Introduction

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known serine hydrolase enzymes responsible for the hydrolysis of acetylcholine (ACh), which is a significant neurotransmitter for regulation of cognition in animals (Saeed et al. 2014; Shah et al. 2016; Chiou et al. 2009; Tampi et al. 2016; Bentley et al. 2011). BChE can also hydrolyse some esters different from Ach, but the role of this enzyme is less known (Groner et al. 2007; Chiou et al. 2009). Inhibition of these enzymes leads to the rise of ACh levels in cholinergic synapses (Groner et al. 2007). Thus, cholinesterase inhibitors are used in the treatment of various neuromuscular disorders which occur as a result of reduced cortical and hippocampal levels of ACh such as Alzheimer’s disease (AD). AD is a complex neurodegenerative disorder characterized by synapse dysfunction, neuronal death, and loss of memory and learning ability (Candeias et al. 2012; Cacabelos 2008; Strelnik et al. 2016; Gilhus et al. 2016). Current treatment approaches for AD continue to be principally symptomatic, with the major therapeutic strategy based on the relationship between the observed cholinergic dysfunction and AD, known as the cholinergic hypothesis, and specifically on cholinesterase inhibition (Anand and Singh 2013; Ballard et al. 2011).

Various AChE inhibitors such as tacrine, donepezil, rivastigmine, and galantamine (Fig. 1) have been used contemporarily for the symptomatic treatment of AD (Mehta et al. 2012; Zha et al. 2016; Williams et al. 2011; Cummings et al. 2007; Costanzo et al. 2016; Romero et al. 2013; Camps et al. 2008). In recent years, novel cholinesterase inhibitors from natural resources or synthetic ways, which have coumarin, benzofuran, berberine, β-carboline, benzophenone, ferulic acid, naphthyridine, triazine, and quinolone scaffolds as the main pharmacophoric groups, have been reported (Saeed et al. 2015; Delogu et al. 2016; Jiang et al. 2011; Otto et al. 2014; Huang et al. 2012; Belluti et al. 2011; Catto et al. 2012; Chen et al. 2012; Samadi et al. 2012).

Fig. 1
figure 1

Molecular structure of tacrine, donepezil, rivastigmine, and galantamine

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Although BChE is sparsely expressed, recent studies proved that AChE inhibition alone may not be sufficient to impair cholinesterase activity and elevate choline levels since BChE makes up for the lost AChE activity. Furthermore, it is known that BChE expression is increased during AD (Mesulam et al. 2002; Li et al. 2008).

With the recent crystallographic studies structural features of AChE and BChE active sites have been clearer and the binding interactions of known inhibitors like tacrine, donepezil, and galantamine with the key binding site residues are well studied (Dvir et al. 2010; Nicolet et al. 2003; Nachon et al. 2013; Leon and Marco-Contelles 2011; Simoni et al. 2012; Nepovimova et al. 2014; Cheung et al. 2012). Donepezil, unlike galantamine and tacrine, interacts with the so-called peripheral anionic site (PAS) residues which line the rim at the entrance of the active gorge as well as the cationic active site (CAS) and acyl-binding pocket (Costanzo et al. 2016; Cheung et al. 2012; Camps et al. 2009; Bourne et al. 2003; Tougu 2001). At the bottom of this gorge are three amino acids, which catalyse the cleavage of acetyl-choline bond, namely S203, E334, and H447, also known as the catalytic triad (Zhou et al. 2010). Ideally, a cholinesterase inhibitor is expected to effectively interact with these sites (Fig. 2). Although the active site gorge residues of BChE and AChE share 65% sequence identity some differences are critical to enzyme specificity such as the volume of the acyl-binding pocket, which is larger in BChE (Brus et al. 2014).

Fig. 2
figure 2

Structural hypothesis for AChE inhibitors

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3(2H)-pyridazinone derivatives were claimed to possess diverse bioactivities such as antibacterial, antifungal, antituberculosis, analgesic, anti-inflammatory, antihypertensive, anticancer, anticholinesterase, and antioxidant activities (Nagle et al. 2014; Utku et al. 2011a, 2011b; Şahin et al. 2004; Siddiqui et al. 2010, 2011; Yamali et al. 2015; Rathish et al. 2012; Abd El-Ghaffar et al. 2011). The contribution to the activity of aryl substituents at the sixth position of 3(2H)-pyridazinone was investigated in many studies (Utku et al. 2011a, 2011b; Şahin et al. 2004; Yamali et al. 2015; Rathish et al. 2012; Abd El-Ghaffar et al. 2011; Xing et al. 2013). In a late study, Xing et al. (2013) identified high affinity AChE inhibitors in 2,6-disubstituted pyridazinone structure. In a previous study, we reported a series of 3(2H)-pyridazinone derivatives bearing ethyl propionate and propyohydrazide at the second position of pyridazinone with AChE/BChE inhibitory activities (Özçelik et al. 2010). Later we reported a series of 6-substituted-3(2H)-pyridazinone derivatives among which 5u and 5v were found to have potent AChE inhibitory activity (Önkol et al. 2013; Utku et al. 2011a, 2011b) (Fig. 3).

Fig. 3
figure 3

Molecular structure of 5u and 5v

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In view of the above mentioned findings we designed and synthesized 18 new 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-acetyl-2-(substitutedbenzal)hydrazon derivatives (5a–t) and in vitro evaluated their AChE and BChE inhibitory effects by using Ellman’s method, which is the most commonly used method to measure cholinesterase activity. It is especially useful in monitoring disulphide bond formation in solution phase reactions. The Ellman method is also useful in testing the quality of peptides containing free sulfhydryl groups (Ellman et al. 1961).

The choline-binding site moiety was designed as a 3,4-dichlorophenyl ring and the structural modifications were focused on substitutions on the phenyl ring of acetobenzalhydrazone moiety. Methyl, methoxy, chloro, bromo, fluoro, nitro, amino, and cyano groups were included as substitutions on this ring at varying positions and combinations. Using molecular docking approach we tried to get insights into binding interactions of our derivatives in comparison with 5u and 5v in the active site of both enzymes and understand the facts that underlie the relationships between structural modifications on these ligands and their efficacy.

Results and discussion

Chemistry

5a–t were synthesized according to the literature methods as outlined in Scheme 1.

Scheme 1
scheme 1

Synthesis of the compounds

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Nucleophilic displacement reaction of commercial 3,6-dichloropyridazine with 3,4-dichlorophenylpiperazine in ethanol afforded 3-chloro-6-substitutedpyridazine (1). 6-4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone (2) was obtained as a result of hydrolysis of 3-chloro-6-[4-(3,4-dichlorophenyl)piperazine-1-yl]pyridazine by heating in glacial acetic acid (Şahin et al. 2004). Ethyl 6-4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetate (3) was obtained by the reaction of 2 with ethyl bromoacetate in the presence of K2CO3 in acetone (Şahin et al. 2004). Then, 6-4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetohydrazide (4) was synthesized by the condensation reaction of 3 with hydrazine hydrate (99%) (Şahin et al. 2004). Ultimately, the title compounds bearing benzalhydrazone structure were obtained by the reaction of 4 with substituted/nonsubstituted benzaldehydes. 2–4 and 5a–t were reported for the first time in this study. The reaction yields of the compounds range approximately from 50 to 90%. Compound 5k was synthesized with the highest yield (93%) while compound 5d was the compound synthesized with the lowest yield (56%). Molecular structures of 2–4 were confirmed by infrared (IR) and 1H-NMR and 5a–t by IR, 1H-NMR, 13C-NMR, and mass spectral data. Molecular structures, yields, melting points, molecular weights, and molecular formula of 5a–t are given in Table 1.

