Synthesis and Antileukemic Activity of Novel 4-(3-(Piperidin-4-yl) Propyl)Piperidine Derivatives

Article abstract of DOI:10.1111/j.1747-0285.2011.01184.x

To explore the anticancer effect associated with the piperidine framework, several (substituted phenyl) {4-[3-(piperidin-4-yl)propyl]piperidin-1-yl} methanone derivatives 3(a-i) were synthesized. Variation in the functional group at N-terminal of the piperidine led to a set of compounds bearing amide moiety. Their chemical structures were confirmed by ¹H NMR, IR and mass spectra analysis. Among these, compounds 3a, 3d and 3e were endowed with antiproliferative activity. The most active compound among this series was 3a with nitro and fluoro substitution on the phenyl ring of aryl carboxamide moiety, which inhibited the growth of human leukemia cells (K562 and Reh) at low concentration. Comparison with other derivative (3h) results shown by LDH assay, cell cycle analysis and DNA fragmentation suggested that 3a is more potent to induce apoptosis. Novel (substituted phenyl){4-[3-(piperidin-4-yl)propyl]piperidin-1-yl} methanone derivatives were identified as potent antiproliferative agents. Compound with nitro and fluoro substitution on the phenyl ring of aryl carboxamide moiety was identified as most potent among the series to induce the apoptosis.

Full text of DOI:10.1111/j.1747-0285.2011.01184.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01184.x
Chem Biol Drug Des 2011; 78: 622–630
Research Article
Synthesis and Antileukemic Activity of Novel
4-(3-(Piperidin-4-yl) Propyl)Piperidine Derivatives
Kambappa Vinaya1,2, Chandagirikoppal V.
past 5 years, the global effort for sequencing human genome has
provided us with an enormous number of potential targets associ-
ated with cancer. The drugs designed are expected to have high
affinity with the novel targets. They not only inhibit the proliferation
but also differentiation of tumor cells and speed up their death.
Cancer chemotherapy is now well established and is considered as
Kavitha3,4, Siddappa Chandrappa1,
Doddakunche S. Prasanna1, Sathees C.
Raghavan3 and Kanchugarakoppal S.
Rangappa1,*
1Department of Studies in Chemistry, University of Mysore,
a highly specialized field.
Manasagangotri, Mysore-570 006, India
2Department of Chemistry, Government First Grade College,
Among the modifications effected to the family of antitumor com-
pounds, heterocyclic organic compounds have been extensively tried
by many groups especially to modify the reactivity profile. Piperidine
is a new class of heterocycle that showed interesting cytotoxicity
profiles.
Kadur-577 548, India
3Department of Biochemistry, Indian Institute of Science, Bangalore-
560 012, India
4Department of Chemistry, Government College for Women,
Mandya-571 401, India
The piperidine nucleus can be found in numerous naturally occurring
alkaloid and synthetic compounds with interesting biological and
pharmacological properties (1–3). Piperidine moiety-containing mole-
cules play an important role in the field of medicinal chemistry.
Several derivatives of this class have found such useful biological
applications as fungicidal, bactericidal, anti-inflammatory, antihista-
minic, wound healing, anticancer, CNS stimulant, and depressant
activities (4–10). Compounds containing amide bond substitution
can alter the chemical properties, disposition, and biological
activities of drugs (11). Various amides are currently used in the
treatment for several diseases, and these include antidepressants,
anti-inflammatory agents, antimalarial drugs, antipsychotic, antiviral
agents, steroids, and general anesthetics (12).
*Corresponding author: Kanchugarakoppal S. Rangappa,
rangappaks@chemistry.uni-mysore.ac.in; rangappaks@gmail.com
To explore the anticancer effect associated with
the piperidine framework, several (substituted
phenyl)
{4-[3-(piperidin-4-yl)propyl]piperidin-1-yl}
methanone derivatives 3(a–i) were synthesized.
Variation in the functional group at N-terminal of
the piperidine led to a set of compounds bearing
amide moiety. Their chemical structures were con-
firmed by 1H NMR, IR and mass spectra analysis.
Among these, compounds 3a, 3d and 3e were
endowed with antiproliferative activity. The most
active compound among this series was 3a with
nitro and fluoro substitution on the phenyl ring of
aryl carboxamide moiety, which inhibited the
growth of human leukemia cells (K562 and Reh) at
low concentration. Comparison with other deriva-
tive (3h) results shown by LDH assay, cell cycle
analysis and DNA fragmentation suggested that
3a is more potent to induce apoptosis.
Literature has several instances, wherein incorporation of two of
the structural features required for activity in a single molecule has
to significantly enhance the activity (13). Because of broad spectrum
of activities reported in the literature so far, we attempted to syn-
thesize a system that combines two biolabile components together
targeting for potent molecules possessing antileukemic activity. In
continuation of previous research on synthesis and anticancer stud-
ies of bioactive heterocycles (14–17), herein, we tried to investigate
the antiproliferative activity and apoptosis induced by the newly
synthesized piperidine analogs against human leukemia cells.
