Synthesis, characterization and in vitro biological studies of novel cyano derivatives of N-alkyl and N-aryl piperazine

Article abstract of DOI:10.1016/j.ejmech.2006.10.009

Cyano derivatives of N-alkyl and N-aryl piperazine have been synthesized and screened for antibacterial and antifungal activities. All the synthesized compounds showed the antibacterial activity against pathogenic strains of Staphylococcus aureus (MTCCB 737), Pseudomonas aeruginosa (MTCCB 741), Streptomyces epidermidis (MTCCB 1824) and Escherichia coli (MTCCB 1652) and antifungal activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) and Aspergillus niger (ITCC 5405). All compounds showed mild to moderate antimicrobial activity. However, compounds 3c, 4a and 6 showed potent antibacterial activity against pathogenic strains used in the study. Compounds 3a, 3b, 4b, and 4d showed mild to moderate antifungal activity against Aspergillus pathogenic strains. The compounds reported in this study were assessed for there cytotoxicity using MTT colorimetric assay on Hela cells. All the compounds showed cell viability more than the control drug gentamicin, with compound 2 having highest i.e. 95% cell viability.

Full text of DOI:10.1016/j.ejmech.2006.10.009

European Journal of Medicinal Chemistry 42 (2007) 471e476
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and in vitro biological studies of novel
cyano derivatives of N-alkyl and N-aryl piperazine
Preeti Chaudhary a, Surendra Nimesh a, Veena Yadav a,
Akhilesh Kr. Verma a, , Rupesh Kumar
*
a,b,**
a Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Mall Road, Delhi-110007, India
b Department of Chemistry, University of Delhi, Delhi-110007, India
Received 20 July 2006; received in revised form 13 October 2006; accepted 19 October 2006
Available online 29 November 2006
Abstract
Cyano derivatives of N-alkyl and N-aryl piperazine have been synthesized and screened for antibacterial and antifungal activities. All the
synthesized compounds showed the antibacterial activity against pathogenic strains of Staphylococcus aureus (MTCCB 737), Pseudomonas
aeruginosa (MTCCB 741), Streptomyces epidermidis (MTCCB 1824) and Escherichia coli (MTCCB 1652) and antifungal activity against
pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) and Aspergillus niger (ITCC 5405). All compounds
showed mild to moderate antimicrobial activity. However, compounds 3c, 4a and 6 showed potent antibacterial activity against pathogenic
strains used in the study. Compounds 3a, 3b, 4b, and 4d showed mild to moderate antifungal activity against Aspergillus pathogenic strains.
The compounds reported in this study were assessed for there cytotoxicity using MTT colorimetric assay on Hela cells. All the compounds
showed cell viability more than the control drug gentamicin, with compound 2 having highest i.e. 95% cell viability.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Piperazine; Cyano; Pathogenic strain; Cytotoxicity; Antibacterial; Antifungal activity; Antibiotics
1. Introduction
against Gram-positive organisms [8]. Oxazolidinone’s antibac-
terial agents are newer class of synthetic antibacterial agents with
activity against Gram-positive bacteria [9]. Cyano derivatives of pi-
perazine have been known for their uses in the synthesis of pharma-
ceutical intermediates, peptide analogues, antibiotics and other
biologically active molecules and drugs [10e12].
The present investigation describes the synthesis of a series
of cyano derivatives of N-alkyl and N-aryl piperazine. The
derivatives so prepared were characterized by employing
various spectroscopic techniques such as 1H NMR, 13C
NMR, mass spectroscopy etc. The derivatives were assessed
for their in vitro antimicrobial activity (zone of inhibition
and minimum inhibitory concentration (MIC) activity) against
a number of bacterial strains and anti-Aspergillus activity
(minimum inhibitory concentration (MIC) activity). The
compounds were further assessed for their in vitro cytotoxicity
on Hela cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide] colorimetric assay.
In recent decades, the problems of multi-drug resistant
microorganisms have reached on alarming level in many coun-
tries around the world [1e3]. Several antibiotics have been
prescribed and found to be effective on various infectious
disorders. For the treatment of these intractable infections,
new anti-infectious agents are needed. Quinolone antibiotics
are widely prescribed drugs because of their safety, good tol-
erance, broad antibacterial spectrum and less resistance [4e7].