Table 1 Molecular structures, yields, melting points, molecular weights, and molecular formula of 5a–t
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Anticholinesterase activity

In vitro AChE and BChE inhibition of 5a–t was determined by the method of Ellman et al. (1961) using galantamine as reference and the activity data of the compounds were summarized as percentage inhibition ± standard deviation (SD) values at 100 µg/ml concentration in Table 2. All compounds showed significant inhibitory activity against AChE and BChE with higher inhibition trend against the former except 5h. The inhibition % of 5g and 5r could not be measured because of collapse. 5f and 5i, 3-methylbenzalhydrazone and 3-metoxybenzalhydrazone derivatives, showed the best AChE inhibition with 75.52 and 71.72%. On the other hand 5h, 2-metoxibenzalhydrazone derivative, and 5f exhibited the highest inhibitory effect against BChE with 67.16 and 62.03% inhibition, respectively. Thus, 5f emerged as a promising dual cholinesterase inhibitor lead molecule. In general, substitution on benzalhydrazone phenyl ring lead to an increase in AChE and BChE inhibition. Substitutions on the meta position of the benzalhydrazone phenyl ring with -OCH3 and –CH3 also improved the anti-AChE activity. Adversely, p-substitution on the benzalhydrazone phenyl ring with strong electron-donating groups such as –OCH3 was observed to reduce the AChE inhibition.

Table 2 AChE and BChE inhibition values (%) of the title compounds at 100 µg/ml
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Molecular docking studies

Glide predicted good fit for 5f in the active site of human AChE (PDB code 4EY7) along the gorge with some characteristic interactions previously defined for some known inhibitors (Fig. 4a). The 3,4-dichlorophenyl moiety of 5f occupied the choline-binding pocket and engaged in strong π–π stacking against W86 aromatic side chain and weak interactions with E202 and the catalytic residue H447. These residues are reportedly involved in ligand–receptor complexes of tacrine, galantamaine, huperzine A, and donepezil (Nachon et al. 2013; Cheung et al. 2012). The piperazine ring occupied the acyl-binding pocket entrance and the constriction site beneath PAS contacting Y124, Y337, and F338 aromatic side chains. Donepezil was reported to form water-mediated H-bonds with Y337 and Y341 via its quaternary N of the piperidine ring (Fig. 4d). Probably the key difference between our derivatives and donepezil is that our derivatives are non-ionized at physiological pH hence the absence of such interaction. Instead, via its carbonyl oxygen of the pyridazinone ring, 5f formed a water-mediated H-bond to R296 backbone NH and a direct H-bond to F295 backbone NH. The latter interaction was reportedly observed with the carbonyl oxygen of the indanone moiety of donepezil (Cheung et al. 2012). The pyridazinone ring of 5f occupied the lower PAS and π–π stacked with Y341 aromatic side chain. In the upper PAS, the 3-methylbenzene ring positioned in the space between W286 and Y72 making hydrophobic contacts.

Fig. 4
figure 4

Binding modes of 5f (a), 5u (b), 5v (c), and donepezil superimposed with the rest in AChE active site gorge. Side views of the active site show ligands and binding-site residues as grey and colour sticks, water molecules as stick and balls; ligand molecular surfaces as solid, partial-charge coloured; H-bonds (yellow) and π–π stacks (turquoise) as dashed lines (color figure online)

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5u and 5v, two structural analogues of 5f, were previously synthesised and their AChE inhibitory activities were screened. 5u was reported to show 74.27 ± 0.78% AChE inhibition at 0.25 mM, which is very close to 5f’s molar inhibition (75.52% at 0.199 mM). On the other hand 5v was reported to show greater inhibition for the same enzyme with 99.76 ± 1.54% at 0.2 mM. Glide predicted 5u’s binding mode and interactions in human AChE active site very similar to 5f (Fig. 4b) whereas 5v’s docking solution differed from those of 5u and 5f to some extent (Fig. 4c). Upon close inspection of these receptor–ligand complexes one can see that the 4-fluorobenzen ring of 5v is slightly flipped and stacked with W337 aromatic side chain and it does not reach as deep to the bottom of the gorge as the 3,4-dichlorobenzene and 4-chlorobenzene of 5f and 5u, respectively. This way the 4-trifluorobenzene ring of 5v lays over the rim of the active site gorge entrance lined by Y72 and T75. The chlorine atoms of 5f and 5u occupying the choline binding pocket, as they are bulkier than the fluorine on 4-fluorobenzene of 5v, make unfavourable VdW interactions, i.e., bad contacts, with some of the residues lining the bottom of the active site gorge (Fig. 5). Thus, 4-F appears a good substitution on this benzene ring with its relatively small volume and high electron affinity. Additionally, Glide scores of these derivatives were in line with their in vitro inhibition percentages (Table 3).

Fig. 5
figure 5

Binding modes of 5f (aquamarine), 5u (yellow), and 5v (orange) in AChE active site gorge. Side view of the gorge showing ligands as colour CPK and the molecular surface of the lower gorge residues as opaque grey (color figure online)

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Table 3 Glide scores (kcal/mol) of 5f, 5u, 5v in AChE and BChE active sites
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We docked these compounds in human BChE active site gorge as well (PDB code 4BDS) and observed similarities with their binding modes in AChE active site (Fig. 6a). Our derivatives lack substitutions to fill the relatively larger acyl-binding pocket and interact with W231, L286, and V288 (Fig. 6b), which might account for their relatively lower BChE inhibition percentages. Indeed, some selective BChE inhibitors with nanomolar IC50 values were reported to effectively occupy this midgorge (Kneza et al. 2015). The 2,4-dichlorobenzene moiety of 5f again occupied the choline-binding subdomain, π–π stacked with W82 aromatic side chain, made π-cation interaction with the imidazolium of H438 (counterpart of H447 in AchE catalytic triad), and formed halogen bond with Y128 side chain via 4-Cl. The carbonyl oxygen of the acetylhydrazone function made a water-mediated H-bond with N70 in PAS.

Fig. 6
figure 6

Binding mode of 5f in BChE active site gorge. Side views of the active site show a 5f and binding-site residues as grey and colour sticks, water molecules as stick and balls; 5f molecular surface as solid, partial-charge coloured; H-bonds (yellow), π–π stacks (turquoise), and cation-π interaction (green) as dashed lines; b 5f in colour CPK, acyl-binding pocket residues in grey sticks, and receptor surface in transparent solid grey. Chlorine atoms are again protruding through the receptor surface (color figure online)

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Conclusion

As a continuation of our research interest in developing novel 3(2H)-pyridazinone derivative anticholinesterase compounds and establishing new relationships between their structure and activity, we designed and synthesized a series of 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)pyridazin-3(2H)-pyridazinone-2-acetyl-3-(p-substituted/ nonsubstituted benzal)hydrazone derivatives and evaluated their AChE and BChE inhibitory activities according to the Ellman’s method. Compounds with significant inhibition at 100 µg/ml were obtained. Compound 5f emerged as a potent dual inhibitor with 75.52 ± 1.76 and 62.03 ± 1.82% inhibition against AChE and BChE, respectively. It was less potent than the reference drug galantamine, yet promising for further modifications as a lead. The molecular docking studies showed similarities in binding interactions in active site gorges of the enzymes with known inhibitors, such as donepezil. However 3,4-dichloro substitution on the phenyl of the 4-phenylpiperazine moiety was sterically less favourable in comparison with 4-fluoro substitution, which is thought to be the reason for 5v’s higher efficacy. Lower inhibition rates against BChE was probably due to the failure in effectively filling the larger acyl-binding pocket of this enzyme’s active site gorge.

