New pyridine derivatives as inhibitors of acetylcholinesterase and amyloid aggregation

Article abstract of DOI:10.1016/j.ejmech.2017.09.022

A new series of pyridine derivatives with carbamic or amidic function has been designed and synthesized to act as cholinesterase inhibitors. The synthesized compounds were tested toward EeAChE and hAChE and toward eqBChE and hBChE. The carbamate 8 was the most potent hAChE inhibitor (IC₅₀ = 0.153 ± 0.016 μM) while the carbamate 11 was the most potent inhibitor of hBChE (IC₅₀ = 0.828 ± 0.067 μM). A molecular docking study indicated that the carbamate 8 was able to bind AChE by interacting with both CAS and PAS, in agreement with the mixed inhibition mechanism. Furthermore, the carbamates 8, 9 and 11 were able to inhibit Aβ₄₂ self-aggregation and possessed quite low toxicity against human astrocytoma T67 and HeLa cell lines, being the carbamate 8 the less toxic compound on both cell lines.

Full text of DOI:10.1016/j.ejmech.2017.09.022

Accepted Manuscript
New pyridine derivatives as inhibitors of acetylcholinesterase and amyloid
aggregation
Fabiana Pandolfi, Daniela De Vita, Martina Bortolami, Antonio Coluccia, Roberto Di
Santo, Roberta Costi, Vincenza Andrisano, Francesco Alabiso, Christian Bergamini,
Romana Fato, Manuela Bartolini, Luigi Scipione
PII:
S0223-5234(17)30736-5
10.1016/j.ejmech.2017.09.022
EJMECH 9737
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 July 2017
Revised Date: 13 September 2017
Accepted Date: 13 September 2017
Please cite this article as: F. Pandolfi, D. De Vita, M. Bortolami, A. Coluccia, R. Di Santo, R. Costi, V.
Andrisano, F. Alabiso, C. Bergamini, R. Fato, M. Bartolini, L. Scipione, New pyridine derivatives as
inhibitors of acetylcholinesterase and amyloid aggregation, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.09.022.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
NEW PYRIDINE DERIVATIVES AS INHIBITORS OF ACETYLCHOLINESTERASE
AND AMYLOID AGGREGATION
Fabiana Pandolfi,a Daniela De Vita,a Martina Bortolami,a Antonio Coluccia,a Roberto Di Santo,b
Roberta Costi,b Vincenza Andrisano,c Francesco Alabiso,d Christian Bergamini,d Romana Fato,d
Manuela Bartolini,d* Luigi Scipionea*
a Department of Chemistry and Technology of Drug, Sapienza University of Rome, Piazzale Aldo
Moro, 5, 00185, Roma, Italy.
b Istituto Pasteur, Fondazione Cenci Bolognetti, Department of Chemistry and Technology of Drug,
Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185, Roma, Italy.
c Department for Life Quality Studies, Alma Mater Studiorum University of Bologna, Corso
d’Augusto 237, 47921 Rimini, Italy.
d Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, Via
Belmeloro 6 /Via Irnerio 48, 40126 Bologna, Italy.
*Corresponding authors: Luigi Scipione (luigi.scipione@uniroma1.it); Manuela Bartolini
(manuela.bartolini3@unibo.it).
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Abstract
A new series of pyridine derivatives with carbamic or amidic function has been designed and
synthesized to act as cholinesterase inhibitors. The synthesized compounds were tested toward
EeAChE and hAChE and toward eqBChE and hBChE. The carbamate 8 was the most potent
hAChE inhibitor (IC50 = 0.153 ± 0.016 µM) while the carbamate 11 was the most potent inhibitor of
hBChE (IC50 = 0.828 ± 0.067µM). A molecular docking study indicated that the carbamate 8 was
able to bind AChE by interacting with both CAS and PAS, in agreement with the mixed inhibition
mechanism. Furthermore, the carbamates 8, 9 and 11 were able to inhibit Aβ42 self-aggregation and
possessed quite low toxicity against human astrocytoma T67 and HeLa cell lines, being the
carbamate 8 the less toxic compound on both cell lines.
Keywords
Acetylcholinesterase inhibitors; butyrylcholinesterase inhibitors; amyloid aggregation inhibitors.
1. Introduction
Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases and accounts for
more than 80 % of dementia in elderly people worldwide. AD leads to the progressive mental,
behavioural, functional decline and loss of the ability to learn [1]. Aetiology of AD is multifactorial
with genetic, environmental, behavioural and developmental components playing a role. Several
hypotheses have been put forward on the basis of the various causative factors, among which the
cholinergic, β-amyloid (Aβ), tau, inflammation, oxidative stress and metal dyshomeostasis
hypotheses [2]. According to the cholinergic hypothesis, behavioural disturbances and memory
impairment are partially related to the observed reduction of the cholinergic activity in the brain.
This hypothesis has led to the development of cholinesterase inhibitors (ChEIs), which, together
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with memantine, an NMDA antagonist of glutamate receptors, are the only drugs available on the
market for the palliative treatment of AD [3, 4].
At the neuronal level, the neurotransmitter acetylcholine (ACh) is degraded by two types of
cholinesterases (ChEs): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). AChE is
the main enzyme involved in ACh hydrolysis. AChE is also thought to be involved in non-
cholinergic mechanism related to neurodegeneration. AChE activity is dominant in the healthy
brain (80 %), while BChE seems to play only a supportive role. However, with the progression of
the disease, brain levels of AChE decline to about 60 % of the normal values while those of BChE
increase to 120 % of normal levels. Thus, the role of BChE as ACh degradating enzyme increases
with the disease progression and BChE seems to play a major role in the severe forms of AD [5, 6].
AChE and BChE have 65 % amino acid sequence identity; their catalytic active site (CAS) is
constituted by a catalytic triad, located at the bottom of the gorge, at 20 Å from the entrance. The
catalytic triad is constituted by the amino acids serine, histidine and glutamic acid: Ser203, His447
and Glu202 in human AChE (hAChE) and Ser198, His438 and Glu197 in human BChE (hBChE)
[7, 8]. The ChEs have a "sub-anionic site" that is constituted by aromatic amino acid Trp86 in
hAChE and Trp82 in hBChE, which, through their indole ring, bind the quaternary ammonium of
the choline moiety of the substrate, by means of cation-π interaction [7, 8, 9].
AChE also features a "peripheral anionic site" (PAS); in hAChE, PAS consists of five residues
(Tyr72, Asp74, Tyr124, Trp286 and Tyr341) located at the entrance of the active site gorge. Apart
being involved in the modulation of the catalytic activity, PAS has been reported to be involved in
the pro-aggregating action of AChE toward β-amyloid peptide [10, 11], another important actor in
the onset and progression of this pathology.
Indeed, despite the continuing debate about the primary role of amyloid β‐protein in AD, several
lines of evidence support the idea that Aβ dyshomeostasis is an initiating factor in AD [12, 13].
Exploitation of amyloid hypothesis has fruitfully led to the discovery of several Aβ-targeting
molecules that have reached clinical trials [12]. Other than specific trial-related reasons for failure,
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the complexity and multifactorial nature of this disease might have contributed to drug candidate
withdrawn from further clinical investigations. In this light, the development of multifunctional
active compounds, i.e. multi-target drug ligands (MTDLs), may offer benefits in term of higher
efficacy, allowing a multi-point intervention along the interconnected net of pathological events.
Based on these considerations, the design of compounds described in this study has been focused on
the synthesis of new molecules that can interact with human cholinesterases and interfere with the
aggregation of β-amyloid. We already reported 2,6-dichloropyridine derivatives, which proved to
act as non competitive inhibitors of AChE and were able to revert the amnesic effects of
scopolamine in rats [14]. Thus, we decided to link a 2,6-dichloropyridine fragment to a benzyl
group by means of a flexible alkyl carbamic chain; these two moieties could be able to establish
stacking interactions with the aromatic amino acid residues of PAS and CAS (Figure 1).
We decided to investigate different chain lengths to cover the distance between CAS and PAS.
Because of the lower precision in predicting the conformational behavior of linear alkyl chains, we
decided to investigate both 10- and 11-unit chain lengths (including methylene units, carbonyl,
nitrogens and oxygen). Moreover, a protonable amine group, that can establish cationic-π
interactions with the aromatic amino acids of the active site gorge, was inserted in the alkyl chain.
Furthermore, in order to obtain information about the structural requirements for optimally targeting
AChE, we replaced the phenyl ring with other aromatic and hetero-aromatic groups with increasing
bulkiness and different polarity and H-bonding properties.
In particular, we chose a 2-methoxyphenyl ring, an indole and two bulkier groups, namely naphthyl
and 4-phenoxybenzyl groups. Indeed, previous studies by different research groups have shown that
the 2-methoxyphenyl residue is a suitable fragment to achieve good AChE inhibition [15, 16, 17,
18]. On the other hand, the indole moiety was selected as it could establish hydrophobic and π–π
interactions with the aromatic residues of PAS [19]. Furthermore, the indole fragment has been
already exploited in several AChE inhibitors [20, 21, 22, 23] and the 6-aminoindole was very
recently shown to be able to interfere with amyloid aggregation [24].
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In order to avoid off-target effects related to the carbamoylation of other serine esterases, a series of
amide analogues was also synthesized. The amide bond should also provide a greater metabolic
stability with respect to the carbamic one [25]. Because of the low nucleophilic reactivity of the
exocyclic nitrogen of the 2,6-dichloro-4-aminopyridine, we chose to synthesize retro-amide
analogues. Finally, to ensure that the distance between the aryl and heteroaryl moieties is
maintained, in the amide analogues the number of methylene units was increased, taking into
account the lower length of the amidic function over the carbamatic one. The anticholinesterase
activity toward the Electric ell (Ee) and human (h) isoforms of AChE was evaluated by Ellman’s
assay [26] as well as the inhibitory activity toward amyloid 1-42 (Aβ42) self-aggregation. Finally, to
ensure compound safety, the cell toxicity in T67 and HeLa cell lines was determined.
FIGURE 1
2. Results and discussion
2.1 Chemistry
The synthetic pathway followed for the preparation of the target compounds 8-13 is illustrated in
the Scheme 1.
SCHEME 1
The synthesis of the isocyanates 1 and 2 were carried out by treatment of the appropriate 4-
aminopyridine, dissolved in dry benzene, with triphosgene, in the presence of triethylamine,
(benzene:triphosgene:triethylamine 1.66:1:6), according to the literature [14]. The reaction mixture
was kept under stirring for 5 hours, at reflux temperature for compound 2, at 35 °C in a sand bath
for 1, to avoid degradation. The reaction was monitored by IR spectroscopy to observe the
appearance of isocyanate group (–NCO) stretching absorption band (2250-2260 cm-1). The obtained
isocyanates were used for the subsequent reactions without purification. The synthesis of imino-
alcohols intermediates (3–7) involved opportune aromatic aldehyde and 5-aminopentan-1-ol or 6-
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aminohexan-1-ol, dissolved in the required amount of dichloromethane. The choice of the suitable
desiccant agent was based on the type of the starting aldehyde, so in the case of compound 6,
instead of potassium carbonate, molecular sieves were used to prevent the acid-base reaction, with
the consequent formation of salt, between the indole -NH and potassium carbonate. The reaction
was monitored by IR spectroscopy to confirm the formation of the imine, by the appearance of the
stretching absorption band around 1640 cm-1, and the disappearance of the aldehyde band at about
1
1700 cm-1. The structure of the synthesized imino-alcohols 3-7 was confirmed by H-NMR:
presence of the singlet signal relative to N=CH imine between 8 and 9 ppm, and by the
disappearance of the singlet signal of the aldehyde O=C-H at about 10 ppm. The obtained imino-
alcohols, without any purification, were solubilized in dichloromethane and the resulting solution
was added to isocyanate 1 or 2, dissolved in dry benzene, at 1:1 molar ratio. The formation of the
carbamate function was monitored by IR spectroscopy (C=O stretching band at 1738 cm-1). The
imino-carbamate intermediates, without purification, were reduced with sodium borohydride. After
the reaction, the excess sodium borohydride was decomposed by lowering the pH of reaction
mixture to 6 or below by addition of aqueous HCl. The residues were purified by chromatography
on a silica gel column to obtain the carbamate products (8-13).
The amide derivatives 15-19 were prepared as reported in Scheme 2.
SCHEME 2
The tert-butyl (6-aminohexyl)carbamate, dissolved in dry dichloromethane, was reacted with 2,6-
dichloropyridine-4-carbonyl chloride, in the presence of triethylamine, according to the molar ratio
1:1:1. The amide formation was confirmed by IR spectroscopy, by the appearance of the C=O
amide stretching band at 1640 cm-1; also the carbamate C=O stretching was observed at 1670 cm-1.
The obtained residue was purified by chromatography on a silica gel column. The structure of the
1
synthesized intermediates was confirmed by H-NMR spectroscopy. The removal of BOC
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protecting group of the tert-butyl(6-((2,6-dichloropyridine-4-carbonyl)amino)hexyl)carbamate was
obtained by treatment with an excess of trifluoroacetic acid in anhydrous dichloromethane. The IR
spectroscopy analysis confirmed the disappearance of carbamate and the presence of the amide
group. The intermediate 14 was obtained as trifluoroacetate salt and was used for the following
reactions. Finally, the compounds 15-19 were synthesized by reaction of the appropriate aromatic
aldehyde and 14 to afford the intermediate imine derivatives. In this case, the potassium carbonate
was used either as a desiccant, either as a base to unlock the amino group of the intermediate 14.
The imine intermediates were treated with sodium borohydride to reduce the imine group and, after
the reaction, the excess of sodium borohydride was decomposed by the addiction of aqueous HCl
lowering the pH below 6. The residues obtained were purified by chromatography on a silica gel
column (15-18) or on preparative layer plates silica gel (19) to provide the required amide products
(15-19). The detailed synthetic procedures, the analytical and spectroscopic data of new synthesized
compounds are reported in the experimental section and are in agreement with the proposed
structures.
2.2 Enzymatic assays
The evaluation of the inhibitory activity toward AChE and BChE was performed according to the
Ellman’s method [26]. Compounds were initially screened against Electrophorus electricus AChE
(EeAChE) and equine butyrylcholinesterase (eqBChE) at three different concentrations in the
presence of 0.0833 UmL-1 of enzyme and 100 µM of acetylthiocholine or butyrylthiocholine. The
results (Table 1) indicate that AChE inhibition is mainly influenced by two features: i) the nature of
the R substituent and ii) the length of the alkyl spacer.
The most active compound on EeAChE, derivative 8, is characterized by an unsubstituted phenyl
ring and a five methylene spacer (91 % inhibition of EeAChE activity, when tested at 0.3 µM). The
presence of a methoxyl group on the aromatic ring (10) decreased the inhibitory activity from 91 %
to 50 % (at 0.3 µM) while the replacement of the phenyl ring with the indole (11) slightly reduced
the inhibitory potency, from 91 to 76 % (at 0.3 µM); on the contrary, the presence of a 4-
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phenoxybenzyl group (12) led to a substantial decrease in the activity (no significant inhibition at
0.3 µM). These data indicate that the nature of the second aromatic/heteroaromatic ring is key
parameter to obtain an optimal inhibition. The bulkiness of the aromatic group seems to play a main
role in EeAChE inhibition, as the presence of a bulkier R moiety caused a reduction of the
inhibitory activity, as observed comparing compound 8 to 10 and 12. For R groups with similar
volumes (10 and 11) other factors should be considered, such as hydrogen bond acceptor/donor
properties, PSA (polar surface area) and the extension of the aromatic system.
TABLE 1
Compounds 8 and 9 contain the simplest aromatic group (phenyl), and therefore are those in which
