Selective Fatty Acid Amide Hydrolase Inhibitors as Potential Novel Antiepileptic Agents

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Research Article

Selective Fatty Acid Amide Hydrolase Inhibitors as Potential Novel

Antiepileptic Agents

Alessandro Grillo, Filomena Fezza, Giulia Chemi, Roberto Colangeli, Simone Brogi, Domenico Fazio,

Stefano Federico, Alessandro Papa, Nicola Relitti, Roberto Di Maio, Gianluca Giorgi, Stefania Lamponi,

Massimo Valoti, Beatrice Gorelli, Simona Saponara, Mascia Benedusi, Alessandra Pecorelli,

Patrizia Minetti, Giuseppe Valacchi, Stefania Butini,* Giuseppe Campiani,* Sandra Gemma,

Mauro Maccarrone, and Giuseppe Di Giovanni

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ABSTRACT: Temporal lobe epilepsy is the most common form

of epilepsy, and current antiepileptic drugs are ineﬀective in many

patients. The endocannabinoid system has been associated with an

on-demand protective response to seizures. Blocking endocanna-

binoid catabolism would elicit antiepileptic eﬀects, devoid of

psychotropic eﬀects. We herein report the discovery of selective

anandamide catabolic enzyme fatty acid amide hydrolase (FAAH)

inhibitors with promising antiepileptic eﬃcacy, starting from a

further investigation of our prototypical inhibitor 2a. When tested

in two rodent models of epilepsy, 2a reduced the severity of the

pilocarpine-induced status epilepticus and the elongation of the

hippocampal maximal dentate activation. Notably, 2a did not aﬀect

hippocampal dentate gyrus long-term synaptic plasticity. These

data prompted our further endeavor aiming at discovering new antiepileptic agents, developing a new set of FAAH inhibitors (3a−

m). Biological studies highlighted 3h and 3m as the best performing analogues to be further investigated. In cell-based studies, using

a neuroblastoma cell line, 3h and 3m could reduce the oxinﬂammation state by decreasing DNA-binding activity of NF-kB p65,

devoid of cytotoxic eﬀect. Unwanted cardiac eﬀects were excluded for 3h (Langendorﬀ perfused rat heart). Finally, the new analogue

3h reduced the severity of the pilocarpine-induced status epilepticus as observed for 2a.

KEYWORDS: Endocannabinoid system, fatty acid amide hydrolase, enzyme inhibitors, selective inhibitors, epilepsy, temporal lobe epilepsy,

seizures

role of AEA was conﬁrmed by the kainic-acid-induced increase

of AEA in the hippocampus to provide, without aﬀecting 2-AG,

“on-demand” protection against acute excitotoxicity. Seizure

activity initiates cellular Ca²⁺inﬂux through NMDA and

voltage-gated channels thus increasing the activity of pro-

oxidant systems.³The ECS can delay or prevent excitotoxic

damage (by rebalancing the excitatory/inhibitory systems) and

OS-related epileptogenesis.⁴On these bases, a safe, promising,

and viable method to attain a therapeutic eﬀect would be the

potentiation of the ECS tone by inhibition of the

endocannabinoid catabolic enzymes, such as the FAAH

INTRODUCTION

■

The endocannabinoid system (ECS) is a modulatory system

orchestrated by endocannabinoids, their receptors, trans-

porters, and biosynthetic and catabolic enzymes. The most

important endocannabinoids, namely, arachidonoylethanola-

mide (or anandamide, AEA) and 2-arachidonoyl-glycerol (2-

AG) are fatty-acid-derived messengers endowed with major

prohomeostatic functions. They are synthesized “on-demand”

from membrane phospholipids precursors and mainly exert

their action by interacting with type 1 and type 2 cannabinoid

receptors (CB1Rs and CB2Rs). Their activity is terminated

after enzymatic degradation that is principally exerted by fatty

acid amide hydrolase (FAAH, for AEA) and monoacylglycerol

lipase (MAGL, for 2-AG).¹The ECS regulates relevant

signaling pathways including excitotoxicity, neurodegeneration,

oxidative stress (OS), and neuroinﬂammation. Particularly, a

reduction of AEA was ascertained in patients aﬀected by

temporal lobe epilepsy (TLE).²However, the neuroprotective

Received: March 29, 2021

Accepted: April 19, 2021

Published: April 23, 2021

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enzyme. This approach would elude the occurrence of

unwanted psychotropic side eﬀects that accompany the

administration of CBR agonists.⁵As a further advantage,

FAAH inhibition could provide higher selectivity, as it would

locally increase the activity of the ECS, at sites where

endocannabinoids are produced.⁶For these reasons, remark-

able eﬀorts have been made both by academia and

pharmaceutical companies to develop potent and selective

FAAH inhibitors.

neuroprotective eﬀects and a rapid increase in AEA levels

during seizures in mice providing a CB1R-mediated protection

against excitotoxicity.²⁹Accordingly, some CB1R agonists

(e.g., Δ9-THC and WIN-55,212)³⁰proved to be antiepileptic.

However, their administration can alter synaptic plasticity with

inhibition of long-term potentiation,³¹likely the cause of

impairment in cognition and the detrimental eﬀects on long-

term memory.^32,33Prolonged use of WIN-55,212 can also lead

to a reduction in its antiepileptic eﬃcacy due to tolerance and

alterations in CB1R expression.³⁴

Epilepsy is a neurological disorder clinically deﬁned by the

International League Against Epilepsy (ILAE).⁷In developed

countries, epilepsy has a prevalence ranging from 4 to 10 cases

per 1000⁸and an incidence of 43.4 per 100 000 people per

year.⁹Developing countries have a higher prevalence with a

range 14−57 cases per 1000¹⁰and also a higher incidence of

68.7 per 100 000 people per year.⁹Although there have been

few epidemiological studies into TLE, they indicate that TLE

accounts for around 60−70% of epilepsy cases¹¹⁻¹³making it

the most common type of focal epilepsy. Seizures have a

myriad of acute eﬀects such as loss of consciousness, tonic

muscle contractions, colonic jerking, and postictal confusion.

There are also comorbidities such as depression and anxiety¹⁴

and long-term eﬀects of epilepsy including neuronal cell loss

with associated memory deﬁcits.¹⁵Mechanisms of brain

damage and neuronal loss associated with epilepsy are

multifactorial including mitochondrial dysfunction, OS, and

inﬂammation (i.e., oxinﬂammation phenomenon^16,17) leading

to seizure-mediated lipoperoxidation damage which may aﬀect

the cellular ionic balance.^18,19

In the context of indirect agonism of the ECS, FAAH

inhibitors such as URB-597 (1, Figure 1)³⁵and hexadecyl

Although cases of epilepsy commonly have a good

prognosis, the disorder is associated with a higher mortality

rate arising from causes such as accidental trauma, drowning,

and suicide.⁸Though some eﬀective antiepileptic drugs exist, a

recent study estimated that about 140 000 patients exist in the

USA with drug-resistant TLE²⁰thus indicating the pressing

need for new therapeutic options for this type of epilepsy.

In the past decades, the ECS has become a focus of research

in many conditions including epilepsy. The endocannabinoids

play a key role in neuromodulation by retrograde signaling.²¹

Activation of CB1R inhibits neurotransmitter release; there-

fore, it may induce depolarization-induced suppression of

inhibition (DSI) or depolarization-induced suppression of

excitation (DSE) depending on if the CB1Rs are on

GABAergic or glutamatergic neurons, respectively.²²Unsur-

prisingly, a neuromodulatory system of such widespread

importance in the brain has been implicated in the etiopatho-

genesis of epilepsy, and changes in the ECS were documented

in animal models of epilepsy, as well as in humans. CB1Rs

expression was found to be reduced in the thalamus of

epileptic rats.²³In human patients with untreated TLE, there is

a reduced amount of AEA in the cerebrospinal ﬂuid.²

Furthermore, the increased CB1R expression on GABAergic

neurons or the increased sprouting of CB1Rs expressing GABA

axons²⁴supports previous knowledge of the involvement of

GABA in seizures.²⁵

Figure 1. Reference compounds (1−2a,b) and title compounds (3a−

m).

sulfonylﬂuoride, namely, AM374,³⁶have previously shown

promising antiepileptic and neuroprotective activities. Other

FAAH inhibitors showed diﬀering eﬀects on memory, which in

many cases have a better proﬁle than CB1R agonists,³⁷as also

recently demonstrated by our team in TLE.³⁸

Recently,³⁹⁻⁴²we identiﬁed a potent and selective FAAH

inhibitor hit, namely, ST3913 (2a, Figure 1). Because the

eﬃcacy of 2a was never measured before in animal models of

epilepsy, this manuscript deals with the in vivo characterization

of the antiepileptic potential of 2a in two rodent models of

epilepsy and the identiﬁcation of a new class of FAAH

inhibitors (3a−m, Figure 1) along with their biological

investigation leading to the selection of 3h to be tested in

vivo to measure its antiepileptic potential.

In this context, the involvement of the endocannabinoids in

excitatory/inhibitory balance mechanisms in the brain, and

their selective impairment in distinct neurological disorders,

oﬀers a glimpse for novel therapeutics.²⁶In fact, as a result of

the neuroprotection oﬀered by the ECS, the latter has become

a target for potential treatment in various neurological

disorders, such as Huntington’s disease,²⁷Alzheimer’s

disease,²⁸and epilepsy.⁵The stimulation of ECS may exert

In particular, after i.p. administration, 2a proved to be

eﬃcacious in the pilocarpine and the maximal dentate

activation murine models of TLE during seizures and using

it as a pretreatment. In addition, we have investigated the eﬀect

on synaptic plasticity (short-term and long-term plasticity

STP-LTP) and neuronal damage resulting from seizures (the

histological study of thiols formations).

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The encouraging data obtained with 2a have prompted us to

further expand the series of phenylpyrrole-based analogues in

the quest to identify further potent and selective FAAH

inhibitors as new antiepileptic agents to be used as a follow-up

of 2a. Accordingly, we initiated a further medicinal chemistry

endeavor to develop a new set of FAAH inhibitors (3a−m,

Figure 1). We herein report the synthesis, molecular modeling,

and biological investigation of this new set of molecules (3a−

m) structurally related to 2a. Computational methods were

used for a preliminary evaluation of the druglike proﬁle and of

the physicochemical properties of the developed compounds

and for a rational analysis of the structure−activity relation-

ships (SARs). The most interesting inhibitors of the new

series, 3g, 3m, and 3h, were selected for further studies.

Selectivity toward CB1R, CB2R, and MAGL was assessed for

the new analogues. For compound 3h, preliminary metabolic

stability studies were performed, by incubation with human

and rat liver microsomes. Cytotoxicity of compounds 3g and

3h was measured on mouse ﬁbroblasts, and the absence of

mutagenicity was observed by the Ames test. Notably,

compounds 3h and 3m demonstrated a potential antioxin-

ﬂammatory proﬁle on the neuroblastoma cell line (IMR32), by

decreasing the LPS-induced activation of the redox-sensitive

transcription factor NF-κB p65. The absence of cardiac toxicity

was determined for compound 3h in the Langendorﬀ isolated

heart model. These data allowed for the selection of the newly

identiﬁed FAAH inhibitor 3h for in vivo studies. Gratifyingly,

the newly developed analogue 3h has replicated the

antiepileptic potential of 2a in the pilocarpine model,

conﬁrming the notable potential for this class of analogues

against seizures.

Scheme 1. Synthesis of the “Inverted” Carbamates 3a−d,

a

Urea 3e, and Amide 3f

a

Reagents and conditions. (a) COCl₂(20% solution in toluene),

pyridine, dry DCM, 25 °C, 12 h, then appropriate alcohol, TEA, dry

THF, reﬂux, 12 h, 14−69%; (b) NaBO₃× 4H₂O, 1,4-dioxane, H₂O,

80 °C, 1.5 h, 25−40%; (c) 6 M NaOH, 30% H₂O₂, EtOH, reﬂux, 12

h, 90%; (d) 6-phenylhexan-1-yl-isocyanate, TEA, dry THF, reﬂux, 12

h, 40−62%; (e) PPh₃, (Cl₃C)₂CO, 6-phenylhexan-1-amine, dry

DCM, TEA, from 0 to 25 °C, 1 h, 30%.

hydrolysis of the nitrile functionality, the carboxamide 13a

was achieved. Finally, the treatment of 13a with 6-phenyl-

hexan-1-yl-isocyanate, followed by reduction of the nitro group

by catalytic hydrogenation, aﬀorded the compound 3g.

For the synthesis of 3k, a convergent approach exploited

phenylpyrrole-3-carboxamide 13b³⁹which, after transforma-

tion into the correspondent chloroformate, by using phosgene,

was reacted with the 2-(4-benzylpiperazin-1-yl)ethan-1-amine

(14) which was prepared as reported.⁴⁵Derivatives 15a,b, and

16 were synthesized as described⁴²and, by reaction with 6-

phenylhexan-1-yl-isocyanate, transformed into the correspond-

ent urethanes 3h−j. The Clauson-Kaas reaction between 3-

aminophenol and 2,5-dimethoxytetrahydrofuran 17 was

performed in the presence of acetic acid under microwave

irradiation allowing a fast, clean, and high-yielding option to

obtain the 1-(3-hydroxyphenyl)pyrrole 18 which substantially

improved the previously reported yields.^46,47The latter was

subjected to a cyanation reaction using chlorosulfonyl

isocyanate in the presence of DMF, aﬀording the cyanoder-

ivative 19. After hydrolysis of 19, the corresponding amide 20

was reacted with 6-phenylhexan-1-yl-isocyanate in order to

obtain compound 3l.

RESULTS AND DISCUSSION

■

Chemistry. The synthesis of compounds 3a−m is reported

in Schemes 1−3. As described in Scheme 1, the 1-(3-

aminophenyl)-1H-pyrrole-3-carbonitile (4)⁴³was identiﬁed as

the key intermediate for the synthesis of the “inverted”

carbamates (3a−d, Table 1) and the urea (3e, Table 1).

Derivative 4 was easily converted, using phosgene, in the

intermediate isocyanate that reacted with 6-phenylhexan-1-ol

(5a) or with the diﬀerently functionalized phenoxyethoxyethyl

alcohols (5b−d),³⁹to aﬀord the corresponding carbamates

(6a−d). From compounds 6a−d, after partial hydrolysis of the

cyano group performed with perborate, compounds 3a−d

were obtained. The hydrolysis of the cyano group of 4, in the

presence of hydrogen peroxide and sodium hydroxide, allowed

for the isolation of 1-(3-aminophenyl)-1H-pirrole-3-carbox-

amide (7⁴³). Compound 7 was identiﬁed as the substrate for a

convergent approach in which it was reacted with 6-

phenylhexan-1-yl-isocyanate³⁹leading to the urea derivative

3e. The amidic derivative 3f was obtained from the acid 8, after

treatment with triphenylphosphine and hexachloroacetone, in

the presence of 6-phenylhexan-1-amine followed by basic

hydrolysis of the cyano portion of the intermediate 9. Acid 8

could be synthesized as previously described by a Clauson-

Kaas reaction,⁴⁰but we herein report a slight elaboration of the

originally employed⁴⁴reaction protocol that has allowed for

the substantial improvement of the reaction yield (see

Experimental Section for details).

