Synthesis of 4-methyl-N′-(3-alkyl-2r,6c-diarylpiperidin-4-ylidene)-1, 2,3-thiadiazole-5-carbohydrazides with antioxidant, antitumor and antimicrobial activities

Article abstract of DOI:10.1039/c4ob00739e

The structures of the newly synthesized 4-methyl-N′-(3-alkyl-2r,6c- diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5a-5l) were confirmed by spectral and elemental analysis. The difference in the potency of activity against various free radicals, human cancer cells and microbial strains has been evaluated by SAR. Compounds with electron-donating methoxy (5i and 5c) and methyl (5h and 5b) substitutions at the para position of the phenyl showed excellent free radical scavenging effects. In the tested compounds, electron withdrawing fluoro (5k and 5e), chloro (5j and 5d), and bromo (5l and 5f) substitution at the para position of the phenyl ring attached to C-2 and C-6 carbons of the piperidine moiety outperformed cytotoxic and antimicrobial activities. Our findings suggest that the antioxidant, anti-tumor and anti-microbial activities of compounds 5a-5l create promising leads for the development of potent anti-tumor and anti-microbial agents. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00739e

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of 4-methyl-N’-(3-alkyl-2r,6c-
diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-
5-carbohydrazides with antioxidant, antitumor
and antimicrobial activities†
Cite this: Org. Biomol. Chem., 2014,
12, 5911
Kodisundaram Paulrasu,a Arul Duraikannu,b Manikandan Palrasu,c
Amirthaganesan Shanmugasundaram,d Murugavel Kuppusamye and
Balasankar Thirunavukkarasu*f
The structures of the newly synthesized 4-methyl-N’-(3-alkyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thia-
diazole-5-carbohydrazide (5a–5l) were confirmed by spectral and elemental analysis. The difference in
the potency of activity against various free radicals, human cancer cells and microbial strains has been
evaluated by SAR. Compounds with electron-donating methoxy (5i and 5c) and methyl (5h and 5b) sub-
stitutions at the para position of the phenyl showed excellent free radical scavenging effects. In the tested
compounds, electron withdrawing fluoro (5k and 5e), chloro (5j and 5d), and bromo (5l and 5f) substi-
tution at the para position of the phenyl ring attached to C-2 and C-6 carbons of the piperidine moiety
outperformed cytotoxic and antimicrobial activities. Our findings suggest that the antioxidant, anti-tumor
and anti-microbial activities of compounds 5a–5l create promising leads for the development of potent
anti-tumor and anti-microbial agents.
Received 8th April 2014,
Accepted 30th May 2014
DOI: 10.1039/c4ob00739e
www.rsc.org/obc
genic process.1–6 A living organism has protective enzymatic
and non-enzymatic antioxidant mechanisms against ROS-
Introduction
Reactive oxygen species (ROS) are essential for an organism’s induced oxidative damage. Nevertheless, these protective
essential activities, such as the regulation of cell proliferation, systems are insufficient to prevent the damage entirely.4–6 Fur-
intracellular signaling and synthesis of biologically active com- thermore, research over the past several decades has demon-
pounds and energy. However, excessive production of ROS strated that antioxidants play a protective role in multistage
causes oxidative stress and chronic diseases such as cardio- carcinogenesis.4–6 Recently, considerable attention has been
vascular disease, diabetes, and cancer. ROS are known to directly focused on identifying synthetic antioxidants that target
interact with all types of biomolecules, including proteins, various signaling pathways that are aberrant in cancer.
lipids, and DNA, resulting in oxidative cellular damage.1–5 Oxi-
The piperidin-4-one nucleus, an important class of pharmaco-
dative stress has been implicated in all stages of the carcino- phore found in a wide variety of natural alkaloids, exhibits a
wide spectrum of biological activities ranging from antibacter-
ial to anticancer.7,8 Many piperidine derivatives possess
pharmacological activities including antimicrobial, anti-
aDepartment of Chemistry, Annamalai University, Annamalainagar, 608002,
Tamilnadu, India
oxidant and anticancer activities and form an essential part of
the molecular structure of important drugs.7–11 Furthermore,
modification of position 3 of the piperidin-4-one nucleus as
well as a substitution of certain functional groups in the para
position of the phenyl ring attached to C-2 and C-6 carbons of
the piperidine moiety would result in compounds with potent
biological activities. Therefore, many researchers have focused
on modifying the piperidin-4-one pharmacophore to achieve
better biological activities.7–9 Hydrazones contain an azo-
methine –NHNvCH– proton that constitutes an important
class of compounds for new drug development. Hydrazide–
hydrazone derivatives received the attention of various medic-
bDepartment of Clinical Neurosciences, University of Calgary, Calgary,
Alberta T2N 4N1, Canada
cDepartment of Pharmacology, University of Maryland School of Medicine,
Baltimore, Maryland 21201
dDepartment of Chemistry, Saveetha School of Engineering, Saveetha University,
Thandalam, Chennai-602 105, Tamilnadu, India
eDepartment of Chemistry, Saveetha Engineering College, Thandalam,
Chennai-602 105, Tamilnadu, India
fChemistry Section, FEAT, Annamalai University, Annamalainagar,
608002, Tamilnadu, India. E-mail: drtbalasankar@rediff.com; Fax: +91 4144 239141;
Tel: +91 9944472276
†Electronic supplementary information (ESI) available: Spectral data (1H NMR,
13C NMR, H–H COSY, NOESY, HSQC, and HMBC) for compounds 5a and 5g. See
DOI: 10.1039/c4ob00739e
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inal chemists as a result of their effectual biological potencies,
viz., antimicrobial, anti-tubercular, and also anticonvulsant
activities.8,10,11 Recently, we have documented a synthesis
Results and discussion
Chemistry
system that combines 3-azabicyclonone and thiadiazole moi- Syntheses
of
4-methyl-N′-(3-methyl-2r,6c-diarylpiperidin-
eties together to produce the corresponding hydrazones with 4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide 5(a–f) and 4-methyl-
promising antimicrobial activities.12 Similarly, the present N′-(3-ethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-
study aims to prove that the addition of hydrazide into the 5-carbohydrazide 5(g–l) were carried out according to the
piperidin-4-one pharmacophore with different modifications steps shown in Fig. 1. A detailed investigation of IR, 1H NMR and
would result in compounds with potent biological activities.
In the present study, a new series of 4-methyl-N′-(3-methyl- to identify and establish the newly-synthesized compounds (5a–l).
