Formal synthesis of ( − )-pereniporin B and ( − )-cinnamosmolide

The paper describes a new pathway for an efficient synthesis of natural and bioactive drimanic compounds ( − )-pereniporin B (1) and ( − )-cinnamosmolide (2) from ketodiol 7, an intermediate obtained before from accessible labdane diterpenoid (+)-larixol (3). The key step involves allylic bromination of acetate 8 with N-bromosuccinimide. The in vitro antimicrobial and antifungal activities of all compounds are also reported. Their structures were confirmed by both spectroscopic data and chemical transformations.

Herein we wish to report a new and efficient pathway for the synthesis of (2 )-pereniporin B (1) and (2 )-cinnamosmolide (2) from (þ )-larixol (3) via key intermediate ketodiol 7. It must be mentioned that previously compound 7 was isolated from natural sources in low amounts (Hayes et al. 1996;Zhou et al. 2011).
It is clear that more applications of dienone 7 for the synthesis of drimanes can be developed, especially when an easy fuctionalisation of the allylic methyl group C-12 can be accomplished. This has been achieved already by the allylic oxidation with selenium oxide (Garlaschelli & Vidari 1989). In our group the same transformation has been carried out using allylic bromination followed by the substitution of bromide by an acetate group as indicated in Scheme 2.
The acetylation of the primary hydroxyl group of 7 -8 under standard conditions was made prior to the allylic bromination in order to prevent undesired oxidation at this position. The allylic bromination of 8 with N-bromosuccinimide (NBS) and subsequent replacement of bromine by treatment with KOAc gave 10 in high yield (Scheme 2). The acetate groups in 10 were hydrolysed leading to the known triol 11 (Urones et al. 1997;Zhou et al. 2011), which after oxidation of the least hindered hydroxyl group with MnO 2 followed by spontaneous cyclisation and oxidation led to lactone 12 (Kubo et al. 1983). The transformation of precursor 12 into pereniporin B (1) was reported earlier (Burke et al. 1991). It includes treatment of lactone 12 with DIBAL-H, followed by Fetizon's oxidation of the resulted lactols. Cinnamosmolide (2) can be prepared from pereniporin B (1) by its acetylation under standard conditions (Canonica et al. 1969).
Compounds 7-12 were screened for their in vitro antifungal and antibacterial activity against pure cultures of three fungi species (Aspergillus niger, Penicillium frequentans, Alternaria alternata) and against both Gram-negative (Pseudomonas aeruginosa) and Grampositive bacteria (Bacillus polymyxa). According to these assays, bromide 9 exhibited good antifungal activity with a minimum inhibitory concentration (MIC) value of 0.85 mg/mL in comparison with the reference compound caspafungin (0.42 mg/mL) and good antimicrobial activity 0.90 mg/mL in comparison with the reference compound kanamycin (0.50 mg/mL). Noteworthy, the antifungal activity of bromide 9 is higher than that reported for cinnamosmolide (2) (Canonica et al. 1969).

Experimental 3.1. General experimental procedure
Melting points (m.p.) were taken on a Boethius (VEB Analytik, DDR) hot stage apparatus. Optical rotations were determined on a Perkin-Elmer 241 polarimeter (Perkin-Elmer, Norwalk, CT, USA) with a 1 dm microcell, in CHCl 3 . IR spectra were obtained on Bio-Rad-Win-IR (Bio-Rad, Cambridge, MA, USA) and Perkin-Elmer spectrometers (Perkin-Elmer, Norwalk, CT, USA). 1 H and 13 C NMR spectra were recorded in CDCl 3 on Bruker AC-E 200 (Bruker BioSpin, Rheinstetten, Germany) and Bruker Avance DRX 400 spectrometers (Bruker BioSpin, Rheinstetten, Germany). Chemical shifts are given in ppm in d scale and referred to CHCl 3 (d H at 7.26 ppm) and to CDCl 3 (d C 77.00 ppm), respectively. Coupling constants (J) are reported in Hertz (Hz). The H, H-COSY, H, C-HSQC and H, C-HMBC experiments were recorded using standard pulse sequences, in the version with z-gradients, as delivered by Bruker Corporation (Bruker BioSpin, Rheinstetten, Germany). Carbon substitution degrees were established by the DEPT pulse sequence. For analytical TLC, Sorbfil silica-gel plates were used. The TLC plates were sprayed with conc. H 2 SO 4 and heated at 808C for 5 min. Column chromatography was carried out on Across silica gel (60 -200 mesh) using petroleum ether (PE) (b.p. 40 -608C) and the gradient mixture of PE and  (Canonica et al. 1969;Burke et al. 1991).
EtOAc. All solvents were purified and dried by standard techniques before use. Solutions in organic solvents were dried over anhydrous Na 2 SO 4 , filtered and evaporated under reduced pressure.

Antimicrobial and antifungal activity
Fungi: A. niger ATCC 53346, P. frequentans ATCC 10110 and A. alternata ATCC 8741, and Gram-negative bacteria P. aeruginosa ATCC 27813 and Gram-positive B. polymyxa were provided by the American Type Culture Collection (ATCC, USA).
Compounds caspafugin and kanamycin, both from Liofilchem (Roseto degli Abruzzi, Italy), were used as standards for antifungal and antibacterial activity testing. After 48 h of incubation, a symmetrical inhibition ellipse centred along the strip was formed. The MIC is read directly from the scale in terms mg/mL, at the point where the edge of the inhibition ellipse intersects with the MIC test strip.
Sample solutions of 0.5%, 1% and 2% concentrations were obtained by dissolution of appropriate amounts of tested compounds 7-12 in fixed volumes of DMSO.
It must be mentioned that for fungi, Sabouraud agar medium with dextrose was used (4%, SDA), and for bacteria a Standard I nutrient agar medium was used, both from Merck (Schwalbach Hesse, Germany).
Microorganism suspensions were prepared using the method of successive agar dilutions according to the standard MIC (Usta et al. 2007) and their cultivation was carried out according to standard procedures (SR-EN 1275:2006 andNCCLS guidelines) (NCCLS 2003). The final charge-stock inoculum was prepared as 1 £ 10 24 mg/mL concentration and inoculated plates were incubated at 318C for 7 days. First observations were made after 48 h and final observations after 7 days of incubation, establishing the MIC. Observations on the results were made by visual analysis, microscopy and photography, using a stereomicroscope Novex Ap-8 Euromex (Olimpus Europa Holding G.m.b.H., Hamburg, Germany) and Olympus SZY 160 microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).