Investigations on 16-Arylideno Steroids as a New Class of Neuroprotective Agents for the Treatment of Alzheimer’s and Parkinson’s Diseases

Article abstract of DOI:10.1021/acschemneuro.6b00313

Neuroinflammatory mechanisms mediated by activated glial and cytokines (TNF-α, IL-1β) might contribute to neuronal degeneration leading to Alzheimer’s (AD) and Parkinson’s disease (PD). Lipopolysaccharide (LPS) is an inflammogen derived from the cell wall of Gram-negative bacteria, which promotes neuroinflammation and subsequent neurodegeneration. Dehydroepiandrosterone (DHEA) and testosterone have been reported as neuroprotective steroids useful for the treatment of various neurodegenerative disorders. In the present study, several 16-arylidene steroidal derivatives have been evaluated as neuroprotective agents in LPS-treated animal models. It was observed that 16-arylidene steroidal derivatives 1a-d and 6a-h considerably improve LPS-induced learning, memory, and movement deficits in animal models. Biochemical estimations of brain serum of treated animals revealed suppression of oxidative and nitrosative stress, acetylcholinesterase activity, and reduction in TNF-α levels, which were induced through LPS mediated neuroinflammatory mechanisms leading to neurodegeneration of brain. Of all the steroidal derivatives, 16-(4-pyridylidene) steroid 1c and its 4-aza analogue 6c were found to be the most active neuroprotective agents and produced effects comparable to standard drug celecoxib at a much lower dose and better than dexamethasone at the same dose in terms of behavioral, biochemical, and molecular aspects.

Full text of DOI:10.1021/acschemneuro.6b00313

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Investigations on 16-arylideno steroids as a new class of neuroprotective
agents for the treatment of Alzheimer’s and Parkinson’s diseases
Ranjit Singh, and Ranju Bansal
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00313 • Publication Date (Web): 24 Oct 2016
Downloaded from http://pubs.acs.org on October 25, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 44
ACS Chemical Neuroscience
1
2
3
4
5
6
Investigations on 16ꢀarylideno steroids as a new class of
neuroprotective agents for the treatment of Alzheimer’s and
Parkinson’s diseases
7
8
9
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
Ranjit Singh 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
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 2 of 44
1
2
3
4
5
6
7
8
9
ABSTRACT: Neuroinflammatory mechanisms mediated by activated glial and cytokines
(TNFꢀα, ILꢀ1β) might contribute neuronal degeneration leading to Alzheimer's (AD) and
Parkinson's disease (PD). Lipopolysaccharide (LPS) is an inflammogen derived from the cell
wall of Gramꢀnegative bacteria, which promotes neuroinflammation and subsequent
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
neurodegeneration. Dehydroepiandrosterone (DHEA) and testosterone have been reported as
neuroprotective steroids useful for the treatment of various neurodegenerative disorders. In
the present study, several 16ꢀarylidene steroidal derivatives have been evaluated as
neuroprotective agents in LPSꢀtreated animal models. It was observed that 16ꢀarylidene
steroidal derivatives 1aꢀd and 6aꢀh considerably improve LPSꢀinduced learning, memory and
movement deficits in animal models. Biochemical estimations of brain serum of treated
animals revealed suppression of oxidative and nitrosative stress, acetylcholinesterase activity
and reduction in TNFꢀα levels, which were induced through LPS mediated
neuroinflammatory mechanism leading to neurodegeneration of brain. Of all the steroidal
derivatives, 16ꢀ(4ꢀpyridylidene) steroid 1c and its 4ꢀaza analogue 6c were found to be the
most active neuroprotective agents and produced effects comparable to standard drug
celecoxib at a much lower dose and better than dexamethasone at the same dose in terms of
behavioural, biochemical and molecular aspects.
Key words: Parkinson’s disease, Alzheimer’s disease, neuroinflammation, 16ꢀarylidene
steroids
ACS Paragon Plus Environment

Page 3 of 44
ACS Chemical Neuroscience
1
2
3
4
INTRODUCTION
Central nervous system (CNS) in response to infections, trauma, stroke, toxins and other
stimuli activate
neuroinflammatory reaction within the brain.1 Chronic neuroinflammation
5
6
7
8
s
9
is usually associated with several neurodegenerative disorders such as Alzheimer's disease
(AD) and Parkinson's disease (PD).2,3 The amyloidꢀβ(Aβ) plaques and neurofibrillary tangles
are the main culprits for the pathology of Alzheimer's Disease (AD), which is characterized
by loss of memory and learning abilities. The neuroinflammatory process in AD leads to
activation of brain cells like microglia and astrocytes and also cytokines, chemokines and the
complement system, which result in neuronal dysfunction and brain cell death. The over
expression of cytokines such as interleukin (IL)ꢀ1β, tumor necrosis factor (TNF)ꢀα and ILꢀ6
enhances amyloid precursor protein (APP) production and the amyloidogenic processing
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
of APP that lead to the formation of amyloid βꢀ42 (Aβꢀ42)
peptide and
hyperphosphorylation of tau (τ) protein.4 Chronic neuroinflammation in PD causes activated
glial and peripheral immune cells mediated degeneration of dopaminergic neurons leading to
motor dysfunction.5 In the striatum of patients with PD, increased concentration of key
mediators of neuroinflammation like TNFꢀα, β2 microglobulin, interleukin 1β, 6 and 2 has been
found. This further results in oxidative stress and cytokineꢀreceptorꢀmediated apoptosis, which
might eventually lead to neurodegeneration via dopaminergic cell death and hence disease
progression.6
Lipopolysaccharide (LPS), a potent bacterial endotoxin elicits multiple
pathological effects in human beings. In CNS, the exact mechanism of action of LPS
remains unknown but it might be acting through the release of cytokines. Recent
evidence shows that chronic infusion of LPS cause neurodegeneration resulting in
impaired spatial memory and locomotor.2,3,5 In addition, the literature reports reveal
binding of LPS to specific transmembrane receptors such as CD14, LPSꢀbinding protein (LBP)
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 4 of 44
1
2
3
4
5
6
7
8
and the tollꢀlike receptor (TLR) in order to release cytokines (ILꢀ1β, TNFꢀα and ILꢀ6), NO, O2ꢀ
and other inflammatory mediators. This leads to neuronal damage or death through
nitrated/oxidative stress, mitochondrial dysfunction and apoptosis.7
9
During the last decades, steroids substituted with heterocycles at Aꢀ or Dꢀ ring of the
steroid skeleton have garnered much attention due to their widespread pharmacological
activities such as antiꢀinflammatory, anticancer and antimicrobial.8ꢀ11 DHEA analogues with
C3ꢀ or C17ꢀ modifications have displayed neuroprotective activity against the neuralꢀcrest
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
derived PC12 cell model of serum deprivationꢀinduced apoptosis.12,13 17βꢀ
OꢀAlkyl ethers of
estradiol exhibited increased protection of neural (HTꢀ22) cells against oxidative stress as
compared to estradiol.14 Testosterone prevent NꢀmethylꢀDꢀaspartate (NMDA)
excitotoxicity and amyloid induced neurotoxicity that are mainly involved in
pathophysiology of neurodegenerative disorders like PD and AD.15,16
16ꢀSubstituted steroids represent a novel class of heterosteroids possessing diversified
biological properties.17ꢀ19 As these compounds are derivatives of DHEA and testosterone,
which are established neuroprotective agents, we became interested in studying the
neuroprotective effects of 16ꢀarylidene steroids and their 4ꢀaza analogues for the treatment
of neurodegenerative disorders like AD, PD and multiple sclerosis. Potent activity of 16ꢀ
arylidene steroids during random screening of steroidal derivatives as neuroprotective agents
further motivated us to pursue research work in this direction.
Literature further reveals the significance of pyridyl moiety for its protective action
against the neurological disorders effecting behaviour and cognition.20 The novel 3ꢀpyridyl
ethers have therapeutic potential as cholinergic channel modulators and can be useful for the
treatment of cognitive impairments in patients with AD. The pyridyl moiety also acts as
novel analogue of choline which promotes the formation and transmission of acetylcholine
resulting in enhanced cognition and memory in patients suffering from neurodegenerative
ACS Paragon Plus Environment

