Synthesis, biological evaluation, and molecular modeling of nitrile-containing compounds: Exploring multiple activities as anti-Alzheimer agents

Article abstract of DOI:10.1002/ddr.21594

Based on the monoamine oxidase (MAO) inhibition properties of aminoheterocycles with a carbonitrile group we have carried out a systematic exploration to discover new classes of carbonitriles endowed with dual MAO and AChE inhibitory activities, and Aβ anti-aggregating properties. Eighty-three nitrile-containing compounds, 13 of which are new, were synthesized and evaluated. in vitro screening revealed that 31, a new compound, presented the best lead for trifunctional inhibition against MAO A (0.34 μM), MAO B (0.26 μM), and AChE (52 μM), while 32 exhibited a lead for selective MAO A (0.12 μM) inhibition coupled to AChE (48 μM) inhibition. Computational analysis revealed that the malononitrile group can find an advantageous position with the aromatic cleft and FAD of MAO A or MAO B. However, the total binding energy can be handicapped by an internal penalty caused by twisting of the ligand molecule and subsequent disruption of the conjugation (32 in MAO B compared to the conjugated 31). Conjugation is also important for AChE as well as the hydrophilic character of malononitrile that allows this group to be in close contact with the aqueous environment as seen for 83. Although the effect of 31 and 32 against Aβ_1–42, was very weak, the effect of 63 and 65, and of the new compound 75, indicated that these compounds were able to disaggregate Aβ_1–42 fibrils. The most effective was 63, a (phenylhydrazinylidene)propanedinitrile derivative that also inhibited MAO A (1.65 μM), making it a potential lead for Alzheimer's disease application.

Full text of DOI:10.1002/ddr.21594

Received: 19 April 2019
DOI: 10.1002/ddr.21594
Revised: 1 July 2019
Accepted: 4 August 2019
R E S E A R C H A R T I C L E
Synthesis, biological evaluation, and molecular modeling
of nitrile-containing compounds: Exploring multiple activities
as anti-Alzheimer agents
Daniel Silva1 | Eduarda Mendes1 | Eleanor J. Summers2 | Ana Neca1
|
Ana C. Jacinto1 | Telma Reis1 | Paula Agostinho3 | Irene Bolea4
M. Luisa Jimeno5 | M. Luisa Mateus1 | Ana M. F. Oliveira-Campos6
|
|
Mercedes Unzeta4 | José Marco-Contelles7 | Magdalena Majekova8
Rona R. Ramsay2 | M. Carmo Carreiras1
|
1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
2Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK
3Faculty of Medicine and Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
4Institut de Neurociències i Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona (UAB), Bellaterra (Barcelona),
Spain
5Centro de Química Orgánica “Lora Tamayo” (CSIC), Madrid, Spain
6Centro de Química, Universidade do Minho, Braga, Portugal
7Laboratory of Medicinal Chemistry, Institute of Organic Chemistry (CSIC), Madrid, Spain
8Center of Experimental Medicine, Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovakia
Correspondence
Abstract
M. Carmo Carreiras, Research Institute for
Medicines (iMed.ULisboa), Faculty of
Based on the monoamine oxidase (MAO) inhibition properties of aminoheterocycles
Pharmacy, Universidade de Lisboa,
with a carbonitrile group we have carried out a systematic exploration to discover
new classes of carbonitriles endowed with dual MAO and AChE inhibitory activities,
and Aβ anti-aggregating properties. Eighty-three nitrile-containing compounds, 13 of
which are new, were synthesized and evaluated. in vitro screening revealed that 31, a
new compound, presented the best lead for trifunctional inhibition against MAO A
(0.34 μM), MAO B (0.26 μM), and AChE (52 μM), while 32 exhibited a lead for selec-
tive MAO A (0.12 μM) inhibition coupled to AChE (48 μM) inhibition. Computational
analysis revealed that the malononitrile group can find an advantageous position with
the aromatic cleft and FAD of MAO A or MAO B. However, the total binding energy
can be handicapped by an internal penalty caused by twisting of the ligand molecule
and subsequent disruption of the conjugation (32 in MAO B compared to the conju-
gated 31). Conjugation is also important for AChE as well as the hydrophilic character
of malononitrile that allows this group to be in close contact with the aqueous envi-
ronment as seen for 83. Although the effect of 31 and 32 against Aβ1–42, was very
weak, the effect of 63 and 65, and of the new compound 75, indicated that these
compounds were able to disaggregate Aβ1–42 fibrils. The most effective was 63, a
Av. Professor Gama Pinto, Lisbon 1649-003,
Portugal.
Email: micarreiras@gmail.con
Rona Ramsay, Biomedical Sciences Research
Complex, University of St. Andrews,
Biomolecular Sciences Building, North Haugh,
St. Andrews KY16 9ST, UK.
Email: rrr@st-andrews.ac.uk
Funding information
European Cooperation in Science and
Technology, Grant/Award Numbers: STSM
CM1103-29512, STSM D34-05989, STSM-
CM1103-29510; Portuguese Foundation for
Science and Technology, Grant/Award
Number: FCT -Project PTDC/SAU-
NEU/64151/2006 (MCC); Slovak Research
and Development Agency (MM); Slovak
Research and Development Agency;
Foundation for Science and Technology
Drug Dev Res. 2019;1–17.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
SILVA ET AL.
(phenylhydrazinylidene)propanedinitrile derivative that also inhibited MAO
A
(1.65 μM), making it a potential lead for Alzheimer's disease application.
K E Y W O R D S
AChE inhibitors, Alzheimer's disease, Aβ1–42 disaggregating agents, in silico study, MAO
inhibitors, nitrile-containing compounds
Monoamine oxidase (MAO) is the major metabolic enzyme
responsible for the deactivation of the monoamine neurotransmitters
generating the corresponding aldehyde, ammonia, and hydrogen per-
oxide as metabolic products (Ramsay, 2016). Since MAO catalytic
activity produces hydrogen peroxide, the increase in MAO B activity
in AD and Parkinson's disease (PD) brain tissues is considered to con-
tribute to the initiation of OS and the consequent deleterious effects
to the cell. Thus, in addition to their ability to elevate the levels of
monoamines in the brains of AD patients, both MAO A and B inhibi-
tors may prevent OS by decreasing the formation of reactive oxygen
species (Ramsay, 2016; Ramsay & Tipton, 2017). MAO B expression is
generally elevated in AD (Kennedy et al., 2003), decreasing mono-
amine levels. Some work proposes that MAO B is also suggested to
associate with β-secretase, γ-secretase, and to regulate neuronal Aβ
peptide levels (Hroudova et al., 2016; Schedin-Weiss et al., 2017).
Elevated MAO could be also associated with the formation of neurofi-
brillary tangles (Hroudova et al., 2016). MAO A also influences mono-
amine levels, particularly serotonin in depression, (Bortolato & Shih,
2011). MAO A levels, regulated by genetic and environmental factors,
including stress, hormonal deregulation, and food influences, are up or
down or unchanged in various brain areas (Naoi et al., 2018). Recent
evidence suggests that MAO-A generated ROS is involved in quality
control signaling in mitochondria (Ugun-Klusek et al., 2019; https://
doi.org/10.1016/j.redox.2018.10.003). However, the role of MAO A
in the trafficking and the health (and hence energy output) of mito-
chondria is still poorly understood. In AD patients, MAO A level
changes seem to involve multiple mechanisms influencing cell survival
(Cai, 2014). Due to the multifactorial nature of AD, design strategies
have been envisaged for the development of different series of
multifunctional drugs. In addition to MAO inhibition, multi-target-
directed ligand (MTDL) properties can include brain permeable iron
chelation, ChEs inhibition, anti-Aβ aggregation, and neuroprotection
among other properties (Unzeta et al., 2016).
1
| INTRODUCTION
Alzheimer's disease (AD), the most common neurodegenerative dis-
ease, is marked by progressive decline in a variety of higher cortical
functions. AD patients share common clinical and neuropathological
features, notably intracellular neurofibrillary tangles and extracellular
senile plaques, the two major pathological hallmarks (Korabecny et al.,
2018). Neurofibrillary tangles are formed by hyperphosphorylated tau
protein, while senile plaques are aggregates of amyloid β (Aβ) peptides
associated with several proteins, metal ions, and enlarged axons and
synaptic terminals. Moreover, reduced levels of acetylcholine (ACh) in
specific brain regions that mediate memory and learning functions,
loss of neurons and synapses, high levels of metal ions like Cu2+, Zn2+
,
Fe3+, mitochondrial dysfunction, inflammation, and the associated oxi-
dative stress (OS) represent features of this multifactorial disease
(Das & Basu, 2017; Jakob-Roetne & Jacobsen, 2009). Acetylcholines-
terase (AChE) hydrolyzes extracellular ACh into choline and acetic
acid, thus preventing the overstimulation of nerve cells. However, in
AD, loss of cholinergic neurons means that stimulation of nerve cells
by ACh is reduced, and neuronal communication is hindered. AChE
inhibitors improve neuronal communication by preventing the break-
down of ACh (Das & Basu, 2017).
Noncatalytic functions of AChE have been suggested in multiple
physiological processes, such as cell adhesion, neurite growth, apopto-
sis, and Aβ aggregation (Wang & Zhang, 2019). AChE interacts with
Aβ by the hydrophobic environment close to the peripheral anionic
site (PAS; De Ferrari et al., 2001; Inestrosa et al., 1996; Johnson &
Moore, 1999), forming stable AChE-Aβ complexes, (Alvarez et al.,
1998; Alvarez et al., 1997) and causing an increase in the neurotoxic-
ity of the Aβ fibrils (Muñoz & Inestrosa, 1999; Reyes et al., 2004). In
addition, AChE activities have also been reported to correlate with
the density of Aβ deposition in the AD brain (Arendt et al., 1992).
Recent experiments suggest that during the early stage of AD, Aβ
enters the mitochondria increasing the generation of free radicals and
might induce OS. Aβ and amyloid precursor protein (APP) were also
reported in the mitochondrial membranes of postmortem brain and
found to be responsible for disruption of electron transport chain to
cause irreversible neurodegeneration and cell damage (Sharma
et al., 2019).
Previous studies have indicated that the MAO inhibitory activ-
ity of several compounds is potentiated by cyanide (Davison, 1957;
Ramadan et al., 2007). The potentiating effect of cyanide can be
ascribed to an activation mechanism whereby cyanide assists the
binding of the inhibitor to the enzyme (Davison, 1957; Juárez-
Jiménez et al., 2014). A series of carbonitrile-containing ami-
noheterocycles were examined experimentally and computationally

