Discovery of potent inhibitors of human and mouse fatty acid amide hydrolases

Article

pubs.acs.org/jmc

Discovery of Potent Inhibitors of Human and Mouse Fatty Acid

Amide Hydrolases

Stefania Butini,^†,‡Margherita Brindisi,^†,‡Sandra Gemma,^†,‡Patrizia Minetti,*^,§Walter Cabri,^§

Grazia Gallo,^§Silvia Vincenti,^§Emanuela Talamonti,^∥,⊥Franco Borsini,^§Antonio Caprioli,^§

Maria Antonietta Stasi,^§Stefano Di Serio,^§Sindu Ros,^†,‡Giuseppe Borrelli,^†,‡Samuele Maramai,^†,‡

Filomena Fezza,^∥,⊥Giuseppe Campiani,*^,†,‡and Mauro Maccarrone^#,⊥

‡

^†European Research Centre for Drug Discovery and Development (NatSynDrugs), Dipartimento Farmaco Chimico Tecnologico,

Universita

^§Sigma-Tau Industrie Farmaceutiche Riunite spa, Via Pontina Km 30400, 00040 Pomezia, Italy

^∥Dipartimento di Medicina Sperimentale e Chirurgia, Universita

degli Studi di Roma “Tor Vergata”, 00133 Rome, Italy

̀

degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy

̀

^⊥Centro Europeo di Ricerca sul Cervello (CERC)/IRCCS Fondazione Santa Lucia, 00143 Rome, Italy

^#Dipartimento di Scienze Biomediche, Universita

̀

degli Studi di Teramo, Piazza Aldo Moro 45, 64100 Teramo, Italy

S

* Supporting Information

ABSTRACT: Fatty acid amide hydrolase (FAAH, EC 3.5.1.99) is

the main enzyme catabolizing endocannabinoid fatty acid amides.

FAAH inactivation promotes beneﬁcial eﬀects upon pain and

anxiety without the side eﬀects accompanying agonists of type-1

cannabinoid receptors. Aiming at discovering new selective FAAH

inhibitors, we developed a series of compounds (5a−u)

characterized by a functionalized heteroaromatic scaﬀold. Partic-

ularly, 5c and 5d were identiﬁed as extremely potent, non-

competitive, and reversible FAAH inhibitors endowed with a

remarkable selectivity proﬁle and lacking interaction with the hERG

channels. In vivo antinociceptive activity was demonstrated for 5c,

5d, and 5n at a dose much lower than that able to induce either

striatal and limbic stereotypies or anxiolytic activity, thus outlining their potential to turn into optimum preclinical candidates.

Aiming at improving pharmacokinetic properties and metabolic stability of 5d, we developed a subset of nanomolar dialyzable

FAAH inhibitors (5v−z), functionalized by speciﬁc polyethereal lateral chains and ﬂuorinated aromatic rings.

approach aims at eliciting the desirable eﬀects of CBRs

activation while avoiding the negative eﬀects of global CB₁R

stimulation. Accordingly, inactivation of FAAH would be

expected to elevate the endogenous concentrations of all its

substrates and consequently prolong and potentiate their

beneﬁcial (therapeutic) eﬀects on pain and anxiety without

eliciting the classical CB₁R agonists side eﬀects (hypomotility,

hypothermia, and catalepsy).¹¹In humans, FAAH was mainly

detected in pancreas, brain, kidney, skeletal muscle, placenta,

and less abundantly in liver.¹²The importance of FAAH was

demonstrated by the generation of faah knockout mice that

showed elevated resting brain concentration of 1 and

manifested: (i) analgesic phenotype in both the carrageenan

model of inﬂammatory pain and in the formalin model of

spontaneous pain,¹³(ii) reduction in inﬂammatory responses,¹⁴

and (iii) improvements in slow wave sleep and memory

acquisition.^15,16Of the two isoforms of the enzyme, FAAH1 is a

INTRODUCTION

■

Endogenous cannabinoids (endocannabinoids, ECs) are a class

of signaling lipids such as N-arachidonoylethanolamine

(anandamide, AEA, 1, Chart 1), oleamide, and 2-arachidonoyl-

glycerol (2-AG), which activate type-1 and type-2 cannabinoid

receptors (CB₁R and CB₂R) to modulate a range of responses

and processes including pain, inﬂammation, appetite, motility,

sleep, thermoregulation, and cognitive and emotional states.^1,2

The actions of these signaling lipids are rapidly terminated by

cellular reuptake through a purported AEA membrane

transporter (AMT),^3,4traﬃcking through intracellular trans-

porters,^5,6and subsequent hydrolysis operated by a number of

enzymes. Among the latter, the fatty acid amide hydrolase

(FAAH, EC 3.5.1.99) was originally identiﬁed as the enzyme

responsible for rat oleamide hydrolysis,⁷while in humans it

preferably hydrolyses 1 and is also the main regulator of the

endogenous tone of 1 in vivo.⁸In addition to FAAH, N-

acylethanolamine acid amidase (NAAA)⁹and monoacylglycerol

lipase (MAGL)¹⁰were later identiﬁed as additional ECs-

metabolizing enzymes. A current therapeutically attractive

Received: May 17, 2012

Published: July 10, 2012

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inhibitors, the major breakthrough was represented by the

development of a series of α-ketoheterocycles.²³⁻²⁵These

ligands were characterized by the presence of a properly

activated ketone functionality, suitable for the active site Ser241

nucleophilic attack. Among them, a potent and selective FAAH

reversible inhibitor (OL135, 4, Chart 1, K_i= 4.7 nM)²⁶was

identiﬁed as a valuable tool for the investigation of FAAH

pharmacology.²⁷

Chart 1. Substrate and Representative Reversible and

Irreversible FAAH Inhibitors and Title Compounds

With the aim of discovering new scaﬀolds for selective FAAH

inhibition, we recently designed, on the model of the known

FAAH inhibitors, a series of ligands where speciﬁc heterocyclic

systems were introduced to modulate potency and lipophilicity.

We herein present a series of new potent and selective FAAH

inhibitors (5a−z, Chart 1, and Tables 1 and 2), characterized

by a phenylheterocyclic structure.²⁸Starting from a small set of

analogues (5a−d), comprising the structure of our 1-phenyl-

pyrrole-based lead compound 5d, the rational SAR studies were

performed by varying and combining: (i) the length and steric

hindrance of the distal chain at the carbamoyl system (R₁of

compounds 5a−r,u of Chart 1 and Table 1: n-butyl, cyclohexyl,

undecynyl, phenylhexyl), (ii) the amide/ester replacement (R₂

of compounds 5e−i, Table 1), (iii) the nature of the heteroaryl

moiety (2-furan, 5j,l,n, 2-thiophene, 5k,m,o, and 2-benzylpyr-

role, 5s−u, Table 1) and the position of the amide functionality

on the heteroaryl system (5p−r, Table 1), and (iv) the

introduction of a hydrophilic lateral chain at the carbamoyl

system ((mono/di/triﬂuoro)phenoxyethoxyethyl substituted

analogues, R₂of compounds 5v−z, Table 2). Synthesis,

molecular modeling, and in vitro and in vivo biological

properties (eﬃcacy and toxicity proﬁles) of the new class of

FAAH inhibitors are discussed in the present article.

Chemistry. The chemistry for the synthesis of compounds

5a−z is described in Schemes 1−5. The isocyanates were

prepared as reported in Scheme 1. Alcohols 6a,b were

submitted to a Mitsunobu reaction in the presence of

phtalimide, triphenylphosphine, and diisopropyl azodicarbox-

ylate (DIAD), and the resulting products were treated with

hydrazine monohydrate in reﬂuxing ethanol to aﬀord amines

7a,b. Reaction of 7a with phosgene furnished the 6-phenyl-1-

hexylisocyanate 8. 11-Isocyanatoundec-1-yne 11 was obtained

from alcohol 9. The latter compound was converted into the

corresponding azide (via the mesylate). Subsequent reduction

of the azido-derivative to amine with iron(III) chloride and

sodium iodide followed by treatment with phosgene led to 11.

Phenoxyethoxyethyl isocyanates 15a−e were obtained starting

from diethylene glycol 12 that was monobrominated in the

presence of carbon tetrabromide and triphenylphosphine to

generate 13. This bromide was reacted with the suitable

phenols in the presence of potassium iodide and potassium

hydroxide to provide alcohols 14a−e. Mitsunobu reaction

followed by treatment with hydrazine monohydrate and then

phosgene led to the desired isocyanates 15a−e.

membrane-bound enzyme belonging to the amidase family.

The analysis of its crystal structure¹⁷revealed a core composed

of a characteristic Ser-Ser-Lys catalytic triad that is buried deep

within the enzyme and can be accessed by two narrow

channels. The right side hydrophobic tunnel, termed acyl

binding channel (ACB), leads to the hydrophilic catalytic triad

from the membrane-bound surface and bifurcates into two

subchannels. The left side channel, known as cytoplasmic

access channel (CA) or cytosolic port (CP), is exposed to the

solvent, emerges at about 80° angle from the ACB, and

represents the back door by which the hydrolysis product leaves

the enzyme.¹⁷

The FAAH inhibitors known so far, depending on their

mechanism of action, can be classiﬁed as irreversible and

reversible inhibitors. While the early irreversible inhibitors (e.g.,

methoxyarachidonoyl ﬂuorophosphonate, MAFP, IC₅₀= 1−3

nM) were substrate-inspired ligands,¹⁸a more recently

characterized class of inhibitors was the carbamate-based

compounds, represented by URB597 (2, Chart 1, IC₅₀= 4.6

nM).¹⁹The early structure−activity relationship (SAR) studies

on the URB series of analogues demonstrated the presence of

an activated carbonyl as a key feature for FAAH inhibition.

Kinetic studies evidenced a noncompetitive and nondialyzable

(thus considered “irreversible”) mechanism of inhibition,

suggesting a covalent binding with FAAH that was later

conﬁrmed by mass spectrometry analysis.^20,21Biochemical

evidence suggest that 2 and related analogues covalently bind

the FAAH active site by adopting an orientation in which the

O-biaryl substituents reside in the CA of the enzyme, where

they would be susceptible to enzyme-catalyzed protonation to

enhance their function as leaving groups.²¹On the basis of this

background, the N-6-phenylhexylcarbamate analogue JP83 (3,

Chart 1, IC₅₀= 14 nM) was later synthesized to conﬁrm the

theoretical binding mode.²²In the context of FAAH reversible

The synthesis of compounds 5a−i and 5v−z is reported in

Scheme 2. 2,5-Dimethoxytetrahydrofuran-3-carboxaldehyde 16,

after treatment with iodine and ammonium hydroxide, led to its

corresponding 3-cyanoderivative 17.²⁹Subsequent Clauson−

Kaas reaction with 3-aminophenol generated the 1-phenyl-3-

cyanopyrrole derivative 18, which was hydrolyzed to the

corresponding amide 19 in the presence of hydrogen peroxide.

Reaction of 19 with the suitable alkylisocyanates aﬀorded the

title compounds 5a−d, whereas reaction with the diﬀerently

functionalized phenoxyethoxyethyl isocyanates aﬀorded the

ﬁnal products 5v−z. Clauson−Kaas reaction of 3-aminophenol

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Table 1. Inhibition Activity towards Mouse Brain FAAH (K_iand IC₅₀, nM) for Compounds 5a−u and towards Human

Recombinant FAAH (IC₅₀, nM) for Selected Compounds

a

b

c

Each value is the mean of at least three experiments (all SD are within 10%); NT not tested; FAAH inhibition as residual FAAH activity % after

d

preincubation with 10 μM of the tested compound (2 inhibition % was 16% at 10 μM); FAAH inhibition as residual FAAH activity % after

preincubation with 25 μM of the tested compound (2 inhibition % was 16% at 10 μM); FAAH inhibition as residual FAAH activity % after

preincubation with 50 μM of the tested compound (2 inhibition % was 16% at 10 μM).; done at Caliper (see: www.CaliperLS.com).

e

f

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Table 2. Determination of the Inhibition Activity towards Mouse Brain FAAH (as K_iand IC₅₀, nM) for Polyethereal

Compounds 5v−z and Calculated Solubility Properties

a

b

Each value is the mean of at least three independent experiments (all SD are within 10%). ACD/Labs V12.0, Toronto, Canada.

a

Scheme 1. Synthesis of Isocyanates 8, 11, and 15a−e

Scheme 2. Synthesis of Compounds 5a−i and 5v−z

a

Reagents and conditions: (a) phthalimide, DIAD, PPh₃, THF, from 0

°C to rt, 10 h; (b) N₂H₄·H₂O, EtOH, reﬂux, 4 h; (c) from 7a: COCl₂

(20% in toluene), NaHCO₃, H₂O, DCM, 0 °C, 15 min; (d) MsCl,

TEA, THF, rt, 15 min; (e) NaN₃, DMF, 85 °C, 4 h; (f) FeCl₃, NaI,

MeCN, reﬂux, 1 h; (g) CBr₄, PPh₃, MeCN, rt, 12 h; (h) appropriate

phenol, KI, KOH, MeCN, 60 °C, 11 h.

a

Reagents and conditions: (a) I₂, NH₄OH, THF, rt, 1 h; (b) 3-

aminophenol, dioxane, 5 N HCl, reﬂux, 10 min; (c) 30% H₂O₂, 6 N

NaOH, EtOH, reﬂux 12 h; (d) appropriate isocyanate, TEA, dry THF,

rt, 16 h; (e) 3-aminophenol, AcOH, H₂O, 100 °C, 15 min; (f) AgNO₃,

MeOH, 2 N NaOH, reﬂux, 1 h; (g) appropriate alcohol, PPh₃, DIAD,

dry THF, 0 °C to rt, 48 h; (h) appropriate isocyanate, TEA, dry THF,

rt, 16 h.

with 2,5-dimethoxytetrahydrofuran-3-carboxaldehyde 16 af-

forded the corresponding 1-phenylpyrrole derivative 20. The

aldehyde functionality was then oxidized using silver nitrate.

The resulting acid 21 was treated with undecyn-1-yl alcohol

and 6-phenyl-1-hexyl alcohol, in the presence of triphenylphos-

phine and DIAD, to aﬀord esters 22 and 23, respectively.

Reaction of the latter compounds with the appropriate

isocyanate yielded the corresponding carbamates 5e−i.

Alkylation of the 1,3-diketone 24 (Scheme 3) with

chloroacetone 25 in the presence of sodium hydride aﬀorded

the key intermediate 26. The microwave (MW)-assisted Paal−

Knorr reaction of the latter substances in the presence of

concentrated hydrochloric acid resulted in the formation of

phenylfuran 27. Analogously, MW-assisted Paal−Knorr reac-

tion of 26 in the presence of Lawesson’s reagent aﬀorded

thiophene-derivative 28.³⁰The hydrolysis of the ester groups

led to the corresponding acids 29 and 30. Subsequent

treatment with thionyl chloride and ammonium hydroxide

aﬀorded the corresponding amides 31 and 32. Methoxy groups

deprotection with boron tribromide followed by reaction of the

corresponding phenols with the appropriate isocyanate aﬀorded

carbamates 5j−o.

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a

Scheme 3. Synthesis of Compounds 5j−o

Scheme 5. Synthesis of Final Compounds 5s−u

a

Reagents and conditions: (a) benzylamine, AcOH, 130 °C, 150 W, 12

min; (b) NaOH, EtOH/THF 1:1 reﬂux, 16 h; (c) amine 7a or 7b,

TEA, HOBt, EDCI, dry DCM, from 0 °C to rt, 16 h; (d) BBr₃, dry

DCM, −78 °C to rt, 12 h; (e) 8, TEA, dry THF, rt, 16 h.

