Lepidotrichilins A and B, New Protolimonoids with Cytotoxic Activity from Trichilia Lepidota (Meliaceae)

Two novel protolimonoids, named lepidotrichilins A (1) and B (2), four known protolimonoids, 21,23-epoxy-7α-21α-dihydroxyapotirucalla-14,24-dien-3-one (3), 21,23-epoxy-7α-21β-dihydroxyapotiru-calla-14,24-dien-3-one (4), dysorone D (5), deoxy-flindissone (6), and the two steroids β-sitosterol (7) and stigmasterol (8) were identified in leaves of Trichilia lepidota subsp. schumanniana (Harms) T.D. Pennington. From wood the coumarin scopoletin (9) was isolated. The structures were established by NMR (1D 1H and 13C-NMR and 2D 1H-1H COSY, HMQC and HMBC), mass spectroscopy and infrared (IR) spectral data. The hexane and methanol extracts of the leaves, the protolimonoids lepidotrichilins A (1) and B (2) (IC50 42.7 µg mL−1) and the protolimonoid deoxy-flindissone (6; IC50 9.3 µgmL−1) exhibited significant cytotoxic activity against the MOLT-4 and U937 leukemic cell lines.


Introduction
Trichilia lepidota subsp.schumanniana (Harms) T.D. Pennington (known popularly as "Cedrinho" in Santa Catarina and Minas Gerais States, Brazil) is a tree of 3-6 meters in height.This wood of this species is used to make furniture [1].The Trichilia genus consists of about 230 species distributed throughout tropical America and other countries ranging from Africa and Asia [2,3].Previous investigations on the chemical constituents of the dichloromethane extract from the leaves of this species revealed the presence of hydrocarbons mixtures (C 29 H 60 , C 31 H 64 and C 33 H 68 ), sesquiterpenes, steroids, amino acids and other compounds derived from the terpene metabolic pathway [4].
Species of the genus Trichilia (Meliaceae) are known to contain varied limonoid structures (tetranortriterpenoids) and other terpenic metabolites which are responsible for various biological properties.Limonoids of various Trichilia with a wide spectrum of biological effects such as potential antiviral, analgesic, insecticidal, and insect growth inhibition activity have been documented [5,6].Previous investigations involving limonoids isolated of another Meliaceae species, namely Melia azedarach, have demonstrated antiproliferative activity against an adenocarcinoma epithelial cell line A549 [7].Additionally, it has been proved that limonoids from Citrus fruits demonstrated in vitro the capacity to induce apoptosis and to inhibit the proliferation of neuroblastoma cells [8].
The structures of the known and new compounds were established on the basis of spectral data, mainly 1 H-and 13 C-NMR (1D and 2D), HRESIMS spectra and by comparison with literature data.This paper also describes the viability of human leukemic lineages cells U937 and MOLT-4 treated with the hexane and methanol extracts, the mixture of the protolimonoids lepidotrichilins A (1) and B (2) and the protolimonoid desoxyflindissone (6).
The mixture of protolimonoids 1 and 2 was obtained as yellow oil.The IR spectrum (KBr disk) for the compounds showed bands at  max 3,438 cm −1 , attributed to a hydroxyl group, 2,925 and 2,855, attributed to methyne and methylene groups, and 1,736 and 1,649 cm −1 , attributed to two carbonyl groups at C-21(ester function) and C-3 (,-conjugated).The HRESIMS spectrum (positive mode) exhibited peaks at m/z 467.3200 ( (1) and nine (2) unsaturation degrees.Details of the molecular structures of compounds 1 and 2 were obtained by analysis of its 1 H-and 13 C-NMR spectra (Table 1) and from the observed 1 H-1 H-COSY, HMQC and HMBC correlations.Table 1. 13 C-(100 MHz) and 1 H-(400 MHz) NMR data of the protolimonoids 1 and 2 in CDCl 3 ,  in ppm and multiplicities and J in Hz (in parenthesis), including results obtained by heteronuclear 2D shift-correlated HMQC ( 1 J HC ) and HMBC ( n J HC n = 2 and 3) *.Comparative analysis of the 13 C-NMR spectra ({ 1 H}-and APT) of the protolimonoids 1 and 2 was used to recognize the signals corresponding to eight non-hydrogenated [four sp 3 and four sp 2 , including two carbonyl groups at  C 204.0 (,-unsaturated C-3 of 1) and 218.0 (C-3 of 2)], ten to 1 (four sp 2   and six sp 3 including two linked to oxygen atom) and eight to 2 (two sp 2 and six sp 3 including two linked to oxygen atom) methine, five to 1 (all sp 3 ) and seven to 2 (all sp 3 ) methylene and seven methyl (1 and 2) carbon atoms, corresponding to 30 carbon signals for each compound (Table 1):  1.
