Development of Potent Inhibitors of Fatty Acid Amide Hydrolase Useful for the Treatment of Neuropathic Pain

Article abstract of DOI:10.1002/cmdc.201800397

The unique role of fatty acid amide hydrolase (FAAH) in terminating endocannabinoid (EC) signaling supports its relevance as a therapeutic target. Inhibition of EC metabolizing enzymes elicits indirect agonism of cannabinoid receptors (CBRs) and therapeutic efficacy devoid of psychotropic effects. Based on our previous ligands, and aiming at the discovery of new selective FAAH inhibitors, we developed a series of 12 new compounds characterized by functionalized tricyclic scaffolds. All the developed compounds display negligible activity on monoacylglycerol lipase (MAGL) and CBRs. The most potent FAAH inhibitors of the newly developed series, 6-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-6-phenylhexylcarbamate (5 h) and 4-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-(6-phenylhexyl)carbamate (5 i) (nanomolar FAAH inhibitors, the latter of which also shows micromolar affinity at the CB₁R), were selected for further studies. Results of cell-based studies on a neuroblastoma cell line (IMR32) demonstrated 5 h, 5 i, and our reference compound 3 ([3-(3-carbamoylpyrrol-1-yl)phenyl] N-(5-phenylpentyl)carbamate) to lack any cytotoxic effect, while all three showed the ability to decrease oxidative stress by reducing the expression of the redox-sensitive transcription factor NF-κB. Encouraged by these data, these compounds were studied in vivo and were dosed orally in a mouse model of neuropathic pain. At 10 mg kg^?1 all the compounds were able to relieve the hypersensitivity induced by oxaliplatin.

Full text of DOI:10.1002/cmdc.201800397

Accepted Article
Title: Development of Potent Inhibitors of Fatty Acid Amide Hydrolase
Useful for the Treatment of Neuropathic Pain
Authors: Margherita Brindisi, Giuseppe Borrelli, Simone Brogi,
Alessandro Grillo, Samuele Maramai, Marco Paolino, Mascia
Benedusi, Alessandra Pecorelli, Giuseppe Valacchi, Lorenzo
Di Cesare Mannelli, Carla Ghelardini, Marco Allarà, Alessia
Ligresti, Patrizia Minetti, Giuseppe Campiani, Stefania Butini,
Vincenzo di Marzo, and Sandra Gemma
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800397
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800397
A Journal of
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Development of Potent Inhibitors of Fatty Acid Amide Hydrolase
Useful for the Treatment of Neuropathic Pain
†,
†,
†,
†,
a]
Margherita Brindisi,[a] Giuseppe Borrelli,[ a] Simone Brogi,[ a] Alessandro Grillo,[ a] Samuele Maramai,[
Marco Paolino,[a] Mascia Benedusi,[b] Alessanda Pecorelli,[c] Giuseppe Valacchi,[b],[c] Lorenzo Di Cesare
Mannelli,[d] Carla Ghelardini,[d] Marco Allarà,[e],[f] Alessia Ligresti,[e] Patrizia Minetti,[g] Giuseppe
Campiani,[a]* Vincenzo di Marzo,[e],[h] Stefania Butini,[a]* Sandra Gemma[a]
[a]
Dr. Margherita Brindisi, Dr. Giuseppe Borrelli, Dr. Simone Brogi, Dr. Alessandro Grillo, Dr. Samuele Maramai, Dr. Marco Paolino, Prof. Giuseppe Campiani,
Prof. Stefania Butini, Prof. Sandra Gemma
European Research Centre for Drug Discovery and Development (NatSynDrugs) and Department of Biotechnology, Chemistry, and Pharmacy (DoE 2018-
2020)
University of Siena
Via Aldo Moro 2, 53100 Siena, Italy
E-mail: : butini3@unisi.it; campiani@unisi.it
†These authors contributed equally
[b]
[c]
[d]
[e]
Prof. Giuseppe Valacchi; Dr. Mascia Benedusi
Department of Life Sciences and Biotechnology
University of Ferrara
Via Borsari 46, 441212, Ferrara, Italy
Prof. Giuseppe Valacchi, Dr. Alessanda Pecorelli
Department of Animal Science
North Carolina State University
NC Research Campus, PHHI Building, 600 Laureate Way, Kannapolis 28081, NC USA
Prof. Carla Ghelardini, Dr. Lorenzo Di Cesare Mannelli
Department of Neuroscience, Psychology, Drug Research and Child Health, Section of Pharmacology and Toxicology (NEUROFARBA)
University of Florence
Viale G. Pieraccini, 6, 50139 Firenze, Italy
Prof. Vincenzo di Marzo, Dr. Alessia Ligresti, Dr. Marco Allarà
Endocannabinoid Research Group, Institute of Biomolecular Chemistry
CNR
Via Campi Flegrei, 80078 Pozzuoli (Napoli), Italy
Dr. Marco Allarà
[f]
EPITECH Group SpA
Via Egadi, 7 - 20144 Milano Italy
[g]
[h]
Dr. Patrizia Minetti
Alfasigma
Via Pontina Km 30,400, 00040 Pomezia, Italy
Prof. Vincenzo di Marzo
Département de Médecine
Université Laval
1050, avenue de la Médecine, Québec City (Québec), QC G1V 0A6, Canada
Supporting information for this article is given via a link at the end of the document.
Abstract. The unique role of fatty acid amide hydrolase (FAAH) in
terminating endocannabinoid (EC) signaling supports its relevance as
therapeutic target. Inhibition of the ECs metabolizing enzymes elicits
indirect agonism of cannabinoid receptors (CBRs), and therapeutic
efficacy, devoid of psychotropic effects. Based on our previous
ligands and aiming at discovering new selective FAAH inhibitors, we
developed a series of new compounds (5a-l) characterized by
functionalized tricyclic scaffolds. All the developed compounds
display negligible activity on MAGL and CBRs. The most potent FAAH
inhibitors of the newly developed series, 5h and 5i (nanomaolar FAAH
inhibitor also some detecting micromolar affinity at CB1R), were
selected for further studies. After cellular studies on a neuroblastoma
cell line (IMR32) 5h,i and our reference compound 3 demonstrated
the lack of any cytotoxic effect and the ability to reduce oxidative
stress by decreasing the expression of the redox sensitive
transcription factor NF-kB. Encouraged by these data, compounds
were studied in vivo and were dosed orally in a mice model of
neuropathic pain. At 10 mg/kg all the compounds were able to relieve
the hypersensitivity induced by oxaliplatin.
Fatty acid amide hydrolase (FAAH, EC 3.5.1.99), an integral
membrane-bound serine hydrolase,[1-2] is the main catabolic
enzyme of the fatty acid ethanolamides (FAEs) such as
anandamide (AEA) and oleamide.[3] Other enzymes that
contribute to the termination of the endocannabinoids’ (EC) action
are N-acylethanolamine acid amidase[4] and monoacylglycerol
lipase (MAGL).[5] FAEs together with 2-arachidonoylglycerol are
the main EC signalling lipids which, by interacting with type-1 and
type-2 cannabinoid receptors (CB1R and CB2R), exert biological
activity and modulate a variety of physiological processes
including pain, inflammation, appetite, motility, sleep,
thermoregulation, cognition and emotional states.[6-7] The
analgesic effect of CB1R and CB2R has been known for
centuries[8] but, as other CBRs agonists, they produce a spectrum
of motor and psychotropic side effects mediated by central CB1R.
Accordingly, a valuable therapeutic alternative aims at eliciting the
desirable effects of CBRs activation, while avoiding the negative
effects of global CB1R stimulation, through indirect receptor
agonism obtained by inactivation of ECBs metabolizing enzymes.
The relevance of FAAH enzyme in the safe management of pain
is supported by the evidence that faah knockout mice display high
Introduction
1
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AEA levels in central nervous system (CNS) and show an
analgesic phenotype, in either models of inflammatory pain
(induced by carrageenan) and of acute pain (induced by
different FAAH inhibitors developed so far, can be clustered in two
families: irreversible (e.g. carbamates 1a[19] and 1b,[20] Figure 1)
and reversible inhibitors (e.g. the α-ketooxazole 2,[21] Figure 1). In
this frame, we recently reported the development of different
classes of compounds as inhibitors of the ECs metabolic
enzymes; potent and selective FAAH[22-23] or MAGL[24] inhibitors
and dual FAAH/MAGL[25] inhibitors useful in different pathological
states.[26-27]
As a continuation of our efforts in the discovery of FAAH inhibitors
we herein describe the development of pyrrole-based analogues
inspired to our previously identified ligands. This new series of
compounds (5a-l, Figure 1 and Table 1) our structural template,
compound 3, was modified in a classical medicinal chemistry
approach which involved a structural rigidification by bridging the
phenylpyrrole scaffold of our lead 3. In particular three types of
tricyclic systems were explored: i) a 6-6-5 system, the pyrrolo[1,2-
a]quinoxaline (also inspired to the structure of previously
developed FAAH/MAGL inhibitors typified by 4[25]); ii) a 6-6-5
system, pyrrolo[1,2-a]quinoxalin-4(5H)-one substituted at C-6 or
C-8; iii) the 6-7-5 systems 4,5-dihydro-6H-benzo[f]pyrrolo[1,2-
a][1,4]diazepin-6-one and 5,6-dihydro-4H-benzo[f]pyrrolo[1,2-
a][1,4]diazepin-4-one heterocycles, substituted at C-9. These
systems were tethered by a piperazinyl urea or a carbamate to
phenylhexyl, undecynyl, or ((monofluoro)phenoxyethoxyethyl
lateral chains (Figure 1 and Table 1). Synthesis, molecular
modelling, in vitro and in vivo biological properties of the new
FAAH inhibitors are discussed in the present article.
formalin,[9] together with
a reduction of the inflammatory
responses,[10] and improved sleep and memory acquisition,[11-12]
supports the relevance of FAAH enzyme in the safe management
of pain. In fact, FAAH inhibition by increasing the endogenous
concentration of its substrates, protracts and potentiates their
beneficial (therapeutic) effects, without eliciting the classical CB1R
agonist side effects (hypomotility, hypothermia, and catalepsy).[13]
In 2016, the serious adverse effects observed in a phase I study
with a FAAH irreversible inhibitor (BIA 10-2474) led to a temporary
suspension of the development of FAAH inhibitors. Shortly after,
a report from FDA[14] concluded that BIA 10-2474 has a peculiar
toxicity profile that does not extend to other FAAH inhibitors. In
fact, an in depth re-investigation uncovered a series of off-target
proteins for the same compound.[15]
Results and Discussion
Chemistry
The synthesis of compounds 5a−l is described in Schemes 1−5.
Reaction of pyrroloquinoxalyl intermediate 6[28-31] (Scheme 1) with
the alkylisocyanates 7a-c afforded the piperazinecarboxamide-
based compounds 5a−c, whereas reaction with the alkyl
bromides afforded the alkylpiperazine-based derivatives 5d,e.[32]
Figure 1. Reference (1-4) and title compounds (5a-l).
Scheme 1. Reagents and conditions: a) R1NCO, TEA, dry THF, reflux, 11 h, 59-
62%; b) R2Br, TEA, MeCN, reflux, 14 h, 71-75%.
