Studies on pyrazine derivatives. XLIX. Synthesis and antibacterial activity of 6-methoxypyrazine-2-carboxylic acid hydrazide derivatives

Article abstract of DOI:10.3987/COM-06-10867

The new 6-methoxy-pyrazine derivatives have been synthesized. 6-Methoxy-pyrazine-2-carboxylic acid hydrazide was used as an initial material to obtain mono- and dithioester of hydrazinecarbodithioic acid (2 and 3). Compound (2) in reaction with ethanolamine gave triazole derivative (8) with β-hydoxyethyl substituent in 4-position and hydroxyl group in 6-position of pyrazine ring. Dithioester (3) in a reaction with morpholine cyclized to 1,3,4-oxadiazole (11). The same substrate with alkyldiamines gave the few following derivatives: 1,3-diazacycloalkane derivatives (9 and 10), S-methyl-1,3,4-oxadiazole derivative (12) and 1,2,4-triazoletetrahydropyrimidine (13). The compounds obtained were tested in vitro for their activity towards pathogenic strains of anaerobic and aerobic bacteria. Derivative (9) was the most active against both types of tested strains.{A figure is presented}. 6-Methoxypyrazine-2-carboxylic Acid Hydrazide Hydrazinecarbodithioic Acid Ester Acid Ester 1,3,4-Oxadiazolethiol 1,2,4-Triazolethione.

Full text of DOI:10.3987/COM-06-10867

HETEROCYCLES, Vol. 68, No. 12, 2006
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HETEROCYCLES, Vol. 68, No. 12, 2006, pp. 2615 - 2626. © The Japan Institute of Heterocyclic Chemistry
Received, 24th August, 2006, Accepted, 23rd October, 2006, Published online, 24th October, 2006. COM-06-10867
STUDIES ON PYRAZINE DERIVATIVES. XLIX.
SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF
6-METHOXYPYRAZINE-2-CARBOXYLIC ACID HYDRAZIDE
DERIVATIVES
Katarzyna Gobis,1* Henryk Foks,1 Aleksandra Żuralska,1 and Anna Kędzia2
1Department of Organic Chemistry, Medical University of Gdansk, 107 Gen.
Hallera Str., 80-416 Gdansk, Poland
2Department of Oral Microbiology, Medical University of Gdansk, 14
Smoluchowskiego Str., 80-214 Gdansk, Poland
Abstract – The new 6-methoxy-pyrazine derivatives have been synthesized.
6-Methoxy-pyrazine-2-carboxylic acid hydrazide was used as an initial material to
obtain mono- and dithioester of hydrazinecarbodithioic acid (2 and 3). Compound
(2) in reaction with ethanolamine gave triazole derivative (8) with β–hydoxyethyl
substituent in 4-position and hydroxyl group in 6-position of pyrazine ring.
Dithioester (3) in a reaction with morpholine cyclized to 1,3,4-oxadiazole (11). The
same substrate with alkyldiamines gave the few following derivatives:
1,3-diazacycloalkane derivatives (9 and 10), S-methyl-1,3,4-oxadiazole derivative
(12) and 1,2,4-triazoletetrahydropyrimidine (13). The compounds obtained were
tested in vitro for their activity towards pathogenic strains of anaerobic and aerobic
bacteria. Derivative (9) was the most active against both types of tested strains.