SILVA ET AL.
3
to explore the role of nitriles in determining the inhibitory activity
against MAO. Dicarbonitrile aminofurans were found to be potent,
selective inhibitors against MAO A (Juárez-Jiménez et al., 2014).
An earlier study examined the inhibition of MAO B by a series of
C4-substituted phthalonitriles and a series of homologs lacking the
nitrile units (Manley-King et al., 2012). It was found that the nitrile
unit was a prerequisite for high binding affinity to both MAO A and
MAO B. The high binding affinities to MAO B were ascribed to the
highly polar nature of the nitrile group. Modeling studies indicated that
the nitrile group interacts with the polar regions in the substrate cavity
of the MAO B enzyme (Chirkova et al., 2015; Manley-King et al., 2012;
Van deer Walt et al., 2012). The carbonitrile function has also been intro-
duced by other research groups in compounds either with the ChEs
inhibitory activity (Samadi et al., 2010; Samadi, Valderas, et al., 2011;
Samadi et al., 2012, 2013) or dual ChEs and MAO inhibitory activities
(Samadi, Chioua, et al., 2011).
recovery (>99%) was observed in both dog and human hepato-
cyte incubations (Gauthier et al., 2008). In most nitrile-containing
drugs, the nitrile group passes through the body metabolically
unaffected. Cimetidine, an H2 receptor antagonist, after both oral
and intravenous doses in man, is oxidized to the sulfoxide
(6–10%) and hydroxymethyl (4%) metabolites and conjugated to
the N-glucuronide (24%), presenting unchanged drug (60–70%) (Strong &
Spino, 1987). The biotransformation of entacapone, a catechol O-
methyltransferase inhibitor was studied in experimental animals and man
by identifying their O-glucuronides in urine and plasma; in rats and dogs,
O-sulfates were also abundant (Wikberg & Vuorela, 1994). Early studies
with radiolabelled cromakalim, an antihypertensive agent, showed the
major urinary metabolites had unchanged nitrile groups (Kudoh, 1990;
Kudoh, Okada, Nakahira,
& Nakamura, 1990). Lersivirine, a non-
nucleoside reverse transcriptase inhibitor, is predominantly cleared by
glucuronidation and cytochrome P450-mediated oxidation whereas
hydrolysis of one of the nitrile moieties to form an amide represented less
than 8% of the total in plasma (Vourvahis et al., 2010). The major urinary
and plasma metabolites of verapamil, a phenethylamine vasodilator, were
the N-dealkylated products which retain the α-isopropylphenylacetonitrile
portion of the molecule (McIlhenny, 1971).
Since the nitrile group may raise concerns regarding cyanide
release, published information on diverse nitrile-containing drugs,
research compounds, and in vivo metabolism of carbonitrile deriva-
tives was closely examined. Structurally diverse nitrile-containing
pharmaceuticals are prescribed for a variety of medical treatments,
for example, the selective serotonin re-uptake inhibitor and antide-
pressant drug citalopram, the nonpurine xanthine oxidase inhibitor
for the treatment of hyperuricemia febuxostat, the aromatase
inhibitor and anticancer agent letrozole, and the non-nucleoside
reverse transcriptase inhibitors and anti-HIV drug etravirine
(Fleming et al., 2010; Jones et al., 2010). Regarding letrozole and
etravirine, both exhibit two nitrile units as does the analog of
etravirine, rilpivirine (Reznicek et al., 2017). In drug design, aro-
matic chloride is very often replaced by an aromatic nitrile since
this exchange can impart desirable properties to the resulting mole-
cule particularly when the potency is similar, or even improved.
Thus, the lipophilicity of the molecule will be significantly reduced
along with enhanced metabolic stability, higher aqueous solubility
and reduced toxicological outcomes (Jones et al., 2010). Due to its
small size, metabolic stability, and ability to act as a hydrogen bond
acceptor, the nitrile functional group has been incorporated into
several pharmaceutical agents (Lindsay-Scott & Gallagher, 2017). A
quantitative assay involving the reaction of nitriles with glutathione
and cysteine has been used as a simple in vitro screen to assess
potential toxicity risk of candidate compounds in drug discovery
and decide whether to progress with compounds in the absence of
radiolabeling studies (MacFaul et al., 2009).
Release of cyanide from aromatic or fully substituted carbons
is not observed (Fleming et al., 2010). Rabbits converted
2,6-dichlorobenzonitrile to an extent of ~2% and 23% into
2,6-dichloro-4-hydroxybenzonitrile and 2,6-dichloro-3-hydroxyben-
zonitrile, respectively. The hydrolysis of the nitrile group appears to
occur to a very minor extent. Only insignificant amounts of the amide
(<0.01%) and benzoic acid (<0.001%) derivatives were found. This was
supported by the observation that rabbits did not convert
2,6-dichlorobenzamide into the corresponding acid (Wit & van Gen-
deren, 1966). However, saturated and β,γ-unsaturated aliphatic nitriles
are hydroxylated in the liver at the α-carbon to form cyanohydrins
with subsequent cyanide release (Hideji & Kazuo, 1984, 1986). Man-
delonitrile, an agent formerly used for the treatment of urinary tract
infections, bears a H atom and OH group at the α-carbon. The cyanide
Regarding metabolic stability, the nitrile group is not particu-
larly electrophilic toward free nucleophiles, even glutathione
(Fleming et al., 2010; MacFaul et al., 2009) and is metabolically
quite robust (Boyd et al., 2009) unless activated by adjacent elec-
tron withdrawing elements (Oballa et al., 2007). Odanacatib, a
selective inhibitor of cathepsin K, exhibited excellent metabolic
stability in hepatocyte incubations across several species. In stan-
dard incubations (2 × 106 cells/ml, 20 μM, 2 hr), a 96% recovery
of the parent compound was found in rat hepatocytes and a 98%
recovery was obtained in rhesus monkey hepatocytes. High
SCHEME 1 General procedure for the Knoevenagel
condensation reaction regarding the syntheses of
arylidenepropanedinitriles of type 3 (Compounds 16–41, Table 1) and
the new Compounds 31, 34, 35, and 36

