Synthesis, biological evaluation and molecular modeling studies of phthalazin-1(2H)-one derivatives as novel cholinesterase inhibitors

Abstract

A new series of donepezil analogues based on the phthalazin-1(2H)-one scaffold was designed and synthesized with the aim of exploring its potential as human ChEIs. Biological results revealed that the structural modifications proposed significantly affected ChE inhibitory potency as well as selectivity for AChE/BuChE. Compound 1d showed promising in vitro inhibition of both enzymes in the μM range. However, most target compounds were significantly more active against AChE than BuChE, specifically 1f, 1h and 1j, with IC₅₀ values in the low micromolar or submicromolar range, the most active compounds in the series. Docking simulations suggested that the most active compounds can recognize the donepezil binding site using a similar interactions network. These results allowed us to rationalize the observed structure–activity relationships. Moreover, the predicted physicochemical and ADME properties were also comparable to those of donepezil.

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1. Introduction

Alzheimer's disease (AD) is a complex and progressive neurodegenerative disorder of the central nervous system that constitutes the most common type of dementia in the world. The prevalence of AD, which increases with age, ranges from 1–2% at age 65 years to 35% or higher at age 85 years.¹ Currently, it is estimated that nearly 40 million people worldwide are affected by AD, and the number of patients will likely increase in future due to increased life expectancy.²

This disorder is characterized by a decreased number of brain cholinergic neurons in the hippocampus and frontal cortex, which is clinically reflected in specific symptoms, such as progressive impairment in memory and intellectual ability to perform basic activities of daily living.¹ Although the etiology of AD is not yet known, extracellular deposits of aberrant proteins, namely β-amyloid (Aβ) and τ-protein, oxidative stress and neurotoxicity, both related to a dysfunction of the glutamate neurotransmission system, dyshomeostasis of biometals and low levels of acetylcholine (ACh) seems to play significant roles in the pathophysiology of the disease.^1,3 The lack of such pivotal information explains why the pharmacological approaches in current use improve symptoms but do not have profound disease-modifying effects.^1,4 They are based on targeting neurotransmitter dysfunctions including acetylcholinesterase inhibitors (AChEIs), and N-methyl-D-aspartic acid (NMDA) glutamate receptor antagonists. Five drugs have been approved by the U.S. Food and Drug Administration (FDA) for AD: four AChEIs—tacrine, donepezil, rivastigmine and galantamine—and one NMDA antagonist, memantine.

Studies developed in recent decades indicate that the acetylcholinesterase (AChE) in AD brains, besides catalysing the hydrolysis of acetylcholine, also plays an important role in Aβ plaques deposition because of its interaction with the Aβ peptide through a set of amino acids located close to the peripheral anionic site (PAS) of the enzyme;⁵ thus AChEIs that bind to PAS could inhibit such processes.⁶ Furthermore, new findings about the roles of neuronal and non-neuronal cholinergic systems on modulation of regional brain blood flow may also contribute to better cholinergic therapies for AD.⁷

In addition, ACh levels are also regulated by butyrylcholinesterase (BuChE), another cholinesterase enzyme with a synaptic ACh hydrolysis role that is less important than AChE in healthy brains. Recent studies indicate that in AD the AChE activity remains unchanged or even declines while activity of BuChE progressively increases, suggesting that BuChE inhibition may also be considered a valid approach for AD therapy.⁸ The BuChE role in regulating cholinergic transmission is not yet fully understood and AChE currently remains as the main target. Nevertheless, this new scenario has encouraged the search for drugs inhibiting both cholinesterase enzymes (ChE), AChE and BuChE.⁹

Donepezil, a selective inhibitor of AChE acting as dual binding-site ligand,¹⁰ is the only one of four FDA-approved AChEIs that simultaneously binds to CAS (catalytic active site) and PAS sites, providing moderate inhibition of Aβ aggregation.¹¹ In this respect, recent clinical trials have shown that continued treatment with donepezil was associated with significant cognitive and functional benefits in patients suffering moderate to severe AD.¹²

X-ray crystallographic data of AChE/donepezil complexes together with structure–activity relationships (SAR) results for donepezil-like compounds, indicate that the N-benzylpiperidine group and the dimethoxy benzoyl fragment are key structural features for both dual binding interaction (CAS and PAS sites) and AChE inhibition.¹³

Taking into account the growing interest in cholinesterase inhibitors,^12,14,15 studying new pharmacophoric scaffolds is desirable. In this regard, pyridazine could be considered a privileged structure because its derivatives have great therapeutic potential.¹⁶ Although several pyridazine derivatives were reported as potential dual binding-site AChEIs, most of them show 3,6-disubstitutions on the pyridazine ring¹⁷ and the structure of only a few includes the pyridazin-3-(2H)-one framework.¹⁸ However, these last pyridazinone analogues do not share any structural similarity with donepezil.

Considering our work as a continuation of abovementioned studies, we have focused on developing enzyme inhibitors potentially effective in aging-related disorders,¹⁹ including the design of a series of donepezil analogues with structure 1 (Fig. 1). In these novel compounds the indanone portion was replaced by a phthalazin-1(2H)-one moiety, in which methoxy groups are both preserved and not preserved, and the linking chain between the new bicyclic fragment and N-benzylpiperidine group ranged from one to three carbon atoms. In addition, the C4 position of this new scaffold could be substituted with groups of different sizes and electronic properties. These structural modifications were performed with the idea of analyzing the ability of the phthalazinone nucleus to modulate AChE and BuChE activities.

Fig. 1 General structure of novel ChE inhibitors and donepezil.

Thus, in the current work we report the synthesis, AChE and BuChE inhibitory activities and molecular modeling of representative molecules for a new family of phthalazin-1(2H)-ones (compounds 1, Fig. 1). The molecular modeling studies were performed in order to compare the binding mode and ADME properties of novel compounds with donepezil.

2. Results and discussion

2.1 Chemistry

The proposed strategy for synthesizing the designed compounds 1a–j involves the N-alkylation of corresponding phthalazinone (compounds 10a–f) with the adequate bromoalkane.²⁰ Phthalazinones 10a,b, precursors of analogues 1a–d and 1i, were commercially available, while phthalazinones 10c–f, required for preparing the derivatives 1e–h and 1j, were synthesized as displayed in Scheme 1. The 4-methylphthalazinone 10c was obtained in good yield by direct cyclization of 2-acethyl benzoic acid 8 with hydrazine hydrate in ethanol, while synthesis of phthalazinones 10d–f, showing one or two methoxy groups on the phenyl ring, was accomplished by adapting several procedures previously described. Using the phthalides 5 as key intermediates via benzylic bromination provided the 3-bromo phthalides 9 suitable for cyclization with hydrazine.²¹

Scheme 1 Reagents and conditions: (i) 5 M NaOH, MeOH reflux, 96%; (ii) Ac₂O reflux, 98%; (iii) NaBH₄, THF, r.t.; 6 M HCl r.t. 33% (5c) and 52% (6); (iv) 6 M HCl reflux, 53% (two steps); (v) 30% HCHO, 37% HCl, 90 °C, 75% (5a) or 30% HCHO, 37% HCl, acetic acid 100 °C, 43% (5b); (vi) NBS, benzoyl peroxide, CCl₄, reflux; (vii) NH₂NH₂·H₂O, EtOH, reflux, 91% (10c), 58% (10d, two steps), 51% (10e, two steps), 65% (10f, two steps).

The 5,6-dimethoxyphthalide 5a and 6-methoxyphthalide 5b were synthesized in good to moderate yield by chloroformylation of 3,4-dimethoxybenzoic and 3-methoxybenzoic acids 7a and 7b, respectively.²² However, a multi-step strategy was required to obtain the 5-methoxyphthalide 5c. The synthetic sequence used involved the alkaline hydrolysis of dimethyl 4-methoxy phthalate 2 to give the dicarboxylic acid 3 and subsequent dehydration of 3 with an excess of acetic anhydride, followed by reduction with NaBH₄ of the phthalic anhydride 4 thus obtained.²³

The reaction of 4-substituted phthalic anhydrides with alkaline borohydrides usually resulted in a mixture of phthalide regioisomers of varying proportions, which is consequence of substituent electronic effects. Thus, when the substituent at C4 is an electron-donating group, a preference was detected for reduction at the m-carbonyl group.²⁴ In contrast with the literature, the treatment of 4-methoxyphthalic anhydride 4 with an equimolecular amount of NaBH₄ gave a mixture of lactone 5c and hydroxymethyl benzoic acid 6 (ratio 5c/6 1 : 1.5, very good yield), both resulting from selective nucleophilic attack by hydride on the m-carbonyl carbon. Although compounds 5c and 6 were initially separated by column chromatography and characterized (method A), the mixture of both compounds was refluxed with hydrochloric acid in order to complete the lactonization of hydroxymethyl benzoic acid 6 (method B). Subsequent radical bromination of phthalides 5a–c with N-bromosuccinimide (NBS) in carbon tetrachloride produced 3-bromophthalides 9, which were directly converted into phthalazinones 10d,f by condensation with hydrazine hydrate in ethanol.

The target compounds 1a–h were then prepared in four steps using phthalazinones 10 and the commercially available N-Boc-4-hydroxyalkylpiperidines 11 as starting material (Scheme 2). First, the N-Boc-4-hydroxyalkylpiperidines 11a,b were transformed into corresponding 4-bromoalkylpiperidines 12a,b by treatment with carbon tetrabromide and triphenylphosphine in dichloromethane at reflux. Then phthalazinones 10a–f were N-alkylated with the appropriate bromoalkyl derivative 12a,b and sodium hydride in dimethylformamide. Finally, the removal of the N-Boc protecting group from the corresponding 2-(N-Boc-4-piperidinylalkyl)phthalazin-1-ones 13a–h by acid hydrolysis with 6 M HCl in ethyl acetate was followed by treatment with benzyl bromide in the presence of sodium hydride to give good to moderate yields of desired compounds 1a–h.

Scheme 2 Reagents and conditions: (i) CBr₄, PPh₃, CH₂Cl₂, reflux, 94% (12a), 89% (12b); (ii) NaH, 12a or 12b, DMF, r.t. 78% (13a), 99% (13b), 99% (13c), 99% (13d), 99% (13e), 79% (13f), 45% (13g), 86% (13h); (iii) 6 M HCl, EtOAc, r.t.; (iv) NaH, BnBr, DMF, r.t. 34% (1a, two steps), 93% (1b, two steps), 30% (1c, two steps), 63% (1d, two steps), 34% (1e, two steps), 66% (1f, two steps), 59% (1g, two steps), 57% (1h, two steps).

