Design and synthesis of cyanamides as potent and selective N-acylethanolamine acid amidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115195

N-acylethanolamine acid amidase (NAAA) inhibition represents an exciting novel approach to treat inflammation and pain. NAAA is a cysteine amidase which preferentially hydrolyzes the endogenous biolipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA is an endogenous agonist of the nuclear peroxisome proliferator-activated receptor-α (PPAR-α), which is a key regulator of inflammation and pain. Thus, blocking the degradation of PEA with NAAA inhibitors results in augmentation of the PEA/PPAR-α signaling pathway and regulation of inflammatory and pain processes. We have prepared a new series of NAAA inhibitors exploring the azetidine-nitrile (cyanamide) pharmacophore that led to the discovery of highly potent and selective compounds. Key analogs demonstrated single-digit nanomolar potency for hNAAA and showed >100-fold selectivity against serine hydrolases FAAH, MGL and ABHD6, and cysteine protease cathepsin K. Additionally, we have identified potent and selective dual NAAA-FAAH inhibitors to investigate a potential synergism between two distinct anti-inflammatory molecular pathways, the PEA/PPAR-α anti-inflammatory signaling pathway,^1–4 and the cannabinoid receptors CB1 and CB2 pathways which are known for their antiinflammatory and antinociceptive properties.^5–8 Our ligand design strategy followed a traditional structure–activity relationship (SAR) approach and was supported by molecular modeling studies of reported X-ray structures of hNAAA. Several inhibitors were evaluated in stability assays and demonstrated very good plasma stability (t_1/2 > 2 h; human and rodents). The disclosed cyanamides represent promising new pharmacological tools to investigate the potential role of NAAA inhibitors and dual NAAA-FAAH inhibitors as therapeutic agents for the treatment of inflammation and pain.

Full text of DOI:10.1016/j.bmc.2019.115195

Journal Pre-proofs
Design and synthesis of cyanamides as potent and selective N-acylethanola-
mine acid amidase inhibitors
Michael S. Malamas, Shrouq I. Farah, Lamani Manjunath, Dimitrios N.
Pelekoudas, Nicholas Thomas Perry, Girija Rajarshi, Christina Yume Miyabe,
Honrao Chandrashekhar, Jay West, Spiro Pavlopoulos, Alexandros
Makriyannis
PII:
S0968-0896(19)31419-1
https://doi.org/10.1016/j.bmc.2019.115195
BMC 115195
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 August 2019
29 October 2019
30 October 2019
Please cite this article as: M.S. Malamas, S.I. Farah, L. Manjunath, D.N. Pelekoudas, N. Thomas Perry, G.
Rajarshi, C. Yume Miyabe, H. Chandrashekhar, J. West, S. Pavlopoulos, A. Makriyannis, Design and synthesis
of cyanamides as potent and selective N-acylethanolamine acid amidase inhibitors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115195
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Design and synthesis of cyanamides as potent and selective N-acylethanolamine acid amidase
inhibitors
*
1
1
1
1
Michael S. Malamas , Shrouq I. Farah , Lamani Manjunath , Dimitrios N. Pelekoudas ,
1
1
1
1
Nicholas Thomas Perry , Girija Rajarshi , Christina Yume Miyabe , Honrao Chandrashekhar ,
1
1
*1
Jay West , Spiro Pavlopoulos and Alexandros Makriyannis .
1Center for Drug Discovery and Departments of Chemistry and Chemical Biology and
Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States
Abstract
N-acylethanolamine acid amidase (NAAA) inhibition represents an exciting novel approach to
treat inflammation and pain. NAAA is a cysteine amidase which preferentially hydrolyzes the
endogenous biolipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA is an
endogenous agonist of the nuclear peroxisome proliferator-activated receptor- (PPAR-), which
is a key regulator of inflammation and pain. Thus, blocking the degradation of PEA with NAAA
inhibitors results in augmentation of the PEA/PPAR- signaling pathway and regulation of
inflammatory and pain processes. We have prepared a new series of NAAA inhibitors exploring
the azetidine-nitrile (cyanamide) pharmacophore that led to the discovery of highly potent and
selective compounds. Key analogs demonstrated single-digit nanomolar potency for hNAAA and
showed >100-fold selectivity against serine hydrolases FAAH, MGL and ABHD6, and cysteine
protease cathepsin K. Additionally, we have identified potent and selective dual NAAA-FAAH
inhibitors to investigate a potential synergism between two distinct anti-inflammatory molecular
1
-4
pathways, the PEA/PPAR- anti-inflammatory signaling pathway , and the cannabinoid
receptors CB1 and CB2 pathways which are known for their antiinflammatory and antinociceptive
5
-8
properties . Our ligand design strategy followed a traditional structure-activity relationship
SAR) approach and was supported by molecular modeling studies of reported X-ray structures of
(
hNAAA. Several inhibitors were evaluated in stability assays and demonstrated very good plasma
stability (t1/2 > 2 hours; human and rodents). The disclosed cyanamides represent promising new
pharmacological tools to investigate the potential role of NAAA inhibitors and dual NAAA-FAAH
inhibitors as therapeutic agents for the treatment of inflammation and pain.

Keywords: N-acylethanolamine acid amidase (NAAA), N-palmitoylethanolamide (PEA),
monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH), nuclear peroxisome
proliferator-activated receptor- (PPAR-), CB1, CB2 cannabinoid receptors, anandamide
(AEA), inflammation, antinociception.
*
Corresponding author. Michael S. Malamas; E-mail address: m.malamas@northeastern.edu.
1
.
Introduction
Notwithstanding the major medical advancements over the past 50-years to treat inflammation and
pain, still there is a great need for safer medications. The classic anti-inflammatory drugs, such as
Nonsteroidal Anti-inflammatory Drugs (NSAIDs) are the most common used medications for the
management of inflammation and pain, however their prolonged use is associated with severe side-
effects, such as gastrointestinal ulcerations, bleeding, renal toxicity and cardiovascular injury. In
order to minimize these undesirable side-effects, new pharmaceutical approaches were undertaken
with drug delivery systems to achieve high drug concentrations at the site of inflammatory stimuli
9
with minimal exposure to normal tissues . While these drugs provided only limited success for the
treatment of inflammatory conditions, as an alternate approach, one can envision the development
of new therapeutic approaches to target the endogenous environment at the site of inflammatory
stimuli. Along these lines, a family of endogenous bioactive lipids the N-acylethanolamines
(
NAEs) is considered to be implicated in the regulation of inflammation and pain10-12. NAEs are
not stored in cells, but rather synthesized “on-demand” upon inflammatory stimuli in most
1
3
mammalian cells . They are produced from membrane phospholipids by the sequential actions of
N-acyltransferase to generate N-acyl phosphatidylethanolamines and phospholipase D (NAPE-
1
4
PLD) . Two endogenous N-acylethanolamines anandamide (AEA) and palmitoylethanolamide
PEA) are well-known to exhibit anti-inflammatory10, 11, 15 and analgesic16-18 properties. AEA
(
suppresses inflammatory processes through stimulation of the cannabinoid receptors CB1 and
6
CB2 , while PEA exerts its anti-inflammatory effects through interaction with the nuclear
1
4
peroxisome proliferator-activated receptor-α (PPARα) . The physiological actions of these lipid
messengers are terminated by two known intracellular lipid amidases fatty acid amide hydrolase
FAAH) and N-acylethanolamine acid amidase (NAAA)20, 21, respectively. Pharmacological
1
9
(

inhibition of FAAH and NAAA augments the endogenous NAEs levels in rodent models
promoting anti-inflammatory and analgesic effects10, 11, 15-18 without any cardiovascular effects and
gastrointestinal hemorrhaging as frequently seen with cyclo-oxygenase-2 (COX-2) inhibitors22-24
,
further supporting their clinical development as an alternative therapeutic strategy against
inflammatory diseases.
1
.2.
Role of NAAA inhibition in inflammation. NAAA is a lysosomal cysteine protease
9
highly expressed in macrophages and peripheral tissues, including lungs and spleen and plays a
central role in the deactivation of biolipid PEA. NAAA is activated by autoproteolysis at acidic
pH ~ 4.5 conditions generating a catalytically competent subunit of the enzyme bearing a cysteine
(Cys131 in mice, Cys126 in humans) as the nucleophilic residue responsible for the hydrolysis of
2
5
13
PEA . PEA is an endogenous biolipid produced on-demand by most mammalian cells and a
growing body of evidence links PEA to the regulation of inflammatory and pain processes. PEA
reduces peripheral inflammation10, 26 and mast cell degranulation and exerts neuroprotective
27
28
1
6
and antinociceptive effects in rats and mice. Local and systemic administration of PEA alleviated
pain behaviors elicited by chemical irritants and was effective even when administered after
induction of acute inflammation11, 17, 29. In recent studies, NAAA inhibition increased the PEA
levels at the site of inflammation in ulcerative colitis preclinical models and alleviated most of the
symptoms of colitis, while significantly downregulated inflammatory cytokines levels at the
3
0
gastrointestinal tract and peripheral tissues . Furthermore, other recent publications have
1
-4
reinforced the use of NAAA inhibitors for the treatment of chronic inflammatory disorders .
Notwithstanding the encouraging pharmacological benefits of NAAA inhibitors in various
inflammatory conditions, only a limited number of NAAA inhibitors with rather sub-optimal
druggability profiles have been discovered to date.4
1
.3.
Endogenous PPAR- activation in inflammation. The molecular thrust of PEA’s anti-
inflammatory effect is dependent on the activation of nuclear receptor peroxisome proliferator-
activated receptor- (PPAR-), which is ubiquitously expressed in the brain, lung, liver, and
intestinal mucosa of the small intestine and colon15, 31. Thus, modulating the PEA/PPAR- anti-
inflammatory signaling pathway with NAAA inhibitors at the localized inflamed-tissue
environment offers the opportunity to intervene locally with drugs and avoid broad receptor