Reagents and conditions: (a) NaH, dry THF, 0 °C, 30 min then rt,

48 h; (b) X = O, concd HCl, EtOH, MW, 10 min; X = S, Lawesson’s

reagent, toluene, MW, 10 min; (c) NaOH, EtOH, H₂O, 7 h; (d)

SOCl₂, reﬂux, 30 min then concd NH₄OH, THF, rt, 7 h; (e) BBr₃, dry

DCM, −78 °C to rt, 8 h; (f) appropriate isocyanate, TEA, dry THF, rt,

16 h.

RESULTS AND DISCUSSION

■

The 1,4-diketo intermediate 37 (Scheme 4) was obtained by

alkylation of ethylacetoacetate 36 with the bromide 35.

Treatment of 37 with concentrated hydrochloric acid under

MW irradiation aﬀorded the phenylfuran 38. After saponiﬁca-

tion of the ester functionality of 38, the resulting acid 39 was

converted into the amide 40 by reaction with thionyl chloride

and ammonium hydroxide. The methoxy group was cleaved by

treatment with boron tribromide to aﬀord the phenol-derivative

41. Reaction of 41 with the appropriate isocyanate gave

compounds 5p−r.

Compounds 5s−u were prepared following Scheme 5. MW-

assisted Paal−Knorr cyclization reaction of 26 in the presence

of benzylamine and acetic acid led to the benzylpyrrole

derivative 42.³⁰After hydrolysis, compounds 5s and 5t were

prepared by coupling the acid 43 with the suitable amines in

the presence of 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide (EDCI) and N-hydroxybenzotriazole (HOBt).

Removal of the methoxy group of compound 5s aﬀorded

phenol 44, which was treated with cyclohexylisocyanate to

generate compound 5u.

Biological Assays. The potency of inhibition of FAAH for

the newly developed compounds (5a−u, Table 1, and 5v−z,

Table 2) was assessed through inhibition studies performed on

the mouse brain enzyme; the relative IC₅₀and K_ivalues are

given in Tables 1 and 2. The inhibitory activity of the most

interesting inhibitors of the series (5c and 5n) was conﬁrmed at

Caliper with the human recombinant enzyme (Table 1).

Molecular modeling studies supported the SAR (Figure 1) and

helped in the rationalization of the observed potencies with

mouse and human enzymes. For compounds 5d, 5o, 5w, and

5y (Table 3 and Figure 2) dialysis studies were performed in

order to evaluate the reversibility of FAAH inhibition. A subset

of compounds (5c, 5d, 5n, and 5o) were further examined for

their selectivity toward CB₁, CB₂, and vanilloid (TRPV₁)

receptors (Table 3). The latter are an additional target of 1.⁶

Moreover, the lack of interference of 5c, 5d, 5n, and 5o with

the biosynthesis of AEA and 2-AG and with the hydrolysis of 2-

AG was assessed as well as the absence of interaction with a

panel of more than 50 CNS monoamine receptors and

transporters (Table 1 of the Supporting Information (SI)).

a

Scheme 4. Synthesis of Compounds 5p

a

Reagents and conditions: (a) NaH, dry THF, 0 °C, 30 min then rt, 8 h; (b) HCl, EtOH, MW, 10 min; (c) NaOH, EtOH, H₂O, 7 h; (d) SOCl₂,

reﬂux, 30 min then NH₄OH, THF, rt, 7 h; (e) BBr₃, dry DCM, −78 °C to rt, 8 h; (f) appropriate isocyanate, TEA, dry THF, rt, 16 h.

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Figure 1. Three-dimensional model of dimeric-FAAH enzyme (turquoise ribbon for A and B chains) in complex with 5c (A) and 5y (B) (purple and

orange ball and sticks, respectively). Ligand−protein interaction plot of 5c (C) and 5y (D) (pink line, H-bond backbone; pink−dot line, H-bond

side chain; green spheres are hydrophobic residues, light-blue are polar residues, violet are positively charged residues, red are negatively charged

residues, and gray ones are Gly residues).

Table 3. Further Characterization of Mouse FAAH Inhibition, Selectivity, and hERG Activity for Selected Compounds

a

b

compd

type of inhibition

CB₁, CB₂, TRPV1

NAPE-PLD, DAGL

MAGL

hERG IC₅₀(μM)

c

d

e

5c

5d

5n

5o

NT

no aﬀ.

no interf.

>10

f

d

e

rev. non comp.

no aﬀ.

no interf.

c

d

e

NT

no aﬀ.

no interf.

f

d

e

rev. non comp.

no aﬀ.

no interf.

>10

a

b

c

d

e

f

Determined by microdialysis studies; done at Cerep; NT not tested. no aﬀ. = no aﬃnity; no interf. = no interference; rev. non comp. =

reversible non competitive.

Figure 2. (Ir)reversible inhibition of FAAH by reference compounds (2, 3, and 4) and selected analogues (5d, 5o, 5w, and 5y) (A) without dialysis,

(B) with dialysis. (Substrate = 10 μM [³H]AEA, control (CT) = 578 16 pmol/min per mg of protein).

Finally, hERG potassium channel activation was assessed for 5c,

5d, and 5n (Table 3).

Moreover, the Irwin test (Table 4) was performed for a

preliminary in vivo characterization of orally administered

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Table 4. In Vivo Preliminary Behavioral Studies and Analgesic Activity Evaluation after Oral Administration in Mice

Irwin test

limbic and striatal stereotypies

elevated plus maze

anxiolytic eﬀect

Randall−Selitto

mg/kg analgesia

compd

mg/kg

60 min

240 min

mg/kg

5c

10

30

+

absent

+

3

10

30

3

absent

10

30

absent

+

100

10

100

10

+

5d

5n

absent

30

+

10

30

3

30

+

100

10

+

100

10

+

absent

+

absent

30

10

30

100

+

Figure 3. Analgesic eﬀect of the selective FAAH inhibitors. Dose−response curves of the analgesic eﬀect of the selective FAAH inhibitors 5d, 5c, and

5n at 10, 30, and 100 mg/kg (A−C, respectively). Area under curve 0−240 min. Kruskal−Wallis one way analysis of variance on ranks. (A) H =

33.431; DF = 3; P = 0.001. Dunn’s test: P < 0.05, 30, and 100 mg/kg vs control. (B) H = 35.063; DF = 3; P = 0.001. Dunn’s test: P < 0.05, 30, and

100 mg/kg vs control. (C) H = 36.183; DF = 3; P = 0.001. Dunn’s test: P < 0.05, 30, and 100 mg/kg vs control. Analgesic eﬀect of 5c, 5d, and 5n at

a dose of 30 mg/kg compared with compound 2 at a dose of 50 mg/kg (D).

compounds 5c, 5d, and 5n, and the Randall−Selitto test (Table

4 and Figure 3) was used for the evaluation of their antalgic

activity.

docking studies were performed in order to establish the

binding mode of the new inhibitors (Figure 1).

(i). Evaluation of the Eﬀect of Varying Length and

Hindrance of the Chain Attached to the Carbamoyl System.

Among the 1-phenylpyrrole-based compounds, all the 1-

phenylpyrrole-3-carboxamido analogues (5a−d) were ex-

tremely potent FAAH inhibitors. In particular, compounds 5c

and 5d, bearing the highly hydrophobic, long, and ﬂexible

chains (undecinyl and phenylhexyl, respectively) were sub-

nanomolar FAAH inhibitors (5c, K_i= 0.18 nM, and 5d, K_i=

0.16 nM, Table 1). Introduction of the more hindering

cyclohexyl moiety lowered the activity by 3 orders of magnitude

(5b, K_i= 32 nM). To further investigate this eﬀect for 1-

phenylpyrrole analogues, the n-butyl analogue 5a was

synthesized and tested, and it was found to be able to inhibit

the enzyme with just half of the potency of the parent analogue

5b. This ﬁnding conﬁrms that length and hydrophobicity of the

chain at the carbamoyl moiety (rather than the steric

hindrance) is of pivotal importance in determining a high

aﬃnity for the FAAH active site.

In Vitro FAAH Inhibition, Structure−Activity Relation-

ship (SAR), and Molecular Modeling Studies. With the

aim of discovering new scaﬀolds for selective FAAH inhibition,

we designed a series of compounds (5a−z, Chart 1, and Tables

1 and 2) characterized by a diﬀerently decorated phenyl-

heterocyclic structure. Starting from a biaryl structure (on the

model of 2 and 3), and on our 1-phenylpyrrole-based lead

compound 5d, SAR studies were performed by varying and

combining: (i) the length and hindrance of the chain attached

to the carbamoyl system (n-butyl, cyclohexyl, undecynyl, and

phenylhexyl, Table 1), (ii) the amide/ester replacement (5e−i,

Table 1), (iii) the nature of the heteroaryl moiety (2-furan,

5j,l,n, 2-thiophene, 5k,m,o, and 2-benzylpyrrole, 5u−z, Table

1), and the position of the amide functionality on the heteroaryl

system (5p−r, Table 1), and (iv) the nature of the lateral chain

attached to the carbamoyl system (phenoxyethoxyethyl, 5v and

mono/di/triﬂuorophenoxyethoxyethyl, 5w−z, Table 2). Also,

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To investigate the binding mode of these new inhibitors, a

docking procedure was applied by using a 3D model of mouse

dimeric-FAAH1 enzyme. Indeed, in line with literature data,

after cocrystallization of MAPF with the enzyme,¹⁷and as

hypothesized for the natural substrate 1, the hydrophobic

interaction engaged by the carbamoyl chain along the FAAH

ACB channel is of crucial importance to potently modulate the

inhibition potency of these analogues. In general, the molecules

bind a pocket that spans from the CP, with the N-heteroaryl

group, to the ACB channel, with the alkyl (or cycloalkyl) chain.

This binding mode suggests the importance of the amide group

making H-bonds with Leu380 and Gln273. Furthermore, the

pyrrole and alkyl chain could favorably interact with Leu192;

this residue is replaced by a Phe in human FAAH1 isoform and

could explain the lower inhibition potency observed when 5c

was tested on the human enzyme (5c, K_ihrFAAH= 170 nM). In

Figure 1A,C, the results of the docking studies performed on

the picomolar analogue 5c are reported. H-Bonds between the

carbamate and protein backbone stabilize the protein−ligand

interaction in the catalytic pocket. In particular, the carbamate

carbonyl O is engaged in an H-bond with Gly239 and Ile238

backbone (in the anionic hole), while the carbamate NH

establishes an H-bond with the Met191 carbonyl. A series of

hydrophobic contacts are possible along the gorge (green

spheres surrounding the alkyl chain in Figure 1C), which may

further contribute to stabilize the proposed binding mode.

Moreover, the presence of an aromatic group at the end of the

O-alkyl chain, as in 5d, could reinforce the binding by means of

π−π stabilization with Phe381.

(ii). Amide/Ester Replacement. Given that FAAH catalytic

residues are accessible by two narrow channels, though slightly

diﬀerent in terms of size and hydrophilicity, we reasoned that

double-chained analogues could shape these channels, thus

giving information about the room available in the proximity of

the amide moiety. Starting from the potent inhibitors 5a−d, we

synthesized and tested a small set of analogues replacing the

amide moiety by the chemically accessible ester functionality.

Although much less eﬃcient than the parent compounds, the

ester analogues (5e−i) disclosed a number of interesting SAR.

The more potent inhibitors were obtained when the phenyl-

hexyl or undecynyl ester chains were combined with a n-butyl

substituent on the carbamoyl moiety. Compound 5e was

identiﬁed as the most active of the series, being equipotent to

compound 2, when tested under the same experimental

conditions; instead, the phenylhexyl analogue 5f was found to

be slightly less potent. Increasing the bulk of the carbamoyl

substituent (cyclohexyl in place of n-butyl, 5g,h) was

detrimental for activity, as already observed for the amide

counterparts. Interestingly, compound 5i, which combines the

presence of the two long chained substituents, appeared to

accommodate quite well both alkyl chains, being slightly less

potent than 2 in inhibiting the enzyme.

phenylhexyl. The three phenylhexyl analogues were indeed the

most potent of the series (5n, K_i= 0.49 nM; 5o, K_i= 0.50 nM;

5r, K_i= 4.80 nM). The activity of 5n was also measured on

human FAAH (5n, K_ihrFAAH= 102 nM). Analysis of the FAAH

inhibition data revealed that the 2-methyl-4,5-difunctionalyzed

furan and thiophene were slightly preferred over the 2-methyl-

3,5-difunctionalyzed furan moiety. The insertion of an extra

volume by means of an N-benzylpyrrole moiety reduced the

inhibitor potency (5u). The same detrimental eﬀect was

observed when replacing the urethane system with a methoxy

group (5s and 5t).

(iv). Eﬀect of the Insertion of Arylpolyethereal Chains

Attached to the Carbamoyl System. As a further step aimed at

improving the hydrophilic and pharmacokinetic properties of

the parent compounds, while retaining their in vitro

pharmacology and selectivity proﬁle, we designed and

synthesized a subset of analogues (5v−z), characterized by

the 1-phenylpyrrole scaﬀold functionalized by speciﬁc poly-

ethereal lateral chains. The latter analogues, as for 5d and 5o,

were also fully characterized as potent, selective, and dialyzable

analogues (Table 3 and Figure 2). Furthermore, although the

pharmacological properties of AEA and other fatty acid amides

underscore the potential therapeutic interest of FAAH

inhibition, the presence of a lipid-like chain may confer to

AEA-like compounds a number of unfavorable biopharmaceut-

ical properties, including low water solubility, high plasma

protein binding, and high accumulation in fat tissues. Moreover,

the marked similarity among the natural substrates of the ECs-

metabolizing enzymes renders selectivity a crucial issue for the

development of a novel drug candidate. Keeping this in mind,

and aiming at improving the hydrophilic and pharmacokinetic

properties of our lead compound 5d while retaining its in vitro

pharmacology and selectivity proﬁles, we developed a subset of

analogues (5v−z, Table 2) where the 1-phenylpyrrole core

structure was maintained and the n-hexyl spacer of the moiety

attached to the carbamoyl functionality of 5d was replaced by a

polyethereal chain of equal length. The distal phenyl system of

5d was also replaced by polyﬂuorinated moieties, placing the F

atoms at ortho and para positions of the phenyl system, which

are known to be the more suitable positions for metabolic

inactivation. Altogether, these modiﬁcations inﬂuenced a series

of parameters (e.g., LogP and LogS, Table 2) and allowed the

identiﬁcation of a subset of potent FAAH inhibitors potentially

endowed with better pharmacokinetic properties and improved

metabolic stability. In particular, the p-F (5w) and the o,p-di-F

(5y) substituted analogues were identiﬁed as the most potent

inhibitors of the series (5w, K_i= 8 nM; 5y, K_i= 7 nM).

Interestingly, the calculated water solubility (ACD/Labs

V12.0,Toronto, Canada) for 5w was also the highest of the

subseries of analogues and was incremented by about one

logarithmic unit with respect to that predicted for 5d (LogS 5w

= −4.72 vs LogS 5d = −5.69, Table 2, ACD/Labs). Notably,

5w maintained FAAH inhibition potency in the nanomolar

range, being only 1 order of magnitude less potent than 5d

(Table 2).

(iii). Evaluation of the Eﬀect of Varying the Nature of the

Heteroaryl Moiety and Diﬀerent Positioning of the

Heteroaryl Amide Functionality. The eﬀect of introducing

diﬀerent ﬁve-membered heteroaryls, in place of the pyrrol-1-yl

system of compounds 5a−d, was widely explored by means of

the design of three subsets of analogues: the 2-methyl-4,5-

difunctionalyzed furans and the thiophene analogues (5j,l,n and

5k,m,o, respectively) and the 2-methyl-3,5-difunctionalyzed

furan-based compounds (5p−r). All the synthesized com-

pounds conﬁrmed the previously observed SAR, and the FAAH

inhibition potency followed the trend n-butyl ≤ cyclohexyl <

Docking studies, in line with the observed FAAH inhibition

potency, conﬁrmed that the polyethereal-chained analogues

share their binding mode with 5c. Accordingly, as reported in

parts B and D of Figure 1, the binding mode of compound 5y

highlights the H-bonds established by both the amide (with

Leu380 and Gln273) and the carbamate moieties (with Ile238,

Met191, and Gly239), which are identical to those already

discussed for 5c.