4
SILVA ET AL.
TABLE 1 IC50 values (μM) for inhibition of human MAO A by arylidenepropanedinitriles 16–41
Compound
X, Y, Z
hMAO AIC50 (μM)
60.7 11.4
>100
16 X,Y,Z = H
17 X,Y = H; Z = CH3
18 X = CH3; Y,Z = H
19 X,Y=H; Z = OCH3
20 X = H; Y,Z = OCH3
21 X,Y,Z = OCH3
71.3 362
21.4 2.70
58.0 12.0
288.0 33.6
11.3 1.08
>100
22 X,Y = H; Z = OCH2CHCH2
23 X,Y = H; Z = OCH2Ph
24 X,Y = H; Z = NO2
25 X,Z = H; Y = NO2
26 X,Y = H; Z = Cl
27 X,Y = H; Z = F
28 X,Y = H; Z = CN
29 X = CN; Y,Z = H
30 X,Y = H; Z = NO2
31 X = H; Y = Cl; Z = OCH3
32 X,Z = H; Y = CF3
33 X,Y,Z = H
>100
9.05 0.091
68.9 4.70
258.0 68.8
27.4 7.40
>100
15.7 1.87
2.67 0.274
1.43 0.265
43.4 10.6
71.7 43.4
15.9 4.07
66.6 59.2
166.0 27.2
27.2 4.47
34 X,Z = H; Y = OCH2Ph
35 X = Br; Y,Z = H
36 X,Y = H; Z = CN
37 X,Z = H; Y = CN
38
39
40
172.0 28.6
13.7 1.06
41
>100
Note: IC50 values were determined spectrophotometrically with kynuramine as substrate.
plus thiocyanate excreted by Wistar rats represented 86% of the dose,
hippuric acid corresponded to 71%, while 13% correlated to mandelic
acid (Singh et al., 1985). Epoxidation of alkenenitriles and ring opening
can potentially liberate cyanide, but epoxidation is synthetically diffi-
cult (Aiai et al., 1995; Miyashita et al., 1987; Pryde et al., 1996) and
metabolism at other sites appears more likely to occur (Fleming
et al., 2010).
and molecular modeling of a number of arylidenepropanedinitriles
(Compounds 16–41, Scheme 1, Tables 1 and 7, and Tables S1, S3,
S8, and S9), ((phenylamino)methylidene)propanedinitriles (Compounds
42–52, Scheme 2, Table 2 and Table S6), 4-amino-1-phenyl-1H-pyr-
role-3-carbonitriles (Compounds 53–57, Scheme 3, Table 3, and
Tables S7 and S10), and 3-amino-1-phenyl-1H-pyrrole-2,4-
dicarbonitriles (Compounds 58–62, Scheme 3, Tables 3 and 7, and
Tables S7 and S10), (phenylhydrazinylidene)propanedinitriles
(Compounds 63–67, Scheme 4, Tables 4 and 7, and Table S4),
4-arylazo-3,5-diamino-1H-pyrazoles (Compounds 68–72, Scheme 5,
Table 5 and Table S5), and (E,E)-4-amino-1-aryl-3-cyano-4-met-
hoxy-2-azabutadienes (Compounds 73–75, Scheme 6, Table 6).
Based on the MAO inhibition properties of aminofurans with the car-
bonitrile group (Juárez-Jiménez et al., 2014), the present study aimed to
identify classes of nitrile-containing compounds endowed with dual
AChE and MAO inhibitory activities, and Aβ anti-aggregating properties.
In this context, we have carried out the synthesis, biological evaluation,

