O-(triazolyl)methyl carbamates as a novel and potent class of Fatty Acid Amide Hydrolase (FAAH) inhibitors

Article abstract of DOI:10.1002/cmdc.201402374

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a coppercatalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC₅₀ values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.

Full text of DOI:10.1002/cmdc.201402374

CHEMMEDCHEM
FULL PAPERS
DOI: 10.1002/cmdc.201402374
O-(Triazolyl)methyl Carbamates as a Novel and Potent
Class of Fatty Acid Amide Hydrolase (FAAH) Inhibitors
Giampiero Colombano,*[a] Clara Albani,[a] Giuliana Ottonello,[a] Alison Ribeiro,[a]
Rita Scarpelli,[a] Glauco Tarozzo,[a] Jennifer Daglian,[b] Kwang-Mook Jung,[b]
Daniele Piomelli,[a, b] and Tiziano Bandiera[a]
Inhibition of fatty acid amide hydrolase (FAAH) activity is under
investigation as a valuable strategy for the treatment of several
disorders, including pain and drug addiction. A number of
potent FAAH inhibitors belonging to different chemical classes
have been disclosed to date; O-aryl carbamates are one of the
most representative families. In the search for novel FAAH in-
hibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate deriv-
atives were designed and synthesized exploiting a copper-
catalyzed [3+2] cycloaddition reaction between azides and
alkynes (click chemistry). Exploration of the structure–activity
relationships within this new class of compounds identified
potent inhibitors of both rat and human FAAH with IC50 values
in the single-digit nanomolar range. In addition, these deriva-
tives showed improved stability in rat plasma and kinetic solu-
bility in buffer with respect to the lead compound. Based on
the results of the study, the novel analogues identified can be
considered to be promising starting point for the development
of new FAAH inhibitors with improved drug-like properties.
Introduction
Fatty acid amide hydrolase (FAAH)[1] is a membrane-bound
serine hydrolase that catalyzes the hydrolytic cleavage of en-
dogenous biologically active fatty acid ethanolamides (FAEs),
such as anandamide (AEA), an agonist of cannabinoid recep-
tors,[2] and palmitoylethanolamide (PEA),[3] and oleoylethanola-
mide (OEA),[4] which are agonists of type-a peroxisome prolifer-
ator-activated receptors (PPAR-a).[5] These natural FAAH sub-
strates may play important roles in the central nervous system
(CNS) and in peripheral tissues, where they are involved in a
variety of physiological processes.[6]
series (Figure 1), exemplified by URB524 (1a),[8a,b,13] URB597
(1b),[8a,b,13] and URB694 (1c),[8c,14] has been extensively investi-
gated.[15] In particular, it was shown that compound 1b exerts
a combination of anxiolytic-like, anti-depressant-like, and anal-
gesic effects, because of its ability to inhibit FAAH activity in
the CNS and peripheral tissues.[16]
The active site of FAAH is characterized by an atypical cata-
lytic triad, consisting of Ser241–Ser217–Lys142, which is capa-
ble of hydrolyzing amide and ester bonds at similar rates.[17]
Several studies, including computational modeling,[18] support-
ed by the resolution of the crystal structure of humanized rat
FAAH in complex with 1b,[19] indicate that O-arylcarbamates
bind covalently to FAAH and cause its irreversible inhibition. In
particular, it has been proposed that this class of molecules is
attacked at the carbonyl group by Ser241, leading to the for-
mation of carbamoylated, catalytically inactive FAAH and re-
leasing the O-biphenyl moiety as the leaving group.
Substantial efforts have been dedicated to the discovery of
potent and selective FAAH inhibitors, with the objective of de-
veloping therapeutic approaches for pathologic conditions
such as pain, drug addiction, anxiety, and depression.[6,7] Differ-
ent classes of molecules are known to increase intracellular
FAE levels through FAAH inhibition, including carbamates[8]
and piperidine/piperazine ureas[9] that covalently bind to
FAAH,[10] and a-keto heterocycle-based inhibitors,[11] which in-
hibit FAAH by reversible hemiketal formation with the catalytic
serine of the enzyme.[12] Among them, the O-arylcarbamate
[a] Dr. G. Colombano,+ C. Albani, Dr. G. Ottonello, Dr. A. Ribeiro,
Dr. R. Scarpelli, Dr. G. Tarozzo, Prof. D. Piomelli, Dr. T. Bandiera
Drug Discovery & Development, Istituto Italiano di Tecnologia
Via Morego 30, 16163 Genova (Italy)
E-mail: giampiero.colombano@iit.it
[b] J. Daglian, Dr. K.-M. Jung, Prof. D. Piomelli
Departments of Anatomy & Neurobiology, Pharmacology & Biological
Chemistry, University of California, Irvine
3216 Gillespie Neuroscience Research Facility, CA 92697-4621 (USA)
[+] Current address: The Institute of Cancer Research, 15 Cotswold Rd, Sutton,
Surrey SM2 5NG (UK); E-mail: giampiero.colombano@icr.ac.uk.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402374.
Figure 1. O-Arylcarbamates fatty acid amide hydrolase (FAAH) inhibitors.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
1
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
The preparation of 1,4-disub-
stituted-1,2,3-triazoles was ac-
complished by [3+2] cycloaddi-
tion reaction between azides
and alkynes, in the presence of
copper(I) salts (click chemis-
try).[20] We designed a versatile
synthetic strategy that allowed
us to generate a first set of mol-
ecules, bearing an O-(1,2,3-tria-
zol-4-yl)methyl moiety. The de-
Figure 2. Side-by-side comparison of URB524 (1a) and O-(1,2,3-triazol-4-yl)alkyl carbamate derivatives.
O-Arylcarbamates such as 1b are selective for FAAH but can
also interact with select liver carboxylesterases, at least at high
concentrations, and have limited plasma stability.[9a] Recently,
however, highly potent O-arylcarbamates with markedly im-
proved selectivity for FAAH were identified.[8c] The insertion of
an electron-donating substituent, such as a hydroxy or amino
group, in the para position of the proximal phenyl ring of 1a
did not significantly affect inhibitory potency in vitro, but
caused a marked increase in the stability of the compounds in
plasma, in comparison to other molecules in the series.
URB694 (1c) was identified as a potent FAAH inhibitor with im-
proved plasma stability, prolonged half-life in vivo, and de-
creased activity towards liver carboxylesterases in comparison
to 1b.
In the search for better FAAH inhibitors with improved sta-
bility, we designed a novel class of carbamates where the O-
aryl moiety is replaced by an O-(triazol-4-yl)alkyl group
(Figure 2). We expected those compounds to be more stable
than their O-aryl analogues, since the aliphatic alcohol result-
ing from the nucleophilic attack on the carbamate is a poorer
leaving group than a phenol. Although carbamate-based FAAH
inhibitors containing an O-(heteroaryl)alkyl moiety have been
reported in the patent literature,[12] to the best of our knowl-
edge, no O-(triazol-4-yl)alkyl carbamate derivatives have been
described to date.
sired compounds (17–30, 32, 33) were synthesized as shown
in Scheme 2.
Aromatic azide 5a was prepared from aniline by a diazota-
tion–azidation protocol,[21] while 5b, 5c, and 6–16 were ob-
tained in good to excellent yields by reacting the correspond-
ing halides with sodium azide.[22] Final compounds 17, 18, and
19 were prepared via click chemistry, starting from prop-2-yn-
1-yl N-cyclohexylcarbamate,[23] prepared by reaction of cyclo-
hexylamine with the commercially available prop-2-ynyl chloro-
formate, and azides 5a, 5b, and 5c, respectively (Scheme 2).
Then, copper catalyzed [3+2] cycloaddition reaction between
azides 5a and 5b with the commercially available but-3-yn-1-
ol, allowed us to obtain compounds 31a and 31b in accepta-
ble yields.[24] Finally, compounds 32 and 33 were prepared by
coupling alcohols 31a and 31b, respectively, with commercial
cyclohexyl isocyanate (Scheme 2). O-(1,2,3-Triazol-4-yl)methyl
carbamate derivatives 20–30 (Scheme 2) were prepared by re-
action of azides 6–16 with prop-2-yn-1-yl N-cyclohexylcarba-
mate, under click chemistry conditions.
A second set of analogues was synthesized, as reported in
Scheme 3, in order to explore region A (Figure 2). Prop-2-ynyl-
N-prop-2-ynyl carbamate,[25] obtained by reaction of the com-
mercially available propargyl amine with prop-2-ynyl chlorofor-
mate, was reacted with aromatic azides 12a and 12b thus af-
fording compounds 34a and 34b, which bear the same sub-
stituent on both aromatic rings (Scheme 3).
Herein, we report the synthesis and characterization of
a series of O-(1,2,3-triazol-4-yl)alkyl carbamates, prepared by
copper-catalyzed [3+2] cycloaddition reaction between azides
and alkynes.[20] The fast and versatile synthesis via click chemis-
try allowed us to prepare a number of analogues in a quick
and reliable manner, and rapidly explore the SAR within this
new class of FAAH inhibitors.
Bis-(triazol-4-yl)methyl carbamates with different substitution
pattern on the aromatic rings were synthesized according to
Scheme 3. Aromatic azides 12a–c were reacted with propargyl
amine, under click chemistry conditions, to give the corre-
sponding aminomethyl triazoles (35a–c), which were subse-
quently coupled with prop-2-ynyl chloroformate to afford N-
(triazol-4-yl)methyl-O-propargyl carbamates 36a–c. The latter
compounds were reacted with aromatic azides 12a–b, under
click chemistry conditions, to afford N-(triazol-4-yl)methyl-O-
(triazol-4-yl)methyl carbamates 37–40.
Results and Discussion
Chemistry
(3-Phenylphenyl)methyl N-cyclo-
hexylcarbamate (4) was pre-
pared from commercially avail-
able 3-phenylbenzoic acid (2) by
lithium aluminum hydride reduc-
tion to 3-phenylbenzyl alcohol
(3) followed by reaction with
commercial cyclohexyl isocya-
nate (Scheme 1).
Scheme 1. Synthesis of (3-phenylphenyl)methyl N-cyclohexylcarbamate (4). Reagents and conditions: a) LiAlH4 (2n
in THF), dry THF, 08C!rt, 2 h; b) cyclohexyl isocyanate, DMAP, dry CH3CN, 808C, 6 h.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
2
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Scheme 2. Synthesis of O-(triazol-4-yl)alkyl carbamates 17–30, 32 and 33. Reagents and conditions: a) prop-2-yn-1-yl N-cyclohexylcarbamate, sodium l-ascor-
bate, CuSO4·5H2O, H2O/t-BuOH (1:1), rt, 3 h; a’) but-3-yn-1-ol, sodium l-ascorbate, CuSO4·5H2O, H2O/t-BuOH (1:1), rt, 3 h; b’) cyclohexyl isocyanate, DMAP, dry
CH3CN, 808C, 5 h.
The compounds bearing a (1,2,4-triazol-3-yl-)methyl moiety,
43 and 46, were synthesized as reported in Scheme 4. Com-
pound 43 was prepared by lithium aluminum hydride reduc-
tion of commercially available 1-phenyl-1,2,4-triazole-3-carbox-
ylic acid 41 followed by reaction with cyclohexyl isocyanate.
Then, commercially available methyl 1H-1,2,4-triazole-3-carbox-
ylate 44 was reacted with benzyl bromide in the presence of
potassium carbonate to obtain compound 45. Reduction by
lithium aluminum hydride followed by reaction with cyclohexyl
isocyanate afforded compound 46.
17, compounds 18–19 and 32–33, to identify the best sub-
stituents at positions 1 and 4 of the triazole ring for FAAH in-
hibition. The results are reported in Table 1.
Replacement of the phenyl group at position 1 of the tria-
zole with a benzyl residue, compound 18 (IC50 =26 nm), led to
an approximate 15-fold increase in potency. It is interesting to
note that this compound was only 4.6-fold less potent than
URB524 (1a). When the methylene linker at position 1 or 4 of
the triazole ring was substituted by an ethylene moiety, as in
compounds 19 and 33, respectively, a drop in potency with re-
spect to 18 was observed. The replacement of the 4-methyl-
ene residue in compound 17 with a 4-ethylene one, leading to
compound 32 (IC50 =1278 nm), resulted in around a threefold
decrease in potency.
Structure–activity relationship (SAR) and stability studies
The compounds were tested for their ability to inhibit the hy-
drolysis of [3H]anandamide by FAAH prepared from rat brain
homogenates. Median inhibitory concentration (IC50) values are
reported in Tables 1–4. In a first attempt to improve the stabili-
ty of O-biphenyl carbamate FAAH inhibitors, we replaced the
O-(3-phenylphenyl) residue of 1a with an O-(3-phenylphenyl)-
methyl group, as in compound 4. This change caused an
almost complete loss of activity, as 4 showed only 65% inhibi-
tion of FAAH activity at 100 mm (Table 1). Interestingly, the sub-
stitution of the O-(3-phenylphenyl)methyl group with an O-(1-
phenyl-1,2,3-triazol-4-yl)methyl residue, as in compound 17
(IC50 =381 nm), recovered the FAAH inhibitory activity. Encour-
aged by this result, we synthesized a set of close analogues of
We then investigated whether 1-phenyl- or 1-benzyl-(1,2,4-
triazol-3yl)methyl moieties work as replacements of the O-(3-
phenylphenyl) residue of 1a and whether they are inter-
changeable with the isomeric 1-phenyl- and 1-benzyl-(1,2,3-tri-
azol-4-yl)methyl residues. Compounds 43 and 46 showed a
dramatic decrease in potency with respect to 1a, and sur-
prisingly, these compounds were much less potent than 17
and 18. We speculate that the loss in potency might be as-
cribed to the different electronic properties of the 1,2,4-triazole
ring with respect to the isomeric 1,2,3-triazole counterpart,
leading to an unfavorable interaction with the active site of
the enzyme.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
3
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
nificantly higher plasma stability than 1a. The latter compound
displayed a half-life of 62 min,[26] but was no longer detectable
after 7 h incubation with rat plasma. In contrast, approximately
90% of the initial amount of compound 17 and 18 was still
detectable after 7 h.