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On the basis of these data, compounds 5w and 5y were

further characterized as potent, noncompetitive, and dialyzable

analogues (Figure 2).

after 1 h and were absent after 4 h with the exception of 5d.

The anxiolytic eﬀect and motor disturbances were absent at any

time of observation. The same dose range was used in the

induced pain model.

Randall-Selitto. Induction of Mechanical Hyperalgesia in

Rats. Mechanical nociceptive thresholds were measured in the

rat paw pressure test by applying increasing pressure to the left

and right hind paws using a Randall−Selitto analgesimeter

(Ugo Basile, Varese, Italy).³²The parameter used to quantify

the nociceptive threshold was deﬁned as the pressure (grams)

at which the rat withdrew its paw.

When 5c, 5d, and 5n (10, 30, and 100 mg/kg) were tested in

a dose−response experiment, all three compounds did exert

analgesic activity at 30 and 100 mg/kg dose regimen, the latter

dosage conferring a higher eﬀect (Figure 3A−C). Notably, oral

treatment with a 30 mg/kg dose of 5c, 5d, and 5n

demonstrated a signiﬁcant analgesic activity, whereas the

reference compound 2 at a dose of 50 mg/kg did not show

any activity (Figure 3D).

Dialysis Studies. As shown in Figure 2, the newly

developed analogues 5d, 5o, 5w, and 5y, as well as reference

irreversible (2 and 3) and reversible (4) FAAH inhibitors, were

incubated with mouse FAAH. The results obtained show that

the nondialyzed enzyme was inhibited by each of the

compounds tested in this study (Figure 2A). After dialysis,

the reversible inhibitors were washed out, and the enzyme

recovered its activity (Figure 2B) unless the compounds

remained ﬁrmly bound to the protein. Comparing the bars

relative to 4 (reversible inhibitor), 2, and 3 (irreversible

inhibitors) with those of the analogues 5d, 5o, 5w, and 5y, it

clearly emerged the reversibility of binding of all the new

compounds tested.

Selectivity Proﬁle. A wide pharmacological in vitro

investigation of compounds 5c, 5d, and 5n (Table 3) on a

variety of receptors (CB₁, CB₂, and TRPV1), metabolic

enzymes (N-acyl phosphatidylethanolamine phospholipase D

(NAPE-PLD), diacylglycerol lipase (DAGL), and MAGL, and

AMT transporter, as well as on a panel of more than 50

proteins of the CNS such as GPCRs and monoamine

transporters (see Table 1, SI), documented the high selectivity

proﬁle of the new compounds 5c and 5d. Notably, the tested

compounds did not show any interference either with AEA and

2-AG biosynthesis by NAPE-PLD and DAGL, respectively, or

with 2-AG hydrolysis by MAGL. We could also assess that 5c,

5d, and 5n fail to interact with hERG channels, thus excluding

interference at the cardiac level, which is a valuable property for

centrally active molecules.

Analgesic in Vivo Activity and Preliminary Safety

Studies. Preliminary Safety Studies. Prior to the in vivo

evaluation of the antalgic properties of the selected compounds

5c, 5d, and 5n, preliminary safety studies were performed for

excluding central eﬀects that could have behavioral implications

and that might interfere with the analgesic evaluation or

negatively aﬀect the therapeutic index. The Irwin test originally

described in 1968³¹is currently employed for assessing the

general eﬀects of a drug or drug candidate and to estimate the

minimum lethal dose of a test substance, the dose range for

CNS responses, and the primary eﬀects on behavior and

physiological functions. Data from the Irwin test were used to

assess the risks associated with the use of each substance (safety

pharmacology) and also to select doses for subsequent tests of

eﬃcacy. Basically, rats or mice are administered the test

substance and then they are observed for the next several hours.

The animals are assessed for behaviors related to neurotoxicity

(such as convulsions and tremor) or related to CNS stimulation

(such as excitation, Straub tail, jumping, hypersensitivity to

external stimuli, stereotypies, and aggressive behavior) and

depression (such as sedation, rolling gait, loss of balance, loss of

traction, motor incoordination, hyposensitivity to external

stimuli, decreased muscle tone, akinesia, catalepsy, and

hypothermia). Eﬀects on autonomic functions (respiration,

pupil diameter, body temperature, salivation, and defecation)

are also noted, along with the eventual lethality of the tested

compounds. Animals are initially administered a large dose of

the tested drug, and the subsequent doses are selected

following the responses to the initial exposure. Accordingly,

preliminary tests demonstrated, as outlined in Table 4, that

after oral administration to mice of 100, 30, and 10 mg/kg of

5c, 5d, and 5n, limbic, and striatal stereotypies were very weak

CONCLUSIONS

■

In summary, we developed a new class of potent and selective

FAAH reversible inhibitors. Besides the high selectivity,

assessed for selected compounds on a large panel of receptors,

including endocannabinoid receptors, enzymes, and trans-

porters, the potent FAAH inhibitors 5c and 5d fail to interact

also with the human ERG channel. The most potent inhibitors

were further analyzed by in vivo studies to evaluate their

antalgic properties. Results indicated that oral administration of

30 mg/kg dose of 5c, 5d, and 5n determined a signiﬁcant

analgesic eﬀect, and the proﬁle was much better than that

obtained with the reference compound 2, which, at a dose of 50

mg/kg, did not show any activity. No behavioral eﬀects were

detected for the tested compounds (in vivo Irwin tests), and

the absence of adverse eﬀects at the analgesic dose highlighted a

wide therapeutic window for the compounds 5c,d and 5n.

Starting from the lead 5c, we rationally synthesized a subset of

analogues which allowed to select potent and reversible FAAH

inhibitors potentially endowed with better solubility properties

and improved metabolic stability. Analogue 5w was the best

performing compound of this subset. Taken together, the data

herein presented evidence a trajectory for the discovery of a

new class of potent, reversible, and safe FAAH inhibitors

potentially useful as valuable pharmacological tools to be added

to the armamentarium of agents potentially useful for the

treatment of (neuropathic) pain.

EXPERIMENTAL PROCEDURES

■

Materials. Chemicals were of the purest analytical grade. AEA,

resinferatoxin (RTX), CP55.940, 1-stearoyl-2-arachidonoyl-sn-glycerol

and 2-oleoylglycerol (2-OG) were from Sigma-Aldrich; N-arachido-

noylphosphatidylethanolamine (NArPE), URB597, OL-135, and JP83

were from Alexis Corporation. [³H]NArPE (200 Ci/mmol), [³H]2-

OG (20 Ci/mmol), [³H-ethanolamine]AEA (60 Ci/mmol), [³H-

arachidonyl]AEA (200 Ci/mmol), and [¹⁴C]DAG (55 mCi/mmol)

were from ARC. [³H]CP55.940 (163 Ci/mmol) and [³H]RTX (43

Ci/mmol) were from Perkin-Elmer Life Sciences.

Chemistry. Reagents were purchased from Aldrich and were used

as received. Reaction progress was monitored by TLC using Merck

Silica Gel 60 F₂₅₄(0.040−0.063 mm) with detection by UV. Merck

Silica Gel 60 (0.040−0.063 mm) was used for column chromatog-

raphy. Melting points were determined in Pyrex capillary tubes using

1

an Electrothermal 8103 apparatus and are uncorrected. H NMR and

¹³C NMR spectra were recorded with a Varian 300 MHz instrument

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using the residual signal of the deuterated solvent as internal standard.

Splitting patterns are described as singlet (s), doublet (d), triplet (t),

quartet (q), and broad (br); the value of chemical shifts (δ) are given

in ppm and coupling constants (J) in hertz (Hz). ES-MS, APCI-MS

spectra were performed by an Agilent 1100 series LC/MSD

spectrometer and by LCQDeca-Thermoﬁnnigan spectrometer. Ele-

mental analyses were performed in a Perkin-Elmer 240C elemental

analyzer, and the results were within 0.4% of the theoretical values,

unless otherwise noted.

Yields refer to puriﬁed products and are not optimized. All

moisture-sensitive reactions were performed under argon atmosphere

using oven-dried glassware and anhydrous solvents. All the organic

layers were dried using anhydrous sodium sulfate.

6-Phenyl-1-hexylamine (7a). 6a (320 μL, 1.68 mmol),

triphenylphosphine (441 mg, 1.68 mmol), and phthalimide (247

mg, 1.68 mmol) were vigorously stirred in 2.4 mL of dry THF while

cooling to 0 °C. DIAD (320 μL, 1.67 mmol) in 2.4 mL of dry THF

was added dropwise to the cooled mixture. After stirring at room

temperature for 12 h, the solvent was removed under reduced

pressure. Column chromatography (25% ethyl acetate in n-hexane)

aﬀorded the pure 2-(6-phenylhexyl)isoindole-1,3-dione (71% yield).

¹H NMR (300 MHz, CDCl₃) δ 1.37 (m, 4H), 1.64 (m, 4H), 2.58 (t,

2H, J = 2.6 Hz), 3.66 (t, 2H, J = 7.2 Hz), 7.15 (m, 3H), 7.23 (m, 2H),

7.68 (m, 2H), 7.82 (m, 2H). ESI-MS m/z [M + H]⁺308 (100). To a

solution of 2-(6-phenylhexyl)isoindole-1,3-dione (364 mg, 1.19 mmol)

in 18.0 mL of ethanol, hydrazine monohydrate (180 μL, 3.71 mmol)

was added. The solution was stirred under reﬂux for 5 h and then

cooled to room temperature, and the white precipitate removed by

ﬁltration. The solvent was removed from the ﬁltrate under reduced

pressure. The residue was dissolved in 1 N sodium hydroxide (50 mL)

and extracted with chloroform (3 × 40 mL). The combined organic

layers were washed with brine, dried over anhydrous sodium sulfate,

ﬁltered, and concentrated. Column chromatography (10% MeOH in

chloroform with 2% of TEA) aﬀorded the pure compound 7a as a

yellow oil (80% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.34 (m, 4H),

1.45 (m, 2H), 1.62 (m, 2H), 2.35 (s, 2H), 2.60 (t, 2H, J = 2.6 Hz),

3.68 (t, 2H, J = 7.0 Hz), 7.16 (m, 3H), 7.26 (m, 2H). ESI-MS m/z [M

+ H]⁺178 (100).

aqueous solution of sodium bicarbonate and with brine and then dried

over anhydrous sodium sulfate, ﬁltered, and concentrated. Column

chromatography (10% ethyl acetate in n-hexane) aﬀorded the pure 11-

azidoundec-1-yne (72% yield). H NMR (300 MHz, acetone-d₆) δ

1.24−1.55 (m, 14H), 1.86 (t, 1H, J = 2.6 Hz), 2.08−2.13 (m, 2H),

3.18 (t, 2H, J = 7.04 Hz).

To a solution of the azide (826 mg, 4.27 mmol) in 44.0 mL of

MeCN, iron(III) chloride (1.04 g, 6.41 mmol) and sodium iodide

(5.77 g, 38.46 mmol) were added. The reaction mixture was reﬂuxed

for 1 h. After cooling, chloroform was added, and the resulting solution

was washed with a saturated aqueous solution of sodium thiosulfate

and then with a saturated aqueous solution of sodium bicarbonate.

The organic layer was dried over anhydrous sodium sulfate, ﬁltered

and concentrated. The crude was puriﬁed by means of ﬂash

chromatography (10% MeOH in chloroform with 0.2% of TEA).

Title compound 10 was obtained as an amorphous pale-yellow solid

(72% yield). ¹H NMR (300 MHz, acetone-d₆) δ 1.30−1.64 (m, 14H),

1.94 (t, 1H, J = 2.6 Hz), 2.15−2.20 (m, 2H), 2.50 (t, 2H, J = 7.6 Hz),

4.72 (brs, 2H). ESI-MS m/z [M + H]⁺194 (100).

1

11-Isocyanatoundec-1-yne (11). Title compound was prepared

as previously described for compound 8 starting from amine 10 and

phosgene. The compound was used in the following step without

1

further puriﬁcation (94% yield). H NMR (300 MHz, acetone-d₆) δ

1.25−1.63 (m, 14H), 1.89−1.91 (m, 1H), 2.13−2.18 (m, 2H), 3.26 (t,

2H, J = 6.7 Hz).

2-(2-Bromoethoxy)ethanol (13). To a solution of 12 (1.79 mL,

18.85 mmol) in 30.0 mL of MeCN, carbon tetrabromide (6.88 g,

20.74 mmol), and triphenylphosphine (4.95 g, 18.85 mmol) were

sequentially added, and the resulting mixture was stirred at room

temperature for 11 h. Then 1 N sodium hydroxide (25 mL) was added

and MeCN was removed by rotary evaporation. The aqueous residue

was extracted with ethyl acetate (3 × 30 mL), dried over anhydrous

sodium sulfate, ﬁltered, and concentrated. The crude residue was

puriﬁed by column chromatography on silica gel (33% n-hexane in

ethyl acetate) to aﬀord the product as yellow oil (61% yield). ¹H NMR

(300 MHz, CDCl₃) δ 2.63 (s, 1H), 3.42 (t, J = 6.0 Hz, 2H), 3.54−3.57

(m, 2H), 3.67−3.70 (m, 2H), 3.75 (t, J = 5.7 Hz, 2H).

2-(2-Phenoxyethoxy)ethanol (14a). To a solution of phenol (52

μL, 0.59 mmol) in 4.0 mL of MeCN, potassium hydroxyde (68 mg,

1.18 mmol) was added and the resultant mixture was heated to 60 °C.

After 2 h, potassium iodide (99 mg, 0.59 mmol) and a solution of

bromide 13 (200 mg, 1.18 mmol) in 3.0 mL of MeCN were added and

the reaction mixture was stirred for additional 10 h at 60 °C. Then the

mixture was diluted with water, extracted with ethyl acetate, and the

organic layers dried over anhydrous sodium sulfate, ﬁltered, and

concentrated. Puriﬁcation by silica gel column chromatography (40%

ethyl acetate in n-hexane) aﬀorded the title compound 14a (72%

6-Phenyl-1-pentylamine (7b). Title compound was prepared as

described for 7a starting from 6b. Column chromatography (10%

MeOH in chloroform with 2% of TEA) aﬀorded the pure compound

1

7b as a yellow oil (80% yield). H NMR (300 MHz, CDCl₃) δ 1.32

(m, 4H), 1.47 (m, 2H), 2.33 (s, 2H), 2.60 (t, 2H, J = 2.5 Hz), 3.68 (t,

2H, J = 7.2 Hz), 7.16 (m, 3H), 7.28 (m, 2H).

(6-Isocyanatohexyl)benzene (8). Compound 7a (165 mg, 0.93

mmol) was dissolved in 9.3 mL of DCM. The solution was cooled to 0

°C, and a saturated aqueous solution of sodium bicarbonate (9.3 mL)

was added. The biphasic mixture was stirred for 10 min at 0 °C, the

layers were allowed to separate, and a solution of phosgene (20% in

toluene, 980 μL, 1.86 mmol) was added directly to the organic layer

via syringe. After 15 min, the aqueous layer was extracted with DCM

(3 × 10 mL). The combined organic layers were dried over anhydrous

sodium sulfate, ﬁltered, and concentrated. The compound was used in

1

yield) as a pale-yellow oil. H NMR (300 MHz, CDCl₃) δ 2.75 (brs,

1H), 3.37−3.67 (m, 2H), 3.73−3.76 (m, 2H), 3.83−3.86 (m, 2H),

3.11−3.14 (m, 2H), 6.91−6.96 (m, 3H), 7.25−7.31 (m, 2H).

