Synthesis and antimicrobial activity of N-alkyl and N-aryl piperazine derivatives

Article abstract of DOI:10.1016/j.bmc.2005.10.032

A series of substituted piperazine derivatives have been synthesized and tested for antimicrobial activity. The antibacterial activity was tested against Staphylococcus aureus (MTCCB 737), Pseudomonas aeruginosa (MTCCB 741), Streptomyces epidermidis (MTCCB 1824) and Escherichia coli (MTCCB 1652), and antifungal activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. All synthesized compounds showed significant activity against bacterial strains but were found to be less active against tested fungi. In vitro toxicity tests demonstrated that compounds 4d and 6a showed very less toxicity against human erythrocytes.

Full text of DOI:10.1016/j.bmc.2005.10.032

Bioorganic & Medicinal Chemistry 14 (2006) 1819–1826
Synthesis and antimicrobial activity of N-alkyl
and N-aryl piperazine derivatives
Preeti Chaudhary,a Rupesh Kumar,a Akhilesh K. Verma,a,* Devender Singh,a
Vibha Yadav,b Anil K. Chhillar,b G. L. Sharmab and Ramesh Chandraa,*
aSynthetic Organic Chemistry Research Laboratory, Dr.B. R. Ambedkar Center for Biomedical Research, University of Delhi,
Delhi 110007, India
bInstitute of Genomics and Integrative Biology, Mall Road, University Campus, Delhi 110007, India
Received 7 August 2005; revised 19 October 2005; accepted 20 October 2005
Available online 9 November 2005
Abstract—A series of substituted piperazine derivatives have been synthesized and tested for antimicrobial activity. The antibacterial
activity was tested against Staphylococcus aureus (MTCCB 737), Pseudomonas aeruginosa (MTCCB 741), Streptomyces epidermidis
(MTCCB 1824) and Escherichia coli (MTCCB 1652), and antifungal activity against Aspergillus fumigatus, Aspergillus flavus and Asper-
gillus niger. All synthesized compounds showed significant activity against bacterial strains but were found to be less active against
tested fungi. In vitro toxicity tests demonstrated that compounds 4d and 6a showed very less toxicity against human erythrocytes.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
albicans from women with candidal vaginitis is resistant
to fluconazole.19 This situation highlights the need for ad-
vent of safe, novel and effective antibacterial and anti-
fungal compounds.
Antimicrobial diseases are now more frequent than dur-
ing the first half of the century, being still difficult to diag-
nose clinically. During the later half of the century,
particularly during the past two decades, a number of dif-
ferent classes of antibacterial1–7 and antifungal agents8–14
have been discovered. Although, since the discovery of
several synthetic and semi-synthetic antibacterial sulfa
drugs, nitrofuranes, penicillins, cephalosporins, tetracy-
clines, macrolides, and oxazolidinones, and antifungal
agents such as fluconazole, ketoconazole and miconazole,
including amphotericin B, there has been much progress
in this field.1–4,11–14 Despite advances in antibacterial
and antifungal therapies, many problems remain to be
solved for most antimicrobial drugs available. For exam-
ple, appearance of multidrug resistant Gram-positive
bacteria, in particular, methicillin-resistant Staphylococ-
cus aureus and vancomycin-resistant Enterococci is caus-
ing a serious menace. The use of amphotericin B, known
as the Ôgold standard,Õ is limited because of its infusion-
related reactions and nephrotoxicity.15,16 Also the use of
azoles, such as fluconazole, ketoconazole and miconaz-
ole, has resulted in clinically resistant strains of Candida
spp.17,18 A 3.6–7.2% of vaginal isolates of Candida
Piperazines and substituted piperazines are important
pharmacophores that can be found in many marketed
drugs, such as the Merck HIV protease inhibitor
Crixivan,20 and drugs under development.21 Piperazi-
nyl-linked ciprofloxacin dimers reported as potent anti-
bacterial agents against resistant strains,22 a novel class
of mixed D2/D4 receptor antagonists,23 dual calcium
antagonist,24 antimalarial agents25 and potential antipsy-
chotic agents.26 Recently, piperazine derivatives contain-
ing tetrazole nucleus have been reported as an antifungal
agent.27Herein, we have described the synthesis of a
series of N-alkyl and N-aryl piperazine derivatives as a
new class of synthetic antimicrobial agents along with
their in vitro antimicrobial activity [zone of inhibition
for antibacterial and minimum inhibitory concentration
(MIC) for antifungal activity].
