Potent α-amino-β-lactam carbamic acid ester as NAAA inhibitors. Synthesis and structure-activity relationship (SAR) studies

Article abstract of DOI:10.1016/j.ejmech.2016.01.046

4-Cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (3b) is a potent, selective and systemically active inhibitor of intracellular NAAA activity, which produces profound anti-inflammatory effects in animal models. In the present work, we describe structure-activity relationship (SAR) studies on 3-aminoazetidin-2-one derivatives, which have led to the identification of 3b, and expand these studies to elucidate the principal structural and stereochemical features needed to achieve effective NAAA inhibition. Investigations on the influence of the substitution at the β-position of the 2-oxo-3-azetidinyl ring as well as on the effect of size and shape of the carbamic acid ester side chain led to the discovery of 3ak, a novel inhibitor of human NAAA that shows an improved physicochemical and drug-like profile relative to 3b. This favourable profile, along with the structural diversity of the carbamic acid chain of 3b, identify this compound as a promising new tool to investigate the potential of NAAA inhibitors as therapeutic agents for the treatment of pain and inflammation.

Full text of DOI:10.1016/j.ejmech.2016.01.046

Accepted Manuscript
Potent α-Amino-β-Lactam Carbamic Acid Ester as NAAA Inhibitors. Synthesis and
Structure–Activity Relationship (SAR) Studies
Andrea Nuzzi, Annalisa Fiasella, Jose Antonio Ortega, Chiara Pagliuca, Stefano
Ponzano, Daniela Pizzirani, Sine Mandrup Bertozzi, Giuliana Ottonello, Glauco
Tarozzo, Angelo Reggiani, Tiziano Bandiera, Fabio Bertozzi, Daniele Piomelli
PII:
S0223-5234(16)30046-0
10.1016/j.ejmech.2016.01.046
EJMECH 8336
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 November 2015
Revised Date: 18 January 2016
Accepted Date: 24 January 2016
Please cite this article as: A. Nuzzi, A. Fiasella, J.A. Ortega, C. Pagliuca, S. Ponzano, D. Pizzirani, S.M.
Bertozzi, G. Ottonello, G. Tarozzo, A. Reggiani, T. Bandiera, F. Bertozzi, D. Piomelli, Potent α-Amino-β-
Lactam Carbamic Acid Ester as NAAA Inhibitors. Synthesis and Structure–Activity Relationship (SAR)
Studies, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.046.
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ACCEPTED MANUSCRIPT
Potent α-Amino-β-Lactam Carbamic Acid Ester as NAAA Inhibitors.
Synthesis and Structure–Activity Relationship (SAR) Studies
†
,§,♯
†,∞,♯
†,♯
†,
†
†
Andrea Nuzzi,
Annalisa Fiasella,
Jose Antonio Ortega, Chiara Pagliuca, Stefano Ponzano,
†
†
†
†,◊
Daniela Pizzirani, Sine Mandrup Bertozzi, Giuliana Ottonello, Glauco Tarozzo, Angelo Reggiani,
†
*,†
*,†,ǁǁ
Tiziano Bandiera, Fabio Bertozzi and Daniele Piomelli
†
Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, I-16163 Genova,
Italy;
ǁǁ
Departments of Anatomy and Neurobiology, Pharmacology and Biological Chemistry, University of
California, Irvine, USA, 92697-4625
1

ACCEPTED MANUSCRIPT
Corresponding Authors
For F.B.: phone, +39-010-71781235; e-mail, fabio.bertozzi@iit.it
For D.P.: phone, +39-010-71781509; fax, +39-010-71781228; e-mail, daniele.piomelli@iit.it
Present Addresses
§
A.N.: Nuffield Department of Medicine Research Building (NDMRB), Target Discovery Institute
(
TDI), University of Oxford, Old Road Campus, Oxford OX3 7FZ, United Kingdom. email:
andrea.nuzzi@ndm.ox.ac.uk
∞
A.F.: Bone Therapeutics S.A., 37 rue Auguste Piccard, 6041 Gosselies, Belgium. email:
afiasella@gmail.com
◊
G.T.: In vitro Pharamacology, Evotec AG., Essener Bogen 7–22419 –Hamburg (Germany). email:
glauco.tarozzo@evotec.com
Author Contributions
♯
A.N., A.F. and J.A.O. contributed equally to this work.
A.N., A.F., J.A.O., C.P., S.P. and D.P. synthesised compounds. S.M.B. and G.O. performed plasma and
metabolic stability tests, as well as solubility analyses. A.R. and G.T. developed fluorogenic human
NAAA in vitro assay. D.P., T.B. and F.B. wrote the manuscript.
2

ACCEPTED MANUSCRIPT
Graphical Abstract
3

ACCEPTED MANUSCRIPT
Abstract
4
-Cyclohexylbutyl-
N
-[(S
)-2-oxoazetidin-3-yl]carbamate (3b) is a potent, selective and systemically
active inhibitor of intracellular NAAA activity, which produces profound anti-inflammatory effects in
animal models. In the present work, we describe structure–activity relationship (SAR) studies on 3-
aminoazetidin-2-one derivatives, which have led to the identification of 3b, and expand these studies to
elucidate the principal structural and stereochemical features needed to achieve effective NAAA
inhibition. Investigations on the influence of the substitution at the β-position of the 2-oxo-3-azetidinyl
ring as well as on the effect of size and shape of the carbamic acid ester side chain led to the discovery
of 3ak, a novel inhibitor of human NAAA that shows an improved physicochemical and drug-like
profile relative to 3b. This favourable profile, along with the structural diversity of the carbamic acid
chain of 3b, identify this compound as a promising new tool to investigate the potential of NAAA
inhibitors as therapeutic agents for the treatment of pain and inflammation.
Keywords
N
-acylethanolamine acid amidase; cysteine hydrolase; inhibitors;
β-lactams; carbamates; structure–
activity relationships
4