2-(2-(4-Fluorophenoxy)ethoxy)ethanol (14b). Title compound

was prepared according to the procedure previously described for 14a

starting from 4-ﬂuorophenol and 13. Compound was obtained as pale-

yellow oil (68% yield). ¹H NMR (300 MHz, CDCl₃) δ 2.87 (brs, 1H),

3.59−3.62 (m, 2H), 3.69−3.73 (m, 2H), 3.76−3.80 (m, 2H), 4.02−

4.05 (m, 2H), 6.79−6.84 (m, 2H), 6.89−6.93 (m, 2H).

1

the following step without further puriﬁcation (94% yield). H NMR

(300 MHz, CDCl₃) δ 1.27 (m, 4H), 1.51 (m, 4H), 2.51 (t, 2H, J = 7.7

Hz), 3.14 (t, 2H, J = 6.6 Hz), 7.07 (m, 3H), 7.17 (m, 2H).

2-(2-(2-Fluorophenoxy)ethoxy)ethanol (14c). Title compound

was prepared according to the procedure previously described for 14a

starting from 2-ﬂuorophenol and 13. Compound was obtained as pale-

yellow oil (61% yield). ¹H NMR (300 MHz, CDCl₃) δ 2.54 (brs, 1H),

3.63−3.67 (m, 2H), 3.71−3.75 (m, 2H), 3.84−3.87 (m, 2H), 4.16−

4.20 (m, 2H), 6.87−7.09 (m, 4H).

Undec-10-ynyl-1-amine (10). To a solution of 9 (1.14 mL, 5.9

mmol) in dry THF (40.0 mL), TEA (910 μL, 6.50 mmol) and

methanesulfonyl chloride (1.37 mL, 17.7 mmol) were added dropwise.

After stirring for 15 min, the suspension was ﬁltered through Celite

and the ﬁltrate was washed with brine. The organic layer was dried

over anhydrous sodium sulfate, ﬁltered, and concentrated. Undec-10-

ynyl methanesulfonate was used in the following step without further

2-(2-(2,4-Diﬂuorophenoxy)ethoxy)ethanol (14d). Title com-

pound was prepared according to the procedure previously described

for 14a starting from 2,4-diﬂuorophenol and 13. Compound was

obtained as pale-yellow oil (75% yield). ¹H NMR (300 MHz, CDCl₃)

δ 2.80 (brs, 1H), 3.59−3.63 (m, 2H), 3.68−3.72 (m, 2H), 3.78−3.82

(m, 2H), 4.10−4.13 (m, 2H), 6.69−6.95 (m, 3H).

1

puriﬁcation. H NMR (300 MHz, acetone-d₆) δ 1.20−1.44 (m, 14H),

1.83 (t, 1H, J = 2.6 Hz), 2.03−2.09 (m, 2H), 2.88 (s, 3H), 4.10 (t, 2H,

J = 6.4 Hz).

To a solution of the mesylate (1.45 g, 5.9 mmol) in 40.0 mL of dry

DMF, sodium azide (1.91 g, 29.0 mmol) was added. The mixture was

stirred at 85 °C for 4 h. After cooling to room temperature, water and

n-hexane were added. The organic layer was washed with a saturated

2-(2-(2,4,6-Triﬂuorophenoxy)ethoxy)ethanol (14e). Title

compound was prepared according to the procedure previously

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described for 14a starting from 2,4,6-triﬂuorophenol and 13.

Compound was obtained as pale-yellow oil (75% yield). H NMR

acetate to 5% MeOH in ethyl acetate). Pure compound 19 was

obtained as a white solid (80% yield). H NMR (300 MHz, acetone-

1

(300 MHz, CDCl₃) δ 2.61 (brs, 1H), 3.56−3.60 (m, 2H), 3.65−3.67

(m, 2H), 3.73−3.76 (m, 2H), 4.16−4.19 (m, 2H), 6.58−6.66 (m, 2H).

2-(2-Phenoxyethoxy)ethanisocyanate (15a). 2-(2-

Phenoxyethoxy)ethanamine was prepared as described for compound

7a. Title compound 15a was prepared according to the procedure

previously described for 8 starting from 2-(2-phenoxyethoxy)-

ethanamine and phosgene. Compound 15a was obtained as pale-

yellow oil and used for the subsequent step without further

puriﬁcation. ¹H NMR (300 MHz, CDCl₃) δ 3.42−3.54 (m, 2H),

3.69−3.73 (m, 2H), 3.87−3.90 (m, 2H), 4.14−4.18 (m, 2H), 6.93−

6.99 (m, 3H), 7.27−7.32 (m, 2H).

d₆) δ 6.47 (brs, 1H), 6.66 (brs, 1H), 6.76−6.84 (m, 2H), 6.99−

7.04(m, 2H), 7.20−7.31 (m, 2H), 7.85 (s, 1H), 9.40 (brs, 1H). ESI-

MS m/z [M − H]⁻201 (100).

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-

butylamine (5a). To a solution of 19 (40 mg, 0.20 mmol) in 4.0 mL

of dry THF, n-butylisocyanate (90 μL, 0.79 mmol) and TEA (110 μL,

0.79 mmol) were added. The reaction mixture was stirred at room

temperature for 16 h, and then the solvent was removed and the

residue was puriﬁed by silica gel column chromatography (2% MeOH

in chloroform) to aﬀord title compound 5a as a white amorphous solid

1

(63% yield). H NMR (300 MHz, acetone-d₆) δ 0.94 (t, 3H, J = 7.2

Hz), 1.34−1.47 (m, 2H), 1.52−1.62 (m, 2H), 3.22 (q, 2H, J = 6.6 Hz),

6.35 (brs, 1H), 6.74 (s, 1H), 6.91 (brs, 2H), 7.08 (d, 1H, J = 7.8 Hz),

7.28−7.50 (m, 4H), 7.85 (s, 1H). ¹³C NMR (75 MHz, acetone-d₆) δ

13.4, 19.9, 32.0, 40.9, 110.6, 114.0, 116.6, 119.7, 120.2, 121.8, 122.7,

130.5, 140.9, 152.9, 154.3, 165.6. Anal. (C₁₆H₁₉N₃O₃): C, H, N.

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-

cyclohexylamine (5b). Title compound was prepared according to

the procedure described for 5a starting from 19, the commercially

available cyclohexylisocyanate, and TEA. Column chromatography

(2% MeOH in chloroform) provided 5b as a white solid (62% yield);

4-Fluoro-1-(2-(2-isocyanatoethoxy)ethoxy)benzene (15b).

Title compound was prepared according to the procedure previously

described for 15a starting from 2-(2-(4-ﬂuorophenoxy)ethoxy)-

1

ethanamine and phosgene, and it was obtained as pale-yellow oil. H

NMR (300 MHz, CDCl₃) δ 3.45−3.48 (m, 2H), 3.70−3.74 (m, 2H),

3.88−3.90 (m, 2H), 4.12−4.16 (m, 2H), 6.92−6.94 (m, 2H), 6.99−

7.03 (m, 2H).

2-Fluoro-1-(2-(2-isocyanatoethoxy)ethoxy)benzene (15c).

Title compound was prepared according to the procedure previously

described for 15a starting from 2-(2-(2-ﬂuorophenoxy)ethoxy)-

ethanamine and phosgene, and pale-yellow oil was obtained. ¹H

NMR (300 MHz, CDCl₃) δ 3.41−3.45 (m, 2H), 3.70−3.74 (m, 2H),

3.88−3.91 (m, 2H), 4.20−4.23 (m, 2H), 6.90−7.08 (m, 4H).

2,4-Diﬂuoro-1-(2-(2-isocyanatoethoxy)ethoxy)benzene

(15d). Title compound was prepared according to the procedure

previously described for 15a starting from 2-(2-(2,4-diﬂuorophenoxy)-

ethoxy)ethanamine and phosgene, and pale-yellow oil was obtained.

¹H NMR (300 MHz, CDCl₃) δ 3.40−3.44 (m, 2H), 3.68−3.72 (m,

2H), 3.85−3.88 (m, 2H), 4.16−4.19 (m, 2H), 6.73−6.99 (m, 3H).

2,4,6-Triﬂuoro-1-(2-(2-isocyanatoethoxy)ethoxy)benzene

(15e). Title compound was prepared according to the procedure

previously described for 15a starting from 2-(2-(2,4,6-

triﬂuorophenoxy)ethoxy)ethanamine and phosgene and pale-yellow

oil was obtained. ¹H NMR (300 MHz, CDCl₃) δ 3.39−3.43 (m, 2H),

3.66−3.70 (m, 2H), 3.81−3.84 (m, 2H), 4.22−4.25 (m, 2H), 6.64−

6.70 (m, 2H).

2,5-Dimethoxytetrahydrofuran-3-carbonitrile (17). Com-

pound 16 (5.0 g, 31.2 mmol) was dissolved in 40.0 mL of dry

THF. Then 10% aqueous ammonium hydroxide (40.0 mL) and iodine

(10.3 g, 40.6 mmol) were sequentially added. The reaction mixture

was stirred at 25 °C for 1.5 h. Then a 10% aqueous solution of sodium

thiosulfate was added to quench the iodine excess. The layers were

separated, and the aqueous phase was extracted with DCM. The

combined organic layers were dried over anhydrous sodium sulfate,

ﬁltered, and concentrated. The crude was puriﬁed by means of ﬂash

chromatography (33% DCM in petroleum ether). Title compound

was obtained as a yellow oil (46% yield). ¹H NMR (300 MHz, CDCl₃)

δ 2.05−2.43 (m, 2H), 3.15 (m, 1H), 3.31 (s, 3H), 3.36 (s, 3H), 5.02−

5.19 (m, 2H).

1-(3-Hydroxyphenyl)-1H-pyrrole-3-carbonitrile (18). To a

solution of 3-aminophenol (632 mg, 5.78 mmol) in 10.0 mL of 1,4-

dioxane, compound 17 (1.0 g, 6.36 mmol) dissolved in 5.0 mL of 1,4-

dioxane was added. The reaction mixture was reﬂuxed for 5 h, and

then 8.0 mL of 5 N hydrochloric acid were added dropwise. The

reaction was reﬂuxed for additional 10 min. After cooling to room

temperature, the reaction mixture was partitioned between water and

DCM. The organic layer was dried over anhydrous sodium sulfate,

ﬁltered, and concentrated. Flash chromatography on silica gel (50% n-

hexane in ethyl acetate) aﬀorded compound 18 as an amorphous white

solid (82% yield). ¹H NMR (300 MHz, CDCl₃) δ 6.57−6.59 (m, 1H),

6.70 (brs, 1H), 6.83−6.92 (m, 3H), 7.01−7.03 (m, 1H), 7.26−7.34

(m, 1H), 7.49−7.51 (m, 1H). ESI-MS m/z [M + Na]⁺207 (100).

1-(3-Hydroxyphenyl)-1H-pyrrole-3-carboxamide (19). Com-

pound 18 (460 mg, 2.50 mmol) was dissolved in 2.0 mL of 6 N

sodium hydroxide and 2.0 mL of 30% aqueous H₂O₂. The reaction

mixture was reﬂuxed for 12 h, and then the solvents were removed in

vacuo. The crude was puriﬁed by ﬂash chromatography (100% ethyl

1

mp 175−176 °C (MeOH). H NMR (300 MHz, CD₃OD) δ 1.19−

1.39 (m, 5H), 1.65 (d, 1H, J = 12.6 Hz), 1.78 (d, 2H, J = 11.7 Hz),

1.95 (d, 2H, J = 10.8 Hz), 3.39−3.48 (m, 1H), 6.72 (s, 1H), 7.07 (d,

1H, J = 7.8 Hz), 7.21−7.38 (m, 3H), 7.47 (t, 1H, J = 7.8 Hz), 7.80 (s,

1H). ¹³C NMR (75 MHz, CD₃OD) δ 25.0 (2), 25.4, 32.8 (2), 50.5,

110.2, 114.2, 116.9, 119.8, 120.5, 121.0, 122.3, 130.4, 140.8, 152.5,

154.7, 168.5. Anal. (C₁₈H₂₁N₃O₃): C, H, N.

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-1-undec-

10-ynylamine (5c). Title compound was prepared according to the

procedure described for 5a starting from 19, 11, and TEA. Column

chromatography (2% MeOH in chloroform) provided compound 5c

1

as a white solid (78% yield); mp 129−130 °C (MeOH). H NMR

(300 MHz, CD₃OD) δ 1.35−1.56 (m, 14H), 2.13 (s, 1H), 2.16 (s,

2H), 3.18 (t, 2H, J = 6.9 Hz), 6.72 (s, 1H), 7.08 (d, 1H, J = 7.2 Hz),

7.22−7.39 (m, 3H), 7.48 (t, 1H, J = 8.1 Hz), 7.80 (s, 1H). ¹³C NMR

(75 MHz, CD₃OD) δ 17.8, 26.7, 28.5, 28.6, 29.0, 29.2, 29.37, 29.5,

40.9, 68.2, 83.9, 110.2, 114.1, 117.0, 119.8, 120.5, 121.0, 122.3, 130.4,

140.9, 152.6, 155.5, 168.5. Anal. (C₂₃H₂₉N₃O₃): C, H, N.

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-(6-

phenylhexyl)amine (5d). Title compound was prepared according

to the procedure described for 5a starting from 19, 8, and TEA.

Column chromatography (2% MeOH in chloroform) provided 5d as a

1

white solid (60% yield); mp 139−140 °C (MeOH). H NMR (300

MHz, CD₃OD) δ 1.38−1.40 (m, 4H), 1.53−1.66 (m, 4H), 2.60 (t,

2H, J = 15.0 Hz), 3.17 (t, 2H, J = 13.8 Hz), 6.72 (s, 1H), 7.05−7.37

(m, 9H), 7.46 (t, 1H, J = 8.4 Hz), 7.79 (s, 1H). ¹³C NMR (75 MHz,

CD₃OD) δ 26.5, 28.8, 29.5, 31.5, 35.6, 40.9, 110.2, 114.1, 117.0, 119.8,

120.5, 121.0, 122.3, 125.5, 128.1 (2), 128.2 (2), 130.4, 140.8, 142.7,

152.5, 155.5, 168.5. ESI-MS m/z [M + H]⁺406, [M + Na]⁺428 (100),

[M + K]⁺444, [2M + H]⁺811, [2M + Na]⁺833. Anal. (C₂₄H₂₇N₃O₃):

C, H, N.

1-(3-Hydroxyphenyl)-1H-pyrrole-3-carbaldehyde (20). To a

solution of 3-aminophenol (588 mg, 5.39 mmol) in acetic acid (10.0

mL) and water (2.0 mL), dimethoxytetrahydrofuran carboxaldehyde

16 (950 mg, 5.93 mmol) in acetic acid (1.0 mL) was added dropwise.

The solution was heated at 100 °C for 15 min. After removal of the

solvent, the dark-brown reaction mixture was diluted with ethyl acetate

and neutralized with an aqueous saturated solution of sodium

carbonate. Then, it was extracted with ethyl acetate (3 × 100 mL),

dried over anhydrous sodium sulfate, ﬁltered, and concentrated.

Column chromatography (50% ethyl acetate in n-hexane) aﬀorded the

1

pure product as a yellow solid (42% yield). H NMR (300 MHz,

acetone-d₆) δ 6.70 (s, 1H), 6.87 (d, 1H, J = 7.8 Hz), 7.08−7.12 (m,

2H), 7.35−7.37 (m, 2H), 8.00 (s, 1H), 8.84 (s, 1H), 9.84 (s, 1H). ESI-

MS m/z [M − H]⁻186 (100).