SILVA ET AL.
5
SCHEME 2 General procedure for
the syntheses of [(phenylamino)
methylidene]propanedinitriles of type
6 (Compounds 42–52, Table 2) and the
new Compounds 45, 48, and 52
1991). Interesting results were also made known regarding their
capacity to activate cell resistance to oxidative stress (Turpaev et al.,
2011). Here we report the MAO and AChE inhibitory properties of a
few arylidenepropanedinitriles and chemical data of the new
arylidenepropanedinitriles 31, 34, 35, and 36 (Scheme 1; Figure 1).
TABLE 2 IC50 values (μM) for inhibition of human MAO A by
((phenylamino)methylidene)propanedinitriles 42–52
hMAO A
3.1.2 | Synthesis of ((phenylamino)methylidene)
X, Y, Z, W, Q
IC50 (μM)
propanedinitriles
42 X,Y,Z,Q,W = H
>100
43 X = CH3; Y,Z,Q,W = H
44 X,Q = CH3; Y,Z,W = H
45 X,Z,Q = CH3; Y,W = H
46 X,Z,Q = H; Y,W= CH3
47 X,W,Q = H; Y,Z = CH3
48 X,W,Q = H; Y,Z = OCH3
49 X,Z,W,Q = H; Y = NO2
50 X,Y,W,Q = H; Z = Cl
51 X,Z = Cl; Y,W,Q = H
52 X,Q = CH3; Y,W = H; Z = Br
>100
((Phenylamino)methylidene)propanedinitriles Type
6
(Compounds
>100
42–52) (Scheme 2) were readily synthesized by the reaction of
malononitrile 2 with ethyl orthoformate 4 and the respective aniline
derivative 5 (Wolfbeis, 1981). The MAO and AChE inhibitory activities
are reported here as well as the data for the new ((phenylamino)met-
hylidene)propanedinitrile derivatives 45, 48, and 52 (Scheme 2; Figure 1).
78.6 70.1
109 72.6
108 24.0
>100
9.65 1.36
>100
3.1.3 | Synthesis of 4-amino-1-phenyl-1H-pyrrole-
3-carbonitriles and 3-amino-1-phenyl-1H-pyrrole-
2,4-dicarbonitriles
>100
197 90.7
Note: IC50 values were determined spectrophotometrically with
The syntheses of both 4-amino-1-phenyl-1H-pyrrole-3-carbonitriles
(Compounds 53–57) and 3-amino-1-phenyl-1H-pyrrole-2,4-dicarbonitriles
(Compounds 58–62) are represented in Scheme 3 (compounds Type 8).
These syntheses were based on a Thorpe-Ziegler cyclization via intermedi-
kynuramine as substrate.
ate
7 using ((phenylamino)methylidene)propanedinitrile derivatives
2
| MATERIALS AND METHODS
6 as precursors (Scheme 2), an α-halo compound (chloroacetonitrile,
chloroacetone, ethyl bromoacetate, α-bromoacetophenone) and
triethylamine. (Salaheldin et al., 2010; Salaheldin et al., 2008; Unverferth
et al., 1998). Pharmacological data are reported concerning MAO inhibi-
tory activities of Type 8 compounds. Additionally, chemical data are
addressed to the new Compounds 59 and 60 (Scheme 3; Figure 1).
Materials and methods for Chemistry and Pharmacology are reported
in Supporting Information. Caution: Storage and safety working condi-
tions regarding malononitrile, arylidenemalononitriles, and some
nitrile-containing chemicals are mentioned in Supporting Information.
3
| RESULTS AND DISCUSSION
3.1.4 | Synthesis of (phenylhydrazinylidene)
propanedinitriles
3.1 | Chemistry
Phenylhydrazonomalononitriles 11 (Compounds 63–67; Scheme 4)
were prepared by diazotization of the appropriate arylamine 9,
followed by condensation of the diazonium salt 10 with
malononitrile (Krystof et al., 2006). Compounds 63 (Krystof et al.,
2006), 64 (Gavlik et al., 2017), 65 (Amer et al., 2004), and 66
(Tsai & Wang, 2005) are reported in the literature while (phe-
3.1.1 | Synthesis of arylidenepropanedinitriles
Arylidenepropanedinitriles 3 (Compounds 16–41; Scheme 1) were
synthesized by straightforward Knoevenagel condensation of the
respective aldehyde 1 with malononitrile (2) (Gazit et al., 1989). Most
compounds are common starting materials or intermediates in chemi-
cal reactions (Ding & Zhao, 2010; Marco et al., 2004; Zayed et al.,
nylhydrazinylidene)propanedinitrile 67 (Scheme 4) is
a new

6
SILVA ET AL.
SCHEME 3 General procedure for
the syntheses of both 4-amino-1-phenyl-
1H-pyrrole-3-carbonitriles and 3-amino-
1-phenyl-1H-pyrrole-2,4-dicarbonitriles of
type 8 (Compounds 53–62, Table 3) and
the new Compounds 59, 60
TABLE 3 IC50 values (μM) for inhibition of human MAO A
by 4-amino-1-phenyl-1H-pyrrole-3-carbonitriles 53–57 and 3-amino-
1-phenyl-1H-pyrrole-2,4-dicarbonitriles 58–62
3.1.6 | Synthesis of (E,E)-4-amino-1-aryl-3-cyano-
4-methoxy-2-azabutadienes
The (E,E)-4-amino-1-aryl-3-cyano-4-methoxy-2-azabutadienes 15
(Compounds 73–75; Scheme 6) were synthesized by reaction of benz-
aldehyde (13: X = H) or its derivatives with aminomalononitrile p-
toluenesulfonate (14; Scheme 6; Gutch, Raza, & Malhotra, 2001). The
synthesis of compound 73 is reported (Gutch et al., 2001). Com-
pounds 74 and 75 (Scheme 6) are new. Here we report the chemical
data and the MAO inhibitory activities for the new Compounds 74
and 75 (Scheme 6; Figure 1).
hMAO A
X, Y, Z, W, Q, R
IC50 (μM)
36.2 6.91
>100
53 X,Y,Z,W,Q = H; R = CO2Et
54 X,Y,Z,W,Q = H; R = COCH3
55 X,Y,W,Q = H; Z = Cl; R = CO2Et
56 X,Y,W,Q = H; Z = Cl; R = COCH3
57 X,Y,W,Q = H; Z = OCH3; R = CO2Et
58 X,Y,Z,W,Q = H; R = CN
Figure 1 represents the chemical structures of all the new com-
pounds (31, 34, 35, 36, 45, 48, 52, 59, 60, 67, 72, 74, 75).
92.5 18.9
>100
Diverse pyrazole derivatives (Compounds 76–81; Silva et al.,
2011; Silva, Samadi, Chioua, Carreiras, & Marco-Contelles, 2010) and
(phenoxyalkoxybenzylidenemalononitriles (Compounds 81 and 82;
Silva et al., 2013) were previously synthesized and their AChE inhibi-
tory activities reported. Here we describe their inhibitory activities in
rat mitochondria (rMAO A and rMAO B; Table S2).
>100
1,464 513
113 17.4
6.21 4.69
241 36.3
>100
59 X,W,Q = H; Y,Z = CH3; R = CN
60 X,Z,Q = H; Y,W=CH3; R = CN
61 X,Y,W,Q = H; Z = Cl; R = CN
62 X,Y,W,Q = H; Z = OCH3; R = CN
Note: IC50 values were determined spectrophotometrically with
3.2 | Pharmacology
kynuramine as substrate.
3.2.1 | Monoamine oxidase inhibition
Compounds were screened for MAO inhibition using various
assays: direct radiometric (rat mitochondrial MAO) or spectropho-
tometric (purified human MAO) assays or coupled fluorescent
assays (any preparation). Initial screening was performed at fixed
concentrations (10 or 25 μM) with 2×KM substrates. Where inhibi-
tion greater than 70% was observed, IC50 values were measured.
The IC50 values for selected inhibitors were re-evaluated in the
coupled assay with horseradish peroxidase, measuring the fluores-
cence of resorufin produced in the presence of 2×KM tyramine as
substrate.
compound which is described here for the first time. Pharmacologi-
cal data are reported concerning MAO inhibitory activities of com-
pounds Type 11. Additionally, chemical data are provided for the
new compound 67 (Scheme 4; Figure 1).
3.1.5 | Synthesis of 4-arylazo-3,5-diamino-1H-
pyrazoles
The 4-arylazo-3,5-diamino-1H-pyrazoles 12 (Compounds 68–72) were
obtained by cyclocondensation of phenylhydrazonomalononitriles 11
(Compounds 63–67) with hydrazine (Krystof et al., 2006). Compounds
68 (Al-Afaleq, 2000; Elgemeie et al., 2001; Krystof et al., 2006), 69 (Al-
Afaleq, 2000; Elgemeie et al., 2001), 70 (Huppatz et al., 1981), and 71
(Zhang et al., 2001) are reported in the literature while diaminopyrazole
72 (Scheme 5; Figure 1) is described here for the first time.
Table 1 shows IC50 values for purified human MAO A obtained
using the spectrophotometic assay with kynuramine as the substrate.
For the arylidenepropanedinitrile derivatives, an electron-rich substit-
uent (F, Cl, NO2) at the para position is detrimental to inhibition,
although m-NO2 is tolerated. Methoxy and propenyloxy substituents
in the para position increase binding (19, 22) but the benzyl ring