ACCEPTED MANUSCRIPT
Abbreviations Used
NAAA,
PEA, palmitoylethanolamide; FAAH, fatty acid amide hydrolase; AC, acid ceramidase; PPAR–
peroxisome proliferator-activated receptor– PAMCA, -(4-methyl-2-oxo-chromen-7-yl)-
N-acylethanolamine acid amidase; FAEs, fatty acid ethanolamides; OEA, oleoylethanolamide;
α,
α
;
N
hexadecanamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; 2-DPC, di-2-pyridyl
carbonate.
5

ACCEPTED MANUSCRIPT
Introduction
Identifying novel molecular targets for the treatment of pain and inflammation is of pivotal therapeutic
importance and remains a major challenge for researchers in industry and academia.[1] Since its
discovery in 2005,[2] the intracellular cysteine amidase
N-acylethanolamine acid amidase (NAAA) has
attracted increasing attention as a potential anti-inflammatory target, due to its essential role in the
regulation of lipid-amide signalling at peroxisome proliferator-activated receptor-
macrophages and other host-defence cells.[2-8]
α (PPAR-α) in
NAAA belongs to the
N-terminal nucleophile (Ntn) family of enzymes and catalyses the deactivating
cleavage of saturated and monounsaturated fatty acid ethanolamides (FAEs) into the corresponding free
fatty acids and ethanolamine,[3, 4, 9,10] thus interrupting their peroxisome proliferator-activated
receptor–
α (PPAR-α) mediated actions. NAAA is primarily localized to macrophages[11] and B-
lymphocytes[8] and, like other Ntn enzymes, is activated by auto-proteolysis, which occurs at acidic
pH and generates a catalytically competent form of the enzyme.[12]
The FAEs are a class of bioactive lipids[13-15[ that contribute to the control of numerous physiological
functions, including pain and inflammation.[16-19] Among these lipids, NAAA preferentially degrades
palmitoylethanolamide (PEA) and oleoylethanolamide (OEA).[2-4] PEA is produced by many
mammalian cells,[20] and has been shown to inhibit peripheral inflammation and mast cell
degranulation[21, 22] and to exhibit antinociceptive properties in rat and mouse models of acute and
chronic pain.[23-25] In addition, evidence suggests that PEA may attenuate skin inflammation and
neuropathic pain in humans.[26-28] These effects are mainly dependent on the ability of PEA to
activate PPAR–α.[19, 29-31] Recent studies have shown that sustaining endogenous PEA levels in
vivo by protection from NAAA-catalysed degradation attenuates lung inflammation[8] and
6

ACCEPTED MANUSCRIPT
hyperalgesic and allodynic states elicited in mice and rats by local inflammation or nerve damage.[6,
3
2]
Despite the encouraging pharmacological benefits achieved by restoring intracellular FAE levels by
inhibition of NAAA, only few chemical classes of potent inhibitors have been discovered.[33] Among
them, compounds featuring an electrophilic warhead, such as
high potency as NAAA inhibitors.[5, 34-37] Topical administration of the potent and selective
lactone NAAA inhibitor, 5-phenylpentyl- -[(2 ,3 )-2-methyl-4-oxo-oxetan-3-yl]carbamate (ARN077,
, Figure 1),[35, 38, 39] was reported to elevate PEA and OEA levels in mouse skin and sciatic nerve
α-amino-β-lactone derivatives, displayed
β
-
N
S R
1
tissues, and to attenuate nociception in mice and rats through a mechanism that required PPAR–
α
activation.[6]
ARN077 (1)
2, X = CH
a, X = O
2
ARN726 (3b)
3
Figure 1. Potent
β
-lactone- (1) and
β
-lactam-based (2, 3a and 3b) NAAA inhibitors.
However,
α
-amino-
β
-lactone derivatives have limited chemical and plasma stability,[36, 40, 41] due to
-lactone ring, which restricts their in vivo use to topical applications.[6,
5] To overcome the limited stability of this class of inhibitors, we investigated compounds in which
an -amino- -lactam (3-aminoazetidin-2-one) group replaces the -lactone ring. These compounds
retain key structural features of the -lactone series, and showed good human-NAAA (h-NAAA)
the hydrolytic cleavage of the
β
3
α
β
β
β
inhibitory activity and favourable physicochemical properties for systemic administration.[42] In
7