Page 5 of 44
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
disorders.21 Therefore in our present work a series of 16ꢀpyridylidene steroids and their 4ꢀaza
analogues have been studied as neuroprotective agents using LPSꢀinduced animal models of
neuroinflammation.
9
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
RESULTS AND DISCUSSION
Chemical Synthesis
The synthesis of 16ꢀarylidene steroids 1aꢀd and 4ꢀazaꢀ16ꢀarylidene steroids 6aꢀh has been
carried out as shown in schemes 1 and 2. Steroids 1aꢀd were synthesized using base catalyzed
Aldol Condensation of dehydroepiandrosterone with requisite aromatic aldehyde as reported
earlier.22,23(Scheme 1)
Results of initial studies indicated significantly better neuroprotective effects with 4ꢀ
azaꢀ16ꢀarylidene analogues in comparison to their deaza counterparts, therefore 4ꢀ
azasteroidal compounds were extensively explored by introducing diversely substituted
arylidene moiety at 16ꢀposition of steroid skeleton. For the synthesis of 4ꢀazaꢀ16ꢀarylidene
steroids 6aꢀh, testosterone (2) was oxidized using permanganate/periodate solution in tert
ꢀ
butanol to obtain an intermediate secoꢀketo acid
reaction to afford 4ꢀazasteroid 4 24
3, which was further subjected to the Leukart
.
Subsequent Jone's oxidation gave 4ꢀazaꢀandrostanꢀ3,17ꢀ
dione (5), which when further subjected to base catalyzed cross Aldol condensation using
various aromatic aldehydes afforded corresponding 4ꢀazaꢀ16ꢀarylidene derivatives 6aꢀh as
depicted in scheme 2.
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 6 of 44
1
2
3
4
O
O
O
5
6
7
8
H-C Ar
CH
Ar
CH3OH/NaOH
OH
OH
9
DHEA
1a-d
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
a
b
c
d
Ar
Scheme 1. Synthesis of 16ꢀarylidene steroids 1aꢀd
OH
OH
OH
NaIO4
KMnO4
HCOOH, NH3
C6H5NO2,180-200 0C, 24 h
O
HOOC
O
O
N
2
H
3
4
CrO3,H2SO4
(CH3)2CO,15 0C
O
O
O
C H
Ar
H-C Ar
CH3OH/NaOH
O
O
N
N
H
H
5
6a-h
a
b
f
c
d
e
g
h
Ar
Scheme 2. Synthesis of 4ꢀazaꢀ16ꢀarylidene steroids 6aꢀh
ACS Paragon Plus Environment

Page 7 of 44
ACS Chemical Neuroscience
1
2
3
4
Biological Activity
5
6
7
8
Neuroinflammation is now recognised as an important pathophysiological feature of various
neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease, however
the exact relationship and mechanism is still unclear. In the current study, we have made an
effort to establish a relationship between LPS induced neuroinflammation and consequent
neurodegeneration leading to impairment of learning, memory and locomotion. It has been
reported recently that systemic LPS injection promotes amyloidogenesis through activation of
β and γ secretase, which cause rise in Aβ 1–42 levels in the cortex and hippocampus. Aβ
oligomers are the principal toxins leading to neurite breakage and neuronal death. LPS
induced Aβ 1–42 generation in both cortex and hippocampus plays a central role in the
pathogenesis of Alzheimer's disease characterized by memory and learning deficits.4
Literature also indicates that subꢀtoxic dose levels of LPS in substantia nigra (SN) induce
neurodegeneration through neuronal loss, microglial activation, and proꢀinflammatory
phenotype with increased production of cytokines that would cause destruction of
dopaminergic markers resulting in impairment of locomotor activity.25
9
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
Neurodegenerative processes initiated with activation of microglial cells may
contribute in the release of proꢀinflammatory and cytotoxic factors mainly TNFꢀα, ILꢀ1β and
finally resulting in memory impairment and motor dysfunction. The antiꢀinflammatory drugs
could be useful in the treatment of neuroinflammation and could help in the prevention of
neurodegeneration induced through so many proꢀinflammatory mediators such as TNFꢀα, ILꢀ
1β, and COXꢀ2. Non steroidal antiꢀinflammatory drugs such as ibuprofen and celecoxib
reduce Aβ levels and brain inflammation in Tg2576 AD mice possibly through the inhibition
of COX enzyme and could reduce the risk of AD.26 Since a long time glucocorticoids are
mainly used as potent antiꢀinflammatory drugs against brain inflammation and spinal cord
injury in clinical neurology. The recent reports prove that dexamethasone produce protective
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 8 of 44
1
2
3
4
5
6
7
8
effects against neuroinflammation and also block the activation of microglia as well as
inflammatory cytokines (TNFꢀα, ILꢀ1β and IFNꢀγ) and is useful against dopaminergic
degeneration in PD.25
9
DHEA and testosterone are established neurosteroids useful against various
neurodegenerative disorders such as AD, PD and multiple sclerosis. DHEA and DHEAS
protect the neurons against oxidative stress and also promote the neurogenesis in CNS.
Mainly DHEA plays a key role to reduce inflammation in brain providing neuroprotection
with improved cognitive function and memory enhancement. DHEA also potentiate
locomotor activity of hemiꢀParkinsonian monkey in MPTPꢀtreated animals model.27
Testosterone received an attractive consideration in the arena by researchers for
improvement in motor and cognition functions in PD and AD, respectively. Testosterone
prevents NMDA excitotoxicity and amyloid induced neurotoxicity. Recent studies have
proven that testosterone supplementation in aged rats decrease the glial fibrilary acidic
protein (GFAP) involved in neurodegenerative disorders through inflammatory cytokine
release and reactive oxidative stress. Testosterone related up regulation of nerve growth
factor (NGF) and its receptors play a promising role against inflammatory mediators
released after brain injury.15,28
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
In light of these observations, we screened neuroprotective effects of the DHEA and
testosterone derived 16ꢀarylideno steroids 1aꢀd and 6aꢀh in LPS induced memory impairment
and locomotor dysfunction. In the present study the administration of LPS in mice (250 µg/kg
i.p.) and rats (10 µg in 2µl of 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl ꢀ 0.0027
M; pH 7.4), intranigrally) resulted in neuroinflammation. Behavioural paradigms for
neurodegeneration including learning, memory and movement were assessed using Morris
water maze, elevated plus maze, photoactometer and block test. Celecoxib and
dexamethasone were used as standard compounds.
ACS Paragon Plus Environment

