Development of novel multipotent compounds modulating endocannabinoid and dopaminergic systems

Article abstract of DOI:10.1016/j.ejmech.2019.111674

Polypharmacology approaches may help the discovery of pharmacological tools for the study or the potential treatment of complex and multifactorial diseases as well as for addictions and also smoke cessation. In this frame, following our interest in the de

Full text of DOI:10.1016/j.ejmech.2019.111674

European Journal of Medicinal Chemistry 183 (2019) 111674
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Development of novel multipotent compounds modulating
endocannabinoid and dopaminergic systems
a
,
1
a
,
1
b
c
Alessandro Grillo , Giulia Chemi , Simone Brogi , Margherita Brindisi ,
a
d
e,
f
g
Nicola Relitti , Filomena Fezza , Domenico Fazio , Laura Castelletti ,
Elisabetta Perdona , Andrea Wong , Stefania Lamponi , Alessandra Pecorelli ,
Mascia Benedusi , Manuela Fantacci , Massimo Valoti , Giuseppe Valacchi
Fabrizio Micheli , Ettore Novellino , Giuseppe Campiani
g
g
a
h
i
j
j
h, i, k
,
g
c
a,
**
, Stefania Butini a
,
, *
f
,
l,
2
a, 2
Mauro Maccarrone
, Sandra Gemma
a
Department of Biotechnology, Chemistry and Pharmacy, DoE Department of Excellence 2018-2022, University of Siena, via Aldo Moro 2, 53100, Siena, Italy
Department of Pharmacy, University of Pisa, via Bonanno 6, 56126, Pisa, Italy
b
c
d
e
f
Department of Pharmacy, University of Napoli Federico II, DoE Department of Excellence 2018-2022, Via D. Montesano 49, 80131, Napoli, Italy
Department of Experimental Medicine, University of Rome Tor Vergata, via Montpellier 1, 00133, Rome, Italy
Faculty of Biosciences and Technology for Food Agriculture and Environment, University of Teramo, via R. Balzarini 1, 64100, Teramo, Italy
Department of Medicine, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21, 00128, Rome, Italy
Aptuit (Verona) Srl, an Evotec Company, Via Alessandro Fleming, 4, 37135, Verona, Italy
NC State University, Plants for Human Health Institute, 600 Laureate Way, Kannapolis, NC, 28081, USA
Department of Biomedical and Specialist Surgical Sciences, University of Ferrara, Via Borsari 46, 44121, Ferrara, Italy
Department of Life Sciences, University of Siena, via Aldo Moro 2, 53100, Siena, Italy
g
h
i
j
k
l
Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea
European Center for Brain Research, Santa Lucia Foundation IRCCS, Via del Fosso di Fiorano snc, 00176, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 June 2019
Received in revised form
Polypharmacology approaches may help the discovery of pharmacological tools for the study or the
potential treatment of complex and multifactorial diseases as well as for addictions and also smoke
cessation. In this frame, following our interest in the development of molecules able to modulate either
the endocannabinoid or the dopaminergic system, and given the multiple and reciprocal in-
terconnections between them, we decided to merge the pharmacophoric elements of some of our early
leads for identifying new molecules as tools able to modulate both systems. We herein describe the
synthesis and biological characterization of compounds 5a-j inspired by the structure of our potent and
30 August 2019
Accepted 1 September 2019
Available online 4 September 2019
Keywords:
2 3
selective fatty acid amide hydrolase (FAAH) inhibitors (3a-c) and ligands of dopamine D or D receptor
Endocannabinoid system
Fatty acid amide hydrolase
Enzyme inhibitors
Selective inhibitors
Dopamine ligands
Polypharmacology
Multitarget compounds
Dopamine receptor ligands
subtypes (4a,b). Notably, the majority of the new molecules showed a nanomolar potency of interaction
with the targets of interest. The drug-likeliness of the developed compounds (5a-j) was investigated in
silico while hERG affinity, selectivity profile (for some proteins of the endocannabinoid system), cyto-
toxicity profiles (on fibroblast and astrocytes), and mutagenicity (Ames test) were experimentally
determined. Metabolic studies also served to complement the preliminary drug-likeliness profiling for
compounds 3a and 5c. Interestingly, after assessing the lack of toxicity for the neuroblastoma cell line
(
IMR 32), we demonstrated a potential anti-inflammatory profile for 3a and 5c in the same cell line.
2019 Elsevier Masson SAS. All rights reserved.
©
1 2
Abbreviations: AEA, anandamide; 2-AG, 2-arachidonoylglycerol; CBR, cannabinoid receptor; CB R, type-1 cannabinoid receptors; CB R, type-2 cannabinoid receptors;
CNS, central nervous system; DCM, dichloromethane; DMF, dimethylformamide; DMSO, dimethylsulfoxide; eCBs, endocannabinoids; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; hERG, human ether-a-go-go related gene; HOBt, N-hydroxybenzo-
triazole; HPLC, high pressure liquid chromatography; LPS, lipopolysaccharide; MAGL, monoacylglycerol lipase; OS, Oxidative stress; PDB, Protein Data Bank; SAR, structure-
activity relationship; TEA, triethylamine; THF, tetrahydrofuran.
*
Corresponding author.
** Corresponding author.
E-mail addresses: campiani@unisi.it (G. Campiani), butini3@unisi.it (S. Butini).
These authors contributed equally to this work.
Equally senior authors.
1
2
https://doi.org/10.1016/j.ejmech.2019.111674
223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
0

2
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
1
. Introduction
some D
for smoking cessation and relapse prevention [15]. There are evi-
dences indicating that D /D receptor density may correlate with
smoking and nicotine dependence. In a recent study in smokers
and non-smoker subjects, by positron emission tomography,
3
receptor antagonists have been engaged in clinical studies
The endocannabinoid system (ECS) is the ensemble of fatty acid-
derived messengers (endocannabinoids, eCBs), their receptors
type-1 and type-2 cannabinoid receptors, CB R and CB R), and the
2
3
(
1
2
enzymatic machinery responsible for the biosynthesis and termi-
nation of the eCBs' signaling (in particular fatty acid amide hy-
drolase, FAAH, and monoacylglycerol lipase, MAGL) [1]. The ECS
plays a modulatory function in several physiological processes,
mainly in the central nervous system (CNS). These effects are
mostly operated via a retrograde signaling where post-synaptically
striatal dopamine D
found that dopamine D
correlated with recent and lifetime smoking and also with nicotine
dependence [16]. This suggests that modulation of the D /D re-
ceptor signaling may represent a potential treatment for nicotine
addiction. In this context, interesting achievements and prospects
2
/D
3
receptor density was measured, and it was
2
3
/D receptor availability was negatively
2
3
released eCBs act at pre-synaptic CB
1
R and modulate a series of
were recently reported through the development of FAAH/D
multiple ligands [17,18].
3
functions (by the suppression of neurotransmitters’ release) [2].
The ECS is involved in several physiological (movement control,
nociception, brain reward, learning and memory, feeding, brain
development etc.) and pathological processes. By virtue of the in-
teractions of the ECS with other neurotransmitter systems, the
range of therapeutic options in which its modulation could be
exploited is dramatically enlarged [3]. Among the various neuro-
transmitters of the CNS, the ECS has been frequently linked to
dopamine [4]. In fact, the ECS operates indirect neuromodulatory
effects on the dopaminergic system. This modulation is mediated
by GABAergic and glutamatergic terminals “innervating” the
dopaminergic neurons [3]. Increased or decreased dopaminergic
tone in the basal ganglia circuitry is associated with hypo- or hyper-
motility, respectively [5]. In the basal ganglia circuitry the tight
interconnection of ECS and dopaminergic system is also confirmed
All these evidences converge to emphasize the high number of
complex and multifactorial diseases and conditions that involve
both the ECS and the dopaminergic system. The development of
multitarget ligands for hitting both systems would be of note for a
potential therapeutic application in different CNS diseases and
cravings. In fact, a polypharmacological approach would offer the
advantage of simultaneously targeting different neurotransmis-
sional systems for attaining higher efficacy and lower toxicity than
the administration of a cocktail of drugs [19].
Since several years we have been deeply involved in the dis-
covery of new CNS agents as potential therapeutics for schizo-
phrenia [20e22], and cocaine-seeking behavior [23]. The
previously developed compounds belong to different structural
classes, and the diverse scaffolds were strategically decorated for
by CB
projection neurons, and by the existence of CB
2A receptor heteromers in the dendritic spines of GABAergic
neurons. Although the exact functional role of these heteromers
needs to be further studied, their existence supports the presence
of an eCB/dopamine bidirectional regulation [3].
As regards the cortico-limbic structures, a “glitch” in the dopa-
minergic system is involved in psychoses such as schizophrenia as
well as in addictive states [3]. In all these cases a normalization of
the dopaminergic transmission is needed for attaining therapeutic
efficacy. In light of the fact that cannabinoids are hypokinetic
substances [3], despite contrasting earlier reports [6], more recent
studies seem to indicate that a beneficial role for eCBs (such as
anandamide, AEA) [7] in the modulation and prevention of the
1
R and D
1
or D
2
receptors co-localization in striatal GABAergic
2 3
modulating their interaction with D and D receptor subtypes, and
1
R, D , and adenosine
2
also with other monoaminergic receptors. More recently, we have
reported the development of indirect agonists of the ECS (e.g. se-
lective inhibitors of FAAH [24e26], MAGL [27], or dual FAAH/MAGL
[28] inhibitors) and we have also studied different potential ap-
plications of FAAH- [29] and MAGL-targeting [30] pharmacological
tools. In the context of a polypharmacological approach involving
the ECS and the dopaminergic system, all the above considerations
motivated us to give our contribution in the development of new
FAAH/dopamine receptors modulators. Inspired by the structure of
well-studied FAAH inhibitors bearing electrophilic centers (e.g.
carbamate) such as 1a [31] and 1b [32], on the bis-aryl structure of 2
[33], and based on our experience in the development of: i) FAAH
A
3
potent reversible inhibitors (typified by 3a-c [24], Fig. 1) ii) D se-
extrapyramidal symptoms elicited by D
ruled out.
2
antagonism could not be
lective ligands (typified by 4a [23], Fig. 1), and iii) multifunctional
compounds for monoaminergic systems (typified by 4b [22], Fig. 1),
we merged the key pharmacophoric elements for the interaction
with the targets of interest and we developed compounds 5a-j
(Fig. 1 and Table 1) as potential dopaminergic D and D receptors
2 3
ligands and FAAH enzyme inhibitors. The designed compounds
bear an electrophilic functionality, needed for potent FAAH inhi-
Further, the ECS signaling is also relevant in brain reward cir-
cuits and regulates the behaviors implied in addiction [8]. In this
frame, a key level of interaction amongst ECS and dopaminergic
system in mesocorticolimbic areas underlies processes like brain
reward and drug abstinence responses. The fact that AEA plays a
crucial role in the reward processes by activation of the dopami-
nergic system (in the mesolimbic area) is known since more than a
decade [9]. Also, it has been demonstrated that FAAH inhibition
contributes to this modulation, through potentiation of the AEA
effects on dopaminergic neurotransmission [9]. The connection of
the ECS to addiction substantiates its targeting also for the treat-
ment of nicotine dependence, and in nicotine reward and relapse
bition, and a phenylpiperazine moiety, a key feature for a potent D
2
/
D
3
interaction. These structures are supported on a versatile bis-
aryl scaffold, where a phenyl system is linked to a 5-membered
amidoheterocycle (Fig. 1 and Table 1). Functionalization of this
scaffold has enabled a systematic structure activity relationship
(SAR) study.
We herein describe the synthesis, molecular modeling and
biological investigation of compounds 5a-j, which were tested on
[
10,11]. In particular, FAAH inhibitors proved effective in reducing
the anxiety induced by nicotine withdrawal [12]. This is a relevant
issue for human health, since nicotine is the major psychoactive
drug of tobacco, and tobacco use is associated with the death of 6
million people each year [13].
2 3
the FAAH enzyme and, for selected analogues, on D and D re-
ceptor subtypes. The drug-like profile and the physicochemical
properties of the developed compounds were preliminarily
assessed by computational methods. Their inhibition profile at
human ether- aꢀ -go-go-Related Gene (hERG) channels was calcu-
As a further matter of fact, the mesolimbic pathway plays a key
role in the control of emotions, and in reward. In particular, D
2
-like
lated by our in-house 3D-QSAR model and for a subset of com-
pounds the values were also experimentally determined. For
compounds 5a and 5c we assessed selectivity towards other ECS
receptor ligands, with a specific emphasis on D receptor antago-
3
nists, have emerged as potential therapeutics for the management
of abuse-related effects and relapse of drugs of abuse [14]. Also
1 2
proteins (CB R and CB R and MAGL enzyme). Cytotoxicity of

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
3
Fig. 1. Reference (1e4) and title compounds (5aej).
compounds 5a and 5c was measured on mouse fibroblasts, and
Ames test was performed to ascertain absence of mutagenicity.
Preliminary metabolic stability studies were performed on 5c, in
comparison to 3a, after incubation with human and rat liver mi-
crosomes in order to gain insights into potential metabolic stability
issues. For compound 5c, we demonstrated a potential anti-
nflammatory profile on the neuroblastoma cell line (IMR32), by
reducing the expression of the lipopolysaccharide (LPS)-induced
encompassing alkylation of phenylpiperazine (6) with bromoace-
tonitrile or bromopropionitrile followed by reduction of the inter-
mediate cyano derivative in the presence of lithium aluminium
hydride. Carbonylimidazolide 8 was synthesized by treatment of
amine 7a with carbonyldiimidazole (CDI) in water. Known phenol
derivative 9 [24], upon treatment with phosgene in the presence of
4-(dimethylamino)pyridine (DMAP), afforded the corresponding
chloroformate which was immediately reacted with amines 7a,b
providing final compounds 5a,i, respectively. For the synthesis of
the urea derivative 5b, dimethoxytetrahydrofuran derivative 10
was submitted to Clauson Kaas reaction with 3-nitroaniline.
Following reduction of the nitro functionality and partial hydrolysis
of the nitrile to amide afforded key aniline derivative 11. Treatment
with phosgene followed by reaction with amine 7a afforded de-
rivative 5b.
activation of the redox-sensitive transcription factor NF-kB.
2
. Results and discussion
2.1. Chemistry
The synthesis of compounds 5a,b,i is shown in Scheme 1.
Amines 7a,b were obtained following a literature procedure [34]
The synthesis of phenylfuran- and phenylthiophene-based