Experimental

Chemistry

All chemicals were purchased from Aldrich, Fluka AG, and E. Merck. The purity of all compounds was checked by thin layer chromatography with Merck Kieselgel F254 plates. Melting points were determined on Electrothermal 9200 melting points apparatus and the values were uncorrected. IR spectra were recorded on a Perkin Elmer Spectrometer by attenuated total reflection (ATR) technique. 1H-NMR and 13C-NMR spectra were recorded on a Brucker Avonce Ultrashield FT-NMR spectrometer in DMSO and CDCl3 at 300 MHz in Inonu University, Malatya. The mass spectra (HRMS) of the compounds were recorded on Waters Acquity Ultra Performance Liquid Chromatography Micromass and combined with LCT PremierTM XE UPLC/MS TOFF spectrophotometer (Waters Corp, Milford, USA) by ESI+ and ESI− techniques.

Synthesis of 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone (2)

A solution of 0.05 mol of a 6-substituted-3-chloropyridazinone derivative (1) in 30 ml glacial acetic acid was refluxed for 6 h. Acetic acid was removed under reduced pressure, the residue was dissolved in water and extracted with chloroform. The organic phase was dried over sodium sulphate and evaporated under reduced pressure. The residue was purified by recrystallization from ethanol (Utku et al. 2011a, 2011b).

Synthesis of ethyl 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetate (3)

A mixture of 0.01 mol 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone (2), 0.02 mol ethyl bromoacetate, and 0.02 mol potassium carbonate in acetone (40 ml) was refluxed overnight. After the mixture was cooled, the organic salts were filtered off, the solvent was evaporated, and the residue was purified by recrystallization with methanol to give the esters (Utku et al. 2011a, 2011b).

Synthesis of ethyl 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)--3(2H)-pyridazinone-2-ylacetohydrazide (4)

Hydrazine hydrate (99%, 3 ml) was added to the solution of 0.01 mol ethyl 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetate (3) in 25 ml methanol and stirred for 3 h at room temperature. The precipitate obtained was filtered off, washed with water, dried, and recrystallized from ethanol (Şahin et al. 2004).

General procedure for the preparation of 6-(4-(3,4-dichlorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(substitutedbenzal)hydrazones (5a–t)

0.01 mol 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-yl acetohydrazide (4) and 0.01 mol substituted/nonsubstituted benzaldehyde were stirred in ethanol (15 ml) and refluxed for 6 h. At the end of the reaction, the mixture was poured into ice water. The precipitate was filtered, dried, and crystallized from methanol/water (Şahin et al. 2004).

6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone (2)

White crystals; yield: 91.67%; mp: 253–255 °C; IR (ν cm−1, ATR): 3444 (N–H), 3020 (C–H aromatic), 2923 (C–H aliphatic), 1679 (C=O), 1586 (C=N), 1238 (C–N), 1006 (C–O) and 831 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 3.34 (4H; t; CH2–N; piperazine b + b′), 3.45 (4H; t; CH2–N; piperazine a + a′), 6.84 (1H; d; pyridazinone H5), 6.87 (1H; d; pyridazinone H4), 7.10–7.32 (3H; m; phenyl protons) and 10.42 (1H; s; –NH). 13C-NMR (DMSO-d 6 , 400 MHz), δ 45.99 (2C; CH2–N), 47.13 (2C; CH2–N), 115.56 (1C; pyridazinone C5), 116.54 (1C; pyridazinone C4), 119.91 (1C; 3,4-dichlorophenyl C6), 127.11 (1C; 3,4-dichloroyphenyl C2) 130.46 (1C; 3,4-dichlorophenyl C5), 131.03 (1C; 3,4-dichlorophenyl C1), 131.50 (1C; pyridazinone C6), 149.05 (1C; 3,4-dichlorophenyl C4), 150.56 (1C; 3,4-dichlorophenyl C3), and 158.88 (1C; pyridazinone C3); C14H14Cl2N4O MS (ESI+) calculated: 324.05, Found: m/e 325.0588 (M+; 100.0%) and 327.0587 (M+2; 67.9%).

Ethyl 6-(4-(3,4-dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetate (3)

White crystals; yield: 57.79%; mp: 212–214 °C; IR (ν cm−1, ATR): 3064 (C–H aromatic), 2981 (C–H aliphatic), 1753 (C=O ester), 1665 (C=O), 1588 (C=N), 1236 (C–N), 1027 (C–O) and 843 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 1.31 (3H; t; –CH3), 3.30 (4H; t; CH2–N; b + b′), 3.41 (4H; t; CH2–N; a + a′), 4.22 (2H; q; –O–CH2–), 4.78 (2H; s; –N–CH2–C=O), 6.78 (1H; d; pyridazinone H5), 6.80 (1H; d; pyridazinone H4) and 6.92–7.34 (3H; m; phenyl protons). 13C-NMR (DMSO-d 6 , 400 MHz), δ 14.13 (1C; CH3), 46.39 (2C; CH2–N), 48.63 (2C; CH2–N), 52.89 (1C; –N–CH2–C=O), 61.54 (1C; –OCH2), 115.98 (1C; pyridazinone C5), 117.94 (1C; 3,4-dichlorophenyl C6), 125.96 (1C; pyridazinone C4), 126.84 (1C; 3,4-dichloroyphenyl C2), 130.62 (1C; 3,4-dichlorophenyl C5), 131.31 (1C; 3,4-dichlorophenyl C1), 132.98 (1C; pyridazinone C6), 148.66 (1C; 3,4-dichlorophenyl C4), 150.01 (1C; 3,4-dichlorophenyl C3), 158.38 (1C; pyridazinone C3) and 167.74 (1C; CH2–N–C=O); C18H20Cl2N4O3 MS (ESI+) calculated: 410.09, Found: m/e 411.0990 (M+; 100.0%) and 413.0984 (M+2; 67.9%).

6-(4-(3,4-Dichlorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetohydrazide (4)

White crystals; yield: 78.23%; mp: 207–209 °C; IR (ν cm−1, ATR): 3308 (N–H), 3056 (C–H aromatic), 2837 (C–H aliphatic), 1682 (C=O), 1590 (C=N), 1235 (C–N) and 846 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 3.27 (4H; t; CH2–N; b + b′), 3.46 (4H; t; CH2–N; a + a′), 4.74 (2H; s; –N–CH2–C=O), 5.15 (2H; s; –NH2), 6.79 (1H; d; pyridazinone H5), 6.81 (1H; d; pyridazinone H4), 6.94–7.35 (3H; m; phenyl protons) and 7.75 (1H; s; –NH–N). 13C-NMR (DMSO-d 6 , 400 MHz), δ 45.67 (2C; CH2–N), 47.07 (2C; CH2–N), 52.16 (1C; –N–CH2–C=O), 116.56 (1C; pyridazinone C5), 119.94 (1C; 3,4-dichlorophenyl C6), 126.55 (1C; pyridazinone C4), 130.64 (1C; 3,4-dichloroyphenyl C2), 131.51 (1C; 3,4-dichlorophenyl C5), 148.51 (1C; 3,4-dichlorophenyl C1), 150.55 (1C; pyridazinone C6), 157.66 (1C; 3,4-dichlorophenyl C4), 158.90 (1C; 3,4-dichlorophenyl C3), 166.25 (1C; CH2–N–C=O) and 168.10 (1C; pyridazinone C3); C16H18Cl2N6O2 MS (ESI+) calculated: 396.09, Found: m/e 397.0909 (M+; 100.0%), 399.0906 (M+2; 67.9%) and 402.0923 (M+4; 10.2%).