The intermediate 21⁴¹(Scheme 3) was subjected to a

bromination reaction aﬀording a mixture of isomers of

monobrominated and dibrominated compounds. After column

chromatography, we could isolate pure isomer 22 as a major

product, which was crystallized from EtOAc and subjected to

X-ray diﬀraction analysis to clarify the exact structure. This

analysis unambiguously indicated the bromine at C α position

of the pyrrole system (C1 of Figure 2). Our eﬀorts to

characterize the other isomers were hampered by the low

amount obtained and by its contamination by the inseparable

2,5-Dimethoxytetrahydrofuran-3-carbonitrile (10) (Scheme

2) was subjected to a Clauson-Kaas reaction with 5-methoxy-2-

nitroaniline to aﬀord the key phenyl-pyrrole intermediate (11).

After cleavage of the anisolic portion (12) and partial

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a

Scheme 2. Synthesis of the Carbamates 3g−l

a

Reagents and conditions. (a) 5-Methoxy-2-nitroaniline, 6 M HCl, 1,4-dioxane, reﬂux, 40 min, 87%; (b) BBr₃, dry DCM, from −78 to 25 °C, 12 h,

50%; (c) 6 M NaOH, 30% H₂O₂, EtOH, reﬂux, 12 h, 50%; (d) 6-phenylhexan-1-yl-isocyanate, TEA, dry toluene/DMF, (4:1), reﬂux, 48 h; (e) H₂,

10% Pd/C, 15 psi, EtOH, 25 °C, 3 h, 45% (over 2 steps); (f) DMAP, COCl₂(20% solution in toluene), THF dry, from 0 to 70 °C, 16 h, 20%; (g)

6-phenylhexan-1-yl-isocyanate, TEA, dry THF, reﬂux, 12 h, 20−79%; (h) 3-aminophenol, AcOH, MW, 170 °C, 10 min, 80%; (i) ClSO₂NCO, dry

MeCN, from −20 to 25 °C, 12 h; then dry DMF, 55 °C, 30 min, 20%.

a

with 6-phenylhexan-1-yl-isocyanate allowed for the isolation of

the tricyclic analogue 3m.

Scheme 3. Synthesis of the Tricyclic Analogue 3m

Structure−Activity Relationship and Molecular Mod-

eling Studies. As a continuation of our endeavors in the

discovery of potent FAAH inhibitors, we have developed a new

series of pyrrole-based analogues (3a−m, Figure 1 and Table

1). In this new series of compounds, our structural hit 2a was

modiﬁed at (i) the electrophilic center, (ii) the lateral chain,

and (iii) the pyrrole moiety. These modiﬁcations were used to

investigate the SARs and to implement the pharmacokinetic

properties such as the metabolic stability (by ﬂuorination and

the use of diﬀerent pyrrole systems) and the solubility proﬁle

(lateral chains and scaﬀold modiﬁcations) of the developed

compounds over the parent 2a. In particular, the original

carbamate moiety of 2a was “inverted” as in compounds 3a−d;

a urea moiety was introduced in derivative 3e, and in

compound 3f, we evaluated the eﬀect of an amide system.

Regarding the lateral chains, we explored the replacement of

the hexamethylene spacer of 2a by a poly ethereal chain (3b−

d) or piperazine containing chain (3k). Furthermore, in the

phenyl system of the lateral chain, some ﬂuorine atoms were

introduced (compounds 3c,d) to improve metabolic stability.

Regarding the central core of the molecules, in 3g, we

a

Reagents and conditions. (a) NBS, dry THF, −78 to 25 °C, 12 h,

45%; (b) CuCN, dry DMF, reﬂux, 12 h, 30%; (c) BBr₃, dry DCM,

from −78 to 25 °C, 12 h, 30%; (d) 6 M NaOH, 30% H₂O₂, EtOH,

reﬂux, 12 h, 40%; (e) 6-phenylhexan-1-yl-isocyanate, TEA, dry THF,

reﬂux, 12 h, 50%.

dibrominated product. Compound 22 was then converted into

its cyano-derivative counterpart 23, which after demethylation,

aﬀorded the phenol 24. Nitrile hydrolysis (25) and reaction

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Table 1. Inhibition Activity towards Mouse Brain FAAH (as

IC₅₀nM) for Compounds 3a−m and Reference Compounds

1 and 2a,b

Figure 2. Crystal structure of the bromo-substituted compound 22.

Ellipsoids enclose 50% probability.

compounds of the series. Our studies indicated that the

potency of the compounds is aﬀected by the nature of the

electrophilic groups, as all the evaluated modiﬁcations (3a−f)

led to less potent compounds when compared to 2a. Among

the modiﬁcations introduced on the lateral chains, our studies

revealed that the introduction of ﬂuorine atoms or phenoxy-

ethoxyethyl groups (as in the subseries of compounds 3b−d)

was detrimental for activity. In fact, compounds 3b−d caused

only 19−56% inhibition of FAAH when tested at 100 μM

(Table 1). As compared to these analogues, the piperazine

containing chain was better tolerated when installed on a

carbamoyl-based analogue, and compound 3k showed 24%

FAAH inhibition when incubated at 100 nM (Table 1). In fact,

the most potent inhibitors belong to the subseries of phenol-

carbamate-based analogues with a phenylhexyl lateral chain

(3g-j,l,m, Table 1). As expected, compound 3g exhibited a

potency of inhibition in the nanomolar range (3g, IC₅₀= 1.10

nM, Table 1) comparable to that of our lead 2a, while

compound 3l displayed an encouraging two-digit nanomolar

potency for enzyme inhibition, demonstrating that the

positioning of the amide at C-2 of the pyrrole system could

be well tolerated by the enzyme. This evidence prompted us to

synthesize the rigidiﬁed tricyclic analogue 3m, which nicely

challenged the activity of 3l. As per our initial interest, we

sought to understand the SARs derived by changing the

pyrrole junctions. Accordingly, we synthesized compounds

3h−j. The 2-phenyl-1,5-dimethyl-1H-pyrrole-3-carboxamide

core, embedded in compound 3h, resulted in the best

performing system when compared to the 2-phenyl-5-methyl-

1H-pyrrole-3-carboxamide of 3i or the 5-phenyl-1-methyl-1H-

pyrrole-2-carboxamide of 3j. Though all the three analogues

behave as potent FAAH inhibitors (Table 1), compound 3h

(IC₅₀= 6.88 nM) resulted in the most potent inhibitor of this

subseries of analogues and was further assayed in biological

tests.

a

Each value is the mean of at least two experiments (95% conﬁdence

intervals are into brackets).

introduced an amino group (aniline) in the phenyl system, and

we varied the junctions with diﬀerently decorated pyrrole

systems (3h-j,l). Following the results of our previous

studies,⁴¹a rigidiﬁcation by bridging the phenyl-pyrrole

scaﬀold of 3l was applied leading to the tricyclic analogue 3m.

The FAAH inhibition potencies of the newly developed

compounds (3a−m) were assessed using enzymatic studies

performed on the mouse brain FAAH and allowed us to

decipher the SARs useful for the selection of the best

To gain information about the possible binding modes of

the tested compounds into the humanized variant of rat FAAH

protein (h/rFAAH) binding site, we performed molecular

docking calculations employing the induced ﬁt docking (IFD)

technique.^41,43Starting with 3a bearing an “inverted”

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carbamate moiety (Figure S1A), we observed a weak

interaction within the binding site of the enzyme, and details

are provided in the Supporting Information. These data appear

to be in line with the micromolar inhibitory potency of the

compound. In compounds 3b−d, the introduction of a

polyether tether resulted in a dramatic decrease of the

inhibitory activity. Accordingly, for these compounds (3b−d,

Figure S1B −D), we detected weak contacts with the active site

of the enzyme (see Supporting Information for details). The

transformation of the electrophilic functionality from the

“inverted” carbamate to urea led to 3e, which with inhibition

potency below 10 μM, improved the eﬃcacy over 3a, as also

outlined by our computational studies (Figure S1E, Supporting

Information). In compound 3f, the change of the electrophilic

functionality into an amide did not produce any improvement

in potency with respect to the previously discussed

compounds. However, the presence of an amide in place of

the urea group made 3f able to establish an H-bond with Q273

(Figure S1F, Supporting Information, for details).

carboxamide moiety formed two H-bonds with the backbone

of Q273, and the central region targeted F192 by a π−π

stacking, while the NH₂substituent H-bound V270. The

carbamate moiety strongly interacted in the catalytic region of

the enzyme with the catalytic S241 and with I238 and G239

(oxyanion hole) by a network of H-bonds. The phenylhexyl

chain established a double π−π stacking with F381 and F432.

Remarkably, the orientation of the carbon of the carbamate

portion and its distance from the catalytic residue S241 (<3 Å)

could be in agreement with a potential nucleophilic attack. In

general, the strong pattern of interaction within the binding

site accounts for an inhibitory activity in the low nanomolar

range (Table 1), making 3g one of the best performing

compounds of the series.

The nanomolar FAAH inhibitors 3h−j bear diﬀerently

decorated pyrrole systems (Figure 4 and Figure S2A,B). For

A carbamate group, as the electrophilic functionality, and a

NH₂in the benzylpyrrole moiety characterize the compound

3g. As reported in Figure 3, 3g made strong interactions with

the active site of the enzyme. This compound, despite the

presence of a NH₂moiety at the para-position with respect to

the electrophilic group, still maintains a good FAAH inhibition

potency, as already observed for other FAAH inhibitors.⁴⁸The

Figure 4. Binding mode of 3h (magenta sticks) into h/rFAAH

enzyme (orange 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 nonpolar hydrogens were removed for clarity. The picture

was generated by means of PyMOL.

3h, we registered one of the best inhibitory proﬁles of the

series (Table 1). In particular, 3h H-bound the backbone of

C269 and V270 by its carboxamide moiety (Figure 4). The

carbamate moiety H-bound S241 and I238 (oxyanion hole).

As for 3g, the distance between the carbon of the carbamate

and the catalytic S241 could be in agreement with a potential

nucleophilic attack. The phenylhexyl chain established a triple

π−π stacking (F192, F381, and F432). Compound 3i diﬀers

from 3h for the absence of methyl in the nitrogen of the

pyrrole ring. This diﬀerence allowed for maintainence of the

Figure 3. Binding mode of 3g (light blue sticks) into h/rFAAH

enzyme (orange 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 nonpolar hydrogens were removed for clarity. The picture

was generated by means of PyMOL.

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same contacts described for 3h, with the exception of a H-

bond with the oxyanion hole (see Supporting Information,

Figure S2A) which may explain the reduction of the inhibitory

potency as compared to 3h. The shifting of the amide system

to position 5 of the pyrrole system led to the analogue 3j that,

in addition to interacting with S241 and I238, also interacted

with S193 (see Supporting Information, Figure S2B).

The introduction of a benzylpiperazine in the nucleus of 2a

led to compound 3k. Few contacts were found with the FAAH

enzyme leading to a decreased aﬃnity (see Supporting

Information, Figure S2C).

The presence of the phenylhexyl lateral chain, combined

with a phenyl-1-pyrrole scaﬀold substituted by a carboxamide

group at C2 of the pyrrole ring as in 3l, allowed for strong

interactions with the key residues of the enzyme. In fact, the

carboxamide group of 3l formed H-bonds with C269 and V270

that, with the other classical contacts (Supporting Information,

Figure S2D), accounted for an inhibitory proﬁle in the

nanomolar range. Finally, a rigidiﬁcation by bridging the

phenylpyrrole scaﬀold of 3l led to the tricyclic analogue 3m.

The docking output (Figure 5) showed the same interactions

found for 3l with the exclusion of the H-bond with S193.

However, compound 3m can target the backbone of T236, due

to its carboxamide group forming an H-bond, and residue

F192, due to its phenylpyrrole moiety establishing a π−π

stacking. The phenylhexyl lateral chain π−π stacks with F381

and F432. This slight improvement in terms of the number of

contacts accounted for a slight increase in the binding aﬃnity

with respect to its parent compound 3l.

For all the new compounds (3a−m), we also calculated the

physicochemical properties using QikProp application. The

output of this calculation is reported in the Supporting

Information (Table S1) along with a brief discussion. In

particular, for compound 3h, we noted that, despite a low

water solubility, the calculated oral absorption was among the

best values of the series. For 3h, we could also predict the

highest value of Caco2 permeability. Taken together with the

aﬃnity and the toxicity data, 3h exhibited favorable properties

to progress for the in vivo studies.

Selectivity toward Cannabinoid Receptors and

MAGL. We next interrogated the selectivity proﬁle of the

analogues 3h and 3g toward the murine receptors mCB1R and

mCB2R (which possess an overall identity of 97.0 and 79.7%

with the human isoforms, respectively). Selectivity for the other

relevant catabolic enzyme of the ECS (MAGL) was also

measured. As depicted in Figure 6, none of the compounds

showed issues of cross-reactivity with the tested proteins.

Cytotoxicity and Mutagenicity Proﬁle Evaluation for

3h and 3g. The potential cytotoxic proﬁle was assessed for

compounds 3h and 3g after incubation with mouse embryonic

ﬁbroblasts (NIH3T3 cell line) and with human glioblastoma

astrocytoma cells (U373MG cell line).

The results, expressed as IC₅₀(μM), are reported in Table 2

and are compared with those obtained for the reference

compounds 2a and 2b. Gratifyingly, when tested on the

NIH3T3 cells, compound 3h displayed low toxicity com-

parable to that of the hit analogues 2a and 2b. Also on the

glioblastoma U373MG cell line, 3h exhibited a 46 μM IC₅₀

value, very similar to that expressed by 2a.

As further proﬁling, we used the Ames test as a reliable and

widely used preliminary assessment for identifying potential

risks of mutagenicity at the early stages of drug development.

We ascertained, for 3g and 3h, the absence of a mutagenic

eﬀect in the Salmonella typhimurium strains TA98 and TA100

(Figure S3). When performed in the presence of the S9

fraction of a rat liver, the assay may also serve for assessing the

potential mutagenicity risks derived from the compound’s

metabolites. By using these conditions, a more in-depth

analysis of compound 3h also allowed us to exclude risks of

toxicity in the presence of the S9 fraction in both TA98 and

TA100 Salmonella typhimurium strains (Figure S3).

Preliminary Metabolic Studies. The metabolic stability

of 3h and 2a was assessed in human and rat liver microsomal

preparations. The plot of nonmetabolized compounds (%)

versus time showed a monoexponential decay relationship

(Figure 7).

For compound 3h, the apparent decay constants k were

0.0328 min⁻¹and 0.0704 min⁻¹for human liver microsomes

(HLMs) and rat liver microsomes (RLMs), respectively. The

resulted half-life time and Cl_intwere, respectively, t_1/2= 21.4

and 10.5 min and Cl_int= 54.8 and 119.7 μL/min. The

reference compound 2a showed a lower intrinsic clearance in

both microsomal preparations and presented a lower micro-

somal stability in humans than that in rat microsomal

preparations. In fact, the calculated Cl_intvalues were 55.5

and 39.2 μL/min in HLM and RLM, respectively.

