Synthesis and biological efficacy of novel piperazine analogues bearing quinoline and pyridine moieties

Article abstract of DOI:10.1134/S1068162015040020

A series of novel piperazine analogues bearing quinolin-8-yloxy-butan-1-ones/pyridin-2-yloxyethanones were synthesized by a simple and convenient approach based on various substituted piperazine incorporating quinoline and pyridine moieties. The analogues were evaluated for in vitro antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferrous ion radical scavenging activities and anti-inflammatory activity by inhibition of Vipera russelli venom (PLA₂) and gastric K⁺/H⁺-ATPase activities. Most of the title compounds exhibited promising activity. Best antioxidant and PLA₂-inhibiting activities were found for piperazine analogues with phenyl and nitro phenyl groups, whereas methoxy group on phenyl piperazine indicated selectivity for the H⁺/K⁺-ATPase.

Full text of DOI:10.1134/S1068162015040020

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 5, pp. 554–561. © Pleiades Publishing, Ltd., 2015.
Synthesis and Biological Efficacy of Novel Piperazine Analogues
Bearing Quinoline and Pyridine Moieties1
M. AlꢀGhorbania, N. D. Rekhab, V. Lakshmi Ranganathaa, c, T. Prashantha, d,
, 2
T. Veerabasappagowdab, and S. A. Khanuma
a Department of Chemistry, Yuvarajas College, University of Mysore, Mysore, Karnataka, India
b Department of Studies in Biotechnology, JSS College of Arts, Commerce and Science, Mysore, Karnataka, India
c PG Department of Chemistry, St. Philomena' s College, Bannimantap, Mysore ꢀ 570015
d Department of Chemistry, The National Institute of Engineering, Manandavadi Road, Mysore 570008
Received August 24, 2014; in final form, January 12, 2015
Abstract—A series of novel piperazine analogues bearing quinolinꢀ8ꢀyloxyꢀbutanꢀ1ꢀones/pyridinꢀ2ꢀyloxyꢀ
ethanones were synthesized by a simple and convenient approach based on various substituted piperazine
incorporating quinoline and pyridine moieties. The analogues were evaluated for in vitro antioxidant activity
against 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH) and ferrous ion radical scavenging activities and antiꢀinflamꢀ
matory activity by inhibition of Vipera russelli venom (PLA2) and gastric K+/H+ꢀATPase activities. Most of
the title compounds exhibited promising activity. Best antioxidant and PLA2ꢀinhibiting activities were found
for piperazine analogues with phenyl and nitro phenyl groups, whereas methoxy group on phenyl piperazine
indicated selectivity for the H+/K+ꢀATPase.
Keywords: piperazine analogues, antioxidant, PLA2, H+/K+ꢀATPase
DOI: 10.1134/S1068162015040020
INTRODUCTION
tion with free radicals has long been considered in the
therapeutic process of various inflammatory disorders.
Antioxidant therapies are gaining importance due
to their ability to retard disease progression by reducꢀ
On the other hand, the inhibition of gastric acid
ing the damage caused by free radical oxidative stress secretion, especially by antagonism of the H2 recepꢀ
in a patient [1]. Physiological levels of reactive oxygen tor, has been proven a powerful tool in the treatment of
species (ROS) play a vital role as signaling molecules
to mediate numerous biological functions causing
alterations in cell growth, gene expression, and host
defense [2]. Under inflammatory conditions, the presꢀ
gastric and duodenal ulcer diseases [5]. Recently,
agents that completely suppress acid secretion by inhiꢀ
bition of the gastric proton pump H+/K+ꢀATPase have
been identified.
ence of excess ROS (O2•–
, )
•OH, H2O2, NO•, ONOO–
Piperazine and their derivatives have been found to
exhibit a variety of biological activities and are well
known for their medicinal importance and recognized
for their use as antidepressive [6], antihistamine [7],
anticancer [8–10], and antioxidant [11] activities.
can initiate damage to nucleic acids, proteins, carboꢀ
hydrates, and lipids in many types of cells, including
macrophages [3].
The association of antioxidants with inflammation
stems from the recognition that free radicals are proꢀ
duced during the inflammatory process by macrophꢀ
ages. It has been reported that ROS are involved in the
cyclooxygenaseꢀ and lipoxygenaseꢀmediated converꢀ
sion of arachidonic acid into proinflammatory interꢀ
mediates and possess the ability to activate PLA2
enzyme, which further augments the inflammatory
process and worsens the disease during chronic
inflammatory conditions [4]. Therefore, suppression
of these proꢀinflammatory lipid mediators in conjuncꢀ
The incorporation of synthetic piperazine is an
important strategy in drug discovery due to its easy
modifiability, proper alkality, water solubility, and the
capacity for the formation of hydrogen bonds and
adjustment of molecular physicochemical properties
[12]. The piperazine nucleus incorporated the quinoꢀ
line and pyridine moiety into a rigid framework. It has
been reported that quinoline and pyridine derivatives
possess appreciable antiꢀinflammatory activity [13].
Moreover, piperazine and related derivatives have
shown special ability to scavenge ROS in processes
involving free radical injury [14] and are effective as
reversible inhibitors of gastric H+/K+ꢀATPase [15].
1
The article is published in the original.
2
Corresponding author: phone: +9901888755; fax: +821 2419239;
eꢀmail: shaukathara@yahoo.co.in.
554

SYNTHESIS AND BIOLOGICAL EFFICACY OF NOVEL PIPERAZINE ANALOGUES
555
1
Based on the above information and continuing synthesized compounds were confirmed by H and
13C NMR, mass spectrometry, and elemental analysis.