6-(4-(3,4-Dicholorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-acetyl-2-(benzal)hydrazon (5a)

White crystals; yield: 70.25%; mp: 278–280 °C; IR (ν cm−1, ATR): 3676 (N–H), 3065 (C–H aromatic), 2901 (C–H aliphatic), 1693 (C=O), 1569 (C=N), 1237 (C–N) and 844 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.51 (4H; t; CH2–N; b + b′), 3.30 (4H; t; CH2–N; a + a′), 5.06 (2H; s; –N–CH2–C=O), 6.93 (1H; d; pyridazinone H5), 6.97 (1H; d; pyridazinone H4), 7.00–7.71 (8H; m; phenyl protons), 8.02 (1H; s; –N=CH–) and 11.66 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.20 (2C; CH2–N), 47.63 (2C; CH2–N), 78.91 (1C; –N–CH2–C=O), 116.08 (1C;=CH), 117.05 (1C; pyridazinone C5), 120.57 (1C; 3,4-dichlorophenyl C6), 127.05 (1C; 3,4-dichlorophenyl C5), 127.31 (1C; pyridazinone C4), 129.22–130.90 (5C; phenyl C2−6), 132.02 (1C; 3,4-dichloroyphenyl C2) 134.41 (1C; phenyl C1), 144.36 (1C; 3,4-dichlorophenyl C1), 148.96 (1C; pyridazinone C6), 150.98 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.40 (1C; pyridazinone C3); C23H22Cl2N6O2 MS (ESI+) calculated: 484.12, Found: m/e 485.1262 (M+; 100.0%), 487.1226 (M+2; 67.9%) and 488.1237 (M+4; 10.2%).

6-(4-(3,4-Dicholorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-acetyl-2-(4-bromobenzal)hydrazon (5b)

White crystals; yield: 87.56%; mp: 271–273 °C; IR (ν cm−1, ATR): 3091 (C–H aromatic), 2953 (C–H aliphatic), 1695 (C=O), 1571 (C=N), 1237 (C–N) and 841 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.51 (4H; t; CH2–N; b + b′), 3.30 (4H; t; CH2–N; a + a′), 5.06 (2H; s; –N–CH2–C=O), 6.94 (1H; d; pyridazinone H5), 6.98 (1H; d; pyridazinone H4), 7.01–7.70 (7H; m; phennyl protons), 7.99 (1H; s; -N=CH-) and 11.74 (1H; s; -NH-N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.23 (2C; CH2–N), 47.56 (2C; CH2–N), 79.01 (1C; –N–CH2–C=O), 114.27 (1C;=CH), 116.21 (1C; pyridazinone C5), 117.02 (1C; 3,4-dichlorophenyl C6), 120.34 (1C; 3,4-dichlorophenyl C5), 127.07 (1C; pyridazinone C4), 130.56 (2C; 4-bromophenyl C2, 6), 130.72 (2C; 4-bromophenyl C3, 5), 130.90 (1C; 3,4-dichloroyphenyl C2), 132.03 (1C; phenyl C1), 144.36 (1C; 3,4-dichloroyphenyl C1), 146.96 (1C; pyridazinone C6), 148.56 (1C; 4-bromophenyl C4), 150.93 (2C; 3,4-dichlorophenyl C3,4), 158.07 (1C; CH2–N–C=O) and 165.70 (1C; pyridazinone C3); C23H21BrCl2N6O2 MS (ESI+) calculated: 564.03, Found: m/e 565.0342 (M+; 100.0%), 567.0299 (M+2; 45.7%) and 568.0392 (M+4; 6.2%).

6-(4-(3,4-Dicholorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-acetyl-2-(2-chlorobenzal)hydrazon (5c)

White crystals; yield: 81.24%; mp: 267–269 °C; IR (ν cm−1, ATR): 3063 (C–H aromatic), 2952 (C–H aliphatic), 1695 (C=O), 1569 (C=N), 1237 (C–N) and 839 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.12 (4H; t; CH2–N; b + b′), 3.30 (4H; t; CH2–N; a + a′), 5.12 (2H; s; –N–CH2–C=O), 6.66 (1H; d; pyridazinone H5), 6.88 (1H; d; pyridazinone H4), 7.10–7.47 (7H; m; phenyl protons), 7.80 (1H; s; –N=CH–) and 11.12 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.61 (2C; CH2–N), 48.32 (2C; CH2–N), 77.12 (1C; –N–CH2–C=O), 115.87 (1C;=CH), 116.85 (1C; pyridazinone C5), 117.21 (1C; 3,4-dichlorophenyl C6), 120.55 (1C; 3,4-dichlorophenyl C5), 126.47 (1C; pyridazinone C4), 130.23 (1C; 2-chlorophenyl C4), 130.56 (1C; 2-chlorophenyl C5), 130.62 (1C; 2-chlorophenyl C3), 130.74 (1C; 2-chlorophenyl C6), 131.90 (1C; 3,4-dichloroyphenyl C2), 135.12 (1C; 2-chlorophenyl C1), 144.36 (1C; 3,4-dichloroyphenyl C1), 146.96 (1C; pyridazinone C6), 148.56 (1C; 2-chlorophenyl C2), 150.93 (2C; 3,4-dichlorophenyl C3,4), 158.07 (1C; CH2–N–C=O) and 165.70 (1C; pyridazinone C3); C23H21Cl3N6O2 MS (ESI+) calculated: 518.08, Found: m/e 519.0864 (M+; 100.0%), 523.0827 (M+4; 30.7%) and 524.0864 (M+6; 3.4%).

6-(4-(3,4-Dicholorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-acetyl-2-(4-chlorobenzal)hydrazon (5d)

White crystals; yield: 56.24%; mp: 275–277 °C; IR (ν cm−1, ATR): 3066 (C–H aromatic), 2919 (C–H aliphatic), 1667 (C=O), 1596 (C=N), 1238 (C–N) and 837 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 3.31 (4H; t; CH2–N; b + b′), 3.50 (4H; t; CH2–N; a + a′), 5.28 (2H; s; –N–CH2–C=O), 6.90 (1H; d; pyridazinone H5), 6.98 (1H; d; pyridazinone H4), 7.02–7.44 (7H; m; phenyl protons), 8.18 (1H; s; –N=CH–) and 10.51 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.17 (2C; CH2–N), 47.59 (2C; CH2–N), 79.14 (1C; –N–CH2–C=O), 112.20 (1C;=CH), 116.08 (1C; pyridazinone C5), 117.03 (1C; 3,4-dichlorophenyl C6), 120.52 (1C; 3,4-dichlorophenyl C5), 128.97 (1C; pyridazinone C4), 130.92 (2C; 4-chlorophenyl C2,6), 131.90 (2C; 4-chlorophenyl C3,5), 134.88 (1C; 3,4-dichloroyphenyl C2), 139.99 (1C; 4-chlorophenyl C1), 143.10 (1C; 3,4-dichloroyphenyl C1), 146.21 (1C; pyridazinone C6), 148.98 (1C; 4-chlorophenyl C4), 150.99 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.49 (1C; pyridazinone C3); C23H21Cl3N6O2 MS (ESI+) calculated: 518.08, Found: m/e 519.0859 (M+; 100.0%), 523.0807 (M+4; 30.7%) and 524.0828 (M+6; 3.4%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-fluorobenzal)hydrazon (5e)

