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

Article abstract of DOI:10.1039/c6ra03841g

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.

Full text of DOI:10.1039/c6ra03841g

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Title: Synthesis, biological evaluation and molecular modeling stuDdOiIe: 1s0.1o03f9/C6RA03841G
phthalazin-1(2H)-one derivatives as novel cholinesterase inhibitors
Noemí Vila,a, b Pedro Besada,a, b Dolores Viña,c Mattia Sturlese,d Stefano Moro,d
Carmen Terána, b,
*
a Departamento de Química Orgánica, Universidade de Vigo, 36310, Vigo, Spain
b Instituto de Investigación biomédica (IBI), Universidade de Vigo, 36310, Vigo, Spain
c
Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS)
Universidade de Santiago de Compostela,15782, Santiago de Compostela, Spain
d Molecular Modeling Section (MMS), Dipartimento di Scienze del Farmaco, Università
degli Studi di Padova, 35131, Padova, Italy
*To whom correspondence should be addressed.
Phone: 34 986 812276
Fax: 34 986 812262
E.mail: mcteran@uvigo.es
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ABSTRACT
A new series of donepezil analogues based on phthalazin-1(2H)-one scaffold was
designed and synthesized with the aim of exploring its potential as human ChEIs. The
biological results revealed that the structural modifications proposed significantly
affected ChE inhibitory potency as well as selectivity AChE/BuChE. Compound 1d
showed promising in vitro inhibition of both enzymes, in the µM range. However, most
of target compounds were significantly more active against AChE than BuChE, being
compounds 1f, 1h and 1j, with IC50 values in the low micromolar or submicromolar
range, the most active compounds in this series.
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. Moreover, the predicted
physicochemical and ADME properties were also comparable with those of donepezil.
Keywords: Phthalazinone derivatives, Acetylcholinesterase, Butyrylcholinesterase,
Molecular modeling, ADME, Alzheimer’s disease
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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 from AD, and the number of patients would be
probably increased over the upcoming years because of the rise of 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 the 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 -amyloid (A ) and -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 this pivotal information explains that 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 US Food and Drug Administration
(FDA) for AD, four AChEIs, tacrine, donepezil, rivastigmine and galantamine, and one
NMDA antagonist, memantine.
However, studies developed in the past decades indicate that the acetylcholinesterase
(AChE) in AD brains, besides catalysing the hydrolysis of acetylcholine, plays also an
important role in A plaques deposition because of its interaction with the A peptide
through a set of amino acids located close to the peripheral anionic site (PAS) of the
enzyme [5], being proved that AChEIs which bind to PAS could inhibit such processes
[6]. Furthermore, the new findings about the roles of the neuronal and nonneuronal
cholinergic systems on modulation of regional brain blood flow may also contribute to
get better potential for cholinergic therapies in AD [7].
In addition, the levels of ACh are also regulated by butyrylcholinesterase (BuChE),
another cholinesterase enzyme with a synaptic ACh hydrolysis role 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
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that BuChE inhibition may also be considered a valid approach for AD therapy [8]. The
BuChE role in regulation of 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 which binds simultaneously to CAS (catalytic
active site) and PAS sites providing moderate inhibition of the A aggregation [11]. In
this respect, recent clinical trials have shown that a 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], it is
desirable to study new pharmacophoric scaffolds. In this regard, pyridazine could be
considered a privileged structure because its derivatives present a large 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 ring [17]
and only a few include in their structure the pyridazin-3-(2H)-one framework [18].
However, these last pyridazinone analogues do not share any structural similarity with
donepezil.
Considering the above mentioned and as a continuation to our work focused on
developing enzyme inhibitors potentially effective in aging-related disorders [19], we
have designed a series of donepezil analogues with structure 1 (Figure 1). In these novel
compounds the indanone portion was replaced by a phthalazin-1(2H)-one moiety,
preserving or not the methoxy groups, and the linking chain between the new bicyclic
fragment and N-benzylpiperidine group was ranged from one to three carbon atoms. In
addition, the C4 position of this new scaffold could be substituted with groups of
different size and electronic properties. These structural modifications were performed
with the idea of analyzing the ability of the phthalazinone nucleus to modulate the
AChE and BuChE activities.