In the 1H NMR spectra of (Va
), two sets of multiplet
signals at .1–4.0 ppm each belonging to the four
protons of two CH2 groups in piperazine ring were
observed. The 1H NMR spectrum of compound (Va
showed the multiplet signals in the region of .8–
our research program aimed at developing simple and
efficient synthesis of pharmacologically useful heteroꢀ
cyclic analogues, we envisage that incorporating quinꢀ
oline and pyridine cores in substituted piperazine
frame could lead to the potent antioxidant activity, as
well as PLA2 and H+/K+ꢀATPase inhibition activities.
–l
δ
3
)
δ
6
7.3 ppm, which appeared due to phenyl protons.
13C NMR spectra also confirmed the piperazine
RESULTS AND DISCUSSION
structure of (Va
range 44.08–67.89. The mass spectra of compounds
Va ) were in agreement with their assigned strucꢀ
tures. The mass spectrum of (Va ) showed molecular
ion peak at 376 ( + 1), which corresponds to the
–l) due to peaks appearance in the
In this study, novel derivatives of piperazine anaꢀ
logues were synthesized in an attempt to find new
compounds having antioxidant activity and antiꢀ
inflammatory related activities, such as inhibition of
PLA2 and gastric H+/K+ꢀATPase activity. The synꢀ
thetic routes for the newly synthesized piperazine anaꢀ
δ
(
–l
M
molecular formula C23H25N3O2. Similarly, the spectral
values for all the compounds and C, H, N analyses are
logues (Va –l) are illustrated and outlined in the
Scheme 1. The structure elucidations of the newly given in the experimental part.
O
K CO , reflux
O
2
Anhyd. acetone
3
n
n
Ar–OH +
(Ia–c)
ArO
X
O
O
(
IIIа–с
)
(
IIа
–b
)
a
b
, X = Br,
, X = Cl;
n
n
= 3
= 1
R
N
OH
TBTU, Et N
3
NaOH
n
N
ArO
+
n
HN
N R
Anhyd. acetonitrile,
inert atmosphere, RT
ArO
Ethanol, reflux
O
O
(
IVa–c)
(Va
–l)
R
R
R
Ar
Ar
Ar
Compd
Compd
Compd
(
Va
= 3
)
(
Ve
= 1
)
(
Vi)
NO2
NO2
n
n
n
= 1
CH3
N
N
N
H3CO
(
Vb
= 3
)
(
Vf)
(Vj
)
–CH3
n
n
= 1
n
= 3
CH3
N
N
N
N
(
Vc
)
(
Vg
)
(
Vk
)
–CH3
n
= 3
n
= 3
n
= 1
CH3
N
N
N
H3CO
(
Vd
= 3
)
(
Vh
=1
)
(Vl
)
–CH3
NO2
n
n
n
= 1
CH3
N
N
H3CO
Scheme 1. Synthesis of piperazine analogues (Va
–l
).
In vitro antioxidant activities were measured DPPH radical scavenging activity evaluation is a rapid
against DPPH and ferrous ion chelating radicals. and convenient technique for screening the antioxiꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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556
ALꢀGHORBANI et al.
)*
Table 1. DPPH radical scavenging activity of the compounds (Va
–l
Concentration,
6
µ
g/mL
Test samples
2
4
8
10
IC50, µg/mL
(
(
(
(
(
(
(
(
(
(
(
(
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vh
)
23.74
29.72
36.63
22.73
26.4
34.23
45.64
48.48
45.51
47.27
53.46
42.57
41.59
43.74
46.58
51.71
41.31
48.46
45.47
59.62
67.1
57.45
73.71
79.65
74.18
78.29
82.35
75.03
84.72
86.51
77.58
83.22
79.14
78.6
77.53
99.4
6.7
4.6
4.1
5.2
4.4
3.5
5.1
4.7
4.5
4.4
3.8
4.6
4.4
)
)
92.65
87.3
)
52.25
65.61
71.12
58.3
)
90.17
92.52
95.24
98.55
98.58
84.22
97.52
93.86
90.46
)
38.31
30.14
21.52
24.59
33.39
39.71
29.56
34.86
)
)
64.61
67.66
66.78
65.64
63.62
65.48
Vi
Vj
Vk
)
)
)
Vl)
BHT
* Results presented here are the mean values from three independent experiments.
Table 2. Ferrous ion radical scavenging activity of the compounds (Va –l)*
Concentration,
12
µ
g/mL
Test samples
4
8
16
20
IC50,
µg/mL
(
(
(
(
(
(
(
(
(
(
Va
Vb
Vc
Vd
Ve
Vf
Vg
)
28.5
48.82
57.45
46.56
37.64
43.44
48.48
42.24
41.92
56.17
41.29
43.5
69.59
67.89
64.5
83.58
79.44
78.51
72.48
78.12
78.35
70.42
76.12
82.84
81.73
86.62
94.7
9.5
7.6
)
33.28
23.6
88.13
94.43
89.14
90.34
91.5
)
9.1
)
19.64
31.46
24.57
30.54
32.65
34.49
21.24
22.65
55.47
66.44
65.45
57.47
54.83
74.88
63.62
64.57
10.9
9.7
)
)
8.6
)
83.59
94.51
94.86
92.54
93.58
10.6
11.2
6.2
Vi)
Vj)
Vl)
9.3
BHT
9.6
* Results presented here are the mean values from three independent experiments.
dant activities. It can be seen from Table 1 that, in
DPPH assay, compounds (Vc), (Vf), and (Vk) with
IC50 values of 4.1, 3.5, and 3.8 µg/mL, respectively,
showed better radical scavenging activities than the
synthetic commercial antioxidant BHT with the highꢀ
est scavenging of 92–97%. Compounds (Ve), (Vi), (Vj),
and (Vl) with IC50 values of 4.4, 4.5, 4.4, and
Metal chelating capacity is important since it
reduces the concentration of the catalyzing transition
metal in lipid peroxidation (thus delaying metalꢀcataꢀ
lyzed oxidation) [16]. Since ferrous ions constitute the
most effective proꢀoxidants in food and biological sysꢀ
tems, the good chelating effect and removal of free
iron from circulation would be beneficial and correct
approach to prevent oxidative stressꢀinduced disorder.