Key words: 4-amino benzoic acid, anticancer agents, apoptosis, cell
death, DNA damage, leukemia
Received 8 September 2010, revised 14 June 2011 and accepted for
publication 9 July 2011
Materials and Methods
Cancer is a serious disease with a complex pathogenesis that
threatens human life greatly. Currently, much efforts have been put
to the identification of novel anticancer targets and the discovery
of anticancer drugs. In recent years, great progress has been made
in the treatment for cancer, and so, the life expectation of cancer
patients has been improving remarkably. Furthermore, during the
Infrared (IR) spectra were recorded using a Jasco FTIR-4100 spec-
trometer in the wave number range of 4000–400 ⁄ cm. Nuclear
magnetic resonance (1H NMR) spectra were recorded on a Bruker
AM 400 MHz spectrometer using DMSO-d6 as solvent and tetra-
methylsilane as an internal standard. The chemical shifts are
622

4-[3-(Piperidin-4-yl) Propyl]Piperidine Derivatives
expressed in d, and the following abbreviations are used: s (sin-
glet), d (doublet), t (triplet), q (quartet), and m (multiplet). The purity
of the compounds was checked by thin layer chromatography (TLC).
Silica gel column chromatography was performed using Merck 7734
silica gel (60–120 mesh) and Merck made TLC plates. All the
reagents and chemicals used were from Sigma Aldrich Chemicals
Pvt Ltd.
Synthesis of (2-amino-1,2,3,4-
tetrahydronaphthalen-2-yl) {4-[3-(piperidin-4-yl)
propyl]piperidin-1-yl}methanone 3b
The product obtained was white solid from 2-amino-1,2,3,4-tetrahy-
dronaphthalene-2-carboxylic acid 2b (0.25 g, 1.30 mmol), isobutyl
chloroformate (0.23 g, 1.69 mmol), N-methyl morpholine (0.39 g,
3.9 mmol), and 4-[3-(piperidin-4-yl)propyl]piperidine
1
(0.27 g,
1
1.30 mmol). H NMR (DMSO-d6, 400 MHz) d: 7.12 (dd, 2H, Ar–H),
6.90 (d, 2H, Ar–H), 3.90 (s, 2H, –NH2), 3.43 (m, 2H, –CH2), 2.95–
2.98 (m, 8H), 2.91 (t, 2H, –CH2), 2.81 (bs, 2H), 2.18 (t, 2H, –CH2),
2.04 (s, 1H, –NH), 1.74 (m, 2H), 1.09–1.26 (m, 12H). MS (ESI) m ⁄ z:
384.3 (M+H+). IR (KBr, ⁄ cm): 3329, 3074, 1656, 1615, 1525, 1169.
Chemistry
For the synthesis of the novel compounds 3(a–i), the reaction
sequences outlined in Scheme 1 were followed. The coupling reac-
tion of 4-[3-(piperidin-4-yl)propyl]piperidine (1) with different substi-
tuted aromatic ⁄ heterocyclic acids 2(a–i) were carried out in the
presence of base N-methyl morpholine, isobutyl chloroformate, and
tetrahydrofuran (THF) as solvent with the yield ranging from 75% to
90%. Synthesized molecules 3(a–i) were structurally characterized
Synthesis of 2-amino-3-(2¢-ethyl-4¢-
methoxybiphenyl-4-yl)-1-{4-[3-(piperidin-4-yl)
propyl]piperidin-1-yl}propan-1-one 3c
1
by H NMR, mass and IR spectroscopic analyses. Compounds 3(a–i)
were confirmed by IR data, which showed disappearance of stretch-
ing frequencies of –COOH at 2995 ⁄ cm. From H NMR spectra, this
showed disappearance of –COOH at 12.03 ppm. The chemical struc-
tures and yield of the synthesized compounds are given in Table 1.
The product obtained was white amorphous solid from 2-amino-3-
(2¢-ethyl-4¢-methoxylbiphenyl-4-yl)propanoic acid 2c (0.25 g,
0.83 mmol), isobutyl chloroformate (0.147 g, 1.08 mmol), N-methyl
morpholine (0.25 g, 2.49 mmol), and 4-[3-(piperidin-4-yl)propyl]piperi-
1
1
dine 1 (0.174 g, 0.83 mmol). H NMR (DMSO-d6, 400 MHz) d: 7.47
(d, 2H, Ar–H), 7.35 (d, 1H, Ar–H), 7.21 (d, 2H, Ar–H), 6.71 (s, 1H,
Ar–H), 6.62 (d, 1H, Ar–H), 3.95 (m, 1H, –CH), 3.92 (s, 2H, –NH2),
3.87 (s, 3H, –OCH3), 3.23 (d, 2H, –CH2), 2.89–2.98 (m, 8H), 2.81 (bs,
2H), 2.62 (m, 2H, –CH2), 2.08 (s, 1H, –NH), 1.74 (m, 2H), 1.23 (t, 3H,
–CH3), 1.09–1.98 (m, 12H). MS (ESI) m ⁄ z: 492.35 (M+H+). IR (KBr,
⁄ cm): 3315, 3052, 1668, 1610, 1538, 1180.