Figure 5. Binding modes of 3m (light gray sticks) into h/rFAAH

enzyme (orange cartoon, PDB ID: 3PPM) binding site. The residues

forming the catalytic triad (K142, S217, and S241) are reported as

sticks, while the other residues of the binding site are reported as

lines. The nonpolar hydrogens were removed for clarity. The picture

was generated by means of PyMOL.

When comparing the CYP-dependent metabolic depletion

of compound 3h compared to that of 2a in RLM, we observed

a higher metabolic liability of 3h. On the contrary, in the

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Figure 6. Selectivity of compounds 3h and 3g toward mCB1R (A), mCB2R (B), and mMAGL (C); data for 2b and the potent MAGL inhibitor

JZL-184 are reported for comparison.

Table 2. Viability of Mouse Fibroblasts NIH3T3 and

Human Glioblastoma Astrocytoma Cells U373-MG after

Incubation with 3h, 3g, and Reference Compounds

In fact, irreversible protein inhibitors, endowed with a longer

duration of action as compared to that of reversible protein

ligands, are less likely to suﬀer from poor pharmacokinetic

parameters such as metabolic stability.⁴⁹In fact, an irreversible

ligand could require less frequent dosing, being characterized

by longer residence times and providing a higher degree of

inactivation of the target protein. In line with these

considerations, when we interrogated the eﬀect of 3h (see

below in the in vivo studies section) in a rodent model of

epilepsy, we observed an eﬃcacy that paralleled that of 2a.

Eﬀect of 3h on Langendorﬀ Perfused Rat Heart. A

major hurdle in the development of new drugs is reducing the

risk of cardiac arrhythmias, such as long QT syndrome. To

evaluate, and possibly exclude, the cardiotoxic potential of

compound 3h, its eﬀect on cardiac mechanical function and

electrocardiogram (ECG) in Langendorﬀ-isolated rat hearts

was assessed following our previously described protocol.⁵⁰

Under control conditions, left ventricle pressure (LVP) and

coronary perfusion pressure (CPP) values of 63 2 and 51

3 mmHg (n = 5), respectively, were obtained. At the maximum

b

compound

NIH3T3 IC₅₀(μM)

U373MG IC₅₀(μM)

3h

3g

2a

2b

69

20

75

5

46

NT

50

NT

c

a

b

As IC₅₀μM. Cell viability was measured by the Neutral Red Uptake

(NRU) test and data normalized as % control; data are expressed as

mean SD of three experiments repeated in six replicates. Values are

c

statistically diﬀerent vs control, p ≤ 0.05. NT = not tested.

concentration tested (10 μM), 3h increased LVP to 74

2

mmHg (n = 5, ** p < 0.01, repeated measures ANOVA and

Dunnett’s post-test) and increased the RR interval, while

reducing HR. However, QTc values did not vary over the drug

range tested (Table 3).

In summary, the collected data did not highlight a

cardiotoxicity predictive of potential risk for a torsade de

pointes for compound 3h at none of the concentrations tested

which were 3 orders of magnitude higher than that eﬀective in

FAAH inhibition. 3h, at the maximum concentration tested,

behaved like a negative chronotropic and a positive inotropic

agent on rat heart preparations.

Figure 7. CYP-dependent metabolic depletion of 5 μM 3h and 2a in

human (HLM, A) and rat liver (RLM, B) microsomal preparations.

Results are presented graphically as the 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.

HLM, we observed comparable proﬁles, even though there was

a slightly higher susceptibility to metabolism for 3h.

These observations are relevant, though they are not an issue

hampering the in vivo eﬃcacy of irreversible enzyme inhibitors.

a

Table 3. Eﬀects of 3h on HR, RR, PQ, QRS, QT, and QTc in Langendorﬀ Perfused Rat Hearts

(μM)

HR (BPM)

RR (ms)

PQ (ms)

QRS (ms)

14 0.5

QT (ms)

QTc (ms)

none

0. 01

0.1

1

10

259

252

241

6

8

9

8

7

232

233

239

250

5

7

8

37

39

1

2

1

2

76

77

78

79

78

4

5

4

76

77

78

77

76

4

3

14

1

14 0.5

14

b

1

a

b

Each value represents mean SEM (n = 5 hearts). p < 0.01, repeated measures ANOVA and Dunnett’s post test.

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Cytotoxicity Determination and Antioxinﬂammatory

Potential Evaluation for 3h and 3m. Oxinﬂammation, a

condition generated by harmful crosstalk between redox

imbalance and inﬂammatory cascade, is a relevant issue in

brain diseases and, in particular, in epilepsy.^17,19To explore the

potential antioxinﬂammatory properties of two structural

templates such as 3h and the tricyclic analogue 3m, ﬁrst an

evaluation of their cytotoxicity proﬁle in IMR32 cell line was

performed using the lactate dehydrogenase (LDH)-release

assay. Diﬀerent concentrations ranging from 0.1 to 50 μM of

the compounds 3h and 3m and reference compound 2a were

tested, and LDH release was determined after 24 h of

treatment. As shown in Figure 8, no signiﬁcant release of LDH

Figure 9. Eﬀect of compounds 3m, 3h, and 2a on an oxinﬂammatory

condition. NF-kB p65 DNA binding activity was evaluated in nuclear

extracts from IMR32 cells pretreated with compounds 3h and 3m and

reference compound 2a for 24 h and challenged with 100 μg/mL LPS

for 30 min. Data are presented as mean SEM of 3 independent

experiments. * p < 0.05 vs control (Ctrl); # p < 0.01 vs LPS

treatment.

discharge duration.⁵¹⁻⁵³We have recently shown that CB

manipulation signiﬁcantly reduced the MDA elongation over

the 4 h period of recording, with the CB1R/CB2R agonist

WIN55,212-2 behaving diﬀerently from the FAAH inhibitor

URB597 (1) in terms of the magnitude of the eﬀect and time-

development.⁵⁴The eﬀects of the vehicle (n = 9) and 2a

treatment (n = 9) on the MDA paradigm are shown in Figure

10. An initial decrease in the time to onset (Figure 10A) was

Figure 8. Cytotoxicity was determined as LDH release in IMR32 cells

treated for 24 h with compounds 3h and 3m and reference compound

2a at diﬀerent concentrations (0.1, 0.5, 1, 5, 10, and 50 μM). Values

represent the average SD of samples performed at least in triplicate.

Data are expressed as a percentage of LDH release as compared to the

maximum release of LDH from Triton X-100-treated cells (100%).

into cell culture media was observed for all three compounds,

demonstrating the lack of cellular toxicity at the concentrations

analyzed. Based on these results, in the next experiment,

IMR32 cells were ﬁrst pretreated with the compounds at the

doses of 0.5 and 1 μM for 24 h and then challenged with 100

μg/mL LPS for 30 min. The ability of the compounds 3h, 3m,

and 2a to prevent the LPS-induced oxinﬂammatory signaling

pathway was assessed by detecting speciﬁc NF-kB p65-DNA

binding activity in IMR32 cells. As expected, the binding of

nuclear NF-kB p65 to consensus oligonucleotide sequences

was signiﬁcantly increased 30 min following LPS challenge

(Figure 9). Pretreatment of IMR32 cells with the three

diﬀerent compounds for 24 h prevented the LPS-induced NF-

kB p65 binding to consensus DNA sequences, reaching the

maximal eﬀect with compound 3m at 1 μM dose (Figure 9). A

concentration-dependent inhibitory eﬀect was evident for all

the compounds (Figure 9). These results suggest that

compounds 3h and 3m and reference compound 2a could

modulate a seizure-induced oxinﬂammatory condition through

regulating the NF-κB p65 signaling pathway that is involved in

epilepsy.¹⁸

Figure 10. Maximal dentate activation (MDA) after tetanic

stimulation of the perforant path (PP). (A) The time to onset of

MDA decreased in both vehicle and 2a-treated animals with no

signiﬁcant diﬀerence between the two groups. (B) In vehicle-treated

animals, the MDA duration increased over time; in animals treated

with 2a, the increase in MDA duration was reduced from 180 min

post-treatment with eﬀects up to 240 min post-treatment. Repeated

measures ANOVA followed by Bonferroni post hoc analysis, ***p <

0.001, *p < 0.05 vs pretreatment, #p < 0.05 vs post-treatment.

seen in vehicle-treated animals before leveling and remaining

more constant. Treatment with 2a had no signiﬁcant eﬀects on

the time to onset of MDA, and the graph followed the same

pattern as with vehicle-treated animals. The duration of MDA

(Figure 10B) saw a steady increase over time in vehicle-treated

animals. Treatment with 2a gave a reduction in the duration of

MDA with signiﬁcant eﬀects 180 min after administration. The

antiepileptic eﬃcacy of 2a during seizures in the MDA model

was somewhat diﬀerent from that observed with CB1R agonist

and similar to that of the FAAH inhibitor URB597 (1).⁵⁴

In Vivo Studies of 2a and 3h on Rodent Models of

Limbic Epilepsy. Eﬀect of 2a on Maximal Dentate

Activation (MDA), a Model of Partial Complex Epilepsy.

MDA is a paroxysmal event characterized by the appearance of

bursts of large amplitude population spikes in the dentate gyrus

that is associated with the production of afterdischarges in

limbic circuits. Repeated elicitation of MDA in the

anesthetized rat produces a progressive increase in after-

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Nevertheless, the 2a eﬀect appeared later than URB597,

although 2a showed a stronger antiepileptic eﬀect that

improved over time similar to another FAAH inhibitor

AM374.³⁶On the other hand, WIN55,212 aﬀects MDA

much faster, immediately following its administration.⁵⁴

Eﬀect of 2a on Hippocampal Dentate Gyrus Long-Term

Potentiation (LTP). Having demonstrated the anticonvulsant

eﬀect of the FAAH inhibitor 2a in the MDA rat model of

epilepsy, we next wanted to exclude any possible alteration of

dentate gyrus excitability and plasticity in normal rats. Indeed,

many exogenous cannabinoids such as WIN55,212-2, which

have shown antiepileptic eﬀects, showed negative eﬀects on

LTP,^54,55learning, and memory³²which are already impaired

in animal models of epilepsy.⁵⁴We, therefore, performed

electrophysiological recordings of the LTP at the level of the

perforant path (PP)-dentate gyrus synapse of the hippocampus

in anesthetized rats. In vehicle-treated animals (n = 9), high-

frequency stimulation (HFS) current delivered to the PP

resulted in potentiation shown by an increase in the population

spike (PS) amplitude (Figure 11). The same potentiation was

Figure 12. Eﬀects of 2a on pilocarpine-induced SE. (A) A dose of 10

mg/kg 2a reduced the severity of seizures and (B) the duration of

seizures compared to all other doses. No other dose had any

signiﬁcant eﬀect on seizure severity or duration. (C) The 10 mg/kg

2a increased the time to onset of SE compared with that of vehicle.

The 2 and 5 mg/kg doses, and a dose of 5 mg/kg, increased the time

to onset of SE compared to that of vehicle and 2 mg/kg dose. The

one-way ANOVA with Bonferroni’s Multiple Comparison ***p <

0.005.

signiﬁcantly increased the time to onset of SE (75.2 3.42; p

< 0.05 vs 0.2 and 5 mg/kg). A dose of 5 mg/kg (n = 6) 2a

signiﬁcantly increased the time to onset of SE compared with

vehicle, while a dose of 2 mg/kg (34.1 2.62 p < 0.05 vs 0.2

and 5 mg/kg) had no signiﬁcant eﬀect on seizure severity or

duration. Similarly, the dose of 2 mg/kg had no signiﬁcant

eﬀects on any seizure measurement.

Figure 11. Eﬀect of high-frequency stimulation (HFS) on long-term

potentiation (LTP) in animals treated with vehicle or 2a. In vehicle-

treated animals, there was a large increase in the population spike

(PS) amplitude. Animals treated with 2a also exhibited the same

eﬀects with no signiﬁcant diﬀerence between the vehicle-treated

animals.

Eﬀect of 2a on Hippocampal Thiol Oxidation Label-

ing. Cannabinoids showed neuroprotective eﬀects against

oxidative damage in pilocarpine-induced SE^58,61in line with

the suggestions that they can give protection against

neurological damage.²⁹

seen in animals (n = 4) given 2a 30 min before HFS, and at no

point, was a signiﬁcant diﬀerence in PS amplitude found

(Figure 11) between the vehicle and 2a groups. 2a does not

aﬀect LTP similarly to 1 in normal conditions,⁵⁴and therefore,

in people with epilepsy, it would be antiepileptic without

exacerbating comorbid memory impairment.

Eﬀect of 2a Pilocarpine-Induced Seizures, a Model of

Limbic Status Epilepticus (SE). Status epilepticus (SE) is a life-

threatening condition characterized by continuous seizures

with serious long-term consequences, such as neuronal death

and the development of TLE.⁵⁶We have recently shown that 2

mg/kg WIN55,212-2 decreased behavioral seizure severity of

pilocarpine-induced SE in CB1R-dependent mechanism.^57,58

Additionally, blockade of the FAAH alleviated behavioral but

not electrographic SE induced by kainic acid.^59,60Here, we

tested whether diﬀerent doses of 2a (2, 5, and 10 mg/kg, i.p.)

were able to aﬀect pilocarpine-induced SE (Figure 12).

Animals treated with vehicle (n = 6) after pilocarpine had

Here, we showed that 2a aﬀected histological thiol staining

in the DG hippocampus 24 h after the pilocarpine

administration. Representative images of the diﬀerential

labeling of thiols are shown in Figure 13A. A lighter shade

of green indicates an increase in oxidized thiol, and lighter red

indicates an increase in reduced thiol. Therefore, in the merged

image, more green shades indicate higher oxidized thiols

(vehicle, 2 and 5 mg/kg 2a), whereas the orange shades signify

a lower amount (vehicle and 10 mg/kg 2a). Ratiometric

histochemistry analysis was performed to analyze these results

quantitatively with total thiol oxidation compared against

vehicle-treated animals (Figure 13B). In animals treated 24 h

before with pilocarpine, there was a signiﬁcant increase in

oxidized thiols (261

13.8%; p < 0.05). There was still a

severe behavioral seizures (Racine scale: 4.33

0.33) that

signiﬁcant increase in oxidized thiols in animals pretreated (15

min before pilocarpine) with 2a doses of 2 mg/kg (268

5.7%; p < 0.05) or 5 mg/kg (273 9.9%; p < 0.05). However,

when pretreated with 10 mg/kg 2a, there was no signiﬁcant

increase in the amount of thiol oxidation in the DG. During

epileptic seizures, a vicious cycle (i.e., oxinﬂammation) starts as

were long-lasting (135 4.73 s) and had a short time to onset

(16.5 2.07 min). Pretreatment with 10 mg/kg (n = 6) 2a

signiﬁcantly reduced the severity of seizures (Racine scale: 1.16

0.31; p < 0.05 vs 0.2 and 5 mg/kg) and the duration of

seizures (67 8.68; p < 0.05 vs 0.2 and 5 mg/kg) and also

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Figure 13. Thiol oxidation in the dentate gyrus of pilocarpine-treated

rats. (A) Diﬀerential labeling of total thiols in the dentate gyrus.