4
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
Table 1
Inhibition Activity of compounds 5a-j towards: Mouse Brain FAAH (as IC50 and K
i
2 3 i
, nM) for Dopaminergic D and D receptors (as K , nM) and for hERG channels (as IC50, mM).
Cmpd
R
X
n
FAAH
FAAH
D
2
D
3
hERG
IC50
IC50 nMa
K
nM
b
K
, nM (pK
)c
K
, nM c (pK
m )
Md (pIC50
i
i
i
i
i
)
1
3
3
3
4
4
5
a
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
1
110 ± 23
0.60 [24]
1.70 [24]
1.90 [24]
NT
NT
NT
8.20 (5.10)
>10 (<5.00)
NT
NT
~10 [22]
5.30 (5.28)
a [24]
b [24]
c [24]
a [23]
b [22]
a
0.16 [24]
0.49 [24]
0.50 [24]
>30000 (<4.50)
>30000 (<4.50)
NT
>10000 [23]
>1000 [22]
186 (6.73 ± 0.13)
>30000 (<4.50)
>30000 (<4.50)
NT
0.18 [23]
0.60 [22]
e
e
e
e
e
e
NT
NT
NT
NT
e
e
58 ± 12 (33e103)
16 ± 4.0
105 (6.98 ± 0.05)
5b
1
1600 ± 600 (1000e2500)
420 ± 100
NTe
NTe
NTe
5
5
c
1
1
0.89 ± 0.11 (0.39e2.08)
26 ± 1.30 (16.7e39.7)
0.23 ± 0.03
6.88 ± 0.34
138 (6.86 ± 0.03)
214 (6.67 ± 0.001)
105 (6.98 ± 0.11)
148 (6.83 ± 0.01)
<0.10 (>7.00)
d
0.28 (6.55)
5e
1
175 ± 40 (92e313)
46 ± 10
NTe
NTe
NTe
5
5
f
1
1
195 ± 25 (123e308)
757 ± 60 (395e1450)
50 ± 6.40
NTe
NTe
NTe
NTe
NTe
NTe
g
194 ± 15.40
5
h
i
1
2
791 ± 25 (587e1066)
94 ± 18 (26.8e299)
203 ± 6.30
25 ± 4.60
56 (7.25 ± 0.06)
66 (7.18 ± 0.07)
76 (7.12 ± 0.01)
34 (7.47 ± 0.07)
6.40 (5.19)
3.50 (5.45)
5
5j
2
62 ± 6.70 (26.9e121)
16 ± 1.80
29 (7.53 ± 0.004)
19 (7.71 ± 0.05)
1.10 (5.97)
a
b
c
Each value is the mean of at least three experiments (all SD are within 10%, 95% confidence intervals are into brackets).
Determined by the Cheng-Prusoff equation.
(
(
all SD are within 10%, pK
pIC50 values are into brackets).
i
values ± SD are into brackets).
d
e
NT not tested.
analogues 5c,d,j is reported in Scheme 2. Reaction of known phe-
nols 12a,b [24] with phosgene in the presence of DMAP led to the
chloroformate intermediates which after reaction with amines 7a,b
provided compounds 5c,d,j.
The synthesis of compounds 5e,f is described in Scheme 3. Paal-
Knorr reaction on dione 13 in the presence of methylamine and
sulfamic acid as the catalyst [35] led to both the 2-aryl-1,5-
dimethylpyrrolic derivative 15, bearing the ester group in 3-
position of the pyrrole ring, and its methylamide counterpart 14.
Ester 15 was hydrolysed to corresponding acid 16, and converted
into amide 17, via classical coupling reaction with ammonium hy-
droxide in the presence of triethylamine (TEA), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), and hydroxybenzo-
triazole (HOBt). Amide 19 was directly obtained by reacting dione
13 with concentrated ammonium hydroxide in the presence of
sulfamic acid. Also in this case, we observed the formation of both
ester and amide derivatives 18 and 19. The methoxyphenyl de-
rivatives 17 and 19 were treated with boron tribromide to afford
their phenolic counterparts 20a,b. In this case, since classical re-
action with phosgene and amine 7a in presence of DMAP led to the

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
5
Scheme 3. Synthesis of compounds 5e,f. Reagents and conditions. a) MeNH
2
(33%
ꢀ
ꢀ
ꢀ
solution in EtOH), NH
2
SO
3
H, ꢁ5 Ce25 C, 2 h, 50%; b) NaOH, EtOH, H
2
O, 65 C, 16 h,
OH, dry DCM, 0 Ce25 C, 16 h, 73%; d) Conc. NH OH,
H, ꢁ5 Ce25 C, 5 h, 36% for 18, 20% for 19; e) BBr (1 M solution in DCM), dry
ꢀ
ꢀ
99%; c) TEA, EDC, HOBt, conc. NH
4
4
ꢀ
ꢀ
2
NH SO
3
3
ꢀ
ꢀ
ꢀ
2
O, 25 C, 5 h, 20e36%.
DCM, ꢁ78 Ce25 C, 12 h, 20e22%; f), carbonylimidazolide 8, H
Scheme 1. Synthesis of compounds 5a,b,i. Reagents and conditions. a) BrCH
2
ꢀ
CN or
ꢀ
ꢀ
BrCH
2
CH
2
CN, Na
2
CO
3
, EtOH, 25 C, 2 h, 65e70%; b) LiAlH
4
, dry THF, 0 Ce25 C, 5 h,
ꢀ
50e60%; c) CDI, H
2
O, 0 C, 1 h, 90%; d) 20% COCl
2
in toluene, DMAP, dry THF, reflux,
commercially available 1-bromo-3-methoxybenzene 28 was con-
verted into its corresponding azide 29, by means of nucleophilic
1
2 h; e) 7a or 7b, dry THF, reflux, 12 h, 20% (over 2 steps); f) 3-nitroaniline, 6 M HCl, 1,4-
dioxane, reflux, 40 min, 92%; g) SnCl
, EtOH, reflux, 48 h, 32%; i) 20% COCl
a, dry THF, reflux, 12 h, 21% (over 2 steps).
2
∙H
2
O, EtOH, reflux, 1.5 h, 95%; h) 6 M NaOH, 30%
ꢀ
H
7
2
O
2
2
in toluene, pyridine, dry DCM, 25 C, 12 h; j)
aromatic substitution, catalysed by copper(I) iodide (CuI) and -
L
proline [36]. The phenyltriazole 30 was then obtained by a con-
ventional click chemistry reaction between 29 and methyl-
propiolate, in presence of CuI [37]. Saponification of the methyl
ester, conversion of the acid 31 into the corresponding amide fol-
lowed by the cleavage of the methoxy functionality, afforded
phenol derivative 32. Compound 32 was then converted, via the
intermediate chloroformate, to the urethane 5h.
Scheme 2. Synthesis of compounds 5c,d,j. Reagents and conditions. a) 20% COCl
2
in
toluene, DMAP, THF, 0 Ce25 C, 30 min; b) amines 7a or 7b dry THF, reflux, 12 h,
0e34% (over 2 steps).
ꢀ
ꢀ
3
isolation of the carbamoylated compound with a cyano group at 3-
position of the pyrrole ring (deriving from amide dehydration), an
alternative strategy involving reaction of phenol derivatives 20a,b
with carbonylimidazolide 8 was employed in order to obtain the
compounds 5e,f.
Scheme 4 describes the synthesis of compound 5g. The
commercially available pyrrole-2-carbaldehyde 21, after protection
as acetal derivative 22, was brominated in 2-position (23) using N-
bromosuccinimide (NBS). By treatment of 23 with 1 M HCl acetal
deprotection aldehyde 24 was obtained. Treatment of the aldehyde
with iodine and ammonium hydroxide provided the cyano deriv-
ative 25 which was submitted to Suzuki coupling with 3-
hydroxyphenylboronic acid to afford phenol derivative 26. Partial
hydrolysis of the nitrile functionality let to amide derivative 27.
Treatment with phosgene followed by reaction with amine 7a
finally provided compound 5g.
Scheme 4. Synthesis of compound 5g. Reagents and conditions. a) p-Toluenesulfonic
ꢀ
ꢀ
acid, 2,2-dimethyl-1,3-propanediol, toluene, 140 C, 24 h, 99%; b) NBS, dry THF, ꢁ20 C,
ꢀ
ꢀ
1
6 h, 81%; c) 1 M HCl, acetone, 50 C, 3 h, 83%; d) I
2
, NH
O, EtOH, toluene, 25 C for 10 min,
, EtOH, reflux, 48 h, 67%; g) 20% COCl in
4
OH, THF, 25 C, 16 h, 91%; e) 3-
ꢀ
hydroxyphenylboronic acid, Pd(PPh
then 80 C for 3 h, 99%; f) 6 M NaOH, 30% H
toluene, DMAP, THF, 0 Ce25 C, 30 min; h) amine 7a, dry THF, reflux, 12 h, 30% (over 2
3
)
4
, Na
2
CO
3
/H
2
ꢀ
2
O
2
2
ꢀ
ꢀ
The synthesis of derivative 5h is described in Scheme 5. The
steps).

6
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
2
.2. Structureeactivity relationship and molecular modeling
although still in the nM range, the inhibition potency of 5b
(K
¼ 450 nM) was 26 fold lower than that of the parent compound
a (K
target the catalytic residues, and formed alternative contacts (H-
bonds, with M191, I238 and G239 located in front of the catalytic
triad). For the other analogues we obtained a binding mode similar
to that found for our previous potent FAAH inhibitors 3a-c [24,26].
Of note, compound 5c (Fig. 2B), the most potent inhibitor of the
series, almost doubled the FAAH inhibition potency of its previously
studies
i
5
i
¼ 26 nM). In line with the drop of efficacy, 5b was not able to
For the new compounds (5a¡j, Table 1) the potency of inhibi-
tion of FAAH was assessed by means of inhibition studies, per-
formed on the mouse brain enzyme, while affinity for D and D
receptors was assessed by competition binding experiments per-
formed on human recombinant receptors.
For gaining dopamine receptor affinity, the developed com-
pounds 5a-j were conceived by introducing a phenylpiperazine
moiety, spaced by a 2 or 3 methylene bridge from the bis-aryl
scaffold (phenyl linked to a 5-membered amido-heterocycle) which
bears the reactive center (carbamate or urea) needed for the
interaction with the FAAH enzyme. Inspired by the structures of our
previously developed FAAH inhibitors 3a-c [24] with a sub-
nanomolar potency (Fig. 1 and Table 1) we observed that in the
new series, the modifications introduced were well tolerated by the
FAAH enzyme and most of the compounds were found to be potent
FAAH inhibitors. The most interesting FAAH inhibitors were then
2
3
developed analogue 3b (5c K
i
¼ 0.23 nM vs 3b K ¼ 0.49 nM,
i
Table 1). This data perfectly matched with the number of relevant
contacts that 5c established within the binding pocket. The
carbamate formed a polar contact with S241, and an additional
contact with G239. Other relevant contacts were also established
by the methylfurancarboxamide moiety that, by its amidic portion
formed polar contacts with the backbones of S190, C269 and V270.
With this accommodation, the pendant phenyl ring of the phe-
nylpiperazine system could interact, by a triple
p-p stacking with
F192, F381 and F432. Several SARs where explored by changing the
nature of the 5-membered heterocycle. Modifying the furan into a
thiophene (5d, Fig. S1B) the tighter bond angle of the ring deter-
mined a different orientation of the molecule within the binding
site, this event projected the amide in a different position where it
could form only an H-bond with V270. The carbamate could
establish H-bonds with S241 and I238, while the phenyl stacked
with F192. By modifying the ring with differently decorated pyrrole
systems (with 1 or 2 methyl substituents) bound at different ring
positions (5a,e,f,h) we observed a loss in the established contacts
with a consequent loss of FAAH inhibition potency with respect to
5c. For 5a (Fig. 2A) the pyrrol-1-yl junction and the presence of the
amide moiety at 3-position, establishing polar contacts with C269
and V270, drove the specific accommodation of the molecule in the
binding cleft where the phenyl system could stack with F192. Thus,
the carbamate of 5a formed two H-bonds with S241 (side chain and
backbone), whereas the aromatic portion of the phenylpiperazine
system stacked with F381 and F432. Compounds 5e and 5f with a
pyrrol-2-yl junction (Figs. S1C and D) were able to enter in the
gorge of the FAAH in a similar fashion to that of 5c. They could
interact with the S241 (by carbamate moieties) and with C269 and
V270 (by the amide moieties), although establishing a lesser
number of contacts with respect to 5c. In these compounds the
methyl groups did not produce any relevant contact. For com-
pounds 5g and 5h (Figs. S1E and F), consistent with their lower
FAAH inhibition potency, we found docked poses belonging to the
most populated clusters that hampered the phenylpiperazine ac-
commodation into the hydrophobic sub-pocket. The introduction
of a triazole, as for 5g, or a 5-substituted pyrrol-2-yl portion (5h),
did not improve inhibition potency, due to the lack of additional
contacts besides that of the carbamate portion with S241. Notably,
this latter interaction was less relevant as the interacting groups
laid at a higher distance potentially precluding the nucleophilic
attack. This observation is supported by the decrease of the
inhibitory potency found for these compounds that were the
weakest inhibitors of the carbamate series. The homologated ana-
logues 5i and 5j (Figs. 2C and D) displayed superimposable binding
modes to that of 5a and 5c. As for the other analogues, also for 5i
and 5j the carbamate drove the accommodation into the catalytic
binding site although a slightly major distance between the
carbamate and the catalytic Ser (S241) was observed. While com-
pound 5j (Fig. 2D) retained the same accommodation of 5c, for 5i
we found a different accommodation of the 3-amidopyrrol-1-yl
portion (compared to 5a) that interacts with Q273 (Fig. 2C) due
to the elongation of the aliphatic chain. Notably this residue was
also targeted by our previously developed highly active compound
3a as observed by us [26].
2 3
selected for being tested against D and D receptor subtypes. In
general, all the selected and tested compounds (5a,c,d,h,i,j, Table 1)
displayed in vitro affinities for the dopamine receptors in the two or
three digit nM concentration range.
In order to gain information about the possible binding modes
of the tested compounds into the humanized variant of rat FAAH
2 3
protein (h/rFAAH) binding site and in human D and D receptors,
we performed a molecular docking calculation employing the
Induced Fit Docking (IFD) technique as previously reported [26,28].
The data were compared with those obtained for our lead 3a
[
24,26]. For each compound the physicochemical properties were
þ
calculated as well as their affinity for the hERG K channel.
The IFD protocol was able to confirm that the presence of an
effective electrophilic center (such as the carbamate) was a key
prerequisite for attaining high FAAH inhibition potency (Figs. 2AeD
and Figs. S1AeF of the Supplementary Material). This moiety was
indeed able, for most of the molecules of the set, to bind the cat-
alytic S241, one of the three residues forming the catalytic triad
(
K142, S217, S241) and also to target one or more residues
belonging to the oxyanion hole of the FAAH binding site (I238,
G239, G240, S241) maintaining a conserved H-bond network. The
modification of this moiety into an urea as in compound 5b
(Fig. S1A) negatively affected binding mode and potency. In fact,
Scheme 5. Synthesis of compound 5h. Reagents and conditions. a) NaN
3
, CuI, L-pro-
ꢀ
ꢀ
line, NaOH, H
2
O, 95 C, 16 h, 47%; b) methylpropiolate, CuI, DIPEA, dry THF, 25 C, 12 h,
ꢀ
99%; c) NaOH, EtOH, H O, 65 C, 16 h, 40%; d) N-methylmorpholine, isobutyl chlor-
2
ꢀ
ꢀ
oformate, conc. NH
dry DCM, ꢁ78 Ce25 C, 12 h, 45%; f) 20% COCl
4
OH, dry THF, ꢁ10 Ce25 C, 2 h, 99%; e) BBr
3
(1 M solution in DCM),
ꢀ
ꢀ
ꢀ ꢀ
2
in toluene, DMAP, THF, 0 Ce25 C,
30 min; g) amine 7a, dry THF, reflux, 12 h, 20% (over 2 steps).