C13 NMR spectral data with CHN analysis (Table 1) was performed
2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydra-
For 5a, all 1H and 13C signals have been assigned unambiguously
zide 5(a–f) and 4-methyl-N′-(3-ethyl-2r,6c-diarylpiperidin- using 1H–1H-COSY, NOESY, HSQC and HMBC spectra.
4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide 5(g–l) was syn-
Structural elucidation of compound 5a
thesized using a reaction of piperidin-4-one with 4-methyl-
1,2,3-thiadiazole-5-carboxylic acid hydrazide in the presence of In H NMR spectra for compound 5a, a broad and more down-
1
acetic acid in methanol. The chemical structures were con- field D2O exchangeable singlet at 10.80 ppm was characteristic of
firmed using IR, 1H-NMR, 13C-NMR and elemental analysis. In the NH amide group. Another, broad singlet signal resonated at
addition, we executed structure–activity relationship studies 2.11 ppm was assigned for the NH proton of the piperidin-4-one
using newly synthesized hydrazone derivatives with potent ring. Signal broadening is due to the faster exchange of the NH
antioxidant, anti-tumour and antimicrobial activities.
proton with solvent moisture than the resonance time scale. Two
Fig. 1 Schematic representation of the synthesis of 4-methyl-N’-(3-methyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide
5(a–f) and 4-methyl-N’-(3-ethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazides 5(g–l).
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Table 1 Analytical data of compounds 5a–5l
Elemental analysis founda (calculated) (%)
Compounds
Molecular formula
22H23N5OS
C24H27N5OS
C24H27N5O3S
Molecular weight
C
H
N
5a
5b
5c
5d
5e
5f
5g
5h
5i
C
405.52
433.57
465.57
474.41
441.50
563.31
419.54
447.60
479.59
487.10
453.16
577.33
65.06 (65.16)
66.42 (66.48)
61.89 (61.92)
55.69 (55.70)
59.03 (59.85)
46.89 (46.91)
65.80 (65.84)
67.02 (67.08)
62.59 (62.61)
56.49 (56.56)
60.59 (60.64)
47.81 (47.85)
5.62 (5.72)
6.26 (6.28)
5.75 (5.85)
4.41 (4.46)
4.74 (4.79)
3.70 (3.76)
6.00 (6.01)
6.49 (6.53)
6.01 (6.09)
4.70 (4.75)
5.03 (5.09)
4.00 (4.02)
17.21 (17.27)
16.11 (16.15)
15.01 (15.04)
14.73 (14.76)
15.81 (15.86)
12.40 (12.43)
16.63 (16.69)
15.62 (15.65)
14.58 (14.60)
14.29 (14.34)
15.33 (15.37)
12.09 (12.13)
C22H21Cl2N5OS
C22H21F2N5OS
C22H21Br2N5OS
C23H25N5OS
C25H29N5OS
C25H29N5O3S
5j
5k
5l
C23H23Cl2N5OS
C23H23F2N5OS
C23H23Br2N5OS
a The observed microanalysis values for C, H and N were within 0.4% of the theoretical values.
doublets were observed in the region of 1.17 and 3.63 ppm due to against various free radicals. Our findings provide evidence
H-2a and the methyl group of C-3 at the piperidin ring. Three that synthetic compounds (5a–5l) showed a concentration-
doublets were observed in the region of 3.43, 2.44 and 3.95 ppm dependent anti-radical activity resulting from reduction of
due to H-5e, H-5a and H-6a. A broad singlet signal appeared at DPPH•, ABTS•+, O•−, OH•, and nitric oxide radicals to their
2.53 ppm, corresponding to three proton integrals attributed to the non-radical forms. IC50 values for the free radical scavenging
methyl group of the thiadiazole ring. A multiple signal appeared at effects of ascorbic acid and various synthetic compounds
2.76 ppm corresponding to one proton integral due to H-3a.
(5a–5l) are shown in Table 2.
In 13C NMR of compound 5a, two downfield resonances at
It is well known that an increase in antioxidant activity is
162.2 and 159.8 ppm were assigned to CvN (C-9) and CvO observed with replacement of alkyl chains such as methyl,
(vN–NH–CO–) carbons, respectively. The carbon resonances ethyl to phenyl rings due to the electron resonance effect of
observed around 142.1 and 142.7 ppm were due to ipso the phenyl group.13 The results of the present study demon-
carbons. However, there were four signals around 69.35, 46.3, strate the presence of an ethyl group substitution in position 3
37.1 and 61.2 ppm, which were conveniently assigned to the of piperidin-4-one compounds (5g–5l) exerts greater inhibitory
C-2, C-3, C-5 and C-6 carbons, respectively. The 13C chemical effects against various free radicals compared to the com-
shift values of the two methyl carbons (C-3 at the piperidin pounds substituted with methyl groups in position 3 of piperi-
ring and C-4 at the thiadiazole ring) were observed at 13.9 and din-4-one (5a–5f). Several studies have demonstrated that
14.8 ppm. The signals at 164.5 and 135.0 ppm were assigned organic molecules incorporating an electron donating group
to C-4 and C-5 of the thiadiazole ring. Taken together, all the (amine, hydroxyl, methoxy and alkyl) at the para position of
above observations substantiate the proposed structure of the phenyl ring can act as free radical trapping agents and are
some 4-methyl-N′-(3-alkyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3- capable of opposing oxidative challenges.7,14–16 Compounds
thiadiazole-5-carbohydrazides (5a–5l).
possessing electron-donating methoxy (5i) and methyl (5h)
substitutions at the para position of the phenyl ring attached
to the C-2 and C-6 carbons of the piperidine moiety showed
excellent free radical scavenging effects compared to standard
antioxidant ascorbic acid, a known antioxidant used as a posi-
tive control. Compounds with electron-donating methoxy (5c)
and methyl (5b) substitutions at the para position of the
phenyl ring attached to the C-2 and C-6 carbons and methyl
substitution at position 3 of piperidin-4-one compounds
showed remarkable activities. These findings confirm reports
by other workers on the in vitro free radical scavenging effects
of organic molecules incorporating an electron donating group
(amine, hydroxyl, methoxy and alkyl) at the para position of
the phenyl ring.14–16
Biological activity
Piperidin-4-one pharmacophores are found to possess a wide
range of pharmacological properties. Molecules containing
azomethine –NHNvCH– groups constitute an important class
of compounds for new drug development.7,8,10 Therefore, we
have developed the system that combines piperidin-4-one
pharmacophore and hydrazide moieties together to produce
the corresponding hydrazones (5a–5l) with the anticipation of
several promising antioxidant, anticancer and antimicrobial
agents emerging. The present study also aimed to investigate
the structure–activity relationship for the antioxidant, cytotoxic
and antimicrobial activities of hybrid molecules containing
the piperidin-4-one pharmacophore and hydrazones.