Macrolide antibiotics are an important [1] therapeutic class
* Corresponding author.
** Corresponding author. Dr. B.R. Ambedkar Center for Biomedical Research,
University of Delhi, Mall Road, Delhi-110007, India. Tel.: þ91 9818901344;
fax: þ91 11 23816312.
E-mail address: chembio005@yahoo.co.in (R. Kumar).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.10.009

472
P. Chaudhary et al. / European Journal of Medicinal Chemistry 42 (2007) 471e476
2. Results and discussion
Pseudomonas aeruginosa (MTCCB 741), Streptomyces
epidermidis (MTCCB 1824) and Escherichia coli (MTCCB
1652). The experimental result of antibacterial activity
indicated variable degree of efficacy of the compounds against
different strains of bacteria (Table 1). The zone of inhibition
and MIC’s values of piperazine derivatives were determined
by disc diffusion method [15] and microbroth dilution
technique [16].
2.1. Chemistry
Herein, we report the synthesis, characterization and
in vitro antimicrobial activity of cyano derivatives of N-alkyl
and N-aryl piperazine. Cyano derivatives of N-alkyl and
N-aryl substituted piperazine 3aee have been prepared by
substituting benzotriazolyl group with cyano anion from the
key benzotriazolyl intermediates 2aee (Scheme 1). The con-
densation of 1-methylpiperazine 1a, 1-phenylpiperazine 1b,
1-benzylpiperazine 1c, 1-(2-methoxyphenyl) piperazine 1d,
and 1-(4-nitrophenyl) piperazine 1e with 1 equiv of benzotria-
zole and 1 equiv of formaldehyde (37% aqueous solution) in
MeOH/H2O (9:1) at 25 ꢀC gave benzotriazolyl intermediate
2aee in 87e94% yields as a sole bt1 isomer [13].
Treatment of benzotriazolyl intermediate 2aee with
1.5 equiv of sodium cyanide in DMSO at room temperature
replaced the benzotriazole group with cyanide group to afford
(4-methylpiperazin-1-yl)-acetonitrile 3a, (4-phenylpiperazin-
1-yl)-acetonitrile 3b, (4-benzylpiperazin-1-yl)-acetonitrile 3c,
[4-(2-methoxyphenyl)-piperazin-1-yl]-acetonitrile 3d and [4-
(4-nitrophenyl)-piperazin-1-yl]-acetonitrile 3e in 76e88%
yields (Scheme 1). The reactive CeN bond of the key benzo-
triazolyl intermediates 2aee allows easy replacement of
benzotriazolyl group with cyano anion to afford cyano
derivatives 3aee in good to excellent yields via nucleophilic
substitutions [13].
We have synthesized these cyano derivatives of N-alkyl and
N-aryl piperazine for antimicrobial activity. (4-Benzyl-pipera-
zin-1-yl)-acetonitrile 3c shows potent antibacterial activity
against S. aureus with MIC value at 19.5 mg/ml and zone of
inhibition at 18 mm, however, it did not show significant effect
on other strains of bacteria used in experiment. Similarly 3-(4-
methyl-piperazin-1-yl)-propanitrile 4a was effective against
P. aeruginosa, E. coli only, the MIC being 19.5 mg/ml and
39.06 mg/ml with zone of inhibition of 19 mm and 18 mm,
respectively (Table 1). 3-[4-(2-Hydroxy-ethyl)-piperazin-1-
yl]-acetonitrile 6 shows potent antibacterial activity against
S. aureus with MIC value 19.5 mg/ml and zone of inhibition
18 mm (Table 1), but moderate activity against P. aeruginosa,
E. coli with MIC at 39.1 mg/ml (Table 1). Compounds 3c and
4a showing better activity in comparison to other compounds
used in study might be due to the presence of benzyl and
methyl groups at 4-position of piperazine nucleus. Enhanced
activity of compound 6 might owe to the presence of hydrox-
yethyl group at 4-position of piperazine nucleus. Other com-
pounds appeared as broad spectrum, as they show mild to
moderate effect on most of the strains used in the experiment.