Finally, Scheme 3 details the strategy adopted for the synthesis of target compounds 1i,j bearing an alkyl chain of three carbons between phthalazinone and N-benzylpiperidine fragments. In this case, the bromoalkane 18 was the reagent involved in N-alkylation of phthalazinones 10a and 10d. The compound 18 was synthesized in good yield starting with the commercially available 3-(pyridin-4-yl)propanol 15. The pyridine ring was reduced via catalytic hydrogenation at atmospheric pressure under acidic conditions and using platinum(IV) oxide as a catalyst to give 16 as a hydrochloride.²⁵ The resulting piperidine derivative was N-benzylated by treatment with benzyl bromide in ethanol in the presence of K₂CO₃ to give 17, which was converted into bromoalkane 18 by reaction with carbon tetrachloride and triphenylphosphine as described for bromo derivatives 12a,b.

Scheme 3 Reagents and conditions: (i) H₂, PtO₂, 4 M HCl dioxane, EtOH, 45 °C, 100%; (ii) BnBr, K₂CO₃, EtOH, r.t. to reflux 76%; (iii) CBr₄, PPh₃, CH₂Cl₂, reflux, 65%; (iv) NaH, 18, DMF, r.t. 70% (1i), 77% (1j).

2.2 Pharmacology

The in vitro activity of compounds 1a–j against AChE and BuChE was determined by Ellman's method²⁶ for evaluating the hydrolysis of acetylthiocholine and butyrylthiocholine, respectively. The activity was measured by the increase in absorbance at 412 nm due to the yellow color of 5-mercapto-2-nitrobenzoic acid produced by reaction of thiocholine with dithiobisnitrobenzoic acid (DNTB). Thiocholine was generated from acetylthiocholine using AChE isolated from human erythrocytes or from butyrylthiocholine using BuChE from human serum.

Assays were performed with a blank containing all components except AChE or BuChE in order to account for nonenzymatic reaction. The reaction rates were compared and the percent of inhibition due to the presence of test compounds was calculated. The ChE inhibitory activity of target compounds 1a–j and reference drugs (tacrine and donepezil) was expressed as IC₅₀ and these data are summarized in Table 1.

Table 1 IC₅₀ values for compounds 1 and reference inhibitors on enzymatic activity of human AChE and BuChE^a

Compound	n	R	R′	R′′	IC50 AChE (μM)	IC50 BuChE (μM)
a Values expressed as mean ± standard error of mean from three experiments (n = 3).
1a	1	H	H	H	>100	46.14 ± 3.08
1b	2	H	H	H	2.58 ± 0.39	64.69 ± 4.13
1c	1	p-Tol	H	H	>100	13.26 ± 0.88
1d	2	p-Tol	H	H	3.45 ± 0.23	5.50 ± 0.37
1e	2	CH3	H	H	2.79 ± 0.19	>100
1f	2	H	OCH3	OCH3	0.67 ± 0.04	39.24 ± 2.62
1g	2	H	H	OCH3	2.40 ± 0.16	27.47 ± 1.83
1h	2	H	OCH3	H	0.55 ± 0.04	53.14 ± 3.54
1i	3	H	H	H	10.29 ± 0.69	62.02 ± 4.13
1j	3	H	OCH3	OCH3	1.07 ± 0.07	>100
Donepezil	—	—	—	—	0.016 ± 0.003	12.01 ± 0.80
Tacrine	—	—	—	—	0.29 ± 0.06	0.15 ± 0.04

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Enzymatic assays revealed that most of the designed compounds showed moderate to good activity with IC₅₀ values for AChE and BuChE inhibition ranged from 10.29 to 0.55 μM and from 64.69 to 5.50 μM, respectively. At the same time the tested compounds, with the exception of compounds 1a, 1c and 1d, were remarkably more active against AChE than BuChE. The methoxy-substituted compounds 1f, 1h and 1j, with IC₅₀ values from the submicromolar to low micromolar range, were the most potent AChEIs of this series, resulting almost equipotent with tacrine but slightly less active than donepezil.

The three kinds of structural modifications proposed—length variation of alkyl chain linking phthalazinone and benzylpiperidine scaffolds, substitution at C4 and inclusion of methoxy groups in C6 or C7—significantly affected ChE inhibitory potency as well as selectivity of AChE/BuChE.

Thus, compounds 1a and 1c (both with n = 1) are inactive against AChE at 100 μM, the highest concentration tested, and moderately active against BuChE, with IC₅₀ values of 46.14 and 13.26 μM, respectively. In contrast, their homologues 1b, 1d and also the analogue 1e (all with n = 2) exhibited inhibitory activity against the AChE at low micromolar concentrations. Compound 1b was the most active with an IC₅₀ value of 2.58 μM. Compound 1e (IC₅₀ = 2.79 μM), showing a methyl group at C4, was equipotent to 1b and more potent than compound 1d (C4 = p-Tol, IC₅₀ = 3.45 μM). In addition, compound 1d was the only inhibitor equipotent for AChE and BuChE enzymes, behaving as a dual cholinesterase inhibitor (AChE IC₅₀ = 3.45 μM and BuChE IC₅₀ = 5.50 μM). However, a further elongation of the alkyl linker, such as in compound 1i (n = 3), had disadvantageous results for AChE inhibition (IC₅₀ = 10.29 μM).

Regarding the role of methoxy substituents at C6 and C7 in this new series, note that when it is placed at C6, the methoxy group provides a good and selective inhibitory effect against AChE. Compounds 1f (IC₅₀ = 0.67), 1h (IC₅₀ = 0.55) and 1j (IC₅₀ = 1.07) were the most potent and selective AChEIs of the series. When it was moved to the C7 position, AChE inhibitory activity seems unaffected (compound 1g, IC₅₀ = 2.40 μM).

2.3 Molecular modeling

Docking studies were carried out to analyze the AChE binding mode of the novel inhibitors with the aim of interpreting experimental affinity data.

To identify the docking protocol and protein–ligand complex that were more suitable for our analogues, we performed a benchmark study based on the self-docking procedure. We selected two protein–ligand complexes from the protein data bank (PDB): the complex of human AChE (hAChE) with donepezil (PDB ID: 4EY7), and the complex of human BuChE (hBuChE) with tacrine (PDB ID: 4BDS). The complex including donepezil was particularly interesting because of its molecular similarity to our analogues. The benchmark study revealed that several protocols were able to reproduce the crystallographic geometries (see the Experimental section for details). Among them, GOLD software when using ChemPLP as the scoring function, gave the best performance (ESI† Fig. 1, panels A and B). In particular, this protocol was able to reproduce both donepezil and tacrine with a RMSD values of 0.34 Å and 0.41 Å, respectively (ESI† Fig. 1, panels C and D).

The selected protocol was used to dock all analogues into hAChE and hBuChE as described in detail in the Experimental section.

All analogues showed a common binding mode similar to the donepezil one (Fig. 2). The N-benzylpiperidine group adopts the same orientation in the active site gorge, mainly interacting with Trp86, Tyr337, Phe338 and Ile451. The phthalazin-1(2H)-one moiety mimics the indanone group of donepezil that is placed in the binding site entrance formed by Tyr72, Tyr124, Trp286 and Tyr341 side chains and the backbone of Phe295 and Arg296.

Fig. 2 Left: Superposition of crystallographic complex donepezil (yellow) bound to AChE and compound 1f (grey) as derived by docking calculation. Molecular surface of protein (PDB ID: 4EY7) is colored according its lipophilicity, using the following color scheme: green (lipophilic region) to violet (hydrophilic region). Part of protein is shown with ribbon representation (grey) to permit clear visualization of ligands. Right: Ligand interaction diagram of 1f bound to AChE.

Based on our docking simulation, all compounds preserve this network of interactions (Fig. 3), as corroborated by the interaction energy values reported in Table 2, with small but significant dissimilarities. While the two important π–π interactions between the indole ring of Trp286 and the phthalazin-1(2H)-one moiety and between the indole ring of Trp86 and N-benzylpiperidine moiety are maintained over the series, one hydrogen bond between the carbonyl oxygen of phthalazin-1(2H)-one and the amide nitrogen of Phe295 is preserved only in compounds with an IC₅₀ less than 10 μM. This hydrogen bond is also present in the crystal structure of donepezil bound to hAChE, mediated by the indanone core.

Fig. 3 Per-residue analysis of protein–ligand interaction for each compound. In panel A, residues considered in analysis are represented as sticks and carbon atoms are grey, while ligand carbon atoms are shown using same representation, but with violet for carbon atoms. Panel B shows interaction energy fingerprints.

Table 2 Calculated docking score values for all synthetized analogues in complex with human AChE and BuChE

Compound	GOLDPLP SCORE hAChE	GOLDPLP SCORE hBuChE
1a	98.2w	68.0
1b	101	74.4
1c	98	70.2
1d	115.6	77.7
1e	111.6	68.3
1f	115.9	70.2
1g	103.3	65.7
1h	104.5	62.6
1j	111.1	67.7
1i	98.3	61.7

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This observation is more evident focusing on the per-residue analysis (Fig. 3), in which contribution to the interaction is computed for each singular residue. In our case, we measured the electrostatic interaction energy and a score taking into account hydrophobic interactions. Fig. 3 shows the per-residue analysis (panel B) for a subset of relevant residues taking into account their interaction and conformation with respect to the most potent analogue (compound 1h, panel A). The minor activity of compounds 1a and 1c could be attributed to the lack of this hydrogen bond. The bulky substituent at the 4-position of the phthalazinone moiety is not well tolerated into the binding pocket in terms of complementary shape and led to a slightly different orientation of the phtalazinone core (Fig. 4, 1c and 1d). However, in the case of compound 1d the longer alkyl linker allows stabilization of the ligand through a similar hydrogen bond with Arg296 instead of Phe295, and at the same time maintains the benzyl group stacked to Trp86, similar to the other analogues without the p-tolyl moiety.

Fig. 4 Binding mode of synthetized compounds. Code of each compound is reported on upper left of each panel. Molecular surface of protein (PDB ID: 4EY7) is colored according lipophilicity, using following color scheme: green (lipophilic region) to violet (hydrophilic region). Part of protein is shown with ribbon representation (grey) to permit clear visualization of ligand (azure stick representation). Molecular surface of ligand (transparent orange) is shown to underline its complementary with binding site.

The effect of the different linker length between compound 1c and 1d is evident upon observing differences in the per-residue profile (residues Trp86 and Arg296) and is in good agreement with experimental IC₅₀ values. On the other hand, the p-tolyl moiety provides an additional hydrophobic interaction with Trp286 and Leu289 (Fig. 3). The per-residue analysis also highlights the role of the methoxy substituent at C6 (compounds 1f, 1h and 1j) and methyl group at C4 (compound 1e). These compounds neither establish hydrophobic interaction by the methyl moiety nor are they involved in hydrogen bond through the ether oxygen atom of methoxy substituent. However, their steric effect improves the orientation of the carbonyl group involved in the hydrogen bond with Phe295, leading to a stronger electrostatic interaction (Fig. 3). The molecular docking studies did not show a clear relationship between the complex conformation and IC₅₀ values only in the case of compound 1i. The ligand orientation is extremely similar to those of the most active analogues but it lacks formation of the H-bond with the Phe295.

