0250-5460 0250-5460 S0250-54602008000100005 Bolivia Sweden Bolivia Bolivia 00 12 2008 00 12 2008 25 1 24 29

MORE INVESTIGATIONS IN POTENT ACTIVITY AND RELATIONSHIP
STRUCTURE OF THE LICHEN ANTIBIOTIC (+)-USNIC ACID AND ITS DERIVATE DIBENZOYLUSNIC ACID

*Marcela Melgarejo1, 2, Olov Sterner2, José Vila Castro1, Patricia Mollinedo1

1 Laboratory of Natural Products, Chemistry Investigations Institute, San Andrés University, Street 27 Cota Cota, P.O. Box 303 La Paz-Bolivia,
2 Division of Organic Chemistry, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden

Key Words: lichens, usnic acid, dibenzoylusnic acid, antibacterial activity, relationship structure, ditch diffusion method.

ABSTRACT

By reacting R-(+)-usnic acid (1) with benzoyl chloride in presence of pyridine gave the dibenzoylusnic acid (2). The antibacterial activity and relationship structure of acid usnic an old lichen drug and its derivate dibenzoylusnic acid was studied against nine ATCC bacteria and one bacterium clinical isolate./ Mediante la reacción del R-(+)-acido úsnico con cloruro de benzoilo en presencia de piridina se obtuvo el acido dibenzoilusnico. Se estudio la relación de actividad antibacteriana y estructura del R-(+)-acido úsnico y su derivado el acido dibenzoilúsnico contra nueve bacterias ATCC y una bacteria clínicamente aislada.

Corresponding author: Marcela.Melgarejo@organic.lu.se


INTRODUCTION

]]> The ever more frequent overuse of antibiotics, in the treatment of bacterial infections, is bringing on an increase of pathogen microorganisms that become resistant to common treatments. Consequently, it is necessary to increase the administered doses or to provide new products1.
Medicinal chemists are still actively seeking new and improved antibacterial agents to combat the worrying ability of bacteria to acquire resistance to current drugs. For example, 60 % of Streptococcus pneumoniae strains are resistant to β-lactams and 60 % of Staphylococcus aureus strains are resistant to methicillin. The last resort in treating S. aureus infections is vancomycin, but resistance is beginning to appear to that antibiotic as well. Some strains of Enterococcus faecalis appearing in urinary and wound infections are resistant to all known antibiotics and are untreatable. If resistance continues to grow, medicine could be plunged back to the 1930s2.
The many functional groups of R-(+)-usnic acid (1) make the molecule a good target for structural modification. The compound reacts with amines, hydrazines and acyl hydrazides to form condensation products, undergoes esterification, and gives numerous degradation derivatives and forms dihydrousnic acid on reduction. Usnic acid salts are usually unstable. A large number of publications dating from 1962 to 1985 bear witness to the extensive work of Japanese researchers on the chemistry of usnic acid and reaction mechanisms (Takani and Takahashi, 1985, last in series). In another excellent series, Canadian scientists (Kutney et al., 1984, last in series) described preparation of numerous usnic acid biodegradation products and intermediate derivates as well as conversion of usnic acid to isousnic acid through base-catalyzed rearrangement3, 4, 5.
In relation to a study aimed at the relationship structure of the R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) endowed with antibacterial activity present here the results obtained investigating the R-(+)-usnic acid (1) as a lead compound.

Lichens

Lichens, symbiotic organisms of fungi and algae, synthesize numerous metabolites, the “lichen substances”, which comprise aliphatic, cycloaliphatic, aromatic, and terpenic compounds. Lichens have been used in Traditional Medicine in many countries over a considerable period time. Lichens and their metabolites have a manifold biological activity: antiviral, antibiotic, allergenic, plant growth inhibitory, antiherbivore, and enzyme inhibitory. Their efficacy is due to the synthesis of unique secondary compounds6.