Compounds 5a and 5g deprived of any substitutes at the
para position of the phenyl groups at the C-2 and C-6 positions
of the piperidine ring showed moderate in vitro free radical
scavenging effects against various free radicals. Compounds
possessing electron-withdrawing bromo (5l and 5f), chloro (5j
In vitro free radical scavenging effects
Twelve different hydrazone derivatives were synthesized and
evaluated for their in vitro free radical scavenging activity
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Table 2 IC50 values for free radical scavenging activity and MTT assaya
IC50 values for free radical scavenging activity (µg ml−1
)
IC50 values for MTT assay (µmol)
Compound
DPPH
ABTS
Superoxide
Hydroxyl
Nitric oxide
HeLa
A549
NL20
Ascorbic acid
4.23
5.85
4.72
3.90
14.96
11.82
8.38
5.53
3.53
3.03
9.53
12.87
7.95
4.52
5.53
4.76
4.19
4.36
3.88
5.53
6.53
5.62
6.32
8.59
7.09
—
—
—
5a
5b
5c
5d
5e
5f
5g
5h
5i
7.91
11.25
4.27
2.28
1.41
3.91
10.20
13.34
4.94
3.10
2.51
4.21
11.28
10.88
6.30
3.40
2.61
5.41
13.71
15.30
8.44
4.57
3.60
6.07
12.35
11.89
7.56
4.63
12.36
8.12
12.45
18.56
9.23
5.90
11.12
7.61
4.12
3.69
5.00
5.92
14.96
11.82
8.38
4.92
3.87
16.83
12.12
8.01
4.13
3.02
14.93
14.89
10.28
5.98
4.59
4.20
10.91
14.82
10.51
17.82
16.23
12.86
7.93
6.12
5.13
13.8
13.40
11.08
3.32
2.21
5j
5k
5l
10.53
11.38
7.18
10.58
14.32
7.02
a IC50 values were determined by plotting dose–response curves of radical scavenging activities vs. the concentration of synthetic compounds
using GraphPad Prism version 4.00 for Windows (GraphPad Software Inc., San Diego, CA).
and 5d), and fluoro (5k and 5e) substitutions at the para posi-
Contrary to reports of a positive correlation between the
tion of the piperidine moiety showed admirable in vitro free cytotoxicity and the antioxidant capacity of natural and syn-
radical scavenging effects against various free radicals. These thetic compounds,21 we found low in vitro antioxidant activity
admirable or less free radical scavenging effects of compounds in synthetic compounds 5e and 5k despite their high cyto-
with bromo, chloro and fluoro substitutions may be due to the toxicity. The strong cytotoxicity with poor antioxidant properties
electron-withdrawing inductive effect of halogens. Our results of synthetic compounds 5e and 5k may be due to the pro-
are in line with other findings.13,16,17 Research over the past oxidant effects by the electron withdrawing halogens. Although
several decades has demonstrated that excessive production of potent antioxidants often possess strong pro-oxidant activity,
toxic radical species is known to cause deleterious changes in we found low cytotoxicity in synthetic compounds with elec-
DNA, lipid, and protein oxidation. Thus, free radicals may tron donating functional groups (–CH3, –OCH3) present on the
serve as a source of mutations that initiate carcinogenesis.4,5 aryl rings attached to piperidones in line with the observations
Our results provide evidence that the ability of synthetic com- of Lee et al.22 However, multiple mechanisms regulating the
pounds to quench O•−, OH•, and nitric oxide radicals is antioxidant and cytotoxic effects of the hybrid molecules need
directly associated with the prevention of oxidative stress and to be further investigated.
free radical-induced carcinogenesis.
We also compared the cytotoxicity of the hydrazone deriva-
tives in A549 lung cancer cells and NL-20 normal lung epi-
thelial cells. Most of the compounds displayed significant
cytotoxic effects in A549 cells, although to different extents;
the synthetic compounds containing electron withdrawing
functional groups (–F, –Cl) exhibited more potent cytotoxic
effects against lung cancer cells. However, these compounds
displayed less cytotoxicity to NL-20 normal lung epithelial
cells. In particular, the results of compounds 5e and 5k seem
to suggest a strikingly differential effect on cancer cells. Other
compounds, which were toxic to lung cancer cells, were toxic
to normal lung cells to more or less the same extent as to
cancer cells. Nevertheless, the mechanisms responsible for the
differential cytotoxic effects of 5e and 5k remain unclear and
require further investigation.
Of the several synthetic compounds, only 5e and 5k, which
were demonstrated to exert potent cytotoxic effects from each
series, were used for testing their antiproliferative effects by
crystal violet blue staining assay in comparison with MMC
(50 ng) (Fig. 2). We have treated the A549 cells with synthetic
compounds 5e and 5k at 2.5, 5, and 10 µM. Compounds 5e
and 5k at 5 and 10 µM showed a greater inhibitory effect on
Anticancer effects
We investigated the cytotoxic effects of newly synthesized
hydrazone derivatives with hydrazide and various substituents
on piperidone-4-one pharmacophore (5a–5l) on human lung
epithelial carcinoma A549 and HeLa cell growth using the
MTT assay in order to validate their anticancer effects and to
correlate their antioxidant activity. Consistent with free radical
scavenging activity, all the synthesized compounds signifi-
cantly inhibited the proliferation of cancer cells in a dose-
dependent manner (0, 3, 6, 9, 12, 15, and 18 µM) after 24 h of
incubation. The inhibitory effects of synthetic compounds
were in the order: 5e > 5k > 5d > 5j > 5f > 5l > 5c > 5i > 5a > 5g
> 5b > 5h. IC50 values for the cytotoxic effects of various syn-
thetic compounds (5a–5l) are shown in Table 2. Generally com-
pounds containing electron withdrawing functional groups
(–F, –Cl) exhibited more potent cytotoxic effects against the
tested cancer cells compared to the electron donating func-
tional groups (–CH3, –OCH3) present on the aryl rings attached
to piperidones. Our results are in line with those of other
workers.7,8,18–20
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Fig. 2 Antiproliferative effects of synthetic compounds 5e and 5k in A549 (A, B and E) and NL 20 (C, D and F) cells by the crystal blue staining
method. A549 cells and NL 20 cells were plated at day 0 with an equal number. After overnight incubation, A549 and NL 20 cells were treated with
compounds 5e (A and C) and 5k (B and D) for 24 h. The representative images on day 1 were shown for cells growing in medium containing synthetic
compounds 5e (A) and 5k (5B) or MMC. IC50 values for the synthetic compounds 5e and 5k in A549 (E) and NL20 (F) cells were measured using the
crystal violet blue staining method.
