Antibacterial Properties of 3 H-Spiro[1-benzofuran-2,1’-cyclohexane] Derivatives from Heliotropium filifolium

A re-examination of cuticular components of Heliotropium filifolium allowed the isolation of four new compounds: 3’-hydroxy-2’,2’,6’-trimethyl-3H-spiro[1-benzo-furan-2,1’-cyclohexane]-5-carboxylic acid (2), methyl 3’-acetyloxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylate (3), methyl 3’-isopentanoyloxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylate (4) and methyl 3’-benzoyloxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylate (5). Compounds 2-5 were identified by their spectroscopic analogies with filifolinol (1), and their structures confirmed by chemical correlation with 1. The antimicrobial properties of the compounds were tested against Gram positive and Gram negative bacteria. Some of them proved to be active against Gram positive, but inactive against Gram negative bacteria. In searching for structure-activity relationships from the obtained MIC values, lipophilicity was shown to be an important variable.

The antimicrobial properties of the compounds were tested against Gram positive and Gram negative bacteria. They all proved to be inactive against the latter, but some exhibited an interesting activity against Gram positive microorganisms.
Compound 3 was obtained as a dextrorotatory oil; the molecular formula C 20 H 26 O 5 was deduced from its exact mass [M + ] 346.1780. The analysis of the NMR spectra (Table 1 and 2) indicated that 3 was the acetylated derivative of filifolinol (1). Confirmation of the structure and the stereochemistry of 3 was done by acetylation of filifolinol (1), with N,N-dimethylaminopyridine and acetyl chloride to yield a pure oil, [α] 20 D = + 0.5°.  (1), esterified with a five-carbon acid, identified as isopentanoic acid. Confirmation of the structure and the stereochemistry of 4 was done by esterification of filifolinol (1) with N,N-dimethylaminopyridine and isopentanoyl chloride to yield a pure oil, [α] 20 D = + 12.0°. Compound 5 was obtained as a dextrorotatory oil; the molecular formula C 25 H 28 O 5 was deduced from its exact mass [M + ] 408.1938. The analysis of the NMR spectra (Table 1), suggested a derivative of filifolinol (1), esterified with a seven-carbon acid, identified as benzoic acid. Confirmation of the structure and the stereochemistry of 5 was done by benzoylation of filifolinol (1) with N,N-dimethylaminopyridine and benzoyl chloride to yield a pure oil, [α] 20 D = + 92.0°. Compounds 1-5 were evaluated as antimicrobial agents against Gram positive and Gram negative bacteria. They all proved to be inactive against the latter, but some exhibited an interesting activity against Gram positive microorganisms (Bacillus subtilis, Bacillus cereus; Micrococcus luteus and Staphylococcus aureus), comparable to that of commercial antibiotics like chloramphenicol, ampicillin or tetracycline. Table 3 lists their MIC values against these microorganisms. In searching for structure-activity trends from the MIC values of Table 3, we investigated the possibility that lipophilicity might be an important factor in the activity of these filifolinol derivatives. This was suggested by the observation that compound 2, the least active member of the series, possessed two hydrophilic groups, a carboxylic and a hydroxyl function. Conversion of either group, or both, into more hydrophobic ester functionalities led in all cases to more active compounds. Our hypothesis was born out by comparison of the activities of compounds 1-5 with their estimated lipophilicities ( Table 3). The only inactive compound against all four tested microorganisms was acid 2, with the smallest lipophilicity, as estimated by its Xlog P value [4]. All other derivatives have higher Xlog P values, and their activity increases in the order 1<3<4< 5, which is the same order of increasing lipophilicities. The good qualitative correlation between lipophilicity and antimicrobial activity does not rule out the importance of other structural factors which may be responsible for the activity of the studied compounds.

General
Optical rotations were measured on a Perkin-Elmer Polarimeter 241. IR spectra were recorded as KBr discs on a Bruker 66v FT-IR spectrometer. EIMS, HREIMS spectra (direct inlet, EI at 70 eV) were recorded with a FISONS VG AUTOSPEC spectrometer. NMR spectra (both 1D and 2D) were obtained on a Bruker AVANCE 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C, respectively) using the residual solvent peaks as internal standard. Column chromatography was carried out on silica gel (Merck, Kieselgel 60, 60-230 mesh). TLC were carried out on silica gel 60 F254 pre-coated plates with detection accomplished by spraying with p-anisaldehy de followed by heating at 105 ºC for 1-2 min.

Antibacterial activity determination in liquid medium
Antibacterial determination in liquid media was carried out by serial two-fold dilutions of the compounds 1-5 and the antibiotics tetracycline, chloramphenicol and ampicillin in 2 mL in a range of 1024 µg/mL to 4 µg/mL. Twenty five microliters of bacteria culture (McFarland 1.0 of B. cereus, B. subtilis, S. aureus and M. luteus) was added to each compound concentration, incubated at 37º C for 18 h and the minimal inhibitory concentration was registered. The MIC determination was carried out in five independent experiments.

Estimated lipophilicity values
Lipophilicity values have been estimated with the aid of the XLOGP3 program, available in the web [4]. The XLOGP3 method estimates log P values for structures related with reference compounds for which experimental log P values are available [5]. The additive model implemented in XLOGP3 uses a total of 87 atom/group types and two correction factors as descriptors. It is calibrated on a training set of 8199 organic compounds with reliable logP data through a multivariate linear regression analysis. It compares favourably with other methods, with average errors in the range of ± 0.24-0.51 units.