2. Results and discussion
2.1. Chemistry
N-Alkyl and N-aryl substituted piperazine derivatives
have been prepared by a well-established three compo-
Keywords: Piperazine; Antibacterial; Antifungal; Cytotoxicity.
*
Corresponding authors. E-mail: akvacbr@rediffmail.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.032

1820
P. Chaudhary et al. / Bioorg. Med. Chem. 14 (2006) 1819–1826
nent condensation reaction of N-alkyl and N-aryl
substituted piperazines 1a-d with benzotriazole and
formaldehyde to construct the key benzotriazolyl
intermediate 2a–d, followed by nucleophilic substitu-
tions of the benzotriazolyl group to perform N-func-
tionalization. The reactive C-N bond of the key
benzotriazolyl intermediate 2a–d allows easy replace-
ment the of benzotriazolyl group with other function-
alities via nucleophilic substitutions, elimination and
reduction.28
2.2. In vitro antibacterial activity
Antibacterial activity of the synthesized compounds
was tested against pathogenic bacterial strains such
as Staphylococcus aureus (MTCCB 737), Pseudomo-
nas aeruginosa (MTCCB 741), Staphylococcus epide-
rmidis (MTCCB 1824) and Escherichia coli
(MTCCB 1652) using the disc diffusion method.29
Gentamicin was used as reference drug for bacteria.
The zone of inhibition (mm) of tested compounds
against pathogenic bacterial strains is shown in Ta-
ble 1. In general, the compounds showed significant
to moderate antibacterial activity, whereas the
compounds 5c and 7b were inactive at the highest
tested concentration against Gram-negative and
Gram-positive bacteria. Diethyl(4-benzylpiperazin-1-
yl)methylphosphonate 4c, diethyl [4-(2-methoxyphe-
nyl)piperazin-1-yl]methylphosphonate 4d and 1-meth-
yl-4-(3-phenylprop-2-ynyl)piperazine 6a had highest
antibacterial activities against pathogenic bacterial
strains with the zone of inhibition in the range of
9–18 mm. The reason for this higher antibacterial
activity might be related to the presence of phos-
phate group in 4c, 4d and triple bond in 6a (Table
1). Compound 4d exhibited significant activity
against S. aureus (with the zone of inhibition,
18 mm), S. epidermidis (16 mm) and P. aeruginosa
(15 mm), but moderate activity against E. Coli
(13 mm). Compound 6a showed significant activity
against S. aureus (with the zone of inhibition,
18 mm), P. aeruginosa (16 mm) and E. coli
(15 mm), but poor activity against S. epidermidis
(9 mm). Diethyl(4-benzylpiperazin-1-yl)methylphosph-
onate 4c showed significant activity against S. aure-
us and S. epidermidis with 16 and 17 mm of the
zone of inhibition, respectively. By considering the
considerable activity of substituted 1,2,4-triazoles
heterocyles against Gram-negative and Gram-positive
bacteria as well as fungi,30–33 a new series of benzo-
triazole derivatives of N-alkyl and N-aryl piperazine
were also synthesized and their antimicrobial prop-
erties evaluated. 1-[(4-Methylpiperazin-1-yl)methyl]-
1H-1,2,3-benzotriazole 2a showed moderate activity
against Gram-negative and Gram-positive bacteria
with the zone of inhibition in the range of 11-
The condensation of 1-methylpiperazine 1a, 1-phenyll-
piperazine 1b, 1-benzylpiperazine 1c and 1-(2-methoxy-
phenyl)piperazine 1d with 1 equiv of benzotriazole and
1 equiv of formaldehyde (37% aqueous solution) in
MeOH/H2O (9:1) at 25 ꢁC gave 1-[(4-methylpiperazin-
1-yl)methyl]-1H-1,2,3-benzotriazole 2a, 1-[(4-phenylpi-
perazin-1-yl)methyl]-1H-1,2,3-benzotriazole 2b, 1-[(4-
benzylpiperazin-1-yl)methyl]-1H-1,2,3-benzotriazole 2c
and 1-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-1H-
1,2,3-benzotriazole 2d in 87-94% yields as a sole ben-
ztoriazol-1-yl (bt1) isomer. Treatment of 2a–d with
2 equiv of sodium borohydride in refluxing THF re-
placed the benzotriazole group with hydrogen to give
1,4-dimethylpiperazine 3a, 1-phenyl-4- methylpiperazine
3b, 1-benzyl-4- methylpiperazine 3c and 1-(2-methoxy-
phenyl)-4-methylpiperazine 3d in 76–88% yields (Scheme
1). The benzotriazolyl group of 2a–d was also replaced by
triethylphosphite in the presence of ZnBr2 to afford
diethyl(4-methylpiperazin-1-yl)methyl phosphonate 4a,
diethyl(4-phenylpiperazin-1-yl)methyl phosphonate 4b,
diethyl(4-benzylpiperazin-1-yl)methyl phosphonate 4c
and diethyl [4-(2-methoxyphenyl)piperazin-1-yl]methyl-
phosphonate 4d in 74–91% yields (Scheme 1).
