Studies on 16,17-Pyrazoline Substituted Heterosteroids as Anti-Alzheimer and Anti-Parkinsonian Agents Using LPS Induced Neuroinflammation Models of Mice and Rats

Article abstract of DOI:10.1021/acschemneuro.7b00303

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common forms of neurodegenerative disorders. Dehydroepiandrosterone (DHEA) has been reported as a neuroprotective steroid useful in the therapeutic management of neurodegenerative disorders such as AD and PD. Herein we report the synthesis and evaluation of a new series of 16,17-pyrazolinyl DHEA analogues 2-4a-d as neuroprotective agents using LPS-induced neuroinflammation animal models. Treatment with the pyrazoline substituted steroids considerably improved the LPS-induced learning, memory and movement deficits in animal models. Suppression of biochemical parameters of oxidative and nitrosative stress, acetylcholinesterase activity, and TNF-α levels was also observed. 16,17-Pyrazolinyl steroids 2c-4c substituted with a 4-pyridyl moiety at the 5-position of the heterocyclic ring were found to be the most potent agents and produced neuroprotective effects better than standard drugs celecoxib and dexamethasone. Of these pyrazoline substituted steroids, the N-acetyl analogue 3c displayed neuroprotective effects better than N-phenyl (4c), which in turn showed potency more than N-unsubstituted analogue 2c.

Full text of DOI:10.1021/acschemneuro.7b00303

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Article
Studies on 16,17-pyrazoline substituted heterosteroids
as anti-Alzheimer and anti-Parkinsonian agents using
LPS induced neuroinflammation models of mice and rats
Ranjit Singh, Sridhar Thota, and Ranju Bansal
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00303 • Publication Date (Web): 11 Oct 2017
Downloaded from http://pubs.acs.org on October 12, 2017
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Studies on 16,17ꢀpyrazoline substituted heterosteroids as antiꢀ
Alzheimer and antiꢀParkinsonian agents using LPS induced
neuroinflammation models of mice and rats
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*
Ranjit Singh, Sridhar Thota and Ranju Bansal
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh
*
Corresponding author:
Prof Ranju Bansal
University Institute of Pharmaceutical Sciences
Panjab University, Chandigarhꢀ160 014, India
Email: ranju29in@yahoo.co.in
Tel.: 0172ꢀ2541142; Fax: 0172ꢀ2543101
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ABSTRACT: Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common
forms of neurodegenerative disorders. Dehydroepiandrosterone (DHEA) has been reported as
a neuroprotective steroid useful in the therapeutic management of neurodegenerative
disorders such as AD and PD. Herein we report the synthesis and evaluation of a new series
of 16,17ꢀpyrazolinyl DHEA analogues 2ꢀ4aꢀd as neuroprotective agents using LPSꢀinduced
neuroinflammation animal models. Treatment with the pyrazoline substituted steroids
considerably improved the LPSꢀinduced learning, memory and movement deficits in animal
models. Suppression of biochemical parameters of oxidative and nitrosative stress,
acetylcholinesterase activity and TNFꢀα levels was also observed. 16,17ꢀPyrazolinyl steroids
2cꢀ4c substituted with a 4ꢀpyridyl moiety at 5ꢀposition of heterocyclic ring were found to be
the most potent agents and produced neuroprotective effects better than standard drugs
celecoxib and dexamethasone. Of these pyrazoline substituted steroids, the Nꢀacetyl analogue
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3c displayed neuroprotective effects better than Nꢀphenyl (4c), which in turn showed potency
more than Nꢀunsubstituted analogue 2c.
KEY WORDS: Alzheimer’s disease, Parkinson’s disease, LPS, 16,17ꢀpyrazolinyl steroids,
neuroinflammation.
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INTRODUCTION
Neurodegeneration is the main cause of the progressive damage of structural or functional
activities of nervous cells resulting in the pathological conditions such as Alzheimer’s disease
1
(AD) and Parkinson’s disease (PD). The oxidative stress, mitochondrial dysfunction,
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excitotoxicity and chronic neuroinflammation are the main mechanisms associated with the
neurodegenerative cascade within the brain. In response to infections, trauma, stroke, toxins
and other stimuli, these mechanisms get activated leading to alteration of neuronal integrity
2
and subsequent neuronal loss. This results in progressive neurodegeneration which is
manifested in the form of various neuropathological disorders including AD and PD.
Lipopolysaccharide (LPS), a potent bacterial endotoxin derived from cell wall of
Gramꢀnegative bacteria elicits multiple pathological effects in human beings. Recent
investigations suggest that chronic infusion of LPS causes neurodegeneration and
3,4
subsequent impaired spatial memory and locomotor functions.
During the last decades, steroids substituted with heterocycles at Aꢀring or Dꢀring of
the skeleton have garnered a great deal of attention as antiꢀinflammatory, anticancer and
5
ꢀ8
antimicrobial agents. Recently much interest has been generated towards the chemical
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ꢀ12
exploration of steroidal skeleton to develop novel neuroprotective agents. Fluasterone (I)
,
O
O
O
H
F
CH
N
CH
N
H
H
OH
O
N
H
(
I)
(II)
(III)
Figure 1. Novel analogues of dehydroepiandrosterone as neuroprotective agents
a novel analog of dehydroepiandrosterone, is considered as a drug candidate that might be
11
useful in the treatment of traumatic brain injury (Figure 1). Recently, neuroprotective
effects of 16 pyridylideneꢀ5ꢀandrostenedione derivatives (II) and their 4ꢀaza analogues (III)
Figure 1) in LPSꢀtreated animal models have been reported from our laboratory.
ꢀ
12
(
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Pyrazoline,
a
five membered nitrogen heterocycle possesses numerous
pharmacological properties such as antimicrobial, antiꢀinflammatory, analgesic,
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antidepressant and anticancer actions. Several pyrazoline analogues have also been reported
as useful candidates for therapeutic management of CNSꢀrelated diseases such as AD and PD
as they possess good radical scavenging capacity and also exhibit potent antiꢀoxidant
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4ꢀ16
activities.
Literature further reveals the neuroprotective actions of pyridyl moiety against
17
the neurological disorders effecting behaviour and cognition. The pyridyl moiety also acts
as a novel analogue of choline which help information and transmission of acetylcholine
leading to enhanced cognition and memory in patients suffering from neuropathological
18
12
disorders. In light of these observations and in continuation with our earlier research work
aimed towards the discovery of potent neuroprotective steroids, synthesis and neuroprotective
studies on a new series of 16,17ꢀpyrazolinyl substituted steroidal analogues derived through a
1
6ꢀpyridylidene moiety have been presented in the current study.
RESULTS AND DISCUSSION
Chemical Synthesis. The synthesis of 16,17ꢀpyrazolinyl steroids has been carried out as
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shown in scheme 1. The 16ꢀarylidene adducts 1aꢀd were prepared as reported earlier.
Further treatment of these aldol products with hydrazine hydrate in refluxing 1,4 dioxane for
h yielded pyrazoline fused steroids 2aꢀd, while reaction with hydrazine hydrate in
refluxing glacial acetic acid for 9 h afforded acetylated steroidal analogues 3aꢀd. Reaction of
6ꢀarylidene steroids 1aꢀd with phenylhydrazine in refluxing anhydrous methanol for 12 h
5
1
afforded corresponding phenylpyrazolinyl substituted derivatives 4aꢀd. The obtained
steroidal pyrazoline derivatives were found to be quite unstable and decompose at ambient
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temperature conditions probably due to the instability associated with pyrazoline nucleus.
While carrying out these reactions, the reflux time should be regulated as the overheating
eventually leads to degradation of the final products. The reaction solvents also play an
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important role in the synthesis of 16,17ꢀpyrazolinyl steroids and should be carefully selected.
Anhydrous 1,4 dioxane and anhydrous methanol remain the most suitable solvents for the
synthesis of pyrazolinyl steroids. The compounds were purified by flash chromatography
(Isolera One, Biotage) as they decompose while crystallization in most of the solvents.
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O
HO
DHEA
O
H-C Ar
CH OH/NaOH
3
O
N
CH3
NH
O
C
N
N
H
H
Ar
CH
Ar
H
NH NH .H O
Ar
NH NH .H O
2
2
2
2
2
2
H
O
C
CH COOH
HO
3
1,4 dioxane
HO
reflux, 9hr
O
2a-d
H C
3
reflux, 5hr
1
a-d
3a-d
NHNH .H O
2
2
reflux, 12hr
anhyd.CH OH
3
N
N
H
Ar
H
HO
4a-d
a
B
c
D
Ar
Scheme1: Synthetic route to the formation ofvarious16,17ꢀpyrazolinyl steroidal derivatives
The structures of all the synthesized compounds were confirmed by various spectral
analyses. 16,17ꢀPyrazoline substituted NꢀH naked steroids 2bꢀd displayed a doublet due to 5'
ꢀ
1
pyrazolinyl proton at ~
δ
4ppm in the H NMR spectra while for compound 2a it resonated as
a multiplet.
N
ꢀacetylated pyrazolinyl steroids 3aꢀd displayed carbonyl stretching vibrations at
ꢀ1
~
1730 and 1670 cm due to the presence of ester and amide functionalities. Presence of two
additional sharp singlets due to the resonating protons of
3 3
NꢀCOCH and OꢀCOCH in the
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proton NMR spectra of steroids 3aꢀd confirmed the acetylation at both the places.
Phenylpyrazoline substituted steroids 4aꢀd exhibited a marginally downfield doublet of 5'
ꢀ
pyrazolinyl proton at ~ 4.6 and a prominent multiplet due to aromatic protons at 6.72ꢀ7.29
δ
1
ppm. The 16
R
and 5’S configurations were assigned on the basis of HNMR spectral data as
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reported earlier. Previous studies indicate that during the condensation of hydrazine hydrate
with the 16ꢀarylidene system after the removal of water molecule and formation of the 17ꢀ
hydrazone, the NH
2
attacks the double bond from αꢀface leading to the formation of 16R. In
׳
addition, the phenyl ring rotates backward forming 5ꢀ
S
center. The deshielded Cꢀ18 methyl
.
protons resonated downfield at ~
δ
1 ppm for all the pyrazoline substituted steroids 2ꢀ4aꢀd
Biological Activity. LPS induced Aβ 1–42 generation in the cortex and hippocampus plays a
19
central role in the pathogenesis of Alzheimer's disease characterized by cognitive deficits.