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
7
Fig. 2. Binding modes of 5a (light pink sticks in panel A), 5c (magenta sticks in panel B), 5i (light blue sticks in panel C), 5j (light green sticks in panel D) into h/rFAAH enzyme (cyan
cartoon, PDB ID: 3PPM) binding site. The residues forming the catalytic triad (K142, S217 and S241) are reported in sticks while the other residues of the binding site are reported as
lines. The non-polar hydrogens were removed for clarity. The pictures were generated by means of PyMOL (The PyMOL Molecular Graphics System, v1.8.4.0, Schr o€ dinger LLC, New
York, 2015). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article).
Docking studies were also performed on the dopaminergic re-
ceptors. For improving the reliability of the in silico approach, we
considered an implicit membrane model during the IFD (see
experimental part for details). As reported in Fig. 3AeF, we
sidechains of Y37 and E95, while 5j with W100 (Fig. 3F). Notably
the mentioned binding modes with the key interactions in the TMs
are similar to those observed for the crystallized ligand (risperi-
done). All the discussed solutions showed a GlideScore < ꢁ10 kcal/
mol in agreement with the nanomolar affinity of the compounds
2
observed at the D receptor a similar binding mode for the tested
molecules. In particular, the phenylpiperazine portion of all the
for the D
2
receptor.
analyzed molecules (5a,c,d,h,i,j) was engaged in a series of in-
The studied compounds docked similarly also in the D receptor
3
teractions (p-p
stacking or hydrophobic contacts) with the hy-
binding site (Fig. 4AeF) and showed a pattern of interaction in
drophobic residues belonging to the seven trans-membrane (TMs)
which the phenylpiperazine system was involved in hydrophobic
helices (TM1-7), identified as reported in Uniprot (Uniprot ID:
interactions (p-p stacking or hydrophobic contacts) with the resi-
P14416 (DRD
2
_HUMAN) https://www.uniprot.org/uniprot/P14416)
dues deeply located in TM3, TM5 and TM6 (V111 in TM3, V189 in
TM5, F345, F346, H349, V350 in TM6). As already observed with the
(
V115 in TM3, F198 in TM5, F382 in TM6, W386 in TM7, F390 in
TM6). The carbamate portion of all the compounds could establish
H-bonds with the key residue D114 (TM3) by its NH group and, for
compounds 5a, 5c and 5i (Fig. 3A,B and E), polar contacts with Y408
2
D receptors, the NH group of the carbamate moiety could, for all
the compounds, establish a H-bond with the key residue D110 on
TM3, while the carbonyl group targeted different residues at TM7
such as Y365 (for 5a and 5j, Fig. 4A and F), T369 (for 5c and 5i,
Fig. 4B and E) and S182 in TM5 (for 5h, Fig. 4D). The phenyl system
of the bis-aryl moiety was engaged in hydrophobic interactions
with Y36 (TM1) and V86 (TM2). The carboxamide moiety of 5a
(
TM7) by their carbonyl group were observed. In general, the
phenyl rings of the studied bis-aryl scaffolds established hydro-
phobic contacts with W100 and Y408. The amide moieties sup-
ported on the heterocycles contacted different D receptor residues
2
lining the extracellular site. In particular, 5a (Fig. 3A) established H-
strongly interacted with residues located at the surface of the D
3
bonds with the sidechains of Y37 (on the surface of TM1) and T412
receptor (Fig. 4A). In particular, 5a formed an H-bond with the
sidechain of E90 (TM2). Compound 5c (Fig. 4B) stacked with Y365
by its furan ring. The same was verified for compound 5d (Fig. 4C),
with the addition of an H-bond with the sidechain of H349. Com-
pound 5h (Fig. 4D) interacted with the sidechain of T369.
(
TM7) and with the backbone of S409 (TM7). Compound 5c is able
to form an H-bond with the sidechain of E95 (TM2) (Fig. 3B) as well
as 5d (Fig. 3C). Compound 5h interacts with the backbone of G98
(
TM2) (Fig. 3D). 5i (Fig. 3E) interacts by H-bonds with the

8
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
Fig. 3. Binding modes of 5a (light pink sticks in panel A), 5c (magenta sticks in panel B), 5d (orange sticks in panel C), 5h (light magenta sticks in panel D), 5i (dark green sticks in
panel E) and 5j (cyan sticks in panel F) into D receptor binding site (grey cartoon, PDB ID: 6CM4). The residues forming the binding site are represented as lines. The non-polar
2
hydrogens were removed for clarity. The pictures were generated by means of PyMOL (The PyMOL Molecular Graphics System, v1.8.4.0, Schr o€ dinger LLC, New York, 2015). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article).
Compound 5i formed H-bonds with the sidechains of S366 (TM7)
and E90 (Fig. 4E), while 5j with the backbone of L89 (TM2) and the
sidechain of E90 (Fig. 4D). Also in this case the solutions showed a
GlideScore < ꢁ10 kcal/mol in agreement with their nM affinity for
cytotoxicity and mutagenicity profile (Table 3 and Fig. 6). We also
performed preliminary metabolic stability studies (Fig. 7 and
Table S2_ and S3).
the D
3
receptor.
2.3.1. Selectivity profile (CB R, CB R, MAGL)
1
2
The selectivity profile was experimentally measured for com-
pounds 5a and 5c towards the mouse cannabinoid receptors and
2.3. Drug-likeliness, selectivity and toxicity profile of selected
analogues
the hydrolytic ECS enzyme MAGL. The mCB
based on sequence alignments (source: uniprot), a high level of
identity with the human isoforms 97.0% for the CB R and 79.7% for
the CB R. As clearly depicted in Fig. 5, these studies allowed us to
exclude, for 5a and 5c, any interaction with either the cannabinoid
receptors or the MAGL enzyme.
1 2
R and mCB R share, as
In order to assess the drug-like profile of the developed com-
1
pounds we evaluated, for selected analogues, some key features
either in silico or by experimental methods. While physicochemical
properties were computationally determined for all the new com-
pounds 5a-j (Table S1 of the Supplementary Material), some key
features were experimentally determined for selected analogues
such as: i) selectivity profile towards proteins of the ECS (Fig. 5); ii)
solubility and chemical stability (by HPLC methods, Table 2), iii)
2
2.3.2. In silico prediction and experimental evaluation of selected
drug-like properties
Some molecular properties were calculated by means of

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
9
Fig. 4. Binding modes of 5a (light pink sticks, panel A), 5c (magenta sticks, panel B), 5d (orange sticks in panel C), 5h (light magenta sticks, panel D), 5i (dark green sticks, panel E)
and 5j (cyan sticks, panel F) into D receptor binding site (purple cartoon, PDB ID: 3PBL). The residues forming the binding site are represented as lines. The non-polar hydrogens
3
were removed for clarity. The pictures were generated by means of PyMOL (The PyMOL Molecular Graphics System, v1.8.4.0, Schr o€ dinger LLC, New York, 2015). (For interpretation of
the references to colour in this figure legend, the reader is referred to the Web version of this article).
QikProp and by our proprietary 3D-QSAR model (3D-chERGi) to
assess the affinity for hERG K channel.
24 h at pH ¼ 3.
þ
As a preliminary safety evaluation, we assessed in silico the
potential interaction of the newly developed compounds with the
The predicted molecular properties evidenced improved
aqueous solubility (cLogS) and cLogP, compared to reference com-
pound 3a. No violations of Lipinski's rules were found, and the
human oral adsorption was considered acceptable.
With these data in hands we decided to undertake in vitro as-
sessments of selected drug-like properties. The solubility (at pH ¼ 3
and pH ¼ 7.4, Table 2) and the chemical stability (at pH ¼ 3, Table 2)
of compound 5c were measured by means of HPLC methods [38].
As predicted, the presence of the basic phenylpiperazine system
improved the solubility profile of 5c over that of 3a. The same was
þ
hERG K channels using the recently developed 3D-QSAR model,
namely 3D-chERGi. It is well established that sustained interaction
with this membrane protein is responsible for a fatal cardiac
arrhythmia (torsade-de-pointes) and this kind of evaluation is
currently a key point in the early drug discovery trajectory. In
general, when tested in silico, the new compounds (Table S1) were
predicted by the 3D-chERGi to possess from moderate to low af-
finity for the hERG channel. Of note, the highest predicted affinities
were found for compounds 5d, 5f and 5i (Table S1).
also true for compound 5a (5a solubility at pH ¼ 3 was 574
m
M).
These results prompted us to experimentally determine the IC50
values for hERG from patch clamp studies (Table 1 last column). As a
whole, these studies were in good agreement with the predicted in
silico data and revealed that amongst the new compounds,
although in general more active than the parent analogues 3a,b, the
Gratifyingly, our analyses also revealed that both 3a and 5c, bearing
a carbamoyl functionality, exhibited a favorable chemical stability
profile at acidic pH. In particular, the chemical stability was almost
quantitative for compound 5c with 99.9% remaining unaltered after

10
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
1 2
Fig. 5. Selectivity of 5a and 5c towards mCB R (Panel A), mCB R (Panel B), and mMAGL (Panel C), data for JZL-184 a potent MAGL inhibitor is reported for comparison.
Table 2
Solubility and chemical stability of compounds 5c and 3a.
from 4 to 100 mM.
As a further profiling, we ascertained for 5a the lack of muta-
genic effect in the Salmonella typhimurium strains TA98 and TA100.
The Ames test is designed to identify potential risks of mutagenicity
at the early stages of drug development. The assay can be per-
formed in presence or not of the S9 fraction of rat liver. This latter
condition is a more in-depth investigation for assessing the po-
tential mutagenicity risks derived from the compound's metabo-
lites. After applying both experimental conditions, we did not
observe any mutagenic effect for compound 5a at all the concen-
Cmpd
Solubility (
m
M) after
Chemical Stability (%) after 24 h
12 h
pH ¼ 3
pH ¼ 7.4
n.c.a
2
pH ¼ 3
3
5
a
c
465
991
92.1
99.9
a
n.d. ¼ not calculable, below the quantitation limit.
trations tested (5e230
m
M) (Fig. 6AeD).
.4. Preliminary metabolic studies
.4.1. In silico and in vitro evaluation of potential site of metabolism
Table 3
Viability of mouse fibroblasts NIH3T3 and human glioblastoma astrocytoma cells
U373-MG after incubation with 5a, 5c and 3a (as IC50
mM).
2
2
Cmpds
NIH3T3a
U373MGa
IC50
(m
M)
IC50 (mM)
of 3a and 5c
In order to further characterize the most promising analogue 5c,
3
5
5
a
a
c
75
5
68
50
NT
23
we computationally investigated the potential sites of metabolism
and 5c in comparison with those of 3a. It is well established that
cytochrome P450 enzymes are crucial in the metabolism of drugs.
For this purpose, we employed P450 site of metabolism (P450 SOM)
application implemented in Schr o€ dinger suite 2018. As previously
a
Cell viability was measured by the Neutral Red Uptake (NRU) test and data
normalized as % control; data are expressed as mean ± SD of three experiment
repeated in six replicates; values are statistically different vs control, p ꢂ 0.05.
demonstrated by us [39,40], this application is very useful in pre-
dicting potential soft spots when aiming at understanding the
liable sites of the molecules.
compounds 5a, 5h and 5i were the best performing of the new
series (exhibiting IC50 values of 5.3, 6.4 and 3.5 mM respectively,
Table 1). On the other hand, 5c and 5d, in line with our in silico
predictions, exhibited submicromolar IC50 values.
We predicted metabolism mediated by isoforms CYP3A4,
CYP2C9, and CYP2D6 of compounds 3a and 5c. The prediction for
isoforms CYP2C9 and CYP2D6 combined IFD (for determining the
compounds’ accessibility to the CYP reactive center) with a rule-
based approach to intrinsic reactivity. In the case of CYP3A4,
which has a highly flexible binding site, only intrinsic reactivity was
used to predict soft spots. Moreover, the MetaSite 6.0.1 prediction
of the metabolites in liver was also performed for the same
compounds.
Based on the calculations (the outputs are reported in Table S2),
we can speculate that the metabolism of 3a and 5c is mainly
mediated by the isoforms CYP3A4 and CYP2C9. For both com-
pounds the para positions on the terminal phenyl rings could
represent the main soft spots, and compound 5c appeared to be
more susceptible to the CYP-mediated metabolism, with respect to
compound 3a, since for 5c extra potential soft spots were found on
the piperazine system. The MetaSite 6.0.1 prediction of the me-
tabolites in liver overlapped the previously described results and it
also evidenced that for compound 3a the site of metabolism was
2.3.2.1. Cytotoxicity and mutagenicity profile evaluation for 3a, 5a,
and 5c. Besides the physico-chemical parameters of the com-
pounds, we experimentally determined additional features that
might contribute to designate the most promising compounds 5a
and 5c as potential hits of the series. To this aim we determined
their potential cytotoxicity profile after incubation with mouse
embryonic fibroblasts (NIH3T3 cell line), and with human glio-
blastoma astrocytoma cells (U373MG cell line). The viabilities of
these cells, after incubation with 5a and 5c, are reported in Table 3
and are expressed as IC50 (mM). The values are compared with those
obtained for the FAAH inhibitor 3a.
We observed that 5a and 5c showed toxicities only in the mM
range. In particular, 5a was found more toxic than 3a on the NIH3T3
cell line, while 5c exhibited a two-digit M toxicity on both cell
m
lines, being as low as that 3a for NIH3T3 cells. All these tests were
performed with concentrations of the tested compounds ranging

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
11
Fig. 6. Ames test performed on S. typhimurium TA98 and TA100 strains for compounds 5a.
Fig. 7. CYP-dependent metabolic depletion of 5
mM 3a (panel A) and 5c (panel B) in human (HLM) and rat liver (RLM) microsomal preparations. Results are presented graphically as
percentage of compound recovery (100% at time 0 min) as a function of incubation time. Data are presented as mean ± SEM, of three different experiments.
preferentially located on the C atom in
a
-position with respect to
apparent decay constants (k) half-life time (t½) and intrinsic
clearance (CLint) are reported in Table 4. Compound 5c presented
very similar kinetic parameters in both preparations. On the con-
trary, compound 3a was slowly metabolized by RLM. In fact, the
resulted t½ were 50.26 min in RLM and 27.77 in HLM, respectively.
However, the incubation of both compounds in HLM gave rise to a
comparable behavior and the intrinsic clearances were quite
the terminal benzene group and in the C atom close to the carba-
moyl N in the aliphatic chain. For compound 5c the most probable
metabolites could derive from hydroxylation on the terminal aro-
matic moieties and/or in the furan system.
To this in silico approach we combined in vitro studies assessing
the metabolic stability of 3a and 5c in human and rat liver micro-
somal preparations (HLM and RLM). The plot of non-metabolized
compound [natural logarithm of % of compound recovery (100%
at time 0 min)] as a function of incubation time showed mono-
exponential decay relationship for both substrates (Fig. 7). The
similar (41.6 and 47.4
respectively).
mL/min/mg protein for 3a and 5c,
These results clearly indicate that the two compounds should be
considered moderate substrates of CYP450 system as resulted from

12
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
their intrinsic clearance value [41]. Interestingly the introduction of
a phenylpiperazine moiety in the molecule, needed for modulating
the pharmacological profile and improving the water solubility, did
not modify the metabolic stability in human liver. The results
suggest that both compounds could possess favorable pharmaco-
kinetic properties, at least regarding their interaction with phase I
enzyme system.
A preliminary qualitative HPLC/ESI-MS analysis for identifying
potential metabolites was performed on the medium obtained after
incubation of the compounds 3a and 5c (with incubation time of
inflammatory potential of compounds 3a and 5c in the IMR 32 cells,
we performed a cytotoxicity assay to exclude any toxic effect on the
same cell line. Accordingly, IMR 32 cells were treated with com-
pounds 3a and 5c in a range of concentrations between 0.1 and
50 mM. As depicted in Fig. 8, both compounds did not affect
significantly the release of LDH from IMR 32 cells at any of the
tested concentrations, thus allowing us to exclude any cytotoxic
effect for the tested compounds. Based on these results we per-
formed the next experiments aiming at evaluating the potential
anti-inflammatory properties at the doses of 0.5 mM and 1 mM.
0
and 30 min at 50
m
M). The data reported in Table S3, show that
To test the anti-inflammatory properties of compounds 3a and
5c, IMR 32 cells were first pre-treated with the compounds of in-
some amount of the tested compounds remains after 30 min of
incubation. Few metabolites were identified that flanked the
presence of the parent compounds and namely: i) the phenol de-
rivative, originated following N-dealkylation and decarboxylation
process, ii) a mono-hydroxylated metabolite, and iii) a phenyl-
piperazine chain was evident for compound 5c. For both analyzed
compounds the mono-hydroxylated analogue was present only in
the medium that was incubated 30 min with the HLM.
terest at the doses of 0.5
treatment, IMR 32 cells were challenged with 100
30 min and the activation of NF- B was evaluated by NF-
binding. As shown in Fig. 9, LPS increased the binding activity of
nuclear extracts to the NF- B-DNA consensus sequence. Pre-
treatment with the compounds 3a and 5c for 24 h significantly
inhibited LPS-inducible NF- B p65 DNA binding in IMR 32 cells as
m
M and 1
m
M for 24 h. After the pre-
g/mL LPS for
B-DNA
m
k
k
k
k
also confirmed by immunoblotting analysis (data not shown).
These experiments allowed us to ascertain a dose dependent in-
hibition of NF-kB p65 DNA binding for both compounds, which
showed a similar anti-inflammatory effect.
2
.5. Evaluation of anti-inflammatory potential for compound 3a
and 5c
Though some treatments are available for nicotine dependence,
there are high rates of relapse. Nicotine dependence has been
frequently associated to stress [42] and several evidences show that
the rewarding properties of nicotine are increased under stress
conditions and that the stress factors can alter neurotransmitter
systems including dopaminergic and serotoninergic systems.
Notably, the mechanisms underlying nicotine dependence and
major depressive disorders appear to involve common alterations
in neurotransmitter pathways [42]. A great body of evidences point
out that nicotine induces oxidative stress (OS) and the production
of reactive oxygen species not only in the periphery but also in CNS
structures involved in emotional and cognitive processes, such as
the prefrontal cortex and hippocampus [43]. In humans, OS
markers have been associated with both depression and smoking
and have been linked to nicotine dependence [43]. The nicotine
pro-oxidative effects have also been demonstrated in animals
in vivo (in liver, kidneys, heart, and in the CNS) [44], where after
acute and subchronic nicotine treatment it induced OS and
inflammation within brain structures responsible for emotional
and cognitive processes [45]. On these bases it was also envisaged
that anti-inflammatory drugs could serve as efficient and novel
treatment for nicotine dependence.
3. Conclusions
We have herein reported the identification and SAR analysis of a
new class of phenyl heterocyclic compounds bearing an arylpi-
perazine moiety behaving as multipotent tools (FAAH inhibitors
and D
terization on the targets of interest, selectivity profile towards
proteins of the ECS (CB R, CB R and MAGL), hERG channel liability
2 3
/D receptor ligands, compounds 5a-j). Biological charac-
1
2
evaluation, together with the in silico assessment of the drug-
likeliness profile allowed the selection of the best performing an-
alogues. The selected analogues, 5a and 5c, in comparison with our
lead FAAH inhibitor 3a, were further assessed in vitro in living cells.
These studies exclude toxic effects on mouse fibroblasts NIH3T3
and human glioblastoma astrocytoma cells U373-MG. Gratifyingly,
as based on the results of the cellular studies in the conditions of
the Ames tests, we could assume the lack of mutagenicity as well.
Preliminary metabolic studies were performed on compounds 3a
and 5c after incubation with RLM and HLM. In line with the in silico
predicted metabolic liabilities, the compounds behaved as moder-
ate substrates of CYP450 system (as indicated by their intrinsic
CLint values). Notably, the introduction of a phenylpiperazine
moiety in the molecule, needed for modulating the pharmacolog-
ical profile and improving the water solubility, did not negatively
affect the metabolic stability in human liver, thus suggesting that 5c
Cigarette smoking induces an excess of pro-inflammatory cy-
tokines, pro-inflammatory gene transcription, and OS [46]. Expo-
sure to OS from cigarette smoke also impacts the NF-
kB pathway
[
47], and activates inflammatory immune response contributing to
additional endogenous oxidants formation [48]. The above con-
siderations point out that OS and inflammation are relevant issues
in most human diseases and are also pivotal in drug and nicotine
addictions [46]. Therefore, we interrogated the ability of one of the
most promising compounds of series (5c) to modulate the
inflammation process and we compared its effect with that induced
by our reference compound 3a. Prior to the evaluation of the anti-
could possess
a favorable pharmacokinetic profile, at least
regarding its interaction with phase I enzyme system. To finally
complement the profile of the compounds 3a and 5c we assessed
their anti-inflammatory properties on IMR 32 cells, where we could
demonstrate their ability to reduce the LPS-induced activation of
the redox-sensitive transcription factor NF-kB.
Table 4
Kinetic parameters and metabolic stability of compounds 3a and 5c.
Cmpd
RLM
HLM
k (min 1
ꢁ
)
CL
(
mL/min/mg prot.)
t1/2 (min)
k (min
ꢁ1
)
CL
t1/2 (min)
int
int
(mL/min/mg prot.)
3
5
a
c
0.01379
0.03482
23.45
59.22
50.26
19.91
0.02496
0.02842
41.60
47.36
27.77
24.39

