Synthesis and Biological Evaluation of Novel Piperazine Containing Hydrazone Derivatives

Some hydrazone derivatives were synthesized and their potential anticholinesterase activities were examined. A series of eleven new compounds of N-(2,4-disubstitutedbenzylidene)-2-(4-(4-nitrophenyl)piperazin-1-yl)acetohydrazide derivatives were obtained via reaction of 2-[4-(4-nitrophenyl)piperazin-1-yl]acetohydrazide with aromatic aldehydes.The chemical structures of the compounds were enlightened by FT-IR, H-NMR, C-NMR, and HRMS (ESI) spectral data. The inhibition potency of the compounds 3a–k against AChE and BuChE was measured and evaluated using a modification of Ellman’s spectrophotometric method. Among the tested compounds, compound 3c was assigned to be the most active derivative. Galantamine was used as a standard drug.


Introduction
Alzheimer's disease (AD) is a chronic neurodegenerative and progressive disease responsible from 60% to 70% of cases of dementia [1] and characterized by progressive impairment of memory and cognition [2].The primary lesions of AD are neurofibrillary tangles (NFT) and the senile plaques that is composed of central core of beta-amyloid [3,4].As the destruction of cholinergic neurons ends up with the decline in acetylcholine (ACh) level, a key aspect of the symptomatic therapy for AD is to increase acetylcholine concentration in presynaptic regions via blocking its metabolic enzyme acetylcholinesterase (AChE).In humans, cholinesterase is classified into two types: acetylcholinesterase (AChE) that hydrolyses acetylcholine selectively and more quickly and butyrylcholinesterase (BuChE) that metabolize butyrylcholine more quickly [5,6].The therapeutic approaches to date have tended to focus on AChE rather than BuChE that is considered to play a minor role in regulating ACh levels in brain.At the present time, reversible acetylcholinesterase inhibitors (AChEIs) like donepezil, rivastigmine, and galantamine are in clinical use in the therapy of AD as well as the N-methyl-D-aspartate-receptor (NMDA) antagonist memantine [7,8].
Reversible AChE inhibitors perform a significant role in pharmacological manipulation of the enzyme activity [9].Despite their efficacy, reversible AChE inhibitors suffer from several major drawbacks such as hepatotoxicity, short half life, and gastrointestinal tract excitement [10].For medicinal chemists, hence, it has been an increasingly important area to discover novel and efficient AChE inhibitors with minimum side effects.
The past decades have seen increasingly rapid advances in the field of developing acetylcholinesterase inhibitors.Hydrazones, thus, have attracted great attention in the light of recent studies that reported compounds bearing hydrazone moiety exhibit various biological activities including acetylcholinesterase activity [11][12][13][14][15][16][17][18].On the other hand, piperazine ring is known to act as bioisosteric replacement for piperidine ring that exist in donepezil and have also been revealed to have acetylcholinesterase activity [19][20][21].Based on these observations, we designed a new series of piperazine containing hydrazone derivatives (Figure 1).This combination of studies provides support for designing novel and efficient agents for treatment of AD.
In view of the above-mentioned findings, herein, we reported the synthesis of and acetylcholinesterase 2 Journal of Chemistry and butyrylcholinesterase inhibitory activity of N  -(2,4disubstitutedbenzylidene)-2-(4-(4-nitrophenyl)piperazin-1yl)acetohydrazide derivatives that may be of value in designing acetylcholinesterase inhibitors.The inhibitory potency of compounds was studied in comparison with the known AChE inhibitor drug galantamine.

Experimental
2.1.Materials.All chemicals were purchased from Sigma-Aldrich Chemical Co.(Sigma-Aldrich Corp., St. Louis, MO, USA) and Merck Chemicals (Merck KGaA, Darmstadt, Germany).All melting points (mp) were determined by Electrothermal 9100 digital melting point apparatus (Electrothermal, Essex, UK) and were uncorrected.All the reactions were monitored by thin-layer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany).Spectroscopic data were recorded with the following instruments: IR, Shimadzu 8400S spectrophotometer (Shimadzu, Tokyo, Japan); NMR, Bruker DPX 500 NMR spectrometer (Bruker Bioscience, Billerica, MA, USA), in DMSO- 6 , using TMS as internal standard; M + 1 peaks were determined by LC-MS-IT-TOF system (Shimadzu, Tokyo, Japan).(1).1-(4-Nitrophenyl)piperazine (0.015 mol) was dissolved in acetone (250 mL).Potassium carbonate (0.018 mol) was added.After addition of ethyl 2-chloroacetate (0.015 mol), the reaction mixture was stirred for 4 hours at room temperature.The solvent was evaporated under reduced pressure and then water was added to wash the resulting solid and the mixture was filtered to give compound 1. (2).Ethyl 2-[4-(4-nitrophenyl)piperazin-1-yl]acetate (0.013 mol) was dissolved in ethanol (250 mL).Hydrazine hydrate (0.013 mol) dissolved in ethanol was added gradually and the mixture was stirred at room temperature.After completion of the reaction, the solvent was evaporated under reduced pressure; then water was added to wash the resulting solid and the mixture was filtered, dried, and recrystallized from ethanol to give compound 2.