ACCEPTED MANUSCRIPT
particular,
N
-[(S)-2-oxoazetidin-3-yl]nonanamide (2, Figure 1) displayed an acceptable inhibitory
potency (IC50 = 0.34 µM) against h-NAAA, good stability in buffer (PBS at pH 7.4 and 5.0) and in
mouse and rat plasma, and good oral bioavailability after single oral administration in rats.[42]
Interestingly, the replacement of the exocyclic amide functionality with a carbamic acid ester, as in
derivative 3a (Figure 1) was beneficial for NAAA inhibitory activity (IC = 0.122 µM).[42,43]
5
0
This prompted us to investigate further the structural and stereochemical features of this new series of
derivatives, leading after a modification on the carbamic acid side chain of 3a to the discovery of 4-
cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (ARN726, 3b, Figure 1), as a novel and potent
NAAA inhibitor.[8] Compound 3b inhibited NAAA activity both in vitro and in vivo as demonstrated
by a competitive activity-based protein profiling (ABPP) study with a structurally similar chemical
probe.[44] Consistent with those findings, 3b exerted marked anti-inflammatory effects in mouse
models of inflammation and suppressed LPS-stimulated inflammatory reactions in human
macrophages.[8]
In the present work, we describe the structure–activity relationship (SAR) work that has led to the
identification of
to achieve -NAAA inhibition with this class of compounds. In particular, we investigated the
influence of the substitution at -position of the 2-oxo-3-azetidinyl ring as well as the effect of the size
β-lactam 3b and the elucidation of key structural and stereochemical features needed
h
β
and shape of the carbamic acid ester side chain. We also outline the selectivity, stability, and solubility
of the best among these novel NAAA inhibitors, which might aid further experimental investigations of
the roles of NAAA.
8

ACCEPTED MANUSCRIPT
Chemistry
The synthesis of differently substituted
an enantio- and diasteroselective fashion by a general strategy consisting of coupling an appropriate 3-
amino- -lactam core with a desired activated alcohol. Initially, a series of -lactam carbamates bearing
a 4-cyclohexylbutyl side chain (3b–10b) was synthesized as reported in Scheme 1.
β-lactam carbamic acid esters was efficiently accomplished in
β
β
(
)3
1
1b
a
(
)3
(
)3
1
3b
1
2b
b
b
b
(
(
(
(
2S,3S)-16
2R,3R)-17
2R,3S)-18
2S,3R)-19
(
(
S)-14
R)-15
(
S)-20
(
R)-21
(
)3
( )3
( )3
(
(
S)-3b
R)-4b
(2S,3S)-5b
(2R,3R)-6b
(S)-22b
(R)-23b
(
(
2R,3S)-7b
2S,3R)-8b
c
(
)3
(
S)-9b
R)-10b
(
9

ACCEPTED MANUSCRIPT
a
Scheme 1. General Synthetic Pathways to
β
-Lactam Carbamic Acid Esters 3b–10b.
a
Reagents and conditions: (a) 2-DPC, DMAP, dry CH
2
Cl
2
2 2
, room temperature, 16 h; (b) DIPEA, dry CH Cl , room
temperature, 16 h; (c) CAN, CH CN/H O (4:1), 0 °C to room temperature, 1 h, then acetone/sat. sol. NaHCO (1:1), room
3
2
3
temperature, 16 h. [TMS = Trimethylsilyl]
The commercially available 4-cyclohexylbutan-1-ol (11b) was reacted with di-2-pyridyl carbonate (2-
DPC) and catalytic DMAP to give an isomeric mixture (1.7:1 ratio) of 4-cyclohexylbutyl 2-pyridyl
carbonate (12b) and 4-cyclohexylbutyl 2-oxopyridine-1-carboxylate (13b).[38,42,45] Then, the
coupling reaction with (
enantiomeric 4-cyclohexylbutyl-
Scheme 1).[8, 42] The reaction of the isomeric mixture 12b and 13b with (2
S
)- or (
-[(S/R)-2-oxoazetidin-3-yl]carbamates (3b and 4b, respectively)
,3 )-(2-methyl-4-oxo-
R)-2-oxoazetidin-3-yl ammonium acetate (14 and 15) afforded
N
(
S S
azetidin-3-yl) ammonium tosylate (16), its enantiomer (2
,3 )-18 and (2 ,3 )-19[46] afforded the desired -methyl
to good yields (Scheme 1). Finally, the coupling reaction with both
lactam amine 20 and its ( )-enantiomer 21 yielded, after oxidative cleavage of the
bis(trimethylsilyl)methyl group, the 2,2-dimethyl -lactam carbamates 9b and 10b (Scheme 1).[47]
R,3R
)-17, and the corresponding epimers
(
2R
S
S
R
β
β-lactam carbamates 5b–8b in moderate
N
-protected 2,2-dimethyl (S)-β-
R
β
(
2
S
,3
sequence,[48,49] starting from the commercially available
converted into the corresponding -methyl hydroxamate derivative (26) in good yield (Scheme 2).
S
)-2-Methyl-4-oxo-azetidin-3-yl ammonium tosylate 16 was prepared following a five-step
N
-Cbz- -threonine, which was initially
L
O
1
0