Page 9 of 44
ACS Chemical Neuroscience
1
2
3
4
Behavioural studies on mice injected with LPS intraperitoneally.
5
6
7
8
The neuroprotective effects of 16ꢀarylidene steroids 1aꢀd and 6aꢀh (5 mg/kg, i.p.) and
standard compounds celecoxib (20 mg/kg, i.p.) and dexamethasone (5 mg/kg, i.p.) on LPS
induced memory impairment and learning disability in mice were studied using Morris
water maze and elevated plus maze models.29 The results are compared with only LPS
treated and control group. The values are shown as mean ± S.E.M and results are expressed
as escape latency and TSTQ (time spent in target quadrant) in Morris water maze and % ITL
(initial transfer latency) in elevated plus maze models (Table 1).
9
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
Table 1. Neuroprotective effects of 16ꢀarylidene steroids on mice injected with LPS
intraperitoneally
Morris Water Maze
Escape Latency (s)
Elevated Plus Maze
% ITL (s)
Compound
Control
LPS
Celecoxib
TSTQ (s)
55 ± 3.4
13 ± 2.9*
44 ± 3#
40 ± 3#
35 ± 4#
32 ± 3#
40 ± 4#
38 ± 3#
37 ± 2#
38 ± 2#
42 ± 4#
36 ± 4#
38 ± 4#
39 ± 3#
37 ± 4#
36 ± 3#
41.5 ± 1.9
73.2 ± 1.6*
52.9 ± 2.4#
56.7 ± 2.5#
62.5 ± 1.8#
65.2 ± 2#
11.4 ± 2.6
91.4 ± 2.9*
24.4 ± 2.2#
31.1 ± 2.4#
37.7± 2.2#
40 ± 2.7#
Dexamethasone
1a
1b
1c
1d
6a
6b
6c
6d
6e
6f
57.9 ± 2#
30.2 ± 4.4#
33.8 ± 2.8#
35.1 ± 3.3#
33.3 ± 3.3#
28.8 ± 2.2#
35.5 ± 2.2#
33.8 ± 1.6#
32 ± 3.3#
60.3 ± 2.3#
61.6 ± 1.4#
63.5 ± 1.9#
55.7 ± 2.0#
65.1 ± 1.4#
62.8 ± 1.5#
59.7 ± 2.3#
66.0 ± 1.4#
66.6 ± 1.6#
34.4 ± 2.2#
35.5 ± 2.2#
6g
6h
*p<0.05 as compared to control; #p<0.05 as 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).
The behavioural assessment for memory impairment in mice treated with LPS
intraperitoneally depicted an increase in escape latency in Morris water maze task (from day
1 to day 5) and % ITL in elevated plus maze due to the LPS induced dysfunction in learning
and memory. Treatment with synthesized derivatives 1aꢀd and 6aꢀh (5mg/kg) resulted in
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 10 of 44
1
2
3
4
5
6
7
8
9
significantly reduced escape latency and % ITL. In probe trial, significantly decreased TSTQ
in LPS treated mice was appreciably reversed on treatment with 16ꢀarylideno steroids 1aꢀd
and 6aꢀh. The data obtained from elevated plus maze model further substantiated the findings
of Morris water maze test as treatment with steroidal derivatives considerably reduced the
otherwise increased % ITL, after 24 h of training period in mice. The 4ꢀpyridylidene steroids
1c and 6c emerged as the most potent compounds and significantly improved the learning and
memory functions at 5 mg/kg, which are comparable with standard drugs celecoxib (20
mg/kg) and better than dexamethasone (5 mg/kg). The effects of steroids 1c and 6c on the
behavioural pattern in mice treated with LPS intraperitoneally have been represented in
Figure 1.
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
100
#
Control
LPS
80
60
40
20
0
*
Celecoxib
#
#
#
#
*
Dexamethasone
#
# # #
1c
6c
# #
#
#
#
*
Figure 1. Behavioural observations for memory and learning deficits in mice treated with
LPS intraperitoneally for compounds 1c and 6c. *p<0.05 as compared to control; #p<0.05 as
compared to LPS (n=5). Oneꢀ way 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.
Behavioural studies on rats injected with LPS intranigrally
The effects of 16ꢀarylidene steroids 1aꢀd and 6aꢀh (2 mg/kg, i.p.) and standard drugs
celecoxib (20 mg/kg, i.p.) and dexamethasone (2 mg/kg, i.p.) on behavioural pattern of
intranigrally LPSꢀtreated rats with respect to locomotor activity and cognition were studied
using actophotometer, elevated plus maze and block test.25,30 The results are compared with
ACS Paragon Plus Environment

Page 11 of 44
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
sham, LPS and vehicle control group. The values are shown as mean ± S.E.M and results are
expressed as ambulation and rearing in locomotor activity, % ITL in elevated plus maze and
degree of catatonia using block test (Table 2).
9
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
Table 2. Neuroprotective effects of 16ꢀarylidene steroids in rats injected with LPS
intranigrally
Locomotor Activity
Ambulation Rearing
Elevated Plus
Maze
% ITL (s)
Catatonic
Response
Degree of
Compound
(min)
(min)
Catatonia
(score)
300 ± 2.5
56.3 ± 3.8*
55.8 ± 4.6#
215 ± 3.8$
200± 3.9$
145 ± 2.0
16.3 ± 3.3*
16.0 ± 2.3#
73.3 ± 2.0$
63± 3.0$
16.6 ± 3.2
83.3 ± 4.4*
82.9 ± 3.1#
31.3 ± 2.0$
37.1± 2.2$
48.3± 3.0$
51.6 ± 4$
0
Sham
LPS + Vehicle
LPS
Celecoxib
Dexamethasone
1.2 ± 0.02*
1.22 ± 0.03#
0.21 ± 0.04$
0.33 ± 0.05$
0.52 ± 0.03$
0.57 ± 0.06$
0.45 ± 0.05$
0.48 ± 0.04$
0.49 ± 0.04$
0.52 ± 0.06$
0.42 ± 0.04$
0.50 ± 0.04$
0.49 ± 0.04$
0.47 ± 0.03$
0.52 ± 0.04$
0.54 ± 0.06$
181.5 ± 2.9$
164 ± 4$
53± 3.2$
1a
1b
1c
1d
6a
6b
6c
6d
6e
6f
47 ± 4$
194.5 ± 4.4$
190 ± 3.5$
192 ± 3.0$
189 ± 2.6$
202.3 ± 4.3$
187 ± 4.3$
193.3 ± 3.9$
200.6 ± 4.1$
183.3 ± 3.4$
176.6 ± 3.5$
63 ± 3.4$
57 ± 2.8$
49.3 ± 4.2$
56 ± 2.7$
67 ± 3.6$
55 ± 3.4$
57± 3.0$
39.7 ± 3.0$
44.6 ± 3.3$
46 ± 2$
48.9 ± 2.9$
37.1 ± 3.3$
52 ± 2.9$
47 ± 3.3$
60 ± 2.8$
53.6 ± 3.2$
50.6 ± 3.2$
42.3 ± 3.0$
51.6 ± 3.2$
55.6 ± 3.4$
6g
6h
*p<0.05 as compared to sham; #p<0.05 as compared to LPS + vehicle; $p<0.05 as
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).
The behavioural assessment for memory and locomotor impairment in rats treated
with LPS intranigrally indicated a significant decline in the locomotor activity in LPS treated
and vehicle control group rats as compared to sham after 21 days. The catatonic score and %
ITL also increased notably in LPS and control group analysed at 21st day of experimental
protocol. The group treated with daily i.p. injections of steroidal derivatives 1aꢀd and 6aꢀh (2
mg/kg) for 21 days showed improvement in the locomotor activity with significant decrease
in degree of catatonia and % ITL as compared to LPS and LPS + vehicle group in the
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 12 of 44
1
2
3
4
5
6
7
8
9
designed period of experimental protocol. The significant improvement in locomotion,
learning ability and catatonia after treatment with compounds 1c and 6c is found to be better
than dexamethasone (2 mg/kg) and comparable to celecoxib (20 mg/kg) as shown in Figures
2 and 3. The improvement in locomotion, catatonia and learning abilities on treatment with 4ꢀ
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
azaꢀ16ꢀarylidiene 6c was found to be more than 4ꢀdeazaꢀ16ꢀarylidiene steroid 1c
.
400
Sham
LPS + Vehicle
300
200
100
0
LPS
$
Celecoxib
$
$ $
Dexamethasone
*
1c
6c
#
$
*
$ $ $
#
*
$ $
$
$
#
$
$
Figure 2. Behavioural changes with compounds 1c and 6c for locomotion and memory
deficits in rats treated with LPS intranigrally. * p<0.05 as compared to sham; #p<0.05 as
compared to LPS + vehicle; $p<0.05 as 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)
1.5
*
1.0
#
#
#
0.5
0.0
#
Figure 3. Behavioural changes with compounds 1c and 6c with respect to catatonic response
in rats treated with LPS intranigrally *p<0.05 as compare to LPS + vehicle; #p<0.05 as
compare 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)
ACS Paragon Plus Environment