Increased oxidized thiols can be observed in vehicle and doses of 2

and 5 mg/kg 2a given after PILO. A dose of 10 mg/kg 2a shows less

oxidized thiols compared to those of other doses. (B) Ratiometric

histochemistry analysis of thiols. Animals treated with PILO showed

signiﬁcant thiol oxidation compared with that of vehicle-treated

controls. Animals pretreated with a dose of 10 mg/kg did not show

this thiol oxidation. One-way ANOVA followed by Tukey’s Multiple

Comparison Test; ***p < 0.005.

Figure 14. Eﬀects of 3h on pilocarpine-induced SE. (A) A dose of 10

mg/kg 3h reduced the severity of seizures, and (B) 5 and 10 mg/kg

decreased the duration of seizures compared against all other doses.

(C) The 5 and 10 mg/kg 3h increased the time to onset of SE

compared with that of the vehicle and 2 mg/kg dose. One-way

ANOVA with Bonferroni’s Multiple Comparison ***p < 0.005, **p <

0.01, **p < 0.05.

the seizure-mediated oxidative damage triggers the activation

of redox-responsive transcription factors including NF-kB that,

in turn, lead to a boost in the oxinﬂammatory state by

producing and releasing other oxidant species and inﬂamma-

tory factors.^17,18,62The FAAH inhibitor 2a demonstrated

antioxinﬂammatory eﬀects by counteracting the loss of reduced

thiols in the pilocarpine model of SE and could also prevent

the activation of the NF-kB pathway. In this regard, the

depletion of the cellular antioxidant pool of thiols is not just a

mere index of oxidative damage caused by the SE but should

be considered as a molecular event able to induce the NF-kB

pathway, increasing the seizure susceptibility.¹⁸

duration. Similar to 2a, the dose of 2 mg/kg 3h had no

signiﬁcant eﬀects on any seizure measurement.

So far, the use of diﬀerent FAAH inhibitors has yielded

varying but promising results in terms of their antiepileptic

properties,^36,54,63,64their neuroprotective qualities in protect-

ing against excitotoxicity,⁶⁵and also on their eﬀect on LTP and

memory.³⁷In conclusion, the newly synthesized FAAH

inhibitors 2a and 3h display antiepileptic eﬀects likely without

impairing LTP and providing protection against oxidative

damage caused by seizures.

In summary, preventing the oxinﬂammatory cycle could in

part contribute to the overall antiepileptic eﬃcacy of FAAH

inhibitors.

CONCLUSIONS

Eﬀect of 3h on Pilocarpine-Induced Seizures, a

Model of Limbic Status Epilepticus (SE). The encouraging

results obtained with 2a prompted us to further explore this

series of FAAH inhibitors by using a medicinal chemistry

approach. A systematic SAR analysis around a series of

phenylpyrrole-based FAAH inhibitors, together with a

combination of biochemical and biological studies, allowed

for the selection of 3h as a suitable candidate for in vivo studies.

Here, we tested whether diﬀerent doses of 3h (2, 5, and 10

mg/kg, i.p.) were able to aﬀect pilocarpine-induced SE (Figure

14) similarly to 2a. Animals treated with vehicle (n = 6), after

pilocarpine, had severe behavioral seizures (Racine scale: 4.16

0.3), which were long-lasting (155 5.52 s) and had a short

time to onset (29.3 5.48 min). Pretreatment with 10 mg/kg

(n = 6) 3h signiﬁcantly reduced the severity of seizures

■

The ECS can delay or prevent excitotoxic damage by

modulating mitochondrial function and ﬁne-tuning the

excitatory/inhibitory stimuli. These eﬀects can prevent OS-

related epileptogenesis. On these bases, we have herein

explored the eﬀect of an indirect activation of the ECS in

epilepsy. When we interrogated the eﬀects of potent and

selective FAAH inhibitors, such as our lead 2a and the new

inhibitor 3h, in rodent models of epilepsy, they demonstrated

promising activities. Our initial studies proved the eﬃcacy of

2a in the pilocarpine and the maximal dentate activation rat

models of TLE during seizures and also its use as a

pretreatment. These results demonstrated the positive eﬀects

on synaptic plasticity (short-term and long-term plasticity

STP-LTP) and neuronal damages resulting from seizures

(histological study of thiols formations). Based on these

outcomes, we accomplished a further medicinal chemistry

approach for identifying innovative compounds for the

potential treatment of epilepsy. A systematic chemical

expansion and SAR analysis around a new series of

phenylpyrrole-based FAAH inhibitors (3a−m), ﬂanked by a

combination of biochemical and biological studies, allowed for

(Racine scale: 1

0.36; p < 0.005) and the duration of

seizures (75 15.06; p < 0.01) and also signiﬁcantly increased

the time to onset of SE (90.6 7.83 p < 0.005). The dose of 5

mg/kg (n = 6) 3h signiﬁcantly reduced the duration of seizures

(96 16.42; p < 0.05) and increased the time to onset of SE

compared with that of vehicle (69.3

10.82 p < 0.01);

however, it had no signiﬁcant eﬀect on seizure severity or

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2-(2-(4-Fluorophenoxy)ethoxy)ethyl (3-(3-Cyano-1H-pyrrol-1-yl)-

phenyl)carbamate (6c). The title compound was prepared according

to the procedure described for 6a, starting from 4 (80 mg, 0.44

mmol) and 5c (106.1 mg, 0.53 mmol). Evaporation and alumina

column chromatography (25−100% Et₂O in petroleum ether)

aﬀorded the title compound as a pale yellow oil (14% yield). ESI-

MS m/z: 432 [M + Na]⁺, 448 [M + K]⁺. ¹H NMR (300 MHz,

CDCl₃): δ 7.69 (br s, 1H), 7.49 (t, J = 1.9 Hz, 1H), 7.35 (t, J = 8.1

Hz, 1H), 7.18−7.11 (m, 1H), 7.07−7.00 (m, 2H), 6.98−6.90 (m,

3H), 6.88−6.79 (m, 2H), 6.56 (s, 1H), 4.52−4.28 (m, 2H), 4.15−

4.06 (m, 2H), 3.88−3.81 (m, 4H).

the selection of 3h as a suitable candidate for in vivo studies. In

brief, for compounds 3g, 3m, and 3h, we demonstrated

selectivity over CB1R, CB2R, and the MAGL enzyme. The

safety proﬁle of 3h was ascertained by combining metabolic

stability studies with cytotoxicity studies and the Ames test.

Notably, compounds 3h and 3m also demonstrated a potential

antioxinﬂammatory proﬁle on the neuroblastoma IMR32 cell

line being able to decrease the LPS-induced activation of NF-

kB p65. The absence of cardiac toxicity was also ascertained for

3h. Gratifyingly, when tested in vivo, the new analogue 3h

replicated the antiepileptic potential of 2a in the pilocarpine

model, conﬁrming the notable potential for this class of

analogues against seizures.

2-(2-(2-Fluorophenoxy)ethoxy)ethyl (3-(3-Cyano-1H-pyrrol-1-yl)-

phenyl)carbamate (6d). The title compound was prepared according

to the procedure described for 6a, starting from 4 (80 mg, 0.44

mmol) and 5d (106.1 mg, 0.53 mmol). Evaporation and silica gel

column chromatography (0−5% EtOAc in DCM) aﬀorded the title

compound as a pale yellow oil (55% yield). ESI-MS m/z: 432 [M +

Na]⁺. ¹H NMR (400 MHz, CDCl₃): δ 7.66 (br s, 1H), 7.45 (d, J = 1.5

Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 7.17−7.11 (m, 1H), 7.04−6.91 (m,

6H), 6.90−6.83 (m, 1H), 6.56−6.49 (m, 1H), 4.40−4.32 (m, 2H),

4.22−4.15 (m, 2H), 3.90−3.86 (m, 2H), 3.83−3.78 (m, 2H).

6-Phenylhexyl (3-(3-Carbamoyl-1H-pyrrol-1-yl)phenyl)-

carbamate (3a). To a solution of 6a (65 mg, 0.17 mmol) in 1,4-

dioxane (12 mL), sodium perborate tetrahydrate (99 mg, 0.65 mmol)

in H₂O (12 mL) was added. The reaction mixture was heated to 80

°C for 20 h. After it was cooled to 25 °C, the reaction mixture was

diluted with distilled H₂O (10 mL) and extracted with EtOAc (3 × 10

mL). The combined organic layers were washed with a saturated

solution of NaCl, dried over anhydrous sodium sulfate, ﬁltered, and

concentrated. Column chromatography (0−5% MeOH in DCM)

aﬀorded the title compound as an amorphous white solid (25% yield)

ESI-MS m/z: 428 [M+Na]⁺. ¹H NMR (300 MHz, acetone-d₆): δ 8.87

(br s, 1H), 7.90 (s, 1H), 7.79 (s, 1H), 7.52−7.35 (m, 3H), 7.31−7.16

(m, 6H), 6.97 (s, 1H), 6.73 (br s, 1H), 6.21 (br s, 1H), 4.14 (t, J = 6.5

Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 1.76−1.33 (m, 8H). ¹³C NMR (75

MHz, acetone-d₆): δ 165.5, 153.9, 142.8, 141.1, 140.8, 130.4, 130.3,

128.5, 128.4, 125.8, 122.5, 121.7, 120.7, 120.1, 116.2, 114.6, 114.3,

110.4, 35.7, 31.5, 29.4, 29.0, 28.9, 25.7. Anal. (C₂₄H₂₇N₃O₃) C, H, N.

2-(2-Phenoxyethoxy)ethyl (3-(3-Carbamoyl-1H-pyrrol-1-yl)-

phenyl)carbamate (3b). Starting from 6b (56 mg, 0.14 mmol), the

title compound was obtained following the same procedure described

for 3a. Silica gel column chromatography (0−5% MeOH in DCM)

aﬀorded the title compound as an amorphous white solid (25% yield).

EXPERIMENTAL SECTION

■

Chemistry. All reagents were purchased from commercial

suppliers and used without further puriﬁcation. All moisture-sensitive

reactions were performed under argon or nitrogen atmosphere using

oven-dried glassware and anhydrous solvents. Dry tetrahydrofuran

(THF) was freshly distilled from sodium/benzophenone, while

dichloromethane (DCM) and N,N-dimethylformamide (DMF)

were freshly distilled from calcium hydride and stored under argon

atmosphere. Flash column chromatography was carried out on silica

gel (Merck: Kieselgel 60, particle size 0.040−0.063 mm). Reverse

phase column chromatography was carried out on Lichroprep RP-18

(25−40 μm, Merck). Reactions’ progression was monitored by thin-

layer chromatography (TLC), carried out using glass-backed plates

coated with Merck Kieselgel 60 GF254. Plates were visualized under

UV light (at 254 nm) or by staining with potassium permanganate,

ninhydrin, or cerium ammonium molybdate followed by heating. MW

reactions were performed in a discovery microwave system (CEM).

¹H NMR and ¹³C NMR spectra were recorded on a Varian 300 MHz

or a Bruker 400 MHz spectrometer using the residual signal of the

deuterated solvent as an internal standard. Coupling constants (J) are

given in hertz (Hz). Splitting patterns are described as singlet (s),

doublet (d), triplet (t), quartet (q), and broad (br); the value of

chemical shifts (δ) are given in parts per million (ppm). Mass spectra

were recorded utilizing an electron spray ionization (ESI) Agilent

1100 Series LC/MSD spectrometer. Yields refer to puriﬁed products

and are not optimized. All compounds that were tested in the

biological assays were analyzed by combustion analysis (CHN) to

conﬁrm the purity >95%.

1

ESI-MS m/z: 410 [M + H]⁺, 432 [M + Na]⁺. H NMR (300 MHz,

acetone-d₆): δ 8.99 (br s, 1H), 7.90 (t, J = 2.0 Hz, 1H), 7.79 (t, J = 1.8

Hz, 1H), 7.53−7.36 (m, 2H), 7.33−7.14 (m, 4H), 7.00−6.84 (m,

3H), 6.79−6.67 (m, J = 2.7, 1.7 Hz, 1H), 6.78−6.66 (m, 1H), 6.20

(br s, 1H), 4.35−4.27 (m, 2H), 4.18−4.11 (m, 2H), 3.90−3.76 (m,

4H). ¹³C NMR (75 MHz, acetone-d₆): δ 166.3, 165.4, 159.2, 153.7,

141.1, 140.8, 130.3, 129.6, 122.5, 121.7, 120.8, 120.1, 117.1, 116.2,

114.7, 114.4, 110.4, 110.2, 69.6, 69.4, 67.5, 64.2. Anal. (C₂₂H₂₃N₃O₅)

C, H, N.

2-(2-(4-Fluorophenoxy)ethoxy)ethyl (3-(3-Carbamoyl-1H-pyrrol-

1-yl)phenyl)carbamate (3c). Starting from 6c (25 mg, 0.06 mmol),

the title compound was obtained following the procedure described

for 3a. The crude was puriﬁed by column chromatography on silica

gel (0−5% MeOH/DCM) aﬀording pure compound 3c as an

amorphous white solid (25% yield). ESI-MS m/z: 450 [M + Na]⁺. ¹H

NMR (300 MHz, acetone-d₆): δ 8.95 (br s, 1H), 7.90 (s, 1H), 7.78 (t,

J = 1.9 Hz, 1H), 7.52−7.36 (m, 2H), 7.28−7.16 (m, 2H), 7.11−6.89

(m, 4H), 6.79−6.69 (m, 1H), 6.40−5.94 (m, 2H), 4.37−4.25 (m,

2H), 4.20−4.05 (m, 2H), 3.87−3.82 (m, 2H), 3.81−3.77 (m, 2H).

¹³C NMR (75 MHz, acetone-d₆): δ 165.7, 165.3, 157.2, 153.7, 141.0,

6-Phenylhexyl (3-(3-Cyano-1H-pyrrol-1-yl)phenyl)carbamate

(6a). To a 0 °C cooled solution of 4 (80 mg, 0.44 mmol) and dry

pyridine (89 μL, 1.1 mmol) in dry DCM (8 mL), a solution of

phosgene (20% in toluene, 279 μL, 0.53 mmol) was added. The

reaction was warmed to 25 °C and stirred for 5 h aﬀording 1-(3-

isocyanatophenyl)-1H-pyrrole-3-carbonitrile; ESI-MS m/z: 242 [M +

Na]⁺. After this time, a solution of 5a (94.5 mg, 0.53 mmol) in dry

DCM (10.0 mL) was added. The resulting mixture was stirred for 20

h at 25 °C. Evaporation and silica gel column chromatography (0−5%

EtOAc in DCM) gave the title compound as a pale oil (yield 46%).

