Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one oximes on human cervical cancer cells

Article abstract of DOI:10.1007/s12039-014-0622-z

A series of fifteen diversified N-benzylpiperidin-4-one oximes were synthesized and characterized by their NMR spectral data. Additionally, single-crystal XRD analysis was performed for the representative symmetrically and unsymmetrically substituted molecules. All the synthesized oximes from unsymmetrical ketones existed as E-isomer as witnessed by their NMR and XRD data. Among the synthesized target compounds that evaluated for their in vitro cytotoxicity against human cervical carcinoma (HeLa) cells, five compounds were potent with IC₅₀ < 17 μM. 1-Benzyl-2,6-bis(4-isopropylphenyl)- 3-methylpiperidin-4-one oxime 3c with an IC₅₀ of 13.88 μM was found to be the best active compound as depicted by the microscopic analysis.

Full text of DOI:10.1007/s12039-014-0622-z

J. Chem. Sci. Vol. 126, No. 3, May 2014, pp. 861–873. ꢀc Indian Academy of Sciences.
Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one
oximes on human cervical cancer cells
a,b
c
d
SOMESHWAR D DINDULKAR , IRA BHATNAGAR , RUPESH L GAWADE ,
d
a
e
VEDAVATI G PURANIK , SE-KWON KIM , DONG HYUN ANH ,
PARAMASIVAM PARTHIBAN and YEON TAE JEONG
Department of Image Science and Engineering, Pukyong National University, Busan 608 737,
Republic of Korea
Institute of MGM’s Jawaharlal Nehru Engineering College, Aurangabad, 431 003, India
Nanotheranostics Laboratory, Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
Center for Materials Characterization, National Chemical Laboratory, Pune 400 008, India
Department of Food Science and Technology, Pukyong National University, Busan 608 737,
f
a,∗
a
b
c
d
e
Republic of Korea
f
Department of Chemistry, Veltech Multitech Dr. Rangarajan Dr. Sakunthala Engineering College, Chennai
6
00 062, India
e-mail: ytjeong@pknu.ac.kr
MS received 28 May 2012; revised 13 January 2014; accepted 17 January 2014
Abstract. A series of fifteen diversified N-benzylpiperidin-4-one oximes were synthesized and characterized
by their NMR spectral data. Additionally, single-crystal XRD analysis was performed for the representative
symmetrically and unsymmetrically substituted molecules. All the synthesized oximes from unsymmetrical
ketones existed as E-isomer as witnessed by their NMR and XRD data. Among the synthesized target com-
pounds that evaluated for their in vitro cytotoxicity against human cervical carcinoma (HeLa) cells, five com-
pounds were potent with IC50 < 17 μM. 1-Benzyl-2,6-bis(4-isopropylphenyl)-3-methylpiperidin-4-one oxime
3
c with an IC50 of 13.88 μM was found to be the best active compound as depicted by the microscopic analysis.
Keywords. N-benzylpiperidin-4-one oximes; cytotoxicity, HeLa cells; single-crystal XRD.
1. Introduction
and anticancer property over a wide range of cancer
11–13
cells.
The structure–activity relationship (SAR)
Cervical cancer, a slow growing squamous cell car-
cinoma, caused by human papillomavirus (HPV) is
the second most common cancer after breast cancer
that affects women.1 Although a number of new and
effective medicines have been implemented in the can-
cer arena, some limitations exist in present therapeu-
tic regime such as target non-specificity, associated side
effects and lack of potency.4 Hence the need of new,
safe and potent anticancer compounds is motivating the
medicinal chemists towards the invention and develop-
ment of effective molecules. Considering the reports
studies from differently substituted piperidones reveal
that the nature of functional groups and position of
the substituents are important to effect the biological
–3
6
–10
actions.
2. Experimental
,5
2.1 NMR experiments
All 1D NMR spectra of the synthesized compounds
were recorded on Jeol JNM ECP 400 NMR spectro-
on piperidin-4-one heterocycles (figure 1a and b) as
1
13
meter at 294 K. The H and C NMR spectra were
measured, respectively 0.03 M and 0.05 M solutions in
good cytotoxic agents,6
–10
we synthesized some N-
benzylated piperidin-4-one oximes (figure 1c) as target
molecules.
In fact, the chalcones with piperidone core (a) are the
heterocyclic analogs of α,β-unsaturated ketones (cur-
cumin). They are reported to possess a strong cytotoxic
CDCl with TMS as internal reference in 5 mm NMR
3
1
tubes. The pulse conditions were as follows: H NMR
spectra: SF 399.78 MHz, AQ 2.73 s, NS 32, DS 0, SW
◦
5
998.8 Hz, pulse 4.65 μs, angle 45 , width 9.3 μs,
DR 0.366 Hz, RD 5 s, RG 13, data points 16384, pre
13
scan delay 1 s; C NMR spectra: SF 100.52 MHz, AQ
∗
For correspondence
1.25 s, NS 250, DS 4, SW 26178.01 Hz, Pulse 3.13 μs,
8
61