Cyano ethyl derivatives of N-alkyl and N-aryl piperazine
4aef (Scheme 2) and 6 (Scheme 3) have been prepared by
aza-Michael reaction of N-alkyl and N-aryl piperazine with ac-
rylonitrile using 10 mol% copper-nanoparticles (14e17 nm)
as a catalyst [14]. The spectral data of all synthesized com-
pounds were in consistency with the reported spectral data.
2.3. In vitro antifungal activity
Nine cyano derivatives of N-alkyl and N-aryl piperazine
have also been examined for antifungal activity against patho-
genic strains of Aspergillus fumigatus (ITCC 4517), Aspergil-
lus flavus (ITCC 5192) and Aspergillus niger (ITCC 5405).
The anti-Aspergillus activity of all the synthesized have been
evaluated by the disc diffusion (DDA), microbroth dilution
(MDA) [17] and percentage spore germination inhibition
(PSGI) assay [18]; the results are given in Table 2.
2.2. In vitro antibacterial activity
The in vitro antibacterial activity was tested by disc diffu-
sion method [15] and microbroth dilution technique [16] using
pathogenic strains of Staphylococcus aureus (MTCCB 737),
i
R
N
N
No
R
R
N
NH
Bt
2a-e
1a-e
a
b
c
d
e
CH3
C6H5
ii
C6H5CH2
2-OMeC6H4
2-NO2C6H4
R
N
N
CN
3a-e
Scheme 1. Reagent and conditions: (i) CH2O/Benzotriazole (1:1), MeOH/H2O (9:1), 25 ꢀC; (ii) NaCN, DMSO, 25 ꢀC.

P. Chaudhary et al. / European Journal of Medicinal Chemistry 42 (2007) 471e476
473
No
R
i
R
N
N
R
N
NH
a
b
c
d
CH3
CN
4a-d
1a-d
C6H5
C6H5CH2
2-OMeC6H4
iii
ii
N
N
HN
N
HN
NH
CN
NC
CN
4f
4e
1f
Scheme 2. Reagent and conditions: (i) and (ii) 1.5 equiv acrylonitrile, 10 mol% Cu-nanoparticles (14e17 nm), THF, 25 ꢀC; (iii) 2.8 equiv acrylonitrile, 15 mol%
Cu-nanoparticles (14e17 nm), THF, 25 ꢀC.
Result of in vitro anti-Aspergillus activity demonstrates that
3a, 3b, 4b and 4d show mild to moderate anti-Aspergillus
activity against pathogenic strains. Compound (4-methylpiper-
azin-1-yl)-acetonitrile 3a has MIC90 at 93.8 mg/disc in DDA
assay against A. flavus and A. niger. The next most active com-
pound is 3-(4-phenyl-piperazin-1-yl)-propanitrile 4b, which
exhibited activity at 93.8 mg/disc in DDA against A. fumigatus.
The structure of compound 3a has methyl group at 1-position
of piperazine nucleus and cyano group at 2-position of side
chain at 4-position of piperazine nucleus. Compound 4b has
phenyl group at 1-position of piperazine nucleus and cyano
group at 3-position of 4-position of piperazine nucleus.
6 prepared in this study have cell viability more than standard
drug gentamicin.
3. Conclusion
Series of cyano derivatives of N-alkyl and N-aryl piperazine
were synthesized and their antimicrobial activities were eval-
uated. Antibacterial activities against antibiotic susceptible
standard and clinically isolated pathogenic both Gram-positive
and Gram-negative strains (S. aureus, P. aeruginosa, S. epider-
midis, E. coli) and antifungal activities against A. fumigatus, A.
flavus and A. niger were used in the study for performing
various experiments. Compounds 3c, 4a and 6 were consid-
ered to be potent antibacterial due to significant antibacterial
activity against bacteria used in the study. Compounds 3a,
3b, 4b and 4d show mild to moderate anti-Aspergillus activity
against pathogenic strains. The in vitro cytotoxicity performed
on Hela cells showed that the compound (1-phenyl-piperazin-
4-yl)-acetonitrile 3b synthesized in this study has 95% cell
viability. The compounds 3b, 3e, 4b, 4d, 4f, and 6 were found
to have low cytotoxicity as compared to control drug gentami-
cin, with cell viability in the range of 60e95%.