1

ESI-MS m/z: 410 [M + Na]⁺. H NMR (400 MHz, CDCl₃): δ 7.70

(br s, 1H), 7.49−7.46 (m, 1H), 7.35−7.32 (m, 4H), 7.30 (d, J = 7.6

Hz, 1H), 7.29−7.25 (m, 3H), 7.22−7.13 (m, 1H), 7.03−6.94 (m,

1H), 6.55 (dd, J = 3.0, 1.6 Hz, 1H), 4.29−4.10 (m, 2H), 2.67−2.52

(m, 2H), 1.74−1.54 (m, 4H), 1.47−1.29 (m, 4H).

2-(2-Phenoxyethoxy)ethyl (3-(3-Cyano-1H-pyrrol-1-yl)phenyl)-

carbamate (6b). The title compound was prepared according to

the procedure described for 6a starting from 4 (80 mg, 0.44 mmol)

and 5b (96.5 mg, 0.53 mmol). Alumina column chromatography

(30−100% Et₂O in petroleum ether) gave the title compound as a

140.8, 130.3, 121.6, 120.9, 120.0, 116.2, 116.0, 115.9, 115.9, 115.6,

115.5, 114.5, 113.6, 69.6, 69.4, 68.3, 64.2. Anal. (C₂₂H₂₂FN₃O₅) C, H,

N.

1

pale yellow oil (yield 33%). ESI-MS m/z: 414 [M + Na]⁺. H NMR

(300 MHz, CDCl₃): δ 7.66 (br s, 1H), 7.46 (d, J = 0.4 Hz, 1H), 7.33

(t, J = 8.1 Hz, 1H), 7.29−7.21 (m, 2H), 7.19−7.12 (m, 2H), 7.04−

6.98 (m, 2H), 6.94−6.86 (m, 3H), 6.56−6.51 (m, 1H), 4.42−4.25

(m, 2H), 4.17−4.04 (m, 2H), 3.90−3.81 (m, 4H).

2-(2-(2-Fluorophenoxy)ethoxy)ethyl (3-(3-Carbamoyl-1H-pyrrol-

1-yl)phenyl)carbamate (3d). Starting from 6d (50 mg, 0.12 mmol),

the title compound was obtained following the same procedure

described for 3a. Silica gel column chromatography (0−2% MeOH in

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DCM) aﬀorded pure compound 3d as an amorphous white solid

dioxane (114 mL), dimethoxytetrahydrofuran-3-carbonitrile 10 (2.2

g, 14.3 mmol) was added; the resulting mixture was reﬂuxed for 20

min; then, 11.8 mL of 6 M HCl were added dropwise, and the

reaction was reﬂuxed for a further 20 min. After it was cooled to 25

°C, the solvent was removed under reduced pressure. The residue was

suspended with H₂O (20 mL) and extracted with DCM (3 × 20 mL).

The combined organic layers were washed with a saturated solution of

NaCl, dried over anhydrous sodium sulfate, ﬁltered, and concentrated.

The crude was puriﬁed by column chromatography on silica gel (20%

EtOAc in petroleum ether) to aﬀord 11 as an amorphous brown solid

(87% yield). ESI-MS m/z: 244 [M + H]⁺; 266 [M + Na]⁺. ¹H NMR

(300 MHz, CDCl₃): δ 8.10 (d, J = 9.2 Hz, 1H), 7.28 (dd, J = 2.2, 1.6

Hz, 1H), 7.06 (dd, J = 9.2, 2.7 Hz, 1H), 6.89 (d, J = 2.7 Hz, 1H), 6.76

(dd, J = 3.0, 2.3 Hz, 1H), 6.60 (dd, J = 3.0, 1.6 Hz, 1H), 3.94 (s, 3H).

1-(5-Hydroxy-2-nitrophenyl)-1H-pyrrole-3-carbonitrile (12). To a

suspension of 11 (500 mg, 2.0 mmol) in dry DCM (20.0 mL), cooled

to −78 °C, boron tribromide (1 M solution in DCM 20.0 mL) was

added. The reaction mixture was warmed to 25 °C and stirred for 96

h. The mixture was treated with 4 M NaOH and extracted with DCM

(3 × 15 mL); the aqueous layer was acidiﬁed to pH = 2 with 6 M HCl

and extracted with EtOAc (3 × 15 mL). The combined organic

extracts were dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure to aﬀord 12 (50% yield). ESI-

1

(25% yield). ESI-MS m/z: 450 [M + Na]⁺. H NMR (300 MHz,

acetone-d₆): δ 8.96 (br s, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 7.54−7.33

(m, 3H), 7.20 (t, J = 12.6 Hz, 3H), 7.09 (t, J = 7.8 Hz, 2H), 6.93 (br

s, 1H), 6.73 (s, 1H), 6.36−5.94 (m, 1H), 4.39−4.28 (m, 2H), 4.26−

4.18 (m, 2H), 3.93−3.85 (m, 2H), 3.83−3.77 (m, 2H). ¹³C NMR (75

MHz, acetone-d₆): δ 165.3, 153.7, 149.0, 141.0, 140.8, 130.3, 127.4,

124.8, 124.7, 121.6, 121.4, 121.3, 116.2, 116.0, 115.5, 114.5, 110.4,

110.2, 69.5, 69.4, 69.0, 64.2. Anal. (C₂₂H₂₂FN₃O₅) C, H, N.

1-(3-Aminophenyl)-1H-pyrrole-3-carboxamide (7). To a solution

of 4 (65.0 mg, 0.35 mmol) in EtOH (22.0 mL), 6N NaOH (867.0

μL) and 30% H₂O₂(867.0 μL) were added. The reaction mixture was

reﬂuxed for 3 h. After it was cooled to 25 °C, a saturated solution of

Na₂S₂O₃(2.0 mL) was added, and the solvent was evaporated under

reduced pressure. The residue was taken up with H₂O and extracted

with EtOAc (3 × 5 mL). The combined organic layers were washed

with a saturated solution of NaCl, dried over anhydrous sodium

sulfate, ﬁltered, and concentrated. The crude was used in the next step

without any further puriﬁcation (yellow solid, 90% yield). ESI-MS m/

1

z: 224 [M + Na]⁺. H NMR (300 MHz, CDCl₃): δ 7.69−7.53 (m,

1H), 7.20 (t, J = 8.0 Hz, 1H), 7.03−6.94 (m, 1H), 6.82−6.73 (m,

1H), 6.68 (d, J = 2.1 Hz, 1H), 6.65−6.58 (m, 1H), 6.56−6.47 (m,

1H), 5.53 (br s, 2H), 3.82 (br s, 2H).

MS m/z: 228 [M − H]⁻. H NMR (400 MHz, acetone-d₆): δ 10.16

1-(3-(3-(6-Phenylhexyl)ureido)phenyl)-1H-pyrrole-3-carboxa-

mide (3e). To a solution of 7 (30 mg, 0.15 mmol) in dry THF (5.0

mL), TEA (83.6 μL, 0.60 mmol) and 6-phenylhexan-1-yl-isocyanate

(121 mg, 0.60 mmol) were sequentially added. The reaction mixture

was stirred at 25 °C for 16 h. Evaporation and silica gel column

chromatography (5% MeOH in DCM) gave the title compound as an

amorphous white solid (62% yield). ESI-MS m/z: 405 [M + H]⁺. ¹H

NMR (300 MHz, acetone-d₆): δ 8.59 (br s, 1H), 7.81−7.69 (m, 2H),

7.43 (s, 1H), 7.36−6.97 (m, 8H), 6.82 (s, 1H), 6.65−6.56 (m, 1H),

6.66−6.57 (m, 1H), 6.21 (t, J = 5.6 Hz, 1H), 3.29−3.24 (m, 2H),

3.12−2.97 (m, 2H), 1.62−1.34 (m, 4H), 1.29−1.21 (m, 4H). ¹³C

NMR (75 MHz, DMSO): δ 165.7, 155.7, 142.9, 142.6, 140.5, 130.6,

128.9 (2), 128.9 (2), 126.3, 122.6, 121.9, 120.5, 116.0, 113.1, 110.9,

109.6, 35.8, 31.7, 31.6, 30.3, 29.1, 26.9. Anal. (C₂₄H₂₈N₄O₂) C, H, N.

3-(3-Cyano-1H-pyrrol-1-yl)-N-(6-phenylhexyl)benzamide (9). To

a 0 °C cooled solution of 8 (200 mg, 0.94 mmol) and PPh₃(495 mg,

1.95 mmol) in dry DCM (30.0 mL), hexachloroacetone (74 μL) was

added, and the reaction was stirred for 30 min. A solution of 6-

phenylhexan-1-amine (111 mg, 0.63 mmol) and TEA (88 μL) in

DCM (5.0 mL), prepared in a diﬀerent ﬂask, was added. The resulting

mixture was stirred for an additional 1 h at 0 °C. After this time,

saturated aqueous NaHCO₃was added, and the aqueous layer was

extracted with DCM (3 × 10 mL). The combined organic layers were

washed with a saturated solution of NaCl, dried over anhydrous

sodium sulfate, ﬁltered, and concentrated. The crude was puriﬁed by

column chromatography on silica gel (40% EtOAc in petroleum

ether) aﬀording the title compound as an amorphous white solid

1

(s, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.66 (m, 1H), 7.12 (m, 1H), 7.03

(m, 2H), 6.60 (m, 1H).

1-(5-Hydroxy-2-nitrophenyl)-1H-pyrrole-3-carboxamide (13a).

Starting from 12 (570 mg, 2.49 mmol), the title compound was

prepared according to the procedure followed for 7. Puriﬁcation by

ﬂash chromatography on silica gel (0−5% MeOH in EtOAc) aﬀorded

the title compound as amorphous orange solid (40% yield). ESI-MS

m/z: 246 [M − H]⁻. ¹H NMR (300 MHz, CD₃OD): δ 8.00 (d, J = 9

Hz, 1H), 7.43 (t, J = 2.1, 1H), 6.95 (dd, J = 9.3, 2.7 Hz, 1H), 6.86 (d,

J = 2.7 Hz, 1H), 6.83 (t, J = 2.4, 1H), 6.70−6.68 (m, 1 H).

4-Amino-3-(3-Carbamoyl-1H-pyrrol-1-yl)phenyl (6-Phenylhexyl)-

carbamate (3g). To a solution of 13a (134 mg, 0,54 mmol) in dry

toluene (12.0 mL) and dry DMF (3.0 mL), 6-phenylhexan-1-yl-

isocyanate(219 mg, 1.08 mmol) and TEA (9 μL, 0.06 mmol) were

sequentially added. The reaction mixture was reﬂuxed for 48 h. After

cooling to 25 °C, solvents were removed in vacuo. The residue was

immediately dissolved in EtOH (10 mL), and a catalytic amount of

10% Pd/C was added. The reaction mixture was stirred at 25 °C

under H₂atmosphere for 4 h. Thereafter, the Pd/C was ﬁltered oﬀ

and EtOH was evaporated under reduced pressure. Silica gel column

chromatography (100% EtOAc) gave the title compound as colorless

1

oil (45% yield). ESI-MS m/z: 421 [M + H]⁺. H NMR (300 MHz,

acetone-d₆): δ 7.43 (t, J = 1.9 Hz, 1H), 7.31−7.10 (m, 5H), 6.97−

6.82 (m, 5H), 6.69 (dd, J = 2.9, 1.7 Hz, 2H), 6.23 (br s, 1H), 4.50 (br

s, 2H), 3.20−3.16 (m2H), 2.61 (t, J = 7.7 Hz, 2H), 1.68−1.48 (m,

4H), 1.48−1.25 (m, 4H). ¹³C NMR (75 MHz, acetone-d₆): δ 165.6,

155.1, 142.9, 142.7, 140.4, 128.5 (2), 128.54 (2), 128.4, 125.9, 125.7,

124.2, 122.5, 121.6, 120.1, 116.3, 109.6, 41.0, 35.8, 29.8, 28.9, 31.6,

26.6. Anal. (C₂₄H₂₈N₄O₃) C, H, N.

1

(30% yield). ESI-MS m/z: 394 [M + Na]⁺. H NMR (300 MHz,

CDCl₃): δ 7.86 (br s, 1H), 7.69 (d, J = 7.3 Hz, 1H), 7.59−7.42 (m,

3H), 7.30−7.02 (m, 6H), 6.59 (d, J = 1.2 Hz, 1H), 6.39 (s, 1H),

3.59−3.28 (m, 2H), 2.60 (t, J = 7.6 Hz, 2H), 1.73−1.49 (m, 4H),

1.40 (m, 4H).

3-(3-Carbamoyl-1H-pyrrol-1-yl)phenyl (2-(4-Benzylpiperazin-1-

yl)ethyl)carbamate (3k). To a solution of 13b (30 mg, 0.15 mmol)

and DMAP (73 mg, 0.60 mmol) in dry THF (12 mL) cooled to 0 °C

a solution of phosgene (20% in toluene, 157 μL, 0.3 mmol) was

added. The reaction mixture was warmed to 25 °C and stirred for 30

min under N₂atmosphere. At this time, the amine 14 (65 mg, 0.30

mmol) was added. The resulting mixture was reﬂuxed for 16 h. After

the evaporation and puriﬁcation by silica gel column chromatography

(2−5% MeOH in AcOEt), the title compound was obtained as an

1-(3-((6-Phenylhexyl)carbamoyl)phenyl)-1H-pyrrole-3-carboxa-

mide (3f). Starting from 9 (85 mg, 0.23 mmol), the title compound

was obtained following the same procedure described for 3a. Column

chromatography on silica gel (5% MeOH in DCM) aﬀorded pure

compound 3j as an amorphous white solid (42% yield). ESI-MS m/z:

412 [M + Na]⁺. ¹H NMR (300 MHz, acetone, d₆): δ 8.06 (br s, 1H),

7.98 (s, 1H), 7.89−7.78 (m, 2H), 7.74−7.69 (m, 1H), 7.64−7.54 (m,

5H), 7.38−7.11 (m, 1H), 7.08−6.90 (br s, 1H), 6.76 (br s, 1H), 6.33

(s, 2H), 3.48−3.28 (m, 2H), 2.65−2.41 (m, 2H), 1.71−1.52 (m, 4H),

1.49−1.33 (m, 4H). ¹³C NMR (75 MHz, acetone-d₆): δ 165.6, 165.4,

142.9, 140.2, 137.2, 130.1, 128.5 (2C), 128.4 (2C), 125.7, 125.2,

122.8, 122.6, 121.9, 120.2, 118.9, 110.6, 39.9, 35.8, 31.6, 29.6, 28.9,

26.9. Anal. (C₂₄H₂₇N₃O₂) C, H, N.

1

amorphous white solid (20% yield). H NMR (300 MHz, CDCl₃): δ

7.64 (s, 1H), 7.41 (t, J = 8.30 Hz, 1H), 7.33−7.21 (m, 7H), 7.09 (d, J

= 8.68 Hz, 1H), 7.02 (s, 1H), 6.55 (s, 1H), 5.86 (brs, 1H), 5.60 (brs,

2H), 3.56 (s, 2H), 3.40 (d, J = 5.27 Hz, 2H), 2.59 (br s, 10H). ¹³C

NMR (75 MHz, CDCl₃): δ 166.1, 154.1, 151.9, 140.5, 130.4, 129.31,

128.31, 127.31, 122.6, 120.6, 119.9, 117.5, 114.6, 109.6, 62.7, 56.6,

52.6, 37.4, 29.7. Anal. (C₂₅H₂₉N₅O₃) C, H, N.

