Withanolides with Antibacterial Activity from Nicandra john-tyleriana

Eleven new withanolides (1−11) were isolated and characterized from the aerial parts of Nicandra john-tyleriana. Five of these withanolides have an unmodified skeleton (1−5), two are acnistins (6, 7), and four are withajardins (8−11). These new isolates were fully characterized using a combination of spectroscopic techniques (including multidimensional NMR) and mass spectrometry. All compounds were evaluated for their antibacterial activity against Bacillus, Enterococcus, Escherichia, Listeria, Pseudomonas, and Staphylococcus strains. W comprise a group of naturally occurring C28 steroids based on an ergostane skeleton, in which C-26 and C-22, or C-26 and C-23, are oxidized in order to form a δor γ-lactone. Biogenetic transformations of withanolides can produce highly modified compounds in both the steroid nucleus and side chain, including the formation of additional rings. Their chemistry and occurrence have been the subject of several reviews. Nicandra Adans. (Solanaceae) is a small genus comprising three species. Two of these are Nicandra john-tyleriana S. Leiva & Pereyra, which grows in the northern Andean region of the Department San Martiń (Prov. Otuzco, ca. 3000 m), and Nicandra yacheriana S. Leiva, from the “lomas” of the Department Arequipa (Prov. Caraveli,́ ca. 600 m), both endemic in Peru. The third species in this group is the wellknown and most widespread species of the genus, Nicandra physalodes (L.) Gaertn., which occurs in a region from Peru to northern Argentina, as well as being found as a ruderal species in tropical and subtropical areas worldwide. Species in this genus are vigorous annual herbs with showy pale violet bellshaped corollas with a white throat and sagittate calyx. The three above-mentioned species are distinguished by their floral and fruit characters. A family of aromatic D-ring withanolides and withanolides with an unmodified skeleton has been isolated from N. physalodes, with some of these compounds having exhibited interesting biological activities such as insecticidal or potential anticancer properties. In the present investigation into the withanolides of the genus Nicandra, reported is the isolation of 11 new withanolides from N. john-tyleriana (1−11). Antibacterial activity has been previously reported for the ethanolic extract of N. john-tyleriana against Escherichia, Pseudomonas, Proteus, and Staphylococcus bacteria. In order to determine the antimicrobial activity, all compounds obtained in the present study were evaluated against different strains of Bacillus, Enterococcus, Escherichia, Listeria, Pseudomonas, and Staphylococcus by utilizing a disk diffusion method and bioautography. Finally, the most active compounds were tested by direct contact against the most sensitive bacteria cells. Received: October 21, 2014 Published: February 10, 2015 Article

