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170 O. Kayser and H. Kolodziej • Structure-Antibacterial Activity of Coumarins
ing to Kayser and Kolodziej (1995), while the re 7-hydroxy-5,6-dimethoxycoumarin and 6,8-dihy-
maining coumarins were available as reference droxy-5,7-dimethoxy-coumarin, respectively, in
samples in the research group of H. K. The iden concentrations of 0.9-2.2 (.im . The initial inoculum
tity of the compounds was proven by spectro was approximately 2 x 105 colony forming units/
scopic techniques. ml. Samples were taken after 0, 2, 4, 8, and 24 h
of incubation, followed by serial 10-fold dilutions
M icroorgan isms and plating on M ueller H inton agar. After 24 h of
incubation at 37 °C, the growth of the bacterium
The above m entioned compounds were tested was determ ined by turbidimetric analysis.
against a panel of microorganisms including, the
Gram-negative bacteria, Escherichia coli ATCC
25922, Klebsiella pneum oniae V 6089, Proteus mir- Results and Discussion
abilis ATCC 14153, Pseudom onas aeruginosa Although coumarins are known to possess anti
ATCC 27853, Haemophilus influenzae ATCC bacterial activity, the effect of oxygenation pat
33379, and the Gram-positive bacteria Staphylo terns on the potency has not yet been investigated
coccus aureus ATCC 25923, beta-hemolytic Strep systematically. In the present study, a series of 14
tococcus 1451, DSMZ, Braunschweig, and Strepto simple coumarins (for structures see Table I) was
coccus pneum oniae (strain 78), Pasteur Institute, tested against a panel of microorganisms, includ
Paris. ing three Gram-positive ( Staphylococcus aureus,
beta-hemolytic Streptococcus, and Streptococcus
Evaluation o f antim icrobial activity pneum oniae ) and five Gram-negative bacteria
For the microdilution assay, standard Mueller ( Escherichia coli , Klebsiella pneumoniae, Pseu
H inton medium was used for most of the microor dom onas aeruginosa, Proteus mirabilis, and
ganisms, with the exception of H. influenzae , S. Haemophilus influenzae ). Bacterial susceptibility
pneum oniae and beta-hemolytic Streptococcus to coumarins was evaluated by determining the
1451 (M ueller Hinton containing 50 ml lysed cit minimal growth inhibitory concentration using the
rate blood/1) (Gersten, 1993). Ethanol (70%) that microdilution broth method. In Table II, the mini
did not affect the growth of any of the microorga mum inhibitory concentrations (MIC) of the test
nisms in the final sample concentrations, was used compounds against the listed bacteria are dis
as solvent for the test compounds. A fter 24 h, played. All samples exhibited antibacterial activity
respectively, 48 h, of incubation at 37 °C and 5% with MICs ranging from 0.9 to >12.4 |_im. The re
C 0 2 (Table II), the antimicrobial effects of sam sults indicated that each com pound showed more
ples were quantitated. Minimum inhibitory con or less pronounced antibacterial potencies, affect
centrations (MICs) of the samples were deter ing both Gram-positive and Gram-negative patho
mined for susceptible microorganisms by a gens. However, the latter group of bacteria ap
standard twofold microdilution broth technique, peared to be more sensitive for most of the
using penicillin G as reference agent (Vanden Ber- coumarins tested, irrespective of the oxygenation
ghe et al., 1991). Inocula were prepared in the pattern. Among the active or potentially active
same medium by diluting microbial suspensions in compounds, the highly oxygenated 7-hydroxy-5,6-
Mueller Hinton broth ( vide infra). Inhibition of dimethoxycoumarin (umckalin) and 6,8-dihy-
growth was judged by comparison with a control droxy-5,7-dimethoxycoumarin represented the
culture prepared without any test sample. MIC most potent candidates with MICs of 0.9-2.1 ^m.