Docking studies performed on the hBuChE revealed an overall decrease in scores (Table 2). In this case, while the N-benzylpiperidine portion still occupies the same site as in hAChE, which is also the binding site of tacrine, the phthalazinone core adopts a different conformation, with the plane of the fused rings having an orthogonal angulation with respect to the conformation assumed in complexes with hAChE (Fig. 5, panel A). The reason for this orientation can be ascribed to the few differences between the two binding sites. Despite the notable similarity between the enzymes (Cα-RMSD 1.36 Å, ESI† Fig. 1, panel E), they show differences in some fundamental residues for binding phthalazinone group. Thus, the Trp286 that establishes the strong stacking interaction is replaced by an alanine. In addition, the amide involved in the hydrogen bond in hAChE (Phe295) is located in a loop that presents a different conformation in hBuChE (Leu286), making it incompatible with the hydrogen bond formation due to a distortion induced by Pro285, which is not present in hAChE (ESI† Fig. 1, panel E and F).

Fig. 5 Binding mode of compound 1f and 1d to hBuChE. Molecular surface of protein (PDB ID: 4BDS) is colored according to its lipophilicity using following color scheme: green (lipophilic region) to violet (hydrophilic region). In (A), conformation of compound 1f (green) is reported as result of molecular docking study. In order to compare binding mode of compound 1f in hAChE, its conformation when docked to hAChE is reported in cyan. In (B), binding mode of most active compound of series for hBuChE, compound 1d, in shown in green.

Interestingly, the analogues showing the p-tolyl moiety at C4 present best IC₅₀ values for hBuChE. The docking studies revealed that the p-tolyl moiety occupy an additional hydrophobic cavity, delimited by Trp231, Phe398, Leu286, and Ala199 (Fig. 5, panel B).

In addition, theoretical calculations were also performed using StarDrop software to predict physicochemical and ADME (absorption, distribution, metabolism and excretion) properties of the novel AChE inhibitors, such as, aqueous solubility (log S), lipophilicity (clog P), cytochrome P450 metabolism (2D6 and 2C9), human ether-à-go-go related gene (hERG) channel inhibition, blood–brain barrier (BBB) penetration, human intestinal absorption (HIA), P-glycoprotein binding (P-gp) and plasma protein binding (PPB90). Interestingly, most of the physicochemical and ADME properties of the synthetized compounds were comparable with those of donepezil, and particularly compound 1b and its methoxy derivatives 1f, 1g, 1h, all potent and selective AChEIs (Table 3).

Table 3 Calculated physicochemical and ADME properties for all synthetized analogues, donepezil and tacrine

Compound	Intravenous CNS profile_score^a	Oral CNS profile_score^b	log S^c	log P^d	2C9 pKi^e	hERG pIC50^f	BBB category^g	HIA category^h	P-gp category^i	2D6 affinity category^j	PPB90 category^k
a Intravenous CNS score: ideal score is 1.b Oral CNS score: ideal score is 1.c Aqueous solubility (log S, μM), preferably >1.d Logarithm of partition coefficient between n-octanol and water (clog P), preferably 0 < clog P < 3.6.e CYP2C9 cytochrome metabolism (CYP2C9 affinity, μM), preferably ≤6.f hERG channel inhibition (pIC50), preferably ≤5.g Blood–brain barrier (BBB) penetration: (+) indicates ratio ≥ 0.5 and (−) indicates ratio < 0.5.h Human intestinal absorption (HIA): (+) indicates absorption ≥ 30% and (−) indicates absorption < 30%.i P-Glycoprotein binding (P-gp): “yes” indicates a substrate and “no” indicates a non-substrate.j CYP2D6 cytochrome metabolism (CYP2D6 affinity, μM), “low” indicates pKi < 5, “medium” indicates 5 < pKi < 6, “high” indicates 6 < pKi < 7, “very high” indicates pKi > 7.k Plasma protein binding (PPB90): “low” indicates <90% of compound bound to plasma proteins, “high” indicates ≥90% of compound bound to plasma proteins. All properties were calculated using StarDrop™, version 6.2 (Optibrium Ltd, 7221 Cambridge Research Park, Beach Drive, Cambridge CB25 9TL, UK).
1a	0.25	0.17	1.71	3.47	4.91	6.59	+	+	Yes	Medium	Low
1b	0.22	0.14	1.77	3.58	4.79	6.69	+	+	Yes	Medium	Low
1c	0.03	0.01	0.16	5.28	5.57	7.15	+	+	Yes	Medium	High
1d	0.05	0.02	0.36	5.40	5.45	7.24	+	+	Yes	Medium	High
1e	0.14	0.09	1.61	3.98	4.90	6.68	+	+	Yes	Medium	Low
1f	0.15	0.09	2.10	3.34	5.13	6.69	−	+	Yes	Medium	High
1g	0.14	0.09	1.91	3.47	4.83	6.69	−	+	Yes	Medium	Low
1h	0.14	0.09	1.91	3.47	4.85	6.70	−	+	Yes	Medium	Low
1i	0.15	0.09	1.64	4.00	4.73	6.81	+	+	Yes	Medium	Low
1j	0.10	0.06	1.98	3.74	4.99	6.81	−	+	Yes	Medium	High
Donepezil	0.25	0.16	1.87	3.54	4.97	6.41	+	+	Yes	Medium	Low
Tacrine	0.32	0.28	2.77	2.57	4.43	4.84	+	+	No	Medium	Low

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3. Conclusions

A series of donepezil analogues based on phthalazin-1(2H)-one scaffold was designed and synthesized with the aim of exploring its potential as ChEIs. In target compounds the indanone was replaced by the phthalazinone nucleus, preserving or not preserving the methoxy groups, and showing the N-benzylpiperidine fragment linked at N2 by an alkyl chain ranging from one to three carbons. Additionally, the C4 position of the phthalazinone scaffold could be substituted with groups of varying sizes and electronic properties.

The biological data indicated that most of the designed compounds showed moderate to good activity as AChE and BuChE inhibitors. In addition, the target compounds, with the exception of 1a and 1c, were remarkably more active against AChE than BuChE. Thus, compounds 1a and 1c (both with n = 1) were inactive against AChE and moderately active against BuChE, while compound 1d (n = 2, C4 = p-Tol) was equipotent toward AChE and BuChE enzymes, behaving as a dual cholinesterase inhibitor (AChE IC₅₀ = 3.45 μM and BuChE IC₅₀ = 5.50 μM).

As can be seen, AChE inhibition was severely affected by the length of the linking chain between the phthalazinone moiety and the N-benzylpiperidine fragment, since the compounds 1b and 1d, homologues of 1a and 1c, respectively, as well as the analogue 1e (all them with n = 2) preferentially inhibited AChE, with IC₅₀ values ranging from 2.58 to 3.45 μM, while a decreased potency was observed for the homologue 1i (IC₅₀ = 10.29, n = 3).

Finally, the role of methoxy substituents in the activity of these new donepezil analogues is noteworthy. When the methoxy group is located at C6, it provides a good and selective inhibitory effect against AChE; compounds 1f (IC₅₀ = 0.67), 1h (IC₅₀ = 0.55) and 1j (IC₅₀ = 1.07) were the most potent and selective AChEIs of this series. They are almost equipotent with tacrine but less potent than donepezil. However, when the methoxy group was placed at C7 position, the AChE inhibitory activity seems unaffected (compound 1g, IC₅₀ = 2.40 μM).

Docking simulations suggested that the most active compounds can recognize the binding site of donepezil using a similar interactions network. These results allowed us to rationalize the observed structure–activity relationships. In addition, docking analysis also indicated a plausible binding mode of dual inhibitor 1d along the active site cavity of AChE and BuChE. Therefore, these novel phthalazinone analogues—and also upon consideration of their predicted physiochemical and ADME properties—can be considered potential drug candidates to develop new ChEIs.

4. Experimental section

4.1 Chemistry

All starting materials and common laboratory chemicals were purchased from commercial sources and used without further purification. All solvents were distilled and dried according to standard procedures. ¹H and ¹³C NMR spectra were recorded on a Bruker ARX400 instrument, using TMS as the internal standard [chemical shifts (δ) in ppm, J in Hz]. Assignment of the signals was performed by COSY, DEPT, and HSQC experiments. High-resolution mass spectra were recorded using a Bruker microTOF focus spectrometer. Silica gel (Merck 60, 230–400 mesh) was used for flash chromatography (FC). Analytical TLC was performed on plates pre-coated with silica gel (Merck 60 F254, 0.25 mm).

4.2 4-Methylphthalazin-1(2H)-one (10c)

Hydrazine hydrate (0.39 mL, 7.84 mmol) was added to a solution of 2-acetylbenzoic acid (8) (1 g, 6.09 mmol) in EtOH (10 mL). The reaction mixture was refluxed overnight. After cooling, the resulting precipitate was filtered and washed with ethanol to give 10c (875 mg, 91%) as a white solid. R_f = 0.5 (hexane/EtOAc, 1 : 1). ¹H NMR (CDCl₃) δ = 8.49–8.45 (m, 1H, H8), 7.89–7.83 (m, 1H, Ar), 7.82–7.77 (m, 2H, Ar), 2.60 (s, 3H, CH₃). ¹³C NMR (CDCl₃) δ = 160.8 (C1), 144.8 (C4), 133.6, 131.6, 130.5, 127.9, 127.0 (C8), 125.2, 18.9 (CH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₉N₂O: 161.07094, found: 161.07100.

4.3 4-Methoxyphthalic acid (3)

A solution of dimethyl 4-methoxyphthalate (2) (1.1 g, 4.76 mmol) in MeOH (5 mL) was treated with aqueous 5 M NaOH (3 mL, 15 mmol). The reaction mixture was refluxed overnight. After cooling at room temperature (r.t.), 3 M HCl (5 mL) was carefully added until pH = 1 and the mixture was extracted with CH₂Cl₂ (3 × 20 mL), the organic layers were collected, dried over Na₂SO₄ and evaporated to dryness to give 3 (766 mg, 96%). ¹H NMR (CD₃OD) δ = 7.91 (d, 1H, J = 8.7 Hz, H6), 7.25 (d, 1H, J = 2.4 Hz, H3), 7.05 (dd, 1H, J = 8.7, 2.4 Hz, H5), 3.85 (s, 3H, OCH₃). ¹³C NMR (CD₃OD) δ = 172.2 (CO), 169.8 (CO), 163.6 (C4), 138.0 (C2), 132.9 (C6), 123.7 (C1), 116.0 (C5), 114.8 (C3), 56.2 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₉O₅: 197.04445, found: 197.04512.