The 1 H and 13 C NMR spectra of 16-oxojaborosalactone D (3) and 16-oxojaborosalactone E (4) were closely related to those of 2 (Tables 1 and 2). The almost identical 13 C NMR data for rings C and D and the side chain of compounds 2−4 indicated that structural differences were restricted to substituents in rings A and B. Furthermore, the presence of a 1-oxo-2-ene functionality in ring A was evident for the three compounds. The 1 H and 13 C NMR data of 3 were consistent with a 5α,6β-diol typical of many withanolides, 11 and the small couplings in the H-6 resonance at δ 3.70 t (J = 2.5 Hz) confirmed the axial orientation (β) of the 6-hydroxy group. Moreover, the 13 C NMR spectrum revealed the expected  chemical shifts for signals of carbons C-5 and C-6 at δ 77.2 and 74.2, respectively. The 1 H and 13 C NMR data of 4 were consistent with a 5α-chloro-6β-hydroxy arrangement. Thus, the signal at δ 4.06 t (J = 2.7 Hz) was assigned to the equatorial H-6, with the unusually high chemical shift observed for H-4β at δ 3.53 (dt, J = 19.9 and 2.8 Hz) being indicative of a chlorine atom at C-5 with an α-orientation. 11 The substitution pattern in ring B was confirmed by the signals at δ 79.9 and 74.3 in the 13 C NMR spectrum, which were assigned to C-5 and C-6, respectively.
Withajardins. The four new withajardins F−I ( 8−11) were isolated from N. john-tyleriana. Withajardins have been previously isolated only from the Deprea 17 and Tubocapsicum 16b,18 genera. They exhibit a bicyclic side chain involving C-21 and the lactone ring, but, in contrast with the acnistins, C-21 is bonded to C-25 instead of C-24.
Withajardin F (8) revealed a molecular formula of C 28 H 38 O 6 by HRESIMS. The 1 H and 13 C NMR data of rings A−D (Tables 2 and 3) were closely related to those of compounds 1 and 6, indicating a 1-oxo-2-ene-5β,6β-epoxy substitution pattern in rings A/B, the presence of a hydroxy group at C-16 with an α-orientation, and the side chain at C-17 with a βorientation. With respect to the side chain, compound 8 exhibited 1 H and 13 C NMR spectra closely related to those of the withajardins tuboanosides A and B. 18 The characteristic NMR spectroscopic data for this side chain were the signals at δ 4.69 (brt, J = 3.2 Hz), 1.13 s, and 1.25 s, assigned to H-22, H 3 -27, and H 3 -28, respectively, in the 1 H NMR spectrum, and with the key cross-correlation peaks observed between the signal corresponding to H-21a (δ 2.15) and the signals at δ 38.5 (C-20), 47.2 (C-25), 61.6 (C-17), and 177.7 (C-26) in the HMBC experiment, thus confirming the characteristic C-21−C-25 bond of the withajardin skeleton. The 13 C NMR spectrum of 8 was in agreement with the structure proposed. Regarding the configuration of C-24, the NOE observed between H 3 -28 and H-12β (δ 1.43 m) supported the S configuration at this position (see the Supporting Information). The 1 H and 13 C NMR spectra of compound 8 were run in pyridine-d 5 in order to correlate the chemical shifts with those of tuboanosigenin pbromobenzoate, a compound for which X-ray analysis has been performed. 18 The observed differences between both spectra were in good agreement with compound 8 having the opposite configuration at C-24 (Table 2). Accordingly, the structure of 8 was elucidated as (17R,20S,22R,24S,25R)-5β,6β-epoxy-16α,24α-dihydroxy-21,25-cycloergost-2-en-1-one.
The 1 H and 13 C NMR spectra of withajardin G (9) and H (10) were closely related to those of 8 (Tables 2 and 3), showing patterns typical of the withajardin arrangement at the side chain, for the resonances of carbons 17−28 and their protons. The almost identical 13 C NMR data for rings C and D and the side chain of compounds 8−10 indicated that structural differences were restricted to substituents in rings A and B. The 1 H and 13 C NMR spectra of 9 revealed a 1-oxo-2-ene-5α,6βdihydroxy substitution pattern from the characteristic signals corresponding to C-1−C-6 and the corresponding protons. Regarding withajardin H (10), the resonances from rings A and B were almost identical to those in compound 4, especially with respect to the unusually high chemical shifts observed for H-4β (δ 3.53 dt, J = 20. 1,2.8 Hz) and the signal at δ 4.03 t (J = 2.7 Hz), which was assigned to the H-6α proton, consistent with a 5α-chloro-6β-hydroxy arrangement in ring B. The 13 C NMR and HSQC spectra of 10 were found to be in agreement with the proposed structure.
Finally, withajardin I (11) revealed closely related 1 H and 13 C NMR spectra to those of compound 8. In fact, the only difference between 11 and 8 was the presence of a carbonyl group at C-16 (δ C 217.0) instead of a hydroxy group (δ H 4.06, δ C 75.9).
The potential antibacterial activity of all the compounds described above was evaluated in vitro against strains of Bacillus, Enterococcus, Escherichia, Listeria, Pseudomonas, and Staphylococcus by different bioassay techniques. Compounds 2 and 6 showed significant antibacterial activity against Bacillus cereus using a disk-diffusion technique and the bioautographic TLC assay. The antibacterial activity of compound 2 was also quantified by direct contact against B. cereus BAC1 cells. This compound exerted bactericidal and bacteriostatic effects at 1000 ppm and close to 750 ppm, respectively ( Figure 1).

■ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-1010 polarimeter. The UV spectra were obtained using a Shimadzu-260 spectrophotometer, and IR spectra were produced using a Nicolet 5-SXC spectrophotometer. NMR spectra were recorded on a Bruker AVANCE II AV-400 operating at 400.13 MHz for 1 H and 100.63 MHz for 13 C, while 2D spectra (COSY, HSQC, HMBC, and NOESY) were obtained using standard Bruker software. Chemical shifts are given in ppm (δ) downfield from the TMS internal standard. HRESIQTOFMS were determined on a Micro TOF II Bruker Daltonics. The chomatographic separations were performed by column chromatography on silica gel 60 (0. 063−0.200 mm) and Sephadex LH-20, and preparative TLC was carried out on silica gel 60 F 245 (0.2 nm thick) plates.