Patent Application
20080090900
Insert
Description of
Compound 1
Physical Properties
Compound 1 was yellow powder with molecular formula of C16H16O2, EI-MS (m/z)=208,161.9,136.9, 109; UV λmaxin CHCl3(nm)=248,193,363; IR vmax(cm−1)=13241,2922,1619.2; soluble in methanol, chloroform and ethyl acetate: insoluble in water and butanol.
Antibacterial Activity
The disc diffusion technique (Bauer A. W, Kirby W, M. M, Sherrisc J C, Turck, M Antibiotic susceptibility testing by a standardized single disc method. Am J Clin Path 1966; 45:493-6) was used to determine the antibacterial activity of Compound 1 against clinical and environmental isolates. Sterile discs containing 150 μg of compound were prepared from a 10 mg/mL stock solution of compound in dimethyl sulfoxide. The Mueller Hinton (MH Oxoid) agar plates were seeded with test organism the prepared discs were placed on to the centre of plates and incubated at 37° C. for 24 hours. Compound 1 was active against Salmonella typhi. Vibrio cholera, Proteus mirabilis, Hafnia alvei, Serratia marcesens, Bacillus subtilis, Staphylococcus aureus (MRSA &MSSA), Staphylococcus epidermids, Enterobacter faecalis but less active against anaerobic bacterium Clostridium sporogenes and Clostridium perfringens. It is not active against Candida albicans.
Minimal Inhibitory Concentration
The minimum inhibitory concentration (MIC) of Compound 1 was determined by the standard microdilution method described in the national committee for clinical laboratory standards (National Committee for Clinical Laboratory Standards, 1997. Method for dilution antimicrobial susceptibility test for bacteria, that grow aerobically, 4th ed., p, 1-29. National Committee for Clinical Laboratory Standards) using Muller Hinton broth medium (Oxoid) incubated at 37° C. for 24 h. The MIC of Compound 1 for Staphylococcus aureus, Staphylococcus epidermidis were 50-100 μg/ml for Enterobacter faecalis 100 μg/ml and for Bacillus subtilis, Bacillus steriothermophilus was 50-60 μg/ml, for Salmonella typhi was 75 μg/ml, for Vibrio cholera, 60 μg/ml, for Proteus mirabilis 75 μg/ml, for Hafnia alvei 100 μg/ml for Serratia marcescens 60 μg/ml. However Compound 1 was less active against anaerobic bacteria Clostridium sporogenes and Clostridium perfringenes and the MIC was 150-200 μg/ml respectively. This substance was slightly active against Streptococcus sp for which the MIC was 200 μg/ml. This substance was not found active against Candida albicans. Table 1 shows the in vitro activity of Compound 1 against environmental and clinical isolates.
staphylococcus aureus
staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Enterococcus faecium
Bacillus subtilis
Bacillus steriothermophilus
Bacillus cereus
Streptococcus group G
Clostridium perfringens
Clostridium sporogenes
Escherichia coli
Vibrio harveyi
Salmonella typhi
Proteus morgani
Proteus mirabilis
Serratia marcescens
Shewanella putrifacians
Hafnia alvei
Klebsiella aerogenes
Candida albicans
asensitivity test by disc diffusion technique
bWidth of inhibition zone: ++++ 18 to 30 mm; +++ 15 to 20; ++ 10 to 15; − no inhibition
cMIC minimal inhibitory concentration.
Heat Stability Test
Solution stock of Compound 1 in methanol was diluted with water, and then heated at 30, 40, 50,60,70,80and 90° C. using a thermostat and at 100° C. by boiling for 10 mm. After the treatment, the liquid in each tube was evaporated completely and Compound 1 was re-dissolved in methanol to give an appropriate concentration. Then antibacterial activity was evaluated in triplicate by the disk diffusion method. The antibacterial activity of Compound 1 was stable between 30 and 100° C. but its antibacterial activity significantly reduced after treatment at 120° C. Activity was completely lost at 200° C.,
Bacteriolytic Assay
The seed culture of Staphylococcus aureus in Luria broth (LB) were washed twice with sterile distilled water containing 0.9% sodium chloride and the absorbance was adjusted to 0.05 at 600 nm. The bacterial cell suspension was divided into aliquots of 5 ml each placed in sterile test tube and exposed to Compound 1 at various concentration, untreated bacterial suspension were used as the negative control. These test tubes were incubated at 30° C. by shaking at 120 rpm the absorbance was measured 0, 60, 120, 180, 240, 300, 360 min. Reduction of the absorbance of the Staphylococcus aureus cell suspension was observed in the presence of Compound 1, in contrast the absorbance was reduced early in the incubation period. These results indicated that Compound 1 lyse Staphylococcus aureus.