4.4 4-Methoxyphthalic anhydride (4)

A solution of compound 3 (300 mg, 1.53 mmol) in acetic anhydride (5 mL) was refluxed for 1 h. After cooling at r.t., the solvent was removed under reduced pressure to furnish anhydride 4 (266 mg, 98%). ¹H NMR (CDCl₃) δ = 7.90 (d, 1H, J = 8.4 Hz, H6), 7.41 (d, 1H, J = 2.2 Hz, H3), 7.35 (dd, 1H, J = 8.4, 2.2 Hz, H5), 3.98 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ = 166.2 (C4), 163.0 (CO), 162.5 (CO), 134.0, 127.3 (C6), 123.1 (C5), 122.8, 109.1 (C3), 56.5 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₇O₄: 179.03389, found: 179.03308.

4.5 5-Methoxyisobenzofuran-1(3H)-one (5c)

4.5.1 Method A. To a solution of NaBH₄ (11 mg, 0.28 mmol) in THF (2 mL) cooled at 0 °C a solution of compound 4 (50 mg, 0.28 mmol) was added dropwise in THF (3 mL). The reaction mixture was stirred for 4 h at r.t., treated with 6 M HCl until pH = 1 and then extracted with Et₂O (3 × 10 mL). The combined organic layers were collected, dried over Na₂SO₄ and concentrated under reduced pressure. The solid obtained was purified by column chromatography on silica gel (gradient elution: hexane/EtOAc 4 : 1 to hexane/EtOAc 1 : 1) to afford 5c (15 mg, 33%) and 6 (26 mg, 52%). Compound 5c: R_f = 0.5 (hexane/EtOAc, 1 : 1). ¹H NMR (CDCl₃) δ = 7.72 (d, 1H, J = 8.5 Hz, H7), 6.97 (dd, 1H, J = 8.5, 2.2 Hz, H6), 6.09–6.88 (m, 1H, H4), 5.19 (s, 2H, H3), 3.85 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ 170.9 (C1), 164.7 (C5), 149.42, 127.1 (C7), 118.0, 116.5 (C6), 106.0 (C4), 69.1 (C3), 55.9 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₉O₃: 165.05462, found: 165.05451. Compound 6: R_f = 0.2 (hexane/EtOAc, 1 : 1). ¹H NMR (CDCl₃) δ = 8.00 (d, 1H, J = 8.7 Hz, H6), 7.23 (d, 1H, J = 2.6 Hz, H3), 6.87 (dd, 1H, J = 8.7, 2.6 Hz, H5), 4.92 (s, 2H, CH₂), 3.87 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ = 169.0 (CO), 163.2 (C4), 146.5, 133.3 (C6), 120.2, 112.6 (C3), 111.3 (C5), 62.5 (CH₂), 54.5 (OCH₃).

4.5.2 Method B. Following the procedure described above and starting from 4 (190 mg, 1.07 mmol), a crude product containing compounds 5c and 6 (160 mg) was obtained, which was dissolved in 6 M HCl (7 mL) and refluxed for 2 h. After cooling, the reaction mixture was extracted with CH₂Cl₂ (6 × 12 mL). The combined organic layers were collected, dried over Na₂SO₄ and concentrated under reduced pressure. The solid residue was purified by column chromatography on silica gel (hexane/EtOAc, 4 : 1) to give 5c (93 mg, 53%).

4.6 5,6-Dimethoxyisobenzofuran-1(3H)-one (5a)

To a solution of 3,4-dimethoxybenzoic acid (7a) (50 mg, 0.27 mmol) in 37% HCl (0.75 mL), 30% formaldehyde (0.13 mL, 1.25 mmol) was added. The reaction mixture was stirred at 90 °C for 10 h. After cooling, the mixture was quenched with H₂O (5 mL) and extracted with EtOAc (4 × 5 mL). The combined organic layers were washed with 2.5 M NaOH (5 mL) and brine (5 mL), dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/EtOAc, 2 : 1) to afford 5a (40 mg, 75%). R_f = 0.4 (hexane/EtOAc, 1 : 2). ¹H NMR (CDCl₃) δ = 7.10 (s, 1H, H7), 6.83 (s, 1H, H4), 5.08 (s, 2H, H3), 3.85 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ = 171.2 (C1), 154.7, 150.2, 141.0, 117.2, 105.7 (C7), 103.5 (C4), 69.0 (C3), 56.2 (OCH₃), 56.0 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₀H₁₁O₄: 195.06519, found: 195.06570.

4.7 6-Methoxyisobenzofuran-1(3H)-one (5b)

To a solution of 3-methoxybenzoic acid (7b) (1 g, 6.44 mmol) in glacial acetic acid (3.3 mL), 37% HCl (4.8 mL, 57.9 mmol) and 30% formaldehyde (1.92 mL, 25.8 mmol) was added, and the reaction mixture was stirred at 100 °C for 1 h. After cooling, a saturated solution of NaHCO₃ was added until pH = 7. The resulting mixture was extracted with CH₂Cl₂ (3 × 20 mL), the combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/EtOAc, 7 : 1) to afford 5b (462 mg, 43%). R_f = 0.4 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 7.36 (d, 1H, J = 8.4 Hz, H4), 7.29 (d, 1H, J = 2.2 Hz, H7), 7.22 (dd, 1H, J = 8.4, 2.2 Hz, H5), 5.24 (s, 2H, H3), 3.84 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ = 171.3 (C1), 160.6 (C6), 138.9, 127.0, 123.1 (C4), 123.0 (C5), 107.5 (C7), 69.6 (C3), 55.8 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₉O₃: 165.05462, found: 165.05455.

4.8 General procedure for preparation of phthalazin-1(2H)-ones (10d–f)

To a solution of the required isobenzofuranone (5a–c) (0.51 mmol) in CCl₄ (15 mL) was added NBS (0.51 mmol) and a catalytic amount of benzoyl peroxide. The reaction mixture was stirred at reflux for 3 h. After cooling, the mixture was filtered and evaporated to dryness to give a residue containing the corresponding 3-bromoisobenzofuranone (9a–c), which were solved in EtOH (5 mL), treated with hydrazine hydrate (1.95 mmol) and refluxed for 14 h. After cooling, the resulting solution was diluted with H₂O (3 mL) and the obtained solid material was filtered and dried to afford the desired phthalazin-1(2H)-one (10d–f).

4.8.1 6,7-Dimethoxyphthalazin-1-(2H)-one (10d). White solid. Yield: 58%. R_f = 0.4 (EtOAc). ¹H NMR (CDCl₃) δ = 8.08 (s, 1H, H4), 7.78 (s, 1H, H8), 7.04 (s, 1H, H5), 4.05 (s, 3H, OCH₃), 4.04 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ: 160.3 (C1), 154.4, 153.1, 138.4 (C4), 125.8, 123.0, 106.4 (C5), 106.2 (C8), 56.7 (OCH₃), 56.5 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₀H₁₁N₂O₃: 207.07642, found: 207.07693.

4.8.2 7-Methoxyphthalazin-1-(2H)-one (10e). White solid. Yield 51%. R_f = 0.4 (hexane/EtOAc, 1 : 1). ¹H NMR (CDCl₃) δ = 10.25 (s, 1H, NH), 8.11 (s, 1H, H4), 7.80 (d, 1H, J = 2.6 Hz, H8), 7.66 (d, 1H, J = 8.7 Hz, H5), 7.40 (dd, 1H, J = 8.7, 2.6 Hz, H6), 3.98 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ = 162.6 (C7), 160.6 (C1), 138.9 (C4), 130.1, 128.4 (C5), 124.5, 124.2 (C6), 106.5 (C8), 56.1 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₉N₂O₂: 177.06585, found: 177.06581.

4.8.3 6-Methoxyphthalazin-1-(2H)-one (10f). White solid. Yield 65%. R_f = 0.4 (hexane/EtOAc, 1 : 1): 0.4. ¹H NMR (CDCl₃) δ = 10.29 (s, 1H, NH), 8.34 (d, 1H, J = 8.8 Hz, H8), 8.10 (s, 1H, H4), 7.33 (dd, 1H, J = 8.8, 2.4 Hz, H7), 7.05 (d, 1H, J = 2.4 Hz, H5), 3.96 (s, 3H, OCH₃). ¹³C NMR (CDCl₃) δ = 163.9 (C6), 160.4 (C1), 138.9 (C4), 132.4, 128.7 (C8), 121.8, 121.1 (C7), 107.4 (C5), 55.9 (OCH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₉N₂O₃: 177.06585, found: 177.06502.

4.9 General procedure for preparation of 4-bromoalkyl-N-Boc-piperidines (12a,b)

To a solution of compound 11a or 11b (1 mmol) in CH₂Cl₂ (5 mL), CBr₄ (2 mmol) and PPh₃ (2 mmol) were added and the reaction mixture was refluxed for 2 h. After solvent removal, the residue was purified by column chromatography on silica gel (hexane/EtOAc, 9 : 1) to afford the desired compound.

4.9.1 4-Bromomethyl-1-tert-butoxycarbonylpiperidine (12a). Colorless oil. Yield: 94%. R_f = 0.7 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 4.22–4.02 (m, 2H, H2), 3.28 (d, 2H, J = 6.2 Hz, H1′), 2.75–2.61 (m, 2H, H2), 1.85–1.72 (m, 3H, H3, H4), 1.44 (s, 9H, 3 × CH₃), 1.24–1.10 (m, 2H, H3). ¹³C NMR (CDCl₃) δ = 154.9 (CO), 79.7 (C(CH₃)₃), 43.8 (C2), 39.0 (C1′), 38.8 (C4), 31.0 (C3), 28.6 ((CH₃)₃). HRMS-ESI: m/z [M + H]⁺ calcd for C₁₁H₂₁BrNO₂: 278.07557, found: 278.07502.

4.9.2 4-(2-Bromoethyl)-1-tert-butoxycarbonylpiperidine (12b). Colorless oil. Yield: 89%. R_f = 0.5 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 4.18–4.01 (m, 2H, H2), 3.44 (t, 2H, J = 6.8 Hz, H2′), 2.74–2.64 (m, 2H, H2), 1.83–1.77 (m, 2H, H1′), 1.70–1.62 (m, 3H, H3, H4), 1.44 (s, 9H, 3 × CH₃), 1.16–1.04 (m, 2H, H3). ¹³C NMR (CDCl₃) δ = 155.0 (CO), 79.5 (C(CH₃)₃), 44.0 (C2), 39.3 (C1′), 34.5 (C4), 31.6 (C3), 31.2 (C2′), 28.6 ((CH₃)₃). HRMS-ESI: m/z [M + H]⁺ calcd for C₁₂H₂₃BrNO₂: 292.09122, found: 292.09021.

4.10 General procedure for preparation of 2-(N-Boc-piperidin-4-ylalkyl)phthalazin-1(2H)-ones (13a–h)

A solution of the corresponding phthalazinone (10a–f) (0.34 mmol) in DMF (0.8 mL) was added to a suspension of NaH (60% dispersion in mineral oil, 0.53 mmol) in DMF (0.8 mL). After stirring at r.t. for 1 h, a solution of 12a or 12b (0.38 mmol) in DMF (0.8 mL) was added. The reaction mixture was stirred at r.t. overnight, followed by quenching with H₂O (15 mL) at 0 °C. The product was extracted with EtOAc (2 × 10 mL), dried over Na₂SO₄, and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/EtOAc, 3 : 1) to obtain the desired compound.