A549 cells and the medium dose of 5 µM was more effective tory effects against HeLa and A549 cells in MTT assays, no
when compared to other doses and MMC, the standard anti- significant difference was observed between a methyl (5e) and
cancer agent. IC50 values for the synthetic compounds 5e and ethyl (5k) substitution at position 3 of piperidin-4-one by
5k using the crystal blue staining assay are shown in Fig. 2E crystal violet blue staining and cell migration assays. These
and 2F. The crystal violet blue staining assay using compounds results demonstrate that the synthetic compounds containing
5e and 5k also strongly supports its antiproliferative effects electron withdrawing fluoro functional groups can inhibit the
against A549 cells. The cytotoxicity of 5e and 5k was also growth and invasive potential of cancer cells and act as potent
tested in normal lung epithelial cells (NL-20). Encouragingly, anticancer agents.
5e and 5k have less cytotoxic effects on cell viability in NL-20
cells as compared to lung cancer cell lines A549. Both
Antibacterial and antifungal activity
compounds showed only approximately 10% reduction in cell
viability in the NL-20 cells.
In vitro antibacterial activity of the synthesized hydrazones was
carried out against B. subtilis, S. aureus, K. pneumoniae
P. aeruginosa and E. coli by the twofold serial dilution method
using Streptomycin as the standard. The MIC values are pre-
sented in Table 3. Analysis of in vitro antimicrobial effects of
all the 4-methyl-N′-(3-alkyl-2,6-diarylpiperidin-4-ylidene)-1,2,3-
thiadiazole-5-carbohydrazides (5a–5l) revealed a diverse range
of inhibitory activity (6.25–200 µg ml−1) against all the patho-
gens except compounds 5a and 5h, which did not show activity
Furthermore, the anti-invasive potential of synthetic com-
pounds was examined by cell a migration assay. Control cells
have a strong invasive potential as revealed by the increased
number of cells (Fig. 3A and D). However, HeLa cells treated
with 5 μM 5e and 5k can mitigate the invasive potential of
cells (Fig. 3B, C and D). Although a methyl substitution at posi-
tion 3 of piperidin-4-one compound 5e exerts greater inhibi-
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Fig. 3 Anti-invasive potential of synthetic compounds 5e and 5k by cell migration assay. Synthetic compounds 5e and 5k block the invasive advan-
tage of HeLa cells. HeLa cells were grown in 5 μM 5e and 5k overnight and plated into transwells the following day in medium containing 5 μM 5e
and 5k. Transwells were processed accordingly 2 hours after plating. Cells were counted with 10 random fields at a magnification of 200×. Repre-
sentative images of the invasive cell density are shown in Fig. 2A. All counted cell numbers were used to plot the relative invasive potential (B).
Control cells have a stronger invasive potential. HeLa cells treated with 5 μM 5e and 5k can mitigate the invasive potential of cells.
against K. pneumoniae and P. Aureus, even at a maximum con-
centration of 200 µg ml−1. The compounds deprived of any
substitutes at the para position of the phenyl groups at the C-2
and C-6 positions of the heterocyclic ring (5a and 5g) hinder
Table 3 In vitro antibacterial (MIC μM ml−1) of compounds 5a–5l by
the 2-fold serial dilution method
the growth of Bacillus subtilis, S. aureus and E. coli at a MIC
value of 50–100 µg ml−1. However, compounds possessing
MIC (μM ml−1
)
para fluoro (5e and 5k) at aryl groups in the piperidine moiety
accounts for the enhanced inhibitory effects against B. subtilis,
P. Aeruginosa, K. pneumoniae, S. aureus and E. coli at MIC
values of 3.13–25 µg ml−1 when compared to the standard anti-
biotic streptomycin. Substitution of electron withdrawing
chloro (5d/5j), and bromo (5f/5l) functional groups at aryl
groups showed modest antibacterial activity against B. subtilis,
P. aeruginosa, K. pneumoniae, S. aureus and E. coli at MIC
values of 12.5–50 µg ml−1 similar to that of standard strepto-
mycin. Several studies have documented that electron with-
drawing substitutes like fluoro, chloro and bromo substituted
2,6-diaryl piperidone derivatives exerted excellent antibacterial
and antifungal activities.7,8,10–12,14,23 Compounds 5b/5h and
5c/5i possessing electron donating methyl and methoxy substi-
tutions, respectively, at the para position of phenyl rings
B.
subtilis
S.
aureus
K.
S.
E.
coli
Compounds
pneumoniae
aeruginosa
5a
5b
5c
5d
5e
5f
5g
5h
5i
50
100
25
200
50
25
12.5
3.12
25
200
100
50
200
100
50
100
200
50
100
200
100
25
6.25
50
100
>200
100
50
12.5
50
12.5
6.25
12.5
50
200
100
12.5
25
25
25
3.12
25
200
>200
50
6.25
50
50
100
100
25
12.5
50
5j
5k
5l
25
6.25
25
50
12.5
25
12.5
Streptomycin 25
12.5
25
12.5
12.5
MIC, minimum inhibitory concentration.
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Table 4 In vitro antifungal (MIC μM ml−1) of compounds 5a–5l by the
2-fold serial dilution method
compounds 5k and 5e in terms of in vitro antioxidant activity,
the synthetic compounds 5k and 5e also possessed significant
antioxidant potential. The strong in vitro free radical scaven-
ging effects of organic molecules (5i, 5h, 5c, and 5b) may be
due to the presence of electron donating groups (methoxy and
alkyl) at the para position of the phenyl ring. However, com-
pounds possessing electron-withdrawing bromo (5l and 5f),
chloro (5j and 5d), and fluoro (5k and 5e), substitutions at the
para position of the two phenyl rings of the piperidine moiety
exerted a wide range of free radical scavenging activities and
showed more cytotoxicity to cancer cells and antimicrobial
activities. This may be due to the presence of azomethine
–NHNvCH– groups as well as electron withdrawing groups at
the para position of the aromatic ring of the piperidine
moiety. In contrast to free radical scavenging effects, the pres-
ence of a methyl group substitution in position 3 of the piperi-
din-4-one compounds (5a–5f) exerts greater inhibitory effects
against various cancer cells and microbes compared to the com-
pounds substituted with ethyl groups in position 3 of piperidin-4-
one (5g–5l). A noteworthy consideration is the fact that the syn-
thetic compounds 5k and 5e selectively exert their cytotoxicity in
cancer cells, but not in normal cells. Among the compounds
tested, 5e and 5k were selected as the lead compounds for further
studies. Studies on the molecular mechanisms by which the syn-
thetic compound exerts its antitumor and antimicrobial activities
are making headway and will be reported on in the future.