1-(5-Methoxy-2-nitrophenyl)-1H-pyrrole-3-carbonitrile (11). To

a solution of 5-methoxy-2-nitroaniline (2.0 g, 11.89 mmol) in 1,4-

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3-(3-Carbamoyl-1,5-dimethyl-1H-pyrrol-2-yl)phenyl (6-

Phenylhexyl)carbamate (3h). To a solution of 15a (30 mg, 0.13

mmol) in dry THF, 6-phenylhexan-1-yl-isocyanate (53 mg, 0.26

mmol) and TEA (72 μL, 0.52 mmol) were sequentially added. The

resulting mixture was reﬂuxed for 12 h. After it was cooled to 25 °C

and after evaporation, silica gel column chromatography (2% MeOH

in DCM) aﬀorded the title compound as a pale-yellow oil (36%

7.09−7.07 (m, 1H), 7.00−6.96 (m, 4H), 6.34−6.32 (m, 1H), 4.65 (br

s, 1H).

1-(3-Hydroxyphenyl)-1H-pyrrole-2-carboxamide (20). Starting

from 19 (70 mg, 0.38 mmol), the title compound was prepared

according to the procedure followed for 7. The crude was puriﬁed by

means of silica gel column chromatography (30% petroleum ether in

EtOAc) to aﬀord 20 as a colorless oil (50% yield). ESI-MS m/z: 203

1

yield). ESI-MS m/z: 434 [M + H]⁺, 456 [M + Na]⁺. H NMR (300

1

[M + H]⁺. H NMR (300 MHz, CD₃OD): δ 7.34−7.27 (m, 1H),

MHz, CDCl₃): δ 7.45 (t, J = 7.5 Hz, 1H), 7.29−7.15 (m, 8H), 6.43 (s,

1H), 5.20 (br s, 3H), 3.26 (s, 3H), 3.24−3.20 (m, 2H), 2.60 (t, J =

7.5 Hz, 2H), 2.23 (s, 3H), 1.65−1.56 (m, 4H), 1.39−1.35 (m, 4H).

¹³C NMR (75 MHz, CDCl₃): δ 166.9, 154.5, 151.6, 142.8, 133.3,

133.2, 130.1, 129.7 (2C), 128.6 (2C), 128.5, 127.9, 125.9, 124.6,

122.4, 115.7, 108.2, 41.5, 36.1, 31.7, 31.5, 29.9, 29.1, 26.1. Anal.

(C₂₆H₃₁N₃O₃) C, H, N.

7.09−7.07 (m, 1H), 7.00−6.96 (m, 4H), 6.34−6.32 (m, 1H).

3-(2-Carbamoyl-1H-pyrrol-1-yl)phenyl (6-Phenylhexyl)-

carbamate (3l). Starting from 20 (10 mg, 0.05 mmol), 6-

phenylhexan-1-yl-isocyanate (40 mg, 0.20 mmol), and TEA (28 μL,

0.20 mmol), the title compound was prepared according to the

procedure followed for 3h. Silica gel column chromatography (1%

MeOH in DCM) aﬀorded 3l as an amorphous white solid (70%

yield). ESI-MS m/z: 428 [M + Na]⁺. ¹H NMR (300 MHz, CD₃OD):

δ 7.39 (t, J = 6 Hz, 1H), 7.25−7.20 (m, 7H), 7.01 (t, J = 3 Hz, 1H),

6.95−6.90 (m, 1H), 6.76−6.71 (m, 1H), 6.25 (m, 1H), 3.15 (t, J =

7.5 Hz, 2 H), 2.62−2.56 (m, 2H), 1.63 (m, 4H), 1.45−1.35 (m, 4H).

¹³C NMR (75 MHz, CD₃OD): δ 164.6, 155.6, 151.6, 142.7, 141.6,

3-(3-Carbamoyl-5-methyl-1H-pyrrol-2-yl)phenyl (6-

Phenylhexyl)carbamate (3i). Starting from 15b (30 mg, 0.14

mmol), 6-phenylhexan-1-yl-isocyanate (57 mg, 0.28 mmol) and

TEA (77 μL, 0.56 mmol), the title compound was prepared according

to the procedure followed for 3h. Column chromatography on silica

gel (1% MeOH in DCM), aﬀording 3i as an amorphous white solid

129.0, 128.4 128.2 (2C), 121.1 (2C), 126.4, 125.4, 122.2, 120.3,

118.9, 115.7, 108.9, 40.8, 35.6, 31.4, 29.5, 28.8, 26.5. Anal.

(C₂₄H₂₇N₃O₃) C, H, N.

1

(20% yield). ESI-MS m/z: 442 [M + Na]⁺, 458 [M + K]⁺. H NMR

(300 MHz, CDCl₃): δ 8.40 (br s, 1H), 7.40−7.34 (m, 2H), 7.29−7.24

(m, 3H), 7.18−7.15 (m, 3H), 7.08−7.06 (m, 1H), 6.28 (s, 1H), 5.35

(br s, 1H), 5.25 (brs, 1H), 5.15 (brs, 1H), 3.26−3.19 (m, 2H), 2.61

(t, J = 7.2 Hz, 2H), 2.24 (s, 3H), 1.65−1.56 (m, 4H), 1.39−1.35 (m,

4H). ¹³C NMR (75 MHz, CDCl₃): δ 167.4, 154.8, 151.4, 142.8,

133.5, 131.6, 129.9, 128.6 (2C), 128.5 (2C), 128.4, 125.9, 122.3,

121.4, 115.8, 108.9, 41.5, 36.1, 31.5, 29.9, 29.1, 26.8, 12.9. Anal.

(C₂₅H₂₉N₃O₃) C, H, N.

3-(5-Carbamoyl-1-methyl-1H-pyrrol-2-yl)phenyl (6-

Phenylhexyl)carbamate (3j). Starting from 16 (35 mg, 0.16

mmol), the title compound was prepared according to the procedure

followed for 3h. Silica gel column chromatography (2% MeOH in

DCM) aﬀorded 3j as a colorless oil (79% yield). ESI-MS m/z: 442

[M + Na]⁺. ¹H NMR (300 MHz, CDCl₃): δ 7.40 (t, J = 7.2 Hz, 1H),

7.30−7.13 (m, 8H), 6.67 (d, J = 3.3 Hz, 1H), 6.18 (d, J = 4.2 Hz,

1H), 5.70 (br s, 2H), 5.18 (br s, 1H), 3.89 (s, 3H), 3.29−3.22 (m,

2H), 2.62 (t, J = 7.2 Hz, 2H), 1.66−1.55 (m, 4H), 1.43−1.39 (m,

4H).¹³C NMR (75 MHz, CDCl₃): δ 164.1, 154.6, 151.3, 142.8, 140.1,

133.6, 129.5, 128.6 (2C), 128.5 (2C), 126.3, 126.3, 125.9, 122.8,

121.4, 113.3, 109.0, 41.5, 36.1, 34.8, 31.6, 30.0, 29.1, 26.8. Anal.

(C₂₅H₂₉N₃O₃) C, H, N.

3-(1H-Pyrrol-1-yl)phenol (18). A solution of 3-aminophenol (100

mg, 0.91 mmol) and 2,5-dimethoxytetrahydrofuran 17 (145 mg, 1.09

mmol) in AcOH (1.0 mL) was placed in a sealed tube and heated to

170 °C in a microwave apparatus to a power of 150 W for 10 min.

After it was cooled to 25 °C, the reaction was treated with water, and

the aqueous layer was extracted with DCM (3 × 5 mL). The

combined organic phases were washed with a saturated solution of

NaCl, dried over anhydrous sodium sulfate, ﬁltered, and concentrated.

Column chromatography on silica gel (2% MeOH in DCM) aﬀorded

the title compound as a pale yellow oil (80% yield). ESI-MS m/z: 158

[M − H]⁻.¹H NMR (300 MHz, CDCl₃): δ 7.27 (t, J = 9 Hz, 1H),

7.09 (t, J = 3 Hz, 2H), 7.00−6.97 (m, 1H), 6.89 (t, J = 3 Hz, 1H),

6.74−6.71 (m, 1H), 6.38 (t, J = 3 Hz, 2H), 6.08 (br s, 1H).

Spectroscopic data were consistent with previously reported ones.^46,47

1-(3-Hydroxyphenyl)-1H-pyrrole-2-carbonitrile (19). To a sol-

ution of compound 18 (50 mg, 0.31 mmol) in dry MeCN (5.0 mL)

cooled to −20 °C, chlorosulfonyl isocyanate (67 mg, 0.47 mmol) was

added, and the mixture was stirred for 5 min at −20 °C and for

another 12 h at 25 °C. Then, 1.0 mL of dry DMF was added, and the

mixture was heated to 55 °C and stirred for an additional 20 min.

Reaction was quenched with water, and the aqueous layer was

extracted with DCM (3 × 5 mL). The combined organic layers were

washed with a saturated solution of NaCl, dried over anhydrous

sodium sulfate, ﬁltered, and concentrated. Column chromatography

on silica gel (20% EtOAc in petroleum ether) aﬀorded the title

compound as an amorphous white solid (20% yield). ESI-MS m/z:

185 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃): δ 7.34−7.27 (m, 1H),

1-Bromo-9-methoxy-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]-

diazepin-4-one (22). To a solution of 21 (821 mg, 3.6 mmol) in dry

THF (20.0 mL) cooled to −70 °C, NBS (725 mg, 4.07 mmol) was

added. The reaction mixture was stirred for 1.5 h at −70 °C and then

gradually warmed to 25 °C and stirred for an additional 12 h.

Evaporation and column chromatography on silica gel (10%

acetonitrile in DCM) and recrystallization from EtOAc aﬀorded the

title compound as a white crystalline solid (45% yield). The mp =

190−193 °C. ESI-MS m/z: 307 [M + H]⁺, 329 [M + Na]⁺. ¹H NMR

(400 MHz, CDCl₃): δ 7.50 (br s, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.07

(d, J = 2.4 Hz, 1H), 7.02 (d, J = 4.0 Hz, 1H), 6.83 (dd, J = 8.4, 2.5 Hz,

1H), 6.49 (d, J = 3.9 Hz, 1H), 4.31 (m, 1H), 3.89 (m, 1H), 3.81 (s,

3H).

9-Methoxy-4-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]-

diazepine-1-carbonitrile (23). To a solution of 22 (496.0 mg, 1.61

mmol) in dry DMF (20.0 mL), CuCN (3,6 g, 25.00 mmol) was

added. The reaction mixture was reﬂuxed for 12 h; then, the crude

was treated with a concentrated solution of NH₄OH and extracted

with DCM. Evaporation and column chromatography on silica gel

(10% acetonitrile in DCM) aﬀorded the title compound as an

amorphous white solid (30% yield). ESI-MS m/z: 254 [M + H]⁺, 276

[M + Na]⁺. ¹H NMR (300 MHz, CDCl₃): δ 7.38 (br s, 1H), 7.29 (s,

1H), 7.18 (d, J = 2.4 Hz, 1H), 7.08−7.03 (m, 2H), 6.92 (d, J = 8.4,

1H), 4.36−4.30 (m, 1H), 4.02−3.94 (m, 1H), 3.87 (s, 3H).

9-Hydroxy-4-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]-

diazepine-1-carbonitrile (24). Starting from 23 (500 mg, 1.97

mmol), title compound was prepared according to the procedure

1

followed for 12 (30% yield). ESI-MS m/z: 238 [M − H]⁻. H NMR

(400 MHz, acetone-d₆): δ 7.74 (br s, 1H), 7.31 (m, 1H), 7.19 (m,

2H), 6.87 (m, 2H), 4.25(m, 1H), 4.03 (m, 1H).

9-Hydroxy-4-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]-

diazepine-1-carboxamide (25). Starting from 24 (45 mg, 0.19

mmol), the title compound was prepared according to the procedure

described for 7. Puriﬁcation on silica gel (DCM/MeOH/AcOH: 9/1/

0.3) aﬀorded 25 as a yellow, pale oil (40% yield). ESI-MS m/z: 256

[M − H]⁻. ¹H NMR (400 MHz, MeOD): δ 7.20 (d, J = 8.3 Hz, 1H),

6.93 (d, J = 4.0 Hz, 1H), 6.89 (d, J = 4.0 Hz, 1H), 6.73 (dd, J = 8.2,

2.3 Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H), 4.33 (d, J = 14.6 Hz, 1H), 3.95

(d, J = 14.6 Hz, 1H).

1-Carbamoyl-4-oxo-5,6-dihydro-4H-benzo[f]pyrrolo[1,2-a][1,4]-

diazepin-9-yl (6-Phenylhexyl)carbamate (3m). Starting from 25 (50

mg, 0.19 mmol), the title compound was prepared according to the

procedure followed for 3h. Puriﬁcation on silica gel (10% MeOH in

DCM) aﬀorded 3m as an amorphous white solid (50% yield). ESI-

MS m/z: 461 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 8.48 (br

s, 1H), 7.87 (br s, 1H), 7.75 (t, J = 5.2 Hz, 1H), 7.39 (d, J = 8.2 Hz,

1H), 7.31 (br s, 1H), 7.22 (m, 2H), 7.15−7.11 (m, 3H), 7.04 (d, J =

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8.3 Hz, 1H), 6.84 (d, J = 3.8 Hz, 1H), 6.79 (d, J = 3.7 Hz, 1H), 6.74

(br s, 1H), 4.15−4.12 (m, 1H), 4.00−3.95 (m, 1H), 2.98 (m, 2H),

2.52 (m, 2H), 1.53 (m, 2H), 1.40 (m, 2H), 1.27−1.20 (m, 4H). ¹³C

NMR (75 MHz, DMSO-d₆): δ 162.7, 161.9, 154.0, 150.8, 142.8,

138.3, 134.0, 132.0, 130.2, 129.1, 129.0 (2C), 128.7, 126.3 (2C),

119.8, 119.0, 115.6, 115.5, 42.0, 35.7, 31.6, 29.7, 28.9, 28.1, 26.5. Anal.

(C₂₆H₂₈N₄O₄) C, H, N.

X-ray Crystallography. A single crystal of compound 22 was

submitted to the X-ray data collection on an Oxford-Diﬀraction

Xcalibur Sapphire 3 diﬀractometer with a graphite monochromated

Mo Kα radiation (λ = 0.710 73 Å) at 293 K. The structure was solved

by direct methods implemented in SHELXS program (Version 2013/

1).⁶⁶The reﬁnement was carried out by full-matrix anisotropic least-

squares on F2 for all reﬂections for non-H atoms by means of the

SHELXL program.⁶⁷

Crystallographic data have been deposited with the Cambridge

Crystallographic Data Centre as supplementary publication no.

CCDC 2008490.

Copies of the data can be obtained, free of charge, on application to

CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44 (0)

1223 336 033; email: deposit@ccdc.cam.ac.uk).

selective FAAH inhibitor URB597 (1). The eﬀect of diﬀerent

compounds on FAAH activity was ascertained by adding each

substance directly to the incubation medium.