The limited decrease in potency of compound 18 versus 1a,
coupled with its significantly higher plasma stability, prompted
us to explore further this chemical class. We first investigated
region C (Figure 2) by preparing a series of compounds bear-
ing variously substituted benzyl residues at position 1 of the
triazole ring. The results are summarized in Table 2.
Replacement of the benzyl residue in 18 with a benzhydryl
moiety, compound 20 (73% inhibition at 100 mm), or a 2-naph-
thylmethyl group, compound 21 (IC50 =2.0 mm), led to a signifi-
cant decrease in FAAH inhibitory potency, indicating that bulky
arylmethyl groups linked to the triazole are not tolerated, most
likely because of steric clash at the active site of the enzyme.
Interestingly, the nature of the substituent on the phenyl
ring appeared to have a limited effect on the potency of the
compounds as FAAH inhibitors. In fact, benzyl residues bearing
both electron-withdrawing (CN, F, Cl) and electron-donating
(Me, OMe) substituents at the ortho or meta position all led to
low-nanomolar inhibitors. Among them, ortho-methoxybenzyl
derivative 26a showed the highest potency (IC50 =1.4 nm). All
of the para-substituted derivatives were less potent than the
corresponding ortho- or meta-substituted analogues, irrespec-
tive of the electronic properties of the substituent. In particu-
lar, compound 22c (IC50 =2282 nm), bearing a para-cyano
group, showed the highest loss in potency within this subset
of analogues. As for the naphthylmethyl compound (21), we
interpret this finding as the result of an unfavorable steric in-
teraction between the para-substituted phenyl ring and the
active site of the enzyme. Together, from this small series of
derivatives, the rank order of potency ortho->meta->para-
substituted compounds clearly emerged.
Scheme 3. Syntheses of compounds 34a,b and 37–40. Reagents and condi-
tions: a) 12a or 12b, prop-2-yn-1-yl N-(prop-2-yn-1-yl)carbamate, sodium
l-ascorbate, CuSO4·5H2O, H2O/t-BuOH (1:1), rt, 3 h; b) prop-2-yn-1-amine,
sodium l-ascorbate, CuSO4·5H2O, H2O/t-BuOH (1:1), rt, 3 h; c) prop-2-ynyl
chloroformate, Et3N, dry CH2Cl2, 08C, 30 min; d) 12a or 12b, sodium l-ascor-
bate, CuSO4·5H2O, H2O/t-BuOH (1:1), rt, 3 h.
The excellent potency of benzyl derivatives bearing a fluoro
or a methoxyl group at the ortho or meta position led us to
synthesize di-substituted compounds 27–30 to verify whether
any additive effect on potency was observed. With the excep-
tion of 2-fluoro-3-methoxy-derivative 30 (IC50 =44.6 nm), all of
the compounds retained an excellent potency, with IC50 values
in the range 10.4–11.9 nm, but none of them improved signifi-
cantly over the corresponding mono-substituted analogue.
The most potent compound, 26a, was effective at inhibiting
FAAH activity ex vivo. One hour after systemic administration
of 26a (3 mgkgꢀ1, intraperitoneally) to CD1 mice, FAAH activity
measured ex vivo in brain tissue was reduced by 78% (n=3)
with respect to the control.
Scheme 4. Syntheses of (1,2,4-triazol-3-yl)methyl N-cyclohexylcarbamates
43and 46. Reagents and conditions: a) BnBr, K2CO3, dry DMF, 808C, 16 h;
b) LiAlH4 (2n in THF), dry THF, 08C!rt, 2 h; c) cyclohexyl isocyanate, DMAP,
dry CH3CN, 808C, 6 h.
As the next step in the investigation of the SAR of this new
class of FAAH inhibitors, we conducted a preliminary explora-
tion of region A (Figure 2). Previous studies on O-arylcarba-
mates showed that replacement of the cyclohexyl group of 1a
with an arylalkyl moiety led to inhibitors of greater potency.[15a]
Exploiting the click chemistry approach, we replaced the cyclo-
hexyl group with a [1-(methoxybenzyl)triazol-4-yl]methyl resi-
due, as in compounds 34a and 34b, and 37–40. The results
are reported in Table 3.
To test our hypothesis that O-(triazol-4-yl)alkyl carbamate de-
rivatives are more stable than O-aryl carbamates, we compared
the rat plasma stability of compounds 1a, 17 and 18. The re-
sults are reported in Table 1. Consistent with our expectation,
O-(1,2,3-triazol-4-yl)methyl carbamates 17 and 18 showed sig-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
4
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
with respect to 34a. Introduc-
tion of a meta-methoxybenzyl
group at position 1 on both tria-
zolyl rings led to the potent in-
hibitor 34b (IC50 =3.9 nm). Inter-
estingly, a para-methoxybenzyl
group at position 1 of the (tria-
zol-4-yl)methyl moiety in re-
gion A was not detrimental for
potency, as compounds 38 and
40 inhibited FAAH activity with
IC50 values of 7.6 and 5.8 nm, re-
spectively.
Table 1. Fatty acid amide hydrolase (FAAH) inhibitory activity and rat plasma stability of 1a, 4, and O-[1-substi-
tuted-(1,2,3-triazol-4-yl)]alkyl carbamates.
Compd
Structure
IC50 [nm][a]
Plasma
Plasma stability
[% at 7 h][b]
1
t [min]
2
1a
5.7ꢁ1.2
62ꢁ19
0
–
4
65%@100 mm
380.6ꢁ62.9
–
–
The most interesting com-
pounds identified from the SAR
exploration were tested for their
inhibitory activity against human
17
90ꢁ9
(h)-FAAH-1.
A comparison be-
18
26.2ꢁ3.8
–
92ꢁ8
tween the inhibitory potency of
selected compounds on rat (r)-
FAAH versus h-FAAH-1 is report-
ed in Table 4.
The series of substituted O-(1-
benzyltriazol-4-yl)methyl N-cyclo-
hexylcarbamate derivatives re-
sulted to be generally less active
at inhibiting h-FAAH-1 than r-
FAAH, displaying a 14- to 240-
fold drop in potency. The only
exception was 27 (IC50 =3.6 nm),
which showed threefold higher
potency on h-FAAH-1 with re-
spect to r-FAAH. Moreover,
19
32
833ꢁ129
1278ꢁ84
–
–
–
–
33
43
2535ꢁ106
–
–
–
–
ortho-substituted
analogues
70%@100 mm
23a, 24a, and 26a, which dis-
played single-digit nanomolar r-
FAAH inhibition, and 2,6-dibusti-
tuted derivative 28, suffered the
most marked loss of potency:
62, 172, 240, and 73-fold respec-
tively. meta-Substituted ana-
logues 23b, 24b, and 26b, and
3,5-disubstituted derivative 29
were more potent inhibitors
than their ortho-substituted ana-
logues, thus reversing the prefer-
46
51500ꢁ1125
–
–
[a] IC50 values are the mean ꢁSEM (n=3). [b] Percent of parent compound remaining after 7 h incubation.
Compound 34a, bearing an ortho-methoxybenzyl residue on
both triazolyl rings turned out to be the least potent derivative
(IC50 =154 nm). Moving the methoxyl group to the meta posi-
tion of the N-(1-benzyltriazol-4-yl)methyl moiety led to deriva-
tive 37 (IC50 =3.2 nm), which showed a 48-fold increase in po-
tency versus 34a and confirmed as the most potent com-
pound of this small series. Consistent with the previous find-
ing, replacement of the ortho-methoxybenzyl residue at posi-
ence for ortho-substituted benzyl residues observed with
r-FAAH.
By contrast, compounds bearing substituted (1-benzyltriazol-
4-yl)methyl residues at both region A and C (Figure 2) dis-
played a minor decrease in activity on h-FAAH-1 compared to
the N-cyclohexylcarbamate derivatives. Indeed, compounds 37,
38, and 39 showed four- to tenfold lower potency, but re-
tained double-digit nanomolar potency on h-FAAH-1. The
most promising molecules in terms of potency were 34b and
40, which possess IC50 values for h-FAAH-1 inhibition of 4.2 nm
and 9.4 nm, respectively. Interestingly, both of them are charac-
tion 1 of the triazole ring in region C with
a meta-
methoxybenzyl group, as in compound 39 (IC50 =9.8 nm), was
accompanied by an approximate 15-fold increase in potency
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
5
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Table 2. Fatty acid amide hydrolase (FAAH) inhibitory activity of O-(triazol-4-yl)methyl carbamates variously substituted at region C.
Compd
R
IC50 [nm][a]
Compd
R
IC50 [nm][a]
21.5ꢁ1.2
20
73%@100 mm
25a
21
2003ꢁ636
19.5ꢁ4.3
25b
25c
24.5ꢁ6.2
500ꢁ44
22a
22b
22c
23a
21.5ꢁ7.9
2282ꢁ585
9.9ꢁ1.9
26a
26b
26c
1.4ꢁ0.2
11.8ꢁ3.7
627ꢁ173
23b
23c
24a
12.1ꢁ0.8
95.5ꢁ22.2
6.2ꢁ1.3
27
28
10.5ꢁ2.9
11.9ꢁ1.0
29
30
10.4ꢁ2.6
24b
24c
13.2ꢁ2.8
215ꢁ31
44.6ꢁ10.3
[a] IC50 values are the mean ꢁSEM (n=3).
terized by a meta-methoxybenzyl group on region C (Figure 2),
suggesting that the binding site on h-FAAH-1 prefers meta-
substituents on that benzyl group.
by compounds 27, 34b, and 40 is in agreement with previous
observations with O-arylcarbamates 1a and 1b.[28]
Finally, compounds 27, 34b, and 40 were further character-
ized by determining their rat plasma and mouse liver microso-
mal (MLM) stability, and their kinetic solubility in buffer. A com-
parison of the overall profile of O-arylcarbamate 1a with com-
pounds 27, 34b, and 40 is reported in Table 5.
The selected O-[(1-benzyltriazol-4-yl)methyl]carbamate deriv-
atives 27, 34b, and 40 showed FAAH inhibitory activity and
MLM stability comparable to those of O-arylcarbamate 1a.
The most potent compounds against both rat and human
FAAH, that is, 27, 34b, and 40, were tested for their selectivity
versus monoacylglycerol lipase (MGL), a serine hydrolase that
inactivates the endocannabinoid 2-arachidonoylglycerol (2-
AG).[27] None of the compounds inhibited MGL activity when
tested at concentrations up to 100 mm (Figure S1 in the Sup-
porting Information). The selective inhibition of FAAH activity
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
6
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
(click chemistry). Exploiting the
same chemistry, we also synthe-
sized carbamates bearing a sub-
Table 3. Fatty acid amide hydrolase (FAAH) inhibitory activity of N-(triazol-4-yl)methyl O-(triazol-4-yl)methyl car-
bamates 34a–40
Compd
Structure
IC50 [nm][a]
stituted
(1-benzyltriazol-4-yl)-
methyl moiety at both the O
and N end. The click chemistry
approach allowed us rapidly to
explore the structure–activity re-
lationships within the class. Sev-
eral single-digit nanomolar in-
hibitors of rat FAAH were ob-
tained, including potent deriva-
tive 26a, which showed an IC50
value of 1.4 nm and inhibited
brain FAAH activity in vivo.
Some of these compounds po-
tently inhibited human FAAH-1.
In particular, compounds 34b
and 40, bearing a [1-[(methoxy-
phenyl)methyl]triazol-4-yl]methyl
group at both the O and N end
of the carbamate function, dis-
played single-digit nanomolar
IC50 values for both rat and
human FAAH. In addition, they
showed a remarkable improve-
ment in rat plasma stability and
kinetic solubility in buffer with
respect to URB524 (1a).
The dramatic decrease in
FAAH inhibitory activity of 1-
phenyl- or 1-benzyl-substituted
O-(1,2,4-triazol-3-yl)mehtyl carba-
mates 43 and 46 demonstrated
that the 1-substituted-(1,2,3-tria-
zol-4-yl)methyl core structure,
easily accessible by click chemis-
try, was essential for obtaining
potent inhibition of FAAH activi-
ty.
34a
154ꢁ14
34b
3.9ꢁ0.7
3.2ꢁ1.2
7.6ꢁ0.6
9.8ꢁ0.1
5.8ꢁ0.6
37
38
39
40
[a] IC50 values are the mean ꢁSEM (n=3).
In
conclusion,
exploiting
a click chemistry approach, we
However, they displayed much higher rat plasma stability than
1a, as approximately 90% of the initial amount of the com-
pounds was still present after 7 h incubation with rat plasma.
Moreover, with the exception of compound 27, the kinetic sol-
ubility in buffer also improved significantly.
prepared a novel series of potent and drug-like FAAH inhibi-
tors containing an O-(1,2,3-triazol-4-yl)alkyl carbamate moiety.
The compounds described in the present study represent
a promising starting point for the development of new FAAH
inhibitors with improved drug-like properties.