1-(3-Hydroxyphenyl)-1H-pyrrole-3-carboxylic Acid (21). To a

solution of 20 (255 mg, 1.36 mmol) in 3.0 mL of MeOH and 3.0 mL

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of 6 N sodium hydroxide, AgNO₃(370 mg, 2.18 mmol) was added.

The reaction mixture was then reﬂuxed for 1 h. After cooling, the

solvent was removed. The residue was diluted with ethyl acetate. The

aqueous layer was then acidiﬁed to pH = 1 using concentrated

hydrochloric acid and subsequently extracted with ethyl acetate (3 ×

20 mL). The organic layer was then dried over anhydrous sodium

sulfate, ﬁltered, and concentrated to aﬀord the pure product as a white

2.01 (d, 2H, J = 9.6 Hz), 2.16 (td, 2H, J = 2.4, 6.9 Hz), 3.54−3.57 (m,

1H), 4.23 (t, 2H, J = 6.6 Hz), 5.06 (d, 1H, J = 7.5 Hz), 6.73 (q, 1H, J =

1.8 Hz), 6.99 (t, 1H, J = 2.7 Hz), 7.08 (d, 1H, J = 8.7 Hz), 7.20−7.22

(m, 2H), 7.40 (t, 1H, J = 8.4 Hz), 7.66 (t, 1H, J = 1.8 Hz). ¹³C NMR

(75 MHz, CDCl₃) δ 18.6, 25.0 (2), 25.6, 26.2, 28.7, 28.9, 29.1, 29.2,

29.5, 29.6, 33.4 (2), 50.5, 64.3, 68.3, 85.0, 111.9, 114.9, 117.7, 118.6,

120.2, 120.7, 124.5, 130.5, 140.8, 152.2, 153.4, 164.9. ESI-MS m/z [M

+ H]⁺479, [M + Na]⁺501 (100), [M + K]⁺517. Anal. (C₂₉H₃₈N₂O₄):

C, H, N

(3-(3-((6-Phenylhexyloxy)carbonyl)-1H-pyrrol-1-yl)-

phenoxycarbonyl)cyclohexylamine (5h). Title compound was

prepared according to the procedure previously described for 5a

starting from 23, cyclohexylisocyanate, and TEA. Column chromatog-

raphy (40% n-hexane in chloroform) aﬀorded 5h as a white solid (66%

yield); mp 112−113 °C (ethyl acetate). ¹H NMR (300 MHz, CDCl₃)

δ 1.22−1.71 (m, 9H), 1.60−1.77 (m, 7H), 2.00−2.04 (m, 2H), 2.62 (t,

2H, J = 7.2 Hz), 3.55−3.58 (m, 1H), 4.24 (t, 2H, J = 6.6 Hz), 5.05 (d,

1H, J = 7.5 Hz), 6.74 (q, 1H, J = 1.5 Hz), 7.01 (t, 1H, J = 2.7 Hz),

7.10−7.27 (m, 8H), 7.40 (t, 1H, J = 8.1 Hz), 7.67 (s, 1H). ¹³C NMR

(75 MHz, CDCl₃) δ 25.0 (2), 25.7, 26.2, 29.1, 29.2, 31.6, 33.4 (2),

36.1, 50.5, 64.3, 112.0, 114.9, 117.7, 118.6, 120.2, 120.8, 124.5, 125.9,

128.5 (2), 128.6 (2), 130.5, 140.8, 142.9, 152.3, 153.4, 165.0. ESI-MS

m/z [M + H]⁺489, [M + Na]⁺511 (100), [M + K]⁺527. Anal.

(C₃₀H₃₆N₂O₄): C, H, N.

1

solid (73% yield). H NMR (300 MHz, acetone-d₆) δ 6.67 (dd, 1H, J

= 1.8, 3.0 Hz), 6.84 (ddd, 1H, J = 7.8, 2.1, 0.9 Hz), 7.04−7.11 (m,

2H), 7.25 (dd, 1H, J = 2.1, 3.0 Hz), 7.34 (t, 1H, J = 8.4 Hz), 7.78 (dd,

1H, J = 1.8, 2.4 Hz,), 8.78 (s, 1H), 10.49 (brs, 1H). ESI-MS m/z [M −

H]⁻202 (100).

Undec-10-ynyl-1-(3-hydroxyphenyl)-1H-pyrrole-3-carboxy-

late (22). To a stirred solution of acid 21 (100 mg, 0.49 mmol) and 9

(90 μL, 0.49 mmol) in 2.0 mL of dry THF at 0 °C, triphenylphosphine

(129 mg, 0.49 mmol), and DIAD (90 μL, 0.49 mmol) were added.

The reaction mixture was stirred at room temperature for 48 h. The

solvent was then removed under reduced pressure. Saturated aqueous

sodium carbonate was added to the residue, and it was extracted with

ethyl acetate (3 × 25 mL), dried over anhydrous sodium sulfate,

ﬁltered, and concentrated. Puriﬁcation by means of ﬂash chromatog-

raphy (10% ethyl acetate in n-hexane) aﬀorded the pure product 22 as

1

a white solid (69% yield). H NMR (300 MHz, CDCl₃) δ 1.32−1.55

(m, 12H), 1.70−1.75 (m, 2H), 1.93 (t, 1H, J = 2.7 Hz), 2.17 (dd, 2H, J

= 2.7, 7.2 Hz), 4.25 (t, 2H, J = 6.9 Hz), 5.52 (brs, 1H), 6.74 (q, 1H , J

= 1.8 Hz), 6.79 (dd, 1H J = 2.1, 8.1 Hz), 6.92 (t, 1H, J = 2.1 Hz),

6.95−7.00 (m, 2H), 7.30 (t, 1H, J = 8.1 Hz), 7.66 (dd, 1H, J = 1.5, 2.1

Hz).

(3-(3-((Undec-10-ynyloxy)carbonyl)-1H-pyrrol-1-yl)-

phenoxycarbonyl)-(6-phenylhexyl)amine (5i). Title compound

was prepared according to the procedure previously described for 5a

starting from 22, 8, and TEA. Column chromatography (40% n-hexane

1

6-Phenylhexyl-1-(3-hydroxyphenyl)-1H-pyrrole-3-carboxy-

late (23). The title compound was prepared according to the

procedure previously described for 22 starting from acid 21, 6a,

triphenylphosphine, and DIAD. Column chromatography (10% ethyl

acetate in n-hexane) aﬀorded the pure product as a white solid (63%

yield). ¹H NMR (300 MHz, CDCl₃) δ 1.38−1.48 (m, 4H), 1.49−1.76

(m, 4H), 2.62 (t, 2H, J = 6.9 Hz), 4.25 (t, 2H, J = 6.9 Hz), 6.73−6.99

(m, 6H), 7.16−7.30 (m, 6H), 7.67−7.68 (m, 1H).

in chloroform) aﬀorded 5i as a colorless viscous oil (85% yield). H

NMR (300 MHz, CDCl₃) δ 1.32−1.75 (m, 22H), 1.94 (t, 1H, J = 2.4

Hz), 2.18 (td, 2H, J = 2.4, 6.9 Hz), 2.62 (t, 2H, J = 7.5 Hz), 3.26 (q,

2H, J = 6.6 Hz), 4.24 (t, 2H, J = 6.6 Hz), 5.16 (brs, 1H), 6.75 (s, 1H),

7.00 (s, 1H), 7.08 (d, 1H, J = 8.7 Hz), 7.16−7.30 (m, 7H), 7.41 (t, 1H,

J = 8.7 Hz), 7.67 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 18.6, 26.3,

26.8, 28.7, 29.0, 29.1 (2), 29.3, 29.5, 29.6, 29.9, 31.6, 36.1, 41.5, 64.4,

68.3, 85.0, 111.9, 114.9, 117.8, 118.7, 120.2, 120.8, 124.5, 125.9, 128.5

(2), 128.6 (2), 130.6, 140.9, 142.8, 152.2, 154.3, 165.0. ESI-MS m/z

[M + H]⁺557, [M + Na]⁺579 (100), [M + K]⁺595. Anal.

(C₃₅H₄₄N₂O₄): C, H, N.

(3-((3-(Undec-10-ynyloxy)carbonyl)-1H-pyrrol-1-yl)-

phenoxycarbonyl)butylamine (5e). Title compound was prepared

according to the procedure previously described for 5a starting from

22, n-butylisocyanate, and TEA. Column chromatography (40% n-

hexane in chloroform) aﬀorded 5e as a white solid (94% yield); mp

52−53 °C (ethyl acetate). ¹H NMR (300 MHz, CDCl₃) δ 0.94 (t, 3H,

J = 7.2 Hz), 1.30−1.74 (m, 18H), 1.93 (s, 1H), 2.16 (dd, 2H, J = 4.5,

6.3 Hz), 3.26 (q, 2H, J = 6.3 Hz), 4.22 (t, 2H, J = 6.3 Hz), 5.22 (brs,

1H), 6.73 (s, 1H), 6.99 (s, 1H), 7.07 (d, 1H, J = 8.1 Hz), 7.19−7.26

(m, 2H), 7.39 (t, 1H, J = 8.1 Hz), 7.65 (s, 1H). ¹³C NMR (75 MHz,

CDCl₃) δ 14.0, 18.6, 20.1, 26.2, 28.7, 28.9, 29.1, 29.2, 29.5, 29.6, 32.1,

41.3, 64.3, 68.3, 85.0, 111.9, 114.8, 117.7, 118.6, 120.2, 120.7, 124.5,

130.5, 140.8, 152.2, 154.3, 165.0. ESI-MS m/z [M + H]⁺453, [M +

Na]⁺475 (100), [M + K]⁺491. Anal. (C₂₇H₃₆N₂O₄): C, H, N.

(3-(3-((6-Phenylhexyloxy)carbonyl)-1H-pyrrol-1-yl)-

phenoxycarbonyl)butylamine (5f). Title compound was prepared

according to the procedure previously described for 5a starting from

23, n-butylisocyanate, and TEA. Column chromatography (40% n-

hexane in chloroform) aﬀorded 5f as a low melting white solid (95%

yield); ¹H NMR (300 MHz, CDCl₃) δ 0.94 (t, 3H, J = 7.2 Hz), 1.34−

1.81 (m, 11H), 2.62 (t, 3H, J = 7.2 Hz), 3.27 (q, 2H, J = 6.6 Hz), 4.24

(t, 2H, J = 6.6 Hz), 5.21 (t, 1H, J = 5.4 Hz), 6.75 (s, 1H), 6.99−7.43

(m, 10H), 7.67 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 14.0, 20.1,

26.2, 29.0, 29.2, 31.6, 32.1, 36.1, 41.3, 64.3, 112.0, 114.9, 117.7, 118.6,

120.2, 120.8, 124.5, 125.8, 128.5 (2), 128.6 (2), 130.6, 140.8, 142.9,

152.2, 154.3, 165.0. ESI-MS m/z [M + H]⁺461, [M + Na]⁺485 (100),

[M + K]⁺501. Anal. (C₂₈H₃₄N₂O₄): C, H, N.

(

)-Ethyl-2-acetyl-4-(3-methoxyphenyl)-4-oxobutanoate

(( )-26). To a suspension of sodium hydride (36 mg, 1.49 mmol) in

8.0 mL of dry THF at 0 °C, 24 (262 μL, 1.35 mmol) was added

dropwise. After 30 min, 25 (119 μL, 1.49 mmol) was added dropwise

at 0 °C and the reaction mixture was stirred at room temperature for

48 h. The reaction was quenched with 1 N hydrochloric acid (3.0 mL),

THF was evaporated, and the residue was extracted with ethyl acetate

(3 × 25 mL). The combined organic layers were dried over anhydrous

sodium sulfate, ﬁltered, and concentrated. Column chromatography

(25% ethyl acetate in n-hexane) aﬀorded the pure product ( )-26

(67% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.14 (t, 3H, J = 6.9 Hz),

2.21 (s, 3H), 3.06 (m, 2H), 3.83 (s, 3H), 4.10 (q, 2H, J = 6.9 Hz), 4.85

(t, 1H, J = 7.0 Hz), 7.09−7.13 (m, 1H), 7.33−7.39 (m, 1H), 7.51 (s,

1H), 7.59 (d, 1H, J = 7.8 Hz).

Ethyl-2-(3-methoxyphenyl)-5-methylfuran-3-carboxylate

(27). Compound ( )-26 (735 mg, 2.60 mmol) was dissolved in 8.0

mL of ethanol. Concentrated hydrochloric acid (0.73 mL) was added,

and the reaction mixture was submitted to MW irradiation (150 W)

(CEM Discovery Microwave System) and reﬂuxed (maximum internal

temperature 100 °C) for 10 min. The reaction mixture was cooled,

diluted with ethyl acetate, neutralized with saturated aqueous sodium

bicarbonate, and extracted with ethyl acetate (3 × 50 mL). The

combined organic layers were dried over anhydrous sodium sulfate,

ﬁltered, and concentrated. Column chromatography (3% ethyl acetate

in n-hexane) aﬀorded the pure product as colorless viscous oil (90%

(3-(3-((Undec-10-ynyloxy)carbonyl)-1H-pyrrol-1-yl)-

phenoxycarbonyl)cyclohexylamine (5g). Title compound was

prepared according to the procedure previously described for 5a

starting from 22, cyclohexylisocyanate, and TEA. Column chromatog-

raphy (40% n-hexane in chloroform) aﬀorded 5g as a white

amorphous solid (83% yield). ¹H NMR (300 MHz, CDCl₃) δ

1.16−1.54 (m, 17H), 1.60−1.72 (m, 5H), 1.93 (t, J = 2.7 Hz, 1H),

1

yield). H NMR (300 MHz, CDCl₃) δ 1.32 (t, 3H, J = 6.9 Hz), 2.34

(d, 3H, J = 0.9 Hz), 3.84 (s, 3H), 4.28 (q, 2H, J = 7.2 Hz), 6.44 (d, 1H,

J = 1.2 Hz), 6.92 (ddd, 1H, J = 0.6, 2.1, 8.1 Hz), 7.32 (t, 1H, J = 7.8

Hz), 7.56−7.59 (m, 1H), 7.62−7.64 (m, 1H). ¹³C NMR (75 MHz,

CDCl₃) δ 13.5, 14.5, 55.5, 60.6, 109.2, 113.5, 115.0, 115.2, 120.7,

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129.2, 131.4, 151.21, 155.8, 159.5, 163.9. ESI-MS m/z [M − OEt]⁺

described for 33 starting from 32 and boron tribromide. Pure product

was obtained as white solid (95% yield). ¹H NMR (300 MHz, acetone-

d₆) δ 2.45 (d, 3H, J = 0.9 Hz), 6.49 (brs, 2H), 6.84 (ddd, 1H, J = 0.9,

2.4, 8.1 Hz), 6.96−7.00 (m, 3H), 7.22 (t, 1H, J = 7.5 Hz), 8.55 (s, 1H).

(3-(3-Carbamoyl-5-methylfuran-2-yl)phenoxycarbonyl)-

butylamine (5j). Title compound was prepared according to the

procedure described for 5a starting from 33, n-butylisocyanate, and

TEA. Column chromatography (1% MeOH in chloroform) aﬀorded

the pure product as a white solid (77% yield); mp 170−171 °C

215, [M + Na]⁺283 (100).