Chronic pain is a major public health problem, with a tremendous
impact on the quality of life of the patients, that produces a
significant economic and social burden.[16] Neuropathic pain (a
severe, debilitating, and persistent, form of chronic pain that may
arise from a dysfunction or damaged peripheral nerves, spinal
cord, or brain[17]) is poorly treated by conventional therapeutics
which also exhibit a series of drawbacks and side effects. All the
above considerations suggest that the use of FAAH inhibitors may
provide a safe and efficient approach for the treatment of painful
syndromes including neuropathies.[17]
The crystal structure of FAAH enabled an in-depth knowledge of
the enzyme features and functioning useful for the development
of selective inhibitors. The catalytic triad (S241, S217, and K142,
human numbering) is accessible through the acyl-binding channel
and the “membrane access channel”.[18] In proximity of the
nucleophilic S241, the oxyanion hole accommodates the carbonyl
oxygen of amide or ester substrates by establishing H-bonds. The
For the synthesis of 5f (Scheme 2) the application of Curtius
rearrangement protocol to the acid 8 with tert-butanol provided
the protected aniline 9. After Boc-removal, the amine 10 was
converted, by
a Clauson-Kaas reaction obtained, in turn
converted into the 1-phenylpyrrole derivative 11. Reduction of the
nitro functionality led to amine 12 that by treatment with
triphosgene provided the quinoxalinone 13. Treatment with boron
tribromide afforded the phenol 14 that was reacted with
phenylhexylisocianate (7b) to achieve 5f.
2
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Scheme 2. Reagents and conditions: a) t-BuOH, DPPA, TEA, reflux, 15 h, 78%;
b) TFA, 0 °C, 1.5 h, 99%; c) 2,5-dimethoxytetrahydrofuran, AcOH, H2O, 100 °C,
15 min, 97%; d) Fe°, CaCl2, EtOH 75%, 80 °C, 2 h, 87%; e) CO(OCCl3)2, toluene,
120 °C, 2 h, 52%; f) BBr3, DCM, from-78 to 25 °C, 12 h, 11%; g) 7b, TEA, THF,
25 °C, 16 h, 30%.
Scheme 4. Reagents and conditions: a) SOCl2, MeOH, 25 °C, 10 h; b)
SnCl2·2H2O, EtOAc, 25 °C, 2 h; c) 5N HCl, 2,5-dimethoxytetrahydrofuran, 1,4-
dioxane, 110 °C, 5 min, 87% (over 3 steps); d) (COCl)2, NH2OH∙HCl, pyridine,
DMF, 1,2-DCE, from 0 to 120 °C, 10 h, 47%; e) NaBH4, CoCl2, MeOH, 25 °C,
30 min, 64%; f) BBr3, DCM, from -78 to 25 °C, 12 h, 50%; g) 7b, TEA, THF,
25 °C, 16 h, 60%.
Nitration of the 3-hydroxyphenylacetamide 15 (Scheme 3) led to
16. Sequential hydrolysis of the acetamido group, Clauson-Kaas
reaction and reduction of the nitro functionality provided the
aminophenol 17. Cyclization reaction carried on with triphogene
afforded derivative 18, later converted into the urethane 5g.
Scheme 5. Reagents and conditions: a) methyl 1H-pyrrole-2-carboxylate,
Cs2CO3, dry DMF, 50 °C, 12 h, 30%; b) NaBH4, CoCl2, MeOH, 25 °C, 30 min,
71%; c) BBr3, dry DCM, from -78 to 25 °C, 12 h, 50%; d) TEA, dry THF, 25 °C,
12 h, 43-55%.
Scheme 3. Reagents and conditions: a) HNO3, AcOH, Ac2O, 25 °C, 8 h, 98%;
b) 12N HCl, 130 °C, 5 h, 95%; c) 5N HCl, 2,5-dimethoxytetrahydrofuran, 1,4-
dioxane, 110 °C, 20 min, 70%; d) SnCl2·2H2O, EtOAc, 25 °C, 2 h, 85%; e)
CO(OCCl3)2, toluene, 120 °C, 2 h, 36%; f) 7b, TEA, THF, 25 °C, 16 h, 10%.
Structure–activity relationship studies and molecular
modeling studies
The inhibition potency of compounds 5a–l for the FAAH
enzyme is reported in Table 1. Selectivity towards MAGL, CB1R
and CB2R was evaluated for the most promising compounds. The
new compounds showed an excellent selectivity profile for FAAH
over MAGL, CB1R, and CB2R with only 5i detecting a one digit
micromolar affinity at CB1R and a two digit micromolar affinity at
CB2R. The data confirmed that the presence of an “activated”
electrophilic center (such as ureido or carbamoyl moieties) is a
crucial prerequisite for potency. In fact compounds 5a-e, where
the electrophilic center is absent (5d,e) or where an urea is
present between the two aliphatic amines (5b-c) were found
inactive. When the urea involved an aniline (5a) as the
carbamoylating moiety, enzyme inhibition was still poor although
slightly higher than that of the other urea-containing analogues of
the series. In line with our previous results, the insertion of a
carbamate in the developed molecules allowed us to identify a
series of selective FAAH inhibitors (5f-l) characterized by the
presence of different scaffolds supporting the phenol moiety. The
influence of polyethereal lateral chains (as the enzyme
carbamoylating entities) was explored, for the most promising and
synthetically accessible scaffolds, which modulated FAAH
inhibition properties (5j-l vs 5i).
After esterification and nitro group reduction of the nitrocarboxylic
acid 19 (Scheme 4) the resulting aniline was subjected to
Clauson-Kaas reaction, to give the intermediate 20. This 1-
phenylpyrrole was converted into the 2-cyanopyrrole derivative 21.
Selective reduction of the nitrile by treatment with sodium
borohydride in the presence of cobalt(II) chloride led to
spontaneous cyclization to the pyrrolobenzo[1,4]diazepinone
derivative 22. Cleavage of the methyl ether functionality followed
by treatment with phenylhexylisocianate provided 5h.
For the preparation of compounds 5i-l (Scheme 5) aryl fluoride 23
was submitted to an aromatic nucleophilic substitution with methyl
1H-pyrrole-2-carboxylate in the presence of cesium carbonate.
The resulting 1-phenylpyrrole 24 was reduced, cyclized and
treated
with
boron
tribromide,
thus
providing
the
pyrrolobenzo[1,4]diazepinone intermediate 25. Reaction with the
isocyanates 7b or 26a-c[22] led to compounds 5i-l.
3
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Table 1. IC50 values on FAAH and MAGL enzymes, CB1R and CB2R for compounds 5a-l, and reference compound 3.
FAAH
rat brain membrane
CB1R
IC50 nM
(displacement % at 50
µM)
CB2R
IC50 nM
(displacement % at 50
µM)
MAGL
Cmpd
Structure
COS cell cytosol
IC50 nM
(displacement %)[a,b]
(displacement %)[a,b]
>50 µM
(15.4)
5a
NT[c]
NT[c]
NT[c]
>50 µM
(7.7)
5b
5c
NT[c]
NT[c]
NT[c]
>50 µM
(7.19)
NTa
NT[c]
NT[c]
>50 µM
(12.7)
5d
NT[c]
NT[c]
NT[c]
>50 µM
(5.8)
5e
5f
NT[c]
NT[c]
NT[c]
NT[c]
>50 µM
(46.2)
>50 µM
(6.6)
520
460
>50 µM
(25.7)
>50 µM
(4.3)
5g
5h
NT[c]
>50 µM
(0.7)
>50 µM
(25.3)
>50 µM
(19.3)
83.5
94.1
>50 µM
(23.7)
5i
5j
6730
24300
>10 µM
(5.7)
>50 µM
(49.0)
>50 µM
(25.5)
2825.3
1672.3
>10 µM
(7.5)
>50 µM
(42.5)
>50 µM
(39.3)
5k
>10 µM
(6.8)
>50 µM
(46.0)
>50 µM
(37.4)
5l
2687.3
0.60
3[22]
NA[d]
NA[d]
NA[d]
[a]Values are means of three experiments (n = 3) and all SD not indicated are within 10%. [b]% of displacement at the maximum concentration tested is reported in
brackets when IC50 is > 10 or 50 µM; tests were performed as previously described [25] with FAAH from rat brain (sequence identity of 82.7% and sequence similarity
of 97.2% with hFAAH) or MAGL COS from cytosol with 30’ or 20’ of incubation time at 37 °C, respectively. [c]NT = Not tested. [d]NA = Not active.
4
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Figure 2. (A,B) IFD pose and ligand interaction diagram of compound 5h (cyan sticks) into FAAH enzyme (PDB ID: 3PPM in orange cartoon). The
catalytic triad of the enzyme is represented by sticks, while the key residues of the FAAH binding site are represented by lines. The H-bonds are
represented by black dotted lines. Pictures generated by PyMOL and Maestro (Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012).
Figure 3. (A,B) IFD pose and ligand interaction diagram of compound 5i (yellow sticks) into FAAH enzyme (PDB ID: 3PPM in orange cartoon). (C)
Superposition between IFD poses of 5i (yellow sticks) and 5j (blue sticks). The catalytic triad of the enzyme is represented by sticks, while the key
residues of the FAAH binding site are represented by lines. The H-bonds are represented by dotted lines. The pictures were generated by PyMOL
and Pictures generated by PyMOL and Maestro (Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012).
To better understand the structure–activity relationships (SARs)
of the developed compounds, we performed molecular docking
studies to assess the interactions of the inhibitors with the FAAH
enzyme at atomic level. We performed an Induced Fit Docking
(IFD) calculation as reported,[33-35] to identify the main contacts
governing the behavior of our compounds into the enzyme. The
data were compared with those obtained by applying the same
protocol to our previously described lead 3 (see Supplementary
Information and Figure S1). In particular 5h, one of the most
potent compounds of this series, interacts with the FAAH active
site by polar and hydrophobic contacts (Figure 2A, B). Polar
contacts were established by the lactam moiety and the
backbones of V270 and C269. In line with the potency of FAAH
inhibition of 5h (IC50 = 83.5 nM, Table 1) the electrophilic moiety
is properly placed in front of the catalytic serine residues (S217
and S241) which can establish H-bonds with the carbamate
phenate oxygen. The orientation of the carbon of the carbamate
portion and its distance from the catalytic S241 (< 3Ǻ) are in
agreement with a potential nucleophilic attack. The carbonyl
group can establish two H-bonds with the I238 and G239.
Furthermore, the phenylhexyl lateral chain could be located in a
sub-hydrophobic pocket, lined by F192, F381 and F432,
producing a double π-π stacking with F192 and F432. Compound
5i displayed similar interactions besides a correct orientation
towards S241 (π-π stackings with F381 and F432, and H-bonds
with C269 and V270, and, the interaction with the catalytic S241)
(Figure 3A, B).
In compound 5j-l, the introduction of a polyethereal tether, for
replacing the hexyl chain of 5i caused a marked decrease of
FAAH inhibition. The docking outputs for 5j-l clearly confirm the
lack of potency (Figure S2A,B, S3A,B, S4A,B). The polyethereal
chain is diverted from the accommodation in the hydrophobic sub-
5
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pocket of the enzyme (see superposition of 5i and 5j, Figure 3C)
and precludes the carbamate to establish interaction with
S241the catalytic serine residue, while being still able to establish
two H-bonds (with I238 and G239 for 5j and with S193 and G239
for 5l and 5k).
translocation with respect to the only LPS treated cells. Notably,
while 5i explicated its higher anti-inflammatory action at 1 µM
concentration, compound 5h exerted an anti-inflammatory effect
inversely proportional to the dose.
In vivo efficacy: effect of acute administration of compounds
3, 5h and 5i on oxaliplatin induced neuropathic pain.
