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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 16,17-pyrazoline substituted heterosteroids as antiAlzheimer and anti-Parkinsonian agents using LPS induced neuroinflammation models of mice and rats
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, Chandigarh-160 014, India Email:
[email protected] Tel.: 0172-2541142; Fax: 0172-2543101
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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 16,17-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 5-position 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 N-acetyl analogue 3c displayed neuroprotective effects better than N-phenyl (4c), which in turn showed potency more than N-unsubstituted analogue 2c. KEY WORDS: Alzheimer’s disease, Parkinson’s disease, LPS, 16,17-pyrazolinyl 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 (AD) and Parkinson’s disease (PD).1 The oxidative stress, mitochondrial dysfunction, 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 and subsequent neuronal loss.2 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 Gram-negative bacteria elicits multiple pathological effects in human beings. Recent investigations suggest that chronic infusion of LPS causes neurodegeneration and subsequent impaired spatial memory and locomotor functions.3,4 During the last decades, steroids substituted with heterocycles at A-ring or D-ring of the skeleton have garnered a great deal of attention as anti-inflammatory, anticancer and antimicrobial agents.5-8 Recently much interest has been generated towards the chemical exploration of steroidal skeleton to develop novel neuroprotective agents.9-12 Fluasterone (I), O H H
F
CH
CH
H OH
(I)
O
O
N
O
(II)
N H
N
(III)
Figure 1. Novel analogues of dehydroepiandrosterone as neuroprotective agents a novel analog of dehydroepiandrosterone, is considered as a drug candidate that might be useful in the treatment of traumatic brain injury (Figure 1).11 Recently, neuroprotective effects of 16-pyridylidene-5-androstenedione derivatives (II) and their 4-aza analogues (III) (Figure 1) in LPS-treated animal models have been reported from our laboratory.12 3 ACS Paragon Plus Environment
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Pyrazoline, pharmacological
a
five
properties
membered
nitrogen
such
antimicrobial,
as
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heterocycle
possesses
numerous
anti-inflammatory,
analgesic,
antidepressant and anticancer actions.13 Several pyrazoline analogues have also been reported as useful candidates for therapeutic management of CNS-related diseases such as AD and PD as they possess good radical scavenging capacity and also exhibit potent anti-oxidant activities.14-16 Literature further reveals the neuroprotective actions of pyridyl moiety against the neurological disorders effecting behaviour and cognition.17 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 disorders.18 In light of these observations and in continuation with our earlier research work 12 aimed towards the discovery of potent neuroprotective steroids, synthesis and neuroprotective studies on a new series of 16,17-pyrazolinyl substituted steroidal analogues derived through a 16-pyridylidene moiety have been presented in the current study.
RESULTS AND DISCUSSION Chemical Synthesis. The synthesis of 16,17-pyrazolinyl steroids has been carried out as shown in scheme 1. The 16-arylidene adducts 1a-d were prepared as reported earlier.12 Further treatment of these aldol products with hydrazine hydrate in refluxing 1,4 dioxane for 5 h yielded pyrazoline fused steroids 2a-d, while reaction with
hydrazine hydrate in