Conclusions
Experimental Section
In the present study, we report the synthesis and characteriza-
tion of O-(1,2,3-triazol-4-yl)alkyl carbamates as a novel class of
FAAH inhibitors. In these compounds, an O-(triazol-4-yl)methyl
group replaces the O-aryl moiety of known and potent FAAH
inhibitors such as compound 1a (URB524) and 1b (URB597). A
number of compounds were prepared by copper-catalyzed
[3+2] cycloaddition reactions between azides and alkynes
Chemistry
Chemicals, materials and methods: Solvents and reagents were
obtained from commercial suppliers and were used without further
purification. Automated column chromatography purifications
were performed by using a Teledyne ISCO apparatus (CombiFlash
Rf) with prepacked silica gel (SiO2) columns of different sizes (4–
120 g). Mixtures of increasing polarity of Cy and EtOAc or CH2Cl2
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
7
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
dC =77.16 ppm; [D6]DMSO: dH =2.50 ppm and dC =39.52 ppm).
UPLC/MS analyses were run on a Waters ACQUITY UPLC/MS system
consisting of a single quadropole detector (SQD) mass spectrome-
ter (MS) equipped with an electrospray ionization (ESI) interface
and a PDA detector. The PDA range was 210–400 nm. ESI in both
the positive and negative mode was applied. Mobile phases:
(A) 10 mm NH4OAc in H2O, pH 5; (B) 10 mm NH4OAc in MeCN/H2O
(95:5), pH 5. Analyses were performed either with method A or B.
Table 4. Comparison of rat versus human fatty acid amide hydrolase
(FAAH)-1 inhibitory activity of selected compounds.
Compd
IC50 [nm][a]
r-FAAH
h-FAAH
1a
5.7ꢁ1.2
9.9ꢁ1.9
12.1ꢁ0.8
6.2ꢁ1.3
13.2ꢁ2.8
1.4ꢁ0.2
11.8ꢁ3.7
10.5ꢁ2.9
11.9ꢁ1.0
10.4ꢁ2.6
3.9ꢁ0.7
3.2ꢁ1.2
7.6ꢁ0.6
9.8ꢁ0.4
5.8ꢁ0.6
3.1ꢁ1.3
619ꢁ138
180ꢁ6
23a
23b
24a
24b
26a
26b
27
28
29
34b
37
38
Method A: gradient 5!95% B over 3 min; flow rate 0.5 mLminꢀ1
;
1068ꢁ144
190ꢁ8
temperature: 408C. Pre-column: Vanguard BEH C18 (1.7 mm, 2.1ꢁ
5 mm). Column: BEH C18 (1.7 mm, 2.1ꢁ50 mm). Method B: gradi-
ent: 50!100% B over 3 min, flow rate 0.5 mLminꢀ1; temperature:
408C. Pre-column: Vanguard BEH C18 (1.7 mm, 2.1ꢁ5 mm). Column:
BEH C18 (1.7 mm, 2.1ꢁ50 mm). High-resolution mass spectrometry
(HRMS) was performed on a Synapt G2 Quadrupole-Tof Instrument
(Waters, USA), equipped with an ESI ion source.
337ꢁ101
158ꢁ12
3.6ꢁ0.9
812ꢁ105
216ꢁ6
4.2ꢁ1.0
30.4ꢁ3.7
30.7ꢁ6.2
39.1ꢁ0.6
9.4ꢁ2.0
All final compounds (4, 17–30, 32, 33, 34a,b, 37–40, 43 and 46)
showed ꢂ95% purity by NMR and UPLC/MS analysis. The synthe-
ses of reaction intermediates 3, 5a–c, 6–16, 31a--b, 35a–c, 36a–c,
41, 42a,b, and 45 are described in the Supporting Information.
39
40
[a] IC50 values are the mean ꢁSEM (n=3).
Abbreviations: acetonitrile (CH3CN), benzyl bromide (BnBr), cyclo-
hexane (Cy), dichloromethane (CH2Cl2), diethyl ether (Et2O), 4-(di-
methylamino)-pyridine (DMAP), ethanol (EtOH), ethyl acetate
(EtOAc), hydrochloric acid (HCl), methanol (MeOH), N,N’-dimethyl-
formamide (DMF), room temperature (rt), sodium sulfate (Na2SO4),
sodium bicarbonate (NaHCO3), sulfuric acid (H2SO4), tert-butanol (t-
BuOH), tetrahydrofuran (THF), triethylamine (Et3N).
and MeOH were used as eluents. NMR experiments were run on
a Bruker Avance III 400 system (400.13 MHz for H; 100.62 MHz for
1
13C), equipped with a BBI probe and Z-gradients. Spectra were ac-
quired at 300 K, using deuterated dimethylsulfoxide ([D6]DMSO) or
deuterated chloroform (CDCl3) as solvents. Chemical shifts (d) for
1H and 13C spectra are reported in parts per million (ppm) relative
to the residual nondeuterated solvent (CDCl3: dH =7.26 ppm and
General procedure (1) for the synthesis of triazoles 17–30, 37–
40: Ethynyl derivative (1 equiv) and azido compound (1 equiv)
Table 5. A comparison of the overall profile of O-arylcarbamate 1a with compounds 27, 34b, and 40.
Compd
Structure
IC50 [nm][a]
r-FAAH
Plasma stab.
[% at 7 h][b]
MLM stab.
Kinetic sol.
[c]
h-FAAH
3.1ꢁ1.3
t [min]
[mm][d]
1
2
1a
5.7ꢁ1.2
0
<5ꢁ0
6
27
10.5ꢁ2.9
3.6ꢁ0.9
91ꢁ3
5ꢁ0
18
34b
3.9ꢁ0.7
5.8ꢁ0.6
4.2ꢁ1.0
9.4ꢁ2.0
88ꢁ11
89ꢁ9
6 ꢁ0
8ꢁ1
174
152
40
[a] IC50 values for FAAH inhibition are the mean ꢁSEM (n=3); [b] Plasma stability; data are the mean ꢁSEM (n=3); [c] Half-life in mouse liver microsomes
(MLM); data are the mean ꢁSD (n=3); [d] Phosphate-buffered saline (pH 7.4), data represent a single determination.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
8
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
were suspended in a solution of water/t-BuOH (1:1). A freshly pre-
pared 1m aq solution sodium ascorbate (0.1 equiv) was added, fol-
lowed by the addition of CuSO4·5H2O (0.01 equiv). The resulting re-
action was vigorously stirred for 3 h at rt. The reaction mixture was
then diluted with water, cooled on ice, and the precipitate was col-
lected by filtration. When addition of water failed to precipitate
the desired triazole, evaporation of the solvent allowed the recov-
ery of the crude product. Purification was performed by column
chromatography.
1.53 (dt, J=12.7, 3.8 Hz, 1H), 1.15 ppm (m, 5H); 13C NMR (100 MHz,
[D6]DMSO): d=155.4, 143.5, 136.5, 129.2 (2C), 128.6, 128.4 (2C),
125.0, 57.2, 53.2, 49.9, 33.0 (2C), 25.6, 25.0 ppm (2C); UPLC-MS:
Method A, tR =2.37 min, ionization: m/z 315 [M+H]+; HRMS-ESI:
m/z [M+H]+ calcd for C17H22N4O2: 315.1821, found: 315.1826.
(1-Phenethyltriazol-4-yl)methyl N-cyclohexylcarbamate (19): The
reaction was carried out following general procedure (1), using
prop-2-ynyl N-cyclohexylcarbamate (0.15 g, 0.82 mmol), 2-azido-
ethylbenzene (0.12 g, 0.82 mmol), sodium ascorbate (0.016,
0.08 mmol), and CuSO4·5H2O (0.002 g, 0.008 mmol) in water/
t-BuOH (1:1; 3 mL). Purification was performed by flash chromato-
graphy (MeOH/CH2Cl2, 0!2%) to afford 19 as a white powder
General procedure (2) for the synthesis of double triazoles car-
bamates 34a,b: Ethynyl derivative (0.5 equiv) and azido com-
pound (1 equiv) were suspended in a solution of water/t-BuOH
1
(1:1).
A
freshly prepared 1m aq solution sodium ascorbate
(0.135 g; 50%): H NMR (400 MHz, [D6]DMSO): d=8.03 (s, 1H), 7.29
(0.1 equiv) was added, followed by the addition of CuSO4·5H2O
(0.01 equiv). The resulting reaction was vigorously stirred for 8 h at
rt. Afterwards, evaporation of the solvent allowed the recovery of
the crude product. Purification was performed by column chroma-
tography.
(m, 2H), 7.21 (m, 3H), 7.13 (d, J=7.9 Hz, 1H), 4.99 (s, 2H), 4.61 (dd,
J=7.9, 6.8 Hz, 2H), 3.24 (m, 1H), 3.16 (t, J=7.4 Hz, 2H), 1.69 (m,
4H), 1.54 (d, J=13.3 Hz, 1H), 1.18 ppm (m, 5H); 13C NMR (100 MHz,
[D6]DMSO): d=154.6, 142.5, 137.5, 128.6 (2C), 128.3 (2C), 126.5,
124.4, 56.6, 50.3, 49.4, 35.6, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-
MS: Method A, tR =2.44 min, ionization: m/z 329 [M+H]+; HRMS-
ESI: m/z [M+H]+ calcd for C18H24N4O2: 329.1978, found: 329.1982.
(3-Phenylphenyl)methyl N-cyclohexylcarbamate (4): (3-Phenyl-
phenyl)methanol (3, 0.125 g, 0.68 mmol) was dissolved in dry
CH3CN (5 mL) while stirring at rt. Then, DMAP (0.08 g, 0.68 mmol)
and cyclohexyl isocyanate (0.09 g, 0.75 mmol) were added, and the
reaction mixture was stirred at 808C for 3 h. Afterwards, the reac-
tion mixture was diluted with EtOAc (20 mL) and washed once
with 2n aq HCl (20 mL), and once with brine (20 mL). The organic
layer was dried over Na2SO4, filtered and concentrated in vacuo.
The crude product was purified by chromatography (MeOH/CH2Cl2,
0!2%) to afford 4 as a white powder (0.13 g; 61%): 1H NMR
(400 MHz, [D6]DMSO): d=7.69–7.61 (m, 3H), 7.60 (dt, J=7.9,
1.4 Hz, 1H), 7.47 (q, J=7.3 Hz, 3H), 7.42–7.31 (m, 2H), 7.19 (d, J=
7.9 Hz, 1H), 5.07 (s, 2H), 3.31–3.22 (m, 1H), 1.76 (dd, J=12.6,
3.6 Hz, 2H), 1.67 (dq, J=12.5, 3.7 Hz, 2H), 1.53 (dq, J=11.5, 3.7 Hz,
1H), 1.31–1.00 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=
155.2, 140.2, 139.9, 138.0, 128.9, 128.9, 127.5, 126.7, 126.6, 126.0
(2C), 65.0, 49.5, 32.6 (2C), 25.1, 24.6 ppm (2C); UPLC-MS: Method B,
tR =1.97 min, ionization: m/z 310 [M+H]+; HRMS-ESI: m/z
[M+Na]+ calcd for C20H23NO2Na: 332.1626, found: 332.1622.
(1-Benzhydryltriazol-4-yl)methyl N-cyclohexylcarbamate (20):
The reaction was carried out following general procedure (1), using
prop-2-ynyl N-cyclohexylcarbamate (0.15 g, 0.82 mmol), [azido-
(phenyl)methyl]benzene (0.17 g, 0.82 mmol), sodium ascorbate
(0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g, 0.008 mmol) in
water/t-BuOH (1:1; 3 mL). Purification was performed by flash chro-
matography (EtOAc/Cy, 0!40%) to afford 20 as a white powder
1
(0.23 g; 71%): H NMR (400 MHz, [D6]DMSO): d=8.08 (s, 1H), 7.39
(m, 6H), 7.31 (s, 1H), 7.21 (m, 4H), 7.14 (d, J=7.9 Hz, 1H), 5.02 (s,
2H), 3.22 (m, 1H), 1.67 (m, 4H), 1.52 (d, J=12.5 Hz, 1H), 1.12 ppm
(m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=155.4, 144.3, 138.6 (2C),
128.7 (4C), 128.2 (2C), 127.9 (4C), 66.5, 56.7, 49.4, 32.5 (2C), 25.1,
24.5 ppm (2C); UPLC-MS: Method A, tR =2.81 min, ionization: m/z
391 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C23H26N4O2:
391.2134, found: 391.2132.
[1-(2-Naphthylmethyl)triazol-4-yl]methyl N-cyclohexylcarbamate
(21): The reaction was carried out following general procedure (1),
using prop-2-ynyl N-cyclohexylcarbamate (0.15 g, 0.82 mmol), 2-
(azidomethyl)naphthalene (0.15 g, 0.82 mmol), sodium ascorbate
(0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g, 0.008 mmol) in
water/t-BuOH (1:1; 3 mL). Purification was performed by flash chro-
matography (MeOH/CH2Cl2, 0!2%) to afford 21 as a white
powder (0.16 g; 54%): 1H NMR (400 MHz, [D6]DMSO): d=8.19 (s,
1H), 7.92 (m, 3H), 7.86 (s, 1H), 7.54 (m, 2H), 7.44 (dd, J=8.5,
1.6 Hz, 1H), 7.10 (d, J=7.8 Hz, 1H), 5.76 (s, 2H), 5.01 (s, 2H), 3.22
(m, 1H), 1.66 (dd, J=26.8, 12.6 Hz, 4H), 1.51 (d, J=12.5 Hz, 1H),
1.11 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=154.9, 143.0,
133.4, 132.7, 132.4, 128.4, 127.7, 127.5, 126.9, 126.5, 126.4, 125.7,
124.6, 56.7, 52.9, 49.4, 32.5 (2C), 25.1, 24.5 ppm (2C); UPLC-MS:
Method A, tR =2.65 min, ionization: m/z 365 [M+H]+; HRMS-ESI:
m/z [M+H]+ calcd for C21H24N4O2: 365.1978, found: 365.1975.