From the results of ferrous ion chelating assay (Table 2),
the compounds (Vb), (Vc), (Vf), (Vj), and (Vl) with IC50
4.6 µg/mL displayed good DPPH radical scavenging
activity. Compounds (Vb), (Vd), (Vg ), and (Vh) showed
moderate radical scavenging abilities, while comꢀ
pound (Va ) showed poor radical scavenging ability in
comparison with the standard.
values of 7.65, 9.12, 8.60, 6.23, and 9.33 µg/mL,
respectively, showed higher radical scavenging activiꢀ
ties than that of the standard BHT. Other compounds
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SYNTHESIS AND BIOLOGICAL EFFICACY OF NOVEL PIPERAZINE ANALOGUES
557
Table 3. PLA2 inhibition activity of compounds (Va –l)
Concentration, µg/mL
Test samples
10
20
30
40
50
60
IC50, µg/mL
(Va )
12.38
37.9
54.37
66.74
78.33
95.63
27.5
(
(
(
(
(
(
(
(
(
(
(
Vb
)
24.26
26.74
25.33
18.91
10.36
8.04
40.04
37.24
34.92
25.43
28.85
17.44
28.5
56.59
46.94
54.42
47.68
47.74
36.94
45.7
68.53
58.13
68.88
59.31
56.74
52.13
63.22
52.87
65.53
52.91
6.94
79.03
75.73
79.52
70.15
75.73
65.63
76.3
93.61
89.03
92.61
81.95
84.72
75.33
89.53
79.36
81.71
38.43
2.61
26
32.4
29
Vc)
Vd)
Ve
Vf
Vg
Vh
)
32.3
31
)
)
38
)
5.31
32
Vi
Vj
Vk
)
17.3
29.89
29.15
67.5
44.38
47.54
60.06
8.75
65.77
73.62
45.77
4.72
40
)
11.36
74.84
12.37
31.2
Inactive
Inactive
5
)
Vl)
10.36
–
Control
100
–
–
–
–
exhibited good to moderate radical scavenging activꢀ
Compounds (Va – l ) were tested for their ability to
ity; compounds (Vh) and (Vk) failed to scavenge ferꢀ inhibit gastric H+/K+ꢀATPase (Table 3). Omeprazole
rous ion radical. Compounds (Vb), (Vc), (Vf), (Vj), and was used as the reference compound. Several comꢀ
(
Vl) demonstrated a marked capacity for iron binding, pounds exhibited potent inhibitory activity on H+/K+
ꢀ
suggesting their role as hydroxyl radical protector. The ATPase. Compounds (Vc), (Vg ), (Vh), and (Vi) having
results reveal that phenyl and nitrophenyl groups in phenyl and methoxyphenyl substituent in the piperaꢀ
the piperazine ring of (Vb), (Vc ), and (Vf), as well as zine ring with IC50 values of 5.2, 4.5, 4.9, and
compounds (Vj), (Vk), and (Vl) with the methyl group 5.3 µg/mL, respectively, indicated selectivity for the
in the piperazine, possess potent DPPH and ferrous H+/K+ꢀATPase. IC50 values for compounds (Vb), (Ve),
ion radical scavenging activities. However, with few and (Vj) could not be calculated as they had no inhibꢀ
exceptions, it has been noted that phenyl, nitrophenyl itory activity. Compound (Vg ) was found to be the most
and Nꢀmethyl piperazine were found to be more suitꢀ active among the tested compounds.
able for reduction of DPPH [14] and ferrous ion
chelating.
From the PLA2 and ATPase results point of view, it
can be pointed out that phenyl and nitrophenyl subꢀ
stituents on the piperazine ring showed promising
venom PLA2 inhibition while methoxyphenyl substitꢀ
uent in the piperazine ring was found to exhibit the sigꢀ
nificant inhibition against H+/K+ꢀATPase. Further
modification of side chain quinoline, particularly the
presence of the electron donating methyl group or steꢀ
reo chemical factors due to ortho substitution, might
be the cause for failed activity associated with the
compounds (Vh) and (Vk) in ferrous ion, (Vk) in
PLA2, and (Vb) and (Ve) in H+/K+ꢀATPase assays. It is
noteworthy that compounds (Vc ) and (Vf) having a
pyridine ring with phenyl and nitrophenyl groups,
respectively, showed potent inhibition in all the assays.
