Cytotoxic Alkaloids from Hippeastrum solandriflorum Lindl .

One new alkaloid, 2a-10ba-dihydroxy-9-O-demethylhomolycorine, in addition to seven others known alkaloids, and 5-(hydroxymethyl)furfural, piscidic acid and eucomic acid, were isolated from the bulbs of Hippeastrum solandriflorum. The structures of all compounds were determined using nuclear magnetic resonance (NMR) spectroscopic techniques: H NMR and C NMR, heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), nuclear Overhauser effect spectroscopy (NOESY), and also the high-resolution electrospray ionization mass spectrometry (EI-HRMS). The cytotoxic activity of all alkaloids was evaluated against three human cancer cell lines (HCT-116, HL-60, OVCAR8 and SF-295) showing IC50 values ranging from 0.01 to 35.7 μM.


Introduction
[8] Hippeastrum is a known ornamental genus, consisting of approximately 70 species, predominantly distributed in Latin America, especially in Brazil, with ca. 30 cataloged species. 9revious investigations carried out with Hippeastrum species have shown that this genus is a prolific source of alkaloids, particularly of the lycorine (pyrrolo[de]phenanthridine) and tazettine (2-benzopyrano[3,4-c] indole) types. 10n our continuing efforts searching for bioactive and/or novel secondary metabolites from plants of the Northeast Brazilian flora, H. solandriflorum was investigated in order to find out new anticancer compounds.In this paper, we report the isolation and characterization of a new homolycorine type alkaloid (1) (Figure 1) from the bulbs of H. solandriflorum, a species widely found in Ceará State.Additionally, seven known alkaloids (2-8) as well as their antiproliferative properties and three phenolic compounds (9-11), are also reported.

Results and Discussion
The chemical investigation of the ethanol extract from bulbs of H. solandriflorum allowed the isolation of eight Vol.26, No. 10, 2015   alkaloids, including a new one.In addition, three known phenolic compounds were also isolated (Figure 1).The chemical characterization was performed by analysis of their nuclear magnetic resonance (NMR) (in one and two dimensions), Fourier transform infrared (FTIR) spectroscopies and high-resolution electrospray ionisation mass spectrometry (EI-HRMS) spectral data.
Compound 1, isolated as an optically active white powder, was determined to have the molecular formula as C 17 H 19 NO 6 (9 degrees of unsaturation) by analysis of its EI-HRMS (m/z: 334.1272 for [M + H] + , calcd.: 334.1285) and the 13 C NMR spectra.The FTIR spectrum showed absorption bands for hydroxyl group at 3461 cm -1 , for conjugated carboxyl ester at 1719 cm -1 and double bonds at 1673-1447 cm -1 , as well as absorption bands for carbonoxygen and carbon-nitrogen at 1247-1034 cm -1 .
The 1 H NMR spectrum showed two singlets at d H 7.30 (H-10) and 7.57 (H-7) for aromatic protons para-positioned, a broad singlet at d H 5.96 (H-3) indicating a trissubstituted double bond, two singlets at d H 4.61 (H-1) and 4.07 (H-2) of oxymethine protons, as well as, a singlet at d H 4.36 (H-4a) of an azomethine proton.In the heteronuclear single quantum coherence (HSQC) spectrum the latest three proton signals showed correlations with the carbons at d C 83.4 (C-1), 72.3 (C-4a) and 68.1 (C-2), respectively.In addition, signals at d H 3.94 (s), for a methoxyl (MeO-8), and at d H 2.62 (s) for a N-methyl (Me-N) were also observed in the 1 H NMR spectrum.These data were consistent with a structure belonging to homolycorine type alkaloids of the Amaryllidaceae. 11,12Further examination of the 13 C NMR and distortionless enhancement by polarization transfer (DEPT) 135 spectral data of 1 (Table 1) showed signals consistent with those of the homolycorine skeleton alkaloids, 12,13 corroborating with the 1 H NMR spectrum.The comparative analysis of the 13 C NMR chemical shifts revealed that 1 shared high structural similarity to 2a-hydroxy-9-Odemethylhomolycorine. 13 Nevertheless, the signal of C-10b (d C 67.3) of 1 showed to be strongly unshielded when compared to the corresponding in 2a-hydroxy-9-Odemethylhomolycorine (d C 40.0). 13This significant difference (∆d 27.3 ppm), was easily justified by the hydroxylation of that carbon.This proposition was confirmed by the heteronuclear multiple bond correlation (HMBC) spectrum through the long-range correlation between H-10 (d H 7.30) with C-10b (d C 67.3).The determination of the relative stereochemistry of compound 1 was accomplished through the nuclear Overhauser effect (NOE) spectra (Figure 2).In CD 3 OD (Table 1) the dipolar coupling between H-1 (d H 4.61) and H-4a (d H 4.36), in addition to the NOE spectra of H-2 (d H 4.07) and the CH 3 -N (d H 2.62) permitted to infer that the relative stereochemistry of H-4a is indeed opposite to H-4a of that of 2a-hydroxy-9-O-demethylhomolycorine. 12In order to confirm the stereochemistry of 1, a new experiment was done with the protonated alkaloid (1) in DMSO-d 6 .
The overall cytotoxic effect of all isolated alkaloids (1-8) was assessed by the [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide] (MMT) assay against the HCT-116 (colon adenocarcinoma), HL-60 (leukemia), OVCAR-8 (ovarian carcinoma) and SF-295 (glioblastoma) cell lines.The data on Table 2 show the high cytotoxicity of narciclasine (8) against the four cell lines tested, with IC 50 values ranging from 0.01 to 0.09 µM.This result is in accordance with other reports, which found a mean IC 50 value of 0.05 µM for 8 against several other cancer cell lines. 23esides 8, only compound 2 can be considered as highly cytotoxic, with IC 50 values on the 1µM range.In general, HCT-116 was the most sensitive cell line, to which five of the eight tested compounds presented IC 50 values below 50 µM.
Data are presented as IC 50 values in µM and as the 95% confidence interval obtained by nonlinear regression for all of the cell lines from two independent experiments, performed in duplicate, after 72 h incubation.

Conclusions
A total of eleven compounds were isolated from H. solandriflorum, among which eight were alkaloids.These finds are in agreement with the chemistry produced by plants of the Amaryllidacea family, a prolific source of alkaloids.

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.FTIR spectra were obtained on a

Extraction and isolation
Fresh bulbs (8.8 kg) of H. solandriflorum were extracted with EtOH (3 × 5.0 L) at room temperature for 24 h, and the resulting solution was concentrated under reduced pressure to give the crude extract (283.0 g).

Figure 1 .
Figure 1.Structure of the compound 1 isolated from H. solandriflorum.

Table 2 .
Cytotoxicity of compounds 1-8 on select tumor cell lines evaluated by the MTT assay after 72 h of exposure Merck), and the spots were visualized by the Dragendorff reagent or by heating (at ca. 100 °C) the plates sprayed with a vanillin/perchloric acid/EtOH solution.Bulbs of H. solandriflorum were collected in Russas County, Ceará State, Brazil, in February 2012, and identified by Dr. Luiz Wilson Lima-Verde of the Departamento de Biologia, Universidade Federal do Ceará.A voucher specimen (# 37956) has been deposited at the Herbário Prisco Bezerra (EAC) of the Universidade Federal do Ceará.
a confidence interval of 95%.