the influence of the aminoalkyl spacer length could be more evident. The presence of a five-
methylene-unit chain resulted in a higher inhibitory activity (compare % inhibition of EeAChE by 8
and 9; see molecular docking section for further discussion). Therefore, we decided to use a 10-unit
spacer, considering both methylene units, the carbamate and the secondary amine functions, as
linker between the two aromatic moieties.
Furthermore, the presence of the 2,6-dichloropyridine moiety appeared to be fundamental to obtain
a higher AChE inhibition (compare 8 with 13).
Derivatives 8-13 bear both a secondary amino group and a carbamate function, thus these
compounds may position into the AChE gorge in two different ways, depending on the group acting
as driving force. Interestingly, the type of inhibition will vary upon the different location of the
carbamate residue (i.e. up part of the gorge or close to the CAS). Indeed, it must be noted that
carbamate derivatives can pseudo-irreversibly inhibit AChE, by carbamoylating the serine residue
located within the AChE catalytic site. In this case, a time dependent inhibition should be observed
[27]. To elucidate the mechanism of inhibition a stop-time assay was carried out by incubating the
AChE for 40 min without any inhibitor, with carbamate derivatives and with carbaryl, a known
carbamoylating agent [28, 29].
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While, in the selected time window, a 65 % increment of inhibition was observed in the presence of
carbaryl (as expected), in the presence of compounds 8-13 no time-dependent AChE inhibition was
observed. These results suggested that these compounds acted as classical reversible inhibitors and
suggested that the benzylamino moiety points down to the bottom of the gorge toward the CAS.
This hypothesis was confirmed by docking studies (see section 2.3).
The inhibition percentages obtained for the amide derivatives (15-19) were quite similar to those
obtained for the carbamates 8-13. The higher inhibition activity of compound 8 with respect to 9
prompted us to synthesize amide derivatives with a ten-unit spacer, thus the number of methylene
groups was increased to six units in order to obtain the same length of the carbamate compounds.
Among the amide derivatives, compound 15 (R = phenyl) presented the highest activity within the
amide series, inhibiting AChE activity by 71 % at 0.3 µM. The replacement of the phenyl with an
indole ring (17) or the introduction of a methoxy group on the phenyl ring (16) produced a small
reduction of the inhibition potency; on the other hand, the presence of bulkier 4-phenoxybenzyl (18)
or naphthyl (19) groups entailed a substantial decrease in the activity (16 % and 15 % inhibition at 3
µM, respectively).
Tested compounds were selective EeAChE inhibitors. Selectivity was mostly related to the weak
inhibitory activity toward eqBChE (% inhibition at 0.30 µM lower than 20 %, Table 1).
For compounds inhibiting EeAChE activity more than 50 % at 3 µM, the inhibition constant (Ki)
and the inhibition mechanism were determined according to Dixon’s method [30]. Obtained data
were confirmed by re-processing according to Lineweaver-Burk analysis [31]. The Dixon’s plot of
the most active compound 8 is shown in Figure 2.
FIGURE 2
TABLE 2
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All tested compounds were found to inhibit EeAChE at submicromolar concentration and showed a
mixed-type inhibition mechanism (Table 2), suggesting that these compounds could interact with
CAS and PAS; thus, if BBB permeation is confirmed, these new molecules could be able to
enhance the cholinergic tone by reducing the hydrolytic activity of AChE and might have the
potential to decrease the deposition of Aβ fibrils induced by AChE.
In general, the carbamate derivatives were more potent inhibitors than the corresponding amides.
Considering the inhibitory potency (as Ki values), the observed trend was: phenyl (8 and 15) <
indole (11 and 17) < 2-methoxyphenyl (10 and 16) substituted derivatives.
All compounds were also studied on human recombinant AChE (hAChE) and BChE from human
serum (hBChE). The inhibition values on human cholinesterases are expressed as half-maximal
inhibitory concentration (IC50) and are listed in Table 3.
TABLE 3
With the exception of 12 and 18, all compounds showed inhibitory potency toward hAChE in the
micromolar and submicromolar range. Considering the subset of carbamic derivative, inhibitory
activity was modulated by the introduction of substituents and modification of the phenyl ring in the
base scaffold of compound 8. In general, the increase of the steric hindrance of the phenyl moiety
led to a decrease of the inhibitory activity, underling that this position allowed only substituents
with limited bulkiness. Indeed, the introduction of a methoxyl group on the 2-position of the phenyl
ring (10) resulted in a forty-fold decrease of the activity; the replacement of the phenyl ring with the
indole (11) reduced the inhibitory activity of four-fold. No inhibition was observed for compound
12, which presents a bulkier 4-phenoxybenzyl group instead of the phenyl ring. As for EeAChE,
compound 11 (R = indole) showed a higher potency than 10 (R = 2-methoxyphenyl). This cannot be
exclusively explained by the bulkiness of R group but it can be likely related to the greater
extension of the aromatic system of the indole, which could better interact with Trp 86 through
stacking interaction.
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Analogously to what observed for EeAChE, compounds with five methylene units in the alkyl chain
performed better than those with a six-methylene units. Indeed, a five-fold decrease in potency was
observed for compound 9, that presents an additional methylene unit with respect to 8.
Although no covalent binding to CAS was detected for the human enzyme (data not shown), the
carbamate derivatives were up to twenty-fold more potent than the corresponding amide, as can be
clearly observed by comparing IC50 values obtained for carbamates 8 and 11 with those obtained for
the amide analogues 15 and 17.
The 2,6-dichloropyridine moiety showed to be important for a suitable interaction with the hAChE
and its substitution with the 3-bromopyridine moiety (carbamate 13) led to a decreased activity of
about twenty-six times (IC50 4.04 µM vs of 0.153 for 8 and 13, respectively).
The bulkiness of the substituents modulated the selectivity toward the two isoforms of the
cholinesterase enzymes. Indeed, compounds 8, 9, 15 and 16 are quite selective hAChE inhibitors,
with one order of magnitude selectivity. The most selective hAChE inhibitor was 8 which was thirty
times a more potent toward hAChEI than hBChEI. Conversely, compounds 10, 11, 13, 17 were
non-selective inhibitor of cholinesterases (SI ranging from 0.77 to 1.88), while compound 19
showed one order of magnitude selectivity for hBChE and 12 inhibited only hBChE with an IC50
value of 8.41 µM. The selectivity of 12 and 19 toward hBChE is probably due to the different
structure of the two enzymes. Indeed, the active gorge is larger in hBChE than in hAChE. The
hAChE active site is lined by 14 aromatic aminoacid residues, and six of them are replaced by
smaller aliphatic residues in hBuChE. In particular, the replacement of two phenylalanine residues
by the smaller amino acids valine and leucine makes the acyl pocket larger and enables bulkier
molecules, as compounds 12 and 19, to fit better into the gorge [32].
2.3 Molecular docking studies
The reported compounds were evaluated by docking studies in order to gain insight into the ligand-
protein interactions and to rationalize the structure activity relationships (SARs). All the newly
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synthesized compounds were docked by Gold [33], Plants [34] and Autodock [35], into the hAChE
structure co-crystallized with Donepezil (pdb code 4EY7) [8].
The proposed binding poses were consistent, independently from the docking software (see
Supporting information for details). Only compound 12 showed an unrelated binding mode (data
not shown). The results from the docking studies allowed to identify some key contacts. Indeed, for
derivative 8 a staking interaction between the phenyl ring and the Trp86 was found; the charged
amine group was involved in cation-aromatic contact with Tyr337 side chain, the carbamoyl oxygen
atoms established two H-bonds with Tyr124 and Phe295 (like Donepezil) side chains, and the 2,6-
dichloropyridine moiety was stabilized into PAS by aromatic interaction with Trp286. This binding
was strengthened by the chlorine atoms (withdrawing effect) [36]. Furthermore, a H-bond between
Ser293 backbone and the closest chlorine atom was observed [37] (Figure 3).
FIGURE 3
Comparing the binding mode of the compound 9 (superior homologue) with that of compound 8 it
was possible to observe that all the described interactions were retained. Nevertheless, for
derivative 9 the positive charged nitrogen atom was located farther from the Tyr337. This was even
more pronounced for the derivative 11. Likely, in order to accommodate the bulkier substituents or
longer linker the charged amino group moved farther from Tyr337 (Figure 4).
FIGURE 4
Similarly, the comparison of the binding poses of compound 8 and the amide analogue 15 showed
that the amine moiety was involved in just one H-bond with Tyr124, whereas all the
hydrophobic/aromatic interactions were retained. (Figure 5).
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Docking studies were also carried out for EeAChE. The binding site identity between the hAChE
and the EeAChE was 100%. Nevertheless, a main difference was observed for Tyr337 position.
Indeed, for EeAChE this residue pointed toward the gorge making it smaller. The compound 8
proposed binding mode by Gold for both enzymes was similar. We had just a slight difference in
the linker position. Indeed, the contacts described for compound 8 at the hAChE were retained also
in the EeAChE (Figure 3S).
Thus, the docking experiments provided us with a general binding mode that justifies the biological
activity of the compounds.
FIGURE 5
2.4. Inhibition of amyloid self-aggregation
Aβ42 is the predominating form of Aβ in senile plaques and accumulation of Aβ aggregates has
been associated to an increased neurotoxicity [38]. Therefore, to assess the inhibitory activity of this
new class of compounds, the most potent cholinesterase inhibitors within the series were evaluated.
Compounds 8, 9 and 11 were screened at an Aβ/inhibitor ratio equals to 1/1, which corresponds to a
screening concentration of 50 µM. An analysis of the results is reported in Table 3. All selected
compounds significantly inhibited Aβ self-aggregation with percentages of inhibition falling in a
close range (24.6-37.7 %). Few considerations can be made on the structural requirements for
amyloid inhibition. Specifically, lengthening the alkyl chain from five to six units did not affect the
inhibitory potency while the substitution of the phenyl ring with the indole ring led to an increase of
the inhibitory potency by about 11 % (compare 8 and 11).
Worth noting the currently approved AChEI are unable to significantly inhibit the Aβ aggregation
[17], thus, although the studied compounds behaving as weak inhibitors of the Aβ42, their activity
towards this AD hallmark was higher than that of the marketed drugs.
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2.5. In vitro cytotoxicity studies
Cell proliferation was assessed by MTT assay on human astrocytoma T67 and HeLa cell lines
(Figure 6). Data showed that compound 8 did not significantly affect cell viability up to 10 µM.
Compound 9 showed a more pronounced cytotoxic effect in comparison to compound 8 (25 % of
cell mortality at 10 µM). Finally, compound 11 at 10 µM induced over 50 % of cell mortality. We
found no significant difference in compound toxicity between the two cell lines. From a simple
comparison of the IC50 values of compounds 8 and 11 (Table 4), it can be concluded that the
presence of the indole residue exerts a clear negative effect on cell safety, being 11 from three to
four times more cytotoxic than 8. Also the spacer chain length seems to have an effect on cell
toxicity (compare 8 and 9).
FIGURE 6
TABLE 4
2.6. Prediction of Blood-Brain Barrier permeation
Blood-brain barrier (BBB) permeation is a fundamental pre-requisite for potential CNS drugs [39].
For early assessment of the ability of the designed compounds to reach CNS, the ADME descriptors
and BBB penetration were predicted by Molinspiration (http://www.molinspiration.com) and
SwissADME [40] respectively. The studied compounds fitted the Lipinski’s Rule of 5 (logP; MW;
H bond donors and acceptors and rotatable bonds; LogS) [41]. Thus, they were usually well
absorbed after oral administration. Furthermore, all selected compounds were expected to be BBB
accessible with the only exception of 11.
TABLE 5
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3. Conclusions
In this paper we described the synthesis of a series of carbamate and amide derivatives of 2,6-
dichloro-4-aminopyridine that were designed as dual binding site AChE inhibitors. The designed
compounds contain two aromatic regions linked by mean of a flexible alkyl chain with variable
length.
Data on EeAChE and eqBChE inhibition indicated that the compounds were able to inhibit both
ChEIs with a moderate selectivity for EeAChE.
Investigation of the inhibition mechanism highlighted a mixed-type inhibition mechanism.
Carbamate 8 and the amide 15 resulted the most active compounds toward EeAChE with Ki values
of 38.6 nM and 52.8 nM, respectively.
Further investigation of on the human isoforms of AChE and BChE showed that, although with
differences related to the enzyme source, both the carbamate and amide derivatives were good to
moderate inhibitors of hChE, being the carbamate 8 the most potent inhibitor of hAChE (IC50 = 153
± 16 nM) and the carbamate 11 the most potent inhibitor of hBChE (IC50 = 828 ± 67 nM).
Molecular docking studies carried out on hAChE (co-crystallized with Donepezil, pdb code 4EY7)
allowed to get further insights into the mode of interaction of the most potent ChEI within the
series. In particular, the study showed that carbamate 8 is able to establish a number of interactions
between the benzyl group and the protonate amine nitrogens with the aminoacids of the CAS
region, while the 2,6-dichloro-pyridine moiety can contact the Trp 286 within the PAS.
In the amyloid self-aggregation assay, the carbamate 8, the most active hAChEI in this study,
showed 26.5 % of Aβ42 peptide self-aggregation at 50 µM; this compound also resulted the less
toxic both T67 and HeLa with IC50 values of 24.1 µM and 20.8 µM respectively.
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In conclusion, from the obtained data, it clearly appears that the carbamate 8 possesses a good
inhibitory potency toward hAChE, with a good selectivity against hBChE and it is also able of
inhibiting the Aβ42 self-aggregation. These data, combined with the low toxicity and with the good
predicted BBB permeation parameters, make this compound an interesting lead for further
development of new multifactorial cholinesterase inhibitors, which may be potentially useful in the
treatment of Alzheimer's disease.
4. Experimental Section
4.1 Chemistry
4.1.1. Materials and methods
All reagents, solvents and deuterated solvents were of high analytical grade and were purchased
from Sigma-Aldrich (Milan, Italy). Benzene was dried just before use by means of Dean Stark
apparatus. Dichloromethane was dried over anhydrous K2CO3. IR spectra were recorded on a FT-IR
1
13
Perkin-Elmer Spectrum One equipped with ATR system; signals are given as ν (cm-1). H and C
NMR spectra were acquired on AVANCE400 Bruker spectrometer, operating at 400 and 100 MHz
respectively, in DMSO-d6, CD3OD and CDCl3 at 27°C; chemical shift values are given in δ (ppm)
relatively to TMS as internal reference. Coupling constants are given in Hz. The following
abbreviation are used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Melting points
were determined on Tottoli apparatus (Buchi) and are uncorrected. Mass spectra were recorded on a
ThermoFinnigan LCQ Classic LC/MS/MS ion trap equipped with an ESI source and a syringe
pump. Samples (10-4-10-5 M in MeOH/H2O 80:20) were infused in the electrospray system at a flow
rate of 5-10 µL min-1. When necessary, 50 µL of 10-2 M HCOOH or 10-2 M NH3 were added to the
sample solutions, in order to promote the analyte ionization. Elemental analyses were obtained by a
PE 2400 (Perkin-Elmer) analyzer and the analytical results were within ± 0.4 % of the theoretical
values for all compounds.
16