We evaluated the effect of compounds 3, 5h and 5i in a mouse
model of neuropathic pain: the chemotherapy-dependent
neuropathy induced by oxaliplatin.[39-40]
In compound 5g the restricted 6-6-5 tricyclic system produces a
binding mode quite different from that found for the 6-7-5 system
of 5h. The tricyclic system can only form a H-bond with the
backbone of C269, and in line with the three digit nanomolar
potency of 5g (Table 1) it lacks a suitable distance for interacting
with S241. The different accommodation of the more planar and
hindering tricyclic system prevents projecting the aliphatic tail into
the hydrophobic sub-pocket (Figure S5A,B). The superposition of
the IFD poses of 5h and 5g clearly traces out the described
differences (Figure S5C).
The same is true for compound 5f (Figure S6A, B). In particular,
the tricyclic moiety establishes only one polar contact with T236.
The carbamate group interacts with S217 and S241 by two H-
bonds and the phenylhexyl tail forms a double π-π stacking with
F192 and F381.
The repeated administration of the neurotoxic anticancer agent
(2.4 mg kg-1, i.p.) evoked an allodynia-like behavior measurable
as increased sensitivity to a non-noxious cold stimulus (Cold plate
test). On day 14, the licking latency decreased to 10.7 ± 0.9 s in
comparison (P<0.01) to control mice (20.1 ± 0.8 s) treated with
vehicle (Figure 4). FAAH inhibitors were tested in oxaliplatin-
treated animals on day 14 and the pain relieving effects were
evaluated over time after a single orally administered dose of the
tested compounds. We initially established that our potent and
selective FAAH inhibitor 3 was able to dose dependently increase
the pain threshold already at 3 and 10 mg kg-1 peaking 30 min
after treatment. On these bases, we decided to test the analogues
5h and 5i (in a dose range of 3 - 30 mg kg-1 p.o.). As shown in
Cytotoxicity determination and antioxidant potential
evaluation for 3, 5h, and 5i.
Figure 4, both compounds induced
a significant relief of
Oxidative stress (OS) plays a crucial role in neuropathic pain[36]
oxaliplatin-induced neuropathic pain starting from 3 mg kg-1.
Higher effects were obtained using 5h and 5i, administered at
doses of 10 and 30 mg kg-1. These dosages were equally effective
between 15 and 30 min after administration. 5h was significantly
effective up to 45 min (Figure 4).
and our working group provided evidence that it plays a crucial
role in oxaliplatin-induced neuropathy.[37] On these bases, we
decided to interrogate the ability of the most potent FAAH
inhibitors of this series in the reduction of OS. Accordingly, the
efficacy of compounds 5h and 5i, in comparison with our lead 3,
was measured in a cellular acute model of OS induced by
hydrogen peroxide. By applying our previously described
model,[38] a preliminary test was performed to assess the effects
of the tested compounds in IMR32 cells morphology. As outlined
in Figure S7 after treating the cells for 24 h with increasing
concentrations of 3 and 5h,i (with ranges between 0.1 to 50 µM),
no changes were observed in cellular morphology.
We then tested the cytotoxicity profile in the same cell line by
measuring the LDH release in the media, as measured by
enzymatic assay (see Supplementary Material for details). As
positive control (LDH amount corresponding to 100% cell death)
IMR32 cells were treated with 1% Triton X-100, according to the
manufacturer’s instructions (Roche, Mannheim, Germany).
Notably, no significant cytotoxic effect was observed for all the
tested compounds at concentrations ranging from 0.1 µM to 50
µM (Figure S8).
These results with 3, 5h and 5i prompted us to evaluate their
potential anti-inflammatory and neuroprotective properties after
treating the cells with pro-inflammatory substances. In order to
find the most appropriate model for probing anti-inflammatory
potential of our molecules in the IMR32 cell line we tested several
pro-inflammatory mediators. Therefore, we first evaluated the
activation of NF-kB by measuring the translocation to the nucleus
of its cytoplasmic subunit p65. Accordingly, we treated the cells
with lipopolysaccharide (LPS 100 µg/mL and 200 µg/mL) for 30,
60 and 90 min. The 100 μg treatment after 30 min was the most
effective, in which we could clearly observe the nuclear
translocation of p65 protein (see in Figure S9 the increment of the
LPS bar with respect to the control bar). Thus, according to our
protocol, we pre-treated the cells with compounds 3, 5h and 5i for
24 h and subsequently with 100 µg/mL of LPS for 30 min. As
shown in Figure S9 the pre-treatment was able to decrease p65
6
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10.1002/cmdc.201800397
ChemMedChem
FULL PAPER
analysis of a series of new analogues (5a-l) bearing functionalized
tricyclic scaffolds and structurally inspired to our previous ligands
3 and 4. The developed compounds showed an excellent
selectivity profile for FAAH over MAGL, CB1R, and CB2R with 5i
detecting some (micromolar) affinity at CBRs. The most potent
FAAH inhibitors of the series 5h and 5i were used for further
studies. When studied on a neuroblastoma cell line (IMR32) 5h,
5i and our reference compound 3 demonstrated the absence of
cytotoxic effects and the ability to reduce OS by decreasing the
expression of the redox-sensitive transcription factor NF-kB. The
efficacy of the same compounds was also assessed in vivo in a
rodent model of neuropathic pain. After oral administration at 10
mg/kg all the compounds (5h, 5i and 3) were able to relieve the
hypersensitivity induced by oxaliplatin.
Experimental Section
Chemistry
Unless otherwise specified, materials were purchased from
commercial suppliers and used without further purification. Reaction
progress was monitored by TLC using silica gel 60 F254 (0.040–0.063
mm) with detection by UV. Silica gel 60 (0.040–0.063 mm) was used
for column chromatography. 1H NMR and 13C NMR spectra were
recorded on a Varian 300 MHz spectrometer 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 (q) and broad (br); the values of chemical
shifts (δ) are given in ppm and coupling constants (J) in Hertz (Hz).
Microwave reactions were performed by a CEM Discovery apparatus.
ESI/MS spectra were performed by an Agilent 1100 Series LC/MSD
spectrometer. HRESIMS were carried out by a Thermo Finningan
LCQ Deca XP Max ion-trap mass spectrometer equipped with
Xcalibur software, operated in positive 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 anhydrous solvents. Final compounds
were analyzed by combustion analysis (CHN) to confirm purity >95%.
N-Phenyl-4-(pyrrolo[1,2-a]quinoxalin-4-yl)piperazine-1-
carboxamide (5a). Compound 6 (60 mg, 0.24 mmol) was dissolved
in dry THF (2.0 mL) and TEA (33 µL, 0.24 mmol) was added. Then
phenyl isocyanate (7a) (52 µL, 0.47 mmol) was added dropwise and
the reaction mixture was heated under reflux for 12 h. After reaction
completion volatiles were removed in vacuo and the residue was
purified. Column chromatography on silica gel (n-hexane/EtOAc 2:1)
provided pure title compound (62% yield) as a pale yellow solid. 1H
NMR (300 MHz, CDCl3) δ 3.74 (m, 4H), 3.91 (m, 4H), 6.45 (br s, 1H),
6.79 (m, 2H), 7.06 (m, 2H), 7.26-7.41 (m, 5H), 7.67-7.76 (m, 2H), 7.85
(m, 1H). 13C-NMR (75 MHz, CDCl3) δ 43.7, 47.5, 106.7, 112.6, 113.3,
114.6, 120.0 (2C), 123.3 (2C), 124.3, 125.2, 125.8, 127.5, 128.9 (2C),
152.3, 155.0. ESI-MS m/z 372 [M + H]+ (100), 394 [M + Na]+.
Elemental analysis calcd (%) for C22H21N5O: C 71.14, H 5.70, N 18.85;
found: C 71.08, H 5.93, N1 8.70.
N-(6-Phenylhexyl)-4-(pyrrolo[1,2-a]quinoxalin-4-yl)piperazine-1-
carboxamide (5b). The title compound was prepared according to
the procedure previously described for 5a starting from 6 (100 mg,
0.40 mmol), TEA (56 µL, 0.40 mmol) and 6-phenyl-1-hexylisocyanate
(7b) (161 mg, 0.79 mmol). Column chromatography on silica gel (n-
hexane/EtOAc 1:1) provided pure title compound (59% yield) as a
pale yellow amorphous solid. 1H NMR (300 MHz, CDCl3) δ 1.28-1.67
(m, 8H), 2.62 (t, J = 7.8 Hz, 2H), 3.26 (m, 2H), 3.59 (m, 4H), 3.84 (m,
4H), 4.64 (br t, 1H), 6.77 (m, 2H), 7.15-7.36 (m, 7H), 7.68 (m, 2H),
7.82 (m, 1H). ESI-MS m/z 456 [M + H]+,(100) 478 [M + Na]+. Elemental
analysis calcd (%) for C28H33N5O: C 73.82, H 7.30, N 15.37; found: C
73.75, H 7.51, N 15.71.
Figure 4. Pain relieving effect of compounds 3, 5h and 5i. Mice were treated
with oxaliplatin (2.4 mg kg-1 i.p., daily for 5 consecutive days every week for 2
weeks) to induce a painful neuropathy. Tested compounds were administered
p.o. on day 14, control animals were treated with vehicles. Pain threshold was
evaluated measuring the licking latency to a cold non-noxious stimulus (Cold
plate test). Each value represents the mean ± S.E.M. of 12 mice performed in 2
different experimental sets. ^P<0.05 and ^^P<0.01 in respect to the value before
treatment.
Conclusions
The FAAH enzyme significantly contributes in terminating EC
signaling and represents a relevant therapeutic target. By
inhibition of the ECs metabolizing enzymes, we can attain indirect
agonism at CBRs, and therapeutic efficacy devoid of psychotropic
effects. Aiming at discovering new selective FAAH inhibitors, in
this manuscript we have described the development and SAR
7
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10.1002/cmdc.201800397
ChemMedChem
FULL PAPER
N-(Undec-10-yn-1-yl)-4-(pyrrolo[1,2-a]quinoxalin-4-yl)piperazine-
1-carboxamide (5c). The title compound was prepared according to
the procedure described for 5a starting from 6 (72 mg 0.29 mmol),
TEA (40 µL, 0.29 mmol) and undec-10-yn-1-yl isocyanate (7c) (110
mg, 0.57 mmol). Column chromatography on silica gel (n-
hexane/EtOAc 2:1) provided pure title compound (60% yield) as a
pale yellow amorphous solid. 1H NMR (300 MHz, CDCl3) δ 1.23-1.54
(m, 14H), 1.94 (m, 1H), 2.18 (m, 2H), 3.26 (m, 2H), 3.60 (m, 4H), 3.84
(m, 4H), 4.53 (br t, 1H), 6.78 (m, 2H), 7.26-7.32 (m, 2H), 7.64-7.74 (m,
2H), 7.83 (s, 1H). ESI-MS m/z 446 [M + H]+, (100) 468 [M + Na]+.
Elemental analysis calcd (%) for C27H35N5O: C 72.78, H 7.92, N 15.72;
found: C 72.73 , H 8.14, N 16.03.
an aqueous saturated solution of Na2CO3. Then, it was extracted with
EtOAc (3 × 100 mL), dried over anhydrous Na2SO4, filtered, and
concentrated. Compound 11 (97% yield) was obtained as a colourless
solid. 1H NMR (300 MHz, CDCl3) δ 3.83 (s, 3H), 6.41 (d, J = 1.5 Hz,
2H), 6.94 (dd, J = 7.5, 1.5 Hz, 1H), 7.26 (d, J = 1.5 Hz, 2H), 7.44 (dd,
J = 7.5, 1.5 Hz, 1H), 7.73 (t, J = 8.2 Hz, 1H). ESI-MS m/z 219 [M +
H]+.