8
62
Someshwar D Dindulkar et al.
NOCH
3
2
.4 General procedure for the synthesis
O
R
R
of 1-benzyl-2,6-diarylpiperidin-4-ones (2a–2o)
X
X
N
R
X
X
A mixture of respective 2,6-diarylpiperidin-4-ones
(0.01 mol), anhydrous potassium carbonate (0.02 mol,
2.76 g) and benzyl bromide (0.015 mol, 1.78 mL) in
DMF (20 mL) was stirred at room temperature for
N
R
(a)
(b)
NOH
12–36 h. Progress and completion of the reactions
R
R
were monitored by the TLC. After the completion,
an excess of ice-cold water was added and extracted
with dichloromethane (3 × 15 mL). The organic layer,
thus separated was thrice washed with brine solution
X = halo/alkyl/alkoxy
R = H/alkyl/alkoxy/acyloxy
N
X
X
(
3 × 10 mL) and dried over anhydrous Na SO . Then
2
4
(
c)
the organic layer was concentrated in rota to obtain
the crude product, followed by purification on silica-
gel (Merck 230–400 mesh), using n-hexane and ethyl
Target Compounds
Figure 1. General structure of literature compounds (a)
and (b) with good cytotoxicity against various cell line and acetate mixture, afforded the pure 1-benzyl-3-alkyl-2,6-
synthesized target compounds (c).
diarylpiperidin-4-ones 2a–2o in good yields 87–92%.
All the N-benzylated piperidine-4-ones were character-
◦
angle 30 , width 9.4 μs, DR 0.798 Hz, RD 1 s, RG
ized by their analytical and spectral data, and confirmed
1
2
5, data points 32768, pre scan delay 1 s. The H and
C chemical shift values are given in δ scale (ppm)
and referred to TMS, via the solvent signals ( H, resid-
18,19
with previous reports.
13
1
13
ual CHCl at 7.26 ppm; C, CHCl at 77.16 ppm). 2.5 General procedure for the synthesis
3
3
Coupling constants J are reported in Hz.
of 1-benzyl-2,6-diarylpiperidin-4-one oximes (3a–3o)
A
mixture of 1-benzyl-2,6-diarylpiperidin-4-one
2.2 Single-crystal XRD
(0.002 mol), hydroxylamine hydrochloride (0.003 mol)
and sodium acetate trihydrate (0.003 mol) were dis-
solved in ethanol (5 mL). The resulting reaction
mixture was refluxed and the formation of the desired
compound monitored by TLC analysis. After com-
pletion of reaction, solvent was evaporated using
rota-vapour, water was added and extracted with ethyl
acetate (3 × 15 mL). The organic layer was dried
The X-ray diffraction quality crystals of 3d and 3n
were obtained by slow evaporation from chloroform
and the crystal structure was determined by the X-ray
diffraction data collected on a Bruker SMART APEX
14
CCD diffractometer. X-ray analysis (3d and 3n) were
performed at 296 K temperature using Mo–Kα radia-
◦
tion (λ = 0.7107 Å ) to a maximum θ range of 25 .
(
anhydrous Na SO ) and the solvent were evaporated
2 4
Crystal to detector distance 6.05 cm, 512 × 512 pix-
◦
on a rota-vapour. In a few cases, further purification
was required; hence, they either recrystallized using
ethanol or purified by column chromatography.
els/frame, oscillation/frame −0.3 , maximum detector
◦
swing angle = −30.0 , beam centre = (260.2, 252.5), in
plane spot width = 1.24, SAINT integration. SHELX-
9
7 (ShelxTL) was used for structure solution and full
2
15
matrix least squares refinement on F . Hydrogen
atoms were included in the refinement as per the riding
model.
2.6 Cytotoxicity assay (MTT assay)
The HeLa cell line was obtained from American
Type Culture Collection (Manassas, VA, USA). Dul-
becco’s Modified Eagle’s Medium (DMEM) was pur-
2.3 General procedure for the synthesis
chased from BioWhittakerꢀ
R
, whereas fetal bovine
of 2,6-diarylpiperidin-4-ones (1a–1o)
serum (FBS) and other cell culture materials were
All the parent 2,6-diarylpiperidin-4-ones were synthe- purchased from Gibco BRL Life Technologies, USA.
sized by adopting the literature precedent of Noller Paraformaldehyde and bisbenzimide Hoechst 33342
16
and Baliah through the condensation of respective stain were procured from Sigma–Aldrich Corp., St.
ketones, aldehydes and ammonium acetate in 1:2:1 Louis, MO, USA, and MTT [3-(4,5-dimethylthiazol-
ratio. All the synthesized piperidin-4-ones are in good 2-yl)-2,5-diphenyltetrasolium bromide] was purchased
17–19
agreement with their NMR data reported earlier.
from Biosesang Inc., Korea.

Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one oximes
863
fluorescence microscope (CTR 6000; Leica, Wetzlar,
Germany).
Mononuclear cells from peripheral blood of healthy
adults was used to isolated PBMCs using Ficoll-
Hypaque (Pharmacia, Freiburg, FRG) density gradient
20
centrifugation. Cells were cultured in T-75 tissue cul-
2.8 Annexin V-PI dual staining to access apoptosis
◦
ture flasks (Nunc, Denmark) at 37 C in a 5% CO
2
humidified incubator using appropriate media supple-
mented with DMEM containing 10% heat-inactivated
FBS, 100 units/mL Penicillin and 100 μg/mL Strepto-
mycin. Cells were seeded in a 96 well microtiter plate
Annexin V-FITC/PI dual staining was performed to
access the level of apoptosis and cell death in HeLa
cells treated with IC50 concentration 13.88 μM of
compound 3c, 24 h post treatment. Annexin V-FITC
and PI staining solutions used were components of
the FITC annexin V Apoptosis Detection Kit (BD
4
containing 100 μL medium at a final density of 2 × 10
cells/well at identical conditions. After overnight incu-
bation, the cells were treated with different concentra-
tions of test compounds (6.25–100 μg/mL) or DMSO
TM
Pharmingen ). The staining procedure was followed
as per manufacturer’s instructions. Briefly, HeLa cells
(carrier solvent) in a final volume of 200 μL. After
◦
incubated at 37 C with 5% CO , 0, 4, 12 and 24 h
2
2
4 h, 10 μL of MTT (5 mg/mL) was added to each
post treatment with 13.88 μM of compound 3c., were
harvested using 1X Trypsin-EDTA solution prepared
in serum free DMEM. Cells were washed twice with
◦
well and the plate was incubated at 37 C in the dark
for 4 h. Then the media along with MTT was removed
and the formazan crystals were solubilised by adding
DMSO (100 μL/well). Finally, the reduction of MTT
was quantified by reading the absorbance at 570 nm by
cold PBS and then resuspended in 1X binding buffer
6
(
provided with the kit) at a concentration of 1 × 10
5
cells/ml.100 μl of this solution (1 × 10 cells)
was transferred to a 5 ml culture tube followed by
addition of 5 μl of FITC Annexin V, gentle vortexing
GENiosꢀ
R
microplate reader (Tecan Austria GmbH).
Effects of the test compounds on cell viability were cal-
culated using cells treated with DMSO as control. The
data were subjected to linear regression analysis and the
regression lines were plotted for the best straight-line
fit.
◦
and incubation for 15 min at room temperature (25 C)
in dark. 2 μl of PI staining solution was then added
to these Annexin V-FITC labelled cells and incubated
further for 5 min under similar conditions. Finally,
400 μl of 1X binding buffer was added to each tube.
2
.7 Altered morphology study
Cells were mounted on glass slides and observed under
fluorescence microscope (CTR 6000; Leica, Wetzlar,
Germany).
5
The altered morphology of exposed cells (1 × 10
cells/well) at IC concentration was studied after
50
2
4 h using phase contrast microscope (DMI6000B,
1
13
2
.9 H and C NMR data of the target compounds
Leica Microsystems, Wetzlar, Germany). Subsequently,
the cells were Hoechst stained to observe the
nuclear/chromosomal condensation that occurred after
treatment with the test compound. For staining the
cells, 96 well cell culture plates were used to cul-
2
.9a 1-Benzyl-3methyl-2,6-diphenylpiperidin-4-one
oxime (3a, C H N O): Faint pink solid; Yield:
9%; m.p.: 176–178 C; H NMR (400 MHz, CDCl ):
25
26
2
◦
1
8
3
δ = 8.59 (s, OH), 7.50 (t, J = 9.7 Hz, 4H), 7.37–7.33
4
ture the cells (1 × 10 cells/well) in three replicates
(
6
3
m, 4H), 7.32–7.24 (m, 2H), 7.10 (t, J = 3.1 Hz, 3H),
to treat with the ideal lead compound 3c. Then the
.83–6.77 (m, 2H), 3.73 (dd, J = 3.5, 11.5 Hz, H-6a),
◦
cells were incubated at 37 C overnight and the media
was removed to wash the cells twice with phosphate
buffered saline (PBS) and fixed with 4% paraformalde-
.57 (d, J = 15.0 Hz, 1H of N-CH ), 3.48–3.36 (m, 3H
2
of H-2a, H-3a and 1H of N-CH merged), 2.58–2.51
2
(
m, H-5a), 2.14 (dd, J = 11.7, 13.9 Hz, H-5e), 0.76 (d,
◦
10
hyde in PBS for one day at −4 C. Further, the cells
13
J = 6.6 Hz, CH at C-3) ppm; C NMR (100.52 MHz,
3
were stained with nuclear binding dye Hoechst 33342
ꢁ
CDCl ): δ = 159.64 (C-4), 144.13 (C-6 ), 142.54
3
(1 μg/mL of the fluorescent DNA-binding dye, bis-
(
1
(
3
C-2’), 136.70 (N-Bn ipso carbon), 130.08, 129.10,
benzimide Hoechst 33342 stain was added to the fixed
cells and incubated for 20 min at room temperature)
to expose the nuclear condensation/aggregation due to
the effect of the compound 3c. Similarly, PI stock
solution (1 mg/mL) was diluted to 1:3000 with 1X
PBS and added to the cells for allowing the stain to
28.73, 128.46, 127.84, 127.64, 127.41, 126.67, 72.36
C-2), 63.96 (C-6), 53.43 (N-CH -Ph), 43.90 (C-3),
5.06 (C-5), 12.84 (CH at C-3) ppm; and HRMS: m/z
calculated: 370.2045, found: 370.4231.
2
3
get permeated by the dead cells. The Hoechst and PI 2.9b 1-Benzyl-3-methyl-2,6-bis(4-methylphenyl)pipe-
stained cells were visualized and photographed under ridin-4-one oxime (3b, C H N O): Off-white solid;
27
30
2