Ethyl-2-(3-methoxyphenyl)-5-methylthiophene-3-carboxy-

late (28). Compound ( )-26 (500 mg, 1.8 mmol) was dissolved in

5.0 mL of toluene. Lawesson’s reagent (2.00 g) was added, and the

reaction mixture was submitted to MW irradiation (150W) (CEM

Discovery Microwave System) and reﬂuxed (maximum internal

temperature 100 °C) for 10 min. After cooling, the mixture was

ﬁltered through Celite and the solvent was removed under reduced

pressure. Flash column chromatography (1% ethyl acetate in n-

1

(MeOH). H NMR (300 MHz, CD₃OD) δ 0.96 (t, 3H, J = 7.5 Hz),

1

hexane) aﬀorded the pure product as colorless oil (48% yield). H

1.36−1.43 (m, 2H), 1.50−1.57 (m, 2H), 2.35 (s, 3H), 3.15−3.21 (m,

2H), 6.38 (d, 1H, J = 0.9 Hz), 7.07 (ddd, 1H, J = 0.9, 2.4, 4.8 Hz), 7.38

(t, 1H, J = 7.8 Hz), 7.63 (t, 1H, J = 1.8 Hz), 7.70 (dt, 1H, J = 1.5, 5.4

Hz). ¹³C NMR (75 MHz, CD₃OD) δ 12.0, 12.9, 19.8, 31.7, 40.5,

107.9, 118.2, 120.2, 121.6, 123.7, 129.0, 131.6, 151.4, 151.5, 151.9,

155.9, 168.0. Anal. (C₁₇H₂₀N₂O₄): C, H, N.

(3-(3-Carbamoyl-5-methylthiophen-2-yl)phenoxycarbonyl)-

butylamine (5k). The title compound was prepared according to the

procedure previously described for 5a starting from 34, n-

butylisocyanate, and TEA. Column chromatography (1% MeOH in

chloroform) aﬀorded 5k as a white solid (64% yield); mp 162−163 °C

(MeOH). ¹H NMR (300 MHz, DMSO-d₆) δ 0.87 (t, 3H, J = 7.5 Hz),

1.26−1.35 (m, 2H), 1.38−1.46 (m, 2H), 2.42 (d, 3H, J = 0.6 Hz), 3.03

(q, 2H, J = 6.9 Hz), 6.93 (d, 1H, J = 1.2 Hz), 7.05 (ddd, 1H, J = 1.2,

2.1, 7.8 Hz), 7.14 (t, 1H, J = 1.8 Hz), 7.24−7.38 (m, 3H), 7.57 (brs,

1H), 7.76 (t, 1H, J = 5.7 Hz). ¹³C NMR (75 MHz, DMSO-d₆) δ 14.3,

15.4, 20.1, 32.0, 41.0, 121.8, 121.9, 125.4, 127.9, 129.9, 135.0, 135.1,

139.2, 139.8, 151.6, 154.8, 167.0. ESI-MS m/z [M + Na]⁺355 (100),

[2M + Na]⁺687. Anal. (C₁₇H₂₀N₂O₃S): C, H, N.

NMR (300 MHz, CDCl₃) δ 1.18 (t, 3H, J = 7.2 Hz), 2.47 (d, 3H, J =

0.9 Hz), 3.82 (s, 3H), 4.17 (q, 2H, J = 6.9 Hz), 6.90 (ddd, 1H, J = 0.9,

2.7, 8.4 Hz), 7.00−7.05 (m, 2H), 7.15 (d, 1H, J = 0.9 Hz), 7.28 (t, 1H,

J = 7.8 Hz). ESI-MS m/z [M − OEt]⁺231, [M + Na]⁺299 (100).

2-(3-Methoxyphenyl)-5-methylfuran-3-carboxylic Acid (29).

To a solution of 27 (42 mg, 0.16 mmol) in ethanol (3.0 mL) and

water (1.0 mL), sodium hydroxide (161 mg, 4.03 mmol) was added

and the reaction mixture was stirred at room temperature for 7 h, then

ethanol was removed and the residue was acidiﬁed with 6 N HCl to

pH = 1. The mixture was extracted with ethyl acetate (3 × 20 mL).

The organic layers were dried over anhydrous sodium sulfate, ﬁltered,

and concentrated. The title compound was used for the following step

1

without further puriﬁcation. H NMR (300 MHz, CDCl₃) δ 2.37 (s,

3H), 3.86 (s, 3H), 6.48 (s, 1H), 6.95 (dd, 1H, J = 7.8, 3.9 Hz,), 7.34 (t,

1H, J = 7.9 Hz), 7.54−7.59 (m, 2H). ESI-MS m/z [M − H]⁻231

(100).

2-(3-Methoxyphenyl)-5-methylthiophene-3-carboxylic Acid

(30). Title compound was prepared according to the procedure

described for 29 starting from 28 and sodium hydroxide. Compound

(3-(3-Carbamoyl-5-methylfuran-2-yl)phenoxycarbonyl)-

cyclohexylamine (5l). The title compound was prepared according

to the procedure previously described for 5a starting from 33,

cyclohexylisocyanate and TEA. Column chromatography (1% MeOH

in chloroform) aﬀorded 5l as a white solid (79% yield); mp 202−203

1

was used in the following step without further puriﬁcation. H NMR

(300 MHz, CDCl₃) δ 2.46 (d, 3H, J = 0.6 Hz), 3.81 (s, 3H), 6.92 (dd,

1H, J = 2.4, 8.4 Hz), 7.03−7.07 (m, 2H), 7.19 (d, 1H, J = 0.9 Hz), 7.28

(t, 1H, J = 8.1 Hz).

1

2-(3-Methoxyphenyl)-5-methylfuran-3-carboxamide (31).

Acid 29 (68 mg, 0.29 mmol) was added portionwise to thionyl

chloride (300 μL) at room temperature. Then the reaction mixture

was reﬂuxed for 30 min and then evaporated to dryness. The residue

was dissolved in THF (3.0 mL), and concentrated ammonium

hydroxide (1.0 mL) was added cautiously at room temperature and the

solution stirred for 1 h. Then the reaction mixture was extracted with

ethyl acetate. The organic layers were dried over anhydrous sodium

sulfate, ﬁltered, and concentrated. Column chromatography on silica

gel (40% ethyl acetate in n-hexane) aﬀorded the pure product as a

white solid (97% yield). ¹H NMR (300 MHz, CDCl₃) δ 2.34 (d, 3H, J

= 0.6 Hz), 3.83 (s, 3H), 5.79 (brs, 1H), 5.90 (brs, 1H), 6.33 (d, 1H, J

= 0.9 Hz), 6.92 (dt, 1H, J = 3.0, 7.5 Hz), 7.26−7.38 (m, 3H).

2-(3-Methoxyphenyl)-5-methylthiophene-3-carboxamide

(32). Title compound was prepared according to the procedure

described for 31 starting from 30, thionyl chloride, and ammonium

hydroxide concentrated solution. Flash column chromatography (40%

ethyl acetate in n-hexane) aﬀorded the pure product as a white solid

(78% yield). ¹H NMR (300 MHz, acetone-d₆) δ 2.46 (s, 3H), 3.82 (s,

3H), 6.50 (brs, 2H), 6.93 (dd, 1H, J = 1.8, 8.4 Hz), 6.99 (d, 1H, J = 1.2

Hz), 7.06−7.11 (m, 2H), 7.32 (t, 1H, J = 8.1 Hz).

°C (MeOH). H NMR (300 MHz, DMSO-d₆) δ 1.13−1.28 (m, 5H),

1.52−1.59 (m, 1H), 1.67−1.70 (m, 2H), 1.79−1.83 (m, 2H), 2.31 (s,

3H), 3.28−3.30 (m, 1H), 6.48 (s, 1H), 7.04 (dd, 1H, J = 0.9, 7.5 Hz),

7.27 (brs, 1H), 7.37 (t, 1H, J = 8.1 Hz), 7.63−7.74 (m, 4H). ¹³C NMR

(75 MHz, DMSO-d₆) δ 13.8, 25.2 (2), 25.8, 33.2 (2), 50.4, 109.3,

119.3, 120.6, 122.3, 123.7, 129.7, 131.8, 150.7, 151.5, 151.6, 154.0,

165.7. ESI-MS m/z [M + Na]⁺365 (100), [2M + Na]⁺707. Anal.

(C₁₉H₂₂N₂O₄): C, H, N.

(3-(3-Carbamoyl-5-methylthiophen-2-yl)phenoxycarbonyl)-

cyclohexylamine (5m). The title compound was prepared according

to the procedure previously described for 5a starting from 34,

cyclohexylisocyanate and TEA. Column chromatography (1% MeOH

in chloroform) aﬀorded 5m as a white solid (72% yield); mp 183−184

1

°C (MeOH). H NMR (300 MHz, CD₃OD) δ 1.22−1.39 (m, 5H),

1.64 (brd, 1H, J = 12.6 Hz), 1.78 (brd, 2H, J = 12.0 Hz), 1.94 (brd,

2H, J = 10.5 Hz), 2.47 (s, 3H), 3.38−3.48 (m, 1H), 6.97 (m, 1H), 7.09

(d, 1H, J = 8.1 Hz), 7.21 (s, 1H), 7.30−7.41 (m, 2H). ¹³C NMR (75

MHz, CD₃OD) δ 13.7, 25.0 (2), 25.4, 32.8 (2), 50.5, 121.5, 122.1,

125.6, 126.8, 129.3, 132.9, 134.7, 139.7, 141.4, 151.5, 155.0, 167.1. ESI-

MS m/z [M + Na]⁺381 (100), [2M + Na]⁺739. Anal.

(C₁₉H₂₂N₂O₃S): C, H, N.

2-(3-Hydroxyphenyl)-5-methylfuran-3-carboxamide (33). To

a suspension of 31 (188.0 mg, 0.81 mmol) in dry DCM (6.0 mL),

boron tribromide (1 M solution in DCM, 2.4 mL, 2.4 mmol) was

added at −78 °C. The reaction mixture was then allowed to warm to

room temperature and stirred for 8 h. A saturated aqueous solution of

sodium carbonate was added to quench the reaction. The residue was

extracted with ethyl acetate (3 × 20 mL). The combined organic

extracts were dried over anhydrous sodium sulfate, ﬁltered, and

concentrated to aﬀord the pure product as a brownish solid (98%

(3-(3-Carbamoyl-5-methylfuran-2-yl)phenoxycarbonyl)-(6-

phenylhexyl)amine (5n). Title compound was prepared according

to the procedure previously described for 5a starting from 33, 8, and

TEA. Column chromatography (1% MeOH in chloroform) aﬀorded

the pure product as a white solid (71% yield); mp 156−157 °C

1

(MeOH). H NMR (300 MHz, CDCl₃) δ 1.38 (brs, 4H), 1.55−1.66

(m, 4H), 2.33 (s, 3H), 2.61 (t, 2H, J = 7.5 Hz), 3.24 (q, 2H, J = 6.6

Hz), 5.10 (brs, 1H), 5.61 (brs, 1H), 5.82 (brs, 1H), 6.32 (s, 1H),

7.11−7.19 (m, 4H), 7.25−7.30 (m, 2H), 7.39 (d, 1H, J = 7.8 Hz), 7.58

(s, 1H), 7.64 (d, 1H, J = 7.8 Hz). ¹³C NMR (75 MHz, CDCl₃) δ 13.6,

26.8, 29.1, 29.9, 31.6, 36.1, 41.5, 108.5, 118.1, 121.2, 122.3, 124.7,

125.9, 128.5 (2), 128.6 (2), 129.8, 131.3, 142.8, 151.3, 151.5, 152.1,

154.6, 165.9. Anal. (C₂₅H₂₈N₂O₄): C, H, N.

1

yield). H NMR (300 MHz, acetone-d₆) δ 2.33 (d, 3H, J = 0.9 Hz),

6.42 (d, 1H, J = 0.9 Hz), 6.56 (brs, 1H), 6.81 (ddd, 1H, J = 0.9, 2.7, 4.8

Hz), 6.94 (brs, 1H), 7.22 (t, 1H, J = 7.8 Hz), 7.45 (dt, 1H, J = 7.8, 1.5

Hz), 7.53 (t, 1H, J = 1.8 Hz), 8.48 (s, 1H).

2-(3-Hydroxyphenyl)-5-methylthiophene-3-carboxamide

(34). Title compound was prepared according to the procedure

(3-(3-Carbamoyl-5-methylthiophen-2-yl)phenoxycarbonyl)-

(6-phenylhexyl)amine (5o). Title compound was prepared

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according to the procedure previously described for 5a starting 34, 8,

and TEA. Column chromatography (1% MeOH in chloroform)

aﬀorded the pure product as a white solid (80% yield); mp 152−153

2H), 2.61 (s, 3H), 3.38 (m, 1H), 7.02 (m, 2H), 7.39 (m, 2H), 7.50 (m,

1H). ¹³C NMR (75 MHz, CD₃OD) δ 12.5, 24.9, 25.4, 32.8, 50.5,

104.9, 116.7, 117.3, 120.1, 120.8, 129.6, 131.5, 150.9, 152.0, 155.0,

157.2, 167.3. ESI-MS m/z [M + Na]⁺365(100), [2M + Na]⁺707.

Anal. (C₁₉H₂₂N₂O₄): C, H, N.

1

°C (MeOH). H NMR (300 MHz, CDCl₃) δ 1.36−1.40 (m, 4H),

1.54−1.69 (m, 4H), 2.45 (d, 3H, J = 1.2 Hz), 2.61 (t, 2H, J = 7.5 Hz),

3.24 (q, 2H, J = 6.9 Hz), 5.13 (t, 1H, J = 5.4 Hz), 5.53 (brs, 2H),

7.12−7.19 (m, 5H), 7.25−7.32 (m, 4H), 7.39 (t, 1H, J = 7.8 Hz). ¹³C

NMR (75 MHz, CDCl₃) δ 15.3, 26.8, 29.1, 29.9, 31.6, 36.1, 41.5,

122.3, 123.1, 125.9, 126.6, 127.9, 128.5 (2), 128.6 (2), 129.9, 132.6,

134.3, 139.5, 141.7, 142.8, 151.4, 154.5, 166.0. Anal. (C₂₅H₂₈N₂O₃S):

C, H, N.

(3-(4-Carbamoyl-5-methylfuran-2-yl)phenoxycarbonyl)-(6-

phenylhexyl)amine (5r). Title compound was prepared according to

the procedure previously described for 5a starting from 41, 8, and

TEA. Column chromatography (10% MeOH in chloroform) aﬀorded

the pure product as a white solid (40% yield); mp 139−140 °C

1

(MeOH). H NMR (300 MHz, CD₃OD) δ 1.37 (m, 4H), 1.52−1.63

(m, 4H), 2.58 (m, 5H), 3.16 (t, 2H, J = 6.9 Hz), 6.99 (m, 2H), 7.12

(m, 3H), 7.22 (m, 2H), 7.35 (m, 2H), 7.46 (m, 1H). ¹³C NMR (75

MHz, CD₃OD) δ 12.6, 26.5, 28.8, 29.5, 31.5, 35.7, 40.9, 104.9, 116.7,

117.3, 120.1, 120.8, 125.5, 128.1, 128.2, 129.7, 131.6, 142.7, 150.9,

152.0, 155.8, 157.2, 167.3. ESI-MS m/z [M + Na]⁺443 (100), [M +

K]⁺459, [2M + Na]⁺863. Anal. (C₂₅H₂₈N₂O₄): C, H, N.

(

)-Ethyl-2-acetyl-4-(3-methoxyphenyl)-4-oxobutanoate

( )-(37). Title compound was prepared according to the procedure

described for ( )-26 starting from 35, 36, and sodium hydride.