2-Methoxy-6-(1H-pyrrol-1-yl)aniline (12). To a solution of 1-(3-
methoxy-2-nitrophenyl)-1H-pyrrole (11) (680 mg, 3.12 mmol) in 75%
ethanol (8.0 mL), calcium chloride (229 mg, 1.56 mmol) and iron
powder (1.23 g, 7.09 mmol) were added. The reaction mixture was
heated under reflux for 2 h, then was filtered through a celite pad. The
4-(4-(6-Phenylhexyl)piperazin-1-yl)pyrrolo[1,2-a]quinoxaline (5d). solvent was removed and the residue was dissolved in chloroform and
6-Phenylhexylbromide (96 mg, 0.40 mmol) was dissolved in MeCN
(HPLC grade, 4.0 mL) and the solution was heated to reflux. Then,
compound 6 (100 mg, 0.40 mmol) was added, followed by TEA (61
µL, 0.44 mmol). The reaction mixture was heated under reflux for 12
washed with water. The organic phase was dried over anhydrous
Na2SO4, filtered and concentrated. The compound was submitted to
the subsequent step without further purification (87% yield). H NMR
(300 MHz, CDCl3) δ 3.83 (s, 3H), 6.27 (brs, 2H), 6.41 (d, J = 1.5 Hz,
2H), 6.70 (t, J = 8.2 Hz, 1H), 6.85 (dd, J = 7.5, 1.5 Hz, 1H), 6.93 (dd,
J = 7.5, 1.5 Hz, 1H), 7.26 (d, J = 1.5 Hz, 2H). ESI-MS m/z 189 [M +
H]+.
6-Methoxypyrrolo[1,2-a]quinoxalin-4(5H)-one (13). To a solution of
12 (1.83 g, 9.73 mmol) in toluene (26.0 mL), triphosgene (778 mg,
2.63 mmol) was added and the resulting solution was heated under
reflux for 2 h. Then the reaction mixture was cooled under nitrogen
flow and the solvent was evaporated. The residue was purified by
silica gel column chromatography (chloroform/EtOAc 2:1) affording
the title compound (52% yield) as colourless solid. 1H NMR (300 MHz,
CDCl3) δ 3.97 (s, 3H), 6.67-6.69 (m, 1H), 6.81 (dd, J = 8.2, 0.9 Hz,
1H), 7.14 (t, J = 8.2 Hz, 1H), 7.25-7.28 (m, 2H), 7.64-7.65 (m, 1H),
8.42 (br s, 1H). ESI-MS m/z 215 [M + H]+.
1
h
and the volatiles were evaporated in vacuo. Column
chromatography on silica gel (n-hexane/EtOAc 2:1) provided pure title
compound (75% yield) as a pale yellow oil. 1H NMR (300 MHz, CDCl3)
δ 1.39-1.69 (m, 8H), 2.42 (m, 2H), 2.64 (m, 6H), 3.85 (m, 4H), 6.78
(m, 2H), 7.19-7.30 (m, 7H), 7.72 (m, 2H), 7.81 (m, 1H). 13C-NMR (75
MHz, CDCl3)  22.8, 23.5, 25.3, 25.3, 27.5, 32.0, 44.0 (2), 49.4 (2),
54.9, 102.8, 108.4, 109.3, 110.4, 116.3, 119.9, 121.2, 121.6, 121.8,
123.5, 124.3 (2C), 124.4 (2C), 132.3, 138.8, 148.6. ESI-MS m/z 413
[M + H]+. Elemental analysis calcd (%) for C27H32N4: C 78.60, H 7.82,
N 13.58; found: C 78.87, H 7.68, N 13.35.
4-(4-(Undec-10-yn-1-yl)piperazin-1-yl)pyrrolo[1,2-a]quinoxaline
(5e). The title compound was prepared according to the procedure
described for 5d starting from 6 (55 mg, 0.22 mmol), undec-10-yn-1-
yl bromide (50 mg, 0.22 mmol) and TEA (33 µL, 0.24 mmol). Column
chromatography on silica gel (n-hexane/EtOAc 2:1) provided pure title
compound (71% yield) as a pale yellow oil. 1H NMR (300 MHz, CDCl3)
δ 1.26-1.56 (m, 14H), 1.94 (m, 1H), 2.20 (m, 2H), 2.41 (m, 2H), 2.64
(m, 4H), 3.84 (m, 4H), 6.76 (m, 2H), 7.22-7.35 (m, 2H), 7.67 (m, 2H),
7.81 (m, 1H). ESI-MS m/z 403 [M + H]+. Elemental analysis calcd (%)
for C26H34N4: C 77.57, H 8.51, N 13.92; found C 77.73, H 8.38, N 14.09.
tert-Butyl 3-methoxy-2-nitrophenylcarbamate (9). To a solution of
3-methoxy-2-nitrobenzoic acid (8) (1 g, 5.08 mmol) in 20.0 mL of tert-
butanol were added diphenylphosphoryl azide (1.15 mL, 5.36 mmol)
and TEA (0.75 mL, 5.42 mmol). The reaction mixture was heated to
reflux for 15 h, then cooled to 25 °C and solvents were removed by
rotary evaporation. Residue was taken up with EtOAc and washed
with a saturated aqueous solution of NH4Cl, water, a saturated
solution of NaHCO3 and brine. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated affording the title
compound (78% yield). 1H NMR (300 MHz, CDCl3) δ 1.38 (s, 9H), 3.83
(s, 3H), 6.68 (dd, J = 7.5, 1.5 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 8.09
(dd, J = 7.5, 1.5 Hz, 1H), 9.15 (br s, 1H). ESI-MS m/z 291 [M + Na]+
(100).
6-Hydroxypyrrolo[1,2-a]quinoxalin-4(5H)-one
(14).
To
a
suspension of 6-methoxypyrrolo[1,2-a]quinoxalin-4(5H)-one (13) (100
mg, 0.47 mmol) in dry DCM (6.0 mL), boron tribromide (1M solution
in DCM, 1.87 mL, 1.87 mmol) was added at −78 °C. The reaction
mixture was then allowed to warm to room temperature and stirred for
12 h. A saturated aqueous solution of Na2CO3 was added to quench
the reaction. The residue was extracted with EtOAc (3 × 20 mL). The
combined organic extracts were dried over anhydrous Na2SO4,
filtered,
and
concentrated.
Column
chromatography
(chloroform/EtOAc 1:1) provided the title compound (11% yield) as a
colourless solid. 1H NMR (300 MHz, CD3OD) δ 6.68-6.70 (m, 1H),
6.79 (d, J = 8.2 Hz, 1H), 7.07 (t, J = 8.2 Hz, 1H), 7.18 (d, J = 2.9 Hz,
1H), 7.34 (d, J = 8.2 Hz, 1H), 7.92 (s, 1H). ESI-MS m/z 199 [M-H]-.
4-Oxo-4,5-dihydropyrrolo[1,2-a]quinoxalin-6-yl
6-
phenylhexylcarbamate (5f). The title compound was prepared
according to the procedure described for 5a starting from 6-
hydroxypyrrolo[1,2-a]quinoxalin-4(5H)-one 14 (10 mg, 0.05 mmol),
phenylhexyl isocyanate 7b (41 mg, 0.20 mmol) and TEA (28 µL, 0.20
mmol). Column chromatography (chloroform) afforded the title
compound (30% yield) as a colourless solid. 1H NMR (300 MHz,
CDCl3) δ 1.36 (m, 4H), 1.57 (m, 4H), 2.55 (t, J = 7.5 Hz, 2H), 3.31 (t,
J = 5.9 Hz, 2H), 6.70 (d, J = 2.3 Hz, 1H), 7.11-7.26 (m, 7H), 7.46 (d, J
= 8.5 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.70 (s, 1H), 10.95 (s, 1H).
13C-NMR (75 MHz, CDCl3)  26.9, 29.1, 30.1, 31.5, 36.1, 41.6, 110.3,
112.4, 113.7, 117.8, 118.7, 120.8, 123.0, 124.2, 125.5, 125.8, 128.5
(2), 128.6 (2), 139.7, 142.9, 153.6, 156.7. ESI-MS m/z 426 [M + Na]+.
Elemental analysis calcd (%) for C24H25N3O3: C 71.44, H 6.25, N
10.41; found: C 71.76, H 6.09, N 10.27.
N-(5-Hydroxy-2-nitrophenyl)acetamide (16). To a solution of 15
(1.0 g, 6.62 mmol) in acetic anhydride (5.0 mL), a mixture of conc.
HNO3 (3.6 mL) and acetic acid (3.6 mL) was added and the reaction
was stirred at 25 °C for 8 h. 2N HCl (5.0 mL) was added to quench
the excess of acetic anhydride and the mixture was diluted with water.
The resulting precipitate was filtered and washed with cold water to
afford the pure title compound (98% yield) as a yellow solid. 1H NMR
3-Methoxy-2-nitroaniline (10). Compound 9 (1.2 g, 4.47 mmol) was
dissolved in 1.4 mL of TFA and the resulting solution was stirred at
0 °C for 1.5 h. Then the solvent was evaporated and the residue was
dissolved in EtOAc and washed with a saturated aqueous NaHCO3.
The organic phase was dried over anhydrous Na2SO4, filtered and
1
concentrated affording the title compound (99% yield). H NMR (300
MHz, CDCl3) δ 3.87 (s, 3H), 4.43 (brs, 2H), 6.30 (dd, J = 8.2, 0.9 Hz,
1H), 6.36 (dd, J = 7.5, 1.5 Hz, 1H), 7.16 (t, J = 8.2 Hz, 1H). ESI-MS
m/z 169 [M + H]+.
1-(3-Methoxy-2-nitrophenyl)-1H-pyrrole (11). To a solution of 3-
methoxy-2-nitroaniline 10 (500 mg, 2.97 mmol) in acetic acid (10.0
mL) and water (2.0 mL), 2,5-dimethoxytetrahydrofuran (385 µL, 2.97
mmol) in acetic acid (1.0 mL) was added dropwise. The solution was
heated at 100 °C for 15 min. After removal of the solvent, the dark-
brown reaction mixture was diluted with EtOAc and neutralized with
8
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10.1002/cmdc.201800397
ChemMedChem
FULL PAPER
(300 MHz, CDCl3) δ 2.24 (s, 3H), 7.07 (d, J = 7.6 Hz, 1H), 7.35 (br s,
1H), 7.46 (s, 1H), 8.07 (d, J = 9.4 Hz, 1H), 10.82 (s, 1H). ESI-MS m/z
195 [M-H]-.
= 8.8 Hz, 1H). ESI-MS m/z 212 [M + H]+ .To a solution of methyl 4-
methoxy-2-nitrobenzoate (5.07 mmol) in EtOAc (10.0 mL) SnCl2∙2H2O
(3.57 g, 15.84 mmol) was added and the reaction was stirred at 25 °C
for 2 h. The reaction was quenched with a saturated solution of
NaHCO3 and the aqueous phase was extracted with EtOAc (3 x 25.0
mL). The combined organic layers were dried over Na2SO4, filtered
and evaporated to afford methyl 2-amino-4-methoxybenzoate
(quantitative yield) as a pale yellow solid which was used in the next
step without any further purification. 1H NMR (300 MHz, CDCl3) δ 3.78
(s, 3H), 3.83 (s, 3H), 5.52 (br s, 2H), 6.11 (d, J = 2.4 Hz, 1H), 6.23 (dd,
J = 9.1, 2.4 Hz, 1H), 7.78 (d, J = 9.1, 1H). ESI-MS m/z 182 [M + H]+,
204 [M + Na]+.