The protolimonoid 6 was obtained as purple crystals.The molecular weight was deduced from a molecular ion peak at m/z 438.3498 allowing the assignment of the molecular formula C 30 H 46 O 2 .The IR spectrum of compound 6 showed absorptions at 1,732 cm −1 , attributed to a carbonyl group (C-3), 800, 959, 1053 and 1,377 cm −1 , attributed to a stretch of the C-O bonds in the ether function.It revealed the presence of one carbonyl functionality at δ C 217.1 (C-3), seven methyl groups, nine methylene groups (CH 2 ), seven methine groups (CH) including a carbon linked to an oxygen atom at (CH-23) δ C 74.7/δ H 4.61 (dt, J = 8.5 and 6.3 Hz) and seven nonhydrogenated carbons (C): four sp 3 and three sp 2 .This compound have two double bonds between δ C 118.3 (CH-7) and δ C 145.7 (C-8); δ C 127.0 (CH-24) and δ C 135.3 (C-25).The protolimonoid 6 was identified as deoxyflindissone previously isolated from Cornus walteri [12] and synthesized by reduction of flindissol with lithium aluminum hydride following the oxidation of the deoxyflindissol [13].Compounds 7, 8 and 9 were were identified as β-sitosterol (7), stigmasterol (8) [14] and scopoletin (9) by comparison of their 1 Hand 13 C-NMR and GC/MS spectra with data from the literature [15].
Cellular viability after treatment with purified compounds and plant extracts was evaluated by MTT metabolization.Reduction in cell viability in both cell lineage submitted to treatment to all extract and isolated compounds were shown on Figure S16 (see the Supplementary Material) and summarized in Table 2.In all cell viability experiments DMSO was used as a normal cell culture control and meaningless cell death was observed, therefore those controls treatments were depicted as 100% of cell viability compared to the cell culture treated with compounds and extracts.Concerning cell death control 15 µgmL −1 of cisplatin was used to treat the cells and 90% of cell death was observed.Based on both leukemia cell viability analyses, our data suggest that protolimonoid 6 would be more active than the protolimonoid compounds 1 and 2.
Our results are in agreement with the activity limonoids isolated from Citrus [8], which were able to reduce viability of neuroblastoma cell line SH-SY5Y.In contrast to our data, the limonoids isolated from Toona ciliata var.pubescens [16] were inactive or had weak activity on cancer and non-cancer cell line, but were able to inhibit CDC25B dual specificity phosphatase.Altogether, these data suggest a range of biological activity associated with a variety of limonoids molecules structures.The lower IC 50 value 9.3 µgmL −1 was observed for protolimonoid 6 for both cells line U937 and MOLT-4.Among the T. lepidota extracts the hexane ones were more efficient than the methanol ones.Comparing the values of IC 50 for LDH release and cellular cytotoxicity we can observe that the sample concentration to induce LDH release was much higher than the sample concentration to induce cytotoxicity.Those data suggested that cell death occurred by apoptosis.Other studies on limonoids showed that these compounds were able to reduce cell viability and induce apoptosis in some cancer cells.Tian et al. showed that four isolated limonoids from Citrus reticulata induced cell death by apoptosis in breast cancer cell line (MCF-7) [17].Additionally neuroblastoma cell culture was shown to die by apoptosis when incubated with limonoids isolated from Citrus [8].
The wood (1.5 kg) was dried, powdered and extracted with methanol (3 L) for three time and ambient temperature.The extract of T. lepidota wood (29.47 g) was first submitted to a liquid-liquid partition with H 2 O-CH 2 Cl 2 (1:3).Part of the dichloromethane fraction (150.0 mg) was fractionated by column chromatography using a gradient of CH 2 Cl 2 -MeOH.Initially 100% dichloromethane was used, and then the composition was changed to 5% methanol in 1% increments, affording coumarin 9 (17.4 mg).

Figure 1 .
Figure 1.Chemical structures of the compounds isolated from T. lepidota.
a *Number of hydrogens bound to carbon atoms deduced by comparative analysis of { 1 H}-and APT-13 C NMR spectra.Chemical shifts and coupling constants (J) obtained of 1D 1 H-NMR spectrum.Superimposed 1 H signals are described without multiplicity and chemical shifts deduced by HMQC, HMBC and 1 H-1 H-COSY spectra.

Table 2 .
Table 2 lists the IC 50 values for cellular viability and LDH release.Analysis of the IC 50 values shows that the isolated protolimonoid 6 was more efficient at inducing cellular cytotoxicity than the new protolimonoids 1 and 2. IC 50 determination for cellular viability and LDH release. U937-