Synthesis and antibacterial activity of new 1,4-disubstituted piperazine derivatives

Article abstract of DOI:10.1080/10426500701640058

In search of antibacterial agents new 4-phenylpiperazine derivatives were obtained. Their structures include some heterocyclic (thiazole, oxadiazole, piperazine, and benzimidazole) systems, as well as functional (thioamide and amidoxime) groups, frequentl

Full text of DOI:10.1080/10426500701640058

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Synthesis and Antibacterial
Activity of New 1,4-
Disubstituted Piperazine
Derivatives
Danuta Pancechowska-Ksepko a , Katarzyna
Spalińska a , Henryk Foks a , Anna Kędzia b , Maria
Wierzbowska b , Ewa Kwapisz b , Mieczysław
Janowiec c , Zofia Zwolska c & Ewa Augustynowicz-
c
Kopeć
a Department of Organic Chemistry, Medical
University of Gdańsk, Gdańsk, Poland
b Department of Oral Microbiology, Medical
University of Gdańsk, 14 Smoluchowskiego, 80-214,
Gdańsk, Poland
c Department of Microbiology, Institute of
Tuberculosis and Pulmonary Diseases, 26 Pocka,
01-138, Warszawa, Poland
Published online: 09 May 2008.
To cite this article: Danuta Pancechowska-Ksepko , Katarzyna Spalińska , Henryk
Foks , Anna Kędzia , Maria Wierzbowska , Ewa Kwapisz , Mieczysław Janowiec , Zofia
Zwolska & Ewa Augustynowicz-Kopeć (2008): Synthesis and Antibacterial Activity of
New 1,4-Disubstituted Piperazine Derivatives, Phosphorus, Sulfur, and Silicon and the
Related Elements, 183:5, 1252-1263
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Phosphorus, Sulfur, and Silicon, 183:1252–1263, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701640058
Synthesis and Antibacterial Activity of New
1,4-Disubstituted Piperazine Derivatives
1
1
´
Danuta Pancechowska-Ksepko, Katarzyna Spalinska,
Henryk Foks,1 Anna Ke¸dzia,2 Maria Wierzbowska,2
Ewa Kwapisz,2 Mieczyslaw Janowiec,3 Zofia Zwolska,3
ꢀ
3
´
and Ewa Augustynowicz-Kopec
1Department of Organic Chemistry, Medical University of Gdan´sk,
Gdan´sk, Poland
2Department of Oral Microbiology, Medical University of Gdan´sk,
14 Smoluchowskiego, 80-214 Gdan´sk, Poland
3Department of Microbiology, Institute of Tuberculosis and Pulmonary
Diseases, 26 Pocka, 01-138 Warszawa, Poland
In search of antibacterial agents new 4-phenylpiperazine derivatives were obtained.
Their structures include some heterocyclic (thiazole, oxadiazole, piperazine, and
benzimidazole) systems, as well as functional (thioamide and amidoxime) groups,
frequently present in biologically active compounds. Thioamides 2b, e and ami-
doximes 3b–e were examined towards 25 strains of aerobic and 25 strains of anaero-
bic bacteria. The derivatives 2e and 3b showed the highest antimicrobial activity to-
wards anaerobes, both Gram-negative and Gram-positive. The compounds studied
were inactive towards the aerobes, however. Their tuberculostatic activity towards
the standard H37Rv strain and two strains isolated from tuberculotic patients was
studied in vitro. The obtained MIC values were within the limits 12.5–100 µg/mL.
Keywords 1,2,4-oxadiazole; 4-Phenylpiperazine derivatives; Antibacterial activity;
Benzimidazoles; Thioamides; Thiazole
INTRODUCTION
Piperazine derivatives appear to be widely applicable in therapeutics.1
The piperazine ring is present in molecules of antibiotics such as
ciprofloxacine, pefloxacine, and rifampycine,2 of antimycotic drugs like
ketokonazole,1,2−4 of circulatory system drugs,5,6 and of antiparasit-
ical agents.1 Novel piperazine containing cephalosporines, active to-
wards both Gram-positive and Gram-negative bacteria, were obtained
Received 16 August 2007; accepted 20 August 2007.
Address correspondence to H. Foks, Department of Organic Chemistry, Medi-
cal University of Gdan´sk, Al. Gen. J. Hallera 107, 80-416 Gdan´sk, Poland. E-mail:
hfoks@amg.gda.pl
1252