4-Amino-3-(1H-pyrrol-1-yl)phenol (17). Compound 16 (1.2 g, 6.49
mmol) was dissolved in concentrated HCl (10.0 mL) and the resulting
suspension was stirred at 110 °C for 5 min. The mixture was cooled
to 25 °C, diluted with water and the aqueous phase was extracted with
EtOAc (3 x 15.0 mL). The combined organic layers were dried over
Na2SO4, filtered, and concentrated to afford the intermediate 3-amino-
4-nitrophenol (95% yield) that was carried on without any further
1
purification. H NMR (300 MHz, acetone-d6) δ 6.24 (dd, J = 9.4, 2.6
Hz, 1H), 6.45 (d, J = 2.4 Hz, 1H), 7.01 (br s, 2H), 7.97 (d, J = 9.4 Hz,
1H), 9.37 (s, 1H). ESI-MS m/z 153 [M-H]-. A stirred solution of 3-
Starting from methyl 2-amino-4-methoxybenzoate (300 mg, 1.70
mmol), the title compound was obtained following the same procedure
reported for 17. The crude was purified by means of chromatography
on silica gel (petroleum ether/chloroform 1:1) to afford pure compound
20 (87% yield) as a withe solid. 1H NMR (300 MHz, CDCl3) δ 3.70 (s,
3H), 3.83 (s, 3H), 6.32 (t, J = 2.2 Hz, 2H), 6.82 (t, J = 2.2 Hz, 2H),
6.87-6.91 (m, 2H), 7.84 (d, J = 8.3 Hz, 1H). ESI-MS m/z 254 [M + Na]+.
Methyl 2-(2-cyano-1H-pyrrol-1-yl)-4-methoxybenzoate (21). To an
ice-cooled solution of dry DMF (1.0 mL) and dry 1,2-DCE (2.0 mL),
(COCl)2 (135 µL, 1.55 mmol) was added. The mixture was allowed to
warm to 25 °C and stirred for 15 min. Then it was cooled to 0 °C and
a solution of 20 (325 mg, 1.41 mmol) in dry 1,2-DCE (1.0 mL) was
added. The reaction was stirred at 25 °C for further 15 min. A solution
of NH2OH∙HCl (108 mg, 1.55 mmol) and pyridine (125 µL, 1.55 mmol)
in dry DMF (1.0 mL) was added and the mixture was stirred at 120 °C
for 10 h. A saturated solution of NaHCO3 was added dropwise and the
aqueous phase was extracted with EtOAc (3 x 10.0 mL). The
combined organic layers were dried over anhydrous Na2SO4, filtered,
and evaporated. The crude was purified by means of chromatography
on silica gel (petroleum ether/diethyl ether 1:1) to afford title
compound (47% yield) as a colourless solid. 1H NMR (300 MHz,
CDCl3) δ 3.72 (s, 3H), 3.90 (s, 3H), 6.33-6.35 (m, 1H), 6.89 (d, J = 2.6
Hz, 1H), 6.92-6.94 (m, 1H), 6.97 (dd, J = 4.0, 1.6 Hz, 1H), 7.03 (dd, J
= 8.8, 2.3 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H). ESI-MS m/z 279 [M + Na]+.
9-Methoxy-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-6(5H)-one (22).
To a stirred suspension of 21 (290 mg, 1.13 mmol) and CoCl2 (294
mg, 2.26 mmol) in MeOH (10.0 mL), NaBH4 (420 mg, 11.30 mmol)
was added and reaction was stirred at 25 °C for 20 min. 2 N HCl was
added and solvent was removed under reduced pressure. The
solution was basified with 2 N NaOH and the aqueous phase was
extracted with ethyl acetate (3 x 10.0 mL). The combined organic
layers were dried over Na2SO4, filtered, and evaporated. The crude
was purified by means of chromatography on silica gel (EtOAc) to
afford title compound (64% yield) as a colourless solid. 1H NMR (300
MHz, CDCl3) δ 3.90 (s, 3H), 4.20 (br d, 2H), 6.12 (s, 1H), 6.28 (s, 1H),
6.87-6.92 (m, 2H), 7.03 (s, 1H), 7.32 (br s, 1H), 7.97 (d, J = 8.8 Hz,
1H). ESI-MS m/z 229 [M + H]+, 251 [M + Na]+.
amino-4-nitrophenol
(950
mg,
6.17
mmol)
and
2,5-
dimethoxytetrahydrofuran (960 µL, 7.41 mmol) in 1,4-dioxane (20.0
mL), was stirred at 120 °C for 15 min before adding 5N HCl (1.5 mL).
The mixture was stirred at 110 °C for further 5 min, then cold water
was added and the resulting white precipitate was filtered and washed
with cold water to afford the pure 4-nitro-3-(1H-pyrrol-1-yl)phenol
(70% yield) as an amorphous solid. 1H NMR (300 MHz, CDCl3) δ 6.36
(d, J = 1.5 Hz, 2H), 6.79 (d, J = 1.5 Hz, 2H), 6.85 (s, 1H), 7.17 (d, J =
2.6 Hz, 1H), 7.92 (d, J = 8.5 Hz). ESI-MS m/z 203 [M-H]-.
To a solution of 4-nitro-3-(1H-pyrrol-1-yl)phenol (400 mg, 1.96 mmol)
in EtOAc (10.0 mL) SnCl2∙2H2O (2.2 g, 9.8 mmol) was added and the
reaction was stirred at 25 °C for 2 h. A saturated solution of NaHCO3
was added and the aqueous phase was extracted with EtOAc (3 x
20.0 mL). The combined organic layers were dried over Na2SO4,
filtered and evaporated. The crude was purified by means of
chromatography on silica gel (petroleum ether/diethyl ether 3:2) to
give pure compound 17 (85% yield) as a colourless solid. 1H NMR
(300 MHz, CDCl3) δ 4.09 (br s, 2H), 6.33 (s, 2H), 6.62-6.67 (m, 3H),
7.80 (s, 2H), 7.16 (s, 1H). ESI-MS m/z 175 [M + H]+.
8-Hydroxypyrrolo[1,2-a]quinoxalin-4(5H)-one (18). Starting from
17 (100 mg, 0.57 mmol), the title compound was obtained following
the same procedure reported for 13. The crude was purified by means
of chromatography on silica gel (MeOH/chloroform 1:20) to provide
title compound (36% yield) as a colourless solid. 1H NMR (300 MHz,
DMSO-d6) δ 6.63 (m, 1H), 6.73-6.76 (m, 1H), 6.70 (m, 1H), 7.09-7.12
(m, 1H), 7.36 (s, 1H), 8.01 (m 1H), 9.57 (s, 1H), 11.00 (s, 1H). ESI-
MS m/z 199 [M-H]-.
4-Oxo-4,5-dihydropyrrolo[1,2-a]quinoxalin-8-yl
6-
phenylhexylcarbamate (5g). Starting from 18 (40 mg, 0.20 mmol)
and 6-phenylhexyl isocyanate (165 mg, 0.80 mmol), the title
compound was obtained following the same procedure reported for
5a. The crude was purified by means of chromatography on silica gel
(diethyl ether/chloroform 2:1) to afford title compound (10% yield) as
1
a colourless solid. H NMR (300 MHz, CD3OD) δ 1.39-1.42 (m, 4H),
1.55-1.67 (m, 4H), 2.58 (t, J = 7.5 Hz, 2H), 3.18 (t, J = 7.0 Hz, 2H),
6.69-6.71 (m, 1H), 7.05 (dd, J = 8.8, 2.4 Hz, 1H), 7.13-7.30 (m, 7H),
7.73 (d, J = 2.4 Hz, 1H), 7.94-7.96 (m, 1H); 13C-NMR (75 MHz,
CD3OD) δ 26.5, 28.8, 29.5, 31.5, 35.7, 40.9, 108.6, 112.3, 113.2,
117.2, 118.2, 119.4, 123.0, 123.7, 125.5 (2C), 128.1 (2C), 128.2 (2C),
142.7, 147.3, 155.9, 156.7. ESI-MS m/z 426 [M + Na]+. Elemental
analysis calcd (%) for C24H25N3O3: C 71.44, H 6.25, N 10.41; found: C
71.37, H 6.51, N 10.58.
Methyl 4-methoxy-2-(1H-pyrrol-1-yl)benzoate (20). To an ice-
cooled solution of 19 (1.0 g, 5.07 mmol) in MeOH (10.0 mL), SOCl2
(740 µL, 10.15 mmol) was added dropwise and the reaction mixture
was stirred at 25 °C for 10 h. Solvent was removed under reduced
pressure and the residue was taken up with EtOAc and washed with
a saturated solution of NaHCO3. The organic phase was dried over
Na2SO4, filtered and evaporated to obtain methyl 4-methoxy-2-
nitrobenzoate (quantitative yield) which was carried on without any
further purification. 1H NMR (300 MHz, CDCl3) δ 3.84 (s, 3H), 3.99 (s,
3H), 7.31 (dd, J = 8.8, 2.6 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 7.88 (d, J
6-Oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-6-
phenylhexylcarbamate (5h). Starting from 22, hydroxy-4H-
benzo[f]pyrrolo[1,2-a][1,4]diazepin-6(5H)-one was obtained following
the same procedure reported for 14. The crude was purified by means
of chromatography on silica gel (chloroform/EtOAc 1:1) to afford
compound hydroxy-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-6(5H)-one
1
(50% yield) as a colourless solid. H NMR (300 MHz, acetone-d6) δ
4.22 (s, 2H), 6.11 (d, J = 2.3 Hz, 1H), 6.20-6.24 (m, 1H), 6.85-6.93 (m,
2H), 7.10-7.12 (m, 1H), 7.81-7.86 (m, 2H), 9.42 (br s, 1H). ESI-MS
m/z 213 [M-H]-. Starting from hydroxy-4H-benzo[f]pyrrolo[1,2-
a][1,4]diazepin-6(5H)-one (14 mg, 0.06 mmol) and 6-phenylhexyl
isocyanate 7b (50 mg, 0.25 mmol), the title compound was obtained
following the same procedure reported for 5a. The crude was purified
by means of chromatography on silica gel (MeOH/DCM 1:20) to afford
title compound 5h (60% yield) as a colourless solid. 1H NMR (300
MHz, CDCl3) δ 1.38-1.41 (m, 4H), 1.57-1.70 (m, 4H), 2.62 (t, J = 7.6
9
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10.1002/cmdc.201800397
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Hz, 2H), 3.27 (q, J = 6.1 Hz, 2H), 4.21 (s, 2H), 5.14 (br s, 1H), 6.11
(br d, 1H), 6.27 (t, J = 2.9 Hz, 1H), 7.01 (br d, 1H), 7.12-7.31 (m, 7H),
7.96 (d, J = 8.5 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 26.8, 29.1, 29.9,
31.6, 36.1, 37.7, 41.6, 107.3, 110.8, 115.7, 119.3, 120.9, 125.9, 128.5
(2C), 128.6 (2C), 132.2, 133.6, 138.6, 142.8, 153.7, 154.6, 169.6. ESI-
MS m/z 418 [M + H]+.
colourless solid. 1H NMR (300 MHz, CDCl3) δ 3.46-3.54 (m, 2H), 3.71
(t, J = 4.8 Hz, 2H), 3.80-3.92 (m, 2H), 4.07-4.25 (m, 4H), 5.66 (t, J =
4.5 Hz, 1H), 6.43 (t, J = 3.3 Hz, 1H), 6.89-6.99 (m, 3H), 7.00-7.17 (m,
4H), 7.20 (s, 1H), 7.23-7.34 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 41.3,
43.2, 67.5, 69.9, 70.1, 111.2, 114.8, 116.8, 118.7, 119.9, 121.4, 125.1,
128.7, 129.0, 129.3, 129.8, 140.0, 151.7, 154.4, 158.8. ESI-MS m/z
422 [M + H]+ (100). Elemental analysis calcd (%) for C23H23N3O5: C
65.55, H 5.50, N 9.97; found: C 65.23, H 5.29, N 9.76.
