Chemical Composition of Aspidosperma ulei Markgr. and Antiplasmodial Activity of Selected Indole Alkaloids

A new indole alkaloid, 12-hydroxy-N-acetyl-21(N)-dehydroplumeran-18-oic acid (13), and 11 known indole alkaloids: 3,4,5,6-tetradehydro-β-yohimbine (3), 19(E)-hunteracine (4), β-yohimbine (5), yohimbine (6), 19,20-dehydro-17-α-yohimbine (7), uleine (10), 20-epi-dasycarpidone (11), olivacine (8), 20-epi-N-nor-dasycarpidone (14), N-demethyluleine (15) and 20(E)-nor-subincanadine E (12) and a boonein δ-lactone 9, ursolic acid (1) and 1D,1O-methyl-chiro-inositol (2) were isolated from the EtOH extracts of different parts of Aspidosperma ulei Markgr. (Apocynaceae). Identification and structural elucidation were based on IR, MS, 1H- and 13C-NMR spectral data and comparison to literature data. The antiplasmodial and antimalarial activity of 1, 5, 6, 8, 10 and 15 has been previously evaluated and 1 and 10 have important in vitro and in vivo antimalarial properties according to patent and/or scientific literature. With the aim of discovering new antiplasmodial indole alkaloids, 3, 4, 11, 12 and 13 were evaluated for in vitro inhibition against the multi-drug resistant K1 strain of the human malaria parasite Plasmodium falciparum. IC50 values of 14.0 (39.9), 4.5 (16.7) and 14.5 (54.3) μg/mL (μM) were determined for 3, 11 and 12, respectively. Inhibitory activity of 3, 4, 11, 12 and 13 was evaluated against NIH3T3 murine fibroblasts. None of these compounds exhibited toxicity to fibroblasts (IC50 > 50 μg/mL). Of the five compounds screened for in vitro antiplasmodial activity, only 11 was active.


Introduction
Malaria continues to be a disease that afflicts the whole World, especially the African continent. However, data from 99 countries reveals that based on the overall number of deaths malaria is in decline [1]. The main antimalarials available today are the quinolines that are structural mimics of the plant-derived natural product quinine and the semi-synthetic derivatives of another plant-derived natural product, artemisinin. Resistance of the malaria parasites to these drugs is an issue of concern and it is important to discover new compounds that may be developed into the next generation of antimalarial drugs [2].
The Aspidosperma spp. (Apocynaceae) comprise trees distributed in Central and South America. Aspidosperma spp. extracts exhibit antimalarial activity and remedies prepared from the bark are used in traditional medicine for the treatment of malaria [3]. Screening of bark extracts representing six Aspidosperma spp. for in vitro inhibition against chloroquine-resistant W2 and chloroquine-sensitive 3D7 strains of the human malaria parasite Plasmodium falciparum revealed good activity (IC 50 = 5.0-65.0 μg/mL). Thus, A. ulei (syn. A. parvifolium) trunk bark EtOH extracts were found to be active, as were the extracts of two other Aspidosperma spp. [4].
The aim of the present work was to perform a compositional study on the extracts of A. ulei and isolate indole alkaloids from this traditionally used antimalarial plant. Several isolated indole alkaloids were evaluated for in vitro antiplasmodial activity and cytotoxicity against fibroblasts as a means to discover new antiplasmodial compounds from this species.