8
64
Someshwar D Dindulkar et al.
◦
1
Yield: 78%; m.p.: 150–152 C; H NMR (400 MHz, 2.9e 1-Benzyl-2,6-bis(4-methoxyphenyl)-3-methyl-
CDCl ): δ = 9.45 (s, OH), 7.40–7.35 (m, 4H), 7.15– piperidin-4-one oxime (3e, C27
H N O ): Pale yellow
30 2 3
3
◦
1
7
1
3
.09 (m, 7H), 6.83–6.82 (m, 2H), 3.67 (dd, J = 3.3, solid; Yield: 86%; m.p.: 142–144 C; H NMR
1.7 Hz, H-6a), 3.56 (d, J = 15.0 Hz, 1H of N–CH ), (400 MHz, CDCl
): δ = 7.40–7.35 (m, 4H), 7.08 (t,
2
3
.47–3.42 (m, 2H of N-CH and H-2a merged), 3.30 (d, J = 2.7 Hz, 3H), 6.87–6.81 (m, 6H), 3.80 (s, 3H),
2
J = 9.8 Hz, H-3a), 2.55–2.47 (m, H-5a), 2.34 (s, 3H), 3.79 (s, 3H), 3.66 (dd, J = 3.3, 11.6 Hz, H-6a), 3.54
2
0
.33 (s, 3H), 2.16–2.09 (unresolved quartet, H-5e), (d, J = 14.6 Hz, 1H of N-CH
), 3.47–3.42 (m, 2H of
2
13
.76 (d, J = 6.6 Hz, CH at C-3) ppm; C NMR H-2a and 1H of N-CH
merged), 3.28 (d, J = 9.8 Hz,
3
2
(
(
100.52 MHz, CDCl ): δ = 159.81 (C-4), 141.12 H-3a), 2.55–2.48 (m, H-5a), 2.13 (t, J = 12.6 Hz,
3
ꢁ
ꢁ
ꢁ
ꢁ
13
C-6 c), 139.45 (C-2 c), 137.09 (C-6 ), 136.86 (C-2 ), H-5e) 0.75 (d, J = 6.6 Hz, CH
at C-3) ppm;
C
3
1
1
4
1
3
36.67 (N-Bn ipso carbon), 130.10, 129.35, 129.07, NMR (100.52 MHz, CDCl
3
): δ = 159.89 (C-4), 158.94
ꢁ
ꢁ
ꢁ
ꢁ
27.51, 71.92 (C-2), 63.55 (C-6), 53.08 (N-CH -Ph), (C-6 c), 158.80 (C-2 c), 137.29 (C-6 ), 136.15 (C-2 ),
3.91 (C-3), 35.27 (C-5), 21.28 (CH at C-6d and C-2d) 134.49 (N-Bn ipso carbon), 129.97, 129.85, 128.84,
2.84 (CH at C-3) ppm; and HRMS: m/z calculated: 127.53, 126.45, 72.10 (C-2), 63.67 (C-6), 55.38
98.2358, found: 398.2833.
2
3
3
ꢁ
ꢁ
(CH
C-3), 35.19 (C-5), 12.81 (CH3 at C-3) ppm;
and HRMS: m/z calculated: 430.2256, found:
30.3451.
O at C-6 c, C-2 c), 53.45 (N-CH -Ph), 43.98
3
2
(
2
.9c 1-Benzyl-2,6-bis(4-isopropylphenyl)-3-methyl-
piperidin-4-one oxime (3c, C H N O): White solid;
4
3
◦
1
38
1
2
Yield: 90%; m.p.: 206–208 C; H NMR (400 MHz,
CDCl ): δ = 7.31 (d, J = 8.0 Hz, 4H), 7.15 (d, 2.9f 1-Benzyl-2,6-bis(4-ethoxyphenyl)-3-methylpipe-
3
J = 7.7 Hz, 2H), 7.10 (unresolved dt, 5H), 6.93 (d, ridin-4-one oxime (3f, C29
H N O ): White solid;
34 2 3
◦
1
J = 6.9 Hz, 2H ), 3.92 (d, J = 6.2 Hz, H-6a), 3.79 (d, Yield: 86%; m.p.: 158–160 C; H NMR (400 MHz,
J = 6.2 Hz, H-2a), 3.69 (d, J = 14.2 Hz, 1H of N- CDCl ): δ = 8.89 (s, OH), 7.35 (d, J = 8.4 Hz, 4H),
3
CH ), 3.59–3.51 (m, 2H of N-CH and H-3a merged), 7.08 (s, 3H), 6.85 (d, J = 6.6 Hz, 4H), 6.82–6.81 (m,
2
2
2
.91–2.81 (m, 2H 2CH of iPr) ), 2.62–2.55 (m, 1H of 2H), 4.04–4.01 (m, 4H), 3.64 (d, J = 8.4 Hz, H-6a),
2
H-5a), 1.23 (t, J = 7.5 Hz, 13H), 1.00 (d, J = 6.9 Hz, 3.54 (d, J = 15.0 Hz, 1H), 3.45–3.41 (m, 2H), 3.27
1
3
CH at C-3) ppm; C NMR (100.52 MHz, CDCl ): δ (d, J = 9.5 Hz, H-3a), 2.50 (t, J = 8.0 Hz, H-5a),
3
3
ꢁ
ꢁ
=
159.96 (C-4), 148.08 (C-6 c), 147.87 (C-2 c), 141.36 2.16–2.08 (m, H-5e), 1.41 (d, J = 3.2 Hz, 6H), 0.75 (d,
ꢁ
ꢁ
13
(
C-6 ), 139.71 (C-2 ), 136.78 (N-Bn ipso carbon), J = 5.4 Hz, CH
3
at C-3) ppm; C NMR (100.52 MHz,
ꢁ
1
29.89, 127.48, 126.59, 126.32, 72.73 (C-2), 64.38 CDCl
3
): δ = 159.70 (C-4), 158.18 (C-6 c), 158.80
ꢁ
ꢁ
ꢁ
(
C-6), 53.89 (N-CH -Ph), 43.82 (C-3), 35.04 (C-5), (C-2 c), 137.64 (C-6 ), 135.87 (C-2 ), 133.83 (N-Bn
2
3
3.90 (CH of iPr at C-2d, C-6d), 24.23 (2CH at ipso carbon), 129.92, 128.83, 127.52, 126.44, 114.56,
3
C-6d), 24.16 (2CH at C-2d), 12.87 (CH at C-3) 114.28, 72.04 (C-2), 63.62 (C-6), 54.13 (CH
CH
-Ph), 44.00 (C-3), 35.15
2
O at
3
3
3
2
ꢁ
ꢁ
ppm; and HRMS: m/z calculated: 454.2984, found: C-6 c, C-2 c), 53.37 (N-CH
ꢁ
ꢁ
454.3362.
(C-5), 15.01 (CH CH O at C-6 c, C-2 c), 12.81 (CH b
3 2 3
at C-3) ppm.
2.9d 1-Benzyl-2,6-bis(4-chlorophenyl)-3-methylpipe-
ridin-4-one oxime (3d, C H Cl N O): Off-white 2.9g 1-Benzyl-2,6-bis(4-benzyloxyphenyl)-3-methyl-
2
5
24
2
2
◦
1
solid; Yield: 82%; m.p.: 174–176 C; H NMR piperidin-4-one oxime (3g, C H N O ): White
3
9
38
◦
2
3
1
(
400 MHz, CDCl ): δ = 7.43–7.39 (m, 4H), 7.30 (d, solid; Yield: 93%; m.p.: 175–177 C; H NMR
3
J = 6.2 Hz, 4H), 7.11(d, J = 5.5 Hz, 3H), 6.78–6.72 (400 MHz, CDCl ): δ = 8.30 (s, OH), 7.44–7.33 (M,
3
(
m, 2H), 3.69 (d, J = 7.3 Hz, H-6a), 3.52–3.32 (m, 14H), 7.08 (d, J = 2.9 Hz, 3H), 6.94 (d, J = 6.2 Hz,
4
2
5
H of H-2a, H-3a and 2H of N-CH merged), 2.53– 4H), 6.81 (m, 2H), 5.05 (s, 2H), 4.99 (s, 2H), 3.65 (d,
2
.45 (m, H-5a), 2.14–2.06 (m, 1H), 0.75 (d, J = J = 10.2 Hz, H-6a), 3.54 (d, J = 15.0 Hz, 1H), 3.45–
1
3
.8 Hz, CH at C-3) ppm; C NMR (100.52 MHz, 3.42 (m, 2H), 3.29 (d, J = 9.8 Hz, H-3a), 2.53–2.49 (m,
3
ꢁ
CDCl ): δ = 158.92 (C-4), 142.30 (C-6 c), 140.75 H-5a), 2.15 (t, J = 12.6 Hz, H-5e), 0.76 (d, J = 6.2 Hz,
3
ꢁ
ꢁ
13
(
1
1
C-2 c), 136.45 (N-Bn ipso carbon), 133.30 (C-6 ), CH at C-3) ppm; C NMR (100.52 MHz, CDCl ):
3
3
ꢁ
ꢁ
ꢁ
33.04 (C-2 ), 130.27, 129.75, 129.10, 128.94, 128.71, δ = 159.95 (C-4), 158.22 (C-6 c), 158.00 (C-2 c),
27.80, 126.86, 71.98 (C-2), 63.55 (C-6), 53.85 (N- 137.36 (N-Bn ipso carbon), 137.20 (O-Bn ipso carbon),
ꢁ
ꢁ
CH -Ph), 43.67 (C-3), 34.72 (C-5), 12.74 (CH at C-3) 136.51 (C-6 ), 134.88 (C-2 ), 130.04, 129.89, 128.73,
2
3
ppm; and HRMS: m/z calculated: 338.1266, found: 128.10, 127.71, 127.67, 127.56, 114.97, 114.69, 72.16
ꢁ
ꢁ
338.1854.
(C-2), 70.23 (O-CH -Ph at C-6 c, C-2 c), 63.74 (C-6),
2

Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one oximes
865
5
4.24 (N-CH -Ph), 43.96 (C-3), 35.15 (C-5), 12.85 2.9k 1-Benzyl-2,6-bis(4-ethoxyphenyl)-3-ethylpipe-
2
(
CH at C-3) ppm.
ridin-4-one oxime (3k, C H N O ): White solid;
Yield: 91%, m.p.: 148–150 C; H NMR (400 MHz,
3
30 36
◦
2
3
1
CDCl ): δ = 7.36 (q, 4H), 7.08 (unresolved triplet, 3H),
3
2
.9h 1-Benzyl-2,6-bis(3-methoxyphenyl)-3-methyl-
6
.84 (d, J = 8.8 Hz, 6H), 4.05–3.99 (m, 4H), 3.72–3.67
piperidin-4-one oxime (3h, C H N O ): Off-white;
Yield: 79%; m.p.: 128–130 C; H NMR (400 MHz,
27
30
1
2
3
(
(
(
m, 1H), 3.55–3.37 (m, 4H), 2.39–2.35 (m, H-5a), 2.16
◦
t, J = 12.6 Hz, H-5e), 1.42–1.38 (m, 6H), 1.25–1.20
CDCl ): δ = 7.26 (t, J = 6.9 Hz, 3H), 7.11–7.04 (m,
3
m, 1H), 1.18–1.13 (m, 1H), 0.75 (t, J = 7.3 Hz, CH
3
6
3
3
H), 6.88–6.85 (m, 2H), 6.81–6.77 (m, 2H), 3.82 (s,
H), 3.81 (s, 3H), 3.68 (dd, J = 2.7, 8.6 Hz, H-6a),
.60 (d, J = 14.6 Hz, 1H), 3.51–3.44 (m, 2H), 3.32
13
of Ethyl at C-3) ppm; C NMR (100.52 MHz, CDCl ):
3
ꢁ
ꢁ
δ = 158.24 (C-4), 158.18 (C-6 c), 158.11 (C-2 c),
37.63 (C-6 ), 136.08 (C-2 ), 134.57 (N-Bn ipso car-
bon), 129.99, 129.76, 128.82, 127.52, 126.37, 114.48,
ꢁ
ꢁ
1
(
d, J = 10.24 Hz, H-3), 2.59–2.51 (m, H-5a), 2.17
13
(q, H-5e) 0.77 (d, J = 6.6 Hz, CH at C-3) ppm;
C
3
1
14.18, 70.06 (C-2), 63.57 (C-6), 53.72 (N-CH -Ph),
2
NMR (100.52 MHz, CDCl ): δ = 159.92 (C-4), 159.77
ꢁ
3
50.94 (C-3), 35.47 (C-5), 20.09 (CH CH O at C-6 c,
3
2
ꢁ
ꢁ
ꢁ
ꢁ
(C-6 c), 159.55 (C-2 c), 145.71 (C-6 ), 144.05 (C-2 ),
ꢁ
C-2 c), 15.01 (CH CH at C-3), 12.10 (CH at C-3)
2
3
3
1
1
1
5
36.90 (N-Bn ipso carbon), 130.03, 129.69, 129.30,
27.56, 126.60, 121.79, 120.24, 114.64, 113, 112.71,
12.62, 72.52 (C-2), 64.04 (C-6), 55.33 (2 O-CH ),
ppm.
3
3.82 (N-CH -Ph), 43.67 (C-3), 34.80 (C-5), 12.81 2.9l 1-Benzyl-3-isopropyl-2,6-diphenylpiperidin-
2
(
CH at C-3) ppm.
4-one oxime (3l, C H N O): Brown semi-solid;
3
27 30 2
1
Yield: 87%; H NMR (400 MHz, CDCl ): δ = 7.56
3
(
3
4
d, J = 7.3 Hz, 2H), 7.39–7.35 (m, 4H), 7.28–7.24 (m,
2
.9i 1-Benzyl-3-ethyl-2,6-bis(4-isopropylphenyl)
H), 7.18–7.09 (m, 4H), 6.93 (dd, J = 2.0, 6.5, 2H),
.31–4.22 (m, 2H), 3.70 (d, J = 14.2 Hz, 1H), 3.55
piperidin-4-one oxime (3i, C H N O): White solid;
Yield: 82%; m.p.: 180–182 C; H NMR (400 MHz,
3
◦
2
40
1
2
(
d, J = 14.2 Hz, 1H), 2.89–2.76 (m, 2H), 2.14 (dd,
CDCl ): δ = 8.16 (s, OH), 7.39–7.34 (m, 4H), 7.15–
3
J = 3.1, 8.9 Hz, H-5e), 1.79–1.71 (m, 1H), 0.96 (d,
7
3
2
.12 (m, 4H), 7.05–7.04 (m, 3H), 6.82-6.80 (m, 2H),
.72 (dd, J = 3.3, 11.3 Hz, H-6a), 3.54–3.38 (m, 4H),
.90–2.84 (m, 2H), 2.45–2.40 (m, 1H), 2.24–2.17 (m,
13
J = 6.6 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H) ppm;
C
NMR (100.52 MHz, CDCl ): δ = 157.66 (C-4), 145.58
3
ꢁ
ꢁ
(C-6 ), 144.65 (C-2 ), 137.87 (N-Bn ipso carbon),
H-5e), 1.46–1.39 (m, 1H), 1.35–1.23 (m, 13H), 0.76
129.66, 128.80, 128.21, 127.98, 127.89, 127.35, 62.83
13
(
t, J = 7.5 Hz, 3H) ppm; C NMR (100.52 MHz,
(
C-2), 60.01 (C-6), 56.15 (N-CH -Ph), 55.68 (C-3),
2
ꢁ
CDCl ): δ = 158.52 (C-4), 147.92 (C-6 c), 147.88
3
3
5.41 (C-5), 30.15 (CH of iPr), 21.72 (CH iPr) 20.63
3
ꢁ
ꢁ
ꢁ
(C-2 c), 141.47 (C-6 ), 140.01 (C-2 ), 138.12 (N-Bn
(
CH iPr) ppm.
3
ipso carbon), 129.70, 128.95, 127.84, 127.49, 126.56,
1
26.58, 70.74 (C-2), 64.40 (C-6), 54.54 (C-3), 50.62
ꢁ
(N-CH -Ph), 35.24 (C-5), 33.90 (CH of iPr at C-6 c, 2.9m 1-Benzyl-3,5-dimethyl-2,6-diphenylpiperidin-4-
2
ꢁ
ꢁ
ꢁ
C-2 c), 24.20 (2CH at C-6 c), 24.13 (2CH at C-2 c), one oxime (3m, C H N O): Off-white solid, Yield:
3
3
26 28
2
◦
1
20.39 (CH -CH at C-3), 12.87 (CH at C-3) ppm.
94%; m.p.: 132–134 C; H NMR (400 MHz, CDCl ):
2
3
3
3
δ = 7.42–7.40 (m, 4H), 7.34–7.11 (m, 9H), 6.96 (d,
J = 7.3 Hz, 2H), 4.00 (d, J = 5.8 Hz, H-6a), 3.80 (d,
J = 6.6 Hz, H-2a), 3.71 (d, J = 14.2 Hz, 1H), 3.58–
2
.9j 1-Benzyl-2,6-bis(4-chlorophenyl)-3-ethylpipe-
ridin-4-one oxime (3j, C H N O): White solid;
Yield: 86%; m.p.: 168–170 C; H NMR (400 MHz,
2
◦
6
26
1
2
3
.55 (m, 2H), 2.62–2.55 (m, H-5a), 1.24 (d, J =
13
6
.9 Hz, 3H), 0.98 (d, J = 7.3 Hz, 3H) ppm; C NMR
CDCl ): δ = 7.42–7.37 (m, 4H), 7.29 (d, J = 8.4 Hz,
3
(
100.52 MHz, CDCl ): δ = 163.05 (C-4), 144.57 (C-
3
4H), 7.15–7.07 (m, 3H), 6.80–6.77 (m, 2H), 3.78 (dd,
ꢁ
ꢁ
2
1
c,6 c), 138.33 (N-Bn ipso carbon), 129.72, 128.48,
J = 3.6, 11.0 Hz, H-6a), 3.58–3.33 (m, 4H), 2.38–2.33
28.22, 127.90, 126.94, 126.78, 69.50 (C-2), 62.11
(m, H-5e), 2.17 (q, H-5e), 1.50–1.39 (m, 1H), 1.18–
(
C-6), 57.06 (N-CH -Ph), 41.87 (C-3), 38.48 (C-5),
2
1
.13 (m, 1H), 0.76 (t, J = 7.3 Hz, CH at C-3) ppm;
3
1
7.67 (CH at C-3), 16.84 (CH at C-5) ppm.
3 3
1
3
C NMR (100.52 MHz, CDCl ): δ = 157.34 (C-4),
3
ꢁ
ꢁ
142.42 (C-6 c), 141.16 (C-2 c), 136.89 (N-Bn ipso
ꢁ
ꢁ
carbon), 133.14 (C-6 ), 133.01 (C-2 ), 130.23, 129.62, 2.9n 1-Benzyl-2,6-bis(4-isopropylphenyl)-3,5-dime-
1
6
29.10, 128.91, 128.64, 127.83, 126.81, 69.75 (C-2), thylpiperidin-4-one oxime (3n, C H N O): White
32
40
◦
2
1
3.37 (C-6), 54.46 (N-CH -Ph), 50.56 (C-3), 34.91 solid; Yield: 92%; m.p.: 158–160 C; H NMR
2
(
C-5), 20.40 (CH at C-3), 12.04 (CH at C-3) ppm.
(400 MHz, CDCl ): δ = 7.31 (d, J = 8.0 Hz, 4H),
2
3
3