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
13
Fig. 8. Cytotoxicity determined as LDH release in IMR 32 cells treated for 24 h with compounds 3a and 5c at different concentrations (0.1, 0.5, 1, 5, 10 and 50
m
M). Values represent
average ± SD of samples performed at least in triplicate. Data are expressed as percentage of LDH release as compared to the maximum release of LDH from Triton X-100- treated
cells (100%).
Fig. 9. NF-
kB-DNA-Binding levels in nuclear extracts from IMR 32 cells pre-treated with compounds 3a and 5c for 24 h and challenged with 100 mg/mL LPS for 30 min. Data are
presented as mean ± SEM of 3 independent experiments. *p < 0.05 vs LPS treatment; **p < 0.01 vs LPS treatment.
4
4
4
. Experimental part
(q) and broad (br); the values of chemical shifts (d) are given in ppm
and coupling constants (J) in Hertz (Hz). HPLC were performed with
a Shimadzu Prominence apparatus equipped with a scanning
absorbance UV-VIS detector (Diode Array SPD-M20A) also equip-
ped with a thermostatic chamber and with Agilent 1100 Series
equipped with UV-VIS detector. ESI-MS spectra were performed by
an Agilent 1100 Series LC/MSD spectrometer. HRESIMS were car-
ried out by a Thermo Finningan LCQ Deca XP Max ion-trap mass
spectrometer equipped with Xcalibur software, operated in posi-
tive ion mode. The yields are referred to purified products and are
not optimized. All moisture-sensitive reactions were performed
under argon atmosphere using oven-dried glassware and anhy-
drous solvents. Final compounds were analyzed by combustion
analysis (CHN) to confirm purity >95%.
.1. Chemistry
.1.1. General procedures
Unless otherwise specified, materials were purchased from
commercial suppliers and used without further purification. Re-
action progress was monitored by TLC using silica gel 60 F254
(
(
0.040e0.063 mm) with detection by UV. Silica gel 60
0.040e0.063 mm) was used for column chromatography. H NMR
1
13
and C NMR spectra were recorded on a Varian 300 MHz spec-
trometer or a Bruker 400 MHz spectrometer by using the residual
signal of the deuterated solvent as internal standard. Splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet

14
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
4
.1.2. 2-(4-Phenylpiperazine-1-yl)ethanamine (7a)
-(4-Phenylpiperazine-1-yl)acetonitrile. To a solution of phenyl-
piperazine 6 (1.27 mL, 8.33 mmol) in EtOH (40.0 mL), Na CO
1324.0 mg, 12.49 mmol) and bromoacetonitrile (580.0 L,
chromatography (100% EtOAc to EtOAc/MeOH 95: 5) afforded the
1
2
title compound as an amorphous white solid (20% yield). H NMR
(300 MHz, CDCl
2
3
3
)
d
7.64 (s, 1H, pyrrol-CH), 7.42 (t, J ¼ 8.7 Hz, 1H,
(
8
m
ArH), 7.30e7.23 (m, 4H, ArH, pyrrol-CH), 7.11 (dd, J ¼ 2.1 Hz, 8.7 Hz,
1H, ArH), 7.03e7.02 (m, 1H, ArH), 6.94 (d, J ¼ 8.4 Hz, 2H, ArH), 6.87
(t, J ¼ 7.2 Hz, 1H, ArH), 6.54-6-52 (m, 1H, pyrrol-CH) 5.71e5.55 (br s,
.33 mmol) were sequentially added. The reaction mixture was
ꢀ
stirred under N
2
atmosphere for 2 h at 25 C. The inorganic salt
precipitate was filtered and washed with EtOH. The filtrate was
concentrated, and the residue taken up with H O. The aqueous
3H, CONH
2
, OCONH), 3.46e3.41 (m, 2H, CH
2
eNH), 3.25e3.22 (m,
piperazine,
3
) d 166.7, 154.4, 152.2, 151.3, 140.8,
2
4H, ArN-(CH
2
)
2
piperazine), 2.69e2.61 (m, 6H, N-(CH
). C NMR (75 MHz, CDCl
2 2
)
13
phase was extracted with EtOAc (3 ꢃ 10 mL). The combined organic
layers were washed with a saturated solution of NaCl, dried over
anhydrous sodium sulphate, filtered and concentrated. The crude
was purified by column chromatography on silica gel (petroleum
ether/EtOAc 4:1). 2-(4-Phenylpiperazine-1-yl)-acetonitrile was
CH
2
130.6, 129.4, 122.9, 120.9, 120.2 (2), 117.7, 116.4, 114.8, 109.8, 57.0,
þ
þ
53.1, 49.2, 37.8. ESI-MS m/z: 434 [MþH] . HRMS-ESI m/z: [MþH]
calcd for C24
28 5 3 27 5 3
H N O 433,2114; found 434,2175. Anal. (C24H N O )
C, H, N.
1
obtained as an amorphous orange solid (70% yield). H NMR
(
3
2
400 MHz, CDCl
.58 (s, 2H, CNeCH
3
)
d
7.30e7.24 (m, 2H, ArH), 6.95e6.86 (m, 3H, ArH),
piperazine),
piperazine). ESI-MS m/z: 189
(3.28 g,
4.1.6. 3-(3-Carbamoyl-1H-pyrrol-1-yl)phenyl (3-(4-
phenylpiperazin-1-yl)propyl)carbamate (5i)
2
), 3.25 (t, J ¼ 4.8 Hz, 4H, ArN-(CH
2
)
2
.77 (t, J ¼ 5.1 Hz, 4H, N-(CH
2
)
2
Compound 5i was prepared following the procedure employed
þ
[
1
MþNa] .
2-(4-Phenylpiperazine-1-yl)-acetonitrile
for the preparation of compound 5a, starting from phenol 9 and
1
6.33 mmol) was dissolved in dry THF (65.3 mL) and the solution
amine 7b. H NMR (300 MHz, CDCl
3
)
d7.62 (pyrrol-CH s, 1H), 7.43 (t,
thus obtained was added dropwise to a suspension of LiAlH
4
J ¼ 8.7 Hz, 1H, ArH), 7.18e7.28 (m, 4H, ArH, pyrrol-CH), 6.98e7.09
ꢀ
(
1.24 g, 32.65 mmol) in dry THF (49.0 mL) at 0 C. The mixture was
(m, 2H, ArH), 6.92 (d, J ¼ 8.4 Hz, 2H, ArH), 6.88 (m, 1H), 6.62 (br s,
ꢀ
then warmed to 25 C and the reaction was stirred under N
2
at-
1H, OCONH), 6.54-6-52 (m, 1H, pyrrol-CH)5.61 (br s, 2H, CONH
2
),
piper-
eN),1.81 (m, 2H,
166.5, 154.3, 152.3, 151.3,
140.8, 130.5, 129.4, 122.9, 120.9, 120.2, 120.1, 117.6, 116.3, 114.9,
109.8, 57.6, 53.5, 49.4, 41.6, 25.5. Anal. (C25 ) C, H, N.
mosphere for 2 h. The reaction was quenched by adding water
3.43e3.38 (m, 2H, CH
2
eNH), 3.23e3.19 (m, 4H, ArN-(CH
piperazine, CH
chain). C NMR (75 MHz, CDCl
2 2
)
ꢀ
dropwise at 0 C. Subsequently, 15% NaOH was added until pH ¼ 9.
azine), 2.65e2.57 (m, 6H, N-(CH
2
)
2
2
13
The resulting precipitate was filtered by means of a Celite pad and
the filtrate washed with EtOAc and then concentrated. The residue
was purified by means of column chromatography on silica gel
CH
2
3
) d
29 5 3
H N O
(
DCM/MeOH, 7:3) to afford the title compound 7a as a white
1
amorphous solid (50% yield). H NMR (400 MHz, CDCl
J ¼ 6.9 Hz, 2H, ArH), 6.92 (d, J ¼ 8.1 Hz, 2H, ArH), 6.85 (t, J ¼ 7.2 Hz,
H, ArH), 3.21e3.18 (m, 4H, ArN-(CH piperazine), 2.82 (t,
J ¼ 6.0 Hz, 2H, CH ), 2.63e2.60 (m, 4H, N-(CH piperazine), 2.48 (t,
J ¼ 5.7 Hz, 2H, CH
3
)
d
7.26 (t,
4.1.7. 1-(3-Aminophenyl)-1H-pyrrole-3-carboxamide (11)
1-(3-Nitrophenyl)-1H-pyrrole-3-carbonitrile was prepared start-
1
2
)
2
ing from 10 and 3-nitroaniline according to a described procedure
1
2
2
)
2
[24] (92% yield, brown oil). H NMR (300 MHz, CDCl
3
)
d
7.41 (s, 1H,
þ
2
). ESI-MS m/z: 206 [MþH] .
pyrrol-CH), 7.18 (t, J ¼ 8.1 Hz, 1H, ArH), 6.96 (t, J ¼ 2.5 Hz, 1H, ArH),
6
.72e6.59 (m, 3H, ArH, pyrrol-CH), 6.54e6.50 (m, 1H, pyrrol-CH).
þ
4
.1.3. 3-(4-Phenylpiperazin-1-yl)propan-1-amine (7b)
Compound 7b was prepared starting from phenylpiperazine and
bromopropionitrile following the same procedure described for 7a.
ESI-MS m/z: 236 [MþNa] .
For the synthesis of 1-(3-aminophenyl)-1H-pyrrole-3-carbonitrile
the previously obtained 1-(3-nitrophenyl)-1H-pyrrole-3-carbonitrile
(153.0 mg, 0.72 mmol) was dissolved in EtOH (10.0 mL) and tin(II)
chloride dihydrate (810.0 mg, 3.59 mmol) was added. The reaction
1
H NMR (400 MHz, CDCl
J ¼ 8.4 Hz, 2H, ArH), 6.86 (t, J ¼ 7.2 Hz, 1H, ArH), 3.19 (t, J ¼ 5.2 Hz,
H, ArN-(CH piper-
azine), 2.65 (t, J ¼ 5.2 Hz, 2H, CH
CH
eN), 1.83e1.76 (q, J ¼ 6.4 Hz, 2H, CH
3
) d 7.27e7.24 (m, 2H, ArH), 6.92e6.90 (d,
ꢀ
4
2
)
2
piperazine), 2.96 (t, J ¼ 6.0 Hz 4H, N-(CH
2
)
2
mixture was refluxed for 2 h. After cooling to 25 C, the mixture was
2
eNH
2
), 2.56 (t, J ¼ 6.4 Hz, 2H,
3
poured in an ice bath, and 10% aqueous NaHCO was added up to
2
2
chain).
pH ¼ 8. After filtration through a Celite pad and EtOH evaporation
under vacuum, the aqueous layer was extracted with DCM
(3 ꢃ 10 mL). The combined organic layers were washed with a
saturated solution of NaCl, dried over anhydrous sodium sulphate,
filtered and concentrated. The obtained 1-(3-aminophenyl)-1H-
4
.1.4. N-(2-(4-Phenylpiperazin-1-yl)ethyl)-1H-imidazole-1-
carboxamide (8)
2
Amine 7a (100.0 mg, 0.49 mmol) was dissolved in H O (35.0 mL)
ꢀ
ꢀ
at 25 C. The solution was cooled to 0 C and CDI (158.0 mg,
0
magnetic stirring at 0 C, until a white precipitate was observed.
After filtration under vacuum, the precipitate was washed with cold
water, affording pure compound as an amorphous white solid (90%
yield). H NMR (300 MHz, CDCl
pyrrole-3-carbonitrile was used in the next step without any further
1
.98 mmol) was added. The resulting mixture was kept under
purification (95% yield, yellow oil). H NMR (300 MHz, CDCl
3
)
d
7.41
ꢀ
(s, 1H, pyrrol-CH), 7.18 (t, J ¼ 8.1 Hz, 1H, ArH), 6.96 (d, J ¼ 2.5 Hz, 1H,
ArH), 6.70e6.60 (m, 3H, ArH, pyrrol-CH), 6.54e6.49 (m, 1H, pyrrol-
þ
CH), 3.96 (br s, 2H, Ar-NH
2
). ESI-MS m/z: 206 [MþNa] .
1
3
)
d
8.11 (s, 1H, imidazole-CH),
To a solution of 1-(3-aminophenyl)-1H-pyrrole-3-carbonitrile
(65.0 mg, 0.35 mmol) in EtOH (22.0 mL), 6 M NaOH (867 L) and
30% H (867 L) were added. The reaction mixture was refluxed
for 3 h. After cooling to 25 C, a saturated solution of Na
(2.0 mL) was added and the solvent was evaporated under reduced
pressure. The residue was taken up with H O and extracted with
7.32e7.25 (m, 3H, ArH), 7.09 (s, 1H, imidazole-CH), 6.94.6.86 (m,
m
3
CH
6
H, ArH, imidazole-CH), 6.55 (br s, 1H, NHCO), 3.59e3.53 (m, 2H,
2
O
2
m
ꢀ
2
eNH), 3.23e3.20 (m, 4H, ArN-(CH
2
)
2
piperazine), 2.70e2.67 (m,
2 2 3
S O
þ
H, N-(CH , CH
2
)
2
2
piperazine). ESI-MS m/z: 322 [MþNa] .
2
4
.1.5. 3-(3-Carbamoyl-1H-pyrrol-1-yl)phenyl (2-(4-
EtOAc (3 ꢃ 5 mL). The combined organic layers were washed with a
saturated solution of NaCl, dried over anhydrous sodium sulphate,
filtered and concentrated. The crude 1-(3-aminophenyl)-1H-pyr-
role-3-carboxamide 11 was used in the next step without any
phenylpiperazin-1-yl)ethyl)carbamate (5a)
To a solution of 9 (50.0 mg, 0.25 mmol) and DMAP (121.0 mg,
ꢀ
0
2
.99 mmol) in dry THF (18.0 mL) at 0 C, phosgene (20% in toluene,
ꢀ
1
43.0
mL, 0.46 mmol) was added. After warming up to 25 C, the
further purification as yellow solid (90% yield). H NMR (300 MHz,
reaction was stirred for 30 min under N
101.5 mg, 0.49 mmol) was then added and the mixture was
refluxed for further 12 h. Evaporation and silica gel column
2
atmosphere. Amine 7a
CDCl
3
)
d
7.69e7.53 (m, 1H, pyrrol-CH), 7.20 (t, J ¼ 8.0 Hz, 1H, ArH),
(
7.03e6.94 (m, 1H, pyrrol-CH), 6.82e6.73 (m, 1H, ArH), 6.68 (d,
J ¼ 2.1 Hz, 1H, ArH), 6.65e6.58 (m, 1H, ArH), 6.56e6.47 (m, 1H,