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4.1.2 General procedure for the synthesis of isocyanate intermediates: 2,6-dichloro-4-
isocyanatopyridine (1), 3-bromo-4-isocyanatopyridine (2)
In 30 mL of anhydrous benzene 2,6-dichloropyridin-4-amine (0.326 g, 2.00 mmol) or 3-
bromopyridin-4-amine (0.346 g, 2.00 mmol) was dissolved, then 7.23 mmol (1.0 mL) of anhydrous
TEA were added. The solution was kept under stirring at rt and 5 mL of benzene, containing 1.20
mmol of triphosgene (0.357 g), were added dropwise. The solution was refluxed for 5 h for the
synthesis of 2, whereas, for compound 1 the solution was stirred at 35 °C for 5 h. The isocyanate
derivatives were used without further purification.
4.1.3. General procedure for the synthesis of imino-alcohols derivatives (3-7)
Method A: To the opportune aminoalcohol and aldehyde (1:1 molar ratio) in CH2Cl2 (20 mL), 2 g
of anhydrous K2CO3 were added. The reaction mixture was stirred for 12 h at room temperature,
filtered on a celite pad and then the CH2Cl2 was evaporated under reduce pressure.
Method B: The opportune aminoalcohol, aldehyde (1:1 molar ratio) and molecular sieves (4 Å) in
CH2Cl2 (20 mL) were stirred at rt for 12 h. The mixture was then filtered on a celite pad and the
CH2Cl2 evaporated under vacuum.
4.1.3.1. 5-((E)-benzylideneamino)pentan-1-ol (3). Compound 3 was prepared using benzaldehyde
(0.203 mL, d=1.045 g/mL, 2.00 mmol), 5-aminopentan-1-ol (0.206 g, 2.00 mmol), following the
method A procedure. The obtained residue was a yellowish oil and it was used without further
1
purification. I.R. (cm-1): νC=N 1645. HNMR (CD3OD) δ (ppm): 8.37 (s, 1H, ArCH=N); 7.76-7.74
(m, 2H, phenyl protons); 7.50-7.43 (m, 3H, phenyl protons); 3.65 (t, J = 7.0 Hz, 2H, CH2OH); 3.58
(t, J = 6.5 Hz, 2H,NCH2); 1.79-1.71 (m = 5, 2H, J = 7.2 Hz, CH2CH2OH); 1.65-1.58 (m = 5, J = 6,9
Hz, 2H,NCH2CH2); 1.49-1.41 (m, 2H, CH2(CH2)2OH).
4.1.3.2. 6-((E)-benzylideneamino)hexan-1-ol (4). Compound 4 was prepared using benzaldehyde
(0.203 mL, d=1.045 g/mL, 2.00 mmol), 6-aminohexan-1-ol (0.234 g, 2.00 mmol), following the
method A procedure. The obtained residue was a white solid and it was used without further
17