Literature also reveals that subꢀtoxic dose levels of LPS in substantia nigra (SN) region
results in increased production of cytokines within the brain resulting in destruction of
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dopaminergic markers, which leads to impairment of locomotor activity.
The neuroprotective effects of the DHEA derived 16,17ꢀpyrazolinyl steroids 2ꢀ4aꢀd
have been investigated in LPS induced neuroinflammation models of mice and rats.
Behavioural paradigms for neurodegeneration that include learning, memory and locomotion
were assessed using Morris water maze, elevated plus maze, photoactometer and block test.
Celecoxib and dexamethasone were used as standard compounds.
AntiꢀAlzheimer’s Activity. The neuroprotective effects of 16,17ꢀpyrazolinyl steroids 2ꢀ4aꢀd
(5 mg/kg, i.p.) and standard drugs celecoxib (20 mg/kg, i.p.) and dexamethasone (5 mg/kg,
i.p.) on the mice suffering from LPS (i.p.) induced memory impairment and learning
1
2
disability were studied using Morris water maze and elevated plus maze models(Table 1).
Treatment with pyrazoline substituted steroidal derivatives 2ꢀ4aꢀd (5mg/kg) considerably
reduced the otherwise increased escape latency and % ITL(initial transfer latency) after 24 h
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Table 1. AntiꢀAlzheimer effects of 16,17ꢀpyrazolinyl steroids on mice treated with LPS
intraperitoneally
Morris Water Maze
Elevated Plus Maze
% ITL (s)
Compound
Escape Latency (s)
TSTQ (s)
Control
LPS
Celecoxib
Dexamethasone
41.5 ± 1.9
55 ± 3.4
11.4 ± 2.6
*
*
*
73.2 ± 1.6
52.9 ± 2.4
56.7 ± 2.5
61.6 ± 1.8
64.0 ± 1.7
56.0 ± 2.9
58.2 ± 1.3
58.0 ± 1.5
61.0 ± 1.3
52.9 ± 1.5
54.4 ± 1.5
61.5 ± 2.4
63.4 ± 1.2
54.8 ± 2.1
13 ± 2.9
91.4 ± 2.9
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
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44 ± 3
24.4 ± 2.2
31.1 ± 2.4
#
#
40 ± 3
#
#
2
a
37 ± 3
35.5 ± 3.3
37.7 ± 2.2
#
#
2
b
35 ± 2
#
#
2c
41 ± 2
28.8 ± 2.2
#
#
2
3
d
a
40 ± 3.2
30 ± 2.3
#
#
39.5 ± 2.5
30 ± 3.3
#
#
3b
3c
37 ± 4
33.3 ± 3.3
26.1 ± 3.8
27.2 ± 3.8
#
#
43 ± 3
#
#
3d
4a
4b
42 ± 3
#
#
38 ± 2.5
32.2 ± 1.9
#
#
36 ± 3
35 ± 3.8
#
#
4c
42 ± 3
27.7 ± 3.3
#
#
4
d
56.1 ± 2.0
41 ± 4
28.8 ± 3.3
#
*
p<0.05 as compared to control; p<0.05as compared to LPS (n=5). Oneꢀ way ANOVA
followed by Tukey’s test was employed to find out the interꢀgroup variation and values were
considered statistically significant (p<0.001).
100
*
Control
LPS
8
0
*
Celecoxib
# #
#
60
#
#
Dexamethasone
#
#
# #
#
2c
3c
4c
40
#
#
# #
#
20
*
0
Escape Latency (s)
TSTQ (s)
%ITL (s)
Figure 2. AntiꢀAlzheimer’s activity of steroids 2cꢀ4c
.
*
p<0.05 as compared to control;
#
p<0.05 as compared to LPS (n=5). Oneꢀway ANOVA followed by Tukey’s test was
employed to find out the interꢀgroup variation and a value of p<0.001 was considered
statistically significant.
of training period in LPS injected mice. In probe trial, significantly decreased TSTQ (time
spent in target quadrant) in only LPS treated mice was appreciably reversed on treatment with
1
6,17ꢀpyrazolinyl steroids 2ꢀ4aꢀd. The 5'ꢀ(4ꢀpyridyl) derived pyrazolinyl steroids 2cꢀ4c
emerged as the most potent neuroprotective agents and produced effects comparable to
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celecoxib at one fourth of dose and better than dexamethasone at the same dose level as
shown in Figure 2. Out of the 4ꢀpyridyl steroids 2cꢀ4c
,
N
ꢀacetyl pyrazolinyl steroid 3c
[
escape latency 52.9 ± 1.5 (s), TSTQ 43 ± 3 (s) and % ITL 26.1 ± 3.8 (s)] was found to be
better than Nꢀphenyl 4c [escape latency 54.8 ± 2.1 (s), TSTQ 42 ± 3 (s) and % ITL 27.7 ±
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
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3.3 (s)], which in turn produced effects superior to NꢀH naked pyrazolinyl steroid 2c [escape
latency 56.0 ± 2.9 (s), TSTQ 41 ± 2 (s) and % ITL 28.8 ± 2.2 (s)] in terms of restoration of
learning and memory functions.
AntiꢀParkinsonian Activity. The antiꢀParkinsonian activity of 16,17ꢀpyrazolinyl steroids 2ꢀ
4aꢀd (2 mg/kg, i.p.) and standard drugs celecoxib (20 mg/kg, i.p.) and dexamethasone (2
mg/kg, i.p.) was studied using actophotometer and block test against the intranigrally LPSꢀ
Table 2. AntiꢀParkinsonian effects of 16,17ꢀpyrazolinyl steroids on rats treated with LPS
intranigrally
Locomotor Activity
Catatonic Response
Compound
Ambulation (min) Rearing (min)
Degree of Catatonia (score)
Sham
LPS + Vehicle
LPS
Celecoxib
Dexamethasone
300 ± 2.5
145 ± 2.0
0
*
*
*
56.3 ± 3.8
55.8 ± 4.6
16.3 ± 3.3
16.0 ± 2.3
73.3 ± 2.0
1.2 ± 0.02
1.22 ± 0.03
0.21 ± 0.04
0.33 ± 0.05
0.40 ± 0.07
0.45 ± 0.09
0.33 ± 0.07
0.38 ± 0.09
0.33 ± 0.14
0.40 ± 0.09
0.23 ± 0.11
0.26 ± 0.11
0.38 ± 0.14
0.45 ± 0.14
0.26 ± 0.11
0.30 ± 0.14
#
#
$
#
$
$
215 ± 3.8
200 ± 3.9
196 ± 3.2
$
$
$
63± 3.0
59.3 ± 2.4
54.3 ± 3.1
68.6 ± 2.7
61.3 ± 3.4
63.3 ± 2.9
58 ± 2.04
72.3 ± 2.8
69 ± 2.3
61.6± 3.5
56.3± 3.7
$
$
$
$
$
2
a
$
$
$
$
2
b
191.6 ± 3.7
205.3 ± 4.9
200.6 ± 2.9
$
2c
2d
$
$
$
$
$
$
3
a
207 ± 4.3
$
$
$
$
3
b
201.3 ± 4.5
216.3 ± 4.6
$
3c
$
$
$
3d
4a
4b
213 ± 4.1
$
$
$
$
206.6 ± 2.8
198.6 ± 4.7
$
$
$
$
$
$
4c
211 ± 3.0
70 ± 1.5
68 ± 3.2
$
$
4d
209.6 ± 3.5
#
$
*
p<0.05 as compared to sham; p<0.05as compared to LPS + vehicle; p<0.05as compared
to LPS (n=5). Oneꢀway ANOVA followed by Tukey’s test was employed to find out the
interꢀgroup variation and values were considered statistically significant (p<0.001).
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00
00
1.5
Sham
#
*
LPS + Vehicle
LPS
3
1.0
$
$
$
$
Celecoxib
Dexamethasone
$
200
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13
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c
0
.5
$
$
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$
$
$
3c
1
00
$
$
$
#
*
4c
* #
#
0.0
0
Ambulation (s)
Rearing (s)
Degree of Catatonia (score)
#
Figure 3. AntiꢀParkinsonian activity of compounds2cꢀ4c. p<0.05 as compared to LPS +
vehicle; p<0.05as compared to LPS (n=5). Oneꢀway ANOVA followed by Tukey’s test was
employed to find out the interꢀgroup variation and values were considered statistically
significant (p<0.001)
$
2
1,22
treated rats with respect to locomotion.
The results obtained are compiled in Table 2. The
groups treated with daily i.p. injections of 16,17ꢀpyrazolinyl steroids 2ꢀ4aꢀd (2 mg/kg) for 21
days showed improvement in the locomotor activity with significant decrease in degree of
catatonia as compared to LPS and LPS + vehicle group. Again, the 5´ꢀ(4ꢀpyridyl) substituted
steroidal pyrazolines 2cꢀ4c (2mg/kg) significantly improved the locomotion abilities and
catatonia. The pyrazoline substituted 4ꢀpyridyl steroids displayed activity in the order
Nꢀ
acetyl (3c >Nꢀphenyl (4c) > –unsubstituted (2c). The neuroprotective effects are
)
N
comparable to celecoxib (20 mg/kg) and much better than dexamethasone (2 mg/kg) as
shown in Figure 3.
Biochemical estimations in brain supernatants of rodents. The levels of biochemical
parameters such as acetylcholinesterase, lipid peroxidation, nitrite and oxidative stress were
evaluated in the brain supernatants of mice (i.p. LPS) and rats (i.g. LPS) after treatment
with 16,17ꢀpyrazolinyl steroids 2ꢀ4aꢀdandstandard drugs celecoxib and dexamethasone as
1
2
reported earlier. The data obtained has been compiled in Tables 3 and 4, respectively.