Chemistry and activity of Acid usnic

R-(+)-usnic acid (1) is a naturally occurring compound and very active lichen substance that is used in pharmaceutical preparations. It can be obtained from different lichens species7. It is a product of the secondary metabolism of the fungal partner and it exists in two en-antiomers. Both the R-(+) and S-(−) forms are known8.A severe limitation to its practical use is its low solubility in organic solvents and in water. Accordingly, an interest arises in the preparation of derivatives maintaining the antibiotic activity or improve the antibiotic activity but characterized by more favorable solubility.
Biological and medical papers on lichens started to appear after the end of the World War II. The most investigated lichen substance is the secondary metabolite, usnic acid, universally known as an antibiotic9.
With the aim to obtain a derivative of usnic acid for pharmacological evaluation, the preparation of the dibenzoylusnic acid (2) for the relationship structure and antibacterial activity studies were investigated starting from R-(+)-usnic acid (1). ]]>

RESULTS

Structure elucidation

R-(+)-usnic acid (1) was obtained yellow crystals. The FABMS of (1) showed a protonated molecular ion at ([M+1] m/z 345), while 1H NMR and 13C NMR spectra indicated the presence of 16 protons and 18 carbon atoms. This was consistent with the molecular formula C18H16O7. The 1H NMR (CDCl3) spectrum of (1) displayed signals for four methyl groups at δ 1.77 s, δ 2.14 s, δ 2.67 s, δ 2.68 s, an aromatic proton at δ 5.98 s and at δ 11.10 s. The 13C NMR spectrum revealed the presence of eighteen carbon atoms four methyls, thirteen non-protonated unsaturated carbons and one protonated unsaturated carbon. In the HMBC spectrum correlations peaks were observed between the following protons and carbons: In the HMBC spectrum, δ 5.98 (1H, s, H-4) correlated with C-4a (δ 179.8) and C-9b (δ 59.5); δ 2.68 (3H, s, CH3CO-6) with C-6 (δ 101.9) and CO (δ 200.8); δ 2.67 (3H, s, CH3CO-2) with C-2 (δ 105.7) and CO (δ 202.2); δ 1.77 (3H, s, CH3-9b) with C-9b (δ 59.5), C-4a (δ 179.8), C-8 (δ 109.7) and C-1 (δ 198.5); δ 2.14 (3H, s, CH3-8) with C-8 (δ 109.7), C-9 (δ 157.9) and C-7 (δ 164.3); δ 11.10 (1H, s, OH-9) with C-8 (δ 109.7) and C9 (δ 157.9). The structure elucidation of R-(+)-usnic acid was confirmed with comparison with literature data3,10(Fig 1).

 

The compound (1) was esterified with benzoyl chloride in presence of chloroform and pyridine gave the dibenzoylusnic acid (2). The 1H NMR (CDCl3) spectrum of (2) showed signals of three methyl groups at δ 1.74 s, δ 1.96 s and δ 2.49 s; the presence of vinyl protons at δ 5.12 (1H, d=1.76 Hz) and δ 5.45 (1H, d=1.76 Hz); the signals of aromatic protons were observed between at δ 7.0 - 8.0 ppm, and two singlets were observed at δ 5.92 s and at δ 10.41 s. The elemental composition of (2) was determined to be C32H24O9 from its FABMS and 1H and 13C-NMR data. In the HMBC spectrum correlations were observed between δ 1.74 (3H, s, CH3-9b) and C-9b (δ 61.0), C-1 (δ 201.2), C-4a (δ 174.2) and C-9a (δ 104.3); between δ 1.96 (3H, s, CH3-8) and C-8 (δ 109.3), C-9 (δ 157.8) and C-7 (δ 164.2); between δ 2.49 (3H, s, CH3CO-6) and C-6 (δ 102.1), CO (δ 200.9) and C-9a (δ 104.3); between δ 5.12 (1H, d=1.76 Hz) and δ 5.45 (1H, d=1.76 Hz) with C-2 (δ 114.9), CH2 (δ 110.2), C (δ 135.0); between δ 5.92 (1H, s, H-4) and C-9b (δ 61.0), C-4a (δ 174.2), C-2 (δ 114.9) and C-3 (δ 165.4) and between δ 10.41 (1H, s, OH- ) and C-9a (δ 104.3) and C-9 (δ 157.8), a significant correlation between δ 7.91 (2H, d, Ar) and CO (δ 163.3) and 46.between δ 7.72 (2H, d, Ar) and CO (δ 164.7). The structure (2) was established as dibenzoylusnic acid7 (Fig. 1)

Antibacterial Activity

We investigated the antibacterial activity and relationship structure of R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) against nine bacteria ATCC and one clinical isolate.
The results obtained in the agar diffusion studies are shown in Table 1.