The Russell viper venom PLA2 is known to induce
presynaptic neurotoxicity and myotoxicity in experiꢀ
mental mice [17]. All these maladies are associated
with PLA2 activity. Compounds (Va – l ) were tested for
in vitro antiꢀinflammatory properties by studying their
indirect hemolytic PLA2 activity (Table 3). Comꢀ
pounds (Va ), (Vb), and (Vd) having phenyl and nitroꢀ
phenyl substituents in the piperazine ring inhibited
snake venom PLA2 in a dose dependent manner with
IC50 values of 27.5, 26, and 29 µg/mL respectively,
while compounds (Vk) and (Vl) having methyl substitꢀ
uent in the piperazine ring failed to inhibit the snake
venom PLA2. On the other hand, the rest of the comꢀ
pounds, (Vc), (Ve), (Vf), (Vh), and (Vj) with IC50 valꢀ
ues of 32.4, 32.3, 31, 32, and 31.2
showed moderate activity and compounds (Vg ) and
Vi) with IC50 values of 38 and 40 g/mL, respectively,
µg/mL, respectively,
EXPERIMENTAL
(
µ
All the chemicals and reagents were purchased
showed low activity. Therefore, compounds (Va ), (Vb), from Sigma Aldrich Chemical Pvt Ltd. All the solvents
and (Vd) are promising antiꢀinflammatory molecules were double distilled before use. Vipera russelli venom
compared to other substances and they tempt to sugꢀ was obtained from IRULA snake catchers, Madras,
gest a role as a candidate for the treatment of snake India. Melting points were determined in open capilꢀ
bite.
lary tubes on an electrothermal melting point apparaꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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558
ALꢀGHORBANI et al.
tus. Absorbance was measured on a Uꢀ2900 (Hitachi) inert atmosphere for 10–24 h; the completion of the
UVꢀVIS spectrophotometer. 1H and 13C NMR spectra reaction was monitored by TLC using chloroform–
were recorded on a Bruker 400 MHz spectrometer in methanol (9 : 1) as eluent. The solvent was evaporated
CDCl3. Chemical shifts (
million (ppm) downfield from tetramethylsilane. water (20 mL), and the obtained solids (Va ), (Vb), (Vc),
Mass spectra were registered on an APIꢀ4000LCꢀ Vd), (Ve), (Vf), (Vi), and (Vk) were filtered, dried, and
MS/MS apparatus. The elemental analysis of the recrystallized from ethanol. In contrast, compounds
compounds was performed on a Perkin Elmer 2400 Vg ), (Vh), (Vj), and (Vl) were obtained by extracting
Elemental Analyzer. The results of elemental analyses with ethyl acetate. The extract was washed successively
δ) are reported in parts per at reduced pressure, quenched by the addition of cold
(
(
were within 0.4% of the theoretical values.
with a solution of 10% HCl (20 mL), 10% NaHCO3
(20 mL), and water (20 mL). The organic layer was
dried over anhydrous sodium sulfate and evaporated,
the crude product was purified by column chromatogꢀ
raphy (eluent: hexane–dichloromethane–acetone,
5 : 3 : 2) to achieve pure piperazine derivatives. The
physical and analytical data of the synthesized title
Synthesis of Compounds
The reaction sequence for different title comꢀ
pounds (Va
ing substituted hydroxyl compounds (Ia
reacted with (IIa ) in the presence of anhydrous
–l) is outlined in the Scheme 1. The startꢀ
–
c
) were
compounds (Va
1ꢀ[4ꢀ(Phenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ4ꢀ(quinolinꢀ8ꢀyloxy)ꢀ
butanꢀ1ꢀone (Va) Yield 78%, white solid, mp112–
C. H NMR: 2.4 (m, 2H, CH2), 2.7 (t, 2H,
–l) are given below.
–b
potassium carbonate and dry acetone at reflux temperꢀ
ature to afford compounds (IIIa–c) [18]. These mateꢀ
rials were subjected to hydrolysis using NaOH solution
.
1
114°
in ethanol to afford the product (IVa
target piperazine analogues (Va ) were obtained in
good yields by stirring of (IVa ) with substituted pipꢀ
erazines in the presence of N,N,N', ꢀtetramethylꢀ
(benzotriazolꢀ1ꢀyl) uroniumtetrafluoroborate
–c). Finally, the
COCH2), 3.1 (m, 4H, piperazine), 3.7 (m, 4H, piperꢀ
azine), 4.4 (t, 2H, OCH2), 7.1–8.9 (m, 6H, quinoline
ArꢀH), 6.8–7.3 (m, 5H, ArꢀH). 13C NMR: 24.41,
29.57, 40.47, 44.25, 46.42, 49.00, 67.58, 76.82, 77.21,
77.26, 109.23, 112.57, 119.69, 122.77, 125.64, 126.47,
129.24, 136.32, 139.85, 140.87, 149.39, 154.65,
–l
–c
N
'
oꢀ
(TBTU) as coupling reagent and triethylamine in dry
acetonitrile at RT under inert atmosphere.
171.46. LCꢀMS:
m/z 376 (M + 1). Anal. calcd. for
C23H25N3O2: C, 73.57; H, 6.71; N, 11.19. Found: C,
73.45; H, 6.54; N, 11.35%.
General synthetic procedure for ethyl butyrate and
ethyl acetate analogues (IIIa–c). Synthetic procedure
for compounds (IIIa–c) has been reported previously
[18, 19].
4ꢀ(Quinolinꢀ8ꢀyloxy)ꢀbutyric acid ethyl ester
(IIIa). mp 62–64°C (ref. 60°C [19]).
1ꢀ(4ꢀPhenylꢀpiperazinꢀ1ꢀyl)ꢀ2ꢀ(2ꢀmethylꢀquinolinꢀ
8ꢀyloxy)ꢀethanone (Vb)
.