activation and minimize potential side-effects. Notably, activation of PPAR- with exogenous
agonists results in global stimulation of the broadly expressed PPAR- receptors and causes severe
side-effects32, 33. PPAR- controls transcriptional processes involved in the development of
inflammation through mechanisms that include direct interactions with the proinflammatory
3
4
transcription factors NF-kB and AP1, and modulation of IkB- function, an inhibitor of NF-kB .
Pharmacological studies have demonstrated that PPAR- agonists are therapeutically effective in
3
5
rodent models of inflammatory and autoimmune diseases . Furthermore, mutant PPAR-
3
5
deficient mice seem to be vulnerable to various inflammatory stimuli , suggesting that
endogenous PPAR- activity negatively regulates the initiation of acute inflammatory responses.
These properties are dependent on PPAR- activation, since they are absent in PPAR- deficient
3
5
mice and blocked by PPAR- antagonists .
1
.4.
Known NAAA inhibitors.
O
O
The reported NAAA inhibitors4, 36-38
O
O
N
O
N
O
O
O
H
H
(Fig. 1) show nanomolar potency for
1
(IC50 7 nM)
2 (IC50 7 nM)
the target, however they exhibit poor
plasma stability (t <5 min) and poor
SO2Me
1
/2
N
O
NH
to moderate metabolic stability39, 40
.
O
N
O
N
H
O
O
H
One can consider the “war-heads” of
these inhibitors being prone to
hydrolytic and metabolic processes.
Reported inhibitor 7 represents one
3
(IC50 340 nM)
4 (IC50 6 nM)
NH
O
O
O
N
H
O
N
O
O
Cl
F
5
(F215; IC50 9 nM)
6 (IC50 33 nM)
4
1
of the most stable compounds . All
the known inhibitors, with the
exception of 7, appeared to form
covalent adducts with the catalytic
cysteine (Cys126) of hNAAA. The
adduct formation is driven by the
NCS
O O O
S
N
N
S
N
7
(IC50 230 nM)
8 (IC50 600 nM)
Figure 1. Known NAAA inhibitors
reactive electrophilic character of the pharmacophoric domain of the molecule. The existing
NAAA inhibitors have served as useful pharmacological tools to study the role of NAAA
inhibition in inflammatory conditions2, 3, 29, 42-44, however their poor drug-like properties rendered

them unfavorable as potential therapeutic candidates. Therefore, new generations of druggable
NAAA inhibitors are needed to delineate the role of NAAA inhibition in inflammatory and pain
processes.
2
.
Chemistry
2
.1 Cyanamides hNAAA inhibitors. In
O
O
the design of a new series of NAAA
inhibitors, we have merged two key
structural pharmacophoric features
comprised of the biphenyl tail of the
O
O
N
O
S
O
H
N
H
N
N
1
9
Cathepsin K (IC50 50 nM)
hNAAA (IC50 7nM)
4
0
known NAAA inhibitor 1 (Fig. 1) and
the azetidine-nitrile (cyanamide)
pharmacophore of cathepsin K inhibitor
945
(Fig. 2). In the design of this new
A
O
N
N
1
7a (Table 1)
Figure 2. Design of cyanamides hNAAA inhibitors
class of NAAA inhibitors, we have considered that both NAAA and cathepsin K are accountable
for similar catalytic processes, considering that both belong to the N-terminal nucleophile (Ntn)
4
6
lysosomal family of enzymes with comparable catalytic sites .
Further, cathepsin K drugs have been pursued as potential therapeutics for the treatment of
4
5
47
osteoporosis. Also, the NSAID agent diacerein used to treat osteoarthritis (OA) , was identified
4
8
as a NAAA inhibitor . Also NAAA inhibitor 5 (Fig. 1; F215) was found to be a therapeutic agent
4
9
for OA , indicative that NAAA may play a role in the pathogenesis of degenerative joint disease.
Our primary objective in this study was to merge the two pharmacophoric features of compounds
1
and 9 (shown dashed-lines in Fig. 2) in an attempt to eliminate the high non-specific reactivity
of the -lactone of 1, which showed high potency for NAAA with an IC value of 7 nM, but poor
5
0
plasma stability (t <1 min). At this stage of ligand design, we made the assumption that both
1
/2
NAAA inhibitor 1 and hybrid inhibitor 17a (Fig. 2, Table 1) are active site inhibitors for NAAA.
Conformational analysis of 1 and 17a resulted in a good overlap of these two molecules (Cartoon
A, Fig. 2). Armed with this information, we have prepared hybrid 17a (synthesized according to
scheme 1), which showed high potency for hNAAA using a fluorometric assay with an IC value
5
0
of 2.8 nM after 90-minute pre-incubation period. At a shorter pre-incubation period (15 min), 17a

had an IC value of about 17 nM, indicative of its irreversible or slow reversible (slow K rate)
5
0
off
binding mode with the enzyme.
To better assess the potency of
1
7a, we used a second order rate
constant derived from the ratio
of kinact/K which has been
I
suggested as the appropriate
way to assess the potency of
irreversible inhibitors. Unlike
IC50
values,
kinact/KI
determinations are independent
of pre-incubation times and
therefore are a better measure of
Fig. 3 Ligand interaction diagram of the lowest binding energy pose
of 17f (Table 1) within the hNAAA binding pocket. The hydrogen
bonds are represented by beige lines. The proximal phenyl group is
forming π-stacking interactions with Tyr177 and Tyr146 represented
potency
for
assessing by green lines.
6
-1 -1
irreversible inhibitors. Compound 17a was found to have a high kinact/K value of 3.03x10 M s
I
(Table 1), indicative of its high potency and its irreversible or very slow reversible binding mode.
2
.2 Computer-assisted drug design. We
were delighted that during our structure-
activity relationship (SAR) studies a
cocrystal structure of hNAAA was
disclosed50 and allowed us to use
structural insights of the NAAA binding
pocket to design new analogs.
Examination of the “precovalent” binding
pose of an early-on synthesized inhibitor
Fig. 4 Covalent docking of 17f (Table 1) with hNAAA crystal
structure with the leaving group no longer attached. A
Connolly surface shown as electrostatic potential (red-white-
blue) was used to represent the shape of the binding pocket.
1
7f (Table 1) revealed that the binding Only the key residues (green) and the ligand (white) are shown.
All other atoms are colored by atom type. The ligand orients
pocket of hNAAA predominately into the binding pocket making several π-stacking interactions
shown in blue dashed lines. Hydrogen bonds are indicated by
yellow dashed lines.
consisted of hydrophobic residues
Tyr177, Trp181, Phe148, Tyr146, Met64 (Fig. 3; ligand interaction diagram of compound 17f
docked with hNAAA). The orientation of 17f within the NAAA binding pocket closely resembled

the endogenous substrate PEA (not shown), which carries a 15 carbon long alkyl tail occupying
the hydrophobic binding pocket of NAAA. We have considered that the electrophilic cyanamide
pharmacophore of compound 17f upon interaction with the active site catalytic cysteine (Cys126)
has triggered the formation of a covalent isothiourea adduct. Covalent docking of 17f with
hNAAA (Fig. 4) showed that the catalytic residue Cys126 of hNAAA was coupled with the
electrophilic nitrile of the cyanamide group to form an isothiourea adduct. The rest of the ligand
appeared to orient very similarly to that of the “precovalent” binding pose of the ligand within the
binding pocket. The newly formed isothiourea adduct is time-dependently reversible45 to
regenerate the active enzyme. This interconversion process can be exploited by modifications of
the ligand’s size (length and bulkiness) and generate analogs with different adduct residence time
(). In contrast, fully irreversible adducts (suicidal inhibitors) have raised serious safety concerns
in drug development because of the relationship between covalent drug binding and the potential
of immunogenic-driven allergic reactions and idiosyncratic drug toxicity51, 52
.
Scheme 1
2
.3. New cyanamides
NAAA
a
R2
N
BOC
MeO C
N
BOC
2
X
HO
10
11
12
b
N
BOC
Br
inhibitors: To
further expand
the scope and
breadth of our
initial findings
HO
N-BOC
14
X = CH O; CH OCH
R2 = H, OMe, CF3
2
2
2
R2
R2
R2
X
X
X
c
d
e
N
NH.TFA
N
BOC
CN
R1
R1
R1
16
17
15
X = CH2O; CH2OCH2
R1 = aryl, heteroaryl
R2 = H, OMe, CF3
X = O, CH O; CH OCH
2 2 2
R1 = aryl, heteroaryl
R2 = H, OMe, CF3
X = CH O; CH OCH
2
R1 = aryl, heteroary
R2 = H, OMe, CF3
2
2
d, e
R2
and
improve
X
f
g
N
HO
N-BOC
MeO SO
N-BOC
2
BOC
Ph
14
potency,
12
13
X = O
R2 = H
selectivity and
the ADME
Reagents: (a) NaBH , MeOH, THF; (b) 4-bromobenzyl bromide, NaH, DMF; (c) trifluoroacetic acid,
CH2Cl2; (d) R1-B(OH)2, K2CO3, Pd(PPh3)4, dioxane, H2O; (e) CNBr, Et3N, CH2Cl2; (f) MeSO2Cl,
Et3N, CH2Cl2; (g) Ph-Ph-OH, NaH, DMF.
4
properties of the molecule, we used molecular modeling strategies allowing us to construct
precovalent” NAAA-ligand poses using Glide/Prime to determine how the inhibitor fits
“
into the binding site with the correct binding geometry. For molecules that geometrically
fit into the NAAA binding pocket, we performed modeling studies to calculate the lowest
free-binding energy of the ligand using the CovDock/Prime programs of the Schrodinger
platform. During the optimization process, we have also employed chemoinformatics