Analysis of Spectral and Physical Data for Isolated Compounds
From the leaf EtOH extract (LEE) of A. ulei, ursolic acid (1), a white solid, m.p. 296.5-297.6 °C and [α] 20 D = +26.0° (c. 0.33, MeOH) was isolated for the first time from this species [18,19]. Stem bark EtOH extracts (SBEE) exhibited a precipitate, methyl-chiro-inositol (2), an amorphous solid, m.p. 150.3-152.2 °C that could be identified based on comparison of its spectral data with that of the literature [20]. Based on acquired spectral data and comparison with data in the literature [21] one of the substances was identified as (+)-3,4,5,6-tetradehydro-β-yohimbine (3, 25.  N-demethyluleine (15) data [22], together with data for β-yohimbine (5) [22]. Alkaloids 5 and 6 were evaluated for in vitro antiplasmodial activity against the chloroquine-resistant Fc M29-Cameroon strain of Plasmodium falciparum and found to present IC 50 values > 1 μg/mL [23]. Several alkaloids, including 6, were cited in a patent on new antimicrobial agents that included antimalarials [24]. The alkaloid 19,20-dehydro-17α-yohimbine (7, 4.0 mg) could be identified by comparison of its NMR data to literature data [25].  (10) and 20-epi-dasycarpidone (11) provided evidence for the difference of the normal series and epi series. In the piperidine ring of uleine the ethyl side chain is in the equatorial position and in 20-epi-uleine this side chain is in the axial position. In the spectra of 11, this difference is evidenced by a 1,3-diaxial γ-effect by the ethyl group on the axial H of C-14, resulting in steric compression of C-14, C-20 and to a lesser extent C-18 and C-19. These C-atoms are more shielded than in the normal series. Olivacine (8, 5,0 mg) was isolated from the root bark through precipitation of the root bark EtOH extract acidic fraction (RBEEAF) and exhibited 1 H and 13 C-NMR data consistent with those found in the literature [35].  20(E)-nor-subincanadine E (12, 36.0 mg) was isolated from the stem bark of A. ulei and its spectral data were similar to those found in the literature [36]. It has been reported as an intermediate in syntheses of Strychnos alkaloids [37,38].
The new indole alkaloid 12-hydroxy-N-acetyl-21(N)-dehydroplumeran-18-oic acid (13, 4.4 mg) was isolated as a resin from the root wood EtOH extract (RWEE) of A. ulei. The IR spectrum exhibited overlapped broad O-H and N-H stretching bands at 3,440 cm −1 and characteristic C=O bands of a conjugated acid and an amide, 1,683 and 1,631 cm −1 , respectively. In the 1 H and 13 C spectra, only three aromatic H signals and three aromatic CH signals were observed. Through long-distance couplings evidenced in the HMBC spectrum it was concluded that the OH group was at the C-12 (δ c 149.2) position thus confirming the monosubstitution of the aromatic ring. Analyses of the HMBC spectrum confirmed the presence of a quaternary N-atom and the C-atom of the iminium (C=N + ) group (signal at  Figure 2. A structural similarity search allowed for models to be obtained for comparison of data [39] together with 1 H and 13 C data from the literature [40].

General Procedures
Melting points were determined on a Digital Microdetermination apparatus (Mettler Toledo) equipped with a FP82HT heating plate and FP90 processing unit. Determinations were performed at a heating velocity of 2 °C/min and were not corrected. IR spectra were acquired on a Perkin-Elmer Spectrum 100 FT-IR spectrometer using a Universal Attenuated Total Reflectance Accessory (UATR) in the range of 400 to 4,000 cm −1 . HPLC analysis of calibration solutions and those of extracts and fractions of A. ulei was performed on a Waters modular chromatograph. This system was controlled by Empower software. The system consisted of a Waters-1525 binary pump and a photo diode array detector (PDA) model 2996. HPLC separations were performed on a Phenomenex RP-18 column (4.6 × 250 mm, 5 μm) and a Phenomenex RP-18 (10 × 250 mm, 10 μm). The samples were eluted with ACN, MeOH and a solution containing ultrapure H 2 O (Milli-Q, Millipore) and trifluoroacetic acid (TFA, 0.1-0.3%). High-resolution mass spectra (ESI-HRMS) were obtained by dissolving samples in suitable solvents and infusing the resulting solutions directly into the electrospray ionizer of a Shimadzu LCMS-IT-TOF (225-07100-34) mass spectrometer. 1D and 2D 1 H and 13 C-NMR spectra such as COSY, HSQC, HMBC and NOESY were obtained on a Bruker Avance DRX500 instrument.

Collection, Botanic Identification and Processing of Plant Materials
Aspidosperma ulei is commonly known as pitiá or piquiá. It was collected in Garapa in the City of Acarape in Ceará State, Brazil. Voucher specimens (registry numbers 30823, 32630 and 34813) were deposited in the Prisco Bezerra Herbarium of the University of Ceará. Botanic identification was performed by Prof. Edson P. Nunes of the Department of Biology of the Federal University of Ceará, Fortaleza, Ceará. Leaves, stem bark, heartwood, root bark and root wood were separately dried and milled. Powdered plant materials were weighed and then extracted as described below.

Preparation of Extracts of A. ulei and Isolation Procedures
Extraction of dry, powdered plant materials was carried out by maceration in EtOH at r.t. for 72 h. The mass of each plant material was extracted a total of three times (3 × 10 L). The EtOH solutions obtained from each extraction were rotary evaporated under reduced pressure and combined to provide each extract ( Table 2).