]]> Structure-Activity Relationships

The natural product R-(+)-usnic acid (1) like a lead compound and its derivate dibenzoylusnic acid (2) have a variety of functional groups present in their structures and the Fig. 2 shows the potential binding interactions that are possible with a target-binding site.

DISCUSSION

The antibacterial activity of the R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) against Escherichia coli ATCC 25922 (sensible), Escherichia coli ATCC 35218 (resistant), Staphylococcus aureus ATCC 25923 (sensible), Staphylococcus aureus ATCC 29213 (resistant), Pseudomona aeruginosa ATCC 27853 (sensible), Shigella flexneri ATCC 12022, Klebsiella pneumoniae ATCC 70063 (resistant), Bacillus subtilis ATCC 6636, Listeria monocytogenes ATCC 7644 and Salmonella typhi (clinical isolate) is shown in Table 1.
The R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) showed inhibitory activity against Staphylococcus aureus ATCC 25923 (sensible), Staphylococcus aureus ATCC 29213 (resistant), Pseudomona aeruginosa ATCC 27853 (sensible), Shigella flexneri ATCC 12022 and Bacillus subtilis ATCC 6636.
The R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) showed similar inhibitory activity against Staphylococcus aureus ATCC 25923 (sensible).
The R-(+)-usnic acid (1) showed less inhibitory activity with a little difference against Staphylococcus aureus ATCC 29213 (resistant) and Pseudomona aeroginosa (sensible) than its derivate dibenzoylusnic acid (2).
The R-(+)-usnic acid (1) showed more considerably inhibitory activity against Shigella flexneri ATCC 12022 and Bacillus subtilis ATCC 6636 than its derivate dibenzoylusnic acid (2). The R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) did not show any inhibitory activity against Escherichia coli ATCC 25922 (sensible), Escherichia coli ATCC 35218 (resistant), Klebsiella pneumoniae ATCC 70063 (resistant), Listeria monocytogenes ATCC 7644 and Salmonella typhi (clinical isolate).


CONCLUSIONS

It can be conclude that the derivate dibenzoylusnic acid (2) shows a similar inhibitory activity against Staphylococcus aureus ATCC 25923 (sensible), significantly lowered antibacterial activity against Staphylococcus aureus ATCC 29213 (resistant) and Pseudomona aeroginosa (sensible), then the modification with an esterification of R-(+)-usnic acid (1) has been important. ]]> It can be conclude too that the derivate dibenzoylusnic acid (2) shows less considerably inhibitory activity against Shigella flexneri ATCC 12022 and Bacillus subtilis ATCC 6636, then the modification with an esterification of R(+)-usnic acid (1) has not been important.
The R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) are pharmacophore that have groups which are important for the antibacterial activity.
The antibacterial activity of the R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) against gram (+) and gram (-) bacteria indicates that both are compounds of broad spectrum.
Despite a shortage of clinical trials, it can be concluded that scientific investigations justify to some extent reputed medicinal effects of R-(+)-usnic acid (1) and derivates.
Inhibitory activity of R-(+)-usnic acid (1) and its derivate dibenzoylusnic acid (2) against Gram-positive and Gramnegative bacteria, including antibiotic-resistant pathogenic strains, definitely seems to merit further study. It should also be remembered that derivatization to potentate antimicrobial activity has in some cases been successful despite a limited number of studies. Results obtained for other types of biological activity could also prove worthy of further pursuit, as could comparison of derivate structure–activity relationships between the R-(+)-acid usnic (1) and other derivates. Furthermore, R-(+)-usnic acid (1) could serve as a lead structure for exploitation in novel therapeutic areas.


GENERAL EXPERIMENTAL PROCEDURES MATERIALS AND METHODS

General

All melting points were recorded with a Reichter microscope. The UV and IR spectra were recorded with a Varian Cary 2290 and a Perkin-Elmer 298 spectrometer, respectively. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded in CDCl3 and MeOD usin a Bruker DRX400 spectrometer with an inverse multinuclear 5-mm probe head equipped with a shielded gradient coil. COSY, HMQC and HMBC experiments were recorded with gradient enhancements using-sine-shape gradient pulses. EIMS were recorded with a JEOL SX102 spectrometer at 70 eV. Column chromatography was\run on Merk silica gel 60 and TLC was carried out on Silica gel GF254.