calculations (i.e. MW, ClogP, tPSA, LiPE) paying close attention to critical “drug-like”
parameters to achieve stable and water-soluble molecules with good oral bioavailability.
5
0
Examination of the reported hNAAA-inhibitor co-crystal revealed that the NAAA active-
site possesses high flexibility allowing several residues lining the binding cavity to attain
adaptable conformations
Scheme 2
and
accommodate
diverse
Br
Br
a
O
CHO
CHO
structurally
ligands.
R2
F
CHO
W
b
O
1
9
Also,
the
18
computational
studies
O
2
2
1a: W = OH; R2 = H, Br
1b: W = Br; R2 = H, Br
c
20
revealed the presence of a
hydrophobic cavity at the
vicinity of the benzylic
region (methoxy linker of
compound 17f Table 1),
which was not occupied
by the ligand (shown in
R2
R2
O
N BOC
O
X
N CN
d
O
e, f
23: R2 = H
22
g
24: R2 = Br
5: R2 = Ar
X = CH O
R2 = H, Br
2
2
Reagents: (a) phenol, K CO , DMF; (b) NaBH , MeOH; (c) PBr , THF; (d)
NaH, DMF; (e) TFA, CH2Cl2; (f) CNBr, Et3N, CH2Cl2 ; (g) R2-B(OH)2, CsF,
Pd(PPh3)4, dimethoxyethane
2
3
4
3
Fig. 4), representing an area for further exploration with new analogs (discussed below).
We have routinely determined the lowest energy conformations of the ligands in the
absence of protein and then compared it to the lowest energy conformers generated during
“
precovalent” docking to determine ligand adaptability within the binding pocket with
minimal energy penalty for synthetic considerations. Structure-activity relationship studies
have produced many compounds, and in this paper, we outline selected analogs in Tables
1
and 2.
2
.4. Synthesis
The compounds needed to delineate the SAR for this study were prepared according to schemes
1
-6.
2
.4.1 Synthesis of alkoxy-linked analogs: In scheme 1, ester 10 was reduced with sodium
borohydride in MeOH to afford alcohol 11. Next, coupling either alcohol 11 or 12 with 1-bromo-4-
bromomethyl) benzene was accomplished by treatment with sodium hydride in N, N-
dimethylformamide to produce 14 (X = CH O, CH OCH ; R = H). Palladium mediated cross-
(
2
2
2
2

coupling reaction between the aryl-bromide 14 (X = CH O, CH OCH ; R = H) and an appropriate
2
2
2
2
boronic acid in the presence of tetrakis (triphenylphosphine) palladium (0), K CO and
2
3
dioxane/water generated the biaryl product 15 (R = aryl, heteroaryl). Unmasking the azetidine
1
nitrogen of 15 with trifluoroacetic acid in dichloromethane afforded amine 16, which upon
treatment with cyanogen bromide and triethylamine in dichloromethane produced cyanamide 17
(
R = aryl, heteroaryl; X = CH O, CH OCH ; R = H).
1 2 2 2 2
2
.4.2 Synthesis of oxygen-linked analogs: In scheme 1, azetidin-3-ol 12 was treated with
methanesulfonyl chloride and triethylamine to afford methane sulfonate 13, which upon coupling
with [1,1'-biphenyl]-4-ol in the presence of sodium hydride produced azetidine 14 (X = O; R =
2
H). Azetidine 14 was converted to the final cyanamide 17 (X = O; R = H) as described above.
2
2
.4.3 Synthesis of pyrrolidine-cyanamides: The pyrrolidine-cyanamides 42 and 43 (Table 1)
were similarly prepared to the
methods described for the synthesis
of the azetidine-cyanamide 17 in
scheme 1 by substituting azetidin-3-
ol 12 with commercially available
chiral (S)-and (R)-pyrrolidin-3-ols.
Scheme 3
BOC
N
HO
R3
R2
a
b
N
BOC
O
N
BOC
O
R3
R3 = alkyl, aryl
28
2
7
26
Br
NH.TFA
R3
R2 = H, F
R2
O
CN
N
e
2
9
d
c,
R2
R1
R1 = aryl
O
R3
28
30
2
.4.4 Synthesis of phenoxy-
CN
N
d,
e
f
R1
cyanamides: In scheme 2, the
R1 = aryl
R2 = H, F
R3 = alkyl, aryl
R2
O
R3
phenoxy-cyanamides 23-25 (R =
2
3
1
Br
H, Br, aryl, X = CH O) were
2
Reagents: (a) RMgBr or Ar-Li, THF; (b) 4N NaOH, Bu NBr, 4-Br-benzyl bromide
4
prepared as followed. Coupling of
or 2-F, 4-Br-benzyl bromide, CH Cl ; (c) R -B(OH) , K CO , Pd(PPh ) , dioxane,
2
2
1
2
2
3
3 4
H O; (d) TFA, CH Cl ; (e) CNBr, Et N, CH Cl ; (f) R -B(OH) , CsF, Pd(PPh ) ,
2
2
2
3
2
2
1
2
3 4
benzaldehyde 18 with phenol in the
dimethoxyethane
presence K CO , in N, N-dimethylformamide afforded product 19. Next, reduction of either
2
3
aldehyde 19 or 20 (commercially available) with sodium borohydride gave benzylic alcohols 21a
R = H, Br), which were treated with phosphorus tribromide in tetrahydrofuran to afford benzyl
(
2
bromides 21b (R = H, Br). Intermediates 21b were converted to cyanamide 23-25 (R = H, Br,
2
2
aryl, X = CH O) similarly to the methods described in scheme 1.
2

2
.4.5 Synthesis of 3,3-substituted azetidine cyanamides: In scheme 3, cyanamide 30 was
prepared by two synthetic routes. (a) Treatment of 3-oxoazetidine 26 with alkyl- or aryl-Grignard
or lithium reagents to produced alcohol 27 (R = alky, aryl). Coupling of alcohol 27 under base-
3
transfer conditions (sodium hydroxide, tetrabutylammonium bromide) with an aralkyl halide
afforded azetidine 28 (R = aryl, heteroaryl; R = H, F; R = alky, aryl). Intermediate 28 was
2
3
converted to azetidine 30 (steps c to e)
similarly to the methods described in
scheme 1. In an alternate route (b), the N-
BOC intermediate 28 was first converted to
cyanamide 31 (steps d and e) and then by
application of the Suzuki coupling protocol
Ph
N
Scheme 4
Ph
Ph
Ph
OH
a
Ph
Ph
+
O
HO
N
34
33
32
Br
Br
CN
N
NH.HCl
c, d
Ph
Ph
O
O
b
3
6
35
(step f) afforded 30, as described in scheme
R1
Br
Reagents: (a) TsOH, CH Cl ; (b) CH CH(Cl)OCOCl, CH Cl ; (c)
CNBr, Et N, CH Cl ; (d) R -B(OH) , CsF, Pd(PPh ) , dimethoxyethane
2
2
3
2
2
2
.
3
2
2
1
2
3 4
2
.4.6 Synthesis of benzhydryl cyanamides: In scheme 4, the benzhydryl-cyanamide 36 was
prepared by coupling of 1-benzhydrylazetidin-3-ol 32 with diphenyl-methanol 33 under acidic
conditions (i.e. p-toluenesulfonic acid) to produce azetidine 34. Unmasking the azetidine with 1-
chloroethyl
chloroformate,
Scheme 5
BOC
N
followed by treatment with
NH
NH
a
N
+
O
N
BOC
Ph
Br
BrCN/Et N and the Suzuki
Y
3
3
7a
R5
26
38a: R1 = Ph; Y = none
38b: R1 = Br; Y = bond
arylation afforded benzhydryl-
cyanamide 36 (R1 = aryl,
heteroaryl), as described in
scheme 2.
CN
N
37b
N
NH.TFA
d
4
0
N
b, c
Y = none
Y
R5
N
N CN
Y = bond
3
3
9a: R1 = Ph; Y = none
9b: R1 = 3-F,4-OMePh; Y = bond
2
.4.7. Synthesis of nitrogen-
linked cyanamides: In scheme
, the amino-cyanamide 40 and
4
1
O
F
Reagents: (a) Na(OAC) BH, AcOH, THF; (b) 3-F,4-OMePhB(OH) , K CO ,
3
2
2
3
Pd(PPh ) , dioxane, H O; (c) TFA, CH Cl ; (d) CNBr, Et N, CH Cl
2
3
4
2
2
2
3
2
5
the isoindoline-cyanamide 41 were prepared as followed. Reductive amination of 3-oxoazetidine
6 with either amine 37a or 37b using sodium triacetoxy borohydride under acidic conditions (i.e.
2

AcOH) produced azetidine 38a (R = Ph; Y = none) and 38b (R = Ph; Y = bond), respectively,
1
1
which were converted to amino-cyanamides 40 and 41 according to the methods described in
scheme 1.
2
.4.8 Synthesis of amide- and
BOC
Scheme 6
N
sulfonamide-linked
Z
R5
N
H N
2
H
cyanamides: In scheme 6,
N
N
BOC
a, b, c
45 (Z = CO)
+
43
46 (Z = SO2)
cyanamides
49-52
were
or
H
N
42
d, b, c
BOC
BOC
O
O
N
prepared as followed. Benzoic
R5 = CO H, SO Cl
47 (Z = CO)
48 (Z = SO2)
2
2
44
Z
N
acid 42 (R5 = CO H) was
2
CN
CN
N
N
coupled with amines 43 and 44
in the presence of HBTU and
diisopropyl ethylamine to afford
amides 45 and 47, respectively.
Sulfonamide 46 and 48 were
prepared from benzene sulfonyl
Z
X
N
b, c
N
H
51 (Z = CO)
2 (Z = SO2)
4
5
9 (Z = CO)
0 (Z = SO2)
5
O
Reagents: (a) HBTU, (i-Pr) EtN, DMF; (b) TFA, CH Cl ; (c) CNBr, Et N, CH Cl ; (d) (i-
2
2
2
3
2
2
Pr) EtN, CH Cl
2
2
2
chloride 42 (R = SO Cl) and amines 43 and 44 in the presence of diisopropyl ethylamine. The
5
2
BOC-protected intermediates 45-48 were converted to the final products 49-52 similarly to
protocols described in scheme 1.
3
.
Results and discussion
All synthesized compounds were assessed in a fluorescence-based assay for NAAA inhibition with
5
3
N-(4-methyl coumarin) palmitamide (PAMCA) as the substrate . Also, k /K determinations
inact
I
were performed for a large set of compounds, however, the SAR development of this series of
inhibitors was primarily based on IC determinations. Compounds that inhibited hNAAA with an
5
0
IC value of < 30 nM were evaluated in selectivity counter screens. They were tested for their
5
0
5
4
55
ability to inhibit human-recombinant FAAH (hFAAH) and purified rat FAAH (rFAAH) using
the fluorogenic substrate arachidonoyl 7-amino-4-methylcoumarin amide (AAMCA)55, 56.
Following similar fluorometric procedures, all compounds were also tested against hydrolytic
5
7
enzymes recombinant human monoacylglycerol lipase (hMGL) and human alpha/beta-hydrolase
5
8
domain containing 6 (hABHD6) for selectivity. Also, selected inhibitors were evaluated in the