The treatment with steroidal pyrazolines 2ꢀ4aꢀd helped to restore the normalcy of the
biochemical mediators along with marked inhibition of reactive oxygen and nitrogen species
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involved in mechanism of neuroinflammation. Of all, the 5´ꢀ(4ꢀpyridyl) substituted steroidal
pyrazolines 2cꢀ4c displayed the best results once again and in concordance with the previous
results. The steroid 3c restored the acetylcholinesterase, lipid peroxidation, nitrite and
oxidative stress levels in the LPSꢀtreated animals in a better manner in comparison to NꢀH
naked pyrazoline 2c and Nꢀphenyl analogue 4c (Figures 4 and 5).
0
1
2
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0
1
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3
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0
Inhibition of TNFꢀα. The estimation of TNFꢀα activity has been carried out in the brain
12,23
serum of rats treated with 16,17ꢀpyrazolinyl steroids using TNFꢀα immunoassay kit.
treatment with Dꢀring fused pyrazolinyl steroids
magnitude of TNFꢀα levels in the brains of LPS injected rats. Again the 5’ꢀ(4ꢀpyridyl)
pyrazolinyl steroids 2cꢀ4c were found to be better than standard drugs in the order ꢀacetyl
3c, 69.6 ± 2.5) > ꢀphenyl (4c, 75.8 ± 2.7) > ꢀunsubstituted analogue (2c, 85.2 ± 2.9) in
reducing TNFꢀα levels (Figure 6).
Docking Studies. The literature reports provide enough evidence that TNFꢀα is one of the
The
2ꢀ4aꢀd resulted in significantly reduced
N
(
N
N
19,24,25
key elements of the molecular mechanism of neuroinflammation.
Observing the
reduction in LPSꢀenhanced TNFꢀα levels on treatment with the 16,17ꢀpyrazolinyl steroids, it
was thought to study their threeꢀdimensional interactions with the receptor using
computational docking studies. Therefore, newly synthesized D ring fused pyrazolinyl
steroids were docked against the crystal structure of TNFꢀα to gain insights into their
mechanism of action as neuroprotective agents. The glide scores of the coꢀcrystallized ligand,
standard drugs (celecoxib and dexamethasone) and pyrazoline substituted steroidal
derivatives are given in Table 6. The 3D and 2D interactions of most active compounds 2cꢀ4c
were compared with coꢀcrystallized ligand. The 2D and 3D interaction diagrams of most
potent pyrazolinyl steroidal derivative 3c at active site of TNFꢀα are represented in Figure 7.
The coꢀcrystallized ligand consisting of trifluoromethylphenylindole and dimethyl chromone
moieties folded back upon each other and the interactions were largely
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Table 3. Estimation of biochemical parameters in brain supernatants of LPS injected (i.p.) mice after treatment with steroids 2ꢀ4aꢀd
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7
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
AchE Assay
µ moles of AchE/
min/mg protein
LPO Assay
n moles of MDA/
mg protein
Nitrite Assay
Nitrite conc.
(µg/ml)
GSH Assay
µ moles of GSH/
mg protein
SOD Assay
SOD units/
mg protein
Catalase Assay
µ moles of H
decomposed/
Compound
2 2
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
min/mg protein
Control
LPS
Celecoxib
Dexamethasone
0.0015 ± 0.0003*
0.0051 ± 0.0003
1.9 ± 0.1
287.5 ± 35
0.0078 ± 0.0003
6.5 ± 1.8
4.9 ± 0.3
*
*
*
*
*
7.5 ± 0.3
730.1 ± 28
0.0034 ± 0.0005
1.5 ± 0.7
1.1 ± 0.4
#
#
#
#
#
#
0.0026 ± 0.0003#
3.0 ± 0.1
434.4 ± 31.1
0.0061 ± 0.0005
4.9 ± 1.6
3.6 ± 0.3
#
#
#
#
#
0.0030 ± 0.0004#
3.5 ± 0.1
#
467.1 ± 32.5
#
0.0056 ± 0.0003
#
4.3 ± 1.3
#
3.2 ± 0.6
#
2
2
2
2
a
b
c
0.0034 ± 0.0003#
3.9 ± 0.2
507.8 ± 18.3
0.0050 ± 0.0003
3.9 ± 2.0
3.0 ± 0.6
#
#
#
#
#
0.0037 ± 0.0004#
4.1 ± 0.1
525.1 ± 17.2
0.0048 ± 0.0003
3.6 ± 2.0
2.8 ± 0.8
#
#
#
#
#
0.0029 ± 0.0006#
0.0030 ± 0.0004#
0.0030 ± 0.0006#
0.0035 ± 0.0006#
3.4 ± 0.2
469.1 ± 18.3
0.0054 ± 0.0005
4.4 ± 2.0
3.3 ± 0.6
#
#
#
#
#
d
3.5 ± 0.2
489.8 ± 16.8
0.0052 ± 0.0004
4.1 ± 1.6
3.2 ± 0.6
#
#
#
#
#
3a
3b
3.4 ± 0.2
471.5 ± 25.0
0.0053 ± 0.0004
4.3 ± 1.6
3.1 ± 0.8
#
#
#
#
#
3.7 ± 0.3
496.8 ± 22.1
0.0050 ± 0.0005
4.0 ± 1.7
2.8 ± 0.8
#
#
#
#
#
3
3
4
4
4
4
c
0.0027 ± 0.0006
3.1 ± 0.1
447.5 ± 23.3
0.0058 ± 0.0004
4.9 ± 1.7
3.6 ± 0.7
#
#
#
#
#
#
d
a
b
c
0.0028 ± 0.0004#
3.3 ± 0.2
466.8 ± 21.9
0.0056 ± 0.0005
4.7 ± 1.5
3.4 ± 0.7
#
#
#
#
#
0.0031 ± 0.0002#
3.5 ± 0.1
#
483.5 ± 22
#
0.0054 ± 0.0003
#
4.1 ± 1.6
#
3.0 ± 0.6
#
0.0034 ± 0.0002
3.8 ± 0.3
502.8 ± 21.4
0.0050 ± 0.0006
3.7 ± 1.1
2.8 ± 0.8
#
#
#
#
#
#
0.0028 ± 0.0006#
3.2 ± 0.2
466.5 ± 19.9
0.0057 ± 0.0005
4.7 ± 1.1
3.5 ± 0.7
#
#
#
#
#
d
0.0029 ± 0.0006
3.4 ± 0.1
476.5 ± 17.7
0.0055 ± 0.0001
4.5 ± 1.8
3.3 ± 0.5
Results are expressed as µmoles of AchE/min/mg protein in AchE Assay, n moles of MDA/mg protein in LPO Assay, Nitrite conc.(µg/ml) in
Nitrite Assay, µmoles of GSH/mg protein in GSH Assay, SOD units/ mg protein in SOD Assay and µ moles of H O decomposed/min/mg
protein in Catalase Assay. The values are shown as mean ± S.E.M. *p<0.05 as compared to control; p<0.05as compared to LPS (n=5 per
group). Oneꢀway ANOVA followed by Tukey’s test was employed to find out the interꢀgroup variationandvalues are considered statistically
significant (p<0.001).
2
2
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Table 4. Estimation of biochemical parameters in brain supernatants of LPS injected (i.g.) rats after treatment with compounds 2ꢀ4aꢀd
AchE Assay
LPO Assay
Nitrite Assay
GSH Assay
SOD Assay
Catalase Assay
µ moles of H
decomposed/
Compound
µ moles of AchE/
min/mg protein
n moles of MDA/
mg protein
Nitrite conc.
(µg/ml)
µ moles of GSH/
mg protein
SOD units/
mg protein
2 2
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
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6
7
8
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2
3
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9
min/mg protein
Sham
0.0032 ± 0.0002*
3.2 ± 0.2
419.5 ± 26.1
0.048 ± 0.002
9.8 ± 1.4
7.0 ± 0.4
2.2 ± 0.4
2.1 ± 0.3
5.5 ± 0.5
5.1 ± 0.3
4.7 ± 0.8
4.4 ± 0.8
5.1 ± 0.8
5.0 ± 0.6
4.9 ± 0.8
4.7 ± 0.8
5.4 ± 0.5
5.3 ± 0.7
4.8 ± 0.4
4.6 ± 0.4
5.3 ± 0.9
*
*
*
*
*
LPS + Vehicle
0.0096 ± 0.0005
9.5 ± 0.4
914.1 ± 25.2
0.021 ± 0.001
2.7 ± 1.0
#
#
#
#
#
#
LPS
0.0097 ± 0.0003$
9.4 ± 0.6
915.8 ± 25.4
0.020 ± 0.001
2.8 ± 1.1
$
$
$
$
$
Celecoxib
Dexamethasone
0.0055 ± 0.0003$
0.0062 ± 0.0007$
0.0065 ± 0.0006$
0.0068 ± 0.0007
5.4 ± 0.4
523.1 ± 20.1
0.036 ± 0.003
6.6 ± 1.5
$
$
$
$
$
6.0 ± 0.3
565.8 ± 27.3
0.033 ± 0.001
6.2 ± 1.7
$
$
$
$
$
2a
2b
2c
6.8 ± 0.5
617.8 ± 23.6
0.030 ± 0.002
5.6 ± 1.5
$
$
$
$
$
7.4 ± 0.6
636.1 ± 21.1
0.028 ± 0.002
5.3 ± 1.3
$
$
$
$
$
$
0.0062 ± 0.0006$
6.2 ± 0.6
570.5 ± 22.9
0.034 ± 0.003
6.3 ± 1.4
$
$
$
$
$
2
3
3
3
3
4
d
a
b
c
d
a
0.0064 ± 0.0008
6.4 ± 0.6
594.5 ± 19.6
0.033 ± 0.004
6.0 ± 1.3
$
$
$
$
$
$
0.0062 ± 0.0007$
6.5 ± 0.6
$
576.8 ± 15.6
$
0.032 ± 0.003
$
6.0 ± 1.2
$
$
0.0065 ± 0.0004
6.8 ± 0.5
5.9 ± 0.4
591.5 ± 19.5
545.1 ± 17.2
0.030 ± 0.002
0.036 ± 0.002
5.8 ± 1.6
6.5 ± 1.5
$
$
$
$
$
$
0.0058 ± 0.0007$
$
$
$
$
$
0.0061 ± 0.0006
6.1 ± 0.4
561.1 ± 10.8
0.035 ± 0.003
6.3 ± 1.4
$
$
$
$
$
$
0.0064 ± 0.0009$
6.6 ± 0.6
592.5 ± 12.5
0.031 ± 0.002
5.8 ± 1.1
$
$
$
$
$
4b
4c
0.0068 ± 0.0005$
0.0057 ± 0.0007
7.1 ± 0.5
616.5 ± 20.7
0.029 ± 0.001
5.6 ± 1.0
$
$
$
$
$
6.1 ± 0.4
551.8 ± 11.5
0.035 ± 0.002
6.3 ± 1.7
$
$
$
$
$
$
4
d
0.0059 ± 0.0005
6.2 ± 0.5
569.5 ± 15.9
0.033 ± 0.001
6.1 ± 1.5
4.1 ± 0.5
Results are expressed as µmoles of AchE/min/mg protein in AchE Assay, n moles of MDA/mg protein in LPO Assay, Nitrite conc.(µg/ml) in
Nitrite Assay, µmoles of GSH/mg protein in GSH Assay, SOD units/ mg protein in SOD Assay and µ moles of H O decomposed/min/mg
2
2
#
protein in Catalase Assay. The values are shown as mean ± S.E.M. *p<0.05 as compared to sham; p<0.05as compared to LPS+ vehicle;
$
p<0.05as compared to LPS (n=5). Oneꢀway ANOVA followed by Tukey’s test was employed to find out the interꢀgroup variation and values
were considered statistically significant (p<0.001).