Page 13 of 44
ACS Chemical Neuroscience
1
2
3
4
Biochemical estimations in brain supernatants of mice and rats
5
6
7
8
Acetylcholine is a major parasympathetic neurotransmitter that inhibits LPSꢀinduced
production of proinflammatory cytokines such as ILꢀ1, TNFꢀα from macrophages and
activated microglia. The neurotransmitter acetylcholine is degraded by the enzyme
acetylcholinesterase leading to memory impairment. The significant increase in the AChE
activity by LPS treatment results in decreased cholinergic activity. In addition, an upꢀ
regulation of proinflammatory cytokines (TNFꢀα and ILꢀ1β) on injecting LPS mainly cause
neurotoxic effects leading to neuron cell death.31 LPS is also suggested to increase iNOS
activation and thereby nitric oxide (NO) production. NO can damage DNA and irreversibly
modifies proteins such as tyrosine nitration and thiol oxidation, which are common
pathogenic mechanisms in several neurodegenerative diseases. LPS administration is also
associated with increased generation of reactive oxygen species (ROS) from highly
metabolically active mitochondria which further stimulate toll like receptor signalling
components to propagate. These ROS such as superoxide anion, hydrogen peroxide and nitric
oxide exert harmful effects on brain homeostasis. Recent studies have revealed that excessive
production and accumulation of NO after LPS administration lead to neurodegeneration. ROS
released from activated microglia or astrocytes may interact with lipid components of cellular
membrane, initiating lipid peroxidation that results in the breakdown of the lipid constituents
into highly reactive byꢀproducts.32 Under LPS induced oxidative stress the mismatch between
the production of free radicals and the ability to scavenge them take place.
Intracerebroventricular administration of LPS causes significant decrease in the activity of
antioxidants such as reduced glutathione, superoxide dismutase and catalase in cerebral
cortex and hippocampus regions of brain.31
9
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
In the current study, the biochemical parameters acetylcholinesterase, lipid
peroxidation, nitrite and oxidative stress were estimated in the brain supernatant of mice
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 14 of 44
1
2
3
4
5
6
7
8
9
(i.p. LPS) and rats (i.g. LPS) treated with 16ꢀarylidene steroids 1aꢀd and 6aꢀh along with
standard drugs celecoxib and dexamethasone as reported earlier.33 Results are expressed as
µmoles of AChE/min/mg protein in AChE Assay, n moles of MDA/mg protein in LPO
Assay, Nitrite concentration (µg/ml) in Nitrite Assay, n moles of GSH/mg protein in GSH
Assay, SOD units/mg protein in SOD Assay and µmoles of H2O2 decomposed/min/mg
protein in Catalase Assay. The values are shown as mean ± S.E.M for mice and rat brain
supernatants in Tables 3 and 4, respectively.
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
A significant increase in AchE, malondialdehyde and nitrite levels along with marked
reduction in reduced glutathione and enzymatic activity of superoxide dismutase and catalase
was observed in the brain supernatants of LPS treated mice and rats. Of all the compounds,
the treatment with 16ꢀ(4ꢀpyridylidene)steroid 1c and its 4ꢀaza analogue 6c helped to restore
maximum normalcy of the biochemical mediators along with marked inhibition of reactive
oxygen and nitrogen species involved in mechanism of neuroinflammation. The biochemical
estimation of the acetylcholinesterase, lipid peroxidation, nitrite and oxidative stress in mice
and rat brain homogenates on treatment with most active compounds 1c and 6c have been
illustrated in Figures 4 and 5.
Inhibition of TNFꢀα
TNFꢀα plays an important role in progression of neuroinflammation as is observed
from the various studies reported in literature. Therefore the estimation of TNFꢀα activity has
been carried out in the brain serum of rats treated with 16ꢀarylidene steroids 1aꢀd and 6aꢀh
and compared with standards celecoxib and dexamethasone using TNFꢀα immunoassay kit.34
The inhibition of TNFꢀα is expressed in pg/mg protein and values are shown as mean ±
S.E.M (Table 5).
ACS Paragon Plus Environment

Page 15 of 44
ACS Chemical Neuroscience
1
2
3
4
5
Table 3. Estimation of biochemical parameters in brain supernatant of mice treated with LPS intraperitonially
6
AchE Assay
µmoles of
LPO Assay
n moles of MDA/mg Nitrite conc.(µg/ml)
protein
Nitrite Assay
GSH Assay
µmoles of GSH/mg
protein
SOD Assay
SOD units/ mg
protein
Catalase Assay
Catalase activity/min
7
8
9
10
11
12
13
14
15 1a
16
17
18
1d
19
Compound
AchE/min/mg protein
0.0015 ± 0.0003
0.0051 ± 0.0003*
0.0026 ± 0.0003#
0.0030 ± 0.0004#
0.0036 ± 0.0005#
0.0039 ± 0.0004#
0.0031 ± 0.0003#
0.0034 ± 0.0004#
0.0034 ± 0.0006#
0.0036 ± 0.0004#
0.0028 ± 0.0004#
0.0033 ± 0.0004#
0.0035 ± 0.0006#
0.0035 ± 0.0003#
0.0034 ± 0.0004#
0.0035 ± 0.0003#
1.9 ± 0.1
7.5 ± 0.3*
3.0 ± 0.1#
3.5 ± 0.1#
3.9 ± 0.2#
4.3 ± 0.2#
3.5 ± 0.2#
3.7 ± 0.2#
3.8 ± 0.1#
4.0 ± 0.2#
3.3 ± 0.2#
3.6 ± 0.2#
3.9 ± 0.2#
3.8 ± 0.2#
3.9 ± 0.1#
4.1 ± 0.1#
287.5 ± 35
0.0078 ± 0.0003
0.0034 ± 0.0005*
0.0061 ± 0.0005#
0.0056 ± 0.0003#
0.0048 ± 0.0004#
0.0044 ± 0.0002#
0.0052 ± 0.0004#
0.0050 ± 0.0005#
0.0050 ± 0.0006#
0.0049 ± 0.0005#
0.0054 ± 0.0004#
0.0051 ± 0.0003#
0.0047 ± 0.0003#
0.0049 ± 0.0003#
0.0047 ± 0.0003#
0.0045 ± 0.0003#
6.5 ± 1.8
4.9 ± 0.3
1.1 ± 0.4*
3.6 ± 0.3#
3.2 ± 0.6#
2.8 ± 0.8#
2.5 ± 0.6#
3.1 ± 0.5#
3.0 ± 0.4#
2.9 ± 0.6#
2.7 ± 0.4#
3.2 ± 0.7#
3.0 ± 0.4#
2.9 ± 0.4#
2.9 ± 0.6#
2.8 ± 0.7#
2.7 ± 0.5#
Control
LPS
Celecoxib
Dexamethasone
730.1 ± 28*
1.5 ± 0.7*
4.9 ± 1.6#
4.3 ± 1.3#
3.4 ± 1.1#
3.0 ± 1.2#
3.9 ± 1.4#
3.7 ± 1.6#
3.7 ± 1.5#
3.5 ± 1.6#
4.1 ± 1.4#
3.8 ± 1.3#
3.4 ± 1.8#
3.5 ± 1.1#
3.5 ± 1.6#
3.3 ± 1.5#
434.4 ± 31.1#
467.1 ± 32.5#
537.8 ± 29.8#
563.5 ± 28.9#
503.6 ± 30.5#
513.4 ± 28.4#
525.8 ± 32.8#
548.2 ± 29.5#
485.8 ± 33.8#
510.4 ± 32.7#
539.7 ± 35#
1b
1c
6a
20
6b
6c
6d
6e
6f
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
531.2 ± 30.5#
535.2 ± 31.3#
544.5 ± 34#
6g
6h
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 Catalase activity/min in Catalase Assay .
The values are shown as mean ± S.E.M. * p<0.05 as compared to control; #p<0.05 as compared to LPS (n=5 per group). Oneꢀ way ANOVA
followed by Tukey’s test was employed to find out the interꢀgroup variation and values are considered statistically significant ( p<0.001).
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 16 of 44
1
2
3
4
5
6
Table 4. Estimation of biochemical parameters in brain supernatants of rats treated with LPS intranigrally
AChE Assay
LPO Assay
Nitrite Assay
GSH Assay
SOD Assay
Catalase Assay
7
8
9
µmoles of
AChE/min/mg protein
0.0032 ± 0.0002
0.0096 ± 0.0005*
0.0097 ± 0.0003#
0.0055 ± 0.0003$
0.0062 ± 0.0007$
0.0068 ± 0.0003$
0.0070 ± 0.0005$
n moles of MDA/mg Nitrite conc.(µg/ml)
protein
µmoles of GSH/mg SOD units/ mg protein Catalase activity/min
protein
Compound
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
3.2 ± 0.2
9.5 ± 0.4*
9.4 ± 0.6#
5.4 ± 0.4$
6.0 ± 0.3$
6.7 ± 0.5$
7.0 ± 0.3$
6.3 ± 0.4$
6.5 ± 0.6$
6.5 ± 0.6$
6.8 ± 0.5$
6.1 ± 0.7$
6.4 ± 0.6$
6.7 ± 0.5$
6.6 ± 0.5$
6.8 ± 0.6$
6.9 ± 0.5$
419.5 ± 26.1
914.1 ± 25.2*
915.8 ± 25.4#
523.1 ± 20.1$
565.8 ± 27.3$
640.5 ± 22.8$
673.8 ± 22.9$
595.8 ± 24.9$
619.4 ± 27.4$
623.1 ± 26.8$
649.3 ± 25.5$
578.5 ± 23.8$
604.5 ± 22.7$
649.5 ± 20.5$
640.1 ± 25.5$
655.3 ± 25.3$
664.1 ± 28.3$
0.048 ± 0.002
0.021 ± 0.001*
0.020 ± 0.001#
0.036 ± 0.003$
0.033 ± 0.001$
0.028 ± 0.003$
0.026 ± 0.002$
0.033 ± 0.002$
0.030 ± 0.004$
0.030 ± 0.003$
0.028 ± 0.005$
0.035 ± 0.002$
0.031 ± 0.003$
0.029 ± 0.002$
0.030 ± 0.004$
0.028 ± 0.004$
0.027 ± 0.004$
9.8 ± 1.4
2.7 ± 1.0*
2.8 ± 1.1#
6.6 ± 1.5$
6.2 ± 1.7$
5.2 ± 1.3$
4.9 ± 1.3$
5.9 ± 1.9$
5.6 ± 1.2$
5.4 ± 1.0$
5.2 ± 1.6$
6.2 ± 1.5$
5.7 ± 1.3$
5.3 ± 1.8$
5.5 ± 1.3$
5.1 ± 1.4$
5.0 ± 1.3$
7.0 ± 0.4
2.2 ± 0.4*
2.1 ± 0.3#
5.5 ± 0.5$
5.1 ± 0.3$
4.3 ± 0.5$
4.0 ± 0.4$
4.9 ± 0.3$
4.6 ± 0.8$
4.5 ± 0.6$
4.3 ± 0.5$
5.1 ± 0.6$
4.7 ± 0.7$
4.4 ± 0.3$
4.5 ± 0.3$
4.4 ± 0.3$
4.2 ± 0.4$
Sham
LPS + Vehicle
LPS
Celecoxib
Dexamethasone
1a
1b
0.0063 ± 0.0003$
0.0065 ± 0.0004$
0.0066 ± 0.0007$
0.0068 ± 0.0004$
0.0059 ± 0.0003$
0.0064 ± 0.0002$
0.0068 ± 0.0005$
0.0067 ± 0.0003$
0.0068 ± 0.0005$
0.0070 ± 0.0004$
1c
1d
6a
6b
6c
6d
6e
6f
6g
6h
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 Catalase activity/min in Catalase Assay .
The values are shown as mean ± S.E.M. * p<0.05 as compared to sham; #p<0.05 as compared to LPS+ vehicle; $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 values were considered statistically
significant (p<0.001).
ACS Paragon Plus Environment