Yield 76%, white solid, mp
1
137–139°C. H NMR: 3.2 (s, 3H, quinoline CH3),
3.5 (m, 4H, piperazine), 3.9 (m, 4H, piperazine),
4.9 (s, 2H, OCH2CO), 6.9–8.4 (m, 5H, quinoline ArꢀH),
6.8–7.3 (m, 5H, ArꢀH). 13C NMR: 24.45, 40.18,
44.54, 46.27, 49.28, 67.41, 76.00, 76.76, 77.87,
109.42, 112.70, 119.95, 121.86, 126.56, 126.89,
129.70, 136.17, 138.66, 140.95, 149.39, 154.48,
General synthetic procedure for butyric acid and
acetic acid analogues (IVa–c). Compounds (IIIa–c
)
(0.02 mol) were dissolved in ethanol (15 mL), sodium
hydroxide (0.035 mol) in water (5 mL) was added, and
the mixture was refluxed for 5–9 h. The reaction mixꢀ
ture was cooled and acidified with 2 N hydrochloric
acid. The precipitate was filtered, washed with water,
and finally recrystallized from methanol to afford
171.87. LCꢀMS:
m/z 362 (M + 1). Anal. calcd. for
C22H23N3O2: C, 73.11; H, 6.41; N, 11.63. Found: C,
73.20; H, 6.19; N, 11.56%.
desired compounds (IVa–c). Compound (IVa) is taken
as a representative example to explain physical and
characterization data.
1ꢀ(4ꢀPhenylꢀpiperazinꢀ1ꢀyl)ꢀ2ꢀ(pyridinꢀ2ꢀyloxy)ꢀ
ethanone (Vc)
.
Yield 64%, white solid, mp 142–
1
144
°C. H NMR: 3.3 (m, 4H, piperazine), 3.9 (m,
4H, piperazine), 4.8 (t, 2H, OCH2), 6.2–7.2 (m, 4H,
pyridine ArꢀH), 6.9–7.4 (m, 5H, ArꢀH). 13C NMR:
40.56, 44.15, 46.07, 49.45, 67.89, 76.20, 77.01, 77.33,
109.29, 112.78, 119.75, 124.47, 136.83, 139.42,
4ꢀ(Quinolinꢀ8ꢀyloxy)ꢀbutyric acid (IVa). Yield
1
80%, pink solid, mp 168–170
°
C. H NMR: 2.1 (m,
2H, CH2), 2.6 (t, 2H, COCH2), 4.2 (t, 2H, OCH2),
.1–8.8 (m, 6H, quinoline ArꢀH), 12.5 (s, 1H,
COOH). LCꢀMS: 232 ( + 1). Anal. calcd. for
7
146.32, 151.42, 171.40. LCꢀMS:
m/z 298 (M + 1).
m/z
M
Anal. calcd. for C17H19N3O2: C, 68.67; H, 6.44; N,
14.13. Found: C, 68.53; H, 6.12; N, 14.27%.
C13H13NO3: C, 67.52; H, 5.67; N, 6.06. Found: C,
67.46; H, 5.53; N, 5.88%.
General synthetic procedure for piperazine anaꢀ
1ꢀ[4ꢀ(4ꢀNitroꢀphenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ4ꢀ(quinolinꢀ
logues (Va – l ). To the mixture of compounds (IVa
–c)
8ꢀyloxy)ꢀbutanꢀ1ꢀone (Vd). Yield 80%, yellow solid, mp
(2 mmol) in dry acetonitrile (15 mL), triethylamine 115–117
°
C.1H NMR: 2.5 (m, 2H, CH2), 2.8 (t, 2H,
(3 mmol) followed by substituted piperazines COCH2), 3.5 (m, 4H, piperazine), 3.9 (m, 4H, piperꢀ
(2 mmol) was added. The mixture was stirred for azine), 4.5 (t, 2H, OCH2), 7.1–8.9 (m, 6H, quinoline
30 min and TBTU (2mmol) was then added and stirꢀ ArꢀH), 6.7–7.6 (m, 4H, ArꢀH). 13C NMR: 24.51,
ring was continued at room temperature under an 29.41, 40.86, 44.62, 46.86, 49.13, 67.77, 76.75, 77.01,
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SYNTHESIS AND BIOLOGICAL EFFICACY OF NOVEL PIPERAZINE ANALOGUES
559
77.26, 109.09, 112.81, 119.75, 121.61, 125.92, 126.83, 119.65, 124.83, 136.83, 139.02 146.70, 151.18, 171.65.
129.73, 136.04, 139.04, 144.35, 149.19, 154.35, LCꢀMS: 328 ( + 1). Anal. calcd. for C18H21N3O3
171.26. LCꢀMS: 421 ( + 1). Anal. calcd. for C, 66.04; H, 6.47; N, 12.84. Found: C, 66.28; H, 6.09;
m/z
M
:
m/z
M
C23H24N4O4: C, 65.70; H, 5.75; N, 13.33. Found: C, N, 12.50%.
65.78; H, 5.89; N, 13.17%.
1ꢀ(4ꢀMethylꢀpiperazinꢀ1ꢀyl)ꢀ4ꢀ(quinolinꢀ8ꢀyloxy)ꢀ
butanꢀ1ꢀone (Vj)
.
Yield 65%, dark yellow oil. 1H NMR:
1ꢀ[4ꢀ(4ꢀNitroꢀphenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ2ꢀ(2ꢀmethylꢀ
quinolinꢀ8ꢀyloxy)ꢀethanon (Ve) Yield 72%, yellow
solid, mp 118–120
C. 1H NMR: 3.3 (s, 3H, quinoline
.