1,4-Disubstituted Piperazine Derivatives
1253
by Heyningen and Brown.7 To the new phenylpiperazine derivatives re-
ported here, thiazole and benzimidazole heterocyclic systems, as well as
thioamide and amidoxime functional groups8,9 were introduced. These
can be found in such well-known medicines, as thiabendazole, ethion-
amide, and prothionamide.10
RESULTS AND DISCUSSION
The preparation of the benzonitriles 1a–e with a phenylpiperazine
group in para position was the initial step of the syntheses. These
compounds were formed upon action of p-fluoro-benzonitrile on the
corresponding piperazines in dimethylsulphoxide-potassium carbonate
medium. The thioamides 2b–e were then obtained by the reaction of
alcoholic solutions of the nitriles 1b–e with hydrogen sulphide. The
amidoximes 3b–e were prepared starting from the nitriles 1b–e by
reaction with hydroxylamine. The 2-substituted benzimidazoles 4c,d
were obtained from the phenyl- and p-fluorophenylpiperazyl substi-
tuted nitriles by reaction with o-phenylenediamine in polyphosphoric
acid (PPA). The pyrazine derivatives 5 and 6 were produced in the re-
action of 3- and 6-chloropyrazine-2-carbonitriles11,12 with benzonitrile
1a. The reaction was conducted in anhydrous benzene in the presence
of triethylamine as hydrogen chloride acceptor. Attempts to transform
the nitrile group in these compounds (5 and 6) into a thioamide or ami-
doxime function failed. Heating of the amidoxime 3b in ethyl ortho-
formate resulted in the formation of the 4,5-dihydro-1,2,4-oxadiazole
derivative 7b.
1-Methyl-4-[4-(5-phenyl-thiazol-2-yl)]-phenylpiperazine hydrobro-
mide 8b was obtained from the reaction of the thioamide 2b with
phenacyl bromide. The reactions are shown in Scheme 1. Characteris-
tics of the compounds obtained are given in Table I.
MICROBIOLOGY
The investigations included 25 strains of obligate anaerobic bacteria
isolated from the oral cavity, respiratory tract and abdominal cavity
as well as 6 standard strains. The anaerobes belonged to the follow-
ing genera: Peptococcus (1 strain), Peptostreptococcus (4), Actinomyces
(2), Propionibacterium (2), Prevotella (6), Porphyromonas (2), Fusobac-
terium (3), Bacteroides (5), and standard strains: Bacteroides fragilis
ATCC 25285, Bacteroides vulgatus ATCC 8482, Bacteroides ovatus
ATCC 8483, Fusobacterium nucleatum ATCC 25586, Peptostreptococ-
cus anaerobius ATCC 27337 and Propionibacterium acnes ATCC 11827.

1254

1255

1256

1,4-Disubstituted Piperazine Derivatives
1257
The susceptibility of the anaerobic bacteria was determined by means
of the plate dilution technique in Brucella agar, supplemented with 5%
sheep’s blood.13,14 The derivatives were dissolved in 1 mL of DMSO
immediately before the experiment. Sterile distilled water was used
for ultimate dilutions. The following concentrations of the piperazine
derivatives were used: 200, 100, 50, 25, 12,5, and 6,2 µg/mL. The in-
oculum containing 106 CFU/spot was applied to the agar plates with
Steers replicator. The inoculated agar plates and compound-free ones
were incubated in anaerobic jars for 48 h at 37◦C in 10% CO2, 10%
H2, and 80% N2 atmosphere with palladium catalyst and indicator of
anaerobiosis. The minimal inhibitory concentration (MIC) was defined
as the lowest concentration of the derivative that inhibited growth of
the anaerobes.
Aerobic Bacteria
The investigations included 25 strains of aerobes isolated from the oral
cavity, respiratory tract and abdominal cavity, as well as 6 standards
strains. The aerobes were as follows: Staphylococcus aureus (4 strains),
Corynebacterium spp. (2), Klebsiella pneumoniae (3), Acinetobacter
baumannii (2), Escherichia coli (6), Pseudomonas aeruginosa (6), Pseu-
domonas stutzeri (2) and 6 standard strains: Staphylococcus aureus
ATCC 25923, Enterococcus faecalis ATCC 29212, Klebsiella pneumo-
niae ATCC 13883, Acinetobacter baumannii ATCC19606, Escherichia
coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. The sus-
ceptibility of the aerobic bacteria was determined by means of agar di-
lution technique with Mueller–Hinton agar.13,14 Sterile distilled water
was used for ultimate dilutions. The inoculum containing 106 CFU/spot
was applied to the agar plates with Steers replicator. The inoculated
agar plates and the derivative-free ones were incubated for 24 h at 37◦C.
The minimal inhibitory concentration (MIC) was defined as the lowest
derivative concentration, that inhibited growth of aerobic bacteria.
Mycobacterium Tuberculosis
The compounds were examined for their tuberculostatic activity to-
wards Mycobacterium tuberculosis H37Rv strain and two “wild” strains
isolated from tuberculotic patients: one (Spec. 210) resistant to p-
aminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), etambutol
(ETB) and rifampicine (RFP), and the other (Spec. 192) fully suscepti-
ble to the drugs administered. In vitro investigations were performed
by a classical test tube method of successive dilutions with Youman’s
liquid medium containing 10% of bovine serum.11