SILVA ET AL.
7
SCHEME 4 General procedure for
the syntheses of (phenylhydrazinylidene)
propanedinitriles of type 11 (Compounds
63–67, Table 4) and the new
Compound 67
TABLE 4 IC50 values (μM) for inhibition of human MAO A by
(phenylhydrazinylidene)propanedinitriles 63–67
hMAO A
X, Y, Z
IC50 (μM)
SCHEME 5 General procedure for the syntheses of 4-arylazo-
3,5-diamino-1H-pyrazoles of type 12 (Compounds 68–72, Table 5)
and the new Compound 72
63 X,Z = H; Y = NO2
64 X = H; Y,Z = OCH3
65 X,Y,Z = H
1.65 0.17
10.7 3.79
9.57 3.60
11.4 6.79
43.5 18.5
66 X,Y = H; Z = Cl
67 X = H; Y,Z = CH3
TABLE 5 IC50 values (μM) for inhibition human MAO A by
4-arylazo-3,5-diamino-1H-pyrazoles 68–72
Note: IC50 values were determined spectrophotometrically with
kynuramine as substrate.
substituent in 23 prevents binding. Changing the core benzyl ring to
furan gives better inhibition (30, 31, and 32).
Only one of the ((phenylamino)methylidene)propanedinitrile deriv-
atives was active, presumably due to interactions conferred by the m-
NO2 group (49 in Table 2).
hMAO A
X, Y, Z
IC50 (μM)
68 X,Y,Z = H
>100
The phenylpyrrole mono- and dicarbonitriles (Table 3) were like-
wise poor inhibitors with only the dicarbonitrile 60 active with an IC50
of 6.2 μM.
69 X,Y=H; Z = Cl
70 X,Z = H; Y = NO2
71 X = H; Y,Z = OCH3
72 X = H; Y,Z = CH3
19.7 17.9
420 250
>100
The (phenylhydrazinylidene)propanedinitrile derivatives 63–67
(Table 4) were more promising with 5 active compounds. Methyl sub-
stitution in 67 was not good but the parent compound 65, the p-Cl 66
and di-methoxy 64 derivatives gave IC50 values around 10 μM. The
best inhibitor in this series was 63, the m-NO2 compound with an
IC50 value of 1.65 μM.
>100
Note: IC50 values were determined spectrophotometrically with
kynuramine as substrate.
as in purified hMAO A (around 60 μM, Table 1), 20 with two methoxy
substituents was a better inhibitor than the others in the RLM MAO A
assay (IC50 15.9 μM). Compounds 24 and 25 also gave similar IC50 values
against both human and rat MAO A. The RLM study allows direct compari-
son of selectivity between MAO A and B (Table S1). Although both 19 and
20 inhibit RLM MAO A, the extra m-OCH3 in 20 removed the inhibition of
MAO B seen with the mono-OCH3 compound (19), making 20 selective
for MAO A. The m-NO2 derivative (25) is the best nonselective inhibitor of
rat MAO, but moving the NO2 to the para position (24) makes the com-
pound selective for MAO B. Thus, in rat 25 is nonselective whereas 20 is
MAO A-selective and 24 is B-selective, suggesting that discrimination
is based on fine steric or electronic interaction in the active sites.
None of the pyrazole derivatives (Compounds 76–81) and the
phenoxyalkoxybenzylidenemalononitriles (Compounds 81, 82) shown in
Table S2 inhibited rMAO in RLM, but the arylidenepropanedinitrile series
Switching the dinitrile to a pyrazole group (Table 5) did not
improve inhibition. The only active compound, the p-Cl derivative 69
was not much changed from the p-Cl derivative in the dinitrile series
(66 in Table 4).
Similarly, for the aminomethoxy compounds in Table 6, only one
active compound was found. The 4-CN derivative 75 gave an IC50
value of 1.92 μM against human MAO A, 100 times better that the
parent compound 73.
Since behavioral tests are performed on rodents, some compounds
were also evaluated on MAO A and MAO B present in rat liver mito-
chondria (RLM) as presented in Supporting Information. Table S1 shows
the IC50 values for arylidenepropanedinitrile derivatives on RLM,
assaying MAO A with [14C]-serotonin (5-HT) and MAO B with [14C]-
β-phenylethylamine (PEA). Although 16, 20, and 26 were poor inhibitors