4
5
cathepsin K assay for selectivity against cysteine proteases . Furthermore, selected compounds
5
9
were evaluated for their ability to bind to CB1 and CB2 receptors using rat brain or HEK293 cell
membranes expressing mouse CB2 (mCB2) or human CB2 (hCB2),60-62 respectively, via
3
61-63
competition-equilibrium binding using [ H]CP-55,940
to minimize/eliminate any potential
cross-reactivity due to potential common pharmacophoric features.
A selection of compounds as described in the synthetic schemes (1-6) are shown in Tables 1 and
2
. First, we studied the
linker region of the biphenyl
analog 17a (Table 1).
Truncation of the methoxy-
linker of 17a by one carbon
afforded phenoxy analog
1
7b, which was about 10-
fold less potent for NAAA,
while elongation by one
carbon (analog 17c) didn’t
affect the ligand’s potency.
Replacement of the oxygen
with the N-Me group
Fig. 5 Induced-fit docking of 17f (Table 1) with hNAAA crystal
structure. A Connolly surface shown as electrostatic potential
(
red-white-blue) was used to represent the shape of the binding
pocket. Only the key residues (green) and the ligand (white) are
shown. All other atoms are colored by atom type. The ligand
orients into the binding pocket making several π-stacking
interactions shown in blue dashed lines. Hydrogen bonds are
indicated by yellow dashed lines
(compound 40, Table 1)
resulted in about 7-fold loss
in potency. Next, we explored modifications at the distal phenyl group exemplified by entries 17d
to 17n (Table 1). As we described above, induced-fit docking of 17f with the hNAAA X-ray crystal
structure (Fig. 5) suggested that there was unoccupied space at the binding pocket close to the
distal phenyl group of the ligand. We have introduced substitutions at the distal phenyl group to
maximize ligand protein interactions. The meta-methoxy analog 17d was found to be 3-fold more
potent than the unsubstituted analog 17a, by exhibiting sub-nanomolar potency for hNAAA. The
longer bezyloxy-analog 17e, the para-methoxy analog 17f and the bulkier 2,3-disubstituted
methoxy analog 17g also showed enhanced potency for NAAA when compared to 17a. In contrast,
the 2,6-disubstituted methoxy analog 17h was about 70-fold less potent than 17g. The 2,6-
dimethoxy substitution was not accommodated well at this region of the binding pocket, primarily

driven by conflict contacts between the ligand’s distal phenyl group and nearby residues of the
binding pocket wall (not shown). The 2,6-dimethoxy substitution pattern of the distal phenyl group
has caused the ring to torque almost perpendicular to the proximal phenyl group of the molecule,
thus positioning the ligand in an unfavorable orientation within the binding pocket and prevented
it from making favorable van der Waals contacts with the protein and also hindered it from forming
an efficient covalent adduct with the catalytic residue Cys126 of NAAA. The para-methyl analog
1
7i was about 3-fold more potent than the analogous para-trifluoromethyl analog 17j, while the
smaller electronegative F-nucleus (analog 17k) exhibited good sub-nanomolar potency.
Replacement of the distal phenyl of 17f with a pyridine moiety (entry 17l) to improve the water
solubility of the molecule resulted in about 40-fold loss of potency. Moving the 4-methoxy
substituent of 17l to the ortho-position (entry 17m) it regained about 7-fold of its potency. The
unsubstituted pyridine analog 17n was about 4-fold less potent than the phenyl analog 17a. We
have postulated that the basic nitrogen of the pyridine’s nucleus formed unfavorable electrostatic
contacts with residues of the binding pocket causing a loss in potency.
Next, we have replaced the azetidine nucleus of 17a with a pyrrolidine ring. Replacement of the
azetidine nucleus with the five-membered pyrrolidine moiety (entry 42) resulted in 10-fold loss of
potency (42 vs 17a). The distomer analog 43 was even less potent (~100-fold) than 17a.
Several potent cyanamides (Table 1) with IC < 30 nM for hNAAA were evaluated in selectivity
5
0
counter screens against serine hydrolases FAAH, MGL, ABHD6 and cysteine protease cathepsin
K. All tested analogs exhibited weak to moderate selectivity (~5-20-fold; data not shown) against
these hydrolases.
Table 1. Mono-substituted cyanamides hNAAA inhibitors
CN
N
R2
R1
X
H
hNAAA
IC50 (nM) ± SD a
Kinact/K M s
I
-1
-1
Compd
R
1
R
2
X
Biphenyl analogs
CH
1
7a
Ph
H
2
O
2.8±0.3
3.03x106

1
7b
7c
7d
7e
7f
7g
7h
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
F
O
29±2
3.4±0.5
0.89±0.19
1.4
-
1
Ph
CH
2
OCH
2
3.04 x106
1
3-OMePh
3-benzyloxyPh
4-OMePh
2,3-di-OMePh
2,6-di-OMePh
4-Me-Ph
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
2
2
2
2
2
2
2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
3.05 x106
1
-
1
0.94±0.06
0.88±0.26
67.5
3.25 x106
1
8.40 x106
1
-
1
7i
0.46±0.26
1.6±0.2
0.68±0.25
40±4
2.24 x106
1
7j
4-CF
3
-Ph
2.74 x106
1
7k
7l
7m
7n
4-F-Ph
4-OMe-m-pyridyl
2-OMe-m-pyridyl
m-pyridyl
2.09 x106
1
-
1
5.8±0.4
12±5
-
1
-
1
7o
7p
7q
3-OMePh
0.47±0.17
0.85±0.21
2.8±0.2
1.73 x106
1.40 x106
-
1
3-OMePh
CF
3
1
3-OMePh
OMe
Benzyloxy analogs
2
3
4
PhO
PhO
PhO
PhO
PhO
PhO
PhO
H
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
O
O
O
O
O
O
O
1.1±0.05
1.03±0.05
8.5±1
1.33 x106
2
Br
-
-
2
5a
Ph
2
5b
5c
5d
5e
2-OMe-Ph
4-OMe-Ph
4-CN-Ph
2-CN-Ph
23.7±2.2
5.7±0.3
2.5±0.3
6.6±0.3
2
-
-
-
2
2
Benzhydryl analogs
CH(Ph)O
3
6a
3-OMePh
H
11.6±0.5
-

3
6b
6c
3,5-isoxazole
H
H
CH(Ph)O
CH(Ph)O
91.2±5.7
-
-
3
N-Me-pyrazole
74.7±7.81
Nitrogen-linked analogs
CN
N
N
4
0
1
20.8
-
-
N
N CN
4
1.6±0.2
O
F
Pyrrolidine cyanamides
N
CN
R
O
4
4
2
3
H
26.4±1.1
315±17.6
-
-
Ph
N
CN
S
O
H
Ph
a
IC50s performed in triplicate and determined from eight concentrations in the hNAAA
inhibition assay. Activity measured after 90 min pre-incubation of the inhibitor and enzyme prior
to the addition of the fluorogenic substrate N-(4-methyl coumarin) palmitamide (PAMCA). IC50
values were calculated using Prism software (GraphPad).
In order to address the weak selectivity profile of this new class of NAAA inhibitors, we have
introduced steric/bulky groups with relative and absolute configuration, as well as conformational
restriction at the vicinity of the azetidine-nitrile pharmacophore. Conformational restriction at this
part of the ligand represents a rational optimization approach to interrogate potency and selectivity
by targeting additional van der Waals contacts to improve potency and also to create
discriminatory ligand/protein interactions with residues against other cysteine proteases and serine
hydrolases and enhance ligand selectivity for the target. As we briefly discussed above, our
molecular modeling studies (Fig. 4) with the hNAAA X-ay structure revealed the presence of a
hydrophobic cavity (formed by residues Phe174, Trp181, Tyr146) at the vicinity of the benzylic
position (linker region) of compound 17f. To take advantage of this unoccupied region, we have
introduced substituents on the molecule projecting toward this hydrophobic cavity of the binding
pocket to fill the space and influence potency and selectivity for the target. To that end, we have
introduced substitutions on the ligand as followed:

1
). Introduced ortho-position substitutions on the proximal-phenyl group (Table 1). The fluoro
(17o) and trifluoromethyl (17p) analogs showed comparable potency to that of 17d, while the
methoxy analog 17q was about 3-fold weaker.
Next, we introduced an oxygen atom between the two phenyl groups (phenoxy analog 23), which
offered a higher degree of rotational flexibility than the parent biphenyl analog 17a. Analog 23
was about 3-fold more potent than 17a. Introduction of bulkier substituents at the ortho-position
of 23 shown in entries 24, 25a-25e (Table 1) exhibited a varied degree of potency for NAAA with
IC values in the range of 1-24 nM. The bromo-analog 24 was the most potent analog with an
5
0
IC value of 1 nM, while the phenyl (25a) and 2-OMe-phenyl (25b) analogs were weaker with 8-
5
0
fold and 22-fold potency loss, respectively. The remaining analogs 25c-25e showed a small
reduction in potency. While the ortho-introduced modifications produced potent inhibitors for
NAAA, they only marginally improved the selectivity (~ 10-20-fold) of the molecule against
FAAH and MGL.
2
). Added substituents at the benzylic-position of ligand 17d (Table 1). We have considered that
such modifications would have been beneficial to the molecule by masking the benzylic position
from any potential CYP-450 oxidative liability. We introduced a phenyl substituent (entry 36a)
at the benzylic position, but unfortunately it was not well tolerated and resulted in about 12-fold
drop in ligand potency (36a vs 17d). Replacement of the distal phenyl of 36a with polar groups
(
entries 36b and 36c) has caused even a larger loss in potency.
). Constrained the methoxy-linker of 17f with the preparation of a fused-isoindoline ring (41,
Table 1). This new bicyclic motif exhibited high potency for NAAA with an IC value of 1.6 nM,
3
5
0
but still lacked good selectivity against the serine hydrolases FAAH and MGL.
). Prepared 3,3 di-substituted-azetidine analogs (Table 2).
First, we introduced a small methyl group (entry 30a) which exhibited 2-fold loss in potency (IC50
1.8 nM) when compared to the monosubstituted analog 17d (Table 1), however it was gratifying
4
=
to observe that 30a was highly selective (~500-fold) against hMGL. Analog 30a also exhibited an
improved selective profile against cathepsin K (~50-fold; data not shown). Unfortunately, 30a
showed no improvement in selectivity against hFAAH. The bulkier phenyl group (analog 30b)
was about 4-fold weaker (IC = 9.1 nM) than the methyl analog 30a for hNAAA, showed good
5
0
selectivity (>100-fold) against MGL, but still lacked selectivity against FAAH (~ 6 fold).
Modifications of the distal phenyl of 30b with polar moieties, dioxole (30c) and dioxine (30d)

were tolerated with IC values of 9.2 and 6.1 nM, respectively. The meta-pyridyl analog 30e has
5
0
retained the hNAAA potency (IC = 4.6 nM), while the cyclopropane 30f analog was found to be
5
0
the most potent hNAAA inhibitor with an IC value of 0.35 nM. Induced-fit docking of 30f with
5
0
the hNAAA crystal structure (Fig. 6) revealed that the cyclopropane group of 30f was buried into
the unoccupied hydrophobic cavity formed by residues Trp181, Tyr146, and Phe174 (not shown).
Hydrophobic van der Waals interactions between the cyclopropane ring and the hydrophobic
residues of this hydrophobic cavity could account for the enhanced affinity of 30f.
Table 2. Di-substituted cyanamides hNAAA inhibitors
CN
N
R2
R1
Y
R3
IC50 (nM) ± SD a
or
%
inhibition @ 1 uM
Compd
R
1
R
2
R
3
Y
hNAAA
hFAAH
hMGL
3
,3-Di-substituted azetidines
3
0a
0b
0c
3-OMePh
3-OMePh
H
H
H
Me
Ph
Ph
CH
CH
CH
2
2
2
O
O
O
1.8±0.3
9.1±0.6
9.2±0.9
0.36±0.02
61±4
1058±47
19.5%
59%
3
3
benzo[d][1,3]dioxole
99%
2
,3-dihydrobenzo[b]
1,4]dioxine
3
0d
H
Ph
CH
2
O
6.1±0.8
99%
13.5%
[
3
0e
0f
4-OMe,3F-Ph
4-OMe,3F-Ph
3-OMePh
F
F
m-pyridyl
cyclopropane
Me
CH
CH
2
2
O
O
4.6±0.4
0.35±0.03
1.58±0.08
3.2±0.2
2.0±0.2
1.0±0.3
23%
66%
472±87
11%
3
4
9
0
H
H
CONH
SO NH
5
3-OMePh
Me
2
17.8±2.2
19%
3
,3-Spiro-azetidines

CN
N
5
5
1
2
3-OMePh
3-OMePh
H
H
6.5±0.4
2.8±0-1
6%
4%
O
O
N
CN
N
10.2±1.9
14%
O
S N
a
IC50s performed in triplicate and determined from eight concentrations in the hNAAA inhibition
assay. Activity measured after 90 min pre-incubation of the inhibitor and enzyme prior to the
addition of the fluorogenic substrate N-(4-methyl coumarin) palmitamide (PAMCA). IC50 values
were calculated using Prism software (GraphPad).
Next, to further restrict the conformational flexibility of the azetidine nucleus attached to the
proximal-phenyl group, we have replaced the methoxy-linker of 30a with amide or sulfonamide
moieties. The amide-linked analog 49 was found to be highly potent for hNAAA (IC = 1.58 nM)
5
0
and gratifyingly exhibited good selectivity (>100-fold) against all tested enzymes hFAAH, hMGL,
hABHD6 and cathepsin
K.
The
analogous
sulfonamide 50 was
found to be 2-fold less
potent than the amide 49.
Interestingly,
the
sulfonamide 50 has
retained good selectivity
(
>100-fold)
against Fig. 6 Induced-fit docking of 30f (Table 2) with hNAAA crystal
structure. A Connolly surface shown as electrostatic potential (red-
hMGL, hABHD6 and
cathepsin K, but showed
white-blue) was used to represent the shape of the binding pocket. Only
the key residues (green) and the ligand (white) are shown. All other
atoms are colored by atom type. The ligand orients into the binding
a
strong
inhibitory pocket making several π-stacking interactions shown in blue dashed
lines. Hydrogen bonds are indicated by yellow dashed lines.
activity for hFAAH (IC50
18 nM).
). Prepared 3,3 spiro-azetidine analogs (Table 2).
=
5
Next, we added additional conformational restriction at the azetidine nucleus of 49 and 50 to
further influence the ligand selectivity for the target. Both spiro[3.3]heptane analogs 51 (amide-

linker) and 52 (sulfonamide-linker) were potent
inhibitors for hNAAA with IC values of 6.5 and stability of cyanamides hNAAA
Table 3. Plasma and gastric fluids
5
0
inhibitors
2
.8 nM, respectively. Again, only the amide analog
Gastric fluids
stability
Plasma stability
5
1 showed good selectivity (>100-fold) against
t1/2 min
t1/2 min
serine hydrolases hFAAH, hMGL, hABHD6 and Compd
Human
204
228
127
902
Rat
189
184
-
1
7d
7o
0f
450
271
-
cysteine peptidase cathepsin K, while the
sulfonamide analog 52 was found to be highly
potent for both hNAAA (IC = 2.8 nM) and FAAH
1
3
4
9
-
-
50
221
458
180
-
-
-
-
-
-
5
0
5
5
1
2
(
IC = 10.2 nM) and selective (>100-fold) against
50
MGL, ABHD6 and cathepsin K, in agreement with
the analogous sulfonamide 50.
We have concluded that the amide-linked inhibitors were highly potent (IC < 10 nM) for hNAAA
5
0
and selective (>100-fold) against serine hydrolases, hFAAH, hMGL and hABHD6 and cysteine
peptidase cathepsin K. In contrast, the sulfonamide-linked inhibitors showed potent dual inhibition
for hNAAA and hFAAH and good selectivity (>100-fold) against hydrolases hMGL, and
hABHD6 and peptidase cathepsin K. A very recent report64
Table 4. Microsomal
stability of cyanamides
outlined the utility of dual NAAA-FAAH inhibitors as a potential
hNAAA inhibitors
therapeutic approach for acute lung injury (ALI) due to
inflammatory progression. We believe that the dual NAAA-
FAAH inhibitors represent an exciting new anti-inflammatory
pharmacological approach by potentially combining two distinct
molecular anti-inflammatory pathways, first, NAAA inhibitors to
augment the PEA/PPAR- anti-inflammatory signaling pathway1-
Microsomal stability
t1/2 min
Compd
7d
7j
7o
0b
Human
11.2
6.8
Rat
10
8.9
7.9
14.5
-
1
1
1
3
5.7
15.3
11.3
14.7
4
0
41
4.4
4,
activates the cannabinoid receptors CB1 and CB2, both known for their strong anti-inflammatory
and secondly FAAH inhibitors to increase the endocannabinoid anandamide (AEA) levels which
5
-8
and antinociceptive properties .
3
.1 Compound properties: We have evaluated a small set of potent (IC < 10 nM) cyanamides
50
in stability assays.
3
.1.1 Plasma and gastric fluids stability: The tested cyanamides exhibited good plasma stability
(
t > 2 hours) in human and rat plasma and in gastric fluids (Table 3).
1
/2