Acid-Base Fractionation of EtOH Extracts
Heartwood EtOH extract (HWEE), RWEE and RBEE (20 g of each) were separately dissolved in 2M HCl (200 mL) with stirring (30 min). Each resulting solution was extracted with DCM (3 × 300 mL). The combined organic phases were dried over anhydrous Na 2 SO 4 , evaporated to dryness and gave rise to the acidic alkaloid fractions of the heartwood, root wood and root bark EtOH extracts (HWEEAF (255 mg), RWEEAF (287 mg) and RBEEAF (384 mg), respectively). Conc. NH 4 OH was added dropwise to each acid fraction until each was pH 9 (Merck 0-14 Indicator Paper). Each fraction was then extracted with DCM (3 × 200 mL). The organic layers were combined, dried over anhydrous Na 2 SO 4 , filtered and totally evaporated to yield basic alkaloid fractions of the heartwood, root wood and root bark EtOH extracts (HWEEBF (363 mg), RWEEBF (302 mg) and RBEEBF (792 mg), respectively).

Isolation of Chemical Components from Acidic Fractions
RBEEAF was subjected to normal-phase CC (10 g silica gel,  = 2.5 cm) using a gradient of increasing polarity of MeOH in DCM as eluents and resulting in 12 chromatographic fractions. Chromatographic fractions 4-9 (331 mg) were combined. The alkaloid olivacine (8, 5.0 mg) was obtained by precipitation from the combined fraction. The combined fraction was further separated by HPLC using a reverse-phase, semi-preparative column (10.0 × 250 mm, 5 μm) that was eluted using 0.1% aq. TFA and MeOH (45:55). The run time was 15 min at a flow rate of 4.5 mL/min. Six fractions were collected using a detector wavelength of 323 nm. This procedure yielded a boonein lactone (9, 10.0 mg) and the alkaloids uleine (10, 40.0 mg) and 20-epi-dasycarpidone (11,26.0 mg).
The fraction HWEEAF was separated by reverse-phase, semi-preparative HPLC (4.6 × 250 mm, 5 μm) using 0.1% aq. TFA and MeOH (70:30) at a flow rate of 3.0 mL/min, a total run time of 20 min and detector running at a wavelength of 300 nm. Four fractions were collected and fraction 4 (43.0 mg) was sufficiently pure for full spectrometric characterization by 1D and 2D 1 H and 13 C-NMR techniques and its structure proved to be that of an indole alkaloid, 20(E)-nor-subincanadine E (12), derived from the stemmadenine skeleton.

In Vitro Culture of Plasmodium Falciparum and in Vitro Antiplasmodial Assay
The multi-drug resistant K1 strains of P. falciparum (Thailand, MRA-159, MR4-ATCC) were maintained in continuous culture [41]. The in vitro antiplasmodial test was performed as previously described [14]. Briefly the substances were diluted in DMSO to a stock concentration of 5 mg/mL and subsequently diluted in complete culture medium to obtain sample solutions having concentrations in the range 100-0.14 µg/mL. Sample solutions were applied to the wells of 96-well test plates containing red blood cell suspension having initial parasitemia of 1.5%. Each sample concentration was tested in triplicate and each test plate was incubated for 48 h at 37 °C. After incubation, the contents of the wells were evaluated by optical microscopy. The inhibition of the growth of parasites (IGP%) was evaluated as a percentage by comparison with controls:

Cell Culture and Cytotoxicity Test Using the Alamar Blue TM Assay
The NHI-3T3 cell line of mouse fibroblasts was grown in DMEN medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/mL streptomycin and 100 U/mL penicillin, and incubated at 37 °C with a 5% atmosphere of CO 2 . For assays, the cells were plated in 96-well plates (10 4 cells per well) and the Alamar Blue TM assay was performed using previously described procedures [42,43]. Briefly, after 24 h, the compounds were dissolved in DMSO and added to each well to give final concentrations of 50 µg/mL. Plates were incubated for 48 h. Control groups had final well concentrations of 0.1% DMSO. Two hours before the end of the incubations, 10 µL of Alamar Blue TM was added to each well. The fluorescent signal was monitored with a multiplate reader using 530-560 nm excitation and 590 nm emission wavelengths.

Conclusions
This work represents a significant contribution to the knowledge of the chemical composition of A. ulei. This included the structural elucidation of a new indole alkaloid, identification of two indole alkaloids not previously reported in Aspidosperma spp. and identification of seven known compounds for the first time in A. ulei. Isolated indole alkaloid 20-epi-dasycarpidone (11) was shown to exhibit moderate inhibitory activity against the K1 strain of P. falciparum. Furthermore, the presence of highly active antimalarial indole alkaloids olivacine and uleine in A. ulei extracts was confirmed in the present study as was the absence of in vitro cytotoxicity of several isolated compounds. Taken together, these results lend further support to earlier reports regarding the antimalarial potential of botanicals prepared from A. ulei and isolated antiplasmodial and antimalarial components.