8
SILVA ET AL.
SCHEME 6 General procedure for the
syntheses of (E,E)-4-amino-1-aryl-3-cyano-
4-methoxy-2-azabutadienes of type 15
(Compounds 73–75, Table 6) and the new
Compounds 74 and 75
at 5 μM) and the phenylpyrrole-aminodicarbonitrile 62 (69% at
10 μM) gave inhibition. Otherwise these series were not inhibitory
toward AChE.
TABLE 6 IC50 values (μM) for inhibition of human MAO
A by (E,E)-4-amino-1-aryl-3-cyano-4-methoxy-2-azabutadienes
73–75
Thus, compound 31 presents a lead for trifunctional inhibition
against MAO A, MAO B, and AChE (Table 7), while 32 offers a lead
for selective MAO A inhibition coupled to AChE inhibition.
After the primary screening in Tables 1–6 and Tables S1–S10, a
set of compounds was re-evaluated against all three human enzyme
targets. Table 7 summarizes the IC50 values for the human MAO and
AChE enzymes with the selected set of active compounds. Adding a
nitrogen to the linker between the phenyl ring and the dicarbonitrile
group (25 vs. 49) loses MAO A inhibition but adding two nitrogens
(63) improves inhibition of both MAO A and B and introduces some
weak inhibition of AChE (IC50 = 44 μM). A furan linker again gives best
lead compounds: 31 is a good inhibitor of both MAO A (0.34 μM) and
MAO B (0.26 μM), whereas the trifluoro derivative (32) is 100-fold
selective for MAO A (0.12 μM vs. 15 μM for MAO B). Both 31 and 32
show some inhibition of AChE (IC50 values of 52 and 48 μM, respec-
tively), so are lead compounds to optimize for multi-target activity.
hMAO A
X, Y, Z
IC50 (μM)
73 X,Y,Z = H
251 95.1
113 130
1.92 0.927
74 X,Y = H; Z = OCH2CHCH2
75 X,Y = H; Z = CN
Note: IC50 values were determined spectrophotometrically with
kynuramine as substrate.
in Table S3 revealed some inhibition. rMAO A was inhibited by the rigid
40 (IC50 = 18 μM) but more effectively by the flexible structures in 22
(3.5 μM) and 30 (0.72 μM). These compounds also inhibited rMAO B. The
most effective on rMAO B were 23 (1.2 μM) and 28 (7.5 μM), neither of
which inhibited MAO A in rat mitochondria.
Returning to human MAO, the inhibition of MAO A and MAO B was
compared for several compound series using the coupled assay for hydro-
gen peroxide production. Table S4 reports the % inhibition at 25 μM and
IC50 values for (phenylhydrazinylidene)propanedinitrile compounds.
Although di-methoxy substitution in 64 gives equal inhibition of MAO A
(IC50 = 22 μM) and MAO B (25 μM), the m-NO2 (63) is 10 times better
against MAO B (0.52 μM) than against MAO A (5.8 μM). In the same range
(IC50 values less than 10 μM), but nonselective, are 68–72 in Table S5,
showing that the diaminopyrazole ring gives better inhibition than the
dinitriles (63–67 in Table S4). Table S6 confirms poor inhibition of human
MAO by ((phenylamino)methylidene)propanedinitriles with none inhibiting
MAO A and only two inhibiting MAO B (45 at 4.27 μM and 52 at
1.66 μM). Table S7 shows that the 4-amino-1-phenyl-1H-pyrrole-
3-carbonitriles did not inhibit human MAO B, but some of the
dicarbonitriles inhibited by about 50% at 25 μM. Dicarbonitrile 60 did not
inhibit MAO B but was the only compound in Table S7 that inhibited MAO
A (44%). Table S8 identified two arylidenepropanedinitriles that almost
completely inhibited MAO A at 25 μM, namely 31 and 32 that share a
furan linker.
3.2.2 | Thioflavin T (ThT) assay to study Aβ
aggregation
For this assay four arylidenepropanedinitriles (31, 32, 33, 37), two (phe-
nylhydrazinylidene)propanedinitriles (63, 65), and one representative of
(E,E)-4-amino-1-aryl-3-cyano-4-methoxy-2-azabutadienes (75) were cho-
sen. The capacity of these compounds to inhibit the formation of Aβ1-42
fibrils (generated as described in Experimental section (Supporting Informa-
tion) was evaluated using the thioflavin T (ThT) fluorescence assay. This
assay measures changes in fluorescence intensity of ThT upon binding to
amyloid fibrils (Le Vine, 1999; Sulatskaya, Kuznetsova, & Turoverov, 2012).
As can be seen in Figure 2, different concentrations (20, 40, and
60 μM) of Compounds 31 (p-OCH3 electron donating group and a m-
Cl electron withdrawing group) and 32 (m-CF3 strong electron with-
drawing group) slightly inhibited the formation of Aβ1–42 fibrils, by
about 25% and an insignificant 15%, respectively.
The pyrrole derivatives 33 and 37 (Figure 2) differed only in the
C5-CN electron withdrawing group in 37. Both compounds were inef-
fective in disaggregating Aβ1–42 fibrils formation, being less adequate
than 31 and 32 (Figure 2).
Some compounds were also screened for inhibition of
AChE (commercially cloned and purified electric eel AChE) at 10 and
5 μM (arylidenepropanedinitriles in Table S9 and phenylpyrrole ami-
nocarbonitriles in Table S10). Only arylidenepropanedinitrile 37 (58%
By contrast, Compounds 63 and 65 (Figure 3), which differ only in
the m-NO2 electron withdrawing group present in 63, were able to
disaggregate Aβ1–42 fibrils. The most effective was 63 (inhibitor effect

SILVA ET AL.
9
TABLE 7 IC50 values (μM) for inhibition of human MAO A, MAO B, and AChE by selected compounds (25, 31, 32, 49, 60, 63, 65, 75)
IC50 (μM) after 5 min preincubation
Comp.
25
Structure
hMAO A
hMAO B
hAChE
44.7 20.6
24.6 9.4
31
32
0.34 0.05
0.12 0.03
0.26 0.11
15.2 5.3
52
48
8
2
49
>100
21.5 2.5
60
63
65
(>100)
(>100)
12.2 4.1
22.9 1.5
3.80 0.31
<10% at 25 μM
44
5
<10% at 100 μM
75
3.41 0.83
3.45 0.52
1.2 0.1b
71 10
ASS234a
0.053 0.004*
0.23 0.02
Note: IC50 values were determined in the coupled assay with tyramine (2×KM).
aBolea et al. (2011).
bWithout preincubation (i.e., reversible binding).
of 50–60%, depending on the concentration), whereas 65 (Figure 3),
and 75 (Figure 3) (p < .05) inhibited Aβ aggregation only at high con-
centrations (40 and 60 μM).
the dicyano group (Figure 6). The value of the positive charge is also
affected by other substituents, for example, for 49 the atomic charge
N (nitro) is diminished to 0.713 (Figure 7).
These specific features impacted the values of total binding ener-
gies (Table 8), which were higher for compound 63 due to a shorter
distance from FAD (3.5 Å for 63 and 3.7 Å for 49) as well as two addi-
tional interactions more (cation–π interaction with Tyr407 and hydro-
phobic interaction with Tyr444).
3.2.3 | Computational chemistry studies
Docking studies on monoamine oxidases
For our study we chose the derivatives 25, 30, 31, 32, 49, 63, and 65.
From these compounds only 49, 63, and 65 form tautomers with the
most probable ones shown in Figure 4. The binding energies values
obtained for individual structures with MAO A and MAO B are repre-
sented in Table 8.
Another important factor affecting the ligand interaction is the
presence and location of water molecules. The original ligand harmine
has water molecules near it in the binding site. The docking procedure
including water molecules is rather peculiar, so water was included
after docking in the procedure of complex optimization. The addition
of water was important to explain the lower activity of 30 (Figure S1).
Although it interacts with Gln215, binding of 30 is hindered by more
effective interaction of water molecule (HOH5342) with FAD.
Inspecting the binding modes of the strongest MAO A inhibitors
31 and 32 (Figure 8), it can be seen that the benzene ring of 31 is situ-
ated near the aromatic cleft of TYR 407 and TYR 444, but the
malononitrile group of 32 is more deeply immersed into this region, at
a distance of 2.99 Å from C10a of flavin. Docking and optimization
studies provided two preferred orientations for both compounds: one
with the malononitrile group near flavin and the second one with
opposite direction. It seems that it is the substitution on the benzene
The analysis of the inhibitor locations in the MAO A model pro-
vided several common features. If present, it is the nitro group, which
appears to be responsible for the interaction of the active derivatives
(Figure 5). The comparison of the structurally similar compounds 49,
63, and 65 showed that two inhibitors (49 and 63) directed the nitro
group toward FAD, whereas for 65 (no nitro group) the dicarbonitrile
group is directed toward FAD.
The main type of the interaction between the active inhibitor and
FAD is a cation–π relationship, which is a kind of electrostatic interre-
lation (cation–quadrupol). Positive charge is located at nitrogen of
nitro group and the π orbital is located at FAD; partial (but much
lower) positive charge is located on the central carbon connected to

