New hydroperoxycycloartane—complete chemical shifts assignment of 13C and 1H—and cytotoxicity evaluation of cycloartanes isolated from Trichilia casaretti

Abstract New cycloartane, 22-hydroxy-25-hydroperoxycycloart-23E-en-3-one (1), along with six known analogues (2–7) and three steroids (8–10), were isolated from the leaves of Trichilia casaretti. Structures were elucidated mainly on the basis of the analysis of 1D and 2D NMR (1H and 13C) and HRESIMS spectroscopic data, involving comparison with data of the literature. The cytotoxic activities of 1–7 and 10 isolated compounds were also evaluated against human leukemia cell line Molt-4 (acute lymphoblastic) and exhibited good cytotoxic activity with IC50 values ranging from 10.62 to 21.14 μM. Graphical Abstract


Introduction
Trichilia genus belongs to the Meliaceae family (also known as Mahogany), consists of 98 species distributed in tropical and subtropical areas (Patr ıcio and Cervi 2005). Researches constantly shown the presence of secondary metabolites of the metabolic pathway of terpenoids, especially limonoids (Ji et al. 2015a;Liu et al. 2016;Wang et al. 2017). These compounds are considered as chemosystematic markers of the family (Vieira et al. 2014). In addition, these species present several biological activities, as in the case of T. americana showed the cytotoxic activities of isolated compounds against five human tumour cell lines (Ji et al. 2015b). In Brazil it has several species of the Trichilia genus. Particularly, Trichilia casaretti (distributed in the Atlantic Forest) has already identified cycloartanes and sesquiterpenes in their chemical composition with biological activity (Vieira et al. 2010;Vieira et al. 2018). In this study, we investigated the constituents from T. casaretti leaf where led to identification of one new cycloartane (1), also six know analogues (2-7) and three steroids (8-10), showed in Figure 1, as well as evaluating cytotoxic activity against leukemia cell line Molt-04.  m/z 495.3450), indicating fourteen hydrogen deficiency (C 30 H 62 O 4 -C 30 H 48 O 4 ¼ 14) corresponding to seven degree of unsaturation. The 13 C-APT NMR spectrum of 1 revealed 13 C signals corresponding to 30 carbon atoms: seven non-hydrogenated [including one sp 2 carbonylic at d C 216.7 (O ¼ C-3) and one sp 3 oxygenated at d C 81.9 (O-C-25)], seven methines [including one sp 3 oxygenated at d C 74.5 (O-CH-22) and two sp 2 olefinics at d C 129.3 (¼CH-23) and 136. (¼CH-24) ¼ HC ¼ CH], nine methylenes [including one cyclopropanic at d C 29.5 (CH 2 -19) above referenced)] and seven methyl groups, allowing to deduce the expanded partial molecular formula C 5 (C ¼ O)(C-O)(CH) 4 (HC-O)(CH ¼ CH)(CH 2 ) 9 (CH 3 ) 7 ¼ C 30 H 46 O 3 requesting two hydrogen atoms and one oxygen atom to confirm the molecular formula C 30 H 48 O 4 established by HRESIMS (Scheme S1). Thus, the seven degree of unsaturation were attributed to one carbonyl group (C-3), one double bond (23-CH ¼ HC-24), one cyclopropane ring and more four rings, compatible with cycloartane skeleton.

Results and discussion
The HSQC spectrum of 1 was possible to confirm 13 C signals from seven non-hydrogenated carbons by the predicted absence of cross-peaks [e. g. carbonyl d C 216.7 (C-3) and an oxygenated quaternary carbon d C 81.9 (C-25)] corresponding to heteronuclear correlations with signals of hydrogen atoms. The cross-peaks involving direct correlations via one bond ( 1 J CH ) of 13 C and 1 H signals were observed to methines [e. g.: double bond: d C /d H 136.0/5.82 (d, J ¼ 15.9 Hz, trans coupling, CH-24) and 129.3/5.73 (dd, J ¼ 15.9 and 6.9 Hz, CH-23)], and oxygenated carbon at d C /d H 74.5/4.26 (dd, J ¼ 6.9 and 3.9 Hz, CH-22)], methylenes and methyls (Table S1). Farther cross-peaks observed in the HSQC of 1 were summarized in Table S1.
The hydroperoxy group (HOOC-25) of 1 was deduced by comparison of the 13 C NMR spectra of 1 and 2 (HOC-25). The protection c effects observed in the 13 C chemical shifts signals of CH-24 (Dd C À 4.4 ppm), CH 3 -26 (Dd C À 5.5 ppm) and CH 3 -27 (Dd C À 5.6 ppm) and deprotection b effect in CH-23 (Dd C þ 4.2 ppm) and C-25 (Dd C þ 11.1 ppm) were used to recognized the presence of the hydroperoxy group no carbon atom C-25 of 1 ( Figure S2). In addition, the molecular formula was also used to confirm the presence of the hydroperoxy group (Scheme S1).  Figure S3.
Subsequently were determined the inhibitory effects of 1-7 and 10 to human leukemia cell line Molt-4 (acute lymphoblastic) and the IC 50 values of these compounds were evaluated based on MTT assay results as summarized in Table S2. As results, all tested compounds exhibited good cytotoxic activity comparable to the cisplatin positive control. Besides, compounds 1 and 3 were the ones that showed the best result with IC 50 values 11.28 and 10.62 mM, respectively, followed to compounds 2 (15.84 mM), mixture of 6 and 7 (12.76 mM), and 10 (14.03 mM). Additionally, compounds 4 (20.42 mM) and 5 (21.14 mM) were the ones that presented results similar to those of cisplatin.

General procedures
Infrared (IR) spectra were recorded with a Shimadzu FT-IR 8300 spectrophotometer. NMR spectra were acquired by using Bruker Ascend 500 (500 MHz for 1 H and 125 MHz for 13 C) in CDCl 3 . Chemical shifts (d) in ppm and coupling constants (J) in Hz. HRESI data were acquired on a micrOTOF-Q II Bruker Daltonics, with the use of the positive ion mode of analysis. Column chromatography (CC) was performed on silica 60 (0.063-0.200 mm, MERCK) and for the preparative thin layer chromatography (PTLC) was used silica gel 60 PF 254 (MERCK) on glass plates. TLC plates contained with silica gel 60 F 254 (MERCK) were used for analytical analysis; detection was under UV (254 and 365 nm) and chromogenic reagent (Vanillin/sulfuric acid). The solvents used were n-Hexane, methanol (MetOH), ethyl acetate (AcOEt), dichloromethane (CH 2 Cl 2 ) and nbutanol purchased from Synth (São Paulo, Brazil).

Plant material
The leaf of Trichilia casaretti C. DC., Meliaceae, were collected in Reserva Natural Vale, Linhares City, Espirito Santo state, Brazil. A voucher specimen (CVRD-449) was deposited in the herbarium of the Reserva Natural Vale.

Antineoplastic activity
The experimental procedures to antineoplastic activity were presented in the supporting information.

Conclusions
An investigation of the leaves of Trichilia casaretti resulted into isolation of a new cycloartane, 22-hydroxy-25-hydroperoxycycloart-23-en-3-one (1) together with nine known compounds (2-10). The structure of 1 was characterized using NMR and Mass spectroscopic data. The compounds have shown good cytotoxic activity against human leukemia cell line Molt-4 (acute lymphoblastic). Besides, the known compounds 2, 3, 6 and 7 are reported for the first time from the genus Trichilia.