Page 17 of 44
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
(A) AChE assay
(B) LPO assay
10
8
0.006
0.004
0.002
0.000
*
*
#
6
#
#
#
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
#
#
#
4
#
2
0
(C) Nitrite assay
(D) GSH assay
0.010
800
600
400
200
0
*
0.008
0.006
0.004
0.002
0.000
#
#
#
#
#
#
#
#
*
(F) Catalase assay
(E) SOD assay
6
4
2
0
8
6
4
2
0
#
#
#
#
#
#
#
#
*
*
Figure 4. Biochemical estimations in brain supernatant of mice treated with compounds 1a
and 6c. *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 variations and
values were considered statistically significant (p<0.001).
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 18 of 44
1
2
3
(A) AChE assay
(B) LPO assay
0.015
0.010
0.005
0.000
15
10
5
4
5
6
7
8
9
#
*
#
*
$
$
$
$
$
$
$
$
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
0
(C) Nitrite assay
(D) GSH assay
0.06
0.04
0.02
0.00
#
*
1000
800
600
400
200
0
$
$
$
$
$
$
$
$
*
#
(E) SOD assay
(F) Catalase assay
15
10
5
$
$
$
$
#
*
0
Figure 5. Biochemical estimation in brain supernatant of rats treated with compounds 1a
and 6c. *p<0.05 as compared to sham; #p<0.05 as compared to LPS + vehicle; $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 values were considered statistically significant (p<0.001).
ACS Paragon Plus Environment

Page 19 of 44
ACS Chemical Neuroscience
1
2
3
4
5
Table 5. Estimation of TNFꢀɑ levels in brain serum of rats
Compound
TNFꢀɑ (pg/mg protein)
6
7
8
9
48.4 ± 1.5
368.8 ± 1.9*
370.2 ± 1.6#
68.2 ± 1.1$
89.6 ± 2.0$
126.8 ± 3.0$
139.2 ± 2.9$
93.6 ± 1.4$
107.2 ± 2.4$
98.8 ± 3.1$
131.2 ± 1.7$
88.6 ± 1.8$
104.8 ± 1.5$
123.2 ± 2.2$
100.6 ± 1.6$
135.2 ± 1.9$
114 ± 1.9$
Sham
LPS + Vehicle
LPS
Celecoxib
Dexamethasone
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
1a
1b
1c
1d
6a
6b
6c
6d
6e
6f
6g
6h
* p<0.05 as compared to sham; #p<0.05 as compared to LPS + vehicle; $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 values were considered statistically significant (p<0.001).
Interestingly, a significant elevation in the levels of TNFꢀα in the brain serum of rats
treated with LPS + vehicle and LPS alone was observed. This indicates that enhancement of
neuroinflammation results into neurodegeneration. LPS significantly increases the magnitude
as well as duration of central and peripheral proꢀinflammatory cytokines (TNFꢀα and ILꢀ1β)
suggesting a role of cytokines in LPSꢀinduced neuroinflammation.31 The treatment with 16ꢀ
arylidene steroids 1aꢀd and 4ꢀazaꢀ16ꢀarylidene steroids 6aꢀh resulted in decreased TNFꢀα
levels in the brains of LPS injected rats. The 16ꢀ(4ꢀpyridylidene)steroid 1c and its 4ꢀaza
counterpart 6c were found to be better than standard drugs in reducing TNFꢀα levels, the
later being more active than the deaza analogue 1c as shown in Figure 6.
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 20 of 44
1
2
3
4
#
400
*
5
6
7
8
300
200
100
0
9
$
$
$
$
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
Figure 6. TNFꢀα levels in the brain serum of rats. *p<0.05 as compared to sham; #p<0.05
as compared to LPS + vehicle; $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 values were
considered statistically significant (p<0.001).
Docking Studies Analysis
TNFꢀα is one of the key elements involved in the molecular mechanism of
neuroinflammation.4ꢀ6 Observing the reduced TNFꢀα levels on treatment with the 16ꢀ
arylidene steroids, it was thought worthwhile to study the three dimensional interactions of
the compounds with the receptor using computational docking studies. The studies might be
helpful in acquiring a tentative idea about one of the possible mechanism of the 16ꢀarylidene
steroidal derivatives as neuroprotective agents. Steroids 1aꢀd and 6aꢀh were docked against
crystal structure of TNFꢀα and compared with reference compounds celecoxib and
dexamethasone. The docking was done on the active site of 10 Å defined around center of coꢀ
crystallized ligand composed of trifluoromethylphenyl indole and dimethyl chromone
moieties linked by a dimethylamine spacer. The docking protocol was validated by reꢀ
docking of the coꢀcrystallized ligand back into the protein. The glide scores of the coꢀ
crystallized ligand, standard drugs (celecoxib and dexamethasone) and synthesized steroidal
derivatives are given in Table 6. The 3D and 2D interactions of most active compounds 1c
and 6c were compared with coꢀcrystallized ligand and are represented in Figures 7 and 8,
respectively.
ACS Paragon Plus Environment