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1
purification. I.R. (cm-1): νC=N 1646. HNMR (CDCl3) δ (ppm): 8.28 (s, 1H, ArCH=N); 7.65-7.52
(m, 2H, phenyl protons); 7.45-7.32 (m, 3H, phenyl protons); 3.58 (m, 2H, CH2OH); 3.40 (t, J = 6.4
Hz, 2H,NCH2); 1.66-1.50 (m, 4H, NCH2CH2(CH2)2CH2CH2OH); 1.36-1.28 (m, 4H,
N(CH2)2CH2CH2(CH2)2OH);
4.1.3.3. 5-((E)-((2-methoxyphenyl)methylidene)amino)pentan-1-ol (5). Compound 5 was
prepared using 2-methoxybenzaldehyde (0.272 g, 2.00 mmol) and 5-aminopentan-1-ol (0.206 g,
2.00 mmol), following the method A procedure. The obtained residue was a yellowish oil and it was
1
used without further purification. I.R. (cm-1): νC=N 1637. HNMR (CD3OD) δ (ppm): 8.73 (s, 1H,
ArCH=N); 7.81-7.77 (m, 1H, phenyl proton); 7.47-7.43 (m, 1H, phenyl proton); 7.07 (d, J = 8.3 Hz,
1H, phenyl proton); 6.99 (t, J = 7.5 Hz, 1H, phenyl proton); 3.90 (s, 3H, OCH3); 3.65-3.61 (m, 2H,
CH2OH); 3.80 (t, J = 6.5 Hz, 2H, NCH2CH2); 1.73 (q, J = 7.5 Hz, 2H, CH2CH2OH); 1.64-1.57 (m,
2H, NCH2CH2); 1.47-1.40 (m, 2H, N(CH2)2CH2).
4.1.3.4. 5-((E)-((1H-indol-3-yl)methylidene)amino)pentan-1-ol (6). Compound 6 was prepared
using 1H-indole-3-carbaldehyde (0.290 g, 2.00 mmol) and 5-aminopentan-1-ol (0.206 g, 2.00
mmol), following the method B procedure. The obtained residue was an orange thick oil and it was
1
used without further purification. I.R. (cm-1): νC=N 1634. HNMR (CD3OD) δ (ppm): 8.52 (s, 1H,
ArCH=N); 8.09 (d, J = 7.8 Hz, 1H, indole proton); 7.72 (s, 1H, indole proton); 7.43 (d, J = 8.0 Hz,
1H, indole proton); 7.23-7.14 (m, 2H, indole protons); 3.62-3.57 (m, 4H, NCH2 + CH2OH); 1.75 (m
= 5, J = 7.5 Hz, 2H, CH2CH2OH); 1.63 (m = 5, J = 6.9 Hz, 2H, NCH2CH2); 1.52-1.45 (m, 2H,
N(CH2)2CH2(CH2)2OH).
4.1.3.5. 5-((E)-((4-phenoxyphenyl)methylidene)amino)pentan-1-ol (7). Compound 7 was
prepared using 4-phenoxybenzaldehyde (0.350 mL, d=1.132 g/mL, 2.00 mmol) and 5-aminopentan-
1-ol (0.206 g, 2.00 mmol), following the method A procedure. The obtained residue was a
yellowish oil and it was used without further purification. I.R. (cm-1): νC=N 1645. 1HNMR (CD3OD)
δ (ppm): 8.31 (s, 1H, ArCH=N); 7.74 (d, J = 8.7 Hz, 2H, aromatic protons); 7.41 (t, J = 7.6 Hz, 2H,
aromatic protons); 7.20 (t, J = 7.4 Hz, 1H, aromatic proton); 7.07-7.01 (m, 4H, aromatic protons);
18