Nucleophilic substitution of 2a–d with various
Grignard reagents 1-propynyl-, phenylethynyl-, p-tolyl-
and isopropyl magnesium bromides in dry
THF furnished 1-but-2-ynyl-4-methylpiperazine 5a,
1-phenyl-4-but-2-ynylpiperazine 5b, 1-benzyl-4-but-2-
ynylpiperazine 5c, 1-but-2-ynyl-4-(2-methoxyphenyl)-
piperazine 5d, 1-methyl-4-(3-phenylprop-2-ynyl)pipera-
zine 6a, 1-phenyl-4-(3-phenylprop-2-ynyl)piperazine
6b, 1-benzyl-4-(3-phenylprop-2-ynyl)piperazine 6c,
1-(2-methoxyphenyl)-4-(3-phenylprop-2-ynyl)piperazine
6d, 1-isobutyl-4-methylpiperazine 7a, 1-phenyl-4-isobu-
tylpiperazine 7b, 1-benzyl-4-isobutylpiperazine 7c,
1-isobutyl-4-(2-methoxyphenyl)piperazine 7d, 1-meth-
yl-4-benzyl-piperazine 8a, 1-phenyl-4-benzyl-piperazine
8b, 1,4-dibenzyl-piperazine 8c and 1-(2-methoxyphe-
nyl)-4-(4-methylbenzyl)piperazine 8d, respectively, in
68–87% yields (Scheme 2).
14 mm.
1-[(4-Benzylpiperazin-1-yl)methyl]-1H-1,2,3-
benzotriazole 2c and 1-{[4-(2-methoxyphenyl) pipera-
zin-1-yl] methyl}-1H-1,2,3-benzotriazole 2d showed
moderate activity against bacterial strains. However,
1-[(4-phenylpiperazin-1-yl)methyl]-1H-1,2,3-benzotriaz-
ole 2b shows mild antibacterial activity. This might
be due to the presence of benzotriazolyl group (tri-
azole fused with benzene nucleus). 1-But-2-ynyl-4-
methylpiperazine 5a and 1-isobutyl-4-(2-methoxyphe-
nyl)piperazine 7d showed significant activity against
antibacterial strains with an inhibition zone diameter
of 11–17 mm. These might be due to the presence
of the triple group in 5a and the isopropyl group
in 7d. The other related compounds in this study
demonstrated weak antibacterial activity. The preli-
minary results of antimicrobial activities indicated
that some of the compounds exhibited a moderate
The condensation of piperazine 9 with 2 equiv of benzo-
triazole and 2 equiv of formaldehyde (37% aqueous
solution) in MeOH/H2O (9:1) at 25 ꢁC gave 1,4-bis-
[1,2,3]benzotriazol-1-ylmethylpiperazine 10 in 92%
yields as a sole bt1 isomer. Varied nucleophiles and
Grignard reagents in varied solvents were tried to substi-
tute the benzotriazolyl group with nucleophiles, but one
failed to obtain substituted compounds due to insolubil-
ity of compound 10 in various solvents (Scheme 3).