Elemental analysis calcd (%) for C25H27N3O3: C 71,92, H 6,52, N
10,06; found: C 72.18, H 6.27, N 10.34.
4-Oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-(2-
(2-(2-fluorophenoxy)ethoxy)ethyl) carbamate (5k). Starting from
25 (10 mg, 0.05 mmol) and isocyanate 26b (14 mg, 0.06 mmol), the
title compound was obtained following the same procedure reported
for 5a. The crude was purified by means of chromatography on silica
gel (MeOH/DCM 1:20) to afford title compound (47% yield) as a
Methyl 1-(2-cyano-5-methoxyphenyl)-1H-pyrrole-2-carboxylate
(24). To a stirred solution of methyl 1H-pyrrole-2-carboxylate (1.0 g,
8.00 mmol) in dry DMF (25.0 mL), Cs2CO3 (13.0 g, 40.00 mmol) and
23 (1.5 g, 9.60 mmol) were added and the reaction was stirred at
50 °C under N2 atmosphere for 12 h. Solvent was removed under
reduced pressure and the crude was taken up with a saturated
solution of NH4Cl. The aqueous phase was extracted with DCM (3 x
25.0 mL) and the combined organic layers were dried over Na2SO4,
filtered, and evaporated. The crude was purified by means of
chromatography on silica gel (ethyl acetate/petroleum ether 1:6) to
afford compound title compound (30% yield) as a colourless solid. 1H
NMR (300 MHz, CDCl3) δ 3.74 (s, 3H), 3.87 (s, 3H), 6.37 (dd, J = 3.8,
2.9 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.94 (q, J = 2.6 Hz, 1H), 7.00
(dd, J = 8.7, 2.5 Hz, 1H), 7.13 (dd, J = 3.9, 1.7 Hz, 1H), 7.64 (d, J =
8.7 Hz, 1H). ESI-MS m/z 257 [M + H]+, 279 [M + Na]+.
9-Hydroxy-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-4-
one (25). Starting from 24 (600 mg, 2.34 mmol), 9-methoxy-5,6-
dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-4-one was obtained
following the same procedure reported for 22. The crude was purified
by means of chromatography on silica gel (ethyl acetate/petroleum
ether 1:1) to afford compound 9-methoxy-5,6-dihydro-4H-
benzo[f]pyrrolo[1,2-a][1,4]diazepin-4-one (71% yield) as a colourless
solid. 1H NMR (300 MHz, CDCl3) δ 3.80 (s, 3H), 4.14 (s, 2H), 6.36-
6.47 (m, 1H), 6.75 (dd, J = 8.3, 1.9 Hz, 1H), 6.83 (d, J = 2.3 Hz, 1H),
7.07-7.15 (m, 1H), 7.15-7.24 (m, 2H), 8.23 (br s, 1H). ESI-MS m/z 229
[M + H]+, 251 [M + Na]+.
1
colourless solid. H NMR (300 MHz, CDCl3) δ 3.49 (q, J = 10.3 Hz,
2H), 3.71 (t, J = 5.0 Hz, 2H), 3.84-3.93 (m, 2H), 4.14-4.26 (m, 4H),
5.65 (t, J = 5.6 Hz, 1H), 6.42 (dd, J = 3.8, 2.9 Hz, 1H), 6.86-6.96 (m,
1H), 6.96-7.11 (m, 4H), 7.11-7.17 (m, 3H), 7.17-7.23 (m, 1H), 7.28 (s,
1H); 13C NMR (75 MHz, CDCl3) δ 41.3, 43.2, 69.3, 69.7, 70.2, 111.3,
115.8, 116.60 (d, JC-F = 18.2 Hz), 116.9, 118.9, 120.0, 121.98 (d, JC-F
= 6.8 Hz), 124.57 (d, JC-F = 3.9 Hz), 125.3, 128.8, 129.3, 140.0, 146.93
(d, JC-F = 10.6 Hz), 151.7, 153.13 (d, JC-F = 245.6 Hz), 154.4, 164.2.
ESI-MS m/z 440 [M + H]+. Elemental analysis calcd (%) for
C23H22FN3O5: C 62.86, H 5.05, N 9.56; found: C 63.12, H 5.18, N 9.47.
4-Oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-(2-
(2-(4-fluorophenoxy)ethoxy)ethyl) carbamate (5l). Starting from 25
(10 mg, 0.05 mmol) and isocyanate 26c (14 mg, 0.06 mmol), the title
compound was obtained following the same procedure reported for
5a. The crude was purified by means of chromatography on silica gel
(MeOH/DCM 1:20) to afford title compound (43% yield) as a
colourless solid. 1H NMR (300 MHz, CDCl3) δ 3.50 (q, J = 5.2 Hz, 2H),
3.70 (t, J = 5.0 Hz, 2H), 3.82-3.89 (m, 2H), 4.07-4.14 (m, 2H), 4.20 (br
d, J = 3.4 Hz, 2H), 5.61 (t, J = 5.7 Hz, 1H), 6.43 (t, J = 3.8 Hz, 1H),
6.81-6.92 (m, 2H), 6.92-7.11 (m, 4H), 7.11-7.17 (m, 2H), 7.17-7.22
(m, 1H), 7.26-7.31 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 41.3, 43.1,
68.2, 69.9, 70.1, 111.3, 115.9 (d, JC-F = 8.0 Hz), 116.1 (d, JC-F = 23.1
Hz), 116.8, 118.8, 119.9, 125.2, 128.6, 128.9, 129.2, 140.0, 151.6,
154.3, 155.0 (d, JC-F = 2.1 Hz), 157.6 (d, JC-F = 238.7 Hz), 164.2. ESI-
MS m/z 440 [M + H]+. Elemental analysis calcd (%) for C23H22FN3O5:
C 62.86, H 5.05, N 9.56; found: C 62.67, H 4.78, N 9.21.
Starting
from
9-methoxy-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-
a][1,4]diazepin-4-one (380 mg, 1.66 mmol), the title compound was
obtained following the same procedure reported for 14. The crude was
purified by means of chromatography on silica gel (EtOAc) to afford
title compound (50% yield) as a colourless solid. 1H NMR (300 MHz,
CD3OD) δ 4.08 (s, 2H), 6.42 (t, J = 3.1 Hz, 1H), 6.74 (dd, J = 8.2, 2.1
Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H), 7.02 (dd, J = 3.8, 1.1 Hz, 1H), 7.21
(d, J = 8.2 Hz, 1H), 7.32-7.40 (m, 1H). ESI-MS m/z 215 [M + H]+, 237
[M + Na]+.
Molecular Docking Studies
a) Ligand preparation
4-Oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-(6-
phenylhexyl)carbamate (5i). Starting from 25 (10 mg, 0.05 mmol)
and 6-phenylhexyl isocyanate 7b (13 mg, 0.06 mmol), the title
compound was obtained following the same procedure reported for
5a. The crude was purified by means of chromatography on silica gel
(MeOH/DCM 1:20) to afford the title compound (52% yield) as a
colourless solid. 1H NMR (300 MHz, CDCl3) δ 1.33-1.44 (m, 4H), 1.48-
1.71 (m, 4H), 2.61 (t, J = 5.1 Hz, 2H), 3.24 (q, J = 6.6 Hz, 2H), 4.17
(br d, 2H), 5.25 (t, J = 4.2 Hz, 1H), 6.42 (t, J = 3.3 Hz, 1H), 7.01 (dd,
J = 8.2, 2.1 Hz, 1H), 7.07-7.22 (m, 6H), 7.22-7.33 (m, 3H), 7.45 (t, J =
5.2 Hz, 1H). ESI-MS m/z 375 [M + H]+; HRMS (ESI) m/z calcd for
Three-dimensional structures of all compounds in this study were built
by means of Maestro (Maestro, version 9.3, Schrödinger, LLC, New
York, NY, 2012). Molecular energy minimizations were performed by
means of MacroModel (MacroModel, version 9.9, Schrödinger, LLC,
New York, NY, 2012) using the Optimized Potentials for Liquid
Simulations-all atom (OPLS-AA) force field 2005.[41] The solvent
effects were simulated using the analytical Generalized-
Born/Surface-Area (GB/SA) model,[42] and no cutoff for nonbonded
interactions was selected. Polak-Ribiere conjugate gradient (PRCG)
method with 1000 maximum iterations and 0.001 gradient
convergence threshold was employed. All compounds reported in this
paper were treated by LigPrep application (LigPrep, version 2.5,
Schrödinger, LLC, New York, NY, 2012), implemented in Maestro
suite 2011, generating the most probable ionization state of any
possible enantiomers and tautomers at cellular pH value (7 ± 0.5) and
also for avoiding potential error in the structures.
+
C25H28N3O3 : 418,2125, found 418,2117 [M + H]+; calcd for:
+
C25H27N3NaO3 : 440,1945 found 440,1936 [M + Na]+, calcd for
+
C50H55N6O6 : 835,4178 found 835,4168 [2M + H]+. Elemental
analysis calcd (%) for C25H27N3O3: C 71.92, H 6.52, N 10.06; found
C 72.11, H 6.35, N 10.20.
b) Protein preparation
The three-dimensional structure of FAAH (PDB ID: 3PPM[43]) was
4-Oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]diazepin-9-yl-(2-
(2-phenoxyethoxy)ethyl) carbamate (5j). Starting from 25 (10 mg,
0.05 mmol) and isocyanate 26a (13 mg, 0.06 mmol), the title
compound was obtained following the same procedure reported for
5a. The crude was purified by means of chromatography on silica gel
(MeOH/DCM 1:20) to afford title compound (55% yield) as a
taken from PDB and imported into Schrodinger Maestro molecular
̈
modeling environment. Water molecules and compounds used for the
crystallization were removed from the available experimental
structure. The obtained enzyme was submitted to protein preparation
10
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10.1002/cmdc.201800397
ChemMedChem
FULL PAPER
wizard implemented in Maestro suite 2012. This protocol through a
series of computational steps, allowed us to obtain a reasonable
starting structure of the protein for molecular docking calculations by
a series of computational steps. In particular, we performed three
steps to (1) add hydrogens, (2) optimize the orientation of hydroxyl
groups, Asn, and Gln, and the protonation state of His, and (3) perform
a constrained refinement with the impref utility, setting the max RMSD
of 0.30. The impref utility consists of a cycles of energy minimization
based on the impact molecular mechanics engine and on the
OPLS_2005 force field.[41]
concentration of compounds (0.1–50 μM). Nonspecific binding was
defined by 10 µM of WIN55,212-2 as the heterologous competitor (Ki
values 9.2 nM and 2.1 nM respectively for CB1 and CB2 receptor).
IC50 values were determined for compounds showing ˃50%
displacement at 10 µM. All compounds were tested following the
procedure described by the manufacturer (Perkin Elmer, Italy).