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3.64-3.56 (m, 4H, NCH2 + CH2OH); 1.74 (m = 5, J = 7.2 Hz, 2H, CH2CH2OH); 1.64-1.40 (m, 4H,
NCH2CH2CH2(CH2)2-OH).
4.1.4. General procedure for the synthesis of carbamate derivatives (8-13)
To a solution of opportune isocyanate (1 or 2), in 35 mL of dry benzene, the appropriate
iminoalcohol (3-7) in 5 mL of anhydrous CH2Cl2 was added (1:1 molar ratio). The reaction mixture
was stirred overnight at rt and, after this time, the obtained orange suspension was filtered, and the
solid was washed with benzene or CH2Cl2. The filtrate and the washings were combined,
concentrated in vacuum to obtain a residue that was dissolved in MeOH (20 mL) and treated with
NaBH4 (1:3 molar ratio) at rt for 1 h. After this time, MeOH was evaporated under reduced
pressure, H2O (5 mL) and HCl 1 N up to pH = 4 were added; after 5 minutes under stirring, the
mixture was alkalinized with a solution of NaOH 2 N until to achieve pH = 11, except for 11 (pH =
7). The aqueous solution was extracted with CH2Cl2 (3 x 20 mL), the combined organic layers were
dried over Na2SO4 and the solvent was removed under vacuum.
4.1.4.1. 5-(benzylamino)pentyl (2,6-dichloropyridin-4-yl)carbamate (8). Compound 8 was
prepared from 2,6-dichloro-4-isocyanatopyridine (1) and 5-((E)-benzylideneamino)pentan-1-ol (3),
following the general procedure described above. The obtained residue was subjected to silica gel
column chromatography using initially CH3CN and subsequently AcOEt/MeOH (1:1) to give a
white solid, mp 134 °C (Rf = 0.9, 32 % yield). IR (cm-1): νC=O 1737. 1HNMR (DMSO-d6) δ (ppm):
7.50 (s, 2H, pyridine protons); 7.32-7.18 (m, 5H, phenyl protons); 4.13 (t, J = 6.4 Hz, 2H,
CH2OCO); 3.67 (s, 2H, ArCH2NH); 2.48 (t, J = 7.1 Hz, 2H, ArCH2NHCH2); 1.66-1.54 (m, 4H,
NHCH2CH2CH2CH2CH2O); 1.44-1.36 (m, 2H, NH(CH2)2CH2). 13CNMR (DMSO-d6) δ (ppm):
153.5, 151.4, 150.1, 141.6, 128.5, 128.3, 126.8, 111.3, 65.8, 53.5, 49.1, 29.8, 28.6, 23.8. ESI-MS
(m/z): (M+H)+ = 382.03 (100); 383.96 (70); 386.02 (15). Anal. (C18H21Cl2N3O2) C, H, N Calcd: C
56.55 %, H 5.54 %, N 10.99 %; Found: C 56.39 %, H 5.53 %, N 11.02 %.
4.1.4.2. 6-(benzylamino)hexyl (2,6-dichloropyridin-4-yl)carbamate (9). Compound 9 was
prepared from 2,6-dichloro-4-isocyanatopyridine (1) and 6-((E)-benzylideneamino)hexan-1-ol (4),
19