4.10.1 2-(1-tert-Butoxycarbonypiperidin-4-ylmethyl)phthalazin-1(2H)-one (13a). Colorless oil. Yield: 78%. R_f = 0.4 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 8.42 (d, 1H, J = 7.7 Hz, H8), 8.15 (s, 1H, H4), 7.83–7.73 (m, 2H, Ar), 7.71–7.67 (m, 1H, Ar), 4.20–4.00 (m, 4H, H1′, H2′′), 2.73–2.60 (m, 2H, H2′′), 2.22–2.10 (m, 1H, H4′′), 1.68–1.58 (m, 2H, H3′′), 1.43 (s, 9H, 3 × CH₃), 1.36–1.22 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.8 (C1), 154.9 (COC(CH₃)₃), 137.8 (C4), 133.3, 131.8, 129.7, 128.0, 126.9, 126.1, 79.4 (C(CH₃)₃), 56.3 (C1′), 43.8 (C2′′), 35.8 (C4′′), 29.8 (C3′′), 28.6 ((CH₃)₃). HRMS-ESI: m/z [M + H]⁺ calcd for C₁₉H₂₆N₃O₃: 344.19687, found: 344.19653.

4.10.2 2-(2-(1-tert-Butoxycarbonylpiperidin-4-yl)ethyl)phthalazin-1(2H)-one (13b). Colorless oil. Yield: 99%. R_f = 0.4 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 8.37 (d, 1H, J = 7.6 Hz, H8), 8.12 (s, 1H, H4), 7.79–7.68 (m, 2H, Ar), 7.67–7.63 (m, 1H, Ar), 4.24 (t, 2H, J = 7.4 Hz, H1′), 4.14–3.96 (m, 2H, H2′′), 2.71–2.56 (m, 2H, H2′′), 1.81–1.68 (m, 4H, H2′, H3′′), 1.51–1.41 (m, 1H, H4′′), 1.40 (s, 9H, 3 × CH₃), 1.19–1.07 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.4 (C1), 155.0 (COC(CH₃)₃), 137.9 (C4), 133.1, 131.8, 129.7, 128.0, 126.8, 126.1, 79.3 (C(CH₃)₃), 48.9 (C1′), 44.1 (C2′′), 35.2 (C2′), 33.7 (C4′′), 32.1 (C3′′), 28.6 ((CH₃)₃). HRMS-ESI: m/z [M + H]⁺ calcd for C₂₀H₂₈N₃O₃: 358.21252, found: 358.21164.

4.10.3 2-(1-tert-Butoxycarbonylpiperidin-4-ylmethyl)-4-p-tolylphthalazin-1(2H)-one (13c). Colorless oil. Yield: 99%. R_f = 0.4 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 8.52–8.47 (m, 1H, H8), 7.78–7.69 (m, 3H, Ar), 7.46 (d, 2H, J = 7.9 Hz, Ar), 7.32 (d, 2H, J = 7.9 Hz, Ar), 4.28–3.98 (m, 4H, H1′, H2′′), 2.75–2.60 (m, 2H, H2′′), 2.43 (s, 3H, CH₃), 2.28–2.15 (m, 1H, H4′′), 1.71–1.62 (m, 2H, H3′′), 1.42 (s, 9H, 3 × CH₃), 1.38–1.25 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.4 (C1), 154.8 (COC(CH₃)₃), 146.9, 139.2, 132.8, 132.3, 131.4, 129.4, 129.1, 128.2, 127.2, 126.8, 79.2 (C(CH₃)₃), 56.3 (C1′), 43.3 (C2′′), 35.8 (C4′′), 29.8 (C3′′), 28.5 ((CH₃)₃), 21.4 (CH₃). HRMS-EI: m/z [M]⁺ calcd for C₂₆H₃₁N₃O₃: 433.2365, found: 433.2379.

4.10.4 2-(2-(1-tert-Butoxycarbonylpiperidin-4-yl)ethyl)-4-p-tolylphthalazin-1(2H)-one (13d). Colorless oil. Yield: 99%. R_f = 0.4 (hexane/EtOAc, 2 : 1). ¹H NMR (CDCl₃) δ = 8.52–8.48 (m, 1H, H8), 7.78–7.70 (m, 3H, Ar), 7.45 (d, 2H, J = 7.9 Hz, Ar), 7.32 (d, 2H, J = 7.9 Hz, Ar), 4.33 (t, 2H, J = 7.5 Hz, H1′), 4.14–3.99 (m, 2H, H2′′), 2.73–2.61 (m, 2H, H2′′), 2.44 (s, 3H, CH₃), 1.87–1.73 (m, 4H, H2′, H3′′), 1.57–1.48 (m, 1H, H4′′), 1.43 (s, 9H, 3 × CH₃), 1.20–1.13 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.1 (C1), 155.0 (COC(CH₃)₃), 147.1, 139.3, 132.8, 132.5, 131.4, 129.5, 129.4, 128.4, 127.3, 126.8, 79.4 (C(CH₃)₃), 49.1 (C1′), 43.9 (C2′′), 35.2 (C2′), 33.9 (C4′′), 32.1 (C3′′), 28.6 ((CH₃)₃), 21.5 (CH₃). HRMS-ESI: m/z [M + H]⁺ calcd for C₂₇H₃₄N₃O₃: 448.25947, found: 448.26072.

4.10.5 2-(2-(1-tert-Butoxycarbonylpiperidin-4-yl)ethyl)-4-methylphthalazin-1(2H)-one (13e). Colorless oil. Yield: 99%. R_f = 0.5 (hexane/EtOAc, 1 : 1). ¹H NMR (CDCl₃) δ = 8.42 (d, 1H, J = 8.0 Hz, H8), 7.80–7.69 (m, 3H, Ar), 4.21 (t, 2H, J = 6.9 Hz, H1′), 4.14–3.95 (m, 2H, H2′′), 2.73–2.60 (m, 2H, H2′′), 2.56 (s, 3H, CH₃), 1.80–1.71 (m, 4H, H2′, H3′′), 1.48–1.40 (m, 10H, H4′′, 3 × CH₃), 1.21–1.11 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.3 (C1), 154.9 (COC(CH₃)₃), 143.5 (C4), 132.8, 131.3, 129.8, 127.8, 127.1, 124.8, 79.2 (C(CH₃)₃), 48.6 (C1′), 43,9 (C2′′), 35.1 (C2′), 33.7 (C4′′), 32.0 (C3′′), 28.5 ((CH₃)₃), 18.9 (CH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₃₀N₃O₃: 372.22817, found: 372.22792.

4.10.6 2-(2-(1-tert-Butoxycarbonylpiperidin4-yl)ethyl)-6,7-dimethoxyphthalazin-1(2H)-one (13f). Colorless oil. Yield: 79%. R_f = 0.5 (EtOAc). ¹H NMR (CDCl₃) δ = 8.04 (s, 1H, H4), 7.75 (s, 1H, H8), 6.99 (s, 1H, H5), 4.28–4.24 (m, 2H, H1′), 4.03–4.00 (m, 8H, H2′′, 2 × OCH₃), 2.70–2.62 (m, 2H, H2′′), 1.81–1.75 (m, 4H, H2′, H3′′), 1.44–1.42 (m, 10H, H4′′, 3 × CH₃), 1.25–1.20 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.0 (C1), 154.9 (COC(CH₃)₃), 153.8, 152.9, 137.0 (C4), 124.9, 122.8, 106.3 (C8), 105.6 (C5), 79.3 (C(CH₃)₃), 56.5 (OCH₃), 56.3 (OCH₃), 48.8 (C1′), 43.6 (C2′′), 35.1(C2′), 33.5 (C4′′), 31.9 (C3′′), 28.4 ((CH₃)₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₃₂N₃O₅: 418.23365, found: 418.23189.

4.10.7 2-(2-(1-tert-Butoxycarbonylpiperidin-4-yl)ethyl)-7-methoxyphthalazin-1(2H)-one (13g). White solid. Yield: 45%. R_f = 0.5 (EtOAc). ¹H NMR (CDCl₃) δ = 8.08 (s, 1H, H4), 7.77 (d, 1H, J = 2.6 Hz, H8), 7.60 (d, 1H, J = 8.7 Hz, H5), 7.34 (dd, 1H, J = 8.7, 2.6 Hz, H6), 4.27 (t, 2H, J = 7.4 Hz, H1′), 4.13–4.00 (m, 2H, H2′′), 3.95 (s, 3H, OCH₃), 2.72–2.61 (m, 2H, H2′′), 1.84–1.75 (m, 4H, H2′, H3′′), 1.44–1.42 (m, 10H, H4′′, 3 × CH₃), 1.21–1.11 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 162.5 (C7), 159.3 (C1), 155.0 (COC(CH₃)₃), 137.6 (C4), 129.8, 128.0 (C5), 123.9, 123.6 (C6), 106.5 (C8), 79.3 (C(CH₃)₃), 56.0 (OCH₃), 48.9 (C1′), 44.3 (C2′′), 35.1 (C2′), 33.68 (C4′′), 32.1 (C3′′), 28.6 ((CH₃)₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₃₀N₃O₄: 388.22308, found: 388.22414.

4.10.8 2-(2-(1-tert-Butoxycarbonylpiperidin-4-yl)ethyl)-6-methoxyphthalazin-1(2H)-one (13h). Colorless oil. Yield: 86%. R_f = 0.5 (hexane/EtOAc, 1 : 1). ¹H NMR (CDCl₃) δ = 8.30 (d, 1H, J = 8.9 Hz, H8), 8.06 (s, 1H, H4), 7.28 (dd, 1H, J = 8.9, 2.4 Hz, H7), 6.99 (d, 1H, J = 2.4 Hz, H5), 4.23 (t, 2H, J = 7.4 Hz, H1′), 4.11–3.99 (m, 2H, H2′′), 3.92 (s, 3H, OCH₃), 2.71–2.60 (m, 2H, H2′′), 1.80–1.72 (m, 4H, H2′, H3′′), 1.45–1.40 (m, 10H, H4′′, 3 × CH₃), 1.20–1.13 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 163.2 (C6), 159.2 (C1), 154.9 (COC(CH₃)₃), 137.4 (C4), 131.7, 128.8 (C8), 121.70, 121.1 (C7), 106.5 (C5), 79.2 (C(CH₃)₃), 55.8 (OCH₃), 48.6 (C1′), 44.3 (C2′′), 35.1 (C2′), 33.6 (C4′′), 31.9 (C3′′), 28.5 ((CH₃)₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₃₀N₃O₄: 388.22308, found: 388.22255.