ACCEPTED MANUSCRIPT
a
Scheme 2. Synthesis of Isomeric 2-Methyl-4-oxo-azetidin-3-yl Ammonium Tosylate Salts 16–19.
Ms
O
Cbz
H O
O
Cbz
HO
O
Cbz
O
O
(2S,3S)-34
b
c
N
a
N
H
N
H
N
H
(
2R,3R)-35
2R,3S)-36
2S,3R)-37
Cbz
O
N
H
(
(
O H
N H
N H
O
O
(
(
(
(
2S,3R)-N-Cbz-L-Thr
2R,3S)-N-Cbz-D-Thr
2S,3S)-24
2R,3R)-25
(1S,2R)-26
(1R,2S)-27
(1S,2S)-28
(1R,2R)-29
(1S,2R)-30
(1R,2S)-31
(1S,2S)-32
(1R,2R)-33
d
HN
e
HN
–
Cbz
N H +3 O Ts
O
O
N
H
(
(
(
(
2S,3S)-16
2R,3R)-17
2R,3S)-18
2S,3R)-19
(2S,3S)-38
(2R,3R)-39
(2R,3S)-40
(2S,3R)-41
a
.
Reagents and conditions: (a) CH ONH HCl, EDC, THF/H O (3:1), room temperature, 3 h; (b) CH S(O) Cl, dry Pyridine,
3
2
2
3
2
-
5 °C to 0 °C, 3h; (c) K CO , Acetone, 100 °C, 3 h; (d) SmI (0.5 M in THF), dry THF, room temperature, 30 min; (e) 1,4-
2 3 2
Cyclohexadiene, 10% Pd on charcoal, EtOH, room temperature, 2 h, then p-TsOH, EtOAc, room temperature, 1 h. [Cbz =
Benzyloxycarbonyl; Ms = Mesyl; Ts = p-Tosyl]
Mesylation of 26 gave compound 30, which was cyclized under basic conditions (K CO ) to afford the
2
3
desired 2-azetidinone (34) in high yields (73%, over 2 steps). Selective
carried out using a fresh solution of samarium iodide, leading to the formation of the
lactam 38.[49] Cbz-deprotection of 38 and in situ trapping of the resulting 2-methyl-3-aminoazetidin-4-
one with -toluensulfonic acid gave the tosylate salt 16 in quantitative yield (Scheme 2).
N
-methoxy deprotection was
β
-methyl β-
p
1
1

ACCEPTED MANUSCRIPT
Tosylate salts 17–19 were stereoselectively obtained using the same procedure, starting either from the
commercially available
N
-Cbz-D-threonine or the synthesized L- and D-N-Cbz-allo-threonine (24 and
2
5), respectively (Scheme 2) (see Supporting Information).
The (S)-3-amino-1-(bis(trimethylsilyl)methyl)-4,4-dimethylazetidin-2-one 20 was prepared by
modifications of previously reported procedures. The synthetic sequence was based on a [2+2]-
cycloaddition reaction between the properly protected imine 44 and the Evans-Sjögren ketene,
generated in situ from the enantiomerically pure oxazolidinone 42 (Scheme 3).[47,50] Final removal of
Evans chiral auxiliary under modified Birch reduction conditions (Na-SG,
Supporting Information), afforded the ( )- -protected 3-amino- -lactam 20.[51] The enantiomeric (
-2,2-dimethyl -lactam amine 21 was obtained following the same synthetic pathway starting from
intermediate ( )-43.[52]
t
-AmOH, EDA, see
S
N
β
R)-
β
β
R
Scheme 3. Synthesis of (
S
)- and (R)-3-Amino-1-[bis(Trimethylsilyl)methyl]-4,4-dimethyl-azetidin-2-
a
one (20,21).
a
4
4
b
c
3
(S)-45
(S)-20
(R)-21
(
(
S)-42
R)-43
(
R)-46
1
2

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) Acetone, room temperature, 12 h; (b) dry Et N, 4Å MS, dry CH Cl , 0 °C to 55 °C, 12 h; (c)
3
2
2
Na-SG(I) 35-40%, ethylendiamine, dry THF, 0 °C, 15 min, then 2-methyl-2-butanol, 0 °C, 5 min, then 45 or 46 in dry THF,
°C to room temperature, 2 h. [TMS = Trimethylsilyl]
0
A series of differently substituted (S)-aminoazetidin-2-one carbamate derivatives (3b–am) was easily
accessed as shown in Scheme 4. Various alcohols (11b–am) were allowed to react with 2-DPC to give
a crude mixture of pyridyl carbonates 12b–am and 2-oxopyridine-1-carboxylates 13b–am, which was
subsequently coupled with the acetate salt (S)-14 (Scheme 4).
a
Scheme 4. Synthesis of (
S)-2-Oxoazetidin-3-yl-carbamic Acid Esters 3b–am.
a
b
11b-am
12b-am
13b-am
3b-am
1
1
u: R = (3-cyclohexylphenyl)methyl
b: R = 4-cyclohexylbutyl
1
1
v: R = (2-cyclohexylphenyl)methyl
c: R = n-octyl
1
1
w: R = (4-cyclohexylphenyl)methyl
d: R = 5-phenylpentyl
1
1
x: R = (4-cyclopentylphenyl)methyl
e: R = (4-phenylphenyl)methyl
1
1
y: R = (4-methylphenyl)methyl
f: R = 3-cyclohexylpropyl
1
1
z: R = (4-ethylphenyl)methyl
g: R = 5-cyclohexylpentyl
1
1
aa: R = (4-propylphenyl)methyl
h: R = 2-(cyclohexylmethoxy)ethyl
1
1
ab: R = (4-n-butylphenyl)methyl
i: R = 3-(cyclohexoxy)propyl
1
1
ac: R = (4-tert-butylphenyl)methyl
j: R = 4-tetrahydropyran-4-yl-butyl
1
1
ad: R = tetralin-6-yl-methyl
k: R = 4-(4,4-difluorocyclohexyl)butyl
1
1
ae: R = indan-5-yl-methyl
l: R = 4-butylcyclohexyl
1
1
af: R = 1-(4-cyclohexylphenyl)ethyl
m: R = 4-cyclopentylbutyl
1
1
ag: R = (4-tetrahydropyran-4-yl-phenyl)methyl
n: R = 4-phenylbutyl
1
1
ah: R = (4-cyclohexoxyphenyl)methyl
o: R = 4-(4-ethylphenyl)butyl
1
1
ai: R = (4-propoxyphenyl)methyl
p: R = 4-(4-methoxyphenyl)butyl
1
1
aj: R = 1,3-benzodioxol-5-yl-methyl
q: R = 4-(4-trifluoromethylphenyl)butyl
1
1
ak: R = (2,2-difluoro-1,3-benzodioxol-5-yl)methyl
r: R = 4-(2-pyridyl)butyl
1
1
al: R = [4-(3-pyridyl)phenyl]methyl
s: R = (E)-4-phenylbut-3-enyl
1
1
am: R = (4-oxazol-4-ylphenyl)methyl
t: R = (Z)-4-phenylbut-3-enyl
a
Reagents and conditions: (a) 2-DPC, DMAP, dry CH Cl , room temperature, 16 h; (b) (S)-14, DIPEA, dry CH Cl , room
2
2
2
2
temperature, 16 h.
1
3