P. Chaudhary et al. / Bioorg. Med. Chem. 14 (2006) 1819–1826
1821
R
No
i
N
R
N
N
H
CH3
R
N
a
b
c
d
Bt
Ph
PhCH2
2-OMeC6H4
2 a-d
1 a-d
R = CH3, Ph, PhCH2, 2-OMeC6H4
ii
iii
R
N
N
Me
O
R
N
4a-d
N
P
3a-d
EtO
OEt
Scheme 1. Reagents and conditions: (i) BtH/CH2O, MeOH/H2O (9:1), 25 ꢁC, 6 h; (ii) NaBH4, THF, reflux, 8–12 h; (iii) triethylphosphite/ZnBr2,
25 ꢁC, 12–16 h.
i
ii
R
N
N
R
N
N
2 a-d
Ph
CH3
6a-d
5a-d
iv
iii
R
a
CH3
Ph
PhCH2
2-OMePh
b
c
d
R
N
N
R
N
N
7a-d
8a-d
Me
Scheme 2. Reagents and conditions: (i) 1-propynyl magnesium bromides, THF, reflux, 8 h; (ii) phenylethynyl magnesium bromides, THF, reflux,
6–8 h; (iii) isopropyl magnesium bromides, THF, reflux, 8–10 h; (iv) p-tolyl-magnesium bromides, THF, reflux, 6–8 h.
Nu
Bt
i
Nu
N
N
N
N
H
N
H
N
Nu
Bt
11
10
9
Scheme 3. Reagents and conditions: (i) BtH/CH2O, MeOH/H2O (9:1), 25 ꢁC, 6 h.
to significant activity against Gram-positive and
Gram-negative strains.
B was used as a standard drug. The minimum inhibitory
concentrations (MICs, mgmlꢀ1, mgdiscꢀ1) of tested
compounds against pathogenic fungi are shown in
Table 2. Results of in vitro anti-Aspergillus activity dem-
onstrate that 2c, 2d, 4c, 6c and 11 show mild to moder-
ate anti-Aspergillus activities against pathogenic strains
used in the experiment. 1-[(4-Phenylpiperazin-1-yl)meth-
yl]-1H-1,2,3-benzotriazole 2b was found to be a poten-
tial inhibitor of the growth of A. fumigatus, A. flavus
and A. niger result of the anti-Aspergillus activity.
1-[(4-Phenylpiperazin-1-yl)methyl]-1H-1,2,3-benzotria-
zole 2b has significant MIC values in the range of
23.43 lg/disc in DDA, 62.50–125.0 lg/ml in MDA and
2.3. In vitro antifungal activity
Antifungal activity of the synthesized compounds was
tested against pathogenic fungal strains such as
Aspergillus fumigatus (ITCC 4517), Aspergillus flavus
(ITCC 5192) and Aspergillus niger (ITCC 5405). The
anti-Aspergillus activity of all the synthesized com-
pounds was evaluated by the disc diffusion (DDA)34
microbroth dilution (MDA),34 and percentage spore
germination inhibition (PSGI) assays.35 Amphotericin

1822
P. Chaudhary et al. / Bioorg. Med. Chem. 14 (2006) 1819–1826
Table 1. Antibacterial activity of substituted piperazine derivatives
1-but-2-ynyl-4-methylpiperazine 6a was investigated
using the haemolytic assay.34,36 Compounds 4d and
6a were found to be less toxic upto the tested con-
centration, that is, 1000 lg/ml and lysed only 10%
4d and 17.4% 6a of human erythrocytes, respectively,
whereas standard drug Gentamicin lysed 31% of
erythrocytes at a concentration of 1000 lg/ml (Figs.
1 and 2).
Compound
Zone of inhibition (mm)
S. aureus S. epidermidis P. aeruginosa E. coli
2a
2b
2c
2d
3c
4c
4d
5a
5b
5c
6a
6c
7b
7d
8d
10
13
9
11
—
14
10
10
17
16
13
6
12
9
14
9
9
9
8
8
10
8
11
7
12
16
18
16
9
14
15
11
7
10
13
14
6
3. Conclusion
—
18
8
—
9
—
16
7
—
15
8
In conclusion, in an effort to discover new piperazine
analogues with antibacterial activity we found that com-
pounds 4c, 4d, 5a, 6a and 7d show significant antibacte-
rial activity. Compounds 2a and 2b were found show
significant anti-Aspergillus activity. Compounds 4d and
6a were found to be less toxic to human erythrocytes
when compared with Gentamicin. The results of the
present study indicate that compounds 2a, 2b, 4c, 5a,
6a and 7d might be of interest for the identification of
new antimicrobial molecules.