General procedure for the synthesis of 4-(3-
(piperidin-4-yl)propyl) piperidine derivatives
3(a–i)
A solution of different substituted aromatic ⁄ heterocyclic acids 2(a–
i) (1.0 eq) in dry THF was taken and cooled to 0–5 ꢀC. Then, isobu-
tyl chloroformate (1.3 eq) and N-methyl morpholine (3.0 eq) were
added to the cold reaction mixture. The reaction mixture was stirred
for 15 min at same temperature, then added 4-[3-(piperidin-4-yl)pro-
pyl]piperidine 1 (1.0 eq) to the reaction mixture, and allowed the
reaction mixture at room temperature for 4–5 h with stirring. The
progress of the reaction was monitored by TLC. After completion of
the reaction, water was added and the reaction mixture was fil-
tered, washed with ether, and dried under vacuum.
Synthesis of [3-(cyclopentyloxy)-4-
methoxyphenyl] {4-[3-(piperidin-4-yl)propyl]
piperidin-1-yl}methanone 3d
The product obtained was pale yellow liquid from 3-(cyclopentyl-
oxy)-4-methoxybenzoic acid 2d (0.25 g, 1.05 mmol), isobutyl chloro-
formate (0.18 g, 1.37 mmol), N-methyl morpholine (0.32 g,
3.17 mmol), and 4-[3-(piperidin-4-yl)propyl]piperidine
1 (0.22 g,
1.05 mmol). 1H NMR (DMSO-d6, 400 MHz) d: 7.45 (d, 1H, Ar–H),
7.32 (s, 1H, Ar–H), 6.87 (d, 1H, Ar–H), 3.79 (s, 3H, –OCH3), 3.72 (m,
1H, –CH), 2.88–2.93 (m, 8H), 2.83 (bs, 2H), 2.12 (m, 4H, –CH2), 2.10
(s, 1H, –NH), 1.72 (m, 2H), 1.59 (m, 4H, –CH2), 1.10–1.23 (m, 12H).
MS (ESI) m ⁄ z: 429.31 (M+H+). IR (KBr, ⁄ cm): 3430, 3042, 1641,
1603, 1366, 1190, 1057.
Synthesis of (4-fluoro-3-nitrophenyl) {4-[3-
(piperidin-4-yl)propyl]piperidin-1-yl} methanone
3a
The product obtained was yellow oily from 4-fluoro-3-nitro benzoic
acid 2a (0.25 g, 1.35 mmol), isobutyl chloroformate (0.239 g,
1.79 mmol), N-methyl morpholine (0.409 g, 4.05 mmol), and 4-[3-
(piperidin-4-yl)propyl]piperidine 1 (0.28 g, 1.35 mmol). 1H NMR
(DMSO-d6, 400 MHz) d: 8.81 (s, 1H, Ar–H), 8.28 (d, 1H, Ar–H), 7.56
(d, 1H, Ar–H), 2.93–3.08 (m, 4H), 2.45–2.60 (m, 4H), 2.04 (s, 1H, –
NH), 1.92 (bs, 2H), 1.60 (m, 2H), 1.09–1.31 (m, 12H). MS (ESI) m ⁄ z:
378.21 (M+H+). IR (KBr, ⁄ cm): 3343, 3092, 1671, 1517, 1335, 1256,
1176, 612.
Synthesis of (4-aminophenyl){4-[3-(piperidin-4-
yl)propyl]piperidin-1-yl}methanone 3e
The product obtained was pale yellow liquid from 4-amino-
benzoic acid 2e (0.25 g, 1.82 mmol), isobutyl chloroformate
(0.32 g, 2.37 mmol), N-methyl morpholine (0.55 g, 5.46 mmol), and
Scheme 1: Synthesis of 4-[3-(piperidin-4-yl)propyl]piperidine derivatives. Reagents and conditions: (i) Aromatic ⁄ heterocyclic acids 2(a–i),
isobutyl chloroformate, N-methylmorpholine, tetrahydrofuran, 5–6 h.
Chem Biol Drug Des 2011; 78: 622–630
623

Vinaya et al.