Displacement curves were generated by incubating drugs with [3H]-
CP-55,940 for 90 minutes at 30 ºC. Ki values were calculated by
applying the Cheng-Prusoff equation to the IC50 values (obtained by
GraphPad) for the displacement of the bound radioligand by
increasing concentrations of the test compound. Data represent mean
values of three separate experiments performed in duplicate and are
expressed as the average of Ki ± SD.
c) Molecular Docking
Molecular docking was carried out using the Schrödinger suite 2012
by applying the IFD protocol (Schrödinger Suite 2012 Induced Fit
Docking protocol; Glide version 5.8, Schrödinger, LLC, New York, NY,
2012; Prime version 3.1, Schrödinger, LLC, New York, NY, 2012).
This procedure induces conformational 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 calculation were built taking into account the
centroid of the co-crystallized ligand for FAAH enzyme. 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. The choice of IFD coupled to XP was done after the
assessment of two docking protocols (IFD-SP and IFD-XP). From the
computational outputs we observed that IFD-XP was able to correctly
accommodate the co-crystallized ligands, belonging to the FAAH
crystal structures 3PPM, 3QJ9 and 2VYA, with lower RMSD than IFD-
SP (data not shown).
Cellular in vitro study
a) Cell culture and treatments
IMR32 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-glutammine
(Lonza, Milan, Italy) and 1% of penicillin/streptomycin antibiotics
(Lonza, Milan, Italy) at 37 °C in 5% CO2. The different formulations (3,
5h, and 5i) were dissolved in DMSO as stock solutions at the final
concentration of 10 mM. Stock solutions were then diluted with cell
culture medium, EMEM with Earle's Balanced Salt Solution, to obtain
an intermediate dose solution (100 μM), to be used for the used
dilutions (50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM and 0.1 µM). Control
vehicle was represented by DMSO ranging from 0.5% to 0.001%.
b) Cytotoxicity determination
IMR32 cells were seeded 100,000 cells/well in 96-wells plate and
were grown to confluence, then were treated with the tested
substances in EMEM medium supplemented with 10% 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). Cytotoxicity was determined by LDH
release in the media, measured by enzymatic assay: in the first step
NADþ is reduced to NADH/Hþ by LDH-catalyzed conversion of lactate
to pyruvate; in the second step the catalyst (diaphorase) transfers
H/Hþ from NADH/Hþ to tetrazolium salt which is reduced to formazan.
For total release of intracellular LDH (positive control of 100% cell
death), a triplicate set of IMR32 cells were treated with 1% Triton X-
100 for 30 min at 37 °C, according to the manufacturer's protocol
(Roche, Mannheim, Germany). The amount of LDH in the supernatant
was determined and calculated according to kit instructions. The
amount of LDH release in each sample was determined by measuring
the absorbance at 490 nm using a microplate spectrophotometer. 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.
Enzymatic assays
FAAH and MAGL activities were detected in cells as previously
described.[44-45] In particular, AEA hydrolysis was measured by
incubating samples with the 10,000x g membrane fraction of rat brain
(70 μg/sample) and synthetic N-arachidonoyl-[14C]-ethanolamine
(110mCi/mmol, ARC St. Louis, MO, USA) properly diluted with AEA
(Tocris Bioscience, Avonmouth, Bristol, UK) in Tris–HCl 50 mM, at pH
9.00–10.00 at 37 °C for 30 min. After incubation, the amount of [14C]-
ethanolamine produced was measured by scintillation counting of the
aqueous phase after extraction of the incubation mixture with 2
volumes of CHCl3/MeOH 1:1 (by vol.). 2-AG hydrolysis was measured
by incubating the 10,000xg cytosolic fraction of COS-7 cells (100
µg/sample), which contains high levels of MAGL, and synthetic 2-
arachidonoyl-[3H]-glycerol (40 Ci/mmol, ARC St. Louis, MO, USA)
properly diluted with 2-AG (Cayman Chemicals, Ann Arbor, MI, USA)
in Tris–HCl 50 mM, at pH 7.0 at 37°C for 20 min. After incubation, the
amount of [3H]-glycerol produced was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with 2 volumes of CHCl3/MeOH 1:1 (by vol.). All data are
expressed as means ± SD of three separate experiments of the
concentration exerting 50% inhibition of [14C]-AEA hydrolysis (IC50)
calculated by fitting sigmoidal concentration response curves by
GraphPad.
c) Nuclear proteins extraction
For nuclear extracts, IMR32 cells were seeded in 100 mm petri (3 x
106 cells). After treatments with different compounds, cells were
detached, washed with ice-cold PBS 1X and cell pellets were
resuspended in hypotonic buffer containing 10 mmol/L HEPES (pH
7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.3% Nonidet P-40, 0.5
mmol/L dithithreitiol, 0.5 mmol/L phenylmethylsulphonyl fluoride and
protease and phosphatase inhibitor cocktails. The lysates were
incubated for 15 minutes on ice with intermitted mixing and then
centrifuged at 24500 x g for 15 min at 4 °C. The supernatant
containing the cytosolic proteins was removed and pellet containing
the nuclei were resuspended in extraction buffer containing 20 mmol/L
HEPES (pH 7.9), 0,6 mol/L KCl, 1.5 mmol/L MgCl2, 20% glycerol, 0.5
mmol/L phenylmethylsulphonyl fluoride and protease and
phosphatase inhibitor cocktails and then incubated for 30 minutes on
Competition Binding Assay
Membranes from HEK-293 cells over-expressing the respective
human recombinant CB1 receptor (Bmax= 2.5 pmol/mg protein) and
human recombinant CB2 receptor (Bmax= 4.7 pmol/mg protein) were
incubated
with
[3H]-CP-55,940
(0.14nM/Kd=0.18nM
and
0.084nM/Kd=0.31nM, respectively for CB1 and CB2 receptor) as the
high affinity ligand. Competition curves were performed as previously
reported[46] by displacing [3H]-CP-55,940 with increasing
11
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10.1002/cmdc.201800397
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FULL PAPER
ice with intermitted mixing. Samples were centrifuged at 21100 x g for
15 minutes to obtain supernatants containing nuclear fractions.
Protein concentration was determined by Bradford analysis (Biorad
protein assay; Biorad, Milan, Italy).
Behavioural measurements were performed on 12 mice for each
treatment carried out in 2 different experimental sets. Results were
expressed as mean ± S.E.M. The analysis of variance of behavioural
data was performed by one way ANOVA, a Bonferroni's significant
difference procedure was used as post-hoc comparison. P values of
less than 0.05 or 0.01 were considered significant. Investigators were
blind to all experimental procedures. Data were analysed using the
“Origin 9” software (OriginLab, Northampton, USA).
d) Western blot analysis
After protein quantification, 60 µg boiled proteins were loaded into
10% sodium dodecyl sulphatepolyacrylamide electrophoresis gels
and separated by molecular size. Gels were electro-blotted onto
nitrocellulose membranes and then blocked for 90 minutes 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: anti-NF-kB, p65 subunit, diluted 1:1000
(Millipore, Billerica, Massachusetts). The membranes were finally
incubated with the peroxidase-conjugated secondary anti-Rabbit
antibody (1:5000) for 1 h. The bound antibodies were detected by
chemiluminescence (Biorad, Milan, Italy). β-Actin was used as loading
control. Images of the bands were digitized using an Epson Stylus
SX405 scanner and the densitometry analysis was performed using
Image-J software.
Acknowledgements
The authors wish to thank Miur PRIN 20088SPEFN_005.
Keywords: endocannabinoid system, fatty acid amide hydrolase,
covalent inhibitors, neuropathic pain, serine hydrolase
Abbreviations. FAAH, fatty acid amide hydrolase; ECs, endocannabinoids;
CBR, cannabinoid receptor; FAEs, fatty acid ethanolamides; AEA, anandamide;
MAGL, monoacylglycerol lipase; CNS, central nervous system; SAR, structure-
activity relationship; THF, tetrahydrofuran; TEA, triethylamine; DMF,
dimethylformamide; 1,2-DCE, 1,2-dichloroethane; DCM, dichloromethane;
DMSO, dimethylsulfoxide; CMC, carboxymethylcellulose; PDB, Protein Data
Bank.
e) Statistical analysis
For each of the variables tested, two-way analysis of variance
(ANOVA) was used. A significant result was indicated by a p
valueꢀ<ꢀ0.05. All the results are expressed as meanꢀ±ꢀ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).
Received: ((will be filled in by the editorial staff))
Published online: ((will be filled in by the editorial staff))
References:
Pharmacological in vivo study
a) Animals
[1]
[2]
[3]
[4]
[5]
[6]
M. P. Patricelli, B. F. Cravatt, Biochemistry 1999, 38, 14125-
14130.
D. K. Giang, B. F. Cravatt, Proc Natl Acad Sci U S A 1997, 94,
2238-2242.
M. P. Patricelli, M. A. Lovato, B. F. Cravatt, Biochemistry 1999, 38,
9804-9812.
K. Tsuboi, Y. X. Sun, Y. Okamoto, N. Araki, T. Tonai, N. Ueda, J
Biol Chem 2005, 280, 11082-11092.
M. Karlsson, J. A. Contreras, U. Hellman, H. Tornqvist, C. Holm, J
Biol Chem 1997, 272, 27218-27223.
W. A. Devane, L. Hanus, A. Breuer, R. G. Pertwee, L. A.
Stevenson, G. Griffin, D. Gibson, A. Mandelbaum, A. Etinger, R.
Mechoulam, Science 1992, 258, 1946-1949.
R. Mechoulam, S. Ben-Shabat, L. Hanus, M. Ligumsky, N. E.
Kaminski, A. R. Schatz, A. Gopher, S. Almog, B. R. Martin, D. R.
Compton, et al., Biochem Pharmacol 1995, 50, 83-90.
J. Zias, H. Stark, J. Sellgman, R. Levy, E. Werker, A. Breuer, R.
Mechoulam, Nature 1993, 363, 215.
A. H. Lichtman, C. C. Shelton, T. Advani, B. F. Cravatt, Pain 2004,
109, 319-327.
F. Massa, G. Marsicano, H. Hermann, A. Cannich, K. Monory, B.
F. Cravatt, G. L. Ferri, A. Sibaev, M. Storr, B. Lutz, J Clin Invest
2004, 113, 1202-1209.
S. Huitron-Resendiz, M. Sanchez-Alavez, D. N. Wills, B. F.
Cravatt, S. J. Henriksen, Sleep 2004, 27, 857-865.
S. A. Varvel, L. E. Wise, F. Niyuhire, B. F. Cravatt, A. H. Lichtman,
Neuropsychopharmacology 2007, 32, 1032-1041.
M. Seierstad, J. G. Breitenbucher, J Med Chem 2008, 51, 7327-
7343.
Y. Miyake, J. J. Keusch, L. Wang, M. Saito, D. Hess, X. Wang, B.
J. Melancon, P. Helquist, H. Gut, P. Matthias, Nat Chem Biol 2016,
12, 748-754.
A. C. M. van Esbroeck, A. P. A. Janssen, A. B. Cognetta, 3rd, D.
Ogasawara, G. Shpak, M. van der Kroeg, V. Kantae, M. P.
Baggelaar, F. M. S. de Vrij, H. Deng, M. Allara, F. Fezza, Z. Lin, T.
van der Wel, M. Soethoudt, E. D. Mock, H. den Dulk, I. L. Baak, B.
I. Florea, G. Hendriks, L. De Petrocellis, H. S. Overkleeft, T.
Hankemeier, C. I. De Zeeuw, V. Di Marzo, M. Maccarrone, B. F.
Cravatt, S. A. Kushner, M. van der Stelt, Science 2017, 356, 1084-
1087.
M. Duenas, B. Ojeda, A. Salazar, J. A. Mico, I. Failde, J Pain Res
2016, 9, 457-467.