Column chromatography (33% diethyl ether in petroleum ether)

1

aﬀorded the pure product as colorless viscous oil (79% yield). H

NMR (300 MHz, CDCl₃) δ 1.29 (t, 3H, J = 7.2 Hz), 2.45 (s, 3H), 3.51

(dd, 1H, J = 18.6, 5.7 Hz), 3.71 (dd, 1H, J = 18.6, 8.3 Hz), 3.85 (s,

3H), 4.23 (m, 3H), 7.12 (m, 1H), 7.37 (m, 1H), 7.47 (m, 1H), 7.58

(m, 1H). ESI-MS m/z [M + H]⁺279, [M + Na]⁺301 (100).

Ethyl-5-(3-methoxyphenyl)-2-methylfuran-3-carboxylate

(38). Title compound was prepared according to the procedure

described for 27 starting from ( )-37 and concentrated hydrochloric

acid. Column chromatography (50% diethyl ether in petroleum ether)

Ethyl-1-benzyl-2-(3-methoxyphenyl)-5-methyl-1H-pyrrole-

3-carboxylate (42). ( )-26 (141 mg, 0.507 mmol) was dissolved in

acetic acid (500 μL) in a 25 mL round bottomed ﬂask. Benzylamine

(277 μL, 2.54 mmol) was added, and the ﬂask was inserted into the

cavity of a CEM Discovery microwave system apparatus and reﬂuxed

(maximum internal temperature 130 °C), employing a microwave

power of 150W for 12 min. Then the reaction mixture was cooled and

diluted with ethyl acetate. It was then neutralized with saturated

sodium bicarbonate aqueous solution and extracted with ethyl acetate

(3 × 50 mL). The combined organic layers were dried over anhydrous

sodium sulfate, ﬁltered, and concentrated. Column chromatography

(20% ethyl acetate in n-hexane) aﬀorded the pure product as pale-

yellow oil (86% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.13 (t, 3H, J =

7.2 Hz), 2.12 (s, 3H), 3.60 (s, 3H), 4.11 (q, 2H, J = 7.2 Hz), 4.92 (s,

2H), 6.50 (s, 1H), 6.73 (s, 1H), 6.84−6.87 (m, 4H), 7.19−7.27 (m,

4H).

1-Benzyl-2-(3-methoxyphenyl)-5-methyl-1H-pyrrole-3-car-

boxylic Acid (43). Compound 42 (123 mg, 0.475 mmol) was

dissolved in 4.0 mL of ethanol/THF in the ratio 1:1. Then, sodium

hydroxide (4 pellets) was added and the reaction mixture was reﬂuxed.

After 16 h, the solvent was removed under reduced pressure. The

residue was partitioned between ethyl acetate and water. The aqueous

layer was then acidiﬁed to pH = 2 with 4 N hydrochloric acid and

extracted with ethyl acetate (3 × 25 mL). The organic layer was dried

over anhydrous sodium sulfate, ﬁltered, and concentrated. The

product was then recrystallized from DCM and hexane and obtained

as a white amorphous solid (98% yield). ¹H NMR (300 MHz, CDCl₃)

δ 2.11 (s, 3H), 3.60 (s, 3H), 4.90 (s, 2H), 6.52 (s, 1H), 6.73 (s, 1H),

6.83−6.86 (m, 4H), 7.17−7.28 (m, 4H).

1-Benzyl-2-(3-methoxyphenyl)-5-methyl-N-(5-phenylpen-

tyl)-1H-pyrrole-3-carboxamide (5s). The title compound was

prepared according to the procedure described for 5s starting from

43 and 7b. Column chromatography (30% ethyl acetate in n-hexane)

aﬀorded the pure product as a white amorphous solid (78% yield.). ¹H

NMR (300 MHz, CDCl₃) δ 1.01−1.11 (m, 2H), 1.21−1.31 (m, 2H),

1.43−1.54 (m, 2H), 2.14 (s, 3H), 2.51 (t, 2H, J = 7.8 Hz), 3.13−3.20

(m, 2H), 3.59 (s, 3H), 4.86 (s, 2H), 5.24−5.26 (m, 1H), 6.53 (s, 1H),

6.71−6.73 (m, 1H), 6.79−6.93 (m, 4H), 7.10−7.29 (m, 9H). ¹³C

NMR (75 MHz, CDCl₃) δ 12.5, 26.6, 29.4, 31.3, 36.0, 39.2, 47.8, 55.3,

108.6, 115.7, 116.0, 117.6, 123.5, 125.8, 125.9, 127.4, 128.5, 128.6,

128.9, 129.4, 130.11, 133.0, 133.5, 138.3, 142.8, 159.9, 165.1. ESI-MS

m/z [M + H]⁺467, [M + Na]⁺489, [M + K]⁺505, [2M + Na]⁺955

(100). Anal. (C₃₁H₃₄N₂O₂): C, H, N.

1-Benzyl-2-(3-methoxyphenyl)-5-methyl-N-(6-phenylhexyl)-

1H-pyrrole-3-carboxamide (5t). To a solution of acid 43 (45 mg,

0.184 mmol) in 4.0 mL of dry DCM at 0 °C were added TEA (52 μL,

0.368 mmol), EDCI (53 mg, 0.276 mmol), and HOBt (40 mg, 0.258

mmol) and the mixture was stirred at 0 °C for 10 min. Then, 7a (39

mg, 0.220 mmol) in 1.0 mL of dry DCM was added. The stirring was

continued at 0 °C for 30 min, and then the reaction mixture was

allowed to warm to room temperature within 16 h. The solvent was

subsequently removed in vacuo. Column chromatography (30% ethyl

acetate in n-hexane) aﬀorded the pure product as a white amorphous

1

aﬀorded the pure product as colorless viscous oil (72% yield). H

NMR (300 MHz, CDCl₃) δ 1.35 (t, 3H, J = 7.2 Hz), 2.62 (s, 3H), 3.81

(s, 3H), 4.29 (q, 2H, J = 7.2 Hz), 6.80 (m, 1H), 6.86 (s, 1H), 7.15−

7.28 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 14.1, 14.6, 55.4, 60.4,

106.0, 109.1, 113.6, 115.6, 116.4, 129.9, 131.5, 151.74, 158.8, 160.1,

164.1.

5-(3-Methoxyphenyl)-2-methylfuran-3-carboxylic Acid (39).

Title compound was prepared according to the procedure described

for 29 starting from compound 38 and sodium hydroxide. Compound

was carried on without further puriﬁcation. ¹H NMR (300 MHz,

CDCl₃) δ 2.69 (s, 3H), 3.86 (s, 3H), 6.84 (m, 1H), 6.92 (s, 1H),

7.18−7.33 (m, 3H), 10.83 (brs, 1H).

5-(3-Methoxyphenyl)-2-methylfuran-3-carboxamide (40).

Title compound was prepared according to the procedure described

for 31 starting from acid 39, thionyl chloride, and ammonium

hydroxide concentrated solution. Column chromatography (30% n-

hexane in ethyl acetate) aﬀorded the pure product as a white solid

1

(74% yield). H NMR (300 MHz, CDCl₃) δ 2.67 (s, 3H), 3.85 (s,

3H), 5.68 (brs, 2H), 6.67 (s, 1H), 6.83 (m, 1H), 7.16 (m, 1H), 7.21

(m, 1H), 7.26−7.32 (m, 1H). ESI-MS m/z [M + Na]⁺254 (100), [M

+ K]⁺270, [2M + Na]⁺485.

5-(3-Hydroxyphenyl)-2-methylfuran-3-carboxamide (41).

Title compound was prepared according to the procedure previously

described for 33 starting from 40 and boron tribromide. Pure product

was obtained as a pale-yellow solid (61% yield). ¹H NMR (300 MHz,

CD₃OD) δ 2.58 (s, 3H), 6.70 (m, 1H), 6.92 (s, 1H), 7.09 (m, 2H),

7.17 (m, 1H). ESI-MS m/z [M + Na]⁺240 (100).

(3-(4-Carbamoyl-5-methylfuran-2-yl)phenoxycarbonyl)-

butylamine (5p). The title compound was prepared according to the

procedure previously described for 5a starting from 41, n-

butylisocyanate, and TEA. Column chromatography (1% MeOH in

chloroform) aﬀorded the pure product as a white solid (47% yield);

1

mp 162−163 °C (MeOH). H NMR (300 MHz, CD₃OD) δ 0.97 (t,

3H, J = 7.4 Hz), 1.37−1.58 (m, 4H), 2.61 (s, 3H), 3.19 (t, 2H, J =

7.1), 7.01 (m, 2H), 7.38 (m, 2H), 7.49 (m, 1H). ¹³C NMR (75 MHz,

CD₃OD) δ 12.5, 12.9, 19.8, 31.7, 40.6, 104.9, 116.7, 117.3, 120.1,

120.8, 129.7, 131.5, 150.9, 152.0, 155.9, 157.2, 167.3. ESI-MS m/z [M

+ H]⁺317, [M + Na]⁺339, [2M + Na]⁺655 (100). Anal.

(C₁₇H₂₀N₂O₄): C, H, N.

(3-(4-Carbamoyl-5-methylfuran-2-yl)phenoxycarbonyl)-

cyclohexylamine (5q). Title compound was prepared according to

the procedure previously described for 5a starting from 41,

cyclohexylisocyanate, and TEA. Column chromatography (10%

MeOH in chloroform) aﬀorded the pure product as a white solid

(34% yield); mp 182−183 °C (MeOH). ¹H NMR (300 MHz,

CD₃OD) δ 1.19−1.39 (m, 5H), 1.65 (m, 1H), 1.79 (m, 2H), 1.96 (m,

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solid (81% yield.). ¹H NMR (300 MHz, CDCl₃) δ 0.98−1.11 (m, 2H),

1.15−1.25 (m, 4H), 1.44−1.57 (m, 2H), 2.13 (s, 3H), 2.55 (t, 2H, J =

7.5 Hz), 3.12−3.21 (m, 2H), 3.57 (s, 3H), 4.85 (s, 2H), 5.21−5.23 (m,

1H), 6.53 (s, 1H), 6.70−6.73 (m, 1H), 6.79−6.89 (m, 4H), 7.14−7.29

(m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 13.5, 26.8, 28.9, 29.4, 31.5,

36.0, 38.2, 48.0, 54.3, 106.6, 115.7, 116.0, 117.6, 123.3, 125.8, 125.9,

127.3, 128.7, 128.8, 128.9, 129.4, 130.2, 133.0, 133.5, 138.3, 142.8,

159.7, 164.5. ESI-MS m/z [M + H]⁺481 (100), [M + Na]⁺503, [2M

+ H]⁺962, [2M + Na]⁺983. Anal. (C₃₂H₃₆N₂O₂): C, H, N.

1-Benzyl-2-(3-hydroxyphenyl)-5-methyl-N-(6-phenylhexyl)-

1H-pyrrole-3-carboxamide (44). The title compound was prepared

according to the procedure previously described for 33 starting from

5s and boron tribromide (99% yield). ¹H NMR (300 MHz, CDCl₃) δ

0.88−1.07 (m, 2H), 1.10−1.19 (m, 4H), 1.40−1.55 (m, 2H), 2.02 (s,

3H), 2.55 (t, 2H, J = 7.5 Hz), 3.11 (m, 2H), 4.82 (s, 2H), 5.41 (m,

1H), 6.53 (s, 1H), 6.65 (m, 1H), 6.70−6.91 (m, 4H), 7.15−7.29 (m,

9H).

from 19, 15d, and TEA. Column chromatography (100% chloroform

to 2% MeOH in chloroform) aﬀorded 5y as a white amorphous solid

(75% yield). H NMR (300 MHz, CDCl₃) δ 3.46−3.52 (m, 2H),

3.68−3.72 (m, 2H), 3.84−3.87 (m, 2H), 4.16−4.20 (m, 2H), 5.72−

5.90 (m, 3H), 6.56 (m, 1H), 6.74−7.07 (m, 5H), 7.17 (m, 2H), 7.35−

7.41 (m, 1H), 7.63 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 41.3, 69.7,

70.1, 70.2, 105.2, 110.0, 110.9, 114.7, 116.7, 117.6, 120.1, 121.0, 122.8,

130.5, 140.7, 143.5, 151.3, 152.2, 154.5, 155.4, 158.6, 166.9. ESI-MS

m/z [M + H]⁺446, [M + Na]⁺468 (100), [M + K]⁺484. Anal.

(C₂₂H₂₁F₂N₃O₅): C, H, N.

1

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-(2-(2-

(2,4,6-triﬂuorophenoxy)ethoxy)ethyl)amine (5z). Title com-

pound was prepared according to the procedure described for 5a

starting from 19, 15e, and TEA. Column chromatography (100%

chloroform to 2% MeOH in chloroform) aﬀorded 5z as a white

1

amorphous solid (78% yield). H NMR (300 MHz, CDCl₃) δ 3.45−

3.53 (m, 2H), 3.65−3.69 (m, 2H), 3.79−3.82 (m, 2H), 4.23−4.26 (m,

2H), 5.67−5.71 (m, 1H), 5.90 (brs, 2H), 6.56 (m, 1H), 6.63−6.73 (m,

2H), 6.98−7.08 (m, 2H), 7.17−7.21 (m, 2H), 7.38 (m, 1H), 7.63 (s,

1H). ¹³C NMR (75 MHz, CDCl₃) δ 41.3, 70.1, 70.3, 74.1, 101.1 (2),

110.0, 114.7, 117.6, 120.1, 120.8, 121.0, 122.8, 130.6, 140.8, 152.2 (2),

154.5, 155.8, 158.0, 158.9, 166.9, ESI-MS m/z [M + H]⁺464, [M +

Na]⁺486 (100). Anal. (C₂₂H₂₀F₃N₃O₅): C, H, N.

(3-(3-(6-Phenylhexylcarbamoyl)-1-benzyl-5-methyl-1H-pyr-

rol-2-yl)phenoxycarbonyl)cyclohexylamine (5u). The title com-

pound was prepared according to the procedure previously described

for 5a starting from 44, cyclohexylisocyanate, and TEA. Column

chromatography (30% ethyl acetate in n-hexane) aﬀorded the pure

1

product as colorless oil (63% yield). H NMR (300 MHz, CDCl₃) δ

1.01−1.39 (m, 12H), 1.43−1.79 (m, 6H), 1.90−2.02 (m, 2H), 2.16 (s,

3H), 2.55 (t, 2H, J = 7.8 Hz), 3.18 (m, 2H), 3.51 (m, 1H), 4.88 (m,

3H), 5.24 (m, 1H), 6.50 (s, 1H), 6.80 (m, 2H), 6.98−7.30 (m, 10H).

¹³C NMR (75 MHz, CDCl₃) δ 12.5, 25.0, 25.6, 26.8, 29.2, 29.5, 29.9,

31.6, 33.4, 36.1, 39.3, 47.9, 50.4, 101.0, 105.0, 108.6, 117.8, 122.3,

124.6, 125.8, 125.9, 127.3, 127.9, 128.5, 128.6, 128.9, 129.8, 132.3,

133.3, 138.1, 143.0, 151.4, 153.4, 159.4, 165.0, 180.7, 183.3, 184.8.

Anal. (C₃₈H₄₅N₃O₃): C, H, N.

Experimental Section for Modeling and Biochemical Assays.

Sequence Alignment, Model Building, and Optimization. The

mouse FAAH1 sequence was taken in fasta format from

UniProtKB/Swiss-Prot [www.uniprot.org/uniprot] (entry: O08914).

A protein−protein BLAST search (applying BLOSUM62 similarity

matrix) [www.ncbi.nlm.nih.gov/blast/Blast.cgi] on the Protein Data

Bank (PDB) [www.pdb.org] was conducted using the target sequence

to identify and align structural homologues. Crystal structure of

FAAH1 from Rattus norvegicus (PDB code: 1MT5, resolution 2.80

Å),¹⁷complexed with the irreversible inhibitor MAPF and sharing 91%

and 95% of sequence identity and similarity, respectively, was chosen

as template.