Table 1: Chemical structures and yield of the synthesized com-
pounds 3(a–i)
1.27 mmol). 1H NMR (DMSO-d6, 400 MHz) d: 6.71 (m, 3H, Ar–H),
4.56 (t, 1H, –CH), 2.71 (t, 2H, –CH2), 2.53 (t, 2H, –CH2), 2.88–2.94
(m, 8H), 2.80 (bs, 2H), 2.12 (s, 1H, –NH), 1.72 (m, 2H), 1.12–1.24
(m, 12H). MS (ESI) m ⁄ z: 389.26 (M+H+). IR (KBr, ⁄ cm): 3352, 3073,
1680, 1340, 1256, 1165, 618.
Compound
R
Yield (%)
3a
F
NO2
Synthesis of 3-(2-oxo-2-{4-[3-(piperidin-4-
yl)propyl]piperidin-1-yl}ethyl)-2-thioxo
thiazolidin-4-one 3g
NH2
3b
3c
79
75
The product obtained was brown solid from 2-(4-oxo-2-thioxothiaz-
olidin-3-yl)acetic acid 2g (0.25 g, 1.30 mmol), isobutyl chloroformate
(0.23 g, 1.7 mmol), N-methyl morpholine (0.39 g, 3.92 mmol), and 4-
[3-(piperidin-4-yl)propyl]piperidine 1 (0.275 g, 1.3 mmol). 1H NMR
(DMSO-d6, 400 MHz) d: 4.15 (s, 2H, –CH2), 3.83 (s, 2H, –CH2-S),
2.90–2.96 (m, 8H), 2.83 (bs, 2H), 2.06 (s, 1H, –NH), 1.76 (m, 2H),
1.16–1.23 (m, 12H). MS (ESI) m ⁄ z: 384.17 (M+H+). IR (KBr, ⁄ cm):
3440, 3087, 1668, 1346, 1251, 1172, 702.
O
NH2
OCH3
O
3d
80
3e
3f
NH2
89
78
Synthesis of 5-{[5-(4-chlorophenyl)furan-2-
yl]methylene}-3-(2-oxo-2-{4-[3-(piperidin-4-
yl)propyl]piperidin-1-yl}ethyl)-2-
F
thioxothiazolidin-4-one 3h
The product obtained was pink solid from 5-{[5-(4-chlorophe-
nyl)furan-2-yl]methylene}-4-oxo-2-thioxothiazolidine-3-carboxylic acid
2h (0.15 g, 0.39 mmol), isobutyl chloroformate (0.07 g, 0.51 mmol),
N-methyl morpholine (0.12 g, 1.18 mmol), and 4-[3-(piperidin-4-
yl)propyl]piperidine 1 (0.083 g, 0.39 mmol). 1H NMR (DMSO-d6,
400 MHz) d: 7.88 (d, 1H, Ar–H), 7.76 (t, 1H, Ar–H), 7.65 (d, 1H,
Ar–H), 7.47 (d, 2H, Ar–H), 7.31 (d, 1H, Ar–H), 4.44 (s, 2H, –CH2),
2.89–2.98 (m, 8H), 2.81 (bs, 2H), 2.05 (s, 1H, –NH), 1.74 (m, 2H),
1.09–1.26 (m, 12H). MS (ESI) m ⁄ z: 559.15 (M+H+). IR (KBr, ⁄ cm):
3454, 3096, 1650, 1368, 1215, 1217, 769.
O
3g
3h
O
S
90
85
N
S
Cl
S
S
N
O
Synthesis of 6-bromo-3-(4-[3-(piperidin-4-
yl)propyl]piperidine-1-carbonyl)-2H-chromen-2-
one 3i
O
O
3i
81
O
The product obtained was pale yellow solid from 6-bromo-2-oxo-2H-
chromene-3-carboxylic acid 2i (0.10 g, 0.37 mmol), isobutyl chloro-
formate (0.066 g, 0.484 mmol), N-methyl morpholine (0.112 g,
1.11 mmol), and 4-[3-(piperidin-4-yl)propyl]piperidine (1) (0.078 g,
Br
1
0.37 mmol). H NMR (DMSO-d6, 400 MHz) d: 7.90 (s, 1H, –CH=C),
4-[3-(piperidin-4-yl)propyl]piperidine 1 (0.38 g, 1.82 mmol). 1H NMR
(DMSO-d6, 400 MHz) d: 7.78 (d, 2H, Ar–H), 6.75 (d, 2H, Ar–H), 4.1
(s, 1H, –NH2), 2.90–2.96 (m, 8H), 2.79 (bs, 2H), 2.10 (s, 1H, –NH),
1.72 (m, 2H), 1.15–1.24 (m, 12H). MS (ESI) m ⁄ z: 330.25 (M+H+). IR
(KBr, ⁄ cm): 3748, 3425, 3062, 1630, 1603, 1342, 1177, 1086.
7.72 (s, 1H, Ar–H), 7.27 (d, 2H, Ar–H), 2.88–2.95 (m, 8H), 2.83 (bs,
2H), 2.11 (s, 1H, –NH), 1.78 (m, 2H), 1.14–1.23 (m, 12H). MS (ESI)
m ⁄ z: 462.13 (M+H+). IR (KBr, ⁄ cm): 3432, 3063, 1609, 1378, 1274,
1172, 576.