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(
B) LPO assay
(A) AChE assay
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0.006
*
8
6
4
2
0
*
0
.004
.002
#
#
#
#
#
#
#
#
#
#
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46
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0
0.000
(C) Nitrite assay (D) GSH assay
8
00
00
00
00
0
*
0
.010
0.008
.006
.004
.002
.000
6
#
#
#
#
#
#
#
#
#
#
0
4
2
0
*
0
0
(E) SOD assay
(F) Catalase assay
8
#
#
6
#
6
4
2
0
#
#
#
#
#
#
#
4
2
0
*
*
Figure 4. Biochemical estimations in brain supernatants of LPS injected (i.p.) mice treated
with compounds 2cꢀ4c. *p<0.05 as compared to control; p<0.05as compared to LPS (n=5).
Oneꢀway ANOVA followed by Tukey’s test was employed to find out the interꢀgroup
variations and values were considered statistically significant (p<0.001).
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6
(
A) AChE assay
(
B) LPO assay
0
.015
1
5
0
5
0
#
*
0.010
#
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*
$
$
$
$
$
$
$
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$
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0
0.005
0.000
(
D) GSH assay
(C) Nitrite assay
#
0.06
1000
00
00
00
00
*
8
$
$
$
0.04
$
$
$
$
$
$
$
6
*
#
4
0.02
0.00
2
0
(E) SOD assay
(F) Catalase assay
1
5
8
1
0
$
$
$
$
$
$
$
$
$
6
4
2
0
$
5
0
#
*
#
*
Figure 5. Biochemical estimation in brain supernatants of LPS injected (i.g.) rats treated
with compounds 2cꢀ4c. *p<0.05 as compared to sham; p<0.05as compared to LPS +
vehicle; p<0.05as compared to LPS (n=5). Oneꢀway ANOVA followed by Tukey’s test was
employed to find out the interꢀgroup variation and values were considered statistically
significant (p<0.001).
#
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Table 5. Estimation of TNFꢀɑ levels in brain serum of rats
Compound
TNFꢀɑ (pg/mg protein)
Sham
LPS + Vehicle
LPS
Celecoxib
Dexamethasone
2a
38.4 ± 1.1
358.8 ± 1.9
372.4 ± 1.6
*
#
$
$
$
68.2 ± 1.1
87.4 ± 1.6
98.6 ± 2.6
105.2 ± 2.2
85.2 ± 2.9
90.8 ± 3.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
$
2
b
$
2
c
$
2d
3a
3b
$
86.4 ± 3.0
91 ± 2.9
$
$
$
$
$
$
$
3
c
69.6 ± 2.5
74.4 ± 2.8
86.8 ± 2.8
92.6 ± 2.4
75.8 ± 2.7
79.8 ± 2.8
3
d
4a
4b
4
c
4
d
#
$
*
p<0.05 as compared to sham; p<0.05as compared to LPS + vehicle; p<0.05as compared
to LPS (n=5). Oneꢀway ANOVA followed by Tukey’s test was employed to find out the
interꢀgroup variation and values were considered statistically significant (p<0.001).
400
#
*
300
200
$
$
$
100
0
$
$
Figure 6. TNFꢀα levels in the brain serum of rats treated with compounds 2cꢀ4c
.
*
p<0.05 as
compared to sham; p<0.05as compared to LPS + vehicle; p<0.05as compared to LPS
n=5). Oneꢀway ANOVA followed by Tukey’s test was employed to find out the interꢀgroup
#
$
(
variation and values were considered statistically significant (p<0.001).
hydrophobic in nature. Coꢀcrystallized ligand produced π–π stacking interactions with the
12,26,27
active site amino acid residues as mentioned earlier.
The observed glide scores of the
most active compound most active compounds 2cꢀ4c were also found comparable to that of
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6
parent coꢀcrystallized ligand. Most of the newly synthesized compounds including standards
(
celecoxib and dexamethasone) did not show binding interactions with the active amino acids
Tyr59, Tyr151 and Tyr119), which are considered essential for TNFꢀα inhibition except
(
phenylpyrazoline substituted steroids 4c and 4d, which showed similar interactions with
tyrosine residue Tyr119 as coꢀcrystallized ligand. The steroids 2a, 2b and 4a displayed the πꢀ
π stacking with the tyrosine residue Tyr59. Docking analysis further revealed that the solvent
accessible surface interactions with the tyrosine residues from both the chains are important
for TNFꢀinhibitory activity. Although the most potent compound 3c did not show binding
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
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4
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0
1
2
3
4
5
6
7
8
9
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2
3
4
5
6
7
8
9
0
Table 6. Glide scores of coꢀcrystallized ligand, celecoxib, dexamethasone and pyrazoline
substituted steroidal derivatives on the active site of TNFꢀα.
Compound
Glide Score
Coꢀcrystallized ligand
Celecoxib
ꢀ4.302
ꢀ4.616
ꢀ4.713
ꢀ4.431
ꢀ5.357
ꢀ4.292
ꢀ4.541
ꢀ3.923
ꢀ3.309
ꢀ3.607
ꢀ3.097
ꢀ5.265
ꢀ3.718
ꢀ4.824
ꢀ4.303
Dexamethasone
2
a
2
b
2c
2d
3a
3b
3c
3d
4a
4
b
4c
4
d
3c
Figure 7. The 2D and 3D interaction diagrams of compound 3c at active site of TNFꢀα
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interactions with the active sites of TNFꢀα, but its solvent accessible surface interactions with
tyrosine residues and its nice fit into the ligand binding pocket of the receptor might be
responsible for TNFꢀα inhibition. However, besides TNFꢀα there are many other factors such
ꢀ
as interleukin 1β, 6 and 2, NO, O2 and several inflammatory mediators, which might be
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5
involved in the progression of neuroinflammation.
CONCLUSION
The current study clearly demonstrates the importance of introducing a 16,17ꢀpyrazoline ring
on the steroid skeleton for improving the neuroprotective effects of heterosteroids. Antiꢀ
Alzheimer’s and antiꢀParkinsonian activity studies on 16,17ꢀpyrazoline substituted
heterosteroids using LPS induced neuroinflammation models of mice and rats depict better
neuroprotective effects in comparison to previously reported 16ꢀarylidene steroids. All the
synthesized pyrazolinyl steroids 2ꢀ4aꢀd significantly improve learning, memory and
movement functions in behavioural models of mice and rats and also suppress LPS induced
oxidative stress, nitrosative stress, acetylcholinesterase activity and enhanced TNFꢀα levels.
In accordance with our previous reports, 4ꢀpyridyl moiety remains a vital substituent in
improving the neuroprotective effects of these heterosteroids in the current study. Among all
the 16,17ꢀpyrazolinyl steroids, 5’ꢀ(4ꢀpyridyl) pyrazoline substituted steroids 2cꢀ4c derived
through
neuroprotective effects better than standard drugs celecoxib and dexamethasone in the order
c>4c>2c. It is observed that ꢀacetylation of the pyrazoline ring fused with the steroidal
a
16ꢀ(4ꢀpyridylidene) moiety displayed highest potency and produced
3
N
nucleus results in higher neuroprotective actions in comparison to the steroids fused with Nꢀ
H naked pyrazoline ring. Further substituting the nitrogen of pyrazoline ring with a phenyl
group also increases the activity but to a lesser extent than
Nꢀacetyl analogue. 16,17ꢀNꢀ
acetylpyrazoline fused steroid 3c derived through a 16ꢀ(4ꢀpyridylidene) moiety represents an
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interesting pharmacophore, which needs further exploration to develop novel and effective
therapeutic interventions against neuroinflammation and neurodegenerative conditions.
EXPERIMENTAL SECTION
Chemistry. General. Melting points were determined on a Veego melting point apparatus
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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2
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8
9
0
ꢀ1
and are uncorrected. IR (wave numbers in cm ) spectra are recorded on Perkin–Elmer
1
13
spectrum RX 1, FTꢀIR spectrophotometer model using KBr pellets. H NMR, C NMR
spectra were recorded on Bruker ACꢀ 300F, 300 MHz and Bruker Advance II 400 MHz using
3 6
deuterated chloroform (CDCl ) or deuterated dimethylsulfoxide (DMSOꢀd ) containing
tetramethylꢀsilane as internal standard (chemical shifts in ppm). Mass spectra were recorded
on a Agilent 1100 LC coupled with Bruker make mass spectrometer model Esquire 3000.