3
.1.2 Microsomal stability: The microsomal stability (t , min) of the tested cyanamides in human
1/2
and rat liver microsomal preparations was moderate with t1/2 values in the range of 5 to 15 min
Table 4).
(
The new discovered cyanamides, encompassing both potent NAAA inhibitors and potent dual
NAAA-FAAH inhibitors represent promising pharmacological tools to investigate their potential
role in anti-inflammatory and pain preclinical models. Their pharmacological evaluation in
preclinical models of inflammation and pain will be presented elsewhere in due course.
4
.
Conclusions
NAAA inhibition represents an exciting novel approach to treat inflammatory conditions
supported by recent publications1-4, 30. In this report, we have described the exploration of the
cyanamide moiety as a new pharmacophore for NAAA inhibition that led to the discovery of
highly potent and selective inhibitors. Key analogs demonstrated single-digit nanomolar potency
for hNAAA and showed >100-fold selectivity for the serine hydrolases FAAH, MGL and ABHD6,
and cysteine protease cathepsin K. We have also identified potent and selective dual NAAA-
FAAH inhibitors, which combine two distinct anti-inflammatory molecular pathways by
activating the PEA/PPAR- anti-inflammatory signaling pathway1-4 via NAAA inhibition, and
also stimulating the cannabinoid receptors CB1 and CB2 via augmentation of the endocannabinoid
AEA through FAAH inhibition. Both CB1 and CB2 receptors are known for their anti-
5
-8
inflammatory and antinociceptive properties . Our ligand design followed a traditional structure-
activity relationship (SAR) approach and was supported by molecular modeling studies of the
5
0
reported X-ay cocrystal structures of NAAA . Several inhibitors were evaluated in stability assays
and demonstrated very good plasma stability (t1/2 >120 min; human and rodents), but rather
moderate microsomal stability (t ~5-15 min) in human and rodent liver microsomal preparations.
1
/2
The disclosed cyanamides represent promising new pharmacological tools to investigate the
potential role of NAAA inhibitors and dual NAAA-FAAH inhibitors as therapeutic agents for the
treatment of inflammation and pain.
5
5
. Experimental section
.1. Chemistry

All solvents and reagents were obtained from commercial sources and were used as received. All
non-aqueous reactions were carried out in oven-dried glassware under out under an atmosphere of
dried argon or nitrogen. All reactions were monitored by thin layer chromatography (TLC plates
F254, Merck) or LC-MS analysis. All products, unless otherwise noted, were purified by flash
chromatography by Biotage Isolera purification system using pre-packed silica cartridges. Proton
nuclear magnetic resonance spectra were obtained on a VARIAN 400 spectrometer at 500 MHz.
Spectra are given in ppm () and coupling constants, J values, are reported in hertz. Splitting
patterns are designated as follows: s, singlet; brs, broad singlet; d, doublet; t, triplet; q, quartet; m,
multiplet. Tetramethylsilane was used as an internal reference standard. Mass spectra were
obtained on a Waters Micromass ZQ spectrometer. HPLC techniques and mass spectrometry were
used to determine the purity of the compounds. Purity of all final products was > 96% as
determined by LC-MS using the following protocol. Mobile Phase A = water, B = acetonitrile
solvent gradient 95/5 to 5/95 A:B in 11 min; flow rate 1.5 mL/min; Waters XTerra MS C8 column
(4.6 × 50 mm) with UV detection at 190-400 nm wavelength.
5
.1.1 Method A (scheme 1).
5
.1.1.1 tert-Butyl 3-((4-bromobenzyl)oxy)azetidine-1-carboxylate (14, X = CH O, R = H).
2
2
Step b). Sodium hydride (60% dispersion in mineral oil; 663.2 mg, 16.58 mmol) was added
portionwise into a cold (0 ºC) solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (3, 2.38 g,
1
3.82 mmol) and DMF (20 mL). After stirring for 1 hour, 1-bromo-4-(bromomethyl)benzene (3.8
g, 15.2 mmol) was added and the new mixture was allowed to come to room temperature and
stirred for 12 h. The mixture was then cooled to 0 ºC and MeOH (4 mL) was added dropwise.
Afterwards, the mixture was poured into aqueous ammonium chloride and extracted twice with
ethyl ether. The organic extracts were dried over anhydrous MgSO . The solvents were removed
4
under vacuum and the residue was purified on silica gel (Biotage; eluting solvents hexanes: EtOAc
4
/1 ratio) to afford tert-butyl 3-((4-bromobenzyl)oxy) azetidine-1-carboxylate as off-white solid
1
(
4.32 g, 92% yield): H NMR (500MHz, CDCl ) δ ppm 7.47 (d, J = 8 Hz, 2H), 7.20 (d, J = 8 Hz,
3
1
2
1
H), 4.41. (s, 2H), 4.28 (m, H), 4.05 (dd, J = 8.5, 5.0 Hz, 2H), 3.85 (dd, J = 8.5, 5.0 Hz, 2H),
.41 (s, 9H); MS (ES) m/z 343.1288 [M+1]+.

5
4
.1.1.2 tert-Butyl 3-((4'-methoxy-[1,1'-biphenyl]-4-yl)methoxy)azetidine-1-carboxylate (15, R =
1
-OMePh, R = H, X = CH O).
2
2
Step c). Into a microwave vessel were added tert-butyl 3-((4-bromobenzyl)oxy)azetidine-1-
carboxylate (200 mg, 0.58 mmol), (4-methoxyphenyl)boronic acid (177 mg, 1.16 mmol), K CO
2
3
(240 mg, 1.74 mmol), dioxane (8 mL) and water (2 mL). Argon gas was passed through the
mixture for 10 minutes and then tetrakis(triphenylphosphine)palladium(0) (6.7 mg 0.0058 mmol)
and the argon flow continued for 5 additional minutes. Then, the vessel was sealed and
microwaved at 100 ºC for 1 h. The mixture was diluted with EtOAc (30 mL) and washed with
water and brine. The organics extracts were dried over anhydrous MgSO . The solvents were
4
removed under vacuum and the residue was purified on silica gel (Biotage; eluting solvents
hexanes: EtOAc 4/1 ratio) to afford tert-butyl 3-((4'-methoxy-[1,1'-biphenyl]-4-
1
yl)methoxy)azetidine-1-carboxylate as viscous oil (189 mg, 88% yield): H NMR (500MHz,
CDCl ) δ ppm 7.53 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 6.95 (d,
3
1
J = 8.5 Hz, 2H), 4.47. (s, 2H), 4.33 (m, H), 4.08 (dd, J = 8.5, 5.0 Hz, 2H), 3.85 (dd, J = 8.5, 5.0
Hz, 2H), 1.41 (s, 9H). MS (ES) m/z 370.2211 [M+1]+.
5
.1.1.3. 3-((4'-Methoxy-[1,1'-biphenyl]-4-yl)methoxy)azetidine.TFA salt (16, R = 4-OMePh, R =
1 2
H, X = CH O).
2
Step d). Trifluoroacetic acid (0.36 mL, 4.7 mmol) was added into a mixture of tert-butyl 3-((4'-
methoxy-[1,1'-biphenyl]-4-yl)methoxy)azetidine-1-carboxylate (175 mg, 0.47 mmol) and CH Cl
2
2
(10 mL). The mixture was stirred at room temperature for 6 h and then the volatiles were removed
under vacuum. The residue was taken (3x) successively in CHCl (10 mL) and the volatiles were
3
removed under vacuum to ensure removal of excess TFA. The crude 3-((4'-methoxy-[1,1'-
biphenyl]-4-yl)methoxy)azetidine.TFA salt (180 mg) was carried to the next step.
5
.1.1.4. 3-((4'-Methoxy-[1,1'-biphenyl]-4-yl)methoxy)azetidine-1-carbonitrile (compound 17f, R1
4-OMePh, R = H, X = CH O; Table 1). Step e). Triethylamine (0.33 mL, 2.35 mmol) was
=
2
2
added into a cold (0 ºC) mixture of 3-((4'-methoxy- [1,1'-biphenyl]-4-yl)methoxy)azetidine.TFA
salt (180 mg, 0.47 mmol), and CH Cl (8 mL). After stirring for 30 minutes cyanogen bromide
2
2
(99.5 mg, 0.94 mmol) was added and the mixture was allowed to come to room temperature and
stirred for 4 h. Then, the mixture was diluted in EtOAc (30 mL) and washed with water and brine.

The organics extracts were dried over anhydrous MgSO . The solvents were removed under
4
vacuum and the residue was purified on silica gel (Biotage; eluting solvents hexanes: EtOAc 3/1
ratio) to afford 3-((4'-methoxy-[1,1'- biphenyl]-4-yl)methoxy)azetidine-1-carbonitrile as white
1
solid (112 mg, 81% yield): H NMR (500MHz, CDCl ) δ ppm 7.55 (d, J = 8 Hz, 2H), 7.51 (d, J =
3
1
8
.5 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 4.47. (s, 2H), 4.41 (m, H), 4.24 (dd,
J = 8.5, 5.0 Hz, 2H), 4.11 (dd, J = 8.5, 5.0 Hz, 2H); MS (ES) m/z 295.4289 [M+1]+; purity 98.5%,
retention time 4.71 min.
5
.1.1.5. tert-Butyl 3-(hydroxymethyl)azetidine-1-carboxylate (11; scheme 1).
Step a). Sodium borohydride (756 mg, 20 mmol) was added portionwise into a solution of 1-(tert-
butyl) 3-methyl azetidine-1,3-dicarboxylate (2.11 g, 10 mml) and THF (10 mL). The mixture was
heated to 80 ºC and then MeOH (2 mL) was added very slowly over a 30 minute period. The
mixture was stirred for 1 hour, cooled to room temperature and poured slowly into ice-cold HCl
(0.5 N). The mixture was extracted (3x) with EtOAc and the organic extracts were dried over
anhydrous MgSO . The solvents were removed under vacuum and the residue was purified on
4
silica gel (Biotage; eluting solvents hexanes: EtOAc 2/1 ratio) to afford tert-butyl 3-
1
(
hydroxymethyl)azetidine-1-carboxylate as oil (1.69 g, 90% yield): H NMR (500MHz, CDCl ) δ
3
1
ppm 3.98 (t, J = 8.3, 2H), 3.77 (m, 2H), 3.68 (dd, J = 8.77, 5.37 Hz, 2H), 2.7 (m, H); MS (ES)
m/z 188.2144 [M+1]+.
5
.1.1.6. 3-(([1,1'-Biphenyl]-4-ylmethoxy)methyl)azetidine-1-carbonitrile (compound 17c, X = O;
R2 = H; Table 1).
1
This compound was prepared according to Method A, steps b-e). H NMR (500MHz, CDCl ) δ
3
ppm 7.6 (m, 4H), 7.44 (m, 2H), 7.39 (m, 3H), 4.21 (t, J = 7.81, Hz, 2H), 4.0 (dd, J = 7.81, 5.86
1
1
+
+
Hz, 2H), 3.6 (d, J = 6.35 Hz, H), 2.98 (m, H); MS (ES) m/z 279.4424 [M 1] ; purity 99.5%,
retention time 4.75 min.
5
.1.2 Method B (scheme 2).
5
.1.2.1. 2-Bromo-4-phenoxybenzaldehyde (19).
Step a). Potassium carbonate (5.98 g, 43.28 mmol) was added into a mixture of phenol (2.31 g,
4.6 mmol), 2-bromo-4-fluorobanzaldehyde (5g, 24.6 mmol) and DMF (50 mL). The mixture was
2