ACCEPTED MANUSCRIPT
Most of the used alcohols were commercially available (11b–f,l,n,p,r,y,z,ab,ac,ae,aj) or prepared by
reduction of the corresponding commercially available precursors (11g,s,v,w,aa,ad,af,ak,al, see
Supporting Information). The remaining alcohols were either obtained following previously reported
protocols (11i,[53] j,[54] t,[55] u,[56] ah,[37] see Supporting Information) or synthesized as described
in Schemes 5 and 6 (11h,k,m,o,q,x,ag,ai,am).
Compound 11h was prepared in a straightforward manner by heating bromo-methylcyclohexane in
ethylene glycol, under strong basic conditions (Scheme 5).[57, 58]
a
Scheme 5. Synthesis of Alcohols 11h,k,m,o,q.
a
Reagents and conditions: (a) ethylene glycol, 25.5 M NaOH, 80 °C, 16 h; (b) [3-benzyloxypropyl-(Ph) P]Br, n-BuLi (2.5
3
M in hexane), dry THF, -30 °C, 45 min, then 47–49, -30 °C, 45 min, then room temperature, 16 h; (c) H , 10% Pd(OH) /C
2 2
(
H-Cube, EtOH, 60 °C, 1.0 bar); (d) [2-(1,3)-dioxolan-2-yl)-ethyl-(Ph)
3
P]Br, n-BuLi (2.5 M in hexane), dry THF, -30 °C,
4
5 min, then cyclopentanecarbaldehyde, -30 °C, 45 min, then room temperature, 16 h; (e) H , 10% Pd/C (H-Cube, EtOH, 35
2
°
C, 1.0 bar); (f) 70% AcOH, THF, 65 °C, 16 h; (g) NaBH , EtOH, room temperature, 30 min.
4
1
4

ACCEPTED MANUSCRIPT
Alcohols 11k,o,q were obtained in moderate to good yields starting from the corresponding aldehydes
(
47–49) via Wittig olefination with a benzyl-protected phosphonium salt, followed by a one-pot
reduction/deprotection reaction, using Pd(OH) in the H-Cube flow reactor (Scheme 5). A similar
synthetic strategy was used for compound 11m, where cyclopentanecarbaldehyde was reacted with 2-
1,3-dioxolan-2-yl)-ethyl-(triphenyl)-phosphonium bromide to give olefin 53 (7:93 E/Z ratio).
2
(
Reduction of the derivative 53, followed by dioxolane cleavage, afforded aldehyde 55 which upon
reduction with NaBH furnished the desired compound (Scheme 5).
4
The synthesis of alcohols 11x,ag was accomplished using minor modifications of a protocol reported
by Fu and co-workers (Scheme 6).[59]
a
Scheme 6. Synthesis of Alcohols 11x,ag,ai,am.
a
b
c
d
b
5
6
11x
57
58
11ag
e
f
g
h
b
59
11ai
6
0
61
11am
a
Reagents and conditions: (a) 4-methoxycarbonyl-phenyl boronic acid, sodium hexamethyldisylazane, trans-2-hydroxy-
cyclohexylamine HCl, NiI , i-PrOH, room temperature, 5 min, then bromocyclopentane, 60 °C, 6 h; (b) LiAlH , dry THF, 0
C, then room temperature, 3 h; (c) phenyl boronic acid, KHMDS, (S)-(+)-prolinol, NiCl -glyme, i-PrOH, room
temperature, 10 min, then 4-chlorotetrahydropyran, 65 °C, 50 h; (d) (COCl) , dry CH Cl , -20 °C, 30 min, then AlCl , -20
2
4
°
2
2
2
2
3
1
5