MIC (μM ml−1
)
Compounds
C. albicans
Rhizopus sp.
A. niger
A. flavus
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
50
50
50
50
6.25
25
50
150
50
25
>200
100
100
12.5
3.12
12.5
>200
200
25
100
200
25
100
>200
25
6.25
6.25
25
100
100
50
25
6.25
12.5
100
150
100
12.5
12.5
50
50
6.25
25
25
12.5
25
25
12.5
12.5
25
Fluconazole
12.5
6.25
MIC, minimum inhibitory concentration.
attached to C-2 and C-6 carbons of the piperidine moiety show
moderate antibacterial activity against all the tested bacterial
strains in the range of 25–200 µg ml−1
.
The results of the present study also provide evidence
for the antifungal effects of an array of 4-methyl-N′-(3-alkyl-2,6-
diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazides
(5a–5l) with the MIC value ranging from 6.25 to 100 µg ml−1
(Table 4). Compounds 5a and 5g that lack any substitutes at
the aryl groups showed mild and comparatively less antifungal
activity against A. flavus (100 µg ml−1) and C. albicans (50 µg
ml−1) compared to fluconazole, a known antifungal agent used
as a positive control.
Materials and methods
Materials and apparatus
Compounds possessing electron withdrawing fluoro substi-
tuents at aryl groups (5e/5k) exerted four/two fold increase in
antifungal activity against C. albicans, Rhizopus and A. flavus at
MIC values of 6.25–12.5 µg ml−1 when compared to the stan-
dard fluconazole. However, compound 5e/5k showed inhibitory
activity against A. niger similar to that of standard fluconazole.
Substitution of electron withdrawing chloro (5d/5j) and bromo
(5f/5l) functional groups at aryl groups exerted modest antifungal
activity against C. albicans, Rhizopus, A. flavus and A. niger at MIC
values of 6.25–50 µg ml−1. Furthermore, a phenyl ring with elec-
tron-donating methoxy and methyl groups at the para position
((5c/5i) and (5b/5h)) hinder the growth of all fungal strains at
All chemicals that were purchased were used without further
purification. All the reported melting points that were
measured in open capillaries were uncorrected. FT-IR analysis
was done by making a pellet of compound with KBr. Both one
and two dimensional NMR spectra were recorded in the NMR
spectrometer. A sample was prepared with a 5 mm diameter
1
tube using DMSO-d6 solvent (10 mg in 0.5 ml). H NMR and
13C NMR data were collected in 400.13 MHz and 100.62 MHz
operating frequency, respectively. Chemical shifts (δ) were
expressed in ppm with respect to TMS. Splitting patterns were
designated as follows: s-singlet, d-doublet, t-triplet, q-quartet
and m-multiplet.
MIC values ranging from 25–100 µg ml−1
.
General procedure for the synthesis of 4-methyl-N′-(3-alkyl-
2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-
carbohydrazides 5(a–l)
Conclusions
Combining piperidin-4-one pharmacophore with hydrazide The 3-alkyl-2r,6c-diaryl piperidin-4-one 3(a–l) were prepared by
moieties is gaining increasing attention as a promising strat- the condensation of appropriate ketones, aldehydes and
egy for the development new hydrazine derivatives (5a–5l) with ammonium acetate in a 1 : 2 : 1 ratio, according to the method
potent biological activities. Synthesized compounds (5a–5l) are described by Noller and Baliah.24 A reaction mixture contain-
examined for their antioxidant, anticancer and antimicrobial ing 3-alkyl-2r,6c-diaryl piperidin-4-one 3(a–l) (1 mmol) and
activities. Although the synthetic compounds with electron- 4-methyl-1,2,3-thiadiazole-5-carboxylic acid hydrazide (1.5 mmol)
donating methoxy (5i) and methyl (5h) substitutions at the was dissolved in the solvent mixture of chloroform and metha-
para position of the phenyl ring attached to the C-2 and C-6 nol (1 : 1 v/v) and acetic acid (2 ml) was added as a catalyst.
carbons of the piperidine moiety were more effective than the The reaction mixture was refluxed for about 3–4 hours. After
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completion of the reaction, the crude product was formed, fil- 68.4 (C-2), 128.0–129.1 (Ar–C), 134.9 (C-5 at thiadiazole ring),
tered and washed with a cold mixture of ethanol and water. 140.3 and 141.0 (ipso carbons), 158.7 (C-4), 162.0 (NHCO) and
The pure compounds 5 (a–l) were obtained by crystallization 164.4 (C-4 at thiadiazole ring).
from distilled ethanol. Analytical data of compounds 5a–5l are
4-Methyl-N′-(3-methyl-2r,6c-bis(p-fluorophenyl)piperidin-4-
shown in Table 1.
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5e). White solid,
4-Methyl-N′-(3-methyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3- yield: 78%, m.p: 188 °C. IR (KBr, νmax cm−1): 1651 (CvN). H
thiadiazole-5-carbohydrazide (5a). White solid, yield: 70%, NMR (δ, 400 MHz-CDCl3, ppm): 1.13 (d, 3H, CH3 at piperidin
m.p: 177 °C. IR (KBr, νmax cm−1): 1653 (CvN). 1H NMR (δ, ring), 2.09 (s, 1H, NH at piperidin ring), 2.73 (m, 1H, C3-1H),
400 MHz-CDCl3, ppm): 1.17 (d, 3H, CH3 at piperidin ring), 2.41 (dd, 1H, C5-1Ha), 2.49 (s, 3H, CH3 at thiadiazole ring),
2.11 (s, 1H, NH at piperidin ring), 2.44 (dd, 1H, C5-1Ha), 2.53 3.42 (dd, 1H, C5-1He), 3.61 (d, 1H, C2-1H), 3.92 (dd, 1H, C6-
(s, 3H, CH3 at thiadiazole ring), 2.76 (m, 1H, C3-1H), 3.44 (dd, 1H), 7.25–7.48 (m, 8H, Ar–H), 10.78 (s, 1H, N–H, amide N–H).