Plant Material, Extraction and Isolation

]]> The Alectoria ochroleuca material was dried and powdered, 56.0 g were extracted with acetone at room temperature. During the extraction, yellow crystals of R-(+)-usnic acid (1) separated out. They were recrystallized from CH2Cl2, precipitated by the addition of methanol yielding 5.1 g corresponding to 9.1 % of the materials dry. The compound(1) was esterified with benzoil chloride in presence of chloroform and pyridine for 24 h at 18oC to give the dibenzoylusnic acid (2).
R-(+)-Usnic acid (1) 13C NMR (CDCl3, 100 MHz); δ 198.5 (C-1), δ 105.7 (C-2), δ 202.2 (C-2-OCCH3), δ 28.3 (C-2-OCCH3), δ 192.1 (C-3), δ 98.4 (C-4), δ 179.8 (C-4a), δ 101.9 (C-6), δ 200.8 (C-6-OCCH3), δ 31.7 (C-6-OCCH3), δ 155.6 (C6a), δ 164.3 (C-7), δ 109.7 (C-8), δ 7.9 (C-8-CH3), δ 157.9 (C-9), δ 105.7 (C-9a), δ 59.5 (C-9b), δ 32.6 (C-9b-CH3). dibenzoylusnic acid (2)13C NMR (CDCl3, 100 MHz); δ 201.2 (C-1), δ 114.9 (C-2), δ 200.9 (C-2-OCCH3), δ 165.4 (C-3), δ 96.9 (C-4), δ 174.2 (C-4a), δ 102.1 (C-6), δ 200.9 (C-6-OCCH3), δ 31.7 (C-6-OCCH3), δ 133.9 (C-6a), δ 164.2 (C-7), δ 109.3 (C-8), δ 8.0 (C-8-CH3), δ 157.8 (C-9), δ 104.3 (C-9a), δ 61.0 (C-9b), δ 31.4 (C-9b-CH3), δ 135.0 (C-2-C), δ 110.2 (C-2-C-CH2), δ 164.7 (C-2-C-OCOPh), δ 163.3 (C-3-O-COPh)

Test microorganisms

The microbial strains studied were Escherichia coli ATCC 25922 (sensible), Escherichia coli ATCC 35218 (resistant), Staphylococcus aureus ATCC 25923 (sensible), Staphylococcus aureus ATCC 29213 (resistant), Pseudomona aeruginosa ATCC 27853 (sensible), Shigella flexneri ATCC 12022, Klebsiella pneumoniae ATCC 70063 (resistant), Bacillus subtilis ATCC 6636, Listeria monocytogenes ATCC 7644 and Salmonella typhi (clinical isolate) and were obtained from INLASA (Instituto Nacional de Laboratorios en Salud) of Bolivia.


Antibacterial assay

Agar ditch diffusion method

Fresh pure bacteria suspensions were obtained from overnight cultures in Muller Hinton Broth (BBLTM trademark of Becton, Dickinson and Company) cultivated at 37°C for 24 h. The bacterial suspensions were adjusted to an inoculum size 108 cells/mL for inoculation of the agar plates. After the medium (Difco Bacto Mueller Hinton Medium) was solidified in the plates, the test strain (0,2 mL) was inoculated into the media. Care was taken to ensure proper homogenization. The suspensions were spread on the medium. Four ditches were made in the plates with the help of a cup-borer (13 mm). For the in vitro studies compounds were dissolved in 100 µL of dimethyl sulfoxide (DMSO). One ditch was used with 100 uL of dimethyl sulfoxide (DMSO) as solvent control, and on other one ditch was used with 100 uL of gentamicin as antibacterial control. The test compounds, the DMSO and the gentamicin were introduced in each ditch and the plates were incubated at 37°C for 24 h. Microbial growth was determined by measuring the diameter of the zone of inhibition11,12. All tests were performed in duplicate 3.


ACKNOWLEDGMENT

]]> The authors express their gratitude to the Swedish International Development Cooperation Agency (SIDA) for developing this research. Chemistry Department of Lund University. Chemistry Investigations Institute-San Andrés University.


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        [ Links ] ]]> 2006 77 156-159 2005 2002 61 729-736 1985 33 2772-2777 1984 62 821-823 1999 86 559-570 1998 53 718-720 1971 29 209 2002 89 137-146 1999 70 113-115 1990 15 113-115 1999 7th 1527-1539