 μM) and MCF-7/ADR (IC50 0.057 μM). Graphical Abstract


Introduction
The Annona (Annonaceae) genus is comprised of about 170 species, distributed in the tropics and subtropics (Leite et al. 2020).Annonaceous acetogenins (ACGs), phytochemicals with a broad range of biological activities, were found almost exclusively in plants of the Annonaceae family, and the isolation of ACGs has been more reported in species of the Annona genus (Neske et al. 2020).Annona squamosa Linn. is a semi-deciduous tree that bears edible fruits in this genus (Ma, Chen, et al. 2017).Its fruit is named Srikayas, due to its shaping like a Buddha's head.According to its sweet and not greasy, Srikayas are extensively used to prepare candies, ice creams and beverages.The seeds, rich in ACGs, are discarded in large quantities as waste from the process (Ma, Chen, et al. 2017).In our previous work (Ma, Wang, et al. 2017;Ma et al. 2019), eleven new ACGs were isolated from seeds of A. squamosa, and showed promising cytotoxicity against multidrugresistant (MDR) cancer cell lines.In this article, we reported the isolation, structural elucidation and cytotoxicity of three new mono-tetrahydrofuran (mono-THF) ACGs.

General experimental procedures
NMR spectroscopic data were acquired on a Bruker ACF-400 spectrometer in deuterated chloroform with TMS as the internal standard.HR-ESI-MS were measured on an AB Sciex Triple-TOF 5600 system.The melting point was determined on a Stuart melting point apparatus SMP10.The IR spectra were acquired on a Thermo Fisher Nicolet iS5 spectrometer.Shimadzu LC-20A system with a Shim-pack PRC-ODS column (20 Â 250 mm, 5 lm) was employed to perform semi-preparative HPLC separations.Silica gel (200-300 mesh) was purchased from Qingdao Marine Chemical Ltd.

Plant materials
The seeds of A. squamosa were collected in January 2015 at Lincang, Yunnan, China, and identified by Dr. Jian-Wei Chen (Pharmaceutical Institute, Nanjing University of Chinese Medicine).A voucher specimen (No. 20150124) was deposited in Pharmaceutical Institute of Nanjing University of Chinese Medicine.

In vitro cytotoxic activity
Compounds 1-3 were evaluated for their cytotoxic activity against three human multidrug-resistance(MDR) cancer lines (SMMC 7721/ADR, MCF-7/ADR, A549/T) by MTT method in 96-well plates.Adriamycin was used as a positive control.IC 50 values of isolated compounds were summarised in Table S2.

Conclusion
In this study, three new mono-THF ACGs were isolated from the seeds of A. squamosa.All isolated compounds showed potent cytotoxic activity against MDR cancer cell lines, and 3 showed significant cytotoxic activities against MDR (SMMC 7721/ADR, MCF-7/ ADR, A549/T) cancer cells with the values of IC 50 at 0.163, 0.057 and 0.064 lM, respectively.
Due to the only difference between compounds 1-3 was the spacer moiety (4, 11 and 13 carbon atoms) linking the THF ring and c lactone ring, the structure-activity relationship was discussed following Miyoshi's strategy (Motoyama et al. 2002).In comparing the cytotoxic values of compounds 1-3 against the SMMC 7721/ADR and MCF-7/ADR cell lines, it appears that the longer length of the spacer moiety is associated with a stronger potency (4-13 carbons), which correlates with Miyoshi's conclusion is consistent (Motoyama et al. 2002).However, this is not the case for A549/T.
peak at m/z 565.4823 in HR-ESI-MS.Its positive reaction to Raymond's reagent indicated the presence of a,b-unsaturated c lactone ring.The signals in 1 H NMR spectrum of 1 (Table