White crystals; yield: 84.03%; mp: 228–230 °C; IR (ν cm−1, ATR): 3001 (C–H aromatic), 2972 (C–H aliphatic), 1687 (C=O), 1585 (C=N), 1236 (C–N) and 836 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 3.10 (4H; t; CH2–N; b + b′), 3.27 (4H; t; CH2–N; a + a′), 5.09 (2H; s; –N–CH2–C=O), 6.65 (1H; d; pyridazinone H5), 6.79 (1H; d; pyridazinone H4), 6.84–7.53 (7H; m; phenyl protons), 7.78 (1H; s; –N=CH–) and 11.10 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.17 (2C; CH2–N), 47.59 (2C; CH2–N), 78.92 (1C; –N–CH2–C=O), 116.10 (1C;=CH), 116.17 (1C; pyridazinone C5), 117.03 (1C; 3,4-dichlorophenyl C6), 120.50 (1C; 3,4-dichlorophenyl C5), 127.09 (1C; pyridazinone C4), 129.60 (2C; 4-fluorophenyl C2,6), 131.00 (2C; 4-fluorophenyl C3,5), 134.88 (1C; 3,4-dichloroyphenyl C2), 143.28 (1C; 4-fluorophenyl C1), 146.42 (1C; 3,4-dichloroyphenyl C1), 149.01 (1C; pyridazinone C6), 151.01 (1C; 4-fluorophenyl C4), 158.34 (2C; 3,4-dichlorophenyl C3,4), 161.86 (1C; CH2–N–C=O) and 168.45 (1C; pyridazinone C3); C23H21Cl2FN6O2 MS (ESI+) calculated: 502.11, Found: m/e 503.1142 (M+; 100.0%), 505.1117 (M+2; 64.9%) and 507.1105 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(3-methylbenzal)hydrazon (5f)

White crystals; yield: 79.28%; mp: 297–299 °C; IR (ν cm−1, ATR): 3059 (C–H aromatic), 2888 (C–H aliphatic), 1693 (C=O), 1570 (C=N), 1237 (C–N) and 845 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.51 (3H; s; –CH3), 3.29 (4H; t; CH2–N; b + b′), 3.37 (4H; t; CH2-N; a + a′), 5.06 (2H; s; –N–CH2–C=O), 7.32 (1H; d; pyridazinone H5), 7.33 (1H; d; pyridazinone H4), 7.34–7.66 (7H; m; phenyl protons), 7.98 (1H; s; –N=CH–) and 11.62 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 21.35 (1C; –CH3), 46.17 (2C; CH2–N), 47.56 (2C; CH2–N), 56.49 (1C; –N–CH2–C=O), 116.08 (1C;=CH), 117.04 (1C; pyridazinone C5), 120.42 (1C; 3,4-dichlorophenyl C6), 124.75 (1C; 3,4-dichlorophenyl C5), 127.67 (1C; pyridazinone C4), 129.17 (1C; 3-methylphenyl C2), 130.95 (1C; 3-methylphenyl C4), 131.99 (1C; 3-methylphenyl C5), 134.38 (1C; 3-methylphenyl C6), 138.54 (1C; 3-methylphenyl C3), 144.42 (1C; 3,4-dichlorophenyl C2), 147.47 (1C; 3-methylphenyl C1), 148.92 (1C; 3,4-dichlorophenyl C1), 151.04 (1C; pyridazinone C6), 158.23 (2C; 3,4-dichlorophenyl C3,4), 163.71 (1C; CH2–N–C=O) and 168.42 (1C; pyridazinone C3); C24H24Cl2N6O2 MS (ESI+) calculated: 498.13, Found: m/e 499.1408 (M+; 100.0%), 501.1368 (M+2; 64.5%) and 502.1409 (M+4; 10.6%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-methylbenzal)hydrazon (5g)

White crystals; yield: 81.79%; mp: 255–257 °C; IR (ν cm−1, ATR): 3086 (C–H aromatic), 2953 (C–H aliphatic), 1684 (C=O), 1568 (C=N), 1237 (C–N) and 840 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 2.24 (3H; s; –CH3), 3.12 (4H; t; CH2–N; b + b′), 3.29 (4H; t; CH2–N; a + a′), 5.11 (2H; s; –N–CH2–C=O), 6.65 (1H; d; pyridazinone H5), 6.68 (1H; d; pyridazinone H4), 6.78–7.43 (7H; m; phenyl protons), 7.78 (1H; s; –N=CH–) and 10.89 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 28.56 (1C; –CH3), 47.21 (2C; CH2–N), 48.32 (2C; CH2–N), 78.57 (1C; –N–CH2–C=O), 114.53 (1C;=CH), 116.03 (1C; pyridazinone C5), 118.56 (1C; 3,4-dichlorophenyl C6), 121.42 (1C; 3,4-dichlorophenyl C5), 130.14 (1C; pyridazinone C4), 130.86 (1C; 4-methylphenyl C2), 130.98 (1C; 4-methylphenyl C4), 131.90 (1C; 4-methylphenyl C5), 132.00 (1C; 4-methylphenyl C6), 134.88 (1C; 4-methylphenyl C3), 137.45 (1C; 3,4-dichlorophenyl C2), 138.99 (1C; 4-methylphenyl C1), 143.10 (1C; 3,4-dichlorophenyl C1), 146.21 (1C; pyridazinone C6), 151.87 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.52 (1C; pyridazinone C3); C24H24Cl2N6O2 MS (ESI+) calculated: 498.13, Found: m/e 499.1443 (M+; 100.0%), 501.1412 (M+2; 64.5%) and 503.1402 (M+4; 10.6%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2-methoxybenzal)hydrazon (5h)

White crystals; yield: 86.32%; mp: 263–265 °C; IR (ν cm−1, ATR): 3063 (C–H aromatic), 2953 (C–H aliphatic), 1695 (C=O), 1568 (C=N), 1236 (C–N), 1091 (C–O) and 839 (C–Cl); 1H-NMR (CDCl3-d, 300 MHz): δ (ppm) 3.05 (4H; t; CH2–N; b + b′), 3.46 (4H; t; CH2-N; a + a′), 4.87 (3H; s; –O–CH3), 5.29 (2H; s; –N–CH2–C=O), 6.82 (1H; d; pyridazinone H5), 6.86 (1H; d; pyridazinone H4), 6.94–7.84 (7H; m; phenyl protons), 8.06 (1H; s; –N=CH–) and 10.59 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.61 (2C; CH2–N), 48.32 (2C; CH2–N), 56.01 (1C; O–CH3), 77.12 (1C; –N–CH2–C=O), 115.87 (1C;=CH), 116.03 (1C; pyridazinone C5), 117.21 (1C; 3,4-dichlorophenyl C6), 120.55 (1C; 3,4-dichlorophenyl C5), 126.47 (1C; pyridazinone C4), 130.23 (1C; 2-methoxyphenyl C4), 130.62 (1C; 2-methoxyphenyl C5), 130.74 (1C; 2-methoxyphenyl C3), 130.98 (1C; 2-methoxyphenyl C6), 131.90 (1C; 3,4-dichlorophenyl C2), 135.12 (1C; 2-methoxyphenyl C1), 144.36 (1C; 3,4-dichlorophenyl C1), 146.96 (1C; pyridazinone C6), 148.56 (1C; 2-methoxyphenyl C2), 150.93 (2C; 3,4-dichlorophenyl C3,4), 158.07 (1C; CH2–N–C=O) and 165.70 (1C; pyridazinone C3); C24H24Cl2N6O3 MS (ESI+) calculated: 514.13, Found: m/e 515.1364 (M+; 100.0%), 517.1365 (M+2; 65.1%) and 518.1400 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(3-methoxybenzal)hydrazon (5i)