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Figure 1 comes about here
Thus, in this work we report the synthesis, the AChE and BuChE inhibitory activities
and the molecular modeling of representative molecules of a new family of phthalazin-
1(2H)-ones (compounds 1, Figure 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 to synthesize 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, the synthesis of phthalazinones
10d-f, showing one or two methoxy groups on the phenyl ring, was accomplished
adapting several procedures previously described and by using the phthalides 5 as key
intermediates, which via benzylic bromination provided the 3-bromo phthalides 9
suitable for cyclization with hydrazine [21].
Scheme 1 comes about here
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].
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The reaction of 4-substituted phthalic anhydrides with alkaline borohydrideDsOIu: 1s0u.1a0l3l9y/C6RA03841G
provided 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, was detected a preference for reduction at m-carbonyl group
[24]. In contrast with literature, the treatment of 4-methoxyphthalic anhydride 4 with an
equimolecular amount of NaBH4 gave a mixture of compounds, lactone 5c and
hydroxymethyl benzoic acid 6 (ratio5c/6 1:1.5, very good yield), both resulting from the
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). The 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.
Then, the target compounds 1a-h were prepared in four steps using as starting material
the phthalazinones 10 and the commercially available N-Boc-4-hydroxyalkylpiperidines
11 (Scheme 2). Thus, firstly, the N-Boc-4-hydroxyalkylpiperidines 11a,b were
transformed into the 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 N-Boc
protecting group from the corresponding 2-(N-Boc-4-piperidinylalkyl)phthalazin-1-ones
13a-h by acid hydrolysis, with 6M HCl in ethyl acetate, was followed by treatment with
benzyl bromide in the presence of sodium hydride to give desired compounds 1a-h in
good to moderate yield.
Scheme 2 comes about here
Finally, in scheme 3 was detailed the strategy adopted for the synthesis of target
compounds 1i,j bearing an alkyl chain of three carbons between phthalazinone and N-
benzylpiperidine fragments.
Scheme 3 comes about here
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In this case, the bromoalkane 18 was the reagent involved in N-alkyDlaOtIi:o10n.103o9/fC6RA03841G
phthalazinones 10a and 10d. The compound 18 was synthesized in good yield starting
from 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 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.
2.2. Pharmacology
The in vitro activity of compounds 1a-j against AChE and BuChE was determined by
Ellman´s method [26] evaluating the hydrolysis of acetylthiocholine and
butyrylthiocholine, respectively. The activity was measured by the increased 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.
The 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.
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 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 kind of structural modifications proposed, length variation of alkyl chain
linking phthalazinone and benzylpiperine scaffolds, substitution at C4 and inclusion of
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methoxy groups in C6 or C7, significantly affected ChE inhibitory potency as well as
selectivity AChE/BuChE.
Thus, compounds 1a and 1c (both with n = 1) are inactive against AChE at 100
the
highest concentration tested, and moderately active against BuChE, with IC50 values of
46.14 and 13.26 µM, respectively. By contrast, their homologues 1b, 1d and also the
analogue 1e (all them with n = 2) exhibited inhibitory activity against the AChE at low
micromolar concentrations, being compound 1b the most active with an IC50 value of
2.58 M. 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 that was 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) resulted disadvantageous for AChE inhibition (IC50 = 10.29 µM).
With regard to the role of methoxy substituents at C6 and C7 in this new series, it can
be noticed that when it is placed at C6, the methoxy group provides a good and selective
inhibitory effect against AChE, being compounds 1f (IC50 = 0.67), 1h (IC50 = 0.55) and
1j (IC50 = 1.07) the most potent and selective AChEIs of this series, while when it was
moved to C7 position the AChE inhibitory activity seems unaffected (compound 1g,
IC50 = 2.40 µM).
Table 1 comes about here
2.3. Molecular Modeling
Dockings studies were carried out in order to analyze the AChE binding mode of the
novel inhibitors with the aim to interpret the experimental affinity data.
To identify which docking protocol and which protein-ligand complex 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 due to the molecular similarity with our
analogues. The benchmark study revealed that several protocols were able to reproduce
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the crystallographic geometries (see the Experimental Section for details). Among them,
GOLD software when using ChemPLP as scoring function, gave the best performance
(SI 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 (SI Fig.1,
panels C and D).