13
—
14
7
—
17
8
10
13
8
—
17
8
10
7
17
8
16
—
10
Gentamicin 16
62.50–125.0 lg/ml in the PSGI assay against A. flavus,
A. niger and A. fumigatus (Table 2). Compound 1-[(4-
methylpiperazin-1-yl)methyl]-1H-1,2,3-benzotriazole 2a
has MIC values in the range of 46.75 lg/disc in DDA,
250–500 lg/ml in MDA and 250 lg/ml in the PSGI
assay against A. flavus, A. niger and A. fumigatus. En-
hanced activity of compounds 2a, 2b might be due to
the presence of the benzotriazolyl group. Compounds
2c and 2d showed moderate anti-Aspergillus activity
with MIC values in the range of 187.5–187.5 lg/disc in
DDA, 500–1000 lg/ml in MDA and 500–500 lg/ml in
PSGI assay against A.flavus, A. niger and A. fumigatus
(Table 2). Although it was not possible to established
the structure–activity relationship with respect to fungi.
4. Experimental
All reagents used were of AR grade. THF was distilled
from sodium/benzophenone prior to use. Melting points
were determined with a Thomas Hoover melting point
1
apparatus and are uncorrected. H (300 MHz) and 13C
NMR (75 MHz) spectra were recorded on a Bruker
300 NMR spectrometer in CDCl3 with TMS for 1H
and chloroform-d for 13C as internal references) unless
otherwise stated. Mass Spectrum was recorded on a Hy-
brid Quadrupole-TOF LCnMSnMS mass spectrometer
(Q. Star XL). Column chromatography was performed
on silica gel (230–400 mesh). Microanalyses were
obtained with an Elemental Analysensysteme GmbH
VarioEL V3.00 element analyser. The reactions were
monitored by thin layer chromatography (TLC) using
aluminium sheets with silica gel 60 F254 (Merck). All
of the reactions were carried out under nitrogen
atmosphere.
2.4. Cytotoxicity study on diethyl [4-(2-methoxyphenyl)
piperazin-1-yl] methylphosphonate (4d) and 1-methyl-4-
(3-phenylprop-2-ynyl)piperazine (6a)
The in vitro cell cytotoxicity of diethyl [4-(2-methoxy-
phenyl) piperazin-1-yl] methylphosphonate 4d and
Table 2. Antifungal activity of substituted piperazine derivatives
Compound
MIC
DDA (lg/disc)
MDA (lg/ml)
PSG1 (lg/ml)
A. flavus
A. niger
A. fumigatus
A. flavus
A. niger
A. fumigatus
A. flavus
A. niger
A. fumigatus
250
62.50
2a
2b
2c
2d
4b
4c
4d
5c
6c
7d
8a
46.75
23.43
187.5
187.5
—
46.75
23.43
375.5
187.5
—
46.75
23.43
187.5
187.5
—
500
62.50
500
500
—
500
125.0
1000
500
—
250
250
62.50
500
500
—
250
125.0
500
500
—
125.0
500
500
500
500
—
—
—
—
—
—
—
—
—
—
—
—
1000
—
—
—
—
—
1000
—
—
—
—
—
—
—
—
—
—
500
—
500
—
250
—
250
—
500
—
500
—
500
—
500
—
500
—
—
—
—
—
—
187.5
2.5
—
—
—
—
—
—
—
—
—
—
11
Amphotericin B
1000
5
500
5
2.5
2.5
5
5
5
5
(—) means no activity.

P. Chaudhary et al. / Bioorg. Med. Chem. 14 (2006) 1819–1826
1823
35
30
25
20
15
10
5
(10 ml) 8–12h, after removal of the solvent in vacuo,
the residue was diluted with EtOAc. The mixture
was washed with 1 N NaOH, and brine and dried
over Na2SO4 and evopration of the solvent in vacuo,
the residue was purified by column chromatography
with hexanes/EtOAc (6:1 to 3:1) as an eluent to give
3a–d in 76–88% yields.
Compound
Gentamicin
Spectral data of the synthesized compounds 3a–d were
consistent with the reported values.
0
0
1
2
3
4
4.3. General procedure for the nucleophilic substitutions
of 2a–d with triethyl phosphite
log conc of compound (µg/ml)
Figure 1. Haemolytic activity of diethyl [4-(2-methoxyphenyl) pipera-
zin-1-yl] methylphosphonate 4d.