Page 21 of 44
ACS Chemical Neuroscience
1
2
3
4
5
Table 6: Glide score of coꢀcrystallized ligand, celecoxib, dexamethasone and steroidal
derivatives on the active site of TNFꢀα.
6
7
8
9
Compound
Coꢀcrystallized ligand
Glide Score
ꢀ4.302
ꢀ4.616
ꢀ4.713
ꢀ6.145
ꢀ6.124
ꢀ4.639
ꢀ6.557
ꢀ4.388
ꢀ4.796
ꢀ3.723
ꢀ4.602
ꢀ4.774
ꢀ4.086
ꢀ4.637
ꢀ3.942
Celecoxib
Dexamethasone
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
1a
1b
1c
1d
6a
6b
6c
6d
6e
6f
6g
6h
The selected TNFꢀα protein is a dimer consisting of 18 contact residues, of which ten
are from chain A (Lys57, Lys58, Tyr59, Ser60, Gly61, Tyr119, Lys120, Gly121, Gly122, and
Tyr151). The remaining eight are a subset of these residues from chain B (Lys57, Tyr59,
Ser60, Gly61, Tyr119, Lys120, Tyr121 and Tyr151). The coꢀcrystallized ligand consisting of
trifluoromethylphenylindole and dimethyl chromone moieties folded back upon each other
and the interactions were largely hydrophobic in nature. Coꢀcrystallized ligand produced
intraꢀmolecular π–π stacking between the aromatic moieties. Six tyrosine residues (Tyr59,
Tyr151 and Tyr119 residues from both chain A and chain B) provide solvent accessible
surface for interacting with the ligand. Tyr119 was notable in being located close to the three
fold symmetry axis of the TNFꢀα and in making contact with coꢀcrystallized ligand.35,36
Steroids 1c and 6a showed the similar interactions with tyrosine residues Tyr119 as coꢀ
crystallized ligand, which are essential for the inhibition TNFꢀα. 4ꢀAzaꢀ16ꢀ(4ꢀpyridylidene)
steroid 6c showed the πꢀπ stacking with the tyrosine residues Tyr151 responsible for the
inhibition of TNFꢀα. 16ꢀarylidene steroids 1d, 6d, 6e and 6h also depicted the πꢀπ stacking
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 22 of 44
1
2
3
4
5
6
7
8
9
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
Coꢀcrystallized ligand
1c
6c
Figure 7. The 3D interaction diagram of coꢀcrystallized ligand, steroidal derivatives 1c and
6c at active site of TNFꢀα
ACS Paragon Plus Environment

Page 23 of 44
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
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
Coꢀcrystallized ligand
1c
6c
Figure 8. The 2D interaction diagram of coꢀcrystallized ligand, steroidal derivatives 1c and
6c at active site of TNFꢀα.
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 24 of 44
1
2
3
4
5
6
7
8
9
and hydrogen bonding interactions with tyrosine residues Tyr151 and Tyr 59, which are also
required for the inhibition of TNFꢀα. Docking analysis further revealed that the solvent
accessible surface interaction with tyrosine residues from both chain is important for TNFꢀ
inhibitory activity. Remaining compounds including standard (celecoxib and dexamethasone)
were not able to bind with the active amino acids (Tyr59, Tyr151 and Tyr 119.) essential for
TNFꢀα inhibition. The observed glide score of most active compounds 1c and 6aꢀh were
comparable to that of parent coꢀcrystallized ligand. It was observed that 4ꢀazasteroids are
more active against TNFꢀα, the main culprit of neuroinflammation. However, besides TNFꢀα
there are many other factors such as interleukin 1β, 6 and 2, NO, O2ꢀ and several inflammatory
mediators, which might be involved in the progression of neuroinflammation.6
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
Overall 16ꢀarylidene steroids 1aꢀd and 6aꢀh significantly improved learning, memory
and movement function and markedly suppressed LPS induced oxidative stress, nitrosative
stress, acetylcholinesterase activity and enhanced TNFꢀα levels. In general, 4ꢀazasteroids 6aꢀ
h displayed superior potential as neuroprotective agents than their corresponding deaza
analogues 1aꢀd. Of all, introduction of 4ꢀpyridylidene ring at 16ꢀposition of the steroid
nucleus seems to be the most appropriate substitution for good neuroprotective activity as 16ꢀ
(4ꢀpyridylidene) steroid 1c and its 4ꢀaza analogue 6c displayed highest potency and produced
effects comparable to the standard drugs.
CONCLUSION
On the basis of behavioural, biochemical and molecular studies, it is concluded that that 16ꢀ
arylidene steroids represent a new class of neuroprotective agents for the treatment of
Alzheimer’s and Parkinson’s diseases. All the synthesized derivatives 1aꢀd and 6aꢀh prevent
the progression of LPSꢀinduced neuroinflammation and subsequent neurodegeneration
possibly by inhibiting TNFꢀα or by restraining the actions of inflammatory mediators. A
significant improvement in LPSꢀinduced learning, memory and movement deficits in animal
ACS Paragon Plus Environment