(1-Phenyltriazol-4-yl)methyl N-cyclohexylcarbamate (17): The re-
action was carried out following general procedure (1), using prop-
2-ynyl N-cyclohexylcarbamate (0.23 g, 1.26 mmol), azidobenzene
(0.15 g, 1.26 mmol), sodium ascorbate (0.02, 0.12 mmol), and
CuSO4·5H2O (0.003 g, 0.01 mmol) in water/t-BuOH (1:1; 3 mL). Pu-
rification was performed by flash chromatography (MeOH/CH2Cl2,
0!2%) to afford 17 as a white powder (0.22 g; 59%): 1H NMR
(400 MHz, [D6]DMSO): d=8.82 (s, 1H), 7.91 (m, 2H), 7.61 (m, 2H),
7.52 (m, 1H), 7.20 (d, J=8.0 Hz, 1H), 5.13 (s, 2H), 3.28 (m, 1H), 1.76
(m, 2H), 1.67 (dt, J=12.2, 3.8 Hz, 2H), 1.54 (m, 1H), 1.18 ppm (m,
5H); 13C NMR (100 MHz, [D6]DMSO): d=155.4, 144.3, 137.0, 130.3
(2C), 129.2, 123.2, 120.6 (2C), 57.0, 50.0, 33.1 (2C), 25.6, 25.0 ppm
(2C); UPLC-MS: Method A, tR =2.42 min, ionization: m/z 301
[M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C16H20N4O2: 301.1665,
found: 301.1666.
[1-[(2-Cyanophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (22a): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 2-(azidomethyl)benzonitrile (0.13 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
(1-Benzyltriazol-4-yl)methyl N-cyclohexylcarbamate (18): The re-
action was carried out following general procedure (1), using prop-
2-ynyl N-cyclohexylcarbamate (0.15 g, 0.82 mmol), azidomethylben-
zene (0.11 g, 0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and
CuSO4·5H2O (0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Pu-
rification was performed by flash chromatography (EtOAc/Cy, 0!
50%) to afford 18 as a white powder (0.18 g; 71%): 1H NMR
(400 MHz, [D6]DMSO): d=8.15 (s, 1H), 7.35 (m, 5H), 7.13 (d, J=
7.9 Hz, 1H), 5.60 (s, 2H), 5.02 (s, 2H), 3.25 (m, 1H), 1.69 (m, 4H),
22a as
a
white powder (0.15 g; 53%): 1H NMR (400 MHz,
[D6]DMSO): d=8.19 (s, 1H), 7.92 (dd, J=7.7, 1.0 Hz, 1H), 7.72 (td,
J=7.7, 1.2 Hz, 1H), 7.57 (td, J=7.7, 0.9 Hz, 1H), 7.36 (d, J=7.8 Hz,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
9
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
1H), 7.14 (d, J=7.8 Hz, 1H), 5.81 (s, 2H), 5.03 (s, 2H), 3.25 (m, 1H),
1.68 (dd, J=27.0, 12.5 Hz, 4H), 1.53 (d, J=12.7 Hz, 1H), 1.13 ppm
(dtd, J=31.1, 24.1, 12.1 Hz, 5H); 13C NMR (100 MHz, [D6]DMSO): d=
154.9, 143.1, 138.7, 133.8, 133.3, 129.4, 129.2, 125.0, 116.9, 111.2,
56.6, 50.9, 49.4, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A,
tR =2.25 min, ionization: m/z 340 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C18H21N5O2: 340.1773, found: 340.1779.
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (EtOAc/Cy, 0!50%) to afford
23b as
a
white powder (0.17 g; 63%): 1H NMR (400 MHz,
[D6]DMSO): d=8.18 (s, 1H), 7.42 (m, 1H), 7.16 (m, 4H), 5.62 (s, 2H),
5.01 (s, 2H), 3.24 (m, 1H), 1.68 (dd, J=26.2, 12.5 Hz, 4H), 1.52 (d,
J=12.5 Hz, 1H), 1.12 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO):
d=162.5 (d, J=244.3 Hz), 155.4, 143.6, 139.2, 131.3 (d, J=8.3 Hz),
125.2, 124.4 (d, J=2.7 Hz), 115.3 (m, 2C), 57.1, 52.5, 49.9, 33.0 (2C),
25.6, 25.0 ppm (2C); UPLC-MS: Method A, tR =2.40 min, ionization:
m/z 333 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C17H21FN4O2:
333.1727, found: 333.1731.
[1-[(3-Cyanophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (22b): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 3-(azidomethyl)benzonitrile (0.13 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
[1-[(4-Fluorophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (23c): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 1-(azidomethyl)-4-fluorobenzene (0.12 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
22b as
a
white powder (0.17 g; 62%): 1H NMR (400 MHz,
[D6]DMSO): d=8.21 (s, 1H), 7.82 (m, 2H), 7.62 (m, 2H), 7.12 (d, J=
7.8 Hz, 1H), 5.67 (s, 2H), 5.01 (s, 2H), 3.24 (m, 1H), 1.68 (dd, J=
25.4, 12.5 Hz, 4H), 1.52 (d, J=12.6 Hz, 1H), 1.13 ppm (m, 5H);
13C NMR (100 MHz, [D6]DMSO): d=154.9, 143.2, 137.5, 132.9, 131.9,
131.6, 130.0, 124.8, 118.3, 111.6, 56.6, 51.7, 49.4, 32.6 (2C), 25.1,
24.5 ppm (2C); UPLC-MS: Method A, tR =2.25 min, ionization: m/z
340 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C18H21N5O2:
340.1773, found: 340.1781.
23c as
a
white powder (0.13 g; 49%): 1H NMR (400 MHz,
[D6]DMSO): d=8.14 (s, 1H), 7.38 (ddd, J=8.4, 5.3, 2.5 Hz, 2H), 7.20
(m, 2H), 7.12 (d, J=7.5 Hz, 1H), 5.58 (s, 2H), 5.00 (s, 2H), 3.23 (m,
1H), 1.67 (m, 4H), 1.52 (d, J=12.5 Hz, 1H), 1.11 ppm (m, 5H);
13C NMR (100 MHz, [D6]DMSO): d=161.8 (d, J=244.5 Hz), 154.9,
143.0, 132.2, 130.2 (d, J=8.4 Hz), 124.5, 115.5 (d, J=21.6 Hz), 56.7,
51.9, 49.4, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =
2.39 min, ionization: m/z 333 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C17H21FN4O2: 333.1727, found: 333.1731.
[1-[(4-Cyanophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (22c): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 4-(azidomethyl)benzonitrile (0.13 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
[1-[(2-Chlorophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (24a): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 1-(azidomethyl)-2-chlorobenzene (0.14 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
22c as
a
white powder (0.21 g; 77%): 1H NMR (400 MHz,
[D6]DMSO): d=8.21 (s, 1H), 7.86 (d, J=8.2 Hz, 2H), 7.45 (d, J=
8.2 Hz, 2H), 7.14 (d, J=7.8 Hz, 1H), 5.72 (s, 2H), 5.02 (s, 2H), 3.24
(m, 1H), 1.69 (dd, J=25.1, 12.6 Hz, 4H), 1.53 (d, J=12.4 Hz, 1H),
1.14 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=154.9, 143.2,
141.4, 132.7 (2C), 128.6 (2C), 124.9, 118.4, 110.9, 56.6, 52.1, 49.4,
32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =2.22 min,
ionization: m/z 340 [M+H]+; HRMS-ESI: m/z [M+Na]+ calcd for
C18H21N5O2Na: 362.1593, found: 362.1594.
24a as
a
white powder (0.19 g; 68%): 1H NMR (400 MHz,
[D6]DMSO): d=8.11 (s, 1H), 7.52 (dd, J=7.7, 1.5 Hz, 1H), 7.38 (m,
2H), 7.22 (dd, J=7.4, 1.7 Hz, 1H), 7.14 (d, J=7.7 Hz, 1H), 5.70 (s,
2H), 5.02 (s, 2H), 3.24 (m, 1H), 1.68 (dd, J=26.2, 12.5 Hz, 4H), 1.53
(m, 1H), 1.11 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=
154.9, 142.9, 133.2, 132.6, 130.4, 130.2, 129.6, 127.6, 124.9, 56.7,
50.5, 49.4, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =
2.49 min, ionization: m/z 349 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C17H21ClN4O2: 349.1431, found: 349.1435.
[1-[(2-Fluorophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (23a): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 1-(azidomethyl)-2-fluorobenzene (0.12 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
[1-[(3-Chlorophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (24b): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 1-(azidomethyl)-3-chlorobenzene (0.14 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (EtOAc/Cy, 0!50%) to afford
23a as
a
white powder (0.17 g; 61%): 1H NMR (400 MHz,
[D6]DMSO): d=8.11 (s, 1H), 7.42 (m, 1H), 7.34 (td, J=7.6, 1.4 Hz,
1H), 7.23 (m, 2H), 7.13 (d, J=7.8 Hz, 1H), 5.66 (s, 2H), 5.00 (s, 2H),
3.24 (m, 1H), 1.67 (m, 4H), 1.52 (d, J=12.5 Hz, 1H), 1.12 ppm (m,
5H); 13C NMR (100 MHz, [D6]DMSO): d=160.0 (d, J=246.7 Hz),
155.0, 143.0, 130.7 (d, J=4.6 Hz), 130.7, 124.8 (d, J=3.4 Hz), 124.7,
122.8 (d, J=14.7 Hz), 115.6 (d, J=20.8 Hz), 56.7, 49.4, 46.8 (d, J=
3.7 Hz), 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =
2.37 min, ionization: m/z 333 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C17H21FN4O2: 333.1727, found: 333.1732.
24b as
a
white powder (0.24 g; 83%): 1H NMR (400 MHz,
[D6]DMSO): d=8.19 (s, 1H), 7.40 (dd, J=6.1, 2.3 Hz, 3H), 7.27 (dq,
J=5.9, 2.8 Hz, 1H), 7.13 (d, J=7.9 Hz, 1H), 5.61 (s, 2H), 5.01 (s, 2H),
3.25 (m, 1H), 1.68 (dd, J=26.4, 12.4 Hz, 4H), 1.52 (d, J=12.5 Hz,
1H), 1.13 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=154.9,
143.1, 138.4, 133.2, 130.6, 128.1, 127.8, 126.6, 124.7, 56.6, 51.9, 49.4,
32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =2.55 min,
ionization: m/z 349 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for
C17H21ClN4O2: 349.1431, found: 349.1436.
[1-[(3-Fluorophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (23b): The reaction was carried out following general pro-
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 1-(azidomethyl)-3-fluorobenzene (0.12 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
[1-[(4-Chlorophenyl)methyl]triazol-4-yl]methyl N-cyclohexylcar-
bamate (24c): The reaction was carried out following general pro-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&10
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
cedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol), 1-(azidomethyl)-4-chlorobenzene (0.14 g, 0.82 mmol),
sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O (0.002 g,
0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was per-
formed by flash chromatography (EtOAc/Cy, 0!50%) to afford
procedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol),
1-(azidomethyl)-2-methoxybenzene
(0.13 g,
0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O
(0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (MeOH/CH2Cl2, 0!2%) to
afford 26a as a white powder (0.18 g; 64%): 1H NMR (400 MHz,
[D6]DMSO): d=7.98 (s, 1H), 7.35 (m, 1H), 7.09 (m, 3H), 6.93 (m,
1H), 5.52 (s, 2H), 4.99 (s, 2H), 3.82 (s, 3H), 3.23 (m, 1H), 1.67 (m,
4H), 1.52 (d, J=12.6 Hz, 1H), 1.12 ppm (m, 5H); 13C NMR (100 MHz,
[D6]DMSO): d=156.8, 155.0, 142.7, 130.0, 129.6, 124.6, 123.5, 120.5,
111.2, 56.7, 55.5, 49.4, 48.2, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-
MS: Method A, tR =2.43 min, ionization: m/z 345 [M+H]+; HRMS-
ESI: m/z [M+H]+ calcd for C18H24N4O3: 345.1927, found: 345.1930.
24c as
a
white powder (0.13 g; 47%): 1H NMR (400 MHz,
[D6]DMSO): d=8.15 (s, 1H), 7.44 (m, 2H), 7.33 (d, J=8.5 Hz, 2H),
7.12 (d, J=7.8 Hz, 1H), 5.60 (s, 2H), 5.00 (s, 2H), 3.23 (m, 1H), 1.67
(m, 4H), 1.52 (m, 1H), 1.11 ppm (m, 5H); 13C NMR (100 MHz,
[D6]DMSO): d=154.9, 143.1, 135.0, 132.8, 129.8 (2C), 128.7 (2C),
124.6, 56.6, 51.9, 49.4, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS:
Method A, tR =2.55 min, ionization: m/z 349 [M+H]+; HRMS-ESI:
m/z [M+H]+ calcd for C17H21ClN4O2: 349.1431, found: 349.1427.