To a solution of benzotriazolyl derivative of N-alkyl
and N-aryl piperazines 2a–d (0.31 g, 0.69 mmol) in
dry CH2Cl2 20 ml) at 0 ꢁC were successively added
ZnBr2 0.186 g, (0.827 mmol) and triethyl phosphite
(0.137 ml, 0.827 mmol). The reaction mixture was stir-
red at 0 ꢁC for 2 h and then at 25 ꢁC for 12–16 h
(completion of the reaction was monitored by TLC),
and the reaction was quenched with H2O. After
extraction with CH2Cl2, the combined organic layers
were washed with 1 N NaOH, brine and dried over
anhydrous Na2SO4. After removal of the solvent in
vacuo, the residue was purified by column chromatog-
raphy with hexanes/EtOAc (4:1) as an eluent to afford
4a–d in 74–91% yields.
35
30
Compound
25
20
15
10
5
Gentamicin
0
0
1
2
3
4
log conc of compound (µg/ml)
4.4. Diethyl(4-phenylpiperazin-1-yl)methyl phosphonate
(4b)
Figure 2. Haemolytic activity of 1-methyl-4-(3-phenylprop-2-ynyl)
piperazine 6a.
The general synthetic method described above affords
4b, as a colourless oil. Yield 98%; H NMR (CDCl3,
1
4.1. General procedure for the synthesis of benzotriazolyl
derivatives of N-alkyl and N-aryl piperazines (2a–d)
300 MHz,): 7.27 (t, J = 9 Hz, 2H), 6.91 (d, J = 9 Hz,
1H), 6.85 (d, J = 6 Hz, 2H), 4.22 (s, 2H), 4.17 (m, 4H),
3.20 (m, 4H), 2.82 (m, 4H), 1.34 (t, J = 6 Hz, 6H); 13C
NMR (75 MHz, CDCl3): 151.15, 129.05, 119.7, 116.02,
62.17, 54.89, 54.77, 49.14, 16.48; LCMS m/z found
312.8 (M+), 160, 175.
To a solution of N-alkyl and N-aryl piperazines 1a–d,
[0.35 g, 2 mmol] and benzotriazole (0.2148 g, 2 mmol)
in CH3OH/H2O (9:1, 10 ml) was added formaldehyde
(37% aqueous solution, 4 mmol). The mixture was stir-
red at room temperature for 6–8 h. The precipitate
formed was filtered and washed with cold Et2O to give
pure product 1-[(4-methylpiperazin-1-yl)methyl]-1H-
1,2,3-benzotriazole 2a, 1-[(4-phenylpiperazin-1-yl)meth-
yl]-1H-1,2,3-benzotriazole 2b, 1-[(4-benzylpiperazin-1-
yl)methyl]-1H-1,2,3-benzotriazole 2c and 1-{[4-(2-meth-
oxyphenyl) piperazin-1-yl] methyl}-1H-1 and 2,3-benzo-
triazole 2d in 87–94% yields as a sole bt1 isomer, which
was directly used for subsequent reactions. For micro-
analysis purposes, the precipitate was recrystallised from
CHCl3/hexanes (1:1).
4.5. Diethyl[4-(2-methoxyphenyl)piperazin-1-yl]meth-
ylphosphonate (4d)
The general synthetic method described above af-
fords 4d as
NMR (300 MHz, CDCl3)
a
colourless oil. Yield 98%; 1H
d
8.07 (d, J = 8.1 Hz,
1H), 7.89 (d, J = 9 Hz, 1H), 7.61–7.69 (m, 1H),
7.36–7.51 (m, 1H), 5.48 (s, 2H), 3.18 (s, 3H),
2.81–2.90 (m, 8H), 1.43–1.46 (m, 4H), 1.24–1.26
(m, 6H); 13C NMR (75 MHz, CDCl3): 141.2,
127.9, 119.2, 118.1, 116.2, 115.2, 77.0, 60.9, 56.0,
54.9, 48.08, 15.098; LCMS: m/z found 341.2
(M+), 342.2 (M+1) peak.
Spectral data of the synthesized compounds 2a–d were
consistent with the reported values.