2.4. In vitro cytotoxicity assay
The in vitro cytotoxicity of synthesized compounds (1-phenyl-
piperazin-4-yl)-acetonitrile 3b, [4-(4-nitro-phenyl)-piperazin-1-
yl]-acetonitrile 3e, 3-(4-phenyl-piperazin-1-yl)-propanitrile 4b,
3-[4-(2-methoxy-phenyl)-piperazin-1-yl]-propanitrile 4d, 3-[4-
(2-cyano-ethyl)-piperazin-1-yl]-propanitrile 4f and 3-[4-(2-
hydroxy-ethyl)-piperazin-1-yl]-propanitrile 6 was estimated
by MTT colorimetric assay [20]. The Hela cells were incubated
with increasing concentration of compounds 3b, 3e, 4b, 4d, 4f, 6
and gentamicin as control drug. Compounds 3e, 4b and 6 have
shown w60% cell viability at 30 mg, which was comparable
to gentamicin at the same concentration. Whereas other com-
pounds 3b, 4d and 4f showed cell viability more than gentamicin
at all the concentrations used for the assay. At a concentration of
30 mg of compound (1-phenyl-piperazin-4-yl)-acetonitrile 3b
gave more than 95% cell viability (Fig. 1). From this investiga-
tion it can be inferred that the compounds 3b, 3e, 4b, 4d, 4f, and
4. Experimental
All reagents used were of AR grade. THF was distilled
from sodium/benzophenone prior to use. Melting points
were determined using a Thomas Hoover melting point appa-
ratus and are uncorrected. 1H (300 MHz) and 13C NMR
(75 MHz) spectra were recorded on a Bruker 300 NMR spec-
trometer in CDCl3 (with TMS for 1H and chloroform-d for 13
C
i
N
N
HN
N
CN
HO
OH
6
5
Scheme 3. Reagent and conditions: 2.5 equiv acrylonitrile, 10 mol% Cu-nanoparticles (14e17 nm), THF, 25 ꢀC.

474
P. Chaudhary et al. / European Journal of Medicinal Chemistry 42 (2007) 471e476
Table 1
benzotriazole 2c and 1-{[4-(2-methoxyphenyl) piperazin-
1-yl] methyl}-1H-1,2,3-benzotriazole 2d in 87e94% yields
as a sole bt1 isomer, which was directly used for subsequent
reactions. For microanalysis purposes, the precipitate was re-
crystallized from CHCl3/hexanes (1:1).
Antibacterial activities of cyano derivatives of N-alkyl and N-aryl piperazine
showing zone of inhibition (mm) and MIC values (mg/ml) against selected
pathogenic strains
Compound
S. aureus
S. epidermidis
P. aeruginosa
E. coli
3a
3b
12/310.5
10/310.5
18/19.5
9/625.0
e
10/310.5
13/156.3
e
8/625.0
12/156.3
10/310.5
8/625.0
e
11/156.3
12/156.3
e
9/625.0
13/156.3
13/156.3
8/625.0
e
19/19.5
9/625.0
e
12/310.5
9/310.5
9/625.0
10/310.5
Spectral data of the synthesized compounds 2aed were
consistent with the reported values.
3c
3d
4.2. General procedure for the replacement of
benzotriazole group from 2aed with sodium cyanide
3e
4a
18/39.1
12/156.3
4b
4c
A solution of benzotriazolyl derivative of N-alkyl and N-
aryl piperazines 2aee (1.0 mmol) and NaCN (2.0 mmol)
was stirred in DMSO (10 ml) for 8e12 h, after removal of
the solvent in vacuo, the residue was diluted with EtOAc.
The mixture was washed with brine and dried over Na2SO4.
Evaporation of the solvent was done in vacuo and the residue
was purified by column chromatography with hexanes/EtOAc
(6:1 to 3:1) as an eluent gave 3aee in 76e88% yields.
Spectral data of the synthesized compounds 3aed were
consistent with the reported values.