[1-(o-Tolylmethyl)triazol-4-yl]methyl
N-cyclohexylcarbama-
[1-[(3-Methoxyphenyl)methyl]triazol-4-yl]methyl N-cyclohexyl-
carbamate (26b): The reaction was carried out following general
procedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
te (25a): The reaction was carried out following general procedure
(1), using prop-2-ynyl N-cyclohexylcarbamate (0.37 g, 2.04 mmol),
1-(azidomethyl)-2-methylbenzene (0.3 g, 2.04 mmol), sodium ascor-
bate (0.040, 0.2 mmol), and CuSO4·5H2O (0.005 g, 0.02 mmol) in
water/t-BuOH (1:1; 3 mL). Purification was performed by flash chro-
matography (MeOH/CH2Cl2, 0!2%) to afford 25a as a white
powder (0.13 g; 47%): 1H NMR (400 MHz, [D6]DMSO): d=8.03 (s,
1H), 7.29–7.12 (m, 4H), 7.07 (d, J=7.5 Hz, 1H), 5.60 (s, 2H), 5.00 (s,
2H), 3.22 (dt, J=10.7, 5.8 Hz, 1H), 2.30 (s, 3H), 1.82–1.58 (m, 4H),
1.52 (d, J=12.5 Hz, 1H), 1.31–0.95 ppm (m, 5H); 13C NMR
(100 MHz, [D6]DMSO): d=155.4, 143.3, 136.7, 134.5, 130.8, 129.1,
128.8, 126.7, 125.1, 57.2, 51.3, 49.9, 33.0 (2C), 25.5, 25.0 (2C),
19.0 ppm; UPLC-MS: Method A, tR =2.5 min, ionization: m/z 329
[M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C18H24N4O2: 329.1978,
found: 329.1977.
0.82 mmol),
1-(azidomethyl)-3-methoxybenzene
(0.13 g,
0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O
(0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (EtOAc/Cy, 0!70%) to afford
26b as
a
white powder (0.14 g; 50%): 1H NMR (400 MHz,
[D6]DMSO): d=8.14 (s, 1H), 7.28 (td, J=7.5, 1.8 Hz, 1H), 7.12 (d, J=
7.8 Hz, 1H), 6.90 (d, J=6.2 Hz, 2H), 6.85 (d, J=7.8 Hz, 1H), 5.55 (s,
2H), 5.00 (s, 2H), 3.73 (s, 3H), 3.24 (d, J=7.6 Hz, 1H), 1.68 (dd, J=
25.7, 12.5 Hz, 4H), 1.52 (d, J=12.4 Hz, 1H), 1.13 ppm (m, 5H);
13C NMR (100 MHz, [D6]DMSO): d=159.4, 154.9, 143.0, 137.4, 129.8,
124.6, 119.9, 113.7, 113.4, 56.7, 55.0, 52.6, 49.4, 32.6 (2C), 25.1,
24.5 ppm (2C); UPLC-MS: Method A, tR =2.38 min, ionization: m/z
345 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C18H24N4O3:
345.1927, found: 345.1929.
[1-(m-Tolylmethyl)triazol-4-yl]methyl
N-cyclohexylcarbamate
(25b): The reaction was carried out following general procedure
(1), using prop-2-ynyl N-cyclohexylcarbamate (0.37 g, 2.04 mmol),
1-(azidomethyl)-3-methylbenzene (0.3 g, 2.04 mmol), sodium ascor-
bate (0.040, 0.2 mmol), and CuSO4·5H2O (0.005 g, 0.02 mmol) in
water/t-BuOH (1:1; 3 mL). Purification was performed by flash chro-
matography (MeOH/CH2Cl2, 0!2%) to afford 25b as a white
powder (0.17 g; 65%): 1H NMR (400 MHz, [D6]DMSO): d=8.12 (s,
1H), 7.25 (t, J=7.8 Hz, 1H), 7.13 (m, 4H), 5.54 (s, 2H), 5.00 (s, 2H),
3.24 (m, 1H), 2.28 (s, 3H), 1.68 (dd, J=25.9, 12.5 Hz, 4H), 1.52 (d,
J=12.5 Hz, 1H), 1.12 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO):
d=154.9, 142.9, 137.9, 135.8, 128.7, 128.6, 128.5, 125.0, 124.5, 56.7,
52.7, 49.4, 32.5 (2C), 25.1, 24.5 (2C), 20.8 ppm; UPLC-MS: Method A,
tR =2.52 min, ionization: m/z 329 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C18H24N4O2: 329.1978, found: 329.1981.
[1-[(4-Methoxyphenyl)methyl]triazol-4-yl]methyl N-cyclohexyl-
carbamate (26c): The reaction was carried out following general
procedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol),
1-(azidomethyl)-4-methoxybenzene
(0.13 g,
0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O
(0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (EtOAc/Cy, 0!60%) to afford
26c as
a
white powder (0.15 g; 54%): 1H NMR (400 MHz,
[D6]DMSO): d=8.08 (s, 1H), 7.29 (d, J=8.6 Hz, 2H), 7.11 (d, J=
7.8 Hz, 1H), 6.92 (d, J=8.6 Hz, 2H), 5.50 (s, 2H), 4.99 (s, 2H), 3.73
(s, 3H), 3.22 (m, 1H), 1.68 (dd, J=25.3, 12.6 Hz, 4H), 1.52 (d, J=
12.6 Hz, 1H), 1.13 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=
159.1, 154.9, 142.9, 129.5 (2C), 127.9, 124.2, 114.0 (2C), 56.7, 55.1,
52.2, 49.4, 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =
2.35 min, ionization: m/z 345 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C18H24N4O3: 345.1927, found: 345.1924.
[1-(p-Tolylmethyl)triazol-4-yl]methyl
N-cyclohexylcarbamate
(25c): The reaction was carried out following general procedure
(1), using prop-2-ynyl N-cyclohexylcarbamate (0.37 g, 2.04 mmol),
1-(azidomethyl)-4-methylbenzene (0.3 g, 2.04 mmol), sodium ascor-
bate (0.040, 0.2 mmol), and CuSO4·5H2O (0.005 g, 0.02 mmol) in
water/t-BuOH (1:1; 3 mL). Purification was performed by flash chro-
matography (MeOH/CH2Cl2, 0!2%) to afford 25c as a white
powder (0.18 g; 67%): 1H NMR (400 MHz, [D6]DMSO): d=8.10 (s,
1H), 7.28–7.08 (m, 5H), 5.53 (s, 2H), 4.99 (s, 2H), 3.30–3.15 (m, 1H),
2.27 (s, 3H), 1.67 (dd, J=24.7, 12.5 Hz, 4H), 1.52 (d, J=12.2 Hz,
1H), 1.15 ppm (dt, J=37.6, 12.2 Hz, 5H); 13C NMR (100 MHz,
[D6]DMSO): d=155.4, 143.4, 137.9, 133.4, 129.7 (2C), 128.4 (2C),
124.9, 57.2, 53.0, 49.9, 33.0 (2C), 25.5, 25.0 (2C), 21.1 ppm; UPLC-
MS: Method A, tR =2.52 min, ionization: m/z 329 [M+H]+; HRMS-
ESI: m/z [M+H]+ calcd for C18H24N4O2: 329.1978, found: 329.1978.
[1-[(3,5-Dimethoxyphenyl)methyl]triazol-4-yl]methyl N-cyclohex-
ylcarbamate (27): The reaction was carried out following general
procedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol),
1-(azidomethyl)-3,5-dimethoxybenzene
(0.16 g,
0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O
(0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (EtOAc/Cy, 0!40%) to afford
1
27 as a white powder (0.17 g; 55%): H NMR (400 MHz, [D6]DMSO):
d=8.14 (s, 1H), 7.12 (d, J=7.8 Hz, 1H), 6.46 (s, 3H), 5.50 (s, 2H),
5.01 (s, 2H), 3.72 (s, 6H), 3.24 (m, 1H), 1.67 (m, 4H), 1.52 (d, J=
12.4 Hz, 1H), 1.12 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=
160.6, 154.9, 143.0, 138.0, 124.6, 106.0, 99.5, 56.7, 55.2, 52.7, 49.4,
32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS: Method A, tR =2.42 min,
ionization: m/z 375 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for
C19H26N4O4: 375.2032, found: 375.2047.
[1-[(2-Methoxyphenyl)methyl]triazol-4-yl]methyl N-cyclohexyl-
carbamate (26a): The reaction was carried out following general
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&
11
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[1-[(2,6-Difluorophenyl)methyl]triazol-4-yl]methyl N-cyclohexyl-
carbamate (28): The reaction was carried out following general
procedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
1H), 7.06 (d, J=8.0 Hz, 1H), 4.25 (t, J=6.7 Hz, 2H), 3.24 (m, 1H),
3.02 (t, J=6.7 Hz, 2H), 1.69 (m, 4H), 1.53 (d, J=13.3 Hz, 1H),
1.15 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=155.7, 145.1,
137.2, 130.3 (2C), 128.9, 121.4, 120.3 (2C), 62.7, 49.8, 33.1 (2C), 26.0,
25.6, 25.0 ppm (2C); UPLC-MS: Method A, tR =2.39 min, ionization:
m/z 315 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C17H22N4O2:
315.1821, found: 315.1829.
0.82 mmol),
2-(azidomethyl)-1,3-difluorobenzene
(0.14 g,
0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O
(0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (EtOAc/Cy, 0!40%) to afford
1
28 as a white powder (0.16 g; 56%): H NMR (400 MHz, [D6]DMSO):
2-(1-Benzyltriazol-4-yl)ethyl N-cyclohexylcarbamate (33): The re-
action was carried out according to the procedure employed for
32, starting from 2-(1-benzyltriazol-4-yl)ethanol (31b, 0.23 g,
1.12 mmol), cyclohexyl isocyanate (0.15 g, 1.23 mmol), and DMAP
(0.14 g, 1.12 mmol) in dry CH3CN (5 mL). Purification was per-
formed by flash chromatography (MeOH/CH2Cl2, 0!2%) to afford
d=8.10 (s, 1H), 7.51 (m, 1H), 7.18 (t, J=8.1 Hz, 2H), 7.13 (d, J=
7.8 Hz, 1H), 5.66 (s, 2H), 4.99 (s, 2H), 3.23 (m, 1H), 1.68 (dd, J=
26.0, 12.6 Hz, 4H), 1.52 (d, J=12.6 Hz, 1H), 1.12 ppm (m, 5H);
13C NMR (100 MHz, [D6]DMSO): d=161.2 (d, J=249.2 Hz), 155.4,
143.3, 132.1 (t, J=10.4 Hz), 125.1, 112.3 (d, J=24.4 Hz), 111.7 (t, J=
19.3 Hz), 57.1, 49.9, 41.2 (t, J=3.7 Hz), 33.0 (2C), 25.6, 25.0 ppm
(2C); UPLC-MS: Method A, tR =2.38 min, ionization: m/z 351
[M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C17H20F2N4O2: 351.1633,
found: 351.1631.
1
33 as a white powder (0.2 g; 54%): H NMR (400 MHz, [D6]DMSO):
d=7.95 (s, 1H), 7.33 (m, 5H), 7.01 (d, J=7.96 Hz, 1H), 5.55 (s, 2H),
4.16 (t, J=6.83 Hz, 2H), 3.21 (m, 1H), 2.90 (t, J=6.82 Hz, 2H), 1.67
(m, 4H), 1.52 (m, 1H), 1.12 ppm (m, 5H); 13C NMR (100 MHz,
[D6]DMSO): d=155.0, 144.1, 136.6, 129.1 (2C), 128.5, 128.3 (2C),
123.1, 99.9, 62.8, 53.1, 49.8, 33.1 (2C), 26.0, 25.6, 25.0 ppm (2C);
UPLC-MS: Method A, tR =2.33 min, ionization: m/z 329 [M+H]+;
HRMS-ESI: m/z [M+H]+ calcd for C18H24N4O2: 329.1978, found:
329.1983.
[1-[(3,5-Difluorophenyl)methyl]triazol-4-yl]methyl N-cyclohexyl-
carbamate (29): The reaction was carried out following general
procedure (1), using prop-2-ynyl N-cyclohexylcarbamate (0.15 g,
0.82 mmol),
1-(azidomethyl)-3,5-difluorobenzene
(0.14 g,
0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and CuSO4·5H2O
(0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (MeOH/CH2Cl2, 0!2%) to
afford 29 as a white powder (0.22 g; 77%): 1H NMR (400 MHz,
[D6]DMSO): d=8.21 (s, 1H), 7.23 (tt, J=9.4, 2.3 Hz, 1H), 7.13 (d, J=
7.8 Hz, 1H), 7.04 (t, J=6.4 Hz, 2H), 5.64 (s, 2H), 5.02 (s, 2H), 3.23
(m, 1H), 1.68 (dd, J=26.4, 12.4 Hz, 4H), 1.52 (d, J=12.6 Hz, 1H),
1.13 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=162.8 (dd, J=
247.1, 13.2 Hz), 155.4, 143.7, 140.6 (t, J=9.4 Hz), 125.4, 111.7 (m),
104.1 (t, J=25.7 Hz), 57.1, 52.1, 49.9, 33.0 (2C), 25.5, 25.0 ppm (2C);
UPLC-MS: Method A, tR =2.46 min, ionization: m/z 351 [M+H]+;
HRMS-ESI: m/z [M+H]+ calcd for C17H20F2N4O2: 351.1633, found:
351.1634.