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
15
pyrrol-CH), 5.53 (br s, 2H, CONH
z: 224 [MþNa] .
2
), 3.82 (br s, 2H, ArNH
2
). ESI-MS m/
ArH), 5.74 (br s, 1H, OCONH), 5.53 (br, 2H, CONH
2H, CH eNH), 3.23e3.20 (m, 4H, AreN(CH
.67e2.60 (m, 6H, N-(CH piperazine), 2.48 (s, 3H, CH
(75 MHz, CD Cl ) 165.6, 154.4, 151.7, 151.5, 141.7, 139.7, 134.5, 132.7,
129.8, 129.2, 127.6, 126.4, 123.0, 122.1, 119.6, 116.1, 56.9, 53.13, 49.3
2
), 3.40e3.38 (m,
þ
2
2
)
2
piperazine),
3
); C NMR
1
3
2
2
)
2
4.1.8. 1-(3-(3-(2-(4-Phenylpiperazin-1-yl)ethyl)ureido)phenyl)-1H-
2
2
pyrrole-3-carboxamide (5b)
þ
þ
Compound 11 (25.0 mg, 0.12 mmol) was dissolved in dry THF
(2), 37.9, 29.9, 15.0. ESI-MS m/z: 465 [MþH] , 487 [MþNa] , 503
ꢀ
þ
(
10.0 mL). Then TEA (67
mL, 0.50 mmol) and, after cooling to 0 C,
[MþK] . Anal. (C25
28 4 3
H N O S) C, H, N.
phosgene (20% in toluene, 130
m
L, 0.25 mmol) were added. The
ꢀ
reaction mixture was stirred at 25 C for 1 h. Amine 7a was then
added and the reaction mixture was stirred for further 12 h at the
same temperature. Evaporation followed by silica gel column
chromatography (DCM/MeOH 9:1) afforded the pure title com-
pound as colourless oil (20% yield). H NMR (400 MHz, CDCl
4.1.12. Ethyl 2-(3-methoxyphenyl)-1,5-dimethyl-1H-pyrrole-3-
carboxylate (15)
In a sealed tube, to a mixture of 13 (300.0 mg, 1.08 mmol) and a
catalytic amount of sulphamic acid, methylamine (33% solution in
1
ꢀ
3
)
d
7.81
EtOH, 15.0 mL) was added at ꢁ6 C. The reaction mixture was
ꢀ
(
(
br s, 1H, ArNHCO), 7.60 (d, J ¼ 29.1 Hz, 1H, pyrrol-CH), 7.39e7.20
m, 5H, ArH, pyrrol-CH), 7.07e6.80 (m, 5H, ArH), 6.48 (s, 1H, pyrrol-
), 3.49e3.47 (m, 2H,
warmed to 25 C and stirred for 2 h. EtOH was removed and the
residue was taken up with EtOAc and washed with a saturated
CH), 6.16 (br s, 1H, NHCO), 5.87 (br s, 2H, CONH
2
aqueous NaHCO
phases were dried over anhydrous Na
3
and then with 2 N HCl. The combined organic
SO , filtered and concen-
CH
2
eNH), 3.24 (br s, 4H, ArN-(CH
2
)
2
piperazine), 2.81e2.65 (m, 6H,
2
4
þ
N-(CH
2
)
N
2
piperazine, CH
) C, H, N.
2
eN). ESI-MS m/z: 433 [MþH] . Anal.
trated. Silica gel column chromatography (n-hexane/EtOAc 9:1)
afforded the title compound as an amorphous white solid (50%
(
C
H
24 28
6
O
2
1
yield). H NMR (300 MHz, CDCl
3
)
d
7.33 (t, J ¼ 7.2 Hz, 1H, ArH), 6.92
4.1.9. 3-(3-Carbamoyl-5-methylfuran-2-yl)phenyl (2-(4-
(t, J ¼ 6.9 Hz, 2H, ArH), 6.87 (s, 1H, ArH), 6.41 (s, 1H, pyrrol-CH), 4.09
-ethyl), 3.81 (s, 3H, OCH ), 3.31 (s, 3H, -
), 2.26 (s, 3H, CeCH
phenylpiperazin-1-yl)ethyl)carbamate (5c)
(q, J ¼ 6.9 Hz, 2H, CH
NeCH
2
3
Compound 12a (50.0 mg, 0.23 mmol) was dissolved in dry THF
3
3
), 1.12 (t, J ¼ 7.5 Hz, 3H, CH
3
-ethyl). ESI-
þ
þ
þ
þ
(
18.6 mL) and DMAP (112.6 mg, 0.92 mmol) was added. After
MS m/z: 274 [MþH] , 296 [MþNa] , 312 [MþK] , 569 [2 M þ Na] .
ꢀ
cooling to 0 C, phosgene (20% solution in toluene, 243
mL,
0
.46 mmol) was added. The reaction mixture was allowed to warm
4.1.13. 2-(3-Methoxyphenyl)-1,5-dimethyl-1H-pyrrole-3-carboxylic
acid (16)
ꢀ
to 25 C and then stirred for 30 min under N
2
atmosphere. Amine
7
a (94.5 mg, 0.46 mmol) was then added and the reaction mixture
Compound 15 (347.0 mg, 1.27 mmol) was dissolved in EtOH
(30 mL) and water (10 mL) and NaOH (1.80 g, 44.49 mmol) was
added. The reaction mixture was stirred for 8 h at 25 C. After
was refluxed for 12 h. The solvent was removed and the crude was
purified by column chromatography on silica gel (form 100% EtOAc
ꢀ
to EtOAc/MeOH 95:5) to obtain 5c as an amorphous white solid
removal of the organic solvent, the aqueous residue was acidified to
pH ¼ 1 by means of 6 M HCl. The aqueous layer was then extracted
with EtOAc (3 ꢃ 10 mL). The combined organic phases were washed
with a saturated solution of NaCl, dried over sodium sulphate,
filtered and concentrated to obtain acid 16 as a yellow solid. Title
compound was obtained enough pure to be used in the next re-
1
(
30% yield). H NMR (300 MHz, CDCl
3
)
d
7.67e7.62 (m, 2H, ArH),
7
8
.41 (t, J ¼ 8.1 Hz, 1 H, ArH), 7.30e7.23 (m, 2H, ArH), 7.14 (dd, J ¼ 1.5,
.1 Hz, 1H, ArH), 6.94 (d, J ¼ 7.8 Hz, 2H, ArH), 6,86 (t, J ¼ 7.2 Hz, 1H,
), 5.49 (br s,
eNH), 3.24e3.21 (m, 4H,
ArH), 6.33 (s, 1H, furan-CH), 5.76e5.65 (br s, 2H, CONH
H, OCONH), 3.45e3.41 (m, 2H, CH
AreN(CH piperazine), 2.68e2.60 (m, 6H N-(CH
CH eN), 2.34 (s, 3H, CH
2
1
2
1
2
)
2
2
)
)
d
2
piperazine,
165.8, 154.6,
action without any further purification (99% yield). H NMR
13
2
3
). C NMR (75 MHz, CDCl
3
(300 MHz, CD
3
OD)
d
7.31 (t, J ¼ 7.8 Hz, 1H, ArH), 6.96e6.92 (m, 1H,
152.2, 151.5, 151.4 (2), 131.3, 129.8, 129.4, 124.7, 122.3, 121.3, 120.1,
ArH), 6.87e6.85 (m, 2H, ArH), 6.33 (s, 1H, pyrrol-H), 3.80 (s, 3H,
1
18.1, 116.3, 108.4, 57.0, 53.2, 49.4, 37.9, 13.6. ESI-MS m/z: 471
OCH
[MþNa] .
3
), 3.30 (s, 3H,-N-CH
3
), 2.25 (s, 3H, CeCH
3
). ESI-MS m/z: 268
þ
þ
[
MþNa] . Anal. (C25
H
28
N
4
O
4
) C, H, N.
4
.1.10. 3-(3-Carbamoyl-5-methylfuran-2-yl)phenyl (3-(4-
4.1.14. 2-(3-Methoxyphenyl)-1,5-dimethyl-1H-pyrrole-3-
carboxamide (17)
phenylpiperazin-1-yl)propyl)carbamate (5j)
The title compound was prepared according to the procedure
described for 5c, starting from phenol 12a and amine 7b. The
product was obtained as an amorphous white solid. (32% yield). H
To a solution of compound 16 (200.0 mg, 0.82 mmol) in dry
DCM, TEA (227
m
L, 1.63 mmol), EDC (234.73 mg, 1.22 mmol) HOBt
1
ꢀ
(154.4 mg, 1.14 mmol) were sequentially added at 0 C. The reaction
mixture was stirred under N
ꢀ
NMR (300 MHz, CDCl
H, ArH), 7.30e7.21 (m, 2H, ArH), 7.12 (m, 1H, ArH), 6.95 (d,
J ¼ 7.8 Hz, 2H, ArH), 6.88 (m, 1H, ArH), 6.50 (br s, 1H, OCONH), 6.32
s, 1H, furan-CH), 5.76 (br s, 1H, CONH ), 5.51 (br s, 1H, CONH ),
.45e3.38 (m, 2H, CH eNH), 3.26e3.18 (m, 4H, AreN(CH
piperazine), 2.69e2.53 (m, 6H, N(CH piperazine), 2.32 (s, 3H,
chain). C NMR (75 MHz, CDCl 165.8,
54.7, 152.1, 151.5, 151.4 (2), 131.2, 129.8, 129.4, 124.6, 122.4, 121.4,
20.1, 118.1, 116.3, 108.5, 57.5, 53.5, 49.4, 31.2, 25.6, 13.6. Anal.
) C, H, N.
3
)
d
7.66e7.52 (m, 2H, ArH), 7.39 (t, J ¼ 8.1 Hz,
2
atmosphere for 10 min at 0 C. Then
1
2.81 mL of conc. NH
30 min at 0 C and for 16 h at 25 C. Evaporation followed by silica
gel column chromatography (petroleum ether/EtOAc 2:3) afforded
4
OH was added and the mixture was stirred for
ꢀ
ꢀ
(
2
2
1
3
2
2
)
2
the title compound as a light yellow solid (73% yield). H NMR
2
)
2
(300 MHz, CDCl
6.47e6.43 (m, 1H, ArH), 5.06 (br s, 2H, CONH
3
)
d
7.40e7.38 (m, 1H, ArH), 7.01e6.91 (m, 3H, ArH),
), 3.83 (s, 3H, OCH ),
13
CH
3
), 1.65 (m, 2H, CH
2
3
)
d
2
3
þ
1
1
3.25 (s, 3H, NeCH
3
), 2.25 (s, 3H, CeCH
3
). ESI-MS m/z: 245 [MþH] ,
þ
þ
þ
267 [MþNa] , 283 [MþK] , 511 [2 M þ Na] .
26 30 4 4
(C H N O
4
.1.15. 2-(3-Methoxyphenyl)-5-methyl-1H-pyrrole-3-carboxamide
(19)
Compound 19 was prepared according to the procedure used for
15 starting from 13 (300.0 mg, 1.08 mmol), a conc. solution of
NH OH (18.0 mL) and a catalytic amount of sulphamic acid, main-
taining the reaction under stirring for 5 h. Mixture was neutralized
by 2 N HCl. Aqueous layer was extracted with EtOAc (3 ꢃ 10 mL).
The combined organic phases were washed with brine, dried over
4
.1.11. 3-(3-Carbamoyl-5-methylthiophen-2-yl)phenyl (2-(4-
phenylpiperazin-1-yl)ethyl)carbamate (5d)
The title compound was prepared according to the procedure
described for 5c, starting from phenol 12b and amine 7a. The
product was obtained as an amorphous white solid. (34% yield). H
NMR (300 MHz, CD
4
1
2 2
Cl ) d 7.45e7.17 (m, 6H, ArH), 7.07 (s, 1H,
tiophen-CH), 6.93 (d, J ¼ 7.8 Hz, 2H, ArH), 6.84 (t, J ¼ 7.2 Hz, 1H,