Elemental analyses were carried out on Vario EL III model CHN analyzer. Plates for TLC
were prepared according to Stahl (E. Merck) using EtOAc as solvent (activated at 110 C for
30 min) and were visualized by exposure to iodine vapors. Anhydrous sodium sulphate was
used as drying agent. All solvents were distilled prior to use according to standard procedure.
Flash chromatography (Isolera One, Biotage) has been used for the purification of the
12
synthesized compounds. 16ꢀArylidene steroids 1aꢀd were prepared as reported earlier.
General procedure for the preparation of 16,17ꢀpyrazolinyl steroids 2aꢀd. Hydrazine hydrate
(0.7 mL, 14.46 mmol) was added to a solution of the requisite 16ꢀarylidene steroid 1aꢀd
(0.5g, 1.32 mmol) in 1,4 dioxane (30 mL). The reaction mixture was heated under reflux for
5 h, reaction being monitored with TLC. On completion, the solvent was removed under
reduced pressure. The solid obtained was filtered off by addition of water, washed repeatedly
with water, dried and purified by flash chromatography to obtain the corresponding steroid
2aꢀd.
5´ꢀ(Pyridinꢀ2ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (2a). Yield: 54 %. Yellow
ꢀ1
colour compound, mp 200ꢀ203
°
C. FTꢀIR (KBr) cm 3379.45 (OꢀH), 3318.10 (NꢀH), 3063.1
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(CꢀH, aromatic), 2933.14 (CꢀH, aliphatic), 1655.40 (C=N), 1592.03, 1436.12, 1376.84,
1
1
307.30, 1052.66. HNMR (400 MHz, DMSOꢀ
d
6
): δ 0.94 (s, 3H, 18ꢀC
H
3
), 0.98 (s, 3H, 19ꢀ
'
C
C
C
H
H
H
3
), 3.16ꢀ3.17 (d, 1H,
, pyrazoline), 5.28 (t, 1H,
, pyridine), 7.57 (d, 1H,
J
= 5.24 Hz, 16ꢀC
H
), 3.23ꢀ3.31 (m, 1H, 3
= 2.4 Hz, 6ꢀC ), 6.81 (brs, 1H, Nꢀ
= 7.8 Hz, 6ꢀC
α
ꢀ
H
), 4.12ꢀ4.16 (m, 1H, 5 ꢀ
J
H
H
), 7.22ꢀ7.29 (m, 1H, 5ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
J
o
H
, pyridine), 7.76 (t, 1H,
J
o
= 7.7 Hz, 4ꢀC
, pyridine). C NMR (100 MHz, CDCl3): 14.25 (CH3),
), 25.89 (CH ), 30.97 (CH ), 31.37 (CH ), 31.83 (CH ), 32.46
CH ), 36.66 (CH), 37.23 (C), 40.05 (C), 42.20 (CH), 50.28 (CH ), 52.92 (CH), 56.22 (CH),
H,
13
pyridine), 8.48ꢀ8.49 (m, 1H, 3ꢀC
9.44 (CH ), 20.21 (CH
H
δ
1
3
2
2
2
2
2
(
2
2
67.07 (CH), 71.53 (CH), 120.94 (ꢀC=CHꢀ), 121.25 (ArCH), 122.73 (ArCH), 136.89 (ArCH),
40.97 (ꢀC=CHꢀ), 149.17 (ArCH), 160.25 (ꢀC=Nꢀ) and 174.57 (ArC).MS: m/z 392.20
1
+
+
[
M+H] and 390.10 [MꢀH] . Anal. Calcd for C25
C, 76.86; H, 8.56; N, 10.98.
5´ꢀ(Pyridinꢀ3ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (2b). Yield: 56 %, white
33 3
H N O: C, 76.69; H, 8.49; N, 10.73. Found:
ꢀ1
colour compound, mp 216ꢀ218
°C. FTꢀIR (KBr) cm 3348.50(OꢀH), 3042.30 (CꢀH,
aromatic), 2936.80 (CꢀH, aliphatic), 1647.20(C=N), 1583.30, 1557.50, 1439.20, 1410.91,
1
1054. H NMR (300 MHz, CDCl
3
):
= 9.8 Hz,
= 5.12 Hz, 6ꢀC
δ
0.89 (s, 3H, 18ꢀC
= 2.52 Hz, 16ꢀC
), 7.41ꢀ7.44 (m, 1H, 6ꢀC
H
3
), 1.02 (s, 3H, 19ꢀC
H
3
), 3.49ꢀ3.54
'
(
m, 1H, 3
αꢀH
), 3.87 (t(d), 1H,
J
J
H
), 4.05 (d, 1H,
J
= 9.84 Hz, 5 ꢀ
C
H
, pyrazoline), 5.30 (d, 1H,
J
H
H
, pyridine), 8.15ꢀ
8.18 (m, 1H, 5ꢀC
H
, pyridine), 8.52ꢀ8.54 (m, 1H, 4ꢀC
m
H, pyridine) and 8.79 ppm (d, 1H,J =
+
+
1.68 Hz, 2ꢀC
H
, pyridine).MS: m/z 392.20 [M+H] and 390.10 [MꢀH] . Anal. Calcd for
C
25
H
33
N
3
O: C, 76.69; H, 8.49; N, 10.73%. Found: C, 76.38; H, 8.26; N, 10.57%.
5´ꢀ(Pyridinꢀ4ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (2c). Yield: 50%, yellow
o
ꢀ1
colour compound, mp 221ꢀ222 C. FTꢀIR (KBr) cm 3285 (OꢀH), 3140 (NꢀH), 2933.90 (Cꢀ
H, aliphatic), 1655.55(C=N), 1604.30, 1566.44, 1456.81, 1420.11, 1376.77, 1068.30, 806.90.
1
H NMR (300 MHz, CDCl
3
):
3 3
δ1.00 (s, 3H, 18ꢀCH ), 1.05 (s, 3H, 19ꢀCH ), 3.34 (t(d), 1H, J =
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'
12.56 Hz and
J
= 3.12 Hz, 16ꢀC
H
), 3.52ꢀ3.55 (m, 1H, 3
α
ꢀ
H
), 4.42 (d, 1H,
= 4.64 Hz and
= 4.48 Hz and = 1.48 Hz, 3ꢀCH
J
= 13.0 Hz and 5 ꢀ
C
H
, pyrazoline), 5.36 (d, 1H, ,
J
= 5.2 Hz, 6ꢀC ), 7.41 (dd, 2H,
H
J
o
J
m
= 1.36
Hz, 2ꢀC
H
and 6ꢀC
H, pyridine) and 8.56 ppm (dd, 2H,
J
o
J
m
+
+
and 5ꢀC
H
, pyridine). MS: m/z 392.20 [M+H] and 390.10[MꢀH] . Anal. Calcd for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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2
3
4
5
6
7
8
9
0
C
25
H
33
N
3
O: C, 76.69; H, 8.49; N, 10.73%. Found: C, 76.46; H, 8.31; N, 10.49%.
5
´ꢀ(4ꢀMethoxyphenyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (2d). Yield: 63 %, white
o
ꢀ1
colour compound, mp 182ꢀ184 C. FTꢀIR (KBr) cm 3429.90(OꢀH), 3301.22 (NꢀH),
2
933.22(CꢀH, aliphatic), 1652.11(C=N), 1610.55, 1511.10, 1241.66 (CꢀOꢀC, asymmetric)
1
and 1046.44 (CꢀOꢀC, symmetric). H NMR (300 MHz, CDCl
3
)δ
1.01 (s, 3H, 18ꢀC
), 3.52ꢀ3.58 (m, 1H, 3
= 5.24
= 9.5
, aromatic).MS: m/z 421.30[M+H] and 419.20 [MꢀH] . Anal. Calcd for
: C, 77.10; H, 8.63; N, 6.66%. Found: C, 76.85; H, 8.47; N, 6.53%.
General procedure for the preparation of 16,17ꢀpyrazolinyl steroids 3aꢀd. Hydrazine hydrate
0.7 mL, 14.46 mmol) was added to a solution of the requisite 16ꢀarylidene steroid 1aꢀd
3
H ), 1.06
(
s, 3H, 19ꢀC
H
3
), 3.37 (t(d), 1H,
), 3.81 (s, 3H, ꢀOC ), 4.38 (d, 1H,
), 6.89 (d, 2H, = 9.6 Hz, 3ꢀC
and 6ꢀC
J
= 12.9 Hz and
J
= 2.56 Hz, 16ꢀC
H
αꢀ
'
H
H
3
J
= 13.08 Hz, 5 ꢀC
H
, pyrazoline), 5.37 (d, 1H,
J
Hz, 6ꢀC
H
J
o
H and 5ꢀCH, aromatic) and 7.42 ppm (d, 2H,J
o
+
+
Hz, 2ꢀC
H
H
27 36 2 2
C H N O
(
(0.5g, 1.32 mmol) in glacial acetic acid (30 mL). The reaction mixture was heated under
reflux for 9 h, reaction being monitored with TLC. On completion, the solvent was removed
under reduced pressure. The solid obtained was filtered off by addition of water, washed
repeatedly with water, dried and purified by flash chromatography to obtain the
corresponding steroid 3aꢀd.
1´ꢀ(Acetyl)ꢀ5´ꢀ(pyridinꢀ2ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀyl
acetate
(3a).