White crystals; yield: 80.32%; mp: 258–260 °C; IR (ν cm−1, ATR): 3080 (C–H aromatic), 2954 (C–H aliphatic), 1695 (C=O), 1569 (C=N), 1236 (C–N), 1042 (C–O) and 844 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.52 (4H; t; CH2–N; b + b′), 3.81 (4H; t; CH2–N; a + a′), 4.68 (3H; s; –O–CH3), 5.08 (2H; s; –N–CH2–C=O), 6.92 (1H; d; pyridazinone H5), 7.02 (1H; d; pyridazinone H4), 7.21–7.68 (7H; m; phenyl protons), 7.99 (1H; s; –N=CH–) and 11.68 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.17 (2C; CH2–N), 47.21 (2C; CH2–N), 55.99 (1C; O–CH3), 78.57 (1C; –N–CH2–C=O), 113.66 (1C;=CH), 116.03 (1C; pyridazinone C5), 117.21 (1C; 3,4-dichlorophenyl C6), 120.52 (1C; 3,4-dichlorophenyl C5), 129.27 (1C; pyridazinone C4), 130.93 (1C; 3-methoxyphenyl C4), 130.98 (1C; 3-methoxyphenyl C5), 131.90 (1C; 3-methoxyphenyl C2), 132.00 (1C; 3-methoxyphenyl C6), 134.88 (1C; 3,4-dichlorophenyl C2), 139.99 (1C; 3-methoxyphenyl C1), 143.10 (1C; 3,4-dichlorophenyl C1), 146.21 (1C; pyridazinone C6), 148.45 (1C; 3-methoxyphenyl C2), 150.99 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.49 (1C; pyridazinone C3); C24H24Cl2N6O3 MS (ESI+) calculated: 514.13, Found: m/e 515.1358 (M+; 100.0%), 517.1332 (M+2; 65.1%) and 518.1354 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-methoxybenzal)hydrazon (5k)

White crystals; yield: 93.86%; mp: 228–230 °C; IR (ν cm−1, ATR): 3065 (C–H aromatic), 2953 (C–H aliphatic), 1683 (C=O), 1574 (C=N), 1237 (C–N), 1032 (C–O) and 826 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.51 (4H; t; CH2–N; b + b′), 3.29 (4H; t; CH2–N; a + a′), 3.81 (3H; s; –O–CH3), 5.04 (2H; s; –N–CH2–C=O), 6.92 (1H; d; pyridazinone H5), 6.98 (1H; d; pyridazinone H4), 7.01–7.67 (7H; m; phenyl protons), 7.96 (1H; s; –N=CH–) and 11.53 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.17 (2C; CH2–N), 47.59 (2C; CH2–N), 53.07 (1C; O–CH3), 79.14 (1C; –N–CH2–C=O), 112.20 (1C;=CH), 117.03 (1C; pyridazinone C5), 117.21 (1C; 3,4-dichlorophenyl C6), 120.52 (1C; 3,4-dichlorophenyl C5), 128.97 (1C; pyridazinone C4), 130.92 (2C; 4-methoxyphenyl C2,6), 132.00 (2C; 4-methoxyphenyl C3,5), 134.88 (1C; 3,4-dichlorophenyl C2), 139.99 (1C; 4-methoxyphenyl C1), 143.10 (1C; 3,4-dichlorophenyl C1), 146.21 (1C; pyridazinone C6), 148.98 (1C; 4-methoxyphenyl C4), 150.99 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.49 (1C; pyridazinone C3); C24H24Cl2N6O3 MS (ESI+) calculated: 514.13, Found: m/e 515.1380 (M+; 100.0%), 517.1362 (M+2; 65.1%) and 518.1405 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2,3-dimethoxybenzal)hydrazon (5l)

White crystals; yield: 75.12%; mp: 265–267 °C; IR (ν cm−1, ATR): 3040 (C–H aromatic), 2969 (C–H aliphatic), 1690 (C=O), 1592 (C=N), 1231 (C–N), 1065 (C–O) and 836 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.27 (4H; t; CH2–N; b + b′), 3.54 (4H; t; CH2–N; a + a′), 3.76 (3H; s; –O–CH3), 3.82 (3H; s; –O–CH3), 5.04 (2H; s; –N–CH2–C=O), 6.98 (1H; d; pyridazinone H5), 7.10 (1H; d; pyridazinone H4), 7.11–7.62 (6H; m; phenyl protons), 8.28 (1H; s; –N=CH–) and 11.59 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.12 (2C; CH2–N), 47.56 (2C; CH2–N), 56.21 (1C; O–CH3), 56.56 (1C; O–CH3), 61.70 (1C; –N–CH2–C=O), 114.65 (1C;=CH), 116.14 (1C; pyridazinone C5), 117.02 (1C; 3,4-dichlorophenyl C6), 117.41 (1C; 3,4-dichlorophenyl C5), 120.48 (1C; pyridazinone C4), 124.94 (1C; 2,3-dimethoxyphenyl C5) 127.78 (1C; 2,3-dimethoxyphenyl C6), 130.99 (1C; 2,3-dimethoxyphenyl C4), 131.98 (1C; 3,4-dichlorophenyl C2), 140.30 (2C; 3,4-dichlorophenyl C1, 2,3-dimethoxyphenyl C1), 148.33 (1C; pyridazinone C6), 149.09 (2C; 2,3-dimethoxyphenyl C2,3), 151.01 (2C; 3,4-dichlorophenyl C3,4), 153.10 (1C; CH2–N–C=O) and 158.44 (1C; pyridazinone C3); C25H26Cl2N6O4 MS (ESI+) calculated: 544.14, Found: m/e 545.1468 (M+; 100.0%), 547.1440 (M+2; 65.4%) and 548.1469 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(3,5-dimethoxybenzal)hydrazon (5m)

White crystals; yield: 65.27%; mp: 271–273 °C; IR (ν cm−1, ATR): 3040 (C–H aromatic), 2960 (C–H aliphatic), 1681 (C=O), 1578 (C=N), 1235 (C–N), 1064 (C–O) and 827 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.27 (4H; t; CH2–N; b + b′), 3.38 (4H; t; CH2–N; a + a′), 3.78 (6H; s; –O–CH3), 5.06 (2H; s; –N–CH2–C=O), 6.86 (1H; d; pyridazinone H5), 6.91 (1H; d; pyridazinone H4), 6.93–7.90 (6H; m; phenyl protons), 8.19 (1H; s; –N=CH–) and 11.62 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.27 (2C; CH2–N), 47.75 (2C; CH2–N), 55.63 (2C; O–CH3), 79.14 (1C; –N–CH2–C=O), 102.53 (1C;=CH), 104.97 (1C; pyridazinone C5), 105.27 (1C; 3,4-dichlorophenyl C6), 120.52 (1C; 3,4-dichlorophenyl C5), 126.89 (1C; pyridazinone C4), 130.76 (2C; 3,5-dimethoxyphenyl C2,6), 130.93 (1C; 3,4-dichlorophenyl C2), 132.12 (1C; 3,5-dimethoxyphenyl C4), 136.37 (1C; 3,5-dimethoxyphenyl C1), 144.06 (1C; 3,4-dichlorophenyl C1), 148.84 (1C; pyridazinone C6), 150.89 (2C; 3,5-dimethoxyphenyl C3,5), 158.25 (2C; 3,4-dichlorophenyl C3,4), 161.06 (1C; CH2–N–C=O) and 168.38 (1C; pyridazinone C3); C25H26Cl2N6O4 MS (ESI+) calculated: 544.14, Found: m/e 545.1475 (M+; 100.0%), 547.1434 (M+2; 65.4%) and 548.1468 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2,4,6-trimethoxybenzal)hydrazon (5n)