8
66
Someshwar D Dindulkar et al.
Single-crystal X-ray diffraction analysis has been
7
.16–7.08 (m, 7H), 6.93 (d, J = 6.9 Hz, 2H), 3.92 (d,
J = 6.2 Hz, H-6a), 3.79 (d, J = 6.2 Hz, H-2a), 3.69 (d, performed representatively for the 3-methyl and 3,5-
J = 14.2 Hz, 1H), 3.57–3.51 (m, 2H), 2.91–2.81 (m, dimethyl compounds 3d and 3n. The puckering para-
two CH of iPr ), 2.62–2.55 (m, H-5a), 1.25–1.21 meters of the compounds were analysed according to
14,15
(
(
(
(
m,15H of 2(CH ) merged with CH at C-3), 1.00 Cremer and Pople and Nardelli.
For the piperidone
3
2
3
13
d, J = 6.9 Hz, 3H of CH at C-5) ppm; C NMR ring C1-C2-C3-C4-C5-N1 of 3d, the smallest displace-
3
100.52 MHz, CDCl ): δ = 163.05 (C-4), 147.42 ment asymmetry parameters q and q are 0.0911 and
3
2
3
ꢁ
ꢁ
ꢁ
ꢁ
C-6 c), 146.72 (C-2 c), 142.00 (C-6 ), 141.61 (C-2 ), -0.5598 Å, respectively. The ring puckering parame-
1
39.02 (N-Bn ipso carbon), 129.60, 128.28, 128.14, ters such as total puckering amplitude ‘Q ’ and phase
T
◦
127.78, 126.50, 126.40, 126.17, 69.74 (C-2), 65.62 angle ‘θ’ are 0.5670 Å and 169.37 . Thus, all puckering
(
C-6), 56.95 (N-CH -Ph), 42.00 (C-3), 38.56 (C-5), parameters strongly support a slightly distorted chair
2
33.88 (2CH iPr), 33.78 (2CH iPr), 24.20 (4CH3 conformation for the piperidone ring.
iPr), 18.16 (CH3 at C-3), 16.70 (CH3 at C-5)
ppm.
The stereochemistry of the substituents are analysed
as follows. The equatorial orientation of the methyl
◦
group is witnessed by its torsion angles 177.4 [C2-
◦
C3-C4-C12] and 176.9 [N1-C1-C2-C12]. The torsion
2.9o 1-Benzyl-2,6-bis(4-ethoxyphenyl)-3,5-dimethyl-
angles of the phenyl groups on both sides of the amino
piperidin-4-one oxime (3o, C H N O ): White
◦
◦
3
0
◦
36
2
3
group are 171.8 [C3-C2-C1-C6] and -178.4 [C3-C4-
C5-C13], which support their equatorial orientation.
The ‘N’ atom of the piperidone molecule shows the
1
solid; Yield: 88%; m.p.; 172–174 C; H NMR (400
MHz, CDCl ): δ = 8.28 (s, OH), 7.28 (t, J = 8.6 Hz,
3
4
6
4
H), 7.15–7.08 (m, 3H), 6.94 (d, J = 5.8 Hz, 2H),
.85 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H),
.05–3.96 (m, 4H), 3.91 (d, J = 6.2 Hz, H-6a), 3.78
3
sp hybridization, which can be evidenced from the
angles around that nitrogen. The N-benzyl group also
adopts an equatorial disposition to the best plane of the
piperidone ring, which is evidenced from the torsion
(d, J = 6.9 Hz, H-2a), 3.69 (d, J = 14.3 Hz, 1H),
3.55–3.49 (m, 2H), 2.59–2.52 (m, H-5a), 1.42–1.37 (m,
◦
angles C19-N1-C5-C4 = 178.4 and C19-N1-C1-C2 =
6
H of 2CH CH O), 1.22 (d, J = 7.3 Hz, 3H of CH at
◦
3
2
3
1
-
176.9 . X-ray analysis of 3d revealed the conforma-
3
C-3), 0.97 (d, J = 6.9 Hz, 3H of CH at C-3) ppm; C
3
tion of the molecule with C1, C2 and C5 having R,
R and S relative configurations, respectively. Overall,
the detailed crystallographic studies such as asymme-
try parameters, ring puckering parameters and torsion
angles calculated for 3d proved that the piperidone ring
exists in a slightly distorted chair conformation with an
equatorial orientation of the methyl group on one of
the active methylene centres and phenyl rings on both
sides of the tertiary amino group along with an equa-
torial orientation of the benzyl group on the tertiary
amino group. ORTEP of the compound 3d is shown in
figure 2.
NMR (100.52 MHz, CDCl ): δ = 163.45 (C-4), 158.19
3
ꢁ
ꢁ
ꢁ
ꢁ
(C-6 c), 157.86 (C-2 c), 138.95 (C-6 ), 136.62 (C-2 ),
1
1
6
36.39 (N-Bn ipso carbon), 129.61, 129.22, 127.83,
26.60, 69.27 (C-2), 64.87 (C-6), 63.48 (OCH ),
2
3.44 (OCH ), 56.81 (N-CH -Ph), 42.06 (C-3), 38.57
2
2
(
C-5), 17.77 (CH at C-3), 16.77 (CH at C-5), 15.04
3
ꢁ
3
ꢁ
(CH CH O at C-6 c, C-2 c) ppm.
3
2
3. Results and discussion
3.1 Chemistry
Single-crystal analysis of 3n clearly indicates that the
molecule exist in a twist-boat conformation with R, R,
S and S relative configurations of C1, C2, C4 and C5,
The highly functionalized 1-benzyl-3,5-dialkyl-2,6-
diarylpiperidin-4-one oximes 3a–o were synthesized
using appropriate piperidin-4-ones,18 ,19 sodium acetate
trihydrate and hydroxylamine hydrochloride in ethanol
as shown in scheme 1. It is interesting to note that it
took only 1–2 h for the i-Pr and Cl-phenyl containing
N-benzylpiperidin-4-ones to be converted to oximes,
while the reaction time for the alkoxy-phenyl contain-
respectively. The ring puckering parameters ‘Q ’ and
T
◦
‘
3
θ’ are 0.7687 Å and 89.38 . ORTEP of the compound
n is shown in figure 3.
21,22
3.2 Cytotoxicity
ing N-benzylpiperidin-4-ones was much longer, about All the synthesized compounds 3a–o were subjected
h. The synthesized compounds were characterized by to MTT assay to evaluate in vitro cytotoxicity against
5
1
13
23
H/ C NMR and single-crystal X-ray diffraction stud- HeLa cells. The IC50 values of all compounds are
ies. All the synthesized oximes from unsymmetrical summarized along with their structure in table 1 for
ketones existed as E-isomer as witnessed by their NMR better structure activity comprehension. Besides, the
24–27
and XRD data.
standard drugs camptothecin and etoposide,
were

Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one oximes
867
O
R3
R2
O
R3
R2
O
O
+
R1
R
R1
R
R1
R
R1
R
a
H H
+
N
H
NH OAc
1a−o
4
b
HO
N
O
N
R3
6'
R2
R3
R2
4
5
6
3
2
6
'a
2'a
R1 6'b
1
2'b R1
R1
R
R1
R
2'
N
c
6
'e
2'e
6'c
2'c
R
R
6
'd
2'd
3a−o
2a−o
(Yield: 80−90%)
Scheme 1. Reagents and conditions: (a) Ethanol, warm. (b) Benzyl bromide, anhydrous K2CO3, THF, rt, 72 h.
c) Hydroxylamine hydrochloride, sodium acetate trihydrate, ethanol, reflux, 1–5 h.
(
Figure 2. The ORTEP of compound 3d. Ellipsoids are
drawn at 50% probability. Crystal analysis clearly proved
that the oxime adopts the E configuration.
Figure 3. The ORTEP of compound 3n. Ellipsoids are
drawn at 40% probability. One of the methyl atoms C25 is
disordered in five different positions with 0.2 occupancy. The
C25 atom has been refined isotropically and no hydrogen
atoms were fixed for this atom.
also analysed under identical conditions and their IC50
values are also reproduced in the table 1.
Analyses of the cytotoxic data from table 1 project
some lead molecules. All the tested compounds 3a–o
exhibited good antiproliferative activity against the
cancer cells with IC50 values ranging from 13.88 to and 3m with unsubstituted phenyl groups on both sides
7.94 μM concentration except compound 3g, which of the tertiary amino group have shown lesser activity
required 93.91 μM. Among them, compounds 3a, 3l (25.23, 34.63 and 37.94 μM, respectively) than their
3