1H, C5-1He), 3.64 (d, 1H, C2-1H), 3.96 (dd, 1H, C6-1H), 13C NMR (δ, 400 MHz-CDCl3, ppm): 13.8 (CH3 at piperidin
7.28–7.51 (m, 10H, Ar–H), 10.80 (s, 1H, N–H, amide N–H). 13C ring), 14.7 (CH3 at thiadiazole ring), 37.1 (C-5), 46.3 (C-3), 61.2
NMR (δ, 400 MHz-CDCl3, ppm): 13.9 (CH3 at piperidin ring), (C-6), 69.3 (C-2), 126.6–128.7 (Ar–C), 134.9 (C-5 at thiadiazole
14.8 (CH3 at thiadiazole ring), 37.1 (C-5), 46.3 (C-3), 61.2 (C-6), ring), 142.0 and 142.6 (ipso carbons), 159.8 (C-4), 162.20 (NH
69.3 (C-2), 126.7–128.8 (Ar–C), 135.0 (C-5 at thiadiazole ring), CO) and 164.4 (C-4 at thiadiazole ring).
1
142.1 and 142.7 (ipso carbons), 159.8 (C-4), 162.2 (NHCO) and
4-Methyl-N′-(3-methyl-2r,6c-bis(p-bromophenyl)piperidin-4-
164.5 (C-4 at thiadiazole ring).
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5f). White solid,
1
4-Methyl-N′-(3-methyl-2r,6c-bis(p-methylphenyl)piperidin-4- yield: 65%, m.p: 220 °C. IR (KBr, νmax cm−1): 1629 (CvN). H
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5b). White solid, NMR (δ, 400 MHz-CDCl3, ppm): 1.13 (d, 3H, CH3 at piperidin
1
yield: 65%, m.p: 170 °C, IR (KBr, νmax cm−1): 1647 (CvN). H ring), 2.17 (s, 1H, NH at piperidin ring), 2.39 (dd, 1H, C5-1Ha),
NMR (δ, 400 MHz-CDCl3, ppm): 1.15 (d, 3H, CH3 at piperidin 2.68 (s, 3H, CH3 at thiadiazole ring), 2.73 (m, 1H, C3-1H), 3.27
ring), 2.07 (s, 1H, NH at piperidin ring), 2.37 (dd, 1H, C5-1Ha), (dd, 1H, C5-1He), 3.59 (d, 1H, C2-1H), 3.93 (d, 1H, C6-1H),
2.38 (s, 6H, CH3 at phenyl ring), 2.75 (s, 3H, CH3 at thiadiazole 7.26–7.49 (m, 8H, Ar–H), 10.13 (s, 1H, N–H, amide
ring), 2.75 (m, 1H, C3-1H), 3.21 (d, 1H, C5-1He), 3.56 (d, 1H, N–H).13C NMR (δ, 400 MHz-CDCl3, ppm): 13.9 (CH3 at piperidin
C2-1H), 3.87 (d, 1H, C6-1H), 7.15–7.36 (m, 8H, Ar–H), 9.96 (s, ring), 14.9 (CH3 at thiadiazole ring), 37.0 (C-5), 46.3 (C-3), 61.2
1H, N–H, amide N–H). 13C NMR (δ, 400 MHz-CDCl3, ppm): (C-6), 69.3 (C-2), 126.7–128.8 (Ar–C), 135.0 (C-5 at thiadiazole
13.8 (CH3 at piperidin ring), 15.0 (CH3 at thiadiazole ring), ring), 142.6 and 142.0 (ipso carbons), 159.5 (C-4), 161.9
21.1 (CH3 at phenyl ring), 36.9 (C-5), 46.2 (C-3), 60.8 (C-6), 69.0 (NHCO) and 164.6 (C-4 at thiadiazole ring).
(C-2), 126.5–129.3 (Ar–C), 135.1 (C-5 at thiadiazole ring), 137.7
and 139.7 (ipso carbons), 159.5 (C-4), 161.5 (NHCO), and 164.5 thiadiazole-5-carbohydrazide (5g). White solid, yield: 70%,
(C-4 at thiadiazole ring).
mp: 189 °C. IR (KBr, νmax cm−1): 1651 (CvN). 1H-NMR (δ,
4-Methyl-N′-(3-ethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-
4-Methyl-N′-(3-methyl-2r,6c-bis(p-methoxyphenyl)piperidin- 400 MHz-CDCl3, ppm): 0.92 (t, 3H, CH3 at piperidin ring), 1.28
4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5c). White solid, (m, 2H, CH2 at piperidin ring), 1.90 (s, 1H, NH at piperidin
yield: 73%, m.p: 160 °C. IR (KBr, νmax cm−1): 1651 (CvN). ring), 2.43 (d, 1H, C5-1Ha), 2.68 (s, 3H, methyl at thiadiazole
1H-NMR (δ, 400 MHz-CDCl3, ppm): 1.14 (d, 3H, CH3 at piper- ring), 2.68 (t, 1H, C3-1H), 3.28 (dd, 1H, C5-1He), 3.75 (d, 1H,
idine ring), 2.04 (s, 1H, NH at piperidin ring), 2.37 (t, 1H, C2-1H), 3.94 (d, 1H, C6-1H), 7.32–7.48 (m, 10H, Ar–H), 10.14
C5-1Ha), 2.69 (s, 1H, CH3 at thiadiazole ring), 2.69 (m, 1H, (s, 1H, N–H, amide NH). 13C (δ, 400 MHz-CDCl3, ppm): 12.41
C3-1H), 3.25 (dd, 1H, C5-1He), 3.54 (d, 1H, C2-1H), 3.80 (dd, (CH3 at piperidin ring), 15.0 (CH3 at thiadiazole), 19.4 (CH2 at
1H, C6-1H), 3.88 (s, 6H, OCH3), 6.86–7.41 (m, 8H, Ar–H), 10.32 piperidin ring), 37.3 (C-5), 52.7 (C-3), 61.1 (C-6), 67.8 (C-2),
(s, 1H, N–H amide N–H). 13C NMR (δ, 400 MHz-CDCl3, ppm): 126.6–128.8 (Ar–C), 135.1 (C-5 at thiadiazole), 142.0 and 142.6
13.8 (CH3 at piperidin ring), 15.0 (CH3 at thiadiazole ring), (ipso carbons) and 158.1 (C-4), 161.6 (NHCO) and 164.6 (C-4 at
37.1 (C-5), 46.4 (C-3), 55.3 (OCH3), 60.5 (C-6), 68.7 (C-2), thiadiazole).