Page 25 of 44
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
models and considerable suppression of LPS induced oxidative stress, nitrosative stress,
acetylcholinesterase activity and enhanced TNFꢀα levels was noticed. Among the 16ꢀ
arylidene steroids, 16ꢀ(4ꢀpyridylidene) steroid 1c and its 4ꢀaza analogue 6c displayed highest
potency and produced neuroprotective effects better than standard drugs celecoxib and
dexamethasone. The studies indicate that the 16ꢀaryldiene steroids should be extensively
explored as promising drug candidates for the treatment of Alzheimer's and Parkinson’s
diseases consequent of neuroinflammatory mechanisms. The findings of this study may help
to develop a novel and effective therapeutic intervention against neuroinflammation and
neurodegenerative conditions.
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
METHODS
Chemistry
General. Melting points were determined on a Veego melting point apparatus and are
uncorrected. IR (wave numbers in cmꢀ1) spectra are recorded on Perkin–Elmer spectrum RX
1
1, FTꢀIR spectrophotometer models using KBr pellets. H NMR, 13C NMR spectra were
recorded on Bruker ACꢀ 300F, 300 MHz using deuterated chloroform (CDCl3) or deuterated
dimethylsulfoxide (DMSOꢀd6) containing tetramethylsilane as internal standard (chemical
shifts in ppm). Mass spectra were recorded on a Vgꢀ11ꢀ250J 70 S model (VG Analytical Ltd.,
Manchester, England). Elemental analyses were carried out on a Perkin Elmerꢀ2400 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. The compounds were purified using flash chromatograph (
Biotage, IsoleraTM system, Sweden)
17βꢀHydroxyꢀ5ꢀoxoꢀ3,5ꢀsecoꢀ4ꢀnorandrostanꢀ3ꢀoic acid (3), 3ꢀOxoꢀ4ꢀazaꢀandrostanꢀ17βꢀol
(4) and 4ꢀazaꢀandrostanꢀ3,17ꢀdione (5) were prepared using a reported procedure.24
General procedure for the preparation of 16ꢀarylidene steroids 1a-d
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 26 of 44
1
2
3
4
5
6
7
8
9
A mixture of dehydroepiandrosterone (5.20 mmol), the appropriate aldehyde and sodium
hydroxide (2.25 g, 56.25 mmol) in methanol (40 ml) was stirred at room temperature for 2 h
until the reaction was completed (monitored by TLC). Cold water was added to the reaction
mixture and the precipitate obtained was filtered, washed with water, dried and crystallized
from methanol to obtain corresponding compounds 1aꢀd.22,23
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
16ꢀ(2ꢀPyridylmethylene)ꢀ17ꢀoxoꢀ5ꢀandrostenꢀ3βꢀol
(1a). Yield: 70%. White colour
crystalline compound, mp 205ꢀ207 °C, mp lit.22 205ꢀ210 °C.
16ꢀ(3ꢀPyridylmethylene)ꢀ17ꢀoxoꢀ5ꢀandrostenꢀ3βꢀol
(1b). Yield: 85%. White colour
crystalline compound. mp 260ꢀ262 °C, mp lit.22 260ꢀ264 °C.
16ꢀ(4ꢀPyridylmethylene)ꢀ17ꢀoxoꢀ5ꢀandrostenꢀ3βꢀol
(1c). Yield: 88%. Yellow colour
crystalline compound. mp 219ꢀ221 °C, mp lit.23 220ꢀ224 °C.
16ꢀ(4ꢀMethoxybenzylidene)ꢀ17ꢀoxoꢀ5ꢀandrostenꢀ3βꢀol
(1d). Yield: 78%. Shining white colour
crystalline compound. mpꢀ 220ꢀ221 °C, mp lit.22 220 °C.
General procedure for the preparation of 4ꢀazaꢀ16ꢀarylidene steroids 6a-d
A mixture of 4ꢀazaandrostanꢀ3,17ꢀdione (0.5 g, 1.72 mmol), the requisite aldehyde and
sodium hydroxide (2.25 g, 56.25 mmol) in methanol (40 ml) was stirred at room temperature
for 2 h until the reaction was completed (monitored by TLC). Cold water was added to the
reaction mixture and the precipitate obtained was filtered, washed with water, dried and
crystallized from methanol to obtain corresponding 4ꢀazaꢀ16ꢀbenzylidene steroids 6aꢀh.
16ꢀ(2ꢀPyridinylmethylene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione
(6a). Yield: 62%. Dark brown colour
compound. mp 305ꢀ307 °C. FTꢀIR (KBr): 3374.01 (NꢀH), 2934.9 (CꢀH, aliphatic), 1715
(C=O), 1654.76 (CONH), 1365.86, 1092.45 (CꢀN) cmꢀ1. 1H NMR: (400 MHz, DMSOꢀd6
0.62 (s, 3H, 18ꢀC 3), 0.65 (s, 3H, 19ꢀC 3), 2.98 (brs, 1H, 5 ), 7.06 (s, 1H, vinylicꢀ
7.11ꢀ7.14 (m, 1H, 5ꢀC , pyridine) 7.48 (d, o = 7.36 Hz, 1H, 6ꢀC , pyridine), 7.64 (t, Jo
)
δ
),
=
H
H
αꢀ
H
H
H
,
J
H
13
6.8 Hz, 1H, 4ꢀC
H
, pyridine), 8.11 (s, 1H, NꢀH), 8.48 ( s, 1H, 3ꢀCH, pyridine). C NMR:
ACS Paragon Plus Environment

Page 27 of 44
ACS Chemical Neuroscience
1
2
3
4
(400 MHz, DMSOꢀd6
)
δ
11.06 (CH3), 14.5 (CH3), 20.0 (CH2), 26.6 (CH2), 28.1 (CH2), 28.4
5
6
7
8
(CH2), 29.1 (CH2), 31.0 (CH2), 32.7 (CH2), 33.6 (CH), 35.1 (C), 47.1 (C), 47.9 (CH), 50.5
(CH), 59.6 (CH), 123.3 (C=CꢀH), 126.7 (ArCH), 130.4 (ArCH), 136.8 (ArCH), 139.7 (C=Cꢀ
H), 149.9 (ArCH), 154.1 (ArC), 170.2 (C=O), 208.8 (C=O). MS: m/z 379.31 [M+H]+. Anal.
Calcd for C24H30N2O2: C, 76.16; H, 7.99; N, 7.40. Found: C, 76.38; H, 7.73; N, 7.59.
9
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
16ꢀ(3ꢀPyridinylmethylene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6b). Yield 73%. Light brown colour
compound. mp 298ꢀ300 °C. FTꢀIR (KBr): 3379.25 (NꢀH), 2936.12 (CꢀH, aliphatic), 1715.44
1
(C=O), 1659.52 (CONH), 1363.15, 1092.03 (CꢀN) cmꢀ1. H NMR: (400 MHz, DMSOꢀd6
)
δ
0.90 (s, 3H, 18ꢀC
(brs, 1H, 6ꢀC , pyridine), 7.43ꢀ7.46 (m, 1H, 5ꢀC
8.55 (d, m = 3.72 Hz, 1H, 4ꢀC , pyridine), 8.78 (d,
(400 MHz, DMSOꢀ 6): δ 11.07 (CH3), 14.1 (CH3), 20.0 (CH2), 26.0 (CH2), 28.0 (CH2), 28.4
H
3), 1.00 (s, 3H, 19ꢀC
H
3), 3.20 (s, 1H, 5
, pyridine), 8.00 (d, = 8.04 Hz, 1H, Nꢀ
m = 1.04, 1H, 2ꢀCH
, pyridine). 13C NMR
αꢀH), 7.22 (s, 1H, vinylicꢀH), 7.30
H
H
J
H),
J
H
J
d
(CH2), 28.6 (CH2), 31.0 (CH2), 32.7 (CH2), 33.6 (CH), 35.1 (C), 47.1 (C), 48.1 (CH), 50.5
(CH), 59.6 (CH), 123.8 (C=CꢀH), 128.4 (ArCH), 130.9 (ArC), 136.6 (ArCH), 138.3 (C=Cꢀ
H), 149. (ArCH), 151.2 (ArCH), 170.2 (C=O ), 207.9 (C=O ). MS: m/z 379.31 [M+H]+.
Anal. Calcd for C24H30N2O2: C, 76.16; H, 7.99; N, 7.40. Found: C, 75.90; H, 8.17; N, 7.28.
16ꢀ(4ꢀPyridinylmethylene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6c). Yield: 65%. White colour
compound. mp 283ꢀ285 °C. FTꢀIR (KBr): 3363.12 (NꢀH), 2935.88 (CꢀH, aliphatic), 1716.84
1
(C=O), 1652.36 (COONH), 1363.5, 1016.21 (CꢀN) cmꢀ1. H NMR (400 MHz, DMSOꢀd6
)
δ
0.97 (s, 3H, 18ꢀC
H
3), 1.05 (s, 3H, 19ꢀC
H
3), 3.27 (s, 1H, 5
α
ꢀ
H
), 7.49 (s, 1H, vinylicꢀ
, pyridine), 9.0 (d, o=6.28 Hz,
11.06 (CH3), 14.09
H), 8.12ꢀ
8.16 (m, 1H, NꢀH), 8.29 (d,
Jo= 6.28 Hz, 2H, 2ꢀC
H
and 6ꢀC
H
J
2H, 3ꢀC
H
and 5ꢀC
H
, pyridine). 13C NMR (400 MHz, DMSOꢀd6
) δ
(CH3), 19.9 (CH2), 26.0 (CH2), 28.03 (CH2), 28.4 (CH2), 28.6 (CH2), 30.9 (CH2), 32.7
(CH2), 33.6 (CH), 35.1 (C), 47.2 (C), 47.9 (CH), 50.5 (CH), 59.6 (CH), 124.1 (C=CꢀH),
129.09 (2xArCH), 140.8 (C=CꢀH), 142.1 (ArC), 150.09 (2xArCH), 170.1 (C=O) and 207.9
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 28 of 44
1
2
3
4
(C=O). MS: m/z 379.31 [M+H]+. Anal. Calcd for C24H30N2O2: C, 76.16; H, 7.99; N, 7.40.
5
6
Found: C, 76.29; H, 8.22; N, 7.33.
7
8
16ꢀ(4ꢀMethoxybenzylidene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6d). Yield: 55%. Yellow colour
9
compound. mp 268ꢀ270 °C. FTꢀIR (KBr): 3329.68 (NꢀH), 2935.98 (CꢀH, aliphatic), 1706.94
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
1
(C=O), 1657.47 (CONH), 1254.27, 1174.69, 1025.65 (CꢀN) cmꢀ1. H NMR: (400 MHz,
DMSOꢀd6
)
δ
0.84 (s, 3H, 18ꢀC
3), 7.02 (d,
H
3), 0.86 (s, 3H, 19ꢀC
H
3), 2.97 (d,
and 6ꢀC
, aromatic), 7.70ꢀ7.76 (m, 1H, NꢀH).
11.06 (CH3), 14.2 (CH3), 20.06 (CH2), 26.0 (CH2), 28.1
J
= 10.08 Hz, 1H, 5
αꢀH ),
3.80 (s, 3H, OC
H
J
o= 8.40 Hz, 2H, 2ꢀC
H
H, aromatic), 7.25 (brs, 1H,
vinylicꢀH), 7.59 (d, Jo = 8.48 Hz, 2H, 3ꢀCH and 5ꢀCH
13C NMR: (400 MHz, DMSOꢀd6
)
δ
(CH2), 28.4 (CH2), 28.6 (CH2), 31.1 (CH2), 32.5 (CH2), 33.6 (CH), 35.1 (C), 46.9 (C), 48.2
(CH), 50.6 (CH), 55.2 (CꢀOCH3), 59.6 (CH), 114.3 (2xArCH), 127.6 (C=CꢀH), 131.7 (ArC),
132.10 (2xArCH), 133.6 (C=CꢀH), 160.1 (ArC), 170.2 (C=O) and 208.3 (C=O). Anal. Calcd
for C26H33NO3: C, 76.62; H, 8.16; N, 3.44. Found: C, 76.35; H, 7.92; N, 3.22.
16ꢀ(4ꢀCyanobenzylidene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6e). Yield: 75%. Brown colour
compound. mp 300ꢀ304 °C. FTꢀIR (KBr): 3393.33 (NꢀH), 2937.28 (CꢀH, aliphatic), 2227.32
(C≡N), 1718.39 (C=O), 1637.83 (CONH), 1089.64 (CꢀN) cmꢀ1. 1H NMR: (400 MHz,
DMSOꢀd6
)
δ
0.88 (s, 3H, 19ꢀC
H
3), 0.92 (s, 3H, 18ꢀC
, 3ꢀC , 5ꢀC and 6ꢀC
11.0 (CH3), 14.0 (CH3), 19.9 (CH2), 20.38 (CH2),
H
3), 3.02 (s, 1H, 5
αꢀH), 7.28 (s, 1H,
vinylicꢀ
H
), 7.74ꢀ7.84 (m, 4H, 2ꢀC
H
H
H
H
, aromatic), 7.94ꢀ7.96 (m, 1H, Nꢀ
H
). 13C NMR: (400 MHz, DMSOꢀd6
) δ
25.9 (CH2), 28.3 (CH2), 28.7 (CH2), 31.03 (CH2), 32.8 (CH2), 33.6 (CH), 35.04 (C), 47.1
(C), 48.1 (CH), 50.6 (CH), 59.7 (CH), 111.3 (ArC), 118.3 (C≡N), 129.9 (C=CꢀH), 130.5
(2xArCH), 132.2 (2xArCH), 139.2 (ArC), 139.6 (C=CꢀH ), 170.3 (C=O ) and 208.3 (C=O).
MS: m/z 403.31 [M + H] +. Anal. Calcd for C26H30N2O2: C, 77.58; H, 7.51; N, 6.96. Found:
C, 77.79; H, 7.78; N, 7.20.
ACS Paragon Plus Environment