4.11 General procedure for the preparation of 2-(piperidin-4-ylalkyl)phthalazin-1(2H)-ones (14a–h)

To a solution of compound 13a–h (0.8 mmol) in EtOAc (0.9 mL), 6 M HCl (0.3 mL) was added. The reaction mixture was stirred at r.t. overnight, followed by addition of saturated aq. NaHCO₃ until pH = 12. The solvent was evaporated, MeOH (15 mL) was added and the insoluble material was removed by filtration. The solvent was concentrated to dryness and after a column chromatography on silica gel (EtOAc/MeOH/NH₃, 90/9.5/0.5) a residue that contains the desired compound was obtained.

4.11.1 2-(Piperidin-4-ylmethyl)phthalazin-1(2H)-one (14a). R_f = 0.3 (CH₂Cl₂/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CD₃OD) δ = 8.39 (s, 1H, H4), 8.38–8.32 (m, 1H, H8), 7.96–7.86 (m, 3H, Ar), 4.23–4.19 (m, 2H, H1′), 3.44–3.38 (m, 2H, H2′′), 2.99–2.91 (m, 2H, H2′′), 2.39–2.29 (m, 1H, H4′′), 1.96–1.88 (m, 2H, H3′′), 1.68–1.56 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 161.5 (C1), 140.2 (C4), 135.0, 133.4, 131.2, 128.6, 128.0, 127.2, 56.4 (C1′), 44.6 (C2′′), 34.9 (C4′′), 27.7 (C3′′). HRMS-EI: m/z [M]⁺ calcd for C₁₄H₁₇N₃O: 243.1372, found: 243.1373.

4.11.2 2-(2-(Piperidin-4-yl)ethyl)phthalazin-1(2H)-one (14b). R_f = 0.3 (CH₂Cl₂/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CD₃OD) δ = 8.33 (s, 1H, H4), 8.28 (d, 1H, J = 7.8 Hz, H8), 7.90–7.79 (m, 3H, Ar), 4.26 (t, 2H, J = 7.1 Hz, H1′), 3.36–3.29 (m, 2H, H2′′), 2.93–2.83 (m, 2H, H2′′), 2.04–1.96 (m, 2H, H3′′), 1.83–1.77 (m, 2H, H2′), 1.65–1.53 (m, 1H, H4′′), 1.49–1.37 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 161.3 (C1), 140.2 (C4), 134.8, 133.3, 131.2, 128.5, 127.9, 127.0, 49.3 (C1′), 45.0 (C2′′), 35.7 (C2′), 32.4 (C4′′), 30.0 (C3′′). HRMS-EI: m/z [M]⁺ calcd for C₁₅H₁₉N₃O: 257.1528, found: 257.1538.

4.11.3 2-(Piperidin-4-ylmethyl)-4-p-tolylphthalazin-1(2H)-one (14c). R_f = 0.3 (CH₂Cl₂/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CD₃OD) δ = 8.46–8.39 (m, 1H, H8), 7.90–7.76 (m, 3H, Ar), 7.51–7.44 (m, 2H, Ar), 7.40–7.31 (m, 2H, Ar), 4.24–4.16 (m, 2H, H1′), 3.23–3.13 (m, 2H, H2′′), 2.78–2.66 (m, 2H, H2′′), 2.44 (s, 3H, CH₃), 2.31–2.19 (m, 1H, H4′′), 1.84–1.74 (m, 2H, H3′′), 1.50–1.40 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 161.1 (C1), 149.4, 140.7, 134.7, 133.3, 133.1, 130.5, 130.4, 130.3, 129.0, 128.2, 127.8, 56.4 (C1′), 44.7 (C2′′), 34.9 (C4′′), 27.7 (C3′′), 21.4 (CH₃). HRMS-EI: m/z [M]⁺ calcd for C₂₁H₂₃N₃O: 333.1841, found: 333.1842.

4.11.4 2-(2-(Piperidin-4-yl)ethyl)-4-p-tolylphthalazin-1(2H)-one (14d). R_f = 0.3 (CH₂Cl₂/MeOH/NH₃, 90/9.5/0.5). ¹H NMR (CD₃OD) δ = 8.45–8.40 (m, 1H, H8), 7.88–7.83 (m, 2H, Ar), 7.80–7.76 (m, 1H, Ar), 7.47 (d, 2H, J = 8.1 Hz, Ar), 7.36 (d, 2H, J = 8.1 Hz, Ar), 4.34 (t, 2H, J = 7.1 Hz, H1′), 3.31–3.24 (m, 2H, H2′′), 2.88–2.78 (m, 2H, H2′′), 2.45 (s, 3H, CH₃), 2.04–1.96 (m, 2H, H3′′), 1.90–1.83 (m, 2H, H2′), 1.69–1.57 (m, 1H, H4′′), 1.43–1.33 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 160.8 (C1), 149.2, 140.7, 134.7, 133.6, 133.1, 130.9, 130.7, 130.5, 129.4, 128.5, 128.0, 45.4 (C1′), 44.8 (C2′′), 36.1 (C2′), 33.1 (C4′′), 30.7 (C3′′), 21.5 (CH₃). HRMS-EI: m/z [M]⁺ calcd for C₂₂H₂₅N₃O: 347.1998, found: 347.2004.

4.11.5 2-(2-(Piperidin-4-yl)ethyl)-4-methylphthalazin-1(2H)-one (14e). R_f = 0.2 (EtOAc/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CD₃OD) δ = 8.32–8.19 (m, 1H, H8), 7.94–7.71 (m, 3H, Ar), 4.25–4.11 (m, 2H, H1′), 3.26–3.20 (m, 2H, H2′′), 2.85–2.71 (m, 2H, H2′′), 2.54 (s, 3H, CH₃), 1.98–1.88 (m, 2H, H3′′), 1.80–1.70 (m, 2H, H2′), 1.59–1.46 (m, 1H, H4′′), 1.41–1.27 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 160.9 (C1), 146.2 (C4), 134.6, 132.9, 130.9, 128.3, 127.4, 126.6, 49.2 (C1′), 45.4 (C2′′), 35.9 (C2′), 32.99 (C4′′), 30.8 (C3′′), 18.9 (CH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₆H₂₂N₃O: 272.17574, found: 272.17602.

4.11.6 2-(2-(Piperidin-4-yl)ethyl)-6,7-dimethoxyphthalazin-1(2H)-one (14f). R_f = 0.3 (EtOAc/MeOH/NH₃, 90/9.5/0.5). ¹H NMR (CD₃OD) δ = 7.79 (s, 1H, H4), 7.17 (s, 1H, H8), 6.85 (s, 1H, H5), 3.89–3.82 (m, 2H, H1′), 3.56 (s, 6H, 2 × OCH₃), 2.98–2.93 (m, 2H, H2′′), 2.56–2.46 (m, 2H, H2′′), 1.65–1.59 (m, 2H, H3′′), 1.44–1.37 (m, 2H, H2′), 1.27–1.15 (m, 1H, H4′′), 1.13–1.00 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 159.3 (C1), 154.3, 153.2, 138.0 (C4), 125.3, 121.7, 106.2 (C5), 105.2 (C8), 55.5 (OCH₃), 55.3 (OCH₃), 47.9 (C1′), 43.6 (C2′′), 34.4 (C2′), 31.1 (C4′′), 28.5 (C3′′). HRMS (EI): m/z [M]⁺ calcd for C₁₇H₂₃N₃O₃: 317.1739, found: 317.1745.

4.11.7 2-(2-(Piperidin-4-yl)ethyl)-7-methoxyphthalazin-1(2H)-one (14g). R_f = 0.3 (EtOAc/MeOH/NH₃, 90/9.5/0.5). ¹H NMR (CD₃OD) δ = 8.28 (s, 1H, H4), 7.82 (d, 1H, J = 8.7, H5), 7.72–7.68 (m, 1H, H8), 7.48–7.43 (m, 1H, H6), 4.30 (t, 2H, J = 6.9 Hz, H1′), 3.97 (s, 3H, OCH₃), 3.41–3.34 (m, 2H, H2′′), 2.98–2.88 (m, 2H, H2′′), 2.08–2.02 (m, 2H, H3′′), 1.85 (c, 2H, J = 6.9 Hz, H2′), 1.68–1.58 (m, 1H, H4′′), 1.52–1.45 (m, 2H, H3′′). ¹³C NMR (CD₃OD) δ = 164.3 (C7), 161.0 (C1), 139.9 (C4), 130.5, 130.0 (C5), 125.3, 124.5 (C6), 107.4 (C8), 56.4 (OCH₃), 49.3 (C1′), 45.1 (C2′′), 35.7 (C2′), 32.4 (C4′′), 29.9 (C3′′). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₆H₂₂N₃O₂: 288.17065, found: 288.17131.

4.11.8 2-(2-(Piperidin-4-yl)ethyl)-6-methoxyphthalazin-1(2H)-one (14h). R_f = 0.2 (EtOAc/MeOH/NH₃, 90/9.5/0.5). ¹H NMR (CDCl₃) δ = 8.32 (s, 1H, H4), 8.23 (d, 1H, J = 8.7 Hz, H8), 7.40 (dd, 1H, J = 8.7, 1.9 Hz, H7), 7.33 (d, 1H, J = 1.9 Hz, H5), 4.28 (t, 2H, J = 6.9 Hz, H1′), 3.97 (s, 3H, OCH₃), 3.39–3.34 (m, 2H, H2′′), 2.95–2.88 (m, 2H, H2′′), 2.06–2.01 (m, 2H, H3′′), 1.84 (c, 2H, J = 6.9 Hz, H2′), 1.65–1.58 (m, 1H, H4′′), 1.52–1.45 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 165.3 (C6), 161.0 (C1), 140.0 (C4), 133.4, 129.2 (C8), 122.8 (C7), 122.1, 108.3 (C5), 56.6 (OCH₃), 49.1 (C1′), 44.9 (C2′′), 35.7 (C2′), 32.4 (C4′′), 29.8 (C3′′). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₆H₂₂N₃O₂: 288.17065, found: 288.17074.

4.12 General procedure for the preparation of 2-(N-benzylpiperidin-4-ylalkyl)phthalazin-1(2H)-ones (1a–h)

A suspension of NaH (60% dispersion in mineral oil, 0.75 mmol) in DMF (3 mL) was added to a suspension of the residue containing 14a–h (0.25 mmol) in DMF (3 mL). After stirring at room temperature for 1 h, BnBr (0.38 mmol) was added. The reaction mixture was stirred at room temperature overnight, followed by quenching with H₂O (15 mL) at 0 °C. The product was extracted with EtOAc (3 × 10 mL) and dried over Na₂SO₄. After the solvent removal, the residue was purified by column chromatography on silica gel (EtOAc → EtOAc/MeOH 9 : 1, compounds 1a–d, hexane/EtOAc 1 : 1 → EtOAc/MeOH 99 : 1, compound 1f and hexane/EtOAc 1 : 1 → 1 : 2 → 1 : 3, compounds 1e, 1g and 1h) to obtain the desired compound.