The selected protocol was used to dock all the analogues into hAChE and hBuChE as
described in detail in the Experimental Section.
All analogues showed a common binding mode similarly 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 sidechains and the backbone of
Phe295 and Arg296.
Based on our docking simulation, all the compounds preserve this network of
interactions (Figure 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 minor
than 10 µM. This hydrogen bond is also present in the crystal structure of donepezil
bound to hAChE, mediated by the indanone core.
This observation is more evident focusing on the per-residue analysis (Figure 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. In Figure 3, is reported the per-residue analysis (panel B) for
a subset of relevant residues taking into account their interaction and their conformation
respect to the most potent analogue (compound 1h, Panel A). The minor activity of
compounds 1a and 1c could be attributed to the lacks 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 shape complementary and lead to a slightly different
orientation of the phtalazinone core (Fig. 4; 1c and 1d). However, in the case the of
compound 1d the longer alkyl linker allows to stabilize the ligand through a similar
hydrogen bond with the Arg296 instead of Phe295, and at the same time to maintain the
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benzyl group stacked to Trp86, similarly to the other analogues without the p-tolyl
moiety. The effect of the different linker length between compound 1c and 1d is evident
observing the difference in the per-residue profile (residues Trp86 and Arg296) and is in
good agreement with the 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 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 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). Only for
compound 1i, the molecular docking studies have not shown a clear relationship
between the complex conformation and the IC50 values; the ligand orientation is
extremely similar to those of the most active analogues but it lacks in the formation of
the H-bond with the Phe295.
The 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 respect to the conformation assumed when complex with hAChE (Fig. 5,
panel A). The reason of this orientation can be ascribed to the few differences between
the two binding sites. Despite the notable similarity between both enzymes (Cα-RMSD
1.36 Å, SI Fig. 1 panel E), they show differences in some fundamental residue for the
binding of the phthalazinone group. Thus, the Trp286 that establish 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) not compatible with the hydrogen bond formation due to a distortion
induced by Pro285, which is not present in hAChE (SI Fig.1, panel E and F).
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 the StarDrop software to
predict physicochemical and ADME (absorption, distribution, metabolism and
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excretion) properties of the novel AChE inhibitors, such as, aqueous solubility (logS),
lipophilicity (clogP), 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 donezepil in particular for
compound 1b and its methoxy derivatives 1f, 1g, 1h, all them potent and selective
AChEIs (Table 3).
Table 3 comes about here
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 the target
compounds the indanone was replaced by the phthalazinone nucleus, preserving or not
the methoxy groups, and showing the N-benzylpiperidine fragment linked at N2 by an
alkyl chain ranged from one to three carbons. Additionally, The C4 position of the
phthalazinone scaffold could be substituted with groups of varying size 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 compounds 1a, 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) resulted
equipotent toward AChE and BuChE enzymes, behaving as a dual cholinesterase
inhibitor (AChE IC50 = 3.45 µM and BuChE IC50 = 5.50 µM).
As it can be seen, the 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 ranged from
2.58 to 3.45 µM, while a decreased potency was observed for the homologue 1i (IC50 =
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10.29, n = 3). Finally, it is noteworthy the role of methoxy substituents in the activity of
these new donepezil analogues. Therefore, when the methoxy group is located at C6
provides a good and selective inhibitory effect against AChE, being compounds 1f (IC50
= 0.67), 1h (IC50 = 0.55) and 1j (IC50 = 1.07), 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 can be
considered potential drug candidates to develop new ChEIs, also considering the
predicted physicochemical and ADME properties.
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
1
and dried according to standard procedures. H and 13C NMR spectra were recorded on
a Bruker ARX400 instrument, using TMS as internal standard [chemical shifts ( ) in
ppm, J in Hz]. The assignment of the signals was performed by COSY, DEPT, 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 precoated with silica
gel (Merck 60 F254, 0.25 mm).
4.2. 4-Methylphthalazin-1(2H)-one (10c)
To a solution of 2-acetylbenzoic acid (8) (1 g, 6.09 mmol) in EtOH (10 mL) was added
hydrazine hydrate (0.39 mL, 7.84 mmol). The reaction mixture was refluxed overnight.