White crystals; yield: 61.86%; mp: 273–275 °C; IR (ν cm−1, ATR): 3083 (C–H aromatic), 2969 (C–H aliphatic), 1667 (C=O), 1592 (C=N), 1238 (C–N), 1061 (C–O) and 836 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.30 (4H; t; CH2–N; b + b′), 3.56 (4H; t; CH2–N; a + a′), 3.79 (3H; s; –O–CH3), 3.82 (6H; s; –O–CH3), 4.94 (2H; s; –N–CH2–C=O), 6.27 (1H; d; pyridazinone H5), 6.28 (1H; d; pyridazinone H4), 6.89–7.65 (5H; m; phenyl protons), 8.19 (1H; s; –N=CH–) and 11.27 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.17 (2C; CH2–N), 47.59 (2C; CH2–N), 55.04 (3C; O–CH3), 79.14 (1C; –N–CH2–C=O), 112.20 (1C;=CH), 115.45 (1C; pyridazinone C5), 117.03 (1C; 3,4-dichlorophenyl C6), 120.52 (1C; 3,4-dichlorophenyl C5), 128.97 (1C; pyridazinone C4), 131.90 (1C; 3,4-dichlorophenyl C2), 134.88 (2C; 2,4,6-trimethoxyphenyl C3,5), 139.99 (1C; 2,4,6-trimethoxyphenyl C1), 143.10 (1C; 3,4-dichlorophenyl C1), 146.21 (1C; pyridazinone C6), 148.98 (1C; 2,4,6-trimethoxyphenyl C4), 149.07 (2C; 2,4,6-trimethoxyphenyl C2,6), 150.99 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.49 (1C; pyridazinone C3). C26H28Cl2N6O5 MS (ESI+) calculated: 574.15, Found: m/e 575.1569 (M+; 100.0%), 577.1545 (M+2; 65.6%) and 579.1544 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(3,4,5-trimethoxybenzal)hydrazon (5o)

White crystals; yield: 67.02%; mp: 286–288 °C; IR (ν cm−1, ATR): 3080 (C–H aromatic), 2938 (C–H aliphatic), 1662 (C=O), 1591 (C=N), 1234 (C–N), 1078 (C–O) and 826 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.28 (4H; t; CH2–N; b + b′), 3.36 (4H; t; CH2–N; a + a′), 3.70 (3H; s; –O–CH3), 3.83 (6H; s; –O–CH3), 5.07 (2H; s; –N–CH2–C=O), 6.99 (1H; d; pyridazinone H5), 7.01 (1H; d; pyridazinone H4), 7.02–7.66 (5H; m; phenyl protons), 7.92 (1H; s; –N=CH–) and 11.66 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.20 (2C; CH2–N), 47.55 (2C; CH2–N), 56.40 (3C; O–CH3), 60.57 (1C; –N–CH2–C=O), 104.79 (1C;=CH), 116.09 (1C; pyridazinone C5), 117.04 (1C; 3,4-dichlorophenyl C6), 120.43 (1C; 3,4-dichlorophenyl C5), 127.18 (1C; pyridazinone C4), 130.10 (1C; 3,4-dichlorophenyl C2), 139.51 (2C; 3,4,5-trimethoxyphenyl C2,6), 139.68 (1C; 3,4,5-trimethoxyphenyl C1), 144.07 (1C; 3,4-dichlorophenyl C1), 147.39 (1C; pyridazinone C6), 153.63 (3C; 3,4,5-trimethoxyphenyl C3,4,5), 158.21 (2C; 3,4-dichlorophenyl C3,4), 163.69 (1C; CH2–N–C=O) and 168.46 (1C; pyridazinone C3); C26H28Cl2N6O5 MS (ESI+) calculated: 574.15, Found: m/e 575.1580 (M+; 100.0%), 577.1530 (M+2; 65.6%) and 578.1566 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-dimethylaminobenzal)hydrazon (5p)

Pale-yellow crystals; yield: 78.30%; mp: 284–286 °C; IR (ν cm−1, ATR): 3091 (C–H aromatic), 2972 (C–H aliphatic), 1660 (C=O), 1590 (C=N), 1235 (C–N) and 843 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 2.97 (6H; s; N–(CH3)2), 3.29 (4H; t; CH2–N; b + b′), 3.31 (4H; t; CH2–N; a + a′), 5.01 (2H; s; –N–CH2–C=O), 6.72 (1H; d; pyridazinone H5), 6.90 (1H; d; pyridazinone H4), 6.98–7.66 (7H; m; phenyl protons), 7.88 (1H; s; –N=CH–) and 11.36 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 42.35 (2C; N–(CH3)2), 46.17 (2C; CH2–N), 47.59 (2C; CH2–N), 79.14 (1C; –N–CH2–C=O), 112.20 (1C;=CH), 116.09 (1C; pyridazinone C5), 117.03 (1C; 3,4-dichlorophenyl C6), 120.52 (1C; 3,4-dichlorophenyl C5), 128.97 (1C; pyridazinone C4), 130.92 (2C; 4-dimethylaminophenyl C2,6), 132.00 (2C; 4-dimethylaminophenyl C3,5), 134.88 (1C; 3,4-dichlorophenyl C2), 139.99 (1C; 4-dimethylaminophenyl C1), 143.10 (1C; 4-dimethylaminophenyl C4), 146.21 (1C; 3,4-dichlorophenyl C1), 148.98 (1C; pyridazinone C6), 150.99 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.46 (1C; pyridazinone C3); C25H27Cl2N7O2 MS (ESI+) calculated: 527.16, Found: m/e 528.1679 (M+; 100.0%), 530.1692 (M+2; 65.0%) and 532.1642 (M+4; 10.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-nitrobenzal)hydrazon (5r)

Yellow crystals; yield: 85.27%; mp: 207–209 °C; IR (ν cm−1, ATR): 3074 (C–H aromatic), 2842 (C–H aliphatic), 1691 (C=O), 1579 (C=N), 1234 (C–N), 926 (N–O) and 833 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.29 (4H; t; CH2–N; b + b′), 3.37 (4H; t; CH2–N; a + a′), 5.11 (2H; s; –N–CH2–C=O), 6.92 (1H; d; pyridazinone H5), 6.98 (1H; d; pyridazinone H4), 7.00–7.97 (7H; m; phenyl protons), 8.12 (1H; s; –N=CH–) and 11.98 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.13 (2C; CH2–N), 48.59 (2C; CH2–N), 78.14 (1C; –N–CH2–C=O), 114.03 (1C;=CH), 116.09 (1C; pyridazinone C5), 118.13 (1C; 3,4-dichlorophenyl C6), 121.52 (1C; 3,4-dichlorophenyl C5), 128.63 (1C; pyridazinone C4), 131.92 (1C; 4-nitrophenyl C2), 132.01 (1C; 4-nitrophenyl C6), 132.90 (1C; 4-nitrophenyl C3), 133.00 (1C; 4-nitrophenyl C5), 134.88 (1C; 3,4-dichlorophenyl C2), 140.07 (1C; 4-nitrophenyl C1), 143.10 (1C; 4-nitrophenyl C4), 147.21 (1C; 3,4-dichlorophenyl C1), 148.98 (1C; pyridazinone C6), 150.99 (2C; 3,4-dichlorophenyl C3,4), 158.31 (1C; CH2–N–C=O) and 168.49 (1C; pyridazinone C3); C23H21Cl2N7O4 MS (ESI+) calculated: 529.10, Found: m/e 530.1080 (M+; 100.0%), 532.1066 (M+2; 64.6%) and 534.1066 (M+4; 11.2%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2-cyanobenzal)hydrazon (5s)