Synthesis of 2-(5-{[5-(4-chlorophenyl)furan-2-
yl]methylene}-4-oxo-2-thioxothiazolidin-3-yl)
acetic acid 2h
A mixture of 3-rhodanine-3-acetic acid 1 (1.0 g, 0.01 mmol), 5-(3-
Chlorophenyl)-furfural 2 (1.08 g, 0.01 mmol), and anhydrous sodium
acetate (1.3 g, 0.03 mmol) were taken in of glacial acetic acid
10 mL. The reaction mixture was heated to 120 ꢀC in an oil bath
for 10 h. Then, the reaction mixture was cooled, filtered, and
Synthesis of (6-fluoro-3,4-dihydro-2H-chromen-
2-yl){4-[3-(piperidin-4-yl)propyl] piperidin-1-
yl}methanone 3f
The product obtained was pale yellow liquid from 6-fluoro-3,4-dihy-
dro-2H-chromene-2-carboxylic acid 2f (0.25 g, 1.27 mmol), isobutyl
chloroformate (0.22 g, 1.65 mmol), N-methyl morpholine (0.38 g,
3.82 mmol), and 4-[3-(piperidin-4-yl)propyl]piperidine
1 (0.26 g,
624
Chem Biol Drug Des 2011; 78: 622–630

4-[3-(Piperidin-4-yl) Propyl]Piperidine Derivatives
washed with ether. Finally, a reddish solid compound (1.75 g, 75%)
was obtained. The schematic representation of the synthesized
compound is shown in Scheme 2. H NMR (DMSO-d6, 400 MHz)d:
10.5 (s, 1H, –COOH), 7.9 (d, 1H, Ar–H), 7.6 (t, 1H, Ar–H), 7.5 (d, 1H,
Ar–H), 7.4 (d, 2H, Ar–H), 7.1 (d, 1H, Ar–H), 4.5 (s, 2H, –CH2). MS:
379.97.
well) were plated in triplicates and incubated with 10, 100, and
250 lM of 3(a–i). Cells were harvested after 48 and 72 h of treat-
ment and incubated with MTT (5 mg ⁄ mL) at 37 ꢀC. The blue MTT
formazan precipitate was then solubilized in detergent (50% final
concentration of N,N-dimethylformamide and 10% of sodium dode-
cyl sulfate). Absorbance was measured at 570 nm using ELISA plate
reader. The mean absorbance of culture medium was used as the
blank and was subtracted. IC50 values (concentration of compound
causing 50% inhibition of cell growth) were estimated after 72 h of
compound treatment using Origin software. The absorbance of
untreated (medium only) cells was taken as 100% viability, and the
values of treated cells were calculated as percentage of control
and presented as histograms (Figure 2).
1
Pharmacology
To assess the antiproliferative activity, leukemic cells growing in log
phase were treated with 10, 100 and 250 lM concentrations of 4-
[3-(piperidin-4-yl)propyl]piperidine derivatives 3(a–i). Because the
compounds were dissolved in DMSO, it was used as vehicle con-
trol. The amount of DMSO used was equal to the DMSO in highest
concentration of compound tested. We conducted trypan blue dye
exclusion assay, MTT assay, LDH assay, and FACS analysis to evalu-
ate the cytotoxicity of these derivatives. In addition, we have also
performed DNA fragmentation assay, which is an indicator of apop-
tosis. Each experiment was repeated a minimum of two times (15).
LDH release assay
Release of lactate dehydrogenase (LDH) is an indicator of mem-
brane integrity and hence cell injury. LDH assay was performed as
per standard protocols to assess the LDH release in the culture
media after treatment with 3a or 3h (10, 50 and 100 lM) on K562
cells for 24 and 48 h. The intracellular LDH was determined after
lysing the cells by freezing at )70 ꢀC and rapid thawing. The LDH
release was measured at an absorbance of 490 nm. The LDH per-
cent release was calculated as follows:
Cell lines and culture
Human cell line, K562 (chronic myelogenous leukemia), was pur-
chased from National Center for Cell Science, Pune, India, and Reh
(B-cell leukemia) cell line was a kind gift from Prof. Michael Lieber,
University of Southern California, USA. Cells were grown in RPMI
1640 supplemented with 10% heat-inactivated fetal bovine serum
(FBS), 100 U ⁄ mL of Penicillin, and 100 lg of streptomycin ⁄ mL and
incubated at 37 ꢀC in a humidified atmosphere containing 5% CO2.
LDH release (%) = (LDH activity in media) ⁄ (LDH activity in med-
ia + LDH activity in total cells) · 100%.