values were determ ined as the lowest concentra This first observation suggested that the antibacte
tion of the samples completely inhibiting macro rial activity of coumarins may be correlated to the
scopic growth of microorganisms. num ber of oxygen substituents, with highly func-
tionalized representatives as the most potent in
hibitory agents. However, the trioxygenated cou-
Bactericidal kinetic assay
marin, 5,6,7-trimethoxycoumarin, was found to be
The bactericidal kinetic assay for E. coli was significantly less effective than the highly oxy
perform ed in Mueller Hinton medium containing genated coumarins 7-hydroxy-5,6-dimethoxy-
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O. Kayser and H. Kolodziej • Structure-Antibacterial Activity of Coumarins 171
Coumarin H H H H
7-Hydroxycoumarin umbelliferone H H OH H
7-Methoxycoumarin herniarin H H och 3 H
8-Hydroxycoumarin H H H OH
6,7-Dihydroxycoumarin esculetin H OH OH H
6-Hydroxy-7-methoxycoumarin scopoletin H OH och 3 H
5,7-Dihydroxycoumarin OH H OH H
6,7-Dimethoxycoumarin H och 3 och 3 H
7-Hydroxy-5-methoxycoumarin och 3 H OH H
7,8-Dihydroxycoumarin H H OH OH
8-Hydroxy-6,7-dimethoxycoumarin tomentin H och 3 och 3 OH
7-Hydroxy-5,6-dimethoxycoumarin umckalin och 3 och 3 OH H
5,6,7-Trimethoxycoumarin och 3 och 3 och 3 H
6,8-Dihydroxy-5,7-dimethoxycoumarin och 3 OH och 3 OH
n
T-2
U73
O
,0
)
/
\
M J f
R 2 6 15
R.
Table II. Antibacterial activity o f simple coumarins using the microdilution method (MIC values o f duplicates in
(j,m; (ig/ml in parentheses).
Compound E. coli K. pneum oniae St. aureus P. aeruginosae Prot. mirabilis ß-hem. Strept. St. pneum oniae H. influenzae
24 h 24 h 24 h 24 h 24 h 48 h 48 h 48 h
Coumarin 2.7 (400) 2.7 (400) 3.4 (500) 3.4 (500) 2.7 (400) 1.7 (250) 1.7 (250) 1.7 (250)
Monosubstituted Derivatives
7-Hydroxycoumarin 6 .2 (1 0 0 0 ) >12.4 (> 2 0 0 0 ) >12.4 (> 2 0 0 0 ) 3.1 (500) >12.4 (> 2 0 0 0 ) >12.4 (> 2 0 0 0 ) >12.4 (> 2 0 0 0 ) >12.4 (> 2 0 0 0 )
(umbelliferon)
7-M ethoxycoumarin 1.4 (250) 1.9 (330) 1.9 (330) 1.9 (330) 1.9 (330) 5.7 (1 0 0 0 ) 5.7 (1 0 0 0 ) 5.7 (1 0 0 0 )
(herniarin)
8 -Hydroxycoumarin 2.3 (375) 2.3 (375) 4.6 (750) 2.3 (375) 4.6 (750) 4.6 (750) 6 .2 (1 0 0 0 ) 4.6 (750)
Disubstituted Derivatives
6,7-Dihydroxycoumarin 2.3 (400) 2 .8 (500) 2 .8 (500) 2.3 (400) 2 .8 (500) 1 1 .2 (2 0 0 0 ) 1 1 .2 (2 0 0 0 ) 1 1 .2 (2 0 0 0 )
(esculetin)
6-Hydroxy-7-methoxycoumarin 2 . 6 (500) 2 .6 (500) 2 .6 (500) 2 .1 (400) 2 .1 (400) 5.2 (1 0 0 0 ) 5.2 (1 0 0 0 ) 5.2 (1 0 0 0 )
(scopoletin)
5,7-Dihydroxycoumarin 1.4 (250) 2 .8 (500) 2 .8 (500 2 .8 (500) 2 .8 (500) 5.6 (1 0 0 0 ) 2 .8 (500) 5.6 (1 0 0 0 )
6,7-Dimethoxycoumarin 4.8 ( 1 0 0 0 ) 4.8 (1 0 0 0 ) 2.4 (500) 4.8 (1 0 0 0 ) 2.4 (500) 2.4 (500) 2.4 (500) 2.4 (500)
7-Hydroxy-5-methoxycoumarin 1.3 (250) 5.2 (1 0 0 0 ) 5.2 (1 0 0 0 ) 5.2 (1 0 0 0 ) 5.2 ( 1 0 0 0 ) 5.2 (1 0 0 0 ) 5.2 (1 0 0 0 ) 5.2 (1 0 0 0 )
7,8-Dihydroxycoumarin >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 ) >5.6 (> 1 0 0 0 )
Trisubstituted Derivatives
8-Hydroxy-6,7-dimethoxy- 1 .0 (2 2 0 ) 2 .2 (500) 2 .2 (500) 2 .2 (500) 4.5 (1 0 0 0 ) 2 .2 (500) 2 .2 (500) 2 .2 (500)
coumarin (tom entin)