[1-[(2-Methoxyphenyl)methyl]triazol-4-yl]methyl N-[[1-[(2-meth-
oxyphenyl)methyl]triazol-4-yl]methyl]carbamate (34a): The reac-
tion was carried out following general procedure (2), using prop-2-
ynyl N-prop-2-ynylcarbamate (0.11 g, 0.767 mmol), 1-(azidomethyl)-
2-methoxybenzene (0.25 g, 1.53 mmol), sodium ascorbate (0.030,
0.15 mmol), and CuSO4·5H2O (0.004 g, 0.002 mmol) in water/t-
BuOH (1:1; 3 mL). Purification was performed by flash chromatog-
raphy (MeOH/CH2Cl2, 0!4%) to afford 34a as a white amorphous
solid (0.2 g; 58%): 1H NMR (400 MHz, [D6]DMSO): d=8.00 (s, 1H),
7.80 (s, 1H), 7.68 (t, J=5.7 Hz, 1H), 7.34 (m, 2H), 7.08 (m, 4H), 6.93
(m, 2H), 5.51 (s, 2H), 5.48 (s, 2H), 5.03 (s, 2H), 4.21 (d, J=5.8 Hz,
2H), 3.81 (s, 3H), 3.80 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO):
d=156.83, 156.8, 155.9, 145.0, 142.5, 130.0, 129.9, 129.6, 129.5,
124.7, 123.6, 123.4, 122.8, 120.5 (2C), 111.2, 111.1, 57.1, 55.5 (2C),
48.2, 48.1, 35.9 ppm; UPLC-MS: Method A, tR =2.18 min, ionization:
m/z 464 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C23H25N7O4:
464.2046, found: 464.2056.
[1-[(2-Fluoro-3-methoxy-phenyl)methyl]triazol-4-yl]methyl N-cy-
clohexylcarbamate (30): The reaction was carried out following
general procedure (1), using prop-2-ynyl N-cyclohexylcarbamate
(0.15 g, 0.82 mmol), 1-(azidomethyl)-2-fluoro-3-methoxybenzene
(0.15 g, 0.82 mmol), sodium ascorbate (0.016, 0.08 mmol), and
CuSO4·5H2O (0.002 g, 0.008 mmol) in water/t-BuOH (1:1; 3 mL). Pu-
rification was performed by flash chromatography (MeOH/CH2Cl2,
0!2%) to afford 30 as a white powder (0.23 g; 77%): 1H NMR
(400 MHz, [D6]DMSO): d=8.09 (s, 1H), 7.14 (m, 3H), 6.84 (m, 1H),
5.64 (s, 2H), 5.00 (s, 2H), 3.83 (s, 3H), 3.24 (m, 1H), 1.68 (dd, J=
25.9, 12.5 Hz, 4H), 1.52 (d, J=12.5 Hz, 1H), 1.12 ppm (m, 5H);
13C NMR (100 MHz, [D6]DMSO): d=154.9, 149.5 (d, J=246.8 Hz),
147.3 (d, J=10.0 Hz), 142.9, 124.7, 124.6 (d, J=4.6 Hz), 123.5 (d, J=
11.9 Hz), 121.1 (d, J=2.0 Hz), 114.0 (d, J=1.3 Hz), 56.7, 56.1, 49.4,
46.7 (d, J=4.6 Hz), 32.6 (2C), 25.1, 24.5 ppm (2C); UPLC-MS:
Method A, tR =2.37 min, ionization: m/z 363 [M+H]+; HRMS-ESI:
m/z [M+H]+ calcd for C18H23FN4O3: 363.1832, found: 363.1834.
[1-[(3-Methoxyphenyl)methyl]triazol-4-yl]methyl N-[[1-[(3-meth-
oxyphenyl)methyl]triazol-4-yl]methyl]carbamate (34b): The reac-
tion was carried out following general procedure (2), using prop-2-
ynyl N-prop-2-ynylcarbamate (0.11 g, 0.767 mmol), 1-(azidomethyl)-
3-methoxybenzene (0.25 g, 1.53 mmol), sodium ascorbate (0.030,
0.15 mmol), and CuSO4·5H2O (0.004 g, 0.002 mmol) in water/t-
BuOH (1:1; 3 mL). Purification was performed by flash chromatog-
raphy (MeOH/CH2Cl2, 0!5%) to afford 34b as a white amorphous
1
solid (0.29 g; 81%): H NMR (400 MHz, [D6]DMSO): d=8.15 (s, 1H),
7.95 (s, 1H), 7.71 (t, J=5.7 Hz, 1H), 7.28 (td, J=8.2, 7.5, 2.1 Hz, 2H),
6.89 (dd, J=5.8, 2.9 Hz, 4H), 6.85 (d, J=7.3 Hz, 2H), 5.55 (s, 2H),
5.51 (s, 2H), 5.04 (s, 2H), 4.22 (d, J=5.8 Hz, 2H), 3.73 ppm (s, 6H);
13C NMR (100 MHz, [D6]DMSO): d=159.4 (2C), 155.9, 145.3, 142.8,
137.5, 137.4, 129.9, 129.8, 124.6, 122.8, 120.0 (2C), 113.7, 113.7,
113.4, 113.4, 57.1, 55.0 (2C), 52.7, 52.6, 36.0 ppm; UPLC-MS:
Method A, tR =2.12 min, ionization: m/z 464 [M+H]+; HRMS-ESI:
m/z [M+H]+ calcd for C23H25N7O4: 464.2046, found: 464.2052.
2-(1-Phenyltriazol-4-yl)ethyl N-cyclohexylcarbamate (32): 2-(1-
Phenyltriazol-4-yl)ethanol (31a, 0.24 g, 1.26 mmol) was dissolved in
dry CH3CN (5 mL) under stirring. Then, DMAP (0.15 g, 1.26 mmol)
and cyclohexyl isocyanate (0.17 g, 1.38 mmol) were added, and the
reaction mixture was stirred overnight at 808C. The mixture was
then diluted with EtOAc (30 mL) and washed once with 2n aq HCl
(30 mL), and once with brine (30 mL). The organic layer was dried
over Na2SO4, filtered and concentrated in vacuo. Purification was
performed by flash chromatography (MeOH/CH2Cl2, 0!2%) to
afford 31a as a white powder (0.25 g; 63%): 1H NMR (400 MHz,
[D6]DMSO): d=8.62 (s, 1H), 7.87 (m, 2H), 7.60 (m, 2H), 7.49 (m,
[1-[(2-Methoxyphenyl)methyl]triazol-4-yl]methyl N-[[1-[(3-meth-
oxyphenyl)methyl]triazol-4-yl]methyl]carbamate (37): The reac-
tion was carried out following general procedure (1), using prop-2-
ynyl N-[[1-[(3-methoxyphenyl)methyl]triazol-4-yl]methyl]carbamate
(36b, 0.15 g, 0.5 mmol), 1-(azidomethyl)-2-methoxybenzene
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&12
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
(0.08 g, 0.5 mmol), sodium ascorbate (0.010, 0.05 mmol), and
CuSO4·5H2O (0.001 g, 0.005 mmol) in water/t-BuOH (1:1; 3 mL). Pu-
rification was performed by flash chromatography (MeOH/CH2Cl2,
0!2%) to afford 37 as a white amorphous solid (0.13 g; 58%):
1H NMR (400 MHz, [D6]DMSO): d=8.00 (s, 1H), 7.95 (s, 1H), 7.70 (t,
J=5.7 Hz, 1H), 7.34 (m, 1H), 7.27 (m, 1H), 7.11 (dd, J=7.5, 1.4 Hz,
1H), 7.05 (d, J=8.2 Hz, 1H), 6.92 (m, 3H), 6.85 (d, J=7.6 Hz, 1H),
5.51 (s, 4H), 5.03 (s, 2H), 4.21 (d, J=5.8 Hz, 2H), 3.80 (s, 3H),
3.73 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=159.8, 157.3,
156.4, 145.8, 142.9, 137.9, 130.4, 130.3, 130.1, 125.2, 123.9, 123.3,
120.9, 120.4, 114.2, 113.8, 111.6, 57.6, 56.0, 55.5, 53.1, 48.7,
36.4 ppm; UPLC-MS: Method A, tR =2.15 min, ionization: m/z 464
[M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C23H25N7O4: 464.2046,
found: 464.2062.
(s, 6H); 13C NMR (100 MHz, [D6]DMSO): d=159.4, 159.1, 155.9,
145.3, 142.8, 137.4, 129.9, 129.6 (2C), 127.9, 124.6, 122.4, 120.0,
114.0 (2C), 113.7, 113.4, 57.1, 55.1, 55.0, 52.6, 52.2, 35.9 ppm; UPLC-
MS: Method A, tR =2.09 min, ionization: m/z 464 [M+H]+; HRMS-
ESI: m/z [M+H]+ calcd for C23H25N7O4: 464.2046, found: 464.2052.
(1-(1-Phenyl-1,2,4-triazol-3-yl)methyl
N-cyclohexylcarbamate
(43): The reaction was carried out following the procedure em-
ployed for 32, starting from (1-phenyl-1,2,4-triazol-3-yl)methanol
(42a, 0.05 g, 0.28 mmol), cyclohexyl isocyanate (0.04 g, 0.30 mmol),
and DMAP (0.03 g, 0.28 mmol) in dry CH3CN (5 mL). Purification
was performed by flash chromatography (EtOAc/Cy, 0!100%) to
afford 43 as a white powder (0.05 g; 58%): 1H NMR (400 MHz,
[D6]DMSO): d=9.26 (s, 1H), 7.89–7.77 (m, 2H), 7.62–7.49 (m, 2H),
7.48–7.37 (m, 1H), 7.26 (d, J=7.8 Hz, 1H), 5.08 (s, 2H), 3.26 (ddd,
J=10.5, 7.2, 3.2 Hz, 1H), 1.86–1.60 (m, 4H), 1.53 (d, J=12.8 Hz,
1H), 1.30–1.01 ppm (m, 5H); 13C NMR (100 MHz, [D6]DMSO): d=
161.0, 155.2, 143.5, 137.0, 130.2, 128.3, 119.7, 58.8, 50.0, 33.1 (2C),
25.6, 25.0 ppm (2C); UPLC-MS: Method A, tR =2.34 min, ionization:
m/z 301 [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for C16H20N4O2:
301.1665, found: 301.1674.
[1-[(2-Methoxyphenyl)methyl]triazol-4-yl]methyl N-[[1-[(4-meth-
oxyphenyl)methyl]triazol-4-yl]methyl]carbamate (38): The reac-
tion was carried out following general procedure (1), using prop-2-
ynyl N-[[1-[(4-methoxyphenyl)methyl]triazol-4-yl]methyl]carbamate
(36c, 0.15 g, 0.5 mmol), 1-(azidomethyl)-2-methoxybenzene (0.08 g,
0.5 mmol), sodium ascorbate (0.010, 0.05 mmol), and CuSO4·5H2O
(0.001 g, 0.005 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (MeOH/CH2Cl2, 0!2%) to
afford 38 as a colorless amorphous solid (0.15 g; 63%): 1H NMR
(400 MHz, [D6]DMSO): d=8.00 (s, 1H), 7.89 (s, 1H), 7.68 (t, J=
5.7 Hz, 1H), 7.34 (m, 1H), 7.28 (d, J=8.5 Hz, 2H), 7.11 (dd, J=7.4,
1.3 Hz, 1H), 7.05 (d, J=8.2 Hz, 1H), 6.92 (m, 3H), 5.51 (s, 2H), 5.46
(s, 2H), 5.03 (s, 2H), 4.20 (d, J=5.8 Hz, 2H), 3.80 (s, 3H), 3.73 ppm
(s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=159.5, 157.3, 156.3,
145.8, 142.9, 130.4, 130.1, 130.0 (2C), 128.4, 125.2, 123.9, 122.9,
120.9, 114.5 (2C), 111.7, 57.6, 56.0, 55.5, 52.7, 48.7, 36.4 ppm; UPLC-
MS: Method A, tR =2.13 min, ionization: m/z 464 [M+H]+; HRMS-
ESI: m/z [M+H]+ calcd for C23H25N7O4: 464.2046, found: 464.2052.
Benzyl-1,2,4-triazol-3-yl)methyl N-cyclohexylcarbamate (46): The
reaction was carried out following the procedure employed for 32,
starting from (1-benzyl-1,2,4-triazol-3-yl)methanol (42b, 0.06 g,
0.32 mmol), cyclohexyl isocyanate (0.044 g, 0.35 mmol), and DMAP
(0.04 g, 0.32 mmol) in dry CH3CN (5 mL). Purification was per-
formed by flash chromatography (EtOAc/Cy, 0!100%) to afford
1
46 as a white powder (0.07 g; 68%): H NMR (400 MHz, [D6]DMSO):
d=8.62 (s, 1H), 7.42–7.25 (m, 5H), 7.17 (d, J=7.7 Hz, 1H), 5.37 (s,
2H), 4.93 (s, 2H), 3.27–3.15 (m, 1H), 1.68 (dd, J=28.7, 12.5 Hz, 4H),
1.52 (d, J=12.6 Hz, 1H), 1.13 ppm (tdd, J=32.8, 24.1, 12.1 Hz, 5H);
13C NMR (100 MHz, [D6]DMSO): d=159.7, 154.7, 145.0, 136.1, 128.6,
127.9, 127.9, 58.4, 52.1, 49.4, 32.5 (2C), 25.1, 24.5 ppm (2C); UPLC-
MS: Method A, tR =2.27 min, ionization: m/z 315 [M+H]+; HRMS-
ESI: m/z [M+H]+ calcd for C17H22N4O2: 315.1821, found: 315.1823.