INTRODUCTION
Our previous research works1-3 as well as chemical literature4-11 have indicated that pyrazine derivatives
exhibit wide range of biological activity, inclusive of antibacterial action. Pyrazinamide and its less toxic
derivative morinamide found an application in tuberculosis chemotherapy.4 In group of sulfonamide drugs
“Kelfizian” distinguishes for effectiveness against infections caused by strepto- and staphylococci.5 Its
molecule possesses methoxy group in 2-position of pyrazine ring and sulfanilamide substituent in
*Address correspondence to Katarzyna Gobis, Department of Organic Chemistry, Medical University of Gdansk
Al. Gen. Hallera 107, 80-416 Gdańsk, Poland
E-mail: katarzyna.gobis@wp.pl

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HETEROCYCLES, Vol. 68, No. 12, 2006
3-position. Natural pyrazine products such as aspergillic and hydroxyaspergillic acids isolated from
Aspergillus flavus culture6 and emimycin from Streptomyces griseochromogenes also demonstrate strong
antibacterial activity.7 Among biologically active pyrazine derivatives many contain methoxy group closed
to pyrazine ring. For example N’-substituted amidrazones8 show high tuberculostatic activity and
sulfonamides obtained by Camerino and Palemidessi9-11 are widely applied as antibacterial drugs in
infections of urinary tract, gall tract, respiratory system inflammation and meningitis caused by
Gram-positive and Gram-negative bacteria.
RESULTS AND DISCUSSION
The intention of this work was to obtain 6-methoxypyrazine-2-carboxylic acid hydrazide and research its
chemical reactivity in reactions leading to gain new derivatives of expected antibacterial activity.
6-Chloropyrazine-2-carbonitrile was used as starting material for that synthesis12 It was transformed into
6-methoxypyrazine-2-carboximidic acid methyl ester in reaction with sodium methoxide in methanol.
Acidification of reaction mixture with hydrochloric acid led to methyl ester formation which was next
changed into hydrazide (1) (Scheme 1).
Cl
N
N
CN
MeO
N
C
O
NH NH2
MeO
N
N
C
OMe
MeO
N
N
C
O
OMe
H2NNH2
MeONa
MeOH
NH
H2O
N
1
Scheme 1
In our previous paper13 we have proved that pyrazine-2-carboxylic acid hydrazide is susceptible to carbon
disulfide addition and the product of that reaction can be useful in synthesis of nitrogen heterocyclic
systems. Within the framework of this work analogical reactions for hydrazide (1) were performed. The
first step concerned an obtainment of hydrazinecarbodithioic acid mono- and dimethylthioesters (2 and 3).
Monothioester (2) was obtained by acting with carbon disulfide on compound (1) in water-methanol milieu
and dimethyl sulfate in the presence of an excess of potassium hydroxide (Scheme 2). A trial of methylation
with methyl iodide led to formation of a mixture consisted of compounds (2 and 3), very difficult to
separate. Using of 2 moles of methyl iodide towards hydrazide (1) and carbon disulfide in alkaline solution
led to pure dithioester (3) formation with high yield.
Significant easiness of dithioester (3) formation prompted us to synthesize cyclic dithioesters (4 and 5) by
exchanging methyl iodide with appropriate dibromoalkanes. 1,2-Dibromoethane and 1,3-dibromopropane
were used in reaction resulting in obtainment of 6-methoxy-pyrazine-2-carboxylic acid

HETEROCYCLES, Vol. 68, No. 12, 2006
2617
[1,3]dithiolan-2-ylidene-hydrazide
(4)
and
6-methoxy-pyrazine-2-carboxylic
acid
[1,3]dithian-2-ylidene-hydrazide (5) appropriately.
S
MeO
N
N
C
O
NH
HO
NH
C
S
SMe
MeO
N
N
C
O
NH
N
X
S
H2NCH2CH2OH
N
2
2KOH
CS2
NH
Nr
X
4
5
4, 5
Me2SO4
H+
-
CH2
N
S
N
Br
CS2
X
8
CH2CH2OH
KOH
N
Br
SMe
MeO
N
N
C
O
NH NH2
C
MeO
N
N
C
O
NH
N
2KOH, 2CS2, 2MeI
SMe
3
2KOH
2CS2
EtOH
1
N
NH
CS2
MeO
N
N
DMF
S
O
N
N
OMe
MeO
N
C
S
NHNH
C
O
C
O
NHNH
N
N
7
MeO
N
N
6
SH
N
O
Scheme 2
A trial to obtain 5-(6-methoxypyrazine-2-yl)-[1,3,4]oxadiazole-2-thiol (7) from hydrazide (1) upon
treatment with carbon disulfide and heating of the adduct formed in that way in DMF was without success.