7-Hydroxy-5,6-dimethoxy- 0.9 (2 0 0 ) 0.9 (2 0 0 ) 0.9 (2 0 0 ) 0.9 (2 0 0 ) 0.9 (2 0 0 ) 2 .2 (500) 2 .2 (500) 2 .2 (500)
coumarin (umckalin)
5,6,7-Trimethoxycoumarin 4.2 (1 0 0 0 ) 4.2 (1 0 0 0 ) 4.2 (1 0 0 0 ) 4.2 (1 0 0 0 ) 4.2 (1 0 0 0 ) 4.2 (1 0 0 0 ) 2 .1 (500) 2 .1 (500)
Tetrasubstituted Derivatives
6,8-Dihydroxy-5,7-dimethoxy- 0.9 (2 2 0 ) 1 .1 (250) 0.9 (2 2 0 ) 0.9 (2 2 0 ) 0.9 (2 2 0 ) 2 .1 (500) 2 .1 (500) 2 .1 (500)-
coumarin
Penicillin G 0.4 (125) 0 .2 (62) 0 .2 (62) 0.4 (125) 0 .2 (62) 0 .1 (32) 0.05 (16) 0.05 (16)
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172 O. Kayser and H. Kolodziej • Structure-Antibacterial Activity of Coumarins
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O. Kayser and H. Kolodziej • Structure-Antibacterial Activity of Coumarins 173
rial activity showed a tendency to increase against methoxy groups such as the 6,7- or 5,7-dimethoxy
those microorganisms which require special arrangement and (ii) at least one additional pheno
growth factors. Also, the presence of a methoxy lic group located either at C-6 , C-7 or C-8 . It should
function in position 5 may enhance inhibitory ac be noted that the pathogens, which require special
tivity provided the remaining pattern of hydroxyl- growth factors, are generally less susceptible when
ation is favourable (7-hydroxy-5,6-dimethoxy- com pared to standard microorganisms (Table I).
coumarin, 6,8-dihydroxy-5,7-dimethoxycoumarin Although the difference in susceptibility can not
and 5,6,7-trimethoxycoumarin vs. 7-hydroxy-5-me- simply be correlated to various cell membrane and
thoxycoumarin). This finding suggests that the cell wall characteristics, Gram-positive and distinc
presence of two methoxy groups is a major con tively encapsulated microorganisms proved to be
tributing factor towards antibacterial activity conspicuously less susceptible than the remaining
against this group of bacteria. The above observa pathogens. Here, the more lipophilic derivatives
tion is in accordance with recent reports in that show slightly greater inhibitory activity. With MICs
increased lipophilicity of compounds may be asso ranging from 0.9-2.2 [.i m , the potentially active
ciated with a greater ease of penetration into highly oxygenated simple coumarins examined
Gram-positive bacteria, but other factors such as were generally only moderately active, when com
shapes and bulkiness have to be considered as well pared with the MICs (0.05-0.4 ja m ) of the medici
(Rauckman et al., 1989). nally used antibiotic, penicillin G.
The relatively high potency of the tri- and tetra- The bactericidal kinetic assay, using E. coli and
oxygenated members against the total spectrum of the potentially active candidates, 7-hydroxy-5,6-di-
test bacteria may be rationalized by a combination methoxycoumarin and 6,8-dihydroxy-5,7-dimeth-
of structural characteristics discussed above and, oxycoumarin, dem onstrated the antibacterial ac
hence, is displayed only by derivatives with (i) two tivity of the coumarins tested to be bacteriostatic.
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174 O. Kayser and H. Kolodziej • Structure-Antibacterial Activity of Coumarins
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