White crystals; yield: 88.73%; mp: 236–8 °C; IR (ν cm−1, ATR): 3068 (C–H aromatic), 2968 (C–H aliphatic), 2227 (C≡N), 1687 (C=O), 1567 (C=N), 1238 (C–N) and 844 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.29 (4H; t; CH2–N; b + b′), 3.37 (4H; t; CH2–N; a + a′), 5.09 (2H; s; –N–CH2–C=O), 7.41 (1H; d; pyridazinone H5), 7.42 (1H; d; pyridazinone H4), 7.59–7.99 (7H; m; phenyl protons), 8.26 (1H; s; –N=CH–) and 11.95 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.17 (2C; CH2–N), 47.56 (2C; CH2–N), 53.22 (1C; C≡N), 56.49 (1C; –N–CH2–C=O), 111.34 (1C;=CH), 118.09 (1C; pyridazinone C5), 120.42 (1C; 3,4-dichlorophenyl C6), 127.25 (1C; 3,4-dichlorophenyl C5), 128.37 (1C; pyridazinone C4), 130.95 (1C; 2-cyanophenyl C4), 131.99 (1C; 2-cyanophenyl C5), 134.03 (1C; 2-cyanophenyl C3), 136.58 (1C; 2-cyanophenyl C6), 137.18 (1C; 3,4-dichlorophenyl C2), 140.40 (1C; 2-cyanophenyl C1), 142.83 (1C; 2-cyanophenyl C2), 149.01 (1C; 3,4-dichlorophenyl C1), 151.03 (1C; pyridazinone C6), 158.25 (2C; 3,4-dichlorophenyl C3,4), 164.16 (1C; CH2–N–C=O) and 168.92 (1C; pyridazinone C3); C24H21Cl2N7O2 MS (ESI+) calculated: 509.11, Found: m/e 510.1203 (M+; 100.0%), 512.1187 (M+2; 64.6%) and 513.1224 (M+4; 10.9%).

6-[4-(3,4-Dicholorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(3-cyanobenzal)hydrazon (5t)

White crystals; yield: 65.03%; mp: 273–275 °C; IR (ν cm−1, ATR): 3093 (C–H aromatic), 2954 (C–H aliphatic), 2230 (C≡N), 1698 (C=O), 1568 (C=N), 1237 (C–N) and 841 (C–Cl); 1H-NMR (DMSO-d 6 , 300 MHz): δ (ppm) 3.28 (4H; t; CH2–N; b + b′), 3.39 (4H; t; CH2–N; a + a′), 5.12 (2H; s; –N–CH2–C=O), 6.88 (1H; d; pyridazinone H5), 6.92 (1H; d; pyridazinone H4), 7.11–8.05 (7H; m; phenyl protons), 8.18 (1H; s; –N=CH–) and 11.80 (1H; s; –NH–N); 13C-NMR (DMSO-d 6 , 300 MHz), δ 46.27 (2C; CH2–N), 47.78 (2C; CH2–N), 53.08 (1C; C≡N), 79.20 (1C; –N–CH2–C=O), 112.66 (1C;=CH), 115.96 (1C; pyridazinone C5), 117.11 (1C; 3,4-dichlorophenyl C6), 120.94 (1C; 3,4-dichlorophenyl C5), 126.88 (1C; pyridazinone C4), 130.18 (1C; 3-cyanophenyl C2), 130.74 (1C; 3-cyanophenyl C4), 130.91 (1C; 3-cyanophenyl C5), 131.48 (1C; 3-cyanophenyl C6), 132.14 (1C; 3,4-dichlorophenyl C2), 133.17 (1C; 3-cyanophenyl C1), 135.81 (1C; 3-cyanophenyl C3), 141.91 (1C; 3,4-dichlorophenyl C1), 148.85 (1C; pyridazinone C6), 150.85 (2C; 3,4-dichlorophenyl C3,4), 158.27 (1C; CH2–N–C=O) and 168.61 (1C; pyridazinone C3); C24H21Cl2N7O2 MS (ESI+) calculated: 509.11, Found: m/e 510.1224 (M+; 100.0%), 512.1208 (M+2; 64.6%) and 513.1225 (M+4; 10.9%).

Anticholinesterase activity

The in vitro inhibition of AChE and BChE for 5a–t was determined by the method of Ellman et al. (1961) using galantamine (CAS 357-70-0) as reference. Electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma, St. Louis, MO, USA) and horse serum BChE (EC 3.1.1.8, Sigma) were employed as the enzyme sources, acetylthiocholine iodide and butyrylthiocholine chloride (Sigma) as substrates, and 5,5′-dithio-bis(2-nitrobenzoic)acid (DTNB) for activity determination. All reagents and conditions were the same as previously described (Orhan et al. 2008). Briefly, in this method, 140 µl 0.1 mM sodium phosphate buffer (pH8.0), 20 µl DTNB, 20 µl test solution and 20 µl AChE/BChE solution were added by multichannel automatic pipette (Gilson pipetman, Middleton, USA) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of 10 µl of acetylthiocholine iodide/butyryl-thiocholine chloride. The hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was determined by the formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm and monitored by a 96-well microplate reader (VersaMax Molecular Devices, North Carolina, USA). The measurements and calculations were evaluated by using Softmax PRO 4.3.2.LS software (Softmax Moleculer Devices, Downingtown, USA). The percentage of inhibition of AChE/BChE was determined by comparison of the reaction rates of the samples relative to the blank sample (ethanol in phosphate buffer, pH = 8) using the formula (E-S)/E × 100, where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. The experiments were done in triplicate and the results were expressed as average values with SD.

Molecular docking

Ligands were sketched, their pKa calculations were performed on MarvinSketch, Marvin 15.5.25.0, 2015, ChemAxon (http://www.chemaxon.com), and they were prepared for docking using LigPrep, version 3.6, Schrödinger, LLC, New York, NY, 2015. OPLS 2005 force field parameters were used for their minimization (Banks et al. 2005). PDB entries were retrieved from Protein Data Bank (http://www.rcsb.org/). Coordinates of the centre of the co-crystallised ligands were picked as the centre of search space for each enzyme and the explicit water molecules in the active site gorges were kept. Molecular docking simulations were performed on Glide, version 6.9, Schrödinger, LLC, New York, NY, 2015 (Friesner et al. 2004, 2006; Halgren et al. 2004) using the following settings: Standard precision with enhanced conformer sampling was set, ligands were kept flexible, 10 poses were written at most per ligand, post-minimization was enabled, and per-residue interaction scores for residues within 12 Å of grid centres were written. Prior to ligand docking, in order to validate our method, we re-docked the co-crystallised ligands, donepezil (PDB ligand code E20) and tacrine (PDB ligand code THA), in AChE and BChE active sites, respectively and were able to reproduce conformers close to the original binding modes (RMSD: 0.82 Å for 4EY7 and 0.85 Å for 4BDS). Docking poses were visually inspected using pose viewer of Maestro, version 10.4, Schrödinger, LLC, New York, NY, 2015.