4.2. General procedure for the reduction of 2a–d with
sodium borohydride
4.6. General procedure for the nucleophilic substitutions
of 2a–d with Grignard reagents (5a–d, 6a–d, 7a–d and
8a–d)
To a solution of benzotriazolyl derivative of N-alkyl
and N-aryl piperazines 2a–d (0.31 g, 1.0 mmol) and
NaBH4 0.076 g, 2.0 mmol) refluxed in dry THF
To a solution of benzotriazolyl derivative of N-alkyl
and N-aryl piperazines 2a–d (1.0 mmol) in dry THF

1824
P. Chaudhary et al. / Bioorg. Med. Chem. 14 (2006) 1819–1826
(10 ml) at 0 ꢁC was added dropwise a solution of an
appropriate Grignard reagent (1.2 mmol). The reaction
mixture was allowed to warm to 25 ꢁC and stirred for
0.5 h. Then, the mixture was refluxed for 6–8 h. After
the mixture was cooled, the reaction was quenched
with water and the mixture was extracted with ether.
The combined extracts were washed with 1 N NaOH,
brine and dried over Na2SO4. After removal of the sol-
vent in vacuo, the residue was purified by column chro-
matography with hexanes/EtOAc (6:1 to 3:1) as an
eluent gave 5a–d, 6a–d, 7a–d and 8a–d in 68–87%
yields.
4.12. 1,4-Bis-[1,2,3]benzotriazol-1-ylmethyl-piperazine
(10)
1
Compound 10 as a white solid. Yield 92%; H NMR
(300 MHz, CDCl3): d 8.04 (d, J = 9 Hz, 2· 1H), 7.64
(d, J = 9 Hz, 2· 1H), 7.48 (t, J = 6 Hz, 2· 1H), 7.35 (t,
J = 6 Hz, 2· 1H), 4.84 (s, 4H), 2.75–2.68 (m, 4H),
2.42–2.23 (m, 4H); 13C NMR (75 MHz, CDCl3): 133.6,
129.6, 127.2, 126.2, 123.6, 118.04, 80.56, 54.52, 49.96;
LCMS: m/z found 348.4 (M+1) as peak.
4.13. Disc diffusion method
The in vitro antibacterial activity was tested by the disc
diffusion method29 using pathogenic strains of S. aureus
(MTCCB 737), P. aeruginosa (MTCCB 741), S. epide-
rmidis (MTCCB 1824) and E. coli (MTCCB 1652).
The concentration 30 lg/disc of compounds was impreg-
nated on the discs. These discs were placed on the sur-
face of the agar plates already inoculated with
pathogenic bacteria. The plates were incubated at
37 ꢁC and examined at 48 h for zone of inhibition, if
any, around the discs. Gentamicin was used in the assay
as a standard control drug. An additional control disc
without any sample but impregnated with an equivalent
amount of solvent (DMSO) was also used in the assay.
The result of antibacterial activity indicated that some
of the compounds exhibited mild to moderate activity.
4.7. 1-But-2-ynyl-4-methylpiperazine (5a)
The general synthetic method described above affords 5a
as a colourless oil. Yield 91%; 1H NMR (300 MHz,
CDCl3) d 2.73–2.69 (m, 6H), 2.54–2.40 (m, 6H), 2.24
(s, 3H); 13C NMR (75 MHz, CDCl3) 77.5, 77.1, 77.0,
52.0, 48.4, 44.2, 1.5; LCMS: m/z found 153.3 (M+1) as
peak.
4.8. 1-But-2-ynyl-4-phenylpiperazine (5b)
The general synthetic method described above affords 5b
as a colourless oil. Yield 92%; 1H NMR (300 MHz,
CDCl3,): 7.23–7.33 (m, 5H), 1.89 (s, 3H), 1.45 (s, 2H),
1.43 (m, 4H), 1.25 (m, 4H); 13C NMR (75 MHz,
CDCl3): 129.1, 118.4, 113.2, 80.5, 57.2, 50.0, 42.6, 1.5;
LCMS m/z found 215.29 (M+1), 175.29, 162.
4.14. Antifungal activity assay
4.9. 1-Benzyl-4-but-2-ynylpiperazine (5c)
The anti-Aspergillus activity of all the compounds was
studied by disc diffusion, microbroth dilution and per-
centage spore germination inhibition assays.34 The path-
ogenic strains of Aspergillus fumigatus (ITCC 4517),
A. flavus (ITCC 5192) and A. niger (ITCC 5405) were
used in the study for performing various experiments.