After cooling, the resulting precipitate was filtered and washed with ethanol to give 10c
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(875 mg, 91%) as a white solid. Rf = 0.5 (hexane/EtOAc, 1:1). H NMR (CDCl3):
=
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): = 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 5M NaOH (3 mL, 15 mmol). The reaction mixture was refluxed
overnight. After cooling at r.t., 3M HCl (5 mL) was carefully added until pH = 1 and the
mixture was extracted with CH2Cl2 (3 x 20 mL), the organic layers were collected, dried
over Na2SO4 and evaporated to dryness to give 3 (766 mg, 96%). 1H NMR (CD3OD):
= 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
13
Hz, H5), 3.85 (s, 3H, OCH3). C NMR (CD3OD) = 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 reduce pressure to
furnish anhydride 4 (266 mg, 98%). 1H NMR (CDCl3): = 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) = 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)
Method A: to a solution of NaBH4 (11 mg, 0.28 mmol) in THF (2 mL) cooled at 0 ºC
was added dropwise a solution of compound 4 (50 mg, 0.28 mmol) in THF (3 mL). The
reaction mixture was stirred for 4 h at r.t., treated with 6M HCl until pH = 1 and then
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extracted with Et2O (3 x 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: Rf =
0.5 (hexane/EtOAc, 1:1). 1H NMR (CDCl3): = 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, ): 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:
1
165.05462, found: 165.05451. Compound 6: Rf = 0.2 (hexane/EtOAc, 1:1). H NMR
(CDCl3) = 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) = 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).
Method B: following the procedure above described 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 6M HCl (7 mL) and refluxed for 2 h. After cooling, the reaction
mixture was extracted with CH2Cl2 (6x12 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) was added 30% formaldehyde (0.13 mL, 1.25 mmol). The reaction mixture was
stirred at 90 ºC for 10 h. After cooling, was quenched with H2O (5 mL) and extracted
with EtOAc (4 x 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%). Rf = 0.4 (hexane/EtOAc, 1:2). 1H NMR (CDCl3): = 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): = 171.2 (C1), 154.7, 150.2, 141.0, 117.2, 105.7 (C7), 103.5 (C4),
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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) was added 37% HCl (4.8 mL, 57.9 mmol) and 30% formaldehyde (1.92 mL, 25.8
mmol) and the reaction mixture was stirred at 100 ºC for 1 h. After cooling, a saturated
solution of NaHCO3 was added until pH = 7. The resulting mixture was extracted with
CH2Cl2 (3 x 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%). Rf = 0.4
(hexane/EtOAc, 2:1). 1H NMR (CDCl3) = 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) = 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 the 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 refux 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%. Rf = 0.4 (EtOAc). 1H NMR (CDCl3) = 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
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(CDCl3) : 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)
1
White solid. Yield 51%. Rf = 0.4 (hexane/EtOAc, 1:1). H NMR (CDCl3) = 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),
13
7.40 (dd, 1H, J = 8.7, 2.6 Hz, H6), 3.98 (s, 3H, OCH3). C NMR (CDCl3) = 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%. Rf = 0.4 (hexane/EtOAc, 1:1): 0.4. 1H NMR (CDCl3) = 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) = 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 the 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)
1
Colorless oil. Yield: 94%. Rf = 0.7 (hexane/EtOAc, 2:1). H NMR (CDCl3): = 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, 3xCH3), 1.24-1.10 (m, 2H, H3). 13C NMR (CDCl3): = 154.9
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(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)
1
Colorless oil. Yield: 89%. Rf = 0.5 (hexane/EtOAc, 2:1). H NMR (CDCl3): = 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,
13
2H, H1´), 1.70-1.62 (m, 3H, H3, H4), 1.44 (s, 9H, 3xCH3), 1.16-1.04 (m, 2H, H3). C
NMR (CDCl3): = 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 the 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 ºC. The product was extracted with EtOAc (2x10
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%. Rf = 0.4 (hexane/EtOAc, 2:1). 1H NMR (CDCl3): = 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, 3xCH3), 1.36-1.22 (m, 2H, H3´´). 13C NMR (CDCl3):
= 159.8 (C1), 154.9 (COC(CH3)3), 137.8 (C4), 133.3, 131.8, 129.7, 128.0, 126.9, 126.1,
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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%. Rf = 0.4 (hexane/EtOAc, 2:1). 1H NMR (CDCl3): = 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, 3xCH3), 1.19-1.07 (m,
13
2H, H3´´). C NMR (CDCl3): = 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%. Rf = 0.4 (hexane/EtOAc, 2:1). 1H NMR (CDCl3): = 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, 3xCH3), 1.38-1.25 (m,
2H, H3´´). 13C NMR (CDCl3): = 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%. Rf = 0.4 (hexane/EtOAc, 2:1). 1H NMR (CDCl3): = 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
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(s, 9H, 3xCH3), 1.20-1.13 (m, 2H, H3´´). 13C NMR (CDCl3): = 159.1 (CD1O)I,: 110.51053.90/C6RA03841G
(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.