o
stirred at 100 C for 18 hours, cooled to room temperature and then poured into water (200 mL).
The mixture was stirred for 30 minutes and the precipitated solid filtered and washed with water.
2
-Bromo-4-phenoxybenzaldehyde as an off-white sold (6.15 g, 90% yield) was collected and used
1
to the next step. H NMR (500MHz, CDCl ) δ ppm 10.24 (s, 1H), 7.9 (d, J = 9.0 Hz, 1H), 7.45-
3
7
9
.42 (m, 2H), 7.27 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 2.5 Hz, 1H), 7.09-7.08 (m, 2H), 6.98 (dd, J =
.0, 2.5 Hz, 1H).
5
.1.2.2. (2-Bromo-4-phenoxyphenyl) methanol (21a).
o
Step b). Sodium borohydride (544.3 mg, 14.4 mmol) was added in portions into a cold (0 C)
mixture of 2-bromo-4-phenoxybenzaldehyde (6.25 g, 22.56 mmol) and anhydrous methanol (30
mL). After the addition the mixture stirred for 30 minutes, and then it was carefully poured into
ice water. The mixture was extracted with ethyl acetate and the organic extracts washed with water
and brine and dried over anhydrous MsSO . The solvents were removed under vacuum and the
4
residue was purified on silica gel (Biotage; eluting solvents hexanes: EtOAc 3/1 ratio) to afford
1
(
2-bromo-4-phenoxyphenyl)methanol as oil (5.92 g, 94.4% yield): H NMR (500MHz, CDCl ) δ
3
ppm 7.42 (d, J = 8.5 Hz, 1H), 7.38-7.34 (m, 4H), 7.2 (d, J = 2.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H),
7
.0 (dd, J = 8.5, 2.5 Hz, 1H), 4.71 (d, J = 6.5 Hz, 2H), 1.97 (t, J = 6.5 Hz, 1H).
5
.1.2.3. 2-Bromo-1-(bromomethyl)-4-phenoxybenzene (21b).
o
Phosphorus tribromide (4.3 mL, 44.74 mmol) was added dropwise into a cold (0 C) mixture of
2-bromo-4-phenoxyphenyl) methanol (4.9 g, 17.56 mmol) and anhydrous tetrahydrofuran (50
(
mL). After the addition the mixture stirred at room temperature for 5 hours, and then it was
carefully poured into ice water. The mixture was extracted with ethyl acetate and the organic
extracts washed with saturated aqueous sodium bicarbonate (three times), water and brine and
dried over anhydrous MgSO . The solvents were removed under vacuum and the residue was
4
purified on silica gel (Biotage; eluting solvents hexanes: EtOAc 30/1 ratio) to afford 2-bromo-1-
1
(
bromomethyl)-4-phenoxybenzene as oil (4.26 g, 71% yield): H NMR (500MHz, CDCl ) δ ppm
3
7
.4-7.36 (m, 3H), 7.19-7.16 (m, 2H), 7.04-7.03 (m, 2H), 6.92 (dd, J = 8., 2.5 Hz, 1H), 4.6 (s, 2H).
.1.2.4. 3-((2-Bromo-4-phenoxybenzyl)oxy)azetidine-1-carbonitrile (compound 24; Table 1).
5

1
This compound was prepared according to Method A, steps b-e). H NMR (500MHz, CDCl ) δ
3
ppm 7.39-7.34 (m, 3H), 7.19 (d, J = 2.5 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.03-7.0 (m, 2H), 6.97
(dd, J = 8.0, 2.5 Hz, 1H), 4.48 (s, 2H), 4.46-4.43 (m, 1H), 4.3-4.27 (m, 2H), 4.16-4.13 (m, 2H);
+
MS (ES) m/z 361.3470 [M+1] ; purity 98%, retention time 5.01 min.
5
.1.2.5. 3-((5-Phenoxy-[1,1'-biphenyl]-2-yl)methoxy)azetidine-1-carbonitrile (compound 25;
Table 1). .
Step f.) Into a microwave vessel were added 3-((2-bromo-4-phenoxybenzyl)oxy)azetidine-1-
carbonitrile (120 mg, 0.33 mmol), phenylboronic acid (60.4 mg, 0.49 mmol), cesium fluoride
(100.3 mg, 0.66 mmol) and anhydrous dimethoxyethane (4 mL). Dry argon gas was passed through
the mixture for 10 minutes and then tetrakis(triphenylphosphine)palladium(0) (12.4 mg 0.0099
mmol) was added and the argon flow continued for 5 additional minutes. Then, the vessel was
o
sealed and microwaved at 100 C for1 hour. The mixture was diluted with EtOAc (30 mL) and
washed with water and brine. The organics extracts were dried over anhydrous MgSO . The
4
solvents were removed under vacuum and the residue was purified on silica gel (Biotage; eluting
solvents hexanes: EtOAc 3/1 ratio) to afford 3-((5-phenoxy-[1,1'-biphenyl]-2-
1
yl)methoxy)azetidine-1-carbonitrile as oil (94.6 mg, 79% yield): H NMR (500MHz, CDCl ) δ
3
ppm 7.44-7.39 (m, 4H), 7.38-7.31 (m, 4H), 7.14 (dt, J = 7.5 Hz, 1.5 Hz, 1H), 7.07-7.05 (m, 2H),
7
.01 (dd, J = 8.0, 2.5 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 4.26 (s, 2H), 4.25-4.2 (m, 1H), 4.14-4.11
+
(
m, 2H), 3.94-3.91 (m, 2H); MS (ES) m/z 357.4797 [M+1] ; purity 98%, retention time 5.18 min.
.
5
.1.3 Method C (scheme 3).
5
.1.3.1. tert-Butyl 3-hydroxy-3-methylazetidine-1-carboxylate (27, R = methyl; scheme 3).
3
Step a). Methyl magnesium bromide (3M in diethyl ether; 2.92 mL, 8.76 mmol) was added
o
portionwise into a cold (0 C) solution of tert-butyl 3-oxoazetidine-1-carboxylate (0.5 g, 2.92
mmol) and anhydrous THF (3 mL). The mixture was allowed to come to room temperature and
stirred for 3 hours and carefully quenched with aqueous ammonium chloride. The mixture was
extracted with ethyl acetate, washed with brine and dried over anhydrous MgSO . The solvents
4
were removed under vacuum and the residue was purified on silica gel (Biotage; eluting solvents
hexanes: EtOAc 1/1 ratio) to afford tert-butyl 3-hydroxy-3-methylazetidine-1-carboxylate as oil

1
(
480 mg, 87% yield): H NMR (500MHz, CDCl ) δ ppm 3.87 (d, J = 9.0 Hz, 2H), 3.82 (d, J = 9.0,
3
2
H), 2.46 (s, 1H), 1.51 (s, 3H), 1.44 (s, 9H).
5
.1.3.2. tert-Butyl 3-((4-bromobenzyl)oxy)-3-methylazetidine-1-carboxylate (28, R = H, R = Me;
2
3
scheme 3)
Step b). Into a mixture of tert-butyl 3-hydroxy-3-methylazetidine-1-carboxylate (480 mg, 2.56
mmol), 1-bromo-4-(bromomethyl)benzene and dichloromethane (5 mL) were added NaOH (4N,
6
1
mL) and tetra-n-butylammonium bromide (82.6 mg, 0.256 mmol). The mixture was refluxed for
6 hours, cooled to room temperature and extracted with ethyl ether. The organic extracts were
washed with water and brine and dried over anhydrous MgSO . The solvents were removed under
4
vacuum and the residue was purified on silica gel (Biotage; eluting solvents hexanes: EtOAc 3/1
ratio) to afford tert-butyl 3-((4-bromobenzyl)oxy)-3-methylazetidine-1-carboxylate as oil (826
1
mg, 90.5% yield): H NMR (500MHz, CDCl ) δ ppm 7.48 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz,
3
2
H), 4.38 (s, 2H), 3.98 (d, J = 9.5 Hz, 2H), 3.74 (d, J = 9.5 Hz, 2H), 1.55 (s, 3H), 1.44 (s, 9H).
5
.1.3.3.
3-((3'-Methoxy-[1,1'-biphenyl]-4-yl)methoxy)-3-methylazetidine-1-carbonitrile
(compound 30a; Table 2).
1
This compound was prepared according to Method A, steps b-e). H NMR (500MHz, CDCl ) δ
3
ppm 7.59 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.36 (t, J = 8.9 Hz, 1H), 7.18-7.16 (m,
1
H), 7.12-7.11 (m, 1H), 6.91-6.89 (m, 1H), 4.46 (s, 2H), 4.26 (d, J = 8.5 Hz, 2H), 3.94 (d, J = 8.5
+
Hz, 2H), 3.87 (s, 3H), 1.64 (s, 3H); MS (ES) m/z 309.4735 [M+1] ; purity 98%, retention time
4
5
5
.82 min.
.1.4 Method D (scheme 4).
.1.4.1. 1-Benzhydryl-3-((4-bromophenyl)(phenyl)methoxy)azetidine (34).
Step a).
A
mixture of 1-benzhydrylazetidin-3-ol 94.54 g, 19.0 mmol), (4-
bromophenyl)(phenyl)methanol (5.9 g, 19.0 mmol), dichloromethane (50 mL) and p-
toluenesulfonic acid (6.5 g, 38 mmol) was refluxed for 1 h under continuous removal of water with
a Dean-Stark trap. The mixture cooled to room temperature, poured into water and extracted with
ethyl acetate. The organic extracts were washed with water and brine and dried over anhydrous
MgSO . The solvents were removed under vacuum and the residue was purified on silica gel
4