8
68
Someshwar D Dindulkar et al.
Table 1. aCytotoxic effect of compounds 3a–o on HeLa cells.
b
Linear regression equation
a
cIC50 in μM
25.23 ± 3.8
Compound
Structure
[log]: Y=A+Bx
R value
3
3
3
3
3
3
3
3
a
b
c
d
e
f
Y = 9.97629+(41.34597)x
Y = −0.90241+(50.11737)x
Y = 12.19751+(47.24709)x
Y = 4.93121+(52.61212)x
Y = 8.459+(43.24166)x
0.98
0.93
0.98
0.97
0.98
0.98
0.96
0.94
25.98 ± 2.04
13.88 ± 0.93
16.39 ± 2.47
21.21 ± 1.42
16.10 ± 3.06
93.91 ± 3.38
32.17 ± 2.37
Y = 17.6655+(37.30817)x
Y = −35.01759+(55.23339)x
Y = −17.78817+(57.67985)x
g
h

Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one oximes
869
Table 1. (contd.)
b
Linear regression equation
a
cIC50 in μM
Compound
Structure
[log]: Y=A+Bx
R value
3
3
3
3
3
3
3
i
Y = 0.59467+(47.01865)x
0.95
23.60 ± 1.20
21.28 ± 2.13
22.07 ± 3.4
34.63 ± 1.43
37.94 ± 1.32
16.08 ± 1.43
16.23 ± 1.65
j
Y = −5.928+(56.84707)x
Y = 3.46962+(45.80483)x
Y = −29.78378+(67.83093)x
Y = −32.48966+(68.4888)x
Y = 7.52381+(48.38217)x
Y = 10.47483+(44.62101)x
0.95
0.98
0.94
0.95
0.99
0.96
k
l
m
n
o
Etoposide
Camptothecin
Y = 2.27708+(41.80905)x
Y = 29.02966+(42.56504)x
0.98
0.99
23.33 ± 0.92
8.93 ± 0.54
a
Exponentially growing cells were treated with different concentrations of test compounds for 24 h and cell growth inhibition
was analysed through MTT assay
Structure drawn on the basis of found relative configuration using Single-crystal XRD analysis (3d and 3n) and 1D NMR
b
c
Mean percent decrease in cell number of five independent experiments was used to calculate the linear regression equation.
Linear regression: Y = A + Bx (A = Y-intercept; B = slope of the line; x = x-scale)
d
IC50 is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control
cultures in the absence of an inhibitor. The values represent the mean ± SD of five individual observations