16
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
sodium sulphate, filtered and concentrated. Crude was purified by
124.7, 122.4, 120.1, 118.6, 116.3, 115.8, 108.2, 56.9, 53.1, 49.3, 37.8,
þ
þ
means of flash chromatography (petroleum ether/EtOAc 5:1)
31.7, 12.6. ESI-MS m/z: 462 [MþH] , 484 [MþNa] . Anal.
1
affording the title compound as a pale-yellow oil (20% yield).
NMR (300 MHz, CDCl
H
(C26H N O ) C, H, N.
31 5 3
3
)
d
8.93 (br s, 1H, pyrrol-NH), 7.27e7.21 (m,
1
H, ArH), 7.05 (s, 2H, ArH), 6.82 (d, J ¼ 8.7 Hz, 1H, ArH), 6.22 (s, 1H,
4.1.20. 2-(5,5-Dimethyl-1,3-dioxan-2-yl)-1-methyl-1H-pyrrole (22)
1-Methyl-1H-pyrrole-3-carbaldehyde 21 (931 L, 9.16 mmol)
was dissolved in toluene (56.0 mL). p-Toluenesulfonic acid (17.4 mg,
.09 mmol) and 2,2-dimethylpropane-1,3-diol (1.91 g, 18.33 mmol)
were sequentially added. The reaction mixture was refluxed in a
Dean-Stark apparatus to remove H O from the reaction. The reac-
tion mixture was refluxed for 24 h. A saturated solution of NaHCO
pyrrol-CH), 5.59 (br s, 2H, CONH
CH
2
), 3.75 (s, 3H, OCH
3
), 2.20 (s, 3H,
m
þ
þ
3
). ESI-MS m/z: 231 [MþH] , 253 [MþNa] .
0
4
(
.1.16. 2-(3-Hydroxyphenyl)-5-methyl-1H-pyrrole-3-carboxamide
20a)
To a suspension of 19 (141.0 mg, 0.61 mmol) in dry DCM
2
3
(
1
10.0 mL), boron tribromide (1 M solution in DCM, 1.8 mL,
was then added to stop the reaction, and toluene was removed
under reduced pressure. The aqueous phase was extracted with
EtOAc (3 ꢃ 10 mL). The combined organic layers were washed with
a saturated solution of NaCl, dried over anhydrous sodium sulphate,
filtered and concentrated. Crude was purified first by column
chromatography on silica gel (petroleum ether/EtOAc 10:1), then by
ꢀ
.82 mmol) was added at ꢁ78 C. The reaction mixture was then
ꢀ
allowed to warm to 25 C and stirred for 96 h. Mixture was treated
with 4 N NaOH and extracted with DCM (3 ꢃ 15 mL). The aqueous
layers were then acidified to pH ¼ 2 with 6 M HCl and extracted
with EtOAc. The combined organic extracts were dried over anhy-
drous sodium sulphate, filtered and concentrated to afford the pure
ꢀ
distillation at 110 C to eliminate the residual aldehyde. Product 22
1
1
product as an amorphous yellow solid (85% yield). H NMR
was obtained as a colorless oil (99%, yield). H NMR (300 MHz,
(
300 MHz, CD
3
OD)
d
7.22e7.14 (m, 1H, ArH), 6.97e6.95 (m, 2H,
3
CDCl ) d 6.56e6.55 (m, 1H, pyrrol-CH), 6.22e6.20 (m, 1H, pyrrol-
ArH), 6.72 (dd, J ¼ 2.7, 6.6 Hz, 1H, ArH), 6.18 (s, 1H, pyrrol-CH), 2.21
CH), 6.04e6.01 (m, 1H, pyrrol-CH), 5.42 (s, 1H, CH), 3.77 (s, 3H,
þ
(
s, 3H, CH
3
). ESI-MS m/z: 239 [MþNa] .
NeCH
3
), 3.74 (s, 2H, CH
2
), 3.62 (s, 2H, CH
2
), 1.30 (s, 3H, CH
3
), 0.79 (s,
þ
þ
3H, CH
3
). ESI-MS m/z: 391 [2 M þ H] , 413 [2 M þ Na] , 429
þ
4.1.17. 2-(3-Hydroxyphenyl)-1,5-dimethyl-1H-pyrrole-3-
[2 M þ K] .
carboxamide (20b)
Title compound was prepared according to the procedure used
for the preparation of 20a starting from 17 (145.0 mg, 0.59 mmol)
4.1.21. 2-Bromo-5-(5,5-dimethyl-1,3-dioxan-2-yl)-1-methyl-1H-
pyrrole (23)
and BBr
was obtained as an amorphous pink solid (99% yield). H NMR
300 MHz, CD OD)
7.31 (t, J ¼ 6.9 Hz, 1H, ArH), 6.89e6.87 (m, 1H,
3
(1 M solution in DCM, 1.7 mL, 1.76 mmol). Compound 20b
Compound 22 (954.0 mg, 4.89 mmol) was dissolved in dry THF
1
ꢀ
(12.0 mL); the solution was cooled to ꢁ20 C and NBS (827.2 mg,
(
3
d
4.64 mmol) was added. The reaction mixture was stirred for 16 h,
ꢀ
ArH), 6.80 (d, J ¼ 7.5 Hz, 1H, ArH), 6.76e6.75 (m, 1H, ArH), 6.31 (s,
under N
2
atmosphere at ꢁ20 C. Evaporation and silica gel column
1
H, pyrrol-CH), 3.25 (s, 3H, NeCH
3
), 2.25 (s, 3H, CeCH
3
). ESI-MS m/
chromatography (petroleum ether/EtOAc 4:1) afforded the title
compound as crystalline white solid (81% yield).
M.p. ¼107e108 C. H NMR (300 MHz, CDCl 6.17 (d, J ¼ 4.0 Hz,
þ
þ
þ
z: 231 [MþH] , 253 [MþNa] , 483 [2 M þ Na] .
a
ꢀ
1
3
) d
4.1.18. 3-(3-Carbamoyl-1,5-dimethyl-1H-pyrrol-2-yl)phenyl (2-(4-
1H, pyrrol-CH), 6.04 (d, J ¼ 4.0 Hz, 1H, pyrrol-CH), 5.31 (s, 1H, CH),
phenylpiperazin-1-yl)ethyl)carbamate (5e)
3.69 (s, 3H, NeCH
CH
3
), 3.67 (s, 4H, CH
2
), 1.27 (s, 3H, CH
3
), 0.79 (s, 3H,
þ
þ
Compound 20a (20.0 mg, 0.07 mmol) was dissolved in H
2
O
3
). ESI-MS m/z: 275 [MþH] , 297 [MþNa] .
(
10.0 mL) and then carbonylimidazolide 8 (15.4 mg, 0.07 mmol)
ꢀ
was added. Mixture was stirred at 25 C for 5 h. The aqueous layer
was extracted with EtOAc (3 ꢃ 10 mL). The combined organic
phases were washeda saturated solution of NaCl, dried over sodium
sulphate, filtered and concentrated. Crude was then triturated with
4.1.22. 5-Bromo-1-methyl-1H-pyrrole-2-carbaldehyde (24)
Compound 23 (2.17 g, 7.90 mmol) was dissolved in acetone
(7.9 mL) and then 1 M HCl (15.8 mL) was added. The reaction
ꢀ
mixture was refluxed for 3 h. After cooling to 25 C, acetone was
Et
2
O to eliminate the 1H-imidazole released by the reaction. Silica
removed and the aqueous residue was extracted with diethyl ether
(3 ꢃ 10 mL). The combined organic layers were washed with a
saturated solution of NaCl, dried over anhydrous sodium sulphate,
filtered and concentrated. Silica gel column chromatography (pe-
gel column chromatography (EtOAc 100% to EtOAc/MeOH 20:1)
afforded compound 5e as an amorphous white solid (20% yield). H
1
NMR (300 MHz, CDCl
ArH), 7.10e7.08 (m, 1H, ArH), 6.93 (d, J ¼ 7.8 Hz, 2H, ArH), 6.86 (t,
), 5.82 (br s, 1H, OCONH),
), 3.47e3.40 (m, 2H, CH eNH), 3.24e3.21 (m,
piperazine), 2.68e2.60 (m, 6H, N-(CH pipera-
167.3, 154,8, 151.4
2), 133.5, 131.5, 129.9, 129.4 (2), 128.6, 125.8, 122.3, 121.5, 120.1,
16.3 (2), 116.0, 109.0, 57.0, 53.2 (2), 49.3 (2), 37.8, 12.9. ESI-MS m/z:
3
) d 8.38 (br s, 1H, pyrrol-H), 7.38e7.25 (m, 5H,
troleum ether/EtOAc 10: 1) afforded the title compound as an
1
J ¼ 7.8 Hz, 1H, ArH), 6.28 (s, 1H, pyrrol-CH
2
amorphous white solid (83% yield). H NMR (300 MHz, CDCl
3
)
5
4
.51 (br s, 2H, CONH
H, AreN(CH
2
2
d
9.35 (s, 1H, CHO), 6.88 (d, J ¼ 4.2 Hz, 1H, pyrrol-CH), 6.32 (d,
2
)
2
2
)
2
J ¼ 4.2 Hz, 1H, pyrrol-CH), 3.96 (s, 3H, NeCH
3
). ESI-MS m/z: 189
13
þ
þ
zine), 2.25 (s, 3H, CH
3
). C NMR (75 MHz, CDCl
3
)
d
[MþH] , 211 [MþNa] .
(
1
4.1.23. 5-Bromo-1-methyl-1H-pyrrole-2-carbonitrile (25)
þ
470 [MþNa] . Anal. (C25
29
H N
5
O
3
) C, H, N.
To a solution of compound 24 (613.0 mg, 3.28 mmol) in THF
(
1.0 mL) and conc. NH
4
OH (5.2 mL), iodine (1.66 g, 6.56 mmol) was
ꢀ
4.1.19. 3-(3-Carbamoyl-5-methyl-1H-pyrrol-2-yl)phenyl (2-(4-
added. The reaction mixture was stirred for 16 h at 25 C. A satu-
phenylpiperazin-1-yl)ethyl)carbamate (5f)
2 2 3
rated solution of Na S O was added to quench the iodine excess
Title compound was obtained according to the procedure fol-
and the aqueous layer was extracted with DCM (3 ꢃ 10 mL). The
combined organic layers were washed with a saturated solution of
NaCl, dried over anhydrous sodium sulphate, filtered and concen-
trated. The crude was purified by means of flash chromatography
1
lowed for 5e (20% yield H NMR (300 MHz, CDCl
3
)
d
7.47 (t,
J ¼ 7.8 Hz, 1H, ArH), 7.30e7.19 (m, 5H, ArH), 6.95e6.92 (m, 2H, ArH),
.87 (t, J ¼ 7.2 Hz, 1H, ArH), 6.44 (s, 1H, pyrrol-CH), 5.69 (br s, 1H,
OCONH), 5.10 (br, 2H, CONH eNH), 3.28
), 3.42 (t, J ¼ 5.9 Hz, 2H, CH
), 3.24e3.21 (m, 4H, ArN(CH piperazine, 2.69e2.61
piperazine), 2.25 (s, 3H, CeCH
166.7, 154.5, 151.6, 149.2, 133.4, 130.1, 129.7, 129.4, 127.9 (2),
6
2
2
(petroleum ether/EtOAc 10: 1) to afford the title compound as
1
(
(
s, 3H, NeCH
m, 6H, N(CH
3
2
)
2
yellow oil (91% yield). H NMR (300 MHz, CDCl
3
)
d
6.76 (d,
13
2
)
2
3
). C NMR (75 MHz,
J ¼ 4.0 Hz, 1H, pyrrol-CH), 6.22 (d, J ¼ 4.3 Hz, 1H, pyrrol-CH), 3.73 (s,
þ
CDCl
3
)
d
3H, NeCH
3
). ESI-MS m/z: 186 [MþH] .

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
17
þ
þ
þ
4
.1.24. 5-(3-Hydroxyphenyl)-1-methyl-1H-pyrrole-2-carbonitrile
OCH
3
). ESI-MS m/z: 137 [MþH] , 160 [MþNa] , 176 [MþK] .
(26)
A
mixture of compound 25 (100.0 mg, 0.54 mmol) and
4.1.28. Methyl 1-(3-methoxyphenyl)-1H-1,2,3-triazole-4-
Pd(PPh
under N
359.8 mg, 3.39 mmol) in H
3
)
4
(31.2 mg, 0.03 mmol) in toluene (3.5 mL), was stirred
carboxylate (30)
ꢀ
2
atmosphere for 10 min at 25 C. Then a solution of Na
2
CO
3
Compound 29 (20.0 mg, 0.13 mmol) and methyl propiolate
(11.3 mg, 0.13 mmol) were dissolved in dry THF (2.0 mL). DIPEA
(
2
O (1.75 mL) and a solution of 3-
hydroxyphenylboronic acid (74.5 mg, 0:54 mmol) in EtOH (1.6 mL)
were sequentially added. The resulting mixture was refluxed under
N atmosphere for 3 h. Volatiles were removed and the residue was
2
taken up with water and extracted with DCM (3 ꢃ 10 mL). The
combined organic layers were washed with a saturated solution of
NaCl, dried over anhydrous sodium sulphate, filtered and concen-
(117
m
L, 0.67 mmol) and CuI (5.1 mg, 0.03 mmol) were sequentially
ꢀ
added. The reaction mixture was stirred at 25 C for 12 h. Evapo-
ration and silica gel column chromatography (petroleum ether/
EtOAc 6:1) afforded 30 as an amorphous white solid (99% yield). H
NMR (300 MHz, CDCl
1H, ArH), 7.34e7.33 (m, 1H, ArH), 7.28e7.25 (m, 1H, ArH), 7.00 (dd,
1
3
)
d
8.50 (s, 1H, triazol-CH), 7.42 (t, J ¼ 8.4 Hz,
trated. Silica gel column chromatography (petroleum ether/EtOAc
J ¼ 2.4, 8.4 Hz,1H, OH), 3.98 (s, 3H, OCH
3
), 3.87 (s, 3H, COOCH
MS m/z: 234 [MþH] , 256 [MþNa] , 489 [2 M þ Na] .
3
). ESI-
1
þ
þ
þ
5
:1) afforded the title compound as an amorphous white solid. H
3
NMR (300 MHz, CDCl ) d 7.33e7.26 (m, 1H, ArH), 6.93e6.89 (m, 3H,
ArH), 6.85 (d, J ¼ 3.6 Hz, 1H, ArH), 6.22e6.20 (m, 2H, pyrrol-CH),
4.1.29. 1-(3-Methoxyphenyl)-1H-1,2,3-triazole-4-carboxylic acid
(31)
-
þ
3
.73 (s, 3H, NeCH
3
). ESI-MS m/z: 197 [M-H] , 199 [MþH] , 221
þ
[
MþNa] .
Compound 30 (30.0 mg, 0.13 mmol) was dissolved in EtOH
2
(2.4 mL) and H O (0.8 mL), and NaOH (129.7 mg, 3.24 mmol) was
4
.1.25. 5-(3-Hydroxyphenyl)-1-methyl-1H-pyrrole-2-carboxamide
added. Mixture was refluxed for 16 h. After evaporation of EtOH, the
residue was taken up with water, acidified with 2 N HCl until pH ¼ 1
and extracted with EtOAc (3 ꢃ 10 mL). The combined organic pha-
(
27)
Title compound was prepared according to the procedure
described for 11, starting from compound 26 (100.0 mg,
.50 mmol), 6 M NaOH (1.7 mL) and 30% aqueous H (1.70 mL).
2 4
ses were dried over anhydrous Na SO , filtered and concentrated to
0
2
O
2
give the title compound as pure amorphous brown solid (40%
1
Crude was purified by flash chromatography on silica gel (petro-
3
yield). H NMR (300 MHz, CD OD) d 9.08 (s, 1H, triazol-CH),
leum ether/EtOAc 1: 2) to obtain pure compound 27 as an amor-
7.52e7.47 (m, 3H, ArH), 7.08 (d, J ¼ 7.5 Hz, 1H, ArH), 3.89 (s, 3H,
1
þ
phous pink solid (67% yield). H NMR (300 MHz, acetone-d
6
)
d
8.68
COOCH
3
). ESI-MS m/z: 220 [MþH] .
(
6
br, 1H, OH), 7.27 (t, J ¼ 7.8 Hz, 1H, ArH), 7.05e6.40 (m, 2H, Ar),
.93e6.88 (m, 3H, Ar), 6.86 (d, J ¼ 1.65, 1H, pyrrol-CH), 6.13 (d,
). ESI-MS m/z: 217
4.1.30. 1-(3-Hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (32)
J ¼ 3.9 Hz, 1H, pyrrol-CH), 3.88 (s, 3H, NeCH
3
1-(3-Methoxyphenyl)-1H-1,2,3-triazole-4-carboxamide.
Com-
þ
þ
þ
[
MþH] , 239 [MþNa] , 255 [MþK] .
pound 31 was dissolved in dry THF and the mixture cooled down
ꢀ
to ꢁ10 C. N-methylmorpholine (25
m
L, 0.23 mmol) and isobutyl
L, 0.23 mmol) were sequentially added. After
formation of a suspension, conc. NH OH (40 L, 0.34 mmol) was
atmosphere for
4.1.26. 3-(4-Carbamoyl-1H-1,2,3-triazol-1-yl)phenyl (2-(4-
chloroformate (30 m
phenylpiperazin-1-yl)ethyl)carbamate (5g)
Title compound was prepared according to the procedure
described for 5c starting from 27 (30.0 mg, 0.14 mmol), phosgene
4
m
added. The reaction mixture was stirred under N
2
ꢀ
ꢀ
30 min at ꢁ10 C and for an additional 1 h at 25 C. Solvent was
removed and the residue was taken up with water and extracted
with EtOAc (3 ꢃ 10 mL). The combined organic phases were dried
(
20% solution in toluene, 146
mL, 0.28 mmol), DMAP (67.2 mg,
0
.55 mmol) and amine 7a (56.8 mg, 0.28 mmol). The solvent was
removed and the crude was purified by means of column chro-
matography on silica gel (100% EtOAc). Title compound 5g was
2 4
over anhydrous Na SO , filtered and concentrated to afford 1-(3-
methoxyphenyl)-1H-1,2,3-triazole-4-carboxamide in quantitative
1
1
obtained as a colorless transparent oil (30% yield). H NMR
yield. H NMR (300 MHz, CDCl
J ¼ 8.1 Hz, 1H, ArH), 7.35e7.25 (m, 2H, ArH), 7.11 (br s, 1H, CONH
7.04e7.01 (m,1H, ArH), 5.71 (br s,1H, CONH ), 3.89 (s, 3H, COOCH
3
) d 8.52 (s, 1H, triazol-CH), 7.45 (t,
(
300 MHz, CDCl
3
)
d
7.41 (t, J ¼ 7.8 Hz, 1H, ArH), 7.30e7.25 (m, 3H,
2
),
).
ArH), 7.17 (t, J ¼ 9.6 Hz, 2H, ArH), 6.94 (d, J ¼ 7.8 Hz, 2H, ArH), 6.87 (t,
J ¼ 7.2 Hz, 1H, ArH), 6.66 (d, J ¼ 4.2 Hz, 1H, pyrrol-CH), 6.18 (d,
J ¼ 3.9 Hz, 1H, pyrrol-CH), 5.71 (br s, 1H, OCONH), 5.52 (br s, 2H,
2
3
þ
þ
ESI-MS m/z: 219 [MþH] , 241 [MþNa] .
Compound 32 was prepared according to the procedure used for
20a starting from 1-(3-methoxyphenyl)-1H-1,2,3-triazole-4-
CONH
2
), 3.89 (s, 3H, NeCH
3
), 3.47e3.41 (m, 2H, CH
2
eNH),
3
.25e3.23 (m, 4H, ArN-(CH
2
)
2
piperazine), 2.70e2.62 (m, 6H,
3
carboxamide (200.0 mg, 0.92 mmol) and BBr (1 M solution in
1
3
N(CH
2
)
2
piperazine, NeCH
2
). C NMR (75 MHz, CDCl
3
)
d
163.8,
DCM, 5.4 mL, 5.40 mmol). Title compound was obtained as an
amorphous white solid enough pure to be used in the next step
1
1
54.7,151.4,151.3,140.1,133.6,129.6,129.4,126.4,126.2,122.8,121.4,
20.2,116.4, 113.2, 109.0, 57.0, 53.2, 49.3, 37.8, 34.8. ESI-MS m/z: 448
1
without any further purification (45% yield). H NMR (300 MHz,
þ
þ
[
MþH] , 470 [MþNa] . Anal. (C25
29
H N
5
O
3
) C, H, N.
CD
3
OD)
d
8.82 (s, 1H, triazol-CH), 7.40e7.35 (m, 1H, ArH), 7.30e7.27
þ
(m, 2H, ArH), 6.94e6.90 (m, 1H, ArH). ESI-MS m/z: 205 [MþH] , 227
þ
4
.1.27. 1-Azido-3-methoxybenzene (29)
In a sealed tube, 1-bromo-3-methoxybenzene 28 (100.0 mg,
.53 mmol), NaN (69.5 mg, 1.07 mmol), CuI (10.2 mg, 0.05 mmol)
-proline (18.5 mg, 0.16 mmol) were sequentially dissolved in a
[MþNa] .
0
and
3
4.1.31. 3-(5-Carbamoyl-1-methyl-1H-pyrrol-2-yl)phenyl (2-(4-
phenylpiperazin-1-yl)ethyl)carbamate (5h)
L
mixture of EtOH/H
Mixture was then refluxed for 16 h. After cooling to 25 C, EtOH was
2
O ¼ 7/3 (740.0
m
L of EtOH and 320.0
m
L of H
2
O).
Compound 5h was synthesized following the same procedure
described for 5c starting from 32 (20.0 mg, 0.09 mmol), phosgene
ꢀ
evaporated and the aqueous layer was extracted with EtOAc
(20% solution in toluene, 100 mL, 0.10 mmol), DMAP (47.6 mg,
(
3 ꢃ 10 mL). The combined organic phases were dried over anhy-
0.39 mmol) and amine 7a (38.9 mg, 0.19 mmol). Crude was purified
by silica gel column chromatography (100% EtOAc to EtOAc/MeOH
drous Na SO , filtered and concentrated. Crude was purified by
2
4
means of flash chromatography (100% petroleum ether) to give
compound 29 as pale-yellow oil (47% yield). H NMR (300 MHz,
100:1) affording title compound as an amorphous white solid (20%
1
1
yield). H NMR (300 MHz, DMSO)
d
9.26 (s,1H, triazole-CH), 7.98 (br
CD
3
OD)
d
7.25 (t, J ¼ 8.4 Hz, 1H, ArH), 6.70 (d, J ¼ 8.4 Hz, 1H, ArH),
s, 1H, OCONH), 7.86e7.75 (m, 3H, ArH), 7.59 (t, J ¼ 8.4 Hz, 2H, ArH),
6
.62 (d, J ¼ 8.4 Hz, 1H, ArH), 6.55e6.53 (m, 1H, ArH), 3.76 (s, 3H,
7.26 (dd, J ¼ 3.0, 8.4 Hz, 1H, ArH), 7.18 (t, J ¼ 7.5 Hz, 2H, ArH), 6.91 (d,