ACCEPTED MANUSCRIPT
following the general procedure described above. The obtained residue was subjected to silica gel
column chromatography using initially CH3CN and subsequently AcOEt/MeOH (1:1) to give a
yellow thick oil (Rf = 0.9, 27 % yield). IR (cm-1): νC=O 1739. 1HNMR (CDCl3) δ (ppm): 7.37 (s, 2H,
pyridine protons); 7.33-7.28 (m, 5H, phenyl protons); 4.18 (t, J = 6.4 Hz, 2H, CH2OCO); 3.80 (s,
2H, ArCH2NH); 2.67 (t, J = 7.1 Hz, 2H, ArCH2NHCH2); 1.67-1.63 (m, 2H, NH(CH2)4CH2CH2O);
13
1.55-1.50 (m, 2H, NHCH2CH2(CH2)4O); 1.39-1.37 (m, 4H, NH(CH2)2CH2CH2(CH2)2O). CNMR
(CDCl3) δ (ppm): 153.0, 151.0, 149.8, 139.0, 128.6, 128.3, 127.4, 111.2, 66.2, 53.7, 49.0, 29.4,
28.5, 26.9, 25.9. ESI-MS (m/z): (M+H)+ = 396.00 (100); 398.07 (60); 399.99 (20). Anal.
(C19H23Cl2N3O2) C, H, N Calcd: C 57.58 %, H 5.85 %, N 10.60 %; Found: C 57.38 %, H 5.87 %, N
10.59 %.
4.1.4.3. 5-(((2-methoxyphenyl)methyl)amino)pentyl (2,6-dichloropyridin-4-yl)carbamate (10).
Compound 10 was prepared from 2,6-dichloro-4-isocyanatopyridine (1) and 5-((E)-((2-
methoxyphenyl)methylidene)amino)pentan-1-ol (5), following the general procedure described
above. The obtained residue was subjected to silica gel column chromatography using initially
CH3CN and subsequently AcOEt/MeOH (1:1) as eluent (Rf = 0.28). Then the obtained residue was
crystallized from AcOEt to give a white solid, mp 138-140°C (223 mg, 27 % yield). IR (cm-1): νC=O
1
1737, finger print 1245, 1218, 1162, 1075, 1028, 940. HNMR (CD3OD δ (ppm): 7.48 (s, 2H,
pyridine protons); 7.28-7.22 (m, 2H, phenyl protons); 6.97 (d, J = 8.2 Hz, 1H, phenyl proton); 6.91
(t, J = 7.4 Hz, 1H, phenyl proton); 4.19 (t, J = 6.4 Hz, 2H, CH2OCO); 3.86 (s, 3H, OCH3); 3.76 (s,
2H, ArCH2NH); 2.58 (t, J = 7.4 Hz, 2H, ArCH2NHCH2); 1.71 (m = 5, J = 7.2 Hz, 2H,
CH2CH2OCO); 1.58 (m = 5, J = 7.8 Hz, 2H, NHCH2CH2); 1.43 (m = 5, J = 7.0 Hz, 2H,
NH(CH2)2CH2). 13CNMR (CD3OD) δ (ppm): 157.8, 153.4, 151.0, 150.5, 129.8, 128.5, 126.7, 120.1,
110.6, 110.1, 65.4, 54.4, 48.4, 48.1, 28.4, 28.3, 23.2. ESI-MS (m/z): (M+H)+ = 412.00 (100);
414.00 (60); 416.00 (10). Anal. (C19H23Cl2N3O3) C, H, N Calcd: C 55.35 %, H 5.62 %, N 10.19 %;
Found: C 55.41 %, H 5.63 %, N 10.18 %.
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4.1.4.4. 5-(((1H-indol-3-yl)methyl)amino)pentyl (2,6-dichloropyridin-4-yl)carbamate (11).
Compound 15 was prepared from 2,6-dichloro-4-isocyanatopyridine (1) and 5-((E)-((1H-indol-3-
yl)methylidene)amino)pentan-1-ol (6), following the general procedure described above. The
obtained residue was subjected to silica gel column chromatography using initially AcOEt/MeOH
(1:1) and subsequently MeOH as eluent (Rf = 0.13). Then the obtained residue was crystallized
from MeOH to give a white solid, mp 124-126°C (84 mg, 10 % yield). IR (cm-1): νC=O 1726, νN-H
3291, 3251, finger print 1255, 1227, 1165, 1069, 990, 944. 1HNMR (DMSO-d6) δ (ppm): 10.82 (s,
1H, -NH indole); 7.59 (d, J = 7.8 Hz, 1H, indole proton); 7.50 (s, 2H, pyridine protons); 7.33 (d, J =
8.1 Hz, 1H, indole proton); 7.20 (d, J = 1.8 Hz, 1H, indole proton); 7.05 (t, J = 7.4 Hz, 1H, indole
proton); 6.95 (t, J = 7.5 Hz, 1H, indole proton); 4.12 (t, J = 6.5 Hz, 2H, CH2OCO); 3.83 (s, 2H,
ArCH2NH); 2.56 (t, J = 6.8 Hz, 2H, ArCH2NHCH2); 1.63 (m = 5, J = 7.1 Hz, 2H, CH2CH2OCO);
1.49-1.36 (m, 4H, NHCH2CH2CH2). 13CNMR (CDCl3) δ (ppm): 153.0, 150.9, 149.8, 136.3, 126.9,
123.2, 122.2, 119.7, 118.3, 113.4, 111.5, 111.2, 66.1, 49.0, 44.4, 29.1, 28.3, 23.9. ESI-MS (m/z):
(M+H)+ = 420.53 (100); 422.47 (65); 424.47 (10). Anal. (C20H22Cl2N4O2) C, H, N Calcd: C 57.01
%, H 5.26 %, N 13.30 %; Found: C 57.13 %, H 5.25 %, N 13.29 %.
4.1.4.5.
5-((4-phenoxybenzyl)amino)pentyl
(2,6-dichloropyridin-4-yl)carbamate
(12).
Compound 12 was prepared from 2,6-dichloro-4-isocyanatopyridine (1) and 5-((E)-((4-
phenoxyphenyl)methylidene)amino)pentan-1-ol (7), following the general procedure described
above. The obtained residue was subjected to silica gel column chromatography using
AcOEt/MeOH (1:1) to give a white solid, mp 126-128°C (Rf = 0.39, 185 mg, 20 % yield). IR (cm-
1
1): νC=O 1734, νN-H 3265, finger print 1227, 1161, 1073, 984, 931. HNMR (CD3OD) δ (ppm): 7.49
(s, 2H, pyridine protons); 7.36-7.32 (m, 4H, aromatic protons); 7.10 (t, J = 7.4 Hz, 1H, aromatic
proton); 6.96 (t, J = 8.2 Hz, 4H, aromatic protons); 4.20 (t, J = 6.5 Hz, 2H, CH2OCO); 3.74 (s, 2H,
ArCH2NH); 2.61 (t, J = 7.3 Hz, 2H, ArCH2NHCH2); 1.73 (m = 5, J = 6.7 Hz, 2H, CH2CH2OCO);
13
1.60 (m = 5, J = 7.2 Hz, 2H,NHCH2CH2); 1.46 (m = 5, J = 7.0 Hz, 2H, NH(CH2)2CH2). CNMR
(CDCl3) δ (ppm): 157.2, 156.5, 152.6, 151.2, 149.3, 134.1, 129.8, 129.7, 123.3, 118.87, 118.86,
21

ACCEPTED MANUSCRIPT
111.1, 66.3, 53.2, 48.9, 29.3, 28.3, 23.8. ESI-MS (m/z): (M+H)+ = 473.67 (100); 475.67 (70);
476.87 (10). Anal. (C24H25Cl2N3O3) C, H, N Calcd: C 60.77 %, H 5.31 %, N 8.86 %; Found: C
60.58 %, H 5.31 %, N 8.89 %.
4.1.4.6. 5-(benzylamino)pentyl (3-bromopyridin-4-yl)carbamate (13). Compound 13 was
prepared from 3-bromo-4-isocyanatopyridine (2) and 5-((E)-benzylideneamino)pentan-1-ol (3),
following the general procedure described above. The obtained residue was subjected to silica gel
column chromatography using AcOEt/MeOH (1:1) as eluent to give a light orange oil (Rf = 0.41,
262 mg, 34 % yield). IR (cm-1): νC=O 1737, finger print 1235, 1202, 1068, 1022. 1HNMR (CD3OD)
δ (ppm): 8.60 (s, 1H, pyridine proton); 8.36 (d, J = 5.7 Hz, 1H, pyridine proton); 8.16 (d, J = 5.7
Hz, 1H, pyridine proton); 7.35-7.32 (m, 4H, phenyl protons); 7.29-7.26 (m, 1H, phenyl proton);
4.25 (t, J = 6.6 Hz, 2H, CH2OCO); 3.77 (s, 2H, ArCH2NH); 2.63 (t, J = 7.4 Hz, 2H,
ArCH2NHCH2); 1.76 (m = 5, J = 7.2 Hz, 2H, CH2CH2CH2OCO); 1.62 (m = 5, J = 7.9 Hz, 2H,
NHCH2CH2CH2); 1.51-1.43 (m, 2H, -NH(CH2)2CH2). 13CNMR (CD3OD) δ (ppm): 153.3, 151.2,
148.3, 144.5, 138.7, 128.3, 128.1, 126.9, 114.7, 110.3, 65.7, 52.9, 48.3, 28.4, 28.3, 23.2. ESI-MS
(m/z): (M-H)- = 391.73 (95); 393.80 (100). Anal. (C18H22BrN3O2) C, H, N Calcd: C 55.11 %, H
5.65 %, N 10.71 %; Found: C 55.30 %, H 5.63 %, N 10.68 %.
4.1.5. Synthesis of tert-butyl (6-((2,6-dichloropyridine-4-carbonyl)amino)hexyl) carbamate (14).
To a solution of tert-butyl (6-aminohexyl)carbamate (671 µL, d=0.962 g/mL, 3.00 mmol) and
distilled TEA (416 µL, d=0.727 g/mL, 3.00 mmol) in 15 mL of dry CH2Cl2, 2,6-dichloropyridine-4-
carbonyl chloride (410 µL, d=1.537 g/mL, 3.00 mmol) was added dropwise and the reaction was
stirred at rt overnight. Afterwards the reaction mixture was washed with aqueous saturated NaHCO3
(30 mL) and extracted with CH2Cl2 (2 x 20 mL). The combined organic layers were dried over
Na2SO4, filtered and the solvent was removed under vacuum. The obtained residue was purified by
silica gel column chromatography using AcOEt/MeOH (9.5:0.5) as eluent to give 14 (Rf = 0.70) as
an off-white waxy solid (972 mg, 83 % yield). IR (cm-1): νC=O amide 1641; νC=O carbamate 1671.
22