8
70
Someshwar D Dindulkar et al.
analogues halo/alkyl/alkoxy substituted phenyl com- cell). There was no significant indication of cell death in
pounds. Compounds 3c, 3d and 3f with isopropyl, these cells even at higher concentration (100 μM) of the
ethoxy and chloro substituents on the phenyl groups at test compounds. This confirmation of non-toxicity on
C-2/C-6 along with the methyl at C-3 showed their best healthy cells can be a chemotherapeutic measure to pro-
activity, below the IC50 of 16.39 μM. Of them, par- pose these compounds as medicinally important drug
ticularly compound 3c, the para-iPr-phenyl compound leads. The IC (inhibition of cell viability to 50%) con-
50
registered the best activity in this series, which inhi- centrations were calculated using the respective regres-
bited the proliferation of cancer cells with an IC50 of sion equation as shown in table 1 and figure 4.
13.88 μM.
In addition to the methyl group at C-3, one more
methyl group was introduced on another active methy- 3.3 Altered morphology study
lene centre at C-5. On the other hand, to improve the
5
efficacy, we replaced the methyl group at C-3 by the The altered morphology of exposed cells (1 × 10
ethyl and isopropyl groups. However, either the replace- cells/well) at IC50 concentration was studied after 24 h
ment of methyl at C-3 by ethyl and isopropyl or incor- using phase contrast microscope (DMI6000B, Leica
poration of another methyl at C-5 did not improve Microsystems, Wetzlar, Germany) and manifested in
the efficacy of compounds 3c, 3d and 3f. Instead, the figure 5.
the 3-ethylated analogues (3i, 3j and 3k, respectively)
The altered morphology of the cells as a rejoinder
of 3c, 3d and 3f required the IC50 between 21.28 to the effect of test compounds was observed under
and 23.60 μM, and the 3,5-dimethylated analogs 3n a phase-contrast microscope. The group that lacks the
(
16.08 μM) and 3o (16.23 μM) exhibited a similar test compound was considered as control. As depicted
range of cytotoxicity. Overall, many compounds viz., in figure 5a, all the control cells have shown normal
c–f, 3j–k and 3n–o are better than one of the stan- healthy and intact nuclei without any cytological abnor-
3
dard drugs Etoposide (23.33 μM) and 3c is compara- malities (figure 5a). The remaining cells were treated
ble to another anticancer drug camptothecin (8.93 μM) with IC50 concentration of highly toxic test compound
under identical conditions. As a reference, MTT assay 3c for 24 h. Number of cytomorphological anomalies
was also performed with lead test compounds using were observed in the test group viz., blebbing of cellu-
normal healthy PBMCs (peripheral blood mononuclear lar membrane, chromatin condensation, fragmentation
Figure 4. The graphs of linear regression analysis representing the
growth inhibition of different test compounds with IC50 values below
2
0 μM.

Design, synthesis and cytotoxicity of novel N-benzylpiperidin-4-one oximes
871
Figure 5. Light and fluorescent micrographs of normal and treated HeLa
cells. Phase contrast micrographs of normal and treated (with the IC50 con-
centration of ideal compound 3c for 24 h) HeLa cells (a and b, respectively).
Fluorescence micrographs of normal and treated (with the IC50 concentration
of ideal compound 3c for 24 h) HeLa cells with Hoechst 33342 stain (c and d,
respectively) and Propidium Iodide (e and f, respectively). The stained cells
were detected by fluorescence light microscope at 360/470 nm and 535/617 nm
excitation/emission for Hoechst stain and PI, respectively. Each inset represents
the magnified image of the corresponding normal and deformed cell/nuclei.
and formation of apoptotic bodies (figure 5b). Most of cells treated with the same concentration as described
the treated cells exhibited the symptoms of apoptosis above. The destructive fragmentation of the nucleus
but the damage was intense in some cells, with the cell (karyorrhexis) resulted in the picknosis of the treated
membrane rupture and the subsequent release of cyto- cells.27,28 When stained with PI, the dead cells took up
plasm as observed in figure 5b (inset). To confirm the the stain to give a clear quantitative picture of the cyto-
light microscopy data and to visualize dead cells in a toxic effect of the test compound at its IC concentra-
50
more proper manner, the cells were exposed to fluores- tion. It is noteworthy here that only the dead cells take
cence microscopy using Hoechst 33342 and PI stains up PI staining thus allowing a better visual analysis of
for control and treated cells (figure 5c and d, respec- the toxicity of the test compound.
tively). Obviously, the bright condensed chromatin was
identified, leading to the deformed nuclear cytoplasmic
consistency followed by the margination of chromatin
3.4 Annexin V-PI dual staining to access appotosis
into a horseshoe shaped structure which was a clear Fluorescence microscopy is a wonderful tool to access
indication of early apoptosis (figure 5d, inset) for the cellular processes and the corresponding morphological

8
72
Someshwar D Dindulkar et al.
Figure 6. Annexin V-FITC/PI dual staining to access the degree of apoptosis. Phase con-
trast (a–d) and fluorescence (e–h) micrographs of control and 3c (13.88 μM) treated HeLa
cells.
changes occurring in the cells in response to exter- Supplementary information
nal stimuli. HeLa cells untreated and treated with
Crystallographic data of compounds 3d (CCDC No.
41850) and 3n (CCDC No. 841849) are provided
as supplementary material. Supplementary crystallo-
graphic data for 3d and 3n can be obtained free of
charge at www.ccdc.cam.ac.uk/conts/retrieving.html.
13.88 μM lead compound 3C were stained with
8
Annexin V labelled with FITC and counter stained
with PI after different time intervals (0, 4, 12, 24 h).
Annexin V specifically binds to the phosphatidylser-
ine in the cells, marking its relocation to the outer-
cell membrane and PI enters only dead cells as it can-
not pass through the intact cell membrane. Dual stain-
ing with Annexin V-FITC and PI simultaneously, gives
a clear indication of early/late apoptosis (with expo-
sure of phosphatidylserine) and cell death. Our results
showed marked apoptosis of 3C treated HeLa cells in
a time dependent manner (figure 6). Early apoptosis
Acknowledgements
This research was supported by Second Phase of Brain
Korea (BK21) Program.
after 4 h of treatment is depicted in figure 6f when the References
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