2.2 (m, 2H, CH2), 2.7 (t, 2H, COCH2), 3.7 (m, 4H,
piperazine), 3.6 (s, 3H, CH3), 4.0 (m, 4H, piperazine),
4.5 (t, 2H, OCH2), 6.9–8.8 (m, 6H, quinoline ArꢀH).
13C NMR: 24.13, 29.60, 40.75, 44.40, 46.48, 48.50,
49.26, 67.43, 76.27, 77.01, 105.76, 109.45, 113.12,
°
CH3), 3.5 (m, 4H, piperazine), 3.9 (m, 4H, piperaꢀ
zine), 4.8 (s, 2H, OCH2CO), 7.0–8.5 (m, 5H, quinoꢀ
line ArꢀH), 6.7–7.6 (m, 4H, ArꢀH). 13C NMR: 25.75,
40.25, 44.11, 46.75, 49.73, 67.89, 76.80, 77.38, 77.46,
109.45, 112.70, 119.45, 121.28, 125.74, 126.57,
129.29, 136.04, 137.77, 144.61, 149.40, 154.78,
139.24, 140.35, 149.46, 154.24, 171.63. LCꢀMS:
+ 1). Anal. Cal. for C18H23N3O2: C, 68.98; H, 7.40;
N, 13.41. Found: C, 69.18; H, 7.28; N, 13.25%.
1ꢀ(4ꢀMethylꢀpiperazinꢀ1ꢀyl)ꢀ2ꢀ(2ꢀmethylꢀquinolinꢀ
8ꢀyloxy)ꢀethanone (Vk) Yield 65%, brown solid, mp
123–125
C. 1H NMR: 3.2 (s, 3H, quinoline CH3), 3.5
m/z 314
(M
171.76. LCꢀMS:
m/z 407 (M + 1). Anal. calcd. for
C22H22N4O4: C, 65.01; H, 5.46; N, 13.78. Found: C,
65.16; H, 5.30; N, 13.62%.
.
°
1ꢀ[4ꢀ(4ꢀNitroꢀphenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ2ꢀ(pyridinꢀ
(s, 3H, CH3), 3.7 (m, 4H, piperazine), 4.0 (m, 4H,
piperazine), 4.7 (s, 2H, OCH2CO), 6.7–8.3 (m, 5H,
quinoline ArꢀH). 13C NMR: 25.25, 40.03, 44.34,
46.62, 48.09, 49.74, 67.89, 76.75, 77.71, 100.26,
109.63, 112.17, 139.57, 140.80, 149.65, 154.87,
2ꢀyloxy)ꢀethanone (Vf)
.
Yield 75%, brown solid, mp
1
184–186
°C. H NMR: 3.3 (m, 4H, piperazine), 3.9
(m, 4H, piperazine), 4.7 (t, 2H, OCH2), 6.2–7.2 (m,
4H, pyridine ArꢀH), 6.9–7.4 (m, 4H, ArꢀH). 13C
NMR: 40.09, 44.59, 46.97, 49.68, 67.25, 76.50, 77.87,
77.30, 109.09, 112.79, 119.06, 124.33, 136.65, 139.34,
146.07, 151.65, 171.16. LCꢀMS:
Anal. calcd. for C17H18N4O4: C, 59.64; H, 5.70; N,
171.09. LCꢀMS:
m/z 300 (M + 1). Anal. calcd. for
C17H21N3O2: C, 68.20; H, 7.07; N, 14.04. Found: C,
68.01; H, 7.32; N, 13.91%.
m/z 343 (M + 1).
1ꢀ(4ꢀMethylꢀpiperazinꢀ1ꢀyl)ꢀ2ꢀ(pyridinꢀ2ꢀyloxy)ꢀ
16.37. Found: C, 59.61; H, 5.99; N, 16.13%.
1
ethanone (Vl)
.
Yield 60%, dark yellow oil. H NMR:
1ꢀ[4ꢀ(2ꢀMethoxyꢀphenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ4ꢀ(quinoꢀ
3.4 (s, 3H, CH3), 3.9 (m, 4H, piperazine), 3.9 (m, 4H,
piperazine), 4.9 (t, 2H, OCH2), 6.2–7.4 (m, 4H, pyriꢀ
dine ArꢀH). 13C NMR: 40.59, 44.08, 46.94, 49.95,
67.37, 76.33, 77.98, 110.17, 124.92, 146.85, 151.74,
linꢀ8ꢀyloxy)ꢀbutanꢀ1ꢀone (Vg). Yield 72%, pale yellow
oil. 1H NMR: 2.4 (m, 2H, CH2), 2.8 (t, 2H, COCH2),
3.4 (m, 4H, piperazine), 3.9 (m, 4H, piperazine),
4.2 (s, 3H, OCH3), 4.6 (t, 2H, OCH2), 7.0–8.9 (m, 6H,
quinoline ArꢀH), 6.6–7.2 (m, 4H, ArꢀH). 13C NMR:
24.36, 29.19, 40.36, 44.29, 46.56, 49.17, 56.5, 67.35,
76.43, 77.22, 77.21, 109.37, 112.78, 119.90, 121.69,
125.73, 126.36, 129.62, 136.37, 139.61, 140.35,
171.38. LCꢀMS:
m/z 236 (M + 1). Anal. calcd. for
C12H17N3O2: C, 61.26; H, 7.28; N, 17.86. Found: C,
61.45; H, 7.04; N, 17.91%.
Antioxidant Assays
149.28, 154.39, 171.72. LCꢀMS:
m/z 406 (M + 1).