4.12.1 2-(N-Benzylpiperidin-4-ylmethyl)phthalazin-1(2H)-one (1a). Colorless oil. Yield: 34% (from 13a). R_f = 0.4 (EtOAc/MeOH, 9 : 1). ¹H NMR (CDCl₃) δ = 8.42 (d, 1H, J = 7.5 Hz, H8), 8.15 (s, 1H, H4), 7.83–7.73 (m, 2H, Ar), 7.71–7.67 (m, 1H, Ar), 7.33–7.28 (m, 3H, Ar), 7.28–7.21 (m, 2H, Ar), 4.14 (d, 2H, J = 7.2 Hz, H1′), 3.51 (s, 2H, CH₂Ph), 2.94–2.86 (m, 2H, H2′′), 2.23–2.11 (m, 1H, H4′′), 2.09–1.93 (m, 2H, H2′′), 1.71–1.62 (m, 2H, H3′′), 1.55–1.43 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.8 (C1), 137.6 (C4), 133.2, 131.8, 129.7, 129.4, 128.3, 127.2, 126.9, 126.1, 63.3 (CH₂Ph), 56.6 (C1′), 53.3 (C2′′), 35.5 (C4′′), 30.0 (C3′′). HRMS-EI: m/z [M]⁺ calcd for C₂₁H₂₃N₃O: 333.1841, found: 333.1845.

4.12.2 2-(2-(N-Benzylpiperidin-4-yl)ethyl)phthalazin-1(2H)-one (1b). Colorless oil. Yield: 93% (from 13b). R_f = 0.3 (EtOAc/MeOH, 9 : 1). ¹H NMR (CDCl₃) δ = 8.42 (d, 1H, J = 7.6 Hz, H8), 8.16 (s, 1H, H4), 7.82–7.73 (m, 2H, Ar), 7.71–7.66 (m, 1H, Ar), 7.33–7.29 (m, 3H, Ar), 7.28–7.21 (m, 2H, Ar), 4.27 (t, 2H, J = 7.5 Hz, H1′), 3.51 (s, 2H, CH₂Ph), 2.94–2.86 (m, 2H, H2′′), 2.02–1.92 (m, 2H, H2′′), 1.84–1.73 (m, 4H, H2′, H3′′), 1.42–1.32 (m, 3H, H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 159.4 (C1), 137.8 (C4), 133.1, 131.7, 129.7, 129.5, 128.3, 128.1, 127.1, 126.8, 126.1, 63.5 (CH₂Ph), 53.8 (C2′′), 49.1 (C1′), 35.2 (C2′), 33.5 (C4′′), 32.1 (C3′′). HRMS-EI: m/z [M]⁺ calcd for C₂₂H₂₅N₃O: 347.1998, found: 347.1995.

4.12.3 2-(N-Benzylpiperidin-4-ylmethyl)-4-p-tolylphthalazin-1(2H)-one (1c). Colorless oil. Yield: 30% (from 13c). R_f = 0.4 (EtOAc/MeOH, 9 : 1). ¹H NMR (CDCl₃) δ = 8.54–8.50 (m, 1H, H8), 7.79–7.71 (m, 3H, Ar), 7.46 (d, 2H, J = 8.0 Hz, Ar), 7.33 (d, 2H, J = 8.0 Hz, Ar), 7.31–7.29 (m, 3H, Ar), 7.28–7.21 (m, 2H, Ar), 4.21 (d, 2H, J = 7.1 Hz, H1′), 3.50 (s, 2H, CH₂Ph), 2.93–2.86 (m, 2H, H2′′), 2.45 (m, 3H, CH₃), 2.11–1.91 (m, 3H, H2′′, H4′′), 1.73–1.65 (m, 2H, H3′′), 1.57–1.44 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.5 (C1), 147.0, 139.3, 132.8, 132.4, 131.4, 129.5, 129.2, 128.4, 128.3, 127.4, 126.8, 63.1 (CH₂Ph), 56.5 (C1′), 53.2 (C2′′), 35.4 (C4′′), 29.7 (C3′′), 21.5 (CH₃). HRMS-EI: m/z [M]⁺ calcd for C₂₈H₂₉N₃O: 423.2311, found: 423.2307.

4.12.4 2-(2-(N-Benzylpiperidin-4-yl)ethyl)-4-p-tolylphthalazin-1(2H)-one (1d). Colorless oil. Yield: 63% (from 13d). R_f = 0.3 (EtOAc/MeOH, 9 : 1). ¹H NMR (CDCl₃) δ = 8.54–8.49 (m, 1H, H8), 7.80–7.71 (m, 3H, Ar), 7.47 (d, 2H, J = 8.2 Hz, Ar), 7.33 (d, 2H, J = 8.2 Hz, Ar), 7.32–7.29 (m, 3H, Ar), 7.28–7.21 (m, 2H, Ar), 4.33 (t, 2H, J = 7.5 Hz, H1′), 3.51 (s, 2H, CH₂–Ph), 2.94–2.86 (m, 2H, H2′′), 2.46 (s, 3H, CH₃), 2.02–1.92 (m, 2H, H2′′), 1.88–1.74 (m, 5H, H2′, H3′′, H4′′), 1.41–1.33 (m, 2H, H3′′). ¹³C NMR (CDCl₃) δ = 159.1 (C1), 147.1, 139.25, 132.7, 132.5, 131.4, 129.5, 129.3, 128.4, 128.3, 127.3, 127.1, 126.8, 63.5 (CH₂–Ph), 53.8 (C2′′), 49.3 (C1′), 35.2 (C2′), 33.6 (C4′′), 32.2 (C3′′), 21.5 (CH₃). HRMS-EI: m/z [M]⁺ calcd for C₂₉H₃₁N₃O: 437.2467, found: 437.2463.

4.12.5 2-(2-(N-Benzylpiperidin-4-yl)ethyl)-4-methylphthalazin-1(2H)-one (1e). Colorless oil. Yield: 34% (from 13e) R_f = 0.6 (EtOAc/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CDCl₃) δ = 8.44 (d, 1H, J = 8.5 Hz, H8), 7.80–7.71 (m, 3H, Ar), 7.31–7.21 (m, 5H, Ar), 4.25–4.19 (m, 2H, H1′), 3.48 (s, 2H, CH₂Ph), 2.91–2.83 (m, 2H, H2′′), 2.57 (s, 3H, CH₃), 1.98–1.90 (m, 2H, H2′′), 1.81–1.74 (m, 4H, H2′, H3′′), 1.39–1.32 (m, 3H, H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 159.2 (C1), 143.4 (C4), 138.4, 132.8, 131.2, 129.7, 129.34, 128.2, 127.8, 127.1, 127.0, 124.7, 63.5 (CH₂Ph), 53.8 (C2′′), 48.9 (C1′), 35.2 (C2′), 33.6 (C4′′), 32.2 (C3′′), 18.9 (CH₃). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈N₃O: 362.22269, found: 362.22152.

4.12.6 2-(2-(N-Benzylpiperidin-4-yl)ethyl)-6,7-dimethoxyphthalazin-1(2H)-one (1f). White solid. Yield: 66% (from 13f). R_f = 0.5 (EtOAc/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CDCl₃) δ = 8.03 (s, 1H, H4), 7.75 (s, 1H, H8), 7.29–7.21 (m, 5H, Ar), 6.97 (s, 1H, H5), 4.24 (t, 2H, J = 7.5 Hz, H1′), 4.02 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 3.46 (s, 2H, CH₂Ph), 2.88–2.82 (m, 2H, H2′′), 1.96–1.88 (m, 2H, H2′′), 1.80–1.73 (m, 4H, H2′, H3′′), 1.35–1.29 (m, 3H, H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 159.0 (C1), 153.8, 152.9, 138.5, 136.8 (C4), 129.3, 128.2, 126.9, 125.0, 123.0, 106.5 (C5), 105.6 (C8), 63.6 (CH₂Ph), 56.6 (OCH₃), 56.4 (OCH₃), 53.8 (C2′′), 49.1 (C1′), 35.2 (C2′), 33.5 (C4′′), 32.3 (C3′′). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₀N₃O₃: 408.22817, found: 408.22756.

4.12.7 2-(2-(N-Benzylpiperidin-4-yl)ethyl)-7-methoxyphthalazin-1(2H)-one (1g). Colorless oil. Yield: 59% (from 13g). R_f = 0.5 (EtOAc/MeOH/NH₃, 90 : 9.5 : 0.5). ¹H NMR (CDCl₃) δ = 8.05 (s, 1H, H4), 7.75 (d, 1H, J = 2.4 Hz, H8), 7.57 (d, 1H, J = 8.7 Hz, H5), 7.32–7.26 (m, 6H, H6, Ar), 4.23 (t, 2H, J = 7.3 Hz, H1′), 3.92 (s, 3H, OCH₃), 3.45 (s, 2H, CH₂Ph), 2.87–2.82 (m, 2H, H2′′), 1.94–1.88 (m, 2H, H2′′), 1.78–1.72 (m, 4H, H2′, H3′′), 1.34–1.29 (m, 3H, H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 162.4 (C7), 159.3 (C1), 138.3, 137.4 (C4), 130.0, 129.4 (C5), 128.2, 127.9, 127.0, 123.8, 123.5 (C6), 106.5 (C8), 63.5 (CH₂Ph), 56.0 (OCH₃), 53.8 (C2′′), 49.2 (C1′), 35.2 (C2′), 33.5 (C4′′), 32.2 (C3′′). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈N₃O₂: 378.21760, found: 378.21775.

4.12.8 2-(2-(N-Benzylpiperidin-4-yl)ethyl)-6-methoxyphthalazin-1(2H)-one (1h). White solid. Yield: 57% (from 13h). R_f = 0.6 (EtOAc/MeOH/NH₃, 90/9.5/0.5). ¹H NMR (CDCl₃) δ = 8.31 (d, 1H, J = 8.9 Hz, H8), 8.06 (s, 1H, H4), 7.31–7.22 (m, 6H, H7, Ar), 6.98 (d, 1H J = 2.4 Hz, H5), 4.23 (t, 2H, J = 7.5 Hz, H2′′), 3.92 (s, 3H, OCH₃), 3.47 (s, 2H, CH₂Ph), 2.89–2.83 (m, 2H, H2′′), 1.97–1.89 (m, 2H, H2′′), 1.80–1.72 (m, 4H, H2′, H3′′), 1.37–1.29 (m, 3H, H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 163.2 (C6), 159.2 (C1), 138.4, 137.3 (C4), 131.7, 129.4, 128.6, 128.2 (C8), 127.0, 121.8, 121.0 (C7), 106.5 (C5), 63.5 (CH₂Ph), 55.8 (OCH₃), 53.9 (C2′′), 48.9 (C1′), 35.2 (C2′), 33.5 (C4′′), 32.2 (C3′′). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈N₃O₂: 378.21760, found: 378.21828.