That reaction gave as a product 6-methoxypyrazine-2-carboxylic acid N’-(N’-6-methoxypyrazinoyl-
2-hydrazinocarbothioyl)–hydrazide (6). IR spectrum of derivative obtained was identical with spectrum of
the compound synthesized in another way.14 That fact confirmed the structure of 6. Desired
1,3,4-oxadiazole (7) was obtained during refluxing of hydrazide (1) with carbon disulfide and potassium
1
hydroxide in alcohol solution for 1.5 h. Double signals for 6-methoxypyrazine protons observed in H
NMR spectrum of that product evidenced that derivative (7) existed in two tautomeric forms of
1,3,4-oxadiazole-2-thiol and 1,3,4-oxadiazole-2-thione.
Previously N’-(pyrazine-carbonyl)-hydrazinecarbodithioic acid methyl ester has been used successfully to
obtain 4-substituted-1,2,4-triazole-thiones in reactions with various aminoalcoholes.15 Analogical trials
were performed for compound (3) without a success. Only with aminoethanol derivative (8)

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HETEROCYCLES, Vol. 68, No. 12, 2006
(6-[4-(2-hydroxy-ethyl)-5-mercapto-4H-[1,2,4]triazol-3-yl]pyrazin-2-ol) was synthesized with rather low
yield. IR and MS spectra of that product indicate a lack of methoxy group in 6-position.
Activity
of
dithioester
(3)
in
reaction
with
morpholine,
1,2-diaminoethane,
2,2-dimethylpropane-1,3-diamine was also investigated. Reaction with morpholine run with cyclization to
1,3,4-oxadiazole (11) with morpholine substituent in 2-position. The course of that reaction was analogical
as described for pyrazine-2-carboxylic acid (bis-methylsulfanyl-methylene)-hydrazide.13 Similar course
was also characteristic for the reactions with diamines carried out in ethanol or dioxane with
6-methoxy-pyrazine-2-carboxylic acid diazacycloalkylhydrazides (9 and 10) as the products.
HN
N
N
MeO
N
N
C
O
NH
N
X
MeO
N
N
N
O
HN
O
9, 10
11
H2N
H2N
HN
O
X
Nr
X
9
10
CMe2
-
SMe
SMe
C
MeO
N
N
C
O
NH
N
H2N
3
Me
Me
H2N
H2N
Me
Me
H2N
diglyme
N
N
N
N
MeO
N
NH
Me
MeO
N
N
SMe
O
N
N
Me
12
13
Scheme 3
Dithioester (3) refluxed in excess of 2,2-dimethyl-propane-1,3-diamine gave compound (13) of two
condensed rings: 1,2,4-triazole and tetrahydropyrimidine. An unexpected effect was gained by refluxing of
compound (3) with the same amine in diglyme. A product remained to be a derivative of
1,3,4-oxadiazole-3-thione (12), and diamine acted as a base catalyzing a cyclization reaction. Close effect
was observed during refluxing of compound (3) in pyridine.
The characteristics of all newly synthesized 6-methoxypyrazine derivatives are presented in Table 1.

HETEROCYCLES, Vol. 68, No. 12, 2006
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HETEROCYCLES, Vol. 68, No. 12, 2006
2621
Microbiology
The investigations of aerobic and anaerobic bacteria susceptibility to the synthesized 6-methoxypyrazine
derivatives are summarized in Table 2A and 2B. The results have been compared with that obtained while
testing the susceptibility of the same bacteria to metronidazole (for anaerobes) and amikacin (for aerobes).