Anal. calcd. for C24H27N3O3: C, 71.09; H, 6.71; N,
10.36. Found: C, 70.97; H, 6.47; N, 10.25%.
DPPH radical scavenging assay
cal scavenging activity of the test compounds (Va –l)
. The DPPH radiꢀ
was evaluated according to the method of [20]. Iniꢀ
tially, 1 mL of the samples at concentrations of 2, 4, 6,
8, and 10 μg/mL were mixed with equal quantity of 0.1
1ꢀ[4ꢀ(2ꢀMethoxyꢀphenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ2ꢀ(2ꢀ
methylꢀquinolinꢀ8ꢀyloxy)ꢀethanone (Vh)
.
Yield 58%,
1
yellow solid, mp 106–108°C. H NMR: 3.3 (s, 3H,
mM DPPH in absolute ethanol. The reaction mixture
was shaken and allowed to stand for 20 min at room
temperature. Absorbance of the resulting solution was
measured at 517 nm with the UVꢀVIS spectrophotomꢀ
eter. Butylated hydroxyl toluene (BHT) was used as a
positive control in all the assays.
quinoline CH3), 3.5 (m, 4H, piperazine), 3.9 (m, 4H,
piperazine), 4.1 (s, 3H, OCH3), 4.8 (s, 2H, OCH2CO),
6
.8–8.4 (m, 5H, quinoline ArꢀH), 6.6–7.2 (m, 4H,
ArꢀH). 13C NMR: 25.82, 40.27, 44.29, 46.92, 49.76,
56.5, 67.24, 76.13, 77.43, 75.98, 109.33, 112.94,
119.74, 121.61, 125.21, 126.61, 129.13, 135.12,
139.79, 144.41, 149.19, 154.80, 169.22. LCꢀMS:
392 ( + 1). Anal. calcd. for C23H25N3O3: C, 70.57; H,
6.44; N, 10.73. Found: C, 70.69; H, 6.23; N, 10.92%.
m/z
Ferrous ion chelating assay. Ferrous ion chelating
ability was measured according to Gordon’s method
[21]. Three sets of test tube were taken. One tube was
M
1ꢀ[4ꢀ(2ꢀMethoxyꢀphenyl)ꢀpiperazinꢀ1ꢀyl]ꢀ2ꢀ(pyridinꢀ taken as control: FeCl3 (200 mM) and K3Fe(CN)6
2ꢀyloxy)ꢀethanone (Vi) Yield 66%, yellow solid, mp (400 mM) were added to the tube and the volume was
C. H NMR: 3.3 (m, 4H, piperazine), 4.0 made up to 1mLby adding distilled water. For the secꢀ
.
1
155–157
°
(m, 4H, piperazine), 4.3 (s, 3H, OCH3), 4.9 (t, 2H, ond tube, EDTA (40 mM), FeCl3 (200 mM), and
OCH2), 6.2–7.0 (m, 4H, pyridine ArꢀH), 6.7–7.4 (m, K3Fe(CN)6 (400 mM) were added and the volume was
4H, ArꢀH). 13C NMR: 40.45, 44.15, 46.58, 49.31, made up to 1mL by adding distilled water. For the
56.5, 67.45, 76.22, 77.01, 77.78, 109.70, 112.18, third one, test compounds (Va –l) with concentrations
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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560
ALꢀGHORBANI et al.
Table 4. K+/H+ꢀATPase inhibition activity of compounds (Va
–l)
Concentration,
µ
g/mL
10
Test samples
2
5
IC50, µg/mL
(
(
(
(
(
(
(
(
(
(
(
(
Va
Vb
)
)
32.61
112.36
29.36
36.24
160.34
33.24
37.24
26.34
22.14
152.34
28.69
39.24
24.65
44.62
108.24
53.4
56.21
147.36
54.36
59.24
47.32
53.5
135.58
51.36
53.27
51.32
68.14
102.36
67.14
75.39
125.36
68.94
79.24
66.34
69.14
115.36
64.28
73.24
78.25
8.5
Inactive
5.2
5.5
Inactive
6.3
Vc)
Vd
)
Ve
Vf
Vg
Vh
)
)
)
4.5
4.9
5.3
)
Vi
Vj
Vk
)
)
Inactive
5.7
)
Vl)
6.2
4.7
Omeprazole
2, 4, 6, 8, and 10
K3Fe(CN)6 (400 mM) were added and the volume was
made up to 1mL by adding distilled water. The tubes
were incubated for 10 min at 20°C, the absorbance at
700 nm was registered, and ion chelating ability was
calculated.
The antioxidant activity of all the compounds was
compared with that of BHT. Radical scavenging activꢀ
ity was expressed as percentage activity using the forꢀ
μg/mL, FeCl3 (200 mM), and Krebs Ringer buffer (pH 7.4) containing 250 mM
sucrose and homogenized with 20 strokes of a mortarꢀ
driven Teflon pestle homogenizer. The tissues were
discarded and the filtrate was subjected to sub cellular
fractionation. The pellets thus obtained were dissolved
in 2 mL of sucroseꢀEGTA buffer and was used as
enzyme sample [23].