Cell cycle analysis
Cellular DNA content was measured by flow cytometry. Flow cytome-
try was performed on FACScan (Becton Dickinson, San Jose, CA,
USA), using CellQuest (Becton Dickinson) software for acquisition and
WinList (Verity, Topsham, ME, USA) software for analysis. Approxi-
mately 7.5 · 104 cells ⁄ mL were cultured and treated with 10, 50,
and 100 lM concentrations of 3a or 3h. Cells were harvested after
48 h of treatment, washed, fixed in 70% ethanol, and incubated with
RNase A (Sigma-Aldrich, St Louis, MO, USA). Propidium iodide (PI,
50 lg ⁄ mL; Sigma-Aldrich) was added half an hour before acquiring
the flow cytometric reading (FACScan; BD Biosciences, New Bedford
MA, USA). A minimum of 10 000 cells were acquired per sample.
Trypan blue exclusion assay
To determine the effect of 4-[3-(piperidin-4-yl)propyl]piperidine ana-
logs 3(a–i) on viability of K562 or Reh cells, approximately
7.5 · 104 cells ⁄ mL were seeded in a 6-well tissue culture plate for
24 h and compounds 3(a–i) were added at a concentration of 10,
100, and 250 lM. Cells were collected at intervals of 24 h and
resuspended in 0.4% trypan blue (viable-unstained and non-viable
blue). The number of viable cells was counted using hemocytometer.
MTT assay
Cell survival was further assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay, which is based on the
ability of viable cells to metabolize yellow tetrazolium salt to violet
formazan. Exponentially growing K562 or Reh cells (1 · 104 cells ⁄
DNA fragmentation assay
To elucidate the mode of action of 3a and 3h, especially with
respect to induction of oligonucleosomal DNA fragmentation, this
Scheme 2: Synthesis of 2-(5-{[5-(4-chlorophenyl)furan-2-yl]methylene}-4-oxo-2-thioxothiazolidin-3-yl) acetic acid. Reagents and conditions:
(i) Gla AcOH ⁄ CH3COONa, 120 ꢀC, 10 h.
Chem Biol Drug Des 2011; 78: 622–630
625

Vinaya et al.
assay was performed. Briefly, K562 cells were cultured in the pres-
ence of 3a at 5, 10, and 20 lM or 3h at 10, 50, and 100 lM for
72 h. Cells were harvested, and genomic DNA was extracted using
standard protocol. DNA was resuspended in 250 lL of TE buffer. To
ensure the equal loading of DNA, pilot gel was run by loading
equal volume of DNA into each well, and then, based on the inten-
sity of bands, concentration of DNA was adjusted. Finally, the DNA
samples were run on 1% agarose gel and visualized by ethidium
bromide staining and photographed.
tested. In trypan blue assay, the cells were counted every day till
the control cells reached stationary phase. Results are represented
as graphs (Figure 1), which Results shows that the addition of 3(a–
i) affected the viability of the cells in both the cell lines investi-
gated (Figure 1 and data not shown). The cytotoxicity induced by
the 4-[3-(piperidin-4-yl)propyl]piperidine s 3(a–i) was dose and time
dependent. It was found that the effect was maintained even after
prolonged incubation periods.
The cytotoxicity was further verified using MTT assay (Figure 2).
The effect of the compounds was expressed as percentage of cell
proliferation. IC50 value was determined after 72 h, and the results
summarized in Table 2 with SD values show that most of the com-
pounds inhibited the growth of both K562 and Reh cells in the
micromolar range. Initially, structure–activity relationship of this ser-
ies of compounds against K562 cell line was investigated. Different
substituents on the N-terminal of the piperidine exhibited different
levels of antitumor activity. On the basis of the data obtained, the
following conclusion may be drawn. In general, the nature of the
substituent at position N-amide of piperidine is crucial in influenc-
ing cytotoxicity. Thus, it was found that incorporation of fluoronitro-
benzene (3a) resulted in more potent molecule with IC50 value of
Results and Discussion
All compounds were evaluated for their cytotoxic activity in vitro
against human leukemia cells, K562 (chronic myeologenous leuke-
mia) and Reh (pre-B cell). Preliminarily, we have used trypan blue
and MTT assays to assess the effect of 4-[3-(piperidin-4-yl)pro-
pyl]piperidine derivatives 3(a–i) on cell viability. For performing the
antiproliferative assay, the compounds were dissolved in DMSO. To
ensure that the solvent per se had no effect on the cell growth,
negative control tests were performed using DMSO at the same
concentration as used for highest concentration of the compound
Figure 1: Dose- and time-dependent effect of 4-[3-(piperidin-4-yl)propyl]piperidines 3(a–i) on K562 cell survival. Approximately
0.75 · 105 cells ⁄ mL were cultured followed by treatment of 3(a–i) after 24 h at 10, 100, and 250 lM. Besides untreated cells, DMSO-treated
cells were used as vehicle control. After every 24 h from the time of addition of compounds, cell viability was determined by trypan blue
exclusion assay and data were represented as a graph. Mean € SD error bars are represented in the figure. Graphs represent the treatment
of compound 3(a–i), respectively, against K562 cells.