Table 2 Antibacterial activity of tested compounds
A
MIC [µg/mL]
Anaerobic bacteria
Metronidazole*
1
4
5
8
9
11
12
13
Gram-positive:
25
Peptococcus niger
Peptostreptococcus magnus
Peptostreptococcus micros
Actinomyces israeli
Actinomyces naeslundii
Propionibacterium
granulosum
≤6.2
≤6.2
≤6.2
≤6.2
≤6.2
≥200
25
50
100 12.5
25
50
25
25
100 100
50 ≥200
≥200 50 ≥200 100
50 50 100 25
≥200 25 ≥200 12.5
100
50
50
50
50
50
100
≥200 ≥200 ≥200 ≥200 ≥200 ≥200 ≥200 ≥200
≥200 ≥200 100 50 ≥200 100 ≥200 ≤6.2
Gram-negative:
Prevotella bivia
Prevotella buccalis
Prevotella intermedia
Prevotella loescheii
Porhyromonas
≤6.2
≤6.2
≤6.2
≤6.2
≤6.2
100 100 ≥200 100
≥200 ≥200 100 ≥200 ≥200 ≥200 ≥200 100
≥200 ≥200 ≥200 ≥200 ≥200 ≥200 ≥200 ≥200
25
25
100 ≥200
100 ≥200 50
50
25
12.5 ≥200 100
100 ≥200 ≥200 ≥200 ≥200 25 ≥200 ≥200
asaccharolytica
Fusobacterium nucleatum
Fusobacterium
≤6.2
≤6.2
12.5 100 ≥200 ≥200 ≥200 ≥200 100
50 ≥200 100 50 ≥200 ≥200 ≥200 25
50
necrophorum
Bacteroides forsythus
Bacteroides uedyticus
B
≤6.2
≤6.2
≥200 ≥200 ≥200 ≥200 ≥200 100 ≥200 ≥200
100 ≥200 50 ≥200 ≥200 ≥200 ≥200 ≥200
MIC [µg/ml]
Aerobic bacteria
Amikacin**
1
4
9
10
12
Gram-positive:
Staphylococcus aureus
Corynebacterium spp.
Gram-negative:
≤6.2
50
≥200
25
≥200
100
≥200
≥200
≥200
25
100
≥200
Klebsiella pneumoniae
Acinetobacter baumannii
Escherichia coli
Pseudomonas aeruginosa
Pseudomonas stutzeri
*Metronidazole (Sigma)
≤6.2
≤6.2
≤6.2
≤6.2
12.5
50
≥200
25
≥200
≥200
50
50
≥200
≥200
≥200
25
≥200
≥200
≥200
25
25
100
≥200
≥200
≥200
≥200
≥200
≥200
≥200
≥200
**Amikacin sulfate salt (Sigma)
Low metronidazole concentrations in range from ≤ 6.2 to 12.5 µg/mL inhibited the growth of
Gram-negative bacteria. Gram-positive rods were the most resistant to metronidazole (MIC > 200 µg/mL).
These results were coincided with those obtained by other authors.16, 17 Activity against anaerobic bacteria

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HETEROCYCLES, Vol. 68, No. 12, 2006
exhibited 8 of 11 tested 6-methoxypirazine derivatives: (1, 4, 5, 8, 9, 11-13). The anaerobes were the most
susceptible at concentrations in range from ≤ 6.2 to 25 µg/mL to derivative (9) (20 % were susceptible) and
to compounds (8 and 11) (16% of susceptible strains). Moreover from 4 to 12 % of anaerobic strains were
susceptible at concentrations in range from ≤ 6.2 to 25 µg/mL to derivatives (4 and 13). The aerobic
bacteria were generally not susceptible to compounds (2, 3 and 10) in mentioned range of concentrations.
Tested 6-methoxypyrazine derivatives at concentrations in range from ≤ 6.2 to 100 µg/mL inhibited growth
of 24 to 48 % of anaerobic strains. All derivatives active towards anaerobic bacteria were more effective to
Gram-positive strains.