4d
4e
12/156.3
e
9/625.0
e
9/625.0
e
11/156.3
4f
11/310.5
18/19.5
20/9.8
9/625.0
11/310.5
18/19.5
11/156.3
15/39.1
19/19.5
10/625.0
15/39.1
18/19.5
6
Gentamicin
e Means no activity.
as internal references) unless otherwise stated. Mass spectrum
was recorded on Hybrid Quadrupole-TOF LC/MS/MS mass
spectrometer (Q. Star XL). Column chromatography was per-
formed on silica gel (230e400 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 sil-
ica gel 60 F254 (Merck). All of the reactions were carried out
under nitrogen atmosphere.
4.2.1. (4-Methyl-piperazin-1-yl)-acetonitrile 3a
The general synthetic method described above affords 3a as
semisolid, yield 88e97%; 1H NMR (300 MHz, CDCl3): d 3.38
(s, 2H), 2.27 (s, 3H), 1.50 (m, 4H), 1.35 (m, 4H); 13C NMR
(75 MHz, CDCl3): d 113.5, 54.9, 50.05, 40.23, 30.02; LCMS
m/z found 140.3 (M þ 1), 113, 138.
4.1. General procedure for the synthesis of
benzotriazolyl derivatives of N-alkyl and N-aryl
piperazines (2aed)
4.2.2. (4-Phenyl-piperazin-1-yl)-acetonitrile 3b
The general synthetic method described above affords 3b as
1
dirty white solid, yield 88%; H NMR (300 MHz, CDCl3):
d 7.31e7.25 (m, 2H), 6.95e6.86 (m, 3H), 3.58 (s, 2H), 3.24
(t, J ¼ 4.8 Hz, 4H), 2.76 (t, J ¼ 4.9 Hz, 4H); 13C NMR
(75 MHz, CDCl3): d 150.9, 129.1, 120.1, 116.3, 114.5, 51.8,
48.9, 45.9; LCMS m/z found 202.3 (M þ 1).
To a solution of N-alkyl and N-aryl piperazines 1aed,
(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 stirred at room
temperature for 6e8 h. The precipitate formed was filtered
and washed with cold Et2O to gave pure product
1-[(4-methylpiperazin-1-yl) methyl]-1H-1,2,3-benzotriazole
2a, 1-[(4-phenylpiperazin-1-yl) methyl]-1H-1,2,3-benzotria-
zole 2b, 1-[(4-benzylpiperazin-1-yl) methyl]-1H-1,2,3-
4.2.3. (4-Benzylpiperazin-1-yl)-acetonitrile 3c
The general synthetic method described above affords 3d as
creamy white solid, yield; 88%; 1H NMR (300 MHz, CDCl3):
d 7.19e7.12 (m, 5H), 3.39 (s, 2H), 3.34 (s, 2H), 2.47e2.46
Table 2
Antifungal activities of cyano derivatives of N-alkyl and N-aryl piperazine
Compound
MIC
DDA (mg/disc)
A. flavus
MDA (mg/ml)
PSGI (mg/ml)
A. niger
A. fumigatus
A. flavus
A. niger
A. fumigatus
A. flavus
A. niger
A. fumigatus
3a
3b
93.8
187.5
e
93.7
187.5
e
187.5
187.5
500
500
e
500
500
e
500
500
e
250
500
e
250
250
e
500
250
3c
4a
e
e
e
e
e
e
e
e
e
4b
4d
187.8
375.5
e
187.5
e
93.8
187.5
e
500
1000
e
500
e
250
500
e
250
500
e
250
e
250
500
4f
4g
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
5
6
Amphotericin B
2.5
2.5
2.5
5
5
5
5
5
e Means no activity.

P. Chaudhary et al. / European Journal of Medicinal Chemistry 42 (2007) 471e476
475
120
100
4.4. Disc diffusion method
The in vitro antibacterial activity was tested by disc diffu-
sion method using pathogenic strains of S. aureus (MTCCB
737), P. aeruginosa (MTCCB 741), S. epidermidis (MTCCB
1824) and E. coli (MTCCB 1652) [15]. Compounds with
30 mg/disc concentration were impregnated on the discs. These
discs were placed on the surface of the agar plates already
inoculated with pathogenic bacteria. The plates were incu-
bated at 37 ꢀC and examined at 48 h for zone of inhibition,
if any, around the discs. Gentamicin was used in assay as
a standard control drug. An additional control disc without
any sample but impregnated with equivalent amount of solvent
(DMSO) was also used in the assay. The result of antibacterial
activity indicated that some of the compound exhibited mild to
moderate activity.