18
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
J ¼ 8.1Hz, 2H, ArH), 6.75 (t, J ¼ 7.2 Hz, 1H, ArH), 3.35e3.22 (m, 2H,
CH eNH), 3.13e3.10 (m, 4H, ArN-(CH piperazine), 2.57e2.54 (m,
H, N-(CH ). C NMR (75 MHz, DMSO) 161.9,
59.1, 151.7, 149.0, 144.4, 138.0, 131.5, 129.6, 125.4, 119.4, 116.7, 116.0,
profiles for promiscuous P450 enzymes that are thought to be
mostly independent of structural restrictions on the binding poses.
The reactivity is predicted with a linear free energy approach based
on the Hammett and Taft scheme, where the reactivity of a given
atom is the sum of a baseline reactivity rate and a series of per-
turbations determined by the connectivity. The IFD approach is a
variation on the normal protocol. The initial sampling is enhanced
by generating multiple starting conformations, so that a wider
range of poses is found in the initial docking stage. The initial
docking includes van der Waals scaling of the receptor and alanine
mutation of the most flexible residues. In the Prime refinement
step, any residue with an atom within 5 Å of any ligand pose is
selected for side-chain prediction. The subsequent minimization
includes the ligand, side chains, and backbones of the flexible
residues. The ligand is then redocked into each of the low-energy
protein conformations, determined by a 40 kcal/mol cut-off. There
is no final scoring stage, because all poses are considered in
determining which atoms are sufficiently accessible to the reactive
heme iron. Any atom within the cut-off distance of 5 Å from the
heme iron is considered as a potential site of metabolism [55].
2
2 2
)
13
6
1
2
)
2
, pipeazine, NeCH
2
d
þ
1
11.5, 108.0, 58.6, 53.4, 48.8, 37.3. ESI-MS m/z: 436 [MþH] , 458
þ
[
MþNa] . Anal. (C22
25 7 3
H N O ) C, H, N.
4.2. Computational studies
4
4
.2.1. Docking studies
.2.1.1. Molecules preparation. Three-dimensional structures of all
compounds in this study were built by means of Maestro (Maestro,
version 10.3, Schr o€ dinger LLC, New York, NY, 2015) applying the
previously adopted protocol [26,28,49].
4
.2.1.2. Proteins preparation. The three-dimensional structures of:
FAAH enzyme (PDB ID: 3PPM [50]), dopamine D (PBD ID: 6CM4
51]) and D (PDB ID: 3PBL [52]) receptors were taken from the PDB
2
[
3
and imported into Schr o€ dinger Maestro molecular modeling envi-
ronment. Water molecules and compounds used for the crystalli-
zation were removed from the available experimental structures.
The obtained enzymes were submitted to protein preparation
wizard implemented in Maestro suite 2015 as previously reported
4
4
.3. Solubility and chemical stability studies
[
22,24,53].
.3.1. HPLC analysis of compounds 3a and 5c
For the HPLC analysis a Chromolith HPLC column RP-18 was
4
.2.1.3. Molecular docking. Molecular docking was carried out us-
employed. The runs were performed by a gradient elution starting
from a mixture 20% MeCN (0.1% TFA as phase modifier) in H O (0.1%
TFA as phase modifier) to 70% MeCN (0.1% TFA) in H O (0.1% TFA) in
5 min. The flow speed was settled at 1.0 mL/min and the tem-
ing the Schr o€ dinger suite 2015 by applying the IFD protocol as
previously reported [22,24,53]. This procedure induces conforma-
tional changes in the binding site to accommodate the ligand and
exhaustively identify possible binding modes and associated
conformational changes by side-chain sampling and backbone
minimization. The protein and the ligands used were prepared as
reported in the previous paragraphs. The boxes for docking calcu-
lation were built taking into account the centroid of the co-
crystallized ligands for all enzymes. Complexes within 30.0 kcal/
mol of minimum energy structure were taken forward for
redocking. The Glide redocking stage was performed by XP (Extra
Precision) methods. The calculations were performed using default
IFD protocol parameters. No hydrogen bonding or other constraints
were used. For dopamine receptors we selected the use of implicit
membrane in the Prime refinement step in order to obtain more
reliable results. The membranes were placed according to the res-
2
2
1
ꢀ
perature was maintained at 25 C. The volume of injection of the
sample was of 10 L and the wavelength selected for the detection
m
was 254 nM. The retention times obtained following this protocol
for compounds 3a and 5c were 12.8 min and 6.58 min, respectively.
ꢀ
4
.3.2. Solubility assay and chemical stability at 25 C
A stock solution for each tested compound was prepared dis-
solving the sample in DMSO to a final concentration of 10 mM.
From the stock solution, three samples were prepared: one was
used as the standard solution and the other two as the test solu-
tions at pH 3.0 and pH 7.4. The samples’ concentration of these
solutions was 250 mM with a DMSO content of 2.5% (v/v).
The standard solution was prepared by dilution of the stock
solution in PBS-buffer solution (MeCN/water, 60:40); the dilution
of the stock solution in 50 mM acetic acid afforded the samples'
solution at pH 3.0; and the dilution of the stock solution in 50 mM
aqueous PBS-buffer afforded the samples’ solution at pH 7.4. These
idues in the transmembrane domain of dopamine D
2
receptor
(
UniProt ID: P14416) and dopamine D receptor (UniProt ID:
3
P00720) as found in UniProtKB database. The selected IFD docking
protocol was able to correctly accommodate the co-crystallized li-
gands, belonging to the proteins used in this study (data not
shown).
ꢀ
suspension/solutions were sealed and left for 24 h at 25 C under
orbital shaking to achieve “pseudothermodynamic equilibrium”.
After that time the solutions were filtered using PTFE filters and
successively diluted 1:2 with the buffer solution used for the
preparation of the samples. Then they were analyzed by HPLC/UV/
DAD, using UV detection at 254 nm for quantitation. Solubility was
calculated by comparing areas of the sample and of the standard:
4
.2.2. Molecular properties prediction
The in silico drug-like features of the designed compounds were
evaluated by means of: i) QikProp implemented in Maestro suite
QikProp, version 4.3, Schr o€ dinger, LLC, New York, NY, 2015) for the
(
assessment of the compounds physico-chemical properties; ii) 3D-
chERGi, an in-house 3D-QSAR model for predicting hERG K
channel affinity) [54]. The outputs of these calculations are re-
ported in Table S1.
þ
Asmp ꢃ FD ꢃ Cst
S ¼
Ast
S ¼ solubility of the compound (
solution; FD ¼ dilution factor (2); Cst ¼ standard concentration
(250
For each sample the analysis was performed in triplicate and the
solubility result reported was obtained from the average of the
three values.
m
M); Asmp ¼ UV area of the sample
4
.2.3. P450 site of metabolism
Compounds 3a and 5c were evaluated for their potential sites of
m
M); Ast ¼ UV area of the standard solution.
metabolism by P450 site of metabolism (P450 SOM) workflow
implemented in Schr o€ dinger suite 2018. For each molecule P450
SOM performed a calculation of intrinsic reactivity coupled to IFD
for the selected isoform of cytochrome. The reactivity rules have
been parametrized in P450 SOM to predict atomic reactivity
The same sample solutions were prepared to evaluate the
ꢀ
chemical stability of the compounds after 24 h at 25 C and

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
19
analyzed by HPLC/UV/DAD, using UV detection at 254 nm for
quantitation. Stability was calculated by comparing the area of the
instructions provided within the BioRad reagent using a BSA
standard curve (Buffer 1 ¼ HEPES 20 mM, EDTA 2 mM pH ¼ 7.4, ice
peak at T
0
and the area of the peak of the same solution after 24 h. A
cold; Buffer 2 ¼ HEPES 20 mM pH ¼ 7.4, NaCl 100 mM, MgCl
2
stability percentage value was calculated by this method at pH 3.0
and pH 7.4 for each compound by applying the following formula:
10 mM, EDTA 1 mM, ice cold).
4.4.2.2.2. Competition binding experiments at hD
2
receptors.
Competition binding experiments were performed in a 96-deep-
well plate at room temperature (23 C) with a final assay volume of
Ac24
ꢀ
%remaining
¼
ꢃ 100
AcT0
1000 mL/well, according to the following protocol: 800 mL of binding
3
buffer were dispensed into each well of the compound plate. [ H]-
Spiperone stock was diluted in binding buffer solution to obtain the
ꢀ
Ac24 ¼ area of the sample after 24 h at 25 C; AcT0 ¼ area of the
sample at T
3
3
0
.
1
0 ꢃ [ H]-spiperone solution (0.8 nM). Then 100
spiperone solution were dispensed into each well of the compound
plate. The competition reaction was started by adding 100 L of
hD -G 16-CHO membrane suspension in binding buffer. The final
membrane concentration/well was 2
mL of 0.8 nM [ H]-
For each sample the analysis was performed in triplicate and the
stability result reported was obtained from the average of the three
values.
m
2
a
3
m
g, and the final [ H]-spiper-
4
.4. Biological data
one concentration was 0.08 nM. The plate was then incubated on a
ꢀ
shaker at 23 C for 120 min. The reaction was terminated by rapid
4
4
.4.1. Enzymatic assays
.4.1.1. FAAH activity assay. FAAH activity was assayed in mouse
brain incubated with 10
7 C for 15 min (pH 9.0). The reaction was stopped with a 2:1 (v/v)
mixture of chloroform/methanol, and the release of [ C]ethanol-
amine in the aqueous phase was measured as reported [56]. Control
experiments were also carried out in the presence of the selective
FAAH inhibitor 1a. The effect of different compounds on FAAH ac-
tivity was ascertained by adding each substance directly to the
incubation medium.
filtration through Unifilter-96 GF/B filter plates presoaked for at
least 1 h in polyethylenimine (PEI) 0.5% (w/v) solution and washed
with 1.0 mL of ice cold 0.9% NaCl before the filtration using a
Packard Cell Harvester. The filter plate was washed 4 times with
1.0 mL of ice-cold 0.9% NaCl and then left to dry for at least 1 h at
14
m
M [ C]AEA (ARC, St. Louis, MO, USA) at
ꢀ
3
14
ꢀ
40 C. The plate was sealed with a back-seal; 50 mL of Microscint-20
were added to each well, and the plate was sealed with a top-seal.
Bound radioactivity was measured using a Microplate Top-Count.
Radioligand concentration was determined as follows: 100
m
L of
[ H]-spiperone solution (5 ꢃ ) and 3 mL of Filter Count were mixed
in the total added vial and read in -Counter TriCarb 2900.
3
b
4
.4.1.2. MAGL activity assay. Mouse brain was thawed and ho-
4.4.2.2.3. Saturation binding experiments at hD2 receptors.
Saturation binding experiments were performed similarly to the
competition binding experiments, with the following deviation:
ꢀ
mogenized at 4 C in sodium phosphate buffer (50 mM, pH ¼ 8.0)
ꢀ
containing 0.32 M sucrose. Homogenates were centrifuged at 4 C
3
sequentially at 800ꢃg, 10,000ꢃg, and 100,000ꢃg. MAGL activity
[ H]-spiperone concentrations were chosen from 0.011 to 3.0 nM in
3
was assayed in final tissue supernatants, incubated with 10
m
M [ H]
a concentrationeresponse curve with 12 points. The reaction was
terminated by rapid filtration through GF/B paper filter presoaked
for 1 h in polyethylenimine (PEI) 0.5% (w/v) solution and washed
with 1.0 mL of ice-cold 0.9% NaCl before the filtration using a
Brandel harvester. The filter was washed 4 times with 1.0 mL of ice-
cold 0.9% NaCl. The filter was put into a pico-vial (PerkinElmer
600252), and 4 mL of Filter Count were added. Bound radioactivity
ꢀ
2
-OG (20 Ci/mmol; ARC, St. Louis, MO) at 37 C for 30 min [56]. The
reaction was stopped with a 2:1 (v/v) mixture of chloroform/
methanol and the release of [ H]glycerol in the aqueous phase was
measured by scintillation counting.
3
4
4
2
.4.2. Receptor binding assays
.4.2.1. CB R and CB R assays. Mouse brain was resuspended in
mM TriseEDTA, 320 mM sucrose, 5 mM MgCl (pH 7.4), then it
1
2
was measured using a b-Counter. Samples of working radioligand
2
solution were taken and measured by traditional liquid scintillation
counting in order to determine the actual concentration of radio-
label added.
was homogenized in a Potter homogenizer and centrifuged three
time at 1000ꢃg (10 min each), and the pellet was discharged. The
supernatant was centrifuged at 18000ꢃg (30 min), and the pellet
was resuspended in assay buffer (50 mM TriseHCl, 2 mM
4.4.2.2.4. Competition binding experiments at hD3 receptors.
Competition binding experiments were performed in a 96-deep
ꢀ
TriseEDTA, 3 mM MgCl
2
, pH 7.4) [56]. These membrane fractions
well plate at room temperature (23 C) with a final assay volume
3
were used in rapid filtration assays with radiolabel agonist [ H]
CP55,940 (PerkinElmer Life Sciences, Boston, MA, USA). In all ex-
periments, nonspecific binding was determined in the presence of
of 500 mL/well according to the following protocol: (a) 300 mL of
binding buffer were dispensed into each well of the compound
3
plate; (b) [ H]-spiperone stock was diluted in binding buffer solu-
3
1
m
M “cold” agonist, and the effect of selective compounds for CB
or CB R was tested by adding each substance directly to the incu-
bation medium.
1
R
tion to obtain the 5 ꢃ [ H]-spiperone solution (1.5 nM); (c) 100
mL of
3
2
1.5 nM [ H]-spiperone solution were dispensed into each well of
the compound plate; (d) the competition reaction was started by
adding 100
m
L of hDRD
3
-CHO membrane suspension in binding
g, and the
4
.4.2.2. Dopamine D
.4.2.2.1. CHO-hD
The frozen pellet of cells recombinantly expressing hD
16-CHO receptors was thawed and homogenized in 10 vol (w/v)
2
and D
and CHO-hD
3
receptor assays
membrane preparation.
and hD
buffer. The final membrane concentration/well was 3.5 m
3
4
3
2
final [ H]-spiperone concentration was 0.3 nM. The plate was then
ꢀ
3
2
-
incubated on a shaker at 23 C for 90 min. The reaction was
G
a
terminated by rapid filtration through Unifilter-96 GF/B filter plates
presoaked for 1 h in polyethylenimine (PEI) 0.5% (w/v) solution and
washed with 1.0 mL of ice-cold 0.9% NaCl before the filtration using
a Packard Cell Harvester. The filter plate was washed 4 times with
in Membrane Preparation Buffer 1 using an Ultraturrax (3 times for
ꢀ
10 s each cycle). The homogenate was centrifuged for 20 min, 4 C,
at 18500 rpm (40000 rcf) in a SL-50T Sorvall rotor and the pellet
resuspended in 10 vol w/v in Membrane Preparation Buffer 1 and
rehomogenized as before. After centrifugation, the pellet was
resuspended in 5 vol of Membrane Preparation Buffer 2. The
1.0 mL of ice-cold 0.9% NaCl and then left to dry for at least 1 h at
ꢀ
40 C. The plate was sealed with a back-seal; 50
m
L of Microscint-20
were added to each well, and the plate was sealed with a top-seal.
Bound radioactivity was measured using a Microplate TopCount.
Radioligand concentration was determined as follows: 100 mL of
ꢀ
resulting suspension was aliquoted and frozen down at ꢁ80 C.
Protein concentration was determined according to the