Page 29 of 44
ACS Chemical Neuroscience
1
2
3
4
16ꢀ(4ꢀNitrobenzylidene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6f). Yield 55%. Dark brown colour
5
6
7
8
compound. mp 238ꢀ240°C. FTꢀIR (KBr): 3386.61 (NꢀH), 2935.17 (CꢀH, aliphatic), 1720.14
(C=O), 1647.79 (CONH), 1518.53 and 1343.67 (NO2), 1099.96 (CꢀN). 1H NMR: (400 MHz,
9
DMSOꢀd6
)
δ
0.90 (s, 3H, 18ꢀC
H
3), 1.00 (s, 3H, 19ꢀC
H
3), 3.20 (s, 1H, 5
2H, vinylicꢀ , aromatic), 8.25 (brs, 2H, 3ꢀC
, aromatic). 13C NMR: (400 MHz, DMSOꢀd6
) δ 13.9 (CH3), 19.6 (CH3), 20.4 (CH2),
α
ꢀ
H
), 7.27ꢀ7.37 (m,
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
H
and NꢀH), 7.87 (brs, 2H, 2ꢀC
H
and 6ꢀC
H
H
and
5ꢀC
H
24.1 (CH2), 25.5 (CH2), 27.5 (CH2), 28.8 (CH2), 31.1 (CH2), 31.7 (CH2), 33.4 (CH), 33.6
(C), 47.0 (C), 48.0 (CH), 49.1 (CH), 57.9 (CH), 123.5 (2xArCH), 129.3 (C=CꢀH), 131.00
(2xArCH), 140.1 (ArC), 141.6 (C=CꢀH), 146.9 (ArC), 170.8 (C=O ) and 207.8 (C=O). MS:
m/z 423.33 [M + H]+. Anal. Calcd for C25H30N2O4: C, 71.07; H, 7.16; N, 6.63. Found: C,
71.33; H, 6.90; N, 6.84.
16ꢀ(4ꢀBromobenzylidene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6g). Yield: 60%. Light yellow colour
compound. mpꢀ 285ꢀ287°C. FTꢀIR (KBr): 3270.83 (NꢀH), 2938.60 (CꢀH, aliphatic), 1720.5
1
(C=O), 1662.53 (CONH), 1395, 1073.20 (CꢀN). HNMR (400 MHz, DMSOꢀd6
)
δ
0.53 (s,
), 7.42ꢀ
, aromatic), 8.03 (brs,
13.2 (CH3), 14.1 (CH3), 15.8 (CH2), 20.3
3H, 18ꢀC
H
3), 0.63 (s, 3H, 19ꢀC
H
3), 2,79 (s, 1H, 5
αꢀH
), 7.37ꢀ7.41 (m, 1H, vinylicꢀ
and 5ꢀC
H
7.47 (m, 2H, 2ꢀC
H
and 6ꢀC
H
, aromatic), 7.54 (m, 2H, 3ꢀC
H
H
1H, NꢀH). 13C NMR: (400 MHz, DMSOꢀd6
)
δ
(CH2), 20.7 (CH2), 25.4 (CH2), 26.05 (CH2), 28.3 (CH2), 30.5 (CH2), 30.8 (CH), 35.2 (C),
42.07 (C), 47.3 (CH), 47.4 (CH), 48.2 (CH), 123.9 (C=CꢀH), 126.2 (ArC), 130.4 (2xArCH),
131.9 (2xArCH), 133..9 (ArC), 141.6 (C=CꢀH), 170.5 (C=O ) and 208.3 (C=O). Anal.
Calcd for C25H30BrNO2: C, 65.79; H, 6.63; N, 3.07. Found: C, 65.49; H, 6.84; N, 3.36.
16ꢀ(2,3ꢀDimethylaminobenzylidene)ꢀ4ꢀazaꢀandrostanꢀ3,17ꢀdione (6h). Yield: 58%. Yellow
colour compound. mp 255ꢀ257°C. FTꢀIR (KBr): 3376.38 (NꢀH), 3096.76 (CꢀH, aromatic),
2937.34 (CꢀH, aliphatic), 1723.8 (C=O), 1662.76 (CONH), 1521, 1346, 1050.36 (CꢀN).
1HNMR: (400 MHz, CDCl3)
δ 0.91 (s, 3H, 18ꢀCH3), 1.13 (s, 3H, 19ꢀCH3), 3.09 (s, 7H, 5αꢀH
ACS Paragon Plus Environment