View Article Online
4.10.5. 2-(2-(1-tert-butoxycarbonylpiperidin-4-yl)ethyl)-4-methylphthalazin-1(2H)-one
(13e)
Colorless oil. Yield: 99%. Rf = 0.5 (hexane/EtOAc, 1:1). 1H NMR (CDCl3): = 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´´, 3xCH3), 1.21–1.11 (m, 2H, H3´´). 13C NMR (CDCl3):
=
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)
1
Colorless oil. Yield: 79%. Rf = 0.5 (EtOAc). H NMR (CDCl3) = 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´´,
2xOCH3), 2.70-2.62 (m, 2H, H2´´), 1.81-1.75 (m, 4H, H2´, H3´´), 1.44-1.42 (m, 10H,
H4´´, 3xCH3), 1.25 – 1.20 (m, 2H, H3´´). 13C NMR (CDCl3) = 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%. Rf = 0.5 (EtOAc). 1H NMR (CDCl3) = 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,
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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´´, 3xCH3), 1.21-1.11 (m,
13
2H, H3´´). C NMR (CDCl3) = 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%. Rf = 0.5 (hexane/EtOAc, 1:1). 1H NMR (CDCl3) = 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´´, 3xCH3), 1.20–1.13 (m, 2H, H3´´). 13C NMR (CDCl3) ) = 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)
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1
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Rf = 0.3 (CH2Cl2/MeOH/NH3, 90:9.5:0.5). H NMR (CD3OD): = 8.39 (s, D1OHI: ,10H.10439)/,C6RA03841G
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): = 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)
1
Rf = 0.3 (CH2Cl2/MeOH/NH3, 90:9.5:0.5). H NMR (CD3OD): =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,
13
2H, H2´), 1.65-1.53 (m, 1H, H4´´), 1.49-1.37 (m, 2H, H3´´). C NMR (CD3OD):
=
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)
1
Rf = 0.3 (CH2Cl2/MeOH/NH3, 90:9.5:0.5). H NMR (CD3OD): = 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): = 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)
1
Rf = 0.3 (CH2Cl2/MeOH/NH3, 90/9.5/0.5). H NMR (CD3OD): = 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´),
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1.69-1.57 (m, 1H, H4´´), 1.43-1.33 (m, 2H, H3´´). 13C NMR (CD3OD): = 160.8 (C1),
View Article Online
DOI: 10.1039/C6RA03841G
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)
1
Rf = 0.2 (EtOAc/MeOH/NH3, 90:9.5:0.5). H NMR (CD3OD): = 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): = 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)
Rf = 0.3 (EtOAc/MeOH/NH3, 90/9.5/0.5). 1H NMR (CD3OD) = 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, 2x 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,
13
H2´), 1.27-1.15 (m, 1H, H4´´), 1.13-1.00 (m, 2H, H3´´). C NMR (CD3OD) = 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)
Rf = 0.3 (EtOAc/MeOH/NH3, 90/9.5/0.5). 1H NMR (CD3OD) = 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) = 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´),
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DOI: 10.1039/C6RA03841G
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)
Rf = 0.2 (EtOAc/MeOH/NH3, 90/9.5/0.5). 1H NMR (CDCl3) = 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) = 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 ºC. The product was extracted with EtOAc (3x10 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-1d, 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). Rf = 0.4 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3):
= 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,
13
2H, H2´´), 1.71-1.62 (m, 2H, H3´´), 1.55-1.43 (m, 2H, H3´´). C NMR (CDCl3):
=
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DOI: 10.1039/C6RA03841G
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). Rf = 0.3 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3):
= 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): = 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). Rf = 0.4 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3):
= 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): = 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). Rf = 0.3 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3):
= 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
13
(m, 2H, H2´´), 1.88-1.74 (m, 5H, H2´, H3´´, H4´´), 1.41-1.33 (m, 2H, H3´´). C NMR
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(CDCl3): = 159.1 (C1), 147.1, 139.25, 132.7, 132.5, 131.4, 129.5, 129.3, 128D.4O,I:1102.1803.V39ie/,wC6ARrtAic0le3O8n4l1inGe
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)
1
Colorless oil. Yield: 34% (from 13e) Rf = 0.6 (EtOAc/MeOH/NH3, 90:9.5:0.5). H NMR
(CDCl3): = 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): = 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). Rf = 0.5 (EtOAc/MeOH/NH3, 90:9.5:0.5). 1H NMR
(CDCl3)
= 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´,
13
H3´´), 1.35-1.29 (m, 3H, H3´´, H4´´). C NMR (CDCl3) = 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]+ calculated for C24H30N3O3: 408.22817, found:
408.22756.