20
A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
[3
H]-spiperone solution (5 ꢃ ) and 3 mL of Filter Count were mixed
MG human glioblastoma astrocytoma were purchased from
American Type Culture Collection (USA). The mutagenicity assay
was supplied by Biologik s.r.l. (Trieste, Italy).
in the total added vial and read in
b
-Counter TriCarb 2900.
4
.4.2.2.5. Saturation binding experiments at hD receptors.
3
Saturation binding experiments were performed similarly to the
competition binding experiments, with the following deviation:
3
[
H]-spiperone concentrations were chosen from 0.015 to 4.0 nM in
4.5.2. Cell cultures and cytotoxicity assay
a concentration response curve with 12 points. The reaction was
terminated by rapid filtration through GF\B paper filter presoaked
for 1 h in polyethylenimine (PEI) 0.5% (w/v) solution and washed
with 1.0 mL of ice-cold 0.9% NaCl before the filtration using a
Brandel harvester. The filter was washed 4 times with 1.0 mL of ice-
cold 0.9% NaCl. The filter was put into a pico-vial (PerkinElmer
NIH3T3 and U-373-MG were utilised for cytotoxicity experi-
ments. Cells were maintained in DMEM at 37 C in a humidified
ꢀ
atmosphere containing 5% CO
mented with 10% fetal calf serum (FCS), 1%
2
. The culture media were supple-
-glutamine-penicillin-
L
streptomycin solution, and 1% MEM Non-Essential Amino Acid
Solution. Once at confluence, cells were washed with PBS 0.1 M,
taken up with trypsin-EDTA solution and then centrifuged at
1000 rpm for 5 min. The pellet was re-suspended in medium so-
lution (dilution 1:15). The stock solution for each compound was
prepared in pure DMSO and diluted with complete culture me-
dium. The solution/suspension obtained was then added to the cell
monolayer. Cell viability after 24 h of incubation with the different
concentrations of each test compound was evaluated by Neutral
Red Uptake by the procedure previously reported [57]. The data
processing included the Student's t-test with p < 0.05 taken as
significance level.
6
00252), and 4 mL of Filter count were added. Bound radioactivity
was measured using a -Counter. Samples of working radioligand
b
solution were taken and measured by traditional liquid scintillation
counting in order to determine the actual concentration of radio-
label added.
4.4.3. Measure of the effect on hERG channel by tail current
recording using in vitro rapid ICE
The potency of the compounds in inhibiting human ERG po-
tassium channel (hERG) tail current was assessed in a recombinant
HEK293 cell line stably transfected with hERG cDNA under an
inducible promoter, using Rapid ICE (rapid ion channel electro-
physiology) assay. Rapid ICE is an automated patch-clamp assay
utilizing the QPatch HTX system (Sophion Bioscience A/S). Briefly,
inducible HEK hERG cells were cultivated in minimum essential
medium supplemented with 10% FBS, 1% nonessential amino acids,
First, the following solutions were prepared in order to deter-
mine the percentage of viable cells:
1. Neutral Red (NR) Stock Solution: 0.33 g NR Dye powder in
100 mL sterile H O
2
2. NR Medium: 1.0 mL NR Stock solution þ99.0 Routine Culture
ꢀ
1
% sodium pyruvate, 2 mM l-glutamine, 15
mg/mL blasticidin, and
Medium pre-warmed to 37 C
1
00 g/mL hygromycin. hERG channel expression induction was
m
3. NR Desorb solution: 1% glacial acetic acid solution þ50%
obtained by adding 10
recordings.
mg/mL tetracycline for 24, 48, or 72 h before
ethanol þ49% H
2
O
On the day of the experiment, cells were detached with TrypLE
and prepared to be loaded on the instrument. Cells were resus-
pended in 7 mL of Serum-Free Media containing 25 mM Hepes and
soybean trypsin inhibitor and immediately placed in the cell stor-
age tank of the machine. The composition of the extracellular buffer
At the end of the incubation the routine culture medium was
removed from each well, and cells were carefully rinsed with 1 mL
of pre-warmed D-PBS. Multiwells were then gently blotted with
paper towels. 1.0 mL of NR Medium was added to each well and
ꢀ
further incubated at 37 C, 95% humidity, 5.0% CO
2
for 3 h. The cells
was (mM): NaCl 137, KCl 4, CaCl
hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) 10, pH
2
1.8, MgCl
2
1.0,
D
-glucose 10, N-2-
were checked during the NR incubation for NR crystal formation.
After incubation, the NR Medium was removed; cells were carefully
rinsed with 1 mL of pre-warmed D-PBS. Then, the PBS was dec-
anted and blotted from the wells and exactly 1 mL of NR Desorb
solution was added to each sample. Multiwells were then put on a
shaker for 20e45 min to extract NR from the cells and form a ho-
mogeneous solution. During this step the samples were covered in
order to protect them from light. After 5 min from the plate shaker
removal the absorbance was read at 540 nm by a UV/visible spec-
trophotometer (Lambda 25, PerkinElmer).
0
7
.4 with 1 M NaOH. The composition of the pipet solution was
mM): KCl 130, MgCl 1.0, ethylene glycol-bis( -aminoethyl ether)-
N,N,N ,N -tetraacetic acid (EGTA) 5, MgATP 5, HEPES 10, pH 7.2 with
M KOH. The voltage protocol included the following steps: step
(
2
b
0
0
1
from ꢁ80 to ꢁ50 mV for 200 m s, þ20 mV for 4.8 s, step to ꢁ50 mV
for 5 s, then step to the holding potential of ꢁ80 mV. Compounds
were dissolved in DMSO and diluted in extracellular buffer to
achieve final test concentrations (0.1, 1, and 10 mM) in 0.1% DMSO.
The voltage protocol was run and recorded continuously during the
experiment. The vehicle, corresponding to 0.1% DMSO in extracel-
lular buffer was then applied for 3 min, followed by the test sub-
stance in triplicate. The standard combined exposure time was
4.5.3. Mutagenicity assay: Ames test
The TA100 and TA98 strains of Salmonella Typhimurium and S9
fraction were utilised for mutagenicity assay. Approximately 10
7
5
min. The average of tail current amplitude values recorded from
four sequential voltage pulses was used to calculate for each cell the
effect of the test substance by calculating the residual current (%
control) compared with vehicle pretreatment. Data were reported
as % inhibition for each concentration tested, and IC50 values were
estimated using QPatch software. At least two cells were tested, and
even more if results diverged.
bacteria were exposed to 6 concentrations of each test compound,
as well as a positive and a negative control, for 90 min in medium
containing sufficient histidine to support approximately two cell
divisions. After 90 min, the exposure cultures were diluted in pH
indicator medium lacking histidine, and aliquoted into 48 wells of a
384-well plate. Within two days, cells which had undergone the
reversion to His grew into colonies. Metabolism by the bacterial
colonies reduced the pH of the medium, changing the colour of that
well. This colour change can be detected visually or by microplate
reader. The number of wells containing revertant colonies were
counted for each dose and compared to a zero dose control. Each
dose was tested in six replicates. The test was performed both with
and without S9 fraction.
4
4
.5. Cytotoxicity and mutagenicity assays
.5.1. Materials
Dulbecco's Modified Eagle's Medium, trypsin solution, and all
the solvents used for cell culture were purchased from Lonza
Switzerland). Mouse immortalized fibroblasts NIH3T3 and U-373-
(

A. Grillo et al. / European Journal of Medicinal Chemistry 183 (2019) 111674
21
4
.6. Analysis of in vitro metabolic stability of 3a and 5c in human
30 min. Then, cells were detached, washed with ice-cold PBS 1X
and cell pellets were resuspended in hypotonic buffer containing
and liver microsomes
2
10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl , 0.3%
The tested compound (3a or 5c), dissolved in MeCN, was incu-
Nonidet P-40, 0.5 mmol/L dithiothreitol, 0.5 mmol/L phenyl-
methylsulphonyl fluoride and protease and phosphatase inhibitor
cocktails. The lysates were incubated for 15 min on ice with inter-
ꢀ
bated at 37 C, at 5
mM concentration in 100 mM phosphate buffer
(
pH 7.4) with 0.3 mg/mL rat and human microsomal preparations
ꢀ
as previously reported [58]. Enzymatic reactions were started by
addition of a NADPH-regenerating system (2 mM NADPH), 66 mM
glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydroge-
mitted mixing and then centrifuged at 24500ꢃg for 15 min at 4 C,
as previously described [62]. The supernatant containing the
cytosolic proteins was removed and pellet containing the nuclei
were suspended in extraction buffer containing 20 mmol/L HEPES
2
nase in 66 mM MgCl ). Reactions were terminated at regular time
intervals (overall range 0e60 min) by adding a 1 mL of MeCN. All
incubations were performed in triplicate. HPLC analysis was per-
formed on Agilent 1100 Series liquid chromatography system
equipped with a EC 150/4.6 nucleosil 100-3 C18 (Macherey-Nagel)
2
(pH 7.9), 0,6 mol/L KCl, 1.5 mmol/L MgCl , 20% glycerol, 0.5 mmol/L
phenylmethylsulphonyl fluoride and protease and phosphatase
inhibitor cocktails and then incubated for 30 min on ice with
intermitted mixing. Samples were centrifuged at 21100ꢃg for
15 min to obtain supernatants containing nuclear fractions. Protein
concentration was determined by Bradford analysis (Biorad protein
assay; Biorad, Milan, Italy).
and coupling with UV-VIS detector, setting at
l 254 nm. Analysis
was carried out using gradient elution of a binary solution; eluent A
was MeCN (MeCN containing 0.1% formic acid), while eluent B
consisting of an aqueous solution of formic acid (0.1%). The analysis
started at 20% A for 3 min, then rapidly increased up to 90% in
4.7.4. NF-
NF- B DNA-binding capability was evaluated using “TransAM
NF- B” ELISA kit (Active Motif, USA). Nuclear protein extracts from
treated IMR 32 cells were incubated with NF- B consensus oligo-
nucleotides (5 -GGG ACT TTCC-3 ) immobilized on 96-well plate for
h at rt. A secondary antibody conjugated with a horseradish
kB nuclear translocation
1
5 min and finally remaining at 90% An until 25 min. The analysis
was performed at flow rate of 0.8 mL/min and injection volume was
L. The intrinsic clearance (Clint) was calculated by the
k
k
2
0
m
k
0
0
equation:
1
Clint ¼ k(min-1)x [V]/[P]
peroxidase provides a colorimetric output, spectrophotometrically
detected at 450 nm.
where k is the rate constant for the depletion of substrate, V is the
volume of incubation in
proteins as reported elsewhere [59].
mL and P is the amount of microsomal
4
.7.5. Western blot analysis
After protein quantification, 40 mg boiled proteins were loaded
into 10% sodium dodecyl sulphate-polyacrylamide electrophoresis
gels and separated by molecular size. Gels were electro-blotted
onto nitrocellulose membranes and then blocked for 90 min in
Tris-buffered saline, pH 7.5, containing 0.5% Tween 20 and 5% (w/v)
skim milk powder. Membranes were incubated overnight at 4 C
with the appropriate primary antibody: antieNFekB, p65 subunit,
diluted 1:1000 (Millipore, Billerica, Massachusetts). The mem-
branes were finally incubated with the peroxidase-conjugated
secondary anti-Rabbit antibody (1:5000) for 1 h. The bound anti-
bodies were detected by chemiluminescence (Biorad, Milan, Italy).
4
.7. Anti-inflammatory properties evaluation on IMR 32 cell line
4
.7.1. Cell culture and treatments
IMR 32 cells line (obtained from American Type Culture
ꢀ
Collection, ATCC), were cultured in EMEM medium supplemented
with 10% fetal bovine serum (Lonza, Milan, Italy) supplemented
with 10% fetal bovine serum (FBS, EuroClone, Milan, Italy), 1% of L-
glutamine (Lonza, Milan, Italy) and 1% of penicillin/streptomycin
antibiotics (Lonza, Milan, Italy) at 37 C in 5% CO
The different formulations (3a and 5c) were dissolved in DMSO
at the final concentration of 10 mM. Stock solutions were then
diluted with cell culture medium, EMEM with Earle's Balanced Salt
ꢀ
2
[60].
b-Actin or Lamin A were used as loading controls. Images of the
bands were digitized using an Epson Stylus SX405 scanner and the
densitometry analysis was performed using Image-J software.
Solution, in order to obtain an intermediate dose solution (100
to be used for the further dilutions (50 M, 10 M, 5 M, 1
.5 M and 0.1 M). Control vehicle was represented by DMSO
ranging from 0.5% to 0.001%. LPS form Escherichia coli serotype
111:B4 was purchased from Sigma (St. Louis, MO) and it was dis-
solved in phosphate buffered saline for use.
m
M),
m
m
m
m
M,
4.7.6. Statistical analysis
0
m
m
For each of the variables tested, two-way analysis of variance
(ANOVA) was used. A significant result was indicated by a p
0
value < 0.05 (*) or by a p value < 0.01 (**). All the results are
expressed as mean ± SD of triplicate determinations obtained in 3
independent experiments. Data were analyzed using the software
GraphPad Prism 4.0 (GraphPad Software, Inc., La Jolla, CA).
4.7.2. Cytotoxicity determination
IMR 32 cells were seeded 100,000 cells/well in 96-wells plate
and were grown to confluence, then were treated with the
mentioned substances in EMEM medium supplemented with 1%
fetal bovine serum. The effects of tested compounds on cellular
morphology were checked after 24 h using a built-in camera in an
inverted Nikon Eclipse microscope (20X magnification) [61].
Cytotoxicity was determined by LDH release in the media, as pre-
viously reported [61]. All tests were performed at least in triplicate.
The absorbance measured from three wells was averaged, and the
percentage of LDH released was calculated as arbitrary unit of
change relative to 1% Triton X-100 treated cells.
Acknowledgements
The Authors wish to thank MIUR-PRIN for financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111674.
References
4
.7.3. Nuclear proteins extraction
After treatments with compounds 3a and 5c at 0.5 and 1 mM
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