Aerobic bacteria were less susceptible to tested compounds than anaerobes. The growth of aerobic bacteria
was inhibited at concentrations in range from 25 to 50 µg/mL. Only 1-2 of tested aerobic strains were
susceptible to derivatives (1, 9, 10 and 12) at concentration 25 µg/mL. Also individual strains of aerobic
bacteria exhibited susceptibility to compound (4) at 50 µg/mL. Other compounds did not inhibit the growth
of aerobic bacteria in the range of tested concentration (≤ 6.2-200 µg/mL). The standard strains of both
types of bacteria exhibited rather high resistance towards tested compounds (MIC ≥ 200 µg/mL). In the
case of anaerobic Bacteroides ovatus ATCC 8483 compounds (11) (MIC 50 µg/mL), (4) (MIC 100 µg/mL),
(5) (MIC 100 µg/mL), (8) (MIC 100 µg/mL) and (9) (MIC 100 µg/mL) were active. Derivative (5) induced
the growth inhibition of Bacteroides vulgatus ATCC 8482 at concentration of 100 µg/mL. Compound (1)
inhibited the growth of Peptostreptococcus anaerobius ATCC 27337 at 100 µg/mL. The same activity
level was exhibited by derivative (13) against Propionibacterium acnes ATCC 11827. Among standard
aerobic strains only Pseudomonas aeruginosa ATCC 27853 was susceptible to compound (9) at
concentration 100 µg/mL.
In summery, derivative (9) was the most active against both types of tested strains. It inhibited 20 % of
anaerobic and 4 % of aerobic strains at concentration 25 µg/mL. That fact makes it an interesting lead
structure for further syntheses. Although only 2 compounds (2 and 3) seemed to be inactive, the MIC values
obtained for the other derivatives were not satisfactory. None of the tested compounds performed better
than metronidazole against anaerobes and amikacin against aerobes.
EXPERIMENTAL
All materials and solvents were of analytical reagent grade. Thin-layer chromatography was performed on
Merck silica gel 60F254 plates and visualized with UV. The results of elemental analyses (%C, H, N) for all
1
the compounds obtained were in good agreement with the data calculated. H NMR spectra in CDCl3 or
DMSO-d6 were recorded on Varian Gemini (200 MHz) instrument. IR Spectra were determined as KBr
pellets of the solids on a Satellite FT-IR spectrophotometer. Mass spectra (MS) for compound (4 and 8)

HETEROCYCLES, Vol. 68, No. 12, 2006
2623
were taken on Finingan MAT 95 (70 eV). Melting points were determined on BOETIUS apparatus and
were uncorrected.
Synthesis of 6-methoxypyrazine-2-carboxylic acid hydrazide (1): Metallic sodium (2.8 g, 0.12 mol) and
dry methanol (60 mL) were placed into round-bottomed flask (250 mL) furnished with condenser and
dropping funnel. After vigorous reaction 12 mL (0.12 mol) of 6-chloropyrazine-2-carbonitrile was added.11
After 15 min, mixture was treated with 40 mL of 20 % hydrochloric acid. Next 30 mL of water was added
until clear solution was obtained. The solution was stirred at room temperature for about 0.5 h. Then the
mixture was poured into 100 mL of ice-water mixture. The solid of 6-methoxypyrazine-2-carboxylic acid
methyl ester was filtered and washed with ice-cold water. Dry ester (10.4 g, 0.06 mol) was dissolved in 30
mL of hot EtOH and 10 mL of 99 % hydrazine hydrate was added, and mixture was refluxed for 1h. The
precipitate was filtered after cooling, washed with ice-cold water and recrystallized. Melting point in
agreement with reference.12
Synthesis of N’-(6-methoxypyrazine-2-carbonyl)hydrazinecarbodithioic acid methyl ester (2):
Compound (1) (5 g, 30 mmol) was treated with a solution of KOH (3.4 g, 60 mmol) in water (20 mL) and
EtOH (30 mL). Next 1.8 mL (30 mmol) of CS2 was added. After 0.5 h, 1.4 mL (15 mmol) of Me2SO4 was
added dropwise. Reaction mixture was stirred at room temperature for 1 h. After that time it was poured in
100 mL of cold water and acidified with 4 mL of AcOH. Precipitate was filtered and recrystalized.