4.12.7. 2-(2-(N-Benzylpiperidin-4-yl)ethyl)-7-methoxyphthalazin-1(2H)-one (1g)
1
Colorless oil. Yield: 59% (from 13g). Rf = 0.5 (EtOAc/MeOH/NH3, 90:9.5:0.5). H
NMR (CDCl3) = 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,
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4H, H2´, H3´´), 1.34-1.29 (m, 3H, H3´´, H4´´). 13C NMR (CDCl3) = 162.4 (C7), 159V.i3ew Article Online
DOI: 10.1039/C6RA03841G
(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). Rf = 0.6 (EtOAc/MeOH/NH3, 90/9.5/0.5). 1H NMR
(CDCl3) = 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´,
13
H3´´), 1.37-1.29 (m, 3H, H3´´, H4´´). C NMR (CDCl3) = 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) was
added 4M HCl in dioxane (90 L, 0.36 mmol) and PtO2 (3 mg). Then was purged with
H2 for 20 min. The reaction mixture was stirred at 45 ºC 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) = 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)
= 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)
To a solution of 16 (41 mg, 0.23 mmol) in EtOH was added BnBr (35 L, 0.29 mmol)
and K2CO3 (178 mg, 1.28 mmol). 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
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was treated with a saturated solution of NaHCO3 (5 mL). The resulting solution was
extracted with CH2Cl2 (3x5 mL) and the combined layers dried over Na2SO4 and
concentrated under reduced pressure to afford 17 (40 mg, 76%) as a colorless oil. Rf =
0.2 (EtOAc/MeOH, 97:3). 1H NMR (CDCl3) = 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´),
13
1.70-1.63 (m, 2H, H3´), 1.60–1.50 (m, 2H, H2), 1.33–1.21 (m, 5H, H3´, H4´, H3). C
NMR (CDCl3) = 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. Rf = 0.4 (EtOAc). 1H NMR (CDCl3) = 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) = 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 3a (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
1
oil. Rf = 0.6 (EtOAc/MeOH 99:1). H NMR (CDCl3) = 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) = 159.4 (C1), 137.8 (C4), 133.1, 131.7, 129.7, 129.6,
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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 3d (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. Rf = 0.3
1
(EtOAc). H NMR (CDCl3) = 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)
=
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 the in vitro ChE activity [26]. The activity was
measured by the increase in absorbance at 412 nm due to the yellow colour 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 AChE from human erytrocytes or 0.005 U/mL BuChE from
human serum (Sigma), and 5 mM substrate (acetylthiocholine or butyrylthiocholine
o
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
o
for 10 min at 37 C (Fluo-Star OptimaTM, 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
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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, ie the
concentration of each drug required to produce a 50% decreased on control value AChE
or BuChE activity.
4.19. Molecular modeling studies
All AChE inhibitors were built and their partial charges calculated after semi-empirical
(PM6) energy minimization [27] 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 structure preparation tool of MOE 2014 [31]. Finally
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 GOLD [33] using ChemPLP [34] and
Goldscore [35] as scoring functions for 4EY7 and 4BDS, respectively, using the virtual
screening tool of DockBench adopting the parameters used in the banchmarck study.
Finally, the obtained conformations with 4bds from the docking were rescored with
ChemPLP.
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