[1-[(3-Methoxyphenyl)methyl]triazol-4-yl]methyl N-[[1-[(2-meth-
oxyphenyl)methyl]triazol-4-yl]methyl]carbamate (39): The reac-
tion was carried out following general procedure (1), using prop-2-
ynyl N-[[1-[(2-methoxyphenyl)methyl]triazol-4-yl]methyl]carbamate
(36a, 0.15 g, 0.5 mmol), 1-(azidomethyl)-3-methoxybenzene (0.08 g,
0.5 mmol), sodium ascorbate (0.010, 0.05 mmol), and CuSO4·5H2O
(0.001 g, 0.005 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (MeOH/CH2Cl2, 0!2%) to
afford 39 as a colorless amorphous solid (0.15 g; 66%): 1H NMR
(400 MHz, [D6]DMSO): d=8.15 (s, 1H), 7.80 (s, 1H), 7.69 (t, J=
5.7 Hz, 1H), 7.34 (t, J=7.8 Hz, 1H), 7.28 (m, 1H), 7.06 (dd, J=14.4,
7.8 Hz, 2H), 6.89 (m, 4H), 5.54 (s, 2H), 5.48 (s, 2H), 5.04 (s, 2H), 4.21
(d, J=5.8 Hz, 2H), 3.80 (s, 3H), 3.73 ppm (s, 3H); 13C NMR
(100 MHz, [D6]DMSO): d=159.4, 156.8, 155.9, 145.0, 142.8, 137.4,
129.9 (2C), 129.5, 124.6, 123.6, 122.8, 120.5, 120.0, 113.7, 113.4,
111.1, 57.1, 55.5, 55.0, 52.7, 48.1, 35.9 ppm; UPLC-MS: Method A,
tR =2.15 min, ionization: m/z 464 [M+H]+; HRMS-ESI: m/z [M+H]+
calcd for C23H25N7O4: 464.2046, found: 464.2044.
Pharmacology
Animals: CD1 male mice were obtained from Charles River Labora-
tories Italia. All procedures were performed in accordance with the
Ethical Guidelines of the International Association for the Study of
Pain, Italian regulations on the protection of animals used for ex-
perimental and other scientific purposes (D.M. 116192) and Europe-
an Economic Community regulations (O.J. of E.C. L 358/1 12/18/
1986). When appropriate, procedures were also approved by the
Institutional Animal Care and Use Committee of the University of
California, Irvine (USA).
In vitro rat FAAH radiometric assay: Rat FAAH was prepared from
male Sprague–Dawley rat brains, homogenized in a potter in
20 mm of Tris HCl (pH 7.4), 0.32m sucrose. The radiometric assay
used to measure FAAH activity was performed in Eppendorf tubes:
total rat brain homogenate (50 mg) were pre-incubated in 445.5 mL
of assay buffer (50 mm Tris-HCl pH 7.4, 0.05% fatty-acid-free
bovine serum albumin (BSA), Sigma–Aldrich) with 4.5 mL of inhibi-
tor (at appropriate concentration in DMSO) or DMSO alone (to
measure FAAH total activity) for 10 min at 378C. The blank (no ac-
tivity control) was prepared using 445.5 mL of assay buffer and
4.5 mL of DMSO without the total rat brain homogenate (50 mg).
After 10 min of pre-incubation with test compound, the reaction
was started by adding substrate (50 mL) and incubating for 30 min
at 378C. The substrate was prepared in assay buffer in order to
achieve the final concentration of 1 mm arachidonoyl ethanolamide
(Cayman Chemical No. 90050) and 0.6 nm anandamide [ethanola-
[1-[(3-Methoxyphenyl)methyl]triazol-4-yl]methyl N-[[1-[(4-meth-
oxyphenyl)methyl]triazol-4-yl]methyl]carbamate (40): The reac-
tion was carried out following general procedure (1), using prop-2-
ynyl N-[[1-[(4-methoxyphenyl)methyl]triazol-4-yl]methyl]carbamate
(36c, 0.15 g, 0.5 mmol), 1-(azidomethyl)-3-methoxybenzene (0.08 g,
0.5 mmol), sodium ascorbate (0.010, 0.05 mmol), and CuSO4·5H2O
(0.001 g, 0.005 mmol) in water/t-BuOH (1:1; 3 mL). Purification was
performed by flash chromatography (MeOH/CH2Cl2, 0!2%) to
afford 40 as a colorless amorphous solid (0.16 g; 68%): 1H NMR
(400 MHz, [D6]DMSO): d=8.15 (s, 1H), 7.89 (s, 1H), 7.69 (t, J=
5.7 Hz, 1H), 7.28 (m, 3H), 6.90 (m, 4H), 6.85 (d, J=7.7 Hz, 1H), 5.55
(s, 2H), 5.46 (s, 2H), 5.04 (s, 2H), 4.20 (d, J=5.8 Hz, 2H), 3.73 ppm
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&13
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
mine-1-3H] (American Radiolabeled Chemicals Inc., ART. 0626, conc.
1 mCimLꢀ1, S.A. 60 Cimmolꢀ1). The reaction was stopped by
adding cold CHCl3/MeOH (1:1). After centrifugation (845 g at 48C
for 10 min), 600 mL of aqueous phase were transferred into scintil-
lation vials previously filled with 3 mL of scintillation fluid (Ultima
GoldTM, PerkinElmer Inc., Cat.6013329). Radioactivity was mea-
sured by liquid scintillation counting (MicroBeta2 LumiJET Perki-
nElmer Inc.).
Monoacylglycerol lipase (MGL) activity assay: Purified rat MGL
(10 ng) was pre-incubated with appropriate test compound for
10 min at 378C in 50 mm Tris-HCl, pH 8.0, containing 0.5 mgmLꢀ1
fatty-acid-free BSA. The final concentration of vehicle (1% DMSO)
had no effect on MGL activity. Then, 2-oleoylglycerol (2-OG, 10 mm
final concentration) was added, and the mixture was incubated for
additional 30 min at 378C. Reactions were stopped by adding
CHCl3/MeOH (2:1 v/v), containing heptadecanoic acid (5 nmol/
sample) as an internal standard. After centrifugation at 2000 g at
48C for 10 min, the organic layers were collected and dried under
a stream of N2. The lipid extracts were then suspended in CHCl3/
MeOH (1:3 v/v) and analyzed by liquid chromatography/mass spec-
trometry (LC/MS).
In vitro human FAAH fluorescent assay: Human recombinant FAAH
was obtained from a HEK-293 cell line stably overexpressing
human FAAH-1 enzyme. Cells were grown in Dulbecco’s modified
Eagle medium (DMEM) containing 10% fetal bovine serum, 1%
penicillin/streptomycin, 1% glutamine, and 500 mgmLꢀ1 G418. To
obtain membrane preparation, cells were scraped off with cold
phosphate-buffered saline (PBS) and collected by centrifugation
(500 g at 48C for 10 min); the cell pellet was re-suspended in
20 mm Tris-HCl pH 7.4, 0.32m sucrose, disrupted by sonication
(10 pulses, 5 times) and centrifuged (800 g at 48C for 15 min); the
collected supernatant was centrifuged at 105000 g for 1 h at 48C,
and the pellet was re-suspended in PBS. The fluorescent assay to
measure FAAH activity was performed in 96-well black plates:
human FAAH-1 membrane preparation (2.5 mg) were pre-incubated
for 50 min at 378C, in 180 mL of assay buffer (50 mm Tris-HCl
pH 7.4, 0.05% fatty-acid-free BSA) with inhibitor (10 mL) or DMSO
(10 mL) to measure FAAH total activity. The background (no activi-
ty) samples were prepared using assay buffer (180 mL) without
human FAAH-1 and DMSO (10 mL). The reaction was then started
by addition of 10 mL of the substrate (, Cayman Chemical,
No. 10005098) dissolved in ethanol and used at a final concentra-
tion of 2 mm. The reaction was carried out for 30 min at 378C, and
fluorescence was measured with a Tecan Infinite M200 nanoquant
plate reader (excitation wavelength 350 nm/emission wavelength
460 nm). The concentration causing half-maximal inhibition (IC50)
was determined by nonlinear regression analysis of the Log[con-
centration]/response curves generated with mean replicate values
using a four-parameter Hill equation curve fitting with GraphPad
Prism5 (GraphPad Software Inc., CA, USA).
In vitro rat plasma stability assay: Compounds were diluted in rat
plasma with 10% DMSO to help solubilization. Plasma was already
pre-heated at 378C (30 min). The final compound concentration
was 1.0 mm. At appropriate time points (immediately after dilution
(0), 30, 60, 120, 240, 360 and 420 min), a 40 mL aliquot of the incu-
bation solution was diluted in 150 mL of cold CH3CN spiked with
200 nm warfarin as an internal standard. After vortexing for 30 s,
the solution was centrifuged at 3500 g for 15 min at 48C, and the
supernatant was transferred for LC/MS analysis on a Waters ACQUI-
TY UPLC/MS TQD system consisting of a triple quadrupole detector
(TQD) mass spectrometer equipped with an ESI interface. Briefly,
an aliquot of the supernatant (3.0 mL) was injected on a reverse-
phase HPLC column (BEH C18 1.7 mm, 2.1ꢁ50 mm) and separated
with a linear gradient of acetonitrile. Compounds were quantified
on the basis of their multiple reaction monitoring (MRM) peak
areas. The response factors, calculated on the basis of the internal
standard peak area, were then plotted over time. For each com-
pound, analyses were conducted in triplicate; the compound re-
maining (%) at 420 min with corresponding standard deviation is
reported.
In vitro mouse liver microsomes (MLM) stability assay: Compounds
were pre-incubated with microsomes in 100 mm TRIS-HCl (pH 7.4)
for 15 min. At time zero, cofactors were added. The final incuba-
tion conditions for each sample were: 1.25 mgmLꢀ1 liver micro-
somes, 5 mm compound (final DMSO 0.1%), NADP 1 mm, G6P
20 mm, MgCl2 2 mm, G6P dehydrogenase 2 U. The mixture was
kept at 378C under shaking. Aliquots (30 mL) were taken at various
time points (typically 0, 5, 15, 30, and 60 min) and crashed with
200 mL of acetonitrile spiked with 200 nm warfarin (internal stan-
dard). A reference incubation, with microsomes but without cofac-
tors, was kept at 378C and sampled at the end of the time course.
After vortexing (5 RCF, 3 min) and centrifugation (3300 RCF,
15 min), an aliquot of supernatant (3 mL) was analyzed by LC-MS/
MS by MRM.
Ex vivo FAAH inhibition assay: CD1 male mice were treated intraper-
itoneally (i.p.) with test compound (3 mgkgꢀ1) or vehicle (1:1:8,
PEG400, Tweenꢂ 80 and Saline 0.9%). After treatment for 1 h, the
animals were killed by decapitation, and the brain and liver were
collected. Samples were homogenized in 1.5 mL of 20 mm Tris-HCl
buffer pH 7.4, containing 0.32m sucrose, and the homogenates
were centrifuged at 1000 g for 10 min at 48C. The supernatants
were collected, and the protein concentration was measured by
Bradford method (Bio Rad Protein assay kit). FAAH activity was
measured using 50 mg of total brain or liver homogenate in 450 mL
of assay buffer (50 mm Tris-HCl pH 7.4, 0.05% fatty-acid-free BSA);
the blank (no activity sample) was prepared with 450 mL of assay
buffer. The reaction was started by adding 50 mL of substrate for
30 min at 378C. The substrate was prepared in assay buffer in
order to obtain a final concentration of 1 mm arachidonoyl-ethano-
lamide (Cayman Chemical, No.90050) and 0.6 nm anandamide
[ethanolamine-1-3H] (American Radiolabeled Chemicals Inc.,
ART.0626, 1 mCimLꢀ1, specific activity 60 CimmoLꢀ1). The reaction
was stopped by adding cold CHCl3/MeOH (1:1). After centrifugation
(845 g at 48C for 10 min), 600 mL of the aqueous phase was trans-
ferred into scintillation vials previously filled with 3 mL of scintilla-
tion fluid (ULTIMA GOLD, Cat.6013329, PerkinElmer). Radioactivity
was measured by liquid scintillation counting (Microbeta2 Lumijet,
PerkinElmer Inc.).
Aqueous kinetic solubility assay: The kinetic solubility in PBS at
pH 7.4 was determined starting from a 10 mm DMSO solution of
the test compound. The study was performed by incubation of an
aliquot (10 mL) of 10 mm DMSO solution in PBS (pH 7.4) at 258C for
24 h, under shaking, followed by centrifugation (21100 RCF,
30 min) and quantification of dissolved compound in the superna-
tant by UPLC/MS. The compound target concentration in the solu-
tion was 250 mm, resulting in a final DMSO concentration of 2.5%.
The supernatant was analyzed by UPLC/MS, and the quantification
of the dissolved compound was determined by monitoring the UV
trace at 215 nm. The kinetic solubility (mm) was calculated by divid-
ing the peak area of the test compound in the supernatant by the
peak area of a reference solution (250 mm) of the test compound
in CH3CN/H2O (1:1), and further multiplied by the concentration of
the test compound reference and the dilution factor. The UPLC/MS
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&14
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Kesten, T. K. Nomanbhoy, B. F. Cravatt, K. Ahn, ACS Med. Chem. Lett.
2011, 2, 91–96.
analyses were performed on a Waters ACQUITY UPLC/MS system
consisting of a SQD mass spectrometer equipped with an ESI inter-
face and a PDA detector. The PDA range was 210–400 nm. The
analyses were run on an ACQUITY UPLC BEH C18 column (50ꢁ2.1
mm ID, particle size 1.7 mm) with a VanGuard BEH C18 pre-column
(5ꢁ2.1 mm ID, particle size 1.7 mm). The mobile phase was 10 mm
NH4OAc in H2O at pH 5 adjusted with AcOH (A) and 10 mm
NH4OAc in CH3CN/H2O (95:5) at pH 5 (B). ESI in the positive and
negative mode was applied in the mass scan range 100–500 Da.