refluxing glacial acetic acid for 9 h afforded acetylated steroidal analogues 3a-d. Reaction of 16-arylidene steroids 1a-d with phenylhydrazine in refluxing anhydrous methanol for 12 h afforded corresponding phenylpyrazolinyl substituted derivatives 4a-d. The obtained steroidal pyrazoline derivatives were found to be quite unstable and decompose at ambient temperature conditions probably due to the instability associated with pyrazoline nucleus.7 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 16,17-pyrazolinyl 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. O
HO
DHEA O H-C Ar CH3OH/NaOH N NH H H Ar HO
CH Ar
NH2NH2.H2O 1,4 dioxane HO reflux, 5hr
2a-d
O CH3 N N C H
O
1a-d
NH2NH2.H2O O CH3COOH reflux, 9hr H C C O 3
H
3a-d
NHNH2.H2O
reflux, 12hr
anhyd.CH3OH
N N H HO
a
H Ar
4a-d
B
c
D
Ar Scheme1: Synthetic route to the formation ofvarious16,17-pyrazolinyl steroidal derivatives The structures of all the synthesized compounds were confirmed by various spectral analyses. 16,17-Pyrazoline substituted N-H naked steroids 2b-d displayed a doublet due to 5'pyrazolinyl proton at ~ δ 4ppm in the 1H NMR spectra while for compound 2a it resonated as a multiplet. N-acetylated pyrazolinyl steroids 3a-d displayed carbonyl stretching vibrations at ~ 1730 and 1670 cm-1 due to the presence of ester and amide functionalities. Presence of two additional sharp singlets due to the resonating protons of N-COCH3 and O-COCH3 in the 5 ACS Paragon Plus Environment
Ar
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proton NMR spectra of steroids 3a-d confirmed the acetylation at both the places. Phenylpyrazoline substituted steroids 4a-d exhibited a marginally downfield doublet of 5'pyrazolinyl proton at ~ δ4.6 and a prominent multiplet due to aromatic protons at 6.72-7.29 ppm. The 16R and 5’S configurations were assigned on the basis of 1HNMR spectral data as reported earlier.5 Previous studies indicate that during the condensation of hydrazine hydrate with the 16-arylidene system after the removal of water molecule and formation of the 17hydrazone, the NH2 attacks the double bond from α-face leading to the formation of 16R. In addition, the phenyl ring rotates backward forming 5׳-S center. The deshielded C-18 methyl protons resonated downfield at ~ δ 1 ppm for all the pyrazoline substituted steroids 2-4a-d. Biological Activity. LPS induced Aβ 1–42 generation in the cortex and hippocampus plays a central role in the pathogenesis of Alzheimer's disease characterized by cognitive deficits.19 Literature also reveals that sub-toxic dose levels of LPS in substantia nigra (SN) region results in increased production of cytokines within the brain resulting in destruction of dopaminergic markers, which leads to impairment of locomotor activity.20 The neuroprotective effects of the DHEA derived 16,17-pyrazolinyl steroids 2-4a-d 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. Anti-Alzheimer’s Activity. The neuroprotective effects of 16,17-pyrazolinyl steroids 2-4a-d (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 disability were studied using Morris water maze and elevated plus maze models(Table 1).12 Treatment with pyrazoline substituted steroidal derivatives 2-4a-d (5mg/kg) considerably reduced the otherwise increased escape latency and % ITL(initial transfer latency) after 24 h 6 ACS Paragon Plus Environment
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Table 1. Anti-Alzheimer effects of 16,17-pyrazolinyl steroids on mice treated with LPS intraperitoneally Morris Water Maze Elevated Plus Maze Escape Latency (s) TSTQ (s) % ITL (s) Compound 41.5 ± 1.9 55 ± 3.4 11.4 ± 2.6 Control * * 73.2 ± 1.6 13 ± 2.9 91.4 ± 2.9* LPS # # 52.9 ± 2.4 44 ± 3 24.4 ± 2.2# Celecoxib 56.7 ± 2.5# 40 ± 3# 31.1 ± 2.4# Dexamethasone # # 61.6 ± 1.8 37 ± 3 35.5 ± 3.3 # 2a # # 64.0 ± 1.7 35 ± 2 37.7 ± 2.2# 2b # # 56.0 ± 2.9 41 ± 2 28.8 ± 2.2# 2c 58.2 ± 1.3# 40 ± 3.2# 30 ± 2.3# 2d # # 58.0 ± 1.5 39.5 ± 2.5 30 ± 3.3# 3a # # 61.0 ± 1.3 37 ± 4 33.3 ± 3.3# 3b # # 52.9 ± 1.5 43 ± 3 26.1 ± 3.8# 3c 54.4 ± 1.5# 42 ± 3# 27.2 ± 3.8# 3d # # 61.5 ± 2.4 38 ± 2.5 32.2 ± 1.9# 4a # # 63.4 ± 1.2 36 ± 3 35 ± 3.8# 4b # # 54.8 ± 2.1 42 ± 3 27.7 ± 3.3# 4c 56.1 ± 2.0# 41 ± 4# 28.8 ± 3.3# 4d # *p