Protein estimation. Protein was measured by
Lowry’s method [24] using bovine serum albumin as
mula [(Control absorbance
bance)/control absorbance)]
– sample absorꢀ
100.
standard (0–75 μg/mL). Eight clean and dry test tubes
×
were taken and aliquots of various concentrations of
the synthesized derivatives were made. To the 7th and
8th test tubes, unknown sample ( and 10 of the
5
µL
μL
Inhibition of Vipera russelli Venom PLA2
cells isolated from the sheep stomach) for which the
protein content had to be estimated was added. In
each test tube, the solution was made up to 1 mL by
adding distilled water, which was followed by the addiꢀ
tion of 5 mL of Lowry’s reagent. All the test tubes were
incubated at room temperature for 10 min. Then
0.5 mL of Folin–Ciocalteu reagent was added, folꢀ
lowed by incubation at room temperature for 30 min.
Absorbance of each solution was read at 670 nm
against the blank solution. A graph was plotted by takꢀ
ing the concentration of protein on Xꢀaxis and optical
density on Yꢀaxis. From the standard graph obtained,
the unknown concentration of protein in the enzyme
sample was calculated and found to contain 21 mg of
protein per 8 g of tissue homogenate.
PLA2 activity was measured by n indirect
hemolytic method of Boman [22]. Briefly, packed
human erythrocytes, egg yolk, and phosphate buffered
saline (1 : 1 : 8 v/v) were mixed. One milliliter of this
suspension was incubated separately with 60
russelli venom for 10 min at 37 . The reaction was
stopped by adding 10 mL of iceꢀcold phosphate buffer
saline and centrifuged at for 10 min at 800 g. The
µg of V.
°C
4°C
amount of hemoglobin released into the supernatant
was measured at 540 nm. The assay was also carried
out in the presence of various concentrations (10, 20,
30, 40, 50, and 60 μg/mL) of compounds (Va –l). Lysis
of erythrocytes by adding 9 ml of distilled water to the
control reaction mixture was taken as 100%.
Inorganic phosphorus estimation. Inorganic phosꢀ
phorus was estimated according to Fiske’s method
[23]. Aliquots of working standard solution
Gastric H+/K+ꢀATPase Activity
Isolation of parietal cells of stomach. The fundic
(40 µg/mL) were added into eight clean and dry test
stomach portion of sheep soon after sacrificing was tubes in the volume of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL;
collected and it was rinsed with Krebs Ringer buffer 2, 5, and 10 µL of the enzyme sample were taken in
(250 mM sucrose, 2 mM MgCl2, 1 mM EGTA, and test tubes 7 and 8, respectively. The volume in the test
2 mM HepesꢀTris, pH 7.4). The upper layer was pined tube was made up to 8.6 mL using 10% TCA. Ammoꢀ
with the help of needles on the dissection Table 4. nium molybdate (1mL) and ANSA reagent (0.4 mL)
Mucosal scrapings were suspended in ten volumes of were added to each test tube. The tubes were allowed
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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2015

SYNTHESIS AND BIOLOGICAL EFFICACY OF NOVEL PIPERAZINE ANALOGUES
561
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vol. 87, pp. 245–313.
developed was read at its λmax of 660 nm.
3. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T.,
H+/K+ꢀATPase activity. ATPase activity was
determined as described in [25]. Basal Mg2+ꢀdepenꢀ
dent ATPase activity was measured in 1.0 mL of the
reaction medium consisting of 2 mM ATP and 50 mM
TrisꢀHCl buffer (pH 7.5), K+ꢀstimulated and
HCO–3 stimulated. ATPase activity was defined as the
activity in the basal medium. The ATPase reaction was
started by the addition of the substrate (ATP), carried
out at 37°C for 15 min, and stopped with 1.0 mL iceꢀ
cold 20% TCA; inorganic phosphate liberated from
ATP was estimated by Fiske–Subbarow method.
All experiments were repeated in triplicates in three
independent sets. Percent inhibition was calculated by
the comparison of compound treated to various conꢀ
trol incubations. The concentration of test compound
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CONCLUSION
A new series of piperazine analogues bearing quinꢀ
oline and pyridine moiety were synthesized and evaluꢀ
ated for antioxidant activity and inhibition of PLA2
and H+/K+ꢀATPase activities. The result of the
present investigations indicates the importance of
these new compounds as potential antioxidants and
PLA2 and H+/K+ꢀATPase inhibitors. The compounds
presented clearly differ in their activities depending on
the type of substitution. Phenyl and nitrophenyl in the
piperazine ring of compounds (Vb), (Vc), and (Vf
)
showed high radical scavenging activities against the
tested DPPH and ferrous ions; also, compounds (Va ),
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(Vb), and (Vd) with same groups showed promising
inhibition against venom PLA2. Compounds (Vg ),
(
Vh), and (Vi) with methoxy group on phenyl piperaꢀ
zine indicated selectivity for the H+/K+ꢀATPase. It is
remarkable that pyridine ring with phenyl and nitroꢀ
phenyl group of (Vc) and (Vf) showed potent inhibition
against all the assays. On the basis of above observaꢀ
tions, we will do further modification to improve the
antioxidant and PLA2 activities of piperazines.
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ACKNOWLEDGMENTS
Talesara, G.L., Arkivoc, 2006, vol. XI, pp. 66–75.
20. Scherer, R. and Godoy, H.T., Food Chem., 2009,
M. AlꢀGhorbani is thankful to the University of
Thamar, Yemen, for providing financial assistance
under the teacher’s fellowship. Dr. S.A. Khanum
gratefully acknowledges the financial support provided
by the Vision Group on Science and Technology, Govꢀ
ernment of Karnataka, under the scheme CISEE,
Department of Information Technology, Biotechnology
and Science & Technology, Bangalore, India.
vol. 112, pp. 654–658.
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antioxidant action in vitro, in Food Antioxidants, Lonꢀ
don: Elsevier Applied Science, 1990.
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