ꢀ1
Yield: 49 %, cream colour compound, mp190ꢀ192 C. FTꢀIR (KBr) cm 3064.71 (CꢀH,
°
aromatic), 2939.47 (CꢀH, aliphatic), 1731.84 (C=O), 1667.33 (C=O), 1577.35, 1408.46,
1
1328ꢀ02, 1245.57, 1035.20. HNMR (400 MHz, CDCl
3
): δ0.97 (s, 3H, 18ꢀC
H
3
), 1.01 (s, 3H,
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19ꢀC
H
3
), 2.01 (s, 3H, NꢀCOC
H
3
), 2.40 (s, 1H, 3H, OꢀCOC
, pyrazoline),5.26ꢀ5.28 (m, 1H, 6ꢀC
, pyridine), 8.02 (t, 1H, = 8 Hz, 4ꢀC
, pyridine). C NMR (100 MHz, CDCl ): 15.25 (CH
), 21.38 (CH ), 22.23 (CH ) 27.69 (CH ), 31.39 (CH ), 31.48 (CH
), 33.78 (CH ), 36.56 (CH), 36.94 (C), 38.02 (C), 45.91 (CH), 47.78 (CH
H
3
), 3.45ꢀ3.50 (m, 1H, 16ꢀC
), 7.25ꢀ7.30 (m, 1H,
, pyridine),
), 19.25
H),
'
4
.47ꢀ4.51 (m, 2H, 3
ꢀC
αꢀH
and 5 ꢀC
H
H
5
H
, pyridine), 7.68ꢀ7.74 (m, 1H, 6ꢀC
H
J
o
H
1
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.60ꢀ8.63 (m, 1H, 3ꢀC
H
3
δ
3
(
(
(
(
CH
3
2
), 20.71 (CH
2
3
3
2
2
2
), 32.21
CH
2
2
), 49.48
CH), 54.41 (CH), 65.82 (CH), 73.82 (CH), 121.52 (ꢀC=CHꢀ), 121.78 (ArCH), 123.91
ArCH), 136.11 (ArCH), 139.73 (ꢀC=CHꢀ), 149.28 (ArCH), 150.73 (ꢀC=Nꢀ), 158.37 (ArC),
+
+
170.49 (C=O) and 171.25 (C=O). MS: m/z 476.60 [M+H] and 474.10 [MꢀH] .Anal. Calcd
: C, 73.23; H, 7.84; N, 8.83%. Found: C, 73.46; H, 7.60; N, 9.09%.
´ꢀ(Acetyl)ꢀ5´ꢀ(pyridinꢀ3ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀyl acetate
37 3 3
for C29H N O
1
(3b)
.
ꢀ1
Yield: 46 %, white colour compound, mp 218ꢀ220
°C.FTꢀIR (KBr) cm 3033.92 (CꢀH,
aromatic), 2940.27 (CꢀH, aliphatic), 1728.97 (C=O),1660.29 (C=O), 1589.10, 1410.87,
1
1247.89, 1031.77. HNMR (400 MHz, CDCl
3
): δ 0.90 (s, 3H, 18ꢀC
), 2.27 (s, 1H, 3H, OꢀCOC ), 3.98ꢀ4.01 (m, 1H, 16ꢀC
, pyrazoline ), 5.22ꢀ5.23 (m, 1H, 6ꢀC ), 7.44ꢀ7.48 (m, 1H, 5ꢀC
, pyridine), 8.59ꢀ8.61 (m, 1H, 4ꢀ
H
3
), 0.96 (s, 3H, 19ꢀC
3
H ),
1.95 (s, 3H, NꢀCOC
H
3
H
3
H
), 4.35ꢀ4.44
'
(m, 2H, 3
αꢀH
and 5 ꢀC
H
H
H,
pyridine), 8.09 (d(t), 1H, Jo = 8.04 Hz and Jm= 1.8 Hz, 6ꢀC
H
13
C
H
, pyridine), 8.90 (d, 1H, Jm= 1.2 Hz, 2ꢀC
H
, pyridine). C NMR (100 MHz, DMSOꢀd6):δ
), 21.42 (CH ), 22.25 (CH ) 27.68 (CH ), 31.15 (CH ),
), 36.55 (CH), 36.80 (C), 38.07 (C), 45.83 (CH), 47.83
14.20 (CH
3
2
), 19.38 (CH
3
), 20.33 (CH
2
2
3
3
2
2
31.31 (CH
), 32.17 (CH
2
), 33.61 (CH
2
(CH ), 50.20 (CH), 54.69 (CH), 64.58 (CH), 73.78 (CH), 121.67 (ꢀC=CHꢀ), 123.58 (ArCH),
136.78 (ArCH), 139.80 (ArC), 140.09 (ꢀC=CHꢀ), 148.18 (ArCH), 149.69 (ArCH), 155.35 (ꢀ
+
+
C=Nꢀ), 170.55 (C=O) and 171.28 (C=O). MS: m/z 476.70 [M+H] and 474.80 [MꢀH] .
Anal. Calcd for C29
8.71%.
37
H N
3
O
3
: C, 73.23; H, 7.84; N, 8.83%. Found: C, 73.35; H, 7.74; N,
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1´ꢀ(Acetyl)ꢀ5´ꢀ(pyridinꢀ4ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀyl acetate (3c).Yield:
ꢀ1
52 %, light yellow colour compound, mp 175ꢀ178
°
C. FTꢀIR (KBr) cm 3030.01 (CꢀH,
aromatic), 2939.39 (CꢀH, aliphatic), 1728.36 (C=O), 1668.91 (C=O), 1596.587, 1410.90,
1
1
373.24, 1245.65, 1032.68. HNMR (400 MHz, CDCl ): δ 0.51 (s, 3H, 18ꢀCH3), 0.97(s, 3H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
19ꢀC
.45ꢀ4.48 (m, 2H, 3
Hz, 2ꢀC and 6ꢀC
NMR (100 MHz, CDCl ): δ14.14 (CH ), 19.48 (CH ), 20.37 (CH ), 21.43 (CH ), 22.24
H
3
), 2.28 (s, 3H, NꢀCOC
H
3
), 2.31 (s, 1H, 3H, OꢀCOC
and 5 ꢀC , pyrazoline), 5.35 (brs, 1H, 6ꢀC
, pyridine), 8.63 (d, 2H, = 5.2 Hz, 3ꢀC
H
3
), 4.05ꢀ4.10 (m, 1H, 16ꢀC
H),
'
4
αꢀ
H
H
H), 7.62 (d, 2H, Jo = 6.04
1
3
H
H
J
o
H
and 5ꢀC
H, pyridine).
C
3
3
3
2
3
(CH ) 27.68 (CH ), 31.21(CH ), 31.49 (CH ), 32.34 (CH ), 33.64 (CH ), 36.61 (CH), 36.93
3
2
2
2
2
2
(C), 37.98 (C), 45.89 (CH), 47.47 (CH
2
), 49.20 (CH), 54.59 (CH), 65.89 (CH), 73.77 (CH),
1
1
20.64 (2xArCH), 121.70 (ꢀC=CHꢀ), 140.59 (ꢀC=CHꢀ), 141.23 (ArC), 150.31 (2xArCH),
+
55.10 (ꢀC=Nꢀ), 169.70 (C=O) and 170.56 (C=O). MS: m/z 476.70 [M+H] and 474.30 [Mꢀ
+
H] . Anal. Calcd for C H N O C, 73.23; H, 7.84; N, 8.83%. Found: C, 73.01; H, 7.98; N,
2
9
37
3
3:
8.69%.
1'ꢀ(Acetyl)ꢀ5´ꢀ(4ꢀmethoxyphenyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀyl acetate (3d).
ꢀ1
Yield: 50 %, white colour compound, mp 227ꢀ230°C. FTꢀIR (KBr) cm 3064.70 (CꢀH,
aromatic), 2937.39 (CꢀH, aliphatic), 1730.40 (C=O), 1648.71 (C=O), 1608.52, 1516.99,
1454.39 1361.77, 1253.25 (CꢀOꢀC, asymmetric), 1175.65, 1030.74 (CꢀOꢀC, symmetric).
1
HNMR (100 MHz, CDCl
3
): δ 0.97 (s, 3H, 18ꢀC
H
3
), 0.99 (s, 3H, 19ꢀC
), 3.83 (s, 1H, ꢀOC ), 3.88ꢀ3.96 (m, 1H,
, pyrazoline), 5.35 (t, 1H, = 2.4 Hz, 6ꢀC
and 6ꢀCH,
H
3
), 2.02 (s, 3H, Nꢀ
= 8.1 Hz,
), 6.91
COC
H
3
), 2.36 (s, 1H, 3H, OꢀCOC
H
3
H
3
J
'
3
αꢀ
H
), 4.47 (d, 1H,
J
= 11.72 Hz, 5 ꢀC
H
J
H
(d, 2H, Jo = 8.8 Hz, 3ꢀC
H
and 5ꢀC
H, aromatic), 7.67 (d, 2H, Jo = 6.8 Hz, 2ꢀC
H
13
aromatic). C NMR (400 MHz, DMSOꢀd6): δ 15.7 (CH ), 19.3 (CH ), 20.9 (CH ), 21.6
3
3
2
(
CH
3
), 22.3 (CH
3
) 27.7 (CH
2
), 31.1 (CH
2
), 31.6 (CH
2
), 32.1 (CH
2
), 36.6 (CH
2
), 36.9 (CH),
37.2 (C), 38.09 (C), 45.6 (CH), 48.1 (CH
2
3
), 49.5 (CH), 55.03 (CH), 55.4 (CH ), 65.10 (CH),
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73.8 (CH), 114.1 (2xArCH), 123.5 (ꢀC=CHꢀ), 128.5 (2xArCH), 140.06 (ꢀC=CHꢀ), 141.2
(ArC), 157.3 (ArC), 161.04 (ꢀC=Nꢀ), 169.1 (C=O) and 170.6 (C=O). MS: m/z 505.60 [M+H]
+
+
and 503.10 [MꢀH] . Anal. Calcd for C31
H
40
2
N O
4: C, 73.78; H, 7.99; N, 5.55 %. Found: C,
73.95; H, 7.80; N, 5.37 %.