ACCEPTED MANUSCRIPT
1HNMR (DMSO-d6) δ (ppm): 8.89 (bt, J = 5.1 Hz, 1H, pyCONH); 7.88 (s, 2H, pyridine protons);
6.79 (bt, J = 5.5 Hz, 1H, NHCOO); 3.25 (q, J = 6.7 Hz, 2H, pyCONHCH2 ); 2.89 (q, J = 6.5 Hz,
2H, CH2NHCOO); 1.52-1.47 (m, 2H, aliphatic protons); 1.37 (s, 9H, aliphatic protons); 1.27 (m,
4H, aliphatic protons).
4.1.6. Synthesis of N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide.
To a solution of tert-butyl (6-((2,6-dichloropyridine-4-carbonyl)amino)hexyl) carbamate (0.176 g,
0.45 mmol) in dry CH2Cl2 (10 mL) TFA was added (729 µL, 9.0 mmol) and the reaction was stirred
at rt for 2 h. After this time the solvent and the excess of TFA were removed under reduce pressure
to give a waxy white solid. IR (cm-1): νC=O amide 1647.
4.1.7. General procedure for the synthesis of amide derivatives (15-19)
The synthesis involves the imine formation from opportune aldehyde and N-(6-aminohexyl)-2,6-
dichloropyridine-4-carboxamide (1:1 molar ratio) in dry CH2Cl2 with 2.0 g of anhydrous K2CO3,
except for 17. N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide was firstly solubilized in dry
CH2Cl2 and 2.0 g of anhydrous K2CO3 were added; then, after elimination of K2CO3 by filtration,
aldehyde and molecular sieves (4 Å) were added. The reaction was stirred at rt for 24 h. The
mixture was then filtered on a celite pad, concentrated under reduce pressure and treated with
NaBH4 (1:2 molar ratio) in MeOH (10 mL) at rt for 1 h. After the reaction MeOH was evaporated
under reduced pressure, was added H2O (5 mL) and then HCl 1 N up to pH = 4. The solution was
stirred at rt for 5 minutes and then was alkalinized with 1 N aqueous NaOH until to pH = 12, except
for compound 17 (pH = 7). The aqueous solution was extracted with CH2Cl2 (3 x 15 mL) and the
combined organic layers were dried over Na2SO4 and the solvent was removed under vacuum.
4.1.7.1. N-(6-(benzylamino)hexyl)-2,6-dichloroisonicotinamide (15). Compound 15 was prepared
from N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide (0.189 g, 0.65 mmol) and
benzaldehyde (0.066 mL, d=1.045 g/mL, 0.65 mmol), following the general procedure described
above. The obtained residue was purified by silica gel column chromatography using initially
AcOEt/MeOH (9:1) and subsequently AcOEt/MeOH (1:1) to give an orange solid, mp 73-74°C (Rf
23

ACCEPTED MANUSCRIPT
= 0.54, 89 mg, 36 % yield). IR (cm-1): νN-H 3290, νC=O 1648, finger print 1165, 1100, 1029. 1HNMR
(DMSO-d6) δ (ppm): 8.89 (t, J = 5.3 Hz, 1H, NHCOpy); 7.88 (s, 2H, pyridine protons); 7.32-7.27
(m, 4H, aromatic protons); 7.22-7.18 (m, 1H, aromatic proton); 3.66 (s, 2H, ArCH2NH); 3.28-3.23
(m, 2H, CH2NHCO); 2.46 (t, J = 7.0 Hz, 2H, ArCH2NHCH2); 1.55-1.49 (m, 2H, aliphatic protons);
1.46-1.39 (m, 2H, aliphatic protons); 1.31-1.29 (m, 4H, aliphatic protons). 13CNMR (CD3OD) δ
(ppm): 163.6, 150.8, 147.6, 138.8, 128.2, 128.1, 126.9, 120.9, 53.0, 48.3, 39.8, 28.7, 28.7, 26.6,
26.4. ESI-MS (m/z): (M+H)+ = 379.93 (100); 381.87 (70); 383.87 (10). Anal. (C19H23Cl2N3O) C, H,
N Calcd: C 60.00 %, H 6.10 %, N 11.05 %; Found: C 60.12 %, H 6.11 %, N 11.07 %.
4.1.7.2. 2,6-dichloro-N-(6-((2-methoxybenzyl)amino)hexyl)isonicotinamide (16). Compound 16
was prepared from N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide (0.105 g, 0.36 mmol)
and 2-methoxybenzaldehyde (0.049 g, 0.36 mmol), following the general procedure described
above. The obtained residue was purified by silica gel column chromatography using initially
AcOEt/MeOH (9:1) and subsequently AcOEt/MeOH (1:1) to give an orange solid, mp 70 °C (Rf =
0.30, 58 mg, 39 % yield). IR (cm-1): νN-H 3278, νC=O 1657, finger print 1242, 1160, 1097, 1028.
1HNMR (CD3OD) δ (ppm): 7.75 (s, 2H, pyridine protons); 7.29-7.23 (m, 2H, aromatic protons);
6.98 (d, J = 8.2 Hz, 1H, aromatic proton); 6.91 (t, J = 7.4 Hz, 1H, aromatic proton); 3.87 (s, 3H,
OCH3); 3.77 (s, 2H, ArCH2NH); 3.37 (t, J = 7.1 Hz, 2H, CH2NHCO); 2.58 (t, J = 7.2 Hz, 2H,
ArCH2NHCH2); 1.64-1.54 (m, 4H, ArCH2NHCH2CH2(CH2)2CH2CH2NHCO); 1.39-1.37 (m, 4H,
13
ArCH2NH(CH2)2CH2CH2(CH2)2NHCO). CNMR (CD3OD) δ (ppm): 163.6, 157.8, 150.8, 147.6,
129.9, 128.7, 126.2, 120.9, 120.1, 110.1, 54.4, 48.2, 46.9, 39.8, 28.7, 28.5, 26.5, 26.4. ESI-MS
(m/z): (M+H)+ = 409.93 (100); 411.93 (70); 413.93 (10). Anal. (C20H25Cl2N3O2) C, H, N Calcd: C
58.54 %, H 6.14 %, N 10.24 %; Found: C 58.36 %, H 6.12 %, N 10.25 %.
4.1.7.3.
N-(6-(((1H-indol-3-yl)methyl)amino)hexyl)-2,6-dichloroisonicotinamide
(17).
Compound 17 was prepared from N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide (0.131 g,
0.45 mmol) and 1H-indole-3-carbaldehyde (0.065 g, 0.45 mmol), following the general procedure
described above. The obtained residue was purified by silica gel column chromatography using
24

ACCEPTED MANUSCRIPT
CH2Cl2/MeOH (9.5:0.5), in these conditions the product remain on the start line (Rf = 0). Once
remove all impurities with Rf greater than 0.22, the silica was extracted with MeOH and
concentrated under reduced pressure to give a yellow oil (73 mg, 39 % yield). IR (cm-1): νN-H 3279,
1
νC=O 1649, finger print 1167, 1100, 1026. HNMR (CD3OD) δ (ppm): 7.75 (s, 2H, pyridine
protons); 7.62 (d, J = 7.9 Hz, 1H, indole proton); 7.27 (s, 1H, indole proton); 7.13 (t, J = 7.1 Hz,
1H, indole proton); 7.05 (t, J = 7.2 Hz, 1H, indole proton); 4.04 (s, 2H, ArCH2NH); 3.38-3.36 (m,
2H, CH2NHCO); 2.74 (t, J = 7.5 Hz, 2H, ArCH2NHCH2); 1.63-1.58 (m, 4H,
ArCH2NHCH2CH2(CH2)2CH2CH2NHCO);
1.39-1.36
(m,
4H,
13
ArCH2NH(CH2)2CH2CH2(CH2)2NHCO). CNMR (CD3OD) δ (ppm): 163.6, 150.8, 147.5, 136.6,
126.8, 124.3, 121.4, 120.9, 118.9, 117.7, 111.1, 109.9, 48.5, 43.1, 39.8, 28.6, 27.9, 26.4, 26.3. ESI-
MS (m/z): (M+H)+ = 418.67 (100); 420.67 (85); 422.67 (10). Anal. (C21H24Cl2N4O) C, H, N Calcd:
C 60.15 %, H 5.77 %, N 13.36 %; Found: C 60.31 %, H 5.77 %, N 13.37 %.
4.1.7.4. 2,6-dichloro-N-(6-((4-phenoxybenzyl)amino)hexyl)isonicotinamide (18). Compound 18
was prepared from N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide (0.160 g, 0.55 mmol)
and 4-phenoxybenzaldehyde (0.096 mL, d=1.132 g/mL, 0.55 mmol), following the general
procedure described above. The obtained residue was purified by silica gel column chromatography
using initially AcOEt/MeOH (9:1) and subsequently AcOEt/MeOH (1:1) to give a yellow oil (Rf =
1
0.50, 60 mg, 23 % yield). IR (cm-1): νN-H 3300, νC=O 1648, finger print 1232, 1164, 1100. HNMR
(CD3OD) δ (ppm): 7.77 (s, 2H, pyridine protons); 7.37-7.33 (m, 4H, aromatic protons); 7.11 (t, J =
7.4 Hz, 1H, aromatic proton); 6.97 (t, J = 8.0 Hz, 4H, aromatic protons); 3.75 (s, 2H, ArCH2NH);
3.38 (t, J = 7.1 Hz, 2H, CH2NHCO); 2.62 (t, J = 7.4 Hz, 2H, ArCH2NHCH2); 1.66-1.56 (m, 4H,
ArCH2NHCH2CH2(CH2)2CH2CH2NHCO);
1.42-1.39
(m,
4H,
13
ArCH2NH(CH2)2CH2CH2(CH2)2NHCO). CNMR (CD3OD) δ (ppm): 163.6, 157.3, 156.6, 150.8,
147.6, 133.7, 129.8, 129.5, 123.0, 120.9, 118.5, 118.4, 52.3, 48.3, 48.2, 39.8, 28.7, 26.6, 26.5. ESI-
MS (m/z): (M+H)+ = 471.86 (100); 473.85 (65); 474.85 (15). Anal. (C25H27Cl2N3O2) C, H, N
Calcd: C 63.56 %, H 5.76 %, N 8.89 %; Found: C 63.38 %, H 5.75 %, N 8.89 %.
25