Time Kill Experiment
The time kill experiment was conducted by the method described by Aeschlimann and Rybak (Aeschlimann, J. R., and M. J. Rybak. 1998. Pharmacodynamic analysis of the activity of quinopristin-dalfopristin against vancomycin-resistant Enterococcus faecium with differing MBCs via time-kill-curve and post antibiotic effect methods. Antimicrob. Agents Chemother. 42:2188-2192). The experiment was conducted in 50 ml Erlenmeyer flasks containing 25 ml of fresh nutrient broth (Oxoid) inoculated with overnight culture of Staphylococcus aureus to give an initial density of 106 cells/ml. The inoculation was carried out immediately after addition of antibacterial compound or test antibiotic at final concentrations that consisted of the MIC, two and four times the MIC. The flasks were further incubated at 30° C. with stirring with a magnetic stirrer at 200 rpm. The viable cell count of Staphylococcus aureus was estimated at various incubation times by the plating method. To minimize the effect of antibiotic carryover the samples were centrifuged at 1600×g for 5 min then the antibiotic medium was replaced with fresh nutrient broth. The cells were re-suspended and plated on to nutrient agar plates, the plates were incubated at 37° C. for 24 h, and colonies were counted. The time kill experiment showed that Compound 1 was able to decrease the counts of Staphylococcus aureus (ATCC33591) at MIC 50 μg/ml. This substance decreased the number of viable bacterial cells alter 8 hr of exposure. A decrease in bacterial cell counts was more readily found when the strains were exposed to Compound 1 at higher concentration. At two times the MIC and four times the MIC the viable count decreased drastically after 2 hand 1 h where as at two and for times of MIC of vancomycin, ampicillin, tetracycline the bacterial cell count decreased gradually as compare to Compound 1 however Compound 1 decreased the bacterial viable counts more significantly than vancomycin, nisin, tetracycline and ampicillin at the same concentration and after the same length of exposure, indicating that the killing rate of Compound 1 for laboratory strain of Staphylococcus aureus (ATCC33591) was much higher then those of test antibiotics.
Spheroplasting Activities
The ability of Compound 1 and known cell wall active antibiotics to induce the formation of osmotically fragile spheroplast was tested in a growing culture of Staphylococcus aureus. Aliquots (100 μl) of an exponential phase culture(A600,0.05) grown in Luria broth supplemented with 0.3M concentration of sucrose were dispensed into test tubes containing different concentration of Compound 1 and 1 μl/ml ampicillin as positive control. The tubes were then incubated at 30° C. with shaking at 140 rpm. After 90 minutes, the cells were examined by phase contrast microscope for any morphological changes or for the presence of spheroplast. To confirm the osmotic fragility of the spheroplast the lytic effect of 2-3 μl of water added under the cover slip was tested. A log phase culture of Staphylococcus aureus growing in osmotically protected medium supplemented with 0.3M sucrose was treated with various concentration of Compound 1. Compound 1 did not induce formation of osmotically fragile spheroplasts similar to those observed with ampicillin. This observation suggests that the bacterial cell wall might not be the site of action of Compound 1.
Phase contrast Microscopy
An exponential phase culture (A600, 0.05) of Bacillus subtilis and Staphylococcus aureus grown in Luria broth was treated with 50 μl/ml of Compound 1 for Bacillus subtilis and 60 μl/ml for Staphylococcus aureus and slides were immediately made in 1% agarose using 4 μl of Compound 1 treated cultures, time lapse images were obtained under phase contrast microscope, particular care was taken to minimize the sample exposure to UV light, image grabbing were performed essentially as described by Edwards and Errrington. (Edwards, D. H., and Errington, J.(1997). The Bacillus subtilis DIvIVA protein targets to the division septum and control the site specificity of cell division. Mol Microbiol 24:905-915). Antibiotic treated cells of Staphylococcus aureus and Bacillus subtilis were observed under phase contrast microscope and time lapse images were captured where a uniform and consistent decrease in size of cells of Bacillus subtilis were observed immediately after treatment with Compound 1. When cells of Staphylococcus aureus were treated with this compound 1 complete lyses was observed immediately alter treatment.
