Oxygenated Cyclohexene Derivatives from the Stem and Root Barks of Uvaria pandensis

Five new cyclohexene derivatives, dipandensin A and B (1 and 2) and pandensenols A–C (3–5), and 16 known secondary metabolites (6–21) were isolated from the methanol-soluble extracts of the stem and root barks of Uvaria pandensis. The structures were characterized by NMR spectroscopic and mass spectrometric analyses, and that of 6-methoxyzeylenol (6) was further confirmed by single-crystal X-ray crystallography, which also established its absolute configuration. The isolated metabolites were evaluated for antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus epidermidis and the Gram-negative bacteria Enterococcus raffinosus, Escherichia coli, Paraburkholderia caledonica, Pectobacterium carotovorum, and Pseudomonas putida, as well as for cytotoxicity against the MCF-7 human breast cancer cell line. A mixture of uvaretin (20) and isouvaretin (21) exhibited significant antibacterial activity against B. subtilis (EC50 8.7 μM) and S. epidermidis (IC50 7.9 μM). (8′α,9′β-Dihydroxy)-3-farnesylindole (12) showed strong inhibitory activity (EC50 9.8 μM) against B. subtilis, comparable to the clinical reference ampicillin (EC50 17.9 μM). None of the compounds showed relevant cytotoxicity against the MCF-7 human breast cancer cell line.

Our recent findings of the bioactive oxygenated cyclohexene derivatives including their chlorinated counterparts from Cleistochlamys kirkii 13 and Monathotaxis trichocarpa 11 inspired a reinvestigation of Uvaria pandensis Verdc., a plant species previously reported to contain similar compounds. 6,36 The species is used in some parts of Tanzania in traditional medicine to treat stomach disorders and fever. 11 Reported herein are the isolation and structure determination of five hitherto unreported oxygenated cyclohexene derivatives (1− 5), which were obtained with 16 known compounds (6−21) from the CH 3 OH extracts of stem and root bark samples. The isolated metabolites were evaluated for antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus epidermidis and the Gram-negative bacteria Enterococcus raf f inosus, Escherichia coli, Paraburkholderia caledonica, Pectobacterium carotovorum, and Pseudomonas putida. They were also assessed for cytotoxicity against the MCF-7 human breast cancer cell line.
In conclusion, 21 natural products including the five new cyclohexene derivatives 1−5 were isolated and characterized from separate CH 3 OH extracts of the stem and root barks of U. pandensis. This is the first report of all but compounds 12 36 and 13 36 from this plant. Polyoxygenated cyclohexenes and Cbenzylated chalcones have restricted occurrence in plants.
They are known to be produced by the members of the Uvariae tribe of the family Annonaceae. Hence, their occurrence in U. pandensis is of chemotaxonomic importance, confirming the placement of this plant in the Uvariae taxon. Some of the isolated compounds showed activity against Gram-positive bacteria, along with low to moderate cytotoxicity.

■ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined using a 341LC OROT polarimeter (589 nm, 20°C), whereas UV measurements were done using a 264 UV−vis spectrophotometer. A MIR 450FT-IR spectrometer was used to record the IR spectra. NMR spectra were acquired on either a Bruker Avance III HD 600/500 or 400 NMR MHz spectrometer and were analyzed with the MestReNova (v10.0.0) software. Structural assignments were based on 1 H NMR, 13 C NMR, COSY, TOCSY, NOESY, HSQC, and HMBC spectra. LC-MS (ESI) spectra were acquired with a PerkinElmer PE SCIEX API 150 EX instrument equipped with a Turbolon spray ion source and a Gemini 5 mm RP-C 18 110 Å column, using a gradient of H 2 O−CH 3 CN (80:20 to 20:80) in the presence of 0.2% HCO 2 H and a separation time of 8 min. HRESIMS were obtained with a Q-TOF-LC/MS spectrometer with a lock mass ESI source (Stenhagen Analysis Lab AB, Gothenburg, Sweden), using a 2.1 × 30 mm 1.7 μm RP-C 18 column and an elution gradient of H 2 O−CH 3 CN (5:95 to 95:5, with 0.2% HCO 2 H). Analytical TLC was performed on aluminum plates precoated with silica gel 60 F254 (Merck). After development with an appropriate solvent system, the plates were evaluated under UV light (254 and 366 nm) and then sprayed with 4-anisaldehyde reagent, prepared by mixing 3.5 mL of 4-anisaldehyde with 2.5 mL of concentrated H 2 SO 4 , 4 mL of glacial HOAc, and 90 mL of CH 3 OH. The plates were then heated for the identification of UV-negative compounds and assessment of the color change of the UV-positive spots. Column chromatography was carried out using silica gel 60 (230−400 mesh), and gel filtration was performed over Sephadex LH-20 (Pharmacia) suspended in CH 2 Cl 2 −CH 3 OH (1:1). Preparative HPLC was performed on a Waters 600E system using Chromulan software (Pikron Ltd.) and an RP- Extraction and Isolation. The stem and root barks of U. pandensis were air-dried for 2 weeks and then powdered to obtain 230.0 and 983.7 g samples, respectively. The ground materials were then soaked in CH 3 OH for 48 h twice for each of the plant parts. The filtrates were concentrated in vacuo on a rotary evaporator at 40°C to obtain 45 g of root bark and 23.0 g of stem bark crude extracts.
The stem bark crude extract (23.0 g) was adsorbed on silica gel and subjected to gravity column chromatography employing gradient elution ranging from 5% EtOAc−isohexane to 10% CH 3 OH−EtOAc, to afford 356 fractions (ca. 200 mL each). Based on TLC analysis, the fractions were combined to obtain 69 fractions, of which fraction 44 (206−208), eluted with 30−50% EtOAc−isohexane, was purified further using reversed-phase HPLC, which afforded a mixture (10.3 mg) of uvaretin (20) and isouvaretin (21) at a retention time of 13.53 min. Fraction 54 (274−278), obtained with 50−70% EtOAc− isohexane, precipitated in CH 3 OH to give benzoic acid 2,3diacetoxy-1,6-dihydroxycyclohex-4-enyl methyl ester (15, 6.4 mg). Fraction 59 (291−294) precipitated in EtOAc to furnish 11.5 mg of lupeol (16). Fraction 47 (246−253), obtained with 50−70% EtOAc− isohexane, was subjected to gel filtration using a Sephadex column (1:1 CH 3 OH−CH 2 Cl 2 ) to give 30 subfractions that on TLC analysis were combined to obtain a further nine subfractions. Subfraction 4 (19−20) was subjected to reversed-phase HPLC for further purification, from which at a retention time of 14.29 min zeylenyl-2,6-diacetate (14,5 X-ray Diffraction Analysis of 6-Methoxyzeylenol (6). The solid state structure of 6 was determined from single crystals of 6, obtained by crystallization from EtOAc. Data were collected on a Bruker D8 APEX-II equipped with a CCD camera using Mo Kα radiation (λ = 0.710 73 Å). Crystals were mounted on a fiber loop and fixated using Fomblin oil. Data reduction was performed with SAINT, 46 and absorption corrections for the area detector were performed using SADABS. 47 The structure was solved in the orthorhombic space group P2 1 2 1 2 1 by direct methods and refined by least-squares methods on F 2 using the SHELX and the OLEX2 software suits. 48−50 The data were collected at 150(2) K. Nonhydrogen atoms were refined anisotropically. Hydrogen atoms were constrained in geometrical positions relative to their parent atoms. A Flack parameter of 0.2(8) precluded determination of the absolute structure based on anomalous dispersion. 51,52 Further details of structure solutions and refinements can be found in the Supporting Information. The X-ray structure data of 6 (CCDC 2105244) have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Antibacterial Assays. The antibacterial activity of the isolated compounds was determined against two Gram-positive bacteria, Bacillus subtilis (NBRC/ATCC #111470) and Staphylococcus epidermidis (ATCC #35984) and five Gram-negative bacteria, Escherichia coli MG1655 (CGSC #6300), Paraburkholderia caledonica (NBRC/ATCC #102488), Pseudomonas putida (NBRC/ATCC #100650), Pectobacterium carotovorum (NBRC/ATCC #3380), and Enterococcus raf f inosus (NBRC/ATCC #100492). The bacteria were cultured as previously described by Mueller and Hinton, and Doyle. Initially, the compounds were dissolved at 10 mg/mL in 100% DMSO, then further diluted 30× in H 2 O and stored at −20°C. For in vitro determination of antibacterial activity, a culture of bacterial cells was grown to OD 600 nm = 0.5. The culture was diluted 10× with prewarmed medium, and the compounds to be tested were added to the culture medium for a final concentration of 30 μg/mL, each at 100 μL in a 96-well microtiter plate, then incubated at 37°C without agitation for 18 h. To measure cell viability, the resazurin-based assay was used, as described previously. To each well was added 12 μL of 10× alamarBlue solution (resazurin solution, ThermoFisher), and the plate incubated at 37°C for 1 h. Next, the fluorescence was measured using a POLARstar Omega microplate-reader from BMG Labtech with the excitation filter set to 544 nm and the emission filter at 590 nm. Cells exposed to an equivalent concentration of DMSO were used as a negative control. Bleed-through of fluorescence from resorufin between wells in the microtiter plate fluorescence reader was measured and found to be <1% between adjacent wells. To check for quenching of fluorescence by any of the investigated compounds, grown bacterial cultures were mixed after 1 h of incubation with resazurin and the compound of interest at the highest concentration to be assayed, and the measured fluorescence was compared with samples without compound added. All tests of compound activity were performed in three independent replicates. Those compounds where a reduction of fluorescence by at least 50% relative to the solvent control was observed in any of the bacterial species tested were followed up by additional tests for more accurate determination of the degree of antibacterial activity in terms of minimum inhibitory concentration (MIC). EC 50 values, from three independent replicate experiments, using 2-fold dilution intervals were also calculated.
Cytotoxicity Assay. The cytotoxicity levels of the isolated compounds were evaluated against human MCF-7 cells grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and kept in exponential growth as previously reported. Before the assay, cells were reseeded into 96-well microtiter plates at a density allowing continued exponential growth and allowed to settle for 24 h. The isolated compounds were added from a stock solution in DMSO, for a final concentration of 0.3% v/v of the solvent in the culture medium. After 24 h of incubation in the presence of the compound, cell viability was assayed using PrestoBlue Cell Viability Reagent (ThermoFisher) according to the manufacturer's instructions. A Polar Star Omega plate reader (BMG Lab Tech) was used to measure resorufin fluorescence at 544 nm excitation/590 nm emissions. Survival was expressed as percentage of the solvent-only control. EC 50 values for each compound were calculated, from three independent replicate experiments, using 2-fold dilution intervals.
NMR, MS, ORD, UV, and IR data for the isolated compounds (PDF) X-ray crystallographic data of compound 6 (CIF)