4.13 3-(Piperidin-4-yl)propanol hydrochloride (16)

To a solution of 3-(pyridin-4-yl)propanol (15) (50 mg, 0.36 mmol) in EtOH (2 mL) 4 M HCl in dioxane (90 μL, 0.36 mmol) and PtO₂ (3 mg) were added. Then the mixture was purged with H₂ for 20 min. The reaction mixture was stirred at 45 °C under hydrogen atmosphere for 5 h. The catalyst was filtered off and the resulting filtrate was concentrated and dried under vacuum to afford 16 (65 mg, 100%) as a white solid. ¹H NMR (CD₃OD) δ = 3.48 (t, 2H, J = 6.4 Hz, H1), 3.34–3.28 (m, 2H, H2′), 2.96–2.86 (m, 2H, H2′), 1.91–1.83 (m, 2H, H3′), 1.57–1.45 (m, 3H, H4′, H2), 1.42–1.26 (m, 4H, H3′, H3). ¹³C NMR (CD₃OD) δ = 62.8 (C1), 45.2 (C2′), 34.5 (C4′), 33.2 (C3), 30.3 (C2), 29.8 (C3′). HRMS (ESI): m/z [(M − Cl) + H]⁺ calcd for C₈H₁₈NO: 144.13829, found: 144.13843.

4.14 3-(1-Benzylpiperidin-4-yl)propanol (17)

BnBr (35 μL, 0.29 mmol) and K₂CO₃ (178 mg, 1.28 mmol) were added to a solution of 16 (41 mg, 0.23 mmol) in EtOH. The reaction mixture was stirred at r.t. for 1 h and refluxed for 2 h. After cooling, was filtered, concentrated under vacuum and the residue was treated with a saturated solution of NaHCO₃ (5 mL). The resulting solution was extracted with CH₂Cl₂ (3 × 5 mL) and the combined layers dried over Na₂SO₄ and concentrated under reduced pressure to afford 17 (40 mg, 76%) as a colorless oil. R_f = 0.2 (EtOAc/MeOH, 97 : 3). ¹H NMR (CDCl₃) δ = 7.34–7.22 (m, 5H, Ar), 3.56 (t, 2H, J = 6.7 Hz, H1), 3.50 (s, 2H, CH₂Ph), 2.92–2.85 (m, 2H, H2′), 1.99–1.90 (m, 2H, H2′), 1.70–1.63 (m, 2H, H3′), 1.60–1.50 (m, 2H, H2), 1.33–1.21 (m, 5H, H3′, H4′, H3). ¹³C NMR (CDCl₃) δ = 137.8 (C, Ar), 129.4, 128.1, 126.9, 63.4 (CH₂Ph), 62.5 (C1), 53.7 (C2′), 35.6 (C4′), 32.6 (C3), 32.7 (C3′), 29.9 (C2). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₅H₂₄NO: 234.18524, found: 234.18520.

4.15 1-Benzyl-4-(3-bromopropyl)piperidine (18)

It was prepared as described for compounds 12 by reaction of alcohol 17 (40 mg, 0.17 mmol), CBr₄ (115 mg, 0.34 mmol) and PPh₃ (91 mg, 0.34 mmol) in CH₂Cl₂ (2 mL). The residue was purified by column chromatography on silica gel (EtOAc/MeOH, 95 : 5) to give 18 (33 mg, 65%) as a white solid. R_f = 0.4 (EtOAc). ¹H NMR (CDCl₃) δ = 7.36–7.26 (m, 5H, Ar), 3.51 (s, 2H, CH₂Ph), 3.41 (t, 2H, J = 6.9 Hz, H3′), 2.93–2.87 (m, 2H, H2), 1.99–1.85 (m, 4H, H2, H2′), 1.70–1.63 (m, 2H, H3), 1.42–1.36 (m, 2H, H1′), 1.32–1.24 (m, 3H, H3, H4). ¹³C NMR (CDCl₃) δ = 138.60 (C, Ar), 129.3, 128.2, 127.0, 63.6 (CH₂Ph), 53.9 (C2), 35.3 (C4), 35.2 (C1′), 34.3 (C3′), 32.4 (C3), 30.3 (C2′). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₅H₂₃BrN: 296.10084, found: 296.10079.

4.16 3-(N-Benzypiperidin-4-yl)propylphthalazin-1(2H)-one (1i)

It was prepared as described for phthalazinones 1a–h by reaction of 10a (11 mg, 0.07 mmol), NaH (60% dispersion in mineral oil, 5 mg, 0.11 mmol) and 18 (25 mg, 0.08 mmol) in DMF (2 mL). The residue was purified by column chromatography on silica gel (hexane/EtOAc 1 : 1 → EtOAc/MeOH 99 : 1) to afford 1i (19 mg, 70%) as a colorless oil. R_f = 0.6 (EtOAc/MeOH 99 : 1). ¹H NMR (CDCl₃) δ = 8.42 (d, 1H, J = 7.4 Hz, H8), 8.15 (s, 1H, H4), 7.83–7.73 (m, 2H, Ar), 7.71–7.66 (m, 1H, Ar), 7.33–7.24 (m, 5H, Ar), 4.21 (t, 2H, J = 7.4 Hz, H1′), 3.51 (s, 2H, CH₂Ph), 2.94–2.84 (m, 2H, H2′′), 2.00–1.91 (m, 2H, H2′′), 1.90–1.80 (m, 2H, H2′), 1.70–1.63 (m, 2H, H3′′), 1.36–1.23 (m, 5H, H3′ H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 159.4 (C1), 137.8 (C4), 133.1, 131.7, 129.7, 129.6, 128.3, 128.0, 127.2, 126.8, 126.0, 63.3 (CH₂Ph), 53.7 (C2′′), 51.4 (C1′), 35.5 (C4′′), 33.5 (C3′), 32.1 (C3′′), 26.0 (C2′). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈N₃O: 362.22269, found: 362.22359.

4.17 3-(N-Benzypiperidin-4-yl)propyl-6,7-dimethoxyphthalazin-1(2H)-one (1j)

It was prepared as described for phthalazinones 1a–h by reaction of 10d (19 mg, 0.09 mmol), NaH (60% dispersion in mineral oil, 6 mg, 0.14 mmol) and 18 (30 mg, 0.1 mmol) in DMF (3 mL). The residue was purified by column chromatography on silica gel (EtOAc → EtOAc/MeOH 97 : 3) to afford 1j (30 mg, 77%) as a white solid. R_f = 0.3 (EtOAc). ¹H NMR (CDCl₃) δ = 8.03 (s, 1H, H4), 7.75 (s, 1H, H8), 7.29–7.21 (m, 5H, Ar), 6.98 (s, 1H, H5), 4.19 (t, J = 7.3 Hz, 2H, H1′), 4.02 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 3.46 (s, 2H, CH₂Ph), 2.87–2.81 (m, 2H, H2′′), 1.94–1.80 (m, 4H, H2′, H2′′), 1.67–1.60 (m, 2H, H3′′), 1.33–1.22 (m, 5H, H3′, H3′′, H4′′). ¹³C NMR (CDCl₃) δ = 159.1 (C1), 153.8, 152.9, 138.4, 136.9 (C4), 129.4, 128.2, 127.0, 125.0, 122.9, 106.5 (C8), 105.6 (C5), 63.5 (CH₂Ph), 56.6 (OCH₃), 56.4 (OCH₃), 53.9 (C2′′), 51.5 (C1′), 35.6 (C4′′), 33.5 (C3′), 32.3 (C3′′), 26.0 (C2′). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₅H₃₂N₃O₃: 422.24382, found: 422.24313.

4.18 Determination of AChE and BuChE activities

Ellman's method was used to determine in vitro ChE activity.²⁶ The activity was measured by the increase in absorbance at 412 nm due to the yellow color of 5-mercapto-2-nitrobenzoic acid produced by the reaction of thiocholine with dithiobisnitrobenzoic acid (DTNB). The assay solution consisted of a 50 mM phosphate buffer pH 7.2, with the addition of 0.25 mM 5,5′-dithio-bis(2-nitrobenzoic acid) (DNTB), 0.01 U mL⁻¹ AChE from human erythrocytes or 0.005 U mL⁻¹ BuChE from human serum (Sigma), and 5 mM substrate (acetylthiocholine or butyrylthiocholine iodide). Test compounds were added to the buffer and preincubated at 37 °C with the enzyme for 5 min followed by the addition of cromogene and substrate. The activity was determined by measuring the increase in absorbance at 412 nm at 1 min intervals for 10 min at 37 °C (Fluo-Star Optima™, BMG LABTECH, Offenburg, Germany). Control experiments were carried out simultaneously by replacing the test drugs (new compounds and reference inhibitors) with appropriate dilutions of the vehicles. The specific absorbance (used to obtain the final results) was calculated after subtraction of the background activity, which was determined from wells containing all components except the AChE or BuChE, which was replaced by a sodium phosphate buffer solution. ChE activity of the test compounds and reference inhibitors is expressed as IC₅₀, that is, the concentration of each drug required to produce a 50% decreased in control value of AChE or BuChE activity.

4.19 Molecular modeling studies

All AChE inhibitors were built and their partial charges calculated after semi-empirical (PM6) energy minimization²⁷ using the MOE2014.²⁸ Two crystallographic structures were selected to perform docking studies: the hAChE in complex with donepezil (PDB ID: 4EY7)²⁹ and BuChE in complex with tacrine (PDB ID: 4BDS).³⁰ Water molecules and all ligands present in the pdb file were removed and the proteins were subjected to the MOE2014 structure preparation tool.³¹ Finally, the protonate 3D tool was used to assign the protomeric state. To identify the more appropriate protocol for the selected complexes, we performed a self-docking benchmark using DockBench 1.01a software,³² which compared the performance of 17 different posing/scoring protocols. The active site was defined using a radius of 12 Å from the centre of mass of the co-crystallized ligand. Each ligand was docked 10 times.

All synthesized analogues were docked using GOLD³³ using ChemPLP³⁴ and Goldscore³⁵ as scoring functions for 4EY7 and 4BDS, respectively, using the virtual screening tool of DockBench adopting the parameters used in the benchmark study. Finally, the obtained conformations with 4 bds from the docking were rescored with ChemPLP.

The predicted ADME and physicochemical properties were calculated using the StarDrop program.³⁶

Acknowledgements

We acknowledge the Xunta de Galicia (CN2012/184 and 10PXIB203303PR) and the Universidade de Vigo for financial support. N.V. thanks the Universidade de Vigo for a pre-doctoral contract. The molecular modeling work coordinated by S.M. was carried out with financial support from the University of Padova, Italy, and the Italian Ministry for University and Research (MIUR), Rome, Italy. We are very grateful to Chemical Computing Group for long and fruitful collaborations.

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Footnote

† Electronic supplementary information (ESI) available: General structure and numbering of phthalazinone derivatives, ¹H and ¹³C NMR spectra and docking figures. See DOI: 10.1039/c6ra03841g

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