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General procedure for the preparation of 16,17ꢀpyrazolinyl steroids 4aꢀd. Phenylhydrazine
(0.15 mL, 1.51 mmol) was added to a solution of the requisite 16ꢀarylidene steroid 1aꢀd
(0.5g, 1.32 mmol) in anhydrous methanol (30 mL) and glacial acetic acid (5mL). The
reaction mixture was heated under reflux for 12 h, reaction being monitored with TLC. On
completion, the solvent was removed under reduced pressure. The ice cold water was added
to the obtained residue and solution was basified with few drops of ammonia solution. The
precipitate obtained was filtered off, washed repeatedly with water, dried and purified by
flash chromatography to obtain the corresponding steroid 4aꢀd.
1´ꢀ(Phenyl)ꢀ5´ꢀ(pyridinꢀ2ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (4a). Yield: 24 %,
ꢀ1
yellow colour compound, mp 269ꢀ270 °C. FTꢀIR (KBr) cm 3335.80 (OꢀH), 3061.46 (CꢀH,
aromatic), 2932.89 (CꢀH, aliphatic), 1594.48, 1487.81, 1341.84, 1056.33, 753.71, 692.83
1
1
cm . HNMR (400 MHz, DMSOꢀ
m, 1H, 3 ), 4.70 (d, 1H, = 12.7 Hz, 5’ꢀC
1H, = 7.30 Hz, 4ꢀC , aromatic), 6.78 (d, 2H,
t, 2H, Jo = 7.94 Hz, 3ꢀC and 5ꢀ C , aromatic), 7.26ꢀ7.29 (m, 1H, 5ꢀC
, pyridine), 7.74 (t(d), 1H, = 7.6 Hz and = 1.7 Hz, 4ꢀC
, pyridine). C NMR (100 MHz, DMSOꢀ ):δ 15.09 (CH
), 27.88 (CH ), 29.89 (CH ), 30.68 (CH ), 31.29 (CH ), 33.82
), 36.35 (CH), 36.73 (C), 42.13 (C), 49.94 (CH), 58.63 (CH ), 62.02 (CH), 64.96 (CH),
9.90 (CH), 75.24 (CH), 114.75 (2xArCH), 119.46 (ArCH), 119.89 (ArCH), 120.38 (ꢀ
d
6
):δ1.00 (s, 3H, 18ꢀC
, pyrazoline), 5.26 (brs, 1H, 6ꢀC
= 8.0 Hz, 2ꢀ C and 6ꢀC , aromatic), 7.07
, pyridine), 7.49 (d,
, pyridine),
),
H
3
), 1.06 (s, 3H, 19ꢀ C
H
3
), 3.35ꢀ3.42
(
α
ꢀ
H
J
H
H
), 6.74 (t,
J
o
H
J
o
H
H
(
H
H
H
1
8
1
H,
.55 (d, 1H,
8.98 (CH ), 20.34 (CH
J
o
= 7.9 Hz, 6ꢀC
H
J
o
J
m
H
13
J
o
= 7.68 Hz, 3ꢀC
H
d
6
3
3
2
2
2
2
2
(CH
2
2
6
C=CHꢀ), 122.50 (ArCH), 128.26 (2xArCH), 137.18 (ArCH), 141.34(ꢀC=CH), 147.75
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5
5
5
5
5
5
5
5
6
+
(ArCH), 149.06 (ArC), 161.20 (ꢀC=Nꢀ) and 172.61 (ArC).MS: m/z 468.30 [M+H] and
+
4
66.10 [MꢀH] .Anal. Calcd for C31
H
37
N
3
O
:
C, 79.62; H, 7.97; N, 8.99 %. Found: C, 79.73;
H, 7.77; N, 8.79%.
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´ꢀ(Phenyl)ꢀ5´ꢀ(pyridinꢀ3ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (4b). Yield: 52%,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
light yellow colour compound, mp185ꢀ188°C. FTꢀIR (KBr) cm 3400.63 (OꢀH), 3043.55 (Cꢀ
H, aromatic), 2933.78 (CꢀH, aliphatic, 1632.56 (C=N), 1453.51, 1097.33, 1058.34, 809.04,
1
7
06.65, HNMR (400 MHz, CDCl
3
): δ0.99 (s, 3H, 18ꢀC
= 11.4 Hz, 5’ꢀC , pyrazoline), 5.39 (m, 1H, 6ꢀC
and 6ꢀC , aromatic), 7.13 (t(br), 2H,3ꢀC and 5ꢀC , aromatic),
, pyridine), 7.83 (d, 1H, , pyridine), 8.56ꢀ8.57(m,
, pyridine), 8.78 (s, 1H, 2ꢀC ):δ 14.19
), 19.48 (CH ), 20.37 (CH ), 27.70 (CH ), 31.51 (CH ),
34.35 (CH ), 36.73 (CH), 37.14 (C), 42.24 (C), 49.72 (CH), 59.46 (CH ), 63.45 (CH), 66.50
H
3
), 1.07 (s, 3H, 19ꢀC
H
3
), 3.51ꢀ3.56
(
m, 1H, 3
α
ꢀ
H
), 4.59 (d, 1H,
,4ꢀC
.35ꢀ7.39 (m, 1H, 5ꢀC
H, 4ꢀC
J
H
H
), 6.76ꢀ
6.87 (m, 3H, 2ꢀC
H
H
H
H
H
7
1
H
J
o
= 7.8 Hz, 6ꢀCH
13
H
H
, pyridine). C NMR (100 MHz, CDCl
3
(
CH
3
3
2
2
), 29.38 (CH
2
), 30.90 (CH
2
2
2
2
(
CH), 68.30 (CH), 73.78 (CH), 116.02 (2xArCH), 118.06 (ArCH), 120.63 (ꢀC=CHꢀ), 123.64
(
ArCH), 129.26 (2xArCH), 136.94 (ArCH), 138.24 (ArC), 140.08 (ꢀC=CH), 141.29 (ArC),
+
1
49.53 (ArCH), 151.01 (ArCH) and 155.34 (ꢀC=Nꢀ). MS: 468.30 [M+H] and 466.20 [Mꢀ
+
H] . Anal. Calcd for C31
37 3 :
H N O C, 79.62; H, 7.97; N, 8.99 %. Found: C, 79.85; H, 8.18; N,
9.14 %.
1
´ꢀ(Phenyl)ꢀ5´ꢀ(pyridinꢀ4ꢀyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (4c). Yield: 41 %,
ꢀ1
yellow colour compound, mp 198ꢀ201
°C. FTꢀIR (KBr) cm 3400.63 (OꢀH), 3060.55 (CꢀH,
1
aromatic), 2933.78 (CꢀH, aliphatic), 1598.57, 1255.04, 1371.92, 1059.35, 815.46. HNMR
(
400 MHz, CDCl
3
):δ0.99 (s, 3H, 18ꢀC
= 12.8 Hz, 5’ꢀC , pyrazoline), 5.38ꢀ5.40 (m, 1H, 6ꢀC
,4ꢀC and 5ꢀC , aromatic), 7.10 (dd, 2H, = 1.18 Hz and
and 6ꢀC
H
3
), 1.07 (s, 3H, 19ꢀ C
H
3
), 3.51ꢀ3.57 (m, 1H, 3
), 6.88 (t, 3H,Jo
= 7.6 Hz, 2ꢀC
α
ꢀ
H
),
=
4
.53 (d, 1H,
.32 Hz, 3ꢀC
J
H
H
7
H
H
H
J
m
J
o
H
and 6ꢀC
H
, aromatic), 7.37 (d, 2H,
J
o
= 5.4 Hz, 2ꢀC
H
H
, pyridine), 8.66 (brs, 2H, 3ꢀ
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C
H
and 5ꢀC
7.2 (CH ), 29.3 (CH
9.5 (CH), 55.5 (CH ), 62.1 (CH), 64.5 (CH), 68.6 (CH), 73.8 (CH), 112.7 (2xArCH), 120.3
H
, pyridine). C NMR (100 MHz, CDCl
3
): δ14.1 (CH
3 3 2
), 19.4 (CH ), 20.3 (CH ),
2
4
2
2
), 30.9 (CH ), 31.2 (CH ), 33.7 (CH
2
2
2
), 36.5 (CH), 37.1 (C), 42.1 (C),
2
(
ArCH), 120.6 (ꢀC=CHꢀ), 123.9 (2xArCH), 129.9 (2xArCH), 140.6 (ꢀC=CHꢀ), 141.2 (ArC),
10
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15
16
17
18
19
20
21
22
23
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27
28
29
30
31
32
33
34
35
36
37
38
39
40
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
+
144.6 (ArC), 150.1 (2xArCH) and 152.3 (ꢀC=Nꢀ). MS: 468.30 [M+H] and 466.20 [MꢀH] .
Anal. Calcd for C31 C, 79.62; H, 7.97; N, 8.99 %. Found: C, 79.49; H, 7.63; N,
37 3 :
H N O
9.20%.
1
´ꢀ(Phenyl)ꢀ5´ꢀ(4ꢀmethoxyphenyl)ꢀandrostꢀ5ꢀenꢀ[17,16ꢀc]ꢀ1´Hꢀpyrazolineꢀ3βꢀol (4d). Yield:
ꢀ1
38 %, white colour compound, mp183ꢀ186°C.°C. FTꢀIR (KBr) cm 3257.11 (OꢀH), 3060.66
(
CꢀH, aromatic), 2934.99 (CꢀH, aliphatic), 1602.84, 1511.89, 1458.28, 1304.04, 1254.11(Cꢀ
ꢀ1
OꢀC, asymmetric), 1174.96, 1371.92, 1059.53, 918.22(CꢀOꢀC, symmetric), 831.75 cm .