Synthesis of 6-methoxypyrazine-2-carboxylic acid (bis-methylsulfanylmethylene)hydrazide (3):
1.68 g (10 mmol) of compound (1) was added to a stirred solution of 1.4 g (25 mmol) of KOH in 25 mL of
water and 15 mL of EtOH. Next 1.5 mL (25 mmol) of CS2 was added and after 15 min 1.6 mL (25 mmol) of
MeI. The reaction mixture was stirred for 0.5 h, poured on 100 g of ice. Yellow precipitate was filtered and
recrystalized.
Synthesis of 6-methoxypyrazine-2-carboxylic acid [1,3]dithiolan-2-ylidenehydrazide (4): Compound
(1) (1.68 g, 10 mmol) was dissolved in a solution of 1.2 g (30 mmol) of KOH in 3 mL of water and 10 mL
of EtOH. Next 0.6 ml (10 mmol) of CS2 was added and after 15 min 0.87 mL (10 mmol) of
1,2-dibromoethane. After 1 h stirring the precipitate (0.16 g) was filtered and filtrate was concentrated and
diluted with 15 mL of water. Next solution was extracted with CHCl3, dried with MgSO4 and evaporated
resulting in another 0.6 g of the product. MS: m/z = 270.1 (100 M+), 210.1 (41.4), 137.1 (22.3), 109.1 (79.1),
66.1 (26.1), 60.0 (44.1), 59.0 (37.2), 45.0 (54.1), 44.0 (39.8), 43.1 (27.3).
Synthesis of 6-methoxypyrazine-2-carboxylic acid [1,3]dithian-2-ylidenehydrazide (5):Compound
(1) (0.84 g, 5 mmol) was dissolved in mixture of EtOH (15 mL) and Et3N (2mL). Next 0.3 mL (5 mmol) of

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HETEROCYCLES, Vol. 68, No. 12, 2006
CS2 and 0.5 mL (5 mmol) of 1,3-dibromopropane were added. Reaction mixture was stirred for 12 h and
then the precipitate was filtered and recrystalized.
Synthesis
of
6-methoxypyrazine-2-carboxylic
acid
N’-(N’-6-methoxypyrazinoyl-2-
hydrazinocarbothioyl)hydrazide (6): 1.68 g (10 mmol) of compound (1) was refluxed in 5 mL of
N,N-dimethylformamide and 2 mL of CS2 for 1 h. After that time the mixture was cooled and diluted with
30 mL of water. The precipitate was filtered and recrystalized.
Synthesis of 5-(6-methoxypyrazin-2-yl)[1,3,4]oxadiazole-2-thiol (7): 0.84 g (5 mmol) of compound (1)
was dissolved in the solution of KOH (0.8 g, 10 mmol), water (2 mL) and EtOH (20 ml). Next 1 mL (10
mmol) of CS2. The mixture was refluxed for 1.5 h, evaporated, diluted with 10 mL of water and acidified
with HCl. The precipitate was filtered and recrystalized.
Synthesis of 6-[4-(2-hydroxyethyl)-5-mercapto-4H-[1,2,4]triazol-3-yl]pyrazin-2-ol (8): Compound
(2) (2.4 g, 10 mmol) was refluxed in 3 mL of ethanolamine. After cooling 10 mL of water was added and
mixture was acidified with 3.5 mL of AcOH. The whole was cooling in freezer for 2 weeks and after that
time the crystals of product were filtered. MS: m/z = 239.1 (9.7 M+), 197.1 (9.2), 196.1 (100), 195.1 (10.9).