80
60
40
20
0
6
G
4b
3b
4d
4f
3e
4.5. Broth dilution method
Fig. 1. In vitro cytotoxicity assay. The compounds are as follows:
(1-phenyl-piperazin-4-yl)-acetonitrile 3b, [4-(2-methoxy-phenyl)-piperazin-1-yl]-
acetonitrile 3e, 3-(4-phenyl-piperazin-1-yl)-propanitrile 4b, 3-[4-(2-methoxy-
phenyl)-piperazin-1-yl]-propanitrile 4d, 3-[4-(2-cyano-ethyl)-piperazin-1-yl]-
The test was performed in 96-well culture plates (Tarsons).
MIC was determined by microbroth dilutions technique using
MuellereHinton broth and 96-well plates for bacteria [16]. Se-
rial nine-fold dilutions ranging from 1250 to 4.8 mg mlꢁ1 were
prepared in media. The inoculum was prepared using a 4e6 h
broth culture of each bacterial strains adjusted to a turbidity
equivalent to a 0.5 McFarland standard, diluted in broth media
to give a final concentration of 3 ꢂ 106 cfu mlꢁ1 for bacteria.
The 96-well plates containing MuellereHinton broth were in-
cubated at 35 ꢀC for 18e20 h. The MIC was defined as the
lowest concentration of compound giving complete inhibition
of visible growth.
propanitrile 4f, 3-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-propanitrile
gentamicin G.
6 and
(m, 4H), 2.39e2.37 (m, 4H); 13C NMR (75 MHz, CDCl3):
d 137.6, 128.6, 127.9, 126.8, 114.5, 62.3, 52.1, 51.4, 45.5;
LCMS m/z found 229.3 (M þ 1).
4.2.4. [4-(2-Methoxyphenyl)piprazin-1-yl]-acetonitrile 3d
The general synthetic method described above affords 3d as
1
pale solid, yield: 89%; H NMR (300 MHz, CDCl3): d 7.89e
7.86 (m, 2H), 6.87e6.89 (m, 2H), 3.869 (s, 3H), 3.57 (s, 2H),
3.14 (m, 4H), 2.81 (m, 4H); 13C NMR (75 MHz, CDCl3):
d 140.8, 125.9, 123.2, 121.0, 118.3, 114.8, 77.0, 55.4, 52.1,
45.9; LCMS m/z found 217.2 (M þ 1).
4.6. Antifungal activity assay
The anti-Aspergillus activity of all the compounds was
studied by disc diffusion, microbroth dilution and percentage
spore germination inhibition assays [17]. The pathogenic
strains of A. fumigatus (ITCC 4517), A. flavus (ITCC 5192)
and A. niger (ITCC 5405) were used in the study for perform-
ing various experiments.
4.2.5. [4-(4-Nitro-phenyl)-piperazin-1-yl]-acetonitrile 3e
The general synthetic method described above affords 3e as
yellow crystals, yield 93%; 1H NMR (300 MHz, CDCl3):
d 7.38e7.24 (m, 5H), 3.57e3.42 (m, 6H), 2.59e2.61 (m,
5H), 2.53e2.48 (m, 4H); 13C NMR (75 MHz, CDCl3):
d 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: 247.3 (M þ 1) peak.
4.7. Microbroth dilution
The test was performed in 96-well culture plates (Nunc,
Nunclon). Various concentrations of synthetic compounds in
the range of 1000e7.81 mg/ml were prepared in the wells by
two-fold dilution method. Assay was performed as per stan-
dard method described earlier [17].
4.3. General procedure for the aza-Michael reaction of
N-alkyl and N-aryl piperazines (Scheme 2)
To a stirred solution of amine (1.0 equiv) and acrylonitrile
(1.2 equiv) in THF (30 ml), Cu-nanoparticles (14e17 nm,
10 mol%) were added at room temperature and stirring was
continued for 8e16 h under nitrogen. After completion of
the reaction (TLC), THF was removed in vacuo, the reaction
mixture was treated with water and extracted with ethyl ace-
tate. The organic layer was dried over Na2SO4 and after the
removal of the solvent in vacuo, the residue was purified by
column chromatography (silica gel 250e400 mesh size).