ACCEPTED MANUSCRIPT
4.1.7.5.
2,6-dichloro-N-(6-((naphthalen-2-ylmethyl)amino)hexyl)isonicotinamide
(19).
Compound 19 was prepared from N-(6-aminohexyl)-2,6-dichloropyridine-4-carboxamide (0.078 g,
0.27 mmol) and naphthalene-2-carbaldehyde (0.042 g, 0.27 mmol), following the general procedure
described above. The obtained residue was purified using PLC (Preparative Layer Plates) Silica gel
20 × 20 cm by eluting initially with AcOEt/MeOH (9:1) and subsequently AcOEt/MeOH (1:1) to
give a yellow oil (Rf = 0.30, 55 mg, 47 % yield). IR (cm-1): νN-H 3299, νC=O 1647, finger print 1163,
1
1100, 987. HNMR (CD3OD) δ (ppm): 7.86-7.84 (m, 3H, aromatic protons); 7.80 (s, 1H, aromatic
proton); 7.76 (s, 2H, pyridine protons); 7.51-7.46 (m, 3H, aromatic protons); 3.94 (s, 2H,
ArCH2NH); 3.37 (t, J = 7.0 Hz, 2H, CH2NHCO); 2.65 (t, J = 7.4 Hz, 2H, ArCH2NHCH2); 1.65-1.59
(m,
4H,
ArCH2NHCH2CH2(CH2)2CH2CH2NHCO);
1.42-1.39
(m,
4H,
13
ArCH2NH(CH2)2CH2CH2(CH2)2NHCO). CNMR (CD3OD) δ (ppm): 163.6, 150.8, 147.6, 136.0,
133.5, 132.8, 127.8, 127.4, 127.3, 126.9, 126.3, 125.8, 125.5, 120.9, 53.0, 48.3, 39.8, 28.7, 28.6,
26.6, 26.4. ESI-MS (m/z): (M+H)+ = 427.87 (100); 429.90 (60); 431.88 (10). Anal. (C23H25Cl2N3O)
C, H, N Calcd: C 64.19 %, H 5.86 %, N 9.76 %; Found: C 64.42 %, H 5.87 %, N 9.75 %.
4.2 Enzymatic assays
4.2.1. Inhibitory activity toward EeAChE and eqBChE
Electric eel AChE (E.C. 3.1.1.7), equine BChE (EC 3.1.1.8), acetylthiocholine iodide,
butyrylthiocholine iodide, 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), donepezil, used as
reference standard, were purchased from Sigma-Aldrich (Milan, Italy). All other chemical and
biological reagents and solvents used were of the highest analytical grade. The assays were carried
out by double beam UV-Vis lambda 40 Perkin Elmer spectrophotometer, using optical polystyrene
cuvettes (10x10x45 mm, 340–800 nm optical transparency). Each measure was repeated at least in
triplicate. Each compound was dissolved in opportune quantity of DMSO in order to obtain a final
DMSO content < 0.05 %, that does not affect the enzyme activity. AChE and BChE were daily
tested to evaluate the effective enzymatic activity. The spectrophotometric method of Ellman [26]
with minor modifications was used to evaluate the inhibition of Cholinesterase enzymes; it is based
26

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on the reaction of released thiocholine to give a coloured product, at wavelength of 412 nm, with a
chromogenic reagent DTNB.
4.2.1.1. Inhibition of EeAChE and eqBChE
3.0 mL of 0.1 M phosphate buffer (pH = 7.4) containing DTNB (0.25 mM) and EeAChE (0.083
UmL-1) or eqBChE (0.083 UmL-1) were placed in a polystyrene cuvette of 1.0 cm path length; 1 µL
of a solution in DMSO of the tested compound was added to obtain in cuvette concentration range
from 30 µM to 30 nM. To start the reaction 30 µL of an aqueous solution in phosphate buffer (pH =
7.4) of acetylthiocoline (10 mM) or butyrylthiocholine (10 mM) were added. The increase in the
absorbance, due to the production of the yellow 5-nitro-2-tio benzoic anion, was recorded at 412 nm
and 25°C between 0.5 and 1.5 min. As a control, an identical solution of the enzyme without the
inhibitor was processed following the same protocol to determine the 100 % of enzyme activity.
Each experiment was repeated at least in triplicate. The potency of each compound to inhibit AChE
or BChE activity was expressed as percent inhibition, calculated using the following equation:
ꢈꢉ − ꢈꢊ
ꢆ ꢇ
ꢀꢁℎꢂꢃꢂꢄꢂꢅꢁ % =
ꢌ 100
ꢈꢋ
where Ai and Ac represent the change in the absorbance in the presence of inhibitor and without
inhibitor, respectively.
4.2.1.2. Time dependent inhibition assay for carbamate derivatives
3.0 mL of 0.1 M phosphate buffer (pH = 7.4) containing DTNB (0.25 mM) and EeAChE (0.083
UmL-1) were mixed in a polystyrene cuvette of 1 cm path length with 1 µL of a solution in DMSO
of the carbamate compounds obtaining a 300 nM final concentration. Immediately after,
acetylthiocoline (30 µL, 10 mM) was added. Then the change in the absorbance was recorded. In
order to obtain a reference enzymatic activity, as a negative control, a solution containing the
enzyme without the inhibitor was processed in the same way. As a positive control of the
carbamoylation reaction, 1 µM carbaryl was used. After 40 minutes of incubation, the inhibition
27

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percentage was measured again, both for EeAChE incubated with inhibitor, for the carbaryl
containing control and for the negative control.
4.2.1.3. Study of kinetic analysis of EeAChE inhibition
The constant and the mechanism of inhibition were determinate for the most active compounds (8-
11, 13, 15-17). 3.0 mL of 0.1 M phosphate buffer (pH = 7.4) containing DTNB (0.25 mM) and
EeAChE (0.083 UmL-1) was mixed with the opportune volume of inhibitor, solubilized in
H2O/DMSO (10:1) in order to obtain the final solutions of inhibitor in cuvette between 50 and 250
nM and the final DMSO content of the assay mixture < 0.05 %. The reaction was started by adding
to the enzyme–inhibitor mixture, the proper volume of an aqueous solution of 10 mM
acetylthiocholine (pH = 7.4, phosphate buffer), in order to obtain a final concentration of
acetylthiocholine ranging between 100 to 400 µM. The production of the yellow anion was
recorded at 412 nm and 25 °C between 0.5 and 1.5 min. In order to determine the inhibition
constants (Ki) and inhibition mechanism we measure the rate of hydrolysis at three different
concentration of substrate (acetylthiocholine 100-400 µM) in presence of five different
concentration of inhibitor. Each determination was performed for five times. The recorded data
were analyzed by the enzyme kinetic module of SigmaPlot (version 8.02) plotting the reciprocal of
rate of hydrolysis (1/v) vs the concentration of inhibitor, according to the Dixon’s method [30], in
order to find the best fitting model of inhibition, as indicated by calculated linear regression
coefficient R2.
4.2.2. Inhibition of hAChE and hBChE
Human recombinant AChE (E.C. 3.1.1.7) lyophilized powder and BChE (E.C. 3.1.1.8) from human
serum were used (Sigma, Italia). The capacity of tested compounds to inhibit hAChE and hBChE
activity was assessed using the Ellman’s method [26]. Initial rate assays were performed at 37 °C
with a Jasco V-530 double beam Spectrophotometer: the rate of increase in the absorbance at 412
nm was followed for 3 min. hAChE stock solution was prepared by dissolving human recombinant
AChE lyophilized powder in 0.1 M phosphate buffer (pH = 8.0) containing Triton X-100 0.1 %.
28