MAGL Activity Assay. Mouse brain was thawed and homogenized

at 4 °C in sodium phosphate buﬀer (50 mM, pH = 8.0) containing

0.32 M sucrose. Homogenates were centrifuged at 4 °C sequentially

at 800 g, 10 000 g, and 100 000 g. MAGL activity was assayed in ﬁnal

supernatants at 100 000 g, incubated with 10 μM [³H] 2-OG (20 Ci/

mmol; ARC, St. Louis, MO) at 37 °C for 30 min. The reaction was

stopped with a 2:1 (v/v) mixture of chloroform/methanol, and the

release of [³H]glycerol in the aqueous phase was measured by

scintillation counting.⁷⁸

Receptor Binding Assays. Mouse brain and spleen for CB1R

and CB2R assay, respectively, was resuspended in 2 mM TrisEDTA,

320 mM sucrose, and 5 mM MgCl₂(pH 7.4); then, it was

homogenized in a Potter homogenizer and centrifuged three times at

1000 g, and the pellet was discharged. The supernatant was

centrifuged at 18 000 g (30 min), and the pellet was resuspended in

assay buﬀer (50 mM TrisHCl, 2 mM TrisEDTA, 3 mM MgCl₂, pH

7.4). These membrane fractions were used in rapid ﬁltration assays

with radiolabel agonist [³H]CP55,940 (PerkinElmer Life Sciences,

Boston, MA, USA), as described previously.⁷⁸In all experiments,

nonspeciﬁc binding was determined in the presence of 1 μM “cold”

agonist, and the eﬀect of selective compounds for CB1R or CB2R was

tested by adding each substance directly to the incubation medium.

Cytotoxicity and Mutagenicity Assays. 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-MG human glioblasto-

ma astrocytoma were purchased from American Type Culture

Collection (USA). The mutagenicity assay was supplied by Biologik

s.r.l. (Trieste, Italy).

Computational Details. Ligands Preparation. Compounds were

̈

built by means of Maestro (Maestro, release 2018, Schrodinger LLC,

New York, NY, 2018). Molecular energy minimizations were

̈

performed by MacroModel (MacroModel, release 2018, Schrodinger

LLC, New York, NY, 2018) using OPLS3 as force ﬁeld.⁶⁸The solvent

eﬀects were simulated using the analytical Generalized-Born/Surface

Area (GB/SA) model,⁶⁹and no cutoﬀ for nonbonded interactions

was selected. Polak−Ribiere conjugate gradient (PRCG) method with

1000 maximum iterations and 0.001 gradient convergence threshold

was used. All derivatives described in this work were treated by

̈

LigPrep application (LigPrep, release 2018, Schrodinger LLC, New

Cell Cultures and Cytotoxicity Assay. NIH3T3 and U-373-MG

were utilized for cytotoxicity experiments. Cells were maintained in

DMEM at 37 °C in a humidiﬁed atmosphere containing 5% CO₂. The

culture media were supplemented with 10% fetal calf serum (FCS),

1% L-glutamine−penicillin−streptomycin solution, and 1% MEM

Non-Essential Amino Acid Solution. Once at the 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

resuspended in medium solution (dilution 1:15). The stock solution

for each compound was prepared in pure DMSO and diluted with

complete culture medium. 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.⁷⁹The data processing included the Student’s t-test with p <

0.05 taken as the signiﬁcance level.

York, NY, 2018), implemented in Maestro suite 2018, generating the

most probable ionization state of any possible enantiomers and

tautomers at cellular pH value (7.4 0.5) and also avoiding potential

error in the structures.^{41,42,70−72}

Protein Preparation. The three-dimensional structure of FAAH

(PDB ID: 3PPM)⁷³was taken from the PDB and imported into

̈

Schrodinger Maestro molecular modeling environment. Water

molecules and compounds used for the crystallization were removed,

and the resulting structure was submitted to the protein preparation

wizard implemented in Maestro suite 2018 as previously

reported.^39,40,74

Molecular Docking. Molecular docking was carried out using the

̈

Schrodinger suite 2018 employing the IFD protocol (Schrodinger

̈

Suite 2018 Induced Fit Docking protocol release 2018, Schrodinger

LLC, New York, NY, 2012) as previously reported.^39,40,75Brieﬂy, 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

cocrystallized ligand for the FAAH enzyme. Complexes within 30.0

kcal/mol of the 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.

First, the following solutions were prepared to determine the

percentage of viable cells:

1. Neutral Red (NR) Stock Solution: 0.33 g of NR Dye powder

in 100 mL of sterile H₂O.

2. NR Medium: 1.0 mL of NR Stock solution + 99.0 Routine

Culture Medium prewarmed to 37 °C.

3. NR Desorb solution: 1% glacial acetic acid solution + 50%

ethanol + 49% H₂O.

At the end of the incubation, the routine culture medium was

removed from each well, and cells were carefully rinsed with 1 mL of

prewarmed D-PBS. Multiwells were then gently blotted with paper

towels. NR Medium (1.0 mL) was added to each well and further

incubated at 37 °C, 95% humidity, and 5.0% CO₂for 3 h. The cells

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 prewarmed D-PBS. Then, the PBS was decanted

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

20−45 min to extract NR from the cells and form a homogeneous

solution. During this step, the samples were covered in order to

protect them from light. Five minutes after the plate shaker removal,

Molecular Properties Prediction. The in silico druglike features of

the designed compounds were evaluated by means of QikProp

̈

implemented in Maestro suite (QikProp, release 2018, Schrodinger,

LLC, New York, NY, 2018).^76,77

Enzymatic Assays. FAAH Activity Assay. Fatty acid amide

hydrolase activity was assayed in homogenate mouse brain incubated

at pH 9.0 with 10 μM [¹⁴C]AEA (ARC, St. Louis, MO, USA) at 37

°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 [¹⁴C]-

ethanolamine in the aqueous phase was measured as reported.⁷⁸

Control experiments were also carried out in the presence of the

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the absorbance was read at 540 nm by a UV/visible spectropho-

tometer (Lambda 25, PerkinElmer).

centrifugation. Cell pellets were processed as previously described.⁸²

The supernatant containing the cytosolic proteins was removed, and

the pellet containing the nuclei was suspended in the nuclear

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 phenyl-methyl-

sulphonyl ﬂuoride, and protease and phosphatase inhibitor cocktails.

After 30 min of incubation on ice with intermitted mixing, samples

were centrifuged at 21 100 g for 15 min to obtain the nuclear protein

fractions. Protein concentration was determined using a Bradford

protein assay (Bio-Rad Protein Assay; Bio-Rad Laboratories, Inc.,

Milan, Italy).

NF-κB p65 DNA Binding Activity in Nuclear Extracts. The DNA-

binding activity of the NF-kB p65 subunit in IMR32 cell nuclear

extracts was quantiﬁed using the TransAM NF-kB p65 Transcription

Factor Assay Kit (Active Motif North America, Carlsbad, CA, USA).

Brieﬂy, 5 μg of nuclear protein extracts were incubated with NF-κB

p65 consensus binding sites (5′-GGG ACT TTCC-3′) immobilized

on a 96-well plate for 1 h at rt. Then, a secondary antibody conjugated

with a horseradish peroxidase provides a colorimetric output,

spectrophotometrically detected at 450 nm. NF-κB p65 binding to

the oligonucleotides was detected by incubation with a primary

antibody speciﬁc for the activated form of p65 (Active Motif North

America), HRP-conjugated secondary antibody, and developing

solution. The absorbance at 450 nm was determined with a

microplate reader. Results were normalized for nuclear extract protein

concentrations.

Statistical Analysis. All the results are expressed as mean SD of

triplicate determinations obtained in three independent experiments.

For each of the variables tested, a two-way analysis of variance

(ANOVA) was used. P values < 0.05 were considered statistically

signiﬁcant. Data were analyzed using the software GraphPad Prism

4.0 (GraphPad Software, Inc., La Jolla, CA).

Langendorﬀ Perfused Rat Heart. Animals. All animal care and

experimental protocols conformed to the European Union Guidelines

for the Care and the Use of Laboratory Animals (European Union

Directive 2010/63/EU) and were approved by the Italian Department

of Health (666/2015-PR). Male Wistar rats (300−350 g, Charles

River Italia, Calco, Italy) were anesthetized (i.p.) with a mixture of

Zoletil 100 (7.5 mg/kg tiletamine HCl + 7.5 mg/kg zolazepam HCl;

Virbac srl, Milano) e Rompun (8 mg/kg xylazine HCl; Bio 98, San

Lazzaro, Bologna), containing heparin (5000 U/kg), decapitated, and

exsanguinated.

Isolated Rat Heart Preparation and Perfusion. The hearts,

spontaneously beating, were rapidly explanted and mounted on a

Langendorﬀ apparatus for retrograde perfusion via the aorta at a

constant ﬂow rate of 10 mL/min with a Krebs−Henseleit solution of

the following composition (mM): NaCl 118, KCl 4.7, CaCl₂2.5,

MgSO₄1.2, NaHCO₃25, KH₂PO₄1.2, glucose 11.5, Na pyruvate 2,

and EDTA 0.5, bubbled with a 95% O₂−5% CO₂gas mixture (pH

7.4) and kept at 37 °C, as described elsewhere.⁸³Hearts were allowed

to equilibrate for at least 20 min before drug exposure.

Heart contractility was measured as left ventricle pressure (LVP)

by means of latex balloon, inserted into the left ventricle via the mitral

valve and connected to a pressure transducer (BLPR, WPI, Berlin,

Germany). The balloon was inﬂated with deionized water from a

microsyringe until a left ventricular end-diastolic pressure of 10

mmHg was obtained. Alteration in coronary perfusion pressure

(CPP), arising from changes in coronary vascular resistance, was

recorded by a pressure transducer (BLPR, WPI, Berlin, Germany)

placed in the inﬂow line.⁸⁴

A surface electrocardiogram (ECG) was recorded at a sampling

rate of 1 kHz by means of two steel electrodes, one placed on the apex

and the other on the left atrium of the heart. The ECG analysis

included the following measurements: RR (cycle length), HR

(frequency), PQ (atrioventricular conduction time), QRS (intra-

ventricular conduction time), and QT (overall action potential

duration).⁸⁵

LVP, CPP, and ECG were recorded with a digital PowerLab data

acquisition system (PowerLab 8/30; ADInstruments, Castle Hill,

Australia) and analyzed by using Chart Pro for Windows software

Mutagenicity Assay: Ames Test. The TA100 and TA98 strains of

Salmonella typhimurium and S9 fraction were utilized for the

mutagenicity assay. Approximately 10⁷bacteria were exposed to six

concentrations of each test compound, as well as a positive and

negative control, for 90 min in a medium containing suﬃcient

histidine to support approximately two cell divisions. After 90 min,

the exposure cultures were diluted in a pH indicator medium lacking

histidine and aliquoted into 48 wells of a 384-well plate. Within two

days, cells that had undergone the reversion to His grew into colonies.

Metabolism by the bacterial colonies reduced the pH of the medium,

changing the color of that well. This color change can be detected

visually or by a microplate reader. The number of wells containing

reverting 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.

Analysis of In Vitro Metabolic Stability of 2a and 3h in

Human and Liver Microsomes. The tested compound (2a or 3h),

dissolved in MeCN, was incubated at 37 °C, at 5 μM concentration in

100 mM phosphate buﬀer (pH 7.4) with 0.3 mg/mL rat and human

microsomal preparations as previously reported.⁸⁰Enzymatic

reactions were started by addition of an NADPH-regenerating system

(2 mM NADPH), 66 mM glucose-6-phosphate, and 0.4 U/mL

glucose-6-phosphate dehydrogenase in 66 mM MgCl₂. Reactions were

terminated at regular time intervals (overall range 0−60 min) by

adding a 1 mL of MeCN. All incubations were performed in triplicate.

HPLC analysis was performed on an Agilent 1100 Series liquid

chromatography system equipped with an EC 150/4.6 nucleosil 100-3

C18 (Macherey-Nagel) and coupling with a UV−vis detector, setting

at λ 254 nm. The analysis was carried out using gradient elution of a

binary solution; eluent A was MeCN (MeCN containing 0.1% formic

acid), while eluent B consisted of an aqueous solution of formic acid

(0.1%). The analysis started at 20% A for 3 min and then rapidly

increased up to 90% in 15 min, and ﬁnally, it remained at 90% A until

25 min. The analysis was performed at a ﬂow rate of 0.8 mL min⁻¹,

and injection volume was 20 μL. The intrinsic clearance (Clint) was

calculated by the equation:

Clint = k(min − 1)x[V]/[P]

where k is the rate constant for the depletion of the substrate, V is the

volume of incubation in microliters (μL), and P is the amount of

microsomal proteins as reported elsewhere.⁸¹

Oxidative Stress. Cell Culture and Treatments. IMR32 cells

(obtained from American Type Culture Collection, ATCC) were

cultured as described in our previous work.⁴²Stock solutions of

compounds 3h, 3m, and 2a were prepared in DMSO and diluted just

prior use into the cell culture medium to reach the ﬁnal

concentrations of the assays (0.1, 0.5, 1, 5, 10, and 50 μM). Control

vehicle was represented by DMSO ranging from 0.5 to 0.001%.

Lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 was

purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO,

USA). LPS was dissolved in phosphate-buﬀered saline (PBS) solution.

Cytotoxicity Determination. Cytotoxic eﬀects of the diﬀerent

compounds on IMR32 cells were evaluated by measurement of LDH

(lactate dehydrogenase) release into the cell culture medium. Brieﬂy,

IMR32 cells were seeded into a 96-well plate at 100 000 cells/well and

allowed to grow at the conﬂuence. Then, cells were treated with

compounds 3h, 3m, and 2a at diﬀerent concentrations (0.1, 0.5, 1, 5,

10, and 50 μM). After 24 h of incubation, LDH assay was performed

according to the manufacturer’s protocol (Euroclone, Milan, Italy).

All tests were performed at least in triplicate. The absorbance

measured from three wells was averaged, and the percentage of LDH

release was calculated as an arbitrary unit of change relative to 2% (v/

v) Triton-X-100-treated cells.

Nuclear Protein Extraction. IMR32 cells were seeded in 100 mm

Petri dishes and allowed to grow at the conﬂuence. After treatment

with compounds 3h, 3m, and 2a at 0.5 and 1 μM doses for 24 h, cells

were challenged with 100 μg/mL LPS for 30 min. Then, cells were

detached, washed with ice-cold PBS 1X, and pelleted by

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(PowerLab; ADInstruments, Castle Hill, Australia). LVP was

calculated by subtracting the left ventricular diastolic pressure from

the left ventricular systolic pressure. As the QT interval is aﬀected by

heart rate changes (e.g., it shortens when the heart rate increases),

Bazett’s formula normalized to average rat RR (QTc = QT/(RR/f)1/

2)⁸⁶was routinely used to correct it, in order to avoid confounding

eﬀects. In our experiments, “f”, the normalization factor according to

the basal RR duration, was 232.18 ms, as it was the average cardiac

cycle length. Analysis of data was accomplished using GraphPad

Prism version 5.04 (GraphPad Software, San Diego, USA). Statistical

analyses and signiﬁcance as measured by repeated measures by

ANOVA (followed by Dunnett’s post-test) were obtained using

GraphPad InStat version 3.06 (GraphPad Software, San Diego, USA).