1258
D. Pancechowska-Ksepko et al.
TABLE II The Minimal Inhibitory Concentration (MIC) for the
Compounds 2b–3e
MIC [µg/mL]
Anaerobic bacteria
Metronidazole
2b
2e
3b
3c
3d
3e
Gram-positive:
Peptococcus niger
Peptostreptococcus
magnus
Peptostreptococcus
micros
Actinomyces
israelii
Actinomyces
neaslundii
≤ 6.2
≤ 6.2
≥ 200 ≥ 200 ≥ 200 ≥ 200
25 ≥ 200
≥ 200
≥ 200
≥ 200
12.5
12.5
100 ≥ 200
12.5 50
25
25
25
≤ 6.2
≤ 6.2
≤ 6.2
≥ 200
12.5
12.5
12.5 ≤ 6.2
12.5 ≥ 200
≥ 200 ≥ 200 ≤ 6.2
≥ 200 25 ≤ 6.2
50
50
100 ≥ 200
100 ≤ 6.2
Propionibacterium
granulosum
Gram-negative:
Prevotella bivia
Prevotella buccalis
Prevotella
≤ 6.2
≤ 6.2
≤ 6.2
≥ 200 ≥ 200 ≥ 200
100 ≥ 200 ≥ 200
≥ 200 ≥ 200 ≥ 200 ≥ 200 ≥ 200 ≥ 200
≥ 200
25
≥ 200
100
100 ≥ 200
25 ≥ 200
intermedia
Prevotella loescheii
Porhyromonas
asaccharolytica
Fusobacterium
nucleatum
Fusobacterium
necrophorum
Bacteroides
≤ 6.2
≤ 6.2
≥ 200 ≥ 200 ≥ 200 ≥ 200
≥ 200
25
50
≥ 200
100
100
100
50
50
≤ 6.2
≤ 6.2
≤ 6.2
≤ 6.2
≥ 200 ≥ 200
12.5
25
25
12.5 ≤ 6.2
100
25
≥ 200
≥ 200
100
25
100 ≥ 200
100
12.5
forsythus
Bacteroides fragilis
100 ≥ 200 ≥ 200 ≥ 200
The susceptibility of anaerobic bacteria towards metronidazole and
piperazine derivatives 2b, e and 3b–e is shown in Table II. Metronida-
zole, the medicine of preference in treatment of the infections breeded or
accompanied by anaerobes, showed the activity at low concentrations
6.2–12.5 µg/mL towards 22 (88%) strains tested. These results were
consistent with those reported elsewhere.16–20 The strains of bacillus
Propionibacterium granulosum (MIC > 200 µg/mL) showed the high-
est refractoriness towards metronidazole. The high susceptibility of the
Gram-positive rod-bacteria towards this medicament was confirmed by
many authors.15,17−20 Among the amidoxime derivatives, compound 3a
showed the highest activity towards anaerobes. Almost 1/3 of the strains
tested (28%) was susceptible to low concentrations ranging from 12.5 to