4.8. Disc diffusion
The disc diffusion assay was performed in radiation steril-
ized Petri plates of 10.0 cm diameter (Tarsons) as described
[17]. Different concentrations in the range of 750e1.46 mg
of the test compounds were impregnated 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

476
P. Chaudhary et al. / European Journal of Medicinal Chemistry 42 (2007) 471e476
inoculated with Aspergillus spores. The plates were incubated
at 37 ꢀC and examined at 24, 48 and 96 h for zone of inhibi-
tion, if any, around the discs. The concentration, which devel-
oped the zone of inhibition of at least 6.0 mm diameter, was
considered as minimum inhibitory concentration (MIC).
viability (%) compared to control cells was calculated by
[abs]sample/[abs]control ꢂ 100.
Acknowledgements
Authors gratefully acknowledge the financial support from
Department of Science and Technology, New Delhi. PC, SN
and RK are thankful to CSIR for fellowships.
4.9. Percentage spore germination inhibition
Various concentrations of the test compounds in 90.0 ml of
culture medium were prepared in 96-well culture plates (Nunc,
Nunclon) by double dilution method [18]. The wells were pre-
pared in triplicates for each concentration. Each well was then
inoculated with 10.0 ml of spore suspension containing
100 ꢃ 5 spores. The plates were incubated at 37 ꢀC for 16 h
and then examined for spore germination with an inverted
microscope (Nikon, Diphot). The number of germinated and
non-germinated spores was counted. The lowest concentration
of the compound, which resulted in >90% inhibition of germi-
nation of spores in the wells, was considered as MIC90.
References
[1] S. Harbart, W. Albrich, D.A. Goldman, J. Huebner, Lancet Infect. Dis. 1
(2001) 251.
[2] L.A. Mitscher, S.P. Pillai, E.J. Gentry, D.M. Shankel, Med. Res. Rev. 19
(1999) 477.
[3] I. Berber, C. Cokmus, E. Atalan, Microbiology 72 (2003) 54.
[4] P.C. Appelbaum, P.A. Hunter, Int. J. Antimicrob. Agents 16 (2000) 5e15.
[5] Y. Mizuki, I. Fujawara, T. Yamaguchi, J. Antimicrob. Chemother. 37
(1996) 41.
[6] V. Snaz-Nebot, I. Valls, D. Barbero, J. Barbosa, Acta Chem. Scand. A 51
(1997) 896e903.
4.10. In vitro cytotoxicity
[7] P. Kurath, P.H. Jones, R.S. Egan, T.J. Perun, Experimentia 27 (1971) 362.
[8] W.A. Gregory, D.R. Britteli, C.-L.J. Wang, M.A. Wuonola,
R.J. McRipley, D.C. Eustice, V.S. Eberly, P.T. Bartholomew, A.M. Sler,
M. Forbes, J. Med. Chem. 32 (1988) 1218.
The toxicity of compounds (1-phenyl-piperazin-4-yl)-aceto-
nitrile 3b, [4-(2-methoxy-phenyl)-piperazin-1-yl]-acetonitrile
3d, 3-(4-phenyl-piperazin-1-yl)-propanitrile 4b, 3-[4-(2-me-
thoxy-phenyl)-piperazin-1-yl]-propanitrile 4d, 3-[4-(2-cyano-
ethyl)-piperazin-1-yl]-propanitrile 4f and 3-[4-(2-hydroxy-
ethyl)-piperazin-1-yl]-propanitrile 6 was studied by using
MTT colorimetric assay [19]. Hela cells were maintained in
Dulbecco’s modified eagle’s medium (DMEM) supplemented
with 10% heat inactivated fetal bovine serum (FBS) and
100 U/ml of streptomycinepenicillin. The cells were cultured
in a humidified 5% CO2 atmosphere at 37 ꢀC. Hela cells were
seeded onto 96-well plates at a density of 8 ꢂ 103 cells/well
and incubated for 16 h for adherence. Afterwards, the media
was aspirated from the wells and the cells were washed once
with DMEM without FBS. The compounds 3b, 3e, 4b, 4d, 4f,
and 6 and gentamicin each increasing in concentration from
10 to 30 mg were diluted with DMEM without FBS to a final vol-
ume of 75 ml and this media was added to separate wells, fol-
lowed by incubation at 37 ꢀC in humidified 5% CO2
atmosphere for 4 h. Then the media containing the compounds
was replaced with 200 ml of normal growth medium and cells
were further incubated for 48 h under same conditions. After
48 h media was replaced by 200 ml of MTT (3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide) (0.5 mg dis-
solved in 1.0 ml of DMEM) and further incubated for 2 h.