The general synthetic method described above affords 5c
as a colourless oil. Yield 93%; 1H NMR (300 MHz,
CDCl3): 7.38–7.24 (m, 5H), 3.57–3.42 (m, 6H), 2.59–
2.61 (m, 5H), 2.53–2.48 (m, 4H); 13C NMR (75 MHz,
CDCl3): 137.9, 132.9, 129.2, 128.1, 125.2, 77.0, 52.8,
52.0, 41.4, 29.7, 3.5; LCMS m/z found: 229.3 (M+1)
peak.
4.15. Microbroth dilution
The test was performed in 96-well culture plates
(Nunc, Nunclon). Various concentrations of synthetic
compounds in the range of 1000–7.81 lg/ml were pre-
pared in the wells by a 2-fold dilution method. Assay
was performed as per the standard method described
earlier.34
4.10. 1-(2-Methoxyphenyl)-4-(3-phenylprop-2-ynyl)piper-
azine (6d)
The general synthetic method described above affords 6d
as a colourless oil. Yield 93%; 1H NMR (300 MHz,
CDCl3) d 7.50–7.89 (m, 5H), 6.29–6.95 (m, 4H), 3.75
(s, 3H), 3.22–3.29 (m, 4H), 2.80 (s, 2H), 2.73–2.79 (m,
4H); 13C NMR (75 MHz, CDCl3) 142.7, 132.3, 129.2,
128.5, 128.3, 127.1, 122.1, 118.9, 116.7, 112.2, 82.5,
77.2, 76.9, 54.4, 50.0, 32.3; LCMS: m/z found 307.4
(M+1) as peak.
4.16. Disc diffusion
The disc diffusion assay was performed in radiation
sterilized petri plates of 10.0 cm diameter (Tarsons)
as described.34 Different concentrations in the range
of 750–1.46 lg of the test compounds were impregnat-
ed on the sterilized discs (5.0 mm in diameter) of
Whatman filter paper No. 1. The discs were placed
on the surface of the agar plates already inoculated
with Aspergillus spores. The plates were incubated at
37 ꢁC and examined at 24, 48 and 96 h for zone of
inhibition, if any, around the discs. The concentration,
which developed the zone of inhibition of at least
6.0 mm diameter, was considered as minimum inhibi-
tory concentration (MIC).
4.11. 1-Benzyl-4-isobutylpiperazine (7c)
The general synthetic method described above affords 7c
as a colourless oil. Yield 91%; 1H NMR (300 MHz,
CDCl3): d 6.84–7.01 (m, 5H), 3.86 (s, 2H), 3.12 (m,
4H), 2.7 (m, 4H), 2.26 (s, 2H), 1.82 (m, 1H), 0.9003 (d,
J = 7.6, 6H); 13C NMR (75 MHz, CDCl3): 138.8,
129.6, 128.4, 127.5, 77.4, 76.5, 62.6, 52.8, 29.6, 20.9;
LCMS m/z found: 233.23 (M+1), 115.4.

P. Chaudhary et al. / Bioorg. Med. Chem. 14 (2006) 1819–1826
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culture medium were prepared in 96-well culture plates
(Nunc, Nunclon) by the double dilution method.35 The
wells were prepared in triplicates for each concentration.
Each well was then inoculated with 10.0 ll of spore sus-
pension containing 100 5 spores. The plates were incu-
bated at 37 ꢁC for 16 h and then examined for spore
germination with an inverted microscope (Nikon, Dip-
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methylphosphonate 4d and 1-methyl -4-(3-phenyl-
prop-2-ynyl) piperazine 6a. Human erythrocytes col-
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washed thrice with PBS and 2.0% (v/v) suspension of
erythrocytes was prepared in phosphate-buffered saline
at pH 7.2. Half millilitres of (2.0%) human erythrocyte
suspension in 16 duplicate sets of tubes was treated
with compound 4c at a concentration of 1.22 lg/ml
for 1 h at 37 ꢁC. After incubation, tubes were centri-
fuged at 5000 rpm for 10 min. The supernatant was
collected and the OD was measured at A415 nm using
a spectrophotometer (UV–vis Spect Lambda Bio 20
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by the compound. Only a buffer of pH 7.2 was used for
background lysis in negative control sets, whereas in
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Acknowledgments
We thankfully acknowledge the CSIR, DST, and Dr. B.
R. Ambedkar Center for Biomedical Research, Univer-
sity of Delhi, Delhi, India. P.C. and R.K. are thankful to
CSIR for JRF and SRF, respectively.
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