ACCEPTED MANUSCRIPT
°
C, 1 h, then room temperature, 1 h, then MeOH, -20 °C, 16 h; (e) 1-bromopropane, NaH, room temperature, 18 h, then 80
+
-
°
C, 3h; (f) LiAlH , dry Et O, 0 °C, then room temperature, 4h; (g) CuBr , EtOAc, reflux, 16 h; (h) NH HCOO , HCOOH,
4 2 2 4
reflux, 7 h.
The Suzuki coupling between bromocyclopentane and 4-methoxycarbonyl-phenyl boronic acid, using
NiI as a catalyst, furnished isopropyl ester 56, which was directly reduced with LiAlH to give alcohol
2
4
1
1x. 4-Phenyl-tetrahydropyranyl derivative 57, obtained via Suzuki reaction between phenyl boronic
acid and 4-chlorotetrahydropyran, was regioselectively acylated via a Friedel-Crafts reaction[60] and
then subjected to LiAlH reduction to give compound 11ag. -Alkylation of ethyl 4-hydroxybenzoate
4
O
with 1-bromopropane led to derivative 59, which upon reduction of the ethyl ester under standard
conditions gave the alcohol 11ai. Finally, alcohol 11am was obtained in a reasonable yield (27%) after
a three steps sequence, which consisted in the formation of the oxazole ring starting from ethyl 4-
acetylbenzoate followed by LiAlH reduction of the ethyl ester (Scheme 6).[61, 62]
4
1
6

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Results and Discussion
In a previous study on NAAA inhibitors we reported that
amide derivatives display a set of pharmacological and physicochemical properties that render them
suitable for systemic administration, overcoming the limited hydrolytic and plasma stability of
amino- -lactone-based NAAA inhibitors, such as ARN077.[42]
The compound -[( )-2-oxoazetidin-3-yl]nonanamide (2, Figure 1) showed acceptable inhibitory
potency (IC50 = 0.34 M) for -NAAA, good stability in buffer (at pH 7.4 and 5.0) and in mouse and
α
-amino-β-lactam (3-aminoazetidin-2-one)
α
-
β
N
S
µ
h
rat plasma, and good oral bioavailability after single oral administration in rats. The replacement of the
exocyclic nonanamide residue with a heptyl carbamate moiety, as in derivative 3a, produced an
increase in NAAA inhibitory potency (IC = 0.122 µM, Table 1).[43]
5
0
a
Table 1. Inhibitory Potencies (IC ) of Compounds 3a,e on h-NAAA Activity.
5
0
Compounds
Structure
IC (µM) ± SD
5
0
3
a
c
0
.122 ± 0.022
.037 ± 0.006
HN
O
3
0
O
N
H
O
3d
0.298 ± 0.037
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7

ACCEPTED MANUSCRIPT
3
e
0.513 ± 0.108
0.073 ± 0.008
3b
a
IC50 values are reported as mean values of three or more determinations.
This result prompted us to expand this new series of derivatives in the attempt to further improve
potency. We first replaced the heptyl group of the carbamic acid ester with residues that in the
amino- -lactone analogue series yielded potent NAAA inhibitors.[38] The introduction of a linear
aliphatic octyl moiety afforded the double-digit nanomolar inhibitor 3c (IC = 0.037 M), while the 5-
-biphenylmethyl group resulted in
M), respectively (Table 1).
α
-
β
µ
5
0
phenylpentyl residue, which is found in compound 1, and the
compounds 3d (IC = 0.298 M) and 3e (IC = 0.51
p
µ
µ
5
0
50
While the increase in potency observed with compound 3c is in accordance with the SAR observed in
-amino- -lactone carbamate derivatives, where long alkyl chains are preferred over short ones,[38]
the detrimental effect of a 5-phenylpentyl or a -biphenylmethyl group differentiates the SAR of β-
α
β
p
lactam carbamate derivatives from that of the corresponding
replacement of the long unsubstituted alkyl chain with a short,
β
-lactone series. We then explored the
ω
-cycloalkyl-substituted chain, a moiety
that led to potent NAAA inhibitors in the α-amino-β-lactone series.[37, 38] The introduction of a 4-
cyclohexylbutyl group yielded compound 3b, which allowed recovering double-digit nanomolar
potency, while reducing the length of the alkyl chain. This compound was preferred over 3c for
1
8

ACCEPTED MANUSCRIPT
conducting further studies because available data on
α-amino-β-lactone and α-amino-β-lactam
derivatives showed that molecules bearing a long alkyl chain have lower selectivity for NAAA versus
acid ceramidase, a cysteine amidase showing 33-34% identity and 70% similarity to NAAA and
catalyses the hydrolysis of ceramide, a pro-inflammatory lipid messenger.[63, 64] Compound 3b was
shown to block NAAA covalently via S-acylation of the catalytic cysteine residue in in vitro
experiments, to suppress lung inflammation in mice via a mechanism that requires PEA- and OEA-
mediated PPAR–
macrophages.[8]
α activation, and to prevent endotoxin-induced inflammatory responses in human
Encouraged by these results, we undertook a structure activity relationship (SAR) study in order to
elucidate the stereochemical and structural requirements necessary for NAAA inhibition by compound
3
b and a series of analogues. We explored both the influence of the β-substitution on the β-lactam ring
and the effect of modifications on the carbamic side chain, taking into account structural changes such
as the introduction of heteroatoms, the length of the side-chain, and the insertion of conformationally
constrained moieties.
All synthesized compounds were tested for their ability to inhibit the hydrolysis of
chromen-7-yl)-hexadecanamide (PAMCA) by recombinant human NAAA heterologously expressed in
HEK293 cells ( -NAAA) (see Supporting Information). Median inhibitory concentration (IC ) values
are reported in Tables 1-5.
N-(4-methyl-2-oxo-
h
5
0
A small set of representative compounds was also evaluated for selectivity, and physicochemical and
drug-like properties, such as solubility in buffer, and mouse and rat plasma and liver microsomal
stability (Table 5).
1
9