Synthesis of 6-methoxypyrazine-2-carboxylic acid imidazolidin-2-ylidenehydrazide (9): 0.84 g (3
mmol) of 3 was refluxed in the mixture of 10 mL of dioxane and 1 mL of 1,2-diaminoethane. After
evaporation of solvent the product was purified by crystallization.
Synthesis of 6-methoxy-pyrazine-2-carboxylic acid (tetrahydro-pyrymidin-2-ylidene)hydrazide
(10): 0.84 g (3 mmol) of 3 was refluxed in the mixture of 10 mL of EtOH and 1 mL of
2,2-dimethylpropane-1,3-diamine for 8 h. After cooling product was filtered and recrystalized.
Synthesis of 4-[5-(6-methoxypyrazin-2-yl)-[1,3,4]oxadiazol-2-yl]morpholine (11): Compound (3) (0.97
g, 5 mmol) was refluxed in 3 mL of morpholine for 2 h. The mixture was cooled, diluted with 20 mL of
water and extracted with CHCl3. The collected CHCl3 fractions were dried with MgSO4. After evaporation
of the solvent residual oil crystallized.
Synthesis of 2-methoxy-6-(5-methylsulfanyl-[1,3,4]oxadiazol-2-yl)pyrazine (12): 0.84 g (3 mmol) of
3 was refluxed in the mixture of 3 mL of diglyme (diethylene glycol dimethyl ether) and 1 mL of
2,2-dimethylpropane-1,3-diamine for 8 h. After cooling 5 mL of water was added, product was filtered and
recrystalized.
Synthesis
of
3-(6-methoxypyrazin-2-yl)-6,6-dimethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]-
pyrimidine (13): 0.84 g (3 mmol) of 3 was refluxed with 4 mL of 2,2-dimethylpropane-1,3-diamine for

HETEROCYCLES, Vol. 68, No. 12, 2006
2625
1h. After 10 min semi-product precipitated and next dissolved gradually. After cooling product was filtered
and recrystalized.
Antibacterial activity
The investigations included 25 strains of anaerobic bacteria and 25 strains of aerobic bacteria isolated from
the oral cavity, respiratory system and abdominal cavity as well as 12 standard strains. The anaerobes
belonged to the following genera: Peptococcus (1 strain), Peptostreptococcus (4 strains), Actinomyces (2),
Propionibacterium (2), Prevotella (6), Porphyromonas (2), Fusobacterium (3), Bacteroides (5), and
standard strains: Bacteroides fragilis ATCC 25285, Bacteroides vulgatus ATCC 8482, Bacteroides ovatus
ATCC 8483, Fusobacterium nucleatum ATCC 25586, Peptostreptococcus anaerobius ATCC 27337 and
Propionibacterium acnes ATCC 11827. There were also the following aerobes: Staphylococcus aureus (4
strains), Corynebacterium spp. (2), Klebsiella pneumoniae (3), Acinetobacter baumannii (2), Escherichia
coli (6), Pseudomonas aeruginosa (6), Pseudomonas stutzeri (2) and 6 standard strains: Staphylococcus
aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Klebsiella pneumoniae ATCC 13883,
Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa
ATCC 27853.
The susceptibility of the anaerobic bacteria was determined by means of the plate dilution technique in
Brucella agar, supplemented with 5 % sheep’s blood.18, 19 For aerobic bacteria experiments agar dilution
technique with Miller-Hinton agar was used. The derivatives were dissolved in 1ml of DMSO immediately
before the experiment. Sterile distilled water was used for further dilutions. The following concentrations
of derivatives were used: 200, 100, 50, 25, 12.5 and 6.2 µg/mL. The inoculum containing 106 CFU/spot
applied to the agar plates with Steers replicator. For aerobes the inoculated agar plates and agar plates
without derivatives were incubated for 24 h at 37 °C. For anaerobes agar plates were incubated in anaerobic
jars for 48 h at 37 °C in 10 % CO2, 10 % H2 and 80 % N2 with palladium catalyst and indicator for
anaerobiosis. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the
derivative that inhibited growth of the anaerobes.
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