1
HNMR (400 MHz, CDCl
3
):δ 0.96 (s, 3H, 18ꢀC
), 4.61 (brs, 1H, 5’ꢀC
and 6ꢀC , aromatic), 7.24 (t(br), 2H,
and 5ꢀC , aromatic), 7.50 (d, 2H,
, aromatic). C NMR (400 MHz, CDCl ):δ14.31 (CH ), 19.39 (CH
(CH ), 29.32 (CH ), 30.94 (CH ), 31.15 (CH ), 32.91 (CH ), 36.83 (CH), 38.11 (C), 42.28
(C), 49.90 (CH), 55.36 (CH ), 58.30 (CH ), 62.0 (CH), 64.67 (CH), 69.03 (CH), 73.75 (CH),
H
3
), 1.08 (s, 3H, 19ꢀC
, pyrazoline), 5.42 (brs, 1H, 6ꢀC
= 8.04 Hz,3ꢀC and 5ꢀC
= 8.16 Hz, 2ꢀC and 6ꢀ
), 20.39 (CH ), 27.71
H
3
), 3.70ꢀ3.7 9 (m, 1H,
3
αꢀ
H
), 3.84 (s, 1H, ꢀOC
H
3
H
H
), 6.78ꢀ
6
.95 (m, 3H, 2ꢀC ,4ꢀC
H
H
H
J
H
H,
aromatic), 7.40 (d(br), 2H, 3ꢀC
H
H
J
o
H
13
C
H
3
3
3
2
2
2
2
2
2
3
2
1
1
1
14.22 (2xArCH), 115.84 (2xArCH), 120.85 (ꢀC=CHꢀ), 121.33 (ArCH), 128.31 (2xArCH),
32.10 (2xArCH), 132.94 (ArC), 133.52 (ArC), 140.07 (ꢀC=CH), 160.48 (ꢀC=Nꢀ) and
+
+
40 2
70.54 (ArC). MS: m/z 497.60 [M+H] and 495.10 [MꢀH] . Anal. Calcd for C33H N O2: C,
79.80; H, 8.12; N, 5.64 %. Found: C, 80.13; H, 8.33; N, 5.39 %.
Biological Activity. Animals. Male Laca mice weighing 25ꢀ30 g and adult Wistar male rats
weighing 180ꢀ250 g were obtained from the central animal house of the institute. The animals
were housed in standard laboratory conditions in groups of three at 25 ± 2°C, humidity of 60
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± 2% and 12 h light: Dark cycle. Animals had a free access to standard laboratory chow diet
and tap water. The animals were acclimatized to the laboratory conditions 1 week prior to
experimentation. All experiments were conducted daily between 09:00 am and 03.00 pm h.
The protocol was approved by Institutional Animal Ethics Committee and the animal
housing, care and handling was followed as per committee guidelines for the purpose of
control and supervision of experiments on animals and for animal care and use.
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Drugs. Lipopolysaccharide (LPS) (serotype E.coli 0111:B4) was procured from Sigma (St.
Louis, MO, USA). Celecoxib and Dexamethasone were received as gift samples from M/s
Panacea Biotech Ltd., Lalru. All other chemicals were of analytical grade.
AntiꢀAlzheimer’s Activity. The mice were randomly divided into 16 groups, each group
consisted of a minimum of 5 animals and daily received LPS as well as combination of LPS
12,20,22
with standards (celecoxib and dexamethasone)
and with synthesized compounds
intraperitoneally (i.p.) for 5 days continuously 4h prior to training and testing sessions. The
groups were divided as i) control group received equivalent volume of vehicles [distilled
water with 2 drops of tween 80 and 1% Phosphate Buffer Solution (PBS)] ii) LPS group
(
250µg/kg) iii) combination of LPS (250µg/kg) with celecoxib (20 mg/kg) iv) combination
of LPS (250µg/kg) with dexamethasone (5 mg/kg) and vꢀxvi) combination of LPS
250µg/kg) with synthesized compounds 2a d, 3a and 4a (5 mg/kg). The behavioural
assessment after intraperitoneal injection of LPS in mice has been carried out using Morris
(
ꢀ
ꢀ
d
ꢀd
1
2
water maze and Elevated plus maze as reported earlier.
AntiꢀParkinsonian Activity. The stereotaxic surgery was performed on the rats anaesthetized
2
0
with thiopentone (45 mg/kg, i.p, Neon Laboratories, India) as reported earlier. The head
was positioned in a stereotaxic frame and a midline sagittal incision was made in the scalp.
Burr holes were drilled in the skull on left sides using the following coordinates: 5.5 mm
posterior, 1.5 mm lateral and 8.3 mm ventral to the bregma. The coordinates for the
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placement of cannulae were determined by using atlas of Paxinos and Watson (2007). LPS
[
10 µg in 2µl of 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl ꢀ 0.0027 M); pH 7.4)]
was infused as a single intranigral injection into the left substantia nigra using the Hamilton
micro syringe. The skin was sutured after the injection followed by daily application of
10
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®
antiseptic powder (Neosporin ). The rats were randomly divided into 17 groups, each group
consisted of a minimum of 5 animals. The animals of all the groups received single
intranigral injection of LPS (10 µg in 2µl of PBS) on day 1 and daily intraperitoneal (i.p.)
injection of standards (celecoxib (20 mg/kg, i.p), dexamethasone (2 mg/kg, i.p) or
synthesized compounds (2 mg/kg, i.p) continuously for 21 days. The groups were divided as
i) sham group received intranigral injection of PBS and equivalent volume of normal saline
(
0.9% NaCl, i.p.) ii) LPS + vehicle group received intranigral injection of LPS and
equivalent volume of vehicle (i.p.) iii) LPS group received intranigral injection of LPS iv)
celecoxib group received intranigral injection of LPS and celecoxib (20 mg/kg, i.p.) v)
dexamethasone group received intranigral injection of LPS and dexamethasone (2 mg/kg,
i.p.) viꢀxvii) intranigral injection of LPS and synthesized compounds
2ꢀ4aꢀd (2 mg/kg,i.p).
The behavioural assessment after intranigral injection of LPS has been carried out using
th
th
st
actophotometer, elevated plus maze and block tests on 7 , 14 and 21 day. As there was
no significant difference in the behavioural observation of animals at day 7 and 14, the
1
2
behavioural data of day 21 is presented as reported in our previous study.
Biochemical Estimations in Brain Supernatants of Rodents. The brain homogenates were
prepared on day 5 for mice and day 21 for rats. The mice (n=5) and rats (n=5) were sacrificed
under deep anaesthesia and brains were rapidly removed and placed on dry ice for isolation
of the cerebral cortex and hippocampus. A 10% (w/v) tissue (cortex and hippocampus)
homogenate was prepared in chilled 0.1M phosphate buffer (pH 7.4) using a Potter
0
Elvenhjem homogenizer. The homogenate was centrifuged at 12000 rpm for 20 min at 4 C to
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obtain the post mitochondrial supernatant (PMS), which was used for further biochemical
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analysis. The biochemical parameters evaluated include: i) Protein Estimation
ii)
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Estimation of Acetylcholinesterase Activity (AChE Assay)
iii) Estimation of Lipid
1
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Peroxidation (LPO Assay)
iv) Plasma Nitrite Estimation (Nitrite Assay)
v) Estimation
0
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0
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of reduced glutathione (GSH Assay)
vi) Estimation of superoxide dismutase (SOD
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Assay)
vii) Estimation of Catalase (Catalase Assay)
Inhibition of TNFꢀα. Quantification of TNFꢀα (R&D Systems Quantikine Rat TNFꢀα
immunoassay kit) was made according to the manufacturer’s instructions. The Quantikine
Rat TNFꢀα immunoassay is a 4.5ꢀh solid phase ELISA designed to measure rat TNFꢀα levels.
The sample values were evaluated for TNFꢀα inhibitory activity as reported earlier and values
12,23
were expressed as mean ± SEM.
Molecular Docking Studies. Docking was done using Glide (Schrodinger, LLC, New York,
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US). TNFꢀα crystal structure was downloaded from Protein Data Bank (PDB ID: 2AZ5).
The ligands were geometrically refined (cleaned) and conformers were generated with
maximum number of conformers per structure as 1000 with force field OPLSꢀ2005 with
RMSD 1.0 A°. Protein was preprocessed, optimized and minimized with force field of
OPLS2005 and RMSD of 0.30 Å using the protein preparation wizard. Grid was generated
using the centroid of workspace of coꢀcrystallized ligand that is composed of
trifluoromethylphenylindole and dimethylchromone moieties linked by a dimethylamine
spacer that inhibited TNFꢀα receptor binding. The docking was performed using Glide with
enabling the “write XP descriptor information” option and keeping the rest default.
Data analysisꢀAll statistical analyses were performed using GraphPad Prism v. 5.03 software
(GraphPad Software, San Diego, CA, USA). Results are expressed as mean ± SEM. The
intergroup variation in behavioural, biochemical and molecular estimations were measured
by one way and two way analysis of variance (ANOVA) followed by Turkey's test. A value
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ACS Chemical Neuroscience
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of p < 0.05 was considered statistically significant.
SUPPORTING INFORMATION
Copies of 1H NMR and Mass spectra of compounds 2bꢀd. Copies of 1H NMR, 13C NMR
and Mass spectra of compounds 2a, 3ꢀ4aꢀd. (DOCX)
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AUTHOR CONTRIBUTIONS
R. S. carried out the synthesis, biological activity and molecular docking studies of the new
compounds 2a and 3ꢀ4aꢀd; S.T. synthesized the compounds 2bꢀd. R.B. supervised the
research work and finalized the manuscript.
ACKNOWLEDGMENTS
The financial support provided by the University Grant Commission (UGC), New Delhi,
India in a Research Fellowship in Science for Meritorious Student (RFSMS) scheme is
gratefully acknowledged. We are also thankful to Sophisticated Advanced Instrumentation
Facilities (SAIF), Panjab University, Chandigarh, India for providing the NMR data.
ABBREVIATIONS
AD, Alzheimer's disease; PD, Parkinson's disease; LPS, lipopolysaccharide;
DHEA,dehydroepiandrosterone; CNS, central nervous system; Aβ, amyloidꢀβ;APP, amyloid
precursor protein; IL, interleukin; TNFꢀα, tumor necrosis factor; NMDA, NꢀmethylꢀDꢀ
aspartate; TSTQ, time spent in target quadrant; % ITL, initial transfer latency; ANOVA,
analysis of variance; S.E.M, standard error mean; AchE, acetylcholinesterase; LPO, lipid
peroxidation; GSH, Glutathione; SOD, superoxide dismutase; SN, substantia nigra; COX,
cyclooxygenase; PBS, phosphate buffer solution.
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