In all comparisons, P < 0.05 was considered signiﬁcant.

between trains was used to induce long-term potentiation (LTP, 200

Hz) at PP-DG synapses. The eﬀect of 2a on LTP was tested in a ﬁrst

set of experiments where HFS was delivered 30 min after 2a

administration. Baseline recordings (10 min) were taken prior to drug

treatment, and LTP was recorded for 120 min after the HFS. Pulse

strength was set to evoke 40−50% of the maximum PS amplitude and

remained unchanged for all stimulations. For baseline and post-HFS

recordings, single pulses were applied every 60 s, and ﬁve responses

were averaged.

Pilocarpine Model of Status Epilepticus. Sprague−Dawley rats

(225−250 g) were treated with vehicle, 2a or 3h (2, 5, or 10 mg/kg,

i.p.); then, after 15 min, they were injected with scopolamine methyl-

hydrate (1 mg/kg in saline, i.p.) to prevent any peripheral cholinergic

side eﬀects. After 45 min, pilocarpine was administered (360 mg/kg in

saline, i.p.). Rats were then recorded for 2 h from the onset of status

epilepticus (SE), and seizures were scored from 0 to 5 on the Racine

scale. The duration of the seizure and the time to onset of SE were

also recorded. Two hours after the ﬁrst seizure induced by PILO

injection, diazepam (10 mg/kg, i.p.) was administered, and SE was

remitted.

Histological Labeling of Thiols. The assay was performed

accordingly with Horowitz et al.⁸⁷Brieﬂy, animals were sacriﬁced

by decapitation 24 h after diazepam treatment to block pilocarpine-

induced SE; their brains were removed and frozen in liquid nitrogen

and stored for 24 h at −80 °C to allow them to freeze

homogeneously. For redox staining, frozen brains were placed in

the ﬁrst alkylating solution (4% paraformaldehyde, 10 μM NEM, 1

μM Alexa-NEM 680 nm, Triton X-100 0.02% in PBS) and protected

from direct light and stored for 5 days at 4 °C. The solution was then

replaced with 30% sucrose solution until brains sank. Brains were then

frozen in dry ice and cut on a microtome, and the slices were placed in

a multiwell plate containing PBS. Slices were then washed with PBS

three times for 5 min each time. Before being exposed to a reducing

solution (50 mM TCEP in PBS), the slices were incubated for 30 min

at room temperature out of direct light. Slices were then exposed to a

second alkylating solution (10 μM N-ethylmaleimide, 1 μM Alexa-N-

ethylmaleimide 540 nm, Triton X-100 0.02% in PBS) before they

were washed with PBS (3 × 5 min) and mounted on slides. An

Olympus Fluoview 1000 laser scanning confocal microscope was used

for the ratiometric confocal analysis of thiols and disulﬁdes. A 1 μm

thick optical slice was taken with a manually generated region-of-

interest (ROI). Signal intensity and the ratio S-S/S-H signal were

measured by the software, and parameters were set in the control

reaction and kept constant. Ratios were taken against control animals

treated with vehicle only.

Compound 3h was dissolved in DMSO. Solvent failed to alter the

response of the preparations (data not shown).

In Vivo Studies on Animal Models of Epilepsy. Animals.

Adult male Sprague−Dawley rats (225−250 g) were housed at

constant temperature (23 °C) and relative humidity (60%), with free

access to food and water and a ﬁxed 12 h light/dark cycle. All animal

use followed the University of Pittsburgh and University of Malta

Institutional Animal Care and Use guidelines and was in strict

accordance with the National Institutes of Health Guide for the Care

and Use of Laboratory Animals, USA, and the EU Directive 2010/63/

EU for animal experiments, respectively.

Drugs. Compounds 2a and 3h were freshly dissolved in a vehicle

(2 mL/kg) of 5% PEG-400 and 5% Tween 80 in saline and

administered intraperitoneally (i.p.). Pilocarpine, scopolamine methyl-

hydrate, and other common salts were bought from SIGMA.

Maximal Dentate Activation (MDA). Surgical procedure and

electrode implantation. Urethane anaesthetized rats (1.2 g/kg, i.p.)

were positioned in a David Kopf stereotaxic frame, and their body

temperature was maintained using a heating pad and temperature

controller unit (Temperature Control Unit HB 101/2, Letica

Scientiﬁc Instruments). One electrode (biﬁlar stainless steel wire,

CFW, CA, USA) was implanted in the entorhinal cortex to stimulate

the perforant pathway (PP; AP = −7.9, L = 4.6, V = 3.4), and another

electrode was implanted in the hilus of the dentate gyrus (DG) of the

hippocampus (AP = −4.8, L = 2.5, V = 3.6) to record ﬁeld potentials.

During the surgery, electrodes were slowly lowered until the optimal

depth to record population spikes (PSs) was reached. Field potentials

were recorded by a NeuroLog ampliﬁer (Digitimer Ltd., high pass =

0.2 Hz, low pass = 5000 Hz, gain = 200). A digitally controlled

current stimulator (Digitimer Ltd., model DS3) was used to apply

square-wave pulses of 0.2 ms at a rate of 1/min. Stimulus intensity

was set to evoke 40−50% of the maximum amplitude of the PSs.

Responses were digitized by a CED 1401 plus analogue−digital

converter (Cambridge Electronic Design Ltd., Cambridge, UK),

stored on a computer and averaged oﬄine using Signal 1.9 software.

The sampling rate was set to 10 kHz. The location of the electrodes

was veriﬁed histologically.

Statistical Analysis. Statistical analysis was performed using

statistical software package GraphPad Prism4 (San Diego, CA). The

data were expressed as means

SEM. Statistical signiﬁcance was

determined at the level of p ≤ 0.05. The data obtained were compared

by the one-way ANOVA analysis of variance followed by Bonferroni

posthoc multiple comparisons or Turkey’s Multiple Comparison tests.

MDA Induction. To initiate MDA, stimulus trains (pulses of 0.3 ms

duration, at 20 Hz) of 10 s were delivered to the PP at 200 μA,

initially. If MDA could not be induced, the stimulus intensity was

increased by 50 μA and redelivered, and the process was repeated

every 5 min until MDA was induced. Stimulus trains were given every

10 min for 4 h (total of 24 stimulus trains), and for each train, the

duration and time to onset of MDA were measured. Measurements of

MDA are shown in Figure S4. The time to onset indicates the time

(in one second) from the beginning of the stimulus train to the point

where PSs appeared with half of the maximal amplitude. The duration

of the MDA was measured from the onset to the end of the after

discharges (ADs). ADs are represented by spontaneous bursts of PSs

that appear after the stimulus train. Duration and time to onset of

MDA were normalized against the ﬁrst stimulus train to give the value

of a change in duration or time to onset so that data from separate

animals could be averaged and compared. 2a was injected after

stimulus train 6.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschemneuro.1c00192.

■

sı

Molecular modeling studies, mutagenicity assays, in silico

measurement of molecular properties for compounds,

binding modes, measurement of MDA parameters, and

elemental analysis (PDF)

AUTHOR INFORMATION

Corresponding Authors

■

Stefania Butini − Department of Excellence of Biotechnology,

Chemistry and Pharmacy, 2018-2022, University of Siena,

53100 Siena, Italy; orcid.org/0000-0002-8471-0880;

Email: butini3@unisi.it

Long-Term Potentiation (LTP). Tetanic high-frequency stimulation

(HFS) consisting of 10 trains of 15 pulses of 200 Hz with a 1 s delay

1732

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Research Article

Giuseppe Campiani − Department of Excellence of

Biotechnology, Chemistry and Pharmacy, 2018-2022,

University of Siena, 53100 Siena, Italy; orcid.org/0000-

0001-5295-9529; Email: campiani@unisi.it

Department of Food and Nutrition, Kyung Hee University,

02447 Seoul, South Korea

Sandra Gemma − Department of Excellence of Biotechnology,

Chemistry and Pharmacy, 2018-2022, University of Siena,

53100 Siena, Italy; orcid.org/0000-0002-8313-2417

Mauro Maccarrone − Department of Biotechnological and

Applied Clinical Sciences, University of L’Aquila, 67100

L’Aquila, Italy; European Center for Brain Research/IRCCS

Santa Lucia Foundation, 00143 Rome, Italy

Authors

Alessandro Grillo − Department of Excellence of

Biotechnology, Chemistry and Pharmacy, 2018-2022,

University of Siena, 53100 Siena, Italy

Giuseppe Di Giovanni − Laboratory of Neurophysiology,

Department of Physiology and Biochemistry, Faculty of

Medicine and Surgery, University of Malta, MSD2080

Msida, Malta; Neuroscience Division, School of Biosciences,

Cardiﬀ University, CF10 3AT Cardiﬀ, United Kingdom;

orcid.org/0000-0003-2006-563X

Filomena Fezza − Department of Experimental Medicine Tor

Vergata, University of Rome, 00121 Rome, Italy

Giulia Chemi − Department of Excellence of Biotechnology,

Chemistry and Pharmacy, 2018-2022, University of Siena,

53100 Siena, Italy; orcid.org/0000-0002-3868-6752

Roberto Colangeli − Laboratory of Neurophysiology,

Department of Physiology and Biochemistry, Faculty of

Medicine and Surgery, University of Malta, MSD2080

Msida, Malta; Department of Experimental and Clinical

Medicine, Section of Neuroscience and Cell Biology,

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschemneuro.1c00192

Author Contributions

̀

Universita Politecnica delle Marche, 60126 Ancona, Italy

M.M., G.D.G., and S.G. are equally senior authors. The

manuscript was written through the contributions of all

authors. All authors approved the ﬁnal version of the

manuscript.

Simone Brogi − Department of Pharmacy, University of Pisa,

56126 Pisa, Italy; orcid.org/0000-0001-9375-6242

Domenico Fazio − European Center for Brain Research/

IRCCS Santa Lucia Foundation, 00143 Rome, Italy

Stefano Federico − Department of Excellence of

Biotechnology, Chemistry and Pharmacy, 2018-2022,

University of Siena, 53100 Siena, Italy; orcid.org/0000-

0002-7478-6128

Notes

The authors declare no competing ﬁnancial interest.

Crystallographic data have been deposited with the Cambridge

Crystallographic Data Centre as supplementary publication no.

CCDC 2008490. Copies of the data can be obtained, free of

charge, on application to CCDC, 12 Union Road, Cambridge

CB2 1EZ, UK (fax: + 44 (0) 1223 336 033; email: deposit@

ccdc.cam.ac.uk).

Alessandro Papa − Department of Excellence of Biotechnology,

Chemistry and Pharmacy, 2018-2022, University of Siena,

53100 Siena, Italy

Nicola Relitti − Department of Excellence of Biotechnology,

Chemistry and Pharmacy, 2018-2022, University of Siena,

53100 Siena, Italy; orcid.org/0000-0001-9783-8966

Roberto Di Maio − Pittsburgh Institute for Neurodegenerative

Diseases and Department of Neurology, University of

Pittsburgh, Pittsburgh 15261, Pennsylvania, United States

Gianluca Giorgi − Department of Excellence of Biotechnology,

Chemistry and Pharmacy, 2018-2022, University of Siena,

53100 Siena, Italy; orcid.org/0000-0002-8817-7745

Stefania Lamponi − Department of Excellence of

Biotechnology, Chemistry and Pharmacy, 2018-2022,

University of Siena, 53100 Siena, Italy

Massimo Valoti − Department of Life Sciences, University of

Siena, 53100 Siena, Italy

Beatrice Gorelli − Department of Life Sciences, University of

Siena, 53100 Siena, Italy

Simona Saponara − Department of Life Sciences, University of

Siena, 53100 Siena, Italy

Mascia Benedusi − Department of Biomedical and Specialist

Surgical Sciences, Section of Medical Biochemistry, Molecular

Biology and Genetics, University of Ferrara, 44121 Ferrara,

Italy

Alessandra Pecorelli − Plants for Human Health Institute,

Animal Science Department, NC Research Campus, NC State

University, Kannapolis 28081, North Carolina, United States

Patrizia Minetti − Sigma Tau, 00040 Pomezia, Italy

Giuseppe Valacchi − Plants for Human Health Institute,

Animal Science Department, NC Research Campus, NC State

University, Kannapolis 28081, North Carolina, United

States; Department of Biomedical and Specialist Surgical

Sciences, Section of Medical Biochemistry, Molecular Biology

and Genetics, University of Ferrara, 44121 Ferrara, Italy;

ACKNOWLEDGMENTS

■

This work was supported by Malta Council of Science and

Technology, grant R&I-2013-14 EPILEFREE to G.D.G. R.C.

was supported by a fellowship funded by EPILEFREE. The

authors wish to thank MIUR-PRIN no. 20175SA5JJ for

ﬁnancial support.

ABBREVIATIONS

■

2-AG, 2-arachidonoyl-glycerol; ADs, after discharges; AEA,

anandamide; CB1R, cannabinoid receptor type 1; CB2R,

cannabinoid receptor type 2; CPP, coronary perfusion

pressure; DCM, dichloromethane; DG, dentate gyrus;

DMAP, 4-(dimethylamino)pyridine; DMF, dimethylforma-

mide; DMSO, dimethyl sulfoxide; DSE, depolarization-

induced suppression of excitation; DSI, depolarization-induced

suppression of inhibition; ECG, electrocardiogram; ECS,

endocannabinoid system; FAAH, fatty acid amide hydrolase;

FCS, fetal calf serum; GB/SA, generalized born/surface area;

HFS, high-frequency stimulation; HLM, human liver micro-

somes; HR, frequency; IFD, induced ﬁt docking; ILAE,

International League Against Epilepsy; LDH, lactate dehydro-

genase; LTP, long-term potentiation; LVP, left ventricle

pressure; MAGL, monoacylglycerol lipase; MDA, maximal

dentate activation; MW, microwave; NBS, N-bromosuccinim-

mide; NRU, Neutral Red Uptake; OS, oxidative stress; PP,

perforant path; PQ, atrioventricular conduction time; PRCG,

Polak-Ribiere conjugate gradient; PS, population spike; QRS,

intraventricular, conduction time; QT, overall action potential

duration; RLM, rat liver microsomes; ROI, region-of-interest;

RR, cycle length; SAR, structure−activity relationship; SE,

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Bach, J. H., Hong, J. S., Yoneda, Y., and Kim, H. C. (2011) Role of

oxidative stress in epileptic seizures. Neurochem. Int. 59 (2), 122−137.

(19) Pecorelli, A., Natrella, F., Belmonte, G., Miracco, C., Cervellati,

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status epilepticus; STP-LTP, short-term and long-term

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thin layer chromatography; TLE, temporal lobe epilepsy; XP,

Extra Precision

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Selective Fatty Acid Amide Hydrolase Inhibitors as Potential Novel Antiepileptic Agents

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