10
SILVA ET AL.
FIGURE 2 Potential disaggregating effect of 31, 32, 33, and 37
on Aβ1-42 fibrils. Different concentrations (20, 40, and 60 μM) of
compounds were added to Aβ1-42 samples (preincubated at 37ꢀC for
24 hr) and incubated at 37ꢀC for an extra 24 hr. The levels of fibrils
were evaluated by the intensity of fluorescence of ThT. The
fluorescence of fibrillary Aβ1–42 (fib), which was incubated at 37ꢀC for
a total time of 48 hr, was also measured; and the fluorescence of
other experimental conditions were indicated as arbitrary units (a.u.)
related to Aβ1–42. The fluorescence of Aβ1–42 fresh solution (N-fib)
was also measured as reference. Data are mean SEM of 3–4
independent experiments
FIGURE 1 The structures of all the new
arylidenepropanedinitriles (31, 34, 35, 36), the ((phenylamino)
methylidene)propanedinitriles (45, 48, 52), the 3-amino-1-phenyl-1H-
pyrrole-2,4-dicarbonitriles (59, 60), the (phenylhydrazinylidene)
propanedinitrile 67, the 4-arylazo-3,5-diamino-1H-pyrazole 72, and
the (E,E)-4-amino-1-aryl-3-cyano-4-methoxy-2-azabutadienes 74
and 75
ring which specifies the orientation of arylidenepropanedinitriles 31
and 32 as well as that for the (phenylhydrazinylidene)propanedinitriles
63, 65 and ((phenylamino)methylidene)propanedinitrile, 49. Nitro and
methoxy groups in positions meta and para of the benzene ring create
sufficiently strong interactions with flavin or the aromatic cleft of the
catalytic center while trifluoromethyl or no substitution permits the
molecule to turn malononitrile group toward flavin. The abundant
numbers of attractive interactions of 31 and 32 are reflected in values
of binding energy (Table 8).
(Milczek, Binda, Rovida, Mattevi, & Edmondson, 2011). However, in
MAO B, the position of the conjugated malononitrile groups in the
vicinity of the aromatic cleft was strengthened by close contact of
one cyano nitrogen with hydroxyl group of Cys172 for 31 and by
the interaction with internal water near FAD for 32. Due to the sur-
rounding residues and water, the furan ring and the malononitrile
group of 32 are visibly turned against the substituted benzene ring
(sum of both deviations is 44ꢀ). This fact breaks the conjugation in
32 and, finally, penalizes the position of 32 in MAO B when com-
pared with 31, which is turned less (22ꢀ).
The inhibition activities of Compounds 31 and 32 in MAO B were
different from those in MAO A. The activity of 31 stayed almost the
same, while IC50 for 32 decreased in two orders from 0.12 to
15.2 μM. (Table 7). Docking calculations and subsequent optimization
revealed the opposite orientation of the molecule 31 in MAO B com-
pared with MAO A (Figures 8 and 9).
The water molecules can provide additional interactions for ligand
and stabilize or destabilize its position. Water found in the binding of
32 in MAO B pocket is positionally similar to the water molecule par-
ticipating in catalysis. So far, there has been no structural work on
MAO to show the positioning of ligands with the malononitrile group
in the active site.
Both compounds interact with the gating residues Ile199 and
Tyr326, which separate entrance and substrate cavities in MAO B

SILVA ET AL.
11
TABLE 8 IC50 values and binding energies Ebin toward hMAO A
for 25, 30, 49, 63, 65, and harmine
MAO A
Ebin
Comp.
Structure
IC50 (μM)
(kJ/mol)
25
321.6
9.05 0.091
15.7 1.87
30
31
338.3
368.2
0.34 0.05
0.12 0.03
32
49
377.6
323.6
9.65 1.36
1.65 0.17
63
346.7
65
9.57 3.60
272.7
395.6
Harmine
5–17 nM
FIGURE 3 Potential disaggregating effect of 63, 65, and 75 on
Aβ1–42 fibrils. Different concentrations (20, 40, and 60 μM) of
compound were added to Aβ1–42 samples (preincubated at 37ꢀC for
24 hr) and for another 24 hr incubation period at 37ꢀC. The levels of
fibrils were evaluated by the intensity of fluorescence of ThT. The
fluorescence of fibrillary Aβ1–42 (fib), which was incubated at 37ꢀC for
a total time of 48 hr, was also measured; and the fluorescence of
other experimental conditions were indicated as arbitrary units
related to Aβ1–42. The fluorescence of Aβ1–42 fresh solution (N-fib)
and of Aβ1-42 incubated with resveratrol (20 μM res), a known anti-
aggregating compound, were also measured as reference. Data are
mean SEM of 3–4 independent experiments. p < .05 statistical
different from Aβ1–42 fib
FIGURE 5 Comparison of the location of 49 (magenta), 63
(element color), and 65 (green) in the proximity of FAD
FIGURE 4 Most probable tautomers of Compounds 49, 63,
and 65
FIGURE 6 Charge distribution of Compound 63

12
SILVA ET AL.
A similar binding mode can be observed for 63. This compound is
depicted in Figure S2 for the binding sites of MAO A and MAO B. The
nitro group of 63 adjacent to FAD in both types of MAO is preferred,
although for both enzymes reverse positions also exist (Figure S3). but
with lower values of binding energy. (The differences in values of
binding energy were 20.5 kJ/mol for MAO A and 4.3 kJ/mol for MAO
B.). Compound 63 is bound in MAO A with Tyr407 by π–π and
cation–π interaction and with FAD by cation-π interaction with dis-
tance 4.1 Å. The position is stabilized by H-bonds with Phe208 and
one surrounding water molecule, as well as a hydrophobic interaction
with Gln215. However, both H-bonds make the position less energet-
ically favorable due to the twisting of malononitrile group. Interaction
FIGURE 7 Charge distribution of Compound 49
FIGURE 8 Interactions of 31 and 32 with monoamine oxidase A (MAO A)
FIGURE 9 Interactions of 31 and 32 with monoamine oxidase B (MAO B)