1,4-Disubstituted Piperazine Derivatives
1259
6.2 µg/mL, and the next five (25%) strains—to 25 µg/mL. For 11 (44%)
strains the growth-inhibiting concentrations were ≥200 µg/mL. More
susceptible to this amidoxime derivatives were the strains of Gram-
positive anaerobes. MIC within the limits of 50 ≤ 6.2 µg/mL inhibited
the growth of 77% of these bacteria, and of only 31% of Gram-negative
strains. Compound 3a inhibited the growth of Propionibacterium gran-
ulosum strains in lower concentrations (25 < MIC ≤ 6.2 µg/mL) than
metronidazole (MIC ≥ 200 µg/mL). Similar activity towards the anaer-
obic bacteria was shown by the derivative 2e. Its growth inhibiting
concentrations towards 4 (16%) strains were within 12.5 ≤ 6.2 µg/mL,
towards 7 (28%) strains—25 µg/mL, towards the next 11 (44%), how-
ever, at least 200 µg/mL. The activity towards the anaerobes, shown by
the compounds 3c and 2b, was even lower. Low concentrations—12.5
µg/mL inhibited the growth of 3 (13%) and 2 (8%) strains respectively,
while concentrations within 25–50 µg/mL the growth of 1 (4%) and 3
(12%), respectively. The growth of the majority of the strains tested (60%
and 76%, respectively) was inhibited by concentrations ≥200 µg/mL.
The least activity, particularly in the range of low concentrations, was
shown by the derivative 3d. The lowest growth-inhibiting concentra-
tions were within 25–50 µg/mL towards only 4 (16%) strains. In the
case of about 1/3 (28%) of the strains these concentrations were 100
µg/mL, and at least 200 µg/mL—toward the remaining strains (56%).
The compounds were, however, more active towards Gram-positive rod-
bacteria of Propionibacterium genus (MIC 100–200 µg/mL), as com-
pared to metronidazole (MIC > 200 µg/mL). The highest activity to-
wards Gram-positive anaerobes from all the amidoximes tested was
demonstrated by the compounds 2e and 3b. Within the range of con-
centrations 6.2 ≥ 25 µg/mL the growth of 7 (77%) strains was inhibited
by metronidazole, and that of 6 (66%) strains by both the derivatives 2e
and 3b. However, the strains of Gram-negative bacteria—most of them
(93%) highly susceptible (MIC ≤ 6.2 µg/mL) towards metronidazole—
appeared to be much less susceptible towards the amidoxime deriva-
tives tested. The highest activity towards these bacteria was shown
by the derivatives 2e and 3b. Within the range of concentrations 6.2–
25 µg/mL the growth-inhibiting effect of 2e was 31% and that of 3b
was 25%. The compounds tested were inactive towards aerobic bacte-
ria within the range of concentrations 6.2–200 µg/mL.
CONCLUSIONS
The derivatives tested showed differentiated activity towards the
anaerobic bacteria. The maximum activity was exhibited by the com-
pounds 3b, 2e, 3e and the lowest by compound 3d. The maximum

1260
D. Pancechowska-Ksepko et al.
TABLE III Tuberculostatic Activity of the
1,4-Disubstituted Piperazines
Bacterial strain
MIC [µg/mL]
H37Rv
192
210
1a
1b
1c
1d
1e
2b
2e
3b
3c
3d
3e
4c
4d
5
100
100
50
50
50
>100
100
100
100
100
25
100
100
50
50
50
100
50
100
50
100
50
100
>100
100
100
50
50
100
100
50
100
50
100
100
100
100
25
50
100
100
100
50
100
100
>100
>100
50
6
7b
8b
12.5
25
12.5
susceptibility towards derivatives 2e and 3b was shown by both Gram-
positive and Gram-negative bacteria. The amidoxime derivatives tested
appeared to be inactive towards the aerobic bacteria used for the investi-
gation. The determined minimum concentrations inhibiting (Table III)
the growth of tuberculous strains (MIC) for most of the compounds ex-
amined were within the limits 12–100 µg/mL. MIC of the most active
compound 8b was 12.5 µg/mL for H37Rv strain and 25 µg/mL for other
strains.
EXPERIMENTAL
Melting points were determined with the Reichert apparatus and are
uncorrected. The IR spectra were recorded with a Satellite spectropho-
tometer. The 1H NMR spectra were obtained with a Varian Gemini 200
spectrometer. Reaction yields and the physical constants of the com-
pounds obtained are given in Table I. The results of elemental analyses
for C and H for all the compounds obtained were in good agreement
(+ 0.4%) with the data calculated.