ACCEPTED MANUSCRIPT
As a first step in our study, we modified the
cyclohexylbutyl carbamate side chain. We explored the role of the stereochemistry at
the effect of mono/di-substitution at -position of the azetidinone ring.
α
-amino
β
-lactam scaffold, while maintaining fixed the 4-
α
-position and
β
A strong stereo-recognition at the NAAA active-site was clearly proved with compound 4b, the (
R)-
enantiomer of 3b, which showed a 40-fold drop in activity (IC = 3.11 µM, Table 2) compared to its
5
0
stereoisomer, in agreement with our previous observation with the 3-aminoazetidin-2-one amide
derivative 2 and its enantiomer.[42] The importance of the ( )-configuration at -position for inhibition
S
α
of NAAA was also confirmed in vivo, as 4b showed no significant anti-inflammatory effect in mouse
models in contrast to its enantiomer 3b.[8]
a
Table 2. Inhibitory Potencies (IC ) of Compounds 3b–10b on h-NAAA Activity.
5
0
Compounds
Structure
IC (µM) ± SD
50
(
S)
3
4
b
b
0.073 ± 0.008
3.11 ± 0.94
(
R)
(
(
S)
S)
5
6
b
b
0.24 ± 0.069
29.36 ± 2.74
2
0

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7
8
b
b
>50
12.91 ± 2.79
9
b
25.42 ± 1.02
17.30 ± 2.23
1
0b
a
IC50 values are reported as mean values of three or more determinations.
Then, as
NAAA inhibition compared to the corresponding unsubstituted derivatives,[35, 38] we turned our
attention to such modification on the -lactam core. Accordingly, we synthesized a small series of 2-
methyl- and 2,2-dimethyl- -lactam carbamic acid esters (Table 2). The insertion of a single methyl
group at C(3) position was in general not beneficial for potency. Among the four 2-methyl- -lactam
diastereoisomers (5b–8b), only compound 5b retained nanomolar activity (IC = 0.24 µM), with the
α-amino β-lactones bearing a methyl group in β-position of the lactone ring showed increased
β
β
β
5
0
other stereoisomers displaying inhibition only in the medium/high micromolar range, with a loss in
potency of >2 orders of magnitude compared to 3b. For these 2-methyl analogues of 3b the ( )-
configuration at -position as well as a defined trans vicinal substitution (as for compound 5b) were
essential to maintain good NAAA inhibition. The insertion of a gem-dimethyl moiety, as for
S
α
2
1

ACCEPTED MANUSCRIPT
compounds 9b and 10b, was detrimental for activity (9b: IC = 25.42 µM; 10b: IC = 17.3 µM, Table
5
0
50
2
).
This first set of modifications of the 3-azetidinone core showed that the configuration of the two
stereogenic centers and the substitution at
of these analogues.
β-position play a key role in the modulation of the potency
Taking into account these results and our previous findings, showing that the
mandatory for the activity,[42] we continued the SAR expansion on -unsubstituted
β
-lactam core is
β
β-lactam carbamic
acid esters. In particular, we decided to synthesize novel analogues of compound 3b to investigate the
effects of the length and conformational flexibility of the aliphatic chain as well as of the insertion of
heteroatoms (Table 3).
a
Table 3. Inhibitory Potencies (IC ) of Compounds 3f–t on h-NAAA Activity.
5
0
Compounds
Structure
IC50 (µM) ± SD
3
f
0.044 ± 0.005
3
g
h
0.049 ± 0.008
1.28 ± 0.122
3
2
2

ACCEPTED MANUSCRIPT
3
3
i
j
2.67 ± 0.343
9.51 ± 1.23
3
k
0.33 ± 0.077
3
l
0.323 ± 0.045
0.033 ± 0.005
1.11 ± 0.342
0.23 ± 0.023
3
m
3
n
o
3
3p
0.52 ± 0.065
2
3

ACCEPTED MANUSCRIPT
3q
0.073 ± 0.003
3
r
30.65 ± 9.56
2.89 ± 0.128
3
s
3
t
1.51 ± 0.17
a
IC50 values are reported as mean values of three or more determinations.
On the basis of the results of the SAR studies on the
β-lactone carbamate[37, 38] and β-lactam
amide[42] classes, we learned that NAAA inhibition could be affected by varying the length of the
aliphatic side chain. A limited exploration of this structural feature was carried out by decreasing (3f)
or increasing (3g) the linear chain of carbamate 3b by a methylene unit. Unexpectedly, both
compounds turned out to be slightly more potent (3f: IC = 0.044 µM; 3g: IC = 0.049 µM, Table 3)
5
0
50
than 3b, differently to what we observed in the β-lactone carbamate series.[37, 38]
Next, we tested whether the replacement of a carbon atom in the side chain with a heteroatom might be
beneficial for both inhibitory activity and physicochemical properties. Disappointingly, the insertion of
2
4