SILVA ET AL.
13
with Phe208 in MAO A or Ile199 in MAO B has been related to selec-
tivity of compounds either in the role of inhibitors (Morón, Campillo,
Perez, Unzeta, & Pardo, 2000) or as substrates (Tsugeno & Ito, 1997).
The stronger inhibition of MAO B with 63 was visualized as a firm
position of nitro group between Tyr398 and Tyr435, H-bond with
Tyr188 and π–π edge-to-edge interaction with FAD (distance 3.7 Å).
The malononitrile group is held by a single H-bond with a water mole-
cule occurring in the cavity. The resulting position keeps the whole
molecule in one plane, contributing thus to the overall benefit of this
mode of interaction.
For further work, the affinity of 63 toward MAO B could be
enhanced by a proper substitution in 2 or 3 position from the nitro
group. An interesting study would be made with a thiol substitution,
as in the proximity of these two places there is Cys172, which could
thus create a disulfide bond with the ligand. Another possibility would
be to introduce a benzyl or phenyl group in Position 1, to obtain the
π–π interaction with Tyr435.
Docking studies on acetylcholinesterase
Docking and optimization calculations were performed to determine
the binding mode of ligands to AChE for Compounds 32 (Table S9),
37 (Table S9, Figure S4), 62 (Salaheldin et al., 2008; Table S10), 82
[IC50 (AChE = 6.0 0.5 μM)] (Silva et al., 2013), (Table S2, Figures 10–
11), and 83 [IC50 (AChE = 2.1 0.5 μM)] (Silva et al., 2013; Table S2;
Figures 10 and 11). In the figures, all the compounds are rendered as
balls and sticks. The side chains conformations of the mobile residues
are illustrated in the same color (light blue) as the ligand. Different
subsites of the active site are colored as follows: catalytic triad (CT) in
green, oxyanion hole (OH) in pink, anionic subsite (AS) in orange
except Trp86, acyl-binding pocket (ABP) in yellow, PAS in cyan, and
the nonactive site residues in gray. Hydrogen bonds are represented
FIGURE 11 Overall view of 82 (blue, up) and 83 (down)
immersed in hAChE protein (4M0F PDB model) viewed through
the gorge of the protein surface. Red circles represent water
molecules
FIGURE 10 Binding mode of 82 and 83 at the active site of hAChE

14
SILVA ET AL.
4-arylazo-3,5-diamino-1H-pyrazoles, and (E,E)-4-amino-1-aryl-3-cyano-
4-methoxy-2-azabutadienes were synthesized and assessed.
From in vitro screening, compound 31 presents the best lead for
trifunctional inhibition against MAO A, MAO B, and AChE (Table 7),
while 32 offers a lead for selective MAO A inhibition coupled to AChE
inhibition.
Computational analysis of the binding modes of the compounds
demonstrated several characteristic features. The malononitrile group,
when not overcome by more attractive groups as nitro or methoxy
groups, can find an advantageous position with the aromatic cleft and
FAD of MAO A or MAO B. The total binding energy can be handicapped
by an internal penalty caused by twisting of ligand molecule and subse-
quent disruption of the conjugation as happens in 32 with MAO B com-
pared to the conjugated 31. Conjugation is also important for AChE. In
addition, the hydrophilic character of malononitrile allows this group to
be in close contact with the water environment in the more open binding
pocket of AChE, as seen for 83 in AChE.
FIGURE 12 Lipophilic potential on the surface of 83. Blue––
hydrophilic values, yellow––hydrophobic
as yellow dashed lines, π–π interactions as red lines, π–cation interac-
tions as blue lines, and hydrophobic contacts as green ones.
Although the effect of 31 and 32 against Aβ1–42 was very weak, the
effect of the other compounds was quite comparable to the action
observed for resveratrol (20 μM), used as a reference disaggregating
compound (Silva et al., 2013). Overall, the data indicated that Com-
pounds 63, 65, and 75 (Figure 3) were able to disaggregate Aβ1–42 fibrils.
The most effective was 63, a (phenylhydrazinylidene)propanedinitrile
derivative that also inhibited MAO A at micromolar concentration, mak-
ing it a potential lead for Alzheimer's disease application.
Table S12 summarizes the values of binding energy (Ebin) together
with the inhibition activities of the chosen compounds. The binding
energy values for 32 and 62 correspond to their lower activities, but
37 should be as strong inhibitor as 82 and 83 according to Ebin value.
This compound is immersed in the ligand cavity with both
malononitrile and cyano groups (Figure S4) bound to Trp86 (π–π inter-
action), Phe297 (hydrophobic), and Glu204 (H-bond). According to
these interactions, 37 should be much stronger inhibitor than it was
measured. The reason for its lower activity could be found in the
value of the solvation energy (Table S11), which is very low and thus
represents quite a big value of desolvation penalty in total ligand
binding.
ACKNOWLEDGMENTS
This collaborative work was enabled by COST Action CM1103, Struc-
ture-based drug design for diagnosis and treatment of neurological dis-
eases. We thank our funding sources: COST Actions D34 and
CM1103 for Short-term Scientific Mission funding (E.M., D.S., M.M.);
the School of Biology at the University of St. Andrews (E.J.S., R.R.R.);
the Research Institute for Medicines (iMed.ULisboa), Faculty of Phar-
macy, Universidade de Lisboa (A.N., A.C.J., T.R., M.C.C.); FCT, the Por-
tuguese Foundation for Science and Technology (Project PTDC/SAU-
NEU/64151/2006 (M.C.C.), and project grant (D.S.) Vega 2/0127/18
and the contract No. APVV-15-0455 of Slovak Research and Devel-
opment Agency (M.M.).
Compounds 82 and 83 contain long aliphatic chains and, for both,
the malononitrile group represents the hydrophilic part in a generally
hydrophobic molecule (Figure 12), which can be observed also in
values of solvation energy (Table S11). The length of the aliphatic
chain fits in the cavity of hAChE more comfortable for 83 than for 82
(Figure 10). Both compounds are anchored to the cavity by strong
interaction between malononitrile group and Trp236 from the PAS
region. Malononitrile groups, directed to the outer space, are also
bound with surrounding water molecules (Figure 11). The compounds
fill major parts of the ligand cavity, 83 more than 82. (Figure 11).
CONFLICT OF INTEREST
The authors declare no conflict of interest in this research work.
4
| CONCLUSIONS
In this paper we have reported the synthesis and biological evaluation of
83 compounds, 13 of which are new. Based on the MAO inhibition proper-
ties of the carbonitrile group we carried out a systematic exploration to dis-
cover new classes of nitrile-containing compounds endowed with dual
AChE and MAO inhibitory activities, and Aβ anti-aggregating properties.
Thus, arylidenepropanedinitriles, ((phenylamino)methylidene)propanedin-
itriles, 4-amino-1-phenyl-1H-pyrrole-3-carbonitriles, 3-amino-1-phenyl-
1H-pyrrole-2,4-dicarbonitriles, (phenylhydrazinylidene)propanedinitriles,
ORCID
M. Carmo Carreiras
https://orcid.org/0000-0001-7096-2449
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How to cite this article: Silva D, Mendes E, Summers EJ,
et al. Synthesis, biological evaluation, and molecular modeling
of nitrile-containing compounds: Exploring multiple activities
as anti-Alzheimer agents. Drug Dev Res. 2019;1–17. https://
doi.org/10.1002/ddr.21594