Chemical Structure of Compound 1
The compound 1 trivially named as 7-(3-furyl)-3,7-dimethyl-7,8-dihydro-1-naphthalenol was isolated from ethyl acetate soluble fraction of Pseudomonas stutzrei. The molecular formula C16H16O2 of Compound 1 was deduced from HR-EIMS at m/z=240.2712. This compound has one aromatic, one cyclohexene and one furane ring. The 1H-NMR spectrum displayed four aromatic protons. The doublet at δ 8.27(d, J=1.2) was meta coupled to the proton at δ 8.34 (dd, J=1.2, 1.4) in 1H NMR spectrum and belongs to the protons (H-2) and (H-4) respectively. The cyclohexene ring showed two vicinal protons in 1H-NMR spectrum at δ 8.44(d, J=8.8, H-5) and at δ 8.42 (d, J=8.8, H-6) respectively. The furan moiety was confirmed by its downfield vicinal proton appearing in 1H-NMR spectra at δ 7.97(dd, J=1.2, 5.9, H-4′) and δ 8.84 (d, J=6.6, H-5′) respectively, while the methylene proton of cyclohexene ring appeared at δ 1.2 s each of these proton showed their singlets in 1H-NMR spectrum. The singlet for the protons of two methyls (C-1″) and (C-1′″) appeared in 1H-NMR spectra at δ 1.37and δ 2.26 respectively. The DEPT experiment displayed sixteen carbon signals out of which seven are methines, two methyles, and one methylene and six quaternary carbons, while the HMBC, HMQC and COSY data confirmed that the structure of Compound 1 is 7-(3-furyl)-3,7-dimethyl-7,8-dihydro-1-naphthalenol. Table 2 lists the NMR spectroscopic parameters of Compound 1.
1H-NMR in CDCl3)
1H NMR
13CNMR
During the lass 10 years, marine microorganisms have provided a large number of new natural products which have been derived from microorganisms. Since Burkholder (Burkholder. P. R P, Fisher, and F. Leitz. 1966. Production of pyrrole antibiotic by a marine bacterium. Appl. Microbiol. 14: 649-653) determined the first structure of a novel antibiotic produced by marine bacteria, there have been growing interest in marine bacteria as a potential source of natural products of pharmaceutical importance. This paper describes the fermentation, purification and elucidation of the chemical structure of antibacterial compound. Compound 1 produced by a marine bacterium active against clinical and environmental isolates. This bacterium was isolated from the gut of ribbonfish caught from Baluchistan coast of Pakistan. Pseudomonas stutzeri is a gram negative pigmented bacterium that produces antibacterial compound. The main product of this strain is an antibacterial compound designated as Compound 1, derived from ethyl acetate extract of the bacterial cells. In this study Compound 1 was determined to be 7-(3-furyl)-3,7-dimethyl-7,8-dihydro-1-naphthalenol and this substance is a new naturally occurring substance. This is also the first report of a marine Pseudomonas that produces this antibacterial compound. Antibacterial aid antifungal compounds by fluorescent Pseudomonas strains have been reported (Fenton, A. M, P. M. Stephens, J. Crowley, M. O'Callaghan arid F O'Gara (1992) Exploitation of gene(s) involved, in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to Pseudomonas strain 58:3873-3878) such as 2,4-Diacetylphloroglucinol(DAPG) produced by Pseudomonas has drawn attention in the medical area because of bacteriolytic activity of DAPG against multi drug resistant Staphylococcus aureus (Isnansetyo, A., and Y. Kamei. 2003. A bactericidal antibiotic produced by a new marine bacterium, Pseudomonas phenolica sp.nov.O-BC30 T, against methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother.47:480-488).
This compound 1 gave excellent activity against Gram-positive bacteria then Gram-negative bacteria and produced very large zones of inhibition and minimal inhibitory concentration (MIC) for Gram-positive bacteria was between 50-75 μg/ml whereas for Gram negative bacteria it was between 75-125 μg/ml. It has been reported that DAPG (2, 4 diacetyl phloroglucinol) produced by Pseudomonas sp AMSN has high in vitro anti-MRSA activity comparable with vancomycin (Isnansetyo, A., M, Horikwa and Y, Kamei. (2001) In vitro antimethicillin resistant Staphylococcus aureus activity of 2, 4, diacetylphloroglucinol produced by Pseudomonas sp. AMSN Isolated from a marine alga. Journal of Antimicrobial Chemotherapy 47:719-730).
Compound 1 demonstrates activity against Salmonella typhi, Vibrio cholera, Proteus mirabilis, Hafnia alvei, Serratia marcescens, Bacillus subtilis, Staphylococcus aureus (MRSA &MSSA), Staphylococcus epidermidis, Enterobacter faecalis but less active against anaerobic bacterium Clostridium sporogenes and Clostridium perfringens. It is not active against Candida albicans.
The time kill experiment indicated that Compound 1 is rapidly bactericidal against Staphylococcus aureus. The killing rate of Compound 1 exhibited concentration dependent bactericidal activity and its killing rate was faster with an increase in the concentration and slower rate was observed at lower concentration. Compound. 1 at 125 μg/ml was able to kill Staphylococcus aureus completely and at 100 μg/ml it completely killed Bacillus subtilis. Compound 1 also killed cells of Staphylococcus aureus in osmotically protected medium which suggest that cell wall might not be the site of action.