1,4-Disubstituted Piperazine Derivatives
1261
4-Piperazin-1-yl-benzonitrile (1a)
4-Fluorobenzonitrile (1 mmol, 0.121 g), piperazine (2 mmol, 0.172 g)
and anhydrous K2CO3 (0.5 g) were refluxed in DMSO (15 mL) for 1.5 h.
On cooling down to ambient temperature, water (25 mL) was added.
The mixture was extracted with diethyl ether (3 × 15 mL), the extracts
dried over anhydrous MgSO4, and the ether was evaporated. The oily
residue crystallized gradually.
4-(4-Methyl-, 4-phenyl-, 4-p-fluorophenyl, and 4-benzyl-
piperazin-1-yl)-benzonitriles (1b–e)
4-Fluorobenzonitrile (1 mmol, 0.121 g) dissolved in DMSO (20 mL) was
treated with the corresponding piperazine (4-methyl-, 4-phenyl-, 4-p-
fluorophenyl-, and benzylpiperazine) (5 mmol) and anhydrous K2CO3
(1 g). The resulting reaction mixture was refluxed for 1.5 h. On cool-
ing down to ambient temperature water was added and the resulting
precipitate was separated by filtration.
4-(4-Methyl- and 4-benzyl-piperazin-1-yl)thioamides (2b, e)
4-(4-Methyl-, or 4-benzyl-piperazin-1-yl)-benzonitrile 1b, e (1 mmol)
was dissolved in warm ethanol (20 mL), concentrated NH4OH (20 mL)
saturated with H2S was added and the reaction mixture was allowed to
stand at ambient temperature for a few days. The resulting precipitate
was filtered off and purified by crystallization from ethanol.
4-(4-Methyl-, 4-phenyl-, 4-p-fluorophenyl- and
4-benzyl-piperazin-1-yl)-N-hydroxybenz-amidines (3b–e)
Two solutions were prepared: 1) hydroxylamine hydrochloride (1.2 g) in
methanol (10 mL); 2) KOH (1.15 g) in methanol (10 mL). The solutions
were mixed, the KCl precipitate was filtered off, the filtrate was treated
with the corresponding nitrile (1 mmol) (1b–e), and the reaction mix-
ture was refluxed for 8 h. On ice cooling the product precipitated and
was separated by filtration.
3-[4-(4-Phenyl- and 4-p-fluorophenyl-piperazin-1-yl)-phenyl]-
1H-benzimidazoles (4c, d)
Compound 1c or 1d (1 mmol) and o-phenylenediamine (4 mmol) were
heated with polyphosphoric acid (15 g) for 2.5 h at a temperature of
180–200◦C. On cooling to ambient temperature ice was added until

1262
D. Pancechowska-Ksepko et al.
the product precipitated completely. The precipitate was filtered and
washed with water.
4-(4-Cyanophenyl)-3,4,5,6-tetrahydro-2H-[1,2’]-bipyrazinyl-3’-
and 6’-carbonitriles (5, 6)
Compound 1a (2 mmol) and 3-chloro- or 6-chloro-pyrazino-2-
carbonitrile (2 mmol) were dissolved in benzene (15 mL) and treated
with triethylamine (0.5 mL). The reaction mixture was refluxed for 0.5
h and then evaporated to dryness. On addition of water (10 mL) and
cooling to 0◦C the product precipitated and was separated by filtration.
1-Methyl-4-[4-(5-ethyl-4,5-dihydro[1,2,4]-oxadiazol-3-
yl)phenyl]-piperazine (7b)
Compound 3b (0.5 mmol, 0.117 g) and ethyl orthoformate (10 mL) were
refluxed for 1 h. The orthoformate was then evaporated and the residue
recrystallized from cyclohexane.
1-Methyl-4-[4-(phenyl-thiazol-2-yl)-phenyl]-piperazine
hydridobromide (8b)
To compound 2b (1 mmol, 0.235 g) dissolved in absolute ethanol (10 mL)
ω-bromo-acetophenone (1 mmol, 0.199 g) was added and the reaction
mixture was refluxed for 1 h. On cooling to ambient temperature the
product 8b precipitated and was separated by filtration.
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