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4-[3-(Piperidin-4-yl) Propyl]Piperidine Derivatives
Figure 2: Determination of the effect of 4-[3-(piperidin-4-yl)propyl]piperidines 3(a–i) on cell proliferation by MTT assay. After 48 and 72 h
of exposure, K562 cells with 3(a–i) (10, 100 and 250 lM) were incubated with MTT (5 mg ⁄ mL) in duplicates and resulting blue formazan pre-
cipitate was dissolved in detergent, and absorbance was measured at 570 nm. Results are presented as percentage of cell proliferation (the
cell viability of vehicle cells was considered as 100%). Mean € SD error bars are represented in the figure. Graphs represent the treatment
of compound 3(a–i), respectively, against K562 cells.
fluoro with amine group in 3e reduced the potency with IC50 values
Table 2: IC50 with mean € SD values (lM) of the synthesized
of 5.1 and 5.5 lM, respectively. This suggests that the anticancer
activity is dependent not only on the nature of the substituent but
also on the nature of the groups attached to the phenyl ring of that
substituent. Similarly, when we compare the potency of compounds
3b (R = amino-1,2,3,4-tetrahydronaphthalene, IC50 = 100.2 lM), 3c
(R = amino-1,2,3,4-tetrahydronaphthalene, IC50 = 50.1 lM), and 3f
(R = fluoro-3,4-dihydro-2H-chromene, IC50 = 50.2 lM), 50% of activ-
ity was reduced in case of 3b. To enhance the inhibitory activity,
we replaced fluoro-3,4-dihydro-2H-chromene (3f, IC50 = 50.2 lM)
with bromo-2-oxo-2H-chromene (3i, IC50 = 125.1 lM). But the
potency to inhibit cell growth got reduced.
compounds 3(a–i) based on MTT assay
Compound
K562
Reh
2.3 € 1.5
107.3 € 9.8
48.0 € 5.2
6 .0 € 1.6
6.0 € 1.8
45.0 € 5.8
18.1 € 4.1
17.2 € 1.9
120.8 € 8.6
3a
3b
3c
3d
3e
3f
2.0 € 1.3
100.2 € 9.2
50.1 € 7.5
5.1 € 1.4
5.5 € 1.5
50.2 € 6.5
20.5 € 4.4
15.0 € 1.6
125.1 € 10.3
3g
3h
3i
It is worthy to mention that previously reported 2-(5-{[5-(4-chlor-
ophenyl)furan-2-yl]methylene}-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid derivatives (18) exhibited weak antiproliferative activity when
compared with 3h (R = 5-{[5-(4-chlorophenyl)furan-2-yl]methylene}-
4-oxo-2-thioxothiazolidine IC50 = 15 lM) having almost same group
as a substituent in the current series. This data clearly show that
the enhanced activity exhibited by compound 3h could be due to
2 lM. The inhibition by compound 3a could be attributed to elec-
tron withdrawing fluoro and nitro groups present on the substituted
benzamide at para and ortho position.
However, the replacement of nitro with cyclopentyloxy and fluoro
with methoxy group in 3d and removal of nitro and replacement of
Chem Biol Drug Des 2011; 78: 622–630
627

Vinaya et al.
Figure 3: Time- and dose-dependent LDH release in K562 cells treated with 3a or 3h. K562 cells were incubated for 24 and 48 h with
different concentrations of 3a or 3h. Release of LDH in the medium was measured at 490 nm. Results are presented as percentage of LDH
release. Mean € SD error bars are represented in the figure.
A
B
C
D
Figure 4: Cell cycle analysis of K562 cells treated with 3a or 3h. K562 cells (0.75 · 105 cells ⁄ mL) were incubated at 37 ꢀC with 3a or
3h (10, 50 and 100 lM). Following 48 h of incubation, cells were fixed and stained with propidium iodide and subjected to FACS analysis.
Panels A and B show histograms comparing the effect of 3a and 3h at specific cell cycle stages. In Panel A and B, the first histogram repre-
sents DMSO-treated cells. Panels C and D show the quantification of cells in different stages of cell cycle after treatment with 3a and 3h,
respectively.
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Chem Biol Drug Des 2011; 78: 622–630

4-[3-(Piperidin-4-yl) Propyl]Piperidine Derivatives
the presence of amide and the 4-[3-(piperidin-4-yl)propyl]piperidine
moiety. The presence of amide group is necessary to lower sys-
temic toxicity and to improve specificity in cancer treatment (11). In
addition to this, compound 3g having thiazolidinone group attached
to the N-terminal of the piperidine also displayed good cytotoxic
activity.
programmed cell death or apoptosis. During the apoptotic process,
activated nucleases degrade the higher-order chromatin structure of
DNA into mono- and oligonucleosomal DNA fragments. Apoptotic
degradation of DNA was analyzed by agarose gel electrophoresis.