Enzymatic Assay. AChE and BuChE inhibitory activity
was determined by Ellman's method with minor modifications [22].Compounds 3a-k were dissolved in 2% DMSO and were tested at final concentrations of 5, 10, 20, 40, and 80 mg/mL.20 mL (1 unit/mL) of AChE (from Electrophorus electricus, electric eel) or BuChE (from equine serum) and 10 mL sample were added to 2.4 mL buffer; the mixture was incubated at 37 ∘ C for 15 min.After the 15 min incubation, 50 mL of 0.01 M DTNB and 20 mL of 75 mM ATCI or 10 mM BTCI were added, and the final mixture was incubated at room temperature for 30 min.A control mixture (blank) was prepared using 10 mL of 2% DMSO instead of the test sample, with all other procedures similar to those used in the case of the sample mixture.Absorbance was measured at 412 nm and 37 ∘ C using polystyrol cuvettes with spectrophotometer (Shimadzu, UV-1700).Experiment was done in triplicate.Data are expressed as mean standard deviation (SD).The inhibition (percent) of AChE and BuChE was calculated using the following equation.The statistical analysis was evaluated using Microsoft Office Excel 2013 and the data were expressed as mean ± SD:  2) with aromatic aldehydes gave the target compounds (3a-k).Some characteristic properties of the compounds were given in Table 1.Structures of the obtained compounds were elucidated by 1 H NMR, 13 C NMR, and HRMS spectral data.

Results and Discussion
In the IR spectra of compounds (3a-k), significant stretching bands belonging to C=O were observed in between 1651-1695 cm −1 , bands belonging to C=N and C=C were observed in between 1599-1471 cm −1 , and bands belonging to NO 2 were observed at about 1597.06 and 1303.88 cm −1 .
In the 1 H NMR spectra of the final compounds (3ak), the hydrazone proton was observed in the area of 10.5-12 ppm.The protons belonging to CO-CH 2 (3.09-3.56ppm), N=CH (7.78-8.10ppm), and CO-NH (11.03-11.13ppm) were detected as paired peaks with respect to the presence of the  and  forms of the compounds [18,19].The other aromatic and aliphatic protons were observed at the expected regions.
In 13 C NMR spectra of the compounds, the carbons belonging to -N=CH-(140-147 ppm), carbonyl (165-170 ppm), and -CH 2 -attached to carbonyl (55-60 ppm) were observed as paired peaks due to  and  isomer forms.Due to the presence of fluoro in compound 3a and 3g, splitting relating to neighbouring atoms was detected in the spectrum.
In the HRMS spectra of final compounds (3a-k), the M + 1 peak was observed in accordance with their molecular formula.The M + 1 peaks were determined in accuracy of 5-16 ppm.

Enzyme Inhibition.
Therapeutic efficacy of novel hydrazone derivatives for the treatment of AD depends on their anticholinesterase activities.Inhibition of AChE and BuChE Only compound 3c with substituent hydroxyl at para position exhibited available inhibitory effect against AChE having IC 50 value of 29.5 ± 2.12.In addition, compound 3a, with substituent fluoro at para position also showed an inhibitory effect against AChE and BuChE.This conclusion supports that the hydroxyl and fluoro substituents on phenyl ring at para positions may have a considerable influence on anticholinesterase activity.It could be considered that hydrogen bonds contribute to the activity of compounds 3a and 3c.Substitution at orto and para positions with chloro did not increase the activity.Compound 3d was found to be nonactive against either AChE or BChE.When compared with reference drugs and other derivatives, compound 3j exhibited the lowest anticholinesterase activity.

Conclusion
In the present paper, we synthesized some hydrazone derivatives and evaluated their AChE and BuChE enzyme inhibitory activities.The consequences verify that compound 3c, with an IC 50 value of 29.5 ± 2.12, is the most promising inhibitory agent of AChE and BuChE among these compounds.As a result, it may be pointed out that hydroxyl and fluoro substituents at para position on the phenyl ring have a vital influence on anticholinesterase activity.Further investigations can be executed on the evaluation of new potent AChE and BuChE inhibitory agents bearing hydrazone moiety by modification of compound 3c.
by synthesized derivatives (3a-k) is compared with galantamine as reference drug and IC 50 values of individual compounds are listed in Table2.The anticholinesterase activities of the compounds were detected via a modification of Ellman's spectrophotometric method.All compounds showed less anticholinesterase potency than reference drugs.