(Biotage; eluting solvents hexanes: EtOAc 10/1 ratio) to afford 1-benzhydryl-3-((4-
1
bromophenyl)(phenyl)methoxy)azetidine as oil (5.1 g, 55.5% yield): H NMR (500MHz, CDCl )
3
δ ppm 7.41-7.4 (m, 2H), 7.36-7.34 (m, 4H), 7.3-7.24 (m, 2H), 7.26-7.22 (m, 7H), 7.18-7.14 (m,
4
H), 5.23 (s, 1H), 4.33 (s, 1H), 4.02-4.17 (m, 1H), 3.44-3.36 (m, 2H), 2.94-2.9 (m, 2H).
5
.1.4.2. 3-((4-Bromophenyl)(phenyl)methoxy)azetidine hydrochloride (35).
Step b). 1-Chloroethyl chloroformate (3.69 g, 25.82 mmol) was added to a cold (0 ºC) mixture of
-benzhydryl-3-((4-bromophenyl)(phenyl)methoxy)azetidine (5.0 g, 10.33 mmol), and
dichloromethane (50 mL). The mixture was allowed to come to room temperature and stirred for
0 hours. The volatiles were removed under vacuum and the residue was dissolved in methanol
1
2
and stirred for 1 hour. The methanol removed under vacuum and the crude product (3.68 g) was
used to the next step.
The following compounds were prepared according to Method C.
5
.1.4.3. 3-((3'-Methoxy-[1,1'-biphenyl]-4-yl)(phenyl)methoxy)azetidine-1-carbonitrile (compound
3
6a; Table 1).
1
This compound was prepared according to Method B, steps e-f). H NMR (500MHz, CDCl ) δ
3
ppm 7.56 (d, J = 8.0 Hz, 2H), 7.37-7.33 (m, 8H), 7.15(dd, J = 8.5, 1.0 Hz, 1H), 7.09 (t, J=2Hz,
1
2
H), 6.09 (dd, J = 8.5, 2.5 Hz, 1H), 5.35 (s, 1H), 4.46-4.44 (m, 1H), 4.16-4.14 (m, 2H), 4.11 (m,
+
H), 3.86 (s, 3H); MS (ES) m/z 371.5464 [M+1] ; purity 99.5%, retention time 5.15 min.
5
.1.5 Method E (scheme 5).
5
.1.5.1. tert-Butyl 3-(([1,1'-biphenyl]-4-ylmethyl)(methyl)amino)azetidine-1-carboxylate (38a, R4
=
Ph, Y = none).
Step a). Into a mixture of 1-([1,1'-biphenyl]-4-yl)-N-methylmethanamine hydrochloride (254 mg,
.09 mmol), tert-butyl 3-oxoazetidine-1-carboxylate (169.6 mg, 0.99 mmol), acetic acid (0.17 mL)
1
and anhydrous tetrahydrofuran (3 mL), was added sodiumtriacetoxy borohydride (292 mg, 1.39
mmol). The mixture was stirred for 5 hours and then was quenched with saturated aqueous sodium
bicarbonate. The mixture was extracted with ethyl acetate, washed with brine and dried over
anhydrous MgSO4. The solvents were removed under vacuum and the residue was purified on

silica gel (Biotage; eluting solvents hexanes: EtOAc 2/1 ratio) to afford tert-butyl 3-(([1,1'-
1
biphenyl]-4-ylmethyl)(methyl)amino)azetidine-1-carboxylate as oil (326 mg, 84.9% yield): H
NMR (500MHz, CDCl ) δ ppm 7.59 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.4 (m, 2H),
3
7
3
.36 -7.34 (m, 3H), 3.97-3.94 (m, 2H), 3.88-3.85 (m, 2H), 3.43 (s, 2), 3.3-3.26 (m, 1H), 2.1 (s,
H), 1.45 (s, 9H).
5
.1.5.2. 3-(([1,1'-Biphenyl]-4-ylmethyl)(methyl)amino)azetidine-1-carbonitrile (compound 40;
Table 1).
1
This compound was prepared according to Method A, steps c-e). H NMR (500MHz, CDCl ) δ
3
ppm 7.6-7.58 (m, 2H), 7.56 (d, J = 8.0, 2H), 7.46 (t, J = 8.0 Hz, 2H), 7.36-7.33 (m, 3H), 4.13-
+
4
.08 (m, 4H), 3.49 (m, 1H), 3.43 (s, 2H), 2.13 (s, 3H MS (ES) m/z 278.4513 [M+1] ; purity 100%,
retention time 4.71 min.
5
.1.6 Method F (scheme 6).
5
.1.6.1. Tert-butyl 3-(3'-methoxy-[1,1'-biphenyl]-4-carboxamido)azetidine-1-carboxylate (45, Z =
CO; scheme 6).
Step a). To a stirred solution of 3'-methoxy-[1,1'-biphenyl]-4-carboxylic acid (228 mg, 1 mmol),
and tert-butyl 3-amino-3-methylazetidine-1-carboxylate (189.2 mg, 1.02 mmol.) and N,N-
diisopropyl-ethylamine (452.4 mg, 3.5 mmol) in DMF (5 mL) was added HBTU (1.32g, 3.48
mmol). The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture
was partitioned between water (30 mL) and ethyl acetate (30 mL) and the aqueous layer was
extracted with ethyl acetate (50 mL). The combined organic extracts were washed with saturated
NaHCO (30 mL), and brine (30 mL). The organic layer was dried over Na SO and concentrated
3
2
4
under reduced pressure. The residue was purified on silica gel (Biotage; eluting solvents hexane:
EtOAc 4/1 ratio) to afford tert-butyl 3-(3'-methoxy-[1,1'-biphenyl]-4-carboxamido)-3-
1
methylazetidine-1-carboxylate as a colorless solid in 80% yield; H NMR (500 MHz, CDCl ) δ
3
7
1
2
.83 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.34 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 7.7 Hz,
H), 7.10 (d, J = 1.7 Hz, 1H), 7.04 (s, 1H), 6.94 – 6.87 (m, 1H), 4.16 (s, 2H), 3.89 (d, J = 7.2 Hz,
H), 3.84 (s, 3H), 1.67 (s, 3H), 1.43 (s, 9H).

5
.1.6.2.
compound 49; Table 2).
This compound was prepared according to Method A, steps c-e). H NMR (500 MHz, CDCl ) δ
N-(1-Cyano-3-methylazetidin-3-yl)-3'-methoxy-[1,1'-biphenyl]-4-carboxamide
(
1
3
7
1
.82 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.2 Hz, 2H), 7.38 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 7.7 Hz,
H), 7.13 (s, 1H), 6.94 (dd, J = 8.1, 2.0 Hz, 1H), 6.52 (s, 1H), 4.56 (d, J = 7.8 Hz, 2H), 4.07 (d, J
+
=
7.9 Hz, 2H), 3.87 (s, 3H), 1.73 (s, 3H); MS (ES) m/z 322.4641 [M+1] ; purity 98%, retention
time 4.42 min.
5
.1.6.3.
Tert-butyl
3-((3'-methoxy-[1,1'-biphenyl])-4-sulfonamido)-3-methylazetidine-1-
carboxylate (46, Z = SO ; scheme 6).
2
Step d). To a stirred solution 3'-methoxy-[1,1'-biphenyl]-4-sulfonyl chloride (141 mg, 0.5mmol),
and tert-butyl 3-amino-3-methylazetidine-1-carboxylate (93 mg, 0.5 mmol) in dichloromethane
(
20 mL) was added N,N-diisopropyl-ethylamine (193 mg, 1.5 mmol). The resulting reaction
mixture was stirred at room temperature for 16 hours. After the completion of the reaction
monitored by TLC) the solvent was removed under vacuum. Water was added (10 mL) and the
(
mixture was extracted with ethyl acetate (3x20 mL). The combined extracts were dried over
Na SO and concentrated under reduced pressure. The residue was purified on silica gel (Biotage;
2
4
eluting solvents hexane: EtOAc 4/1 ratio) to give tert-butyl 3-((3'-methoxy-[1,1'-biphenyl])-4-
1
sulfonamido)-3-methylazetidine-1-carboxylate in as a colorless solid (212 mg, 98% yield); H
NMR (500 MHz, CDCl ) δ 7.92 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.40 (t, J = 8.0 Hz,
3
1
H), 7.19 (d, J = 7.7 Hz, 1H), 7.14 – 7.11 (m, 1H), 6.97 (dd, J = 8.2, 1.9 Hz, 1H), 4.60 (d, J = 8.1
Hz, 2H), 3.92 (d, J = 9.6 Hz, 2H), 3.88 (s, 3H), 3.80 (s, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.45 (s, 9H).
The following compound was prepared according to Methods A and F.
5
.1.6.4.
3-((3'-Methoxy-[1,1'-biphenyl]-4-yl)sulfonyl)-3-methylazetidine-1-carbonitrile
(compound 50; Table 2).
1
H NMR (500 MHz, CDCl ) δ 7.93 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 7.9
3
Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.01 – 6.95 (m, 1H), 4.92 (s, 1H), 4.31 (d, J = 8.1
Hz, 2H), 3.91 (d, J = 8.2 Hz, 2H), 3.88 (s, 3H), 1.53 (s, 3H).; MS (ES) m/z 358.4389 [M+1]+;
purity 100%, retention time 4.51 min.