113.8–128.8 (Ar–C), 134.3 and 134.9 (ipso carbons), 135.1 (C-5
4-Methyl-N′-(3-ethyl-2r,6c-bis(p-methylphenyl)piperidin-4-
at thiadiazole ring), 159.4 (C-4), 161.8 (NHCO) and 164.5 (C-4 ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5h). White solid,
1
at thiadiazole ring).
yield: 65%, m.p: 150 °C. IR (KBr, νmax cm−1): 1651 (CvN). H
4-Methyl-N′-(3-methyl-2r,6c-bis(p-chlorophenyl)piperidin-4- NMR (δ, 400 MHz-CDCl3, ppm): 0.91 (t, 3H, CH3 at piperidin
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5d). White solid, ring), 1.46 (m, 2H, CH2 at piperidin ring), 2.01 (s, 1H, NH at
1
yield: 70%, m.p: 204 °C. IR (KBr, νmax cm−1): 1651 (CvN). H piperidin ring), 2.34 (s, 6H, CH3 at phenyl ring), 2.39 (dd, 1H,
NMR (δ, 400 MHz-CDCl3, ppm): 1.14 (s, 3H, CH3 at piperidin C5-1Ha), 2.60 (m, 1H, C3-1H), 2.68 (s, 3H, CH3 at thiadiazole
ring), 2.08 (s, 1H, NH at piperidin ring), 2.36 (dd, 1H, C5-1Ha), ring), 3.25 (d, 1H, C5-1He), 3.70 (d, 1H, C2-1H), 3.89 (d, 1H,
2.64 (s, 3H, CH3 thiadiazole ring), 2.69 (m, 1H, C3-1H), 3.35 C6-1H), 7.00–7.36 (m, 8H, Ar–H), 10.16 (s, 1H, N–H amide
(dd, 1H, C5-1He), 3.62 (d, 1H, C2-1H), 3.93 (dd, 1H, C6-1H), N–H). 13C NMR (δ, 400 MHz-CDCl3, ppm): 12.4 (CH3 at piperi-
7.32–7.44 (m, 8H, Ar–H), 10.51 (s, 1H, N–H, amide N–H). 13C din ring), 15.0 (CH3 at thiadiazole ring), 19.4 (CH2 at piperidin
NMR (δ, 400 MHz-CDCl3, ppm): 13.7 (CH3 at piperidin ring), ring), 21.2 (CH3 at phenyl group), 37.4 (C-5), 52.7 (C-3), 60.9
14.9 (CH3 at thiadiazole ring), 36.9 (C-5), 46.2 (C-3), 60.4 (C-6), (C-6), 67.5 (C-2), 126.5–129.3 (Ar–C), 135.1 (C-5 at thiadiazole
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ring), 139.1–139.7 (ipso carbons), 158.4 (C-4), 161.6 (NHCO) (C-6), 69.9 (C-2), 128.3–132.0 (arc), 135.0 (C-5 at thiadiazole
and 164.6 (C-4 at thiadiazole ring). ring), 140.8 and 141.4 (ipso carbons), 157.0 (C-4), 161.6
4-Methyl-N′-(3-ethyl-2r,6c-bis(p-methoxyphenyl)piperidin-4- (NHCO) and 164.6 (C-4 at thiadiazole ring).
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5i). White solid,
In vitro free radical scavenging assays
yield: 73%, m.p: 151 °C. IR (KBr, νmax cm−1): 1649 (CvN).
1H-NMR (δ, 400 MHz-CDCl3, ppm): 0.91 (t, 3H, CH3 at piper- The free radical scavenging capacity was evaluated by the
idine ring), 1.46 (m, 2H, CH2 at piperidin ring), 2.17 (s, 1H, DPPH assay described by Blois.25 The total antioxidant poten-
NH at piperidin ring), 2.39 (t, 1H, C5-1Ha), 2.60 (t, 1H, C3-1H), tial was measured by the ABTS assay that measures the relative
2.60 (s, 1H, CH3 at thiadiazole ring), 3.31 (dd 1H, C5-1He), ability of antioxidants to scavenge the ABTS•+ cation radical
3.67 (d, 1H, C2-1H), 3.82 (dd, 1H, C6-IH), 3.82 (s, 6H, OCH3), generated in the aqueous phase.26 Hydroxyl radical scavenging
7.26–7.39 (m, 8H, Ar–H), 10.60 (s, 1H, N–H amide N–H). 13C activity was determined by the method of Halliwell et al.27
NMR (δ, 400 MHz-CDCl3, ppm): 12.5 (CH3 at piperidin ring), on the basis of the ability to compete with deoxyribose for
15.0 (CH3 at thiadiazole ring), 19.4 (CH2 at piperidin ring), hydroxyl radicals. The nitric oxide radical inhibition activity
37.7 (C-5), 53.0 (C-3), 55.3 (OCH3), 60.6 (C-6), 67.2 (C-2), was evaluated according to the method of Nishimiki et al.28
113.9–128.9 (Ar–C), 134.3 (C-5 at thiadiazole ring), 134.9 and Superoxide anions derived from dissolved oxygen by a PMS/
135.1 (ipso carbons) and 158.9 (C-4), 162.0 (NHCO), and 164.5 NADH coupling reaction reduced nitro blue tetrazolium (NBT),
(C-4 at thiadiazole ring).
which was measured by the method of Garrat using Griess
4-Methyl-N′-(3-ethyl-2r,6c-bis(p-chlorophenyl)piperidin-4- reagent.29
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5j). White solid,
yield: 70%, m.p: 169 °C. IR (KBr, νmax cm−1): 1645 (CvN). H
1
Cell culture and maintenance
NMR (δ, 400 MHz-CDCl3, ppm): 0.91 (t, 3H, CH3 at piperidin HeLa cells derived from cervical cancer cells, adenocarcinoma
ring), 1.45 (m, 2H, CH2 at piperidin ring), 2.00 (s, 1H, NH at human alveolar basal epithelial cells (A549) and normal lung
piperidin ring), 2.37 (t, 1H, C5-1Ha), 2.56 (t, 1H, C3-1H), 2.69 epithelial cells (NL20) were obtained from the National Centre
(s, 3H, CH3 at thiadiazole ring), 3.28 (dd, 1H, C5-1He), 3.72 (d, for Cell Sciences (NCCS), Pune, India. The cells were main-
1H, C2,-1H), 3.91 (dd, 1H, C6-1H), 7.26–7.43 (m, 8H, Ar–H), tained in minimum essential medium, Dulbecco’s modified
10.23 (s, 1H, N–H, amide N–H). 13C NMR (δ, 400 MHz-CDCl3, Eagle’s medium (DMEM), and Ham’s F12 medium sup-
ppm): 12.4 (CH3 at piperidin ring), 15.0 (CH3 at thiadiazole plemented with 10% fetal bovine serum (FBS) (Sigma
ring), 19.4 (CH2 at piperidin ring), 37.3 (C-5), 52.7 (C-3), 60.4 Chemical Co., St Louis, USA), penicillin (100 U ml−1) and
(C-6), 66.9 (C-2), 128.0–129.2 (arc), 133.9 (C-5 at thiadiazole streptomycin (100 µg ml−1) as antibiotics (Himedia, Mumbai,
ring), 141.0 and 140.4 (ipso carbons), 157.4 (C-4), 161.9 India) under a humidified atmosphere of 5% CO2 and 95% air
(NHCO) and 164.5 (C-4 at thiadiazole ring).