[10] J. P. Alexander, B. F. Cravatt, Chem. Biol. 2005, 12, 1179–1187.
[11] a) D. L. Boger, H. Sato, A. E. Lerner, M. P. Hedrick, R. A. Fecik, H. Miyauchi,
G. D. Wilkie, B. J. Austin, M. P. Patricelli, B. F. Cravatt, Proc. Natl. Acad. Sci.
USA 2000, 97, 5044–5049; b) K. Otrubova, C. Ezzili, D. L. Boger, Bioorg.
Med. Chem. Lett. 2011, 21, 4674–4685; c) K. Otrubova, D. L. Boger, ACS
Chem. Neurosci. 2012, 3, 340–348; d) K. Otrubova, M. Brown, M. S. Mc-
Cormick, G. W. Han, S. T. O’Neal, B. F. Cravatt, R. C. Stevens, A. H. Licht-
man, D. L. Boger, J. Am. Chem. Soc. 2013, 135, 6289–6299; e) K. Otrubo-
va, B. F. Cravatt, D. L. Boger, J. Med. Chem. 2014, 57, 1079–1089.
[12] M. Feledziak, D. M. Lambert, J. Marchand-Brynaert, G. G. Muccioli, Recent
Pat. CNS Drug Discov. 2012, 7, 49–70.
[13] S. Kathuria, S. Gaetani, D. Fegley, F. Valino, A. Duranti, A. Tontini, M. Mor,
G. Tarzia, G. La Rana, A. Calignano, A. Giustino, M. Tattoli, M. Palmery, V.
Cuomo, D. Piomelli, Nat. Med. 2002, 9, 76–81.
[14] G. Tarzia, A. Duranti, G. Gatti, G. Piersanti, A. Tontini, S. Rivara, A. Lodola,
P. V. Plazzi, M. Mor, S. Kathuria, D. Piomelli, ChemMedChem 2006, 1,
130–139.
[15] a) M. Mor, A. Lodola, S. Rivara, F. Vacondio, A. Duranti, A. Tontini, S. San-
chini, G. Piersanti, J. R. Clapper, A. R. King, G. Tarzia, D. Piomelli, J. Med.
Chem. 2008, 51, 3487–3498; b) F. Vacondio, C. Silva, A. Lodola, A. Fioni,
S. Rivara, A. Duranti, A. Tontini, S. Sanchini, J. R. Clapper, D. Piomelli, M.
Mor, G. Tarzia, ChemMedChem 2009, 4, 1495–1504; c) J. R. Clapper, G.
Moreno-Sanz, R. Russo, A. Guijarro, F. Vacondio, A. Duranti, A. Tontini, S.
Sanchini, N. R. Sciolino, J. M. Spradley, A. G. Hohmann, A. Calignano, M.
Mor, G. Tarzia, D. Piomelli, Nat. Neurosci. 2010, 13, 1265–1270; d) F. Va-
condio, C. Silva, A. Lodola, C. Carmi, S. Rivara, A. Duranti, A. Tontini, S.
Sanchini, J. R. Clapper, D. Piomelli, G. Tarzia, M. Mor, Eur. J. Med. Chem.
2011, 46, 4466–4473.
Acknowledgements
The authors thank Sine Mandrup Bertozzi for determination of
compound solubility in buffer and for HRMS analyses, Dr. Andrea
Armirotti for discussions on the rat plasma stability data, Luca
Goldoni for NMR technical support, and Silvia Venzano for com-
pounds handling (all at the Istituto Italiano di Tecnologia,
Genova, Italy). This study was partially supported by grants from
the US National Institutes on Drug Abuse (R01-DA-012413 and
DP1DA031387 to D.P.).
Keywords: click chemistry
· fatty acid amide hydrolase
(FAAH) · inhibitors · O-(triazolyl)methyl carbamates · structure–
activity relationships
[16] a) G. Gobbi, F. R. Bambico, R. Mangieri, M. Bortolato, P. Campolongo, M.
Solinas, T. Cassano, M. G. Morgese, G. Debonnel, A. Duranti, A. Tontini,
G. Tarzia, M. Mor, V. Trezza, S. R. Goldberg, V. Cuomo, D. Piomelli, Proc.
Natl. Acad. Sci. USA 2005, 102, 18620–18625; b) D. Piomelli, G. Tarzia, A.
Duranti, A. Tontini, M. Mor, T. R. Compton, O. Dasse, E. P. Monaghan,
J. A. Parrott, D. Putman, CNS Drug Rev. 2006, 12, 21–38; c) R. Russo, J.
Loverme, G. La Rana, T. R. Compton, J. Parrott, A. Duranti, A. Tontini, M.
Mor, G. Tarzia, A. Calignano, D. Piomelli, J. Pharmacol. Exp. Ther. 2007,
322, 236–242.
[1] a) F. Desarnaud, H. Cadas, D. Piomelli, J. Biol. Chem. 1995, 270, 6030–
6035; b) C. J. Hillard, D. M. Wilkison, W. S. Edgemond, W. B. Campbell,
Biochim. Biophys. Acta Lipids Lipid Metab. 1995, 1257, 249–256; c) N.
Ueda, Y. Kurahashi, S. Yamamoto, T. Tokunaga, J. Biol. Chem. 1995, 270,
23823–23827; d) B. F. Cravatt, D. K. Giang, S. P. Mayfield, D. L. Boger,
R. A. Lerner, N. B. Gilula, Nature 1996, 384, 83–87.
[2] a) W. A. Devane, L. Hanus, A. Breuer, R. G. Pertwee, L. A. Stevenson, G.
Griffin, D. Gibson, A. Mandelbaum, A. Etinger, R. Mechoulam, Science
1992, 258, 1946–1949; b) V. Di Marzo, A. Fontana, H. Cadas, S. Schinelli,
G. Cimino, J. C. Schwartz, D. Piomelli, Nature 1994, 372, 686–691.
[3] T. Bisogno, S. Maurelli, D. Melck, L. DePetrocellis, V. DiMarzo, J. Biol.
Chem. 1997, 272, 3315–3323.
[17] a) M. K. McKinney, B. F. Cravatt, Annu. Rev. Biochem. 2005, 74, 411–432;
b) K. Ahn, M. K. McKinney, B. F. Cravatt, Chem. Rev. 2008, 108, 1687–
1707.
[18] a) A. Lodola, M. Mor, J. C. Hermann, G. Tarzia, D. Piomelli, A. J. Mulhol-
land, Chem. Commun. 2005, 4399–4401; b) A. Lodola, M. Mor, J. Zurek,
G. Tarzia, D. Piomelli, J. N. Harvey, A. J. Mulholland, Biophys. J. 2007, 92,
L20–L22; c) E. I. Chudyk, E. Dyguda-Kazimierowicz, K. M. Langner, W. A.
Sokalski, A. Lodola, M. Mor, J. Sirirak, A. J. Mulholland, J. Phys. Chem. B
2013, 117, 6656–6666; d) A. Lodola, L. Capoferri, S. Rivara, G. Tarzia, D.
Piomelli, A. Mulholland, M. Mor, J. Med. Chem. 2013, 56, 2500–2512.
[19] M. Mileni, S. Kamtekar, D. C. Wood, T. E. Benson, B. F. Cravatt, R. C. Ste-
vens, J. Mol. Biol. 2010, 400, 743–754.
[4] a) F. Rodrꢃguez de Fonseca, M. Navarro, R. Gomez, L. Escuredo, F. Nava,
J. Fu, E. Murillo-Rodriguez, A. Giuffrida, J. LoVerme, S. Gaetani, S. Kathu-
ria, C. Gall, D. Piomelli, Nature 2001, 414, 209–212; b) D. Piomelli,
Trends Endocrinol. Metab. 2013, 24, 332–341.
[5] a) J. Fu, S. Gaetani, F. Oveisi, J. Lo Verme, A. Serrano, F. R. De Fonseca, A.
Rosengarth, H. Luecke, B. Di Giacomo, G. Tarzia, D. Piomelli, Nature
2003, 425, 90–93; b) J. Lo Verme, J. Fu, G. Astarita, G. La Rana, R. Russo,
A. Calignano, D. Piomelli, Mol. Pharmacol. 2005, 67, 15–19.
[6] V. Di Marzo, Nat. Rev. Drug Discovery 2008, 7, 438–455.
[20] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40,
2004–2021; Angew. Chem. 2001, 113, 2056–2075; b) V. V. Rostovtsev,
L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41,
2596–2599; Angew. Chem. 2002, 114, 2708–2711; c) C. W. Tornøe, C.
Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–3064; d) H. C.
Kolb, K. B. Sharpless, Drug Discovery Today 2003, 8, 1128–1137.
[21] G. Colombano, C. Travelli, U. Galli, A. Caldarelli, M. G. Chini, P. L. Canoni-
co, G. Sorba, G. Bifulco, G. C. Tron, A. A. Genazzani, J. Med. Chem. 2010,
53, 616–623.
[22] C. Fischer, S. L. Zultanski, H. Zhou, J. L. Methot, W. C. Brown, D. M. Mam-
preian, A. J. Schell, S. Shah, H. Nuthall, B. L. Hughes, N. Smotrov, C. M.
Kenific, J. C. Cruz, D. Walker, M. Bouthillette, G. N. Nikov, D. F. Savage,
V. V. Jeliazkova-Mecheva, D. Diaz, A. A. Szewczak, N. Bays, R. E. Middle-
ton, B. Munoz, M. S. Shearman, Bioorg. Med. Chem. Lett. 2011, 21, 4083–
4087.
[7] a) D. M. Lambert, G. G. Muccioli, Curr. Opin. Clin. Nutr. Metab. Care 2007,
10, 735–744; b) K. Ahn, D. S. Johnson, B. F. Cravatt, Expert Opin. Drug
Discovery 2009, 4, 763–784; c) H. S. Hansen, Exp. Neurol. 2010, 224, 48–
55.
[8] a) G. Tarzia, A. Duranti, A. Tontini, G. Piersanti, M. Mor, S. Rivara, P. V.
Plazzi, C. Park, S. Kathuria, D. Piomelli, J. Med. Chem. 2003, 46, 2352–
2360; b) M. Mor, S. Rivara, A. Lodola, P. V. Plazzi, G. Tarzia, A. Duranti, A.
Tontini, G. Piersanti, S. Kathuria, D. Piomelli, J. Med. Chem. 2004, 47,
4998–5008; c) J. R. Clapper, F. Vacondio, A. R. King, A. Duranti, A. Tonti-
ni, C. Silva, S. Sanchini, G. Tarzia, M. Mor, D. Piomelli, ChemMedChem
2009, 4, 1505–1513.
[9] a) K. Ahn, D. S. Johnson, L. R. Fitzgerald, M. Liimatta, A. Arendse, T. Ste-
venson, E. T. Lund, R. A. Nugent, T. K. Nomanbhoy, J. P. Alexander, B. F.
Cravatt, Biochemistry 2007, 46, 13019–13030; b) D. S. Johnson, K. Ahn,
S. Kesten, S. E. Lazerwith, Y. Song, M. Morris, L. Fay, T. Gregory, C. Stiff,
J. B. Dunbar, Jr., M. Liimatta, D. Beidler, S. Smith, T. K. Nomanbhoy, B. F.
Cravatt, Bioorg. Med. Chem. Lett. 2009, 19, 2865–2869; c) D. S. Johnson,
C. Stiff, S. E. Lazerwith, S. R. Kesten, L. K. Fay, M. Morris, D. Beidler, M. B.
Liimatta, S. E. Smith, D. T. Dudley, N. Sadagopan, S. N. Bhattachar, S. J.
[23] R. Ramesh, R. G. Bhat, S. Chandrasekaran, J. Org. Chem. 2005, 70, 837–
840.
[24] J. I. Sarmiento-Sanchez, A. Ochoa-Teran, I. A. Rivero, ARKIVOC 2011,
2011, 177–188.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&15
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[25] T. P. Curran, R. S. H. Yoon, B. R. Volk, J. Organomet. Chem. 2004, 689,
4837–4847.
[28] A. G. Hohmann, R. L. Suplita, N. M. Bolton, M. H. Neely, D. Fegley, R.
Mangieri, J. F. Krey, J. M. Walker, P. V. Holmes, J. D. Crystal, A. Duranti, A.
Tontini, M. Mor, G. Tarzia, D. Piomelli, Nature 2005, 435, 1108–1112.
[26] The half-life of URB524 determined here is comparable with the value
reported in Ref. [15b] for the same compound in 80% rat plasma.
[27] T. P. Dinh, T. F. Freund, D. Piomelli, Chem. Phys. Lipids 2002, 121, 149–
158.
Received: September 3, 2014
Published online on && &&, 0000
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&16
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
Searching FAAH and wide: O-(Triazo-
lyl)methyl carbamates were designed
and synthesized via click chemistry to
investigate their inhibitory activity
against fatty acid amide hydrolase
(FAAH), a validated target in the treat-
ment of several disorders, including
pain and drug addiction. The results
highlighted the importance of 1,4-di-
substituted-1,2,3-triazoles for activity.
G. Colombano,* C. Albani, G. Ottonello,
A. Ribeiro, R. Scarpelli, G. Tarozzo,
J. Daglian, K.-M. Jung, D. Piomelli,
T. Bandiera
&& – &&
O-(Triazolyl)methyl Carbamates as
a Novel and Potent Class of Fatty Acid
Amide Hydrolase (FAAH) Inhibitors
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 17
&17
&
ÞÞ
These are not the final page numbers!