Afterwards, the supernatant was aspirated, and the formazan
crystals so formed were suspended in 100 ml iso-propanol
containing 0.06 M HCl and 0.5% SDS. Aliquots were drawn
from each well and the intensity of colour was measured spec-
trophotometrically in an ELISA plate reader at 540 nm. Un-
treated cells were taken as control with 100% viability and
cells without addition of MTT were used as blank to calibrate
the spectrophotometer to zero absorbance. The relative cell
[9] (a) S.J. Brickner, D.K. Hutchinson, M.R. Barbachyn, S.A. Garman, K.C.
Grega, S.K. Hendges, P.R. Manninem, D.S. Topps, D.A. Wanowicz, J.D.
Killburn, S. Glicknam, G.E. Zurenko, C.W. Ford, 35th Interscience
Conference on Antimicrobial Agents and Chemotherapy, San Francisco,
September 1995, F-208, p. 149.
(b) S.J. Brickner, D.K. Hutchinson, M.R. Barbaachyn, P.R. Manninem,
D.A. Wanowicz, S.A. Garmon, K.C. Grega, S.K. Hendges, D.S. Topps,
C.W. Ford, G.E. Zurenko, J. Med. Chem. 39 (1996) 673e679.
[10] M.M. McNeil, S.L. Nash, R.A. Hajjeh, M.A. Phelan, L. Conn,
B.D. Plikaytis, D.W. Warnock, Clin. Infect. Dis. 33 (2001) 641e647.
´
[11] A. Orjales, L. Alonso-Cires, L. Labeaga, R. Corcostegui, J. Med. Chem.
38 (1995) 1273e1277.
[12] Y.E. Ahmad, E. Laurent, P. Maillet, A. Talab, J.F. Teste, R. Dohkan,
G. Tran, V. Ollivier, J. Med. Chem. 40 (1997) 952e960.
[13] Y. Torisawa, K. Shinhama, T. Nishi, J. Minamikawa, Bioorg. Med.
Chem. Lett. 13 (2003) 65.
[14] (a) A.R. Katritzky, X. Lan, J.Z. Yang, O.V. Denisko, Chem. Rev. 98
(1998) 409;
(b) A.R. Katritzky, K. Yannakopoulou, P. Lue, D. Rasala, L. Urogdi,
J. Chem. Soc. Perkin Trans. 1 (1989) 225;
(c) A.R. Katritzky, K. Suzuki, H.-Y. He, J. Org. Chem. 67 (2002) 3109;
(d) A.R. Katritzky, S.K. Singh, H.Y. He, Synthesis 12 (2002) 1646;
(e) A.R. Katritzky, H.Y. He, J. Wang, J. Org. Chem. 67 (2002) 4951.
[15] A.K. Verma, R. Kumar, P. Chaudhary, A. Saxena, R. Shankar,
S. Mozumdar, R. Chandra, Tetrahedron Lett. 46 (2005) 5229.
[16] P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolke, Manual
of Clinical Microbiology, seventh ed. ASM, Washington, 1999 pp.
1527e1539.
[17] National Committee for Clinical Laboratory Standards, Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically Approved Standard M7-A5 NCCLS, fifth ed., Wayne, 2000.
[18] V. Yadav, R. Mandhan, R. Dabur, A.K. Chhillar, J. Gupta, G.L. Sharma,
J. Med. Microbiol. 54 (2005) 75.
[19] R. Dabur, G.L. Sharma, J. Ethnopharmacol. 80 (2002) 193.
[20] T. Mosmann, Rapid colorimetric assay for cellular growth and survival
application to proliferation and cytotoxicity assays, J. Immunol. Methods
65 (1983) 55.