in a CO2 incubator.
4-Methyl-N′-(3-ethyl-2r,6c-bis(p-fluorophenyl)piperidin-4-
Cell viability assay by MTT
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5k). White solid,
1
yield: 78%, m.p: 168 °C. IR (KBr, νmax cm−1): 1641 (CvN). H Cell survival was assessed by the MTT assay. HeLa, A549 and
NMR (δ, 400 MHz-CDCl3, ppm): 0.91 (t, 3H, CH3 at piperidin NL20 cells, grown to approximately 80% confluence, were tryp-
ring), 1.42 (m, 2H, CH2 at piperidin ring), 1.99 (s, 1H, NH at sinized, counted, seeded in 96-well plates with an average
piperidin ring), 2.54 (t, 1H, C5-1Ha), 2.57 (t, 1H, C3-1H), 2.65 population of 1000 cells per well, incubated overnight, and
(s, 3H, CH3 at thiadiazole ring), 3.29 (dd, 1H, C5-1He), 3.73 (d, then treated for 24 h with compounds 5a–5l (0, 3, 6, 9, 12, 15,
1H, C2-1H), 3.92 (dd, 1H, C6-1H), 7.00–7.46 (m, 8H, Ar–H), and 18 µM). All experiments were done using three replicates.
10.38 (s, 1H, N–H, amide N–H). 13C NMR (δ, 400 MHz-CDCl3, Untreated cells were used as controls.
ppm): 12.4 (CH3 at piperidin ring), 15.0 (CH3 at thiadiazole
ring), 19.4 (CH2 at piperidin ring), 37.5 (C-5), 52.9 (C-3), 60.4
Cell proliferation assay by the crystal blue staining method
(C-6), 66.9 (C-2), 128.3–137.7 (arc), 135.0 (C-5 at thiadiazole We have treated the A549 cells with synthetic compounds 5e
ring), 138.4 and 138.4 (ipso carbons), 157.8 (C-4), 161.9 and 5k at 2.5, 5, and 10 µM. Compounds 5e and 5k at 5 and
(NHCO) and 164.5 (C-4 at thiadiazole ring).
10 µM showed a greater inhibitory effect on A549 cells and the
4-Methyl-N′-(3-ethyl-2r,6c-bis(p-bromophenyl)piperidin-4- medium dose of 5 µM was more effective when compared to
ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5l). White solid, other doses and MMC, the standard anticancer agent. There-
1
yield: 65%, m.p: 177 °C. IR (KBr, νmax cm−1): 1647 (CvN). H fore, we have used 5 and 10 µM doses of the synthetic com-
NMR (δ, 400 MHz-CDCl3, ppm): 0.91 (t, 3H, CH3 at piperidin pounds 5e and 5k for crystal violet blue staining and cell
ring), 1.27 (m, 2H, CH2 at piperidin ring), 2.01 (s, 1H, NH at migration assay. A549 and NL20 cells, grown to approximately
piperidin ring), 2.35 (t, 1H, C5-1Ha), 2.56 (t, 1H, C3-1H), 2.72 80% confluence, were trypsinized, counted, and seeded in
(s, 3H, CH3 at thiadiazole ring), 3.24 (dd, 1H, C5-1He), 3.71 (d, 12-well plates with an average population of 4000 cells per
1H, C2-1H), 3.90 (d, 1H, C6-1H), 7.26–7.52 (m, 8H, Ar–H), well, incubated overnight, and then treated for 24 h with com-
10.07 (s, 1H, N–H amide N–H). 13C NMR (δ, 400 MHz-CDCl3, pounds 5e and 5k (5 and 10 μM). All experiments were done
ppm): 12.4 (CH3 at piperidin ring), 15.1 (CH3 at thiadiazole using three replicates. Untreated cells were used as controls.
ring), 19.5 (CH2 at piperidin ring), 37.1 (C-5), 52.6 (C-3), 60.4 MMC (50 ng ml−1) was used as a positive control.
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IC50 values for the synthetic compounds 5e and 5k in A549 Drug standards are streptomycin for anti-bacterial activity and
and NL20 cells was also confirmed using the crystal blue stain- fluconazole for fungal studies.
ing assay by the method of Itagaki et al.30 A549/NL20 cells
were seeded in 96-well microplates which contained serially
diluted synthetic compounds 5e and 5k (0, 1.5/3, 3/6, 6/9, 9/12,
12/15, 15/18, and 18.21 µM). The cells were incubated for 24 h,
Acknowledgements
and then fixed, stained with crystal violet. The cells were The authors would like to thank the Department of Chemistry,
washed with PBS two times and a third to half the total well Annamalai University, India for the recording of the NMR
volume of straight methanol was added to solubilize the dye. spectra. We are grateful to CECRI, Karaikudi, for providing
The intensity of the dye colours was read at 540 nm. After analytical data. The authors extend their thanks to
spectrophotometric measurement, the concentrations of test the RMMCH, Annamalai University for the antimicrobial
materials that inhibited the absorbance to 50% of the control studies.
level (IC50) were determined. All experiments were done using
three replicates. Untreated cells were used as controls.
Notes and references
Cell migration assay
HeLa cells were grown in 5 μM 5e and 5k for 24 h and plated
into a chamber with transwells the following day in a medium
containing 5 μM 5e and 5k in triplicate as described in the pro-
cedure provided by the manufacturer. The chamber transwell
was taken out of the chamber 2 h after seeding, and was fixed
and stained with DAPI. The number of cells per microscope
field was calculated by averaging 10 fields randomly selected.
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