B. K. Lau, C. W. Vaughan, Front Pharmacol 2014, 5, 28.
M. H. Bracey, M. A. Hanson, K. R. Masuda, R. C. Stevens, B. F.
Cravatt, Science 2002, 298, 1793-1796.
Male CD-1 albino mice (Envigo, Varese, Italy) weighing approximately
22–25 g at the beginning of the experimental procedure, were used.
Animals were housed in CeSAL (Centro Stabulazione Animali da
Laboratorio, University of Florence) and used at least 1 week after
their arrival. Ten mice were housed per cage (size 26 × 41 cm);
animals were fed a standard laboratory diet and tap water ad libitum,
and kept at 23 ± 1 °C with a 12 h light/dark cycle, light at 7 a.m. All
animal manipulations were carried out according to the Directive
2010/63/EU of the European parliament and of the European Union
council (22 September 2010) on the protection of animals used for
scientific purposes. The ethical policy of the University of Florence
complies with the Guide for the Care and Use of Laboratory Animals
of the US National Institutes of Health (NIH Publication No. 85-23,
revised 1996; University of Florence assurance number: A5278-01).
Formal approval to conduct the experiments described was obtained
from the Animal Subjects Review Board of the University of Florence.
Experiments involving animals have been reported according to
ARRIVE guidelines.All efforts were made to minimize animal suffering
and to reduce the number of animals used.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
b) Oxaliplatin-induced neuropathic pain model and compound
administration.
Oxaliplatin (2.4 mg kg-1) was dissolved in 5% glucose solution and i.p.
administered for 5 consecutive days every week for 2 weeks.[40] On
day 14, compounds 3, 5h and 5i were administered p.o.[47-48] using 1%
carboxymethylcellulose (CMC) as vehicle.
[15]
c) Cold plate test
The animals were placed in a stainless steel box (12 cm × 20 cm × 10
cm) with a cold plate as floor. The temperature of the cold plate was
kept constant at 4 °C ± 1 °C. Pain-related behavior (licking of the hind
paw) was observed and the time (s) of the first sign was recorded.
The cut-off time of the latency of paw lifting or licking was set at 60
s.[49] Measurements were performed before and 15, 30, 45 and 60 min
after compound administration.
[16]
[17]
[18]
[19]
G. Tarzia, A. Duranti, A. Tontini, G. Piersanti, M. Mor, S. Rivara, P.
V. Plazzi, C. Park, S. Kathuria, D. Piomelli, J Med Chem 2003, 46,
2352-2360.
d) Statistical analysis
12
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FULL PAPER
[20]
[21]
J. P. Alexander, B. F. Cravatt, Chem Biol 2005, 12, 1179-1187.
S. Schenone, A. Giordani, L. Di Francesco, P. Patrignani, M.
Biava, Bioorg Med Chem 2013, 21, 3695-3701.
D. L. Boger, H. Miyauchi, W. Du, C. Hardouin, R. A. Fecik, H.
Cheng, I. Hwang, M. P. Hedrick, D. Leung, O. Acevedo, C. R.
Guimaraes, W. L. Jorgensen, B. F. Cravatt, J Med Chem 2005, 48,
1849-1856.
[48]
[49]
M. Anzini, S. Valenti, C. Braile, A. Cappelli, S. Vomero, S. Alcaro,
F. Ortuso, L. Marinelli, V. Limongelli, E. Novellino, L. Betti, G.
Giannaccini, A. Lucacchini, S. Daniele, C. Martini, C. Ghelardini, L.
Di Cesare Mannelli, G. Giorgi, M. P. Mascia, G. Biggio, J Med
Chem 2011, 54, 5694-5711.
L. Di Cesare Mannelli, A. Pacini, F. Corti, S. Boccella, L. Luongo,
E. Esposito, S. Cuzzocrea, S. Maione, A. Calignano, C. Ghelardini,
PLoS One 2015, 10, e0128080.
[22]
[23]
[24]
S. Butini, M. Brindisi, S. Gemma, P. Minetti, W. Cabri, G. Gallo, S.
Vincenti, E. Talamonti, F. Borsini, A. Caprioli, M. A. Stasi, S. Di
Serio, S. Ros, G. Borrelli, S. Maramai, F. Fezza, G. Campiani, M.
Maccarrone, J Med Chem 2012, 55, 6898-6915.
S. Butini, S. Gemma, M. Brindisi, S. Maramai, P. Minetti, D.
Celona, R. Napolitano, F. Borsini, W. Cabri, F. Fezza, L. Merlini, S.
Dallavalle, G. Campiani, M. Maccarrone, Bioorg Med Chem Lett
2013, 23, 492-495.
M. Brindisi, S. Maramai, S. Gemma, S. Brogi, A. Grillo, L. Di
Cesare Mannelli, E. Gabellieri, S. Lamponi, S. Saponara, B.
Gorelli, D. Tedesco, T. Bonfiglio, C. Landry, K. M. Jung, A.
Armirotti, L. Luongo, A. Ligresti, F. Piscitelli, C. Bertucci, M. P.
Dehouck, G. Campiani, S. Maione, C. Ghelardini, A. Pittaluga, D.
Piomelli, V. Di Marzo, S. Butini, J Med Chem 2016, 59, 2612-2632.
M. Brindisi, S. Brogi, S. Maramai, A. Grillo, G. Borrelli, S. Butini, E.
Novellino, M. Allara, A. Ligresti, G. Campiani, V. Di Marzo, S.
Gemma, RSC Adv 2016, 6, 64651-64664.
[25]
[26]
[27]
[28]
J. Lim, M. Igarashi, K. M. Jung, S. Butini, G. Campiani, D. Piomelli,
Neuropsychopharmacology 2016, 41, 1329-1339.
R. Colangeli, M. Pierucci, A. Benigno, G. Campiani, S. Butini, G. Di
Giovanni, Sci Rep 2017, 7, 11152.
G. Campiani, A. Cappelli, V. Nacci, M. Anzini, S. Vomero, M.
Hamon, A. Cagnotto, C. Fracasso, C. Uboldi, S. Caccia, S.
Consolo, T. Mennini, J Med Chem 1997, 40, 3670-3678.
S. Butini, R. Budriesi, M. Hamon, E. Morelli, S. Gemma, M.
Brindisi, G. Borrelli, E. Novellino, I. Fiorini, P. Ioan, A. Chiarini, A.
Cagnotto, T. Mennini, C. Fracasso, S. Caccia, G. Campiani, J Med
Chem 2009, 52, 6946-6950.
E. Morelli, S. Gemma, R. Budriesi, G. Campiani, E. Novellino, C.
Fattorusso, B. Catalanotti, S. S. Coccone, S. Ros, G. Borrelli, M.
Persico, I. Fiorini, V. Nacci, P. Ioan, A. Chiarini, M. Hamon, A.
Cagnotto, T. Mennini, C. Fracasso, M. Colovic, S. Caccia, S.
Butini, J Med Chem 2009, 52, 3548-3562.
[29]
[30]
[31]
[32]
A. Cappelli, S. Butini, A. Brizzi, S. Gemma, S. Valenti, G. Giuliani,
M. Anzini, L. Mennuni, G. Campiani, V. Brizzi, S. Vomero, Curr
Top Med Chem 2010, 10, 504-526.
M. Brindisi, S. Butini, S. Franceschini, S. Brogi, F. Trotta, S. Ros,
A. Cagnotto, M. Salmona, A. Casagni, M. Andreassi, S. Saponara,
B. Gorelli, P. Weikop, J. D. Mikkelsen, J. Scheel-Kruger, K.
Sandager-Nielsen, E. Novellino, G. Campiani, S. Gemma, J Med
Chem 2014, 57, 9578-9597.
[33]
[34]
[35]
S. Butini, M. Brindisi, S. Brogi, S. Maramai, E. Guarino, A. Panico,
A. Saxena, V. Chauhan, R. Colombo, L. Verga, E. De Lorenzi, M.
Bartolini, V. Andrisano, E. Novellino, G. Campiani, S. Gemma,
ACS Med Chem Lett 2013, 4, 1178-1182.
M. Chioua, M. Perez, O. M. Bautista-Aguilera, M. Yanez, M. G.
Lopez, A. Romero, R. Cacabelos, R. P. de la Bellacasa, S. Brogi,
S. Butini, J. I. Borrell, J. Marco-Contelles, Mini Rev Med Chem
2015, 15, 648-658.
M. Y. Wu, G. Esteban, S. Brogi, M. Shionoya, L. Wang, G.
Campiani, M. Unzeta, T. Inokuchi, S. Butini, J. Marco-Contelles,
Eur J Med Chem 2016, 121, 864-879.
[36]
[37]
[38]
C. Carrasco, M. Naziroglu, A. B. Rodriguez, J. A. Pariente, Front
Physiol 2018, 9, 95.
L. Di Cesare Mannelli, M. Zanardelli, P. Failli, C. Ghelardini, J Pain
2012, 13, 276-284.
S. Brogi, A. Ramunno, L. Savi, G. Chemi, G. Alfano, A. Pecorelli,
E. Pambianchi, P. Galatello, G. Compagnoni, F. Focher, G.
Biamonti, G. Valacchi, S. Butini, S. Gemma, G. Campiani, M.
Brindisi, Eur J Med Chem 2017, 138, 438-457.
[39]
[40]
L. Di Cesare Mannelli, A. Pacini, L. Bonaccini, M. Zanardelli, T.
Mello, C. Ghelardini, J Pain 2013, 14, 1585-1600.
G. Cavaletti, G. Tredici, M. G. Petruccioli, E. Donde, P. Tredici, P.
Marmiroli, C. Minoia, A. Ronchi, M. Bayssas, G. G. Etienne, Eur J
Cancer 2001, 37, 2457-2463.
[41]
[42]
[43]
[44]
W. L. Jorgensen, D. S. Maxwell, J. TiradoRives, J Am Chem Soc
1996, 118, 11225-11236.
W. C. Still, A. Tempczyk, R. C. Hawley, T. Hendrickson, J Am
Chem Soc 1990, 112, 6127-6129.
M. Mileni, J. Garfunkle, C. Ezzili, B. F. Cravatt, R. C. Stevens, D. L.
Boger, J Am Chem Soc 2011, 133, 4092-4100.
S. Montanari, L. Scalvini, M. Bartolini, F. Belluti, S. Gobbi, V.
Andrisano, A. Ligresti, V. Di Marzo, S. Rivara, M. Mor, A. Bisi, A.
Rampa, J Med Chem 2016, 59, 6387-6406.
[45]
[46]
G. Ortar, T. Bisogno, A. Ligresti, E. Morera, M. Nalli, V. Di Marzo, J
Med Chem 2008, 51, 6970-6979.
N. A. Osman, A. Ligresti, C. D. Klein, M. Allara, A. Rabbito, V. Di
Marzo, K. A. Abouzid, A. H. Abadi, Eur J Med Chem 2016, 122,
619-634.
[47]
C. Battilocchio, G. Poce, S. Alfonso, G. C. Porretta, S. Consalvi, L.
Sautebin, S. Pace, A. Rossi, C. Ghelardini, L. Di Cesare Mannelli,
13
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Entry for the Table of Contents
By rational design, aiming at discovering selective FAAH inhibitors to exploit the therapeutic effects against neuropathic pain, we develop a
series of compounds characterized by a functionalized tricyclic scaffolds. The most potent non toxic FAAH inhibitors 5h and 5i reduced oxidative
stress by decreasing the expression of NF-kB, showed efficacy in a mice model of neuropathic pain relieving the hypersensitivity induced by
oxaliplatin.
14
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