To verify this, chromosomal DNA was extracted after 72 h from the
K562 cells treated with increasing concentrations of 3a (5, 10, and
20 lM) or 3h (10, 50, and 100 lM) as also from vehicle control-
treated cells. This DNA was used for agarose gel electrophoresis.
The results showed fragmentation of DNA leading to a smear in
the lanes in which cells were treated with 3a or 3h (Figure 5).
The observed smear is the result of DNA breakage at multiple posi-
tions across the chromosomal DNA. The intensity of smear
increased with the dose in both 3a- and 3h-treated cells. In 3a-
treated cells, maximum smear was observed even at 10 lM. With
respect to the DNA damage, we find that the extent of DNA strand
breakage by 3a is similar to that by 3h. However, the concentra-
tions required by 3a to achieve this are much lesser compared with
those by 3h. These results further suggest that 3a having fluoro
and nitro phenyl ring induces fragmentation of chromosomal DNA
leading to apoptosis more significantly than 3h having 5-{[5-(4-
chlorophenyl)furan-2-yl]methylene}-4-oxo-2-thioxothiazolidine group.
Furthermore, the potency of both 3a and 3h was verified by LDH
assay, cell cycle analysis, and DNA fragmentation against K562
cells. The release of LDH is an indication of cell injury and hence
cell death. Upon treatment with 3a or 3h, the LDH gets released
into media, which was measured. Data show that the release of
LDH was time and dose dependent (Figure 3). In case of 3a treated
with 100 lM, maximum release of LDH was observed at 48 h. This
further confirmed that 3a is more potent.
Flow cytometry was used to evaluate the effects of 3a and 3h on
the growth and division of K562 cells, by measuring the DNA con-
tent of both treated and untreated cells stained by propidium
iodide. Interestingly, upon addition of 3a or 3h to K562 cells, a
concentration-dependent change was observed in the cell cycle pat-
tern (Figure 4). As evident from Figure 4, that 3a caused remark-
able accumulation of sub-G1 cells (mostly dead cells) at
concentration of 10 lM and higher with almost complete decline of
both S and G2 ⁄ M phase cells, which indicated the sign of apopto-
sis. However, such a drastic change was not observed when the
cells were treated with compound 3h at the same concentration
levels. The histogram of vehicle control (DMSO treated) cells
showed a standard cell cycle pattern, which include G0 ⁄ G1 and
G2 ⁄ M peaks separated by S phase peak with almost no dead cells.
Therefore, the studies further confirmed that remarkable growth
inhibition caused by 3a could be due to apoptosis.
Conclusion
In summary, this study demonstrates that the 4-[3-(piperidin-4-yl)pro-
pyl]piperidine derivatives exhibit interesting antileukemia properties,
depending on a delicate balance between the piperidine substitu-
tion pattern and nature of the acid moieties. They exhibited IC50
values of 2–125 lM. While 3a was highly potent, a few others
(3d, 3e, 3g and 3h) exhibited moderate activity. The substituents
at the N-amide of the pyridine ring were shown to be vital for
potency. Work is in progress to optimize antileukemia activity by
optimizing the N-substituted piperidine, to establish the mechanism
of action.
Finally, we were interested to ascertain whether 3a or 3h caused
DNA strand breaks or not, which is a characteristic feature of the
Figure 5: Detection of 3a or
3h induced DNA damage in K562
cells. The chromosomal DNA was
extracted from K562 cells following
treatment with different concentra-
tions of 3a (A) and 3h (B). The
purified DNA was then resolved on
a 1% agarose gel at 30 V for 6 h.
In both panels, Lane 1: DMSO;
Lane 2, 3 and 4: K562 cells treated
with 5, 10, and 20 lM of 3a, and
10, 50 and 100 lM of 3h, respec-
tively. 'M' is Marker.
Chem Biol Drug Des 2011; 78: 622–630
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Vinaya et al.
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Acknowledgements
Authors are grateful to UGC, Govt. of India for financial support to
KSR under the project UGC-SAP (DRS-II) vide No. F. 540 ⁄ 10 ⁄ 2004-
05. SCR acknowledges support from Lady Tata Memorial Trust inter-
national award for leukemia research (London). Two of the authors
KV and DSP are grateful to Council of Scientific and Industrial
Research (CSIR), New Delhi, for financial support under CSIR SRF
order No. 09 ⁄ 119(0172)2K8 EMR-I and 09 ⁄ 119(0173)2K8 EMR-I,
respectively. Authors are thankful to Dr. C. S. Ananda Kumar and
Dr. S. B. Benaka Prasad for their help while preparing the manu-
script.
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