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-(2-(2-

phenoxyethoxy)ethyl)amine (5v). Title compound was prepared

according to the procedure described for 5a starting from 19, 15a, and

TEA. Column chromatography (100% chloroform to 2% MeOH in

1

Before 3D model building step, the template structure was

optimized using Protein Preparation Wizard tool implemented in

chloroform) aﬀorded 5v as a white amorphous solid (65% yield). H

NMR (300 MHz, CDCl₃) δ 3.46−3.52 (m, 2H), 3.69−3.73 (m, 2H),

3.85−3.88 (m, 2H), 4.10−4.16 (m, 2H), 5.85−6.00 (m, 3H), 6.55 (m,

1H), 6.92−7.03 (m, 4H), 7.06 (m, 1H), 7.15−7.19 (m, 2H), 7.25−

7.39 (m, 3H), 7.61 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 41.3, 67.4,

69.8, 70.1, 110.0, 114.7, 114.8 (2), 117.6, 120.1, 120.7, 121.1, 121.3,

122.7, 129.8 (2), 130.5, 140.8, 152.1, 154.5, 158.8, 166.8. ESI-MS m/z

[M + Na]⁺432 (100). Anal. (C₂₂H₂₃N₃O₅): C, H, N.

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-(2-(2-(4-

ﬂuorophenoxy)ethoxy)ethyl)amine (5w). Title compound was

prepared according to the procedure described for 5a starting from 19,

15b, and TEA. Column chromatography (100% chloroform to 2%

MeOH in chloroform) aﬀorded 5w as a white amorphous solid (73%

yield). ¹H NMR (300 MHz, CDCl₃) δ 3.44−3.53 (m, 2H), 3.69−3.72

(m, 2H), 3.84−3.87 (m, 2H), 4.10−4.13 (m, 2H), 5.65−5.77 (m, 3H),

6.55 (m, 1H), 6.89−7.08 (m, 6H), 7.18−7.26 (m, 2H), 7.37−7.43 (m,

1H), 7.63 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 41.3, 68.2, 69.9,

70.1, 109.9, 114.8, 115.8, 115.9 (2), 116.0, 116.3, 117.7, 120.1, 120.9,

122.8, 130.6, 140.8, 152.1, 154.4, 154.9, 159.2, 166.6. ESI-MS m/z [M

+ Na]⁺447 (100). Anal. (C₂₂H₂₂FN₃O₅): C, H, N.

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-(2-(2-(2-

ﬂuorophenoxy)ethoxy)ethyl)amine (5x). Title compound was

prepared according to the procedure described for 5a starting from 19,

15c, and TEA. Column chromatography (100% chloroform to 2%

MeOH in chloroform) aﬀorded 5x as a white amorphous solid (78%

yield). ¹H NMR (300 MHz, CDCl₃) δ 3.48−3.52 (m, 2H), 3.69−3.73

(m, 2H), 3.86−3.90 (m, 2H), 4.20−4.23 (m, 2H), 5.79−5.99 (m, 3H),

6.56 (m, 1H), 6.85−7.19 (m, 8H), 7.36 (m, 1H), 7.63 (s, 1H). ¹³C

NMR (75 MHz, CDCl₃) δ 41.3, 69.2, 69.7, 70.1, 109.9, 114.8, 115.8,

116.5, 116.7, 120.1, 120.9, 122.0 (2), 122.8, 124.6 (2), 130.5, 140.8,

146.9, 152.2, 154.5, 166.6. ESI-MS m/z [M + Na]⁺447 (100). Anal.

(C₂₂H₂₂FN₃O₅): C, H, N.

Maestro (version 9.1, Schrodinger, LLC, New York, NY, 2010). Also, a

̈

missing atom building step was performed using the Prime-Refinement

tool (Side Chain Prediction):³³beginning with the ﬁrst residue to be

predicted, the side-chain rotamer library was used to ﬁnd rotamers

with the lowest energy while keeping all other side chains ﬁxed. Then,

the 3D model of mouse FAAH1 was built by Prime. The inhibitor

MAPF was incorporated in the model generation process to preserve

catalytic site conformation. Side chain rotamers of conserved residues

were retained and all side chains were optimized. Amino acid side

chains not derived from the template were minimized as well. The

reﬁned model was subjected to structural check program (PRO-

CHECK^34,35) to gauge its quality. To optimize the CA channel, a

mFAAH1/transition intermediate (TI) of 2 complex was built. The TI

of 2 was made based on crystallized ligand MAPF, being the

phosphonate group an analogue of the tetrahedral intermediate. The

inhibitor was orientated as described according to biochemical

evidence that carbamates covalently modify the active site of FAAH,

by an irreversible (or slowly reversible) mechanism involving

carbamylation of the nucleophile Ser241²²and on theoretical QM/

MM calculations by Lodola et al.³⁶

A molecular dynamic was carried out at 300 K with time step of 1.5

fs: the bounded-ligand and a set of side chain around 8 Å from it were

allowed to move. The system was equilibrated for a period of 10 ps,

and then a production run of 1 ns was carried out. One hundred

sampled structures were cauterized and minimized. The lowest energy

one was used for homology model validation. All the calculations were

carried out by using MacroModel, version 9.8 (Schrodinger, LLC:

̈

New York, 2010). The bounded-2 atoms were removed, and the

hydrogen atoms of the catalytic site residues were reassigned, restoring

the native state of the catalytic triad (Ser241, Ser217, and Lys142).

The 3D structures of all the molecules used in this study were built

(3-(3-Carbamoyl-1H-pyrrol-1-yl)phenoxycarbonyl)-(2-(2-

(2,4-diﬂuorophenoxy)ethoxy)ethyl)amine (5y). Title compound

was prepared according to the procedure described for 5a starting

using Maestro (version 9.1, Schrodinger, LLC, New York, NY, 2010)

̈

and minimized by MacroModel (version 9.8, Schrodinger, LLC, New

̈

6912

dx.doi.org/10.1021/jm300689c | J. Med. Chem. 2012, 55, 6898−6915

Journal of Medicinal Chemistry

Article

York, NY, 2010) using the OPLS-2005 as force ﬁeld. All molecules

Basile, Varese, Italy). The parameter used to quantify the nociceptive

threshold was deﬁned as the pressure (grams) at which the rat

withdrew its paw. The day before testing, rats received three training

sessions. Pressure was gradually applied to the right hind paw, and paw

withdrawal thresholds (PWTs) were assessed as the pressure (grams)

required for eliciting paw withdrawal. A cutoﬀ point of 250 g was used

to prevent any tissue damage to the paw.

were then pretreated by LigPrep application (version 2.4, Schrodinger,

̈

LLC, New York, NY, 2010), implemented in Maestro, generating the

most probable ionization state and tautomers at physiological pH and

any possible enantiomer. Compound 2 and our inhibitors were docked

using Glide sp protocol (version 5.5, Schrodinger, LLC, New York,

̈

NY, 2009), and bounded-2 was used as reference ligand.

Elevated Plus Maze. The apparatus was of gray Plexiglas and

consisted of two open and two closed arms linked by a common

central platform. The maze was elevated 40 cm above ﬂoor level and

dimly lit. Animals were individually placed on the central platform of

the maze facing an open arm. A standard 5 min test was employed.

The amount of time spent by each animal in either open or closed arm

was recorded by an Any-maze video tracking system (Ugo Basile), as

well as the number of entries and the total distance walked by each

animal into either arm.

FAAH Assay. The activity of FAAH (E.C.3.5.1.4) was assayed by

measuring the release of [³H]ethanolamine from 10 μM [³H-

ethanolamine]AEA.³⁷Mouse brain homogenates (40 μg per test)

were preincubated for 20 min with each compounds, and then they

were incubated with [³H-ethanolamine]AEA for 15 min at 37 °C, in

500 μL of 50 mM Tris−HCl buﬀer (pH 9.0). The reaction was

stopped by the addition of 800 μL ice-cold methanol/chloroform (2:1,

v/v) with vortexing. The mixture was centrifuged at 3000g for 5 min,

the upper aqueous layer was put in a vial containing liquid scintillation

cocktail (Ultima Gold XR, Perkin-Elmer Life Sciences), and

radioactivity was quantiﬁed in a β-counter. FAAH activity was

expressed as pmol [³H]ethanolamine released/min per mg of protein.

Kinetic Analysis of FAAH. The IC₅₀(half-maximal inhibitory

concentration) values of the compounds toward FAAH activity were

calculated through nonlinear regression analysis of dose−response

curves, performed with the Prism4 program (GraphPAD Software for

Science, San Diego, CA). The type of inhibition and the apparent

inhibition constant (K_i) values of the compounds were ascertained

using diﬀerent concentrations of substrate ([³H-ethanolamine]AEA,

0−20 μM range) and inhibitor (0−5 μM range). The kinetic data were

ﬁtted to Michaelis−Menten curves, which were subjected to nonlinear

regression analysis through the Prism4 program in order to calculate

apparent Michaelis−Menten constant (K_m), maximal velocity (V_max),

and K_ivalues. The same Michaelis−Menten curves were also analyzed

by Lineweaver−Burk diagrams (double reciprocal plots) in order to

further visualize the type of inhibition.³⁷

Dialysis of FAAH. Dialysis was performed on mouse brain

homogenates (40 μg per test), preincubated for 20 min with selected

compounds, and then dialyzed for 18 h at 4 °C against an excess (2 L)

of 20 mM Tris-HCl buﬀer (pH 7.0).³⁷Then, FAAH activity was

assayed as described above.

Assays of Other ECS Elements. In all assays, homogenates were

preincubated for 20 min with each compounds, then activity was

measured as detailed below. The synthesis of AEA through the activity

of NAPE-PLD was assayed with mouse brain homogenates (200 μg

per test), using 100 μM [³H]NArPE as substrate.³⁷The uptake of 500

nM [³H-arachidonyl]AEA was measured in intact human keratinocytes

(HaCaT cells) (5 × 10⁵per test), and the synthesis of 2-AG by DAGL

was assayed with mouse brain homogenates (100 μg protein per test)

using 100 μM [¹⁴C]DAG as substrate.³⁷The hydrolysis of 2-AG by

MAGL was measured with mouse brain supernatants (100 μg protein

per test) using 10 μM [³H]2-OG as substrate.³⁷

Receptor Binding Assays. Membrane fractions were isolated from

mouse brain (as a source of CB₁R) or mouse spleen (as a source of

CB₂R), and were used in rapid ﬁltration assays (100 μg per test) with

the synthetic cannabinoid [³H]CP55.940 (500 pM). Membrane

fractions prepared from mouse brain were used in rapid ﬁltration

assays (100 μg per test) also to test the eﬀect of the compounds on the

binding of [³H]RTX (500 pM) to TRPV1. In all experiments,

unspeciﬁc binding was determined in the presence of an excess (1

μM) of “cold” agonist (CP55.940 or RTX, respectively).³⁷

Experimental Details for the in Vivo Tests. Irwin Test. General

behavioral observations were recorded by camera in CD-1 mice

(Charles River, Calco, Italy), 5−6 week old, (4 animals/group) 60 and

240 min after oral 10, 30, and 100 mg/kg 5c, 5d, and 5n

administration by Irwin test²⁸. Limbic stereotypies as sniﬃng and

grooming while striatal as licking and biting were considered. The

compounds were suspended in a solution containing 0.1% Tween 80

in 0.5 CMC (medium viscosity) (Sigma-Aldrich, Milan), which was

used as vehicle, and administered in a volume of 10 mL/kg in mice.

Randall−Selitto. Mechanical nociceptive thresholds were measured

in the rat paw pressure test³²by applying increasing pressure to the left

and right hind paws using a Randall−Selitto analgesimeter (Ugo

hERG (Automated Patch-Clamp). CHO-K1 cells were originally

obtained from American Tissue Culture Collection (ATCC). hERG

cDNA (GenBank sequence NM_000238) was subcloned into pSI

vector (Promega) at Cerep. The CHO-K1 cells were cotransfected

with this construct and pPUR (containing puromycin selective marker,

BD Bioscience). After selection in puromycin for 10 days, single

colonies were selected and veriﬁed with hERG potassium currents.

The stably transfected cells were used in this study. The stably

transfected cells were cultured in F-12 Kaighn’s Nutrient Mixture

medium (Invitrogen) + 10% FBS at 37 °C for 1−3 days. Cells were

kept at 30 °C for 12−24 h before patch clamp experiment in order to

increase the hERG current amplitude. Subsequently, the cells were

harvested by trypsination, and kept in serum-free medium for up to 6 h

at room temperature before recording. The cells were washed and

resuspended in extracellular solution before being applied to the patch

clamp site. After whole cell conﬁguration was achieved, the cells were

held at −80 mV. A 50 ms pulse to −40 mV was delivered to measure

the leaking current, which was subtracted from the tail current on line.

Then the cell was depolarized at +20 mV for 2 s, followed by a 1 s

pulse to −40 mV to reveal hERG tail current. This paradigm was

delivered once every 5 s to monitor the current amplitude. The

extracellular solution (control) was applied ﬁrst, and the cell was

stabilized in extracellular solution for 5 min. Then the test compound

was applied from low concentrations to high concentrations

cumulatively. The cell was incubated with each test concentration

for 5 min. During the incubation, the cell was repetitively stimulated

using the voltage protocol described above, and the tail current

amplitude was continuously monitored. The degree of inhibition (%)

was obtained by measuring the tail current amplitude before and after

drug incubation.

ASSOCIATED CONTENT

* Supporting Information

■

S

Biological assays to deﬁne the selectivity proﬁle of compounds

5c and 5d on a panel of diﬀerent targets. Elemental analysis

results for ﬁnal compounds. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

■

*For G.C.: phone, 0039-0577-234172; fax, 0039-0577-234333;

E-mail, campiani@unisi.it. For P.M.: phone, 0039-06-

91393906; fax, 0039-06-91393638; E-mail, patrizia.minetti@

sigma-tau.it.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful to Dr. Chiara De Simone for her assistance in

the preliminary assays of FAAH activity. This study was

supported by grants from MIUR (Rome, grant PRIN 2008 to

6913

dx.doi.org/10.1021/jm300689c | J. Med. Chem. 2012, 55, 6898−6915

Journal of Medicinal Chemistry

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ABBREVIATIONS USED

■

ECs, endocannabinoids; AEA, anandamide; CBR, cannabinoid

receptor; 2-AG, 2-arachidonoylglycerol; AMT, AEA membrane

transporter; FAAH, fatty acid amide hydrolase; MAGL,

monoacylglycerol lipase; ACB, acyl binding channel; CA,

cytoplasmic access; CP, cytosolic port; CNS, central nervous

system; PNS, peripheral nervous systems; MAFP, methoxyar-

achidonoyl ﬂuorophosphonate; MW, microwave; SAR, struc-

ture−activity relationship; THF, tetrahydrofuran; TEA, triethyl-

amine; DMF, dimethylformamide; DCM, dichloromethane;

DMSO, dimethylsulfoxide; DIAD, diisopropyl azodicarboxy-

late; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

HOBt, N-hydroxybenzotriazole; SAR, structure−activity rela-

tionship; TRPV, vanilloid receptor; hERG, human ether-a-go-

go related gene; CT, control; NAPE-PLD, N-acyl phosphati-

dylethanolamine phospholipase D; DAGL, diacylglycerol

lipase; PWTs, paw withdrawal thresholds; AA, arachidonic

acid; RP-HPLC, reverse phase high pressure liquid chromatog-

raphy; RTX, resinferatoxin; 2-OG, 2-oleoylglycerol; PDB,

Protein Data Bank; TI, transition intermediate

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Discovery of potent inhibitors of human and mouse fatty acid amide hydrolases

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