ACCEPTED MANUSCRIPT
an oxygen atom in
β
- (3h),
α
- (3i) or 4’-position (3j) of the cyclohexyl ring was not tolerated, as all the
compounds showed only micromolar activity against NAAA (3h: IC = 1.28 µM; 3i: IC = 2.67 µM,
5
0
50
3
j: IC = 9.51 µM, Table 3). On the contrary a gem-di-fluoro moiety in 4’-position of the cyclohexyl
50
ring (3k, IC50 = 0.33 µM) was well accommodated, showing a marginal 4-fold drop in potency
compared to carbamate 3b.
Switching the 4-cyclohexylbutyl chain of 3b into a more conformationally rigid 4’-butylcyclohexanol-
derived carbamate, as in analogue 3l, resulted only in a limited reduction of NAAA inhibition (IC =
5
0
0
.32 µM).
To further evaluate the role of the terminal cyclohexyl ring, we replaced this group with either a
smaller cyclopentyl residue (3m) or a planar phenyl ring (3n). Whereas a slight improvement in
activity was observed for
β-lactam 3m (IC = 0.033 µM), the phenyl substituted derivative displayed a
50
decrease in potency of ca. 15-fold (3n: IC = 1.11 µM). To test whether substituents on the terminal
5
0
phenyl ring could recover potency, we synthesized compounds 3o–q featuring moieties with different
stereo-electronic properties. Although the 4-ethyl (3o) and 4-methoxy (3p) substituted phenyl
derivatives turned out to be more active than the corresponding unsubstituted analogue 3n (IC50 = 0.23
µM and IC50 = 0.52 µM, respectively), their activity was still 3- to 8-fold lower than that of the
cyclohexyl substituted carbamate 3b. Interestingly, the insertion of a 4-trifluoromethyl group (3q) was
well tolerated, showing similar activity to 3b (IC50 = 0.073 µM). This recovery of activity for
compound 3q could be explained by the more lipophilic nature of the trifluoromethyl group, which
possibly finds better accommodation in the enzyme hydrophobic active site. Finally, while the insertion
of a nitrogen atom in the aromatic ring, as for pyridine derivative 3r, resulted in an almost complete
loss of activity (IC50 = 30.65 µM), rigidifying the side chain with a E- or Z-configured double bond
2
5

ACCEPTED MANUSCRIPT
conjugated to the phenyl ring did not affect inhibitory potency compared to the linear saturated
analogue 3n (3s: IC50 = 2.89 µM; 3t: IC50 = 1.51 µM, Table 3).
Overall, the results reported in Table 3 indicated a clear preference for lipophilic, linear side chains,
and highlighted a limited possibility to capture further interactions by introducing heteroatoms. These
outcomes further support the idea that lipophilic moieties, which resemble the enzyme natural
substrate, are preferentially accommodated in the NAAA active site.
To obtain additional insights into the SAR of this novel chemical class, we evaluated the influence of
the flexibility of the aliphatic side chain on NAAA inhibition. We explored the possibility to fix the 4-
cycloexylbutyl moiety of 3b in a more rigid conformation and then we examined a series of less
conformationally flexible analogues (Table 4).
a
Table 4. Inhibitory Potencies (IC ) of Compounds 3u–am on h-NAAA Activity.
5
0
Compounds
Structure
IC (µM) ± SD
50
3u
1.72 ± 0.24
3
v
12.83 ± 0.21
2
6

ACCEPTED MANUSCRIPT
3
w
0.041 ± 0.009
0.071 ± 0.011
0.513 ± 0.108
3
3
x
e
3
y
z
1.04 ± 0.26
0.16 ± 0.061
0.052 ± 0.018
0.023 ± 0.002
3
3
aa
ab
3
2
7

ACCEPTED MANUSCRIPT
3
ac
0.31 ± 0.056
3
ad
ae
0.14 ± 0.02
3
0.13 ± 0.027
3
af
4.47 ± 1.11
3
3
ag
ah
1.37 ± 0.121
2.07 ± 0.061
0.25 ± 0.049
3
ai
2
8

ACCEPTED MANUSCRIPT
3
aj
2.19 ± 0.433
0.085 ± 0.011
3
ak
3al
8.11 ± 1.99
3am
1.88 ± 0.257
a
IC50 values are reported as mean values of three or more determinations.
First, we replaced the 4-cyclohexylbutyl moiety with a meta-cyclohexyl substituted benzyl carbamate,
as in -lactam 3u. This modification turned out to be detrimental for NAAA inhibition (IC = 1.72
β
5
0
µM), leading to a 25-fold drop in potency compared to 3b. To assess the effect of regiochemistry for
this type of substitution, ortho- (3v) and para- (3w) regioisomers were synthesized and tested. While
compound 3v displayed inhibitory activity only in the high micromolar range (IC = 12.83 µM), the
5
0
para-cyclohexyl benzyl carbamate 3w exhibited a slight increase in potency (IC = 0.041 µM) with
5
0
respect to the more flexible analogue 3b. The finding that a para-substituted benzyl group retained a
higher NAAA inhibitory activity compared to the corresponding ortho- and meta- isomers prompted us
to focus our subsequent efforts on the synthesis of para-substituted benzyl carbamates. We evaluated
2
9